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https://openalex.org/W2894832711	https://europepmc.org/articles/pmc6172778?pdf=render	English	null	A tribute to Ernest E. “Gene” Moore	World journal of emergency surgery	2,018	cc-by	969	-Lord Jonathan Sacks -Lord Jonathan Sacks The executive board, leadership, and membership of WSES take great pride in congratulating Professor Ernest E. “Gene” Moore on the news that the Rocky Mountain Trauma Center at Denver Health Medical Center was named the Ernest E. Moore Shock Trauma center at a ceremony in Denver, Colorado, July 10, 2018, and that a special ceremony in his honor will be held in Denver on September 29, 2018. In the international arena, WSES takes great pride in recognizing Gene’s dedication and commitment to the establishment of our organization. In 2006, Gene joined with Fausto Catena and Luca Ansaloni to establish the World Journal of Emergency Surgery, for which he served as the Editor in Chief for 5 years. In 2009, the same group founded the World Society of Emergency Surgery. Gene’s dedication and commitment to our organization has helped catapult WJES into a leading journal in the field of emergency surgery, with a growing impact factor, and his suggestions and guidance have helped promote WSES as a key player in the establishment of guidelines for the field. Prof. Moore’s ongoing legacy is an inspiration to all. His illustrious career in Trauma and Acute Care Surgery began in 1976 when he, as a young surgeon that had completed his training in Vermont under Dr. John Davis (editor, J. Trauma, 1975–1994), chose to move west and start a new life in Colorado. The young Dr. Moore quickly found himself with big shoes to fill, assuming the leadership of the Trauma services at Denver General Hospital from the illustri- ous Professor Ben Eiseman. Clearly, Dr. Eiseman saw in Dr. Moore a young, talented, and academically in- spired young man who would carry on his trailblazing approach to academic pursuits, patient care, and ad- vancement of the profession. But Gene’s support and commitment did not end with his initial vision and support. He has remained an active participant in our organization, providing constant guidance and relevant recommendations to- wards the growth of our group. Simply said, when Gene rises to speak, everyone listens and takes his re- marks to heart. As truly the father of our specialty, he embodies what an academic surgeon should be, combining a busy clinical practice with outstanding academic pursuits. Dr. Eiseman was not to be disappointed. He lived to see Dr. EDITORIAL Open Access Kashuk et al. World Journal of Emergency Surgery (2018) 13:46 https://doi.org/10.1186/s13017-018-0206-1 Kashuk et al. World Journal of Emergency Surgery (2018) 13:46 https://doi.org/10.1186/s13017-018-0206-1 com edicine, Tel Aviv, Israel © The Author(s). 2018 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. A tribute to Ernest E. “Gene” Moore Jeffry Kashuk, Walter Biffl, Yoram Kluger, Luca Ansaloni and Fausto Catena eral, thoracic, vascular, and cardiac injuries, with the parallel emerging field of Surgical Critical Care, cata- pulted the Denver group into a position of being one of the founding institutions of the new emerging spe- cialty of Acute Care Surgery. Gene had the vision and clarity to drive this agenda on the national stage, helping to create academic viability for the field and then, in later years, transforming the Journal of Trauma into the Journal of Trauma and Acute Care Surgery, highlighting the importance of the emerging specialty of Acute Care Surgery. Good Leaders create followers. Great leaders create leaders -Lord Jonathan Sacks Moore assumes international repute and builds the Denver Health Medical Center into one of the world’s most respected centers. Denver Health’s unique setting where the trauma team cared for a wide variety of issues, including gen- Correspondence: jeffrykashuk@gmail.com Tel Aviv University Sackler Faculty of Medicin * Correspondence: jeffrykashuk@gmail.com Tel Aviv University Sackler Faculty of Medicine, Tel Aviv, Israel * Correspondence: jeffrykashuk@gmail.com Tel Aviv University Sackler Faculty of Medicine, Tel Aviv, Israel © The Author(s). 2018 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 waive (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise state Competing interests h h d l h Competing interests h h d l h p g The authors declare that they have no competing interests. The authors declare that they have no competing interests. Authors’ contributions Authors’ contributions Authors’ contributions All authors read and approved the final manuscript. All authors read and approved the final manuscript. Page 2 of 2 Kashuk et al. World Journal of Emergency Surgery (2018) 13:46 Kashuk et al. World Journal of Emergency Surgery (2018) 13:46 Kashuk et al. World Journal of Emergency Surgery (2018) 13:46 WSES, therefore, takes this opportunity to applaud Gene on this tremendous honor. Your colleagues, stu- dents, and friends remain eternally grateful for your con- tributions and look forward to your continuing contributions to our organization. Indeed, we all deserve and look forward to MOORE!!!!!!! With much respect, admiration, and heartfelt congratulations. The Board and Readership of WSES and World Journal of Emergency Surgery Authors’ contributions All authors read and approved the final manuscript. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Received: 18 September 2018 Accepted: 18 September 2018
https://openalex.org/W2806244589	https://europepmc.org/articles/pmc6011050?pdf=render	English	null	The Genetic Diversity and Geographic Differentiation of the Wild Soybean in Northeast China Based on Nuclear Microsatellite Variation	International journal of genomics	2,018	cc-by	6,333	Hindawi International Journal of Genomics Volume 2018, Article ID 8561458, 9 pages https://doi.org/10.1155/2018/8561458 Hindawi International Journal of Genomics Volume 2018, Article ID 8561458, 9 pages https://doi.org/10.1155/2018/8561458 1Key Laboratory of Vegetation Ecology, Ministry of Education, Institute of Grassland Sciences, Northeast Norm Changchun 130024, China 2Institute of Crop Germplasms, Jilin Academy of Agricultural Sciences, Gongzhuling 136100, China 3Institute of Soybean Research, Jilin Academy of Agricultural Sciences, Changchun 130033, China orrespondence should be addressed to Jixun Guo; gjixun666@163.com and Yingshan Dong; ysdong@cjaas.com Received 25 February 2018; Accepted 8 May 2018; Published 6 June 2018 Received 25 February 2018; Accepted 8 May 2018; Published 6 June 2018 Academic Editor: Marco Gerdol Academic Editor: Marco Gerdol Copyright © 2018 Hongkun Zhao 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 this study, the genetic diversity and population structure of 205 wild soybean core collections in Northeast China from nine latitude populations and nine longitude populations were evaluated using SSR markers. A total of 973 alleles were detected by 43 SSR loci, and the average number of alleles per locus was 22.628. The mean Shannon information index (I) and the mean expected heterozygosity were 2.528 and 0.879, respectively. At the population level, the regions of 42°N and 124°E had the highest genetic diversity among all latitudes and longitudes. The greater the diﬀerence in latitude was, the greater the genetic distance was, whereas a similar trend was not found in longitude populations. Three main clusters (1N, <41°N-42°N; 2N, 43°N-44°N; and 3N, 45°N–>49°N) were assigned to populations. AMOVA analysis showed that the genetic diﬀerentiation among latitude and longitude populations was 0.088 and 0.058, respectively, and the majority of genetic variation occurred within populations. The Mantel test revealed that genetic distance was signiﬁcantly correlated with geographical distance (r = 0 207, p < 0 05). Furthermore, spatial autocorrelation analysis showed that there was a spatial structure (ω = 119 58, p < 0 01) and the correlation coeﬃcient (r) decreased as distance increased within a radius of 250 km. Hongkun Zhao ,1,2 Yumin Wang,3 Fu Xing,1 Xiaodong Liu,3 Cuiping Yuan,3 Guangxun Qi,2 Jixun Guo ,1 and Yingshan Dong 2,3 Hongkun Zhao ,1,2 Yumin Wang,3 Fu Xing,1 Xiaodong Liu,3 Cuiping Yuan,3 Guangxun Qi,2 Jixun Guo ,1 and Yingshan Dong 2,3 1Key Laboratory of Vegetation Ecology, Ministry of Education, Institute of Grassland Sciences, Northeast Normal Univer Changchun 130024, China 1. Introduction Northeast China was probably a very important diversity center for wild soybeans [6–8]. The annual wild soybean (Glycine soja Sieb. and Zucc.), the direct progenitor of the cultivated soybean (Glycine max (Linn.) Merr.), is a predominantly self-pollinated annual plant species [1, 2]. It is widely distributed across most prov- inces of China, with the exceptions of Qinghai, Xinjiang, and Hainan [3]. In Northeast China, the wild soybean is well known for its abundant populations, high population den- sity, and rich phenotypic types [4]. A total of 48 wild soy- bean in situ reserves have been established in China, 14 of which are located in Northeast China. A total of 8518 wild soybean accessions have been ex situ conserved in the National Gene Bank, and nearly half were collected from Northeast China [5]. Some reports have suggested that Genetic diversity is essential to population stability and is the basis of the evolution of species [9]. The genetic diver- sity and population structure of wild soybeans have been described in many reports [10–13]. The wild soybean in Northeast China, which is a very important ecotype, has been used in many previous studies [14–18]. The genetic diversity of wild soybeans in Northeast China was often compared with that in other areas using morphological traits and various molecular markers. Alternatively, wild soybeans from some speciﬁc areas have been analyzed for their distri- bution patterns, origin, evolution, classiﬁcation, and so on. Accordingly, results have not been consistent because diﬀer- ent accessions (or populations), numbers of samples, and International Journal of Genomics 2 Heilongjiang Jilin Liaoning Figure 1: Geographic distribution map of wild soybean accessions in Northeast China. ■, ●, and ▲represent the wild soybean core collections from Heilongjiang (HLJ), Jilin (JL), and Liaoning (LN), respectively. 2 International Journal of Genomics Figure 1: Geographic distribution map of wild soybean accessions in Northeast China. ■, ●, and ▲represent the wild soybean core collections from Heilongjiang (HLJ), Jilin (JL), and Liaoning (LN), respectively. analysis methods have been used. SSR markers have proven to be a reliable tool for determining the diversity of the wild soybean [19]. However, regarding wild soybeans in Northeast China as a single population, their genetic diver- sity and population structure have not been fully understood using molecular markers; in particular, the genetic diﬀerenti- ation of geographical populations of this region has not been well investigated. 1. Introduction The 25 μL PCR reaction buﬀer consisted of 2.5 μL 10x PCR buﬀer (100 mmol/L), Tris-HCl (pH 8.3), 500 mmol/L KCl, 200 mmol/L MgCl2 (0.001% gelatin, 0.1% Np-40), 0.4 μL dNTP (2.5 mmol/L), 2.0 μL each of forward and reverse primers (10 pmol/L), 2.0 μL total DNA (20 ng/μL), 1.0 μL Taq DNA polymerase (2 U/μL), and 15.1 μL ddH2O. PCR was performed using the T100™thermal cycler (Bio-Rad, USA) with the following cycle conditions: an initial denaturing at 95°C for 5 min, followed by 35 cycles of 95°C denaturing for 45 s, 52–57°C annealing for 45 s, 68°C exten- sion for 45 s, and a ﬁnal extension at 72°C for 10min. The objectives of this work were to determine the extent of genetic variation in the wild soybean in Northeast China, to elucidate the geographical structure and genetic diﬀerenti- ation within and between latitude or longitude populations, and, ultimately, to provide valuable information for the scientiﬁc protection and eﬃcient utilization of wild soybean resources in Northeast China. Ampliﬁed products were fractionated by electrophoresis through 6% denaturing polyacrylamide gels and stained with silver staining, which could detect 60 bp to 500 bp fragments and had a high resolution of 2 bp. The size of the stained band was analyzed based on its migration distance relative to the 100 bp DNA ladder (MBI Fermentas) using AlphaView soft- ware (version 1.3.0.7). 2. Materials and Methods 2.1. Plant Material. All the wild soybean accessions in this study were preserved in the Gene Bank of Jilin Academy of Agricultural Sciences. A total of 205 wild soybean acces- sions from Northeast China were selected from the core collection established by Zhao et al. [20]. The collection sites of these samples were located within the region of 39–52°N and 119–133°E, covering 84 counties or districts. Nine latitude populations and nine longitude populations were divided based on a degree interval when the sample size was higher than 10; otherwise, the samples were combined into adjacent latitude or longitude population (see Figure 1 and Supplementary Table 1). 2.3. Data Analysis. The ampliﬁcation fragments of genomic DNA by each SSR marker were scored based on the migra- tion diﬀerence. The data format was converted accordingly in Microsoft Excel. The number of alleles (Na), Shannon- Weaver index (I), expected heterozygosity (He), observed heterozygosity (H0), ﬁxation index (Fis), genetic diﬀerentia- tion coeﬃcient (Fst), genetic identity and genetic distance, molecular variation analysis of variance (AMOVA), Mantel tests, and spatial autocorrelation coeﬃcients were com- puted by GenAIEx v6.5 [23]. The Shannon-Weaver index (I) and expected heterozygosity (He) for evaluating the diversity were measured according to the formulas I = −1 × sum pi × Ln pi and He = 1 – sum pi 2, where pi is the frequency of the ith allele. H0 is generally lower than He due to inbreeding, and Fis = 1 −H0 /He. Outcrossing rate ( t) was calculated using the equation t = 1 −Fis / 1 + Fis [24]. Based on the matrix of Nei’s genetic distance [25], the dendrogram was constructed using NTSYSpc21 [26]. Spatial autocorrelation analysis was performed using “spatial-single 2.2. SSR Analysis. One single fresh leaf was used to extract genomic DNA for each accession using a modiﬁed CTAB method [21]; 43 SSR markers (see Supplementary Table 2), developed from 60 core loci [22] on 20 genetic linkage groups, were used to detect nuclear DNA variation. The primer sequences, with their linkage group locations, are available at https://www.soybase.org/dlpages/#soybasedata. International Journal of Genomics International Journal of Genomics 3 pop”; spatial distance was set as 100 km and the number of The population genetic structure was predicted by Table 1: Genetic diversity of 205 wild soybean accessions by 43 nSSRs. 2. Materials and Methods Number Primer LG Na I H0 He Fis t 1 satt005 Dlb + W 28 2.862 0.025 0.924 0.973 0.014 2 satt022 N 23 2.593 0.010 0.890 0.989 0.006 3 satt099 L 17 2.257 0.020 0.862 0.976 0.012 4 satt112 E 19 2.571 0.010 0.909 0.989 0.006 5 satt146 F 21 2.582 0.034 0.902 0.962 0.019 6 satt168 B2 21 2.743 0.000 0.915 1.000 0.000 7 satt180 C1 18 2.145 0.005 0.813 0.994 0.003 8 satt184 Dla + Q 24 2.658 0.005 0.900 0.995 0.003 9 satt197 B1 31 2.633 0.005 0.876 0.994 0.003 10 satt216 Dlb + W 26 2.295 0.005 0.835 0.994 0.003 11 satt226 D2 22 2.477 0.005 0.888 0.994 0.003 12 satt236 A1 17 2.495 0.015 0.903 0.984 0.008 13 satt239 I 22 2.295 0.005 0.822 0.994 0.003 14 satt242 K 22 2.580 0.005 0.882 0.994 0.003 15 satt243 O 26 2.951 0.015 0.937 0.984 0.008 16 satt267 Dla + Q 18 2.440 0.059 0.890 0.934 0.034 17 satt268 E 18 2.284 0.005 0.859 0.994 0.003 18 satt279 H 28 2.815 0.005 0.916 0.995 0.003 19 satt281 C2 29 2.869 0.005 0.920 0.994 0.003 20 satt286 C2 36 3.124 0.020 0.941 0.979 0.011 21 satt300 A1 20 2.540 0.005 0.903 0.995 0.003 22 satt307 C2 26 2.877 0.061 0.923 0.934 0.034 23 satt308 M 27 2.718 0.015 0.893 0.983 0.009 24 satt309 G 13 1.424 0.005 0.586 0.992 0.004 25 satt334 F 20 2.384 0.010 0.862 0.988 0.006 26 satt345 O 26 2.827 0.005 0.921 0.995 0.003 27 satt346 M 16 2.075 0.005 0.787 0.994 0.003 28 satt352 G 24 2.675 0.010 0.902 0.989 0.006 29 satt373 L 22 2.311 0.005 0.847 0.994 0.003 30 satt386 D2 21 2.489 0.005 0.891 0.994 0.003 31 satt390 A2 25 2.593 0.000 0.894 1.000 0.000 32 satt429 A2 26 2.786 0.005 0.916 0.995 0.003 33 satt431 J 23 2.628 0.005 0.893 0.995 0.003 34 satt434 H 19 2.394 0.000 0.878 1.000 0.000 35 satt453 B1 20 2.301 0.005 0.847 0.994 0.003 36 satt462 L 23 2.618 0.010 0.895 0.989 0.006 37 satt487 O 19 2.279 0.005 0.866 0.994 0.003 38 satt530 N 19 2.330 0.005 0.868 0.994 0.003 39 satt571 B2 21 2.520 0.026 0.897 0.971 0.015 40 satt586 F 33 2.994 0.005 0.932 0.995 0.003 41 satt588 K 26 2.619 0.000 0.896 1.000 0.000 42 satt590 M 22 2.570 0.029 0.899 0.967 0.017 43 satt596 J 16 2.071 0.005 0.835 0.994 0.003 Mean 22.628 2.528 0.011 0.879 0.987 0.007 LG = linkage group. 3. Results 3.1. SSR Polymorphism and Geographic Variation. Two hun- dred ﬁve representative wild soybean accessions across Northeast China were used in this study. A total of 973 alleles were detected from 43 SSR loci, and the percentage of poly- morphic loci was 100%. The number of alleles per SSR marker varied from 13 (Satt309) to 36 (Satt286), with an average of 22.628. The mean Shannon information index (I) and expected heterozygosity (He) were 2.528 and 0.879, respectively (see Table 1). At the population level (see Table 2), the Shannon infor- mation index (I) of nine latitude populations ranged from 1.300 to 2.419, with an average of 1.716; the expected hetero- zygosity (He) ranged from 0.661 to 0.884, with an average of 0.756. The region of 42°N had the highest genetic diversity among all latitudes. The Shannon information index (I) of nine longitudes varied from 1.659 to 2.368, averaging 1.919; the expected heterozygosity (He) varied from 0.751 to 0.880, averaging 0.805. The region of 124°E had the highest genetic diversity among all longitudes. As shown in Tables 3 and 4, the smaller the latitude diﬀerence was, the higher the genetic identity was; however, similar trends were not found in longitude populations. Two major geographical clustering groups (N and S) can be seen in the UPGMA dendrogram. Group N consists of four northern latitude groups (46°N–>49°N), and group S consists of ﬁve southern latitude groups (<41°N–45°N) (see Figure 2). The results indicate that the genetic diversity of wild soybean accessions in Northeast China is related to their latitudinal origin. The Fst value was used to evaluate the genetic diﬀerenti- ation of wild soybean populations at the scales of latitude and longitude in Northeast China (see Tables 3 and 4). Pairwise Fst values for latitude populations ranged from 0.040 to 0.183, and pairwise Fst values for longitude populations ranged from 0.034 to 0.132. In general, moderate diﬀerentia- tion (0.05 < Fst <0.15) [29] was observed between most latitude and longitude populations, and the genetic diﬀeren- tiation among adjacent groups was relatively low. These results were conﬁrmed by AMOVA; most of the genetic var- iations were found within latitude and longitude populations (see Table 6). The Mantel test indicated that there was a positive corre- lation between geographic and genetic distance (r = 0 207, p < 0 05), which suggests that geographic distance limits gene ﬂow among populations and inﬂuences the genetic structure. 3. Results For further analyses, spatial autocorrelation analysis was per- formed by distance classes of 100 km, and a general decline was found in the correlation coeﬃcient (r) with distance. The correlation values were negative and signiﬁcant up to 250 km. This revealed that there is a clinal spatial structure in wild soybeans in Northeast China (ω = 119 58, p < 0 01) (see Figure 3). 3.2. Population Structure and Genetic Diﬀerentiation. The STRUCTURE procedure was run to predict genetic struc- ture for each predeﬁned latitude and longitude population. When k was 3, Δk was the highest, which indicated that 3 main clusters had been identiﬁed (see Table 5). For the lati- tude population, 83.8% of individuals from the <41°N region and 50.2% of individuals from the 42°N region were assigned to Cluster1N, most individuals from the 43°N to 44°N region to Cluster2N, and individuals from the 45°N to >49°N regions to Cluster3N. These results were roughly consistent with those of hierarchical cluster analysis (see Figure 2). Some admixtures were found among the 41°N–45°N regions, which probably were important transitional areas. For the longitude population, most of the individuals from the <122°E region were separated from all others and formed a cluster (Cluster1E); three regions of 123°E, 125°E, and 129°E were assigned to Cluster2E, and three regions of 127°E, 128°E, and 130°E to Cluster3L. Admixtures were found widely among 9 predeﬁned longitude populations. The regions of 42°N, 124°E, and 126°E were special, as they were not dominated (<60%) by the three clusters. 2. Materials and Methods Table 1: Genetic diversity of 205 wild soybean accessions by 43 nSSRs. Table 1: Genetic diversity of 205 wild soybean accessions by 43 nSSRs. pop”; spatial distance was set as 100 km, and the number of permutations and the bootstraps of the selection mode were set as 999 times. pop”; spatial distance was set as 100 km, and the number of permutations and the bootstraps of the selection mode were set as 999 times. The population genetic structure was predicted by STRUCTURE 2.3.4 [27]. The default k value was set to 1 to 12, and ten runs were performed for each value of k to test 4 International Journal of Genomics Table 2: Genetic diversity of diﬀerent geographical populations in Northeast China. stability of the results. The MCMC (Markov chain Monte Carlo) value was set as 100,000 burn-in with 200,000 iterations. The correct number of genetic clusters was inferred according to a value of Δk Δk = mean lnP k + 1 −2lnP k + lnP k −1 /Sd lnP k [28]. Longitude Pop. I He Longitude Pop. I He <41°N 1.915 0.808 <122°E 1.959 0.820 42°N 2.419 0.884 123°E 1.808 0.789 43°N 2.063 0.816 124°E 2.368 0.880 44°N 1.621 0.751 125°E 2.067 0.833 45°N 1.882 0.790 126°E 2.094 0.827 46°N 1.302 0.661 127°E 1.771 0.780 47°N 1.521 0.733 128°E 1.661 0.769 48°N 1.300 0.662 129°E 1.880 0.797 >49°N 1.419 0.699 >130°E 1.659 0.751 Total 1.716 0.756 Total 1.919 0.805 Pop. = population. 4. Discussion In our study, the results indicate that the region of 42°N and 124°E has the highest level of genetic diversity Table 4: Nei’s genetic distance and genetic diﬀerentiation among longitude populations. Group <122°E 123°E 124°E 125°E 126°E 127°E 128°E 129°E >130°E <122°E — 0.103 0.049 0.082 0.096 0.124 0.106 0.095 0.132 123°E 0.974 — 0.055 0.052 0.044 0.066 0.075 0.065 0.083 124°E 0.535 0.535 — 0.036 0.039 0.060 0.055 0.049 0.069 125°E 0.818 0.438 0.404 — 0.036 0.052 0.065 0.042 0.076 126°E 0.929 0.345 0.374 0.306 — 0.035 0.035 0.037 0.034 127°E 1.304 0.504 0.563 0.426 0.286 — 0.060 0.061 0.072 128°E 0.980 0.572 0.520 0.537 0.294 0.460 — 0.050 0.056 129°E 0.897 0.500 0.479 0.364 0.298 0.468 0.397 — 0.068 >130°E 1.168 0.545 0.530 0.533 0.227 0.463 0.377 0.443 — Pop. = population; genetic diﬀerentiation coeﬃcient (Fst) (above diagonal); Nei’s genetic identity (below diagonal). 41°N 42°N 43°N 44°N 45°N 46°N 47°N 48°N 49°N 0.43 0.50 0.57 Coefficient 0.64 0.72 Figure 2: UPGMA dendrogram based on Nei’s genetic identity among the latitudes. Table 4: Nei’s genetic distance and genetic diﬀerentiation among longitude populations. Group <122°E 123°E 124°E 125°E 126°E 127°E 128°E 129°E >130°E <122°E — 0.103 0.049 0.082 0.096 0.124 0.106 0.095 0.132 123°E 0.974 — 0.055 0.052 0.044 0.066 0.075 0.065 0.083 124°E 0.535 0.535 — 0.036 0.039 0.060 0.055 0.049 0.069 125°E 0.818 0.438 0.404 — 0.036 0.052 0.065 0.042 0.076 126°E 0.929 0.345 0.374 0.306 — 0.035 0.035 0.037 0.034 127°E 1.304 0.504 0.563 0.426 0.286 — 0.060 0.061 0.072 128°E 0.980 0.572 0.520 0.537 0.294 0.460 — 0.050 0.056 129°E 0.897 0.500 0.479 0.364 0.298 0.468 0.397 — 0.068 >130°E 1.168 0.545 0.530 0.533 0.227 0.463 0.377 0.443 — Pop. = population; genetic diﬀerentiation coeﬃcient (Fst) (above diagonal); Nei’s genetic identity (below diagonal). Table 4: Nei’s genetic distance and genetic diﬀerentiation among longitude populations. 41°N 42°N 43°N 44°N 45°N 46°N 47°N 48°N 49°N 0.43 0.50 0.57 Coefficient 0.64 0.72 Figure 2: UPGMA dendrogram based on Nei’s genetic identity among the latitudes. 41°N 42°N 43°N 44°N 45°N 46°N 47°N 48°N 49°N 0.43 0.50 0.57 Coefficient 0.64 0.72 Figure 2: UPGMA dendrogram based on Nei’s genetic identity among the latitudes. Figure 2: UPGMA dendrogram based on Nei’s genetic identity among the latitudes. The wild soybean is widely distributed in Northeast Asia. The higher its genetic diversity is, the greater its habitat- expansion capacity and environmental adaptation are [38]. 4. Discussion The wild soybean in Northeast China is an important ecotype, and its genetic diversity has been widely studied by using phenotypic traits [7, 30, 31] and molecular markers [17]. The common view has been that the wild soybean in this region possesses a high genetic variation. In this study, the average allele number, Shannon index (I), and expected het- erozygosity (He) were 22.628, 2.528, and 0.879, respectively, which were signiﬁcantly higher than previously reported results [32–34]. The rich genetic variation could be attrib- uted to the fact that the samples in this study were selected from the core collection, which has been deﬁned as a subset of a crop species preserved with the most abundant repeti- tiveness [35, 36]. On the other hand, the large sample size and various geographical origins might also result in high diversity; 205 samples used in this study, accounting for International Journal of Genomics 5 Table 3: Nei’s genetic distance and genetic diﬀerentiation among latitude populations. Group <41°N 42°N 43°N 44°N 45°N 46°N 47°N 48°N >49°N <41°N — 0.042 0.079 0.101 0.111 0.183 0.150 0.178 0.162 42°N 0.447 — 0.040 0.061 0.062 0.120 0.091 0.119 0.105 43°N 0.674 0.346 — 0.049 0.066 0.139 0.111 0.140 0.119 44°N 0.790 0.489 0.334 — 0.064 0.156 0.120 0.142 0.126 45°N 0.941 0.499 0.435 0.402 — 0.096 0.080 0.118 0.112 46°N 1.428 0.801 0.818 0.825 0.446 — 0.093 0.155 0.163 47°N 1.441 0.788 0.800 0.784 0.486 0.417 — 0.086 0.098 48°N 1.432 0.858 0.886 0.762 0.615 0.640 0.414 — 0.074 >49°N 1.491 0.896 0.823 0.77 0.674 0.780 0.550 0.349 — Pop. = population; genetic diﬀerentiation coeﬃcient (Fst) (above diagonal); Nei’s genetic identity (below diagonal). Table 3: Nei’s genetic distance and genetic diﬀerentiation among latitude populations. 85% of core collections in Northeast China, were selected from 242 core collections developed by Zhao et al. [20]. These accessions may have a continuous distribution in Northeast China (see Supplementary Table 1). Furthermore, 43 SSR primers covering 20 linkage groups might also be important causes for the detection of richer genetic variation [37]. The wild soybean is widely distributed in Northeast Asia. The higher its genetic diversity is, the greater its habitat- expansion capacity and environmental adaptation are [38]. Therefore, it also forms a speciﬁc natural distribution pat- tern [39]. 4. Discussion Therefore, it also forms a speciﬁc natural distribution pat- tern [39]. In our study, the results indicate that the region of 42°N and 124°E has the highest level of genetic diversity 85% of core collections in Northeast China, were selected from 242 core collections developed by Zhao et al. [20]. These accessions may have a continuous distribution in Northeast China (see Supplementary Table 1). Furthermore, 43 SSR primers covering 20 linkage groups might also be important causes for the detection of richer genetic variation [37]. International Journal of Genomics 6 Table 5: Inferred population structure based on latitude populations and longitude populations. Table 5: Inferred population structure based on latitude populations and longitude populations. Pop. Inferred clusters Pop. Inferred clusters Cluster1N Cluster2N Cluster3N Cluster1E Cluster2E Cluster3E <41°N 0.158 0.004 0.838 <122°E 0.891 0.106 0.003 42°N 0.431 0.066 0.502 123°E 0.078 0.690 0.232 43°N 0.916 0.019 0.065 124°E 0.462 0.360 0.179 44°N 0.943 0.054 0.002 125°E 0.122 0.714 0.164 45°N 0.246 0.741 0.013 126°E 0.029 0.433 0.539 46°N 0.034 0.962 0.003 127°E 0.002 0.303 0.695 47°N 0.021 0.977 0.003 128°E 0.053 0.305 0.642 48°N 0.052 0.946 0.002 129°E 0.110 0.614 0.276 >49°N 0.151 0.847 0.002 >130°E 0.003 0.203 0.795 Pop. = population. Table 6: AMOVA analysis of diﬀerent geographical populations. Table 6: AMOVA analysis of diﬀerent geographical populations. Group Source of variation SS MS Est. Var. Percentage of variation Fst p Latitude Among pops 723.151 90.394 1.695 9% 0.088 0.001 Within pops 7058.358 17.602 17.602 91% Longitude Among pops 539.012 67.376 1.111 6% 0.058 0.001 Within pops 7242.498 18.061 18.061 94% Probability, p (rand ≥data), for Fst is based on standard permutation across the full data set. Fst = Est. Var. among pops/(Est. Var. among pops + Est. Var. within pops); SS = the sums of squares; MS = the mean sums of squares; Est. Var. = the estimated variance. Probability, p (rand ≥data), for Fst is based on standard permutation across the full data set. Fst = Est. Var. among pops/(Est. Var. among pops + Est. Var. within pops); SS = the sums of squares; MS = the mean sums of squares; Est. Var. = the estimated variance. Probability, p (rand ≥data), for Fst is based on standard permutation across the full data set. Fst = Est. Var. among pops/(Est. Var. among pops + Est. Var. 4. Discussion within pops); SS = the sums of squares; MS = the mean sums of squares; Est. Var. = the estimated variance. −0.040 −0.020 0.000 0.020 0.040 0.060 0.080 0 100 200 300 400 500 600 700 800 900 r Distance class (start point) Results of spatial structure analysis r U L Figure 3: Results of spatial structure analysis. r: solid lines represent spatial autocorrelation coeﬃcients; U and L: dashed lines represent 95% conﬁdence interval. −0.040 −0.020 0.000 0.020 0.040 0.060 0.080 0 100 200 300 400 500 600 700 800 900 r Distance class (start point) Results of spatial structure analysis Results of spatial structure analysis Figure 3: Results of spatial structure analysis. r: solid lines represent spatial autocorrelation coeﬃcients; U and L: dashed lines represent 95% conﬁdence interval. (see Table 2), and the results roughly agree with those of pre- vious studies based on morphological traits [7]. The results support the view that the genetic diversity of wild soybeans in Northeast China is related to latitude but not to longitude; three evolutionarily signiﬁcant units were distinguished by latitude, corresponding to regions of <41°N-42°N, 43°N- 44°N, and >45°N (see Tables 3–5). Moderate diﬀerentiation among the latitude populations (the mean Fst value was 0.088) and longitude populations (the mean Fst value was 0.058) occurred (see Table 6). This implies that natural selection might be the main cause of genetic structure [8, 29]. Previous studies have revealed that wild soybean genotypes exhibit regional distributions at diﬀerent geo- graphical scales [15, 40, 41], which are especially associated with latitudinal origin. However, some studies have also reported that the genetic diﬀerentiations were associated with longitude origins; for example, Leamy et al.’s results showed that the four genetic groups (Central China, Northern China, Korea, and Japan) diﬀered more in longi- tude than in latitude [13]. Possible explanations for those 7 International Journal of Genomics Supplementary Materials The Mantel test revealed that there was a positive rela- tionship between geographic and genetic distance (r = 0 207, p < 0 05), which indicates that geographical isolation has also been an important factor in forming the current genetic structure of wild soybeans in Northeast China. Spatial autocorrelation analysis revealed that the correla- tion between geographical distance and genetic distance is limited. Supplementary Table 1: geographic origin and grouping information of wild soybean core collections in Northeast China. Supplementary Table 2: list of SSR loci. (Supplementary Materials) References The wild soybean in Northeast China has become one of the most severely endangered wild plant species due to the interference of human activities [45]. The genetic diversity of wild soybeans is high, indicating that the wild soybean in this region has great potential for evolution, and in situ con- servation is preferable. Although wild soybean accessions ex situ conserved in the National Gene Bank are more substan- tial than those from other areas, the collection in this area is still very limited, so further investigation and collection works in this region are necessary. According to distribution patterns of the genetic diversity of wild soybeans in Northeast China, the conservation strategy should emphasize individ- ual protection, and protection in areas with high genetic diversity should be prioritized. [1] S. L. Broich and R. G. Palmer, “A cluster analysis of wild and domesticated soybean phenotypes,” Euphytica, vol. 29, no. 1, pp. 23–32, 1980. [2] J. Abe, A. Hasegawa, H. Fukushi, T. Mikami, M. Ohara, and Y. Shimamoto, “Introgression between wild and cultivated soybeans of Japan revealed by RFLP analysis for chloroplast DNAs,” Economic Botany, vol. 53, no. 3, pp. 285–291, 1999. [3] B. C. Zhuang, Biological Studies on Wild Soybean in China, Science press, Beijing, China, 1999. [4] F. S. Li, “Study on origin and evolution of soybean,” Soybean Science, vol. 13, pp. 61–66, 1994. [5] K. J. Wang and X. H. Li, “Exploration and studies of wild soy- bean germplasm resources in the China Genebank during recent decade,” Journal of Plant Genetic Resources, vol. 13, no. 508, pp. 507–514, 2012. [6] Y. Fukuda, “Cytogenetical studies on the wild and cultivated Manchurian soybeans (Glycine L),” Japanese Journal of Bot- any, vol. 6, pp. 489–506, 1933. Acknowledgments This work was supported by the National Science Foun- dation of China (31300282), the Agricultural Science and Technology Innovation Project (CXGC2017JQ018, CXGC2017ZD014), and the National Key Research and Development Program (2016YFD0100201-19). Data Availability results may include small sample size, large geographic span, strait isolation, and diverse ecosystems. All the wild soybean accessions in this study were preserved in the Gene Bank of Jilin Academy of Agricultural Sciences, and the geographical information for all samples is attached in Supplementary Table 1. The primer sequences with their linkage group locations are available at https://www.soybase .org/dlpages/#soybasedata. Genetic structure was mainly determined by the breeding system, gene ﬂow, distance isolation, and so on [42]. Wild soybean is a strictly self-pollinating plant, with limited pollen ﬂow. In general, for self-pollination-dominated plants, with an average Gst = 0 51, the total genetic variation among the populations accounts for more than half of the genetic struc- ture; for out-crossing-dominated species Gst = 0 10, 90% of genetic variation occurs within populations [43]. In the pres- ent study, most of the genetic variation was found between individuals within populations, with less than 10% among populations (see Table 6). This suggests that the genetic diﬀerentiation among latitude or longitude populations in Northeast China is similar to that of out-crossing- dominated species [17, 40]. Zhao speculated that this phe- nomenon could be explained by out-crossing rates and long-distance gene ﬂow [44]. Our results show that the out- crossing rate of Northeast China wild soybeans is only 0.7% (Fis = 0 987) (see Table 1), which conﬁrms its selﬁng mating reproductive system and plays an important role in keeping a strong genetic structure. Conflicts of Interest All authors have reported no ﬁnancial interests or potential conﬂicts of interest regarding the publication of this article. International Journal of Genomics 16, no. 2, pp. 133–142, 2008. [15] D. Xu, J. Abe, J. Gai, and Y. Shimamoto, “Diversity of chloro- plast DNA SSRs in wild and cultivated soybeans: evidence for multiple origins of cultivated soybean,” TAG Theoretical and Applied Genetics, vol. 105, no. 5, pp. 645–653, 2002. [33] Y. N. Liu, L. I. Xiang-Hua, and K. J. Wang, “Analysis of the genetic variability for the mini-core collection of Chinese wild soybean (Glycine soja) collection in the National Gene Bank based on SSR markers,” Journal of Plant Genetic Resources, vol. 10, pp. 211–217, 2009. [16] Z. Li and R. L. Nelson, “RAPD marker diversity among culti- vated and wild soybean accessions from four Chinese prov- inces,” Crop Science, vol. 42, no. 5, pp. 1737–1744, 2002. [17] X. H. Li, K. J. Wang, and J. Z. Jia, “Genetic diversity and diﬀer- entiation of Chinese wild soybean germplasm (G. soja Sieb. & Zucc.) in geographical scale revealed by SSR markers,” Plant Breeding, vol. 128, no. 6, pp. 658–664, 2009. [34] H. Fan, T. J. Zhao, Y. L. Ding, G. N. Xing, and J. Y. Gai, “Genetic analysis of the characteristics and geographic diﬀer- entiation of Chinese wild soybean population,” Scientia Agri- cultura Sinica, vol. 45, pp. 414–425, 2012. [18] Y. Wang, F. Ghouri, M. Q. Shahid, M. Naeem, and F. S. Baloch, “The genetic diversity and population structure of wild soy- bean evaluated by chloroplast and nuclear gene sequences,” Biochemical Systematics and Ecology, vol. 71, pp. 170–178, 2017. [35] O. Frankel, Genetic Perspectives of Germplasm Conservation. Genetic Manipulation: Impact on Man and Society, Cambridge University Press, Cambridge, UK, 1984. [36] A. H. D. Brown, “Core collections: a practical approach to genetic resources management,” Genome, vol. 31, no. 2, pp. 818–824, 1989. [19] Y. Kuroda, N. Tomooka, A. Kaga, S. M. S. W. Wanigadeva, and D. A. Vaughan, “Genetic diversity of wild soybean (Glycine soja Sieb. et Zucc.) and Japanese cultivated soybeans [G. max (L.) Merr.] based on microsatellite (SSR) analysis and the selection of a core collection,” Genetic Resources and Crop Evo- lution, vol. 56, no. 8, pp. 1045–1055, 2009. [37] M. A. Nawaz, S. H. Yang, H. M. Rehman et al., “Genetic diver- sity and population structure of Korean wild soybean (Glycine soja Sieb. and Zucc.) inferred from microsatellite markers,” Biochemical Systematics and Ecology, vol. 71, pp. 87–96, 2017. [38] L. F. International Journal of Genomics 8 [25] M. Nei, “Genetic distance between populations,” The Ameri- can Naturalist, vol. 106, no. 949, pp. 283–292, 1972. [10] Y. H. Li, W. Li, C. Zhang et al., “Genetic diversity in domesti- cated soybean (Glycine max) and its wild progenitor (Glycine soja) for simple sequence repeat and single-nucleotide poly- morphism loci,” New Phytologist, vol. 188, no. 1, pp. 242– 253, 2010. [26] F. J. Rohlf, NTSYSps: Numerical Taxonomy System, ver.2.20. Setauket, Exeter publishing Ltd., NY, USA, 2008. [27] J. K. Pritchard, M. Stephens, and P. Donnelly, “Inference of population structure using multilocus genotype data,” Genet- ics, vol. 155, no. 2, pp. 945–959, 2000. [11] S. He, Y. Wang, S. Volis, D. Li, and T. Yi, “Genetic diversity and population structure: implications for conservation of wild soybean (Glycine soja Sieb. et Zucc) based on nuclear and chloroplast microsatellite variation,” International Jour- nal of Molecular Sciences, vol. 13, no. 12, pp. 12608–12628, 2012. [28] G. Evanno, S. Regnaut, and J. Goudet, “Detecting the number of clusters of individuals using the software STRUCTURE: a simulation study,” Molecular Ecology, vol. 14, no. 8, pp. 2611–2620, 2005. [12] J. Guo, Y. Liu, Y. Wang et al., “Population structure of the wild soybean (Glycine soja) in China: implications from microsatel- lite analyses,” Annals of Botany, vol. 110, no. 4, pp. 777–785, 2012. [29] S. Wright, Variability within and among Natural Populations. Evolution and the Genetics of Populations, University of Chi- cago Press, Chicago, USA, 1978. [30] X. Yan, H. Zhao, X. Liu et al., “Phenotypic traits and diversity of diﬀerent leaf shape accessions of the wild soybean (Glycine soja Sieb. et Zucc.) in China,” Canadian Journal of Plant Sci- ence, vol. 94, no. 2, pp. 397–404, 2014. [13] L. J. Leamy, C.-R. Lee, Q. Song et al., “Environmental versus geographical eﬀects on genomic variation in wild soybean (Glycine soja) across its native range in northeast Asia,” Ecol- ogy and Evolution, vol. 6, no. 17, pp. 6332–6344, 2016. [31] L. Sun, H. K. Zhao, F. Zhao, and Y. S. Dong, “Analysis of genetic diversity of Glycine soja in Northeast China,” Soybean Science, vol. 34, pp. 355–360, 2015. [14] J. J. Doyle, “5S ribosomal gene variation in the soybean and its progenitor,” Theoretical and Applied Genetics, vol. 75, no. 4, pp. 621–624, 1988. [32] D. Yanlai, Z. Tuanjie, and G. Junyi, “Genetic diversity and eco- logical diﬀerentiation of Chinese annual wild soybean (Glycine soja),” Biodiversity Science, vol. 5. Conclusions [7] Y. S. Dong, B. C. Zhuang, L. M. Zhao, H. Sun, and M. Y. He, “The genetic diversity of annual wild soybeans grown in China,” TAG Theoretical and Applied Genetics, vol. 103, no. 1, pp. 98–103, 2001. In summary, the genetic diversity and geographic population structure of the wild soybean in Northeast China were fully investigated as a single population, or at diﬀerent latitude or longitude populations for the ﬁrst time. The distribution pattern of genetic variation is related to latitude, and the highest level of genetic diversity was found at 42°N, and pro- tection in areas with higher genetic diversity should be prior- itized. This study disclosed that natural selection to adapt temperature and photoperiod, selﬁng mating reproductive system, and distance isolation resulted in the current popula- tion structure of wild soybean in Northeast China. [8] Z. Wen, Y. Ding, T. Zhao, and J. Gai, “Genetic diversity and peculiarity of annual wild soybean (G. soja Sieb. et Zucc.) from various eco-regions in China,” Theoretical and Applied Genet- ics, vol. 119, no. 2, pp. 371–381, 2009. [9] P. Taberlet, L. Fumagalli, A.-G. Wust-Saucy, and J.-F. Cosson, “Comparative phylogeography and postglacial colonization routes in Europe,” Molecular Ecology, vol. 7, no. 4, pp. 453– 464, 1998. International Journal of Genomics Hunneke, “Ecological implications of genetic variation in plant populations,” in Genetics and Conservation of Rare Plants, D. A. Falk and K. E. Holsinger, Eds., pp. 31–44, Oxford University Press, New York, 1991. [20] Z. Limei, D. Yingshan, L. Bao, H. Shui, W. Kejing, and L. Xianghua, “Establishment of a core collection for the Chi- nese annual wild soybean (Glycine soja),” Chinese Science Bul- letin, vol. 50, no. 10, p. 989, 2005. [39] X. Li, K. Wang, F. Li, and M. Yan, “Research progress of wild soybean (Glycine soja) and suggestions for improving its eﬀec- tive utilization and protection,” Soybean Science, vol. 24, pp. 305–309, 2005. [21] S. O. Rogers and A. J. Bendich, “Extraction of DNA from mil- ligram amounts of fresh, herbarium and mummiﬁed plant tis- sues,” Plant Molecular Biology, vol. 5, no. 2, pp. 69–76, 1985. [22] H. Xie, R. Z. Chang, Y. S. Cao, M. H. Zhang, Z. F. Feng, and L. J. Qiu, “Selection of core SSR loci by using Chinese autumn soybean,” Scientia Agricultura Sinica, vol. 36, pp. 360–366, 2003. [40] Y. T. Kiang, Y. C. Chiang, and N. Kaizuma, “Genetic diversity in natural populations of wild soybean in Iwate Prefecture, Japan,” Journal of Heredity, vol. 83, no. 5, pp. 325–329, 1992. [41] W. An, H. K. Zhao, S. Ying et al., “Genetic diversity in annual wild soybean (Glycine soja Sieb. et Zucc.) and cultivated soy- bean (g. Max. Merr.) from diﬀerent latitudes in China,” Paki- stan Journal of Botany, vol. 41, no. 5, pp. 2229–2242, 2009. [23] R. Peakall and P. E. Smouse, “GenALEx6.5: genetic analysis in Excel. Population genetic software for teaching and research- an update,” Bioinformatics, vol. 28, no. 19, pp. 2537–2539, 2012. [42] R. Z. Qu, L. Hou, H. L. Lü, and H. Y. Li, “The gene ﬂow of pop- ulation genetic structure,” Hereditas, vol. 26, no. 3, pp. 377– 382, 2004. [24] B. S. Weir, Genetic Data Analysis II, Sinauer Associates, Sun- derland, MA, 1996. 9 9 International Journal of Genomics [43] J. L. Hamrick and M. J. W. Godt, “Allozyme diversity in plant species,” in Plant Population Genetics, Breeding, and Genetic Resources, A. H. D. Brown, M. T. Clegg, A. L. Kahler, and B. S. Weir, Eds., Sinauer Associates Inc. Sunderland, Massachu- setts, USA, 1990. [44] R. Zhao, “Exploring basic problems in conservation genetics of wild soybean (Glycine soja L.). Ph D thesis,” Fudan University, China, 2007. [45] S.-L. He, Y.-S. Wang, D.-Z. Li, and T.-S. Yi, “Environmental and historical determinants of patterns of genetic diﬀerentia- tion in wild soybean (Glycine soja Sieb. et Zucc),” Scientiﬁc Reports, vol. 6, no. 1, article 22795, 2016.
https://openalex.org/W4297916202	https://zenodo.org/record/5384797/files/216758_CRV.pdf	English	null	Towards Multi-Criteria Prioritization of Best Practices in Research Artifact Sharing	arXiv (Cornell University)	2,021	cc-by	2,692	Open peer review artifacts for this paper are available at https://zenodo.org/communities/ opensciense2021 Towards Multi-Criteria Prioritization of Best Practices in Research Artifact Sharing 1Faculty of Science (iCIS) - Radboud University Nijmegen, the Netherlands 2Sao Carlos Engineering School (EESC) - University of Sao Paulo (USP), Brazil 3Universidad Adolfo Ib´a˜nez (UAI), Chile {d.damasceno,d.strueber}@cs.ru.nl, isotilia@gmail.com {d.damasceno,d.strueber}@cs.ru.nl, isotilia@gmail.com Abstract. Research artifact sharing is known to strengthen the transparency of scientiﬁc studies. However, in the lack of common discipline-speciﬁc guidelines for artifacts evaluation, subjective and conﬂicting expectations may happen and threaten artifact quality. In this paper, we discuss our preliminary ideas for a framework based on quality management principles (5W2H) that can aid in the establishment of common guidelines for artifact evaluation and sharing. Also, using the Analytic Hierarchy Process, we discuss how research communities could join efforts to aid the guidelines’ adequacy to research priorities. These combined methodologies constitute a novelty for software engineering research which can foster research software sustainability. 2. Proposed Framework According to the Project Management Institute (PMI), project management is deﬁned as the application of knowledge, skills, tools, and techniques to project activities to meet the project requirements [PMI 2017]. Among the project management knowledge areas, quality management is fundamental as it applies to all projects, regardless of the nature of their deliverables. The objectives of project quality management are to incorporate the organization’s quality policy regarding planning, managing, and controlling project and product quality requirements so that stakeholders’ expectations are met. In SE research, artifact stakeholders may include funding agencies, research collaborators, artifact users, or AEC members. Artifact quality requirements are speciﬁc to the type of artifact being produced and the research domain. Thus, they should be identiﬁed and documented a priori. The plan quality management is the process of identifying quality requirements and/or standards for a project and its artifacts and documenting how a project shall demonstrate compliance with such quality requirements and/or standards. Otherwise, it may have serious negative consequences for the project stakeholders. With this in mind, in the next sections, we in- troduce a framework for designing and prioritizing quality guidelines for artifact sharing. In Figure 1, we illustrate a schema of our proposed framework. Figure 1. Proposed Framework for Design and Prioritization of Quality Guidelines Figure 1. Proposed Framework for Design and Prioritization of Quality Guidelines 1. Introduction Research artifacts are known to allow others to build ideas upon existing knowledge, adopt novel ideas in practice, and increase the likelihood of citations. By ”artifact” we mean any digital object created to be used as part of a study or generated in an experiment [ACM 2020]. This deﬁnition covers software systems, scripts used to run experiments, input datasets, raw data collected in the experiment, or scripts used to analyze results. While artifact sharing is known to be a social good [Timperley et al. 2021], the creation and maintenance of high-quality research artifacts can be challenging as re- searchers may have different expectations toward artifact quality [LCRDM 2021]. Also, the current lack of discipline-speciﬁc guidelines for research data management (RDM) [Marjan Grootveld et al. 2018] opens the opportunity to misunderstandings on the poten- tial of artifacts and threats to the sustainability of research projects [Hermann et al. 2020]. To address these issues, we envision the need for studies experimenting with project man- agement principles for artifact quality management in software engineering (SE) research. In this paper, we present initial thoughts on a generic framework for SE research artifact quality management. Our framework is envisioned as a guide for artifact stake- holders (e.g., users, authors, reviewers, artifact evaluation communities - AECs) to iden- tify essential concerns (i.e., research practices and frequently asked questions) in artifact quality management and prioritize them based on project constraints. Particularly, we aim to extend the work of [Damasceno and Str¨uber 2021] beyond its initial scope. We envision that their work [Damasceno and Str¨uber 2021] can be im- proved with prioritization principles, such as the Analytic Hierarchy Process (AHP) [Saaty and Vargas 2012]. The AHP can provide a participatory model that accommo- dates the interests of the research community and AEC members in a mutually prioritized and agreed-upon guideline. Thus, we can encompass the research community and AEC members’ viewpoints of what concerns should be given higher priority in artifact cre- ation and review. Finally, we believe that the work of [Damasceno and Str¨uber 2021] has the potential to help in other types of research artifacts and domains, e.g., datasets for machine learning [Lindauer and Hutter 2020], data collected in surveys [Hui et al. 2019]. 2.2. Community Survey and Prioritization of Practices Once a set of useful factual questions is agreed upon and documented, time and resource constraints may limit the investments in creating and maintaining an artifact. Thus, pri- oritization techniques could be helpful to rank artifact quality requirements, as done in traditional software requirements management [Pitangueira et al. 2013]. In the prioritization literature, the Analytic Hierarchy Process (AHP) stands out as a practical tool that can incorporate users’ preferences for decision making through the pair-wise judgment of possible solutions for a given problem [Yoo et al. 2009]. Particu- larly, the AHP constitutes an interesting model where artifact stakeholders can join efforts to categorize different quality concerns (i.e., factual questions) and research practices ac- cording to their relative importance towards the goal of sharing high-quality artifacts. Developed in the 1970s, the AHP is a structured multi-criteria decision-making approach that is underpinned by psychology and mathematics principles. In the AHP, individual domain-speciﬁc experts (e.g., AEC members, SE research community) esti- mate the relative importance of factors through pair-wise comparison (i.e., how much important is it to answer a given factual question?). Then, this prioritization criteria can be constrained to attend the preferences of reviewers or a research community by using mathematical consistency assumptions [Saaty and Vargas 2012, Colin 2000]. In our framework, we envision the AHP as a practical means for ranking Artifact quality concerns and Research best practices. In Figure 2, we provide a schema of our AHP-based solution to prioritizing artifact quality requirements and research practices. In our framework, we propose the AHP as a tool to ﬁnd out “What is the hier- archical importance priority of the artifact quality concerns established through 5W2H method?”. These quality concerns should be agreed upon among AEC members as be- ing useful questions that artifact authors should provide answers to. Once this set of quality concerns is agreed upon, AEC members shall individually indicate the relative importance of those questions through pair-wise comparisons. As recommended by the AHP method, experts should assign grades from one to nine to say which alternative out of a pair they consider as more important [Saaty and Vargas 2012, Colin 2000]. Second, the AHP shall be applied again to identify “What is the hierarchical im- portance priority of a list of best practices for artifact sharing?”. This should be answered by SE community members, also through pair-wise comparisons of research best prac- tices. 2.1. Identiﬁcation of Research Practices and Artifact Quality Concerns To understand the concept of artifact quality requirements, there is an extensive toolbox of methods [Tague 2005] that can be used. Among them, the 5W2H (an acronym for What, Where, Why, Who, When, How, and How Much) constitutes a simple but powerful method for inquiring questions about a problem, as well as for research planning, analysis, and reviews. We see the 5W2H as a useful method to identify and document factual questions that artifact stakeholders may frequently inquire about an artifact and indicate What con- cerns should artifact stakeholders keep in mind?. For example, some factual questions that could be asked are: What is the context of the development of this artifact? Where is this artifact hosted? Why was this artifact created? Who are the artifact authors? When did changes in this artifact happen? How to reproduce the experiment? How much RAM does it require?. To address these 5W2H questions, we assume there is a list of research best practices explicitly documented or implicitly known by a given research community. This list of practices could be cataloged in collaboration with domain experts, e.g., via community surveys, one-to-one interviews, or by means of literature review. 2.2. Community Survey and Prioritization of Practices As in the ACM SIGSOFT Empirical Standards [Ralph et al. 2020], community Figure 2. The AHP-Based Prioritization of Quality Guidelines Figure 2. The AHP-Based Prioritization of Quality Guidelines members can be asked to rank best practices according to their relative priority levels, e.g., Essential, Desirable, Extraordinary. One advantage of segmenting this hierarchy is that AEC members can indepen- dently establish domain-speciﬁc or SE-wide artifact quality concerns, while the SE com- munity can establish their own best practices used to address a given quality concern. On the other hand, considerations on the extenuation of experts during extensive pair-wise analysis should also be raised. To surpass this issue, AHP variants that rely on a re- duced number of comparisons [Leal 2020], inter-cluster prioritization [Yoo et al. 2009], or metaheuristics [Bose 2020] could be explored as alternative prioritization methods. In summary, using this framework, AEC members could tackle the need for high- quality research artifacts by bringing into the spotlight the most important research best practices for artifact sharing and factual questions that researchers should concern about an artifact. The AHP is possibly an adequate approach once it promotes a hierarchical representation of the problem, considering different users’ judgments and preferences through the mathematical aggregation of priorities; and fosters the community engage- ment on reaching an agreement of what research practices and artifact quality concerns are of utmost importance for research software sustainability [LCRDM 2021]. 3. Preliminary Results In the SE literature, there is a lack of domain-speciﬁc quality guidelines for artifact shar- ing. To the best of our knowledge, [Damasceno and Str¨uber 2021] have been the ﬁrst to investigate quality guidelines for Model-Driven Engineering (MDE) research artifacts. In MDE research, there is a limited number of data sets of benchmark models of diverse modeling languages and application domains [Basciani et al. 2015]. Moreover, the need for consolidated artifact sharing practices in MDE research has recently become more pronounced, as the community targets a broader use of artiﬁcial intelligence (AI) techniques. To beneﬁt from the advances in AI and deep learning, MDE researchers need to have access to larger open data sets and high conﬁdence measures for their quality. Thus, having more systematic artifact sharing practices would help to increase the im- pact of MDE research and support the thorough evaluation of MDE research tools and techniques. To contribute towards the quality of MDE research artifacts, [Damasceno and Str¨uber 2021] introduced a set of guidelines for artifact sharing speciﬁcally tailored to MDE research. To design these guidelines, they systematically analyzed general-purpose research practices for artifact sharing used by major SE venues, categorized them according to the 5W2H method, and tailored them to MDE research artifacts. Subsequently, the authors conducted an online survey among 90 researchers and practitioners with expertise in MDE. In this survey, participants were asked to rank each item of the proposed guideline as essential, desirable, or unnecessary; and evaluate them with respect to clarity, completeness, and relevance. In each of these dimensions, the proposed guidelines were assessed positively by more than 92% of the participants. We believe that the results by [Damasceno and Str¨uber 2021] can be extended by considering the multi-criteria prioritization of their guidelines. Prioritization techniques, as the AHP, may be helpful to manage artifact quality based on the relevance of both practices and quality concerns. Besides, we believe that their proposed framework could also be applied to other research domains, not exclusively MDE projects, and investigate how it could support artifact quality management in artifact evaluation. 4. Final Remarks In this paper, we bring into the spotlight the limited knowledge on SE research artifact quality management. As we discuss, artifact quality management has an impact on the sustainability of research artifacts. Hence, research artifact quality concerns and practices for artifact sharing should receive more attention from the SE research community. To ﬁll this gap, we propose the combined application of quality management principles, the 5W2H method, and the AHP for designing and prioritizing artifact quality requirements. For future works, we recommend a community-wide initiative towards identifying useful research practices and factual questions about artifact quality concerns, applying this proposed framework. Once the community develops such a catalog of quality con- cerns and practices, AEC members could move towards a ranking of agreed-upon quality concerns and adopt the proposed guidelines in their daily routine and yearly conferences. This combined methodology constitutes a novelty for SE research project management that can foster more open and sustainable SE research and artifact development. References [ACM 2020] ACM (2020). Artifact Review and Badging - Current. [ACM 2020] ACM (2020). Artifact Review and Badging - Current. [Basciani et al. 2015] Basciani, F., Di Rocco, J., Di Ruscio, D., Iovino, L., and Pierantonio, A. (2015). Model repositories: Will they become reality? In CloudMDE@ MoDELS, pages 37–42. [Bose 2020] Bose, A. (2020). Using genetic algorithm to improve consistency and retain authenticity in the analytic hierarchy process. OPSEARCH, 57(4):1070–1092. [Colin 2000] Colin, E. C. (2000). Pesquisa Operacional. 170 Aplicac¸˜oes em Estrat´egia, Financ¸as, Log´ıstica, Produc¸˜ao, Marketing e Vendas. Atlas. [Damasceno and Str¨uber 2021] Damasceno, C. D. N. and Str¨uber, D. (2021). Quality guide- lines for research artifacts in model-driven engineering. In MoDELS’21: ACM/IEEE 24th International Conference on Model Driven Engineering Languages and Systems, [Damasceno and Str¨uber 2021] Damasceno, C. D. N. and Str¨uber, D. (2021). Quality guide- lines for research artifacts in model-driven engineering. In MoDELS’21: ACM/IEEE 24th International Conference on Model Driven Engineering Languages and Systems, Virtual Event, Japan, 10-15 October, 2021. ACM. http://arxiv.org/abs/ 2108.04652. [Hermann et al. 2020] Hermann, B., Winter, S., and Siegmund, J. (2020). Community ex- pectations for research artifacts and evaluation processes. In Proceedings of the 28th ACM Joint Meeting on European Software Engineering Conference and Symposium on the Foundations of Software Engineering. ACM. [Hui et al. 2019] Hui, W., Lui, S. M., and Lau, W. K. (2019). A reporting guideline for IS survey research. 126:113136. [LCRDM 2021] LCRDM (2021). Research software sustainability in the Nether- lands: Current practices and recommendations. Technical report, [Zenodo] DOI:10.5281/zenodo.4543569. [Leal 2020] Leal, J. E. (2020). AHP-express: A simpliﬁed version of the analytical hierar- chy process method. MethodsX, 7:100748. [Lindauer and Hutter 2020] Lindauer, M. and Hutter, F. (2020). Best practices for scien- tiﬁc research on neural architecture search. Journal of Machine Learning Research, 21(243):1–18. [Marjan Grootveld et al. 2018] Marjan Grootveld, Leenarts, E., Jones, S., Hermans, E., and Fankhauser, E. (2018). OpenAIRE and FAIR Data Expert Group survey about Horizon 2020 template for Data Management Plans. [Pitangueira et al. 2013] Pitangueira, A. M., Maciel, R. S. P., de Oliveira Barros, M., and Andrade, A. S. (2013). A systematic review of software requirements selection and prioritization using sbse approaches. In Ruhe, G. and Zhang, Y., editors, Search Based Software Engineering, pages 188–208, Berlin, Heidelberg. Springer Berlin Heidelberg. [PMI 2017] PMI (2017). A guide to the project management body of knowledge. PMBOK guide. References Project Management Institute (PMI), Newtown Square, PA, 6th edition. [Ralph et al. 2020] Ralph, P., Baltes, S., Bianculli, D., Dittrich, Y., Felderer, M., Feldt, R., Filieri, A., Furia, C. A., Graziotin, D., He, P., Hoda, R., Juristo, N., Kitchenham, B., Robbes, R., Mendez, D., Molleri, J., Spinellis, D., Staron, M., Stol, K., Tamburri, D., Torchiano, M., Treude, C., Turhan, B., and Vegas, S. (2020). ACM SIGSOFT Empirical Standards. arXiv:2010.03525 [cs]. arXiv: 2010.03525. [Saaty and Vargas 2012] Saaty, T. L. and Vargas, L. G. (2012). Models, Methods, Concepts & Applications of the Analytic Hierarchy Process, volume 175 of International Series in Operations Research & Management Science. Springer US. Tague 2005] Tague, N. R. (2005). The quality toolbox. ASQ Quality Press, 2nd edition. [Timperley et al. 2021] Timperley, C. S., Herckis, L., Le Goues, C., and Hilton, M. (2021). Understanding and improving artifact sharing in software engineering research. Em- pirical Software Engineering, 26(4):67. [Yoo et al. 2009] Yoo, S., Harman, M., Tonella, P., and Susi, A. (2009). Clustering test cases to achieve effective and scalable prioritisation incorporating expert knowledge. In Proceedings of the 18th International Symposium on Software Testing and Analysis, ISSTA ’09. ACM.
https://openalex.org/W2017719254	https://dash.harvard.edu/bitstream/1/41288156/1/49587%20aam%20nihms158880.pdf	English	null	Is workplace smoking policy equally prevalent and equally effective among immigrants?	Journal of epidemiology and community health	2,009	cc-by	9,623	Terms of Use This article was downloaded from Harvard University’s DASH repository, and is made available under the terms and conditions applicable to Other Posted Material, as set forth at http:// nrs.harvard.edu/urn-3:HUL.InstRepos:dash.current.terms-of-use#LAA Citation Osypuk, T L, S V Subramanian, I Kawachi, and D Acevedo-Garcia. 2009. “Is Workplace Smoking Policy Equally Prevalent and Equally Effective among Immigrants?” Journal of Epidemiology & Community Health 63 (10): 784–91. https://doi.org/10.1136/jech.2008.079475. Permanent link http://nrs.harvard.edu/urn-3:HUL.InstRepos:41288156 http://nrs.harvard.edu/urn-3:HUL.InstRepos:41288156 1Corresponding author & current affiliation: Theresa L. Osypuk; Assistant Professor, Northeastern University, Bouvé College of Health Sciences, Department of Health Sciences, 360 Huntington Avenue, Robinson 316, Boston, MA 02115 USA. Phone: 617-373-3667, Fax: 617-373-2968, tosypuk@neu.edu, Work undertaken while Dr. Osypuk was a doctoral student at Harvard School of Public Health; Department of Society, Human Development, & Health; Boston, MA USA. NIH Public Access Author Manuscript NIH-PA Author Manuscript Published in final edited form as: J Epidemiol Community Health. 2009 October ; 63(10): 784–791. doi:10.1136/jech.2008.079475. "The Corresponding Author has the right to grant on behalf of all authors and does grant on behalf of all authors, an exclusive license (or non exclusive for government employees) on a worldwide basis to the BMJ Publishing Group Ltd and its Licensees to permit this article to be published in Journal of Epidemiology & Community Health editions and any other BMJPGL products to exploit all subsidiary rights, as set out in our license (http://jech.bmj.com/ifora/licence.pdf)" Theresa L. Osypuk, SD, SM1, S.V. Subramanian, PhD2, Ichiro Kawachi, MD, PhD3, a Dolores Acevedo-Garcia, PhD, MPA-URP2 2Associate Professor of Society, Human Development, and Health; Harvard School of Public Health; Department of Society, Human Development, & Health, Boston, MA USA 3Professor of Social Epidemiology; Harvard School of Public Health; Department of Society, Human Development, & Health, Boston, MA USA NIH-PA Author Manuscript Share Your Story The Harvard community has made this article openly available. Please share how this access benefits you. Submit a story . The Harvard community has made this article openly available. Please share how this access benefits you. Submit a story . Accessibility NIH-PA Author Manuscript Is Workplace Smoking Policy Equally Prevalent and Equally Effective Among Immigrants? r Manuscript NIH-PA Author Manuscript Theresa L. Osypuk, SD, SM1, S.V. Subramanian, PhD2, Ichiro Kawachi, MD, PhD3, and Dolores Acevedo-Garcia, PhD, MPA-URP2 MeSH Keywords MeSH Keywords NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Auth Environmental Tobacco Smoke; Environmental Smoke Pollution; Tobacco; Immigrants; Public Policy; Inequalities Environmental Tobacco Smoke; Environmental Smoke Pollution; Tobacco; Immigrants; Public Policy; Inequalities A substantial body of evidence suggests that Environmental Tobacco Smoke (ETS) is a cause of morbidity and mortality.[1,2] Increasing the strength and reach of tobacco control policy is one explicit path to reducing tobacco-related disease,[3] the single leading preventable cause of US deaths.[4] Although workplace smokefree laws were originally enacted to protect workers from ETS, their major benefit is that they discourage smoking among smokers as well. [5] Tobacco use regulations do not protect everyone equally, however. Those with lower education, and those in blue collar, service or agricultural jobs are less likely to be covered by smokefree policies than college graduates and white collar/professional workers.[5–7] Moreover, smokefree policies may be more weakly enforced in blue-collar occupations.[8] African Americans and Hispanics are also less likely to be covered by a workplace smokefree policy than whites,[5,7] and more likely to experience high smoke exposure.[3,9] As a growing segment of the U.S. population,[1,10] racial/ethnic minorities carry a higher burden compared to whites of developing and dying from cancer and other tobacco-related disease.[1,11–13] Although the causes explaining these racial/ethnic differentials in disease burden remain poorly understood,[11] exposure to second-hand smoke may be one contributing factor. NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA A Although it is often conflated with racial/ethnicity in health analyses, immigrant status (or nativity) is a distinct dimension of social inequality and may partially explain racial/ethnic smoking-related disparities. Together, immigrants and their children comprise over 22% of the US population,[14–16] and disproportionately contribute to the low-wage and low-skilled end of the labor force.[17,18] Immigrants are less likely than the US-born to enjoy workplace benefits including union membership or union contract coverage,[19] or health insurance coverage.[20] Given this evidence, immigrants may also be less likely to be covered by workplace smoking policies. In this analysis, we explore how tobacco ETS policy is differentially prevalent among, and may differentially affect the smoking behavior of, different nativity groups in the US. We examine two hypotheses: that workplace smokefree policy is (1) less prevalent among immigrants, and (2) less effective for immigrants (with respect to smoking) than for the US- born population. The literature is currently limited for addressing these research questions. J Epidemiol Community Health. Author manuscript; available in PMC 2010 October 1. Abstract Background—We examined whether immigrants were less likely to be covered by a smokefree workplace policy, as well as whether workplace smoking policies garnered comparable associations with smoking for immigrants and the US-born, in the US. Methods—We applied the 2001/02 Current Population Survey Tobacco Use Supplement among US indoor workers (n=85,784) using multiple logistic regression analyses. First, we examined whether nativity (immigrants vs. US-born) was independently associated with smokefree policy coverage. Second, we examined whether the smokefree policy association with current smoking was differential by nativity (effect modification). Results—Immigrants were less likely to work in smokefree workplaces than the US-born; however occupation and industry accounted for these disparities. Employment in a workplace that was not smokefree was associated with higher odds of smoking (vs. smokefree workplaces), both before (OR=1.83, 95% CI:1.74–1.92) and after (OR=1.36, (1.29–1.44)) covariate adjustment among the US-born, but associations were weaker among immigrants (OR=1.39(1.20–1.61) unadjusted, OR=1.15(0.97–1.35) adjusted). Worker industry partly explained (16% of) the weaker policy- smoking association among immigrants, while other socioeconomic variables reduced the policy- smoking association without explaining the disparity. NIH-PA Author Manuscript NIH-PA Author Manuscript Conclusions—The patchwork of US workplace smoking restriction policy at different governmental levels, combined with a voluntary regime among some employers, generates coverage inequalities. Workplace smokefree policies may be less effective for immigrants, and this is related to differential coverage by such policies due to occupational segregation. Understanding the complex patterns of the social context of smoking is important for understanding how policy interventions might have heterogeneous effects for different demographic groups. Page 2 NIH-PA Author Manuscript Osypuk et al. METHODS We used the 2001–02 Tobacco Use Supplement (TUS) of the Current Population Survey (CPS) [27,28] which has valid and reliable tobacco use questions, high response rates, yielding nationally-representative estimates for the US non-institutionalized, civilian population aged 15 years and older. The CPS is conducted by the Census Bureau and employs a multistage, stratified probability sample. TUS questions determining smoking status are identical to those used by the National Center for Health Statistics.[6,29,30] MeSH Keywords Some articles have reported point estimates of workplace smoking policy prevalence for different racial groups,[6,7,21] but have not examined prevalence among nativity subgroups, or have not examined policy associations with smoking. Although a few studies have examined racial differences in smokefree policy-smoking associations with multivariate analyses, they did not control for nativity (immigrant vs. US-born), or model interactions of race/ethnicity and gender.[5,21,22] NIH-PA Author Manuscript NIH-PA Author Manuscript Modeling demographic interactions are important since gender, race/ethnicity, and nativity interact powerfully for smoking patterns. For instance, the protective effects of being foreign- born for smoking are much more pronounced for racial minority groups than whites, and for women than men, even after adjusting for demographic and economic covariates.[23] Moreover, whites and the US-born are numerically dominant in nationally representative datasets. If there is heterogeneity of policy effects on smoking, a statistical model that omits demographic interactions produces estimates essentially for US-born whites, masking differential patterns for other groups. Osypuk et al. Osypuk et al. Page 3 NIH-PA Author Manuscript Immigrants may exhibit a weaker effect of smokefree policy than native-born counterparts for four reasons, including confounding by prior common causes (#3–4): (1) if smokefree workplace policies are most influential for groups with the highest prevalence and daily consumption of cigarettes,[5] (2) if other causes are more powerful than workplace policy for influencing smoking among immigrants, such as anti-tobacco socialization and social norms, [23–25] (3) if immigrants have fewer resources to facilitate quitting,[26] or (4) if immigrants are more likely to be in blue collar and service jobs[17] or industries which have lower smokefree policy coverage[5–7] or weaker enforcement.[8] Immigrants may exhibit a weaker effect of smokefree policy than native-born counterparts for four reasons, including confounding by prior common causes (#3–4): (1) if smokefree workplace policies are most influential for groups with the highest prevalence and daily consumption of cigarettes,[5] (2) if other causes are more powerful than workplace policy for influencing smoking among immigrants, such as anti-tobacco socialization and social norms, [23–25] (3) if immigrants have fewer resources to facilitate quitting,[26] or (4) if immigrants are more likely to be in blue collar and service jobs[17] or industries which have lower smokefree policy coverage[5–7] or weaker enforcement.[8] NIH-PA Author Manuscript Variables NIH-PA Author Manuscript Current smoking was modeled dichotomously, defined as having smoked 100 cigarettes in lifetime and currently smoking every day or some days; non smokers were referents.[31] We excluded values missing for the smoking variable (n=1512,0.6% of sample). Workplace smoking policy was a dichotomous variable (smokefree vs. non-smokefree workplace), assessed using 3 items. Those answering no to this question (“Does your place of work have an official policy that restricts smoking in any way?”) had no policy at work regarding smoking, and were coded as non-smokefree. Those answering yes were asked 2 questions to ascertain their workplace’s smoking policy for work areas, and for indoor public or common areas, such as lobbies, rest rooms, lunch rooms. The possible answers to these questions were: smoking is not allowed in any areas; smoking is allowed in some areas; or smoking is allowed in all areas. We classified people in smokefree workplaces if they answered yes to the first question, and “smoking is not allowed in any areas” to the 2 subsequent questions.[5,7] We classified other responses as non-smokefree workplaces. As discussed elsewhere[6,7,32] the TUS asks the workplace smoking policy questions only among indoor workers who were self-respondents, thus this group comprises our sample. We excluded those missing data for these policy questions (n=2192). Our final sample size was 85,784. NIH-PA Author Manuscript NIH-PA Author Manuscript Nativity was modeled dichotomously: either foreign-born or US-born. Those born in the US, in Puerto Rico, or born abroad to US citizens were classified as US-born by the TUS. Foreign born is used interchangeably with the term immigrant throughout the manuscript. We modeled two additional immigrant-related variables including language of interview (English or other), and immigrant length of stay in the US (described further in Table 1). Race/ethnicity was self- reported, restricted to the four largest groups in the US (excluding Native Americans, due to few foreign-born), and coded: non-Hispanic (NH) white, NH black, NH Asian/Pacific Islander and Hispanics of any race. Routine labor force questions were used by the Bureau of Labor Statistics to determine employment status, occupation, and worker industry classification.[6,7,27] For regression, we collapsed occupation into 6 standard categories, and collapsed industrial sector into 8 standard categories.[7] Please see the notes in Table 2 for occupation/industry variable detail in regressions. Education was measured as highest level of school completed or degree received. J Epidemiol Community Health. J Epidemiol Community Health. Author manuscript; available in PMC 2010 October 1. Variables Author manuscript; available in PMC 2010 October 1. Osypuk et al. Page 4 NIH-PA Author Manuscript Income was measured as total annual household earnings, modeled in $10,000 groups, including a missing category. We additionally adjusted for gender, age (age, age2, both centered at 45), marital status, and state of residence (fixed effects). Please see Table 1 for additional covariate coding detail. Income was measured as total annual household earnings, modeled in $10,000 groups, including a missing category. We additionally adjusted for gender, age (age, age2, both centered at 45), marital status, and state of residence (fixed effects). Please see Table 1 for additional covariate coding detail. NIH-PA Author Manuscript NIH-P Analytic Methods We used SAS 9.1.3 for univariate and bivariate analyses; we used SUDAAN 10.0 for multiple logistic regression analyses to adjust for the complex survey design by applying sample weights and replicate weights, created using a Balanced Repeated Replication method (personal communication, Anne Hartman, NCI, 3/23/05 and 8/12/07). We produced bivariates and calculated chi-squared tests among demographic variables with smoking prevalence, smokefree policy coverage, and immigrant composition. In logistic regression models, we first regressed smokefree policy coverage on nativity, before and after adjusting for covariates (Model 1 A-P). In these models (Models 1), we predicted the odds of being in a smokefree workplace (1) vs. a non-smokefree workplace (0). We next regressed current smoking on smokefree policy (Models 2–6), to test the main effect of policy (Models 2), and to test nativity interactions with policy (Models 3) in unstratified models. We then stratified by nativity (Models 4–5), and then by nativity, race/ethnicity and gender (Model 6), to examine policy associations with smoking. In Models 2–6, smokefree policy was reverse coded from Model 1, so that in Models 2–6 we modeled the association between the odds of smoking for those in a non-smokefree workplace (1) compared with those in a smokefree workplace (0). In model 3, the logit interaction coefficient represents the differential policy effectiveness among foreign born; a negative coefficient denotes a weaker policy association with smoking for immigrants compared to the US born. The 95% confidence intervals were adjusted for design effects. We present parallel models across Models 1–5. Models A were unadjusted. Models B-N built on Model A to add one variable at a time, to examine changes in the nativity (Model 1) and/or policy (Models 2–5) coefficients. Models P adjusted for all covariates simultaneously. NIH-PA Author Manuscript Approval to conduct this study was provided by the Harvard School of Public Health Human Subjects Committee (#P11687-101). J Epidemiol Community Health. Author manuscript; available in PMC 2010 October 1. Logistic Regression Results NIH-PA Author Manuscript NIH-PA Au Association of Nativity with Smokefree Policies—Table 2 demonstrates that immigrants have significantly lower odds of workplace smokefree policy coverage compared with the US-born in unadjusted models (Model 1A, OR=.90, 95%CI: 0.83,0.96), and in several models adjusted for certain demographic variables one at a time. However, there are no significant independent associations of nativity with smokefree workplaces after addition of occupation or industry (models 1B–1C), after addition of social class, race, or nativity-related variables, or in models adjusting for all covariates simultaneously (Model 1P). These results suggest that immigrants’ differential employment by occupation and industry category accounts for their lower probability of smokefree policy coverage. Association of Smokefree Policies with Current Smoking—Table 3 demonstrates that workplace smoking policy was associated with smoking in the expected direction both before and after covariate adjustment, and all smoking policy associations were significant at the .10 level. In unstratified main effect models, we observed higher odds of smoking in non- smokefree workplaces compared to smokefree workplaces in unadjusted models (OR=1.77, 95% CI: (1.69,1.85), Model 2A). The odds ratios of smoking declined with addition of potential confounding variables; addition of occupation, industry, income and education to each model (2–5, C–F) contributed to the largest declines in the smokefree policy-smoking odds ratio. For example, with the addition of occupation (2C), the smoking odds ratio associated with a non- smokefree worksite declined 34% to 1.51 compared to the unadjusted model 2A. In a model adjusted for all factors, the smoking odds ratio associated with working in a non-smokefree worksite was 1.34 (1.27,1.41) (Model 2P). The policy-smoking associations from Model 2 were similar to the results for the US-born in the interaction models (Model 3) and in the stratified models (Model 4). NIH-PA Author Manuscript NIH-PA Author Manuscript The association between workplace policy and smoking was weaker among immigrants compared to the US-born on average (Table 3,Model 5). The smoking odds ratio for immigrant workers in a non-smokefree environment was only 1.39 in unadjusted models (CI:1.20,1.61, Model 5A) compared to 1.83 among the US-born (Model 4A); this policy-smoking association among immigrants declined to 1.15 (0.97,1.35) but remained significant in fully adjusted models (Model 5P), compared to 1.36 among US-born (4P). Although confounding factors (e.g. Descriptive Analysis Table 1 presents descriptive statistics. All chi-squared tests of differences were significant at p<.001. Twenty-one percent of US indoor workers were current smokers in 2001–2002. Those born in the US were more likely to smoke than were US immigrants. The majority of US indoor employees worked in smokefree workplaces (70.5%). We observed lower smoking prevalence in smokefree workplaces (18.2%) compared with non-smokefree workplaces (28.2%). Figure 1 presents the variation in prevalence of smokefree policies by employee race/ethnicity, gender, and nativity. Foreign-born Hispanic men are least likely to be covered by a smokefree policy at work – only 58% are in smokefree workplaces vs. 65% for US-born Hispanic men. NIH-PA Author Manuscript Table 1 also shows that workers experience differential smokefree policy coverage depending on occupation and industry category, in addition to demographic attributes. For example, workers in professional specialty occupations and professional industries (education, public administration) experience the highest coverage by workplace smokefree policies. These industries also have an underrepresentation of immigrants (compared to the 12.3% prevalence of immigrants in the US). Conversely, service and blue collar occupations, as well as service, construction, manufacturing, and agricultural industries have low coverage of smokefree policies and are overrepresented by immigrants. Page 5 Page 5 NIH-PA Author Manuscript Osypuk et al. Logistic Regression Results occupation, income) reduced the odds ratio between policy and smoking among both the US born and the foreign born, the logit interaction for foreign born was affected little across all models (Model 3), except for industry. Addition of industry to the model did reduce the nativity-policy interaction coefficient by 16% (from −27 in Model 3B to −23 Model 3D). This suggests that some of the weaker policy association for foreign born was due to confounding by industry. NIH-PA Author Manuscript In models stratified by nativity, race/ethnicity and gender, (Model 6,Table 4) we found differential associations between smoking and smoking policy across subgroups. Although most US-Born and immigrant groups experienced beneficial smoking associations of being in smokefree workplaces (Models 6P), the associations for immigrants were generally weaker than the associations observed among the US born. We lastly observed some unexpected inverse effects for foreign-born Black women, and null associations for US-born Asians and black men, and for most immigrant groups. J Epidemiol Community Health. Author manuscript; available in PMC 2010 October 1. DISCUSSION This study has three principal findings. First, immigrants are less likely to work in jobs where they are covered by a smokefree policy, and this disparity is accounted for by the industries and occupations where immigrants disproportionately work. Second, individuals in non- smokefree workplaces exhibited higher odds of smoking than their counterparts in smokefree Osypuk et al. Osypuk et al. Page 6 NIH-PA Author Manuscript workplaces, and ETS policy appeared to be more strongly associated with smoking among the US-born than among immigrants on average. Third, there is heterogeneity of the ETS policy effect with smoking across racial and gender subgroups, signifying that the “average” effect of policy from unstratified models was driven by patterns among US-born whites. We discuss these patterns below. NIH-PA Author Manuscript NIH-PA A Our manuscript extends prior literature focusing on workplace smoking policy disparities by race/ethnicity and gender,[6,7,21] to document that immigrants are less likely to be covered by smokefree policies than the US-born on average, and that this disparity is attributable to occupational and industry segregation. Blue collar and service workplaces are less likely to voluntarily enact smokefree policies compared to white collar workplaces. [6] Moreover, clean indoor air policies often exempt jobs with higher concentrations of minorities, e.g. manufacturing jobs [8] or service jobs in restaurants, bars, and hotels.[6,33] Since some workplaces enact their smoking policies based on employee request,[8] doing so may disadvantage employees in industries or occupations dominated by immigrants, since immigrants may be less vocal to advocate for workers’ rights if they are illegal residents and/ or not citizens. A voluntary policy regime will therefore be less effective for covering certain minority groups because occupational segregation will induce systematic disparities. Mandating smokefree workplaces must therefore be a matter of federal policy, rather than relegated to individual states or municipalities, or to voluntary bans by the private sector – the situation which currently describes the US context of tobacco control policy. Such decentralized policy context generates inequalities in smokefree-policy coverage; moreover, when smoking bans are applied across the board nationally, it helps to eliminate race/ethnic and socioeconomic disparities in smokefree workplaces. J Epidemiol Community Health. Author manuscript; available in PMC 2010 October 1. DISCUSSION [34] NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA A We found evidence that tobacco policy exhibits weaker average associations with smoking among foreign-born respondents compared to native born counterparts.[23] Smokefree policies may be more effective for the US-born since they have higher smoking prevalence and consumption than the foreign-born,[35] and these patterns generally held in our study. Among immigrants, other factors may be more influential for smoking than workplace tobacco policy. Family context or cultural norms in an immigrant’s country of origin may play a more important role to influence smoking among immigrants, although we could not test those variables with our data, including family anti-tobacco socialization or norms that smoking is socially unacceptable for women, e.g. among Asian immigrants,[36] gender roles and concomitant access to power or status (e.g. being confined to traditional gender roles),[37] socialization with smoking peers,[38] or the stage of the tobacco epidemic in the sending country.[23,39] Alternately, if immigrants have higher job instability and therefore a shorter duration of coverage by smokefree workplace policies, this may explain their weaker associations with smoking. NIH-PA Author Manuscript NIH-PA Author Manuscript Although we posited weaker or null associations among immigrants between workplace tobacco policy and smoking, we did not expect inverse associations, which we found among black immigrant women. We believe that results among black immigrants were due to very small cell sizes (few smokers in non-smokefree workplaces), which is related to the low prevalence of smoking among black immigrants. The heterogeneous policy-smoking associations observed across these demographic subgroups demand further attention in future studies, since our analysis could not sort out the reasons underlying the heterogeneity. With our data, we could test several hypotheses to explain weaker policy associations with smoking among immigrants. We found immigrant-related variables did not affect policy- smoking associations. We found some evidence that differential industries accounted for 16% of the weaker policy effects among immigrants, but occupational adjustment had little effect J Epidemiol Community Health. Author manuscript; available in PMC 2010 October 1. Osypuk et al. Page 7 on the differential. Adding covariates did, however, reduce the coefficients for the policy main effects, indicating social class and demographic characteristics did confound the association of policy with smoking for both nativity groups. Strengths and Limitations One of the strengths of this analysis is that the CPS data is representative of the US population, which is especially important for examination of health disparities, considering the small populations of some minority/nativity groups. Second, our explicit focus on demographic subgroups builds on the inequality literature aiming to ameliorate disparities in tobacco use and tobacco-related disease. Third, we appropriately accounted for the complex survey design of the CPS by applying survey replicate weights in SUDAAN, which accounted for the differential sampling fractions across states, non-response, and survey-induced clustering. However, the analysis has several limitations. The cross sectional study design limits the extent to which we may draw causal conclusions. Selection is the most serious threat to validity present in observational studies, although we attempted to minimize this via multiple regression. Selection may confound workplace smoking policy associations with smoking behavior since smokers may be attracted to firms where they can smoke; workplaces with low smoking prevalence may be more likely to enact smokefree policies; or we may misattribute lower smoking to the effect of smoking bans, instead of to other policies or employer-offered programs that may affect smoking.[5,6] However methodologically stronger analyses find that omitted variables do not dramatically alter conclusions from single-equation estimation.[40] NIH-PA Author Manuscript NIH-PA Author Manuscript Measurement error may be present in the smokefree policy variable (including because it was self-reported) or occupation-related variables (e.g. duration of employment in smokefree workplaces, or differential enforcement of policies in immigrant workplaces), and such mismeasurement could have been differential by nativity or by smoking status. With the increasing diversity of the US, understanding smoking patterns and tobacco-control policy disparities among racial minorities and immigrant groups will be crucial for ameliorating smoking disparities. DISCUSSION Therefore differential smokefree coverage across certain industries and occupations does contribute to why smokefree policies are associated with current smoking, even if they do not explain the entire association. NIH-PA Author Manuscript Acknowledgments Financial support for this study was provided to Dr. Osypuk by a 2-year Association of Schools of Public Health (ASPH) and American Legacy Foundation (Legacy) STEP-UP to Tobacco Control Dissertation Grant L2010-02. Dr. Acevedo-Garcia was supported by National Cancer Institute grant 1 R03 CA093198-01 and a grant from the ASPH and Legacy (L4002-01/03; Dolores Acevedo-Garcia, PI). The conclusions do not necessarily represent the views of ASPH, Legacy Foundation, Legacy Foundation staff, or Legacy's Board of Directors. Dr. Subramanian was supported by the National Institutes of Health Career Development Award (National Institute Heart Lung and Blood Institute, 1 K25 HL081275). Results from parts of this manuscript were presented at the National Tobacco or Health Conference, May 5, 2005, Chicago IL, as well as at “STEP UP to Tobacco Control: Advancing the Role of Public Health and Public Health Professionals” Meeting, April 14–16, 2004, St. Louis, MO. NIH-PA Author Manuscript J Epidemiol Community Health. Author manuscript; available in PMC 2010 October 1. REFERENCES 1. U.S. DHHS. The Health Consequences in Involuntary Smoking. A Report of the Surgeon General. Washington DC: U.S. DHHS, Public Health Service, Centers for Disease Control; 1986. 2. National Cancer Institute. Health Effects of Exposure to Environmental Tobacco Smoke: A Report of the California Environmental Protection Agency, Smoking and Tobacco Control, Monograph 10. Bethesda, MD: U.S. Department of Health & Human Services; 1999. 3. U.S. Department of Health & Human Services. Healthy People 2010: Understanding and Improving Health. Washington, DC: Government Printing Office; 2000. J Epidemiol Community Health. Author manuscript; available in PMC 2010 October 1. Osypuk et al. Osypuk et al. Page 8 NIH-PA Author Manuscript 4. CDC. State-Specific Prevalence of Current Cigarette Smoking among Adults and Attitudes about Secondhand Smoke -- United States 2000. MMWR 2001;50:1101–1106. [PubMed: 11794619] NIH-PA Author Manuscript NIH- 5. Farrelly MC, Evans WN, Sfekas AES. The impact of workplace smoking bans: results from a national survey. Tob Control 1999;8:272–277. [PubMed: 10599571] 6. Gerlach K, Shopland D, Hartman A, et al. Workplace smoking policies in the United States: results from a national survey of more than 100,000 workers. Tob Control 1997;6:199–206. [PubMed: 9396104] 7. Sweeney CT, Shopland DR, Hartman AM, et al. Sex differences in workplace smoking policies: results from the current population survey. Journal of the American Medical Womens Association 2000;55:311–315. 8. Aakko E, Schafer E, Gyarmathy V, et al. Smoking policies in manufacturing and assembly workplaces, Wisconsin 1999. Wis Med J 2001;100:67–69. 9. Pirkle JL, Bernert JT, Claudill SP, et al. Trends in the Exposure of Nonsmokers in the U.S. Population to Secondhand Smoke: 1988–2002. Environmental Health Perspectives 2006;114:853–858. [PubMed: 16759984] 10. Smelser, N.; Wilson, W.; Mitchell, F. American Becoming: Racial Trends and their Consequences. Vol. Volumes I and II. Washington DC: National Academy Press; 2001. 11. Kagawa-Singer M. From genes to social science: impact of the simplistic interpretation of race, ethnicity, and culture on cancer outcome. Cancer 2001;91:226–232. [PubMed: 11148584] 12. Caraballo RS, Giovino GA, Pechacek TF, et al. Racial and ethnic differences in serum cotinine levels of cigarette smokers: Third National Health and Nutrition Examination Survey, 1988–1991. JAMA 1998;280:135–139. [comment]. [PubMed: 9669785] NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript N 13. Wong MD, Shapiro MF, Boscardin WJ, et al. Contribution of Major Diseases to Disparities in Mortality. N Engl J Med 2002;347:1585–1592. [PubMed: 12432046] 14. Dixon, D. The Second Generation in the United States. Migration Policy Institute; 2006. 15. REFERENCES U.S. Census Bureau. 2005. American Community Survey, Table R0501: Percent of People Who Are Foreign Born: 2005. Washington, DC: 2006. 16. Rumbaut RG, Foner N, Gold SG. Immigration and immigration research in the United States. American Behavioral Scientist 1999;42:1258–1263. 17. Capps, R.; Fortuny, K.; Fix, M. Trends in the Low-Wage Immigrant Labor Force, 2000–2005. Washington, DC: The Urban Institute; 2007. p. 9 18. U.S. Department of Commerce, Economics and Statistics Administration, Bureau of the Census. We the American Foreign Born (Report #WE-7). US Census Bureau; 1993. 19. Grieco, E. Migration Policy Institute Immigration Facts. Washington DC: Migration Policy Institute; 2004. Immigrant Union Members: Numbers and Trends; p. 8 20. Grieco, E. Migration Policy Institute Immigration Facts. Washington DC: Migration Policy Institute; 2004. Health Insurance Coverage of the Foreign BOrn in the United States: Numbers and Trends; p. 9 NIH-PA Author Manuscript NIH-PA Author Manuscript 21. Shavers VL, Fagan P, Jouridine Alexander LA, et al. Workplace and home smoking restrictions and racial/ethnic variation in the prevalence and intensity of current cigarette smoking among women by poverty status, TUS-CPS 1998–1999 and 2001–2002. J Epidemiol Community Health 2006;60:ii34– ii43. 22. Shavers VL, Lawrence D, Fagan P, et al. Racial/ethnic variation in cigarette smoking among the civilian US population by occupation and industry, TUS-CPS 1998–1999. Preventive Medicine 2005;41:597–606. [PubMed: 15917058] 23. Acevedo-Garcia D, Pan J, Jun H-J, et al. The effect of immigrant generation on smoking. Social Science & Medicine 2005;61:1223–1242. [PubMed: 15970233] 24. Jackson C, Henriksen L. Do as I say: Parent smoking, antismoking socialization, and smoking onset among children. Addictive Behaviors 1997;22:107–114. [PubMed: 9022876] 25. Hoving C, Reubsaet A, de Vries H. Predictors of smoking stage transitions for adolescent boys and girls. Preventive Medicine 2007;44:485–489. [PubMed: 17363048] J Epidemiol Community Health. Author manuscript; available in PMC 2010 October 1. Osypuk et al. Page 9 Osypuk et al. Page 9 NIH-PA Author Manuscript 26. Weden MM, Astone NM, Bishai D. Racial, ethnic, and gender differences in smoking cessation associated with employment and joblessness through young adulthood in the US. Social Science & Medicine 2006;62:303–316. [PubMed: 16029919] NIH-PA Author Manuscript NIH 27. U.S. Census Bureau. Current Population Survey, June 2001, November 2001, And February 2002: Tobacco Use Supplement File Technical Documentation CPS-02. Washington DC: U.S. Census Bureau; 2002. 28. Unicon Research Corporation. CPS Utilities Software. Tobacco Use Supplement. 2001–02 29. Trosclair A, Husten C, Pederson LL, et al. Cigarette Smoking Among Adults. J Epidemiol Community Health. Author manuscript; available in PMC 2010 October 1. REFERENCES Morbidity and Mortality Weekly Report 2002;51:642–645. [PubMed: 12186222] 30. Bang KM, Kim JH. Prevalence of Cigarette Smoking by Occupation and Industry in the United States. American Journal of Industrial Medicine 2001;40:233–239. [PubMed: 11598969] 31. Giovino, GA.; Pederson, LL.; Trosclair, A. The Prevalence of Selected Cigarette Smoking Behaviors by Occupational Class in the United States. Presented at the Organized Labor, Public Health, and Tobacco Control Policy Conference; 2000. 32. Shopland DR, Gerlach KK, Burns DM, et al. State-Specific Trends in Smoke-Free Workplace Policy Coverage:The Current Population Survey Tobacco Use Supplement, 1993–1999. J Occup Environ Med 2001;43:680–686. [PubMed: 11515250] 33. Maryland Occupational Safety and Health. COMAR 09.12.23 and Chapter 5, Acts of 1995 Maryland Occupational Safety and Health. Baltimore, MD: 1995. Prohibition on Smoking in an Enclosed Workplace - Compliance Guidelines for the Hospitality Industry. 34. Edwards R, Thomson G, Wilson N, et al. After the smoke has cleared: evaluation of the impact of a new national smoke-free law in New Zealand. Tob Control 2008;17:e2. [PubMed: 18218788] NIH-PA Author Manuscript NIH-PA Author Manuscript 35. Baluja KF, Park J, Myers D. Inclusion of Immigrant Status in Smoking Prevalence Statistics. Am J Public Health 2003;93:642–646. [PubMed: 12660211] 36. Averbach AR, Lam D, Lam L-P, et al. Smoking behaviours and attitudes among male restaurant workers in Boston's Chinatown: a pilot study. Tob Control 2002;11 34ii-7. 37. Hu, S. Osypuk PCwT. ed.. Chicago, IL: 2005. 38. Georgiades K, Boyle MH, Duku E, et al. Tobacco use among immigrant and nonimmigrant adolescents: individual and family level influences. Journal of Adolescent Health 2006;38:443. e1- e7. [PubMed: 16549306] 39. Lopez AD, Collishaw NE, Piha T. A descriptive model of the cigarette epidemic in developed countries. Tob Control 1994;3:242–247. 40. Evans WN, Farrelly MC, Montgomery E. Do workplace smoking bans reduce smoking? The American Economic Review 1999;89:728–747. NIH-PA Author Manuscript J Epidemiol Community Health. Author manuscript; available in PMC 2010 October 1. Page 10 Page 10 Osypuk et al. NIH-PA Author Manuscript Figure 1. Prevalence of smokefree workplaces by nativity, race/ethnicity, and gender (2001/02 TUS) NOTES for Figure 1: Sample sizes for each of the demographic subgroups correspond to those listed in Table 4. Racial groups White, Black, and Asian exclude Hispanics. Self-response weights applied. NIH-PA Author Manuscript NIH-PA Author Manuscript Figure 1. J Epidemiol Community Health. Author manuscript; available in PMC 2010 October 1. REFERENCES Page 11 Table 1 urrent Population Survey 2001–02 Workplace Smoking Policy Sample % current smokers % in smokefree workplace % foreign born Category or Sub-category n % of sample (weighted)1 (weighted)1 (weighted)1 85,784 100.00% 21.12% 70.53% 12.29% Smokefree 61,302 71.46% 18.17% -- 11.94% Not smokefree 24,482 28.54% 28.18% -- 13.13% US Born 77,561 90.41% 22.19% 70.81% -- Foreign Born (Immigrant) 8,223 9.59% 13.52% 68.51% -- US-Born (reference group) 77,561 90.41% 22.19% 70.81% 0.00% Immigrant, 0–4 years 1,202 1.40% 18.08% 66.65% 100.00% Immigrant, 5–9 years 1,663 1.94% 13.14% 64.24% 100.00% Immigrant, 10–14 years 1,195 1.39% 12.23% 65.81% 100.00% Immigrant, 15–19 years 1,250 1.46% 10.94% 69.29% 100.00% Immigrant, 20+ years 2,913 3.40% 13.57% 72.69% 100.00% English 83,908 97.81% 21.31% 71.03% 10.09% Other 1,794 2.09% 15.15% 54.06% 86.03% 1st generation (foreign born) 8,223 9.59% 13.52% 68.51% 100.00% 2nd generation (US-born of foreign born 5,464 6.37% 17.55% 72.13% 0.00% 3rd generation and higher (US born of US- 72,097 84.04% 22.58% 70.70% 0.00% Non-Hispanic White 68,254 79.56% 22.76% 71.32% 3.90% Non-Hispanic Black 7,828 9.13% 18.50% 69.17% 9.95% Non-Hispanic Asian 3,181 3.71% 12.47% 71.94% 77.80% Hispanic 6,521 7.60% 16.23% 65.82% 46.82% Male 35,789 41.72% 22.12% 65.81% 14.53% Female 49,995 58.28% 20.27% 74.58% 10.37% $0–$9,999 2,782 3.24% 30.65% 60.90% 16.16% $10,000–$19,999 6,233 7.27% 31.19% 60.35% 17.40% $20,000–$29,999 9,379 10.93% 28.22% 64.62% 15.93% $30,000–$39,999 10,346 12.06% 26.06% 67.83% 12.77% $40,000–$49,999 8,608 10.03% 23.12% 68.41% 11.08% Osypuk et al. REFERENCES Prevalence of smokefree workplaces by nativity, race/ethnicity, and gender (2001/02 TUS) NOTES for Figure 1: Sample sizes for each of the demographic subgroups correspond to those listed in Table 4. Racial groups White, Black, and Asian exclude Hispanics. Self-response weights applied. NIH-PA Author Manuscript J Epidemiol Community Health. Author manuscript; available in PMC 2010 October 1. Osypuk et al. Page 11 Table 1 Descriptive Statistics, Tobacco Use Supplement-Current Population Survey 2001–02 Workplace Smoking Policy Sample % current smokers % in smokefree workplace % foreign born Variable Category Category or Sub-category n % of sample (weighted)1 (weighted)1 (weighted)1 Total 85,784 100.00% 21.12% 70.53% 12.29% Workplace Smoking Policy Smokefree 61,302 71.46% 18.17% -- 11.94% Not smokefree 24,482 28.54% 28.18% -- 13.13% Nativity US Born 77,561 90.41% 22.19% 70.81% -- Foreign Born (Immigrant) 8,223 9.59% 13.52% 68.51% -- Immigrant length of stay in the US US-Born (reference group) 77,561 90.41% 22.19% 70.81% 0.00% Immigrant, 0–4 years 1,202 1.40% 18.08% 66.65% 100.00% Immigrant, 5–9 years 1,663 1.94% 13.14% 64.24% 100.00% Immigrant, 10–14 years 1,195 1.39% 12.23% 65.81% 100.00% Immigrant, 15–19 years 1,250 1.46% 10.94% 69.29% 100.00% Immigrant, 20+ years 2,913 3.40% 13.57% 72.69% 100.00% Interview Language English 83,908 97.81% 21.31% 71.03% 10.09% Other 1,794 2.09% 15.15% 54.06% 86.03% Immigrant generation 1st generation (foreign born) 8,223 9.59% 13.52% 68.51% 100.00% 2nd generation (US-born of foreign born 5,464 6.37% 17.55% 72.13% 0.00% 3rd generation and higher (US born of US- 72,097 84.04% 22.58% 70.70% 0.00% Race/Ethnicity Non-Hispanic White 68,254 79.56% 22.76% 71.32% 3.90% Non-Hispanic Black 7,828 9.13% 18.50% 69.17% 9.95% Non-Hispanic Asian 3,181 3.71% 12.47% 71.94% 77.80% Hispanic 6,521 7.60% 16.23% 65.82% 46.82% Sex Male 35,789 41.72% 22.12% 65.81% 14.53% Female 49,995 58.28% 20.27% 74.58% 10.37% Income $0–$9,999 2,782 3.24% 30.65% 60.90% 16.16% $10,000–$19,999 6,233 7.27% 31.19% 60.35% 17.40% $20,000–$29,999 9,379 10.93% 28.22% 64.62% 15.93% $30,000–$39,999 10,346 12.06% 26.06% 67.83% 12.77% $40,000–$49,999 8,608 10.03% 23.12% 68.41% 11.08% J Epidemiol Community Health. Author manuscript; available in PMC 2010 October 1. Osypuk et al. REFERENCES Page 11 Table 1 Descriptive Statistics, Tobacco Use Supplement-Current Population Survey 2001–02 Workplace Smoking Policy Sample % current smokers % in smokefree workplace % foreign born Variable Category Category or Sub-category n % of sample (weighted)1 (weighted)1 (weighted)1 Total 85,784 100.00% 21.12% 70.53% 12.29% Workplace Smoking Policy Smokefree 61,302 71.46% 18.17% -- 11.94% Not smokefree 24,482 28.54% 28.18% -- 13.13% Nativity US Born 77,561 90.41% 22.19% 70.81% -- Foreign Born (Immigrant) 8,223 9.59% 13.52% 68.51% -- Immigrant length of stay in the US US-Born (reference group) 77,561 90.41% 22.19% 70.81% 0.00% Immigrant, 0–4 years 1,202 1.40% 18.08% 66.65% 100.00% Immigrant, 5–9 years 1,663 1.94% 13.14% 64.24% 100.00% Immigrant, 10–14 years 1,195 1.39% 12.23% 65.81% 100.00% Immigrant, 15–19 years 1,250 1.46% 10.94% 69.29% 100.00% Immigrant, 20+ years 2,913 3.40% 13.57% 72.69% 100.00% Interview Language English 83,908 97.81% 21.31% 71.03% 10.09% Other 1,794 2.09% 15.15% 54.06% 86.03% Immigrant generation 1st generation (foreign born) 8,223 9.59% 13.52% 68.51% 100.00% 2nd generation (US-born of foreign born 5,464 6.37% 17.55% 72.13% 0.00% 3rd generation and higher (US born of US- 72,097 84.04% 22.58% 70.70% 0.00% Race/Ethnicity Non-Hispanic White 68,254 79.56% 22.76% 71.32% 3.90% Non-Hispanic Black 7,828 9.13% 18.50% 69.17% 9.95% Non-Hispanic Asian 3,181 3.71% 12.47% 71.94% 77.80% Hispanic 6,521 7.60% 16.23% 65.82% 46.82% Sex Male 35,789 41.72% 22.12% 65.81% 14.53% Female 49,995 58.28% 20.27% 74.58% 10.37% Income $0–$9,999 2,782 3.24% 30.65% 60.90% 16.16% $10,000–$19,999 6,233 7.27% 31.19% 60.35% 17.40% $20,000–$29,999 9,379 10.93% 28.22% 64.62% 15.93% $30,000–$39,999 10,346 12.06% 26.06% 67.83% 12.77% $40,000–$49,999 8,608 10.03% 23.12% 68.41% 11.08% Osypu Table 1 Descriptive Statistics, Tobacco Use Supplement-Current Population Survey 2001–02 Workplace Smoking Policy Sample Page 11 Osypuk et al. NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA A NIH-PA Author Manuscript NIH-PA Author Manuscript J Epidemiol Community Health. Author manuscript; available in PMC 2010 October 1. NIH-PA Author Manuscript Page 12 Osypuk et al. Page 12 % current smokers % in smokefree workplace % foreign born Variable Category Category or Sub-category n % of sample (weighted)1 (weighted)1 (weighted)1 $50,000–$59,999 9,075 10.58% 21.08% 70.88% 10.14% $60,000–$74,999 10,118 11.79% 17.97% 73.70% 9.61% $75,000+ 22,040 25.69% 13.21% 77.91% 10.41% Education Missing 7,203 8.40% 19.45% 69.22% 14.22% Less than High School Grad 7,918 9.23% 26.59% 56.86% 26.16% High School Grad 24,376 28.42% 29.64% 63.78% 9.88% Some College/Associates 25,569 29.81% 22.78% 70.71% 8.14% Bachelors or Higher 27,921 32.55% 10.39% 80.76% 13.56% Occupation White Collar Executive, administrative, & managerial 14,317 16.69% 17.41% 75.19% 8.88% Professional specialty 17,109 19.94% 11.41% 84.02% 12.09% Technicians & related support 3,684 4.29% 18.52% 77.79% 12.14% Sales 9,521 11.10% 21.59% 69.21% 9.63% Service Administrative support including clerical 15,296 17.83% 21.37% 76.12% 8.26% Private household service 56 0.07% 13.12% 44.33% 43.32% Protective service 1,148 1.34% 21.12% 69.60% 7.96% Blue Collar Service, except protective & household 10,138 11.82% 28.74% 58.29% 17.95% Precision production, craft, & repair 5,919 6.90% 30.63% 52.16% 14.78% Machine operators, assemblers, inspectors 4,776 5.57% 30.71% 53.65% 21.76% Transportation & material moving 944 1.10% 31.37% 50.10% 16.08% Handlers, equipment cleaners, helpers, & laborers 2,540 2.96% 27.66% 60.55% 16.06% Agricultural, forestry, fisheries Farming, forestry, & fishing 336 0.39% 21.99% 46.84% 24.49% Industry Agriculture 462 0.54% 25.26% 53.39% 15.37% Mining 283 0.33% 19.85% 63.29% 6.84% Construction 1,778 2.07% 27.27% 48.77% 14.31% Manufacturing Durable Goods 8,349 9.73% 25.45% 58.26% 15.43% Nondurable Goods 5,383 6.28% 24.91% 64.85% 16.56% Transportation, communications, & other public utilitites Transportation 2,531 2.95% 23.73% 67.58% 12.26% Communications 1,405 1.64% 19.29% 81.95% 9.55% Wholesale trade Utilities & sanitary service 752 0.88% 15.00% 72.46% 4.34% J Epidemiol Community Health. Author manuscript; available in PMC 2010 October 1. Osypu Table 1 Descriptive Statistics, Tobacco Use Supplement-Current Population Survey 2001–02 Workplace Smoking Policy Sample % current smokers % in smokefree workplace % foreign born Variable Category Category or Sub-category n % of sample (weighted)1 (weighted)1 (weighted)1 $50,000–$59,999 9,075 10.58% 21.08% 70.88% 10.14% $60,000–$74,999 10,118 11.79% 17.97% 73.70% 9.61% $75,000+ 22,040 25.69% 13.21% 77.91% 10.41% Education Missing 7,203 8.40% 19.45% 69.22% 14.22% Less than High School Grad 7,918 9.23% 26.59% 56.86% 26.16% High School Grad 24,376 28.42% 29.64% 63.78% 9.88% Some College/Associates 25,569 29.81% 22.78% 70.71% 8.14% Bachelors or Higher 27,921 32.55% 10.39% 80.76% 13.56% Occupation White Collar Executive, administrative, & managerial 14,317 16.69% 17.41% 75.19% 8.88% Professional specialty 17,109 19.94% 11.41% 84.02% 12.09% Technicians & related support 3,684 4.29% 18.52% 77.79% 12.14% Sales 9,521 11.10% 21.59% 69.21% 9.63% Service Administrative support including clerical 15,296 17.83% 21.37% 76.12% 8.26% Private household service 56 0.07% 13.12% 44.33% 43.32% Protective service 1,148 1.34% 21.12% 69.60% 7.96% Blue Collar Service, except protective & household 10,138 11.82% 28.74% 58.29% 17.95% Precision production, craft, & repair 5,919 6.90% 30.63% 52.16% 14.78% Machine operators, assemblers, inspectors 4,776 5.57% 30.71% 53.65% 21.76% Transportation & material moving 944 1.10% 31.37% 50.10% 16.08% Handlers, equipment cleaners, helpers, & laborers 2,540 2.96% 27.66% 60.55% 16.06% Agricultural, forestry, fisheries Farming, forestry, & fishing 336 0.39% 21.99% 46.84% 24.49% Industry Agriculture 462 0.54% 25.26% 53.39% 15.37% Mining 283 0.33% 19.85% 63.29% 6.84% Construction 1,778 2.07% 27.27% 48.77% 14.31% Manufacturing Durable Goods 8,349 9.73% 25.45% 58.26% 15.43% Nondurable Goods 5,383 6.28% 24.91% 64.85% 16.56% Transportation, communications, & other public utilitites Transportation 2,531 2.95% 23.73% 67.58% 12.26% Communications 1,405 1.64% 19.29% 81.95% 9.55% Wholesale trade Utilities & sanitary service 752 0.88% 15.00% 72.46% 4.34% J Epidemiol Community Health. Author manuscript; available in PMC 2010 October 1. NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA A Category or Sub-category NIH-PA Author Manuscript J Epidemiol Community Health. Author manuscript; available in PMC 2010 October 1. Osypuk et al. % current smokers % in smokefree workplace % foreign born Variable Category Category or Sub-category n % of sample (weighted)1 (weighted)1 (weighted)1 Retail trade 2,742 3.20% 23.29% 62.61% 12.63% Finance, insurance, & real estate 15,020 17.51% 26.52% 60.74% 12.70% Personal services including private households 6,579 7.67% 18.17% 77.07% 9.47% Business, auto & repair services 67 0.08% 13.09% 41.72% 41.99% Personal services except private households 4,889 5.70% 24.47% 65.75% 17.99% Entertainment & recreation services 2,212 2.58% 29.18% 55.21% 24.25% Professional & related services 1,389 1.62% 22.68% 56.12% 10.58% Hospitals 4,753 5.54% 15.53% 85.05% 12.30% Medical services, except hospitals 5,399 6.29% 21.87% 80.12% 11.21% Educational services 10,132 11.81% 10.87% 90.06% 8.69% Social services 2,370 2.76% 18.83% 81.62% 8.27% Other professional services 4,544 5.30% 12.66% 76.84% 9.48% Forestry & fisheries 63 0.07% 18.16% 79.08% 0.93% Public administration 4,682 5.46% 16.34% 81.00% 5.85% Marital Status Married 48,660 56.72% 17.05% 72.72% 14.03% Divorced/Separated/Widowed 15,206 17.73% 30.20% 71.05% 9.31% Single/Never married 21,918 25.55% 23.48% 66.27% 10.79% Age 15–19 3,483 4.06% 17.57% 62.12% 6.59% 20–29 16,171 18.85% 24.53% 65.34% 12.61% 30–39 21,883 25.51% 20.88% 70.90% 15.29% 40–49 22,651 26.40% 22.27% 72.80% 11.95% 50–59 15,875 18.51% 18.90% 75.42% 10.35% 60+ 5,721 6.67% 13.85% 72.87% 11.03% All P-values were less than 0.01. P values reported using chi-square statistical tests. Sample excludes Native Americans and those with indeterminate smoking status, and those not in universe for determining workplace smoking policy Page 13 Page 13 Osypuk et al. Category or Sub-category NIH-PA Author Manuscript % current smokers % in smokefree workplace % foreign born Variable Category Category or Sub-category n % of sample (weighted)1 (weighted)1 (weighted)1 Retail trade 2,742 3.20% 23.29% 62.61% 12.63% Finance, insurance, & real estate 15,020 17.51% 26.52% 60.74% 12.70% Personal services including private households 6,579 7.67% 18.17% 77.07% 9.47% Business, auto & repair services 67 0.08% 13.09% 41.72% 41.99% Personal services except private households 4,889 5.70% 24.47% 65.75% 17.99% Entertainment & recreation services 2,212 2.58% 29.18% 55.21% 24.25% Professional & related services 1,389 1.62% 22.68% 56.12% 10.58% Hospitals 4,753 5.54% 15.53% 85.05% 12.30% Medical services, except hospitals 5,399 6.29% 21.87% 80.12% 11.21% Educational services 10,132 11.81% 10.87% 90.06% 8.69% Social services 2,370 2.76% 18.83% 81.62% 8.27% Other professional services 4,544 5.30% 12.66% 76.84% 9.48% Forestry & fisheries 63 0.07% 18.16% 79.08% 0.93% Public administration 4,682 5.46% 16.34% 81.00% 5.85% Marital Status Married 48,660 56.72% 17.05% 72.72% 14.03% Divorced/Separated/Widowed 15,206 17.73% 30.20% 71.05% 9.31% Single/Never married 21,918 25.55% 23.48% 66.27% 10.79% Age 15–19 3,483 4.06% 17.57% 62.12% 6.59% 20–29 16,171 18.85% 24.53% 65.34% 12.61% 30–39 21,883 25.51% 20.88% 70.90% 15.29% 40–49 22,651 26.40% 22.27% 72.80% 11.95% 50–59 15,875 18.51% 18.90% 75.42% 10.35% 60+ 5,721 6.67% 13.85% 72.87% 11.03% All P-values were less than 0.01. P values reported using chi-square statistical tests. Sample excludes Native Americans and those with indeterminate smoking status, and those not in universe for determining workplace smoking policy J Epidemiol Community Health. Author manuscript; available in PMC 2010 October 1. NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA A NIH-PA Author Manuscript Variable Variable J Epidemiol Community Health. Author manuscript; available in PMC 2010 October 1. NIH-PA Author Manuscript Page 14 Page 14 Osypuk et al. Table 2 Table 2 Table 2 Relative Odds of working in a Smokefree Workplace (compared to non-smokefree workplace n=85,784 Model 1: Foreign Born Coefficient 1 Model Variables in Model OR (95% CI) p Unadjusted model 2 A Foreign born only 0.90 (0.83, 0.96) Models adjusted for one variable at a time 3 B Foreign born + Occupation 4 1.02 (0.95, 1.10) C Foreign born + Industry 5 1.02 (0.94, 1.10) D Foreign born + Income 0.95 (0.88, 1.02) E Foreign born + Education 0.94 (0.87, 1.02) F Foreign born + Gender 0.93 (0.87, 1.00) # G Foreign born + Race/ethnicity 0.95 (0.88, 1.03) H Foreign born + Marital status 0.88 (0.82, 0.95) J Foreign born + Age 0.90 (0.84, 0.97) K Foreign born + Length of stay 1.09 (0.93, 1.27) L Foreign born + Language of interview 1.06 (0.98, 1.15) M Foreign born + State fixed effects 0.77 (0.71, 0.83) * Fully adjusted model 6 P Foreign born + all other variables (Final Model) 1.03 (0.88, 1.21) # Odds of working in a Smokefree Workplace (compared to non-smokefree workplace) by nativity. NIH-PA Author Manuscript NIH-PA Author Manuscript uscript NIH-PA Author Manuscript NIH-PA Author Manuscript 1Nativity is modeled as a 2-level variable, foreign born compared to the reference group of USborn. 1Nativity is modeled as a 2-level variable, foreign born compared to the reference group of USborn. 2Model A, the unadjusted model, includes only the nativity variable. 3Models B-M include nativity and one other variable at a time. 3Models B-M include nativity and one other variable at a time. 4Occupation was coded in 5 categories for regression: professional specialty; executive administrative & managerial; technicians & related support, sales, and administrative support including clerical; Service; Blue Collar. 5Industry was coded in 8 categories for regression: trade; manufacturing; services; professional and related services; finance, insurance, and real estate; public administration; transportation and communications; agricultural, forestry and fishing, mining, and construction. 6Model P is the fully adjusted (final) model including nativity, occupation, industry, income, education, gender, race/ethnicity, marital status, age, state fixed effects, length of stay among immigrants, and language of interview. J Epidemiol Community Health. Author manuscript; available in PMC 2010 October 1. Page 15 Osypuk et al. Page 15 NIH-PA Author Manuscript Odds of current smoking, by workplace smoking policy and nativity. Table 2 Model 2 Model 3 3 Model 4 Model 5 Unstratified model: Main Effect of Policy Unstratified model: Nativity Interaction with Policy Stratified model: US Born Stratified model: Foreign Born unweighted sample size --> n=85,784 n=85,784 n=77,561 n=8,223 Variable reported --> Policy is Not Smokefree Policy is Not Smokefree Foreign Born Non Smokefree PolicyForeign Born Interaction Policy is Not Smokefree Policy is Not Smokefree Mo del Variables in model OR (95% CI) p OR (95% CI) p OR (95% CI) p Logit Coeff p OR 2 OR (95% CI) p OR (95% CI) p A Policy only 1.77 (1.69, 1.85) -- -- -- -- -- -- -- -- -- 1.83 (1.74, 1.92) * 1.39 (1.20, 1.61) *** One variable added at a time. Table 2 3For model 3, the odds ratios for the main effect of policy is interpreted as the policy association for the US-born, while the interaction logit coefficient is the differential policy association for the foreign born (on the logit scale). 4Occupation was coded in 5 categories: professional specialty; executive administrative & managerial; technicians & related support, sales, and administrative support including clerical; Service; Blue Collar. 5Industry was modeled in 8 categories: trade; manufacturing; services; professional and related services; finance, insurance, and real estate; public administration; transportation and communications; agricultural, forestry and fishing, mining, and construction. 6Immigrant length of stay was included only in Models 2M and 5M to maintain consistency of the meaning of the nativity variable. tio for the interaction was calculated as the odds ratio of being in a non-smokefree workplace among foreign born (compared to the foreign born in smokefree workplaces). del 3, the odds ratios for the main effect of policy is interpreted as the policy association for the US-born, while the interaction logit coefficient is the differential policy association for the foreign born (on the logit scale). tion was coded in 5 categories: professional specialty; executive administrative & managerial; technicians & related support, sales, and administrative support including clerical; Service; Blue Collar. y was modeled in 8 categories: trade; manufacturing; services; professional and related services; finance, insurance, and real estate; public administration; transportation and communications; agricultural, forestry and fishing, mining, and construction. Model 2 Model 3 3 Model 4 Model 5 Unstratified model: Main Effect of Policy Unstratified model: Nativity Interaction with Policy Stratified model: US Born Stratified model: Foreign Born unweighted sample size --> n=85,784 n=85,784 n=77,561 n=8,223 Variable reported --> Policy is Not Smokefree Policy is Not Smokefree Foreign Born Non Smokefree PolicyForeign Born Interaction Policy is Not Smokefree Policy is Not Smokefree Mo del Variables in model OR (95% CI) p OR (95% CI) p OR (95% CI) p Logit Coeff p OR 2 OR (95% CI) p OR (95% CI) p State Variables N Policy + State Dummies 1 1.73 (1.65, 1.81) 1.77 (1.69, 1.86) * 0.65 (0.59, 0.71) * −0.27 1.36 1.77 (1.69, 1.86) * 1.36 (1.16, 1.59) * Multivariate Model Building P Policy + all covariates 1.34 (1.27, 1.41) * 1.37 (1.30, 1.44) * 0.71 (0.56, 0.88) −0.23 1.09 1.36 (1.29, 1.44) *** 1.15 (0.97, 1.35) # #p-value: p=<.10 p=<.05 p=<.01 p=<.001 OR = Odds Ratio. Table 2 1For models 2 & 3, all unstratified models (B-P) also contain the variable Nativity. 2Odds ratio for the interaction was calculated as the odds ratio of being in a non-smokefree workplace among foreign born (compared to the foreign born in smokefree workplaces). 3For model 3, the odds ratios for the main effect of policy is interpreted as the policy association for the US-born, while the interaction logit coefficient is the differential policy association for the foreign born (on the logit scale). 4Occupation was coded in 5 categories: professional specialty; executive administrative & managerial; technicians & related support, sales, and administrative support including clerical; Service; Blue Collar. 5Industry was modeled in 8 categories: trade; manufacturing; services; professional and related services; finance, insurance, and real estate; public administration; transportation and communications; agricultural, forestry and fishin 6Immigrant length of stay was included only in Models 2M and 5M to maintain consistency of the meaning of the nativity variable. J Epidemiol Community Health. Author manuscript; available in PMC 2010 October 1. Table 2 Demographic/Socioeconomic Variables B Policy + Foreign Born 1.78 (1.71, 1.87) * 1.83 (1.74, 1.92) * 0.60 (0.55, 0.65) * −0.27 * 1.39 -- -- C Policy + Occupation 1,4 1.51 (1.45, 1.58) * 1.55 (1.48, 1.63) * 0.55 (0.50, 0.60) * −0.28 * 1.17 1.54 (1.47, 1.62) * 1.26 (1.07, 1.47) D Policy + Industry 1,5 1.56 (1.49, 1.64) * 1.60 (1.52, 1.68) * 0.55 (0.50, 0.61) * −0.23 1.27 1.60 (1.52, 1.68) * 1.27 (1.09, 1.47) E Policy + Income 1 1.65 (1.57, 1.72) * 1.69 (1.61, 1.78) * 0.56 (0.51, 0.61) * −0.31 * 1.24 1.68 (1.60, 1.77) * 1.33 (1.14, 1.55) * F Policy + Education 1 1.54 (1.47, 1.61) * 1.58 (1.50, 1.66) * 0.60 (0.55, 0.66) * −0.3 * 1.17 1.56 (1.49, 1.64) * 1.32 (1.13, 1.55) * G Policy + Gender 1 1.77 (1.69, 1.85) * 1.81 (1.73, 1.90) * 0.59 (0.54, 0.65) * −0.27 1.38 1.83 (1.74, 1.92) * 1.32 (1.13, 1.54) * H Policy + Race/ethnicity 1 1.80 (1.72, 1.88) * 1.84 (1.75, 1.93) * 0.72 (0.66, 0.80) * −0.27 1.41 1.84 (1.75, 1.93) * 1.39 (1.20, 1.61) * J Policy + Marital 1 1.76 (1.69, 1.84) * 1.81 (1.78, 1.89) * 0.62 (0.56, 0.68) * −0.25 1.41 1.81 (1.72, 1.89) * 1.39 (1.20, 1.62) * K Policy + Age 1 1.78 (1.70, 1.86) * 1.82 (1.74, 1.91) * 0.59 (0.53, 0.64) * −0.28 * 1.38 1.83 (1.74, 1.91) * 1.38 (1.19, 1.60) * L Policy + Interview Language 1.79 (1.71, 1.87) * 1.83 (1.74, 1.92) * 0.60 (0.54, 0.66) * −0.27 1.40 1.83 (1.75, 1.92) * 1.37 (1.18, 1.60) * M Policy + Immigrant Length of Stay 1,6 1.79 (1.71, 1.87) * -- -- -- -- 1.39 (1.19, 1.62) *** J Epidemiol Community Health. Author manuscript; available in PMC 2010 October 1. Odds of current smoking, by workplace smoking policy and nativity. J Epidemiol Community Health. Author manuscript; available in PMC 2010 October 1. NIH-PA Author Manuscript Osypuk et al. Page 16 NIH-PA Author Manuscript NIH-PA Author M 2Odds ratio for the interaction was calculated as the odds ratio of being in a non-smokefree workplace among foreign born (compared to the foreign born in smokefree workplaces). Table 2 Model 2 Model 3 3 Model 4 Model 5 Unstratified model: Main Effect of Policy Unstratified model: Nativity Interaction with Policy Stratified model: US Born Stratified model: Foreign Born unweighted sample size --> n=85,784 n=85,784 n=77,561 n=8,223 Variable reported --> Policy is Not Smokefree Policy is Not Smokefree Foreign Born Non Smokefree PolicyForeign Born Interaction Policy is Not Smokefree Policy is Not Smokefree Mo del Variables in model OR (95% CI) p OR (95% CI) p OR (95% CI) p Logit Coeff p OR 2 OR (95% CI) p OR (95% CI) p State Variables N Policy + State Dummies 1 1.73 (1.65, 1.81) 1.77 (1.69, 1.86) * 0.65 (0.59, 0.71) * −0.27 1.36 1.77 (1.69, 1.86) * 1.36 (1.16, 1.59) * Multivariate Model Building P Policy + all covariates 1.34 (1.27, 1.41) * 1.37 (1.30, 1.44) * 0.71 (0.56, 0.88) −0.23 1.09 1.36 (1.29, 1.44) *** 1.15 (0.97, 1.35) # J Epidemiol Community Health Author manuscript; av Model 2 Model 3 3 Model 4 Model 5 Unstratified model: Main Effect of Policy Unstratified model: Nativity Interaction with Policy Stratified model: US Born Stratified model: Foreign Born unweighted sample size --> n=85,784 n=85,784 n=77,561 n=8,223 Variable reported --> Policy is Not Smokefree Policy is Not Smokefree Foreign Born Non Smokefree PolicyForeign Born Interaction Policy is Not Smokefree Policy is Not Smokefree Mo del Variables in model OR (95% CI) p OR (95% CI) p OR (95% CI) p Logit Coeff p OR 2 OR (95% CI) p OR (95% CI) p State Variables N Policy + State Dummies 1 1.73 (1.65, 1.81) 1.77 (1.69, 1.86) * 0.65 (0.59, 0.71) * −0.27 1.36 1.77 (1.69, 1.86) * 1.36 (1.16, 1.59) * Multivariate Model Building P Policy + all covariates 1.34 (1.27, 1.41) * 1.37 (1.30, 1.44) * 0.71 (0.56, 0.88) −0.23 1.09 1.36 (1.29, 1.44) * 1.15 (0.97, 1.35) # J Epidemiol Community Health. Author manuscript; av J Epidemiol Community Health. Author manuscript; available in PMC 2010 October 1. Page 17 Page 17 Osypuk et al. NIH-PA Author Manuscript Table 4 Odds ratios of current smoking for non-smokefree (vs. smokefree) workplaces, stratified by race/ethnicity, gender, and nativity (Models 6). J Epidemiol Community Health. Author manuscript; available in PMC 2010 October 1. Table 2 US-Born Women Men NH white women NH black women NH asian women hispanic women NH white men NH black men NH asian men hispanic men n=38532 n=4721 n=557 n=2113 n=27447 n=2371 n=432 n=1388 Model OR (95% CI) p OR (95% CI) p OR (95% CI) p OR (95% CI) p OR (95% CI) p OR (95% CI) p OR (95% CI) p OR (95% CI) p Unadjusted 6A 1.88 (1.74, 2.03) * 1.51 (1.24, 1.84) * 1.38 (0.74, 2.57) 1.54 (1.16, 2.03) 2.01 (1.89, 2.15) * 1.19 (0.95, 1.49) 1.27 (0.68, 2.37) 1.71 (1.26, 2.31) * Adjusted 6P 1.41 (1.30, 1.52) * 1.37 (1.10, 1.69) 1.12 (0.52, 2.41) 1.56 (1.16, 2.08) 1.45 (1.34, 1.56) * 1.02 (0.80, 1.30) 0.76 (0.36, 1.61) 1.42 (1.04, 1.95) * Foreign Born Women Men NH white women NH black women NH asian women hispanic women NH white men NH black men NH asian men hispanic men n=1172 n=409 n=1107 n=1384 n=1103 n=327 n=1085 n=1636 OR (95% CI) p OR (95% CI) p OR (95% CI) p OR (95% CI) p OR (95% CI) p OR (95% CI) p OR (95% CI) p OR (95% CI) p Unadjusted 6A 0.95 (0.62, 1.46) 0.21 (0.01, 3.21) 1.98 (1.01, 3.89) * 1.27 (0.75, 2.14) 1.74 (1.19, 2.55) 0.39 (0.13, 1.23) 1.72 (1.19, 2.47) 1.27 (0.95, 1.69) Adjusted 6P 0.76 (0.51, 1.15) 0.09 (0.01, 0.85) * 1.97 (0.96, 4.07) # 1.30 (0.75, 2.24) 1.27 (0.84, 1.93) 0.35 (0.08, 1.47) 1.49 (0.98, 2.27) # 1.18 (0.86, 1.63) # J Epidemiol Community Health. Author manuscript; available in PMC 2010 October 1. J Epidemiol Community Health. Author manuscript; available in PMC 2010 October 1. J Epidemiol Community Health. Author manuscript; available in PMC 2010 October 1. NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Auth Page 18 Page 18 Osypuk et al.
https://openalex.org/W4385474887	https://zenodo.org/records/8207181/files/NACORE%20P132.pdf	English	null	A Concise Review On E-Commerce Website For Visually Impaired	Zenodo (CERN European Organization for Nuclear Research)	2,023	cc-by	1,994	International Journal on Emerging Research Areas (IJERA) International Journal on Emerging Research Areas (IJERA) ISSN:2230-9993 A Concise Review On E-Commerce Website For Visually Impaired Rekha KS Assistant Professor, Department of CSE College of Engineering Kidangoor Kottayam, Kerala, India rekhaks@ce-kgr.org Aleena Jomon, Maria Joy, NaveenV, Sarath P O Department of CSE College of Engineering Kidangoor Kottayam, Kerala, India naveenv99611@gmail.com Incorporating voice-enabled systems into web applications not only provides more options for users but also enhances the usability of the applications for all users. Incorporating voice-enabled systems into web applications not only provides more options for users but also enhances the usability of the applications for all users. Abstract— E-commerce has revolutionized the way people shop for goods and services, making it easier and more convenient to purchase products from the comfort of their own homes. However, visually impaired individuals face significant challenges in accessing and navigating e-commerce websites due to their reliance on visual cues. This can lead to a frustrating and isolating shopping experience, limiting their ability to make informed purchases and participate in online commerce. The purpose of this paper is to explore the challenges faced by visually impaired individuals when using e-commerce websites and to propose a solution to make e-commerce more accessible and inclusive. We aim to develop a voice-controlled e-commerce platform that uses natural language processing and machine learning to create a user-friendly interface for visually impaired users. This solution will enable visually impaired individuals to independently navigate e-commerce websites, browse products, and make purchases with ease. As more companies shift their focus to digital platforms, it is crucial to ensure that individuals with disabilities can access these resources with ease. By integrating Speech Recognition Systems (SRS) into web applications, users can navigate and interact with the platform using natural language, making it more convenient and accessible for everyone. However, many existing solutions have limited accuracy due to frequent misinterpretation. Therefore, this paper focuses on creating an e-commerce website based on Alan AI, Voice API, and NLP to improve accessibility for handicapped and visually impaired customers, allowing them to access platform services without relying on others. Keywords—Speech Recognition, Natural Language Processing, Voice API, Alan AI I. INTRODUCTION Speech recognition is the process of converting human speech into text that are assigned meaning to some defined actions. As the world continues to transition to a digital environment, it is essential for everyone to keep up with the changes. However, visually impaired individuals have not benefited from technological advancements as much as the general population has. While technologies like screen readers and braille keyboards have made it easier for them to access certain applications, they still face challenges accessing many critical resources and often require third-party assistance. To help address these issues, various web assistive technologies have been developed over the years, such as screen readers, special browsers, and screen magnification techniques. These systems enable users to comprehend web page contents through reading, voice commands, or screen magnification. II. LITERATURE SURVEY All the related works that have been done by other researchers that are related to the current research problem are summarized in this section. International Journal on Emerging Research Areas (IJERA) The authors discuss the various techniques used in speech recognition, such as Hidden Markov Models (HMMs), Artificial Neural Networks (ANNs), and Deep Neural Networks (DNNs). They also discuss the different approaches to speech recognition, such as the acoustic modeling approach, the language modeling approach, and the hybrid approach. browsing, reading and writing text. The application combines: a) client-- commerce application. side scripts (JavaScript and HTML) used to present information to the users. b) a back Python)end application powered by serverside scripts (JavaScript and c) a database system, where the latter two allows storing and retrieving information and generating responses to send back to the client.. The paper also discusses the challenges faced in developing voice-controlled web applications for different languages and accents. The authors highlight the importance of designing speech recognition systems that can handle a variety of accents and dialects, and provide examples of techniques used to improve the accuracy of speech recognition in different languages. The rest of the paper presents a background of the concepts, components, and applications of SRSs. 1. NLP And Machine Learning For Voice Synthesis This paper presents a study of the state-of-the-art speech recognition systems and propose a taxonomy of SRS. Mandeep, Farhana, Haruna presented a voice-controlled e- commerce application using IBM Watson speech-to-text service as a part of a comparative study with other speech-to- text systems such as Google and Amazon [1]. IBM Watson speech-to-text service uses advanced NLP and machine learning technologies for voice synthesis and text conversion. They claimed that their web-application takes a voice command, converts it to text, extracts meaning from the text, and then performs a wide variety of tasks including searching, IJERA ,2023,Volume 3,Issue 1 IJERA ,2023,Volume 3,Issue 1 IJERA ,2023,Volume 3,Issue 1 10.5281/zenodo.8207181 10.5281/zenodo.8207181 308 ISSN:2230-9993 International Journal on Emerging Research Areas (IJERA) IJERA ,2023,Volume 3,Issue 1 4. Alan AI The Alan AI is a powerful voice assistant platform that enables developers to add voice-enabled capabilities to their web and mobile applications[4]. The platform is designed to help developers create conversational interfaces using natural language processing (NLP) and machine learning technologies. Alan AI is easy to integrate into existing applications and can be customized to meet the specific needs of individual businesses. One of the key benefits of Alan AI is its flexibility. The platform supports a wide range of languages and dialects, making it accessible to users from all over the world. It also allows developers to create custom voice commands, which can be used to trigger specific actions or responses within an application. This gives users a more intuitive and personalized experience, which can lead to higher levels of engagement and satisfaction. Voice API has got some limitations in context of e-commerce applications. JavaScript libraries also will be great use to implement Speech To Text (STT) and Text To Speech (TTS) services. Above all the mentioned techniques, usage of online AI tool such as Alan AI is found to be more convenient to integrate with e-commerce applications. In this section we present a comparison of the methodologies used in each literature surveys. In this section we present a comparison of the methodologies used in each literature surveys. Fig 5: Comparison of surveys Fig 3: Data Flow Diagram[2] Fig 5: Comparison of surveys Fig 4: System Flow Diagram[2] Fig 4: System Flow Diagram[2] In a voice controlled e-commerce web application, the results can be summarized as follows. In terms of quality, Google proved to be superior to the other systems as it was able to identify 73.3% of the text with only 15.8% WER(Word Error Rate) and 73.3% PRR(Phrase Recogniton Rate). Cloud based SRS can be used, but not necessary. ISSN:2230-9993 Alan AI also includes a powerful analytics dashboard, which provides developers with valuable insights into how users are interacting with their applications. This data can be used to identify areas for improvement and optimize the user experience over time. Fig 3: Data Flow Diagram[2] Fig 4: System Flow Diagram[2] Fig 3: Data Flow Diagram[2] 3. Text to Speech and Speech to Text Fig 1:Taxonomy of SRS [1] 2. Artificial Neural Networks (ANNs), and Deep Neural Networks (DNNs) Fig 1:Taxonomy of SRS [1] The hands-free approach provided by the system goes to a great length and makes the user interact more often as the user usually prefers to use voice command rather than giving commands by typing [2]. One of the biggest advantages of the proposed system is that the voice recognition is not limited to just mobile phones, laptops or computers but voice recognition is being installed in all type of devices that users interact with like smart televisions, smart watches etc. S. Usharani application. , P. Manju Bala, R. Balamurugan[2] suggested a voice based form filling The application contains Google voice recognition, Text to Speech, Speech to Text, Voice access, Button mapping technology. The app is developed to fill the form by using voice. After filling the details, the mobile is connected with the printer by using the OTG adaptor and takes print of the form. With the help of headset button people can control the app button. If you click the headset button at the first time it asks the question next If you click the second button the app will get the input from your voice. 2. Artificial Neural Networks (ANNs), and Deep Neural Networks (DNNs) This paper[3] is all about speech recognition systems and their applications in developing voice-controlled web applications. The authors discuss the existing literature on speech recognition systems and their limitations, as well as the potential for developing more sophisticated systems that can accurately recognize and interpret natural language commands. They presented a new web based service that is a fusion of the revolutionary new Alan Studio, News API and React. The service provides all the components required for a user to be able to use voice and speech as a medium to find and look for news about his/her choice and the option to go through the news in a very concise manner. ANN and DNN are carried by Alan Studio[4] which construct complex and trustworthy in-- adds an entire serverless environment to app voice assistants and chat bots.React enable users to construct encapsulated parts that manage their own state, and then combine them to make dynamic Uis. Fig 2: Initial App View[2] Fig 2: Initial App View[2] IJERA ,2023,Volume 3,Issue 1 IJERA ,2023,Volume 3,Issue 1 309 International Journal on Emerging Research Areas (IJERA) IJERA ,2023,Volume 3,Issue 1 III. COMPARATIVE STUDY In this section we present a comparison of the methodologies used in each literature surveys. International Journal on Emerging Research Areas (IJERA) commerce website by their own. The main motivation for development of such a website is the lack of availability of visually impaired friendly e-commerce website. The website has linear navigation through entire website and processes voice output to instruct users about each step and the inputs to be provided so that it is easier for navigation. By following the best practices and staying up-to-date with the latest technology standards, we could provide a trustworthy voice-based shopping experience for the customers. In future, we may implement other similar voice-controlled applications. IV. CONCLUSION A voice based e-commerce website can provide a convenient and user-friendly experience for the customers. The main focus was on the blind people who cannot use the E- IJERA ,2023,Volume 3,Issue 1 IJERA ,2023,Volume 3,Issue 1 310 ISSN:2230-9993 International Journal on Emerging Research Areas (IJERA) International Journal on Emerging Research Areas (IJERA) International Journal on Emerging Research Areas (IJERA) References [1] A Voice Controlled E-commerce web application” by Mandeep Singh Kandhari;Farhana Zulkemine;Haruna Isah . 2018 IEEE 9th Annual Information Technology, Electronics and Mobile Communication Conference (IEMCON). [2] “Voice Based Form Filling System for Visually Challenged People”, year 2020, by S. Usharani P. Manju Bala;R. Balamurugan. [3] “Voice Controlled News Web Application with Speech Recognition using Alan Studio” by Aaditya Chaprana, Ranjeet Kumar, Ajay Saini, Akash Kumar, International Journal of Computer Applications,May 2021. [4] Ramu Sunkare, Alan AI documentation, accessed 20 April 2023, https://alan.app/docs/usage/how-works/about/ [5] “Blindness and vision impairment,” World Health Organization, 11-Oct- 2018..Available:https://www.who.int/news-room/factsheets/detail/ blindness-and-visual-impairment. [6] Jonathan Lazar ,Aaron Allen, Jason Kleinman Chris Malarkey, “What Frustrates Screen Reader Users on the Web: A Study of 100 Blind Users,” International Journal of Human–Computer Interaction ; vol 22, 2007. [7] Farnendes, Carvalho, Almeida, Simoes, “Transcoding for Web Accessibility for the Blind: Semantics from Structure,” Digital Spectrum: Integrating Technology and Culture - Proceedings of the 10th International Conference on Electronic Publishing held in Bansko - ELPUB 2006, Bansko, Bulgaria, June 14-16, 2006. [8] Mandeep, Farhana, Haruna, “A Voice Controlled E-Commerce Web Application”. Published in 2018 [9] Youhao Yu, “Research on Speech Recognition Technology and Its Application”. Published in 2012 [10] Phoemporn Lakhanawannakun, Chaluemwut Noyunsan, “Speech Recognition using Deep Learning”. Published in 2019. IJERA ,2023,Volume 3,Issue 1 IJERA ,2023,Volume 3,Issue 1 IJERA ,2023,Volume 3,Issue 1 311
https://openalex.org/W1947305501	https://kops.uni-konstanz.de/bitstreams/b6bcff6b-0ecd-4f0f-8652-215cea96bdd9/download	English	null	Induced responses to grazing by an insect herbivore (<i>Acentria ephemerella</i>) in an immature macrophyte (<i>Myriophyllum spicatum</i>): an isotopic study	Ecology and evolution	2,015	cc-by	7,397	Erschienen in: Ecology and Evolution ; 5 (2015), 17. - S. 3657-3665 Erschienen in: Ecology and Evolution ; 5 (2015), 17. - S. 3657-3665 Funding Information The study was supported by a startup grant (Young Scholar fund) of the University of Konstanz to EY. Received: 11 May 2015; Revised: 27 June 2015; Accepted: 8 July 2015 Received: 11 May 2015; Revised: 27 June 2015; Accepted: 8 July 2015 Received: 11 May 2015; Revised: 27 June 2015; Accepted: 8 July 2015 Ecology and Evolution 2015; 5(17): 3657–3665 doi: 10.1002/ece3.1624 doi: 10.1002/ece3.1624 Baldwin 1997; Kempel et al. 2011). Such mechanisms are fairly well documented in freshwater angiosperms (ref as given in Fornoff and Gross 2014, p.174). Keywords Acentria, freshwater macrophytes, herbivory, Myriophyllum, nitrogen sectoriality, nutrient reallocation, stable isotope. While the mechanisms by which adult terrestrial plants deploy constitutive and induced responses to grazing pressure are well known, the means by which young aquatic plants defend themselves from herbivory are little studied. This study addresses nitrogen transport in the aquatic angiosperm Myriophyllum spi- catum in response to herbivore exposure. Nitrogen tracers were used to moni- tor nitrogen uptake and reallocation in young plants in response to grazing by the generalist insect herbivore Acentria ephemerella. Total nitrogen content (N %) and patterns of nitrogen uptake and allocation (d15N) were assessed in vari- ous plant tissues after 24 and 48 h. Following 24 h exposure to herbivore dam- age (Experiment 1), nitrogen content of plant apices was signiﬁcantly elevated. This rapid early reaction may be an adaptation allowing the grazer to be sated as fast as possible, or indicate the accumulation of nitrogenous defense chemi- cals. After 48 h (Experiment 2), plants’ tips showed depletion in nitrogen levels of ca. 60& in stem sections vulnerable to grazing. In addition, nitrogen uptake by grazed and grazing-prone upper plant parts was reduced and nutrient alloca- tion into the relatively secure lower parts increased. The results point to three conclusions: (1) exposure to an insect herbivore induces a similar response in immature M. spicatum as previously observed in mature terrestrial species, namely a rapid (within 48 h) reduction in the nutritional value (N%) of vul- nerable tissues, (2) high grazing intensity (100% of growing tips affected) did not limit the ability of young plants to induce resistance; and (3) young plants exposed to herbivory exhibit different patterns of nutrient allocation in vulnera- ble and secure tissues. These results provide evidence of induced defense and resource reallocation in immature aquatic macrophytes which is in line with the responses shown for mature aquatic macrophytes and terrestrial plants. Correspondence Correspondence Elizabeth Yohannes, Limnological Institute, University of Konstanz, Mainaustrasse 252, D-78464 Konstanz, Germany. Tel: ++49 7531 882916; Fax: ++49 7531 883533; E-mail: Elizabeth.yohannes@uni-konstanz.de Correspondence Elizabeth Yohannes, Limnological Institute, University of Konstanz, Mainaustrasse 252, D-78464 Konstanz, Germany. Tel: ++49 7531 882916; Fax: ++49 7531 883533; E-mail: Elizabeth.yohannes@uni-konstanz.de Correspondence Elizabeth Yohannes, Limnological Institute, University of Konstanz, Mainaustrasse 252, D-78464 Konstanz, Germany. Tel: ++49 7531 882916; Fax: ++49 7531 883533; E-mail: Elizabeth.yohannes@uni-konstanz.de E-mail: Elizabeth.yohannes@uni-konstanz.de Present address Felix Fornoff, Naturschutz & Landschafts€okologie, Albert-Ludwigs- Universit€at, Freiburg, Germany Funding Information The study was supported by a startup grant (Young Scholar fund) of the University of Konstanz to EY. Present address Felix Fornoff, Naturschutz & Landschafts€okologie, Albert-Ludwigs- Universit€at, Freiburg, Germany Funding Information The study was supported by a startup grant (Young Scholar fund) of the University of Konstanz to EY. Present address Felix Fornoff, Naturschutz & Landschafts€okologie, Albert-Ludwigs- Universit€at, Freiburg, Germany ª 2015 The Authors. Ecology and Evolution published by John Wiley & Sons Ltd. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. Introduction The twin imperatives of defense against herbivory and competitive growth mean that both aquatic and terrestrial plants arrive at an optimal strategy through condition- dependent selection of either induced or constitutive defenses (Ito and Sakai 2009; Rasmann and Agrawal 2009). Thus, plants respond to herbivore attack in vari- able and complex ways that balance competition and defense. Induced mechanisms of defense are employed by a number of grazing-prone angiosperms to limit damage and include increased plant toughness, reduced nitrogen content, and thus lower nutritional values. Such responses have been shown to both increase plant ﬁtness (induced defense) and reduce growth of the herbivore (induced resistance) (e.g., Meldau et al. 2012). Increasing nutrient storage on the other hand, for example, by elevating concentrations of amino acids, enhances the nutritional quality of plant tissues and thereby their attractiveness to Constitutive resistance may be used to circumvent the severity of herbivore damage by either decreasing herbivore ﬁtness or increasing plant ﬁtness (Karban and 3657 3657 ª 2015 The Authors. Ecology and Evolution published by John Wiley & Sons Ltd. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. ª 2015 The Authors. Ecology and Evolution published by John Wiley & Sons Ltd. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. Konstanzer Online-Publikations-System (KOPS) URL: http://nbn-resolving.de/urn:nbn:de:bsz:352-0-303735 Konstanzer Online-Publikations-System (KOPS) URL: http://nbn-resolving.de/urn:nbn:de:bsz:352-0-303735 Induced Grazing Response In a Macrophyte K.-O. Rothhaupt et al. K.-O. Rothhaupt et al. herbivores (Mattson 1980; McClure 1980; Slansky and Rodriguez 1987). Thus, sectoriality in plant nutrient stor- age, whereby nutrient levels are reduced in important tis- sues subjected to grazing and elevated in less vulnerable parts of the plant, is an adaptive response to herbivory. immature specimens subject to short-term intensive grazing by A. ephemerella, which is frequently employed as a biological control agent. We conducted experiments using single element stable isotope 15N as nutrient tracer in young specimens of M. spicatum, which are more susceptible to damage than mature plants. In two similar laboratory-based setups, we examined the effect of grazing by A. emphemerella larvae on total plant nitrogen, nitrogen uptake, and nitrogen allocation. Introduction In order to estimate induced nitrogen realloca- tion, we analyzed the total nitrogen content (N%) of vari- ous tissues obtained from young plants after exposure to grazing. Using isotopic labeling with 15N, we investigated nitrogen uptake during grazing (d15N) and tracked the fate of nutrients within plants, including the delocaliza- tion of nitrogen-containing compounds in upper and lower plant sections. We expected immature M. spicatum subjected to grazing to show visible signs of damage and to respond by reallocating nitrogen content between grazed and nongrazed tissues. Most investigations of herbivory-induced plant defenses have focused on terrestrial angiosperms and illuminate the principal characteristics of defenses, including mecha- nisms of actions, effects on herbivores, and how herbi- vores react in turn. In contrast, induced defenses against herbivory in aquatic plants are hardly studied. Aquatic macrophytes differ from terrestrial counterparts in that they usually exist in two spatially distinct nutrient envi- ronments, with their roots in bottom sediment or soil and their vegetative parts submerged in the water column. Such heterogeneous abiotic resource distribution ought to permit the plant some ﬂexibility in nutrient uptake and chemical-related defense strategies (Smith and Barko 1990). Earlier studies indicate grazing pressure to be higher in aquatic than in terrestrial ecosystems (Gliwicz and Hill- bricht-Ilkowska 1972; Valiela 1984; Ricklefs 1990; Cry and Face 1991), and the rate of grazing damage on submersed leaves is reported to be higher than on ﬂoating leaves (Cronin et al. 1998). This has led to an assumption that defenses in aquatic plants might be uncommon and that losses are instead compensated for by increased primary production (Eppley 1981; McQueen et al. 1986; Sager and Richman 1991). However, the ecological assessment of spatiotemporal defense mechanisms by Karban (2011) suggests that plastic, induced defense should be particu- larly advantageous (e.g., over permanent constitutive defense) in variable situations where plants can access cues to predict future conditions, where defenses are costly and when defense is not necessarily essential. These circumstances apply convincingly to freshwater angios- perms such as Myriophyllum spicatum, which reproduces by fragmentation and inhabits aquatic systems with highly variable and heterogeneous spatiotemporal distributions of abiotic resources and herbivores. Experiment 1 Thirty M. spicatum shoots were planted in a single water tank (38 w 9 24 l 9 20 d cm) ﬁlled with lake water at 7°C (Fig. 1A). In order to create rooting substrate for the plants and supply microorganisms and small invertebrates similar to the mesocosm from which the immature shoots of M. spicatum were obtained about 50 mL of sieved (5 mm mesh size) sediment was added. The sediment was obtained from the same mesocosm where the plants were collected. To reduce leaching of nutrients from the sedi- ment into the water column, the top of the sediment was covered with a 2 cm layer of clean, ﬁne sand. The tank was then placed in a climate chamber with a 14 h/10 h light and temperature regime of 140/0 lmol quantam1s1 and 24/18°C, respectively. A recent experimental study investigating the responses of M. spicatum to the generalist insect herbivore Acentria ephemerella sought to address the gap in knowledge per- taining to aquatic plant defenses and yielded the ﬁrst evi- dence of both constitutive and induced mechanisms in a freshwater angiosperm (Fornoff and Gross 2014). The study was conducted using mature M. spicatum in both controlled and in seminatural ﬁeld conditions. The results indicated diverse responses to grazing by A. ephemerella, including reallocation of N-containing metabolites. Given the invasive nature of M. spicatum outside its native range, and its ability to reproduce readily by fragmentation, this study sought similar induced defense mechanisms in Materials and Methods Immature shoots of M. spicatum were obtained from a herbivore-free stand growing in a concrete mesocosm (2 w 9 2 l 9 1 d meter) at the Limnological Institute, University of Konstanz, Germany. Ninety individual shoots with tips of equivalent leaf density, maturity (no ﬂowers and no side shoots), and length (ca. 8–10 cm) were collected at random and transported to the labora- tory. Experiment 2 Sixty 100-mL crimp-top glass vials were ﬁlled with about 20 mL of sieved sediment (5 mm mesh size), covered with ca. 5 mL of ﬁne sand and carefully topped up with lake water. A single M. spicatum shoot was planted in each vial, and all vials were placed in a shared water tank (38w 9 24 l 9 20 d cm) ﬁlled with lake water at 7°C. The tank was then maintained in a climate chamber with a 14 h/10 h light and temperature regime of 140/0 lmol quantam1s1 and 24°/18°C, respectively. Plants were incubated for 8 days, after which they had reached a mean height (total length) of ca. 18.5 cm, of which ca. 10.2 cm (4.4, SD) was inside the vial and ca. 8.3 cm (3.5, SD) was growth emerging into main body of the tank. At the end of the incubation period, individual vials were removed gently from the shared tank and the top of the vial was sealed using a soft rubber bung (3 cm diame- ter 9 0.5 cm thickness) with a 3 mm slit and a punched aperture at the center allowing the plant stem to be inserted without damage (Fig. 1B). In order to prevent water exchange between the tank and the vial, the plant stem was dried at the point it passed through the ring using soft paper, and the slit and aperture of the rubber bung and the dried plant stem were smeared with silica gel, ensuring a watertight seal between the water of the tank and the vial. The size of the aperture and the soft- ness of the silica gel allowed unimpeded transport of plant ﬂuids and growth of the stem. Following this proce- dure, plants were able to continue growing with their upper and lower parts isolated in two separate water envi- ronments: the lower stems and roots in individual 100 mL crimp-top vials and the upper stems in the shared tank water. Figure 1. Schematic representation of stable isotope tracer experiment (A) Experiment 1 “Whole tank”) isotope tracer and (B) Experiment 2 “sectorial” isotope tracer experiment. Laboratory, 98% atom %) was added to the tank water. Plants had reached a mean height (total length) of ca. 41.39 cm (5.4, SD). Individual larvae of A. ephemerella (instar II, total length of ca. 4–5 mm) were then placed at the apex of every second plant. Experiment 2 Samples of plant tissues were collected for stable isotope analysis after 24 h expo- sure. Plant tips were examined for the presence of clear herbivory signs: feeding scars, feeding larvae, or larval shelters. Based on the presence or absence of all three characteristics, plants were classiﬁed as either grazed (G) or nongrazed (NG). Plants with less emphatic signs of damage, including only one or two of these three charac- teristics were excluded from further analysis. If the herbi- vore consumed greater portions of the tip or had removed the entire tip, the plant was excluded from the analysis. “Whole tank” stable isotope tracer After allowing plants to take root and grow for 2 weeks, nitrogen tracer (2 mgL1 15NH4Cl Cambridge Isotope ª 2015 The Authors. Ecology and Evolution published by John Wiley & Sons Ltd. 3658 Induced Grazing Response In a Macrophyte Induced Grazing Response In a Macrophyte K.-O. Rothhaupt et al. Figure 1. Schematic representation of stable isotope tracer experiment (A) Experiment 1 “Whole tank”) isotope tracer and (B) Experiment 2 “sectorial” isotope tracer experiment. ª 2015 The Authors. Ecology and Evolution published by John Wiley & Sons Ltd. “Sectorial” stable isotope tracer Two liters of tank water was removed, mixed with 1 mgL1 15NH4Cl and returned to the tank, allowing the upper sections of experimental plants to be exposed to the tracer compound, while the lower parts remained iso- lated in the unlabeled water of the vials. Four days later, a single larva of A. ephemerella (instar II, total length of ca. 4–5 mm) was placed at the apical shoot of every sec- ond plant. After 48 h, plant-tip (T) and upper stem M) samples were classiﬁed as well grazed (G) and nongrazed (NG), as described in Experiment 1, with slightly dam- aged tissues excluded from analysis. Individual plants were gently removed from the sedi- ment and washed with tracer-free lake water. Senescent leaves and roots were removed, and tissue samples were prepared from three locations for stable isotope and nitrogen content analysis. Tip sections (T) comprised the apex including the apical meristem and all nodules (in- cluding leafs) separated by internodes shorter than 2 mm (total size of resulting tip was ca. 1 cm³), while the remaining stem was halved into two pieces of equal size yielding upper stem (M) and lower stem (L) sections. For sampling of plant tissues, each vial containing an individual plant was removed from the water tank and processed individually. Plant upper sections were cut off at the lowest internode above the rubber seal. The rubber seal was then removed, the plants were cut again at the 3659 ª 2015 The Authors. Ecology and Evolution published by John Wiley & Sons Ltd. K.-O. Rothhaupt et al. Induced Grazing Response In a Macrophyte (A) 15 20 5 10 N % T-NG T-G M-NG M-G L-NG L-G 0 (B) 30 10 20 N % T-G R-G 0 T-NG S1-NG S1-G S2-NG S2-G S3-NG S3-G S-NG4 S4-G R-NG Plant sections Plant sections Figure 2. Nitrogen content (N%) of different plant sections of Experiment 1 (A) and 2 (B). Experiment 1: tip (T), upper stem (M), and lower stem (L). Experiment 2: tip (T), upper half of upper stem (S1), lower half of upper stem (S2), upper halve of lower stem (S3), lower half of the lower stem (S4), and root (R). “Sectorial” stable isotope tracer (A) 15 20 5 10 N % T-NG T-G M-NG M-G L-NG L-G 0 Pl t ti ﬁrst internode below the closure, and the lower section of the plant, complete with its roots, was gently removed from the sediment. Each plant part was washed with fresh lake water and kept separately. None of the stem parts tunneling though the rubber showed any signs of damage. The upper parts, which had been exposed to nutrient tra- cer, were separated into tip samples comprising apices and internodes up to 2 mm in length (T), upper halves of upper stems (S1), and lower halves of upper stems (S2). After removing the root, the lower sections which had not been exposed directly to tracer were cut into two halves ca. 4 cm each. Lower plant samples included the upper halves of lower stems (S3), the lower halves of lower stems (S4), and roots (R). Plant sections Plant sections (B) 30 10 20 N % T-G R-G 0 T-NG S1-NG S1-G S2-NG S2-G S3-NG S3-G S-NG4 S4-G R-NG Plant sections Stable isotope All samples were dried at 50°C for 48–72 h and pulver- ized before being subject to stable isotope analysis. Sam- ples were weighed in small tin cups to the nearest 0.001 mg, using a micro-analytical balance and combusted in a vario Micro cube elemental analyzer (Ele- mentar Analysensysteme, Germany). The resulting N2 was separated by gas chromatography and passed into a Micromass isotope ratio mass spectrometer (IRMS, Isoprime Ltd., Manchester, UK) for determination of 15N/14N ratios. Stable isotope values (d15N) are reported in d notation (per mill) where d = (1000 9 [Rsample/Rstandard]1)&; relative to atmo- spheric N2 for nitrogen in parts per thousand deviations (&). Two sulfanilamide (Iso-prime internal standards) and two Casein were used as laboratory standards for every 10 unknowns in sequence. Replicate assays of inter- nal laboratory standards indicated measurement errors (SD) of 0.15&. Plant sections Plant sections Plant sections Figure 2. Nitrogen content (N%) of different plant sections of Experiment 1 (A) and 2 (B). Experiment 1: tip (T), upper stem (M), and lower stem (L). Experiment 2: tip (T), upper half of upper stem (S1), lower half of upper stem (S2), upper halve of lower stem (S3), lower half of the lower stem (S4), and root (R). Comparisons between three tissues showed signiﬁcant dif- ferences between T and M (t = 10.08; P < 0.001) and between T and L (t = 11.55; P < 0.001). Mean N% data indicated that 24 h after herbivore addition, the nitrogen content of grazed tips was slightly elevated (mean N % SE: 18.49 1.37) compared to that of nongrazed tips (mean N% SE: 14.10 1.08), Fig. 2A. Compar- isons between M and L plant sections showed no signiﬁ- cant difference (P = 0.16). However, grazing alone did not have a signiﬁcant effect on the overall plant N% (F1,33 = 2.70.22; P = 0.12). Experiment 1 and Experiment 2 For each separate experiment, Two-way analyses of vari- ance were conducted to test for the effects of herbivory (G or NG) and plant section (T, M, L) or (T, S1, S2, S3, S4, R) on nitrogen (N%) or d15N. Bonferroni’s multiple test was used for all pairwise comparisons. “Sectorial” stable isotope tracer In Experiment 2, individual plants were incubated in such a way that isotopic tracers imparted “sectorial” d15N labeling only to the upper parts of the plant (T, S1, and S2) (Fig. 1B). This allowed us to detect shifts in d15N induced by 48 h of exposure to herbivory. Overall, the isotope values of the exposed upper plant parts were more enriched than those of the isolated lower stems and roots, as shown in Fig. 3B. (A) T-NG T-G M-NG M-G L-NG L-G 0 1000 2000 3000 Plant sections Plant sections 15N (B) T-NG T-G S1-NG S1-G S2-NG S2-G S3-NG S3-G S4-NG S4-G R-NG R-G 0 100 200 300 400 500 600 δ 15N δ Figure 3. Stable nitrogen isotope (d15N) values of different plant sections of Experiment 1 (A) and 2 (B). Experiment 1: tip (T), upper stem (M), and lower stem (L). Experiment 2: tip (T), upper half of upper stem (S1), lower half of upper stem (S2), upper halve of lower stem (S3), lower half of the lower stem (S4), and root (R). (A) T-NG T-G M-NG M-G L-NG L-G 0 1000 2000 3000 Plant sections 15N δ Nitrogen uptake (d15N) over 24 h – experiment 1 Plant sections Nitrogen content (N%) after 24 h – experiment 1 The effect of tissue type was also signiﬁ- cant (F5,156 = 15.60; P < 0.001). For almost all plant tis- sues, N% in grazed plants was lower than in nongrazed plants (Fig. 2B). Unlike for grazed and nongrazed tip tis- sues (t = 5.30; P < 0.001), Bonferroni’s pairwise compar- isons exhibited no signiﬁcant difference between grazed and nongrazed S1, S2, S3, S4, and R (P > 0.05). In grazed plants, tip tissues (T) exhibited signiﬁcantly higher N% than S1, S2, S3, S4, and R tissues (P < 0.02, for all Bon- ferroni’s pairwise comparisons). All other between-tissue comparisons showed no signiﬁcance difference (P > 0.05). Similarly, in nongrazed plants, N% in T sec- tions was signiﬁcantly higher than in S1, S2, S3, S4, and R. However, mean (SE) differences between the nitro- gen content (N%) in T and other plant sections were much higher in nongrazed plants (12.1% 0.6) than in grazed plants (5.5% 0.3). These results, together with those of Experiment 1 (i.e., change in N% in apices within 24 h), conﬁrm that grazing by A. ephemerella induces time- and tissue-based variation in nutrient allo- cation between plant tissues of immature M. spicatum. Therefore, in the subsequent nitrogen uptake (d15N) investigations, we questioned whether nitrogen uptake would also be affected by grazing. Although not statistically signiﬁcant, 24 h after herbivore addition, d15N of nongrazed tips was elevated compared to grazed tips (Fig. 3A). These results indicate that after 24 h of exposure to grazing, nitrogen transport to damaged tips is reduced relative to in nongrazed plants but that nitrogen uptake by damaged tips was not completely suspended. Nitrogen content (N%) after 24 h – experiment 1 Results of ANOVA did show a signiﬁcant effect of plant sections on N% (F2,33 = 7909; P < 0.001) (Fig. 2A). After 48 h of exposure to grazing, two-way ANOVA revealed a signiﬁcant effect of herbivory (F1,156 = 15.60; After 48 h of exposure to grazing, two-way ANOVA revealed a signiﬁcant effect of herbivory (F1,156 = 15.60; 3660 ª 2015 The Authors. Ecology and Evolution published by John Wiley & Sons Ltd. Induced Grazing Response In a Macrophyte K.-O. Rothhaupt et al. P < 0.001) on apical N%. However, the effect on tip nitrogen content was reversed compared to that observed at 24 h, with grazed tips showing signiﬁcantly reduced nitrogen (up to 50%) content (mean N% SE: 8.55 0.75) relative to nongrazed tips (mean N% SE: 17.66 3.16). The effect of tissue type was also signiﬁ- cant (F5,156 = 15.60; P < 0.001). For almost all plant tis- sues, N% in grazed plants was lower than in nongrazed plants (Fig. 2B). Unlike for grazed and nongrazed tip tis- sues (t = 5.30; P < 0.001), Bonferroni’s pairwise compar- isons exhibited no signiﬁcant difference between grazed and nongrazed S1, S2, S3, S4, and R (P > 0.05). In grazed plants, tip tissues (T) exhibited signiﬁcantly higher N% than S1, S2, S3, S4, and R tissues (P < 0.02, for all Bon- ferroni’s pairwise comparisons). All other between-tissue comparisons showed no signiﬁcance difference (P > 0.05). Similarly, in nongrazed plants, N% in T sec- tions was signiﬁcantly higher than in S1, S2, S3, S4, and R. However, mean (SE) differences between the nitro- gen content (N%) in T and other plant sections were much higher in nongrazed plants (12.1% 0.6) than in grazed plants (5.5% 0.3). These results, together with those of Experiment 1 (i.e., change in N% in apices within 24 h), conﬁrm that grazing by A. ephemerella induces time- and tissue-based variation in nutrient allo- cation between plant tissues of immature M. spicatum. Therefore, in the subsequent nitrogen uptake (d15N) investigations, we questioned whether nitrogen uptake would also be affected by grazing. P < 0.001) on apical N%. However, the effect on tip nitrogen content was reversed compared to that observed at 24 h, with grazed tips showing signiﬁcantly reduced nitrogen (up to 50%) content (mean N% SE: 8.55 0.75) relative to nongrazed tips (mean N% SE: 17.66 3.16). ª 2015 The Authors. Ecology and Evolution published by John Wiley & Sons Ltd. “Whole tank” stable isotope tracer Plant sections (B) T-NG T-G S1-NG S1-G S2-NG S2-G S3-NG S3-G S4-NG S4-G R-NG R-G 0 100 200 300 400 500 600 15N δ Our analyses revealed a signiﬁcant effect of grazing by A. ephemerella on d15N (F2,33 = 5.81; P = 0.025), which varied between plant sections (F2,33 = 109.26; P < 0.001) However, Bonferroni’s pairwise comparisons between grazed and nongrazed plant sections showed no signiﬁ- cant difference in nitrogen levels after a 24 h of exposure to herbivory (T: t = 1.63; P = 0.12; M: t = 1.36; P = 0.19; L: t = 1.18; P = 0.25). In both grazed and nongrazed plants, a signiﬁcant reduction in plant d15N values was evident with increased distance from the apex. Immature shoots of M. spicatum obtained from the mesocosm stand at the Limnological Institute (University of Konstanz) exhibit a natural d15N of ca. 5& (authors unpublished data). Following the addition of nitrogen trac- ers, all analyzed plant tissues showed a pronounced shift in d15N values, indicating that tracers had been successfully incorporated (in all plant sections, including roots) as a nutrient source for plant growth and competition. Plant sections Figure 3. Stable nitrogen isotope (d15N) values of different plant sections of Experiment 1 (A) and 2 (B). Experiment 1: tip (T), upper stem (M), and lower stem (L). Experiment 2: tip (T), upper half of upper stem (S1), lower half of upper stem (S2), upper halve of lower stem (S3), lower half of the lower stem (S4), and root (R). 3661 ª 2015 The Authors. Ecology and Evolution published by John Wiley & Sons Ltd. Induced Grazing Response In a Macrophyte K.-O. Rothhaupt et al. Following 48 h of exposure, there was no signiﬁcant effect of herbivory on tissue d15N (F1,156 = 1.38; P = 0.24), but a signiﬁcant effect of tissue section on d15N was apparent (F5,156 = 52.58; P < 0.001). On aver- age, the d15N values of grazed upper stems sections (S1) were depleted by ca. 60& relative to nongrazed S1 stems (Fig. 4). This is the part of the plant at closest proximity to the herbivore-damaged tips is therefore likely to be most vulnerable to further grazing. In contrast, the mid- lower stems (S3) of grazed plants, which are less prone to herbivore damage, showed an enrichment of d15N (mean = ca. 10&) compared to nongrazed S3 specimens. Nitrogen content (N%) In the ﬁrst experiment, after 24 h of exposure to A. ephe- merella, plant tips with characteristic signs of grazing damage exhibited higher nitrogen levels. After 48 h, how- ever, this initial response was reversed and grazed tips exhibited a ca. 50% decline in nitrogen levels compared to nongrazed apices. These time-regulated and tissue- (stem section-) dependent responses could be a mecha- nism whereby young M. spicatum are able to express both induced defense and induced resistance, reducing attrac- tiveness to herbivores while maintaining competitive growth. Results of Bonferroni’s multiple test for all pairwise comparisons (following two-way ANOVA) on the effects of herbivory (A. ephemerella) and plant section nitrogen content (N%) and nitrogen stable isotope (d15N) for Experiments 1 and 2 are given as supplementary materials a and b. g Similar experiments using mature M. spicatum and longer exposure to A. ephemerella have previously resulted in clear induced responses, including changes in the appearance of grazed apices, withdrawal of nitrogen, and increased nitrogen levels in lower parts of grazed shoots (Fornoff and Gross 2014). In mature M. spicatum, nitro- gen levels in the apices of grazed plants declined by up to 10%, while that of the lower sections was elevated by 14%. The reduction in N% in young plant tips grazed for 48 h in the current study is up to ﬁve times greater than observed previously in mature plants (Fornoff and Gross 2014), but the lower sections of immature plants showed no substantial increase in nitrogen levels in either grazed or nongrazed specimens. Despite the lack of elevated nitrogen in secure lower tissues, the stable nitrogen iso- tope data yielded by the current study (d15N) do imply a faster incorporation of d15N into lower plant sections (see results nitrogen uptake (d15N) – Experiments 1 and 2). It might be expected that large, mature plants with complex structures and multiple growing tips will express a more complex suite of defensive mechanisms incorporating dif- ferential uptake, routing, and allocation of nutrients than seen in single-tip immature plants like those in the cur- rent investigation. Our isotopic study examined responses to herbivory induced within a maximum of 48 h of graz- ing exposure. The possibility that longer exposure times might facilitate detection of further induced responses, affecting both uptake and reallocation (storage) of nitro- gen-containing compounds in immature plants, remains unexplored. “Whole tank” stable isotope tracer A minor increase in d15N was also notable in the lowest stem sections (S4) (mean = ca. 4.5&) of grazed plants. These results indicate an overall reduction in nutrient uptake by grazed and grazing-prone plant parts and a reallocation of nutrient to more secure tissues. on the nitrogen content (N%) of most tissues. In agree- ment with Hempel et al. (2009), our data also indicated reduction in tissue nitrogen levels with increased distance from the growing tips. ª 2015 The Authors. Ecology and Evolution published by John Wiley & Sons Ltd. Nitrogen uptake (d15N) Our results conﬁrm that exposure to an insect herbivore can result a relatively rapid alteration in nutrient content and nutrient allocation in apical tissues of immature aqua- tic macrophytes. Within only 24 h of grazer introduction, nongrazed apices were enriched by up to 280& compared to grazed shoots (experiment 1, d15N). However, after 48 h of grazing exposure, d15N values were equivalent in grazed and nongrazed apices. The levels of 15N enrichment are possibly but not necessarily in the range where simple diffusion and equilibration could result. Nevertheless, the results indicate a reduced rate of nitrogen uptake in grazed plants compared to ungrazed plants and a net transport of nutrient to affected plant parts. This could be interpreted as evidence of concurrent induced defense and induced resistance responses whereby an attacked plant may be able to (1) prevent loss of growing apical meristems, resulting in increased ﬁtness of the plant and (2) limit the nutri- tional value of the attacked tissues, thereby making the apices less attractive as dietary sources and resulting in decreased ﬁtness of the attacking herbivore. In summary, herbivory places additional demands on plants, through direct appropriation of resources and by altering the plant’s functional physiology. Induced with- drawal of nutrients from grazed tissues has implications for the ﬁtness of both the plant and the herbivore. Plants might render themselves less attractive to herbivores by reducing the availability of stored nitrogen and thus the nutritional value of particular tissues. This reduced nutri- ent intake will impair or halt growth and fecundity in individual herbivores, but it may also encourage higher consumption, which can only be tolerated by fast-growing macrophytes (Orians et al. 2011). A second possibility, when a plant is not resource limited, is for some nutrient to be reallocated from the most important tissues (such as apical meristems) to other tissues, leaving adequate nutrient in targeted tissues for herbivores to feed without causing signiﬁcant damage or driving it to colonize other sections of the plant. The second argument is more likely to hold if the plant is growing slowly and if grazed or grazing-prone tissue is particularly essential for survival. In our experiment, we used young plants undergoing rapid development, in which nutrient reduction likely to be the most cost-efﬁcient defense mechanism for with- standing damage without a reduction in ﬁtness. Discussion Using inorganic nitrogen isotopes as tracers, we measured plant nitrogen levels (N%) and tracked nitrogen uptake (d15N) by immature submerged specimens of the aquatic macrophyte M. spicatum following exposure to an insect herbivore, A. ephemerella. Experimentally, grazed plants exhibited three characteristic signs of grazing damage (feeding scars, detectable larvae, and larval shelters), con- ﬁrming that they had been subject to direct attack. Gener- ally, results showed a negative effect of sustained grazing Figure 4. Mean d15N (&) enrichment or depletion values of different plant sections after 48 h herbivore exposure and tracer addition (Experiment 2). Previous studies in Lake Constance report that up to 82% of apical meristems in M. spicatum specimens with 20–50 tips were damaged or missing, due to grazing by Acentria (Gross et al. 2002). Our experimentally grazed plants all had just one tip (simulating the establishment Figure 4. Mean d15N (&) enrichment or depletion values of different plant sections after 48 h herbivore exposure and tracer addition (Experiment 2). 3662 ª 2015 The Authors. Ecology and Evolution published by John Wiley & Sons Ltd. Induced Grazing Response In a Macrophyte K.-O. Rothhaupt et al. plants. Nitrogen transport to plant sections closest to the site of herbivore damage was reduced. Secondly, nutrient reallocation to lower sections of the stem was enhanced. Coupling the results of the whole-plant and sectoral stable isotope-labeling experiments, we see that herbivory induces changes to resource allocation and sequestration of nutrient into lower stems. These tissues may serve as secure storage for the limiting resource of nitrogen, including complex nitrogen compounds and nitrogen-rich chlorophyll molecules, while simultaneously making the attacked plants less appealing to herbivores. phase of natural propagules), and each individual plant was exposed to just one Acentria larva. This could be interpreted as 100% exposure in the grazed treatment, with implications in respect of biocontrol of M. spicatum during its establishment and propagation as an alien inva- der. However, despite this grazing pressure, apices of all experimental plants remained intact, and while stem parts exhibited visible feeding scars, the damage was not severe and there was no fragmentation of apical meristems. Thus, in our experiments, nutrient transport was main- tained even during herbivore attack, ensuring the poten- tial for nitrogen-containing metabolites to be allocated to growing meristems and storage organs. The sequestration of nutrient resources in less vulnera- ble plant parts may bestow both growth and competitive advantages on young plants. Nitrogen uptake (d15N) Presumably, the observed short-term response to onset of grazing reﬂects an almost immediate uptake or accumu- lation of nitrogen-rich defensive chemicals such as alka- loids in apical meristems. By acting as a deterrent to the herbivore, such a reaction might be expected to minimize injury and prevent loss of growing apical meristems. Alter- natively, a herbivory-induced response that increases N% in apices could also confer defense by sating the herbivore faster, while the potential for rapid compensatory growth by young M. spicatum may render localized induced nutri- ent uptake a cost-effective defense against A. ephemerella. There are increasing evidences on the ecological func- tion of interplant volatile signaling in plant–herbivore interactions (e.g., Heil and Karban 2010; Pearse et al. 2013). Our experimental set-up allows the potential for organic compounds released by the grazed plants to be detected by the ungrazed ones. As the importance of interplant signaling in plant–herbivore interactions has barely been studied in aquatic systems, the direction of its Discussion The suite of induced responses have previously observed in mature M. spica- tum (Fornoff and Gross 2014), included nutrient seques- tration, and similar strategies are documented in terrestrial plants (e.g., Verges et al. 2008; Orians et al. 2011; Meldau et al. 2012) and in another submerged macrophyte, Potamogeton perfoliatus, following grazing by A. ephemerlla larvae (Miler and Straile 2010). Herbivore-induced changes in resource allocation and sequestration Comparisons between six different tissue sections con- ﬁrmed major responses in nutrient allocation in grazed 3663 ª 2015 The Authors. Ecology and Evolution published by John Wiley & Sons Ltd. Induced Grazing Response In a Macrophyte K.-O. Rothhaupt et al. potential effect on the results presented in this study could be a question of future research. Cook, C. D. K. 1999. The number and kinds of embr bearing plants which have become aquatic: a survey Perspect. Plant Ecol. Evol. Syst. 2:79–102. Cook, C. D. K. 1999. The number and kinds of embryo- bearing plants which have become aquatic: a survey. l l l Finally, while patterns of constitutive chemical resis- tance in aquatic ecosystems have been well studied (e.g., Prusak et al. 2005; Agrawal and Fishbein 2006; Erhard et al. 2007), detailed comparisons between induced resis- tance mechanisms described in freshwater plants are lim- ited. This is because generalizations about herbivory in aquatic systems have to be based on relatively few data (e.g., Morrison and Hay 2011; Fornoff and Gross2014). Thus, far, induced defense has been reported in two recent studies of marine angiosperms (Steele and Valen- tine 2012; Darnell and Heck 2013) and in a limited num- ber of earlier studies of freshwater angiosperms (Jeffries 1990; Bolser et al. 1998; Lemoine et al. 2009; Morrison and Hay 2011; Fornoff and Gross 2014). Perspect. Plant Ecol. Evol. Syst. 2:79–102. Cronin, G., K. D. Wissing, and D. M. Lodge. 1998. Cronin, G., K. D. Wissing, and D. M. Lodge. 1998. Comparative feeding selectivity of herbivorous insects on water lilies: aquatic vs. semiterrestrial insects and submersed vs. ﬂoating leaves. Freshw. Biol. 39:243–257. Cry, H., and M. L. Face. 1991. Magnitude and patterns of herbivory in aquatic and terrestrial ecosystems. Nature 361:148–150. Darnell, K. M., and K. L. Jr Heck. 2013. Species-speciﬁc effects of prior grazing on the palatability of turtle grass. J. Exp. Mar. Biol. Ecol. 440:225–232. Eppley, R. W. 1981. Autotrophic production of particulate matter. Pp. 343–361 in A. R. Longhurst, ed. Analysis of marine ecosystems. Academic Press, London. Erhard, D., G. Pohnert, and E. M. Gross. 2007. Chemical defense in Elodea nuttallii reduces feeding and growth of aquatic herbivorous Lepidoptera. J. Chem. Ecol. 33:1646– 1661. Herbivore-induced changes in resource allocation and sequestration Although all aquatic angiosperms share common ances- tors with terrestrial plants and are secondary adapted to water, it is not yet to know whether they exhibit an equivalent capacity for induced and constitutive defense (Cook 1999). This study provides additional isotopic evi- dence for rapid induced responses to grazing by an insect herbivore (A. ephemerella) in an immature macrophyte (M. spicatum). This adaptive response could be a key trait supporting the species’ successful establishment as an invasive alien in North America. The current work also highlights that accurate interpretation of macrophyte nitrogen content and nitrogen stable isotope values requires a cautious assessment herbivore pressure, as even very short periods of exposure to grazing are sufﬁcient to induce a defensive response. Fornoff, F., and E. M. Gross. 2014. Induced defense mechanisms in an aquatic angiosperm to insect Herbivory. Oecologia 175:173–185. Gliwicz, Z. M., and A. Hillbricht-Ilkowska. 1972. Efﬁciency of the utilization of nannoplankton primary productivity by communities of ﬁlter feeding animals measured in situ. Verhandlungen der Internationale Vereinigung f€ur Theoretische und Angewandte Limnologie 18:197–203. Gross, E. M., C. Feldbaum, and C. Choi. 2002. High abundance of herbivorous Lepidoptera larvae (Acentria ephemerella Denis & Schiffermuller) on submersed macrophytes in Lake Constance (Germany). Arch. Hydrobiol. 155:1–21. Acknowledgments Heil, M., and R. Karban. 2010. Explaining evolution of plant communication by airborne signals. Trends Ecol. Evol. 25:137–144. We appreciate the help of Martin Wolf and Birgit Beck during sample collection, preparation, and analysis. We thank Amy-Jane Beer, Ioanna Salvarina, Corinna Waider, and members of the stable isotope laboratory for valuable editorial and scientiﬁc comments, discussions, and sug- gestions. The study was supported by a startup grant (Young Scholar fund) of the University of Konstanz to EY. Hempel, M., H. P. Grossart, and E. M. Gross. 2009. Community composition of bacterial bioﬁlms on two submerged macrophytes and an artiﬁcial substrate in a pre- alpine lake. Aquat. Microb. Ecol. 58:79–94. Ito, K., and S. Sakai. 2009. Optimal defense strategy against herbivory in plants: conditions selecting for induced defense, constitutive defense, and no-defense. J. Theor. Biol. 260:453–459. None declared. Karban, R. 2011. The ecology and evolution of induced resistance against herbivores. Funct. Ecol. 25:339–347. Conﬂict of Interest Jeffries, M. 1990. Evidence of induced plant defences in a pondweed. Freshw. Biol. 23:265–269. None declared. Agrawal, A. A., and M. Fishbein. 2006. Plant defense syndromes. Ecology 87:132–149. Bolser, R. C., M. E. Hay, N. Lindquist, W. Fenical, and D. Wilson. 1998. Chemical defenses of freshwater macrophytes against crayﬁsh herbivory. J. Chem. Ecol. 24:1639–1658. References Valiela, I. 1984. Marine ecological processes. Springer, New York, NY. Morrison, W. E., and M. E. Hay. 2011. Induced chemical defenses in a freshwater macrophyte suppress herbivore ﬁtness and the growth of associated microbes. Oecologia 165:427–436. Verges, A., M. Perez, T. Alcoverro, and J. Romero. 2008. Compensation and resistance to herbivory in seagrasses: induced responses to simulated consumption by ﬁsh. Oecologia 155:751–760. Orians, C. M., A. Thorn, and S. Gomez. 2011. Herbivore- induced resource sequestration in plants: why bother? Oecologia 167:1–9. References Karban, R., and I. T. Baldwin. 1997. Induced responses to herbivory. Univ. of Chicago Press, Chicago, IL. Agrawal, A. A., and M. Fishbein. 2006. Plant defense syndromes. Ecology 87:132–149. Kempel, A., M. Sch€adler, T. Chrobock, M. Fischer, and M. van Kleunen. 2011. Tradeoffs associated with constitutive and induced plant resistance against herbivory. Proc. Natl Acad. Sci. USA 108:5685–5689. Bolser, R. C., M. E. Hay, N. Lindquist, W. Fenical, and D. Wilson. 1998. Chemical defenses of freshwater macrophytes against crayﬁsh herbivory. J. Chem. Ecol. 24:1639–1658. 3664 ª 2015 The Authors. Ecology and Evolution published by John Wiley & Sons Ltd. K.-O. Rothhaupt et al. Induced Grazing Response In a Macrophyte and antagonism between resistance traits. Curr. Opin. Plant Biol. 12:473–478. Lemoine, D. G., M.-H. Barrat-Segretain, and A. Roy. 2009. Morphological and chemical changes induced by herbivory in three common aquatic macrophytes. Int. Rev. Hydrobiol. 94:282–289. Ricklefs, R. 1990. Ecology, 3rd edn. Freeman, New York, NY. Sager, P. E., and S. Richman. 1991. Functional interaction of phytoplankton and zooplankton along the trophic gradient in Green Bay, Lake Michigan. Can. J. Fish Aquat. Sci. 48:116–122. Mattson, W. J. Jr. 1980. Herbivory in relation to plant nitrogen content. Annu. Rev. Ecol. Syst. 11:119–161. Slansky, F., and J. G. Rodriguez. 1987. Nutritional ecology of dung- and carrion-feeding insects. Pp. 837–884 in F. Slansky, F. G. Rodriguez, eds. Nutritional ecology of insects, mites, spiders, and related invertebrates. John Wiley & Sons, New York, NY. McClure, M. S. 1980. Foliar nitrogen: a basis for host suitability for elongate hemlock scale, Fiorinia externa (Homoptera: Diaspididae). Ecology 61:72–79. dung and carrion feeding insects. Pp. 837 884 in F. Slansky, F. G. Rodriguez, eds. Nutritional ecology of insects, mites, spiders, and related invertebrates. John Wiley & Sons, New York, NY. (Homoptera: Diaspididae). Ecology 61:72–79. McQueen, D. J., J. R. Post, and E. L. Mills. 1986. Trophic relationship in freshwater pelagic ecosystem. Can. J. Fish Aquat. Sci. 43:1571–1581. Smith, C. S., and J. Barko. 1990. Ecology of Eurasian watermilfoil. J. Aquat. Plant Manag. 28:55–64. Meldau, S., M. Erb, and I. T. Baldwin. 2012. Defence on demand: mechanisms behind optimal defence patterns. Ann Bot. 110:1503–1514. Steele, L., and J. F. Valentine. 2012. Idiosyncratic responses of seagrass phenolic production following sea urchin grazing. Mar. Ecol. Prog. Ser. 466:81–92. Miler, O., and D. Straile. 2010. How to cope with a superior enemy? Plant defense strategies in response to annual herbivore outbreaks. J. Ecol. 98:900–907. ª 2015 The Authors. Ecology and Evolution published by John Wiley & Sons Ltd. Supporting Information Pearse, S. I., K. Hughes, K. Shiojiri, S. Ishizaki, and R. Karban 2013. Interplant volatile signaling in willows: revisiting the original talking trees. Oecologia 172:869–875. Additional Supporting Information may be found in the online version of this article: Appendix S1. Bonferroni’s Multiple Test for all Pairwise comparisons (following Two-way ANOVA) on the effects of herbivore (A. ephemerella) and plant section nitrogen content (N%) and nitrogen stable isotope (d15N) for experiment 1 and 2. Prusak, A. C., J. O’Neal, and J. Kubanek. 2005. Prevalence of chemical defenses among freshwater plants. J. Chem. Ecol. 31:1145–1160. Rasmann, S., and A. A. Agrawal. 2009. Plant defense against herbivory: progress in identifyingsynergism, redundancy, 3665 ª 2015 The Authors. Ecology and Evolution published by John Wiley & Sons Ltd.
https://openalex.org/W2733544057	https://revistas.ucm.es/index.php/IJHE/article/download/56516/51079, https://eprints.ucm.es/46870/1/17-4-1%2863-73%29.pdf, https://estudogeral.sib.uc.pt/bitstream/10316/43668/1/The%20internationalization%20of%20economic%20ideas.pdf, https://eprints.ucm.es/id/eprint/46870/1/17-4-1%2863-73%29.pdf	es		The internationalization of economic ideas. A search for connecting principles	Iberian journal of the history of economic thought	2,017	cc-by	7,094	ARTÍCULOS Iberian Journal of the History of Economic Thought ISSN-e 2386-5768 http://dx.doi.org/10.5209/IJHE.56516 The internationalization of economic ideas. A search for connecting principles1 Vítor Neves2 Received: 16/02/2017 / Accepted: 26/04/2017 Abstract. The processes of internationalization of economic ideas, in particular those associated with the transmission, assimilation and appropriation in scientific peripheral and semi peripheral countries of ideas originally produced in other spaces, are an important aspect of how economics as a science develops at a global scale. However, in spite of its relevance, knowledge of these processes is still relatively incipient. Explaining the international diffusion of economic ideas entails choosing some sort of connecting principle(s). This paper discusses this issue and attempts to put forward a broader connecting principle than the ones currently available, which is based on the idea that economics is a network of institutionally situated conversations. Keywords: Connecting principles, international diffusion of ideas, assimilation/appropriation, intellectual peripheries, economics, conversation JEL Classification: B00 [es] La internacionalización de las ideas económicas. Una búsqueda de principios de conexión Resumen. Los procesos de internacionalización de ideas económicas, en particular aquellos asociados con la transmisión, la asimilación y la apropiación de ideas en los países científicos periféricos y semi-periféricos producidas originalmente en otros lugares, son un aspecto importante de cómo la economía como ciencia se desarrolla a escala global. Sin embargo, a pesar de su importancia, el conocimiento de estos procesos es todavía relativamente incipiente. La explicación de la difusión internacional de ideas económicas requiere la selección de algún tipo del principio(s) conector(es). Este artículo trata este tema e intenta proponer un más amplio principio de unión que los actualmente disponibles, basado en la idea que la economía es una red de conversaciones institucionalmente situadas. Palabras clave: Principios conectores, difusión de ideas internacionales, asimilación/apropiación, periferias intelectuales, economía, conversación Clasificación JEL: B00 I have always known that the words of others help me to think. Quotations (and misquotations), asides, seemingly dead ends, explorations and rummagings, retracing one’s steps and leaping ahead — all seem to me valid instruments for inquiry. Alberto Manguel, Curiosity (Yale University Press, 2015, 83) 1 2 Faculdade de Economia / Centro de Estudos Sociais. Universidade de Coimbra, Portugal. vneves@fe.uc.pt Acknowledgements: This work was supported by the Portuguese Foundation for Science and Technology, namely through the Project PTDC/IVCHFC/3826/2014- POCI-01-0145-FEDER-016871. Special thanks are due to Carlos Bastien, José Luís Cardoso, John B. Davis, Javier San Julián Arrupe and the participants in the Third Lisbon International Conference on Philosophy of Science: Contemporary Issues (14-16 December 2016, Lisboa) and in a workshop held at the Department of Economic History, Institutions, Policy and World Economy of the University of Barcelona (29 March 2017) for their valuable and insightful comments and suggestions. The usual disclaimer applies. Iber. hist. econ. thought. 4(1) 2017: 63-73 63 64 1. Introduction History of economics textbooks are, in general, histories of the contributions considered to have been decisive in the formation of economics as a scientific discipline, i.e. they are largely histories of the scientific contributions to knowledge at the core of the economics profession. The histories of economics in peripheral and semi peripheral countries, like Portugal or Spain, are usually neglected. The implicit assumption is that economics is a “universal”, increasingly global science, and that, as such, giving attention to the national realities of the intellectual peripheries would be somewhat expendable. This paper starts from a different belief. The processes of internationalization of economic ideas, in particular those associated with the transmission, reception, assimilation and appropriation in scientific peripheral and semi peripheral countries of ideas originally produced in other spaces, are an important aspect of how economics as a science develops at a global scale. Histories of economics in those countries are relevant, not only from a national point of view, but as an input for the historiography of economics in general. Place, travel and assimilation/appropriation are fundamental keywords —and, in the end, as I will show, institutions, networks and conversations. The making of economics at intellectual peripheries is, to a large extent, a history of international transmission of economic ideas (doctrines, theories, methods/techniques and policy recommendations), practices and institutions (Mäki, 1996). The study of their histories thus provides a better knowledge of how economic ideas and practices circulate at an international scale and allow us to check how globalization is having an impact at the national level. Unfortunately, in spite of a significant amount of work already done, our knowledge of these matters is still relatively incipient. Several studies have attempted to model processes of international diffusion and appropriation, but we are far from a comprehensive (“general”) historiographical framework allowing for an explanation of the occurrences of invention, importation, acceptance, rejection and assimilation/modification/appropriation of economic ideas in intellectually peripheral or semi peripheral countries. In particular: Neves, V. Iber. hist. econ. thought. 4(1) 2017: 63-73 — What factors in general affect knowledge circulation? — Why are some foreign influences accepted (to varying degrees) and others rejected? — Under what circumstances is the original content of imported theories modified or retained? — How do “internal” and “external” factors condition (promoting, prohibiting or hindering) the international dissemination of economic knowledge? A mere description of “facts” is not enough. As Vicent Llombart (1995, 32) noted, “We need the help of theoretical models with a capacity to explain the phenomenon of the spread of ideas”. Historical writing is an explanatory endeavor (Mäki, 1996). It is a search for the mode of production of phenomena —looking for its real causes (determining factors) or conditions of possibility and its generating mechanisms. In the human realm this entails identifying and understanding the social structures, relationships, capacities and other real conditions that govern, facilitate, or in some way produce, actual relevant social events and states of affairs. This raises the crucial issue of the role and significance of the connecting principles one chooses to construct models. Adam Smith ([1795]1980) and, more recently, Brian Loasby (1991, 1999, 2005) have taught us that, in order to make sense of what we experience, we invent connecting principles, i.e. organizing ideas and concepts, interpretative frameworks, conjectures about reality that link together phenomena in our minds. I will try to show that each model of the international diffusion of economic ideas presupposes a different set of (often merely implicit) connecting principles (and metaphors), hence the relevance of focusing attention on them. In a certain sense this is an exercise in putting old wine in new bottles. However, it will, it is hoped, contribute to redirect attention to a fundamental issue in the endeavor of explaining the international diffusion and appropriation of economic thought: the connecting principles underlying the models we build. Moreover, it is expected that it may contribute to guide empirical research on this important issue. The paper is structured as follows. In the next section, the national/global tension in economics occupies center stage. Afterwards, in Neves, V. Iber. hist. econ. thought. 4(1) 2017: 63-73 65 section 3, a brief overview of the relevant literature on the diffusion of economic ideas is provided. In section 4 I show why the choice of connecting principles is so crucial in the modeling of the processes of international diffusion and appropriation of economic ideas. In section 5the “economics as conversation” metaphor is explored as a basis for a broader framework for the analysis of those processes. Some concluding remarks in section 6 close the paper. 2. The national/global tension in economics Economics is a plural and complex science, subject to different methodological approaches and constructions not independent from the historical, social and cultural circumstances conditioning them. The vision of economics as a “universal” science, with a unified set of true and absolute concepts and universal procedures of analysis, is misleading. Economics is a heterogeneous space where multiple languages, metaphors, and conversations converge and compete. Economic theories are often (but not always)3developed at a “core”, which is itself “locally” shaped, they travel and are assimilated/appropriated in other, peripheral and semi peripheral, contexts and settings4. As Almodovar and Cardoso (1998, 2) nicely put the issue: 3 4 The structuralist approach originated in Latin America with authors such as Raul Prebisch and Celso Furtado being a case in point. The terms “periphery” and “semi periphery” are usually applied to classify the economic, political and intellectual realities of some countries and regions. Often, they are used in a very loose way. Almodovar and Cardoso (1998) and Cardoso (2002) considered that a country is intellectually peripheral in economics if it occupies a permanently or quasi-permanently subordinate position, never reaching, or only very episodically drawing close to, the front line of the creation of economic theories. In turn, Bastien and Cardoso(2003, 39) referred to semi-peripheral countries as those intermediate situations between the two extreme types (“core” and “periphery”) whose distinctive character is “their willingness to accept influences from both sides, preserving a certain degree of autonomy and identity”. More precisely, and drawing also on Mäki (1996), we may say that identifying a country as part of the intellectual periphery or semi periphery in economics within a given period of time has to be decided in terms of, simultaneously, its (i) propensity to import ideas; (ii) the time lag it takes between the adoption of ideas in the originating and in the importing country; (iii) the degree of modification (appropriation) of ideas in the importing country; (iv) the willingness of the country to accept influences from both sides; and (v) its degree of autonomy and identity. It is thus not simply a matter of being a net importer or exporter of ideas; the specific contribution of the receiving country and its capacity also to generate influence abroad are crucial. conomic science has no homeland, though it E is represented and conveyed through different tongues, anthems and flags. But, as it is the work of scientists, we cannot ignore the fact that this homeless quest creates links between a huge number of people (researchers, teachers). Although these people are united by a common pursuit of knowledge, they are nevertheless spread all around the world, giving factual existence to schools and other particular institutional environments where science is actually produced and nurtured. Economics is institutionally situated. Economic ideas are “locally” produced, assimilated and appropriated, in accordance with social, economic, political, cultural, academic and educational conditions, and are expressed in different “locally” shaped ways. Economic ideas circulate internationally and this is not just a matter of transmission of ideas produced at the core of the profession to more or less passive receivers on the periphery(a mere case of unidirectional export of ideas from the core to the periphery). Ideas tend to be actively appropriated (and as such somewhat produced) in peripheral and semi peripheral countries (Gavroglu et al., 2008). They are institutionally-specific and acquire a national dimension. It is therefore imperative, as Cardoso (1997, 208) claimed, to analyze the processes that are at the origin of a diversity of national forms and contents of economic thought5. However, either an exclusively transmission-focused approach (a “historiography of transmission”) or a renovated “historiography of appropriation” (Gavroglu et al., 2008) would be partial and biased. As Marion Fourcade (2006) rightly noted, although we tend to see the internationalization of economics largely as a unidirectional process going from the core to the periphery, a trend towards transnationalization/ globalization is taking place in economics with the very nature of economic knowledge and the jurisdictional control of the economics profession being dynamically reshaped and transformed both at the core and on the periphery. The end result of this process, it might be expected, will be “a form of institutional and intellectual convergence between the econom5 As Argemí (2006, 167) accurately pointed out, the “national” term should be understood here as related to the economic thought of a given cultural, political or legal area in relation to the development of economic thinking in the rest of the world, not to any specific entity of political right. Neves, V. Iber. hist. econ. thought. 4(1) 2017: 63-73 66 ics professions in peripheral countries and those at the center, as foreigners and foreign-trained professionals increasingly penetrate local institutions, and as these institutions try to emulate dominant foreign models” (Fourcade, 2006, 152). Anyway, this does not exclude the asymmetric nature of the relationships established at an international scale. Economics must be understood as a complex intellectual and institutional system of interacting ideas and practices in which the asymmetric connections between the “core” and the “peripheries” (and their respective conditions of production of economic ideas) are paramount. 3. Modeling the international diffusion of economic ideas: the state of the art Let us now proceed with a brief overview of the relevant literature on the international diffusion of economic ideas. The first systematic incursion into this subject (Letiche, 1955) took place in the mid-1950sat a session of the annual conference of the American Economic Association specifically organized to discuss the topic, with papers by T. W. Hutchison (1955), J. Dorfman (1955), and comments by J. Letiche, G. Hildebrand and W. Jaffé. These works provided heuristic guidelines for the study of the conditions and factors (favorable, accelerating or obstructive) influencing the diffusion of economic ideas (Cardoso, 2003). Hutchison’s views are particularly noteworthy as he believed that “[w]ith the vastly increased number of translations and of widely circulating specialist journals, including international journals, and with the increasingly mathematical character of advanced economic analysis”, it would be “very unlikely that good new ideas, whenever or wherever they do arise, will not have a reasonably fair chance of being heard and of making their way” (Hutchison, 1955, 14-15). Such an optimistic (and “universalistic”) view was contested at the time (e.g. Letiche, 1955) and on other occasions thereafter (see Lluch 1999). Several constraints to the smooth exchange of economic ideas, namely those related to the development of the media of transmission, the existence of enduring disequilibrium relationships between exporting and importing countries, and the specificities of economic realities, social and political insti- tutions, and scientific environments of the latter countries, have been pointed out (Cardoso, 2003, 625). Since the 1950s several works and case studies have been published on the international flow of economic ideas. This is not the time or the place to recapitulate all this literature6. For my purpose here, it suffices to look, in a very selective way, to a few representative instances in order to assess its nature and highlight the relevance of choosing appropriate connecting principles for an explanation of the processes of internationalization of economic ideas, practices and institutions. Coats and Colander’s (1989) The Spread of Economic Ideas is a landmark in this endeavor although it does not give direct relevance to the issue of the international diffusion of ideas. In the introduction to this book, their editors consider three models for the study of the spread of economic ideas: (i) the infectious disease model; (ii) the model of the market for ideas; and (iii) the information theory model. In the infectious disease (or epidemiological) model the dissemination of ideas is likened to the spread of a disease (e.g. AIDS). The focus is on the contacts between individuals and groups (as if “points of contagion”) and on the incentives and barriers to the propagation of ideas. The model of the market for ideas considers the dissemination of ideas in terms of supply of and demand for ideas, and the competition among them, as if these were commodities transacted in a market. Economists are seen as optimizing agents pursuing some goal —be it truth, attention, recognition, access to funding, status, income, fame or some such— which they try to maximize under constraints (costs). The focus is again on the incentives to the dissemination and reception of ideas, but now based on the application of the economists’ conventional framework to the analysis of a reputational science. The information theory model sees the processes of transmission, selection and adaptation of economic ideas in terms of information theory. The focus in this case goes to the analysis of the four elements of a communication process: the source of ideas; the receiver (and 6 For those interested, Cardoso (2003; 2009) are excellent references. Neves, V. Iber. hist. econ. thought. 4(1) 2017: 63-73 its milieu); the content transmitted and the media (or channel) of transmission. Mäki (1996) provides an important development of this model, namely with his emphasis on the need to distinguish internal and external factors at different levels (theoretical/ non theoretical within the cognitive aspect of science; cognitive/non cognitive within science; and science/non science within society), each of them leading to the consideration of a different object of historiographical explanation and, of course, to a different approach —“cognitive”, “scientific” or “societal”— to the analysis of the role of internal versus external factors in the development of economic thought. Previously, Ernest Lluch (1980) had already taken a significant step forward in the study of the international flow of economic ideas by linking it to a variety of national processes of assimilation and appropriation, which justify the relevance given to the national histories of economic thought (Lluch, 1999, develops and improves this initial innovative move). José Luís Cardoso must also be mentioned as a key reference here. For a long time now (e.g. 1997, 2002, 2003and 2017), he has been emphasizing the need to adopt a comparative approach to the analysis of different national experiences. In his view, “it is in the diagnosis of national problems and in their resolution that innovative and genuinely national forms of economic thought emerge” (Cardoso, 1997, 214). The national perspective, Cardoso and Lluch (1999, 478) jointly stated, “introduces a number of additional details and makes it possible to develop and modify the theories and doctrines that are appropriated and used.” Cardoso (1997, 226-227, and 2002, 143) explicitly detailed a whole program of research, which included: (i) Assessing the levels of understanding, familiarization and misrepresentation in the importing countries of the concepts, principles and theoretical relationships developed at the core; (ii) Analyzing the processes of reception, assimilation, adaptation and social appropriation of the economic discourse produced abroad, taking into account the social and economic specificities of the recipient country; (iii) Studying the institutional and technical mechanisms for the dissemination and access to economic discourse (quantity and quality of translations; circulation and reading rates of national and foreign bibliography; 67 ease or otherwise of access to relevant journals; mastery of foreign languages; conditions for the establishment of international contacts; linguistic adaptations, etc.); (iv) Analyzing the conditions of production and dissemination of economic knowledge and practices; (v) Studying the processes of formation and gradual enrichment of a tradition (or various traditions) of economic thought and explaining its repercussions over time. Several empirical works have been developed over the past few decades on the study of the diffusion and appropriation of economic ideas at an international scale (e.g. Hall, 1989; Llombart, 1995; Bastien and Cardoso, 2003; Girón, 2006; Montecinos and Markoff, 2009; or, more recently, Cunha and Suprinyak, 2017). Llombart’s work was particularly noteworthy. It analyses the reception of physiocracy in Spain explicitly based on a market for ideas approach (complemented by other contributions, namely those coming from the information theory model). In this study the reasons why economic ideas circulate —being accepted, ignored, modified or rejected in a given historical period— are found in the logic of a demand-driven “special” market for ideas. These are considered to be exogenously determined and to have characteristics of a “pure public good” (as such not having a price).It is assumed that ideas spread according to a logic of “consumer’s choice”, based on considerations of utility and transaction costs (conditioned by “institutional, political and administrative variables” as well as by “feelings and passions”). A second study that I would like to emphasize here is the one by Carlos Bastien and José Luís Cardoso (2003)7 as it studies a more unusual case —the diffusion of ideas and techniques from the periphery (Latin American) to a semi peripheral European country (Portugal)— and adopts a substantively different approach. Bastien and Cardoso highlight, through a comparative analysis of Latin American structuralist and developmentalist ideas, concepts, analytical tools and policy recommendations and the corresponding ones in Portugal, the proximities and differences between the economic thought at the origin and at the importing country and outline an explanation of the diffusion process based on the political, economic and academic conditions and features of the latter. This study 7 See also Cardoso(2009). Neves, V. Iber. hist. econ. thought. 4(1) 2017: 63-73 68 can be thought of as an instance of what Bastien, in his PhD thesis (Bastien, 1989), called a “Critical and Explanatory” or “Sociological” History. According to Bastien8, national cases should be understood as “fields” (in the sense of Bourdieu)9, and studied as socially and culturally determined, demand-driven markets for ideas. By highlighting the working of those scientific fields we should be able to define the needs and conditions for the import of ideas. Export of ideas would, obviously, follow different rules. Taking stock of all this literature, we may say that, in spite of all relevant developments —both theoretical and empirical— we are far from a “general theory, or even specific theory, about how ideas spread”10. However, against Llombart’s (1995, 32) claim, “[t]he lack of generally accepted models, and even skepticism as to their full viability often shown by the principal scholars in the subject”, instead of being “a serious difficulty”, is, as I will try to show in the next section, inevitable. A “general” theory may be just a utopia that leads us to go on walking and searching for better connecting principles. 4. The structuring role of connecting principles Knowing is establishing connections, creating categories and imagining patterns and causal linkages between them as representations of the world. As Loasby has consistently argued over a considerable time span, “knowledge grows through a fallible process of making connections” (2001, 398) and “wherever we start there are, in principle, very many directions in which we may look for connections” (ibid., 401, emphasis added), “each move opens up a new set of possibilities” (ibid.), that is, we work “in large combinatorial spaces” (2003, 301). Knowing is a fallible and plural connecting process. Our knowledge is inevitably partial and incomplete. Error is unavoidable and coordination of different contributions to knowledge indispensable. A well-developed science is a dense network of inter-connected propositions about a set of phenomena, a set of connections that grows by making novel connections (sometimes involving destroying and 8 9 10 Personal exchange (email dated from 7 March 2017). See Bourdieu (1976). Intentionally, I use here the words that Coats and Colander (1989, 15) wrote to assess the state of the art at the time. The situation, in this regard, has not changed significantly. substituting other connections). Like a rope, such a network is stronger than each of its constituting links (“any one strand breaking will not bring down the edifice” [Dow, 2012, 222]). Thus, Loasby’s (2001, 401) conclusion that “the best way to improve knowledge is to encourage many people to imagine connections, and to try to arrange that different people will imagine different connections “seems appropriate. Of course, such a pluralist approach does not dispense with the need to decide which options should be accepted and developed and which must simply be discarded or modified. Crucial here is the way we organize ideas in order to make sense of the world in which we live, that is, what concepts and connecting principles (or interpretative frameworks) we imagine(and adopt) in order to link together phenomena in our minds. The choice of connecting principles is obviously central in the modeling of the processes of international exchange of economic ideas. Let us see why by returning to the three models of the spread of economic ideas considered in Coats and Colander (1989). It is manifest that each of these models presupposes a different set of connecting principles, a different interpretative framework. Ideas are in turn likened to a “disease”, “information” or “commodities” and treated as such. As a result we are led to “see” different things. We build different “realities”. Each model highlights a relevant aspect (or aspects) of the international diffusion and “local” appropriation of economic ideas. However, it is also the case that each of them conceals or distorts significant aspects or dimensions of these processes. The analogy made between the diffusion of an idea and the propagation of a disease, for example, obscures the fact that while a disease spreads regardless of our will, ideas can be voluntarily accepted, modified or rejected. The epidemiological model excludes the volitional dimension involved in the spread of ideas. Also, in treating ideas as commodities and by putting the emphasis on the metaphor of economists as consumers and adopting categories such as marginal utility, transaction costs and consumer’s choice, the model of the market for ideas, although catching relevant dimensions of scientific activity, misses, distorts and even corrupts some important features, experiences and meanings of the practice of economics as a science, as also happens when the market metaphor is applied to nonmarket decisions such as constituting a Neves, V. Iber. hist. econ. thought. 4(1) 2017: 63-73 family, becoming involved in politics, donating blood or relating to friends. The point I wish to emphasize here is that by adopting different connecting principles (and metaphors), that is, different ways of organizing phenomena—different sets of elements and/or different links between them— we are led to different ways of dealing with the international circulation of actors, worldviews, scientific theories and models, professional practices and policy recommendations. Their discussion is therefore of utmost importance. 5. Towards a broader connecting principle: economics as a network of institutionally situated conversations A set of principles should be as comprehensive as possible11. Also, if our theories are to have some sort of connection with reality, our principles should be conceived so that a maximum degree of ontological integrity12 is preserved. Hence, in this section an attempt is made to explore a broader (and, it is hoped, ontologically sounder) framework, based on the idea that economics is everywhere a “bunch of conversations” (Klamer, 2007, 15) and increasingly a global network of institutionally situated conversations. The conversation metaphor, as developed by Klamer (2007), is obviously focused on the idea of “conversation “but, I submit, has the potential to capture the essential institutional and intellectual dimensions of the processes we want to describe and explain. It puts the emphasis on the social, cultural and relational as well as the rhetorical and hermeneutic features of the practice of economics and allows us (or rather, it demands) to consider its institutional settings —the “local “specificities and the connections (the internal and the external factors that promote/ inhibit/constrain the international travelling and assimilation/appropriation of ideas and practices). The conversation metaphor may raise understandable skepticism, not only because of its colloquial connotations, as Klamer himself recognized, but because it would apparently 11 12 “The more extensive the range of a set of principles […] the better” (Loasby, 1991, 7). Ontological integrity is meant here to express the adequacy of our theorizing to the fundamental nature of the phenomena observed (see Oakley, 1999). 69 miss the foundations underpinning conversations. In spite of its undeniable charm, it would be, in the end, just “abundant foam that quickly fades”13. However, I believe, the conversation metaphor (put in the context of relevant networks, institutions and relations of power)14 allows for a very broad framework. As Klamer (2007) shows, it involves the idea of science as a social process and, at the same time, draws attention to the linguistic, discursive and relational aspects of science. It allows us to look at the practice of building knowledge, but also taking into consideration a world of “passions, discriminations, incriminations and abuse” (ibid., 15), as Klamer (1984) and the interviews contained in that famous book so vividly showed. A conversation encompasses both formal and informal forms of interaction, conversations in print (books and journal articles) as well as participation in conferences, writing and reading, rigorous formal (usually mathematical) argumentation but also gossiping, sharing of ideas (cooperation) and controversy/dissent (tension and conflict).To be part of a conversation requires a range of skills, the adequate diplomas, “the mastery of econspeak” (ibid., 16) —that is, the use of the right vocabulary and rhetorical devices, a certain way of arguing— and, in the end, the ability to attract attention, be recognized and appreciated in the relevant network(s). It involves an art of speaking (rhetoric) and an art of listening and reading (hermeneutics). Ideas only get attention if in a conversation. Conversations are attention spaces (Klamer, 2007, 55). Taking part in a conversation entails being part of a social and intellectual community. Scientists cluster in universities, set up barriers to entry, organize professional associations in order to organize conferences and issue journals, constitute schools, subscribe to research programs, develop specialized research communities which will organize specialized conferences and issue specialized journals, and form networks of like-minded souls. All these institutions help to define, bolster or protect a space of 13 14 José Luís Cardoso (private conversation, email dated from 3 February 2017). More on this below. Neves, V. Iber. hist. econ. thought. 4(1) 2017: 63-73 70 attention, that is, a concentration and intensification of signals interchanged. It helps explain why innovations in science are geographically localized and not evenly dispersed throughout the world (Klamer and van Dalen, 2002, 302). Conversation, networks and institutions are the relevant keywords here. Conversation in economics takes place in the context of various (and often overlapping) networks, that is, more or less hierarchical, fluid and dynamic structures of connections linking more or less far away nodes (individuals, schools, organizations, cultural, political or legal geographic areas) whose relative importance evolves over time. Those conversations usually reproduce and reinforce the networks in which they occur, but sometimes lead to dynamic instability and change. They are a source of continuity and transformation in the profession. Conversations are institutionally molded. Institutions are here understood as “systems of established and prevalent social rules15 that structure social interactions” (Hodgson, 2006, 2)16. Institutions “enable ordered thought, expectation, and action by imposing form and consistency on human activities” (ibid.). That is, they constrain behavior (through social rules) but, at the same time, they enable choices and actions that otherwise would not be possible. They are a special type of enduring social structures with the potential to mold individual aspirations, capacities and purposes through the rules embedded in shared habits of thought and behavior. Since institutions constrain and mold individual action —“any single individual is born into a pre-existing institutional world” (ibid., 7)— they are a source of continuity in social life (“they have strong self-reinforcing and self-perpetuating characteristics” (ibid.). But, at the same time, they depend, for their existence, upon the thoughts and activities of individuals, so they are not unchangeable. Institutions are simultaneously objective realities “out there” and inter-subjective mental models “in here” (shared, or at least mutually consistent, cognitive processes and habits of thought). Individual action and institutional structure, as Hodgson has made clear, although distinct, are 15 16 A social rule is “a socially transmitted and customary normative injunction or immanently normative disposition, that in circumstances X do Y” (Hodgson, 2006, 3). Language, money, law, systems of weights and measures, table manners, firms and other organizations are the examples Hodgson (ibid, 2) provides. “connected in a circle of mutual interaction and interdependence” (ibid., 8). In spite of its significance, Klamer’s approach basically provides an inward-looking perspective of economics as a science. The political context in which the practices of economics occur, the implications of the increasing marketization of academia and the commodification of knowledge (tighter university budgets, pressures to fierce intra- and interuniversity competition, metric-based research assessment patterns), the molding role of funding structures and agencies, the structures of power conditioning economic research (not only within academia), the complex links established with governments, international organizations (such as the IMF, World Bank, OECD, ECB, etc.), think tanks and corporations, are all largely absent (or at least substantially neglected) from his writings. Analyzing the international diffusion of economic ideas from a “network of conversations” point of view should not ignore these aspects. It must look at how economic conversations work and develop (on the national and the international scale) and this requires, if a proper explanation of these conversations is to be achieved, taking into account the economic, political, social and disciplinary institutional settings in which they occur. The emphasis then goes to the characterization of “local” conversations and the kind of networks they demand, promote or prevent. This includes attention to the following aspects: i. The identification of relevant individuals and organizations17, their characteristics and positions in the global system of knowledge production and of the structure and dynamics of the (more or less hierarchical) relationships (the patterns of connections) individuals and organizations establish among them. ii. The explanation of how institutions, understood as the rules of the game18 —the overall institutional structure in which economic research takes place, including the structures of power (academic, economic, political) and 17 18 As Hodgson (2006, 8) maintained, “[o]rganizations are special institutions that involve (a) criteria to establish their boundaries and to distinguish their members from nonmembers, (b) principles of sovereignty concerning who is in charge, and (c) chains of command delineating responsibilities within the organization”. Klamer and van Dalen(2002, 301) rightly acknowledge that “[i]nteraction based theories are […] not sufficient to understand science, one also has to explain how institutions —the rules by which the game of science is played— come about and change.” Neves, V. Iber. hist. econ. thought. 4(1) 2017: 63-73 the funding schemes affecting the way economic research and teaching is organized and practiced— affect the conversation process. iii. The identification in each country of the relevant “attention spaces” for economists’ work and the explanation of how these attention spaces condition their work, both at the core and on the peripheries. Understanding how attention is formed and distributed in the community of scientists is, as Klamer and van Dalen maintain, “the key to understanding the creation and diffusion of ideas” (2002, 296-297). iv. An analysis of the elements of communication19, with a particular focus on the rhetoric and hermeneutic aspects of discourses (including attention to what fosters or hinders the construction of relationships at an international scale). v. The meanings and values economists attribute to things and activities and the meanings and values they realize with their actions20. An approach such as this calls for a research strategy based on a variety of methods, from social network analysis to interviews and content analysis. Of course, it is only at the level of practice, of undertaking specific pieces of empirical research, that such a strategy may be further detailed. 6. Concluding remarks In order to make sense of our world we cannot avoid inventing connecting principles. These are the basis of any explanatory endeavor. In this paper I have tried to show their relevance in building models of the international diffusion and appropriation of economic ideas, practices and institutions. Some structuring ideas have been put forward in the previous pages: i. Economics is a complex, “locally”-shaped system of interacting ideas, practices and institutions. The connections established between the “core” and the “peripheries” of the profession are an important aspect of how economics as a science develops on a global scale. ii. A “general” framework to explain the international diffusion/appropriation of economic ideas is still far from our reach and may be even unreachable. 19 20 As pointed out by the information theory model. “[T]he main purpose of studying the behavior of people is to sort out, interpret and characterize the meanings and values that people attribute to things and activities, and the meanings and values that they realize with their actions” (Klamer, 2016, 18) 71 iii. Knowing is a fallible and plural connecting process —a pluralist approach to knowledge is, then, justified. iv. Any model of the international diffusion of economic ideas presupposes a given set of connecting principles. Different models usually entail different connecting principles. v. Connecting principles should be as comprehensive as possible and conceived in order to preserve the maximum degree of ontological integrity. vi. The idea of economics as a network of institutionally situated conversations may constitute a powerful basis for a broader and richer explanatory framework than the alternatives used so far. Approaching the internationalization of economic ideas from a “network of institutionally situated conversations” point of view is worthy of further elaboration and may provide relevant heuristic guidelines for new empirical work. vii. Obviously, the “proof of the pudding” —the usefulness of the proposed framework— can only occur at the empirical level. Actually, it is my view that a richer and broader framework for the analysis of the historical process of internationalization of economics must come from a dynamic movement back and forth between theoretical-methodological elaboration and comparative analyses of empirical case studies. Be that as it may, the exploratory nature of the present endeavor should be emphasized. Other connecting principles might be considered —it is the case of thinking the diffusion and appropriation of economic ideas as part of a complex system in which economics is seen as “a dynamic entity, which generates a self-reproducing, evolving, complex system of interacting ideas” (Colander et al., 2004, 486)— and specific models developed. In this exercise the purpose was basically to discuss the significance of the connecting principles one adopts in the endeavor to build an adequate framework for the explanation of the processes of internationalization of economic ideas, practices and institutions. The approach here espoused is pluralist. It does not preclude consideration of other possibilities. The advantage of starting from the idea of economics as a network of institutionally situated conversations (as outlined in this paper) is that it allows for the adoption of a very broad, multidimensional perspective. 72 Neves, V. Iber. hist. econ. thought. 4(1) 2017: 63-73 7. References Almodovar, António; Cardoso, J. Luís.1998. A history of Portuguese Economic Thought. London and New York, Routledge. Argemí, Lluís.2006. El pensamiento económico en Cataluña. In: Girón, P. (ed.). Variaciones sobre la Historia del Pensamiento Economico Mediterraneo, El Ejido (Almería): Fundación Cajamar, pp. 167-181 (Available at: http://www.publicacionescajamar.es/pdf/publicaciones-periodicas/mediterraneo-economico/9/mediterraneo-economico-9.pdf). Bastien, Carlos. 1989. Para a história das ideias económicas no Portugal contemporâneo: a crise dos anos 1945-1954. 2 vols. PhD Thesis in economics, Instituto Superior de Economia. Bastien, Carlos; Cardoso, J. Luís. 2003. Structuralism and Development Economics in the European Semi-Periphery: the case of Portugal. Notas Económicas, 17, 36-51. Bourdieu, Pierre. 1976. Le champ scientifique. Actes de la recherche en sciences sociales, 2 (2-3), 88-104. Cardoso, J. Luís. 1997. Pensar a Economia em Portugal: Digressões Históricas. Lisboa, Difel. Cardoso, J. Luís.2002. The history of economic thought in Spain and Portugal: a brief survey. History of Political Economy, 34, 137-147. Cardoso, J. Luís.2003. The international diffusion of economic thought. In: Samuels, W.; Biddle, J.;Davis, J. (eds.). A Companion to the History of Economic Thought, pp. 622-633, Oxford and New York, Blackwell. Cardoso, J. Luís.2009. Reflexões periféricas sobre a difusão internacional do pensamento económico. Nova Economia, 19(2), 251-65. Cardoso, J. Luís.2017. Circulating economic ideas: Adaptation, appropriation, translation. In: Cunha, A.; Suprinyak, C. (eds.) The Political Economy of Latin American Independence. London and New York, Routledge, pp. 32-40. Cardoso, J. Luís; Lluch, Ernest.1999. Las teorías económicas contempladas a través de una óptica nacional. In: Quintana, E. F. (ed.). Economia y Economistas Españoles. Barcelona, Galaxia Gutemberg, v. 1, pp. 477-484. Coats, A. W.; Colander, David (eds.). 1989. The spread of economic ideas. Cambridge, Cambridge University Press. Colander, David; Holt, R.; Barkley Rosser Jr. 2004.The changing face of mainstream economics, Review of Political Economy, 16(4), 485-499. Cunha, Alexandre; Suprinyak, Carlos. (eds.). 2017. The Political Economy of Latin American Independence. London and New York, Routledge. Dorfman, Joseph. 1955. The role of the German Historical School in American economic thought. American Economic Review, 45(2), 17-28. Dow,Sheila. 2012. Foundations for New Economic Thinking: A Collection of Essays. Basingstoke and New York, Palgrave Macmillan. Fourcade, Marion. 2006. The Construction of a Global Profession: The Transnationalization of Economics. American Journal of Sociology, 112(1), 145-94. Gavroglu, K. et al. 2008.Science and Technology in the European Periphery: Some Historiographical Reflections, History of Science, xlvi, 153-175. Girón, Pedro. (ed.). 2006. Variaciones sobre la Historia del Pensamiento Económico Mediterráneo, El Ejido (Almería): Fundación Cajamar (Available at: http://www.publicacionescajamar.es/pdf/publicaciones-periodicas/mediterraneo-economico/9/mediterraneo-economico-9.pdf). Hall, Peter. (ed.). 1989. The Political Power of Economic Ideas: Keynesianism Across Nations. Princeton, Princeton University Press. Hodgson, Geoffrey.2006. What are Institutions?, Journal of Economic Issues, XL(1), 1-25. Hutchison, T. W. 1955. Insularity and cosmopolitanism in economic ideas. American Economic Review, 45(2), 1-16. Klamer, Arjo. 1984. The New Classical Macroeconomics. Conversations with the New Classical Economists and their Opponents. Brighton, Wheatsheaf Books. Klamer, Arjo. 2007. Speaking of Economics: How to get in the conversation. London and New York, Routledge. Klamer, Arjo. 2016. Doing the Right Thing: A value based economy. London, Ubiquity Press. Klamer, Arjo; van Dalen, Hendrik. 2002. Attention and the art of scientific publishing. Journal of Economic Methodology, 9(3), 289-315. Neves, V. Iber. hist. econ. thought. 4(1) 2017: 63-73 73 Letiche, J.M. 1955. Discussion. American Economic Review, 45(2), 29-34. Llombart, Vicent. 1995. Market for ideas and reception of physiocracy in Spain: some analytical and historical suggestions. The European Journal of the History of Economic Thought, 2(1), 29-51. Lluch, Ernest. 1980. Sobre la historia nacional del pensamiento economico. Introduction to A. Florez Estrada. In: Curso de Economia Política (1828). Madrid: Instituto de Estudios Fiscales (ed. Fundación Ernest Lluch, Sección Aragonesa y Gobierno de Aragón, Departamento de Economía, Hacienda y Empleo, 2007). Lluch, Ernest.1999. Las historias nacionales del pensamiento económico y España. In: Quintana, E. F. (ed.). Economia y Economistas Españoles. Barcelona, Galaxia Gutemberg, v. 1, pp. 435-476. Loasby, Brian. 1991. Equilibrium and evolution: an exploration of connecting principles in economics, Manchester, Manchester University Press. Loasby, Brian. 1999. Knowledge, Institutions and Evolution in Economics, London and New York, Routledge. Loasby, Brian. 2001. Time, knowledge and evolutionary dynamics: why connections matter. Journal of Evolutionary Economics, 11(4), 393-412. Loasby, Brian. 2003. Closed models and open systems, Journal of Economic Methodology, 10(3), 285-306. Loasby, Brian. Making Connections.2005. Econ Journal Watch, 2(1), 56-65. Mäki, Uskali.1996. Economic Thought on the Outskirts: Toward a Historiographical Framework for Studying Intellectual Peripheries. Research in the History of Economic Thought and Methodology, 14, 307323. Montecinos, Verónica; Markoff, John (eds.).2009. Economists in the Americas. Cheltenham, UK and Northampton, MA, USA: Edward Elgar. Oakley, Allen. 1999. Situational analysis and agent rationality: Shackle contra Popper. In: Sardoni, C.; Kriesler, P. (eds.), Keynes, PostKeynesianism and Political Economy: Essays in Honour of Geoff Harcourt, v. III, London and New York, Routledge. Smith, Adam. [1795]1980.The Principles Which Lead and direct Philosophical Enquiries: Illustrated by the History of Astronomy. In Essays on Philosophical Subjects, ed. W. P. D. Wightman. Oxford: Oxford University Press, pp. 33-105.
https://openalex.org/W2961556035	https://kops.uni-konstanz.de/bitstream/123456789/46681/3/Ziegler_2-ulp96534pyqs1.pdf	English	null	Coral bacterial community structure responds to environmental change in a host-specific manner	Nature communications	2,019	cc-by	12,153	ARTICLE Results l Sample and sequencing overview. After 21 months of reciprocal transplantations between 5 reef sites representing different levels of anthropogenic impact in the vicinity of Jeddah, Saudi Arabia, 135 coral fragments and 20 seawater samples were collected and their bacterial communities investigated (Fig. 1). MiSeq sequen- cing of bacterial 16S rRNA gene amplicons resulted in 10,857,521 sequences from 131 samples and 2 negative controls (Supplementary Data 1). After quality control and removal of unwanted sequences, 2,509,787 sequences with a mean length of 291 bp were retained (31,920 distinct sequences). A. hemprichii samples contained an average of 16,267 sequences (range: 189–45,628), P. verrucosa samples 26,930 sequences (range 5–67,805), and seawater samples 4,993 sequences (range 3,101–9,801) (Supplementary Data 1). After subsampling to 3,101 sequences per sample, we retained 48 samples of P. ver- rucosa and 54 samples of A. hemprichii (Table 1). Clustering to 97% similarity yielded 7,032 distinct OTUs (Source Data, Sup- plementary Data 2). The mean number of OTUs per sample was 237 (range 58–466) for A. hemprichii, 159 (range 45–427) for P. verrucosa, and 174 (range 129–249) for seawater (Source Data). At present, it is unclear whether the potential for microbiome restructuring is a conserved trait across coral species or whether species-speciﬁc differences exist. For instance, the coral Pocillo- pora verrucosa shows a globally conserved association with its main bacterial symbiont Endozoicomonas14 that remains unchanged even under conditions of bleaching and mortality24. P. verrucosa further was shown to maintain a stable Symbiodinia- ceae community during a cross-transplantation experiment over depth25 and between seasons and reefs, while Porites lutea sam- pled under the same conditions had a highly ﬂexible Symbiodi- niaceae community26. Therefore, it appears that the ability of corals to associate with distinct microbial associates may depend on location, environmental setting, and coral host species. A. hemprichii P. verrucosa A B C D E Unimpacted Local wasterwater and nutrients Municipal sewage and sedimentation and oil Collection N 20 km Jeddah Red Sea Fragmentation Reciprocal transplantation 21 months 15 × 15 × 1 2 3 5 fragments per colony 3 colonies per site Fig. 1 Design of transplantation experiment. Back-transplantation and cross-transplantation of coral fragments from Acropora hemprichii and Pocillopora verrucosa across sites of differing environmental impact close to the Saudi Arabian city of Jeddah in the Red Sea to test for microbiome ﬂexibility. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-10969-5 NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-10969-5 S Our study shows that the degree of bacterial community restructuring upon transplantation to reef sites with different levels of anthropogenic impact differs between host species. A. hemprichii harbors a highly ﬂexible bacterial community that is characterized by many differentially abundant bacterial taxa between impacts. In contrast, the bacterial community of P. verrucosa remains remarkably stable between impacts. In addi- tion, microbial communities recovered to their original states upon cross-transplantation to unaffected sites. Thus, distinct degrees of host-associated bacterial community restructuring exist, but their role in holobiont adaptation to environmental change is currently unknown. S cleractinian corals live in close association with endo- symbiotic dinoﬂagellates of the family Symbiodiniaceae and a diverse community of bacteria (among other micro- organisms), collectively referred to as the microbiome1. The community of the coral host and its associated microbiome comprises a metaorganism and is referred to as the coral holo- biont2. While most corals depend on Symbiodiniaceae to meet their energetic demands by the transfer of photosynthetically ﬁxed carbon3,4, bacterial microbiome members fulﬁll a range of other functions including nitrogen ﬁxation, sulfur cycling, and protection against pathogenic bacteria5–9. Accordingly, restruc- turing of the microbiome is proposed to contribute to coral holobiont plasticity and adaptation1,10–12. Recent studies have found differences in the degree to which coral microbiomes vary over environmental gradients or experi- mental treatments13–15. For example, microbiomes of Ctenactis echinata varied between different reef habitats to the degree that abundance of coral host species was associated with the presence/ absence of speciﬁc bacteria16. Further, microbial diversity was shown to increase with depth in several coral species, possibly allowing corals to access a broader range of food sources17. In addition, some studies have found seasonal ﬂuctuations in coral- associated microbiomes16,18,19 and tide-related shifts on much shorter time scales20, while other corals maintain temporally stable microbiomes21. Unidirectional transplantation experiments of the coral species Acropora muricata22 and Porites cylindrica23 from a pristine site to impacted or modiﬁed sites further illustrate that microbiomes change under adverse environmental condi- tions. In contrast, a transplantation experiment between different thermal habitats showed that microbiomes of heat tolerant Acropora hyacinthus can be acquired by heat sensitive corals upon environmental transplantation over the course of 17 months11. Notably, these corals exhibited increased thermo- tolerance in a subsequent heat stress experiment, harboring a more robust and stable microbiome. ARTICLE ARTICLE Coral bacterial community structure responds to environmental change in a host-speciﬁc manner Maren Ziegler 1,2,8, Carsten G. B. Grupstra1,3,8, Marcelle M. Barreto1, Martin Eaton4 Khalid Zubier5, Abdulmohsin Al-Sofyani5, Adnan J. Turki5, Rupert Ormond 4,5 & Ch The global decline of coral reefs heightens the need to understand how corals respond to changing environmental conditions. Corals are metaorganisms, so-called holobionts, and restructuring of the associated bacterial community has been suggested as a means of holobiont adaptation. However, the potential for restructuring of bacterial communities across coral species in different environments has not been systematically investigated. Here we show that bacterial community structure responds in a coral host-speciﬁc manner upon cross-transplantation between reef sites with differing levels of anthropogenic impact. The coral Acropora hemprichii harbors a highly ﬂexible microbiome that differs between each level of anthropogenic impact to which the corals had been transplanted. In contrast, the micro- biome of the coral Pocillopora verrucosa remains remarkably stable. Interestingly, upon cross- transplantation to unaffected sites, we ﬁnd that microbiomes become indistinguishable from back-transplanted controls, suggesting the ability of microbiomes to recover. It remains unclear whether differences to associate with bacteria ﬂexibly reﬂects different holobiont adaptation mechanisms to respond to environmental change. Konstanzer Online-Publikations-System (KOPS) URL: http://nbn-resolving.de/urn:nbn:de:bsz:352-2-ulp96534pyqs1 onstanze Online ublikations System ( O S) URL: http://nbn-resolving.de/urn:nbn:de:bsz:352-2-ulp96534pyqs1 1 Red Sea Research Center, Division of Biological and Environmental Science and Engineering (BESE), 4700 King Abdullah University of Science and Technology (KAUST), Thuwal 23955, Saudi Arabia. 2 Department of Animal Ecology & Systematics, Justus Liebig University, Heinrich-Buff-Ring 26–32 IFZ, 35392 Giessen, Germany. 3 BioSciences Department, Rice University, 6100 Main Street, Houston, TX 77005, USA. 4 Centre for Marine Biology and Biodiversity, Institute for Earth and Life Sciences, Heriot-Watt University, Riccarton, Edinburgh EH14 4AS, UK. 5 Faculty of Marine Science, King Abdulaziz University, PO Box 80207Jeddah 21589, Saudi Arabia. 6 Department of Marine Biology, Faculty of Environmental Science and Marine Biology, Hadhramout University, Al-Mukalla, Republic of Yemen. 7 Department of Biology, University of Konstanz, 78457 Konstanz, Germany. 8These authors contributed equally: Maren Ziegler, Carsten G. B. Grupstra. Correspondence and requests for materials should be addressed to M.Z. (email: maren.ziegler@bio.uni-giessen.de) or to C.R.V. (email: christian.voolstra@uni-konstanz.de) 1 NATURE COMMUNICATIONS \| (2019) 10:3092 \| https://doi.org/10.1038/s41467-019-10969-5 \| www.nature.com/naturecommunications Results l Three coral colonies of each species at each site were collected and fragmented into ﬁve fragments each per colony to allow for reciprocal transplantation across all sites. GPS locations for site A: N 21°52′22.83″ E 38°58′01.61″, site B: N 21°47′08.23″, E 39°02′28.56″, site C: N 21°36′ 54.53″, E 39°06′17.92″, site D: N 21°34′34.21″, E 39°06′27.27″, site E: N 21°26′21.41″, E 39°06′28.49″ A. hemprichii P. verrucosa A B C D E Unimpacted Local wasterwater and nutrients Municipal sewage and sedimentation and oil Collection N 20 km Jeddah Red Sea Fragmentation Reciprocal transplantation 21 months 15 × 15 × 1 2 3 5 fragments per colony 3 colonies per site 2 3 To assess potential differences in ﬂexibility of bacterial asso- ciation across coral species, we conducted a long-term large-scale reciprocal transplantation experiment using the coral species Acropora hemprichii and P. verrucosa. Based on previous studies, these coral genera were suspected to differ in the ﬂexibility of their association with different microbial communities across environmental gradients14,24,27,28. The use of a reciprocal trans- plant design between reef sites subjected to different levels of anthropogenic impact allowed us to assess whether environ- mental differences align with distinct bacterial communities and whether the ability to adapt bacterial community composition differs between coral species. As coral microbiomes have pre- viously been shown to recover from stress events, such as bleaching29 or disease30, we were interested to elucidate whether coral microbiomes can recover from chronic pollution, i.e. return to a state that resembles conspeciﬁc microbiomes at unaffected sites upon transplantation of coral fragments from affected to pristine sites. Jeddah Fig. 1 Design of transplantation experiment. Back-transplantation and cross-transplantation of coral fragments from Acropora hemprichii and Pocillopora verrucosa across sites of differing environmental impact close to the Saudi Arabian city of Jeddah in the Red Sea to test for microbiome ﬂexibility. Three coral colonies of each species at each site were collected and fragmented into ﬁve fragments each per colony to allow for reciprocal transplantation across all sites. GPS locations for site A: N 21°52′22.83″ E 38°58′01.61″, site B: N 21°47′08.23″, E 39°02′28.56″, site C: N 21°36′ 54.53″, E 39°06′17.92″, site D: N 21°34′34.21″, E 39°06′27.27″, site E: N 21°26′21.41″, E 39°06′28.49″ NATURE COMMUNICATIONS \| (2019) 10:3092 \| https://doi.org/10.1038/s41467-019-10969-5 \| www.nature.com/naturecommunications 2 ARTICLE NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-10969-5 Table 1 Overview of transplanted coral fragments of A. hemprichii and P. Results l Seawater samples were dominated by the bacterial families Pelagibacter- aceae (38–54%) and OCS 155 (14–31%) (Supplementary Fig. 2). Other abundant families included Flavobacteriaceae (4–13%) and Halomonadaceae (4–12%). Five additional taxa that were present at abundances >1% included Rhodobacteraceae (2–6%), Alter- omonadaceae (0–10%), Rhodospirillaceae (1–2%), unclassiﬁed Alphaproteobacteria (1–6%), and unclassiﬁed Bacteria (0–3%). Microbial communities of the seawater were distinct from those of the two coral species (Supplementary Fig. 3). Of the 1,105 OTUs identiﬁed in water samples, only 172 (<16%) were shared with at least one of the coral species. In order to focus on dif- ferences between coral microbiomes, seawater samples were excluded from subsequent analyses. signiﬁcantly different from each other (Table 2). The differential pattern of bacterial microbiome restructuring between coral species was also evident when sites were pooled by impacts (Supplementary Data 3). Notably, analyzing the dataset using a 99% OTU similarity cutoff to account for putative differences at a higher phylogenetic resolution conﬁrmed observed patterns across sites and impacts (pooled sites) (Supplementary Data 3–4). Similarly, excluding Endozoicomonadaceae from the dataset to rule out that patterns were largely driven by dominant association with Endozoicomonas also reproduced that patterns are different between both coral species when considering bacterial micro- biome composition across sites and impacts (pooled sites) (Supplementary Data 3, 5). The site of origin, where the coral fragments were originally collected, had no signiﬁcant effect on coral microbiome structuring (Table 2, Supplementary Figs. 5–8). Differentially abundant taxa align with microbiome ﬂexibility. In A. hemprichii pronounced microbiome restructuring between different impact levels was aligned with large shifts in abundance of 60 speciﬁc bacterial lineages, whereas P. verrucosa was asso- ciated with a much more stable and less variant microbial community, only 5 signiﬁcantly different taxa being recorded between impacts (Fig. 3). A. hemprichii colony fragments transplanted to unimpacted sites had higher abundances of the bacterial family Endozoicomonadaceae (31% compared to 1–5%, Fig. 4a), as highlighted by OTU00003 (99% identical to Endo- zoicomonas acroporae GenBank accession no. NR_158127.1), which was signiﬁcantly more abundant at unimpacted sites (LEfSe, LDA = 5.2, p < 0.05; Fig. 3a). Further bacterial taxa that occurred at higher abundance at unimpacted sites included Alteromonadales (23% compared to 11–26%) and Simkaniaceae (2% compared to 0–1%) (Fig. 4a). In contrast, several bacterial families showed impact-speciﬁc increases in their relative abundance. Results l verrucosa Destination impact (site) Unimpacted (A) Unimpacted (B) Local wastewater and nutrients (C) Local wastewater and nutrients (D) Municipal sewage and sedimentation and oil (E) Origin impact (site): A. hemprichii Unimpacted (A) 3 0 1 2 1 Unimpacted (B) 3 3 1 2 3 Local wastewater and nutrients (C) 2 3 3 0 3 Local wastewater and nutrients (D) 3 3 1 2 3 Municipal sewage and sedimentation and oil (E) 3 3 1 2 3 Samples per destination site 14 12 7 8 13 Samples per destination impact 26 15 13 Samples per origin site 7 12 11 12 12 Samples per origin impact 19 23 12 Origin impact (site): P. verrucosa Unimpacted (A) 3 2 0 1 3 Unimpacted (B) 3 2 1 2 3 Local wastewater and nutrients (C) 3 3 2 1 2 Local wastewater and nutrients (D) 1 2 1 1 2 Municipal sewage and sedimentation and oil (E) 3 2 1 1 3 Samples per destination site 13 11 5 6 13 Samples per destination impact 24 11 13 Samples per origin site 9 11 11 7 10 Samples per origin impact 20 18 10 Shown are numbers of analyzed coral fragments after 21 months of reciprocal transplantation between 5 sites exposed to different levels of anthropogenic impact in the central Red Sea. Paired sites correspond to distinct impacts: A/B = unimpacted, C/D = Local wastewater and nutrients, E/lost site F = Municipal sewage and sedimentation and oil ble 1 Overview of transplanted coral fragments of A. hemprichii and P. verrucosa Shown are numbers of analyzed coral fragments after 21 months of reciprocal transplantation between 5 sites exposed to different levels of anthropogenic impact in the central Red Sea. Paired sites correspond to distinct impacts: A/B = unimpacted, C/D = Local wastewater and nutrients, E/lost site F = Municipal sewage and sedimentation and oil Shown are numbers of analyzed coral fragments after 21 months of reciprocal transplantation between 5 sites exposed to different levels of anthropogen correspond to distinct impacts: A/B = unimpacted, C/D = Local wastewater and nutrients, E/lost site F = Municipal sewage and sedimentation and oil Microbial communities in water and coral samples are distinct. Bacterial communities in seawater samples were distinct between individual sites, with the exception of sites B and E (PERMA- NOVA, F = 2.2, PMC = 0.001; Supplementary Fig. 1). NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-10969-5 All plots based on non-metric multidimensional scaling (nMDS) of Bray-Curtis distances of bacterial community compositions associated with coral samples (after 21 months of reciprocal transplantation). a corals A. hemprichii and P. verrucosa. b only coral A. hemprichii with sites indicated. c only coral P. verrucosa with sites indicated. A/B unimpacted sites; C/D local wastewater and nutrients sites; E municipal sewage and sedimentation and oil site. Ellipses denote 90% conﬁdence intervals. Source data are provided as a Source Data ﬁle Table 2 Statistical overview over bacterial microbiome differences across sites Table 2 Statistical overview over bacterial microbiome differences across sites Two-way PERMANOVA test statistics Pairwise comparisons by destination site Factor Model-F PMC A B C D E Species: A. hemprichii A – 3.4 3.62 3.7 3.28 Destination site 2.86 <0.001 B 0.01 – 2.55 1.1 3.33 Origin site 1.1 0.14 C 0.01 0.01 – 1.36 2.02 Destination × Origin site 0.98 0.6 D 0.01 0.01 0.01 – 2.17 E 0.01 0.01 0.01 0.01 – Species: P. verrucosa A – 2.48 1.76 1.9 2.14 Destination site 1.82 <0.001 B 0.01 – 1.54 1.4 1.92 Origin site 1.1 0.18 C 0.08 0.11 – 1.19 1.22 Destination × Origin site 1.07 0.17 D 0.17 0.26 1.00 – 1.26 E 0.04 0.03 1.00 1.00 – Signiﬁcant comparisons are printed in bold PMC: Monte-Carlo permuted p-value Pairwise comparisons: lower triangle p-values, upper triangle Model-F values Tests were conducted separately for the corals Acropora hemprichii and Pocillopora verrucosa after 21 months of reciprocal transplantation Two-way PERMANOVA test statistics Coral core microbiome. To determine a putative core micro- biome, all bacterial OTUs that were found at all sites in ≥75% of samples per site for a given coral host were considered. This led us to identify 7 bacterial taxa consistently associated with A. hemprichii. Among these were the second most abundant OTU0002 (Melitea sp.) as well as OTU0007 (Caulobacter sp.) and OTU0017 (Flavobacterium sp.), which were both present in all samples of A. hemprichii. For P. verrucosa, we identiﬁed 6 consistently associated taxa, of which the most abundant OTU0001 (Endozoicomonadaceae) was present in all samples of P. verrucosa. We further found a second Endozoicomona- daceae OTU consistently associated with P. verrucosa, but at much lower abundances. Three taxa were shared between the core microbiomes of the two corals (Supplementary Table 1). to Caulobacter sp. Genbank AB470462.1, LEfSe, LDA = 4.3, p < 0.05) and Pelomonas sp. Results l For instance, Caulobacteraceae and Comamonada- ceae (both 2–6%) with 1 and 4 OTUs, respectively, including their representatives Caulobacter sp. (OTU00007, 99% identical Coral microbiomes differ in their ﬂexibility between sites. A total of 4,704 bacterial OTUs were identiﬁed for A. hemprichii and 3,023 OTUs for P. verrucosa. Of these, 1,628 OTUs were shared between the two coral species. Bacterial assemblages were highly species-speciﬁc and signiﬁcantly different in their multi- variate dispersion between coral species (ANOVA, F = 16.01, p < 0.001; Fig. 2a). Overall, the bacterial communities of A. hem- prichii samples were more variable than those of P. verrucosa, as evidenced by signiﬁcantly higher distances to centroids (A. hemprichii mean = 0.55, SD = 0.05; P. verrucosa mean = 0.51, SD = 0.06, Supplementary Fig. 4, Source Data). After 21 months of reciprocal transplantations between 5 reef sites, the bacterial community of A. hemprichii differed signiﬁcantly between all individual sites (Fig. 2b; Table 2). By comparison, differences in the bacterial community of P. verrucosa between sites were far less pronounced, with only fragments transplanted to the most highly impacted site (municipal sewage and sedimentation and oil —site E) being signiﬁcantly different from coral fragments at the unimpacted sites A and B (Fig. 2c). The latter were also TURE COMMUNICATIONS \| (2019) 10:3092 \| https://doi.org/10.1038/s41467-019-10969-5 \| www.nature.com/naturecommunications 3 ARTICLE NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-10969-5 NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-10969-5 Acropora hemprichii Pocillopora verrucosa a b c 2D stress: 0.2 2D stress: 0.18 2D stress: 0.21 Acropora hemprichii Destination site:A B C D E Destination site: A B C D E Pocillopora verrucosa Fig. 2 Bacterial community structure and relative dispersion of the coral species A. hemprichii and P. verrucosa. All plots based on non-metric multidimensional scaling (nMDS) of Bray-Curtis distances of bacterial community compositions associated with coral samples (after 21 months of reciprocal transplantation). a corals A. hemprichii and P. verrucosa. b only coral A. hemprichii with sites indicated. c only coral P. verrucosa with sites indicated. A/B unimpacted sites; C/D local wastewater and nutrients sites; E municipal sewage and sedimentation and oil site. Ellipses denote 90% conﬁdence intervals. Source data are provided as a Source Data ﬁle b 2D stress: 0.18 Acropora hemprichii Destination site:A B C D E Acropora hemprichii Pocillopora verrucosa a 2D stress: 0.2 c 2D stress: 0.21 Destination site: A B C D E Pocillopora verrucosa a structure and relative dispersion of the coral species A. hemprichii and P. verrucosa. All plots based on non-metric Fig. 2 Bacterial community structure and relative dispersion of the coral species A. hemprichii and P. verrucosa. All plots based on non-metric multidimensional scaling (nMDS) of Bray-Curtis distances of bacterial community compositions associated with coral samples (after 21 months of reciprocal transplantation). a corals A. hemprichii and P. verrucosa. b only coral A. hemprichii with sites indicated. c only coral P. verrucosa with sites indicated. A/B unimpacted sites; C/D local wastewater and nutrients sites; E municipal sewage and sedimentation and oil site. Ellipses denote 90% conﬁdence intervals. Source data are provided as a Source Data ﬁle Fig. 2 Bacterial community structure and relative dispersion of the coral species A. hemprichii and P. verrucosa. All plots based on non-metric multidimensional scaling (nMDS) of Bray-Curtis distances of bacterial community compositions associated with coral samples (after 21 months of reciprocal transplantation). a corals A. hemprichii and P. verrucosa. b only coral A. hemprichii with sites indicated. c only coral P. verrucosa with sites indicated. A/B unimpacted sites; C/D local wastewater and nutrients sites; E municipal sewage and sedimentation and oil site. Ellipses denote 90% conﬁdence intervals. Source data are provided as a Source Data ﬁle Fig. 2 Bacterial community structure and relative dispersion of the coral species A. hemprichii and P. verrucosa. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-10969-5 (OTU00026, 100% identical to Pelo- monas sp. GenBank MF400787.1, LEfSe, LDA = 4.0, p < 0.05) were signiﬁcantly more abundant at local wastewater and nutrients sites (Figs. 3a, 4a). Furthermore, Flavobacteriaceae (2–4%, 3 OTUs, incl. OTU0017, 100% identical to Flavobacter- ium sp. GenBank MK216896.1, LEfSe, LDA = 3.7, p < 0.05) and Rhodobacteraceae (5–15%, 3 OTUs, incl. OTU0178, 100% identical to Roseovarius sp. GenBank NR_108232.1, LEfSe, LDA = 3.1, p < 0.05) were signiﬁcantly more abundant at the muni- cipal sewage and sedimentation and oil site. In contrast, the stable microbiome of P. verrucosa was dominated by the bacterial family Endozoicomonadaceae at all sites (56–71%, Fig. 4b). The only dominant bacterial family that noticeably changed in abundance was the Simkaniaceae with a decrease from 17% at unimpacted sites to 5 and 0% at impacted sites (OTU0005, 100% identical to Simkania sp. GenBank NR_074932.1, LEfSe, LDA = 5.0, p < 0.05, Figs. 3b, 4b). Coral microbiomes differ in their diversity and evenness. Bacterial diversity of A. hemprichii and P. verrucosa showed dif- ferent patterns in response to anthropogenic impacts. While bacterial community richness and diversity of A. hemprichii responded to the degree of observed anthropogenic impacts, bacterial community richness and diversity in P. verrucosa was stable (Fig. 5). A. hemprichii had a 30% lower OTU richness (Chao estimate) at local wastewater and nutrients sites than at other sites (Fig. 5a; Kruskal–Wallis, H = 12.51, p < 0.005; pairwise tests N > 7.7, p < 0.01). Moreover, the bacterial communities at unimpacted sites (A-B) were more even than at impacted sites (Fig. 5b; Kruskal–Wallis, H = 15.17, p < 0.001), with signiﬁcantly lower evenness at the local wastewater and nutrients sites and the Microbial indicator taxa. In line with substantial differences in the bacterial association of A. hemprichii across sites, and a rather stable microbiome of P. verrucosa, we identiﬁed 62 bacterial indicator taxa (indicspecies analysis) for impacts of destination in A. hemprichii, nearly nine times as many as for P. verrucosa (Supplementary Data 6). When considering bacterial indicator taxa for the impact of origin, only 5 taxa were identiﬁed for A. hemprichii and none for P. verrucosa. NATURE COMMUNICATIONS \| (2019) 10:3092 \| https://doi.org/10.1038/s41467-019-10969-5 \| www.nature.com/naturecommunications 4 ARTICLE NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-10969-5 NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-10969-5 A/B C/D E A/B C/D E A/B C/D E A/B C/D E A/B C/D E A/B C/D E A/B Unimpacted C/D Local wastewater and nutrients E Municipal sewage and sedimentation and oil A/B Unimpacted C/D Local wastewater and nutrients E Municipal sewage and sedimentation and oil 0 25 50 75 100 Bacterial community composition (%) Origin Destination impact 0 25 50 75 100 Bacterial community composition (%) a b Acropora hemprichii Pocillopora verrucosa Other (< %1) Alphaproteobacteria (p) Bacteria (k) Erythrobacteraceae Rickettsiales (o) Entomoplasmataceae Moraxellaceae Sphingomonadaceae Caulobacteraceae Flavobacteriaceae Comamonadaceae Rhodobacteraceae Rhizobiales (o) Gammaproteobacteria (c) Simkaniaceae Alteromonadales (o) Endozoicomonadaceae Fig. 4 Bacterial community composition of the corals A. hemprichii and P. verrucosa. Bacterial communities shown per coral species a A. hemprichii and b P. verrucosa across sites of differing anthropogenic impact near Jeddah, Saudi Arabia. Stacked column plots display the most abundant bacterial families (>1%, determined separately for each coral species). Samples were collected after 21 months of reciprocal transplantation. A/B unimpacted sites; C/D local wastewater and nutrients sites; E municipal sewage and sedimentation and oil site. Source data are provided as a Source Data ﬁle b a Bacterial community composition of the corals A. hemprich Fig. 4 Bacterial community composition of the corals A. hemprichii and P. verrucosa. Bacterial communities shown per coral species a A. hemprichii and b P. verrucosa across sites of differing anthropogenic impact near Jeddah, Saudi Arabia. Stacked column plots display the most abundant bacterial families (>1%, determined separately for each coral species). Samples were collected after 21 months of reciprocal transplantation. A/B unimpacted sites; C/D local wastewater and nutrients sites; E municipal sewage and sedimentation and oil site. Source data are provided as a Source Data ﬁle 0 250 500 750 Chao OTU richness A. hemprichii P. verrucosa a 15 13 24 11 13 N = 26 Unimpacted Lo 0 250 500 750 Chao OTU richness 0.00 0.25 0.50 0.75 Simspon evenness index 0 2 4 6 Shannon diversity index A. hemprichii P. verrucosa A. hemprichii P. verrucosa A. hemprichii P. verrucosa a b c 15 13 24 11 13 N = 26 Unimpacted Local wastewater and nutrients Municipal sewage and sedimentation and oil Fig. 5 Bacterial diversity of the corals A. hemprichii and P. verrucosa across impacts. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-10969-5 Samples were collected after 21 months of reciprocal transplantation across different impacts (“Unimpacted”, “Local wastewater and nutrients”, “Municipal sewage and sedimentation and oil”) near Jeddah, Red Sea and are displayed by destination impact. a Chao Index of bacterial richness, b Simpson Index of bacterial community evenness, c Shannon Index of bacterial diversity. Boxes of box plots represent the 25th to 75th percentile, lines show medians, error bars represent smallest/largest values to a maximum of 1.5 * IQR. N of each group indicated in a, Kruskal–Wallis tests signiﬁcance level: p < 0.01. Source data are provided as a Source Data ﬁle 0 2 4 6 Shannon diversity index A. hemprichii P. verrucosa c 0.00 0.25 0.50 0.75 Simspon evenness index A. hemprichii P. verrucosa b ** cal wastewater and nutrients Mun b c a A. hemprichii P. verrucosa Municipal sewage and sedimentation and oil Local wastewater and nutrients Unimpacted Fig. 5 Bacterial diversity of the corals A. hemprichii and P. verrucosa across impacts. Samples were collected after 21 months of reciprocal transplantation across different impacts (“Unimpacted”, “Local wastewater and nutrients”, “Municipal sewage and sedimentation and oil”) near Jeddah, Red Sea and are displayed by destination impact. a Chao Index of bacterial richness, b Simpson Index of bacterial community evenness, c Shannon Index of bacterial diversity. Boxes of box plots represent the 25th to 75th percentile, lines show medians, error bars represent smallest/largest values to a maximum of 1.5 * IQR. N of each group indicated in a, Kruskal–Wallis tests signiﬁcance level: *p < 0.01. Source data are provided as a Source Data ﬁle Fig. 5 Bacterial diversity of the corals A. hemprichii and P. verrucosa across impacts. Samples were collected after 21 months of reciprocal transplantation across different impacts (“Unimpacted”, “Local wastewater and nutrients”, “Municipal sewage and sedimentation and oil”) near Jeddah, Red Sea and are displayed by destination impact. a Chao Index of bacterial richness, b Simpson Index of bacterial community evenness, c Shannon Index of bacterial diversity. Boxes of box plots represent the 25th to 75th percentile, lines show medians, error bars represent smallest/largest values to a maximum of 1.5 IQR. N of each group indicated in a, Kruskal–Wallis tests signiﬁcance level: **p < 0.01. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-10969-5 NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-10969-5 a Otu0477_Sinobacteraceae Otu0453_Solirubrobacterales Otu0384_Nakamurellaceae Otu0372_Solibacteraceae Otu0339_EB1017 Otu0313_Chitinophagaceae Otu0299_Cytophagaceae Otu0275_Sphingomonadaceae Otu0260_Syntrophobacteraceae Otu0254_Rhodobacteraceae Otu0240_Corynebacteriaceae Otu0236_Bacteroidaceae Otu0233_unclassiﬁed Acidimicrobiales Otu0223_Lachnospiraceae Otu0219_Ruminococcaceae Otu0218_Rhodocyclaceae Otu0210_Flavobacteriaceae Otu0196_Spirochaetaceae Otu0194_[Weeksellaceae] Otu0190_Carnobacteriaceae Otu0178_Rhodobacteraceae Otu0170_Sphingomonadaceae Otu0169_Ruminococcaceae Otu0167_Sphingobacteriaceae Otu0152_Ruminococcaceae Otu0145_C111 Otu0142_Microbacteriaceae Otu0129_Alteromonadaceae Otu0108_Rhodobacteraceae Otu0062_Comamonadaceae Otu0055_Pseudomonadaceae Otu0051_Flavobacteriaceae Otu0038_Moraxellaceae Otu0017_Flavobacteriaceae Otu0237_Flavobacteriaceae Otu0151_unclassiﬁed actinomycetales Otu0125_ACK−M1 Otu0117_Oxalobacteraceae Otu0116_Comamonadaceae Otu0109_Microbacteriaceae Otu0102_Methylophilaceae Otu0093_Microbacteriaceae Otu0092_Oxalobacteraceae Otu0087_Flavobacteriaceae Otu0081_Cytophagaceae Otu0071_Sinobacteraceae Otu0061_Bradyrhizobiaceae Otu0056_Comamonadaceae Otu0049_Polyangiaceae Otu0031_Staphylococcaceae Otu0027_unclassiﬁed Ucp1540 Otu0026_Comamonadaceae Otu0021_Comamonadaceae Otu0014_Sphingomonadaceae Otu0007_Caulobacteraceae Otu0005_Simkaniaceae Otu0321_Endozoicomonadaceae Otu0132_unclassiﬁed Gammaproteobacteria Otu0079_Flavobacteriaceae Otu0003_Endozoicomonadaceae b Pocillopora verrucosa Destination site A B C D E Destination site Destination impact A B C D E Unimpacted Local wastewater and nutrients Municipal sewage and sedimentation and oil 4− 2− 0 2 4 Z scores (row): Otu0107_Propionibacteriaceae Otu0050_Moraxellaceae Otu0072_unclassiﬁed Proteobacteria Otu0327_Endozoicomonadaceae Otu0005_Simkaniaceae Fig. 3 Differentially abundant bacterial taxa (OTUs) over impacts. Bacterial taxa were associated with the corals a A. hemprichii and b P. verrucosa ac sites of differing anthropogenic impact near Jeddah, Saudi Arabia. The ﬁrst cluster in each panel is signiﬁcantly more abundant at unimpacted sites second cluster at sites exposed to local wasetwater and nutrients, and the third cluster at sites with municipal sewage and sedimentation and oil (L method, all LDA >2.0, p < 0.05). Sites and impacts marked with horizontal bars above the heatmaps. Source data are provided as a Source Data ﬁ b Pocillopora verrucosa Destination site Destination impact A B C D E Unimpacted Local wastewater and nutrients Municipal sewage and sedimentation and oil 4− 2− 0 2 4 Z scores (row): Otu0107_Propionibacteriaceae Otu0050_Moraxellaceae Otu0072_unclassiﬁed Proteobacteria Otu0327_Endozoicomonadaceae Otu0005_Simkaniaceae b Pocillopora verrucosa Destination site Destination impact A B C D E Unimpacted Local wastewater and nutrients Municipal sewage and sedimentation and oil 4− 2− 0 2 4 Z scores (row): Otu0107_Propionibacteriaceae Otu0050_Moraxellaceae Otu0072_unclassiﬁed Proteobacteria Otu0327_Endozoicomonadaceae Otu0005_Simkaniaceae b Local wastewater and nutrients Z scores (row): Fig. 3 Differentially abundant bacterial taxa (OTUs) over impacts. Bacterial taxa were associated with the corals a A. hemprichii and b P. verrucosa across sites of differing anthropogenic impact near Jeddah, Saudi Arabia. The ﬁrst cluster in each panel is signiﬁcantly more abundant at unimpacted sites, the second cluster at sites exposed to local wasetwater and nutrients, and the third cluster at sites with municipal sewage and sedimentation and oil (LEfSe method, all LDA >2.0, p < 0.05). Sites and impacts marked with horizontal bars above the heatmaps. Source data are provided as a Source Data ﬁle NATURE COMMUNICATIONS \| (2019) 10:3092 \| https://doi.org/10.1038/s41467-019-10969-5 \| www.nature.com/naturecommunications 5 ARTICLE Discussion Changes in microbial community structure represent a poten- tially fast and ﬂexible mechanism that may facilitate coral holo- biont adaptation and broaden plasticity1,31,32. In this study we tested two coral species for their capacity to undergo microbiome restructuring in response to changing environmental conditions during a long-term reciprocal transplantation experiment. We found that the A. hemprichii microbiome is highly ﬂexible and more variable, whereas the P. verrucosa microbiome is fairly stable and overall less variable in response to changing environ- mental conditions. These ﬁndings suggest that coral species exhibit different degrees of ﬂexibility in holobiont structure and composition. The differences we observe in bacterial assemblages between A. hemprichii and P. verrucosa across different sites argue for dif- ferent degrees of microbiome ﬂexibility. Thus, these potentially represent differences in the underlying strategy employed by the two species to cope with environmental stress. Importantly, it has to be considered that corals are in general long-lived, sessile animals that are unable to escape changes in their environment. And because of their long generation times, evolutionary change is supposedly slow43. Strategies to cope with and survive rapid environmental change are therefore critical. One mechanism by which corals may adjust more rapidly to change may be through their association with different bacterial taxa, whereby selection occurs for the most advantageous and beneﬁcial microbiome in a particular environment1,10. It is therefore striking to ﬁnd in A. hemprichii a readily “responding” microbiome, in contrast to which the bacterial community of P. verrucosa seems rather “inert”. However, this ﬁnding is not entirely unexpected. Previous studies have shown highly stable Symbiodiniaceae26,44 and bacterial14,24,28 communities both in P. verrucosa and also its close relative Pocillopora acuta45, even under conditions of mortality. In comparison the microbial communities of Acropora are ﬂexible and seem to align with environmental patterns13,29,46. In line with these studies, we found the bacterial microbiome of A. hemprichii to be highly variable between sites and impacts and also ﬂexible upon reciprocal transplantation. In contrast, P. ver- rucosa harbored a far less variable bacterial microbiome that showed less ﬂexibility upon transplantation. p A. hemprichii underwent strong microbiome restructuring when transplanted from unimpacted sites to impacted ones. This was characterized by increased bacterial diversity and decreased evenness (i.e., the loss of dominant taxa from the unimpacted site). Discussion An increase in bacterial diversity in coral microbiomes often accompanies the holobiont stress response as a result of emerging opportunistic taxa that are otherwise absent or suppressed33,34. Also, increased bacterial diversity has been repeatedly observed in diseased coral microbiomes35,36. Notably, the changes in bacterial communities of A. hemprichii at impacted sites were character- ized by decreased relative abundances and potential loss of Endozoicomonadaceae, the bacterial family containing the enig- matic coral symbiont genus Endozoicomonas37,38. Moreover, bacterial communities of A. hemprichii fragments transplanted to impacted sites had higher abundances of bacterial families that have previously been characterized as opportunists and that have been associated with coral disease. For instance, taxa within the Rhodobacteraceae have been found on corals with white plague disease35 and the family Flavobacteraceae also contains potentially pathogenic taxa36. Nonetheless, other bacterial families that were more abundant in A. hemprichii at the impacted sites (Erythrobacteraceae, Comamonadaceae, Oxalo- bacteraceae, Moraxellaceae) have also been isolated from healthy corals, which illustrates that shifts in microbial abundances do not exclusively or necessarily reﬂect a pathobiome, but rather an environmentally selected, putatively more beneﬁcial micro- biome39–41. Indeed, the notion of an environmentally explicit set of bacterial taxa that ﬁll speciﬁc functional niches through changes in the microbiome of A. hemprichii17 is supported by the high number of speciﬁc taxa found under different impacts in our LEfSe and indicspecies analyses. In contrast, the number of such environmentally explicit taxa in P. verrucosa was an order of magnitude lower (5 and 7 taxa in P. verrucosa vs. 60 and 62 taxa in A. hemprichii for the indicspecies and LEfSe analyses, respec- tively). This is also in line with the current notion that holobiont composition (or metaorganism structure for that matter) is not static, but rather dependent on age, development, sex, and environment, among other factors42. As such, consistent or close association of bacteria with their animal hosts is not a sensu stricto criterion for functional relevance42. Furthermore, the microbiome of P. verrucosa was remarkably consistent between sites, with only small changes between impact levels in bacterial diversity and evenness. The most abundant Endozoicomonas OTU in P. verrucosa dominated most samples and, in contrast to the loss of Endozoicomonadaceae in A. hemprichii at impacted sites, was consistent. Generally, there were no major abundance changes in bacterial taxa between impact levels in P. verrucosa, with the exception of the Simkaniaceae family, whose role we can only speculate on. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-10969-5 NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-10969-5 of less abundant members), or whether it is simply the indication of an inﬂexible host-microbial association, remains to be determined. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-10969-5 Source data are provided as a Source Data ﬁle municipal sewage and sedimentation and oil site, compared to the unimpacted sites (pairwise tests N = 11.63, p < 0.001 and H = 8.7, p < 0.005, respectively). Consequently, overall OTU diversity (Shannon diversity) in A. hemprichii was signiﬁcantly different between impact levels (Fig. 5c; Kruskal–Wallis, H = 18.31, p < 0.001) with signiﬁcantly lower diversity at unimpacted sites compared to local wastewater and nutrients sites (pairwise test N = 12.57, p < 0.001), and the municipal sewage and sedimenta- tion and oil site (pairwise test N = 11.33, p < 0.001). In contrast, differences in bacterial diversity between impacts were minor in P. verrucosa (Fig. 5). Overall, OTU richness in P. verrucosa was similar between environmental impacts (Fig. 5a; Kruskal–Wallis, H = 0.58, p > 0.05). Similarly, community evenness in P. verrucosa was similar between impacts (Fig. 5b; Kruskal–Wallis, H = 3.48, p > 0.05). Further, community evenness was much higher than for A. hemprichii (Fig. 5b), resulting in overall low bacterial diversity estimates (Fig. 5c; Kruskal–Wallis, H = 0.57, p > 0.05). Notably, re-analyses of the dataset at a 99% OTU similarity cutoff recap- tured the observed patterns (Supplementary Fig. 9, Source Data). However, it should be noted that because the Endozoicomona- daceae are a dominant feature in the P. verrucosa microbiome, excluding bacteria of this family changed the evenness and diversity of these microbial communities (Supplementary Fig. 9; Source Data). NATURE COMMUNICATIONS \| (2019) 10:3092 \| https://doi.org/10.1038/s41467-019-10969-5 \| www.nature.com/naturecommunications 6 6 ARTICLE NATURE COMMUNICATIONS \| (2019) 10:3092 \| https://doi.org/10.1038/s41467-019-10969-5 \| www.nature.com/naturecommunications Methods E i t 10, Source Data) paired with unauthorized local wastewater outfalls that are estimated to release 99,000 m3 d−1 of untreated wastewater into the nearshore area along 30 km of coastline and lead to elevated nitrate levels (Supplementary Fig. 11, Source Data) 63,64, while levels of THC are comparable to the unimpacted sites (Supplementary p p pp y Fig. 12, Source Data). Both sites were characterized by relatively high cover of Xenia spp., known to opportunistically invade degraded reefs65. Site E represents the most severe impact level being located within Jeddah Bay, in proximity to the industrial port, which generates intermediate levels of oil pollution (Supplementary Fig. 12, Source Data). In addition, site E was less than ﬁve km from the three main discharge points of Jeddah’s sewage treatment facilities, which regularly discharge extensive amounts (35,000, 68,000, and 300,000 m3 d−1, respectively) of untreated or only partially treated sewage that lead to intermittent increases in nitrate levels (Supplementary Fig. 11, Source Data)66,67. Hence, this site is subjected to elevated turbidity and highest sedimentation loads (Supplementary Fig. 10, Source Data). Hard coral cover at site E is similar to sites C and D with soft coral cover being intermediate, with lower counts of Xenia spp.28. y A ﬁnal point arising from results of cross-transplantation and back-transplantation is that the bacterial communities of both coral species were similar to their local back-transplanted con- speciﬁcs after cross-transplantation to unimpacted sites. This ﬁnding may hold the promise of microbiome “recovery”. In other words, coral fragments transplanted from impacted to control sites did not, after 21 months, continue to share similarities with fragments of the same colonies that remained at the impacted sites. These ﬁndings suggest that stress-induced microbiome alterations may be reverted upon removal of chronic and long- term stressors, similar to the recovery observed after coral bleaching29 or disease30. Following the notion that coral micro- biomes contribute to coral health, our results indicate that reducing and removing sources of pollution and sedimentation may result in the reversal of microbiomes. Hence, anthropogenic pollution may not irreversibly disrupt microbiomes in supporting coral health1,60,61. Our results are in line with recent studies, which report that increases in coral disease caused by experi- mental nutrient enrichment were reversed 6–10 months after termination of the experimental treatment62. Methods E i t Taken together, our data create an additional incentive to reduce sources of anthro- pogenic pollution and sedimentation close to coral reefs, even if the corals on the target reefs already appear stressed and in poor condition. , pp In total, 36 coral colonies (18 per species) were each fragmented into 6 pieces resulting in 216 coral fragments that were reciprocally transplanted. Speciﬁcally, at each of the six intial sites (A–F), three visually healthy colonies of each of A. hemprichii and P. verrucosa were collected at 5–10 m depth, fragmented into six or more pieces (about 10 cm max length) each, and suitable pieces then transported to the King Abdulaziz University marine laboratory at Sharm Obhur for measurement and weighing. The fragments were allowed to recover for 2–3 weeks at a depth of 5 m deep on a shallow reef adjacent to the laboratory, and then cross- and back-transplanted across all sites during July/August 2013 (reciprocal transplantation design, Fig. 1). This design allowed fragments from each colony to be assessed at each location, including one fragment per colony that was back- transplanted to its respective site of origin. Of note, the back-transplanted fragments (to their site of origin) act as experimental and handling “controls” in that any microbiome changes would arguably be a result of the transplantation procedure. These fragments also aid in correcting for possible changes of the microbiome that may be related to time or age of the fragments, in that fragments from the same colonies (with the same age, sharing the same life history) were investigated at all sites68. Coral fragments were attached by cable-ties to a frame suspended in mid-water at 5–10 m depth to prevent sediment cover and predation by benthic predators. Experiment sites were then visited approximately every three months to monitor growth and check the integrity of the structures. Coral fragments were retrieved after a further 21 months (April/May 2015), when they were rinsed with ﬁltered seawater, placed individually in sterile polyethylene bags, and transported back to the Sharm Obhur laboratory on ice, before being frozen for temporary storage at −80 °C. Using cubitainers, four water samples (1 L) were also collected from the same depth as the coral samples at each site, and likewise transported in dark portable coolers back to Sharm Obhur, where they were ﬁltered using 0.22 μm Durapore PVDF ﬁlters (Millipore, Billerica, MA, USA). Discussion Additional analyses excluding the Endozoico- monadaceae family further support the notion of a more stable and less variable microbiome of P. verrucosa compared to A. hemprichii. Whether dominance of a particular bacterial lineage promotes a generally more stable microbiome (inducing stability y p p Consequent to these ﬁndings, we argue that coral species differ in their ability to associate ﬂexibly with different bacterial assemblages. In analogy to strategies for coping with osmotic stress47, Acropora might be referred to as a “microbiome con- former” (showing microbial adaptation to the surrounding environment), whereas Pocillopora might be referred to as a “microbiome regulator” (showing microbial regulation that maintains a constant microbiome). Hence we propose the term “microbiome ﬂexibility” to describe a coral species’ potential for dynamic microbiome restructuring in the face of environmental change. It remains to be investigated how pervasive these patterns of microbiome ﬂexibility are across and within coral taxa, as illustrated by instances of reversed patterns of relatively high microbiome ﬂexibility in Pocillopora41 and relative low micro- biome ﬂexibility in Acropora27 (although each of the studies tested only one species separately, rendering cross-species com- parisons difﬁcult). It further remains to be determined whether differences in microbiome ﬂexibility represent distinct holobiont adaptation mechanisms to environmental change. High micro- biome ﬂexibility presumably supports holobiont adaptation to environmental change and follows a generalist strategy albeit with the risk of losing important associates/functions or acquiring pathogens. Conversely, low microbiome ﬂexibility helps to maintain stable and robust relationships with conserved micro- bial functions reﬂecting a more specialized strategy at the expense of a putatively low capacity for microbiome adaptation and potential susceptibility to rapid environmental change. The degree of microbiome ﬂexibility may be linked to life history strategy of the host48. A. hemprichii and P. verrucosa both have wide distribution ranges49,50 and inhabit the shallow to mid reef slope50. However, the microbiome conformer A. hemprichii has a relatively slow growth rate51 and, in contrast to Pocillo- porids, Acroporids generally have a competitive strategy, with longer generation times52. Indeed, the microbiome regulator P. verrucosa shows a rather limited physiological plasticity and ecological niche space, being adapted to high-light wave-exposed 7 ARTICLE NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-10969-5 environments near the reef crest, in particular in the Red Sea44. Pocilloporids generally favor an opportunistic colonization strategy that is characterized by fast growth, high reproduction rates, and relatively rapid generation times53. Discussion Given that a limited diversity in the diet of deep-sea corals has been linked to reduced diversity within the microbiome48, the mainly autotrophic life- style and low heterotrophic capacity of P. verrucosa may also partially explain its low microbiome ﬂexibility48. However, it remains to be determined whether low microbiome ﬂexibility is a cause or consequence of this strategy. distinct degrees of microbiome ﬂexibility exist, potentially reﬂecting different holobiont adaptation mechanisms to envir- onmental change. Thus, bacterial community structures may respond in a host-speciﬁc manner (not “one-size-ﬁts-all”), a situation which would hamper elucidation of a universal core microbiome. Importantly, altered microbial community struc- tures of corals from impacted sites recovered, being indis- tinguishable from local conspeciﬁcs when transplanted back into an unimpacted control environment. This ﬁnding holds the promise of microbiome recovery, encouraging the reduction of anthropogenic pollution, even in reef areas where coral assem- blages are already degraded. The presence of varying degrees of microbiome ﬂexibility among different coral taxa may also help to resolve the dis- crepancy regarding the absence of a consistent coral core microbiome. While the concept of a core microbiome may be debatable, especially in the light of the coral probiotic hypothesis1,10, the expectation is that at least some bacterial associates are intimately and tightly associated with a coral and not expendable14,17,54,55. A recent study54, after examining two coral species across a wide range of habitats, proposed seven distinct bacterial phylotypes as universal coral core microbiome members. Surprisingly, these core OTUs were nevertheless not ubiquitous, the threshold used to qualify as a core microbiome member being comparably low at ≥30% of samples. Our data suggest that attempts to identify a universal coral core micro- biome might result in only a small number of bacterial taxa, if different coral species are compared that display such high or low microbiome ﬂexibility. Potentially relevant here is that coral phylogeny is characterized by a deep phylogenetic split (between “complex” and “robust” clades) that dates back >245 mya56,57. This led to substantial genomic divergence58 and consequences for host-microbe pairings that may be shaped by phylo- symbiosis59. Such a separation might arguably underlie not only differences in microbial association, but also differences in microbiome ﬂexibility, such as observed here. NATURE COMMUNICATIONS \| (2019) 10:3092 \| https://doi.org/10.1038/s41467-019-10969-5 \| www.nature.com/naturecommunicatio Methods E i t Methods Experimental design and sample collection. Six study sites (A–F) were selected as described in the previous work of Ziegler et al.28 (Fig. 1). The experimental setup at site F was destroyed during the experiment and hence results from ﬁve sites are reported hereafter (A–E). These sites represent a range of anthropogenic impacts with differences in abiotic factors, benthic community composition, seawater microbial communities, and coral-associated bacterial community compositions. Sites A and B were relatively unimpacted and represent almost pristine control conditions. Both locations were characterized by comparatively low sedimentation loads (Supplementary Fig. 10, Source Data), low inorganic nitrate concentrations (Supplementary Fig. 11, Source Data), and low levels of total hydrocarbons (THC), measured against a standard of Light Arabian Crude Oil (Supplementary Fig. 12, Source Data; see Supplementary Methods for details on measurements). During previous surveys a high stony coral cover and diversity as well as low abundances of soft corals was recorded at these sites28. Sites C and D were located along the fringing reef of the heavily developed Jeddah Corniche and represent an intermediate impact level. The area is exposed to chronic turbidity and intermediate sedimentation loads from inﬁlling (Supplementary Fig. 10, Source Data) paired with unauthorized local wastewater outfalls that are estimated to release 99,000 m3 d−1 of untreated wastewater into the nearshore area along 30 km of coastline and lead to elevated nitrate levels (Supplementary Fig. 11, Source Data) 63,64, while levels of THC are comparable to the unimpacted sites (Supplementary y p Sites A and B were relatively unimpacted and represent almost pristine control conditions. Both locations were characterized by comparatively low sedimentation loads (Supplementary Fig. 10, Source Data), low inorganic nitrate concentrations (Supplementary Fig. 11, Source Data), and low levels of total hydrocarbons (THC), measured against a standard of Light Arabian Crude Oil (Supplementary Fig. 12, Source Data; see Supplementary Methods for details on measurements). During previous surveys a high stony coral cover and diversity as well as low abundances of soft corals was recorded at these sites28. Sites C and D were located along the fringing reef of the heavily developed Jeddah Corniche and represent an intermediate impact level. The area is exposed to chronic turbidity and intermediate sedimentation loads from inﬁlling (Supplementary Fig. Data availability Sequence data determined in this study are available under NCBI BioProject ID PRJNA491299. Abundant coral bacterial microbiome OTU reference sequences are available under GenBank accession numbers MK736129 to MK736265 for Acropora hemprichii and under GenBank accession numbers MK736048 to MK736100 for Pocillopora verrucosa. Source data underlying ﬁgures and statistical analyses are provided as a Source Data File. indexing adapters (Illumina, USA). Samples were then cleaned and normalized (to 25 ng DNA per sample) using the Invitrogen SequalPrep Normalization Plate Kit (Thermo Fisher Scientiﬁc, USA). Normalized samples were pooled and sequenced at the KAUST Bioscience Core Lab (BCL) on the Illumina MiSeq platform using 2 × 300 bp paired-end v3 chemistry at 8 pM with 10% PhiX. Received: 24 September 2018 Accepted: 12 June 2019 Sequence data processing and analysis. Sequence data processing and analysis were done using mothur v. 1.41.170. Speciﬁcally, after forward and reverse reads were assembled into contigs, ambiguous reads were removed, and sequences were quality trimmed and pre-clustered71. Singletons were discarded and the remaining sequences were aligned against the SILVA database72. Chimeric sequences were excluded using VSEARCH73 and chloroplast, mitochondrial, archaeal, and eukaryotic sequences were removed. The remaining sequences were then classiﬁed against the Greengenes database74. Three bacterial taxa that comprised >95% of sequences in the DNA extraction negative control and the PCR negative control were removed from all samples for subsequent analyses using the remove.lineages() command in mothur. These taxa were an unclassiﬁed Brachybacterium in the family Dermabacteraceae, an unclassiﬁed Dietzia in the family Dietziaceae, and the bacterium Brevibacterium casei. The latter two have been reported previously as common laboratory contaminants75. After removal of putative contaminants, samples were subsampled to 3,101 sequences and again classiﬁed against the Greengenes database. Stacked column plots representing bacterial community compositions at the taxonomic family level were constructed based on these phylogenetically classiﬁed sequences. For all subsequent analyses, Operational Taxonomic Units (OTUs) were built based on sequence clustering at a 97% similarity cutoff. Because this cutoff can be considered rather conservative, a comparative analysis of a second dataset using a 99% similarity cutoff for OTU clustering was also conducted. This analysis yielded similar sample groupings and results (Supplementary Data 3–4). Given that the microbiome of P. verrucosa was dominated by a single bacterial taxon (OTU0001, Endozoicomonadaceae), a third dataset was created that excluded the Endozoicomonadaceae family with the command remove.lineages() in mothur as detailed above. ARTICLE ARTICLE NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-10969-5 DNA extraction and 16S rRNA gene amplicon sequencing. Between 2 and 4 ml PBS were applied to each frozen coral fragment before tissue was sprayed off into sterile zip lock bags using sterile ﬁlter pipette tips (1000 μl barrier tips, Neptune, USA) connected via a rubber hose to a bench top air pressure valve. 0.5 M EDTA was added to the resulting coral tissue slurry to a ﬁnal concentration of 1.25 mM before storage at −80 °C. DNA was extracted with the DNeasy Plant Mini Kit (Qiagen, Hilden, Germany) following the manufacturer’s protocol and using a volume of 100 μl coral tissue slurry suspended in 300 μl AP1 lysis buffer. For DNA extraction from water samples, each ﬁlter was cut into three pieces using sterile surgical blades. One piece was sliced into 2–4 mm wide strips, suspended in 400 μl AP1 buffer in a 1.5 ml centrifuge tube, and incubated for 30 min on a rotating wheel at a 45° angle. The DNeasy Plant Mini Kit protocol was then used to extract DNA. DNA concentrations of all samples were measured using a NanoDrop 2000C spectrophotometer (Thermo Fisher Scientiﬁc, Waltham, MA, USA). sites for seawater samples with the “adonis” function. Two-factorial PERMANOVAs were run with 9,999 permutations to test for effects of origin and destination sites for each coral species separately in a fully crossed design. This analysis was repeated with pooled sites per impact. The Linear discriminant analysis Effect Size (LEfSe) method79 was used with Kruskal–Wallis and Wilcoxon tests to identify shifts in the abundance of OTUs between impacts (p = 0.05; logarithmic LDA threshold = 2) as implemented in mothur. Alpha diversity indices (Chao1, Simpson Evenness, Shannon Diversity) were calculated for the bacterial community of each sample in mothur and analyzed for differences between destination impact levels using Kruskal–Wallis tests in R. Bacterial indicator taxa analyses were conducted to identify bacteria that associate with corals at speciﬁc impact destinations and to investigate whether corals originating from the same impact continue harboring speciﬁc taxa even after transplantation (p-value threshold = 0.01). The analysis was conducted in R using the indicspecies package with the command “multipatt”80. We also investigated the members of the microbiome that were broadly present and presumably stably associated with A. hemprichii and P. verrucosa. References 1. Rosenberg, E., Koren, O., Reshef, L., Efrony, R. & Zilber-Rosenberg, I. The role of microorganisms in coral health, disease and evolution. Nat. Rev. Microbiol. 5, 355–362 (2007). 2. Rohwer, F., Seguritan, V., Azam, F. & Knowlton, N. Diversity and distribution of coral-associated bacteria. Mar. Ecol. Prog. Ser. 243, 1–10 (2002). 3. Muscatine, L. in Ecosystems of the World. Coral Reefs (eds. Dubinsky, Z.) (Elsevier, 1990). 4. Muscatine, L., Porter, J. W. & Kaplan, I. R. Resource partitioning by reef corals as determined from stable isotope composition. Mar. Biol. 100, 185–193 (1989). 5. Glasl, B., Herndl, G. J. & Frade, P. R. The microbiome of coral surface mucus has a key role in mediating holobiont health and survival upon disturbance. ISME J. 10, 2280–2292 (2016). 6. Lema, K. A., Willis, B. L. & Bourne, D. G. Corals form characteristic associations with symbiotic nitrogen-ﬁxing bacteria. Appl. Environ. Microbiol. 78, 3136–3144 (2012). 7. Lesser, M. P., Mazel, C. H., Gorbunov, M. Y. & Falkowski, P. G. Discovery of symbiotic nitrogen-ﬁxing cyanobacteria in corals. Science 305, 997–1000 (2004). 8. Raina, J. B., Tapiolas, D., Willis, B. L. & Bourne, D. G. Coral-associated bacteria and their role in the biogeochemical cycling of sulfur. Appl. Environ. Microbiol. 75, 3492–3501 (2009). 9. Ritchie, K. B. Regulation of microbial populations by coral surface mucus and mucus-associated bacteria. Mar. Ecol. Prog. Ser. 322, 1–14 (2006). 10. Reshef, L., Koren, O., Loya, Y., Zilber-Rosenberg, I. & Rosenberg, E. The coral probiotic hypothesis. Environ. Microbiol. 8, 2068–2073 (2006). 11. Ziegler, M., Seneca, F. O., Yum, L. K., Palumbi, S. R. & Voolstra, C. R. Bacterial community dynamics are linked to patterns of coral heat tolerance. Nat. Commun. 8, 14213 (2017). All data analyses were performed in R version 3.5.076; for multivariate statistics the package vegan was used77 and for illustrations the package ggplot78. For multivariate analyses, OTU count data was square-root transformed. To test microbiome ﬂexibility, the “betadisper” function was used to calculate multivariate dispersion of samples (Bray-Curtis distances) between coral species and between impacts within species. Homogeneity of multivariate dispersions were tested with ANOVAs, followed where applicable by Tukey’s Honest Signiﬁcant Differences post-hoc tests and visualized with boxplots. 12. Buddemeier, R. W. & Fautin, D. G. Coral bleaching as an adaptive mechanism —a testable hypothesis. Bioscience 43, 320–326 (1993). 13. Grottoli, A. G. et al. Coral physiology and microbiome dynamics under combined warming and ocean acidiﬁcation. ARTICLE Such putative core microbiome members were determined for each species separately by querying all bacterial OTUs that were present in ≥75% of samples at each site, ensuring even distribution of these core taxa across the sample set55. For PCR reactions between 5 and 50 ng template DNA was used for coral samples and between 4 and 10 ng template DNA for water samples. PCRs were performed in triplicate 10 μl reactions using the QIAGEN Multiplex PCR kit and ﬁnal primer concentrations of 0.5 μM. Variable regions 5 and 6 of the 16S rRNA gene were ampliﬁed using primers 784F [5′TCGTCGGCAGCGTCAGATGTGTA TAAGAGACAGAGGATTAGATACCCTGGTA′3] and 1061 R [5′GTCTCGTGG GCTCGGAGATGTGTATAAGAGACAGCRRCACGAGCTGACGAC′3]69 with Illumina adapter overhangs (underlined above). The PCR conditions consisted of initial denaturing at 95 °C for 15 min, followed by 27 cycles of 95 °C for 30 s, 55 °C for 90 s, and 72 °C for 30 s, and a ﬁnal extension step at 72 °C for 10 min. PCR ampliﬁcation was visually conﬁrmed using 1% agarose gels with 5 μl PCR product. The triplicate PCR amplicons were pooled, 5 μl of each pooled sample was aliquoted, and samples were then cleaned using illustra ExoProStar 1-step (GE Healthcare Life Sciences USA) Indexing was performed using Nextera XT For PCR reactions between 5 and 50 ng template DNA was used for coral samples and between 4 and 10 ng template DNA for water samples. PCRs were performed in triplicate 10 μl reactions using the QIAGEN Multiplex PCR kit and ﬁnal primer concentrations of 0.5 μM. Variable regions 5 and 6 of the 16S rRNA gene were ampliﬁed using primers 784F [5′TCGTCGGCAGCGTCAGATGTGTA TAAGAGACAGAGGATTAGATACCCTGGTA′3] and 1061 R [5′GTCTCGTGG GCTCGGAGATGTGTATAAGAGACAGCRRCACGAGCTGACGAC′3]69 with Illumina adapter overhangs (underlined above). The PCR conditions consisted of initial denaturing at 95 °C for 15 min, followed by 27 cycles of 95 °C for 30 s, 55 °C for 90 s, and 72 °C for 30 s, and a ﬁnal extension step at 72 °C for 10 min. PCR ampliﬁcation was visually conﬁrmed using 1% agarose gels with 5 μl PCR product. The triplicate PCR amplicons were pooled, 5 μl of each pooled sample was aliquoted, and samples were then cleaned using illustra ExoProStar 1-step (GE Healthcare Life Sciences, USA). Indexing was performed using Nextera XT indexing adapters (Illumina, USA). Samples were then cleaned and normalized (to 25 ng DNA per sample) using the Invitrogen SequalPrep Normalization Plate Kit (Thermo Fisher Scientiﬁc, USA). Data availability This dataset was then analyzed using the same procedures and conﬁrmed the observed differences in microbiome ﬂexibility between the two species (Source Data). For statistical ana- lyses, samples were grouped by sample type and coral host species (i.e., seawater, A. hemprichii, P. verrucosa) as well as origin and/or destination sites (A, B, C, D, E) or impact levels (unimpacted—sites A, B; local wastewater & nutrients—sites C, D; municipal sewage and sedimentation and oil—site E). ARTICLE Normalized samples were pooled and sequenced at the KAUST Bioscience Core Lab (BCL) on the Illumina MiSeq platform using 2 × 300 bp paired-end v3 chemistry at 8 pM with 10% PhiX. Reporting summary. Further information on research design is available in the Nature Research Reporting Summary linked to this article. NATURE COMMUNICATIONS \| (2019) 10:3092 \| https://doi.org/10.1038/s41467-019-10969-5 \| www.nature.com/naturecommunications Methods E i t Coral samples and water ﬁlters were transported frozen to King Abdullah University of Science and Technology (KAUST) and kept at −80 °C until further analysis. Changes in the microbiome of plant and animal hosts are increasingly being associated with the potential for acclimatiza- tion and adaptation in multicellular organisms. In particular, stony corals seem to be strongly reliant on their microbial associates, as highlighted by their obligate endosymbiosis with algal dinoﬂagellates. However, the potential for differences in microbiome ﬂexibility across coral species had not until now been systematically investigated. Our study supports the notion that NATURE COMMUNICATIONS \| (2019) 10:3092 \| https://doi.org/10.1038/s41467-019-10969-5 \| www.nature.com/naturecommunications 8 ARTICLE Changes in coral- associated microbial communities during a bleaching event. ISME J. 2, 350–363 (2008). 58. Voolstra, C. R. et al. Comparative analysis of the genomes of Stylophora pistillata and Acropora digitifera provides evidence for extensive differences between species of corals. Sci. Rep. 7, 17583 (2017). between species of corals. Sci. Rep. 7, 17583 (2017). 30. Sweet, M., Smith, D., Bythell, J. & Craggs, J. Changes in microbial diversity associated with two coral species recovering from a stressed state in a public aquarium system. JZAR 1, 52–60 (2013). 59. Pollock, F. J. et al. Coral-associated bacteria demonstrate phylosymbiosis and cophylogeny. Nat. Commun. 9, 4921 (2018). 60. Krediet, C. J., Ritchie, K. B., Paul, V. J. & Teplitski, M. Coral-associated micro- organisms and their roles in promoting coral health and thwarting diseases. Proc. R. Soc. B 280, 20122328 (2013). 31. Theis, K. R. et al. Getting the hologenome concept right: an eco-evolutionary framework for hosts and their microbiomes. mSystems 1, e00028-16 (2016). 61. Mao-Jones, J., Ritchie, K. B., Jones, L. E. & Ellner, S. P. How microbial community composition regulates coral disease development. PLoS Biol. 8, e1000345 (2010). 32. Bang, C. et al. Metaorganisms in extreme environments: do microbes play a role in organismal adaptation? Zoology 127, 1–19 (2018). g p gy 33. Vega Thurber, R. et al. Metagenomic analysis of stressed coral holobionts. Environ. Microbiol. 11, 2148–2163 (2009). 62. Vega Thurber, R. L. et al. Chronic nutrient enrichment increases prevalence and severity of coral disease and bleaching. Glob. Change Biol. 20, 544–554 (2014). 34. Zaneveld, J. R., McMinds, R. & Vega Thurber, R. Stress and stability: applying the Anna Karenina principle to animal microbiomes. Nat. Microbiol. 2, 17121 (2017). 63. Peña-García, D., Ladwig, N., Turki, A. J. & Mudarris, M. S. Input and dispersion of nutrients from the Jeddah Metropolitan Area, Red Sea. Mar. Pollut. Bull. 80, 41–51 (2014). 35. Roder, C., Arif, C., Daniels, C., Weil, E. & Voolstra, C. R. Bacterial proﬁling of White Plague disease across corals and oceans indicates a conserved and distinct disease microbiome. Mol. Ecol. 23, 965–974 (2014). 64. Risk, M., Sherwood, O., Nairn, R. & Gibbons, C. Tracking the record of sewage discharge off Jeddah, Saudi Arabia, since 1950, using stable isotope records from antipatharians. Mar. Ecol. Prog. Ser. 397, 219–226 (2009). 36. Sunagawa, S. et al. Bacterial diversity and White Plague disease-associated community changes in the Caribbean coral Montastraea faveolata. ISME J. ARTICLE 3, 512–521 (2009). 65. Benayahu, Y. & Loya, Y. Settlement and recruitment of a soft coral: why is Xenia macrospiculata a successful colonizer? Bull. Mar. Sci. 36, 177–188 (1985). 37. Neave, M. J., Apprill, A., Ferrier-Pages, C. & Voolstra, C. R. Diversity and function of prevalent symbiotic marine bacteria in the genus Endozoicomonas. Appl. Microbiol. Biotechnol. 100, 8315–8324 (2016). 66. Basaham, A. S., Rifaat, A. E., El-Mamoney, M. H. & El Sayed, M. A. Re- evaluation of the impact of sewage disposal on coastal sediments of the Southern Corniche, Jeddah, Saudi Arabia. JKAU 20, 109–126 (2009). 38. Bartz, J.-O. et al. Parendozoicomonas haliclonae gen. nov. sp. nov. isolated from a marine sponge of the genus Haliclona and description of the family Endozoicomonadaceae fam. nov. comprising the genera Endozoicomonas, Parendozoicomonas, and Kistimonas. Syst. Appl. Microbiol. 41, 73–84 (2018). 67. Mudarris, M. S. & Turki, A. J. Sewage water quality and its dilution in the coastal waters of South Corniche, Jeddah, Red Sea. JKAU 17, 115–128 (2006). 67. Mudarris, M. S. & Turki, A. J. Sewage water quality and its dilution in the coastal waters of South Corniche, Jeddah, Red Sea. JKAU 17, 115–128 (2006). 68. Williams, A. D., Brown, B. E., Putchim, L. & Sweet, M. J. Age-related shifts in bacterial diversity in a reef coral. PLoS ONE 10, e0144902 (2015). 68. Williams, A. D., Brown, B. E., Putchim, L. & Sweet, M. J. Age-related shifts in bacterial diversity in a reef coral. PLoS ONE 10, e0144902 (2015). 39. Kvennefors, E. C. E., Sampayo, E., Ridgway, T., Barnes, A. C. & Hoegh- Guldberg, O. Bacterial communities of two ubiquitous Great Barrier Reef corals reveals both site-and species-speciﬁcity of common bacterial associates. PLoS ONE 5, e10401 (2010). 69. Andersson, A. F. et al. Comparative analysis of human gut microbiota by barcoded pyrosequencing. PLoS ONE 3, e2836 (2008). 40. Sunagawa, S., Woodley, C. M. & Medina, M. Threatened corals provide underexplored microbial habitats. PLoS ONE 5, e9554 (2010). 70. Schloss, P. D. et al. Introducing mothur: open-source, platform-independent, community-supported software for describing and comparing microbial communities. Appl. Environ. Microbiol. 75, 7537–7541 (2009). 41. van Oppen, M. J. H. et al. Adaptation to reef habitats through selection on the coral animal and its associated microbiome. Mol. Ecol. 27, 2956–2971 (2018). 71. Huse, S. M., Welch, D. M., Morrison, H. G. & Sogin, M. L. ARTICLE Richards, Z. et al. Acropora hemprichii. The IUCN Red List of Threatened Species (2008). 51. Guzner, B., Novoplansky, A. & Chadwick, N. E. Population dynamics of the reef-building coral Acropora hemprichii as an indicator of reef condition. Mar. Ecol. Prog. Ser. 333, 143–150 (2007). p g p 23. Garren, M., Raymundo, L., Guest, J., Harvell, C. D. & Azam, F. Resilience of coral-associated bacterial communities exposed to ﬁsh farm efﬂuent. PLoS ONE 4, e7319 (2009). g 52. Darling, E. S., Alvarez-Filip, L., Oliver, T. A., McClanahan, T. R. & Côté, I. M. Evaluating life-history strategies of reef corals from species traits. Ecol. Lett. 15, 1378–1386 (2012). 24. Pogoreutz, C. et al. Dominance of Endozoicomonas bacteria throughout coral bleaching and mortality suggests structural inﬂexibility of the Pocillopora verrucosa microbiome. Ecol. Evol. 8, 2240–2252 (2018). 53. Lenihan, H. S. & Edmunds, P. J. Response of Pocillopora verrucosa to corallivory varies with environmental conditions. Mar. Ecol. Prog. Ser. 409, 51–63 (2010). 25. Ziegler, M., Roder, C. M., Büchel, C. & Voolstra, C. R. Limits to physiological plasticity of the coral Pocillopora verrucosa from the central Red Sea. Coral Reefs 33, 1115–1129 (2014). 54. Ainsworth, T. et al. The coral core microbiome identiﬁes rare bacterial taxa as ubiquitous endosymbionts. ISME J. 9, 2261–2274 (2015). f 26. Ziegler, M., Roder, C., Büchel, C. & Voolstra, C. R. Niche acclimatization in Red Sea corals is dependent on ﬂexibility of host-symbiont association. Mar. Ecol. Prog. Ser. 533, 149–161 (2015). 55. Sweet, M. J. & Bulling, M. T. On the importance of the microbiome and pathobiome in coral health and disease. Front. Mar. Sci. 4, 9 (2017). g 27. Jessen, C. et al. In-situ effects of eutrophication and overﬁshing on physiology and bacterial diversity of the Red Sea coral Acropora hemprichii. Plos One 8, e62091 (2013). 56. Park, E. et al. Estimation of divergence times in cnidarian evolution based on mitochondrial protein-coding genes and the fossil record. Mol. Phylogen Evol. 62, 329–345 (2012). 28. Ziegler, M. et al. Coral microbial community dynamics in response to anthropogenic impacts near a major city in the central Red Sea. Mar. Pollut. Bull. 105, 629–640 (2016). 57. Simpson, C., Kiessling, W., Mewis, H., Baron-Szabo, R. C. & Müller, J. Evolutionary diversiﬁcation of reef corals: a comparison of the molecular and fossil records. Evolution 65, 3274–3284 (2011). 29. Bourne, D., Iida, Y., Uthicke, S. & Smith-Keune, C. ARTICLE NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-10969-5 16. Roder, C., Bayer, T., Aranda, M., Kruse, M. & Voolstra, C. R. Microbiome structure of the fungid coral Ctenactis echinata aligns with environmental differences. Mol. Ecol. 24, 3501–3511 (2015). 44. Sawall, Y. et al. Extensive phenotypic plasticity of a Red Sea coral over a strong latitudinal temperature gradient suggests limited acclimatization potential to warming. Sci. Rep. 5, 8940 (2015). g p 45. Epstein, H. E., Torda, G. & van Oppen, M. J. H. Relative stability of the Pocillopora acuta microbiome throughout a thermal stress event. Coral Reefs 38, 373–386 (2019). 17. Hernandez-Agreda, A., Leggat, W., Bongaerts, P. & Ainsworth, T. D. The microbial signature provides insight into the mechanistic basis of coral success across reef habitats. mBio 7, e00560–00516 (2016). 46. Morrow, K. M. et al. Natural volcanic CO2 seeps reveal future trajectories for host-microbial associations in corals and sponges. ISME J 9, 894–908 (2015). 18. Koren, O. & Rosenberg, E. Bacteria associated with mucus and tissues of the coral Oculina patagonica in summer and winter. Appl. Environ. Microbiol 72, 5254–5259 (2006). 47. Mayﬁeld, A. B. & Gates, R. D. Osmoregulation in anthozoan–dinoﬂagellate symbiosis. Comp. Biochem. Phys. A 147, 1–10 (2007). 19. Li, J. et al. Bacterial dynamics within the mucus, tissue and skeleton of the coral Porites lutea during different seasons. Sci. Rep. 4, 7320 (2014). 48. Meistertzheim, A.-L. et al. Patterns of bacteria-host associations suggest different ecological strategies between two reef building cold-water coral species. Deep Sea Res Part I 114, 12–22 (2016). g p 20. Sweet, M. J., Brown, B. E., Dunne, R. P., Singleton, I. & Bulling, M. Evidence for rapid, tide-related shifts in the microbiome of the coral Coelastrea aspera. Coral Reefs 36, 815–828 (2017). , J , , , , , g , g, for rapid, tide-related shifts in the microbiome of the coral Coelastrea aspera. Coral Reefs 36, 815–828 (2017). 49. Pinzón, J. H. et al. Blind to morphology: genetics identiﬁes several widespread ecologically common species and few endemics among Indo-Paciﬁc cauliﬂower corals (Pocillopora, Scleractinia). J. Biogeogr. 40, 1595–1608 (2013). f 21. Chu, N. D. & Vollmer, S. V. Caribbean corals house shared and host-speciﬁc microbial symbionts over time and space. Environ. Microbiol. Rep. 8, 493–500 (2016). 22. Casey, J. M., Connolly, S. R. & Ainsworth, T. D. Coral transplantation triggers shift in microbiome and promotion of coral disease associated potential pathogens. Sci. Rep. 5, 11903 (2015). 50. References PLoS ONE 13, e0191156 (2018). 14. Neave, M. J. et al. Differential speciﬁcity between closely related corals and abundant Endozoicomonas endosymbionts across global scales. ISME J. 11, 186–200 (2017). Differences between groups were visualized using non-metric MultiDimensional Scaling (nMDS). Because dispersion of samples was signiﬁcantly different between groups (function “betadisper”, see above), multivariate statistical analyses were conducted per sample type (i.e., separately for seawater, P. verrucosa, and A. hemprichii samples). One-factorial PERmutational MANOVA (PERMANOVA) was run with 9,999 permutations to test for differences between 15. van de Water, J. A. J. M. et al. Comparative assessment of Mediterranean Gorgonian-associated microbial communities reveals conserved core and locally variant bacteria. Micro. Ecol. 73, 466–478 (2017). and A. hemprichii samples). One-factorial PERmutational MANOVA (PERMANOVA) was run with 9,999 permutations to test for differences between p p ) (PERMANOVA) was run with 9,999 permutations to test for differences between 9 Competing interests: The authors declare no competing interests. g 77. Oksanen, J. et al. vegan: Community Ecology Package (2018). 77. Oksanen, J. et al. vegan: Community Ecology Package (2018) 78. Wickham, H. ggplot2: Elegant Graphics for Data Analysis. (Springer, New York, 2016). Reprints and permission information is available online at http://npg.nature.com/ reprintsandpermissions/ Reprints and permission information is available online at http://npg.nature.com/ reprintsandpermissions/ 79. Segata, N. et al. Metagenomic biomarker discovery and explanation. Genome Biol. 12, R60 (2011). Peer review information: Nature Communications thanks Sebastian Fraune, Michael Sweet and other anonymous reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available. 80. Cáceres, M. D. & Legendre, P. Associations between species and groups of sites: indices and statistical inference. Ecology 90, 3566–3574 (2009). Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional afﬁliations. Acknowledgements We would like to thank Adam Porter (University of Exeter, UK) for assistance with the ﬁeldwork and Craig Michell (King Abdullah University of Science and Technology, KAUST) for help with sequencing library preparation. We acknowledge the KAUST Bioscience Core Lab for assistance with MiSeq sequencing. Research reported in this publication was supported by baseline funds to CRV from KAUST and undertaken as part of the Chair’s Program in Coastal Marine Conservation at King Abdulaziz Uni- versity, Jeddah, funded by HRH Prince Khaled bin Sultan. The research was further supported by scholarships under the KAUST visiting student research program (VSRP) to C.G.B.G. and M.M.B. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/ licenses/by/4.0/. Supplementary Information accompanies this paper at https://doi.org/10.1038/s41467- 019-10969-5. Supplementary Information accompanies this paper at https://doi.org/10.1038/s41467- 019-10969-5. 76. R Core Team. R: A Language and Environment for Statistical Computing. (Foundation for Statistical Computing, Vienna, Austria, 2016). Competing interests: The authors declare no competing interests. ARTICLE Ironing out the wrinkles in the rare biosphere through improved OTU clustering. Environ. Microbiol. 12, 1889–1898 (2010). 42. Jaspers, C. et al. Resolving structure and function of metaorganisms through a holistic framework combining reductionist and integrative approaches. Zoology 133, 81–87 (2019). 72. Pruesse, E. et al. SILVA: a comprehensive online resource for quality checked and aligned ribosomal RNA sequence data compatible with ARB. Nucleic Acids Res. 35, 7188–7196 (2007). gy 43. Torda, G. et al. Rapid adaptive responses to climate change in corals. Nat. Clim. Change 7, 627–636 (2017). 10 NATURE COMMUNICATIONS \| (2019) 10:3092 \| https://doi.org/10.1038/s41467-019-10969-5 \| www.nature.com/naturecommunicati ARTICLE NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-10969-5 and C.G.B.G. wrote the manuscript which R.O. edited; M.Z., C.R.V. revised the manu- script with input from R.O., C.G.B.G.; all authors read and approved the ﬁnal manuscript. 73. Rognes, T., Flouri, T., Nichols, B., Quince, C. & Mahé, F. VSEARCH: a ersatile open so rce tool for metagenomics PeerJ 4 e2584 (2016) 73. Rognes, T., Flouri, T., Nichols, B., Quince, C. & Mahé, F. VSEARCH: a versatile open source tool for metagenomics. PeerJ 4, e2584 (2016). versatile open source tool for metagenomics. PeerJ 4, e2584 (201 74. McDonald, D. et al. An improved Greengenes taxonomy with explicit ranks for ecological and evolutionary analyses of bacteria and archaea. ISME J. 6, 610–618 (2012). Additional information 75. Salter, S. J. et al. Reagent and laboratory contamination can critically impact sequence-based microbiome analyses. BMC Biol. 12, 87 (2014). Author contributions M.Z., R.O., and C.R.V. designed and conceived the experiment; M.E., J.B.O., A.A.S., and R.O. collected and transplanted the samples; A.J.T. collected environmental data; K.Z., A.A.S., and C.R.V. provided reagents/tools; C.G.B.G., M.Z., and M.M.B. generated the molecular data; M.Z., C.G.B.G., M.M.B., and C.R.V. analyzed the data; M.Z., C.R.V., 11 NATURE COMMUNICATIONS \| (2019) 10:3092 \| https://doi.org/10.1038/s41467-019-10969-5 \| www.nature.com/naturecommunications
https://openalex.org/W4205127581	https://www.researchsquare.com/article/rs-185088/latest.pdf	English	null	Long-term surgical oncological and functional outcome of large petroclival and cerebellopontine angle epidermoid cysts: a multicenter study	Neurosurgical review	2,022	cc-by	8,373	Long Term Surgical Oncological and Functional Outcome of Large Petroclival and Cerebellopontine Angle Epidermoid Cysts: A Multicenter Study. Aurore Sellier (  aurore.sellier@live.fr ) HIA Sainte Anne https://orcid.org/0000-0003-2655-8515 Research Article Research Article Page 1/26 Page 1/26 Keywords: Epidermoid Cyst, Petroclival Area, Cerebellopontine Angle, Cranial Nerve, Functional Outcome, Microsurgery Posted Date: November 1st, 2021 DOI: https://doi.org/10.21203/rs.3.rs-185088/v1 DOI: https://doi.org/10.21203/rs.3.rs-185088/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Version of Record: A version of this preprint was published at Neurosurgical Review on January 10th, 2022. See the published version at https://doi.org/10.1007/s10143-021-01702-2. Page 2/26 Abstract Objective: To assess the long-term surgical results on cranial nerve (CN) function and tumor control in patients harboring cerebellopontine angle (CPA) and petroclival area (PCA) epidermoid cysts (EC). Methods: This is a retrospective cohort study about 56 consecutive patients operated on for a CPA or PCA EC between January 2001 and July 2019 in six participating French cranial base referral centers. Results: Sixteen patients (29%) presented a PCA EC, and 40 a CPA EC (71%). The median clinical and radiological follow-up was 46 months (range 0-409). Preoperative CN disorders were present in 84% of patients (n=47), 72% of them experienced CN deficits improvement at last follow-up consultation (n=34) : 60% of cochlear and vestibular deficits (n=9/15 in both groups), 67% of trigeminal neuralgia (n=10/15), 53% of trigeminal hypoesthesia (n=8/15), 44% of lower cranial nerve disorders (n=4/9), 38% of facial nerve deficits (n=5/8), and 43% of oculomotor deficits (n=3/7) improved or were cured after surgery. New postoperative CN deficits occurred in 48% of patients (n=27). Most of them resolved at last follow-up, except for cochlear deficits which improved in only 14% of cases (n = 1/7). Twenty-six patients (46 %) showed evidence of tumor progression after a median duration of 63 months (range 7-210). Extent of resection, tumor location and tumor size were not associated with the occurrence of new postoperative CN deficit nor tumor progression. Conclusion: A functional nerve-sparing resection of posterior fossa EC is an effective strategy to optimize the results on preexisting CN deficits and reduces the risk of permanent de novo deficits. Introduction Epidermoid cysts (EC), also known as pearly tumors or cholesteatomas, are rare benign tumors that represent 0.2 to 1.8% of all intracranial tumors.[5] They are congenital lesions caused by abnormal trapping of epidermal elements during the processes of neural groove closure and disjunction of surface ectoderm, which occur between the 3rd and 5th weeks of fetal life.[6] They display a thin epithelial-lined capsule, and their growth is mainly due to the accumulation of desquamated epithelial cells, forming shiny pearly debris, in addition to the secretion of cholesterol and keratin into the cyst. Cerebellopontine angle (CPA) cysts, being the most common location for posterior fossa EC, constitute approximately 40– 50% of all intracranial EC. They represent the third most common CPA tumors after acoustic neuromas and meningiomas, comprising approximately 7% of CPA pathology.[7, 17] EC grow slowly and may penetrate to every accessible space around its origin. EC originating from the petroclival area (PCA) and the CPA share common behavior and surgical challenges. Cranial nerve (CN) disorders are the foremost symptoms in both locations. Microsurgical resection is the only effective treatment for these lesions when eligible for an active treatment [15]. Previous reports have already studied the correlation between total removal and tumor recurrence. Some authors found a higher rate of recurrence after non-total resection, [1, 7, 19, 22] while other reports found no difference between GTR and PR.[13, 20] Moreover, little is known about the kinetic of regrowth after incomplete surgical resection. Page 3/26 Page 3/26 Some other studies have focused on functional outcomes and the course of CN deficits after surgery.[3, 4, 9, 13, 14] However, there is no existing report presenting a detailed monitoring of the evolution of CN deficits over time. The aim of this multicenter study was to assess the long-term oncological and functional surgical outcome in CPA/PCA EC patients. Methods All consecutive patients who underwent surgical treatment for a CPA or PCA EC between January 2001 and July 2019 in six participating French cranial base referral centers were included in this observational retrospective cohort study. The centers involved were North University Hospital, Grenoble University Hospital, Pitié Salpêtrière Hospital, Rennes University Hospital, Angers University Hospital, La Timone Hospital. This study has been approved by the French Neurosurgical College Institutional Review Board (reference: IRB00011687 College de neurochirurgie IRB #1: 2020/04). Data collection Demographics, clinical manifestations (presenting signs and symptoms, duration of symptoms, CN function), radiological investigations (location, extension, laterality, size, existence of a diffusion-weighted magnetic resonance imaging (DWI) sequence, complications), surgical procedure (surgical approach, extent of removal), postoperative imagery, postoperative complications (occurring within 30 days after surgery), neurological outcome, radiological progression/recurrence and possible second surgery were retrospectively reviewed. Tumor size was assessed based on their largest diameter in MRI. PCA cysts were distinguished from CPA cysts by their primary origin in the upper two-thirds of the clivus or the petrous apex, medial to the trigeminal nerve. Both could then extend forward or backward to the cavernous sinus, the Meckel’s cave, the sellar region, the incisure of the tentorium, the porus and the ventral edge of the foramen magnum. Postoperative Assessment Clinico-radiological follow-up was planned at 3, 6 and 12 months after resection, and subsequently at 2, 3, 5, 7 & 10 years after surgery, and then once every 3 years thereafter. The function of each CN involved in the posterior fossa was analyzed in the postoperative period, and at every follow up visit. The Barrow Neurological Institute (BNI) classification [18] was used to assess the trigeminal neuralgia, and the facial nerve function was scored according to the House and Brackmann (HB) classification.[11] Preoperative CN deficit improvement was defined as the reduction or resolution of one or more preoperative CN deficit(s) between the preoperative period and the end of follow up. The patient’s functional status evolution was assessed with the World Health Organization performance status score (WHO PS) [16] at each follow up consultation. The postoperative cystic residue was assessed by MRI scan at 3 months after surgery. Extent of resection (EOR) was considered total (Gross Total Resection, GTR) if keratinous debris and the entire tumor capsule were removed, confirmed with the absence of postoperative cystic residue in DWI on the first postoperative MRI. Subtotal Resection (STR) was defined as complete content removal and incomplete capsule removal, with a small residue on postoperative MRI. Partial Resection (PR) was defined as incomplete resection of both cyst contents and capsule, with postoperative residue on postoperative MRI. Progression or recurrence of the lesion was based on the apparition or increase of the EC size on the DWI sequences of the follow-up MRI scan. Surgical protocol: Functional-sparing technique The main goal of the surgery was to achieve optimal and safe cyst resection, without compromising neurovascular structures (functional sparing technique). The intraoperative decision to interrupt the resection was based on the evidence of critical adhesion of the tumor capsule to CN, vascular structures or brain stem. In this situation, small fragments of capsule were left in place to avoid the risk of neurological deficits. The surgical approach was customized for location and cyst extension and depended on the experience of each center. Neuronavigation and CN neurophysiological monitoring could be used by the surgeon according to his preoperative evaluation. The suboccipital retrosigmoid (RS) (Figure 1) and the epidural anterior transpetrosal (TP) approach (Figure 2) were the most frequently used. In case of cyst extension above the tentorium, the resection could be combined with a pterional or a Page 4/26 Page 4/26 subtemporal approach. A median suboccipital telovelar approach could also be performed when the lesion had spread into the fourth ventricle. subtemporal approach. A median suboccipital telovelar approach could also be performed when the lesion had spread into the fourth ventricle. Population study Population study A total of 56 consecutive patients were enrolled in this study, including 29 (52%) females and 27 (48%) males, with a median age of 38 years (20–71) at the time of diagnosis. Six patients (11%) had previously been operated on in other centers. The median duration of symptoms before diagnosis was 6 months (0- 300). Clinical and radiological features (Table 1) Fifty-four patients (96%) were symptomatic at the time of the diagnosis. Preoperative CN disorders were present in 47 patients (84%). The foremost symptoms were the cochleo-vestibular impairment (25 patients - 45%): 15 patients reported hearing loss, and 15 patients presented with vestibular deficits (balance disorder/tinnitus/vertigo). Fifteen patients (27%) suffered from trigeminal neuralgia, and 15 patients (27%) presented with facial hypoesthesia. Lower cranial nerve (LCN) disorders were observed in 9 patients (16%). Facial nerve deficits were present in 8 patients (14%) preoperatively, mostly HB grade II (11%, n = 6). Seven patients (13%) presented one or more oculomotor dysfunction in the preoperative period: there were 3 CN III (5%), 3 CN IV (5%) and 5 CN VI (9%) deficiencies. Sixteen EC (29%) were mainly localized in the PCA, and 40 EC (71%) were localized in the CPA. Sixteen EC (29%) were mainly localized in the PCA, and 40 EC (71%) were localized in the CPA. Surgical Features (Table 2) RS approach was the most frequently performed, especially for the CPA location: 75% of CPA EC were removed through this approach, 3% were resected through a pterional approach, 10% through a TP approach, 10% through a subtemporal approach and 3% through a suboccipital approach (n = 30, 1, 4, 4, 1/40, respectively). In the PCA location, the surgical approach was less homogeneous. A RS approach was performed in 38% of them, a pterional approach in 44% of them, a TP approach in 13% of them, and a subtemporal approach in 7% of them (n = 6, 7, 2 and 1/16, respectively). GTR, STR and PR were performed in respectively 14% (n = 8), 61% (n = 34) and 23% (n = 13) of patients. The EOR was assessed by the surgeon at the end of the procedure in all cases, according to the criteria defined above. The surgical perioperative evaluation was also confirmed by a DWI sequence on the 3-month postoperative MRI when available (42 patients -75 %). For the remaining 14 patients, the surgery occurred before 2010 and no 3-month postoperative MRI was performed then. The most frequent postoperative complications were aseptic meningitis (13%, n = 7), and hydrocephalus (7%, n = 4). A 28-year-old woman died in the postoperative period from a postoperative cerebellar hematoma. Statistical analysis All statistical analyses were conducted using IBM Corp. Released 2012. IBM SPSS Statistics for Windows, Version 25.0. Armonk, NY: IBM Corp. Categorical variables were presented as numbers and percentages, and continuous variables were presented as mean and standard deviation or median and range (minimum-maximum), as appropriate. Analyses were tailored to address associations among surgical data (surgical approach, EOR, tumor location and tumor size) and the occurrence of preoperative CN deficit improvement, new postoperative CN deficit and tumor progression/recurrence. Univariate analyses were performed using the Pearson’s chi- squared test or Fisher’s exact test for categorical variables, and Mann Whitney U test for continuous variables, as appropriate. The progression free survival after surgery and the time between progression and the second surgery were presented as Kaplan-Meier plots. A two-sided p-value of less than 0.05 was considered to indicate statistical significance. Adjusted p-value <0,004 was considered statistically significant after Bonferroni correction for multiple comparison tests. Page 5/26 Page 5/26 Functional outcome 1. Preoperative CN deficit 1. Preoperative CN deficit Page 6/26 Page 6/26 Course of preoperative CN deficit (Table 3) Course of preoperative CN deficit (Table 3) Among the 47 patients with CN disorders before surgery, 34 experienced a CN improvement during follow up (72%, Figure 3A). At the end of follow-up, 60% of the cochlear and vestibular preoperative dysfunction improved or were completely resolved (n = 9/15 in both groups). Six patients retained hearing loss or deafness, and 6 had a persistent vestibular deficit at the end of the follow-up. At the end of follow-up, 60% of the cochlear and vestibular preoperative dysfunction improved or were completely resolved (n = 9/15 in both groups). Six patients retained hearing loss or deafness, and 6 had a persistent vestibular deficit at the end of the follow-up. Respectively 67 and 53% of trigeminal preoperative neuralgia and hypoesthesia were resolved at the last follow up control (n = 10/15 and n = 8/15). Among the remaining five persistent neuralgia (9%), three (5%) decreased according to the BNI classification (Figure 4). Preoperative LCN disorders disappeared throughout the follow-up in 44% of cases (n = 4/9). In the same way, an improvement of preexisting facial nerve deficits was seen in 38% of cases (n = 5/8). Forty-three percent of patients with preoperative oculomotor nerve deficit were asymptomatic at the end of follow up (n = 3/7), while 57% of patients (n = 4/7) retained an oculomotor deficit (CN VI: n = 2, CN IV: n = 1and CN III: n = 1). Forty-three percent of patients with preoperative oculomotor nerve deficit were asymptomatic at the end of follow up (n = 3/7), while 57% of patients (n = 4/7) retained an oculomotor deficit (CN VI: n = 2, CN IV: n = 1and CN III: n = 1). Prediction of preoperative CN deficit improvement at last follow-up consultation Eighty-eight percent (n = 7/8) of patients who received GTR experienced an improvement in their initial CN deficit, compared to 53% (n = 18/34) for STR and 69% (n = 9/13) for PR (p = 0.196). Patients who experienced postoperative improvement of their CN deficit harbored smaller tumor volumes than those who remained stable over time (median 40mm (range 17-72) vs median 45mm (range 24-82); p = 0,127). There was no significant association between the surgical approach or the EC location and the improvement of preoperative deficit (respectively 67% of improvement for TP vs 64% for RS approach (p = 1.00); and 65% of improvement for CPA vs 50% for PCA location (p = 0.369)). 2. New postoperative deficit 2. New postoperative deficit Tumor control No patient was lost to follow-up in the first one-year postoperative period, except for deceased patient. The median clinical and radiological follow-up was 46 months (0-409). Twenty-six patients (46 %) showed evidence of tumor progression or recurrence during the follow-up period, after a median duration of 63 months (7-210). Fifty percent (n=13) of the patients presenting with EC progression underwent a second surgery. Re-operated patients were symptomatic in 85% of cases before second surgery (n = 11/13). The remaining two patients were operated on again because of the rapidity of the cyst expansion. The median delay between the recurrence and the new surgery was 5 months (0-41) (Figure 5). RS approach and smaller tumor size tended to be associated with less recurrences (p = 0.672 and 0.245 respectively, see Table 5). Postoperative residual volume was not associated with tumor progression or recurrence: 50% of GTR patients experienced tumor regrowth, as well as 50% of STR patients and 31% of PR patients (p = 0.394). Similarly, CPA or PCA location of the EC did not influence the recurrence rate (p = 1). Prediction of early postoperative CN deficit In the univariate analysis, patients who underwent a TP approach had an increased risk of new postoperative deficit compared to the ones treated with a RS approach (100% vs 42%, p = 0.020). All of them were oculomotor deficit, including 2 CN III deficits (1 transitory deficit and 1 persistent at discharge), 5 CN IV deficits (4 transitory deficits, and only one persistent at discharge), and 2 CN VI transitory deficits. When a RS approach was performed, the new postoperative oculomotor rate was only 22% versus 100% after a TP approach (p = 0.001). After Bonferroni correction, significant differences (adjusted p value <0.004) only existed between TP approach and oculomotor rate. Tumor size, location and extent of resection were not statistically associated with de novo early postoperative CN deficits (Table 5). Functional status At last follow-up examination, 49 patients (88%) were fully independent (WHO PS 0 and 1) compared to 45 patients (80%) in the postoperative period. The number of ambulatory patients unable to work (WHO PS at 2) decreased over time, with 3 patients (5%) at the end of follow-up compared to 8 patients (14%) in the early postoperative period. Only 2 patients displayed poor general condition (WHO PS 3 and 4) at the end of follow-up (4%). Finally, two patients died during follow-up: one in the postoperative period as described previously, and the other after 45 days of intensive care for postoperative cerebral ischemia. To note, the second comatose patient survived and was ambulatory at the end of follow up (WHO PS at 2). Course of new postoperative CN deficit (Table 4) Course of new postoperative CN deficit (Table 4) New neurological CN deficits occurred during the immediate postoperative course in 27 (48%) patients (Figure 3B). Among the patients with oculomotor CN, vestibular CN and LCN postoperative disorders, only 2 still experienced an oculomotor deficit at the end of follow-up, and none retained LCN or vestibular CN impairment anymore. In contrast, only 14% of patients with postoperative cochlear deficit improved at the end of follow-up (n = 1/7). To resume, 44 patients were free of cochlear deficits at the last follow up examination, including 9 preoperative cochlear deficit and the new postoperative deficit that improved. Page 7/26 CN preoperative deficit We report here the very good evolution of preoperative CN disorders after surgery, with an improvement of 72% of preexisting deficits, regardless of the approach or the location (PCA or CPA) of the cyst. Both the inflammatory effect induced by the cystic content and the direct mechanical compression over the cisternal segment of the CN by the tumor, may explain preoperative CN disorders favorable outcome after surgery, even with small volume cysts. Contrary to schwannoma or meningioma cases wherein the CN are shift by the boundaries of the tumor capsule, EC invade the cisternal space by adapting their shape to the local morphology, encompassing nerves and vessels. Thus, surgery might improve symptoms by reducing cystic content and inflammatory process over the nerve in addition to reducing the mass effect. In this way, GTR has a greater impact than STR and PR on the improvement of pre-operative CN deficits in our series (88% of improvement VS 53% and 69%, p=0.196): maximal resection of cystic content and capsule fragments results in a higher reduction of local irritation. The effects of local irritation by the cholesterol seeping through the cyst wall have already been reported in previous study, in cases of hyperactive dysfunction such as trigeminal neuralgia or hemifacial spasm. [5, 9] Vascular compression of the nerve, either by a displaced artery or by nerve displacement toward the artery by the tumor has also been relieved.[5, 13] The beneficial effects of surgery on these preoperative CN deficits had also been demonstrated in several series. Of the 17 CPA EC reported by Czernicky et al, 11 patients experienced improvement or resolution of their preoperative deficits, in particular with trigeminal neuralgia, LCN deficits and facial nerve deficits.[4] In their cohort of 37 EC patients, Gopalakrishnan et al demonstrated a significant improvement in trigeminal and lower cranial nerve dysfunction after surgery, and half of the CN VIII and oculomotor deficits [7]. Of the 21 patients with preoperative CN dysfunction reviewed by Schiefer and Link, 33% were resolved (n = 7) after the surgery and 43% were improved (n = 9).[20] In Vernon et al series of 139 patients, 74% of them improved compared with their preoperative clinical status. Prior to our series, none of these studies attempted to analyze the association between these CN improvements and surgical approach or EOR. Discussion The kinetics of postoperative recovery of CN disorders is an original aspect of this work, which demonstrates the favorable effect of surgery on the functional outcome of CN function, with a low rate of Page 8/26 Page 8/26 ong-term morbidity since postoperative CN deficits were mostly transient. This study is one of the largest surgical series of CPA-PCA EC published so far, with a total of 56 patients. The literature dealing with the same topic is scarce and only a few retrospective cohort studies include more than 30 patients.[5, 7, 12, 13, 19, 23, 24] This study is one of the largest surgical series of CPA-PCA EC published so far, with a total of 56 patients. The literature dealing with the same topic is scarce and only a few retrospective cohort studies include more than 30 patients.[5, 7, 12, 13, 19, 23, 24] CN preoperative deficit Of note, none of them included PCA location of the cyst, and the TP approach was not used. Postoperative new CN deficit New CN deficits occurred during the immediate postoperative course in almost 50% of our patients. Most of them, apart from cochlear impairment, tended to resolve during the follow-up period. The unique cisternal cytoarchitecture of the VIII CN (i.e. centrally myelinated) could explain its higher surgical Page 9/26 Page 9/26 vulnerability in comparison to the other CN. Moreover, postoperative CN VIII impairment is related to direct nerve dissection or vasculature damage during surgery because of the adhesion between the lesion and the nerve and is therefore less likely to recover.[8] TP approach tended to increase postoperative impairment, especially oculomotor deficits (p = 0.001). However, most of them were transient and TP approach remains advantageous for some PCA locations, particularly when the cyst crosses the midline or straddles the basilar artery. Thus, this approach must remain systematically considered to ensure the best surgical exposure. The high rate of postoperative CN injury, resulting from the adhesion of the tumor capsule to the nerve, is a well-known complication of this surgery.[12] Very few studies have detailed the evolution of CN deficit over time.[4, 5, 7, 12, 13, 20, 23]. Vernon et al reported 41% of new postoperative deficits, which resolved completely on long-term follow-up in 21% patients, improved significantly in 10% patients, and remained at an unchanged level in 9% patients. Czernicki et al provided comparable results in term of frequency and evolution of CN deficit, with 58% of new postoperative CN injury (n = 10) and persistent cochlear deficits during follow-up. Postoperative new CN deficit Similar to our series, the EOR had no effect on the occurrence of new postoperative deficits.[4] In Gopalakrishnan series of CPA EC, only 13% (n = 5) of the 38 new postoperative deficits persisted at long term follow-up.[7] They reported a higher incidence of new neurological deficits in patients undergoing total removal compared to subtotal removal, but at the same time, the former group experienced a better improvement in preoperative neurological deficits compared to the latter one.[7] Two series have reported a lower rate of postoperative deficit.[5, 12] The cohort of 30 patients undergoing retrosigmoid surgery associated with whole course neuroendoscopy of Hu et al experienced only 7% of new deficits.[12] Finally, two papers have reported a higher rate of new postoperative deficit, but with a very good improvement over time.[13, 20] Despite these differences in term of postoperative CN deficit, our cohort covered a larger group of accurately and sequentially monitored patients than previously reported, which was the main objective of this work, and strengthen our findings. General Condition Long term general condition was good to excellent for most of patients. Only 2 patients presented a WHO PS score at 3 or 4 (capable of limited self-care or completely disabled) at the end of follow-up. In the literature, two other series have recorded long-term general condition, and their results were similar.[4, 20] Eighty-eight percent of patients were able to carry out all usual activities (modified Rankin score of 0 or 1) in Czernicki‘s cohort, and 84% for Schiefer’s cohort. These results are linked to a cautious surgical strategy, avoiding maximalist resection of the fragments adhering to the brain stem, vessels or nerves. The fact that EC are mostly managed in young and healthy patients favorably outweigh the outcome. Tumor control and onco-functional balance Twenty-six patients (46 %) showed evidence of tumor progression during the follow-up period, after a median duration of 63 months. The factors that might predict a stable behavior instead of a keep Page 10/26 Page 10/26 growing ones couldn’t be unveiled by our study. Based on our experience, patients with postoperative residual lesions had no increased risk of progression or recurrence during follow-up (p = 0.394). This result could be due to many biases inherent to the design of the study, and to the short follow-up of only 46 months. In our work, GTR rate (14%) is lower and recurrence rate (46%) higher than those previously reported for posterior fossa EC: Farhoud et al (32 patients, 59% GTR rate, 0 recurrence), Samii et al (40 patients, 75% GTR rate, 8% recurrence rate), Kobata et al (30 patients, 57% GTR rate, 7% recurrence rate), Vernon et al (139 patients, 73% GTR rate, 8 % recurrence rate), Yawn et al (47 patients, 46% GTR rate, 8% recurrence rate) or Schiefer et al (24 patients, 54% GTR rate, 25% recurrence rate) for instance.[5, 13, 19, 20, 23, 24] Recurrence rate in Gopalakrishnan et al series was higher after long term follow up : 45% of patients showed evidence of tumor recurrence after a mean duration of 9.3 year.[7] Indeed, our definitions of GTR and recurrence were strict, and non-total resections and recurrence rate could have been overestimated. Postoperative tumor control results are heterogeneous in the literature. Like our study, some reports found no difference in tumor progression after complete or incomplete resection, even after longer follow-up periods (respectively 11,5 years and 4,3 years of follow up).[13, 20] In contrast, some authors found a higher rate of recurrence after non-total resection [7, 19, 22]. A recent large meta-analysis including 691 patients with intracranial epidermoid tumors found that STR was associated with a 7-times higher rate of regrowth than tumors that underwent GTR.[22] However, the analyses were not stratified according to the intracranial location of the cysts, such as infratentorial sites. Moreover, capsule adherence to the neurovascular structures in PCA and CPA locations makes total removal extremely challenging. Additionally, the propensity of EC for regional spreading toward neighbor cisterns hampers the ability to expose the full volume of the cyst “behind the corner” using regular approaches. Tumor control and onco-functional balance The use of angled endoscopes (endoscopic assisted microsurgery) could be of help to check for fragments that could be overlooked under microscope, as suggested above.[10, 12] These findings confirm that altogether, operative findings and high field MR doesn’t reach the level of sensibility to insure the cure of the disease. EC grow linearly, not exponentially,[2] and the overlooked micro-fragments of cyst walls have the propensity to regrowth which is empirically known.[4, 5, 9, 12, 14, 20, 21] Our results (see Kaplan Meier plot, Figure 5A) underline that recurrence or regrowth might be expected in the ultra-late period, regardless the EOR of the cyst. Patients should be aware and a clinic-radiological sequential follow-up must be planned in long term. Only half of the growing residual tumors justified additional surgery (see Kaplan Meier plot, Figure 5B). The expectation for clinical symptomatology in 85% of reoperated patients can explain the half-year delay before considering surgery. Weaknesses Page 11/26 This is a retrospective study, and so there may be missing data in our work. Also, the center effect has not been tested. Multivariate analysis and cox-regression analysis was not performed, because not appropriate to this limited patient sample. We deliberately merged the findings of PCA and CPA EC in our Page 11/26 Page 11/26 work; these locations are not supposed to carry the same risks when surgically approached. However, testing this variable did not impact the functional nor the oncological results. Finally, it would have been interesting to obtain precise volumes and growth rates of the preoperative and residual EC. DWI sequence is essential for the diagnosis of EC and differentiation from other lesions, as the content of the epidermoid cysts shows prominent diffusion restriction due to layered microstructure of the debris [4]. However, this sequence is mediocre in term of anatomical resolution, and did not allow precise calculation of cyst size. The interest of new sequences coupled to the DWI in order to increase the three-dimensional resolution while keeping an important sensitivity and specificity for the diagnosis of EC is an axis to develop in the future. Last, the length of follow-up did not reach the long-term which weaken the analysis of tumor growth potential over time. Conclusion This multicenter retrospective cohort study of large posterior fossa EC demonstrates the favorable impact of a functional sparing surgery policy over preexisting CN deficits. In addition, the high rate of early new postoperative CN deficits observed in this study underlines the complexity of such surgery. Most of these disturbances recovered, except for the cochlear nerve. The counterpart of this surgical strategy was the evidence of tumor remnant or recurrence in half of the study group, which was demonstrated by a regular sequential MR follow-up protocol. The linear progression of EC and the need of additional surgery in half of recurrent patients supports the need for a lifetime DWI MR surveillance, which need to be early mentioned to the patient. Abbreviations Page 12/26 Abbreviations BNI = Barrow Neurological Institute CPA = Cerebellopontine angle CN = Cranial nerve(s) DWI = Diffusion-weighted magnetic resonance imaging EC = Epidermoid cyst EOR = Extent of resection GTR = Gross total resection HB = House and Brackmann LCN = Lower cranial nerves BNI = Barrow Neurological Institute BNI = Barrow Neurological Institute CN = Cranial nerve(s) Page 12/26 Page 12/26 Authors' contributions: Authors' contributions: Author contributions to the study and manuscript preparation include the following: Conception and design: Roche. Acquisition of data: Sellier, Baumgarten, Caudron, Bretonnier, Gallet, Boissonneau. Analysis and interpretation of data: all authors. Drafting the article: Sellier, Troude and Roche. Critically revising the article: all authors. Reviewed submitted version of manuscript: all authors. Approved the final version of the manuscript on behalf of all authors: Roche. Study supervision: Roche. Code availability: Not applicable Ethics approval: This study has been approved by the French Neurosurgical College Institutional Review Board (reference: IRB00011687 College de neurochirurgie IRB #1: 2020/04). Ethics approval: This study has been approved by the French Neurosurgical College Institutional Review Board (reference: IRB00011687 College de neurochirurgie IRB #1: 2020/04). Consent to participate: No informed consent was required, as this is a retrospective analysis without any traceable patient data. Consent to participate: No informed consent was required, as this is a retrospective analysis without any traceable patient data. Consent for publication: Not applicable. Authors' contributions: Declarations Conflicts of interest/Competing interests: The authors report no conflict of interest. Conflicts of interest/Competing interests: The authors report no conflict of interest. Availability of data and material: Data will be available upon reasonable request from a qualified investigator. Availability of data and material: Data will be available upon reasonable request from a qualified investigator. References Page 13/26 Page 13/26 1. Aboud E, Abolfotoh M, Pravdenkova S, Gokoglu A, Gokden M, Al-Mefty O (2015) Giant intracranial epidermoids: is total removal feasible? J Neurosurg 122:743–756. doi: 10.3171/2014.11.JNS1481 2. Alvord EC (1977) Growth rates of epidermoid tumors. Ann Neurol 2:367–370. doi: 10.1002/ana.410020504 3. Berger MS, Wilson CB (1985) Epidermoid cysts of the posterior fossa. J Neurosurg 62:214–219. doi: 10.3171/jns.1985.62.2.0214 4. Czernicki T, Kunert P, Nowak A, Wojciechowski J, Marchel A (2016) Epidermoid cysts of the cerebellopontine angle: Clinical features and treatment outcomes. Neurol Neurochir Pol 50:75–82. doi: 10.1016/j.pjnns.2015.11.008 5. Farhoud A, Khedr W, Aboul-Enein H (2018) Surgical Resection of Cerebellopontine Epidermoid Cysts: Limitations and Outcome. J Neurol Surg Part B Skull Base 79:167–172. doi: 10.1055/s-0037- 1606220 6. Forbes JA, Banu M, Lehner K, Ottenhausen M, La Corte E, Alalade AF, Ordóñez-Rubiano EG, Greenfield JP, Anand VK, Schwartz TH (2018) Endoscopic endonasal resection of epidermoid cysts involving the ventral cranial base. J Neurosurg 1–10. doi: 10.3171/2017.12.JNS172575 7. Gopalakrishnan CV, Ansari KA, Nair S, Menon G (2014) Long term outcome in surgically treated posterior fossa epidermoids. Clin Neurol Neurosurg 117:93–99. doi: 10.1016/j.clineuro.2013.11.025 8. Guclu B, Meyronet D, Simon É, Streichenberger N, Sindou M, Mertens P (2009) Anatomie structurelle des nerfs crâniens (V, VII, VIII, IX, X). Neurochirurgie 55:92–98. doi: 10.1016/j.neuchi.2009.02.001 9. Hasegawa M, Nouri M, Nagahisa S, Yoshida K, Adachi K, Inamasu J, Hirose Y, Fujisawa H (2016) Cerebellopontine angle epidermoid cysts: clinical presentations and surgical outcome. Neurosurg Rev 39:259–267. doi: 10.1007/s10143-015-0684-5 9. Hasegawa M, Nouri M, Nagahisa S, Yoshida K, Adachi K, Inamasu J, Hirose Y, Fujisawa H (2016) Cerebellopontine angle epidermoid cysts: clinical presentations and surgical outcome. Neurosurg Rev 39:259–267. doi: 10.1007/s10143-015-0684-5 10. Hitti FL, Lee JYK (2019) Endoscopic Resection of a Cerebellopontine Angle Epidermoid Cyst via a Retrosigmoid Approach. J Neurol Surg Part B Skull Base 80:S330. doi: 10.1055/s-0039-1677852 10. Hitti FL, Lee JYK (2019) Endoscopic Resection of a Cerebellopontine Angle Epidermoid Cyst via a Retrosigmoid Approach. J Neurol Surg Part B Skull Base 80:S330. doi: 10.1055/s-0039-1677852 11. House JW, Brackmann DE (1985) Facial nerve grading system. Otolaryngol--Head Neck Surg Off J Am Acad Otolaryngol-Head Neck Surg 93:146–147. doi: 10.1177/019459988509300202 11. House JW, Brackmann DE (1985) Facial nerve grading system. Otolaryngol--Head Neck Surg Off J Am Acad Otolaryngol-Head Neck Surg 93:146–147. doi: 10.1177/019459988509300202 12. References doi: 10.3171/jns.1996.84.1.0014 20. Schiefer TK, Link MJ (2008) Epidermoids of the cerebellopontine angle: a 20-year experience. Surg Neurol 70:584–590. doi: 10.1016/j.surneu.2007.12.021 20. Schiefer TK, Link MJ (2008) Epidermoids of the cerebellopontine angle: a 20-year experience. Surg Neurol 70:584–590. doi: 10.1016/j.surneu.2007.12.021 21. Schroeder HWS, Oertel J, Gaab MR (2004) Endoscope-assisted microsurgical resection of epidermoid tumors of the cerebellopontine angle. J Neurosurg 101:227–232. doi: 10.3171/jns.2004.101.2.0227 21. Schroeder HWS, Oertel J, Gaab MR (2004) Endoscope-assisted microsurgical resection of epidermoid tumors of the cerebellopontine angle. J Neurosurg 101:227–232. doi: 10.3171/jns.2004.101.2.0227 22. Shear BM, Jin L, Zhang Y, David WB, Fomchenko EI, Erson-Omay EZ, Huttner A, Fulbright RK, Moliterno J (2020) Extent of resection of epidermoid tumors and risk of recurrence: case report and meta-analysis. J Neurosurg 133:291–301. doi: 10.3171/2019.4.JNS19598 22. Shear BM, Jin L, Zhang Y, David WB, Fomchenko EI, Erson-Omay EZ, Huttner A, Fulbright RK, Moliterno J (2020) Extent of resection of epidermoid tumors and risk of recurrence: case report and meta-analysis. J Neurosurg 133:291–301. doi: 10.3171/2019.4.JNS19598 23. Vernon V, Naik H, Guha A Surgical management of cerebellopontine angle epidermoid cysts: an institutional experience of 10 ye. 11 24. Yawn RJ, Patel NS, Driscoll CL, Link MJ, Haynes DS, Wanna GB, Thompson RC, Carlson ML (2016) Primary Epidermoid Tumors of the Cerebellopontine Angle: A Review of 47 Cases. Otol Neurotol Off Publ Am Otol Soc Am Neurotol Soc Eur Acad Otol Neurotol 37:951–955. doi: 10.1097/MAO.0000000000001085 References Hu Z, Guan F, Kang T, Huang H, Dai B, Zhu G, Mao B, Kang Z (2015) Whole Course Neuroendoscopic Resection of Cerebellopontine Angle Epidermoid Cysts. J Neurol Surg Part Cent Eur Neurosurg 77:381–388. doi: 10.1055/s-0035-1558818 12. Hu Z, Guan F, Kang T, Huang H, Dai B, Zhu G, Mao B, Kang Z (2015) Whole Course Neuroendoscopic Resection of Cerebellopontine Angle Epidermoid Cysts. J Neurol Surg Part Cent Eur Neurosurg 77:381–388. doi: 10.1055/s-0035-1558818 13. Kobata H, Kondo A, Iwasaki K (2002) Cerebellopontine angle epidermoids presenting with cranial nerve hyperactive dysfunction: pathogenesis and long-term surgical results in 30 patients. Neurosurgery 50:276–286 13. Kobata H, Kondo A, Iwasaki K (2002) Cerebellopontine angle epidermoids presenting with cranial nerve hyperactive dysfunction: pathogenesis and long-term surgical results in 30 patients. Neurosurgery 50:276–286 14. Miller ME, Mastrodimos B, Cueva RA (2012) Hearing preservation in management of epidermoids of the cerebellopontine angle: CPA epidermoids and hearing preservation. Otol Neurotol 33:1599–1603 14. Miller ME, Mastrodimos B, Cueva RA (2012) Hearing preservation in management of epidermoids of the cerebellopontine angle: CPA epidermoids and hearing preservation. Otol Neurotol 33:1599–1603 14. Miller ME, Mastrodimos B, Cueva RA (2012) Hearing preservation in management of epidermoids of the cerebellopontine angle: CPA epidermoids and hearing preservation. Otol Neurotol 33:1599–1603 15. de Morais MV, Mota RA de AA, Marques TA, Loduca RD de S, Melo PM de P (2019) Epidermoid Cyst in the Cerebellopontine Angle: Technical Description Video. J Neurol Surg Part B Skull Base 80:S325–S326. doi: 10.1055/s-0038-1676997 15. de Morais MV, Mota RA de AA, Marques TA, Loduca RD de S, Melo PM de P (2019) Epidermoid Cyst in the Cerebellopontine Angle: Technical Description Video. J Neurol Surg Part B Skull Base 80:S325–S326. doi: 10.1055/s-0038-1676997 Page 14/26 16. Oken M, Creech R, Tormey D, Horton J, Davis T, McFadden E, Carbone P (1982) Toxicity and response criteria of the Eastern Cooperative Oncology Group. Am J Clin Oncol 5:649–656 17. Pojskić M, Arnautović KI (2019) Microsurgical Resection of the Epidermoid Tumor in the Cerebellopontine Angle. J Neurol Surg Part B Skull Base 80:S327–S328. doi: 10.1055/s-0038- 1677499 18. Rogers CL, Shetter AG, Fiedler JA, Smith KA, Han PP, Speiser BL (2000) Gamma knife radiosurgery for trigeminal neuralgia: the initial experience of the Barrow Neurological Institute. Int J Radiat Oncol 47:1013–1019. doi: 10.1016/S0360-3016(00)00513-7 19. Samii M, Tatagiba M, Piquer J, Carvalho GA (1996) Surgical treatment of epidermoid cysts of the cerebellopontine angle. J Neurosurg 84:14–19. Complications are considered postoperative when occurring within 30 days after surgery. RS = Retrosigmoid Tables Table 1. Clinical data and tumor characteristics Page 15/26 Page 15/26 Number of patients (%) – CI95% Characteristics Number of patients (%) – CI95% Initial signs and symptoms Facial weakness 7 (12.5%) CI95% (5.4 ; 21.4) Hemifacial spasm 4 (7.1 %) CI95% (1.8 ; 14.3) Facial hypoesthesia 15 (26.8) CI95%(15.8 ; 40.3) Trigeminal neuralgia 15 (26.8) CI95%(15.8 ; 40.3) Hearing impairment 15 (26.8) CI95%(15.8 ; 40.3) Diplopia 8 (14.3) CI95%(5.4 ; 25) Tinnitus 9 (16.1) CI95%(7.1 ; 26.8) Vertigo 15 (26.8) CI95%(15.8 ; 40.3) Gait disturbance 13 (23.2) CI95%(12.5 ; 33.9) Axial sign 12 (21.4) CI95%(10.7 ; 32.1) Intracranial hypertension (ICH) 8 (14.3) CI95%(5.4 ; 23.2) Seizures 3 (5.4) CI95%(0 ; 12.5) Dysphagia/ Phonation Disorders 9 (16.1) CI95%(7.1 ; 26.8) Main location of the lesion CPA PCA 16 (28.6) CI95%(17.9 ; 41.1) 40 (71.4) CI95%(58.9 ; 82.1) Accessory location Meckel Foramen Magnum Supratentorial Suprasellar 11 (19.6) CI95%(10.7 ; 30.4) 2 (3.6) CI95%(0 ; 8.9) 12 (21.4) CI95%(10.7 ; 32.1) 3 (5.4) CI95%(0 ; 12.5) Side Left Right 27 (48.2) CI95%(35.7 ; 60.7) 29 (51.8) CI95%(39,3 ; 64.3) Largest diameter (mm) 44 (IQR 30 – 50, range 17-82) Hydrocephalus 5 (8.9) CI95%(1.8 ; 16.1) Preoperative exploration by DWI sequence 39 (69.6) CI95%(57.1; 80.4) ival area cal and postoperative data Characteristics Number of patients (%) – CI95% Surgical Approach RS TP Pterional Subtemporal Suboccipital median 36 (64.3) - CI95%(51.8 ; 76.8) 6 (10.7) - CI95%(3.6 ; 19.6) 8 (14.3) CI95%(5.4 ; 23.2) 4 (7.1) CI95%(1.8 ; 14.3) 1 (1.8) - CI95%(0 ; 5.4) Extent of resection GTR STR PR 8 (14.3) CI95%(5.4 ; 23.2) 34 (60.7) CI95%(48.2 ; 73.2) 13 (23.2) CI95%(12.5 ; 35.7) Postoperative complications Aseptic Meningitis 7 (12.5) - CI95%(5.4 ; 21.4) Hydrocephalus 4 (7.1) - CI95%(1.8 ; 14.3) Cerebral Ischemia 3 (5.4) - CI95%(0 ; 12.5) Infield hematoma 3 (5.4) - CI95%(0 ; 12.5) Septic meningitis 3 (5.4) - CI95%(0 ; 125) CSF leakage 3 (5.4) - CI95%(0 ; 12.5) Coma/ Intensive Care Management 2 (3.6) - CI95%(0 ; 8.9) Bed rest complication 2 (3.6) - CI95%(0 ; 8.9) Scar disorder 2 (3.6) - CI95%(0 ; 8.9) Keratitis/ corneal ulceration 1 (1.8) - CI95%(0 ; 5.4) Death 1 (1.8) - CI95%(0 ; 5.4) ns are considered postoperative when occurring within 30 days after surgery. id Table 2. Surgical and postoperative data Number of patients (%) – CI95% Page 17/26 TP = Transpetrosal GTR = Gross Total Resection STR = Subtotal Resection PR = Partial Resection CSF = Cerebro-spinal fluid Table 3. Preoperative CN deficits evolution after the surgery CN Deficit Preoperatively, n (%) Immediate postoperative care, n (%) 6 Months postoperative, n (%) 1 Year postoperative, n (%) Last Follow-Up, n (%) Oculomotor 7(12.5%) 6(10.7%) 5(8.9%) 4 (7.1%) 4(7.1%) Trigeminal Neuralgia BNI2 8(14.3%) 5(8.9%) 4(7.1%) 2(3.6%) 2(3.6%) BNI3 4(7.1%) 5(8.9%) 4(7.1%) 1(1.8%) 1(1.8%) BNI4 3(5.4%) 0 0 2(3.6%) 2(3.6%) Total 15(26.8%) 10(17.9%) 8(14.3%) 5(8.9%) 5(8.9%) Hypoesthesia 15 (26.8%) 15 (26.8%) 9 (16.1%) 7 (12.5%) 7 (12.5%) Facial H&B II 6(10.7%) 4(7.1%) 2(3.6%) 2(3.6%) 2(3.6%) H&B III 1(1.8%) 1(1.8%) 1(1.8%) 1(1.8%) 1(1.8%) H&B IV-VI 1(1.8%) 1(1.8%) 1(1.8%) 0 0 Total 8(14.2%) 6(10.7%) 4(7.1%) 3(5.4%) 3(5.4%) Cochlear Hearing loss Deafness 15 (26.8%) 0 9 (16.1 %) 2 (3.6%) 6 (10.7 %) 2 (3.6%) 5 (8.9 %) 2 (3.6%) 4 (7.1%) 2 (3.6%) Vestibular 15 (26.8%) 9 (16.1%) 6 (10.7%) 6 (10.7%) 6 (10.7%) BNI =Barrow Neurological Institute Classification Table 4. New CN postoperative deficits and their evolution p p CN Deficit Preoperatively, n (%) Immediate postoperative care, n (%) 6 Months postoperative, n (%) 1 Year postoperative, n (%) Last Follow-Up, n (%) Oculomotor 18 (32.1%) 6 (10.7%) 2(3.6%) 2 (3.6%) CN III NA 7 (13.2%) 1 (1.9%) 1 (1.9%) 2 (3.8%) CN IV NA 10 (18.9%) 4 (7.5%) 1 (1.9%) 1 (1.9%) CN VI NA 6 (11.8%) 2 (3.9%) 0 0 Trigeminal Neuralgia NA 0 0 0 0 Hypoesthesia NA 4(7.1%) 1(1.8%) 1(1.8%) 1(18%) Facial NA 2(3.6%) 1(1.8%) 0 0 Cochlear Hearing loss NA 4 (7.2 %) 4 (7.2 %) 4 (7.2 %) 3 (5.4%) Deafness NA 3 (5.4%) 3 (5.4%) 3 (5.4%) 3 (5.4%) Total NA 7(12.5%) 7(12.5%) 7(12.5%) 6(10.7%) Vestibular NA 2(3.6%) 1(1.8%) 0 0 LCN NA 6(10.7%) 1(1.8%) 1(1.8%) 0 BNI =Barrow Neurological Institute Classification BNI =Barrow Neurological Institute Classification LCN = Lower Cranial Nerves NA = not applicable LCN = Lower Cranial Nerves Table 5. Predictive factors * Univariate analyses: Pearson’s chi-squared test, Fisher’s exact test or Mann Whitney U test, as appropriate * Univariate analyses: Pearson’s chi-squared test, Fisher’s exact test or Mann Whitney U test, as appropriate Page 19/26 Predictive factors New postoperative deficit Progression of the lesion Absence n = 29 Presence n = 27 P- value* No n = 30 Yes n = 26 P- value* Surgical approach 0.020 TP 0 6 3 3 0.672 RS 21 15 22 14 Extent of resection 0.681 GTR 5 3 4 4 0.394 STR 17 17 17 17 PR 7 6 9 4 Location CPA PCA 21 8 19 8 1 21 9 19 7 1 Largest diameter (median, range) 42 (19- 72) 45 (17- 82) 0.478 45 (19- 72) 34 (17- 82) 0.245 RS = Retrosigmoid TP = Transpetrosal GTR = Gross Total Resection STR = Subtotal Resection PR = Partial Resection CPA = Cerebellopontine Angle PCA = Petroclival area Figures Predictive factors New postoperative deficit Progression of the lesion Absence n = 29 Presence n = 27 P- value* No n = 30 Yes n = 26 P- value* Surgical approach 0.020 TP 0 6 3 3 0.672 RS 21 15 22 14 Extent of resection 0.681 GTR 5 3 4 4 0.394 STR 17 17 17 17 PR 7 6 9 4 Location CPA PCA 21 8 19 8 1 21 9 19 7 1 Largest diameter (median, range) 42 (19- 72) 45 (17- 82) 0.478 45 (19- 72) 34 (17- 82) 0.245 RS = Retrosigmoid Figures Page 20/26 Page 21/26 Figure 1 Illustrative case of a cerebello-pontine angle epidermoid cyst operated on through a retrosigmoid approach A & B: The preoperative DWI Diffusion and T2-weighted MRI showed a left cerebello- pontineIllustrative case of a cerebello-pontine angle epidermoid cyst operated on through a retrosigmo approach A & B: The preoperative DWI Diffusion and T2-weighted MRI showed a left cerebello-pontine angle epidermoid cyst extended to the petroclival region in a 30 years-old woman. The tumor was 21 26 Illustrative case of a cerebello-pontine angle epidermoid cyst operated on through a retrosigmoid approach A & B: The preoperative DWI Diffusion and T2-weighted MRI showed a left cerebello- pontineIllustrative case of a cerebello-pontine angle epidermoid cyst operated on through a retrosigmoid approach A & B: The preoperative DWI Diffusion and T2-weighted MRI showed a left cerebello-pontine angle epidermoid cyst extended to the petroclival region in a 30 years-old woman. The tumor was Page 21/26 Page 21/26 incidentally detected in imaging studies after a traumatic brain injury. The patient was free from any symptom before surgery. C: Peroperative view. The tumor was resected through the corridor between the acoustico-facial bundle and the lower CNs. The cranial nerves are exposed. D & E: No CN disturbances occurred in the postoperative period. The DWI Diffusion and T2-weighted MR performed 3 months after surgery did not reveal any tumor residue, and remained stable 2 years after surgery. angle epidermoid cyst extended to the petroclival region in a 30 years-old woman. The tumor was incidentally detected in imaging studies after a traumatic brain injury. The patient was free from any symptom before surgery. C: Peroperative view. The tumor was resected through the corridor between the acoustico-facial bundle and the lower CNs. The cranial nerves are exposed. D & E: No CN disturbances occurred in the postoperative period. The DWI Diffusion and T2-weighted MR performed 3 months after surgery did not reveal any tumor residue, and remained stable 2 years after surgery. incidentally detected in imaging studies after a traumatic brain injury. The patient was free from any symptom before surgery. C: Peroperative view. The tumor was resected through the corridor between the acoustico-facial bundle and the lower CNs. The cranial nerves are exposed. D & E: No CN disturbances occurred in the postoperative period. Figures The DWI Diffusion and T2-weighted MR performed 3 months after surgery did not reveal any tumor residue, and remained stable 2 years after surgery. angle epidermoid cyst extended to the petroclival region in a 30 years-old woman. The tumor was incidentally detected in imaging studies after a traumatic brain injury. The patient was free from any symptom before surgery. C: Peroperative view. The tumor was resected through the corridor between the acoustico-facial bundle and the lower CNs. The cranial nerves are exposed. D & E: No CN disturbances occurred in the postoperative period. The DWI Diffusion and T2-weighted MR performed 3 months after surgery did not reveal any tumor residue, and remained stable 2 years after surgery. Page 22/26 P 23/26 Figure 2 Illustrative case of a petroclival epidermoid cyst operated on through a transpetrosal approach. A & B: The preoperative T2-weighted MRI showed a right petroclival epidermoid cyst (Figure 2A) in a 44 years- old man. He presented with a slight numbness of CN V2 & 3, and reported trigeminal neuralgia for 2 years. The superior pole of the tumor extended to the perimesencephalic cistern (Figure 2B). The tumor was responsible for a mass effect on the brainstem and the ipsilateral middle cerebellar peduncle. C & D: Illustrative case of a petroclival epidermoid cyst operated on through a transpetrosal approach. A & B: The preoperative T2-weighted MRI showed a right petroclival epidermoid cyst (Figure 2A) in a 44 years- old man. He presented with a slight numbness of CN V2 & 3, and reported trigeminal neuralgia for 2 years. The superior pole of the tumor extended to the perimesencephalic cistern (Figure 2B). The tumor was responsible for a mass effect on the brainstem and the ipsilateral middle cerebellar peduncle. C & D: Page 23/26 Page 23/26 Peroperative views. The operative window after epidural anterior transpetrosal approach offered an exposure from the trigeminal nerve to the basilar and the superior cerebellar artery. E & F: The patient presented postoperative transient diplopia due to CN IV disturbances, and retained long term CN V3 facial hypoesthesia. The DWI Diffusion and T2-weighted MR performed 3 months after surgery showed small residual fragments of tumor located at the level of perimesencephalic cistern. Those remnants experienced asymptomatic regrowth 5 years after surgery. A Wait-and-reScan policy was decided. Figures Figure 3 Evolution of cranial nerves deficit A: Evolution of cranial nerves preoperative deficit Line chart representing the proportion of patients with CN deficit during follow up among those with preoperative CN disorders. Only the most frequent CN deficits are illustrated: oculomotor (.), trigeminal hypoesthesia (+), trigeminal neuralgia (x) and cochlear nerve ( v ). B: Evolution of new postoperative cranial nerves deficit Line chart representing the proportion of patients with new CN deficit during follow up. Only the most frequent CN deficits are illustrated: oculomotor (.), cochlear (x) and lower cranial nerves (+). Figure 3 Evolution of cranial nerves deficit A: Evolution of cranial nerves preoperative deficit Line chart representing the proportion of patients with CN deficit during follow up among those with preoperative CN disorders. Only the most frequent CN deficits are illustrated: oculomotor (.), trigeminal hypoesthesia (+), trigeminal neuralgia (x) and cochlear nerve ( v ). B: Evolution of new postoperative cranial nerves deficit Line chart representing the proportion of patients with new CN deficit during follow up. Only the most frequent CN deficits are illustrated: oculomotor (.), cochlear (x) and lower cranial nerves (+). Page 24/26 Page 24/26 Figure 4 Evolution of preoperative trigeminal neuralgia Bar Chart illustrating the evolution of preoperative trigeminal neuralgia during follow-up, according to the Barrow Neurological Institute (BNI) Classification (from Grade 1 to Grade 5). Grade 1 defines asymptomatic patients, who are not shown is this figure. No Grade 5 patients was identified in our cohort. Figure 4 Evolution of preoperative trigeminal neuralgia Bar Chart illustrating the evolution of preoperative trigeminal neuralgia during follow-up, according to the Barrow Neurological Institute (BNI) Classification (from Grade 1 to Grade 5). Grade 1 defines asymptomatic patients, who are not shown is this figure. No Grade 5 patients was identified in our cohort. Evolution of preoperative trigeminal neuralgia Bar Chart illustrating the evolution of preoperative trigeminal neuralgia during follow-up, according to the Barrow Neurological Institute (BNI) Classification (from Grade 1 to Grade 5). Grade 1 defines asymptomatic patients, who are not shown is this figure. No Grade 5 patients was identified in our cohort. Evolution of preoperative trigeminal neuralgia Bar Chart illustrating the evolution of preoperative trigeminal neuralgia during follow-up, according to the Barrow Neurological Institute (BNI) Classification (from Grade 1 to Grade 5). Grade 1 defines asymptomatic patients, who are not shown is this figure. No Grade 5 patients was identified in our cohort. Page 25/26 Figure 5 Tumor control A: Kaplan-Meier plot illustrating progression-free survival. The shaded areas indicate 95% CI. B: Kaplan-Meier plot illustrating the delay between progression/recurrence and a second resection surgery. The shaded areas indicate 95% CI. Page 26/26
https://openalex.org/W2757772641	https://link.springer.com/content/pdf/10.1007%2Fs40820-017-0156-2.pdf	English	null	MemBrain: An Easy-to-Use Online Webserver for Transmembrane Protein Structure Prediction	Nano-micro letters	2,017	cc-by	5,465	MemBrain: An Easy-to-Use Online Webserver for Transmembrane Protein Structure Prediction Xi Yin1,2 . Jing Yang1,2 . Feng Xiao1,2 . Yang Yang3,4 . Hong-Bin Shen1,2 Received: 11 July 2017 / Accepted: 26 August 2017 / Published online: 27 September 2017 The Author(s) 2017. This article is an open access publication Xi Yin and Jing Yang have contributed equally to this study. & Hong-Bin Shen hbshen@sjtu.edu.cn 1 Institute of Image Processing and Pattern Recognition, Shanghai Jiao Tong University, Shanghai 200240, People’s Republic of China 2 Key Laboratory of System Control and Information Processing, Ministry of Education of China, Shanghai 200240, People’s Republic of China 3 Department of Computer Science, Shanghai Jiao Tong University, Shanghai 200240, People’s Republic of China 4 Key Laboratory of Shanghai Education Commission for Intelligent Interaction and Cognitive Engineering, Shanghai 200240, People’s Republic of China Nano-Micro Lett. (2018) 10:2 https://doi.org/10.1007/s40820-017-0156-2 Nano-Micro Lett. (2018) 10:2 https://doi.org/10.1007/s40820-017-0156-2 ARTICLE ARTICLE Highlights • MemBrain is a fully automatic online tool for transmembrane protein structure prediction, which is able to predict the irregular half-transmembrane helix. M B i ’ h i di i id i l d i l f f h l b i • MemBrain is a fully automatic online tool for transmembrane protein structure prediction, which is able to predict the irregular half-transmembrane helix. • MemBrain’s theoretic predictions provide timely and important clues for further wet-lab ex Abstract Membrane proteins are an important kind of proteins embedded in the membranes of cells and play crucial roles in living organisms, such as ion channels, transporters, receptors. Because it is difﬁcult to determinate the membrane protein’s structure by wet-lab experiments, accurate and fast amino acid sequence-based computa- tional methods are highly desired. In this paper, we report an online prediction tool called MemBrain, whose input is the amino acid sequence. MemBrain consists of specialized modules for predicting transmembrane helices, residue– residue contacts and relative accessible surface area of a-helical membrane proteins. MemBrain achieves a prediction accuracy of 97.9% of ATMH, 87.1% of AP, 3.2 ± 3.0 of N-score, 3.1 ± 2.8 of C-score. MemBrain- Contact obtains 62%/64.1% prediction accuracy on train- ing and independent dataset on top L/5 contact prediction, respectively. And MemBrain-Rasa achieves Pearson cor- relation coefﬁcient of 0.733 and its mean absolute error of 13.593. These prediction results provide valuable hints for revealing the structure and function of membrane proteins. MemBrain web server is free for academic use and avail- able at www.csbio.sjtu.edu.cn/bioinf/MemBrain/. Xi Yin and Jing Yang have contributed equally to this study. & Hong-Bin Shen hbshen@sjtu.edu.cn 1 Institute of Image Processing and Pattern Recognition, Shanghai Jiao Tong University, Shanghai 200240, People’s Republic of China 2 Key Laboratory of System Control and Information Processing, Ministry of Education of China, Shanghai 200240, People’s Republic of China 3 Department of Computer Science, Shanghai Jiao Tong University, Shanghai 200240, People’s Republic of China 4 Key Laboratory of Shanghai Education Commission for Intelligent Interaction and Cognitive Engineering, Shanghai 200240, People’s Republic of China Xi Yin and Jing Yang have contributed equally to this study. Keywords Transmembrane a-helices Structure prediction Machine learning Contact map prediction Relative accessible surface area 123 12 3 3 2 Page 2 of 8 Nano-Micro Lett. (2018) 10:2 portion of the membrane proteins are transmembrane pro- teins, which have one or multiple hydrophobic transmem- brane segments. 1.2 MemBrain-Contact: Residue–Residue Contact Map Prediction For the past 10 years, we are developing an online predictor named MemBrain (as shown in Fig. 2) that can predict a-helical membrane protein structure [6–8]. Cur- rently, this predictor consists of the following three func- tional modules: When two residues are close enough in the space (e.g., \8 A˚ ), they are generally acknowledged as ‘contact.’ The contact map prediction is to generate a 2D map marking the contacted residue pairs. Although the TMH structure predictions can help ﬁguring out the general structure topology of a-helical membrane protein, it is not enough to build the 3D structure of a membrane protein. The residues contact map provides spatial constraints for constructing tertiary structure models of TMH proteins, which has recently been a hot topic in protein structure prediction [12–15]. The existing methods for predicting residue– residue contacts of a-helix proteins and TMH–TMH interactions from the primary sequences can be generally divided into two categories: (1) machine learning-based methods, (2) statistical-based coevolution mining methods. Our results show that these two branches of methods highly complement each other [7]. The machine learning-based engines need the training process and highly depend on the distributions of training dataset. Hence, the prediction outputs of machine learning-based models have higher preference to match the distribution of the training set, resulting in a relatively lower generalization and coverage of the predictions. Training process is not needed in the coevolution mining methods, which align the query sequence against a large protein sequence pool to calculate 1 Introduction Signiﬁcant advancement of sequencing technologies has resulted in an explosion of protein amino acid sequences in various databases such as the UniProt (as shown in Fig. 1). However, due to the difﬁculties of wet-lab experiments, the gap between the numbers of known sequences and their corresponding experimentally solved structures keeps growing [1]. Thus, the development of the fast and accurate computational approaches for predicting structures from the amino acid sequences has attracted more and more attention. Membrane proteins constitute approximately 30% of the proteins in both prokaryotic and eukaryotic genomes [2], due to the crucial functions of them, and more than 60% current drug targets are membrane proteins [3]. The 3D structures of membrane proteins will provide important insights for membrane protein-orientated drug design. For instance, the binding mechanisms of membrane protein-drug ligand can be modeled with the 3D structures. However, solving membrane protein structures through the wet-lab experiments is extremely difﬁcult. The reason is that membrane proteins usually have one or more trans- membrane segments, which are very hydrophobic making the chances for crystallization of membrane proteins small [4, 5]. In such a case, computational bioinformatics algo- rithms are highly desired, which will provide fast and accurate membrane protein structure predictions. Highlights Transmembrane proteins have two types: a-helical and b-barrels proteins. The former proteins are the major membrane proteins and the latter one only account for 30% in membrane proteins. We also devel- oped a method for predicting spanning segments for b- barrels [9]. One of the important steps for the membrane protein structure prediction is to identify the transmem- brane segments from the amino acid sequence, e.g., TMH. The initial methods of TMH structure prediction employed the amino acid hydrophobicity analysis; later, beneﬁtting from the rapid expansion of structural database, machine learning methods have been widely applied to automati- cally learn the rules for classifying the TMH residues from the solved structures (training samples). Such TMH topology structure predictors include HMM-based approach like TMHMM [10], SVM-based methods like SVMtm [11], the OET-KNN-based MemBrain [6], etc. The prediction of irregular half TMHs is a challenging topic in the transmembrane TMH predictions. In our MemBrain- TMH model, the multi-scale modeling and dynamic threshold approach are incorporated to improve its pre- diction performance. 2 MemBrain Prediction Functions In a 3D structure, some residues are buried into the internal core making them hard to be reached by other ligands. The relative solvent accessibility is a quantitative measurement of the visibility of the residues in a structure. Although many computational methods have been developed to predict the residues’ Rasa in soluble proteins [16, 17], Fig. 2 A screenshot of the submission interface of MemBrain web server (www.csbio.sjtu.edu.cn/bioinf/MemBrain/) Fig. 2 A screenshot of the submission interface of MemBrain web server (www.csbio.sjtu.edu.cn/bioinf/MemBrain/) relatively few approaches are available for the membrane proteins. The reason is that the solved membrane protein structures are much fewer than the soluble proteins, making the training samples difﬁcult to collect. The module of MemBrain-Rasa software is a combination of machine learning-based engine and the segment template-based module, which can solve the prediction preference problem caused by the pure machine learning-based model. the residue pair potential coevolution score. And because such statistical approaches are unsupervised methods, they will have predictions of wider coverage, but with higher false positives at the same time. Our MemBrain model is a consensus predictor of the two branches of engines, so its prediction accuracy is higher than a single independent model. 1.1 MemBrain-TMH: Transmembrane a-Helical Segment (TMH) Prediction A TMH is a segment of residues along the sequence which spans the membrane. The prediction of TMHs is labeling the residue positions of inside/outside membrane. A large Number of known sequences Number of solved structures 500000 400000 300000 200000 100000 0 Number of data 1990 2000 2005 2010 2015 2017 1995 Year Fig. 1 The gap between known protein sequences and structures is rapidly expanding 1 Number of known sequences Number of solved structures 500000 400000 300000 200000 100000 0 Number of data 1990 2000 2005 2010 2015 2017 1995 Year Our results show that these two branches of methods highly complement each other [7]. The machine learning-based engines need the training process and highly depend on the distributions of training dataset. Hence, the prediction outputs of machine learning-based models have higher preference to match the distribution of the training set, resulting in a relatively lower generalization and coverage of the predictions. Training process is not needed in the coevolution mining methods, which align the query sequence against a large protein sequence pool to calculate Fig. 1 The gap between known protein sequences and structures is rapidly expanding 3 Page 3 of 8 2 Nano-Micro Lett. (2018) 10:2 g. 2 A screenshot of the submission interface of MemBrain web server (www.csbio.sjtu.edu.cn/bioinf/MemBrain/) 2.1.1 Multi-scale Predictors Modeling The input features are amino acid evolution information from optimized sliding windows with different lengths. We built a proﬁle for a query sequence with L residues by the position speciﬁc scoring matrix (PSSM) implemented by PSI-BLAST [18] program. The PSSM contains amino acid evolutionary information from multiple sequence align- ment searching against the SWISS-PROT database [19]. 2.1.1 Multi-scale Predictors Modeling The proﬁle has L rows and 20 columns, where the ith row C-terminal Position specific scoring matrix α-helical bundle N-terminal SWISS-PROT database Query sequence PSI-BLAST TMH #1: 10-31 TMH #2: 41-63 TMH #3: 86-106 TMH #4: 118-126 TMH #5: 141-155 Predicted TMH Dynamic threshold 1.0 0.5 0 0.5 150 200 Median filter OET-KNN with sliding window size W=13 OET-KNN with sliding window size W=15 A 3 5 −2 −2 −4 −2 −1 −3 4 −3 3 −1 −2 −1 −2 1 R −2 −3 −4 −2 −4 −1 −4 −3 −3 7 −2 −4 −4 −4 −3 −2 N −1 −2 −4 0 −4 3 −2 −3 −3 −2 −1 −2 −4 −2 −4 3 D −1 −2 −5 6 −5 −2 −3 −4 −4 −3 −2 −3 −4 −3 −4 −1 C −1 −2 −2 −5 −4 −4 −4 −4 −2 −5 −2 −4 −2 −3 −3 −2 Q −1 −2 −4 −1 −4 −1 −3 −3 −3 0 −1 −3 −4 −3 −3 −1 E −1 0 −4 3 −4 −1 −3 −3 −3 −1 −2 −3 −4 −3 −3 −2 G −1 3 −5 −3 −4 −3 7 −4 −2 −4 −1 7 −5 6 −4 −2 H −2 −3 −4 −2 −2 9 −3 1 −2 −1 −2 −3 −4 −3 −4 −2 I −3 −3 2 −4 −2 −5 −5 −3 −1 −4 −3 −5 5 1 2 −3 L −3 −3 0 −5 −1 −4 −5 −2 −1 −4 −4 −5 0 −3 −1 −3 K −1 −2 −4 −2 −4 0 −3 −3 −3 1 −2 −3 −4 −3 −3 −2 M −2 −3 −1 −4 −1 −3 −4 −2 4 −3 −3 −4 0 −3 4 −2 F −3 −4 4 −5 9 −3 −5 2 4 −4 −4 −5 −2 −4 −1 −4 P −2 −2 −4 −3 −5 −3 −3 −4 −3 −3 −3 −3 −4 −3 −1 −2 S 4 1 −3 1 −4 0 −2 −3 −1 −2 5 −2 −3 −2 −3 4 T 0 −1 −2 −2 −3 −2 −3 −3 −2 −2 0 −3 0 −3 1 4 W −4 −4 −3 −5 4 −4 −4 1 −2 −4 −4 −4 5 −4 8 −4 Y −2 −3 −1 −4 4 0 −4 9 3 −3 −3 −4 −2 −4 −1 −3 V −2 −2 5 −4 −2 −4 −5 −2 0 −4 −2 −5 4 −3 3 −2 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 S G F E F H G Y A R S G V I M N V F Q Y W A D S Q D V V D A S W Y S A S SS O d b C t i l l Fig. 2.1 MemBrain-TMH: Prediction of TMHs in Membrane Proteins Accurate TMH prediction is a long-term interest in trans- membrane protein structure prediction. At the very 123 123 2 Page 4 of 8 Nano-Micro Lett. (2018) 10:2 2 represents the probabilities of the ith residue in the protein sequence being mutated to 20 native residues during the evolution process. The sequence evolution knowledge encoded in the PSSM helps to remove the potential noise caused by mutations. beginning of methodology development in this problem, motivated by the fact that transmembrane residues are usually highly hydrophobic, average hydrophobic scores were used for detecting the hydrophobic segments. Later, more studies have revealed that this task is much more complicated than initially thought. For instance, very short (\10 residues) and very long ([35 residues) irregular TMH helices have been found and some loop regions linking the neighboring TMH segments can be very short (e.g., 2 residues). These structure complexities have posed signif- icant difﬁculties for prediction methodology development. Considering the irregular lengths of the TMH, we designed the multi-scale model with different sliding win- dow sizes. The size of the sliding window for extracting input feature has a great impact on the prediction outcome. If the sliding window is too small, the prediction accuracy would suffer from the loss of neighborhood sequence information; on the contrary, if it is too large, much redun- dant information will be included especially for the cases of short TMHs. We tried different lengths of windows for fusing the global and local sequence parameters, and at last we combined two window sizes to minimize the bias induced by a single window size, i.e., W = 13 and W = 15. This strategy makes current MemBrain approach capable of predicting half TMHs or tight turns shorter than 15 residues. The MemBrain also employs a powerful machine learning technique, the optimized evidence-theoretic K-nearest neighbor (OET-KNN) algorithm, which will output a propensity of residue belonging to TMH segments. The ﬁnal obtained TMH propensity is averaged over the results of lengths 13 and 15 for each residue along the sequence. In our MemBrain-TMH module (as shown in Fig. 3) [8], two typical strategies are adopted to enhance the TMH predictions. 2.1.1 Multi-scale Predictors Modeling 3 The pipeline of MemBrain for predicting transmembrane a-helices. For a query sequence, we generate the position speciﬁc scoring matrix as input features by searching against SWISS-PROT database using the PSI-BLAST tool. The OET-KNN algorithm is employed as the classiﬁer with fused different sizes of sliding window for extracting features. Median ﬁlter is applied to smooth the proﬁle of predicted probabilities. Finally, the dynamic threshold is effectively used to optimize the results of prediction SWISS-PROT database S SS O d b SWISS-PROT database S SS O d b PSI-BLAST Query sequence Fig. 3 The pipeline of MemBrain for predicting transmembrane a-helices. For a query sequence, we generate the position speciﬁc scoring matrix as input features by searching against SWISS-PROT database using the PSI-BLAST tool. The OET-KNN algorithm is employed as the classiﬁer with fused different sizes of sliding window for extracting features. Median ﬁlter is applied to smooth the proﬁle of predicted probabilities. Finally, the dynamic threshold is effectively used to optimize the results of prediction 12 2.1.2 Dynamic Threshold Decision Therefore, the contiguous TMH segments linking with short loops or tight turns are often misclassiﬁed as a long one. This indicates that the optimal threshold for deﬁning two TMHs separated by long loops is very different from the threshold required for identifying TMHs separated by short loops. To solve this problem, we exploit the dynamic threshold strategy for identiﬁcation of TMHs from the propensity scores. First, we set an initial threshold as 0.4, i.e., residues with propensity greater than or equal to 0.4 are considered as TMH. Second, we gradually increase the initial value of T with step size of 0.05 up to ﬁnd the plot valley to decide whether we need to split the initial seg- ments into two by a set of pre-learned rules. The results show that the dynamic threshold method not only improves the localization prediction of THM residues, but also enhances the correct number of TMH predictions. For a query sequence, a plot of predicted TMH propensity scores gives an overview of the residue-speciﬁc TMH propensity. In order to optimize the accuracy, we adopt the median ﬁlter technique to smooth the predicted TMH propensity proﬁle for reducing noise and avoid the burr phenomena. The ﬁnal TMHs are determined by the smoothed propensity plot. A threshold will be needed for classifying them into TMHs or non-TMHs, i.e., if the predicted scores of residues are higher than the threshold, they are predicted as TMH residues. A ﬁxed threshold is often used for this purpose, which may be problematic for segmenting two TMHs linked by short loops. Many high-resolution membrane protein 3D structures have shown that two adjacent TMHs could often be con- nected by very short loops, e.g.,\2 residues. In such cases, the predicted TMH propensity scores corresponding to the short loop residues will also be very high due to the sliding window technique used for extracting features. Taking W = 13 as an example, if the short loop is composed by 2 residues, then 11 residues belong to TMH in the window making the TMH features dominate for loop residues. 123 123 Nano-Micro Lett. (2018) 10:2 Page 5 of 8 2 2.2 MemBrain-Contact: TMH–TMH Residue Contact Map Prediction Based on the determined TMHs, the prediction of TMH– TMH residue contacts can provide crucial spatial con- straints for accurately modeling tertiary structures of Predicted contact map Residue contact probabilities OPM and PDBTM database TMH location α-helix topology Protein sequence PSI-BLAST UniRef90 database Multiple sequence alignment (MSA) PSICOV D A W F S V V G V Q S Q A Q S P W Y W Y Q S A W F S G P SVM1 SVM2 Ensemble SVM5 OET- KNN1 Ensemble Helix-helix interaction OET- KNN2 OET- KNN5 0 100 200 300 400 0 5 2 0 0 2 0 0 1 0 5 0 0 250 200 150 100 50 0 150 100 200 300 400 60 50 40 30 20 10 0 Fig. 4 The pipeline of MemBrain-Contact for predicting TMH–TMH contact map. We extract the TMH locations and topologies from protein database to build the training dataset. The coevolved mutation analysis by PSICOV using multiple sequence alignment generated by PSI-BLAST and machine learning-based algorithm outputs are combined to generate the ﬁnal predictions OPM and PDBTM database TMH location α-helix topology Protein sequence PSI-BLAST UniRef90 database Multiple sequence alignment (MSA) PSICOV D A W F S V V G V Q S Q A Q S P W Y W Y Q S A W F S G P SVM1 SVM2 Ensemble SVM5 OET- KNN1 Ensemble OET- KNN2 OET- KNN5 TMH location OPM and PDBTM database UniRef90 database α-helix topology Helix-helix interaction Helix-helix interaction Helix-helix interaction Fig. 4 The pipeline of MemBrain-Contact for predicting TMH–TMH contact map. We extract the TMH locations and topologies from protein database to build the training dataset. The coevolved mutation analysis by PSICOV using multiple sequence alignment generated by PSI-BLAST and machine learning-based algorithm outputs are combined to generate the ﬁnal predictions 12 3 2 Page 6 of 8 Nano-Micro Lett. (2018) 10:2 SVR-based classifier Threshold Predicted profile of Rasa G P F D Input sequence G Feature Vector Extraction Q S N 1 2 3 j L 1. Position specific scoring matrix 2. Evolution rate 3. Z-coordinate score 4. Secondary structure prediction 5. Physical parameters 6. 2.2 MemBrain-Contact: TMH–TMH Residue Contact Map Prediction Sequence length BLAST Rasa database Template similarity A Y W 60 50 40 30 20 10 0 Predictions calculated by D G V (∑rASAexist)/numexist FFG G G CV V AA A A A A M A G G G QWE I L LT W I I Y SV V S F LK V Fig. 5 The ﬂowchart of MemBrain-Rasa prediction protocol. For a protein sequence, we extract six kinds of sequential features, which will be fed into the SVR classiﬁer. We also designed a segment template similarity-based prediction engine for searching similar segments as templates for the target sequence against a locally constructed structure data pool. The outputs of the two engines are combined together to improve the prediction of relative accessible surface area SVR-based classifier G P F D Input sequence G Feature Vector Extraction Q S N 1 2 3 j L 1. Position specific scoring matrix 2. Evolution rate 3. Z-coordinate score 4. Secondary structure prediction 5. Physical parameters 6. Sequence length BLAST Rasa database Template similarity Predictions calculated by (∑rASAexist)/numexist G P F D Input sequence Q S N 1 2 3 j L Rasa database G Threshold Predicted profile of Rasa A Y W 60 50 40 30 20 10 0 D G V FFG G G CV V AA A A A A M A G G G QWE I L LT W I I Y SV V S F LK V Fig. 5 The ﬂowchart of MemBrain-Rasa prediction protocol. For a protein sequence, we extract six kinds of sequential features, which will be fed into the SVR classiﬁer. We also designed a segment template similarity-based prediction engine for searching similar segments as templates for the target sequence against a locally constructed structure data pool. The outputs of the two engines are combined together to improve the prediction of relative accessible surface area membrane proteins [15, 20]. The MemBrain-Contact pre- diction module is constructed by combining statistical machine learning algorithms and biological residue coevolution analysis from multiple sequence alignments as shown in Fig. 4 [7]. The machine learning-based prediction algorithm was implemented by applying multiple random under-samplings so that strong diversities can be produced via different learning methods in various spaces. The coevolved mutation scores from multiple sequence align- ments were generated by PSICOV algorithm [12]. predictions but with relatively high false positives. 2.2 MemBrain-Contact: TMH–TMH Residue Contact Map Prediction The machine learning-based engine is a supervised learning approach, whose outputs are dependent on the training samples, and hence has a relatively low coverage of pre- dictions. The combination of the two approaches will not only improve the prediction coverage but also reduce the false positives, resulting in an overall performance improvement. 123 2.3 MemBrain-Rasa: Relative Accessible Surface Area Prediction Fusing the coevolution-based engine and machine learning-based engine is a typical advantage of MemBrain- Contact module. We found that these two engines highly complement to each other. The coevolution-based engine does not need the training process, which is an unsuper- vised approach and hence can result in a wide coverage of Prediction of RASA in a-helical transmembrane proteins provides the relative accessibilities of the residues which are helpful to 3D structure prediction. In MemBrain-Rasa, we designed a segment template similarity-based 123 123 Nano-Micro Lett. (2018) 10:2 Page 7 of 8 2 signiﬁcantly enhanced compared to either the machine learning-based or template-based engines. prediction engine, which is effectively combined with the machine learning engine to improve the performance. In order to take the advantage of the solved structures, we organized a local database of residue relative solvent accessibility surface area from the protein data bank (PDB), which is applied to search similar segments as templates for the target sequence against the local data pool. The template similarity-based prediction is then fused with the output of support vector regression (SVR) using a designed knowledge rule. Figure 5 shows the MemBrain- Rasa prediction protocol [8]. 2.4 Prediction Performance of MemBrain On a test dataset including 70 helical membrane proteins consisting of 378 TMHs, MemBrain achieves a prediction accuracy of 97.9% of ATMH, 87.1% of AP, 3.2 ± 3.0 of N- score, 3.1 ± 2.8 of C-score, where ATMH denotes the rate of correctly predicted TMHs, AP denotes the ratio of cor- rectly predicted proteins (all predicted TMHs are success- ful), and N-score and C-score are the accuracy scores of predicted ends of TMH segments. Acknowledgements This work was supported by the National Nat- ural Science Foundation of China (Nos. 61671288, 91530321, 61603161), and Science and Technology Commission of Shanghai Municipality (Nos. 16JC1404300, 17JC1403500, 16ZR1448700). Acknowledgements This work was supported by the National Nat- ural Science Foundation of China (Nos. 61671288, 91530321, 61603161), and Science and Technology Commission of Shanghai Municipality (Nos. 16JC1404300, 17JC1403500, 16ZR1448700). Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://crea tivecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. Two benchmark datasets are used to evaluate the per- formance of MemBrain-Contact module, i.e., a training dataset consists of 60 a-helical proteins and an independent dataset with 21 a-helical proteins. Both of the two datasets have a sequence identity cutoff at 40% among pairwise sequence for reducing protein homology similarity redun- dancy. Their TMH locations and native topologies were extracted from the databases of TOPDB [21], PDBTM [22] and OPM [23]. For top L/5 contact predictions, prediction accuracies are 62%/64.1% on the training and independent datasets, respectively, where L is the length of sequence. The experimental results on 13 solved G protein-coupled receptors have shown that the predictions of MemBrain- Contact engine have helped increase the TM-score of the I-TASSER models by 37% in the transmembrane region. O b h k d i i f 52 b 3 Conclusions and Future Development MemBrain is a fully automated online server and is free to academic use, which is available at http://www.csbio.sjtu. edu.cn/bioinf/MemBrain/. For a query protein, the user simply needs to input its amino acid sequence and select the corresponding prediction functions, and then submit it to the server. Prediction results will be sent back to the user’s email address when the task is ﬁnished. Usually, MemBrain is very fast, depending on the length of protein sequence, and it will automatically send back the results in 5 min of most cases. MemBrain theoretic predictions have provided useful information to the wet-lab studies of membrane proteins [24–26]. A typical merit of MemBrain-Rasa is its hierarchical prediction model by combining supervised SVR model with a segment template similarity-based approach as the whole computational framework to deal with RASA pre- diction problem. The results show that for many long protein sequences, it is very hard to ﬁnd homology struc- ture templates of the full chains. However, when we only consider short segments, many existing structure templates can be found, which provide important complement to the pure machine learning-based predictions. In the future, we will keep on updating MemBrain to make it more powerful. One of the potential directions is developing the deep learning-based modules, which are expected to be highly complementary to current engines. Deep learning algorithms represent a new progress in the statistical machine learning ﬁeld [27, 28] which is expected to provide more opportunities for further enhancing the prediction performance of MemBrain. References 1. H.M. Berman, J. Westbrook, Z. Feng, G. Gilliland, T.N. Bhat, H. Weissig, I.N. Shindyalov, P.E. Bourne, The protein data bank. Nucleic Acids Res. 28(1), 235–242 (2000). doi:10.1093/nar/28.1. 235 2. M. Cserzo¨, E. Wallin, I. Simon, G. von Heijne, A. Elofsson, Prediction of transmembrane alpha-helices in prokaryotic mem- brane proteins: the dense alignment surface method. Protein Eng. 10(6), 673–676 (1997). doi:10.1093/protein/10.6.673 p 3. A.L. Hopkins, C.R. Groom, The druggable genome. Nat. Rev. Drug Discov. 1(9), 727–730 (2002). doi:10.1038/nrd892 On a benchmark dataset consisting of 52 membrane proteins composed of 80 chains with pairwise sequence identity \20% to avoid homology redundancy, the Mem- Brain-Rasa achieves a Pearson correlation coefﬁcient of 0.733 and mean absolute error of 13.593, which are 4. H.B. Shen, J. Yang, K.C. Chou, Fuzzy KNN for predicting membrane protein types from pseudo-amino acid composition. J. Theor. Biol. 240(1), 9–13 (2006). doi:10.1038/nrd897 5. K.C. Chou, H.B. Shen, MemType-2L: a web server for predicting membrane proteins and their types by incorporating evolution 5. K.C. Chou, H.B. Shen, MemType-2L: a web server for predicting membrane proteins and their types by incorporating evolution 12 3 2 Page 8 of 8 Nano-Micro Lett. (2018) 10:2 2 information through Pse-PSSM. Biochem. Biophys. Res. Com- mun. 360(2), 339–345 (2007). doi:10.1016/j.bbrc.2007.06.027 17. E. Durham, B. Dorr, N. Woetzel, R. Staritzbichler, J. Meiler, Solvent accessible surface area approximations for rapid and accurate protein structure prediction. J. Mol. Model. 15(9), 1093–1108 (2009). doi:10.1007/s00894-009-0454-9 6. H.B. Shen, J.J. Chou, MemBrain: improving the accuracy of predicting transmembrane helices. PLoS ONE 3(6), e2399 (2008). doi:10.1371/journal.pone.0002399 18. S.F. Altschul, T.L. Madden, A.A. Schaffer, J. Zhang, Z. Zhang, W. Miller, D.J. Lipman, Gapped BLAST and PSI-BLAST: a new generation of protein database search. Nucleic Acids Res. 25(17), 3389–3402 (1997). doi:10.1093/nar/25.17.3389 7. J. Yang, R. Jang, Y. Zhang, H.B. Shen, High-accuracy prediction of transmembrane inter-helix contacts and application to GPCR 3D structure modeling. Bioinformatics 29(20), 2579–2587 (2013). doi:10.1093/bioinformatics/btt440 19. A. Bairoch, R. Apweiler, The SWISS-PROT protein sequence database and its supplement TrEMBL in 2000. Nucleic Acids Res. 28(1), 45–48 (2000). doi:10.1093/nar/28.1.45 8. F. Xiao, H.B. Shen, Prediction enhancement of residue real-value relative accessible surface area in transmembrane helical proteins by solving the output preference problem of machine learning- based predictors. J. Chem. Inf. Model. 55(11), 2464–2474 (2015). doi:10.1021/acs.jcim.5b00246 20. J. Yang, B.J. He, R. Jang, Y. Zhang, H.B. Shen, Accurate disulﬁde-bonding network predictions improve ab initio structure prediction of cysteine-rich proteins. References Bioinformatics 31(23), 3773–3781 (2015). doi:10.1093/bioinformatics/btv459 9. X. Yin, Y.Y. Xu, H.B. Shen, Enhancing the prediction of trans- membrane b-barrel segments with chain learning and feature sparse representation. IEEE/ACM Trans. Comput. Biol. 13(6), 1016–1026 (2016). doi:10.1109/TCBB.2016.2528000 21. G.E. Tusnady, L. Kalmar, I. Simon, TOPDB: topology data bank of transmembrane proteins. Nucleic Acids Res. 36(suppl_1), D234–D239 (2007). doi:10.1093/nar/gkm751 g 22. G.E. Tusna´dy, Z. Doszta´nyi, I. Simon, PDB_TM: selection and membrane localization of transmembrane proteins in the protein data bank. Nucleic Acids Res. 33, 275–278 (2005). doi:10.1093/ nar/gki002 10. A. Krogh, B. Larsson, H.G. Von, E.L. Sonnhammer, Predicting transmembrane protein topology with a hidden Markov model: application to complete genomes. J. Mol. Biol. 305, 567–580 (2001). doi:10.1006/jmbi.2000.4315 g 23. M.A. Lomize, A.L. Lomize, I.D. Pogozheva, OPM: orientations of proteins in membranes database. Bioinformatics 22(5), 623–625 (2006). doi:10.1093/bioinformatics/btk023 11. Z. Yuan, J.S. Mattick, R.D. Teasdale, SVMtm: support vector machines to predict transmembrane segments. J. Comput. Chem. 25, 632–636 (2004). doi:10.1002/jcc.10411 12. D.T. Jones, D.W.A. Buchan, D. Cozzetto, M. Pontil, PSICOV: precise structural contact prediction using sparse inverse covari- ance estimation on large multiple sequence alignments. Bioin- formatics 28(2), 184–190 (2012). doi:10.1093/bioinformatics/ btr638 24. M.S. Taylor, T.R. Ruch, P.Y. Hsiao, Y. Hwang, P.F. Zhang et al., Architectural organization of the metabolic regulatory enzyme ghrelin O-acyltransferase. J. Biol. Chem. 288(45), 32211–32228 (2013). doi:10.1074/jbc.M113.510313 25. F. Kallenberg, S. Dintner, R. Schmitz, S. Gebhard, Identiﬁcation of regions important for resistance and signalling within the antimi- crobial peptide transporter BceAB of Bacillus subtilis. J. Bacteriol. 195(14), 3287–3297 (2013). doi:10.1128/JB.00419-13 13. A. Fuchs, A. Kirschner, D. Frishman, Prediction of helix–helix contacts and interacting helices in polytopic membrane proteins using neural networks. Proteins 74, 857–871 (2009). doi:10.1002/ prot.22194 26. G.A. Morrill, A.B. Kostellow, L.J. Liu, R.K. Gupta, Evolution of the a-Subunit of Na/K-ATPase from Paramecium to Homo sapiens: invariance of transmembrane helix topology. J. Mol. Evol. 82(4–5), 183–198 (2016). doi:10.1007/s00239-016-9732-1 14. N. Timothy, D.T. Jones, Predicting transmembrane helix packing arrangements using residue contacts and a force-directed algo- rithm. PLoS Comput. Biol. 6, e1000714 (2010). doi:10.1371/ journal.pcbi.1000714 27. P.D. Lena, K. Nagata, P. Baldi, Deep architecture for protein contact map prediction. Bioinformatics 28(19), 2449–2457 (2012). doi:10.1093/bioinformatics/bts475 15. J. Yang, Q.Y. Jin, B. Zhang, H.B. Shen, R2C: improving ab initio residue contact map prediction using dynamic fusion strategy and Gaussian noise ﬁlter. Bioinformatics 32(16), 2435–2443 (2016). doi:10.1093/bioinformatics/btw181 28. S. Wang, S. Sun, Z. Li, R. Zhang, J. References Xu, Accuracy de novo prediction of protein contact map by ultra-deep learning model. PLoS Comput. Biol. 13(1), e1005324 (2012). doi:10.1371/jour nal.pcbi.1005324 16. J. Sim, S.Y. Kim, J. Lee, Prediction of protein solvent accessi- bility using fuzzy k-nearest neighbor method. Bioinformatics 21(12), 2844–2849 (2005). doi:10.1093/bioinformatics/bti423 123 123 123
https://openalex.org/W2044699478	https://europepmc.org/articles/pmc4444795?pdf=render	English	null	Cross-sectional imaging of common and unusual complications after endoscopic retrograde cholangiopancreatography	Insights into imaging	2,015	cc-by	9,621	M. Tonolini (): A. Pagani: R. Bianco Department of Radiology, BLuigi Sacco^ University Hospital, Via G.B. Grassi 74, 20157 Milan, Italy e-mail: mtonolini@sirm.org Insights Imaging (2015) 6:323–338 DOI 10.1007/s13244-015-0393-1 Insights Imaging (2015) 6:323–338 DOI 10.1007/s13244-015-0393-1 PICTORIAL REVIEW Massimo Tonolini & Alessandra Pagani & Roberto Bianco Received: 25 November 2014 /Revised: 19 January 2015 /Accepted: 30 January 2015 /Published online: 26 February 2015 # The Author(s) 2015. This article is published with open access at Springerlink.com imaging pancreatitis and abnormal collections without the use of ionising radiation. Abstract Endoscopic retrograde cholangiopancreatography (ERCP) is currently a primarily therapeutic procedure that is extensively employed to treat several biliopancreatic disorders. Although widely considered a safe procedure, ERCP is associated with a non- negligible morbidity and occasional mortality. Due to the number and complexity of operative ERCPs per- formed, radiologists are increasingly faced with urgent requests for investigation of suspected post-procedural complications, which often have similar clinical and lab- oratory manifestations. This pictorial essay reviews the usual post-procedural CT findings, the clinical features and imaging appearances of common and unusual post- ERCP occurrences including interstitial oedematous and necrotising acute pancreatitis, haemorrhages, retroperito- neal and intraperitoneal duodenal perforations, infections and stent-related complications. Emphasis is placed on the pivotal role of multidetector CT, which is warranted after complex or prolonged ERCP procedures as it rep- resents the most effective modality to detect and grade ERCP-related complications and to monitor nonsurgically treated patients. Timely diagnosis and op- timal management require a combination of clinical and laboratory data with imaging appearances; therefore, this article aims to provide an increased familiarity with in- terpretation of early post-ERCP studies, particularly to triage those occurrences that require interventional or surgical treatment. In selected patients MRI allows Teaching Points Teaching Points • Endoscopic retrograde cholangiopancreatography (ERCP) allows treating many biliopancreatic disorders. • Due to the number and complexity of procedures, post- ERCP complications are increasingly encountered. • Main complications include acute pancreatitis, haemorrhages, duodenal perforation and infections. • Diagnosis and management of complications rely on com- bined clinical, laboratory and imaging data. • Multidetector CT is most effective to diagnose, categorise and monitor post-ERCP complications. Keywords Endoscopic retrograde cholangiopancreatography (ERCP) . Complications . Computed tomography (CT) . Acute pancreatitis . Duodenal perforation Cross-sectional imaging of common and unusual complications after endoscopic retrograde cholangiopancreatography Massimo Tonolini & Alessandra Pagani & Roberto Bianco Aim According to the guidelines issued by the American Society for Gastrointestinal Endoscopy (ASGE) and the World Society of Emergency Surgery (WSES), diagnosis and management of ERCP-related complications should rely upon a combination of clinical, laboratory and im- aging data. Since iatrogenic complications represent a non-negligible cause of litigation and lawsuits, early recognition and prompt appropriate intervention are es- sential in optimising patient management and outcome [6, 11, 12]. Based upon 8 years of personal experience at a university-based general hospital where approximately 230 ERCPs are performed each year, this pictorial essay reviews and illustrates the normal post-procedural ap- pearances observed shortly after ERCP, and the imaging findings associated with common and unusual complica- tions, aiming to provide radiologists with an increased familiarity with interpretation of early post-procedural studies for an optimal triage of complications. Most of the emphasis is placed on the pivotal role of multide- tector CT, which represents, according to the WSES guidelines for management of intra-abdominal infec- tions, the preferred and most effective modality to com- prehensively investigate the abdomen and pelvis to search for possible iatrogenic complications [6, 12–15]. Furthermore, endoscopic positioning of biliary pros- theses allows minimally invasive management of ob- structive jaundice from benign and neoplastic causes with high technical (over 90 %) and clinical (approxi- mately 80 %) success rates. Plastic stents are commonly used to treat benign strictures, postoperative bile leaks and pancreatic diseases. Conversely, self-expanding met- al stents (SEMS) may provide effective palliation of malignant biliary obstruction [4, 5]. g y However, ERCP is associated with a non-negligible post-procedural morbidity (estimated in the range 4– 10 % overall). The most common short-term (occurring within 3 days) complications include more or less se- vere cardiopulmonary problems (hypoxia, arrhythmia and aspiration) commonly associated with medications used for sedation and analgesia. The reported incidence of ERCP-specific complications ranges from 5 to 40 %, depending on the underlying diagnosis, patient age and comorbidities, complexity of the procedure, and operator experience. The most common occurrences include post- ERCP acute pancreatitis (PEAP, 2–9 %), haemorrhage (1.3–3.7 %), infection (1.9–3.6 %) and duodenal perfo- ration (DP) in descending order of frequency. Addition- ally, a variety of rare complications have been occasion- ally reported, including pneumothorax, portal venous air embolism, splenic injury and perforation of colonic di- verticula [2, 6–9]. Background Due to the widespread availability of non-invasive imaging tech- niques such as magnetic resonance cholangiopancreatography (MRCP) and multidetector computed tomography (CT), during the last decade endoscopic retrograde cholangiopancreatography (ERCP) has evolved from a diagnostic tool towards a primarily therapeutic procedure. Currently indispensable in surgical prac- tice, ERCP is extensively used to treat several disorders of the biliopancreatic system so that over 500,000 procedures are per- formed yearly in the USA [1]. 324 Insights Imaging (2015) 6:323–338 The growing number of indications for ERCP encom- pass retained or recurrent choledocholithiasis, benign and malignant biliary strictures, ampullary stenosis, sphincter of Oddi dysfunction (SOD), postoperative and traumatic ductal injuries, acute gallstone and chron- ic pancreatitis. Widely considered a safe procedure that often obviates the need for surgery (particularly in frail patients with poor performance status), ERCP has lim- ited contraindications such as upper aerodigestive ob- struction, severe coagulopathy, oesophageal and/or gas- tric varices, anaphylactic reaction to iodinated contrast medium (CM), acute nonbiliary pancreatitis, severe car- diopulmonary impairment and recent myocardial infarc- tion. ERCP is technically challenging in patients with pancreatico-duodenectomy or Billroth type II surgical reconstruction, unfeasible with the previous Roux-en-Y anastomosis. Following identification and cannulation of the ampullary orifice and CM injection under fluorosco- py, current endoscopic equipment allows performing sphincterotomy, extraction of common bile duct (CBD) stones, lithotripsy, biliary drainage, stricture dilatation, brush cytology and biopsy [1–3]. biliary stents. Mostly related to operative procedures, ERCP-related mortality (0.5–1.4 %) may result from any of the above-mentioned complications and is partic- ularly high in elderly patients with comorbidities and in centres with limited caseloads [2, 6–8, 10]. Clinical features and indications for imaging Clinical features and indications for imaging In the vast majority of cases, the clinical suspicion of early iatrogenic complications is based on a combination of intraprocedural findings (difficult or repeated cannu- lation, suspected or confirmed DP), symptoms and physical signs (sudden or worsening abdominal pain, distension, fever, haemodynamic impairment) and labo- ratory data (decreasing haemoglobin, elevated leukocyte count, acute phase reactants, serum lipase or amylase). The key role of CT imaging to investigate patients who become acutely ill hours or days after ERCP results from the limited value of physical examination (due to the retroperitoneal location of most iatrogenic changes) and the similar clinical and laboratory manifestations The risk of complications is increased by operative techniques such as use of balloons and dilating cathe- ters, tissue sampling, mechanical lithotripsy and wire baskets for stone extraction, and plastic and metallic 325 Insights Imaging (2015) 6:323–338 observed in most complications, particularly AP and DP [2, 6, 12]. observed in most complications, particularly AP and DP [2, 6, 12]. Multiplanar CT reformations (Figs. 2 and 3) provide high-resolution visualisation of biliary endoprostheses, whether of plastic or metallic reticular Bmesh^ material, and of their anatomical relationships. When interpreting post-procedural studies, the stent type should be report- ed along with the integrity, patency and position includ- ing proximal and distal extremities [13, 17]. Furthermore, ERCP duration may represent a surro- gated marker for challenging cannulation and/or opera- tive manoeuvres that can lead to papillary oedema and compromise ductal outflow, thus resulting in an in- creased risk and severity of complications, particularly PEAP. According to both the literature and personal experience, planned CT may be warranted in patients after prolonged ERCP procedures, particularly with sphincterotomy or multiple cannulations [16]. In up to 29 % of asymptomatic patients, limited ret- roperitoneal air observed within 24 h from ERCP may result from excessive insufflation during and after endo- scope withdrawal. Although this occurrence is categorised as type IV perforation according to the Stapfer classification (Table 1), we suggest not to em- phasise the term Bperforation^ in the radiological report, since its does not require treatment [8, 18, 19]. CT technique, usual post-procedural findings and reactive changes A preliminary unenhanced CT acquisition without intra- venous and peroral CM is helpful to visualise extraluminal air (particularly using image review at lung or bone window settings) and hyperattenuating (35–70 Hounsfield units, HU) fresh blood. Clinical features and indications for imaging Afterwards, follow- ing standard-dose intravenous CM injection we recom- mend a biphasic (arterial-dominant and venous) acquisi- tion, particularly to identify pancreatic necrosis and CM extravasation indicating active bleeding. Additionally, in selected cases gastroduodenal opacification by means of peroral ingestion of diluted enteral CM may be benefi- cial to confirm or exclude leakage indicating DP (Fig. 1) [6, 13–15]. Finally, reversible oedematous-type mural thickening consistent with acute duodenitis (Fig. 3) may be ob- served following therapeutic ERCP [20]. Role and limitations of ultrasound and magnetic resonance imaging (MRI) 2 Normal findings observed hours after operative ERCP in a 75-year-old male investigated with unenhanced CT (c, d) to verify correct placement of a plastic biliary stent, including de- pendent contrast medium (arrows) and air (arrowheads) in the intrahepatic bile ducts. In a different patient, a 44-year-old female patient suffering from acute post-procedural pain, contrast-enhanced CT (a, b) 2 days after ERCP excluded signs of acute complications and showed stratified iodinated con- trast medium (), bile and air (thin arrow) in the gallbladder Fig. 2 Normal findings observed hours after operative ERCP in a 75-year-old male investigated with unenhanced CT (c, d) to verify correct placement of a plastic biliary stent, including de- pendent contrast medium (arrows) and air (arrowheads) in the intrahepatic bile ducts. In a different patient, a 44-year-old female patient suffering from acute post-procedural pain, contrast-enhanced CT (a, b) 2 days after ERCP excluded signs of acute complications and showed stratified iodinated con- trast medium (), bile and air (thin arrow) in the gallbladder complemented with MRCP sequences to assess the bil- iary and pancreatic ducts and with additional dynamic fat-suppressed T1-weighted gradient echo sequences during intravenous gadolinium contrast when pancreatic necrosis is suspected. Furthermore, diffusion-weighted MRI may be more sensitive in detecting cases of mild pancreatic inflammation. However, despite recent techni- cal advancements MRI remains limited in acutely ill or uncooperative patients, and the presence or pneumobilia often renders MRCP sequences uninformative or confus- ing [13, 22, 23]. two of the three following criteria: (1) consistent new- onset or worsening abdominal pain (persistent, severe, epigastric pain often radiating to the back); (2) new or prolongation of hospitalisation for at least 2 days; (3) abnormally elevated (at least three times above the up- per normal limit) serum lipase or amylase 24 h after the procedure [2, 6, 21]. The multifactorial pathogenesis of PEAP involves mechanical factors (such as direct trauma from endosco- py, difficult bile duct cannulation and multiple pancre- atic duct injections) along with enzymatic, microbiolog- ical and patient-related factors including history of aller- gy [24–26]. The role of iodinated CM as one of the cofactors in causing PEAP has been extensively studied. In experimental models of acute pancreatitis, digestive enzyme activation occurs rapidly within acinar cells fol- lowing an initiating event: secretion blockage leads to accumulation of zymogen granules within cells, their fusion into large vacuoles, enzyme activation and finally cellular injury [24]. Role and limitations of ultrasound and magnetic resonance imaging (MRI) Besides the reduced pancreatic blood flow due to CM hyperviscosity, controversy exists on the role of the chemical effect of CM osmolality. Earlier studies reported reduced rates of pancreatic dam- age and decreased with newer low- or iso-osmolar com- pared to high-osmolar contrast medium, but other Role and limitations of ultrasound and magnetic resonance imaging (MRI) Compared to CT, sonographic investigation of patients with suspected post-ERCP complications is strongly limited by the usual abdominal distension due to ileus, pneumoperitoneum or pneumoretroperitoneum and by its inability to assess pancreatic necrosis and bleeding. Practically, ultrasound may be useful for rapid assess- ment of the gallbladder and biliary tree (with or without stents) and for follow-up of known collections, and its use is not recommended by current practice guidelines [6, 12, 14, 21]. After ERCP the presence of air in the biliary tree is an expected, common finding. Intra- and extrahepatic pneumobilia is visible in the majority of patients studied with CT within a few weeks from the procedure and may persist for months or years in patients who underwent sphincterotomy (Fig. 2). Shortly after ERCP retained CM is commonly seen in the biliary tree and gallbladder, often with a characteristic stratified appear- ance (Fig. 2) [13, 15]. An increasingly attractive alternative modality, MRI can image PEAP and abnormal collections without use of ionising radiation, and it is particularly beneficial in young patients. Unenhanced T1- and T2-weighted se- quences with and without fat suppression may be of enteral contrast medium (CM) and viewed at the bone window setting showed a well-opacified gastroduodenal lumen without extraluminal CM leak, consistent with sealed DP. Note the persistent extensive retroperito- neal air (+) and metallic clips (arrowhead in B) Fig. 1 During endoscopic retrograde cholangiopancreatography (ERCP), a 65-year-old female with sphincter of Oddi dysfunction expe- rienced duodenal perforation (DP), which was recognised intraprocedurally and treated with endoluminal clipping. Twenty-four hours later, unenhanced multidetector CTobtained after peroral ingestion of enteral contrast medium (CM) and viewed at the bone window setting showed a well-opacified gastroduodenal lumen without extraluminal CM leak, consistent with sealed DP. Note the persistent extensive retroperito- neal air (+) and metallic clips (arrowhead in B) Fig. 1 During endoscopic retrograde cholangiopancreatography (ERCP), a 65-year-old female with sphincter of Oddi dysfunction expe- rienced duodenal perforation (DP), which was recognised intraprocedurally and treated with endoluminal clipping. Twenty-four hours later, unenhanced multidetector CTobtained after peroral ingestion of enteral contrast medium (CM) and viewed at the bone window setting showed a well-opacified gastroduodenal lumen without extraluminal CM leak, consistent with sealed DP. Note the persistent extensive retroperito- neal air (+) and metallic clips (arrowhead in B) 326 Insights Imaging (2015) 6:323–338 Fig. Acute pancreatitis Arguably the most common serious adverse event, PEAP has been reported with variable incidence (2– 9 % overall, approximately 3.5 % in a meta-analysis of several prospective studies, and up to 30 % of high risk cases) according to patient selection. A transient asymptomatic increase in serum pancreatic enzymes oc- curs in the majority (70–75 %) of patients within 4 h after ERCP and resolves within 4 days. According to the widely used consensus definition adopted by the ASGE guidelines, diagnosis of PEAP requires at least 327 Insights Imaging (2015) 6:323–338 Fig. 3 Acute reversible duodenitis following ERCP. A 78-year-old male with common bile duct (CBD) carcinoma causing concentric stenosis of the proximal choledochus suffered from acute diffuse abdominal pain without peritonitis 24 h after ERCP including cytological brushing and positioning of a 10-French plastic biliary stent. Urgent contrast-enhanced CT (a, b) showed a correctly placed biliary stent, appearance of moderate ascites () and of marked circumferential thickening of the duodenum from the Vaterian papilla to the Treitz angle, with enhancing mucosa and hypoattenuating oedematous submucosa (Btarget sign^, thin arrows), and minimal associated inflammatory changes in the periduodenal fat. Imaging findings, laboratory data and the subsequent course excluded iatrogenic pancreatitis, haemorrhage and DP. Three weeks later, the plas- tic biliary stent was removed and replaced with a self-expanding metal stent (SEMS). Follow-up CT (d) showed imaging resolution of both bil- iary obstruction and acute duodenal inflammatory changes [partly reprinted from Ref. 20] and hypoattenuating oedematous submucosa (Btarget sign^, thin arrows), and minimal associated inflammatory changes in the periduodenal fat. Imaging findings, laboratory data and the subsequent course excluded iatrogenic pancreatitis, haemorrhage and DP. Three weeks later, the plas- tic biliary stent was removed and replaced with a self-expanding metal stent (SEMS). Follow-up CT (d) showed imaging resolution of both bil- iary obstruction and acute duodenal inflammatory changes [partly reprinted from Ref. 20] Fig. 3 Acute reversible duodenitis following ERCP. A 78-year-old male with common bile duct (CBD) carcinoma causing concentric stenosis of the proximal choledochus suffered from acute diffuse abdominal pain without peritonitis 24 h after ERCP including cytological brushing and positioning of a 10-French plastic biliary stent. Acute pancreatitis 4 Oedematous acute pancreatitis (AP) in a 77-year-old female 3 days after endoscopic treatment of choledocholithiasis including sphincterotomy. Unenhanced (a) and post-contrast (b) CT images showed persistent moderately hyperattenuating contrast medium () in the gall- bladder, oedema and fluid in the pancreatico-duodenal groove (arrows), minimal ascites (+) consistent with clinical and laboratory features of mild AP, and excluded abnormal collections and non-enhancing paren- chyma indicating pancreatic necrosis. The patient had an uneventful course with conservative treatment minimal ascites (+) consistent with clinical and laboratory features of mild AP, and excluded abnormal collections and non-enhancing paren- chyma indicating pancreatic necrosis. The patient had an uneventful course with conservative treatment Fig. 4 Oedematous acute pancreatitis (AP) in a 77-year-old female 3 days after endoscopic treatment of choledocholithiasis including sphincterotomy. Unenhanced (a) and post-contrast (b) CT images showed persistent moderately hyperattenuating contrast medium () in the gall- bladder, oedema and fluid in the pancreatico-duodenal groove (arrows), collections (APFCs). Containing a mixture of exudates, necrosis and blood, APFCs are adjacent to the pancreas and confined by normal fascial planes, and they display homogeneous low (0–30 HU) attenuation without a dis- cernible wall and enhancement [22, 23]. Treatment is analogous to that of non-iatrogenic AP, and mor- tality reaches 3 % of cases [2, 6, 21]. In patients with consistent above-mentioned clinical and laboratory criteria following ERCP, early imaging is generally unnecessary to diagnose PEAP, and during the first week organ failure and systemic inflammatory response syndrome (SIRS) may be present with few local complications. Although not specifically developed for iatrogenic occurrences, the revised Atlanta classifica- tion differentiates morphologically interstitial oedematous (IEP) from necrotising acute pancreatitis (NAP), the latter associated with a high probability of infection, subsequent organ failure and increased mortal- ity (12–30 % versus below 3 %). CT diagnosis of IEP relies on diffuse or localised enlargement of the pancre- atic parenchyma with preserved homogeneous enhance- ment (to 80–150 HU) after intravenous CM, accompa- nied by peripancreatic fat stranding or by hypoattenuating (0–30 HU) acute peripancreatic fluid Nevertheless, in our experience CT is requested with- in 24–72 h in most patients with clinical and laboratory features indicating PEAP to exclude other complications (particularly DP) with similar manifestations. Therefore, a majority of patients with IEP are imaged with near- normal or subtle CT findings, including a homoge- neously enhancing pancreas, fat stranding and minimal fluid around the head and neck of the pancreas (Figs. Acute pancreatitis Urgent contrast-enhanced CT (a, b) showed a correctly placed biliary stent, appearance of moderate ascites () and of marked circumferential thickening of the duodenum from the Vaterian papilla to the Treitz angle, with enhancing mucosa and hypoattenuating oedematous submucosa (Btarget sign^, thin arrows), and minimal associated inflammatory changes in the periduodenal fat. Imaging findings, laboratory data and the subsequent course excluded iatrogenic pancreatitis, haemorrhage and DP. Three weeks later, the plas- tic biliary stent was removed and replaced with a self-expanding metal stent (SEMS). Follow-up CT (d) showed imaging resolution of both bil- iary obstruction and acute duodenal inflammatory changes [partly reprinted from Ref. 20] Whereas the risk of bleeding and perforation is similar to that of younger patients, the elderly are less likely to experience PEAP. Clinical guidelines increasingly suggest pharmacolog- ic prophylaxis with nonsteroidal anti-inflammatory drugs, glyceryl trinitrate or somatostatin; furthermore, in patients with SOD, the use of temporary pancreatic duct stenting has been proposed to limit the risk and severity of PEAP. Whereas the risk of bleeding and perforation is similar to that of younger patients, the elderly are less likely to experience PEAP. Clinical guidelines increasingly suggest pharmacolog- ic prophylaxis with nonsteroidal anti-inflammatory drugs, glyceryl trinitrate or somatostatin; furthermore, in patients with SOD, the use of temporary pancreatic duct stenting has been proposed to limit the risk and severity of PEAP. reports including a meta-analysis did not confirm a sig- nificantly different risk and outcome of PEAP [25–28]. Probably unavoidable even in the hands of experienced endoscopists, PEAP is associated with established risk condi- tions including patient-specific (age below 55 years, female gender, SOD, history of AP) and procedure-related factors (difficult cannulation, standard or pre-cut sphincterotomy). reports including a meta-analysis did not confirm a sig- nificantly different risk and outcome of PEAP [25–28]. Table 1 Classification of duodenal perforations according to Stapfer et al. (Ref. 8) Type Description Comment I Endoscope-related lateral or medial duodenal wall perforations Often large and distant from the ampulla, requires surgical intervention in most cases II Retroperitoneal peri-Vaterian perforations resulting from (pre-cut) sphincterotomy Of variable severity but most often discrete and amenable to conservative management III Distal common bile duct injuries secondary to guidewire insertion or instrumentation for stone extraction Amenable to conservative management IV Isolated retroperitoneal air from excessive insufflation Does not require specific treatment 328 Insights Imaging (2015) 6:323–338 Fig. Fig. 5 Oedematous AP with markedly increased serum lipase 48 h after ERCP treatment of choledocholithiasis in a 55-year- old male with previous history of biliary AP and laparoscopic cho- lecystectomy. Unenhanced CT (a) and T2-weighted MRI (b) images show mild peripancreatic and ret- roperitoneal fascial fluid (thin arrows) without abnormal collec- tions. Clinical symptoms and laboratory changes regressed within 72 h Acute pancreatitis Differentiation from APFCs is very difficult in the early phase and is generally based on the presence or absence of glandular necrosis. PNPFCs may become progressively more organised and encapsu- lated later and are termed walled-off pancreatic necrosis after 4 weeks of symptom onset (Fig. 7) [13, 15, 16, 22, 23]. account the presence of pancreatic and/or peripancreatic inflammatory changes, APFCs, necrosis (<30 %, 30– 50 % and over 50 %) and extrapancreatic complications such as pleural effusion, ascites, vascular or gastrointes- tinal involvement. The only large specific series graded PEAP severity according to M-CTSI scores as mild (≤2 points), moderate (4–6) and severe (≥8) in 53.6, 42.8 and 3.6 % of cases respectively [13, 15, 16, 22, 23]. Furthermore, peripancreatic and glandular changes consistent with PEAP are well demonstrated by MRI (Figs. 5 and 6). Typical MRI findings of AP include var- iable degrees of glandular enlargement with mildly in- creased T2-weighted signal intensity corresponding to oe- dema, normal signal on fat-saturated T1-weighted acqui- sitions, peripancreatic inflammatory fat stranding, fluid or collections. Similarly to CT, pancreatic enhancement may appear homogeneous (in IEP), diminished or heteroge- neous (in NAP) after intravenous gadolinium CM. In the setting of gallstones, it has been proposed that a limited noncontrast MRI protocol has high correlation with Therefore, the radiologist’s role mostly relies in the identification of the presence and extent of necrosis. Severity of AP may be graded according to the modi- fied CT severity index (M-CTSI), which takes into Fig. 7 Necrotising AP plus retroperitoneal air in a 79-year-old male occurring 48 h after ERCP treatment of choledocholithiasis, including sphincterotomy. Contrast-enhanced CT (a, b) showed an enlarged, mark- edly inhomogeneous, poorly enhancing pancreas with fascial (thin arrow in a), peripancreatic and infrahepatic (+ in b) fluid, gas bubbles along the superior mesenteric vessels and in the periduodenal region. Pleural effu- sion was present bilaterally. Urgent laparotomic surgery confirmed severe necrotic-haemorrhagic pancreatitis. During his prolonged stay in the in- tensive care unit, follow-up unenhanced CT (c) showed the appearance of a vast walled-off postnecrotic collection () Fig. 7 Necrotising AP plus retroperitoneal air in a 79-year-old male occurring 48 h after ERCP treatment of choledocholithiasis, including sphincterotomy. Acute pancreatitis 4 and 5). Borrowing from experience with gallstone AP, early contrast-enhanced CT obtained within 2–3 days from the onset of symptoms may underestimate the se- verity of PEAP including confident exclusion of necro- sis so that repeated scanning may be required during post-ERCP hospitalisation [13, 15, 16, 22, 23]. Fig. 5 Oedematous AP with markedly increased serum lipase 48 h after ERCP treatment of choledocholithiasis in a 55-year- old male with previous history of biliary AP and laparoscopic cho- lecystectomy. Unenhanced CT (a) and T2-weighted MRI (b) images show mild peripancreatic and ret- roperitoneal fascial fluid (thin arrows) without abnormal collec- tions. Clinical symptoms and laboratory changes regressed within 72 h 329 Insights Imaging (2015) 6:323–338 Fig. 6 Necrotising AP in a 73-year-old male after ERCP including en- doscopic treatment of mild bleeding after papillotomy. Urgent MRI (T2- weighted image in a) requested because of increased C-reactive protein (CRP), leukocyte count and serum lipase showed a markedly enlarged pancreatic gland () with peripancreatic oedema. Contrast-enhanced CT (b, c) confirmed severe AP with inhomogeneous enhancement of the enlarged pancreas due to diffuse peripancreatic necrosis (+). The patient finally recovered after 2 weeks of in-hospital conservative treatment pancreatic gland () with peripancreatic oedema. Contrast-enhanced CT (b, c) confirmed severe AP with inhomogeneous enhancement of the enlarged pancreas due to diffuse peripancreatic necrosis (+). The patient finally recovered after 2 weeks of in-hospital conservative treatment Fig. 6 Necrotising AP in a 73-year-old male after ERCP including en- doscopic treatment of mild bleeding after papillotomy. Urgent MRI (T2- weighted image in a) requested because of increased C-reactive protein (CRP), leukocyte count and serum lipase showed a markedly enlarged pancreatic gland () with peripancreatic oedema. Contrast-enhanced CT (b, c) confirmed severe AP with inhomogeneous enhancement of the enlarged pancreas due to diffuse peripancreatic necrosis (+). The patient finally recovered after 2 weeks of in-hospital conservative treatment Conversely, NAP includes fatty tissue necrosis in ei- ther the pancreatic parenchyma or peripancreatic tissue and corresponds at imaging to pancreatic enlargement, heterogeneous because of single or multiple areas of diminished or absent enhancement consistent with ne- crosis (Figs. 6 and 7). In NAP, post-necrotic peripancreatic fluid collections (PNPFCs) are commonly observed in the pancreas, peripancreatic tissue or both. PNPFCs contain fluid, necrosis and/or loculation in varying degrees. Acute pancreatitis Arterial-phase acquisition completed with maximum- intensity projection (MIP) reconstructions at vascular window settings may effectively show active contrast extravasation in the duodenum, indicating ongoing bleeding (Fig. 9). Furthermore, following operative ERCP bleeding may occasionally form intrahepatic or subcapsular hepatic haematomas with associated haemoperitoneum (Fig. 10) [13, 31, 32]. therapeutic anticoagulation, haemodialysis, acute cholangitis or papillary stenosis, pre-cut or extended sphincterotomy [2, 6]. Although clinically significant haemorrhage requiring angiographic, endoscopic or surgical intervention is rare (less than 1/1,000 sphincterotomies), CT imaging may help in the diagnosis by showing hyperattenuating fluid consistent with blood in the choledochus (Fig. 8) or duodenal lumen. Residual diluted CM in the duodenal or jejunal lumen may sometimes be confused with fresh haemorrhage. Alternatively, intramural bleeding may ap- pear as high-attenuation duodenal wall thickening. Arterial-phase acquisition completed with maximum- intensity projection (MIP) reconstructions at vascular window settings may effectively show active contrast extravasation in the duodenum, indicating ongoing bleeding (Fig. 9). Furthermore, following operative ERCP bleeding may occasionally form intrahepatic or subcapsular hepatic haematomas with associated haemoperitoneum (Fig. 10) [13, 31, 32]. Acute pancreatitis Contrast-enhanced CT (a, b) showed an enlarged, mark- edly inhomogeneous, poorly enhancing pancreas with fascial (thin arrow in a), peripancreatic and infrahepatic (+ in b) fluid, gas bubbles along the superior mesenteric vessels and in the periduodenal region. Pleural effu- sion was present bilaterally. Urgent laparotomic surgery confirmed severe necrotic-haemorrhagic pancreatitis. During his prolonged stay in the in- tensive care unit, follow-up unenhanced CT (c) showed the appearance of a vast walled-off postnecrotic collection () 330 Insights Imaging (2015) 6:323–338 Fig. 8 Haemobilia following ERCP in a 57-year-old female with benign papillary stenosis and previous cholecystectomy, manifesting with ab- dominal pain and mild haemoglobin drop. Unenhanced (a, b) and post- contrast (c) CT images showed moderate dilatation and hyperattenuating (45 HU) content of the CBD (arrowheads) corresponding to endoluminal haemorrhage but initially misinterpreted as retained CM injected during the procedure, without signs of active bleeding. This complication was self-limiting and resolved on conservative treatment (45 HU) content of the CBD (arrowheads) corresponding to endoluminal haemorrhage but initially misinterpreted as retained CM injected during the procedure, without signs of active bleeding. This complication was self-limiting and resolved on conservative treatment Fig. 8 Haemobilia following ERCP in a 57-year-old female with benign papillary stenosis and previous cholecystectomy, manifesting with ab- dominal pain and mild haemoglobin drop. Unenhanced (a, b) and post- contrast (c) CT images showed moderate dilatation and hyperattenuating (45 HU) content of the CBD (arrowheads) corresponding to endoluminal haemorrhage but initially misinterpreted as retained CM injected during the procedure, without signs of active bleeding. This complication was self-limiting and resolved on conservative treatment contrast-enhanced CT in the assessment of mild AP. Fur- thermore, the use of diffusion-weighted MRI including measurement of apparent diffusion coefficients (ADCs) may allow differentiation among normal, inflamed and necrotic pancreas before or without CM administration [22, 23, 29, 30]. therapeutic anticoagulation, haemodialysis, acute cholangitis or papillary stenosis, pre-cut or extended sphincterotomy [2, 6]. Although clinically significant haemorrhage requiring angiographic, endoscopic or surgical intervention is rare (less than 1/1,000 sphincterotomies), CT imaging may help in the diagnosis by showing hyperattenuating fluid consistent with blood in the choledochus (Fig. 8) or duodenal lumen. Residual diluted CM in the duodenal or jejunal lumen may sometimes be confused with fresh haemorrhage. Alternatively, intramural bleeding may ap- pear as high-attenuation duodenal wall thickening. Haemorrhage The classification system proposed by Stapfer et al. (Table 1) categorises DP into four classes in descending order of severity, on the basis of the mechanism and anatomical location, in order to predict the need for surgery [8]. Although controversy exists about the optimal ap- proach, the management of ERCP-related DP should be individualised according to its type, clinical picture and imaging findings. Whereas type I (endoscope- related) duodenal wall perforations invariably require early surgery, the majority (approximately 70 %) of pa- tients with peri-Vaterian (type II) DP tend to seal spon- taneously and thus are amenable to endoclipping (Fig. 1) or conservative management (nasogastric drain- age, nil-by-mouth and parenteral nutrition, intravenous hydration and antibiotics). Currently, the therapeutic ap- proach to management of stable patients is increasingly 331 Insights Imaging (2015) 6:323–338 331 Fig. 10 A 39-year-old male with previous Bismuth type II iatro- genic bile duct injury during lap- aroscopic cholecystectomy 10 months earlier, shown in a coronal thick-slab MR cholangi- ography (MRCP) image (thin ar- rows in a), which required imme- diate reintervention to remove the surgical clips plus temporary plastic biliary stenting. Note haematoma in the gallbladder fossa (§). Twenty-four hours after endoscopic positioning of the covered metallic biliary stent (b), he suffered from sudden abdomi- nal pain, vomiting and hypoten- sion, and unenhanced (c) CT im- ages detected a large hyperattenuating subcapsular liv- er haematoma () and haemoperitoneum (+) attributed to guidewire manoeuvres, which progressed at repeated contrast- enhancement CT on the next day (d), thus dictating immediate sur- gical evacuation The CT detection of active bleeding represents an indication for endoscopic, interventional or surgical treatment. The generally accepted first-line treatment to control bleeding is repeated endoscopy with several haemostatic methods such as flushing with epinephrine solution, balloon tamponade, haemostatic (fibrin glue) injection, hemoclip placement, electrocoagulation or temporary stent placement. When endoscopic procedures fail, bleeding control requires interventional radiological embolisation or surgery [33–36]. In a variable proportion (26–48 %) of patients DP is suspected or verified during ERCP by means of CM injection through the endoscope showing extraluminal CM leakage (Fig. 11). Alternatively, clinical presenta- tion occurs hours or days later, may be closely similar to those associated with PEAP and is not unusually mild compared to the imaging findings [2, 6–8, 14, 37, 38]. The classification system proposed by Stapfer et al. Haemorrhage (Table 1) categorises DP into four classes in descending order of severity, on the basis of the mechanism and anatomical location, in order to predict the need for surgery [8]. Haemorrhage Primarily a complication related to sphincterotomy rather than to diagnostic ERCP, bleeding may be im- mediate or delayed up to 2 weeks (in up to 50 % of cases) and is clinically suggested by haematemesis, melaena or haemoglobin drop. Whereas variable de- grees of bleeding occur in approximately 1–2 % of treated patients, haemorrhage is graded as mild (ac- cording to blood loss and transfusion need) in approx- imately 70 % of cases, with occasional mortality (0.3 %). Specific risk factors include coagulopathy, Fig. 9 Acute intraluminal bleeding in the duodenum manifesting with acute abdominal pain and impending haemodynamic shock, hours after an unsuccessful endoscopic attempt to relieve the intrahepatic biliary obstruction in 77-year-old female with recently diagnosed infiltrating gallbladder carcinoma. Operative ERCP was interrupted after sphincterotomy because of duodenal haemorrhage, which was treated with epinephrine injection and endoluminal clipping. Comparison of unenhanced (a) and arterial-phase (b, c) CT images detected active con- trast extravasation in the duodenal lumen (arrowheads) abutting the me- tallic clips (thin arrows), consistent with persistently ongoing arterial haemorrhage. Endovascular therapeutic embolisation of the gastroduode- nal artery was necessary to control bleeding with epinephrine injection and endoluminal clipping. Comparison of unenhanced (a) and arterial-phase (b, c) CT images detected active con- trast extravasation in the duodenal lumen (arrowheads) abutting the me- tallic clips (thin arrows), consistent with persistently ongoing arterial haemorrhage. Endovascular therapeutic embolisation of the gastroduode- nal artery was necessary to control bleeding Fig. 9 Acute intraluminal bleeding in the duodenum manifesting with acute abdominal pain and impending haemodynamic shock, hours after an unsuccessful endoscopic attempt to relieve the intrahepatic biliary obstruction in 77-year-old female with recently diagnosed infiltrating gallbladder carcinoma. Operative ERCP was interrupted after sphincterotomy because of duodenal haemorrhage, which was treated represents an al or surgical ne treatment to y with several th epinephrine c (fibrin glue) coagulation or pic procedures nal radiological rence, DP com- ssociated with a s mostly associ- ome catheter) or y extending be- uidewire manip- perforation in- D, presence of h II surgery [2, In a variable proportion (26–48 %) of patients DP is suspected or verified during ERCP by means of CM injection through the endoscope showing extraluminal CM leakage (Fig. 11). Alternatively, clinical presenta- tion occurs hours or days later, may be closely similar to those associated with PEAP and is not unusually mild compared to the imaging findings [2, 6–8, 14, 37, 38]. Duodenal perforation During ERCP, fluoroscopy (a) showed opacification of dilated intrahepatic and common bile ducts and of a sizeable extraluminal CM collection (thin arrows) from lateral duodenal wall perforation (type I according to the Stapfer classification system). A few hours later CT (b– d) showed diffuse gaseous bowel distension from insufflation during endoscopy, presence of posterior pneumomediastinum, perihepatic and right parietocolic pneumoperitoneum (), and extensive retroperitoneal emphysema in the right perirenal, posterior and anterior pararenal spaces (+). Note persistently opacified intrahepatic and common bile ducts with residual lithiasis fragments (arrowheads), persistently extravasated CM (arrow in d). Imaging findings and worsening clinical conditions with peritonitis dictated urgent laparotomy, which confirmed intra-abdominal free air, and included opening and toilette of common bile duct and positioning of a Kehr T-tube. The patient finally recovered after a prolonged hospital stay right parietocolic pneumoperitoneum (), and extensive retroperitoneal emphysema in the right perirenal, posterior and anterior pararenal spaces (+). Note persistently opacified intrahepatic and common bile ducts with residual lithiasis fragments (arrowheads), persistently extravasated CM (arrow in d). Imaging findings and worsening clinical conditions with peritonitis dictated urgent laparotomy, which confirmed intra-abdominal free air, and included opening and toilette of common bile duct and positioning of a Kehr T-tube. The patient finally recovered after a prolonged hospital stay nonsurgical and proves successful in approximately two- thirds of cases: unfortunately, delayed recognition of type I perforations and failure of conservative treatment are associated with a high death rate (50 %) from sep- sis. As from the WSES guidelines, accepted indications for surgical treatment include clinical features such as sepsis and peritonitis, imaging features (free intraperito- neal air, periduodenal or retroperitoneal fluid collections, contrast extravasation at ERCP, CT or upper gastrointes- tinal study with water-soluble CM, retained stones or basket/wire endoscopic instruments) and failure of con- servative management. Surgery may include perforation closure or duodenal exclusion, retroperitoneal drainage, CBD exploration and T-tube insertion [2, 6, 8, 12, 14, 38–42]. duodenal wall, but most commonly dissects through the ret- roperitoneal compartments. Due to the anatomic location of the CBD, the typical appearance of a type II DP includes air collecting posteriorly to the duodenum and pancreatic head, in the anterior pararenal and right perirenal space, surrounding the inferior vena cava, portal vein and splanchnic vessels, and sometimes crossing the midline (Figs. 11, 12, 13 and 14). Duodenal perforation Although controversy exists about the optimal ap- proach, the management of ERCP-related DP should be individualised according to its type, clinical picture and imaging findings. Whereas type I (endoscope- related) duodenal wall perforations invariably require early surgery, the majority (approximately 70 %) of pa- tients with peri-Vaterian (type II) DP tend to seal spon- taneously and thus are amenable to endoclipping (Fig. 1) or conservative management (nasogastric drain- age, nil-by-mouth and parenteral nutrition, intravenous hydration and antibiotics). Currently, the therapeutic ap- proach to management of stable patients is increasingly Representing the rarest yet most feared occurrence, DP com- plicates 0.1–1 % of ERCP procedures and is associated with a non-negligible mortality rate (9–18 %). DP is mostly associ- ated with standard (using an electrical papillotome catheter) or pre-cut (with a needle knife) sphincterotomy extending be- yond the intramural CBD portion and with guidewire manip- ulation. Other risk factors for ERCP-related perforation in- clude dilated CBD, stricture dilatation, SOD, presence of peripapillar diverticula and previous Billroth II surgery [2, 6–8, 14, 19, 37, 38]. 332 Insights Imaging (2015) 6:323–338 Fig. 11 An elderly 80-year-old male with multiple comorbidities underwent endoscopic treatment of choledocholithiasis, including pre- cut sphincterotomy of the Vaterian papilla and stone retrieval using a basket device. During ERCP, fluoroscopy (a) showed opacification of dilated intrahepatic and common bile ducts and of a sizeable extraluminal CM collection (thin arrows) from lateral duodenal wall perforation (type I according to the Stapfer classification system). A few hours later CT (b– d) showed diffuse gaseous bowel distension from insufflation during endoscopy, presence of posterior pneumomediastinum, perihepatic and right parietocolic pneumoperitoneum (), and extensive retroperitoneal emphysema in the right perirenal, posterior and anterior pararenal spaces (+). Note persistently opacified intrahepatic and common bile ducts with residual lithiasis fragments (arrowheads), persistently extravasated CM (arrow in d). Imaging findings and worsening clinical conditions with peritonitis dictated urgent laparotomy, which confirmed intra-abdominal free air, and included opening and toilette of common bile duct and positioning of a Kehr T-tube. The patient finally recovered after a prolonged hospital stay Fig. 11 An elderly 80-year-old male with multiple comorbidities underwent endoscopic treatment of choledocholithiasis, including pre- cut sphincterotomy of the Vaterian papilla and stone retrieval using a basket device. tions as well as to monitor the patient until recovery [2, 6, 12, 13, 15]. tions as well as to monitor the patient until recovery [2, 6, 12, 13, 15]. develop peritonitis, fever or impending shock. However, negative physical findings do not reliably exclude sur- gery since severe DP may be masked clinically by the retroperitoneal site especially in elderly or chronically ill patients. Extraluminal CM leak (Fig. 11), pneumoperito- neum (Fig. 14) and fluid collections represent the key imaging findings, which usually shift the therapeutic approach towards surgery. During conservative treat- ment, repeat CT may be warranted after a 48–72-h in- terval to confirm sealed leaks and absent fluid collec- Duodenal perforation Occasionally, air may track cranially in the posterior medias- tinum [2, 8, 9, 13, 38]. However, the presence and amount of retroperitoneal air at imaging do not linearly correlate with the severity of the injury or with the need for invasive treatment since it rather reflects the degree of continuous endo- scopic air insufflation and manipulation after an unde- tected injury occurred. Therefore, successful nonopera- tive management of type II DP with extensive retroper- itoneal air is possible, provided that the patient does not The CT imaging hallmark of DP is represented by extraluminal air, which may sometimes be located in the Insights Imaging (2015) 6:323–338 333 Fig. 12 Severe retroperitoneal (type II) DP in an 80-year-old fe- male following ERCP with sphincterotomy, incomplete re- moval of choledocholithiasis and positioning of a plastic CBD prosthesis (arrowhead). Post- procedural unenhanced CT viewed at both soft-tissue (a, b) and bone (c, d) window settings showed extensive retroperitoneal air (+) predominantly located in the right peri- and pararenal spaces, around the descending duodenum and tracking along the inferior vena cava and portal vein (thin arrows). Surgical explora- tion dictated by sepsis failed to detect a perforation site, and the patient recovered fully Fig. 13 Failed conservative treatment of DP in an 83-year-old male with chronic duodenal stric- ture who underwent ERCP to prevent further episodes of infec- tious cholangitis. The next day, contrast-enhanced CT (a) showed moderate right-sided retroperito- neal gas (). Follow-up CT (b, c) 4 days later showed formation of multiple confluent periduodenal and right parietocolic fluid-like infected collections (+), with ex- tensive occupation of the ipsilat- eral posterior pararenal space, which required percutaneous drainage (d) to relieve sepsis 334 Insights Imaging (2015) 6:323–338 Fig. 14 Stapfer type I DP with moderate retroperitoneal air (+) plus pneumoperitoneum extending to the scrotum () shown by emergency unenhanced CT in a 62-year-old male undergoing ERCP treatment of choledocholithiasis. Laparotomic surgery included duodenotomy and suture of a posterior duodenal wall discontinuity, toilette and drainage of the peritoneal cavity. The subsequent course was uneventful and re- peated CT with oral CM at discharge (not shown) excluded extraluminal contrast leak Fig. 14 Stapfer type I DP with moderate retroperitoneal air (+) plus pneumoperitoneum extending to the scrotum () shown by emergency unenhanced CT in a 62-year-old male undergoing ERCP treatment of choledocholithiasis. Fig. 15 Acute cholecystitis in a 66-year-old female manifesting with fever and transverse upper abdominal pain 3 days after ERCP with sphincterotomy and biopsy of ampullary adenoma in- cluding antibiotic prophylaxis. Multiplanar contrast-enhanced CT images show a thickened gallbladder wall (arrows) with focal perforation (arrowhead in c) and localised collection (), which required emergency laparotomic cholecystectomy. Duodenal perforation Laparotomic surgery included duodenotomy and suture of a posterior duodenal wall discontinuity, toilette and drainage of the peritoneal cavity. The subsequent course was uneventful and re- peated CT with oral CM at discharge (not shown) excluded extraluminal contrast leak suture of a posterior duodenal wall discontinuity, toilette and drainage of the peritoneal cavity. The subsequent course was uneventful and re- peated CT with oral CM at discharge (not shown) excluded extraluminal contrast leak Fig. 14 Stapfer type I DP with moderate retroperitoneal air (+) plus pneumoperitoneum extending to the scrotum () shown by emergency unenhanced CT in a 62-year-old male undergoing ERCP treatment of choledocholithiasis. Laparotomic surgery included duodenotomy and Post-procedural infections Although the current guidelines recommend antibiotic prophylaxis in known or suspected biliary obstruction, bacteraemia is a common event (15–27 %) after diag- nostic or therapeutic ERCP. The reported incidence of Fig. 15 Acute cholecystitis in a 66-year-old female manifesting with fever and transverse upper abdominal pain 3 days after ERCP with sphincterotomy and biopsy of ampullary adenoma in- cluding antibiotic prophylaxis. Multiplanar contrast-enhanced CT images show a thickened gallbladder wall (arrows) with focal perforation (arrowhead in c) and localised collection (), which required emergency laparotomic cholecystectomy. 335 Insights Imaging (2015) 6:323–338 s is limited (1– cause of death. e stenting across dice, combined mary sclerosing drainage [2, 6]. The spectrum of post-ERCP infectious complications encompasses cholecystitis, septic cholangitis, liver ab- scess and systemic sepsis. Occurring in approximately 0.2–0.5 % of patients, cholecystitis has been correlated to cholelithiasis and intraprocedural filling of the gall- bladder with CM, and it is heralded by the appearance Fig. 16 Cholangitis and sepsis in an 81-year-old female following endoscopic stone removal using a Fogarty catheter. Multiplanar contrast-enhanced CT images showed minimal pneumoperito- neum (arrowheads) and peritone- al effusion, thickened enhancing walls of CBD and main intrahepatic ducts (thin arrows). Additionally, left-sided pneumo- nia (* in c) and a limited renal infarct (arrow in d) were seen. Worsening clinical conditions dictated surgery, which revealed biliary peritonitis from contained cystic duct injury. The patient re- covered after a long intensive care unit stay The spectrum of post-ERCP infectious complications encompasses cholecystitis, septic cholangitis, liver ab- scess and systemic sepsis. Occurring in approximately 0.2–0.5 % of patients, cholecystitis has been correlated to cholelithiasis and intraprocedural filling of the gall- bladder with CM, and it is heralded by the appearance clinically significant iatrogenic infections is limited (1– 3 %), but sepsis represents a common cause of death. Specific risk factors for infection include stenting across tumours, obstructed ducts and jaundice, combined percutaneous-endoscopic procedures, primary sclerosing cholangitis, incomplete or failed biliary drainage [2, 6]. Fig. 17 Perihepatic abscess diagnosed 10 days after endoscopic stent positioning through a cytologically benign distal CBD stricture in a 78- year-old female. Contrast-enhanced CT (a) requested because of fever and right hypochondrium pain revealed a fluid-like collection () with an enhancing peripheral rim and minimal extraluminal air (+) predomi- nantly in the right-sided retroperitoneum. Urgent surgery included chole- cystectomy and abscess drainage. Postoperatively, unenhanced T2- weighted MRI (b) depicted resolution of the abscess (arrowheads) Fig. Post-procedural infections 17 Perihepatic abscess diagnosed 10 days after endoscopic stent positioning through a cytologically benign distal CBD stricture in a 78- year-old female. Contrast-enhanced CT (a) requested because of fever and right hypochondrium pain revealed a fluid-like collection () with an enhancing peripheral rim and minimal extraluminal air (+) predomi- nantly in the right-sided retroperitoneum. Urgent surgery included chole- cystectomy and abscess drainage. Postoperatively, unenhanced T2- weighted MRI (b) depicted resolution of the abscess (arrowheads) 336 Insights Imaging (2015) 6:323–338 m and ffered a) and HU) e sur- hance- basal 3 l of stinking pus. Note the correctly positioned metallic CBD stent (arrowheads), and clinical, laboratory and imaging (* in c) resolution was obtained. During endoscopic replacement, the biliary stent was not found anymore (d), displaced and probably lost with stools. Endoscopic cholangiogram (e) confirmed persistent iatrogenic biliary stricture (arrow). Follow-up MRI (T2-weighted image in f) showed resolved subphrenic abscess with low signal intensity consistent with fibrosis () One month after evacuation of the haemoperitoneum and ar hepatic haematoma, the same patient in Fig. 10 suffered t-sided thoraco-abdominal pain and fever. Arterial- (a) and hase (b) CT showed a huge, fluid-attenuating (15 HU) c collection () with inflammatory enhancement of the sur- compressed liver parenchyma (+) and thin peripheral enhance- sistent with an abscess. Ipsilateral pleural effusion and basal ctasis were associated. Percutaneous drainage yielded 3 l of stinking pus. Note the correctly positioned metallic CBD stent (arrowheads), and clinical, laboratory and imaging (* in c) resolution was obtained. During endoscopic replacement, the biliary stent was not found anymore (d), displaced and probably lost with stools. Endoscopic cholangiogram (e) confirmed persistent iatrogenic biliary stricture (arrow). Follow-up MRI (T2-weighted image in f) showed resolved subphrenic abscess with low signal intensity consistent with fibrosis () stinking pus. Note the correctly positioned metallic CBD stent (arrowheads), and clinical, laboratory and imaging ( in c) resolution was obtained. During endoscopic replacement, the biliary stent was not found anymore (d), displaced and probably lost with stools. Endoscopic cholangiogram (e) confirmed persistent iatrogenic biliary stricture (arrow). Follow-up MRI (T2-weighted image in f) showed resolved subphrenic abscess with low signal intensity consistent with fibrosis () Fig. 18 One month after evacuation of the haemoperitoneum and subcapsular hepatic haematoma, the same patient in Fig. 10 suffered form right-sided thoraco-abdominal pain and fever. Fig. 19 In a 60-year-old male with severe chronic calcific pan- creatitis, after ERCP positioning of the metallic pancreatic stent (arrowhead in a) contrast- enhanced CT showed minimal intraperitoneal air (), which was treated conservatively with suc- cess. Note pneumobilia (arrows) and retained CM in the gallbladder Post-procedural infections Arterial- (a) and venous-phase (b) CT showed a huge, fluid-attenuating (15 HU) subphrenic collection () with inflammatory enhancement of the sur- rounding, compressed liver parenchyma (+) and thin peripheral enhance- ment, consistent with an abscess. Ipsilateral pleural effusion and basal lung atelectasis were associated. Percutaneous drainage yielded 3 l of References 22. Zaheer A, Singh VK, Qureshi RO et al (2013) The revised Atlanta classification for acute pancreatitis: updates in imaging terminology and guidelines. Abdom Imaging 38:125–136 1. Adler DG, Baron TH, Davila RE et al (2005) ASGE guideline: the role of ERCP in diseases of the biliary tract and the pancreas. Gastrointest Endosc 62:1–8 23. Sheu Y, Furlan A, Almusa O et al (2012) The revised Atlanta classi- fication for acute pancreatitis: a CT imaging guide for radiologists. Emerg Radiol 19:237–243 2. Silviera ML, Seamon MJ, Porshinsky B et al (2009) Complications related to endoscopic retrograde cholangiopancreatography: a com- prehensive clinical review. J Gastrointestin Liver Dis 18:73–82 24. Pezzilli R, Romboli E, Campana D et al (2002) Mechanisms involved in the onset of post-ERCP pancreatitis. JOP 3:162–168 3. Glomsaker TB, Hoff G, Kvaloy JT et al (2013) Patient-reported out- c o m e m e a s u r e s a f t e r e n d o s c o p i c r e t r o g r a d e cholangiopancreatography: a prospective, multicentre study. Scand J Gastroenterol 48:868–876 25. Pfau PR, Mosley RG, Said A et al (2006) Comparison of the effect of non-ionic and ionic contrast agents on pancreatic histology in a ca- nine model. JOP 7:27–33 26. Johnson GK, Geenen JE, Johanson JF et al (1997) Evaluation of post- ERCP pancreatitis: potential causes noted during controlled study of differing contrast media. Midwest Pancreaticobiliary Study Group. Gastrointest Endosc 46:217–222 4. Siriwardana HP, Siriwardena AK (2005) Systematic appraisal of the role of metallic endobiliary stents in the treatment of benign bile duct stricture. Ann Surg 242:10–19 5. van Boeckel PG, Vleggaar FP, Siersema PD (2009) Plastic or metal stents for benign extrahepatic biliary strictures: a systematic review. BMC Gastroenterol 9:96 27. Goebel C, Hardt P, Doppl W et al (2000) Frequency of pancreatitis after endoscopic retrograde cholangiopancreatography with iopromid or iotrolan: a randomized trial. Eur Radiol 10:677–680 6. Anderson MA, Fisher L, Jain R et al (2012) Complications of ERCP. Gastrointest Endosc 75:467–473 28. George S, Kulkarni AA, Stevens G et al (2004) Role of osmolality of contrast media in the development of post-ERCP pancreatitis: a met- analysis. Dig Dis Sci 49:503–508 7. Kapral C, Duller C, Wewalka F et al (2008) Case volume and out- come of endoscopic retrograde cholangiopancreatography: results of a nationwide Austrian benchmarking project. Endoscopy 40:625– 630 29. Stent-related complications of luminal overdistension and circumferential mural thickening (Fig. 15). Septic cholangitis is suggested by thickened enhancing CBD and intrahepatic duct walls (Fig. 16). Accessory signs of sepsis such as atelectasis/ pneumonia, splenic or renal infarcts may be observed (Fig. 16). Alternatively, sepsis may result from superin- fection of PNPFCs or abnormal collections from DP (Fig. 13). Abscess collections usually display enhancing mural thickening, similar attenuation to that of non- infected fluid collections and sometimes internal gas bubbles (Figs. 17 and 18) [6, 13, 15]. Relatively rare, early complications after biliary stenting in- clude haemorrhage (Fig. 10), PEAP, stent misplacement, per- foration (Fig. 19) and injury to the CBD or main pancreatic duct [13, 17]. Chronic stent-related complications are more common and include stent obstruction (25–35 % of patients), migration, fracture or collapse, infections such cholangitis, liver abscess (Fig. 18) or sepsis. Biliary stent dislodgement (Fig. 18) occurs after a variable time interval in up to 6 % of patients Fig. 19 In a 60-year-old male with severe chronic calcific pan- creatitis, after ERCP positioning of the metallic pancreatic stent (arrowhead in a) contrast- enhanced CT showed minimal intraperitoneal air (), which was treated conservatively with suc- cess. Note pneumobilia (arrows) and retained CM in the gallbladder 337 Insights Imaging (2015) 6:323–338 10. Glomsaker T, Hoff G, Kvaloy JT et al (2013) Patterns and predictive factors of complications after endoscopic retrograde cholangiopancreatography. Br J Surg 100:373–380 and is more frequent with plastic stents compared to SEMS. Displacement may be proximal, particularly in patients with malignant obstruction, or alternatively dis- tal to the intestine, the latter not unusually asymptomat- ic [4, 5, 17, 43, 44]. and is more frequent with plastic stents compared to SEMS. Displacement may be proximal, particularly in patients with malignant obstruction, or alternatively dis- tal to the intestine, the latter not unusually asymptomat- ic [4, 5, 17, 43, 44]. 11. Cotton PB (2006) Analysis of 59 ERCP lawsuits; mainly about indi- cations. Gastrointest Endosc 63:378–382, quiz 464 12. Sartelli M, Viale P, Catena F et al (2013) 2013 WSES guidelines for management of intra-abdominal infections. World J Emerg Surg 8:3 13. Pannu HK, Fishman EK (2001) Complications of endoscopic retro- grade cholangiopancreatography: spectrum of abnormalities demon- strated with CT. Radiographics 21:1441–1453 Conclusion 14. Paspatis GA, Dumonceau JM, Barthet M et al (2014) Diagnosis and management of iatrogenic endoscopic perforations: European Society of Gastrointestinal Endoscopy (ESGE) position statement. Endoscopy 46:693–711 Diagnosis and management of post-ERCP complications should combine clinical and laboratory data with imaging ap- pearances. Urgent CT imaging is crucial to promptly identify and categorise the most common and unusual complications after ERCP, particularly to triage those occurrences that re- quire prolonged in-hospital treatment or surgical reoperation. Knowledge of ERCP procedural details, the appropriate CT acquisition technique and familiarity with normal appearances are needed to diagnose and grade oedematous or necrotic PEAP, bleeding, DP and infections, which may have similar, nonspecific clinical and laboratory manifestations [2, 6, 12]. 15. Wax BN, Katz DS, Badler RL et al (2006) Complications of abdom- inal and pelvic procedures: computed tomographic diagnosis. Curr Probl Diagn Radiol 35:171–187 16. Woods RW, Akshintala VS, Singh VK et al (2014) CT severity of post-ERCP pancreatitis: results from a single teriary medical center. Abdom Imaging 39:1162–1168 17. Catalano O, De Bellis M, Sandomenico F et al (2012) Complications of biliary and gastrointestinal stents: MDCT of the cancer patient. AJR Am J Roentgenol 199:W187–W196 18. Dubecz A, Ottmann J, Schweigert M et al (2012) Management of ERCP-related small bowel perforations: the pivotal role of physical investigation. Can J Surg 55:99–104 19. Zissin R, Shapiro-Feinberg M, Oscadchy A et al (2000) Retroperitoneal perforation during endoscopic sphincterotomy: im- aging findings. Abdom Imaging 25:279–282 Open Access This article is distributed under the terms of the Creative Commons Attribution License which permits any use, distribution, and reproduction in any medium, provided the original author(s) and the source are credited. 20. Tonolini M (2014) Reversible acute duodenitis as a complication of endoscopic biliary stenting. J Gastrointestin Liver Dis 23:8 21. Dumonceau JM, Andriulli A, Elmunzer BJ et al (2014) Prophylaxis of post-ERCP pancreatitis: European Society of Gastrointestinal Endoscopy (ESGE) Guideline-updated June 2014. Endoscopy 46: 799–815 References Miller FH, Keppke AL, Dalal K et al (2004) MRI of pancreatitis and its complications: part 1, acute pancreatitis. AJR Am J Roentgenol 183:1637–1644 8. Stapfer M, Selby RR, Stain SC et al (2000) Management of duodenal perforation after endoscopic retrograde cholangiopancreatography and sphincterotomy. Ann Surg 232:191–198 30. de Freitas Tertulino F, Schraibman V, Ardengh JC et al (2014) Diffusion-weighted magnetic resonance imaging indicates the sever- ity of acute pancreatitis. Abdom Imaging 9. Kwon CI, Song SH, Hahm KB et al (2013) Unusual complications related to endoscopic retrograde cholangiopancreatography and its endoscopic treatment. Clin Endosc 46:251–259 31. Horton KM, Jeffrey RB Jr, Federle MP et al (2009) Acute gastroin- testinal bleeding: the potential role of 64 MDCT and 3D imaging in the diagnosis. Emerg Radiol 16:349–356 338 Insights Imaging (2015) 6:323–338 32. Jaeckle T, Stuber G, Hoffmann MH et al (2008) Acute gas- trointestinal bleeding: value of MDCT. Abdom Imaging 33: 285–293 38. Miller R, Zbar A, Klein Y et al (2013) Perforations following endo- scopic retrograde cholangiopancreatography: a single institution ex- perience and surgical recommendations. Am J Surg 206:180–186 33. So YH, Choi YH, Chung JW (2012) Selective embolization for post- endoscopic sphincterotomy bleeding: technical aspects and clinical efficacy. Korean J Radiol 13:73–81 39. Avgerinos DV, Llaguna OH, Lo AY et al (2009) Management of endoscopic retrograde cholangiopancreatography: related duodenal perforations. Surg Endosc 23:833–838 34. Katsinelos P, Kountouras J, Chatzimavroudis G (2010) Endoscopic hemostasis using monopolar coagulation for postendoscopic sphincterotomy bleeding refractory to injection treatment. Surg Laparosc Endosc Percutan Tech 20:84–88 40. Machado NO (2012) Management of duodenal perforation post- endoscopic retrograde cholangiopancreatography. When and whom to operate and what factors determine the outcome? A review article. JOP 13:18–25 35. Tsou YK, Lin CH, Liu NJ (2009) Treating delayed endoscop- ic sphincterotomy-induced bleeding: epinephrine injection with or without thermotherapy. World J Gastroenterol 15:4823– 4828 41. Polydorou A, Vezakis A, Fragulidis G et al (2011) A tailored ap- proach to the management of perforations following endoscopic ret- rograde cholangiopancreatography and sphincterotomy. J Gastrointest Surg 15:2211–2217 36. Maleux G, Bieden J, Laenen A et al (2014) Embolization of post- biliary sphincterotomy bleeding refractory to medical and endoscopic therapy: technical results, clinical efficacy and predictors of outcome. Eur Radiol 24:2779–2786 42. References Rabie ME, Mir NH, Al Skaini MS et al (2013) Operative and non- o p e r a t i v e m a n a g e m e n t o f e n d o s c o p i c r e t r o g r a d e cholangiopancreatography-associated duodenal injuries. Ann R Coll Surg Engl 95:285–290 43. Matlock J, Freeman ML (2005) Endoscopic therapy of benign biliary strictures. Rev Gastroenterol Disord 5:206–214 37. Kim J, Lee SH, Paik WH et al (2012) Clinical outcomes of patients who experienced perforation associated with endo- scopic retrograde cholangiopancreatography. Surg Endosc 26: 3293–3300 44. Judah JR, Draganov PV (2007) Endoscopic therapy of benign biliary strictures. World J Gastroenterol 13:3531–3539
https://openalex.org/W2903052179	https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0207880&type=printable	English	null	Infected cyst in patients with autosomal dominant polycystic kidney disease: Analysis of computed tomographic and ultrasonographic imaging features	PloS one	2,018	cc-by	7,963	RESEARCH ARTICLE Materials & methods The institutional review board approved this retrospective study. Fifty-one episodes with proven cyst infection in forty-three ADPKD patients were included. Two experienced abdominal radiologists reviewed CT and US images and evaluated the following imaging features in consensus: cyst size, location, cyst shape, intracystic attenuation, intracystic echogenicity, intracystic heterogeneity, wall thickness, the presence of fluid-fluid level, sep- tation, intracystic gas, pericystic fat infiltration, and pericystic hyperemia. Intracystic attenua- tion was measured for all infected cysts and two presumed normal cysts and compared using the Wilcoxon rank-sum test. Editor: Giovanna Valenti, Universita degli Studi di Bari Aldo Moro, ITALY Copyright: © 2018 Oh et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. OPEN ACCESS To investigate the imaging features of cyst infection in autosomal dominant polycystic kid- ney disease (ADPKD) patients using computed tomography (CT) and ultrasonography (US). Citation: Oh J, Shin C-I, Kim SY (2018) Infected cyst in patients with autosomal dominant polycystic kidney disease: Analysis of computed tomographic and ultrasonographic imaging features. PLoS ONE 13(12): e0207880. https://doi. org/10.1371/journal.pone.0207880 Infected cyst in patients with autosomal dominant polycystic kidney disease: Analysis of computed tomographic and ultrasonographic imaging features Jiseon OhID1,2, Cheong-Il ShinID1,2, Sang Youn Kim1,2 Jiseon OhID1,2, Cheong-Il ShinID1,2, Sang Youn Kim1,2 1 Department of Radiology, Seoul National University Hospital, Seoul, Korea, 2 Department of Radiology, Seoul National University College of Medicine, Seoul, Korea 1 Department of Radiology, Seoul National University Hospital, Seoul, Korea, 2 Department of Radiology, Seoul National University College of Medicine, Seoul, Korea a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 * cheongil.tree@gmail.com * cheongil.tree@gmail.com Results On CT scans, the median size of infected cysts was 5.5 cm (range: 2.3–18.8 cm) and 46 of 51 (90.2%) infected cysts were located in the subcapsular region. Most (48 of 51, 94.1%) infected cysts showed lobulated, focal bulging or irregular shape. Discernible wall thickening (84.1%) was the most frequently found imaging feature of infected cysts followed by rela- tively higher intracystic attenuation compared to normal cysts (79.1%) and pericystic fat infil- tration (52.9%). Fluid/fluid level was found in 3 of 51 (5.9%) infected cysts and intracystic gas was found in 3 of 51 (5.9%) infected cysts, respectively. For hepatic cysts, 11 of 14 (78.6%) infected cysts showed pericystic hyperemia. Intracystic attenuation was signifi- cantly higher in infected cysts (median; 19.0 HU) than in presumed normal cysts (median; 8.5 HU) (P<0.001), and exceeded 25 HU in 18 (35.3%) of 51 infected cysts. Among the 41 Imaging features of infected cysts in ADPKD infected cysts for which US images were available, 35 (85.1%) showed heterogeneous echogenicity. infected cysts for which US images were available, 35 (85.1%) showed heterogeneous echogenicity. percutaneous catheter drainage; US, ultrasonography; WBC, white blood cell. percutaneous catheter drainage; US, ultrasonography; WBC, white blood cell. percutaneous catheter drainage; US, ultrasonography; WBC, white blood cell. percutaneous catheter drainage; US, ultrasonography; WBC, white blood cell. Conclusion Minute imaging features such as minimal wall thickening or relatively high attenuation com- pared to normal cysts would be helpful to detect infected cysts in ADPKD patients. Data Availability Statement: All relevant data are within the manuscript. Funding: The authors received no specific funding for this work. Funding: The authors received no specific funding for this work. Competing interests: The authors have declared that no competing interests exist. Abbreviations: ADPKD, Autosomal dominant polycystic kidney disease; CRP, C-reactive protein; CT, computed tomography; GFR, glomerular filtration rate; HU, Hounsfield units; PCD, PLOS ONE \| https://doi.org/10.1371/journal.pone.0207880 December 5, 2018 1 / 16 Introduction Autosomal dominant polycystic kidney disease (ADPKD) is the most common hereditary renal disease, affecting 1 in 500 to 1 in 1,000 live births and 8–10% of patients with end-stage renal disease [1]. It is characterized by the development of numerous cysts in the kidney and liver parenchyma, which arise from various renal tubular segments and biliary ducts, which lead to increased kidney and liver size, respectively. Cysts are also associated with some of the most common complications of ADPKD such as intracystic hemorrhage, gross hematuria, obstruction mainly caused by liver cysts, and infections [2, 3]. Urinary tract infection, includ- ing cystic infection, is one of the major complications of polycystic kidney disease and occurs with an annual frequency of 0.01 episodes per patient [4]. Early diagnosis of cystic infection is clinically important, because cystic infection can be treated with antibiotics only if it is diag- nosed early; in addition, the patient may die from sepsis if proper drainage is not performed in severe cases [5, 6]. The diagnosis of cyst infection is confirmed by extracting cyst fluid from the suspected cyst to help identify the microorganism or inflammatory findings such as neutrophils debris [4]. However, it is challenging to detect infected cysts in ADPKD patients with numerous cysts in the kidney or liver. Computed tomography (CT), which is a readily available and widely acces- sible cross-sectional imaging modality with a high spatial resolution, can be used. However, CT is believed to have a limited value in diagnosing infected cysts in patients with ADPKD because of a low sensitivity of less than 25% [7–9]. Positron emission tomography with fluoro- deoxyglucose (FDG-PET) has recently been investigated as a promising diagnostic tool [8, 10– 12] along with magnetic resonance imaging (MRI), especially diffusion-weighted imaging (DWI) as an alternative diagnostic modality for infected cysts [13, 14]. CT diagnostic criteria of enhanced wall thickening and/or perilesional inflammation that have been described in most previous studies are somewhat subjective and unclear [4, 7, 8, 10, 11]. In addition, imag- ing feature analysis of cyst infection in ADPKD patients using CT has rarely been performed. Therefore, we investigated the CT imaging features of cyst infection in ADPKD patients. The imaging features of ultrasonography (US) were also investigated in cases for which US data were available, given that US features of cyst infection have also not been reported in a case series or case-control studies. Diagnosis of cyst infection The diagnosis of cyst infection was established with the aspiration of cyst fluid and made in 1 of 3 ways: (1) cyst fluid showing complete pus-like or turbid features and/or (2) cyst fluid showing neutrophils debris and/or (3) cyst fluid showing microorganism [6]. Among 51 episodes with infected cysts, renal cysts and hepatic cysts comprised 31 and 20 episodes, respectively. Patients The institutional review board approved this retrospective study (Seoul National University Hospital Institutional Review Board; H-1706-012-855), with a waiver of informed consent. From January 2008 to December 2016, 93 patients with ADPKD were admitted to the hospital for clinically suspected cyst infection (Fig 1). Among a total of 104 episodes, the episodes that PLOS ONE \| https://doi.org/10.1371/journal.pone.0207880 December 5, 2018 2 / 16 Imaging features of infected cysts in ADPKD Fig 1. Flowchart for inclusion and exclusion of patients. Note: ADPKD = autosomal dominant polycystic kidney disease; PCD = Percutaneous catheter drainage. https://doi.org/10.1371/journal.pone.0207880.g001 Fig 1. Flowchart for inclusion and exclusion of patients. Note: ADPKD = autosomal dominant polycystic kidney disease; PCD = Percutaneous catheter drainage. https://doi.org/10.1371/journal.pone.0207880.g001 https://doi.org/10.1371/journal.pone.0207880.g001 met all three of the following criteria were included for CT features analysis in infected cysts: (1) the patient should have been undergone percutaneous catheter drainage (PCD) or aspira- tion for suspected infected cysts, (2) pre-procedural abdominal CT images should have been obtained less than 1 week before PCD or aspiration, and (3) histological reports of cyst fluid are available. Exclusion criteria as follows: (1) episodes with a history of recent PCD insertion within 2 weeks previously (n = 4), (2) episodes in which aspirated cysts were not localized on CT images (n = 12), (3) episodes of renal abscess (n = 2), and (4) episodes without fulfilment of the diagnostic criteria for infected cyst (n = 9). Finally, 51 episodes of 43 ADPKD patients with proven cyst infection formed the study group for the CT feature analysis in infected cysts (12 men and 39 women; age 54.7 ± 10.4 years, age range 28–74 years). US images used as procedure guidance were available for 41 episodes in 37 patients. In addition, CT imaging features were assessed for the nine episodes excluded because aspirates showed no evidence of cyst infection; however, statistical analysis was not performed because of the small number of cases. Imaging protocol All CT examinations were performed using multi-detector computed tomography (MDCT) scanners (Brilliance 64, Philips Medical Systems, Cleveland, Ohio; Aquilion ONE, Toshiba Medical Systems, Otawara, Japan; Sensation 16 Speed 4D, Siemens Medical Solutions, For- chheim, Germany). Scanning parameters for MDCT scanners included a gantry rotation time of 0.5 seconds, a pitch of 1.0 to 1.5, 150 to 230 mAs, 100 to 120 kVp, and a 512 x 512 matrix. The reconstruction parameters were a 3 mm slice thickness and a 2 mm or 3 mm reconstruc- tion interval, both of which are standard for axial mages. Of the 51 episodes, 7 episodes have only pre-contrast images, 8 episodes have only portal venous phase images, and the remaining 36 cases have pre-contrast, arterial and portal venous/delayed images. Post-contrast CT scans were obtained after the administration of 1.5 mL/kg of nonionic contrast material with 350 mgI/mL for 30 seconds at a rate of 2.5 to 4.0 mL/s using a power injector. This injection was followed by a saline flush of 40 mL with the same injection rate as that of the contrast material. For arterial phase scanning, a 15-second delay was used after the attenuation of the descending aorta reached 100 Hounsfield Unit (HU) using bolus tracking. The portal venous phase was attained 35 seconds after the acquisition of the arterial phase or 70 seconds after the beginning of the contrast injection. Delayed phase (equilibrium phase) images were obtained 180 seconds after the start of contrast administration. Clinical variables Patient medical records were reviewed; in addition, demographic and clinical data including age, sex, dialysis status, white blood cell (WBC) count, serum level of creatinine and C-reactive protein (CRP), glomerular filtration rate (GFR), and results from the cyst fluid/blood/urine culture study were collected. The highest WBC count and serum CRP level within 1 week after the symptom onset were recorded. Serum creatinine level within 2 weeks before admission was recorded and the GFR PLOS ONE \| https://doi.org/10.1371/journal.pone.0207880 December 5, 2018 3 / 16 Imaging features of infected cysts in ADPKD was estimated by using the modified isotope dilution mass spectrometry (IDMS)-traceable 4-variable Modification of Diet in Renal Disease (MDRD) study equation. Blood and urine culture samples were cultured from all patients at the time of admission; however, some patients had already received antibiotics. PLOS ONE \| https://doi.org/10.1371/journal.pone.0207880 December 5, 2018 Image analysis CT and US images were assessed by a consensus reading of two radiologists with 11 years (C.I. S) and 12 years (S.Y.K) of experience in abdominal imaging. On CT images, we evaluated the following imaging features: cyst size measured using maximum axial diameter; cyst shape classified into a round, lobulated, and focal bulging/ irregular shape; and cyst location classified into subcapsular, parenchymal, and hilar. Intra- cystic attenuation (HU) of the infected cyst was also measured on pre-contrast or post-con- trast images. To compare attenuation difference between an infected and normal cyst, attenuation was measured in two presumed normal cysts that showed no discernible wall, water attenuation, and no evidence of perilesional inflammation. In addition, we assessed whether each cyst appeared relatively hyperdense compared to surrounding normal cysts in the abdominal soft tissue window setting in a subjective manner but in consensus, and whether it showed heterogeneous attenuation. Pericystic hyperemia was evaluated only for hepatic cysts and defined as gross hyperenhancement outside of the cyst regardless of shape (wedge shape or circumferential) on arterial phase images. When a cyst wall was not imper- ceptible, it was considered to have discernible wall thickening, and in the 4× magnified image, wall thickness was measured three times and the largest measured value was recorded. We also evaluated whether each cyst showed fluid-fluid level, intracystic gas, and pericystic fat infiltration. On US images, intracystic echogenicity was classified into anechoic versus echoic type. In terms of homogeneity, homogeneous echogenicity was defined as uniform intracystic echo- genicity, and all other cases were considered heterogeneous echogenicity. We also evaluated whether each cyst showed fluid-fluid level and septation. 4 / 16 PLOS ONE \| https://doi.org/10.1371/journal.pone.0207880 December 5, 2018 Imaging features of infected cysts in ADPKD Statistical analysis Continuous variables are expressed as the mean ± standard deviation (SD) or median with range, according to the distribution of the data which was tested using the Shapiro–Wilk normality test. Categorical data are expressed as numbers (percentages). The attenuation of infected and normal cysts was compared using the Wilcoxon rank-sum test. Probability (P) value less than 0.05 was considered to indicate statistical significance. All statistical analyses were performed using software (MedCalc for Windows, version 17.6; MedCalc, Mariakerke, Belgium). https://doi.org/10.1371/journal.pone.0207880.t001 Clinical characteristics Among 51 episodes, 25 patients had 31 episodes of renal cyst infection, and 18 patients had 20 episodes of hepatic cyst infection. Median WBC count was 9,100/mm3 (range: 2,600–21,000/ mm3) and median serum level of CRP was 14.2 mg/dL (range: 2.6–35.0 mg/dL). Table 1 sum- marizes the demographic and clinical information. Microbiological documentation was available for 28 of 51 episodes (54.9%) (Table 2); posi- tive cyst fluid culture was noted in 24 episodes, positive blood culture was noted in 13 episodes, and positive urine culture was noted in 17 episodes. Escherichia coli accounted for 18 (64.3%) of 28 retrieved bacterial strains. Cyst fluid culture was positive in all cases of 19 renal cyst infec- tion except one. In fluid culture-negative case, blood and urine cultures were positive and the diagnosis of cyst infection was made because the aspirate was pus. There was no episode that was positive only in the urine culture. In addition, of the 9 episodes of hepatic cyst infection, 3 episodes were positive only in the blood culture (Escherichia coli, Klebsiella pneumoniae, and Table 1. Clinical characteristics of 51 episodes of ADPKD patients with cyst infection. Episodes Gender, n (%) Male 12 (23.5%) Female 39 (76.5%) Age (years) mean ± SD 54.7 ± 10.4 eGFR (ml/min per 1.73 m2) median (range) 20.7 (3.0–113.8) Patients on Dialysis, n (%) Yes 32 (62.8%) No 19 (37.2%) WBC Count (x103/mm3) median (range) 9.1 (2.6–21.7) CRP (mg/dL) median (range) 14.2 (2.6–35.0) Culture study of cystic fluid, n (%) Positive 24 (47.1%) Negative 27 (52.9%) Note: Continuous data expressed as the mean and SD (in case of the normal distribution) or the median and range (in case of violation of the normality assumption). Note: ADPKD = autosomal dominant polycystic kidney disease; SD = standard deviation; eGFR = estimated glomerular filtration rate; WBC = white blood cell; CRP = C-reactive protein https://doi.org/10.1371/journal.pone.0207880.t001 Table 1. Clinical characteristics of 51 episodes of ADPKD patients with cyst infection. Note: Continuous data expressed as the mean and SD (in case of the normal distribution) or the median and range (in case of violation of the normality assumption). PLOS ONE \| https://doi.org/10.1371/journal.pone.0207880 December 5, 2018 Clinical characteristics Note: ADPKD = autosomal dominant polycystic kidney disease; SD = standard deviation; eGFR = estimated glomerular filtration rate; WBC = white blood cell; CRP = C-reactive protein Note: Continuous data expressed as the mean and SD (in case of the normal distribution) or the median and range (in case of violation of the normality assumption). Note: ADPKD = autosomal dominant polycystic kidney disease; SD = standard deviation; eGFR = estimated glomerular filtration rate; WBC = white blood cell; CRP = C-reactive protein 5 / 16 PLOS ONE \| https://doi.org/10.1371/journal.pone.0207880 December 5, 2018 Imaging features of infected cysts in ADPKD Table 2. Bacterial strains retrieved during 51 episodes of cyst infection in 43 patients with ADPKD. Infection (episodes) Positive culture Cyst Fluid Blood Urine Renal cyst infection (19 of 31 episodes, 61.3%) Total (18 of 31 episodes, 58.1%) Total (8 of 31 episodes, 25.8%) Total (17 of 31 episodes, 54.8%) Escherichia coli (14) Staphylococcus aureus (1) Enterococcus faecium (1) Klebsiella pneumoniae (1) Klebsiella oxytoca (1) Escherichia coli (6) Staphylococcus aureus (1) Enterococcus faecium (1) Escherichia coli (12) Staphylococcus aureus (1) Staphylococcus aureus (1) Enterococcus faecium (1) Klebsiella pneumoniae (1) Klebsiella oxytoca (1) Hepatic cyst infection (9 of 20 episodes, 45.0%) Total (6 of 20 episodes, 30%) Total (5 of 20 episodes, 25%) Escherichia coli (3) Klebsiella pneumoniae (2) Pseudomonas aeruginosa (1) Escherichia coli (1) Klebsiella pneumoniae (1) Escherichia coli (1) Klebsiella pneumoniae (1) Enterococcus faecium (1) Enterococcus faecium), not in the cyst fluid. Seventeen percent of Escherichia coli (3 of 18) and twenty percent of Klebsiella species (1 of 5) were extended-spectrum beta-lactamase (ESBL)- producing strains. In addition, fifty percent of Staphylococcus aureus (one of two) and Entero- coccus species (one of two) strains were resistant to methicillin and amoxicillin. Enterococcus faecium), not in the cyst fluid. Seventeen percent of Escherichia coli (3 of 18) and twenty percent of Klebsiella species (1 of 5) were extended-spectrum beta-lactamase (ESBL)- producing strains. In addition, fifty percent of Staphylococcus aureus (one of two) and Entero- coccus species (one of two) strains were resistant to methicillin and amoxicillin. CT imaging features Morphologic evaluation. Fig 2 and Table 3 summarize the CT findings. The median sizes of the infected renal and hepatic cysts were 5.5 cm (range: 2.9–11.6 cm) and 5.5 cm (range: 2.3–18.8 cm), respectively. Most infected cysts (30 of 31 renal cysts and 18 of 20 hepatic cysts) showed a lobulated, focal bulging or irregular shape. All 31 episodes of infected renal cysts and most of infected hepatic cysts (15 of 20 episodes, 75%) were located in the subcapsular region, and the remaining infected hepatic cysts (5 of 20 episodes, 25%) were located in the interstitial region. Fluid/fluid level was found in 3 of 51 (5.9%) infected cysts and intracystic gas was found in 3 of 51 (5.9%) infected cysts, respectively. In addition, 23 of 31 (74.2%) infected renal cysts and 4 of 20 (20%) infected hepatic cysts had pericystic fat infiltration (Fig 3). Attenuation evaluation. The median attenuation of infected cysts was 19 HU (range: 5–67 HU) on pre-contrast images and 21 HU (range: 7–79 HU) on post-contrast images. Among the 51 episodes, intracystic attenuation exceeded 25 HU in 18 episodes (35.3%) and 50 HU in 4 episodes (7.8%). Infected cysts also appeared to have relatively high attenuation com- pared to the surrounding normal cysts in 34 of 43 episodes (79.1%) on pre-contrast images and 26 of 44 episodes (59.1%) on post-contrast images. Of the 36 infected cysts for which both pre-contrast and post-contrast images were available, 28 episodes matched each other and 7 and 1 episodes showed subjective higher attenuation than the surrounding normal cysts on only pre-contrast and post-contrast images, respectively (Fig 4). The median attenuation value of the 36 infected cysts was 18.0 HU and 19.5 HU in pre-contrast and post-contrast images, respectively. Measuring the attenuation difference between the infected cyst and presumed normal cyst revealed that intracystic attenuation was significantly higher in the infected cysts than in the presumed normal cysts for both pre-contrast and post-contrast images (Fig 5 and Table 4). 6 / 16 PLOS ONE \| https://doi.org/10.1371/journal.pone.0207880 December 5, 2018 Imaging features of infected cysts in ADPKD Mapping for CT imaging features in 51 episodes of cyst infection in 43 patients with ADPKD. Note: = autosomal dominant polycystic kidney disease; Pre = pre-contrast images; AP = arterial phase images; rtal venous phase images; DP = delayed phase images; R, renal cyst; H, Hepatic cyst. PLOS ONE \| https://doi.org/10.1371/journal.pone.0207880 December 5, 2018 CT imaging features oi.org/10.1371/journal.pone.0207880.g002 Mapping for CT imaging features in 51 episodes of cyst infection in 43 patients w D = autosomal dominant polycystic kidney disease; Pre = pre-contrast images; AP = rtal venous phase images; DP = delayed phase images; R, renal cyst; H, Hepatic cys Mapping for CT imaging features in 51 episodes of cyst infection in 43 patients with ADPKD. Note: D = autosomal dominant polycystic kidney disease; Pre = pre-contrast images; AP = arterial phase images; rtal venous phase images; DP = delayed phase images; R, renal cyst; H, Hepatic cyst. oi.org/10.1371/journal.pone.0207880.g002 Fig 2. Mapping for CT imaging features in 51 episodes of cyst infection in 43 patients with ADPKD. Note: ADPKD = autosomal dominant polycystic kidney disease; Pre = pre-contrast images; AP = arterial phase images; PP = portal venous phase images; DP = delayed phase images; R, renal cyst; H, Hepatic cyst. https://doi.org/10.1371/journal.pone.0207880.g002 Fig 2. Mapping for CT imaging features in 51 episodes of cyst infection in 43 patients with ADPKD. Note: ADPKD = autosomal dominant polycystic kidney disease; Pre = pre-contrast images; AP = arterial phase images; PP = portal venous phase images; DP = delayed phase images; R, renal cyst; H, Hepatic cyst. 7 / 16 PLOS ONE \| https://doi.org/10.1371/journal.pone.0207880 December 5, 2018 peremia is evaluated only in hepatic cyst. Note: Wall thickness is measured only when there is discernible wall thickening mean and SD (in case of the normal distribution) or median and range (in case of violation of the normality assumption); Note: Continuous data expressed as the mean and SD (in case of the normal distribution) or median and range (in case of violation of the normality assumption); HU = Hounsfield Unit. Note: Perilesional hyperemia is evaluated only in hepatic cyst. Note: Wall thickness is measured only when there is discernible wall thickening https://doi.org/10.1371/journal.pone.0207880.t003 Imaging features of infected cysts in ADPKD Table 3. CT findings from 51 episodes of cyst infection in 43 patients with ADPKD. Pre or Post-contrast Available episodes, n Renal cyst infection Hepatic cyst infection Total 31 20 51 Size (cm) median (range) 5.5 (2.9–11.6) 5.5 (2.3–18.8) 5.5 (2.3–18.8) Shape, n (%) Round 1 (3.2%) 2 (10%) 3 (5.9%) Lobulating 10 (32.3%) 11 (55%) 21 (41.2%) Focal bulging or irregular 20 (64.5%) 7 (35%) 27 (52.9%) Location, n (%) Subcapsular 31 (100%) 15 (75%) 46 (90.2%) Interstitial 0 (0%) 5 (25%) 5 (9.8%) Fluid/fluid level, n (%) Yes 1 (3.2%) 2 (10%) 3 (5.9%) No 30 (96.8%) 18 (90%) 48 (94.1%) Presence of intracystic gas, n (%) Yes 2 (6.5%) 1 (5%) 3 (5.9%) No 29 (93.5%) 19 (95%) 48 (94.1%) Heterogeneous attenuation, n (%) Yes 10 (32.3%) 5 (25%) 15 (29.4%) No 21 (67.7%) 15 (75%) 36 (70.6%) Presence of pericystic fat infiltration, n (%) Yes 23 (74.2%) 4 (20%) 27 (52.9%) No 5 (16.1%) 4 (20%) 9 (17.6%) N/A 3 (9.7%) 12 (60%) 15 (29.4%) Pre-contrast Available episodes, n Renal cyst infection Hepatic cyst infection Total 26 17 43 Intracystic attenuation (HU) median (range) 19.0 (9–67) 19.0 (5–43) 19.0 (5–67) Relatively high attenuation, n (%) Yes 19 (73.1%) 15 (88.2%) 34 (79.1%) No 7 (26.9%) 2 (11.8%) 9 (20.9%) Arterial phase Available episodes, n Renal cyst infection Hepatic cyst infection Total 22 14 36 Perilesional hyperemia, n (%) Yes 0 (0%) 11 (78.6%) 11 (30.6%) No 0 (0%) 2 (14.3%) 2 (5.5%) N/A 22 (100%) 1 (7.1%) 23 (63.9%) Portal or delayed phase Available episodes, n Renal cyst infection Hepatic cyst infection Total 27 17 44 Intracystic attenuation (HU), n (%) median (range) 22.0 (7–79) 20.0 (7–44) 21.0 (7–79) Relatively high attenuation, n (%) Yes 17 (63.0%) 9 (52.9%) 26 (59.1%) No 10 (37.0%) 8 (47.1%) 18 (40.9%) Discernible wall thickening, n (%) Yes 26 (96.3%) 11 (64.7%) 37 (84.1%) No 1 (3.7%) 6 (35.3%) 7 (15.9%) Wall thickness (mm) median (range) 2.3 (1.5–3.9) 1.9 (1.5–3.3) 2.1 (1.5–3.9) Note: Continuous data expressed as the mean and SD (in case of the normal distribution) or median and range (in case of violation of the normality assumption); Table 3. CT findings from 51 episodes of cyst infection in 43 patients with ADPKD. Note: Continuous data expressed as the mean and SD (in case of the normal distribution) or median and range (in case of violation of the normality assumption); HU = Hounsfield Unit. Note: Perilesional hyperemia is evaluated only in hepatic cyst. Note: Wall thickness is measured only when there is discernible wall thickening d as the mean and SD (in case of the normal distribution) or median and range (in case of violation of the normality assumpt rilesional hyperemia is evaluated only in hepatic cyst. Note: Wall thickness is measured only when there is discernible wall th Note: Continuous data expressed as the mean and SD (in case of the normal distribution) or median and range (in case of violation of the normality assumption); HU = Hounsfield Unit. Note: Perilesional hyperemia is evaluated only in hepatic cyst. Note: Wall thickness is measured only when there is discernible wall thickening PLOS ONE \| https://doi.org/10.1371/journal.pone.0207880 December 5, 2018 8 / 16 Imaging features of infected cysts in ADPKD Fig 3. A 65-year-old female patient with an infected cyst (R10 in Fig 2). (A, B) Pre-contrast CT image shows a 4.5 cm sized cyst with a lobulated shape in left kidney lower pole (dotted arrows). The cyst has pericystic infiltration (arrows). (C) On US scan, the cyst (arrows) appears heterogeneous and echogenic. Fig 3. A 65-year-old female patient with an infected cyst (R10 in Fig 2). (A, B) Pre-contrast CT image shows a 4.5 cm sized cyst with a lobulated shape in left kidney lower pole (dotted arrows). The cyst has pericystic infiltration (arrows). (C) On US scan, the cyst (arrows) appears heterogeneous and echogenic. https://doi.org/10.1371/journal.pone.0207880.g003 Enhancement-related evaluation. On post-contrast CT images, 37 of 51 (84.1%) infected cysts had discernible wall thickening with a median wall thickness of 2.1 mm (range: 1.5–3.9 mm) (Fig 6). All infected renal cysts except one showed discernible wall thickening, while 11 of 17 infected hepatic cysts delineated wall thickening. On arterial phase CT images, 11 of 14 infected hepatic cysts (78.6%) showed pericystic hyperemia (Fig 7). p y p y yp g Combined imaging feature. Forty-four of 51 (86.3%) episodes of infected cysts showed relatively higher attenuation on pre-contrast or post-contrast CT images. Among 14 infected Fig 4. A 59-year-old female patient with an infected cyst (H12 in Fig 2). (A) The pre-contrast CT image shows a 5.5 cm sized cyst with a lobulated contour in segment 6 of the liver (arrow). The cyst appears to have a relatively high attenuation compared to the surrounding cysts. (B) The arterial phase image shows arterial hyperenhancement (arrows) in segment 6 of the liver; however, perilesional hyperemia could not be definitely diagnosed due to the intervening cysts between the lesion and hyperemia site. (C) The cyst shows discernible wall thickening in the portal venous phase. Of note, intracystic attenuation seems equal in contrast to the pre- contrast scan. (D) Ultrasonography shows a heterogeneous hyperechoic cyst with internal septation (arrow). https://doi.org/10.1371/journal.pone.0207880.g004 Fig 4. A 59-year-old female patient with an infected cyst (H12 in Fig 2). (A) The pre-contrast CT image shows a 5.5 cm sized cyst with a lobulated contour in segment 6 of the liver (arrow). The cyst appears to have a relatively high attenuation compared to the surrounding cysts. (B) The arterial phase image shows arterial hyperenhancement (arrows) in segment 6 of the liver; however, perilesional hyperemia could not be definitely diagnosed due to the intervening cysts between the lesion and hyperemia site. (C) The cyst shows discernible wall thickening in the portal venous phase. Of note, intracystic attenuation seems equal in contrast to the pre- contrast scan. (D) Ultrasonography shows a heterogeneous hyperechoic cyst with internal septation (arrow). https://doi org/10 1371/journal pone 0207880 g004 https://doi.org/10.1371/journal.pone.0207880.g004 PLOS ONE \| https://doi.org/10.1371/journal.pone.0207880 December 5, 2018 9 / 16 Imaging features of infected cysts in ADPKD Fig 5. Intracystic attenuation (HU) of infected cysts and presumed normal cysts. (A) Intracystic attenuation was measured on pre-contrast images in 43 episodes. (B) Intracystic attenuation was measured on post-contrast images in 44 episodes. Note: HU = Hounsfield units; IC = infected cyst; N = presumed normal cyst. https://doi org/10 1371/journal pone 0207880 g005 Fig 5. Intracystic attenuation (HU) of infected cysts and presumed normal cysts. (A) Intracystic attenuation was measured on pre-contrast images in 43 episodes. (B) Intracystic attenuation was measured on post-contrast images in 44 episodes. Note: HU = Hounsfield units; IC = infected cyst; N = presumed normal cyst. Fig 5. Intracystic attenuation (HU) of infected cysts and presumed normal cysts. (A) Intracystic attenuation was measured on pre-contrast images in 43 episodes. (B) Intracystic attenuation was measured on post-contrast images in 44 episodes. Note: HU = Hounsfield units; IC = infected cyst; N = presumed normal cyst. https://doi.org/10.1371/journal.pone.0207880.g005 https://doi.org/10.1371/journal.pone.0207880.g005 hepatic cysts for which arterial phase images were available, 13 (92.9%) had perilesional hyper- emia or pericystic fat infiltration. On post-contrast CT images, 37 of 44 (84.1%) infected cysts had discernible wall thickening, and 5 of the remaining 7 cases showed perilesional hyperemia or pericystic fat infiltration, and 6 of the remaining 7 cases showed relatively high attenuation than the surrounding normal cysts on pre-contrast and post-contrast images. In addition, 27 of 44 (61.4%) infected cysts had both discernible wall thickening and evidence of perilesional fl ( l l h f f l ) hepatic cysts for which arterial phase images were available, 13 (92.9%) had perilesional hyper- emia or pericystic fat infiltration. On post-contrast CT images, 37 of 44 (84.1%) infected cysts had discernible wall thickening, and 5 of the remaining 7 cases showed perilesional hyperemia or pericystic fat infiltration, and 6 of the remaining 7 cases showed relatively high attenuation than the surrounding normal cysts on pre-contrast and post-contrast images. In addition, 27 of 44 (61.4%) infected cysts had both discernible wall thickening and evidence of perilesional inflammation (i.e. perilesional hyperemia or pericystic fat infiltration) on post-contrast CT images, and 22 of 44 (50%) infected cysts had both discernible wall thickening and relatively higher than the surrounding normal cysts. Consequently, one case out of 51 episodes showed no evidence of cyst infection on CT, and in this case, cyst aspiration was performed at the site where the patient complained of tenderness. Table 4. Attenuation of infected cysts and presumed normal cysts. Pre-contrast Available episodes Renal cyst (n = 26) Hepatic cyst (n = 17) Total (n = 43) Infected cysts (HU) median (range) 19.0 (9–67) 19.0 (5–43) 19.0 (5–67) Presumed normal cysts (HU) median (range) 8.8 (-5–17.5) 7.5 (-4.5–15.5) 8.5 (-5–17.5) Median difference ([95% CI], P-value) 15.0 ([10.5–25.8], P<0.001) 13.8 ([9.6–19.5], P<0.001) 14.5 ([11.3–19.5], P<0.001) Post-contrast Available episodes Renal cyst (n = 27) Hepatic cyst (n = 17) Total (n = 44) Infected cysts (HU) median (range) 22.0 (7–79) 20.0 (7–44) 21.0 (7–79) Presumed normal cysts (HU) median (range) 9.5 (1.5–20) 9.5 (-6.5–24) 9.5 (-6.5–24) Median difference ([95% CI], P-value) 11.8 ([8.3–15.5], P<0.001) 12.0 ([6.2–17.0], P = 0.002) 12.0 ([9.3–14.8], P<0.001) Note: CI = confidence interval Table 4. Attenuation of infected cysts and presumed normal cysts. PLOS ONE \| https://doi.org/10.1371/journal.pone.0207880 December 5, 2018 10 / 16 Imaging features of infected cysts in ADPKD Fig 6. A 65-year-old female patient with an infected cyst (R24 in Fig 2). (A) Pre-contrast CT image shows a 9.7 cm sized round cyst that has higher attenuation than the surrounding cysts in right kidney upper pole (arrow). (B, C) On the arterial and portal venous phase images, the cyst shows discernible wall thickening with prominent wall enhancement and pericystic fat infiltration (arrows). Fig 6. A 65-year-old female patient with an infected cyst (R24 in Fig 2). (A) Pre-contrast CT image shows a 9.7 cm sized round cyst that has higher attenuation than the surrounding cysts in right kidney upper pole (arrow). (B, C) On the arterial and portal venous phase images, the cyst shows discernible wall thickening with prominent wall enhancement and pericystic fat infiltration (arrows). https://doi.org/10.1371/journal.pone.0207880.g006 CT findings of cysts without infection. There were 9 episodes from 7 patients that showed no evidence of cyst infection on aspirates. Among them, three episodes were acute hemorrhagic renal cysts with 69 HU, 61 HU, and 26 HU of intracystic attenuation, respec- tively, and one was a recent hemorrhagic renal cyst. Of the eight cysts for which we were able to assess wall thickness, only one (12.5%) with acute hematoma showed discernible wall thick- ening. CT findings were listed in Fig 8. US imaging features Table 5 and Fig 2 summarize the US findings. Most infected cysts (22 of 24 renal cysts and 15 of 17 hepatic cysts) appeared hyperechoic and echogenic. When comparing intracystic hetero- geneity on CT and US images, all 13 cysts showing heterogeneous attenuation on CT revealed heterogeneous echogenicity and 6 homogeneously attenuating cysts showed homogeneous Fig 7. A 65-year-old female patient with an infected cyst (H10 in Fig 2). (A) On the pre-contrast CT image, a proven 6 cm infected cyst (arrow) shows no remarkable finding. (B) On the arterial phase image, there is perilesional hyperemia around the cyst (arrows). (B, C) On the arterial or delayed phase image, no discernible wall thickening is noted. In this case, arterial hyperemia is the only clue for cyst infection. Fig 7. A 65-year-old female patient with an infected cyst (H10 in Fig 2). (A) On the pre-contrast CT image, a proven 6 cm infected cyst (arrow) shows no remarkable finding. (B) On the arterial phase image, there is perilesional hyperemia around the cyst (arrows). (B, C) On the arterial or delayed phase image, no discernible wall thickening is noted. In this case, arterial hyperemia is the only clue for cyst infection. https://doi.org/10.1371/journal.pone.0207880.g007 https://doi.org/10.1371/journal.pone.0207880.g007 11 / 16 PLOS ONE \| https://doi.org/10.1371/journal.pone.0207880 December 5, 2018 Imaging features of infected cysts in ADPKD h i i H h i i f d i h l i Fig 8. Mapping for CT imaging features in 9 episodes of noninfected cysts in 9 patients with ADPKD. Note: ADPKD = autosomal dominant polycystic kidney disease; Pre = pre-contrast images; AP = arterial phase images; PP = portal venous phase images; DP = delayed phase images; NR = noninfected renal cyst; NH = noninfected hepatic cyst. https://doi.org/10.1371/journal.pone.0207880.g008 h i it H t h i it f d i 22 h l tt ti t Fig 8. Mapping for CT imaging features in 9 episodes of noninfected cysts in 9 patients with ADPKD. Note: ADPKD = autosomal dominant polycystic kidney disease; Pre = pre-contrast images; AP = arterial phase images; PP = portal venous phase images; DP = delayed phase images; NR = noninfected renal cyst; NH = noninfected hepatic cyst. https://doi.org/10.1371/journal.pone.0207880.g008 Fig 8. Mapping for CT imaging features in 9 episodes of noninfected cysts in 9 patients with ADPKD. Discussion The most common CT feature for infected cysts in ADPKD in this study was discernible wall thickening, which was observed in 84.1% of cases; this has also been regarded as a suggestive finding for infected cysts in previously reported studies. The median wall thickness of infected cysts was 2.1 mm, ranging from 1.5 mm to 3.9 mm; therefore, a definition of wall thickening 3 mm used in a previously reported study [15], might be misleading considering our results. Discernible wall thickening was found to be more common in renal cyst infection (96.3%) than in hepatic cyst infection (64.7%). One possible explanation would be that the liver in patients with ADPKD often retains cyst-free parenchyma, whereas in most cases, the kidney is completely replaced by cysts, so the wall of hepatic cysts may be less perceptible because the tis- sue contrast between the cyst wall and the surrounding liver parenchyma would be lower than the tissue contrast between the renal cyst and adjacent cysts. Salle´e et al.[6] proposed that cyst infection is a probable diagnosis in the concurrent mani- festation of four conditions: fever (temperature >38˚C for >3 days), abdominal tenderness in the kidney or liver area, increased level of C-reactive protein (CRP) (>5 mg/dL) and the absence of CT augmentation for recent intracystic bleeding. A previous study, which com- pared clinical features of cyst infection and hemorrhage in AKPKD patients, defined the cutoff value for intracystic attenuation to distinguish cyst hemorrhage from infected cysts as 25 HU [15]. However, 25 HU cannot be the threshold for differentiating hemorrhagic cysts from infected cysts considering the fact that our results showed that the intracystic attenuation of the infected cysts had a wide range from 5 HU to 67 HU with a median value of 19.0 HU. Moreover, some infected cysts showed attenuation greater than 50 HU, strongly suggesting an acute hemorrhagic cyst, so even if the cyst appears as an acute hemorrhagic cyst, the possibility of combined infection cannot be ruled out. With regard to intracystic attenuation, a relatively high attenuation based on the visual perception by the reviewer was a common feature in infected cysts. Interestingly, it was more frequently noted in the pre-contrast scan than in the post-contrast scan, although the mean attenuation of the cyst was slightly higher in the post-contrast scan. US imaging features Note: ADPKD = autosomal dominant polycystic kidney disease; Pre = pre-contrast images; AP = arterial phase images; PP = portal venous phase images; DP = delayed phase images; NR = noninfected renal cyst; NH = noninfected hepatic cyst. https://doi.org/10.1371/journal.pone.0207880.g008 echogenicity. Heterogeneous echogenicity was found in 22 homogeneously attenuating cysts; conversely, there was no homogeneously echoic cyst that showed heterogeneous attenuation. Fluid/fluid level and intracystic septation were found in 6 out of 41 (14.6%) and 11 out of 41 (26.8%) infected cysts, respectively. 12 / 16 PLOS ONE \| https://doi.org/10.1371/journal.pone.0207880 December 5, 2018 Imaging features of infected cysts in ADPKD Table 5. US findings from 41 episodes of cyst infection in 37 patients with ADPKD. Renal cyst infection (24 episodes) Hepatic cyst infection (17 episodes) Total (41 episodes) Intracystic echogenicity, n (%) hypoechoic or hyperechoic 22 (91.7%) 15 (88.2%) 37 (90.2%) anechoic 2 (8.3%) 2 (11.8%) 4 (9.8%) Heterogeneous echogenicity, n (%) Yes 22 (91.7%) 13 (76.5%) 35 (85.4%) No 2 (8.3%) 4 (23.5%) 6 (14.6%) Fluid-fluid level, n (%) Yes 1 (4.2%) 5 (29.4%) 6 (14.6%) No 23 (95.8%) 12 (70.6%) 35 (85.4%) Septation, n (%) Yes 7 (29.2%) 4 (23.5%) 11 (26.8%) No 17 (70.8%) 13 (76.5%) 30 (73.2%) https://doi.org/10.1371/journal.pone.0207880.t005 Table 5. US findings from 41 episodes of cyst infection in 37 patients with ADPKD. Imaging features of infected cysts in ADPKD Perilesional hyperemia is one of the signs of inflammation. Arterial phase scans sometimes provide diagnostic clues, because perilesional hyperemia around a certain cyst could be the only finding indicating infected hepatic cyst. In addition, the post-contrast CT scan would be recommended in the diagnosis of an infected cyst if a patient is not at risk for contrast- induced nephropathy, because discernible wall thickening is a common feature that can often be assessed on enhanced images. However, the pre-contrast scan also has value because several features, such as relatively high or heterogeneous attenuation and the presence of intracystic gas, can be evaluable on CT without enhancement. In our study, 22 infected cysts with homogeneous attenuation on CT showed heteroge- neous echogenicity on US, indicating that US can demonstrate intracystic complicated fluid content more sensitively. Although not investigated in this study, US also has advantages of no radiation hazard and a crude evaluation of vascularity without contrast enhancement. How- ever, it would be difficult to detect a complicated cyst using US as a first imaging modality because kidney or liver affected by ADPKD is usually markedly enlarged, sometimes measur- ing more than 20 cm in size. Most infected cysts were shown to be located in the subcapsular region. Rather than being an indication that infected cysts occur more frequently in the subcapsular region, this observa- tion reflects that cysts in the subcapsular location could be more easily aspirated than deeply seated cysts. CT features of infected cysts investigated in this study are not specific and could be seen in hemorrhagic cysts or infrequently in cysts with sterile inflammation. Case-control study design would be better to assess the diagnostic performance of CT features; however, the num- ber of non-infected cysts was thought to be too small for statistical analysis and this is a major limitation of our study. However, the problem of CT diagnosis for infected cysts has been low sensitivity rather than low specificity; therefore, we think that it is notable that all infected cysts, except one lesion, showed at least one positive finding in the CT features. Discussion This phenomenon was interpreted to be because a subtle attenuation difference can be better recognized in pre-con- trast images than in post-contrast images, which have a wider window width and higher win- dow level than pre-contrast images due to enhanced high-attenuating parenchyma of the liver or kidneys. 13 / 16 PLOS ONE \| https://doi.org/10.1371/journal.pone.0207880 December 5, 2018 Discernible wall thickening, the most frequent CT feature, was rarely noted in non-infected cysts; there- fore, if there is a cyst showing discernible wall thickening among numerous cysts on CT scan, careful investigation would be helpful to detect an infected cyst and may reduce the need for high cost diagnostic modality. Nevertheless, the matter of specificity in imaging features drawn in this study still remains complicating confident clinical application, so further studies with a control group are warranted to prove or disprove the usefulness of these imaging fea- tures in the diagnosis of infected cysts in ADPKD patients. There were several other limitations in this study. First, there was a substantial number of cases that were excluded, and this could result in a bias; approximately 15% (12/78) were excluded because aspirated cysts were not localized on CT due to lack of a guiding US image or no written record about the aspiration site. This problem was an inherent limitation in this retrospective study. Second, there was no comparison with the emerging promising diagnostic modalities of FDG-PET or MRI. Therefore, further studies with a larger number of cases including a control group are warranted to evaluate the diagnostic performance of CT, hope- fully with a comparison to FDG-PET or MRI. Third, for patients with ADPKD, the use of con- trast media can be a problem although much information was obtained from the post-contrast scan. In our study group, 32 of 51 episodes (62.8%) were cases of ADPKD patients on dialysis, which allowed us to perform contrast-enhanced CT. However, considering that a larger pro- portion of ADPKD cases progress to ESRD compared to those with other forms of chronic kidney disease [16, 17], it is clinically challenging to perform contrast-enhanced CT in all APDKD patients with suspected cyst infection. In conclusion, minute findings such as minimal wall thickening, pericystic fat infiltration, or relatively higher attenuation compared to normal cysts could be suggestive of infected cyst PLOS ONE \| https://doi.org/10.1371/journal.pone.0207880 December 5, 2018 14 / 16 Imaging features of infected cysts in ADPKD in ADPKD patients. In hepatic cysts, perilesional hyperemia may be the only finding sugges- tive of cyst infection. Therefore, it will be helpful to evaluate CT or US imaging features more carefully and comprehensively if a patient with ADPKD has symptoms that raise a suspicion of cyst infection. in ADPKD patients. Writing – review & editing: Cheong-Il Shin, Sang Youn Kim. Writing – review & editing: Cheong-Il Shin, Sang Youn Kim. In hepatic cysts, perilesional hyperemia may be the only finding sugges- tive of cyst infection. Therefore, it will be helpful to evaluate CT or US imaging features more carefully and comprehensively if a patient with ADPKD has symptoms that raise a suspicion of cyst infection. Conceptualization: Cheong-Il Shin. Conceptualization: Cheong-Il Shin. Data curation: Jiseon Oh. Formal analysis: Jiseon Oh, Sang Youn Kim. Investigation: Cheong-Il Shin. Methodology: Jiseon Oh, Cheong-Il Shin, Sang Youn Kim. Supervision: Cheong-Il Shin. Writing – original draft: Jiseon Oh. Data curation: Jiseon Oh. Writing – original draft: Jiseon Oh. Author Contributions Conceptualization: Cheong-Il Shin. References 1. Torres VE, Harris PC, Pirson Y. Autosomal dominant polycystic kidney disease. Lancet (London, England). 2007; 369(9569):1287–301. Epub 2007/04/17. https://doi.org/10.1016/s0140-6736(07) 60601-1 PMID: 17434405. 2. Grantham JJ. Clinical practice. Autosomal dominant polycystic kidney disease. The New England jour- nal of medicine. 2008; 359(14):1477–85. Epub 2008/10/04. https://doi.org/10.1056/NEJMcp0804458 PMID: 18832246. 3. Christophe JL, van Ypersele de Strihou C, Pirson Y. Complications of autosomal dominant polycystic kidney disease in 50 haemodialysed patients. A case-control study. The U.C.L. Collaborative Group. Nephrology, dialysis, transplantation: official publication of the European Dialysis and Transplant Asso- ciation—European Renal Association. 1996; 11(7):1271–6. Epub 1996/07/01. PMID: 8672022. 4. Jouret F, Lhommel R, Devuyst O, Annet L, Pirson Y, Hassoun Z, et al. Diagnosis of cyst infection in patients with autosomal dominant polycystic kidney disease: attributes and limitations of the current modalities. Nephrology, dialysis, transplantation: official publication of the European Dialysis and Trans- plant Association—European Renal Association. 2012; 27(10):3746–51. Epub 2012/11/02. https://doi. org/10.1093/ndt/gfs352 PMID: 23114901. 5. Schwab SJ, Bander SJ, Klahr S. Renal infection in autosomal dominant polycystic kidney disease. The American journal of medicine. 1987; 82(4):714–8. Epub 1987/04/01. PMID: 3565428. 6. Sallee M, Rafat C, Zahar JR, Paulmier B, Grunfeld JP, Knebelmann B, et al. Cyst infections in patients with autosomal dominant polycystic kidney disease. Clinical journal of the American Society of Nephrol- ogy: CJASN. 2009; 4(7):1183–9. Epub 2009/05/28. https://doi.org/10.2215/CJN.01870309 PMID: 19470662. 7. Neuville M, Hustinx R, Jacques J, Krzesinski JM, Jouret F. Diagnostic Algorithm in the Management of Acute Febrile Abdomen in Patients with Autosomal Dominant Polycystic Kidney Disease. PloS one. 2016; 11(8):e0161277. Epub 2016/08/17. https://doi.org/10.1371/journal.pone.0161277 PMID: 27529555. 8. Bobot M, Ghez C, Gondouin B, Sallee M, Fournier PE, Burtey S, et al. Diagnostic performance of [(18) F]fluorodeoxyglucose positron emission tomography-computed tomography in cyst infection in patients with autosomal dominant polycystic kidney disease. Clinical microbiology and infection: the official pub- lication of the European Society of Clinical Microbiology and Infectious Diseases. 2016; 22(1):71–7. Epub 2015/10/11. https://doi.org/10.1016/j.cmi.2015.09.024 PMID: 26454062. 9. Balbo BE, Sapienza MT, Ono CR, Jayanthi SK, Dettoni JB, Castro I, et al. Cyst infection in hospital- admitted autosomal dominant polycystic kidney disease patients is predominantly multifocal and asso- ciated with kidney and liver volume. Brazilian journal of medical and biological research = Revista brasi- leira de pesquisas medicas e biologicas. 2014; 47(7):584–93. Epub 2014/06/12. https://doi.org/10. 1590/1414-431X20143584 PMID: 24919173. PLOS ONE \| https://doi.org/10.1371/journal.pone.0207880 December 5, 2018 15 / 16 Imaging features of infected cysts in ADPKD 10. PLOS ONE \| https://doi.org/10.1371/journal.pone.0207880 December 5, 2018 References Kwon HW, Lee HY, Hwang YH, Park HC, Ahn C, Kang KW. Diagnostic performance of 18F-FDG- labeled white blood cell PET/CT for cyst infection in patients with autosomal dominant polycystic kidney disease: a prospective study. Nuclear medicine communications. 2016; 37(5):493–8. Epub 2016/03/26. https://doi.org/10.1097/MNM.0000000000000466 PMID: 27014954. 11. Piccoli GB, Arena V, Consiglio V, Deagostini MC, Pelosi E, Douroukas A, et al. Positron emission tomography in the diagnostic pathway for intracystic infection in adpkd and "cystic" kidneys. a case series. BMC nephrology. 2011; 12:48. Epub 2011/10/01. https://doi.org/10.1186/1471-2369-12-48 PMID: 21957932. 12. Jouret F, Lhommel R, Beguin C, Devuyst O, Pirson Y, Hassoun Z, et al. Positron-emission computed tomography in cyst infection diagnosis in patients with autosomal dominant polycystic kidney disease. Clinical journal of the American Society of Nephrology: CJASN. 2011; 6(7):1644–50. Epub 2011/06/28. https://doi.org/10.2215/CJN.06900810 PMID: 21700816. 13. Ichioka K, Saito R, Matsui Y, Terai A. Diffusion-weighted magnetic resonance imaging of infected renal cysts in a patient with polycystic kidney disease. Urology. 2007; 70(6):1219. Epub 2007/12/26. https:// doi.org/10.1016/j.urology.2007.09.040 PMID: 18158052. 14. Katano K, Kakuchi Y, Nakashima A, Takahashi S, Kawano M. Efficacy of diffusion-weighted magnetic resonance imaging in detecting infected cysts in a case of polycystic kidney disease. Clinical nephrol- ogy. 2011; 75 Suppl 1:24–6. Epub 2011/01/29. PMID: 21269589. 15. Suwabe T, Ubara Y, Sumida K, Hayami N, Hiramatsu R, Yamanouchi M, et al. Clinical features of cyst infection and hemorrhage in ADPKD: new diagnostic criteria. Clinical and experimental nephrology. 2012; 16(6):892–902. Epub 2012/06/13. https://doi.org/10.1007/s10157-012-0650-2 PMID: 22688273. 16. Zhang QL, Rothenbacher D. Prevalence of chronic kidney disease in population-based studies: system- atic review. BMC public health. 2008; 8:117. Epub 2008/04/15. https://doi.org/10.1186/1471-2458-8- 117 PMID: 18405348. 17. Gilbertson DT, Liu J, Xue JL, Louis TA, Solid CA, Ebben JP, et al. Projecting the number of patients with end-stage renal disease in the United States to the year 2015. Journal of the American Society of Nephrology: JASN. 2005; 16(12):3736–41. Epub 2005/11/04. https://doi.org/10.1681/ASN. 2005010112 PMID: 16267160. 16 / 16
https://openalex.org/W4294531553	https://journalretinavitreous.biomedcentral.com/counter/pdf/10.1186/s40942-022-00413-0	English	null	Outcomes of Heavy Silicone Oil (Densiron) compared to Silicone Oil in primary rhegmatogenous retinal detachment: a multivariable regression model	International journal of retina and vitreous	2,022	cc-by	5,552	International Journal of Retina and Vitreous Open Access © The Author(s) 2022. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativeco mmons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Abstract Purpose: To measure the visual outcomes, proliferative vitreoretinopathy (PVR) and retinectomy rates following pri- mary rhegmatogenous retinal detachment (RRD) repair, comparing silicone oil (SO) and heavy SO (Densiron). Methods: Retrospective, continuous comparative study from January 2017 to May 2021 of all primary RRD. Multivari- able linear (logMAR gain) and binary-logistic (PVR-C and retinectomy rate) regression models to compare tamponade were performed. Covariates included age, gender, ocular co-morbidities, high myopia, macula-status, giant-retinal- tear (GRT), pre-op vision, PVR-C, oil type, perfluorocarbon-use, combined scleral buckle/vitrectomy, combined phaco-vitrectomy, 360-degrees-endolaser and oil duration. Cases with trauma or less than six-month follow-up were excluded. Results: A total of 259 primary RD were analysed. There were 179 SO patients and 80 Densiron patients that had six- month primary re-detachment in 18 (10.1%) and 8 (10.0%) respectively (p = 1.000). No difference in logMAR gain was detected between tamponade choice on multivariable linear regression. Subsequent glaucoma surgery was 5 (2.8%) and 4 (5.0%) for SO and Densiron patients respectively (p = 0.464). On multivariate binary-logistic regression we found no difference in development of PVR-C between oil tamponades. However, SO had significantly higher subsequent retinectomy rate compared to Densiron (odds ratio 15.3, 95% CI 1.9–125.5, p = 0.011). Duration of oil tamponade was not linked to differences in logMAR gain, PVR-C formation or increased retinectomy rate. Conclusions: We report no difference in primary anatomical success, number of further RRD surgeries, subsequent glaucoma surgery, visual outcomes, PVR-C between both tamponades on multivariable models. Densiron oil was found to be more retinectomy sparing relative to SO. Keywords: Retinal detachment, Silicone oil, Heavy oil, Heavy silicone oil, Densiron, Outcomes, Vitreoretinal, Retina, Glaucoma, Retinectomy George Moussa1,2* , Maria Tadros2, Soon Wai Ch’ng2, Ash Sharma2, Kim Son Lett2, Arijit Mitra2, Ajai K. Tyagi2 and Walter Andreatta2,3,4 George Moussa1,2* , Maria Tadros2, Soon Wai Ch’ng2, Ash Sharma2, Kim Son Lett2, Arijit Mitra2, Ajai K. Tyagi2 and Walter Andreatta2,3,4 Moussa et al. International Journal of Retina and Vitreous (2022) 8:61 https://doi.org/10.1186/s40942-022-00413-0 Moussa et al. International Journal of Retina and Vitreous (2022) 8:61 https://doi.org/10.1186/s40942-022-00413-0 International Journal of Retina and Vitreous Outcomes of Heavy Silicone Oil (Densiron) compared to Silicone Oil in primary rhegmatogenous retinal detachment: a multivariable regression model George Moussa1,2* , Maria Tadros2, Soon Wai Ch’ng2, Ash Sharma2, Kim Son Lett2, Arijit Mit Ajai K. Tyagi2 and Walter Andreatta2,3,4 Introduction While gas tamponades are often utilised in the treatment of rhegmatogenous retinal detachments (RRD), specific characteristics of RRD can lead to the clinical decision to use silicone oil (SO) tamponade agents. These include, but are not limited to, factors such as their chronicity, Inclusion/exclusion criteria and definitions Primary RRDs repaired by pars plana vitrectomy (PPV) were selected to reduce confounding factors of prior re- detachments and risk adjusted for case complexity to allow more meaningful comparison between both SO and Densiron. Exclusion criteria include post-traumatic RRD and lack of follow up (due to patients being referred back to other peripheral hospital units).i Primary failure was defined as a detachment under oil or the decision for permanent oil tamponade. Patients that were awaiting ROSO, with stable examination find- ings were not defined as failure. This was particularly Surgical technique All RRD All RRD surgery was performed with transconjunctival 23-gauge PPV and retinopexy undertaken with cryo- therapy, endolaser, a combination of both with or with- out three-sixty barrier laser. Surgery could be combined with a scleral buckle, involve PVR peeling for PVR Grade C, retinectomy and the use of PFCL when necessary. The choice of tamponade was a clinical decision based on the operating surgeon, including number, location and mor- phology of retinal breaks [especially giant retinal tears (GRTs)], the presence of PVR Grade C, RRD location and chronicity. Patients with reduced ability to posture or with inferior retinal detachments were more likely to receive Densiron compared to SO in our unit. Our SO was supplied by FCI Ophthalmics (FCI S.A.S. – France Chirurgie Instrumentation, 20–22 rue Louis Armand, 75,015 Paris, France). Methods l A single centre, retrospective, continuous and compara- tive study, to analyse all patients that had primary RRD performed at the Birmingham and Midland Eye Centre (BMEC). The study period covers 4.5 consecutive years from January 2017 to May 2021. © 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. Moussa et al. International Journal of Retina and Vitreous (2022) 8:61 Page 2 of 8 important due to increased waiting list pressures due to the COVID-19 pandemic would falsely raise the six- month failure statistics. the location and morphology of breaks, presence of pro- liferative vitreoretinopathy (PVR), and the likelihood of strict posturing by patients. However, as SOs are of lower density than water and have limited ability to sup- port the inferior retina, tamponade agents with heavier- than-water properties, such as heavy SO (HSO) may be selected to support these areas of pathology [1]. Den- siron®68 (Fluoron Co, Neu-Ulm, Germany) is a HSO that is a solution of 30.5% perfluorohexyloctane (F6H8) and 69.5% SO 5000cs [2]. It is an effective tamponade for inferior retinal detachments and particularly useful in patients that cannot perform prone posturing. Data collection All the data were extracted from electronic patient records (EPR, Medisoft Ophthalmology, Medisoft Lim- ited, Leeds, UK). Data collection included: I. Baseline demographics and characteristics [age, gender, pre-operative lens status, laterality, the presence of high myopia (defined as greater than six dioptres of myopia), pre-operative visual acu- ity (VA), macula status and ocular co-morbidities (including macular degeneration, vein occlusion, corneal pathology, glaucoma, However, HSOs have their own hypothesised risk pro- files. Heimann et al. postulated a foreign-body reaction to emulsification of droplets of HSO that may result in superior PVR-type membranes [3, 4].i II. It is thought that dispersion, or emulsification of drop- lets, leads to a heightened inflammatory response con- centrated superiorly above the main bubble, resulting in the formation of precipitates, fibrin, PVR and epiretinal membranes that can potentially increase the retinectomy rate [5].fi gy g II. Intra-operative and post-operative factors (choice of tamponade, post-operative lens status, use of perfluorocarbon liquid (PFCL), requirement of PVR peel and/or retinectomy, retinal detachment re-operation rate (excluding routine removal of SO), rate of subsequent PVR C and retinectomy in patients with and without initial PVR C and reti- nectomy, rate of SO, Densiron and gas tampon- ade if subsequent RRD surgery was required, rate of post-operative glaucoma surgery and epiretinal membrane (ERM) peel, and duration of SO/Den- siron tamponade). Patients must have had a mini- mum of six-month follow up to be included. However, it can be difficult to distinguish complica- tions attributable to the tamponade agent from sequelae of complex retinal pathology, particularly when multi- ple different tamponade agents are included in analysis. Therefore, we conducted this retrospective study to pri- marily assess the visual outcomes following primary repair of RRD with SO and Densiron. Our secondary outcomes include the retinectomy, PVR and glaucoma surgery rate following SO and Densiron tamponade. Results In our cohort, 259 patients were analysed. The SO group has 179 patients and Densiron group has 80 patients. As expected, there are several significant differences in base- line characteristics and outcomes of patients with dif- ferent tamponade agents (Table 1). Similarly, there are significant differences between pre and postoperative visual outcomes by tamponade agent (Fig. 1). Statistical analysis All statistical analysis was performed using IBM SPSS Statistics for Windows, Version 28.0 (IBM Corp, Armonk NY). Statistical significance was defined as p < 0.05. Prior Moussa et al. International Journal of Retina and Vitreous (2022) 8:61 Page 3 of 8 to analysis, continuous variables were assessed using the Shapiro–Wilk test and found not to be normally distrib- uted. Hence, data are primarily reported as medians and interquartile ranges (IQRs) throughout. For univariate comparisons, Mann Whitney U test was used to com- pare two groups respectively (age, VA and duration of oil tamponade). Wilcoxon signed rank test was used for two- paired VA data. Fisher exact test and Chi-Squared test were used for nominal variables. to analysis, continuous variables were assessed using the Shapiro–Wilk test and found not to be normally distrib- uted. Hence, data are primarily reported as medians and interquartile ranges (IQRs) throughout. For univariate comparisons, Mann Whitney U test was used to com- pare two groups respectively (age, VA and duration of oil tamponade). Wilcoxon signed rank test was used for two- paired VA data. Fisher exact test and Chi-Squared test were used for nominal variables.if such as age, gender, high myopia, glaucoma rate, and ocular co-morbidities were similar between both groups (Table 1). To enable risk adjusted comparisons; various multivari- able regression models were conducted. Primary outcome bl l d Table 2 includes a multivariable linear regression models for logMAR gain following primary RRD repair, includ- ing only pseudophakic patients to reduce the confounder of cataracts (n = 187). In this model, 187 patients (70.7%) were included, 127 (71.8%) and 60 (77.9%) of total SO and Densiron patients respectively. Low logMAR gain was found to be significantly associated with better pre- operative logMAR and a combined scleral buckle with vitrectomy with no difference detected between SO and Densiron tamponade. Due to significant differences in case complex- ity between cases, we undertook several multivariable regression analyses. To investigate our primary outcome of visual outcomes following primary RRD repair, we conducted a multivariate linear regression analysis on logMAR gain (pre-operative logMAR minus post-oper- ative logMAR) on visual outcome including only pseu- dophakic patients (to reduce lens opacities and aphakic patients as confounders). As covariates, we included (i) baseline demographics characteristics: age, gender, high myopia, presence of ocular comorbidities (other than high myopia), macula status, presence of GRT, pre-oper- ative visual acuity and presence of PVR C, (ii) intraopera- tive characteristics: tamponade choice (SO or Densiron), retinectomy performed, combined scleral buckle with PPV, 360 degrees endolaser retinopexy perfomed, com- bined phacovitrectomy performed and (iii) post-opera- tive characteristics: duration of oil tamponade. Secondary outcomes On univariate analysis, SO had significantly higher rates of retinectomy than Densiron in patients that did not have initial retinectomy (p = 0.005). Figure 2 includes multivariable binary logistic regres- sion models for assessing risks for (A) further PVR C rate and (B) further retinectomy rate. SO had a trend but a non-significant increase in subsequent prolifera- tive vitreoretinopathy C rate compared to Densiron, and no significant risk factors were identified. SO (compared to Densiron) was the only factor found to significantly increase subsequent retinectomy rate. Initial retinectomy was not found to be a significant risk factor for further retinectomy. Combined buckle with vitrectomy had a trend towards significance for further retinectomy rate.h Best corrected VA was used and records in Snellen were converted to logMAR. Low VA, corresponding to count fingers (CF), hand movements (HM), perception of light (PL) and no PL (NPL) were substituted with 2.10, 2.40, 2.70 and 3.00 LogMAR, respectively, in keeping with previous publications from the national ophthal- mology database group [6], using a tool by Moussa et al. [7] For our secondary outcomes, we also carried out mul- tivariable binary logistic regression analyses with sub- sequent retinectomy rate and PVR C rate as dependent variables. i The outcomes of primary RRD are found in Table 3. Densiron, compared to SO, had shorter duration of oil tamponade (p < 0.001) and had higher rates of successful ROSO (p = 0.030). We found no difference in subsequent RRD rate, or in rates of glaucoma surgery (for those without and with history of glaucoma pre-operatively, Table 3). If patients required subsequent RRD surgery, gas tamponade was more likely to be used for Densiron than SO tamponade (p = 0.004). Discussionh This study is the largest comparative case series in the lit- erature at the time of publication between SO and Den- siron and is the only manuscript involving multivariable regression analyses comparisons. Our data demonstrate that although there was no dif- ference in logMAR gain between SO and Densiron in pseudophakic patients at final VA on risk adjusted mul- tivariable linear regression analysis, there was a signifi- cantly higher retinectomy rate for SO on both univariate Compared to Densiron, the SO group had higher pro- portion of macular off retinal detachment (p = 0.008), higher rates of PVR C (p = 0.005), lower pre-operative VA (p < 0.001), and higher rates of combined scleral buckle and PPV (p = 0.001). However, baseline characteristics Moussa et al. Discussionh International Journal of Retina and Vitreous (2022) 8:61 Page 4 of 8 Table 1 Baseline clinical and operative characteristics of primary retinal detachments Age is reported as median (interquartile range) and Kruskal Wallis test used to compare continuous variables Chi Squared test to compare more than two nominal groups Total Silicone Oil Densiron p Value Total 259 179 80 - Baseline characteristics Age (years, IQR) 61 (49 to 71) 61 (49 to 70) 60 (49 to 73) 0.829 Gender (% Male) 187 (72.2%) 131 (73.2%) 56 (70.0%) 0.653 Laterality (% Right) 137 (52.9%) 92 (51.4%) 45 (56.3%) 0.502 Ocular Co-morbidities 97 (37.5%) 72 (40.2%) 25 (31.3%) 0.211 High Myope (% Yes) 22 (8.5%) 13 (7.3%) 9 (11.3%) 0.336 Glaucoma (% Yes) 8 (3.1%) 3 (1.7%) 5 (6.3%) 0.112 Preoperative lens* Phakic 142 (62.6%) 101 (63.9%) 41 (59.4%) 0.680 Pseudophakic 78 (34.4%) 53 (33.5%) 25 (36.2%) Aphakic 7 (3.1%) 4 (2.5%) 3 (4.3%) Macula status Off 201 (78.8%) 147 (83.5%) 54 (68.4%) 0.008 On 54 (21.2%) 29 (16.5%) 25 (31.6%) Giant retinal tear 11 (4.2%) 8 (4.5%) 3 (3.8%) 1.000 PVR C 65 (25.1%) 54 (30.2%) 11 (13.8%) 0.005 Pre-operative VA (logMAR) 1.50 (0.60 to 2.40) 1.60 (0.60 to 2.40) 0.80 (0.35 to 1.85) < 0.001 Surgical Characteristics Retinectomy Performed 22 (8.5%) 19 (10.6%) 3 (3.8%) 0.090 Perfluorocarbon used 64 (24.7%) 53 (29.6%) 11 (13.8%) 0.008 Combined Buckle/PPV 19 (7.3%) 19 (10.6%) 0 (0.0%) 0.001 Combined Phacovitrectomy 19 (7.3%) 15 (8.4%) 4 (5.0%) 0.443 Epiretinal Membrane Peeled 2 (0.8%) 2 (1.1%) 0 (0.0%) 1.000 Retinopexy Cryotherapy only 33 (12.9%) 16 (9.1%) 17 (21.5%) 0.009 Endolaser only 139 (54.5%) 105 (59.7%) 34 (43.0%) 0.015 Cryotherapy and endolaser 83 (32.5%) 55 (31.3%) 28 (35.4%) 0.564 Three hundred and sixty laser 120 (46.3%) 87 (48.6%) 33 (41.3%) 0.284 Table 1 Baseline clinical and operative characteristics of primary retinal detachments and binary logistic multivariable analyses. No difference in subsequent retinal detachment rate was found between the two tamponade agents. Our study also found no dif- ference in subsequent PVR, glaucoma procedures, or ERM peels between the tamponade agents. Interest- ingly we found a trend toward significance for increased retinectomy rate in the combined scleral buckle / PPV group on multivariable regression. As a combined pro- cedure should be retinectomy sparing, this suggests that inserting a buckle in SO cases does not reduce the risk of requiring a retinectomy. Discussionh associated with an elevated intraocular pressure in the early post-operative period. This difference was initially clinically significant up to day 14, however at week four, the intraocular pressure difference between the groups was no longer significant (P = 0.17) [8]. i In a case series of 180 eyes in 2010, Romano et al. found that the use of Densiron as an endotamponade in PPV was not significantly associated with higher intraocular pressure [9]. Our study consolidates this finding clini- cally, as we did not find a difference in rate of glaucoma surgery between groups. Our results bring to light data which may reassure sur- geons when considering alternatives to SO for patients. Wong et al. in 2009 had concluded that Densiron was Semeraro et al. evaluated the inflammation associ- ated with Densiron and standard silicone oil by measur- ing the aqueous IL-1a and prostaglandin-E2 levels. They Page 5 of 8 Moussa et al. International Journal of Retina and Vitreous (2022) 8:61 Moussa et al. International Journal of Retina and Vitreous (20 Page 5 of 8 Fig. 1 Box and Whisker Plot of Visual Acuity Baseline and Outcomes by Tamponade. Box and Whisker plot. ‘X’ denotes mean. Mann Whitney U-test. Statistical significance in bold Table 2 Multivariable linear regression model for logMAR gain following primary retinal detachment repair Only Pseudophakic patients at final visual acuity were included, n = 187 Significance defined as p < 0.05. Discussionh Significant values in bold A Low logMAR gain was found to be significantly associated with better pre-operative logMAR and combined scleral buckle with vitrectomy Independent variable B Coefficient (95% CI) p Value Demographics & baseline characteristics Age 0.002 (− 0.005 to 0.008) 0.613 Male Gender (vs Female) 0.095 (− 0.122 to 0.312) 0.390 Ocular Comorbidities − 0.166 (− 0.395 to 0.063) 0.154 High Myopia − 0.029 (− 0.386 to 0.328) 0.873 Macular Status = ON 0.052 (− 0.232 to 0.336) 0.716 Giant Retinal Tear 0.269 (− 0.249 to 0.786) 0.307 Pre-Op Visual Acuity (logMAR) 0.754 (0.616 to 0.893) < 0.001 Proliferative Vitreoretinopathy C 0.030 (− 0.271 to 0.331) 0.845 Intraoperative characteristics Silicone oil tamponade (REF Densiron) 0.165 (− 0.057 to 0.388) 0.143 Perfluorocarbon − 0.154 (− 0.404 to 0.095) 0.223 Retinectomy − 0.506 (− 1.031 to 0.020) 0.059 Combined PPV / Buckle − 0.651 (− 1.061 to − 0.241) 0.002 Combined phacovitrectomy 0.137 (− 0.390 to 0.664) 0.608 Three Sixty EndoLaser − 0.021 (− 0.225 to 0.184) 0.843 Postoperative characteristics Duration of Oil 0.000 (− 0.001 to 0.001) 0.585 Table 2 Multivariable linear regression model for logMAR gain following primary retinal detachment repair Independent variable B Coefficient (95% CI) Duration of Oil Only Pseudophakic patients at final visual acuity were included, n = 187 Page 6 of 8 Moussa et al. International Journal of Retina and Vitreous (2022) 8:61 Moussa et al. International Journal of Retina and Vitreous (2022) 8:61 Moussa et al. International Journal of Retina and Vitreous Page 6 of 8 Fig. 2 Forest Plots of Multivariable binary logistic regression model following primary retinal detachment repair. Significance defined as p < 0.05. Significant values in bold. A No significant risk factors were identified to increase risk for proliferative vitreoretinopathy formation (PVR). Combined Buckle / PPV had a had a trend toward significance (p = 0.068). B Silicone oil relative to Densiron was significantly associated with increased retinectomy rate (p = 0.011). Combined buckle / PPV had a trend toward significance (p = 0.054) Fig. 2 Forest Plots of Multivariable binary logistic regression model following primary retinal detachment repair. Significance defined as p < 0.05. Significant values in bold. A No significant risk factors were identified to increase risk for proliferative vitreoretinopathy formation (PVR). Combined Buckle / PPV had a had a trend toward significance (p = 0.068). B Silicone oil relative to Densiron was significantly associated with increased retinectomy rate (p = 0.011). Combined buckle / PPV had a trend toward significance (p = 0.054) location and extent of detachment. Despite this, our study has several strengths. A retrospective analysis allowed us to collate a large case series with adequate numbers in one unit to compare outcomes between SO and Densiron tamponade with risk adjusted multivari- able regression analyses, to demonstrate its safety profile of each tamponade relative to each other. concluded that Densiron caused a more severe inflam- matory reaction in comparison [10]. Our cohort reflects a lack of clinical difference despite these findings, with no evidence that Densiron is more inflammatory than silicone oil, or that there is increased PVR or retinectomy rate. Overall, although we report a primary success rate of 90.0% at six months, on longer follow up in median 411 (interquartile range] 207 to 729) days, we find 71.0% did not require subsequent RRD surgery at the final follow up visit, with no difference between both tamponades. Duration of Oil In the literature there is a wide range of primary success using oil tamponade in primary RRD repair (63.0% to 87.6%) [1, 11–14], reflecting the heterogeneity between studies, in inclusion criteria, follow up duration and defi- nition of primary failure. Conclusion We report on our experience in using Densiron as a primary tamponade relative to SO. Despite reports of raised glaucoma, increased inflammation, and the risk of increased superior retinectomy compared to SO, we found significantly lower retinectomy rate and a trend towards significance for reduced PVR and better visual outcomes compared to SO. Although patients requiring glaucoma surgery was higher in Densiron, this was non- significant. We find that Densiron is as safe as SO for pri- mary RD repair for visual outcomes, glaucoma surgery rate and PVR formation, albeit, with a lower retinectomy rate. Declarations Patients and the public were not involved in this study due to its retrospective design. Ethical approval and consent to participation This study was registered and approved by our local clinical effectiveness team (Clinical Effectiveness Department, Sandwell General Hospital: reference number: 1595). As this was a retrospective anonymized study, as per our local protocol from our Clinical Effectiveness Department, and as per national guidelines from the National Code of Clinical Research, and the Health Research Authority (HRA), this study has ethical approval exemption and no patient consent was required for participation. All procedures were completed prior to the design of this study. Patients were diagnosed and treated accord- ing to local guidelines and agreements and written consent from patients Acknowledgements Nil Data are available upon reasonable request. Author contributions All authors have made substantial contributions to the following: (1) the conception and design of the study, or acquisition of data, or analysis and interpretation of data, (2) drafting the article or revising it critically for impor- tant intellectual content, (3) final approval of the version to be submitted. All authors read and approved the final manuscript. Study limitations and strengthsh International Journal of Retina and Vitreous (2022) 8:61 Page 7 of 8 Table 3 Outcomes of primary retinal detachment surgery by oil type Age is reported as median (interquartile range) and Kruskal Wallis test used to compare continuous variables Chi Squared test to compare more than two nominal groups Total Silicone Oil Densiron p Value Total 259 179 80 Postoperative Lens Phakic 48 (18.9%) 34 (19.2%) 14 (18.2%) 0.307 Pseudophakic 187 (73.6%) 127 (71.8%) 60 (77.9%) Aphakic 16 (6.3%) 14 (7.9%) 2 (2.6%) Duration of oil (days) 133 (77 to 222) 163 (91 to 239) 85 (61 to 133) < 0.001 Days Follow up 411 (207 to 729) 447 (237 to 765) 300 (178 to 675) Removal of tamponade (%) 193 (74.5%) 126 (70.4%) 67 (83.8%) 0.030 Primary Failure at six-months (%) 26 (10.0%) 18 (10.1%) 8 (10.0%) 1.000 Further RRD Surgery (% Yes) 75 (29.0%) 51 (28.5%) 24 (30.0%) 0.882 0 184 (71.0%) 128 (71.5%) 56 (70.0%) 0.844 1 53 (20.5%) 37 (20.7%) 16 (20.0%) ≥ 2 22 (8.5%) 14 (7.8%) 8 (10.0%) Further PVR C (% Yes) 42 (16.2%) 33 (18.4%) 9 (11.3%) 0.201 No initial PVR C (% Yes) 26 (13.4%) 21 (16.8%) 5 (7.2%) 0.078 Initial PVR C (% Yes) 16 (24.6%) 12 (22.2%) 4 (36.4%) 0.442 Further Retinectomy rate 23 (8.9%) 22 (12.3%) 1 (1.3%) 0.003 No initial retinectomy (% Yes) 20 (8.4%) 19 (11.9%) 1 (1.3%) 0.005 Initial retinectomy (% Yes) 3 (13.6%) 3 (15.8%) 0 (0.0%) 1.000 Further ERM Surgery 12 (4.7%) 9 (5.1%) 3 (3.8%) 0.759 Required Glaucoma procedures (all) 9 (3.5%) 5 (2.8%) 4 (5.0%) 0.464 No History of Glaucoma 9 (3.5%) 5 (2.8%) 4 (5.2%) 0.459 Known Glaucoma Patient 0 (0.0%) 0 (0.0%) 0 (0.0%) - Required Glaucoma Surgery (tube/trab- eculectomy/cyclodiode) 7 (2.7%) 4 (2.2%) 3 (3.8%) 0.680 Subsequent Tamponade - Oil 49 (65.3%) 34 (66.7%) 15 (62.5%) 0.797 Densiron 17 (22.7%) 14 (27.5%) 3 (12.5%) 0.237 Gas 18 (24.0%) 7 (13.7%) 11 (45.8%) 0.004 Post-Operative VA (logMAR) 0.80 (0.50 to 1.60) 1.00 (0.60 to 1.60) 0.50 (0.30 to 1.00) < 0.001 LogMAR Gain 0.20 (− 0.30 to 0.80) 0.30 (− 0.30 to 0.90) 0.20 (− 0.25 to 0.65) 0.859 Table 3 Outcomes of primary retinal detachment surgery by oil type Availability of data and materials Study limitations and strengthsh The limitations of our study include its retrospective nature and lack of case randomization including the Page 7 of 8 Moussa et al. International Journal of Retina and Vitreous (2022) 8:61 Moussa et al. No funding applicable. Author details 1 1 Birmingham and Midland Eye Centre and Academic Unit of Ophthalmol- ogy, University of Birmingham, Birmingham, UK. 2 Birmingham and Midland Eye Centre, Sandwell and West, Birmingham Hospitals NHS Trust, Dudley Road, Birmingham B18 7QH, UK. 3 Kantonsspital Winterthur, Brauerstrasse 15, 8400 Winterthur, Switzerland. 4 University of Zurich, Rämistrasse 71, 8006 Zurich, Switzerland. Received: 24 June 2022 Accepted: 22 August 2022 Received: 24 June 2022 Accepted: 22 August 2022 Competing interests Springer Nature remains neutral with regard to jurisdictional claims in pub- lished maps and institutional affiliations. All authors have no conflict of interest in the production of this manuscript. There are no external funders that have played a role in study design, data col- lection and analysis, decision to publish, or preparation of the manuscript. Funding f d No funding applicable. No funding applicable. Moussa et al. International Journal of Retina and Vitreous (2022) 8:61 Moussa et al. International Journal of Retina and Vitreous (2022) 8:61 Page 8 of 8 (2022) 8:61 Moussa et al. International Journal of Retina and Vitreous 14. Stappler T, Heimann H, Wong D, Gibran SK, Groenewald C, Pearce IA. Heavy tamponade 2 Densiron 68® in routine clinical practice: ana- tomical and functional outcomes of a consecutive case series. Eye. 2008;22(10):1360–5. 14. Stappler T, Heimann H, Wong D, Gibran SK, Groenewald C, Pearce IA. Heavy tamponade 2 Densiron 68® in routine clinical practice: ana- tomical and functional outcomes of a consecutive case series. Eye. 2008;22(10):1360–5. 14. Stappler T, Heimann H, Wong D, Gibran SK, Groenewald C, Pearce IA. Heavy tamponade 2 Densiron 68® in routine clinical practice: ana- tomical and functional outcomes of a consecutive case series. Eye. 2008;22(10):1360–5. 14. Stappler T, Heimann H, Wong D, Gibran SK, Groenewald C, Pearce IA. Heavy tamponade 2 Densiron 68® in routine clinical practice: ana- tomical and functional outcomes of a consecutive case series. Eye. 2008;22(10):1360–5. was acquired prior to all procedures as clinically indicated. This study does not report on the use of new or experimental protocols. References Romano MR, Angi M, Romano V, Parmeggiani F, Campa C, Valldeperas X, et al. Intraocular pressure changes following the use of silicone oil or Densiron® 68 as endotamponade in pars plana vitrectomy. Clin Ophthal- mol. 2010;4:1391. 9. Romano MR, Angi M, Romano V, Parmeggiani F, Campa C, Valldeperas X, et al. Intraocular pressure changes following the use of silicone oil or Densiron® 68 as endotamponade in pars plana vitrectomy. Clin Ophthal- mol. 2010;4:1391. • fast, convenient online submission • thorough peer review by experienced researchers in your ﬁeld • rapid publication on acceptance • support for research data, including large and complex data types • gold Open Access which fosters wider collaboration and increased citations maximum visibility for your research: over 100M website views per year • At BMC, research is always in progress. Learn more biomedcentral.com/submissions Ready to submit your research Ready to submit your research ? Choose BMC and benefit from: ? Choose BMC and benefit from: • fast, convenient online submission • thorough peer review by experienced researchers in your ﬁeld • rapid publication on acceptance • support for research data, including large and complex data types • gold Open Access which fosters wider collaboration and increased citations maximum visibility for your research: over 100M website views per year • At BMC, research is always in progress. Learn more biomedcentral.com/submissions Ready to submit your research Ready to submit your research ? Choose BMC and benefit from: ? Choose BMC and benefit from: 10. Semeraro F, Russo A, Morescalchi F, Gambicorti E, Vezzoli S, Parmeggiani F, et al. Comparative assessment of intraocular inflammation following standard or heavy silicone oil tamponade: a prospective study. Acta Ophthalmol. 2019;97(1):e97–102. 11. Herbrig E, Sandner D, Engelmann K. Anatomical and functional results of endotamponade with heavy silicone oil—Densiron® 68—in complicated retinal detachment. Ophthalmic Res. 2007;39(4):198–206. 12. Berker N, Batman C, Ozdamar Y, Eranil S, Aslan O, Zilelioglu O. Long- term outcomes of heavy silicone oil tamponade for complicated retinal detachment. Eur J Ophthalmol. 2007;17(5):797–803. 12. Berker N, Batman C, Ozdamar Y, Eranil S, Aslan O, Zilelioglu O. Long- term outcomes of heavy silicone oil tamponade for complicated retinal detachment. Eur J Ophthalmol. 2007;17(5):797–803. 13. Joussen AM, Rizzo S, Kirchhof B, Schrage N, Li X, Lente C, et al. Heavy sili- cone oil versus standard silicone oil in as vitreous tamponade in inferior PVR (HSO Study): interim analysis. Acta Ophthalmol. 2011;89(6):e483–9. 13. References 1. Kocak I, Koc H. Comparison of Densiron 68 and 1000 cSt silicone oil in the management of rhegmatogenous retinal detachment with inferior breaks. Int J Ophthalmol. 2013;6(1):81. 1. Kocak I, Koc H. Comparison of Densiron 68 and 1000 cSt silicone oil in the management of rhegmatogenous retinal detachment with inferior breaks. Int J Ophthalmol. 2013;6(1):81. p 2. Caporossi T, Franco F, Finocchio L, Barca F, Giansanti F, Tartaro R, et al. Densiron 68 heavy silicone oil in the management of inferior retinal detachment recurrence: analysis on functional and anatomical outcomes and complications. Int J Ophthalmol. 2019;12(4):615. 3. Hiscott P, Magee RM, Colthurst M, Lois N, Wong D. Clinicopathologi- cal correlation of epiretinal membranes and posterior lens opacifica- tion following perfluorohexyloctane tamponade. Br J Ophthalmol. 2001;85(2):179–83. 4. Li W, Zheng J, Zheng Q, Wu R, Wang X, Xu M. Clinical complications of Densiron 68 intraocular tamponade for complicated retinal detachment. Eye. 2010;24(1):21–8. 5. Heimann H, Stappler T, Wong D. Heavy tamponade 1: a review of indica- tions, use, and complications. Eye. 2008;22(10):1342–59. 6. Day AC, Donachie PHJ, Sparrow JM, Johnston RL. The Royal College of Ophthalmologists’ National Ophthalmology Database study of cataract surgery: report 1, visual outcomes and complications. Eye (Basingstoke). 2015;29:552–60. 6. Day AC, Donachie PHJ, Sparrow JM, Johnston RL. The Royal College of Ophthalmologists’ National Ophthalmology Database study of cataract surgery: report 1, visual outcomes and complications. Eye (Basingstoke). 2015;29:552–60. 7. Moussa G, Bassilious K, Mathews N. A novel excel sheet conversion tool from Snellen fraction to LogMAR including ‘counting fingers’, ‘hand move- ment’, ‘light perception’ and ‘no light perception’ and focused review of literature of low visual acuity reference values. Acta Ophthalmol. 2021;99:aos.14659. https://doi.org/10.1111/aos.14659. 7. Moussa G, Bassilious K, Mathews N. A novel excel sheet conversion tool from Snellen fraction to LogMAR including ‘counting fingers’, ‘hand move- ment’, ‘light perception’ and ‘no light perception’ and focused review of literature of low visual acuity reference values. Acta Ophthalmol. 2021;99:aos.14659. https://doi.org/10.1111/aos.14659. 8. Wong D, Kumar I, Quah SA, Ali H, Valdeperas X, Romano MR. Comparison of postoperative intraocular pressure in patients with Densiron-68 vs con- ventional silicone oil: a case-control study. Eye. 2009;23(1):190–4. 8. Wong D, Kumar I, Quah SA, Ali H, Valdeperas X, Romano MR. Comparison of postoperative intraocular pressure in patients with Densiron-68 vs con- ventional silicone oil: a case-control study. Eye. 2009;23(1):190–4. 9. • fast, convenient online submission • thorough peer review by experienced researchers in your ﬁeld • rapid publication on acceptance • support for research data, including large and complex data types • gold Open Access which fosters wider collaboration and increased citations maximum visibility for your research: over 100M website views per year • At BMC, research is always in progress. Learn more biomedcentral.com/submissions Ready to submit your research Ready to submit your research ? Choose BMC and benefit from: ? Choose BMC and benefit from: References Joussen AM, Rizzo S, Kirchhof B, Schrage N, Li X, Lente C, et al. Heavy sili- cone oil versus standard silicone oil in as vitreous tamponade in inferior PVR (HSO Study): interim analysis. Acta Ophthalmol. 2011;89(6):e483–9.
https://openalex.org/W2982511147	https://research.rug.nl/files/110249621/s41467_019_12760_y.pdf	English	null	GWAS for systemic sclerosis identifies multiple risk loci and highlights fibrotic and vasculopathy pathways	Nature communications	2,019	cc-by	14,948	GWAS for systemic sclerosis identifies multiple risk loci and highlights fibrotic and vasculopathy pathways European Scleroderma Group† Published in: Nature Communications DOI: 10.1038/s41467-019-12760-y IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite from it. Please check the document version below. Document Version Publisher's PDF, also known as Version of record Publication date: 2019 Link to publication in University of Groningen/UMCG research database Citation for published version (APA): European Scleroderma Group† (2019). GWAS for systemic sclerosis identifies multiple risk loci and highlights fibrotic and vasculopathy pathways. Nature Communications, 10(1), Article 4955. https://doi.org/10.1038/s41467-019-12760-y GWAS for systemic sclerosis identifies multiple risk loci and highlights fibrotic and vasculopathy pathways European Scleroderma Group† Published in: Nature Communications DOI: 10.1038/s41467-019-12760-y IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite from it. Please check the document version below. Document Version Publisher's PDF, also known as Version of record Publication date: 2019 Link to publication in University of Groningen/UMCG research database Citation for published version (APA): European Scleroderma Group† (2019). GWAS for systemic sclerosis identifies multiple risk loci and highlights fibrotic and vasculopathy pathways. Nature Communications, 10(1), Article 4955. https://doi.org/10.1038/s41467-019-12760-y GWAS for systemic sclerosis identifies multiple risk loci and highlights fibrotic and vasculopathy pathways European Scleroderma Group† IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite from it. Please check the document version below. Document Version Publisher's PDF, also known as Version of record Link to publication in University of Groningen/UMCG research database Citation for published version (APA): European Scleroderma Group† (2019). GWAS for systemic sclerosis identifies multiple risk loci and highlights fibrotic and vasculopathy pathways. Nature Communications, 10(1), Article 4955. https://doi.org/10.1038/s41467-019-12760-y Citation for published version (APA): European Scleroderma Group† (2019). GWAS for systemic sclerosis identifies multiple risk loci and highlights fibrotic and vasculopathy pathways. Nature Communications, 10(1), Article 4955. https://doi.org/10.1038/s41467-019-12760-y Copyright Other than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons). The publication may also be distributed here under the terms of Article 25fa of the Dutch Copyright Act, indicated by the “Taverne” license. More information can be found on the University of Groningen website: https://www.rug.nl/library/open-access/self-archiving-pure/taverne- amendment. Take-down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. Take-down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons the number of authors shown on this cover page is limited to 10 maximum. ARTICLE ARTICLE ARTICLE ARTICLE NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-12760-y (Pcond) < 5 × 10−6) (Table 1, Fig. 1, Supplementary Fig. 2). Hence, a total of 27 independent signals associated with SSc were identiﬁed. R h d a R heumatic diseases are one of the main causes of physical disability of non-mental origin in the Western world according to the World Health Organization. Rheumatic diseases have a marked impact on the quality of life of patients. Among them, systemic sclerosis (SSc) has one of the highest mortality rates1. SSc is a chronic autoimmune disease (AD) that affects the connective tissue, with very heterogeneous clinical manifestations. The pathogenesis of the disease involves extensive ﬁbrosis of the skin and internal organs, vascular damage, and immune imbalance, including autoantibody production2,3. Lung involvement—both pulmonary hypertension and/or pulmonary ﬁbrosis—is the leading cause of death4. The two independent signals identiﬁed in the DNASE1L3 genomic region (3p14.3) (rs4076852, rs7355798) were intronic variants at PXK and FLNB, respectively. PXK-rs4076852 is in high linkage disequilibrium (LD) with PXK-rs2176082 (r2 = 0.92), which was reported to be associated with SSc in Martin et al.13 However, two Immunochip studies conducted by Mayes et al.9 and Zochling et al.12 showed that the primary association in this genomic region was with the nonsynonymous SNP DNASE1L3-rs35677470 (R206C), not present in our SNP panel. Mayes et al.9 showed that the PXK-rs2176082 association was dependent on the rs35677470 (R206C). Therefore, although we could not analyze the dependence in our GWAS data, we presumed that PXK-rs4076852 signal was also dependent on DNASE1L3-rs35677470 on the basis of previous evidence. Regarding the intronic signal in FLNB (rs7355798), we could not estimate whether it was dependent on DNASE1L3- rs35677470 or not. However, given its role in vascular injury repair14, FLNB may be an interesting SSc locus and should be the object of future research. In the case of the STAT4 genomic region (2q32.2-q32.3), we observed three independent signals, of which the third was an intronic variant at NAB1 (rs16832798). This ﬁnding—added to further functional evidence provided below— revealed NAB1 as a new SSc risk locus. We also observed that the genome-wide signal in GSDMB (17q21.1; rs883770) was inde- pendent (Pcond = 1.27 × 10−7) from the recently reported signal at GSDMA (rs3894194), which is located in the same genomic region10. As most ADs, SSc has a complex genetic component and its etiology is poorly understood. ARTICLE Genome-wide association studies (GWASs) have been successful in the identiﬁcation of thousands of genetic variants associated with the susceptibility of complex traits. Moreover, GWASs provide invaluable information on disease aetiopathogenesis and contribute to drug discovery and repurposing5,6. Several GWASs of SSc have been published, which greatly contributed to the understanding of SSc patho- genesis, and pointed out to relevant pathways for the disease, such as the interferon pathway, the interleukin 12 pathway, and apoptosis7–12. Nonetheless, the rate of discovery of previous studies was limited owing to the relatively small sample sizes of the study cohorts. To continue unraveling the partially known genetic back- ground of SSc, we perform a powerful meta-GWAS in European population that includes ~ 10,000 patients. We also hypothesize that an integrative approach combining all SSc association sig- nals, ﬁne-mapping, and the identiﬁcation of target genes based on chromatin contacts would provide further insights into the biol- ogy of the disease. Fine-mapping of SSc-associated loci in a Bayesian framework. The identiﬁcation of the causal SNPs driving the association signals remains an open question after completion of a GWAS. To address this question, Bayesian ﬁne-mapping was performed to deﬁne 95% credible sets (the smallest set of variants that summed together at least a 95% probability of including the likely causal variant) in each of the independently associated loci (the two independent signals in IRF5-TNPO3 were excluded as ﬁne- mapping was not feasible). To improve SNP prioritization accu- racy, the probabilistic method integrated association strength with functional annotation data (Methods). Eighteen (72%) and 12 (48%) out of the 25 loci were ﬁne-mapped to ≤10 and to <5 plausible causal variants, respectively (Table 2, Supplementary Data 2). In six loci, the 95% credible set comprised a single variant (ARHGAP31, BLK, CD247, TNIP1, CSK, STAT4-a), and for four others the credible set contained two SNPs (DGKQ, NUP85-GRB2, STAT4-b, IL12RB1). Moreover, in 64% of the credible sets, the index SNP showed the maximum posterior probability (PPmax) of being causal. The SNPs with PPmax were intergenic, intronic, or noncoding RNA intronic (ncRNA intro- nic) variants, although the remaining credible set SNPs involved additional SNP categories, namely: UTR3′, downstream, exonic synonymous, and exonic nonsynonymous (Table 2). GWAS for systemic sclerosis identiﬁes multiple risk loci and highlights ﬁbrotic and vasculopathy pathways Elena López-Isac et al.# Systemic sclerosis (SSc) is an autoimmune disease that shows one of the highest mortality rates among rheumatic diseases. We perform a large genome-wide association study (GWAS), and meta-analysis with previous GWASs, in 26,679 individuals and identify 27 independent genome-wide associated signals, including 13 new risk loci. The novel asso- ciations nearly double the number of genome-wide hits reported for SSc thus far. We deﬁne 95% credible sets of less than 5 likely causal variants in 12 loci. Additionally, we identify speciﬁc SSc subtype-associated signals. Functional analysis of high-priority variants shows the potential function of SSc signals, with the identiﬁcation of 43 robust target genes through HiChIP. Our results point towards molecular pathways potentially involved in vasculopathy and ﬁbrosis, two main hallmarks in SSc, and highlight the spectrum of critical cell types for the disease. This work supports a better understanding of the genetic basis of SSc and provides directions for future functional experiments. email: eisac.csic@gmail.com; javiermartin@ipb.csic.es NATURE COMMUNICATIONS \| (2019) 10:4955 \| https://doi.org/10.1038/s41467-019-12760-y \| www.nature.com/naturecommunicatio 1 The new genome-wide signiﬁcant loci for systemic sclerosis are highlighted in bold. NAB1-rs16832798 p value conditioned on conditioned on STAT4-rs3821236 and STAT4-rs4853458. For those intronic or regulatory SNPs that are located in a high gene density region, the gene they lie in was underlined Bp base pair, Chr chromosome, MAF minor allele frequency in the 1000 Genome Project European Population, N number of cohorts, OR odds ratio, Pcond p value conditioned on index SNP, Ref. reference allele, SNP single-nucleotide polymorphism ture.com/nat Table 1 Twenty-seven signals independently associated with systemic sclerosis in the meta-GWAS Chr Locus Bp SNP Index SNP Ref. The new genome-wide signiﬁcant loci for systemic sclerosis are highlighted in bold. NAB1-rs16832798 p value conditioned on conditioned on STAT4-rs3821236 and STAT4-rs4853458. For those intronic or regulatory SNPs that are located in a high gene density region, the gene they lie in was underlined Bp base pair, Chr chromosome, MAF minor allele frequency in the 1000 Genome Project European Population, N number of cohorts, OR odds ratio, Pcond p value conditioned on index SNP, Ref. reference allele, SNP single-nucleotide polymorphism ture.com/nat MAF N P value OR Q I Pcond Func refgene 1 IL12RB2 67814440 rs3790566 Yes T 0.24 13 3.84E-10 1.16 0.80 0 - Intronic 1 CD247 167420425 rs2056626 Yes G 0.39 6 1.31E-11 0.81 0.57 0 - Intronic 1 TNFSF4-LOC100506023-PRDX6 173238736 rs2022449 No T 0.23 12 6.28E-08 1.15 0.90 0 6.63E-08 Regulatory region 1 TNFSF4-LOC100506023-PRDX6 173332629 rs1857066 Yes A 0.25 13 5.02E-09 0.87 0.84 0 - ncRNA intronic 2 NAB1 191534372 rs16832798 Yes C 0.14 14 5.20E-09 1.18 0.41 3.79 3.84E-07 Intronic 2 STAT4 191902758 rs3821236 Yes A 0.20 12 1.94E-23 1.31 0.03 48.21 - Intronic 2 STAT4 191959489 rs4853458 No A 0.23 9 4.86E-18 1.35 0.42 1.79 5.58E-08 Intronic 3 FLNB-DNASE1L3-PXK 58131515 rs7355798 No T 0.24 13 1.24E-08 1.14 0.14 30.5 7.42E-07 Intronic 3 FLNB-DNASE1L3-PXK 58375286 rs4076852 Yes G 0.26 13 1.04E-10 1.16 0.71 0 - Intronic 3 POGLUT1-TIMMDC1-CD80-ARHGAP31 119116150 rs9884090 Yes A 0.16 13 1.89E-10 0.83 0.92 0 - Intronic 3 IL12A 159733527 rs589446 Yes T 0.35 11 1.95E-10 0.86 0.85 0 - ncRNA intronic 4 DGKQ 965779 rs11724804 Yes A 0.44 12 5.31E-11 1.17 0.24 21.04 - Intronic 4 NFKB1 103449041 rs230534 Yes T 0.34 10 5.38E-09 1.15 0.92 0 - Intronic 5 TNIP1 150455732 rs3792783 Yes G 0.16 14 2.42E-12 1.20 0.03 47.41 - Intronic 6 ATG5 106734040 rs633724 Yes T 0.35 14 2.84E-09 1.13 0.31 13.41 - Intronic 7 IRF5-TNPO3 128651522 rs36073657 Yes T 0.10 12 3.10E-21 1.40 0.21 23.35 - Intronic 7 IRF5-TNPO3 128658739 rs12155080 No G 0.37 13 2.87E-13 0.85 0.69 0 2.22E-07 Intronic 8 FAM167A-BLK 11343973 rs2736340 Yes T 0.24 14 3.33E-21 1.24 0.17 26.76 - Intergenic 8 RAB2A-CHD7 61564964 rs685985 Yes T 0.47 11 3.82E-08 0.87 0.15 30.84 - Intergenic 11 CDHR5-IRF7 618172 rs6598008 Yes A 0.44 4 1.97E-08 0.80 0.16 42.27 - Intronic 11 TSPAN32,CD81-AS1 2348619 rs2651804 Yes T 0.17 12 2.54E-10 0.82 0.67 0 - Intergenic 11 DDX6 118639353 rs11217020 Yes A 0.20 14 2.08E-11 0.84 0.80 0 - Intronic 15 CSK 75077367 rs1378942 Yes C 0.39 13 1.84E-14 1.18 0.90 0 - Intronic 16 IRF8 85971922 rs11117420 Yes C 0.19 12 3.82E-15 0.81 0.47 0 - Intergenic 17 IKZF3-GSDMB 38063381 rs883770 Yes T 0.50 14 4.79E-09 1.13 0.75 0 - Intronic 17 NUP85-GRB2 73224639 rs1005714 Yes G 0.20 13 1.87E-08 0.85 0.68 0 - Intronic 19 IL12RB1 18193191 rs2305743 Yes A 0.20 12 4.64E-10 0.83 0.28 16.88 - Intronic The new genome-wide signiﬁcant loci for systemic sclerosis are highlighted in bold. The new genome-wide signiﬁcant loci for systemic sclerosis are highlighted in bold. NAB1-rs16832798 p value conditioned on conditioned on STAT4-rs3821236 and STAT4-rs4853458. For those intronic or regulatory SNPs that are located in a high gene density region, the gene they lie in was underlined Bp base pair, Chr chromosome, MAF minor allele frequency in the 1000 Genome Project European Population, N number of cohorts, OR odds ratio, Pcond p value conditioned on index SNP, Ref. reference allele, SNP single-nucleotide polymorphism ture.com/nat NAB1-rs16832798 p value conditioned on conditioned on STAT4-rs3821236 and STAT4-rs4853458. For those intronic or regulatory SNPs that are located in a high gene density region, the gene they lie in was underlined Bp base pair, Chr chromosome, MAF minor allele frequency in the 1000 Genome Project European Population, N number of cohorts, OR odds ratio, Pcond p value conditioned on index SNP, Ref. reference allele, SNP single-nucleotide polymorphism Results T Twenty-seven signals independently associated with SSc. We performed genome-wide association analyses in 14 independent European cohorts comprising a total of 26,679 individuals (9,095 SSc patients and 17,584 healthy controls). Nine out of the 14 SSc GWAS cohorts were so far unreported, whereas 5 had been previously published7,8 (Supplementary Data 1). After correcting for sex and the ﬁrst ﬁve principal components (PCs) (Methods), we did not observe genomic inﬂation in any of the independent GWAS cohorts, with the exception of the Italian cohort, which remained with residual inﬂation (Supplementary Data 1 and Supplementary Fig. 1). Overall, the meta-analysis showed a genomic inﬂation factor (λ) of 1.10, with a rescaled λ1000 of 1.008 for an equivalent study of 1000 cases/1000 controls (Supple- mentary Data 1). We undertook an inverse variance-weighted meta-analysis with a high-density genotyped and imputed SNP panel (4.72 million SNPs) to combine all independent GWASs. We considered all the SNPs that were shared by at least two data sets to avoid SNP data loss. This approach yielded 431 signiﬁcantly associated SNPs (association test p value ≤5 × 10−8) excluding the well-known HLA region. Signiﬁcant signals involved 23 genomic regions, of which 13 were new genome-wide signiﬁcant loci for SSc and 10 corresponded to previously reported GWAS signals (Table 1, Fig. 1). Functional annotation of SNPs from credible sets. Since most of the likely causal variants were linked to regulatory functions rather than affecting the function of proteins encoded by sur- rounding genes, we further explored their regulatory effects. For this purpose, we performed functional annotation of SNPs from credible sets through eQTL analysis (Methods). In addition, we explored overlap with histone marks of active promoters and active enhancers (H3K9ac, H3K4me1, and H3K27ac) of cell types relevant to the disease using data from the Roadmap Epigenomics Project15 (Supplementary Table 1) (Methods). The presence of independent signals in the genomic regions that showed signiﬁcant associations was investigated by stepwise conditional analysis using summary statistics from the meta- analysis (Methods). Four genomic regions—TNFSF4 (1q25.1), STAT4 (2q32.2-q32.3), DNASE1L3 (3p14.3), and IRF5-TNPO3 (7q32.1)—showed additional signiﬁcant signals after conditioning on the lead SNP of each locus (conditional association test p value NATURE COMMUNICATIONS \| (2019) 10:4955 \| https://doi.org/10.1038/s41467-019-12760-y \| www.nature.com/naturecommunications 2 ARTICLE NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-12760-y NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-12760-y The lowest p value was observed within the MHC region for rs6457617 (association test p = 3.25 × 10−43). b Locuszoom to depict independent association signals in IRF5-TNPO3. From left to right, locuszoom of the association signals in IRF5-TNPO3 for the global meta-analysis; association signals conditioned on the lead SNP (rs36073657), and conditioned on rs36073657 and the secondary signal at the locus (rs12155080) Supplementary Figure 3 summarizes the results of the functional characterization of credible set SNPs. When the 95% credible set was not well resolved (credible sets that contained > 15 likely causal variants), we selected the SNP with PPmax and the index SNP. In the case of IRF5-TNPO3, where the credible set was not feasible, we selected the two independent signals identiﬁed at this locus. We obtained a ﬁnal reduced list of credible set SNPs containing a total of 81 variants. As it can be observed in Supplementary Fig. 3, the vast majority of the likely causal variants overlapped with promoter and enhancer histone marks in the cell types interrogated. These observations suggest that most of the genetic variations involved in the susceptibility to SSc modulate transcriptional regulatory mechanisms. In this regard, we found that 61 out of the 81 interrogated variants (75.31% of the 81 listed credible set SNPs) represent eQTLs, thus altering gene expression in different tissues and cell types (Supplementary Data 3). In fact, the credible set SNPs were signiﬁcantly enriched for eQTLs in blood and non-blood tissues (odds ratio (OR) = 3.05, Fisher’s exact test P = 5.65 × 10−6; OR = 1.61, Fisher’s exact test P = 4.48 × 10−2, respectively) (Sup- plementary Table 2). Many SNPs were shown to impact the expression of the closest gene (a priori candidate gene) (Supple- mentary Data 3). In addition, we also found genetic variants affecting the expression of a priori candidate genes and other genes. However, some SNPs only showed eQTL signals for genes other than the closest one. As an example, the SNP rs9884090—which is an intronic variant at ARHGAP31—was found to alter the expression of POGLUT1 and TIMMDC1 in several tissues (Supplementary Data 3). These results highlight that assigning association signals to the nearby gene is not always the most appropriate strategy and the functional role of certain SSc signals may expand to different target genes. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-12760-y From left to right, locuszoom of the association signals in IRF5-TNPO3 for the global meta-analysis; association signals conditioned on the lead SNP (rs36073657), and conditioned on rs36073657 and the secondary signal at the locus (rs12155080) 30 a 25 20 15 –log10(p) 10 5 0 1 IL12RB2 CD247 NAB1 FLNB-DNASE1L3-PXK IL12A NFKB1 DGKQ TNIP1 ATG5 RAB2A-CHD7 CDHR5-IRF7 TSPAN32,CD81-AS1 DDX6 CSK IKZF3-GSDMB NUP85-GRB2 IL12RB1 IRF8 IRF5-TNPO3 FAM167A-BLK POGLUT1-TIMMDC1-CD80-ARHGAP31 STAT4 TNFSF4-LOC100506023-PRDX6 2 3 4 5 6 7 Chromosome 8 9 10 11 12 13 14 15 17 19 21 a 30 a 25 20 15 –log10(p) 10 5 0 1 IL12RB2 CD247 NAB1 FLNB-DNASE1L3-PXK IL12A NFKB1 DGKQ TNIP1 ATG5 RAB2A-CHD7 CDHR5-IRF7 TSPAN32,CD81-AS1 DDX6 CSK IKZF3-GSDMB NUP85-GRB2 IL12RB1 IRF8 IRF5-TNPO3 FAM167A-BLK POGLUT1-TIMMDC1-CD80-ARHGAP31 STAT4 TNFSF4-LOC100506023-PRDX6 2 3 4 5 6 7 Chromosome 8 9 10 11 12 13 14 15 17 19 21 20 0.8 0.6 0.4 0.2 Plotted SNPs P 15 r 2 –log10(p value) 10 5 0 128 MGC27345 LINC01000 FLNC TNPO3 TPI1P2 SMO LOC407835 TSPAN33 AHCYL2 STRIP2 SMKR1 NRF1 KCP IRF5 FAM71F2 FAM71F1 CALU OPN1SW CCDC136 ATP6V1F RBM28 PRRT4 IMPDH1 HILPDA METTL28 128.2 128.4 rs36073657 128.6 Position on chr7 (Mb) 128.8 129 129.2 100 b 80 Recombination rate (cM/Mb) 60 40 20 0 1 gene omitted b Plotted SNPs 0.8 0.6 0.4 0.2 r 2 MGC27345 LINC01000 FLNC TNPO3 TPI1P2 SMO LOC407835 TSPAN33 AHCYL2 STRIP2 SMKR1 NRF1 KCP IRF5 FAM71F2 FAM71F1 CALU OPN1SW CCDC136 ATP6V1F RBM28 PRRT4 IMPDH1 HILPDA METTL28 –log10(p value) 20 15 10 5 0 100 80 Recombination rate (cM/Mb) 60 40 20 0 1 gene omitted 128 128.2 128.4 128.6 Position on chr7 (Mb) 128.8 129 129.2 Plotted SNPs 0.8 0.6 0.4 0.2 r 2 –log10(p value) MGC27345 LINC01000 FLNC TNPO3 TPI1P2 SMO LOC407835 TSPAN33 AHCYL2 STRIP2 SMKR1 NRF1 KCP IRF5 FAM71F2 FAM71F1 CALU OPN1SW CCDC136 ATP6V1F RBM28 PRRT4 IMPDH1 HILPDA METTL28 20 15 10 5 0 100 80 Recombination rate (cM/Mb) 60 40 20 0 1 gene omitted 128 128.2 128.4 128.6 Position on chr7 (Mb) 128.8 129 129.2 Fig. 1 Association signals for systemic sclerosis in a large meta-GWAS. a Manhattan plot representing the meta-GWAS results. The −log10 of the p values are plotted against their physical chromosomal position. The red and blue lines represent the genome-wide level of signiﬁcance (p < 5 × 10−8) and p value threshold at p < 1 × 10−5, respectively. The plot has been truncated at p < 1 × 10−30. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-12760-y 20 0.8 0.6 0.4 0.2 Plotted SNPs Plotted SNPs Plotted SNPs 15 r 2 0.8 0.6 0.4 0.2 r 2 0.8 0.6 0.4 0.2 r 2 –log10(p value) –log10(p value) 10 5 0 128 MGC27345 LINC01000 FLNC TNPO3 TPI1P2 SMO LOC407835 TSPAN33 AHCYL2 STRIP2 SMKR1 NRF1 KCP IRF5 FAM71F2 FAM71F1 CALU OPN1SW CCDC136 ATP6V1F RBM28 PRRT4 IMPDH1 HILPDA METTL28 MGC27345 LINC01000 FLNC TNPO3 TPI1P2 SMO LOC407835 TSPAN33 AHCYL2 STRIP2 SMKR1 NRF1 KCP IRF5 FAM71F2 FAM71F1 CALU OPN1SW CCDC136 ATP6V1F RBM28 PRRT4 IMPDH1 HILPDA METTL28 MGC27345 LINC01000 FLNC TNPO3 TPI1P2 SMO LOC407835 TSPAN33 AHCYL2 STRIP2 SMKR1 NRF1 KCP IRF5 FAM71F2 FAM71F1 CALU OPN1SW CCDC136 ATP6V1F RBM28 PRRT4 IMPDH1 HILPDA METTL28 128.2 128.4 rs36073657 128.6 Position on chr7 (Mb) 128.8 129 129.2 100 30 a b 25 20 15 –log10(p) 10 5 0 1 IL12RB2 CD247 NAB1 FLNB-DNASE1L3-PXK IL12A NFKB1 DGKQ TNIP1 ATG5 RAB2A-CHD7 CDHR5-IRF7 TSPAN32,CD81-AS1 DDX6 CSK IKZF3-GSDMB NUP85-GRB2 IL12RB1 IRF8 IRF5-TNPO3 FAM167A-BLK POGLUT1-TIMMDC1-CD80-ARHGAP31 STAT4 TNFSF4-LOC100506023-PRDX6 2 3 4 5 6 7 Chromosome 8 9 10 11 12 13 14 15 17 19 21 20 15 10 5 0 80 Recombination rate (cM/Mb) 60 40 20 0 –log10(p value) 100 20 15 10 5 0 80 Recombination rate (cM/Mb) 60 40 20 0 100 80 Recombination rate (cM/Mb) 60 40 20 0 1 gene omitted 1 gene omitted 1 gene omitted 128 128.2 128.4 128.6 Position on chr7 (Mb) 128.8 129 129.2 128 128.2 128.4 128.6 Position on chr7 (Mb) 128.8 129 129.2 Fig. 1 Association signals for systemic sclerosis in a large meta-GWAS. a Manhattan plot representing the meta-GWAS results. The −log10 of the p values are plotted against their physical chromosomal position. The red and blue lines represent the genome-wide level of signiﬁcance (p < 5 × 10−8) and p value threshold at p < 1 × 10−5, respectively. The plot has been truncated at p < 1 × 10−30. The lowest p value was observed within the MHC region for rs6457617 (association test p = 3.25 × 10−43). b Locuszoom to depict independent association signals in IRF5-TNPO3. 16832798 p value conditioned on conditioned on STAT4-rs3821236 and STAT4-rs4853458. For those intronic or regulatory SNPs that are located in a hig European Population, N number of cohorts, OR odds ratio, Pcond p value conditioned on index SNP, Ref. reference allele, SNP single-nucleotide polymo 3 NATURE COMMUNICATIONS \| (2019) 10:4955 \| https://doi.org/10.1038/s41467-019-12760-y \| www.nature.com/naturecommunicati ARTICLE NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-12760-y NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-12760-y Five 95% credible sets comprised exonic nonsynonymous variants or contained SNPs in high-to-moderate LD with exonic nonsynonymous variants (r2 ≥0.8, r2 ≥0.6, respectively) (ARH- GAP31, IRF7, GSDMB, NUP85-GRB2, and IL12RB1) (Supple- mentary Fig. 3, Supplementary Data 4). However, based on SIFT and PolyPhen, none of these exonic variants showed a clear consensus to be deleterious16,17 (Supplementary Data 4). Finally, we assessed pleiotropic effect of our signals by determining whether the likely causal SNPs were also risk factors for other diseases. The results showed extensive overlap especially with other two ADs: systemic lupus erythematosus and primary biliary cholangitis (Supplementary Data 5). These ﬁndings were consistent with previous reports that identiﬁed shared risk loci for SSc and other immune-mediated diseases11,13. H3K27ac HiChIP in T cells expands and reﬁnes target genes. As stated above, assigning disease-associated variants to the clo- sest gene is not always an appropriate strategy to determine the potential mechanistic effect of association signals. With the aim of identifying the putative drivers of SSc association hits on the basis of functional evidence, we performed an analysis of experimen- tally derived high-resolution maps of enhancer-promoter inter- actions generated by H3K27ac HiChIP experiments in human CD4 + T cells18 (Methods). HiChIP interactions were detected in 18 out of the 27 (66.67%) independently associated loci, using the SNPs with PPmax as anchor points (Table 3). Several intronic variants were linked to the target gene promoter in which they are mapped. This was the case of CD247-rs2056626, which showed a strong H3K27ac HiChIP signal to the CD247 promoter (Fig. 2). Other relevant NATURE COMMUNICATIONS \| (2019) 10:4955 \| https://doi.org/10.1038/s41467-019-12760-y \| www.nature.com/naturecommunications 4 ARTICLE ARTICLE NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-12760-y Table 2 Posterior probabilities of systemic sclerosis ﬁne-mapped loci Chr Credible set locus SNPs Cred. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-12760-y examples of this type of interactions were found in IL12RB2 and NFKB1. The intronic variants in STAT4, rs3821236 and rs4853458, showed strong normalized HiChIP signal to STAT4 and STAT1 promoters (Fig. 2). We also observed HiChIP contacts that linked intergenic SNPs to the closest genes. For example, rs11117422, located ~40 kb downstream of IRF8 transcriptional start site, showed interactions with the promoter region of IRF8 (Fig. 2). In addition, several other enhancer- promoter interactions linked intronic and intergenic SNPs to distant genes. In total, H3K27ac HiChIP signals nominated 155 target genes from 18 SSc likely causal variants (~8 genes per SNP on average) (Table 3). pp In total, we provided strong evidence to nominate 43 genes as robust SSc target genes in CD4 + T cells (Table 3). Interestingly, some of them pinpointed to new mechanistic insights relevant for the diseases (see Discussion). Chromatin interaction analyses in other relevant cell types. It is noteworthy that the epigenomic proﬁles are cell type spe- ciﬁc19,20. Considering that the HiChIP analyses were performed in CD4+ T cells, and that the pathogenesis of SSc is not only mediated by T cells, we also explored chromatin interaction maps derived from promoter capture Hi-C experiments in additional immune cell types21,22 (Methods). These analyses identiﬁed promoter interactions not observed in CD4+ T cells, which targeted new genes (Table 3, Supplementary Data 6). For example, we observed that the FLNB-intronic variant rs7355798 interacted with FLNB and PXK promoters in B cells and mac- rophages. Moreover, some of the interactions observed with H3K27ac HiChIP in CD4+ T cells were also found in other immune cell types. Subsequently, we further validated the functional relevance of H3K27ac HiChIP results by investigating whether the explored SNPs were eQTLs for the nominated target genes. Forty enhancer- target gene relationships showed overlap with SSc eQTL genes (eGenes) (OR = 10.1, Fisher’s exact test P = 2.92 × 10−19, Supple- mentary Table 3). Although no eQTL to IRF8, STAT4, and STAT1 signals were found, the enrichment of the HiChIP signal observed at these loci (q value < 1e-60, Methods) over a global background of distance-matched interactions, and the crucial role of these genes in the immune response, provided evidence to prioritize them as candidate genes. Remarkably, the third independent association signal observed in the STAT4 genomic region (2q32.2-q32.3)—mapped in a NAB1 intron—was linked to NAB1 promoter by our H3K27ac HiChIP analysis. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-12760-y Set Index SNP PP Index SNP PP Max PP Max Func.refgene SNP PP Max Func.refgene in the 95% credible set 1 IL12RB2 6 rs3790566 0.195 rs3790567 0.321 Intronic Intronic 1 CD247 1 rs2056626 0.999 rs2056626 0.999 Intronic - 1 TNFSF4- LOC100506023-PRDX6 6 rs2022449 0.659 rs2022449 0.659 ncRNA Intronic ncRNA_intronic 1 TNFSF4- LOC100506023-PRDX6 43 rs1857066 0.046 rs11576547 0.265 ncRNA intronic ncRNA_intronic 2 NAB1 11 rs16832798 0.191 rs716254 0.242 Intronic Intronic; intergenic; downstream 2 STAT4-a1 1 rs3821236 1.000 rs3821236 1.000 Intronic - 2 STAT4-b2 2 rs4853458 0.905 rs4853458 0.905 Intronic Intronic 3 FLNB-DNASE1L3-PXK 6 rs7355798 0.365 rs7355798 0.365 Intronic Intronic 3 FLNB-DNASE1L3-PXK 27 rs4076852 0.123 rs7653734 0.292 Intronic Intronic; intergenic 3 POGLUT1-TIMMDC1- CD80-ARHGAP31 1 rs9884090 0.956 rs9884090 0.956 Intronic - 3 IL12A 23 rs589446 0.385 rs589446 0.385 ncRNA intronic ncRNA_intronic 4 DGKQ 2 rs11724804 0.793 rs11724804 0.793 Intronic Intronic 4 NFKB1 6 rs230534 0.200 rs230517 0.329 Intronic Intronic 5 TNIP1 1 rs3792783 0.999 rs3792783 0.999 Intronic - 6 ATG5 3 rs633724 0.588 rs633724 0.588 Intronic Intronic 8 FAM167A-BLK 1 rs2736340 1.000 rs2736340 1.000 Intergenic - 8 RAB2A-CHD7 80 rs685985 0.003 rs6987084 0.139 Intronic Intronic; UTR3; intergenic 11 CDHR5-IRF7 4 rs6598008 0.760 rs6598008 0.760 Intronic Intronic; exonic synonymous SNV; UTR3; exonic nonsynonymous 11 TSPAN32,CD81-AS1 20 rs2651804 0.184 rs2651804 0.184 Intergenic Intergenic 11 DDX6 7 rs11217020 0.021 rs10892286 0.775 Intronic Intronic; intergenic 15 CSK 1 rs1378942 0.993 rs1378942 0.993 Intronic - 16 IRF8 6 rs11117420 0.202 rs11117422 0.54 Intergenic Intergenic 17 IKZF3-GSDMB 17 rs883770 0.032 rs9303277 0.157 Intronic Intergenic; intronic; exonic synonymous SNV; exonic nonsynonymous 17 NUP85-GRB2 2 rs1005714 0.940 rs1005714 0.940 Intronic Intronic 19 IL12RB1 2 rs2305743 0.944 rs2305743 0.944 Intronic Intronic Chr chromosome, PP posterior probability, SNP single-nucleotide polymorphism 1Name of the credible set that comprised the index SNP from STAT4 genomic region (2q32.2-q32.3) 2Name of the credible set that comprised the secondary association signal in STAT4 genomic region (2q32.2-q32.3) Table 2 Posterior probabilities of systemic sclerosis ﬁne-mapped loci interaction was validated by an eQTL signal (Supplementary Data 3). These results supported NAB1 as a new SSc risk locus. In total, we provided strong evidence to nominate 43 genes as robust SSc target genes in CD4 + T cells (Table 3). Interestingly, some of them pinpointed to new mechanistic insights relevant for the diseases (see Discussion). interaction was validated by an eQTL signal (Supplementary Data 3). These results supported NAB1 as a new SSc risk locus. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-12760-y The The functional relevance of the observed chromatin interac- tions by promoter capture Hi-C analyses was also validated by eQTLs analysis. In total, these analyses nominated 25 additional target genes for SSc (Table 3). NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-12760-y Interestingly, our large panel of reference epigenomes allowed us to identify cell type speciﬁc patterns of enrichment. This speciﬁcity was especially relevant for some tissue/cell types that showed enrichment for a single histone mark. For example, dermal ﬁbroblast primary cells (SKIN.NHDFAD) only showed signiﬁcant enrichment for H3K4me2 (OR = 7.91, Penr = 6.54 × 10−6). Speciﬁc patterns of associations for the main SSc subtypes. We performed GWAS stratiﬁed analyses considering the main SSc clinical subtypes (limited cutaneous SSc (lcSSc) or diffuse cuta- neous SSc (dcSSc)) and autoantibody status according to the presence of anticentromere (ACA), antitopoisomerase (ATA), and anti-RNA polymerase III (ARA) autoantibodies (Supple- mentary Table 4) (Methods). Tissue-speciﬁc enrichment of SSc loci in epigenetic marks. The majority of credible set SNPs overlapped with epigenetic marks related to active regions (Supplementary Fig. 3). To quantify the extent of this overlap, we investigated whether SSc associations were non-randomly distributed in histone marks of active pro- moters (H3K9ac, H3K4me2, H3K4me3, H3K4ac), active enhan- cers (H3K27ac, H3K4me1, H2BK20ac), and active (or at least accessible) genes (H3K79me1, H2BK15ac) across the 127 refer- ence epigenomes available from the Roadmap Epigenomics Consortium and the Encyclopedia of DNA Elements (ENCODE) projects15,24. We used a nonparametric approach (GAR- FIELD25,26) to compute ORs and estimate the signiﬁcance of functional enrichment at various GWAS p value cutoffs (5 × 10−6, 5 × 10−7, and 5 × 10−8) (Methods). y A total of 18 and 5 non-HLA signiﬁcant signals were identiﬁed for lcSSc and dcSSc, respectively, representing 15 new genome- wide signiﬁcant signals for lcSSc and 3 for dcSSc (Supplementary Data 9). Among the associations, there were loci that yielded stronger associations and larger effect size in the subtype analyses than in the global meta-analysis; all despite the reduction of the sample and, consequently, of statistical power. To assess whether the more powerful genetic signals were randomly observed, we performed 10,000 permutation analyses (Methods) and computed empirical p values (p) taking into account the proportion of permuted genetic signals that were at least as extreme as the observed signals. As an example, DNASE1L3 genomic region (3p14.3, rs7652027) was associated with the global disease with an OR of 1.15, whereas the OR observed for the lcSSc subtype was 1.20. Permutation analysis showed signiﬁcant empirical p value (p = 1.9 × 10−3) for DNASE1L3-rs7652027 thus conﬁrming a larger effect of this risk factor in lcSSc patients (Supplementary Data 9, Supplementary Fig. 4). NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-12760-y ARTICLE Normalized signal Naive ATAC TH17 ATAC TREG ATAC Naive ATAC TH17 ATAC TREG ATAC Naive ATAC TH17 ATAC TREG ATAC Normalized signal Normalized signal 40 6 12 10 8 6 4 2 0 5 4 3 2 1 0 30 20 10 0 –100 kb RefSeq genes <CD247 <STAT1 <STAT4 IRF8> RefSeq genes RefSeq genes chr. 1 5 kb res. Naive TH17 TREG Naive TH17 TREG Naive TH17 TREG chr. 16 5 kb res. chr. 2 5 kb res. +200 kb 167.42 Mb –300 kb –100 kb +200 kb +100 kb 85.97 Mb 191.9 Mb Fig. 2 H3K27ac HiChIP signals at systemic sclerosis loci in human CD4+ T cells. The SNPs with maximum Posterior Probabilities in each locus were set as anchor points to assess promoter-enhancer chromatin interactions. Representation of overlap with ATAC-seq peaks is included. Chr, chromosome; Kb, kilo base; Mb, mega base; Res, resolution Normalized signal Naive ATAC TH17 ATAC TREG ATAC 40 30 20 10 0 –100 kb RefSeq genes <CD247 chr. 1 5 kb res. Naive TH17 TREG +200 kb 167.42 Mb Naive ATAC TH17 ATAC TREG ATAC Normalized signal 6 5 4 3 2 1 0 IRF8> RefSeq genes Naive TH17 TREG chr. 16 5 kb res. –300 kb +100 kb 85.97 Mb Naive ATAC TH17 ATAC TREG ATAC Normalized signal 12 10 8 6 4 2 0 <STAT1 <STAT4 RefSeq genes Naive TH17 TREG chr. 2 5 kb res. –100 kb +200 kb 191.9 Mb Fig. 2 H3K27ac HiChIP signals at systemic sclerosis loci in human CD4+ T cells. The SNPs with maximum Posterior Probabilities in each locus were set as anchor points to assess promoter-enhancer chromatin interactions. Representation of overlap with ATAC-seq peaks is included. Chr, chromosome; Kb, kilo base; Mb, mega base; Res, resolution Candidate genes prioritized by DEPICT. In addition, we also conducted gene prioritization by means of DEPICT (Data-driven Expression-Prioritized Integration for Complex Traits) (http:// www.broadinstitute.org/mpg/depict/index.html), which pinpoints the most likely candidate gene/s in associated loci based on predicted gene functions23. Signiﬁcant gene prioritization p values were found in 19 of the 27 queried loci (Table 3, Supplementary Data 7). Most of the prioritized genes were previously nominated by the chromatin interaction analyses. In addition, this method nominated TNFSF4, TMEM194B, FLNB-AS1, CD80, ARHGAP31, C8orf14, TSPAN32, and MAST3. osteoblasts, intestinal mucosa, and esophagus, among others (Fig. 3, Supplementary Data 8). NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-12760-y 5 5 TURE COMMUNICATIONS \| (2019) 10:4955 \| https://doi.org/10.1038/s41467-019-12760-y \| www.nature.com/naturecommunications ARTICLE NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-12760-y 3 H3K27ac HiChIP target genes and nominated target genes for the 27 systemic sclerosis association signals Locus SNP PP Max HiChIP target genes with SNP PP Max Nominated genes by H3K27ac HiChIP + eQTL validation Nominated genes by CHi-C + eQTL validation Prioritized genes by DEPICT/other criteria IL12RB2 rs3790567 IL12RB2, IL23R IL12RB2 IL12RB2, SERBP1 IL12RB2 CD247 rs2056626 CD247, POU2F1, TADA1, GPA33, MAEL, DUSP27, CREG1, RCSD1, MPZL1, DCAF6, MPC2 CD247 CD247 CD247 TNFSF4- LOC100506023-PRDX6 rs2022449 TNFSF4 TNFSF4- LOC100506023-PRDX6 rs11576547 TNFSF4a NAB1 rs716254 NAB1, GLS, TMEM194B, MFSD6 NAB1 NAB1, TMEM194B STAT4 rs3821236 STAT4, STAT1, GLS, MYO1B, NABP1, SDPR STAT4, STAT1 STAT4 STAT4 rs4853458 STAT4, STAT1, GLS, MYO1B, NABP1, SDPR STAT4, STAT1 FLNB-DNASE1L3-PXK rs7355798 PXK, FLNB, RPP14 FLNB-AS1 FLNB-DNASE1L3-PXK rs7653734 PXK, RPP14 PXK, DNASE1L3b POGLUT1-TIMMDC1- CD80-ARHGAP31 rs9884090 POGLUT1, TIMMDC1 CD80, ARHGAP31, POGLUT1 IL12A rs589446 IL12A IL12A IL12A DGKQ rs11724804 DGKQ, GAK, TMEM175 NFKB1 rs230517 NFKB1, MANBA, UBE2D3, CISD2, SLC9B1, SLC39A8 NFKB1, MANBA NFKB1, MANBA, BDH2 NFKB1 TNIP1 rs3792783 TNIP1, GPX3, CD71, RPS14, RMB22, DCTN4, IRGM, SMIM3,ANXA6, GM2A, SLC36A3 TNIP1, ANXA6 TNIP1, ANXA6 TNIP1 ATG5 rs633724 ATG5b IRF5-TNPO3* rs36073657 TNPO3, IRF5 IRF5 IRF5, FAM71F2 IRF5-TNPO3* rs12155080 TNPO3, IRF5 TNPO3, IRF5 TNPO3 IRF5 FAM167A,BLK rs2736340 BLK, RP11-481A20.11, FDFT1, NEIL2 BLK, C8orf14 RAB2A-CHD7 rs6987084 CHD7, RAB2A, CLVS1 RAB2A CDHR5-IRF7 rs6598008 IRF7, RIC8A, BET1L, PSMD13, SIRT3, NLRP6, PTDSS2, RNH1, HRAS, RASSF7, PHRF1, DEAF1, DRD4, TALDO1, PDDC1, CEND1, EPS8L2 IRF7, RNH1, HRAS, PHRF1, DRD4, EPS8L2 DRD4, C11orf35, IRF7, PHRF1, RNH1 IRF7 TSPAN32,CD81-AS1 rs2651804 CD81, ASCL2 TSPAN32 DDX6 rs10892286 DDX6, KMT2A, TREH, CXCR5, BCL9L, UPK2, ATP5L, TRAPPC4, FOXR1, VPS11 DDX6, TREH PHLDB1, TREH CSK rs1378942 CSK, PML, STOML1, ARID3B, SEMA7A, CLK3, EDC3, CYP1A1, LMAN1L, CPLX3, ULK3, SCAMP2, MPI, COX5A, RPP25, PPCDC, GOLGA6C, MAN2C1, NEIL1, SIN3A, SNUPN, SNX33 CSK, CYP1A1, LMAN1L, CPLX3, ULK3, SCAMP2, MPI, RPP25, PPCDC CPLX3, CSK, CYP1A1, FAM219B, LMAN1L, MPI, PPCDC, RPP25, SCAMP2, SCAMP5, ULK3 CSK, SCAMP2, ULK3 IRF8 rs11117422 IRF8, COX4I1, EMC8, FOXF1 IRF8** IRF8 IKZF3-GSDMB rs9303277 GSDMB, IKZF3, ZPBP2, GRB7, MED1, CDK12, PPP1R1B,PNMT, PGAP3, ERBB2, ORMDL3, NEUROD2, STARD3, TCAP, MIEN1, RPL19, CACNB1, FBXL20, LRRC3C, GSDMA, PSMD3, THRA, NR1D1, MSL1, RARA, GJD3, TNS4, IGFBP4, CCR7, SMARCE1, KRTAP17, KRT33A GSDMB, IKZF3, ZPBP2, PNMT, PGAP3, ORMDL3, GSDMA, PSMD3 IKZF3, ZPBP2, GSDMB, ORMDL3 GSDMB, IKZF3, ORMDL3 NUP85-GRB2 rs1005714 NUP85, MRPS7, ATP5H, ICT1 NUP85, MRPS7 ARMC7, GGA3, SLC16A5, MRPS7, NT5C, NUP85 ARMC7, GGA3, NT5C IL12RB1 rs2305743 IL12RB1, MAST3, ARRDC2, ISYNA1, ELL, SSBP4, LSM4, JUND, RAB3A, PDE4C, KIAA1683, MPV17L2, IFI30, PIK3R2, B3GNT3, FCHO1, INSL3, JAK3, RPL18A, SLC5A IL12RB1, KIAA1683 KIAA1683 IL12RB1, MAST3 ‘HiChIP Target Genes withSNP PP Max’, genes with HiChIP and eQTL signals are highlighted in bold mosome; PP, posterior probability from the statistical ﬁne-mapping; SNP, single-nucleotide polymorphism set not feasible TL signals found but strong HiChIP signal minated by proximity ominated according to the results from the ﬁrst Immunochip in systemic sclerosis (9) 6 NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-12760-y 7 7 NATURE COMMUNICATIONS \| (2019) 10:4955 \| https://doi.org/10.1038/s41467-019-12760-y \| www.nature.com/naturecommunications ARTICLE NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-12760-y BLD.GM12878 1.25e–05 < p < 1.25e–04 1.25e–06 < p < 1.25e–05 1.25e–07 < p < 1.25e–06 1.25e–08 < p < 1.25e–07 p < 1.25e–08 Cell types Tissue Enhancer H2BK20ac Enhancer H3K4me1 Enhancer H3K27ac Promotor H3K4ac Promotor H3K9ac Active H2BK15ac Active H3K79me1 Promotor H3K4me2 Promotor H3K4me3 Blood Spleen Thymus Bone Skin Connective Intestine Other Lung BLD.CD19.PPC BLD.CD56.PC BLD.CD4.CD25M.IL17M.PL.TPC BLD.CD4.CD25M.IL17P.PL.TPC BLD.CD3.PPC BLD.CD4.CD25.CD127M.TREGPC BLD.CD4.MPC BLD.CD4.CD25M.CD45RO.MPC BLD.CD4.CD25I.CD127.TMEMPC BLD.CD4.CD25M.CD45RA.NPC BLD.CD4.CD25M.TPC BLD.CD19.CPC BLD.CD4.NPC BLD.CD8.MPC BLD.CD14.MONO BLD.PER.MONUC.PC BLD.CD8.NPC BLD.CD3.CPC BLD.CD34.CC BLD.CD14.PC BLD.CD15.PC BLD.CD34.PC BLD.MOB.CD34.PC.F BLD.MOB.CD34.PC.M SPLN THYM.FET THYM BONE.OSTEO STRM.CHON.MRW.DR.MSC SKIN.PEN.FRSK.KER.02 SKIN.NHDFAD SKIN.PEN.FRSK.FIB.01 SKIN.PEN.FRSK.FIB.02 SKIN.PEN.FRSK.KER.03 SKIN.PEN.FRSK.MEL.01 SKIN.NHEK ESDR.H1.MSC ESDR.H1.BMP4.MESO STRM.MRW.MSC GI.DUO.MUC GI.CLN.SIG GI.ESO GI.STMC.GAST GI.RECT.MUC.29 GI.RECT.SM.MUS MUS.HSMM GI.DUO.SM.MUS VAS.HUVEC BRN.ANT.CAUD BRN.ANG.GYR BRN.HIPP.MID BRN.DL.PRFRNTL.CRTX BRN.SUB.NIG BRN.NHA BRST.MYO BRST.HMEC.35 LNG.IMR90 LNG Moreover, the associated variant (rs3761700), affects the expres- sion of MERTK in whole blood (p value = 1.22 × 10−35). p Regarding dcSSc, we found an association signal in the ANKRD12 genomic region (18p11.22) (association test p value = 3.97 × 10−08, OR = 1.22). This locus did not show signiﬁcant associations in the global meta-analysis (association test p value = 2.30 × 10−05, OR = 1.10) nor in the lcSSc subtype (association test p value = 0.034, OR = 1.06) (Supplementary Data 9 and Supplementary Fig. 5) (Supplementary Fig. 4, p* = 4.0 × 10−4). ANKRD12 encodes a member of the ankyrin repeats-containing cofactor family involved in the modulation of gene transcription. The associated variant—rs4798783—is an eQTL for TWSG1 (p value = 6.44 × 10−06) in transformed ﬁbroblasts. Interestingly, TWSG1 enhances TGF-beta signaling (which proﬁbrotic role is well-known) in activated T lympho- cytes29. These ﬁndings and the speciﬁc association with dcSSc are of utmost importance, given the more aggressive and rapidly progressing ﬁbrosis observed in this clinical subtype2,3. When data were stratiﬁed by patients positive for ACA, nine genome-wide signiﬁcant signals were found, of which two had been previously reported (IRF5-TNPO3, and DNASE1L3) and seven were novel associations (Supplementary Data 10). Notably, the locus CDHR5-IRF7 was strongly associated with this autoanti- body presentation (association test p value = 5.32 × 10−09, OR = 0.73) and showed stronger effect as compared to the global meta-analysis (association test p value = 1.97 × 10−08, OR = 0.80) (Supplementary Fig. 4, p* = 2.1 × 10−3). Regarding the ATA- positive subgroup, we replicated the previously reported associa- tion with IRF5-TNPO3 (Supplementary Data 10)30. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-12760-y Finally, in the case of the RNA pol III-positive SSc patients, we did not observe any signal at the genome-wide signiﬁcance level outside the HLA region. Nonetheless, we found suggestive associations in FAM167A-BLK, GUSBP1-CDH12, and STEAP2 (Supplementary Data 10). Overall, our ﬁndings highlights how performing GWASs in more homogeneous group of patients can increase the success of case–control studies by improving association strengths, thus avoiding reduction of statistical power owing to phenotypic heterogeneity31. Moreover, consistent with previous studies, suggesting genetic differences in the susceptibility to SSc subtypes9,32 the identiﬁcation of speciﬁc patterns of association in each SSc subphenotype emphasizes the importance of classiﬁcation biomarkers to predict more accurately the best therapeutic approach in each group of patients. Drug target enrichment analysis. The advantage of using human genetic evidence in drug discovery and repurposing has been comprehensively addressed in the last years5,6. In this line, we assessed whether any of the 78 SSc target genes identiﬁed in the present study (genes from the last three columns of Table 3) encode proteins that are drug targets in any phase of develop- ment. Seven out of the 78 genes (9%) overlapped with pharma- cological active targets (CD80, BLK, TNFSF4, IL12A, DRD4, PSMD3, FDFT1) in the Open Targets Platform33 (Supplementary Data 11). Among them, CD80 and BLK were targets of drugs for SSc in any phase of clinical trial (i.e., abatacept and dasatinib, respectively). We assessed the signiﬁcance of the overlap and found that our SSc target genes were signiﬁcantly enriched in pharmacological active targets for SSc (OR = 6.0; Fisher’s exact test P = 4.7 × 10−2) (Supplementary Table 5). Fig. 3 Tissue-speciﬁc enrichment for systemic sclerosis associations in epigenetic marks. The heatmap displays the signiﬁcant enrichment (p value < 1.25 × 10−4) in 59 out of the 127 cell and tissue types in Roadmap Epigenomics Consortium and the Encyclopedia of DNA Elements (ENCODE) projects. The enrichment p values are plotted with different colors according to the strength of the signiﬁcance. Since the enrichments were computed at various GWAS p value cutoffs (5 × 10−6, 5 × 10−7, 5 × 10−8), the most signiﬁcant p value was selected if a cell type/epigenetic mark combination showed more than one signiﬁcant enrichment across the different cutoffs. Supplementary Data 13 provides the correspondence between cell codes and cell types NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-12760-y Our results showed 363 signiﬁcant enrichments (p value < 1.25 × 10−4) in 59 out of the 127 cell and tissue types analyzed (Fig. 3, Supplementary Data 8). Most of the signiﬁcant enrichments were found in immune cells. SSc-associated variants displayed the most signiﬁcant enrichments in H2BK15ac and H2BK20ac marks in the GM12878 lymphoblastoid cell line (OR = 37.33, enrichment p value (Penr) = 4.84 × 10−14; OR = 12.79, Penr = 2.40 × 10−10, respectively), followed by H3K79me1 in primary Natural Killer (NK) cells (BLD.CD56. PC) (OR = 12.23, Penr = 5.73 × 10−09) and primary T cells (BLD. CD3.PPC) (OR = 12.58, Penr = 9.78 × 10−09). The spleen also showed a strong functional enrichment in H2BK15ac (OR = 14.69, Penr = 9.81 × 10−09). There were signiﬁcant enrich- ments of associations with SSc within H3K27ac, H3K4me1, H3K4me2, and H3K9ac marks—among others—of several CD4+ T cells (T helper, T regulatory, etc), CD8+ T cells, primary B cells, monocytes, primary neutrophils, and thymus (Fig. 3, Supple- mentary Data 8). Moreover, the SSc association signals showed different epigenetic enrichment patterns in non-immune cell/ tissue types, such as lung, ﬁbroblasts, chondrocytes, keratinocytes, pp y g Remarkably, we observed two subtype-speciﬁc signals that did not show statistical signiﬁcance in the global analysis. In the case of lcSSc, there was a signiﬁcant association in the MERTK genomic region (2q13) (association test p value = 1.04 × 10−08, OR = 1.15) that was not signiﬁcant in the global meta-analysis (association test p value = 3.49 × 10−05, OR = 1.09) nor in dcSSc sub-analysis (association test p value = 0.503, OR = 1.02) (Sup- plementary Data 9 and Supplementary Fig. 5) (Supplementary Fig. 4, p* = 1.0 × 10−4). MERTK is a tyrosine kinase member of the MER/AXL/TYRO3 receptor kinase family that is associated with multiple sclerosis27 and hepatitis C-induced liver ﬁbrosis28. ARTICLE NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-12760-y number of genome-wide association signals reported for SSc, bringing the total number of SSc risk loci up to 28. number of genome-wide association signals reported for SSc, bringing the total number of SSc risk loci up to 28. The identiﬁcation of target genes for GWAS signals is one of the most challenging questions. In the present study, aggregate analysis of chromatin interaction maps in a wide spectrum of immune cell lines and eQTLs provided strong support to nomi- nate 68 genes as robust SSc target genes (Table 3). Interestingly, the function of some of these experimentally nominated target genes are related to relevant pathways or biological pro- cesses in SSc. For example, DDX6 encodes a RNA helicase essential for efﬁcient miRNA-induced gene silencing. De Vries et al. demonstrated the role of DDX6 in the regulation of vascular endothelial growth factor under hypoxic conditions37. Therefore, this hit may establish a link between vasculopathy and SSc unknown so far. g g In the present study, some of the cohorts were genotyped using different genotyping platforms between cases and controls. To control for the potential spurious associations that this fact may lead owing to the possibility of differential imputation quality for the SNPs, we applied additional steps along with the standard QC procedures. Prior to the imputation, we carefully excluded multi- allelic, or A/T-C/G variants with MAF > 0.4 from all the data sets. After imputation, we applied an in-house Perl script that com- pares the genotypic frequencies between cases and controls and excluded all SNPs showing genotypic inconsistencies. In addition, manual inspection of the individual Manhattan plots from the 14 independent cohorts was performed and any suspicious false positive signal was carefully analyzed and removed, if necessary. In addition, the genome-wide signiﬁcant signals identiﬁed in the present study were the results of combining the effect of the signals across several independent cohorts. Moreover, no sig- niﬁcant heterogeneity of the ORs was observed. Other two new loci providing relevant mechanistic insights are RAB2A and GSDMB. RAB2A belongs to the Rab family, a group of membrane-bound proteins, involved in vesicular fusion and trafﬁcking. Speciﬁcally, RAB2A has been proposed to be a key factor in autophagosome clearance38, thus it is another SSc risk locus involved in autophagy apart from the previously described ATG59. These results reinforce the role of autophagy in SSc pathogenesis. ARTICLE In regard to GSDMB, it encodes a member of the gasdermin-domain containing protein family. The functional mechanism of gasdermin proteins is not clearly understood yet. However, recent evidence demonstrated that some gasdermin-N domains—including GSDMB—play a role in the induction of pyroptosis39,40, an inﬂammatory form of cell death that is crucial for the immune response. In line of these observations, our results also suggest a role of defective pyroptosis in SSc. g y Applying a statistical ﬁne-mapping approach, we reduced associated signals to 95% credible sets of 10 likely causal SNPs or fewer for 18 loci (72%). Notably, 95% credible sets comprised a single variant in 6 loci (ARHGAP31, BLK, CD247, TNIP1, CSK, STAT4-a). In other four loci, the credible sets contained two SNPs (DGKQ, NUP85-GRB2, STAT4-b, IL12RB1). Functional annota- tion of likely causal variants from credible sets revealed that all variants with PPmax were intronic, intergenic, or ncRNA intronic SNPs. These observations suggest that most of the genetic var- iations underlying SSc susceptibility are related to transcriptional regulatory mechanisms, including mRNA processing or stability mediated by ncRNAs. Our results are in accordance with emer- ging evidence that suggest a role of ncRNAs in autoimmunity34. Moreover, the exonic nonsynonymous variants included in the 95% credible sets or in high LD to credible set SNPs did not show clear evidence of being damaging mutations, as in the case of CDHR5-rs2740375 located close to IRF7. As an exception, it is worth mentioning that the nonsynonymous variant rs35677470 (R206C) in DNASE1L3, not present in our SNP panel, was reported to impact the DNase activity of the encoded protein in in vitro studies35. This fact is consistent with the result of our statistical ﬁne-mapping since the 95% credible set for this locus was not well resolved (27 likely causal variants comprised the credible set). Further functional studies will be needed to conﬁrm that rs35677470 (R206C) is the actual causal variant underlying the association or whether there are secondary signals that also inﬂuence the role of this genomic region in SSc susceptibility. Enrichment analyses of SSc loci in epigenetic marks of active gene regulation showed a strong immune signature. We identiﬁed relevant cell types and tissues for disease pathogenesis. Note- worthy, primary NK cells represented one of the highest enrichment signals across almost the entire panel. Our results are consistent with previous reports linking NK cells to SSc41. ARTICLE In a very recent publication, Benyamine et al.42 reported a particular expression proﬁle of NK cells in SSc and showed that this cell type induced endothelial activation42. These ﬁndings may provide a link between vascular damage and the immune imbalance in SSc. The inclusion of a wide panel of tissue and cell types captured cell type-speciﬁc patterns of enrichment. For example, there were some cell types that showed enrichment for a single histone mark. It is important to note that these results add valuable information to design future functional studies on the basis of accurately and well-chosen cell types or tissues, thus increasing the rate of suc- cess of the experiment. g g p y As expected, the results from gene expression data (eQTLs) suggested that the functional role of certain SSc signals may expand to several target genes. This hypothesis was conﬁrmed through the experimentally derived high-resolution maps of enhancer-promoter interactions generated by H3K27ac HiChIP in human CD4+ T cells. On average, HiChIP results found physical interactions for approximately eight genes per SNP across the 18 SSc likely causal variants that were mapped in the H3K27ac HiChIP analysis, consistent with previous ﬁndings for other ADs20. Strong interactions were observed in relation to some SNPs. For example, CD247-rs2056626 (intronic SNP with PPmax = 0.99 in the ﬁne-mapping) showed a strong normalized signal of HiChIP interaction to the CD247 promoter, suggesting that the SNP affects an intronic regulatory element that controls gene expression. This interaction between the SSc risk SNP and the promoter of the CD247 gene in CD4 + T-cells was also observed by the promoter capture Hi-C technique21,36, further supporting that the SNP may be involved in the transcriptional regulation of this gene. In fact, rs2056626 is a cis-eQTL for CD247 (p value = 2.411 × 10−48; FDR = 0) in whole blood. Finally, it has been demonstrated that human genetic evidence positively impacts the success rate in clinical development5. The drug target enrichment found in the identiﬁed SSc target genes supports that our results might also be informative in drug repurposing. As an example, the present study supports the possibility to consider ustekinumab for SSc treatment, which is a drug currently approved for related diseases, such as psoriasis, active psoriatic arthritis, and Crohn’s disease. Discussion The large cohort of SSc included in the present study allowed us to identify 13 new risk loci for the disease, almost doubling the NATURE COMMUNICATIONS \| (2019) 10:4955 \| https://doi.org/10.1038/s41467-019-12760-y \| www.nature.com/naturecommunications 8 ARTICLE Functional annotation of SNPs from credible sets. Functional characterization of the SNPs included in credible sets was performed by assessing SNP functional categories by means of wANNOVAR using default parameters53. Then we explored overlap with eQTLs, epigenetic histone marks of active promoters and active enhancers (H3K9ac, H3K4me1, and H3K27ac), and the presence of exonic non- synonymous variants in high or moderate LD (r2 ≥0.8, r2 ≥0.6, respectively) using HaploReg v4.154. For eQTL interrogation, we used blood eQTL from Westra et al.55, the Geuvadis data set56—which contains expression data from lympho- blastoid cell lines—and the Genotype–Tissue Expression (GTEx) project57—which provides RNA sequencing-based eQTL for a wide range of human tissues. Overlap of SNPs with chromatin marks was interrogated in selected cell lines from the Roadmap Epigenomics Project15 (Supplementary Table 1). Cell lines were selected according to the results of the functional enrichment analysis from ‘Enrichment analysis of SSc risk loci in epigenetic marks and cell types’ for H3K9ac, H3K4me1, and H3K27ac histone marks. Stratiﬁed analysis in clinical and serological SSc subtypes. Stratiﬁcation of patients according to SSc subtype (lcSSc, dcSSc) or autoantibody status (ACA, ATA, and ARA) was performed to conduct stratiﬁed genome-wide association analyses using the same procedure as for global analysis. All sub-analyses included the 14 independent cohorts, with the exception of the GWAS analysis for ARA- positive patients, which included Spain 2, USA 2, Italy, and UK cohorts according to data availability. Permutation analysis for subphenotype hits. A number of loci exhibited stronger genetic signals in stratiﬁed analysis (lcSSc, dcSSc, ACA) as compared with SSc as a whole despite the loss of statistical power caused by smaller numbers of the subphenotypes. To investigate whether these outcomes could have occurred by chance, we randomly shufﬂed 10,000 times a number of cases from each cohort (while keeping controls constant) and reran association testing and subsequent meta-analysis on the reshufﬂed data sets. The p values were converted to z scores to generate a null distribution of this test statistic. In detail, for each subtype, the number of cases randomly selected was determined by the prevalence of the subtype observed in the present study: we selected 62.52% of cases for lcSSc, 27.75% for dcSSc, and 36.77% for ACA +. ARTICLE A probability threshold of 0.9 was set for merging genotypes using GTOOL. Imputed data sets were also QC-ﬁltered by removing SNPs with call rates < 0.98, with MAFs < 0.01 and those that deviated from HWE (p < 0.001). In addition, singleton SNPs (which are not informative for phasing) and those that showed genotypic inconsistency between cases and controls were also excluded from analysis using an in house Perl script. The selection of functional annotations for PAINTOR ﬁne-mapping was carried out by stratiﬁed information enrichment calculations using GARFIELD25,26 (http:// www.ebi.ac.uk/birney-srv/GARFIELD/) (method explained in more detail in ‘Enrichment analysis of SSc risk loci in epigenetic marks and cell types’) with the annotation panel distributed in GARFIELD package. The purpose was to systematically select annotations relevant to SSc on the basis of functional enrichment analysis. GARFIELD tests its robustness by calculating functional enrichment for at least four signiﬁcance cutoffs (p value < 1e −5/−6/−7/−8) applied to the variants. GARFIELD analysis was carried out in our genome-wide SNP panel by setting default parameters and omitting SNPs from chromosome 6 between Mb25 and Mb34. We determined a set of nine annotations to be used for ﬁne-mapping that showed: A signiﬁcant enrichment (FDR < 0.05) of GWAS SNPs for at least two out of the four signiﬁcance cutoffs analyzed (p value < 1e −5/−6/ −7/−8); and b) A low inter-annotation correlation as suggested by PAINTOR (median inter-annotation Pearson correlation < 0.35) (Supplementary Data 12). Genome-wide association analysis. Genome-wide association analyses were performed in PLINK46 using a logistic regression model of additive effects, including sex and the ﬁve ﬁrst PCs as covariates in each of the 14 independent European cohorts. Genomic inﬂation factor (λ) was calculated by cohort and rescaled for an equivalent study of 1000 cases and 1000 controls when necessary (λ1000). Quantile–quantile (Q–Q) plots were generated and plotted with an in house R script to compare genome-wide distribution of the test statistic with the expected null distribution (Supplementary Fig. 1). We conducted a ﬁxed effects inverse variance meta-analysis in PLINK46 to combine the ORs obtained in each independent GWAS study. Heterogeneity values (I2 and Q) were cal- culated with PLINK46 to evaluate possible OR heterogeneity across the 14 individuals cohorts. Novel signals of associations were deﬁned as the genome- wide signiﬁcant associations (p value ≤5 × 10−8) that did not overlap with previously SSc reported signals at the genome-wide signiﬁcance level of association. ARTICLE Samples showing > 4 standard deviations from the cluster centroids of each cohort were considered outliers and removed from further analyses. The total number of individuals that remained in the ﬁnal ﬁltered data sets after PC analysis and identiﬁcation of outliers. To identify ancestry outliers, ~100,000 quality-ﬁltered independent SNPs were selected from each case-control GWAS h C l f d d GCTA d b f Fine-mapping of SSc-associated loci in a Bayesian framework. After the assessment of independent signals in signiﬁcant loci from the meta-analysis, statistical ﬁne-mapping was carried out using PAINTOR (Probabilistic Anno- tation INTegratOR) v3.050,51 searching for one causal SNP per independent associated region. PAINTOR performs probabilistic inference and computes posterior probabilities (PP) for SNPs to be casual considering the strength of association (Z score) and the LD pattern across genomic regions. The association strength was quantiﬁed using Wald statistic (“Equation (1)”) from ref. 50, and the LD information was provided by a LD matrix containing pairwise Pearson correlations coefﬁcients between each SNP. In addition, PAINTOR leverages functional annotation data as a prior probability to improve SNP prioritization. Finally, the method uses Bayes theorem to obtain PP for SNPs to be casual, which in turn were used to generate 95% credible sets (the smallest list of variants that jointly have a probability of including the causal variant ≥95%). Associated regions that contained more than one independent signal were split to obtain regions containing only one independent signal by integrating local LD information as well as the recombination rates using the online-tool LDlink (https://ldlink.nci.nih.gov/)52. q y p cohort. PC analysis was performed using PLINK and GCTA64 and R-base software under GNU Public license v.2. The ﬁrst ten PCs per individual were calculated and plotted. Samples showing > 4 standard deviations from the cluster centroids of each cohort were considered outliers and removed from further analyses. y The total number of individuals that remained in the ﬁnal ﬁltered data sets after this procedure was 26,679 (9095 SSc patients and 17,584 healthy controls). Imputation. QC-ﬁltered GWAS data sets were subjected to whole-genome geno- type imputation using IMPUTE247 and the 1000 Genome Project Phase III (1KGPh3) data as reference panel48. GTOOL was used to convert data sets into the ﬁle format used by IMPUTE2. SNPs that were duplicated, multi-allelic, or A/T-C/G with MAF > 0.4 were excluded. Imputation was done separately for each inde- pendent study. ARTICLE NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-12760-y clinical features are shown in Supplementary Table 4. This study complied with all relevant ethical regulations. CSIC’s Ethics Committee approved the study protocol, and written informed consent was obtained in accordance with the tenets of the Declaration of Helsinki. between SNPs estimated from a reference sample set. Conditional analysis of each associated locus was performed within a standard region of 1.5 Mb-win- dow centered on the most associated SNP (index or lead SNP), with the exception of DNASE1L3 region, where we explored the locus to 2 Mb owing to the extent of the haplotype block. LD patterns were estimated using genotype data from the 14 individual cohorts as reference. Conditional association ana- lysis was performed including the lead SNP as covariate. Any SNP showing a conditional association p value < 5 × 10−6 was considered as independent signal and was further included in a new round of conditional analysis. This process was repeated until no SNP with p value < 5 × 10−6 remained in any of the genomic regions explored. The observed independent signals were conﬁrmed using PLINK46 by dependence analysis at cohort level scans through stepwise logistic regression with adjustment for the most associated signals in each locus, followed by inverse variance weighted meta-analysis under a ﬁxed effects model. Genome-wide genotyping was undertaken using the arrays speciﬁed in detail in Supplementary Table 1. Stringent QC measures were applied to all GWAS data sets as follows: SNPs with call rates < 0.98; minor allele frequencies (MAFs) < 0.01; and those that deviated from Hardy-Weinberg equilibrium (HWE; p < 0.001 in both case and control subjects) were ﬁltered out from further analysis; samples with call rates < 0.95 were removed. The presence of relatives and/or duplicates was assessed by computing identity-by-descent (IBD) estimation using PLINK46. An individual from each pair of relatives (Pi_Hat > 0.45) or duplicates (Pi_Hat > 0.99) was removed Additionally duplicate/relatedness testing was also performed between removed. Additionally, duplicate/relatedness testing was also performed between different GWAS data sets with the same country origin. PC analysis and identiﬁcation of outliers. To identify ancestry outliers, ~100,000 quality-ﬁltered independent SNPs were selected from each case-control GWAS cohort. PC analysis was performed using PLINK and GCTA64 and R-base software under GNU Public license v.2. The ﬁrst ten PCs per individual were calculated and plotted. Methods St d h Study cohorts and GWAS quality control. This study included 14 independent epidemiological cohorts comprising a total of 28,179 unrelated and genome-wide genotyped individuals (9846 SSc) patients and 18,333 healthy controls), after genotyping quality control (QC) steps. In brief, nine new SSc GWAS cohorts and ﬁve previously published SSc GWAS cohorts7,8 of European ancestry were included (Spain 1, Germany 1, The Netherlands 1, USA 1, France, Spain 2, Germany 2, The Netherlands 2, USA 2, Italy, UK, Sweden, Norway and Australia/UK) (Supple- mentary Table 1). SSc patients fulﬁlled the 1980 American College of Rheuma- tology classiﬁcation criteria for this disease or the criteria proposed by LeRoy and Medsger for early-SSc43,44. In addition, patients were classiﬁed as having lcSSc or dcSSc, as described in LeRoy et al.45 Patients were also subdivided by autoantibody status according to the presence of ACA, ATA, or ARA autoantibodies. The main NATURE COMMUNICATIONS \| (2019) 10:4955 \| https://doi.org/10.1038/s41467-019-12760-y \| www.nature.com/naturecommunications 9 9 References The SNPs with maximum PPs in each of the independent associated loci were used as anchor points to explore physical interactions between restriction fragments containing the variants and gene promoters. 9. Mayes, M. D. et al. Immunochip analysis identiﬁes multiple susceptibility loci for systemic sclerosis. Am. J. Hum. Genet. 94, 47–61 (2014). 10. Terao, C. et al. Transethnic meta-analysis identiﬁes GSDMA and PRDM1 as susceptibility genes to systemic sclerosis. Ann. Rheum. Dis. 76, 1150–1158 (2017). 11. Lopez-Isac, E. et al. Brief report: IRF4 newly identiﬁed as a common susceptibility locus for systemic sclerosis and rheumatoid arthritis in a cross- disease meta-analysis of genome-wide association studies. Arthritis Rheumatol. 68, 2338–2344 (2016). Enrichment of SSc loci in epigenetic marks and cell types. To assess whether our SSc GWAS SNPs were not randomly distributed among functional or regulatory elements in the genome, we performed functional enrichment ana- lysis of non-MHC SNPs using GARFIELD v2.025,26. This method estimates enrichment of overlap on functional information computing ORs at various GWAS p value cutoffs, and tests the signiﬁcance of the enrichment under a generalized linear model. GARFIELD accounts for major sources of con- founding factors by incorporating high-LD proxies (r2 > 0.8), MAF, and tran- scription start site distance as categorical covariates in the logistic regression model. Enrichment was tested on independent SNPs after pruning of GWAS SNPs (r2 > 0.1). We omitted SNPs of chromosome 6 between Mb25 and Mb34 to avoid bias. 12. Zochling, J. et al. An Immunochip-based interrogation of scleroderma susceptibility variants identiﬁes a novel association at DNASE1L3. Arthritis Res. Ther. 16, 438 (2014). 13. Martin, J. E. et al. A systemic sclerosis and systemic lupus erythematosus pan- meta-GWAS reveals new shared susceptibility loci. Hum. Mol. Genet. 22, 4021–4029 (2013). 14. Zhou, X. et al. Filamin B deﬁciency in mice results in skeletal malformations and impaired microvascular development. Proc. Natl. Acad. Sci. U S A 104, 3919–3924 (2007). GARFIELD provides an annotation panel that includes 1005 annotations (genetic annotations, chromatin states, histone modiﬁcations, DNase I hypersensitive sites and transcription factor binding sites in different cell lines) from ENCODE, GENCODE and Roadmap Epigenomics projects15,24,64. Moreover, GARFIELD can be run using a custom annotation panel. References tools dump command was used to extract the chromosome of interest from the.hic ﬁle60,61. The interaction proﬁle of a speciﬁc 5 kb or 10 kb bin containing the anchor was then plotted in R. Replicate reproducibility was visualized with the mean proﬁle shown as a line and the shading surrounding the mean representing the standard deviation between replicates. We explored chromatin interactions of the most likely causal variants by setting as anchor points the SNPs with maximum PPs in each of the independent associated loci. To identify the connectivity of candidate SNPs to target genes, we called interactions by manual inspection of individual SNP virtual 4 C interaction ﬁles and subset these interactions to those containing a transcription start site and SNP18,20. Fit-Hi-C algorithm was used to identify statistically signiﬁcant (q value ≤1e-60) distance-matched enrichment of interaction over background18,62. 1. Barnes, J. & Mayes, M. D. Epidemiology of systemic sclerosis: incidence, prevalence, survival, risk factors, malignancy, and environmental triggers. Curr. Opin. Rheumatol. 24, 165–170 (2012). p 2. Gabrielli, A., Avvedimento, E. V. & Krieg, T. Scleroderma. N. Engl. J. Med. 360, 1989–2003 (2009). 3. Denton, C. P. & Khanna, D. Systemic sclerosis. Lancet 390, 1685–1699 (2017). 4. Steen, V. D. & Medsger, T. A. Changes in causes of death in systemic sclerosis, 1972–2002. Ann. Rheum. Dis. 66, 940–944 (2007). 5. Nelson, M. R. et al. The support of human genetic evidence for approved drug indications. Nat. Genet. 47, 856–860 (2015). 6. Okada, Y. et al. Genetics of rheumatoid arthritis contributes to biology and drug discovery. Nature 506, 376–381 (2014). g The functional relevance of the H3K27ac HiChIP ﬁndings was further validated by evaluating whether the explored SNPs were eQTLs for the HiChIP nominated target genes. 7. Radstake, T. R. et al. Genome-wide association study of systemic sclerosis identiﬁes CD247 as a new susceptibility locus. Nat. Genet. 42, 426–429 (2010). . Radstake, T. R. et al. Genome-wide association study of syst 8. Allanore, Y. et al. Genome-wide scan identiﬁes TNIP1, PSORS1C1, and RHOB as novel risk loci for systemic sclerosis. PLoS Genet. 7, e1002091 (2011). Promoter capture Hi-C analysis. Chromatin interaction maps obtained by pro- moter capture Hi-C experiments in a wide spectrum of immune cell types21,22 were assessed using the web-based tool Capture Hi-C Plotter (CHiCP) (https://www. chicp.org/)63. References 45, 124–130 (2013). 20. Mumbach, M. R. et al. Enhancer connectome in primary human cells identiﬁes target genes of disease-associated DNA elements. Nat. Genet. 49, 1602–1612 (2017). 21. Javierre, B. M. et al. Lineage-speciﬁc genome architecture links enhancers and non-coding disease variants to target gene promoters. Cell 167, 1369–1384 e19 (2016). Drug-target gene enrichment analysis. Target genes nominated in the present study were used to query the Open Target Platform33 in order to assess whether any of the genes encode proteins that are drug targets in any phase of clinical trial (phase I–IV). Enrichment of overlap between SSc target genes with pharmacolo- gical active targets for the diseases was calculated by Fisher’s exact test. 22. Mifsud, B. et al. Mapping long-range promoter contacts in human cells with high-resolution capture Hi-C. Nat. Genet. 47, 598–606 (2015). 23. Pers, T. H. et al. Biological interpretation of genome-wide association studies using predicted gene functions. Nat. Commun. 6, 5890 (2015). g p g 24. Consortium, E. P. An integrated encyclopedia of DNA elements in the human genome. Nature 489, 57–74 (2012). Reporting summary. Further information on research design is available in the Nature Research Reporting Summary linked to this article. 25. Iotchkova, V. et al. GARFIELD - GWAS Analysis of Regulatory or Functional Information Enrichment with LD correction. bioRxiv. https://doi.org/10.1101/ 085738 (2016). 26. Iotchkova, V. et al. Discovery and reﬁnement of genetic loci associated with cardiometabolic risk using dense imputation maps. Nat. Genet. 48, 1303–1312 (2016). ARTICLE ARTICLE NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-12760-y ARTICLE The empirical p value (p*) was calcu- lated as the number of permuted z scores that were at least as extreme as the actual z score + 1 divided by the number of permutations + 131. Finally, we assessed pleiotropic effect of our signals by determining whether the SNPs included in the credible sets had been reported to be associated with other ADs. For this, we interrogated the new NHGRI-EBI GWAS Catalog (https://www. ebi.ac.uk/gwas/)58 through the web tool FUMA GWAS (http://fuma.ctglab.nl/)59. H3K27ac HiChIP analysis in human CD4+ T cells. Experimentally derived high- resolution maps of enhancer-promoter interactions generated by H3K27ac HiChIP experiments20 were explored to identify target genes of SSc-associated variants. HiChIP was developed by Mumbach et al.18 for the analysis of protein-directed chromosome conformation in a very efﬁcient and sensitive way. The H3K27ac HiChIP experiments were performed by Mumbach et al. in human CD4 + T cells from healthy donors: Primary human naïve T cells (CD4+CD45RA+CD25 −CD127hi), regulatory T (Treg) cells (CD4+CD25+CD127lo) and T helper 17 (Th17) cells (CD4+CD45RA−CD25−CD127hiCCR6+CXCR5−)20. Virtual 4 C plots were generated from dumped matrices generated with Juicebox. The Juicebox Stepwise conditional analysis in SSc-associated loci. The presence of inde- pendent signals in the genomic regions with signiﬁcant signals in the meta- analysis was assessed by joint conditional analysis by GCTA49. This method uses summary-level statistics from meta-analysis and applies LD correction 10 NATURE COMMUNICATIONS \| (2019) 10:4955 \| https://doi.org/10.1038/s41467-019-12760-y \| www.nature.com/naturecommunications References The second option was selected for our enrichment analysis using annotations for 127 reference epigenomes (Supplementary Data 13) and 9 epigenetic marks (H2BK20ac, H3K27ac, H3K4me1, H3K4me2, H3K4me3, H3K9ac, H3K4ac, H3K79me1, H2BK15ac) obtained from the Roadmap Epigenomics Consortium and the Encyclopedia of DNA Elements (ENCODE) projects15,24. Annotations used were Imputed Narrow Peaks as generated by the software Chrom-Impute65 and obtained from https://egg2.wustl.edu/roadmap/data/byFileType/peaks/ consolidatedImputed/narrowPeak/. The estimated ORs were computed at various GWAS p value cutoffs (5 × 10−6, 5 × 10−7, 5 × 10−8) and the R code Garﬁeld-Meff- Padj.R provided by GARFIELD was used to calculate an enrichment p value threshold adjusted for multiple testing (P value = 1.25 × 10−4) on the effective number of annotations (Meff = 400.4454). GARFIELD provides an annotation panel that includes 1005 annotations (genetic annotations, chromatin states, histone modiﬁcations, DNase I hypersensitive sites and transcription factor binding sites in different cell lines) from ENCODE, GENCODE and Roadmap Epigenomics projects15,24,64. Moreover, GARFIELD can be run using a custom annotation panel. The second option was selected for our enrichment analysis using annotations for 127 reference epigenomes (Supplementary Data 13) and 9 epigenetic marks (H2BK20ac, H3K27ac, H3K4me1, H3K4me2, H3K4me3, H3K9ac, H3K4ac, H3K79me1, H2BK15ac) obtained from the Roadmap Epigenomics Consortium and the Encyclopedia of DNA Elements (ENCODE) projects15,24. Annotations used were Imputed Narrow Peaks as generated by the software Chrom-Impute65 and obtained from https://egg2.wustl.edu/roadmap/data/byFileType/peaks/ consolidatedImputed/narrowPeak/. The estimated ORs were computed at various GWAS p value cutoffs (5 × 10−6, 5 × 10−7, 5 × 10−8) and the R code Garﬁeld-Meff- Padj.R provided by GARFIELD was used to calculate an enrichment p value threshold adjusted for multiple testing (P value = 1.25 × 10−4) on the effective number of annotations (Meff = 400.4454). 15. Roadmap Epigenomics, C. et al. Integrative analysis of 111 reference human epigenomes. Nature 518, 317–330 (2015). 16. Sim, N. L. et al. SIFT web server: predicting effects of amino acid substitutions on proteins. Nucleic Acids Res. 40, W452–W457 (2012). 17. Adzhubei, I., Jordan, D. M. & Sunyaev, S. R. Predicting functional effect of human missense mutations using PolyPhen-2. Curr. Protoc. Hum. Genet. Chapter 7, Unit 7.20 (2013). p 18. Mumbach, M. R. et al. HiChIP: efﬁcient and sensitive analysis of protein- directed genome architecture. Nat. Methods. 13, 919–922 (2016). 19. Trynka, G. et al. Chromatin marks identify critical cell types for ﬁne mapping complex trait variants. Nat. Genet. 45, 124–130 (2013). complex trait variants. Nat. Genet. Received: 28 February 2019; Accepted: 30 September 2019; Additional information 48. Genomes Project, C. et al. A global reference for human genetic variation. Nature 526, 68–74 (2015). Supplementary information is available for this paper at https://doi.org/10.1038/s41467- 019-12760-y. Supplementary information is available for this paper at https://doi.org/10.1038/s41467- 019-12760-y. 49. Yang, J. et al. Conditional and joint multiple-SNP analysis of GWAS summary statistics identiﬁes additional variants inﬂuencing complex traits. Nat. Genet. 44, 369–375 S1-3. (2012). Correspondence and requests for materials should be addressed to E.L.-I. or J.M. 50. Kichaev, G. et al. Improved methods for multi-trait ﬁne mapping of pleiotropic risk loci. Bioinformatics 33, 248–255 (2017). Peer review information Nature Communications thanks Wilson Liao and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. 51. Kichaev, G. & Pasaniuc, B. Leveraging functional-annotation data in trans- ethnic ﬁne-mapping studies. Am. J. Hum. Genet. 97, 260–271 (2015). Reprints and permission information is available at http://www.nature.com/reprints 52. Machiela, M. J. & Chanock, S. J. LDlink: a web-based application for exploring population-speciﬁc haplotype structure and linking correlated alleles of possible functional variants. Bioinformatics 31, 3555–3557 (2015). Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional afﬁliations. f 53. Chang, X. & Wang, K. wANNOVAR: annotating genetic variants for personal genomes via the web. J. Med. Genet. 49, 433–436 (2012). 54. Ward, L. D. & Kellis, M. HaploReg: a resource for exploring chromatin states, conservation, and regulatory motif alterations within sets of genetically linked variants. Nucleic Acids Res. 40, D930–D934 (2012). Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/ licenses/by/4.0/. 55. Westra, H. J. et al. Systematic identiﬁcation of trans eQTLs as putative drivers of known disease associations. Nat. Genet. 45, 1238–1243 (2013). 56. ARTICLE NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-12760-y 31. Sham, P. C. & Purcell, S. M. Statistical power and signiﬁcance testing in large- scale genetic studies. Nat. Rev. Genet. 15, 335–346 (2014). 60. Rao, S. S. et al. A 3D map of the human genome at kilobase resolution reveals principles of chromatin looping. Cell 159, 1665–1680 (2014). p p p g 61. Durand, N. C. et al. Juicebox provides a visualization system for Hi-C contact maps with unlimited zoom. Cell Syst. 3, 99–101 (2016). g 32. Gorlova, O. et al. Identiﬁcation of novel genetic markers associated with clinical phenotypes of systemic sclerosis through a genome-wide association strategy. PLoS Genet. 7, e1002178 (2011). 62. Ay, F., Bailey, T. L. & Noble, W. S. Statistical conﬁdence estimation for Hi-C data reveals regulatory chromatin contacts. Genome Res. 24, 999–1011 (2014). gy 33. Carvalho-Silva, D. et al. Open Targets Platform: new developments and updates two years on. Nucleic Acids Res. 47, D1056–D1065 (2018). 63. Schoﬁeld, E. C. et al. CHiCP: a web-based tool for the integrative and interactive visualization of promoter capture Hi-C datasets. Bioinformatics 32, 2511–2513 (2016). 34. Gupta, B. & Hawkins, R. D. Epigenomics of autoimmune diseases. Immunol Cell Biol. 93, 271–276 (2015). 64. Harrow, J. et al. GENCODE: the reference human genome annotation for The ENCODE Project. Genome Res. 22, 1760–1774 (2012). 35. Ueki, M. et al. Caucasian-speciﬁc allele in non-synonymous single nucleotide polymorphisms of the gene encoding deoxyribonuclease I-like 3, potentially relevant to autoimmunity, produces an inactive enzyme. Clin. Chim. Acta. 407, 20–24 (2009). ENCODE Project. Genome Res. 22, 1760–1774 (2012). 65. Ernst, J. & Kellis, M. Large-scale imputation of epigenomic datasets for systematic annotation of diverse human tissues. Nat. Biotechnol. 33, 364–376 (2015). 36. Martin, P. et al. Capture Hi-C reveals novel candidate genes and complex long-range interactions with related autoimmune risk loci. Nat. Commun. 6, 10069 (2015). Acknowledgements 37. de Vries, S. et al. Identiﬁcation of DEAD-box RNA helicase 6 (DDX6) as a cellular modulator of vascular endothelial growth factor expression under hypoxia. J. Biol. Chem. 288, 5815–5827 (2013). We thank Soﬁa Vargas, Sonia García, and Gema Robledo for their excellent technical assistance and all the patients and control donors for their essential collaboration. We thank National DNA Bank Carlos III (University of Salamanca, Spain) that supplied part of the control DNA samples from Spain, WTCCC and EIRA Consortiums, and PopGen 2.0 network. This work was supported by Spanish Ministry of Economy and Competi- tiveness (grant ref. SAF2015-66761-P), Consejeria de Innovacion, Ciencia y Tecnologia, Junta de Andalucía (P12-BIO-1395), Ministerio de Educación, Cultura y Deporte through the program FPU, Juan de la Cierva fellowship (FJCI-2015-24028), Red de Investigación en Inﬂamación y Enfermadades Reumaticas (RIER) from Instituto de Salud Carlos III (RD16/0012/0013), and Scleroderma Research Foundation and NIH P50- HG007735 (to H.Y.C.). H.Y.C. is an Investigator of the Howard Hughes Medical Insti- tute. PopGen 2.0 is supported by a grant from the German Ministry for Education and Research (01EY1103). M.D.M and S.A. are supported by grant DoD W81XWH-18-1- 0423 and DoD W81XWH-16-1-0296, respectively. yp 38. Lorincz, P. et al. Rab2 promotes autophagic and endocytic lysosomal degradation. J. Cell Biol. 216, 1937–1947 (2017). g 39. Ding, J. et al. Pore-forming activity and structural autoinhibition of the gasdermin family. Nature 535, 111–116 (2016). 40. Chao, K. L., Kulakova, L. & Herzberg, O. Gene polymorphism linked to increased asthma and IBD risk alters gasdermin-B structure, a sulfatide and phosphoinositide binding protein. Proc. Natl. Acad. Sci. USA 114, E1128–E1137 (2017). 41. Barranco, C. Systemic sclerosis: the future is CD56-bright. Nat. Rev. Rheumatol. 12, 624 (2016). 42. Benyamine, A. et al. Natural killer cells exhibit a peculiar phenotypic proﬁle in systemic sclerosis and are potent inducers of endothelial microparticles release. Front. Immunol. 9, 1665 (2018). 43. Anon, M. C. Preliminary criteria for the classiﬁcation of systemic sclerosis (scleroderma). Subcommittee for scleroderma criteria of the American Rheumatism Association Diagnostic and Therapeutic Criteria Committee. Arthritis Rheum. 23, 581–590 (1980). Author contributions E.L.I., L.B.C., S.A., M.D.M. and J.M. contributed to the conception and study design. ELI., M.A.H., M.K., F.D.C. and G.O. contributed to data collection, QC, and imputation. A.F., C.W., T.V., Y.A., M.A.B. and T.R.D.J.R contributed to control and/or case GWAS data collection. E.L.I., M.A.H., M.K., A.T.S., J.G., M.R.M. and H.Y.C. contributed to data analysis. All co-authors made substantial contributions to data acquisition, data inter- pretation, and revised the work critically for important intellectual content. 44. LeRoy, E. C. & Medsger, T. A. Criteria for the classiﬁcation of early systemic sclerosis. J. Rheumatol. 28, 1573–1576 (2001). 45. LeRoy, E. C. et al. Scleroderma (systemic sclerosis): classiﬁcation, subsets and pathogenesis. J. Rheumatol. 15, 202–205 (1988). 46. Purcell, S. et al. PLINK: a tool set for whole-genome association and population-based linkage analyses. Am. J. Hum. Genet. 81, 559–575 (2007). Competing interests H.Y.C. is a co-founder of Accent Therapeutics and advisor to 10x Genomics and Spring Discovery. All other authors declare no competing interests. 47. Howie, B. N., Donnelly, P. & Marchini, J. A ﬂexible and accurate genotype imputation method for the next generation of genome-wide association studies. PLoS Genet. 5, e1000529 (2009). Data availability Summary statistics of the meta-GWAS analyzed in the current study will be made available through the NHGRI-EBI GWAS Catalog (https://www.ebi.ac.uk/gwas/ downloads/summary-statistics) (please use ‘Systemic Sclerosis’ and/or ‘Lopez-Isac/ Martin’ as search terms). Individual-level genotype data are not publicly available owing to them containing information that could compromise research participant privacy or informed consent. All other data are contained in the article ﬁle and its supplementary information or available upon reasonable request to the corresponding authors. Epigenetic annotation panel used in this study were Imputed Narrow Peaks obtained from https://egg2.wustl.edu/roadmap/data/byFileType/peaks/consolidatedImputed/ narrowPeak/. 27. International Multiple Sclerosis Genetics, C. et al. Genetic risk and a primary role for cell-mediated immune mechanisms in multiple sclerosis. Nature 476, 214–219 (2011). 28. Patin, E. et al. Genome-wide association study identiﬁes variants associated with progression of liver ﬁbrosis from HCV infection. Gastroenterology 143, 1244–1252 e12 (2012). Epigenetic annotation panel used in this study were Imputed Narrow Peaks obtained from https://egg2.wustl.edu/roadmap/data/byFileType/peaks/consolidatedImputed/ narrowPeak/. 29. Tzachanis, D. et al. Twisted gastrulation (Tsg) is regulated by Tob and enhances TGF-beta signaling in activated T lymphocytes. Blood 109, 2944–2952 (2007). 30. Bossini-Castillo, L., Lopez-Isac, E. & Martin, J. Immunogenetics of systemic sclerosis: deﬁning heritability, functional variants and shared-autoimmunity pathways. J Autoimmun 64, 53–65 (2015). Received: 28 February 2019; Accepted: 30 September 2019; 11 NATURE COMMUNICATIONS \| (2019) 10:4955 \| https://doi.org/10.1038/s41467-019-12760-y \| www.nature.com/naturecommunications 24Department of Internal Medicine, Virgen de las Nieves Hospital, Granada, Spain. 25Department of Rheumatology, Virgen de la Victoria Hospital, Málaga, Spain. 26Department of Internal Medicine, Carlos Haya Hospital, Málaga, Spain. 27Department of Rheumatology, Carlos Haya Hospital, Málaga, Spain. 28Department of Immunology, Virgen del Rocío Hospital, Sevilla, Spain. 29Department of Internal Medicine, Virgen del Rocío Hospital, Sevilla, Spain. 30Department of Rheumatology, Reina Sofía/IMIBIC Hospital, Córdoba, Spain. 31Department of Rheumatology, San Carlos Clinic Hospital, Madrid, Spain. 32Department of Rheumatology, Madrid Norte Sanchinarro Hospital, Madrid, Spain. 33Department of Rheumatology, La Princesa Hospital, Madrid, Spain. 34Department of Rheumatology, Puerta de Hierro Hospital-Majadahonda, Madrid, Spain. 35Department of Rheumatology, Gregorio Marañón University Hospital, Madrid, Spain. 36Department of Internal Medicine, Clinic Hospital, Barcelona, Spain. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-12760-y 63Royal Adelaide Hospital and University of Adelaide, Adelaide, SA, Australia. 64St. Vincent’s Hospital, Melbourne, VIC, Australia. 65The University of Melbourne at St. Vincent’s Hospital, Melbourne, VIC, Australia. 72Department of Medical and Molecular Genetics, King’s College London, London, UK. 73Centre for Musculoskeletal Research, The University of Manchester, Salford Royal NHS Foundation Trust, Manchester Academic Health Science Centre, Manchester, UK. 74NIHR Manchester Biomedical Research Centre, Manchester, UK. 75Centre for Rheumatology, Royal Free and University College Medical School, London, United Kingdom. 76Department of Rheumatology A, Cochin Hospital, INSERM U1016, Paris Descartes University, Paris, France. 77Institute of Health and Biomedical Innovation, Queensland University of Technology, Translational Research Institute, Princess Alexandra Hospital, Brisbane, QLD, Australia. 78Department of Rheumatology & Clinical Immunology, Laboratory of Translational Immunology, department of Immunology, University Medical Center Utrecht, Utrecht, The Netherlands. 79These authors contributed equally: Marialbert Acosta-Herrera and Martin Kerick. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-12760-y ARTICLE NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-12760-y Elena López-Isac1, Marialbert Acosta-Herrera1,79, Martin Kerick1,79, Shervin Assassi2, Ansuman T. Satpathy 3,4 Jeffrey Granja3,4, Maxwell R. Mumbach3,4, Lorenzo Beretta5, Carmen P. Simeón6, Patricia Carreira7, Norberto Ortego-Centeno8, Ivan Castellvi9, Lara Bossini-Castillo10, F. David Carmona 11, Gisela Orozco 12, Nicolas Hunzelmann13, Jörg H.W. Distler14, Andre Franke 15, Claudio Lunardi16, Gianluca Moroncini17, Armando Gabrielli17, Jeska de Vries-Bouwstra18, Cisca Wijmenga19, Bobby P.C. Koeleman20, Annika Nordin21, Leonid Padyukov 21, Anna-Maria Hoffmann-Vold22, Benedicte Lie23, European Scleroderma Group†, Susanna Proudman63, Wendy Stevens64, Mandana Nikpour65, Australian Scleroderma Interest Group (ASIG), Timothy Vyse 72, Ariane L. Herrick73,74, Jane Worthington12, Christopher P. Denton75, Yannick Allanore 76, Matthew A. Brown 77, Timothy R.D.J. Radstake78, Carmen Fonseca75, Howard Y. Chang 3,4, Maureen D. Mayes 2 & Javier Martin1 1Institute of Parasitology and Biomedicine López-Neyra, IPBLN-CSIC, Granada, Spain. 2The University of Texas Health Science Center–Houston, Houston, USA. 3Center for Personal Dynamic Regulomes, Stanford University School of Medicine, Stanford, CA, USA. 4Howard Hughes Medical Institute, Stanford University, Stanford, CA, USA. 5Referral Center for Systemic Autoimmune Diseases, Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico di Milano, Milan, Italy. 6Department of Internal Medicine, Valle de Hebrón Hospital, Barcelona, Spain. 7Department of Rheumatology, 12 de Octubre University Hospital, Madrid, Spain. 8Department of Internal Medicine, San Cecilio Clinic University Hospital, Granada, Spain. 9Department of Rheumatology, Santa Creu i Sant Pau University Hospital, Barcelona, Spain. 10Wellcome Trust Sanger Institute, Hinxton, UK. 11Department of Genetics and Institute of Biotechnology, University of Granada, Granada, Spain. 12Arthritis Research UK Centre for Genetics and Genomics, Centre for Musculoskeletal Research, Faculty of Biology, Medicine and Health, Manchester Academic Health Science Centre, The University of Manchester, Oxford Road, Manchester, UK. 13Department of Dermatology, University of Cologne, Cologne, Germany. 14Department of Internal Medicine 3, Institute for Clinical Immunology, University of Erlangen-Nuremberg, Erlangen, Germany. 15Institute of Clinical Molecular Biology, Christian-Albrechts-University of Kiel, Kiel, Germany. 16Department of Medicine, Università degli Studi di Verona, Verona, Italy. 17Clinica Medica, Department of Clinical and Molecular Science, Università Politecnica delle Marche and Ospedali Riuniti, Ancona, Italy. 18Department of Rheumatology, Leiden University Medical Center, Leiden, The Netherlands. 19Department of Genetics, University Medical Center Groningen, University of Groningen, Groningen, Netherlands. 20University Medical Center Utrecht, Utrecht, The Netherlands. 21Division of Rheumatology, Department of Medicine, Karolinska University Hospital, Karolinska Institute, Stockholm, Sweden. 22Department of Rheumatology, Oslo University Hospital, Oslo, Norway. 23Department of Medical Genetics, and the Department of Immunology, University of Oslo and Oslo University Hospital, Oslo, Norway. 1Institute of Parasitology and Biomedicine López-Neyra, IPBLN-CSIC, Granada, Spain. 2The University of Texas Health Science Center–Houston, Houston, USA. 3Center for Personal Dynamic Regulomes, Stanford University School of Medicine, Stanford, CA, USA. 4Howard Hughes Medical Institute, Stanford University, Stanford, CA, USA. 5Referral Center for Systemic Autoimmune Diseases, Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico di Milano, Milan, Italy. 6Department of Internal Medicine, Valle de Hebrón Hospital, Barcelona, Spain. 7Department of Rheumatology, 12 de Octubre University Hospital, Madrid, Spain. 8Department of Internal Medicine, San Cecilio Clinic University Hospital, Granada, Spain. 9Department of Rheumatology, Santa Creu i Sant Pau University Hospital, Barcelona, Spain. 10Wellcome Trust Sanger Institute, Hinxton, UK. 11Department of Genetics and Institute of Biotechnology, University of Granada, Granada, Spain. 12Arthritis Research UK Centre for Genetics and Genomics, Centre for Musculoskeletal Research, Faculty of Biology, Medicine and Health, Manchester Academic Health Science Centre, The University of Manchester, Oxford Road, Manchester, UK. 13Department of Dermatology, University of Cologne, Cologne, Germany. 14Department of Internal Medicine 3, Institute for Clinical Immunology, University of Erlangen-Nuremberg, Erlangen, Germany. 15Institute of Clinical Molecular Biology, Christian-Albrechts-University of Kiel, Kiel, Germany. 16Department of Medicine, Università degli Studi di Verona, Verona, Italy. 17Clinica Medica, Department of Clinical and Molecular Science, Università Politecnica delle Marche and Ospedali Riuniti, Ancona, Italy. 18Department of Rheumatology, Leiden University Medical Center, Leiden, The Netherlands. 19Department of Genetics, University Medical Center Groningen, University of Groningen, Groningen, Netherlands. 20University Medical Center Utrecht, Utrecht, The Netherlands. 21Division of Rheumatology, Department of Medicine, Karolinska University Hospital, Karolinska Institute, Stockholm, Sweden. 22Department of Rheumatology, Oslo University Hospital, Oslo, Norway. 23Department of Medical Genetics, and the Department of Immunology, University of Oslo and Oslo University Hospital, Oslo, Norway. 63Royal Adelaide Hospital and University of Adelaide, Adelaide, SA, Australia. 64St. Vincent’s Hospital, Melbourne, VIC, Australia. 65The University of Melbourne at St. Vincent’s Hospital, Melbourne, VIC, Australia. 72Department of Medical and Molecular Genetics, King’s College London, London, UK. 73Centre for Musculoskeletal Research, The University of Manchester, Salford Royal NHS Foundation Trust, Manchester Academic Health Science Centre, Manchester, UK. 74NIHR Manchester Biomedical Research Centre, Manchester, UK. 75Centre for Rheumatology, Royal Free and University College Medical School, London, United Kingdom. 76Department of Rheumatology A, Cochin Hospital, INSERM U1016, Paris Descartes University, Paris, France. 77Institute of Health and Biomedical Innovation, Queensland University of Technology, Translational Research Institute, Princess Alexandra Hospital, Brisbane, QLD, Australia. 78Department of Rheumatology & Clinical Immunology, Laboratory of Translational Immunology, department of Immunology, University Medical Center Utrecht, Utrecht, The Netherlands. 79These authors contributed equally: Marialbert Acosta-Herrera and Martin Kerick. Additional information Lappalainen, T. et al. Transcriptome and genome sequencing uncovers functional variation in humans. Nature 501, 506–511 (2013). 57. Consortium, G. T. Human genomics. The Genotype-Tissue Expression (GTEx) pilot analysis: multitissue gene regulation in humans. Science 348, 648–660 (2015). 58. MacArthur, J. et al. The new NHGRI-EBI Catalog of published genome-wide association studies (GWAS Catalog). Nucleic Acids Res. 45, D896–D901 (2017). 59. Watanabe, K., Taskesen, E., van Bochoven, A. & Posthuma, D. Functional mapping and annotation of genetic associations with FUMA. Nat. Commun. 8, 1826 (2017). © The Author(s) 2019 12 NATURE COMMUNICATIONS \| (2019) 10:4955 \| https://doi.org/10.1038/s41467-019-12760-y \| www.nature.com/naturecommunicatio NATURE COMMUNICATIONS \| (2019) 10:4955 \| https://doi.org/10.1038/s41467-019-12760-y \| www.nature.com/naturecommunications ARTICLE ARTICLE 66Menzies Research Institute Tasmania, University of Tasmania, Hobart, TAS, Australia. 67Department Rheumatology, Monash Medical Centre, Melbourne, VIC, Australia. 68Rheumatology, Royal Perth Hospital, Perth, WA, Australia. 69Research Unit, Sunshine Coast Rheumatology, Maroochydore, QLD, Australia. 70Canberra Rheumatology, Canberra, ACT, Australia. 71Department Rheumatology, The Queen Elizabeth Hospital, Woodville, SA, Australia European Scleroderma Group† R. Ríos8, J.L. Callejas8, J.A. Vargas-Hitos24, R. García-Portales25, M.T. Camps26, A. Fernández-Nebro27, M.F. González-Escribano28, F.J. García-Hernández29, M.J. Castillo29, M.A. Aguirre30, I. Gómez-Gracia30, B. Fernández-Gutiérrez31, L. Rodríguez-Rodríguez31, P. García de la Peña32, E. Vicente33, J.L. Andreu34, M Fernández de Castro34, F.J. López-Longo35, L. Martínez35, Fonollosa V6, A. Guillén6, G. Espinosa36, C. Tolosa37, A. Pros38, M. Rodríguez-Carballeira39, F.J. Narváez40, M. Rubio-Rivas41, Ortiz-Santamaría V42, A.B. Madroñero43, M.A. González-Gay44, B. Díaz45, L. Trapiella45, A. Sousa46, M.V. Egurbide47, P. Fanlo-Mateo48, L. Sáez-Comet49, F. Díaz50, Hernández V50, E. Beltrán51, J.A. Román-Ivorra52, E. Grau52, J.J. Alegre-Sancho53, M. Freire54, F.J. Blanco-García55, N. Oreiro55, T. Witte56, A. Kreuter57, G. Riemekasten58 P. Airó59, C. Magro18, A.E. Voskuyl60, M.C. Vonk61 & R. Hesselstrand62 13 NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-12760-y NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-12760-y 37Department of Internal Medicine, Parc Tauli Hospital, Sabadell, Spain. 38Department of Rheumatology, Hospital Del Mar, Barcelona, Spain. 39Department of Internal Medicine, Hospital Universitari Mútua Terrasa, Barcelona, Spain. 40Department of Rheumatology, Bellvitge University Hospital, Barcelona, Spain. 41Department of Internal Medicine, Bellvitge University Hospital, Barcelona, Spain. 42Department of Rheumatology, Granollers Hospital, Granollers, Spain. 43Department of Internal Medicine, Hospital General San Jorge, Huesca, Spain. 44Epidemiology, Genetics and Atherosclerosis Research Group on Systemic Inﬂammatory Diseases, DIVAL, University of Cantabria, Santander, Spain. 45Department of Internal Medicine, Hospital Central de Asturias, Oviedo, Spain. 46Infectious Diseases Unit, Department of Internal Medicine, Hospital Xeral-Complexo Hospitalario Universitario de Vigo, Vigo, Spain. 47Department of Internal Medicine, Hospital Universitario Cruces, Barakaldo, Spain. 48Department of Internal Medicine, Hospital Virgen del Camino, Pamplona, Spain. 49Department of Internal Medicine, Hospital Universitario Miguel Servet, Zaragoza, Spain. 50Department of Rheumatology, Hospital Universitario de Canarias, Tenerife, Spain. 51Department of Rheumatology, Hospital General Universitario de Valencia, Valencia, Spain. 52Department of Rheumatology, Hospital Universitari i Politecnic La Fe, Valencia, Spain. 53Department of Rheumatology, Hospital Universitari Doctor Peset, Valencia, Spain. 54Department of Internal Medicine, Thrombosis and Vasculitis Unit, Complexo Hospitalario Universitario de Vigo, Vigo, Spain. 55Department of Rheumatology, INIBIC-Hospital Universitario A Coruña, La Coruña, Spain. 56Department of Clinical Immunology, Hannover Medical School, Hannover, Germany. 57Department of Dermatology, Josefs-Hospital, Ruhr University Bochum, Bochum, Germany. 58Clinic of Rheumatology, University of Lübeck, Lübeck, Germany. 59Service of Rheumatology and Clinic Immunology Spedali Civili, Brescia, Italy. 60Department of Rheumatology, VU University Medical Center, Amsterdam, The Netherlands. 61Department of Rheumatology, Radboud University Nijmegen Medical Center, Nijmegen, Netherlands. 62Department of Rheumatology, Lund University, Lund, Sweden Australian Scleroderma Interest Group (ASIG) Australian Scleroderma Interest Group (ASIG) J. Zochling66, J. Sahhar67, J. Roddy68, P. Nash69, K. Tymms70, M. Rischmueller71 & S. Lester71 J. Zochling66, J. Sahhar67, J. Roddy68, P. Nash69, K. Tymms70, M. Rischmueller71 & S 66Menzies Research Institute Tasmania, University of Tasmania, Hobart, TAS, Australia. 67Department Rheumatology, Monash Medical Centre, Melbourne, VIC, Australia. 68Rheumatology, Royal Perth Hospital, Perth, WA, Australia. 69Research Unit, Sunshine Coast Rheumatology, Maroochydore, QLD, Australia. 70Canberra Rheumatology, Canberra, ACT, Australia. 71Department Rheumatology, The Queen Elizabeth Hospital, Woodville, SA, Australia NATURE COMMUNICATIONS \| (2019) 10:4955 \| https://doi.org/10.1038/s41467-019-12760-y \| www.nature.com/naturecommunications 14 NATURE COMMUNICATIONS \| (2019) 10:4955 \| https://doi.org/10.1038/s41467-019-12760-y \| www.nature.com/naturecommunicatio
https://openalex.org/W4244645683	https://www.qeios.com/read/GUSCVE/pdf	English	null	Sodium Dodecylsulfate-Polyacrylamide Gel Electrophoresis	Definitions	2,020	cc-by	114	Open Peer Review on Qeios Open Peer Review on Qeios Sodium Dodecylsulfate-Polyacrylamide Gel Electrophoresis National Cancer Institute Qeios ID: GUSCVE · https://doi.org/10.32388/GUSCVE Qeios · Definition, February 2, 2020 Open Peer Review on Qeios Source National Cancer Institute. Sodium Dodecylsulfate-Polyacrylamide Gel Electrophoresis. NCI Thesaurus. Code C18150. Sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis is a procedure for estimating molecular weights of polypeptides, and uses SDS, a detergent that dissociates and unfolds oligomeric proteins into its subunits. The SDS binds to the polypeptides to form complexes with fairly constant charge to mass ratios. The electrophoretic migration rate through a gel is therefore determined only by the size of the complexes. Qeios ID: GUSCVE · https://doi.org/10.32388/GUSCVE 1/1
https://openalex.org/W2526247390	https://www.nature.com/articles/srep34303.pdf	English	null	The structure of a furin-antibody complex explains non-competitive inhibition by steric exclusion of substrate conformers	Scientific reports	2,016	cc-by	6,595	The structure of a furin-antibody complex explains non-competitive inhibition by steric exclusion of substrate conformers Sven O. Dahms1,2, John W. M. Creemers3, Yvonne Schaub1, Gleb P. Bourenkov4, Thomas Zögg2,†, Hans Brandstetter2 & Manuel E. Than1 received: 25 May 2016 accepted: 09 September 2016 Published: 27 September 2016 received: 25 May 2016 accepted: 09 September 2016 Published: 27 September 2016 Proprotein Convertases (PCs) represent highly selective serine proteases that activate their substrates upon proteolytic cleavage. Their inhibition is a promising strategy for the treatment of cancer and infectious diseases. Inhibitory camelid antibodies were developed, targeting the prototypical PC furin. Kinetic analyses of them revealed an enigmatic non-competitive mechanism, affecting the inhibition of large proprotein-like but not small peptidic substrates. Here we present the crystal structures of furin in complex with the antibody Nb14 and of free Nb14 at resolutions of 2.0 Å and 2.3 Å, respectively. Nb14 binds at a site distant to the substrate binding pocket to the P-domain of furin. Interestingly, no major conformational changes were observed upon complex formation, neither for the protease nor for the antibody. Inhibition of furin by Nb14 is instead explained by steric exclusion of specific substrate conformers, explaining why Nb14 inhibits the processing of bulky protein substrates but not of small peptide substrates. This mode of action was further supported by modelling studies with the ternary factor X-furin-antibody complex and a mutation that disrupted the interaction interface between furin and the antibody. The observed binding mode of Nb14 suggests a novel approach for the development of highly specific antibody-based proprotein convertase inhibitors. Furin1 belongs to the family of the calcium-dependent proprotein convertases (PCs). These endoproteinases share structural homology of their catalytic domains with subtilisin. However, in contrast to subtilisin they are highly specific enzymes, activating a large number of secreted and membrane-associated secretory proteins by limited proteolysis. Substrate proteins include blood coagulation factors, hormones, growth factors, matrix met- alloproteases as well as viral capsid proteins and bacterial toxins1,2. The classical PCs cleave after basic residue motifs, with furin preferentially recognising the motif R-X-K/R-R↓ (where “↓” represents the scissile peptide bond)3,4. Besides the subtilisin-like catalytic domain, all PCs require the so-called proprotein convertase domain (P-domain, for some family members also called Homo B domain) for catalytic activity5. The P-domain is located C-terminal to the catalytic domain and adopts a β-barrel-like fold. These proteases are also involved in a large number of pathologies, including bacterial and viral infections as well as cancer progression and metastasis2. www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports Results C Co-crystal structure of furin and Nb14. We have crystallised the ternary complex of human furin with decanoyl-RVKR-chloromethylketone (RVKR-CMK) and the inhibitory camelid antibody Nb14. Even optimised crystals grew as needle clusters. Using a whole needle-cluster, initial diffraction data were obtained to a limit- ing resolution of 2.9 Å at the synchrotron beamline BL14.1 of the Helmholtz-Zentrum Berlin (HZB) (Fig. S1a, Table S1). These data verified the presence of well diffracting mono-crystalline fragments within the mounted crystal bundles. The structure was solved by molecular replacement revealing the presence of two copies of the furin:RVKR-CMK:Nb14 complex in the asymmetric unit. To obtain better defined crystallographic data, rather small crystal fragments with approx. sizes of only 5 × 5 × 150 μm3 were separated from these needle clusters and were used for single crystal diffraction data collection at the micro-focus beamline P14 at the European Molecular Biology Laboratory (EMBL) in Hamburg (Fig. S1b, Table S1). The latter dataset was subsequently used for refine- ment of the furin:RVKR-CMK:Nb14 complex at 2.0 Å resolution at high quality with final R/Rfree factors of 16.3/19.7% (Fig. 1, Table S1). ( g , ) Nb14 was bound to the P-domain of furin rather than to the catalytic domain (Figs 1 and S2), which in addition was covalently inhibited by RVKR-CMK. The interaction interface at the P-domain is located distant to the active site cleft of the protease, involving amino acids Pro458-Asp460, Thr492-Asn496, Ala525-Asn529 and Asn558-Thr564 of furin. The complementary region at the surface of Nb14 involves the amino acid seg- ments Tyr32-Tyr35, Trp47-Arg53, Ser56-Asp59, Val98-Ala102 as well as Trp106. Assembly analysis with the PISA-server28 revealed an average covered surface area of ~685 A2. The interaction interface (~700 A2) is some- what smaller than for the HGFA-inhibiting Fabs Ab58 (~900 A2) and Ab75 (~1000 A2). However, it corresponds well to the average size observed for protein-antigen complexes (varying from ~400 A2-~1000 A2 29) and to canonical kunitz-type trypsin inhibitors like amyloid precursor protein protease inhibitor domain (APPI or KPI, ~700 A2) and basic pancreatic trypsin inhibitor (BPTI, ~800 A2)30. The calculated average solvation free energy gain P-value (ΔiG-P) and complexation significance score (CSS) values of 0.199 and 0.293 of this inter- face underline the significance of the observed interaction. All other molecular interactions, either mediated by non-crystallographic or crystallographic contacts, are classified as non-significant. The structure of a furin-antibody complex explains non-competitive inhibition by steric exclusion of substrate conformers Sven O. Dahms1,2, John W. M. Creemers3, Yvonne Schaub1, Gleb P. Bourenkov4, Thomas Zögg2,†, Hans Brandstetter2 & Manuel E. Than1 Consequently, inhibitors of furin and other PCs are promising drug candidates6 and inhibitory molecules of a different chemical nature are currently being investigated in various labs7. Inhibitors of furin were successfully applied to inhibit the cell motility and invasiveness of cancer cells8, to impair carcinoma cell growth9 and to inhibit activation of HIV-1 glycoprotein gp16010. However, although inhibitors with high affinity have been devel- oped11, obtaining specificity between the PC-family members remains challenging12. Most inhibitors target the substrate binding cleft of the PCs, which is highly conserved between these proteases13,14. gt g y p Crystal structures of inhibitor-bound murine4 and human11,15 furin as well as of the yeast homolog Kex2p16,1 nd modelling approaches13,14 gave hints as to how substrates and substrate-derived inhibitors bind to the PCs 1Protein Crystallography Group, Leibniz Institute on Aging-Fritz Lipmann Institute (FLI), Beutenbergstr. 11, 07745 Jena, Germany. 2Department of Molecular Biology, University of Salzburg, Billrothstrasse 11, A-5020 Salzburg, Austria. 3Department of Human Genetics, KU Leuven, Herestraat 49, B-3000 Leuven, Belgium. 4European Molecular Biology Laboratory, Hamburg, Germany. †Present address: VUB Vrije Universiteit Brussel, VIB Dept. Molecular and Cellular Interactions, Pleinlaan 2,B-1050 Brussels, Belgium. Correspondence and requests for materials should be addressed to S.O.D. (email: sdahms@fli-leibniz.de) or M.E.T. (email: than@fli-leibniz.de) Scientific Reports \| 6:34303 \| DOI: 10.1038/srep34303 1 www.nature.com/scientificreports/ The commonly accepted notion is that the minimal working unit of the PCs consists of two consecutive struc- tural units, the catalytic domain and the P-domain. PCs bind their cognate substrates and inhibitors via recog- nition at several subsites at the catalytic domain, typically involving multiple tight contacts and hydrogen bonds. The P-domain is hereby closely associated with the catalytic domain and is essential for its stabilisation, but it does not seem to be involved in the ultimate subsite recognition. Antibodies can act as highly specific protease inhibitors18. Camelid antibodies (or their minimal active subfragments, the variable heavy chain (VH) domains; as isolated proteins often called VHH-fragments or “nanobodies”) as well as antigen binding fragments (Fabs) have been successfully applied to effectively inhibit pharmacological targets like human growth factor activator (HGFA)19, matriptase20, tumour necrosis factor-α-converting enzyme (TACE)21 or the trypsin-like serine pro- tease HtrA-122. Nanobodies are highly versatile tools for research, diagnostic and therapeutic applications. They can be easily manipulated to change their half-life or to link them to another polypeptide like a toxin, a reporter, or a peptide inhibitor23. The structure of a furin-antibody complex explains non-competitive inhibition by steric exclusion of substrate conformers Sven O. Dahms1,2, John W. M. Creemers3, Yvonne Schaub1, Gleb P. Bourenkov4, Thomas Zögg2,†, Hans Brandstetter2 & Manuel E. Than1 Structural analyses showed that antibody binding often blocks the active site cleft of tar- get proteases (e.g. refs 19 and 20) or induces conformational changes and thus inhibits proteolysis allosterically (e.g. refs 24–26). Recently, nanobodies were developed which specifically target human and mouse furin27. They inhibited cleavage of diphtheria toxin and effectively protected cells from diphtheria-toxin-induced cytotoxic- ity. Interestingly, only the turnover of protein substrates by furin was inhibited, whereas the hydrolysis of short fluorogenic peptides remained unaltered. As the kinetic analysis revealed a non-competitive mode of inhibition, it was concluded that these nanobodies do not seem to directly interfere with the catalytic mechanism or bind- ing of substrates to the active site cleft27, calling for a structural characterization of their rather unusual mode of inhibition. To unravel the binding epitope and the mode-of-action of the furin-inhibiting nanobody Nb14 we solved its structure in isolation as well as in complex with the target protease. Our structural and biochemical data explain its unusual inhibitory properties and suggest strategies for the future of inhibitor development. Results C An unusually high number of tyrosine side chains is found in the interaction interface of furin and Nb14, including Tyr32, Tyr35, Tyr109 of Nb14 and Tyr560 of furin. These tyrosines form specific hydrogen bonds and contribute to the hydrophobic character of the surface. Analysis with the PISA server28 revealed an average solvation free energy gain (ΔiG) of −28.5 kJ/mol, indicating a highly hydrophobic interface. Mutation of the interaction interface abrogates inhibition by Nb14. To analyse the impact of the observed molecular contact, a point mutation was introduced to furin changing Thr562 into arginine. Thr562 is located at a central position of the interaction interface (inset in Fig. 1) Substitution by a bulky arginine sidechain, however, is expected to clash with the complementary surface of Nb14 and to disrupt its interaction with furin. The conservation of this amino acid in evolution is very low and its substitution should therefore not affect pro- teolysis13. Indeed, we measured very similar specific activities of 53.3 ± 1.2 μmol 7-Amino-4-methylcoumarin (AMC)/h/mg and 54.3 ± 0.5 μmol AMC/h/mg for purified wild type (FurinWT) and mutated human furin (FurinT562R), respectively. ( ) p y Next, we analysed the binding properties of FurinWT and FurinT562R in analytical gel permeation chromatog- raphy (GPC) experiments. If an equimolar mixture of FurinWT and Nb14 is subjected to gel permeation chro- matography, the proteins form the expected complex and co-elute in a single peak (Fig. 2a). Pre-mixed Nb14 and mutant FurinT562R, however, elute as two separated peaks from the GPC-column (Fig. 2b). In conclusion, the mutation Thr562Arg at the interaction interface of furin disrupts complex formation with Nb14. Scientific Reports \| 6:34303 \| DOI: 10.1038/srep34303 2 www.nature.com/scientificreports/ Figure 1. Structure of the furin:RVKR-CMK:Nb14 complex. Human furin is shown as surface representation. The catalytic domain and the P-domain are coloured in yellow and brown, respectively. The dec- RVKR-CMK inhibitor is shown as a ball and stick model with magenta carbons. Nb14 is displayed as a cartoon representation (blue) with the central disulphide bond highlighted as spheres. The inset shows a part of the interaction interface of furin and Nb14. The surface of the P-domain is shown as a partially transparent surface. The peptide stretch Gly561-Thr562-Leu563 is shown as a stick model. The 2Fo−Fc electron density composite omit map (green mesh) is contoured at 1.0 σ. Figure 1. Structure of the furin:RVKR-CMK:Nb14 complex. Human furin is shown as surface representation. Results C The catalytic domain and the P-domain are coloured in yellow and brown, respectively. The dec- RVKR-CMK inhibitor is shown as a ball and stick model with magenta carbons. Nb14 is displayed as a cartoon representation (blue) with the central disulphide bond highlighted as spheres. The inset shows a part of the interaction interface of furin and Nb14. The surface of the P-domain is shown as a partially transparent surface. The peptide stretch Gly561-Thr562-Leu563 is shown as a stick model. The 2Fo−Fc electron density composite omit map (green mesh) is contoured at 1.0 σ. Figure 1. Structure of the furin:RVKR-CMK:Nb14 complex. Human furin is shown as surface representation. The catalytic domain and the P-domain are coloured in yellow and brown, respectively. The dec- RVKR-CMK inhibitor is shown as a ball and stick model with magenta carbons. Nb14 is displayed as a cartoon representation (blue) with the central disulphide bond highlighted as spheres. The inset shows a part of the interaction interface of furin and Nb14. The surface of the P-domain is shown as a partially transparent surface. The peptide stretch Gly561-Thr562-Leu563 is shown as a stick model. The 2Fo−Fc electron density composite omit map (green mesh) is contoured at 1.0 σ. Figure 2. Interaction of Nb14 with FurinWT or FurinT562R. The UV280 absorption is shown as a function of the elution volume of the GPC column. Fractions under the peaks (black bars) were analysed by SDS-PAGE (insets). The premixed GPC sample was loaded as control to the SDS-PAGE (S). (a) GPC run of Nb14 and FurinWT, marked in the inset as open and filled arrowheads, respectively. (b) GPC run of Nb14 and FurinT562R, marked in the inset as open and filled arrowheads, respectively. Figure 2. Interaction of Nb14 with FurinWT or FurinT562R. The UV280 absorption is shown as a function of Figure 2. Interaction of Nb14 with FurinWT or FurinT562R. The UV280 absorption is shown as a function of the elution volume of the GPC column. Fractions under the peaks (black bars) were analysed by SDS-PAGE (insets). The premixed GPC sample was loaded as control to the SDS-PAGE (S). (a) GPC run of Nb14 and FurinWT, marked in the inset as open and filled arrowheads, respectively. (b) GPC run of Nb14 and FurinT562R, marked in the inset as open and filled arrowheads, respectively. Nb14 inhibits furin cleavage of large substrates via steric exclusion. An overall comparison o h h f b d b h l d h f ( 11) l d l Nb14 inhibits furin cleavage of large substrates via steric exclusion. An overall comparison of the human furin structure bound to Nb14 with isolated human furin (PDB-ID: 4RYD11) revealed a low average Cα-root mean square deviation (r.m.s.d.) of 0.32 Å. Furin is found in its catalytically active state forming a typical covalent complex with the chloromethylketone compound (Fig. S3a,b) that is very similar to non-covalently inhibited human furin15 and covalently inhibited mouse furin4. To investigate the effect of furin binding on the structure of the antibody, we also solved the structure of Nb14 in its free state (Table S1, Fig. S3c, see Supplementary Information for details). Also here, no significant structural alteration could be found as also indicated by the low Cα-r.m.s.d. of 0.20 Å (Fig. S3c). y α ( g ) Analysing the complex between furin and Nb14, we found that the shortest distance observed between Nb14 (Asp62, side chain carboxylate) and the decanoylamide-C9 of the inhibitor measures approx. 19 Å. Canonical binding of peptides up to the S5 or S6 pockets is thus completely unperturbed by Nb14 and the placement of an elongated peptide chain to this region is sterically not hindered. In addition, the catalytic cleft and the active site of furin are clearly not blocked by the binding of Nb14, and its inactivity towards the cleavage of small peptidic substrates27 is perfectly conceivable. p y Globular folded parts of substrate proteins do however show an additional level of access control to the active site of proteases, as they are largely sterically restricted from the substrate binding cleft. Therefore, the binding of protein- aceous substrates to furin and their cleavage should only be possible if their overall conformation fits to the specific topology of the furin:Nb14 complex. This hypothesis was tested by means of modelling studies with the coagulation factor X (Figs 4 and S4). Proteolysis occurs here C-terminal to the recognition sequence Arg181-[P3]-Lys183-Arg184-↓. The P1-P4 amino acids are estimated to bind in a similar conformation as observed for the RVKR-CMK inhibitor com- plex. For our modelling studies this peptide stretch was aligned immediately C-terminal to the EGF2-domain of factor Xa (PDB-ID:2GD433), creating an extended factor Xa model, Xa* (catalytic domain including the furin recognition motif). Nb14 inhibits furin cleavage of large substrates via steric exclusion. An overall comparison o h h f b d b h l d h f ( 11) l d l The whole catalytic domain of factor Xa* was hereby treated as a rigid body, varying initially only the relative conformation of the amino acids between Thr178 and Arg181 and later also of the peptide stretch Cys174 to Glu180. Using this approach we modelled 13 different conformers of furin-bound factor Xa* that fit to the substrate binding pocket (Figs 4 and S4a). During modelling we realised that the conformation of Xa* is largely restricted by the deep sub- strate binding cleft of furin and that the region between Thr178 and Arg181 requires a rather extended conformation to avoid steric clashes of furin and factor Xa. In presence of the Nb14 the conformational space is even more restricted and 12 out of the 13 initially modelled Xa* conformers now show main-chain clashes with the antibody (Figs 4 and S4b). These clashes exclusively occur at sites distant to the active site cleft of furin. Consequently, the modelling studies sug- gest a mode of action of Nb14 involving conformational restriction of the substrate. Binding and turnover of substrate proteins is still possible if this antibody is bound to furin, but now only a subset of all possible substrate conformers is still allowed to bind to and to be cleaved by the Nb14:furin complex. Results C Inhibition of furin by Nb14 can conveniently be monitored in limited proteolysis experiments with the furin substrate coagulation factor X31. Maturation of it requires cleavage by furin in between the catalytic domain and the EGF-2 domain. For cleavage assays we used the inactive Ser195Ala mutant (factor XS195A), showing largely reduced self-degradation compared to the wild type protease32. After incubation of both proteins a band shift Inhibition of furin by Nb14 can conveniently be monitored in limited proteolysis experiments with the furin substrate coagulation factor X31. Maturation of it requires cleavage by furin in between the catalytic domain and the EGF-2 domain. For cleavage assays we used the inactive Ser195Ala mutant (factor XS195A), showing largely reduced self-degradation compared to the wild type protease32. After incubation of both proteins a band shift Scientific Reports \| 6:34303 \| DOI: 10.1038/srep34303 3 www.nature.com/scientificreports/ Figure 3. Proteolysis of factor XS195A by FurinWT or FurinT562R and its inhibition by Nb14. Factor XS195A was subjected to limited proteolysis by FurinWT or FurinT562R and analysed by SDS-PAGE. The bands of uncleaved factor XS195A (FX), cleaved factor XS195A (FX(cleaved)) and Nb14 are marked with arrowheads. Figure 3. Proteolysis of factor XS195A by FurinWT or FurinT562R and its inhibition by Nb14. Factor XS195A was subjected to limited proteolysis by FurinWT or FurinT562R and analysed by SDS-PAGE. The bands of uncleaved factor XS195A (FX), cleaved factor XS195A (FX(cleaved)) and Nb14 are marked with arrowheads. from 42 to 38 kDa is observed (Fig. 3). FurinWT and FurinT562R essentially showed the same activity in the factor XS195A cleavage assay (Fig. 3). Apparently the introduced mutation affects neither the turnover of small peptidic substrates (see above) nor the proteolysis of protein substrates like factor XS195A. Cleavage of factor XS195A by FurinWT is largely reduced in the presence of 1 μM Nb14 indicating inhibition of furin by the antibody (Fig. 3). FurinT562R, however, performs very similarly with and without Nb14 in the reaction mixture (Fig. 3). Discussion We have investigated the structural basis of inhibition of human furin by the highly specific inhibitory camelid antibody Nb1427. The structure of the furin-nanobody complex revealed a binding epitope at the P-domain dis- tant to the active site cleft. These data imply a unique inhibition mechanism based on steric exclusion of certain conformers during substrate binding. Using a micrometre-sized X-ray beam and a high-precision diffractometer at the PETRAIII beamline P14 was hereby essential to solve the crystallographic structure as respective complex crystals could only be obtained in the form of small needles. Scientific Reports \| 6:34303 \| DOI: 10.1038/srep34303 4 www.nature.com/scientificreports/ Figure 4. Modelling of the furin-factor X enzyme-substrate complex. The furin:Nb14 complex is shown as surface representation and coloured in yellow (furin, catalytic domain), brown (furin, P-domain) and blue (Nb14). For modelling, the catalytic domain of factor X (cartoon representation) was linked to the N-terminus of the P1-P4 tetrapeptide (stick representation, magenta) of the co-crystallised RVKR-CMK inhibitor. (a) The one conformer that fits to the furin:Nb14 complex is coloured in magenta. (b) Conformers that do not fit to the furin:Nb14 complex are shown in grey. Figure 4. Modelling of the furin-factor X enzyme-substrate complex. The furin:Nb14 complex is shown as surface representation and coloured in yellow (furin, catalytic domain), brown (furin, P-domain) and blue (Nb14). For modelling, the catalytic domain of factor X (cartoon representation) was linked to the N-terminus of the P1-P4 tetrapeptide (stick representation, magenta) of the co-crystallised RVKR-CMK inhibitor. (a) The one conformer that fits to the furin:Nb14 complex is coloured in magenta. (b) Conformers that do not fit to the furin:Nb14 complex are shown in grey. The mode of action observed for Nb14 is largely different to other nanobodies that inhibit proteases by either blocking the active site cleft (e.g. refs 19 and 20) and/or by inducing conformational changes that inter- fere with the catalytic mechanism (e.g. refs 24–26). It also suggests how inhibitory specificity between the highly homologous PCs can be achieved. Nb14 was shown to bind exclusively to furin27. Other PC family members, e.g. paired basic amino acid cleaving enzyme 4 (PACE4), proprotein convertase 1 (PC1) and propro- tein convertase 2 (PC2), are not recognised by Nb1427. Discussion Interestingly it binds to a region of furin that was previ- ously shown to have a low degree of conservation13, as the surface residues of the P-domain and especially the region around Thr562 are largely variable among the PC family members (Fig. S5). Notably, a point mutation in furin at Thr562 also completely abolished binding of Nb14. In contrast, peptide-based inhibitors and related small molecules directly interact with the core of the substrate binding pocket that is much more conserved between the different family members. The specificity of such compounds for individual PC-family members is correspondingly much lower12.h p g y The structural features observed for the furin:RVKR-CMK:Nb14 complex also nicely explain the reported kinetic properties of Nb14. Binding of the nanobody occurs independently of the occupation of the substrate binding cleft. In previous studies we demonstrated that covalently and non-covalently inhibited furin showed very similar structures10. Therefore a structural influence of the bound inhibitor on the interaction interface of Nb14 is unlikely. As observed for the RVKR-CMK peptide, small fluorogenic substrates can bind to the protease and are hydrolysed with unaltered efficacy with and without bound Nb1427. Proteinaceous substrates, however, occupy a large space in the non-primed region of furin’s active site cleft as shown by modelling studies with factor X. In the furin:Nb14 complex especially, this space is restricted and thus specific substrate conformers are excluded from binding, reducing the effective substrate concentration. Previous studies have shown that the inhi- bition strength of Nb14 indeed varied between substrates, e.g. a stronger inhibition was observed for glypican 3 (GPC3) in comparison to tumour necrosis factor β (TGFβ)27. These data are very much in line with the structural Scientific Reports \| 6:34303 \| DOI: 10.1038/srep34303 5 www.nature.com/scientificreports/ features of the furin:RVKR-CMK:Nb14 complex and with the modelled furin:Xa:Nb14 enzyme substrate com- plex. Our data suggest that inhibition by Nb14 also seems to depend on the specific steric properties of furin’s substrates.i features of the furin:RVKR-CMK:Nb14 complex and with the modelled furin:Xa:Nb14 enzyme substrate com- plex. Our data suggest that inhibition by Nb14 also seems to depend on the specific steric properties of furin’s substrates In conclusion, Nb14 seems to facilitate the specific inhibition of furin over other PC family members. In addition, it probably also allows some specificity for certain substrate proteins or substrate classes. This potential substrate specificity of furin inhibiting antibodies must be characterised in detail by future studies. Referencesh References 1. Thomas, G. Furin at the cutting edge: from protein traffic to embryogenesis and disease. Nat Rev Mol Cell Biol 3, 753–766 (2002).h 1. Thomas, G. Furin at the cutting edge: from protein traffic to embryogenesis and disease. Nat Rev Mol Cell Biol 3, 753–766 (2002). 2. Artenstein, A. W. & Opal, S. M. Proprotein convertases in health and disease. The New England journal of medicine 365, 2507–2518 (2011). h g g pfi y g 2. Artenstein, A. W. & Opal, S. M. Proprotein convertases in health and disease. The New England journal of medicine 365, 2507–251 (2011). 3. Seidah, N. G., Sadr, M. S., Chretien, M. & Mbikay, M. The multifaceted proprotein convertases: their unique, redundant, complementary, and opposite functions. J Biol Chem 288, 21473–21481 (2013).hi p y pp ( ) 4. Henrich, S. et al. The crystal structure of the proprotein processing proteinase furin explains its stringent specificity. Nat Struct Bio 10, 520–526 (2003). 5. Creemers, J. W., Siezen, R. J., Roebroek, A. J., Ayoubi, T. A., Huylebroeck, D. & Van de Ven, W. J. Modulation of furin-mediated proprotein processing activity by site-directed mutagenesis. J Biol Chem 268, 21826–21834 (1993).h 6. Seidah, N. G. & Prat, A. The biology and therapeutic targeting of the proprotein convertases. Nat Rev Drug Discov 11, 367–383 (2012). 7. Basak, A. Inhibitors of proprotein convertases. J Mol Med 83, 844–855 (2005). 7. Basak, A. Inhibitors of proprotein convertases. J Mol Med 83, 844–855 (2005). p p 8. Coppola, J. M., Bhojani, M. S., Ross, B. D. & Rehemtulla, A. A small-molecule furin inhibitor inhibits cancer cell motility and invasiveness. Neoplasia 10, 363–370 (2008). p 9. Lopez de Cicco, R., Bassi, D. E., Zucker, S., Seidah, N. G. & Klein-Szanto, A. J. Human carcinoma cell growth and invasiveness is impaired by the propeptide of the ubiquitous proprotein convertase furin. Cancer research 65, 4162–4171 (2005). y 10. Hallenberger, S., Bosch, V., Angliker, H., Shaw, E., Klenk, H. D. & Garten, W. Inhibition of fu HIV-1 glycoprotein gp160. Nature 360, 358–361 (1992). 10. Hallenberger, S., Bosch, V., Angliker, H., Shaw, E., Klenk, H. D. & Garten, W. Inhibition of furin-mediated cleavage activation of HIV-1 glycoprotein gp160. Nature 360, 358–361 (1992). g y p gp , ( ) 11. Hardes, K. et al. Novel Furin Inhibitors with Potent Anti-infectious Activity. ChemMedChem (2015). 11. Hardes, K. et al. Novel Furin Inhibitors with Potent Anti-in 12. Discussion Nonetheless, this outstanding feature of Nb14 is of great interest for pharmacological applications. Potential therapeutic side effects might be reduced by targeting the conversion of only specific substrates rather than by complete inhibi- tion of furin or even several PCs at the same time. Therapeutic antibodies might be raised to mainly inhibit only specific substrates of furin (e.g. hemagglutinin to treat acute influenza infections). Molecular tools are available (e.g. phage display34) allowing protein- and epitope-specific selection of antibodies. Therefore, similar molecules like Nb14 may be developed for inhibition of other PC family members (e.g. PACE4) or other protease classes. Methodsh The expression of camelid antibody Nb1427, human furin15 and recombinant coagulation factor XS195A 32 as well as the activity test of furin were described previously. Prior to crystallisation Nb14 and dec-RVKR-CMK-inhibited human furin were mixed at equimolar ratios and the resulting complex was purified by GPC. Details of the puri- fication procedure and activity assays of the enzymes used are described in Supplementary Information.h i p y y y pp y The furin:RVKR-CMK:Nb14 complex (~9 mg/ml in in 10 mM Hepes, pH 7.5, 100 mM NaCl and 2 mM CaCl2) was crystallised in 0.1 M sodium acetate, pH 5.6, 16–18% PEG 3350. Crystals of isolated Nb14 were grown using a controlled dry-out procedure. Crystallisation drops (200 nl) of protein solution (10 mg/ml in 10 mM Hepes, pH 7.5, 100 mM NaCl and 1mM CaCl2) were pipetted without any reservoir solution. The plates were sealed and crystals appeared after several days and were stable for several weeks. Diffraction data were collected at BL 14.1 of BESSY-II, Helmholtz-Zentrum Berlin (HZB)35, and beamline P14, EMBL Hamburg (Table S1). Data processing was performed in XDS36 and programs of the CCP4-suite37. The structures were solved by molecular replace- ment in PHASER38 using the structures of the isolated Nb14 and human furin (PDB-ID 4RYD11). COOT39 and PHENIX40 were applied for model building and refinement, respectively. MAIN41 was used for modelling of the furin:Nb14:factor X complex. Details of data collection, structure solution, model building and refinement pro- cedures are described in Supplementary Information. Analytical gel permeation chromatography runs were performed on a Superdex 200 5/150 GL column (GE Healthcare) in GPC buffer (10 mM Hepes, pH 7.5, 100 mM NaCl, 1 mM CaCl2) using an Aekta Micro FPLC system (GE Healthcare). The proteins were premixed at an equimolar ratio or mixed with GPC buffer for control runs at final concentrations of ~23μM each and subsequently subjected to GPC at 0.2 ml/min. The peak fractions were analysed by SDS-PAGE. GPC binding assays were repeated three times. y y g y p Cleavage and inhibition assays with factor XS195A were performed in 100 mM Hepes, 5 mM CaCl, 0.5% TritonX100, pH7.0 at 25 °C in 20 μl reaction setups. Samples contained 2.8 μg recombinant factor XS195A, 4 ng FurinWT or FurinT562R and 1 μM Nb14. For control samples the respective protein buffers were added to the reactions. Referencesh Couture, F., D’Anjou, F. & Day, R. On the cutting edge of proprotein convertase pharmacology: from molecular concepts to cli applications. Biomolecular concepts 2, 421–438 (2011).h 13. Henrich, S., Lindberg, I., Bode, W. & Than, M. E. Proprotein convertase models based on the crystal structures of furin and kexin: explanation of their specificity. J Mol Biol 345, 211–227 (2005).fi i y 4. Lu, Y. et al. Peptidomimetic furin inhibitor MI-701 in combination with oseltamivir and ribavirin efficiently blocks propagation o highly pathogenic avian influenza viruses and delays high level oseltamivir resistance in MDCK cells. Antiviral Res 120, 89–100 (2015). , pfi y k p p g highly pathogenic avian influenza viruses and delays high level oseltamivir resistance in MDCK cells. Antiviral Res 120, 89–100 (2015). 15 Dahms S O Hardes K Becker G L Steinmetzer T Brandstetter H & Than M E X ray Structures of Human Furin in Complex (2015). 15. Dahms, S. O., Hardes, K., Becker, G. L., Steinmetzer, T., Brandstetter, H. & Than, M. E. X-ray Structures of Human Furin in Complex i h C i i I hibi ACS Ch Bi l 9 1113 1118 (2014) ( ) 5. Dahms, S. O., Hardes, K., Becker, G. L., Steinmetzer, T., Brandstetter, H. & Than, M. E. X-ray Structures of Human Furin in Complex with Competitive Inhibitors. ACS Chem Biol 9, 1113–1118 (2014).f p 6. Holyoak, T., Kettner, C. A., Petsko, G. A., Fuller, R. S. & Ringe, D. Structural basis for differences in substrate selectivity in Kex2 and furin protein convertases. Biochemistry 43, 2412–2421 (2004).f p y 7. Wheatley, J. L. & Holyoak, T. Differential P1 arginine and lysine recognition in the prototypical proprotein convertase Kex2. Pro Natl Acad Sci USA 104, 6626–6631 (2007). 17. Wheatley, J. L. & Holyoak, T. Differential P1 arginine and lysine recognition in the prototypical proprotein convertase Kex2. Proc Natl Acad Sci USA 104, 6626–6631 (2007). 18. Ganesan, R., Eigenbrot, C. & Kirchhofer, D. Structural and mechanistic insight into how antibodies inhibit serine proteases. Biochem J 430, 179–189 (2010). y yf g y g p yp p p Natl Acad Sci USA 104, 6626–6631 (2007). 18. Ganesan, R., Eigenbrot, C. & Kirchhofer, D. Structural and mechanistic insight into how antibodies inhibit serine proteases. Biochem Natl Acad Sci USA 104, 6626–6631 (2007). 18. Ganesan, R., Eigenbrot, C. & Kirchhofer, D. Structural and mechanistic insight into how antibodies inhibit serine proteases. Author Contributions S.O.D. and M.E.T. designed the experiments, analysed data, interpreted results and co-wrote the paper. S.O.D., Y.S., G.P.B. and T.Z. performed experiments and analysed data. J.W.M.C., T.Z. and H.B. provided new reagents. All authors contributed to the interpretation of the results and assisted in writing the paper. Acknowledgements g We would like to thank the Helmholtz Zentrum Berlin BESSY II for providing synchrotron radiation at the beamline BL 14.1 and the scientific staff for their assistance. Funding was provided by FWO Vlaanderen, the federal government of Germany and the free state of Thuringia. Referencesh Biochem J 430, 179–189 (2010). , ( ) 18. Ganesan, R., Eigenbrot, C. & Kirchhofer, D. Structural and mechanistic insight into how antibodies inhibit serine proteases. Biochem J 430, 179–189 (2010). Scientific Reports \| 6:34303 \| DOI: 10.1038/srep34303 6 www.nature.com/scientificreports/ 9. Wu, Y. et al. Structural insight into distinct mechanisms of protease inhibition by antibodies. Proc Natl Acad Sci USA 104 19784–19789 (2007).i ( ) arady, C. J., Egea, P. F., Schneider, E. L., Darragh, M. R. & Craik, C. S. Structure of an Fab-protease complex reveals a highly specific ( ) 20. Farady, C. J., Egea, P. F., Schneider, E. L., Darragh, M. R. & Craik, C. S. Structure of an Fab-protease complex reveals a highly sp non-canonical mechanism of inhibition J Mol Biol 380 351–360 (2008) 20. Farady, C. J., Egea, P. F., Schneider, E. L., Darragh, M. R. & Craik, C. S. Structure of an Fab-protease complex revea non-canonical mechanism of inhibition. J Mol Biol 380, 351–360 (2008). 21. Tape, C. J. et al. Cross-domain inhibition of TACE ectodomain. Proc Natl Acad Sci USA 108, 5578–5583 (2011).h 21. Tape, C. J. et al. Cross-domain inhibition of TACE ectodomain. Proc Natl Acad Sci USA 108, 5578–5583 (2011). 22. Ciferri, C. et al. The trimeric serine protease HtrA1 forms a cage-like inhibition complex with an anti-HtrA1 antibody. Bioche 22. Ciferri, C. et al. The trimeric serine protease HtrA1 forms a cage-like inhibition complex with an anti-HtrA1 antibody. Bioch 472, 169–181 (2015). 3. De Meyer, T., Muyldermans, S. & Depicker, A. Nanobody-based products as research and diagnostic tools. Trends in biotechnology 32, 263–270 (2014). , ( ) 4. Koschubs, T. et al. Allosteric antibody inhibition of human hepsin protease. Biochem J 442, 483–494 (2012). al. Allosteric antibody inhibition of human hepsin protease. Bioche . Koschubs, T. et al. Allosteric antibody inhibition of human hepsin 5. Ganesan, R. et al. Unraveling the allosteric mechanism of serine protease inhibition by an antibody. Structure 17, 1614–1624 (2009) 25. Ganesan, R. et al. Unraveling the allosteric mechanism of serine protease inhibition by an antibody. Structure 17, 1614–1624 (2009). 26. Oyen, D., Srinivasan, V., Steyaert, J. & Barlow, J. N. Constraining enzyme conformational change by an antibody leads to hyperbolic inhibition. J Mol Biol 407, 138–148 (2011). 27. Zhu, J. et al. Generation and characterization of non-competitive furin-inhibiting nanobodies. Biochem J 448, 73–82 (2012). Referencesh McCoy, A. J., Grosse-Kunstleve, R. W., Adams, P. D., Winn, M. D., Storoni, L. C. & Read, R. J. Phaser crystallographic software. 37. Winn, M. D. et al. Overview of the CCP4 suite and current developments. Acta Crystallogr D Biol Crystallogr 67, 235–242 (2011). nn, M. D. et al. Overview of the CCP4 suite and current developme 37. Winn, M. D. et al. Overview of the CCP4 suite and current developments. Acta Crystallogr D Biol Crystallogr 67, 235–242 (201 38. McCoy, A. J., Grosse-Kunstleve, R. W., Adams, P. D., Winn, M. D., Storoni, L. C. & Read, R. J. Phaser crystallographic softw Journal of applied crystallography 40, 658–674 (2007). f pp y g p y 39. Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. Features and development of Coot. Acta Crystallogr D Biol Crystallogr 66, 486–501 (2010). f pp y g p y 9. Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. Features and development of Coot. Acta Crystallogr D Biol Crystallogr 66 486–501 (2010). 0. Adams, P. D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr D Bio Crystallogr 66, 213–221 (2010). 40. Adams, P. D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr D Biol Crystallogr 66, 213–221 (2010). y g 1. Turk, D. MAIN software for density averaging, model building, structure refinement and validation. Acta Crystallogr D Bio Crystallogr 69, 1342–1357 (2013). Referencesh l k d h ( ) l f f l h d 8. Krissinel, E. & Henrick, K. Secondary-structure matching (SSM), a new tool for fast protein structure alignment in three dimensions Acta Crystallogr D Biol Crystallogr 60, 2256–2268 (2004). y g y g ( ) 29. Decanniere, K., Transue, T. R., Desmyter, A., Maes, D., Muyldermans, S. & Wyns, L. Degenerate interfaces in antigen-antibody complexes. J Mol Biol 313, 473–478 (2001).f 30. Scheidig, A. J., Hynes, T. R., Pelletier, L. A., Wells, J. A. & Kossiakoff, A. A. Crystal structures of bovine chymotrypsin and trypsin complexed to the inhibitor domain of Alzheimer’s amyloid beta-protein precursor (APPI) and basic pancreatic trypsin inhibitor (BPTI): engineering of inhibitors with altered specificities. Protein science: a publication of the Protein Society 6, 1806–1824 (1997).hfi g g pi p f y 31. Griessner, A., Zögg, T. & Brandstetter, H. The activation peptide of coagulation factor IX and X serves as a high affinity recept cationic ligands. Thrombosis and Haemostasis 110, 620–622 (2013). h 32. Sichler, K., Kopetzki, E., Huber, R., Bode, W., Hopfner, K. P. & Brandstetter, H. Physiological fIXa activation involves a cooperative conformational rearrangement of the 99-loop. J Biol Chem 278, 4121–4126 (2003). h 32. Sichler, K., Kopetzki, E., Huber, R., Bode, W., Hopfner, K. P. & Brandstetter, H. Physiologi conformational rearrangement of the 99-loop. J Biol Chem 278, 4121–4126 (2003). g p 33. Johnson, D. J., Li, W., Adams, T. E. & Huntington, J. A. Antithrombin-S195A factor Xa-heparin structure reveals the allosteric mechanism of antithrombin activation. The EMBO journal 25, 2029–2037 (2006).f h j 4. Bradbury, A. R., Sidhu, S., Dubel, S. & McCafferty, J. Beyond natural antibodies: the power of in vitro display technologies. Nature biotechnology 29, 245–254 (2011). gy 35. Mueller, U. et al. Facilities for macromolecular crystallography at the Helmholtz-Zentrum Berlin. Journal of synchrotron radiation 19, 442–449 (2012). 36. Kabsch, W. Xds. Acta Crystallogr D Biol Crystallogr 66, 125–132 (2010). y g y g 37. Winn, M. D. et al. Overview of the CCP4 suite and current developments. Acta Crystallogr D Biol Crystallogr 67, 235–242 (2011). 38. McCoy, A. J., Grosse-Kunstleve, R. W., Adams, P. D., Winn, M. D., Storoni, L. C. & Read, R. J. Phaser crystallographic software. Journal of applied crystallography 40, 658–674 (2007). 37. Winn, M. D. et al. Overview of the CCP4 suite and current developments. Acta Crystallogr D Biol Crystallogr 67, 235–242 (2011). 38. Additional Information Accession codes: Structure factors and coordinates of the complex structure Nb14:Furin as well as of the structure of the isolated Nb14 have been deposited in the protein databank (PDB) with accession codes 5JMO and 5JMR, respectively. Accession codes: Structure factors and coordinates of the complex structure Nb14:Furin as well as of the structure of the isolated Nb14 have been deposited in the protein databank (PDB) with accession codes 5JMO and 5JMR, respectively. Supplementary information accompanies this paper at http://www.nature.com/srep Supplementary information accompanies this paper at http://www.nature.com/srep Competing financial interests: The authors declare no competing financial interests. How to cite this article: Dahms, S. O. et al. The structure of a furin-antibody complex explains non-competitive nhibition by steric exclusion of substrate conformers. Sci. Rep. 6, 34303; doi: 10.1038/srep34303 (2016). How to cite this article: Dahms, S. O. et al. The structure of a furin-antibody complex explains non-competitive inhibition by steric exclusion of substrate conformers. Sci. Rep. 6, 34303; doi: 10.1038/srep34303 (2016). 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https://openalex.org/W2130036919	https://lilloa.univ-lille.fr/bitstream/20.500.12210/72523/3/https%3a//hal.archives-ouvertes.fr/hal-01150899/document	English	null	Early spring mesopelagic carbon remineralization and transfer efficiency in the naturally iron-fertilized Kerguelen area	Biogeosciences	2,015	cc-by	18,687	To cite this version: S. H. M. Jacquet, Frank Dehairs, A. Lefevre, Anne-Julie Cavagna, Frédéric Planchon, et al.. Early spring mesopelagic carbon remineralization and transfer eﬀiciency in the naturally iron-fertilized Ker- guelen area. Biogeosciences, European Geosciences Union, 2015, 12 (6), pp.1713-1731. 10.5194/bg- 12-1713-2015. hal-01150899 Early spring mesopelagic carbon remineralization and transfer eﬀiciency in the naturally iron-fertilized Kerguelen area S. H. M. Jacquet, Frank Dehairs, A. Lefevre, Anne-Julie Cavagna, Frédéric Planchon, Urania Christaki, L. Monin, Luc André, Ivia Closset, Damien Cardinal S. H. M. Jacquet1, F. Dehairs2, D. Lefèvre1, A. J. Cavagna2, F. Planchon3, U. Christaki4, L. Monin5, L. André5, I. Closset6, and D. Cardinal6 1Aix Marseille Université, CNRS/INSU, IRD, Mediterranean Institute of Oceanography (MIO), UM110, 13288 Marseille, France 2Vrije Universiteit Brussel, Analytical, Environmental & Geo-Chemistry and Earth System Sciences, Brussels, Belgium 3Laboratoire des Sciences de l’Environnement Marin (LEMAR), Université de Brest, CNRS, IRD, UMR6539, IUEM; Technopôle Brest Iroise, Place Nicolas Copernic, 29280 Plouzané, France 13288 Marseille, France 2Vrije Universiteit Brussel, Analytical, Environmental & Geo-Chemistry and Earth System Sciences, Brussels, Belgium 3Laboratoire des Sciences de l’Environnement Marin (LEMAR), Université de Brest, CNRS, IRD, UMR6539, IUEM; Technopôle Brest Iroise, Place Nicolas Copernic, 29280 Plouzané, France 2Vrije Universiteit Brussel, Analytical, Environmental & Geo-Chemistry and Earth System Sciences, Brussels, Belgium 3Laboratoire des Sciences de l’Environnement Marin (LEMAR), Université de Brest, CNRS, IRD, UMR6539, IUEM; Technopôle Brest Iroise, Place Nicolas Copernic, 29280 Plouzané, France p p 4INSU-CNRS, UMR8187 LOG, Laboratoire d’Océanologie et de Géosciences, Université du Littoral Côte d’Opale, ULCO, 32 avenue Foch, 62930 Wimereux, France 5 p p 4INSU-CNRS, UMR8187 LOG, Laboratoire d’Océanologie et de Géosciences, Université du Littoral Côte d’Opale, ULCO, 32 avenue Foch, 62930 Wimereux, France 5 5Earth Sciences Department, Royal Museum for Central Africa, Leuvensesteenweg 13, Tervuren, 3080, Belgium 6Sorbonne Universités (UPMC, Univ Paris 06)-CNRS-IRD-MNHN, LOCEAN Laboratory, 4 place Jussieu, 75005 Paris, France 5Earth Sciences Department, Royal Museum for Central Africa, Leuvensesteenweg 13, Tervuren, 3080, Belgium 6Sorbonne Universités (UPMC, Univ Paris 06)-CNRS-IRD-MNHN, LOCEAN Laboratory, 4 place Jussieu, 75005 Paris, France 5Earth Sciences Department, Royal Museum for Central Africa, Leuvensesteenweg 13, Tervuren, 3080, Belgium 6Sorbonne Universités (UPMC, Univ Paris 06)-CNRS-IRD-MNHN, LOCEAN Laboratory, 4 place Jussieu, 75005 Paris France Correspondence to: S. H. M. Jacquet (stephanie.jacquet@mio.osupytheas.fr) Correspondence to: S. H. M. Jacquet (stephanie.jacquet@mio.osupytheas.fr) Received: 6 May 2014 – Published in Biogeosciences Discuss.: 16 June 2014 Received: 6 May 2014 – Published in Biogeosciences Discuss.: 16 June 2014 Revised: 13 February 2015 – Accepted: 23 February 2015 – Published: 17 March 2015 Received: 6 May 2014 – Published in Biogeosciences Discuss.: 16 June 2014 Revised: 13 February 2015 – Accepted: 23 February 2015 – Published: 17 March 2015 y g Revised: 13 February 2015 – Accepted: 23 February 2015 – Published: 17 March 2015 Abstract. We report on the zonal variability of mesopelagic particulate organic carbon remineralization and deep car- bon transfer potential during the Kerguelen Ocean and Plateau compared Study 2 expedition (KEOPS 2; October– November 2011) in an area of the polar front support- ing recurrent massive blooms from natural Fe fertilization. S. H. M. Jacquet1, F. Dehairs2, D. Lefèvre1, A. J. Cavagna2, F. Planchon3, U. Christaki4, L. Monin5, L. André5, I. Closset6, and D. Cardinal6 Mesopelagic carbon remineralization (MR) was assessed us- ing the excess, non-lithogenic particulate barium (Baxs) in- ventories in mesopelagic waters and compared with bacterial production (BP), surface primary production (PP) and ex- port production (EP). Results for this early season study are compared with the results obtained during a previous study (2005; KEOPS 1) for the same area at a later stage of the phytoplankton bloom. Our results reveal the patchiness of the seasonal advancement and of the establishment of remineral- ization processes between the plateau (A3) and polar front sites during KEOPS 2. For the Kerguelen plateau (A3 site) we observe a similar functioning of the mesopelagic ecosys- tem during both seasons (spring and summer), with low and rather stable remineralization ﬂuxes in the mesopelagic col- umn (150–400 m). The shallow water column (∼500 m), the lateral advection, the zooplankton grazing pressure and the pulsed nature of the particulate organic carbon (POC) trans- fer at A3 seem to drive the extent of MR processes on the plateau. For deeper stations (> 2000 m) located on the mar- gin, inside a polar front meander, as well as in the vicinity of the polar front, east of Kerguelen, remineralization in the upper 400 m in general represents a larger part of surface car- bon export. However, when considering the upper 800 m, in some cases, the entire ﬂux of exported carbon is remineral- ized. In the polar front meander, where successive stations form a time series, two successive events of particle transfer were evidenced by remineralization rates: a ﬁrst mesopelagic and deep transfer from a past bloom before the cruise, and a second transfer expanding at mesopelagic layers during the cruise. Regarding the deep carbon transfer efﬁciency, it ap- peared that above the plateau (A3 site) the mesopelagic rem- ineralization was not a major barrier to the transfer of organic matter to the seaﬂoor (close to 500 m). There, the efﬁciency of carbon transfer to the bottom waters (> 400 m) as assessed by PP, EP and MR ﬂuxes comparisons reached up to 87 % of the carbon exported from the upper 150 m. In contrast, at the deeper locations, mesopelagic remineralization clearly lim- ited the transfer of carbon to depths of > 400 m. For sites at the margin of the plateau (station E-4W) and the polar front (station F-L), mesopelagic remineralization even exceeded HAL Id: hal-01150899 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 Distributed under a Creative Commons Attribution\| 4.0 International License Biogeosciences, 12, 1713–1731, 2015 www.biogeosciences.net/12/1713/2015/ doi:10.5194/bg-12-1713-2015 © Author(s) 2015. CC Attribution 3.0 License. 1 Introduction While numerous artiﬁcial (Boyd et al., 2000, 2004; Gervais et al., 2002; Buesseler et al., 2004, 2005; de Baar et al., 2005; Hoffmann et al., 2006; Boyd et al., 2012; Smetacek et al., 2012) and natural (Blain et al., 2007; Pollard et al., 2009; Zhou et al., 2010, 2013) ocean iron-fertilization experiments in the Southern Ocean have demonstrated the role of iron in enhancing the phytoplankton biomass and production in high-nutrient low-chlorophyll (HNLC) regions, determining to what extent fertilization could modify the transfer of par- ticulate organic carbon (POC) to the deep ocean is far from being comprehensively achieved (Lampitt et al., 2008; Mor- ris and Charette, 2013; Le Moigne et al., 2014; Robinson et al., 2014). This is partly due to the short term over which the observations were made, precluding extrapolation to longer timescales. Moreover, when assessing whether Fe-supply could induce vertical POC transfer, the magnitude of the ex- port from the surface is not the only important parameter to take into account. Indeed, POC fate in the mesopelagic zone (deﬁned as the 100–1000 m depth layer) is often largely over- looked although these depth layers are responsible for the remineralization of most of the POC exported from the sur- face layer (Martin et al., 1987; Longhurst, 1990; Lampitt and Antia, 1997; François et al., 2002; Buesseler et al., 2007b; Buesseler and Boyd, 2009). Only few studies have consid- ered mesopelagic carbon (C) remineralization rates (Bues- seler et al., 2007a; Jacquet et al., 2008a, b, 2011a, b; Salter et al., 2007) to estimate the response of deep POC export to fertilization. Assessing mesopelagic C remineralization is pivotal for evaluating remineralization length scale as well as the timescale of the C storage in the deep ocean. Indeed the typical depth of the main thermocline, 1000 m (IPCC, WG1, 2007, chapter 5) is often referred to as the horizon, clearly re- moved from the surface ocean and atmosphere (Passow and Carlson, 2012). Overall, assessing mesopelagic C reminer- alization will allow one to better quantify the ocean’s bio- logical carbon pump and its efﬁciency in the global C cycle which holds large uncertainty and is currently under debate (e.g. from 5 Gt yr−1 in Henson et al., 2011 to 21 Gt C yr−1 in Laws et al., 2000 and 13 Gt yr−1 in the IPCC WG1 re- port (2013, chapter 6)). Published by Copernicus Publications on behalf of the European Geosciences Union. S. H. M. Jacquet et al.: Early season mesopelagic carbon remineralization 1714 upper 150 m export, resulting in a zero transfer efﬁciency to depths > 800 m. In the polar front meander (time series), the capacity of the meander to transfer carbon to depth > 800 m was highly variable (0 to 73 %). The highest carbon transfer efﬁciencies in the meander are furthermore coupled to in- tense and complete deep (> 800 m) remineralization, result- ing again in a near-zero, deep (> 2000 m) carbon sequestra- tion efﬁciency there. ineralization and zonal variability in the Kerguelen area (Southern Ocean). Here, C remineralization was assessed from particulate biogenic Ba (hereafter called excess Ba or Baxs; mainly forms as barite BaSO4 crystals) contents in the mesopelagic water column. The link between barite and C remineralization depends on the fact that this mineral precip- itates in oversaturated micro-environments (biogenic aggre- gates) during the process of prokaryotic degradation of sink- ing POC (Dehairs et al., 1980, 1992, 1997, 2008; Stroobants et al., 1991, Cardinal et al., 2001, 2005; Jacquet et al., 2007, 2008b, 2011a; Planchon et al., 2013; Sternberg et al., 2007, 2008a, 2008b). Once the aggregates have been remineral- ized, barites are released and spread through the mesopelagic layer. Overall, earlier work highlights the fact that suspended barite in mesopelagic waters builds up over the growing sea- son and reﬂects past remineralization activity integrated over several days to weeks (Dehairs et al., 1997; Cardinal et al., 2005; Jacquet et al., 2007, 2008b). An algorithm linking mesopelagic Baxs contents to oxygen consumption (Shopova et al., 1995; Dehairs et al., 1997) allowed remineralization of POC ﬂuxes to be estimated for the mesopelagic layer. Combined with surface C production and export estimates, mesopelagic Baxs also highlights the deep carbon transfer ef- ﬁciency of the system. From earlier studies, the efﬁciency of C transfer through the mesopelagic layer was reported to in- crease under artiﬁcially induced (EIFEX; Strass et al., 2005; Smetacek et al., 2012) and natural (KEOPS; Blain et al., 2007) Fe-replete conditions (Jacquet et al., 2008a, b; Savoye et al., 2008) compared to Fe-limited, non-bloom HNLC ref- erence stations in the Southern Ocean. In contrast, C transfer efﬁciency through the mesopelagic layer was reported to be lower in natural Fe-replete locations during the SAZ-Sense (Sub-Antarctic Zone Sensitivity to Environmental Change) cruise off Tasmania (Jacquet et al., 2011a, b). Published by Copernicus Publications on behalf of the European Geosciences Union. ﬂagellates, type of diatoms) were suggested as possible causes of such discrepancies in C transfer efﬁciency through the mesopelagic layer (Jacquet et al., 2008a, 2011a, b). Also, differences in integration timescales for the processes that control the carbon ﬂuxes in artiﬁcially vs. naturally Fe fertil- ized systems may yield an incomplete picture of the C trans- fer potential and lead to misleading conclusions. Published by Copernicus Publications on behalf of the European Geosciences Union. Differences in plankton community structure and composition (e.g. diatoms vs. ﬂagellates, type of diatoms) were suggested as possible causes of such discrepancies in C transfer efﬁciency through the mesopelagic layer (Jacquet et al., 2008a, 2011a, b). Also, differences in integration timescales for the processes that control the carbon ﬂuxes in artiﬁcially vs. naturally Fe fertil- ized systems may yield an incomplete picture of the C trans- fer potential and lead to misleading conclusions. Here we examine changes in mesopelagic POC reminer ineralization and zonal variability in the Kerguelen area (Southern Ocean). Here, C remineralization was assessed from particulate biogenic Ba (hereafter called excess Ba or Baxs; mainly forms as barite BaSO4 crystals) contents in the mesopelagic water column. The link between barite and C remineralization depends on the fact that this mineral precip- itates in oversaturated micro-environments (biogenic aggre- gates) during the process of prokaryotic degradation of sink- ing POC (Dehairs et al., 1980, 1992, 1997, 2008; Stroobants et al., 1991, Cardinal et al., 2001, 2005; Jacquet et al., 2007, 2008b, 2011a; Planchon et al., 2013; Sternberg et al., 2007, 2008a, 2008b). Once the aggregates have been remineral- ized, barites are released and spread through the mesopelagic layer. Overall, earlier work highlights the fact that suspended barite in mesopelagic waters builds up over the growing sea- son and reﬂects past remineralization activity integrated over several days to weeks (Dehairs et al., 1997; Cardinal et al., 2005; Jacquet et al., 2007, 2008b). An algorithm linking mesopelagic Baxs contents to oxygen consumption (Shopova et al., 1995; Dehairs et al., 1997) allowed remineralization of POC ﬂuxes to be estimated for the mesopelagic layer. Combined with surface C production and export estimates, mesopelagic Baxs also highlights the deep carbon transfer ef- ﬁciency of the system. From earlier studies, the efﬁciency of C transfer through the mesopelagic layer was reported to in- crease under artiﬁcially induced (EIFEX; Strass et al., 2005; Smetacek et al., 2012) and natural (KEOPS; Blain et al., 2007) Fe-replete conditions (Jacquet et al., 2008a, b; Savoye et al., 2008) compared to Fe-limited, non-bloom HNLC ref- erence stations in the Southern Ocean. In contrast, C transfer efﬁciency through the mesopelagic layer was reported to be lower in natural Fe-replete locations during the SAZ-Sense (Sub-Antarctic Zone Sensitivity to Environmental Change) cruise off Tasmania (Jacquet et al., 2011a, b). Differences in plankton community structure and composition (e.g. diatoms vs. www.biogeosciences.net/12/1713/2015/ 1 Introduction Here, we examine changes in mesopelagic POC reminer- alization during the early spring (October–November 2011) KEOPS 2 expedition to the naturally iron-fertilized area east of Kerguelen Islands. The hydrographic structure of the Ker- guelen area generates contrasted environments that are dif- ferently impacted by iron availability and mesoscale activ- ity. The speciﬁc objectives of the present work are to as- sess the zonal variability of mesopelagic C remineraliza- tion and deep C transfer potential, and to identify possible causes of this variability. The same area was visited earlier in 2005 during summer at a late stage of the bloom (KEOPS 1; January–February 2005), offering a unique opportunity to es- timate the main carbon ﬂuxes over most of the growth sea- The present work aims at understanding the impact of a natural iron-induced bloom on the mesopelagic POC rem- Biogeosciences, 12, 1713–1731, 2015 www.biogeosciences.net/12/1713/2015/ S. H. M. Jacquet et al.: Early season mesopelagic carbon remineralization Jacquet et al.: Early season mesopelagic carbon remineralization 1715 !" !"#$ #$"#%&"'()$"+,(-." /)0$1234-"'56." /,7234-"'58." %&'()$ %&'($ +,$ -(.$ /(0$ 1(21$ 1(23$ 1(4$ 567"8$-869:$;69<$ 567"8$-869:$$ +9:"8=>=$;69<$ ?<8@A<7<9$BC7"9D$ %13(E$ /)0$1234-"'56." /,7234-"'58." 1()$ 1(0$ 1(,$ %&'(E$ %13(,$ !F#$ 9)(,:";:)02"" 567"8$-869:$;69<$ +9:"8=>=$;69<$ <" =" &" >" ?" ;,@0"A:)3$B" C3::-02" Figure 1. (a) Kerguelen Island area in the Southern Ocean with KEOPS 2 sampling zones and MODIS chlorophyll concentrations (mg m−3) (Chl a map from 11 November 2011, courtesy of F. d’Ovidio) superimposed. 1 refers to station A3; 2 to stations E; 3 to the south–north transect; 4 to the west–east transect; 5 to station F-L and 6 to reference station R-2; (b) Corresponding stations location. Colours indicate stations with similar θ −S and Chl a characteristics. !" !"#$ #$"#%&"'()$"+,(-." /)0$1234-"'56." /,7234-"'58." 9)(,:";:)02"" 567"8$-869:$;69<$ +9:"8=>=$;69<$ <" =" &" >" ?" ;,@0"A:)3$B" C3::-02" %&'()$ %&'($ +,$ -(.$ /(0$ 1(21$ 1(23$ 1(4$ 567"8$-869:$;69<$ 567"8$-869:$$ +9:"8=>=$;69<$ ?<8@A<7<9$BC7"9D$ %13(E$ /)0$1234-"'56." /,7234-"'58." 1()$ 1(0$ 1(,$ %&'(E$ %13(,$ !F#$ Figure 1. (a) Kerguelen Island area in the Southern Ocean with KEOPS 2 sampling zones and MODIS chlorophyll concentrations (mg m−3) (Chl a map from 11 November 2011, courtesy of F. d’Ovidio) superimposed. 1 refers to station A3; 2 to stations E; 3 to the south–north transect; 4 to the west–east transect; 5 to station F-L and 6 to reference station R-2; (b) Corresponding stations location. Colours indicate stations with similar θ −S and Chl a characteristics. 1 Introduction of the study area revealed the presence of different Chl a rich plumes (D’Ovidio et al., 2015) (Fig. 1a; e.g. Chl a map from 11 November 2011). Stations were sampled in distinct zones covering these different bloom patterns (Fig. 1a) (cor- responding stations are reported in Fig. 1b): (a) on the shal- low plateau (station A3; see 1 in Fig. 1a). Note that station A3 coincides with a site studied during the KEOPS 1 cruise, and that it was sampled twice over a 27-day period; (b) in a meander formed by a quasi-permanent retroﬂection of the polar front (PF) and topographically steered by the eastern escarpment (Gallieni Spur) of the Kerguelen Plateau (mainly stations E, sampled as a quasi-Lagrangian temporal series) (see 2 in Fig. 1a); (c) along a north–south transect (referred to as TNS stations; see 3, grey line in Fig. 1a) and a east– west transect (referred to as TEW stations; see 4, grey line in Fig. 1a), both crossing the PF; and (d) in the polar front zone (PFZ) in the vicinity (east) of the PF (station F-L; see 5 in Fig. 1a). Furthermore we also sampled a reference HNLC, non-bloom, non-Fe-fertilized station southwest of the plateau (station R-2; see 6 in Fig. 1a). Station locations are given in Table 1. son. Mesopelagic C remineralization estimates are compared to particle and biological parameters as reported in other papers included in this issue (Cavagna et al., 2014; Chris- taki et al., 2014; Dehairs et al., 2014; Lasbleiz et al., 2014; Laurenceau-Cornec et al., 2015; Planchon et al., 2014; van der Merve et al., 2015) and in Blain et al. (2007), Christaki et al. (2008), Jacquet et al. (2008a), Park et al. (2008) and Savoye et al. (2008). www.biogeosciences.net/12/1713/2015/ 2.1 Study area (a) Potential temperature θ–salinity S plots and isopycnals for KEOPS 2 proﬁles, (b) Focus on the upper 200 m water column. AASW: Antarctic Surface Waters, AAIW: Antarctic Intermediate Waters, WW: winter waters, UCDW and LCDW: Upper and Lower Cir- cumpolar Deep Water, AABW: Antarctic Bottom Water. Graph constructed using Ocean Data View (Schlitzer, 2002; Ocean Data View; http://www.awi-bremerhaven.de/GEO/ODV). ((/'$ 12324$ /56789:$ .26;<$ /25;<$ =>?'$ @>?'$ ((1'$ ''$ C"B$),$ C"B$)B+$ -./"$ D"@$)A0$ D"@$$)A&$ -!'"0$ /8EFGF;H$ $$$$$$$$$$$$$$$$$$I2;:GJ8E$;:4K:68;56:$ L8M$ !"#$ !"%!$ !"&$ !"%'$ (&$)+,$ (&$)%$ -./"0$ -./"A$ -!'"&$ !"$)B,$ !"$)&+$ /8EFGF;H$ $$$$$$$$I2;:GJ8E$;:4K:68;56:$ LNM$ $$I2;:GJ8E$;:4K:68;56:$ L8M$ LNM$ Figure 2. (a) Potential temperature θ–salinity S plots and isopycnals for KEOPS 2 proﬁles, (b) Focus on the upper 200 m water column. AASW: Antarctic Surface Waters, AAIW: Antarctic Intermediate Waters, WW: winter waters, UCDW and LCDW: Upper and Lower Cir- cumpolar Deep Water, AABW: Antarctic Bottom Water. Graph constructed using Ocean Data View (Schlitzer, 2002; Ocean Data View; http://www.awi-bremerhaven.de/GEO/ODV). Table 1. Station locations, cast numbers and bottom depths during KEOPS 2. Depth-weighted average (DWA) values of mesopelagic Baxs- (pM) and Baxs-based mesopelagic POC remineralization (MR; mgCm−2 day−1) integrated between 150–400 and 150–800 m depths. See text for further information on calculation. Table 1. Station locations, cast numbers and bottom depths during KEOPS 2. Depth-weighted average (DWA) values of mesopelagic Baxs- (pM) and Baxs-based mesopelagic POC remineralization (MR; mgCm−2 day−1) integrated between 150–400 and 150–800 m depths. See text for further information on calculation. Table 1. Station locations, cast numbers and bottom depths during KEOPS 2. Depth-weighted average (DWA) values of mesopelagic Baxs- (pM) and Baxs-based mesopelagic POC remineralization (MR; mgCm−2 day−1) integrated between 150–400 and 150–800 m depths. See text for further information on calculation. Station CTD Long Lat Date of Seaﬂoor DWA DWA MR MR Std. MR MR Std. cast no. 2.1 Study area The KEOPS 2 (Kerguelen Ocean and Plateau compared Study) cruise was conducted in austral spring at the onset of the bloom from 10 October to 20 November aboard the R/V Marion Dufresne (TAAF/IPEV). The KEOPS 2 expe- dition studied the Kerguelen Plateau area (Indian sector of the Southern Ocean) which is characterized by the passage of the polar front (PF), as illustrated in Fig. 1a. The Ker- guelen Plateau is surrounded by the Antarctic Circumpolar Current (ACC) whose main branch circulates to the north of the plateau (Park et al., 2008). A second branch of the ACC circulates to the south of Kerguelen Islands to later join a branch of the Fawn Trough Current (FTC). The FTC has a main northeast direction, but a minor branch splits away northwestward to join the eastern side of the Kergue- len Plateau (Park et al., 2008; Fig. 1a). These particular hy- drographic features generate a mosaic of recurrent massive bloom patterns in the northeastern part of the plateau, and the possible sources and mechanisms for fertilization were investigated during ANTARES 3 (1995; Blain et al., 2001) and the KEOPS 1 cruise (January–February 2005, late sum- mer conditions; Blain et al., 2007, 2008). During KEOPS 2 the evolution of Chl a data based on multi-satellite imagery Detailed descriptions of the complex physical structure of the area, circulation, water masses and fronts are given in Park et al. (2014). Brieﬂy, the main hydrodynamic features observed during the cruise are the following (see θ −S di- agram, Fig. 2a): (1) north of the PF, stations in the PFZ (TNS-1, TEW-8 and F-L) present Antarctic Surface Wa- ters (AASW; θ ≃4 ◦C and density < 27); θ −S character- istics between 150 to 400 m at station F-L (and to a lesser extent at station TNS-1) reveal the presence of interleav- ing with waters from northern (subantarctic) origin, cen- tred between the 27.2 and 27.5 kg m−3 density curves, where Antarctic Intermediate Waters (AAIW) are usually found. This contrasts with the conditions at station TEW-8, where there is no evidence of interleaving; (2) stations south of www.biogeosciences.net/12/1713/2015/ Biogeosciences, 12, 1713–1731, 2015 S. H. M. Jacquet et al.: Early season mesopelagic carbon remineralization 1716 !"#$ !"%!$ !"&$ !"%'$ (&$)+,$ (&$)%$ -./"0$ ((/'$ 12324$ /56789:$ .26;<$ /25;<$ =>?'$ @>?'$ ((1'$ ''$ -./"A$ -!'"&$ !"$)B,$ !"$)&+$ C"B$),$ C"B$)B+$ -./"$ D"@$)A0$ D"@$$)A&$ -!'"0$ /8EFGF;H$ /8EFGF;H$ $$$$$$$$$$$$$$$$$$I2;:GJ8E$;:4K:68;56:$ $$$$$$$$I2;:GJ8E$;:4K:68;56:$ L8M$ LNM$ Figure 2. 2.2 Sampling and analyses The rate of oxygen consumption and particulate organic car- bon remineralization rate in the mesopelagic layer (later re- ferred to as MR) can be estimated using an algorithm relating mesopelagic Baxs contents and oxygen consumption based on earlier observations in the Southern Ocean (Shopova et al., 1995; Dehairs et al., 1997, 2008). The detailed calcula- tions are described in Jacquet et al. (2008a, 2011a). Brieﬂy, we use the following equations: Twenty-two CTD (conductivity, temperature, and depth) casts (surface to 500–2000 m) were sampled for particu- late barium (Table 1) using a CTD rosette equipped with twenty-two 12 L Niskin bottles. Deep particulate Ba proﬁles (> 1000 m) were not systematically obtained from the same CTD cast, but from successive casts sampled closely in time and space and having similar θ −S data proﬁles. In the fol- lowing, we use both the station and CTD numbers to refer to stations. JO−2 = (Baxs −Baresidual)/17 450, (1) Crespired = Z × JO2 × RR, (2) (1) (2) Four to 7 L of seawater were ﬁltered onto 47 mm polycar- bonate membranes (0.4 µm porosity) under slight overpres- sure supplied by ﬁltered air (0.4 µm). The ﬁlters were rinsed with Milli-Q grade water (< 5 mL) to remove sea salt, dried (at 50 ◦C) and stored in Petri dishes for later analysis. In the laboratory, we performed a total digestion of samples using a tri-acid (0.5 mL HF / 1.5 mL HCl / 1 mL HNO3; all Suprapur grade) mixture in closed Teﬂon beakers overnight at 90 ◦C in a clean pressurized room. After evaporating until nearly dry, samples were re-dissolved into around 13 mL of 2 % HNO3. The solutions were analysed for Ba and other major and mi- nor elements using a ICP-QMS (inductively coupled plasma quadrupole mass spectrometer; X Series 2 Thermo Fisher) equipped with collision cell technology (CCT). To correct instrumental drift and matrix effects, internal standards and matrix-matched calibrations were used. We analysed several certiﬁed reference materials which consisted of dilute acid- digested rocks (BHVO-1, JB-3 and JGb-1), natural river- ine water (SLRS-5) and multi-element artiﬁcial solutions for these external calibrations. Based on analyses of these ex- ternal standards, accuracy and reproducibility are both bet- ter than ±5 %. For more details on sample processing and analysis we refer to Cardinal et al. (2001). Among all ele- ments analysed, we were particularly interested in Ba and Al. S. H. M. Jacquet et al.: Early season mesopelagic carbon remineralization S. H. M. Jacquet et al.: Early season mesopelagic carbon remineralization Based on the θ −S characteristics (Fig. 2a, b) and surface phytoplankton biomasses, we can schematically group the stations as follows. The R-2 HNLC reference station (white dot in Fig. 1b) is characterized by a very low biomass (with low iron contents; Quéroué et al., 2015). Stations TEW-3 and TNS-8 (black dots) are characterized by low to moderate biomass and Fe contents. Stations A3 and E-4W (red dots; south of the PF) as well as stations TNS-1, F-L and TEW-8 (blue dots; north of the PF) are characterized by high biomass and iron contents. Stations in the core of the PF meander (green dots; stations TNS-6, E-1, E-2, E-3, E-4E and E-5 considered as a temporal series) are characterized by mod- erate biomass and iron contents. from around 5 L of Milli-Q water, dissolved in a ﬁnal vol- ume of 13 mL as for the samples. Biogenic barium (hereafter called excess Ba or Baxs) was calculated as the difference be- tween total particulate Ba and lithogenic Ba using Al as the lithogenic reference element (Dymond et al., 1992; Taylor and McLennan, 1985). At most sites and depths, the biogenic Baxs represented > 95 % of total particulate Ba. Lithogenic Ba reached up to 20 % of total particulate Ba at some depths in the upper 80–100 m mainly at station R-2 and stations north of the polar front (i.e. TEW-8, F-L and TNS-1). The standard uncertainty (Ellison et al., 2000) of Baxs data ranges between 5 and 5.5 %. Baxs and Al data are reported in Ap- pendix A. 2.2 Sampling and analyses The presence of sea salt was checked by analysing Na and the sea-salt particulate Ba contribution was found to be negligible. Average detection limits equal 0.6 nM for Al and 3 pM for Ba. Detection limits were calculated as 3 times the standard deviation of the blank measured on board and then normalized to an average dilution factor of 385, i.e. particles (2) where JO2 is the O2 consumption (µmol L−1 day−1) and Crespired is the mineralization rate of organic carbon (in mmol C m−2 day−1; MR); Baxs is the depth-weighted average (DWA) Baxs value, i.e. the Baxs inventory divided by the depth layer considered Z, Baresidual is the residual Baxs sig- nal (or Baxs background) at zero oxygen consumption and RR is the Redﬁeld C / O2 molar ratio (127/175; Broecker et al., 1985). DWA Baxs values were calculated for both layers at 150 to 400 m (plateau and deep stations) and 150 to 800 m (deep stations only; see details further below). The residual Baxs is considered as “preformed” Baxs, left over after par- tial dissolution and sedimentation of Baxs produced during a previous phytoplankton growth event. In BaSO4, saturated waters, such as the ones ﬁlling the whole ACC water col- umn (Monnin et al., 1999), this background Baxs value was considered to reach 180 to 200 pM, which is rather character- istic for the deep ocean (> 1000 m) (see Dehairs et al., 1997; Jacquet et al., 2008a, 2011). In the present study we used a Baxs background of 180 pM. where JO2 is the O2 consumption (µmol L−1 day−1) and Crespired is the mineralization rate of organic carbon (in mmol C m−2 day−1; MR); Baxs is the depth-weighted average (DWA) Baxs value, i.e. the Baxs inventory divided by the depth layer considered Z, Baresidual is the residual Baxs sig- nal (or Baxs background) at zero oxygen consumption and RR is the Redﬁeld C / O2 molar ratio (127/175; Broecker et al., 1985). DWA Baxs values were calculated for both layers at 150 to 400 m (plateau and deep stations) and 150 to 800 m (deep stations only; see details further below). The residual Baxs is considered as “preformed” Baxs, left over after par- tial dissolution and sedimentation of Baxs produced during a previous phytoplankton growth event. 2.1 Study area (◦E) (◦S) sampling [m] Baxs[pM] Baxs[pM] 150–400 m Uncertainty 150–800 m Uncertainty 150–400 m 150–800 m [mgCm−2 day−1] % [mgCm−2 day−1] % Plateau A3-1 4a 72.080 50.629 20 Oct 2011 530 316 – 14 4 – – A3-2 107a 72.056 50.624 16 Nov 2011 527 267 – 11 5 – – TEW-3 38 71.018 48.799 31 Oct 2011 560 324 – 28 8 – – Meander time series TNS-6 10 72.277 48.779 22 Oct 2011 1885 427 389 31 7 69 17 E-1 27/30 72.187 48.458 29, 30 Oct 2011 2056 387 325 26 6 48 14 E-2 43 72.077 48.523 1 Nov 2011 2003 301 309 15 5 42 13 E-3 50/55 71.967 48.702 3, 4 Nov 2011 1915 258 286 10 4 35 12 E-4E 94/97 72.563 48.715 13, 14 Nov 2011 2210 395 357 27 7 58 15 E-5 113/114 71.900 48.412 18 Nov 2011 1920 402 380 28 7 66 17 Polar front zone TNS-1 15 71.501 46.833 23 Oct 2011 2280 350 315 22 6 45 14 TEW-8 47 74.999 48.471 2 Nov 2011 2786 199 240 2 4 20 11 F-L 63/68 74.659 48.532 6, 7 Nov 2011 2695 345 328 21 6 49 14 Polar front E-4W 81/87 71.425 48.765 11, 12 Nov 2011 1384 468 411 36 8 76 18 Antarctic zone R-2 (reference site) 17/20 66.717 50.359 25, 26 Oct 2011 2300 572 456 50 10 91 20 TNS-8 8 72.240 49.463 21 Oct 2011 1030 473 358 37 8 59 15 a Station A3 (CTD no. 4 and 107); integration up to 354 and 405 m. a Station A3 (CTD no. 4 and 107); integration up to 354 and 405 m. lar Deep Water (UCDW and LCDW), and the Antarctic Bot- tom Water (AABW). These water masses are present roughly in the following depth intervals: 700 m < UCDW < 1500 m; 1500 m < LCDW < 2500 m; AABW > 2500 m. the PF exhibit subsurface temperature minima character- istic of winter waters (WW); below the WW, three wa- ter masses can be identiﬁed, namely: the Upper (tempera- ture maximum) and Lower (salinity maximum) Circumpo- Biogeosciences, 12, 1713–1731, 2015 www.biogeosciences.net/12/1713/2015/ 1717 www.biogeosciences.net/12/1713/2015/ S. H. M. Jacquet et al.: Early season mesopelagic carbon remineralization few days to weeks for Baxs) and by the fact that KEOPS 1 occurred at the decline of the bloom (late summer; low organic substrates), which would explain the lower JO2 rates as estimated by the incubation method. zone from Niskin bottles and placed into 125 cm3 borosil- icate glass bottles according to the WOCE (World Ocean Circulation Experiment) procedure, and oxygen concentra- tion was determined by Winkler titrations using a photomet- ric endpoint detector (Williams and Jenkinson, 1982). By integrating DCR data with the water column, we estimated the rate of oxygen consumption (referred to as JO2-W). We compared JO2-W obtained from incubated oxygen samples with the rate of oxygen consumption based on KEOPS 1 mesopelagic Baxs contents (Eq. 1; later referred to as JO2- Ba). Dissolved oxygen was measured three times at station A3 (same location as during KEOPS2) over a 19-day period (A3 CTD no. 32, 74 and 119). Dissolved oxygen was also measured at station C11, located off-shelf in less productive HNLC waters (51.65◦S, 78.00◦E; not shown in Fig. 1) and was sampled two times over a 10-day period (C11 CTD no. 42 and 83). Figure 3 compares JO2-W and JO2-Ba for repeat stations A3 (no. 32, 74 and 119) and C11 (no. 42 and 83) (integration between 150–300 m). JO2-W rates range from 0.082 to 0.208 mmol m−2 day−1 at station A3 and from 0.292 to 0.528 mmol m−2 day−1 at station C11. Although JO2-Ba rates (from 0.846 to 1.555 mmol m−2 day−1) are slightly higher than JO2-W, JO2 rates are of the same order of mag- nitude and present the same trend. We observed a signiﬁ- cant positive correlation between the JO2 rates (R2 =0.90; p < 0.01), with a slope of 0.64. The difference in oxygen consumption rates can be explained by the integration time of both methods (a few hours for the incubations vs. few days to weeks for Baxs) and by the fact that KEOPS 1 occurred at the decline of the bloom (late summer; low organic substrates), which would explain the lower JO2 rates as estimated by the incubation method. mmol m-‐2 day-‐1 (150-‐300 m) Figure 3. Rates of oxygen consumption (mmol m−2 day−1) dur- ing KEOPS 1 as directly measured (JO2-W) and from mesopelagic Baxs contents (JO2-Ba). Rates are integrated between 150–300 m. degradation of sinking POC (Martin et al., 1987; Sarmiento et al., 1993; Buesseler et al., 2007b). S. H. M. Jacquet et al.: Early season mesopelagic carbon remineralization For KEOPS 2 we observed that Baxs concentrations generally increase below 150 m (i.e. they increase above the background level set at 180 pM), but some sites have ocean surface Baxs contents signiﬁcantly larger than background (E-1, 896 pM at 21 m; E4-E, 563 pM at 93 m). Such values are not unusual, and very high surface values have been observed occasionally in ear- lier Southern Ocean studies. During KEOPS 1, surface Baxs maxima at the three A3 repeat stations ranged from 1354 to 5930 pM at 50 m, likely associated with phytoplankton- derived particles (Jacquet et al., 2008a). Overall, these results highlight the need for further con- straining spatial and temporal variability of deep ocean oxy- gen utilization via a combination of direct rate measurements and the Baxs proxy. In the present work, O2 consumption and POC remineralization were assessed from Baxs inventories and Eqs. (1) and (2). C remineralization rates are given in Table 1. Relative standard uncertainties (Ellison et al., 2000) of C remineralization ranged between 4 and 20 %. The following part focuses on the mesopelagic zone where most of the remineralization of exported organic matter takes place. The Baxs proﬁle for station R-2 (CTD no. 17) dis- played a characteristic mesopelagic Baxs maximum, reaching up to 834 pM at 304 m, which is actually one of the highest values observed for the whole study (Fig. 4a). Baxs proﬁles for stations A3 above the Kerguelen Plateau (A3-1 CTD no. 4 and A3-2 CTD no. 107; Fig. 4b) had lower mesopelagic Baxs content, with values ranging from about 80 to 350 pM. For both A3 visits, Baxs values increased close to the seaﬂoor, reaching up to 1108 pM (A3-1, 474 m) and 1842 pM (A3-2, 513 m). In contrast, station E-4W (located further north along the margin in deeper waters, but with similar θ −S and Chl a characteristics as station A3) displayed a large mesopelagic Baxs maximum reaching up to 627 pM at 252 m (Fig. 4c). Station TEW-3 (located on the Kerguelen Plateau, in waters with similar θ −S and Chl a characteristics as station TNS- 8) had a proﬁle similar to the one observed at station A3-2, but compared to plateau sites A3-1 and A3-2, no increased Baxs contents were observed in bottom water (Fig. 4d). The other stations of the study area (Fig. 4d–g) have Baxs pro- 2.2 Sampling and analyses In BaSO4, saturated waters, such as the ones ﬁlling the whole ACC water col- umn (Monnin et al., 1999), this background Baxs value was considered to reach 180 to 200 pM, which is rather character- istic for the deep ocean (> 1000 m) (see Dehairs et al., 1997; Jacquet et al., 2008a, 2011). In the present study we used a Baxs background of 180 pM. We take the opportunity here to also compare O2 con- sumption rates for the KEOPS 1 expedition (D. Lefèvre, un- published data) with KEOPS 1 Baxs data published earlier (Jacquet et al., 2008a). No such O2 consumption data are available for KEOPS 2. During KEOPS 1, dark community respiration (DCR) was estimated from changes in the dis- solved oxygen concentration over 72 h incubations. Discrete samples were collected at three depths in the mesopelagic www.biogeosciences.net/12/1713/2015/ www.biogeosciences.net/12/1713/2015/ Biogeosciences, 12, 1713–1731, 2015 S. H. M. Jacquet et al.: Early season mesopelagic carbon remineralization 1718 R² = 0.90 y = 0.6443x -‐ 0.4867 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.2 0.7 1.2 1.7 2.2 2.7 JO2 -‐W JO2 -‐Ba mmol m-‐2 day-‐1 (150-‐300 m) C11 #42 C11 #83 A3 #74 A3 #119 A3 #32 Figure 3. Rates of oxygen consumption (mmol m−2 day−1) dur- ing KEOPS 1 as directly measured (JO2-W) and from mesopelagic Baxs contents (JO2-Ba). Rates are integrated between 150–300 m. R² = 0.90 y = 0.6443x -‐ 0.4867 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.2 0.7 1.2 1.7 2.2 2.7 JO2 -‐W JO2 -‐Ba mmol m-‐2 day-‐1 (150-‐300 m) C11 #42 C11 #83 A3 #74 A3 #119 A3 #32 zone from Niskin bottles and placed into 125 cm3 borosil- icate glass bottles according to the WOCE (World Ocean Circulation Experiment) procedure, and oxygen concentra- tion was determined by Winkler titrations using a photomet- ric endpoint detector (Williams and Jenkinson, 1982). By integrating DCR data with the water column, we estimated the rate of oxygen consumption (referred to as JO2-W). We compared JO2-W obtained from incubated oxygen samples with the rate of oxygen consumption based on KEOPS 1 mesopelagic Baxs contents (Eq. 1; later referred to as JO2- Ba). Dissolved oxygen was measured three times at station A3 (same location as during KEOPS2) over a 19-day period (A3 CTD no. 32, 74 and 119). Dissolved oxygen was also measured at station C11, located off-shelf in less productive HNLC waters (51.65◦S, 78.00◦E; not shown in Fig. 1) and was sampled two times over a 10-day period (C11 CTD no. 42 and 83). Figure 3 compares JO2-W and JO2-Ba for repeat stations A3 (no. 32, 74 and 119) and C11 (no. 42 and 83) (integration between 150–300 m). JO2-W rates range from 0.082 to 0.208 mmol m−2 day−1 at station A3 and from 0.292 to 0.528 mmol m−2 day−1 at station C11. Although JO2-Ba rates (from 0.846 to 1.555 mmol m−2 day−1) are slightly higher than JO2-W, JO2 rates are of the same order of mag- nitude and present the same trend. We observed a signiﬁ- cant positive correlation between the JO2 rates (R2 =0.90; p < 0.01), with a slope of 0.64. The difference in oxygen consumption rates can be explained by the integration time of both methods (a few hours for the incubations vs. S. H. M. Jacquet et al.: Early season mesopelagic carbon remineralization 4b), DWA Baxs was calculated for the layer between 150 and 354 m for A3-1 (CTD no. 4) and between 150 and 405 m for A3-2 (CTD no. 107). For station TEW-3 (CTD no. 38), DWA Baxs was calculated for the water layer between 150 and 400 m (Fig. 4d). For the deep sites, we considered both the 150–400 and the 150–800 m depth intervals when calculating the DWA Baxs contents. Depth-weighted aver- age Baxs values were translated into carbon remineralization rates using Eqs. (1) and (2) given above. These rates ranged from 2 to 91 mg C m−2 day−1 (Table 1). ﬁles similar to the one at station E-4W, showing the char- acteristic Baxs maximum between 200 and 500 m. Note that for most of the stations, Baxs concentrations in waters below the mesopelagic maximum did not systematically decrease to reach the Baxs background level (180 pM; see above). In some cases Baxs contents signiﬁcantly higher than residual Baxs were observed until below 1000 m (see Appendix A). This is particularly salient at stations TNS-6, E-1, E-2 and F- L where Baxs values below 1000 m reach 410 pM at 1886 m (TNS-6) and 436 pM at 1498 m (E-1). These cases of high, deep Baxs contents clearly contrasted with the values ob- served at station E4-E (Fig. 4h). DWA Baxs values range from 199 to 572 pM (Table 1) and ﬁt within the range reported for polar front areas dur- ing previous studies (Cardinal et al., 2001, 2005; Jacquet et al., 2005, 2008a, b, 2011; Planchon et al., 2013). For the KEOPS 2 cruise the main observed features are as follows: S. H. M. Jacquet et al.: Early season mesopelagic carbon remineralization S. H. M. Jacquet et al.: Early season mesopelagic carbon remineralization 1719 0 100 200 300 400 500 600 700 800 0 400 800 1200 Depth [m] Particulate biogenic Baxs [pM] A3-1 CTD 4 A3-2 CTD 107 BKG !"#"$% &'(% )%+"%,-.,%/% 0 100 200 300 400 500 600 700 800 0 400 800 1200 Depth [m] Particulate biogenic Baxs [pM] E-4E CTD 94 E-5 CTD 114 BKG &0(% 0 100 200 300 400 500 600 700 800 0 400 800 1200 Depth [m] Particulate biogenic Baxs [pM] E-1 CTD 27 E-2 CTD 43 E-3 CTD 50 TNS-6 CTD 10 BKG &1(% 0 100 200 300 400 500 600 700 800 0 400 800 1200 Depth [m] Particulate biogenic Baxs [pM] E-4W CTD 81 BKG &2(% 0 100 200 300 400 500 600 700 800 0 400 800 1200 Depth [m] Particulate biogenic Baxs [pM] TEW-8 CTD 47 F-L CTD 63 TNS-1 CTD 15 BKG &3(% 0 100 200 300 400 500 600 700 800 0 400 800 1200 Depth [m] Particulate biogenic Baxs [pM] TEW-3 CTD 38 TNS-8 CTD 8 BKG &4(% !"#"$% 0 100 200 300 400 500 600 700 800 0 400 800 1200 Depth [m] Particulate biogenic Baxs [pM] R-2 CTD 17 BKG &5(% 0 500 1000 1500 2000 2500 0 400 800 1200 Depth [m] Particulate biogenic Baxs [pM] TNS06 CTD 10 E1 CTD 27/30 E4-E CTD 94/97 BKG &6(% Figure 4. Particulate biogenic Baxs proﬁles (pM) in the upper 800 m (Fig. 4a–g) and in the upper 2500 m (Fig. 4h). Stations are identiﬁed by CTD cast numbers. BKG: Baxs background (180 pM). S. H. M. Jacquet et al.: Early season mesopelagic carbon remineralization 0 100 200 300 400 500 600 700 800 0 400 800 1200 Depth [m] Particulate biogenic Baxs [pM] R-2 CTD 17 BKG &5(% 0 100 200 300 400 500 600 700 800 0 400 800 1200 Depth [m] Particulate biogenic Baxs [pM] TEW-3 CTD 38 TNS-8 CTD 8 BKG &4(% !"#"$% Particulate biogenic Baxs [pM] 0 100 200 300 400 500 600 700 800 0 400 800 1200 Depth [m] Particulate biogenic Baxs [pM] E-4W CTD 81 BKG &2(% 0 100 200 300 400 500 600 700 800 0 400 800 1200 Depth [m] Particulate biogenic Baxs [pM] A3-1 CTD 4 A3-2 CTD 107 BKG !"#"$% &'(% )%+"%,-.,%/% Particulate biogenic Baxs [pM] Particulate biogenic Baxs [pM] Particulate biogenic Baxs [pM] 0 500 1000 1500 2000 2500 0 400 800 1200 Depth [m] Particulate biogenic Baxs [pM] TNS06 CTD 10 E1 CTD 27/30 E4-E CTD 94/97 BKG &6(% 0 100 200 300 400 500 600 700 800 0 400 800 1200 Depth [m] Particulate biogenic Baxs [pM] E-1 CTD 27 E-2 CTD 43 E-3 CTD 50 TNS-6 CTD 10 BKG &1(% 0 100 200 300 400 500 600 700 800 0 400 800 1200 Depth [m] Particulate biogenic Baxs [pM] E-4E CTD 94 E-5 CTD 114 BKG &0(% 0 100 200 300 400 500 600 700 800 0 400 800 1200 Depth [m] Particulate biogenic Baxs [pM] TEW-8 CTD 47 F-L CTD 63 TNS-1 CTD 15 BKG &3(% Particulate biogenic Baxs [pM] Particulate biogenic Baxs [pM] Particulate biogenic Baxs [pM] Depth [m] Figure 4. Particulate biogenic Baxs proﬁles (pM) in the upper 800 m (Fig. 4a–g) and in the upper 2500 m (Fig. 4h). Stations are identiﬁed by CTD cast numbers. BKG: Baxs background (180 pM). (Fig. 4b), DWA Baxs was calculated for the layer between 150 and 354 m for A3-1 (CTD no. 4) and between 150 and 405 m for A3-2 (CTD no. 107). For station TEW-3 (CTD no. 38), DWA Baxs was calculated for the water layer between 150 and 400 m (Fig. 4d). For the deep sites, we considered both the 150–400 and the 150–800 m depth intervals when calculating the DWA Baxs contents. Depth-weighted aver- age Baxs values were translated into carbon remineralization rates using Eqs. (1) and (2) given above. These rates ranged from 2 to 91 mg C m−2 day−1 (Table 1). (Fig. 3.1 Particulate biogenic Baxs proﬁles Baxs proﬁles in the upper 800 m are reported in Fig. 4. The complete whole water column data set is given in Ap- pendix A. From previous studies we know that Baxs in sur- face waters is distributed over different, mainly non-barite biogenic phases (see Stroobants et al., 1991; Jacquet et al., 2007; Cardinal et al., 2005; Sternberg et al., 2005). As such, these do not reﬂect POC remineralization processes, in con- trast to mesopelagic waters where Baxs is mainly composed of barite (Dehairs et al., 1980) formed during prokaryotic Biogeosciences, 12, 1713–1731, 2015 www.biogeosciences.net/12/1713/2015/ 3.2 Depth-weighted average Baxs content of mesopelagic waters Since the base of the mixed layer was generally shallower than 150 m, this depth is taken as the upper boundary of the mesopelagic domain. The depth-weighted average (DWA) Baxs contents, calculated for the 150–400 and 150–800 m depth intervals, are given in Table 1. For the proﬁles on the plateau (500 m water column), bottom waters with evidence of sediment resuspension were not taken into account when calculating DWA Baxs values (≥400 m). Particle size spec- tra indicated that sediment resuspension occurred especially at stations A3 and TEW-3 (Jouandet et al., 2014; Lasbleiz et al., 2014; van der Merve et al., 2015;). Thus, at site A3 a. Unexpectedly, the highest DWA Baxs value of the whole study area (572 pM; 150–400 m) was observed at the reference R-2 site. For R-2, Bowie et al. (2014), Quéroué et al. (2015) and van der Merve et al. (2015) re- ported local maxima in particulate and dissolved trace metals at 500 m and deeper, reﬂecting lateral trans- port of lithogenic matter possibly originating from the Leclaire Rise (a large seamount located west of R- 2). Similarly, Lasbleiz et al. (2014) observed a max- imum of lithogenic silica (LSi) at 500 m, conﬁrm- ing lithogenic inputs there. However, we note that the S. H. M. Jacquet et al.: Early season mesopelagic carbon remineralization ent from those reported in Jacquet et al. (2008a). At the other depths, the lithogenic Ba contribution at A3 (KEOPS 2) was only minor; ent from those reported in Jacquet et al. (2008a). At the other depths, the lithogenic Ba contribution at A3 (KEOPS 2) was only minor; mesopelagic Baxs maximum at R-2 occurs at shallower depths, around 300 m, and that there is no evidence for elevated values at 500 m where the previous authors re- ported higher trace element and silica concentrations. Also, as reported above (see Sect. 2.2 and Appendix A), the higher lithogenic Ba fractions at R-2 (up to 20 % of the total Ba) occur only in the upper 80 m. Moreover, we note that surface waters at R-2 has already experi- enced some nitrate consumption as compared to subsur- face winter waters (Tmin waters). Indeed, surface wa- ters had 10 % less nitrate than winter water (26 µM at 5 m vs. 29 µM at 200 m), and the isotopic enrichment of this surface nitrate conﬁrmed a suggestion of uptake (see Dehairs et al., 2014). Also, Lasbleiz et al. (2014) reported relatively low Si : C and Si : N ratios for sur- face ocean suspended matter), pointing to the develop- ment of a diatom assemblage just prior to the sampling, consistent with the high dissolution rates of biogenic sil- ica (BSi) that Closset et al. (2014) reported for R-2 sur- face waters. It is therefore likely that the mesopelagic Baxs content at R-2 indeed reﬂects remineralization of organic material that was fuelled by an important past early spring production and export event. Similarly, dur- ing late winter (November 1993) F. Dehairs (unpub- lished results) observed the presence of signiﬁcant num- bers of barite microcrystals in mesopelagic waters at the KERFIX time series station (50◦40′ S, 68◦25′ E) located east of R-2. Results would thus suggest the occurrence in this HNLC area of recurrent brief early spring di- atom productive period pulses and subsequent export and remineralization activity in the underling layers. Chl a satellite images (Giovanni – Interactive Visualiza- tion and Analysis, NASA GES DISC) corroborate that the R-2 and KERFIX area is occasionally subject to en- hanced biomass during early spring; c. The time series stations in the polar front meander had DWA Baxs contents ranging from 258 to 427 pM (150–400 m), so reaching values exceeding those on the plateau. S. H. M. Jacquet et al.: Early season mesopelagic carbon remineralization For these time series, stations’ values de- creased between day 0 (TNS-6) and 12 (E-3), and then increased again at days 22 (E-4E) and 27 (E-5). Sta- tions E-4W and TNS-8, above the plateau but in deeper waters close to the Kerguelen margin, at the edge the high biomass plume (Fig. 1), had the highest DWA Baxs values (468 and 473 pM, respectively; 150–400 m), not considering the R-2 reference station. The polar front F-L site, although located within the eastern part of the high biomass plume, had a smaller DWA Baxs value of 345 pM (150–400 m) and the nearby station TEW-8 had the lowest DWA Baxs value of the whole study area (199 pM; 150–400 m). c. The time series stations in the polar front meander had DWA Baxs contents ranging from 258 to 427 pM (150–400 m), so reaching values exceeding those on the plateau. For these time series, stations’ values de- creased between day 0 (TNS-6) and 12 (E-3), and then increased again at days 22 (E-4E) and 27 (E-5). Sta- tions E-4W and TNS-8, above the plateau but in deeper waters close to the Kerguelen margin, at the edge the high biomass plume (Fig. 1), had the highest DWA Baxs values (468 and 473 pM, respectively; 150–400 m), not considering the R-2 reference station. The polar front F-L site, although located within the eastern part of the high biomass plume, had a smaller DWA Baxs value of 345 pM (150–400 m) and the nearby station TEW-8 had the lowest DWA Baxs value of the whole study area (199 pM; 150–400 m). 4.1 Mesopelagic Baxs and bacterial production Previous studies revealed that the shape of the column- integrated bacterial production (BP) proﬁle (i.e. the attenu- ation length scale) was important in setting the Baxs signal in the mesopelagic zone (Dehairs et al., 2008; Jacquet et al., 2008a, 2011a). Mesopelagic Baxs content is smaller when most of the column-integrated BP is restricted to the upper mixed layer (indicating an efﬁcient, near-complete reminer- alization within the surface), compared to situations where a signiﬁcant part of integrated BP was located deeper in the water column (reﬂecting signiﬁcant deep bacterial activity and POC export). During KEOPS 2 the incorporation of 3H- leucine was used to estimate bacterial production. BP data are described in Christaki et al. (2014). In Fig. 5 we compare column-integrated BP at 150 m over 400 m (BP150 / 400) and DWA Baxs for the 150–400 m depth interval, along with the relationship obtained during KEOPS 1 (BP200 / 125 and 150–450 m DWA Baxs; Jacquet et al., 2008a; Christaki et al., 2008). Excluding stations A3, E-1, E-2 and E-3, KEOPS 2 data presented a signiﬁcant correlation (R2 =0.88; p < 0.01) and a similar trend to the one reported for KEOPS 1. A simi- lar picture was obtained when integrating DWA Baxs and BP up to 800 m (not shown). The time series “E” stations in the meander revealed a shift from stations E-1, E-2 and E-3 to stations E-4E and E-5, i.e. towards the trend reported above. A shift was also apparent at station A3 from KEOPS 2 (early spring) to KEOPS 1 (late summer). It is thus possible that results reﬂect the occurrence of different stages of bloom ad- vancement. The large variability of the Baxs and BP relation- ship during KEOPS 2, especially at A3 site and in the me- b. The two successive visits (27 days apart) at site A3 yielded relatively low DWA Baxs values of 267 and 316 pM, and a quite similar value was observed for the shallow station TEW-3 (324 pM), located further north on the plateau and north of the PF. Note that for com- parison purposes, we recalculated the DWA Baxs and MR values of KEOPS 1 by considering upper and lower mesopelagic layer boundaries of 150 and 400 m rather than 125 and 450 m, as in Jacquet et al. (2008a). www.biogeosciences.net/12/1713/2015/ www.biogeosciences.net/12/1713/2015/ Biogeosciences, 12, 1713–1731, 2015 1720 S. H. M. Jacquet et al.: Early season mesopelagic carbon remineralization 1721 Table 2. Comparison of mesopelagic POC remineralization (MR) with primary production (PP) and export production (EP). All ﬂuxes in mgCm−2 day−1. r ratio is the ratio of Mr over EP. The C sequestration (or transfer) efﬁciency at 400 and 800 m (T400, T800) is the fraction of C export (EP) at 150 m exiting through the 400 m (T400) or the 800 m (T800) horizons. See text for further information on calculation. Table 2. Comparison of mesopelagic POC remineralization (MR) with primary production (PP) and export production (EP). All ﬂuxes in mgCm−2 day−1. r ratio is the ratio of Mr over EP. The C sequestration (or transfer) efﬁciency at 400 and 800 m (T400, T800) is the fraction of C export (EP) at 150 m exiting through the 400 m (T400) or the 800 m (T800) horizons. See text for further information on calculation. Station CTD MLD Ezb PPc Ez EPd MR MR EP/PP r ratio r ratio T400 T800 [m] [m] [mgCm−2 day−1] 150 m 150–400 m 150–800 m 150–400 m 150–800 m [mgCm−2 day−1] [mgCm−2 day−1] [mgCm−2 day−1] Plateau A3-1 4a 161 – – 47 14 – – 0.29 – 0.70 – A3-2 107a 165 38 2172 85 11 – 0.04 0.13 – 0.87 – Reference site R-2 17/20 111 92 132 30 50 91 0.23 1.65 3.02 0 0 Meander time series E-1 27/30 84 64 578 100 26 48 0.17 0.26 0.48 0.74 0.52 E-3 50/55 41 68 748 130 10 35 0.17 0.08 0.27 0.92 0.73 E-4E 94/97 77 34 1037 48 27 58 0.05 0.57 1.21 0.43 0 E-5 113/114 36 54 1064 84 28 66 0.08 0.33 0.78 0.67 0.22 Polar front zone F-L 63/68 21 29 3380 43 21 49 0.01 0.48 1.13 0.52 0 Polar front E-4W 81/87 67 31 3287 54 36 76 0.02 0.67 1.41 0.33 0 a Station A3 (CTD4 and 107); MR integrated up to 354 and 405 m. b Ez euphotic layer (until 1 % PAR level). c PP data from Cavagna et al. (2015). d EP data from Planchon et al. (2015). !"#$#%&''# %&(# %&)# %&# %&'# %&+# ,# ,&,# ,'%# -'%# .'%# /'%# ('%# )'%# !"#$%&'%()+,-*+% &./0%1234%5678%()9-*%+% 0.# 1,21-21.# 1/121(# 324# !2-# 1/25# 61789#,# 0.#:61789#,;# 9<=>?@# 0AB=@C<D<@E# Figure 5. S. H. M. Jacquet et al.: Early season mesopelagic carbon remineralization We remind the reader here that MR ﬂuxes based on mesopelagic Baxs reﬂect past remineralization activity integrated over sev- eral days to a few weeks (Dehairs et al., 1997; Cardinal et al., 2005; Jacquet et al., 2007, 2008b). In order to compare Figure 5. Regression of the ratio of integrated bacterial production (BP) in the upper 150 m over integrated BP in the upper 400 m ver- sus depth-weighted average (DWA) mesopelagic Baxs (pM; 150– 400 m) during KEOPS 2. KEOPS 1 data (dots) are reported for comparison (Christaki et al., 2008; Jacquet et al., 2008a). ander, could reﬂect the temporal evolution and patchiness of the establishment of mesopelagic remineralization processes in this polar front area. S. H. M. Jacquet et al.: Early season mesopelagic carbon remineralization Regression of the ratio of integrated bacterial production (BP) in the upper 150 m over integrated BP in the upper 400 m ver- sus depth-weighted average (DWA) mesopelagic Baxs (pM; 150– 400 m) during KEOPS 2. KEOPS 1 data (dots) are reported for comparison (Christaki et al., 2008; Jacquet et al., 2008a). !"#$#%&''# %&(# %&)# %&# %&'# %&+# ,# ,&,# ,'%# -'%# .'%# /'%# ('%# )'%# !"#$%&'%()+,-+% &./0%1234%5678%()9-*%+% 0.# 1,21-21.# 1/121(# 324# !2-# 1/25# 61789#,# 0.#:61789#,;# 9<=>?@# 0AB=@C<D<@E# al., 2014; Schneider et al., 2008). To address these ques- tions, we deﬁned two ratios: (1) the mesopelagic C rem- ineralization efﬁciency (r ratio in Table 2), which is the ra- tio of mesopelagic C remineralization (MR, based on the DWA Baxs concentrations) over C export (EP) from the 150 m horizon (based on 234Th, see Planchon et al., 2014), and (2) the C transfer efﬁciency at 400 and 800 m (i.e. T400, T800 in Table 2), which is the fraction of C ex- port (EP) at 150 m passing through the 400 m (T400) or the 800 m (T800) horizons (e.g. T400 = EP400 / EP150 = 1– (MR / EP150), with MR / EP150 = r ratio; see above). This approach is similar to the one developed by Buesseler and Boyd (2009) stating that a conventional curve-ﬁtting of par- ticle ﬂux data (i.e. power law or exponential) skews our inter- pretation of the mesopelagic processes. They recommended the use of combined metrics to capture and compare dif- ferences in ﬂux attenuation. In the following, we compare MR ﬂuxes for the different KEOPS 2 areas (reference site; plateau sites; polar front and polar front meander) and dis- cuss remineralization and transfer efﬁciencies for those sites for which MR, primary production (PP) and/or EP data (Ta- ble 2) were available. PP data were estimated from uptake experiments including 24 h incubations at different PAR lev- els over the euphotic layer, i.e. up to the 0.01 % PAR level (Cavagna et al., 2014). EP data were estimated from 234Th activities and 234Th / POC ratios and are discussed in Plan- chon et al. (2014). The thorium method integrates POC ex- port over a 1-month period (234Th half live is 24.1 days). 4.1 Mesopelagic Baxs and bacterial production Also, in the aforementioned study the high Baxs contents ob- served near the seaﬂoor were not excluded from the calculations, while they are here. These increased ben- thic boundary layer Baxs contents (observed also during KEOPS 2) are due to sediment resuspension which ex- tended up to 70 m above the seaﬂoor during KEOPS 1 (Blain et al., 2008; Venchiarutti et al., 2008; Armand et al., 2008). Because of these slightly different depth intervals over which Baxs values were integrated, the KEOPS 1 values discussed here will be slightly differ- b. The two successive visits (27 days apart) at site A3 yielded relatively low DWA Baxs values of 267 and 316 pM, and a quite similar value was observed for the shallow station TEW-3 (324 pM), located further north on the plateau and north of the PF. Note that for com- parison purposes, we recalculated the DWA Baxs and MR values of KEOPS 1 by considering upper and lower mesopelagic layer boundaries of 150 and 400 m rather than 125 and 450 m, as in Jacquet et al. (2008a). Also, in the aforementioned study the high Baxs contents ob- served near the seaﬂoor were not excluded from the calculations, while they are here. These increased ben- thic boundary layer Baxs contents (observed also during KEOPS 2) are due to sediment resuspension which ex- tended up to 70 m above the seaﬂoor during KEOPS 1 (Blain et al., 2008; Venchiarutti et al., 2008; Armand et al., 2008). Because of these slightly different depth intervals over which Baxs values were integrated, the KEOPS 1 values discussed here will be slightly differ- www.biogeosciences.net/12/1713/2015/ Biogeosciences, 12, 1713–1731, 2015 4.2 Fate of exported organic C in the mesopelagic zone and deep water column An important question relates to the fate of the exported POC: how much of this POC is respired in the mesopelagic waters and how much escapes remineralization and is ex- ported to deeper layers where longer-term sequestration is likely (see e.g. Passow and Carlson, 2012; Robinson et Biogeosciences, 12, 1713–1731, 2015 4.2.1 Reference station R-2 Since station R-2 had the highest DWA Baxs content, it yielded the highest MR ﬂux of the whole study area (91 mg C m−2 day−1; 150–800 m; Table 2). In contrast, both PP and EP ﬂuxes at R-2 were very low (132 and 10 mg C m−2 day−1, respectively) and the calculated MR ﬂux exceeded EP (Ta- ble 2). The resulting export efﬁciency (EP / PP) was high, and T400 and T800 values (the fraction of EP exported deeper than 400 and 800 m, as deﬁned above) equal 0 (i.e. no ex- port of POC beyond 400 and 800 m; note that > 100 % val- ues, i.e. MR > EP, were set to zero in Fig. 7a and Table 2). The fact that MR exceeds EP therefore implies a non-steady state condition at the R-2 site. As reported above, R-2 proba- bly experienced a brief early spring diatom production pulse days to a few weeks before the start of the KEOPS 2 cruise, followed by subsequent export and very important reminer- alization activity in the underling layers as depicted by MR data. It is important to underline the fact that MR at station A3 was only slightly higher in summer than in spring, especially considering the large differences in export efﬁciency between seasons. According to results from sediment traps deployed over 1 year at the A3 site, Rembauville et al. (2014) re- ported that 60 % of the annual POC export at the base of the mixed layer occurred over a short periods of time repre- senting < 4 % of the year and was composed of small highly siliciﬁed, fast-sinking, resting spores of diatoms that bypass grazing pressure. According to these authors, the pulses are linked to nutrient depletion dynamics inducing resting spore formation. During the rest of the year, the ﬂux was com- posed of small diatoms (empty frustules) and small fecal pellets, with efﬁcient C retention in the surface layer or transfer to trophic levels. If we consider that export con- ditions during KEOPS 2 are more similar to those prevail- ing most of the year, it is surprising that during KEOPS 1 (which would reﬂect an export event toward the end of the growth season) MR is not more important. This would in- dicate that fast-sinking, highly siliciﬁed and pulsed material was directly transferred to the bottom without major rem- ineralization. 4.2.1 Reference station R-2 Note for example that at the complex R-2 ref- erence station, a small export event (Laurenceau-Cornec et al.; 2015) held heavily siliciﬁed diatoms (Lasbleiz et al., 2014) and that the material was efﬁciently remineralized in the upper mesopelagic layer as witnessed by the high MR values we observed for that station. For the KEOPS 2 A3 site, Laurenceau-Cornec et al. (2015) reported that the sink- ing ﬂux collected in the upper layer using gel-ﬁlled sediment traps was composed of phytodetrital aggregates that held slightly siliciﬁed diatoms (Lasbleiz et al., 2014). Even con- sidering the shift from slightly to highly siliciﬁed material transfer between spring (KEOPS2) and summer (KEOPS 1), MR only slightly increases between both periods. Also, the mesozooplankton biomass at A3-2 was one of the highest of the KEOPS2 cruise, with a doubling from KEOPS 2 (early spring) to KEOPS 1 (late summer) (Carlotti et al., 2015). It is thus possible that at A3 the export event reported above, combined with a lasting grazing pressure, could have induced this rather low and enduring mesopelagic remineralization. www.biogeosciences.net/12/1713/2015/ S. H. M. Jacquet et al.: Early season mesopelagic carbon remineralization S. H. M. Jacquet et al.: Early season mesopelagic carbon remineralization 1722 EP with MR (r ratio and transfer efﬁciency), we consider EP ﬂuxes from 150 m. Results are compared with late sum- mer KEOPS 1 results. For KEOPS 1, PP data are detailed in Lefèvre et al. (2008) and Mosseri et al. (2008), EP data are detailed in Savoye et al. (2008) and Baxs data are described in Jacquet et al. (2008a). suggested by Christaki et al. (2014). Note that a negative relationship between primary productivity and surface car- bon export efﬁciency has already been reported from previ- ous studies in the Southern Ocean (Lam et al., 2007; Mor- ris et al., 2007; Savoye et al., 2008; Jacquet et al., 2011a, b). Among possible explanations for the occurrence of high- productivity low export efﬁciency regimes in high-latitude systems Maiti et al. (2013) mentioned differences in trophic structure, grazing intensity, recycling efﬁciency, high bacte- rial activity or increase in DOC export, but the exact rea- son remain unclear. In contrast, during KEOPS 1 (summer), EP ﬂuxes reached 250 mg C m−2 day−1 at 125 m (14–31 % of PP), while PP ranged from 865 to 1872 mg C m−2 day−1, reﬂecting enhanced export efﬁciency (Jacquet et al., 2008a; Savoye et al., 2008). www.biogeosciences.net/12/1713/2015/ S. H. M. Jacquet et al.: Early season mesopelagic carbon remineralization 0.0 0.1 0.2 0.3 0.4 0.5 0.0 0.2 0.4 0.6 0.8 1.0 EP/PP T400 KEOPS 1 KEOPS 2 1% 5% 10% 20% 30% 40% 30% A3 #119 A3 #32 A3 #76 A3-1 #4 A3-2 #107 E-4W X E-4W (T800) F-L (T800) F-L !"#$ R-2 0.0 0.1 0.2 0.3 0.4 0.5 0.0 0.2 0.4 0.6 0.8 1.0 EP/PP Transfer efficiency T400 T800 1% 5% 10% 20% 30% 40% 30% E-1 E-3 E-1 E-3 E-5 E-5 E-4E E-4E !%#$ !%#$ !"#$ Figure 7. y axis: EP / PP = POC ﬂux at 150 m (EP150) as a fraction of primary production (PP); x axis: EPx / EP150 = POC ﬂux at deﬁned depths (EPx; here 400 and 800 m) as a fraction of POC ﬂux at 150 m (EP150). The green cross (Fig. 5a) is for station A3-1 (KEOPS-2). Since no PP data are available for that station, the EP / PP value has been set to 0. Isolines represent the modelled 1, 5, 10, 20 and 30 % of PP export to depths > at 400 or 800 m, and represent export efﬁciency. In Fig. 7a, the ratio of EP to PP (export efﬁciency) vs. the fraction of EP exported deeper than 400 m (i.e. T400; de- ﬁned above) is shown for both KEOPS cruises. Note that for station A3-1 (KEOPS 2), there are no PP data. The A3 site shows increasing EP / PP ratios from spring (KEOPS 2) to late summer (KEOPS 1), and so do the T400 values (A3-1: 70 %; A3-2: 87 %; KEOPS 1 A3 site: 92 ± 1 %). Station E- 4W is located in waters with similar θ −S and Chl a charac- teristics as the A3 plateau site but has a deeper water column (1384 m has PP and EP ﬂuxes of the same order of magnitude (Table 2)). However, MR values (36 mg C m−2 day−1; 150– 400 m) are larger at E-4W, resulting in a lower T400 value of around 33 %, compared to 87 % for A3-2 (Fig. 7a). When integrating between 150 and 800 m, T800 at E-4W equals 0 (i.e. no export of POC beyond 800 m; Fig. 7a and Table 2). We also wonder if the shallow water column at A3 combined with lateral advection above the plateau plays a role in trig- gering the mesopelagic POC remineralization activity and in setting its efﬁciency. S. H. M. Jacquet et al.: Early season mesopelagic carbon remineralization KEOPS 2 KEOPS 1 (Early spring 2011) (Late summer 2005) A3-1 A3-2 Mean of the 3 repeats PP Not available 2172 864-1872 - 4% EP 47 85 250 29% 13% MR 14 11 17-23 All fluxes in mgC m day -2 -1 Blue values: ratio, mesopelagic remineralization efficiency (MR/EP) Green values: EP/PP Red values: MR/PP !"#$"%" &'"#(&"%" &#)%" &%" #" r Mean of the 3 repeats 864-1872 Mean of the 3 repeats 864-1872 11 11 All fluxes in mgC m day -2 -1 Blue values: ratio, mesopelagic remineralization efficiency (MR/EP) Green values: EP/PP Red values: MR/PP r Figure 6. Schematic, comparing the fate of POC at station A3 during KEOPS 1 and KEOPS 2 cruises. PP: primary production, EP: export production at 150 m depth and MR: mesopelagic POC remineralization deduced from the Baxs maxima and integrated between 150–400 m; all ﬂuxes in mgC m−2 day−1. EP / PP (green values), MR / PP (red values) and MR / EP (r ratio, blue values) ratios shown as percentage. 0.0 0.1 0.2 0.3 0.4 0.5 0.0 0.2 0.4 0.6 0.8 1.0 EP/PP T400 KEOPS 1 KEOPS 2 1% 5% 10% 20% 30% 40% 30% A3 #119 A3 #32 A3 #76 A3-1 #4 A3-2 #107 E-4W X E-4W (T800) F-L (T800) F-L !"#$ R-2 0.0 0.1 0.2 0.3 0.4 0.5 0.0 0.2 0.4 0.6 0.8 1.0 EP/PP Transfer efficiency T400 T800 1% 5% 10% 20% 30% 40% 30% E-1 E-3 E-1 E-3 E-5 E-5 E-4E E-4E !%#$ Figure 7. y axis: EP / PP = POC ﬂux at 150 m (EP150) as a fraction of primary production (PP); x axis: EPx / EP150 = POC ﬂux at deﬁned depths (EPx; here 400 and 800 m) as a fraction of POC ﬂux at 150 m (EP150). The green cross (Fig. 5a) is for station A3-1 (KEOPS-2). Since no PP data are available for that station, the EP / PP value has been set to 0. Isolines represent the modelled 1, 5, 10, 20 and 30 % of PP export to depths > at 400 or 800 m, and represent export efﬁciency. 4.2.2 Station A3 on the plateau The MR ﬂuxes on the plateau varied little between the two visits 27 days apart (Table 1) and, moreover, as discussed below they were similar to summer values obtained during KEOPS 1 (see Jacquet et al., 2008a) when the same A3 site was sampled three times over a 19-day period. While dur- ing KEOPS 2 (spring) MR ﬂuxes at A3 ranged from 11 to 14 mg C m−2 day−1 (with a standard uncertainty of around 5 %), they were slightly larger during KEOPS 1 (summer; 17 to 23 mg C m−2 day−1) (Fig. 5). We observed differences in the mesopelagic POC remineralization efﬁciency between the two seasons (r ratio, blue values in Fig. 6, Table 2). Dur- ing KEOPS 1, r ratios (MR / EP) remained low, ranging from 7 to 9 % of EP at A3, while during KEOPS 2, r ratios were slightly higher but decreased from 29 % (A3-1; ﬁrst visit) to 13 %, 27 days later (A3-2; second visit). This variation in r ratio during KEOPS 2 is mostly due to an increase of EP (from 47 to 85 mg C m−2 day−1; Planchon et al., 2014) over the same period while MR showed little change. Al- though at this early stage of the season (spring) PP at A3- 2 had already reached 2172 mg C m−2 day−1 (Cavagna et al., 2014), EP remained relatively low (85 mg C m−2 day−1). Here EP accounted for only about 4 % of PP (low export ef- ﬁciency; see green data points in Fig. 5). These conditions suggested that phytoplankton biomass had accumulated in the surface waters without signiﬁcant export at that point, or that C had been channelled to higher trophic levels as Biogeosciences, 12, 1713–1731, 2015 www.biogeosciences.net/12/1713/2015/ 1723 www.biogeosciences.net/12/1713/2015/ S. H. M. Jacquet et al.: Early season mesopelagic carbon remineralization For KEOPS 1, Venchiarutti et al. (2008) reported that lateral advection over the plateau could sig- niﬁcantly impact particle dynamics. During KEOPS 1, sta- tion B1 (CTD68), located on the plateau upstream from A3 according to the plateau circulation (Park et al., 2008), ex- hibited a very similar Baxs distribution as station A3: low mesopelagic Baxs and important bottom resuspension (not shown here; see Jacquet et al., 2008a). These strong sim- ilarities in Baxs proﬁle shapes would indicate that next to the pulsed nature of the events, the dynamics on the shal- low plateau play an important role in limiting the extent of mesopelagic POC remineralization processes. 4.3 Stations in the meander Therefore, the high, deep (> 1000 m) Baxs contents at TNS-6 and E-1 most likely reﬂects the fact that here signiﬁcant remineralization of POC material actually did occur in the bathypelagic domain and even down to the seaﬂoor. Note that suspended particles in the depth range containing the deep Baxs maxima were dom- inated by the < 2 µm size fraction (M. Zhou, personal com- munication, 2014). When integrating the Baxs contents from 150 m to the seaﬂoor at stations TNS-6 and E-1, MR ﬂuxes increase to 156 and 184 mg C m−2 day−1, respectively. Such C ﬂuxes were similar to the EP values (maximum value of 130 mg C m−2 day−1 at E-3) and suggested that the exported Station F-L (in the vicinity of the PF; 74.7◦E) appears to function in a similar way as observed for E-4W (71.4◦E). PP at station F-L is relatively high (3380 mg C m−2 day−1), while EP is quite low (43 mg C m−2 day−1), reﬂecting the fact that the biomass was not yet exported from the surface waters or was transported to higher trophic levels. Since MR ﬂuxes are slightly lower (21 mg C m−2 day−1; 150–400 m) at F-L than at E-4W, resulting T400 values are higher (52 %) there. Station F-L (in the vicinity of the PF; 74.7◦E) appears to function in a similar way as observed for E-4W (71.4◦E). PP at station F-L is relatively high (3380 mg C m−2 day−1), while EP is quite low (43 mg C m−2 day−1), reﬂecting the fact that the biomass was not yet exported from the surface waters or was transported to higher trophic levels. Since MR ﬂuxes are slightly lower (21 mg C m−2 day−1; 150–400 m) at F-L than at E-4W, resulting T400 values are higher (52 %) there. Overall, during KEOPS 2, it appears that biomass at sta- tions A3, E-4W and F-L (sites of high productivity) had been accumulating in surface waters (e.g. transfer to higher trophic levels) and export had not yet started, considering the early stage of the season during KEOPS 2. Our observations allow us to conclude the following: 1. Both seasons (KEOPS 1 and KEOPS 2) showed a sim- ilar functioning of the mesopelagic ecosystem at A3. 4.3 Stations in the meander Temporal short-term changes for the stations TNS-6, E-1, E- 2, E-3, E-4E and E-5, located in the polar front meander, will be discussed in this section. Note that no PP or EP data exist for TNS-6. From Table 2 it appears that PP almost doubled between E-1 and E-5, but this increase was not paralleled by an increase of EP and MR, except for the 30 % EP increase from E-1 to E-3. In fact, overall EP shows a decreasing trend with time, while MR (150–400 m) stays rather constant, ex- cept for the decrease between E-1 and E-3 (Table 2). As re- ported above such a mismatch may result from differences in timescales characterizing the different processes that were compared. The most likely explanation is that in this early stage of the growth season, phytoplankton biomass accumu- lated in the surface layer and export lagged behind. Figure 8. Temporal evolution of particulate biogenic Ba (Baxs; pM) in the upper 2000 m water column in the polar front meander. Graph constructed using Ocean Data View (Schlitzer, 2002; Ocean Data View; http://www.awi-bremerhaven.de/GEO/ODV). The ratio of EP to PP vs. T400 and T800 showed a large variability in transfer efﬁciency inside the meander (Fig. 7b). PP and EP ﬂuxes increased by about 30 % from E-1 to E-3, but a concomitant decrease of mesopelagic MR yielded to an enhanced transfer efﬁciency, from 74 to 92 %, through the 400 m boundary, and from 52 to 73 % through the 800 m boundary. This suggests that signiﬁcant remineraliza- tion should have occurred at greater depths (even > 1000 m), and it is also reﬂected by the presence of Baxs maxima below 1000 m (see Fig. 4h and Appendix A). This was particularly salient when plotting Baxs contents vs. depths over the 27- day observation period (Fig. 8). The high, deep water Baxs values in Fig. 8 were not taken into account when integrating TNS-6 and E-1 proﬁles between 150 and 400 or even 150 and 800 m (Fig. 5e). Considering that the seaﬂoor in the meander area is at about 2000 m depth, it seems unlikely that these high Baxs contents at depths > 1000 m were due to sediment resuspension. Also, particle spectra for these sites do not re- veal any bottom resuspension (Jouandet et al., 2014; Lasbleiz et al., 2014; van der Merve et al., 2015). S. H. M. Jacquet et al.: Early season mesopelagic carbon remineralization 1724 ciency of C transfer beyond 400 m to 33 and 52 %, re- spectively, and to zero for both stations beyond 800 m. Bottom depth, lateral advection, zooplankton grazing pressure and the pulsed nature of the POC transfer at A3 were the particular conditions that could drive the differences in C transfer efﬁciency between A3 and E4- W and F-L and limit the extent of MR processes at A3. E)(+,-(./ day% 0")$% 123% !(/4%1-3% !"#$%& '$(& '$)& &&&&&&&&&'$'&&&&&&&&&&&&&&&&&&&&&&'$+& '$,& Figure 8. Temporal evolution of particulate biogenic Ba (Baxs; pM) in the upper 2000 m water column in the polar front meander. Graph constructed using Ocean Data View (Schlitzer, 2002; Ocean Data View; http://www.awi-bremerhaven.de/GEO/ODV). E)(+,-(./ day% 0")$% 123% !(/4%1-3% !"#$%& '$(& '$)& &&&&&&&&&'$'&&&&&&&&&&&&&&&&&&&&&&'$+& '$,& !(*/4%1-3% www.biogeosciences.net/12/1713/2015/ Biogeosciences, 12, 1713–1731, 2015 S. H. M. Jacquet et al.: Early season mesopelagic carbon remineralization 5 Conclusions Based on spatially and temporally well-resolved mesopelagic excess particulate Ba inventories, this work estimated mesopelagic POC remineralization above the Kerguelen Plateau and inside a permanent meander of the polar front to the east of plateau. The observed variability of mesopelagic remineralization reﬂects differences in the fate of the biomass that is exported to the deep ocean between the plateau and polar front. Results also reveal the patchiness of the seasonal advancement and of the establishment of rem- ineralization processes between these sites. Our results indi- cate that a few days to weeks before the start of the cruise, the reference station R-2 experienced an export event that was efﬁciently remineralized in the upper mesopelagic layer. In terms of deep ocean carbon transfer efﬁciency, our results highlight that above the plateau (A3 site), mesopelagic rem- ineralization is not a major barrier to organic matter trans- fer to the seaﬂoor, with carbon transfer beyond 400 m reach- ing up to 87 % of EP during KEOPS 2, while in the polar front meander, remineralization of exported organic carbon in the upper 400 m is more efﬁcient than above the plateau. In the meander area, remineralization may even balance export when including its effect in the deeper waters (up to 800 m and even deeper), thus resulting in a near-zero carbon trans- fer to sediment. A similar condition is also observed for sites at the margin of the plateau (E-4W) and the polar front (F-L). Overall, the temporal pattern of mesopelagic remineraliza- tion described above reﬂects two successive events of particle transfer: a ﬁrst transfer from a previous bloom (occurred be- fore visiting TNS-6 and enduring at E-1) and a second trans- fer from E-4E to E-5. The ﬁrst transfer was evidenced by the downward (to the bottom) propagation of the mesopelagic Baxs maximum signal, which mostly weakens at E-2. The second event was reﬂected by the occurrence again of impor- tant mesopelagic Baxs build-up at E-4E and E-5. Overall, our results indicated the large capacity of the polar front meander to transfer POC material to depth, but in contrast to station A3 on the plateau, this transfer was coupled to intense and near-complete POC remineralization (as also observed at E- 4W and F-L). Between-site changes in mesopelagic carbon remineralization due to unequal biomass productivity and iron fertilization over the Kerguelen Plateau were thus rel- atively complex. S. H. M. Jacquet et al.: Early season mesopelagic carbon remineralization S. H. M. Jacquet et al.: Early season mesopelagic carbon remineralization POC was entirely remineralized in the water column leaving no C for transfer to the sediments. POC was entirely remineralized in the water column leaving no C for transfer to the sediments. 4.3 Stations in the meander The rather low and enduring MR ﬂuxes under high pro- duction and variable export regimes (high export efﬁ- ciency during KEOPS 1 and low export efﬁciency dur- ing KEOPS 2) indicated that here mesopelagic reminer- alization does not represent a major resistance to or- ganic matter transfer to the seaﬂoor at A3. On average (considering both seasons, but excluding A3-1), the C transfer efﬁciency into the deep (> 400 m) as assessed by PP, EP and MR ﬂuxes comparisons reached 91 ± 3 % at A3; 2. In contrast to A3, E-4W and F-L showed important mesopelagic remineralization rates, reducing the efﬁ- Biogeosciences, 12, 1713–1731, 2015 www.biogeosciences.net/12/1713/2015/ 1725 5 Conclusions Furthermore, the conditions in the meander area seems to corroborate results obtained in the iron-replete Subantarctic Zone east of the Tasman Plateau (Australian sector of the Southern Ocean; SAZ-Sense cruise; Jacquet et al., 2011a, b), where the mesopelagic remineralization efﬁ- ciency reported was relatively high (on average 91 %) and the deep (> 600 m) carbon transfer weak (< 10 %). Finally, the important Baxs contents reported between 1000 and 2000 m during the ﬁrst stages of the meander time-series support re- cent results indicating for the Southern Ocean that 1000 m is insufﬁcient as an ocean-wide reference for carbon transfer and sequestration potential (Robinson et al., 2014). Biogeosciences, 12, 1713–1731, 2015 Biogeosciences, 12, 1713–1731, 2015 www.biogeosciences.net/12/1713/2015/ www.biogeosciences.net/12/1713/2015/ Appendix A Table A1. Excess particulate biogenic Ba (Baxs; pM) and particu- late Al (nM) during KEOPS 2. Station A3 Station RK2 A3-1 CTD4 A3-2 CTD 107 R-2 CTD17 R-2 CTD 20 Niskin Depth Baxs Al Niskin Depth Baxs Al Niskin Depth Baxs Al Niskin Depth Baxs Al no. www.biogeosciences.net/12/1713/2015/ www.biogeosciences.net/12/1713/2015/ S. H. M. Jacquet et al.: Early season mesopelagic carbon remineralization 1726 Appendix A [m] [pM] [nM] [m] [pM] [nM] [m] [pM] [nM] [m] [pM] [nM] 23 11 224 35 23 11 122 16 24 21 110 107 17 356 546 12 21 42 217 64 21 40 140 12 23 40 0 693 15 507 239 17 19 104 345 65 19 81 141 10 22 80 95 49 13 609 226 7 17 152 234 19 17 126 82 27 20 100 131 27 11 812 267 3 15 173 244 19 15 151 119 14 18 126 168 5 10 1011 189 2 13 204 333 17 13 176 199 9 16 151 205 4 8 1520 201 4 11 227 235 8 11 202 186 14 14 203 334 3 6 1832 184 2 9 253 315 6 9 277 323 24 13 253 616 6 1 2473 286 3 7 279 480 8 7 303 359 32 12 304 834 16 5 354 216 21 5 405 247 19 10 404 573 9 1 474 1108 155 1 513 1842 186 9 507 430 10 7 608 367 4 5 708 337 10 1 911 368 13 Station E E-1 CTD 27 E-1 CTD 30 E-2 CTD 43 E-3 CTD 50 Niskin Depth Baxs Al Niskin Depth Baxs Al Niskin Depth Baxs Al Niskin Depth Baxs Al [m] [pM] [nM] [m] [pM] [nM] [m] [pM] [nM] [m] [pM] [nM] 24 21 896 166 17 303 424 30 23 11 192 43 24 11 129 45 23 41 221 131 16 353 195 25 21 41 93 152 23 42 117 93 22 81 190 161 15 455 143 6 18 102 143 17 22 71 130 28 20 101 172 102 13 505 268 6 16 153 215 57 20 102 160 22 18 125 150 10 11 636 343 7 14 204 408 9 18 125 201 11 16 151 290 9 10 808 138 2 12 254 311 6 16 153 225 18 14 182 375 5 8 1011 442 4 10 305 227 4 14 203 193 3 13 253 450 12 6 1498 436 9 8 406 353 5 13 252 210 2 12 303 402 9 1 2042 326 7 7 507 371 9 12 304 309 6 10 403 327 10 6 609 271 10 10 404 316 7 9 505 230 6 5 813 297 14 9 505 419 64 7 605 305 10 4 1016 350 35 7 606 320 14 5 707 298 10 1 2020 302 11 5 707 193 12 1 913 309 7 1 912 265 5 E-3 CTD 55 E-4W CTD 81 E-4W CTD 87 E-4E CTD 94 Niskin Depth Baxs Al Niskin Depth Baxs Al Niskin Depth Baxs Al Niskin Depth Baxs Al [m] [pM] [nM] [m] [pM] [nM] [m] [pM] [nM] [m] [pM] [nM] 17 404 185 5 24 10 101 16 17 304 277 9 24 20 116 32 16 455 272 5 23 41 134 17 16 354 350 10 23 51 260 11 15 505 176 6 22 70 152 5 15 453 233 7 22 93 563 223 13 605 378 5 20 94 86 18 13 606 182 9 20 103 170 5 11 810 258 3 18 126 84 7 11 811 186 5 18 126 215 9 10 910 172 2 16 153 193 8 10 910 187 7 16 152 210 6 8 1012 184 3 14 203 312 4 8 1011 268 61 14 181 247 7 6 1214 228 6 13 252 628 17 6 1214 249 29 13 253 547 4 1 1908 237 9 12 304 488 12 1 1384 250 30 12 305 403 78 10 406 594 11 10 404 408 26 9 507 272 11 9 505 403 26 7 607 418 12 7 606 298 13 5 708 338 21 5 706 285 8 1 909 294 14 1 912 245 65 Station E (continued) E-4E CTD 97 E-5 CTD 113 E-5 CTD 114 Niskin Depth Baxs Al Niskin Depth Baxs Al Niskin Depth Baxs Al [m] [pM] [nM] [m] [pM] [nM] [m] [pM] [nM] 21 404 199 2 10 911 111 3 24 11 210 5 18 505 242 3 8 1011 266 16 23 41 196 14 13 706 175 1 6 1214 256 15 22 82 245 4 8 1012 212 11 1 1922 208 5 20 102 264 14 7 1265 189 2 18 126 131 6 6 1518 149 2 16 152 153 5 5 1827 225 43 14 202 181 2 4 2027 212 12 13 252 469 6 1 2212 254 9 12 302 606 9 10 404 377 13 9 507 422 11 7 606 425 7 5 707 281 12 1 910 281 6 Table A1. Appendix A Excess particulate biogenic Ba (Baxs; pM) and particu- late Al (nM) during KEOPS 2. Table A1. Excess particulate biogenic Ba (Baxs; pM) and particu- late Al (nM) during KEOPS 2. Table A1. Excess particulate biogenic Ba (Baxs; pM) and particu- late Al (nM) during KEOPS 2. Biogeosciences, 12, 1713–1731, 2015 S. H. M. Jacquet et al.: Early season mesopelagic carbon remineralization 1 Table A1. Continued. Appendix A West–east transect Station F-L TEW-3 CTD38 TEW-8 CTD 47 F-L CTD 63 F-L CTD 68 Niskin Depth Baxs Al Niskin Depth Baxs Al Niskin Depth Baxs Al Niskin Depth Baxs Al [m] [pM] [nM] [m] [pM] [nM] [m] [pM] [nM] [m] [pM] [nM] 23 16 133 40 23 10 196 31 24 11 146 37 17 405 264 6 21 41 107 112 21 41 0 251 23 35 97 41 16 456 233 9 19 61 209 45 18 102 92 41 22 61 0 228 15 506 339 12 17 76 148 20 16 152 169 45 20 82 141 36 13 607 265 3 13 112 128 13 14 202 134 9 18 101 134 5 11 910 718 7 11 183 235 8 12 254 164 5 16 126 185 5 10 1013 257 5 9 253 391 11 10 304 268 12 14 151 221 5 8 1215 316 8 7 277 348 9 8 405 217 5 13 202 280 7 6 1772 225 7 5 404 356 8 7 507 319 5 12 252 399 8 1 2741 2999 131 1 545 242 13 6 609 209 8 10 303 420 7 5 809 330 14 9 404 305 26 4 1011 334 22 7 506 408 7 1 2812 11179 826 5 707 247 10 1 911 282 11 North–south transect TNS-1 CTD15 TNS-6 CTD 10 TNS-8 CTD8 Niskin Depth Baxs Al Niskin Depth Baxs Al Niskin Depth Baxs Al [m] [pM] [nM] [m] [pM] [nM] [m] [pM] [nM] 23 11 262 62 23 35 182 26 23 12 478 45 21 41 30 90 21 42 141 12 21 41 258 53 18 102 93 15 18 103 143 14 18 102 303 32 16 153 225 17 16 184 413 11 16 150 1008 33 14 202 289 5 14 204 461 8 14 205 341 10 12 253 228 2 12 255 298 5 12 254 447 6 10 304 521 4 10 306 505 7 10 305 481 4 8 405 346 3 8 407 474 4 8 405 312 3 7 506 230 1 7 509 464 9 7 505 208 3 6 607 352 13 6 610 315 15 6 606 283 7 5 809 234 4 5 813 269 9 5 707 325 11 4 1520 127 6 4 1526 362 13 4 910 376 35 1 2282 211 19 1 1886 410 21 1 1000 294 28 S. Appendix A H. M. Jacquet et al.: Early season mesopelagic carbon remineralization 1727 Biogeosciences, 12, 1713–1731, 2015 Biogeosciences, 12, 1713–1731, 2015 www.biogeosciences.net/12/1713/2015/ www.biogeosciences.net/12/1713/2015/ References Buesseler, K. O. and Boyd, P. W.: Shedding light on processes that control particle export and ﬂux attenuation in the twilight zone, Limnol. Oceanogr., 54, 1210–1232, 2009. Armand, L. K., Crosta, X., Quéguiner, B., Mosseri, J., and Garcia, N.: Diatoms preserved in surface sediments of the northeastern Kerguelen Plateau, Deep- Sea Res. Pt. II, 55, 677–692, 2008. Blain, S., Tréguer, P., Belviso, S., Bucciarelli, E., Denis, M., Desabre, S., Fiala, M., Martin Jézéquel, V., Le Fèvre, J., Mayzaud, P., Marty, J.-C., and Razouls, S.: A biogeochemical study of the island mass effect in the context of the iron hypothe- sis: Kerguelen Islands, Southern Ocean, Deep-Sea Res. Pt. I, 48, 163–187, 2001. Armand, L. K., Crosta, X., Quéguiner, B., Mosseri, J., and Garcia, N.: Diatoms preserved in surface sediments of the northeastern Kerguelen Plateau, Deep- Sea Res. Pt. II, 55, 677–692, 2008. Buesseler, K. O., Andrews, J. E., Pike, S. M., and Charette, M. A.: The effect of iron fertilization on carbon sequestration in the Southern Ocean, Science, 304, 414–417, 2004. Blain, S., Tréguer, P., Belviso, S., Bucciarelli, E., Denis, M., Desabre, S., Fiala, M., Martin Jézéquel, V., Le Fèvre, J., Mayzaud, P., Marty, J.-C., and Razouls, S.: A biogeochemical study of the island mass effect in the context of the iron hypothe- sis: Kerguelen Islands, Southern Ocean, Deep-Sea Res. Pt. I, 48, 163–187, 2001. Buesseler, K. O., Andrews, J. E., Pike, S. M., Charette, M. A., Gold- son, L. E., Brzezinski, M. A., and Lance, V. P.: Particle export during the Southern Ocean Iron Experiment (SOFeX), Limnol. Oceanogr., 50, 311–327, 2005. Buesseler, K. O., Antia, A. N., Chen, M., Fowler, S. W., Gardner, W. D., Gustaffson, Ö., Harada, K., Michaels, A. F., Rutgers van der Loeff, M., Sarin, M., Steinberg, D. K., and Trull, T.: An as- sessment of the use of sediment traps for estimating upper ocean particle ﬂuxes, J. Mar. Res., 65, 345–416, 2007a. Blain, S., Queguiner, B., Armand, L., Belviso, S., Bombled, B., Bopp, L., Bowie, A., Brunet, C., Brussaard, C., Carlotti, F., Christaki, U., Corbiere, A., Durand, I., Ebersbach, F., Fuda, J. -L., Garcia, N., Gerringa, L., Grifﬁths, B., Guigue, C., Guillerm, C., Jacquet, S., Jeandel, C., Laan, P., Lefevre, D., Lo Monaco, C., Malits, A., Mosseri, J., Obernosterer, I., Park, Y. References -H., Picheral, M., Pondaven, P., Remenyi, T., Sandroni, V., Sarthou, G., Savoye, N., Scouarnec, L., Souhaut, M., Thuiller, D., Timmer- mans, K., Trull, T., Uitz, J., van Beek, P., Veldhuis, M., Vincent, D., Viollier, E., Vong, L., and Wagener, T.: Effect of natural iron fertilization on carbon sequestration in the Southern Ocean, Na- ture, 446, 1070–1074, 2007. Buesseler, K. O., Lamborg, C. H., Boyd, P. W., Lam, P. J., Trull, T. W., Bidigare, R. R., Bishop, J. K. B., Casciotti, K. L., Dehairs, F. , Elskens, M., Honda, M. , Karl, D. M., Siegel, D. A., Silver, M. W., Steinberg, D. K. , Valdes, J., Van Mooy, B., and Wilson, S.: Revisiting carbon ﬂux through the ocean’s twilight zone, Sci- ence, 316, 567–569, 2007b. Cardinal, D., Dehairs, F., Cattaldo, T., and André, L.: Constraints on export and advection in the Subantarctic and Polar Front Zones, south of Australia from the geochemistry of suspended particles, J. Geophys. Res.-Oceans, 106, 31637–31656, 2001 Blain, S., Quéguiner, B., and Trull, T.: The natural iron fertiliza- tion experiment keops (kerguelen ocean and plateau compared study): An overview, Deep-Sea Res. Pt. II, 55, 559–565, 2008. Cardinal, D., Savoye, N., Trull., T.W., André, L., Kopczynska, E., and Dehairs, F.:, Particulate Ba distributions and ﬂuxes suggest latitudinal variations of carbon mineralization in the Southern ocean, Deep-Sea Res. Pt. I, 52, 355–370, 2005. Boyd, P. W., Watson, A. J., Law, C. S., Abraham, E. R., Trull, T., Murdoch, R., Bakker, D. C. E., Bowie, A. R., Buesseler, K. O., Chang, H., Charette, M., Croot, P., Downing, K., Frew, R., Gall, M., Hadﬁeld, M., Hall, J., Harvey, M., Jameson, G., LaRoche, J., Liddicoat, M., Ling, R., Maldonado, M. T., McKay, R. M., Nobber, S., Pickmere, S., Pridmore, R., Rintoul, S., Saﬁ, K., Sut- ton, P., Strzepek, R., Tanneberger, K., Turner, S., Waite, A., and Zeldis, J.: Phytoplankton bloom upon mesoscale iron fertiliza- tion of polar Southern Ocean water, Nature, 407, 695–702, 2000. Carlotti, F., Jouandet, M.-P., Nowaczyk, A., Harmelin-Vivien, M., Lefèvre, D., Guillou, G., Zhu, Y., and Zhou, M.: Mesozooplank- ton structure and functioning during the onset of the Kerguelen Bloom during Keops2 survey, Iogeosci. Iscuss., 12, 2381–2427, 2015. Cavagna, A. S. H. M. Jacquet et al.: Early season mesopelagic carbon remineralization 1728 fate of an iron-induced subarctic phytoplankton bloom, Nature, 428, 549–553, 2004. Acknowledgements. We thank the ofﬁcers and crew of R/V Marion Dufresne for their assistance during our work at sea. We are indebted to chief scientist S. Blain and voyage leader B. Quéguiner for their skillful leadership during the cruise and to the CTD team for managing rosette operation and CTD data. This research was supported by a French National Research Agency grant (project KEOPS 2, no. ANR-10-BLAN-0614), the Belgian Science Policy (BELSPO) project BIGSOUTH (SD/CA/05A), Flanders Research Foundation (FWO Project G071512N), the European Union Seventh Framework Programme (Marie Curie CIG MuSiCC, under grant agreement no. 294146 to D. Cardinal) and the Strategic Research Programme at Vrije Universiteit Brussel. fate of an iron-induced subarctic phytoplankton bloom, Nature, 428, 549–553, 2004. Boyd, P. W., Bakker, D. C. E., and Chandler, C.: A new database to explore the ﬁndings from large-scale ocean iron enrichments experiments, Oceanography, 25, 64–71, doi:10.5670/oceanog.2012.104, 2012. Bowie, A. R., van der Merwe, P., Quéroué, F., Trull, T., Fourquez, M., Planchon, F., Sarthou, G., Chever, F., Townsend, A. T., Obernosterer, I., Sallée, J.-B., and Blain, S.: Iron budgets for three distinct biogeochemical sites around the Kerguelen archipelago (Southern Ocean) during the natural fertilisation experiment KEOPS-2, Biogeosciences Discuss., 11, 17861–17923, doi:10.5194/bgd-11-17861-2014, 2014. Edited by: S. Blain Edited by: S. Blain Broecker, W. S., Takahashi, T., and Takahashi, T.: Sources and ﬂow patterns of deep-ocean waters as deduced from potential temper- ature, salinity and initial phosphate concentration, J. Geophys. Res., 90, 6925–6939, 1985. S. H. M. Jacquet et al.: Early season mesopelagic carbon remineralization M., Dehairs, F., Cardinal, D., Navez, J., and Delille, B.: Barium distribution across the Southern Ocean Frontal sys- tem in the Crozet-Kerguelen Basin, Mar. Chem., 95, 149–162, 2005. Jacquet, S. H. M., Dehairs, F., Elskens, M., Savoye, N., and Cardi- nal, D.: Barium cycling along WOCE SR3 line in the Southern Ocean, Mar. Chem., 106, 33–45, 2007. Jacquet, S. H. M., Dehairs, F., Savoye, N., Obernosterer, I., Chris- taki, U., Monnin, C., and Cardinal, D.: Mesopelagic organic carbon mineralization in the Kerguelen Plateau region tracked by biogenic particulate Ba, Deep-Sea Res. Pt. II, 55, 868–879, 2008a. Dehairs, F., Chesselet, R., and Jedwab, J.: Discrete suspended par- ticles of barite and the barium cycle in the open ocean, Earth Planet. Sc. Lett., 49, 40–42, 1980. Jacquet, S. H. M., Savoye, N Dehairs, F., Strass, V., and Cardinal, D.: Mesopelagic carbon mineralization during the European Iron Fertilization Experiment (EIFEX), Glob. Biogeochem. Cy., 22, GB1023, doi:10.1029/2006GB002902, 2008b. Dehairs, F., Baeyens, W., and Goeyens, L.: Accumulation of sus- pended barite at mesopelagic depths and export production in the Southern Ocean, Science, 258, 1332–1335, 1992. Jacquet, S. H. M., Dehairs, F., Becquevort, S., Dumont, I., Cavagna, A., and Cardinal, D.: Twilight zone organic car- bon remineralization in the PFZ and SAZ south of Tasma- nia (Southern Ocean), Deep-Sea Res. Pt. II, 58, 2222–2234 doi:10.1016/j.dsr2.2011.05.029, 2011a. Dehairs, F., Shopova, D., Ober, S., Veth, C., and Goeyens, L.: Par- ticulate barium stocks and oxygen consumption in the Southern Ocean mesopelagic water column during spring and early sum- mer: Relationship with export production, Deep-Sea Res. Pt. II, 44, 497–516, 1997. Jacquet, S. H. M., Lam, P., Trull ,T., and Dehairs, F.: Carbon export production in the Polar front zone and Subantarctic Zone south of Tasmania, Deep-Sea Res. Pt. II, 58, 2277–2292, 2011b. Dehairs, F., Jacquet, S. H. M., Savoye, N., van Mooy, B., Buesseler, K., Bishop, J., Lamborg, C., Elskens, M., Baeyens, W., Casciotti K., and Monnin, C.: Barium in twilight zone suspended matter as proxy for organic carbon mineralization: results for the North Paciﬁc, Deep-Sea Res. Pt. II, 55, 1673–1683, 2008. Jouandet, M. P., Jackson, G. A., Carlotti, F., Picheral, M., Stem- mann, L., and Blain, S.: Rapide formation of large aggre- gates during the spring bloom of Kerguelen Island: observa- tion and model comparisons, Biogeosciences, 11, 4393–4406, doi:10.5194/bg-11-4393-2014, 2014 . Dehairs, F., Fripiat, F., Cavagna, A.-J., Trull, T. S. H. M. Jacquet et al.: Early season mesopelagic carbon remineralization 1729 M., and Williams, A., Second edition ISBN 0948926 15 5, 120 pp., 2000. Closset, I., Lasbleiz, M., Leblanc, K., Quéguiner, B., Cavagna, A.- J., Elskens, M., Navez, J., and Cardinal, D.: Seasonal evo- lution of net and regenerated silica production around a natu- ral Fe- fertilized area in the Southern Ocean estimated from Si isotopic approaches, Biogeosciences, 11, 5827–5846, doi:10.5194/bg-11- 5827-2014, 2014. François, R., Honjo, S., Krishﬁeld, R., and Manganini, S.: Fac- tors controlling the ﬂux of organic carbon to the bathy- pelagic zone of the ocean, Global Biogeochem. Cy., 16, 1087, doi:10.1029/2001GB001722, 2002. Christaki, U., Obernosterer, I., VanWambeke, F., Veldhuis, M., Gar- cia, N., and Catala, P.: Microbial food web structure in a naturally iron fertilized area in the southern ocean (Kerguelen plateau), Deep-Sea Res. Pt. II, 55, 706–719, 2008. Gervais, F., Riebesell, U., and Gorbunov, M. Y.: Changes in primary productivity and chlorophyll a in response to iron fertilization in the southern Polar Frontal Zone, Limnol. Oceanogr., 47, 1324– 1335, 2002. Henson, S., Sanders, R., Madsen, E., Morris, P., Le Moigne, F., and Quartly, G.: A reduced estimate of the strength of the ocean’s biological carbon pump, Geophys. Res. Lett., 38, L04606, 2011. Christaki, U., Lefèvre, D., Geoges, C., Colombet, J., catala, P., Courties, C., Sime-Ngando, T., Blain, S., and Obernosterer, I.: Microbial food web dynamics during spring phytoplankton blooms in the naturally iron-fertilized Kerguelen area (South- ern Ocean), Biogeosciences, 11, 6739–3753, doi:10.5194/bg-11- 6739-2014, 2014. Hoffmann, L., Peeken, I., Lochte, K., Assmy, P., and Veldhuis, M.: Different reactions of Southern Ocean phytoplankton size classes to iron fertilization, Limnol. Oceanogr, 51, 1217–1229, 2006. de Baar, H. J. W., Boyd, P. W., Coale, K. H., Landry, M. R., Tsud, A., Assmy, P., Bakker, D. C. E, Bozec, Y., Barber, R. T., Brzezin- ski, M. A., Buesseler, K. O., Boyé, M., Croot, P. L., Gervais, F., Gorbunov, Y., Harrison, P. J., Hiscock, W. T., Laan, P., Lancelot, C., Law, C. S., Levasseur, M., Marchetti, A., Millero, F. J., Nishika, J., Nojiri, Y., van Oijen, T., Riebesell, U., Rijkenberg, M. J. A., Saito, H., Takeda, S., Timmermans, K. R., Veldhuis, J. W., Waite, A. M., and Wong, C. S.: Synthesis of iron fertilization experiments: From the iron age in the age of enlightenment, J. Geophys. Res., 110, C09S16, doi:10.1029/2004JC002601, 2005. Jacquet, S. H. References J., Fripiat, F., Elskens, M., Dehairs, F., Mangion, P., Chirurgien, L., Closset, I., Lasbleiz, M., Flores–Leiva, L., Car- dinal, D., Leblanc, K., Fernandez, C., Lefèvre, D., Oriol, L., Blain, S., and Quéguiner, B.: Biological productivity regime and associated N cycling in the vicinity of Kerguelen Island area, Southern Ocean, Biogeosciences Discuss., 11, 18073–18104, doi:10.5194/bgd-11-18073-2014, 2014. Boyd, P. W., Law, C. S., Wong, C. S., Nojiri, Y., Tsuda, A., Lev- asseur, M., Takeda, S., Rivkin, R., Harrison, P. J., Strzepek, R., Gower, J., McKay, R. M., Abraham, E., Arychuk, M., Barwell- Clarke, J., Crawford, W., Crawford, D., Hale, M., Harada, K., Johnson, K., Kiyosawa, H., Kudo, I., Marchetti, A., Miller, W., Needoba, J., Nishioka, J., Ogawa, H., Page, J., Robert, M., Saito, H., Sastri, A., Sherry, N., Soutar, T., Sutherland, N., Taira, Y., Whitney, F., Wong, S. K. E., and Yoshimura, T.: The decline and H., Sastri, A., Sherry, N., Soutar, T., Sutherland, N., Taira, Y., Whitney, F., Wong, S. K. E., and Yoshimura, T.: The decline and Biogeosciences, 12, 1713–1731, 2015 www.biogeosciences.net/12/1713/2015/ www.biogeosciences.net/12/1713/2015/ S. H. M. Jacquet et al.: Early season mesopelagic carbon remineralization 1730 ern Ocean, Biogeosciences, 11, 5931–5955, doi:10.5194/bg-11- 5931-2014, 2014. len area of the Southern Ocean using 234Th-based approach, Biogeosciences Discuss., 11, 15991–16032, doi:10.5194/bgd- 11-15991-2014, 2014. Laurenceau-Cornec, E. C., Trull, T. W., Davies, D. M., Bray, S. G., Doran, J., Planchon, F., Carlotti, F., Jouandet, M.-P., Cav- agna, A.-J., Waite, A. M., and Blain, S.: The relative impor- tance of phytoplankton aggregates and zooplankton fecal pel- lets to carbon export: insights from free-drifting sediment trap deployments in naturally iron-fertilised waters near the Kergue- len plateau, Biogeosciences, 12, 1007–1027, doi:10.5194/bg-12- 1007-2015, 2015. Porris, P. J., Sanders, R., Turnewithsch, R., and Thomalla, S.: 234T h-derived particulate organic carbon export from an island- induced phytoplankton blomm in the Southern Ocean,Deep-Sea Res. Pt. II, 24, 2208–2232, 2007. Quéroué, F., Sarthou, G., Planquette, H. F., Bucciarelli, E., Chever, F., van der Merwe, P., Lannuzel, D., Townsend, A. T., Cheize, M., Blain, S., d’Ovidio, F., and Bowie, A. R.: High vari- ability of dissolved iron concentrations in the vicinity of Kergue- len Island (Southern Ocean), Biogeosciences Discuss., 12, 231– 270, doi:10.5194/bgd-12-231-2015, 2015. Laws, E. A., Falkowski, P. G., Smith, W. O., Ducklow, H., and Mc- Carthy, J. J.: Temperature effects on export production the ocean, Global Biogeochem. Cy., 14, 1231–1246, 2000. Rembauville, M., Blain, S., Armand, L., Quéguiner, B., and Salter, I.: Export ﬂuxes in a naturally fertilized area of the South- ern Ocean, the Kerguelen Plateau: ecological vectors of carbon and biogenic silica to depth (Part 2), Biogeosciences Discuss., 11, 17089–17150, doi:10.5194/bgd-11-17089-2014, 2014. Lefèvre, D., Guigue, C., and Obernosterer, I.: The metabolic bal- ance at two contrasting sites in the Southern Ocean: the iron- fertilized Kerguelen area and HNLC waters, Deep-Sea Res. Pt. II, 55, 766–776, 2008. Le Moigne, F. A. C., Moore, C. M., Sanders, R. J., Villa-Alfageme, M., Steigenberger, S., and Achterberg, E. P.: Sequestration ef- ﬁciency in the iron-limited North Atlantic: Implications for iron supply mode to fertilized blooms, Geophys. Res. Lett., 41, 4619– 4627, doi:10.1002/2014GL060308, 2014. Robinson, J., Popova, E. E., Yool, A., Srokosz, M. A., Lampitt, R. S., and Blundell, J. R.: How deep is deep enough? Ocean iron fer- tilization and carbon sequestration in the Southern Ocean, Geo- phys. Res. Lett., 41, 2489–2495, 2014. Salter, I., Lampitt, R. S., Sanders, R., Poulton, A., Kemp, A. E. S. H. M. Jacquet et al.: Early season mesopelagic carbon remineralization S., Boorman, B., Saw, K., and Pearce, R.: Estimating carbon, sil- ica and diatom export from a naturally fertilised phytoplankton bloom in the Southern Ocean using PELAGRA: a novel drifting sediment trap, Deep-Sea Res. Pt. II, 54, 2233–2259, 2007. Longhurst, A. R., Bedo, A. W., Harrison, W. G., Head, E. J. H., and Sameoto, D. D.: Vertical ﬂux of respiratory carbon by oceanic diel migrant biota, Deep-Sea Res, 37, 685–694, 1990. Maiti, K., Charette, M., Buesseler, K., and Kahru, M.: An inverse re- lationship between production and export efﬁciency in the South- ern Ocean, Geophys. Res. Lett., 40, 1–5, 2013. Sarmiento, J. L., Slater, R. D., Fasham, M. J. R., Ducklow, H. W., and Toggweiler, J. R.: A seasonal three-dimensional ecosystem model of nitrogen cycling in the North Atlantic photic zone, Global Biogeochem. Cy., 7, 417–450, 1993. Martin, J. H., Knauer, G. A., Karl, D. M., and Broenkow, W. W.: VERTEX: carbon cycling in the NE Paciﬁc, Deep-Sea Res., 34, 267–285, 1987. Monnin, C., Jeandel, C., Cattaldo, T., and Dehairs, F.: The marine barite saturation state of the world’s oceans, Mar. Chem., 65, 253–261, 1999. Savoye, N., Trull, T., Jacquet, S. H. M., Navez, J., and Dehairs, F.: 234Th-derived export ﬂuxes during a natural iron fertilization experiment (KEOPS), Deep-Sea Res. Pt. II, 55, 841–855, 2008. Schlitzer, R.: Ocean Data View, http://www.awi-bremerhaven.de/ GEO/ODV, 2002. Morris, P. J. and Charette, M. A.: A synthesis of upper oceancar- bon and dissolved iron budgets for Southern Ocean natural iron fertilization studies, Deep-Sea Res., 90, 147–157, 2013. Schneider, B., Bopp, L., and Gehlen, M.: Assessing the sensitivity of modeled airsea CO2 exchange to the remineralization depth of particulate organic and inorganic carbon, Global Biogeochem. Cy., 22, GB3021, doi:10.1029/2007GB003100, 2008. Mosseri, J., Quéguiner, B., Armand, L. K., and Cornet-Barthaux, V.: Impact of iron on silicon utilization by diatoms in the Southern Ocean: a case study of Si / N cycle decoupling in a naturally iron- enriched area, Deep-Sea Res. Pt. II, 55, 801–819, 2008. Shopova, D., Dehairs, F., and Baeyens, W.: A simple model of bio- geochemical element distribution in the oceanic water column, J. Mar. Sy., 6, 331–344, 1995. Park, Y.-H., Roquet, F., Durand, I., and Fuda, J.-L.: Large-scale cir- culation over and around the Northern Kerguelen Plateau, Deep- Sea Res. Pt. II, 55, 566–581, 2008. Smetacek, V., Klass, C., Strass, V. S. H. M. Jacquet et al.: Early season mesopelagic carbon remineralization W., Fernandez, C., Davies, D., Roukaerts, A., Fonseca Batista, D., Planchon, F., and Elskens, M.: Nitrogen cycling in the Southern Ocean Ker- guelen Plateau area: evidence for signiﬁcant surface nitriﬁcation from nitrate isotopic compositions, Biogeosciences Discuss., 11, 13905–13955, doi:10.5194/bgd-11-13905-2014, 2014. Lam, P. J. and Bishop, J. K. B.: High biomass, low export regimes in the Southern Ocean, Deep-Sea Res. Pt. II, 54, 601–638, 2007. Lampitt, R. S. and Antia, A. N.: Particle ﬂux in deep seas: regional characteristics and temporal variability, Deep-Sea Res. Pt. I, 44, 1377–1403, 1997. D’Ovidio, F., Della Penna, A., Trull, T. W., Nencioli, F., Pu- jol, I., Rio, M. H., Park, Y. H., Cotté, C., Zhou, M., and Blain, S.: The biogeochemical structuring role of horizontal stir- ring: Lagrangian perspectives on iron delivery downstream of the Kerguelen plateau, Biogeosciences Discuss., 12, 779–814, doi:10.5194/bdg-12-779-2015, 2015. Lampitt, R. S., Achterberg, E. P., Anderson, T. R., Hughes, J. A., Iglesisas-Rodriguez, M. D., Kelly-Gerreyn, B. A., Lucas, M., Popova, E. E., Sanders, R., Shepherd, J. G., Smythe-Wright, D., and Yool, A.: Ocean fertilization: A potential means of geoengi- neering?, Philos. Trans. R. Soc. A, 366, 3919–3945, 2008. Dymond, J. R., Suess, E., and Lyle, M.: Barium in deep-sea sedi- ment: a geochemical proxy for paleoproductivity, Paleoceanog- raphy, 7, 163–181, 1992. g Lasbleiz, M., Leblanc, K., Blain, S., Ras, J., Cornet-Barthaux, V., Hélias Nunige, S., and Quéguiner, B.: Pigments, elemental com- position (C, N, P, and Si), and stoichiometry of particulate matter in the naturally iron fertilized region of Kerguelen in the South- Ellison, Eurachem/CITAC Guide CG4, Quantifying Uncertainty in Analytical Measurement, edited by: Ellison, S.L.R., Rosslein, Biogeosciences, 12, 1713–1731, 2015 www.biogeosciences.net/12/1713/2015/ S. H. M. Jacquet et al.: Early season mesopelagic carbon remineralization H., Assmy, P., Montresor, M., Cisewki, B., Savoye, N., Webb, A., d’Ovidio, F., Arrieta, J. M., Bathmann, U., Bellerby, R., Mine Berg, G., Croot, P., Gonza- lez, S., Jenjes, J., Herndl, G. J., Hoffmann, L.J., Leach, H., Losh, M., Mills, M. M., Neill, C., Peeken, I., Rottgers, R., Sachs, O., Sauter, E., Schmidt, M. M., Schwarz, J., Terbruggen, A., and Wolf-Gladrow, D.: Deep carbon export from a Southern Ocean iron-fertilized diatom bloom, Nature, 487, 313–319, 2012. Park, Y. H., Durand, I., Kestenare, E., Rougier, G., Zhou, M., d’Ovidio, F., Cotté, C., and Lee J. H.: Polar front around the Ker- guelen islands: An up-to-date determination and associated cir- culation of surface/subsurface water, J. Geophys. Res. Oc., 119, 6575–6592, doi:10.1002/2014JC010061, 2014. Passow, U. and Carlson, C. A.: The biological pump in a high CO2 world, Mar. Ecol. Prog. Ser., 470, 249–271, 2012. Planchon, F., Cavagna A. J., Cardinal, D., André, L., and De- hairs, F.: Late summer particulate organic carbon export and twi- light zone remineralisation in the Atlantic sector of the Southern Ocean, Biogeosciences, 10, 803–820, doi:10.5194/bg-10-803- 2013, 2013. Sternberg, E., Jeandel, C., Miquel, J.-C., Gasser, B., Souhaut, M., Arraes-Mescoff, R., and Francois R.: Particulate barium ﬂuxes and export production in the northwestern Mediterranean, Mar. Chem. 105, 281–295, 2007. Sternberg, E., Jeandel, C., Robin, E., and Souhaut, M.: Seasonal cycle of suspended barite in the Mediterranean Sea, Geochim. Cosmochim. Ac., 72, 4020–4034, 2008a. Planchon, F., Ballas, D., Cavagna A. J., Van Der Merwe, P., Bowie, A., Trull, T., Laurenceau-Cornec, E., Davis, D., and De- hairs, F.: Carbon export in the naturally iron fertilized Kergue- Biogeosciences, 12, 1713–1731, 2015 www.biogeosciences.net/12/1713/2015/ www.biogeosciences.net/12/1713/2015/ S. H. M. Jacquet et al.: Early season mesopelagic carbon remineralization 1731 Venchiarutti, C., Jeandel, C., and Roy-Barman, M.: Particle dynam- ics study in the wake of Kerguelen Island using thorium isotopes, Deep-Sea Res. Pt. I, 55, 1343–1363, 2008. Sternberg, E., Tang, D., Ho, T. Y., Jeandel, C., and Morel, M. M..: Barium uptake and adsorption in diatoms, Geochim. Cos- mochim. Ac., 69, 2745–2752, 2008b. Strass, V., Cisewski, B., Gonzales, S., Leach, H., Loquay, K.-D., Prandke, H., Rohr, H., and Thomas, M.: The physical setting of the European Iron Fertilization Experiment “EIFEX” in the Southern Ocean, Reports Pol. Mar. Res., 500, 15–49, 2005. Williams, P. J. and Jenkinson, N. W.: A transportable microprocessor-controlled precise Winkler titration suitable for ﬁeld station and shipboard use, Limnol. Oceanogr., 27, 576–585, 1982. Zhou, M., Zhu, Y., Dorland, R. D., and Measures, C. I.: Dynamics of the current system in the southern Drake Passage, Deep-Sea Res. Pt. I, 57, 1039–1048, 2010. Stroobants, N., Dehairs, F., Goeyens, L., Vanderheijden, N., and Van Grieken, R.: Barite formation in the Southern Ocean water column, Mar. Chem., 35, 411–422, 1991. Zhou, M., Zhu, Y., Measures, C. I., Hatta, M., Charette, M. A., Gille, S. T., Frants, M., Jiang, M., and Mitchell, B. G.: Winter mesoscale circulation on the shelf slope region of the southern Drake Passage, Deep-Sea Res. Pt. II, 90, 4–14, 2013. Taylor, S. R. and McLennan, S. M.: The continental crust: its com- position and evolution, Blackwell Scientif. Publicat., 312 pp., 1985. van der Merwe, P., Bowie, A. R., Quéroué, F., Armand, L., Blain, S., Chever, F., Davies, D., Dehairs, F., Planchon, F., Sarthou, G., Townsend, A. T., and Trull, T.: Sourcing the iron in the naturally- fertilised bloom around the Kerguelen Plateau: particulate trace metal dynamics, Biogeosciences, 12, 739–755, doi:10.5194/bg- 12-739-2015, 2015. Biogeosciences, 12, 1713–1731, 2015 Biogeosciences, 12, 1713–1731, 2015 www.biogeosciences.net/12/1713/2015/ www.biogeosciences.net/12/1713/2015/
https://openalex.org/W3083638644	https://apcyccv.org.pe/index.php/apccc/article/download/12/8	Spanish; Castilian	null	Experiencia en Cirugía de Miocardiopatía Hipertrófica Obstructiva en un Centro de Referencia Nacional	Archivos peruanos de cardiología y cirugía cardiovascular	2,020	cc-by	3,579	Correo: Responsabilidades éticas: Protección de personas y animales. Los autores declaran que para esta investigación no se ha realizado experimentos en seres humanos ni en animales. Conclusiones: Nuestra experiencia en cirugía de MHO es adecuada. El tratamiento simultáneo de los componentes miocárdicos y valvular permite reducir el GTSVI y corregir la IM. Confidencialidad de los datos: Los autores declaran que han seguido los protocolos de su centro de trabajo sobre la publi- cación de datos de pacientes. Derecho a la privacidad y con- sentimiento informado: Los autores declaran que en este artículo no aparecen datos de pacientes. Experience in Obstructive Hyperthrophic Myocardiopathy Artículo Original Experiencia en Cirugía de Miocardiopatía Hipertrófica Obstructiva en un Centro de Referencia Nacional Víctor Robles,1* Yemmy Pérez,1 Josías Ríos1 Recibido 10 de marzo de 2020 Aceptado 8 de abril de 2020 Objetivos: Identificar las características clínicas, analizar los resultados y mostrar la eficacia del tratamiento quirúrgico de la miocardiopatía hipertrófica obstructiva (MHO), en un hospital de referen- cia nacional Afiliaciones de los Autores: 1 Servicio de Cirugía Cardiovascular Adulto - Instituto Nacional Cardio- vascular INCOR. Lima, Perú. Material y métodos: Se realizó un estudio descriptivo, retrospectivo, tipo serie de casos, de pacientes operados en el Instituto Nacional Cardiovascular con el diagnóstico de MHO, entre diciembre del 2016 y enero del 2019. Se analizó la evolución posoperatoria de la sintomatología, clase funcional (CF), gra- diente del tracto de salida del ventrículo izquierdo (GTSVI) y de la insuficiencia mitral (IM). * Correspondencia: Instituto Nacional Cardiovascular INCOR. Jr. Coronel Zegarra 417. Jesús María. Lima 11. Perú. Telef. 01-4111560, anexo 5931. Resultados: Se evaluaron 13 casos con MHO sometidos a miectomía septal extendida. Un 31% fueron mujeres y la edad media fue de 39.6 años. Antes del tratamiento quirúrgico 85% se encontraba en CF III-IV, 85% de pacientes presentaba IM severa, el grosor medio del septum interventricular era de 27 mm (rango de 19 a 39 mm) y el GTSVI medio, de 111 mmHg (rango de 60 a 150 mmHg). Luego del tratamiento quirúrgico se observó mejoría de la CF (69% en CF I)y del grado de IM (92% con IM nula o mínima), y reducción de GTSVI media a 16 mmHg (rango de 6 a 35 mmHg). En 7 pacientes (54%) se realizó cirugía simultánea de la válvula mitral. Material y Método Se realizó un estudio descriptivo, retrospectivo, tipo serie de casos, de pacientes con MHO, intervenidos quirúrgi- camente en el Instituto Nacional Cardiovascular - INCOR EsSalud del Perú entre diciembre del 2016 a enero del 2019. La recolección de datos se realizó mediante la revisión de las historias clínicas. Se utilizó para el análisis y elaboración de resultados las siguientes variables: edad, sexo, antecedentes familiares, estudios diagnósticos realizados en el preoperato- rio, signos y síntomas preoperatorios y postoperatorios, clase funcional preoperatorio y postoperatorio, grado de insufi- ciencia mitral (IM) en el preoperatorio y postoperatorio, frac- ción de eyección del ventrículo izquierdo (FEVI) preoperato- rio, grosor del septum interventricular pre y postoperatoria, gradiente del tracto de salida del ventrículo izquierdo (GTSVI) pre y postoperatorio, técnica quirúrgica realizada, cirugía de la válvula mitral, uso de desfibrilador automático implantable (DAI) y estancia hospitalaria. Las variables numéricas se expre- saron en media y desviación estándar, mediana y rango inter- cuartil y las categóricas en porcentajes. Se analizaron las diferencias entre grupos con la prueba de T de Student para muestras independientes o con la prueba en U de Mann- Whitney para variables continuas y la prueba de chi cuadrado para variables categóricas. Se consideró estadísticamente significativo el valor de p < 0.05. Los datos fueron sometidos al análisis en el programa estadístico STATA 15. La fisiopatología de la MHO, está caracterizada por una obstrucción dinámica, de intensidad variable, normal- mente localizada en posición subvalvular aórtica, entre el septum interventricular hipertrofiado y la válvula mitral, aso- ciada con frecuencia a un movimiento sistólico anterior (MAS) de la vávula mitral que produce diversos grados de insufi- ciencia mitral (IM).1 El diagnóstico de la MHO se basa en la detección de un aumento del grosor de la pared del ventrículo izquierdo (VI) mediante cualquier modalidad de imagen (ecocardio- grafía, resonancia magnética, tomografía computarizada). Material y Método En el adulto se define por un grosor de la pared ≥ de 15 mmm en uno o más segmentos miocárdicos del VI, que no pueda ex- plicarse únicamente por condiciones de carga.4 La obstruc- ción del tracto de salida del ventrículo izquierdo (OTSVI) se define como un gradiente doppler instantáneo máximo del tracto de salida del ventrículo izquierdo ≥ 30 mmHg, pero el umbral a considerar para el tratamiento invasivo es de ≥ 50 mmHg.4 En la evolución clínica de la MHO, la mayoría no pre- sentan síntomas clínicos y/o eventos adversos relevantes, no requieren tratamiento y cursan con una esperanza de vida normal. Un 5% de los pacientes evolucionan con síntomas persistentes y son candidatos a una terapia invasiva, ya sea con el desfibrilador automático implantable (DAI), ablación miocárdica septal transluminal percutánea o cirugía.5 Experiencia en Cirugía de Miocardiopatía Hipertróﬁca Obstructiva en un Centro de Referencia Nacional Experiencia en Cirugía de Miocardiopatía Hipertróﬁca Obstructiva en un Centro de Referencia Nacional E E studios epidemiológicos basados en estudios eco- cardiográficos muestran una prevalencia de la miocardiopatía hipertrófica obstructiva (MHO) de 1 caso por 500 personas en la población general, con ligero predominio del sexo masculino.1,2 Etiológicamente, en un 60% de los adolescentes y adultos con MHO la enfermedad tiene un rasgo autosómico dominante, causado por muta- ciones en más de 11 genes que codifican los componentes proteicos del sarcómero.3 El objetivo de este trabajo fue analizar los resultados del tratamiento quirúrgico en pacientes con MHO que reci- bieron terapia farmacológica máxima sin mejoría clínica, en el Instituto Nacional Cardiovascular INCOR del Perú. Experience in Obstructive Hyperthrophic Myocardiopathy Surgery at a National Reference Center Objectives: To identify the clinical characteristics, analyze the results and show the efficacy of surgical treatment of hypertrophic obstructive cardiomyopathy (HOC), in a national reference institute. Methods: A descriptive, retrospective, case series of patients with the diagnosis of HOC operated at the National Cardiovascular Institute, was performed between December 2016 and January 2019. We analyzed the postoperative evolution of symptomatology, functional class (FC), left ventricular outflow tract gradient (LVOTG) and mitral regurgitation (MR). Results: Thirteen cases with HOC undergoing extended septal myectomy were evaluated. 31% were women and the average age was 39.6 years. Before surgical treatment, 85% were in functional class (FC) III-IV, 85% of patients had severe MR, the mean septum thickness was 27 mm (range from 19 to 39 mm), and mean LVOTG was 111 mmHg (range from 60 to 150 mmHg). After surgical treatment we found improvement of the functional class (69% in FC I) and the degree of MR (92% with zero or mini- mal), and reduction of mean LVOTG to 16 mmHg (range from 6 to 35 mmHg). Simultaneous surgery of the mitral valve was performed in 7 patients (54%). Conclusions: Our experience in HOC surgery is good. The treatment of both myocardial and valve components allows reducing LVOTG and correcting MR. Keywords: hypertrophic obstructive cardiomyopathy • surgery • septal myectomy EsSalud 43 Experiencia en Cirugía de Miocardiopatía Hipertróﬁca Obstructiva en un Centro de Referencia Nacional Resultados Entre diciembre del 2016 y enero del 2019 se inter- vinieron quirúrgicamente 13 pacientes con el diagnóstico de MHO en el Instituto Nacional Cardiovascular INCOR - EsSalud. Once pacientes (85%) presentaban antecedentes familiares de MHO. La edad de los pacientes fluctuó entre los 14 años y los 67 años, y la mayoría de ellos fueron varones (69%). Todos los pacientes tenían terapia farmacológica máxima sin mejoría de la sintomatología y 9 pacientes (69%) eran porta- dores de DAI. (Tabla 1) El tratamiento invasivo para reducir la OTSVI se debe considerar en pacientes con un gradiente ≥ 50 mmHg, sín- tomas de moderados a graves (clase funcional NYHA III – IV), o síncope de esfuerzo recurrente a pesar de recibir tratamiento farmacológico a dosis máximas toleradas.6 Por la complejidad del sustrato anatómico que produce la obstrucción en la MHO se describe diferentes estrategias quirúrgicas que se han repor- tado en los últimos años.7,8 El tratamiento quirúrgico más usual para tratar la OTSVI es la miectomía ventricular (procedimiento de Morrow).9 Un grupo de pacientes requiere cirugía mitral concomitante como la sustitución de la válvula mitral, reali- neación, escisión parcial o movilización de los músculos papi- lares, plicatura o extensión de la valva anterior.10 Otra opción quirúrgica es la miectomía septal mínimamente invasiva trans- mitral con reparación y/o reemplazo de la válvula mitral.11 El estudio ecocardiográfico se realizó en el 100% de los pacientes y el cateterismo cardíaco en 46%. Los síntomas más frecuentes de presentación fueron disnea, angina y sín- cope. El grado de IM fue evaluado en el preoperatorio, intra- operatorio y postoperatorio por medio de ecografía transtorácica y transesofágica. En 12 pacientes se realizó la miectomía septal extendida según la técnica clásica (Morrow) 44 \| EsSalud Arch Per Card Cir Card 2020;1(1):43-47 y en 1 paciente se realizó miectomía septal transmitral por mini toracotomía anterolateral derecha videoasistida, cuyos resultados quirúrgicos fueron similares a la técnica clásica. Edad 39.6 (± 17.4) Varones 9 (69) Antecedente familiar de MHO 11 (85) Disnea 13 (100) Angina 7 (54) Síncope 4 (31) Portador de DAI 9 (69) Se reporta medias (desviación estándar) y frecuencias (porcentaje) para variables cuantitativas y categóricas, respectivamente. MHO: miocardiopatía hipertrófica obstructiva; DAI: defibrilador automático implantable. Tabla 1. Características basales Desenlaces El 85% de los pacientes en el preoperatorio se encon- traban en CF III-IV y luego del tratamiento quirúrgico, el 100% de los pacientes sobrevivientes pasaron a CF I-II. El gradiente medio del tracto de salida del VI disminuyó de 111.4 mmHg (preoperatorio) a 15.7 mmHg (postoperatorio). En cuanto a la IM, el 85% de los pacientes presentaba IM severa y en el post- operatorio el 92% tuvo IM leve o nula. (Tabla 3) Procedimiento quirúrgico Características ecográficas pre y postoperatorias Pre Operatorio Post Operatorio Septum basal 27.1 (± 6.1) 19.3 (± 8.3) Gradiente en TSVI 111.4 (± 35.9) 15.7 (± 9.8) Insuficiencia mitral moderada o severa 13 (100) 1 (8.0) Se reporta medias (desviación estándar) y frecuencias (porcentaje) para variables cuantitativas y categóricas, respectivamente. TSVI: Tracto de salida de ventrículo izquierdo Tabla 3. Características ecográficas pre y postoperatorias Procedimiento quirúrgico La cirugía se realizó, en todos los casos, con control de ecocardiografía transesofágica. En 12 pacientes se realizó una esternotomía media, canulación en aorta ascendente y de ambas cavas, heparinización y entrada a circulación extracor- pórea, llevando al paciente a una temperatura de 32°C. La parada cardíaca con protección miocárdica se realizó usando cardioplejia sanguínea. Se realizó una aortotomía oblicua dirigida hacia el velo aórtico no coronariano para acceder al septum interventricular. Se separó cuidadosamente los velos aórticos y con un bisturí número 15 se practicó una resección de un fragmento del septum interventricular por medio de dos incisiones paralelas, la primera debajo del nadir del velo coronariano derecho y la segunda (en contra de las agujas del reloj) debajo de la comisura del velo coronariano derecho e izquierdo, respetando 5 mm debajo del anillo valvular aórtico. La resección se prolongó distalmente hasta la base de la im- plantación de los músculos papilares (miectomía septal ex- tendida). (Tabla 2) Tiempo de pinzamiento de aorta (minutos) 82.6 (± 36.5) Tiempo de circulación extracorpórea (minutos) 138.2 (± 60) Plastía de la válvula mitral (pacientes) 3 (23.1) Reemplazo de la válvula mitral (pacientes) 4 (30.8) Tiempo en cuidado intensivo (días) 2.9 (± 1.1) Se reporta medias (desviación estándar) y frecuencias (porcentaje) para variables cuantitativas y categóricas, respectivamente. Tabla 2. Características del intraoperatorio En 6 pacientes se realizó miectomía aislada (46%) y en 7 pacientes (54%) se realizó cirugía de válvula mitral concomi- tante (3 valvuloplastias y 4 implantes de prótesis valvular) con abordaje por atrio izquierdo. En el único caso de miectomía septal transmitral mínimamente invasiva, se hizo con canu- lación arterial y venosa vía femoral, y el abordaje fue por una minitoracotomía anterolateral derecha videoasistida. Al ter- minar la circulación extracorpórea se realizó en todos los ca- sos el control ecocardiográfico transesofágico para compro- bar la reducción del GTSVI y la corrección de la IM. Se reporta medias (desviación estándar) y frecuencias (porcentaje) para variables cuantitativas y categóricas, respectivamente. Pre Operatorio Post Operatorio Septum basal 27.1 (± 6.1) 19.3 (± 8.3) Gradiente en TSVI 111.4 (± 35.9) 15.7 (± 9.8) Insuficiencia mitral moderada o severa 13 (100) 1 (8.0) Se reporta medias (desviación estándar) y frecuencias (porcentaje) para variables cuantitativas y categóricas, respectivamente. TSVI: Tracto de salida de ventrículo izquierdo Tabla 3. Experiencia en Cirugía de Miocardiopatía Hipertróﬁca Obstructiva en un Centro de Referencia Nacional Experiencia en Cirugía de Miocardiopatía Hipertróﬁca Obstructiva en un Centro de Referencia Naciona cardíaca.13 Los betabloqueadores y calcioantagonistas pueden dar mejoría sintomática a muchos pacientes con MHO, especialmente en aquellos sin obstrucción u obstruc- ción latente, pero hay considerables variaciones en la respuesta individual a las drogas.14 El reemplazo valvular mitral, posiblemente el trata- miento más radical de la MHO para conseguir una mayor reducción de la GTSVI y corregir la IM, debe realizarse rese- cando toda la válvula y el aparato subvalvular, incluidos los músculos papilares.23 Si desaparece el velo anterior no existe posibilidad de MAS. Sin embargo, esta debe ser una alternati- va en casos seleccionados, debido a las complicaciones propias de las prótesis.24 El manejo depende de la sintomatología del paciente. En los sintomáticos se debe usar betabloqueadores y cal- cioantagonistas, y como última opción queda el manejo inva- sivo que se divide en dos modalidades: ablación con alcohol y la miectomía quirúrgica que disminuye el grosor del septum, corrige la OTSVI y la IM. En un 5% de los pacientes la sintoma- tología es refractaria al tratamiento farmacológico y se debe considerar terapia invasiva.6,15 En nuestra experiencia, la cirugía logró una reducción significativa del GTSVI, de la IM y una mejoría sintomática. Las cifras del GTSVI mostradas se encuentran dentro de los límites de otros estudios internacionales (que oscilan entre 4.5 y 16 mmHg).22,25 No se ha tenido ningún caso de comunicación interventricular ni insuficiencia aórtica iatrogénica, debido al respeto de los límites quirúrgicos ya descritos. Las cifras de mortalidad hospitalaria de la miectomía en centros de gran experiencia es menor al 2%, el riesgo puede ser mayor en paciente ancianos y en aquellos con comorbilidades exis- tentes.26,27 Para los pacientes con MHO y OTSVI, el tratamiento quirúrgico da mejoría sustancial de los síntomas y puede mejorar el pronóstico.16,17 La miectomía septal transaórtica como procedimiento estándar fue desarrollado por Morrow et al,9 es excelente en liberar la OTSVI y es altamente repro- ducible. Otras alternativas quirúrgicas como la miectomía septal mínimamente invasiva se proponen seguras y re- quieren experiencia e instrumental especializado.11,18 La mayor limitación del presente estudio es el número pequeño de pacientes sometidos a cirugía y las dificultades en el seguimiento a largo plazo por ser nuestro instituto un centro de referencia nacional y debido a que el paciente postoperado, una vez resuelta la patología quirúrgica, retorna a su hospital de origen. Conclusión La miectomía septal extendida en pacientes con MHO, corrige la obstrucción del tracto de salida del VI, la IM y se observa una mejoría en la capacidad funcional. El tratamiento debe abarcar el componente miocárdico y valvu- lar. Los resultados obtenidos en el presente estudio muestran que en nuestro instituto el tratamiento quirúrgico de la MHO es adecuado y se encuentra dentro de los parámetros inter- nacionales. Experiencia en Cirugía de Miocardiopatía Hipertróﬁca Obstructiva en un Centro de Referencia Nacional La miectomía septal extendida incrementa el diámetro del tracto de salida del ventrículo izquierdo con la disminución del gradiente y la velocidad de flujo, y corrige la IM funcional. Sin embargo, el tratamiento debe considerarse no solo para el septum hipertrófico, sino también para la válvula mitral y el aparato subvalvular pues la presencia de alteraciones anatómicas de la válvula mitral en pacientes con MHO está bien documentada.19,20 Los velos mitrales son ha- bitualmente más grandes de lo normal y los músculos papi- lares adoptan una posición más anterior dentro de la cavidad ventricular. En ese sentido, la plicatura transversal,21 y la movi- lización y escisión parcial de los papilares22 se describen como técnicas quirúrgicas para el manejo de patología mitral. Estas técnicas deben limitarse a aquellos con MAS, IM severa y ve- los grandes y redundantes. Discusión Se encontraron dos eventos intrahospitalarios post cirugía: un paciente portador de DAI falleció al decimosegun- do día postoperatorio por una arritmia sin respuesta al tratamiento. En otro paciente el control ecocardiográfico al primer día postoperatorio encontró IM severa a pesar de haberse realizado la valvuloplastia mitral por lo que se pro- cedió a una segunda intervención para reemplazo valvular mitral sin complicaciones. La elección del tratamiento para la MHO es diversa y la historia natural, impredecible. Diversos estudios retrospec- tivos han estimado una mortalidad anual aproximada menor del 1%2,12 y muchas de las muertes son súbitas, presumible- mente causadas por arritmias ventriculares.1 Otras complica- ciones que pueden ocurrir incluyen fibrilación auricular, endocarditis infecciosa y estadios finales de insuficiencia EsSalud \| 45 EsSalud \| 45 45 Referencias Bibliográficas The Task Force for the Diagnosis and Management of Hyper- trophic Cardiomyopathy of the European Society of Cardiology (ESC). European Heart Journal 2014;35:2733-79. 1. Maron BJ. Clinical Course and Management of Hypertrophic Cardiomyopathy. N Engl J Med 2018;379:655-68. The Task Force for the Diagnosis and Management of Hyper- trophic Cardiomyopathy of the European Society of Cardiology (ESC). European Heart Journal 2014;35:2733-79. The Task Force for the Diagnosis and Management of Hyper- trophic Cardiomyopathy of the European Society of Cardiology (ESC). European Heart Journal 2014;35:2733-79. 2. Gersh BJ, Maron BJ, Bonow RO, et al. 2011 ACCF/AHA guideline for the diagnosis and treatment of hypertrophic cardiomyopathy: executive summary: a report of the American College of Cardiol- ogy Foundation/American Heart Association Task Force on Prac- tice Guidelines. Circulation 2011;124:2761-96. 5. Spirito P, Seidman CF, McKenna WJ, et al. The management of hypertrophic cardiomiopathy. N Engl J Med 1997;336:775-85. 6. Maron BJ, Mathenge R, Casey SA, et al. Clinical profile of hyper- trophic cardiomyopathy identified de novo in rural communities. J Am Coll Cardiol. 1999;33:1590-5. 3. Alcalai R, Seidman JG, Seidman CE. Genetic basis of hypertrophic cardiomyopathy: From bench to the clinics. J Cardiovasc Electro- physiol. 2008;19:104-10. 7. Jahangiri M, Nicholson IA, Del Nido PJ, et al. Surgical Manage- ment of complex and tunel-like subaortic stenosis. Eur J Cardio- thorac Surg. 2000;17:637-42. 4. Elliott PM, Anastasakis A, Borger MA, et al. 2014 ESC Guidelines on diagnosis and management of hypertrophic cardiomyopathy. 46 \| EsSalud Arch Per Card Cir Card 2020;1(1):43-47 19. Maron MS, Maron BJ. Clinical impact of contemporary cardiovas- cular magnetic resonance imaging in hypertrophic cardiomyopa- thy. Circulation 2015;132: 292-8. 8. Candela-Navarro G, Aguilar-Jiménez JM, Valencia-Díaz YF, et al. Tratamiento quirúrgico de la miocardiopatía hipertrófica obstruc- tiva mediante la técnica de Konno modificada. Cir Cardiov. 2013;20(1):42-5. 20. Sherrid MV, Balaram S, Kim B, et al. The mitral valve in obstructive hypertrophic cardiomyopathy: a test in context. J Am Coll Cardiol 2016; 67:1846-58. 9. Morrow AG, Brockenbrough EC. Surgical Treatment of Idiopathic Subaortic Stenosis. Technic and Hemodynamic Results of Subaortic Ventriculomyotomy. Annals of Surgery 1961;154(2): 181-9. 21. Sherrid MV, Chaudhry FA, Swistel DG. Obstructive hypertrophic cardiomyopathy: echocardiography, pathophysiology, and the continuing evolution of surgery for obstruction. Ann Thorac Surg 2003;75:620-32. . 10. Messmer BJ. Extended Myectomy for Hypertrophic Obstructive Cardiomyopathy. Ann Thorac Surg 1994;58:575-7. 22. Schoendube FA, Klues HG, Reith S, et al. Referencias Bibliográficas Long-term clinical and echocardiographic follow-up after surgical correction of hyper- trophic obstructive cardiomyopathy with extended myectomy and reconstruction of the subvalvular mitral apparatus. Circula- tion 1995;92(Suppl II):122-7. . 11. Gilmanov DS, Bevilacqua S, Solinas M, et al. Minimally Invasive Septal Myectomy for the Treatment of Hypertrophic Obstructive Cardiomyopathy and Intrinsic Mitral Valve Disease. Innovations 2015;10:106-13. 12. Maron BJ. Hypertrophic cardiomyopathy: a systematic review. JAMA 2002;287:1308-20. 23. Cooley DA, Leachman RD, Wukasch DC. Mitral valve replacement for idiopathic hypertrophic cardiomyopathy. J Cardiovasc Surg 1976;17:380-7. 13. Maron BJ, Bonow RO, Cannon RO III, et al. Hypertrophic car- diomyopathy: interrelations of clinical manifestations, patho- physiology, and therapy. N Engl J Med 1987;316:844-52. 24. Vesey JM, Otto CM. Complications of prosthetic heart valves. Curr Cardiol Rep. 2004;6(2):106-11. 14. Rowin EJ, Maron MS, Chan RH, et al. Interaction of adverse dis- ease-related pathways in hypertrophic cardiomyopathy. Am J Cardiol 2017; 120: 2256-64. 25. Schulte HD, Borisov K, Gams E, Gramsch-Zabel H, Losse B, Schwartzkopff B. Management of symptomatic hypertrophic obstructive cardiomyopathy-long term results after surgical therapy. Thorac Cardiovasc Surg 1999;47:213-8. 15. Vriesendorp PA, Liebregts M, Steggerda RC, et al. Long-term outcomes after medical and invasive treatment in patients with hypertrophic cardiomyopathy. JACC Heart Fail 2014; 2: 630-6. 26. Maron BJ, Dearani JA, Ommen SR, et al. Low operative mortality achieved with surgical septal myectomy: role of dedicated hyper- trophic cardiomyopathy centers in the management of dynamic subaortic obstruction. J Am Coll Cardiol. 2015;66:1307–08. 16. Dearani JA, Ommen SR, Gersh BJ, et al. Surgery insight: Septal myectomy for obstructive hypertrophic cardiomyopathy - the Mayo Clinic experience. Nat Clin Pract Cardiovasc Med. 2007;4:503–11. 27. Desai MY, Bhonsale A, Smedira NG, et al. Predictors of Long-Term Outcomes in Symptomatic Hypertrophic Obstructive Cardiomy- opathy Patients Undergoing Surgical Relief of Left Ventricular Outflow Tract Obstruction. Circulation. 2013;128:209-16. 17. Castedo E, Cabo RA, Núñez I, et al. Tratamiento quirúrgico de la miocardiopatía hipertrófica obstructiva. Rev Esp Cardiol 2004;57(8):751-6. 18. Matsuda H. Transatrial and transmitral myectomy for hyper- trophic obstructive cardiomyopathy of the left ventricle. Oper Tech Thorac Cardiovasc Surg. 2004;9:304-9. EsSalud \| 47
https://openalex.org/W4376139788	https://ijpbms.com/index.php/ijpbms/article/download/277/181	English	null	A Comprehensive Review of Organotin Complexes: Synthesis and Diverse Applications	International journal of pharmaceutical and bio-medical science	2,023	cc-by	2,777	1.1.3 Transmetallation Transmetallation involves the exchange of ligands between different metal complexes. Organotin complexes can be synthesized using transmetallation reactions where a tin- containing compound is reacted with a metal complex, resulting in the transfer of the tin atom to the metal complex and the formation of the organotin complex (9,10). The general equation for ligand substitution in an organotin complex can be represented as: [R3Sn(L1)(L2)...Ln] + L' → [R3Sn(L1)(L2)...(L' or Ln)] [ 3S ( )( 2) n] [ 3S ( 1)( 2) ( n)] In this equation, R represents an organic group such as alkyl or aryl, and L1, L2, ... Ln represent the existing ligands in the complex. The ligand substitution reaction involves the replacement of one or more of the existing ligands (L1, L2, ... Ln) with a new ligand (L'). The resulting product is a new organotin complex with a different combination of ligands. The general equation for a transmetallation reaction between a metal complex A and a tin-containing compound B can be written as: A-M + B-SnR3 → A-SnR3 + B-M where M represents the metal in complex A, R represents an organic group attached to the tin atom in compound B, and the arrow indicates the direction of the reaction. The result of the reaction is the formation of a new metal complex A-SnR3, which contains the tin atom transferred from compound B, and a new metal complex B-M, which may or may not be of interest in the context of the reaction. The exact mechanism and specific reaction conditions may vary depending on the nature of the organotin complex and the ligand being substituted (4,5). 1.1 Synthetic Methods 1.1 Synthetic Methods The general equation for a redox reaction involving a tin salt and a reducing agent can be represented as: Organotin complexes can be synthesized using a variety of methods. Some of the commonly employed synthetic routes include: g g p SnX2 + 2e- + 2H+ → SnR2 + 2HX where SnX2 is the tin salt, e- represents electrons, H+ represents protons, SnR2 is the organotin complex formed, and HX is the acid produced during the reaction. The reducing agent used in the reaction will depend on the specific system and can vary widely. where SnX2 is the tin salt, e- represents electrons, H+ represents protons, SnR2 is the organotin complex formed, and HX is the acid produced during the reaction. The reducing agent used in the reaction will depend on the specific system and can vary widely. 1.1.1 Ligand Substitution In this method, a pre-formed organotin complex is reacted with a suitable ligand to replace one or more of the existing ligands. This method allows for the modification of the coordination environment around the tin atom and provides access to a wide range of organotin complexes with different properties (1-3). ABSTRACT Organotin complexes are a class of coordination compounds that contain tin as a central metal atom bonded to organic ligands. These complexes have been extensively studied for their synthetic versatility and wide range of applications in various fields. Here is a review of organotin complexes, including their synthetic methods and applications. Available on: https://ijpbms.com/ KEYWORDS: Organotin, synthetic, applications, ligands. Corresponding Author: Angham G. Hadi International Journal of Pharmaceutical and Bio-Medical Science ISSN(print): 2767-827X, ISSN(online): 2767-830X Volume 03 Issue 05 May 2023 Page No: 181-184 International Journal of Pharmaceutical and Bio-Medical Science ISSN(print): 2767-827X, ISSN(online): 2767-830X Volume 03 Issue 05 May 2023 Page No: 181-184 International Journal of Pharmaceutical and Bio-Medical Science ISSN(print): 2767-827X, ISSN(online): 2767-830X Volume 03 Issue 05 May 2023 International Journal of Pharmaceutical and Bio-Medical Science ISSN(print): 2767-827X, ISSN(online): 2767-830X Volume 03 Issue 05 May 2023 Page No: 181-184 DOI: https://doi.org/10.47191/ijpbms/v3-i5-02, Impact Factor: 6.858 A Comprehensive Review of Organotin Complexes: Synthesis and Diverse Applications Zainab Al Talebi1, Ahmed Saleh Farhood2, Angham G. Hadi3 1,2,3 Department of Chemistry, College of Science, Babylon University, Babil 51002, Iraq 1. INTRODUCTION higher oxidation state to a lower oxidation state, leading to the formation of organotin complexes (6-8). higher oxidation state to a lower oxidation state, leading to the formation of organotin complexes (6-8). The general equation for a redox reaction involving a tin salt and a reducing agent can be represented as: SnX2 + 2e- + 2H+ → SnR2 + 2HX where SnX2 is the tin salt, e- represents electrons, H+ represents protons, SnR2 is the organotin complex formed, and HX is the acid produced during the reaction. The reducing agent used in the reaction will depend on the specific system and can vary widely. 1.2.2 Medicinal Chemistry Organotin complexes have shown potential as anti-cancer agents and anti-microbial agents. Some organotin complexes exhibit selective toxicity towards cancer cells, making them promising candidates for the development of anti-cancer drugs. Additionally, organotin complexes have been studied for their anti-fungal, anti-bacterial, and anti-viral activities, which can be utilized in the development of new anti- microbial agents (17-19). Organotin compounds are a type of 1.2 Applications Organotin complexes find diverse applications in various fields due to their unique properties. Some of the major applications of organotin complexes include: 1.2.1 Catalysis Organotin complexes serve as catalysts in a wide range of chemical reactions. For example, they are used as catalysts in cross-coupling reactions, such as the Stille coupling, Suzuki coupling, and Sonogashira coupling, which are important methods for the synthesis of organic compounds. Organotin complexes also exhibit catalytic activity in other types of reactions, such as hydrosilylation, hydroselenation, and polymerization reactions (14-16). 2 R-NCO + HO-R'-OH + dibutyltin dilaurate → R- NHCOOR'-OCOOR-SnR2R2 + HX where R and R' are organic groups, NCO is an isocyanate functional group, HO-R'-OH is a diol, and HX is a hydrogen halide byproduct. This reaction is known as the urethane reaction and is used extensively in the synthesis of polyurethanes. Organotin complexes have been found to be effective photostabilizers for a variety of materials, including polymers and coatings. These complexes are typically composed of an organotin compound, such as dibutyltin dilaurate, and a ligand that acts as a stabilizing agent. When added to a material, organotin complexes can absorb and dissipate ultraviolet radiation, protecting the material from degradation and discoloration caused by sunlight exposure. Additionally, organotin complexes can act as radical scavengers, intercepting free radicals generated by the photodegradation process and preventing further degradation (24-26). Due to their effectiveness and versatility, organotin complexes have become an important component in the design of photostable materials for a variety of industrial applications. However, their use has been limited in certain cases due to environmental concerns regarding the toxicity of organotin compounds. The general equation for the Stille coupling reaction catalyzed by an organotin complex can be written as: Ar-X + R-SnR3 → Ar-R + R-SnR3-X where Ar is an aryl group, X is a leaving group, R is an alkyl group, and SnR3 is an organotin catalyst. This reaction involves the formation of a new carbon-carbon bond between the aryl group and the alkyl group in the presence of the organotin catalyst. Similarly, the Suzuki coupling and Sonogashira coupling reactions also involve the formation of new carbon-carbon bonds and can be catalyzed by organotin complexes. The specific equations for these reactions depend on the choice of reactants and catalyst. A Comprehensive Review of Organotin Complexes: Synthesis and Diverse Applications chemical compound containing tin atoms bonded to organic groups, and they have been studied for their potential use as anti-cancer and anti-microbial agents. Some organotin complexes have shown promising results in preclinical studies, exhibiting selective toxicity towards cancer cells while sparing healthy cells. The exact mechanism of action of these complexes is not fully understood, but it is thought to involve the disruption of cellular processes crucial for cancer cell survival (20,21). 1.2.3 Materials Science R-SnR' + Ar-X → Ar-R' + R-SnR'X Organotin complexes are used in the synthesis of a variety of materials, including polymers, nanoparticles, and thin films. Organotin complexes are used in the synthesis of a variety of materials, including polymers, nanoparticles, and thin films. These materials have diverse applications in fields such as electronics, optics, and coatings. For example, organotin complexes can be used as precursors in the synthesis of organometallic polymers, which have unique properties such as high thermal stability, electrical conductivity, and optical properties (22,23). where R and R' are organic groups attached to the tin atom, Ar is an aryl group, and X is a leaving group, typically a halide. The reaction is catalyzed by a palladium complex, often Pd(PPh3)4, which coordinates with both the organotin compound and the organic substrate to facilitate the formation of the carbon-carbon bond. The reaction is typically carried out in an organic solvent such as tetrahydrofuran (THF) or toluene under an inert atmosphere. One example of an organotin complex used in materials science is dibutyltin dilaurate, which has the chemical formula (C4H9)2Sn(OOCCH3)2. This complex is commonly used as a catalyst in the synthesis of polyurethanes, which are versatile materials used in a variety of applications including adhesives, coatings, and foams. The reaction can be represented by the following equation: 1.1.4 Stille Coupling Stille coupling is a popular method for the synthesis of organotin complexes, particularly in organometallic chemistry. It involves the reaction of an organotin compound with an organic substrate in the presence of a palladium catalyst, resulting in the formation of a new carbon-carbon bond (11-13). The general equation for Stille coupling can be written as follows: 1.1.2 Redox Reactions Organotin complexes can also be synthesized through redox reactions involving tin salts and suitable reducing agents. These reactions typically result in the reduction of tin from a Corresponding Author: Angham G. Hadi 181 Volume 03 Issue 05 May Corresponding Author: Angham G. Hadi CONCLUSION XI. Jieping Zhu, Stille Coupling Reactions: Recent Progress in Palladium-Catalyzed Cross-Coupling Reactions, Angewandte Chemie International Edition, 1998. Organotin complexes are versatile coordination compounds with a wide range of synthetic methods and applications. They are widely studied for their potential in catalysis, medicinal chemistry, materials science, and agriculture. XII. Zvi Rappoport and Ilan Marek, Organotin Compounds in Modern Technology," G. P. Maruzen Petrochemical Co., Ltd., Japan, 2003. CONFLICTS OF INTEREST XIV. Lin, W. & Hayashi, T. Organotin compounds as versatile catalysts for various organic transformations. Chemical Society Reviews, 2015, 44, 1791–1802 https://doi.org/10.1039/C4CS00351H No conflict of interest. No conflict of interest. A Comprehensive Review of Organotin Complexes: Synthesis and Diverse Applications V. D. D. Kim, K. H. Kim, and Y. S. Chang, Organotin Compounds in Modern Technology: Applications and Health Effects, Journal of Hazardous Materials, vol. 195, pp. 1-12, 2011. effectiveness in controlling plant diseases caused by fungi and bacteria, making them important tools in modern agriculture (27-29). One example of an organotin complex used as a fungicide in agriculture is tributyltin oxide (TBTO), which has the chemical formula (C4H9)3SnO. Its use as a fungicide is based on its ability to inhibit the growth of fungi and bacteria by disrupting their cell membranes. VI. Kaur, M., Kumar, V., & Gupta, R. Recent Advances in Organotin(IV) Chemistry: An Overview. Applied Organometallic Chemistry, 2019, 33(1), e4504. https://doi.org/10.1002/aoc.4504 The mode of action of TBTO involves binding to the sulfhydryl groups (-SH) of enzymes involved in the synthesis of ergosterol, a vital component of fungal cell membranes. VII. Gopalakrishnan, R., Dey, A., & Dinda, R. Organotin(IV) Complexes: Synthesis, Characterization and Biological Applications. Inorganica Chimica Acta, 2019,485, 54-75. https://doi.org/10.1016/j.ica.2018.10.027 The mode of action of TBTO involves binding to the sulfhydryl groups (-SH) of enzymes involved in the synthesis of ergosterol, a vital component of fungal cell membranes. This binding results in the inhibition of ergosterol synthesis, which disrupts the integrity of the fungal cell membrane, leading to the leakage of cellular contents and ultimately, fungal death (30,31). This binding results in the inhibition of ergosterol synthesis, which disrupts the integrity of the fungal cell membrane, leading to the leakage of cellular contents and ultimately, fungal death (30,31). VIII. Maurya, M. R., & Singh, R. V. Organotin(IV) Complexes with Schiff Bases: Synthesis, Characterization and Biological Applications. Applied Organometallic Chemistry, 2017, 31(5), e3578. https://doi.org/10.1002/aoc.3578 The effectiveness of TBTO as a fungicide has been demonstrated in various crops such as rice, wheat, and soybeans, among others. In addition, TBTO has also been used as a bactericide in animal husbandry to control bacterial infections in livestock (32). IX. H. R. Allcock. Contemporary Polymer Chemistry. Prentice Hall, 2003. X. R. B. King, Ed. Encyclopedia of Inorganic Chemistry, Vol. 4. Chichester: Wiley, 1994. ACKNOWLEDGMENTS The authors would like to thank Babylon University and Ibn -Al-Haitham University for kind support. XIII. "The Chemistry of Organic Tin Compounds," John Wiley & Sons, 2007. 1.2.4 Agriculture Organotin complexes are used as fungicides and bactericides in agriculture to protect crops from fungal and bacterial diseases. Some organotin complexes have shown high 182 Volume 03 Issue 05 May Corresponding Author: Angham G. Hadi Corresponding Author: Angham G. Hadi 184 Volume 03 Issue 05 May REFERENCES I. Wang, J. Y.; Lu, J. M.; Zhang, H. L.; Song, Y. C.; Wang, H. L. Synthesis, crystal structure and biological activity of organotin(IV) complexes with pyridine carboxylic acids. J. Inorg. Biochem. 2006, 100, 1529–1535. XV. Manna, S., Bhattacharyya, A. & Sarkar, T. Organotin(IV) complexes: their synthesis, structure, and applications in organic transformations. RSC Advances, 2015, 5, 26917–26946. https://doi.org/10.1039/C5RA02711B XVI. Yin, S., Chen, J., Zhang, J. et al. Organotin compounds in organic synthesis: recent advances. Chemistry Central Journal, 2017, 11, 20. https://doi.org/10.1186/s13065-017-0247-5 II. Chohan, Z. H.; Pervez, H.; Rauf, A.; Khan, K. M.; Supuran, C. T. Synthesis and biological evaluation of organotin(IV) complexes of sulfonamide Schiff bases derived from 4-aminoantipyrine. Eur. J. Med. Chem. 2008, 43, 1732–1740. XVII. Sreeja R. K., Sathyanarayana S., Nair M. S., Kumar R. R., Nair C. G. R., Jose G. P. Organotin compounds in cancer chemotherapy: an update. Future Science OA, 2018, 4(10), FSO336. doi: 10.4155/fsoa-2018- 0079 III. Ferreira, R. B.; Gomes, C. S. B.; Teixeira, L. R.; Paz, F. A. A.; Correia, I.; Pombeiro, A. J. L. Synthesis, characterization and DFT studies of some new organotin(IV) derivatives of 1-phenyl-3-methyl-4- (2′-pyridyl)-5-pyrazolone. Polyhedron 2011, 30, 571–579. XVIII. Zhang, W., Zheng, K., Liu, Y., Jiang, J., Huang, J., & Zhong, Z. (2019). Organotin(IV) complexes as anticancer agents: A review. European Journal of Medicinal Chemistry, 2019, 180, 562-581. doi: 10.1016/j.ejmech.06.011 IV. M. L. Scudder and R. T. Baker, Organotin Compounds: Chemistry and Applications, John Wiley & Sons, Inc., 2004. 183 Volume 03 Issue 05 May Corresponding Author: Angham G. Hadi A Comprehensive Review of Organotin Complexes: Synthesis and Diverse Applications XIX. Jain, R., & Jain, N. Antimicrobial activity of organotin(IV) compounds: A review. Applied Organometallic Chemistry, 2017, 31(5), e3575. doi: 10.1002/aoc.3575 XXVI. Arraq, R. R., Hadi, A. G., Ahmed, D. S., El-Hiti, G. A., Kariuki, B. M., Husain, A. A., ... & Yousif, E. Enhancement of Photostabilization of Poly (Vinyl Chloride) in the Presence of Tin–Cephalexin Complexes. Polymers, 2023, 15(3), 550. XX. Al-Majedy YK, Al-Jumaili AS, Al-Rubaye AA, Al- Salihi NAA. Synthesis, characterization, and biological activity of some organotin(IV) complexes with Schiff bases derived from 2-hydroxy-1- naphthaldehyde and amino acids. J Coord Chem. 2021;74(9):1629-1650. doi:10.1080/00958972.2021.1920892 XXVII. Xu, Y., Li, J., Liu, X., Zhang, L., & Wu, Y. Recent progress in the development of organotin compounds as pesticides. Current Opinion in Chemical Biology, 2020, 57, 58-66. doi: 10.1016/j.cbpa.2020.04.013 XXVIII. Zhang, Y., Zheng, M., Wang, Q., Zhao, X., & Yu, X. Recent advances in organotin(IV) complexes with antimicrobial and antifungal activities. Coordination Chemistry Reviews, 2021, 443, 214033. doi: 10.1016/j.ccr.2021.214033 XXI. Singh V, Rawat DS. Organotin compounds as anticancer agents: A review. Chem Biol Drug Des. 2018;92(4):1627-1644. doi:10.1111/cbdd.13368 XXII. Guo, S., Song, S., & Dai, J. Organotin(IV) Complexes with Schiff Base Ligands: Synthesis, Characterization, and Applications. Molecules, 2018, 23(8), 2026. doi: 10.3390/molecules23082026 XXIX. Alcantara, A. F., Navarro, M. C., Martínez-Máñez, R., Sancenón, F., & Rurack, K. Recent advances in the design and application of chemosensors and biosensors for the detection of organotin compounds. TrAC Trends in Analytical Chemistry, 2021, 137, 116219. doi: 10.1016/j.trac.2021.116219 XXIII. Liao, Y., Wang, L., & Han, B. Synthesis and Applications of Organotin Polymers. Polymer Reviews, 2016, 56(3), 405-432. doi: 10.1080/15583724.2015.1125155 XXX. Eiichi Terao, Toshikatsu Arai, and Shigeru Ishizaki, Mode of Action of Tri-n-Butyltin Oxide on Fungi, Pesticide Science, 1997. XXIV. HADI, Angham G., et al. Photostabilization of poly (vinyl chloride) by organotin (IV) compounds against photodegradation. Molecules, 2019, 24.19: 3557. XXXI. S.K. Sahoo, K.K. Hazra, and S. Kundu, Evaluation of the efficacy of tri-n-butyltin oxide against sheath blight disease in rice, Crop Protection, 2017. XXV. Hadi, A. G.;Yousif, E.; El-Hiti, G. A.; Ahmed, D. S.; Jawad, K.; Alotaibi, M. H.; Hashim, H. Long-term effect of ultraviolet irradiation on poly (vinyl chloride) films containing naproxen diorganotin (IV) complexes. Molecules 2019,24, 2396. http://dx.doi.org/10.3390/molecules24132396 XXXII. K. Suzuki, K. Endo, and T. Kaneko, In vitro bactericidal activity of tri-n-butyltin oxide against veterinary pathogens, Journal of Veterinary Medical Science, 2003.
https://openalex.org/W2524981236	https://europepmc.org/articles/pmc5265968?pdf=render	English	null	Risk factors for early mortality after hepatectomy for hepatocellular carcinoma	Medicine	2,016	cc-by	8,313	Abstract Abstract Despite advances in surgical technique and medical care, liver resection for hepatocellular carcinoma (HCC) remains a high-risk major operation. The present study evaluated the risk factors for early mortality after hepatectomy. We retrospectively reviewed records of patients undergoing liver resection for HCC between 1983 and 2015. A point score (Risk Assessment for early Mortality (RAM) score) for hepatectomy was developed based on multivariate analyses. Three hundred eighty-three patients (11.3%) expired within 6 months after the operation. Logistic regression analyses identiﬁed that operative duration >270minutes and blood loss >800cc were signiﬁcant predictors of major surgical complications (P=0.013 and 0.002, respectively). On the other hand, diabetes mellitus, albumin 3.5g/dL, a-fetoprotein (AFP) >200ng/mL, major surgical procedure, blood loss >800cc, and major surgical complications were independent risk factors for early mortality after hepatectomy (P=0.019, <0.001, <0.001, 0.006, 0.018, and <0.001, respectively). Risk Assessment for early Mortality score (RAM score) identiﬁed 3 subgroups of patients with distinct 6-month mortality rate, with Class III (score 10) having highest risk of early mortality. identiﬁed 3 subgroups of patients with distinct 6-month mortality rate, with Class III (score 10) having highest risk of early mortality. Our study demonstrated that meticulous surgical techniques to minimize blood loss and avoid prolonged operative time may help decrease the occurrence of major surgical complications. In addition to major surgical complications, diabetes mellitus, hypoalbuminemia, high AFP, massive blood loss, and major surgical procedure are also associated with early mortality after liver resection. Further study is warranted to validate the utility of RAM score as a bedside scoring system to predict postoperative outcome. Our study demonstrated that meticulous surgical techniques to minimize blood loss and avoid prolonged operative time may help decrease the occurrence of major surgical complications. In addition to major surgical complications, diabetes mellitus, hypoalbuminemia, high AFP, massive blood loss, and major surgical procedure are also associated with early mortality after liver resection. Further study is warranted to validate the utility of RAM score as a bedside scoring system to predict postoperative outcome. Abstract Abbreviations: AFP = a-fetoprotein, AJCC = American Joint Committee on Cancer, ALT = alanine aminotransferase, AST = aspartate aminotransferase, CT = computed tomography, DFS = disease-free survival, ECOG = Eastern Cooperative Oncology Group, ER = emergency room, HBV = hepatitis B virus, HCC = hepatocellular carcinoma, HCV = hepatitis C virus, ICG-15 = indocyanine green retention at 15min, ICU = intensive care unit, MRI = magnetic resonance imaging, OS = overall survival, RAM score = Risk Assessment for early Mortality score, TACE = transarterial chemoembolization. Keywords: hepatectomy, hepatocellular carcinoma, hepatoma, liver resection, mortality, RAM score, risk factors Risk factors for early mortality after hepatectomy for hepatocellular carcinoma Chao-Wei Lee, MDa,b,c, Hsin-I Tsai, MDc,d, Chang-Mu Sung, MDa,e, Chun-Wei Chen, MDe, Shu-Wei Huang, MDe, Wen-Juei Jeng, MD, PhDe, Tsung-Han Wu, MDa, Kun-Ming Chan, MDa,b, Ming-Chin Yu, MDa,b,c,∗, Wei-Chen Lee, MDa,b, Miin-Fu Chen, MDa,b Chao-Wei Lee, MDa,b,c, Hsin-I Tsai, MDc,d, Chang-Mu Sung, MDa,e, Chun-Wei Chen, MDe, Shu-Wei Huang, MDe, Wen-Juei Jeng, MD, PhDe, Tsung-Han Wu, MDa, Kun-Ming Chan, MDa,b, Ming-Chin Yu, MDa,b,c,∗, Wei-Chen Lee, MDa,b, Miin-Fu Chen, MDa,b Chao-Wei Lee, MDa,b,c, Hsin-I Tsai, MDc,d, Chang-Mu Sung, MDa,e, Chun-Wei Chen, MDe, Shu-Wei Huang, MDe, Wen-Juei Jeng, MD, PhDe, Tsung-Han Wu, MDa, Kun-Ming Chan, MDa,b, Ming-Chin Yu, MDa,b,c,∗, Wei-Chen Lee, MDa,b, Miin-Fu Chen, MDa,b Medicine ® OPEN Observational Study ∗Correspondence: Ming-Chin Yu, Department of Surgery, Chang Gung Memorial Hospital, No. 5, Fuxing St., Guishan Dist., Taoyuan City 333, Taiwan (R.O.C.) (e-mail: mingchin2000@gmail.com). 1. Introduction it is the second most common cause of cancer death and causes more than 8000 deaths each year.[2] Surgical resection remains the most effective therapy in selected patients, but the coexisting underlying liver diseases, such as chronic hepatitis B or C and alcoholic liver disease, had limited the extent and feasibility of liver resection. Earlier before 1980s, liver resections in the presence of liver cirrhosis was associated with a relatively high mortality rate in the range of 10% to 30%, and were therefore largely limited to minor resections.[3–10] With improvements in patient selection, surgical techniques, and postoperative care, the mortality rate has improved dramatically in recent decades.[3,11–13] The unexpected occurrence of death after the operation, however, is still catastrophic to both the patients’ family and surgeon. As a result, the identiﬁcation of potential risk factors for mortality before operation is of paramount importance. Previous studies had demonstrated multiple risk factors for perioperative morbidity and mortality after liver resection[3,12,14]; neverthe- less, the study end-point in most series was set at 30 days after the operation. Since many patients could survive the ﬁrst postoperative month but still suffered from mortality several months after the operation, the aforementioned recognized risk factors may be less applicable. The purpose of this study was therefore to unravel the potential risk factors for in-hospital and 6-month mortality and propose a scoring system in an attempt to predict postoperative outcome and risk of early mortality immediately after the operation. Hepatocellular carcinoma (HCC) is the most common primary malignancy of the liver with an estimated annual death incidence of approximately 700,000 worldwide.[1] In Taiwan, Editor: Maria Kapritsou. Funding: This study was supported by Chang Gung Memorial Hospital (CMRPG3D0291, to CWL). The authors have no conﬂicts of interest to disclose. Supplemental Digital Content is available for this article. a Department of Surgery, Chang Gung Memorial Hospital, Linkou, b College of Medicine, Chang Gung University, Guishan, Taoyuan, c Graduate Institute of Clinical Medical Sciences, Chang Gung University, Guishan, Taoyuan, d Department of Anesthesiology,Chang Gung Memorial Hospital, Linkou, e Department of Gastroenterology and Hepatology, Chang Gung Memorial Hospital, Linkou, Taiwan (R.O.C.). ∗Correspondence: Ming-Chin Yu, Department of Surgery, Chang Gung Memorial Hospital, No. 5, Fuxing St., Guishan Dist., Taoyuan City 333, Taiwan (R.O.C.) (e-mail: mingchin2000@gmail.com). Copyright © 2016 the Author(s). Published by Wolters Kluwer Health, Inc. All rights reserved. Editor: Maria Kapritsou. Editor: Maria Kapritsou. Funding: This study was supported by Chang Gung Memorial Hospital (CMRPG3D0291, to CWL). The authors have no conﬂicts of interest to disclose. Supplemental Digital Content is available for this article. a Department of Surgery, Chang Gung Memorial Hospital, Linkou, b College of Medicine, Chang Gung University, Guishan, Taoyuan, c Graduate Institute of Clinical Medical Sciences, Chang Gung University, Guishan, Taoyuan, d Department of Anesthesiology,Chang Gung Memorial Hospital, Linkou, e Department of Gastroenterology and Hepatology, Chang Gung Memorial Hospital, Linkou, Taiwan (R.O.C.). ∗Correspondence: Ming-Chin Yu, Department of Surgery, Chang Gung Memorial Hospital, No. 5, Fuxing St., Guishan Dist., Taoyuan City 333, Taiwan (R.O.C.) (e-mail: mingchin2000@gmail.com). Copyright © 2016 the Author(s). Published by Wolters Kluwer Health, Inc. All rights reserved. This is an open access article distributed under the Creative Commons Attribution License 4.0 (CCBY), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Medicine (2016) 95:39(e5028) Received: 8 June 2016 / Received in ﬁnal form: 31 August 2016 / Accepted: 1 September 2016 http://dx.doi.org/10.1097/MD.0000000000005028 Funding: This study was supported by Chang Gung Memorial Hospital (CMRPG3D0291, to CWL). Received: 8 June 2016 / Received in ﬁnal form: 31 August 2016 / Accepted: 1 September 2016 2.3. Deﬁnition Preoperative, intraoperative, and postoperative data were retrieved from a prospectively collected database. Major liver resection deﬁned resection of three or more liver segments.[20] Major surgical complications comprised grade III and grade IV surgical complications,[21] which included postoperative bleeding requiring angiographic embolization or reoperation, major biliary complications requiring drainage or endoscopic interven- tion, intestinal obstruction requiring operation, upper gastroin- testinal bleeding requiring endoscopic hemostasis, massive ascites or pleural effusion requiring paracentesis, sepsis of any etiology, liver failure, renal failure, respiratory failure, or any condition dictating ICU care. Thirty-day mortality was deﬁned as the occurrence of death within 30 days after the operation. In- hospital mortality was deﬁned as death during the same hospital stay, and 6-month, or early mortality was deﬁned as the occurrence of death within 6 months after the operation. The cause of early mortality included HCC recurrence/metastasis, hepatic failure due to liver cirrhosis, and postoperative surgical complications. Overall survival (OS) was deﬁned by the time elapsing from the date of diagnosis to either the date of death or the date of the last contact. 2.4. Statistical analysis The statistical analysis was performed with IBM SPSS Statistics Version 21 for Windows (IBM Corporation, NY). Fisher exact test or Pearson x2 test was used to analyze categorical data. Student t test was used to analyze continuous variables. Statistical signiﬁcance was deﬁned as P values<0.05 in 2-sided tests. Signiﬁcant variables in the univariate analysis were then subjected into a stepwise logistic regression analysis (conditional forward selection) as candidate variables. The regression coefﬁcients of the independent variables identiﬁed by logistic regression model were multiplied by two and rounded to integer in order to calculate the Risk Assessment for early Mortality score (RAM score). The inﬂuence of the RAM score on OS and early mortality was then examined by Kaplan–Meier analysis and Log rank test. All operations were performed by experienced hepatobiliary surgeons in the same surgical department. In all patients, intra- operative exploration by both manual palpation and ultrasonog- raphy was conducted to deﬁne the extent of the tumor(s), any invasion of the portal or hepatic veins, the texture of liver parenchyma, and the size of future liver remnant. Inﬂow control, which included Pringle maneuver, Glissonian pedicle control,[18,19] selective vascular control, and total vascular exclusion, was applied whenever necessary according to individual surgeon’s discretion. Parenchymal transection was performed by using either crush clamp technique, ultrasonic dissector, or other energy device based on surgeon’s preference. Hemostasis was achieved and bile leakage was repaired meticulously in each operation. 2.2. Preoperative assessment, surgical technique, and postoperative management Preoperative diagnosis of HCC was established by characteristic features on imaging by either triphasic computed tomography (CT), magnetic resonance imaging (MRI), hepatic arteriography, and/or a serum a-fetoprotein (AFP) level greater than 200ng/mL. Resection criteria were constant over the entire study period, including a lack of cancerous thrombi in the main trunk of the portal vein, no distant metastasis to other organs, a technically operable main tumor in the preoperative evaluation, and a adequate liver functional reserve. Liver function was routinely assessed preoperatively by Child–Pugh classiﬁcation and indoc- yanine green retention test. A previous study identiﬁed an indocyanine green retention at 15minutes (ICG-15) of less than 14% as the safety limit for major hepatic resection.[16] In our institute, an ICG-15 10% was the prerequisite for major hepatic resection. On the other hand, in patients with higher ICG- 15, extensive hepatectomy could also be performed if the liver biochemistry was satisfactory and the size of the future liver remnant was considered adequate according to preoperative CT and intraoperative assessment.[17] 2.1. Patients From January 1983 to January 2015, records of patients with histologically proven primary HCC from the Cancer Registry of the Cancer Center, Chang Gung Memorial Hospital, Linkou, Taiwan were retrospectively reviewed. Only patients who underwent curative hepatectomy by our surgical team were included in the study. Patients who underwent only exploratory laparotomy for liver tumor biopsy, who had multiple distant metastases before operation, who did not have detailed preoperative/intraoperative clinical records, or who did not have regular postoperative out-patient follow-up were excluded from our study. A total of 3383 patients were evaluated eventually, and their clinicopathological data were retrieved from the prospectively collected database. Our primary study endpoints were risk factors for major complication and 6-month (early) mortality. The RAM (Risk Assessment for early Mortality) score was established based on risk factors for 6-month mortality. The secondary endpoints were risk factors for 30-day and in-hospital mortality. The study end date was December 31, 2015, and tumor staging was based on the American Joint Committee on Cancer (AJCC) TNM staging system for HCC.[15] This study was approved by the Institutional Review Boards of Chang Gung Memorial Hospital (CGMH IRB No: 201600359B0). 2. Materials and methods individual patient’s condition. The duration of ICU stay depended on individual patient’s clinical condition. Intravenous crystalloid ﬂuids were given to maintain ﬂuid requirement. First line broad-spectrum antibiotics were administered intravenously for 3 to 7 days in all patients. Fresh frozen plasma or albumin was given if the plasma albumin level was lower than 3.0g/dL. Hemogram and biochemical liver function tests were examined 2 and 7 days after liver resection. Oral feeding or enteral nutrition was encouraged and resumed as early as possible after operation. Parenteral nutrition was provided if patient was malnourished or enteral nutrition could not be resumed 5 to 7 days after operation. All patients received blood tests and triphasic CT examination 1 to 2 months after operation. Out-patient follow-up with serial lab tests and image study was arranged every 2 to 3 months after hospital discharge. 1. Introduction This is an open access article distributed under the Creative Commons Attribution License 4.0 (CCBY), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Medicine (2016) 95:39(e5028) Received: 8 June 2016 / Received in ﬁnal form: 31 August 2016 / Accepted: 1 September 2016 http://dx.doi.org/10.1097/MD.0000000000005028 Editor: Maria Kapritsou. Funding: This study was supported by Chang Gung Memorial Hospital (CMRPG3D0291, to CWL). The authors have no conﬂicts of interest to disclose. Supplemental Digital Content is available for this article. a Department of Surgery, Chang Gung Memorial Hospital, Linkou, b College of Medicine, Chang Gung University, Guishan, Taoyuan, c Graduate Institute of Clinical Medical Sciences, Chang Gung University, Guishan, Taoyuan, d Department of Anesthesiology,Chang Gung Memorial Hospital, Linkou, e Department of Gastroenterology and Hepatology, Chang Gung Memorial Hospital, Linkou, Taiwan (R.O.C.). ∗Correspondence: Ming-Chin Yu, Department of Surgery, Chang Gung Memorial Hospital, No. 5, Fuxing St., Guishan Dist., Taoyuan City 333, Taiwan (R.O.C.) (e-mail: mingchin2000@gmail.com). Copyright © 2016 the Author(s). Published by Wolters Kluwer Health, Inc. All rights reserved. This is an open access article distributed under the Creative Commons Attribution License 4.0 (CCBY), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Medicine (2016) 95:39(e5028) Received: 8 June 2016 / Received in ﬁnal form: 31 August 2016 / Accepted: 1 September 2016 http://dx.doi.org/10.1097/MD.0000000000005028 The authors have no conﬂicts of interest to disclose. This is an open access article distributed under the Creative Commons Attribution License 4.0 (CCBY), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Medicine (2016) 95:39(e5028) Received: 8 June 2016 / Received in ﬁnal form: 31 August 2016 / Accepted: 1 September 2016 http://dx.doi.org/10.1097/MD.0000000000005028 1 Lee et al. Medicine (2016) 95:39 Medicine 3.1. Patient demographics and operative variables A total of 3386 patients with HCC underwent liver resection during the study period. The median follow-up time was 39.3 months. Their demographic and clinical data are summarized in Patients were monitored and cared postoperatively in either intensive care unit (ICU) or general wards depending on 2 Lee et al. Medicine (2016) 95:39 www.md-journal.com Table 1 Demographic data of patients with hepatocellular carcinoma undergoing hepatectomy (n=3386). Variables ∗ No. (%) Variables ∗ Mean±SE Age (65 vs >65 year-old) 2360 (69.7) vs 1026 (30.3) Age, year-old 57.28±0.229 Gender (male vs female) 2647 (78.2) vs 739 (21.8) ICG-15, % 11.09±0.20 Comorbidity (yes) 1034 (31.2) Hemoglobin, g/dL 13.25±0.35 Diabetes mellitus (yes) 630 (19) Albumin, g/dL 4.03±0.01 Hypertension (yes) 486 (30.1) Platelet, 1000/mL 178.46±1.44 ESRD (yes)† 68 (2.1) INR 1.08±0.002 Old stroke (yes) 42 (2.6) ALT, U/L 58.17±1.20 Smoking (yes) 1015 (30.6) Bilirubin total, mg/dL 0.93±0.02 Alcohol (yes) 698 (21.8) Alkaline phosphatase, U/L 100.7±3.33 HBV surface antigen (positive) 2018 (65.3) Preoperative a-fetoprotein, ng/mL 8249.4588±1699.2 Hepatitis C virus (positive) 963 (35.7) Preoperative CEA, ng/mL 6.05±1.45 Non-B Non-C (yes) 363 (11.2) Preoperative CA-199, U/mL 457.99±265.76 Child–Pugh classiﬁcation (A/B/C) 3156 (95.5)/146 (4.4)/2 (0.1) OP duration, min 270.45±1.83 Symptoms (yes)‡ 1370 (40.5) Blood loss, mL 790.03±21.09 Preoperative treatment (yes) 274 (8.1) Tumor size, cm 5.488±0.72 Procedure (major resection), %x 1348 (41.6) Inﬂow control, yes 1475 (75.8) Procedure type, % Surgical complications (major vs minor/none)¶ 215 (11.0) vs 1742 (89.0) Right lobectomy 452 (13.9) Left lobectomy 230 (7.1) Extended right lobectomy 134 (4.1) Extended left lobectomy 53 (1.6) Trisegmentectomyjj 479 (14.8) Bisegmentectomy ∗∗ 1114 (34.4) Single segmentectomy†† 733 (22.6) Wedge resection 48 (1.5) ALT=alanine aminotransferase, CEA=carcinoembryonic antigen, CA 19-9=carbohydrate antigen 19-9, HBV=hepatitis B virus, ICG-15=indocyanine green retention at 15min, INR=international normalized ratio, OP=operation, SD=standard error. ∗Only patients with available data were analyzed. † End-stage renal disease. ‡ Include HCC presenting with anemia, jaundice, palpable mass, abdominal pain or ascites. x Includes trisegmentectomy, right/left lobectomy, and extended right/left lobectomy. ¶ Major surgical complications include grade III–IV surgical complications. jj Include central lobectomy. ∗∗Include left lateral sectionectomy. †† Include nonanatomical partial hepatectomy g ‡ Include HCC presenting with anemia, jaundice, palpable mass, abdominal pain or ascites. x Includes trisegmentectomy, right/left lobectomy, and extended right/left lobectomy g y, g y, g ¶ Major surgical complications include grade III–IV surgical complications. ∗∗Include left lateral sectionectomy. †† Include nonanatomical partial hepatectomy. †† Include nonanatomical partial hepatectomy. 3.1. Patient demographics and operative variables intensive critical care with organ support systems were employed in these patients. Table 1. Among patients who did not receive liver resection immediately after diagnosis, transarterial chemoembolization (TACE) was the most common preoperative treatment (254 patients, 92.7%), followed by percutaneous ethanol injection (3.8%) and radiofrequency ablation (2.9%). Major resection was conducted in 1348 patients (41.6%), with trisegmentectomy being the most common major resection procedure (14.8%), followed by right hepatectomy (13.9%), left hepatectomy (7.1%), extended right hepatectomy (4.1%), and extended left hepatectomy (1.6%). Pringle maneuver (87.7% of all inﬂow control) remained the most common form of hepatic inﬂow control during operation. For risk factor analysis, statistical analysis was conducted and the results are summarized in Table 2. Eighteen preoperative variables and 4 operative variables that may potentially affect the operative outcome were included in the initial univariate analysis. Variables with a P-value less than 0.05 by univariate analysis were subjected to a stepwise multivariate logistic regression analysis. The result showed that operative duration greater than 270minutes (hazard ration (HR) 1.946; P=0.013) and blood loss greater than 800mL (HR 2.299; P=0.002) were 2 independent risk factors for the occurrence of major surgical complications after hepatectomy. 3.3. Risk factors for 30-day and in-hospital mortality Among 3386 patients, 1957 had detailed records of their postoperative complications and were analyzed. Among them, 1250 patients (63.9%) were either uneventful or experiencing only grade I complications during postoperative period. Grade II complications occurred in 491 patients (25.1%) and they can be treated with pharmacological therapy, parenteral nutrition, or blood transfusion. Grade III complications happened in 147 patients (7.5%), and these patients required either surgical, endoscopic, or radiological interventions. Life-threatening, or grade IV complications developed in 68 patients (3.4%), and Sixty-one patients (1.8%) died within 30 days after hepatectomy. The mean hospital stay for these patients was 12.5 days (range 0–30 days). Statistical analysis of 18 preoperative variables and 6 operative variables was conducted and is summarized in Table 3. On the other hand, 97 patients (2.9%) died during the same hospitalization of hepatectomy. The mean length of survival of these patients was 24.5 days (range 0–123 days). Risk factors for in-hospital mortality were analyzed and are summa- rized in Table 4. 3 Lee et al. Medicine (2016) 95:39 Medicine Table 2 Univariate and multivariate analyses of risks factors for major complications after hepatectomy for hepatocellular carcinoma (n=1957). 3.3. Risk factors for 30-day and in-hospital mortality Univariate Multivariate Variables Cases with major complication (%) Odds ratio P Hazard ratio (95% CI) P Age (≥80 vs <80 year-old) 20 (27.4) vs 195 (10.4) 3.269 <0.001 2.289 (0.937–5.592) 0.069 Gender (male vs female) 178 (11.6) vs 37 (8.6) 1.393 0.080 Diabetes mellitus (yes vs no) 65 (15.9) vs 147 (9.5) 1.798 <0.001 1.638 (0.960–2.795) 0.070 Hypertension (yes vs no) 69 (15.8) vs 93 (9.6) 1.769 0.001 1.560 (0.938–2.594) 0.086 End-stage renal disease (yes vs no) 8 (20) vs 204 (10.7) 2.092 0.061 Old stroke (yes vs no) 8 (21.1) vs 154 (11.3) 2.099 0.063 Smoking (yes vs no) 55 (12) vs 160 (10.7) 1.146 0.413 Alcohol (yes vs no) 35 (11.9) vs 180 (10.8) 1.113 0.585 HBs Ag (positive vs negative) 98 (9.1) vs 82 (12.5) 0.699 0.024 1.160 (0.727–1.850) 0.534 Hepatitis C virus (positive vs negative) 60 (10.5) vs 113 (11.1) 0.945 0.739 ICG-15 (>10 vs 10), % 86 (13.2) vs 112 (9.5) 1.449 0.015 1.464 (0.920–2.332) 0.108 Hemoglobin (10 vs >10), g/dL 29 (24.2) vs 186 (10.1) 2.828 <0.001 1.561 (0.704–3.463) 0.273 Albumin (3.5 vs >3.5), g/dL 57 (25.4) vs 152 (9.0) 3.453 <0.001 1.780 (0.986–3.215) 0.056 Platelet (100 vs >100), 1000/mL 34 (13.8) vs 181 (10.6) 1.349 0.136 INR (>1.4 vs 1.4) 3 (50) vs 209 (10.8) 8.249 0.002 NA 0.999 ALT (>40 vs 40), U/L 108 (12.2) vs 103 (9.8) 1.271 0.100 Bilirubin total (>1.5 vs 1.5), mg/dL 22 (22.2) vs 191 (10.3) 2.488 <0.001 1.213 (0.482–3.051) 0.682 a-fetoprotein (>200 vs 200), ng/mL 75 (13.5) vs 135 (9.8) 1.444 0.016 1.088 (0.661–1.791) 0.740 Procedure (major vs minor), % ∗ 111 (18.4) vs 100 (7.5) 2.794 <0.001 1.644 (0.956–2.828) 0.072 OP duration (>270 vs 270), min 136 (16.8) vs 70 (6.3) 3.014 <0.001 1.946 (1.152–3.286) 0.013 Blood loss (>800 vs 800), mL 78 (29.2) vs 123 (7.6) 5.006 <0.001 2.299 (1.358–3.892) 0.002 Inﬂow control (yes vs no) 149 (10.9) vs 30 (6.9) 1.650 0.015 1.222 (0.685–2.180) 0.497 Tumor size (>10 vs 10), cm 56 (23.3) vs 153 (9.0) 3.075 <0.001 1.594 (0.856–2.968) 0.141 ALT=alanine aminotransferase, CI=conﬁdence interval, HBs Ag=hepatitis B surface antigen, ICG-15=indocyanine green retention at 15min, INR=international normalized ratio, OP=operation. ∗Includes trisegmentectomy, right/left lobectomy, and extended right/left lobectomy. Table 3 Univariate and multivariate analyses of risks factors for 30-day mortality after hepatectomy for hepatocellular carcinoma. ALT=alanine aminotransferase, CI=conﬁdence interval, HBs Ag=hepatitis B surface antigen, ICG-15=indocyanine green retention at 15min, INR=international normalized ratio, OP=operation. ∗Includes trisegmentectomy, right/left lobectomy, and extended right/left lobectomy. ALT=alanine aminotransferase, CI=conﬁdence interval, HBs Ag=hepatitis B surface antigen, ICG-15=indocyanine green retention at 15min, INR=international normalized ratio, OP=operation. ∗Includes trisegmentectomy, right/left lobectomy, and extended right/left lobectomy. † Major surgical complications include grade III–IV surgical complications. t lobectomy, and extended right/left lobectomy. e grade III–IV surgical complications. ALT=alanine aminotransferase, CI=conﬁdence interval, HBs Ag=hepatitis B surface antigen, ICG-15=indocyanine green retention at ∗Includes trisegmentectomy, right/left lobectomy, and extended right/left lobectomy. 3.3. Risk factors for 30-day and in-hospital mortality Variables Univariate Multivariate Cases with 30-day mortality (%) Odds ratio P Hazard ratio (95% CI) P Age (≥80 vs <80 year-old) 3 (3.9) vs 58 (1.8) 2.272 0.162 Gender (male vs female) 46 (1.7) vs 15 (2.0) 0.853 0.598 Diabetes mellitus (yes vs no) 17 (2.7) vs 43 (1.6) 1.701 0.064 Hypertension (yes vs no) 10 (2.1) vs 32 (2.8) 0.718 0.364 End-stage renal disease (yes vs no) 3 (4.4) vs 57 (1.8) 2.580 0.104 Old stroke (yes vs no) 2 (4.8) vs 40 (2.5) 1.913 0.374 Smoking (yes vs no) 19 (1.9) vs 41 (1.8) 1.052 0.855 Alcohol (yes vs no) 15 (2.1) vs 46 (1.8) 1.170 0.601 HBs Ag (positive vs negative) 37 (1.8) vs 21 (2.0) 0.934 0.804 Hepatitis C virus (positive vs negative) 25 (2.6) vs 27 (1.6) 1.684 0.061 ICG-15 (>10 vs 10),% 25 (2.4) vs 17 (1.1) 2.300 0.007 1.359 (0.557–3.317) 0.501 Hemoglobin (10 vs >10), g/dL 8 (3.1) vs 53 (1.7) 1.858 0.102 Albumin (3.5 vs >3.5), g/dL 34 (6.5) vs 19 (0.7) 9.711 <0.001 2.791 (1.154–6.748) 0.023 Platelet (100 vs >100), 1000/mL 19 (3.8) vs 40 (1.4) 2.766 <0.001 2.281 (0.765–6.796) 0.139 INR (>1.4 vs 1.4) 4 (9.5) vs 54 (1.8) 5.735 <0.001 117952551.3 0.999 ALT (>40 vs 40), U/L 36 (2.3) vs 20 (1.2) 1.904 0.020 1.148 (0.487–2.706) 0.752 Bilirubin total (>1.5 vs 1.5), mg/dL 16 (5.9) vs 44 (1.4) 4.372 <0.001 1.225 (0.287–5.235) 0.784 a-fetoprotein (>200 vs 200), ng/mL 27 (2.5) vs 31 (1.4) 1.770 0.030 1.372 (0.582–3.235) 0.470 Procedure (major vs minor), % ∗ 34 (2.6) vs 20 (1.0) 2.470 0.001 1.801 (0.654–4.954) 0.255 OP duration (>270 vs 270), min 27 (2.0) vs 24 (1.3) 1.512 0.141 Blood loss (>800 vs 800), mL 28 (3.3) vs 22 (1.0) 3.565 <0.001 1.292 (0.527–3.168) 0.575 Inﬂow control (yes vs no) 25 (1.7) vs 4 (0.9) 2.009 0.189 Tumor size (>10 vs 10), cm 10 (1.9) vs 42 (1.5) 1.268 0.503 Surgical complications (major vs minor/none)† 36 (16.7) vs 3 (0.2) 116.581 <0.001 55.775 (15.97–194.77) <0.001 ALT=alanine aminotransferase, CI=conﬁdence interval, HBs Ag=hepatitis B surface antigen, ICG-15=indocyanine green retention at 15min, INR=international normalized ratio, OP=operation. ∗Includes trisegmentectomy, right/left lobectomy, and extended right/left lobectomy. † Major surgical complications include grade III–IV surgical complications. 4 Lee et al. Medicine (2016) 95:39 www.md-journal.com Table 4 Univariate and multivariate analyses of risks factors for in-hospital mortality after hepatectomy for hepatocellular carcinoma. 4. Discussion Despite improvements in surgical technique, operative instru- ments, and postoperative care, liver resection still carries substantial risks in modern era. Recent studies reported perioperative mortality rates of 2.6% to 8.4%[3,12,22,23] for HCC patients undergoing major liver resection. The deﬁnition of perioperative mortality in most published studies was death in hospital or death within 30 days after the operation. In the present study, the 30-day mortality rate was 1.8% and in- hospital mortality rate was 2.9%, which were comparable to those of previous studies. The 1.1% difference between in- hospital mortality rate and 30-day mortality rate in our study may indicate that even if the patient could live for more than 30 days postoperatively, death could still occur 1 month after the operation. Likewise, some patients, even after discharge, required frequent emergency room (ER) visits or rehospitalization to deal with postoperative complications. And still several of these patients eventually expired several months after the operation 3.4. Risk factors for 6-month (early) mortality distinct early mortality rates were identiﬁed, with score 0 to 6, 7 to 9, and 10 as 3 different groups. These 3 group of patients were then designated as RAM class I, II, and III, respectively, and their 6-month survival curves are illustrated in Fig. 1B. As shown in Table 6 and Fig. 1B, RAM class I had only 6% risk of early mortality, while one-third of patients died within 6 months if they were RAM class III. In addition to early mortality, the RAM score was also signiﬁcantly associated with OS. The OS curves are illustrated in Supplemental Figure 1, http://links.lww.com/MD/ B304. Furthermore, the RAM score was still predictive of OS even if we excluded patients with in-hospital mortality (Supplemental Figure 2, http://links.lww.com/MD/B304). Three hundred eighty-three patients (11.3%) died within 6 months after hepatectomy. The mean survival of these patients after operation was 81 days (range 0–179 days). Their risk factor analysis is summarized in Table 5. The signiﬁcant risk factors identiﬁed by univariate analysis were subjected to a stepwise multivariate logistic regression analysis. The result showed that diabetes mellitus (HR 1.743; P=0.019), albumin 3.5g/dL (HR 2.998; P<0.001), AFP >200ng/dL (HR 2.731; P<0.001), major surgical procedure (HR 2.014; P=0.006), blood loss >800mL (HR 1.874; P=0.018), and major surgical complica- tions (HR 5.522; P<0.001) were 6 independent risk factors for the occurrence of mortality within 6 months after hepatectomy for HCC. 3.3. Risk factors for 30-day and in-hospital mortality Univariate Multivariate ALT=alanine aminotransferase, CI=conﬁdence interval, HBs Ag=hepatitis B surface antigen, ICG-15=indocyanine green retention at 15min, INR=international normalized ratio, OP=operation. ∗Includes trisegmentectomy, right/left lobectomy, and extended right/left lobectomy. † Major surgical complications include grade III IV surgical complications † Major surgical complications include grade III–IV surgical complications. 3.3. Risk factors for 30-day and in-hospital mortality Univariate Multivariate Variables Cases with in-hospital mortality (%) Odds ratio P Hazard ratio (95% CI) P Age (≥80 vs <80 year-old) 5 (6.5) vs 92 (2.8) 2.426 0.054 Gender (male vs female) 74 (2.8) vs 23 (3.1) 0.894 0.646 Diabetes mellitus (yes vs no) 27 (4.3) vs 66 (2.5) 1.776 0.013 1.014 (0.486–2.117) 0.970 Hypertension (yes vs no) 21 (4.3) vs 41 (3.6) 1.199 0.507 End-stage renal disease (yes vs no) 4 (5.9) vs 89 (2.7) 2.213 0.122 Old stroke (yes vs no) 4 (9.5) vs 58 (3.7) 2.739 0.053 Smoking (yes vs no) 30 (3.0) vs 65 (2.8) 1.049 0.832 Alcohol (yes vs no) 25 (3.6) vs 72 (2.9) 1.250 0.345 HBs Ag (positive vs negative) 49 (2.4) vs 38 (3.6) 0.676 0.072 Hepatitis C virus (positive vs negative) 33 (3.4) vs 46 (2.7) 1.299 0.259 ICG-15 (>10 vs 10), % 34 (3.3) vs 37 (2.3) 1.434 0.132 Hemoglobin (10 vs >10), g/dL 14 (5.4) vs 83 (2.7) 2.107 0.010 1.111 (0.400–3.085) 0.841 Albumin (3.5 vs >3.5), g/dL 44 (8.4) vs 41 (1.5) 5.906 <0.001 2.555 (1.219–5.356) 0.013 Platelet (100 vs >100), 1000/mL 26 (5.2) vs 69 (2.4) 2.201 0.001 2.308 (0.973–5.478) 0.058 INR (>1.4 vs 1.4) 5 (11.9) vs 82 (2.7) 4.797 <0.001 4.615 (0.207–102.994) 0.334 ALT (>40 vs 40), U/L 50 (3.2) vs 40 (2.4) 1.317 0.199 Bilirubin total (>1.5 vs 1.5), mg/dL 20 (7.4) vs 75 (2.4) 3.211 <0.001 1.410 (0.431–4.613) 0.570 a-fetoprotein (>200 vs 200), ng/mL 46 (4.2) vs 48 (2.2) 1.964 0.001 2.175 (1.102–4.293) 0.025 Procedure (major vs minor), % ∗ 54 (4.1) vs 35 (1.8) 2.260 <0.001 1.023 (0.474–2.208) 0.955 OP duration (>270 vs 270), min 53 (3.8) vs 30 (1.6) 2.412 <0.001 1.114 (0.464–2.673) 0.809 Blood loss (>800 vs 800), mL 47 (5.6) vs 34 (1.5) 3.944 <0.001 2.812 (1.365–5.791) 0.005 Inﬂow control (yes vs no) 45 (3.1) vs 8 (1.7) 1.817 0.118 Tumor size (>10 vs 10), cm 20 (3.8) vs 66 (2.4) 1.631 0.057 Surgical complications (major vs minor/none)† 58 (27.0) vs 7 (0.4) 91.565 <0.001 60.526 (24.39–150.22) <0.001 ALT=alanine aminotransferase, CI=conﬁdence interval, HBs Ag=hepatitis B surface antigen, ICG-15=indocyanine green retention at 15min, INR=international normalized ratio, OP=operation. ∗Includes trisegmentectomy, right/left lobectomy, and extended right/left lobectomy. † Major surgical complications include grade III–IV surgical complications. Table 4 Univariate and multivariate analyses of risks factors for in-hospital mortality after hepatectomy for hepatocellular carcinoma. 3.5. The development of RAM score The respective calculated regression coefﬁcient (B-value) of the 6 independent risk factors was multiplied by 2 and rounded to integer in order to formulate a scoring system predictive of early mortality (6-month mortality) after hepatectomy. The “Risk Assessment for early Mortality (RAM)” score for hepatectomy for HCC was developed (Table 6). The RAM score was the summation of scores of 6 independent variables. Only patients (1935 patients) with complete informa- tion of all 6 variables had their RAM score calculated. Among them, 145 patients died within 6 months after operation. As shown in Table 6, the RAM score was signiﬁcantly associated with 6-month mortality, with higher score indicating higher risk of early mortality (AUC 0.725, P<0.001). Their respective 6- month survival curve is illustrated in Fig. 1A. After visual inspection of the Kaplan–Meier curves, 3 groups of patients with 5 Lee et al. Medicine (2016) 95:39 Medicine Table 5 Univariate and multivariate analyses of risks factors for 6-month mortality after hepatectomy for hepatocellular carcinoma. 3.5. The development of RAM score Univariate Multivariate Variables Cases with 6-mo mortality (%) Odds ratio P Hazard ratio (95% CI) B ∗ P Age (≥80 vs <80 year-old) 14 (18.2) vs 369 (11.2) 1.769 0.054 Gender (male vs female) 303 (11.5) vs 80 (10.8) 1.063 0.643 Diabetes mellitus (yes vs no) 92 (14.6) vs 277 (10.3) 1.483 0.002 1.743 (1.095–2.776) 0.556 0.019 Hypertension (yes vs no) 52 (10.7) vs 100 (8.9) 1.231 0.249 End-stage renal disease (yes vs no) 11 (16.2) vs 358 (11.0) 1.554 0.183 Old stroke (yes vs no) 7 (16.7) vs 145 (9.2) 1.963 0.105 Smoking (yes vs no) 150 (14.8) vs 218 (9.5) 1.657 <0.001 1.442 (0.866–2.403) 0.160 Alcohol (yes vs no) 116 (16.6) vs 242 (9.7) 1.856 <0.001 1.235 (0.689–2.213) 0.478 HBs Ag (positive vs negative) 237 (11.7) vs 109 (10.2) 1.173 0.192 Hepatitis C virus (positive vs negative) 102 (10.6) vs 188 (10.9) 0.973 0.836 ICG-15 (>10 vs 10), % 133 (12.7) vs 125 (7.8) 1.737 <0.001 1.097 (0.703–1.712) 0.684 Hemoglobin (10 vs >10), g/dL 49 (19.1) vs 334 (10.7) 1.965 <0.001 1.082 (0.518–2.260) 0.833 Albumin (3.5 vs >3.5) g/dL 141 (26.9) vs 212 (7.9) 4.280 <0.001 2.998 (1.804–4.982) 1.098 <0.001 Platelet (100 vs >100), 1000/mL 85 (17.0) vs 289 (10.2) 1.808 <0.001 1.536 (0.876–2.695) 0.134 INR (>1.4 vs 1.4) 16 (38.1) vs 305 (10.2) 5.425 <0.001 4.143 (0.222–77.380) 0.341 ALT (>40 vs 40), U/L 210 (13.3) vs 142 (8.6) 1.633 <0.001 1.188 (0.778–1.814) 0.424 Bilirubin total (>1.5 vs 1.5), mg/dL 58 (21.6) vs 318 (10.3) 2.391 <0.001 1.159 (0.513–2.618) 0.723 a-fetoprotein (>200 vs 200), ng/mL 182 (16.7) vs 172 (7.9) 2.351 <0.001 2.731 (1.777–4.198) 1.005 <0.001 Procedure (major vs minor), %† 219 (16.4) vs 125 (6.6) 2.803 <0.001 2.014 (1.221–3.322) 0.700 0.006 OP duration (>270 vs 270), min 198 (14.4) vs 148 (8.1) 1.917 <0.001 1.438 (0.873–2.368) 0.153 Blood loss (>800 vs 800), mL 188 (22.3) vs 150 (6.5) 4.122 <0.001 1.874 (1.113–3.156) 0.628 0.018 Inﬂow control (yes vs no) 117 (7.9) vs 31 (6.6) 1.217 0.347 Tumor size (>10 vs 10), cm 125 (23.8) vs 238 (8.6) 3.342 <0.001 1.130 (0.639–1.997) 0.674 Surgical complications (major vs minor/none)‡ 71 (33.0) vs 87 (5.0) 9.374 <0.001 5.522 (3.458–8.118) 1.709 <0.001 ALT=alanine aminotransferase, CI=conﬁdence interval, HBs Ag=hepatitis B surface antigen, ICG-15=indocyanine green retention at 15min, INR=international normalized ratio, OP=operation. ∗The regression coefﬁcients (B) were multiplied by 2 and rounded in order to calculate the RAM score. 3.5. The development of RAM score † Includes trisegmentectomy, right/left lobectomy, and extended right/left lobectomy. ‡ Major surgical complications include grade III–IV surgical complications. ALT=alanine aminotransferase, CI=conﬁdence interval, HBs Ag=hepatitis B surface antigen, ICG-15=indocyanine green retention at 15min, INR=international normalized ratio, OP=operation. ∗The regression coefﬁcients (B) were multiplied by 2 and rounded in order to calculate the RAM score. † Includes trisegmentectomy, right/left lobectomy, and extended right/left lobectomy. ‡ Major surgical complications include grade III–IV surgical complications. 24% to 70% was reported in published studies.[3,12,14,22–24] In our study, 36% of patients suffered from grade II or more surgical complications, which was comparable to most of the published series. Since the most common postoperative complications after liver resection for HCC were ascites and pleural effusion,[3,22,25] which,althougha nuisancefor surgeonsandpatients,couldmostly be controlled by pharmacotherapy and were rarely life-threaten- ing,itmay bemore practicalclinically to analyze the risk factors for signiﬁcant or life-threatening complications. In present study, the incidence of major surgical complications were 10.9%, which was comparable to other published studies.[3,26] without evidence of tumor recurrence. To minimize medical costs and optimize surgical outcome, it is of paramount signiﬁcance to clarify signiﬁcant risk factors contributing to major postoperative complications and early mortality after liver resection for HCC. Our study, to the present date, is the only research that analyzed the surgical outcome from 1 month to 6 months after the operation. With more than 3300 patients operated in a single institute, our study is also by far one of the largest reports dealing with the surgical results in the English literature. The surgical complication rate may differ among literatures due to different deﬁnition or criteria. An overall complication rate of Table 6 Risk Assessment for early Mortality (RAM) score for hepatectomy for hepatocellular carcinoma. Variables Score allocation ∗ Total score No. (% of total) 6-mo mortality (%) Total score No. † Includes trisegmentectomy, right/left lobectomy, and extended right/left lobectomy. ∗The regression coefﬁcients (B) were multiplied by 2 and rounded to integer in order to calculate the RAM score. ‡ Major surgical complications include grade III–IV surgical complications. 3.5. The development of RAM score (B) Predictive signiﬁcance of the RAM class. RAM class I had only 6% risk of early mortality, while one-third of patients died within 6 months after hepatectomy if they were RAM class III. It was reported that preoperative platelet count, serum aspartate aminotransferase (AST)/alanine aminotransferase (ALT) level, intraoperative blood loss, portal clamping, periop- erative blood transfusion, comorbid illness, Child–Pugh classiﬁ- cation, ICG retention test, additional procedures, the American Society of Anaesthesiology (ASA) score, extent of resection, and presence of hepatic steatosis were risk factors for the occurrence of postoperative complications after liver resection for HCC.[3,12,23–26] Because most (96%) patients in our study were Child–Pugh A, the impact of Child–Pugh classiﬁcation on the occurrence of major complications may be biased and under- estimated; as a result, we decided not to include this parameter into our ﬁnal analysis. We found in the present study that operative duration greater than 270minutes and blood loss greater than 800mL were 2 independent risk factors for the occurrence of major surgical complications after hepatectomy. Old age, comorbidity such as diabetes mellitus or hypertension, high ICG-15, major procedure, anemia, hypoalbuminemia, coagulopathy, and large tumor size were not independently related to major complications. Our results indicated that even for patients with suboptimal preoperative clinical data, surgeons can still conduct liver resections as long as we extreme our every effort to minimize blood loss and shorten operative duration. Massive blood loss often required blood transfusions, which had been shown to be associated with adverse effects on the immune system, leading to an increased risk of postoperative infection.[27] Furthermore, prolonged abdominal operations have been proved to be at higher risk for surgical site infections.[28] All these studies supported that liver surgeons should try to reduce blood loss and operative duration by employing any form of vascular control, vessel-sealing device, and parenchymal transection technique during hepatectomy. Since inﬂow control was not a signiﬁcant risk factor for major surgical complications in present study, and previous research showed that Pringle maneuver during liver transection resulted in less blood loss,[29] we believe that the use of portal clamping in selected patients is justiﬁed. However, our study did not suggest liberal performance of liver resections in patients with poor liver function and/or reserve. 3.5. The development of RAM score (% of total) 6-mo mortality (%) Diabetes mellitus 1 0 36 (1.8) 1 (2.8) 6 203 (10.5) 27 (13.3) Albumin 3.5g/dL 2 1 36 (1.8) 2 (5.6) 7 112 (5.7) 21 (18.75) a-fetoprotein >200ng/mL 2 2 532 (27.5) 11 (2.1) 8 76 (3.9) 12 (15.8) Major resection† 1 3 308 (15.9) 10 (3.2) 9 39 (2.0) 9 (25.6) Blood loss >800mL 1 4 288 (14.9) 20 (6.9) 10 6 (0.3) 2 (33.3) Major surgical complications‡ 3 5 299 (15.5) 30 (10) Total 1935 (100) 145 (7.5) RAM scorex Score 6-mo mortality (%) Class I 0–6 101 (5.9) P<0.001 Class II 7–9 42 (18.5) Class III 10 2 (33.3) ∗The regression coefﬁcients (B) were multiplied by 2 and rounded to integer in order to calculate the RAM score. † Includes trisegmentectomy, right/left lobectomy, and extended right/left lobectomy. ‡ Major surgical complications include grade III–IV surgical complications. x AUC=0.725, P<0.001. When cutoff score is 4.5, the sensitivity and speciﬁcity for 6-month mortality was 0.705 and 0.648, respectively. Major surgical complications include grade III IV surgical complications. x AUC=0.725, P<0.001. When cutoff score is 4.5, the sensitivity and speciﬁcity for 6-month mortality was 0.705 and 0.648, respectively. 6 Lee et al. Medicine (2016) 95:39 www.md-journal.com Figure 1. (A and B) Six-month Kaplan–Meier survival curves and predictive signiﬁcance of the RAM score. (A) Predictive signiﬁcance of the single point scores. The higher the individual RAM score, the higher the risk of 6-month mortality after hepatectomy for HCC. The development of a trichotomized RAM score was achieved by visual inspection of the Kaplan–Meier survival curves. Three groups of patients with distinct 6-month survival were identiﬁed, with score 0 to 6, 7 to 9, and 10 as 3 different groups. (B) Predictive signiﬁcance of the RAM class. RAM class I had only 6% risk of early mortality, while one-third of patients died within 6 months after hepatectomy if they were RAM class III. Figure 1. (A and B) Six-month Kaplan–Meier survival curves and predictive signiﬁcance of the RAM score. (A) Predictive signiﬁcance of the single point scores. The higher the individual RAM score, the higher the risk of 6-month mortality after hepatectomy for HCC. The development of a trichotomized RAM score was achieved by visual inspection of the Kaplan–Meier survival curves. Three groups of patients with distinct 6-month survival were identiﬁed, with score 0 to 6, 7 to 9, and 10 as 3 different groups. Medicine Medicine [5] Bismuth H, Houssin D, Ornowski J, et al. Liver resections in cirrhotic patients: a Western experience. World J Surg 1986;10:311–7. In addition to patient and tumor factors, viral factors (hepatitis B virus (HBV) or hepatitis C virus (HCV)) also inﬂuenced the treatment strategy and outcome of HCC patients. Previous studies have shown that high baseline HBV viral load was associated with shorter disease-free survival (DFS) and OS after hepatectomy, and adequate antiviral therapy after hepatectomy could prolong both DFS and OS.[31–35] The RAM score as a result, should take either viral load factor or antiviral therapy into consideration. Nevertheless, many of our patients could not receive either viral load examination or antiviral therapy during the study period, these 2 important factors were not incorporated into our ﬁnal analysis. Further study regarding viral factors is thus warranted to more precisely predict patient outcome after operation. [6] Thompson HH, Tompkins RK, Longmire WPJr. Major hepatic resection. A 25-year experience. Ann Surg 1983;197:375–88. [7] Fortner JG, Kim DK, Maclean BJ, et al. Major hepatic resection for neoplasia: personal experience in 108 patients. Ann Surg 1978;188:363–71. [8] Howat JM. Major hepatic resections in infancy and childhood. Gut 1971;12:212–7. [9] Lai EC, Fan ST, Lo CM, et al. Hepatic resection for hepatocellular carcinoma. An audit of 343 patients. Ann Surg 1995;221:291–8. [10] Matsumata T, Kanematsu T, Shirabe K, et al. Decreased morbidity and mortality rates in surgical patients with hepatocellular carcinoma. Br J Surg 1990;77:677–80. [11] Lin HM, Lei LM, Zhu J, et al. Risk factor analysis of perioperative mortality after ruptured bleeding in hepatocellular carcinoma. World J Gastroenterol 2014;20:14921–6. [12] Yang T, Zhang J, Lu JH, et al. Risk factors inﬂuencing postoperative outcomes of major hepatic resection of hepatocellular carcinoma for patients with underlying liver diseases. World J Surg 2011;35:2073–82. The RAM score is clinically relevant for several reasons. First, it is simple and easily applicable in a real-life clinical setting without requirement of massive calculations. Second, the classiﬁcation can inform the surgeon and patient the likelihood of early mortality after hepatectomy for HCC. Third, it can exclude patients from risky operations if the preoperative RAM score is already high. Last but not the least, it can help identify which patients may require more intensive postoperative care and should not be discharged as usual routine. Medicine The RAM score, as a result, can be applied to clinical practice to predict the outcome in the perioperative period. [13] Fan ST, Lo CM, Liu CL, et al. Hepatectomy for hepatocellular carcinoma: toward zero hospital deaths. Ann Surg 1999;229:322–30. [14] Farges O, Malassagne B, Flejou JF, et al. Risk of major liver resection in patients with underlying chronic liver disease: a reappraisal. Ann Surg 1999;229:210–5. [15] Brierley JD, Gospodarowicz MK, Wittekind C. International Union Against Cancer (UICC): TNM Classiﬁcation of Malignant Tumours, 7 th edition. Wiley-Blackwell. New Jersey, USA. 2009. [16] Lau H, Man K, Fan ST, et al. Evaluation of preoperative hepatic function in patients with hepatocellular carcinoma undergoing hepatectomy. Br J Surg 1997;84:1255–9. However, since our study is a retrospective analysis based on clinical data from a single institute, incomplete data were inevitable when reviewing records from a very long time ago. The lack of validation group is another ﬂaw of present study. Further research by either an external cohort or prospective trial is thus warranted to validate the ﬁndings of our study. g ; [17] Lam CM, Fan ST, Lo CM, et al. Major hepatectomy for hepatocellular carcinoma in patients with an unsatisfactory indocyanine green clearance test. Br J Surg 1999;86:1012–7. [18] Yamamoto M, Katagiri S, Ariizumi S, et al. Glissonean pedicle transection method for liver surgery (with video). J Hepatobiliary Pancreat Sci 2012;19:3–8. [19] Takasaki K. Glissonean pedicle transection method for hepatic resection: a new concept of liver segmentation. J Hepatobiliary Pancreat Surg 1998;5:286–91. In conclusion, our study demonstrated that meticulous surgical techniques to minimize blood loss and avoid prolonged operative time may help decrease the occurrence of major surgical complications after hepatectomy. In addition to major surgical complications, we should aware that diabetes mellitus, hypo- albuminemia, high AFP, massive blood loss, and major surgical procedure are independently associated with early mortality for patients undergoing liver resection. RAM score is an effective beside tool to predict the short-term outcome immediately after hepatectomy. Further study by either an external cohort or prospective trial is warranted to validate the utility of RAM score as a predicting tool for postoperative outcome. [20] Pang YY. The Brisbane 2000 terminology of liver anatomy and resections. HPB 2000; 2:333–339. HPB 2002;4:99author reply 99–100. [21] Dindo D, Demartines N, Clavien P-A. Classiﬁcation of surgical complications. Ann Surg 2004;240:205–13. [22] Benzoni E, Molaro R, Cedolini C, et al. Medicine Liver resection for HCC: analysis of causes and risk factors linked to postoperative complications. Hepatogastroenterology 2007;54:186–9. [23] Cescon M, Vetrone G, Grazi GL, et al. Trends in perioperative outcome after hepatic resection: analysis of 1500 consecutive unselected cases over 20 years. Ann Surg 2009;249:995–1002. y g [24] Sun HC, Qin LX, Wang L, et al. Risk factors for postoperative complications after liver resection. Hepatobiliary Pancreat Dis Int 2005;4:370–4. [25] Taketomi A, Kitagawa D, Itoh S, et al. Trends in morbidity and mortality after hepatic resection for hepatocellular carcinoma: an institute’s experience with 625 patients. J Am Coll Surg 2007;204:580–7. Acknowledgment [26] Belghiti J, Hiramatsu K, Benoist S, et al. Seven hundred forty-seven hepatectomies in the 1990s: an update to evaluate the actual risk of liver resection. J Am Coll Surg 2000;191:38–46. We are grateful to all our colleagues and authors in the Department of Cancer Center, Department of Gastroenterology and Hepatology, Department of Surgery, Department of Anesthesiology, and Chang Gung University for their technical assistance. [27] Blumberg N, Heal JM. Effects of transfusion on immune function. Cancer recurrence and infection. Arch Pathol Lab Med 1994;118:371–9. [28] Haley RW, Morgan WM, Culver DH, et al. Update from the SENIC project. Hospital infection control: recent progress and opportunities under prospective payment. Am J Infect Control 1985;13:97–108. 3.5. The development of RAM score Since all of our patients were Child–Pugh A or early B, ECOG 0, and had adequate future liver remnant, we believe that prudent patient selection and comprehensive preoperative preparation may be as important as excellent surgical technique in terms of surgical outcome. As for risk factors for mortality, we found in the present study that, regardless of study end point, albumin 3.5g/dL and major surgical complications were 2 independent risk factors for the occurrence of mortality. The pathological variables (i.e., vascular invasion, daughter nodules, etc.) were not related to 6-month mortality (data not shown). Massive blood loss and high AFP, although not signiﬁcant at 30-day study point, became signiﬁcantly important when study endpoint was in-hospital or 6-month mortality. Likewise, diabetes mellitus and major surgical procedure became independent risk factors for mortality when endpoint was set at 6 months. Since diabetes mellitus, procedural type, and AFP level are essentially inherent to the patient or tumor, careful patient selection, adequate preoperative nutritional support to restore albumin, and meticulous surgical technique to avoid massive blood loss and major complications were of paramount importance for liver resections for HCC as a result. In order to predict the short-term outcome of hepatectomy, we proposed the RAM scoring system by incorporating the signiﬁcant risk factors of 6-month mortality. The results turned out that the higher the RAM score/class, the higher the risk of 6- month mortality. In addition, the RAM score was signiﬁcantly associated with OS, indicating that the perioperative variables would also inﬂuence the long-term oncological outcome and should not be overlooked. Indeed, massive blood loss usually required blood transfusion, and it was reported that blood transfusion was an independent adverse prognostic factor of long-term disease-free and OS after hepatectomy.[30] By minimizing blood loss and avoiding major complications, we surgeons could improve surgical as well as oncological outcomes as a result. 7 Lee et al. Medicine (2016) 95:39 [35] Miao RY, Zhao HT, Yang HY, et al. Postoperative adjuvant antiviral therapy for hepatitis B/C virus-related hepatocellular carcinoma: a meta- analysis. World J Gastroenterol 2010;16:2931–42. [33] Chong CC, Wong GL, Lai PB. Impact of antiviral therapy on post- hepatectomy outcome for hepatitis B-related hepatocellular carcinoma. World J Gastroenterol 2014;20:6006–12. hepatocellular carcinoma following curative resection (Review). Oncol Lett 2015;9:527–34. [33] Chong CC, Wong GL, Lai PB. Impact of antiviral therapy on post- hepatectomy outcome for hepatitis B-related hepatocellular carcinoma. World J Gastroenterol 2014;20:6006–12. hepatocellular carcinoma following curative resection (Review). Oncol Lett 2015;9:527–34. [34] Yu LH, Li N, Cheng SQ. The role of antiviral therapy for HBV-related hepatocellular carcinoma. Int J Hepatol 2011;2011:416459. [34] Yu LH, Li N, Cheng SQ. The role of antiviral therapy for HBV-related hepatocellular carcinoma. Int J Hepatol 2011;2011:416459. [35] Miao RY, Zhao HT, Yang HY, et al. Postoperative adjuvant antiviral therapy for hepatitis B/C virus-related hepatocellular carcinoma: a meta- analysis. World J Gastroenterol 2010;16:2931–42. References [29] Man K, Fan ST, Ng IO, et al. Prospective evaluation of Pringle maneuver in hepatectomy for liver tumors by a randomized study. Ann Surg 1997;226:704–11. discussion 11–13. [1] Torre LA, Bray F, Siegel RL, et al. Global cancer statistics, 2012. CA Cancer J Clin 2015;65:87–108. [30] Fan ST, Ng IO, Poon RT, et al. Hepatectomy for hepatocellular carcinoma: the surgeon’s role in long-term survival. Arch Surg 1999;134:1124–30. [2] Department of Health, Taiwan, ROC. Report of leading cancer-related death in Taiwan in 2014. Department of Health, Taiwan, ROC. 2015. [3] Wei AC, Tung-Ping Poon R, Fan ST, et al. Risk factors for perioperative morbidity and mortality after extended hepatectomy for hepatocellular carcinoma. Br J Surg 2003;90:33–41. [31] Chen JL, Lin XJ, Zhou Q, et al. Association of HBV DNA replication with antiviral treatment outcomes in the patients with early-stage HBV- related hepatocellular carcinoma undergoing curative resection. Chin J Cancer 2016;35:28. [4] Gozzetti G, Mazziotti A, Cavallari A, et al. Clinical experience with hepatic resections for hepatocellular carcinoma in patients with cirrhosis. Surg Gynecol Obstet 1988;166:503–10. [32] Zuo C, Xia M, Wu Q, et al. Role of antiviral therapy in reducing recurrence and improving survival in hepatitis B virus-associated 8 Lee et al. Medicine (2016) 95:39 www.md-journal.com www.md-journal.com www.md-journal.com [34] Yu LH, Li N, Cheng SQ. The role of antiviral therapy for HBV-related hepatocellular carcinoma. Int J Hepatol 2011;2011:416459. 9 9
https://openalex.org/W2943018079	https://europepmc.org/articles/pmc6481280?pdf=render	English	null	Organizational Meeting Orientation: Setting the Stage for Team Success or Failure Over Time	Frontiers in psychology	2,019	cc-by	10,842	Organizational Meeting Orientation: Setting the Stage for Team Success or Failure Over Time Joseph E. Mroz1, Nicole Landowski2, Joseph Andrew Allen2* and Cheryl Fernandez3 1 2 Joseph E. Mroz1, Nicole Landowski2, Joseph Andrew Allen2* and Cheryl Fernandez3 Joseph E. Mroz1, Nicole Landowski2, Joseph Andrew Allen2* and Cheryl Fernandez3 1 Denison Consulting, Ann Arbor, MI, United States, 2 Department of Psychology, University of Nebraska Omaha, Omaha NE, United States, 3 Gallup Inc., Omaha, NE, United States 1 Denison Consulting, Ann Arbor, MI, United States, 2 Department of Psychology, University of Nebraska Omaha, Omaha, NE, United States, 3 Gallup Inc., Omaha, NE, United States Teams are an integral tool for collaboration and they are often embedded in a larger organization that has its own mission, values, and orientations. Speciﬁcally, organizations can be oriented toward a variety of values: learning, customer service, and even meetings. This paper explores a new and novel construct, organizational meeting orientation (the set of policies and procedures that promote or lead to meetings), and its relationship to perceived team meeting outcomes and work attitudes. An organization’s policies, procedures, and overall orientation toward the use of team meetings—along with the quality and perceived effectiveness of those meetings—set the stage for how teams develop and collaborate. Across two exploratory studies, we demonstrate that perceptions of an organization’s orientation toward meetings is associated with the perceived quality and satisfaction of team meetings, along with work engagement and intentions to quit. Employees who feel meetings lack purpose or are overused tend to be less engaged with their work and more likely to consider leaving the organization. Based on the ﬁndings, we conclude with a robust discussion of how meeting orientation may set the stage for team interactions, inﬂuencing how their team operates over time on a given project or series of projects. An organization’s orientation toward meetings is a new construct that may exert an inﬂuence on team dynamics at the organizational level, representing a factor of the organization that affects how and when teams meet and collaborate. Edited by: Eduardo Salas, Rice University, United States Eduardo Salas, Rice University, United States Reviewed by: Ricardo Martinez Cañas, University of Castilla–La Mancha, Spain Mario Arias-Oliva, University of Rovira i Virgili, Spain Correspondence: Reviewed by: Ricardo Martinez Cañas, University of Castilla–La Mancha, Spain Mario Arias-Oliva, University of Rovira i Virgili, Spain Correspondence: Joseph Andrew Allen josephallen@unomaha.edu Reviewed by: Ricardo Martinez Cañas, University of Castilla–La Mancha, Spain Reviewed by: Ricardo Martinez Cañas, University of Castilla–La Mancha, Spain Mario Arias-Oliva, University of Rovira i Virgili, Spain C Mario Arias-Oliva, University of Rovira i Virgili, Spain *Correspondence: Joseph Andrew Allen josephallen@unomaha.edu Specialty section: This article was submitted to Organizational Psychology, a section of the journal Frontiers in Psychology Keywords: meetings, groups, teams, job attitudes, time ORIGINAL RESEARCH published: 17 April 2019 doi: 10.3389/fpsyg.2019.00812 INTRODUCTION Workplace meetings are essential to both the functioning of organizations and employees’ workplace experiences. Of the estimated 55 million meetings occurring daily in the United States, managers in large organizations are dedicating over three-quarters of their time preparing for, attending, leading, and processing meeting results (Keith, 2015). Among the various reasons to call a meeting, workplace meetings can be used to share information (McComas, 2003), brainstorm (Reinig and Shin, 2002), socialize (Horan, 2002), and solve problems (e.g., McComas et al., 2007). Being that meetings are an integral part of organizations, ﬁrms may have a unique culture of policies, procedures, and practices that promote, emphasize, and result in meetings – that is, a meeting orientation (Hansen and Allen, 2015). Meeting orientation is a relatively unexplored topic in meeting science, and no empirical studies have looked at its relationship to employee attitudes concerning meetings or their broader work environments Received: 31 October 2018 Accepted: 26 March 2019 Published: 17 April 2019 Citation: Mroz JE, Landowski N, Allen JA and Fernandez C (2019) Organizational Meeting Orientation: Setting the Stage for Team Success or Failure Over Time. Front. Psychol. 10:812. doi: 10.3389/fpsyg.2019.00812 April 2019 \| Volume 10 \| Article 812 Frontiers in Psychology \| www.frontiersin.org 1 Meeting Orientation Mroz et al. (Allen and Hansen, 2011; Hansen and Allen, 2015). An organization’s overall culture toward meetings (i.e., meeting orientation) may have important consequences for how groups and teams develop over time by, for instance, inﬂuencing how often, when, and under what circumstances group members come together to work and discuss problems. (Allen and Hansen, 2011; Hansen and Allen, 2015). An organization’s overall culture toward meetings (i.e., meeting orientation) may have important consequences for how groups and teams develop over time by, for instance, inﬂuencing how often, when, and under what circumstances group members come together to work and discuss problems. good meetings. Likewise, low meeting orientation organizations may hold fewer meetings, and meetings are not necessarily higher or lower quality than in organizations with a diﬀerent meeting orientation. For example, meetings may be viewed negatively when a meeting culture inhibits employees from doing their job because they attend too many group and team meetings. Alternatively, additional meetings that provide employees the opportunity to pose questions to executive management can be viewed positively (Hansen and Allen, 2015). Depending on the context, these meeting cultures may be advantageous or disadvantageous. Across two studies, we propose that there are a number of ways in which individuals’ belief about the meeting orientation of their organization may inﬂuence how people view various meeting and organizational outcomes, which can subsequently inﬂuence team development over time. Speciﬁcally, building upon the original theory and conceptualization by Hansen and Allen (2015), we argue that meeting orientation is related to employees’ satisfaction with meetings and the perceived eﬀectiveness of meetings, along with broader work-related attitudes such as intentions to quit (ITQ) and work engagement. Consistent with other theories of and empirical evidence for organizational orientations (e.g., market orientation; Kirca et al., 2005), we believe meeting orientation will relate to both proximal (team meeting satisfaction) and distal (work engagement) individual outcomes. Citation: After establishing meeting orientation as an important construct of interest in meeting science and for organizations, we provide a discussion and testable propositions for future research regarding how meeting orientation, and a ﬁrm’s overall cultural toward meetings, can inﬂuence how teams develop and grow over time. Meeting orientation is composed of four facets: policy focus, rewards for meetings, strategic use of meetings, and overuse of meetings (Hansen and Allen, 2015). Policy focus refers to the strength of formal policies and procedures at the organizational level with respect to meetings. Rewards for meeting speaks to how much organizational members believe that the organization rewards people who attend, lead, or organize meetings. Strategic use of meetings deals with how much an organization relies on meetings to gather, disseminate, or respond to information. Finally, meeting overuse refers to how much an organization utilizes meetings too often or holds meetings that are too long. Despite the potential relevance and impact that an organization’s meeting orientation may have on the way employees interact, no published research has empirically evaluated the relation between meeting orientation and meeting outcomes. As previously mentioned, a high or low meeting orientation does not necessarily provide an indication as to the quality of an organization’s meetings or how satisﬁed employees are with their group and team meetings at work. However, based on the nature of several meeting orientation facets, there are a number of ways in which individuals’ beliefs about the meeting orientation of their organizations may inﬂuence how people view their meetings. Further it may inﬂuence how they view their organization and it may enable or constrain their team’s ability to function over time. Organizational Orientations and the Meeting Orientation g Organizational orientations provide a potential competitive advantage for ﬁrms and examples include a market orientation or entrepreneurial orientation (Kirca et al., 2005; Rauch et al., 2009). A particularly relevant organizational characteristic that may aﬀect team meeting processes and outcomes, as well as employee attitudes toward the organization, is an organization’s meeting orientation, or the policies, procedures, and practices that emphasize, promote, or leads to meetings (Hansen and Allen, 2015). As market, entrepreneurial, and learning orientations aﬀect how an organization structures itself and operates (e.g., Matsuno et al., 2005), a meeting orientation describes the value that an organization places on meetings (i.e., team meetings) and how often meetings are used as a collaborative tool. The meeting orientation serves as the mode by which other organizational orientations permeate and are enacted across the organization. That is, unlike other organizational orientations, meeting orientation is a process focused orientation speciﬁc to how people in the organization interact with one another in, through, and around their group and team meetings. Frontiers in Psychology \| www.frontiersin.org Overview of Studies We conducted two studies to investigate the concept of meeting orientation and its relation to team meeting and organizational outcomes. These were exploratory studies designed to be a “ﬁrst look” at the concept of a meeting orientation and how it may be related to organizationally relevant employee attitudes. Our ﬁrst study sought to explore whether policy focus, rewards, strategic usage, and potential overuse were advantageous or disadvantageous to perceptions of team meeting quality. Given that meetings are events that can be strategically used to foster employee engagement (Allen and Rogelberg, 2013), in Study 2 we explored whether the facets of meeting orientation were related to work-related outcomes such as employee engagement and ITQ. The degree to which an organization is oriented toward the use of group and team meetings is best represented on a continuum from low to high (Hansen and Allen, 2015). Organizations with a high meeting orientation implicitly or explicitly encourage employees to use group and team meetings as an important form of interaction and the overall work process. Therefore, high meeting orientation organizations may hold many workplace meetings, but those group and team meetings are not necessarily STUDY 1 The four facets of meeting orientation will likely diﬀerentially relate to team meeting outcomes. First, one facet of meeting orientation is group and team meeting overuse, or how much April 2019 \| Volume 10 \| Article 812 2 Meeting Orientation Mroz et al. an organizational member thinks that the organization has too many meetings, has meetings that are too long, or routinely holds meetings just because meetings are scheduled. Individuals who believe that their organization overuses group and team meetings are likely to think that, in general, meetings are not eﬀective or satisfying. One aspect of an eﬀective meeting is having and achieving goals. Routine or “standing” meetings, and other meetings generally, may have no clear goals, making it diﬃcult for the meeting to be eﬀective. Likewise, people tend to dislike meetings (Tracy and Dimock, 2004), and this dislike may intensify if individuals believe that their organizations have too many meetings. Finally, people may not trust their group or team meeting leader’s managerial abilities or capacity to “do the right thing” if meeting attendees think the organization has too many meetings. Employees may view managers, who typically lead team meetings at work, as embodiments of the organization (Eisenberger et al., 1986), and if the organization overuses meetings, then the manager overuses group and team meetings. Therefore, we hypothesize the following: the purpose is readily apparent and aligns with important, widely held assumptions about what a work meeting should be (Allen et al., 2014). Policy focus and rewards may also inﬂuence how supported group and team meeting attendees feel from the organization. Support in this case derives from perceived organizational support (POS) theory (Eisenberger et al., 1986), which refers to the extent to which employees believe that their work organization cares about their wellbeing and values their contribution. A team meeting leader is supportive by valuing contributions of attendees and by fostering a caring atmosphere in their group or team meetings. If an organization has an orientation toward the strategic use of meetings and the organization rewards the use of meetings, team meeting attendees may feel that the meeting leader is supportive. For instance, if a meeting has a purpose for information sharing and the organization encourages these sorts of group and team meetings, meeting leaders may become adept at conducting these meetings by supporting and encouraging the participation of all attendees. STUDY 1 Likewise, if group and team meetings are overused and lack purpose, attendees may not feel supported because their meeting role is unclear or the meeting is generally unnecessary. Hypothesis 1: Overuse will be negatively related to team meeting eﬀectiveness (1a) and team meeting satisfaction (1b). The other three facets should have a markedly diﬀerent relationship to meeting outcomes. Strategic use of meetings, or how much meeting attendees believe their organizations use group and team meetings to gather, exchange, and act on information, may be positively related with both team meeting eﬀectiveness and team meeting satisfaction. People who believe that their organizations have meetings for a purpose, namely to interact with information, are likely to believe that those group and team meetings are eﬀective and satisfying because Hypothesis 2: Policy focus (2a), rewards (2b), and strategic use of meetings (2c) will be positively related to team meeting satisfaction. Hypothesis 3: Policy focus (3a), rewards (3b), and strategic use of meetings (3c) will be positively related to team meeting eﬀectiveness. Figure 1 includes hypothesized relationships in Study 1. FIGURE 1 \| Hypothesized relationships in Study 1. FIGURE 1 \| Hypothesized relationships in Study 1. FIGURE 1 \| Hypothesized relationships in Study 1. Frontiers in Psychology \| www.frontiersin.org April 2019 \| Volume 10 \| Article 812 3 Meeting Orientation Mroz et al. meetings,” “rewards those who lead meetings,” and “rewards those who organize meetings.” For strategic use, items were my ﬁrm “holds meetings to gather information,” “holds meetings to disseminate (share) information with attendees,” and “holds meetings to respond to (gathered) information.” Lastly, overuse was measured with the following items: my ﬁrm “has more meetings than what is required,” “has longer meetings than what is required,” and “holds meetings for meetings sake.” Participants responded to all items on a scale ranging from 1 (strongly disagree) to 5 (strongly agree). Hansen and Allen (2015) conducted a factor analysis of the scale and found that the four-factor solution ﬁt the data best and explained 79% of the variability in the rotated sum of square factor loadings. Further, average variance extracted for each factor exceed 0.71 for all factors and Cronbach’s alpha was 0.79 or greater. In the current study, rewards (0.85), strategic use (0.67), and overuse (0.77) demonstrated acceptable internal consistency as assessed by Cronbach’s alpha, whereas the internal consistency of the policy focus measure was somewhat low (0.58). Results Descriptive statistics, alpha estimates of internal consistency, and correlations between study variables are included in Table 1. Hierarchical regression analyses were used to test each hypothesis. All hypotheses related to each outcome were tested concurrently in the same regression models. Team Meeting Satisfaction Hypotheses 1a and 2a,b predicted that overuse would be negatively related to team meeting eﬀectiveness, whereas policy focus, rewards, and strategic use of group and team meetings would be positively related to team meeting satisfaction. In order to separate the inﬂuence of demographic factors on meeting satisfaction, the ﬁrst step of the regression model included age, number of meetings attended per week, supervisory status, and job level as control variables, following best practice recommendations for statistical controls (Becker, 2005). Meeting load, or the number of meetings participants attend within a given period, has been demonstrated to aﬀect employee job attitudes (Luong and Rogelberg, 2005). This step accounted for a signiﬁcant amount of variance in meeting team satisfaction, F(4, 153) = 4.47, p = 0.002, R2 = 0.11. Meeting and demographic variables Meeting and demographic variables Participants reported on several factors of their last workplace meeting including meeting type, purpose (Allen et al., 2014), and number of attendees. Demographic variables included age, race/ethnicity, education level, job status, job tenure, and job level. Team meeting satisfaction Meeting satisfaction was measured using a 6-item measure developed by Rogelberg et al. (2010). Participants read a question stem (“My last meeting was. . .”) followed by series of adjectives and indicated how well each one described their last meeting (e.g., “stimulating” and “boring”) from 1 (strongly disagree) to 5 (strongly agree). Cronbach’s alpha estimate of internal consistency was 0.85. Team meeting eﬀectiveness Participants indicated how eﬀective they felt their last meeting was across six areas (e.g., “Achieving your own work goals” and “Providing you with an opportunity to acquire useful information”) using a 5-point Likert scale (1 = very ineﬀective; 5 = very eﬀective). Cronbach’s alpha for this measure was 0.83. Participants and Procedure In exchange for course credit, students in an undergraduate psychology course recruited working adults to participate in the study through Qualtrics, an online survey tool. A total of 22 students sent invitations to potential participants, 174 of whom ﬁnished the survey. Thus, the ﬁnal sample consisted of 174 well-educated adults (59% held a four-year degree) who ranged from 19 to 68 years old (M = 38.72, SD = 13.03). Of participants who provided information, 30% were men. Respondents worked in a variety of industries such as healthcare, education, and the military. Workers who supervised at least one employee comprised 48% of the sample. Due to the cross-sectional nature of the design, we implemented several procedures to mitigate concerns of common method bias (Podsakoﬀet al., 2003). Adhering to the recommendations proposed by Podsakoﬀet al. (2003), which are aimed at reducing demand characteristics and evaluation apprehension, participants were assured that they would be provided with anonymity, and that their responses would not be considered right or wrong. We also followed recommendations suggested by Conway and Lance (2010), which include utilizing counterbalancing of measures and demonstrating adequate evidence of measure reliability. In an eﬀort to mitigate concerns of item-context-induced mood states, priming eﬀects, and biases related to the order of measures or individual items, all measures and items were counterbalanced via randomization. Furthermore, each item utilized simple and precise language, addressing one particular concept, as suggested by Tourangeau et al. (2000). Meeting orientation Meeting satisfaction Meeting effectiveness Variable Model 1 Model 2 Model 1 Model 2 Controls Age 0.24∗ 0.23∗ 0.03 0.02 Meetings/week 0.12 0.10 0.09 0.04 Supervisory status −0.17 −0.22∗ 0.02 −0.05 Job level −0.05 −0.10 0.09 0.02 Focal variables Policy focus −0.01 −0.01 Rewards 0.10 −0.01 Strategic use 0.36∗∗ 0.53∗∗ Overuse −0.20∗ −0.22∗∗ F 4.47∗ 7.46∗∗ 0.72 8.60∗∗ Adjusted R2 0.11 0.29 0.02 0.31 1R2 0.18 0.29 Standardized regression coefﬁcients are displayed. N = 158. ∗p < 0.05, ∗∗p < 0.001. Meeting orientation Allen and Hansen’s (2011) meeting orientation scale consists of four facets: policy focus, rewards, strategic use, and overuse. Three items comprise each facet. Participants indicated their agreement or disagreement to statements for each facet. Items for policy focus included my ﬁrm “has policies that promote meetings,” “has a lot of standard procedures associated with meetings,” and “has what could be called a meeting orientation.” Items for rewards were my ﬁrm “rewards those who attend In the second step of the analysis, the meeting orientation dimensions were jointly added to the model in order to test the relationships of interest and accounted for an additional 18% of variance in team meeting satisfaction, F(8, 149) = 7.46, p < 0.001. Results indicated that overuse (β = −0.20, p = 0.007) and strategic use of meetings (β = 0.36, p < 0.001) were signiﬁcantly related to meeting satisfaction, thus providing support for hypotheses 1a April 2019 \| Volume 10 \| Article 812 Frontiers in Psychology \| www.frontiersin.org 4 Meeting Orientation Mroz et al. TABLE 1 \| Descriptive statistics and correlations of focal variables in study 1. Variable M SD 1 2 3 4 5 6 7 1. Meetings per week 3.37 3.82 − 2. Rewards 2.71 0.87 0.02 (0.85) 3. Strategic use 3.75 0.68 0.21∗ 0.39∗∗ (0.67) 4. Overuse 2.82 0.95 0.17∗ 0.08 0.12 (0.77) 5. Policy 3.04 0.76 0.07 0.36∗∗ 0.45∗∗ 0.32∗∗ (0.58) 6. Team meeting effectiveness 3.65 0.67 0.09 0.22∗ 0.51∗∗ −0.18∗ 0.17∗ (0.83) 7. Team meeting satisfaction 3.53 0.75 0.17∗ 0.26∗∗ 0.36∗∗ −0.18∗ 0.09 0.48∗∗ (0.85) N = 158. Diagonal values represent internal consistency estimates. ∗p < 0.05, ∗∗p < 0.001. TABLE 1 \| Descriptive statistics and correlations of focal variables in study 1. TABLE 2 \| Hierarchical multiple regression analyses predicting meeting satisfaction and meeting effectiveness in study 1. and 2c. Policy focus (β = −0.01, p = 0.88) and rewards (β = 0.10, p = 0.18) were not related to meeting satisfaction so hypotheses 2a and 2b were not supported. Team Meeting Effectiveness The analytic strategy described for team meeting eﬀectiveness as the outcome variable was followed to test hypotheses related to team meeting eﬀectiveness. Hypothesis 1b predicted that overuse would be negatively related to team meeting eﬀectiveness, and hypothesis 3a,c proposed that policy focus, rewards, and strategic use of meetings would be positively related to team meeting eﬀectiveness. As in the earlier test of meeting satisfaction, the ﬁrst step of the regression model included age, number of meetings attended per week, supervisory status, and job level as control variables. These demographic variables did not account for a signiﬁcant portion of the variability in meeting eﬀectiveness, F(4, 156) = 0.72, p = 0.56, R2 = 0.02. The meeting orientation facets were then added to the model in the second step and explained an additional 29% of meeting eﬀectiveness variance, F(8, 152) = 8.60, p < 0.001. Overuse (β = −0.22, p = 0.002) and strategic use of meetings (β = 0.53, p < 0.001) were signiﬁcantly related to meeting eﬀectiveness, which provided support for hypotheses 1b and 3c. Policy focus (β = −0.01, p = 0.89) and rewards (β = −0.01, p = 0.88) were not related to meeting satisfaction so hypotheses 3a and 3b were not supported. Complete results analyses are displayed in Table 2. commitment, turnover intentions, actual turnover, and job performance (Graen and Uhl-Bien, 1995). However, certain facets of meeting orientation may be advantageous or disadvantageous relative to employee attitudes. For instance, employees who believe that their organization overuses group and team meetings—meeting overuse is a negative facet of meeting orientation that refers to the degree to which employees believe the organizations has too many meetings—may have poor work attitudes. Building from social exchange theory and POS theory, if employees believes that the organization does not value their time and wastes it on unnecessary group and team meetings, the employees are likely to have less favorable work attitudes. These positive (or negative) interactions may represent something beyond the dyadic relationship because leaders represent a proxy for the organization (Graen and Uhl-Bien, 1995). Subordinates who perceive their supervisors to be supportive may construe this interaction as an extension of the organization’s support. Through social exchange mechanisms, subordinates may further identify with the organization’s goals and care about organizational outcomes (Eisenberger et al., 1986). Therefore, we propose the following hypotheses: Participants and Procedure Participants in this study were recruited through a snowball sampling technique. Undergraduate students attending a large southeastern university enrolled in a psychology course were given a description of the study and Qualtrics link to share with full-time working adults in exchange for course extra credit. At the end of the survey, participants were encouraged to forward the survey link to other working adults who might be interested in participating. Participants were required to be employees in the United States who attend at least one work meeting per week. The sample consisted of 213 primarily White (66%) working adults, nearly split between males (48%) and females (52%). Measures Hypothesis 6: Policy focus (6a), rewards (6b), and strategic use of meetings (6c) will be negatively related to ITQ. Hypothesis 7: Policy focus (7a), rewards (7b), and strategic use of meetings (6c) will be positively related to work engagement. Hypothesis 6: Policy focus (6a), rewards (6b), and strategic use of meetings (6c) will be negatively related to ITQ. Hypothesis 6: Policy focus (6a), rewards (6b), and strategic use of meetings (6c) will be negatively related to ITQ. Meeting orientation The 12-item meeting orientation scale (Allen and Hansen, 2011) described in Study 1 was used in Study 2. Estimates of internal consistency as assessed by Cronbach’s alpha exceed 0.79 for all scales. Hypothesis 7: Policy focus (7a), rewards (7b), and strategic use of meetings (6c) will be positively related to work engagement. Hypothesis 7: Policy focus (7a), rewards (7b), and strategic use of meetings (6c) will be positively related to work engagement. Although we expect that an organization’s meeting orientation is related to various job attitudes, such as ITQ and work engagement, additional team factors seem relevant in the context of this framework. That is, if meeting orientation is optimal or suboptimal, there are team factors that may strengthen positive job attitudes or reduce negative job attitudes. One good condition for teamwork, perceptions of voice, may promote good team behaviors (Gorden and Infante, 1991). Work engagement Employee work engagement was assessed using the Utrecht Work Engagement Scale (Schaufeli and Bakker, 2003). The scale consists of 17 items that measure three dimensions of work engagement: vigor, dedication, and absorption. Sample items include “At my work, I feel bursting with energy” (vigor), “I ﬁnd the work that I do full of meaning and purpose” (dedication), and “I am immersed in my work” (absorption). Participants responded using a 7-point scale to indicate how often they feel each way at work from never to always. Engagement is typically examined as one factor due to high inter-correlations between the three dimensions (Allen and Rogelberg, 2013), as is the case in the present study. Internal consistency for this measure was 0.94. Voice refers to the degree in which employees feel as if they have voice and freedom to discuss their concerns (Gorden and Infante, 1991). Traditionally, this concept has been used as an important variable for employees who feel the need to change dissatisfying working conditions (Hirschman, 1970). Employees that perceive themselves to have a high voice may feel that: their ideas are valuable, they may share such ideas with others, and they may feel like they can actively participate in solving problems rather than simply acknowledging to decisions made by management (Gorden and Infante, 1991). In the context of meeting orientation, voice may serve as a resource that augments the eﬀect of meeting orientation on positive workplace attitudes and depresses the eﬀect of meeting orientation on negative workplace attitudes. In other words, we expect that the act of allowing dissenting views, ideas, or opinions in meetings may STUDY 2 Hypothesis 9: Voice in team meetings moderates the relationship between policy focus (9a) and strategic use of meetings (9b) and engagement, such that the relationships will be more strongly positive when voice is high compared to low. Figure 2 includes all hypothesized relationships tested in Study 2. In contrast, the group and team meeting context may also allow employees to engage in withdrawal behaviors—temporarily or permanently separating from their work roles (Harrison et al., 2006). For example, there are a variety of counterproductive team meeting behaviors that precipitously decrease employees’ attitudes related to their meetings and their organization overall (Lehmann-Willenbrock et al., 2016). As meetings are repeatedly held in contexts that are not conducive to the team’s best interests, individuals may feel drained and burned out since they are relying on this form of collaboration to facilitate the accomplishment of their goals. Thus, we believe that supervisors that exemplify the positive aspects of an organizations meeting orientation will enable engagement and reduce feelings related to quitting. The following are hypothesized: STUDY 2 The dimensions of meeting orientation may uniquely relate to employee work-related attitudes. According to Hansen and Allen’s (2015) theoretical propositions, meeting orientation should impact the culture, structure, and resources within an organization. Workplace meetings provide a setting in which supervisors and subordinates come together and interact in meaningful ways. Therefore, organizations with a high meeting orientation allow employees more opportunities for such meaningful interactions. High quality interactions are associated with trust, loyalty, respect, and obligation (Cropanzano and Mitchell, 2005). As a result, high quality leader-member exchange can result in organizational outcomes including: organizational April 2019 \| Volume 10 \| Article 812 Frontiers in Psychology \| www.frontiersin.org 5 Meeting Orientation Mroz et al. Hypothesis 4: Overuse will be positively related to ITQ. Hypothesis 5: Overuse will be negatively related to work engagement. build a context of openness that empowers employees to take ownership of their work; in turn, this should promote feelings of engagement and reduce ITQ. Thus, we hypothesize: Hypothesis 8: Voice in team meetings moderates the relationship between policy focus (8a) and strategic use of meetings (8b) and ITQ, such that the relationships will be more strongly negative when voice is low compared to high. Hypothesis 8: Voice in team meetings moderates the relationship between policy focus (8a) and strategic use of meetings (8b) and ITQ, such that the relationships will be more strongly negative when voice is low compared to high. Hypothesis 9: Voice in team meetings moderates the relationship between policy focus (9a) and strategic use of meetings (9b) and engagement, such that the relationships will be more strongly positive when voice is high compared to low. An organization’s emphasis on meeting orientation may contribute to both employee engagement and ITQ. Previous research demonstrated that employee engagement can be fostered in the context of workplace meetings (Allen and Rogelberg, 2013). Speciﬁcally, eﬀectively managed group and team meetings create the conditions necessary for employees to engage in their work. Organizations with a stronger meeting orientation may provide employees with group and team meeting opportunities that assist with their ability to perform at optimal levels, connect with their role in the organization, and become fully immersed in their work (Bakker and Shaufeli, 2008). Frontiers in Psychology \| www.frontiersin.org Intentions to Quit Hypotheses 4 stated that overuse would be positively related to ITQ, whereas Hypotheses 6a,c proposed that policy focus, rewards, and strategic use of meetings would be negatively related to ITQ. Our control, number of meetings per week did not explain a signiﬁcant amount of variability in ITQ, F(1, 211) = 0.02, p = 0.88, R2 = 0.00. The meeting orientation facets were jointly added to the model in the second step and accounted for an additional 19% of variance in ITQ, F(5, 207) = 9.81, p < 0.05, R2 = 0.19. Overuse (β = 0.32, p < 0.001) and policy focus (β = −0.29, p < 0.05) were signiﬁcantly related to ITQ, which supported Hypothesis 4 and 6a. Rewards (β = 0.07, p = 0.30) and strategic use of meetings Intentions to quit A 3-item measure developed by Landau and Hammer (1986) was used to capture employees’ ITQ their work organization. Along a 7-point scale, participants reported the extent to which they agree with the statements (e.g., “I am actively looking for a job outside my current company”) from not at all to extremely. This measure demonstrated acceptable internal consistency with a Cronbach’s alpha of 0.88. April 2019 \| Volume 10 \| Article 812 6 Mroz et al. Meeting Orientation FIGURE 2 \| Hypothesized relationships in Study 2. FIGURE 2 \| Hypothesized relationships in Study 2. each hypothesis, and complete results of the ﬁnal models are displayed in Table 4. Voice Voice was assessed using a 5-item measure from Gorden and Infante (1991) focusing on the degree to which employees felt they had voice and freedom to discuss concerns in their company or organization. Sample items included: “there was fear of expressing your true feelings on work issues” and “employees were penalized if they openly disagreed with management practices.” Ratings were made on a 7-point scale ranging from 1 (never) to 7 (always). Internal consistency for this measure was 0.75. Frontiers in Psychology \| www.frontiersin.org April 2019 \| Volume 10 \| Article 812 Results 1 2 3 4 5 6 7 Low Policy High Policy Intentions to Quit Meeting Orientation: Policy Focus Low Voice High Voice FIGURE 4 \| Policy focus interacted with voice such that the negative relationship between ITQ and policy focus was stronger when voice was low compared to high. 1 2 3 4 5 6 7 Low Strategic Use High Strategic Use Intentions to Quit Meeting Orientation: Strategic Use Low Vocie High Vocie FIGURE 3 \| Strategic use of meetings interacted with voice such that using meetings strategically was most beneﬁcial in reducing intentions to quit when voice was low (1 SD below the mean) compared to high (1 SD above the mean). 1 2 3 4 5 6 7 Low Strategic Use High Strategic Use Intentions to Quit Meeting Orientation: Strategic Use Low Vocie High Vocie FIGURE 3 \| Strategic use of meetings interacted with voice such that using meetings strategically was most beneﬁcial in reducing intentions to quit when voice was low (1 SD below the mean) compared to high (1 SD above the mean). 1 2 3 4 5 6 7 Low Policy High Policy Intentions to Quit Meeting Orientation: Policy Focus Low Voice High Voice N = 230. Standardized regression coefﬁcients are displayed. N = 192. ∗p < 0.05, ∗∗p < 0.001. 1R2 is from the model that included all variables aside from the interaction term. (β = −0.08, p = 0.28) were not related to ITQ, which did not support Hypotheses 6b or 6c. We also hypothesized that the relationship between policy focus and strategic use of meetings and ITQ would be moderated by voice, such that the relationships would be stronger when voice was high compared to low. First, we calculated an interaction term between policy and strategic use of meeting sand ITQ. For the regression analyses, the ﬁrst step contained the control, number of meetings per week, the second step contained voice, the third step contained the four meeting orientations, and the interaction term was entered in the ﬁnal step. The interaction term between policy and voice was signiﬁcant and accounted for a signiﬁcant portion of variance in ITQ, 1R2 = 0.02, β = 0.66, p < 0.05, within the context of the entire model, F(7, 205) = 13.30, p < 0.05, R2 = 0.31, supporting Hypothesis 8a. Results Descriptive statistics, alpha estimates of internal consistency, and correlations between study variables are included in Table 3. Hierarchical regression analyses were used to test TABLE 3 \| Descriptive statistics and correlations of focal variables in study 2. Variable M SD 1 2 3 4 5 6 7 8 1. Meetings per week 2.69 2.90 − 2. Reward 3.59 1.63 0.07 (0.91) 3. Strategic use 5.04 1.31 0.16∗ 0.38∗∗ (0.84) 4. Overuse 3.87 1.62 0.26∗∗ 0.16∗ 0.12 (0.84) 5. Policy 4.49 1.35 0.08 0.34∗∗ 0.53∗∗ 0.20∗ (0.79) 6. Voice 4.80 1.26 0.04 −0.08 0.16∗ −0.35∗∗ 0.08 (0.75) 7. Engagement 4.80 1.11 0.02 −0.15∗ 0.36∗∗ −0.03 0.38∗∗ 0.22∗ (0.94) 8. Intention to quit 3.39 1.85 −0.01 −0.16∗ −0.24∗ 0.23∗ −0.30∗∗ −0.44∗∗ −0.48∗∗ (0.88) N = 213. Diagonal values represent internal consistency estimates. ∗p < 0.05, ∗∗p < 0.01. Frontiers in Psychology \| www.frontiersin.org 7 Meeting Orientation Mroz et al. TABLE 4 \| Hierarchical multiple regression analyses predicting intentions to quit and work engagement in study 2. Intentions to quit Work engagement Variable Model 1 Model 2 Model 1 Model 2 Meetings per week −0.01 −0.02 −0.03 −0.03 Policy focus −0.73∗∗ −0.26∗∗ 0.18 0.27∗∗ Rewards −0.12 −0.11 0.01 0.01 Strategic use −0.01 −0.56∗ 0.20∗ 0.24 Overuse 0.18∗ 0.19∗ −0.05 −0.05 Voice −0.78∗∗ −0.89∗∗ 0.07 0.19 Voice x policy focus 0.66∗ − 0.13 − Voice x strategic use − 0.83∗ − −0.06 F 13.29∗∗ 13.38∗∗ 7.68∗∗ 7.65∗∗ Adjusted R2 0.29 0.29 0.18 0.18 1R2 0.02∗ 0.02∗ 0.01 < 0.01 N = 230. Standardized regression coefﬁcients are displayed. N = 192. ∗p < 0.05, ∗∗p < 0.001. 1R2 is from the model that included all variables aside from the interaction term. TABLE 4 \| Hierarchical multiple regression analyses predicting intentions to quit and work engagement in study 2. 1 2 3 4 5 6 7 Low Strategic Use High Strategic Use Intentions to Quit Meeting Orientation: Strategic Use Low Vocie High Vocie FIGURE 3 \| Strategic use of meetings interacted with voice such that using meetings strategically was most beneﬁcial in reducing intentions to quit when voice was low (1 SD below the mean) compared to high (1 SD above the mean). Work Engagement Hypotheses 5 proposed that overuse of meetings would be negatively related to work engagement, and Hypotheses 7a,c stated that policy focus, rewards, and strategic use of meetings would be positively associated with work engagement. The ﬁrst step with the control variable, number of meetings per week, did not explain a signiﬁcant amount of variance in work engagement, F(1, 211) = −0.08, p = 0.78, R2 = 0.00. Results Similarly, the interaction term between strategic use in meetings and voice was signiﬁcant, 1R2 = 0.02, β = 0.07, p < 0.05, within the context of the entire model, F(7, 205) = 13.38, p < 0.05, R2 = 0.31, supporting Hypothesis 8b. The interactions are depicted in Figures 3, 4. FIGURE 4 \| Policy focus interacted with voice such that the negative relationship between ITQ and policy focus was stronger when voice was low compared to high. 7c were supported. Overuse (β = −0.11, p = 0.09) and rewards (β = −0.01, p = 0.86), however, were not related to ITQ, which did not support Hypothesis 5 or 7b. We also hypothesized that the relationship between policy focus and strategic use of meetings and engagement would be moderated by voice, such that the relationship would be stronger for those with greater policy focus or strategically focused orientations. First, we calculated an interaction term between policy and strategic use of meeting sand ITQ. For the regression analyses, the ﬁrst step contained the control, number of meetings per week, the second step contained voice, the third step contained the four meeting orientations, and the interaction term was entered in the ﬁnal step. The interaction term was not signiﬁcant for either policy (1R2 = 0.00, β = 0.13, p = 0.70) or strategic use (1R2 = 0.00, β = −0.06, p = 0.88). GENERAL DISCUSSION The four meeting orientation facets were added to the model in the second step and accounted for an additional 19% of variance in work engagement, F(5, 207) = 9.97, p < 0.05, R2 = 0.19. Policy (β = 0.28, p < 0.05) and strategic use of meetings (β = 0.23, p < 0.05) were signiﬁcantly related to work engagement in the appropriate directions so Hypotheses 7a and This paper represents the ﬁrst empirical investigation of the meeting orientation construct. As the ﬁrst, exploratory step in a broader investigation of organizational meeting orientation, the results of this study conﬁrm a series of hypotheses that relate facets of meeting orientation, policy focus, rewards, strategic use, April 2019 \| Volume 10 \| Article 812 Frontiers in Psychology \| www.frontiersin.org 8 Meeting Orientation Mroz et al. and potential overuse, to perceived team meeting eﬀectiveness and team meeting satisfaction as well as ITQ and work engagement. In Study 1 which included all variables, strategic use was positively related to perceived team meeting eﬀectiveness and satisfaction; overuse, on the other hand, was negatively related to perceived team meeting eﬀectiveness and satisfaction, whereas rewards and policy were not related to either outcome. Extending our ﬁndings from Study 1, we explored the extent to which an organization’s orientation toward meetings inﬂuences employee attitudes toward the organization. We found that employees in ﬁrms with a stronger, positive meeting orientation (deﬁned as high on strategy, policy, and rewards and low on overuse) were more engaged in their work than employees in ﬁrms with a weak or negative meeting orientation. Policy, rewards, and strategic use were positively related to engagement, whereas meeting overuse was negatively related. Similarly, our ﬁndings indicate that meeting orientation is also related to employee ITQ. Greater meeting overuse was associated with higher turnover intentions, whereas strategic use of meetings was negatively related to ITQ. meeting outcomes. For instance, prior research has demonstrated that satisfaction with meetings is a unique component of overall job satisfaction, even controlling for all traditional predictors of job satisfaction (Rogelberg et al., 2010). Across the two studies reported in this paper, organizational meeting orientation explained 33% of the variability in team meeting eﬀectiveness, 20% of team meeting satisfaction, 19% of ITQ, and 19% of employee engagement. GENERAL DISCUSSION Much research on improving group and team meetings focuses on individual meeting practices, such as using an agenda, which may be helpful in improving the meetings of speciﬁc managers, but does not address meeting processes and procedures fostered at the organizational level. Second, a variety of meeting scholars (cf. Allen et al., 2015) have suggested that technological advances in the workplace have nearly made informational meetings, or meetings in which people gather and exchange information, irrelevant, and that these irrelevant and unnecessary team meetings have contributed to the negative view of meetings in popular culture. The results of the study, however, indicate that people are more satisﬁed and believe that their group and team meetings are more eﬀective when the organization supports and extensively utilizes information sharing in team meetings. In Study 2, we expanded our focus to an important variable related to group dynamics: perceived voice in meetings. Employees who believe they have high voice in meetings are more likely to speak up to voice their concerns, thoughts, and opinions during a group meeting context (Gorden and Infante, 1991). Indeed, we found that voice moderated the relationship between some facets of meeting orientation and ITQ. In general, a stronger organizational meeting orientation toward strategic use of team meetings for sharing, reacting to, and action upon information and having speciﬁc policies for the use of group and meetings was more beneﬁcial to lower ITQ when voice was low compared to high. These ﬁndings illustrate that, in the absence of productive climates toward group interactions, factors speciﬁc to the organizational team meeting context can compensate, thereby leading to a more favorable employee attitude. Third, group and team meetings may serve as an important tool which allows for the facilitation of employee-supervisor interactions; guided by an organizational meeting orientation, these exchanges can be advantageous and disadvantageous toward work attitudes. For instance, if an employee evaluates the dyadic relationship positively, they may construe the interactions as an extension of the organization’s support, thus, may be more motivated to accomplish work tasks (Eisenberger et al., 1986). However, if an employee feels as if their supervisor requires attendance to too many irrelevant team meetings, the employee may evaluate these interactions negatively, thus, engage in withdrawal behaviors (Allen and Rogelberg, 2013). The eﬀects of these interactions may ripple across work attitudes. Practical Implications Organizations may have various organizational-level orientations (e.g., market, customer, technology) meant to advance the topic of interest (Hansen and Allen, 2015). Although meeting orientation is not an overarching business aim like those previously mentioned, there are potentials for positive outcomes related to employee engagement, transfer of knowledge, and dynamic capabilities (i.e., response to change) as explained by Hansen and Allen (2015) in their theoretical framework. Being that policy and overuse meeting orientations are related to these job outcomes, there seem to be high costs associated with overuse and turnover intentions but gains related to policy and managerial support. Our ﬁndings warrant several managerial and organizational implications. GENERAL DISCUSSION g p y Despite the strong pattern of results linking aspects of meeting orientation to group and team meeting outcomes and employees’ work attitudes, several of our hypotheses were not supported. Controlling for number of meetings attended per week and the unique contribution of each facet of meeting orientation, policy focus and rewards explained unique variability only in work engagement. One reason for the relatively small contributions of these facets may be that these facets are more nebulous and less concrete than the others. For example, many organizations may not have speciﬁc policies that promote group and team meetings that employees can readily identify, meaning that the policy focus aspect of meeting orientation may not be useful or that the scale needs to be modiﬁed. Similarly, employees may have diﬃculty recalling speciﬁc rewards that their organizations oﬀer to people who attend, lead, or organize team meetings. Limitations The ﬁndings of the study are an encouraging ﬁrst step in the exploration of organizational level attitudes toward team meetings that can aﬀect individual level outcomes, but a number of limitations must be considered when interpreting these ﬁndings. Most importantly, data examined in this study is cross-sectional in nature, which precludes drawing causal connections between variables, especially considering the scant literature and theorizing on meeting orientation generally. Furthermore, the cross-sectional, same-source data also makes the ﬁndings less potent. Although the models in this study depict meeting orientation leading to team meeting eﬀectiveness, team meeting satisfaction, ITQ, and work engagement, it is entirely plausible that the opposite is true. For example, perhaps people who think their meetings are eﬀective and satisfying believe that the organization strategically uses (and does not overuse) meetings. Future research should examine meeting orientation using a variety of data sources, such as objective, behaviorally based measures of team meeting eﬀectiveness or quality, and relate these two ratings of meeting orientation. Future research on team meeting orientation should focus on the measurement of full teams given that perceptions of meeting quality may be driven by the role held by the meeting participant (e.g., leader, attendee). Decades of organizational research have compared self, peer, and supervisor ratings on perceptions of traits, skills, abilities, and performance levels; at best, self-ratings demonstrate a moderate relationship to objective measures (Mabe and West, 1982; Harris and Schaubroeck, 1988; Bass and Yammarino, 1991). Team meetings may serve as another context in which there are discrepant ratings between roles, driven by various biases (e.g., Greenwald, 1980; Goethals, 1986). In fact, Cohen et al. (2011) noted that employees in higher positions of power tended to rate their meetings as higher quality compared to others. Perhaps these discrepant meeting perceptions are more complicated than a role diﬀerences but also a function of meeting type. For instance, status update meetings may be more valuable to the project manager than the attendees, however, a strategic planning meeting may be valuable to all attendees involved. Second, participants in this study represented a wide variety of organizations and were therefore each rating diﬀerent organizations and diﬀerent meetings. This is both a strength (i.e., increases generalizability) and limitation (i.e., hard to make speciﬁc predictions) of the studies. Theoretical Implications The results of these studies have several implications. First, although the fact of being unstudied does not necessarily warrant research into a new area, this paper provided preliminary evidence that facets of organizational meeting orientation are related, and in some cases quite strongly, to important team In terms of managerial implications, our ﬁndings suggest that meeting leaders have the discretion to capitalize on planning and leadership behaviors associated with the various meeting orientation dimensions. First, managers should consider whether it is necessary to schedule a team meeting; if the information April 2019 \| Volume 10 \| Article 812 Frontiers in Psychology \| www.frontiersin.org 9 Meeting Orientation Mroz et al. can easily be shared through email or one-on-one conversations, managers should take advantage of these alternative forms of communication rather than holding pointless meetings. Second, when calling employees for a necessary group or team meeting, leaders should only invite people for which the content is relevant. For instance, rather than a manager calling their entire team, managers can make decisions as to which collaborators are essential to accomplish the meeting’s purpose. Third, to respect everyone’s time, meeting leaders should use an agenda as a roadmap to guide and end the team meeting when the items are completed. Fourth, it is crucial that meeting leaders utilize group and team meetings as a strategic tool to gather, disseminate, and respond to information relevant to all attendees. level factor, of interest to meeting researchers should be how organizations with diﬀerent meeting orientations conduct and approach group and team meetings, and another area that he may be how individuals with in those organi zations perceive their meetings. Third, we implemented several strategies to mitigate concerns of common method variance given the cross-sectional nature of these studies (Podsakoﬀet al., 2003). To reduce demand characteristics and evaluation apprehension, we assured participants that their responses would remain anonymous and that there were no right or wrong answers. To mitigate order eﬀects, priming eﬀects, and item-context-induced mood states, we counterbalanced the measures and items through randomization (Podsakoﬀet al., 2003; Conway and Lance, 2010). To optimize comprehension, each item was simple, speciﬁc, and concise. In terms of organizational implications, our ﬁndings suggest that organizations can use meeting orientation as a competitive advantage to guide skills, behaviors, and processes of leaders and employees. Theoretical Implications First, organizations should assess where they fall within the four dimensions of meeting orientation; if necessary, organizations should make adjustments to the policies, procedures, and practices surrounding their meeting usage. Second, since group and team meetings may be perceived as interruptions from daily work tasks, organizational leaders should instruct on when it is appropriate to hold team meetings. Third, organizations should institute policies, procedures, or training programs to instruct managers on good team meeting practices (e.g., temporal, physical, cross-cultural considerations). Future Directions and Propositions for Teams Over Time Although the forgoing studies substantiate the existence of meeting orientation, they cannot directly speak to how meeting orientation impacts teams at initial formation and over time as they work in the organization. However, an organization’s orientation toward the use of team meetings in each of the four facets could have implications for the ways in which teams develop and evolve over time. In our approach to meeting orientation, a “positive” orientation includes high levels of strategic use, policy focus, and rewards, whereas a negative orientation is low on those facets and high on overuse. Based on the ﬁndings reported in this paper, we develop several propositions below regarding meeting orientation. With respect to how teams develop over time, a positive meeting orientation may play an important role in establishing the working environment of new teams, acclimating new team members to the team and organization’s culture, fostering high-quality interactions with co-workers, enhancing commitment to the team and organization, and creating more stable team memberships. Frontiers in Psychology \| www.frontiersin.org ETHICS STATEMENT The institutional review board (IRB) for the University of Nebraska Medical Center and the University of Nebraska at Omaha approved an exempt IRB protocol for the forgoing study. In this case, consent was given by participation in the surveys provided and completion of the survey was that consent and no identifying information was asked on the survey. Proposition 2: Teams will experience less member change over time in organizations with a positive compared to a negative meeting orientation. AUTHOR CONTRIBUTIONS A critical role of meetings in team functioning is to act as a space for knowledge transfer among team members (Allen et al., 2014). Knowledge transfer includes passing information between individuals, groups, or organizations (Argote and Ingram, 2000), All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication. CONCLUSION Unlike other organizational orientations (e.g., entrepreneurial), no empirical studies have investigated the consequences of meeting orientation. Studies 1 and 2 suggest that meeting orientation is related to individual perceptions of team meeting eﬀectiveness, team meeting satisfaction, ITQ, and employee engagement even when controlling for several demographic variables. Although meeting orientation is not a predominant business aim, we see potential costs associated with meeting overuse but potential gains associated with strategic usage. Additionally, meeting orientation is an organizational level environmentally constraining construct with implications for new teams and for established teams. Over time, the meeting orientation of an organization has the potential to enable or constrain team performance and our hope is that the studies and propositions here will spur additional work by researchers on this important meeting science domain. Proposition 1a: Newly constituted teams will perform better in organizations with a positive compared to a negative meeting orientation. Proposition 1b: Newly constituted teams performance will be optimized over time in an organization with a positive meeting orientation compared to a negative meeting orientation. Team member change is one of the most common forms of changes in teams (Summers et al., 2012). Team member change can occur for a variety of reasons, but member change can often lead to, or be, a disruptive event (Olekalns et al., 2003). Member change has been conceptualized as a possible stimulant of team creativity as new members bring new ideas (Choi and Thompson, 2005), as a disruptive event that can lead to teams examining their processes and interaction strategies with an eye toward improvement (Zellmer-Bruhn, 2003), or as an opportunity for knowledge transfer and team functioning to decrease if core members change (Summers et al., 2012). We anticipate that team members will change less frequently as employees are less likely to think about quitting the organization entirely, and are more engaged in their work, when they perceive the organization to have a positive meeting orientation. Limitations To strengthen the design, future research on meeting orientation should contain a combination of individual and organizational levels of analyses, such that multiple data points are collected within each organization to make comparisons across organizations possible. As meeting orientation is inherently an organizational April 2019 \| Volume 10 \| Article 812 10 Mroz et al. Meeting Orientation Organizational leaders are often hiring new employees and launching new teams targeting projects of interest (Lester et al., 2002). Team comprised predominantly of new organizational members enter an environment where newcomer challenges exist (Chen and Klimoski, 2003), socialization to the organization is needed (Allen et al., 1999), and meeting orientation essentially deﬁnes how the team operates from a team meeting perspective. Given these challenges, it is likely that a positive meeting orientation as just deﬁned would facilitate team performance generally, while a negative meeting orientation may hinder such progress in these newly formed and newly constituted teams. Further, over time, we anticipate that although team performance of new teams general improves with familiarity and codiﬁcation of group processes, the stable meeting orientation (positive or negative) will create an artiﬁcial boundary condition on team performance either enabling maximal performance (i.e., positive meeting orientation) or constraining performance to a less than optimal level (i.e., negative meeting orientation). Thus, the following propositions are suggested: Organizational leaders are often hiring new employees and launching new teams targeting projects of interest (Lester et al., 2002). Team comprised predominantly of new organizational members enter an environment where newcomer challenges exist (Chen and Klimoski, 2003), socialization to the organization is needed (Allen et al., 1999), and meeting orientation essentially deﬁnes how the team operates from a team meeting perspective. Given these challenges, it is likely that a positive meeting orientation as just deﬁned would facilitate team performance generally, while a negative meeting orientation may hinder such progress in these newly formed and newly constituted teams. Further, over time, we anticipate that although team performance of new teams general improves with familiarity and codiﬁcation of group processes, the stable meeting orientation (positive or negative) will create an artiﬁcial boundary condition on team performance either enabling maximal performance (i.e., positive meeting orientation) or constraining performance to a less than optimal level (i.e., negative meeting orientation). Thus, the following propositions are suggested: and knowledge/information sharing is a positive predictor of team performance (Mesmer-Magnus and DeChurch, 2009). Limitations As team members share information more frequently, the pool of information available for other team members to use increases, which can improve team performance (Hackman, 1987). When team meetings are used strategically and when necessary, teams may engage in increased information sharing behaviors, which may result in increased performance over time. Therefore, we propose: Proposition 3: There is a positive a relationship between team information sharing over time and an organization’s meeting orientation. Allen, J. A., Lehmann-Willenbrock, N., and Rogelberg, S. G. (2015). The Cambridge Handbook of Meeting Science. New York, NY: Cambridge University Press. Allen, J. A., and Rogelberg, S. G. (2013). Manager-led group meetings: a context for promoting employee engagement. Group Organ. Manag. 38, 543–569. doi: 10.1177/1059601113503040 Allen, T. D., McManus, S. E., and Russell, J. E. (1999). Newcomer socialization and stress: formal peer relationships as a source of support. J. Vocat. Behav. 54, 453–470. doi: 10.1006/jvbe.1998.1674 Allen, T. D., McManus, S. E., and Russell, J. E. (1999). Newcomer socialization and stress: formal peer relationships as a source of support. J. Vocat. Behav. 54, 453–470. doi: 10.1006/jvbe.1998.1674 REFERENCES Allen, J. A., Beck, T., Scott, C. W., and Rogelberg, S. G. (2014). Understanding workplace meetings: a qualitative taxonomy of meeting purposes. Manag. Res. Rev. 37, 791–814. doi: 10.1108/MRR-03-2013-0067 Allen, J. A., and Hansen, J. (2011). Meeting orientation: conceptualization, antecedents, and outcomes. Paper Presented at the INGRoup Conference, Minneapolis, MN. April 2019 \| Volume 10 \| Article 812 Frontiers in Psychology \| www.frontiersin.org 11 Mroz et al. Meeting Orientation Argote, L., and Ingram, P. (2000). Knowledge transfer: a basis for competitive advantage in ﬁrms. Organ. Behav. Hum. Decis. Process. 82, 150–169. doi: 10. 1006/obhd.2000.2893 Lehmann-Willenbrock, N., Allen, J. A., and Belyeu, D. (2016). Our love/hate relationship with workplace meetings: relating good and bad meeting behaviors to meeting outcomes, engagement, and exhaustion. Manag. Res. Rev. 39, 1293–1312. doi: 10.1108/MRR-08-2015-0195 Bakker, A. B., and Shaufeli, W. B. (2008). Positive organizational behavior: engaged employees in ﬂourishing organizations. J. Organ. Behav. 29, 147–154. doi: 10. 1002/job.515 Lester, S. W., Meglino, B. M., and Korsgaard, M. A. (2002). The antecedents and consequences of group potency: a longitudinal investigation of newly formed work groups. Acad. Manag. J. 45, 352–368. doi: 10.5465/ 3069351 Bass, B. M., and Yammarino, F. J. (1991). Congruence of self and others’ leadership ratings of naval oﬃcers for understanding successful performance. Appl. Psychol. Int. Rev. 40, 437–454. doi: 10.1111/j.1464-0597.1991.tb01002.x Luong, A., and Rogelberg, S. G. (2005). Meetings and more meetings: the relationship between meeting load and the daily well-being of employees. Group Dyn. Theory Res. Pract. 9, 58–67. doi: 10.1037/1089-2699.9.1.58 Becker, T. E. (2005). Potential problems in the statistical control of variables in organizational research: a qualitative analysis with recommendations. Organ. Res. Methods 8, 274–289. doi: 10.1177/1094428105278021 Mabe, P. A., and West, S. G. (1982). Validity of self-evaluation of ability: a review and meta-analysis. J. Appl. Psychol. 67, 280–296. doi: 10.1037/0021- 9010.67.3.280 Chen, G., and Klimoski, R. J. (2003). The impact of expectations on newcomer performance in teams as mediated by work characteristics, social exchanges, and empowerment. Acad. Manag. J. 46, 591–607. doi: 10.5465/30040651 Matsuno, K., Mentzer, J. T., and Rentz, J. O. (2005). A conceptual and empirical comparison of three market orientation scales. J. Bus. Res. 58, 1–8. doi: 10.1016/ S0148-2963(03)00075-4 Choi, H. -S., and Thompson, L. (2005). Old wine in a new bottle: impact of membership change on group creativity. Organ. Behav. Hum. Decis. Process. 98, 121–132. doi: 10.1016/j.obhdp.2005.06.003 McComas, K. A. (2003). REFERENCES Citizen satisfaction with public meetings used for risk communication. J. Appl. Commun. Res. 31, 164–184. doi: 10.1080/ 0090988032000064605 Cohen, M. A., Rogelberg, S. G., Allen, J. A., & Luong, A. (2011). Meeting design characteristics and attendee perceptions of staﬀ/team meeting quality. Group Dyn. Theor. Res. Pract. 15, 90–104. doi: 10.1037/a0021549 McComas, K. A., Tuit, L. S., Waks, L., and Sherman, L. A. (2007). Predicting satisfaction and outcome acceptance with advisory committee meetings: the role of procedural justice. J. Appl. Soc. Psychol. 37, 905–927. doi: 10.1111/j.1559- 1816.2007.00192.x Conway, J. M., and Lance, C. E. (2010). What reviewers should expect from authors regarding common method bias in organizational research. J. Bus. Psychol. 25, 325–334. doi: 10.1007/s10869-010-9181-6 Cropanzano, R., and Mitchell, M. S. (2005). Social exchange theory: an interdisciplinary review. J. Manag. 31, 874–900. doi: 10.1177/ 0149206305279602 Mesmer-Magnus, J. R., and DeChurch, L. A. (2009). Information sharing and team performance: a meta-analysis. J. Appl. Psychol. 94, 535–546. doi: 10.1037/ a0013773 Eisenberger, R., Huntington, R., Hutchison, S., and Sowa, D. (1986). Perceived organizational support. J. Appl. Psychol. 71, 500–507. doi: 10.1037/0021-9010. 71.3.500 Olekalns, M., Brett, J. M., and Weingart, L. R. (2003). Phases, transitions and interruptions: modeling processes in multi-party negotiations. Int. J. Conﬂ. Manag. 14, 191–211. doi: 10.1108/eb022898 Podsakoﬀ, P. M., MacKenzie, S. B., Lee, J. Y., and Podsakoﬀ, N. P. (2003). Common method biases in behavioral research: a critical review of the literature and recommended remedies. J. Appl. Psychol. 88, 879–903. doi: 10.1037/00219010. 88.5.879 Goethals, G. R. (1986). “Fabricating and ignoring social reality: Self-serving estimates of consensus,” in Social Comparison and Relative Deprivation: The Ontario Symposium, eds J. M. Olson, C. P. Herman, and M. P. Zhanna (Hillsdale, NJ: Erlbaum), 135–157. Gorden, W. I., and Infante, D. A. (1991). Test of a communication model of organizational commitment. Commun. Quart. 39, 144–155. doi: 10.1080/ 01463379109369792 Rauch, A., Wiklund, J., Lumpkin, G. T., and Frese, M. (2009). Entrepreneurial orientation and business performance: an assessment of past research and suggestions for the future. Entrepreneursh. Theor. Pract. 33, 761–787. doi: 10. 1111/j.15406520.2009.00308.x Graen, G. B., and Uhl-Bien, M. (1995). Relationship-based approach to leadership: development of leader-member exchange (LMX) theory of leadership over 25 years: applying a multi-level multi-domain perspective. Leadersh. Quart. 6, 219–247. doi: 10.1016/1048-9843(95)90036-5 Reinig, B. A., and Shin, B. (2002). The dynamic eﬀects of group support systems on group meetings. J. Manag. Inform. Syst. 19, 303–325. doi: 10.1080/07421222. 2002.11045728 Greenwald, A. G. (1980). REFERENCES The totalitarian ego: fabrication and revision of personal history. Am. Psychol. 35, 603–618. Rogelberg, S. G., Allen, J. A., Shanock, L., Scott, C., and Shuﬄer, M. (2010). Employee satisfaction with meetings: a contemporary facet of job satisfaction. Hum. Res. Manag. 49, 149–172. doi: 10.1002/hrm.20339 Greenwald, A. G. (1980). The totalitarian ego: fabrication and revision of personal history. Am. Psychol. 35, 603–618. Hackman, J. R. (1987). “The design of work teams,” in Handbook of Organizational Behavior, Ed J. W. Lorsch (Upper Saddle River, NJ: Prentice Hall), 315–342. Hackman, J. R. (1987). “The design of work teams,” in Handbook of Organizational Behavior, Ed J. W. Lorsch (Upper Saddle River, NJ: Prentice Hall), 315–342. Schaufeli, W. B., and Bakker, A. B. (2003). UWES–Utrecht Work Engagement Scale: Test Manual. Utrecht: Department of Psychology, Utrecht University. Hansen, J., and Allen, J. A. (2015). “An organizational meeting orientation: The construct, scales, and research propositions,” in The Cambridge Handbook of Meeting Science, eds J. A. Allen, N. Lehmann-Willenbrock, and S. G. Rogelberg (New York: NY: Cambridge University Press), 347–383. Summers, J. K., Humphrey, S. E., and Ferris, G. R. (2012). Team member change, ﬂux in coordination, and performance: eﬀects of strategic core roles, information transfer, and cognitive ability. Acad. Manag. J. 55, 314–338. doi: 10.5465/amj.2010.0175 Harris, M. M., and Schaubroeck, J. (1988). A meta-analysis of self-supervisor, self- peer, and peer-supervisor ratings. Pers. Psychol. 41, 43–62. doi: 10.1111/j.1744- 6570.1988.tb00631.x Tourangeau, R., Rips, L. J., and Rasinski, K. (2000), The Psychology of Survey Response, New York, NY: Cambridge University Press. Harrison, D. A., Newman, D. A., and Roth, P. L. (2006). How important are job attitudes? meta-analytic comparisons of integrative behavioral outcomes and time sequences. Acad. Manag. J. 49, 305–325. doi: 10.5465/AMJ.2006.20786077 Tracy, K., and Dimock, A. (2004). Meetings: Discursive sits for building and fragmenting community. Commun. Yearbook 28, 127–165. doi: 10.1207/ s15567419cy2801_4 Zellmer-Bruhn, M. E. (2003). Interruptive events and team knowledge acquisition. Manag. Sci. 49, 514–528. doi: 10.1287/mnsc.49.4.514. 14423 Hirschman, A. O. (1970). Exit, Voice, and Loyalty: Responses to Decline in Firms, Organizations, and States. Cambridge, MA: Harvard University Press. , ( ) , , y y p , Organizations, and States. Cambridge, MA: Harvard University Press. Horan, A. P. (2002). An eﬀective workplace stress management intervention: Organizations, and States. Ca b dge, : a va d U ve s ty ess. Horan, A. P. (2002). An eﬀective workplace stress management intervention: Horan, A. P. (2002). REFERENCES An eﬀective workplace stress management intervention: Chicken soup for the soul at work employee groups. Work 18, 3–13. Chicken soup for the soul at work employee groups. Work 18, 3–13. Conﬂict of Interest Statement: The authors declare that the research was conducted in the absence of any commercial or ﬁnancial relationships that could be construed as a potential conﬂict of interest. Conﬂict of Interest Statement: The authors declare that the research was conducted in the absence of any commercial or ﬁnancial relationships that could be construed as a potential conﬂict of interest. Keith, E. (2015). 55 Million: A Fresh Look at the Number, Eﬀectiveness, and Cost of Meetings in the U.S. [Blog Post]. Available at: https://blog.lucidmeetings.com/ blog/fresh-look-number-eﬀectiveness-cost-meetings-in-us (accessed October 20, 2018). Copyright © 2019 Mroz, Landowski, Allen and Fernandez. 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. Kirca, A. H., Jayachandran, S., and Bearden, W. O. (2005). Market orientation: a meta-analytic review and assessment of its antecedents and impact on performance. J. Mark. 69, 24–41. doi: 10.1509/jmkg.69.2.24.60761 Landau, J., and Hammer, T. H. (1986). Clerical employees’ perceptions of intraorganizational career opportunities. Acad. Manag. 29, 385–404. doi: 10. 2307/256194 April 2019 \| Volume 10 \| Article 812 Frontiers in Psychology \| www.frontiersin.org 12
https://openalex.org/W2913118035	https://www.frontiersin.org/articles/10.3389/fphys.2019.00052/pdf	English	null	Book Review: The Prevention of Cardiovascular Disease Through the Mediterranean Diet	Frontiers in physiology	2,019	cc-by	924	Esther Udo Asamudo* and Chidinma Adanna Okolo Department of Physiology, School of Biomedical Sciences, University of Otago, Dunedin, New Zealand Keywords: Mediterranean diet (MD), cardiovascular health, Mediterranean countries, nutrition, cardiovascular disease BOOK REVIEW published: 07 February 2019 doi: 10.3389/fphys.2019.00052 BOOK REVIEW Reviewed by: Reviewed by: Antonio Marcus de Andrade Paes, Universidade Federal do Maranhão, Brazil Reviewed by: Antonio Marcus de Andrade Paes, Universidade Federal do Maranhão, Brazil Graziela Scalianti Ceravolo, State University of Londrina, Brazil The book is made up of 12 chapters which gave in-depth explanation of what MD is. The composition as well as methods with which to achieve and adapt to MD, with the aim of preventing cardiovascular diseases, were highlighted. Graziela Scalianti Ceravolo, State University of Londrina, Brazil Graziela Scalianti Ceravolo, State University of Londrina, Brazil *Correspondence: Esther Udo Asamudo esther.asamudo@ postgrad.otago.ac.nz The introductory chapter talks about the traditional healthy MD pyramid, the biological and epidemiological evidences regarding the beneﬁts of MD. This chapter discusses the several clinical trials and results involving the adherence to MD together with how it has impacted on the risk of cardiovascular diseases. The authors described the positive results from several clinical trials and mentioned that the signiﬁcant PREDIMED trial showed ∼30% decrease in cardiovascular disease risk factors. They also highlighted the fact that other lifestyle-related factors could be aﬀecting the potential eﬀect of MD on the improvement of cardiovascular health. Specialty section: This article was submitted to Integrative Physiology, a section of the journal Frontiers in Physiology Chapters 3 to 8 revealed the importance of major components of MD; fats, oil, mixed nuts, fruits and vegetables, cereals and legumes, ﬁsh and meats. Chapter 9 highlights the beneﬁts of moderate consumption of red wine to the body. Chapter 10 lays emphasis on the relationship between high life expectancy rates, low mortality rates and Mediterranean countries with regards to adopting a social lifestyle. Chapter 10 also elaborates on how climate conditions can impact on cardiovascular health and stresses the importance of mental well-being in maintaining a healthy lifestyle. Chapter 11 covers healthy diet and eﬀect of MD on heart and brain function and lists diﬀerent studies (between 2006 and 2017) which have attempted to show association between MD and improvement of cognitive function. Received: 10 November 2018 Accepted: 17 January 2019 Published: 07 February 2019 The Prevention of Cardiovascular Disease Through the Mediterranean Diet Almudena Sánchez-Villegas and Ana Sánchez-Tainta, (London; San Diego, CA: Academic Press) 2018, 240 Pages, ISBN: 978-0-12-811259-5. doi: 10.1016/C2016-0-00845-8 This book describes the Mediterranean diet (MD), explored its nutritional constituents and its beneﬁcial health eﬀects. It explains in detail how consumption of MD helps to prevent cardiovascular diseases. It also delineates how much wrong diet contributes to development and progression of heart diseases. Edited by: Kesia Palma-Rigo, Universidade Estadual de Maringá, Brazil In this book, MD was deﬁned as a healthy eating model, dating back to early 1960s and observed in Greece and Southern Italy. It was also described as a traditional diet obtained from Mediterranean countries, consisting of foods such as grains, vegetables, fruits, olive oil, nuts, ﬁsh, dairy products, eggs, meat, herbs, and spices. Citation: Citation: Asamudo EU and Okolo CA (2019) Book Review: The Prevention of Cardiovascular Disease Through the Mediterranean Diet. Front. Physiol. 10:52. doi: 10.3389/fphys.2019.00052 Citation: Asamudo EU and Okolo CA (2019) Book Review: The Prevention of Cardiovascular Disease Through the Mediterranean Diet. Front. Physiol. 10:52. doi: 10.3389/fphys.2019.00052 The 12th Chapter which is the concluding chapter provides details of 30 short and simple recipes containing the diﬀerent classes of food in MD. February 2019 \| Volume 10 \| Article 52 Frontiers in Physiology \| www.frontiersin.org Asamudo and Okolo Mediterranean Diet and Disease Prevention susceptible and healthy individuals, aiming to maintain a healthy lifestyle. Each chapter contained illustrations, in form of ﬁgures and tables highlighting more details about the subject. For instance, the Mediterranean Diet pyramid which showed details of types of foods and the amount to be consumed. This is helpful for readers who understand better with visuals. Frontiers in Physiology \| www.frontiersin.org AUTHOR CONTRIBUTIONS EA and CO read the book and drafted the review summary together. EA typed the manuscript and edited. CO proofread and made ﬁnal corrections. In this book, some of the illustrated ﬁgures appeared blur due to low image quality. We also observed that the pattern in each chapter was slightly diﬀerent which could be due to the diﬀerent contributing authors. However, all chapters included references which would be very helpful for anyone interested in researching more about the Mediterranean Diet. 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. In summary, this book emphasized the fact that MD is a highly recommended eating model to be adopted toward the prevention of cardiovascular diseases. We do recommend this text for students, physicians, and researchers in areas such as cardiovascular and nutritional sciences. It contains guidelines on dietary patterns that can help improve the health of Copyright © 2019 Asamudo and Okolo. 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. February 2019 \| Volume 10 \| Article 52 Frontiers in Physiology \| www.frontiersin.org 2
https://openalex.org/W2507126781	https://www.nature.com/articles/srep31137.pdf	English	null	Fibroproliferative response to urothelial failure obliterates the ureter lumen in a mouse model of prenatal congenital obstructive nephropathy	Scientific reports	2,016	cc-by	11,035	Fibroproliferative response to urothelial failure obliterates the ureter lumen in a mouse model of prenatal congenital obstructive nephropathy Amanda J. Lee1, Noemi Polgar1, Josephine A. Napoli1, Vanessa H. Lui1, Kadee-Kalia Tamashiro1, Brent A. Fujimoto1, Karen S. Thompson2 & Ben Fogelgren1 received: 05 May 2016 accepted: 12 July 2016 Published: 11 August 2016 Amanda J. Lee1, Noemi Polgar1, Josephine A. Napoli1, Vanessa H. Lui1, Kadee-Kalia Tamashiro1, Brent A. Fujimoto1, Karen S. Thompson2 & Ben Fogelgren1 Congenital obstructive nephropathy (CON) is the most prevalent cause of pediatric chronic kidney disease and end-stage renal disease. The ureteropelvic junction (UPJ) region, where the renal pelvis transitions to the ureter, is the most commonly obstructed site in CON. The underlying causes of congenital UPJ obstructions remain poorly understood, especially when they occur in utero, in part due to the lack of genetic animal models. We previously showed that conditional inactivation of Sec10, a central subunit of the exocyst complex, in the epithelial cells of the ureter and renal collecting system resulted in late gestational bilateral UPJ obstructions with neonatal anuria and death. In this study, we show that without Sec10, the urothelial progenitor cells that line the ureter fail to differentiate into superficial cells, which are responsible for producing uroplakin plaques on the luminal surface. These Sec10-knockout urothelial cells undergo cell death by E17.5 and the urothelial barrier becomes leaky to luminal fluid. Also at E17.5, we measured increased expression of TGFβ1 and genes associated with myofibroblast activation, with evidence of stromal remodeling. Our findings support the model that a defective urothelial barrier allows urine to induce a fibrotic wound healing mechanism, which may contribute to human prenatal UPJ obstructions. Congenital anomalies of the kidney and urinary tract (CAKUT) are a heterogeneous spectrum of disorders that include renal dysplasia, aplasia, ureteral duplication, horseshoe kidney, and obstructions along the urinary tract. Within CAKUT, congenital obstructive nephropathy (CON) is the most common cause of chronic renal failure and end stage renal disease for infants and children1–3. The obstructed outflow of urine leads to hydronephrosis, which is the enlargement of the kidney due to a build up of urine in the renal pelvis. Detection of CON often occurs during prenatal development, and hydronephrosis is detected by ultrasound in ~1% of pregnancies4–6. Although about 70% of prenatal hydronephrosis cases spontaneously resolve, the remaining 30% of cases can worsen and cause lasting renal damage6. The severe cases require surgical correction, such as stent placement or a pyeloplasty. The most common site of obstruction in CON is at the ureteropelvic junction (UPJ), which is where the kidney pelvis transitions into the ureter7–9. www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports received: 05 May 2016 accepted: 12 July 2016 Published: 11 August 2016 Fibroproliferative response to urothelial failure obliterates the ureter lumen in a mouse model of prenatal congenital obstructive nephropathy Amanda J. Lee1, Noemi Polgar1, Josephine A. Napoli1, Vanessa H. Lui1, Kadee-Kalia Tamashiro1, Brent A. Fujimoto1, Karen S. Thompson2 & Ben Fogelgren1 p We recently generated a novel transgenic mouse model of prenatal UPJ obstructions by knocking out Sec10 in ureteric bud-derived epithelial cells, including the kidney distal tubules and collecting duct, renal pelvis, and the ureter18. Sec10 is a central subunit of the octameric exocyst protein complex, which mediates the targeting and docking of intracellular vesicles to some subcellular locales19. Previous studies have shown that Sec10 links Sec15, the exocyst subunit that binds specific Rab GTPases on the surface of secretory vesicles, to the rest of the exocyst complex at the plasma membrane20,21. As previously reported, we crossed the floxed Sec10 mice with the Ksp-Cre mouse strain22,23, where Cre recombinase expression is driven by a 1.3 kb promoter fragment of kidney-specific cadherin (Ksp-cadherin; cadherin 16). During nephrogenesis, Ksp-cadherin is expressed in the ureteric bud and the epithelial cells derived from the ureteric bud, allowing us to investigate the role of Sec10 in these cells during urinary tract development. We showed that 95% of the Sec10 knockout pups (Sec10FL/FL; Ksp-Cre) died hours after birth with severe bilateral hydronephrosis and complete anuria18.h pt y p p These mice had bilateral UPJ obstructions late in gestation, between E17.5 and E18.5, due to a cellular over- growth that filled the ureter lumen18. This overgrowth was composed of mesenchymal shaped cells positive for smooth muscle actin (SMA), with an almost complete disappearance of E-cadherin-positive urothelial cells. Analysis of the Sec10FL/FL;Ksp-Cre ureters at E17.5 identified an absence of uroplakin-3 (Upk3) on the superficial surface of the urothelium. In addition, a higher proliferation rate of SMA-positive cells was measured at the UPJ region in these ureters at E17.5, prior to the obstruction of the ureter lumen.h g p The purpose of this study was to identify the cellular mechanism that causes the in utero UPJ obstructions after the conditional inactivation of Sec10 in epithelial cells of the ureteric bud. Here, we show that the urothe- lium in Sec10FL/FL;Ksp-Cre ureters fails to develop a superficial cell layer and luminal uroplakin plaques between E16.5 and E17.5. In these developing mutant ureters, we measured almost no uroplakin gene expression, and a highly decreased expression of peroxisome proliferator-activated receptor gamma (PPARγ) at E16.5. PPARγ is a key transcriptional activator of the uroplakin gene family, and has been shown to be critical for urothelial dif- ferentiation24–26. This suggested that the terminal differentiation of superficial cells is impaired in these mutant embryos. Fibroproliferative response to urothelial failure obliterates the ureter lumen in a mouse model of prenatal congenital obstructive nephropathy Amanda J. Lee1, Noemi Polgar1, Josephine A. Napoli1, Vanessa H. Lui1, Kadee-Kalia Tamashiro1, Brent A. Fujimoto1, Karen S. Thompson2 & Ben Fogelgren1 The underlying molecular causes of congenital UPJ obstructions are poorly understood, as are the contributing environmental factors and natural variability. The gap in knowl- edge about CON and UPJ obstructions is in part due to the lack of genetic, non-surgical animal models to study these diseases8,10. Ureter development in mice begins at embryonic day 10.5 (E10.5) when the ureteric bud grows from the nephric duct in response to signals from the metanephric mesenchyme. As the tip of the ureteric bud grows and branches into the metanephric mesenchyme to become the kidney, the stalk elongates to form the ureter. The base of the ureteric bud migrates down the nephric duct and eventually connects directly with the bladder11. Although 1Department of Anatomy, Biochemistry and Physiology, John A. Burns School of Medicine, University of Hawaii at Manoa, HI 96813, USA. 2Department of Pathology, John A. Burns School of Medicine, University of Hawaii at Manoa, HI 96813, USA. Correspondence and requests for materials should be addressed to B.F. (email: fogelgre@hawaii. edu) Scientific Reports \| 6:31137 \| DOI: 10.1038/srep31137 1 www.nature.com/scientificreports/ the ureteric bud starts as a single monolayer of epithelial cells, it induces development of an outer smooth muscle layer and then differentiates into a multilayered transitional epithelium called the urothelium. Lining the renal pelvis, ureter, bladder and urethra, the urothelium is both flexible and fluid impermeable to allow stretching while preventing urine from escaping the lumen of the urinary tract. In order to do this, the lumen-facing superficial cells of the urothelium produce very high levels of transmembrane proteins called uroplakins, which form hexag- onal plaques on the luminal surface12–15. These uroplakin plaques cover a majority of the apical plasma membrane and are connected by flexible hinge regions. The four members of the uroplakin family (Upk1a, Upk1b, Upk2, and Upk3) are transmembrane proteins that assemble into heterodimers in the ER (Upk1a-Upk2 and Upk1b-Upk3) before being trafficked through the Golgi and to the apical surface in discoid/fusiform vesicles13. A pool of these uroplakin-containing vesicles are maintained under the apical surface of the mature superficial cells, and the dynamic exocytosis and endocytosis allows the urothelial surface area to expand and contract in response to mechanical stretch16,17. High expression of the uroplakin family members and formation of these plaques is crit- ical for the development and maintenance of the urothelial barrier. Fibroproliferative response to urothelial failure obliterates the ureter lumen in a mouse model of prenatal congenital obstructive nephropathy Amanda J. Lee1, Noemi Polgar1, Josephine A. Napoli1, Vanessa H. Lui1, Kadee-Kalia Tamashiro1, Brent A. Fujimoto1, Karen S. Thompson2 & Ben Fogelgren1 By E17.5, Sec10 mutant urothelial cells started to undergo cell death and detached from the wall of the ureter, and had largely disappeared by E18.5. Concomitant with the failure of the urothelial barrier by E17.5, we saw increased levels of TGF-β1 and other fibrotic markers, invasion of the lumen by fibroblastic cells, as well as rearrangement of the basement membrane with an increased ECM deposition. These results show that in our Sec10FL/FL;Ksp-Cre mouse model of prenatal CON, the failure of urothelial differentiation precedes a fibroprolif- erative wound healing response that occludes the lumen at the UPJ. Results P l Prenatal UPJ obstructions in Sec10FL/FL;Ksp-Cre mice are preceded by a loss of ureter urothelium. As previously reported, we crossed our novel floxed Sec10 mouse line with the Ksp-Cre mouse strain to con- ditionally knockout the Sec10 gene in epithelial cells of the urinary tract derived from the ureteric bud. The Sec10FL/FL;Ksp-Cre mice developed bilateral in utero UPJ obstructions, severe hydronephrosis (Fig. 1A,B), with neonatal anuria and death, with a 95% penetrance18. We observed that the ureter lumen became obstructed at the UPJ region between E17.5 and E18.5, but the underlying basis of the blockage was unclear. By immunos- taining for E-cadherin, we saw that epithelial cells had largely disappeared from the obstructed UPJ by E18.5. Representative cross sections of E18.5 ureters stained with Alcian blue show a normal multilayered ureter with a patent lumen in littermate controls (Fig. 1C), but show that Sec10FL/FL;Ksp-Cre ureters were completely obstructed by E18.5 (Fig. 1D). From histological analysis, the Sec10FL/FL;Ksp-Cre ureters had completely lost the urothelial cell layer by E18.5, with what looked like granulation tissue filling the lumen of the ureters. We utilized a tdTomato reporter mouse strain to confirm Cre activity and to track Sec10 knockout cells in the urothelium. We previously showed that Cre is activated in the Ksp-Cre ureteric bud cells prior to E13.518, confirming an early deletion of the Sec10 gene during nephrogenesis. As expected, newborn control mice with both Ksp-Cre and tdTomato alleles exhibited strong red fluorescence in the urothelium of the pelvis and throughout the entire length of the ureter (Fig. 1E). However, in newborn Sec10FL/FL;Ksp-Cre mice, red fluorescent cells were visible only in the upper-most ureter (Fig. 1F). As the renal pelvis transitions into the ureter at the UPJ, tdTomato labeling of the urothelial cells revealed an abrupt disappearance of these cells in the Sec10FL/FL;Ksp-Cre ureters (Fig. 1F). Whole mount images of younger tdTomato-labeled ureters (E16.5–E18.5) also showed that the number of urothelial cells in Sec10FL/FL;Ksp-Cre;To ureters was significantly decreased at E17.5, and by E18.5 there were very few urothelial cells remaining (Fig. 1G–J). This shows that the loss of Sec10 in urothelial cells leads to degeneration of the urothelial layer prior to the formation of the UPJ obstruction. Also, these data showed that epithelial-mesenchy- mal transition (EMT) does not contribute to the obstruction in this mouse model, since we did not detect any tdTomato-labeled mesenchymal cells among the tissue filling the ureter lumens. Results P l Scientific Reports \| 6:31137 \| DOI: 10.1038/srep31137 2 www.nature.com/scientificreports/ Figure 1. Sec10FL/FL;Ksp-Cre ureters form complete UPJ obstructions by E18.5 with loss of urothelial cells starting at E17.5. (A,B) Representative H&E-stained histological sections demonstrate substantial hydronephrosis in Sec10FL/FL;Ksp-Cre newborn kidneys (B), not present in Sec10FL/FL control littermates (A). (C,D) Alcian blue staining of cross-sections from representative Sec10FL/FL and Sec10FL/FL;Ksp-Cre ureters at the UPJ region. Bar = 20 μm. (E,F) Fluorescence imaging merged with differential interference contrast (DIC) microscopy of representative P0 Ksp-Cre;To control and Sec10FL/FL;Ksp-Cre;To mutant ureters. Loss of tdTomato-labeled urothelial cells is evident at and below the UPJ obstruction in Sec10FL/FL;Ksp-Cre;To ureters. (G-J) Fluorescence microscopy of whole mount E16.5 Ksp-Cre;To control ureters, and of E16.5-E18.5 Sec10FL/FL; Ksp-Cre;To mutant ureters, showing progressive loss of tdTomato-labeled urothelial cells starting after E16.5. Figure 1. Sec10FL/FL;Ksp-Cre ureters form complete UPJ obstructions by E18.5 with loss of urothelial cells starting at E17.5. (A,B) Representative H&E-stained histological sections demonstrate substantial hydronephrosis in Sec10FL/FL;Ksp-Cre newborn kidneys (B), not present in Sec10FL/FL control littermates (A). (C,D) Alcian blue staining of cross-sections from representative Sec10FL/FL and Sec10FL/FL;Ksp-Cre ureters at the UPJ region. Bar = 20 μm. (E,F) Fluorescence imaging merged with differential interference contrast (DIC) microscopy of representative P0 Ksp-Cre;To control and Sec10FL/FL;Ksp-Cre;To mutant ureters. Loss of tdTomato-labeled urothelial cells is evident at and below the UPJ obstruction in Sec10FL/FL;Ksp-Cre;To ureters. (G-J) Fluorescence microscopy of whole mount E16.5 Ksp-Cre;To control ureters, and of E16.5-E18.5 Sec10FL/FL; Ksp-Cre;To mutant ureters, showing progressive loss of tdTomato-labeled urothelial cells starting after E16.5. Sec10 is necessary for normal differentiation of the superficial urothelial cells in the developing ureter. Urothelial cell differentiation and stratification during ureter development is critical for the formation of the mature urothelial barrier against urine. The urothelial progenitor epithelial cells, derived from the stalk of the ureteric bud, are initially present as a monolayer, but as they respond to morphogens from surrounding mesenchymal tissue they stratify into three urothelial cell types: basal cells, intermediate cells, and superficial cells11,27. We used transmission electron microscopy (TEM) to compare the ultrastructure of the ureter’s urothe- lium in E16.5 and E17.5 Sec10FL/FL and Sec10FL/FL;Ksp-Cre embryos. At E16.5, control ureters had a single urothe- lial layer with microvilli extending into the lumen of the ureter (Fig. 2A), which looked similar in Sec10FL/FL; Ksp-Cre ureters except for a distinct reduction in apical microvilli (Fig. 2B). Results P l (E) Measurements of urothelial layer thickness in Sec10FL/FL;Ksp-Cre versus Sec10FL/FL ureters at E16.5 and E17.5 (p < 0.01). (F) Measurements of smooth muscle layer thickness in E16.5 and E17.5 ureters based on E-cadherin and smooth muscle actin immunostaining18. No significant changes were detected in thicknesses of smooth muscle layers. Figure 2. Ultrastructural analysis reveals failure of Sec10FL/FL;Ksp-Cre urothelial cells to stratify between E16.5 and E17.5. (A,B) Transmission electron microscopy (TEM) of the urothelial layer of E16.5 ureters of Sec10FL/FL;Ksp-Cre and Sec10FL/FL control littermates. Dashed line marks the basement membrane. Bar = 4 μm. (C,D) TEM of the urothelial layer of E17.5 ureters of Sec10FL/FL;Ksp-Cre and Sec10FL/FL control littermates. Bar = 10 μm. (E) Measurements of urothelial layer thickness in Sec10FL/FL;Ksp-Cre versus Sec10FL/FL ureters at E16.5 and E17.5 (p < 0.01). (F) Measurements of smooth muscle layer thickness in E16.5 and E17.5 ureters based on E-cadherin and smooth muscle actin immunostaining18. No significant changes were detected in thicknesses of smooth muscle layers. Confocal imaging of E16.5 ureter cross sections after immunohistochemistry revealed that members of the exocyst complex, Sec10 and Sec3, localize at the apical (luminal) plasma membrane in Sec10FL/FL urothelial cells (Fig. 3A,C). As expected, in Sec10FL/FL;Ksp-Cre mutant ureters, Sec10 was completely absent, but also Sec3 lev- els were significantly decreased (Fig. 3B,D). Degradation of other exocyst subunits was previously measured in Sec10-knockdown MDCK cells28, but this is the first in vivo evidence that the Sec10 protein may be required for the expression or stability of the other exocyst subunits. These data also showed that the localization of the exocyst in these monolayered urothelial cells at E16.5 differs from previous reports of the exocyst in other mon- olayered epithelial cells, where it localized to sites of cell-cell contact and had been associated with basolateral membrane delivery21,29,30. The decrease in exocyst protein at the apical plasma membrane in E16.5 Sec10FL/FL; Ksp-Cre urothelial cells coincided with highly decreased number of microvilli on the apical surface (Fig. 3E,F). The transcription factor p63 is highly expressed in the progenitor ureteric bud and has been shown to be important in the growth and differentiation of epithelial tissues31,32. In the mature urothelium, it is highly expressed in the basal urothelial cells and is critical for the maintenance the basal layer26,33. Immunohistochemistry of Sec10FL/FL and Sec10FL/FL;Ksp-Cre ureters showed similar p63 levels and localization at E16.5 (Fig. 3G,H). Results P l In both E16.5 Sec10FL/FL;Ksp-Cre and control ureters, projections were visible between the urothelial cells, an early sign of epithelial stratification. At E17.5, control ureter cross sections revealed a two-layered urothelium with a characteristic scalloped structure with hinge regions on the luminal membrane of the superficial cells (Fig. 2C). However, E17.5 Sec10FL/FL;Ksp-Cre ureters showed a highly abnormal single urothelial layer, with gaps in the epithelium and cells pulling away from the basement membrane toward the center of the lumen (Fig. 2D). These Sec10-knockout urothelial cells had lost large amounts of cytoplasm, and showed irregular disrupted plasma membranes and unusual distributions of electron-dense material in the nuclei. From immunostaining E-cadherin and SMA in E16.5 and E17.5 ureter cross sections, we measured the widths of both urothelial and smooth muscle layers in Sec10FL/FL;Ksp-Cre and Sec10FL/FL ureters. We confirmed that at E16.5, Sec10FL/FL;Ksp-Cre ureters had no significant difference in the average width of the urothelial layer compared to Sec10FL/FL ureters, but by E17.5, the Sec10-knockout urothelial layer was about half the width of control urothelium (Fig. 2E). No significant changes in the width of the smooth muscle layer at either E16.5 or E17.5 were measured (Fig. 2F). Scientific Reports \| 6:31137 \| DOI: 10.1038/srep31137 3 www.nature.com/scientificreports/ Figure 2. Ultrastructural analysis reveals failure of Sec10FL/FL;Ksp-Cre urothelial cells to stratify between E16.5 and E17.5. (A,B) Transmission electron microscopy (TEM) of the urothelial layer of E16.5 ureters of Sec10FL/FL;Ksp-Cre and Sec10FL/FL control littermates. Dashed line marks the basement membrane. Bar = 4 μm. (C,D) TEM of the urothelial layer of E17.5 ureters of Sec10FL/FL;Ksp-Cre and Sec10FL/FL control littermates. Bar = 10 μm. (E) Measurements of urothelial layer thickness in Sec10FL/FL;Ksp-Cre versus Sec10FL/FL ureters at E16.5 and E17.5 (*p < 0.01). (F) Measurements of smooth muscle layer thickness in E16.5 and E17.5 ureters based on E-cadherin and smooth muscle actin immunostaining18. No significant changes were detected in thicknesses of smooth muscle layers. igure 2. Ultrastructural analysis reveals failure of Sec10FL/FL;Ksp-Cre urothelial cells to stratify between Figure 2. Ultrastructural analysis reveals failure of Sec10FL/FL;Ksp-Cre urothelial cells to stratify between E16.5 and E17.5. (A,B) Transmission electron microscopy (TEM) of the urothelial layer of E16.5 ureters of Sec10FL/FL;Ksp-Cre and Sec10FL/FL control littermates. Dashed line marks the basement membrane. Bar = 4 μm. (C,D) TEM of the urothelial layer of E17.5 ureters of Sec10FL/FL;Ksp-Cre and Sec10FL/FL control littermates. Bar = 10 μm. Results P l Loss of Sec10 in urothelial cells results in defective urothelial cell differentiation and stratification. (A–D) Immunostaining and confocal microscopy of exocyst members Sec10 and Sec3 in E16.5 ureters revealed exocyst was localized at the apical/luminal plasma membrane. The Sec10 protein was absent in E16.5 Sec10FL/FL;Ksp-Cre urothelial cells compared to Sec10FL/FL ureters (A,B), and Sec3 was also significantly decreased in Sec10FL/FL;Ksp-Cre ureters. (E,F) TEM of the apical plasma membrane of E16.5 Sec10FL/FL;Ksp- Cre and Sec10FL/FL urothelial cells at 6000x. Bar = 500nm. (G,H) Immunohistochemistry of p63 (red) in E16.5 Sec10FL/FL;Ksp-Cre and Sec10FL/FL ureters showed no detectable differences. (I) Real time qPCR measurement of PPARγ gene expression in E16.5–E18.5 Sec10FL/FL;Ksp-Cre and Sec10FL/FL ureters (p < 0.05; **p < 0.001). Ct values for each gene were normalized against beta actin. Figure 4. Electron microscopy and real time qPCR of E17.5 Sec10FL/FL;Ksp-Cre ureters confirms an absence of uroplakin plaques on the luminal surface of urothelial cells. (A,B) TEM of E17.5 Sec10FL/FL control ureters detected scallop-shaped uroplakin plaques on the apical plasma membrane (arrow heads) and fusiform vesicles being trafficked to the apical surface (arrows), both characteristic of superficial cells. In Sec10FL/FL;Ksp-Cre ureters, no uroplakin plaques or vesicles were detected. Bar = 0.5 μm. (C,D) Scanning electron microscopy (SEM) of the luminal surfaces of E17.5 Sec10FL/FL control ureters showed hallmark hexagonal uroplakin plaques covered the surface (indicated by arrows), with well-established tight cell-cell junctions (indicated by arrow heads). SEM of the luminal surface of E17.5 Sec10FL/FL;Ksp-Cre ureters showed a complete absence of uroplakin plaques and damaged cells pulling away from the urothelial layer (indicated by arrows) along with poor cell- cell junctions (indicated by arrow heads). Bar = 5 μm. (E–H) Real time qPCR measurement of Upk1a, Upk1b, Upk2, and Upk3a gene expression in Sec10FL/FL;Ksp-Cre and Sec10FL/FL ureters collected from E15.5–E18.5 embryos (p < 0.05, p < 0.01, p < 0.001, **p < 0.0001). Ct values for each gene were normalized against beta actin. Figure 4. Electron microscopy and real time qPCR of E17.5 Sec10FL/FL;Ksp-Cre ureters confirms an absence of uroplakin plaques on the luminal surface of urothelial cells. (A,B) TEM of E17.5 Sec10FL/FL control ureters detected scallop-shaped uroplakin plaques on the apical plasma membrane (arrow heads) and fusiform vesicles being trafficked to the apical surface (arrows), both characteristic of superficial cells. In Sec10FL/FL;Ksp-Cre ureters, no uroplakin plaques or vesicles were detected. Bar = 0.5 μm. Results P l PPARγ is a nuclear receptor that is a critical activator of uroplakin gene expression and has been reported to be necessary for the differentiation of superficial urothelial cells24–26. With qPCR, we measured a large decrease in PPARγ gene expres- sion in Sec10FL/FL;Ksp-Cre ureters compared to Sec10FL/FL ureters from E16.5 to E18.5 (Fig. 3I). That PPARγ was 80% decreased in Sec10FL/FL;Ksp-Cre ureters as early at E16.5, prior to gross morphological changes, supports the hypothesis that Sec10 is required for the differentiation of superficial urothelial cells in embryonic ureters. Sec10-knockout urothelial cells fail to produce uroplakin plaques on the luminal surface. Differentiation of superficial urothelial cells has been shown, in the bladder, to be an early event in urothelial maturation33. Normal mature superficial cells are characterized by the presence of numerous intracellular vesicles carrying uroplakins at the luminal plasma membrane, which establishes and maintains the watertight barrier against urine. Previously, we detected an absence of Upk3 protein in the Sec10FL/FL;Ksp-Cre urothelium at E17.5 via immunohistochemistry18. Here we investigated production of uroplakins in Sec10FL/FL;Ksp-Cre urothelium in more detail. TEM of control ureters at E17.5 showed numerous intracellular vesicles that clustered towards the apical membrane of the superficial urothelial cells (arrows, Fig. 4A). However, in the E17.5 Sec10FL/FL;Ksp-Cre urothelial cells, there was a complete absence of these apical vesicles (Fig. 4B), correlating with the absent urop- lakin mRNA gene expression that we measured by qPCR (Fig. 4E–H). Using scanning electron microscopy (SEM) of E17.5 ureter lumens, we could clearly visualize the hexagonal uroplakin plaques on the luminal membrane Scientific Reports \| 6:31137 \| DOI: 10.1038/srep31137 4 www.nature.com/scientificreports/ Figure 3. Loss of Sec10 in urothelial cells results in defective urothelial cell differentiation and stratification. (A–D) Immunostaining and confocal microscopy of exocyst members Sec10 and Sec3 in E16.5 ureters revealed exocyst was localized at the apical/luminal plasma membrane. The Sec10 protein was absent in E16.5 Sec10FL/FL;Ksp-Cre urothelial cells compared to Sec10FL/FL ureters (A,B), and Sec3 was also significantly decreased in Sec10FL/FL;Ksp-Cre ureters. (E,F) TEM of the apical plasma membrane of E16.5 Sec10FL/FL;Ksp- Cre and Sec10FL/FL urothelial cells at 6000x. Bar = 500nm. (G,H) Immunohistochemistry of p63 (red) in E16.5 Sec10FL/FL;Ksp-Cre and Sec10FL/FL ureters showed no detectable differences. (I) Real time qPCR measurement of PPARγ gene expression in E16.5–E18.5 Sec10FL/FL;Ksp-Cre and Sec10FL/FL ureters (p < 0.05; **p < 0.001). Ct values for each gene were normalized against beta actin. Figure 3. Results P l (C,D) Serial sections of a representative E17.5 Sec10FL/FL;Ksp-Cre ureter (dotted line depicts the basement membrane) demonstrated that although the urothelium was a damaged single layer (H&E staining, C), very few cells were positive for activated caspase-3 (red, D). Bar = 20 μm. (E) Measurement of cleaved caspase-3 and cleaved PARP levels in multiple Sec10FL/FL and Sec10FL/FL;Ksp-Cre ureters did not detect any significant increase in these apoptotic markers at E17.5. Columns represent the means of relative fluorescent units (RFU), with error bars representing SEM. (F,G) In E17.5 embryos, the left renal pelvis was injected with FITC-dextran which was allowed to flow to the bladder, then tissue was frozen and cryosectioned (ureters are circled, arrows note left ureter). Normal Sec10FL/FL controls (F) had very little FITC- dextran retained in the injected ureter (arrow), but Sec10FL/FL;Ksp-Cre mice (G) had a much larger retention of the dextran in the injected ureter tissue, indicating leakiness of the urothelial barrier. surface of Sec10FL/FL control ureters and tight cell-cell contacts (Fig. 4C). SEM also confirmed that uroplakin plaques were completely missing on the luminal surface of E17.5 Sec10FL/FL;Ksp-Cre ureters (Fig. 4D). We also noted that the Sec10FL/FL;Ksp-Cre urothelial cells did not show tight cell-cell contacts at E17.5, which confirms findings with TEM that many of these cells are damaged and are becoming detached from the ureter wall. Necrosis of urothelial cells contributes to the loss of barrier integrity in Sec10FL/FL;Ksp-Cre ureters. Analysis of E17.5 Sec10FL/FL;Ksp-Cre TEM images revealed that the damaged cells pulling away from the basement membrane had morphological characteristics similar to necrosis rather than apoptosis (Fig. 5A,B). Apoptotic cells via TEM exhibit membrane blebbing and nuclear fragmentation, but necrotic cells showed a darkening of the nucleus, uncontrolled swelling of the cell, rough irregular plasma membranes, and an absence of bleb- bing. Immunohistochemistry of E17.5 ureter cross-sections confirmed that very few urothelial cells were positive for cleaved caspase 3 (Fig. 5C,D), a marker for apoptotic activation. Protein analysis of Sec10FL/FL;Ksp-Cre and Sec10FL/FL ureters also confirmed no significant differences between the levels of cleaved caspase-3 or cleaved PARP, another marker of apoptosis (Fig. 5E). Results P l (C,D) Scanning electron microscopy (SEM) of the luminal surfaces of E17.5 Sec10FL/FL control ureters showed hallmark hexagonal uroplakin plaques covered the surface (indicated by arrows), with well-established tight cell-cell junctions (indicated by arrow heads). SEM of the luminal surface of E17.5 Sec10FL/FL;Ksp-Cre ureters showed a complete absence of uroplakin plaques and damaged cells pulling away from the urothelial layer (indicated by arrows) along with poor cell- cell junctions (indicated by arrow heads). Bar = 5 μm. (E–H) Real time qPCR measurement of Upk1a, Upk1b, Upk2, and Upk3a gene expression in Sec10FL/FL;Ksp-Cre and Sec10FL/FL ureters collected from E15.5–E18.5 embryos (p < 0.05, p < 0.01, p < 0.001, **p < 0.0001). Ct values for each gene were normalized against beta actin. Scientific Reports \| 6:31137 \| DOI: 10.1038/srep31137 5 www.nature.com/scientificreports/ Figure 5. Loss of urothelial cells at E17.5 is primarily due to necrosis and not apoptosis. (A,B) Representative TEM of the urothelial layer of E17.5 Sec10FL/FL and Sec10FL/FL;Ksp-Cre ureters. Most of the remaining attached urothelial cells in Sec10FL/FL;Ksp-Cre ureters displayed characteristics of cell damage and necrosis, but not apoptosis. Bar = 4 μm. (C,D) Serial sections of a representative E17.5 Sec10FL/FL;Ksp-Cre ureter (dotted line depicts the basement membrane) demonstrated that although the urothelium was a damaged single layer (H&E staining, C), very few cells were positive for activated caspase-3 (red, D). Bar = 20 μm. (E) Measurement of cleaved caspase-3 and cleaved PARP levels in multiple Sec10FL/FL and Sec10FL/FL;Ksp-Cre ureters did not detect any significant increase in these apoptotic markers at E17.5. Columns represent the means of relative fluorescent units (RFU), with error bars representing SEM. (F,G) In E17.5 embryos, the left renal pelvis was injected with FITC-dextran which was allowed to flow to the bladder, then tissue was frozen and cryosectioned (ureters are circled, arrows note left ureter). Normal Sec10FL/FL controls (F) had very little FITC- dextran retained in the injected ureter (arrow), but Sec10FL/FL;Ksp-Cre mice (G) had a much larger retention of the dextran in the injected ureter tissue, indicating leakiness of the urothelial barrier. Figure 5. Loss of urothelial cells at E17.5 is primarily due to necrosis and not apoptosis. (A,B) Representative TEM of the urothelial layer of E17.5 Sec10FL/FL and Sec10FL/FL;Ksp-Cre ureters. Most of the remaining attached urothelial cells in Sec10FL/FL;Ksp-Cre ureters displayed characteristics of cell damage and necrosis, but not apoptosis. Bar = 4 μm. Results P l However, Sec10FL/FL;Ksp-Cre ureters exhibited a complete loss of the epithelial layer, previously shown by immunostain- ing for epithelial markers such as E-cadherin18. Instead, the lumen was filled with migrating fibroblastic cells and deposits of ECM (Fig. 6B), suggesting the presence of a fibrotic wound healing response at the obstruction. Immunohistochemistry of collagen IV in Sec10FL/FL ureters showed a distinct basement membrane attached to the basal layer of the urothelium separating the mucosa from the smooth muscle cells (Fig. 6C). In contrast, Sec10FL/FL; Ksp-Cre ureters had a reorganization of collagen IV throughout the new tissue in the lumen of the ureter, indicat- ing a disrupted and expanded basement membrane that allowed the surrounding mesenchyme to penetrate the lumen (Fig. 6D).i healing of the skin, the fibroproliferative response seen in granulation tissue can also occur in internal epithelial tissues to obliterate lumens, such as in bronchiolitis obliterans34. Here, we expanded on our original findings and performed ultrastructure morphological and molecular analysis at the UPJ obstruction to evaluate the degree of fibrotic response. TEM analysis of control Sec10FL/FL ureters at E18.5 confirmed a mature multilayered structure with numerous uroplakin vesicles (Fig. 6A), and a distinct basement membrane (data not shown). However, Sec10FL/FL;Ksp-Cre ureters exhibited a complete loss of the epithelial layer, previously shown by immunostain- ing for epithelial markers such as E-cadherin18. Instead, the lumen was filled with migrating fibroblastic cells and deposits of ECM (Fig. 6B), suggesting the presence of a fibrotic wound healing response at the obstruction. Immunohistochemistry of collagen IV in Sec10FL/FL ureters showed a distinct basement membrane attached to the basal layer of the urothelium separating the mucosa from the smooth muscle cells (Fig. 6C). In contrast, Sec10FL/FL; Ksp-Cre ureters had a reorganization of collagen IV throughout the new tissue in the lumen of the ureter, indicat- ing a disrupted and expanded basement membrane that allowed the surrounding mesenchyme to penetrate the lumen (Fig. 6D).i ( g ) TGF-β1 is a critical mediator of wound healing and fibrosis, and was found in a previous study to be increased in human UPJ samples35. We hypothesized that the TGFβ pathway may be activated from urinary leakage into the tissue underlying the disrupted urothelium in Sec10FL/FL;Ksp-Cre ureters. We measured TGF-β1 mRNA expression using real time qPCR in E16.5-E18.5 Sec10FL/FL;Ksp-Cre ureters and compared to Sec10FL/FL controls. TGF-β1 had a five-fold expression increase in Sec10FL/FL;Ksp-Cre at E17.5 and E18.5 compared to Sec10FL/FL ureters (Fig. Results P l Obstructed Sec10FL/FL;Ksp-Cre ureters have stromal remodeling and increased expression of TGFβ1 and other fibroblastic markers. (A,B) TEM of ureter cross sections at E18.5. Sec10FL/FL sections show mature superficial urothelial cells with uroplakin plaques on the apical membrane. In Sec10FL/FL;Ksp-Cre ureters, the lumen has been filled with fibroblastic cells and extracellular matrix deposits. Arrows mark collagen fibers in the filled lumen. Bar = 4 μm. (C) Immunohistochemistry of collagen IV in a Sec10FL/FL E18.5 ureter cross section revealed a distinct basement membrane separating the epithelial and smooth muscle layers. (D) Collagen IV immunohistochemistry in Sec10FL/FL;Ksp-Cre E18.5 ureter cross sections revealed a remodeled basement membrane and no distinction between cell layers. Bar = 20 μm. (E–H) Real time qPCR analysis of TGFβ1, S100A4 (fibroblast specific protein), periostin, and desmin relative gene expression in Sec10FL/FL;Ksp-Cre and Sec10FL/FL embryonic ureters. p < 0.05, p < 0.01, p < 0.001, **p < 0.0001. Ct values for each gene were normalized against beta actin. Figure 6. Obstructed Sec10FL/FL;Ksp-Cre ureters have stromal remodeling and increased expression of TGFβ1 and other fibroblastic markers. (A,B) TEM of ureter cross sections at E18.5. Sec10FL/FL sections show mature superficial urothelial cells with uroplakin plaques on the apical membrane. In Sec10FL/FL;Ksp-Cre ureters, the lumen has been filled with fibroblastic cells and extracellular matrix deposits. Arrows mark collagen fibers in the filled lumen. Bar = 4 μm. (C) Immunohistochemistry of collagen IV in a Sec10FL/FL E18.5 ureter cross section revealed a distinct basement membrane separating the epithelial and smooth muscle layers. (D) Collagen IV immunohistochemistry in Sec10FL/FL;Ksp-Cre E18.5 ureter cross sections revealed a remodeled basement membrane and no distinction between cell layers. Bar = 20 μm. (E–H) Real time qPCR analysis of TGFβ1, S100A4 (fibroblast specific protein), periostin, and desmin relative gene expression in Sec10FL/FL;Ksp-Cre and Sec10FL/FL embryonic ureters. p < 0.05, p < 0.01, p < 0.001, **p < 0.0001. Ct values for each gene were normalized against beta actin. healing of the skin, the fibroproliferative response seen in granulation tissue can also occur in internal epithelial tissues to obliterate lumens, such as in bronchiolitis obliterans34. Here, we expanded on our original findings and performed ultrastructure morphological and molecular analysis at the UPJ obstruction to evaluate the degree of fibrotic response. TEM analysis of control Sec10FL/FL ureters at E18.5 confirmed a mature multilayered structure with numerous uroplakin vesicles (Fig. 6A), and a distinct basement membrane (data not shown). Results P l The absence of apoptotic characteristics, and the cell morphology observed via TEM, indicated that Sec10-knockout urothelial cells in the developing ureter were dying largely due to necrosis.l Based on TEM images of the disrupted urothelium at E17.5, we performed fluorescein isothiocyanate dex- tran (FITC-dextran) injections into the renal pelvis of the kidney to determine if there was any sign of urothe- lial barrier dysfunction in the ureter. In control E17.5 ureters (injected ureter marked by arrow, Fig. 5F), the FITC-dextran was not retained in the ureters and passed through the ureters and into the bladders, indicating a strong luminal barrier. In contrast, E17.5 Sec10FL/FL;Ksp-Cre ureters retained a very strong green fluorescence in the injected ureters (arrow, Fig. 5G), indicating the retention of the FITC-dextran in the tissue. Thus, the disrupted differentiation of the Sec10-knockout urothelium has a functional consequence of compromising the urothelial barrier by E17.5. Urothelial degeneration in the developing Sec10FL/FL;Ksp-Cre ureter induces a fibroproliferative response that rapidly occludes the lumen. In histological sections of our Sec10 knockout ureter cross sections, we noted that the tissue in the UPJ obstruction looked similar to granulation tissue, characteristic of wound healing. Granulation tissue includes the presence of myofibroblasts, extracellular matrix (ECM) deposi- tion and remodeling, inflammatory cells, and newly formed capillaries. Although often associated with wound Scientific Reports \| 6:31137 \| DOI: 10.1038/srep31137 6 www.nature.com/scientificreports/ Figure 6. Obstructed Sec10FL/FL;Ksp-Cre ureters have stromal remodeling and increased expression of TGFβ1 and other fibroblastic markers. (A,B) TEM of ureter cross sections at E18.5. Sec10FL/FL sections show mature superficial urothelial cells with uroplakin plaques on the apical membrane. In Sec10FL/FL;Ksp-Cre ureters, the lumen has been filled with fibroblastic cells and extracellular matrix deposits. Arrows mark collagen fibers in the filled lumen. Bar = 4 μm. (C) Immunohistochemistry of collagen IV in a Sec10FL/FL E18.5 ureter cross section revealed a distinct basement membrane separating the epithelial and smooth muscle layers. (D) Collagen IV immunohistochemistry in Sec10FL/FL;Ksp-Cre E18.5 ureter cross sections revealed a remodeled basement membrane and no distinction between cell layers. Bar = 20 μm. (E–H) Real time qPCR analysis of TGFβ1, S100A4 (fibroblast specific protein), periostin, and desmin relative gene expression in Sec10FL/FL;Ksp-Cre and Sec10FL/FL embryonic ureters. p < 0.05, p < 0.01, p < 0.001, ***p < 0.0001. Ct values for each gene were normalized against beta actin. Figure 6. Obstructed Sec10FL/FL;Ksp-Cre ureters have stromal remodeling and increased expression of i Figure 6. Discussion In this study, we identify the underlying cellular basis of the in utero UPJ obstruction, and a timeline of ureter maldevelopment, in a Sec10 conditional knockout mouse. We provide evidence that Sec10 is necessary for proper differentiation of superficial urothelial cells and establishment of the uroplakin barrier. It is not clear what exactly causes the cell death observed in E17.5 Sec10FL/FL;Ksp-Cre urothelium, but our data shows that it is primarily necrotic in origin. Since the Sec10 deletion occurs in these cells prior to E13.518, we hypothesize that the cell death after E16.5 is either in response to a failure of superficial differentiation or the lack of protection against the grow- ing urine flow, or likely a combination of multiple factors. We also hypothesize that the leakage of urine through the urothelial barrier induces the observed fibrotic wound healing response from the surrounding mesenchyme. We measured no change in TGF-β1 expression at E16.5 in Sec10FL/FL;Ksp-Cre ureters, prior to the urothelial cell death and barrier degeneration. But beginning at E17.5, we measured an increase in TGF-β1 expression (Fig. 6E), as well as an increase in the fibroblast specific gene S100A4 (Fig. 6F). We also measured increased expression of periostin at E18.5, which is now recognized to be a critical regulator of the activation of fibroblast during wound healing and fibrosis36–38. This correlates with a decrease in the relative expression of desmin, by the smooth muscle cells. Although the surrounding mesenchyme includes both fibroblasts and smooth muscle cells, our data sug- gest the resident fibroblast population becomes activated myofibroblasts that rapidly proliferate and migrate to obstruct the ureter lumen between E17.5 and E18.5. The progression of kidney disease arising from CON has been studied for decades, but despite this, we still know very little about the genetic and cellular basis of human UPJ obstructions. The unilateral ureter obstruc- tion (UUO) animal model, which requires surgery to ligate one ureter, has been the most widely studied model of CON39. This model has greatly advanced the current understanding of the stages of renal pathology after the ureter obstruction and after correction of the obstruction. The obstructed kidney develops parenchymal loss and interstitial fibrosis that leads to loss of renal function40, a pathology common to many renal diseases. Discussion However, surgical models like the UUO are labor intensive with some degree of technical variability, and also limited in that they cannot be used to investigate the natural causes of human UPJ obstructions. Additionally, with the exception of large animals models like sheep41, they cannot be used to study ureter obstructions that occur in utero.i g y y Few genetic models of CON have been identified and characterized. One of these models is the megablad- der mouse, in which the mice develop hydronephrosis and lower urinary tract obstructions secondary to a non-functional over-distended bladder42. Other mouse models of CON have arisen from targeted deletion of genes necessary for growth or differentiation of ureter smooth muscle cells or pacemaker cells43,44. However, these models are non-obstructive, with a failure of ureter muscle tension that leads to hydroureter and hydrone- phrosis. The models that display UPJ obstructions typically involve inducing over proliferation of the surround- ing smooth muscle, but without urothelial degeneration or fibrotic infiltration of the lumen45,46. None of these mouse models of CON are neonatal lethal and they typically have wide-ranging variability and penetrance. In comparison, this Sec10FL/FL;Ksp-Cre model is the most consistently severe, with ~95% of the embryos developing lumen-obliterating bilateral UPJ obstructions with neonatal anuria and death18.t Clinical cases of UPJ obstructions, often detected as hydronephrosis via prenatal ultrasound, are highly vari- able in severity and progression4–6. In addition, the lack of prognostic indicators or biomarkers requires ongoing surveillance, which is a burden to the patient and costly47–49. Typically, cases of UPJ obstructions can be classified as intrinsic or extrinsic, where the more common intrinsic factors involve malformation of the ureter or cellular overgrowth in the ureter lumen, while extrinsic causes include pressure on the ureter from other tissues such as crossing vessels. One histological study demonstrated that surgically removed human samples of intrinsic obstructed UPJ regions had excessive collagen fibers that replaced smooth muscle in the ureter50. Another study of congenital UPJ obstructions from 25 patients (versus 15 age-matched control samples) showed an increase in TGF-β1 mRNA expression and protein levels in the stenotic segments35. Taken together, these studies show that at least for a percentage of human UPJ obstructions, there is a significant increase in fibrosis at the stenotic region. Results P l 6E). At E16.5 however, prior to the observed urothelial degeneration, we measured no change in TGF-β1 expression. We had previously reported an over proliferation of cells positive for SMA in the surrounding mes- enchyme at E17.5 prior to the onset of the obstruction18. However, multiple cell types express SMA, including smooth muscle cells and activated myofibroblasts. The presence of activated fibroblasts (myofibroblasts) is one of the key characteristics of the wound healing response. Quantitative PCR analysis showed a significant increase in expression of S100A4 (fibroblast specific protein, FSP) at E17.5 and E18.5 (Fig. 6F). There was also a significantly increased level of periostin expression at E18.5 (Fig. 6G), but decreased desmin expression in the ureters at E17.5 and E18.5 (Fig. 6H). Periostin is a matricellular protein that has been shown to promote myofibroblast prolifera- tion and differentiation during wound healing and fibrosis37–39. In contrast, desmin is specific to smooth muscle cells and is not expressed in myofibroblasts. This expression data indicated that the cells invading the ureter lumen in Sec10FL/FL;Ksp-Cre embryos were activated myofibroblasts, rather than an expanded smooth muscle cell population. In summary, the evidence supports our hypothesis that the UPJ obstruction arises in these Sec10 mutant mice from a fibroproliferative wound healing response after degeneration of the urothelium in the embry- onic ureter. Scientific Reports \| 6:31137 \| DOI: 10.1038/srep31137 7 www.nature.com/scientificreports/ Discussion Thus, the molecular and histological findings in our Sec10 mouse model of UPJ obstructions are consistent with the ureter pathology of a significant portion of human clinical cases of UPJ obstructions. p gy gi p Based on crosses with our tdTomato reporter mice, we know Cre-mediated inactivation occurs prior to E13.5 in Sec10FL/FL;Ksp-Cre embryos18. However, the first abnormalities that we have detected did not occur until ~E16.5, which demonstrates that Sec10 knockout in these epithelial cells does not de facto cause cell death. At E16.5, there was a significant decrease in PPARγ expression (Fig. 3I), which is normally expressed at high levels during urothelial differentiation of the superficial cells and regulates transcription of the uroplakin gene family through FOXA1 and IRF-1 mediators24,25. A PPARγ conditional knockout mouse, created using the Hoxb7-Cre strain that targets the same ureteric bud derived epithelial cells as the Ksp-Cre mice, showed that although PPARγ is specifically important for differentiation of superficial urothelial cells, but knockout of PPARγ did not cause urothelial cell death or prenatal UPJ obstructions. Both Upk2 and Upk3 knockout mice have also been reported, and they had severe defects in uroplakin plaque formation at the apical surface and did not have proper urothelial barrier function51,52. However, these knockout mice also did not display the in utero urothelial degeneration and fibroproliferative UPJ obstructions seen in our Sec10 knockout model. This suggests that failure of superficial dif- ferentiation, or failure to produce uroplakin plaques, is not sufficient to produce our UPJ obstruction phenotype. Thus, the exocyst likely has additional roles in urothelial cells or their progenitors making this Sec10 knockout model a unique tool to bridge the gap between urothelial differentiation and the onset of UPJ obstructions.i q g g pf Significant decrease in mRNA expression of the four members of the uroplakin gene family in the Sec10FL/FL; Ksp-Cre ureters starting at E16.5 (Fig. 4) indicate that the exocyst regulates superficial cell differentiation. It is also possible that the exocyst plays a direct role in uroplakin vesicle trafficking to the apical membrane. The decreased microvilli on the apical surface of E16.5 Sec10FL/FL;Ksp-Cre urothelial cells (Fig. 3E,F) indicates dis- rupted membrane trafficking toward the apical plasma membrane where Sec10 and Sec3 are localized in control ureters (Fig. 3A,C). Materials and Methods Animals. All animal procedures and protocols were carried out in accordance with IACUC specifications approved by the University of Hawaii Animal and Veterinary Services. Dr. Fogelgren’s IACUC approved protocol is #11-1094, and the University of Hawaii has an Animal Welfare Assurance on file with the Office of Laboratory Animal Welfare (OLAW), assurance number is A3423-01. Adult mice were housed under standard conditions with 12-hr light cycle and supplied with water and food ad libitum. The floxed Sec10 mouse strain (Sec10FL/FL) was generated and used as previously described18. The Ksp-Cre mouse strain was obtained from Jackson Laboratories22,23. The B6.Cg-Gt(ROSA)26Sortm9(CAG-tdTomato)Hze/J reporter mouse strain (here designated tdTomato h g p g or To) was kindly provided by Dr. Michelle Tallquist at University of Hawaii and was used to detect Cre recom- binase activity through Cre-activated expression of the tdTomato red fluorescent protein62. All mice were of C57Bl/6J inbred background. For timed matings, females mated with a male overnight were examined for a vag- inal plug the following morning and constituted gestational day E0.5 if present. Both male and female embryos were obtained between days E13.5 and E18.5 and were subsequently staged using Theiler staging criteria (TS) to ensure the developmental stage of each embryo was similar to the conception day (E) designation63. Only animals of the same E designation and TS were compared. Electron Microscopy. Dissected kidneys and ureters were fixed with 2.5% glutaraldehyde and in 0.1 M sodium cacodylate buffer, pH 7.2, washed in 0.1 M cacodylate buffer for 3 × 15 min, followed by post fixation with 1% OsO4 in 0.1 M cacodylate buffer for 1 hour. The tissue was dehydrated in a graded ethanol series (30%, 50%, 70%, 85%, 95%, 100%), substituted with propylene oxide, and either embedded in LX112 epoxy resin for transmission electron microscopy (TEM) or dried in a Tousimis Samdri-795 critical point dryer for scanning electron microscopy (SEM). For TEM, ultrathin sections (60–80 nm) were obtained on a Reichert Ultracut E ultramicrotome, double stained with uranyl acetate and lead citrate, viewed on a Hitachi HT7700 TEM at 100 kV, and photographed with an AMT XR-41B 2k × 2k CCD camera. For SEM, tissues were mounted on aluminum stubs with double stick tape and ureters were opened using a razor blade, and subsequently coated with gold/ palladium in a Hummer 6.2 sputter coater. Discussion Although some evidence has suggested the exocyst is primarily a basolateral vesicle regulator Scientific Reports \| 6:31137 \| DOI: 10.1038/srep31137 8 www.nature.com/scientificreports/ in polarized epithelial cells21,29,30, other studies have shown apical trafficking in some epithelial cell types can uti- lize the exocyst53–55. Several previous studies have also firmly established that the exocyst subunit Sec15 interacts directly with Rab11 and Rab8 GTPases to promote exocytosis of vesicles bound to those proteins53,56–58. Rab8 and Rab11 are both present on uroplakin-containing discoidal/fusiform vesicles in bladder superficial cells, and both have been shown to be critical for the stretch-induced exocytosis of new uroplakin plaques59,60. This estab- lished connection between two Rab GTPases, which directly regulate uroplakin exocytosis and recycling, and the exocyst complex suggests that the exocyst also has a direct role in uroplakin exocytosis and dynamic recycling. Utilizing inducible Cre mouse strains that avoid urothelial differentiation defects could allow investigations of uroplakin trafficking in mature Sec10-knockout superficial cells. pfi g pi Our histological analysis of the obstructed ureter at the UPJ region identified pathological changes similar to granulation tissue, with a robust and rapid fibroproliferative response between E17.5 and E18.5. This included invasion of the lumen by cells with a myofibroblastic appearance, deposition of ECM, and disruption of the basement membrane underlying the epithelium. Based on our tdTomato labeling of the Sec10-knockout urothe- lial cells, and the absence of any red-labeled fibroblastic cells in the obstructed lumens, we ruled out EMT as a contributor to the fibrotic response. We hypothesize the pathogenesis in our model of in utero UPJ obstructions shares similarities with other diseases of epithelial injury and aberrant stromal remodeling of internal tissues, such as obliterative bronchiolitis. In this pulmonary pathology commonly associated with lung transplants, the epithelial cell layer lining the bronchioles is damaged, triggering a fibroproliferative response of the underlying stroma that obstructs the airway lumen34. Our Sec10FL/FL;Ksp-Cre mouse model shows that this mechanism may also contribute to prenatal UPJ obstructions, which may be triggered by leakage of the urine into the ureter’s interstitial tissue. This mechanism for congenital UPJ obstructions was previously hypothesized based on histo- logical observations of human pyeloplasty samples. Although Bartoli et al. Discussion first published this hypothesis 20 years ago61, our model provides the first experimental evidence that defective urothelial maturation during prenatal development can induce a fibroproliferative response from the underlying mesenchyme.i pi p p y g y Future investigations will aim to identify exactly how the exocyst intersects with specific genetic signaling pathways and morphogens known to regulate urothelial differentiation and ureter development. Key to our understanding of the role of the exocyst in these processes will be identifying which proteins are trafficked by the exocyst complex in urothelial cells. We will likely find the exocyst is multifunctional in this cell type, as this complex has already been implicated in extremely diverse cellular processes, depending on the cell type and environment. Additionally, it will be important to investigate how the urine may damage the tissue underlying the leaky Sec10FL/FL;Ksp-Cre urothelium at E17.5, and test how this damage induces the fibroproliferative response that rapidly occludes the lumen. This has direct implications to clinical cases of both intrinsic and extrinsic UPJ obstructions, and we may be able to use the Sec10FL/FL;Ksp-Cre mouse model to screen potential therapeutics that ameliorate this type of response, or identify potential diagnostic or predictive biomarkers. Collectively, findings presented in this study demonstrate this Sec10FL/FL;Ksp-Cre mouse model may be highly valuable for extending our understanding of the etiology of human congenital UPJ obstructions and the associated renal disease, as well as identifying novel approaches for treatments. Materials and Methods Tissues were viewed and digital images were obtained with a Hitachi S-4800 Field Emission Scanning Electron Microscope at an accelerating voltage of 5 kV. Quantitative real time PCR analysis. Gene expression in embryonic tissues using real time quantita- tive PCR (qPCR) was performed as described previously, using the 2^(−ΔΔCt) method of analysis to calculate fold changes of expression64. Briefly, ureter segments from the UPJ region from embryos of various stages were Scientific Reports \| 6:31137 \| DOI: 10.1038/srep31137 9 www.nature.com/scientificreports/ dissected and placed immediately into RNAlater (Sigma) and stored at −20 °C. Ureters from three animals of the same stage and with the same genotype were pooled and extracted using the RNeasy micro kit (Qiagen). cDNA was generated from extracted RNA using the iSCRIPT reverse transcriptase (Bio-Rad) and qPCR was performed using SYBR green (Bio-Rad) with a CFX96 Real Time System (Bio-Rad), as per the manufacturer’s recommended instructions. Please refer to Supplementary Information for a complete list of primer sequences used for qPCR. Expression data was analyzed using Graphpad Prism software. Differences between means for any parameter measured in two groups of age-matched mice were evaluated using Student’s t-tests. Histology and Immunohistochemistry. Caudal torsos of Sec10 knockout and control animals were dissected, the abdominal cavity opened, immediately placed in freshly prepared 4% formaldehyde in PBS, and fixed overnight with rocking at 4 °C. Some samples were embedded in paraffin according to standard methods, and some samples were instead subjected to cryosectioning. The tissues were sectioned into 5 μm sections and staining and immunohistochemistry procedures were performed as previously reported18. Primary antibodies used for immunostaining were: anti-cleaved caspase-3 at 1:400 (Cat # 9664, Cell Signaling Tech.); anti-p63 at manufacturer’s prepared dilution (Cat # API 3050 G3, Biocare Medical); anti-collagen IV at 1:200 (Cat # ab6586, Abcam); anti-E-cadherin at 1:200 (Cat # 3195, Cell Signaling Tech.); anti-smooth muscle actin at 1:800 (Cat # A2547, Sigma). Stained sections were analyzed using a fluorescent Olympus BX41 microscope or an Olympus Fluoview1000 confocal microscope. Image processing, quantification of cell layer widths, and cell counts were done using Image J software (NIH). For ureter injections, fluorescein isothiocyanate (FITC) labeled dextran (aver- age molecular weight 10,000, Cat # FD10S, Sigma) was dissolved in PBS to make a stock solution of 25 mg/ml. The FITC-dextran solution was injected into the renal pelvis (left kidney only) of genitourinary tracts and allowed to flow to the bladder. Materials and Methods Injected tissues were frozen in OCT, cryosectioned, dried, and analyzed with a fluorescent Olympus BX41 microscope. Protein Analysis. Ureters were dissected from E17.5 and E18.5 embryos and immediately frozen. Proteins were extracted using standard RIPA lysis buffer with phosphatase and protease inhibitors. Protein quantification was performed using the Bradford Assay and 1.0 μg/μl of protein lysate was placed onto each chamber of the PathScan Intracellular signaling array (Cell Signaling Technology #7744). Assay conditions and procedures fol- lowed the manufacturer’s recommendations. Florescence readout was performed using the Licor Odyssey Imager. PathScan data analysis was quantified using ImageStudio software provided by Licor Biosciences. The relative flu- orescence unit (RFU) of each antibody spot was quantified according to the Cell Signaling protocol and normal- ized to the positive control antibody spots (also provided on the Cell Signaling array). Student t-test comparisons were performed using Prism GraphPad software. Referencesi 1. Benfield, M. R., McDonald, R. A., Bartosh, S., Ho, P. L. & Harmon, W. Changing trends in pediatric transplantation: 2001 Annua Report of the North American Pediatric Renal Transplant Cooperative Study. Pediatric transplantation 7, 321–335 (2003).h 2. Woolf, A. S. & Thiruchelvam, N. Congenital obstructive uropathy: its origin and contribution to end-stage renal disease in children. Advances in renal replacement therapy 8, 157–163 (2001). 3. Collins, A. J. et al. Excerpts from the United States Renal Data System 2007 annual data report. Am J Kidney Dis 51, S1–320, doi 10.1053/j.ajkd.2007.11.001 (2008). j j 4. Roth, J. A. & Diamond, D. A. Prenatal hydronephrosis. Current opinion in pediatrics 13, 138–141 (2001). 5. Woodward, M. & Frank, D. Postnatal management of antenatal D. Postnatal management of antenatal hydronephrosis. BJU intern g y p 6. Mesrobian, H. G. & Mirza, S. P. Hydronephrosis: a view from the inside. Pediatric clinics of North America 59, 839–851, doi: 10.1016/j.pcl.2012.05.008 (2012). 7. Roth, K. S., Koo, H. P., Spottswood, S. E. & Chan, J. C. Obstructive uropathy: an important cause of chronic renal failure in children Clinical pediatrics 41, 309–314 (2002). p 8. Klein, J. et al. Congenital ureteropelvic junction obstruction: human disease and a experimental pathology 92, 168–192, doi: 10.1111/j.1365-2613.2010.00727.x (2011). p 8. Klein, J. et al. Congenital ureteropelvic junction obstruction: human disease and animal models. International journal of experimental pathology 92, 168–192, doi: 10.1111/j.1365-2613.2010.00727.x (2011). p p gy j ( ) 9. Ingraham, S. E. & McHugh, K. M. Current perspectives on congenital obstructive nephropathy. Pediatr Nephrol 26, 1453–1461, doi: 10.1007/s00467-011-1799-8 (2011). 0. Bascands, J. L. & Schanstra, J. P. Obstructive nephropathy: insights from genetically engineered animals. Kidney Int 68, 925–937, doi 10.1111/j.1523-1755.2005.00486.x (2005). j 11. Woolf, A. S. & Davies, J. A. Cell biology of ureter development. J Am Soc Nephrol 24, 19–25, doi: 10.1681/ASN.2012020127 (2013). 12 Kong X T et al Roles of uroplakins in plaque formation umbrella cell enlargement and urinary tract diseases J Cell Biol 167 j 11. Woolf, A. S. & Davies, J. A. Cell biology of ureter development. J Am Soc Nephrol 24, 19–25, doi: 10.1681/ASN.2012020127 (2013). 12. Kong, X. T. et al. Roles of uroplakins in plaque formation, umbrella cell enlargement, and urinary tract diseases. J Cell Biol 167, 11. Woolf, A. S. & Davies, J. A. Cell biology of ureter developmen 1. Woolf, A. S. & Davies, J. A. Cell biology of ureter development. www.nature.com/scientificreports/ The expression of epidermal growth factor and transforming growth factor-bet the stenotic tissue of congenital pelvi-ureteric junction obstruction in children. Journal of pediatric surgery 38, 1656–1660 (20 6. Elliott, C. G. et al. Periostin modulates myofibroblast differentiation during full-thickness cutaneous wound repair. J Cell Sci 125 121–132, doi: 10.1242/jcs.087841 (2012).lih 37. Huang, Y. et al. Matricellular protein periostin contributes to hepatic inflammation and fibrosis. The American journal of pathology 185, 786–797, doi: 10.1016/j.ajpath.2014.11.002 (2015).ii j jp 8. Crawford, J., Nygard, K., Gan, B. S. & O’Gorman, D. B. Periostin induces fibroblast proliferation and myofibroblast persistence in hypertrophic scarring. Experimental dermatology 24, 120–126, doi: 10.1111/exd.12601 (2015).hi 9. Chevalier, R. L., Forbes, M. S. & Thornhill, B. A. Ureteral obstruction as a model of renal interstitial fibrosis and obstructive nephropathy. Kidney Int 75, 1145–1152, doi: 10.1038/ki.2009.86 (2009).h p p y y 40. Chevalier, R. L., Thornhill, B. A., Forbes, M. S. & Kiley, S. C. Mechanisms of renal injury and progression of renal disease in congenital obstructive nephropathy. Pediatr Nephrol 25, 687–697, doi: 10.1007/s00467-009-1316-5 (2010).h g y p 41. Peters, C. A., Gaertner, R. C., Carr, M. C. & Mandell, J. Fetal compensatory renal growth due to unilateral ureteral obstruction Journal of urology 150, 597–600 (1993). f gy 42. Singh, S. et al. Identification of a unique transgenic mouse line that develops megabladder, obstructive uropathy, and renal dysfunction. J Am Soc Nephrol 18, 461–471, doi: 10.1681/ASN.2006040405 (2007). y p 3. Chang, C. P. et al. Calcineurin is required in urinary tract mesenchyme for the development of the pyeloureteral peristaltic machinery. The Journal of clinical investigation 113, 1051–1058, doi: 10.1172/JCI20049 (2004). h 44. Cain, J. E., Islam, E., Haxho, F., Blake, J. & Rosenblum, N. D. GLI3 repressor controls functional development of the mouse u The Journal of clinical investigation 121, 1199–1206, doi: 10.1172/JCI45523 (2011).fi h f g 45. Aoki, Y. et al. Id2 haploinsufficiency in mice leads to congenital hydronephrosis resembling that in humans. Genes to cells : de to molecular & cellular mechanisms 9, 1287–1296, doi: 10.1111/j.1365-2443.2004.00805.x (2004). j ( ) 46. Tripathi, P., Wang, Y., Casey, A. M. & Chen, F. Absence of canonical Smad signaling in ureteral and bladder mesenchyme ca ureteropelvic junction obstruction. J Am Soc Nephrol 23, 618–628, doi: 10.1681/ASN.2011060566 (2012).h 7. Chevalier, R. L. Biomarkers of congenital obstructive nephropathy: past, present and future. The Journal of urology 172, 852–857 doi: 10.1097/01.ju.0000129542.22043.ef (2004). j 8. Decramer, S. www.nature.com/scientificreports/ 22. Shao, X., Johnson, J. E., Richardson, J. A., Hiesberger, T. & Igarashi, P. A minimal Ksp-cadherin promoter linked to a green fluorescent protein reporter gene exhibits tissue-specific expression in the developing kidney and genitourinary tract. J Am Soc Nephrol 13, 1824–1836 (2002).i p 3. Shao, X., Somlo, S. & Igarashi, P. Epithelial-specific Cre-lox recombination in the developing kidney and genitourinary tract. J Am Soc Nephrol 13, 1837–1846 (2002).f p , ( ) 24. Varley, C. L. et al. PPARgamma-regulated tight junction development during human urothelial cytodifferentiation. Journal of cellular physiology 208, 407–417, doi: 10.1002/jcp.20676 (2006). p y gy j p 5. Varley, C. L., Bacon, E. J., Holder, J. C. & Southgate, J. FOXA1 and IRF-1 intermediary transcriptional regulators of PPARgamma induced urothelial cytodifferentiation. Cell death and differentiation 16, 103–114, doi: 10.1038/cdd.2008.116 (2009). yf ff 6. Weiss, R. M. et al. Brg1 determines urothelial cell fate during ureter development. J Am Soc Nephrol 24, 618–626, doi: 10.1681 ASN.2012090902 (2013). 27. Yamany, T., Van Batavia, J. & Mendelsohn, C. Formation and regeneration of the urothelium. Current opinion in organ transplantation 19, 323–330, doi: 10.1097/MOT.0000000000000084 (2014).h p ( ) 28. Zuo, X., Guo, W. & Lipschutz, J. H. The Exocyst Protein Sec10 Is Necessary for Primary Ciliogenesis and Cystogenesis In Vitro. Mol Biol Cell 20, 2522–2529 (2009).hf 29. Lipschutz, J. H., Lingappa, V. R. & Mostov, K. E. The exocyst affects protein synthesis by acting on the translocation machinery o endoplasmic reticulum. J Biol Chem 278, 20954–20960 (2003). p 30. Choi, S. Y. et al. Exocyst Sec10 Is Involved in Basolateral Protein Translation and Translocation in the Endoplasmic Reticulum. Nephron Exp Nephrol 120, e134–e140, doi: 10.1159/000342366 (2012). p p p 31. Yang, A. et al. p63 is essential for regenerative proliferation in limb, craniofacial and epithelial development. Nature 398, 714– doi: 10.1038/19539 (1999). ( ) 2. Mills, A. A. et al. p63 is a p53 homologue required for limb and epidermal morphogenesis. Nature 398, 708–713, doi: 10.1038/19531 (1999).i 3. Gandhi, D. et al. Retinoid signaling in progenitors controls specification and regeneration of the urothelium. Developmental cell 26 469–482, doi: 10.1016/j.devcel.2013.07.017 (2013). 34. Sato, M. & Keshavjee, S. Bronchiolitis obliterans syndrome: alloimmune-dependent and -independent injury with aberrant tissue remodeling. Seminars in thoracic and cardiovascular surgery 20, 173–182, doi: 10.1053/j.semtcvs.2008.05.002 (2008).h g g y j 35. Yang, Y., Zhou, X., Gao, H., Ji, S. J. & Wang, C. Referencesi J Am Soc Nephrol 24, 19–25, doi: 10.1681/ASN.2012020127 (2013) 2. Kong, X. T. et al. Roles of uroplakins in plaque formation, umbrella cell enlargement, and urinary tract diseases. J Cell Biol 167 1195–1204, doi: 10.1083/jcb.200406025 (2004). j 13. Wu, X. R., Kong, X. P., Pellicer, A., Kreibich, G. & Sun, T. T. Uroplakins in urothelial biology, function, and disease. Kidney Int 75, 1153–1165, doi: 10.1038/ki.2009.73 (2009). 14. Khandelwal, P., Abraham, S. N. & Apodaca, G. Cell biology and physiology of the uroepithelium. Am J Physiol Renal Physiol 297, F1477–F1501, doi: 10.1152/ajprenal.00327.2009 (2009).h jp 15. Liang, F. X. et al. Organization of uroplakin subunits: transmembrane topology, pair formation and plaque composition Biochemical journal 355, 13–18 (2001). j 16. Apodaca, G. Stretch-regulated exocytosis of discoidal vesicle j 16. Apodaca, G. Stretch-regulated exocytosis of discoidal vesicles in urinary bladder epithelium. Urology 57, 103–104 (2001). 7. Truschel, S. T. et al. Stretch-regulated exocytosis/endocytosis in bladder umbrella cells. Mol Biol Cell 13, 830–846, doi: 10.1091 mbc.01-09-0435 (2002). 8. Fogelgren, B. et al. Urothelial Defects from Targeted Inactivation of Exocyst Sec10 in Mice Cause Ureteropelvic Junction Obstructions. PloS one 10, e0129346, doi: 10.1371/journal.pone.0129346 (2015).fi j p 19. Heider, M. R. & Munson, M. Exorcising the Exocyst Complex. Traffic, doi: 10.1111/j.1600-0854.2012.01353.x (2012).hf fi 0. Guo, W., Roth, D., Walch-Solimena, C. & Novick, P. The exocyst is an effector for Sec4p, targeting secretory vesicles to sites o exocytosis. The EMBO journal 18, 1071–1080 (1999). h 21. Lipschutz, J. H. et al. Exocyst Is involved in cystogenesis and tubulogenesis and acts by modulating synthesis and delivery of basolateral plasma membrane and secretory proteins. Mol Biol Cell 11, 4259–4275 (2000). Scientific Reports \| 6:31137 \| DOI: 10.1038/srep31137 10 www.nature.com/scientificreports/ www.nature.com/scientificreports/ et al. Predicting the clinical outcome of congenital unilateral ureteropelvic junction obstruction in newborn by urinary proteome analysis. Nature medicine 12, 398–400, doi: 10.1038/nm1384 (2006).h p y 9. Mesrobian, H. G. et al. Candidate urinary biomarker discovery in ureteropelvic junction obstruction: a proteomic approach. The Journal of urology 184, 709–714, doi: 10.1016/j.juro.2010.03.061 (2010). srobian, H. G. et al. Candidate urinary biomarker discovery in ur 50. Yurtcu, M., Gurbuzer, N., Findik, S., Avunduk, M. C. & Gunel, E. Investigation of histopathologic changes in pelviureteral junction obstruction. Renal failure 32, 162–166, doi: 10.3109/08860220903541101 (2010). f areb, T. et al. Alterations in bladder function associated with urot f 1. Aboushwareb, T. et al. Alterations in bladder function associated with urothelial defects in uroplakin II and IIIa knockout mice Neurourology and urodynamics 28, 1028–1033, doi: 10.1002/nau.20688 (2009).l gy y 2. Hu, P. et al. Ablation of uroplakin III gene results in small urothelial plaques, urothelial leakage, and vesicoureteral reflux. J Cell Bio 151, 961–972 (2000). 53. Bryant, D. M. et al. A molecular network for de novo generation of the apical surface and lumen. Nat Cell Biol 12, 1035–1045, doi: 10.1038/ncb2106 (2010).fi 4. Oztan, A. et al. Exocyst requirement for endocytic traffic directed toward the apical and basolateral poles of polarized MDCK cells Mol Biol Cell 18, 3978–3992 (2007).h 5. Blankenship, J. T., Fuller, M. T. & Zallen, J. A. The Drosophila homolog of the Exo84 exocyst subunit promotes apical epithelia identity. J Cell Sci 120, 3099–3110, doi: 10.1242/jcs.004770 (2007).i 56. Essid, M., Gopaldass, N., Yoshida, K., Merrifield, C. & Soldati, T. Rab8a regulates the exocyst-mediated kiss-and-run discharge of the Dictyostelium contractile vacuole. Mol Biol Cell 23, 1267–1282, doi: 10.1091/mbc.E11-06-0576 (2012).f 57. Zhang, X. M., Ellis, S., Sriratana, A., Mitchell, C. A. & Rowe, T. Sec15 is an effector for the Rab11 GTPase in mammalian cells. J Biol Chem 279, 43027–43034, doi: 10.1074/jbc.M402264200 (2004).f j 8. Wu, S., Mehta, S. Q., Pichaud, F., Bellen, H. J. & Quiocho, F. A. Sec15 interacts with Rab11 via a novel domain and affects Rab11 localization in vivo. Nat Struct Mol Biol 12, 879–885, doi: 10.1038/nsmb987 (2005). 9. Khandelwal, P. et al. A Rab11a-Rab8a-Myo5B network promotes stretch-regulated exocytosis in bladder umbrella cells. Mol Biol Cel 24, 1007–1019, doi: 10.1091/mbc.E12-08-0568 (2013). 60. Khandelwal, P. et al. Rab11a-dependent exocytosis of discoidal/fusiform vesicles in bladder umbrella cells. Proc Natl Acad Sci USA 105, 15773–15778, doi: 10.1073/pnas.0805636105 (2008). p ( ) 1. Acknowledgements We are grateful to the University of Alabama at Birmingham’s Hepato/Renal Fibrocystic Diseases Core Center (5P30DK074038) and Transgenic Mouse Facility (Dr. R. Kesterson) for generation of the floxed Sec10 mice. At the University of Hawaii, we thank Tina Carvalho at the Biological Electron Microscope Facility for her expertise in electron microscopy, and the RCMI-BRIDGES Histopathology Core (supported by NIH G12MD007601 and P30GM103341) for outstanding histology services. This work was supported by grants from the National Institutes of Health [grant numbers K01DK087852, R03DK100738, P20GM103457-06A1-8293 to B.F.]; Hawaii Community Foundation [grant number 12ADVC-51347 to B.F.]; the University of Alabama at Birmingham (UAB) Hepato/Renal Fibrocystic Diseases Core Center (HRFDCC) [grant number 5P30DK074038, Pilot award to B.F.); the March of Dimes [Basil O’Connor Starter Scholar Research Award, grant number #5-FY14-56 to B.F.]; and the University of Hawaii at Manoa Research Centers in Minority Institute, BRIDGES program [grant number 5G12MD007601, Pilot award to B.F.]. www.nature.com/scientificreports/ Bartoli, F. A. et al. Urothelium damage as the primary cause of ureteropelvic junction obstruction: a new hypothesis. Urologica research 24, 9–13 (1996). Scientific Reports \| 6:31137 \| DOI: 10.1038/srep31137 11 www.nature.com/scientificreports/ 62. Madisen, L. et al. A robust and high-throughput Cre reporting and characterization system for the whole mouse brain. N neuroscience 13, 133–140, doi: 10.1038/nn.2467 (2010).hh , , ( ) 63. Theiler, K. The house mouse: atlas of embryonic development., (Springer-Verlag, 1989).i , ( ) 63. Theiler, K. The house mouse: atlas of embryonic development., (Springer-Verlag, 1989).i hh f y p p g g 64. Fogelgren, B. et al. Deficiency in Six2 during prenatal development is associated with reduced nephron number, chronic renal failure, and hypertension in Br/+ adult mice. Am J Physiol Renal Physiol 296, F1166–F1178 (2009). Author Contributions A.J.L., N.P. and B.F. conceived and designed the research and wrote the paper. A.J.L., N.P., J.A.N., V.H.L., B.A.F. and B.F. executed experiments, including data collection and analysis. A.J.L., N.P., J.A.N., K.S.T. and B.F. interpreted results and provided key insights. All authors were involved in editing the manuscript and approving the final submitted version. Scientific Reports \| 6:31137 \| DOI: 10.1038/srep31137 Additional Information Supplementary information accompanies this paper at http://www.nature.com/srep Competing financial interests: The authors declare no competing financial interests. How to cite this article: Lee, A. J. et al. Fibroproliferative response to urothelial failure obliterates the ureter lumen in a mouse model of prenatal congenital obstructive nephropathy. Sci. Rep. 6, 31137; doi: 10.1038/ srep31137 (2016). How to cite this article: Lee, A. J. et al. Fibroproliferative response to urothelial failure obliterates the ureter lumen in a mouse model of prenatal congenital obstructive nephropathy. Sci. Rep. 6, 31137; doi: 10.1038/ srep31137 (2016). This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/ © The Author(s) 2016 Scientific Reports \| 6:31137 \| DOI: 10.1038/srep31137 12
https://openalex.org/W2799144112	https://hal.inria.fr/hal-01854162/document	English	null	Low Latency FPGA Implementation of Izhikevich-Neuron Model	IFIP advances in information and communication technology	2,017	cc-by	3,032	To cite this version: Vitor Bandeira, Vivianne L. Costa, Guilherme Bontorin, Ricardo Reis. Low Latency FPGA Imple- mentation of Izhikevich-Neuron Model. 5th International Embedded Systems Symposium (IESS), Nov 2015, Foz do Iguaçu, Brazil. pp.210-217, 10.1007/978-3-319-90023-0_17. hal-01854162 Distributed under a Creative Commons Attribution 4.0 International License HAL Id: hal-01854162 https://inria.hal.science/hal-01854162v1 Submitted on 6 Aug 2018 L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. Distributed under a Creative Commons Attribution 4.0 International License Low Latency FPGA Implementation of Izhikevich-Neuron Model Vitor V. Bandeira1, Vivianne L. Costa1,2, Guilherme Bontorin1, and Ricardo A. L. Reis1 1 Universidade Federal do Rio Grande do Sul PGMicro/PPGC – Instituto de Informática 2 Universidade Federal do Paraná PPGMNE {vvbandeira,gbontorin,reis}@inf.ufrgs.br; vlcosta@ufpr.br; Low Latency FPGA Implementation of Izhikevich-Neuron Model Vitor V. Bandeira1, Vivianne L. Costa1,2, Guilherme Bontorin1, and Ricardo A. L. Reis1 1 Universidade Federal do Rio Grande do Sul PGMicro/PPGC – Instituto de Informática 2 Universidade Federal do Paraná PPGMNE {vvbandeira,gbontorin,reis}@inf.ufrgs.br; vlcosta@ufpr.br; Abstract. The Izhikevich’s simple model (ISM) for neural activity presents a good compromise between waveform quality and computational cost. FPGAs (Field Programmable Gate Array) are powerful, ﬂexible, and inexpensive digital hardware that can implement such model. In this pa- per, we present a highly combinational, low latency implementation of ISM for FPGA. In the absence of oﬃcial benchmark to compare diﬀer- ent implementations, we propose two diﬀerent metrics to compare the technical literature with our implementation. In this benchmark, we can implement a system that, when compared to the literature, has almost 1.5 times the number of digital neurons (DN), and latency more than 56 times smaller. This shows that our implementation is best suited for hy- brid network systems and presents a fair performance for only-artiﬁcial networks. 1 Introduction The human brain has about 1011 neurons, and each one can have more than 104 synaptic connections with others neurons [8]. As the most inspiring and powerful computing machine we know at present, it is normal to try breaking the code and understand how it works. We believe its computer capacity comes from a three level complexity: (a) the number of adaptable cells, the neurons; (b) the capability of conﬁgurable connections, the synapses; and (c) the waveform that is at the same time robust against noise and capable of encoding information, the spike or action potential. In terms of the waveform, the literature presents various spike models, each one with a respective biological plausibility and computational complexity. The Izhikevich’s Simple Model (ISM) [9] presents one of the best compromises be- tween waveform quality and computational cost at the moment. It is composed of a system of two ordinary diﬀerential equations of the ﬁrst order that can be easily digitalized. In terms of capability of conﬁguration connections and the number of cells, it is important to ﬁnd a hardware that can at the same time be powerful, ﬂexible, and inexpensive. FPGAs (Field Programmable Gate Array) seem to ﬁll all these requirements as reprogrammable digital circuits. Some papers describe diﬀerent implementations of ISM in FPGA [1,4–6,10, 11]. They diﬀer from how serial or parallel the computations are implemented and the number of pipeline stages used. Our implementation is highly combina- tional and present low latency. No other paper before has proposed any benchmark. To compare our work with others, we propose two diﬀerent metrics. The ﬁrst one is neural lattice network. It estimates the maximum number of cells we can simulate in a single hardware. The second metric we propose is the latency of one neuron. It is the time a variation on the input takes to propagate to the output. This metric has a direct correlation to how parallel an implementation is. Depending on the application, this performance can or cannot be important. Hybrid neural networks, like [2], are an example of systems where such a performance is fundamental. These are systems where the whole network is composed of the real-time communication between an artiﬁcial and a living part. Low and reliable latency is fundamental to ensure the real-time communication integrity between networks. 1 Introduction As biological neurons have latency slower than digital ones, we expect to reuse the hardware, virtualizing a greater number of neurons. In Section 2, we review the simple model proposed by Izhikevich and adapt its equation for digital computing. Section 3 presents the hardware implementa- tion of the neuron and shows a lattice network for comparison reasons. Section 4 shows the hardware results of the implementation to compare it to current lit- erature. Section 5 concludes on the potential of this work and comments about future projects. Table 1. PARAMETERS FOR EACH NEUROCOMPUTATIONAL FEATURE AND INJECTED CURRENT USED IN THE IMPLEMENTATION Neural Behaviour Tonic Phasic Tonic Phasic Mixed Spike-frequency spiking spiking bursting bursting mode adaptation Parameters a* 0.015625 0.015625 0.015625 0.015625 0.015625 0.0078125 b* 0.15625 0.1953125 0.15625 0.1953125 0.1953125 0.1953125 c -65 -65 -50 -55 -55 -65 d* 4.6875 4.6875 1.56125 0.0390625 3.125 6.25 Input I* 10.9375 5 11.71875 4.6875 9.375 23.4375 2.1 Equations Model Ensuring some biological plausibility, the ISM reduces the Hodgkin-Huxley model in two-dimensional system of ordinary diﬀerential equations [9]: dv dt = 0.04v2 + 5v + 140 −u + I (1) (1) du dt = a(bv −u) (2) (2) with the auxiliary after-spike resetting: with the auxiliary after-spike resetting: v ≥30mV =⇒ v ←c u ←u + d (3) (3) where v is the membrane potential of the neuron and u is the membrane recovery variable, both in millivolts (mV); t is the time in milliseconds (ms); I is the total injected currents in nanoamperes (nA); a, b, c, and d are parameters to set the desired waveform or the neuronal activity. The parameter a describes the time scale of the recovery variable u. The parameter b represents a sensitivity of the recovery variable u to the subthreshold ﬂuctuations of the membrane potential v. The parameter c describes the after- spike reset value of the membrane potential v. The parameter d represents after- spike reset of the recovery variable u. Diﬀerent choices of the parameters a, b, c, and d result in diﬀerent intrinsic ﬁring patterns. 2.2 Change of Variables In order to facilitate the model implementation in a digital circuit, it is possible to rewrite Equations (1) to (3) as: hdv dt = 1 32v2 + 3.90625v + 109.375 −u∗+ I∗ (4) hdu∗ dt = a∗(b∗v −u∗) (5) v ≥30mV =⇒ v ←c u∗←u∗+ d (6) (4) (5) (6) where h = 0.78125, u∗= hu, and I∗= hI; the parameters a, b, and d are replaced by a, b, and d, respectively, each one also multiplied by h. This transformation is suggested in [4] and [1], but both neglect the factor h. The new system of diﬀerential Equations (4) to (6) ensures the same behavior of Equations (1) to (3). We can solve by Euler’s Method [3], a numerical method of the ﬁrst order, which produces accurate results. This approach results on: vn+1 = vn + ∆t 1 32v2 n + 3.90625vn + 109.375 −u∗ n + I∗ n = vn + ∆t.kv (7) (8) u∗ n+1 = u∗ n + ∆t [a∗(b∗vn −u∗ n)] = u∗ n + ∆t.ku (8) u∗ n+1 = u∗ n + ∆t [a∗(b∗vn −u∗ n)] = u∗ n + ∆t.ku (8) where ∆t is the time increment of the Euler’s Method. Moreover, in Equation (7) we also approximate 3.90625v ≈4v [4]. kv and ku are used further in the imple- mentation and they represent the variation for each iteration. The parameters in Table 1 are adapted from the original publication [9] considering the factor h, and it depends on the type of the simulated neuron. The choice of parameters is beyond the scope of this paper, as it is a current research topic. The input current is set to an appropriated input to reveal a realistic behavior. In the next section, we show the methodology for ISM implementation on FPGA. 3.1 One Neuron For our implementation, we use combinational logic for the most of the circuit. The circuitry is as parallel as possible, optimized for latency rather than the area with no reuse of any adder or multiplier. Figure 1 presents a single neuron, and Figure 2 the computation for the new value of v, u as well as the activity log. For the calculation of the next v and u, we opted for two parallel operations, one for the case with a spike and other without a spike and select between them afterward. Fig. 1. Implementation of One Neuron Fig. 1. Implementation of One Neuron Each neuron receives the parameters (a, b, c, d) from a top module and stores locally the initial values for v and u. We use an 18-bit ﬁxed point two’s complement representation: 1 sign bit, 9 bits for the integer part and 8 bits for the fractionary part. This representation is better suited for digital implementations than ﬂoating point, and 18 bits uses more eﬃciently the available hardware without compromising the accuracy as presented on [1]. Fig. 2. Schematics of one neuron, the operations to compute Equations (4) to (6). All variables and parameters in this ﬁgure already account for the variable change presented in Section 2. Fig. 2. Schematics of one neuron, the operations to compute Equations (4) to (6). All variables and parameters in this ﬁgure already account for the variable change presented in Section 2. The initial values of v and u are, respectively -70 mV and -15.63 mV. The time incremental is ∆t = h = 0.78125 ms (milliseconds). The parameters and injected currents are exhibited in Table 1. 3.2 Network for Tests and Metrics We have chosen a lattice network: one neuron is directly connected to the next, by I[N] and I[N+1], Figure 3. Even though this has low biological meaning, it can be used estimate the maximal number of neurons that can be implemented on a single FPGA chip. Fig. 3. Schematics of the lattice network used. Fig. 3. Schematics of the lattice network used. We use an Altera’s DE4 Board (EP4SGX230KF40C2) to estimate the num- ber of cells that we could implement, and measure latency. Figures 4 and 5 represent about 200 ms in biological time and about 1.8 microseconds in FPGA with a 250-MHz clock. The maximum and minimum tensions are, respectively, +32 mV and -70 mV. Table 1 contains the parameters used for the implementa- tion as well as the input current. These results were obtained with the SignalTap II, provided in the Quartus II software, and will be presented in the next section. tion as well as the input current. These results were obtained with the SignalTap II, provided in the Quartus II software, and will be presented in the next section. Fig. 4. Simulation results of the lattice network used. Fig. 4. Simulation results of the lattice network used. Fig. 5. Measurement of the lattice network used. Fig. 5. Measurement of the lattice network used. Fig. 5. Measurement of the lattice network used. Table 2. COMPARISON OF OUR IMPLEMENTATION WITH LITERATURE Table 2. COMPARISON OF OUR IMPLEMENTATION WITH LITERATURE Ref Digital HW Time Representation FPGA Use Performance (bits) Neurons FF LUT Clock Pipeline Latency Total Integer Vendor Family (MHz) Stages (ns) Decimal [5] 32 28% 44% 50 0 320 - Xilinx Spartan [4] 64% 78% 40 5 150 18 10.8 [1] 1 1% 1,5% 84.81 7 94.33 Virtex-4 [10] 25 79% 198 23 121.21 44 32.12 [6] 32 32% 36% 110.47 6 63.37 18 9.8 Virtex-5 [11] 256 3.39% 307 96 315.96 32 - 7.04% 214 147 453.27 64 This 364 93% 250 0 8 18 10.8 Altera Stratix IV Figure 4 shows the simulation of our Verilog description of lattice network using ModelSimTM. Figure 5 shows the measurement of the lattice network on the FPGA, with only the ﬁrst and last neuron being presented. The data for Figure 5 was obtained using the SignalTap II tool from Quartus II Software. This tool implements a circuit on the FPGA that acquires data directly on the logic circuits and then send it through a JTAG connection to the computer. The data can be displayed and handled on SignalTap II or exported to other software. Only one neuron activity is shown, but we have tested the activity of the six neurons presented on Table 1. With a lattice network, we can ﬁt 364 digital neurons (DN) on an FPGA. This is 1.5 times more than previous from the literature, [11], which presented 256 DN (Table 2). Our estimative is from a lattice network, but this number alone is expressive. And it is important to estimate how many realistic DN we can implement in a realistic physical network. We consider that there is no coherence of network implementations and avail- able data in the literature, since each paper implements a diﬀerent network. Therefore, we do not compare values for virtual neurons at the network level. Indeed, we have not found any paper comparing it either. There is much evidence of biological data from experiments indicating that the information in brain structures can be coded, among other ways, in the time interval between spikes [12]. Because of this we have considered paramount to our implementation to have a high precision on the spike timestamp, which is the instant that the spike occurred. That is achieved with low latency and reliable system. Table 2. COMPARISON OF OUR IMPLEMENTATION WITH LITERATURE The latency is the time that the cell takes to provide a valid output from a variation on the input. We have shown that our latency (8 ns) is more than 56 times smaller than the literature, the best comparison being with [11] (453.27 ns), Table 2. The pipelines presented in the literature do not show a parallel load, causing to have a bigger latency. And also it implies an approximation of the spike times- tamp as high as the pipeline extension. We suppose that such an approximation do not interfere with their applications [1, 4, 6, 10, 11], but the same cannot be said to all applications. 5 Conclusion In this paper, we presented a highly-combinational low-latency implementation for Izhikevich’s Neuron Model in FPGA. This approach is better suited for hybrid network applications as it has the best latency in the literature, Table 2. Some other implementations can be better suited for emulation of networks purely artiﬁcial with less precision in spike timestamp, as they use fewer resources than ours. We could also implement more DN in a single FPGA board than the liter- ature when we consider our lattice network. Although as in many cases with bioinspired circuits, these implementations are very particular, and a fair com- parison between two diﬀerent networks is near impossible. Future works include: (a) to implement a network with more biological mean- ing; (b) to reuse logic blocks and to implement a pipeline for some calculations to achieve a better speed without compromising latency; (c) to use precomputed values in auxiliary shared memory to reduce computation time and latency; and (d) to explore the parallelism technique for multiple virtual neurons, increasing the maximum size of a network in a single FPGA chip. As our application is to study and interface with natural living neural net- works, the FPGA implementation is preferable for it has easier conﬁgurability, reconﬁgurability, and test. Other implementations such as artiﬁcial networks implemented on a full-custom analog [7], or digital integrated circuits may be interesting to implement the short-term objectives (a) through (d) are achieved. Such long-term implementation can improve power consumption, timing perfor- mance, and area occupation at the expense of conﬁgurability. Acknowledgement This work is funded by the following agencies: Federal Agency for Support and Evaluation of Higher Education of Brazil (CAPES), the National Council for Technological and Scientiﬁc Development (CNPq), and the Foundation for Re- search of the State of Rio Grande do Sul (FAPERGS). The authors thank the Macnica-DHW Ltda for the FPGAs boards and technical support. ( ) Macnica-DHW Ltda for the FPGAs boards and technical support. 12. Wennberg, R., Velazquez, J.L.P.: Coordinated Activity in the Brain : Measure- ments and Relevance to Brain Function and Behavior. Springer-Verlag New York, New York, NY (2009) References 1. Ambroise, M., Levi, T., Bornat, Y., Saighi, S.: Biorealistic spiking neural network on fpga. In: Information Sciences and Systems (CISS), 2013 47th Annual Confer- ence on. pp. 1–6 (March 2013) 2. Bontorin, G., Renaud, S., Garenne, A., Alvado, L., Le Masson, G., Tomas, J.: A real-time closed-loop setup for hybrid neural networks. In: Engineering in Medicine and Biology Society, 2007. EMBS 2007. 29th Annual International Conference of the IEEE. pp. 3004–3007 (Aug 2007) 3. Burden, R.L., Faires, J.D.: Numerical Analysis. Brooks/Cole Publishing Company, Boston, 9 edn. (2011) 4. Cassidy, A., Andreou, A.: Dynamical digital silicon neurons. In: Biomedical Cir- cuits and Systems Conference, 2008. BioCAS 2008. IEEE. pp. 289–292 (Nov 2008) 5. Cassidy, A., Denham, S., Kanold, P., Andreou, A.: Fpga based silicon spiking neural array. In: Biomedical Circuits and Systems Conference, 2007. BIOCAS 2007. IEEE. pp. 75–78 (Nov 2007) 6. Cheung, K., Schultz, S., Leong, P.: A parallel spiking neural network simulator. In: Field-Programmable Technology, 2009. FPT 2009. International Conference on. pp. 247–254 (Dec 2009) ( ) 7. Indiveri, G., Horiuchi, T.K.: Frontiers in neuromorphic engineering. Frontiers in neuroscience 5 (2011) 8. Izhikevich, E.M.: Neural Excitability, Spiking and Bursting. International Journal of Bifurcations and Chaos 10(6), 1171–1266 (2000) 9. Izhikevich, E.M.: Simple model of spiking neurons. IEEE 14, 1569– 1572 (2003) 10. Rice, K.L., Bhuiyan, M., Taha, T., Vutsinas, C.N., Smith, M.: Fpga implementation of izhikevich spiking neural networks for character recognition. In: Reconﬁgurable Computing and FPGAs, 2009. ReConFig ’09. International Conference on. pp. 451–456 (Dec 2009) 11. Thomas, D.B., Luk, W.: Fpga accelerated simulation of biologically plausible spik- ing neural networks. In: Pocek, K.L., Buell, D.A. (eds.) FCCM. pp. 45–52. IEEE Computer Society (2009)
https://openalex.org/W3101727960	https://iopscience.iop.org/article/10.1088/1367-2630/8/1/013/pdf	English	null	Quantum superposition and entanglement of mesoscopic plasmons	New journal of physics	2,006	cc-by	5,224	This content was downloaded from IP address 195.242.1.103 on 24/10/2024 at 06:43 Quantum superposition and entanglement of mesoscopic plasmons Telecom wavelength single photon sources Xin Cao, Michael Zopf and Fei Ding - To cite this article: Sylvain Fasel et al 2006 New J. Phys. 8 13 You may also like Telecom wavelength single photon sources Xin Cao, Michael Zopf and Fei Ding - Nonlocal quantum superpositions of bright matter-wave solitons and dimers Bettina Gertjerenken and Christoph Weiss - Surface state decoherence in loop quantum gravity, a first toy model Alexandre Feller and Etera R Livine - You may also like You may also like Telecom wavelength single photon sources Xin Cao, Michael Zopf and Fei Ding - Nonlocal quantum superpositions of bright matter-wave solitons and dimers Bettina Gertjerenken and Christoph Weiss - Surface state decoherence in loop quantum gravity, a first toy model Alexandre Feller and Etera R Livine - You may also like Telecom wavelength single photon sources Xin Cao, Michael Zopf and Fei Ding - Nonlocal quantum superpositions of bright matter-wave solitons and dimers Bettina Gertjerenken and Christoph Weiss - Surface state decoherence in loop quantum gravity, a first toy model Alexandre Feller and Etera R Livine - 1 Author to whom any correspondence should be addressed. PII: S1367-2630(06)13970-1 © IOP Publishing Ltd and Deutsche Physikalische Gesellschaft New Journal of Physics 8 (2006) 13 1367-2630/06/010013+8$30.00 To cite this article: Sylvain Fasel et al 2006 New J. Phys. 8 13 To cite this article: Sylvain Fasel et al 2006 New J. Phys. 8 13 Surface state decoherence in loop quantum gravity, a first toy model Alexandre Feller and Etera R Livine - View the article online for updates and enhancements. This content was downloaded from IP address 195.242.1.103 on 24/10/2024 at 06:43 Sylvain Fasel1, Matthäus Halder, Nicolas Gisin and Hugo Zbinden Group of Applied Physics, University of Geneva, CH-1211 Geneva 4, Switzerland E-mail: sylvain.fasel@physics.unige.ch (Figure inspired by [12].) Institute of Physics DEUTSCHE P 2 Figure 1. Schematic structure of the electric ﬁeld E of a SP propagating on a metallic surface in the x-direction. The ﬁeld strength decreases exponentially with distance z from the surface. H: direction of the magnetic ﬁeld; + and −indicate areas with lower and higher density of electrons respectively. (Figure inspired by [12].) entanglement between two remote SPs existing at the same time and without photons being present simultaneously. In a second experiment, we create temporal superposition states at extreme scales. SPs have a ﬁnite lifetime: they are created, propagate and eventually die. We create SPs with photons in superposition of two time-bins and consequently this plasmonic process is in a coherent superposition of occurring at two times that differ by much more than its lifetime. At a macroscopic level, this would superpose a cat living at two epochs that differ by much more than a cat’s lifetime. One should stress that the investigated system involves a mesoscopic number of electrons coding a single-quantum degree of freedom; hence they are not proper Schrödinger cat states. SPs correspond to the propagation of an electromagnetic quantum ﬁeld, but with major differences with respect to the propagation of photons in a ﬁbre. The structure of the electric ﬁeld is indeed very different (see ﬁgure 1) in the two cases. Moreover, the implication of the free electron plasma at the surface of the metal is essential for the generation of a SP state and its quantum properties are completely deﬁned by the geometrical and electromagnetical characteristics of the solid state of matter that support them. Therefore the existence of SPs is a truly mesoscopic phenomenon and is very bound to matter, in contrast to the propagation of photons. p LR-SPs are symmetric low loss propagating modes, that can be excited on thin metallic stripes sandwiched between dielectrics. A particular geometry of these structures allows the direct in and out coupling of the 1550 nm light mode of a standard telecom ﬁbre to the LR-SPs mode, with very low insertion losses [15]. These devices are called plasmonic stripe waveguides (PSWs). In the ﬁrst experiment presented in this paper, we focus on the entanglement of distant SPs that fulﬁl the following two criteria. Firstly, the two SPs being transient processes, care must be taken to excite them at the same time within the SPs lifetime. Sylvain Fasel1, Matthäus Halder, Nicolas Gisin and Hugo Zbinden Group of Applied Physics, University of Geneva, CH-1211 Geneva 4, Switzerland E-mail: sylvain.fasel@physics.unige.ch Sylvain Fasel1, Matthäus Halder, Nicolas Gisin and Hugo Zbinden Group of Applied Physics, University of Geneva, CH-1211 Geneva 4, Switzerland E-mail: sylvain.fasel@physics.unige.ch New Journal of Physics 8 (2006) 13 Received 7 December 2005 Published 30 January 2006 Online at http://www.njp.org/ doi:10.1088/1367-2630/8/1/013 Abstract. Quantum superpositions and entanglement are at the heart of the quantum information science. There have been only a few investigations of these phenomena at the mesoscopic level, despite the fact that these systems are promising for quantum state storage and processing. Here, we present two novel experiments with surface plasmons propagating on cm-long metallic stripe waveguides. We demonstrate that two plasmons can be entangled at remote places. In addition, we create a single plasmon in a temporal superposition state: it exists in a superposition of two widely separated moments. These quantum states, created using photons at telecom wavelength, are collectively held by a mesoscopic number of electrons coding a single-quantum bit of information; they are shown to be very robust against decoherence. Quantum superpositions and entanglement are widely recognized as the core of quantum physics, with all its counterintuitive features. They are also essential for the coming age of quantum technology, as needed for instance for quantum information processing. Entanglement of several photons [1]–[5] or ions [6]–[10] is nowadays common in laboratories, but not much is known at the mesoscopic level. Surface plasmons (SPs) are propagating charge density waves, involving about 1010 free electrons at the surface of metals [11, 12]. SPs can be excited using light, and are thus good candidates to test the robustness of quantum superpositions and entanglement at a mesoscopic level. Previous experiments focused on the conservation of entanglement for photon–plasmon–photon conversion using metallic subwavelength hole-arrays [13] and long- range SPs (LR-SPs) device [14]. In this paper, we use cm-long plasmon guides to experimentally investigate these aspects of quantum physics. To begin, we demonstrate for the ﬁrst time PII: S1367-2630(06)13970-1 © IOP Publishing Ltd and Deutsche Physikalische Gesellschaft New Journal of Physics 8 (2006) 13 1367-2630/06/010013+8$30.00 New Journal of Physics 8 (2006) 13 1367-2630/06/010013+8$30.00 Institute of Physics DEUTSCHE PHYSIKALISCHE GESELLSCHAFT Figure 1. Schematic structure of the electric ﬁeld E of a SP propagating on a metallic surface in the x-direction. The ﬁeld strength decreases exponentially with distance z from the surface. H: direction of the magnetic ﬁeld; + and −indicate areas with lower and higher density of electrons respectively. New Journal of Physics 8 (2006) 13 (http://www.njp.org/) Sylvain Fasel1, Matthäus Halder, Nicolas Gisin and Hugo Zbinden Group of Applied Physics, University of Geneva, CH-1211 Geneva 4, Switzerland E-mail: sylvain.fasel@physics.unige.ch Secondly, the SPs propagation distance must be larger than the coherence length of the exciting photons, in order to clearly destroy the photon during the process. Hole-array-based experiments did not meet these requirements, but this is achieved in our experiment using LR-SPs. New Journal of Physics 8 (2006) 13 (http://www.njp.org/) Institute of Physics DEUTSCHE PHYSIKALISCHE GESELLSCHAFT 2ν 1544 nm 1548 nm 773 nm TAC IF2 1cm long 9.2dB losses PSW2 PC PC C2 C1 PPLN CWL BG2 BG1 FM FM ∆φ PSW1 0.5 cm long 6dB losses Dump Plasmon conversions D1 (passive) D2 (gated) IF1 gate trigger Figure 2. Scheme of the experimental setup used for entangling two SPs on PSW1 and PSW2. CWL: continuous wave laser at 773 nm; C1, C2: circulators; BG1, BG2: tuneable Bragg ﬁlters; PC: polarization controller; FM: Faraday mirrors; IF1, IF2: unbalanced Michelson interferometers; TAC: time to amplitude converter; D1, D2: InGaAs avalanche photodiode (APD) single photon detectors cooled at −45 ◦C; ϕ: controllable phase shifter. D1 is operated in passive mode (passively quenched by a resistance of 1.3 M) at 7% quantum efﬁciency with 5 kHz of noise and a signal of 20 kHz. D1 triggers D2, working in gated mode at 15% efﬁciency. A successful detection of the corresponding photon leads to a mean coincidence rate of 12 per second. The dead-time of the detection system is about 10 µs. 3 Institute of Physics DEUTSCHE PHYSIKALISCHE GESELLSCHAFT Institute of Physics DEUTSCHE PHYSIKALISCHE GESELLSCHAF 2ν 1544 nm 1548 nm 773 nm TAC IF2 1cm long 9.2dB losses PSW2 PC PC C2 C1 PPLN CWL BG2 BG1 FM FM ∆φ PSW1 0.5 cm long 6dB losses Dump Plasmon conversions D1 (passive) D2 (gated) IF1 gate trigger Institute of Physics DEUTSCHE PHYSIKALISCHE GESELLSCHAFT Institute of Physics D1 (passive) Figure 2. Scheme of the experimental setup used for entangling two SPs on PSW1 and PSW2. CWL: continuous wave laser at 773 nm; C1, C2: circulators; BG1, BG2: tuneable Bragg ﬁlters; PC: polarization controller; FM: Faraday mirrors; IF1, IF2: unbalanced Michelson interferometers; TAC: time to amplitude converter; D1, D2: InGaAs avalanche photodiode (APD) single photon detectors cooled at −45 ◦C; ϕ: controllable phase shifter. D1 is operated in passive mode (passively quenched by a resistance of 1.3 M) at 7% quantum efﬁciency with 5 kHz of noise and a signal of 20 kHz. D1 triggers D2, working in gated mode at 15% efﬁciency. New Journal of Physics 8 (2006) 13 (http://www.njp.org/) Sylvain Fasel1, Matthäus Halder, Nicolas Gisin and Hugo Zbinden Group of Applied Physics, University of Geneva, CH-1211 Geneva 4, Switzerland E-mail: sylvain.fasel@physics.unige.ch A successful detection of the corresponding photon leads to a mean coincidence rate of 12 per second. The dead-time of the detection system is about 10 µs. We use two distant similar PSWs inserted in between an energy-time entangled photon pair source and two interferometers, to create and verify the entanglement of SP pairs. The corresponding setup is presented in ﬁgure 2. We use two distant similar PSWs inserted in between an energy-time entangled photon pair source and two interferometers, to create and verify the entanglement of SP pairs. The corresponding setup is presented in ﬁgure 2. The source is a spontaneous parametric down conversion (SPDC) source consisting of a periodicallypoledlithiumniobate(PPLN)waveguide(HCPhotonics)pumpedwithacontinuous- wave laser diode. The phase matching conditions of the process are such that it takes place at the degeneracy point, i.e. the two photons are created around the same average central wavelength of about 1546 nm. Their spectral width is about 80 nm. The photon pairs and the remaining pump power are butt coupled into a single-mode ﬁbre. By energy conservation, (and provided that the spectrum of the pump laser is of negligible width), whenever one photon is measured at a given wavelength, its twin photon can only be detected at a wavelength such that the sum of both energies are equal to the pump photons energy. We use this property to separate the paired photons with high efﬁciency and controllability. This is done with tunable ﬁbred Bragg gratings (AOS GmbH) and circulators. Photons coming from the source with a ﬁrst chosen wavelength are reﬂected by the ﬁrst Bragg grating BG1 and launched into PSW1 via a ﬁrst circulator C1. The remaining light passes through this ﬁlter and reaches the Bragg grating BG2. This ﬁlter is tuned such that only the photons that are energetically complementary of the ﬁrst ones are in turn back-reﬂected, and then launched into the second PSW via circulator C2. This setup ensures New Journal of Physics 8 (2006) 13 (http://www.njp.org/) Institute of Physics DEUTSCHE PHYSIKALISCHE GESELLSCHAFT phase φ + A φB SS+LL φΑ Difference of detection time Events ∆t LS SS+LL SL ∆t L S φB ∆t L S SPDC source TAC pump laser Figure 3. Franson-type interferometers-source arrangement. S: short path; L: long path; t: time difference between photon propagations through long and short arm; ϕA,B: phases applied on photons. Sylvain Fasel1, Matthäus Halder, Nicolas Gisin and Hugo Zbinden Group of Applied Physics, University of Geneva, CH-1211 Geneva 4, Switzerland E-mail: sylvain.fasel@physics.unige.ch Two photons are emitted simultaneously, and independently travel through long or short arms of the interferometers. Only three distinguishable two-photon detection events are possible. Indeed, when photons both choose the short or long arms, the two detections occur with the same time differences, and are thus quantum mechanically undistinguishable, under the condition that the pump laser coherence length is larger than the L–S path difference. The detection rate corresponding to these events is discriminated using a time-window. This rate is modulated by quantum interferences as a function of the sum of the phases applied in the interferometers, and is recorded as sinusoidal fringes. This is the signature of the energy-time entanglement, and their visibility is directly related to the degree of entanglement. Institute of Physics DEUTSCHE PHYSIKALISCHE GESELLSCHAFT phase φ + A φB SS+LL φΑ Difference of detection time Events ∆t LS SS+LL SL ∆t L S φB ∆t L S SPDC source TAC pump laser 4 4 Figure 3. Franson-type interferometers-source arrangement. S: short path; L: long path; t: time difference between photon propagations through long and short arm; ϕA,B: phases applied on photons. Two photons are emitted simultaneously, and independently travel through long or short arms of the interferometers. Only three distinguishable two-photon detection events are possible. Indeed, when photons both choose the short or long arms, the two detections occur with the same time differences, and are thus quantum mechanically undistinguishable, under the condition that the pump laser coherence length is larger than the L–S path difference. The detection rate corresponding to these events is discriminated using a time-window. This rate is modulated by quantum interferences as a function of the sum of the phases applied in the interferometers, and is recorded as sinusoidal fringes. This is the signature of the energy-time entanglement, and their visibility is directly related to the degree of entanglement. Figure 3. Franson-type interferometers-source arrangement. S: short path; L: long path; t: time difference between photon propagations through long and short arm; ϕA,B: phases applied on photons. Two photons are emitted simultaneously, and independently travel through long or short arms of the interferometers. Only three distinguishable two-photon detection events are possible. Indeed, when photons both choose the short or long arms, the two detections occur with the same time differences, and are thus quantum mechanically undistinguishable, under the condition that the pump laser coherence length is larger than the L–S path difference. New Journal of Physics 8 (2006) 13 (http://www.njp.org/) Sylvain Fasel1, Matthäus Halder, Nicolas Gisin and Hugo Zbinden Group of Applied Physics, University of Geneva, CH-1211 Geneva 4, Switzerland E-mail: sylvain.fasel@physics.unige.ch New Journal of Physics 8 (2006) 13 (http://www.njp.org/) Institute of Physics DEUTSCHE PHYSIKALISCHE GESELLSCHAFT 0 2 4 6 8 10 12 14 0 20 40 60 80 100 120 coincidences/5sec. phase [arbitrary units] noise level Figure 4. Interference fringes measured with the two PSWs in the path of the photons. The visibility, obtained through sinusoidal ﬁtting (solid curve), is 96.5 ± 1.6%, while the visibility of the reference fringes measured without PSWs (fringes not shown) is 97.4 ± 1.2%. Institute of Physics DEUTSCHE PHYSIKALISCHE GESELLSCHAFT 5 Institute of Physics DEUTSCHE PHYSIKALISCHE GESELLSCHAFT 0 2 4 6 8 10 12 14 0 20 40 60 80 100 120 coincidences/5sec. phase [arbitrary units] noise level Figure 4. Interference fringes measured with the two PSWs in the path of the photons. The visibility, obtained through sinusoidal ﬁtting (solid curve), is 96.5 ± 1.6%, while the visibility of the reference fringes measured without PSWs (fringes not shown) is 97.4 ± 1.2%. The result of this measurement is presented in ﬁgure 4. In the same way, we measured the entanglement of the original photon pairs emitted by the source as a reference. The interference fringes recorded after photon–plasmon–photon conversion and the reference exhibit the same high visibility (inside the error uncertainty) of about 97% in both cases. The result of this measurement is presented in ﬁgure 4. In the same way, we measured the entanglement of the original photon pairs emitted by the source as a reference. The interference fringes recorded after photon–plasmon–photon conversion and the reference exhibit the same high visibility (inside the error uncertainty) of about 97% in both cases. This result demonstrates that the degree of entanglement is not lowered with respect to the initial two-photon entanglement. At some point, the entanglement is therefore entirely carried by two SPs since the experimental conditions fulﬁl the required criteria. To our knowledge, this is achieved for the ﬁrst time. We demonstrated the creation of entangled SP pairs, and hence the entanglement of two distant mesoscopic systems (separated by about 1 m) constituted by the very large number of free electrons at the surface of the two PSWs. These systems, although consisting of many particles, could code entangled qubits (e.g. time-bin qubits [20]) in a simpler way than other experiments relying on different type of plasmonic devices [13] or atomic ensembles [21]–[23]. Sylvain Fasel1, Matthäus Halder, Nicolas Gisin and Hugo Zbinden Group of Applied Physics, University of Geneva, CH-1211 Geneva 4, Switzerland E-mail: sylvain.fasel@physics.unige.ch The detection rate corresponding to these events is discriminated using a time-window. This rate is modulated by quantum interferences as a function of the sum of the phases applied in the interferometers, and is recorded as sinusoidal fringes. This is the signature of the energy-time entanglement, and their visibility is directly related to the degree of entanglement. that only paired photons are injected into the PSWs. It also enables us to ﬁlter out the remaining pump light that is directed to a dump in order to avoid back reﬂections. The Bragg grating ﬁlters are tuned by stretching the ﬁbre in which the grating is inscribed. The spectral width of these ﬁlters is 0.8 nm, thus the coherence length of the ﬁltered photons is 0.9 mm (coherence time of 4.25 ps), i.e. much smaller than the PSWs. The optical lengths of the two paths from the source to PSW1 and PSW2 are equalized with less than 1 mm uncertainty using optical frequency domain reﬂectometry (OFDR) [16]. The uncertainty on the path lengths from the source to the PSWs is small compared to the length of the shorter PSW (5 mm). The simultaneous existence of both SPs is thus ensured since SPs on PSWs are not faster than photons in ﬁbres. Photons are collected back at the output of the PSWs into single-mode ﬁbres and sent to two matched unbalanced ﬁbre Michelson interferometers having a path length difference between the two arms corresponding to 1.2 ns. They are stabilized in temperature and the relative phase between the two arms of IF2 can be scanned by a piezoelectric actuator. InGaAs APD detectors are connected at the output of each interferometer. Note that the ﬁrst InGaAs APD (D1) (Epitaxx) is operated in passive mode [17, 18] and its detection signal triggers the detection gate of the second (D2), which is operated in gated mode (IdQuantique). In order to maximize the signal-to-noise ratio, which means maximizing the probability of getting a corresponding count at D2 conditioned on a photon detection at D1, the more lossy path is connected to the passive APD D1. The difference of the detection times is recorded using a time to amplitude converter (TAC). Using this setup, we performed a conventional Franson-type entanglement measurement [19] (details in ﬁgure 3). New Journal of Physics 8 (2006) 13 (http://www.njp.org/) Sylvain Fasel1, Matthäus Halder, Nicolas Gisin and Hugo Zbinden Group of Applied Physics, University of Geneva, CH-1211 Geneva 4, Switzerland E-mail: sylvain.fasel@physics.unige.ch L: pulsed laser at 1550 nm; repetition rate: 5 Mhz; pulse length: 1.2 ns; A: variable attenuator; D: InGaAs peltier cooled photodiode single photon counter; BS: 50/50 beam splitter; PM: phase modulator; PC: polarization controller; PSW: surface plasmons stripe waveguide; PBS: polarization beam splitter; S: standard ﬁbre spool of several kilometres; FM: Faraday mirror. Figure 5. Scheme of the experimental setup for the coherent superposition of plasmons at two instants of existence. All ﬁbres are polarization maintaining, except the ﬁbre spool and the ﬁbres of the plasmon conversion part. L: pulsed laser at 1550 nm; repetition rate: 5 Mhz; pulse length: 1.2 ns; A: variable attenuator; D: InGaAs peltier cooled photodiode single photon counter; BS: 50/50 beam splitter; PM: phase modulator; PC: polarization controller; PSW: surface plasmons stripe waveguide; PBS: polarization beam splitter; S: standard ﬁbre spool of several kilometres; FM: Faraday mirror. two paths of equal lengths. One corresponds to pulses that ﬁrst choose the Mach–Zehnder long arm, undergo photon–plasmon–photon conversion on the PSW (and thus achieve the full SP creation, propagation and recollection), travel back and forth through the ﬁbre spool, and then take the short arm of the Mach–Zehnder. The other path corresponds to pulses that ﬁrst choose the short arm, travel back and forth, and then excite a SP in the long arm. The SP conversion thus does not occur at the same time for the two paths, but at instants separated by the time needed for pulses to travel twice inside the ﬁbre spool (i.e. twice 5 µs multiplied by the length of the ﬁbre in kilometres). Pulses are ﬁnally detected at one output of the interferometer. The two paths are undistinguishable and we are thus in presence of a coherent superposition, but only as long as the PSW does not introduce distinguishability between them.At this condition only, one can observe interference at the detector, and the detection probability is a sinusoidal function of the phase shift which is applied in synchronization with the returning pulses. These interference fringes are recorded by sending several light pulses and applying different phase values. The visibility of these fringes represents a direct indication of the coherence of the created superposition state for the path, and thus for the existence time of the plasmonic processes. We recorded interference fringes with and without PSW in the path of the photons. Sylvain Fasel1, Matthäus Halder, Nicolas Gisin and Hugo Zbinden Group of Applied Physics, University of Geneva, CH-1211 Geneva 4, Switzerland E-mail: sylvain.fasel@physics.unige.ch The present setup is working at standard telecom wavelength and with a form of entanglement which is robust for transmission in ﬁbres [24]. In the second experiment, we create a single SP in a coherent superposition of two widely separated instants of existence.We verify whether the coherence is preserved even if the temporal separation of the two instants of existence is several orders of magnitude larger than the SP lifetime. For this purpose, we used the auto-compensating interferometer described in ﬁgure 5, consisting of an unbalanced Mach–Zehnder interferometer connected by a spool of ﬁbre to a Faraday mirror. The timescale for a change in the length of the interferometer due to temperature variation is slower than the time needed for the two-way travel of the light, and the whole system behaves like a single very large and symmetric interferometer (when applied to quantum cryptography, this conﬁguration is known as the ‘Plug’ & ‘Play’ system) [25]. A PSW of 1 cm length is placed in the long arm of the Mach–Zehnder interferometer. Pulses can propagate following New Journal of Physics 8 (2006) 13 (http://www.njp.org/) Institute of Physics DEUTSCHE PHYSIKALISCHE GESELLSCHAFT FM BS PBS L S PM PC PC PSW D A Plasmon conversion 1cm long 9.2dB losses Figure 5. Scheme of the experimental setup for the coherent superposition of plasmons at two instants of existence. All ﬁbres are polarization maintaining, except the ﬁbre spool and the ﬁbres of the plasmon conversion part. L: pulsed laser at 1550 nm; repetition rate: 5 Mhz; pulse length: 1.2 ns; A: variable attenuator; D: InGaAs peltier cooled photodiode single photon counter; BS: 50/50 beam splitter; PM: phase modulator; PC: polarization controller; PSW: surface plasmons stripe waveguide; PBS: polarization beam splitter; S: standard ﬁbre spool of several kilometres; FM: Faraday mirror. Institute of Physics DEUTSCHE PHYSIKALISCHE GESELLSCHAFT 6 Institute of Physics DEUTSCHE PHYSIKALISCHE GESEL FM BS PBS L S PM PC PC PSW D A Plasmon conversion 1cm long 9.2dB losses FM BS PBS L S PM PC PC PSW D A Plasmon conversion 1cm long 9.2dB losses Figure 5. Scheme of the experimental setup for the coherent superposition of plasmons at two instants of existence. All ﬁbres are polarization maintaining, except the ﬁbre spool and the ﬁbres of the plasmon conversion part. Sylvain Fasel1, Matthäus Halder, Nicolas Gisin and Hugo Zbinden Group of Applied Physics, University of Geneva, CH-1211 Geneva 4, Switzerland E-mail: sylvain.fasel@physics.unige.ch We ﬁrst recorded fringes with an average number of photons per pulse set to 1. More precisely, this value is adjusted (using the variable attenuator) so that the sum of the average number of photons in the short arm and in the long arm just before the PSW is 1. The results of this measurement for a short delay t is presented in ﬁgure 6. For larger delays, we increased the launched pulse energy in order to obtain good statistics on the detection counts. We repeated the experiment many times for various pulse powers and for several different delays ranging from 0.27 to 1.24 ms (corresponding to spooled ﬁbre length from 27 to 124 km). In every cases, we consistently found visibilities higher than 99%. The time needed for a SP to propagate from the input to the output of the PSW (i.e. the ‘lifetime’ of the SP), is of the order of 50 ps. The maximal delay we used in our experiment (1.24 ms) is therefore more than 107 times larger. We thus demonstrated that SPs can be in a coherent superposition state of existing at two times separated by a large delay, even if this delay is much larger than their lifetime. New Journal of Physics 8 (2006) 13 (http://www.njp.org/) Institute of Physics DEUTSCHE PHYSIKALISCHE GESELLSCHAFT 0 1 2 3 4 0 20 40 60 80 100 120 140 noise level Number of detections phase [a.u.] Figure 6. Interference fringes measured with the PSW in the path of the photons. Theaveragenumberofphotonsperpulsewas1,andthedelayt wasabout270 µs (this corresponds to a ﬁbre length of 27 km). The visibility, obtained through sinusoidal ﬁtting (solid curve), is 99.4 ± 1.1% in this case, compatible with the visibility of the reference fringes measured without PSW (fringes not shown). Institute of Physics DEUTSCHE PHYSIKALISCHE GESELLSCHAFT 7 Institute of Physics DEUTSCHE PHYSIKALISCHE GESELLSCHAFT Institute of Physics D Institute of Physics DEUTSCHE PHYSIKALISCHE GESELLSCHA 0 1 2 3 4 0 20 40 60 80 100 120 140 noise level Number of detections phase [a.u.] Figure 6. Interference fringes measured with the PSW in the path of the photons. Theaveragenumberofphotonsperpulsewas1,andthedelayt wasabout270 µs (this corresponds to a ﬁbre length of 27 km). The visibility, obtained through sinusoidal ﬁtting (solid curve), is 99.4 ± 1.1% in this case, compatible with the visibility of the reference fringes measured without PSW (fringes not shown). Sylvain Fasel1, Matthäus Halder, Nicolas Gisin and Hugo Zbinden Group of Applied Physics, University of Geneva, CH-1211 Geneva 4, Switzerland E-mail: sylvain.fasel@physics.unige.ch The two presented experiments demonstrate that quantum superpositions and entanglement can be surprisingly robust. This adds to the growing experimental evidence that robust manipulation of entanglement is feasible [1]–[10] with today’s technology. It stresses that quantum bits can be carried by collective modes of a mesoscopic number of particles, here electrons. However, one should emphasize that in the reported experiments, as in similar ones [13], [21]–[23], the many particles collectively code for only a very limited number of degrees of freedom. These results are thus not in conﬂict with the well-established theory of decoherence. Entangling many degrees of freedom, or equivalently many quantum bits, remains a challenge; however, the present results are encouraging. Acknowledgments We would like to thank Peter W Madsen and Thomas Nikolajsen from Micro Managed Photons A/S (Denmark) for providing PSW samples and expert advices, and Franck Robin, Daniel Erni and Esteban Moreno for stimulating discussions. Financial support by the Swiss NCCR Quantum Photonics is acknowledged. New Journal of Physics 8 (2006) 13 (http://www.njp.org/) References [1] Pan J-W, Bouwmeester D, Daniell M, Weinfurter H and Zeilinger A 2000 Experimental test of quantum nonlocality in three-photon Greenberger-Horne-Zeilinger entanglement Nature 403 515–8 [2] Zhao Z et al 2004 Experimental demonstration of ﬁve-photon entanglement and open-destination teleportation Nature 430 54–8 [3] Thew R T, Ac´ın A, Zbinden H and Gisin N 2004 Bell-type test of energy-time entangled qutrits Phys. Rev. Lett. 93 010503 [4] Ursin R et al 2004 Quantum teleportation across the Danube Nature 430 849 [5] de Riedmatten H et al 2005 Long-distance entanglement swapping with photons from separated sources Phys. Rev. A 71 050302 (R) [6] Sackett C A et al 2000 Experimental entanglement of four particles Nature 404 256–9 [7] Schmidt-Kaler F et al 2003 Realization of the Cirac–Zoller controlled-NOT quantum gate Nature 4 [7] Schmidt-Kaler F et al 2003 Realization of the Cirac–Zoller controlled-NOT quantum gate Nature 422 408–11 [8] Blinov B B, Moehring D L, Duan L-M and Monroe C 2004 Observation of entanglement between a single trapped atom and a single photon Nature 428 153–7 [8] Blinov B B, Moehring D L, Duan L-M and Monroe C 2004 Observation of entanglement between a single trapped atom and a single photon Nature 428 153–7 [9] Barrett M D et al 2004 Deterministic quantum teleportation of atomic qubits Nature 429 737–9 [10] Riebe M et al 2004 Deterministic quantum teleportation with atoms Nature 429 734–7 [11] Raether H 1988 Surface Plasmons ed G Hohler (Berlin: Springer) [12] Barnes W L, Dereux A and Ebbesen T W 2003 Surface plasmon subwavelength optics Nature 424 824–30 [13] Altewischer E, van Exter M P and Woerdman J P 2002 Plasmon-assisted transmission of entangled photons Nature 418 304–6 [14] Fasel S et al 2005 Energy-time entanglement preservation in plasmon-assisted light transmission Phys. Rev. Lett. 94 110501 [15] NikolajsenT, Leosson K, Salakhutdinov I and Bozhevolnyi S I 2003 Polymer-based surface-plasmon-polariton stripe waveguides at telecommunication wavelengths Appl. Phys. Lett. 82 668–70 [16] Mussi G, Passy R, Von Der Weid J-P and Gisin N 1997 On the characterization of optical ﬁbe components with OFDR J. Light. Technol. 15 1–11 [17] Halder M et al 2005 Photon-bunching measurement after two 25-km-long optical ﬁbers Phys. Institute of Physics DEUTSCHE PHYSIKALISCHE GESELLSCHAFT 8 References 042335 [18] Rarity J G, Wall T E, Ridley K D, Owens P C M and Tapster P R 2000 Single-photon counting for the 1300 1600-nm range by use of peltier-cooled and passively quenched InGaAs avalanche photodiodes Appl. Opt. 39 6746–53 [19] Franson J D 1989 Bell inequality for position and time Phys. Rev. Lett. 62 2205–8 [20] Brendel J, Tittel W, Zbinden and Gisin N 1999 Pulsed energy-time entangled twin-photon source for quantum communication Phys. Rev. Lett. 82 2594–7 [21] Julsgaard B, Kozhekin A and Polzik E S 2001 Experimental long-lived entanglement of two macroscopic objects Nature 413 400–3 [22] Sherson J, Julsgaard B and Polzik E S 2004 Distant entanglement of macroscopic gas samples Preprint quant-ph/0408146 q p [23] Matsukevich D N 2005 Entanglement of a photon and a collective atomic excitation Phys. Rev. Lett. [23] Matsukevich D N 2005 Entanglement of a photon and a collective atomic excitation Phys. Rev. Lett. 95 040405 [24] Thew R T, Tanzilli S, Tittel W, Zbinden H and Gisin N 2002 Experimental investigation of the robustness of partially entangled qubits over 11 km Phys. Rev. A 66 062304 [25] Ribordy G, Gautier J-D, Gisin N, Guinnard O and Zbinden H 2000 Fast and user-friendly quantum key distribution J. Mod. Opt. 47 517–31 New Journal of Physics 8 (2006) 13 (http://www.njp.org/)
https://openalex.org/W3113973786	https://journals.iucr.org/d/issues/2021/01/00/ba5309/ba5309.pdf	English	null	<i>Phasertng</i>: directed acyclic graphs for crystallographic phasing	Acta crystallographica. Section D, Structural biology	2,021	cc-by	10,749	ISSN 2059-7983 Airlie J. McCoy,a* Duncan H. Stockwell,a‡ Massimo D. Sammito,a‡ Robert D. Oeffner,a Kaushik S. Hatti,a,b Tristan I. Crolla and Randy J. Reada Airlie J. McCoy,a* Duncan H. Stockwell,a‡ Massimo D. Sammito,a‡ Robert D. Oeffner,a Kaushik S. Hatti,a,b Tristan I. Crolla and Randy J. Reada aDepartment of Haematology, Cambridge Institute for Medical Research, University of Cambridge, Hills Road, Cambridge CB2 0XY, United Kingdom, and bDrug Discovery Unit, Wellcome Centre for Anti-Infectives Research, School of Life Sciences, University of Dundee, Dow Street, Dundee DD1 5EH, United Kingdom. *Correspondence e-mail: ajm201@cam.ac.uk Received 16 January 2020 Accepted 6 November 2020 Received 16 January 2020 Accepted 6 November 2020 Received 16 January 2020 Accepted 6 November 2020 Crystallographic phasing strategies increasingly require the exploration and ranking of many hypotheses about the number, types and positions of atoms, molecules and/or molecular fragments in the unit cell, each with only a small chance of being correct. Accelerating this move has been improvements in phasing methods, which are now able to extract phase information from the placement of very small fragments of structure, from weak experimental phasing signal or from combinations of molecular replacement and experimental phasing information. Describing phasing in terms of a directed acyclic graph allows graph-management software to track and manage the path to structure solution. The crystallographic software supporting the graph data structure must be strictly modular so that nodes in the graph are efﬁciently generated by the encapsulated functionality. To this end, the development of new software, Phasertng, which uses directed acyclic graphs natively for input/output, has been initiated. In Phasertng, the codebase of Phaser has been rebuilt, with an emphasis on modularity, on scripting, on speed and on continuing algorithm development. As a ﬁrst application of phasertng, its advantages are demon- strated in the context of phasertng.xtricorder, a tool to analyse and triage merged data in preparation for molecular replacement or experimental phasing. The description of the phasing strategy with directed acyclic graphs is a general- ization that extends beyond the functionality of Phasertng, as it can incorporate results from bioinformatics and other crystallographic tools, and will facilitate multifaceted search strategies, dynamic ranking of alternative search pathways and the exploitation of machine learning to further improve phasing strategies. ‡ These authors contributed equally. Keywords: Phaser; Phasertng; molecular replacement; SAD phasing; directed acyclic graphs; maximum likelihood; machine learning. research papers research papers Keywords: Phaser; Phasertng; molecular replacement; SAD phasing; directed acyclic graphs; maximum likelihood; machine learning. 1. Introduction Our Phaser crystallographic software for phasing macro- molecular crystal structures based on maximum likelihood and multivariate statistics (Bricogne, 1992, 1997; Read, 2001) has been an asset to the crystallographic community, having solved tens of thousands of macromolecular crystal structures in the Protein Data Bank (PDB; Burley et al., 2019). The focus of our developments has been phasing by molecular replace- ment (MR; Huber, 1965; Read, 2001) and single-wavelength anomalous dispersion (SAD; Hendrickson & Teeter, 1981; Pannu & Read, 2004) because these methods are similar in having the relative ease of requiring only a single (merged) data set, because they are both amenable to rigorous like- lihood treatments and because single-wavelength data collection can require a lower total radiation dose than multiple-wavelength methods. Most of the structures depos- ited in the PDB are currently phased by one or the other of these two methods (Burley et al., 2019). However, both MR and SAD phasing can fail for unavoidable reasons, and phasing by multiple isomorphous replacement (MIR; Green et 2. Directed acyclic graphs In addition, the introduction of the expected log-likelihood gain (eLLG; McCoy et al., 2017) in Phaser has brought about a fundamental change in MR strategies. With the eLLG, it has become possible to calculate the probability that MR with a given model will succeed, replacing the ad hoc rules that have guided the attempts of crystallographers to predict the outcome of MR, and to prove that rules based solely on minimum percentages of sequence identity between the model and the target are not sufﬁcient for good prediction across all resolution ranges and model sizes. Using the eLLG as a guide, minimal models can be prepared with the conﬁdence that a solution is possible within the resources available. For phasing problems that are amenable to this approach, successful MR can be achieved with models consisting of small units of secondary structure (Glykos & Kokkinidis, 2003; Robertson et al., 2010), conserved cores of structurally divergent proteins (Bernstein et al., 1997) or reliable fragments of ab initio models (Qian et al., 2007). Fragment-based approaches to model generation and MR have proven to be highly effective (Rodrı´guez et al., 2009; Bibby et al., 2012) and the use of small fragments for MR, with many such fragments needing to be placed, is now well established. A directed acyclic graph (DAG) is a type of graph that describes an aetiological network linking causes to effects. Formally, a DAG (Fig. 1) is a ﬁnite graph in which the edges Figure 1 (a) Path, (b) tree, (c) directed acyclic graph. The root node is shown in red, leaf nodes are shown in green and intermediate are shown in blue, except that all nodes with two parents are shown in purple. 1 MR has also beneﬁtted from advances in homology modelling and ab initio modelling (Kryshtafovych et al., 2019). Utility for MR was a scoring criterion in CASP13 (Read et al., 2019; Croll et al., 2019), with contributors being encouraged to deposit not only coordinates but also estimates of coordi- nate error. Accurate coordinate-error estimates have been demonstrated to improve success in MR calculations (Bunko´czi et al., 2015). When only very poor templates are available, CASP13 showed that the best homology models are better than the best template or even the best ensemble from PDB entries (Wallner, 2020; Croll et al., 2019). research papers research papers model the target sufﬁciently for a MR signal to be obtained. With the multi-trial approach to MR, data tracking becomes a signiﬁcant part of the phasing strategy. al., 1954; Blow & Crick, 1959), multiple-wavelength anom- alous dispersion (MAD; Hendrickson, 2014; Hendrickson & Teeter, 1981) or multiple isomorphous replacement with anomalous scattering (MIRAS; Vonrhein et al., 2007; Ross- mann, 1961) are alternatives to MR and SAD that should be included in broader phasing strategies. These approaches of extracting solutions from many phasing attempts, each individually with a low probability of success, but with a high probability of overall success, have also partly been driven by Moore’s law rates of increase in processing speed and the increasing number of CPUs avail- able on the desktop (Waldrop, 2016). Highlights of the ongoing development in Phaser have been the fast maximum-likelihood rotation function, the fast maximum-likelihood translation function, SCEDS domain analysis through normal-mode perturbation, ensemble variance estimation and reﬁnement, translational noncrys- tallographic symmetry (TNCS) expected intensity-correction terms, twinning detection in the presence of translational noncrystallographic symmetry, the log-likelihood gain on intensity, single-atom MR, gyre and gimble reﬁnement, Phassade substructure determination, information content and translational noncrystallographic symmetry detection [for a review, see McCoy (2017) and citations therein]. We have come to realize that further development of our phasing strategies will require a step change in the software from our laboratory. We describe here how the source code of Phaser has been rebuilt as Phasertng in order to make use of advances in computing and to meet user expectations of faster and more automated software that can optimally explore a wide range of structure-solution strategies. Acta Cryst. (2021). D77, 1–10 Acta Cryst. (2021). D77, 1–10 https://doi.org/10.1107/S2059798320014746 Acta Cryst. (2021). D77, 1–10 Acta Cryst. (2021). D77, 1–10 3.1. DAG modularity Our choice of directed acyclic graphs for describing phasing pathways is supported by our experience of automation in Phaser. The tree-search-with-pruning strategy for MR and SAD in Phaser (McCoy et al., 2007) makes effective use of the strength of the maximum-likelihood functions in using prior information in the search for additional components in the asymmetric unit: either MR models or anomalously scattering atoms. The tree-search-with-pruning strategy is formally a directed acyclic graph. We retain the term ‘mode’ previously used to refer to Phaser’s different executable blocks, but whereas in Phaser a ‘mode’ does not cleanly represent a single functionality, in Phasertng the software is strictly modular. Each ‘mode’ generates a branch on the DAG, and can be initiated with the information contained in, and only in, the DAG nodes. The strict modularity means that data-preparation steps do not need to be repeated in subsequent steps, and the restarting of structure solution from a halted pathway is trivial and trans- parent. We deﬁne the nodes of the DAG as hypotheses for the unit cell, with the edge direction describing increasing information about the position of atoms within. The data encompass information about the crystallization-drop contents, data processing, crystal symmetry, SAD substructures, partial or full poses of models during MR and validated atomic models of the asymmetric unit. The data structure of the node is extensible. Nodes also contain information about the relia- bility or ranking of the hypothesis. research papers of verbosity, keywords that toggle the writing of ﬁles and callbacks to Python to provide updates on progress. of verbosity, keywords that toggle the writing of ﬁles and callbacks to Python to provide updates on progress. are directed and there are no directed cycles, and nodes can have more than one parent. DAGs underly dataﬂow programming, where ‘the ordering of the operations is not speciﬁed by the programmer, but . . . is implied by the data interdependencies’ (Sharp, 1992). The DAG describes the connections between operations rather than the order in which they should occur; upon the execution of a dataﬂow program, the computer infers the order of operation from the connections given in the DAG. An early application of DAGs in computing was the visual programming language Prograph (Matwin & Pietrzykowski, 1985) written for the Apple Macintosh. Phasertng differs from Phaser in four major ways: a modular architecture to encapsulate the functionality that generates DAG nodes; the use of Phil ﬁles for input and output (Echols et al., 2012) to support scripting; the use of enhanced features of C++11 over C++98, including the use of the C++11 stan- dard threading library (ISO, 1998, 2011) to increase speed; and last, but certainly not least, improved algorithms. We expand on these four ways below. 3. Development of Phasertng 3. Development of Phasertng The core functionality in Phaser has been reconﬁgured as Phasertng, principally to support the DAG framework, but the opportunity to rebuild the codebase has allowed us to make other improvements. The development of new algorithms has continued throughout this process. 3.3. Speed The Phasertng code has been parallelized using the ‘thread’ and ‘future’ libraries introduced with the C++11 ISO standard (ISO, 2011). The threading is implemented where a compu- tationally expensive function evaluation must be calculated for each reﬂection; the threading is over the reﬂection loop. Although parts of the Phaser code were parallelized with the nonstandard OpenMP library (Dagum & Menon, 1998), the granularity of the parallelization was coarser than that in Phasertng, owing to the overhead in initializing OpenMP threads, and the threading was not implemented over reﬂec- tion loops. Proﬁling of the source code with Gprof (Graham et al., 2004) has led to further increases in speed. 3.2. Scripting Phasertng has input and output in the Python-based hier- archical interchange language Phil (Echols et al., 2012). By using Phil for output, results are available in Python. By also using Phil ﬁles as input, parameters set by one mode can be used by another without the need for reformatting. Keyword documentation (including information about the defaults) is generated from a master Phil ﬁle, which ensures that the code and documentation are synchronized. Coordinate data input/ output in Phasertng uses PDB-format ﬁles, and reﬂection data are input/output using MTZ-format ﬁles (Winn et al., 2011). Nodes with more than one parent can arise in several different scenarios in phasing. Perhaps the most signiﬁcant is at the stage of placing components by MR, where several poses of (the same or different) components, identiﬁed by independent rotation and translation functions and therefore independent hypotheses for the contents of the asymmetric unit, may be brought together (on the same origin) to build a combined hypothesis for the contents of the asymmetric unit. Other examples of scenarios where nodes are combined from two parents include the validation of MR model placements with independently determined SAD substructures, or where placed MR components are substituted with homologous components and rescored to ﬁnd the best components for phasing. 2. Directed acyclic graphs Contributing to these improvements has been the incorporation of evolu- tionary-covariance information in the modelling process (Simkovic et al., 2016). The key to these implementations of MR strategy is the generation of many models, each slightly perturbed from the others, so that as a group they sample conformational space ﬁnely enough that at least one is able to Figure 1 (a) Path, (b) tree, (c) directed acyclic graph. The root node is shown in red, leaf nodes are shown in green and intermediate are shown in blue, except that all nodes with two parents are shown in purple. Figure 1 (a) Path, (b) tree, (c) directed acyclic graph. The root node is shown in red, leaf nodes are shown in green and intermediate are shown in blue, except that all nodes with two parents are shown in purple. 2 McCoy et al. Phasertng Acta Cryst. (2021). D77, 1–10 research papers 4.1. DAG The results of the phasertng.xtricorder tool are recorded as nodes in a DAG data structure. There are no cycles and so the data structure is a ‘tree’ subgroup of the DAG (Fig. 1). By comparison, Phaser’s NCS mode uses only the most probable TNCS order for TNCS correction and does not expand to subgroups if twinning is detected, and so the results can be described as a ‘path’ subgroup of the DAG (Fig. 1), although the results are not reported in this form. Fig. 2 shows an example of the DAG nodes generated by phasertng.xtricorder for PDB entry 4n3e (Sliwiak et al., 2014), which is a case of a highly pathological crystal with tetarto- hedral twinning and sevenfold TNCS. This structure was solved by taking the data merged in P422, and after suspecting twinning, expanding the data to P1 for MR. MR was performed with TNCS of order 7 and ﬁnding 56 monomers in the P1 asymmetric unit. The space group was determined as C2 after examining the symmetry of the calculated structure factors, so that there were 28 monomers in the C2 asymmetric unit. The phasertng.xtricorder tool reads the data from an input merged MTZ-format data ﬁle; analyses the data to determine whether the French and Wilson procedure (French & Wilson, 1978) has been applied to the data and generates reﬂection intensities; performs anisotropy correction; ﬁnds the possible TNCS order(s); performs TNCS correction for the TNCS order(s); estimates the probability that the data are twinned; and expands the data to subgroups if twinning is detected. In the general case, the result of running phasertng.xtricorder is a set of MTZ-format data ﬁles with different TNCS orders, TNCS corrections and space-group expansions. In the simplest case, where TNCS and twinning are absent, there will only be a single MTZ-format data ﬁle. These data ﬁles are ready for taking forward into MR and SAD phasing trials with maximum-likelihood functions. General descriptions of the nodes generated by phasertng. xtricorder are given below. 4.1.1. Crystal. The DAG is rooted in the information about the molecules present in the crystallization drop: the sequence(s) of protein, DNA, RNA, ligands and small mole- cules from the crystallization conditions. A list of anomalously scattering elements (e.g. sulfur or selenium) is explicitly included. If known, the experimentally determined oligomeric association of the components in the unit cell will also be part of the hypothesis. research papers Read & McCoy, 2016) and the addition of TNCS-correction terms (Sliwiak et al., 2014) throughout the source code. Most of the functions are not only used for function evaluation but are also used as reﬁnement targets. In implementing functions for reﬁnement, the parameterization of the functions is important and the parameterizations have been stringently tested for robust convergence and numerical stability. The minimization code itself has also been developed to handle the speciﬁc features of the likelihood functions, which include correlated parameters and parameters on very different scales (Stockwell et al., 2020). Thus, the public release of the source code is the most complete, up-to-date and exhaustive form of publication of our methods. performing space-group expansion. In addition, phasertng. xtricorder has modiﬁcations to details of the anisotropy correction [the replacement of Newton’s method of reﬁne- ment with BFGS reﬁnement (Fletcher, 1987) and changes to the restraint terms at low resolution], changes to the para- meterization of the TNCS correction (effective molecular radius of volume related by TNCS, r.m.s. deviation between TNCS-related components and resolution-dependent fraction of the scattering related by TNCS) and modiﬁcations to the minimiser code (Stockwell et al., 2020). In phasertng. xtricorder, the reﬂection loops for the calculation of the anisotropy-correction terms, the TNCS correction terms and the outlier rejection have been parallelized with the C++11 threading library. We illustrate the advantages of Phasertng over Phaser in two different ways: ﬁrstly by showing the tracking capabilities of the DAG infrastructure and secondly by providing speed comparisons. 3.4. Improved algorithms Mathematical derivations of the functions included in Phasertng were published approximately contemporaneously with their release in Phaser; however, inspection of the source code in Phaser and Phasertng will show variation from the functions as published. Contributing to the variation has been the adoption of the log-likelihood gain on intensity (LLGI; Phasertng retains the popular features of Phaser, including the ability to run either as binary executables or as Python modules. Almost unchanged from Phaser is the method of logging text output, including logﬁle output of different levels McCoy et al. Phasertng 3 Acta Cryst. (2021). D77, 1–10 4. phasertng.xtricorder As an example of the functionality of Phasertng, we describe the implementation of phasertng.xtricorder, which is a data- analysis and preparation tool that provides some functionality overlapping with phenix.xtriage (Zwart et al., 2005) and TRUNCATE in CCP4 (Winn et al., 2011). In the context of our phasing strategies in development, phasertng.xtricorder is needed in order to provide appropriate data preparation so as to optimize the capabilities of our maximum-likelihood phasing; the underlying maximum-likelihood functions are highly dependent on appropriate multi-step preparation of the data for optimal performance. Since the introduction of the LLGI target (Read & McCoy, 2016), data for maximum- likelihood calculations should preferentially be entered as intensities and should not have been through the French and Wilson procedure (French & Wilson, 1978) that yields posterior expectations. Externally applied anisotropic data truncation is extremely problematic for our algorithms, because the truncation hampers correct normalization of the data. Where the LLGI target is employed, low information- content reﬂections can be excluded internally, on-the-ﬂy, in the interest of speed (Jamshidiha et al., 2019). research papers The anisotropic correction terms are unique for the point-group symmetry, with the anisotropy tensor constrained to that symmetry. If twinning is suspected and the data are expanded to lower symmetry (see below) the aniso- tropy correction need not be repeated in the lower symmetry space group with fewer constraints on the tensor, since the intensities will retain the higher symmetry. 4.1.6. Twinning. The probability of twinning is best deter- mined after TNCS analysis, because accounting for the statistical effects of TNCS can unmask the effect of twinning 4.1.6. Twinning. The probability of twinning is best deter- mined after TNCS analysis, because accounting for the statistical effects of TNCS can unmask the effect of twinning Figure 2 (a) Schematic for the DAG resulting from user input and phasertng.xtricorder with a colouring scheme as in Fig. 1. Nodes outlined in orange are those generated by phasertng.xtricorder. Multiple arrows indicate where the DAG may branch. (b) Schematic of DAG for structure solution of PDB entry 4n3e (Sliwiak et al., 2014). The crystal was obtained by co-crystallization of Hyp-1 with an eightfold molar excess of the ligand 8-anilino-1-naphthalene sulfonate (ANS). Strong ﬂuorescence under UV illumination conﬁrmed the presence of ANS in the crystals. Data were collected from a single crystal at a wavelength of 1.00 A˚ . The data extended to 2.4 A˚ resolution. There were no systematic absences of reﬂections along the axes and the highest symmetry in which the data merged was space group P422. Signiﬁcant anisotropy was present. TNCS of order 7 was suspected as a result of analysis of the Patterson map, with the absence of TNCS maintained as a hypothesis. Twinning was detected in the intensity statistics after TNCS corrections for order 7. Since overmerging is possible in the presence of twinning, data were expanded to all subgroups of P422. Twinning was not detected in the absence of TNCS. At the conclusion of phasertng.xtricorder data analysis there are eight hypotheses for the contents of the asymmetric unit; solutions can be obtained for two of these hypotheses. g (a) Schematic for the DAG resulting from user input and phasertng.xtricorder with a colouring scheme as in Fig. 1. Nodes outlined in orange are those generated by phasertng.xtricorder. Multiple arrows indicate where the DAG may branch. (b) Schematic of DAG for structure solution of PDB entry 4n3e (Sliwiak et al., 2014). research papers forms or for a single crystal form. Data collected from a single radiation-damaged crystal can be merged taking data up to different dosages, balancing damage against completeness and multiplicity. A multi-crystal data collection can result in data sets merged by including different subsets of crystals, balan- cing non-isomorphism against completeness and multiplicity. The processing of the diffraction data gives merged intensities, their errors, the unit-cell dimensions, the wavelength of data collection and the Laue symmetry. forms or for a single crystal form. Data collected from a single radiation-damaged crystal can be merged taking data up to different dosages, balancing damage against completeness and multiplicity. A multi-crystal data collection can result in data sets merged by including different subsets of crystals, balan- cing non-isomorphism against completeness and multiplicity. The processing of the diffraction data gives merged intensities, their errors, the unit-cell dimensions, the wavelength of data collection and the Laue symmetry. 4.1.4. TNCS order. Hypotheses about the TNCS can be ranked based on inspection of the Patterson map. The absence of TNCS is always considered, even when TNCS is indicated by the presence of large peaks in the Patterson map, as the results of our analysis indicate that large peaks in the Patterson map can be caused by crystal pathologies other than TNCS (Rye et al., 2007; Dauter et al., 2005). 4.1.5. TNCS correction. Systematic modulations of the intensities from an isotropic Wilson distribution caused by TNCS are accounted for by the application of expected intensity factors for each reﬂection derived from a model of the TNCS represented by the effective molecular radius, the r.m.s. deviation between TNCS-related components and the fraction of the scattering related by TNCS. The TNCS- correction terms depend on the hypothesis about the TNCS order. 4.1.5. TNCS correction. Systematic modulations of the intensities from an isotropic Wilson distribution caused by TNCS are accounted for by the application of expected intensity factors for each reﬂection derived from a model of the TNCS represented by the effective molecular radius, the r.m.s. deviation between TNCS-related components and the fraction of the scattering related by TNCS. The TNCS- correction terms depend on the hypothesis about the TNCS order. 4.1.3. Anisotropy. Systematic modulations of the intensities from an isotropic Wilson distribution caused by diffraction anisotropy are corrected by the application of anisotropic scaling factors. 4.1. DAG Note that, at the root, the copy number of each component in the asymmetric unit is not part of the hypothesis and that the data analysis in phasertng.xtricorder is independent of the number of copies. In work to come, adding hypotheses about copy numbers will be a branch point on the DAG. The phasertng.xtricorder tool is most closely related to Phaser’s NCS mode. The phasertng.xtricorder tool expands on the functionality in Phaser’s NCS mode by carrying out the Padilla–Yeates L-test (Padilla & Yeates, 2003) using the TNCS intensity-correction terms (Sliwiak et al., 2014) and 4.1.2. Data. More than one set of data may be obtained for a given set of crystal components, either for different crystal 4 McCoy et al. Phasertng Acta Cryst. (2021). D77, 1–10 research papers research papers on intensity statistics (Padilla & Yeates, 2003; Read et al., 2013). If twinning is indicated, then the data may have been merged in a higher symmetry than the crystal symmetry, and subgroups of the space-group symmetry should be considered (as discussed below). is also a parameter in generating the TNCS-correction terms. The orientational difference is reﬁned starting from exact alignment (no angular perturbation) and four small initial perturbations from perfect TNCS translation, giving ﬁve starting angles for reﬁnement. In Phaser’s NCS mode, there is no parallelization in the anisotropy correction or the outlier rejection and no paralle- lization for TNCS correction with TNCS of order greater than 2. For TNCS of order 2, Phaser’s NCS mode has coarse- grained parallelization of the TNCS correction over the ﬁve starting angles for reﬁnement, leading to a maximum ﬁvefold increase in speed even when more than ﬁve cores are avail- able. Since the parallelization in Phasertng is over the reﬂec- tions, Phasertng can utilize all available cores for parallel execution (assuming that the number of cores is fewer than the number of reﬂections). 4.1.7. Space group. The space group is a hypothesis on a branch of the DAG. Ambiguities of space group within the Laue group arise from theoretical considerations (for example if the space group has subgroups and/or an enantiomorph) or on experimental grounds (for example if axial reﬂections were not recorded and hence systematic absences cannot be inspected). For SAD phasing in the case of an enantiomorphic space group, the enantiomorph of the space group is ambig- uous when the anomalous substructure consists of a single type of anomalously scattering atom. For MR using single atoms as the model, the enantiomorph of the space group will not be resolved until the enantiomer of the structure can be interpreted. Perfect twinning can further complicate space- group determination by adding symmetry to the measured intensities. Apart from the introduction of reﬂection-wise threading of anisotropy, outlier rejection and TNCS corrections, algo- rithmic changes also contributed to speed enhancements. Changes in the parameterization of the TNCS correction terms gave better convergence and also generally improved the results (Table 1). Changes to the minimization methods also improved the speed of convergence (Stockwell et al., 2020) for the TNCS and anisotropy corrections. research papers The crystal was obtained by co-crystallization of Hyp-1 with an eightfold molar excess of the ligand 8-anilino-1-naphthalene sulfonate (ANS). Strong ﬂuorescence under UV illumination conﬁrmed the presence of ANS in the crystals. Data were collected from a single crystal at a wavelength of 1.00 A˚ . The data extended to 2.4 A˚ resolution. There were no systematic absences of reﬂections along the axes and the highest symmetry in which the data merged was space group P422. Signiﬁcant anisotropy was present. TNCS of order 7 was suspected as a result of analysis of the Patterson map, with the absence of TNCS maintained as a hypothesis. Twinning was detected in the intensity statistics after TNCS corrections for order 7. Since overmerging is possible in the presence of twinning, data were expanded to all subgroups of P422. Twinning was not detected in the absence of TNCS. At the conclusion of phasertng.xtricorder data analysis there are eight hypotheses for the contents of the asymmetric unit; solutions can be obtained for two of these hypotheses. McCoy et al. Phasertng 5 Acta Cryst. (2021). D77, 1–10 research papers Ten cases exercised the code for no TNCS (PDB entries 2gw3, 1cb7, 1t70, 4nd5, 2afx, 2gtl, 4dpv, 5ej8, 3ux1 and 1za7), ten exercised the code for research papers Because these details of the algorithms are not strictly the same, it is possible that when running without threading users may discover individual cases in which the runtime for Phasertng is slightly longer than that for Phaser. 4.1.8. Space-group expansion. Perfect twinning may mask the crystal symmetry by making the observed intensities consistent with a symmetry higher than that given by the crystal symmetry alone. The intensities can be merged in a Laue group higher than that of the Laue group of the crystal. The data may be expanded to the crystal symmetry without having to integrate the data using the crystal symmetry; there will be no loss of information if the data are perfectly twinned. After expanding the data, MR will give a set of solutions related by the twinning operator(s), which all model the intensities equally well. g 4.2.1. Database. Speed tests were performed using a data- base of 30 test cases selected from the PDB where diffraction data had been deposited. All entries are found in the PDB- REDO database (Joosten et al., 2012) and therefore the data can reproduce the published R factor within ten percentage points. Cases were selected following the curation of the data in Caballero et al. (2021). The entries in this database were sorted by number of reﬂections, and the selection was made by including some of those with the largest number of reﬂections in order to maximize the proportion of execution time spent in threaded sections of code (Fig. 3). Ten cases exercised the code for no TNCS (PDB entries 2gw3, 1cb7, 1t70, 4nd5, 2afx, 2gtl, 4dpv, 5ej8, 3ux1 and 1za7), ten exercised the code for 4.2.1. Database. Speed tests were performed using a data- base of 30 test cases selected from the PDB where diffraction data had been deposited. All entries are found in the PDB- REDO database (Joosten et al., 2012) and therefore the data can reproduce the published R factor within ten percentage points. Cases were selected following the curation of the data in Caballero et al. (2021). The entries in this database were sorted by number of reﬂections, and the selection was made by including some of those with the largest number of reﬂections in order to maximize the proportion of execution time spent in threaded sections of code (Fig. 3). 4.2. Speed In future work, the optimal number of threads of phasertng.xtricorder will be set by embedding it in a Phaser Phasertng Second moments P-value Second moments P-value PDB code TNCS order Centric Acentric Untwinned Twin < 5% Centric Acentric Untwinned Twin < 5% 1hto 2 3.09 2.03 0.008 1 1 3.05 2.03 0.006 1 1 1o04 2 2.94 1.98 0.008 0.00266 1 2.95 1.95 0.006 2.51 1020 1 1upp 2 2.52 1.72 0.021 6.16 1040 1.41 1010 2.62 1.70 0.014 1.26 10102 6.89 1049 2a8y 2 — 2.03 0.010 1 1 — 2.02 0.006 1 1 2fuq 2 3.03 2.00 0.024 1 1 2.97 1.98 0.013 0.0516 1 2ign 2 3.02 1.99 0.009 0.178 1 2.87 1.87 0.006 2.15 10117 1.29 109 3n80 2 3.03 2.03 0.009 1 1 3.02 2.03 0.009 1 1 3uio 2 — 1.65 0.007 0 3.44 10272 — 1.65 0.007 0 3.58 10278 4qrn 2 3.22 2.07 0.011 1 1 3.22 2.07 0.011 1 1 4ttg 2 2.96 2.03 0.009 1 1 2.99 2.04 0.006 1 1 1e94 6 3.71 2.31 0.028 1 1 3.75 2.33 0.028 1 1 1h6d 3 3.53 2.33 0.017 1 1 3.58 2.28 0.008 1 1 2x86 4 3.48 2.51 0.015 1 1 3.47 2.46 0.010 1 1 3g5g 7 2.75 2.06 0.034 1 1 2.58 2.01 0.025 1 1 3lk4 3 — 2.35 0.014 1 1 — 2.43 0.007 1 1 3ts3 4 3.65 2.19 0.020 1 1 3.62 2.12 0.013 1 1 4fj6 6 — 2.01 0.019 1 1 — 1.95 0.010 1.95 107 1 4n3e 7 7.13 3.21 0.014 1 1 4.44 2.47 0.009 1 1 4y0m 6 3.20 2.05 0.017 1 1 3.19 2.04 0.017 1 1 5dp4 4 4.18 2.67 0.056 1 1 4.29 2.63 0.056 1 1 Phaser Phasertng Second moments P-value Second moments P-value PDB code TNCS order Centric Acentric Untwinned Twin < 5% Centric Acentric Untwinned Twin < 5% 1hto 2 3.09 2.03 0.008 1 1 3.05 2.03 0.006 1 1 1o04 2 2.94 1.98 0.008 0.00266 1 2.95 1.95 0.006 2.51 1020 1 1upp 2 2.52 1.72 0.021 6.16 1040 1.41 1010 2.62 1.70 0.014 1.26 10102 6.89 1049 2a8y 2 — 2.03 0.010 1 1 — 2.02 0.006 1 1 2fuq 2 3.03 2.00 0.024 1 1 2.97 1.98 0.013 0.0516 1 2ign 2 3.02 1.99 0.009 0.178 1 2.87 1.87 0.006 2.15 10117 1.29 109 3n80 2 3.03 2.03 0.009 1 1 3.02 2.03 0.009 1 1 3uio 2 — 1.65 0.007 0 3.44 10272 — 1.65 0.007 0 3.58 10278 4qrn 2 3.22 2.07 0.011 1 1 3.22 2.07 0.011 1 1 4ttg 2 2.96 2.03 0.009 1 1 2.99 2.04 0.006 1 1 1e94 6 3.71 2.31 0.028 1 1 3.75 2.33 0.028 1 1 1h6d 3 3.53 2.33 0.017 1 1 3.58 2.28 0.008 1 1 2x86 4 3.48 2.51 0.015 1 1 3.47 2.46 0.010 1 1 3g5g 7 2.75 2.06 0.034 1 1 2.58 2.01 0.025 1 1 3lk4 3 — 2.35 0.014 1 1 — 2.43 0.007 1 1 3ts3 4 3.65 2.19 0.020 1 1 3.62 2.12 0.013 1 1 4fj6 6 — 2.01 0.019 1 1 — 1.95 0.010 1.95 107 1 4n3e 7 7.13 3.21 0.014 1 1 4.44 2.47 0.009 1 1 4y0m 6 3.20 2.05 0.017 1 1 3.19 2.04 0.017 1 1 5dp4 4 4.18 2.67 0.056 1 1 4.29 2.63 0.056 1 1 order greater than 2 (Fig. 5. Discussion Phasertng has supplanted Phaser as our platform for imple- menting novel phasing algorithms and bringing the most effective approaches to the crystallographic community. The change between Phaser and Phasertng can be summarized as pivoting the focus of the software from algorithms that generate results in ad hoc data structures, which must then be interpreted by automation pipelines, to an extensible graph database structure describing automation, whose nodes are ﬁlled with data by the software. Our goal remains achieving the best possible initial electron-density map for model building by MR and SAD phasing. 4.2. Speed 5). The fold speedup is proportional to the number of threads for up to ﬁve threads (Fig. 5). In Phaser, the fold speedup cannot increase with more than ﬁve threads owing to the coarse-grained parallelization discussed previously. In Phasertng, the fold speedup can increase with more than ﬁve threads, although the fold speedup does not continue to increase almost linearly with thread number owing to thread-initialization overheads and nonthreaded code running in serial mode. In future work, the optimal number of threads of phasertng.xtricorder will be set by embedding it in a control structure that can take account of the number of reﬂections and the overall load balance on their system, after benchmarking. TNCS of order 2 (PDB entries 2fuq, 1upp, 1hto, 2a8y, 4ttg, 2ign, 1o04, 3uio, 3n80 and 4qrn) and ten exercised the code for TNCS of order greater than 2 [PDB entries 5dp4 (4), 3ts3 (4), 3g5g (7), 1h6d (3), 4fj6 (6), 3lk4 (4), 2x86 (4), 1e94 (6), 4y0m (4) and 4n3e (7), where the TNCS order is given in parenth- eses]. TNCS of order 2 (PDB entries 2fuq, 1upp, 1hto, 2a8y, 4ttg, 2ign, 1o04, 3uio, 3n80 and 4qrn) and ten exercised the code for TNCS of order greater than 2 [PDB entries 5dp4 (4), 3ts3 (4), 3g5g (7), 1h6d (3), 4fj6 (6), 3lk4 (4), 2x86 (4), 1e94 (6), 4y0m (4) and 4n3e (7), where the TNCS order is given in parenth- eses]. 4.2.2. Hardware and OS. Calculations were performed on a multiprocessing workstation with two eight-core hyper- threaded Intel Xeon processors W-2145 at 3.70 GHz and 128 GB RAM with operating system Centos 7. Compilation was with GCC 4.8.5 (C++11 ﬂag) with optimization level 3. Differences between runtimes for Phaser executables compiled on Linux and Windows operating systems, with and without patches for the ‘meltdown’ bug, have recently been explored (Oeffner, 2018) and we would expect these conclu- sions to also hold for Phasertng. 4.2. Speed In order to compare the speed of Phaser and Phasertng, the phasertng.xtricorder algorithm was reduced to the function- ality of Phaser’s NCS mode by removing the Padilla–Yeates L-test, propagating only the most probable TNCS order to the TNCS correction and not expanding the space group if twin- ning was detected. The speed was compared for three different cases of TNCS (no TNCS, TNCS of order 2 and TNCS of order greater than The speed was compared for three diff (no TNCS, TNCS of order 2 and TNCS o 2) because each of these uses different TNCS-correction algorithms. With no TNCS, the only corrections to the intensities are anisotropic scaling terms. With TNCS of order greater than 2, in addition to the anisotropic scaling correction, TNCS-correction terms are derived from parameters for the TNCS order, the translation vector, the effective molecular radius, the r.m.s. deviation between TNCS-related components and the fraction of the scattering related by TNCS. With TNCS of order 2, in addition to these para- meters, the orientational difference between the TNCS-related components Figure 3 Number of reﬂections in each test case for the database of 30 test cases used for the speed tests in Figs. 4 and 5. PDB identiﬁers are shown for data with no TNCS (orange), for data with TNCS of order 2 (light blue) and data with TNCS of order greater than 2 (dark blue). Figure 3 Number of reﬂections in each test case for the database of 30 test cases used for the speed tests in Figs. 4 and 5. PDB identiﬁers are shown for data with no TNCS (orange), for data with TNCS of order 2 (light blue) and data with TNCS of order greater than 2 (dark blue). g Number of reﬂections in each test case for the database of 30 test cases used for the speed tests in Figs. 4 and 5. PDB identiﬁers are shown for data with no TNCS (orange), for data with TNCS of order 2 (light blue) and data with TNCS of order greater than 2 (dark blue). 6 McCoy et al. Phasertng Acta Cryst. (2021). D77, 1–10 research papers ison of the translational noncrystallographic symmetry (TNCS)-correction algorithms in Phaser and Phasertng. 4.2. Speed Second moments of the intensity distributions and P-value for twinning after TNCS expected intensity-factor correction terms have been applied for the ten cases of TNCS of order 2 and of TNCS of order greater than 2. The expected values of the second moments for untwinned acentric and centric data are 2.0 and 3.0, respectively; the corresponding values for perfectly twinned data are 1.5 and 2.0, respectively. Second moments of the intensity distributions and P-value for twinning after TNCS expected intensity-factor correction terms have been applied for the ten cases of TNCS of order 2 and of TNCS of order greater than 2. The expected values of the second moments for untwinned acentric and centric data are 2.0 and 3.0, respectively; the corresponding values for perfectly twinned data are 1.5 and 2.0, respectively. TNCS of order 2 (PDB entries 2fuq, 1upp, 1hto, 2a8y, 4ttg, 2ign, 1o04, 3uio, 3n80 and 4qrn) and ten exercised the code for TNCS of order greater than 2 [PDB entries 5dp4 (4), 3ts3 (4), 3g5g (7), 1h6d (3), 4fj6 (6), 3lk4 (4), 2x86 (4), 1e94 (6), 4y0m (4) and 4n3e (7), where the TNCS order is given in parenth- eses]. 4.2.2. Hardware and OS. Calculations were performed on a multiprocessing workstation with two eight-core hyper- threaded Intel Xeon processors W-2145 at 3.70 GHz and 128 GB RAM with operating system Centos 7. Compilation order greater than 2 (Fig. 5). The fold speedup is proportional to the number of threads for up to ﬁve threads (Fig. 5). In Phaser, the fold speedup cannot increase with more than ﬁve threads owing to the coarse-grained parallelization discussed previously. In Phasertng, the fold speedup can increase with more than ﬁve threads, although the fold speedup does not continue to increase almost linearly with thread number owing to thread-initialization overheads and nonthreaded code running in serial mode. 4.3. Results The improved algorithms, memory management and C++11 threading implemented over reﬂection loops discussed above resulted in signiﬁcant speed enhancements in Phasertng over Phaser (Fig. 4). Calculations were performed using one and ﬁve threads, where ﬁve was chosen because of the upper limit on the increase in speed possible with the Phaser threading (see above). Average speed improvements are shown in Table 2. The Phasertng code without threading runs between threefold and eightfold faster than the broadly equivalent Phaser code. With threading, the total runtime (which includes the execution of small sections of non-threaded code) runs between ﬁvefold and 30-fold faster when using ﬁve threads. The dependence of runtime on the number of threads is shown for one case each of no TNCS, TNCS of order 2 and TNCS of Data tracking and job management are major components of the two software distributions through which Phaser is distributed: CCP4 (Winn et al., 2011) and Phenix (Liebschner et al., 2019). In CCP4, Phaser has been integrated into the data-tracking systems in ccp4i (no longer supported; Potterton et al., 2003), ccp4i2 (Potterton et al., 2018) and CCP4 Cloud 7 McCoy et al. Phasertng Acta Cryst. (2021). D77, 1–10 research papers Table 2 Comparison of the average fold speedup of Phasertng over Phaser for the test cases shown in Fig. 3. Without threading, one core With threading, ﬁve cores No TNCS 4.1 1.6 6.4 2.5 TNCS of order 2 5.1 2.3 21.6 8.1 TNCS of order greater than 2 5.9 2.0 18.8 7.2 (Krissinel et al., 2018), while in Phenix there are several Phaser interfaces (Echols et al., 2012). Support for directed acyclic graphs in Phasertng will supplement these data-tracking and job-management systems by giving them a new tool with which to report complicated Phasertng-dependent phasing Table 2 Comparison of the average fold speedup of Phasertng over Phaser for the test cases shown in Fig. 3. interfaces (Echols et al., 2012). Support for directed acyclic graphs in Phasertng will supplement these data-tracking and job-management systems by giving them a new tool with which to report complicated Phasertng-dependent phasing strategies. In particular, directed acyclic graphs have mathe- matical properties that allow the execution of useful algo- rithms over their nodes, for example topological sorting, the ability to compute a path between any pair of nodes and fast algorithms for calculating the shortest path (Cormen et al., 1990). We hope that shifting to the Phasertng codebase will also beneﬁt software that is currently dependent on Phaser. In the Phenix suite there are phenix.automr (Zwart et al., 2008) and phenix.mr_rosetta (Terwilliger et al., 2012); in the CCP4 suite 8 McCoy et al. Phasertng Acta Cryst. (2021). D77, 1–10 Comparison of the average fold speedup of Phasertng over Phaser for the test cases shown in Fig. 3. Without threading, one core With threading, ﬁve cores No TNCS 4.1 1.6 6.4 2.5 TNCS of order 2 5.1 2.3 21.6 8.1 TNCS of order greater than 2 5.9 2.0 18.8 7.2 Figure 4 Comparison of the fold speedup of wall time for Phaser and Phasertng with and without threading over ﬁve threads. (a) Data with no TNCS, (b) data with TNCS of order 2 and (c) data with TNCS of order greater than 2. Four times are shown for each PDB identiﬁer: Phaser without threading (blue), Phaser threaded on ﬁve cores (red), Phasertng without threading (green) and Phasertng threaded on ﬁve cores (purple). research papers The longest runtime for each PDB identiﬁer is shown in seconds above each column group, which is for Phaser without threading in every case. Figure 5 Comparison of the fold speedup of wall time for Phaser (red) and Phasertng (purple) with threading for between one and ﬁve threads. The elapsed wall time is shown above each column in seconds for (a) data with no TNCS (PDB entry 1za7), (b) data with TNCS of order 2 (PDB entry 1hto) and (c) data with TNCS of order greater than 2 (PDB entry 4n3e). graphs in Phasertng will supplement these data-tracking and job-management systems by giving them a new tool with which to report complicated Phasertng-dependent phasing Figure 4 Comparison of the fold speedup of wall time for Phaser and Phasertng with and without threading over ﬁve threads. (a) Data with no TNCS, (b) data with TNCS of order 2 and (c) data with TNCS of order greater than 2. Four times are shown for each PDB identiﬁer: Phaser without threading (blue), Phaser threaded on ﬁve cores (red), Phasertng without threading (green) and Phasertng threaded on ﬁve cores (purple). The longest runtime for each PDB identiﬁer is shown in seconds above each column group, which is for Phaser without threading in every case. strategies. In particular, directed acyclic graphs have mathe- matical properties that allow the execution of useful algo- rithms over their nodes, for example topological sorting, the ability to compute a path between any pair of nodes and fast algorithms for calculating the shortest path (Cormen et al., 1990). We hope that shifting to the Phasertng codebase will also beneﬁt software that is currently dependent on Phaser. In the Phenix suite there are phenix.automr (Zwart et al., 2008) and phenix.mr_rosetta (Terwilliger et al., 2012); in the CCP4 suite A t C t (2021) D77 1 10 Figure 5 Comparison of the fold speedup of wall time for Phaser (red) and Phasertng (purple) with threading for between one and ﬁve threads. The elapsed wall time is shown above each column in seconds for (a) data with no TNCS (PDB entry 1za7), (b) data with TNCS of order 2 (PDB entry 1hto) and (c) data with TNCS of order greater than 2 (PDB entry 4n3e). Figure 4 Figure 5 Figure 4 Figure 4 Comparison of the fold speedup of wall time for Phaser and Phasertng with and without threading over ﬁve threads. Funding information The following funding is acknowledged: Wellcome Trust Principal Research Fellowship (grant No. 209407/Z/17/Z to RJR); NIH (grant No. P01GM063210 to RJR). MDS grate- fully acknowledges fellowship support from the European Union’s Horizon 2020 research and innovation program under the Marie Skłodowska-Curie grant (number 790122). KSH was supported by funding from CCP4. A simplistic approach to phasing is best described as a disjoint union of DAGs: every MR or SAD phasing trial is treated independently. More sophisticated search strategies simultaneously consider results from searches with different MR models or SAD substructures or both, as in MR-SAD. The phenix.MRage pipeline processes many MR models in parallel and if a solution is found with one model then all models are superimposed on the solution and rescored, so that the best model can be used to phase the map put forward for model building (Bunko´czi et al., 2013). The ARCIMBOLBO software makes high-level use of persistence of solutions while the model is systematically varied (Rodrı´guez et al., 2009; Sammito et al., 2013, 2014, 2015; Milla´n et al., 2018). The molecular-replacement parameter matrix (MRPM) procedure uses the anomalous substructure derived from MR-SAD to verify MR substructures (Pedersen et al., 2016). Other exam- ples of complicated phasing strategies include ab initio phasing using molecular averaging (Tsao et al., 1992), phase improvement by cross-crystal averaging after MR (Isupov et al., 2004) or experimental phasing (Crennell et al., 2000; Chen et al., 2005; Su et al., 2010), and phasing with electron-micro- scopy reconstructions (Wynne et al., 1999). These approaches will be simpliﬁed with native support for the DAG data structure in Phasertng. References Bernstein, B. E., Michels, P. A. M. & Hol, W. G. J. (1997). Nature, 385, 275–278. Bibby, J., Keegan, R. M., Mayans, O., Winn, M. D. & Rigden, D. J. (2012). Acta Cryst. D68, 1622–1631. ( ) y Blow, D. M. & Crick, F. H. C. (1959). Acta Cryst. 12, 794–802. Bricogne, G. (1992). Proceedings of the CCP4 Study Weekend. Molecular Replacement, edited by W. Wolf, E. J. Dodson & S. Gover, pp. 62–75. Warrington: Daresbury Laboratory. Bricogne, G. (1997). Methods Enzymol. 276, 361–423. Bunko´czi, G., Echols, N., McCoy, A. J., Oeffner, R. D., Adams, P. D. & Read, R. J. (2013). Acta Cryst. D69, 2276–2286. Bunko´czi, G., Wallner, B. & Read, R. J. (2015). Structure, 23, 397–406. Burley, S. K., Berman, H. M., Bhikadiya, C., Bi, C., Chen, L., Costanzo, L., Christie, C., Duarte, J. M., Dutta, S., Feng, Z., Ghosh, S., Goodsell, D. S., Green, R. K., Guranovic, V., Guzenko, D., Hudson, B. P., Liang, Y., Lowe, R., Peisach, E., Periskova, I., Randle, C., Rose, A., Sekharan, M., Shao, C., Tao, Y., Valasatava, Y., Voigt, M., Westbrook, J., Young, J., Zardecki, C., Zhuravleva, M., Kurisu, G., Nakamura, H., Kengaku, Y., Cho, H., Sato, J., Kim, J. Y., Ikegawa, Y., Nakagawa, A., Yamashita, R., Kudou, T., Bekker, G., Suzuki, H., Iwata, T., Yokochi, M., Kobayashi, N., Fujiwara, T., Velankar, S., Kleywegt, G. J., Anyango, S., Armstrong, D. R., Berrisford, J. M., Conroy, M. J., Dana, J. M., Deshpande, M., Gane, P., Ga´borova´, R., Gupta, D., Gutmanas, A., Kocˇa, J., Mak, L., Mir, S., Mukhopadhyay, A., Nadzirin, N., Nair, S., Patwardhan, A., Paysan-Lafosse, T., Pravda, L., Salih, O., Sehnal, D., Varadi, M., Varˇekova´, R., Markley, J. L., Hoch, J. C., Romero, P. R., Baskaran, K., Maziuk, D., Ulrich, E. L., Wedell, J. R., Yao, H., Livny, M. & Ioannidis Y E (2019) Nucleic Acids Res 47 D520–D528 S., Goodsell, D. S., Green, R. K., Guranovic, V., Guzenko, D., Hudson, B. P., Liang, Y., Lowe, R., Peisach, E., Periskova, I., Randle, C., Rose, A., Sekharan, M., Shao, C., Tao, Y., Valasatava, Y., Voigt, M., Westbrook, J., Young, J., Zardecki, C., Zhuravleva, M., Kurisu, G., Nakamura, H., Kengaku, Y., Cho, H., Sato, J., Kim, J. Y., Ikegawa, Y., Nakagawa, A., Yamashita, R., Kudou, T., Bekker, G., Suzuki, H., Iwata, T., Yokochi, M., Kobayashi, N., Fujiwara, T., Velankar, S., Kleywegt, G. J., Anyango, S., Armstrong, D. R., Berrisford, J. M., Conroy, M. research papers (Winn et al., 2011) there are MrBUMP (Keegan & Winn, 2007, 2008), SIMBAD (Simpkin et al., 2018), AMPLE (Bibby et al., 2012; Thomas et al., 2015) and MRparse (https://github.com/ rigdenlab/MrParse); in the ARCIMBOLDO suite there are ARCIMBOLDO, ARCIMBOLDO_LITE, ARCIMBOLDO_ BORGES and ARCIMBOLDO_SHREDDER (Rodrı´guez et al., 2009; Sammito et al., 2013, 2014, 2015; Milla´n et al., 2018). Auto-Rickshaw (Panjikar et al., 2005) automates structure solution by experimental phasing and MR using Phaser. Phaser is also used as the engine behind the UCLA Diffraction Anisotropy Server (Strong et al., 2006) and the SBGrid wide- search MR server (Stokes-Rees & Sliz, 2010). Diamond Light Source has the Phaser-dependent difference-map pipeline DIMPLE for ligand screening (Wojdyr, 2018). We expect that there are bespoke pipelines using Phaser for speciﬁc purposes in laboratory and synchrotron settings of which we are not aware. aim to improve the efﬁciency of phasing and discover unex- pected dependencies between DAG node data elements. Details of the phaser.voyager pipeline will be published else- where. Phasertng, phasertng.xtricorder and phaser.voyager will be made available through the Phenix (Liebschner et al., 2019) and CCP4 (Winn et al., 2011) software distributions. Acknowledgements Acknowledgements We thank Isabel Uso´n for helpful suggestions and discussion. We thank Isabel Uso´n for helpful suggestions and discussion. research papers (a) Data with no TNCS, (b) g Comparison of the fold speedup of wall time for Phaser and Phasertng with and without threading over ﬁve threads. (a) Data with no TNCS, (b) data with TNCS of order 2 and (c) data with TNCS of order greater than 2. Four times are shown for each PDB identiﬁer: Phaser without threading (blue), Phaser threaded on ﬁve cores (red), Phasertng without threading (green) and Phasertng threaded on ﬁve cores (purple). The longest runtime for each PDB identiﬁer is shown in seconds above each column group, which is for Phaser without threading in every case. Comparison of the fold speedup of wall time for Phaser and Phasertng with and without threading over ﬁve threads. (a) Data with no TNCS, (b) data with TNCS of order 2 and (c) data with TNCS of order greater than 2. Four times are shown for each PDB identiﬁer: Phaser without threading (blue), Phaser threaded on ﬁve cores (red), Phasertng without threading (green) and Phasertng threaded on ﬁve cores (purple). The longest runtime for each PDB identiﬁer is shown in seconds above each column group, which is for Phaser without threading in every case. g Comparison of the fold speedup of wall time for Phaser (red) and Phasertng (purple) with threading for between one and ﬁve threads. The elapsed wall time is shown above each column in seconds for (a) data with no TNCS (PDB entry 1za7), (b) data with TNCS of order 2 (PDB entry 1hto) and (c) data with TNCS of order greater than 2 (PDB entry 4n3e). 8 McCoy et al. Phasertng Acta Cryst. (2021). D77, 1–10 8 research papers research papers Sammito, M., Milla´n, C., Frieske, D., Rodrı´guez-Freire, E., Borges, R. J. & Uso´n, I. (2015). Acta Cryst. D71, 1921–1930. ISO (2011). ISO/IEC 14882:2011. Information Technology – Programming Languages – C++. https://www.iso.org/standard/ 64029.html. Sammito, M., Milla´n, C., Rodrı´guez, D. D., de Ilarduya, I. M., Meindl, K., De Marino, I., Petrillo, G., Buey, R. M., de Pereda, J. M., Zeth, K., Sheldrick, G. M. & Uso´n, I. (2013). Nat. Methods, 10, 1099– 1101. Isupov, M. N., Brindley, A. A., Hollingsworth, E. J., Murshudov, G. N., Vagin, A. A. & Littlechild, J. A. (2004). Acta Cryst. D60, 1879– 1882. Jamshidiha, M., Pe´rez-Dorado, I., Murray, J. W., Tate, E. W., Cota, E. & Read, R. J. (2019). Acta Cryst. D75, 342–353. Sharp, J. A. (1992). Dataﬂow Computing: Theory and Practice, edited by J. A. Sharp, pp. 1–15. Norwood: Ablex Publishing Corp. Joosten, R. P., Joosten, K., Murshudov, G. N. & Perrakis, A. (2012). Acta Cryst. D68, 484–496. Simkovic, F., Thomas, J. M. H., Keegan, R. M., Winn, M. D., Mayans, O. & Rigden, D. J. (2016). IUCrJ, 3, 259–270. egan, R. M. & Winn, M. D. (2007). Acta Cryst. D63, 44 Simpkin, A. J., Simkovic, F., Thomas, J. M. H., Savko, M., Lebedev, A., Uski, V., Ballard, C., Wojdyr, M., Wu, R., Sanishvili, R., Xu, Y., Lisa, M.-N., Buschiazzo, A., Shepard, W., Rigden, D. J. & Keegan, R. M. (2018). Acta Cryst. D74, 595–605. Keegan, R. M. & Winn, M. D. (2008). Acta Cryst. D64, 119–124. Krissinel, E., Uski, V., Lebedev, A., Winn, M. & Ballard, C. (2018). Acta Cryst. D74, 143–151. Kryshtafovych, A., Schwede, T., Topf, M., Fidelis, K. & Moult, J. (2019). Proteins, 87, 1011–1020. Sliwiak, J., Jaskolski, M., Dauter, Z., McCoy, A. J. & Read, R. J. (2014). Acta Cryst. D70, 471–480. ( ) Liebschner, D., Afonine, P. V., Baker, M. L., Bunko´czi, G., Chen, V. B., Croll, T. I., Hintze, B., Hung, L.-W., Jain, S., McCoy, A. J., Moriarty, N. W., Oeffner, R. D., Poon, B. K., Prisant, M. G., Read, R. J., Richardson, J. S., Richardson, D. C., Sammito, M. D., Sobolev, O. V., Stockwell, D. H., Terwilliger, T. C., Urzhumtsev, A. G., Videau, L. L., Williams, C. J. & Adams, P. D. (2019). Acta Cryst. D75, 861–877. Stockwell, D. H., McCoy, A. J. & Read, R. J. (2020). Comput. Crystallogr. Newsl. 11, 23–31. Liebschner, D., Afonine, P. V., Baker, M. L., Bunko´czi, G., Chen, y g Stokes-Rees, I. research papers Read, R. J. (2001). Acta Cryst. D57, 1373–1382. Read, R. J. (2001). Acta Cryst. D57, 1373–1382. Echols, N., Grosse-Kunstleve, R. W., Afonine, P. V., Bunko´czi, G., Chen, V. B., Headd, J. J., McCoy, A. J., Moriarty, N. W., Read, R. J., Richardson, D. C., Richardson, J. S., Terwilliger, T. C. & Adams, P. D. (2012). J. Appl. Cryst. 45, 581–586. Read, R. J., Adams, P. D. & McCoy, A. J. (2013). Acta Cryst. D69, 176– 183. Read, R. J. & McCoy, A. J. (2016). Acta Cryst. D72, 375–387. , R. J. & McCoy, A. J. (2016). Acta Cryst. D72, 375–387 P. D. (2012). J. Appl. Cryst. 45, 581 586. Fletcher, R. (1987). Practical Methods of Optimization, 2nd ed. Chi h t J h Wil & S Fletcher, R. (1987). Practical Methods of Optimization, 2nd ed. Chichester: John Wiley & Sons. Read, R. J., Sammito, M. D., Kryshtafovych, A. & Croll, T. I. (2019). Proteins, 87, 1249–1262. ( ) f p Chichester: John Wiley & Sons. y nch, S. & Wilson, K. (1978). Acta Cryst. A34, 517–525 Robertson, M. P., Chi, Y.-I. & Scott, W. G. (2010). Methods, 52, 168– 172. ( ) y Glykos, N. M. & Kokkinidis, M. (2003). Acta Cryst. D59, 709–718. Rodrı´guez, D. D., Grosse, C., Himmel, S., Gonza´lez, C., de Ilarduya, I. M., Becker, S., Sheldrick, G. M. & Uso´n, I. (2009). Nat. Methods, 6, 651–653. Graham, S. L., Kessler, P. B. & McKusick, M. K. (2004). ACM SIGPLAN Notices, 39(4), 49–57. ( ) Green, D., Ingram, V. M. & Perutz, M. F. (1954). Proc. R. Soc. A Math. Phys. Sci. 225, 287–307. Rossmann, M. G. (1961). Acta Cryst. 14, 383–388. y , Hendrickson, W. A. (2014). Q. Rev. Biophys. 47, 49–93. Rye, C. A., Isupov, M. N., Lebedev, A. A. & Littlechild, J. A. (2007). Acta Cryst. D63, 926–930. Hendrickson, W. A. & Teeter, M. M. (1981). Nature, 29 Hendrickson, W. A. & Teeter, M. M. (1981). Nature, 290, 107–113. H b R (1965) A C 19 353 356 Hendrickson, W. A. & Teeter, M. M. (1981). Nature, 290, 107–113. Huber, R. (1965). Acta Cryst. 19, 353–356. ( ) Huber, R. (1965). Acta Cryst. 19, 353–356. y Sammito, M., Meindl, K., de Ilarduya, I. M., Milla´n, C., Artola- Recolons, C., Hermoso, J. A. & Uso´n, I. (2014). FEBS J. 281, 4029– 4045. ISO (1998). ISO/IEC 14882:1998. Programming Languages – C++. https://www.iso.org/standard/25845.html. research papers & Sliz, P. (2010). Proc. Natl Acad. Sci. USA, 107, 21476–21481. Strong, M., Sawaya, M. R., Wang, S., Phillips, M., Cascio, D. & Eisenberg, D. (2006). Proc. Natl Acad. Sci. USA, 103, 8060–8065. Videau, L. L., Williams, C. J. & Adams, P. D. (2019). Acta Cryst. D75, 861–877. Su, J., Li, Y., Shaw, N., Zhou, W., Zhang, M., Xu, H., Wang, B.-C. & Liu, Z.-J. (2010). Protein Cell, 1, 453–458. Matwin, S. & Pietrzykowski, T. (1985). Comput. Languages, 10, 91– 126. Terwilliger, T. C., DiMaio, F., Read, R. J., Baker, D., Bunko´czi, G., Adams, P. D., Grosse-Kunstleve, R. W., Afonine, P. V. & Echols, N. (2012). J. Struct. Funct. Genomics, 13, 81–90. McCoy, A. J. (2017). Methods Mol. Biol. 1607, 421–453. McCoy, A. J., Grosse-Kunstleve, R. W., Adams, P. D., Winn, M. D., Storoni, L. C. & Read, R. J. (2007). J. Appl. Cryst. 40, 658–674. Thomas, J. M. H., Keegan, R. M., Bibby, J., Winn, M. D., Mayans, O. & Rigden, D. J. (2015). IUCrJ, 2, 198–206. McCoy, A. J., Oeffner, R. D., Wrobel, A. G., Ojala, J. R. M., Tryggvason, K., Lohkamp, B. & Read, R. J. (2017). Proc. Natl Acad. Sci. USA, 114, 3637–3641. Tsao, J., Chapman, M. S. & Rossmann, M. G. (1992). Acta Cryst. A48, 293–301. Vonrhein, C., Blanc, E., Roversi, P. & Bricogne, G. (2007). Methods Mol. Biol. 364, 215–230. Milla´n, C., Sammito, M. D., McCoy, A. J., Nascimento, A. F. Z., Petrillo, G., Oeffner, R. D., Domı´nguez-Gil, T., Hermoso, J. A., Read, R. J. & Uso´n, I. (2018). Acta Cryst. D74, 290–304. Waldrop, M. M. (2016). Nature, 530, 144–147. Wallner, B. (2020). Acta Cryst. D76, 285–290. Oeffner, R. D. (2018). Comput. Crystallogr. Newsl. 9, 25–44. Padilla, J. E. & Yeates, T. O. (2003). Acta Cryst. D59, 1124–1130. ( ) y g Padilla, J. E. & Yeates, T. O. (2003). Acta Cryst. D59, 1124–1130. Winn, M. D., Ballard, C. C., Cowtan, K. D., Dodson, E. J., Emsley, P., Evans, P. R., Keegan, R. M., Krissinel, E. B., Leslie, A. G. W., McCoy, A., McNicholas, S. J., Murshudov, G. N., Pannu, N. S., Potterton, E. A., Powell, H. R., Read, R. J., Vagin, A. & Wilson, K. S. (2011). Acta Cryst. D67, 235–242. W jd M (2018) DIMPLE h // di d k/I / Panjikar, S., Parthasarathy, V., Lamzin, V. S., Weiss, M. S. & Tucker, P. A. (2005). Acta Cryst. D61, 449–457. Pannu, N. References J., Dana, J. M., Deshpande, M., Gane, P., Ga´borova´, R., Gupta, D., Gutmanas, A., Kocˇa, J., Mak, L., Mir, S., Mukhopadhyay, A., Nadzirin, N., Nair, S., Patwardhan, A., Velankar, S., Kleywegt, G. J., Anyango, S., Armstrong, D. R., Phasertng is central to the development of phaser.voyager, which will leverage the solution-tracking capabilities of the DAG in sophisticated and exhaustive phasing pathways. The DAG, paired with a formal database architecture for efﬁcient model storage and retrieval, will streamline the termination and restarting of phasing pathways in order to simplify user intervention in search strategies. Wrapping Phasertng and the DAG data structure in phaser.voyager enables us to make optimal use of the maximum-likelihood and multivariate statistics for the preparation and selection of the data, the choice of SAD or MR as the primary phasing strategy, the generation of ensemble models customized to both the data and the hypothesis of the contents of the unit cell, tracking the persistence of solutions, managing coordinate editing and reﬁnement. By applying graph analysis and data-mining methods to databases of phaser.voyager-generated DAGs, we P., Ga´borova´, R., Gupta, D., Gutmanas, A., Kocˇa, J., Mak, L., Mir, K., Maziuk, D., Ulrich, E. L., Wedell, J. R., Yao, H., Livny, M. & K., Maziuk, D., Ulrich, E. L., Wedell, J. R., Yao, H., Livny, Ioannidis, Y. E. (2019). Nucleic Acids Res. 47, D520–D528. Caballero, I., Sammito, M., Afonine, P. V. Uso´n, I., Read, R. J. & McCoy, A. J. (2021). Submitted. y ( ) Chen, B., Vogan, E. M., Gong, H., Skehel, J. J., Wiley, D. C. & Harrison, S. C. (2005). Structure, 13, 197–211. Cormen, T. H., Leiserson, C. E., Rivest, R. L. & Stein, C. (1990). Introduction to Algorithms, 1st ed. Cambridge: MIT Press. Crennell, S., Takimoto, T., Portner, A. & Taylor, G. (2000). Nat. Struct. Biol. 7, 1068–1074. Croll, T. I., Sammito, M. D., Kryshtafovych, A. & Read, R. J. (2019). Proteins, 87, 1113–1127. Dagum, L. & Menon, R. (1998). IEEE Comput. Sci. Eng. 5, 46–55. Dagum, L. & Menon, R. (1998). IEEE Comput. Sci. Eng. 5, 46–55. Dauter, Z., Botos, I., LaRonde-LeBlanc, N. & Wlodawer, A. (2005). Acta Cryst. D61, 967–975. 9 McCoy et al. Phasertng 9 Acta Cryst. (2021). D77, 1–10 research papers research papers S. & Read, R. J. (2004). Acta Cryst. D60, 22–27. Pedersen, B. P., Gourdon, P., Liu, X., Karlsen, J. L. & Nissen, P. (2016). Acta Cryst. D72, 440–445. Wojdyr, M. (2018). DIMPLE. https://www.diamond.ac.uk/Instruments/ Mx/I03/I03-Manual/Data-Analysis/Automated-Software-Pipeline/ Dimple.html. Potterton, E., Briggs, P., Turkenburg, M. & Dodson, E. (2003). Acta Cryst. D59, 1131–1137. y , Potterton, L., Agirre, J., Ballard, C., Cowtan, K., Dodson, E., Evans, Wynne, S. A., Crowther, R. A. & Leslie, A. G. W. (1999). Mol. Cell, 3, 771–780. Potterton, L., Agirre, J., Ballard, C., Cowtan, K., Dodson, E., Evans, P. R., Jenkins, H. T., Keegan, R., Krissinel, E., Stevenson, K., Lebedev, A., McNicholas, S. J., Nicholls, R. A., Noble, M., Pannu, N. S., Roth, C., Sheldrick, G., Skubak, P., Turkenburg, J., Uski, V., von Delft, F., Waterman, D., Wilson, K., Winn, M. & Wojdyr, M. (2018). Acta Cryst. D74, 68–84. Zwart, P. H., Afonine, P. V., Grosse-Kunstleve, R. W., Hung, L.-W., Ioerger, T. R., McCoy, A. J., McKee, E., Moriarty, N. W., Read, R. J., Sacchettini, J. C., Sauter, N. K., Storoni, L. C., Terwilliger, T. C. & Adams, P. D. (2008). Methods Mol. Biol. 426, 419–435. ( ) y Qian, B., Raman, S., Das, R., Bradley, P., McCoy, A. J., Read, R. J. & Baker, D. (2007). Nature, 450, 259–264. ( ) Zwart, P. H., Grosse-Kunstleve, R. W. & Adams, P. D. (2005). CCP4 Newsl. Protein Crystallogr. 43, 27–35. 10 McCoy et al. Phasertng Acta Cryst. (2021). D77, 1–10
https://openalex.org/W2323751316	http://old.scielo.br/pdf/bdj/v27n1/1806-4760-bdj-27-01-00009.pdf	English	null	Study of Anatomical Relationship between Posterior Teeth and Maxillary Sinus Floor in a Subpopulation of the Brazilian Central Region Using Cone-Beam Computed Tomography - Part 2	Brazilian Dental Journal	2,016	cc-by	6,601	Brazilian Dental Journal (2016) 27(1): 9-15 http://dx.doi.org/10.1590/0103-6440201600679 Brazilian Dental Journal (2016) 27(1): 9-15 http://dx.doi.org/10.1590/0103-6440201600679 ISSN 0103-6440 1Department of Stomatologic Sciences, Dental School, UFG - Universidade Federal de Goiás, Goiânia, GO, Brazil 2Department of Oral Sciences, Dental School, UNIC - Universidade de Cuiabá, Cuiabá, MT, Brazil 3Department of Restorative Dentistry, Dental School, USP - Universidade de São Paulo, Ribeirão Preto, SP, Brazil Correspondence: Professor Carlos Estrela, Praça Universitária s/n, Setor Universitário, 74605-220 Goiânia, GO, Brasil. Tel: +55-62-3209-6254. e-mail: estrela3@terra.com.br Study of Anatomical Relationship between Posterior Teeth and Maxillary Sinus Floor in a Subpopulation of the Brazilian Central Region Using Cone- Beam Computed Tomography – Part 2 Carlos Estrela1, Carla A. B. C. M. Nunes1, Orlando Aguirre Guedes2, Ana Helena G. Alencar1, Cynthia R. A. Estrela1, Ricardo Gariba Silva3, Jesus Djalma Pécora3, Manoel Damião Sousa-Neto3 This study evaluated the anatomical relationship between posterior teeth root apices and maxillary sinus floor (MSF) on 202 cone beam computed tomography (CBCT) exams. The distance between the root apices and the MSF, as well as the MSF thickness of the cortical bone closest to root apices and furcation regions were measured. The vertical and horizontal relationships of the MSF with the molar roots were classified into categories adapted from the criteria proposed by Kwak et al. (14). The shortest distances between MSF and the root apices were observed in the mesiobuccal root of the second molar (0.36±1.17 mm) and the palatal root of the first molar (0.45±1.10 mm) and the widest in buccal roots of the first premolars (5.47±4.43 mm). Significant differences were observed between the distance of MSF to the root apices of single-rooted first and second premolars. The cortical thickness ranged from 0.65±0.41 mm over the mesiobuccal root of the second molar to 1.28±0.42 mm over the buccal root of the first premolar. The most observed vertical and horizontal relationships were type II and 2H, respectively. The maxillary molar roots showed greater proximity to the MSF. The thickness of the MSF cortical bone closest to the apices and furcation regions was found to be similar only for premolars. Key Words: anatomy, maxillary sinus floor, maxillary sinusitis, periapical lesion, cone beam computed tomography. 1Department of Stomatologic Sciences, Dental School, UFG - Universidade Federal de Goiás, Goiânia, GO, Brazil 2Department of Oral Sciences, Dental School, UNIC - Universidade de Cuiabá, Cuiabá, MT, Brazil 3Department of Restorative Dentistry, Dental School, USP - Universidade de São Paulo, Ribeirão Preto, SP, Brazil Material and Methods Study Sample For the premolars, the following items were measured: SR: the distance between the apex of single-rooted teeth and the inferior wall of the MSF; BR: the distance between the apex of the buccal root and the inferior wall of the MSF; PR: the distance between the apex of the palatal root and the inferior wall of the MSF; CTSR: the cortical thickness of the inferior wall of the MSF nearest to the apex of single-rooted tooth; CTBR: the cortical thickness of the inferior wall of the MSF closest to the apex of the buccal root; CTPR: the cortical thickness of the inferior wall of the MSF nearest to the apex of the palatal root; CTF: the cortical thickness of the inferior wall of the MSF closest to the furcation area (Figs. 1A and 1B). The present study was performed as a retrospective analysis of CBCT exams selected from the database of a private radiologic center (TCO, Goiânia, GO, Brazil). The inclusion criteria were CBCT exams of the maxilla presenting fully erupted first and second premolars and first and second molars with fully formed apices. Excluded from the study sample were exams presenting image of a device or apparatus of orthodontic retention and presence of external resorption of the root apex, apical periodontitis, bone changes associated with systemic diseases and benign and/or malignant tumors in the posterior area of the maxilla and/or MS. Two hundred and two CBCT exams met the inclusion criteria and were included in this study. Among the selected participants, 128 were females (63.37%) and 74 were males (36.63%), with a mean age of 40.7 years (range: 15-80 years). One thousand and two-hundred maxillary posterior teeth were evaluated (300 first premolars, 300 second premolars, 300 first molars and 300 second molars). Two hundred and sixty-six premolars were single-rooted and 334 were bi-rooted. All molars were tri-rooted teeth. The protocol for the study was approved by the Research Ethical Committee of the Federal University of Goiás (Process number 391.886). Introduction (1-3,8,9). Periapical radiographs were unable to determine the risk of perforation of the maxillary sinus floor (MSF) during periapical surgery (8). The limitation resulting from the two-dimensional images prevents the correct interpretation of the periapical lesions relationship with the MSF (9). Periapical and panoramic radiographs offer little accuracy to the morphometric analysis of the relationship of bone structures with teeth (10). The clinical application of cone beam computed tomography (CBCT) as an aid in the diagnosis and planning has contributed to establish effective therapeutic protocols (11-13). The importance of CBCT scans in the analysis of the morphological characteristics of the MS and its relationship with the roots of the maxillary posterior teeth has been shown (10,14-19). Infection of the root canal system may spread through the periapical tissues and reach important anatomical structures resulting in several complications. The anatomical proximity of the root apices of the maxillary posterior teeth to the maxillary sinus floor (MSF) may favor the development of inflammatory, infectious and/or traumatic alterations in the maxillary sinus (MS) (1-4). In addition, operative procedural errors during root canal therapy (overinstrumentation, overirrigation and overfilling) and aggressive surgical procedures represent potential risk factors for introduction of foreign material into the MS (5). The diagnosis of sinus disease of odontogenic origin is not simple, confounding both patient and the medical and dental professionals (6). The most common causes of odontogenic sinus disease are dental abscesses and periodontal disease that perforated the Schneiderian membrane, and irritation and secondary infection promoted by intra-antral foreign bodies and sinus perforation during tooth extraction (7). The biological constitution of different populations has a variety of genetic characters, which can determine distinct anatomical and topographical relationships. The anatomical knowledge of the structures that compose the middle and lower thirds of the face, especially the MS and its relation with posterior teeth, is of utmost importance not only for the accurate diagnosis of inflammatory alterations that may be established in both the MS and periapical region, but also for the correct establishment of therapeutic, surgical and rehabilitation plans. Thus, the aim of this study was Conventional radiographic exams are commonly used in the study of the anatomical relationship between maxillary posterior teeth and the MS. Introduction However, these exams have limitations that may jeopardize this analysis Braz Dent J 27(1) 2016 to evaluate the anatomical relationship between maxillary posterior teeth root apices and the MSF in a subpopulation of the Brazilian central region by CBCT images. between the inferior wall of the MSF and the root apices of the posterior teeth and the MSF cortical thickness in the region closest to the root apices and in the furcation areas. For the measurements, a specific tool of the I-CAT program was used, and the measurements were performed on the cross-sectional images with slice thickness of 1 mm. C. Estrela et al. The protocol for the study was approved by the Research Ethical Committee of the Federal University of Goiás (Process number 391.886). Material and Methods Study Sample For the molars, the following items were measured: MBR: the distance between the apex of the mesiobuccal root and the inferior wall of the MSF; DBR: the distance between the apex of the distobuccal root and the inferior wall of the MSF; PR: the distance between the apex of the palatal root and the inferior wall of the MSF; CTMBR: the cortical thickness of the inferior wall of the MSF nearest to the apex of the mesiobuccal root; CTDBR: the cortical thickness of the inferior wall of the MSF closest to the apex of the distobuccal root; CTPR: the cortical thickness of the inferior wall of the MSF nearest to the apex of the palatal root; CTF - the cortical thickness of the inferior wall of the MSF closest to the furcation area (Figs. 1C-E). C. Estrela et al. CBCT Image Acquisition and Analysis g q y All CBCT images were acquired using the I-CAT Cone Beam 3D imaging system (Imaging Sciences International, Hatfield, PA, USA) using a 16 cm x 6 cm field of view (FOV). Image volume was reconstructed with isotropic-isometric 0.25 x 0.25 x 0.25 mm voxels. The tube voltage was 120 KVp, tube current was 3.8 mA, and an exposure time of 40 s was used. The images in DICOM format were processed, interpreted and measured by the proprietary software of the CBCT machine (Xoran version 3.1.62; Xoran Technologies, Ann Arbor, MI, USA). The PC workstation used Windows® 7 professional 32-bit with XP Mode operating system (Microsoft Corporation, Redmond, WA, USA) with 2nd Generation Intel® CoreTM i5-2400, 3.1 GHz up to 3.4 GHz with Intel Turbo Boost 2.0, 4 Threads 6 MB Cache (Intel Corporation, USA), card video nVidia GeForce GT610 1 GB, 64-bit (NVIDIA Corporation, USA) and Dell monitor E2211H 21.5 inches – Widescreen resolution of 1920 x 1080 pixels (Dell Corporation, Round Rock, Texas, USA). The vertical relationship between the MSF and the roots of the maxillary molars was evaluated on the CBCT cross-sectional images and classified into five categories according to the criteria described by Kwak et al. (14): Type I: the MSF was located above the level connecting the buccal and palatal root apices; Type II: the MSF was located below the level connecting the buccal and palatal root apices, without an apical protrusion over the MSF; Type III: an apical protrusion of the buccal root apex was observed over the MSF; Type IV: an apical protrusion of the palatal root apex was observed over the MSF; Type V: apical protrusions of the buccal and palatal root apices were observed over the MSF (Figs. 2A-E). The horizontal relationship between the MSF and the roots of the maxillary molars was evaluated on the CBCT cross-sectional images and classified into five categories adapted from the criteria proposed by Kwak et al. (14): Type 1H: the alveolar recess of the MSF was located more towards the buccal side than towards the buccal root; Type 2H: the alveolar recess of the MSF was located between the buccal The anatomical relationship between MSF and maxillary posterior teeth was evaluated by measuring the distances 10 Braz Dent J 27(1) 2016 Figure 1. CBCT Image Acquisition and Analysis A,B: CBCT cross-sections of maxillary premolars with measurements (mm) of the distance between the maxillary sinus floor and the root apices and the maxillary sinus floor cortical thickness in the region closest to the root apices and in the furcation. A: SR and CTSR in single-rooted; B: BR, PR, CTBR, CTPR and CTF bi-rooted. C-E: CBCT cross-sections of maxillary molars with measurements (mm) of the distance between the maxillary sinus floor and the root apices and the maxillary sinus floor cortical thickness in the region closest to the root apices and in the furcation. C: MBR and CTMBR; D: DBR, CTDBR and CTF; E: PR and CTPR. Figure 1. A,B: CBCT cross-sections of maxillary premolars with measurements (mm) of the distance between the maxillary sinus floor and the root apices and the maxillary sinus floor cortical thickness in the region closest to the root apices and in the furcation. A: SR and CTSR in single-rooted; B: BR, PR, CTBR, CTPR and CTF bi-rooted. C-E: CBCT cross-sections of maxillary molars with measurements (mm) of the distance between the maxillary sinus floor and the root apices and the maxillary sinus floor cortical thickness in the region closest to the root apices and in the furcation. C: MBR and CTMBR; D: DBR, CTDBR and CTF; E: PR and CTPR. Figure 1. A,B: CBCT cross-sections of maxillary premolars with measurements (mm) of the distance between the maxillary sinus floor and the root apices and the maxillary sinus floor cortical thickness in the region closest to the root apices and in the furcation. A: SR and CTSR in single-rooted; B: BR, PR, CTBR, CTPR and CTF bi-rooted. C-E: CBCT cross-sections of maxillary molars with measurements (mm) of the distance between the maxillary sinus floor and the root apices and the maxillary sinus floor cortical thickness in the region closest to the root apices and in the furcation. C: MBR and CTMBR; D: DBR, CTDBR and CTF; E: PR and CTPR. 11 Figure 2. CBCT images. Vertical relationship. A: Type I; B: Type II; C: Type III; D: Type IV; E: Type V. Horizontal relationship. F: Type 1H, G: Type 2H, H: Type 3H, I: Type 4H, J: Type 5H. Adapted from Kwak et al. 2004 (14). Figure 2. CBCT images. Vertical relationship. A: Type I; B: Type II; C: Type III; D: Type IV; E: Type V. Horizontal relationship. CBCT Image Acquisition and Analysis F: Type 1H, G: Type 2H, H: Type 3H, I: Type 4H, J: Type 5H. Adapted from Kwak et al. 2004 (14). 11 Braz Dent J 27(1) 2016 and palatal roots; Type 3H: the alveolar recess of the MSF was located more towards the palatal side than towards the palatal root; Type 4H: the alveolar recess of the MSF passes over the roots without establishing relationship with them; Type 5H: the alveolar recess of the MSF is located towards the buccal side and towards the palatal side, and may or may not also extend between the roots (Figs. 2F-J). premolars (p<0.05). The shortest distance was observed for single-rooted second premolar (1.71±2.81 mm) (Table 1). No significant difference was observed between the distance of MSF to the buccal and palatal root apices of bi-rooted first and second premolars (p>0.05). With regards to the molars, the greatest proximity was observed in the mesiobuccal root of the second left (0.36±1.17 mm) and second right (0.44±1.07 mm) molars and the palatal root of the first left molar (0.45±1.10 mm) (Table 2). All measurements and analyzes were performed by two oral and maxillofacial radiologists, with experience in interpreting CBCT exams. The examiners were trained and calibrated using 10% of the sample in a pilot study. In absence of consensus, a third examiner, with the same qualification (oral and maxillofacial radiologist), was called for a final decision. The mean and standard deviation values (mm) of the MSF cortical thickness in the region of the root apices and the furcation area of the maxillary premolars and molars are shown in Tables 3 and 4. The cortical thickness of the MSF inferior wall nearest to the root apices ranged from 0.65±0.41 mm over the mesiobuccal root of the second molar to 1.28±0.42 mm over the buccal root of the first premolar. A statistically significant difference was observed between the cortical thickness of the MSF inferior wall and the root apices of single-rooted first and second premolars. Considering individually each premolar, no statistically significant difference was observed in the mean value of the cortical thickness of the inferior wall of the MSF nearest to the root apex (single, buccal and palatal roots) and the furcation area in all premolars (Table 3). With regards to the molars, significant differences were observed regarding only the cortical bone thickness over the mesiobuccal and distobuccal roots (p>0.05). CBCT Image Acquisition and Analysis Considering each molar individually, statistically significant differences were observed in the mean value of the cortical thickness of the MSF inferior wall nearest to the root apex (mesiobuccal, distobuccal and palatal) and the furcation area in all molars (Table 4). C. Estrela et al. C. Estrela et al. Statistical Analysis The mean and standard deviation of the distances between the root apices and the MSF; and the thickness of the MS cortical bone were obtained. The differences between the distances, as well as between the thicknesses were evaluated by Kruskal-Wallis test. The statistical differences between the types of vertical and horizontal relationships were evaluated by Chi-square test. All statistical analyses were carried out with the Statistical Package for Social Sciences (IBM SPSS 20, IBM Co., New York, NY, USA). Results The mean and standard deviation values (mm) of the distances between the root apices of the maxillary premolars and molars and MSF are shown in Tables 1 and 2. CBCT analysis revealed that the mean value of the distance from the root apices to the MSF ranged from 0.36±1.17 mm for the mesiobuccal root of the second molar to 5.47±4.43 mm for the buccal root of the first premolar. A statistically significant difference was obtained between the distance of root apices to the MSF of single-rooted first and second The frequency distributions of the vertical and horizontal relationships between MSF and roots of maxillary molars are shown in Tables 5 and 6. The most frequent vertical and horizontal relationships were types II and 2H, respectively (p<0.05). Table 2. The mean distances values in mm (SD) between the maxillary molar root apices and MSF Tooth n MBR (X ± SD) DBR (X ± SD) PR (X ± SD) 16 150 0.96 ± 1.79A, b 0.97 ± 1.87A, a 0.79 ± 1.58A, ab 17 150 0.44 ± 1.07A, a 0.74 ± 1.52AB, a 1.00 ± 1.72B, b 26 150 0.75 ± 1.43A, ab 0.66 ± 1.21AB, a 0.45 ± 1.10B, a 27 150 0.36 ± 1.17A, a 0.62 ± 1.53AB, a 0.73 ± 1.63B, ab n = number of teeth; X = mean; SD = standard deviation. Different capital letters indicate significant differences in horizontal lines and different lowercase letters indicate significant differences in the vertical lines; p <0.05 (Kruskal-Wallis); MBR: mesiobuccal root, DBR: distobuccal root, PR: palatal root. Table 1. The mean distances and standard deviation values in mm between the maxillary premolar root apices and MSF Tooth N SR (X ± SD) BR (X ± SD) PR (X ± SD) 14 150 4.25 ± 4.52A, b 5.12 ± 4.14A, b 4.89 ± 4.45A, b 15 150 1.80 ± 2.86A, a 3.19 ± 3.68A, a 2.20 ± 2.90A, a 24 150 4.98 ± 4.97A, b 5.47 ± 4.43A, b 4.39 ± 4.59A, ab 25 150 1.71 ± 2.81A, a 3.31 ± 4.90A, a 2.65 ± 4.36A, ab n = number of teeth; X = mean; SD = standard deviation. Different capital letters indicate significant differences in horizontal lines and different lowercase letters indicate significant differences in the vertical lines; p <0.05 (Kruskal-Wallis); SR: single root, BR: buccal root, PR: palatal root. Table 2. Discussion Jung & Cho (23) analyzed the relationship of the maxillary molars and adjacent structures by CBCT. The authors performed measurements on a sample of 83 patients/332 molars and found that the shortest distance between the root apex and the MS was in the MB root of the second molar. Pagin et al. (22) evaluated qualitatively the close relationship between the MSF and the root apices of the posterior teeth in a Brazilian population by CBCT images. Their sample was composed by 100 MS, 315 teeth, and 601 root apices. Close proximity was observed in 216 roots. Among them, 130 presented root apices in close contact with the MSF with no root protrusion within the MS and no elevation in the sinus floor trajectory. The opposite was observed in 86 roots. The mesiobuccal root of the second molar was frequently found in close proximity to the MSF. With regards to the largest distances, the results of the present study showed that the root apices of first premolars are frequently far away from the MSF, which agrees with the study conducted by Kilic et al. (21). loor e V 67%) 67%) 33%) 33%) 00%) n = number of teeth; X = mean; SD = standard deviation. Different capital letters indicate significant differences in horizontal lines and different lowercase letters indicate significant differences in the vertical lines; p <0.05 (Kruskal-Wallis); CTSR: cortical thickness single root, CTBR: cortical thickness buccal root, CTPR: cortical thickness palatal root, CTF: cortical thickness furcation. n = number of teeth; X = mean; SD = standard deviation. Different capital letters indicate significant differences in horizontal lines and different lowercase letters indicate significant differences in the vertical lines; p <0.05 (Kruskal-Wallis); CTSR: cortical thickness single root, CTBR: cortical thickness buccal root, CTPR: cortical thickness palatal root, CTF: cortical thickness furcation. Table 4. Results The mean distances values in mm (SD) between the maxillary molar root apices and MSF Table 2. The mean distances values in mm (SD) between the maxillary molar root apices and MSF Table 1. The mean distances and standard deviation values in mm between the maxillary premolar root apices and MSF Table 1. The mean distances and standard deviation values in mm between the maxillary premolar root apices and MSF 12 Braz Dent J 27(1) 2016 Braz Dent J 27(1) 2016 Table 3. The mean cortical thickness values in mm (SD) of the maxillary sinus floor in the region of root apices and the furcation area of the maxillary premolars Discussion Molar roots compared to the premolars showed a closer relationship with the MSF. The shortest distance between the root apex and the MSF was observed for the mesiobuccal root of the second left molar. Tooth CTSR (X ± SD) CTBR (X ± SD) CTPR (X ± SD) CTF (X ± SD) 14 1.26 ± 0.37A,b 1.28 ± 0.42A,b 1.15 ± 0.47A,b 1.18 ± 0.33A,b 15 0.92 ± 0.47A,a 1.01 ± 0.52A,a 0.92 ± 0.53A,ab 1.00 ± 0.52A,ab 24 1.17 ± 0.51A,b 1.14 ± 0.34A,ab 1.13 ± 0.47A,b 1.13 ± 0.31A,b 25 0.96 ± 0.49A,a 0.94 ± 0.41A,a 0.77 ± 0.49A,a 0.88 ± 0.35A,a n = number of teeth; X = mean; SD = standard deviation. Different capital letters indicate significant differences in horizontal lines and different lowercase letters indicate significant differences in the vertical lines; p <0.05 (Kruskal-Wallis); CTSR: cortical thickness single root, CTBR: cortical thickness buccal root, CTPR: cortical thickness palatal root, CTF: cortical thickness furcation. The results of the present study are in agreement with those from previous studies that have used computed tomography (CT) (10,14) and CBCT images (20-22). Eberhardt et al. (10) measured the distance between the root apices of posterior teeth and the MS using CT in 38 patients (12 specimens) and obtained results similar to the present study. Jung & Cho (23) analyzed the relationship of the maxillary molars and adjacent structures by CBCT. The authors performed measurements on a sample of 83 patients/332 molars and found that the shortest distance between the root apex and the MS was in the MB root of the second molar. Pagin et al. (22) evaluated qualitatively the close relationship between the MSF and the root apices of the posterior teeth in a Brazilian population by CBCT images. Their sample was composed by 100 MS, 315 teeth, and 601 root apices. Close proximity was observed in 216 roots. Among them, 130 presented root apices in close contact with the MSF with no root protrusion within the The results of the present study are in agreement with those from previous studies that have used computed tomography (CT) (10,14) and CBCT images (20-22). Eberhardt et al. (10) measured the distance between the root apices of posterior teeth and the MS using CT in 38 patients (12 specimens) and obtained results similar to the present study. Discussion The mean cortical thickness values in mm (SD) of the maxillary sinus floor in the region of root apices and the furcation area of the maxillary molars floor in the region of root apices and the furcation area of the maxillary molars Tooth CTMBR (X ± SD) CTDBR (X ± SD) CTPR (X ± SD) CTF (X ± SD) 16 0.88 ± 0.45AB, b 0.85 ± 0.45AB, b 0.78 ± 0.43A, a 0.96 ± 0.22B, a 17 0.71 ± 0.42A, a 0.76 ± 0.42AB, ab 0.81 ± 0.38B, a 0.98 ± 0.27C, a 26 0.85 ± 0.48A, b 0.75 ± 0.39AB, ab 0.72 ±0.43B, a 0.95 ± 0.24AC, a 27 0.65 ± 0.41A, a 0.72 ± 0.45AB, a 0.77 ± 0.46B, a 0.95 ± 0.25C, a n = number of teeth; X = mean; SD = standard deviation. Different capital letters indicate significant differences in horizontal lines and different lowercase letters indicate significant differences in the vertical lines. p <0.05 (Kruskal-Wallis). CTMBR: cortical thickness mesiobuccal root, CTDBR: cortical thickness distobuccal root, CTPR: cortical thickness palatal root, CTF: cortical thickness furcation. Posterior teeth and maxillary sinus n = number of teeth; X = mean; SD = standard deviation. Different capital letters indicate significant differences in horizontal lines and different lowercase letters indicate significant differences in the vertical lines. p <0.05 (Kruskal-Wallis). CTMBR: cortical thickness mesiobuccal root, CTDBR: cortical thickness distobuccal root, CTPR: cortical thickness palatal root, CTF: cortical thickness furcation. n = number of teeth; X = mean; SD = standard deviation. Different capital letters indicate significant differences in horizontal lines and different lowercase letters indicate significant differences in the vertical lines. p <0.05 (Kruskal-Wallis). CTMBR: cortical thickness mesiobuccal root, CTDBR: cortical thickness distobuccal root, CTPR: cortical thickness palatal root, CTF: cortical thickness furcation. Table 5. The vertical relationship between maxillary molar roots and maxillary sinus floor Tooth n Type I Type II Type III Type IV Type V 16 150 39 (26.00%) 67 (44.67%) 12 (8.00%) 25 (16.57%) 7 (4.67%) 17 150 47 (31.33%) 43 (28.67%) 41 (27.33%) 12 (8.00%) 7 (4.67%) 26 150 27 (18.00%) 82 (54.67%) 10 (6.67%) 23 (15.33%) 8 (5.33%) 27 150 38 (25.33%) 52 (34.67%) 37 (24.67%) 15 (10.00%) 8 (5.33%) Total 600 151 (25.16%) 244 (40.67%) 100 (16.67%) 75 (12.50%) 30 (5.00%) n = number of teeth; Chi-square test (p =0.05). Other studies (14,20,21) showed different results from those observed in this study. Kwak et al. (14) analyzed the clinical and morphological features of the MS in a Korean population using CT. The shortest distance between the root apex and the MSF was observed in the distobuccal root of the second molar. Kilic et al. (21) also found a shorter distance between the root apex of distobuccal root of the second molar and the MS, after analyzing 87 right and 89 left MS of Table 6. The horizontal relationship between maxillary molar roots and MSF Tooth n Type 1H Type 2H Type 3H Type 4H Type 5H 16 150 7 (4.67%) 93 (62.00%) 9 (6.00%) 39 (26.00%) 2 (1.33%) 17 150 20 (13.33%) 78 (52.00%) 7 (4.67%) 44 (29.33%) 1 (0.67%) 26 150 7 (4.67%) 106 (70.67%) 8 (5.33%) 29 (19.33%) 0 (0.00%) 27 150 30 (20.00%) 78 (52.00%) 5 (3.33%) 36 (24.00%) 1 (0.67%) Total 600 64 (10.67%) 355 (59.17%) 29 (4.83%) 148 (24.67%) 4 (0.67%) n = number of teeth; Chi-square test (p = 0.00). 13 13 Braz Dent J 27(1) 2016 92 patients using CBCT. Yoshimine et al. Posterior teeth and maxillary sinus (20) analyzed the anatomical characteristics of premolars, molars and MS for planning of dental implant treatment in 30 patients (120 teeth). The shortest distance was found for the palatal root of the first molar. The present results showed that after the mesiobuccal root of the second molar, the root with greater proximity to the MS was the palatal root of the first molar (p>0.05). These results highlight the care that is required in case of periapical surgery involving this area. Maillet et al. (24) noted that odontogenic sinus disease is frequent in patients presenting apical periodontitis in the palatal root of first molar and mesiobuccal root of second molar. the region of the greatest cortical thickness closer to the apex (first premolars), and the region of shortest distance between the apex and the MSF coincided with the lowest cortical thickness closer to the apex (second molars). This could be an indication of a greater chance of spreading infections of dental origin to the MS in the second molars region. Further studies with specific criteria for sample selection are likely to corroborate this hypothesis. All first and second molars (600 teeth) had classified their vertical and horizontal relationship with the MSF. The vertical relationship Type II (MSF located below the level connecting the buccal and palatal root apices without an apical protrusion over the MSF) was the most common. However, contrary to the present results, the vertical relationship most observed in the studies developed by Kwak et al. (14) (Korea) and Kilic et al. (21) (Turkey) were type I (MSF located above the level connecting the buccal and palatal root apices). The difference between these studies may be attributed not only to methodological differences, but also to the characteristics of ethnicity, since the analyzed populations were diverse. A common feature to the I and II types is the absence of protrusion of the roots into the sinus floor. The absence of projection of the roots into the sinus was also observed with high prevalence in other studies (10,15,22). However, Jung and Cho (23) projection of the roots into the MS was the most commonly observed vertical relationship. C. Estrela et al. Posterior teeth and maxillary sinus Some of the studies that analyzed the relationship of the roots of the maxillary posterior teeth with the MS used as reference the presence of protrusion of the roots in MS and assigned negative values for the distance between the apex and MSF, considering the lower portion of the alveolar recess adjacent to the protrusion (21,23). In this study, it was considered that even with the protrusion of the roots into the MS the presence of cortical bone and the mucosa overlying the MSF must be investigated. So, this measure was considered as 0.00 mm when the apex had contact with the floor and also when there was a root protrusion into the MS. For further research, was measured the thickness of the cortical bone of the sinus floor in the region closest to the apex and in the furcation area. Kwak et al. (14) also made measurements of the MSF cortical bone thickness in nearby regions of the root apex and furcation area and pointed out that the thickness of the MSF cortical bone and its relationship to the adjacent teeth is important in determining the prognosis of the orthodontic tooth movement. According to these authors, this information can provide a more appropriate basis for controlling orthodontic tooth movement and forecasting the degree of tooth movement during orthodontic procedures. Yoshmine et al. (20), in a study of the topography of the upper posterior teeth and the MS using CBCT, considered important to know the thickness of the MS cortical bone, in the closest region to the buccal root apex of the posterior teeth. This knowledge helps in the planning of dental implants and obtaining successful aesthetic treatment. In this study, the greatest cortical thickness of the MS floor was found in the region of the first premolar (1.13±0.62 mm) and the smallest in the region of the first molars (0.82±0.28 mm). A similar result was obtained in the present study in which the greatest thickness of the cortical bone of the MS floor was observed in the region of the first premolar apex (1.28±0.42 mm) and the smallest in the region of the second molars apex (0.65±0.41 mm). Although there was no statistically significant difference for these results in this study, it is interesting to note that the region of greatest distance between the apex and the MSF coincided with C. Estrela et al. The type 2H horizontal relationship (alveolar recess of the MSF located between the buccal and palatal roots) was the most frequent. These results are in agreement with the of previous studies (14,23). The Type 1H (alveolar recess of the MSF located towards the buccal side rather than towards the buccal root), showed a higher frequency in the second molars than first molars, agreeing with Jung and Cho (23), and contrasting the results of Kwak et al. (14). The presence of vertical Type II and horizontal Type 2H relationships may contribute to a rapid dissemination of odontogenic infectious processes to the MS and still provide the alveolar extension post extraction, which may jeopardize future rehabilitation by dental implants. Considering the spread of infections originating from maxillary teeth, Obayashi et al. (4) observed that 65.7% of the analyzed cases showed alterations in alveolar cortical bone involving these teeth, the buccal cortical bone being the most affected. Despite these changes being most evident in the anterior teeth, 59% of the analyzed first molars and 42% of second molars showed infection spreading to these cortical plates. Thus, depending on the type of the horizontal relationship, a greater possibility of MS alterations could be present in cases of extensions towards the buccal and palatal sides (Types 1H, 3H, 5H), due to the spread of odontogenic infection. 14 Braz Dent J 27(1) 2016 JD. Characterization of successful root canal treatment. Braz Dent J 2014;25:3-11. JD. Characterization of successful root canal treatment. Braz Dent J 2014;25:3-11. Rational application of CBCT for the evaluation of different aspects involved in an infectious process between the posterior teeth and the MS, and analysis of periapical lesions has favored a better treatment plan, accurate diagnosis compared to conventional radiography and consequently a better therapeutic option (9,13,25). All relationships between teeth and adjacent anatomical structures that may serve to ease spreading an infection should be well studied, especially considering the different features between populations. It was verified (25) that maxillary posterior teeth with periapical radiolucent lesions had the highest frequency of sinus changes. A close spatial relationship between periapical lesion and sinus resulted most frequently in sinus abnormalities. 6. Maloney PL, Doku HC. Maxillary sinusitis of odontogenic origin. J Can Dent Assoc 1968;34:591–603. 7. Brook I. Sinusits of odontogenic origin. Otolaryngol Head Neck Surg 2006; 135:349-355. 8. Oberli K, Bornstein MM, von Arx T. Resumo Avaliou-se a relação anatômica entre dentes posteriores e o soalho do seio maxilar (SSM) por meio da tomografia computadorizada de feixe cônico (TCFC) em 202 exames. A distância entre os ápices radiculares e o SSM, bem como a espessura do osso cortical do SSM próximo dos ápices radiculares e áreas de bifurcação foram medidas. As relações verticais e horizontais do SSM com as raízes dos molares foram classificados em categorias adaptadas a partir dos critérios propostos pelo Kwak et al. (14). A menor distância entre o SSM e os ápices dentários foi observada na raiz mesiovestibular do segundo molar (0,36±1,17 mm) e na raiz palatina do primeiro molar (0,45±1,10 mm), e a maior na raiz vestibular do primeiro pré-molar (5,47±4.43 mm). Diferenças significantes foram observadas entre a distância do SSM e os ápices dentários de primeiros e segundos pré-molares unirradiculares. A espessura da cortical óssea variou de 0,65±0,41 mm na região da raiz mesiovestibular do segundo molar a 1,28±0,42 na raiz vestibular do primeiro pré-molar. As relações vertical e horizontal mais prevalentes foram do tipo II e 2H, respectivamente. As raízes dos molares superiores apresentaram maior proximidade com o SSM. A espessura da cortical óssea do SSM nas regiões mais próximas dos ápices e área de furca foi similar apenas para os pré-molares. 15. Sharan A, Madjar D. Correlation between maxillary sinus floor topography and related root position of posterior teeth using panoramic and cross-sectional computed tomography imaging. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2006;102:375-381. 16. Koymen R, Gocmen-Mas N, Karacayli U, Ortakoglu K, Ozen T, Yazici AC. Anatomic evaluation of maxillary sinus septa: surgery and radiology. Clinical Anatomy 2009; 22:563-570. 17. Park Y-B, Jeon H-S, Shim J-S. Analysis of the anatomy of the maxillary sinus septum using 3-dimensional computed tomography. J Oral Maxillofac Surg 2011;69:1070-1078. 18. Low KMT, Dula K, Bürgin W, von Arx T. Comparison of periapical radiography and limited cone-beam tomography in posterior maxillary teeth referred for apical surgery. J Endod 2008;34:557–562. 19. Lemagner F, Maret D, Peters OA, Arias A, Coudrais E, Georgelin-Gurgel M. Prevalence of apical bone defects and evaluation of associated factors detected with cone-beam computed tomographic images. J Endod 2015;41:1043-1047. 20. Yoshimine S, Nishihara K, Nozoe E, Yoshimine M, Nakamura N. Topographic analysis of maxillary premolars and molars and maxillary sinus using cone beam computed tomography. Implant Dentistry 2012;21:528-535. C. Estrela et al. Periapical surgery and the maxillary sinus; radiographic parameters for clinical outcome. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2007;103:848-853. 9. Estrela C, Porto OCL, Costa NL, Garrote MS, Decurcio DA, Bueno MR, et al.. Large reactional osteogenesis in maxillary sinus associated with secondary root canal infection detected using cone-beam computed tomography. J Endod 2015;41:2068–2078. 10. Eberhardt JA, Torabinejad M, Christiansen E. A computed tomographic study of the distances between the maxillary sinus floor and the apices of the maxillary posterior teeth. Oral Surg Oral Med Oral Pathol 1992;73:345-346. 11. Mozzo P, Procacci C, Taccoci A, Martini PT, Andreis IA. A new volumetric CT machine for dental imaging based on the cone-beam technique: preliminary results. Eur Radiol 1998;8:1558-1564. In conclusion, the roots of the maxillary molars showed greater proximity with the MS when compared with premolars; the thickness of the cortical bone of the MS floor in the region closest to the apex and furcation area was found to be similar only for premolars. 12. Arai Y, Tammisalo E, Iwai K, Hashimoto K, Shinoda K. Development of a compact computed tomographic apparatus for dental use. Dentomaxillofac Radiol 1999;28:245-248. 13. Estrela C, Bueno MR, Leles CR, Azevedo B, Azevedo JR. Accuracy of cone beam computed tomography and panoramic and periapical radiography for detection of apical periodontitis. J Endod 2008;34: 273-279. 14. Kwak HH, Park HD, Yoon HR, Kang MK, Koh KS, Kim HJ. Topographic anatomy of the inferior wall of the maxillary sinus in Koreans. Int J Oral Maxillofac Surg 2004;33:382-388. Acknowledgements 21. Kilic C, Kamburoglu K, Yuksel S P, Ozen T. An assessment of the relationship between the maxillary posterior teeth root tips using dental cone-beam computerized tomography. Eur J Dent 2010;4:462-467. This study was supported in part by grants from the National Council for Scientific and Technological Development (CNPq - 306394/2011-1 to C.E.). 22. Pagin O, Centurion BS, Rubira-Bullen IRF, Capelozza ALA. Maxillary sinus and posterior teeth: accessing close relationship by cone-beam computed tomographic scanning in a Brazilian Population. J Endod 2013;39:748-751. References 1. Haumman CHJ, Chandler NP, Tong DC. Endodontic implications of the maxillary sinus: a review. Int Endod J 2002;35:127-141. 1. Haumman CHJ, Chandler NP, Tong DC. Endodontic implications of the maxillary sinus: a review. Int Endod J 2002;35:127-141. 23. Jung Y-H, Cho B-H. Assessment of the relationship between the maxillary molars and adjacent structures using cone beam computed tomography. Imaging Sci Dent 2012;42:219-224. 2. Maillet M, Bowles WR, McClanahan SL, John MT, Ashmad M. Cone- beam computed tomography evaluation of maxillary sinusitis. J Endod 2011;37:753-757. 2. Maillet M, Bowles WR, McClanahan SL, John MT, Ashmad M. Cone- beam computed tomography evaluation of maxillary sinusitis. J Endod 2011;37:753-757. 24. Maillet M, Bowles WR, McClanahan SL, John MT, Ashmad M. Cone- beam computed tomography evaluation of maxillary sinusitis. J Endod 2011;37:753-757. 3. Nair UP, Nair MK. Maxillary sinusitis of odontogenic origin: cone-beam volumetric computerized tomography-aided diagnosis. Oral Sur Oral Med Oral Pathol Oral Radiol Endod 2010;110:e53-e57. 25. Nunes CABCM, Guedes OA, Alencar AHG, Peters OA, Estrela CRA, Estrela C. Evaluation of periapical lesions and their association with maxillary sinus abnormalities on cone-beam computed tomographic images. J Endod 2016;42:42-46. 4. Obayashi N, Ariji Y, Goto M, Izumi M, Naitoh M, Kurita K, et al.. Spread of odontogenic infection originating in the maxillary teeth: Computerized tomographic assessment. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2004;98:223-231. Received August 2, 2015 Accepted December 10, 2015 Received August 2, 2015 Accepted December 10, 2015 5. Estrela C, Holland R, Estrela CRA, Alencar AHG, Sousa-Neto MD, Pecora 15
https://openalex.org/W2534421350	https://aacr.figshare.com/articles/journal_contribution/Supplemental_Figure_Legends_from_Benefits_of_Multifaceted_Chemopreventives_in_the_Suppression_of_the_Oral_Squamous_Cell_Carcinoma_OSCC_Tumorigenic_Phenotype/22533659/1/files/39996920.pdf	English	null	Benefits of Multifaceted Chemopreventives in the Suppression of the Oral Squamous Cell Carcinoma (OSCC) Tumorigenic Phenotype	Cancer prevention research	2,017	cc-by	1,417	Supplemental Figure 1. Supplemental Figure 1.A. Clinical stage of parent OSCC tumors and corresponding cell line characterization. Immunofluorescent staining revealed tumor derived cell lines uniformly demonstrate cytokeratin with variable co-expression of the intermediate filament marker vimentin. Cytokeratin and vimentin coexpression supports cells have undergone the epithelial to mesenchymal transition (EMT); findings consistent with the perineural, lymphovascular and lymph node invasion noted in tumor tissues. Supplemental Figure 1.B. pSTAT3 and pEGFR expression in normal and corresponding JSCC tumor tissues. Clinically and histologically normal oral mucosa demonstrates modest pSTAT3 nuclear staining that is restricted to basal layer keratinocytes with no pEGFR expression. In contrast, OSCC tumor tissues show pSTAT3 expression, often observed at the periphery of the tumor islands. While JSCC1 and JSCC2 tumor tissues exhibit robust membranous and cytosolic pEGFR staining, JSCC3 levels are more modest. (100X image scale larger photo, 200X image scale inset). Supplemental Figure 2. Figure 2.A. Effects of treatment on DNA binding in specific cell lines. While STAT3-DNA binding was significantly inhibited in the JSCC2 cells (n=5) by both 4-HPR+ TOC and triple treatment, only the triple treatment suppressed STAT3-DNA binding in the JSCC1 cells (n=3). Kruskal Wallis with Dunn’s post-hoc comparion. Supplemental Figure 2.B. Effects of treatment on sIL-6R in specific cell lines Combination of 4-HPR+2-ME+TOC significantly inhibits sIL-6R production in the three cell lines with constitutive STAT3 activation (JSCC1, JSCC2 and 2095sc). Treatment group legend: 1) VCtrl (vehicle control 0.01% DMSO), 2) 2-methoxyestradiol (2-ME, 2.5 µM), 3) fenretinide (4-HPR, 5 µM), 4) tocilizumab (TOC, 1 µg/ml), 2 + T (2.5 µM 2-ME + 1 µg/ml TOC), 4 + T (5 µM 4- HPR+ 1 µg/ml TOC), 2 + 4 (2.5 µM 2-ME+5 µM 4-HPR), Triple (2.5 µM 2-ME+5 µM 4- HPR+1 µg/ml TOC). Kruskal Wallis with Dunn’s post-hoc comparion. While all cell lines release sIL6R, inter-line, approximately 2.8 fold differences are noted. The JSCC3 cells, which do not demonstrate constitutive STAT3 activation, revealed highest sIL-6R basal and post-stimulated (10 ng/mL of IL-6 and 5 ng/mL of TGF-α) release. Treatment group legend: 1) VCtrl (vehicle control 0.01% DMSO), 2) 2-methoxyestradiol (2-ME, 2.5 µM), 3) fenretinide (4-HPR, 5 µM), 4) tocilizumab (TOC, 1 µg/ml), 2 + T (2.5 µM 2-ME + 1 µg/ml TOC), 4 + T (5 µM 4-HPR+ 1 µg/ml TOC), 2 + 4 (2.5 µM 2-ME+5 µM 4-HPR), Triple (2.5 µM 2-ME+5 µM 4-HPR+1 µg/ml TOC). Kruskal Wallis with Dunn’s post-hoc comparion. Supplemental Figure 2.C. Supplemental Figure 1. Presence of the BCR-ABL fusion oncoprotein in OSCC cells. Cells were cultured in sera-free optimal media for 24 hours, followed by harvest for Western 1 immunoblotting. The K562 cells, which are derived from pleural effusion of a patient with chronic myelogenous leukemia in blast crisis, serve as the BCR-ABL positive controls. The 2095sc cells also demonstrate the presence of BCR-ABL, albeit at lower levels that the CML control cells. The JSCC1 and JSCC3 cells demonstrate no expression, while BCR-ABL is equivocal in the JSCC2 cells. Supplemental Figure 3. 24-hour sera deprived OSCC cell lines were challenged for 15 minutes with 50 ng/ml TNF-α, followed by harvest to assess binding of the NF-κB subunits p50 and p65 to cognate κB DNA sites. One hour prior to stimulation (50 ng/ml TNF-α, added to all groups except vehicle control), 24-h sera-deprived cells were treated with: 1) VCtrl and Stim (vehicle control 0.01% DMSO), 2) 2-methoxyestradiol (2-ME, 2.5 µM), 3) fenretinide (4-HPR, 5 µM), 4) tocilizumab (TOC, 1 µg/ml), 2 + 4 (2.5 µM 2-ME+5 µM 4-HPR), 2 + T (2.5 µM 2-ME + 1 µg/ml TOC), 4 + T (5 µM 4-HPR+ 1 µg/ml TOC), Triple (2.5 µM 2-ME+5 µM 4-HPR+1 µg/ml TOC). The 2095sc cells were refractory to TNF-α stimulation; findings that likely reflect high constitutive NF-κB activation. Treatment effects were cell line dependent. Only the 2095sc and JSCC2 lines showed modest therapeutic effects as seen in reduced DNA binding of the gene activating p65 subunit. Supplemental Figure 4. A. c-Src (cellular Src) is one of a class of non-receptor tyrosine kinase which also includes: Yes, Fyn, Fgr, Yrk, Lyn, Blk, Hck and Lck. The protein can adopt one of two distinct conformations. In the “inactive” form where SH2, SH3 and the kinase regions are “closed” configuration as typified by 2SRC (A). In the “active” form the SH2 and SH3 regions “unlatch” from the kinase and adopt an extended conformation. Both conformations were analyzed with a common set of ligands for binding at the “ATP” binding site. Again, after deleting water and ligands, the proteins were optimized in Yasara [S1] using the default minimization algorithm. All ligands were constructed in Spartan 10 [S2] and minimized using MMFF [S3]. The optimized protein structure and ligands were docked using AutoDock Vina [27] using an exhaustiveness of 100. Each calculation was repeated three times to ensure a thorough exploration of the binding site. Supplemental Figure 1. Previous results have shown that flexible amino acid side chains provide for better results [21] therefore the side chains for amino acids: 273, 275, 277, 278, 280- 283, 291, 295, 310, 339-343, 345 and 404 were made flexible to ensure a more realistic binding mode for all calculations. B. Abl can adopt one of two conformations, the inactive DFG-in and active DGF-out as depicted in Figure B. DFG-in and out conformations were analyzed with the same common set of ligands used for c-Src for binding at the “ATP” binding site. Again, after deleting water and ligands, the proteins were optimized in Yasara [S1] using the default minimization algorithm. All ligands were constructed in Spartan 10 [S2] and minimized using MMFF [S3]. The optimized protein structure and ligands were docked using AutoDock Vina [27] using an exhaustiveness of 100. Each calculation was repeated three times to ensure a thorough exploration of the binding site. Previous results have shown that flexible amino acid side chains provide for better results [21] therefore the side chains for amino acids: 248, 253, 256, 271, 285, 2 286, 289, 290, 293, 299, 313, 315-318, 359-362, 370, 380-382 in both 1M52 and 1IEP were made flexible to ensure a more realistic binding mode for all calculations. 286, 289, 290, 293, 299, 313, 315-318, 359-362, 370, 380-382 in both 1M52 and 1IEP were made flexible to ensure a more realistic binding mode for all calculations. S1. Krieger E, Koraimann G, Vriend G. Increasing the precision of comparative models with YASARA NOVA—a self-parameterizing force field. Proteins 2002;47: 393–402 S2. Shao Y, Molnar LF, Jung Y, Kussmann J, Ochsenfeld C, Brown ST, et al. Advances in methods and algorithms in a modern quantum chemistry program package. Phys Chem Chem Phys 2006;8:3172-91. S3. Halgren TA. Merck molecular force field. I. Basis, form, scope, parameterization, and performance of MMFF94. J Comp Chem 1996;17:490-519. S4. Rahuel J, Garcia-Echeverria C, Furet P, Strauss A, Caravatti G, Fretz H, et al. Structural basis for the high affinity of amino-aromatic SH2 phosphopeptide ligands. J Mol Biol 1998;279:1013-1022. S5. O’Hare T, Pollock R, Stoffregen EP, Keats JA, Abdullah OM, Moseson EM, et al. Inhibition of wild-type and mutant Bcr-Abl by AP23464, a potent ATP-based oncogenic protein kinase inhibitor: implications for CML. Blood 2004;104:2532-9. S6. Green TP, Fennell M, Whittaker R, Curwen J, Jacobs V, Allen J, et al. Supplemental Figure 1. Preclinical anticancer activity of the potent, oral Src inhibitor AZD0530. Mol Oncol 2009;3:248-61. S7. Daud AI, Krishnamurthi SS, Saleh MN, Gitlitz BJ, Borad MJ, Gold PJ, et al. Phase I study of bosutinib, a src/abl tyrosine kinase inhibitor, administered to patients with advanced solid tumors. Clin Cancer Res 2012;18:1092-100. S8. Das J, Chen P, Norris D, Padmanabha R, Lin J, Moquin RV, et al. 2-aminothiazole as a novel kinase inhibitor template. Structure-activity relationship studies toward the discovery of N-(2- chloro-6-methylphenyl)-2-[[6-[4-(2-hydroxyethyl)-1-piperazinyl)]-2-methyl-4- pyrimidinyl]amino)]-1,3-thiazole-5-carboxamide (dasatinib, BMS-354825) as a potent pan-Src kinase inhibitor. J Med Chem 2006;49:6819-32. S9. Weisberg E, Manley PW, Breitenstein W, Brüggen J, Cowan-Jacob SW, Ray A, et al. Characterization of AMN107, a selective inhibitor of native and mutant Bcr-Abl. Cancer Cell 2005;7:129-41. S10. Nagar B, Bornmann WG, Pellicena P, Schindler T, Veach DR, Miller WT, et al. Crystal structures of the kinase domain of c-Abl in complex with the small molecule inhibitors PD173955 and imatinib (STI-571). Cancer Res 2002;62:4236-43. S11. Duan Y, Chen L, Chen Y, Fan XG. c-Src binds to the cancer drug Ruxolitinib with an active conformation. PLoS One 2014;9:e106225. S12. Cowan-Jacob SW, Fendrich G, Manley PW, Jahnke W, Fabbro D, Liebetanz J, et al. The crystal structure of a c-Src complex in an active conformation suggests possible steps in c-Src activation. Structure 2005;13:861-71. 3 3
https://openalex.org/W2101900155	https://npg.copernicus.org/articles/15/773/2008/npg-15-773-2008.pdf	English	null	Mitigation of energetic electrons in the magnetosphere by amplified whistler wave under double cyclotron resonances	Nonlinear processes in geophysics	2,008	cc-by	8,293	Mitigation of energetic electrons in the magnetosphere by ampliﬁed whistler wave under double cyclotron resonances S. P. Kuo Department of Electrical and Computer Engineering, Polytechnic Institute of New York University, 6 MetroTech Center, Brooklyn, NY 11201, USA Received: 9 July 2008 – Revised: 5 September 2008 – Accepted: 5 September 2008 – Published: 22 October 2008 ways which can mitigate unexpected radiation enhancement to keep satellite systems less vulnerable. Abstract. An optimal approach reducing the population of MeV electrons in the magnetosphere is presented. Under a double resonance condition, whistler wave is simultaneously in cyclotron resonance with keV and MeV electrons. The injected whistler waves is ﬁrst ampliﬁed by the background keV electrons via loss-cone negative mass instability to be- come effective in precipitating MeV electrons via cyclotron resonance elevated chaotic scattering. The numerical results show that a small amplitude whistler wave can be ampliﬁed by more than 25 dB. The ampliﬁcation factor reduces only about 10 dB with a 30 dB increase of the initial wave inten- sity. Use of an ampliﬁed whistler wave to scatter 1.5 MeV electrons from an initial pitch angle of 86.5◦to a pitch angle <50◦is demonstrated. The ratio of the required wave mag- netic ﬁeld to the background magnetic ﬁeld is calculated to be about 8×10−4. Whistler waves can be ducted in an L-shell of the magne- tosphere to continuously interact with the energetic electrons trapped in the same L-shell. The motions of energetic elec- trons are adversely affected by the wave ﬁelds which scatter some of them into loss cones (Helliwell et al., 1973). In- duced electron precipitation (Voss et al., 1984; Arnoldy and Kintner, 1989; Imhof et al., 1994; Pradipta et al., 2007) by whistler waves has been observed. The Doppler shifted elec- tron cyclotron resonance interaction (Kennel and Petschek, 1966; Villalon and Burke, 1991; Albert, 2000) has been sug- gested to be a likely electron precipitation mechanism. The numerical results show that the electron cyclotron resonance interaction can diffuse energetic electrons, with their initial pitch angles close to the loss cone angle, into the loss cone, via small angle scattering process (Albert, 2000). However, the number of electrons resonant with the wave at a given frequency is small. Moreover, the resonance condition is anisotropic, which makes it difﬁcult to explain the observa- tion of precipitation events occurring simultaneously at ge- omagnetic conjugate regions due to a single lightning ﬂash (Burgess and Inan, 1990). Nonlin. Processes Geophys., 15, 773–782, 2008 www.nonlin-processes-geophys.net/15/773/2008/ © Author(s) 2008. This work is distributed under the Creative Commons Attribution 3.0 License. Nonlin. Processes Geophys., 15, 773–782, 2008 www.nonlin-processes-geophys.net/15/773/2008/ © Author(s) 2008. This work is distributed under the Creative Commons Attribution 3.0 License. Nonlin. Processes Geophys., 15, 773–782, 2008 www.nonlin-processes-geophys.net/15/773/2008/ © Author(s) 2008. This work is distributed under the Creative Commons Attribution 3.0 License. Nonlinear Processes in Geophysics Mitigation of energetic electrons in the magnetosphere by ampliﬁed whistler wave under double cyclotron resonances S. P. Kuo Department of Electrical and Computer Engineering, Polytechnic Institute of New York University, 6 MetroTech Center, Brooklyn, NY 11201, USA Received: 9 July 2008 – Revised: 5 September 2008 – Accepted: 5 September 2008 – Published: 22 October 2008 Published by Copernicus Publications on behalf of the European Geosciences Union and the American Geophysical Union. 3 Ampliﬁcation of whistler waves We are interested in wave ampliﬁcation in time; moreover, the ampliﬁcation mainly proceeds in the region near the mag- netic equator, inferred by the common static source region of chorus to be near magnetic equator deduced from corre- lated chorus elements of different frequency/time character- istics measured by Cluster Wideband Data (WBD) receiver (Breneman et al., 2007). Thus, the formulation can be sim- pliﬁed by assuming a local uniform magnetic ﬁeld B0=B0ˆz. The electron plasma in the magnetosphere consists of three components: 1) cold background (γ =γ0∼1), 2) energetic (in keV range) electrons (γ =γ1), and 3) very energetic (in MeV range) electrons (γ =γ2). The background electron plasma is nonrelativistic (γ ∼1) and determines the propagation char- acteristics of the whistler wave, which has the dispersion re- lation In this paper, the theoretical basis of this optimal approach is presented and the feasibility of the approach is examined. In Sect. 2, the double cyclotron resonance mechanism is ex- plained. In Sect. 3, the formulation of the nonlinear instabil- ity theory is presented; a ﬁfth order differential equation gov- erning the temporal evolution of the whistler ﬁeld amplitude is derived. Numerical results are also presented. Section 4 devotes to the formulation and analysis of chaotic scattering. An example of chaotic scattering under double cyclotron res- onances is given in Sect. 5. Summary is presented in Sect. 6. ω = 0c2k2/ω2 pb (2) (2) 1 Introduction Chorus is basically discrete VLF emissions; on the other hand, the amplitude of the ampliﬁed whistler wave oscillates continuously in time. 1982; Helliwell, 1983) that trapped energetic (keV) elec- trons in the magnetosphere can signiﬁcantly amplify whistler waves. The anisotropic relativistic plasma can also excite electromagnetic instability through the electron cyclotron resonance interaction (Tsurutani and Smith, 1974; Nunn et al., 1997; Trakhtengerts, 1999; Trakhtengerts et al., 2004). VLF wave generation by energetic electrons is evidenced by the natural event of chorus (Sazhin and Hayakawa, 1992) oc- curring in the inner magnetosphere and by the appearance of large amplitude whistler-mode waves in radiation belts (Cat- tell et al., 2008). The experimental observations also indicate some intrinsic differences between emission and ampliﬁca- tion processes. Chorus is basically discrete VLF emissions; on the other hand, the amplitude of the ampliﬁed whistler wave oscillates continuously in time. sumed; ω<0 for whistler waves; ω and 0=eB0/m are the wave frequency and the nonrelativistic electron cyclotron fre- quency. For a small γ , i.e., ω<0/γ , the resonant electrons are moving oppositely to the wave propagation direction. Because of the γ dependence, this condition leads to a quadratic equation for Pz as AP 2 z + 2BPz + C = 0 where A=(1–ω2/k2c2), B=m0/k, and C=(m/k)2 [2 0−ω2(1+P 2 ⊥/m2c2)]. This quadratic equation has two real solutions Pz=[−B±(B2–AC)1/2]/A, subject to the condition B2≥AC. The double solutions suggest that the wave can be simultaneously resonant with two dif- ferent groups of electrons. The coefﬁcients A and C of the quadratic equation are positive because ω/kc<1 and 0/γ1,2>ω are considered; thus both Pz are negative, i.e., the two groups of electrons, which can resonantly interact with the wave, move opposite to the propagation direction of the wave. The relativistic cyclotron resonance condition is a quadratic equation in the electron momentum, thus, there ex- ists a double resonance situation (Kuo et al., 2007), namely, a whistler wave is simultaneously in cyclotron resonance with the keV electrons and with the MeV electrons. This sug- gests an optimal approach, which applies the chaotic scat- tering process under a double resonance condition, for the control of the population of MeV electrons trapped in the magnetosphere. This approach ﬁrst uses keV electrons (hav- ing a loss-cone velocity distribution) to energize the incident whistler waves, which become more effective to precipitate MeV electrons into loss cones, via cyclotron resonance en- hanced chaotic scattering. 1 Introduction In the magnetosphere, energetic electrons are trapped by the Earth’s magnetic dipole ﬁeld to undergo a bouncing motion about the geomagnetic equator. Energetic electrons in the MeV range have a strong impact on passing satellite systems. Satellites are designed to survive a certain amount of radia- tion (ionizing) dose accumulated during their lifetimes. Un- expected enhancement of the radiation ﬂuxes caused by, for example, very strong solar storms, will signiﬁcantly increase the total radiation dose to the satellites. Consequently, the ra- diation damage on active electronics and detectors of satellite systems will accumulate faster than that designed for. As the damage exceeds a threshold level, satellite systems become incapable of performing their mission. It is essential to ﬁnd The trajectories of trapped energetic electrons in the pres- ence of whistler waves can become chaotic, subject to that the whistler wave intensity (Faith et al., 1996, 1997a, b; Kuo et al., 2004) and/or the initial electron energy (Khazanov et al., 2008) exceed threshold levels. Once chaos occurs, many of electrons can wander into both loss cones. This chaotic (large angle) scattering process precipitates electrons to both loss cones simultaneously. Cyclotron resonance can reduce the threshold wave ﬁeld for the commencement of chaos in the electron trajectories. Magnetospheric energetic electrons have an anisotropic velocity distribution, which is potentially unstable to elec- tromagnetic waves (Tsytovich and Stenﬂo, 1983). Indeed, it has been observed (Helliwell et al., 1980; Helliwell and Inan, Correspondence to: S. P. Kuo (skuo@duke.poly.edu) Correspondence to: S. P. Kuo (skuo@duke.poly.edu) Published by Copernicus Publications on behalf of the European Geosciences Union and the American Geophysical Union. Published by Copernicus Publications on behalf of the European Geosciences Union and the Ame 774 S. P. Kuo: Whistler wave-electron interaction in the magnetosphere 1982; Helliwell, 1983) that trapped energetic (keV) elec- trons in the magnetosphere can signiﬁcantly amplify whistler waves. The anisotropic relativistic plasma can also excite electromagnetic instability through the electron cyclotron resonance interaction (Tsurutani and Smith, 1974; Nunn et al., 1997; Trakhtengerts, 1999; Trakhtengerts et al., 2004). VLF wave generation by energetic electrons is evidenced by the natural event of chorus (Sazhin and Hayakawa, 1992) oc- curring in the inner magnetosphere and by the appearance of large amplitude whistler-mode waves in radiation belts (Cat- tell et al., 2008). The experimental observations also indicate some intrinsic differences between emission and ampliﬁca- tion processes. S. P. Kuo: Whistler wave-electron interaction in the magnetosphere The normalized equation has the form The collective result in electron-wave resonance inter- action can be demonstrated through a phase average on 1ω=1ω0−(k2c2/ω−ω0)(γ1−γ10)/γ1, where 1ω0=ω−ω0 is the initial mismatch frequency and ω0=0/γ10−kvz0. In carrying out phase average, energy conservation has to be satisﬁed. The energy conservation equation is given by [d3 ξ −48Xfcd2 ξ +(1+bX2+48fsX)dξ]X =16[1−g(X2−X2 0)]fc [d3 ξ −48Xfcd2 ξ +(1+bX2+48fsX)dξ]X =16[1−g(X2−X2 0)]fc (8) (8) where X0=X(0); fc= ∫ξ 0 X(ξ′)cosϕ(ξ, ξ′)dξ′ and fs=–∫ξ 0 X(ξ′)sinϕ(ξ, ξ′)dξ′; ϕ(ξ, ξ′)= ∫ξ ξ′ {1−2g[X2(ξ′′)–X2 0]}dξ′′; b=[41ω2 pεk2c2/γ10(εr−1) \|⟨1ω0⟩\|3ω][(1+vz0ω/kc2)+3(ω0/γ10k2c2)–(9/4)α2 0 (ω0/γ10k2c2)2]∼=192[(εr+1)/(εr–1)](ωγ10/α2 00); b and g are constant coefﬁcients; Eq. (8) is subjected to the initial conditions: X(0)=X0, dξX(0)=√3X0, d2 ξ X(0)=3X0. where X0=X(0); fc= ∫ξ 0 X(ξ′)cosϕ(ξ, ξ′)dξ′ and fs=–∫ξ 0 X(ξ′)sinϕ(ξ, ξ′)dξ′; ϕ(ξ, ξ′)= ∫ξ ξ′ {1−2g[X2(ξ′′)–X2 0]}dξ′′; b=[41ω2 pεk2c2/γ10(εr−1) \|⟨1ω0⟩\|3ω][(1+vz0ω/kc2)+3(ω0/γ10k2c2)–(9/4)α2 0 (ω0/γ10k2c2)2]∼=192[(εr+1)/(εr–1)](ωγ10/α2 00); b and g are constant coefﬁcients; Eq. (8) is subjected to the initial conditions: X(0)=X0, dξX(0)=√3X0, d2 ξ X(0)=3X0. where X0=X(0); fc= ∫ξ 0 X(ξ′)cosϕ(ξ, ξ′)dξ′ and fs=–∫ξ 0 X(ξ′)sinϕ(ξ, ξ′)dξ′; ϕ(ξ, ξ′)= ∫ξ ξ′ {1−2g[X2(ξ′′)–X2 0]}dξ′′; b=[41ω2 pεk2c2/γ10(εr−1) \|⟨1ω0⟩\|3ω][(1+vz0ω/kc2)+3(ω0/γ10k2c2)–(9/4)α2 0 (ω0/γ10k2c2)2]∼=192[(εr+1)/(εr–1)](ωγ10/α2 00); b and g are constant coefﬁcients; Eq. (8) is subjected to the initial conditions: X(0)=X0, dξX(0)=√3X0, d2 ξ X(0)=3X0. 1nεmc2d⟨γ1⟩ε/dt + ε0[(1 + εr)/2]dE2 0(t)/dt = 0 (6) where ⟨⟩ε represents an average over the initial random phases of those energetic electrons, which are involved in (near) resonant interaction with the wave; 1nε=N1– N2∼=j−1/2nε[j−1/2f ′ ε⊥(P⊥1)fεz(Pz1)−fε⊥(P⊥1)f ′ εz(Pz1)] ×(2γ1m1ω0/k)3; d⟨γ1⟩ε2/dt=−d⟨γ1⟩ε1/dt (i.e., ⟨cosφ⟩ε2∼=−⟨cosφ⟩ε1 is assumed); εr=1+ω2 pb/ω0 is the dielectric function of background plasma responding to the whistler wave and 1nε (≪nε≪nb) is the net density of energetic electrons transferring energy to the wave through the resonant interaction with the wave. An integration of Eq. (6) leads to 0 b and g are constant coefﬁcients; Eq. (8) is subjected to the initial conditions: X(0)=X0, dξX(0)=√3X0, d2 ξ X(0)=3X0. ( ) ξ ( ) √ ξ ( ) The background parameters give b=1440 and g=2×107 (i.e., εr∼4 and\|⟨1ω0⟩\|/ω∼5×10−4); setting X0=3.58×10−4, Eq. (8) is solved by an ODE solver. The re- sult is presented in Fig. 1, showing the temporal evolution of the ﬁeld amplitude E0(t). The dB-scale plot in Fig. 1 is for a direct comparison with the early Siple experimental result (Helliwell et al., 1980; Helliwell and Inan, 1982; Helliwell, 1983). S. P. Kuo: Whistler wave-electron interaction in the magnetosphere 775 The distribution in Pz is a Maxwellian having the form The distribution in Pz is a Maxwellian having the form fεz(Pz) = π−1/21P −1 jε exp(−P 2 z /1P 2 jε) (4) and the distribution in P⊥has a loss cone form fε⊥(P⊥)=(2/j!)(1Pjε)−(2j+2)P 2j+1 ⊥ exp(−P 2 ⊥/1P 2 jε) (5) Fig. 1. Temporal evolution of the amplitude of a whistler wave propagating in the magnetosphere. (4) fε⊥(P⊥)=(2/j!)(1Pjε)−(2j+2)P 2j+1 ⊥ exp(−P 2 ⊥/1P 2 jε) (5) In the following, an instability process to amplify whistler waves by keV electrons having a loss cone momentum dis- tribution in the magnetosphere is studied. The relativistic ef- fect, through the Doppler shifted cyclotron resonance (Eq. 1), provides essential nonlinearity for the phase bunching (Kuo and Cheo, 1985) of those electrons in near cyclotron reso- nant interaction with the wave. The bunched electrons then excite the loss-cone negative mass instability for the wave ampliﬁcation (Kuo and Lee, 1986). Speciﬁcally, the reso- nant interaction of a whistler wave with a particular group of electrons in the magnetosphere will be formulated. Since these electrons are practically collisionless, a single electron system will be considered to ﬁrst derive the resonant trajec- tory equations for a single electron in the wave ﬁelds. This derivation is presented in Appendix A. The results are then averaged over the electron’s random phase angle (with re- spect to the wave ﬁeld) to obtain the collective effect for wave ampliﬁcation, which is described physically in Appendix B. Fig. 1. Temporal evolution of the amplitude of a whistler wave propagating in the magnetosphere. Eq. (7) indicates that the mismatch frequency \|⟨1ω⟩\| de- creases as the wave amplitude E0 increases, a positive feed- back for energy transfer from resonant electrons to the wave. The governing Eq. (C11) for the self-consistent ﬁeld amplitude E0(t) is now analyzed numerically. We ﬁrst normalize Eq. (C11) to a dimensionless form by intro- ducing X=[ε0(εr–1)\|⟨1ω0⟩\| /4ωγ101nεmc2]1/2E0(t), ξ=\|⟨1ω0⟩\|t, and g=(nε/1nε)(εr+1)ωω2 pb/0(εr–1) ⟨1ω0⟩2=(nε/1nε)(εr+1)(ω/⟨1ω0⟩)2. The normalized equation has the form The governing Eq. (C11) for the self-consistent ﬁeld amplitude E0(t) is now analyzed numerically. We ﬁrst normalize Eq. (C11) to a dimensionless form by intro- ducing X=[ε0(εr–1)\|⟨1ω0⟩\| /4ωγ101nεmc2]1/2E0(t), ξ=\|⟨1ω0⟩\|t, and g=(nε/1nε)(εr+1)ωω2 pb/0(εr–1) pb ⟨1ω0⟩2=(nε/1nε)(εr+1)(ω/⟨1ω0⟩)2. The normalized equation has the form pb ⟨1ω0⟩2=(nε/1nε)(εr+1)(ω/⟨1ω0⟩)2. 2 Relativistic effect for double cyclotron resonances where ωpb=(4πnbe2/m)1/2; nb is the background cold elec- tron density. Cyclotron resonance is an effective process to enhance the interaction between wave and charge particles and is essen- tial to whistler wave ampliﬁcation. Thus the possibility of a double cyclotron resonance situation, under which the wave is simultaneously in cyclotron resonances with keV electrons for ampliﬁcation and with MeV electrons to instigate precip- itation, is explored in the following. The Doppler shifted cyclotron resonance condition in the relativistic case is given by Energetic electrons in keV range are weakly relativistic (e.g., γ1∼1.1 for 50 keV electrons) and have considerable density nε to amplify whistler waves. These electrons have a loss cone distribution given by fε(P⊥, Pz) = 2πP⊥Fε(P⊥, Pz) = nε(2π−1/2/j!)(1Pjε)−(2j+3)P 2j+1 ⊥ exp[−(P 2 ⊥+ P 2 z )/1P 2 jε] = nεfε⊥(P⊥)fεz(Pz) (3) ω = 0/γ + kPz/γ m (1) (1) (3) where 1Pjε=[mTeε/(1/2+j/3)]1/2; Teε≫Teb; subscripts ε and b stand for “energetic” and “background”, respectively. where γ =(1+P 2 ⊥/m2c2+P 2 z /m2c2)1/2 is the relativistic fac- tor of the electron, P⊥=γ mv⊥, Pz=γ mvz, and k=ˆzk is as- where γ =(1+P 2 ⊥/m2c2+P 2 z /m2c2)1/2 is the relativistic fac- tor of the electron, P⊥=γ mv⊥, Pz=γ mvz, and k=ˆzk is as- Nonlin. Processes Geophys., 15, 773–782, 2008 Nonlin. Processes Geophys., 15, 773–782, 2008 Nonlin. Processes Geophys., 15, 773–782, 2008 www.nonlin-processes-geophys.net/15/773/2008/ S. P. Kuo: Whistler wave-electron interaction in the magnetosphere 775 S. P. Kuo: Whistler wave-electron interaction in the magnetosphere S. P. Kuo: Whistler wave-electron interaction in the magnetosphere In Siple experiments (Helliwell et al., 1980), injected Siple signals of 3 kHz, propagating along the L∼=4 shell, were often ampliﬁed by 10 to 30 dB and oscillated in time. The numerical result presented in Fig. 1 also indicates that whistler wave amplitude can indeed be ampliﬁed more than 25 dB by keV electrons through loss-cone negative mass instability, and also oscillates in time in a similar fashion. A good agreement between the numerical and experimental results is obtained. It is worth to point out that the wave amplitude oscillation feature observed in Siple experiments 1nεmc2(⟨γ1⟩ε−⟨γ10⟩ε)=−ε0[(1+εr)/2][E2 0(t)−E2 0(0)] With the aid of this relation, the average of 1ω=1ω0−(k2c2/ω−ω0)(γ1−γ10)/γ1 becomes With the aid of this relation, the average of 1ω=1ω0−(k2c2/ω−ω0)(γ1−γ10)/γ1 becomes ⟨1ω⟩= ⟨1ω0⟩+(ω2 pb/0)[ε0(1+εr)/2γ0 1nεmc2][E2 0(t)−E2 0(0)] (7) (7) where the dispersion relation εr=(kc/ω)2 has been used; ⟨1ω0⟩=1/2 (⟨1ω01⟩–⟨1ω02⟩)∼⟨1ω01⟩. Since ⟨1ω0⟩<0, where the dispersion relation εr=(kc/ω)2 has been used; ⟨1ω0⟩=1/2 (⟨1ω01⟩–⟨1ω02⟩)∼⟨1ω01⟩. Since ⟨1ω0⟩<0, www.nonlin-processes-geophys.net/15/773/2008/ www.nonlin-processes-geophys.net/15/773/2008/ Nonlin. Processes Geophys., 15, 773–782, 2008 S. P. Kuo: Whistler wave-electron interaction in the magnetosphere 776 (a) (b) Fig. 2. (a) Wave amplitude verses time for four different incident wave intensities and (b) amplitude gain G of ampliﬁed whistler wave verses the incident wave intensity. dition, where the wave frequency is in the range to achieve double cyclotron resonances. This is exempliﬁed by con- sidering a case with the following background conditions: 0/ω=8, γ10=1.1, and εr=41; \|⟨1ω0⟩\|/ω∼1.287×10−3 and v/c=0.417; setting v⊥/c=0.32, leads to α0=0.173, b=428, and g=1.06×107. Presented in Fig. 2a are the plots of wave intensity verse time for four different intensity levels of the incident wave. As shown both the gain and the oscillat- ing period of the wave intensity decrease as the initial wave intensity increases. The gain function G(Iin) is plotted in Fig. 2b, where Iin is proportional to E2 in. It shows that with a 30 dB increase of Iin, G reduces only about 10 dB; in other words, the ampliﬁcation process remains effective for a large increase of Iin. (a) 4 Formulation and analysis of chaotic scattering process Interaction between trapped energetic electrons (hundreds of keV to MeV) and a large amplitude whistler wave is con- sidered. These electrons are trapped by the magnetic mirror of the geomagnetic ﬁeld and bounce back and forth about the geomagnetic equator. Since these electrons are practi- cally collisionless, single particle approach will be adopted. To simplify the formulation while retaining the essential physics, the magnetic dipole ﬁeld is modeled by a parabolic scalar potential, ϕ=−mω2 bz2/2e, superimposed over a uni- form magnetic ﬁeld, B0=B0ˆz, where z is the distance from the equatorial plane. This parabolic potential characterized by a bounce frequency ωb simulates the mirror effect of the magnetic dipole ﬁeld (Ho et al., 1994). Justiﬁcation for this simpliﬁcation of the background magnetic ﬁeld conﬁgura- tion is given in Appendix D. (b) Fig. 2. (a) Wave amplitude verses time for four different incident wave intensities and (b) amplitude gain G of ampliﬁed whistler wave verses the incident wave intensity. Nonlin. Processes Geophys., 15, 773–782, 2008 4.1 Formulation When the sign of Q changes from positive to negative between two time steps, the tra- jectory between these two points is interpolated to the Q=0 plane, and the resulting point recorded. The time integra- tion continues for about 1000 bounce periods. The initial conditions (Q0, z0, P0, pz0) of Eqs. (15–17) are determined as follows. Consider an electron having an initial energy γ20 and pitch angle θ20 at equator, where θ20=tan−1(P0/pz0), and set Q0=0=z0, hence, γ20=1/2(4+P 2 0 +p2 z0)1/2. We can then obtain the remaining two initial conditions P0=2(γ 2 20– 1)1/2sinθ20 and pzo=2(γ 2 20–1)1/2cos θ20. The trajectories of different electrons represented by different initial energies γ20 are examined by the surface of section technique. As these electrons interact with the wave having normalized am- plitude 1, the behavior of each trajectory, which is regular or chaotic, is expected to depend strongly on two quantities, the value of γ20 and the wave amplitude represented by 1. 0 ωt=t′→t, and ωb/ω=ω′ b→ωb, we arrive at the normalized relativistic equations of motion convenient for later numeri- cal analysis: dP/dt = −0(0/γ2 −1)Q + (01/γ2) sin z (14) dQ/dt = (0/γ2 −1)(P/0) + (1/γ2) cos z (15) dz/dt = pz/γ2 (16) dpz/dt = −ω2 bz + (1/γ2)(P sin z + 0Q cos z) (17) where γ2=(ω/kc)[(kc/ω)2+P 2+p2 z+2 0Q2+2 1+21(Pcosz– 1 0Qsinz)]1/2, is the relativistic factor; ω/kc, the normalized phase velocity of whistler wave, will be taken to be 1/2 in the numerical analysis. This value of the phase velocity corresponds quite well to the case of the magnetosphere. Since K, as given by Eq. (13), does not depend on t ex- plicitly, it is a constant of motion, i.e., dK/dt=∂K/∂t=0, which reduces the degree of freedom of the system by one. Thus the set of Eqs. (14–17) describes trajectories in a three- dimensional space. A surface of section technique is used to further reduce the three-dimensional continuous time sys- tem to a two-dimensional map, where we can examine the chaoticity of the system graphically. We now use surface of section approach to characterize the interaction of a 3 kHz whistler wave with energetic elec- trons in L=3.3 shell. The parameters of the system are nor- malized to be 0=8 and ωb=0.1. 4.1 Formulation pair (Q, P) by introducing the generating function (Faith et al., 1997a, b) pair (Q, P) by introducing the generating function (Faith et al., 1997a, b) F1(x, Q, t) = 1/2m0(x2 + Q2)cotωt −m0xQcscωt (11) Equation (11) is applied for the canonical transformation: px=∂F1/∂x and P=−∂F1/∂Q, to determine the new coor- dinates P = px cos ωt + m0x sin ωt, Q = −(px/m0) sin ωt + x cos ωt (12) d h il i P = px cos ωt + m0x sin ωt, Q = −(px/m0) sin ωt + x cos ωt (12) (12) and the new Hamiltonian and the new Hamiltonian K = H + ∂F1/∂t = c n P 2 + p2 z + (m0Q)2 + m2c2 + (m1/k)2 + 2m(1/k)[P cos kz −m0Q sin kz]} 1/2 +1/2mω2 bz2 −1/2ω(m0Q2 + P 2/m) (13) Fig. 3. Surface of section plot from the trajectories of four electrons with initial energies γ0=1.2 (in blue), 1.5 (in green), 2 (in orange), and 3 (in red), and with the same initial pitch angle θ0=60◦and wave amplitude 1=0.08 in a system with 0=8 and ωb=0.1. The unperturbed trajectories (1=0) represented by elliptical curves are superimposed in the same plot. (13) where 1=eB/m. From Eq. (13), the Hamilton’s equa- tions of motion, dr/dt=∇pK and dp/dt=−∇K, can be derived. Use of the normalizations: k2K/mω2=K′→K, kQ=Q′→Q, kP/mω=P ′→P, kz=z′→z, kpz/mω=p′ z→pz, 0/ω=′ 0→0, 1/ω=′ 1→1, ωt=t′→t, and ωb/ω=ω′ b→ωb, we arrive at the normalized relativistic equations of motion convenient for later numeri- cal analysis: where 1=eB/m. From Eq. (13), the Hamilton’s equa- tions of motion, dr/dt=∇pK and dp/dt=−∇K, can be derived. Use of the normalizations: k2K/mω2=K′→K, kQ=Q′→Q kP/mω=P ′→P kz=z′→z kQ Q →Q, kP/mω P →P, kz z →z, kpz/mω=p′ z→pz, 0/ω=′ 0→0, 1/ω=′ 1→1, ′ ′ z/mω=p′ z→pz, 0/ω=′ 0→0, 1/ω=′ 1→1 ′ d / ′ i h project trajectories onto the z−pz plane or graphically de- picting the chaoticity of the system. It is noted that for a given K, both P and dQ/dt are double valued. Hence, the surface of section is separated into dQ/dt<0 and dQ/dt>0 cases. We choose only to present the dQ/dt<0 case be- cause the results of the two cases are mirror images of each other. Equations (15–17) are integrated in time using a ﬁfth order Runge Kutta ODE solver. 4.1 Formulation The dependence of the chaoticity of the system on the electron energy, i.e., γ20, is examined by mapping the trajectories of four electrons hav- ing γ20=1.2, 1.5, 2, and 3, in the same surface of section 4.1 Formulation is different from the pattern of pulsations appearing in the natural event of chorus, which has been simulated by the quasi-periodic ELF/VLF generation model (Pasmanik et al., 2004). The total vector potential in the system, contributed by both the wave and static ﬁelds, is given by A =Aw+ ˆyB0x, where Aw, the vector potential of the whistler wave ﬁelds, is ex- pressed explicitly to be The dependence of the wave amplitude gain G=20 log (E0m/Ein) on the density of energetic electrons is through the parameter g in Eq. (8), where E0m and Ein are the maximum amplitude of the ampliﬁed wave and the amplitude of the incident wave; g∝(nε/1nε)(ω/⟨1ω0⟩)2∝nε/1n5/3 ε ∝n−2/3 ε . The numerical analysis shows that (E0m/Ein)2∝nε, i.e., the gain G increases linearly with the logarithm of the density of energetic electrons. Aw = (B/k)[ ˆx cos(kz −ωt) + ˆy sin(kz −ωt)] (9) (9) With both of the potentials given and let p be the canonical momentum, the electron relativistic Hamiltonian H (r, p), is given by With both of the potentials given and let p be the canonical momentum, the electron relativistic Hamiltonian H (r, p), is given by H = c[(p + eA)2 + m2c2] 1/2 −eϕ (10) (10) It is noted that the ﬁeld amplitude Ein of the incident whistler wave in Fig. 1 is rather low (to be consistent with that of previous experiments (Helliwell et al., 1980)). In practical application for achieving signiﬁcant electron pre- cipitation, the incident wave ﬁeld has to increase consider- ably. Thus, it is important to realize how the gain G varies with the incident wave intensity for a ﬁxed background con- This Hamiltonian yields trajectories in a six-dimensional phase space. However, it can be simpliﬁed to reduce the de- gree of freedom. The y-coordinate is cyclic, py=constant, which can be set equal to zero without losing the generality. We next transform the canonical coordinates (x, px) to a new Nonlin. Processes Geophys., 15, 773–782, 2008 www.nonlin-processes-geophys.net/15/773/2008/ www.nonlin-processes-geophys.net/15/773/2008/ S. P. Kuo: Whistler wave-electron interaction in the magnetosphere 777 Fig. 3. Surface of section plot from the trajectories of four electrons with initial energies γ0=1.2 (in blue), 1.5 (in green), 2 (in orange), and 3 (in red), and with the same initial pitch angle θ0=60◦and wave amplitude 1=0.08 in a system with 0=8 and ωb=0.1. The unperturbed trajectories (1=0) represented by elliptical curves are superimposed in the same plot. 4.2 Numerical analysis The constant of the motion, K=constant, allows us to elim- inate one of the four variables in Eqs. (14–17). In the fol- lowing analysis, we choose to eliminate the variable P, i.e., Eq. (14). We also choose Q=0 as the surface of section to Nonlin. Processes Geophys., 15, 773–782, 2008 www.nonlin-processes-geophys.net/15/773/2008/ S. P. Kuo: Whistler wave-electron interaction in the magnetosphere 778 Fig. 4. Temporal evolution of the pitch angle of a 1.5 MeV elec- tron interacting with a whistler wave at Doppler shifted cyclotron resonance. Plots A and B correspond to wave magnetic ﬁeld B1=7×10−4 B0 and 8.1×10−4B0 cases, respectively. the bounce frequency of MeV electrons; n∼=(ω2 pb/ω0)1/2 is the index of refraction of the background cold plasma on the whistler wave; L is the number of earth radii, i.e., L value of a magnetic ﬂux tube; θ1=tan−1(j)1/2 is the initial pitch an- gle of keV electrons which contribute to wave ampliﬁcation, thus cos θ1=1/(1+j)1/2; γ1∼1.1 is the relativistic factor of ∼50 keV electrons; θ2 is the initial pitch angle of MeV elec- trons which are being precipitated; the normalized phase ve- locity of the wave V =vp/c=(0/ωp)ξ−1/2, where ξ=0/ω. p p With the aid of some known background parameters: nb∼280 cm−3 at L=4.9, B0∼0.25 Gauss at L=1, and ωb/0∼(1/3)×10−2 for γ2=3 and L=2, we can obtain the functional dependence of the background parameters on L as ωpb=2π×1.63×106/L3/2, 0=2π×7×105/L3, and ωb/0=(ωb/ω)/(0/ω)=7.07×10−3 (1–γ −2 2 )−1/2/L. The resonance conditions (1) and (18) and the dispersion relation lead to 0/ω=γ2+n(γ 2 2 −1)1/2 cos θ2=ξ=γ1+n(γ 2 1 −1)1/2 cos θ1 (19) n=2.33L3/2ξ1/2=2.33L3/2[γ2+n(γ 2 2 −1)1/2 cos θ2]1/2 (20) Fig. 4. Temporal evolution of the pitch angle of a 1.5 MeV elec- tron interacting with a whistler wave at Doppler shifted cyclotron resonance. Plots A and B correspond to wave magnetic ﬁeld B1=7×10−4 B0 and 8.1×10−4B0 cases, respectively. Equation (19) leads to n=(γ2–γ1)/[(γ 2 1 –1)1/2cosθ1–(γ 2 2 – 1)1/2cosθ2], which is then used to re-express Eq. (19) as Equation (19) leads to n=(γ2–γ1)/[(γ 2 1 –1)1/2cosθ1–(γ 2 2 – 1)1/2cosθ2], which is then used to re-express Eq. (19) as 0/ω→0 = [γ2(γ 2 1 −1)1/2 cos θ1 −γ1(γ 2 2 −1)1/2 cos θ2]/[(γ 2 1 −1)1/2 cos θ1−(γ 2 2 −1)1/2 cos θ2] plot. Presented in Fig. 3 is the one corresponding to 1=0.08 (1% of the background magnetic ﬁeld) and the same initial pitch angle θ20=60◦. The elliptical curves in Fig. 4.2 Numerical analysis 3 represent the unperturbed trajectories (1=0 case), which are super- imposed in the plot for a comparison. As shown, the tra- jectories for γ20=1.5, 2, and 3 are mapped in a large phase space region, suggesting the occurrence of signiﬁcant chaotic pitch angle scattering. Indeed, it is found that the electron equatorial pitch angle θ, in the three chaotic cases, can be- come as low as that less than 30◦. However, the trajectory in γ20=1.2 case remains regular, demonstrating that the elec- tron energy is important to the type of motion the electron undergoes. The wave amplitude required causing an elec- tron trajectory to become chaotic is lower for those elec- trons with energy >100 keV. This conﬁrms that one can use the lower energy (<100 kev) electrons to amplify whistler waves, which become more effective in precipitating higher energy (>1 MeV) electrons. Equations (19) and (20) are solved to yield Equations (19) and (20) are solved to yield cos θ2 = 1/2[(γ 2 1 −1)/(γ 2 2 −1)]1/2 ×{(γ2/γ1 + 1) cos θ1 −(γ2/γ1 −1)[cos2 θ1 +4γ1/5.43L3(γ 2 1 −1)]1/2} (21) (21) The normalized (to ω) bounce frequency of electrons and normalized (to c) phase velocity of the wave are given by ωb=7.07×10−3 (1–γ −2 2 )−1/20/L and V =0.432L−3/2−1/2 0 . 0 We now consider the γ2=4 case that precipitates 1.5 MeV electrons. From Eq. (21), θ2=86.5◦. We then have 0=8.6038, V =0.0517, and ωb=2.945×10−2. Equa- tions (15) to (17) are now integrated numerically to evaluate the pitch angle scattering, resulting from the wave-electron resonance interaction. Presented in Fig. 4 is a result that dou- ble resonance condition is satisﬁed. 6 Summary (A7) where α=kv⊥/0 and φ=kz0+[20+8(t)]−∫t 0 1ω′dt′; 1ω=1ω0+0(γ1−γ10)/γ1γ10+k(vz−vz0), 1ω0=ω−ω0, and ω0=0/γ10−kvz0; 20=tan−1(vy0/vx0), 8 accounts for the phase shift in the electron gyration due to interaction with the wave ﬁelds, γ10 is the initial rel- ativistic factor of the resonant electrons. The ratio of Eqs. (A6) and (A7) leads to an invariant relation γ1(kc2/ω+vz)=const.=γ10(kc2/ω+vz0), which is used to obtain 1ω=1ω0−(k2c2/ω−ω0)(γ1−γ10)/γ1. It is noted that k2c2/ω−ω0>0 for whistler waves considered in the present case, i.e., 1ω increases as electron loses energy to the wave and vice versa. Finally, it should be pointed out that this optimal approach, relying on electron cyclotron resonance interaction, requires that the wave have a broad frequency spectrum, so that a con- siderable fraction of the very energetic electrons can be pre- cipitated simultaneously. Collective effect The keV electron plasma has a loss-cone distribution given by Eq. (3) and its distribution in Pz is yet a Maxwellian given by Eq. (4) and shown in Fig. B1. Only a fraction of total electrons, e.g., in the two shade regions in Fig. B1, are close to Doppler shifted cyclotron resonance with the wave. In fact, the wave is experiencing cyclotron damping to the elec- trons which are initially at exact cyclotron resonance with the wave, i.e., 1ω0=0. On the other hand, the wave can exchange energy with other electrons having a mismatch frequency 5 Double cyclotron resonances for effective precipita- tion of MeV electrons by whistler waves As shown, with 1=6×10−3 (i.e., the wave magnetic ﬁeld B1=7×10−4B0), the electron trajectory is chaotic but the pitch angle (plot A) of the scattered electron remains larger than 60◦. However, as the wave magnetic ﬁeld B1 increases slightly to 8.1×10−4B0 (i.e., 1=7×10−3), sig- niﬁcantly large pitch angle scattering occurs. As shown, electron is scattered to a pitch angle <50◦(plot B), less than the loss cone angle. The required wave mag- netic ﬁeld amplitude is calculated to be about 0.08% (i.e., 1/0=0.007/8.6038=0.0008) of the background magnetic ﬁled. This is an example that a whistler wave, with proper A wave resonant with MeV electrons (γ =γ2) satisﬁes a res- onance condition, similar to that given by Eq. (1), ω = 0/γ2 + kPz2/γ2m (18) (18) ω = 0/γ2 + kPz2/γ2m We now ﬁnd the initial conditions of electrons such that Eqs. (1) and (18) can be satisﬁed simultaneously. The re- lationships and notations to be applied are ﬁrst introduced as follows: 0∝L−3; ωpb∝L−3/2; ωb∝(1−γ −2 2 )−1/20/L is www.nonlin-processes-geophys.net/15/773/2008/ Nonlin. Processes Geophys., 15, 773–782, 2008 Nonlin. Processes Geophys., 15, 773–782, 2008 779 S. P. Kuo: Whistler wave-electron interaction in the magnetosphere are given by parameters, can be ampliﬁed by the keV electrons and si- multaneously scatters MeV electrons, both processes via the cyclotron resonance interaction. The results show that cy- clotron resonant interaction reduces the required ﬁeld ampli- tude, for achieving effective chaotic scattering, by a factor more than 20. On the other hand, the required interaction time is also increased by an order of magnitude. dr/dt = p/γ1m (A1) dp/dt = −e[E + v × (B + ˆzB0)] (A2) mc2dγ1/dt = eE · v (A3) (A1) (A2) (A3) where γ1=(1+p2/m2c2)1/2, p=γ1mv, and the whistler wave ﬁelds E and B are E=E0(t) ˆu and B=(k/ω)E0(t)ˆv; ˆu=[ ˆxcos(kz−ωt)−ˆysin(kz−ωt)] and ˆv=[ ˆx sin(kz−ωt)+ ˆycos(kz−ωt)]; ω and k are related by the whistler wave dispersion relation ω=0k2c2/ω2 pb given in Eq. (2); ε0 is the free-space permitivity. 6 Summary Small pitch angle scatterings in cyclotron resonance interac- tion with a whistler wave can diffuse MeV electrons, with pitch angles close to the loss cone angle, into loss cones (Al- bert, 2000). On the other hand, it will need a chaotic scatter- ing process to precipitate those deeply trapped electrons; the wave ﬁeld has to exceed a threshold (Kuo et al., 2004). When the wave frequency ω is near the Doppler shifted electron cyclotron frequency, ω∼=0/γ1−kvz, the electron trajectory is mainly governed by the cyclotron resonance in- teraction. After removing all the fast oscillating components in the Lorentz force, Eqs. (A1–A3) reduce to a set of self- consistent governing equations for the slowly time varying functions γ1, v⊥, vz and 8 (Kuo and Cheo, 1985) Ampliﬁcation of whistler wave by (tens of keV) energetic electrons in the magnetosphere through loss-cone negative mass instability is studied. The theory is formulated and the numerical result is shown to agree well with the experimental result, both qualitatively and quantitatively. Such ampliﬁca- tion reduces considerably the required ﬁeld intensity of the incident whistler wave for the purpose of precipitating MeV electrons in the magnetosphere. dα/dt = −(k/0)(eE0/m)(1 + kvz/ω) cos φ (A4) dφ/dt=−1ω+(k/0)(eE0/m)(1+kvz/ω)α−1 sin φ (A5) dvz/dt=(0/k)(eE0/mc2)(α/γ 2 1 )(kc2/ω+vz) cos φ (A6) dγ1/dt = −(0/k)(eE0/mc2)(α/γ1) cos φ (A7) (A4) The feasibility to invoke a double cyclotron resonance sit- uation for an optimal approach to reduce the population of MeV electrons trapped in the magnetosphere is then demon- strated. In this approach, the wave is ﬁrst ampliﬁed by (tens of keV) energetic electrons; once the wave ﬁeld exceeds the threshold for the commencement of chaos, cyclotron resonance-enhanced chaotic scattering can precipitate deeply trapped MeV electrons into loss cones. The numerical re- sults demonstrate that a 1.5 MeV electron can be scattered from an initial pitch angle of 86.5◦to a pitch angle <50◦by a whistler wave with the magnetic ﬁeld amplitude of 0.08 % of the background magnetic ﬁeld, which is about 20 times smaller than that without invoking cyclotron resonance. This percentage converts to about 3100 pT at L=2, and 200 pT at L=5, which reduce to 550 pT and 36 pT, respectively, after taking advantage of the 15 dB gain. Resonant trajectory equations Resonant trajectory equations The equations for the electron motion in a dc magnetic ﬁeld ˆzB0 and right-hand circularly polarized wave ﬁelds E and B www.nonlin-processes-geophys.net/15/773/2008/ www.nonlin-processes-geophys.net/15/773/2008/ Nonlin. Processes Geophys., 15, 773–782, 2008 780 S. P. Kuo: Whistler wave-electron interaction in the magnetosphere S. P. Kuo: Whistler wave-electron interaction in the magnetosphere Fig. B1. Momentum distribution of energetic electrons and the re- gions close to cyclotron resonant interaction with the wave. where Jpc=−e1nε(0/k)⟨(α/γ1)cosφ⟩ε1∼=-P1⟨cosφ⟩ε1, and Jps=−e2nε(0/k)⟨(α/γ1)sinφ⟩ε1∼=−P2⟨sinφ⟩ε1; nε=(N1+N2)/2 and P2=2nεe(0/k)(α0/γ10); ⟨sinφ⟩ε2∼=⟨sinφ⟩ε1 is assumed. where Jpc=−e1nε(0/k)⟨(α/γ1)cosφ⟩ε1∼=-P1⟨cosφ⟩ε1, and Jps=−e2nε(0/k)⟨(α/γ1)sinφ⟩ε1∼=−P2⟨sinφ⟩ε1; nε=(N1+N2)/2 and P2=2nεe(0/k)(α0/γ10); ⟨sinφ⟩ε2∼=⟨sinφ⟩ε1 is assumed. Comparing Eq. (6) with the average of Eq. (A7), leads to ⟨(α/γ )cosφ⟩ε=(k/1nεe0)[ε0(1+εr)]dtE0, which is ap- proximated to be ⟨cos φ⟩ε ∼= [ε0(1 + εr)/P1]dtE0 (C3) (C3) where P1=1nεe(0/k)(α0/γ0) and the notation dt=d/dt is used. where P1=1nεe(0/k)(α0/γ0) and the notation dt=d/dt is used. Fig. B1. Momentum distribution of energetic electrons and the re- gions close to cyclotron resonant interaction with the wave. Substitute E=E0(t) ˆu in Eq. (C1) and with the aid of Eq. (C2) and the dispersion relation εr=1+ω2 p/ω=k2c2/ω2, Eq. (C1) is reduced to d2 t E0 ∼= −ε−1 0 (dtJpc −ωJps) (C4) ω(1 + εr)dtE0 ∼= −ε−1 0 (dtJps + ωJpc) (C5) (C4) 1ω0 slightly different from zero. Depending on the initial phases of those electrons, the interaction can cause them ei- ther to gain energy from, or lose energy to, the wave, initially. In region 1, 1ω01<0, one half of the electrons will lose en- ergy to the wave initially, those electrons also reduce the mis- match frequency in the interaction (because γ1−γ10<0); it results in the increase of the interaction period of losing en- ergy to the wave. The other half of the electrons, which gain energy from the wave initially, will increase their mismatch frequencies (because γ1−γ10>0) and reduce the interaction period of gaining energy from the wave. Therefore, those electrons with 1ω01<0 will lose energy to the wave on aver- age. (C5) Using the relation Jpc=−ε0(1+εr) dt E0, Eq. (C5) reduces to dtJps∼=0, i.e., ⟨(dtφ)cosφ⟩ε1∼=0, which, with the aid of Eq. (A6), leads to the relation sE0⟨sin 2φ⟩ε1 ∼= 2⟨1ω0⟩⟨cos φ⟩ε1 (C6) (C6) where s=(k/0)(e/m)(1+kvz0/ω)α−1 0 , and Eq. (C4) reduces to Jps=−(ε0εr/ω)d2 t E0, which leads to ⟨sin φ⟩ε1 = (ε0εr/ωP2)d2 t E0 (C7) (C7) For those electrons in region 2 with 1ω0=1ω02>0, the above dynamic interaction process is reversed; on average, those electrons will gain energy from the wave. Resonant trajectory equations Using the deﬁnition 1ω0=ω−ω0=ω−0/γ10+kvz0, 1ω01<1ω02 leads to vz01<vz02, which indicates that there are more elec- trons in the 1ω01<0 region than in the 1ω02>0 region (i.e., N1>N2 as shown in Fig. B1). Overall, the wave will gain energy from electrons and be ampliﬁed. Furthermore, with the aid of Eqs. (C7), (A4) and (A5) can be combined to obtain Furthermore, with the aid of Eqs. (C7), (A4) and (A5) can be combined to obtain sE0⟨cos 2φ⟩ε1 ∼= ω(P2/P1)(1 + ε−1 r )⟨sin φ⟩ε1 (C8) (C8) Jpc is the source current density of the radiation; it is usually governed by a second order differential equation. Introduc- ing the relation d2 t ⟨(α/γ1) cos φ⟩= −⟨[(α/γ1)d2 t φ+2dt(α/γ1)dtφ] sin φ⟩ −⟨[(α/γ1)(dtφ)2−d2 t (α/γ1)] cos φ⟩(C9) Phase average relations and the governing equation of the wave amplitude With the aid of Eqs. (A4–A7), the right hand side terms of Eq. (C9) can be expressed explicitly in terms of the function E0(t) and its integrals and derivatives as follows The wave equation governs the self-consistent wave ﬁeld [c2∂2/∂z2 −∂2/∂t2]E = ε−1 0 ∂(Je + Jp)/∂t (C1) (α/γ1)(d2 t φ) sin φ⟩∼= (α0/γ10)(s/2) n dtE0(1 −⟨cos 2φ⟩) −(A0E0/2)(Ic⟨sin φ⟩+ Is⟨cos φ⟩) +s[1 + (α2 0/2)(1 + vz0ω/kc2)]E2 0⟨cos φ⟩−[⟨1ω0⟩ +α2 0ω(1 + kvz0/ω) ] E0⟨sin 2φ⟩ o Modeling a magnetic dipole ﬁeld: The geomagnetic ﬁeld localized around the electron guiding center resembles a parabolic mirror ﬁeld. This mirror ﬁeld may be expressed in local cylindrical coordinates (r, θ, z) as Bg = B0[1 + (z2 −r2/2)/L2]ˆz −(B0zr/L2)ˆr where L is the scale length of the magnetic ﬁeld and Bg satisﬁes both ∇·Bg=0 and ∇×Bg=0 as required. The z- component of the equation of motion of an electron gyrating about the z-axis in this mirror magnetic ﬁeld is given by (C10) (C10) where A0=(ω2 pb/0) (b1α0/γ 2 10), b1=(0/k) (e/mc2); Ic= ∫t 0 E0(t′)cos⟨1φ(t−t′)⟩ε1dt′ and Is=∫t 0 E0(t′)sin⟨1φ(t−t′)⟩ε1dt′; ⟨1φ(t−t′)⟩ε1= −∫t t′⟨1ω(τ)⟩ε1dτ=−⟨1ω0⟩∫t t′ {1+(ω2 pb/0⟨1ω0⟩) 2 2 2 where A0=(ω2 pb/0) (b1α0/γ 2 10), b1=(0/k) (e/mc2); Ic= ∫t 0 E0(t′)cos⟨1φ(t−t′)⟩ε1dt′ and Is=∫t 0 E0(t′)sin⟨1φ(t−t′)⟩ε1dt′; ⟨1φ(t−t′)⟩ε1= −∫t t′⟨1ω(τ)⟩ε1dτ=−⟨1ω0⟩∫t t′ {1+(ω2 pb/0⟨1ω0⟩) [ε0(1+εr)/2γ101nεmc2] [E2 0(τ)–E2 0(0)]}dτ; the dispersion relation k2c2/ω–ω∼=ω2 pb/0 is used; the higher order terms ⟨cos3φ⟩ε1 and ⟨sin3φ⟩ε1 are neglected and the approxima- tions ⟨cos(φ−φ′)⟩ε1∼=cos⟨(φ−φ′)⟩ε1∼=cos⟨1φ(t−t′)⟩ε1 and ⟨sin(φ−φ′)⟩ε1∼=sin⟨(φ−φ′)⟩ε1∼=sin⟨1φ(t−t′)⟩ε1 are used in the derivation. dvz/dt = d2z/dt2 = evθBgr/m = −∂(µBgz)/∂z = −eB0zrvθ/mL2 = −µB0z/mL2 = −ω2 bz where µ=mv2 θ0/2B0 is the magnetic dipole moment of the electron; vθ0=vθ(z=0) and Bgz∼=B0 is assumed; r=rL=(vθ0/vθ)rL0 is equal to the Larmour radius; rL0=mvθ0/eB0 is the electron Larmour radius at equa- tor; and ωb=(µB0/mL2)1/2 is the bounce frequency and is assumed to be a constant. Thus a parabolic potential may be used to simulate the mirroring effect of a magnetic dipole ﬁeld. The approximations that ωb=const. and Bgz∼=B0 are justiﬁed as long as the magnetic moment µ varies slowly in time, and z2/L2 and r2/L2≪1. [ε0(1+εr)/2γ101nεmc2] [E2 0(τ)–E2 0(0)]}dτ; the dispersion relation k2c2/ω–ω∼=ω2 pb/0 is used; the higher order terms ⟨cos3φ⟩ε1 and ⟨sin3φ⟩ε1 are neglected and the approxima- tions ⟨cos(φ−φ′)⟩ε1∼=cos⟨(φ−φ′)⟩ε1∼=cos⟨1φ(t−t′)⟩ε1 and ⟨sin(φ−φ′)⟩ε1∼=sin⟨(φ−φ′)⟩ε1∼=sin⟨1φ(t−t′)⟩ε1 are used in the derivation. With the aid of the phase average relations (C3) and (C6– C10), the source terms on the right hand side of Eq. (C1) can be expressed explicitly in terms of the self-consistent ﬁeld amplitude E0(t) and its derivatives and integrations. In essence, this is a procedure to combine the three ﬁrst order differential Eqs. (A4) to (A6) to the wave Eq. (C1). It leads to a differential-integral equation for E0 (Kuo et al., 2004) Acknowledgements. The author is grateful to James T. Huynh, Steven S. Kuo, and Paul Kossey for collaborative works. Modeling a magnetic dipole ﬁeld: This work was supported by the High Frequency Active Auroral Research Program (HAARP), Air Force Research Laboratory at Hanscom AFB, Massachusetts, and by the Ofﬁce of Naval Research, Grant No. ONR-N00014-05-1-0109. Part of the ﬁnancial support was arranged through NorthWest Research Associates, Inc. [d3 t + 20d2 t + (1ω2 + C)dt]E0 = a0{1+(nε/1nε)(ω2 pb/0⟨1ω0⟩)[ε0(1+εr)/4γ101nεmc2] ×[E2 0(t) −E2 0(0)]} t ∫ 0 E0(t′) cos⟨1φ(t −t′)⟩ε1dt′ (C11) Edited by: L. Zelenyi Reviewed by: A. G. Demekhov, L. Stenﬂo and another anonymous referee Edited by: L. Zelenyi Reviewed by: A. G. Demekhov, L. Stenﬂo and another anonymous referee where dt stands for d/dt; 0∼=–[3εr/8 (1+εr)] (b1α0/γ 2 10)×(A0E0Ic/ω) and 1ω2=⟨1ω0⟩2+α2 0{s2 [(1+vz0ω/kc2)+3(ω/kc)2(1+kvz0/ω)– (9/4)α2 0(ω/kc)4(1+kvz0/ω)2]E2 0–ω⟨1ω0⟩(1+kvz0/ω)}– (3b1α0/4γ 2 10–2⟨1ω0⟩2/sE2 0)A0IsE0; where dt stands for d/dt; 0∼=–[3εr/8 (1+εr)] (b1α0/γ 2 10)×(A0E0Ic/ω) and 1ω2=⟨1ω0⟩2+α2 0{s2 [(1+vz0ω/kc2)+3(ω/kc)2(1+kvz0/ω)– (9/4)α2 0(ω/kc)4(1+kvz0/ω)2]E2 0–ω⟨1ω0⟩(1+kvz0/ω)}– (3b1α0/4γ 2 10–2⟨1ω0⟩2/sE2 0)A0IsE0; where dt stands for d/dt; 0∼=–[3εr/8 (1+εr)] (b1α0/γ 2 10)×(A0E0Ic/ω) and 1ω2=⟨1ω0⟩2+α2 0{s2 [(1+vz0ω/kc2)+3(ω/kc)2(1+kvz0/ω)– (9/4)α2 0(ω/kc)4(1+kvz0/ω)2]E2 0–ω⟨1ω0⟩(1+kvz0/ω)}– (3b1α0/4γ 2 10–2⟨1ω0⟩2/sE2 0)A0IsE0; 2 2 10 (1+vz0ω/kc2)+3(ω/kc)2(1+kvz0/ω)– z / z (9/4)α2 0(ω/kc)4(1+kvz0/ω)2]E2 0–ω⟨1ω0⟩(1+kvz0/ω)}– (3b1α0/4γ 2 10–2⟨1ω0⟩2/sE2 0)A0IsE0; Appendix D Appendix D (C1) where Je=−enbve is the linear current density induced by the wave ﬁelds in the background plasma with ve=−(eE0/m0)ˆv and Jp, the induced polarization current density associated with the resonant electrons, is given by e ( 0 0) Jp, p density associated with the resonant electrons, is given by ⟨2[dt(α/γ1)](dtφ) sin φ)⟩∼= (α0/2γ10)(s −b1α0/γ 2 10) [2⟨1ω0⟩E0⟨sin 2φ⟩+ A0E0(Ic⟨sin φ⟩ +Is⟨cos φ⟩) −sE2 0⟨cos φ⟩] ⟨(α/γ1)(dtφ)2 cos φ⟩∼= (α0/γ10) ⟨2[dt(α/γ1)](dtφ) sin φ)⟩∼= (α0/2γ10)(s −b1α0/γ 2 10) [2⟨1ω0⟩E0⟨sin 2φ⟩+ A0E0(Ic⟨sin φ⟩ +Is⟨cos φ⟩) −sE2 0⟨cos φ⟩] ⟨(α/γ1)(dtφ)2 cos φ⟩∼= (α0/γ10) Jp = −e[N1⟨( ˆxvx + ˆyvy)⟩ε1 + eN2⟨( ˆxvx + ˆyvy)⟩ε2] = −e{N1⟨{ ˆuv⊥cos φ + ˆvv⊥sin φ}⟩ε1 + N2⟨{ ˆuv⊥cos φ +ˆvv⊥sin φ}⟩ε2} = ˆuJpc + ˆvJps (C2) (C2) Nonlin. Processes Geophys., 15, 773–782, 2008 Nonlin. Processes Geophys., 15, 773–782, 2008 Nonlin. Processes Geophys., 15, 773–782, 2008 www.nonlin-processes-geophys.net/15/773/2008/ www.nonlin-processes-geophys.net/15/773/2008/ S. P. Kuo: Whistler wave-electron interaction in the magnetosphere S. P. Kuo: Whistler wave-electron interaction in the magnetosphere S. P. Kuo: Whistler wave-electron interaction in the magnetosphere 781 n [⟨1ω0⟩2 + s2E2 0/4 + (A2 0/4)(I 2 c + I 2 s )]⟨cos φ⟩ +(A0/2)(⟨1ω⟩+ ⟨1ω0⟩)Ic −(sA0E0/2) (Ic⟨sin φ⟩+ Is⟨cos φ⟩) −sE0⟨1ω0⟩⟨sin 2φ⟩ +⟨1ω0⟩A0(Ic⟨cos 2φ⟩+ Is⟨sin 2φ⟩) o ⟨[d2 t (α/γ1)] cos φ⟩∼= −(α0/2γ10) n (s −bα0/γ 2 10) [dtE0(1 + ⟨cos 2φ⟩) + (A0E0/2)(Ic⟨sin φ⟩ +Is⟨cos φ⟩) −1/2(s −9b1α0/γ 2 10)E2 0⟨cos φ⟩ +E0⟨1ω0⟩⟨sin 2φ⟩] + (3s2α2 0/2) (1 + vz0ω/kc2)E2 0⟨cos φ⟩ o (C1 References 10 0 C=[1ω2 pε/(1+εr)γ10][(1+kvz0/ω)–1/2(0α0/γ10kc)2], a0=– [1ω2 pε2 0⟨1ω0⟩α2 0/(1+εr)γ 3 10ω], and 1ω2 pε=1nεe2/mε0. In essence, Eq. (C11) is a ﬁfth order ordinary differential equation (ODE). It is linearized to obtain the relation \|⟨1ω0⟩\|3=1ω2 pε2 0α2 0/16(1+εr)γ 3 10ω for determining \|⟨1ω0⟩\|. Albert, J. M.: Gyroresonant interactions of radiation belt particles with a monochromatic electromagnetic wave, J. Geophys. Res., 105, 21 191–21 205, 2000. Arnoldy, R. L. and Kintner, P. M.: Rocket observations of the pre- cipitation of electrons by ground VLF transmitters, J. Geophys. Res., 94, 6825–6832, 1989. Breneman, A., Kletzing, C. A., Chum, J., Santolik, O., Gurnett, D., and Pickett, J.: Multispacecraft observations of chorus dis- persion and source location, J. Geophys. Res., 112, A05221, doi:10.1029/2006JA012058, 2007. Burgess, W. C. and Inan, U. S.: Simultaneous disturbance of conju- gate ionospheric regions in association with individual lightning ﬂashes, Geophys. Res. Lett., 17, 259–262, 1990. www.nonlin-processes-geophys.net/15/773/2008/ Nonlin. Processes Geophys., 15, 773–782, 2008 S. P. Kuo: Whistler wave-electron interaction in the magnetosphere S. P. Kuo: Whistler wave-electron interaction in the magnetosphere S. P. Kuo: Whistler wave-electron interaction in the magnetosphere 782 Cattell, C., Wygant, J. R., Goetz, K., Kersten, K., Kellogg, P. J., von Rosenvinge, T., Bale, S. D., Roth, I., Temerin, M., Hudson, M. K., Mewaldt, R. A., Wiedenbeck, M., Maksimovic, M., Ergun, R., Acuna, M., and Russell, C. T.: Discovery of very large am- plitude whistler-mode waves in Earth’s radiation belts, Geophys. Res. Lett. 35, L01105, doi:10.1029/2007GL032009, 2008. Kuo, S. P. and Lee, M. C.: Saturation of cyclotron maser instability driven by an electron loss-cone distribution, Int. J. Infrared Mill., 7, 623–633, 1986. Kuo, S. P., Kossey, P., Huynh, J., and Kuo, S. S.: Ampliﬁcation of whistler waves for the precipitation of trapped relativistic elec- trons in the magnetosphere, IEEE Trans. Plasma Sci., 32, 362– 369, 2004. Res. Lett. 35, L01105, doi:10.1029/2007GL032009, 2008. Faith, J., Kuo, S. P., and Huang, J.: Chaotic electron motion driven by whistler waves in the magnetosphere, Comments Plasma Phys. Contr. F., 17, 173–182, 1996. Kuo, S. P., Kuo, S. S., Huynh, J. T., and Kossey, P.: Precipitation of trapped relativistic electrons by ampliﬁed whistler waves in the Magnetosphere, Phys. Plasmas, 14, 009706(1–7), 2007. Faith, J., Kuo, S. P., and Huang, J.: Electron precipition caused by chaotic motion in the magnetosphere due to large amplitude whistler waves, J. Geophys. Res., 102, 2233–2241, 1997. References Nunn, D., Omura, Y., Matsumoto, H., Nagano, I., and Yagitani, S.: The numerical simulation of VLF chorus and discrete emissions observed on the Geotail satellite using a Vlasov code, J. Geo- phys. Res., 102, 27 083–27 097, 1997. Faith, J., Kuo, S. P., Huang, J., and Schmidt, G.: Precipitation of magnetospheric electrons caused by relativistic effect enhanced chaotic motion in the whistler wave ﬁelds, J. Geophys. Res., 102, 9631–9638, 1997. Pasmanik, D. L., Titova, E. E., Demekhov, A. G., Trakhtengerts, V., Santolik., O., Jiricek, F., Kudela, K., and Parrot, M.: Quasi- periodic ELF/VLF wave emissions in the Earth’s magnetosphere: comparison of satellite observations and modeling, Ann. Geo- phys., 22, 4351–4361, 2004, http://www.ann-geophys.net/22/4351/2004/. Helliwell, R. A., Katsufrakis, J. P., and Trimpi, M. L.: Whistler induced amplitude perturbation in VLF propagation, J. Geophys. Res., 78, 4679–4688, 1973. Helliwell, R. A., Carpenter, D. L., and Miller, T. R.: Power thresh- old for growth of coherent VLF signals in the magnetosphere, J. Geophys. Res., 85, 3360–3366, 1980. Pradipta, R., Labno, A., Lee, M. C., Burke, W. J., Cohen, J. A., Bur- ton, L. M., Kuo, S. P., and Rokusek, D.: Electron precipitation from the inner radiation belt above Arecibo, Geophys. Res. Lett., 34, L08101, doi:10.1029/2007GL029807, 2007. Helliwell, R. A. and Inan, U. S.: VLF wave growth and discrete emission triggering in the magnetosphere: A feedback model, J. Geophys. Res., 87, 3537–3550, 1982. Sazhin, S. S. and Hayakawa, M.: Magnetospheric chorus emissions: A review, Planet. Space Sci., 40, 681–697, 1992. Helliwell, R. A.: Controlled stimulation of VLF emissions from Siple Station, Antarctica, Radio Sci., 6, 801–814, 1983. Trakhtengerts, V.: A generation mechanism for chorus emission, Ann. Geophys., 17, 95–100, 1999, http://www.ann-geophys.net/17/95/1999/. Ho, A. Y., Kuo, S. P., and Schmidt, G.: Chaotic proton motion driven by kinetic Alfven waves in the magnetosphere leading to polar and equatorial proton precipitations, J. Geophys. Res., 99, 11 087–11 093, 1994. Trakhtengerts, V., Demekhov, A. G., Titova, E. E., Kozelov, B. V., Santolik., O., Gurnett, D. A., and Parrot, M.: Interpretation of Cluster data on chorus emissions using the backward wave oscil- lator model, Phys. Plasmas, 11, 1345–1351, 2004. Imhof, W. L., Robinson, R. M., Colin, H. L., Wygant, J. R., and Anderson, R. R.: Simultaneous measurements of waves and pre- cipitating electrons near the equator in the outer radiation belt, J. Geophys. Res., 99, 2415–2427, 1994. Tsurutani, B. T. and Smith, E. J.: Postmidnight chorus: a substorm phenomenon, J. Nonlin. Processes Geophys., 15, 773–782, 2008 References Geophys. Res., 79, 118–127, 1974. p y Kennel, C. F. and Petschek, H. E.: Limit on stably trapped particle ﬂux, J. Geophys. Res., 71, 1–28, 1966. Tsytovich, V. N. and Stenﬂo, L.: Electromagnetic wave instability in a plasma with relativistic particles in a random magnetic ﬁeld, Astrophys. Space Sci., 89, 233–235, 1983. Khazanov, G. V., Tel’nikhin, A. A., and Kronberg, T. K.: Dynamic theory of relativistic electrons stochastic heating by whistler mode waves with application to the Earth magnetosphere, J. Geo- phys. Res., 113, A03207, doi:10.1029/2007JA012488, 2008. Villalon, E. and Burke, W. J.: Near-equatorial pitch angle diffu- sion of energetic electrons by oblique whistler waves, J. Geo- phys. Res., 96, 9655–9667, 1991. Kuo, S. P. and Cheo, B. R.: Relativistic adiabatic invariants of elec- tron motion under ECRH, Phys. Lett. A, 109, 39–42, 1985. Voss, H. D., Imhof, W. L., Mobilia, J., Gaines, E. E., Walt, M., Inan, U. S., Helliwell, R. A., Carpenter, D. L., Katsufrakis, J. P., and Chang, H. C.: Lightning-induced electron precipitation, Nature, 312(20/27), 740–742, 1984. Nonlin. Processes Geophys., 15, 773–782, 2008 www.nonlin-processes-geophys.net/15/773/2008/
https://openalex.org/W4240963753	https://sciencepubco.com/index.php/ijet/article/download/8992/16604	English	null	Optimizing performance of fuzzy decision support system with multiple parameter dependency for cloud provider evaluation	International journal of engineering & technology	2,017	cc-by	4,893	Optimizing performance of fuzzy decision support system with multiple parameter dependency for cloud provider evaluation Uma Rani 1 , Surjeet Dalal 2, Jugnesh Kumar 2 1 Department of Computer Science & Engineering, SRM University Sonipat, Haryana, India 2 Department of Computer Science & Engineering, St. Andrews Institute of Technology and Management, Haryana Corresponding author E-mail: profsurjeetdalal@gmail.com 1 Department of Computer Science & Engineering, SRM University Sonipat, Haryana, India 2 Department of Computer Science & Engineering, St. Andrews Institute of Technology and Management, Haryana *Corresponding author E-mail: profsurjeetdalal@gmail.com Abstract In today’s world, technological trend offers computing resources as services through the internet in on-demand or pay-as-you-go ap- proach. These services are provided by different cloud service providers. Due to which trust on the any service provider is a choice of any customer. In order to choose a reputed cloud service provider a new method using the concept of fuzzy has proposed in this paper. This method enhanced the customer’s satisfaction level of using cloud services by avoiding ambiguities in fuzzy interface system (FIS) through optimization. Proposed fuzzy rule-based decision support system is collaborating with advanced fuzzy system optimized using a swarm intelligent firefly algorithm that facilitates the consumers in selecting right CSP based upon their rating value. It conducts three different reviews of three different components, i.e. customer review, service provider review and public review. Results are carried out on the basis of both simple and the optimized fuzzy, and it is found that the optimized fuzzy surpasses the simple fuzzy logic. Keywords: Decision Support System; Fuzzy Decision Making; Cloud Service Provider. Let us assume that a user wants to reject the resources after completing the work, so there has to be a truthful and flexi- ble commitment so that user can easily rollback from the al- located resources. Let us assume that a user wants to reject the resources after completing the work, so there has to be a truthful and flexi- ble commitment so that user can easily rollback from the al- located resources. 1. Introduction Cloud computing is a distributed computing model in which the computer facilities and several other resources are provided to the user on the basis of pay as you go criteria [1]. This On-demand model mainly intended to increase the opportunities for the users by using the cloud infrastructure and application hosted in the cloud on the leased basis structure by using these facilities with the help of internet from anywhere. The different kind of infor- mation and services are offered by cloud computing in order to expand the visualization of the different IT services [2-3]. e) Measure of Service: A proper system of framework has to be present in order to estimate the usage based on the indi- vidual user handling of the resources. Concluding this part, a cloud service provider is accountable for making the resources accessible on the ondemand basis for the multiple users, accessing that cloud and manages the available resources in a proficient manner in order to achieve the user con- straints [10]. A flawless computing can only be achieved in the cloud environ- ment by satisfying the below written requirements A flawless computing can only be achieved in the cloud environ- ment by satisfying the below written requirements International Journal of Engineering & Technology, 7 (1.2) (2018) 61-65 Copyright © 2018 Uma Rani et al. This is an open access article distributed under the Creative Commons Attribu unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. opyright © 2018 Uma Rani et al. This is an open access article distributed under the Creative Commons Attribution License, which perm restricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Keywords: Decision Support System; Fuzzy Decision Making; Cloud Service Provider. 1.2. Benefits The Fuzzy inference is a definite procedure from the offered input to the output by utilizing the logic and mapping of fuzzy, that gives the base on the basis of that base the choice has been done, or pattern can be predicted. It is utilized to reproduce or simulate the decision making of human that depends on the fuzzy control rules. To relate the yields of the illative tenets, low-high inference technique is utilized [19] [20]. Carbo [5] in his work developed a trust management system using a fuzzy logic, which prevents unclear, inaccurate and unsure in- formation. Simulation has performed through combining two fuzzy sets named as S and R in which S represents the satisfaction ensures by the merchant and R represents the reputation ensures by the service provider. Thus, proposed model rotates around the reputation of different merchants and its predictions have been evaluated through buyers of the system. One aspect, i.e. independ- ent view about the behavior of the merchant is vague in this work. Bharadwaj et al. [6] discussed a model in his paper this model is based on the reputation and trust which is basically dependent on the parameters as reciprocity and an experience. Both parameters were evaluated simultaneously, in which reputation is the key parameter to make out similarities and also to discover its neigh- bors. And the parameter of trust has been evaluated on which the filtering of the neighbors has been done in order to achieve the set of person recommend their services. The proposed models here have been imitated to the movie recommender system, i.e. Movie- Lens, so as to perform compared with the BETA and EBAY repu- tation models. As a result proposed model only focuses on the trust and reputation of the entities, but deficient in taking non- biased opinion about the entities of the system. q Lofti A. Zadeh has given the methodology of the fuzzy sets firstly. He presumes that all the issues present in reality could be effort- lessly solved with effective and logical methods or by the elec- tronic PCs [15]. It is concluded from several research reports that the service pro- vider state has a high impact on the online sell-off rates, particu- larly for the high -valued items [1]. The reliance between the cus- tomers and service provider‟s can get influenced by the reputation construed from agents in their framework or system. 2. Related work Fuzzy logics have been used by different models in order to pro- vide a reliable solution to the online services. Many of the re- searchers have proposed different ideas, collaborated with the fuzzy system for evaluation of trust on a particular service provid- er. y p Initially, Lik Mui [23] has surveys existing literatures based upon trust, reputation and reciprocity. Consequently, a computational model was proposed which relies on the sociological and biologi- cal understandings of reviewing concepts. This proposed model can be applied in real system scenario for calculations of agents‟ trust and the reputation scores in future. For illustration, Falcon et al. [3] proposed a model of social- cognitive approach which evaluates the credibility of a service. It has been used with different components for evaluation and can be altered according to the required situations. Considering the other approach model implemented by Sabater and Sierra [4] defines a fuzzy logic based approach. This approach is used to analyze the relationship of different service users in e-marketplaces or elec- tronic marketplaces. In order to provide secure transmission to the users in the architecture, a mechanism has been used named as the reputation mechanism. This mechanism uses in different e-market systems such as Alibaba and Snapdeal. Accordingly, models have been developed on the basis of trust and reputation, in this paper to provide reliable and secure transmission in e-marketplaces. Alternatively, it does not offer security to the providers and the services consumers. Simone A. Ludwig [25] developed A trust approach using three sources, based on the fuzzy. A valuation of the proposed system is performed that resulted into the effectiveness and stoutness of the fuzzy approach. In [18], V. introduced a method which improved the accessibility of the cloud using the concept of fuzzy. This method enhanced the customer‟s satisfaction level of using cloud services by avoiding ambiguities in fuzzy interface system (FIS). The most important aspect of these proposed methods is that they all are based on the fuzzy evaluation and their trust on the past behaviors of the users. But the question of worry is that how a customer can choose a right or reputed CSP by taking a smart decision based on the trust. In this article, a Md Whaiduzzaman [17] this work analyzed the applications of the MCDA in the area of the selection of services in the Cloud Com- puting. 2. Related work Along with this, several techniques involved in the same approach were discussed and identified as per the case study de- pendency. Figures and results are analyzed for the approach de- fined in the cloud computing. The work of author mainly focused on the brief of cloud computing service selection by working on different case study and scenarios. Along with this a comparative study and analysis of different MCDA approaches was done for the evaluation of the results. Also the study defines the different state of MCDA techniques Decision Support System based on the fuzzy rules has been de- signed which evaluates rating of five different service providers based on Support, Feasibility, Uptime and value parameters which able the customer to pick right CSP. 1.1. Trending approaches of inference system ment by satisfying the below written requirements a) Resource Pooling: A service provider providing the cloud services should support the multitenancy of the resource, in order to maximize the efficiency of the infrastructure. For example, it should be able to dynamically assign the re- source according to the demand of the consumer. Trust becomes an important factor while using the distributed system. In such a changing environment, trust is the major concern in order to enhance the interaction between the resource user and the resource providers. b) On-demand self-service: It focuses on the available re- sources that are required by the user. These resources can be the power of the Central Processing Unit or the network re- quirements. As it is a self service, it is recommended that there is no human interference. The process of the trust evaluation is clear and uncomplicated for the user base due to the presence of fuzzy and the unique individ- ual values. It guides to the way of a clear picture of the narrative method to give the trust value in a way that can be understood and represented easily. To solve this problem fuzzy logic can be a handy approach [10]. The approach of using the fuzzy logic is a better method in order to describe the human observation. Hence, the approach of fuzzy logic is proposed in this paper in order to estimate the credibility of the service provider that is providing the different resources on the basis of different rating given by the customers. c) A broad network access: As the internet behaves as an in- teresting medium for user and the system, the availability of the broad network access becomes important for the flaw- less experience of the user while using the application avail- able on the different cloud platform. d) Rapid Elasticity: It means the flexibility in order to access the computing resource on the basis of user requirement. International Journal of Engineering & Technology 62 computing, which helps to protect the user‟s data through trust quantification of different cloud services. In this paper, the exist- ing trust concept based on dynamic requirements was enhanced. Some cloud service attributes were introduced to study layered service representation for the trust, preference and then applied fuzzy comprehensive evaluation theory to perform the trust quan- tification. 1.2. Benefits By the inves- tigating and designing these systems, this inference has been often hand-waived [1]. In addition, many reviews don't account the probability of extortion and doubt [1]. In his work, Akerlof pointed the primary issue about the data in- consistency between the purchasers and the merchants. The pur- chasers know their own particular exchanging conduct and the quality of their items they are offering. And the dealers or mer- chants can assume that what the purchasers know about them from data collected, for example, their status and reputation. The repu- tation of the trading partners is used with each other to diminish this data asymmetry to encourage the unquestioning of the ex- changing connections. [2] p y The fact of uncertainty in the e-transactions in cloud platform are considered by Nafi et al. [7] in their work. E-commerce architec- ture has proposed, which introduced a secured trust model based on the encryption and fuzzy logic. This architecture aided in less- ening the problems of the existing techniques. The Trustworthi- ness of the system is calculated through the reputation of the com- pany where a broker is considered as a third party. He is responsi- ble for maintaining the information regarding different companies and provides it to the different clients as well. Consequently, di- rect experiences of different clients were not considered in the model which lacks the system‟s performance. In this paper, an approach based on the fuzzy interface system has developed which explains how the reputation value of the particu- lar service provider can evaluate and how a customer can choose the bestreputed service provider based upon the rating values. • Direct experience of customer with different Cloud Service Providers 3. Proposed fuzzy rule based expert system The customer calculates customer re- view, the Service Provider calculates service provider‟s review, and Auditor calculates Auditor reviews. The result acquired after teaming up three components declared above is termed as the Final rating. In the given model, the rating based on each CSP is getting evaluated on the basis of three par- g g p ties, specifically, first the Cloud Customer, then the Service Pro- viders, and the Auditors. The customer calculates customer re- view, the Service Provider calculates service provider‟s review, and Auditor calculates Auditor reviews. ties, specifically, first the Cloud Customer, then the Service Pro- viders, and the Auditors. The customer calculates customer re- view, the Service Provider calculates service provider‟s review, and Auditor calculates Auditor reviews. • It is an approach for handling uncertainties in a case where firefly makes a choice in choosing best CSP through fuzzy rules. As control strategies of fireflies are best implemented through fuzzy rules. Based on these reviews, optimization algorithm has applied to obtain a rating of each component individually, such as customer rating, service provider rating and auditor‟s rating. Based on these reviews, optimization algorithm has applied to obtain a rating of each component individually, such as customer rating, service provider rating and auditor‟s rating. The main idea behind proposing a new method is the evaluation of reputed cloud service providers among various CSPs. A proposed method has been divided into three levels such as: • Firefly algorithm will help in choosing the right component based on attractiveness and desirability property or on the basis of rating using fuzzy sets. • Firefly algorithm will help in choosing the right component based on attractiveness and desirability property or on the basis of rating using fuzzy sets. • Level 1 (collaboration of reviews with fuzzy) These are the components involved in the proposed technique that provides collaborated experience for evaluation of the final rating value. • Level 2 (fuzzy with optimization) • Level 3 (Fuzzy with final rating) The central phase of this model relates the information with the preceding behaviors of different customers. The data basis on this was carried as a reputation of those users concerning different parameters such as support, features, uptime and value in the case of customer and service provider‟s review, data centers, support, monitoring and APIs in the case of auditor‟s reviews. 3. Proposed fuzzy rule based expert system These re- views are used to rate the Cloud Service provider where customers and providers rate each other using statistical ratings. This rating will be considered as a reputable report for an individual user which suggests them whether to proceed with the particular pro- vider or not. Consider an example of Amazon, which uses the scales of 1, as positive, 0 as neutral and -1 as negative respective- ly. Different customers use this parameter to evaluate the rating of different service providers on the basis of their experiences. 4. Results and discussion The implementation has been conceded out in the MATLAB software. An online survey conducted in which different users of cloud were approached in order to rate individual Cloud Service Provider based upon four different parameters such as, support, uptime, features and value on a scale of 1– 5, whereas one is con- sidered as Very Poor, two is considered as below average, three is used for the average, and our is considered above average and at last the five rating is for Excellent. Obtained results of the ratings given by the customers get stored in a form of a matrix in the software. After that Simulation is performed and the acquired results are mentioned below. 3. Proposed fuzzy rule based expert system The proposed system appraises reputation of the Cloud Service Providers based upon three components involved such as: • Direct experience of customer with different Cloud Service Providers Another scenario presented by [8] for the trust quantification was based on a fuzzy comprehensive evaluation theory for the cloud International Journal of Engineering & Technology 63 Thus, above reasons clears that optimization algorithm can work well with fuzzy systems. Moreover, from the initial stages, there are a number of customers, service providers and auditor reviews are involved, which will be difficult for the system to attain an individual rating of different components. Defined rules for the different components reviews will be extreme in numbers and becomes complicated to evaluate. But optimization algorithm can resolve the issue by providing a unique solution. From the differ- ent optimization algorithms, Firefly algorithm has been chosen because of: Thus, above reasons clears that optimization algorithm can work well with fuzzy systems. Moreover, from the initial stages, there are a number of customers, service providers and auditor reviews are involved, which will be difficult for the system to attain an individual rating of different components. Defined rules for the different components reviews will be extreme in numbers and becomes complicated to evaluate. But optimization algorithm can resolve the issue by providing a unique solution. From the differ- ent optimization algorithms, Firefly algorithm has been chosen because of: • The reputation of the Providers providing the cloud re- sources • The reputation of the Providers providing the cloud re- sources • The reputation of the Providers providing the cloud re- sources • An independent review of the Cloud Auditor The result acquired after teaming up three components declared above is termed as the Final rating. In the given model, the rating based on each CSP is getting evaluated on the basis of three par- ties, specifically, first the Cloud Customer, then the Service Pro- viders, and the Auditors. The customer calculates customer re- view, the Service Provider calculates service provider‟s review, and Auditor calculates Auditor reviews. The result acquired after teaming up three components declared above is termed as the Final rating. In the given model, the rating based on each CSP is getting evaluated on the basis of three par- ties, specifically, first the Cloud Customer, then the Service Pro- viders, and the Auditors. 3.1. Agenda of proposed technique Fig. 1: 3-D Shaded Surface Plots of Reviews Rating. Above figure represents the surf graph of the proposed model in which different component‟s review such as customer reviews and service provider‟s review has been shown. These two reviews ar input to the system, whereas final rating is the output symbolized as „z‟. On the basis of inputs, output will be obtained, i.e., aggre gated rating of three components. Fig. 1: 3-D Shaded Surface Plots of Reviews Rating. A proposed technique is not a single component; it has aggregated with different techniques to offer a reliable solution to the custom- ers. The Components involved in the proposed techniques are: Cloud computing has evolved into several areas, especially in business ideas where CSP provides services to the users as a value which needs to be paid accordingly. Numerous service providers of cloud are available for last few years, thus the customers need to make a careful decision about choosing a reputed CSP based on several parameters. In addition to this, the fuzzy comprehensive evaluation is about the analysis and the synthesis of fuzzy rela- tionships that involve one or more factors which deals with the vagueness and the subjective judgment of multiple factors accord- ing to their importance. g p A fuzzy-based trust approach has proposed which is totally based on the rating of the users and making of a wise decision among several service providers. The Cloud Service providers combined with the fuzzy interface system based on the reviews given by the users. As fuzzy is the system which is able to compute uncertain values and provides reliable outputs. Moreover, fuzzy can evaluate the human mind in an efficient way which resulted in efficient results. For the proposed work, fuzzy has combined with the op- timization algorithm as: • A single objective algorithm cannot serve the actual purpose of finding fitness value in multilevel or multi criteria data item forms as clusters. Fig. 1: 3-D Shaded Surface Plots of Reviews Rating. Fig. 1: 3-D Shaded Surface Plots of Reviews Rating. Above figure represents the surf graph of the proposed model in which different component‟s review such as customer reviews and service provider‟s review has been shown. These two reviews are input to the system, whereas final rating is the output symbolized as „z‟. On the basis of inputs, output will be obtained, i.e., aggre- gated rating of three components. 3.1. Agenda of proposed technique Above figure represents the surf graph of the proposed model in which different component‟s review such as customer reviews and service provider‟s review has been shown. These two reviews are input to the system, whereas final rating is the output symbolized as „z‟. On the basis of inputs, output will be obtained, i.e., aggre- gated rating of three components. • To define the notion of optimality in multi criteria optimiza- tion. • To translate the distributed representation into a single structure. • To translate the experts‟ advice into a symbolic representa- tion of fuzzy. • To evaluate simple fuzzy rule base method rather than a complex mathematical model in the optimization. International Journal of Engineering & Technology 64 Fig. 2: Rating of User 1 for Five Service Providers. Fig. 3: Best Rating of Proposed Work over Actual Rating. Fig. 2: Rating of User 1 for Five Service Providers. [2] G. Akerlof, “The Market for „Lemons‟: Qualitative Uncertainty and the Market Mechanism,” Quarterly Journal of Economics, vol. 84, pp. 488-500, 1970. https://doi.org/10.2307/1879431. pp p g [3] R. Falcone, Giovanni Pezzulo and Cristiano Castelfranchi, "A fuzzy approach to a belief-based trust computation," Trust, reputation, and security: theories and practice, pp. 55-60, 2003. [4] J. Sabater and C. Sierra, "Reputation and social network analysis in multi-agent systems," pp. 475-482, 2002. https://doi.org/10.1145/544741.544854. g [5] Carbo, J., Molina, J.M., Davila, J.: Trust management through fuzzy reputation. International Journal of Coop. Inf. Syst. Vol. 12, no. 01, Pp. 135–155, 2003. https://doi.org/10.1142/S0218843003000681. [6] Bharadwaj, K.K., Al-Shamri, M.Y.H.: Fuzzy computational models for trust and reputation systems. Electron. Commer. Res. Appl., vol. 8, no. 1, pp. 37–47, 2009. https://doi.org/10.1016/j.elerap.2008.08.001. [7] Nafi, K.W., Kar, T.S., Hossain, M., Hashem, M.M.A.: A new trust- ed and secured E-commerce architecture for cloud computing. In: Proceedings of International Conference on Informatics, Electron- ics & Vision (ICIEV). IEEE, pp. 1–6, 2013. Fig. 3: Best Rating of Proposed Work over Actual Rating. Fig. 3: Best Rating of Proposed Work over Actual Rating. [8] Robert H. Bonczek, Clyde W. Holsapple and Andrew B. Whinston, “The evolving roles of models in decision support systems”, Vol. 11, No. 2, Pp. 337-356, April 1980. https://doi.org/10.1111/j.1540- 5915.1980.tb01143.x. [9] Ramesh Sharda, Steve H. Barr and James C. MCDonnell, “Deci- sion Support System Effectiveness: A Review and an Empirical Test”, Management science, vol. 34, No. 2, Pp. 139-159, February 1988. https://doi.org/10.1287/mnsc.34.2.139. 3.1. Agenda of proposed technique [10] Mohammed Alhamad, Tharam Dillon, and Elizabeth Chang, “A Trust-Evaluation Metric for Cloud applications”, IJMLC, Vol. 1, No. 4, Pp. 416, October 2011. https://doi.org/10.7763/IJMLC.2011.V1.62. [11] M. Balazinski, E. Czogala and S. Gravelle, “Automatic Tool Selec- tion using a Fuzzy Decision Support System”, IEEE, Pp. 615-620, 1995. https://doi.org/10.1109/FUZZY.1995.409748. Fig. 3: Best Rating of Proposed Work over Actual Rating. Fig. 3: Best Rating of Proposed Work over Actual Rating. [12] Javier Puente, Raul Pino, Paolo Priore and David de la Fuente, “A Decision Support System for applying failure mode and effects analysis”, International Journal of Quality and Reliability manage- ment, Vol. 19, No. 2, Pp. 137-150, 2002. https://doi.org/10.1108/02656710210413480. Rating about individual service provider has taken from the differ- ent consumers or users. Each user rates particular service provider accordingly and rating of each customer has shown in terms of bar graphs in figure2. Rating before optimization regarding each com- pany declares as traditional and after optimization affirms as pro- posed. [13] Ludmil Mikhailov and M.G. Singh, “Fuzzy Analytic Network Pro- cess and its Application to the Development of Decision Support Systems”, Vol. 33, No. 1, Pp. 33-41, February 2003. https://doi.org/10.1109/TSMCC.2003.809354. Figure 3 below explains the best rating of the proposed work over the actual rating. Thus, the graph contains five service providers with respect to best rating. For each customers rating value, best rating is evaluated. [14] Nader Naderpajouh, A. Afshar and S.A. Mirmohammadsadeghi, “Fuzzy Decision Support System for Application of Value Engineering in Construction Industry”, International Journal of Civil En- gineering, Vol. 4, No. 4,Pp. 261-273, December 2006. [15] M. Baran Pouyan R. Yousefi, S. Ostadabbas and M. Nourani, “A Hybrid Fuzzy-Firefly Approach for Rule-Based Classification”, Proceedings of the Twenty-Seventh International Florida Artificial Intelligence Research Society Conference, Pp. 357-362. 5. Conclusion The reputation estimation plays an important role in the distribut- ed service oriented environment. Because of the qualities of the service oriented environment and the nature of the trust in the fuzzy, development of a fuzzy-based approach of reputation was created.The developed model calculates the overall rating of the output by taking the three parameters into account as the input parameters i.e customer review, service provider review and the auditor review as the input parameters and. With the joint effort of every segments survey, final rating value has attained. Simulations have been performed, where traditional and proposed rating has compared with each other and it has been concluded that the pro- posed optimization method is better and feasible for cloud compu- ting. Moreover, on the basis of these ratings of each customer, a decision can be taken in an effective manner regarding a best CSP. In future multiple QoS metrics can be aimed for ensuring the trust of service provider to the customer. [16] Xiaohui Li, Jingsha He, Bin Zhao, Jing Fang, Yixuan Zhang and Hongxing Liang, “A Method for Trust Quantification in Cloud Computing Environments”, IJDSN, February 2016. https://doi.org/10.1155/2016/5052614. [17] Md Whaiduzzaman Abdullah Gani, Nor Badrul Anuar, Muhammad Shiraz, Mohammad Nazmul Haque, and Israat Tanzeena Haque, “Cloud Service Selection Using Multicriteria Decision Analysis”, HINDAWI, Vol. 2014, Pages 10, 2014. https://doi.org/10.1155/2014/459375. [18] V. Prasath, Nithya Bharathan, Neetha N.P, N. Lakshmi and M.Nathiya, “Fuzzy Logic In Cloud Computing”, IJERT, Vol. 2, No. 3, March 2013. E-ISSN: 2278-0181. [19] Qi Zhang, Lu Cheng and Raouf Boutaba, “Cloud computing: state- of-the-art and research challenges”, Journal of Internet Services and Applications, vol. 1, no. 1, Pp. 7-18, May 2010. https://doi.org/10.1007/s13174-010-0007-6. p g [20] Ilango Sriram and Ali Khajeh-Hosseini, “Research Agenda in Cloud Technologies”, 2007. [21] Srinivas Sethi, Anupama Sahu and Suvendu Kumar Jena, “Efficient load Balancing in Cloud Computing using Fuzzy Logic”, IOSR- JEN, Vol. 2, No. 7, Pp. 65-71, July, 2012. ISSN: 2250-3021. [1] D. E. Houser and John wooders, “Reputation in Internet Auctions: Theory and Evidence from eBay,” Journal of Economics & Man- agement Strategy. https://doi.org/10.1111/j.1530- 9134.2006.00103.x. [24] Sini Ruohomaa and Lea Kutvonen, “Trust Management Survey”, SPRINGER, vol. 3477, pp. 77-92, 2005. [26] A. Hegyi, B. De Schutter, S. Hoogendoorn, R. Babuˇska, H. van Zuylen, and H. Schuurman, “A Fuzzy Decision Support System for Traffic Control Centers”, Pp. 358-363, August 2001. International Journal of Engineering & Technology [24] Sini Ruohomaa and Lea Kutvonen, “Trust Management Survey”, SPRINGER, vol. 3477, pp. 77-92, 2005. [25] Ludwig, S.A. Venkat Pulimi and Andriy Hnativ, “Fuzzy approach for the evaluation of trust and reputation of services”, IEEE interna- tional conference on Fuzzy systems, pp. 115-120, 2009. https://doi.org/10.1109/FUZZY.2009.5277061. [26] A. Hegyi, B. De Schutter, S. Hoogendoorn, R. Babuˇska, H. van Zuylen, and H. Schuurman, “A Fuzzy Decision Support System for Traffic Control Centers”, Pp. 358-363, August 2001. [25] Ludwig, S.A. Venkat Pulimi and Andriy Hnativ, “Fuzzy approach for the evaluation of trust and reputation of services”, IEEE interna- tional conference on Fuzzy systems, pp. 115-120, 2009. https://doi.org/10.1109/FUZZY.2009.5277061. References [22] Rachna Satsang, Dr. Pankaj Dashore and Dr.Nishith Dubey, “Risk Management in Cloud Computing Through Fuzzy Logic”, Vol. 1, No. 4, Pp. 1-4, December 2012. ISSN 2319-4847. [1] D. E. Houser and John wooders, “Reputation in Internet Auctions: Theory and Evidence from eBay,” Journal of Economics & Man- agement Strategy. https://doi.org/10.1111/j.1530- 9134.2006.00103.x. [23] Lik Mui and Ari Halberstadt, “A Computational Model of Trust and Reputation”, IEEE, 2002. 65
W4247751123.txt	https://www.cambridge.org/core/services/aop-cambridge-core/content/view/8EFFC2C3A3C84C553F1995198B9371BB/S0955603600018444a.pdf/div-class-title-new-attention-on-children-who-abuse-div.pdf	en		New attention on children who abuse	Psychiatric bulletin of the Royal College of Psychiatrists/Psychiatric bulletin	1,991	cc-by	533	Psychiatric Bulletin ( 1991 ), 15, 39 The times Private medical insurers- independent hospitals seek better communications Private medical insurers and providers of health care services need to work more closely together so that patients receive the best care possible, say the Psychiatric and Substance Abuse Standing Com mittee of the Independent Healthcare Association. Patients need to know their entitlements to health care services under their private medical insurance policies. Doctors who use independent hospitals' facilities can be confident that high standards of health care are being maintained. IH A Members will be working towards this through a working party. Commenting on these developments, David Wakefield, Chairman of the IHA Psychiatric and Substance Abuse Standing Committee and Manag ing Director of Priory Hospitals Group said, "I am delighted that providers belonging to IHA are taking steps to improve communications with the private medical insurers. It can only be of benefit to patients both in terms of quality of care and in keeping costs down". need to know whether private medical insurers rec ognise their qualifications. What is needed is an agreed set of criteria for acceptance of doctors' quali fications so that patient, doctor, hospital and insurer Psychiatric Bulletin ( 1991 ), 15, 39 New attention on children who abuse A Committee of Enquiry has been established, under the Chairmanship of National Children's Home Members of the Committee are: Rosemary Arkley, DoH Inspectorate (Observer); Ann Doyle, Rainer Foundation; Dr Arnon Bentovim, Consultant Child Psychiatrist, Great Ormond Street; Irene Bloomfield. Social Work Practitioner, Dundee; David Glasgow, Psychologist/Researcher, Liverpool University; Alan Holden, Director Harrow Social Services/Secretary Children and Family Committee ADSS; Valerie Howarth, Director, Childline; Rupert Hughes, DoH Assistant Secretary (Observer); Des McGinn, Salford Probation Service; Phillip Noyes, Head of Public Policy, NSPCC; Jennifer Temkin, Professor of Law, Buckingham University; Dr Eileen Vizard, Consult ant Child Psychiatrist, London Borough of Newham; Tom White (Chair), Chief Executive, NCH. (NCH) Chief Executive, Tom White CBE, on the issue of children and young people who abuse other children. The Committee met for the first time on 1 October 1990 and will meet over the next year on about ten occasions. The aim is to prepare a report, to include rec ommendations, by October 1991. Some of the meet ings will take the form of 'consultations' where specialists in certain fields relating to the subject of the enquiry will be invited to share their experience with the Committee. The Committee has the following terms of reference: ( 1) to investigate the problem of children and young people under 18 who sexually abuse other children, having regard to: (a) known incidence (b) extent of existing treatment facilities (c) the management of case investigation (d) appropriate forms of intervention to change and/or modify such behaviour (e) appropriate management processes needed to maximise effective service (2) to make recommendations. Secretariat: Jan van Wagtendonk (NCH Development Consult ant, Child Sexual Abuse); Cathy Cooper (NCH Policy Unit). If anyone would like to present evidence to the Committee, or has experience in this subject area, please write to Cathy Cooper, NCH, 85 Highbury Park, London N5 1UD (telephone 071 226 2053; fax 0712262537). See Correspondence, p. 43 39 https://doi.org/10.1192/pb.15.1.39-a Published online by Cambridge University Press
W2004856967.txt	https://zenodo.org/records/2187474/files/article.pdf	en		Notes on the Evidences of Human Remains from Jacobs Cavern	Science	1,903	public-domain	2,142	SCIENCE. of gain and the metabolism of the food given. From the results so far attained with thirty-five animals fed in series of five each, it would appear that there is a definite relation between the amount of lime and magnesia that enters into the animal organism., I n the case of added lime, especially with the calcium phosphate, there is an increase i n the .rate of gain with a reduction of the amount of food required per pound of increase. A very small addition of a magnesium salt also appears advantageous when fed with a lime compound, though the point of benefit is easily passed. Where the magnesium is given regularly in any excess the gain in -weight is reduced to a minimum. Though the health of the animal may be apparently unaffected and the coat noticeably smooth and glossy, there is but little gain in weight from the food consumed. Another instance of the action of magnesia may be here noted. I n fattening beef cattle for market they are fed large quantities of grain and occasionally become surfeited or, as it is termed, get 'off feed' for several days. By giving an animal in such cases magnesium sulphate it is quickly brought back to its regular ration. I n human medicine calcined magnesia is given when the system has become overcharged with food as in some cases of dyspepsia and gout. As the concentrated feeds such as grains are rich in magnesia as compared with lime, while in the growing plant the opposite is true, i t seems reasonable to suppose that magnesia operates favorably in the assimilation of food materials when present in the proper proportion, especially in heavy feeding. The results from the presence of lime and magnesia in the animal body in excessive amounts may be somewhat understood from their well-known physiological tendencies, i.e., the lime compounds are constipating, while the magnesium salts are laxative i n their ~rature. From the effect of lime compounds in the animal body from both a medical and a dietetic standpoint this element may be said to be constructive and .fixative i n its results. On the other hand, magnesia is more movable in its relation, serving to carry assimilable phosphoric acid, which i t gives up readily, and is thereby enabled to repeat the process many times. Therefore, a too small amount of magnesia is less detrimental than a deficiency of lime. If, however, with magnesia in excess there is a tendency for the lime as the stronger base to unite with the acid of the magnesium salt and the magnesia to form magnesium nucleo-proteids, such a disturbance would result in the elimination of products rather than in a further constructive effort. I n the physiological effect of an excess of magnesia in the animal organism we find such a result indicated. D. W. MAY. KENTUCKY STATION. EXPERIMENT NOTES ON THE EVIDENCES O F HUMAN REMAINS FROM JACOBS' CAVERN. BY the courtesy of Dr. Charles Peabody, Director of the department of archeology, Phillips Academy, Andover, Mass., and of Professor Warren K. Moorehead, curator of this department, the writer was permitted during the month of May, 1903, to examine and assist in excavating a cave in southwest Missouri, in which were found numerous evidences of human occupancy. The cave is located on the north bluff of Little Sugar Creek two and one half miles southwest of Pineville, the county seat of McDonald County, four miles from the Arkansas line and fourteen east of the Indian Territory line. I n honor of the discoverer, Mr. E. H. Jacobs, an enthusiastic archeologist of Bentonville, Ark., the cave has been name& Jacobs' cavern. To each of these gentlemen are due my sincere thanks for many kindnesses extended during a week's visit to their camp. The hills along Sugar Creek are composed of massive ledges of limestone containing a large amount of flint and chert, in the form either of regular layers or of nodular concret i o y To this formation the name Boone chert has been applied by geologists. It is the rock that outcrops extensively in the southwest part of Missouri, the northeastern part of the Indian Territory and northern Arkansas. I n the lower part of the Boone, flint is often absent and the rocks consist of massive gray limestone arranged in definite 152 SCIENCE. layers. To this part of the formation the name St. Joe limestone has been applied. The St. Joe is sometimes sixty feet or more thick, and often weathers into characteristic precipitous or overhanging bluffs extending for miles along the streams. Immediately beneath the St. Joe limestone is a mass of shales sometimes attaining a thickness of fifty feet, known as the Eureka shales. These shales are usually black and papyraceous, weathering into thin flakes or tablets. Throughout the region thousands of springs issue from between the Eureka and the St. Joe. Jacobs' cavern is located in the St. Joe limestone some forty feet above the level of Little Sugar Creek. The cave faces southwest, overlooking the narrow valley. The bluff above the cave continues to the height of one hundred and fifty feet or more, the upper part being composed of Boone chert. The cave is in fact but a rock shelter, irregxlarly TT-shaped in outline, with floor, walls and roof of limestone. The flat top is composed of a single stratum of limestone, and stratification lines are well exhibited on the sides of the cave. Along the front the entire length of the rock shelter is approximately seventy feet; the extreme depth is fifty feet. Before removing any of the contents the height was from four to seven feet. The floor was covered, however, by two deposits, one of clay and one of ashes, aggregating six feet thick, so that the distance from the limestone floor to the limestone roof is approximately twelve feet. The rock floor was covered to the depth of three feet with clay, usually yellowish-brown in color, containing numerous fragments of limestone. This clay was probably formed by the disintegration of the limestone and so far as noticed has never been disturbed. On the clay was a layer of wood ashes averaging three feet in thickness. Throughout the greater part of the cave these ashes were so loose and dry that the men engaged in removing them were obliged to use sponges i11 order to avoid breathing the ashes. I n fact several of the men were unable to continue [N. S. VOL. XVIII. NO. 448. the work on this account. Mingled with the ashes and sonletimes extending into the subjacent clay were slabs and blocks of limestone fallen from the roof. At the back of the cave there is a fissure extending upward to the height of ten feet or more, separating the roof of the cave from the rear wall. This fissure, which is probably a master joint in the limestone, is from eighteen inches to three feet wide and continues for some distance beyond the main part of the cave, where it divides into a lower and an upper part separated by a block of limestone. All along this fissure and also along part of the back of the cave beyond the point where the fissure extends, there are numerous stalactites, stalagmites and pilasters formed by water dripping from the roof. I n places the entire fissure above the level of the roof is filled with this material. The col?tinued dripping of water carrying CaCO, on the ashes covering the floor of the cave has formed a sort of stala-mitip ash breccia often enclosing flint flakes, implements and bones. I n these stalagmites charcoal is often present. That this ash breccia was formed gradually and after the deposition of the ashes is proved by the peculiar toadstool-like shape of some of the pillars. I t seems from the shape that ashes were first laid down, then the dripping of the water formed the brecciated mass, then other ashes were deposited, other breccia formed, then further deposits of ashes, and so on till the entire pillar was formed. The clay beneath the ashes near the back part of the cave is in many places cemented by the action of lime forming a clay and limestone breccia. Scattered about in the ashes and enclosed in the stalagniitic breccia at the back of the cave were found a number of objects which point to the fact that the cave has been occupied by man. These objects may be divided into eleven groups, of which seven may be considered as witnessing to human occupancy, and four may or may not bear such testimony. The objects are as follows: A. Objects witnessing to the human occupancy of Jacobs7 cavern : (1) Human bones, (2) pottery, (3) flint implements, (4) stone SCIENCE. implements, (5) bone implements, (6) clam shells, ( 7 ) ashes and charcoal. B. Objects which do not certainly bear testimony to human occupancy: (8) Flint flakes, (9) animal bones, (10) sandstone fragments, (11) polished rocks. I t will be obviously impossible in a paper of this kind to do more than simply mention these objects. All detailed study must be reserved for those more skilled in the discussion of such data. Human Bones.-Fra,ments of at least four human skeletons were discovered in the ashes. One of these skeletons, including a skull in a good state of preservation, was nearly complete. Pottery.-Fra,qents of at least six vessels were found, including one handle. Several of the fragments were decorated. Flilzt Implements.-Chipped flint implements are quite common, more than one hundred specimens having been found. These implements include arrow points, drills, spear points, knives, scrapers, etc., as well as cores from which knives were obtained. The flint is in most cases similgr to that found on the hills near by, but in some cases it is believed to have been carried for considerable distances. Stone Implements.-One large stone mortar was found, as well as hammer stones, a stone hatchet and stones used for sharpening implements. Bone Implements.-Several awls, needles, scrapers and other implements fashioned from bone were secured. Clam #hells.-A number of shells of Unio were taken from the ashes. At least two genera are represented, both probably being found at the present time in Sugar Creek. Ashes and Charcoal.-As stated above, the floor of the cave was covered to the depth of three feet with ashes. A comemative estimate would place the amount of ashes at 5,000 cubic feet. Intermingled with the ashes was a large amount of charcoal varying in size from small specks to lumps the size of a walnut. I t was in the ashes that the other objects mentioned in this paper were found. Flint Flakes.-Thousands of flakes of flint were found in the ashes and embedded in the stalagmites. This flint varies in size from sinall slivers to pieces the size of the hand. Careful search was made along the walls and roof of the cave to detect the presence of flint in the limestone, but without success. , There is plenty of flint at a horizon fifty feet higher, but so far as known there is none in the strata in which the cave is located. For this reason it is believed that the flint was carried into the cave. Animal Bones.-Great numbers of bbnes of various animals, including mammals, birds and turtles, were found among the ashes and embedded in the stalagmites. These bones have not yet been identified but i t is probable that a large part of them are those of living species. #andstone Fragments.-A number of small pieces of unshaped sandstone were obtained. The nearest point, so far as known, where sandstone outcrops is four miles distant from the cave, in the viciniti of White Rock. I t seems probable that the sandstone has been carried into -tLe cave. Pobislzed Lirne~tone.-~4 number of flat limestone slabs that have fallen from above, both just within the cave's mouth and particularly along the foot of the bluff a few feet distant, have been polished or glazed apparently by the friction or contact of greasy bodies. These polished rocks are invariably in such a position as most readily to serve as seats or reclining places for the inhabitants of the cave. There are more than twenty of these slabs that exhibit this peculiar structure. CHARLES NEWTON GOULD. THEUNIVERSITY OF OKLAHOMA, May 16, 1903. KEW TERMS IN CHEMISTRY. I , may ~ not be out of placeto attestion to several new terms which have recently been to the English-speaking scientific world and to discuss their merits. However reluctant we may be, in view of possible misunderstandings, to accept new words and phrases, the need of them is often unquestionable, and it only remains for us to determine the proper forms which the words shall talre.
https://openalex.org/W4284970969	http://qspace.qu.edu.qa/bitstream/10576/42695/1/s41598-023-29655-0.pdf	English	null	Greater Traditionalism Predicts COVID-19 Precautionary Behaviors Across 27 Societies	null	2,022	cc-by	11,932	www.nature.com/scientificreports www.nature.com/scientificreports Greater traditionalism predicts COVID‑19 precautionary behaviors across 27 societies OPEN Theodore Samore 1, Daniel M. T. Fessler 2, Adam Maxwell Sparks 45, Colin Holbrook 3, Lene Aarøe 4, Carmen Gloria Baeza 5, María Teresa Barbato 5, Pat Barclay 6, Renatas Berniūnas 7, Jorge Contreras‑Garduño 8, Bernardo Costa‑Neves 9,10, Maria del Pilar Grazioso 11,12, Pınar Elmas 13, Peter Fedor 14, Ana Maria Fernandez 5, Regina Fernández‑Morales 15,16, Leonel Garcia‑Marques 17,18, Paulina Giraldo‑Perez 19, Pelin Gul 20, Fanny Habacht 21, Youssef Hasan 22, Earl John Hernandez 23, Tomasz Jarmakowski 24, Shanmukh Kamble 25, Tatsuya Kameda 26,27,28, Bia Kim 29, Tom R. Kupfer 30, Maho Kurita 26, Norman P. Li 31, Junsong Lu 32, Francesca R. Luberti 33, María Andrée Maegli 34, Marinés Mejia 12, Coby Morvinski 35, Aoi Naito 26,36, Alice Ng’ang’a 37, Angélica Nascimento de Oliveira 6, Daniel N. Posner 38, Pavol Prokop 14,39, Yaniv Shani 40, Walter Omar Paniagua Solorzano 34, Stefan Stieger 21, Angela Oktavia Suryani 41, Lynn K. L. Tan 31, Joshua M. Tybur 42, Hugo Viciana 43, Amandine Visine 44, Jin Wang 32 & Xiao‑Tian Wang 32 People vary both in their embrace of their society’s traditions, and in their perception of hazards as salient and necessitating a response. Over evolutionary time, traditions have offered avenues for addressing hazards, plausibly resulting in linkages between orientations toward tradition and orientations toward danger. Emerging research documents connections between traditionalism and threat responsivity, including pathogen-avoidance motivations. Additionally, because hazard- mitigating behaviors can conflict with competing priorities, associations between traditionalism and pathogen avoidance may hinge on contextually contingent tradeoffs. The COVID-19 pandemic provides a real-world test of the posited relationship between traditionalism and hazard avoidance. Across 27 societies (N = 7844), we find that, in a majority of countries, individuals’ endorsement of tradition positively correlates with their adherence to costly COVID-19-avoidance behaviors; accounting for some of the conflicts that arise between public health precautions and other objectives further strengthens this evidence that traditionalism is associated with greater attention to hazards. 1Department of Anthropology, Center for Behavior, Evolution, and Culture, University of California, Los Angeles, CA 90095, USA. 2Department of Anthropology, Center for Behavior, Evolution, and Culture, Bedari Kindness Institute, University of California, Los Angeles, CA 90095, USA. 3Department of Cognitive and Information Sciences, University of California, Merced, CA 95343, USA. 4Department of Political Science, Aarhus University, 8000 Aarhus C, Denmark. 5Laboratorio de Evolución y Relaciones Interpersonales, Universidad de Santiago de Chile, Santiago, Chile. www.nature.com/scientificreports/ 28Center for Experimental Research in Social Sciences, Hokkaido University, Sapporo 060‑0810, Japan. 29Department of Psychology, Pusan National University, Busan, South Korea. 30Department of Psychology, Nottingham Trent University, Nottingham NG1 4FQ, UK. 31School of Social Sciences, Singapore Management University, Singapore 188065, Singapore. 32School of Humanities and Social Science, The Chinese University of Hong Kong (Shenzhen), Shenzhen 518172, China. 33Department of Psychology, Nipissing University, North Bay, ON P1B 8L7, Canada. 34Department of Psychology, Universidad del Valle de Guatemala, Guatemala City 01015, Guatemala. 35Department of Management, Ben-Gurion University of the Negev, Be’er Sheva, Israel. 36Japan Society for the Promotion of Science, Tokyo 102‑0083, Japan. 37Lazaridis School of Business and Economics, Wilfrid Laurier University, Waterloo, ON N2L 3C5, Canada. 38Department of Political Science, University of California, Los Angeles, CA 90095, USA. 39Institute of Zoology, Slovak Academy of Sciences, 845 06 Bratislava, Slovakia. 40Coller School of Management, Tel Aviv University, Tel Aviv, Israel. 41Atma Jaya Catholic University of Indonesia, Jakarta 12930, Indonesia. 42Department of Experimental and Applied Psychology, Vrije Universiteit Amsterdam, Amsterdam 1081 HV, The Netherlands. 43Departamento de Filosofía y Lógica y Filosofía de la Ciencia, Universidad de Sevilla, 41018 Seville, Spain. 44L’Institut Agro Montpellier, 34060 Montpellier, France. 45Adam Maxwell Sparks is an independent scholar. email: theo.samore@gmail.com Traditionalism—the tendency to embrace what are perceived to be the longstanding norms and values of one’s group, while rejecting changes to them—varies across individuals1. Given the centrality of sociality and culture for humans, individuals’ orientations toward traditions have important downstream consequences. These include the tendency to embrace or reject innovations in the face of environmental change2, the ability to coordinate actions with fellow group members3, and the shaping of political attitudes and ideologies in democratic contexts4. It is therefore vital to understand factors that contribute to variation in traditionalism.f Emerging research demonstrates associations between individual differences in traditionalism and varia- tion in the propensity to attend, and respond, to hazards3,5. Initial evidence indicates that individual variation in traditionalism may in part associate with variation in pathogen avoidance, the motivation to take actions to alleviate the costs of potential pathogen threats3,6–10. Hence, what is termed the traditional norms account7 identifies pathogen avoidance as an important factor relating to traditionalism. www.nature.com/scientificreports/ Consistent with the traditional norms account, multiple evolutionary pathways may lead individuals to leverage adherence to tradition as a way of ameliorating danger3,7.i g g First, as a result of cultural evolutionary processes favoring beliefs and practices that benefit individual and group fitness11, some traditions may have instrumental value for addressing particular pathogen threats12. While it is possible that individuals explicitly or implicitly understand the connections between some instrumental norms and their outcomes, the functionality of norms is frequently opaque to those who adopt them13,14. If the average instrumental benefit of adhering to traditions when confronting danger outweighs the costs of impreci- sion resulting from causal opacity, then individuals may be motivated to broadly embrace traditions in pursuit of safety. Co-evolution may have resulted in psychological adaptations or reaction norms connecting traditions to threat if the above cost–benefit structure was common over evolutionary time.hi i y The benefits of sociality generate a second pathway by which an association between traditionalism and the salience of pathogen threats could arise. Adherence to traditional norms might provide broad payoffs via increased social support, for example by signaling in-group identity in cooperative exchanges and systems of indirect reciprocity, and/or by facilitating in-group coordination15–17. Such benefits might plausibly include cost amelioration in the face of pathogen threats, for example by obtaining care and resources during periods of illness18. For all of the above possibilities, natural selection could have produced either (a) stable dispositional linkages between pathogen-threat concerns and long-term preferences for tradition, (b) facultative plasticity, such that individuals prophylactically upregulate their embrace of tradition in response to cues indicating an increased risk of disease, or (c) both. Together, these considerations generate the prediction that, ceteris paribus, relative to individuals less invested in tradition, those who evince greater traditionalism will be more inclined to attempt to diminish the risk of acquiring transmissible disease. Note that the theorized connection between traditionalism and threat avoidance mirrors a similar putative relationship between social conservatism and threat avoidance, where socially conservative beliefs reflect sup- port for traditionsee19,20 in contexts where people hold political ideologies. Indeed, much of the theoretical work connecting traditional attitudes with threat reactivity comes out of political psychology, where extensive prior research has long recognized the role that motives to mitigate uncertainty, fear, and threat—particularly disease threats and threats to the stability of the social system—play in shaping socially conservative ideology19,21–23. www.nature.com/scientificreports/ University of Groningen, Campus Fryslân, Groningen, The Netherlands. 21Division of Psychological Methodology, Department of Psychology and Psychodynamics, Karl Landsteiner University of Health Sciences, Krems an der Donau, Austria. 22Psychology Program, Department of Social Sciences, College of Arts and Sciences, Qatar University, 2713 Doha, Qatar. 23College of Arts and Sciences, Partido State University, Goa 4422, Camarines Sur, Philippines. 24Institute of Psychology, Nicolaus Copernicus University in Toruń, 87‑100 Toruń, Poland. 25Department of Psychology, Karnatak University, Dharwad, Karnataka 580003, India. 26Department of Social Psychology, The University of Tokyo, Tokyo 113‑0033, Japan. 27Brain Science Institute, Tamagawa University, Tokyo 194‑8610, Japan. 28Center for Experimental Research in Social Sciences, Hokkaido University, Sapporo 060‑0810, Japan. 29Department of Psychology, Pusan National University, Busan, South Korea. 30Department of Psychology, Nottingham Trent University, Nottingham NG1 4FQ, UK. 31School of Social Sciences, Singapore Management University, Singapore 188065, Singapore. 32School of Humanities and Social Science, The Chinese University of Hong Kong (Shenzhen), Shenzhen 518172, China. 33Department of Psychology, Nipissing University, North Bay, ON P1B 8L7, Canada. 34Department of Psychology, Universidad del Valle de Guatemala, Guatemala City 01015, Guatemala. 35Department of Management, Ben-Gurion University of the Negev, Be’er Sheva, Israel. 36Japan Society for the Promotion of Science, Tokyo 102‑0083, Japan. 37Lazaridis School of Business and Economics, Wilfrid Laurier University, Waterloo, ON N2L 3C5, Canada. 38Department of Political Science, University of California, Los Angeles, CA 90095, USA. 39Institute of Zoology, Slovak Academy of Sciences, 845 06 Bratislava, Slovakia. 40Coller School of Management, Tel Aviv University, Tel Aviv, Israel. 41Atma Jaya Catholic University of Indonesia, Jakarta 12930, Indonesia. 42Department of Experimental and Applied Psychology, Vrije Universiteit Amsterdam, Amsterdam 1081 HV, The Netherlands. 43Departamento de Filosofía y Lógica y Filosofía de la Ciencia, Universidad de Sevilla, 41018 Seville, Spain. 44L’Institut Agro Montpellier, 34060 Montpellier, France. 45Adam Maxwell Sparks is an independent scholar. *email: theo.samore@gmail.com University of Groningen, Campus Fryslân, Groningen, The Netherlands. 21Division of Psychological Methodology, Department of Psychology and Psychodynamics, Karl Landsteiner University of Health Sciences, Krems an der Donau, Austria. 22Psychology Program, Department of Social Sciences, College of Arts and Sciences, Qatar University, 2713 Doha, Qatar. 23College of Arts and Sciences, Partido State University, Goa 4422, Camarines Sur, Philippines. 24Institute of Psychology, Nicolaus Copernicus University in Toruń, 87‑100 Toruń, Poland. 25Department of Psychology, Karnatak University, Dharwad, Karnataka 580003, India. 26Department of Social Psychology, The University of Tokyo, Tokyo 113‑0033, Japan. 27Brain Science Institute, Tamagawa University, Tokyo 194‑8610, Japan. Greater traditionalism predicts COVID‑19 precautionary behaviors across 27 societies OPEN 6Department of Psychology, University of Guelph, Guelph, ON N1G 2W1, Canada. 7Institute of Psychology, Vilnius University, Vilnius, Lithuania. 8Escuela Nacional de Estudio Superiores, Universidad Nacional Autónoma de México, Unidad Morelia, 58190 Morelia, Michoacán, Mexico. 9Lisbon Medical School, University of Lisbon, 1649‑028 Lisbon, Portugal. 10Centro Hospitalar Psiquiátrico de Lisboa, 1749‑002 Lisbon, Portugal. 11Centro Integral de Psicología Aplicada, Universidad del Valle de Guatemala, Guatemala City 01015, Guatemala. 12Proyecto Aiglé Guatemala, Guatemala City, Guatemala. 13Department of Psychology, Adnan Menderes University, Aydın, Turkey. 14Department of Environmental Ecology and Landscape Management, Faculty of Natural Sciences, Comenius University, 842 15 Bratislava, Slovakia. 15Facultad de Humanidades, Universidad Rafael Landivár, Guatemala City 01016, Guatemala. 16Departamento de Psicología, Universidad Francisco Marroquín, Guatemala City, Guatemala. 17CICPsi Research Center for Psychological Science, Universidade de Lisboa, Lisbon, Portugal. 18School of Psychology, Universidade de Lisboa, Lisbon, Portugal. 19The School of Biological Sciences, The University of Auckland, Auckland 1010, New Zealand. 20Department of Sustainable Health, \| https://doi.org/10.1038/s41598-023-29655-0 Scientific Reports \| (2023) 13:4969 www.nature.com/scientificreports/ www.nature.com/scientificreports/ Hence, at present, the extent to which traditionalism and threat-avoidance behaviors are related across the highly variable traditional practices and beliefs of diverse societies is not fully known. p y Encouragingly, research has begun to take the costs of pathogen avoidance into account10,28,29. Likewise, though relying on hypothetical scenarios, a recent study examined the relationship between disgust sensitivity and traditionalism in a large cross-cultural sample7. However, to date, no large-scale international investiga- tion has addressed the relationship between pathogen avoidance and traditionalism in a real-world context, or assessed the potential for conflicts between pathogen avoidance and competing goals to impact said relationship. The COVID-19 pandemic affords such research.h h pf The pandemic involves a pathogen threat that is both salient for much of the world’s population30 and has had marked effects on behavior31. Further, these real-world behaviors are inherently costly32, and may epitomize the kinds of cost–benefit tradeoffs individuals face when various priorities are perceived to clash. Moreover, individuals are influenced by their information environments, which can in turn shape perceptions of costs and benefits regardless of the actual underlying distribution. Concordantly, from an error management perspective33, individuals must balance the relative costs and frequencies of type 1 and type 2 errors when it comes to disease threats (i.e. the cost of taking insufficient precautions against a hazardous disease versus the social and opportu- nity costs entailed by being overly cautious). Indeed, individuals appear to be influenced by decision processes that reduce the probability of committing the more costly error in the context of disease avoidance34,35. In addi- tion to the tradeoffs between disease avoidance and social opportunities, in social ecologies wherein COVID-19 precautions are positively or negatively moralized, error-management considerations will likely also include the reputational costs of locally counter-normative behavior.h p y The traditional norms account of the relationship between traditionalism and threat avoidance predicts that, all else equal, precautionary COVID-19 health behaviors should correlate with traditionalism, given that such behaviors can accurately index general pathogen avoidance motivations by virtue of occurring in a real- world context. Specifically, if traditionalism and pathogen avoidance motivations are linked, then the extent to which individuals engage in COVID-19 prophylaxis should correspond with the extent to which they embrace traditions. www.nature.com/scientificreports/ (compare to the proximate motivation to reduce threat), epistemic motivations (compare to the potential instru- mental value of traditionalism/conservatism in reducing threat), and relational motivations (compare to the potential sociality benefit of traditionalism/conservatism in reducing threat) that underly political ideologies24.i (compare to the proximate motivation to reduce threat), epistemic motivations (compare to the potential instru- mental value of traditionalism/conservatism in reducing threat), and relational motivations (compare to the potential sociality benefit of traditionalism/conservatism in reducing threat) that underly political ideologies24.i p yi g ) y p g Although adherence to tradition can provide benefits, it can also entail costs. In addition to political consid- erations, there are often tangible costs to sticking to the tried-and-true—most notably because innovations may generate higher payoffs than existing practices. Any given manifestation of a linkage between threat-mitigating behavior and traditionalism may therefore depend in part on how individuals assign weights to the cost–benefit structure characterizing the specific context, and exceptions to that connection should be expected when com- peting priorities arise. Moreover, behaviors that mitigate the costs of a threat may lead to costs in other areas, either directly, or indirectly due to the zero-sum nature of the time, attention, and resources available. Taken in sum, the relationship between traditionalism and pathogen avoidance may not be straightforward if responses to pathogen threats are perceived to clash with other priorities. p g p p Much of the previous literature on the relationship between traditionalism and pathogen avoidance does not take account of the costs of the latter. Investigators often rely on subjective responses to hypothetical scenarios(e.g.25,26)—for example, feeling sick after witnessing someone vomit—that do not distinguish the real- world contexts, conflicting goals, or costs of the relevant behaviors (such as opportunity costs, allocation trade- offs between—or vulnerabilities to—different threats, etc.). Using hypothetical scenarios is sensible in research that aims to measure emotional and/or behavioral tendencies—which may correlate with general behavioral tendencies27—while holding contextual factors equal. However, hypotheticals cannot capture the specific trade- offs that likely determine how such propensities play out in consequential real-world decision making.i f y p p p y q g Past research predominantly employs samples from a narrow range of societies. Given that cost–benefit structures are likely culturally variant, the observed associations between traditionalism and pathogen avoidance may be rooted in aspects of particular practices, values, or beliefs within those societies. www.nature.com/scientificreports/ In the present research, our focus is on traditionalism writ large rather than social conservatism in particular. Political ideologies are culturally relevant in some contexts but not others. In contrast, by virtue of their translat- ability across cultural and political contexts, attitudinal antecedents such as traditionalism are better suited for large-scale cross-cultural investigation. That said, the underlying evolutionary logic presented here draws on, and is consistent with, seminal theoretical perspectives in political psychology that identify the existential motivations Scientific Reports \| (2023) 13:4969 \| https://doi.org/10.1038/s41598-023-29655-0 www.nature.com/scientificreports/ q Do COVID‑19 health precautions, as potential manifestations of general pathogen avoidance tendencies, positively correlate with traditionalism across diverse societies? Our primary goal Consistent with the importance of tradeoffs in shaping the relationship between pathogen avoidance and traditionalism, we expect such suppression to occur when competing priorities that also associate with traditionalism, such as personal liberties, are perceived to clash with COVID-19 precautions. p p It is an open question whether, in other societies, individuals similarly weight the components of the cost–benefit tradeoffs previously identified in the U.S. On the one hand, many aspects of the U.S.’ socio-political environment are unlikely to generalize beyond its borders. On the other hand, pathogen avoidance precautions— particularly in the case of COVID-19—may commonly be perceived to clash with benefits derived from social interaction, including both economic and community activity. Therefore, in the present study, we also sought to investigate the extent to which the suppressive dynamics identified in previous research in the U.S.10 emerge across a much broader range of socio-political contexts. g Drawing on the previous research conducted in the U.S., we tested seven theoretically relevant variables that may suppress traditionalism-precautions relationships in some cultural contexts. First, we measured concerns over personal liberties and the economy, as well as perceived tradeoffs between personal liberties, the economy, and the practice of traditions on the one hand, and COVID-19 public health precautions on the other. Here, we explicitly pitted public health precautions against priorities that have been commonly perceived to clash in some societal contexts. Second, we measured trust in scientists regarding COVID-19 information. Because many scientific explanations for natural phenomena are incompatible with many traditional explanations thereof, trust in scientists may negatively correlate with traditionalism in many cultural contexts. If this is the case, and if COVID-19 public health precautions are perceived to derive from the advice of scientists, traditionalists may discount these precautions, resulting in suppression of any direct positive relationships between traditionalism and COVID-19 precautions. The precise configuration, however, will depend on culturally parochial relation- ships between traditional and scientific meaning systems. pi g y Finally, related to the logic regarding trust in scientists, we included a measure of SDO. SDO contributes to distrust in both scientists and various scientific findings, likely because scientists are more likely to be viewed as actors seeking to disrupt the social hierarchies preferred by individuals with higher SDO42. This may be particularly true when hierarchy-promoting authoritarian leaders denounce the legitimacy of scientists in the context of COVID-19, or imply that their recommended practices are only for the weak. q Do COVID‑19 health precautions, as potential manifestations of general pathogen avoidance tendencies, positively correlate with traditionalism across diverse societies? Our primary goal q Do COVID‑19 health precautions, as potential manifestations of general pathogen avoidance tendencies, positively correlate with traditionalism across diverse societies? Our primary goal tendencies, positively correlate with traditionalism across diverse societies? Our primary goal was to assess whether the hypothesis that traditionalism and pathogen avoidance covary at the individual level obtains across a wide array of cultural contexts. Specifically, we were interested in whether individuals’ choices to adopt precautionary COVID-19 behaviors positively associated with their own endorsement of traditional- ism. We used individuals’ self-reports of their actual COVID-19 precautionary health behaviors (such as mask wearing, social distancing, and supplement taking) as a complex, real-world manifestation of pathogen avoid- ance behavior. We selected precautionary behaviors that had been widely adopted across the globe, and that had been plausibly viewed as medically- or public health-derived preventative measures by experts and/or laypeople. The actual efficacy of the precautions in question varied. In contrast to previous methods that left the costs of pathogen avoidance unspecified, individuals’ decisions about COVID-19 precautions intrinsically embody the kinds of tradeoff calculations discussed above.i f Because specific traditions and cultural practices vary substantially across societies, to measure traditional- ism, we examined individuals’ general tendency to endorse or reject the traditional norms and values of their society writ large, rather than the specific content of those traditions themselves. This allowed us to measure traditionalism in a relatively consistent manner across study sites, affording comparisons despite wide variation in the contents of traditions. Testing the individual-level relationship between traditionalism and COVID-19 precautions across many cultural contexts was important for at least two reasons. First, given claims of an evolved link between traditional- ism and general pathogen avoidance, it is critical to determine whether that relationship is evident across a broad swath of humanity. Second, given that clashes between pathogen avoidance and other priorities are likely often parochial as a function of different cultural values and beliefs, examining the individual-level traditionalism- pathogen avoidance relationship across many societies affords identification of overarching patterns despite local variation. Do perceived tradeoffs between health precautions and other priorities influence the tradi- tionalism‑precautions relationship? Parochial factors interacting with individual preferences may con- ceal direct relationships between pathogen avoidance and traditionalism. For example, a recent study found evi- dence that, in the U.S., greater economic conservatism, greater social dominance orientation (SDO), and lower trust in science statistically suppressed the direct precautions-traditionalism relationship10. www.nature.com/scientificreports/ conservatism in the U.S., Poland, and the U.K. following the start of the pandemic8,37,38, but see39. However, these results come from only three societies, and may be contingent on the parochial conditions obtaining therein, notably including the extensive politicization of the pandemic in the U.S. and Poland40,41. We therefore investi- gated the relationship between COVID-19 precautions and traditionalism across 27 countries, examining both the zero-order relationships and the direct relationships after statistically accounting for indirect effects (i.e., mediation or suppression) of variables related to the perception that COVID-19 precautions exacerbate other threats or otherwise conflict with competing priorities. www.nature.com/scientificreports/ Despite the apparent simplicity of the above prediction, all else may not be equal in the case of reactions to the current pandemic, as group-level and individual-level contextual factors may parochially shape the perceived cost–benefit structure of COVID-19 health precautions. For example, at the group level, precautions promulgated by public health authorities may be seen as threatening economic prosperity or personal liberty to a greater extent in some cultural contexts than in others. Individual assessments of those countervailing tradeoffs, shaped by the social and political environment, will likely vary as well. Furthermore, some public health precautions may directly interfere with traditional practices; for example, social distancing restrictions preclude the kinds of ritual gatherings that are often important for religious services and other activities central to in-group identity. Finally, as stated above, individuals’ characterizations of the cost–benefit structures may or may not be accurate: miscalculations or erroneous beliefs can arise. In particular, for politically, ideologically, and socially salient issues such as the pandemic, individuals’ information environments may shape inaccurate beliefs about such tradeoffs. In sum, these clashes potentially reduce, or even reverse, the observed relationship between pathogen avoidance behaviors—in this case, COVID-19 health precautions—and traditionalism.f p Recent research has found support for both the traditional norms account and the presence of tradeoffs. At the national level, consistent with the logic connecting traditions and threat mitigation, researchers have found that greater cultural tightness (i.e. stronger and more heavily enforced social norms and constraints) correlated negatively with COVID-19 incidence rates36. At the individual level, two recent studies in the U.S10 found that variables such as greater economic conservatism and lower trust in scientists statistically suppressed the traditionalism-COVID-19 precautions relationship. Concordantly, consonant with the close relationship between traditionalism and social conservatism4, other research provides evidence for an increase in social Scientific Reports \| (2023) 13:4969 \| https://doi.org/10.1038/s41598-023-29655-0 www.nature.com/scientificreports/ www.nature.com/scientificreports/ some socio-political contexts44,45, SDO might act as a statistical suppressor of any direct relationship between traditionalism and COVID-19 precautions when the above conditions are met.if We did not make specific predictions about the effects of each of the above variables at each of the study sites, and we did not expect to find suppression across all countries given the likelihood that many of these tradeoff dynamics are parochial. Further, this was not an exhaustive test of every possible dynamic that may be relevant to the zero-order relationship across individual societies. Rather, we sought to explore the generalizability of the extent to which the particular factors operating in the U.S. also exert suppressive effects elsewhere, perhaps reflec- tive of some relatively common ways in which pathogen avoidance behaviors can clash with competing priorities. q Do COVID‑19 health precautions, as potential manifestations of general pathogen avoidance tendencies, positively correlate with traditionalism across diverse societies? Our primary goal Likewise, SDO may reflect preferences for fewer constraints on individual liberties regardless of their effects on public goods43. Because traditionalism also intersects with preferences for authoritarian leaders1, and associates with SDO in Scientific Reports \| (2023) 13:4969 \| https://doi.org/10.1038/s41598-023-29655-0 www.nature.com/scientificreports/ Results Baseline relationships between COVID‑19 precautions and traditionalism across study sites. Treating each study site as a separate sample, we conducted a random effects meta-analysis to test the extent to which overall indices of COVID-19 precautions and traditionalism were related across study sites (see Figs. 1 and 2). At the majority of study sites (16 of 27), the relationship between traditionalism and COVID-19 precautions was positive and significant, as was the overall meta-analyzed point estimate representing a weighted average of the effects found for each study site (r = 0.19, 95% confidence interval [0.14, 0.24]; note that the 95% confidence interval for the overall estimate does not overlap with zero). There was also substantial variation Dotted lines are study site-specific product-moment correlations between traditionalism and COVID-19 health precautions. The solid thick line is the unweighted product-moment correlation in the pooled sample across all study sites. Dots show individual data points, jittered along the x- and y-axes to aid interpretability. Density plots along the x- and y-axes represent the raw distributions of the traditionalism and COVID-19 health precautions composites. Thin grey lines show density distributions at individual study sites, whereas the thick black lines show the overall distribution in the pooled sample across all study sites. Study sites are unlabeled to improve readability. For labeled study-site specific correlations and density distributions, see Figs. S2–S4 in the Supplement. Figure 2. Graphical visualization of the country-specific correlations listed in Fig. 1. Dotted lines are study site-specific product-moment correlations between traditionalism and COVID-19 health precautions. The solid thick line is the unweighted product-moment correlation in the pooled sample across all study sites. Dots show individual data points, jittered along the x- and y-axes to aid interpretability. Density plots along the x- and y-axes represent the raw distributions of the traditionalism and COVID-19 health precautions composites. Thin grey lines show density distributions at individual study sites, whereas the thick black lines show the overall distribution in the pooled sample across all study sites. Study sites are unlabeled to improve readability. For labeled study-site specific correlations and density distributions, see Figs. S2–S4 in the Supplement. across study sites, as indicated by observed levels of heterogeneity (I2 = 78.34%; 95% prediction interval [− 0.03, 0.41]); concordantly, the 95% prediction interval overlapped with zero, suggesting that if similar nations were randomly added to the sample, some of their true effect sizes would be null, or even negative46.h y pf g These results were robust to the inclusion of demographic controls—including age and education—as well as COVID-19-related covariates, such as participants’ estimates of COVID-19 prevalence (see Fig. S5; see “Methods” section for details on COVID-19-related covariates). Additionally, the reliability of the traditionalism composite varied widely across study sites (αs 0.39–0.88, mean α = 0.74; see Table S8). Figure 1. Results of a random effects, restricted maximum likelihood meta-analysis in which each study site was treated as a separate sample. Plot shows zero-order product-moment correlations between traditionalism and COVID-19 health precautions at each study site, ordered by effect size. For the individual country estimates, the location of the square along the x-axis corresponds with the correlation coefficient, the size of the square corresponds with the weight of that study site in the meta-analysis, and bands are 95% confidence intervals. At the bottom of the plot, an overall meta-analyzed point estimate is provided. The midpoint of the diamond corresponds with that point estimate, the width of the diamond corresponds with the 95% CI, and the dotted bands correspond with the 95% prediction interval. On the right side of the plot, weights, correlation coefficients, and 95% CIs respectively are numerically listed for both the site-specific correlations, as well as the overall estimate. Note that for the overall meta-analyzed point estimate, the 95% confidence interval does not overlap with zero, while the 95% prediction interval does. Figure 1. Results of a random effects, restricted maximum likelihood meta-analysis in which each study site was treated as a separate sample. Plot shows zero-order product-moment correlations between traditionalism and COVID-19 health precautions at each study site, ordered by effect size. For the individual country estimates, the location of the square along the x-axis corresponds with the correlation coefficient, the size of the square corresponds with the weight of that study site in the meta-analysis, and bands are 95% confidence intervals. At the bottom of the plot, an overall meta-analyzed point estimate is provided. The midpoint of the diamond corresponds with that point estimate, the width of the diamond corresponds with the 95% CI, and the dotted bands correspond with the 95% prediction interval. On the right side of the plot, weights, correlation coefficients, and 95% CIs respectively are numerically listed for both the site-specific correlations, as well as the overall estimate. Note that for the overall meta-analyzed point estimate, the 95% confidence interval does not overlap with zero, while the 95% prediction interval does. Figure 1. Results of a random effects, restricted maximum likelihood meta-analysis in which each study site was treated as a separate sample. Plot shows zero-order product-moment correlations between traditionalism https://doi.org/10.1038/s41598-023-29655-0 Scientific Reports \| (2023) 13:4969 \| www.nature.com/scientificreports/ Figure 2. Graphical visualization of the country-specific correlations listed in Fig. 1. www.nature.com/scientificreports/ www.nature.com/scientificreports/ In order to use the same set of candidate suppressors for each study site in the meta-analysis, we first identified suppressors in a pooled sample across all study sites. Using bootstrapping procedures to determine confidence intervals, we utilized mixed-effects mediation analyses with study site set as a random effect to test whether any of the seven candidate variables were suppressing the precautions-traditionalism relationship in the pooled sam- ple. Of the seven variables, we identified five suppressors in the pooled sample (see Table S1): SDO; distrust in scientists; and perceived tradeoffs between COVID-19 public health efforts and personal liberties, the economy, and personal traditions, respectively. See Supplement pages S66–S71 for information on mean levels of each suppressor variable across study sites.fi pp y Next, we assessed the combined effects of all five suppressors at each study site (see Table S2). We observed a wide range of indirect effects across study sites, ranging from suppression in slightly less than half of the study sites, all the way to partial mediation at three of the sites. This suggests that while the suppression effects originally observed in the U.S. are shared with some other societies, the effects of these five variables on the traditionalism- precautions relationship are parochial, and contingent on socio-political dynamics and perceptions that vary widely across societies.f We then ran a new set of random effect meta-analyses examining the relationship between traditionalism and overall COVID-19 health precautions, adjusting for the joint effects of the five aforementioned variables (see Fig. 3). While the overall meta-analyzed point estimate was conceptually indistinguishable from the effect size of the zero-order meta-analysis, accounting for the five variables resulted in the following observations: (a) the amount of heterogeneity in effect sizes across study sites was substantially reduced (I2 = 56.39%; 95% prediction interval [0.08, 0.33]); (b) the 95% prediction intervals suggest that if similar nations were randomly Figure 3. Results of a random effects, restricted maximum likelihood meta-analysis in which each study site was treated as a separate sample. The plot shows semi-partial correlations54,55 between traditionalism and COVID-19 health precautions at each study site, after adjusting for the effects of the five identified suppressor variables in multiple linear regressions where health precautions were regressed on traditionalism and each of Figure 3. Results of a random effects, restricted maximum likelihood meta-analysis in which each study site was treated as a separate sample. To address this, (a) we performed item-by-item meta-analyses using each item from the traditionalism composite separately (see Supplement page S44)—results were conceptually unchanged compared to the composite, and were similar across items, with some variation in effect size—and (b) given the possibility of measurement error contributing to unreliability, we per- formed random effects meta-analyses using the traditionalism composite that disattenuated for unreliability47, see Fig. S7. These analyses used averages of raw scores to create composite indices for traditionalism and COVID-19 precautions, where item inclusion was based on the results of factor analyses (see “Methods” section and Supple- ment pages S30 and S38 for details). While averaged composites are easier to interpret, they may make unrealistic assumptions about the relative weights of each item in the composites. We therefore tested whether using factor scores instead of raw averages for the traditionalism and COVID-19 precautions indices conceptually altered the results. Factor scores were highly correlated with the raw average composites (marginal R2s = 0.96–0.98), and using them in place of the raw average composites did not conceptually change results (see Supplement page S62 for details). Finally, country-specific estimates of COVID-19 prevalence at the time of data collection did not explain any of the variance in effect sizes between study sites when tested in a meta-regression (see Supple- ment page S23), although the reliability of officially reported prevalence numbers may vary across study sites. Exploring the effects of potential suppressor variables:. To test the generalizability of suppression phenomena originally observed in the U.S. socio-political context, we examined the extent to which the poten- tial suppressor variables assessed in those studies affected the zero-order precautions-traditionalism relationship across study sites. Here, suppression refers to variables that result in a negative indirect relationship between traditionalism and health precautions in a mediation analysis, such that accounting for them in a regression increases (rather than decreases, as in a traditional mediation analysis) the effect size of the direct positive tra- ditionalism-precautions relationship48. We therefore conducted a second random effects meta-analysis on the traditionalism-precautions relationship accounting for the effects of potential suppressor variables. https://doi.org/10.1038/s41598-023-29655-0 Scientific Reports \| (2023) 13:4969 \| www.nature.com/scientificreports/ added to the sample, their true effect sizes would be positive and significant if adjusted for the five variables; and (c) the traditionalism-precautions relationship was now positive and significant in 21 out of 27 study sites. Taken together, these results suggest that the suppressive effects of these five variables emerge in a variety of socio-political contexts across the countries included in this study, and adjusting for their effects reveals a more consistent positive relationship in the direct pathway between pathogen avoidance and traditionalism across societies in our models. Note that these results remain robust after accounting for the same demographic and COVID-19-related covariates used previously (see Fig. S6), as well as when disattenuating for scale unreliability (see Fig. S8); when using factor scores in place of raw average composites (see Supplement page S62); and when conducting item-by-item analyses of the traditionalism composite items (see Supplement page S44). External‑facing versus internal‑facing precautions. As discussed in the “Methods” section, explora- tory factor analysis revealed that the COVID-19 health precaution items can be decomposed into two factors, interpretable as distinguishing between actions in which other actors are salient, and which are often publicly visible (e.g., mask wearing and social distancing; hereafter external-facing precautions), versus actions in which other actors are not salient, and which often occur in private (e.g., hand washing and surface disinfection; here- after internal-facing precautions). Because we did not predict this factor structure in advance, and therefore did not have a priori predictions about how it would affect the precautions-traditionalism relationship, the following analyses are exploratory. y p y To examine whether the relationship between traditionalism and COVID-19 precautionary behaviors varies as a function of whether precautions are external- or internal-facing, we assessed whether subscale moderated the traditionalism-precautions relationship in a mixed linear regression. We found that the strength of the Figure 4. Results of a restricted maximum likelihood moderated mixed linear regression in which COVID- 19 health precautions were regressed on traditionalism, a health precautions indicator variable (e.g., either internal-facing or external-facing), and the interaction between those two variables in the pooled sample. The model included participants nested within study sites as random effects. To test this interaction, there were two observations for each participant; the first observation contained each participants’ internal-facing precautions score, and the second their external-facing precautions score. We simultaneously created an indicator variable specifying which health precautions subscale corresponded with each observation. www.nature.com/scientificreports/ The plot shows semi-partial correlations54,55 between traditionalism and COVID-19 health precautions at each study site, after adjusting for the effects of the five identified suppressor variables in multiple linear regressions where health precautions were regressed on traditionalism and each of those five variables. Covariates were identical across study sites. Note that the semi-partial correlations indicate the variance in health precautions uniquely explained by the aspects of traditionalism separate from the five suppressor variables, and the effect sizes can be interpreted using the same metrics applied to product-moment correlations. See Fig. 1 for a description of how to interpret the forest plot. For the overall meta-analyzed point estimate, neither the 95% confidence interval nor the 95% prediction interval overlap with zero. Figure 3. Results of a random effects, restricted maximum likelihood meta-analysis in which each study site was treated as a separate sample. The plot shows semi-partial correlations54,55 between traditionalism and COVID-19 health precautions at each study site, after adjusting for the effects of the five identified suppressor variables in multiple linear regressions where health precautions were regressed on traditionalism and each of those five variables. Covariates were identical across study sites. Note that the semi-partial correlations indicate the variance in health precautions uniquely explained by the aspects of traditionalism separate from the five suppressor variables, and the effect sizes can be interpreted using the same metrics applied to product-moment correlations. See Fig. 1 for a description of how to interpret the forest plot. For the overall meta-analyzed point estimate, neither the 95% confidence interval nor the 95% prediction interval overlap with zero. https://doi.org/10.1038/s41598-023-29655-0 Scientific Reports \| (2023) 13:4969 \| www.nature.com/scientificreports/ Discussion Consistent with a postulated link between traditionalism and motivations to mitigate dangers, across 27 nations, we found evidence that at the individual level, traditionalism associates positively with health precautions aimed at a global pathogen threat. In addition, in some socio-political contexts, perceived tradeoffs between health precautions and priorities concerning the economy, personal liberties, and the ability to practice traditions statistically suppressed the zero-order relationship between traditionalism and COVID-19 precautions, as did low trust in scientists and high social dominance orientation. Importantly, accounting for the effects of the suppressor variables resulted in a more consistent positive correlation between traditionalism and COVID-19 precautions. This suggests that when individuals’ weightings of the costs, benefits, and tradeoffs of pathogen- threat mitigation and competing priorities—many of which are themselves tied to traditionalism—are taken into account, statistical associations between traditionalism and pathogen avoidance are more likely to be detected within any given cultural context.h y g These results both support the traditional norms account of the relationship between traditionalism and threat avoidance, and underscore the importance of parochial, countervailing preferences, many of which con- cern competing threat responses. Understanding the weights accorded to the costs and benefits of particular pathogen-avoidance behaviors in the real world is thus critical when assessing the extent to which traditional- ism and pathogen avoidance covary among individuals. As expected, we found considerable heterogeneity in effect sizes across study sites, further highlighting the importance of parochial factors, and the contribution of cultural variation in shaping traditionalism-pathogen avoidance relationships. Indeed, given the nested rela- tionship between cultural evolution and the production of traditional norms, any evolutionary explanation for relationships between pathogen avoidance and traditionalism must take into account the possibility of substan- tial variation within cultures across contexts, and across cultures. For example, the extent to which traditions protect against pathogen threats may depend in part upon the content of those traditions, either via traditions’ instrumental effects, or via the effects of adherence on ingroup cooperation and/or coordination.f ff g p p Consistent with prior research on the tradeoffs attending COVID-19 prophylactic behaviors29, we found that the relationships between traditionalism and COVID-19 precautions were stronger for internal-facing precautions (e.g., hand washing) than for external-facing precautions (e.g., mask wearing). This may owe to differences in the extent to which these two types of precautions are constrained by factors outside of personal control. Discussion Because external-facing precautions are more likely to be regulated by government rules—such as mask mandates—individuals may have less leeway to align their behavior with their preferences. Alternately, because they are more likely to conflict with the pursuit of a wide variety of benefits obtained through sociality, external-facing precautions may reflect valuation of the latter to a greater extent than internal-facing precautions. Indeed, external-facing precautions are probably more likely to clash with traditions, as precautions such as social distancing will often interfere with activities such as traditional religious practices. There are thus multiple plausible potential reasons why traditionalism covaries with external-facing precautions to a lesser extent than with internal-facing ones.h g This study has multiple limitations. First, samples were recruited on the basis of convenience, and were not representative of their countries more broadly. In particular, given that participants needed access to computing devices and internet connectivity, and because some samples were comprised of students, socio-economic status and levels of formal education are not representative. Of equal importance, in addition to a lack of representa- tiveness within study sites, the countries included were not globally representative. Countries from the Global North were overrepresented, while countries from Africa and South America were especially underrepresented. In both cases, our sampling procedures limit the generalizability of our findings. In particular, the relatively high frequency at which suppression was observed using a limited variable set derived from prior work conducted in the U.S. may reflect the over-representation of countries having shared cultural and political histories.hf yl p g p The effect sizes that we observed, though analogous in magnitude to those obtained in similar previous research7,10, are relatively small. This likely owes in part to the fact that traditionalism is complex and multide- termined, and variation in it is not solely explained by pathogen-avoidance motivations. The same logic applies with regard to COVID-19 health precautions. Other sources of measurement error are also possible, such as the translatability and coherence of folk concepts and terminologies across societies and languages. In particular, our use of a broad but shallow assessment of traditionalism was likely one source of noise. We measured the general proclivity to endorse one’s society’s traditions without examining the actual con- tent of those traditions. www.nature.com/scientificreports/ Simple slopes were then plotted in the figure. There was an interaction between health precautions subscale and traditionalism (B = 0.16, SE = 0.01, t(7,535) = 12.76, p < 0.001). A simple slopes analysis revealed that the correlation between traditionalism and internal-facing precautions (B = 0.29, SE = 0.01, t(7,535) = 23.17, p < 0.001) was about twice as strong as the correlation between traditionalism and external-facing precautions (B = 0.14, SE = 0.01, t(7,535) = 10.84, p < 0.001). Note that these results were robust to the inclusion of demographic and COVID- 19-related covariates, and they were not conceptually affected when the five suppressor variables were included as covariates (see Supplement page S26). Further, results did not conceptually change when using factor scores instead of averaged composites (see Supplement page S63). Finally, we considered the possibility that the presence—or lack of presence—of planning precautions may be confounding our interpretation of the external- and internal-facing precautions subscales. Specifically, the internal-facing subscale has more items related to planning precautionary behaviors (such as the importance of obtaining prophylactic supplies), whereas the external-facing subscale has more items related to actual precautionary behavior (such as wearing a mask when outside the home). To address this possibility, we created a modified internal-facing precautions composite that excluded all planning-related precautions. Using the planning-less internal-precautions composite did not conceptually affect these results (see Supplement S26), suggesting that planning behaviors versus actual behaviors are not confounding our explanation for the moderating effect of external- versus internal-facing precautions. https://doi.org/10.1038/s41598-023-29655-0 Scientific Reports \| (2023) 13:4969 \| www.nature.com/scientificreports/ traditionalism-precautions relationship was greater for internal-facing precautions relative to external-facing precautions (see Fig. 4). traditionalism-precautions relationship was greater for internal-facing precautions relative to external-facing precautions (see Fig. 4). www.nature.com/scientificreports/ If so, are these contingent on the nature of distinct threat domains? (E.g., are the components of traditionalism driving associations with pathogen avoidance distinct from components associated with threat responses to intergroup conflict?) Future work should examine which aspects encompassed by the superordinate construct of tradition are most strongly linked with pathogen-threat responsivity, as well responsivity to contrastive threats. Such work may require focusing on fewer societies to allow more detailed consideration of the relative contributions of parochial beliefs and practices.i p p Ours is the first study to systematically investigate the relationship between traditionalism and avoidance of a specific infectious disease across a wide range of societies, attending to the kinds of costly, real-world behav- iors that reflect the tradeoffs that shape actual decision making. Examining these phenomena at a global scale, we required methods that were coarse with regard to the particulars of the pandemic and its interactions with traditions in any one cultural setting. Despite this lack of granularity, consistent with the thesis that individual differences in the propensity to adhere to traditions are driven in part by differences in threat responsivity, we found evidence of a positive direct relationship between traditionalism and avoidance of a specific disease. When the individual and/or social contexts facilitated the alignment of traditionalism and health precautions, we observed that relationship at the zero order without needing to take other factors into account. When other preferences were perceived to clash with public health measures against COVID-19, stronger positive relation- ships between traditionalism and health precautions were detected in many cases after the effects of those clashing objectives were held constant.i j Our findings have practical relevance for public health authorities and clinicians seeking to promulgate behavior changes that slow the spread of a disease that has claimed over six million victims worldwide. Whereas casual reflection might suggest that those who adhere to values and practices rooted in the past would be more hesitant to change behaviors or utilize new medical resources in the service of protecting themselves and others from a novel illness, in actuality, these may be the very people for whom, all else being equal, threats such as those posed by COVID-19 evoke mitigating action. The challenge may be that the same disposition to respond to this pathogen threat may also incline traditionalists to respond to other threats having conflicting mitiga- tion requirements. www.nature.com/scientificreports/ may have obtained in specific study sites but not others, although we encourage future research that delves into particular social contexts more deeply, as well as possible culture-level moderators. may have obtained in specific study sites but not others, although we encourage future research that delves into particular social contexts more deeply, as well as possible culture-level moderators. We examined only a relatively narrow set of possible suppressor variables, selected on the basis of their effects in previous research in the U.S. Our intention was to use these variables to probe whether, across diverse cultural contexts, cost–benefit tradeoffs and conflicting attitudes could influence traditionalism-pathogen avoid- ance relationships, rather than to exhaustively document all such possible tradeoffs. The latter would have been impractical in the present project given the large number of study sites and the diverse parochial factors germane to tradeoffs, and subjective weightings of those tradeoffs, entailed by COVID-19 precautions. Future studies, focused more narrowly on one or a small number of societies, should explore such tradeoffs in detail, including the extent to which politicization influences how individuals perceive cost–benefit structures. pl pi Future work should elucidate the proximate mechanisms linking traditionalism and threat responsivity. Are traditionalists prone to perceive threats as relatively more attention-grabbing, and/or important, and/or suscep- tible to resolution through threat-mitigating action? Or, given the established links between traditionalism and respect for authority figures1, might traditionalists simply be more adherent to the directives of relevant leaders in times of crisis? Relatedly, traditionalism may be linked with a propensity for collective coalitional action which facilitates threat-responsive behaviors in concert with others. The extent to which any or all of these comple- mentary potential pathways contribute to the link between traditionalism and pathogen-avoidance is currently unknown. More broadly, whereas we have focused here on a real-world pathogen threat, might comparable dynamics obtain with regard to traditionalism and the propensity to take action in response to threats in other domains, such as intergroup conflict or resource scarcity?i g pl y We have approached the construct of traditionalism in an underspecified manner loosely isomorphic with a folk concept of “tradition” that recurs reliably across societies. Having found a cross-culturally replicable association, we encourage investigators to explore the particular facets of traditionalism driving the relationship with COVID-19 precautions. Are there specific in-group practices and/or beliefs of perceived antiquity (i.e., traditions) more closely associated with threat responsivity? Discussion This facilitated comparison across study sites irrespective of the particulars of any given society’s traditions; point estimates indicate the relationships between traditionalism and precautions as construed at each particular study site. Nevertheless, by leaving the content of those traditions unspecified, this approach is unable to explore the rich cultural particulars that may importantly drive variation across study sites. Such particulars likely vary markedly across social contexts and across cultures. Hence, we think it is inappropriate to closely compare the magnitudes of precise point estimates between the 27 study sites, or test causal explanations for heterogeneity in those estimates, especially given the issue of non-independence in country-level analyses49. Additionally, our samples were collected on a convenience basis, and none can be considered nationally representative. Although putative cultural dimensions such as tightness-looseness and collectivism-individualism might plausibly moderate the individual-level relationship between traditionalism and COVID-19 precautions36,50, for all of the aforementioned reasons, these data are not structured in such a way as to test nation-level hypotheses. Relatedly, it is beyond the purview of this project to unpack why effects https://doi.org/10.1038/s41598-023-29655-0 Scientific Reports \| (2023) 13:4969 \| www.nature.com/scientificreports/ www.nature.com/scientificreports/ It is thus crucial to recognize and address potential conflicts or tradeoffs that may inhibit tradition-minded individuals from adopting vital prophylactic and treatment practices beneficial to themselves, their societies, and the global community. More broadly, understanding the relationship between traditionalism and the extent to which danger prompts corrective action may prove vital as humanity confronts worldwide threats, from emerging pandemics to climate change, that can only be overcome through innovation and the adoption of new practices. www.nature.com/scientificreports/ participants were unpaid volunteers, recruitment and compensation schemes varied across study sites. A mix of non-student and student populations were used, depending on the study site. See Table S3 in the supplementary materials for a summary of study sites, study site-specific Ns, exclusions, as well as full information on survey languages, recruitment procedures, and participant demographics for each study site. Data were prescreened for minimum completeness and correct answers to attention checks. participants were unpaid volunteers, recruitment and compensation schemes varied across study sites. A mix of non-student and student populations were used, depending on the study site. See Table S3 in the supplementary materials for a summary of study sites, study site-specific Ns, exclusions, as well as full information on survey languages, recruitment procedures, and participant demographics for each study site. Data were prescreened for minimum completeness and correct answers to attention checks. Measures. Measures were consistent across study sites, with some small deviations where necessary (e.g., items addressing education levels differed across study sites according to the local education structure). A full list of these differences can be found on the OSF repository (see link above). COVID‑19 health precautions. COVID-19 health precautions were measured with a 13-item scale examining participants’ self-reported real-world behaviors. Questions addressed behaviors which, at the time, were widely thought by public health authorities to have significant protective value against COVID-19 (e.g., the frequency of mask wearing, hand washing, and social distancing, as well as the importance to the participant of stocking up on supplies such as hand sanitizer). Items were rated on 7-point scales, either from “never” to “as often as pos- sible”, or from “not important at all”, to “extremely important”. Based on the results of an exploratory factor analy- sis (see Table S4), a composite COVID-19 health precautions variable was created for the purposes of analysis by averaging across the thirteen items. The factor analysis also revealed that this scale can be subdivided into two subscales: external-facing health precautions (e.g., observing mask wearing and social distancing), and internal- facing health precautions (e.g., washing hands). These factors are consistent with results from prior research on COVID-19 precautions29. Main text analyses report results using the combined composite, unless otherwise noted. See Supplement page S31 for details on scale development and factor analysis. Traditionalism. www.nature.com/scientificreports/ Because we were unable to identify a culturally neutral traditionalism scale in the prior litera- ture, we drew upon two instruments that had previously been deployed in large-scale cross-cultural research. These scales jointly assessed the concept of traditionalism, or the tendency to endorse and place importance on the practice of traditional norms. To increase comparability across study sites, questions were designed to measure participants’ general tendency to endorse or reject their own society’s traditional social norms and val- ues. The two scales were the conventionalism subscale of the Aggression-Submission-Conventionalism scale51, which measures the general tendency to endorse one’s society’s traditional social norms without specifying the content of those traditions (e.g., “Traditions are the foundation of a healthy society and should be respected”), as well as items from the authority subscale from the Moral Foundations Questionnaire Short Version52,53, which similarly assesses whether individuals respect traditions and authorities, both generally (e.g., “To what extent are the following considerations relevant to your thinking… Whether or not someone conformed to the tradi- tions of society”), and in relation to specific values regarding gender and age roles (e.g., “Respect for authority is something all children need to learn”). Items were rated on 7-point scales, either from “Not at all relevant” to “Extremely relevant”, or from “Strongly Disagree” to “Strongly Agree”. After conducting an exploratory factor analysis on items from both scales jointly (see Table S7), a six-item averaged composite traditionalism variable was computed for analyses involving traditionalism. See Supplement page S39 for details on scale development and factor analysis. Potential suppressor variables. We included seven variables related to potential perceived conflicts between COVID-19 health precautions and other priorities: distrust in science regarding the COVID-19 pandemic; SDO (measured using the 4-item short form scale30); concern about the effects of the COVID-19 pandemic on the economy and personal liberties; and perceptions that COVID-19 health precautions were clashing with personal liberties, one’s own traditions, and the health of the economy, respectively. Unless otherwise noted, these vari- ables were measured using single items. Demographics, COVID‑19‑related covariates, and attention checks. Participants indicated their gender identity and age, and their income relative to others in their country. Education was also measured, but because differ- ent countries in the study have different educational systems, levels of education examined varied across study sites. www.nature.com/scientificreports/ For the purposes of analysis, education was therefore coded into a universal four-level structure: primary school, secondary school, undergraduate-level, and postgraduate-level. We also measured a number of covari- ates relevant to the pandemic itself, including perceived COVID-19 prevalence in participants’ local communi- ties; the population density of those communities; whether participants’ jobs required that they leave the home; and whether participants had certain pre-existing medical conditions that may put them at higher risk for severe disease. Finally, we included several attention checks. Methods Methods Project overview. This study was approved by the UCLA Office of the Human Research Protection Pro- gram, and all methods were performed in accordance with relevant guidelines and regulations. Informed con- sent was obtained before participation. Complete questionnaire in English, translations, datasets, analysis code, and preregistrations of predictions and methods are available at https://osf.io/6vu5b/?view_only=873259d429 c346d2912303fc44df5079. See Supplement page S1 for a list of questionnaire items and composite scales. Adult participants were recruited online for an observational, cross-sectional survey-based study between October 2020 and July 2021 in 27 countries, with a final N of 7844. Countries were selected on a convenience basis, and both the range of possible study sites and the representativeness of samples recruited at each were constrained by our use of remote internet-mediated interactions for recruitment and participation. Nevertheless, we endeavored to collect data in a wide range of societies, selected from diverse major culture areas; see Fig. S1. Where appropriate, survey materials were translated from English by fluent bilingual speakers. While most Project overview. This study was approved by the UCLA Office of the Human Research Protection Pro- gram, and all methods were performed in accordance with relevant guidelines and regulations. Informed con- sent was obtained before participation. Complete questionnaire in English, translations, datasets, analysis code, and preregistrations of predictions and methods are available at https://osf.io/6vu5b/?view_only=873259d429 c346d2912303fc44df5079. See Supplement page S1 for a list of questionnaire items and composite scales. Adult participants were recruited online for an observational, cross-sectional survey-based study between October 2020 and July 2021 in 27 countries, with a final N of 7844. Countries were selected on a convenience basis, and both the range of possible study sites and the representativeness of samples recruited at each were constrained by our use of remote internet-mediated interactions for recruitment and participation. Nevertheless, we endeavored to collect data in a wide range of societies, selected from diverse major culture areas; see Fig. S1. Where appropriate, survey materials were translated from English by fluent bilingual speakers. While most https://doi.org/10.1038/s41598-023-29655-0 Scientific Reports \| (2023) 13:4969 \| www.nature.com/scientificreports/ Data availability Data availability All relevant data are openly available via the Open Science Framework at the following link: https://osf.io/6vu5b/? view_only=873259d429c346d2912303fc44df5079. Received: 11 October 2022; Accepted: 8 February 2023 Received: 11 October 2022; Accepted: 8 February 2023 www.nature.com/scientificreports/ D. & Fessler, D. M. T. Normative bias and adaptive challenges: A relational approach to coalitional psychology and a critique of terror management theory. Evol. Psychol. 3, 147470490500300 (2005).h g y y 16. Fessler, D. M. T. Steps toward an evolutionary psychology of a culture-dependent species. and Cognition (eds Carruthers, P. et al.) 61–77 (Oxford University Press, 2006). 16. Fessler, D. M. T. Steps toward an evolutionary psychology of a culture-dependent species. In The Innate Mind: Volume 2: Culture and Cognition (eds Carruthers, P. et al.) 61–77 (Oxford University Press, 2006). 17. Mcelreath, R., Boyd, R. & Richerson, P. J. Shared norms and the evolution of ethnic markers. Curr. Anthropol. 44, 122–129 (2 17. Mcelreath, R., Boyd, R. & Richerson, P. J. Shared norms and the evolution of ethnic markers. Curr. Anthropol. 44, 122–129 (2003). 18. Sugiyama, L. S. Illness, injury, and disability among Shiwiar forager-horticulturalists: implications of health-risk buffering for the evolution of human life history. Am. J. Phys. Anthropol. 123, 371–389 (2004). y p 18. Sugiyama, L. S. Illness, injury, and disability among Shiwiar forager-horticulturalists: implications of health-risk buffering for the evolution of human life history. Am. J. Phys. Anthropol. 123, 371–389 (2004). 19. Jost, J. T., Glaser, J., Kruglanski, A. W. & Sulloway, F. J. Political conservatism as motivated social cognition. Psychol. Bull. 129, 339–375 (2003).hi 20. Conover, P. J. & Feldman, S. The Origins and Meaning of Liberal/Conservative Self-Identifications. Am. J. Polit. Sci. 25, 617 (1981).h ( ) 21. Wilson, G. D. The Psychology of Conservatism Vol. xv, 277 (Academic Press, 1973).h Wilson, G. D. The Psychology of Conservatism Vol. xv, 277 (Academ h k l Th b h l D. The Psychology of Conservatism Vol. xv, 277 (Academic Press, 1 h 2. Terrizzi, J. A., Shook, N. J. & McDaniel, M. A. The behavioral immune system and social conservatism: A meta-analysis. Evol Hum. Behav. 34, 99–108 (2013).h 22. Terrizzi, J. A., Shook, N. J. & McDaniel, M. A. The behavioral immune system and Hum. Behav. 34, 99–108 (2013).h 23. Jost, J. T., Stern, C., Rule, N. O. & Sterling, J. The politics of fear: Is there an ideological asymmetry in existential motivation? Cogn. 35, 324–353 (2017). g 24. Jost, J. T. Ideological asymmetries and the essence of political psychology. Polit. Psychol. 38, 167–208 (2017). 24. Jost, J. T. Ideological asymmetries and the essence of political 5. Duncan, L. A., Schaller, M. & Park, J. H. www.nature.com/scientificreports/ Perceived vulnerability to disease: Development and validation of a 15-item self-repor instrument. Personal. Individ. Differ. 47, 541–546 (2009).i ff 6. Olatunji, B. O. et al. Confirming the three-factor structure of the disgust scale—Revised in eight countries. J. Cross-Cult. Psychol 40, 234–255 (2009).h 7. Karinen, A., Tybur, J. M. & de Vries, R. E. The disgust traits: Self-other agreement in pathogen, sexual, and moral disgust sensitivity and their independence from HEXACO personality. https://doi.org/10.1037/emo0000795 (2019).f p p y p g 28. Tybur, J. M., Lieberman, D., Fan, L., Kupfer, T. R. & de Vries, R. E. Behavioral immune trade-offs: Interpersonal value relaxes social pathogen avoidance. Psychol. Sci. 31, 1211–1221 (2020).f p g y 9. Gul, P. et al. Disease avoidance motives trade-off against social motives, especially mate-seeking, to predict social distancing Evidence from the COVID-19 pandemic. Soc. Psychol. Personal. Sci. https://doi.org/10.1177/19485506211046462 (2021). p y p g Ritchie, H. et al. Coronavirus pandemic (COVID-19). Our World Data (2020). p y p 30. Ritchie, H. et al. Coronavirus pandemic (COVID-19). Our World Data (2020). 1. Hensel, L. et al. Global behaviors, perceptions, and the emergence of social norms at the onset of the COVID-19 pandemic. J. Econ Behav. Organ. 193, 473–496 (2022). g ( ) 32. Czeisler, M. É. et al. Mental health, substance use, and suicidal ideation during the COVID-19 pandemic—United States, June 24–30, 2020. MMWR Morb. Mortal. Wkly. Rep. 69, 1049–1057 (2020). y p 3. Haselton, M. G. & Buss, D. M. Error management theory: A new perspective on biases in cross-sex mind reading. J. Pers. Soc Psychol. 78, 81–91 (2000).h y 4. Galperin, A. & Haselton, M. G. Error management and the evolution of cognitive bias. In Social Thinking and Interpersonal Behavior 45–63 (Psychology Press, 2013). y gy 35. Neuberg, S. L., Kenrick, D. T. & Schaller, M. Human threat management systems: Self-protection and disease avoidance. Neurosci. Biobehav. Rev. 35, 1042–1051 (2011).h 6. Gelfand, M. J. et al. The relationship between cultural tightness–looseness and COVID-19 cases and deaths: A global analysis Lancet Planet. Health 5, e135–e144 (2021). 7. Karwowski, M. et al. When in danger, turn right: Does COVID-19 threat promote social conservatism and right-wing presidentia candidates?. Hum. Ethol. 35, 37–48 (2020). 38. Rosenfeld, D. L. & Tomiyama, A. J. Can a pandemic make people more socially conservative? Political ideology, gender roles, and the case of covid-19. J. Appl. Soc. Psychol. 51, 425–433 (2021). y 39. Leeuwen, F. www.nature.com/scientificreports/ www.nature.com/scientificreports/ 2. Giuliano, P. & Nunn, N. Understanding Cultural Persistence and Change. w23617 http://www.nber.org/papers/w23617.pdf (2017) https://doi.org/10.3386/w23617.h p g 3. Claessens, S., Fischer, K., Chaudhuri, A., Sibley, C. & Atkinson, Q. The dual evolutionary foundations of political ideology. Nat Hum. Behav. 4, 1–10 (2020).fi 4. Jost, J. T., Federico, C. M. & Napier, J. L. Political ideology: Its structure, functions, and elective affinities. Annu. Rev. Psychol. 60 307–337 (2009).f 5. Hibbing, J. R., Smith, K. B. & Alford, J. R. Differences in negativity bias underlie variations in political ideology. Behav. Brain Sci 37, 297–307 (2014).h gf g y p gy 37, 297–307 (2014). 6. Murray, D. R. & Schaller, M. Threat(s) and conformity deconstructed: Perceived threat of infectious disease and its implications 37, 297–307 (2014). 6. Murray, D. R. & Schaller, M. Threat(s) and conformity deconstructed: Perceived threat of infectious disease and its implications for conformist attitudes and behavior Eur J Soc Psychol 42 180–188 (2012) 6. Murray, D. R. & Schaller, M. Threat(s) and conformity deconstructed: Perceived threat of infectious disease and its implications for conformist attitudes and behavior. Eur. J. Soc. Psychol. 42, 180–188 (2012). y 7. Tybur, J. M. et al. Parasite stress and pathogen avoidance relate to distinct dimensions of political ideology across 30 nations. Proc Natl. Acad. Sci. 113, 12408–12413 (2016).l 8. Fischer, K., Chaudhuri, A. & Atkinson, Q. Responses to the COVID-19 Pandemic Reflect the Dual Evolutionary Foundations of Political Ideology. https://psyarxiv.com/qeap8/ (2020) doi:https://doi.org/10.31234/osf.io/qeap8. 9. Makhanova, A., Plant, E. A., Monroe, A. E. & Maner, J. K. Binding together to avoid illness: Pathogen avoidance and moral world- views. Evol. Behav. Sci. 13, 182–204 (2019). 0. Samore, T., Fessler, D. M. T., Sparks, A. M. & Holbrook, C. Of pathogens and party lines: Social conservatism positively associates with COVID-19 precautions among U.S. Democrats but not Republicans. PLoS ONE 16, e0253326 (2021).h g y y with COVID-19 precautions among U.S. Democrats but not Republicans. PLoS ONE 16, e0253326 (2021).h 12. Murray, D. R., Fessler, D. M. T., Kerry, N., White, C. & Marin, M. The kiss of death: Three tests of the relationship between disease threat and ritualized physical contact within traditional cultures. Evol. Hum. Behav. 38, 63–70 (2017). p y 13. Henrich, J. A Cultural Species: How Culture Drove Human Evolution. https://doi.org/10.1037/e519392012-002 14. Zwirner, E. & Thornton, A. Cognitive requirements of cumulative culture: Teaching is useful but not essential. Sci. Rep. 5 (2015). 5. Navarrete, C. References erences Duckitt, J., Bizumic, B., Krauss, S. W. & Heled, E. A tripartite approach to right-wing authoritarianism: The authoritarianism- onservatism-traditionalism model. Polit. Psychol. 31, 685–715 (2010). Scientific Reports \| (2023) 13:4969 \| https://doi.org/10.1038/s41598-023-29655-0 www.nature.com/scientificreports/ ,h p ,h y pf J , ( 55. Pituch, K. A. & Stevens, J. P. Applied Multivariate Statistics for the Social Sciences (Routledge, 2016). Acknowledgements g We thank Mariam Baghdasaryan, Sarah Durham, Neinel Estapanians, Alexa Henrie, Emma Raffman, and Mor- gan Speer for research assistance, the UCLA Experimental Biological Anthropology (XBA)s group for feedback on this research, and two anonymous reviewers for their valuable input. T.S. benefited from support by the Templeton Religion Trust/Issachar Fund project “Science and Religion: An Evolutionary Perspective” while this research was conducted. Author contributions T.S. designed the study, analyzed the data, contributed to data curation and visualization, and wrote the original manuscript. D.M.T.F. and C.H. designed the study, and contributed to writing the original manuscript. A.M.S. designed the study, contributed to writing the original manuscript, and contributed to data curation and visu- alization. L.A. collected data, translated study materials, and contributed to editing the original manuscript. P.B., D.N.P., and J.M.T. collected data and contributed to editing the original manuscript. C.G.B., M.T.B., R.B., J.C., B.C., M.D.P.G., P.E., P.F., A.M.F., R.F., L.G., P. Giraldo-Perez, P. Gul, F.H., Y.H., T.J., T.K., B.K., T.R.K., M.K., J.L., F.R.L., M.A.M., M.M., C.M., A. Naito., P.P., Y.S., W.O.P.S., S.S., A.O.S., H.V., A.V., J.W., and X.T.W. collected data and translated study materials. A.N.O., E.J.H., S.K., N.P.L., A. Ng’ang’a, and L.K.L.T. collected data. All authors reviewed the manuscript. www.nature.com/scientificreports/ van, Jaeger, B., Sleegers, W. & Petersen, M. B. Do experimental manipulations of pathogen avoidance motivations influence conformity? https://doi.org/10.31234/osf.io/t3bcw (2021). l 40. Pennycook, G., McPhetres, J., Bago, B. & Rand, D. G. Beliefs about COVID-19 in Canada, the United Kingdom, and the United States: A novel test of political polarization and motivated reasoning. Pers. Soc. Psychol. Bull. https://doi.org/10.1177/0146167221 1023652 (2021). 1. Carothers, T. & O’Donohue, A. Polarization and the Pandemic. Carnegie Endowment for International Peace https://carnegieen dowment.org/2020/04/28/polarization-and-pandemic-pub-81638 (2020). Kerr, J. R. & Wilson, M. S. Right-wing authoritarianism and social dominance orientation predict rejection of science and scientists. Group Process. Intergroup Relat. 24, 550–567 (2021).f 42. Kerr, J. R. & Wilson, M. S. Right-wing authoritarianism and social dominance orientation predict rejection of science and scientists. Group Process. Intergroup Relat. 24, 550–567 (2021).f p g p ratto, F. et al. Social dominance in context and in individuals: Contextual moderation of robust effects of social dominance rientation in 15 languages and 20 countries. Soc. Psychol. Personal. Sci. 4, 587–599 (2013).i 43. Pratto, F. et al. Social dominance in context and in individuals: Contextual moderation of robust effects of social dominance orientation in 15 languages and 20 countries. Soc. Psychol. Personal. Sci. 4, 587–599 (2013).i g g y ( ) Roccato, M. & Ricolfi, L. On the correlation between right-wing authoritarianism and social dominance orientation. Basic Appl. oc. Psychol. 27, 187–200 (2005). g g y ( ) 4. Roccato, M. & Ricolfi, L. On the correlation between right-wing authoritarianism and social dominance orientation. Basic Appl Soc. Psychol. 27, 187–200 (2005). https://doi.org/10.1038/s41598-023-29655-0 Scientific Reports \| (2023) 13:4969 \| Competing interests h p g The authors declare no competing interests. www.nature.com/scientificreports/ www.nature.com/scientificreports/ 45. Wilson, M. S. & Sibley, C. G. Social dominance orientation and right-wing authoritarianism: Additive and interactive effects on political conservatism: SDO, RWA, and conservatism. Polit. Psychol. 34, 277–284 (2013).f p y 46. Spineli, L. M. & Pandis, N. Prediction interval in random-effects meta-analysis. Am. J. Orthod. Dentofacial Orthop. 157, 586 (2020). ( ) 7. Hunter, J. & Schmidt, F. Methods of Meta-analysis Corrected Error and Bias in Research Findings. Educational Researcher vol. 20 (2004).f ( ) 8. MacKinnon, D. P., Krull, J. L. & Lockwood, C. M. Equivalence of the mediation, confounding and suppression effect. Prev. Sci Off. J. Soc. Prev. Res. 1, 173 (2000).h ff 49. Claessens, S. & Atkinson, Q. The non-independence of nations and why it matters. https://doi.org/10.31234/osf.io/m6bsn (2 h y g 0. Maaravi, Y., Levy, A., Gur, T., Confino, D. & Segal, S. “The tragedy of the commons”: How individualism and collectivism affected the spread of the COVID-19 pandemic. Front. Public Health 9, 627559 (2021).h 50. Maaravi, Y., Levy, A., Gur, T., Confino, D. & Segal, S. “The tragedy of the commons”: How the spread of the COVID-19 pandemic. Front. Public Health 9, 627559 (2021).h p p 1. Dunwoody, P. & Funke, F. The aggression-submission-conventionalism scale: Testing a new three factor measure of authoritarian- ism. J. Soc. Polit. Psychol. 4, 571–600 (2016). y 52. Graham, J. et al. Mapping the moral domain. J. Pers. Soc. Psychol. 101, 366–385 (2011).h y 52. Graham, J. et al. Mapping the moral domain. J. Pers. Soc. Psychol. 101, 366–385 (2011).h 52. Graham, J. et al. Mapping the moral domain. J. Pers. Soc. Psychol. 101, 366 385 (2011). 53. Graham, J., Haidt, J. & Nosek, B. A. Questionnaires \| Moral Foundations Theory. https://moralfoundations.org/questionnaires/ (2008). Al A Th C G Th h f l ff S S k ( ) 53. Graham, J., Haidt, J. & Nosek, B. A. Questionnaires \| Moral Foundations Theory. https://moralfoundations.org/questionna (2008).hhf 54. Aloe, A. M. & Thompson, C. G. The synthesis of partial effect sizes. J. Soc. Soc. Work Res. 4, 390–405 (2013). 55 Pituch K A & Stevens J P Applied Multivariate Statistics for the Social Sciences (Routledge 2016) 54. Aloe, A. M. & Thompson, C. G. The synthesis of partial effect sizes. J. Soc. Soc. Work Res. 4, 390–405 (2013). 55. Pituch, K. A. & Stevens, J. P. Applied Multivariate Statistics for the Social Sciences (Routledge, 2016). Additional informationh Additional information Supplementary Information The online version contains supplementary material available at https://doi.org/ 10.1038/s41598-023-29655-0. Correspondence and requests for materials should be addressed to T.S. Reprints and permissions information is available at www.nature.com/reprints. Reprints and permissions information is available at www.nature.com/reprints. Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and nstitutional affiliations. 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. 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https://openalex.org/W4304014422	https://link.springer.com/content/pdf/10.1140/epjd/s10053-022-00514-3.pdf	English	null	Demonstrating quantum properties of triple photons generated by $$\chi ^3$$ processes	The European physical journal. D, Atomic, molecular and optical physics/The European physical journal. D, Atomic, molecular, optical and plasma physics	2,022	cc-by	17,018	Demonstrating quantum properties of triple photons generated by χ3 processes Kamel Bencheikh1 , Marina F. B. Cenni2, Enky Oudot2, V´eronique Boutou3, Corinne F´elix3, Joel Compte Prades2, Augustin Vernay3, Julien Bertrand3, Florent Bassignot4, Mathieu Chauvet4, F´elix Bussi`eres5, Hugo Zbinden5, Ariel Levenson1, and Benoˆıt Boulanger3,a 1 Centre de Nanosciences et de Nanotechnologies, CNRS/Universit´e Paris-Saclay, 91120 Palaise 2 1 Centre de Nanosciences et de Nanotechnologies, CNRS/Universit´e Paris-Saclay, 91120 Palaiseau, France 2 ICFO - Institut de Ciencies Fotoniques, The Barcelona Institute of Science and Technology, 08860 Castelldefels, Barcelona, Spain Centre de Nanosciences et de Nanotechnologies, CNRS/Universite Paris Saclay, 91120 Palaiseau, France 2 ICFO - Institut de Ciencies Fotoniques, The Barcelona Institute of Science and Technology, 08860 Castelldefels, Barcelona, Spain p CNRS, Institut N´eel, Univ. Grenoble Alpes, 38000 Grenoble, France 3 CNRS, Institut N´eel, Univ. Grenoble Alpes, 38000 Grenoble, France 4 FEMTO-ST Institute, UMR CNRS 6174, Universit´e de Bourgogne Franche-Comt´e, 25000 Besan¸con, France 5 U i i ´ d G ` GAP Q T h l i G ` 4 S i l d 4 FEMTO-ST Institute, UMR CNRS 6174, Universit´e de Bourgogne Franche-Comt´e, 25000 Besan¸con, France 5 Universit´e de Gen`eve GAP-Quantum Technologies, Gen`eve 4, Switzerland , , g g 5 Universit´e de Gen`eve GAP-Quantum Technologies, Gen`eve 4, Switzerland Received 24 May 2022 / Accepted 20 September 2022 / Published online: 10 October 2022 © The A tho (s) 2022 co ected blicatio 2022 Received 24 May 2022 / Accepted 20 September 2022 / Published online: 10 October 2022 © The Author(s) 2022, corrected publication 2022 Received 24 May 2022 / Accepted 20 September 2022 / Published online: 10 October 2022 © The Author(s) 2022, corrected publication 2022 Abstract. Triple-photon generation (TPG) is based on a third-order nonlinear optical interaction, which is the most direct way to produce pure quantum three-photon states. These states can exhibit three-body quantum correlations, and their statistics cannot be reproduced by any Gaussian statistics of coherent sources or optical parametric twin-photon generator, making them potentially useful for quantum infor- mation processing tasks such as quantum state distillation, quantum error-correction and universal quan- tum computing. Furthermore, the generation of entangled photon pairs heralded by the detection of a third photon can be used in advanced quantum communication protocols. We made the ﬁrst experimental demonstration of TPG in 2004 using a bi-stimulation scheme in a bulk KTP crystal, followed by the quan- tum theory. Demonstrating quantum properties of triple photons generated by χ3 processes The new challenges are now to achieve a spontaneous TPG and the corresponding quantum experiments and protocols using oriented ridge KTP waveguides, which ensures both birefringence phase- matching and light conﬁnement. The waveguides are cut by a precision dicing saw. We recently performed their characterization using third-harmonic generation measurements, which showed their good quality. A rate of about 5 triplets per second is expected when pumping a 5-cm-long waveguide with a 5-W 532 nm beam in the CW regime. Such a spontaneous TPG exhibits low rate of triple photons, which makes the certiﬁcation of quantum features hard. In this article, we review our theoretical and experimental work on TPG and the associated quantum modeling. We also develop theoretical tools for the certiﬁcation of quantum features of spontaneous triple-photon states. a e-mail: benoit.boulanger@neel.cnrs.fr (corresponding author) Eur. Phys. J. D (2022) 76:186 https://doi.org/10.1140/epjd/s10053-022-00514-3 Eur. Phys. J. D (2022) 76:186 https://doi.org/10.1140/epjd/s10053-022-00514-3 THE EUROPEAN PHYSICAL JOURNAL D THE EUROPEAN PHYSICAL JOURNAL D 1 Introduction TPG over three, two or one modes of the triplet that has to be generated, or the spontaneous TPG, where there is no stimulation at all. Figure 1 shows the three last cases. In the two stimulated cases, the generated pho- tons come from the triplets as well as from the residual photons of the stimulation, as shown in Fig. 1a, b. Twin photons have deeply inﬂuenced the history of non- linear and quantum optics by their wide range of appli- cations and the paradigmatic place they stand in gener- ating new quantum states of light [1]. As regards triple- photon generation (TPG), the story is only starting. TPG is based on the third-order optical nonlinearity, i.e., the third-order electric susceptibility χ(3) [2,3]: it is a process which can directly generate a 3-photon (3P) state. During TPG, three highly correlated photons at energies ℏω1, ℏω2 and ℏω3 are created in a nonlinear medium from the annihilation of a higher energy pho- ton at ℏω0, with the energy conservation being fulﬁlled: ℏω0 = ℏω1+ℏω2+ℏω3. Four conﬁgurations are possible regarding the level of stimulation of TPG: a stimulated At the opposite, the spontaneous scheme shown in Fig. 1c allows to generate a pure 3P state, which corre- sponds to the third-order spontaneous parametric down conversion (SPDC). These three conﬁgurations are all interesting from the quantum point of view regarding the context of continuous or discrete variables. Indeed, these three conﬁgurations will all generate states, which exhibit quantum features such as entanglement. In 2004, we made the ﬁrst experimental demonstra- tion of a TPG. We considered a two-photon stimulation scheme, as shown in Fig. 1a, using a phase-matched bulk KTiOPO4 crystal [4]. More recently, we proposed a waveguide approach to boost the generation eﬃciency 12 3 3 Eur. Phys. J. D (2022) 76:186 186 Page 2 of 21 86 age o u ys J ( 0 ) 76 86 (a) (b) (c) Fig. 1 The three schemes of generation of a 3-photon state at energies ℏω1, ℏω2 and ℏω3 in a third-order nonlinear medium pumped at ℏω0: a stimulation over two modes, at ℏω2 and ℏω3 for example; b stimulation over one mode, at ℏω1 for example; c no stimulation. The energy levels are described by continuous lines for the matter and a dashed line for the electromagnetic ﬁeld (b) (a) (a) (b) (b) (c) (c) Fig. 1 Introduction 1 The three schemes of generation of a 3-photon state at energies ℏω1, ℏω2 and ℏω3 in a third-order nonlinear medium pumped at ℏω0: a stimulation over two modes, at ℏω2 and ℏω3 for example; b stimulation over one mode, at ℏω1 for example; c no stimulation. The energy levels are described by continuous lines for the matter and a dashed line for the electromagnetic ﬁeld [5,6]. TPG has also been an active ﬁeld of research for many groups around the world [7–15]. This new corpus has thus opened new exciting opportunities in quantum optics. numerical solution to this problem that has not known analytical solution. Finally, we propose new tools in order to characterize quantum features of TPG in Sect. 4, a diﬃcult and important problem under active inves- tigation in several international teams. Now we wish to overcome a new obstacle by aiming at experimentally generating the 3P state of light by mono-stimulated or spontaneous TPG. This is a real tour de force given both the low eﬃciency of these two conﬁgurations and the fact that the eﬃciency, contrary to the second-order nonlinear process, increases with the injection intensity. From this step, it will be then possible to open new avenues in quantum information. Indeed, TPG can provide a new powerful resource for advanced quantum information protocols. For example, spontaneous TPG can be considered to generate her- alded two photons states, which can be used in a qubit ampliﬁer or in a device-independent quantum key dis- tribution protocol [16]. Our choice for the pump and 3P wavelengths is directly conditioned by this application context: λ0 = 532 nm, which corresponds to the second harmonic of the Nd:YAG laser, and λ1, λ2, λ3 ranging from 1500 nm to 1600 nm, that is to say in the telecom range. 3 2.1.1 Theory We are considering a practical case where the four inter- acting waves propagate in the same direction. The cor- responding wave vectors are then written ⃗ki = ki⃗s, i =(0,1,2,3), with ki = 2π λi n(λi) where n(λi) is the refrac- tive index of the wave at λi in the considered direction ⃗s. The electric ﬁelds are taken linearly polarized and are expressed as ⃗Ei = ⃗eiEi(Z)expj(kiZ) where ⃗ei is the unit vector, namely the light polarization, Ei(Z) the complex amplitude, and Z the spatial coordinate of the laboratory frame along ⃗s. This article is organized as follows: we ﬁrst review our theoretical and experimental work on TPG in Sect. 2 going from bulk to waveguided conﬁgurations, followed by the review of the quantum modeling of TPG in Sect. 3 where we additionally implement an easily accessible The resolution of Maxwell equations written at each angular frequency ωi leads to the following coupled amplitude equations [17]: 123 123 Eur. Phys. J. D (2022) 76:186 Page 3 of 21 186 ⎧ ⎪ ⎪ ⎪ ⎪ ⎪ ⎪ ⎪ ⎪ ⎪ ⎪ ⎪ ⎨ ⎪ ⎪ ⎪ ⎪ ⎪ ⎪ ⎪ ⎪ ⎪ ⎪ ⎪ ⎩ ∂E0(Z) ∂Z = j · π n (λ0) λ0 cos2 (ρ0)χ(3) eﬀE1(Z)E2(Z)E3(Z) exp(−jΔkZ) ∂E1(Z) ∂Z = j · π n (λ1) λ1 cos2 (ρ1)χ(3) eﬀE0(Z)E∗ 2(Z)E∗ 3(Z) exp(jΔkZ) ∂E2(Z) ∂Z = j · π n (λ2) λ2 cos2 (ρ2)χ(3) eﬀE0(Z)E∗ 1(Z)E∗ 3(Z) exp(jΔkZ) ∂E3(Z) ∂Z = j · π n (λ3) λ3 cos2 (ρ3)χ(3) eﬀE0(Z)E∗ 1(Z)E∗ 2(Z) exp(jΔkZ). (1) ⎧ ⎪ ⎪ ⎪ ⎪ ⎪ ⎪ ⎪ ⎪ ⎪ ⎪ ⎪ ⎨ ⎪ ⎪ ⎪ ⎪ ⎪ ⎪ ⎪ ⎪ ⎪ ⎪ ⎪ ⎩ ∂E0(Z) ∂Z = j · π n (λ0) λ0 cos2 (ρ0)χ(3) eﬀE1(Z)E2(Z)E3(Z) exp(−jΔkZ) ∂E1(Z) ∂Z = j · π n (λ1) λ1 cos2 (ρ1)χ(3) eﬀE0(Z)E∗ 2(Z)E∗ 3(Z) exp(jΔkZ) ∂E2(Z) ∂Z = j · π n (λ2) λ2 cos2 (ρ2)χ(3) eﬀE0(Z)E∗ 1(Z)E∗ 3(Z) exp(jΔkZ) ∂E3(Z) ∂Z = j · π n (λ3) λ3 cos2 (ρ3)χ(3) eﬀE0(Z)E∗ 1(Z)E∗ 2(Z) exp(jΔkZ). (1) (1) The parameter ρi, with i=(0,1,2,3), is the spatial walk- oﬀangle in the considered direction of propagation [18]. χ(3) eﬀis the eﬀective nonlinear coeﬃcient expressed as χ(3) eﬀ= χ(3) (ω0 = ω1 + ω2 + ω3) ::−→ e0 ⊗−→ e1 ⊗−→ e2 ⊗−→ e3 where :: stands for the four-rank contracted product. 2.1.1 Theory The parameter Δk is deﬁned by Δk = k0−(k1+k2+k3): this spatial phase term corresponds to the phase mis- match between the third-order nonlinear polarization and the radiated ﬁeld. The dispersion coeﬃcients are given in Table 1 for λ given in (µm). The parameter ρi, with i=(0,1,2,3), is the spatial walk- oﬀangle in the considered direction of propagation [18]. χ(3) eﬀis the eﬀective nonlinear coeﬃcient expressed as χ(3) eﬀ= χ(3) (ω0 = ω1 + ω2 + ω3) ::−→ e0 ⊗−→ e1 ⊗−→ e2 ⊗−→ e3 where :: stands for the four-rank contracted product. The parameter Δk is deﬁned by Δk = k0−(k1+k2+k3): this spatial phase term corresponds to the phase mis- match between the third-order nonlinear polarization and the radiated ﬁeld. given in (µm). From Eq. (3) and Table 1, we identiﬁed that angle non-critical phase-matching, i.e., ρ0 = ρ1 = ρ2 = ρ3 = 0, is possible at a pump wavelength λ0 = 532 nm when the four interacting waves propagate along the x-axis of the dielectric frame (O,x,y,z), i.e., ⃗s ⃗ Ox. According to Eq. 2, it means that the principle refractive indices verify ny(λ0) λ0 − nz(λ1) λ1 + ny(λ2) λ2 + nz(λ3) λ3 = 0. The cor- responding phase-matching curve is given in Fig. 2: it shows that the triplet (λ1, λ2, λ3) is spread over a broad continuum, from 1000 to 4500 nm, that is to say up to the infrared cutoﬀof KTP. This speciﬁc feature of TPG is due to the fact that there are only two coupled rela- tions, the energy and momentum conservations, for the determination of three unknown values. Note that it is completely diﬀerent from twin photon generation for which there is a unique couple of solutions for the sig- nal and idler wavelengths once the pump wavelength and the direction of propagation are ﬁxed. Actually, in this well-known case of second-order SPDC, there is the same number of equations as solutions to be deter- mined. An alternative to avoid any wavelength spread- ing, and so any energy spreading, is to ﬁx the value of the wavelength of at least one photon of the triplet: that can be done by stimulating at one or two of the three wavelengths, as in Fig. 1a, b, respectively. Con- cerning the later scheme, Fig. 2.1.1 Theory I0(L) = I0(0) [γ3 + γ0] cn2(aL \| 1 −m) γ3msn2(aL \| 1 −m) + [γ3 + γ0] cn2(aL \| 1 −m) I1(L) = γ3γ0sn2(aL \| 1 −m) γ3msn2(aL \| 1 −m) + [γ3 + γ0] cn2(aL \| 1 −m) I2(L) = I2(0) [γ3 + γ0] (sn2(aL \| 1 −m) + cn2(aL \| 1 −m) γ3msn2(aL \| 1 −m) + [γ3 + γ0] cn2(aL \| 1 −m) I3(L) = I3(0) [γ3 + γ0] (m sn2(aL \| 1 −m) + cn2(aL \| 1 −m)) γ3msn2(aL \| 1 −m) + [γ3 + γ0] cn2(aL \| 1 −m) , (5) with a = Λ 2 γ3 (γ0 + γ2) Λ = μ0 ε0 4πχ(3) eﬀ n (λ0) n (λ1) n (λ2) n (λ3) λ1 λ0λ2λ3 I0(L) = I0(0) [γ3 + γ0] cn2(aL \| 1 −m) γ3msn2(aL \| 1 −m) + [γ3 + γ0] cn2(aL \| 1 −m) I1(L) = γ3γ0sn2(aL \| 1 −m) γ3msn2(aL \| 1 −m) + [γ3 + γ0] cn2(aL \| 1 −m) I2(L) = I2(0) [γ3 + γ0] (sn2(aL \| 1 −m) + cn2(aL \| 1 −m) γ3msn2(aL \| 1 −m) + [γ3 + γ0] cn2(aL \| 1 −m) I3(L) = I3(0) [γ3 + γ0] (m sn2(aL \| 1 −m) + cn2(aL \| 1 −m)) (5) I0(L) = I0(0) [γ3 + γ0] cn2(aL \| 1 −m) γ3msn2(aL \| 1 −m) + [γ3 + γ0] cn2(aL \| 1 −m) I1(L) = γ3γ0sn2(aL \| 1 −m) γ3msn2(aL \| 1 −m) + [γ3 + γ0] cn2(aL \| 1 −m) I2(L) = I2(0) [γ3 + γ0] (sn2(aL \| 1 −m) + cn2(aL \| 1 −m) γ3msn2(aL \| 1 −m) + [γ3 + γ0] cn2(aL \| 1 −m) I3(L) = I3(0) [γ3 + γ0] (m sn2(aL \| 1 −m) + cn2(aL \| 1 −m)) γ3msn2(aL \| 1 −m) + [γ3 + γ0] cn2(aL \| 1 −m) , (5) with g ( ) Figure 3 shows that phase-matching is possible from 1000 to 3000 nm for the three triplet wavelengths. But we can expect to have a wider range, up to 4500 nm, as it is the case in Fig. 2 for bulk KTP, when using proper dispersion equations beyond 3000 nm. Figure 3 also shows that the partially degenerated conﬁguration (λ1 = 1550 nm, λ2 = λ3 = 1619 nm) is allowed in this ridge. 123 2.1.1 Theory The indices x, y and z stand for the dielectric axes of the crystal, and λ1, λ2 and λ3 for the triplet wavelengths The system described by Eq. 1 can be analytically solved using the sn(u\|m) and cn(u\|m) Jacobi elliptic functions [21,22]. Given the boundary conditions in terms of energy at the entrance of the nonlinear medium (Z=0), this resolution is only feasible if there is a stim- ulation at the three wavelengths of the triplet, or at two wavelengths as in the scheme described by Fig. 1a. In this latter case, the boundary conditions are: E0(Z = 0) ̸= 0, E1(Z = 0) = 0, E2(Z = 0) ̸= 0 and E3(Z = 0) ̸= 0. Knowing that the intensity is expressed as Ii(Z) = n(λi) 2 ε0 μ0 \|Ei(Z)\|2, it comes for the four intensities at the exit of the nonlinear medium of length Z = L: (neﬀ)i = Ai × λBi + Ci 10−6 × λDi −Ei −Fi × 10−6 × λGi Hi + Ii, (4) (4) where the wavelength λ is expressed in nanometer (nm). The dispersion coeﬃcients depend on the transverse dimension of the optical waveguide and are given in Table 2 for d × d = 6 × 6 µm2. The phase-matching curves of Fig. 3 are calculated using Eq. (4) and Table 2 . The infrared limit of 3000 nm is ﬁxed by the range of validity of Eq. (4). 2.1.1 Theory D (2022) 76:186 186 Page 4 of 21 186 Page 4 of 21 Fig. 3 TPG phase-matching curve in a ridge y-cut KTP crystal pumped at 532 nm with transverse dimension d×d = 6×6µm2. The indices x, y and z stand for the dielectric axes of the crystal Table 1 Dispersion coeﬃcients of bulk KTP crystal at room temperature [19] j Aj Bj Cj Dj x 3.0065 0.03901 0.04251 0.01327 y 3.0333 0.04154 0.04547 0.01408 z 3.3134 0.05694 0.05658 0.01682 The indices x, y and z stand for the dielectric axes of the crystal Table 1 Dispersion coeﬃcients of bulk KTP crystal at room temperature [19] Fig. 2 TPG phase-matching curve in a bulk x-cut KTP crystal pumped at λ0 = 532 nm. The indices x, y and z stand for the dielectric axes of the crystal, and λ1, λ2 and λ3 for the triplet wavelengths Fig. 3 TPG phase-matching curve in a ridge y-cut KTP crystal pumped at 532 nm with transverse dimension d×d = 6×6µm2. The indices x, y and z stand for the dielectric axes of the crystal tion of the intersection point corresponding to the par- tial degeneracy, e.g. (λ1 = 938 nm, λ2 = 2457 nm) for d × d = 4 × 4 µm2 and (λ1 = 2456 nm, λ2 = λ3 = 1345 nm) for d × d = 16 × 16 µm2, as it is shown in Fig. 4 where it also appears that the full degeneracy (λ1 = λ2 = λ3 = 1596 nm) is possible for d × d = 6.12 × 6.12 µm2. Figure 4 shows well that the geometry of the waveguide is a ﬁne parameter of tun- ability. Fig. 2 TPG phase-matching curve in a bulk x-cut KTP crystal pumped at λ0 = 532 nm. The indices x, y and z stand for the dielectric axes of the crystal, and λ1, λ2 and λ3 for the triplet wavelengths Fig. 2 TPG phase-matching curve in a bulk x-cut KTP crystal pumped at λ0 = 532 nm. The indices x, y and z stand for the dielectric axes of the crystal, and λ1, λ2 and λ3 for the triplet wavelengths Fig. 2 TPG phase-matching curve in a bulk x-cut KTP crystal pumped at λ0 = 532 nm. 2.1.1 Theory 2 shows that there exists a partially degenerate scenario where the two injection wavelengths are equal, i.e., λ2 = λ3 = 1681 nm for λ1 = 1449nm, which can be interesting from the exper- imental point of view as it will be seen in the next sec- tion. Note that it is also obviously possible to stimulate over the three wavelengths. In order to maximize the derivatives ∂Ei/∂Z in Eq. (1), which corresponds to the maximization of energy exchange between the four waves, it is necessary to maximize the amplitude of χ(3) eﬀand to get Δk = 0, i.e., Δk = 2π n (λ0) λ0 − n (λ1) λ1 + n (λ2) λ2 + n (λ3) λ3 = 0. (2) (2) Equation 2 is called the phase-matching relation that ensures a constructive interference between the non- linear polarization and the radiated ﬁeld over the full interacting length Z. This condition also corresponds to the full momentum conservation of the photons in the quantum picture. Then, the design of an optimal TPG requires to ﬁnd a material with a high eﬀective nonlinear coeﬃcient, low spatial walk-oﬀangles, and allowing phase-matching at the targeted wavelengths. We identiﬁed the biaxial crys- tal KTiOPO4 (KTP) as the good platform, under two diﬀerent technologies: a bulk crystal [4] and a ridge opti- cal waveguide crystal [6]. Then, the strategy is to per- form a birefringence phase-matching in both cases. The same kind of calculations can be done in the case of ridge KTP crystals, knowing that in that case the refractive index has to be replaced by the eﬀective index of the optical modes. For a given dimension of the square waveguide (d × d), the eﬀective index of the fundamental guided mode is calculated using Comsol for x-, y- and z-polarized light, by considering wave- lengths varying between 500 and 3000 nm. Dispersion equations giving the eﬀective indices are retrieved by ﬁtting those numerical data assuming a Sellmeier-like form. The best equation we found is expressed as [20]: The phase-matching properties are calculated from the refractive indices over the full transparency range of the crystal. For bulk KTP, the best dispersion equa- tions in the visible and near infrared of the principal refractive indices nj(λ), with respect to the dielectric axes, are [19]: nj(λ) = Aj + Bj λ2 −Cj −Djλ2. (3) (3) 12 3 Eur. Phys. J. 2.1.1 Theory 4 Partially degenerated (λ1 ̸= λ2 = λ3) and fully degenerated (λ1 = λ2 = λ3) TPG phase-matching curve as a function of the transverse dimension of a ridge y-cut KTP crystal pumped at 532 nm. The indices x, y and z stand for the dielectric axes of the crystal / When the TPG eﬃciency is so weak that the pump and stimulation ﬁelds can be considered as constant over L, i.e., E0(Z) ≃E0(0) ̸= 0, E2(Z) ≃E2(0) ̸= 0 and E3(Z) ≃E3(0) ̸= 0, the integration of Eqs. 1 is immediate. For Δk = 0, it leads to: I0(L) ≃I0(0) I2(L) ≃I2(0) I3(L) ≃I3(0) I1(L) ≃μ0 ε0 2π λ1 2 χ(3) eﬀL 2 n (λ0) n (λ1) n (λ2) n (λ3) ×I0(0)I2(0)I3(0). ( γ0 = λ0 λ1 I0(0) γ2 = λ2 λ1 I2(0) γ3 = λ3 λ1 I3(0) m = γ2 (γ0 + γ3) γ3 (γ0 + γ2). (6) (6) (8) Figure 5 gives the corresponding curves for a propa- gation along the x-axis of a bulk KTP crystal pumped at λ0 = 532 nm. The point of partial degeneracy shown in Fig. 2, i.e., (λ1 = 1449 nm, λ2 = λ3 = 1681 nm), is taken for the calculation. In that case, the numerical values of the refractive indices are n(λ0) = ny(λ0) = 1.7902, n(λ1) = nz(λ1) = 1.8182, n(λ2) = ny(λ2) = 1.7345 and n(λ3) = nz(λ3) = 1.8128; from Eq. 3 and Table 1, the third-order nonlinear eﬀective coeﬃcient is χ(3) eﬀ= 9.0 × 10−22 m2V−2 using Miller’s rule from the magnitude of the third-order electric susceptibility coeﬃcient χ(3) yzyz given in Ref.[23]. The incident inten- sities that are used are: I0(0) = 250 GW/cm2 and I2(0) = I3(0) = 3.25 GW/cm2. These values corre- spond to the intensities considered in the experiments described in section 2.1.2. The comparison of the behavior of I1(L) given in Fig. 5 with that calculated from Eq. 8 using the same bound- ary conditions is shown in Fig. 6. It appears that the undepleted pump and stimulation approximation are valid below a crystal length of about 5 mm, but it underestimates the generated intensity at the level of the ﬁrst maximum of the Jacobi elliptic function and it overestimates the intensity at longer interacting lengths. 2.1.1 Theory It is important to notice that the walk-oﬀangle is nil along the y-axis, which ensures a perfect spatial overlap between the interacting waves. (5) with a = Λ 2 γ3 (γ0 + γ2) Λ = μ0 ε0 4πχ(3) eﬀ n (λ0) n (λ1) n (λ2) n (λ3) λ1 λ0λ2λ3 The phase-matching curves calculated for other val- ues of transverse section (d × d) have all the same shapes and extensions. They mainly diﬀer by the loca- 123 Eur. Phys. J. D (2022) 76:186 Page 5 of 21 186 Page 5 of 21 186 Table 2 Dispersion coeﬃcients at room temperature of a ridge KTP crystal with a transverse dimension d×d = 6×6µm2 [20] j Aj Bj Cj Dj Ej Fj Gj Hj Ij x 0.4488 0.01948 0.5288 2.277 0.4939 0.88 1.615 0.07128 0.7808 y 1.017 0.004481 1.47 2.323 0.6407 1.242 1.653 0.07207 0.7373 z 2.236 0.006506 0.8399 2.14 0.1814 0.5619 1.811 0.1125 0.7183 The indices x, y and z stand for the dielectric axes of the crystal The indices x, y and z stand for the dielectric axes of the crystal The indices x, y and z stand for the dielectric axes of the crystal Fig. 4 Partially degenerated (λ1 ̸= λ2 = λ3) and fully degenerated (λ1 = λ2 = λ3) TPG phase-matching curve as a function of the transverse dimension of a ridge y-cut KTP crystal pumped at 532 nm. The indices x, y and z stand for the dielectric axes of the crystal tal length, which is suited for an experiment leading to the extremum I1(L = 1.27 cm) = 91.7 GW/cm2. The generation at λ1 and the ampliﬁcation at λ2 and λ3 are due to the generation of 3P states. Then from the knowledge of I1 assuming a Gaussian spatial and temporal shapes under the parallel beam assumption, it is easy to access to N3P that is the number of triple photons, N3P (L) = 1 ℏω1 I1(L) π 2 3/2 τ1 2 (w1)2 , (7) (7) where τ1 and w1 are the full-width pulse duration and radius at 1/e2, respectively. In the example of bulk KTP, by taking τ1 = 88 ps and w1 = 66 µm for exam- ple, we obtain N3P = 2.57 × 1015 triplets/pulse. Fig. 2.2 Experimental demonstration of bi-stimulated triple-photon generation An example of experimental setup of a TPG stimulated over two modes is shown in Fig. 7 [22]. The nonlinear medium was a 2-cm-long bulk KTP crystal cut along the x-axis. In order to minimize the number of stimu- lation beams, we choose the partially degenerated case, i.e., λ2 = λ3, the two corresponding waves being orthog- onally polarized. Figure 5 shows well the periodic character of Jacobi elliptic functions, with several values of the crystal length for which there is the full pump depletion, i.e., I0(L) = 0. It is then suﬃcient to consider the ﬁrst value, i.e., L = 1.27 cm, for ﬁxing the optimal crys- 12 3 186 Page 6 of 21 Eur. Phys. J. D (2022) 76:186 Fig. 5 Intensities of phase-matched TPG, pumped at λ0 and stimulated at λ2 and λ3, as a function of the crystal length L of a bulk x-cut KTP crystal pumped at 532 nm Fig. 5 Intensities of phase-matched TPG, pumped at λ0 and stimulated at λ2 and λ3, as a function of the crystal length L of a bulk x-cut KTP crystal pumped at 532 nm Fig. 6 Intensity generated at λ1 by phase-matched TPG in a bulk x-cut KTP crystal pumped at 532 nm using the general modeling (red curve of Fig. 5) and the undepleted pump and stimulation approximation 1681 nm) according to Fig. 2, the diﬀerences being due to a small inaccuracy of the refractive indices that are used for the calculation. Using a pump intensity I0(L = 0) = 250 GW/cm2 and a stimulation intensi- ties I2(L = 0) = I3(L = 0) = 3.25 GW/cm2, which corresponds to the intensity values taken for plotting the curves of Figs. 5 and 6, we obtained I1(L = 2cm) = 0.85 GW/cm2 at λ1 = 1474 nm, which cor- responds to N3P = 3.7 × 1013 triplets/s according to Eq. (7) [22]. The calculation using Fig. 5 at L = 2 cm gives N3P = 6.16 × 1013 triplets/s. This small diﬀer- ence with the measurement has been explained by the Kerr eﬀect due to the high intensities that are used [24]. Note also that according to Figs. 5 and 6, a crys- tal with the optimal length L = 1.27 cm would lead to I1(L = 1.27 cm) = 91.7 GW/cm2 at λ1 = 1474 nm and so to N3P = 2.57 × 1015 triplets/s. 123 2.2 Experimental demonstration of bi-stimulated triple-photon generation The triplets are mixed with residual photons at λ2 and λ3, their numbers corresponding simply to those at the entrance of the KTP crystal, i.e., N2(L = 0) = N3(L = 0) = 4.25 × 1014 photons/s, the number of incident pump photons being N0(L = 0) = 6.15 × 1015 photons/s. Fig. 6 Intensity generated at λ1 by phase-matched TPG in a bulk x-cut KTP crystal pumped at 532 nm using the general modeling (red curve of Fig. 5) and the undepleted pump and stimulation approximation The use of bulk KTP as described above allows the beams to be strongly conﬁned over a limited length ranging around one centimeter. Actually, it corresponds to the typical value of the Rayleigh length associated with the focusing conditions that are considered. In order to overcome this limitation, which is a crucial point in the case of a spontaneous TPG, we proposed in 2018 to explore the feasibility of a new technology taking advantage of both birefringence phase-matching and conﬁnement. The idea is to use ridge waveguides where the direction of propagation is along a phase- matching direction of a KTP crystal. By this way, the pump and triplet waves can exhibit the same propa- gation modes so that the overlap will be optimal. KTP ridge waveguides are fabricated using a technique based on precise diamond blade dicing [6]. A picture of such a waveguide is shown in Fig. 8. It was necessary to use very intense pump and stimu- lation beams, typically several GW/cm2 because of the weakness of the amplitude of the third-order nonlinear- ity. This is why we used the picosecond regime. The pump beam at λ0=532 nm was the second-harmonic of a 5 Hz picosecond Ekspla SL312-P Nd:YAG laser. The stimulation beam at λ2 = λ3 was generated by a home- made optical parametric oscillator emitting at 1665 nm, which exactly corresponds to the experimental phase- matching, i.e., (λ1 = 1474 nm, λ2 = λ3 = 1665 nm), that had been determined before from the pioneer experiment thanks to a tunable source for the stimu- lation beam at λ2 = λ3 [4]. Note that these experi- mental phase-matching wavelengths are very close to the calculated values, i.e., (λ1 = 1449 nm, λ2 = λ3 = 123 Page 7 of 21 186 Eur. Phys. J. D (2022) 76:186 Fig. 2.2 Experimental demonstration of bi-stimulated triple-photon generation 7 Experimental setup of a phase-matched TPG in a bulk x-cut KTP crystal pumped at 532 nm and stimulated at λ2 = λ3 = 1665 nm with orthogonal polarizations in the picosecond regime. The dashed lines with double arrows stand for the direction of polarization of the diﬀerent interacting beams. The values of pulse durations (τ) and waist radius (w) are taken at 1/e2 Fig. 7 Experimental setup of a phase-matched TPG in a bulk x-cut KTP crystal pumped at 532 nm and stimulated at λ2 = λ3 = 1665 nm with orthogonal polarizations in the picosecond regime. The dashed lines with double arrows stand for the direction of polarization of the diﬀerent interacting beams. The values of pulse durations (τ) and waist radius (w) are taken at 1/e2 Fig. 8 Electron microscopy image of a KTP ridge waveguide obtained using diamond blade dicing. (x, y, z) is the dielectric frame of KTP. The total length along the y-axis is equal to 8.6 mm Fig. 8 Electron microscopy image of a KTP ridge waveguide obtained using diamond blade dicing. (x, y, z) is the dielectric frame of KTP. The total length along the y-axis is equal to 8.6 mm It is then a step index waveguide, the upper and side faces being in contact with air and the lower face being coated with a silica layer. The transverse aver- age section was found to be of about 38 µm2, non- constant along the ridge axis: it corresponds to an average square waveguide of side d = 6.17 µm. We performed a preliminary validation of this technology by achieving high-eﬃciency third-harmonic generation (THG: ω + ω + ω →3ω) in the waveguide depicted in Fig. 8 [6]. THG is particularly interesting since it is the exact reverse of TPG that is degenerated in energy, i.e., 3ω →ω + ω + ω. As a consequence, their phase-matching properties are exactly the same. But the advantage of THG is that the conversion eﬃciency is higher by several orders of magnitude, leading to a much easier way to study phase-matching of TPG. The experiments were carried out using a TOPAS optical parametric generator to deliver the pump beam, with a pulse duration of 15 ps, a repetition rate of 10Hz, and a wavelength that is tunable around 1600 nm. 2.2 Experimental demonstration of bi-stimulated triple-photon generation By measuring the third-harmonic (TH) intensity as a func- tion of the fundamental wavelength, we found that 12 3 186 Page 8 of 21 Page 8 of 21 Eur. Phys. J. D (2022) 76:186 186 P 186 the phase-matching was achieved at λω = 1594 nm [6]. The calculated value is λω = 1594.2 nm from Eq. (2), with λ0 = λ3ω and λ1 = λ2 = λ3 = λω , and using Eq. (4) and Table 1. These two values are very close and assuredly within the accuracy of measure- ment that is at ±1 nm. In these phase-matching con- ditions, we found that the TH energy conversion eﬃ- ciency was E3ω Eω = 3.4 % when the fundamental energy is Eω = 2 µJ [6]. The calculation gives 2.6 %, which is a little bit lower than the measurement, but the two values are suﬃciently close for a validation of the mag- nitude of the nonlinear coeﬃcient that is excited here, i.e., χ(3) xzxz(1594 nm/3) = 8.0 × 10−22 m2V−2 [6]. Thus, these preliminary measurements of THG allow us to prepare at best the design of spontaneous TPG experi- ments in KTP ridge waveguides thanks to the validation of the dispersion equations of both the eﬀective index and third-order nonlinear coeﬃcient. The waveguides of the current generation are 3 cm long. negligible value. As we are limited in the photon num- ber used in the quantum approach, the expected eﬀects during the interaction described by Eq. (10) will not be observed. In our approach and to gain insights on the quantum properties of TPG, we will instead consider κ = 0.02, corresponding to χ(3) ∼1 × 10−18 m2V−2, a value far above the third-order nonlinearities we can reach in present materials. With this value, a pump ﬁeld with an average 100 photons is suﬃcient to observe sig- niﬁcant evolution of the quantum system. In the follow- ing, we will thus consider TPG evolution under Hamil- tonian given by Eq. (9) with a pump ﬁeld in a coherent state \|α0⟩, where \|α0\|2 = ⟨n0⟩= 100. Such a coherent state has a Poisson distribution and can be expanded in the Fock-basis up to 200 with very good ﬁdelity. As the generated modes are far weaker, we have used a Fock representation with an expansion up to 50, keeping the calculation times reasonable. 3 Quantum description The starting point of the quantum description of triple-photon generation is the interaction Hamiltonian describing the nonlinear process, given by ˆHnl = iℏκ(ˆa0ˆa† 1ˆa† 2ˆa† 3 −ˆa† 0ˆa1ˆa2ˆa3). (9) (9) where ˆa† 1, ˆa† 2 and ˆa† 3 refer to the creation operators corresponding to the three modes and ˆa† 0 the creation operator of the pump mode. The nonlinear coeﬃcient κ is proportional to the eﬀective third-order nonlinear susceptibility. The evolution of the quantum system is given in the Heisenberg picture, where the operators follow where ˆa† 1, ˆa† 2 and ˆa† 3 refer to the creation operators corresponding to the three modes and ˆa† 0 the creation operator of the pump mode. The nonlinear coeﬃcient κ is proportional to the eﬀective third-order nonlinear susceptibility. The evolution of the quantum system is given in the Heisenberg picture, where the operators follow Figure 9 shows the evolution of the diﬀerent modes computed numerically considering the three initial states. In the travelling wave conﬁguration, the space evolution can be inferred from the time evolution of Eq. (10) by multiplying the time by the speed v of the propagating modes in the nonlinear material. For sim- plicity, we considered in our analysis that v = 1 and we integrated Eq. (10) up to t = 1. It is very interesting to notice that the ampliﬁcation of the mode which is initially in vacuum depends on the number of seeded modes. Indeed in the case of the double seeding conﬁg- uration, at t = 1, the mean photon number for mode 1 is ⟨n⟩= 1.76, whereas it is lower, ⟨n⟩= 0.27, for modes 1 and 2 in the single seeding regime. These val- ues represent the number of triple photons generated during the interaction. Indeed, we have checked that for the excited modes, the added photon number is exactly Δn = 1.76 at modes 2 and 3 for the double seeded case, and Δn = 0.27 at mode 3 for the single seeded interaction. dˆal dt = i ℏ[ ˆHnl, ˆal], (10) (10) which reduces to dˆal dt = κˆa0ˆa† mˆa† n, (11) (11) using the Hamiltonian described by Eq. (9). This set of equations is equivalent to the classical ones given in Eq. (1). However, from the quantum mechanics point of view, they have no known analytical solution. We have instead considered numerical methods using the QuTiP package [25,26]. 2.2 Experimental demonstration of bi-stimulated triple-photon generation We start our analysis with the double seeding conﬁg- uration, where two of the triplet modes are excited with a coherent state containing in average ⟨n⟩= \|α0\|2 = 5. The quantum system is then in the initial state \|ψin⟩= \|α0, 0, α, α⟩. Next, we consider the single seeding con- ﬁguration \|ψin⟩= \|α0, 0, 0, α⟩, and ﬁnally the spon- taneous triple-photon generation for which the initial state is \|ψin⟩= \|α0, 0, 0, 0⟩. Before proceeding further, it is worth noting here that the double stimulation case describes indeed a displacement operator acting on the mode which is initially in vacuum, under the assump- tion of a classical undepleted pump in the Hamilto- nian Eq. (9). In the single stimulation case and under the classical pump approximation too, the Hamiltonian Eq. (9) reduces to the one describing the well-known spontaneous-parametric down conversion. 123 3 Quantum description This is the rea- son why, despite the fact that more than 1014 triplets are generated in the double seeded conﬁguration as observed in [22], the spontaneous TPG emission has not yet been reported. Figure 9 shows also the associated quantum ﬂuctu- ations of the diﬀerent triple-photon modes. For each mode, we have calculated the variances ⟨Δˆq2⟩and ⟨Δˆp2⟩of the two quadratures ˆq = (ˆa + ˆa†) and ˆp = i(ˆa−ˆa†). For coherent states and vacuum, which are at the shot noise limit, the variances are ⟨Δˆq2⟩= ⟨Δˆp2⟩= 1. Our analysis shows that in the case of TPG, the three modes have excess noise and all the variances are greater than unity. Moreover, in the case of the double seeded interaction, the quantum ﬂuctuations are not equally distributed, being larger along the ˆq than the ˆp quadrature. The generation of triple photons fulﬁlls two con- ditions: the energy and momentum conservations, as explained in Sect. 2. In the case of spontaneous emis- sion, where only the pump is ﬁxed through its optical frequency ω0 and its k0 wave-vector, we end up with two equations with three unknown parameters: ω1, ω2 and ω3. The system has thus an inﬁnity of triplet solutions as already mentioned in Sect. 2. q Now, we consider the case of a triple-seeded conﬁgu- ration where the three modes are excited by a coherent state with a mean photon number \|α\|2 = 5. In this particular situation, we should also consider the rel- ative phase between the pump and the triple modes given by Δφ = \|ψ1 + ψ2 + ψ3 −ψ0\|. For sake of sim- plicity, we take φ0 = 0 and φ1 = φ2 = φ3 = φ. Our analysis reveals two interesting situations: an ampliﬁ- cation for φ = π/2, 7π/6 and 11π/6, where the pump photons are converted into triplets; an attenuation for φ = π/6, 5π/6 and 3π/2, which corresponds to a regime where the triple photons are converted back into pump photons. These behaviors are depicted in Fig. 10, rep- resenting in the left plot the evolution of mean photon number for φ = π/2 (blue) and for φ = π/6 (red). The inset is the mean photon number at t = 1 as a function of the phase in a polar plot. 3 Quantum description This approach is convenient as long as a small number of photons are considered in order to keep the computation time reasonable, due to the repre- sentation of the states and the operators in the Hilbert space of Fock states. To have a signiﬁcant eﬀect, it is thus necessary to compensate the low pumping ﬁeld with a higher nonlinear interaction eﬃciency κ. Indeed, considering the results obtained in [23] and reported in section B, we can infer κ\|α0\| ∼1.75×10−6 ≪1, a rather A similar analysis with a weaker seeding, \|α\|2 = 1, shows that the generated mean photon numbers are even smaller, indicating that the generation rate of triple photons depends not only on the strength of the pumping excitation, but also on the number and strength of the modes excited prior to the interaction. This is unlike the behavior of optical twin-photon para- 123 Eur. Phys. J. D (2022) 76:186 Page 9 of 21 186 Fig. 9 Top: Evolution of the average photon number of the diﬀerent modes under TPG. Bottom : Corresponding quadra- ture variances. For this analysis, κ = 0.02, \|α0\|2 = 100, and \|α\|2 = 5 Fig. 9 Top: Evolution of the average photon number of the diﬀerent modes under TPG. Bottom : Corresponding quadra- ture variances. For this analysis, κ = 0.02, \|α0\|2 = 100, and \|α\|2 = 5 curves represent the variances in the case of ampliﬁ- cation, and the red ones correspond to the attenua- tion case. Like in the partially seeded conﬁgurations, the quantum ﬂuctuations are always above the shot noise limit. Moreover, as depicted in the contour plot, they exhibit a phase dependence, similar to the one of the mean photon number. The behavior of the fully seeded TPG is comparable to the phase-sensitive twin- photon parametric interaction. It has a dependency to the relative phase between the pump and the generated modes and it exhibits noisy modes, which makes them robust against optical losses. The diﬀerences between the twin-photon and triple-photon states are related to the dependence on the seeding intensity and more importantly to the non-Gaussian nature of their statis- tics. This point will be addressed in the next section. metric ampliﬁcation. In the spontaneous conﬁguration, the mean photon number generated is about ⟨n⟩≃0.04, even lower, and it obviously only depends on the pump intensity and the nonlinear coeﬃcient κ. 3 Quantum description In the plot on the right, we have reported the quantum ﬂuctuations of the two conjugate quadratures ˆq and ˆp. The blue We should thus reconsider our quantum approach by taking into account this broadband generation. A more suitable approach in this case is to consider the space evolution under the nonlinear momentum ˆGnl = ℏ dω0dω1dω2Γ(ω0, ω1, ω2) ˆa† 0ˆa1ˆa2ˆa3e−iΔkz + H.c, (12) (12) where Γ(ω0, ω1, ω2) = ℏχ(3) 4ϵ0c2S ω0ω1ω2(ω0 −ω1 −ω2) n(ω0)n(ω1)n(ω2)n(ω0 −ω1 −ω2), (13) Γ(ω0, ω1, ω2) (13) 123 123 Eur. Phys. J. D (2022) 76:186 186 Page 10 of 21 186 Fig. 10 Left: Evolution of the average photon number of the diﬀerent modes under TPG interaction when all modes are seeded and when the phase of the seeding is π/2 (blue) and π/6 (red). For this analysis, κ = 0.02, \|α0\|2 = 100, and \|α\|2 = 5. The horizontal dashed black line indicates the initial mean photon number \|α\|2 = 5. The inset shows ⟨n⟩at t = 1 in a parametric plot as a function of the seeding phase ψ. The dashed black circle indicates once more the initial mean photon number \|α\|2 = 5. Right: The corresponding quadrature variances for the same seeding phase. The horizontal dashed black line shows the shot noise. The inset is the ˆq and ˆp variances at t = 1 as a function of the phase of the seeding. The shot noise is highlighted by the black dashed circle Fig. 10 Left: Evolution of the average photon number of the diﬀerent modes under TPG interaction when all modes are seeded and when the phase of the seeding is π/2 (blue) and π/6 (red). For this analysis, κ = 0.02, \|α0\|2 = 100, and \|α\|2 = 5. The horizontal dashed black line indicates the initial mean photon number \|α\|2 = 5. The inset shows ⟨n⟩at t = 1 in a parametric plot as a function of the seeding phase ψ. The dashed black circle indicates once more the initial mean photon number \|α\|2 = 5. Right: The corresponding quadrature variances for the same seeding phase. The horizontal dashed black line shows the shot noise. The inset is the ˆq and ˆp variances at t = 1 as a function of the phase of the seeding. 3 Quantum description The shot noise is highlighted by the black dashed circle and Δk = k(ω0)−k(ω1)−k(ω2)−k(ω0−ω1−ω2), where we have replaced the frequency ω3 of the third photon by ω3 = ω0 −ω1 −ω2 using the energy conservation condition. In the following, a reasonable assumption is to consider that the pump spectral bandwidth is very narrow in comparison with the bandwidth of the triple photons, especially if a CW laser is used. We can also consider that the pump is strong and undepleted and can be treated as a complex classical amplitude. We obtain the following evolution equations contributions of triple photons that fulﬁll the phase- matching condition and the energy conservation. For example, in the KTP waveguides, these two conditions are fulﬁlled over almost the full transparency window of the nonlinear crystal as shown in Fig. 3. Figure 11 shows the spectral density distribution \|ψ(ω0, ω, ωt)\|2 as a function of λ1 and λ2. It is obtained for the KTP waveg- uide considered in Fig. 3 and using the corresponding dispersion relations of the eﬀective index Eq. 4. Integrating \|ψ(ω0, ω, ωt)\|2 over λ2-axis, we obtain the mean photon number ⟨n1(ω, L)⟩per second as a func- tion of λ1. Similar calculations can be hold to compute ⟨n2(ω, L)⟩and ⟨n3(ω, L)⟩. The expected mean photon number is higher than the one calculated by the sin- gle mode model described by the Hamiltonian given by Eq. (9). The total mean photon number that we expect is further estimated by integrating Eq. (16) over the full spectrum, i.e., ∂ˆal(ω) ∂z = −i dωtΓ(ω0, ω, ωt)A0ˆa† m(ωt)ˆa† n (ω0 −ω −ωt)e+iΔkz, (14) (14) where A0 is the real amplitude of the pump electric ﬁeld. Moreover, considering a weak interaction, which is reasonable for spontaneous TPG, we can solve Eq. (14) to the ﬁrst-order of the Baker–Hausdorﬀexpan- sion, which gives where A0 is the real amplitude of the pump electric ﬁeld. Moreover, considering a weak interaction, which is reasonable for spontaneous TPG, we can solve Eq. (14) to the ﬁrst-order of the Baker–Hausdorﬀexpan- sion, which gives N3P (L) = dω⟨ni(ω, L)⟩. (17) (17) ˆal(ω, z) = ˆal(ω, 0) −i dωtψ(ω0, ω, ωt)ˆa† m(ωt, 0)ˆa† n(ω0 −ω −ωt, 0), (15) ˆal(ω, z) = ˆal(ω, 0) −i dωtψ(ω0, ω, ωt)ˆa† m(ωt, 0)ˆa† n(ω0 −ω −ωt, 0), Indeed, considering the full phase-matching bandwidth of the KTP ridge waveguide as shown in Fig. 4 Non-classical features of TPG We now focus on spontaneous TPG, and we consider the non-degenerate case where the Hamiltonian is ˆHnd = iξ(ˆa† 1ˆa† 2ˆa† 3 −ˆa1ˆa2ˆa3) (18) (18) and the degenerate case characterized by the following Hamiltonian: ˆu = 3 i=1 hiˆpi, ˆv = 3 i=1 giˆqi, (21) (21) ˆHd = iξ(ˆa†3 −ˆa3). (19) (19) where hi and gi are arbitrary real numbers. Thus, the non-separability criterion is easily accessible experimen- tally using standard balanced homodyne detections. The criterion is deﬁned as S = ⟨Δˆu2⟩+ ⟨Δˆv2⟩. We can show that when S < 2 min(\|hkgk\| + \|hlgl + hmgm\|) for any permutation of k, l, m = 1, 2, 3, the triplets exhibit genuine entanglement. However, when S > 2 (\|hkgk\| + \|hlgl\| + \|hmgm\|), the system is completely separable. Surprisingly, we found that the spontaneous triple pho- tons do not exhibit three-body quantum entanglement in the continuous variable (CV) regime, always fulﬁll- ing the last inequality. Entanglement has only been predicted in the diﬀerent seeded cases as depicted in Fig. 12. The diﬀerent results are obtained following the analysis reported in our work [27]. In both cases, we consider a pump with a high photon number so that it can be treated as a classical state. We also deﬁne ξ = κα0 where \|α0\| is the amplitude of the pump. The generation of TPG quantum states has been per- formed in the double-seeded conﬁguration in [24], and in the single seeded and spontaneous cases in [15]. In those experiments, the authors show strong evidence that the states are generated by a TPG; however, the experimental demonstration of quantum features for such states remains, up to our knowledge, to be done. In this section, we develop the theoretical tools that allow to demonstrate the diﬀerent quantum features of the states generated by all the possible conﬁgurations of TPG, i.e., stimulation over one, two or three modes, as well as no stimulation. We ﬁrst note that all quantum features can be inferred from the density matrix of the state or equivalently from a phase space distribution. In the present case, we expect 4.9 triplets per second, which makes a full quantum tomography totally out of reach. We thus focus on tools which require a min- imum information about the state but are still able to conclude about quantum features. 4.1 Entanglement A state is said to be GME if it cannot be written as a biseparable state for any bipartition. A general bisepa- rable state is a mixture of states that are product states for some bipartition, i.e., a partition of all modes into two groups. Formally, it is given by: ϱbisep = G1\|G2 p(G1\|G2) ρG1\|G2. (20) (20) (20) Here, the sum runs over all 3 partitions G1\|G2 of the 3 parties where G1 ∪G2 = {1, 2, 3} and G1 ∩G2 = ∅. The probabilities of diﬀerent partitions sum up to one, G1\|G2 p(G1\|G2) = 1, and ρG1\|G2 is a separable state with respect to the partition G1\|G2. We aim to demon- strate entanglement using homodyne detection, so that we deﬁne a general homodyne measurement on the mode i as ˆXθ i = (ˆa†eiθ + ˆae−iθ)/2 and write ˆXπ/2 i = ˆpi and ˆX0 i = ˆqi. Our ﬁrst attempt to describe the quantum properties of triple photons, especially their three-body quantum entanglement, was in 2018 [27] using the exist- ing tools dedicated to characterize the inseparability of multi-body quantum states. Our analysis is based on P. van Loock and A. Furusawa non-separability crite- rion S [28], relying on the evaluation of the quantum ﬂuctuation of the generalized quadratures Fig. 11 Spectral density distribution \|ψ(ω0, ω, ωt)\|2 as a function of the phase-matching wavelengths λ1 and λ2 3 Quantum description 3, i.e., from 1 to 3 µm, and a CW pump power of 5 W at 532 nm, we estimate N3P (L = 5cm) ≃4.9 triplets/s. Such a pump level is a reasonable target regarding the expected improvements of the losses and surface quality of the waveguides. Note that the rate of triplets remains the same in the pulsed regime than in the CW regime if the average power is kept at the same value. But the number of triplets per pulse will depend on both the pulse duration and repetition rate. For example a pump of 5W at 532 nm with a pulse duration of 11.3 ps and a repetition rate of 88 MHz, gives N3P (L = 5cm) = 4.9 triplets/s, corresponding to 5.6×10−8 triplets/pulse. (15) where ψ = Γ(ω0, ω, ωt)LA0sinc(ΔkL/2)e+iΔkL/2. where ψ = Γ(ω0, ω, ωt)LA0sinc(ΔkL/2)e+iΔkL/2. It is now easier to calculate the mean photon number deﬁned as ⟨n⟩= ⟨ψin\|ˆa†ˆa\|ψin⟩. With the initial state in vacuum, \|ψin⟩= \|0, 0, 0⟩, we obtain: ⟨ni(ω, L)⟩= dωt\|ψ(ω0, ω, ωt)\|2. (16) (16) This indicates that the mean photon number at mode i = 1, 2, 3 at frequency ω is the integral over all the 123 Eur. Phys. J. D (2022) 76:186 Page 11 of 21 186 Page 11 of 21 186 Fig. 11 Spectral density distribution \|ψ(ω0, ω, ωt)\|2 as a function of the phase-matching wavelengths λ1 and λ2 4 Non-classical features of TPG In the ﬁrst part, we focus on the states generated by the Hamiltonian (18) and propose tools to demonstrate genuine multipartite entanglement (GME) of such states using homodyne measurement. We then focus on the state generated by the Hamiltonian (19) and build tools in order to demon- strate non-classical features of such states. [ ] In fact, TPG is a third-order nonlinear process with non-Gaussian statistics. This statement is obvious in the degenerate case when ˆa1 = ˆa2 = ˆa3 and the Hamil- tonian (18) becomes (19). It will be shown hereafter in Fig. 16 that the Wigner function exhibits interfer- ences and negativities, which is a clear signature of the non-Gaussian nature of the triple-photon mode a. In the non-degenerate case (Hamiltonian (18)), we have recently shown that even though the Wigner function associated with each mode looks Gaussian, the quadra- ture probability distribution is super-Gaussian [29]. The S criterion is thus not anymore a relevant parameter to analyze and reveal the entanglement properties of the triple-photon states. Indeed, the S criterion is based 12 12 3 3 186 Page 12 of 21 Eur. Phys. J. D (2022) 76:186 Fig. 12 Evolution of the entanglement criterion S for dif- ferent interactions TPG conﬁgurations. Red: fully seed TPG with a relative phase of π/2. Blue: double-seed TPG. Green: One mode seeding conﬁguration and S calculated for the two unseeded modes. \|α\|2 is the mean photon number per second of each seeding beam Fig. 13 Red: logarithmic negativity of the spontaneous TPG excited by a coherent pump with a mean photon num- ber \|α0\|2 = 10. Black: Evolution of the pump mean photon number. Blue: Evolution of the mean photon number of the generated triplets Eur. Phys. J. D (2022) 76:186 186 Page 12 of 21 186 Page 12 of 21 Fig. 12 Evolution of the entanglement criterion S for dif- ferent interactions TPG conﬁgurations. Red: fully seed TPG with a relative phase of π/2. Blue: double-seed TPG. Green: One mode seeding conﬁguration and S calculated for the two unseeded modes. \|α\|2 is the mean photon number per second of each seeding beam Fig. 13 Red: logarithmic negativity of the spontaneous TPG excited by a coherent pump with a mean photon num- ber \|α0\|2 = 10. Black: Evolution of the pump mean photon number. Blue: Evolution of the mean photon number of the generated triplets Fig. 4 Non-classical features of TPG 12 Evolution of the entanglement criterion S for dif- ferent interactions TPG conﬁgurations. Red: fully seed TPG with a relative phase of π/2. Blue: double-seed TPG. Green: One mode seeding conﬁguration and S calculated for the two unseeded modes. \|α\|2 is the mean photon number per second of each seeding beam Fig. 13 Red: logarithmic negativity of the spontaneous TPG excited by a coherent pump with a mean photon num- ber \|α0\|2 = 10. Black: Evolution of the pump mean photon number. Blue: Evolution of the mean photon number of the generated triplets ment for the state \|ψ3⟩. Moreover, their witness failed to detect entanglement in the seeded conﬁguration, which highlights the diﬀerence between the two states. We brieﬂy review their witness. The authors of [31] show that any biseparable state satisﬁes on the second-order moments, i.e., variances, which are suﬃcient to describe a Gaussian distribution. When at least one of the triplet modes is seeded, the statistics become again Gaussian, and hence, using Furusawa and Van Loock criterion, S is suﬃcient. The failure of the S criterion to describe the entanglement in the case of spontaneous TPG due to its super-Gaussian nature pushed us to further explore the entanglement nature of the triplet, in order to claim that the triple photons are indeed entangled despite the results based on S. Hence, we have used the logarithmic negativity deﬁned as EN = ln \|\|ρTi\|\|, where ρ is the density matrix of the triple photons and ρTi is its partial transpose over mode i = 1, 2 or 3 [29,30]. A quantum system is said to be entangled whenever EN > 1. Figure 13 shows the calculated EN in the case of spontaneous TPG, start- ing from the Hamiltonian Eq. (9) and using a numeri- cal approach to solve the Heisenberg equation. For this simulation, we considered a coherent pump ﬁeld with a mean photon number ⟨n0⟩= 10. The logarithmic nega- tivity EN increases with the nonlinear parameter κ\|α0\|, reaching EN ≃2 for κ\|α0\| = 1, which clearly demon- strates the three-body entanglement of the triple pho- tons. In the same ﬁgure, we have also reported the evo- lution of ⟨n0⟩and ⟨n3P ⟩, respectively, the pump and triplets mean photon numbers. \|⟨ˆa1ˆa2ˆa3⟩\| ≤ ⟨ˆn1⟩⟨ˆn2ˆn3⟩+ ⟨ˆn2⟩⟨ˆn1ˆn3⟩ + ⟨ˆn3⟩⟨ˆn2ˆn1⟩ (22) (22) where ˆni is the number operator for mode i. The state \|ψ3⟩does not satisfy (22) and is thus GME. 123 4 Non-classical features of TPG Non-classicality is a necessary feature for a state to exhibit any quantum advantage, for example in quantum metrology where any advantage over classi- cal metrology requires non-classicality. A state is classical if its GS distribution P(α) can be interpreted as a classical probability distribution [34]. Non-classicality is a necessary feature for a state to exhibit any quantum advantage, for example in quantum metrology where any advantage over classi- cal metrology requires non-classicality. Fig. 14 GME certiﬁcation parameter w with respect to the eﬃciency η for ϵ = ξtint = 10−2 The second layer of quantum features is quantum non-Gaussianity (QNG). A Gaussian state is by deﬁ- nition a state with a Gaussian Wigner function. Hud- son’s theorem [35] stipulates that all non-Gaussian pure states are Wigner negative; nevertheless, it is not the case for mixed states. Any pure Gaussian state can be generated by the action of the squeezing operator ˆS(s) = eˆa2s∗−ˆa†2s following by displacement operator D(α) = eαˆa†−α∗ˆa on the vacuum : \|s, α⟩= S(s)D(α)\|0⟩. Also, any state can be written in terms of Gaussian states, i.e., The terms ˆniˆnj being hard to measure experimentally, we bound them according to ∀{i, j} ∈{1, 3} ⟨ˆniˆnj⟩≤1 2(⟨ˆn2 i ⟩+ ⟨ˆn2 j⟩) (25) (25) where ⟨ˆn2 i ⟩can be measured locally by combining second- and fourth-order moments of phase average ﬁeld quadrature, that is to say: ρ = p(α, s)\|s, α⟩⟨s, α\|. (30) ˆn2 i = 16 6 1 2π dθ ( ˆXθ i )4 −1 2 1 2π dθ ( ˆXθ i )2 . (26) We deﬁne the quantity Ci,j,k = ⟨ˆni⟩ρT P G(η)(1 2(⟨ˆn2 j⟩ρT P G(η) + ⟨ˆn2 k⟩ρT P G(η)) (27) (30) ˆn2 i = 16 6 1 2π dθ ( ˆXθ i )4 −1 2 1 2π dθ ( ˆXθ i )2 . (26) A state is Gaussian if p(α, s) can be interpreted as a probability distribution. Non-Gaussian quantum states are essential to a variety of quantum information pro- cessing tasks such as quantum state distillation [36], quantum error-correction [37] or quantum computa- tional speedup [38]. We deﬁne the quantity We deﬁne the quantity Ci,j,k = ⟨ˆni⟩ρT P G(η)(1 2(⟨ˆn2 j⟩ρT P G(η) + ⟨ˆn2 k⟩ρT P G(η)) (27) (27) [ ] The third layer of quantum features is Wigner neg- ativity. 4 Non-classical features of TPG The Wigner function is a representation of a single mode state ρ in terms of the a quasi-probability distribution given as [39]: where ⟨.⟩ρT P G(η) is the expectation value of ′′.′′ on the state ρT P G. We can certify the presence of GME for the state ρT P G if the quantity Wρ(α) = 2 π Tr(D(α)(−1)a†aD(α)† ρ). (31) w = \|⟨ˆa† 1ˆa† 2ˆa† 3⟩ρT P G(η) + ⟨ˆa1ˆa2ˆa3⟩ρT P G(η)\| 2 −(C1,2,3 + C2,1,3 + C3,2,1) (28) (31) (28) Wigner negativity is arguably the strongest form of quantum feature for a single mode state and is a neces- sary condition to perform eﬃcient quantum computing using CV states [40]. We plot in Fig. 16 the Wigner function of state \|ψ⟩as a function of its canonical quadratures q and p. is positive. We plot in Fig. 14 w with respect to the eﬃciency η. We see a violation even for low eﬃciency, which makes the detection of GME for the state ρT P G robust against losses when homodyne detection is used. We can observe diﬀerent areas of negativities in the Wigner function, which implies that the TPG state is Wigner negative, non-Gaussian and non-classical. We note that this function is not phase-invariant, and as a consequence the presence of such negativities can- not be detected by only photon counting strategies. The reconstruction of the Wigner function requires a 4 Non-classical features of TPG We want to formulate a relaxation of this witness only in terms of local homodyne measurement. In order to formulate our relaxation, we ﬁrst note that (22) implies that any biseparable state satisﬁes: \|⟨ˆa† 1ˆa† 2ˆa† 3⟩\| ≤ ⟨ˆn1⟩⟨ˆn2ˆn3⟩+ ⟨ˆn2⟩⟨ˆn1ˆn3⟩ + ⟨ˆn3⟩⟨ˆn2ˆn1⟩. (23) (23) By summing (22) and (23) and using the triangular inequality, we end up with By summing (22) and (23) and using the triangular inequality, we end up with \|⟨ˆa† 1ˆa† 2ˆa† 3⟩+ ⟨ˆa1ˆa2ˆa3⟩\| 2 ≤ ⟨ˆn1⟩⟨ˆn2ˆn3⟩+ ⟨ˆn2⟩⟨ˆn1ˆn3⟩ + ⟨ˆn3⟩⟨ˆn2ˆn1⟩ (24) Even though the logarithmic negativity has the abil- ity to reveal the entanglement of a quantum system, it is very hard to measure in practice, as it requires a full tomography of the state. One instead can use a witness of genuine entanglement, i.e., an observable ˆO such that ⟨ˆO⟩≤0 for all biseparable state. As stated before, the state \|ψ3⟩associated with non-degenerate TPG has non-Gaussian entanglement, which implies that a witness of entanglement (or GME) for the state \|ψ3⟩requires at least a third-order ﬁeld operator. The authors of [31] derive a non-Gaussian witness of GME, which allows the demonstration of genuine entangle- (24) which holds for all biseparable states. Interestingly, the left-hand side of this previous equality can be measured using local homodyne measurement , i.e., ˆa† 1ˆa† 2ˆa† 3 + ˆa1ˆa2ˆa3 = −2( ˆX π 2 1 ˆX π 2 2 ˆX0 3 + ˆX0 1 ˆX π 2 2 ˆX π 2 3 + ˆX π 2 1 ˆX0 2 ˆX π 2 3 −ˆX0 1 ˆX0 2 ˆX0 3). 123 Eur. Phys. J. D (2022) 76:186 Page 13 of 21 186 Page 13 of 21 186 186 Fig. 14 GME certiﬁcation parameter w with respect to the eﬃciency η for ϵ = ξtint = 10−2 assume to be single mode. There exists a hierarchy of quantum features for a single mode of light in Fig. 15. The ﬁrst layer of quantum features is the non- classicality, which is deﬁned using the Glauber– Sudarshan (GS) or P distribution [32,33]. Any state ρ can be written in terms of coherent states \|α⟩as fol- lows: ρ = dα2P(α)\|α⟩⟨α\|. (29) (29) A state is classical if its GS distribution P(α) can be interpreted as a classical probability distribution [34]. 4.2 A hierarchy of quantum features for single mode of light We focus on the degenerate state \|ψ⟩, which we take to be the outcoming state created by the process associ- ated with the Hamiltonian of Eq. (19), and which we 12 3 186 Page 14 of 21 Eur. Phys. J. D (2022) 76:186 Fig. 15 Hierarchy of quantum features for single mode state of light Fig. 17 Experimental setup consisting of three balanced beam-splitters (t = r = 1/2) and four detectors D1-4 186 Page 14 of 21 Eur. Phys. J. D (2022) 76:186 Fig. 15 Hierarchy of quantum features for single mode state of light Fig. 15 Hierarchy of quantum features for single mode state of light e of light Fig. 17 Experimental setup consisting of three balanced beam-splitters (t = r = 1/2) and four detectors D1-4 Fig. 15 Hierarchy of quantum features for single mode state of light Fig. 16 Wigner function W of the state \|ψ⟩as a function of the canonical quadratures q and p. The positivity plane (colored in lighter color) divides the Wigner function axis between positive and negative values Fig. 17 Experimental setup consisting of three balanced beam-splitters (t = r = 1/2) and four detectors D1-4 ent modes and ii) the setup in Fig. 17 does not allow us to acquire information about the coherence terms of \|ψ⟩, then we can consider a single-mode state with the same number of photons than that of the expected mul- timode state in order to correctly model our experiment (see Appendix A). Fig. 16 Wigner function W of the state \|ψ⟩as a function of the canonical quadratures q and p. The positivity plane (colored in lighter color) divides the Wigner function axis between positive and negative values ( ) In Fig. 17, an input state ρi of the ith experimen- tal run is split into four spatially separated modes using three balanced beam splitters and sent to four non-photon number resolving (NPNR) detectors. We do not want to make assumptions about the eﬃciency of the NPNR detectors, so we consider perfect detec- tion and that any ineﬃciencies can be mapped into the input states ρi. A NPNR detector with unit eﬃciency can be modeled by a two element positive operator- valued measure (POVM) as the set {E•, E◦} = {1 − \|0⟩⟨0\|, \|0⟩⟨0\|}, corresponding to the single detector events click and no click, respectively. 4.2 A hierarchy of quantum features for single mode of light high number of experimental runs, which is not avail- able in the present experiment. Witnesses of Wigner negativity can be systematically derived using hierar- chy of semi-deﬁnite programs [41], but those witnesses require again a number of measurement runs that is much higher than the one available in our case, and for those reasons we proceed by focusing on non-classicality and non-Gaussianity only. 123 4.3 Witnessing non-classicality of the degenerate state \|ψ⟩ The number of runs that is needed to estimate the value of the witness, with a pre- cision 3 times smaller than the distance to the classical bound, can thus be estimated by ﬁnding the number of runs Nopt such that: Therefore, we can conclude that the maximum of the RHS of (34) is achieved by a pure coherent state. With this simpliﬁcation, we have that wc(θ) = max ∀ \|α⟩(⟨α\| ˆW(θ)\|α⟩). (36) (36) qopt = 3σWs. (41) (41) We ﬁnd that this maximization equivalent to a prob- lem of ﬁnding the roots of a third-order polynomial (see Appendix A) and can thus be simply performed ana- lytically. The vector ˆθ = (θ1, θ2, θ3) is associated with the weights of the diﬀerent events deﬁned by the opera- tors ˆPi on the witness ˆW. The witness is now arranged such that if for a given state ρ, it exists one vector ˆθ for which Tr( ˆW(θ)ρ) > wc(θ), then the non-classicality of the state ρ can be demonstrated using the setup of Fig. 17. We plot Nopt with respect to the eﬃciency in the inset of Fig. 19. With 4.9 triplets/s and a 5-W laser at 88 MHz, we ﬁnd that 18 seconds of experiment are enough to certify the non-classicality of the state for an eﬃciency of 50%. We plot Nopt with respect to the eﬃciency in the inset of Fig. 19. With 4.9 triplets/s and a 5-W laser at 88 MHz, we ﬁnd that 18 seconds of experiment are enough to certify the non-classicality of the state for an eﬃciency of 50%. 4.3 Witnessing non-classicality of the degenerate state \|ψ⟩ The set of non-classical states is convex (29), so that the set of the diﬀerent probabilities of clicks achieved by classical states is also convex, by linearity of the trace. We can thus consider a linear combination of probabil- ity operators corresponding to the events of click and no click on the detector arrangement, A witness of non-classicality can be represented by an observable ˆW together with the maximum expectation value of ˆW on a classical state. The most widely used witness of non-classicality is probably the second-order correlation function g2(0) [42] together with its classi- cal bound 1. Several criteria that analyze matrices of moments of annihilation and creation operators have also been derived [43–45]. In this section, we focus on the setup of Fig. 17 and derive a witness tailored to the state \|ψ⟩and that is based on photodetection events. We consider a single-mode case for the sake of clar- ity. The results are holding in the multimode case and, since i) the detector cannot distinguish between diﬀer- ˆW(θ) = ci ˆPi, (32) (32) where ˆPi are the POVM elements of the arrangement, which correspond to the event where i detectors click while the others do not click, as for example: ˆP4 = E(1) • E(2) • E(3) • E(4) • = (1 −\|0⟩⟨0\|)⊗4, and represent an 123 123 Page 15 of 21 186 Page 15 of 21 186 Eur. Phys. J. D (2022) 76:186 qopt = max θ (q(θ)). (38) event where all four detectors click. The coeﬃcients ci are parametrized such that the vector c(θ) is normal- ized: event where all four detectors click. The coeﬃcients ci are parametrized such that the vector c(θ) is normal- ized: (38) We plot in Fig. 18 qopt with respect to the overall eﬃ- ciency η of the setup for ϵ = ξt = 0.01. We ﬁnd that, in the present case, the optimal q is always found when wc(θ) = 0. We also ﬁnd a positive qopt for the range of all eﬃciencies, which proves the detection of non- classicality of the state \|ψ⟩using the setup of Fig. 17 to be robust against losses. We plot in Fig. 18 qopt with respect to the overall eﬃ- ciency η of the setup for ϵ = ξt = 0.01. We ﬁnd that, in the present case, the optimal q is always found when wc(θ) = 0. 4.3 Witnessing non-classicality of the degenerate state \|ψ⟩ We also ﬁnd a positive qopt for the range of all eﬃciencies, which proves the detection of non- classicality of the state \|ψ⟩using the setup of Fig. 17 to be robust against losses. c(θ) = ⎛ ⎜ ⎝ sin θ1 sin θ2 cos θ1 sin θ3 cos θ2 cos θ1 cos θ3 cos θ2 cos θ1, ⎞ ⎟ ⎠. (33) (33) The next step is to compute the maximum expec- tation value of the observable constructed in Eq. (32) that any classical state can achieve, i.e., In the last part of this section, we turn to give an estimation of the number of experimental runs that would be necessary to estimate the quantity Ws = Tr(ρT P G(η) ˆW(θ)). We assume that the POVM ele- ments ˆPi are independent quantities that are measured N times. At each run, we evaluate a random variable Xi, associated with ˆPi, which takes the value 1 when i detectors click and i −4 do not click, and the value 0 otherwise. An unbiased estimator of Ws after N runs is given by: wc(θ) = max ∀ ρc(Tr ˆW(θ)ρc ). (34) (34) We note that Tr ˆW(θ)ρc is linear in ρc, and since any arbitrary classical state can be written as a mixture of pure classical states according to Eq. (29), we ﬁnd that: Tr ˆW(θ)ρc = Tr ˆW(θ) dα2P(α)\|α⟩⟨α\| = dα2P(α)Tr ˆW(θ)\|α⟩⟨α\| ≤ dα2P(α) max α (Tr ˆW(θ)\|α⟩⟨α\| ) = max α (Tr ˆW(θ)\|α⟩⟨α\| ). (35) ¯ Ws = N k=1 4 i=1 ci Xk i N . (39) (39) We bound the standard deviation of ¯ Ws as follows: We bound the standard deviation of ¯ Ws as follows: σWs = i=1 \|ci\|σXi N , (40) (40) (35) where σXi = ⟨Xi⟩(1 −⟨Xi⟩) is the standard devia- tion of a binary variable. The number of runs that is needed to estimate the value of the witness, with a pre- cision 3 times smaller than the distance to the classical bound, can thus be estimated by ﬁnding the number of runs Nopt such that: where σXi = ⟨Xi⟩(1 −⟨Xi⟩) is the standard devia- tion of a binary variable. 4.4 Demonstrating non-Gaussianity of the degenerate state \|ψ⟩ In the lower inset, we zoom in on the region of eﬃciency η from 0 to 45%, showing that we ﬁnd positive values of gopt for all eﬃciencies Fig. 18 Optimized mean values of qopt. The optimization is performed numerically over the set of parameter coeﬃ- cients θ for a range of eﬃciencies η for ϵ = 10−2. Inset: Number of experimental runs Nopt necessary for certiﬁca- tion Fig. 19 Optimized mean values of gopt. The optimization is performed numerically over the set of parameter coeﬃ- cients θ for a range of eﬃciencies η and for ϵ = 10−2. In the upper inset, we plot the expected minimum number of runs Nopt necessary for non-Gaussianity certiﬁcation. In the lower inset, we zoom in on the region of eﬃciency η from 0 to 45%, showing that we ﬁnd positive values of gopt for all eﬃciencies two as in [46]. In order to witness such non-Gaussianity, we again need to specialize the witness over the free parameters of the coeﬃcients c(θ). A ﬁrst step is deﬁn- ing and computing the maximum mean value wg of the witness operator over all Gaussian states, i.e., Figure 19 shows the values of gopt obtained by numer- ical optimizations performed over a range of eﬃciencies η of the setup. We ﬁnd that a violation of the Gaussian bound can be witnessed by the auto-correlation witness of the form of Eq. (32) over the range of all eﬃciencies, which again demonstrates the robustness of the method against photon losses. In order to estimate the number Nopt of experimental runs needed for the certiﬁcation, we one more time set wg(θ) = max ∀ ρG(Tr ˆW(θ)ρG ). (42) (42) We note one more time that this expression is linear in ρG, and thus, we can conclude in the same manner that its maximum is achieved by a pure state and that it is then suﬃcient to perform the optimization over the set of pure Gaussian states. In this case, as opposed to the witnessing of non-classicality via wc(θ), the expression for wg(θ) is more intricated and the optimization was performed numerically over all states of the form gopt = 3σWs, (45) (45) where σWs is given by Eq. (39), but now using the coef- ﬁcients ci optimized for the non-Gaussianity witness. This estimation is plotted in the upper inset Fig. 19 for the range of eﬃciencies η. 4.4 Demonstrating non-Gaussianity of the degenerate state \|ψ⟩ ρG = ˆSs ˆDα\|0⟩, (43) (43) i.e., squeezed coherent states, in which ˆDα and ˆSs are the canonical displacement and squeezing operators. More details can be found in Appendix B. The sec- ond and last step is performing a further optimization, now over the set of the coeﬃcient parameters θ, of the diﬀerence 4.4 Demonstrating non-Gaussianity of the degenerate state \|ψ⟩ A ﬁrst step is deﬁn- ing and computing the maximum mean value wg of the witness operator over all Gaussian states, i.e., wg(θ) = max ∀ ρG(Tr ˆW(θ)ρG ). (42) We note one more time that this expression is linear in ρG, and thus, we can conclude in the same manner that its maximum is achieved by a pure state and that it is then suﬃcient to perform the optimization over the set of pure Gaussian states. In this case, as opposed to the witnessing of non-classicality via wc(θ), the expression for wg(θ) is more intricated and the optimization was performed numerically over all states of the form Fig. 19 Optimized mean values of gopt. The optimization is performed numerically over the set of parameter coeﬃ- cients θ for a range of eﬃciencies η and for ϵ = 10−2. In the upper inset, we plot the expected minimum number of runs Nopt necessary for non-Gaussianity certiﬁcation. In the lower inset, we zoom in on the region of eﬃciency η from 0 to 45%, showing that we ﬁnd positive values of gopt for all eﬃciencies Figure 19 shows the values of gopt obtained by numer- ical optimizations performed over a range of eﬃciencies η of the setup. We ﬁnd that a violation of the Gaussian bound can be witnessed by the auto-correlation witness of the form of Eq. (32) over the range of all eﬃciencies, which again demonstrates the robustness of the method against photon losses. In order to estimate the number Nopt of experimental runs needed for the certiﬁcation, we one more time set gopt = 3σWs, (45) where σWs is given by Eq. (39), but now using the coef- ﬁcients ci optimized for the non Gaussianity witness Fig. 18 Optimized mean values of qopt. The optimization is performed numerically over the set of parameter coeﬃ- cients θ for a range of eﬃciencies η for ϵ = 10−2. Inset: Number of experimental runs Nopt necessary for certiﬁca- tion Fig. 19 Optimized mean values of gopt. The optimization is performed numerically over the set of parameter coeﬃ- cients θ for a range of eﬃciencies η and for ϵ = 10−2. In the upper inset, we plot the expected minimum number of runs Nopt necessary for non-Gaussianity certiﬁcation. 4.4 Demonstrating non-Gaussianity of the degenerate state \|ψ⟩ In practice, the state \|ψ⟩will experience losses; we then deﬁne the state A witness operator ˆW in the same general form of Eqs. (32–33) associated with the linear optical setup of Fig. 17 proves to be useful to the demonstration of the non- Gaussianity of the one-mode state \|ψ⟩as well, meaning that, by analyzing the mean value of this operator on this state, we can assert that \|ψ⟩cannot be written as a convex mixture of pure Gaussian states. Witnesses of non-Gaussianity for arbitrary Fock states using linear optics have been derived in [46]. The aim of this section is to derive a witness of non-Gaussianity tailored to the state \|ψ⟩where the full knowledge of the probabilities distributions of each detector is used, and not only of (37) ρT P G(η) = TrB(Uη\|ψ⟩⟨ψ\| ⊗\|0⟩⟨0\|BU † η), (37) where Uη is a unitary corresponding to a beam-splitter with transmitivity η, referring to the eﬃciency, and the quantity q(θ) = Tr(ρT P G(η) ˆW(θ))−wc(θ), that will be positive when the witness can detect non-classicality for the lossy state ρT P G(η). In order to ﬁnd the opti- mal witness of the state for a given eﬃciency, we can compute: 12 3 186 Page 16 of 21 Eur. Phys. J. D (2022) 76:186 Fig. 18 Optimized mean values of qopt. The optimization is performed numerically over the set of parameter coeﬃ- cients θ for a range of eﬃciencies η for ϵ = 10−2. Inset: Number of experimental runs Nopt necessary for certiﬁca- tion Fig. 19 Optimized mean values of gopt. The optimization is performed numerically over the set of parameter coeﬃ- cients θ for a range of eﬃciencies η and for ϵ = 10−2. In the upper inset, we plot the expected minimum number of runs Nopt necessary for non-Gaussianity certiﬁcation. In the l i i h i f ﬃi f 0 Eur. Phys. J. D (2022) 76:186 186 Page 16 of 21 186 Page 16 of 21 Eur. Phys. J. D (2022) 76:186 Fig. 18 Optimized mean values of qopt. The optimization is performed numerically over the set of parameter coeﬃ- cients θ for a range of eﬃciencies η for ϵ = 10−2. Inset: Number of experimental runs Nopt necessary for certiﬁca- tion two as in [46]. In order to witness such non-Gaussianity, we again need to specialize the witness over the free parameters of the coeﬃcients c(θ). 5 Discussion and conclusion In this article, we have reviewed our past work, both experimental and theoretical, from bulk crystal to KTP waveguide and from a simplistic quantum anal- ysis extrapolated from second-order nonlinear process, to a deep understanding of peculiarities of quantum behavior introduced by the third-order nonlinear inter- action. We thus highlighted these diﬀerences, such as the dependence of the classical and quantum behavior on the seeding intensity, and the emergence and con- straints induced by the non-Gaussianity. Then, we pre- sented original theoretical results and ﬁxed one of the remaining important problems: how to experimentally g(θ) = Tr(ρT P G(η) ˆW(θ)) −wg(θ), (44) (44) where ρT P G is the lossy state given by Eq. (37), in order to ﬁnd gopt = maxθ(g(θ)). Finding a positive value of gopt certiﬁes that the mean value of the witness in state ρT P G breaks the Gaussian bound and thus that the state \|ψ⟩cannot be represented by a mixture of states of the form of Eq. (43), meaning that this state is non- Gaussian. 123 Eur. Phys. J. D (2022) 76:186 Page 17 of 21 186 BB, EO and KB wrote the manuscript with the help of JB and MFBC for plotting ﬁgures and critical feedback from all authors. conclude at the quantumness and the non-Gaussianity of the generated triple-photon states engaging a mini- mal resource budget. We have thus introduced quantum witnesses using the minimum number of photocounting detectors, optimizing thus losses and complexity. The spontaneous generation of triple photon statistics by the third-order nonlinear interaction is still elusive, but we feel now closer. Data Availability Statement This manuscript has no associated data or the data will not be deposited. [Authors’ comment: Data underlying the results presented in this paper are not publicly available at this time but may be obtained from the authors upon reasonable request.] The following step is to demonstrate the ability of these states to exhibit stronger form of quantum cor- relation such as Wigner negativity or non-locality. It is then possible to use these behaviors in quantum infor- mation protocols. The ability of generating heralded pairs of photons can be used in quantum repeater pro- tocols. Moreover, using photon detectors preceded by displacement operation on one of the three modes of the state \|ψ⟩3, it is then possible to herald a state of the form \|00⟩+ ϵ\|11⟩that has the ability to violate Bell inequalities [47]. Declarations Competing interests Authors declare to disclose interests that are directly or indirectly related to the work submitted for publication. Open Access This article is licensed under a Creative Com- mons 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 arti- cle 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 statu- tory 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://creativecomm ons.org/licenses/by/4.0/. Appendix A: Non-classicality witness Acknowledgements We thank Nicolas Sangouard for fruitfull discussions. This work was supported by Agence Nationale de la Recherche and Fonds National Suisse ANR/FNS Projets de recherche collaborative – Interna- tional (PRCI 16707). It has also been partly supported by the French RENATECH network and its FEMTO-ST technological facility and by the EIPHI Graduate school (contract ”ANR-17-EURE-0002”). E.O. acknowledges sup- port from the Government of Spain (FIS2020-TRANQI and Severo Ochoa CEX2019-000910-S), Fundaci´o Cellex, Fundaci´o Mir-Puig, Generalitat de Catalunya (CERCA, AGAUR SGR 1381) and from the ERC AdGCERQUT. A pure coherent state can be written in the Fock or number state basis as \|α⟩= exp −\|α\|2 2 ∞ n=0 αn √ n! \|n⟩, (A1) (A1) where α is a parameter deﬁning the speciﬁc state. The action of balanced beam-splitters at coherent states given by Eq. (A1) is straightforwardly computable. Actually, after a beam splitting, the state is represented by two pure coherent states attenuated as α ′ →α/ √ 2 at each output path, so that the probabilities Pi of observing i clicks at the detectors of Fig. 17 can be written as 5 Discussion and conclusion Time-bin genuine entanglement and non-locality can be demonstrated on the state \|ψ⟩3 using Franson-type measurement. Indeed, all the pho- tons can be sent to a single imbalanced interferome- ter and look at threefold coincidences at the outputs of the interferometer. Then, if the arm length diﬀer- ence of the interferometers is smaller than the coherence length of the pump, it is by principle impossible to tell if all three photons have taken the short or the long arm, eﬀectively generating a three-mode Greenberger- Horne-Zeilinger state (GHZ state). The long-term aim is to add TPG process to the quantum optics toolbox for quantum communication and computation. Author contributions By com- puting the value of W(θ) at values α associated with each of the found roots, and verifying which of those points consist of W(θ) maxima ( ∂2W (θ) ∂2α < 0), the max- imization of Eq. (34) can thus be completely computed analytically. ˆPi = 4 i (ˆI −ˆP0)i( ˆP0)(4−i), (B2) (B2) where ˆP0 = \|0⟩⟨0\| is the projector onto vacuum at each detector. We use the fact that any Gaussian state \|G⟩ can be written as a squeezed coherent state and, with- out loss of generality, we can choose s to be real while keeping α generally complex. Let us focus on the multimode case, since we cannot have access to the coherence on the setup. A general multimode classical state can be written as: Alternatively, and for convenience of calculation, we can reinterpret Eq. (B1) as a reverse evolution U † acting on the ˆPi operators and compute the expec- tation value of U † ˆPiU in the incoming general pure Gaussian state \|G⟩. In accordance with Eq. (B2), this can be performed by deriving all the operators of the form U †( ˆP0)k(ˆI)4−kU, each of those associated to hav- ing 0 clicks at k detectors without mention of what is observed at the other 4−k detectors. This can be done as follows. ρm = λ p(λ) ϱλ = λ p(λ) k ρ[k] λ (A5) (A5) where ρ[k] λ = \|αk⟩⟨αk\|. The POVM associated with the event ”i detector click and 4-i does not clicks” can be written as: ˆPim = 4 i (1 − k ˆP0)⊗i ⊗( k ˆP0)⊗(4−i) (A6) For a Fock state \|n⟩, the probability of detecting a vacuum state (no click) at a detector placed after two balanced beam-splitters, without mention to what is observed at any remaining detectors, can be written as: The maximum of Tr(W(θ)ρm) is achieved by pure mul- timode states for which we have The maximum of Tr(W(θ)ρm) is achieved by pure mul- timode states for which we have P0,n = 1 2 n n k=0 n k 1 2 k = 3 4 n . Author contributions (B3) Tr( ˆPim\|αk⟩⟨αk\|⊗k) = 4 i 1 − k i=1 e− \|αk\|2 4 i k i=1 e− \|αk\|2 4 (4−i) (A7) = 4 i 1 −e− k i=1 \|αk\|2 4 i e− k i=1 \|αk\|2 4 (4−i) (A8) (B3) For any convex combination of Fock states \|n⟩, the associated projector onto vacuum at this detector (U †( ˆP0)1(ˆI)3U) can then be written in terms of the number operator a†a as: which is the same expression than (A2) if we take \|α\| = k i=1 \|αk\|2. Thus, the bound for single mode holds for the multimode case. ˆP0,n = 3 4 a†a . (B4) (B4) The operators associated with the detection of two or three vaccum states after the beam-splitters can be derived in the same manner, and are given, respectively, by: Author contributions BB developed the nonlinear theoretical framework on TPG and designed the experiments on bulk crystals. KB, AL and BB developed the quantum theoretical framework on TPG. EO, KB JCP and MFBC devel- oped the models and simulations on quantum proper- ties. BB, VB, CF, AL, KB, AV, JB and MC designed and implemented THG and TPG experiments on opti- cal waveguides, data analysis and simulations. MC and FBa designed and fabricated the optical waveguides. FBu and HZ have been working on the development of SNSPD detectors for future quantum experiments. BB, AL and HZ contributed to the design of the research. Pi = 4 4 −i exp(−\|α\|2 4 ) i 1 −exp(−\|α\|2 4 ) i , (A2) (A2) for i = 1, 2, 3, 4. The non-classicality witness will then simply be given by the summation W(θ) = 4 i=1 ci(θ)Pi, (A3) (A3) 123 12 3 3 Page 18 of 21 Eur. Phys. J. D (2022) 76:186 Eur. Phys. J. D (2022) 76:186 186 after the unitary evolution U representing the beam- splitters, and for a general pure Gaussian state \|G⟩ arriving in the setup of Fig. 17 this can be written as: with ci(θ) deﬁned by Eq. (33), and the maximization of wc(θ) of Eq. (34) will consist of a maximization over the sole parameter α. If we ﬁrst deﬁne the change of parameters x = exp(−\|α\|2 4 ), the value of α which max- imizes Eq. (A3) can be found by computing the roots of after the unitary evolution U representing the beam- splitters, and for a general pure Gaussian state \|G⟩ arriving in the setup of Fig. 17 this can be written as: Pi = ⟨G\|U † ˆPiU\|G⟩ = ⟨0\| ˆD† α ˆS† sU † ˆPiU ˆSs ˆDα\|0⟩ = ⟨α\| ˆS† sU † ˆPiU ˆSs\|α⟩, (B1) (B1) ∂W(θ) ∂x = 4 i=1 ci(θ)∂Pi ∂x = 0, (A4) (A4) where ˆDα and ˆSs are the canonical displacement and squeezing operators, with displacement and squeezing parameters equal to α and s, respectively, and with ˆPi given by so that αopt will be given by ± −4 ln(x), with x ade- quately picked among the found roots of Eq. (A4). As can easily be seen, due to the form of the Pi, Eq. (A4) is, in turn, a third-order polynomial in the new variable x, and thus, its roots can be routinely found. References Hayat, M. Orenstein, Photon conversion processes in dispersive microcavities: quantum-ﬁeld model. Phys. Rev. A 77, 013830 (2008). https://doi.org/10.1103/ PhysRevA.77.013830 where Ss = sinh (s), Cs = cosh (s), Ts = tanh (s) and Fs = e−kC2 s −ekS2 s, and, to be consistent with ref. [49] results, we have to take s to −s. By taking values for the parameter k in accordance with each of the projectors of Eqs. (B4) and (B5), we will have all needed terms of the form S†(s)U †( ˆP0)k(ˆI)4−kUS(s), and we are ready to compute their expectation values on the coherent state \|α⟩: as Eq. (B7) is normal ordered in the ﬁeld operators, it can be directly applied between ⟨α\| and \|α⟩in Eq. (B1) using ⟨α\|a†2\|α⟩= α∗2, ⟨α\|a2\|α⟩= α2 and ⟨α\|a†a\|α⟩= \|α\|2. where Ss = sinh (s), Cs = cosh (s), Ts = tanh (s) and Fs = e−kC2 s −ekS2 s, and, to be consistent with ref. [49] results, we have to take s to −s. By taking values for the parameter k in accordance with each of the projectors of Eqs. (B4) and (B5), we will have all needed terms of the form S†(s)U †( ˆP0)k(ˆI)4−kUS(s), and we are ready to compute their expectation values on the coherent state \|α⟩: as Eq. (B7) is normal ordered in the ﬁeld operators, it can be directly applied between ⟨α\| and \|α⟩in Eq. (B1) using ⟨α\|a†2\|α⟩= α∗2, ⟨α\|a2\|α⟩= α2 and ⟨α\|a†a\|α⟩= \|α\|2. 9. M. Corona, K. Garay-Palmett, A.B. U’Ren, Experi- mental proposal for the generation of entangled photon triplets by third-order spontaneous parametric down- conversion in optical ﬁbers. Opt. Lett. 36(2), 190–192 (2011). https://doi.org/10.1364/OL.36.000190 ( ) p // g/ / 10. M.V. Chekhova, O.A. Ivanova, V. Berardi, A. Garuc- cio, Spectral properties of three-photon entangled states generated via three-photon parametric down-conversion in a χ(3) medium. Phys. Rev. A 72, 023818 (2005). https://doi.org/10.1103/PhysRevA.72.023818 // / / 11. A. Cavanna, F. Just, X. Jiang, G. Leuchs, M.V. Chekhova, P.S.J. Russell, N.Y. Joly, Hybrid photonic- crystal ﬁber for single-mode phase matched generation of third harmonic and photon triplets. Optica 3(9), 952– 955 (2016). https://doi.org/10.1364/OPTICA.3.000952 ⟨\| \| ⟩ \| \| The witness of Eq. (32) of a Gaussian state can then be readily computed by summing the results as: ⟨G\| ˆ W(θ)\|G⟩= 4 i=1 ci⟨G\| ˆPi\|G⟩ (B8) (B8) 12. C.C. Evans, K. Shtyrkova, O. Reshef, M. Moebius, J.D.B. Bradley, S. Griesse-Nascimento, E. Ippen, E. References (B5) 1. A. Aspect, P. Grangier, G. Roger, Experimental tests of realistic local theories via bell’s theorem. Phys. Rev. Lett. 47, 460–463 (1981). https://doi.org/10.1103/ PhysRevLett.47.460 Finally, the operator associated with all four detections not clicking ( ˆP0000,n = U †( ˆP0)4U) will just be the vac- uum operator \|0⟩⟨0\|, with associated probability being the modulus squared of the overlap of the incoming Gaussian state with the vaccum, that is ⟨G\|0⟩⟨0\|G⟩= \|⟨0\|G⟩\|2. As derived in ref.[48], we have that: Finally, the operator associated with all four detections not clicking ( ˆP0000,n = U †( ˆP0)4U) will just be the vac- uum operator \|0⟩⟨0\|, with associated probability being the modulus squared of the overlap of the incoming Gaussian state with the vaccum, that is ⟨G\|0⟩⟨0\|G⟩= \|⟨0\|G⟩\|2. As derived in ref.[48], we have that: 2. S.L. Braunstein, R.I. McLachlan, Generalized squeez- ing. Phys. Rev. A 35, 1659–1667 (1987). https://doi. org/10.1103/PhysRevA.35.1659 3. K. Banaszek, P.L. Knight, Quantum interference in three-photon down-conversion. Phys. Rev. A 55, 2368–2375 (1997). https://doi.org/10.1103/PhysRevA. 55.2368 ⟨0\|G⟩= ⟨0\| ˆSs ˆDα\|0⟩= exp −1 2\|α\|2 + 1 2α2 tanh (s) . (B6) 4. J. Douady, B. Boulanger, Experimental demonstration of a pure third-order optical parametric downconversion process. Opt. Lett. 29(23), 2794–2796 (2004). https:// doi.org/10.1364/OL.29.002794 (B6) (B6) Again, instead of directly computing the expectation value of such projection operators in a general Gaus- sian state, in accordance with Eq. (B1), we can apply the reversed squeezing operator to these projectors and compute their expectation values on a coherent state \|α⟩. Following the result in Eq. (9) of ref. [49], and the fact that e(ka†a)=:e((ek−1)a†a) :, where :: represents nor- mal ordering, we have 5. S. Richard, K. Bencheikh, B. Boulanger, J.A. Levenson, Semiclassical model of triple photons generation in opti- cal ﬁbers. Opt. Lett. 36(15), 3000–3002 (2011). https:// doi.org/10.1364/OL.36.003000 6. A. Vernay, V. Boutou, C. F´elix, D. Jegouso, F. Bassig- not, M. Chauvet, B. Boulanger, Birefringence phase- matched direct third-harmonic generation in a ridge optical waveguide based on a ktiopo4 single crystal. Opt. Express 29(14), 22266–22274 (2021). https://doi.org/ 10.1364/OE.432636 ˆS† seka†a ˆSs = e−k 2 (e Ts 2 ( e2k C2s −S2s e2k −1)a†2 × : e(e−ln Fs−1)a†a : ×e Ts 2 ( e2k C2s −S2s e2k −1)a2 )/ Fs, (B7) 7. T. Jennewein, C. Simon, G. Weihs, H. Weinfurter, A. Zeilinger, Quantum cryptography with entangled pho- tons. Phys. Rev. Lett. 84, 4729–4732 (2000). https:// doi.org/10.1103/PhysRevLett.84.4729 8. A. Appendix B: Non-gaussianity witness The expectation value of the probability Pi of observ- ing i out of 4 detectors clicking is associated with the projector operator ˆPi of Eq. (32) acting on the state ˆP00,n = 1 2 a†a , 123 Eur. Phys. J. D (2022) 76:186 Page 19 of 21 186 ˆP000,n = 1 4 a†a . (B5) References References Mazur, Multimode phase-matched third-harmonic gen- eration in sub-micrometer-wide anatase tio2 waveg- uides. Opt. Express 23(6), 7832–7841 (2015). https:// doi.org/10.1364/OE.23.007832 Finally, we witness non-Gaussianity of state \|ψ⟩when- ever we ﬁnd θ so that: 13. M.G. Moebius, F. Herrera, S. Griesse-Nascimento, O. Reshef, C.C. Evans, G.G. Guerreschi, A. Aspuru- Guzik, E. Mazur, Eﬃcient photon triplet generation in integrated nanophotonic waveguides. Opt. Express 24(9), 9932–9954 (2016). https://doi.org/10.1364/OE. 24.009932 g(θ) = ⟨ψ\| ˆW(θ)\|ψ⟩−max ∀ α,s ×(⟨G(α, s)\| ˆW(θ)\|G(α, s)⟩) > 0. (B9) g(θ) = ⟨ψ\| ˆW(θ)\|ψ⟩−max ∀ α,s ×(⟨G(α, s)\| ˆW(θ)\|G(α, s)⟩) > 0. (B9) (B9) Moreover, we can optimize the witness by computing gopt = maxθ(g(θ)). Moreover, we can optimize the witness by computing gopt = maxθ(g(θ)). 12 3 Eur. Phys. J. D (2022) 76:186 186 Page 20 of 21 67, 052315 (2003). https://doi.org/10.1103/PhysRevA. 67.052315 67, 052315 (2003). https://doi.org/10.1103/PhysRevA. 67.052315 14. M. Akbari, A.A. Kalachev, Third-order spontaneous parametric down-conversion in a ring microcavity. Laser Phys. Lett. 13(11), 115204 (2016). https://doi.org/10. 1088/1612-2011/13/11/115204 29. D. Zhang, Y. Cai, Z. Zheng, D. Barral, Y. Zhang, M. Xiao, K. Bencheikh, Non-Gaussian nature and entangle- ment of spontaneous parametric nondegenerate triple- photon generation. Phys. Rev. A 103, 013704 (2021). https://doi.org/10.1103/PhysRevA.103.013704 15. C.W. Sandbo Chang, C. Sab´ın, P. Forn-D´ıaz, F. Qui- jandr´ıa, A.M. Vadiraj, I. Nsanzineza, G. Johansson, C.M. Wilson, Observation of three-photon spontaneous parametric down-conversion in a superconducting para- metric cavity. Phys. Rev. X 10, 011011 (2020). https:// doi.org/10.1103/PhysRevX.10.011011 30. G. Vidal, R.F. Werner, Computable measure of entan- glement. Phys. Rev. A 65, 032314 (2002). https://doi. org/10.1103/PhysRevA.65.032314 16. J. Kolody´nski, A. M´attar, P. Skrzypczyk, E. Wood- head, D. Cavalcanti, K. Banaszek, A. Ac´ın, Device- independent quantum key distribution with single- photon sources. Quantum 4, 260 (2020). ISSN 2521- 327X. https://doi.org/10.22331/q-2020-04-30-260. 31. A. Agust´ı, C.W. Sandbo Chang, F. Quijandr´ıa, G. Johansson, C.M. Wilson, C. Sab´ın, Tripartite genuine non-gaussian entanglement in three-mode spontaneous parametric down-conversion. Phys. Rev. Lett. 125, 020502 (2020). https://doi.org/10.1103/PhysRevLett. 125.020502 17. A. Dot, A. Borne, B. Boulanger, K. Bencheikh, J.A. Levenson, Quantum theory analysis of triple photons generated by a χ(3) process. Phys. Rev. A 85, 023809 (2012). https://doi.org/10.1103/PhysRevA.85.023809 32. E.C.G. Sudarshan, Equivalence of semiclassical and quantum mechanical descriptions of statistical light. Phys. Rev. Lett 10, 277 (1963). https://doi.org/10. 1103/PhysRevLett.10.277 18. B. Boulanger, J. Zyss, Non-linear optical properties. Chapter 17 in International Tables for Crystallography. D Phys. Prop. Crystals:181–222 (2013) 33. R.J. Glauber, Coherent and incoherent states of the radiation ﬁeld. Phys. Rev. 131, 2766 (1963). References https:// doi.org/10.1103/PhysRev.130.2529 19. K. Kato, Parametric oscillation at 3.2 μ m in ktp pumped at 1.064 mu m. IEEE J. Quant. Electron. 27(5), 1137–1140 (1991). https://doi.org/10.1109/3.83367 34. U.M. Titulaer, R.J. Glauber, Correlation functions for coherent ﬁelds. Phys. Rev. 140, B676–B682 (1965). https://doi.org/10.1103/PhysRev.140.B676 20. V. Boutou, A. Vernay, C. F´elix, F. Bassignot, M. Chau- vet, D. Lupinski, B. Boulanger, Phase-matched second- harmonic generation in a ﬂux grown ktp crystal ridge optical waveguide. Opt. Lett. 43(15), 3770–3773 (2018). https://doi.org/10.1364/OL.43.003770 35. R. L. Hudson, When is the wigner quasi- probability density non-negative? Rep. Math. Phys. 6(2), 249–252 (1974). ISSN 0034-4877. https://doi.org/10.1016/0034-4877(74)90007-X. URL https://www.sciencedirect.com/science/article/pii/ 003448777490007X 21. J.-P. F`eve, B. Boulanger, J. Douady, Speciﬁc proper- ties of cubic optical parametric interactions compared to quadratic interactions. Phys. Rev. A 66, 063817 (2002). https://doi.org/10.1103/PhysRevA.66.063817 36. G. Giedke, J. Ignacio Cirac, Characterization of gaus- sian operations and distillation of gaussian states. Phys. Rev. A 66, 032316 (2002). https://doi.org/10.1103/ PhysRevA.66.032316 22. F. Gravier, B. Boulanger, Triple-photon generation: comparison between theory and experiment. J. Opt. Soc. Am. B 25(1), 98–102 (2008). https://doi.org/10. 1364/JOSAB.25.000098 37. J. Niset, J. Fiur´aˇsek, N.J. Cerf, No-go theorem for gaus- sian quantum error correction. Phys. Rev. Lett. 102, 120501 (2009). https://doi.org/10.1103/PhysRevLett. 102.120501 23. B. Boulanger, I. Rousseau, G. Marnier, Cubic optical nonlinearity of ktiop04. J. Phys. B: Atom. Mol. Opt. Phys. 32(2), 475–488 (1999). https://doi.org/10.1088/ 0953-4075/32/2/026 38. S.D. Bartlett, B.C. Sanders, S.L. Braunstein, K. Nemoto, Eﬃcient classical simulation of continu- ous variable quantum information processes. Phys. Rev. Lett. 88, 097904 (2002). https://doi.org/10.1103/ PhysRevLett.88.097904 24. A. Dot, A. Borne, B. Boulanger, P. Segonds, C. F´elix, K. Bencheikh, J. Ariel Levenson, Energetic and spectral properties of triple photon downconversion in a phase- matched ktiopo4 crystal. Opt. Lett. 37(12), 2334–2336 (2012). https://doi.org/10.1364/OL.37.002334 39. A. Royer, Wigner function as the expectation value of a parity operator. Phys. Rev. A 15, 449–450 (1977). https://doi.org/10.1103/PhysRevA.15.449 25. J.R. Johansson, P.D. Nation, F. Nori, Qutip: an open- source python framework for the dynamics of open quantum systems. Comput. Phys. Commun. 183(8), 1760–1772 (2012). https://doi.org/10.1016/j.cpc.2012. 02.021 40. A. Mari, J. Eisert, Positive wigner functions render clas- sical simulation of quantum computation eﬃcient. Phys. Rev. Lett. 109, 230503 (2012). https://doi.org/10.1103/ PhysRevLett.109.230503 26. J.R. Johansson, P.D. Nation, F. Nori, Qutip 2: a python framework for the dynamics of open quantum systems. Comput. Phys. Commun. 184(4), 1234–1240 (2013). https://doi.org/10.1016/j.cpc.2012.11.019 41. U. Chabaud, P.E. Emeriau, F. Grosshans, Witness- ing wigner negativity. Quantum 471, 230503 (2021). https://doi.org/10.22331/q-2021-06-08-471 42. D.F. References Walls, G.J. Milburn, Quantum Optics. Springer, Berlin, Heidelberg, 2 edition (2008). ISBN 978-3-540- 28574-8 https://doi.org/10.1016/j.cpc.2012.11.019 27. E.A. Rojas Gonz´alez, A. Borne, B. Boulanger, J.A. Lev- enson, K. Bencheikh, Continuous-variable triple-photon states quantum entanglement. Phys. Rev. Lett. 120(4), 043601 (2018). https://doi.org/10.1103/PhysRevLett. 120.043601 43. A. Miranowicz, M. Bartkowiak, X. Wang, Y. Liu, F. Nori, Testing nonclassicality in multimode ﬁelds: a uni- ﬁed derivation of classical inequalities. Phys. Rev. A 82, 013824 (2010). https://doi.org/10.1103/PhysRevA. 82.013824 28. P. van Loock, A. Furusawa, Detecting genuine multi- partite continuous-variable entanglement. Phys. Rev. A 3 Eur. Phys. J. D (2022) 76:186 Page 21 of 21 186 48. J.J. Gong, Expansion coeﬃcients of a squeezed coherent state in the number state basis. Am. J. Phys. 58(10), 1003 (1990). https://doi.org/10.1119/1.16337 44. T. Richter, W. Vogel, Nonclassicality of quantum states: a hierarchy of observable conditions. Phys. Rev. Lett. 89, 283601 (2002). https://doi.org/10.1103/ PhysRevLett.89.283601 49. P. Sekatski, B. Sanguinetti, E. Pomarico, N. Gisin, C. Simon, Cloning entangled photons to scales one can see. Phys. Rev. A 82, 053814 (2010). https://doi.org/10. 1103/PhysRevA.82.053814 45. E. Shchukin, T. Richter, W. Vogel, Nonclassicality cri- teria in terms of moments. Phys. Rev. A 71, 011802 (2005). https://doi.org/10.1103/PhysRevA.71.011802 46. I. Straka, L. Lachman, J. Hlouˇsek, M. Mikov´a, M. Miˇcuda, M. Jeˇzek, R. Filip, Quantum non-gaussian mul- tiphoton light. npj Quant. Inf. 4, 13 (2018). https://doi. org/10.1038/s41534-017-0054-y / / 47. V. Caprara Vivoli, P. Sekatski, J.-D. Bancal, C.C.W. Lim, A. Martin, R.T. Thew, H. Zbinden, N. Gisin, N. Sangouard, Comparing diﬀerent approaches for generat- ing random numbers device-independently using a pho- ton pair source. New J. Phys. 17(2), 023023 (2015). https://doi.org/10.1088/1367-2630/17/2/023023 12 3
https://openalex.org/W3212020417	https://link.springer.com/content/pdf/10.1007/s00204-021-03190-1.pdf	English	null	An in vitro-based hazard assessment of liquid smoke food flavourings	Archives of toxicology	2,021	cc-by	7,815	Abstract Liquid smoke products are widely used as a food additive to create a desired smoke flavour. These products may contain hazardous chemicals generated during the wood-burning process. However, the toxic effects of these types of hazardous chemicals constituting in the commercially available products are largely unknown. Therefore, a test battery of cell-based in vitro methods, covering different modes of actions of high relevance to human health, was applied to study liquid smoke products. Ten liquid smoke flavourings were tested as non-extracted and extracted. To assess the potential drivers of toxic- ity, we used two different solvents. The battery of in vitro methods covered estrogenicity, androgenicity, oxidative stress, aryl hydrocarbon receptor activity and genotoxicity. The non-extracted samples were tested at concentrations 0.002 to 1 μL liquid smoke flavouring/mL culture medium, while extracted samples were tested from 0.003 to 200 μL/mL. Genotoxicity was observed for nearly all non-extracted and all hexane-extracted samples, in which the former had higher potency. No genotoxicity was observed for ethyl acetate-extracted samples. Oxidative stress was activated by almost all extracted and non-extracted samples, while approximately half of the samples had aryl hydrocarbon receptor and estrogen receptor activi- ties. This study used effect-based methods to evaluate the complex mixtures of liquid smoke flavourings. The increased bioactivities seen upon extractions indicate that non-polar chemicals are driving the genotoxicity, while polar substances are increasing oxidative stress and cytotoxic responses. The differences in responses indicate that non-extracted products contain chemicals that are able to antagonize toxic effects, and upon extraction, the protective substances are lost. Keywords Smoke flavouring · Commercial liquid smoke flavouring · Food additives · Bioassays · Bioanalytical tool · Effect-based methods Keywords Smoke flavouring · Commercial liquid smoke flavouring · Food additives · Bioassays · Bioanalytical tool · Effect-based methods An in vitro‑based hazard assessment of liquid smoke food flavourings Erica Selin1 · Geeta Mandava1 · Alexandra‑Livia Vilcu1 · Agneta Oskarsson1 · Johan Lundqvist1 Received: 12 August 2021 / Accepted: 4 November 2021 / Published online: 20 November 2021 © The Author(s) 2021 IN VITRO SYSTEMS IN VITRO SYSTEMS 1 Department of Biomedical Science and Veterinary Public Health, Swedish University of Agricultural Sciences, Box 7028, 750 07 Uppsala, Sweden Archives of Toxicology (2022) 96:601–611 https://doi.org/10.1007/s00204-021-03190-1 Archives of Toxicology (2022) 96:601–611 https://doi.org/10.1007/s00204-021-03190-1 Introduction In 2010–2012, EFSA published a number of safety assessments of smoke flavouring primary products where they concluded that there were safety concerns for the proposed uses and levels for several products, whereas oth- ers were of no safety concern (EFSA Panel on Food Contact Materials 2011a, b, 2012). The smoke flavouring primary products, evaluated by EFSA, are mainly used in the food industry. However, there are also smoke flavouring products commercially available directly to consumers. The products available on the market and the primary products are supposedly produced in a simi- lar manner by pyrolysis, but from different sorts of woods. To differentiate the products, we have tested in this study from the smoke flavouring primary products evaluated by EFSA, the tested products will hereafter be called: liquid smoke flavourings. l Liquid smoke flavourings are characterized by hav- ing a high variability and complex chemical composition with limited information on toxicity of individual chemi- cal constituents, a large number of unidentified chemicals, and potential interaction between chemicals in the mixture (EFSA FAF Panel 2021). Thus, alternative methods for toxicity testing would be useful (Montazeri et al. 2013). Effect-based methods, often based on cultured mammalian cells that have been modified to respond to key molecular events early in toxicity pathways, have proven to be valuable to evaluate highly complex mixtures (Escher et al. 2021; Rosenmai et al. 2017; Selin et al. 2021). were transferred into Eppendorf tubes and stored at + 4 °C until analysis. The liquid smoke flavourings were also extracted by two different solvents, namely hexane (Hex, log Kow = 3.8) and ethyl acetate (EA, log Kow = 0.7), to investigate to what extent polar or non-polar substances are driving the toxic effects. f Samples were extracted with either solid-phase extraction (SPE) or liquid–liquid extraction (LLE), the latter using both hexane and ethyl acetate. We wanted to compare the simpler and more traditionally used extraction method LLE against the more automated SPE method. SPE was performed with Oasis HLB 20 cc cartridges that are able to extract a wide range of compounds with pH ranging from 0 to 14. The extraction procedures are described in the Supplementary Information (SI-1, Sect. 1). In short, samples were either extracted by SPE with hexane or LLE using either hexane or ethyl acetate (Table 1). Introduction After extraction, samples were evap- orated to dryness using nitrogen and resuspended in 0.5 mL of DMSO before being transferred into Eppendorf tubes for bioanalysis. Hickory samples 1, 2, and 5 were not dissolved in DMSO due to their oily composition and were diluted in cell culture media instead of DMSO. Only two samples were successfully extracted through LLE, namely hickory sample 4 and 5, since a clear separable solvent phase was not obtained for the rest of the samples. The concentrations of extracted samples are given as μL liquid smoke flavouring used for the extraction per mL cell culture medium, to enable a comparison of effect concentrations between non-extracted and extracted samples. The extracted samples were stored at – 20 °C. In this study, we used a panel of in vitro bioassays to evaluate effects of hazardous chemicals in ten commercially available liquid smoke flavourings. The liquid smoke fla- vourings were tested as non-extracted and extracted, and potential drivers of toxicity were tested by using two differ- ent solvents for the extraction. Endpoints covered were estro- genicity, androgenicity, oxidative stress, aryl hydrocarbon receptor activity (AhR), and genotoxicity. Introduction could pose a risk to public health, e.g., polycyclic aromatic hydrocarbons (PAHs) like benzo[a]pyrene (BaP) (Šimko 2018; Yabiku et al. 1993). Smoke flavourings are specifi- cally regulated according to Regulation (EC) No 2065/2003, which focuses on the usage of smoke flavourings on or in food items (European Parliament, Council of the European Union 2003). There are currently ten primary smoke flavour- ings authorized to be used in or on food items (Council of the European Union 2013). Primary products are the pri- mary smoke condensates and primary tar fractions, which are further processed to produce the smoke flavourings applied in food. European Food Safety Authority (EFSA) recently issued an updated guidance document for appli- cation on smoke flavouring primary products (EFSA FAF Panel 2021). The initial toxicity studies needed focus on potential genotoxic properties of the smoke flavours, and a tiered approach is applied by combining in silico, in vitro and in vivo evaluations of genotoxic properties. In addition, tier I safety data for developmental and reproductive toxicity While smoking of foods traditionally has been performed mainly as a mean of preservation, it is today also used to cre- ate foods with a desired flavour of smoke. This has resulted in the development of smoke flavouring products, which are adding smoke flavour to food without actual smoking of the food item. Smoke flavourings are produced by thermal treat- ment of wood in the absence of oxygen (pyrolysis), followed by condensation of the vapours and fractionation of the liq- uid products, resulting in a complex mixture of compounds (EFSA FAF Panel 2021; Sikorski 2004). It is well known that this process also produces hazardous compounds that (0123 1 3456789) 3 Archives of Toxicology (2022) 96:601–611 602 Table 1 Sample ID analysed as non-extracted and extracted liquid smoke flavourings Hex hexane, EA ethyl acetate Smoke flavour- ing Extraction method Non-extracted SPE (Hex) LLE (Hex) LLE (EA) Apple A1 A1 SPE Hickory H1 H1 SPE H2 H2 SPE H3 H3 SPE H4 H4 SPE H4 Hex H4 EA H5 H5 SPE H5 Hex H5 EA Mesquite M1 M1 SPE M2 M2 SPE Oak O1 O1 SPE Pecan P1 P1 SPE Table 1 Sample ID analysed as non-extracted and extracted liquid smoke flavourings are required for new authorizations (EFSA FAF Panel 2021, Appendix E). Effect‑based in vitro methods vehicle controls without DHT, followed by normalization to the max effect of the vehicle control exposed to DHT. Standard curves for the nuclear receptors were created in GraphPad Prism 9 Software (San Diego, California, USA) using non-linear (log logistic) sigmoidal curve fit. For oxi- dative stress response, the activity was normalized to the vehicle control, since no max effect can be reached (Escher et al. 2018). The response was therefore fitted to a linear regression dose–response curve. Effect-based tests that covered reactive, non-specific, and specific modes of actions were applied (Escher et al. 2021). The methods assessed activation of AhR, androgenicity (AR), estrogenicity (ER), oxidative stress (Nrf2), and geno- toxicity (micronucleus test) (Table 2). Detailed information of the methods is presented in the Supplementary Informa- tion (Table SI-1).i The limit of detection (LOD) was calculated as three times the standard deviation of the vehicle control. The cut- off limit was based on the LOD and used to define a sample as bioactive. The cut-off was set as the even number above the LOD for agonistic activity and below the LOD for antag- onistic activity (Escher et al. 2018, Table SI-1).f For all assays, a specific reference compound was used as a standard to validate each run. The vehicle controls con- sisted of cell medium for the non-extracted smoke flavour- ings and hickory samples 1, 2, as well as 5, and DMSO was used for the remaining extracted samples. Cytotoxicity was evaluated by MTS and ATPase assay, as described in the Supplementary Information (SI, Sect. 1.5), and by ethidium monoazide stain (EMA) in the genotoxicity (micronucleus) assay.l The cut-off was set at 70% for antagonistic samples; thus, samples with activities at or below 70% were considered bioactive. The effect concentration 20% (EC20) or inhibitory concentration 30% (IC30) was calculated for agonistic and antagonistic activity for all bioactive samples, respectively. Effect concentration induction ratio 1.5 (ECIR1.5) was calculated for samples in the oxidative stress Nrf2 assay. Nrf2 activity was presented as fold change compared to the vehicle controls. The cut-off was set at 70% for antagonistic samples; thus, samples with activities at or below 70% were considered bioactive. Liquid smoke flavourings Ten liquid smoke flavourings marketed to consumers were purchased from different online stores, and were pro- duced from apple, hickory, mesquite, oak, and pecan wood (Table 1). All samples were used within their expiration date. Information of the ingredients and recommended doses are provided in Table SI-2. No information on the flavour- ing ingredient other than the wood was given on the product itself.l For the non-extracted liquid smoke flavouring, 2 mL was filtered using a 0.22 μm syringe. Thereafter, the samples 1 3 1 3 Archives of Toxicology (2022) 96:601–611 603 Effect‑based in vitro methods The effect concentration 20% (EC20) or inhibitory concentration 30% (IC30) was calculated for agonistic and antagonistic activity for all bioactive samples, respectively.f The non-extracted smoke flavouring samples were tested at concentrations 0.002–1 μL liquid smoke flavouring/mL cell culture medium and the extracted samples were tested at concentrations from 0.003 to 200 μL liquid smoke fla- vouring/mL cell culture media. The concentrations used in the bioassays were decided from the effects on cytotoxic- ity to ensure that bioactivity was assessed at non-cytotoxic concentrations. Samples were analysed in either twofold or fivefold dilutions. Effect concentration induction ratio 1.5 (ECIR1.5) was calculated for samples in the oxidative stress Nrf2 assay. Nrf2 activity was presented as fold change compared to the vehicle controls. For genotoxicity, the micronucleus formation data were analysed in GraphPad Prism 9 using one-way ANOVA with Dunnett’s multiple comparison test. Samples were defined as bioactive if the responses were statistically significant compared to the vehicle control value (p value < 0.05). Cytotoxicity The linear dose–response of the standard tBHQ is pre- sented in the Supplementary Information (Fig. SI-2). For all non-extracted bioactive samples, defined by the cut-off limit of 1.5-fold change, BEQ values were calculated as mg tBHQ equivalent concentrations (eq) per serving size/dose. The BEQ values ranged from 0.9 to 452 mg tBHQeq per 5 mL sample (Table SI-3). Hickory sample 2, 4, and 5 retrieved the highest BEQ values of 452, 384, and 345 mg tBHQeq/5 mL sample, respectively (Table SI-3). Cell viability was investigated after 24 h exposure of MCF7 AREc32, DR-Ecoscreen, VM7Luc4E2, and AR-EcoScreen with glucocorticoid receptor knockout mutant 1 (GR-KO MI) cells to liquid smoke flavourings (Figs. SI-1, SI-3, SI-5, SI-7). Treatments that reduced the viability with more than 20% were considered cytotoxic and were excluded from fur- ther testing. Non-extracted samples exhibited higher cytotoxicity in comparison to all extracted samples. For the majority of the non-extracted samples, cytotoxicity was observed at the highest concentration tested (Figs. 2A, SI-1, SI-3, SI-5, SI-7). Non-extracted hickory samples 2, 4, and 5 retrieved the highest potency of all tested samples in MCF7 AREc32, DR-EcoScreen GR-KO M1, VM7Luc4E2, and AR-Eco- Screen cell lines (Figs. SI-1, SI-3, SI-5, SI-7). Genotoxicity Samples inducing oxidative stress to a high degree were investigated in the micronucleus test (MN) (Fig. 2). Liq- uid smoke samples detected as cytotoxic, as indicated by a fourfold increase in % EMA events compared to the control, were excluded for MN assessment (Fig. 2A). A similar trend of cytotoxicity was seen for ethyl acetate LLE samples, where higher potencies were obtained for ethyl acetate-extracted samples than for hexane-extracted samples (Figs. 2A, SI-1C, SI-3C SI-5C, SI-7C). The non-extracted tested hickory sample 2, 5 and mes- quite sample 2 showed a statistically significant increase in the micronuclei formation (Fig. 2B). In agreement with the oxidative stress assay, hickory sample 4 and 5 extracted with SPE also caused a statistically significant increase in the genotoxic response (Fig. 2B). Extraction with ethyl acetate did not affect the micronuclei formation. Genotoxicity was observed for both non-extracted and SPE mesquite sample 2, but the potency was higher in the non-extracted sample (Fig. 2B). SPE samples exhibited varying cytotoxicity, although to a considerable lower degree compared to the non-extracted samples (Figs. 2A, SI-1, SI-3, SI-5, SI-7). Oxidative stress (Nrf2) Oxidative stress, measured as Nrf2 activity, was induced by all non-extracted hickory-smoke product samples 1–5 in a dose-related manner (Fig. 1A). Highest potency was obtained for H2, H4 and H5. H2 was the most potent of all non-extracted samples tested, causing a 25-fold induction at a concentration of 0.04 μL/mL. This specific sample was even bioactive at the lowest concentration of 0.003 μL/mL (Fig. 1A). The positive control mitomycin C caused an elevated genotoxic response in a dose-dependent manner, in which the highest concentration of 200 nM caused the highest MN formation. Data evaluation For the manufacture that stated that the recommended serv- ing size was a few drops, we estimated that one drop was 0.05 mL and that three drops would be representative as the serving dosage. Data evaluation The bioanalytical equivalent concentration (BEQ) was calculated, to relate the effect of the sample to a known refer- ence compound, according to the following formula (Escher et al. 2015): Cell viability results were normalized to the vehicle con- trol, which was set as 100%. Samples causing more than 20% reduction were considered cytotoxic, except for the micronucleus test where the cytotoxicity limit was defined as fourfold increase in %EMA-positive events compared to the vehicle control. BEQ = EC20orECIR1.5orIC30(referencecompound) EC20orECIR1.5orIC30(sample) . BEQ = EC20orECIR1.5orIC30(referencecompound) EC20orECIR1.5orIC30(sample) . For nuclear receptor agonistic response, the activity was first normalized to the vehicle control, and then normalized to the maximum (max) effect of the standard. The antag- onistic receptor activities of samples were normalized to The BEQ was then multiplied with the recommended serving size from the manufacturers to retrieve the esti- mated exposure in bioequivalents of reference compound Table 2 Summary of the effect-based in vitro methods MMC was used as a positive control In vitro method Cell line Reference compound Concentration (µM) Cytotoxicity All cell lines mentioned below N/A N/A Aryl hydrocarbon receptor activity DR-EcoScreen 2,3,7,8-Tetrachlorodibenzodioxin (TCDD) 1 × 10–8 to 3 × 10–4 Androgen receptor agonistic activity AR-EcoScreen GR-KO M1 Dihydrotestosterone (DHT) 1 × 10–9 to 1 × 10–3 Androgen receptor antagonistic activity AR-EcoScreen GR-KO M1 Hydroxyflutamide (OHF) 1 × 10–5 to 1 × 101 Estrogen receptor agonistic activity VM7Luc4E2 Estradiol (E2) 4 × 10–7 to 4 × 10–4 Oxidative stress response MCF7 AREc32 Tert-Butylhydroquinone (tBHQ) 8 × 10–1 to 2.5 × 101 Micronucleus test TK6 Mitomycin C (MMC) 1 × 10–1 and 2 × 10–1 Table 2 Summary of the effect-based in vitro methods Archives of Toxicology (2022) 96:601–611 604 the highest activity and was bioactive at all concentrations tested, ranging from 0.1 to 6.3 μL/mL (Fig. 1C). In contrast, samples extracted with hexane showed a very low induction of Nrf2 (Fig. 1C). Hickory sample 4 exhibited a similar Nrf2 efficacy when tested non-extracted, extracted through SPE and LLE with ethyl acetate, however, at different concentra- tions, 0.04, 100, and 6.3 μL/mL, respectively (Fig. 1A–C). A similar oxidative stress response was seen for non-extracted and SPE mesquite sample 1 at the highest concentration tested, although the former being 1000 times less concen- trated (Fig. 1D, E). per serving size/portion. If the recommended serving size was not stated, it was assumed to be 5 mL (Table SI-3). 1 3 AhR activity Activation of AhR, defined by the cut-off limit of 15% of max effect, was observed for both non-extracted and SPE extracted samples. The sample being the most potent and having the highest efficacy was hickory sample 2 of all non- extracted samples, while hickory sample 4 was the most potent of all extracted samples (Fig. 3A, B). The highest concentration of SPE extracted hickory sample 2 had 23% of max effect and 40% with hickory sample number 4 (Fig. 3B). Nearly all SPE samples (8/10) were bioactive at the high- est concentration tested, however, at considerable higher concentrations (12.5–200 μL/mL) than in the non-extracted samples. Mesquite sample 2 caused oxidative stress to the highest degree, reaching a 31-fold induction at 200 μL/mL (Fig. 1E). LLE samples extracted with ethyl acetate induced Nrf2 with a higher potency than SPE extracted (Fig. 1C). Hickory sample 5, extracted with ethyl acetate, showed 1 3 605 Archives of Toxicology (2022) 96:601–611 Non-extracted and SPE extracted mesquite sample 1 and 2 induced AhR activity even at the lower tested concentrations (Fig. 3D, E). The AhR response drastically increased upon SPE extraction, whereas it remained inactive when tested non-extracted or extracted with hexane and ethyl acetate through liquid–liquid (Fig. 3A–C). Still, worth mentioning is that the effect for the majority of the samples was only visible at the highest non-cytotoxic concentration. The only exceptions were for SPE mesquite sample 1 and 2 (Fig. 3E). Hickory sample 1, 5 and apple sample 1 remained inactive when tested non-extracted and extracted (Fig. 3A–E). Pecan sample 1 evoked a higher AhR response upon SPE extrac- tion likely due to the higher concentration used and the TCDD was used as standard and the non-linear dose response is shown in the Supplementary Information (Fig. SI-4). The BEQ values ranged from 14,000 to 300,000 pg TCDDeq per 5 mL sample for the non-extracted samples, in which hickory sample 2 obtained the highest BEQ value (Table SI-3). Estrogenicity We observed estrogenic activity in three out of ten non- extracted samples (Figs. SI-6A, 6D). The activity was only seen at the highest concentration tested for hickory sample Fig. 1 Nrf2 response (fold change compared to control) upon 24 h exposure of MCF7 AREc32 cells to liquid smoke flavourings: non- extracted (A, D), SPE extracted (B, E), and LLE extracted (C). AhR activity Con- centrations on the x-axis are expressed as μL liquid smoke flavouring/ mL cell culture medium. Data illustrate mean ± SD (n = 4), and the dotted line represents the induction ratio of 1.5-fold change, defined as the cut-off limit of bioactivity Archives of Toxicology (2022) 96:601–611 605 Fig. 1 Nrf2 response (fold change compared to control) upon 24 h exposure of MCF7 AREc32 cells to liquid smoke flavourings: non- extracted (A, D), SPE extracted (B, E), and LLE extracted (C). Con- mL cell culture medium. Data illustrate mean ± SD (n = 4), and the dotted line represents the induction ratio of 1.5-fold change, defined as the cut-off limit of bioactivity Fig. 1 Nrf2 response (fold change compared to control) upon 24 h exposure of MCF7 AREc32 cells to liquid smoke flavourings: non- extracted (A, D), SPE extracted (B, E), and LLE extracted (C). Con- centrations on the x-axis are expressed as μL liquid smoke flavouring/ mL cell culture medium. Data illustrate mean ± SD (n = 4), and the dotted line represents the induction ratio of 1.5-fold change, defined as the cut-off limit of bioactivity TCDD was used as standard and the non-linear dose response is shown in the Supplementary Information (Fig. SI-4). The BEQ values ranged from 14,000 to 300,000 pg TCDDeq per 5 mL sample for the non-extracted samples, in which hickory sample 2 obtained the highest BEQ value (Table SI-3). Non-extracted and SPE extracted mesquite sample 1 and 2 induced AhR activity even at the lower tested concentrations (Fig. 3D, E). The AhR response drastically increased upon SPE extraction, whereas it remained inactive when tested non-extracted or extracted with hexane and ethyl acetate through liquid–liquid (Fig. 3A–C). Still, worth mentioning is that the effect for the majority of the samples was only visible at the highest non-cytotoxic concentration. The only exceptions were for SPE mesquite sample 1 and 2 (Fig. 3E). Hickory sample 1, 5 and apple sample 1 remained inactive when tested non-extracted and extracted (Fig. 3A–E). Pecan sample 1 evoked a higher AhR response upon SPE extrac- tion, likely due to the higher concentration used and the activity reached a max effect of 30% (Fig. 3E). Estrogenicity Concentrations on the x-axis are expressed as μL liquid smoke flavouring/mL cell culture medium. Data illustrate mean ± SD (n = 4) and the dotted line represents the % max effect of 15, defined as the cut-off limit of bioactivity are expressed as μL liquid smoke flavouring/mL cell culture medium. Data illustrate mean ± SD (n = 4) and the dotted line represents the % max effect of 15, defined as the cut-off limit of bioactivity Fig. 3 AhR activity (% of max effect) after 24 h exposure of DR-Eco- Screen cells to liquid smoke flavourings: non-extracted (A, D), SPE extracted (B, E) and LLE extracted (C). Concentrations on the x-axis No ER activity was observed for extracted hickory sample 1 and 2 (Fig. SI-6B). On the contrary, mesquite sample 2 had a drastically increased activity, between 60 and 108% of the max effect, after SPE extraction (Figs. SI-6B, 6E). while LLE extraction by hexane and ethyl acetate caused an elevated response of the estrogen receptor (Figs. SI-6C). E2 was used as a standard and the non-linear dose–response curve is found in the Supplementary Infor- mation (Fig. SI-6F). Only one BEQ value was obtained for the non-extracted samples, which was 1.6 ng E2eq/5 mL for mesquite sample 2, as the remaining samples either remained inactive or were below the detection limit (Table SI-3). f ER activity was highly increased in several of the extracted products, this was especially true for SPE extracted samples (Figs. SI-6B, 6E). When comparing the extraction techniques, samples extracted with SPE elicited higher estro- genic response for hickory sample 4 compared to LLE hex- ane samples (Figs. SI-6B, SI-6C). Estrogenicity We observed estrogenic activity in three out of ten non- extracted samples (Figs. SI-6A, 6D). The activity was only seen at the highest concentration tested for hickory sample 1 and 2, as well as mesquite sample 2. 1 3 1 3 3 3 Archives of Toxicology (2022) 96:601–611 606 606 Archives of Toxicology (2022) 96:601 611 Fig. 2 Cytotoxic and geno- toxic response (fold change of micronuclei events compared to control) upon 24 h exposure of TK6 cells to liquid smoke flavourings: cytotoxicity (A) and micronuclei events (B). Concentrations on the x-axis are expressed as μL liquid smoke flavouring/mL cell culture medium. The graph demon- strates mean ± SD, n = 12 for controls and n = 4 for samples. Mitomycin C (MMC) was used as a positive control at concentrations 100 and 200 nM. Samples that were statistically significantly different from the control are indicated with an asterisks (p value < 0.05) Fig. 2 Cytotoxic and geno- toxic response (fold change of micronuclei events compared to control) upon 24 h exposure of TK6 cells to liquid smoke flavourings: cytotoxicity (A) and micronuclei events (B). Concentrations on the x-axis are expressed as μL liquid smoke flavouring/mL cell culture medium. The graph demon- strates mean ± SD, n = 12 for controls and n = 4 for samples. Mitomycin C (MMC) was used as a positive control at concentrations 100 and 200 nM. Samples that were statistically significantly different from the control are indicated with an asterisks (p value < 0.05) 1 3 3 607 Archives of Toxicology (2022) 96:601–611 N ER ti it b d f t t d hi k l hil LLE t ti b h d th l t t d Fig. 3 AhR activity (% of max effect) after 24 h exposure of DR-Eco- Screen cells to liquid smoke flavourings: non-extracted (A, D), SPE extracted (B, E) and LLE extracted (C). Concentrations on the x-axis are expressed as μL liquid smoke flavouring/mL cell culture medium. Data illustrate mean ± SD (n = 4) and the dotted line represents the % max effect of 15, defined as the cut-off limit of bioactivity Archives of Toxicology (2022) 96:601–611 607 Fig. 3 AhR activity (% of max effect) after 24 h exposure of DR-Eco- Screen cells to liquid smoke flavourings: non-extracted (A, D), SPE extracted (B, E) and LLE extracted (C). Androgenicity SPE caused a higher induction, likely explained by being more concentrated (Figs. SI-6B, SI-6C). Hickory sample 4 extracted with ethyl acetate did not induce estrogenicity, in comparison to the hexane extractions which showed a strong estrogenic response. On the other hand, hickory sam- ple 5 exerted no response as non-extracted or SPE extracted, SPE caused a higher induction, likely explained by being more concentrated (Figs. SI-6B, SI-6C). Hickory sample 4 extracted with ethyl acetate did not induce estrogenicity, in comparison to the hexane extractions which showed a strong estrogenic response. On the other hand, hickory sam- ple 5 exerted no response as non-extracted or SPE extracted, The non-extracted and SPE hickory samples did not activate the androgen receptor, defined by the cut-off limit of 4% of max effect (Figs. SI-8A, SI-8B). The lack of response could be explained by the usage of low concentrations, as higher 1 3 Archives of Toxicology (2022) 96:601–611 608 drivers of oxidative stress, as discussed above for cyto- toxicity. As oxidative stress may be associated with geno- toxicity, four of the samples which induced Nrf2 activity were tested for genotoxic potential by a micronucleus test. Non-extracted samples had a higher potency compared to hexane-extracted samples, except for one non-extracted sample (H4), which was not genotoxic at non-cytotoxic concentrations. Interestingly, ethyl acetate-extracted sam- ples did not increase micronuclei formation, which may be explained by the low concentrations used as higher con- centration caused toxicity, or by the fact that polar sub- stances do not drive genotoxicity. concentrations exerted cytotoxicity. Nevertheless, upon SPE extraction, the oak sample 1 and pecan sample 1 elicited agonistic response of the androgen receptor (Figs. SI-8D, SI-8E). Furthermore, the agonistic response of hickory sam- ple 5 drastically increased in a dose-related manner after LLE with hexane, but it remained inactive when extracted with ethyl acetate (Fig. SI-8C). No antagonistic mode of action on the androgen receptor was observed when cells were exposed to the non-extracted liquid smoke flavourings (Figs. SI-9A, SI-9D). A few sam- ples exhibited antagonistic effects, but the sudden drop in activity suggests that these effects more likely can be explained by undetected cytotoxicity and should therefore be interpreted with caution (Figs. SI-9B, SI-9E). A similar profile of antagonistic effect was seen for hickory sample 4 that was liquid–liquid extracted with ethyl acetate (Fig. SI-9C). Previous studies have shown increased DNA single-strand breaks (Ohshima et al. Discussion In this study, we used a panel of effect-based methods to retrieve information on potential toxicity of the mixture of chemicals that defines liquid smoke flavourings. The specific endpoints studied were oxidative stress, genotoxicity, aryl hydrocarbon, estrogen, and androgen receptor activities in addition to general cytotoxicity. The formation of PAHs is of human health concern and has to be analysed for authorization of liquid smoke flavour- ings (Commission Regulation EC No 627/2006 2006). Meta- bolic activation is needed for PAHs to exert DNA damaging effects. In this study, we did not include a metabolic activa- tion system and the results therefore suggest that the geno- toxicity observed is mediated through other chemicals. Fur- thermore, the specificity of TK6 cells to distinguish between clastogen and aneugen modes of action seems to be lower in comparison to when other cell lines are used (Bryce et al. 2011; Smart et al. 2020). The results in this study together with previous studies show that a variety of commercially available liquid smoke flavourings may have genotoxic prop- erties in vitro, which needs to be further investigated.i A high cytotoxicity was observed in all but two of the non-extracted samples. For several of the samples, cytotox- icity was already seen at 1 μL liquid smoke flavouring per 1 mL cell culture medium, and for some even at 0.2 μL/ mL. Cytotoxicity was considerably reduced after SPE and LLE with hexane, thus allowing higher concentrations to be tested, and therefore, higher effects were seen in comparison to non-extracted products. The results indicate that cytotox- icity mainly originates from polar substances. This is further supported by the higher cytotoxicity in samples after LLE with ethyl acetate, where cytotoxicity was almost as high as in the non-extracted samples. The results emphasize the impact of extraction procedure in bioanalysis (Abbas et al. 2019).l Exposure of cells to liquid smoke flavourings induced oxidative stress response, determined as Nrf2 activity. All hickory and the two mesquite samples induced oxidative stress especially in non-extracted but also in extracted samples. Extraction procedure had a main impact on the oxidative stress response, and induction of Nrf2 activ- ity was pronounced upon extraction with ethyl acetate, supporting the suggestion that polar substances are main AhR activity was induced in five of the ten non-extracted samples and in seven of the SPE samples, although at much higher concentrations. Androgenicity 1989), altered pyloric glands in rats after oral exposure to hickory-smoke condensate (Shichino et al. 1992) and mutation in human lymphocytes in vitro after exposure to aqueous wood smoke flavourings (Braun et al. 1987). A more recent study confirmed the genotoxic potential of commercially available liquid smoke flavourings in a human p53 reporter gene cell line, and reported higher p53 response in hickory than mesquite samples (Hossain et al. 2013). Additionally, increased γ-H2AX, p21 and p53 protein levels were detected. However, other studies failed to detect genotoxicity or obtained inconclusive results in the Ames test (Braun et al. 1987; Putnam et al. 1999). The lack of effect in the Ames test may be explained by the low sen- sitivity of the test and/or usage of different smoke flavoured products (Kirkland et al. 2014). PAHs are generated during the smoke formation and are thought to covalently bind to protein and nucleic acids, forming DNA adducts that may be carcinogenic (Luo et al. 2008; Oz 2020; Šimko 2011). The non-linear dose–response curves of DHT and OHF are available in the Supplementary Information (Figs. SI-8F, 9F). No BEQ values were obtained for agonistic and antago- nistic androgen receptor response (Table SI-3). 1 3 Discussion It was concluded that less cytotoxicity in the neutral red uptake (NRU) assay and no mutagenicity (Ames test) or genotoxicity (MN test) was seen for E-cigarettes, com- pared to the reference cigarette. However, it is not agreed within the research field that e-cigarettes should be consid- ered as safer, as these liquids may contain genotoxicants (Barhdadi et al. 2021). Bioactivities varied widely between the various prod- ucts. Some products exhibited no or a low activity in all assays (H1, O1), while others had a high activity in sev- eral of the assays (H2, M2). The bioactivities depend on the concentrations of the individual bioactive compounds and interactions between the compounds in the mixture, which are unknown factors. Wood type, burning conditions, purification, pH-, total acid, chemical, and water content are factors influencing the chemical composition of smoke flavourings (Budaraga et al. 2016; Sikorski 2004; Šimko 2005). The commercially available liquid smoke flavour- ings investigated in the present study had limited informa- tion on manufacturing and identity, compared to the regis- tered smoke flavourings (Council of the European Union 2013). However, it is supposed that the smoke flavourings in general should be regarded as safer than smoke products generated directly from the traditional smoking procedure, as toxic chemicals can be removed during the filtration and purification processes (European Parliament, Council of the European Union 2003). The bioequivalent concentrations corresponding to the observed bioactivity for the non-extracted products in each assay was calculated and expressed as bioequivalents of the reference compound per serving size, to allow a compari- son to the estimated intake via food or drinking water. For estrogenic activity, the only sample with a BEQ value was M2, corresponding to 1.6 ng E2eq per serving. This can be compared to the WHO benchmark value for drinking water of 1 ng E2/L (WHO 2017). The daily consumption of drink- ing water is estimated to 2 L, which means that the exposure of E2eq from one serving size of M2 is below the benchmark value of E2 in drinking water. The same controversy can be said for liquid smoke fla- vourings in comparison to the traditional smoking of food. A similar approach to use and generate in vitro data of E-ciga- rettes would be recommended to be applied to liquid smoke flavourings (Moore et al. 2020). Discussion This was most obvious for hickory sample 2, where the non-extracted sample had the highest efficacy of all tested liquid smoke samples, and the activity was drastically reduced after SPE extraction. AhR activ- ity can be induced by numerous chemicals, for example by PAHs (Boonen et al. 2020). 1 3 Archives of Toxicology (2022) 96:601–611 609 Apart from a single positive sample and two at the cut-off level, no ER activity was observed in the non-extracted sam- ples. However, higher concentrations could be tested than of the non-extracted samples due to cytotoxicity, and seven of the SPE samples exhibited estrogenic activities. Boonen et al. (2020) reported ER activity by BaP in bioassays. No AR activities, agonistic or antagonistic, were detected in the non-extracted samples, while two of the SPE samples were active in the highest concentration. PAHs have been shown to induce antagonistic androgen receptor activity in water samples (Xu et al. 2019). obviously is not induced by dioxins or planar PCBs, but rather by other chemicals with different toxicokinetics and toxicodynamics. The calculated TCDD equivalents from the liquid smoke flavourings ranged from 14,000 to 300,000 pg per serving, and greatly exceeded the toler- able weekly intake (TWI) established by EFSA of 2 pg/kg body weight, corresponding to 140 pg/person/week (EFSA Panel on Contaminants in the Food Chain (CONTAM) et al. 2018). ) Information on potential toxic effects of the com- mercially available liquid smoke flavourings is scarce. In contrast, toxicity of smoke products in E-cigarettes has attracted more attention. Smoke flavourings as food additives and in E-cigarettes are both based on the same concept, namely to remove the most toxic substances produced from natural combustion, while retaining fla- vouring compounds. Cell-based bioassays have been used for hazard evaluation of cigarette smoke constituents, but we are not aware of a similar approach for hazard evalu- ation of liquid smoke flavourings (Barhdadi et al. 2021; Moore et al. 2020; Rudd et al. 2020; Stabbert et al. 2017). Even if the route of exposure differs between E-cigarettes and liquid smoke flavourings, both will reach the systemic circulation after absorption. Rudd et al. (2020) reported that E-cigarettes should be considered as a safer option to cigarette smoke, which likely can be explained by the fact that the flavour and nicotine are received through aerosoli- zation of E-cigarettes, compared to burning of tobacco in cigarette smoke, allowing fewer toxicants to be formed. References European Food Safety Authority (EFSA) (2004) Opinion of the Sci- entific Panel on food additives, flavourings, processing aids and materials in contact with food (AFC) on a request from the Com- mission related to tertiary- Butylhydroquinone (TBHQ). EFSA J 2:84. https://doi.org/10.2903/j.efsa.2004.84 Abbas A, Schneider I, Bollmann A et al (2019) What you extract is what you see: optimising the preparation of water and wastewater samples for in vitro bioassays. Water Res 152:47–60. https://doi. org/10.1016/j.watres.2018.12.049 European Parliament, Council of the European Union (2003) Regu- lation (EC) No 2065/2003 of the European Parliament and of the Council of 10 November 2003 on smoke flavourings used or intended for use in or on foods Barhdadi S, Rogiers V, Deconinck E, Vanhaecke T (2021) Toxicity assessment of flavour chemicals used in e-cigarettes: current state and future challenges. Arch Toxicol 95:2879–2881. https://doi. org/10.1007/s00204-021-03080-6 Hossain MZ, Gilbert SF, Patel K et al (2013) Biological clues to potent DNA-damaging activities in food and flavoring. Food Chem Toxi- col 55:557–567. https://doi.org/10.1016/j.fct.2013.01.058 Boonen I, Van Heyst A, Van Langenhove K et al (2020) Assessing the receptor-mediated activity of PAHs using AhR-, ERα- and PPARγ- CALUX bioassays. Food Chem Toxicol 145:111602. https://doi.org/10.1016/j.fct.2020.111602 Kirkland D, Zeiger E, Madia F et al (2014) Can in vitro mammalian cell genotoxicity test results be used to complement positive results in the Ames test and help predict carcinogenic or in vivo geno- toxic activity? I. Reports of individual databases presented at an EURL ECVAM Workshop. Mutat Res Toxicol Environ Mutagen 775–776:55–68. https://doi.org/10.1016/j.mrgentox.2014.10.005 Braun AG, Busby WF, Jackman J, Halpin PA, Thilly WG (1987) Commercial hickory-smoke flavouring is a human lymphoblast mutagen but does not induce lung adenomas in newborn mice. Food Chem Toxicol 25:331–335. https://doi.org/10.1016/0278- 6915(87)90131-1 Luo XJ, Chen SJ, Mai BX et al (2008) Distribution, source apportion- ment, and transport of PAHs in sediments from the Pearl River Delta and the Northern South China Sea. Arch Environ Contam Toxicol 55:11–20. https://doi.org/10.1007/s00244-007-9105-2 Bryce SM, Avlasevich SL, Bemis JC, Dertinger SD (2011) Miniatur- ized flow cytometry-based CHO K1 micronucleus assay discrimi- nates aneugenic and clastogenic modes of action. Environ Mol Mutagen 52:280–286. https://doi.org/10.1002/em.20618 Montazeri N, Oliveira ACM, Himelbloom BH et al (2013) Chemical characterization of commercial liquid smoke products. Food Sci Nutr 1:102–115. https://doi.org/10.1002/fsn3.9 g p g Budaraga IK, Arnim A, Marlida Y, Bulanin U (2016) Liquid smoke production quality from raw materials variation and different pyrolysis temperature. Int J Adv Sci Eng Inf Technol 6:306–315. Consent to participate and for publication Not applicable. Consent to participate and for publication Not applicable. Consent to participate and for publication Not applicable. Open Access This article is licensed under a Creative Commons Attri- bution 4.0 International License, which permits use, sharing, adapta- tion, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. EFSA Panel on Food Contact Materials, E, Flavourings and Process- ing Aids (CEF) (2012) Scientific opinion on the safety of smoke flavouring primary product SmokEz C-10 - 2012 Update. EFSA J 10:2830. https://doi.org/10.2903/j.efsa.2012.2830f Escher BI, Neale PA, Leusch FDL (2015) Effect-based trigger values for in vitro bioassays: reading across from existing water qual- ity guideline values. Water Res 81:137–148. https://doi.org/10. 1016/j.watres.2015.05.049 Escher BI, Neale PA, Villeneuve D (2018) The advantages of linear concentration-response curves for in vitro bioassays with environ- mental samples: linear CRC. Environ Toxicol Chem. https://doi. org/10.1002/etc.4178 Escher BI, Neale PA, Leusch F (2021) Bioanalytical tools in water quality assessment. IWA Publ. https://doi.org/10.2166/97817 89061987 Discussion l In this study, we have tested ten commonly used liquid smoke flavourings and used two different solvents to inves- tigate if polar or non-polar substances are driving the toxic effects. The increased bioactivities upon extraction indi- cate that non-polar substances are driving the genotoxicity, whereas polar substances are driving the oxidative stress and cytotoxicity. The usage of effect-based methods allowed testing of the complex whole mixture, enabling us to study interactive effects of the product. Findings in this study indi- cate that liquid smoke flavourings contain compounds with hazardous properties and to ensure that these widely used products are safe further studies should be carried out. For oxidative stress, the BEQ values ranged from 0.9 to 452.0 mg tBHQeq/serving. This value can be compared to the acceptable daily intake (ADI) of tBHQ provided by EFSA, which is 0.7 mg/kg bw/day, corresponding to 49 mg/ day at a body weight of 70 kg (EFSA 2004). Six of the ten liquid smoke flavourings resulted in intakes above the ADI for one serving size, of which hickory samples 2, 4, and 5 had the highest BEQ values. The calculated guidance value for AhR activity was in this case not appropriate, as the liquid smoke products 1 3 610 Archives of Toxicology (2022) 96:601–611 Council of the European Union (2013) Commission Implementing Regulation (EU) No 1321/201 Council of the European Union (2013) Commission Implementing Regulation (EU) No 1321/201 Supplementary Information The online version contains supplemen- tary material available at https://doi.org/10.1007/s00204-021-03190-1. Supplementary Information The online version contains supplemen- tary material available at https://doi.org/10.1007/s00204-021-03190-1. Commission Regulation EC No 627/2006 (2006) Implementing Regu- lation (EC) No 2065/2003 of the European Parliament and of the Council as regards quality criteria for validated analytical meth- ods for sampling, identification and characterisation of primary smoke productsil Funding Open access funding provided by Swedish University of Agri- cultural Sciences. The work was financially supported by the Swedish University of Agricultural Sciences Early Career Grant awarded to Johan Lundqvist. EFSA Panel CEF (2011a) Scientific opinion on the safety of smoke fla- vour primary product Fumokomp—2011a update. EFSA J 9:2308. https://doi.org/10.2903/j.efsa.2011.2308il Availability of data and materials All data are included in the manu- script or in the Supplementary Material. Further data or information will be supplied upon request. EFSA Panel CEF (2011b) Scientific opinion on the safety of smoke fla- vour primary product Zesti smoke code 10–2011b update. EFSA J 9:2307. https://doi.org/10.2903/j.efsa.2011.2307 Declarations EFSA Panel on Contaminants in the Food Chain (CONTAM), Knut- sen HK, Alexander J, Barregård L et al (2018) Risk for animal and human health related to the presence of dioxins and dioxin- like PCBs in feed and food. EFSA J 16:e05333. https://doi.org/ 10.2903/j.efsa.2018.5333 Conflict of interest The authors have no conflicts of interest to declare. Ethics approval Not applicable. EFSA Panel on Food Additives and Flavourings (FAF), Younes M, Aquilina G, Castle L et al (2021) Scientific Guidance for the preparation of applications on smoke flavouring primary prod- ucts. EFSA J 19:e06435. https://doi.org/10.2903/j.efsa.2021. 6435 References https://doi.org/10.18517/ijaseit.6.3.737 Moore MM, Clements J, Desai P et al (2020) Workshop series to iden- tify, discuss, and develop recommendations for the optimal gener- ation and use of in vitro assay data for tobacco product evaluation: 1 3 Archives of Toxicology (2022) 96:601–611 611 phase 1 genotoxicity assays. Appl Vitro Toxicol 6:49–63. https:// doi.org/10.1089/aivt.2020.0004 phase 1 genotoxicity assays. Appl Vitro Toxicol 6:49–63. https:// doi.org/10.1089/aivt.2020.0004 flavorings. Mol Nutr Food Res 49:637–647. https://doi.org/10. 1002/mnfr.200400091 Šimko P (2011) 19 - Heat and processing generated contaminants in processed meats. In: Kerry JP, Kerry JF (eds) Processed meats. Woodhead Publishing, pp 478–507. https://doi.org/10.1533/97808 57092946.3.478 Ohshima H, Furihata C, Matsushima T, Bartsch H (1989) Evidence of potential tumour-initiating and tumour-promoting activities of hickory smoke condensate when given alone or with nitrite to rats. Food Chem Toxicol 27:511–516. https://doi.org/10.1016/ 0278-6915(89)90046-X Šimko P (2018) Chapter seven—modern procedures for removal of hazardous compounds from foods. In: Toldrá F (ed) Advances in food and nutrition research. Academic Press, pp 241–285. https:// doi.org/10.1016/bs.afnr.2017.12.004 ( ) Oz E (2020) Effects of smoke flavoring using different wood chips and barbecuing on the formation of polycyclic aromatic hydrocarbons and heterocyclic aromatic amines in salmon fillets. PLoS ONE 15:e0227508–e0227508. https://doi.org/10.1371/journal.pone. 0227508 doi.org/10.1016/bs.afnr.2017.12.004 Smart DJ, Helbling FR, Verardo M, Huber A, McHugh D, Vanscheeu- wijck P (2020) Development of an integrated assay in human TK6 cells to permit comprehensive genotoxicity analysis in vitro. MRGTEM 849:503129. https://doi.org/10.1016/j.mrgentox.2019. 503129 Putnam KP, Bombick DW, Avalos JT, Doolittl DJ (1999) Comparison of the cytotoxic and mutagenic potential of liquid smoke food flavourings, cigarette smoke condensate and wood smoke conden- sate. Food Chem Toxicol 37:1113–1118. https://doi.org/10.1016/ S0278-6915(99)00104-0f Stabbert R, Dempsey R, Diekmann J et al (2017) Studies on the con- tributions of smoke constituents, individually and in mixtures, in a range of in vitro bioactivity assays. Toxicol Vitro 42:222–246. https://doi.org/10.1016/j.tiv.2017.04.003 Rosenmai AK, Bengtström L, Taxvig C et al (2017) An effect-directed strategy for characterizing emerging chemicals in food contact materials made from paper and board. Food Chem Toxicol. https://doi.org/10.1016/j.fct.2017.05.061 WHO (2017) Drinking water Parameter Cooperation Project. Support to the revision of Annex I Council Directive 98/83/EC on the Quality of Water Intended for Human Consumption (Drinking Water Directive). https://ec.europa.eu/environment/water/water- drink/pdf/WHO_parameter_report.pdf. Accessed 11 Aug 2021 Rudd K, Stevenson M, Wieczorek R et al (2020) Chemical composi- tion and in vitro toxicity profile of a pod-based e-cigarette aerosol compared to cigarette smoke. Appl Vitro Toxicol 6:11–41. https:// doi.org/10.1089/aivt.2019.0015 /pdf/WHO_parameter_report.pdf. References Accessed 11 Aug 2021 Xu S, Zhou S, Xing L et al (2019) Fate of organic micropollutants and their biological effects in a drinking water source treated by a field-scale constructed wetland. Sci Total Environ 682:756–764. https://doi.org/10.1016/j.scitotenv.2019.05.151 Selin E, Svensson K, Gravenfors E et al (2021) Food contact materials: an effect-based evaluation of the presence of hazardous chemicals in paper and cardboard packaging. Food Addit Contam Part A. https://doi.org/10.1080/19440049.2021.1930200f Yabiku HY, Martins MS, Takahashi MY (1993) Levels of benzo [a] pyrene and other polycyclic aromatic hydrocarbons in liquid smoke flavour and some smoked foods. Food Addit Contam 10:399–405. https://doi.org/10.1080/02652039309374163 Shichino Y, Tatematsu M, Ohshima H et al (1992) Effects of hickory- smoke condensate on development of pepsinogen 1-altered pyloric glands in rats. Food Chem Toxicol 30:859–864. https://doi.org/ 10.1016/0278-6915(92)90051-L Publisher's Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Sikorski ZE (2004) SMOKING \| traditional. In: Jensen WK (ed) Encyclopedia of meat sciences. Elsevier, Oxford, pp 1265–1272. https://doi.org/10.1016/B0-12-464970-X/00191-4 p g Šimko P (2005) Factors affecting elimination of polycyclic aro- matic hydrocarbons from smoked meat foods and liquid smoke p g Šimko P (2005) Factors affecting elimination of polycyclic aro- matic hydrocarbons from smoked meat foods and liquid smoke 1 3 1 3 1 3
https://openalex.org/W4321601211	https://www.frontiersin.org/articles/10.3389/fimmu.2023.1127588/pdf	English	null	MAIT cells and the microbiome	Frontiers in immunology	2,023	cc-by	18,860	OPEN ACCESS OPEN ACCESS EDITED BY Laurent Gapin, University of Colorado Denver, United States REVIEWED BY Thierry Mallevaey, University of Toronto, Canada Olivier Gasser, Victoria University of Wellington, New Zealand Liyen Loh, University of Colorado, United States CORRESPONDENCE Timothy S. C. Hinks timothy.hinks@ndm.ox.ac.uk SPECIALTY SECTION This article was submitted to T Cell Biology, a section of the journal Frontiers in Immunology RECEIVED 19 December 2022 ACCEPTED 14 February 2023 PUBLISHED 23 February 2023 CITATION Jabeen MF and Hinks TSC (2023) MAIT cells and the microbiome. Front. Immunol. 14:1127588. doi: 10.3389/fimmu.2023.1127588 COPYRIGHT © 2023 Jabeen and Hinks. 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. Maisha F. Jabeen 1,2 and Timothy S. C. Hinks 1,2 1Respiratory Medicine Unit, Experimental Medicine Division, Nufﬁeld Department of Medicine, University of Oxford, John Radcliffe Hospital, Oxford, United Kingdom, 2National Institute for Health Research Oxford Biomedical Research Centre, John Radcliffe Hospital, Oxford, United Kingdom Mucosal associated invariant T (MAIT) cells are innate-like T lymphocytes, strikingly enriched at mucosal surfaces and characterized by a semi-invariant ab T cell receptor (TCR) recognizing microbial derived intermediates of riboﬂavin synthesis presented by the MHC-Ib molecule MR1. At barrier sites MAIT cells occupy a prime position for interaction with commensal microorganisms, comprising the microbiota. The microbiota is a rich source of riboﬂavin derived antigens required in early life to promote intra-thymic MAIT cell development and sustain a life-long population of tissue resident cells. A symbiotic relationship is thought to be maintained in health whereby microbes promote maturation and homeostasis, and in turn MAIT cells can engage a TCR- dependent “tissue repair” program in the presence of commensal organisms conducive to sustaining barrier function and integrity of the microbial community. MAIT cell activation can be induced in a MR1-TCR dependent manner or through MR1-TCR independent mechanisms via pro-inﬂammatory cytokines interleukin (IL)-12/-15/-18 and type I interferon. MAIT cells provide immunity against bacterial, fungal and viral pathogens. MAIT cell, microbiome and dysbiosis, tissue homeostasis, airways diseases, inﬂammatory bowel conditions, metabolic syndromes, stem cell transplant (SCT) KEYWORDS MAIT cell, microbiome and dysbiosis, tissue homeostasis, airways diseases, inﬂammatory bowel conditions, metabolic syndromes, stem cell transplant (SCT) TYPE Review PUBLISHED 23 February 2023 DOI 10.3389/fimmu.2023.1127588 MAIT cells and the microbiome OPEN ACCESS EDITED BY Laurent Gapin, University of Colorado Denver, United States REVIEWED BY Thierry Mallevaey, University of Toronto, Canada Olivier Gasser, Victoria University of Wellington, New Zealand Liyen Loh, University of Colorado, United States *CORRESPONDENCE Timothy S. C. Hinks timothy.hinks@ndm.ox.ac.uk SPECIALTY SECTION This article was submitted to T Cell Biology, a section of the journal Frontiers in Immunology RECEIVED 19 December 2022 ACCEPTED 14 February 2023 PUBLISHED 23 February 2023 CITATION Jabeen MF and Hinks TSC (2023) MAIT cells and the microbiome. Front. Immunol. 14:1127588. doi: 10.3389/fimmu.2023.1127588 COPYRIGHT © 2023 Jabeen and Hinks. 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 Introduction model and single cell RNA sequencing, it has been shown that positive selection of 5-OP-RU : MR1 speciﬁc thymocytes can occur on different cell types destined for divergent outcomes. These are heat stable antigen high (HSAhi) precursors undergoing positive selection by thymic epithelial cells (TEC) and differentiating into naïve CD4+ T cells or, thymocytes selected by hematopoietic cells differentiating into CD44hi effector cells (7). The seminal discovery that the mucosal associated invariant T (MAIT) T cell receptor (TCR) recognizes riboﬂavin metabolites derived from bacteria, mycobacteria and fungi (1), revealed a prime role in sensing and responding to the microbiome at mucosal surfaces. The MAIT TCR is a semi-invariant TCR-a chain (typically TRAV1-2-TRAJ33 or TRAV1-2-TRAJ12 or TRAV1-2- TRAJ20), predominantly associated with the b-chains TRBV20 or TRBV6 in humans and TRBV19 or TRBV13 in mice and speciﬁcally recognizes the naturally-occurring activating ligand 5- (2-oxopropylideneamino)-6-dribityllumazine (5-OP-RU) presented on MHC-related protein 1 (MR1) (2). Whilst initially these cells were understood to play a role in antimicrobial host defense, the more recent discoveries of separate antiviral and ‘tissue repair’ responses have revealed a more nuanced complexity in their functional repertoire. Nonetheless the microbiome remains absolutely essential to the development and peripheral expansion of MAIT cells as a source of TCR ligand, such that the nature of the early life microbiome can mediate life-long changes in the MAIT cell repertoire. In this review we therefore make a speciﬁc focus on the role of the microbiome in the ontogeny of MAIT cells. We then review how microbial dysbiosis, often marked by compositional shifts in speciﬁc phyla, alongside changes in intestinal permeability and inﬂammatory cytokine milieus, are together involved in the potential pathogenicity of MAIT cells. Though human data on the inﬂuence of MAIT cell deﬁciency on the microbiome are relatively scarce, we explore the insights given by speciﬁc clinical instances of acquired MAIT cell deﬁciency, in particular those of hematopoietic stem cell transplantation and of human immunodeﬁciency virus (HIV)-induced MAIT cell loss, which provide experimental windows into MAIT cell biology. We review human data from the gut, lung and skin, which comprise the body’s largest barrier surfaces, and conclude with thoughts on the potential for therapeutic manipulation of the microbiome or MAIT cells populations directly. OPEN ACCESS However, MAIT cells may have deleterious effects through insufﬁcient or exacerbated effector activity and have been implicated in autoimmune, inﬂammatory and allergic conditions in which microbial dysbiosis is a shared feature. In this review we summarize the current knowledge on the role of the microbiota in the development and maintenance of circulating and tissue resident MAIT cells. We also explore how microbial dysbiosis, alongside changes in intestinal permeability and imbalance between pro- and anti-inﬂammatory components of the immune response are together involved in the potential pathogenicity of MAIT cells. Whilst there have been signiﬁcant improvements in our understanding of how the microbiota shapes MAIT cell function, human data are relatively lacking, and it remains unknown if MAIT cells can conversely inﬂuence the composition of the microbiota. We speculate whether, in a human population, differences in microbiomes might account for the heterogeneity observed in MAIT cell frequency across mucosal sites or between individuals, and response to therapies targeting T cells. Moreover, we speculate whether manipulation of the microbiota, or harnessing MAIT cell ligands within the gut or disease-speciﬁc sites could offer novel therapeutic strategies. 01 01 Frontiers in Immunology frontiersin.org 10.3389/ﬁmmu.2023.1127588 Jabeen and Hinks Introduction Regardless of their mode of selection there is resultant expression of the survival factor Bcl2 with induction of Ccr7 and loss of Ccr9 expression to suggest migration of these cells from the thymic cortex to the medulla. Following positive selection in mouse and human, MAIT cells progress through three stages of intrathymic development summarized in Figure 1. Like iNKT cells (8), effector differentiation of murine MAIT cell precursors selected by hematopoietic cells in the thymus is reliant upon expression of the signaling lymphocyte activation molecule (SLAM) adaptor protein (SAP) (4, 7, 9). This shared characteristic reﬂects ZBTB16 expression (encoding the master transcription factor PLZF) in both cell types, conferring innate-like functionality. PLZF suppresses the naïve T cell program and is required for expression of effector genes, alongside CD44 expression (3, 4). There is concurrent downregulation of Bach2 (a transcription factor seen in conventional T lymphocytes) and then Klf2, involved in regulating thymic egress (4). The population of naïve MR1-restricted T cells, having undergone positive selection on MR1 expressing TECs, patrol secondary lymphoid organs whereas their counterparts selected on MR1 expressing hematopoietic cells undergo proliferation and differentiation in preparation for migration to peripheral tissue to support mucosal immunity (7). In mice two clear MAIT cell subsets with distinct effector properties are identiﬁed by their expression factors T-bet (MAIT1) and RORgt (MAIT17) enabling them to readily produce IFN-g and IL-17 respectively (7). Whist single cell RNAseq has been informative in describing early transcriptional event in MAIT cell development, the signaling pathways and mechanisms responsible for adoption of a MAIT1 versus MAIT17 phenotype are yet to be fully elucidated. It has been speculated that choice of lineage may be directed at random by positively selected MAIT cell precursors, shaped by environmental cues such as cytokines or relate to TCR afﬁnity for the selecting ligand but this remains to be conﬁrmed experimentally (10). MAIT1 and MAIT17 cells express distinct patterns of chemokine receptors and integrins; MAIT1 cells express Cxcr3 and Itga1, while MAIT17 cells express Ccr6, Itga5 and Itgb3 and respond more efﬁciently to TCR-stimulation (7, 11, 12). PLZF expression in MAIT cells confers a broad tissue homing capacity whereas the transcription factors T-bet and RORgt likely ﬁne tune tissue targeting, promoting residency of MAIT1 cells preferentially in spleen and liver, and MAIT17 cells in barrier tissue including the lung, skin and gut (11, 12). Ontogeny of MAIT cells Whilst conventional CD4+ or CD8+ T cells exit the thymus as naïve cells, gaining effector functionality following antigen exposure in secondary lymphoid organs, MAIT cells acquire effector functions intra-thymically (3–5). We review the ontogeny of MAIT cells in mouse and human and consider how commensal derived bacterial metabolites are needed at each stage of their development and acquisition of antimicrobial functionality. Frontiers in Immunology Human MAIT cells arise in the thymic cortex following the development of CD4+CD8+ double positive (DP) thymocytes possessing a T cell receptor (TCR) speciﬁc to the MR1:5-OP-RU complex, surveying their vicinity for subsequent positive selection of MR1-expressing cells (6). Using a murine bone marrow chimera Mature T cell development begins in utero in humans. MAIT cell precursors appear early during gestation at low frequencies in the thymus and cord blood (5, 13). MR1 is expressed on hematopoietic double positive thymocytes suggesting a similar 02 frontiersin.org 10.3389/ﬁmmu.2023.1127588 Jabeen and Hinks FIGURE 1 MAIT cell ontogeny. Mouse (top) and human (bottom) mucosal associated invariant T (MAIT) cell development follows three stages, largely within the thymus. Microbially derived 5-OP-RU is trafﬁcked to the thymus and loaded onto MR1 expressed on double positive (DP) thymocytes. MAIT cells are subsequently positively selected on DP thymocytes in a process reliant upon signaling lymphocytic activation molecule (SLAM) interactions in mice (stage 1). During stage 2 and 3 MAIT cells acquire effector functions and their prototypic phenotype driven by PLZF expression. This occurs intrathymically in mice, but in human stage 3 can also occur peripherally. Distinguishing markers and important co-factors regulating differentiation are shown. FIGURE 1 MAIT cell ontogeny. Mouse (top) and human (bottom) mucosal associated invariant T (MAIT) cell development follows three stages, largely within the thymus. Microbially derived 5-OP-RU is trafﬁcked to the thymus and loaded onto MR1 expressed on double positive (DP) thymocytes. MAIT cells are subsequently positively selected on DP thymocytes in a process reliant upon signaling lymphocytic activation molecule (SLAM) interactions in mice (stage 1). During stage 2 and 3 MAIT cells acquire effector functions and their prototypic phenotype driven by PLZF expression. This occurs intrathymically in mice, but in human stage 3 can also occur peripherally. Distinguishing markers and important co-factors regulating differentiation are shown. manner of selection to mouse MAIT cells (14). In human cord blood a minority of Va7.2+CD161+ have high avidity for the MR1:5-OP-RU complex; only these cells go on to acquire a memory phenotype in the ﬁrst few weeks of life and expand to provide the adult MAIT cell pool over the following 5 to 6 years (13). Thus, the adult MAIT cell clonal size is antigen driven and results in a restricted TCR repertoire in adults (13). Key similarities and differences between mouse and human MAIT cell development are discussed below. Human transcription factors are expressed mutually exclusively in mouse (giving way to MAIT1 and MAIT17 cells) (5). Peripheral MAIT cell expression of transcriptions factors Eomesederin (EOMES) and Blimp-1 (PRDM1) further support type-1 responses, whereas STAT3 supports type-17 responses. Human thymic MAIT cells possess cytotoxic capacity (4) but limited ability to secrete TNF and IFN-g in response to PMA and ionomycin stimulation when compared to peripheral blood MAIT cells (3). Therefore, in human MAIT cells terminal maturation appears to occur in the periphery following birth suggesting additional peripheral mechanisms support acquisition of full effector functionality. As seen in mouse, human MAIT cells preferentially locate to non-lymphoid tissue. Thymic MAIT cells express tissue-homing molecules including CCR6, CCR5 and CXCR6 (4). In addition, as they mature, thymic MAIT cells upregulate the transcription factors C/EBPd (4), important for MAIT cell trafﬁcking (15), and RUNX3 (4), also required by conventional CD8+ tissue resident memory cells to establish niches in diverse tissue environments (16). The shared transcription factors PLZF (ZBTB16), RORgt (RORC) and T-bet (TBX21) are induced differentially in both mouse and human during thymic development (5), but expression of SAP is not necessary for human MAIT cells (5, 7). Mature tetramer positive (MR1:5-OP-RU restricted) thymocytes express PLZF, CD161 and IL-18Ra. However, unlike thymic MAIT cells in mouse, which display a memory phenotype at the point of egress, mature thymic and cord blood MAIT cells in human are negative for the memory marker CD45RO (5, 13). In addition, human MAIT cells are largely T-bet+RORgt+ as they exit the thymus (12) whereas these Microbes and MAIT cell development MAIT cell reliance on the microbiome is evidenced by their relative deﬁciency in the periphery (21) and thymus (3) of germ free (GF) mice, compared with speciﬁc pathogen free animals. This pattern of deﬁciency is similar to other innate and memory T cell populations in GF and antibiotic treated mouse models (22). Metabolites from riboﬂavin-synthesizing commensals are needed for most stages of MAIT intra-thymic development and subsequent peripheral expansion (3, 23). Riboﬂavin metabolites secreted by the microbiota travel rapidly to the thymus. In fact 5-OP-RU is trafﬁcked and detected in the thymus within an hour of topical or oral administration (23). MAIT cell development can be promoted following mono-colonization of GF mice with riboﬂavin synthesizing bacterial species such as Proteus mirabilis or Escherichia coli, but not with species deﬁcient in this biosynthetic pathway such as Lactobacillus johnsonii (21, 23). Using mutant E. coli strains deﬁcient in riboﬂavin enzymes either upstream (DRibD) or downstream (DRibE) of 5-A-RU to colonize GF mice has identiﬁed the essential role for RibD, and thus 5-OP-RU, in thymic MAIT cell development. The non-stimulatory MR1-ligand Acetyl-6-formyl-pterin (Ac-6-FP) cannot support MAIT cell development (23). Following recolonization of GF mice, there is selective restoration of RORgt+ MAIT17 cells reliant on TCR- triggering for proliferation and function (23). Of note, GF mice retain a small residual population of thymic MAIT cells whereas Mr1-/- are completely lacking in MAIT cells strongly supporting an essential role for MR1 in positive selection of these cells (7). The microbiome is diverse and heterogenous, varying between mucosal sites with distinct microenvironments. A large in vitro screen of microbiota-associated bacterial species found that the capacity to stimulate MAIT cells correlated with riboﬂavin secretion as measured by mass spectrometry (31). High stimulator species belonged to Bacteroidetes and Proteobacteria phyla, whereas Actinobacteria and Firmicutes were poor stimulators. Vb2+ MAIT cells were most activated. Conventional human T cell subsets were able to present MR1-ligand but induced a weaker cytokine response compared to professional APCs. This suggests a capacity for MAIT cells to discriminate between members of the microbiota by TCR signal strength based on antigen load and presenting cell (31). Microbial diversity has been shown to reduce MAIT cell activation in vitro, correlating with net riboﬂavin secretion in a human intestinal model community. Higher diversity resulted in greater riboﬂavin consumption and thus less antigen presentation to MAIT cells. Microbes and MAIT cells at barrier sites: Development and homeostasis The microbiome has a fundamental role in the induction, development, and homeostatic function of the host immune system. A symbiotic relationship between the host immune system and microbiota is required to balance regulatory pathways conferring tolerance to innocuous antigens and protective immunity against pathogens (17). Following their development in the thymus MAIT cells are equipped with a transcriptional program and homing markers to support tissue residency. They localize to sites including the oropharynx, respiratory and GI tracts, skin and female genital Frontiers in Immunology 03 frontiersin.org 10.3389/ﬁmmu.2023.1127588 Jabeen and Hinks restricted reconstitution of MAIT cell populations may be competition for a shared niche imposed by similar innate T cell subsets. MAIT cell frequencies positively correlate with mouse and human iNKT cells (13, 24) and gdT cells (24), all sharing overlapping functions and a reliance on the microbiome. Competitive regulation of individual populations is supported by increased frequency of iNKT and MAIT cells seen in Tcrd-deﬁcient mice (21) and increased splenic and thymic MAIT cells in Cd1d- deﬁcient mice (3). In humans, the R9H mutation in MR1prevents its binding to 5-OP-RU but retains afﬁnity for Ac-6-FP (25); in a rare patient with a homozygous R9H mutation in MR1, MAIT cell deﬁciency is observed with an expanded gdT cell (Vd2+) population, again suggesting a compensatory interaction between innate T cell subsets (25). MAIT cells, iNKT and gdT cells do not compete for the same antigen, thus competition for immunological space may be imposed through alternative mechanisms orchestrated by immunoregulatory cytokines (e.g. IL-7, IL-15) (26–28), or host and dietary metabolites regulating shared transcriptional pathways (e.g. via the aryl hydrocarbon receptor) (20, 29). It is yet to be determined if population pressures and temporal restrictions apply to restoring human MAIT cell frequencies. Partial reconstitution of MAIT cells is seen following allogenic hematopoietic stem cell transplantation (HSCT) and correlates with the diversity of gut microbiota (30). It is tempting to speculate that the microbiome offers a key to regulating MAIT cells, however it has not yet been fully elucidated how this interaction could be skewed by neighboring innate T cells or the effects of age and disease. mucosa, also hosting uniquely adapted microbial communities (18). MAIT cells thereby occupy a prime position for crosstalk with commensal microorganisms which uniquely synthesize riboﬂavin at mammalian barrier surfaces (18). Microbes and MAIT cells at barrier sites: Development and homeostasis The co-evolution of MR1 and TRAV1, and accumulation of MR1 mutations in species following loss of TRAV1, supports the idea that the main function of MR1 is to present antigen to MAIT cells (19). By extension this provides an insight into conserved mechanisms through which barrier surfaces can imprint mucosal immunity. These interactions are further shaped by cellular networks, environmental and metabolic factors within the microenvironment (20). With broad anti-bacterial speciﬁcity and a capacity for tissue repair, MAIT cells may be key in restoring homeostasis following infection or tissue injury thereby offering protection from invading pathogens and preserving the microbiome. Frontiers in Immunology Microbes and tissue repair MAIT cells can engage a “tissue repair” program associated with accelerated wound repair in the context of commensal organisms. We and others have described the transcriptome of activated MAIT cells following transcriptomic analysis of MR1:5-OP-RU tetramer positive cells in mouse and human (34–36). Alongside expected pro- inﬂammatory responses, we identiﬁed a TCR-mediated and activation-driven expression of the tissue repair program previously reported in murine skin homing H2-M3 restricted Tc17 cells induced by commensal ﬂora and accelerating repair in an epithelial wound model (35, 37). Key genes expressed in both species included TNF, CSF2, HIF1A, FURIN, VEGFB, PTGES2, PDGFB, TGFB1, MMP25, and HMGB1 (35). Accelerated wound healing could be observed in an intestinal epithelial cell line system following treatment with supernatants from TCR-stimulated MAIT cells and blocked with anti-MR1 antibodies (36). This MAIT tissue repair program is observed following TCR ligation but not cytokine mediated stimulation alone (34, 36). Thus, similarly to H2-M3 restricted Tc17 cells in mouse skin (37) and gdT cells in the lung and gut (38–40), MAIT TCR signaling appears to play a role in tissue homeostasis. In humans, peripheral blood MAIT cells respond differently from tissue-derived MAIT cells originating from intestinal mucosa (45), oropharynx (46), nasopharynx (47), lung (48) and female genital tract (49) following TCR ligation, also suggesting tissue- speciﬁc imprinting. Colonic MAIT cells acquire a primed phenotype, compared with their peripheral blood counterparts, proportionately to accumulation of antigenic metabolites derived from the microbiome (50). Overall, these adaptive mechanisms are speculated to be favorable for long term residency in tissue. In parabiotic pairs most spleen, liver and lung (except some RORgt- cells) MAIT cells did not recirculate over 5 weeks, implying persistent tissue residency (11). In human, whether MAIT cells are permanently resident or leave tissue and recirculate remains unclear. MAIT cells from matched thoracic duct lymph and blood samples have a shared TCR repertoire but are CCR7-, this could indicate transit through tissue between the two compartments or a CCR7-independent migration mechanism (51). The tissue residency markers CD69 and CD103 are widely expressed by MAIT cells at mucosal surfaces, but rare in blood MAIT cells (20). Therefore, there may be a small pool of recirculating cells in health, although its role in disease is not known. Murine skin-resident MAIT cells also engage a distinct tissue repair transcriptional signature (21) reminiscent of H2-M3 restricted Tc17 cells reactive to S. epidermidis derived N-formylated peptides (37). Microbes and tissue repair To unpick the role of MAIT cells from H2-M3 restricted Tc17 or gdT cells, given their overlapping properties, Tcrd-/- mice and S. epidermidis strain incapable of inducing H2-M3 Tc17 cells were utilized (21). This revealed a MAIT cell-dependent tissue repair response to S. epidermidis with accelerated epidermal tongue length growth in a skin punch biopsy model compared with MAIT cell deﬁcient Mr1-/-Tcrd-/-mice. A further observationfrom this study was that direct topical application of the MAIT cell ligand 5-OP-RU prior to skin injury, in the presence or absence of additional cytokines, was sufﬁcient to induce local expansion of MAIT cells and accelerate tissue repair (21). The importance of this role at human barrier sites and mechanisms through which MAIT cell mediators might exert their homeostatic function on the local environment is yet to be elucidated. Microbes and MAIT cell development Interestingly, introducing microbial stress through environmental acidiﬁcation reduced activation by impairing availability of riboﬂavin (32). MAIT cells have also been shown to exhibit microbe-speciﬁc responses to bacterial and fungal organisms with differential TCR b-chain bias and MR1-dependent activation, suggesting a further dimension of functional heterogeneity (33). Microbial recolonization of adult GF mice restores thymic MAIT cell development, but fails to populate peripheral tissue such as the skin (21) or lung (23) with newly differentiated MAIT cells. There is a narrow neonatal window (within ﬁrst 3 weeks of life) when recolonization of GF mice can restore the MAIT cell population (21). In addition, topical administration of 5-OP-RU is sufﬁcient for MAIT cell development and skin homing in neonates but not adults (21, 23). Thus, adult MAIT cell development is reliant on microbiome-derived co-stimulation. However, preservation of MAIT cells in MyD88- and TLR3- deﬁcient mice rules out TLR or IL-1 receptor family members (IL-1, IL-18 or IL- 33) as likely drivers (23). A further explanation for this temporally- Microbial diversity varies by tissue, and notably between health and disease, typically marked by dysbiosis with reduced diversity. As discussed below, this may be a mechanism through which MAIT cells contribute to pathology and equally offer a therapeutic opportunity to manipulate MAIT cell function. It is worthwhile Frontiers in Immunology 04 frontiersin.org Jabeen and Hinks 10.3389/ﬁmmu.2023.1127588 considering barrier homeostasis alongside microbial diversity. Pathogen invasion disrupts the mucosa and induces an inﬂammatory response. Co-stimulation of MAIT cells via TCR and cytokine has been shown in vitro to engage the full antimicrobial repertoire in MAIT cells (34). Therefore, where MAIT cells are implicated in disease pathogenesis it is important to consider any changes to the microbiome in parallel. Further studies, particularly in human, are needed to address this and consider speciﬁc mechanisms which shift MAIT cell function from homeostatic to pro-inﬂammatory. likely to be closely intertwined with barrier integrity and its effects on the microbiota. likely to be closely intertwined with barrier integrity and its effects on the microbiota. Tissue localization and MAIT cell phenotype Microbiome-derived signals are likely to contribute to the establishment of tissue resident MAIT cell populations as murine lung and skin MAITcell frequencies at steady state are cage dependent (21). It is unknown if this is shaped by antigen load or other innate signals.Cytokinesappeartohaveavariedrole;inskinIL-23signalingis necessary for sustaining a MAIT17 cell population (21), yet in the lung normal MAIT cell frequencies are maintained not only in Il23-/- mice but also Ifng-/-, Il6-/-, Il18-/- and Il12-/- deﬁcient animals (43). Cytokine reliancemaybeimprintedinthethymusasIl23rexpressionishigherin theMAIT17thymicsubset(4,44).TissueMAITcellsundergoterminal differentiationintissuewithuniquetranscriptomicprogramsobserved between the lung versus the spleen and liver (11), or indeed skin compared with spleen, lung and liver (21). Frontiers in Immunology Microbial dysbiosis and MAIT cells in immune mediated diseases MAIT cell dysfunction and dysbiosis often feature together in immune mediated diseases driven by autoimmune, atopic, and metabolic processes, alongside chronic infection. We review how MAIT cell-microbial interactions change from homeostatic to potentially harmful within this context and summarise this in Figure 2. Conditions characterised by shifts in the gut microbiome that provide insight into the potential symbiosis between microbiota and MAIT cell biology are considered ﬁrst, before exploring other mucosal niches and their potential effects on MAIT cell phenotype. MAIT cell-microbiome interactions in Murine studies have demonstrated reduced intestinal microbial diversity in MR1 deﬁcient animals, which may result from altered IL-17A signaling downregulating tight junction protein expression (41). In non-obese diabetic (NOD) mice deﬁcient in MR1 there is impaired intestinal barrier integrity (42). Further work is needed to understand if human MAIT cells can shape the composition of the healthy microbiome. As discussed below, in disease states this is 05 frontiersin.org 10.3389/ﬁmmu.2023.1127588 Jabeen and Hinks speciﬁc autoimmune conditions is reviewed more extensively elsewhere (52), signals following the resultant barrier compromise, however their direct role in pathological processes caused by dysbiosis is yet to be fully elucidated. Most mechanistic studies exploring MAIT cell-microbiome interactions have utilized animal models, often comparing to MR1-/- strains devoid of MAIT cells. The fecal microbiota in MR1-/- mice is distinct from wild-type animals with unique organisms belonging to Bacteroidaceae, Desulfovibrionaceae and unclassiﬁed Burkholderiales families, conferring greater overall richness. It is also resistant to antibacterial killing and Clostridium difﬁcile colonization (56). Furthermore, intestinal barrier function has been reported to be compromised in the absence of MR1 (41, 42). Gut microbiota vary between mouse strains (57) and to date no studies have compared animals with MAIT cell deﬁciency or excess across common background laboratory strains to determine if shifts in microbiome are linked to their genetic background. Thus challenges arise when elucidating MAIT cell mechanisms in animal models and it is important to corroborate ﬁndings in human studies given fundamental differences not only in microbiome but also MAIT cell tissue distribution. The gut microbiome and MAIT cells The gut microbiome has been most widely studied in health and disease. It is estimated that the bacterial density of the colon is 1011- 1012/milliliter making it the most densely populated microbial habitat in the human body (53). Human gut microbiota is composed of Firmicutes, Bacteroidetes, Actinobacteria, Proteobacteria, Fusobacteria, and Verrucomicrobia, with Firmicutes and Bacteroidetes accounting for 90% of total species (54). Its principal function is to protect against colonization of exogenous pathogens and potentially pathogenic indigenous organisms, through competition for ﬁnite nutrients and modulation of the host immune response (55). Throughout life the gut microbiota is required for the development of innate and adaptive arms of the immune response, providing education in host/ pathogen discrimination and sustaining barrier homeostasis (55). As discussed, MAIT cell development is reliant on the gut microbiome. Bacteroidetes, highly abundant in the gut, are the strongest stimulators of MAIT cells and likely further inﬂuence their phenotype in the intestinal mucosa (31). In dysbiosis (related to localized pathology or systemic inﬂammation) MAIT cells, enriched in the lamina propria, are likely to be one of the ﬁrst cells exposed to translocated gut microbes and inﬂammatory The gut-liver axis and chronic liver diseases IL-17 is reported to have an additional protective role in the intestinal mucosa (69, 70) and clinical trials with anti-IL-17 therapy (secukinumab) have lacked efﬁcacy in CD (71). IL-12 and IL-18 are upregulated in the intestinal mucosa of CD patients and polymorphisms of IL-23R, NLRP3, IL-18R and IL-12B2 signiﬁcantly associate with CD implicating these cytokines in its pathophysiology (72). It remains to be seen if phenotypic changes reported in tissue MAIT cells in IBD are an epiphenomenon or directly implicated in pathogenesis. Taken together with barrier disruption and dysbiosis in IBD, MAIT cell may need to balance conﬂicting roles; their capacity to produce IL-17 and engage a tissue repair program following exposure to microbial antigens provides homeostatic capacity (35), however in a dysbiotic landscape with high antigen burden and pro-inﬂammatory cytokine co-stimulus a pro-inﬂammatory program may be engaged (36). In animal models the liver has been shown to remain sterile in the presence of an intact intestinal mucosa, with immune responses to gut commensals conﬁned to the mesenteric lymphoid system. In the context of infection or inﬂammation the liver acts as an immunological ‘ﬁrewall’, clearing bacteria or their derived products breaching intestinal or vascular barriers (73). MAIT cells are the dominant population of innate like T cells in the liver (up to 50% of CD3+ cells). They are adapted for tissue homing (high expression of CXCR6 and CCR6) and poised for host defense (CD69, HLA-DR, CD38high). Compared with mucosal barrier MAIT cells, their steady state responses are less skewed to type 17 functionality and require IL-7 licensing for sustained IL-17 production following TCR ligation (74). This distinction may be driven by the lack of interaction with commensal organisms in health. Below we consider changes to the microbiome in prevalent chronic liver diseases in which MAIT cells have been implicated. Fatty liver diseases associated with alcohol, obesity or metabolic syndromes continue to grow in prevalence. High fat diet and alcohol induced dysbiosis can disrupt host-microbe interactions through metabolic dysregulation and mucosal barrier disruption. In this setting MAIT cell activating bacteria and microbe derived metabolites can translocate to the liver (75). Non-alcoholic fatty liver disease (NAFLD) affects approximately 40% of all adults worldwide and can range from benign hepatic steatosis to progressive non-alcoholic steatohepatitis (NASH) (76). Peripheral blood MAIT cells are depleted in NAFLD, with their enrichment in the liver (77). Inﬂammatory bowel diseases Taken together with barrier disruption and dysbiosis in IBD, MAIT cell may need to balance conﬂicting roles; their capacity to produce IL-17 and engage a tissue repair program following exposure to microbial antigens provides homeostatic capacity (35) however in a dysbiotic landscape with The gut-liver axis and chronic liver diseases chronic inﬂammatory conditions of the gastrointestinal tract associated with dramatic changes to the microbiota and local metabolic landscape (58). Several studies have investigated the relationship between genetic and immune susceptibility to IBD alongside the role of the gut microbiome in pathogenesis. This has been reviewed elsewhere (59–61). MAIT cells and their derived cytokines, particularly IL-17, have been considered as pathogenic drivers however the evidence for this is conﬂicting. MAIT cells are depleted in children with early onset inﬂammatory bowel disease under the age of six (62). Adult peripheral blood MAIT cell are activated (Ki67+) and decline in frequency in CD and UC (63, 64), unrelated to therapeutic intervention (anti-TNF or corticosteroids). There is a concurrent enrichment of ileal mucosa MAIT cells in CD and colonic mucosa MAIT cells in UC, correlating with disease activity (45). Blood MAIT cells in CD demonstrate a shift in cytokine production with greater IL-17 and reduced IFN-g secretion following ex vivo stimulation (64), while UC MAIT cells upregulate CD69 (45) and secrete more IL-22 (64) and IL-17 (45). In oxazolone colitis, a murine model of UC which histologically resembles UC and is predominantly mediated by type-2 cytokines, it has been proposed that MAIT cell play a directly pathogenic role as disease severity is reduced in MR1-/- animals or with MR-1 antagonist isobutyl 6-formyl pterin (65). However, oxazolone can induce a very severe colitis with a systemic inﬂammatory response resembling sepsis, thus likely to reﬂect only the most severe forms of disease, making it challenging to draw parallels with the full spectrum of IBD (66). A recent study has shown that whilst a signiﬁcant expansion is seen in the Tc17 population during active CD, this is largely due to induction of conventional T cells and not MAIT cells. Disease associated Tc17 cells acquire a distinct phenotype (CD6high, CD39, CD69, PD-1, CD27low). MAIT cells were the major IL-17 producing CD8+ population in blood during health or remission but not active CD, when their frequency declines in blood but is maintained in tissue (67). A subpopulation of predominantly CD8+ Crohn’s-associated invariant T (CAIT) cells have also been described, resembling NKT type II cells and enriched in blood of CD patients concurrently with decline in MAIT cell clonotypes. This could represent a compensatory expansion of an innate-like cell population, but the role of these cells is yet to determined (68). Inﬂammatory bowel diseases Inﬂammatory bowel diseases (IBD), consisting of the subtypes Crohn’s disease (CD) and ulcerative colitis (UC), are multifactorial FIGURE 2 MAIT cells and the microbiome in health and disease. Constituents of the human microbiome in health, at different barrier sites, and interactions with tissue resident MAIT cells are shown (left). Key compositional shifts in human microbiota and their resultant effects in a range of disease states are summarized by barrier site (right), data are derived from human studies except within obesity where mouse studies have been included and highlighted within the ﬁgure. There is relative paucity of data on direct MAIT cell effect on the microbiome. CRSwNP, chronic rhinosinusitis with nasal polyposis; COPD, chronic obstructive pulmonary disease; GVHD, graft versus host disease; HSCT, hematopoietic stem cell transplant; HCV, hepatitis C virus; HIV, human immunodeﬁciency virus; IBD, inﬂammatory bowel disease. FIGURE 2 MAIT cells and the microbiome in health and disease. Constituents of the human microbiome in health, at different barrier sites, and interactions with tissue resident MAIT cells are shown (left). Key compositional shifts in human microbiota and their resultant effects in a range of disease states are summarized by barrier site (right), data are derived from human studies except within obesity where mouse studies have been included and highlighted within the ﬁgure. There is relative paucity of data on direct MAIT cell effect on the microbiome. CRSwNP, chronic rhinosinusitis with nasal polyposis; COPD, chronic obstructive pulmonary disease; GVHD, graft versus host disease; HSCT, hematopoietic stem cell transplant; HCV, hepatitis C virus; HIV, human immunodeﬁciency virus; IBD, inﬂammatory bowel disease. 06 Frontiers in Immunology frontiersin.org Jabeen and Hinks 10.3389/ﬁmmu.2023.1127588 chronic inﬂammatory conditions of the gastrointestinal tract associated with dramatic changes to the microbiota and local metabolic landscape (58). Several studies have investigated the relationship between genetic and immune susceptibility to IBD alongside the role of the gut microbiome in pathogenesis. This has been reviewed elsewhere (59–61). MAIT cells and their derived cytokines, particularly IL-17, have been considered as pathogenic drivers however the evidence for this is conﬂicting. MAIT cells are depleted in children with early onset inﬂammatory bowel disease under the age of six (62). Adult peripheral blood MAIT cell are activated (Ki67+) and decline in frequency in CD and UC (63, 64), unrelated to therapeutic intervention (anti-TNF or corticosteroids). Inﬂammatory bowel diseases There is a concurrent enrichment of ileal mucosa MAIT cells in CD and colonic mucosa MAIT cells in UC, correlating with disease activity (45). Blood MAIT cells in CD demonstrate a shift in cytokine production with greater IL-17 and reduced IFN-g secretion following ex vivo stimulation (64), while UC MAIT cells upregulate CD69 (45) and secrete more IL-22 (64) and IL-17 (45). In oxazolone colitis, a murine model of UC which histologically resembles UC and is predominantly mediated by type-2 cytokines, it has been proposed that MAIT cell play a directly pathogenic role as disease severity is reduced in MR1-/- animals or with MR-1 antagonist isobutyl 6-formyl pterin (65). However, oxazolone can induce a very severe colitis with a systemic inﬂammatory response resembling sepsis, thus likely to reﬂect only the most severe forms of disease, making it challenging to draw parallels with the full spectrum of IBD (66). A recent study has shown that whilst a signiﬁcant expansion is seen in the Tc17 population during active CD, this is largely due to induction of conventional T cells and not MAIT cells. Disease associated Tc17 cells acquire a distinct phenotype (CD6high, CD39, CD69, PD-1, CD27low). MAIT cells were the major IL-17 producing CD8+ population in blood during health or remission but not active CD, when their frequency declines in blood but is maintained in tissue (67). A subpopulation of predominantly CD8+ Crohn’s-associated invariant T (CAIT) cells have also been described, resembling NKT type II cells and enriched in blood of CD patients concurrently with decline in MAIT cell clonotypes. This could represent a compensatory expansion of an innate-like cell population, but the role of these cells is yet to determined (68). IL-17 is reported to have an additional protective role in the intestinal mucosa (69, 70) and clinical trials with anti-IL-17 therapy (secukinumab) have lacked efﬁcacy in CD (71). IL-12 and IL-18 are upregulated in the intestinal mucosa of CD patients and polymorphisms of IL-23R, NLRP3, IL-18R and IL-12B2 signiﬁcantly associate with CD implicating these cytokines in its pathophysiology (72). It remains to be seen if phenotypic changes reported in tissue MAIT cells in IBD are an epiphenomenon or directly implicated in pathogenesis. Obesity and metabolic syndromes Intestinal microbiota has also been linked to susceptibility in alcoholic liver disease (ALD) (80). The gut microbiome shifts with disease progression from steatohepatitis, through to ﬁbrosis then cirrhosis and with patterns of alcohol consumption (binge drinking vs chronic consumption). The microbiome had been largely characterized in animal models, with limited human studies (81). Overall, there is lower abundance of Bacteroidetes and a higher proportion of Enterobacteriaceae and Proteobacteria (75, 81). Humanized germ-free mice, following transplantation of intestinal microbiota from patients with severe alcoholic hepatitis, develop hepatitis and intestinal barrier impairment (80). In a murine model of acute binge on the background of chronic alcohol exposure, MAIT cells became depleted in the intestine, liver and lung. Liver and lung MAIT cells upregulated CD69, IFN-g, TNF and the transcription factor T-bet. It is worth noting that female animals were exclusively used in this study, given the variable effects of ethanol consumption and differences in microbiome driven by sex (82). Alcohol exposure reduced intestinal microbiome beta diversity (a measure of community variation between animals), with resultant reduction in riboﬂavin production capacity. Adoptive transfer of cecal microbiota to antibiotic pre-treated alcohol naïve mice produced a decline in pulmonary and hepatic MAIT cell frequency, with increased IFN-g+/ T-bet+ cells at these sites and TNF+ cells in the lung. This change in MAIT cell frequency could be abolished with antibiotic therapy following alcohol exposure. Serum levels of intestinal fatty acid binding protein (iFABP), a biomarker of intestinal epithelial damage, and bacterial 16S rRNA gene copies were elevated following alcohol exposure. In vitro treatment of human MAIT cells with this serum reduced viability, driving apoptotic death and upregulation of CD38, IFN-g and granzyme B. Direct ethanol exposure did not produce the same effect (82). Similarly, blood MAIT cells are depleted in patients with severe alcoholic hepatitis and alcohol related cirrhosis with altered transcriptional programming (reduced RORgt and PLZF expression), activated phenotype (CD69high) and altered responses (reduced IL-17 and granzyme B with E. coli challenge). Plasma endotoxin and D- lactate (measure of gut permeability) are increased with ALD. Faecal extracts derived from patient with ALD reproduce the abnormal MAIT cell phenotype in vitro with accelerated cell death, upregulation of CD69 and HLA-DR and diminished antibacterial capacity (reduced IFN-g, TNF, IL-17, granzyme B and perforin with E. coli infection) (83). The gut-liver axis and chronic liver diseases A negative correlation has been reported between circulating MAIT cell frequency and serum glycated hemoglobin (HbA1c), gamma-glutyl transferase or total triglycerides. These cells upregulate PD-1, CCR5 and CXCR6 (77). Upon stimulation, an IL- 4 dominant response is seen over IFN-g and TNF production. In vitro, activated MAIT cells can induce monocyte/macrophage differentiation into a M2 phenotype (77). Furthermore, in a mouse model of NASH, induced with methionine and choline deﬁcient diet, MAIT cells localize to the liver and display a Th2 proﬁle (IL-4+ and IL-10+ > IFN-g+ cells) in wild-type animals. In the MR1-/- counterparts there was greater steatohepatitis with an accompanying increase in the proportion of CD11c+ pro- inﬂammatory M1 macrophages relative to CD206+ M2 macrophages suggesting a potentially protective role for MAIT cells (77). However, in the context of ﬁbrosis, a more pro- inﬂammatory and ﬁbrogenic function has been proposed given the observation of more progressive carbon tetrachloride (CCl4) induced liver ﬁbrosis in animal models using MAIT cell enriched (Va19TCRTg) over MAIT cell deﬁcient (MR1-/-) mice (78). Both in alcoholic and non-alcoholic fatty liver disease MAIT cells have been shown to accumulate in liver ﬁbrotic septa and demonstrate increased proliferative capacity with upregulation of Ki-67 (78). With progression to cirrhosis these cells are closely positioned to ﬁbrogenic myoﬁbroblasts; in vitro co-culture experiments have shown MR1 and contact-dependent mitogenic effects of MAIT cells on human myoﬁbroblasts (78). Blood MAIT frequency Frontiers in Immunology 07 frontiersin.org Jabeen and Hinks 10.3389/ﬁmmu.2023.1127588 10.3389/ﬁmmu.2023.1127588 declines with liver ﬁbrosis, and remaining MAIT cells are activated (CD25 and CD69 high). Interestingly long-term prophylactic antibiotic therapy (norﬂoxacin or rifaximin) is signiﬁcantly associated with less decline in MAIT cell frequency and lower CD25 expression (78). The human gut microbiome in NAFLD and NASH is devoid of Bacteroidetes, with expansion of Prevotella and Porphyromonas species. In NASH the proportion of ethanol producing bacteria also increases (75). Rifaxamin is the most widely used antimicrobial prophylaxis agent for the prevention of hepatic encephalopathy in end stage liver disease. It reduces bacterial translocation, has anti-inﬂammatory properties and modulates the microbiota, speciﬁcally reducing the abundance of harmful bacteria (e.g. Klebsiella, Streptococcus, Clostridium) relative to probiotic organisms (e.g. Bacteroides) (79). The role of MAIT cells in chronic liver disease pathogenesis is yet to be fully deﬁned; indeed, their deﬁciency in advanced disease could contribute to increased susceptibility towards systemic infection, particularly with shifts seen in the microbiome. The gut-liver axis and chronic liver diseases Conversely these changes in the context of wider inﬂammatory and pro-ﬁbrotic signals could account for MAIT cell phenotypes observed in cirrhosis. It is not known if MAIT cells directly interact with and shape the microbiome in homeostasis, by extension further studies are needed to determine if following barrier disruption MAIT cells are bystanders responding to changes in their microenvironment or lose an innate capacity to support commensal communities. Haematopoietic stem cell transplantation In a similar pattern to the systemic inﬂammatory conditions discussed above, chronic infection with HIV (101–104) and HCV (105–107) cause a decline in circulating MAIT cell frequency (108) which cannot be fully restored with treatment (101, 109) and is associated with intestinal dysbiosis (108). Circulating MAIT cells upregulate perforin and granzyme B in HIV and HCV infection, with high CD69 and PD-1 expression in viral co-infection. Following ex vivo stimulation with E. coli there is impaired IFN-g, TNF and IL-17 generation in presence of mono- or co-infection, compared with health. Virally-infected patients have relatively low endotoxin core antibodies, implying a diminished capacity to control translocating bacteria. There is a compositional change in the fecal microbiome with higher abundance of Bacteroidetes and lower abundance of Firmicutes following viral infection. Bacteroides spp. abundance has been shown to correlate positively with MAIT cell frequency but negatively with TNF production following bacterial challenge, whereas Firmicutes abundance negatively correlated with PD-1+ MAIT cell frequency (108). Gut dysbiosis is not reversed with antiretroviral therapy (ART) in HIV (110) and there is partial recovery following HCV eradication (111) except in cirrhotic patients (112). The lung microbiome is also less diverse in HIV and only partially restored with ART (113). Intriguingly, compared with blood, lung MAIT cells better retain function and transcriptional features in HIV, including a tissue repair capacity (48). These studies further support a role for the microbiome in shaping MAIT cell function; circulating MAIT cells are likely reﬂective of the state of the gut microbiome given its association with barrier disruption and potential microbial translocation into circulation, whereas at different barrier sites (e.g. lung) the local microbiome has the capacity to uniquely modify tissue resident MAIT cell phenotype. It also raises further questions regarding the requirements for reconstitution of MAIT cells following their depletion – how essential are individual constituents of the microbiome and their capacity for riboﬂavin synthesis? What other co-stimulatory signals are required and potentially deﬁcient in these viral infections? And, is complete re-population of tissue with MAIT cells restricted by a temporal window as in mice? These questions need to be addressed in human studies to best translate therapeutic strategies for re-establishing tissue homeostasis with MAIT cells. Haematopoietic stem cell transplantation Allogenic HSCT can offer curative therapy for a wide range of hematological disorders but can be complicated by inappropriate immune reconstitution, resulting in inﬂammatory sequelae such as acute or chronic graft versus host disease (GVHD), and increased risk of infection (94). Post-transplantation intestinal microbiome is associated with survival and complications related to HSCT (95– 97). Through their interaction, the intestinal microbiome and mucosal T cells, particularly human MAIT cells, are thought to play a protective role post-transplant (30, 94, 98). In a murine major MHC (class I and II) mismatched allogenic stem cell transplant model, MAIT cell frequency was higher in tissue compared with peripheral blood (41). MAIT cells preferentially localized to the colon, producing high concentration of IL-17A to maintain barrier integrity and limit alloantigen presentation after bone marrow transplantation. By comparison IL-17A-/- and MR1-/- animals displayed relatively accelerated GVHD, associated with altered fecal microbiota and downregulation of tight junction proteins claudin 4 and claudin 8 (41). In human HSCT, a diverse intestinal microbiome early after transplant is associated with a higher MAIT cell frequency (30), reduced incidence of acute GVHD (99, 100) and improved survival (100). Circulating Vd2 cell frequency correlated with MAIT cells and intestinal alpha diversity, a measure of species diversity and abundance (100). Speciﬁcally, a higher abundance of Bacteroidetes was seen with higher MAIT cell numbers, whereas abundance of Firmicutes was associated with fewer MAIT cell (100). Blautia spp. abundance is also predictive of MAIT reconstitution (98). No increased abundance of the riboﬂavin biosynthesis pathways is however observed between individuals based on MAIT cell frequency (100). Single cell RNA sequencing of peripheral blood MAIT cells revealed a pro-inﬂammatory phenotype, upregulating genes linked to effector function (GNLY, PRF1, CCL4) and migration (ITGB2) with concurrent downregulation of NfkB signaling inhibitors (NFKBIA, TNFAIP3). Vd2 cells also display a complementary activated phenotype. As expected, a tissue repair signature was not detected in peripheral blood MAIT cells (100). High MAIT cell frequency in infused grafts is linked to higher abundance of intestinal ﬂora post-transplant and lower incidence of acute GVHD. Following the onset of GVHD, circulating MAIT cell frequency declines as these cells become activated (upregulating CD69, CXCR3, CXCR4 and transcription factors RORgt and T- bet). This coincides with a decline in riboﬂavin synthesising gut microbiota, perhaps reﬂecting a component of microbial regulation by MAIT cells in health (99). Obesity and metabolic syndromes There is evidence that changes in the gut microbiome may contribute to the development of obesity (84), with murine and human studies suggesting gut microbe-derived lipopolysaccharide and translocating gut bacteria may contribute to systemic inﬂammation in obesity (85–87), which can drive insulin resistance associated with the development of type 2 diabetes (88). Obesity is associated with a decrease in overall circulating MAIT cells (89–91), but an increase in circulating, activated, IL-17 producing MAIT cells, which may be correlated with translocated bacteria such as Bacteroidetes (89, 90). Obesity is also associated with reduction in glycolytic metabolism, mTORC1 signaling, and SLC7A5 aa transport in circulating MAIT cells (92). IL-17- producing and Granzyme B+ MAIT cells are increased in omental adipose tissue in human obesity (89, 91). Moreover, whilst circulating MAIT cells are deﬁcient in obesity, weight loss after bariatric surgery is associated with both a restoration of circulating MAIT cell numbers, and an increased diversity of the gut microbiome, including increased in Bacteroidetes and Fusobacteria (93). Most human studies are correlative, but in murine models, with leptin deﬁcient or high-fat diet-fed mice, MAIT cells were likewise decreased in peripheral blood and ileal and epididymal adipose tissue, due to a shift towards a pro-apoptotic phenotype, with relative increase in activated, IL-17-producing MAIT cells (88). These changes were correlated with a decreased expression of MR1 ligands and of riboﬂavin pathway genes within the cecal microbiome. These MAIT cells may have been contributing to insulin resistance, as MR1-/- mice had greater insulin sensitivity to oral insulin tolerance testing. Adipose MAIT cells were also contributing to enhanced pro-inﬂammatory cytokine and chemokine signaling in adipose tissue, and to a reduction in FoxP3+ Treg and a shift towards M1 rather than M2 polarization of macrophages (88). The presence of MAIT cells was also associated with decreased gut epithelial barrier integrity, measured by FITC-dextran translocation and by expression of tight junction proteins, which would favor bacterial translocation. Thus, these data suggest that MAIT cells promote inﬂammation in obesity. Might these changes in MAIT cells also affect the gut microbiome in a deleterious manner? Haematopoietic stem cell transplantation These studies provide insight into the interdependence of MAIT cells and the microbiome in a unique Obesity and metabolic syndromes In the same mouse model fecal transfer was performed into C57BL/6 mice from MAIT- Frontiers in Immunology 08 frontiersin.org Jabeen and Hinks 10.3389/ﬁmmu.2023.1127588 deﬁcient MR1-/- or MAIT over-expressing Va19+/- mice, and showed MAIT cells promoted a microbiome lower in Biﬁdobacteriaceae and Lactobacteriaceae, and these changes were again associated with a decrease in gut epithelial barrier integrity and in frequencies of mucosal Treg, innate lymphoid cell(ILC)2 and ILC3 cells, which implies the MAIT-dependent effects on the microbiome are not simple epiphenomena, but can have signiﬁcant immunological effects. setting of immune reconstitution in human adults. However, due to the lack of tissue MAIT cell proﬁling several questions remain unanswered including whether MAIT cells can repopulate tissues at similar baseline frequencies and if their phenotype is altered upon their return thereby affecting homeostatic functions, barrier integrity and microbiome composition. The lower airway The oropharynx harbors a diverse microbiome characterized by the genera Streptococcus, Neisseria, Rothia, and anaerobes, including Veillonella., Prevotella and Leptotrichia (114). It is thought to be the primary source of lung microbiota, introduced through subclinical microaspiration (118). MAIT cells are present in the buccal mucosa (up to 50% CD8aa T cells), displaying a tissue resident effector memory phenotype (CD69, CD103, HLA-DR and PD-1 high) and IL-17 skewed response to PMA-ionomycin stimulation (46). One study has considered oromucosal MAIT cell function in relation to the microbiome in apical periodontitis, characterized by inﬂammation of the periodontal tissue leading to translocation and dissemination of opportunistic organisms. In this condition a MAIT cell signature appears in affected tissue with increased MAIT TCR, TNF, IFN-g and IL-17A transcripts compared to healthy adjacent gingiva. There is an expansion of riboﬂavin producing taxa in the local microbiome and using a sparse partial least squares discriminant analysis the authors report several bacterial genera negatively correlated with MAIT TCR and IL-17A transcripts (119). Whilst this is an interesting approach, a large variation in abundance and diversity of bacterial taxa was observed in this study within a small population (n=25), thus there is limited power to derive conclusions regarding mechanisms of microbiome-MAIT cell interaction. The healthy adult lung microbiome is dominated by the genera Prevotella, Veillonella, and Streptococcus (123). These belong to the phyla Bacteroidetes and Firmicutes, both stimulators of MAIT cells, with a stronger capacity in the former group (31). In lung diseases there are notable changes to the microbiome likely provoked by host inﬂammatory responses often leading to increased airway wall permeability and mucus production modifying growth conditions (124–126). A sustained bidirectional relationship between the mucosal immune system and disordered respiratory microbiota is likely to be a key driver of disease progression. We propose that the shift in community membership towards species with greater MAIT cell antigenic load, immunogenicity and capacity for epithelial disruption can overwhelm MAIT cell homeostatic barrier defenses, particularly with numeric and functional deﬁciencies seen in these conditions as exempliﬁed below. Airways diseases carry a huge global burden; asthma is the commonest chronic respiratory disease affecting 262 million people worldwide (127) and chronic obstructive pulmonary (COPD) is the third leading cause of mortality (128). These conditions have uniquely disordered airway microbiome but share a pulmonary MAIT cell deﬁciency proportionate to inhaled corticosteroid (ICS) dose (116, 129), a mainstay of therapy with increasing disease severity. The respiratory microbiome and MAIT cells The respiratory tract encompasses the upper (anterior nares, nasal passages, paranasal sinuses, nasopharynx, oropharynx, and laryngeal segment proximal to the vocal cords) and lower (larynx Frontiers in Immunology 09 frontiersin.org 10.3389/ﬁmmu.2023.1127588 Jabeen and Hinks distal to vocal cords, trachea, small airways, and alveoli) tracts. The total surface area of the airways is approximately 70m2 making it the second largest barrier site after the gut mucosa in human (114). Culture independent methods of microbial detection have dispelled the long-held theory of lung sterility and instead demonstrated the presence of diverse communities of microbiota in the lower airway (115). MAIT cells are enriched in the airways and form the largest population of antibacterial T cells in the lungs (116); they express tissue residency markers (CD69 and CD103) and display polycytotoxic potential (117). Thus, MAIT cells occupy a prime position to support pulmonary immunity, and there has been growing interest in studying their role in airways diseases. Airways inﬂammation and common treatments (corticosteroids and antimicrobials) signiﬁcantly modify the local microbiome and alter barrier function, therefore careful consideration of the interdependence between MAIT cells and the microbiome is crucial when studying pulmonary pathology as discussed below. disease severity (47). Allergic rhinitis and nasal polyposis are often accompanied by asthma. In these conditions, changes to the nasopharyngeal microbiome have been reported with expansion of MAIT cell activating organisms belonging to Bacteroidetes and Proteobacteria taxa in asthma (120) and predominantly Firmicutes, Proteobacteria and Actinobacteria in chronic rhinosinusitis with nasal polyposis. Interestingly in children, nasal Corynebacterium sp. and S. epidermidis abundance is associated with absence of pet allergen sensitization (121); Corynebacterium have been shown to negatively correlate with inﬂammatory gene expression in the nose (122) whilst S. epidermidis can engage a tissue repair program in skin resident murine MAIT cells (21). It is tempting to speculate that the microbiome in health supports a homeostatic phenotype in MAIT cells whereas disease driven changes to the microbiome supply the antigenic and co-stimulatory signals to sustain an inﬂammatory IL-17 dominant response. It is yet to be determined if MAIT cells can in fact interact with the microbiome in this manner and indeed if any potentially pathogenic activity can be reversed by manipulating the microbial community. frontiersin.org Frontiers in Immunology The lower airway In a large bronchoscopy study no evidence of increased IL-17A in serum, sputum or BAL was found in asthma nor was there an increase in IL-17+ T cell populations (Th17 or gdT cells). This study identiﬁed a striking deﬁciency in MAIT cell frequency with increasing disease severity and ICS use (116). Corticosteroid exposure has beendemonstratedtoimpair MAITcellIFN-gresponsesinvitroto non-typeable H. inﬂuenzae (NTHi), the most prevalent strains linked to exacerbations of airways diseases (129). Sputum MAIT cells are CD69 and PD-1 high, and peripheral blood cells from patients with asthma are skewed towards IL-17/TNF production (over IFN-g) following activation. IL-7 levels in sputum and serum are elevated in neutrophil dominant airways inﬂammation and induces greater IL-17 response to PMA-Ionomycin ex vivo stimulation (146). MAIT cell frequencies havebeen reported tocorrelatewithNK,ILC1,ILC2,ILC3 cells in severe asthma, declining with airﬂow obstruction (reduction in FEV1%) (147). In a murine model of allergic airway inﬂammation using Alternaria inhalation, MAIT cells are proposed to repress ILC2 driven inﬂammation and airway hyperresponsiveness via expression of interleukin-4-induced gene 1 (IL4I1) (148). In asthma, MAIT cells are thus depleted and exposed to a Proteobacteria dominated microbiome in the setting of epithelial barrier disruption. Translocation of bacterial antigen and products may account for the activated MAIT cell phenotype however ICS-disabled anti-bacterial responses could cause susceptibility to airways infection seen in severe asthma. Moreover, their absence could deprive the airway of tissue repair and type-2 immune regulatory mechanisms. The lines of causality in severe treatment refractory asthma, particularly with dominance of pathogenic organisms in the airway and neutrophilic inﬁltration, are verycomplex. Itlikelyinvolves barrierdysfunctionand mucosal immune disarmament; in the case of MAIT cells it is yet to be determined if a pathogenic role can be ascribed or if these cells are stripped of their homeostatic antibacterial function due to the wider spanning inﬂammatory landscape or indeed treatment. Future studies therefore need to examine their function in well characterized patient cohorts and compare tissue resident cells in disease affected and unaffected locations with due consideration to the local microbiome. COPD is also heterogenous in its pathogenesis and manifests with predominantly neutrophilic airways inﬂammation, mucus hypersecretion, emphysema and variable vascular dysfunction (149). Unlike asthma, alterations in airway microbiome appear late ith more se ere disease The microbiome declines in di ersit biopsies of corticosteroid treated (but not ICS naïve) COPD patients (129). The lower airway inﬂuenzae (NTHi), the most prevalent strains linked to exacerbations of airways diseases (129). Sputum MAIT cells are CD69 and PD-1 high, and peripheral blood cells from patients with asthma are skewed towards IL-17/TNF production (over IFN-g) following activation. IL-7 levels in sputum and serum are elevated in neutrophil dominant airways inﬂammation and induces greater IL-17 response to PMA-Ionomycin ex vivo stimulation (146). MAIT cell frequencies havebeen reported tocorrelatewithNK,ILC1,ILC2,ILC3 cells in severe asthma, declining with airﬂow obstruction (reduction in FEV1%) (147). In a murine model of allergic airway inﬂammation using Alternaria inhalation, MAIT cells are proposed to repress ILC2 driven inﬂammation and airway hyperresponsiveness via expression of interleukin-4-induced gene 1 (IL4I1) (148). In asthma, MAIT cells are thus depleted and exposed to a Proteobacteria dominated microbiome in the setting of epithelial barrier disruption. Translocation of bacterial antigen and products may account for the activated MAIT cell phenotype however ICS-disabled anti-bacterial responses could cause susceptibility to airways infection seen in severe asthma. Moreover, their absence could deprive the airway of tissue repair and type-2 immune regulatory mechanisms. The lines of causality in severe treatment refractory asthma, particularly with dominance of pathogenic organisms in the airway and neutrophilic inﬁltration, are verycomplex. Itlikelyinvolves barrierdysfunctionand mucosal immune disarmament; in the case of MAIT cells it is yet to be determined if a pathogenic role can be ascribed or if these cells are stripped of their homeostatic antibacterial function due to the wider spanning inﬂammatory landscape or indeed treatment. Future studies therefore need to examine their function in well characterized patient cohorts and compare tissue resident cells in disease affected and unaffected locations with due consideration to the local microbiome. hypopharyngeal microbiome can predict development of allergic asthma in childhood (134, 140). Circulating MAIT cell frequency at 1 year of age is associated with reduced risk of asthma diagnosis within the ﬁrst 7 years of life and a Th1 dominant response in CD4+ T cells (141). In pediatric asthma, small studies have reported increased frequency of IL-17+ MAIT cells in bronchoalveolar lavage (142) and blood (143) of patients presenting with severe exacerbations, however no comparisons were made with health when sampling the lower airway (142). In adult disease Haemophilus inﬂuenzae has emerged as the commonest potentially pathogenic organism in the airway, associated with sputum neutrophilia and altered microbial diversity, namely reduction in Streptococcus, Gemella and Porphyromonas taxa (135, 136, 144, 145). The lower airway A further study has reported MAIT cell depletion in blood with enrichment in lung parenchyma and accumulation around alveolar epithelial cells in less clinically severe disease, however a limitation of these data is the lack of reporting on ICS use in lung tissue donors (152). There is a higher frequency of IL- 17+ lung MAIT cells in COPD compared to health, and blood MAIT cells generate IL-17 (over IFN-g) following PMA-ionomycin stimulation in vitro with diminishing magnitude of response seen with worsening airﬂow obstruction (152). Lower MAIT cell frequencies are associated with elevated serum C-reactive protein (CRP) levels (153) and increased frequency of exacerbations requiring hospitalization (154). At the time of exacerbation peripheral blood MAIT cells are activated with upregulation of CD38 and LAG-3 (154). The above studies hint towards an impaired antibacterial defense with MAIT cell deﬁciency in COPD sufﬁcient to cause clinical exacerbation events as disease severity increases. The relationship with ICS in COPD and asthma is important and raises the question whether we should supplement such therapies with counteractive strategies to boost barrier MAIT cell frequencies either directly with ligand (5-OP-RU) or indirectly by manipulating the microbiome (e.g. with probiotics). Further work is also needed to understand the signiﬁcance of MAIT cell derived IL-17 in the setting of neutrophilic airways inﬂammation – is it a sufﬁcient signal to perpetuate neutrophil recruitment or are MAIT cells attempting to re-establish tissue homeostasis? These questions need to be considered in the context of the microbiome and dysregulated epithelium (155). Frontiers in Immunology The lower airway The nasopharynx is similarly enriched with MAIT cells (47) but is home to a unique microbiome compared with the oropharynx and lower respiratory tract. As a transition site between keratinized squamous epithelium and stratiﬁed squamous epithelium it hosts skin colonizers from the genera Staphylococcus, Propionibacterium and Corynebacterium alongside Moraxella, Corynebacterium, Dolosigranulum, Haemophilus and Streptococcus (114). As seen in the oropharynx, sinonasal MAIT cells possess a tissue resident effector memory phenotype. They have been linked to disease severity in allergic rhinitis with nasal polyposis; in this condition MAIT cell appear more activated with CD38 upregulation and IL- 17A skewed response to stimulation. Both markers correlate with Asthma is a clinically and immunologically heterogenous condition. Treatment approaches in asthma have been revolutionized by identifying and targeting ‘treatable traits’ (130), notably in type-2 cytokine (IL-5, IL-4, IL-13) mediated eosinophilic airways inﬂammation for which novel biologics have emerged (131, 132). ‘T2-low’ non-eosinophilic disease is refractory to corticosteroids and existing biologics (130). It affects ~30% of severe asthmatics (133), and is associated with airways neutrophilia alongside high IL-17 (133) expression, and may be driven by chronic bacterial airways infection (118, 134–138). Epithelial barrier disruption is central to pathogenesis, regardless of inﬂammatory phenotype (139). An altered naso-/ Frontiers in Immunology frontiersin.org 10 10.3389/ﬁmmu.2023.1127588 Jabeen and Hinks hypopharyngeal microbiome can predict development of allergic asthma in childhood (134, 140). Circulating MAIT cell frequency at 1 year of age is associated with reduced risk of asthma diagnosis within the ﬁrst 7 years of life and a Th1 dominant response in CD4+ T cells (141). In pediatric asthma, small studies have reported increased frequency of IL-17+ MAIT cells in bronchoalveolar lavage (142) and blood (143) of patients presenting with severe exacerbations, however no comparisons were made with health when sampling the lower airway (142). In adult disease Haemophilus inﬂuenzae has emerged as the commonest potentially pathogenic organism in the airway, associated with sputum neutrophilia and altered microbial diversity, namely reduction in Streptococcus, Gemella and Porphyromonas taxa (135, 136, 144, 145). In a large bronchoscopy study no evidence of increased IL-17A in serum, sputum or BAL was found in asthma nor was there an increase in IL-17+ T cell populations (Th17 or gdT cells). This study identiﬁed a striking deﬁciency in MAIT cell frequency with increasing disease severity and ICS use (116). Corticosteroid exposure has beendemonstratedtoimpair MAITcellIFN-gresponsesinvitroto non-typeable H. The skin microbiome and MAIT cells decreased S. epidermidis abundance have been observed (158), this pattern is also reported in atopic dermatitis (157). MAIT cells can mount a cytotoxic response to S. aureus infected dendritic cells in an IL-12 reliant and partially MR-1 dependent manner, with IFN-g and Granzyme B upregulation (159). Thus, they are equipped to provide cutaneous antibacterial defenses against S. aureus. MAIT cells originally garnered interest in psoriasis as a source of IL-17A, which is a key driver of inﬂammation; but Teunissen et al. have shown that the majority of IL-17A+CD8+ T cells are in fact conventional CD8+ T cells rather than MAIT cells (160). In atopic dermatitis abundance of S. aureus induces a host transcriptomic signature characterized by upregulation of genes encoding antimicrobial factors, tryptophan metabolites, immune activation (IL1B, CCL2, CCL19) and Th2 signaling mediators (IL4R, IL5, IL13, PI3, TNFRSF4, CCR4) (161). Interestingly in a murine model of atopic dermatitis MAIT cells have been implicated in eosinophil activation and recruitment of IL-4/-13 producing type 2 effector cells in a MR1 dependent fashion (162). The skin therefore reﬂects another major barrier site at which the local microbiome may shape not only antimicrobial type 1 immunity exercised by MAIT cells but also inﬂuence the regulation of type 2 allergic inﬂammation. There is a relative paucity of human data examining the balance between tissue repair and proinﬂammatory functionality in MAIT cells in the context of primary skin disorders or cutaneous manifestations of systemic inﬂammatory disorders. Murine and human in vitro data have shown successful harnessing of MAIT cells to accelerate wound healing through TCR ligation (21, 36). It is yet to be seen if this capacity can be utilized as an add- on therapy to address barrier disruption in skin diseases through direct application of MAIT cell ligand or manipulation of the skin/ gut microbiome with targeted topical or oral probiotics respectively. This approach may even be relevant within the female genital tract, as an extension of the skin barrier and a mucosal site at which MAIT cells are enriched (49). Conversely in contexts of chronic mucosal infection or epithelial damage, therapeutic stimulation of MAIT cells would be anticipated to promote beneﬁcial antibacterial responses and activate tissue repair programs promoting epithelial wound repair. The use of activating ligands would require selection of synthetic molecules with much greater stability than naturally- occurring 5-OR-RU which degenerates rapidly in aqueous solution. The skin microbiome and MAIT cells The skin microbiome is critical for homeostasis and shaping of the mucosal immune system. As previously discussed, cutaneous commensals have been shown to induce a homeostatic tissue repair program and functionality in murine skin MAIT cells. In human skin, MAIT cells are a tissue resident population upregulating skin homing marker CLA and CD103 and not enriched in common skin lesions (except dermatitis herpetiformis) (156). The colonizing microbial population in human skin is composed of a core group of species andvariation is seen with changein topology, introducedby structures such as hair follicles, sebaceous glands and ducts, alongside environmental factors (20, 157). The core phyla comprising the epidermal microbiome are Actinobacteria (up to 50% of organisms), Firmicutes, followed by Proteobacteria and Bacteroidetes. The gut microbiome has also been implicated in shaping skin health given common cutaneous manifestations of GI disorders such as IBD and coeliac disease. The mechanisms underlying gut-skin microbial interactions are not known but it is postulated that gut dysbiosis and resultant systemic inﬂammation or intestinal microbial translocation may contribute to disrupted skin homeostasis (157). COPD is also heterogenous in its pathogenesis and manifests with predominantly neutrophilic airways inﬂammation, mucus hypersecretion, emphysema and variable vascular dysfunction (149). Unlike asthma, alterations in airway microbiome appear late with more severe disease. The microbiome declines in diversity, it is depleted of Bacteroidetes with a relative expansion in Proteobacteria, particularly Haemophilus and Moraxella (123, 150, 151). MAIT cells are depleted in blood and endobronchial Skin dysbiosis is recognized in multiple chronic inﬂammatory conditions including psoriasis, atopic dermatitis, rosacea and acne vulgaris. In psoriasis there are complex patterns of change in microbial composition with variation in reporting between studies (158). Within psoriatic lesions increased S. aureus abundance and Frontiers in Immunology 11 frontiersin.org 10.3389/ﬁmmu.2023.1127588 Jabeen and Hinks conceptual proof of principle using exogenous oral administration of the synthetic inhibitory ligand acetyl-6-FP in obese mice. This reduced obesity-associated ileal inﬂammation and decreased MAIT cell IL-17 production (88). Furthermore, this ligand also led to alteration of the gut microbiome, inducing an increase in Bacteroidetes abundance. From these data a picture emerges of MAIT cells whose functions are very context dependent, and likewise the intent of therapeutic manipulation will be context dependent. The skin microbiome and MAIT cells In the situation of obesity, changes in the microbiome lead to reduced tissue MAIT cell frequencies, likely through increased apoptosis, but also to increased activation of MAIT cells promoting type 1 biased chronic inﬂammation, and consequent metabolic dysfunction. In such a situation therapeutic inhibition of MAIT cells has the potential to reduce dysbiosis, improve gut integrity and ameliorate systemic inﬂammation and metabolic dysfunction. decreased S. epidermidis abundance have been observed (158), this pattern is also reported in atopic dermatitis (157). MAIT cells can mount a cytotoxic response to S. aureus infected dendritic cells in an IL-12 reliant and partially MR-1 dependent manner, with IFN-g and Granzyme B upregulation (159). Thus, they are equipped to provide cutaneous antibacterial defenses against S. aureus. MAIT cells originally garnered interest in psoriasis as a source of IL-17A, which is a key driver of inﬂammation; but Teunissen et al. have shown that the majority of IL-17A+CD8+ T cells are in fact conventional CD8+ T cells rather than MAIT cells (160). In atopic dermatitis abundance of S. aureus induces a host transcriptomic signature characterized by upregulation of genes encoding antimicrobial factors, tryptophan metabolites, immune activation (IL1B, CCL2, CCL19) and Th2 signaling mediators (IL4R, IL5, IL13, PI3, TNFRSF4, CCR4) (161). Interestingly in a murine model of atopic dermatitis MAIT cells have been implicated in eosinophil activation and recruitment of IL-4/-13 producing type 2 effector cells in a MR1 dependent fashion (162). The skin therefore reﬂects another major barrier site at which the local microbiome may shape not only antimicrobial type 1 immunity exercised by MAIT cells but also inﬂuence the regulation of type 2 allergic inﬂammation. There is a relative paucity of human data examining the balance between tissue repair and proinﬂammatory functionality in MAIT cells in the context of primary skin disorders or cutaneous manifestations of systemic inﬂammatory disorders. Murine and human in vitro data have shown successful harnessing of MAIT cells to accelerate wound healing through TCR ligation (21, 36). It is yet to be seen if this capacity can be utilized as an add- on therapy to address barrier disruption in skin diseases through direct application of MAIT cell ligand or manipulation of the skin/ gut microbiome with targeted topical or oral probiotics respectively. This approach may even be relevant within the female genital tract, as an extension of the skin barrier and a mucosal site at which MAIT cells are enriched (49). The skin microbiome and MAIT cells A key aspect of MAIT cell biology which remains to be elucidated is how it is determined whether the effects of MAIT cell stimulation lead to a dominant pro-inﬂammatory antimicrobial response, or to a more homeostatic tissue repair activity favoring restoration of epithelial integrity. It is likely that a critical determinant of MAIT cell response is the integrity of the epithelial barrier itself. A damaged epithelium will release a number of factors which may inﬂuence MAIT cells directly, including alarmins, such as IL-33, whose receptor IL1R1/ST2 is highly expressed on lung MAIT cells during acute bacterial infection (35). MAIT cells also highly express IL17RE in mice and humans (35), the receptor for IL-17C. IL-17C is abundantly released by epithelia after stimulation by IL-1b, TNF, various pathogens or through cell damage via TLR2 and TLR5, promoting a proinﬂammatory, Th17 response from T cells (163). A damaged epithelium will also allow translocation of pathogens into proximity to the MAIT cells, directly furnishing danger signals such as lipopolysaccharide, which will activate membrane TLR2, which is particularly highly expressed on stimulated human MAIT cells (35). Conversely, in situations where riboﬂavin producing commensals are abundant, but the epithelium is intact, the MAIT cell will receive TCR stimulation alone, promoting a dominant homeostatic tissue repair response (35, 36). Therefore, approaches to manipulate MAIT cell biology may need to simultaneously target these danger signal pathways in addition to the MAIT TCR, for instance combining inhibition of TLR2 or IL17RE with inhibitory MAIT cell ligands to reduce inﬂammation. Theoretically MAIT TCR ligands could be used sequentially or at different stages of an inﬂammatory response, favoring antagonistic ligands with or without additional immunosuppressants to reduce overt inﬂammation, followed subsequently by agonistic ligands to support restored barrier integrity. Extensive modelling in experimental systems would be required before clinical trials could be considered. Frontiers in Immunology 2. Corbett AJ, Eckle SB, Birkinshaw RW, Liu L, Patel O, Mahony J, et al. T-Cell activation by transitory neo-antigens derived from distinct microbial pathways. Nature (2014) 509:361–5. doi: 10.1038/nature13160 1. Kjer-Nielsen L, Patel O, Corbett AJ, Le Nours J, Meehan B, Liu L, et al. MR1 presents microbial vitamin b metabolites to MAIT cells. Nature (2012) 491:717–23. doi: 10.1038/nature11605 Publisher’s note In human airways disease, long term antibiotic therapy with macrolide antibiotics has been shown to reduce inﬂammation and exacerbations of asthma (165). The mechanism is unknown, but it is postulated that reduction in bacteria such as Haemophilus inﬂuenzae colonizingtherespiratorymucosamayleadtoameliorationofmucosal inﬂammation. It is known that MAIT cells are deﬁcient in airways 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. Discussion and future directions A common theme within this expanding body of data is that dysbiosis at any mucosal surface can affect MAIT cell frequency and function, both locally and at distant sites, with dysbiosis typically associated with a decrease in circulating MAIT cell frequencies, but a relative increase in activated, IL-17-producing, pro-inﬂammatory MAIT cells. Conversely changes in MAIT cell frequency have also been shown to drive changes in the host microbiome, with evidence these can in turn have direct immunological consequences. Given the incredible diversity of bacteria within human microbiomes, full understanding of these complex interactions with the mucosal immune system is hard to achieve, and further research focusing in vivo on changes in deﬁned microbial species and their consequences for the host immune response are warranted. Nonetheless, already there is scope for investigating the potential for therapeutic manipulation of the microbiome or MAIT cell compartment. An alternative approach to therapeutic manipulation of MAIT cells would be altering the microbiome could be altered directly by Manipulation of the gut microbiome could be achieved using simple MR1 ligands. Indeed an in vivo murine study showed 12 frontiersin.org Jabeen and Hinks 10.3389/ﬁmmu.2023.1127588 the administration of probiotics which favor MAIT ligand producing microbiomes, and might thereby enhance the overall riboﬂavin-synthetic capacity of the fecal microbial community. The net effects of such an intervention are, however, unpredictable, due to complex symbiotic relationships between microbes, such that increasing riboﬂavin availability might, paradoxically, favor the growth of otherwise less dominant species which lack this synthetic pathway. The potential impact of a modulated MAIT microbiome to impact the host immune response was demonstrated in work by Toubal et al. (88) which showed that feces from MR1-/- mice triggered more MAIT cell activation than feces from MR1 sufﬁcient mice, suggesting MAIT cells are able to differentially sense microbiome assemblages which differ in riboﬂavin synthesis. The use of probiotics is controversial as a large proportion of bacteria fail to survive the gastric and upper GI environment, or to become signiﬁcantly established amongst the complex, diverse microbiome of the lower GI tract which compete for the same niche. Nonetheless it could be possible to engineer bacterial species to over-express riboﬂavin pathways to enhance MAIT cell activation of the entire microbial assemblage, which might for instance help accelerate reconstitution of mucosal immune cell populations after HSCT or HIV. Funding This work was supported by grants from MRC-Oxford Doctoral Training Programme (MJ), the Wellcome Trust (104553/z/14/z, 211050/Z/18/z) (TH) and the National Institute for Health Research (NIHR) Oxford Biomedical Research Centre(BRC) (MJ and TH). The views expressed are those of the authors and not those of the NHS or NIHR. Within the airways administration of MAIT cell ligands might be manipulated to enhance immune responses during vaccination, as a component of aerosolized vaccines, or as an adjunctive treatment for persistent microbial infection. Indeed, preliminary work has explored this in the context of chronic infection with Mycobacterium tuberculosis (M.tb) (164). During acute infection administration of 5- OP-RU did not enhance protective responses, but surprisingly rather delayed T cell priming through mechanisms dependent on MAIT cells and TGF-b. However, conversely, during chronic infection intrapulmonary 5-OP-RU administration drove a 30-fold MAIT cell expansion and an IL-17A-dependent 10-fold reduction in pulmonary bacterial loads. Of note this protective effect was local to the mucosa and did not affect bacterial load in the liver, implying the potential to manipulate mucosal MAIT cell populations selectively. 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. Author contributions MJ and TH jointly conceived the review, conducted the literature review, and drafted the manuscript. TH created the ﬁgures. All authors contributed to the article and approved the submitted version. Discussion and future directions The expected clinical effect of these will need to be determined using in vivo models because the ultimate effect on MAIT cell activation will be inﬂuenced by a number of factors which are hard to predict from in vitro experiments, including differing efﬁciencies of Rib pathway enzymes, interactions between riboﬂavin producing and riboﬂavin scavenging species and the extent to which other microbial factors trigger concomitant innate immune signaling. diseases (116, 129) so studies using direct airway tissue sampling should explore whether the MAIT cell populations are restored by antibiotics and whether this is associated with restoration of a homeostatic rather than proinﬂammatory MAIT cell phenotype. Simpler than both these approaches, and more amenable to very simple clinical trials would be assessment of the utility of topical application of MAIT cell ligands or riboﬂavin-synthesizing commensal bacteria of low invasive capacity to accelerate wound healing, as has already been demonstrated in mice with H2-M3 restricted commensal bacteria (37) and with topical 5-OP-RU (21). In the decade since the ﬁrst MR1 ligands were discovered (1) our knowledge of MAIT cell biology has expanded rapidly. Though there remain many unanswered questions, we should expect to see the ﬁeld start to progress to the next stage of clinical translation over the next 5 years. Simpler than both these approaches, and more amenable to very simple clinical trials would be assessment of the utility of topical application of MAIT cell ligands or riboﬂavin-synthesizing commensal bacteria of low invasive capacity to accelerate wound healing, as has already been demonstrated in mice with H2-M3 restricted commensal bacteria (37) and with topical 5-OP-RU (21). In the decade since the ﬁrst MR1 ligands were discovered (1) our knowledge of MAIT cell biology has expanded rapidly. Though there remain many unanswered questions, we should expect to see the ﬁeld start to progress to the next stage of clinical translation over the next 5 years. Frontiers in Immunology References Gold MC, Eid T, Smyk-Pearson S, Eberling Y, Swarbrick GM, Langley SM, et al. Human thymic MR1-restricted MAIT cells are innate pathogen-reactive effectors that adapt following thymic egress. Mucosal Immunol (2013) 6:35–44. doi: 10.1038/ mi.2012.45 37. Linehan JL, Harrison OJ, Han SJ, Byrd AL, Vujkovic-Cvijin I, Villarino AV, et al. Non-classical immunity controls microbiota impact on skin immunity and tissue repair. Cell (2018) 172:784–796.e718. doi: 10.1016/j.cell.2017.12.033 15. Lee CH, Zhang HH, Singh SP, Koo L, Kabat J, Tsang H, et al. C/EBPdelta drives interactions between human MAIT cells and endothelial cells that are important for extravasation. Elife (2018) 7. doi: 10.7554/eLife.32532 38. Chen Y, Chou K, Fuchs E, Havran WL, Boismenu R. Protection of the intestinal mucosa by intraepithelial gamma delta T cells. Proc Natl Acad Sci U.S.A. (2002) 99:14338–43. doi: 10.1073/pnas.212290499 16. Milner JJ, Toma C, Yu B, Zhang K, Omilusik K, Phan AT, et al. Runx3 programs CD8(+) T cell residency in non-lymphoid tissues and tumours. Nature (2017) 552:253– 7. doi: 10.1038/nature24993 39. King DP, Hyde DM, Jackson KA, Novosad DM, Ellis TN, Putney L, et al. Cutting edge: protective response to pulmonary injury requires gamma delta T lymphocytes. J Immunol (1999) 162:5033–6. doi: 10.4049/jimmunol.162.9.5033 17. Belkaid Y, Hand TW. Role of the microbiota in immunity and inﬂammation. Cell (2014) 157:121–41. doi: 10.1016/j.cell.2014.03.011 40. Guo XJ, Dash P, Crawford JC, Allen EK, Zamora AE, Boyd DF, et al. Lung gammadelta T cells mediate protective responses during neonatal inﬂuenza infection that are associated with type 2 immunity. Immunity (2018) 49:531–544.e536. doi: 10.1016/j.immuni.2018.07.011 18. Mondot S, Boudinot P, Lantz O. MAIT. MR1, microbes and riboﬂavin: a paradigm for the co-evolution of invariant TCRs and restricting MHCI-like molecules? Immunogenetics (2016) 68:537–48. doi: 10.1007/s00251-016-0927-9 41. Varelias A, Bunting MD, Ormerod KL, Koyama M, Olver SD, Straube J, et al. Recipient mucosal-associated invariant T cells control GVHD within the colon. J Clin Invest (2018) 128:1919–36. doi: 10.1172/JCI91646 19. Boudinot P, Mondot S, Jouneau L, Teyton L, Lefranc MP, Lantz O. Restricting nonclassical MHC genes coevolve with TRAV genes used by innate-like T cells in mammals. Proc Natl Acad Sci U.S.A. (2016) 113:E2983–2992. doi: 10.1073/ pnas.1600674113 42. Rouxel O, Da Silva J, Beaudoin L, Nel I, Tard C, Cagninacci L, et al. Cytotoxic and regulatory roles of mucosal-associated invariant T cells in type 1 diabetes. Nat Immunol (2017) 18:1321–31. doi: 10.1038/ni.3854 20. Amini A, Pang D, Hackstein CP, Klenerman P. References Molecular mechanisms of lineage decisions in metabolite-speciﬁc T cells. Nat Immunol (2019) 20:1244–55. doi: 10.1038/s41590-019-0465-3 32. Krause JL, Schape SS, Schattenberg F, Muller S, Ackermann G, Rolle-Kampczyk UE, et al. The activation of mucosal-associated invariant T (MAIT) cells is affected by microbial diversity and riboﬂavin utilization in vitro. Front Microbiol (2020) 11:755. doi: 10.3389/fmicb.2020.00755 8. Gapin L. Development of invariant natural killer T cells. Curr Opin Immunol (2016) 39:68–74. doi: 10.1016/j.coi.2016.01.001 9. Krovi SH, Loh L, Spengler A, Brunetti T, Gapin L. Current insights in mouse iNKT and MAIT cell development using single cell transcriptomics data. Semin Immunol (2022) 60:101658. doi: 10.1016/j.smim.2022.101658 33. Dias J, Leeansyah E, Sandberg JK. Multiple layers of heterogeneity and subset diversity in human MAIT cell responses to distinct microorganisms and to innate cytokines. Proc Natl Acad Sci U.S.A. (2017) 114:E5434–43. doi: 10.1073/ pnas.1705759114 10. Salou M, Legoux F, Lantz O. MAIT cell development in mice and humans. Mol Immunol (2021) 130:31–6. doi: 10.1016/j.molimm.2020.12.003 34. Lamichhane R, Schneider M, de la Harpe SM, Harrop TWR, Hannaway RF, Dearden PK, et al. TCR- or cytokine-activated CD8(+) mucosal-associated invariant T cells are rapid polyfunctional effectors that can coordinate immune responses. Cell Rep (2019) 28:3061–3076.e3065. doi: 10.1016/j.celrep.2019.08.054 11. Salou M, Legoux F, Gilet J, Darbois A, du Halgouet A, Alonso R, et al. A common transcriptomic program acquired in the thymus deﬁnes tissue residency of MAIT and NKT subsets. J Exp Med (2019) 216:133–51. doi: 10.1084/jem.20181483 12. Rahimpour A, Koay HF, Enders A, Clanchy R, Eckle SB, Meehan B, et al. Identiﬁcation of phenotypically and functionally heterogeneous mouse mucosal- associated invariant T cells using MR1 tetramers. J Exp Med (2015) 212:1095–108. doi: 10.1084/jem.20142110 35. Hinks TSC, Marchi E, Jabeen M, Olshansky M, Kurioka A, Pediongco TJ, et al. Activation and In vivo evolution of the MAIT cell transcriptome in mice and humans reveals tissue repair functionality. Cell Rep (2019) 28:3249–3262.e3245. doi: 10.1016/ j.celrep.2019.07.039 13. Ben Youssef G, Tourret M, Salou M, Ghazarian L, Houdouin V, Mondot S, et al. Ontogeny of human mucosal-associated invariant T cells and related T cell subsets. J Exp Med (2018) 215:459–79. doi: 10.1084/jem.20171739 36. Leng T, Akther HD, Hackstein CP, Powell K, King T, Friedrich M, et al. TCR and inﬂammatory signals tune human MAIT cells to exert speciﬁc tissue repair and effector functions. Cell Rep (2019) 28:3077–3091.e3075. doi: 10.1016/ j.celrep.2019.08.050 14. References MAIT cells in barrier tissues: Lessons from immediate neighbors. Front Immunol (2020) 11:584521. doi: 10.3389/ ﬁmmu.2020.584521 43. Wang H, Kjer-Nielsen L, Shi M, D'Souza C, Pediongco TJ, Cao H, et al. IL-23 costimulates antigen-speciﬁc MAIT cell activation and enables vaccination against bacterial infection. Sci Immunol (2019) 4. doi: 10.1126/sciimmunol.aaw0402 21. Constantinides MG, Link VM, Tamoutounour S, Wong AC, Perez-Chaparro PJ, Han SJ, et al. MAIT cells are imprinted by the microbiota in early life and promote tissue repair. Science (2019) 366. doi: 10.1126/science.aax6624 44. Lee DJ, Schnitzlein CW, Wolf JP, Vythilingam M, Rasmusson AM, Hoge CW. Psychotherapy versus pharmacotherapy for posttraumatic stress disorder: Systemic review and meta-analyses to determine ﬁrst-line treatments. Depress Anxiety (2016) 33:792–806. doi: 10.1002/da.22511 22. Kennedy EA, King KY, Baldridge MT. Mouse microbiota models: Comparing germ-free mice and antibiotics treatment as tools for modifying gut bacteria. Front Physiol (2018) 9:1534. doi: 10.3389/fphys.2018.01534 45. Haga K, Chiba A, Shibuya T, Osada T, Ishikawa D, Kodani T, et al. MAIT cells are activated and accumulated in the inﬂamed mucosa of ulcerative colitis. J Gastroenterol Hepatol (2016) 31:965–72. doi: 10.1111/jgh.13242 23. Legoux F, Bellet D, Daviaud C, El Morr Y, Darbois A, Niort K, et al. Microbial metabolites control the thymic development of mucosal-associated invariant T cells. Science (2019) 366:494–9. doi: 10.1126/science.aaw2719 46. Sobkowiak MJ, Davanian H, Heymann R, Gibbs A, Emgard J, Dias J, et al. Tissue-resident MAIT cell populations in human oral mucosa exhibit an activated proﬁle and produce IL-17. Eur J Immunol (2019) 49:133–43. doi: 10.1002/ eji.201847759 24. Gherardin NA, Souter MN, Koay HF, Mangas KM, Seemann T, Stinear TP, et al. Human blood MAIT cell subsets deﬁned using MR1 tetramers. Immunol Cell Biol (2018) 96:507–25. doi: 10.1111/imcb.12021 47. Rha MS, Yoon YH, Koh JY, Jung JH, Lee HS, Park SK, et al. IL-17A-producing sinonasal MAIT cells in patients with chronic rhinosinusitis with nasal polyps. J Allergy Clin Immunol (2022) 149:599–609.e597. doi: 10.1016/j.jaci.2021.07.037 25. Howson LJ, Awad W, von Borstel A, Lim HJ, McWilliam HEG, Sandoval- Romero ML, et al. Absence of mucosal-associated invariant T cells in a person with a homozygous point mutation in MR1. Sci Immunol (2020) 5. doi: 10.1126/ sciimmunol.abc9492 48. Khuzwayo S, Mthembu M, Meermeier EW, Prakadan SM, Kazer SW, Bassett T, et al. MR1-restricted MAIT cells from the human lung mucosal surface have distinct phenotypic, functional, and transcriptomic features that are preserved in HIV infection. Front Immunol (2021) 12:631410. doi: 10.3389/ﬁmmu.2021.631410 26. References Frontiers in Immunology frontiersin.org 13 10.3389/ﬁmmu.2023.1127588 Jabeen and Hinks Jabeen and Hinks 3. Koay HF, Gherardin NA, Enders A, Loh L, Mackay LK, Almeida CF, et al. A three-stage intrathymic development pathway for the mucosal-associated invariant T cell lineage. Nat Immunol (2016) 17:1300–11. doi: 10.1038/ni.3565 28. Martin CE, Spasova DS, Frimpong-Boateng K, Kim HO, Lee M, Kim KS, et al. Interleukin-7 availability is maintained by a hematopoietic cytokine sink comprising innate lymphoid cells and T cells. Immunity (2017) 47:171–182.e174. doi: 10.1016/ j.immuni.2017.07.005 28. Martin CE, Spasova DS, Frimpong-Boateng K, Kim HO, Lee M, Kim KS, et al. Interleukin-7 availability is maintained by a hematopoietic cytokine sink comprising innate lymphoid cells and T cells. Immunity (2017) 47:171–182.e174. doi: 10.1016/ j.immuni.2017.07.005 4. Koay HF, Su S, Amann-Zalcenstein D, Daley SR, Comerford I, Miosge L, et al. A divergent transcriptional landscape underpins the development and functional branching of MAIT cells. Sci Immunol (2019) 4. doi: 10.1126/sciimmunol.aay6039 29. Kedia-Mehta N, Finlay DK. Competition for nutrients and its role in controlling immune responses. Nat Commun (2019) 10:2123. doi: 10.1038/s41467-019-10015-4 29. Kedia-Mehta N, Finlay DK. Competition for nutrients and its role in controlling immune responses. Nat Commun (2019) 10:2123. doi: 10.1038/s41467-019-10015-4 5. Martin E, Treiner E, Duban L, Guerri L, Laude H, Toly C, et al. Stepwise development of MAIT cells in mouse and human. PloS Biol (2009) 7:e54. doi: 10.1371/ journal.pbio.1000054 30. Konuma T, Kohara C, Watanabe E, Takahashi S, Ozawa G, Suzuki K, et al. Reconstitution of circulating mucosal-associated invariant T cells after allogeneic hematopoietic cell transplantation: Its association with the riboﬂavin synthetic pathway of gut microbiota in cord blood transplant recipients. J Immunol (2020) 204:1462–73. doi: 10.4049/jimmunol.1900681 30. Konuma T, Kohara C, Watanabe E, Takahashi S, Ozawa G, Suzuki K, et al. Reconstitution of circulating mucosal-associated invariant T cells after allogeneic hematopoietic cell transplantation: Its association with the riboﬂavin synthetic pathway of gut microbiota in cord blood transplant recipients. J Immunol (2020) 204:1462–73. doi: 10.4049/jimmunol.1900681 6. Treiner E, Duban L, Bahram S, Radosavljevic M, Wanner V, Tilloy F, et al. Selection of evolutionarily conserved mucosal-associated invariant T cells by MR1. Nature (2003) 422:164–9. doi: 10.1038/nature01433 31. Tastan C, Karhan E, Zhou W, Fleming E, Voigt AY, Yao X, et al. Tuning of human MAIT cell activation by commensal bacteria species and MR1-dependent T-cell presentation. Mucosal Immunol (2018) 11:1591–605. doi: 10.1038/s41385-018-0072-x 7. Legoux F, Gilet J, Procopio E, Echasserieau K, Bernardeau K, Lantz O. Jabeen and Hinks References Massier L, Chakaroun R, Tabei S, Crane A, Didt KD, Fallmann J, et al. Adipose tissue derived bacteria are associated with inﬂammation in obesity and type 2 diabetes. Gut (2020) 69:1796–806. doi: 10.1136/gutjnl-2019-320118 62. Dou Y, Maurer K, Conrad M, Patel T, Shraim R, Sullivan KE, et al. Mucosal invariant T cells are diminished in very early-onset inﬂammatory bowel disease. J Pediatr Gastroenterol Nutr (2021) 73:529–36. doi: 10.1097/MPG.0000000000003189 86. Anhe FF, Jensen BAH, Varin TV, Servant F, Van Blerk S, Richard D, et al. Type 2 diabetes inﬂuences bacterial tissue compartmentalisation in human obesity. Nat Metab (2020) 2:233–42. doi: 10.1038/s42255-020-0178-9 63. Rosati E, Pogorelyy MV, Dowds CM, Moller FT, Sorensen SB, Lebedev YB, et al. Identiﬁcation of disease-associated traits and clonotypes in the T cell receptor repertoire of monozygotic twins affected by inﬂammatory bowel diseases. J Crohns Colitis (2020) 14:778–90. doi: 10.1093/ecco-jcc/jjz179 87. Jayashree B, Bibin YS, Prabhu D, Shanthirani CS, Gokulakrishnan K, Lakshmi BS, et al. Increased circulatory levels of lipopolysaccharide (LPS) and zonulin signify novel biomarkers of proinﬂammation in patients with type 2 diabetes. Mol Cell Biochem (2014) 388:203–10. doi: 10.1007/s11010-013-1911-4 64. Serriari NE, Eoche M, Lamotte L, Lion J, Fumery M, Marcelo P, et al. Innate mucosal-associated invariant T (MAIT) cells are activated in inﬂammatory bowel diseases. Clin Exp Immunol (2014) 176:266–74. doi: 10.1111/cei.12277 88. Toubal A, Kiaf B, Beaudoin L, Cagninacci L, Rhimi M, Fruchet B, et al. Mucosal- associated invariant T cells promote inﬂammation and intestinal dysbiosis leading to metabolic dysfunction during obesity. Nat Commun (2020) 11:3755. doi: 10.1038/ s41467-020-17307-0 65. Yasutomi Y, Chiba A, Haga K, Murayama G, Makiyama A, Kuga T, et al. Activated mucosal-associated invariant T cells have a pathogenic role in a murine model of inﬂammatory bowel disease. Cell Mol Gastroenterol Hepatol (2022) 13:81–93. doi: 10.1016/j.jcmgh.2021.08.018 89. Magalhaes I, Pingris K, Poitou C, Bessoles S, Venteclef N, Kiaf B, et al. Mucosal- associated invariant T cell alterations in obese and type 2 diabetic patients. J Clin Invest (2015) 125:1752–62. doi: 10.1172/JCI78941 66. Meroni E, Stakenborg N, Gomez-Pinilla PJ, De Hertogh G, Goverse G, Matteoli G, et al. Functional characterization of oxazolone-induced colitis and survival improvement by vagus nerve stimulation. PloS One (2018) 13:e0197487. doi: 10.1371/journal.pone.0197487 90. Li Y, Yang Y, Wang J, Cai P, Li M, Tang X, et al. Bacteroides ovatus-mediated CD27(-) MAIT cell activation is associated with obesity-related T2D progression. Cell Mol Immunol (2022) 19:791–804. doi: 10.1038/s41423-022-00871-4 67. References Park JY, Won HY, DiPalma DT, Kim HK, Kim TH, Li C, et al. In vivo availability of the cytokine IL-7 constrains the survival and homeostasis of peripheral iNKT cells. Cell Rep (2022) 38:110219. doi: 10.1016/j.celrep.2021.110219 49. Gibbs A, Leeansyah E, Introini A, Paquin-Proulx D, Hasselrot K, Andersson E, et al. MAIT cells reside in the female genital mucosa and are biased towards IL-17 and IL-22 production in response to bacterial stimulation. Mucosal Immunol (2017) 10:35– 45. doi: 10.1038/mi.2016.30 27. Baccala R, Witherden D, Gonzalez-Quintial R, Dummer W, Surh CD, Havran WL, et al. Gamma delta T cell homeostasis is controlled by IL-7 and IL-15 together with subset-speciﬁc factors. J Immunol (2005) 174:4606–12. doi: 10.4049/ jimmunol.174.8.4606 Frontiers in Immunology frontiersin.org 14 10.3389/ﬁmmu.2023.1127588 Jabeen and Hinks Jabeen and Hinks 10.3389/ﬁmmu.2023.1127588 75. Betrapally NS, Gillevet PM, Bajaj JS. Changes in the intestinal microbiome and alcoholic and nonalcoholic liver diseases: Causes or effects? Gastroenterology (2016) 150:1745–1755.e1743. doi: 10.1053/j.gastro.2016.02.073 50. Schmaler M, Colone A, Spagnuolo J, Zimmermann M, Lepore M, Kalinichenko A, et al. Modulation of bacterial metabolism by the microenvironment controls MAIT cell stimulation. Mucosal Immunol (2018) 11:1060–70. doi: 10.1038/s41385-018-0020-9 51. Voillet V, Buggert M, Slichter CK, Berkson JD, Mair F, Addison MM, et al. Human MAIT cells exit peripheral tissues and recirculate via lymph in steady state conditions. JCI Insight (2018) 3. doi: 10.1172/jci.insight.98487 76. Riazi K, Azhari H, Charette JH, Underwood FE, King JA, Afshar EE, et al. The prevalence and incidence of NAFLD worldwide: a systematic review and meta-analysis. Lancet Gastroenterol Hepatol (2022) 7:851–61. doi: 10.1016/S2468-1253(22)00165-0 52. Mechelli R, Romano S, Romano C, Morena E, Buscarinu MC, Bigi R, et al. And microbiota in multiple sclerosis and other autoimmune diseases. Microorganisms (2021) 9. doi: 10.3390/microorganisms9061132 77. Li Y, Huang B, Jiang X, Chen W, Zhang J, Wei Y, et al. Mucosal-associated invariant T cells improve nonalcoholic fatty liver disease through regulating macrophage polarization. Front Immunol (2018) 9:1994. doi: 10.3389/ ﬁmmu.2018.01994 53. Ley RE, Turnbaugh PJ, Klein S, Gordon JI. Microbial ecology: human gut microbes associated with obesity. Nature (2006) 444:1022–3. doi: 10.1038/4441022a 78. Hegde P, Weiss E, Paradis V, Wan J, Mabire M, Sukriti S, et al. Mucosal- associated invariant T cells are a proﬁbrogenic immune cell population in the liver. Nat Commun (2018) 9:2146. doi: 10.1038/s41467-018-04450-y 54. Arumugam M, Raes J, Pelletier E, Le Paslier D, Yamada T, Mende DR, et al. Enterotypes of the human gut microbiome. References Nature (2011) 473:174–80. doi: 10.1038/ nature09944 79. Yu X, Jin Y, Zhou W, Xiao T, Wu Z, Su J, et al. Rifaximin modulates the gut microbiota to prevent hepatic encephalopathy in liver cirrhosis without impacting the resistome. Front Cell Infect Microbiol (2021) 11:761192. doi: 10.3389/ fcimb.2021.761192 55. Chu H, Mazmanian SK. Innate immune recognition of the microbiota promotes host-microbial symbiosis. Nat Immunol (2013) 14:668–75. doi: 10.1038/ni.2635 56. Smith AD, Foss ED, Zhang I, Hastie JL, Giordano NP, Gasparyan L, et al. Microbiota of MR1 deﬁcient mice confer resistance against clostridium difﬁcile infection. PloS One (2019) 14:e0223025. doi: 10.1371/journal.pone.0223025 80. Llopis M, Cassard AM, Wrzosek L, Boschat L, Bruneau A, Ferrere G, et al. Intestinal microbiota contributes to individual susceptibility to alcoholic liver disease. Gut (2016) 65:830–9. doi: 10.1136/gutjnl-2015-310585 57. Guo J, Song C, Liu Y, Wu X, Dong W, Zhu H, et al. Characteristics of gut microbiota in representative mice strains: Implications for biological research. Anim Model Exp Med (2022) 5:337–49. doi: 10.1002/ame2.12257 81. Mutlu EA, Gillevet PM, Rangwala H, Sikaroodi M, Naqvi A, Engen PA, et al. Colonic microbiome is altered in alcoholism. Am J Physiol Gastrointest Liver Physiol (2012) 302:G966–978. doi: 10.1152/ajpgi.00380.2011 58. Franzosa EA, Sirota-Madi A, Avila-Pacheco J, Fornelos N, Haiser HJ, Reinker S, et al. Gut microbiome structure and metabolic activity in inﬂammatory bowel disease. Nat Microbiol (2019) 4:293–305. doi: 10.1038/s41564-018-0306-4 82. Gu M, Samuelson DR, Taylor CM, Molina PE, Luo M, Siggins RW, et al. Alcohol-associated intestinal dysbiosis alters mucosal-associated invariant T-cell phenotype and function. Alcohol Clin Exp Res (2021) 45:934–47. doi: 10.1111/ acer.14589 59. Lee M, Chang EB. Inﬂammatory bowel diseases (IBD) and the microbiome- searching the crime scene for clues. Gastroenterology (2021) 160:524–37. doi: 10.1053/ j.gastro.2020.09.056 83. Riva A, Patel V, Kurioka A, Jeffery HC, Wright G, Tarff S, et al. Mucosa- associated invariant T cells link intestinal immunity with antibacterial immune defects in alcoholic liver disease. Gut (2018) 67:918–30. doi: 10.1136/gutjnl-2017-314458 60. Qiu P, Ishimoto T, Fu L, Zhang J, Zhang Z, Liu Y. The gut microbiota in inﬂammatory bowel disease. Front Cell Infect Microbiol (2022) 12:733992. doi: 10.3389/ fcimb.2022.733992 84. Turnbaugh PJ, Ley RE, Mahowald MA, Magrini V, Mardis ER, Gordon JI. An obesity-associated gut microbiome with increased capacity for energy harvest. Nature (2006) 444:1027–31. doi: 10.1038/nature05414 61. Glassner KL, Abraham BP, Quigley EMM. The microbiome and inﬂammatory bowel disease. J Allergy Clin Immunol (2020) 145:16–27. doi: 10.1016/j.jaci.2019.11.003 85. References Globig AM, Hipp AV, Otto-Mora P, Heeg M, Mayer LS, Ehl S, et al. High- dimensional proﬁling reveals Tc17 cell enrichment in active crohn's disease and identiﬁes a potentially targetable signature. Nat Commun (2022) 13:3688. doi: 10.1038/s41467-022-31229-z 91. Carolan E, Tobin LM, Mangan BA, Corrigan M, Gaoatswe G, Byrne G, et al. Altered distribution and increased IL-17 production by mucosal-associated invariant T cells in adult and childhood obesity. J Immunol (2015) 194:5775–80. doi: 10.4049/ jimmunol.1402945 68. Rosati E, Rios Martini G, Pogorelyy MV, Minervina AA, Degenhardt F, Wendorff M, et al. A novel unconventional T cell population enriched in crohn's disease. Gut (2022) 71:2194–204. doi: 10.1136/gutjnl-2021-325373 92. O'Brien A, Loftus RM, Pisarska MM, Tobin LM, Bergin R, Wood NAW, et al. Obesity reduces mTORC1 activity in mucosal-associated invariant T cells, driving defective metabolic and functional responses. J Immunol (2019) 202:3404–11. doi: 10.4049/jimmunol.1801600 69. O'Connor WJr., Kamanaka M, Booth CJ, Town T, Nakae S, Iwakura Y, et al. A protective function for interleukin 17A in T cell-mediated intestinal inﬂammation. Nat Immunol (2009) 10:603–9. doi: 10.1038/ni.1736 93. Fukuda N, Ojima T, Hayata K, Katsuda M, Kitadani J, Takeuchi A, et al. Laparoscopic sleeve gastrectomy for morbid obesity improves gut microbiota balance, increases colonic mucosal-associated invariant T cells and decreases circulating regulatory T cells. Surg Endosc (2022) 36:7312–24. doi: 10.1007/s00464-022-09122-z 70. O'Connor WJr., Zenewicz LA, Flavell RA. The dual nature of T(H)17 cells: shifting the focus to function. Nat Immunol (2010) 11:471–6. doi: 10.1038/ni.1882 71. Hueber W, Sands BE, Lewitzky S, Vandemeulebroecke M, Reinisch W, Higgins PD, et al. Secukinumab in crohn's disease study g. secukinumab, a human anti-IL-17A monoclonal antibody, for moderate to severe crohn's disease: unexpected results of a randomised, double-blind placebo-controlled trial. Gut (2012) 61:1693–700. doi: 10.1136/gutjnl-2011-301668 94. Andrlova H, van den Brink MRM, Markey KA. An unconventional view of T cell reconstitution after allogeneic hematopoietic cell transplantation. Front Oncol (2020) 10:608923. doi: 10.3389/fonc.2020.608923 95. Peled JU, Gomes ALC, Devlin SM, Littmann ER, Taur Y, Sung AD, et al. Microbiota as predictor of mortality in allogeneic hematopoietic-cell transplantation. N Engl J Med (2020) 382:822–34. doi: 10.1056/NEJMoa1900623 72. Treiner E. Mucosal-associated invariant T cells in inﬂammatory bowel diseases: bystanders, defenders, or offenders? Front Immunol (2015) 6:27. doi: 10.3389/ ﬁmmu.2015.00027 96. Jenq RR, Taur Y, Devlin SM, Ponce DM, Goldberg JD, Ahr KF, et al. Intestinal blautia is associated with reduced death from graft-versus-Host disease. Biol Blood Marrow Transplant (2015) 21:1373–83. Jabeen and Hinks References Lancet (2014) 384:691–702. doi: 10.1016/ S0140-6736(14)61136-3 102. Leeansyah E, Ganesh A, Quigley MF, Sonnerborg A, Andersson J, Hunt PW, et al. Activation, exhaustion, and persistent decline of the antimicrobial MR1-restricted MAIT-cell population in chronic HIV-1 infection. Blood (2013) 121:1124–35. doi: 10.1182/blood-2012-07-445429 125. Schmidt A, Belaaouaj A, Bissinger R, Koller G, Malleret L, D'Orazio C, et al. Neutrophil elastase-mediated increase in airway temperature during inﬂammation. J Cyst Fibros (2014) 13:623–31. doi: 10.1016/j.jcf.2014.03.004 126. Worlitzsch D, Tarran R, Ulrich M, Schwab U, Cekici A, Meyer KC, et al. Effects of reduced mucus oxygen concentration in airway pseudomonas infections of cystic ﬁbrosis patients. J Clin Invest (2002) 109:317–25. doi: 10.1172/JCI0213870 103. Eberhard JM, Hartjen P, Kummer S, Schmidt RE, Bockhorn M, Lehmann C, et al. CD161+ MAIT cells are severely reduced in peripheral blood and lymph nodes of HIV-infected individuals independently of disease progression. PloS One (2014) 9: e111323. doi: 10.1371/journal.pone.0111323 127. Diseases GBD, Injuries C. Global burden of 369 diseases and injuries in 204 countries and territories, 1990-2019: a systematic analysis for the global burden of disease study 2019. Lancet (2020) 396:1204–22. doi: 10.1016/S0140-6736(20)30925-9 104. Fernandez CS, Amarasena T, Kelleher AD, Rossjohn J, McCluskey J, Godfrey DI, et al. MAIT cells are depleted early but retain functional cytokine expression in HIV infection. Immunol Cell Biol (2015) 93:177–88. doi: 10.1038/icb.2014.91 128. Agusti A, Vogelmeier CF, Halpin DMG. Tackling the global burden of lung disease through prevention and early diagnosis. Lancet Respir Med (2022) 10:1013–5. doi: 10.1016/S2213-2600(22)00302-2 105. Hengst J, Strunz B, Deterding K, Ljunggren HG, Leeansyah E, Manns MP, et al. Nonreversible MAIT cell-dysfunction in chronic hepatitis c virus infection despite successful interferon-free therapy. Eur J Immunol (2016) 46:2204–10. doi: 10.1002/ eji.201646447 129. Hinks TS, Wallington JC, Williams AP, Djukanovic R, Staples KJ, Wilkinson TM. Steroid-induced deﬁciency of mucosal-associated invariant T cells in the chronic obstructive pulmonary disease lung. implications for nontypeable haemophilus inﬂuenzae infection. Am J Respir Crit Care Med (2016) 194:1208–18. doi: 10.1164/ rccm.201601-0002OC 106. BolteFJ,O'KeefeAC,WebbLM,SertiE,RiveraE,LiangTJ,etal.Intra-hepaticdepletion of mucosal-associated invariant T cells in hepatitis c virus-induced liver inﬂammation. Gastroenterology (2017) 153:1392–1403.e1392. doi: 10.1053/j.gastro.2017.07.043 130. Pavord ID, Beasley R, Agusti A, Anderson GP, Bel E, Brusselle G, et al. After asthma: redeﬁning airways diseases. Lancet (2018) 391(10118):350–400. doi: 10.1016/ S0140-6736(17)30879-6 107. Barathan M, Mohamed R, Vadivelu J, Chang LY, Saeidi A, Yong YK, et al. Peripheral loss of CD8(+) CD161(++) TCRValpha7.2(+) mucosal-associated invariant T cells in chronic hepatitis c virus-infected patients. References Eur J Clin Invest (2016) 46:170–80. doi: 10.1111/eci.12581 131. Singhania A, Rupani H, Jayasekera N, Lumb S, Hales P, Gozzard N, et al. Altered epithelial gene expression in peripheral airways of severe asthma. PloS One (2017) 12:e0168680. doi: 10.1371/journal.pone.0168680 108. Merlini E, Cerrone M, van Wilgenburg B, Swadling L, Cannizzo ES, d'Arminio Monforte A, et al. Association between impaired Valpha7.2+CD161++CD8+ (MAIT) and Valpha7.2+CD161-CD8+ T-cell populations and gut dysbiosis in chronically HIV- and/or HCV-infected patients. Front Microbiol (2019) 10:1972. doi: 10.3389/ fmicb.2019.01972 132. Woodruff PG, Boushey HA, Dolganov GM, Barker CS, Yang YH, Donnelly S, et al. Genome-wide proﬁling identiﬁes epithelial cell genes associated with asthma and with treatment response to corticosteroids. Proc Natl Acad Sci U.S.A. (2007) 104:15858–63. doi: 10.1073/pnas.0707413104 109. Khlaiphuengsin A, Chuaypen N, Sodsai P, Reantragoon R, Han WM, Avihingsanon A, et al. Successful direct-acting antiviral therapy improves circulating mucosal-associated invariant T cells in patients with chronic HCV infection. PloS One (2020) 15:e0244112. doi: 10.1371/journal.pone.0244112 133. Bullone M, Carriero V, Bertolini F, Folino A, Mannelli A, Di Stefano A, et al. Elevated serum IgE, oral corticosteroid dependence and IL-17/22 expression in highly neutrophilic asthma. Eur Respir J (2019) 54. doi: 10.1183/13993003.00068-2019 134. Bisgaard H, Hermansen MN, Buchvald F, Loland L, Halkjaer LB, Bonnelykke K, et al. Childhood asthma after bacterial colonization of the airway in neonates. N Engl J Med (2007) 357:1487–95. doi: 10.1056/NEJMoa052632 110. Ishizaka A, Koga M, Mizutani T, Parbie PK, Prawisuda D, Yusa N, et al. Unique gut microbiome in HIV patients on antiretroviral therapy (ART) suggests association with chronic inﬂammation. Microbiol Spectr (2021) 9:e0070821. doi: 10.1128/ Spectrum.00708-21 135. Simpson JL, Daly J, Baines KJ, Yang IA, Upham JW, Reynolds PN, et al. Airway dysbiosis: Haemophilus inﬂuenzae and tropheryma in poorly controlled asthma. Eur Respir J (2016) 47:792–800. doi: 10.1183/13993003.00405-2015 111. Honda T, Ishigami M, Yamamoto K, Takeyama T, Ito T, Ishizu Y, et al. Changes in the gut microbiota after hepatitis c virus eradication. Sci Rep (2021) 11:23568. doi: 10.1038/s41598-021-03009-0 136. Green BJ, Wiriyachaiporn S, Grainge C, Rogers GB, Kehagia V, Lau L, et al. Potentially pathogenic airway bacteria and neutrophilic inﬂammation in treatment resistant severe asthma. PloS One (2014) 9:e100645. doi: 10.1371/journal.pone.0100645 112. Bajaj JS, Sterling RK, Betrapally NS, Nixon DE, Fuchs M, Daita K, et al. HCV eradication does not impact gut dysbiosis or systemic inﬂammation in cirrhotic patients. Aliment Pharmacol Ther (2016) 44:638–43. doi: 10.1111/apt.13732 137. References Goleva E, Jackson LP, Harris JK, Robertson CE, Sutherland ER, Hall CF, et al. The effects of airway microbiome on corticosteroid responsiveness in asthma. Am J Respir Crit Care Med (2013) 188:1193–201. doi: 10.1164/rccm.201304-0775OC 113. Twigg HL, Knox KS, Zhou J, Crothers KA, Nelson DE, Toh E, et al. Effect of advanced HIV infection on the respiratory microbiome. Am J Respir Crit Care Med (2016) 194:226–35. doi: 10.1164/rccm.201509-1875OC 138. Singhania A, Wallington JC, Smith CG, Horowitz D, Staples KJ, Howarth PH, et al. Multi-tissue transcriptomics delineates the diversity of airway T cell functions in asthma. Am J Respir Cell Mol Biol (2017) 58(2):261–70. doi: 10.1165/rcmb.2017- 0162OC 114. Man WH, de Steenhuijsen Piters WA, Bogaert D. The microbiota of the respiratory tract: Gatekeeper to respiratory health. Nat Rev Microbiol (2017) 15:259–70. doi: 10.1038/nrmicro.2017.14 139. Carlier FM, de Fays C, Pilette C. Epithelial barrier dysfunction in chronic respiratory diseases. Front Physiol (2021) 12:691227. doi: 10.3389/fphys.2021.691227 115. Hilty M, Burke C, Pedro H, Cardenas P, Bush A, Bossley C, et al. Disordered microbial communities in asthmatic airways. PloS One (2010) 5:e8578. doi: 10.1371/ journal.pone.0008578 140. Teo SM, Mok D, Pham K, Kusel M, Serralha M, Troy N, et al. The infant nasopharyngeal microbiome impacts severity of lower respiratory infection and risk of asthma development. Cell Host Microbe (2015) 17:704–15. doi: 10.1016/ j.chom.2015.03.008 116. Hinks TS, Zhou X, Staples KJ, Dimitrov BD, Manta A, Petrossian T, et al. Innate and adaptive T cells in asthmatic patients: Relationship to severity and disease mechanisms. J Allergy Clin Immunol (2015) 136:323–33. doi: 10.1016/j.jaci.2015.01.014 141. Chandra S, Wingender G, Greenbaum JA, Khurana A, Gholami AM, Ganesan AP, et al. Development of asthma in inner-city children: Possible roles of MAIT cells and variation in the home environment. J Immunol (2018) 200:1995–2003. doi: 10.4049/jimmunol.1701525 117. Meermeier EW, Zheng CL, Tran JG, Soma S, Worley AH, Weiss DI, et al. Human lung-resident mucosal-associated invariant T cells are abundant, express antimicrobial proteins, and are cytokine responsive. Commun Biol (2022) 5:942. doi: 10.1038/s42003-022-03823-w 142. Lezmi G, Abou-Taam R, Garcelon N, Dietrich C, Machavoine F, Delacourt C, et al. Evidence for a MAIT-17-high phenotype in children with severe asthma. J Allergy Clin Immunol (2019) 144:1714–1716 e1716. doi: 10.1016/j.jaci.2019.08.003 118. Dickson RP, Erb-Downward JR, Freeman CM, McCloskey L, Beck JM, Huffnagle GB, et al. Spatial variation in the healthy human lung microbiome and the adapted island model of lung biogeography. References doi: 10.1016/j.bbmt.2015.04.016 73. Balmer ML, Slack E, de Gottardi A, Lawson MA, Hapfelmeier S, Miele L, et al. The liver may act as a ﬁrewall mediating mutualism between the host and its gut commensal microbiota. Sci Transl Med (2014) 6:237ra266. doi: 10.1126/ scitranslmed.3008618 97. Taur Y, Jenq RR, Perales MA, Littmann ER, Morjaria S, Ling L, et al. The effects of intestinal tract bacterial diversity on mortality following allogeneic hematopoietic stem cell transplantation. Blood (2014) 124:1174–82. doi: 10.1182/blood-2014-02- 554725 74. Kurioka A, Walker LJ, Klenerman P, Willberg CB. MAIT cells: new guardians of the liver. Clin Transl Immunol (2016) 5:e98. doi: 10.1038/cti.2016.51 Frontiers in Immunology 15 frontiersin.org 10.3389/ﬁmmu.2023.1127588 10.3389/ﬁmmu.2023.1127588 Jabeen and Hinks Jabeen and Hinks 98. Bhattacharyya A, HanaﬁLA, Sheih A, Golob JL, Srinivasan S, Boeckh MJ, et al. Graft-derived reconstitution of mucosal-associated invariant T cells after allogeneic hematopoietic cell transplantation. Biol Blood Marrow Transplant (2018) 24:242–51. doi: 10.1016/j.bbmt.2017.10.003 120. Fazlollahi M, Lee TD, Andrade J, Oguntuyo K, Chun Y, Grishina G, et al. The nasal microbiome in asthma. J Allergy Clin Immunol (2018) 142:834–843.e832. doi: 10.1016/j.jaci.2018.02.020 121. Chun Y, Do A, Grishina G, Arditi Z, Ribeiro V, Grishin A, et al. The nasal microbiome, nasal transcriptome, and pet sensitization. J Allergy Clin Immunol (2021) 148:244–249 e244. doi: 10.1016/j.jaci.2021.01.031 99. Gao MG, Hong Y, Zhao XY, Pan XA, Sun YQ, Kong J, et al. The potential roles of mucosa-associated invariant T cells in the pathogenesis of gut graft-Versus-Host disease after hematopoietic stem cell transplantation. Front Immunol (2021) 12:720354. doi: 10.3389/ﬁmmu.2021.720354 122. Chun Y, Do A, Grishina G, Grishin A, Fang G, Rose S, et al. Integrative study of the upper and lower airway microbiome and transcriptome in asthma. JCI Insight (2020) 5. doi: 10.1172/jci.insight.133707 100. Andrlova H, Miltiadous O, Kousa AI, Dai A, DeWolf S, Violante S, et al. MAIT and Vdelta2 unconventional T cells are supported by a diverse intestinal microbiome and correlate with favorable patient outcome after allogeneic HCT. Sci Transl Med (2022) 14:eabj2829. doi: 10.1126/scitranslmed.abj2829 123. Dickson RP, Erb-Downward JR, Martinez FJ, Huffnagle GB. The microbiome and the respiratory tract. Annu Rev Physiol (2016) 78:481–504. doi: 10.1146/annurev- physiol-021115-105238 101. Cosgrove C, Ussher JE, Rauch A, Gartner K, Kurioka A, Huhn MH, et al. Early and nonreversible decrease of CD161++ /MAIT cells in HIV infection. Blood (2013) 121:951–61. doi: 10.1182/blood-2012-06-436436 124. Dickson RP, Martinez FJ, Huffnagle GB. The role of the microbiome in exacerbations of chronic lung diseases. Frontiers in Immunology Jabeen and Hinks References Ann Am Thorac Soc (2015) 12:821–30. doi: 10.1513/AnnalsATS.201501-029OC 143. Lezmi G, Abou Taam R, Dietrich C, Chatenoud L, de Blic J, Leite-de-Moraes M. Circulating IL-17-producing mucosal-associated invariant T cells (MAIT) are associated with symptoms in children with asthma. Clin Immunol (2018) 188:7–11. doi: 10.1016/j.clim.2017.11.009 119. Davanian H, Gaiser RA, Silfverberg M, Hugerth LW, Sobkowiak MJ, Lu L, et al. Mucosal-associated invariant T cells and oral microbiome in persistent apical periodontitis. Int J Oral Sci (2019) 11:16. doi: 10.1038/s41368-019-0049-y 119. Davanian H, Gaiser RA, Silfverberg M, Hugerth LW, Sobkowiak MJ, Lu L, et al. Mucosal-associated invariant T cells and oral microbiome in persistent apical periodontitis. Int J Oral Sci (2019) 11:16. doi: 10.1038/s41368-019-0049-y Frontiers in Immunology 16 frontiersin.org 10.3389/ﬁmmu.2023.1127588 10.3389/ﬁmmu.2023.1127588 Jabeen and Hinks Jabeen and Hinks 144. Taylor SL, Leong LEX, Choo JM, Wesselingh S, Yang IA, Upham JW, et al. Inﬂammatory phenotypes in patients with severe asthma are associated with distinct airway microbiology. J Allergy Clin Immunol (2018) 141:94–103.e115. doi: 10.1016/ j.jaci.2017.03.044 155. Huber ME, Larson E, Lust TN, Heisler CM, Harriff MJ. Chronic obstructive pulmonary disease and cigarette smoke lead to dysregulated MAIT cell activation. Am J Respir Cell Mol Biol (2022) 68(1):90–102. doi: 10.1101/2022.02.28.482383 156. Li J, Reantragoon R, Kostenko L, Corbett AJ, Varigos G, Carbone FR. The frequency of mucosal-associated invariant T cells is selectively increased in dermatitis herpetiformis. Australas J Dermatol (2017) 58:200–4. doi: 10.1111/ajd.12456 145. Jabeen MF, Sanderson ND, Foster D, Crook DW, Cane JL, Borg C, et al. Identifying bacterial airways infection in stable severe asthma using Oxford nanopore sequencing technologies. Microbiol Spectr (2022) 10:e0227921. doi: 10.1128/spectrum.02279-21 157. Sinha S, Lin G, Ferenczi K. The skin microbiome and the gut-skin axis. Clin Dermatol (2021) 39:829–39. doi: 10.1016/j.clindermatol.2021.08.021 146. Wen X, Nian S, Wei G, Kang P, Yang Y, Li L, et al. Changes in the phenotype and function of mucosal-associated invariant T cells in neutrophilic asthma. Int Immunopharmacol (2022) 106:108606. doi: 10.1016/j.intimp.2022.108606 158. Chen L, Li J, Zhu W, Kuang Y, Liu T, Zhang W, et al. Skin and gut microbiome in psoriasis: Gaining insight into the pathophysiology of it and ﬁnding novel therapeutic strategies. Front Microbiol (2020) 11:589726. doi: 10.3389/ fmicb.2020.589726 147. Ishimori A, Harada N, Chiba A, Harada S, Matsuno K, Makino F, et al. Circulating activated innate lymphoid cells and mucosal-associated invariant T cells are associated with airﬂow limitation in patients with asthma. Allergol Int (2017) 66:302–9. doi: 10.1016/j.alit.2016.07.005 159. References Cooper AJR, Clegg J, Cassidy FC, Hogan AE, McLoughlin RM. Human MAIT cells respond to staphylococcus aureus with enhanced anti-bacterial activity. Microorganisms (2022) 10. doi: 10.3390/microorganisms10010148 148. Ye L, Pan J, Pasha MA, Shen X, D'Souza SS, Fung ITH, et al. Mucosal- associated invariant T cells restrict allergic airway inﬂammation. J Allergy Clin Immunol (2020) 145:1469–1473.e1464. doi: 10.1016/j.jaci.2019.12.891 160. Teunissen MBM, Yeremenko NG, Baeten DLP, Chielie S, Spuls PI, de Rie MA, et al. The IL-17A-producing CD8+ T-cell population in psoriatic lesional skin comprises mucosa-associated invariant T cells and conventional T cells. J Invest Dermatol (2014) 134:2898–907. doi: 10.1038/jid.2014.261 149. Hoenderdos K, Condliffe A. The neutrophil in chronic obstructive pulmonary disease. Am J Respir Cell Mol Biol (2013) 48:531–9. doi: 10.1165/rcmb.2012-0492TR 161. Fyhrquist N, Muirhead G, Prast-Nielsen S, Jeanmougin M, Olah P, Skoog T, et al. Microbe-host interplay in atopic dermatitis and psoriasis. Nat Commun (2019) 10:4703. doi: 10.1038/s41467-019-12253-y 150. Diver S, Richardson M, Haldar K, Ghebre MA, Ramsheh MY, Bafadhel M, et al. Sputummicrobiomicclusteringinasthmaandchronicobstructivepulmonarydiseaserevealsa haemophilus-predominant subgroup. Allergy (2020) 75:808–17. doi: 10.1111/all.14058 151. Mayhew D, Devos N, Lambert C, Brown JR, Clarke SC, Kim VL, et al. Longitudinal proﬁling of the lung microbiome in the AERIS study demonstrates repeatability of bacterial and eosinophilic COPD exacerbations. Thorax (2018) 73:422–30. doi: 10.1136/thoraxjnl-2017-210408 162. Naidoo K, Woods K, Pelleﬁgues C, Cait A, O'Sullivan D, Gell K, et al. MR1- dependent immune surveillance of the skin contributes to pathogenesis and is a photobiological target of UV light therapy in a mouse model of atopic dermatitis. Allergy (2021) 76:3155–70. doi: 10.1111/all.14994 152. Qiu W, Kang N, Wu Y, Cai Y, Xiao L, Ge H, et al. Mucosal associated invariant T cells were activated and polarized toward Th17 in chronic obstructive pulmonary disease. Front Immunol (2021) 12:640455. doi: 10.3389/ﬁmmu.2021.640455 163. Hynes GM, Hinks TSC. The role of interleukin-17 in asthma: A protective response? ERJ Open Res (2020) 6. doi: 10.1183/23120541.00364-2019 164. Sakai S, Kauffman KD, Oh S, Nelson CE, Barry CE, Barber DL. MAIT cell- directed therapy of mycobacterium tuberculosis infection. Mucosal Immunol (2021) 14:199–208. doi: 10.1038/s41385-020-0332-4 153. Kwon YS, Jin HM, Cho YN, Kim MJ, Kang JH, Jung HJ, et al. Mucosal- associated invariant T cell deﬁciency in chronic obstructive pulmonary disease. COPD (2016) 13:196–202. doi: 10.3109/15412555.2015.1069806 165. Gibson PG, Yang IA, Upham JW, Reynolds PN, Hodge S, James AL, et al. Jabeen and Hinks References Effect of azithromycin on asthma exacerbations and quality of life in adults with persistent uncontrolled asthma (AMAZES): A randomised, double-blind, placebo-controlled trial. Lancet (2017) 390(10095):659–68. doi: 10.1016/S0140-6736(17)31281-3 154. Pincikova T, Parrot T, Hjelte L, Hogman M, Lisspers K, Stallberg B, et al. MAIT cell counts are associated with the risk of hospitalization in COPD. Respir Res (2022) 23:127. doi: 10.1186/s12931-022-02045-2 17 17 Frontiers in Immunology frontiersin.org
W1973094511.txt	https://zenodo.org/records/2147917/files/article.pdf	en		Über das Chiteninon	Berichte der Deutschen Chemischen Gesellschaft. Abteilung B, Abhandlungen	1,922	public-domain	2,221	39,31 nach 18ngerer Behandlung mit konz. SchwefelsBm-e und h t i l l n t i o n iiber : = 1.44182; 11. Frakt. (Sdp. 240-2500) n”,”= 1.459O;i. Natrium n Die aus Kohlenwasserstoffen bestehenden Fraktionen zeigten folgendes Verhalten : 1. Mit K a l i u n i p e r m a n g a n a t und SodalBsung tritt bei den iiicht vltrbehandellen Deslillalen Entflrbung ein ; waren diese jedoch mit Irma. Schwefelslure geschiittelt worden, so blieb die Reaktion pus. Die Hauptmenge bestand also aus g e s i t t i g l e n Kohlenwasserstoffen. 2. Eine LBsung von B r q r n in TetrachlorBohlenstoff wurde von dein glcsichfalls i n Tetrachlorkohlenstoff gcldsten Kohlenwasserstoff in der Kilte 11111 langsam bei zerstreutem Tagerjliclit cntfsrbt; bairn Schfitteln irn Sonnenlicht verschwand dagegen die Bromfarbe bereils bei Zimrnerteingeratur schnell, wihreqd glcichzeitig Bromwasersloff entwich. 3. A m m o n i a 1i a1 i sc 11 e S i 1b e r n i t r a 1- Ldsung wurde nicht reduziert. Beim Schttteln irn Rohr mit Silbernitrat und Eisesig bei 1800 srhied sirh Silher ab; es war also Oxydation des Kohlcnwasserstoffes eingc treten. 4. Mil B r s m - a n i l ( T e t r a b r o n i - p - c h i n a n ) i n Eisessig-L6sung cnlstand k i n e Dunkelfirbung oder Abscheidung von Iirystallenl). Ihn s o wen i g honnte die Bildung einer Molcktilverbindung mit Pikrinslurc Iiwbachtrt werden. 431. hligmund Frlinkel, Charlotte Tritt-Zirming und L i l y Gottesmann-Grauer: Cfber das Chiteninon. [.4us d. Laburat. d. 1 , i i d w i g - S p i e g l r r - S t i f t u n g in Widn.] (Eingegangen ani 31. Olitobcr 1922.) Uber die E i n w i r k u n g v o n W a s s e r s t o f f s u p e r o x y d a u f C h i n i n liegen in der Literatur zwei Ansaben vor: N i e r e n s t e i n * ) hat hierbei das C h i t e n i n erhalten, welches bis dahin nur bei der Einwirkung von Kaliurnperinangannt auf Chinin yon S k r a u p und seinen Schiilern gewonnen worden war. Bekanntlich hat S k r a u p zeigen konnen, da13 dieser Korper urn 1 KohlenstoffAtom Brmer und um 2 Sauerstoff-Atome reicher ist als das Ausgangsmaterial, niithin die Vinyl-Seitenkette des Chinins zur Carboxylgruppe und zu Ameisensaure osydiert worden war. In jungster Zeit erhielten E. S p e y e r und A. G. B e c k e r 3 ) bei der EinwirBung yon 3O-proz. Wasserstoffsuperoxyd auf die Chinin-Base nuf dem Wasserbade unter heftigcm Aufschiiumen eine a t h e r - n n I o s l i c h e Base, die sich als Chinin-N-oxyd erwies. vergl. P. I ’ f e i l f e r , 1. c ’J) Biuchem. Journ. 1Q. 572 [1!)30]. (C 3 ) R . 5 5 , 1321 ;lS22] 1) 1921. I 27). Bei unseren schon vor langerer Zeit unternommenen Versuchen, Was se r s t o I f s u p e r o x y d bei Gegenwart eines K a t a 1 y s a t o r s auf C h i n i n einwirken zh lassen, fanden wir eine Verbindung, welche sich in ihren Eigenschaftcn, im Schmelzpunkt und in den Loslichkeitsverhaltnissen von Chitenin unterschied, deren Elementarzu~alysie aber nur wenig dif€erente Zahlen dem Chitenin gegenuber gab, so dalj wir die Vermutung hatten, da8 dieser Korper das ICe t o 11 sei, melches dem sekundaren Alkohol entspricht. Wir haben diese Substanz nach erfolgter Konstitiitionsermittlung C h i t e n i n o n benannt. Durch die Untersuchung von R a b e wurde gezeigt, dalj bei der Oxydation von Cinchonin mit Chromsaure eine urn zwci Wassersloffe armere Base entsteht, und da13 die beiden Verbindungen im Verhiiltnis von sekundarem Alkohol zu Keton stehenl). Wir haben Chinin-Sulfat in schwefelsaurer Losung mit Wasserstoffsuperosyd und ICupfersulfat oder Eisenvitriol als ICatalysntor osydiert und das bei 1560 schmelzende Chiteninon crhalten. Als wir diese Untersuchungen unter verschiedenen Bedingunsen wiederholten, ergab sich, daI3 die neaktion gar nicht so einfach verliiuft, selb'st wenn man die genau berechnete Menge, Wnssersto~fsupero?cyc~ venvendet, sondern dalj eine Reihe von Zwischenprodukten enlsteht, zu denen auch anscheinend das C h i n i n - o x y d gehort. E s ist nicht unwahrscheinlich, da13 der Reaktionsverlauf abhiingig ist von der 'Pemperatur der' Umgebung, da schon bei geringen Schwankungen der AuBentemperatur die Ausbeuten an den verschiedenen Produkten gegeneinander wechseln. Wendet nian iliese Oxydationsmethode nicht auf Chinin, sondern auf C h i t e n i n an, so gelangt man nach unseren Versuchen ebenfslls zum Chiteninon, so daB das Chitenin als Zwischenprodukt dieser Reaktion auftritt, wodurch die Resultate von N i e r e n s t e i n erklart werdcn konnen. Um die K o n s t i t u t i o n d e s C h i t e n i n o n s zu erweison, wurde vorerst der E s t e r clargestellt, welcher sich von dem ebenfalls von uns dargestellten C h i t e n i n - e s t e r unterschied. Dainit war die Gegenwart der Carboxylgruppe erwiesen. Von den gewohnlichen Keton-Reagenzien wirken Phenyl-h ydrazin, p-Nitrophenyl-hydrazin und Semicarbazid-Chlorhydrat auf Cinchoninon nicht ein, ebensowenig auf das Chiteninon. H h g e p reagiert in alkalischer Losung Cinchoninon mit Hydroxylamin. In gleicher Weise konnten wir das 0 s i m d c s C h i t e n i n o 11 s ,dars tellen. 1) B. 40, 3658 [1907], 41, 82 [I'JOU]. h i - c h Variicroiig dcr llcalitioiisdaucr rind dcr 1Iciige des Wasscrstoi rsuperoxydcs lidmi wir eiuc llcihe von Vcrbintluilgeii dnrgestcllt, die i m Vergleich ziiiii Chinin. ziiin Chitcniii uiid zuiii Chiteninon niedrige Kolllciistoff- und selir hohe Sauerstoff-Werte bei dcr 'Annlyse ergabeti. Sie warcii alle in Ather nnl6slich uiid l i e h i sicli atis .\lkohol gilt urnlrrystallisierrn. Es haiidclt sicli nnscheinciitl iini Geiiiciige voii Verbindungen, die sirh durch wiederhaltes fraBtiooierIes Uiii1;ig~tnllisicrcn niclit treiinen lassrn. Die einzige gut definierte Verbindung, die wir itti p t c r .iusbeute erhielt~n, war des BtlierlOsliche Chitcniiion. Beschreibung der Vcrsuche. D a r s tellung d e s C h i t e n i n o n s a u s Chinin. 5 g C h i n i n -Base werden in der erhrderlichcn Menge \ w d . Schwefels%ure gelijst und mit 27 g 30-proz. JV a s s e r s t o f f s u p e r o x y d und einem Krystiillclien K u p f e r s u l f a t uls Katalysalor versetzt. Das Reaktionsgcmisch lHDt inan 6 Tage in ciner dunlclcn Flasche bei Zimmertemperatur stehen. D a m schiittet man die Losung in einen Sctieidetrichter uiid iiberschichtet mit Bthcr. Man 1iiBt langsam verd. Ammoniak unter stetem Umschiitteln ZNflieBen, bis eine schwach alkalische Reaktion a u h i t t . Bei zu starker Erwiirmung wirft man kleine Eisstuclce in den Scheidctrichtcr. Das C h i t e n i n o n holt man init Ather heraus und filtriert von dem voluminBsen Niederschlsg der iihrigcn Basen. Die iitlierischc Losung trocknc t man iiber Glaubersalz, verdunstet tlvn Ather und e r h d t nadelf6rmige Krystalle, die man aus bthyl- oder Methylalkohol umkrys,tallisiert. Die Verbindung schmilzt bei 1560. 14 531 111g Sbst. (bei 1100 getrocknet;: 35.695 mg CO,, 7 f i L :ng I I , O . - 1.725 mg SSst.: 0.1S1 cciii N (1S0, 7% mm). C131-12,0,Np. Ber. C 67.01, FI 5.02, N 8.23. Gcf. o 67.01, )) 5.89, n S.17. Darstellung des Chiteninons a u s Chitenin. Die gleiche Substanz wurde aus C h i t e n i n erhalten: 2 g Chitenin wurden in Alkohol geliist, mit verd. Schwefelsiiure angesauert und 10 g konz. Wasserstoffsuperoxyd hinzugefiigt. Als Katalysator wurde ein Krystallchen Ihpfersulfat Iiinzugegeben. Nach S-tigigern Stehen in einer dunklen Flasche bei Zimmerlemperatur wurde die L8sung in einen Scheidetrichter geschuttelt und nrit Ather iiberschichtet. Dann wurde langsam unter jedesmaligcm Umschiitteln verd. Ainmonialr his zur schwach alkalischen Reaktion hinzugefiigt. Hierbei muB man jede Erwiirmung vermeidm. Das Chiteninon gcht in den Ather, wiihrcnd ein weifier, volunii- aiiiser, in Ather unlijslicher Iiiirper znriicltblcibt. Aus dern A h e r krystallisiert Chiteninon, das man aus Alkohol umkrystallisiert. Es crweist sich als identisch mit dem Oxydationsprodukt des Chinins durch den Schmelzpunkt von 1560 und durch den MischSchmelzpunkt nach T h i e le. 7.142 nig Slist.: 17.620 nig 8.115 ccrn N (“5 0 , 756 mm). COP, 3.870 mg H p O . C I nt12004N2. Ber. C 67.04, H 5.02, Gef. )) G7.30, )) G.06: - 1.985 mg Sbst.: N 8.23. )) 8.16. P i l i r a 1: U:is Chilciiinon gibt, in allcoholischrr L f i r h g init allcoholisclier Piltrinsiure v e m t z t , einc krystallisierle Vcrbindung, die, aus Essigs5tire uinkqstnllisiert, dcn Schmp. 1 4 0 0 zeigt. C h i t e xi i n o n m e t h y I e s t e r - P i k r at. ~ 2 g Chiteninon wurden in 20 g absol. RiIcthylalkohol gelost nnd 6 Stdn. lnng ein tiackner Strom Salzsauregas uriter Kuhlung uirrgeleil.et. Uic Liisiing wurde dann auE Eis und Soda gegossen uiid ausgeathert und die atherische Losung nach dem Trocknen iitm Glaubersalz iin Vakuum eingeengt. Ua der Ester weder ails A h e r nocli aus Mcthylalkohol krystallisi~erk,murde cr in sein Pikrnt iibergefiihrt. Der nach. dem Abdunsten des Athers hinterblcibendv Sirup murde init alkoholischer Piltrinsiiure-Liisung nngerieben und dcr Krystallisation tiherlassen. Dieses Pikrat zeigle nach zweinialigern Umkrystallisieren aus Allrohol den Zersetzungspunkt 2700. 3.498 nig Slist.: 6.010 nig CO,, 1.112 rng H20. - 3.293 nig Sbst.: 5.701 mg CO,, 1.00G nig H?O. - 3318 nig Sbst.: 0.483 ccin N ( 2 6 0 , 7-12inm). 3.371 mg Sl~st.:0.-1:13cciii N (280, 516mni). C,o H??0, 3 2 , 2 C, H3 0 7 N,. Iler. C 47.20, f l 13.45, N 13.80. Gel. x 47.11, 47.23, )) 13.65, 13.41, )) 13.63: 12.76. C 11 i 1 c n i n’on - ni t h y 1e s t c r -D i c h 1 o rli y d r a I. (3 2 g Cliilcuinun cviirden in 20 g alxol. Alctl~ylail~oliolgelOst uiirl - wic w r h i n angegebcn - vercstert, arrf Bis uiid Soda gegossen und tlic Massc aiisgeltliert. In dic 2tlicrischc LOsung wurde nach dern Tmclinen fiber Glaiihersalz eiiiigc Miniiten lang cin troclincr Salzsiure-Strorn eingcleitel. (Ins ausfallcnde Clilorhydrat wiirdc nus Allcohol uinlirystallisiert. Es srliinilzt bei 1810. 7.856 n7g Sbst.: 1G.243mg CO,, 3.840 mg H ? O . - 5.1RG m g Sbsl.: 11.109rng CO?, 2.581nig H.0. .- 4.043mg Sbst.: 0.310ccm N (170, 554mrn!. - 2.820 rng Sbst.: 0.149 ccrn N (150, 751 nini). C,,HzzO1N,. 2IICl. Ber. C 5F.21, 1-1 5.62, N 0.55. Ccl. )) 5G.30, 56.10, )) 5.17, 5.33, x G.32, G.22. \ ' c r s u c h c z u r D a r s t c l l u n g d c s Oxims. Wir vcrsuchten das Chiteninon sowohl mit Hydraziu-hydrat, als ancli mit Phenyl-hydrazin und p-Nitrophenyl-hydrazin zur Reaklion zu bringen. A b e r aus dem Rcaklionsprodukte lionnle man die gewiinschte Verbindung nicht isoberen. hiit Hydroxylamin-Chlorhydrat erhielten wir ein Gemenge dcs Oxims niit unvcrinderter Base. 1 g Chilcninon wurde in Alltohol gelost und eine Losung von 1g Natriumcarbonat und 1.4 g Hydroxylamin Chlorhydrat zugefiigt. Nach 4 Tagen wurde aiisgeatliert und dcr Ather iiber Glaubersalz getrocknet. Das nacli dem Abdunsten des Athers iiberbleibende 01 wurde mit alliohol. I'ikrinslure nngeriebcn iind d e r Krystallisation iiberlassen. Dos Pilirat \vurde aus vcrd. Alkohsl umkrystallisiert: - , 3.971mg Sbst.: 0.4G7ccm N (270, 742mm). - 4.102nig Sbst.: 0.479ccm N (280, 7JGmm). Gef. N 12.58, 12.51, u:ilirend I'ur clas Pilirat dcs Osinis 11.720/, N bercclmcl sind. Es verhalt sich also das Chiteninon Ihnlich, wie das von P. R a b e beschriebene Cinchimn. Wir wrsuchten nun auch, wie .t:s R a b e mit Vorteil getan, mit Hydroxylamin-Chlorhydrat in stark nlkalischer Losung zu arbeiten und gelangten auE di,esem JVege nnch zum Oxim. C h i tenin,on-oxim - P i k r a t . 1 g Chiteninon wurde in Alkohol gelost und 1g HydroxylaminChlorhydrat zugekgt, dann wurde eine Losung von 5 g Natron (1:3) eingetragen und 1Stde. auf dem Wasserbade erwarmt. Nach dern vollstiindigen Erkalten wurde ausgeathert, der Ather uber Glaubersalz getrocknet und abdunsten gelassen. Es hlieb ein Sirup zuriick, der mit alkohol. Pikrinsaure-Losung angerieben und der Krystallisation uberlassen wurde. Die Krystalle hatten nach 2-maligem Umkrystallisieren aus verd. Alkohol den Schmp. 1260. 3.508111s Sbst.: 6.592mg CO,, 1.201 mg H,O. - 7.036 mg Sbst.: 14.576mg CO,, 2.640mg HpO. - 2.257mg Sbst.: 0.3llccm N (270, 747mni). - 4.015mg Sbst.: 0.545ccm N (260, 747mm). Cig Hzi 0 4 NJ:cs Hs 07 Ns. N 14.72. Ber. C 51.37, 1-1 3.7G, Cef. D 51.27, 51.19, )) 3.83, 3.72, )) 14.81, 14.65. Beriohte 6 D. Chem. Gesellachafk Jahrg. LP. 252
https://openalex.org/W2560704913	https://www.duo.uio.no/bitstream/10852/53255/1/13063_2016_Article_1723.pdf	English	null	Abdominal Septic Shock – Endotoxin Adsorption Treatment (ASSET) – endotoxin removal in abdominal and urogenital septic shock with the Alteco® LPS Adsorber: study protocol for a double-blinded, randomized placebo-controlled trial	Trials	2,016	cc-by	6,474	Lipcsey et al. Trials (2016) 17:587 DOI 10.1186/s13063-016-1723-4 Lipcsey et al. Trials (2016) 17:587 DOI 10.1186/s13063-016-1723-4 STUDY PROTOCOL Open Access Abdominal Septic Shock – Endotoxin Adsorption Treatment (ASSET) – endotoxin removal in abdominal and urogenital septic shock with the Alteco® LPS Adsorber: study protocol for a double-blinded, randomized placebo-controlled trial Miklos Lipcsey1,2† , Jyrki Tenhunen2†, Jan Sjölin3, Robert Frithiof2, Stepani Bendel4, Hans Flaatten5, Rafael Kawati2, Anne Kuitunen6, Tor Inge Tønnessen7 and Sten Rubertsson2 Open Access Correspondence: Miklos.lipcsey@surgsci.uu.se †Equal contributors 1Hedenstierna Laboratory, Department of Surgical Sciences/Anaesthesiology and Intensive Care Medicine, Uppsala University, Uppsala, Sweden 2Department of Surgical Sciences/Anaesthesiology and Intensive Care Medicine, Uppsala University, Akademiska sjukhuset, 751 85 Uppsala, Sweden Full list of author information is available at the end of the article Abdominal Septic Shock – Endotoxin Adsorption Treatment (ASSET) – endotoxin removal in abdominal and urogenital septic shock with the Alteco® LPS Adsorber: study protocol for a double-blinded, randomized placebo-controlled trial Miklos Lipcsey1,2† , Jyrki Tenhunen2†, Jan Sjölin3, Robert Frithiof2, Stepani Bendel4, Hans Flaatten5, Rafael Kawati2, Anne Kuitunen6, Tor Inge Tønnessen7 and Sten Rubertsson2 Background Gram-negative sepsis. Case reports and series from pa- tients with septic shock suggest that endotoxin removal could be achieved in clinical practice with LPS Ad- sorber [14, 15]. However, no randomized controlled trial has investigated the potential benefits of this LPS Adsorber. LPS Adsorber is described as biocompatible and no specific side effects related to it have been reported. Gram-negative sepsis. Case reports and series from pa- tients with septic shock suggest that endotoxin removal could be achieved in clinical practice with LPS Ad- sorber [14, 15]. However, no randomized controlled trial has investigated the potential benefits of this LPS Adsorber. LPS Adsorber is described as biocompatible and no specific side effects related to it have been reported. Although severe sepsis and septic shock are common in intensive care and carry high mortality rates no specific treatment has been established to counteract the asso- ciated inflammatory response. Current management may be considered supportive only and consists mainly of effective antimicrobial therapy, removal or drainage of infections (source control) and support of failing or- gans [1]. Therefore, development of new treatment methods for sepsis and septic shock is crucial for med- ical, humanitarian and health-economic reasons. We hypothesized that in patients with Gram-negative infections, early and extensive removal of endotoxin may limit the inflammatory response that characterizes septic shock. This LPS Adsorber can be used for endotoxin re- moval and, in this way, add to the currently available strategies for sepsis treatment. Endotoxins are molecules found in the outer mem- brane of the cell wall in Gram-negative bacteria. They are potent activators of the inflammatory system through the innate immune system and are presumably the key triggers of the systemic inflammatory response [2–4]. Hence, attenuating the effect of endotoxins seems to be a logical and desirable strategy in the treat- ment of severe sepsis and septic shock. However, so far the use of anti-endotoxin strategies has been disap- pointing [5–8]. On the other hand, adsorber therapy was proven beneficial in early sepsis patients due to intra-abdominal infections and added substantially to improved outcome in conjunction with conventional interventions. Improved outcome was associated with reduced endotoxin levels in blood [9, 10]. Therefore, it is reasonable to hypothesize that using adsorption membranes in the treatment of selected Gram-negative septic shock in the early phase, with a strictly defined time-frame since the onset of clinical symptoms, may offer therapeutic benefits. Background Several extracorporeal endotoxin- removal devices, based on various endotoxin-binding mechanisms, have been investigated with diverging results [9, 11, 12]. Currently, at least one trial is investigating the effect of endotoxin removal with polymyxin B cartridges [13]. The present clinical investigation aims to collect infor- mation on the feasibility and safety of LPS Adsorber therapy in patients with septic shock of presumed ab- dominal or urogenital origin. Secondary aims are to re- port the performance of the LPS Adsorber. Specifically, we wish to study the extent of endotoxin removal by the LPS Adsorber, its effects on organ failure development and the extent of organ support, as well as its possible impact on the inflammatory response. This trial should contribute to new knowledge since it investigates a high-capacity peptide-binding-based endotoxin removal that can be run for longer periods and at higher blood flow that other endotoxin ad- sorbers. Moreover, unlike in other device trials, the LPS Adsorber will be compared to an identical LPS Ad- sorber cartridge without active LPS-binding peptide. Thus, a double-blinded randomized controlled trial will be possible to perform. Finally, the ultimate objective of this pilot investigation is to provide the necessary information on the magni- tude of the clinical effects of the LPS Adsorber, thus providing the basis for further investigations in a larger population. Another endotoxin removal device based on a differ- ent concept is a novel, high-capacity adsorber system (from Alteco® Medical AB, Lund, Sweden) with a high- endotoxin-affinity-peptide endotoxin-binding mechan- ism (hereafter referred to as the lipopolysaccharide (LPS) Adsorber) that has been clinically available for some years. This adsorber putatively binds endotoxin and thus decreases endotoxin load in patients with Abstract Background: Severe sepsis and septic shock are common in intensive care and carry high mortality rates. In patients with Gram-negative infections, early and extensive removal of endotoxin may limit the inflammatory response that characterizes septic shock. The Alteco® LPS Adsorber (hereafter referred to cited as the lipopolysaccharide (LPS) Adsorber) can be used for endotoxin removal and attenuate the deleterious inflammatory and clinical responses seen in septic shock. Methods/design: The Abdominal Septic Shock – Endotoxin Adsorption Treatment (ASSET) trial is a pilot study investigating the feasibility and safety of LPS Adsorber therapy. This pilot, multicenter, stratified, parallel, double-blinded, randomized, phase IIa, feasibility clinical investigation will be performed in five Scandinavian intensive care units. Thirty-two subjects with early septic shock and organ failure, following adequate resuscitation, will be randomized to receive either: extracorporeal veno-venous hemoperfusion therapy with the LPS Adsorber or veno-venous hemoperfusion therapy with a placebo adsorber (without active LPS-binding peptide). Patients will be stratified by infection focus such that 20 subjects with an abdominal focus (stratum A) and 12 subjects with a urogenital focus (stratum B) will be included in a parallel design. Thereafter, an interim analysis will be performed and an additional 12 patients may be included in the study. The study is designed as adaptive a priori: the patients from this study can be included in a later phase IIb study. The aim of the study is to investigate the feasibility of LPS Adsorber therapy commenced early in the time-course of septic shock. The primary endpoint will be a characterization of all reported unanticipated serious adverse device effects and anticipated serious adverse device effects. Secondary outcomes are decrease in endotoxin plasma concentration, impact on clinical outcome measures and impact on inflammatory response by LPS Adsorber therapy, as well as detailed description of the relevant mediators bound to the LPS Adsorber. Recruitment of patients will start in September 2015. (Continued on next page) © 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. Lipcsey et al. Abstract Trials (2016) 17:587 Page 2 of 10 (Continued from previous page) Discussion: The ASSET trial will give insight into the feasibility and safety of this LPS Adsorber therapy and preliminary data on its potential clinical effects in septic shock. Moreover, this pilot trial will provide with necessary data for designing future studies. Trial registration: ClinicalTrials.gov Identifier NCT02335723. Registered on 28 November 2014. Keywords: Septic shock, Endotoxins, Hemoperfusion, Gram-negative bacteria Methods/design This is a pilot, multicenter, stratified, parallel, double- blinded, randomized, phase IIa, feasibility clinical investigation and is reported according to the Standard Protocol Items: Recommendations for Interventional Trials (SPIRIT) guidelines [16]. Subjects will be Lipcsey et al. Trials (2016) 17:587 Page 3 of 10 Page 3 of 10 will be randomly allocated to the active LPS Adsorber group (Interventional Medical Device group, IMD group): current best practice in combination with LPS Adsorber therapy or placebo device group (control group): current best practice in combination with pla- cebo adsorber therapy (identical LPS Adsorber cartridge without active LPS-binding peptide). The random alloca- tion to either therapy arm in each stratum will be per- formed in a 1:1 ratio. enrolled in accordance with an adaptive design, with an interim analysis allowing enrollment of additional patients until an adequate sample size is attained, to show the safety and the feasibility or lack thereof of LPS Adsorber therapy in the target patient population. The study will be conducted in five general intensive care units (ICU) in Uppsala, Sweden, Bergen and Oslo, Norway, and Tampere and Kuopio, Finland. These ICUs will be screened around the clock to identify potential patients. Initially, 32 subjects admitted to the ICU with confirmed septic shock will be stratified in accordance with the origin of their suspected endotoxemia, i.e., 20 septic shock subjects with an abdominal focus (stratum A) and 12 subjects with a urogenital focus (stratum B) An interim analysis will be performed after all subjects in their corresponding stratum have completed their participation in the investigation to reveal whether 12 additional subjects are required. The enrollment plan in the clinical investigation is summarized in Fig. 1. Fig. 1 Enrollment plan for each stratum and interim analysis. Optional enrollment of sepsis subjects (of abdominal, urogenital or both origins) after interim analysis Fig. 1 Enrollment plan for each stratum and interim analysis. Optional enrollment of sepsis subjects (of abdominal, after interim analysis Lipcsey et al. Trials (2016) 17:587 Lipcsey et al. Trials (2016) 17:587 Page 4 of 10 Treatment criteria Treatment criteria The study has an adaptive design and thus the investi- gators have made an a-priori decision to include the pa- tients from this study in a later phase IIb study aiming to investigate and report possible outcome benefits of the therapeutic intervention. 6. Illness severity criteria At enrollment, subjects must meet the inclusion criteria #1 through #5 listed below to be eligible to enter the clinical investigation: 1. Subjects must have suspected infection of abdominal or urogenital origin for which the subject is receiving intravenous antimicrobial therapy 2. Subjects must have systemic inflammatory response syndrome (SIRS), i.e., they must meet at least two of the criteria defined below during the 36 h prior to clinical investigation entry: (a) Abnormal body temperature: (a) Abnormal body temperature: i.) Fever: core temperature (measured via rectum, urinary bladder, central catheter) above 38 ° C (100.4 ° F), or ii.) Hypothermia: core temperature (measured via rectum, urinary bladder, central catheter) below 36 ° C (96.8 ° F) 12. Subjects must be able to initiate the clinical investigation intervention within 12 h of fulfillment of the Illness severity criteria (b) Heart rate above 90 beats/min (b) Heart rate above 90 beats/min (c) Spontaneous respiratory rate above 20 breaths/ min or PaCO2 < 32 mmHg (4.3 kPa) or concurrent use of mechanical ventilation for an acute process Inclusion criteria Upon enrollment (i.e., pretreatment phase), subjects ad- mitted to the ICU with suspected endotoxemia will be screened for fulfillment of the “Illness severity criteria” confirming early stage severe sepsis. 8. Subjects must score a Sequential Organ Failure Assessment (SOFA) score of 10 or higher, and a Simplified Acute Physiology Score (SAPS II) of 58 or higher Within 6 h of enrollment, subjects who also fulfill the “Treatment criteria” confirming septic shock will be eli- gible for randomization. 9. Appropriate vascular access must have been obtained The subjects have to meet all of the following criteria to be eligible to enter the clinical investigation: 10. Subjects must have received ≥30 mL/kg of intravenous fluid within the 6 h prior to randomization 11. Subjects must have a continuous requirement for vasopressor support (at least one of the vasopressors must be given as listed below) for at least 2 h prior to randomization to maintain mean arterial pressure (MAP) >65 mmHg or systolic arterial pressure >90 mmHg with reasonable attempts made to wean the subject from vasopressor support. Careful evaluation is made so that vasopressor dependency is not only related to sedation. Required vasopressor support includes at least one of the following vasopressors administered as described below (and allows for combination therapy where other vasopressors are given with modified administration regimen): (a) Norepinephrine ≥0.07 μg/kg/min (b) Dopamine ≥10 μg/kg/min (c) Phenylephrine ≥35 μg/kg/min (d) Epinephrine ≥0.07 μg/kg/min (e) Vasopressin ≥0.03 units/min 11. Subjects must have a continuous requirement for vasopressor support (at least one of the vasopressors must be given as listed below) for at least 2 h prior to randomization to maintain mean arterial pressure (MAP) >65 mmHg or systolic arterial pressure >90 mmHg with reasonable attempts made to wean the subject from vasopressor support. Careful evaluation is made so that vasopressor dependency is not only related to sedation. Required vasopressor support includes at least one of the following vasopressors administered as described below (and allows for combination therapy where other vasopressors are given with modified administration regimen): (a) Norepinephrine ≥0.07 μg/kg/min (b) Dopamine ≥10 μg/kg/min (c) Phenylephrine ≥35 μg/kg/min (d) Epinephrine ≥0.07 μg/kg/min (e) Vasopressin ≥0.03 units/min Methods/design Prior to randomization, subjects must meet all inclusion criteria (#7 through #11) listed below to be assigned to a treatment group: 7. Subjects must have septic shock according to the modified septic shock criteria, i.e., Illness severity criteria #1 through #3 are still fulfilled Exclusion criteria Subjects who meet any of the exclusion criteria listed below will not be permitted to enter the clinical investigation: (d) White blood cell count (WBC) of >12,000 cells/ mm3 or <4000 cells/mm3 or a >10% increase in immature neutrophils Sepsis-induced organ dysfunction for longer than 12 h prior to the time point for achieving “Illness severity criteria fulfilled” 3. Subjects must have clinical signs consistent with organ dysfunction, i.e., they must meet at least one of the following criteria during the 6 h prior to clinical investigation entry: Vasopressor therapy (at any dose) for longer than 8 h (not including the time spent in the operation theatre) prior to the start of the therapy with the investigational device 4. Subjects, independent of gender, must be aged 18 years or older 5. Subjects or legally acceptable representatives, as appropriate, are willing and able to provide signed informed consent Preexisting irreversible medical conditions such as: ○Poorly controlled neoplasms or hematologic disease (i.e., indication of disseminated cancer Lipcsey et al. Trials (2016) 17:587 Page 5 of 10 Page 5 of 10 outside the suspected primary tumor and hematologic disease not in remission) screened for fulfillment of the “Illness severity criteria” confirming the early stages of severe sepsis. Within 6 h of enrollment, subjects who also fulfill the “Treatment criteria” confirming septic shock will be eligible for randomization. Randomization to either of the treatment groups will be performed as close as possible to the start of treatment with the LPS Adsorber or placebo device. outside the suspected primary tumor and hematologic disease not in remission) ○End-stage cardiac disease ○Cardiac arrest requiring cardiopulmonary resuscitation or with pulseless electrical activity or asystole within the past 7 days ○End-stage lung disease; end-stage liver disease ○HIV/AIDS with known end-stage processes ○Other uncorrectable medical condition(s) deemed by the clinical investigator to hinder the subject to adhere to the fulfillment of the activities described in the Clinical Investigation Plan (CIP) ○Cardiac arrest requiring cardiopulmonary resuscitation or with pulseless electrical activity or asystole within the past 7 days ○End-stage lung disease; end-stage liver disease ○HIV/AIDS with known end-stage processes Treatment with the LPS Adsorber or placebo device must be initiated within 6 h (day 1) following fulfillment of the “Treatment criteria” and given for 6 h. Exclusion criteria A second device treatment will be performed 24 h after the end of the first device treatment on day 2, as long as there is no evidence that treatment with the investigational device will not be beneficial or will indicate an unnecessary risk for subjects (for example, if the subject is free of vaso- pressor support). ○Other uncorrectable medical condition(s) deemed by the clinical investigator to hinder the subject to adhere to the fulfillment of the activities described in the Clinical Investigation Plan (CIP) Extreme illness, i.e., subject is moribund and death is perceived to be imminent (within 24 h) Recent or current participation (≤30 days) in another interventional sepsis trial Recent or current treatment (≤30 days) with an adsorption product, including the LPS Adsorber The treatment with the LPS Adsorber or placebo can be discontinued at any time on the decision of the at- tending physician. At least one 2-h treatment session is required to fulfill the treatment protocol. Treatment with an investigational medicinal product for any indication within the last 30 days before enrollment in the clinical investigation Pregnancy After treatment with the LPS Adsorber or placebo is finalized (post-treatment phase), subjects will be moni- tored until they are ready for ICU discharge or deceased. Discharge from hospital and mortality at 28 days since enrollment will also be collected. Figure 2 and Table 1 summarize the participant timeline of the clinical investigation. Pregnancy Contraindications to the use heparin or protamine, as in case of, but not limited to: ○Prior heparin-induced thrombocytopenia II (HIT II) ○Prior intracranial bleeding of any cause up to 90 days before inclusion in this clinical investigation Concomitant therapy ○Prior neurosurgery up to 90 days before inclusion in this clinical investigation. Prior hemorrhagic stroke up to 90 days before inclusion in this clinical investigation ○ h ll Medication, which is considered necessary for the sub- jects’ safety and wellbeing, may be given at the discretion of the investigator. Relevant medications or other inter- ventions include, but are not limited to current best practice for the treatment of septic shock as described in the Surviving Sepsis Campaign guidelines [1]. Other abdominal inflammatory conditions such as, but not limited to: Administration of concomitant medication/therapy during the conduct of the investigation may lead to withdrawal of the subject from the clinical investigation. Prohibited medications/therapies include: ○Suspected pancreatitis (serum amylase (s-amylase) level at least three times the upper limit of normal (ULN)) ○Suspected fulminant hepatitis Medications without a marketing approval in the country of the site ○Suspected gastric/duodenal ulcer-related gastrointestinal perforation Colloids (with the exception of albumin) for use in fluid resuscitation Perforation of hollow organ linked to trauma within 48 h before enrollment in the clinical investigation Laparotomy reveals isolated gastric ulcer The use of renal replacement therapy (RRT) concomitantly with the use of the LPS Adsorber or placebo device is allowed between device therapies on day 1 and day 2, and after the end of therapy phase on day 2 Subjects and/or their immediate family are directly affiliated to investigative site personnel in this clinical investigation (immediate family is defined as a spouse, parent, child or sibling, whether biological or legally adopted) Primary endpoint Primary endpoint is characterization of all reported un- anticipated serious adverse device effects and anticipated serious adverse device effects. Secondary objective The secondary objectives are: To investigate the ability of the LPS Adsorber to reduce levels of endotoxin in plasma during and directly after treatment with the investigational device To investigate the impact of the LPS Adsorber on clinical outcome To investigate the impact of the LPS Adsorber on the inflammatory response in septic shock Intervention This is a double-blinded study. Data is collected by an independent contract research organization (TFS Trial Form Support AB, Lund, Sweden). Neither health care Upon enrollment (i.e., pre-treatment phase), subjects ad- mitted to the ICU with suspected endotoxemia will be Page 6 of 10 Lipcsey et al. Trials (2016) 17:587 Fig. 2 Overall clinical investigational design. Treatment with IMD or placebo device will be repeated on Day 2, i.e. 24 h after end of treatment on day 1. § Randomization will be performed as close as possible to start of treatment start (time-point O h) on Day 1 Fig. 2 Overall clinical investigational design. *Treatment with IMD or placebo device will be repeated on Day 2, i.e. 24 h after end of treatment on day 1. § Randomization will be performed as close as possible to start of treatment start (time-point O h) on Day 1 with presumed endotoxemia of abdominal or urogenital origin. providers nor Steering Committee members, investiga- tors or statisticians will have access to group allocation of subjects. The LPS Adsorber contains a designed syn- thetic peptide developed for endotoxin adsorption. The placebo comparator device differs from the LPS Ad- sorber only in that no peptide component (i.e, active component) has been attached to the matrix. Both the LPS Adsorber and the placebo device will be relabelled for the purpose of this clinical investigation. The investi- gational site(s) will be provided with emergency code break envelopes for all subjects. Randomization Screening will be web-based. Patients are randomized to either the LPS Adsorber or placebo adsorber arms by being treated with a blinded cartridge on site starting with the lowest serial number. The abdominal focus (stratum A) and the urogenital focus (stratum B) pa- tients are kept apart by different serial numbers. This re- sults in double-blinding, stratification by sepsis focus and randomization in blocks. To investigate the content bound to the LPS Adsorber in a subset of subjects after treatment e To investigate the content bound to the LPS d b b f b f To investigate the content bound to the LPS Adsorber in a subset of subjects after treatment end g Adsorber in a subset of subjects after treatment end Primary objective The primary objective of this clinical investigation is to investigate the feasibility, safety and possible benefits of the LPS Adsorber in treating patients with septic shock Relative change from baseline in plasma endotoxin (p-endotoxin) levels during (i.e., at 2 h) and Page 7 of 10 Lipcsey et al. Trials (2016) 17:587 Table 1 The timeline of the study according to the Standard Protocol Items: Recommendations for Interventional Trials (SPIRIT) guidelines Assessments Pre-treatment phase Treatment phase Post-treatment phase Screening Day 1 Day 2 Days 3–6 ICU discharge 28-day follow-up Illness criteria Treatment criteria Baseline Following start of treatment 1 End of treatment 1 Prior to treatment 2 start Start of treatment 2 End of treatment 2 up to −12 h up to −6 h Before 0 h 0 h 6 h Before 30 h 0 h 6 h ~48–120 h Max 28 days Enrollment Informed consent X Demography, medical history X X Vital signs, organ support Assessment X X X X X X Physical examination X X X X X X X X Laboratory assessments X X X X X X X X Illness severity scoring X X X X X X X X X X X Eligibility criteria #1 X Eligibility criteria #2 X Randomization X Intervention Investigational/placebo device treatment X X Assessments Laboratory assessments X X X X X X X X Illness severity scoring X X X X X X X X X X X Prior or concomitant therapy X X X X X X X X X X AEs X X X X X X X X ADEs X X X X X X X X Device deficiencies X X X X ICU-LOS X Hospital-LOS X Mortality (ICU and 28-day) X X ADEs adverse device effects, AEs adverse events, ICU intensive care unit, LOS length of stay Lipcsey et al. Trials (2016) 17:587 Page 8 of 10 Need of other intervention, for medical reasons, that leads to the interruption of the sepsis treatment. Participant withdrawal Participation in the clinical investigation is voluntary and subjects (or their legally accepted representa- tives) may discontinue their participation at any time. Subjects may be withdrawn from investigational treat- ment and assessments at any time if deemed necessary by the investigator. Amendments Protocol amendments will be made by the Steering Committee and submitted to the ethical boards in each participating country for approval. The protocol pre- sented is version 5 from 2 March 2016. Primary objective Examples: immediately after end (i.e., at 6 h) of therapy with device, on both day 1 and day 2 immediately after end (i.e., at 6 h) of therapy with device, on both day 1 and day 2 immediately after end (i.e., at 6 h) of therapy with device, on both day 1 and day 2 y y Monitoring of clinical outcome parameters during stay at ICU: ○Imminent need of surgery ○Relative change from baseline in SOFA score (total and organ specific) measured once daily and up to 120 h (or time for ICU discharge, whichever comes first) after start of therapy with device ○Imminent need of continuous renal replacement therapy (CRRT), defined as Plasma potassium >6.5 mmol/L despite other therapeutic options Life-threatening fluid overload ○Relative change from baseline in the monitoring of renal function ○Computed tomography (CT) scan ○Computed tomography (CT) scan Pregnancy ○Computed tomography (CT) scan Pregnancy ○Relative change from baseline in the monitoring of liver function Subject is lost to follow-up ○Relative change from baseline in circulatory support Severe adverse reactions ○Relative change from baseline in respiratory support ○Relative change from baseline in respiratory support ○ICU mortality ○ICU length-of-stay (LOS) up to 28 days The LPS Adsorber is a class IIa medical device and does not include any drugs or toxic substances that enter the blood circulation. All components in the LPS Adsorber are biocompatible. The capturing-peptide component is nontoxic, and cannot be released during rinsing or ther- apy procedures as it is covalently bound to the matrix. The LPS Adsorber is part of an extracorporeal hemoper- fusion system which is supplied sterile and only for sin- gle use. p y pp ○ICU mortality ○ICU length-of-stay (LOS) up to 28 days Monitoring of clinical outcome parameters during stay at hospital following ICU discharge: ○The total extension of renal support ○28-day mortality ○Hospital-LOS up to 28 days Monitoring of inflammatory response biomarkers: ○Inflammatory triggers and markers ○Acute phase protein markers ○Plasma cytokines All serious adverse events and serious adverse device effects are promptly reported by the investigation site to Clinical Drug Safety Support at the contract research organization (TFS Trial Form Support AB, Lund, Sweden) within 24 h of learning about the event, regard- less of the time that may have elapsed from the time the event occurred and these will be evaluated and recorded. Determination of the molecular components extracted from blood circulation and captured in the LPS Adsorber Characterization of all reported adverse events (regardless of attribution), adverse device effects and device deficiencies Acquisition of data An eCRF (electronic Case Report Form) system will be used. Data validation and data queries will be handled by the contract research organization‘s Data Management Team (TFS Trial Form Support AB, Lund, Sweden). Potential reasons for withdrawal of subjects from this clinical investigation are: Screening failure (if any inclusion criterion is not fulfilled or if any exclusion criterion is fulfilled) Data ownership and publication policy participation in the investigation to assess whether 12 additional subjects are required. The investigators and the sponsor will be joint owners of all materials and all results. Any publication shall be subject to the prior review and approval of the sponsor, such approval is not to be unreasonably withheld. The sponsor shall not have a right for veto in any correct fac- tual part of any planned publication. Abbreviations ALT Al ALT: Alanine aminotransferase; AST: Aspartate aminotransferase; CIP: Clinical investigation plan; CRRT: Continuous renal replacement therapy; CT: Computed tomography; eCRF: Electronic Case Report Form; HIT II: Heparin-induced thrombocytopenia II; ICU: Intensive care unit; IMD: Interventional Medical Device; LOS: Length-of-stay; MAP: Mean arterial pressure; RRT: Renal replacement therapy; SAPS II: Simplified Acute Physiology Score; SIRS: Systemic inflammatory response syndrome; SOFA: Sequential Organ Failure Assessment Funding The study is sponsored by institutional grants from Uppsala University and by ALTECO Medical AB. RF was supported by funds from the Swedish Research Council (grant 523-2014-2569). Consent for publication The signed Informed Consent Form will be obtained by the investigator prior to inclusion in the study and scientific use of acquired data. Should subjects be incapable of giving informed consent (e.g., subjects may be unconscious or obviously disoriented), the clinical investigator will request informed consent from the legally acceptable representative(s). As soon as the subject’s medical condition allows, they will be informed about the clinical trial and asked to provide informed consent for continued participation. Competing interests SR and ML have attended a scientific meeting at the sponsor’s expense. The other author(s) declare that they have no competing interests. The Safety Set will consist of all randomized subjects who started therapy. Safety summaries will be performed on the Safety Set. Availability of data and materials The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. Nonfulfillment of all inclusion criteria Data handling and record keeping A monitor from the contract research organization (TFS Trial Form Support AB, Lund, Sweden) will perform monitoring activities at the assigned clinical investiga- tional site(s) in accordance with the monitoring plan. Management and storage of data will be handled by the contract research organization. The decision of a subject or legally acceptable representative(s), as appropriate, to withdraw from the clinical investigation (including if the subject withdraws informed consent) Administration of concomitant medication or procedure prohibited by this investigation plan Lipcsey et al. Trials (2016) 17:587 Page 9 of 10 Authors’ contributions Fulfilling at least one exclusion criteria ML and JT drafted the manuscript based on the protocol and had equal contribution to the manuscript and the protocol. All authors contributed to and approved the manuscript. ML, JT, RF, JS and SR designed the study and wrote the protocol with contributions from RK, TIT, HF, AK and SB. SR is the principal investigator. The site investigators are TIT, HF, AK and SB. The FAS is considered as the primary analysis dataset, and will be used for all performance analyses. The analy- sis(es) that will be repeated using the PPS will be further described in the SAP. Major discrepancies between the results from the FAS and the PPS analyses will be com- pared and discussed. Power calculation Since this is a pilot study, no sample size calculation was possible or has been performed. The sample size was chosen for practical reasons, and aimed to allow recruit- ment of all subjects in the first enrollment period (i.e., stratum A and stratum B) within a year, and within six add- itional months for subjects in the second enrollment period. Data from this pilot investigation will provide infor- mation for designing future studies with the LPS Adsorber. The key goal of this study is to identify patients who might benefit from endotoxin removal. This goal is achieved by including severely ill septic shock patients with presumed endotoxemia and initiating endotoxin re- moval therapy early. The objective of the ASSET study is to investigate the safety and feasibility of LPS Adsorber therapy and to provide data for further studies. Acknowledgements The Per Protocol Set (PPS) will consist of subjects from the FAS who completed at least 2 h of therapy with the investigational device or the placebo device on day 1 and on day 2. Additionally, the subjects in the PPS will not have any major CIP violations that will affect the evaluation. Major CIP violations will include but are not limited to the following: We thank the TFS Trial Form Support AB for editing the protocol. Endpoints No primary performance variables will be collected in the present clinical investigation. The statistical analyses of secondary endpoints will be descriptive. Discussion To date, there is no specific therapy for the inflammatory response induced by bacteria in sepsis. Given its role in triggering inflammatory responses, endotoxin could play a central role in inducing organ failure in Gram-negative sepsis. Endotoxin removal with an endotoxin-specific, high-affinity, high-capacity adsorber system is, therefore, an attractive therapeutic option. The LPS Adsorber used in this study fulfills these characteristics. Analysis sets The following analysis sets will be used for the statistical analysis: The Full Analysis Set (FAS) will consist of all random- ized subjects who were treated with the investigational device or placebo device for at least 2 h on day 1. Trial status R i Recruitment of patients started in September 2015. Interim analysis An interim analysis will be performed after all subjects in their corresponding stratum have completed their Page 10 of 10 Page 10 of 10 Lipcsey et al. Trials (2016) 17:587 Lipcsey et al. Trials (2016) 17:587 Ethics approval and consent to participate doi:10.1159/ 000330323. participation. Participation in the clinical investigation is voluntary and subjects (or their legally accepted representatives) may discontinue their participation at any time. 15. Duszynska W, Smiechowicz J, Adamik B, Zielinski S, Kubler A. Advanced therapeutic methods for the treatment of meningococcal septic shock – case report. Anaesthesiol Intensive Ther. 2012;44(4):212–6. Ethics approval and consent to participate randomized controlled trial. JAMA. 2009;301(23):2445–52. doi:10.1001/jama. 2009.856. randomized controlled trial. JAMA. 2009;301(23):2445–52. doi:10.1001/jama. 2009.856. The clinical investigation will be conducted in compliance with applicable international standards (ISO 14155:2011) and regulatory requirements, as well as with the ethical principles of the latest revision of the Declaration of Helsinki as adopted by the World Medical Association. The study has been approved by Regionala etikprövningsnämnden i Uppsala, Sweden (Regional Ethical Review Board in Uppsala; 2014/370, ALT C1-01), Regionale Komiteer for Medisinsk og Helsefaglig Forskningsetikk, Norway (Regional Committees for Medical and Health Research Ethics; 2014/1059/REK vest) and Tampereen Yliopistollisen Sairaalan Erityisvastuualueen Alueellinen Eettinen Toimikunta, Finland (Regional Ethical Committee of the Tampere University Hospital; R14130). Each patient will be consented according to the regulations of the country where the patient is included in the study. The signed Informed Consent Form will be obtained by the investigator prior to inclusion in the study. Should subjects be incapable of giving informed consent (e.g., sub- jects may be unconscious or obviously disoriented), the clinical investigator will request informed consent from the legally acceptable representative(s). As soon as the subject’s medical condition allows, they will be informed about the clinical trial and asked to provide informed consent for continued participation. Participation in the clinical investigation is voluntary and subjects (or their legally accepted representatives) may discontinue their ti i ti t ti 10. Yaroustovsky M, Abramyan M, Popok Z, Nazarova E, Stupchenko O, Popov D, et al. Preliminary report regarding the use of selective sorbents in complex cardiac surgery patients with extensive sepsis and prolonged intensive care stay. Blood Purif. 2009;28(3):227–33. doi:10.1159/000231988. 11. Bengsch S, Boos KS, Nagel D, Seidel D, Inthorn D. Extracorporeal plasma treatment for the removal of endotoxin in patients with sepsis: clinical results of a pilot study. Shock. 2005;23(6):494–500. 12. Payen DM, Guilhot J, Launey Y, Lukaszewicz AC, Kaaki M, Veber B, et al. Early use of polymyxin B hemoperfusion in patients with septic shock due to peritonitis: a multicenter randomized control trial. Intensive Care Med. 2015; 41(6):975–84. doi:10.1007/s00134-015-3751-z. 13. Klein DJ, Foster D, Schorr CA, Kazempour K, Walker PM, Dellinger RP. The EUPHRATES trial (Evaluating the Use of Polymyxin B Hemoperfusion in a Randomized controlled trial of Adults Treated for Endotoxemia and Septic shock): study protocol for a randomized controlled trial. Trials. 2014;15:218. doi:10.1186/1745-6215-15-218. 14. Ala-Kokko TI, Laurila J, Koskenkari J. A new endotoxin adsorber in septic shock: observational case series. Blood Purif. 2011;32(4):303–9. Author details 1H d ti L 16. Chan AW, Tetzlaff JM, Altman DG, Dickersin K, Moher D. SPIRIT 2013: new guidance for content of clinical trial protocols. Lancet. 2013;381(9861):91–2. doi:10.1016/S0140-6736(12)62160-6. 1Hedenstierna Laboratory, Department of Surgical Sciences/Anaesthesiology and Intensive Care Medicine, Uppsala University, Uppsala, Sweden. 2Department of Surgical Sciences/Anaesthesiology and Intensive Care Medicine, Uppsala University, Akademiska sjukhuset, 751 85 Uppsala, Sweden. 3Section of Infectious Diseases, Department of Medical Sciences, Uppsala University, Uppsala, Sweden. 4Department of Intensive Care, Kuopio University Hospital, Kuopio, Finland. 5Department of Clinical Medicine, Haukeland University Hospital, UiB, Bergen, Norway. 6Critical Care Medicine Research Group, Tampere University Hospital, PO Box 200033521 Tampere, Finland. 7Division of Emergencies and Critical Care, Oslo University Hospital and Institute of Clinical Medicine, 0450 Oslo, Norway. Received: 26 November 2015 Accepted: 23 November 2016 References 1. Dellinger RP, Levy MM, Rhodes A, Annane D, Gerlach H, Opal SM, et al. Surviving Sepsis Campaign: international guidelines for management of severe sepsis and septic shock, 2012. Intensive Care Med. 2013;39(2):165– 228. doi:10.1007/s00134-012-2769-8. 1. Dellinger RP, Levy MM, Rhodes A, Annane D, Gerlach H, Opal SM, et al. Surviving Sepsis Campaign: international guidelines for management of severe sepsis and septic shock, 2012. Intensive Care Med. 2013;39(2):165– 228. doi:10.1007/s00134-012-2769-8. 2. Annane D, Bellissant E, Cavaillon JM. Septic shock. Lancet. 2005;365(9453): 63–78. doi:10.1016/S0140-6736(04)17667-8. 3. Lipcsey M, Larsson A, Eriksson MB, Sjölin J. Inflammatory, coagulatory and circulatory responses to logarithmic increases in the endotoxin dose in the anaesthetised pig. J Endotox Res. 2006;12(2):99–112. 4. Suffredini AF, Fromm RE, Parker MM, Brenner M, Kovacs JA, Wesley RA, et al. The cardiovascular response of normal humans to the administration of endotoxin. N Engl J Med. 1989;321(5):280–7. 5. McCloskey RV, Straube RC, Sanders C, Smith SM, Smith CR. Treatment of septic shock with human monoclonal antibody HA-1A. A randomized, double-blind, placebo-controlled trial. CHESS Trial Study Group. Ann Intern Med. 1994;121(1):1–5. 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: 6. Angus DC, Birmingham MC, Balk RA, Scannon PJ, Collins D, Kruse JA, et al. E5 murine monoclonal antiendotoxin antibody in gram-negative sepsis: a randomized controlled trial. E5 Study Investigators. JAMA. 2000;283(13): 1723–30. • 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 and we will help you at every step: 7. Rice TW, Wheeler AP, Bernard GR, Vincent JL, Angus DC, Aikawa N, et al. A randomized, double-blind, placebo-controlled trial of TAK-242 for the treatment of severe sepsis. Crit Care Med. 2010;38(8):1685–94. doi:10.1097/ CCM.0b013e3181e7c5c9. 8. Opal SM, Laterre PF, Francois B, LaRosa SP, Angus DC, Mira JP, et al. Effect of eritoran, an antagonist of MD2-TLR4, on mortality in patients with severe sepsis: the ACCESS randomized trial. JAMA. 2013;309(11):1154–62. doi:10. 1001/jama.2013.2194. 9. Cruz DN, Antonelli M, Fumagalli R, Foltran F, Brienza N, Donati A, et al. Early use of polymyxin B hemoperfusion in abdominal septic shock: the EUPHAS
https://openalex.org/W1993203025	https://europepmc.org/articles/pmc3870959?pdf=render	English	null	Effect of tai chi on musculoskeletal health-related fitness and self-reported physical health changes in low income, multiple ethnicity mid to older adults	BMC geriatrics	2,013	cc-by	8,117	RESEARCH ARTICLE Open Access © 2013 Manson 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. Effect of tai chi on musculoskeletal health-related fitness and self-reported physical health changes in low income, multiple ethnicity mid to older adults James Manson1, Michael Rotondi2, Veronica Jamnik3, Chris Ardern4 and Hala Tamim5 Abstract Background: Tai Chi (TC) has proven to be effective at improving musculoskeletal fitness by increasing upper and lower body strength, low back flexibility and overall physical health. The objectives of this study were to examine changes in musculoskeletal health-related fitness and self-reported physical health after a 16 week TC program in a low income multiple ethnicity mid to older adult population. Methods: Two hundred and nine ethnically diverse mid to older community dwelling Canadian adults residing in low income neighbourhoods were enrolled in a 16 week Yang style TC program. Body Mass Index and select musculoskeletal fitness measures including upper and lower body strength, low back flexibility and self-reported physical health measured by SF 36 were collected pre and post the TC program. Determinants of health such as age, sex, marital status, education, income, ethnicity of origin, multi-morbidity conditions, weekly physical activity, previous TC experience as well as program adherence were examined as possible musculoskeletal health-related fitness change predictors. Results: Using paired sample t-tests significant improvements were found in both upper and lower body strength, low back flexibility, and the SF 36 physical health scores (p < 0.05). Based on multiple linear regression analyses, no common health determinants explained a significant portion of the variation in percent changes of the musculoskeletal fitness and SF 36 measures. Conclusions: These results reveal that TC has the potential of having a beneficial influence on musculoskeletal health- related fitness and self-reported physical health in a mid to older low socioeconomic, ethnically diverse sample. Keywords: Ageing, Community dwelling, Ethnicity of origin, Health-related fitness, Low income, Tai Chi Correspondence: jmanson@yorku.ca 1York University, 341 Bethune College, 4700 Keele Street, M3J 1P3 North York, Ontario, Canada Full list of author information is available at the end of the article Manson et al. BMC Geriatrics 2013, 13:114 http://www.biomedcentral.com/1471-2318/13/114 Manson et al. BMC Geriatrics 2013, 13:114 http://www.biomedcentral.com/1471-2318/13/114 Manson et al. BMC Geriatrics 2013, 13:114 Background Dundas and Spadina was a lo- gical choice since it is an area that is particularly dense in adults of Chinese origin (a purposeful decision to ex- plore research questions of ethnic origin affiliations with TC) as well as being socio-economically similar to the Jane-Finch community [13]. Almost 50% of Chinese Canadians live in Ontario, and the Dundas-Spadina area of Toronto, is identified as the center of one of the lar- gest Chinese communities in North America [18]. and musculoskeletal fitness, arthritis and psychosocial behavior [5]. In TC, the body is comfortably moved and relaxed, the mind is focused on the moment, and body movements are slow, smooth, and well-coordinated as the various forms are undertaken [6]. TC exercise has the abil- ity to produce balanced movements between natural phys- ical and metabolic processes in the body; in a slow, meditative, and relaxed way. These sequential graceful movements emphasize the smooth integration of trunk rotation, weight shifting, and coordination, along with a progressive narrowing of one’s stance or base of support. The intensity of TC is moderate and approximately equivalent to a walking speed of 3.7 mph [7]. As powerful as TC is as a good, low-intensity exercise, it is important to emphasize the social benefits as part of the participa- tion structure that helps keep the mind engaged, com- bined with this, evidence has demonstrated that being active with people of similar age, ability and outlook highly influences the social rewards that are a significant factor for adherence to long-term practice [8]. Physically TC is highly appropriate for an aging population since it can be practiced by participants with one or more chronic condi- tions due to its low intensity, steady rhythm and low phys- ical and mental demands but it can also specifically influence balance and motor control to reduce falls in this at risk aging population group [9]. Although several studies to date have reported muscu- loskeletal health-related fitness benefits for mid to older adults who practice TC (community living and institu- tionalized) [10], they have not specifically focused on low socioeconomic status (SES) and ethnically diverse aging populations who could also benefit substantially from an inexpensive, low impact PA program such as TC. Factors such as age, sex, marital status, among others, may individually or collectively influence these health-related musculoskeletal fitness outcomes [11]. Background them close to important thresholds of physical ability that straddles the line between independence and de- pendence [2] it is important to research and implement appropriate community based activity programs. Adding further layers to this challenge are health-related fitness concerns that are unique to specific populations. Data has consistently shown low socioeconomic status (SES), ethnic minorities and new immigrants have lower activ- ity levels than White or non-immigrant groups [3] as well as living in poorer social environments that add to the barriers of activity overall [4]. As the Canadian population both ages and increases in number musculoskeletal health-related fitness mainten- ance concerns become a higher priority. Physical activity (PA) has been shown to be effective in the prevention and management of cardiovascular disease, stroke, hypertension, breast cancer, colon cancer, type 2 diabetes and osteoporosis [1]. Since many older adults must deal with multiple chronic health conditions that may place * Correspondence: jmanson@yorku.ca 1York University, 341 Bethune College, 4700 Keele Street, M3J 1P3 North York, Ontario, Canada Full list of author information is available at the end of the article Tai Chi (TC) has been shown to be a successful pro- gram for reduction in falls, health-related cardiovascular Manson et al. BMC Geriatrics 2013, 13:114 http://www.biomedcentral.com/1471-2318/13/114 Page 2 of 10 the Greater Toronto Area of Ontario, Canada; Jane and Finch as well as Dundas and Spadina. These two loca- tions were chosen for their diversity of ethnic groups represented and their low SES. The city of Toronto has specific areas that have a significant proportion of so- cially and economically vulnerable population groups, the two areas chosen for this study were almost identical in low income rates [13]. The Dundas and Spadina and Jane and Finch area have a population average income of about $26,771.00 [14]. Ethnic or visible minorities in Canada are defined as persons, other than Aboriginal peoples, who are non-Caucasian in race or non-white in color [15]. The Jane-Finch community is ethnically di- verse and has approximately 86,000 immigrants with 63% of the total population is a visible minority [16]. Poverty and various forms of discrimination, including racism, have been identified as negatively affecting the quality of life of the community’s families, and as signifi- cant risk factors for poor physical and mental health [17]. Moreover, the SES of the Jane-Finch population is modest compared to many other areas of Toronto and Ontario as a whole [13]. Background The above notwithstanding, the goal with any PA modal- ity is to improve overall health-related fitness. In this sense, the maintenance of adequate musculoskeletal fit- ness allows older adults to perform normal daily activ- ities in a safe and independent fashion without undue fatigue or pain [12]. Eligibility for participation was limited to those indi- viduals who were 50 years of age and older (male/fe- male), residing in both target community, and who had the medical capability to be involved in an physical ac- tivity program. This capability was measured by Physical Activity Readiness Questionnaire (PAR-Q) which is a self-screening tool that if any participant answers yes to one of the seven health questions they must be cleared by their doctor via the Physical Activity Readiness Medical Examination (PAR-Med-X)[19]. To facilitate enrollment and to increase access to the TC programs, two focus groups (male/female) were con- ducted for each cohort to identify barriers and pro- moters to participation in a community based TC program. The focus group attendees were recruited from the local community and key contacts from community housing agencies in the geographical area. Information attained from participants of these focus groups helped identify information dissemination strategies (and areas) in the Jane-Finch and Dundas-Spadina neighborhood for targeted recruitment. In addition to these strategies, fur- ther recruitment of study participants was achieved through networking and invitations from focus group par- ticipants. Three cohorts of participants were followed; The primary objective of the current study was to exam- ine the effect of a 16 week TC program on musculoskel- etal health-related fitness and self-reported physical health changes in a sample of low SES, ethnically diverse mid to older community dwelling Canadian adults. As a second- ary objective we aimed to identify factors related to overall changes in musculoskeletal health-related fitness and SF 36 physical health improvement. Methods The study targeted community dwelling mid to older adults of various ethnicities of origin in two locations in Page 3 of 10 Manson et al. BMC Geriatrics 2013, 13:114 http://www.biomedcentral.com/1471-2318/13/114 Manson et al. BMC Geriatrics 2013, 13:114 http://www.biomedcentral.com/1471-2318/13/114 kilograms was measure to the nearest 0.1 kg. Grip strength was taken using a dynamometer using each in- dividual hand allowing for two trials with the maximum of each combined together. The arm curl test in 30 sec- onds was done with the participant sitting on a chair with back straight and feel flat on the floor and the dom- inant side of the body close to the edge of the seat. For men the weight was 3.63 kg pounds and for women the weight was 2.27 kg and was held in the dominant hand, perpendicular to the floor with a handshake grip. Partici- pants were allowed two repetitions without the weight to ensure proper form before the test of as many curls as possible in 30 seconds. The chair stand test in 30 sec- onds involved the participant siting in the middle of the chair with back straight, feet flat on the floor, arms crossed at the wrist and held against the chest. On the signal “go” the participant rose to a full stand, then returned to a fully seated position, the participant was allowed two stands to ensure proper form. The timed 8- foot up and go test had the participant sitting in a chair that was placed against a wall and facing a cone marker exactly 8 feet away, on the signal “go” the participant got up from the chair, walked as quickly as possible around either side of the cone and sat back down in the chair. The participant was allowed one test practice and then two test trials. The sit and reach test involved a small warm up of a modified hurdle stretch held for 20 sec- onds for each leg before they placed their feet, without shoes, against the flexometer. The participants was coa- ched to reach forward along the flexometer ruler while breathing out and extending their arms, palms down to a comfortable limit that was held for two seconds. This test was repeated twice. Program h For each of the three cohorts, a TC program was offered free of charge to the participants. The TC program con- sisted of 6-7 classes given throughout the week where participants were advised to attend two classes per week for 16 consecutive weeks. Classes for cohort 1 took place at a Toronto Community Housing building whereas classes for cohorts 2 and 3 took place at local commu- nity centers in their respective area. A professional Tai Chi master facilitated the classes. Each class was 60 mi- nutes long and consisted of 15 minutes of a Qigong warm up followed by 45 minutes of Yang style TC. A re- search assistant monitored participation in the TC clas- ses so that exact attendance could be recorded. Methods The SF-36 scales of physical functioning, role-physical, bodily pain and general health were assessed pre- and post-TC program were used to provide an overall summary measure of self-reported physical health [20]. cohorts 1 and 3 were recruited from the Jane and Finch area and were followed from August through December 2009, and from October 2011 through April 2012 respect- ively. Participants for cohort 2 were recruited from the Dundas and Spadina area and were followed from February through August 2011. For cohort 1, participant recruit- ment was completed prior to commencement of the TC program whereas for cohorts 2 and 3, participants were enrolled on an ongoing basis, which accounts for the 6-month duration. All participants were exposed to 16 consecutive weeks of TC. Study variables Socio-demographics and physical health-related fitness characteristics were collected at baseline and included information on sex, age, education, smoking/drinking status, marital status, income and chronic conditions. Weekly PA, based on the CPAFLA Healthy Physical Ac- tivity Participation that examines frequency, intensity and perceived fitness, - and previous lifetime TC partici- pation of more than one year, were also recorded [19]. Pre- and post-TC program musculoskeletal health- related fitness characteristic testing was conducted by qualified exercise personnel and were assessed pre- and post-TC program by employing a combination of the Canadian Physical Activity Fitness and Lifestyle Ap- proach and the Senior Fitness Test [12,19]. These mea- sures included anthropometrics (height and weight which was used to calculate body mass index), upper body (overall grip strength, arm curl test in 30 seconds), lower body (chair stand test in 30 seconds, timed 8-foot up and go test) and lower back flexibility measure (sit- and-reach). Height was measured using a wall mounted tape measure without footwear, standing erect, arms hanging by the sides with feet together, the heels and back in contact with the wall using a set square the measure was made to the nearest 0.5 cm. Weight was measured using a calibrated scale on a wooden surface with the participant wearing light clothing, the weight in Statistical analysis Diff i b Differences in baseline socio-demographic, physical health related characteristics, musculoskeletal health and SF-36 physical scales among participants by cohort was performed using chi square tests and a one-way ANOVA. To compare the health-related musculoskeletal fitness measures and SF 36 physical scales for the pre versus post TC program values pair samples t-tests were performed for both the individual cohorts and the com- bined cohorts, effect size was determined using a Cohen’s D calculation. To examine the determinants of health predictors of changes for these outcomes, a multi- variate linear regression model was performed for each of the health-related musculoskeletal fitness dependent variables and the overall summary measure of the SF 36 scales. For each of the regression models the dependent Manson et al. BMC Geriatrics 2013, 13:114 http://www.biomedcentral.com/1471-2318/13/114 Manson et al. BMC Geriatrics 2013, 13:114 http://www.biomedcentral.com/1471-2318/13/114 Page 4 of 10 Tables 1 and 2 summarize the socio-demographic char- acteristics, physical characteristics, health-related muscu- loskeletal fitness characteristics and SF 36 physical scales of the 209 study participants. There were initially 79.9% female and 20.1% male participants and the overall mean age of the participants at enrollment was 68.1 years (range 50-87 years). The ethnicity of origin of participants in- cluded China (36.1%), South America (26.3%), Caribbean (6.3%), Europe (16.1%), South Asia (4.9%), Canada (6.2%) and other (3.9%). variable was the percent change (calculated as the post minus the pre score divided by the pre score and multi- plied by 100) and the independent variables included age, sex, marital status, education, income, ethnicity of origin (defined by Chinese versus non-Chinese origin), attendance, previous TC experience, weekly physical ac- tivity and multi-morbidity influences. Standardized beta coefficients and R2 were reported. Significance was set at an alpha of 0.05. The study was approved by the human participants ethics review committee of York University. variable was the percent change (calculated as the post minus the pre score divided by the pre score and multi- plied by 100) and the independent variables included age, sex, marital status, education, income, ethnicity of origin (defined by Chinese versus non-Chinese origin), attendance, previous TC experience, weekly physical ac- tivity and multi-morbidity influences. Standardized beta coefficients and R2 were reported. Significance was set at an alpha of 0.05. The study was approved by the human participants ethics review committee of York University. Several differences existed between cohorts notably with cohort 2 having a lower mean age of 63.8 years of age. Statistical analysis Diff i b Cohort 3 also had a higher prevalence of (greater than high school) education (30.4%), whereas cohort 2 had a higher percentage of married participants (64.9%). Finally, cohort 1 had the greatest proportion of lower in- come status (less than $14,000) participants (90.4%). Figure 1 Flowchart of study. Results A total of 209 participants were recruited for this study (78, 80, and 51 for cohorts 1, 2, and 3 respectively). Figure 1 shows the study flow, recruitment, enrollment and loss to follow up. Of the 209 overall sample re- cruited, 56 (26.7%) did not complete the study and were lost to follow-up. Reasons for loss to follow-up included health reasons not related to the TC program, travel, busy or inaccessible for post TC program data collection and unknown reasons. Baseline musculoskeletal fitness characteristics for the overall cohort can be seen in Table 2. Differences be- tween musculoskeletal fitness characteristics between cohorts were also observed, cohort 1 having a lower Figure 1 Flowchart of study. Figure 1 Flowchart of study. Manson et al. Results BMC Geriatrics 2013, 13:114 Page 6 of 10 http://www.biomedcentral.com/1471-2318/13/114 Manson et al. BMC Geriatrics 2013, 13:114 http://www.biomedcentral.com/1471-2318/13/114 Manson et al. BMC Geriatrics 2013, 13:114 Page 6 of 10 Table 1 Baseline socio-demographic & physical characteristics of program participants (Continued) Table 1 Baseline socio-demographic & physical characteristics of program participants (Continued) cWeekly physical activity Needs improvement/fair 35 (23.3) 14 (18.4) 15 (20.0) 6 (12.5) Good 16 (7.9) 8 (10.5) 4 (5.3) 4 (8.3) .606 Very good/excellent 150 (74.6) 56 (71.0) 56 (74.7) 38 (79.6) Mean (SD) 6.7 (3.1) 6.4 (2.9) 6.6 (3.1) (79.6) .196 dPrevious Tai Chi 38 (18.1) 0 (0.0) 32 (40.0) 6 (11.8) <0.001 aCOPD: chronic obstructive lung disease. bMulti-morbidity: two or more chronic conditions. cPhysical Activity: based on the Healthy Physical Activity Participation Questionnaire. dPrevious Tai Chi: Previous Tai Chi participation more than one year. Note: Totals may vary due to missing responses. significant improvements were observed in musculoskel- etal health measures of overall grip strength, arm curl in 30 seconds, chair stand in 30 seconds, 8-foot up and go test and sit and reach as well as physical functioning, general health, and the aggregate summary measure of physical health in the SF 36 (p < 0.05). upper body strength (overall grip strength 46.8 ± 16.8 kg and arm curl test in 30 seconds 11.9 ± 4.0). Cohort 1 also was below the other cohorts in lower body musculoskel- etal strength (chair stand test in 30 seconds 10.0 ± 3.1 and timed 8-foot up and go 8.8 ± 4.2). As well in the SF 36 physical summary measure cohort 1 had a lower mean score of 46.9 ± 8.8. Table 4 shows results of the multivariate linear regres- sion models. Overall, no common health determinants explained a significant portion of the variation in percent changes of the musculoskeletal health-related fitness and SF 36 physical health measure. Percent change in body mass index, overall grip strength, arm curl in 30 seconds, chair stand in 30 seconds and the 8-foot up and go test all showed a significant linear association (p < 0.05) with a For the overall sample, mean attendance was 1.1 ± 0.94 sessions per week with 0.9 ± 0.72, 0.9 ± 1.44, and 1.3 ± 0.86 for cohorts 1, 2 and 3 respectively. Table 3 summarizes the differences in effect size using Cohen’s D for all outcomes. Results BMC Geriatrics 2013, 13:114 http://www.biomedcentral.com/1471-2318/13/114 Page 5 of 10 aphic & physical characteristics of program participants mbined cohorts N (%) Cohort 1 N (%) Cohort 2 N (%) Cohort 3 N (%) P 209 (100.0) 78 (37.3) 80 (38.2) 51 (24.4) 42 (20.1) 17 (21.8) 16 (20.0) 9 (17.6) 167 (79.9) 61 (78.2) 64 (80.0) 42 (82.4) .847 73 (35.3) 12 (15.4) 46 (57.5) 15 (30.6) 86 (41.5) 41 (52.6) 28 (35.0) 17 (34.7) <0.001 48 (23.2) 25 (32.1) 6 (7.5) 17 (34.7) 68.1 (8.62) 71.3 (6.7) 63.8 (7.7) 70 (9.8) <0.001 74 (36.1) 0 (0) 73 (91.3) 1 (2.0) 54 (26.3) 45 (60.8) 2 (2.5) 7 (13.7) 13 (6.3) 9 (12.2) 1 (1.3) 3 (5.0) <0.001 33 (16.1) 7 (9.5) 2 (2.5) 24 (47.1) 10 (4.9) 10 (13.5) 0 (0) 0 (0) 13 (6.3) 1 (1.4) 0 (0) 12 (23.5) 8 (3.9) 2 (2.6) 2 (2.5) 4 (7.8) 94 (46.5) 47 (61.0) 33 (41.2) 14 (28.6) 79 (39.1) 25 (32.5) 34 (44.7) 20 (40.8) <0.001 29 (14.3) 5 (6.5) 9 (11.8) 15 (30.4) 4 (1.9) 2 (2.6) 1 (1.3) 1 (1.9) .833 45 (21.4) 17 (21.8) 9 (11.3) 19 (36.5) .003 112 (54.9) 59 (75.6) 27 (35.1) 26 (52.0) <0.001 90 (44.1) 18 (23.1) 50 (64.9) 23 (48.0) 135 (71.4) 66 (90.4) 52 (70.3) 17 (40.5) 35 (18.5) 5 (6.8) 12 (16.2) 18 (42.9) <0.001 19 (10.1) 2 (2.7) 10 (13.5) 7 (16.7) 105 (50.0) 52 (49.4) 27 (33.8) 26 (50.0) <0.001 102 (48.6) 38 (51.3) 39 (48.8) 25 (48.1) .997 45 (21.4) 28 (19.5) 9 (11.3) 8.(15.4) <0.001 54 (25.7) 20 (25.6) 21 (26.3) 13 (25.0) .987 31 (14.8) 14 (17.9) 5 (6.3) 12 (23.1) .017 26 (12.4) 9 (11.5) 9 (11.3) 8 (15.4) .749 14 (7.2) 9 (11.5) 4 (5.0) 1 (1.9) .090 12 (5.7) 8 (10.3) 3 (3.8) 1 (1.9) .097 10 (4.8) 4 (5.1) 2 (2.5) 4 (7.7) .385 5 (2.4) 1 (1.3) 0 (0.0) 4 (7.7) .013 133 (63.3) 56 (71.8) 41 (51.2) 36 (69.2) .016 18 (8.6) 13 (16.7) 0 (0.0) 5 (9.6) .001 Table 1 Baseline socio-demographic & physical characteristics of program participants Combined cohorts N (%) Cohort 1 N (%) Cohort 2 N (%) Cohort 3 N (%) P Totals 209 (100.0) 78 (37.3) 80 (38.2) 51 (24.4) Sex Male 42 (20.1) 17 (21.8) 16 (20.0) 9 (17.6) Female 167 (79.9) 61 (78.2) 64 (80.0) 42 (82.4) .847 Age groups 50-64 years 73 (35.3) 12 (15.4) 46 (57.5) 15 (30.6) 65-74 years 86 (41.5) 41 (52.6) 28 (35.0) 17 (34.7) <0.001 75+ years 48 (23.2) 25 (32.1) 6 (7.5) 17 (34.7) Mean (SD) 68.1 (8.62) 71.3 (6.7) 63.8 (7.7) 70 (9.8) <0.001 Ethnicity of origin Chinese 74 (36.1) 0 (0) 73 (91.3) 1 (2.0) South American 54 (26.3) 45 (60.8) 2 (2.5) 7 (13.7) Caribbean 13 (6.3) 9 (12.2) 1 (1.3) 3 (5.0) <0.001 European 33 (16.1) 7 (9.5) 2 (2.5) 24 (47.1) South Asian 10 (4.9) 10 (13.5) 0 (0) 0 (0) Canadian 13 (6.3) 1 (1.4) 0 (0) 12 (23.5) Other 8 (3.9) 2 (2.6) 2 (2.5) 4 (7.8) Education < High School 94 (46.5) 47 (61.0) 33 (41.2) 14 (28.6) High School 79 (39.1) 25 (32.5) 34 (44.7) 20 (40.8) <0.001 > High School 29 (14.3) 5 (6.5) 9 (11.8) 15 (30.4) Smoking Status-Yes 4 (1.9) 2 (2.6) 1 (1.3) 1 (1.9) .833 Drinking Status-Yes 45 (21.4) 17 (21.8) 9 (11.3) 19 (36.5) .003 Marital status Single 112 (54.9) 59 (75.6) 27 (35.1) 26 (52.0) <0.001 Married 90 (44.1) 18 (23.1) 50 (64.9) 23 (48.0) Income <$14,000 per year 135 (71.4) 66 (90.4) 52 (70.3) 17 (40.5) $14,000-$30,000 35 (18.5) 5 (6.8) 12 (16.2) 18 (42.9) <0.001 >$30,000 19 (10.1) 2 (2.7) 10 (13.5) 7 (16.7) Chronic conditions Hypertension 105 (50.0) 52 (49.4) 27 (33.8) 26 (50.0) <0.001 Arthritis 102 (48.6) 38 (51.3) 39 (48.8) 25 (48.1) .997 Diabetes Mellitus 45 (21.4) 28 (19.5) 9 (11.3) 8.(15.4) <0.001 Sleep Disturbance 54 (25.7) 20 (25.6) 21 (26.3) 13 (25.0) .987 Depression 31 (14.8) 14 (17.9) 5 (6.3) 12 (23.1) .017 Hearing Impairment 26 (12.4) 9 (11.5) 9 (11.3) 8 (15.4) .749 Disorientation 14 (7.2) 9 (11.5) 4 (5.0) 1 (1.9) .090 Heart Disease 12 (5.7) 8 (10.3) 3 (3.8) 1 (1.9) .097 aCOPD 10 (4.8) 4 (5.1) 2 (2.5) 4 (7.7) .385 Tumour 5 (2.4) 1 (1.3) 0 (0.0) 4 (7.7) .013 bMulti-Morbidity 133 (63.3) 56 (71.8) 41 (51.2) 36 (69.2) .016 Walking Assistance 18 (8.6) 13 (16.7) 0 (0.0) 5 (9.6) .001 Table 1 Baseline socio-demographic & physical characteristics of program participants line socio-demographic & physical characteristics of program participants Manson et al. Note: # designates repetitions completed. Results Participation in the 16-week TC program showed no significant change in body mass index measures, role physical and bodily pain. However, Table 2 Baseline health-related musculoskeletal fitness characteristics and SF 36 physical scales and physical summary measure of study participants Combined cohorts mean (SD) Cohort 1 mean (SD) Cohort 2 mean (SD) Cohort 3 mean (SD) P Anthropometric measures Body Mass Index (kg/m2) 26.7 (4.8) 28.3 (4.8) 24.3 (3.7) 28.2 (5.2) <0.001 Upper body musculoskeletal measures Overall Grip Strength (kg) 54.3 (17.7) 46.8 (16.8) 59.8 (16.5) 57.7 (16.8) <0.001 Arm Curl Test in 30 Seconds (#) 15.5 (5.4) 11.9 (4.0) 17.9 (4.4) 17.6 (5.9) <0.001 Lower body musculoskeletal measures Chair Stand Test in 30 Seconds (#) 12.2 (4.1) 10.0 (3.1) 13.9 (4.1) 13.2 (3.8) <0.001 Timed 8-Foot Up and Go (secs) 7.6 (3.2) 8.8 (4.2) 6.4 (1.7) 7.8 (2.2) <0.001 Low back flexibility measures Sit and Reach (cm) 26.4 (9.1) 25.3 (8.2) 26.2 (9.6) 28.7 (9.1) .141 SF 36 physical scales Physical Functioning (PF) 75.0 (21.6) 67.2 (23.8) 80.9 (16.4) 78.0 (22.1) <0.001 Role-Physical (RP) 80.9 (25.6) 74.4 (29.5) 87.0 (21.1) 81.8 (23.1) .009 Bodily Pain (BP) 68.7 (24.8) 62.8 (27.2) 74.1 (22.7) 69.4 (22.2) .017 General Health (GH) 64.8 (20.5) 64.9 (21.1) 60.5 (20.2) 71.7 (18.3) .012 SF 36 physical summary measure Physical Health (PCS) 49.2 (7.9) 46.9 (8.8) 50.7 (5.9) 50.3 (8.6) .009 Note: # designates repetitions completed. h-related musculoskeletal fitness characteristics and SF 36 physical scales and physical summary icipants elated musculoskeletal fitness characteristics and SF 36 physical scales and physical summary e health-related musculoskeletal fitness characteristics and SF 36 physical scales and physical summ dy participants Table 2 Baseline health-related musculoskeletal fitness characteristics and SF 36 physical scales a measure of study participants Manson et al. Results BMC Geriatrics 2013, 13:114 http://www.biomedcentral.com/1471-2318/13/114 Page 7 of 10 Page 7 of 10 Table 3 Mean difference of health-related musculoskeletal fitness measures, SF 36 physical scales and summary measure Combined cohorts Cohen’s D P Cohort 1 Cohen’s D P Cohort 2 Cohen’s D P Cohort 3 Cohen’s D P Anthropometric measures Body Mass Index (kg/m2) −0.048 .450 0.100 .906 −0.273 .004 −0.778 .002 Upper body musculoskeletal measures Overall Grip Strength (kg) 0.312 <0.001 0.420 .002 0.188 .262 0.298 .133 Arm Curl Test in 30 Seconds (#) 0.673 <0.001 0.895 <0.001 0.661 <0.001 0.404 .034 Lower body musculoskeletal measures Chair Stand Test in 30 Seconds (#) 0.717 <0.001 0.957 <0.001 0.790 <0.001 0.676 .001 Timed 8-Foot Up & Go Test (secs) −0.300 .001 −0.214 .321 −0.533 <0.001 −0.231 .047 Low back flexibility measures Sit & Reach (cm) 0.268 .004 0.155 .315 0.188 .167 0.575 .009 SF 36 physical scales Physical Functioning (PF) 0.326 <0.001 0.355 .006 0.326 .004 0.276 .180 Role Physical (RP) 0.037 .663 −0.069 .696 0.004 .918 0.368 .046 Bodily Pain (BP) 0.047 .587 −0.087 .557 0.051 .655 0.300 .069 General Health (GH) 0.241 .005 0.252 <0.001 0.209 <0.001 0.284 <0.001 SF 36 physical summary measure Physical Health (PCS) 0.192 .033 0.067 .582 0.209 .025 0.487 .055 Cohen’s D: mean difference calculated as post measure minus pre measure divided by standard deviation. Note: # designates repetitions completed. Table 3 Mean difference of health-related musculoskeletal fitness measures, SF 36 physical scales and summary measure limited number of predictors. The sit and reach test and the physical health summary measure showed no signifi- cant linear associations’. characteristics differed at baseline. Cohort 1 was based in a Toronto Community Housing building with an onsite auditorium while cohort 2 and 3 were both based in community centers that needed some participants to use some form of transportation to attend. Cohort 2 in the Chinese community had a wider age range of partici- pants that used the community center and were moti- vated to partake in multiple social programs throughout the day. Toronto can have a variety of weather that can also influence attendance to any community program. Cohort 1 occurred summer into fall, whereas cohort 2 took place between spring into summer and cohort 3 oc- curred fall into winter. Results However despite these influences, the TC program showed consistent improvements across all cohorts in both health-related musculoskeletal fitness and SF 36 summary physical health measures. Discussion Finally when looking at the psychometric based physical scales from the SF 36 and the pre post differences in our study we found that physical function (PF), general health (GH) and the over- all physical health summary measure (PCS) were signifi- cant. Our findings were similar with other Tai Chi studies that also showed significant changes in the phys- ical scales of the SF 36 [34]. length of hospital stay [25]. Multiple TC studies have found an increase in overall hand-grip strength [26] as did this present study. Similarly, evidence continues to build on the beneficial effects of musculoskeletal fitness in the prevention of chronic diseases and in combination with performance of activities of daily living [27]. Upper and lower body musculoskeletal fitness is important in executing many normal everyday activities such as household chores, carrying groceries, lifting objects and picking up grandchildren [24] as well as performance variables such as gait, stair climbing, rising from a chair, and balance [28]. Multiple TC studies have also shown upper and lower body strength improvements in older adults in line with our findings [29,30]. In Canada the fall-related injury rate is nine times greater among se- niors than among those less than 65 years of age [31] thus the importance to prioritize programs that improve balance, mobility and strength. The timed 8 foot Up and Go test was specifically designed to test motor agility and dynamic balance for older adults [24]. In our study there was a significant improvement for the timed 8 foot Up and Go test similar to recent TC studies [32]. As older adults age their range of motion decreases and in- sufficient hamstring flexibility is associated with low back pain, increased susceptibility to injury and in- creased risk of falling [24]. Once again in our study there was a significant change in flexibility similar to other findings in the TC literature [33]. Finally when looking at the psychometric based physical scales from the SF 36 and the pre post differences in our study we found that physical function (PF), general health (GH) and the over- all physical health summary measure (PCS) were signifi- cant. Our findings were similar with other Tai Chi studies that also showed significant changes in the phys- ical scales of the SF 36 [34]. Discussion This study examined the effect of a 16 week TC program on musculoskeletal health-related fitness changes and self-reported physical health in a community based pro- gram with low income mid to older adults from multiple ethnicities of origin. This study is the first TC study in Canada to look at a community-based program in this specific population. Results showed that a 16 week pro- gram aimed at 2 sessions per week participation has the potential for being beneficial on improving health- related musculoskeletal fitness and SF 36 measures, re- sults could not be explained by traditional determinants of health. These benefits may be particularly valuable given that many participants attendance averaged less than 2 sessions a week, yet still showed improvements in health- related musculoskeletal measures and self-perceived phys- ical health. The ability of aging adults to maintain quality of life through activities of daily living is important in a coun- try that has an ever increasing maturing population [22]. To enhance health and empower self-management in this area accessible PA is an important factor for creat- ing and sustaining well-being at all ages and especially so in aging adults [23]. The basic body functions, such as strength, endurance, balance and flexibility in upper and lower extremities, are all important to maintain physical independence in older age [24]. Overall grip strength has been found to be a reliable tool for the pre- dictor of mortality, disability, health complications and Research in community based settings often deal with complexities of health challenges (as well as environ- mental challenges) that can influence the research goals and program practices [21]. Factors such as geography, time of year, population density, population demograph- ics plus cultural influences combine to create different and unique cohort influences. In this study multiple Manson et al. Discussion BMC Geriatrics 2013, 13:114 http://www.biomedcentral.com/1471-2318/13/114 Page 8 of 10 Table 4 Multivariate linear regression for relationship between musculoskeletal health, SF 36 physical summary measure and determinates of health Body mass index (kg/m2) Overall grip strength (kg) Arm curl test in 30 seconds (#) Chair stand test in 30 seconds (#) Time 8-foot up and go test (secs) Sit and reach (cm) SF 36 physical health Beta P Beta P Beta P Beta P Beta P Beta P *Beta P Age −0.032 .783 0.124 .290 0.274 .024 0.269 .025 −0.224 .046 −0.237 .060 0.192 .120 aGender 0.070 .470 0.090 .363 0.112 .261 0.009 .930 0.016 .868 −0.042 .703 0.107 .302 bMarital status 0.098 .377 −0.263 .018 0.021 .852 0.003 .977 −0.017 .872 −0.038 .752 0.084 .302 Education: cHigh school −0.125 .235 −0.063 .545 0.215 .043 0.112 .294 −0.299 .004 −0.075 .752 0.197 .069 c > High school 0.158 .192 0.082 .498 0.127 .303 0.193 120 -0.328 .006 0.013 .924 0.142 .278 dIncome: $15,000-$30,000 −0.031 .766 −0.63 .545 −0.112 .291 0.096 .361 0.139 .165 −0.066 .564 0.050 .642 >$30,000 0.071 .513 -0.052 .630 -0.024 .303 -0.060 .592 0.241 023 0.136 .266 -0.092 .426 Ethnicity: Chinese origin −0.062 .651 0.134 .328 0.079 .572 0.327 .019 −0.248 .06 −0.04 .761 0.140 .313 eTC attended −0.004 .973 −0.118 .328 −0.049 .648 0.038 .723 0.018 .858 −0.025 .827 −0.173 .111 fPrevious TC experience −0.153 .204 −0.118 .329 −0.068 .587 −0.047 .702 0.046 .688 −0.038 .769 −0.070 .569 gPhysical activity −0.223 .036 0.057 .577 −0.073 .479 0.056 .588 0.065 .504 −0.004 .974 0.058 .579 hMulti-morbidity 0.066 .506 0.009 .929 0.036 .716 −0.027 .784 0.120 .202 −0.054 .616 0.011 .913 R2 .166 .130 .107 .102 .177 .069 .086 aReference group male Table 4 Multivariate linear regression for relationship between musculoskeletal health, SF 36 physical summary measure and determinates of health Up and Go test similar to recent TC studies [32]. As older adults age their range of motion decreases and in- sufficient hamstring flexibility is associated with low back pain, increased susceptibility to injury and in- creased risk of falling [24]. Once again in our study there was a significant change in flexibility similar to other findings in the TC literature [33]. Discussion Even though this present study was a unique combin- ation of low SES with multiple ethnicities there have been some TC studies that have used low SES popula- tions. One study was done in a community center based in the United States with a lower income Chinese ethni- city of origin population [29]. This group of 39 mixed gender older adults were enrolled in a 12 week Yang Manson et al. BMC Geriatrics 2013, 13:114 http://www.biomedcentral.com/1471-2318/13/114 Page 9 of 10 validity of results. These limitations include uncontrolled program induced changes in daily physical activities, sea- sonal variations in health status and mood, lifestyle fac- tors and self-reporting bias. However it should be noted the strength of this study are the real world outcomes demonstrated in a high at risk population that is under researched and over exposed to stressors from both aging and lower income. style TC program and demonstrated significant results in muscular strength similar to our study [29]. Another group of low income Caucasian seniors living in the United States were enrolled in a 6 month TC study that focused on physical functioning as the primary outcome [35]. This study also showed significant improvements in all aspects of physical functioning [35]. Since this was a preliminary analysis targeting multiple ethnicities of origin and a low SES population, our goal was to explore potential relationships while attempting to control for known confounders. In this present study we found that no overall independent variables were strong predictors of health-related musculoskeletal fit- ness and SF 36 changes, however there were select indi- vidual variables that had predictive influences. It is also important to note that despite select significant vari- ables, R2 values in the multivariate linear regression models were consistently small throughout all models. However since the duration of the program was only 4 months this was possibly too short a time to fully dis- cover potential mediators. Future studies should be de- signed specifically to explore these mediators further. The few predictors that showed some potentially small influential effects such as initial physical activity levels, marital status, multi-morbidity and age are worth some consideration. As community PA programs continue to grow all populations, especially older adults, can in- crease their health benefits when participating in these programs even with small changes in BMI regardless of their initial PA levels [36]. Conclusion h f d These findings suggest that a community based TC pro- gram with low income mid to older adult participants consisting of multiple ethnicities has the potential to be beneficial in improving health-related musculoskeletal fitness changes through strength, flexibility improve- ments and SF 36 physical scales. This program was ef- fective with a wide range of participants with multiple chronic conditions ranging from metabolic to orthopedic that influenced a large range of functional abilities. It is important to note that a community program such as this can be offered at a minimal cost making it an ac- cessible and sustainable approach to maintaining and/or enhancing health-related fitness in a wide variety of participants. Authors’ contributions JM E l f i i JM: Enrolment of participants, data collection, data analysis, write-up of manuscript. MR: Statistician lead and critical revision of the manuscript for important intellectual Content. VJ: Research methods and critical revision of the manuscript for important intellectual Content. CA: Design of the project, research methods and critical revision of the manuscript for important intellectual Content. HT: Conception and design of the project, critical revision of the manuscript for important intellectual Content. All authors read and approved the final manuscript. Author details 1York University, 341 Bethune College, 4700 Keele Street, M3J 1P3 North York, Ontario, Canada. 2University of Western Ontario York University, 341 Bethune College, 4700 Keele Street, M3J 1P3 North York, Ontario, Canada. 3University of Toronto, York University, 341 Bethune College, 4700 Keele Street, M3J 1P3 North York, Ontario, Canada. 4Queen’s University, York University, 341 Bethune College, 4700 Keele Street, M3J 1P North York, Ontario, Canada. 5McGill University, York University, 341 Bethune College, 4700 Keele Street, M3J 1P3 North York, Ontario, Canada. Received: 25 March 2013 Accepted: 19 September 2013 Published: 28 October 2013 Competing interests Th fi i l p g There are on financial competing interests. No stocks or shares are held that are associated with this publication. No currently applying patents are involved. There are no non-financial competing interests. Acknowledgements The project was funded by Social Sciences and Humanities Research Council of Canada. Sport Canada Research Initiative. Sport Canada Research Initiative. Sport Canada Research Initiative. Discussion Although research has ex- plored marital status and longevity [37] the relationship between marital status and grip strength would be inter- esting to explore in futures studies. Grip strength has become a powerful tool for predicting frailty and in- creased risk for early morbidity and mortality and even despite increases in chronic conditions, as seen in our sample population, strength can be improved [25]. Fi- nally as the population ages it is important to under- stand that this increase in years does not mean that strength, agility and power cannot be positively modified by physical activity programs like TC and that increased age also highlights the importance of physical activity programs as an anti-aging intervention [38]. In our ana- lysis no predictors were associated with the sit and reach test and the summary measure of physical health in the SF 36. Despite the absence of strong predictors this could potentially point in the direction of the broad adaptability of TC on the musculoskeletal system. Since TC is a complex, multicomponent intervention that in- tegrates effects on multiple systems [39] being unable to narrow outcomes to specific predictors could simply be reinforcing its strong multivariate influence. Unlike a randomized controlled study, the current study design has known limitations related to internal 1. Warburton DER, Charlesworth S, Ivey A, Nettlefold L, Bredin SSD: A systematic review of the evidence for Canada’s Physical Activity Guidelines for Adults. Int J Behav Nutr Phys Act 2010, 7:39. References 1. Warburton DER, Charlesworth S, Ivey A, Nettlefold L, Bredin SSD: A systematic review of the evidence for Canada’s Physical Activity Guidelines for Adults. Int J Behav Nutr Phys Act 2010, 7:39. Unlike a randomized controlled study, the current study design has known limitations related to internal Page 10 of 10 Manson et al. BMC Geriatrics 2013, 13:114 http://www.biomedcentral.com/1471-2318/13/114 31. Health M: Preventing falls and fall-related injuries in. A review of best practices. Impact: Manitoba; 2005. 2. Paterson DH, Warburton DER: Physical activity and functional limitations in older adults: a systematic review related to Canada's Physical Activity Guidelines. Int J Behav Nutr Phys Act 2010, 7:38. 32. Lovecchio N, Bettoni M, Merati M, Eid L: Physical improvement in elderly female after training: sit up and speed gait. Sport Sci Rev 2012:247–261. XIX. 3. Dawson AJ, Sundquist J, Johansson SE: The influence of ethnicity and length of time since immigration on physical activity. Ethn Health 2005, 10(4):293–309. 33. Yu DH, Yang HX: The effect of Tai Chi intervention on balance in older males. J Sport Health Sci 2012, 1(1):57–60. 34. Chen K-M, Snyder M, Krichbaum KRN: Tai Chi and Well-Being of Taiwanese 34. Chen K-M, Snyder M, Krichbaum KRN: Tai Chi and Well-Being of Taiwanese Community-Dwelling Elders. Clin Gerontol 2002, 24(3-4):137–156. 4. Marshall SJ, Jones DA, Ainsworth BE, Reis JP, Levy SS, Macera CA: Race/ ethnicity, social class, and leisure-time physical inactivity. Med Sci Sports Exerc 2007, 39(1):44–51. 35. Fuzhong L, Peter H, Edward M, Duncan TE, Duncan SC, Nigel C: An evaluation of the effects of Tai Chi exercise on physical function among older persons: a randomized controlled trial. Ann Behav Med 2006, 23(2):139–146. 5. Taylor-Piliae RE: The Effectiveness of Tai Chi Exercise in Improving Aerobic Capacity J Cardiovasc Nurs 2004 19(1):48–57 Aerobic Capacity. J Cardiovasc Nurs 2004, 19(1):48–57. 6. Hong Y, Xian J: Biomechanics of Tai Chi: A Review. Sport Biomech 2007, 6(3):453–464. 36. Bean JF, Ariana V, Walter RF: Benefits of exercise for community-dwelling older adults. Arch Phys Med Rehabil 2004, 85(Suppl 3):S31–S42. 7. Zhou D, Shepard RJ, Plyley M, Davis GM: Cardiorespiratory and Metabolic Responses during Tai Chi Chuan Exercise. Can J Appl Sports Sci 1984, 9:7–10. 37. Kaplan RM, Kronick RG: Marital status and longevity in the United States population. J Epidemiol Community Health 2006, 60(9):760–765. 38. Castillo GMJ, Ortega PFB, Ruiz JR: Improvement of physical fitness as anti- aging intervention. Med Clín 2005, 124(4):146. 8. References Physical activity and older adults: World Health Organization. [http://www. who.int/dietphysicalactivity/factsheet_olderadults/en/index.html] g p p p p p p j 23. Physical activity and older adults: World Health Organization. who.int/dietphysicalactivity/factsheet_olderadults/en/index.html] 24. Langhammer BA, Stangehelle JK: Functional fitness in elderly Norwegians measured with the Senior Fitness Test. Adv Physiother 2011, 13:137–144. 24. Langhammer BA, Stangehelle JK: Functional fitness in elderly N 24. Langhammer BA, Stangehelle JK: Functional fitness in elderly Norwegians measured with the Senior Fitness Test. Adv Physiother 2011, 13:137–144. y , 25. Bohannon RW: Hand-grip dynamometry predicts future outcomes in aging adults. J Geriatr Phys Ther 2008, 31(1):3. 25. Bohannon RW: Hand-grip dynamometry predicts future outcomes in aging adults. J Geriatr Phys Ther 2008, 31(1):3. 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: 26. Kim H, So H, Song R: Effects of Tai Chi exercise on physical fitness, bone mineral density, and fracture risk in institutionalized elderly. J Korean Acad Fundam Nurs 2010, 17(3):334–342. 26. Kim H, So H, Song R: Effects of Tai Chi exercise on physical fitness, bone mineral density, and fracture risk in institutionalized elderly. J Korean Acad Fundam Nurs 2010, 17(3):334–342. 27. Wolfe RR: The underappreciated role of muscle in health and disease. Am J Clin Nutr 2006, 84:475–482. 27. Wolfe RR: The underappreciated role of muscle in health and disease. Am J Clin Nutr 2006, 84:475–482. • Convenient online submission 28. References Chen KM, Snyder M, Krichbaum K: Facilitators and barriers to elders’ practice of tai chi: a mind-body, low-intensity exercise. J Holistic Nurs 2001, 19:238–255. 39. Wayne PM, Kaptchuk TJ: Challenges inherent to T'ai Chi research: part I-T 'ai Chi as a complex multicomponent intervention. JAltern Complement Med 2008, 14(1):95–102. 9. Li Y, Cheri NC, Van Oteghen S: Effects of extended tai chi intervention on balance and selected motor functions of the elderly. Am J Chin Med 2007, 35(03):383–391. doi:10.1186/1471-2318-13-114 Cite this article as: Manson et al.: Effect of tai chi on musculoskeletal health-related fitness and self-reported physical health changes in low income, multiple ethnicity mid to older adults. BMC Geriatrics 2013 13:114. doi:10.1186/1471-2318-13-114 Cite this article as: Manson et al.: Effect of tai chi on musculoskeletal health-related fitness and self-reported physical health changes in low income, multiple ethnicity mid to older adults. BMC Geriatrics 2013 13:114. 10. Lin MR, Hwang HF, Wang YW, Chang SH, Wolf SL: Community-based tai chi and its effect on injurious falls, balance, gait, and fear of falling in older people. Phys Ther 2006, 86(9):1189–1201. 11. Konopack JF, Marquez DX, Hu L, Elavsky S, McAuley E, Kramer AF: Correlates of Functional Fitness in Older Adults. Int J Behav Med 2008, 15:311–318. 12. Rikli RE, Jones CJ: Assessing physical performance in independent older adults: Issues and guidelines. J Aging Phys Act 1997, 5:244–261. 13. Profile of Low Income in the City of Toronto 2010. 2010. [http://www. toronto.ca/demographics/pdf/poverty_profile_2010.pdf] 14. Social Planning Toronto 2009. 2009. [http://socialplanningtoronto.org/wp- content/uploads/2009/01/what-was-heard_alexandra-park.pdf] 15. Canadian census 2001. 2001. [http://www.fin.gov.on.ca/en/economy/ demographics/census/cenhi6.html] 16. Statistics Canada 2006. 2006. [http://www12.statcan.gc.ca/census- recensement/2006/dp-pd/prof/92-591/index.cfm?Lang=E] 17. United Way Toronto: Poverty by Postal Code 2. Toronto. [http://www. unitedwaytoronto.com/whatWeDo/reports/verticalPovertyReport.php] 18. Statistic Canada 2007. 2007. [http://www45.statcan.gc.ca/2007/ cgco_2007_000_e.htm] 19. Gledhill N, Jamnik V: Canadian physical activity, fitness and lifestyle approach. Ottawa, Canada: Canadian Society for Exercise Physiology; 2003. 20. Brazier JE, Harper R, Jones NM, O'Cathain A, Thomas KJ, Usherwood T, Westlake L: Validating the SF-36 health survey questionnaire: new outcome measure for primary care. BMJ 1992, 305(6846):160. 21. Minkler M: Community-based Research Partnerships: Challenges and Opportunities. J Urban Health 2005, 82(2):ii3–ii12. 22. Day JC: National population projections. U. S. Census Bureau. [http://www. census.gov/population/www/pop-profile/natproj.html] 22. Day JC: National population projections. U. S. Census Bureau. [http://www. census.gov/population/www/pop-profile/natproj.html] g p p p p p p j 23. Physical activity and older adults: World Health Organization. [http://www. who.int/dietphysicalactivity/factsheet_olderadults/en/index.html] g p p p p p p j 23. Manson et al. BMC Geriatrics 2013, 13:114 http://www.biomedcentral.com/1471-2318/13/114 References Brown M, Sinacore DR, Host HH: The relationship of strength to function in the older adult. J Gerontol 1995, 50A(Special Issue):55–59. 29. Taylor-Piliae RE, Haskell WL, Stotts NA, Froelicher ES: Improvement in balance, strength, and flexibility after 12 weeks of Tai chi exercise in ethnic Chinese adults with cardiovascular disease risk factors. Altern Ther Health Med 2006, 12(2):50–58. 30. Lia J-X, Xub DQ, Hong Y: Changes in muscle strength, endurance, and reaction of the lower extremities with Tai Chi intervention. J Biomech 2009, 42(8):967–971. 30. Lia J-X, Xub DQ, Hong Y: Changes in muscle strength, endurance, and reaction of the lower extremities with Tai Chi intervention. J Biomech 2009, 42(8):967–971.
https://openalex.org/W4386587960	https://jprs.gov.iq/index.php/jprs/article/download/729/511	English	null	Petrography and Geochemistry of Zubair Shale Formation in Rumaila Oilfield, Southern Iraq: Implications for Provenance and Tectonic Setting	Mağallaẗ al-buḥūṯ wa-al-dirāsāt al-nafṭiyyaẗ	2,023	cc-by	10,285	Journal of Petroleum Research and Studies Journal of Petroleum Research and Studies P- ISSN: 2220-5381 E- ISSN: 2710-1096 P- ISSN: 2220-5381 E- ISSN: 2710-1096 Open Access No. 40, September 2023, pp. 19-40 Open Access No. 40, September 2023, pp. 19-40 Abstract A detailed sequential analysis which included thin section petrography, X-ray diffraction, and X- ray fluorescence was applied to investigate the mineral, chemical classifications, provenance, paleo-weathering, paleoclimate, and maturity features of such Zubair oil shale in the Rumaila oilfield in southern Iraq. In core samples and thin sections, the analyzed shales are primarily silty, flaky to subflaky, micaceous, calcareous, well-sorted, poorly cemented, and weakly to moderately compacted silt-grade sandy mudstone. According to XRD analysis, the main mineral is quartz, which is followed by kaolinite, while calcite and dolomite are less common and Illite, Illite/smectite, and pyrite are rarely abundant. Petrographic analysis of the Zubair shales revealed four lithofacies: silty clayey laminated mudstone lithofacies, mica-rich mudstone lithofacies, clay-rich siliceous mudstone lithofacies, and clay-bearing calcareous mudstone lithofacies. Major and trace element concentrations reveal that the oil shales have been formed from felsic rocks such as granodiorite, tonalite, and granite. A passive margin setting was revealed by the tectonic discrimination diagram. Weathering index values like the (CIA) chemical index of alteration, the (PIA) plagioclase index of alteration, and the (CIW) chemical index of weathering imply extensive chemical weathering in the source area. Zubair shales' K2O/Na2O ratio and (ICV) index of compositional variation are uniform with their high maturity. The compositions of mineral and trace elemental ratios, as well as the climatic index "C," indicate a warm to humid subtropical climate with deposition in a shallow oxic and dysoxic environment. eywords: Paleoclimate, Paleo-weathering, Provenance, Tectonic setting, Zubair shales. Petrography and Geochemistry of Zubair Shale Formation in Rumaila lfield, Southern Iraq: Implications for Provenance and Tectonic Setting This work is licensed under a Creative Commons Attribution 4.0 International License. P- ISSN: 2220-5381 E- ISSN: 2710-1096 Such a formation is comprised of multiple depositional cycles. So every cycle represents the forming of a deltaic lobe (construction phase), then by a prolonged break after which the delta margin has been subjected to tidal action and wave (destruction phase). Finally, the cycle was completed by the formation of a marine transgressive shale. However, when growth of a deltaic lobe has been renewed above a shale base, a new cycle starts [3]. The Zubair Formation is dominated by sequences of siliciclastic lithostratigraphic, with lim. constrained towards the Formation's upper portion, which represents a transgressive phase. The shale packs thin out toward the west of the research, whereas the coarse clastic packs thin out toward the east [4]. The Zubair Formation's upper contact was defined by the presence of shale following the Shuaiba Carbonate Formation, while the lower contact was defined by the presence of the Ratawi Limestone Formation (Fig. 1B). [5] studied trialing and assessment of shale stabilization for Zubair Shale Formation. [6] investigated the geochemistry of the Zubair Formation (upper shale member) in both northern and southern Rumaila oilfield also compared between the major and trace elements in both fields. Based on mineralogical and geochemical features, the study aims to examine the mineral, chemical classifications, paleo-weathering, provenience, paleoclimate and maturity features of the upper, middle and lower oil shale members in the research areas. This makes it possible to research oil shale's composition, quality parameters, and formation process. Additionally, since 1953 in Rumaila Oilfields, the Zubair oil shale's industrial quality has been evaluated. It's also necessary to re-assess the efficiency of Zubair oil shale, to provide new parameters for the future assessment of oil shale in the Rumaila Oilfield in the context of improving current experimental technology. P- ISSN: 2220-5381 E- ISSN: 2710-1096 P- ISSN: 2220-5381 E- ISSN: 2710-1096 Open Access No. 40, September 2023, pp. 19-40 can be considered an important reservoir in southern Iraq and neighboring countries [1]. It is interesting to note that the shale constituents are the source lithology within the formation, even though they contain some rare elements and pyrite, which demonstrate a depositional environment that is reductive and acidic. Rocks known as source rocks have an abundance of organic material that, when heated and compressed over time, can yield hydrocarbons. Shales and limestones are the most common source rocks (i.e., sedimentary rocks). This evidence backs up the hypothesis that the source rock created in the Zubair Formation [2]. Such a formation is comprised of multiple depositional cycles. So every cycle represents the forming of a deltaic lobe (construction phase), then by a prolonged break after which the delta margin has been subjected to tidal action and wave (destruction phase). Finally, the cycle was completed by the formation of a marine transgressive shale. However, when growth of a deltaic lobe has been renewed above a shale base, a new cycle starts [3]. The Zubair Formation is dominated by sequences of siliciclastic lithostratigraphic, with lim. constrained towards the Formation's upper portion, which represents a transgressive phase. The shale packs thin out toward the west of the research, whereas the coarse clastic packs thin out toward the east [4]. The Zubair Formation's upper contact was defined by the presence of shale following the Shuaiba Carbonate Formation, while the lower contact was defined by the presence of the Ratawi Limestone Formation (Fig. 1B). [5] studied trialing and assessment of shale stabilization for Zubair Shale Formation. [6] investigated the geochemistry of the Zubair Formation (upper shale member) in both northern and southern Rumaila oilfield also compared between the major and trace elements in both fields. can be considered an important reservoir in southern Iraq and neighboring countries [1]. It is interesting to note that the shale constituents are the source lithology within the formation, even though they contain some rare elements and pyrite, which demonstrate a depositional environment that is reductive and acidic. Rocks known as source rocks have an abundance of organic material that, when heated and compressed over time, can yield hydrocarbons. Shales and limestones are the most common source rocks (i.e., sedimentary rocks). This evidence backs up the hypothesis that the source rock created in the Zubair Formation [2]. 1. Introduction: Lower Cretaceous deposits, particularly in Southern Iraq, are extremely valuable due to significant hydrocarbon accumulations but also reservoirs (Fig. 1A). The Zubair Formation is part of the Late Berriasian-Albian cycle formations, due to its excellent reservoir properties, it 19 Journal of Petroleum Research and Studies P- ISSN: 2220-5381 E ISSN: 2710 1096 Open Access No 40 September 2023 pp 19 40 Journal of Petroleum Research and Studies P- ISSN: 2220-5381 E- ISSN: 2710-1096 Open Access No. 40, September 2023, pp. 19-40 Journal of Petroleum Research and Studies P- ISSN: 2220-5381 E- ISSN: 2710-1096 P- ISSN: 2220-5381 E- ISSN: 2710-1096 P- ISSN: 2220-5381 E- ISSN: 2710-1096 Open Access No. 40, September 2023, pp. 19-40 subzones comprise the Mesopotamian Zone. This research's proposal is Zubair subzones, and the area is defined by latitude 30°00'-30°45'N and longitude 47°00'-47°30'E. The Zubair Subzone has been identified by numerous enlarged folds extending from the north to the northwest to the southeast, representing Basra province's vast oilfields [7]. These folds, which were eventually confined in the Late Cretaceous, were defined by narrow and linear anticlines with few clues of faulted basement stone [4], Rumaila North (R) and Rumaila South (Ru) are the oil fields in this subzone of interest. The formation in Zubair Subzone is classified: Upper shale member, Upper sandstone member, Middle shale member, Lower sandstone member, and Lower shale member. subzones comprise the Mesopotamian Zone. This research's proposal is Zubair subzones, and the area is defined by latitude 30°00'-30°45'N and longitude 47°00'-47°30'E. The Zubair Subzone has been identified by numerous enlarged folds extending from the north to the northwest to the southeast, representing Basra province's vast oilfields [7]. These folds, which were eventually confined in the Late Cretaceous, were defined by narrow and linear anticlines with few clues of faulted basement stone [4], Rumaila North (R) and Rumaila South (Ru) are the oil fields in this subzone of interest. The formation in Zubair Subzone is classified: Upper shale member, Upper sandstone member, Middle shale member, Lower sandstone member, and Lower shale member. Fig. (1): (A) Location map of the investigated wells and oil fields, (B) stratigraphic column in southern Iraq for the study area [8]. Fig. (1): (A) Location map of the investigated wells and oil fields, (B) stratigraphic column in southern Iraq for the study area [8]. 2. Geological setting The area of the current study is located in the north and south Rumaila oilfield, which is part of the Mesopotamian zone of the stable shelf division (Figure 1). Zubair, Euphrates, and Tigris 20 Journal of Petroleum Research and Studies P- ISSN: 2220-5381 Open Access Journal of Petroleum Research and Studies P- ISSN: 2220-5381 E- ISSN: 2710-1096 Open Access No. 40, September 2023, pp. 19-40 subzones comprise the Mesopotamian Zone. This research's proposal is Zubair subzones, and the area is defined by latitude 30°00'-30°45'N and longitude 47°00'-47°30'E. The Zubair Subzone has been identified by numerous enlarged folds extending from the north to the northwest to the southeast, representing Basra province's vast oilfields [7]. These folds, which were eventually confined in the Late Cretaceous, were defined by narrow and linear anticlines with few clues of faulted basement stone [4], Rumaila North (R) and Rumaila South (Ru) are the oil fields in this subzone of interest. The formation in Zubair Subzone is classified: Upper shale member, Upper sandstone member, Middle shale member, Lower sandstone member, and Lower shale member. Journal of Petroleum Research and Studies P- ISSN: 2220-5381 E- ISSN: 2710-1096 4. Results and Discussions 4. Results and Discussions P- ISSN: 2220-5381 E- ISSN: 2710-1096 P- ISSN: 2220-5381 E- ISSN: 2710-1096 Open Access No. 40, September 2023, pp. 19-40 the main components and determine the primary lithofacies, 35 thin sections of Zubair shales were prepared. Powder XRD mineralogical analysis was performed on 50 grams of the sample. the main components and determine the primary lithofacies, 35 thin sections of Zubair shales were prepared. Powder XRD mineralogical analysis was performed on 50 grams of the sample. The objective is to identify the mineralogy and semi-quantitatively analyze it using the ICDD database and the High Score Plus software. The powdered specimens for geochemical analyses have been heated at 105oC in the presence of oxygen to remove any remaining volatile components and oxidized Fe. At 1000oC, the loss on ignition (L.O.I.) of samples was determined. X-ray fluorescence (XRF) was used to determine the concentrations of major and trace elements. Table (1) The wells and samples number of Zubair shales. Table (1) The wells and samples number of Zubair shales. Well No. Sample No. Formation Depth (m) Oilfield Sample type R-31 23 Zubair 3030-3350 North Rumaila cores R-24 25 Zubair 3160-3360 North Rumaila cores Ru-15 27 Zubair 3100-3357 South Rumaila cores Ru-39 24 Zubair 3095- 3350 South Rumaila cutting 3. Materials and Methods Samples of Zubair Formation were collected from four boreholes drilled by the South Oil Company in the north and south Rumaila Oilfield in the Zubair Subzone, Southern Iraq. These wells are R-31 (3030-3350 m sampling depth), R-24 (3160-3360 m sampling depth), Ru-15 (3100-3357 m sampling depth), and Ru-39 (3095-3350 m sampling depth) (Table 1). To examine 21 Journal of Petroleum Research and Studies P- ISSN: 2220-5381 E- ISSN: 2710-1096 Open Access No. 40, September 2023, pp. 19-40 the main components and determine the primary lithofacies, 35 thin sections of Zubair shales were prepared. Powder XRD mineralogical analysis was performed on 50 grams of the sample. Journal of Petroleum Research and Studies P- ISSN: 2220-5381 E- ISSN: 2710-1096 P- ISSN: 2220-5381 E- ISSN: 2710-1096 P- ISSN: 2220-5381 E- ISSN: 2710-1096 Open Access No. 40, September 2023, pp. 19-40 clastic origin or the result of weathering of mica-rich igneous or metamorphic rocks. With the availability of K+, illite also forms from kaolinite in diagenesis after its transfer to the marine environment, or by increasing the depth of burial, the montmorillonite is completely transformed into illite, explaining the absence of montmorillonite in the ancient sediments [11]. Illite/Smectite is formed by the partial filtration of K+ or Mg(OH)2 between layers of illite or chlorite, followed by partial adsorption by layers of montmorillonite or vermiculite. This effect usually occurs during weathering or in the sedimentation basin, but it can also be hydrothermal in nature. Chlorites are established when physical and chemical changes occur in mafic minerals. Chlorites, which are common in low-temperature hydrothermal alteration and low-grade metamorphism, are not among the most reactive minerals to introduce into a sedimentary system. Chlorite is a prevalent detrital mineral throughout sedimentary systems since it weathers and changes slowly in the hinterlands, soils, also during transport [11]. clastic origin or the result of weathering of mica-rich igneous or metamorphic rocks. With the availability of K+, illite also forms from kaolinite in diagenesis after its transfer to the marine environment, or by increasing the depth of burial, the montmorillonite is completely transformed into illite, explaining the absence of montmorillonite in the ancient sediments [11]. Illite/Smectite is formed by the partial filtration of K+ or Mg(OH)2 between layers of illite or chlorite, followed by partial adsorption by layers of montmorillonite or vermiculite. This effect usually occurs during weathering or in the sedimentation basin, but it can also be hydrothermal in nature. Chlorites are established when physical and chemical changes occur in mafic minerals. Chlorites, which are common in low-temperature hydrothermal alteration and low-grade metamorphism, are not among the most reactive minerals to introduce into a sedimentary system. Chlorite is a prevalent detrital mineral throughout sedimentary systems since it weathers and changes slowly in the hinterlands, soils, also during transport [11]. Calcite and dolomite have low percentages in the samples, (4.74-9.42%), and (0-2.69%), respectively, dolomite is only present in a few of them (Figure 3 C, D & E). This indicates that the Zubair Formation's sedimentation environment is shallow, with a decrease in carbonate minerals and an increase in clastic minerals. 4.1 Mineralogy The mineral composition of five Zubair shale samples was examined using XRD experiments, which shows with petrographical study that the average quartz content (monocrystalline) in samples is 41.54 - 47.23 % (Fig.2). Quartz is a common auxiliary mineral connected with siliceous eruptions such as plutonic granite (Fig.3 A, E & F). Participating in all stages of metamorphism and possessing an elevated resistance to chemical weathering. This mineral is abundant in sedimentary rocks, along with oil shales [9]. The shale samples were rich in pore- filling detrital clays, (41.14 - 45.53 %), which including kaolinite, illite, illite/smectite and chlorite. The alteration of feldspar and muscovite produces kaolinite. There are two types of kaolin deposits: primary and secondary. Secondary kaolin is sedimentary in origin, while primary results from residual weathering or hydrothermal alteration. Kaolinite spreads in river and lake sediments where leaching processes predominate, but it quickly turns into illite in marine environments due to its low stability. Because kaolinite grains are relatively coarse, the fluctuation process is faster, and it is therefore found in deltaic areas [10]. Illite could be of the 22 Journal of Petroleum Research and Studies P- ISSN: 2220-5381 Open Access Journal of Petroleum Research and Studies P- ISSN: 2220-5381 E- ISSN: 2710-1096 Open Access No. 40, September 2023, pp. 19-40 Journal of Petroleum Research and Studies P- ISSN: 2220-5381 E- ISSN: 2710-1096 P- ISSN: 2220-5381 E- ISSN: 2710-1096 Pyrite has a percentage of 2.0- 5.24%, is abundant in both ancient and recent sedimentary rocks and is the most stable iron sulfide mineral in anoxic environments at low temperatures. Framboidal pyrites have been made up of clusters of subspheroidal to spheroidal pyrite microcrystals with a raspberry-like appearance (Figure 4 A & B). Pyrite framboids have such a unique framework composed of invariable pyrite microcrystals (euhedral-shaped), which is not even interrupted by overburden pressure. Euhedral pyrite, on the other hand, is made up of a single pyrite crystal or a grouping of pyrite crystals of varying sizes (Figure 3 A). The mineral composition of Zubair shales was also plotted using a ternary graph [12], where the total contents of (QFM) quartz, feldspar, and mica, (Carbonates) calcite and dolomite, and (Clays) kaolinite, illite, illite/smectite, and chlorite all are equivalent to 100%. The findings (Fig. 5a) show that the Zubair shale in the study region is relatively rich in quartz & clay minerals but low in carbonates, implying that it is a Silica-rich argillaceous mudstone. This mineral composition is ideal for hydraulic fracturing due to the presence of a high concentration of brittle minerals. The minor differences in shale composition between wells are depicted in Figure (5b). 23 23 Journal of Petroleum Research and Studies Journal of Petroleum Research and Studies P- ISSN: 2220-5381 E- ISSN: 2710-1096 Open Access No. 40, September 2023, pp. 19-40 Fig. (2): Annotated XRD diffractograms of geologic source samples. Peaks are highlighted in pink, and labels above the peaks indicate d-spacing in Aº. For a Cu radiation source with a K-alpha wavelength of 1.540, the X-axis is marked by degrees 2-theta. P- ISSN: 2220-5381 E- ISSN: 2710-1096 Open Access No. 40, September 2023, pp. 19-40 P- ISSN: 2220-5381 E- ISSN: 2710-1096 P- ISSN: 2220-5381 E- ISSN: 2710-1096 Fig. (2): Annotated XRD diffractograms of geologic source samples. Peaks are highlighted in pink, and labels above the peaks indicate d-spacing in Aº. For a Cu radiation source with a K-alpha wavelength of 1.540, the X-axis is marked by degrees 2-theta. Journal of Petroleum Research and Studies P- ISSN: 2220-5381 E- ISSN: 2710-1096 P- ISSN: 2220-5381 E- ISSN: 2710-1096 4.2 Petrography Throughout the sequence, irregular laminae, lenses, and mottles of sandstones and siltstones in shale ground mass can be found. The mineralogy and textures of Zubair shale samples were studied using petrographic analysis of thin sections. The shale samples examined range in color from gray to black, and highly bituminous shales also are prevalent. The Zubair shale does have a laminated structure composed of well-sorted, badly cemented, loosely to moderately compacted, silt-grade sandy mudstone, white grains (calcite mineral), heavy minerals, black assemblies (residual hydrocarbons or pyrite), kaolinite booklets, illite, and chlorite. There is no visual macroporosity and only a little fracture (mainly 10-20 µm wide black lines) that run primarily along the bedding plane. The petrographic analysis identifies four lithofacies as follows: 1- Silty-clayey laminated mudstone lithofacies: it displays lenticular to wavy laminations of silt-sized grains also with clay minerals in size much smaller than 0.05mm (Figure 3B). The silt-size grains mostly are sub-angular to angular quartz grains, with a few pyrite and mica grains thrown in for good measure (Figure 3A). The laminations are clearly defined by the conversely of dark and light color as well as fine and coarse grain sizes. Organic particles are more abundant in clay laminae than in silt laminae. It is mostly found in the Zubair Formation's middle shale member. 24 Journal of Petroleum Research and Studies P- ISSN: 2220-5381 E- ISSN: 2710-1096 Open Access No. 40, September 2023, pp. 19-40 Journal of Petroleum Research and Studies P- ISSN: 2220-5381 E- ISSN: 2710-1096 Open Access No. 40, September 2023, pp. 19-40 Open Access No. 40, September 2023, pp. 19-40 Open Access No. 40, September 2023, pp. 19-40 2- Mica-rich mudstone lithofacies: a strong compaction past has occurred in the merging of mica and clay particles (primarily muscovite) around the silica detrital particles, with organic particles defining the boundaries of these merging (Figure 3C, D). It mostly occurs in the lower shale member of the Zubair Formation. 2- Mica-rich mudstone lithofacies: a strong compaction past has occurred in the merging of mica and clay particles (primarily muscovite) around the silica detrital particles, with organic particles defining the boundaries of these merging (Figure 3C, D). It mostly occurs in the lower shale member of the Zubair Formation. 3- Clay-rich siliceous mudstone lithofacies: It's mostly brown and black, with light laminations of clay particles and silt-sized quartz grains. A few mica flakes and organic particles have been scattered, but there are no carbonate minerals to be found (Figure 3E, F). It is mostly noticed in the Zubair Formation's middle shale member. 4- Clay bearing calcareous mudstone lithofacies: it's mostly brown, with some silt-sized quartz grains and light laminations of the dominant clay particles. A few pyrite and organic particles are scattered (Figure 3G, H). This lithofacies contains common fecal pellets with preferred orientation (Figure 4C, D). It is mostly found in the Zubair Formation's upper shale member. 25 25 Journal of Petroleum Research and Studies P- ISSN: 2220-5381 E- ISSN: 2710-1096 P- ISSN: 2220-5381 E- ISSN: 2710-1096 E- ISSN: 2710-1096 No. 40, September 2023, pp. 19-40 Fig. (3): Photomicrographs of shale samples of Zubair Fm. from Rumaila oilfield. (A) Silty- clayey laminated mudstone displays (Q) silt sized quartz grain dispersed in clay (ground mass), and (P) pyrite. (B) Silty-clayey laminated mudstone with (light brown) silt size, (dark brown) clay laminae and (black) organic particles (O) aligned horizontally. (C) Mica- rich lithofacies display a compacted merging of elongated mica (M), clay (Cl), and calcite (Ca), residual inter-granular pores. (D) Mica-rich lithofacies display Ca (calcite) and D (dolomite) in clay ground mass (Cl). (E) Clayey-rich lithofacies display the dispersed nature of quartz detritus (Q), mica (M), and pyrite framboid (P) in clay groundmass. (F) (G) and (H) a gentle lamination pattern. Fig. (3): Photomicrographs of shale samples of Zubair Fm. from Rumaila oilfield. P- ISSN: 2220-5381 E- ISSN: 2710-1096 (A) Silty- clayey laminated mudstone displays (Q) silt sized quartz grain dispersed in clay (ground mass), and (P) pyrite. (B) Silty-clayey laminated mudstone with (light brown) silt size, (dark brown) clay laminae and (black) organic particles (O) aligned horizontally. (C) Mica- rich lithofacies display a compacted merging of elongated mica (M), clay (Cl), and calcite (Ca), residual inter-granular pores. (D) Mica-rich lithofacies display Ca (calcite) and D (dolomite) in clay ground mass (Cl). (E) Clayey-rich lithofacies display the dispersed nature of quartz detritus (Q), mica (M), and pyrite framboid (P) in clay groundmass. (F) (G) and (H) a gentle lamination pattern. 26 Journal of Petroleum Research and Studies P- ISSN: 2220-5381 E- ISSN: 2710-1096 P- ISSN: 2220-5381 E- ISSN: 2710-1096 E- ISSN: 2710-1096 No. 40, September 2023, pp. 19-40 Fig. (4) Photomicrographs of shale samples of Zubair Fm. from Rumaila oilfield. (A) calcite with mica grain and framboidal pyrites. (B) Framboidal pyrites. (C) & (D) facial pellets. Fig. (4) Photomicrographs of shale samples of Zubair Fm. from Rumaila oilfield. (A) calcite with mica grain and framboidal pyrites. (B) Framboidal pyrites. (C) & (D) facial pellets. Fig. (5): (a) The lithotype classification relies on a ternary graph of the mineral composition of Zubair shale samples from the Rumaila oilfield, as modified by [12]; (b) XRD and thin- section point counting charts depicting stratigraphic variation in the composition of shale samples from the R31, R24, Ru15, and Ru39 wells. Fig. (5): (a) The lithotype classification relies on a ternary graph of the mineral composition of Zubair shale samples from the Rumaila oilfield, as modified by [12]; (b) XRD and thin- section point counting charts depicting stratigraphic variation in the composition of shale samples from the R31, R24, Ru15, and Ru39 wells. 4.3 Geochemistry (Major and Trace Elements) Major & trace elements concentrations for Zubair shale samples (R31, R24, Ru15, Ru39; n= 12) are shown in Tables (2) and (3). Similar concentrations can be found in the oil shales from the Rumaila oilfield. The SiO2, Al2O3, and Fe2O3 abundances differ from 52.88 to 70.33 wt. % (av.= 60.87), 12.25 to 21.91 wt. % (av.= 16.04), and 1.66 to 6.42 wt. % (av.= 3.92), respectively. The abundance of clay minerals is associated with the high content of Al2O3. TiO2 content of Zubair shales ranges from 0.78 to 1.28 wt. % (ave.= 1.06). The average of alkali (Na+, K+), alkaline-earth (Ca2+, Mg2+), and SO3 contents in the shales are 1-2 wt. %. 27 Journal of Petroleum Research and Studies P- ISSN: 2220-5381 E- ISSN: 2710-1096 Table (2) Shows the major elements (weight percentages) by XRF, and some elemental ratios of the Zubair shale samples. CIA (%) = [Al2O3/ (Al2O3 + Na2O+ K2O+ CaO)] × 100 from [13] PIA (%) = [Al2O3-K2O/ (Al2O3 + Na2O+ K2O+ CaO)] × 100 from [14] CIW (%) = [Al2O3/ (Al2O3 + Na2O+CaO)] × 100 from [15] ICV (%) = (Fe2O3+ CaO+K2O+ MgO +Na2O+ TiO2+MnO)/Al2O3 from [16]. In which CaO* correction was used, the procedure of [17] was followed. Sample No. R31-1 R31-2 R31-3 R24-1 R24-2 R24-3 Ru15-1 Ru15-2 Ru15-3 Ru39-1 Ru39-2 Ru39-3 Mean ∑ Depth m. 4.3 Geochemistry (Major and Trace Elements) 3031 3307 3345 3165 3315 3360 3110 3330 3355 3095 3315 3349 SiO2 59.55 54.23 60.41 53.80 64.06 52.88 66.77 67.22 61.34 55.71 64.25 70.33 60.87 Al2O3 18.23 15.93 14.67 15.87 15.26 14.65 13.93 12.25 19.32 21.91 16.98 13.49 16.04 Fe2O3 3.27 4.97 5.62 6.23 6.42 4.76 3.30 3.24 2.33 1.66 3.26 2.00 3.92 CaO 0.21 0.36 1.67 1.23 0.11 2.45 0.12 1.02 0.98 1.32 1.97 1.78 1.10 MgO 1.02 2.65 1.49 1.87 1.41 2.45 2.56 2.17 1.28 0.94 1.24 1.98 1.75 Na2O 1.15 1.05 1.03 0.99 1.12 0.93 1.09 0.52 1.10 0.29 1.02 0.98 0.94 K2O 2.96 3.75 2.15 2.19 2.64 2.71 1.21 1.93 2.46 1.80 2.16 1.14 2.25 TiO2 1.02 1.12 1.00 1.12 0.99 1.24 0.95 0.78 1.28 1.27 1.09 0.88 1.06 SO3 1.26 1.32 1.55 0.98 1.45 1.85 0.77 1.66 1.16 1.09 1.57 0.98 1.30 L.O.I 10.85 14.4 9.66 15.48 6.43 15.53 9.06 8.95 8.45 13.88 5.53 6.42 10.38 Total 99.52 99.78 99.25 99.76 99.89 99.45 99.76 99.74 99.70 99.87 99.07 99.98 99.61 K2O/Na2O 2.57 3.57 2.08 2.21 2.35 2.91 1.11 3.71 2.23 6.2 2.11 1.16 2.68 Al2O3/TiO2 17.87 14.22 14.67 14.16 15.41 11.81 14.66 15.7 15.09 17.25 15.57 15.32 15.14 SiO2/Al2O3 3.26 3.4 4.11 3.39 4.19 3.6 4.79 5.48 3.17 2.54 3.78 5.21 3.91 CIA 80.84 75.53 75.15 78.25 79.76 70.63 85.19 77.92 80.97 86.53 76.72 77.57 78.75 PIA 91.82 89.62 82.26 86.03 88.54 82.37 91.31 87.01 89.01 92.58 83.21 81.73 87.12 CIW 93.05 91.86 84.45 87.72 92.54 81.25 92 88.83 90.28 93.15 85.02 83.01 88.59 ICV 0.52 0.87 0.88 0.85 0.83 0.99 0.66 0.78 0.48 0.33 0.63 0.65 0.70 e (2) Shows the major elements (weight percentages) by XRF, and some elemental ratios of the Zubair shale samples. Table (3) shows the trace elements (ppm) by XRF, and elemental ratios of Zubair shale samples. Sample No. R31-1 R31-2 R31-3 R24-1 R24-2 R24-3 Ru15-1 Ru15-2 Ru15-3 Ru39-1 Ru39-2 Ru39-3 Mean ∑ Depth m. P- ISSN: 2220-5381 E- ISSN: 2710-1096 P- ISSN: 2220-5381 E- ISSN: 2710-1096 Open Access No. 40, September 2023, pp. 19-40 between K2O, Na2O, L.O.I, Fe2O3, TiO2, and SO3 with Al2O3 suggests that silicates are primarily responsible for their distribution [18]. between K2O, Na2O, L.O.I, Fe2O3, TiO2, and SO3 with Al2O3 suggests that silicates are primarily responsible for their distribution [18]. The Zubair Shale Formation appears in shale fields on the [19] diagram (Figure 7a, b). Samples schemed in a shale field could reflect the clay minerals correlation, and source rocks can be felsic to intermediate in composition. Table (3) displays the trace element concentrations obtained from 12 oil shale specimens. Transitional trace element concentrations (for example, Mn, V, Ni, Cr, Zn, Cu, Zr, and Co) varied little in core shale. The existence of calcite and dolomite may explain the Sr concentration (25.25 to 300.66 ppm) in Zubair shale. The occurrence of large-ion lithosphere components (e.g., Sr, Ba, and Rb) in sheet silicates such as illite and illite-smectite suggests incorporation, as well as adsorbent on to the clay mineral substrates. Figure (8) depicts the elemental constituents of the Zubair shale normalized to chondrite and (PAAS) Post Archean Australian Shale [20]. The proportion of the major and trace elements at various specimens is equivalent to that of chondrite and PAAS. Al2O3, Na2O, K2O, and TiO2 are enriched comparative to chondrite, as are Sr, Zr, and Rb, with a prominent enrichment in Ba. In comparison to chondrite, CaO, MgO, Mn, Ni, Cr, and Co are depleted (Figure 8a). Normalized trace and major element components against PAAS display a minor enrichment in Ni, Zn, Zr, & Co with a depletion in CaO, Mn, Cr, Cu, and Rb (Figure 8b). 4.3 Geochemistry (Major and Trace Elements) 3031 3307 3345 3165 3315 3360 3110 3330 3355 3095 3315 3349 Mn 466 76 238 62 175 29 52 38 22 87 27 63 111.25 Sr 144.54 220.43 90.69 100.55 62.78 300.66 25.25 55.89 46.78 34.98 85.42 64.99 102.74 Ba 166.5 143.7 88.3 195.7 163.8 90.4 275 140.5 290 416 300 130 199.99 V 77.5 81 90 50 100 62 57 40 35 27 56 77 62.70 Ni 173 125 72 94 115 130 67 74 90 106 15 59 95 Cr 56 34 147 112 44 19 23 36 56 34 65 18 53.66 Zn 167 120 472 309 190 227 32 78 67 191 234 333 201.66 Cu 52 79 42 35 136 84 15 32 13 49 41 28 50.50 Zr 334 556 398 457 589 479 178 259 364 119 168 78 131.58 Rb 21 39 13 29 18 33 15 12 11 18 23 27 21.58 Co 20 29 40 38 44 18 23 27 13 67 9 17 28.75 Rb/Sr 0.14 0.17 0.14 0.28 0.28 0.11 0.59 0.21 0.23 0.51 0.26 0.41 0.21 Sr/Cu 2.77 2.79 2.15 2.87 0.46 3.57 1.68 1.74 3.59 0.71 2.08 2.32 2.03 Ni/Co 8.65 4.31 1.8 2.47 2.61 7.22 2.91 2.74 6.92 1.58 1.66 3.47 3.30 C 0.57 0.66 0.75 0.63 0.78 0.82 0.72 0.76 0.81 0.74 0.68 0.64 0.71 Table (3) shows the trace elements (ppm) by XRF, and elemental ratios of Zubai samples. The shales also have a high (L.O.I) loss on ignition, that ranges from 5.53 to 15.53 wt.% (av.= 10.38 wt.%) and is due to the presence of clay minerals and organic matter. Figure (6) depicts the relationship between Al2O3 and major elements. The inverse relationship between SiO2 and Al2O3 is a result of the fact that quartz contains the majority of the silica. The positive correlation 28 Journal of Petroleum Research and Studies P- ISSN: 2220-5381 E- ISSN: 2710-1096 P- ISSN: 2220-5381 E- ISSN: 2710-1096 P- ISSN: 2220-5381 E- ISSN: 2710-1096 Open Access No. 40, September 2023, pp. 19-40 This felsic granitic provenance was also supported by [27] major elemental discriminant illustration, which shows that samples are mostly felsic in origin (Fig. 9a). This felsic provenance is associated with granite, whereas the intermediate provenance is associated with an andesitic mixture or the Arabian Shield's amphibolite, granitic, and magmatic gneisses. This felsic granitic provenance was also supported by [27] major elemental discriminant illustration, which shows that samples are mostly felsic in origin (Fig. 9a). This felsic provenance is associated with granite, whereas the intermediate provenance is associated with an andesitic mixture or the Arabian Shield's amphibolite, granitic, and magmatic gneisses. Clastic sediments can be derived from a variety of tectonic settings and have distinct geochemical signatures due to parent rock geochemical composition. Clastic sediments can be derived from a variety of tectonic settings and have distinct geochemical signatures due to parent rock geochemical composition. Fig. (6): Al2O3 diagram versus selected major oxides. R² = 0.2621 -1 0 1 2 3 4 5 0 5 10 15 20 25 K2O (Wt. %) b R² = 0.4201 0 10 20 30 40 50 60 70 80 0 5 10 15 20 25 SiO2 (Wt. %) a R² = 0.1214 0 0.5 1 1.5 2 2.5 0 5 10 15 20 25 Na2O (Wt. %) c R² = 0.1564 0 5 10 15 20 0 5 10 15 20 25 L.I.O (Wt. %) d R² = 0.1401 0 2 4 6 8 10 0 5 10 15 20 25 30 Fe2O3 (Wt. %) e R² = 0.1 0 0.5 1 1.5 2 2.5 3 0 5 10 15 20 25 30 MgO (Wt. %) f R² = 0.325 0 0.5 1 1.5 2 2.5 3 0 10 20 30 TiO2 (Wt. %) Al2O3 (Wt. %) g R² = 0.0613 0 0.5 1 1.5 2 2.5 3 0 10 20 30 SO3 (Wt. %) Al2O3 (Wt. %) h R² = 0.4201 0 10 20 30 40 50 60 70 80 0 5 10 15 20 25 SiO2 (Wt. %) a R² = 0.2621 -1 0 1 2 3 4 5 0 5 10 15 20 25 K2O (Wt. %) b b a R² = 0.1564 0 5 10 15 20 0 5 10 15 20 25 L.I.O (Wt. 4.4 Provenance and Tectonic Setting The provenance and tectonic setting of the Zubair oil shale have been defined using elemental ratios and discriminant functions [21] [22] [23] [24]. [25] determined Al2O3/ TiO2 ratios to differentiate mafic Al2O3/ TiO2 14 and felsic Al2O3/ TiO2 from 19 to 28 source rocks, which could be Arabian Shield granodiorite, tonalite, and granites. This ratio (11.81 to 17.87) is high in Zubair shale, indicating a felsic source with few contributors of an intermediate source (Table 2). [26] K2O/Na2O ratio is also used as a provenance marker in clastic sedimentary rocks; a reduced value indicates that the source is mafic rather than felsic. The existence of illite and illite- smectite in oil shale samples confirms their origins from an intermediate and felsic source, and K2O/Na2O ratios range from 1.11 to 3.71. The zircon content of sediments can also be used to determine their provenance [25]. A binary graph of Zr vs TiO2 implies that the Zubair oil shale's source rocks are intermediate to felsic igneous rocks sourced from a stable cratonic setting (Figure 7b). 29 Journal of Petroleum Research and Studies P- ISSN: 2220-5381 E- ISSN: 2710-1096 Open Access No. 40, September 2023, pp. 19-40 Journal of Petroleum Research and Studies P- ISSN: 2220-5381 E- ISSN: 2710-1096 P- ISSN: 2220-5381 E- ISSN: 2710-1096 P- ISSN: 2220-5381 E- ISSN: 2710-1096 %) d d R² = 0.1214 0 0.5 1 1.5 2 2.5 0 5 10 15 20 25 Na2O (Wt. %) c c R² = 0.1 0 0.5 1 1.5 2 2.5 3 0 5 10 15 20 25 30 MgO (Wt. %) f R² = 0.1401 0 2 4 6 8 10 0 5 10 15 20 25 30 Fe2O3 (Wt. %) e f e R² = 0.0613 0 0.5 1 1.5 2 2.5 3 0 10 20 30 SO3 (Wt. %) Al2O3 (Wt. %) h h R² = 0.325 0 0.5 1 1.5 2 2.5 3 0 10 20 30 TiO2 (Wt. %) Al2O3 (Wt. %) g g Fig. (6): Al2O3 diagram versus selected major oxides. 30 Journal of Petroleum Research and Studies P- ISSN: 2220-5381 E- ISSN: 2710-1096 Open Access No. 40, September 2023, pp. 19-40 Fig. (7): (a) Geochemical classification of Zubair oil shale focused on the log (SiO2/ Al2O3) vs the log (Fe2O3/ K2O) diagram after [19] and (b) Provenance of major and trace elements, as well as a tectonic diagram based on TiO2 versus Zr [25]. Fig. (8): Normalized multi-element graph for the major element & identified the trace element compositions normalized comparable to (a) chondrite [31] & (b) PAAS [20]. Journal of Petroleum Research and Studies P- ISSN: 2220-5381 E- ISSN: 2710-1096 Open Access No. 40, September 2023, pp. 19-40 Fig. (7): (a) Geochemical classification of Zubair oil shale focused on the log (SiO2/ Al2O3) vs the log (Fe2O3/ K2O) diagram after [19] and (b) Provenance of major and trace elements, as well as a tectonic diagram based on TiO2 versus Zr [25]. Journal of Petroleum Research and Studies Journal of Petroleum Research and Studies P- ISSN: 2220-5381 E- ISSN: 2710-1096 Open Access Fig. (7): (a) Geochemical classification of Zubair oil shale focused on the log (SiO2/ Al2O3) vs the log (Fe2O3/ K2O) diagram after [19] and (b) Provenance of major and trace elements, as well as a tectonic diagram based on TiO2 versus Zr [25]. Fig. (8): Normalized multi-element graph for the major element & identified the trace element compositions normalized comparable to (a) chondrite [31] & (b) PAAS [20]. Fig. P- ISSN: 2220-5381 E- ISSN: 2710-1096 Open Access No. 40, September 2023, pp. 19-40 The unique discriminant function graphs rely on the major oxides that have been adjusted to 100% volatile-free [22]. For the low-silica tectonic discrimination [(SiO2)adj= 52.88- 70.33% (m2)]sediments, discrimination diagrams were proposed [28] [29]. In the collision field, all samples were plotted (Fig. 9b). The unique discriminant diagram (passive & active) suggested relied on the mixture for ten major (SiO2 to L.O.I) and eleven trace elements (Mn to Co) on a volatile-free basis, adjusted to 100% confirm collision setting for Zubair shales. Mostly all samples on this plot are schemed in the passive margin, except one in active margin settings (Fig. 10). In accordance with the geological precursor and geochemical features of the oil shale metamorphic, plutonic, and sedimentary rocks from the Late Berriasian-Albian cycle. The plutonic and metamorphic rocks that drive the Zubair shale formed in collision settings (passive margin) and signified the beginning of the Zubair shale deposit's lower Cretaceous deposit. These hydrocarbons had (in part) charged the Tertiary and Cretaceous reservoirs, particularly the Zubair Formation, formed by Alpine collision traps 15-10 million years ago, which formed folds in the Mesopotamian Basin and closed the Tethys Ocean between the Arabian and Eurasian Plates. This interpretation fits with the geological history of the Zubair oil shale [30]. Fig. (9): Major & trace element provenance and tectonic graphs. (a) Discriminant function (F) diagram after [27] where F1 (Discriminant Function 1) = (0.607 × Al2O3) + (− 1.773 × TiO2) + (− 1.5 × MgO) + (0.76×Fe2O3) + (0.509 × Na2O) + (0.616 × CaO) + (K2O × (− 1.22)) + (− 9.09). F2 (Discriminant Function 2) = (Al2O3× 0.07) + (0.445 × TiO2) + (MgO × (− 1.142)) +( − 0.25 × Fe2O3) + (Na2O ×1.475) + (0.438 × CaO) + (1.426 × K2O) + (− 6.861); (b) Major elements tectonic setting discrimination diagram [28]. The low-silica diagram is represented by the subscript m2 in DF1 and DF2, where DF1 (Col - Rift -Arc) m2 = (− 1.854 × ln(Al2O3/SiO2)adj) + (0.608 × ln (TiO2/SiO2)adj) + (− 0.550 × ln(MnO/SiO2)adj) + (0.299 × ln(Fe2O3t/SiO2)adj) + (0.194 × ln(CaO/SiO2)adj) + (0.1120 × ln(MgO/SiO2)adj) +(1.941×ln(K2O/SiO2)adj) + (−1.510 × ln(Na2O/SiO2)adj) + (0.003 × ln(P2O5/SiO2)adj) − 0.294. P- ISSN: 2220-5381 E- ISSN: 2710-1096 DF2 (Arc-Rift-Col) m2 = (− 0.995 × ln(Al2O3/ SiO2)adj) + (− 0.554 × ln(TiO2/SiO2)adj) + (− 1.391 × ln(MnO/SiO2)adj) + (1.765 × ln(Fe2O3t/SiO2)adj) +(0.225 × ln(CaO/SiO2)adj) + (− 1.034 × ln(MgO/SiO2)adj) + (0.330 × ln(K2O/SiO2)adj) + (0.713 × ln(Na2O/SiO2)adj) + (0.637 × ln(P2O5/ SiO2)adj) − 3.631. Fig. (9): Major & trace element provenance and tectonic graphs. (a) Discriminant function (F) dia Fig. (9): Major & trace element provenance and tectonic graphs. (a) Discriminant function (F) diagram after [27] where F1 (Discriminant Function 1) = (0.607 × Al2O3) + (− 1.773 × TiO2) + (− 1.5 × MgO) + (0.76×Fe2O3) + (0.509 × Na2O) + (0.616 × CaO) + (K2O × (− 1.22)) + (− 9.09). F2 (Discriminant Function 2) = (Al2O3× 0.07) + (0.445 × TiO2) + (MgO × (− 1.142)) +( − 0.25 × Fe2O3) + (Na2O ×1.475) + (0.438 × CaO) + (1.426 × K2O) + (− 6.861); (b) Major elements tectonic setting discrimination diagram [28]. The low-silica diagram is represented by the subscript m2 in DF1 and DF2, where DF1 (Col - Rift -Arc) m2 = (− 1.854 × ln(Al2O3/SiO2)adj) + (0.608 × ln (TiO2/SiO2)adj) + (− 0.550 × ln(MnO/SiO2)adj) + (0.299 × ln(Fe2O3t/SiO2)adj) + (0.194 × ln(CaO/SiO2)adj) + (0.1120 × ln(MgO/SiO2)adj) +(1.941×ln(K2O/SiO2)adj) + (−1.510 × ln(Na2O/SiO2)adj) + (0.003 × ln(P2O5/SiO2)adj) − 0.294. DF2 (Arc-Rift-Col) m2 = (− 0.995 × ln(Al2O3/ SiO2)adj) + (− 0.554 × ln(TiO2/SiO2)adj) + (− 1.391 × ln(MnO/SiO2)adj) + (1.765 × ln(Fe2O3t/SiO2)adj) +(0.225 × ln(CaO/SiO2)adj) + (− 1.034 × ln(MgO/SiO2)adj) + (0.330 × ln(K2O/SiO2)adj) + (0.713 × ln(Na2O/SiO2)adj) + (0.637 × ln(P2O5/ SiO2)adj) − 3.631. Fig. (9): Major & trace element provenance and tectonic graphs. (a) Discriminant function (F) diagram after [27] where F1 (Discriminant Function 1) = (0.607 × Al2O3) + (− 1.773 × TiO2) + (− 1.5 × MgO) + (0.76×Fe2O3) + (0.509 × Na2O) + (0.616 × CaO) + (K2O × (− 1.22)) + (− 9.09). F2 (Discriminant Function 2) = (Al2O3× 0.07) + (0.445 × TiO2) + (MgO × (− 1.142)) +( − 0.25 × Fe2O3) + (Na2O ×1.475) + (0.438 × CaO) + (1.426 × K2O) + (− 6.861); (b) Major elements tectonic setting discrimination diagram [28]. P- ISSN: 2220-5381 E- ISSN: 2710-1096 (7): (a) Geochemical classification of Zubair oil shale focused on the log (SiO2/ Al2O3) th l (F O / K O) di ft [19] d (b) P f j d t l t : (a) Geochemical classification of Zubair oil shale focused on the log (SiO2/ Al2O3) Fig. (7): (a) Geochemical classification of Zubair oil shale focused on the log (SiO2/ Al2O3) vs the log (Fe2O3/ K2O) diagram after [19] and (b) Provenance of major and trace elements, as well as a tectonic diagram based on TiO2 versus Zr [25]. Fig. (7): (a) Geochemical classification of Zubair oil shale focused on the log (SiO2/ Al2O3) vs the log (Fe2O3/ K2O) diagram after [19] and (b) Provenance of major and trace elements, Fig. (8): Normalized multi-element graph for the major element & identified the trace element compositions normalized comparable to (a) chondrite [31] & (b) PAAS [20]. Fig. (8): Normalized multi-element graph for the major element & identified the trace element compositions normalized comparable to (a) chondrite [31] & (b) PAAS [20]. Fig. (8): Normalized multi-element graph for the major element & identified the trace element compositions normalized comparable to (a) chondrite [31] & (b) PAAS [20]. 31 31 Journal of Petroleum Research and Studies P- ISSN: 2220-5381 E- ISSN: 2710-1096 P- ISSN: 2220-5381 E- ISSN: 2710-1096 P- ISSN: 2220-5381 E- ISSN: 2710-1096 The low-silica diagram is represented by the subscript m2 in DF1 and DF2, where DF1 (Col - Rift -Arc) m2 = (− 1.854 × ln(Al2O3/SiO2)adj) + (0.608 × ln (TiO2/SiO2)adj) + (− 0.550 × ln(MnO/SiO2)adj) + (0.299 × ln(Fe2O3t/SiO2)adj) + (0.194 × ln(CaO/SiO2)adj) + (0.1120 × ln(MgO/SiO2)adj) +(1.941×ln(K2O/SiO2)adj) + (−1.510 × ln(Na2O/SiO2)adj) + (0.003 × ln(P2O5/SiO2)adj) − 0.294. DF2 (Arc-Rift-Col) m2 = (− 0.995 × ln(Al2O3/ SiO2)adj) + (− 0.554 × ln(TiO2/SiO2)adj) + (− 1.391 × ln(MnO/SiO2)adj) + (1.765 × ln(Fe2O3t/SiO2)adj) +(0.225 × ln(CaO/SiO2)adj) + (− 1.034 × ln(MgO/SiO2)adj) + (0.330 × ln(K2O/SiO2)adj) + (0.713 × ln(Na2O/SiO2)adj) + (0.637 × ln(P2O5/ SiO2)adj) − 3.631. 32 Journal of Petroleum Research and Studies P- ISSN: 2220-5381 E- ISSN: 2710-1096 Open Access No. 40, September 2023, pp. 19-40 Fig. (10): Diagram of a multidimensional discriminant function discriminating the (A) active and (P)Passive margin settings [29], where DF(A-P)MT = (Ilr1TiO2MT × 3.2683)+ (5.3873 × Ilr2Al2O3MT) + (1.5546 × Ilr3Fe2O3MT) + (3.2166 × Ilr4MnOMT) + (4.7542 × Ilr5MgOMT) + (2.0390 × Ilr6CaOMT)+(4.0490× Ilr7Na2OMT) + (3.1505 × Ilr8K2OMT) + (2.3688 × Ilr9P2O5MT)+( Ilr10CrMT × 2.8354) + (Ilr11NbMT × 0.9011) + (Ilr12NiMT ×1.9128)+( Ilr13VMT × 2.9094) + (Ilr14YMT × 4.1507 ) + (Ilr15ZrMT × 3.4871)– 3.2088 Journal of Petroleum Research and Studies Journal of Petroleum Research and Studies P- ISSN: 2220-5381 E- ISSN: 2710-1096 Open Access No. 40, September 2023, pp. 19-40 P- ISSN: 2220-5381 E- ISSN: 2710-1096 P- ISSN: 2220-5381 E- ISSN: 2710-1096 Open Access g. (10): Diagram of a multidimensional discriminant function discriminating the (A Fig. (10): Diagram of a multidimensional discriminant function discriminating the (A) active and (P)Passive margin settings [29], where DF(A-P)MT = (Ilr1TiO2MT × 3.2683)+ (5.3873 × Ilr2Al2O3MT) + (1.5546 × Ilr3Fe2O3MT) + (3.2166 × Ilr4MnOMT) + (4.7542 × Ilr5MgOMT) + (2.0390 × Ilr6CaOMT)+(4.0490× Ilr7Na2OMT) + (3.1505 × Ilr8K2OMT) + (2.3688 × Ilr9P2O5MT)+( Ilr10CrMT × 2.8354) + (Ilr11NbMT × 0.9011) + (Ilr12NiMT ×1.9128)+( Ilr13VMT × 2.9094) + (Ilr14YMT × 4.1507 ) + (Ilr15ZrMT × 3.4871)– 3.2088 active and (P)Passive margin settings [29], where DF(A-P)MT = (Ilr1TiO2MT × 3.2683)+ (5.3873 × Ilr2Al2O3MT) + (1.5546 × Ilr3Fe2O3MT) + (3.2166 × Ilr4MnOMT) + (4.7542 × Ilr5MgOMT) + (2.0390 × Ilr6CaOMT)+(4.0490× Ilr7Na2OMT) + (3.1505 × Ilr8K2OMT) + (2.3688 × Ilr9P2O5MT)+( Ilr10CrMT × 2.8354) + (Ilr11NbMT × 0.9011) + (Ilr12NiMT ×1.9128)+( Ilr13VMT × 2.9094) + (Ilr14YMT × 4.1507 ) + (Ilr15ZrMT × 3.4871)– 3.2088 P- ISSN: 2220-5381 E- ISSN: 2710-1096 P- ISSN: 2220-5381 E- ISSN: 2710-1096 Open Access No. 40, September 2023, pp. 19-40 the maturity of shales. ICV > 1 sediments have abundant unweathered detrital minerals and are compositionally immature, whereas ICV < 1 sediments are composed of weathered minerals (e.g. clay minerals) and are compositionally mature [16]. The ICV values of the examined shales range from 0.33 to 0.99 (av.= 0.70%), implying texturally mature shales deposited inside a tectonically active environment. The ratios of K2O/Na2O for such core shales range from 1.11 to 6.2 (av.= 2.68), indicating that the shales are mature. The ratio of SiO2/Al2O3 is frequently used to assess the maturity of the textural of shales, and it ranges from 2.54 to 5.48 (av.=3.91), showed the highest compositional maturity, most likely due to feldspar alteration [13]. The ICV versus CIA binary graph [13] [16] proves the severe weathering and elevated maturity of shales (Fig.11b). As a result, the chemical weathering degree is primarily determined by the source area's climate and erosion. The recycled shale input could represent distal facies deposited in low relief. the maturity of shales. ICV > 1 sediments have abundant unweathered detrital minerals and are compositionally immature, whereas ICV < 1 sediments are composed of weathered minerals (e.g. clay minerals) and are compositionally mature [16]. The ICV values of the examined shales range from 0.33 to 0.99 (av.= 0.70%), implying texturally mature shales deposited inside a tectonically active environment. The ratios of K2O/Na2O for such core shales range from 1.11 to 6.2 (av.= 2.68), indicating that the shales are mature. The ratio of SiO2/Al2O3 is frequently used to assess the maturity of the textural of shales, and it ranges from 2.54 to 5.48 (av.=3.91), showed the highest compositional maturity, most likely due to feldspar alteration [13]. The ICV versus CIA binary graph [13] [16] proves the severe weathering and elevated maturity of shales (Fig.11b). As a result, the chemical weathering degree is primarily determined by the source area's climate and erosion. The recycled shale input could represent distal facies deposited in low relief. Fig. (11): Diagrams of weathering and maturity for Zubair oil shales. (a) Ternary graph (in mole fraction) of A-CN-K, sample placement specifying severe weathering (chemical). (b) ICV vs CIA chart [13] [16] displaying shales maturity. Fig. (11): Diagrams of weathering and maturity for Zubair oil shales. (a) Ternary graph (in mole fraction) of A-CN-K, sample placement specifying severe weathering (chemical). 4.5 Paleo-weathering Conditions and Shale Maturity Many research’s and studies have used various weathering indexes & elemental proportions to assess paleo-weathering mechanisms and also the weathering intensity of source rock [22] [32]. The chemical weathering intensity within the source area is assessed using CIA, PIA, and CIW in this study. The triangular plot A-CN-K (Al2O3-CaO* + Na2O-K2O) as well specifies a weathering degree and possible source rocks composition [13]. CaO* values have been accepted just when CaO < Na2O, if CaO > Na2O, the CaO concentration is assumed to be equivalent to that of Na2O [17]. The values of CIA for Zubair shale samples range from 70.63 to 86.53% (av.= 78.75%), implying that such source rocks have undergone extensive chemical weathering. High PIA (av. = 87.12%) and CIW (av. = 88.59%) values support this strong chemical weathering (Table 2). The mineralogical analysis also showed a predominance of clay minerals as well as a higher amount of iron oxide coatings, which could be the result of diagenetic alteration or severe weathering inside the area of the source. The existence of kaolinite and illite implies that feldspar and muscovite, which are mineral phases derived from of (granitic) source area, were chemically weathered. As a result, the A-CN-K figure shows that each sample map is high above the feldspar section near kaolinite & illite, chlorite, and gibbsite fields, and follows the A-CN meet the weathering trend towards A-apex (Fig.11a), indicating granites as the primary source rock but as well as reflecting fairly stable conditions of weathering. The difference in value systems for the chemical index in deposits is related to the source rock content. This implication assumes that shales from the Zubair Formation, which were deposited in the collision stage, were probably subjected to tectonic activity. The ICV can be used to determine the maturity of shales [16]. Chemical weathering tends to increase with alumina content and may frequently represent 33 Journal of Petroleum Research and Studies P- ISSN: 2220-5381 Open Access Journal of Petroleum Research and Studies P- ISSN: 2220-5381 E- ISSN: 2710-1096 Open Access No. 40, September 2023, pp. 19-40 Journal of Petroleum Research and Studies P- ISSN: 2220-5381 E- ISSN: 2710-1096 P- ISSN: 2220-5381 E- ISSN: 2710-1096 P- ISSN: 2220-5381 E- ISSN: 2710-1096 Open Access No. 40, September 2023, pp. 19-40 Open Access No. 40, September 2023, pp. 19-40 P- ISSN: 2220-5381 E- ISSN: 2710-1096 (b) ICV vs CIA chart [13] [16] displaying shales maturity. 34 Journal of Petroleum Research and Studies P- ISSN: 2220-5381 E- ISSN: 2710-1096 4.6 Paleoclimate and Depositional Setting (12): Binary graph of (a) C value vs Sr/Cu ratio, proposing semi- arid to semi- humid paleoclimatic condition; (b) Al2O3 vs V. 4.6 Paleoclimate and Depositional Setting Weathering index values (CIA and PIA), as well as trace elements (Rb, Sr, and Cu) also their ratios (Sr/Cu and Rb/Sr) are all be regarded a suitable tool for investigating the source area's paleoenvironment. A few research findings have been using Rb/Sr & Sr/Cu ratios to deduce the paleoclimatic conditions that generated the deltaic environment [33] [34]. A humid and warm environment is indicated by low Rb/Sr & Sr/Cu ratios. Clays could indicate climate changes within source areas. Kaolinite is created by severe weathering of feldspars in a subtropical humid climate, while illite is created in a cold climate and very little precipitation. The greater CIA value shows the sediments were deposited in a whole subtropical warm and humid climate. The ratios of Rb/Sr range between 0.11 to 0.59 (ave. 0.21) and the ratios of Sr/Cu range between 0.46 to 3.59 (ave. 2.03), indicating a minimal change throughout the years (Table 3). Climate index C value [where C= ∑ (Mn + Fe + Ni + Cr + Co + V / ∑ (Mg + Ca + Ba + Sr + Na + K)] has effectively defined paleoclimatic conditions. Sediment C values range from 0.57 to 0.82, implying lesser oscillating paleoclimatic conditions, inferring semi-humid to semi-arid environments (Fig.12a). This suggests that the Zubair oil shale sediments were not subjected to considerable paleoclimatic differences. The elements of Al2O3 and V can be used to infer paleoenvironmental conditions in fluvial-deltaic, deltaic, and shallow marine environments. Vanadium concentration in marine facies is relatively better than in freshwater deposits. As clarified by the V-Al2O3 plot, sediments have been deposited in fluvial and shallow marine environments, according to binary diagram of paleoenvironmental modeling (Fig.12b). The shallow marine & fluvial depositional environments of shales were oxic and dysoxic, as indicated by Ni/Co ratios ranging from 1.58 to 8.65 (av.=3.30) (Table 3). Moreover, a ratio of Ni/Co for 5 implies an oxic environment, while a ratio of Ni/Co greater than 5 implies an anoxic environment. 35 35 Journal of Petroleum Research and Studies P- ISSN: 2220-5381 E- ISSN: 2710-1096 Open Access No. 40, September 2023, pp. 19-40 Fig. (12): Binary graph of (a) C value vs Sr/Cu ratio, proposing semi- arid to semi- humid paleoclimatic condition; (b) Al2O3 vs V. Journal of Petroleum Research and Studies Journal of Petroleum Research and Studies P- ISSN: 2220-5381 E- ISSN: 2710-1096 Fig. References [1] R. M. Idan, F. A. Al-Musawi, A. L. M. Salih, and S. A. F. Al-Qaraghuli, “The Petroleum System of Zubair Formation in Zubair Subzone, Southern Iraq”, Journal of Petroleum Research and Studies, vol. 9, no. 4, pp. 57-73, Dec. 2019. DOI: https://doi.org/10.52716/jprs.v9i4.322 [2] R.M. Idan, R.F. Faisal, M.E. Nasser, T.K. AL-Ameri, and D. AL-Rawi, "Hydrocarbon potential of Zubair Formation in the south of Iraq, "Arabian Journal of Geosciences, Vol. 8, no.7, pp. 4805–4817, 2015 b, DOI: https://doi.org/10.1007/s12517-014-1569-6 [3] F.K. Bahman, F.H. Abdullah, A. Saleh and H. Alimi, "Organic geochemical and petrographical characteristics of the major lower cretaceous petroleum source rock (Makhul Formation) in Kuwait, " Kuwait J. Sci., vol.49, no. 1, pp. 1-25, 2022, [1] R. M. Idan, F. A. Al-Musawi, A. L. M. Salih, and S. A. F. Al-Qaraghuli, “The Petroleum System of Zubair Formation in Zubair Subzone, Southern Iraq”, Journal of Petroleum Research and Studies, vol. 9, no. 4, pp. 57-73, Dec. 2019. DOI: https://doi.org/10.52716/jprs.v9i4.322 [1] R. M. Idan, F. A. Al-Musawi, A. L. M. Salih, and S. A. F. Al-Qaraghuli, The Petroleum System of Zubair Formation in Zubair Subzone, Southern Iraq”, Journal of Petroleum Research and Studies, vol. 9, no. 4, pp. 57-73, Dec. 2019. DOI: https://doi.org/10.52716/jprs.v9i4.322 [2] R.M. Idan, R.F. Faisal, M.E. Nasser, T.K. AL-Ameri, and D. AL-Rawi, "Hydrocarbon potential of Zubair Formation in the south of Iraq, "Arabian Journal of Geosciences, Vol. 8, no.7, pp. 4805–4817, 2015 b, DOI: https://doi.org/10.1007/s12517-014-1569-6 [2] R.M. Idan, R.F. Faisal, M.E. Nasser, T.K. AL-Ameri, and D. AL-Rawi, "Hydrocarbon potential of Zubair Formation in the south of Iraq, "Arabian Journal of Geosciences, Vol. 8, no.7, pp. 4805–4817, 2015 b, DOI: https://doi.org/10.1007/s12517-014-1569-6 [3] F.K. Bahman, F.H. Abdullah, A. Saleh and H. Alimi, "Organic geochemical and petrographical characteristics of the major lower cretaceous petroleum source rock (Makhul Formation) in Kuwait, " Kuwait J. Sci., vol.49, no. 1, pp. 1-25, 2022, DOI: https://doi.org/10.48129/kjs.v49i1.10277 [4] S.Z. Jassim and J.C. Goff, "Geology of Iraq, " Dolin, Prague and Moravian Museum, Brno, Czech Republic, p. 341, 2006. [5] A.K. Abbas, R.E. Flori and M. Alsaba, "Testing and Evaluation of Shale Stability for Zubair Shale Formation, " Society of Petroleum Engineers, 192274, pp. 1–9, 2018d, https://doi.org/10.2118/192274-MS [6] H.K. Almayyahi, M.H. Aljaberi and H. almalikee, " Geochemical Study for the Upper Shale Member – Zubair Formation in Rumaila Oilfield, South Iraq, "Int. J. of Mining Science, vol. 4, no. 4, pp. 56-75, 2018, DOI: http://dx.doi.org/10.20431/2454-9460.0404006 [7] A.F. 5. Conclusions 1- Silty-clayey laminated mudstone lithofacies, mica-rich mudstone lithofacies, clay-rich siliceous mudstone lithofacies, and clay-bearing calcareous mudstone lithofacies were identified through petrographic analysis. 2- Zubair oil shale is a silica-rich argillaceous mudstone type with a high organic matter content. The shales are made up of kaolinite, illite, and smectite, besides quartz, calcite, dolomite, and pyrite. 2- Zubair oil shale is a silica-rich argillaceous mudstone type with a high organic matter content. The shales are made up of kaolinite, illite, and smectite, besides quartz, calcite, dolomite, and pyrite. 3- The geochemical composition of Zubair shales demonstrates that they were formed from felsic rocks such as granites to minor quantities of intermediate igneous rocks. Discrimination diagrams indicate passive margin settings (collision) to similar maturity levels. 3- The geochemical composition of Zubair shales demonstrates that they were formed from felsic rocks such as granites to minor quantities of intermediate igneous rocks. Discrimination diagrams indicate passive margin settings (collision) to similar maturity levels. 4- According to the findings of this study, oil shales are strongly weathered and deposition occurs in a warm to the semi-humid environment. 36 Journal of Petroleum Research and Studies P- ISSN: 2220-5381 E- ISSN: 2710-1096 Open Access No. 40, September 2023, pp. 19-40 References Nader, R.J. Muhammad, W.M. Saleh, M.S. Abdullah and A.Q. Atwan, "Evaluation of main pay- Zubair Formation after operations re-injection of produced water directly in Rumaila Oil Field norths under matrix condition, " J. of Petroleum Research & Studies, no.35, pp.13- 26, 2022, DOI: https://doi.org/10.52716/jprs.v12i2.655 [8] M.K. Al-Jafar and M.H. Al-Jaberi, "Determination of clay minerals using gamma ray spectroscopy for the Zubair Formation in Southern Iraq. Journal of Petroleum Exploration and Production Technology, " 2021. https://doi.org/10.1007/s13202-021-01371-3 [9] W. Bleam, "Soil and Environmental Chemistry, " Elsevier, Amsterdam, 2nd Edition, 573p., 2017. [10] H.G. Dill, "Kaolin: Soil, rock and ore: From the mineral to the magmatic, sedimentary and metamorphic environments, "Earth- Science Reviews, vol. 161, pp. 16-129, 2016, https://doi.org/10.1016/j.earscirev.2016.07.003 37 Journal of Petroleum Research and Studies P- ISSN: 2220-5381 E- ISSN: 2710-1096 P- ISSN: 2220-5381 E- ISSN: 2710-1096 P- ISSN: 2220-5381 E- ISSN: 2710-1096 P- ISSN: 2220-5381 E- ISSN: 2710-1096 Open Access No. 40, September 2023, pp. 19-40 Open Access No. 40, September 2023, pp. 19-40 [11] F. Bergaya, B.K.G. Theng and G. Lagaly, "Handbook of Clay Science, " Elsevier, 1st Edition, 1224p., 2006. [12] K.S. Glaser, C.K. Miller, G.M. Johnson, R.L. Kleinberg and W.D. Pennington, "Seeking the sweet Spot: Reservoir and completion quality in organic shales, " Oilﬁeld Rev., vol. 25, pp. 16–29, 2014. [13] H.W. Nesbitt and G.M. Young, "Prediction of some weathering trends of plutonic and volcanic rocks based on thermodynamic and kinetic considerations, "Geochimica et Cosmochimica Acta, vol. 48, pp.1523–1534, 1984, https://doi.org/10.1016/0016- 7037(84)90408-3 [14] C.M. Fedo, H. Nesbitt and G.M. Young, "Unraveling the effects of potassium metasomatism in sedimentary rocks and paleosols, with implications for paleoweathering conditions and provenance, " Geol., vol. 23, pp. 921–924, 1995, https://doi.org/10.1130/0091-7613(1995)023<0921:UTEOPM>2.3.CO;2 [15] L. Harnois, "The CIW index: a new chemical index of weathering, " Sediment. Geol., vol. 55, pp.319–322, 1988, https://doi.org/10.1016/0037-0738(88)90137-6 [16] R. Cox, D.R. Lowe and R.L. Cullers, "The influence of sediment recycling and basement composition on evolution of mudrock chemistry in the southwestern United States, " Geochim Cosmochim Acta, vol.59, pp. 2919–2940, 1995, DOI: 10.1016/0016- 7037(95)00185-9 [17] S.M. McLennan, S.R. Taylor, M.T. McCulloch and J.B. Maynard, "Geochemical and Nd/Sr isotopic composition of deep-sea turbidites: crustal evolution and plate tectonic associations, " Geochim Cosmochim Acta, vol. 54, pp. 2015–2050, 1990, DOI:10.1016/0016- 7037(90)90269-Q [18] K. Yan, C.L. Wang, S. Mischke and J.Y. Wang, "Major and trace‑element geochemistry of Late Cretaceous clastic rocks in the Jitai Basin, southeast China, " Scientific Reports, vol.11,13846, 2021, Doi: https://doi.org/10.1038/s41598-021-93125-8. [19] M.M. Herron, "Geochemical classification of terrigenous sands and shales from core and log data," Journal of Sedimentary Petrology, vol. 58, pp. 820‒829, 1988, https://doi.org/10.1306/212F8E77-2B24-11D7-8648000102C1865D 38 38 Journal of Petroleum Research and Studies P- ISSN: 2220-5381 E- ISSN: 2710-1096 P- ISSN: 2220-5381 E- ISSN: 2710-1096 P- ISSN: 2220-5381 E- ISSN: 2710-1096 Open Access No. 40, September 2023, pp. 19-40 Open Access No. 40, September 2023, pp. 19-40 [20] S.R. Taylor and S.M. McLennan, "The continental crust: its composition and evolution, " Oxford: Blackwell, 312 p, 1985, https://doi.org/10.1002/gj.3350210116 [20] S.R. Taylor and S.M. McLennan, "The continental crust: its composition and evolution, " Oxford: Blackwell, 312 p, 1985, https://doi.org/10.1002/gj.3350210116 [20] S.R. Taylor and S.M. McLennan, "The continental crust: its composition and evolution, " Oxford: Blackwell, 312 p, 1985, https://doi.org/10.1002/gj.3350210116 [21] G. Ngueutchoua, A.Z.E. Bessa, J.T. Eyong and D.D. Zandjio, "Geochemistry of cretaceous [21] G. Ngueutchoua, A.Z.E. Bessa, J.T. Eyong and D.D. Zandjio, "Geochemistry of cretaceous fine-grained siliciclastic rocks, Douala sub-basin, SW Cameroon: implications for weathering intensity, provenance, paleoclimate, redox condition, and tectonic setting, " J. Afr. Earth Sci., vol.152, pp. 215–236, 2019, https://doi.org/10.1016/j.jafrearsci.2019.02.021 [22] A.Z. Ekoa Bessa, P.D. Ndjigui, G.C. Fuh and J.S. Armstrong-Altrin, "Mineralogy and geochemistry of the Ossa lake Complex sediments, Southern Cameroon: implications for paleoweathering and provenance, " Arab. J. Geosci., vol. 14, 322, 2021, DOI: 10.1007/s12517-021-06591-9 [23] M.A. Farooqui, K.U. Rehman and A. Yaseen, "Petrography, geochemistry and depositional model of Ispikan Conglomerate, Southwest Pakistan, " Kuwait J. Sci., vol. 49, no.1, pp. 1- 25, 2022, https://doi.org/10.48129/kjs.v49i1.10486 [24] T. Boschetti, S.M. Awadh, H.S. Al-Mimar, P. Iacumin, L. Toscani, E. Selmo, Z.M. Yaseen, "Chemical and isotope composition of the oilfield brines from Mishrif Formation (southern Iraq): Diagenesis and geothermometry," Marine and Petroleum Geology, 1;122: 104637, 2020, https://doi.org/10.1016/j.marpetgeo.2020.104637 [25] K.I. Hayashi, H. Fujisawa, H.D. Holland and H. Ohmoto, "Geochemistry of∼ 1.9 Ga sedimentary rocks from northeastern Labrador, Canada, "Geochim Cosmochim Acta, vol. 61, pp. 4115–4137, 1997, https://doi.org/10.1016/S0016-7037(97)00214-7 [26] P.E. Potter, "Petrology and chemistry of modern big river sands, " J. Geol., vol. 86, pp. 423–449, 1978, https://doi.org/10.1086/649711 [27] B.P. Roser and R.J. Korsch, "Determination of tectonic setting of sandstone-mudstone suites using SiO2 content and K2O/Na2O ratio, " J. Geol., vol. 94, pp. 635–650, 1986, https://doi.org/10.1086/629071 [28] S.P. Verma and J.S. Armstrong-Altrin, "New multi-dimensional diagrams for tectonic discrimination of siliciclastic sediments and their application to Precambrian basins, " Chem. Geol., vol. 355, pp.117–133, 2013, https://doi.org/10.1016/j.chemgeo.2013.07.014 [29] S.P. Verma and J.S. Armstrong-Altrin, "Geochemical discrimination of siliciclastic sediments from active and passive margin settings, " Sediment. Geol., vol. 332, pp.1–12, 2016, https://doi.org/10.1016/j.sedgeo.2015.11.011 39 Journal of Petroleum Research and Studies P- ISSN: 2220-5381 E- ISSN: 2710-1096 P- ISSN: 2220-5381 E- ISSN: 2710-1096 P- ISSN: 2220-5381 E- ISSN: 2710-1096 P- ISSN: 2220-5381 E- ISSN: 2710-1096 Open Access No. 40, September 2023, pp. 19-40 Open Access No. 40, September 2023, pp. 19-40 [30] M.F. Al-Shahwan and F.M. Al-Najm, "Tectonic evolution of southern part of the Mesopotamian foredeep basin, " J. of Petroleum Research & Studies, vol.30, no.3, pp.1- 17, 2021, https://doi.org/10.52716/jprs.v11i1.425 [30] M.F. Al-Shahwan and F.M. Al-Najm, "Tectonic evolution of southern part of the Mesopotamian foredeep basin, " J. of Petroleum Research & Studies, vol.30, no.3, pp.1- 17, 2021, https://doi.org/10.52716/jprs.v11i1.425 [31] W.F. McDonough and S.S. Sun, "The composition of the earth, " Chem. Geol., vol.120, pp. 223–253, 1995, https://doi.org/10.1016/0009-2541(94)00140-4 p p , f , , , pp , 2021, https://doi.org/10.52716/jprs.v11i1.425 [31] W.F. McDonough and S.S. Sun, "The composition of the earth, " Chem. Geol., vol.120, pp. 223–253, 1995, https://doi.org/10.1016/0009-2541(94)00140-4 [31] W.F. McDonough and S.S. Sun, "The composition of the earth, " Chem. Geol., vol.120, pp. 223–253, 1995, https://doi.org/10.1016/0009-2541(94)00140-4 [32] A.Z. Ekoa Bessa, G. Ngueutchoua and P.D. Ndjigui, "Mineralogy and geochemistry of sediments from Simbock Lake, Yaoundé area (southern Cameroon): provenance and environmental implications, " Arab J. Geosci, vol.11, 710, 2018, DOI: 10.1007/s12517-018- 4061-x [33] J.S. Armstrong-Altrin, Y.I. Lee and J.J. Kasper-Zubillaga, "Mineralogy and geochemistry of sands along the Manzanillo and El Carrizal beach areas southern Mexico: implications for palaeoweathering, provenance and tectonic setting, " Geol. J., vol. 52, pp. 559–582, 2017, https://doi.org/10.1002/gj.2792 [34] J.S. Armstrong-Altrin, A.V. Botello and S.F. Villanueva, "Geochemistry of surface sediments from the northwestern Gulf of Mexico: implications for provenance and heavy metal contamination, "Geol. Q, vol. 63, pp. 522–538, 2019, DOI: http://dx.doi.org/10.7306/gq.1484 40 40
https://openalex.org/W4223899927	https://zenodo.org/records/6449730/files/%D0%90%D0%A0%D0%93%D0%A3%D0%9C%D0%95%D0%9D%D0%A2%D0%98%20%D0%A1%D0%A3%D0%A7%D0%90%D0%A1%D0%9D%D0%9E%D0%87%20%D0%A4%D0%86%D0%9B%D0%9E%D0%9B%D0%9E%D0%93%D0%86%D0%87%202022%20%D0%B2%D0%B8%D0%BF%D1%80-%D0%BA%D0%BE%D0%BD%D0%B2%D0%B5%D1%80%D1%82%D0%B8%D1%80%D0%BE%D0%B2%D0%B0%D0%BD-95-99.pdf	Ukrainian	null	КОНЦЕПТ ЖІНОЧНОСТІ У ТВОРЧОСТІ А. АВЕРЧЕНКА ПЕРІОДУ ЕМІГРАЦІЇ (НА ПРИКЛАДІ ТВОРІВ «КАНИТЕЛЬ» ТА «ЗВИЧАЙНА ЖІНКА»)	Zenodo (CERN European Organization for Nuclear Research)	2,022	cc-by	1,494	Аргументи сучасної філології. Образ Жінки : «жіноче», «феміністське», «фемінне» Аргументи сучасної філології. Образ Жінки : «жіноче», «феміністське», «фемінне» ________________________________________________________________________________ Sapir-Whorf hypothesis in Hoijer, H. (1954). Language in culture: Conference on the interrelations of language and other aspects of culture. Chicago: University of Chicago Press. 92–105. Sapir-Whorf hypothesis in Hoijer, H. (1954). Language in culture: Conference on the interrelations of language and other aspects of culture. Chicago: University o Chicago Press. 92–105. Abstract The femininity of the heroines of Averchenko`s emigration stories is an integral part of their portraits. The understanding of the real essence of this bright feature of a real woman was distorted by the townsfolk who understood femininity as weakness, insignificance and stupidity. Femininity for them was a shameful trait, opposed to the respected courage of men. In the story «Gimp» Averchenko ridicules those character traits of the heroines, which are mistakenly identified by the philistine with the concept of femininity. The writer portrayed the frivolous flirtatious behavior of two women constantly creating artificial problems. Arkady Averchenko describes the disdainful attitude of a man towards a woman in the story «An Ordinary Woman». The heroine thinks about the aspects of mutual respect and responsibility of a man and a woman as the basis of their relationship. Plain and simple at first glance, Zoya is able to feel the deep moods of her beloved man, whom she sincerely admires. She tries to support him and inspire him with hope for the best. It depicts true femininity, incomprehensible to the average middlebrows. The concept of femininity was illuminated through the comic and satirical effects by means of grotesque, caricature, travesty and burlesque. 95 Аргументи сучасної філології. Образ Жінки : «жіноче», «феміністське», «фемінне» Аргументи сучасної філології. Образ Жінки : «жіноче», «феміністське», «фемінне» ________________________________________________________________________________ Keywords: the femininity, a woman, Arkady Averchenko, «Gimp», «An Ordinary Woman», the comic and satirical effects, the story. У творчості А. Аверченка періоду еміграції простежується уважне ставлення до жінки, прихильність та розуміння особливостей її становища у суспільстві, життєвих проблем. Жіночність героїнь творів письменника є невід’ємною складовою портрету справжньої жінки. Вона робить жіночу натуру магнетичною та унікальною. Розуміння дійсної суті цієї яскравої риси справжньої жінки спотворювалося обивателями, які вважали жіночність слабкістю, нікчемністю та дурістю, ганебною рисою, яка протиставлялася шанованій мужності чоловіків. У творі «Канитель» Аркадій Аверченко виносить до епіграфу ніби суто гумористичний, але дійсно, дуже характерний з приводу вищезгаданого, епізод спілкування судді та звинувачуваного на тему дефініції жіночності: «жіночна… це означає: дурепа» (Averchenko, 2007). І далі автор засобами яскравого гумору характеризує саме ті риси характерів героїнь твору, які обивателями помилково ототожнюються з поняттям жіночності. Визначаючи основні елементи комічного у творі Аверченка, можемо акцентувати на відображенні явищ, які мають у собі певні логічні невідповідності (Leinonen, 2015). Композиція цього твору (персонажі, сюжетні зв’язки, монтаж частин, повтори, інверсія, градація та ін.), як і багатьох інших оповідань А. Аверченка, відзначається як кільцева, коли дія ніби закручується по спіралі вверх через повторення ситуації. Це повторення відіграє важливу роль у створенні комічного ефекту. У «Канителі», дійсно, такі кільцеві повторення сварок та примирень двох подруг відразу налаштовують читача на гумористичне сприйняття сюжету. Комічний ефект також талановито створюється письменником через невідповідність певних зовнішніх ознак взаємодій персонажів (довірливі взаємовідносини, сповнені зворушливої відданості та зізнань у дружній любові 96 Аргументи сучасної філології. Образ Жінки : «жіноче», «феміністське», «фемінне» ________________________________________________________________________________ брюнетки та блондинки) та їхніх несподіваних сварок, за якими йдуть раптові бурхливі примирення. Оповідач відноситься до знайомих дам та їхніх штучних «перипетій» грайливо та доволі легковажно, на що, вони, власне, і заслуговують, з огляду на сміхотворні взаємні образи та претензії, кокетливу поведінку, не зовсім розумні як для дорослих зрілих жінок розмови, що помилково ототожнюються обивателями саме із жіночністю. Зверхнє, насмішкувате і зневажливе відношення чоловіка до жінки Аверченко змальовує в оповіданні «Звичайна жінка» (Averchenko, 2007). На відміну від незначущих за своєю сутністю «проблем», які турбують жіноцтво у «Канителі», героїня цього твору Зоя серйозно замислюється над питаннями життєвої філософії, як то: взаємоповага чоловіка і жінки у якості фундаменту їхніх стосунків, соціальні ролі з огляду на гендерні відмінності, відповідальність у парі один перед одним та перед нащадками. Письменник досягає більшої довіри читача описом того, що відбувається, знову ж таки, від першої особи. Аргументи сучасної філології. Образ Жінки : «жіноче», «феміністське», «фемінне» На початку оповіді він характеризує жінку, з якою живе герой твору, як витончене, ніжне та невагоме створіння, «золотисте, все наскрізь пронизане жовтими променями сонця». Саме це внутрішнє світло і було тим чинником, яке зумовлювало істинну жіночність Зої. Чоловік не сприймає жінку за рівну собі: «Але я ніяк не міг позбутися думки, що вона не справжня людина, потай дивився на неї, як на забавну іграшку». Коли Зоя запитує чоловіка, чи поважає він її, тому стає смішно від абсурдності самої ідеї про те, що жінка може очікувати поваги до себе: «Ну, за що тебе шанувати, скажи на милість?» Сприйняття жінки відбувається як людини, зовсім не гідної рівності з чоловіком: «розглядав її близько-близько, як дослідник природи – рідкісного звірка, і мені було смішно-смішно». 97 Для Зої ж її обранець – найгідніший з усіх чоловіків, вона щиро захоплюється ним, палко переконує у його надзвичайних якостях: «Знаєш, який ти? Я тебе опишу: у тебе очі горять, як дві зірочки, посмішка твоя туманить 97 Аргументи сучасної філології. Образ Жінки : «жіноче», «феміністське», «фемінне» ________________________________________________________________________________ голову, а твій голос проникає в саме серце і прямо перевертає його. Знаєш, на кого ти схожий? На срібного тигра, ось на кого». ________________________________________________________________________________ голову, а твій голос проникає в саме серце і прямо перевертає його. Знаєш, на кого ти схожий? На срібного тигра, ось на кого». Навіть пряма критика на адресу жінки з приводу, начебто, відсутності в неї логіки, притаманної чоловіку, і унеможливлення самої здатності Зої бути розумною та гідною бути на одному рівні з ним, не загасило в неї бажання відчути духовну єдність з коханою людиною: «Вона підняла на мене страждаючі, заплакані очі. — Це все дрібниці, всі зовнішні відмінності, а я говорю про духовну спорідненість. — На жаль, де вона?.. Чоловік майже завжди духовно та розумово перевершує жінку…» Насправді ж тонка і чутлива натура Зої була за духовним рівнем набагато вищою, ніж бачилося герою оповідання. Він відчував самовідданість жінки та її почуття до нього, в яких не було дріб’язкової прискіпливості, грубості, запеклості, мстивості за особисті образи. Натомість Зоя була сповнена любові та ніжності, прощення, намагання зрозуміти та бажання підтримати та упевнити свого чоловіка у його особливості. Чи ж не була ця жінка насправді жіночною? Герой твору відчував беззахисність вразливої Зоїної натури, шкодував її, навіть не розуміючи, який міцний стрижень у характері має це ефірне створіння, думаючи, що вона нікому не потрібна – така непристосована до життя та захоплена. Аргументи сучасної філології. Образ Жінки : «жіноче», «феміністське», «фемінне» Аргументи сучасної філології. Образ Жінки : «жіноче», «феміністське», «фемінне» Концепт жіночності було влучно висвітлено письменником у притаманному йому яскравому авторському стилі, зокрема, у творах «Канитель» та «Звичайна жінка», за допомогою створення комічного та сатиричного ефекту засобами гротеску, карикатури, травестування з елементами бурлеску (Lіteraturoznavchii slovnik-dovіdnik, 2007). Спираючись на розподіл Д. Левицьким жанрових різновидів творчості А. Аверченка на побутові оповідання та гротески, де перші характеризуються сюжетами, в основі яких лежать спостереження автора за довколишньою дійсністю, а другі ґрунтуються на комічних ситуаціях і характерах, створених фантазією автора, можемо зазначити, що оповідання «Канитель» можна віднести до другої, а «Звичайна жінка» – до першої групи [Levitskiy, 1999]. Бачимо, що розкриває Аркадій Аверченко образ жінки у контексті притаманних їй рис і якостей, серед яких провідна роль належить саме жіночності, з прихильністю, симпатією, та розумінням. При цьому навіть деякі гумористичні зауваження щодо жіночої логіки та поведінки не змінюють цього загалом чутливого ставлення митця. Аргументи сучасної філології. Образ Жінки : «жіноче», «феміністське», «фемінне» А вона по-доброму, поблажливо сама заспокоювала чоловіка-майбутнього батька, щоби той не хвилювався: «Ми, жінки, набагато сміливіші, мужніші за вас». В образі простої, як на перший погляд, тендітної Зої, здатної відчувати навіть глибинні настрої коханого чоловіка, підтримувати його та вселяти у нього світлу радість на надію на краще, Аверченко зобразив справжню жіночність. Трагічний фінал оповідання – це не крапка, а продовження процесу духовного дозрівання героя оповідання та усвідомлення ним образу втраченої назавжди жінки. 98 References Averchenko, A. (2007). Sobranie sochinenii: V 6 t. T. 4. Sornye travy [Weeds]. Moscow: TERRA-Knizhnyi klub [in Russian]. Averchenko, A. (2007). Sobranie sochinenii: V 6 t. T. 4. Sornye travy [Weeds]. Moscow: TERRA-Knizhnyi klub [in Russian]. Averchenko, A. (2007). Sobranie sochinenii: V 6 t. T. 6. Otdykh na krapive [Rest on nettles]. Moscow: TERRA-Knizhnyi klub [in Russian]. Averchenko, A. (2007). Sobranie sochinenii: V 6 t. T. 6. Otdykh na krapive [Rest on nettles]. Moscow: TERRA-Knizhnyi klub [in Russian]. Leinonen Anastasia. Huumorin ilmaisukeinot A. Averčenko nvalituissa kertomuksissa. Itä-Suomen yliopisto, 2015. URL: http://urn.fi/urn:nbn:fi:uef- 20160106 [in Finnish]. Leinonen Anastasia. Huumorin ilmaisukeinot A. Averčenko nvalituissa kertomuksissa. Itä-Suomen yliopisto, 2015. URL: http://urn.fi/urn:nbn:fi:uef- 20160106 [in Finnish]. Levitskiy, D. (1999). Zhizn' i tvorcheskii put' Arkadiya Averchenko [Life and creative path of Arkady Averchenko]. Moscow: Novyi put [in Russian]. Levitskiy, D. (1999). Zhizn' i tvorcheskii put' Arkadiya Averchenko [Life and creative path of Arkady Averchenko]. Moscow: Novyi put [in Russian]. p y y p Lіteraturoznavchii slovnik-dovіdnik [Literary dictionary-reference book] (2007). Kyiv: VTS «AkademіYA» [in Ukrainian]. Lіteraturoznavchii slovnik-dovіdnik [Literary dictionary-reference book] (2007). Kyiv: VTS «AkademіYA» [in Ukrainian]. 99
https://openalex.org/W4286005715	https://usiena-air.unisi.it/bitstream/11365/1220954/1/brainsci-12-00950.pdf	English	null	A Retrospective Evaluation to Assess Reliability of Electrophysiological Methods for Diagnosis of Hearing Loss in Infants	Brain sciences	2,022	cc-by	8,428	Marco Mandalà 1 , Luca Mazzocchin 2, Bryan Kevin Ward 3, Francesca Viberti 1,* , Ilaria Bindi 1 , Lorenzo Salerni 1 , Giacomo Colletti 4, Liliana Colletti 5 and Vittorio Colletti 6 Marco Mandalà 1 , Luca Mazzocchin 2, Bryan Kevin Ward 3, Francesca Viberti 1,* , Ilaria Bindi 1 , Lorenzo Salerni 1 , Giacomo Colletti 4, Liliana Colletti 5 and Vittorio Colletti 6 andalà 1 , Luca Mazzocchin 2, Bryan Kevin Ward 3, Francesca Viberti 1,* , Ilaria Bindi 1 , alerni 1 , Giacomo Colletti 4, Liliana Colletti 5 and Vittorio Colletti 6 1 Otolaryngology Department, University of Siena, 53100 Siena, Italy; mandal@unisi.it (M.M.); ilaria.bindi88@gmail.com (I.B.); lorenzosalerni@gmail.com (L.S.) 1 Otolaryngology Department, University of Siena, 53100 Siena, Italy; mandal@unisi.it (M.M.); 1 Otolaryngology Department, University of Siena, 53100 Siena, Italy; m ilaria.bindi88@gmail.com (I.B.); lorenzosalerni@gmail.com (L.S.) 1 Otolaryngology Department, University of Siena, 53100 Siena, Italy ilaria.bindi88@gmail.com (I.B.); lorenzosalerni@gmail.com (L.S.) ilaria.bindi88@gmail.com (I.B.); lorenzosalerni@gmail.com (L.S.) ilaria.bindi88@gmail.com (I.B.); lorenzosalerni@gmail.com (L.S.) 2 Ospedale Sacro Cuore-Don G. Calabria, Negrar, 37100 Verona, Italy; kaed@libero.it 3 Department of Otolaryngology-Head and Neck Surgery, John Hopkins Hospital, Baltimore, MD 21218, USA; bward15@jhmi.edu 4 Department of Maxillo-Facial Surgery, University of Milan, 20122 Milan, Italy; giacomo.colletti@gmail.com 5 ENT Department, Biomedical Sciences for Health, University of Milan, 20122 Milan, Italy; liliana.colletti@unimi.it 6 International Center for Performing and Teaching Auditory Brainstem Surgery in Children, 20121 Milan, Italy; vittoriocolletti@yahoo.com * Correspondence: francesca.vibe@gmail.com Abstract: Background: An electrophysiological investigation with auditory brainstem response (ABR), round window electrocochleography (RW-ECoG), and electrical-ABR (E-ABR) was performed in children with suspected hearing loss with the purpose of early diagnosis and treatment. The effectiveness of the electrophysiological measures as diagnostic tools was assessed in this study. Methods: In this retrospective case series with chart review, 790 children below 3 years of age with suspected profound hearing loss were tested with impedance audiometry and underwent electro- physiological investigation (ABR, RW-ECoG, and E-ABR). All implanted cases underwent pure-tone audiometry (PTA) of the non-implanted ear at least 5 years after surgery for a long-term assessment of the reliability of the protocol. Results: Two hundred and fourteen children showed bilateral severe-to-profound hearing loss. In 56 children with either ABR thresholds between 70 and 90 dB nHL or no response, RW-ECoG showed thresholds below 70 dB nHL. In the 21 infants with bilateral profound sensorineural hearing loss receiving a unilateral cochlear implant, no statistically signiﬁcant differences were found in auditory thresholds in the non-implanted ear between electrophysiological measures and PTA at the last follow-up (p > 0.05). Citation: Mandalà, M.; Mazzocchin, L.; Ward, B.K.; Viberti, F.; Bindi, I.; Salerni, L.; Colletti, G.; Colletti, L.; Colletti, V. A Retrospective Evaluation to Assess Reliability of Electrophysiological Methods for Diagnosis of Hearing Loss in Infants. Brain Sci. 2022, 12, 950. https:// doi.org/10.3390/brainsci12070950 Academic Editor: Manuel Sánchez Malmierca Academic Editor: Manuel Sánchez Malmierca Received: 2 June 2022 Accepted: 17 July 2022 Published: 20 July 2022 Received: 2 June 2022 Accepted: 17 July 2022 Published: 20 July 2022 Keywords: infants; auditory electrophysiological investigation; cochlear implant; auditory brainstem implant; reliability Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional afﬁl- iations. Marco Mandalà 1 , Luca Mazzocchin 2, Bryan Kevin Ward 3, Francesca Viberti 1,* , Ilaria Bindi 1 , Lorenzo Salerni 1 , Giacomo Colletti 4, Liliana Colletti 5 and Vittorio Colletti 6 Eight implanted children showed residual hearing below 2000 Hz worse than 100 dB nHL and 2 children showed pantonal residual hearing worse than 100 dB nHL (p > 0.05). Conclusion: The audiological evaluation of infants with a comprehensive protocol is highly reliable. RW-ECoG provided a better deﬁnition of hearing thresholds, while E-ABR added useful information in cases of auditory nerve deﬁciency. brain sciences brain sciences brain sciences 1. Introduction Infant hearing loss is associated with multiple adverse sequelae, including poor speech and language acquisition, decreased global quality of life, and increased societal cost for the care of deaf people. Cochlear implantation (CI) is one of the most common procedures to address this problem, together with auditory brainstem implantation (ABI), with steadily improving outcomes. Yet, despite the success of cochlear implantation, several important questions remain; speciﬁcally, the early identiﬁcation of which infants would beneﬁt from cochlear implantation [1–12]. Low rates of anesthetic and perioperative complications in young children [13] in association with improved outcomes in earlier implanted children Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). https://www.mdpi.com/journal/brainsci Brain Sci. 2022, 12, 950. https://doi.org/10.3390/brainsci12070950 Brain Sci. 2022, 12, 950 2 of 11 have led to dramatic increases in the number of infants being implanted at younger than 12 months of age [14–19]. The beneﬁts of earlier implantation increase the need to accurately diagnose hearing loss in younger infants. g g y g The primary aims of electrophysiological investigation in children with suspected hearing loss are: (1) the determination of hearing levels, and subsequently, in combination with radiological investigations, (2) the determination of candidacy for CI or ABI. Several electrophysiological tests have been proposed [20–23]. The most widely used methods for screening newborns for hearing loss are otoacoustic emissions, automated ABR and ABR during sleep, and behavioral audiograms. However, these methods may be unreliable in determining a precise hearing threshold in infants. g p g Since 1998, we have been developing a comprehensive electrophysiologic testing protocol. In addition to auditory brainstem responses (ABR), round window electro- cochleography (RW-ECoG) and round window electrical auditory brainstem responses (RW-EABR) have been applied in a standardized manner. RW-ECoG, a near-ﬁeld cochlear potential, has the advantage over ABR of better deﬁning hearing thresholds and residual hearing levels. RW-EABR can provide important information regarding the excitability of the auditory nerve, the site of the hearing loss, and therefore the candidacy for CI or ABI. y g y The aim of the present study is to evaluate this auditory electrophysiological testing protocol in deaf children for the selection of candidates for auditory implantation by reviewing the long-term follow-up of the non-implanted ear. With our testing protocol, we e al ofou d e o i eu al hea i Electrophysiological Investigation: eral profound sensorineural hearing loss before 6 months of age between 2002 and 2009 and were fitted with a unilateral cochlear implant (Cochlear Nucleus Series, Cochlear Ltd., Sydney, Australia) before the age of 1 year. This group of implanted infants was used to test the efficacy of the protocol below 1 year of age through long-term audiologi- cal follow-up. All children were implanted by the senior author (VC). All patients un- derwent pure-tone audiometry (PTA) and speech perception testing in quiet (mono/disyllabic word lists) of the non-implanted ear at least 5 years after implantation. Exclusion criteria were children who were unable to complete PTA testing due to cognitive disabilities or had developmental delay; diagnosed with auditory neuropathy; whose parents did not consent to CI or ABI even after diagnosis (n = 6). Electrophysiological Investigation: Evoked potentials were recorded either with the Amplaid MK12 or Medelec Syner- gy N-EP, Viasys system. The initial tests in the protocol included impedance audiometry Evoked potentials were recorded either with the Amplaid MK12 or Medelec Synergy N-EP, Viasys system. The initial tests in the protocol included impedance audiometry and ABR to clicks. ABR outcomes determined the continuation of the investigation with RW-ECoG. In the presence of a normal ABR threshold, i.e., better than 30 dB nHL, no further investigation was performed. When the ABR thresholds indicated moderate, severe, or profound hearing loss, children underwent RW-ECoG. For RW-ECoG, a myringotomy was performed under otomicroscopy from the malleus umbo to the posterior annulus of the tympanic membrane to visualize the round window niche. Any effusion was evacuated. A cotton-wick recording electrode was then placed on the round window niche under direct view (Figure 1). The electrode consisted of a string of 7 silver wires insulated with Teﬂon and soldered at the distal end to a cotton wick of about 1.5 mm diameter. A small pledget of wet Surgicel was placed over the electrode to ensure stability and improve electrode impedance. Contralateral masking was used during ABR and RW-ECoG. gy , y y p p y and ABR to clicks. ABR outcomes determined the continuation of the investigation with RW-ECoG. In the presence of a normal ABR threshold, i.e., better than 30 dB nHL, no further investigation was performed. When the ABR thresholds indicated moderate, se- vere, or profound hearing loss, children underwent RW-ECoG. 2. Materials and Methods Since 1998, we have used an electrophysiological testing protocol for determining hearing thresholds in children suspected of having profound hearing loss. This protocol was adopted in a tertiary referral center after newborn hearing screening test failure (otoacoustic emission and/or ABR during sleep). The protocol involves a series of tests including ABR, RW-ECoG, and RW-EABR performed under general anesthesia in a single recording session. The aim of the testing protocol is to exclude possible residual functional hearing, determine the site of the hearing loss, and subsequently, in association with imaging studies, select candidates for CI or ABI. If the electrophysiological data indicate that a child is a suitable candidate for CI or ABI, he or she additionally undergoes computed tomography (CT) and magnetic resonance imaging (MRI) to conﬁrm the diagnosis and evaluate for inner ear malformations. All subjects underwent genetic, pediatric, and neuropsychiatric evaluation, in order to understand if any associated disabilities (developmental delay, cognitive disorders) or genetic factors for hearing loss were present. A ﬂow chart for the testing protocol is shown in Scheme 1. g p With our testing protocol, we identiﬁed 21 infants who were diagnosed with bilateral profound sensorineural hearing loss before 6 months of age between 2002 and 2009 and were ﬁtted with a unilateral cochlear implant (Cochlear Nucleus Series, Cochlear Ltd., Sydney, Australia) before the age of 1 year. This group of implanted infants was used to test the efﬁcacy of the protocol below 1 year of age through long-term audiological follow-up. All children were implanted by the senior author (VC). All patients underwent pure-tone audiometry (PTA) and speech perception testing in quiet (mono/disyllabic word lists) of the non-implanted ear at least 5 years after implantation. Exclusion criteria were children who were unable to complete PTA testing due to cognitive disabilities or had developmental delay; diagnosed with auditory neuropathy; whose parents did not consent to CI or ABI even after diagnosis (n = 6). 3 of 11 3 of 11 Brain Sci. 2022, 12, 950 Brain Sci. 2022, 12, x FO Scheme 1. Flow chart of testing children in a single session. Scheme 1. Flow chart of testing children in a single session. Scheme 1. Flow chart of testing children in a single session. Scheme 1. Flow chart of testing children in a single session. With our testing protocol, we e al ofou d e o i eu al hea i Electrophysiological Investigation: Thereafter, ABR was repeated using 10 dB steps and then 5 dB steps until the threshold was established. EVIEW 4 of 11 windows of 10–15 ms. The threshold was deﬁned as the minimum stimulating level where an ABR response was both identiﬁable and repeatable. Starting at 120 dB nHL, clicks were presented twice for each sound intensity level in order to ensure reproducible responses. The level of stimulation was decreased in 20 dB nHL steps until no response was present. Thereafter, ABR was repeated using 10 dB steps and then 5 dB steps until the threshold was established. EVIEW 4 of 11 Figure 1. Cotton-wick electrode placed on the round window. Figure 1. Cotton-wick electrode placed on the round window. Figure 1. Cotton-wick electrode placed on the round window. Children with ABR and ECoG thresholds between 3 to an audiologist for a hearing aid trial. If RW-ECoG conf ing loss, i.e., a RW-ECoG threshold worse than 75 dB formed to differentiate a cochlear from a retrocochlear sit ton-wick electrode previously used for recording was th in order to test the viability of the cochlear nerve fibers b recording (Figure 2). Upon the completion of testing, th approximating the incision margins and placing a tympa when deemed necessary by the surgeon. Figure 1. Cotton-wick electrode placed on the round window. Figure 1. Cotton-wick electrode placed on the round window. Figure 1. Cotton-wick electrode placed on the round window. Children with ABR and ECoG thresholds between to an audiologist for a hearing aid trial. If RW-ECoG con ing loss, i.e., a RW-ECoG threshold worse than 75 dB formed to differentiate a cochlear from a retrocochlear s ton-wick electrode previously used for recording was t in order to test the viability of the cochlear nerve fibers recording (Figure 2). Upon the completion of testing, t approximating the incision margins and placing a tymp when deemed necessary by the surgeon. Figure 1. Cotton-wick electrode placed on the round window. Figure 1. Cotton-wick electrode placed on the round window. pp g g p g y p when deemed necessary by the surgeon. Figure 1. Cotton-wick electrode placed on the round window. Figure 1. Cotton-wick electrode placed on the round window. pp g g p g y p when deemed necessary by the surgeon. Children with ABR and ECoG thresholds between 30 and 75 dB nHL were referred to an audiologist for a hearing aid trial. With our testing protocol, we e al ofou d e o i eu al hea i Electrophysiological Investigation: If RW-ECoG confirmed severe or profound hear- ing loss, i.e., a RW-ECoG threshold worse than 75 dB nHL, RW-EABR was also per- formed to differentiate a cochlear from a retrocochlear site of hearing loss. The same cot- ton-wick electrode previously used for recording was then used to perform RW-EABR in order to test the viability of the cochlear nerve fibers by means of contralateral EABR recording (Figure 2). Upon the completion of testing, the myringotomy was closed by approximating the incision margins and placing a tympanic membrane resorbable patch when deemed necessary by the surgeon. Figure 2. ABR and RW-ECOG thresholds in a 5-month-old child. (A) ABR threshold in the right ear set at 110 dB nHL. (B) RW-ECOG threshold in the same ear observed at 70 dB nHL. (C) ABR in the left ear does not allow observation of the hearing threshold. (D) RW-ECOG threshold in the same ear at 70 dB nHL. Figure 2. ABR and RW-ECOG thresholds in a 5-month-old child. (A) ABR threshold in the right ear set at 110 dB nHL. (B) RW-ECOG threshold in the same ear observed at 70 dB nHL. (C) ABR in the left ear does not allow observation of the hearing threshold. (D) RW-ECOG threshold in the same ear at 70 dB nHL. Children with ABR and ECoG to an audiologist for a hearing aid ing loss, i.e., a RW-ECoG thresho formed to differentiate a cochlear f ton-wick electrode previously use in order to test the viability of the recording (Figure 2). Upon the co approximating the incision margin when deemed necessary by the sur G thresholds between 30 and 75 dB trial. If RW-ECoG confirmed severe old worse than 75 dB nHL, RW-EA from a retrocochlear site of hearing d for recording was then used to p cochlear nerve fibers by means of c ompletion of testing, the myringoto ns and placing a tympanic membran rgeon. trial to an i Figure 2. ABR and RW-ECOG thresholds in a 5-month-old child. (A) ABR threshold in the right ear set at 110 dB nHL. (B) RW-ECOG threshold in the same ear observed at 70 dB nHL. (C) ABR in the left ear does not allow observation of the hearing threshold. (D) RW-ECOG threshold in the same ear at 70 dB nHL. Figure 2. ABR and RW-ECOG thresholds in a 5-month-old child. With our testing protocol, we e al ofou d e o i eu al hea i Electrophysiological Investigation: For RW-ECoG, a myrin- gotomy was performed under otomicroscopy from the malleus umbo to the posterior annulus of the tympanic membrane to visualize the round window niche. Any effusion was evacuated. A cotton-wick recording electrode was then placed on the round win- dow niche under direct view (Figure 1). The electrode consisted of a string of 7 silver wires insulated with Teflon and soldered at the distal end to a cotton wick of about 1.5 Children with ABR and ECoG thresholds between 30 and 75 dB nHL were referred to an audiologist for a hearing aid trial. If RW-ECoG conﬁrmed severe or profound hearing loss, i.e., a RW-ECoG threshold worse than 75 dB nHL, RW-EABR was also performed to differentiate a cochlear from a retrocochlear site of hearing loss. The same cotton-wick electrode previously used for recording was then used to perform RW-EABR in order to test the viability of the cochlear nerve ﬁbers by means of contralateral EABR recording (Figure 2). Upon the completion of testing, the myringotomy was closed by approximating the incision margins and placing a tympanic membrane resorbable patch when deemed necessary by the surgeon. wires insulated with Teflon and soldered at the distal end to a cotton wick of about 1.5 mm diameter. A small pledget of wet Surgicel was placed over the electrode to ensure stability and improve electrode impedance. Contralateral masking was used during ABR and RW-ECoG. Auditory Brainstem Responses (ABR). For ABR testing, needle electrodes were placed subcutaneously with the standard vertex to pre-tragus montage. Electrode impedance was smaller than 3 kΩ. Electrodes were connected to an evoked potential system and responses were band-pass ﬁltered between 100 and 3000 Hz. Clicks of 100 µs duration, produced by a 100 µs electrical square wave, were presented via TDH-39 headphones at a rate of 21 per second with alternating polarity. Click signals were calibrated in dB nHL. At each presentation, the number of sweeps was set at 1024. Data were collected in time Brain Sci. 2022, 12, 950 4 of 11 windows of 10–15 ms. The threshold was deﬁned as the minimum stimulating level where an ABR response was both identiﬁable and repeatable. Starting at 120 dB nHL, clicks were presented twice for each sound intensity level in order to ensure reproducible responses. The level of stimulation was decreased in 20 dB nHL steps until no response was present. With our testing protocol, we e al ofou d e o i eu al hea i Electrophysiological Investigation: (A) ABR threshold in the right ear set at 110 dB nHL. (B) RW-ECOG threshold in the same ear observed at 70 dB nHL. (C) ABR in the left ear does not allow observation of the hearing threshold. (D) RW-ECOG threshold in the same ear at 70 dB nHL. Round Window Electrocochleography (RW-ECoG). RW-ECoG was performed us- ing the cotton-wick electrode over the round window niche, placed as described above Brain Sci. 2022, 12, 950 5 of 11 (Figure 1). The electrode was connected to the positive input of the preampliﬁer and referenced to a needle electrode placed at the ipsilateral pre-tragus area. The same ﬁlter settings and recording parameters as for ABR recording were used. Stimuli were presented with alternated polarity to cancel the microphonic potentials. At each presentation level, 128 to 256 sweeps were averaged. Acoustic monophasic clicks of 100 µs duration were presented with the same protocol as used for ABR recording. Cochlear microphonics (CM) recordings were obtained with positive and negative polarity clicks. Action potential (AP) responses were evoked with clicks of alternating polarity. Short 4 ms tone bursts or logon of different frequencies were then presented to selectively stimulate regions of the basilar membrane, and the AP was recorded. Four frequencies were used to assess the cochlear responses: 500, 1000, 2000, and 4000 Hz. The stimulus was selected to be brief enough to evoke a synchronized response, but of sufﬁcient duration to provide frequency speciﬁcity. Stimuli had the following characteristics: 500 Hz—rise and fall of 1 cycle with no plateau; 1000 Hz—rise and fall of 1 cycle and a plateau of 2 cycles; 2000 Hz—rise and fall of 2 cycles and a plateau of 4 cycles; 4000 Hz— rise and fall of 4 cycles and a plateau of 8 cycles. The thresholds for clicks and tone burst stimuli were obtained in a similar manner to that described for ABR testing above. g Round window electrical auditory brainstem responses (RW-EABR). After being used as a recording electrode for RW-ECoG, the same cotton-wick electrode placed on the round window niche was used to deliver electrical stimuli to test the viability of the cochlear nerve ﬁbers. To minimize stimulus artifacts, biphasic rectangular pulses were delivered by a constant current stimulator to the cotton-wick electrode. The stimulating electrode was referenced to a sub-dermal needle electrode placed in the ipsilateral pre-tragus area. 3. Results 3.1. Population With our testing protocol, we e al ofou d e o i eu al hea i Electrophysiological Investigation: The electrical stimulus was a biphasic pulse of 100 µs per phase and its intensity ranged from 1 to 1.5 mA. EABR were recorded using a contralateral electrode montage with the positive electrode inserted at the vertex, the negative electrode at the contralateral pre-tragus area, and the ground at the forehead. Each recorded waveform consisted of 1024 samples of EEG activity over a 10 ms time base, and the results were band-pass ﬁltered between 1 and 2500 Hz. The EABR threshold was deﬁned as the minimum current level required to evoke an identiﬁable wave V. The best threshold identiﬁed with ABR or ECoG testing was used to deﬁne the preoperative electrophysiological threshold for the frequency of the stimulus used. Before undergoing implantation, children underwent a trial with hearing aids, vibrotactile devices, or both with no improvement. Statistical analysis was performed using the paired T-test and Mann–Whitney test as appropriate (we compared the implanted ear with the non-implanted ear). Statistical signiﬁcance was set at p < 0.05. 3.1. Population A total number of 790 subjects underwent the diagnostic protocol, including 401 males and 389 females. The median age at the time of diagnosis was 13.4 ± 9.6 months. g g Out of the 790 tested children, 214 (27%) showed bilateral severe-to-profound hearing loss. Among these children, 13 (6.1%) also had a diagnosis of auditory neuropathy (absent ABR associated with normal CM) [24–26]. One hundred and ﬁfty-eight children (20%) received a CI and ﬁfty (6%) received an ABI. Six subjects did not perform surgery due to their parents’ refusal of the proposed treatment. 3.2. Outcomes of the Electrophysiological Investigation In 56 subjects (6.8%) with either ABR thresholds between 70 and 90 dB nHL or no response, RW-ECoG showed thresholds below 75 dB nHL (see Figure 2 for an example). RW-EABR showed reliable responses in 161 subjects, of whom 158 received a CI (Figure 3). RW-EABR showed no response in 50 children. These children had various inner ear malformations diagnosed by MR and CT (e.g., cochlear nerve deﬁciency, cochlear malformation, or both), and received an ABI. In 56 subjects (6.8%) with either ABR thresholds between 70 and 90 dB nHL or no response, RW-ECoG showed thresholds below 75 dB nHL (see Figure 2 for an example). RW-EABR showed reliable responses in 161 subjects, of whom 158 received a CI (Figure 3). RW-EABR showed no response in 50 children. These children had various in- ner ear malformations diagnosed by MR and CT (e.g., cochlear nerve deficiency, cochle- ar malformation, or both), and received an ABI. In 56 subjects (6.8%) with either ABR thresholds between 70 and 90 dB nHL or no In 56 subjects (6.8%) with either ABR thresholds between 70 and 90 dB nHL or no p g p RW-EABR showed reliable responses in 161 subjects, of whom 158 received a CI (Figure 3). RW-EABR showed no response in 50 children. These children had various inner ear malformations diagnosed by MR and CT (e.g., cochlear nerve deﬁciency, cochlear malformation, or both), and received an ABI. e po e, E o o e e o e o ( ee igu e o a e a p e) RW-EABR showed reliable responses in 161 subjects, of whom 158 received a CI (Figure 3). RW-EABR showed no response in 50 children. These children had various in- ner ear malformations diagnosed by MR and CT (e.g., cochlear nerve deficiency, cochle- ar malformation, or both), and received an ABI. Figure 3. RW-EABR sample. Test (gray line) and retest (black line) of the electrical stimulus. Figure 3. RW-EABR sample. Test (gray line) and retest (black line) of the electrical stimulus. Figure 3. RW-EABR sample. Test (gray line) and retest (black line) of the electrical stimulus. Figure 3. RW-EABR sample. Test (gray line) and retest (black line) of the electrical stimulus. No short-term or long-term complications (e.g., persistent tympanic membrane per- foration) due to either the testing protocol or general anesthesia were identified. Test protocol duration was assessed in a subset of recording sessions (163 subjects). 3.2. Outcomes of the Electrophysiological Investigation 3.2. Outcomes of the Electrophysiological Investigation Electrophysiological thresholds obtained with ABR and RW-ECoG tests are presented in Table 1. Electrophysiological thresholds obtained with ABR and RW-ECoG tests are presented in Table 1. Brain Sci. 2022, 12, 950 6 of 11 re pre- Table 1. Assessment of hearing thresholds with ABR, RW-ECoG, and RW-EABR. Investigation Hearing Threshold Number of Patients (%) ABR (790 subjects) Normal (<30 dB nHL) 436 (55.2%) Mild to Moderate (30–50 dB nHL) 48 (6.1%) Moderate to Severe (55–70 dB nHL) Severe (75–90 dB nHL) Profound (>90 dB nHL) 39 (4.9%) 96 (12.2%) 171 (21.6%) RW-ECoG (354 subjects) Normal (<30 dB nHL) Mild to Moderate (30–50 dB nHL) 11 (3.1%) 42 (11.8%) Moderate to Severe (55–70 dB nHL) Severe (75–90 dB nHL) Profound (>90 dB nHL) 87 (24.6%) 70 (19.8%) 144 (40.7%) RW-EABR Present 161 (76.2%) Absent 53 (24.8%) In 56 subjects (6.8%) with either ABR thresholds between 70 and 90 dB nHL or no response, RW-ECoG showed thresholds below 75 dB nHL (see Figure 2 for an example). RW-EABR showed reliable responses in 161 subjects, of whom 158 received a CI (Figure 3). RW-EABR showed no response in 50 children. These children had various inner ear malformations diagnosed by MR and CT (e.g., cochlear nerve deﬁciency, cochlear malformation, or both), and received an ABI. Table 1. Assessment of hearing thresholds with ABR, RW-ECoG, and RW-EABR. Investigation Hearing Threshold Number of Patients (%) ABR (790 subjects) Normal (<30 dB nHL) 436 (55.2%) Mild to Moderate (30–50 dB nHL) 48 (6.1%) Moderate to Severe (55–70 dB nHL) Severe (75–90 dB nHL) Profound (>90 dB nHL) 39 (4.9%) 96 (12.2%) 171 (21.6%) RW-ECoG (354 subjects) Normal (<30 dB nHL) Mild to Moderate (30–50 dB nHL) 11 (3.1%) 42 (11.8%) Moderate to Severe (55–70 dB nHL) Severe (75–90 dB nHL) Profound (>90 dB nHL) 87 (24.6%) 70 (19.8%) 144 (40.7%) RW-EABR Present 161 (76.2%) Absent 53 (24.8%) In 56 subjects (6.8%) with either ABR thresholds between 70 and 90 dB nHL or no response, RW-ECoG showed thresholds below 75 dB nHL (see Figure 2 for an example). RW-EABR showed reliable responses in 161 subjects, of whom 158 received a CI (Figure 3). RW-EABR showed no response in 50 children. These children had various in- ner ear malformations diagnosed by MR and CT (e.g., cochlear nerve deficiency, cochle- ar malformation, or both), and received an ABI. Table 1. 3.2. Outcomes of the Electrophysiological Investigation Test pro- No short-term or long-term complications (e.g., persistent tympanic membrane perfo- ration) due to either the testing protocol or general anesthesia were identiﬁed. Test protocol duration was assessed in a subset of recording sessions (163 subjects). Test protocol dura- tion for ABR is related to the number of intensity levels tested and ranged from 4 min to 15 min (mean 9 ± 4 min). The RW-ECoG portion, including myringotomy and placement of the electrode, lasted between 8 min and 20 min (mean 14 ± 5 min). RW-EABR testing lasted between 4 and 11 min (mean 8 ± 3 min). Total electrophysiological protocol duration ranged from 28 to 65 min (mean 41 ± 13 min). 3.2. Outcomes of the Electrophysiological Investigation Assessment of hearing thresholds with ABR, RW-ECoG, and RW-EABR. Investigation Hearing Threshold Number of Patients (%) ABR (790 subjects) Normal (<30 dB nHL) 436 (55.2%) Mild to Moderate (30–50 dB nHL) 48 (6.1%) Moderate to Severe (55–70 dB nHL) Severe (75–90 dB nHL) Profound (>90 dB nHL) 39 (4.9%) 96 (12.2%) 171 (21.6%) RW-ECoG (354 subjects) Normal (<30 dB nHL) Mild to Moderate (30–50 dB nHL) 11 (3.1%) 42 (11.8%) Moderate to Severe (55–70 dB nHL) Severe (75–90 dB nHL) Profound (>90 dB nHL) 87 (24.6%) 70 (19.8%) 144 (40.7%) RW-EABR Present 161 (76.2%) Absent 53 (24.8%) Table 1. Assessment of hearing thresholds with ABR, RW-ECoG, and RW-EABR. Investigation Hearing Threshold Number of Patients (%) ABR (790 subjects) Normal (<30 dB nHL) 436 (55.2%) Mild to Moderate (30–50 dB nHL) 48 (6.1%) Moderate to Severe (55–70 dB nHL) Severe (75–90 dB nHL) Profound (>90 dB nHL) 39 (4.9%) 96 (12.2%) 171 (21.6%) RW-ECoG (354 subjects) Normal (<30 dB nHL) Mild to Moderate (30–50 dB nHL) 11 (3.1%) 42 (11.8%) Moderate to Severe (55–70 dB nHL) Severe (75–90 dB nHL) Profound (>90 dB nHL) 87 (24.6%) 70 (19.8%) 144 (40.7%) RW-EABR Present 161 (76.2%) Absent 53 (24.8%) Table 1. Assessment of hearing thresholds with ABR, RW-ECoG, and RW-EABR. Table 1. Assessment of hearing thresholds with ABR, RW-ECoG, and RW-EABR I ti ti H i Th h ld N b In 56 subjects (6.8%) with either ABR thresholds between 70 and 90 dB nHL or no response, RW-ECoG showed thresholds below 75 dB nHL (see Figure 2 for an example). RW-EABR showed reliable responses in 161 subjects, of whom 158 received a CI (Figure 3). RW-EABR showed no response in 50 children. These children had various inner ear malformations diagnosed by MR and CT (e.g., cochlear nerve deﬁciency, cochlear malformation, or both), and received an ABI. In 56 subjects (6.8%) with either ABR thresholds between 70 and 90 dB nHL or no response, RW-ECoG showed thresholds below 75 dB nHL (see Figure 2 for an example). RW-EABR showed reliable responses in 161 subjects, of whom 158 received a CI (Figure 3). RW-EABR showed no response in 50 children. These children had various in- ner ear malformations diagnosed by MR and CT (e.g., cochlear nerve deficiency, cochle- ar malformation, or both), and received an ABI. 3.3. Pure Tone Audiometry To further assess the efﬁcacy of our protocol, hearing thresholds obtained at initial diagnosis by electrophysiological methods were compared to PTA at least 5 years after the implantation. Twenty-one infants diagnosed with bilateral profound sensorineural hearing loss before 6 months of age and ﬁtted with a unilateral cochlear implant before 12 months underwent pure-tone audiometry (PTA) of the non-implanted ear at least 5 years after the implantation. Demographics and clinical data of these cases, including the etiology of hearing loss if known, are presented in Table 2. Brain Sci. 2022, 12, 950 7 of 11 Table 2. Demographics and clinical data of implanted subjects. Number of Patients (%) Number of subjects 21 Sex (male/female) 10/11 Etiologies - Genetic - Cytomegalovirus - Perinatal anoxia - Unknown Age at diagnosis Age at implantation Age at PTA of non-implanted ear 7 (33.3%) 3 (14.3%) 3 (14.3%) 8 (38.1%) 3.5 ± 1.1 months 6.8 ± 3.0 months 8.3 ± 2.6 months None of the implanted children had functional hearing in the non-operated ear, deﬁned as at least one threshold better than or equal to 75 dB nHL for 250, 500, or 1000 Hz. • In 11 children (52.5%), PTA conﬁrmed the previous diagnosis of profound hearing loss (no responses up to 120 dB nHL), with no differences between pre- and post-operative PTA in the non-operated ear. W 8 of 11 • In 11 children (52.5%), PTA conﬁrmed the previous diagnosis of profound hearing loss (no responses up to 120 dB nHL), with no differences between pre- and post-operative PTA in the non-operated ear. W 8 of 11 p • Eight children (38%) had hearing worse than 100 dB nHL at frequencies below 2000 Hz and two children (9.5%) showed a ﬂat residual hearing worse than 100 dB nHL. No statistically signiﬁcant differences in the paired T-test and Mann–Whitney test were identiﬁed between auditory thresholds of the non-implanted ear between the results at diagnosis (115.8 ± 4.9 dB nHL) and at the last follow-up (113 ± 6.2 dB nHL) among the 8 subjects (Figure 4; p > 0.05) with residual hearing below 2 kHz (0.5 and 1 kHz) (Table 3). able 3. Mean hearing threshold at diagnosis and at the last follow-up on the non-implanted side the 8 subjects with residual hearing below 2 kHz. 4. Discussion As a result of the critical importance of auditory input early in life for speech and language development and the ability to intervene with hearing devices (hearing aids, CI, and ABI), many countries have adopted mandatory newborn hearing screening. Children deprived of auditory information in the ﬁrst 3–5 years of life will have difﬁculty in oral com- munication even if their hearing is restored because their brain mechanisms for developing auditory pattern recognition will have passed the critical period of brain plasticity [27,28]. For many years, it was also thought that RW-ECoG was not reliable enough to predict hearing status and thus that the risks of the procedure (notably, general anesthesia and myringotomy) did not justify the limited beneﬁt. However, as the present results show, RW-ECoG does have the reliability to diagnose severe and profound hearing loss in infants. This objective electrophysiological procedure, combined with RW-EABR, can be used to establish candidacy for CI or ABI in infants. RW-ECoG represents cochlear microphonics and summation potentials and is related to hair cell presence and health. RW-EABR, in contrast, is an evoked neural potential and indicates the presence and health of the auditory nerve. Recording these potentials from the round window provides a clear signal that has high reliability, selectivity, and speciﬁcity for hearing diagnosis. Children with damaged hair cells but an intact auditory nerve can beneﬁt from a CI, while children with no response on either measure require an ABI because this indicates the absence of an auditory nerve, so a CI would be of no beneﬁt. This lack of auditory nerve can be cross-checked with MRI results. Buchmann et al. (2011) [19] showed that children with auditory nerve deﬁciency as shown on MRI as well as poor EABR measures performed in the lower 10%-ile of CI outcomes, even after 10 years of use. Furthermore, RW-ECoG demonstrated moderate hearing loss (<70 dB nHL) in 53/790 (6.8%) subjects with an ABR threshold between 70 and 90 dB nHL or no response. All these children were referred to the audiologist for hearing aid evaluation. Conversely, extratympanic ECoG seems less effective [29,30]. RW-ECoG has been demonstrated as reli- able in monitoring hearing function during CI in children [22] and adults [31,32]. Recently, early implantation has also been associated with an improved rate of residual hearing preservation [33,34]. 3.3. Pure Tone Audiometry Hearing Threshold (dB nHL) p-Value ean Hearing Threshold (0,5 kHZ) at diagnosis 115.5 ± 5.5 0.38 ean Hearing Threshold (0,5 kHZ) at last follow-up 1 112.5 ± 5.9 ean Hearing Threshold (1 kHZ) at diagnosis 116 ± 4.6 0 41 ( ) Residual hearing was not detectable on the implanted side in any children. Speech discrimination scores at the last follow-up with the cochlear implant off were 0%. ea i g e o ( ) a iag o i 6 6 0.41 Hearing Threshold (1 kHZ) at last follow-up 1 113.5 ± 6.7 hearing threshold at the last follow-up on the non-implanted side. ( ) • Residual hearing was not detectable on the implanted side in any children. Speech discrimination scores at the last follow-up with the cochlear implant off were 0%. g ( ) g 0.41 ean Hearing Threshold (1 kHZ) at last follow-up 1 113.5 ± 6.7 Mean hearing threshold at the last follow-up on the non-implanted side. gure 4. Electrophysiological auditory measurements (before 6 months of age; RW-ECoG tone urst at 500 and 1000 Hz, better thresholds between tone burst at 1000, 2000 Hz and click) vs. PTA utcomes (later than 5 years after surgery) in the 10 children with residual hearing. Discussion Figure 4. Electrophysiological auditory measurements (before 6 months of age; RW-ECoG tone burst at 500 and 1000 Hz, better thresholds between tone burst at 1000, 2000 Hz and click) vs. PTA outcomes (later than 5 years after surgery) in the 10 children with residual hearing. ure 4. Electrophysiological auditory measurements (before 6 months of age; RW-ECoG tone st at 500 and 1000 Hz, better thresholds between tone burst at 1000, 2000 Hz and click) vs. PTA comes (later than 5 years after surgery) in the 10 children with residual hearing. Figure 4. Electrophysiological auditory measurements (before 6 months of age; RW-ECoG tone burst at 500 and 1000 Hz, better thresholds between tone burst at 1000, 2000 Hz and click) vs. PTA outcomes (later than 5 years after surgery) in the 10 children with residual hearing. Brain Sci. 2022, 12, 950 8 of 11 Table 3. Mean hearing threshold at diagnosis and at the last follow-up on the non-implanted side in the 8 subjects with residual hearing below 2 kHz. 3.3. Pure Tone Audiometry Hearing Threshold (dB nHL) p-Value Mean Hearing Threshold (0,5 kHZ) at diagnosis 115.5 ± 5.5 0.38 Mean Hearing Threshold (0,5 kHZ) at last follow-up 1 112.5 ± 5.9 Mean Hearing Threshold (1 kHZ) at diagnosis 116 ± 4.6 0.41 Mean Hearing Threshold (1 kHZ) at last follow-up 1 113.5 ± 6.7 1 Mean hearing threshold at the last follow-up on the non-implanted side. The mean speech perception outcome at the last follow-up for the 21 children im- planted (implant ON) was 85.2 ± 10.7. 4. Discussion Another possible use of RW-ECoG and RW-EABR, in combination with MRI, concerns the selection of the children suitable for an ABI, prior to CI use and subsequent poor outcomes, avoiding the loss of time for speech development [35]. The combination of the presented extensive electrophysiological and radiological (CT and MRI) protocols is helpful in improving the efﬁcacy of diagnosis, despite being mildly invasive. The extra information that RW-ECoG provided can be fundamental in view of a better threshold deﬁnition as well as to rule out auditory neuropathy. In cases of cochlear nerve hypoplasia, RW-EABR could add important information regarding the candidacy between CI and ABI. Brain Sci. 2022, 12, 950 9 of 11 9 of 11 The audiological evaluation of infants with a comprehensive protocol seems to be highly effective in subjects with profound hearing loss who are candidates for cochlear implantation below 1 year of age and even before 6 months. Residual hearing was detected in half of the 21 subjects who underwent CI, but no subjects showed serviceable residual hearing for hearing aid ﬁtting. Our results agree with Vlastarakos et al., who underline the safety and efﬁcacy of the diagnosis and, eventually, CI in infants [3,13]. Regarding ABR outcomes, multifrequency response seems reliable for deﬁning CI candidates only if the result is “no responce” [29]. Aimoni et al. [20], recently described a series of 23 infants who demonstrated signiﬁcant improvement in hearing after 6 months for term-born children and up to 11 months in severe prematurity. Among these subjects, only 10 demonstrated profound bilateral hearing loss at the ﬁrst assessment, and only 1 among the 8 described in detail in the text performed transtympanic ECoG under general anesthesia. In our opinion, these results are comparable with the outcomes of the present study in light of the fact that RW-ECoG seems to be the most sensitive test to deﬁne hearing levels in children due to the nearer ﬁeld recording and the chance to improve middle ear effusion with suction. The major limitations of the present study are the small number of children below 1 year of age implanted and that the non-implanted ear has been considered as the control group since they had substantially the same hearing threshold. Finally, as in all studies concerning cochlear implants, the heterogeneity of the population and multiple covariates must be taken into consideration. 4. Discussion In our experience, RW-ECoG and RW-EABR are at present the most reliable electro- physiologic techniques for establishing hearing thresholds and the site of lesion in infants with severe to profound hearing loss, furnishing useful indications for candidacy to CI or ABI in a quick, inexpensive, cost-effective, and reproducible way. The entirety of the proce- dure lasts on average 40′, a duration that may be achieved thanks to general anesthesia. The important information obtained from RW-ECoG and RW-EABR can avoid placing an unnecessary CI as well as placing a cochlear implant in a “dead” cochlea unsuitable for electrical stimulation. 5. Conclusions A comprehensive electrophysiological evaluation of infants is highly reliable in identi- fying children with profound hearing loss who may be candidates for CI or ABI. Severe-to- profound loss was detected in half of the examined cohort, and some had minor residual hearing, but none showed residual hearing sufﬁcient for hearing aid use. We propose RW- ECoG as a reliable method for diagnosing and deﬁning hearing loss and for determining whether a hearing aid, CI, or ABI is the most appropriate management approach. Author Contributions: Conceptualization: M.M., V.C.; methodology, M.M., B.K.W., V.C., F.V.; formal analysis, M.M., L.M., F.V.; investigation, L.M., M.M., L.C., G.C., V.C.; resources: V.C., M.M., L.M.; data curation, M.M., L.M., L.S.; writing—original draft preparation, B.K.W., M.M., I.B., F.V.; writing— review and editing, I.B., B.K.W., F.V. All authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding. Funding: This research received no external funding. Institutional Review Board Statement: Ethics approval was obtained from the University ethics committees (Comitato etico per la Sperimentazione Clinica delle Province di Verona e Rovigo, CESC VR-RO). Informed Consent Statement: All parents were duly informed regarding the aim and protocol of the study and gave their consent. Informed Consent Statement: All parents were duly informed regarding the aim and protocol of the study and gave their consent. Data Availability Statement: Not applicable. Data Availability Statement: Not applicable. Acknowledgments: We thank University of Siena for supporting research and logistic assistance. Acknowledgments: We thank University of Siena for supporting research and logistic assistance. Conﬂicts of Interest: The authors declare no conﬂict of interest. Conﬂicts of Interest: The authors declare no conﬂict of interest. 10 of 11 Brain Sci. 2022, 12, 950 References [CrossRef] [PubMed] 13. Vlastarakos, P.V.; Candiloros, D.; Papacharalampous, G.; Tavoulari, E.; Kampessis, G.; Mochloulis, G.; Nikolopoulos, T.P. Diagnos- tic challenges and safety considerations in cochlear implantation under the age of 12 months. Int. J. Pediatr. Otorhinolaryngol. 2010, 74, 127–132. [CrossRef] [PubMed] 14. Colletti, L.; Mandalà, M.; Colletti, V. Cochlear implants in children younger than 6 months. Otolaryngol. Head Neck Surg. 2012, 147, 139–146. [CrossRef] [PubMed] 15. Tait, M.; De Raeve, L.; Nikolopoulos, T.P. Deaf children with cochlear implants before the age of 1 year: Comparison of preverbal communication with normally hearing children. Int. J. Pediatr. Otorhinolaryngol. 2007, 71, 1605–1611. [CrossRef] 16. Valencia, D.M.; Rimell, F.L.; Friedman, B.J.; Oblander, M.R.; Helmbrecht, J. Cochlear implantation in infants less than 12 months of age. Int. J. Pediatr. Otorhinolaryngol. 2008, 72, 767–773. [CrossRef] 17. Miyamoto, R.T.; Hay-McCutcheon, M.J.; Kirk, K.I.; Houston, D.M.; Bergeson-Dana, T. Language skills of who received cochlear implants under 12 months of age: A preliminary study. Acta Otolaryngol. 2008, 17. Miyamoto, R.T.; Hay-McCutcheon, M.J.; Kirk, K.I.; Houston, D.M.; Bergeson-Dana, T. Language skills of profoundly deaf children who received cochlear implants under 12 months of age: A preliminary study. Acta Otolaryngol. 2008, 128, 373–377. [CrossRef] 18 N ij K S K i E D S hi R Sh h PV K l A B H B R h id A L D J S i R i f 18. Noij, K.S.; Kozin, E.D.; Sethi, R.; Shah, P.V.; Kaplan, A.B.; Herrmann, B.; Remenschneider, A.; Lee, D Nontumor Pediatric Auditory Brainstem Implant Outcomes. Otolaryngol. Head Neck Surg. 2015, 153, 73 19. Buchman, C.A.; Teagle, H.F.; Roush, P.A.; Park, L.R.; Hatch, D.; Woodard, J.; Zdanski, C.; Adunka, O.F. Cochlear implantation in children with labyrinthine anomalies and cochlear nerve deﬁciency: Implications for auditory brainstem implantation. Laryngoscope 2011, 121, 1979–1988. [CrossRef] 20. Aimoni, C.; Ciorba, A.; Bovo, R.; Trevisi, P.; Busi, M.; Martini, A. Hearing threshold assessment in young children with electrocochleography (EcochG) and auditory brainstem responses (ABR): Experience at the University Hospital of Ferrara. Auris Nasus Larynx. 2010, 37, 553–557. [CrossRef] y 21. Ciorba, A.; Hatzopoulos, S.; Petruccelli, J.; Mazzoli, M.; Pastore, A.; Kochanek, K.; Skarzynski, P.; Wlodarczyk, A.; Skarzynski, H. Identifying congenital hearing impairment: Preliminary results from a comparative study using objective and subjective audiometric protocols. Acta Otorhinolaryngol. Ital. 2013, 33, 29–35. [PubMed] p y g 22. References 1. Nikolopoulos, T.P. Neonatal hearing screening: What we have achieved and what needs to be improved. Int. J. Pediatr. Otorhinolaryngol. 2015, 79, 635–637. [CrossRef] [PubMed] [ ] [ ] 2. Dettman, S.J.; Dowell, R.C.; Choo, D.; Arnott, W.; Abrahams, Y.; Davis, A.; Dornan, D.; Leigh, J.; Constantinescu, G.; Cowan, R.; et al. Long-term Communication Outcomes for Children Receiving Cochlear Implants Younger Than 12 Months: A Multicenter Study. Otol. tol Neurotol. 2016, 37, e82–e95. [CrossRef] y 3. Vlastarakos, P.V.; Proikas, K.; Papacharalampous, G.; Exadaktylou, I.; Mochloulis, G.; Nikolopoulos, T.P. Cochlear implantation under the ﬁrst year of age: The outcomes. A critical systematic review and meta-analysis. Int. J. Pediatr. Otorhinolaryngol. 2010, 74, 119–126. [CrossRef] 4. Colletti, L.; Mandala‘, M.; Zoccante, L.; Shannon, R.V.; Colletti, V. Infants versus older children ﬁtted with cochlear implants: Performance over 10 years. Int. J. Pediatr. Otorhinolaryngol. 2011, 75, 504–509. [CrossRef] 5. Lammers, M.J.; Jansen, T.T.; Grolman, W.; Lenarz, T.; Versnel, H.; van Zanten, G.A.; Topsakal, V.; Lesinski-Schiedat, A. The inﬂuence of newborn hearing screening on the age at cochlear implantation in children. Laryngoscope 2015, 125, 985–990. [CrossRef] 6. Waltzman, S.B.; Roland, J.T., Jr. Cochlear implantation in children younger than 12 months. Pediatrics 2005, 116, e487–e493. [CrossRef] [PubMed] 7. Dettman, S.J.; Pinder, D.; Briggs, R.J.; Dowell, R.C.; Leigh, J.R. Communication development in children who receive the cochlear implant younger than 12 months: Risks versus beneﬁts. Ear Hear. 2007, 28, 11S–18S. [CrossRef] [PubMed] 8. Colletti, L.; Shannon, R.V.; Colletti, V. The development of auditory perception in children after auditory brainstem implantation. Audiol. Neurootol. 2014, 19, 386–394. [CrossRef] 9. Kanji, A.; Khoza-Shangase, K.; Moroe, N. Newborn hearing screening protocols and their outcomes: A systematic review. Int. J. Pediatr. Otorhinolaryngol. 2018, 115, 104–109. [CrossRef] 10. Farinetti, A.; Raji, A.; Wu, H.; Wanna, B.; Vincent, C. International consensus (ICON) on audiological a in children. Eur. Ann. Otorhinolaryngol. Head Neck Dis. 2018, 135, S41–S48. [CrossRef] ; Wu, H.; Wanna, B.; Vincent, C. International consensus (ICON) on audiological assessment of hearing loss n. Otorhinolaryngol. Head Neck Dis. 2018, 135, S41–S48. [CrossRef] y g 11. O’Neill, C.; Archbold, S.M.; O’Donoghue, G.M.; McAlister, D.A.; Nikolopoulos, T.P. Indirect costs, cost-utility variations and the funding of paediatric cochlear implantation. Int. J. Pediatr. Otorhinolaryngol. 2001, 58, 53–57. [CrossRef] g p p y g 12. Colletti, L.; Mandalà, M.; Shannon, R.V.; Colletti, V. Estimated net saving to society from cochlear implantation in infants: A preliminary analysis. Laryngoscope 2011, 121, 2455–2460. References Formeister, E.J.; McClellan, J.H.; Merwin, W.H., 3rd; Iseli, C.E.; Calloway, N.H.; Teagle, H.F.; Buchman, C.A.; Adunka, O.F.; Fitzpatrick, D.C. Intraoperative round window electrocochleography and speech perception outcomes in pediatric cochlear implant recipients. Ear Hear. 2015, 36, 249–260. [CrossRef] [PubMed] p p 23. Kileny, P.R.; Zwolan, T.A. Perioperative, transtympanic electric abr in paediatric cochlear implant candidates. Cochlear Implant. Int. 2004, 5 (Suppl. 1), 23–25. [CrossRef] 24. Santarelli, R.; Arslan, E. Electrocochleography in auditory neuropathy. Hear. Res. 2002, 170, 32–47. 24. Santarelli, R.; Arslan, E. Electrocochleography in auditory neuropathy. Hear. Res. 2002, 170, 32–47. [CrossRef] 25. Santarelli, R.; Starr, A.; Michalewski, H.J.; Arslan, E. Neural and receptor cochlear potentials obtained by transtympanic electrocochleography in auditory neuropathy. Clin. Neurophysiol. 2008, 119, 1028–1104. [CrossRef] 11 of 11 11 of 11 Brain Sci. 2022, 12, 950 26. De Siati, R.D.; Rosenzweig, F.; Gersdorff, G.; Gregoire, A.; Rombaux, P.; Deggouj, N. Auditory Neuropathy Spectrum Disorders: From Diagnosis to Treatment: Literature Review and Case Reports. J. Clin. Med. 2020, 9, E1074. [CrossRef] g p J [ ] 27. Cardon, G.; Campbell, J.; Sharma, A. Plasticity in the developing auditory cortex: Evidence from children with sensorineural hearing loss and auditory neuropathy spectrum disorder. J. Am. Acad. Audiol. 2012, 23, 396–495. [CrossRef] [PubMed] g y p y p 28. Harrison, R.V.; Gordon, K.A.; Mount, R.J. Is there a critical period for cochlear implantation in congenitally deaf children? Analyses of hearing and speech perception performance after implantation. Dev. Psychobiol. 2005, 46, 252–261. [CrossRef] 29. Hang, A.X.; Roush, P.A.; Teagle, H.F.; Zdanski, C.; Pillsbury, H.C.; Adunka, O.F.; Buchman, C.A. Is “no response” on diagnostic auditory brainstem response testing an indication for cochlear implantation in children? Ear Hear. 2015, 36, 8–13. [CrossRef] 29. Hang, A.X.; Roush, P.A.; Teagle, H.F.; Zdanski, C.; Pillsbury, H.C.; Adunka, O.F.; Buchman, C.A. Is “no response” on diagnostic auditory brainstem response testing an indication for cochlear implantation in children? Ear Hear. 2015, 36, 8–13. [CrossRef] 30. Fjermedal, O. 1.; Laukli, E.; Mair, I.W. Auditory brainstem responses and extratympanic electrocochleography. A threshold comparison in children. Scand. Audiol. 1988, 17, 231–235. [CrossRef] y g 30. Fjermedal, O. 1.; Laukli, E.; Mair, I.W. Auditory brainstem responses and extratympanic electrococh comparison in children. Scand. Audiol. 1988, 17, 231–235. [CrossRef] ti, L.; Tonoli, G.; Colletti, V. Electrocochleography during cochlear implantation for hearing preservation ck Surg. 2012, 146, 774–781. [CrossRef] p 31. Mandalà, M.; Colletti, L.; Tonoli, G.; Colletti, V. [ ] [ ] 35. Colletti, L.; Colletti, G.; Mandalà, M.; Colletti, V. The therapeutic dilemma of cochlear nerve deﬁciency: Cochlear or brainstem implantation? Otolaryngol. Head Neck Surg. 2014, 151, 308–314. [CrossRef] [PubMed] References Electrocochleography during cochlear implantation Otolaryngol. Head Neck Surg. 2012, 146, 774–781. [CrossRef] 32. Haumann, S.; Imsiecke, M.; Bauernfeind, G.; Büchner, A.; Helmstaedter, V.; Lenarz, T.; Salcher, R.B. Monitoring of the Inner Ear Function During and After Cochlear Implant Insertion Using Electrocochleography. Trends Hear. 2019, 23, 2331216519833567. [CrossRef] [PubMed] [ ] [ ] 33. Giardina, C.K.; Brown, K.D.; Adunka, O.F.; Buchman, C.A.; Hutson, K.A.; Pillsbury, H.C.; Fitzpatrick, D.C. Intracochlear Electrocochleography: Response Patterns During Cochlear Implantation and Hearing Preservation. Ear Hear. 2019, 40, 833–848. [CrossRef] [PubMed] [ ] [ ] 34. Anagiotos, A.; Hamdan, N.; Lang-Roth, R.; Gostian, A.O.; Lüers, J.C.; Hüttenbrink, K.B.; Beutner, D. Young age is a positive prognostic factor for residual hearing preservation in conventional cochlear implantation. Otol. Neurotol. 2015, 36, 28–33. [CrossRef] [PubMed] [ ] [ ] 35. Colletti, L.; Colletti, G.; Mandalà, M.; Colletti, V. The therapeutic dilemma of cochlear nerve deﬁciency: Cochlear or brainstem implantation? Otolaryngol. Head Neck Surg. 2014, 151, 308–314. [CrossRef] [PubMed]
https://openalex.org/W2792316956	https://ascimaging.springeropen.com/track/pdf/10.1186/s40679-018-0050-0	English	null	A non-rigid registration method for the analysis of local deformations in the wood cell wall	Advanced structural and chemical imaging	2,018	cc-by	7,630	© The Author(s) 2018. 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. A non‑rigid registration method for the analysis of local deformations in the wood cell wall Patera1,2, Stephan Carl3, Marco Stampanoni1,5, Dominique Derome3* and Jan Carmeliet3,4 Alessandra Patera1,2, Stephan Carl3, Marco Stampanoni1,5, Dominique Derome3* and J Abstract This paper concerns the problem of wood cellular structure image registration. Given the large variability of wood geometry and the important changes in the cellular organization due to moisture sorption, an affine-based image registration technique is not exhaustive to describe the overall hygro-mechanical behaviour of wood at microme- tre scales. Additionally, free tools currently available for non-rigid image registration are not suitable for quantifying the structural deformations of complex hierarchical materials such as wood, leading to errors due to misalignment. In this paper, we adapt an existing non-rigid registration model based on B-spline functions to our case study. The so-modified algorithm combines the concept of feature recognition within specific regions locally distributed in the material with an optimization problem. Results show that the method is able to quantify local deformations induced by moisture changes in tomographic images of wood cell wall with high accuracy. The local deformations provide new important insights in characterizing the swelling behaviour of wood at the cell wall level. K d I i t ti B li f ti F f d f ti S d X t h E i l t Keywords: Image registration, B-spline function, Free form deformation, Spruce wood, X-ray tomography, Equivalent strain mage registration, B-spline function, Free form deformation, Spruce wood, X-ray tomography, Equiva Patera et al. Adv Struct Chem Imag (2018) 4:1 https://doi.org/10.1186/s40679-018-0050-0 Patera et al. Adv Struct Chem Imag (2018) 4:1 https://doi.org/10.1186/s40679-018-0050-0 Open Access Correspondence: dominique.derome@empa.ch 3 EMPA, Swiss Federal Laboratories for Materials Science and Technology, Laboratory for Multiscale Studies in Building Physics, Überlandstrasse 129, 8600 Dübendorf, Switzerland Full list of author information is available at the end of the article Background 1 a Cross-sectional view of spruce wood sample consisting in two tissues with different porosity, i.e. latewood and earlywood. The ROI used for analysis is shown with the red box. b 3D comparison of the volumes at the two RH states: 25%RH in yellow and 85%RH in red i In image registration, the deformed image, also called moving image IM(x), is transformed to be aligned to the reference or original image, called fixed image IF(x). Both images have dimensions s and are defined in their spa- tial domain: ΩF ⊂Rd and ΩM ⊂Rd for fixed and mov- ing images, respectively. In general, the transformation is defined as a mapping from the moving to the fixed image, i.e. T : ΩF ⊂Rd →ΩM ⊂Rd. The transformation that matches IM(x) to IF(x) is defined as: Fig. 1 a Cross-sectional view of spruce wood sample consisting in two tissues with different porosity, i.e. latewood and earlywood. The ROI used for analysis is shown with the red box. b 3D comparison of the volumes at the two RH states: 25%RH in yellow and 85%RH in red (1) T(x) = x + U(x) (1) where U(x) is the displacement that makes IM(x + U(x)) to be aligned to IF(x). The goodness of alignment is evalu- ated by a distance or similarity measure S, such as the correlation ratio, the sum of squared differences (SSD), or the mutual information (MI). study. However, the same method can be applicable to a variety of materials that present a complex structure such as wood. Before describing our approach for resolving quantitatively the local deformation in wood cell wall, a general overview of the documented image registration algorithms is briefly given in the following paragraph. Furthermore, the B-spline based non-rigid registration technique is detailed described prior defining the sug- gested improvements to make it suitable for material sci- ence applications. The method is then validated by means of finite element modelling comparison and applied on images of wood cell wall acquired at two relative humid- ity states. Background or is accompanied by local deformations. Tridimensional characterization of swelling strains in wood tissues is rarely documented. X-ray tomography, a non-invasive method, is retained to image tridimensionally the cel- lular structure, at different states of swelling or shrink- age induced by relative humidity changes, an example of wood structure with earlywood and latewood, scanned at two relative humidity (RH) values, i.e. 25% RH and 85% RH, is shown in Fig. 1. In Derome et al. [5] and in Pat- era et al. [25], the swelling/shrinkage of wood has been documented in 3D by adopting an affine registration model as good approximation for describing the defor- mations of wood cell wall due to moisture changes. How- ever, this model fails to identify the local deformations, thus resulting not exhaustive in the characterization of the hygro-mechanical behavior of wood. This paper pre- sents a quantitative approach to precisely capture the local deformations of wood cellular structure in high- resolution tomographic datasets. The proposed method represents a modification of an existing non-rigid image registration algorithm to be adapted to the specific case Wood, a cellular biological material, swells or shrinks due to the adsorption or desorption of water in the hygro- scopic domain. While sorption occurs within the cell wall material, the cellular organization of wood leads to signif- icant deformations in the transversal directions of wood, i.e. tangentially and radially to the growth rings, with very little swelling in the longitudinal direction. Growth rings are made of low density spring wood and denser summer wood, yielding cell lumen and wall dimensions to vary over more than one order of magnitude. Most cells, the tracheids, are vertical in the trunk but 5–10% of the cells, named rays, are positioned horizontally linking radially the cells across growth rings. Given this geomet- rical variability, an analysis of wood deformations at the cellular scale could elucidate whether swelling is regular Correspondence: dominique.derome@empa.ch 3 EMPA, Swiss Federal Laboratories for Materials Science and Technology, Laboratory for Multiscale Studies in Building Physics, Überlandstrasse 129, 8600 Dübendorf, Switzerland Full list of author information is available at the end of the article Page 2 of 11 Patera et al. Adv Struct Chem Imag (2018) 4:1 at capturing the local deformations of cellular materi- als, such as wood, a general mathematical description of image registration method is first given. Fig. Background Commonly, registration is a problem of optimization in which a cost function C has to be minimized, resulting in ˆT: (2) ˆT = arg min T C(T; IF, IM) (2) where ˆT represents the set of points of the transforma- tion between fixed and moving images for which the given cost function attains its minimum value.hi The problem of the identification of the non-rigid transformation T becomes easily ill-posed and, therefore, a regularisation or penalty term P constraining the trans- formation T is introduced. Then the cost function is as follows: Image registration problem: a general overviewf Image registration is a method to map two different images, which are acquired with the same or different experimental setups. Due to the importance of image registration in various application areas and given its complicated nature, a large number of image registration algorithms have been developed in the past. An exhaus- tive review of general-purpose image registration meth- ods can be found in Brown [2] and in Wyawahare et al. [33]. Applications of image registration in the medical field include combining data from different modalities e.g., computer tomography (CT) and magnetic resonance imaging (MRI), to obtain more complete information about the patient, monitoring tumour growth [33], treat- ment verification [11, 12, 30], computer-aided diagnosis and disease following-up [15]; surgery simulation [22]; atlas building and comparison [13]; radiation therapy [8, 17]; anatomy segmentation [4, 7, 9, 10, 16, 20, 34] and image subtraction for contrast-enhanced images [19]. In contrast, much less algorithms for image registration are nowadays available for material applications. To allow introducing the basic idea of the new algorithm, aimed (3) C(T; IF, IM) = −S(T; IF, IM) + γ P(T) (3) where γ weights similarity against regularity. The cost function is described by the similarity term when γ tends to zero. A similarity measure is a function that takes two input images as parameters and computes a numerical value that quantifies the extent to which the two images are similar. The regularity term P(T) is designed to penalize control points displacements that potentially lead to naturally implausible deformations. Transformations used to align two images can be global or local. A global transformation is given by a sin- gle equation, which maps the entire image. One global method is the affine registration model, which allows to quantify the affine strains along the three orthotropic directions of wood. However, this model fails to identify the local deformations [5, 25]. In this paper, an approach to detect and quantify local deformations in the cellular tissues using a non-rigid registration model is proposed. Any plane through wood cellular structure shows a form, Patera et al. Adv Struct Chem Imag (2018) 4:1 Page 3 of 11 u = x/nx −⌊x/nx⌋, v = y/ny − y/ny w = z/nz−⌊z/nz⌋. B1 represents the lth basis function of the B-spline, as defined below: which can be represented by a free-form surface with the aim of tracking its deformation during free swelling. Image registration problem: a general overviewf This freeform surface can be determined using control points connected together by a mesh. The surface is approxi- mated using a control mesh guaranteeing a certain level of smoothness. Many representations of free-form sur- face exist in the literature [29] and an approach of rep- resenting free-form deformations based on B-splines is used [28]. The approach proposed in this work has been adapted to the specific case study of wood cellular mate- rial and implemented in Matlab. It is based on an existing method implemented by Rueckert, as described in the following. (5) B0(u) = (1 −u)3/6 B1(u) = (3u3 −6u2 + 4)/6 B2(u) = (−3u3 + 3u2 + 3u + 1)/6 B3(u) = u3/6 (5) The control points Πi,j,k are the unknown parameters of the B-spline FFD. The level of the non-rigid trans- formation depends on the resolution of the mesh of the control points. The spacing between the control points determines the resolution of non-rigid registration, i.e. a large spacing or low resolution results in a more global estimation of the deformations, compared to a smaller spacing (higher resolution) which models highly local deformations. At the same time, the number of control points determines the number of degrees of freedom and the computational complexity. The B-spline grid is con- structed with the method of Lee et al. [18].hi the wood case study In general, the deformation of wood contains a non-rigid component so that affine transformations alone are not sufficient to describe local deformations in wood tissues, subjected to free swelling due to water vapour adsorp- tion, as identified, but not quantified yet, in Derome et al. [5] and Patera et al. [25]. Therefore, the transformation analysis includes both the affine and non-rigid com- ponents, as shown in Eq. 6. The global transformation Tglobal(x, y, z) is the affine transformation resulting from the affine registration model, as described in Derome et al. [5] and Patera et al. [25].h more global deformations in the material. ‘Registration 2’ is performed directly on the original images with increas- ing the freedom of the B-spline grid, i.e. decreasing the weight coefficient γ of the penalty. In this way, the local misalignment can be more easily detected, although more artefacts coming from the high freedom of the B-spline grid can arise. To prevent these artefacts problems, the local deformations are defined by subtracting ‘Registra- tion 2’ from ‘Registration 1’ and the local transformation is calculated. However, there are cases in which these methods do not give the optimal solution. For this rea- son, a third registration technique is introduced. The third approach for non-rigid registration is called point- based registration or ‘Registration P’. In this case, the input consists in a set of points in the two images. Dif- ferent techniques for the detection of control point pairs in the images are used, which are named Manual, Map, Skeleton, Harris or Edges techniques. The manual selec- tion technique consists in selecting the initial pairs in the two images manually. All the other techniques are auto- matic. Map is a simple procedure of tracking the borders of features using binary images and giving the coordi- nates of the borders as output. Skeleton follows a skel- etonization procedure consisting in the extraction of a region-based shape feature which represents the general structure of an object. Harris is a method for calculating and displaying the feature points as Harris corners [14] and, finally, edges is based on the Canny edges detec- tion method [3]. One of these methods is initially used The algorithm, proposed by Rueckert et al. [28] and described above, is modified to improve the performance of the FFD for describing local deformations in complex cellular materials, such as wood. B‑splines based non‑rigid registration method: an elegant formulationh The local transformation Tlocal(x, y, z) captures the local deformations in the object. An elegant way to describe local deformations avoiding difficult parameterization methods has been introduced by Rueckert et al. [28]. The model is based on B-splines [31] and it is known as the Free Form Deformation (FFD) model. FFD is a tech- nique for manipulating any shape in a free-form manner, as shown in Fig. 2. Basically, an object is deformed by manipulating an underlying mesh of control points. The local transformation is described in the volume domain Ω= (x, y, z)\|0 ≤x < X, 0 ≤y < Y , 0 ≤z < Z as a mesh of control points Πi,j,k with uniform spacing δ and with number of elements N = nx × ny × nz. The transfor- mation function Tlocal(x, y, z) is written as the 3-D tensor product of 1-D cubic B-splines and is defined in terms of the control points Πi,j,k: The total transformation in the object is defined as the sum of the global and local transformation. (6) T x, y, z = Tglobal x, y, z + Tlocal x, y, z (6) The parameters of the global and local transformations are determined by solving the optimization problem, i.e. by minimising the cost function defined in Eq. 3. A penalty term is introduced in Eq. 3 to constrain the local transformation and ensure smoothness. This term is described by Wahba [32] as follows: (7) P(T) = 1 V X 0 Y 0 Z 0 ∂2T ∂x2 2 + ∂2T ∂y2 2 + ∂2T ∂z2 2 + 2 ∂2T ∂xy 2 +2 ∂2T ∂xz 2 + 2 ∂2T ∂yz 2 dx dy dz (4) Tlocal x, y, z = 3 l=0 3 m=0 3 n=0 Bl(u)Bm(v)Bn(w)Πi+l,j+m,k+n (4) where i = ⌊x/nx⌋−1, j = y/ny −1, k = ⌊z/nz⌋−1 and where ⌊⌋ means the floor of the num- ber (i.e. the largest integer less than or equal to the number). The variables u, v, w are defined as: (7) Fig. 2 Example of deformable mesh in which each point is locally controlled, as shown, for example, in the blue, yellow and green boxes Fig. 2 Patera et al. Adv Struct Chem Imag (2018) 4:1 Page 4 of 11 Fig. 3 Overview of the non-rigid registration algorithm with the three methods described in the text with V denoting the volume of the image domain. B‑splines based non‑rigid registration method: an elegant formulationh The penalty term of a cost function is equal to zero in the case of an affine transformation. In Eq. 3, the similarity term is evaluated by comparing the histogram, hist, of IF and IM. As a general rule, mutual information (e.g., the degree of dependence of one image with respect to the other one, Pluim et al. [27]) is applied if (8) hist(IM) −hist(IF) > 0.25 (8) In the specific case study of this work, the difference between the two histograms of grey levels of the ref- erence and moving images is smaller than 0.25 and the goodness of alignment cannot, therefore, be evaluated with the mutual information. Thus, the similarity meas- ure is evaluated with the SSD. the wood case study Most of the algorithms presented in literature, such as the one applied in this work, are based on the histogram of grey levels. The basic and simple idea of this modified version is to introduce some morphological operations in the original method to guide the algorithm in recognising typical features in complex structures, such as wood. As an overview of the non-rigid registration method, a flowchart is given in Fig. 3. Three approaches for non- rigid registration are used to register the moving image IM(x) and the fixed image IF(x). The two first registration approaches perform registration directly on the images, i.e., on the intensity of the grey values. We refer to these two approaches as ‘Registration 1’ and ‘Registration 2’. ‘Registration 1’ is a rough and fast estimation of non-rigid deformations performed on smaller size images, where the B-spline functions are largely constrained (increasing the weight γ of the penalty term in Eq. 3) to describe the Page 5 of 11 Patera et al. Adv Struct Chem Imag (2018) 4:1 A polynomial or affine transformation is used to trans- form the artificial grid matching Πj(GF) with Πl(GM). The initial pairs or feature points in the two images are selected in such a way to ensure a matching between image features using correlation. A graphical illustration of the point registration method, illustrating how the fea- ture points are added to the artificial grid coordinates, is given in Fig. 4. to extract feature points in both the fixed and moving images. The registration algorithm is performed on pair of control points instead of the image histograms. After detection, it is important to check that corresponding feature coordinates are found in both images. A nor- malized cross-correlation function is thus introduced to adjust each pair of control points. The algorithm moves the position of a control point by up to four pixels, to adjust the coordinates with an accuracy up to one-tenth of a pixel. One of the major drawbacks of such non-rigid reg- istration method is related to the high-degree of free- dom-inducing artefacts which is given to the B-spline functions to capture all potential local deformations in the structure. A way to prevent such artefacts is to include more constraints on the transformation. How- ever, this becomes at finer grid resolutions. the wood case study To overcome these difficulties, the solution proposed in this work is to make a comparison between two registration types and to consider, as final result, the image difference between Then, the initial image pairs Πi(IF), Πk(IM) i,k=1...n are added, in both images, to the points of the artificial grids Πj(GF), Πl(GM) j,l=1...m to obtain ΠF and ΠM, as: (9)    F = i(IF), j(GF) i=1...n j=1...m F = k(IM), l(GM) k=1...n l=1...m (9)    F = i(IF), j(GF) i=1...n j=1...m F = k(IM), l(GM) k=1...n l=1...m (9) Fig. 4 Graphical illustration of the different steps representing the two key-features of non-rigid registration algorithm based on points. On the right, a zoom on a squared region of interest is presented to visualise the described procedure. a The feature points (or coordinates) are extracted on the reference and moving images. b An artificial grid is added on both images and, finally, c the features points are summed to the nodes of the artificial grid in both, reference and moving images Fig. 4 Graphical illustration of the different steps representing the two key-features of non-rigid registration algorithm based on points. On the right, a zoom on a squared region of interest is presented to visualise the described procedure. a The feature points (or coordinates) are extracted on the reference and moving images. b An artificial grid is added on both images and, finally, c the features points are summed to the nodes of the artificial grid in both, reference and moving images Fig. 4 Graphical illustration of the different steps representing the two key-features of non-rigid registration algorithm based on points. On the right, a zoom on a squared region of interest is presented to visualise the described procedure. a The feature points (or coordinates) are extracted on the reference and moving images. b An artificial grid is added on both images and, finally, c the features points are summed to the nodes of the artificial grid in both, reference and moving images Patera et al. Adv Struct Chem Imag (2018) 4:1 Page 6 of 11 Page 6 of 11 ‘Registration 1’, considered as the reference since it includes more constraints, and ‘Registration 2’ or ‘Regis- tration P’, depending on the case study.hif ∇C ≤χ for some small positive value of χ. the wood case study The mini- mization loop based on the steepest descent technique is implemented within a line search strategy. As line search strategy, two methods are used: the first is a simple one based on a parametric function; the second is a normal line search method with Wolfe conditions. A detailed description of line search strategy can be found in Noce- dal and Wright [24]. The error is defined as the pixel difference between the two images after non-rigid registration. The pair of reg- istration images with the smallest difference is consid- ered for the final calculation of the local deformations and non-rigid strains. This step ensures the selection of the best non-rigid registration method for each specific region of interest studied in the volume. Once the opti- mization problem is solved by minimization of the cost function, the displacement field is determined and the local strain fields are evaluated and plotted.h As mentioned above, the algorithm is implemented in 2D as the deformations in wood occur almost only along the tangential and radial directions. Therefore, before applying the non-rigid registration algorithm, a set of slices at the same plane of fixed and moving images are selected. Then, the optimal parameters for the three reg- istrations methods, ‘Registration 1’, ‘Registration 2’ and ‘Registration P’, are determined. Finally, the algorithm runs in a loop over the whole stack of slices. i The algorithm runs for a stack of images in a loop, per- forming a 2D registration image by image. In the case study of swelling deformations in softwood tissues, a 2D registration is sufficient to define the local deforma- tions as wood swelling is predominantly occurring along tangential and radial directions. For each registration step (i.e. ‘Registration 1’, ‘Registration 2’ and ‘Registra- tion P’), the non-rigid (briefly, n.a. to differentiate from the affine a.) strain fields are calculated from the gradi- ent of the displacement field (Ux, Uy) in each direction (εn.a.x, εn.a.xy, εn.a.y) and the equivalent strain is calculated using a simplification of the von Mises relationship (von Mises [23]), valid in this case study: Validation of the algorithm by comparison with a finite element modelh The performance of the algorithm is validated in 2D using a pair of images, e.g. a fixed image IF and a mov- ing image IM, generated specifically for this validation exercise. A homogeneous sample of square configura- tion undergoes bending, which is a pure non-rigid defor- mation, and the deformations are calculated by finite element method, as shown in Fig. 5a where a grid is drawn for visualisation of the deformation. In this exam- ple, the steepest descend method is selected to solve the optimization problem, since a better solution is found with this method compared to line search meth- ods of optimization. Additionally, γ = 0.01 in ‘Registra- tion 1’ and γ = 0.001 in ‘Registration 2’. In ‘Registration P’, ‘Map’ is chosen as the method for point extraction. When the algorithm finds a minimum, the refinement loop is ended. The error between the reference and the deformed images is calculated for each registration type. In this case, the error is expressed in terms of number of pixels, normalised over the total amount of pixels in the region. The error obtained for ‘Registration P’, 0.006, is smaller than the error calculated with ‘Registration 2’, 0.009. Therefore, ‘Registration P’ is chosen for the calcu- lation of the deformation fields and of the local strains shown in Fig. 5d, e. (10) εn.a. = εn.a.2x + 2 × εn.a.2xy + εn.a.2y (10) The equivalent strain is used to describe the deforma- tion intensity in wood. More details on strain tensors cal- culation can be found in Abd-Elmoniem et al. [1]. As previously described, the non-rigid registration problem can be then solved by performing first the inten- sity-based methods (‘Registration 1’ and ‘Registration 2’) and then the point-based method (‘Registration P’). The two intensity-based registrations methods are the critical steps of the algorithm as they incorporate the initial opti- misation and minimisation problem in a sequential loop. Rueckert et al. [28] describe the optimization prob- lem in terms of minimizing a cost function, as given in Eq. 3, where the optimization proceeds in several steps to improve the computational efficiency. First, the aff- ine transformation Tglobal(x,y,z) is optimized, which cor- responds to optimizing the similarity between the two images, where the penalty term of the cost function in (7) is zero. During the subsequent stage, the non-rigid registration parameters are optimized. Results and discussionh deformation is seen between the CT datasets acquired at start and end of the sorption–desorption sequences. The aim of this study is to quantify these deformations also locally using non-rigid registration. The non-rigid registration algorithm presented in this paper is used for documenting the occurrence of local deformations during swelling of the complex cellu- lar structure of softwood. The focus is to investigate the moisture-induced deformations in tissues from spruce wood, namely Picea abies (L. Karst). The algo- rithm is applied on tomographic datasets of wood with a voxel pitch equal to 0.8 μm, acquired at the Centre for X-Ray Tomography of the Ghent University (UGCT) in Belgium [6, 21]. The analysis is performed on a wood sample of cross-section dimensions of approximately 500 × 700 μm2 which presents a combination of tissues, named earlywood and latewood, with different porosities (≈ 78% for earlywood and 45% for latewood) and hygro- mechanical behaviour (anisotropic swelling in earlywood and more isotropic swelling in latewood). The sample is scanned at two relative humidity (RH) values, referred as dry state, i.e. 25% RH, and wet state, i.e. 85% RH, as shown in Fig. 1b. Wood swells when exposed to an increase in RH and the typical deformations observed in the cellular structure of wood samples has been described globally. From previous [5, 25] and recent work [26], it is known that these X-ray measurements are reproducible as no From the 3D datasets, 200 cross-sectional slices are selected and further cropped to a region of interest (ROI) of the cellular structure, as shown in Fig. 1a. Two datasets are thus obtained, using the images acquired at 25% RH as the fixed images and the ones at 75% RH at the moving images. After the affine registration, the non-rigid registration proceeds as follows. The registra- tion approach based on the grey values intensity with the steepest descend method allows to compare the perfor- mance of the B-spline algorithm in two cases: the first on the resized volumes for a fast estimation (‘Registration 1’), where the B-spline functions are constrained to less freedom using a value of γ = 1.0E−2, while the second case, ‘Registration 2’, consists in giving more freedom to the functions with a γ = 1.0E−4. Additionally, the point- based registration algorithm (‘Registration P’) is applied with the method named ‘Map’ used for point extraction. Validation of the algorithm by comparison with a finite element modelh In each stage, a simple iterative steepest descent technique is used step- ping in the direction of the gradient vector with a certain step size. The algorithm stops when a local minimum of the cost function is found, given by the condition that The local strains calculated with the modified algo- rithm can be compared with the results of the simulation with finite element (Fig. 5f) obtained with the software Abaqus FEA. A good agreement between the results of simulation and registration algorithm is observed. The strains show in the same range of values and distribu- tion over the surface. Only is the non-rigid strains, in the y-direction, we see some fluctuations. Page 7 of 11 Patera et al. Adv Struct Chem Imag (2018) 4:1 Example of application of the algorithm on an artificial homogeneous image (a) where the grid is imposed to highlight the deformations smatches. The difference between reference and moving images is shown before registration (b), then after non-rigid registration (c). In on-rigid displacement fields in the two directions and in e the non-rigid strains in the x–y plane are mapped on the reference image. f i elds plotted on the deformed image as result of finite element simulation, under the assumption of homogeneous material. The strain in ion (E11) is on the left side, the strain calculated in y-direction (E22) is on right side Fig. 5 Example of application of the algorithm on an artificial homogeneous image (a) where the grid is imposed to highlight the deformations and mismatches. The difference between reference and moving images is shown before registration (b), then after non-rigid registration (c). In d the non-rigid displacement fields in the two directions and in e the non-rigid strains in the x–y plane are mapped on the reference image. f Strain fields plotted on the deformed image as result of finite element simulation, under the assumption of homogeneous material. The strain in x-direction (E11) is on the left side, the strain calculated in y-direction (E22) is on right side Page 8 of 11 Page 8 of 11 Patera et al. Adv Struct Chem Imag (2018) 4:1 Results and discussionh The non-rigid differences (errors) calculated as the differ- ence due to misalignment between reference and moving images are reported for each slice in Fig. 6a. ‘Registration Fig. 6 a Errors between reference and registered images after non-rigid registration for each slices of the volume, using an intensity-based method for R1 (grey) and R2 (black) estimation and a point-based registration RP (orange). b Displacement fields in one slice of the wood sample in pixels (1 pixel = 0.8 μm), c 2D map of total strains (affine plus non-rigid) on the reference images Fig. 6 a Errors between reference and registered images after non-rigid registration for each slices of the volume, using an intensity-based method f R ( ) d R (bl k) ti ti d i t b d i t ti R ( ) b Di l t fi ld i li f th d l i i l (1 Fig. 6 a Errors between reference and registered images after non-rigid registration for each slices of the volume, using an intensity-based method for R1 (grey) and R2 (black) estimation and a point-based registration RP (orange). b Displacement fields in one slice of the wood sample in pixels (1 pixel = 0.8 μm), c 2D map of total strains (affine plus non-rigid) on the reference images Fig. 6 a Errors between reference and registered images after non-rigid registration for each slices of the volume, using an intensity-based method for R1 (grey) and R2 (black) estimation and a point-based registration RP (orange). b Displacement fields in one slice of the wood sample in pixels (1 pixel = 0.8 μm), c 2D map of total strains (affine plus non-rigid) on the reference images Patera et al. Adv Struct Chem Imag (2018) 4:1 Page 9 of 11 Fig. 7 a Three-dimensional map of equivalent von Mises non-rigid strains on the wood cell wall of sample ELWd,1 for 200 slices, as indicated in the bar. b Cross-section cuts in the position indicated in red in the bar 2’ gives the best approximation, showing less non-rigid errors compared with ‘Registration P’. Therefore, we use ‘Registration 2’ for calculating the non-rigid displace- ment and strain fields, by subtracting the displacements obtained with ‘Registration 1’ from the ones calculated with ‘Registration 2’. Results and discussionh Figure 6b shows the deformations in pixel, where a pixel measures 0.8 μm, in the tangential (x-) and radial (y-) directions for one slice of the datasets. The deforma- tions are calculated over the whole area of the ROI, and thus deformations are calculated independently from the actual cellular structure. In the next step, shown in Fig. 6c, the total local strain (affine plus non-rigid), are presented. In this sample, a combination of positive (red) and negative (blue) total strains in Exx are observed in the region between earlywood and latewood. This com- bined effect indicates a bending of the cell structure. For Eyy, a negative strain (blue) is observed along the loca- tion of ray cells in the earlywood cell wall, surrounded by regions in earlywood with positive (red) strains. This observation could indicate to a kind of slip behaviour between rays and surrounding material. This result sug- gests the restraining role of ray cells on the cellular struc- ture of soft materials, such as wood. Fig. 7 a Three-dimensional map of equivalent von Mises non-rigid strains on the wood cell wall of sample ELWd,1 for 200 slices, as indicated in the bar. b Cross-section cuts in the position indicated in red in the bar Finally, the equivalent non-rigid (von Mises) com- ponents are calculated slice by slice and plotted in 3D respecting the cellular structure in Fig. 7. Figure 7 shows that high values of equivalent non-rigid strains occur in latewood cells close to the latewood/earlywood inter- face and close to the ray cells, especially in the early- wood layer. Wood samples combined by high density (named latewood) and low density (earlywood) swell in general less than the single tissues. In addition, it was found in Patera et al. [25] that the swelling becomes less anisotropic between radial and tangential directions in combined samples compared to single earlywood and latewood tissues. This means that, due to the restraining effect of latewood on earlywood and vice versa, swelling of combined wood reduces and becomes less anisotropic. In the analysis of the non-rigid strain components, it is possible to observe positive swelling strain values in late- wood cells located at the interface latewood/earlywood and shrinkage strains in soft earlywood cells. This effect is especially visible in the tangential direction of wood. Results and discussionh This means that, in these regions of combined compres- sive/tensile stresses, a local bending with respect to the interface late-/earlywood occurs. Such local bending effects or high strain effects around restraining rays can- not be observed by global affine analysis. Further applica- tions of this proposed non-rigid registration method are presented in Patera et al. [26].h Fig. 7 a Three-dimensional map of equivalent von Mises non-rigid strains on the wood cell wall of sample ELWd,1 for 200 slices, as indicated in the bar. b Cross-section cuts in the position indicated in red in the bar capturing the deformations of complex cellular and bio- logical materials. Such information cannot be recovered using other types of image registration techniques, such as, for example, the affine registration. Funding Funding This work was supported of the Swiss National Science Foundation (SNF) [Grant Number 125184]. Availability of data and materials 13. Gooya, A., Biros, G., Davatzikos, C.: deformable registration of glioma images using EM algorithm and diffusion reaction modeling. IEEE Trans. Med. Imaging 30(2), 375–390 (2011). https://doi.org/10.1109/ TMI.2010.2078833 Once accepted for publication, the data of this publication will be made avail- able on the ETHZ website. Once accepted for publication, the data of this publication will be made avail- able on the ETHZ website. Competing interests 12. Gering, D.T., Nabavi, A., Kikinis, R., Hata, N., O’Donnell, L.J., Grimson, W.E.L., Wells, W.M.: An integrated visualization system for surgical planning and guidance using image fusion and an open MR. J. Magn. Reson. Imaging 13(6), 967–975 (2001). https://doi.org/10.1002/jmri.1139 The authors declare that they have no competing interests. The authors declare that they have no competing interests. Author details 1 8. Foskey, M., Davis, B., Goyal, L., Chang, S., Chaney, E., Strehl, N., Tomei, S., Rosenman, J., Joshi, S.: Large deformation 3D image registration in image-guided radiation therapy. Phys. Med. Biol. 50(24), 5869–5892 (2005) 1 Swiss Light Source, Paul Scherrer Institute, Villigen, Switzerland. 2 Cen- tre d’Imagerie BioMedicale, Ecole Polytechnique Federale de Lausanne, 1015 Lausanne, Switzerland. 3 EMPA, Swiss Federal Laboratories for Materi- als Science and Technology, Laboratory for Multiscale Studies in Building Physics, Überlandstrasse 129, 8600 Dübendorf, Switzerland. 4 ETH Zurich, Chair of Building Physics, Stefano‑Franscini‑Platz 1, Zürich Hönggerberg, 8093 Zurich, Switzerland. 5 ETH Zurich, Institute for Biomedical Engineering, Gloriastrasse 35, 8092 Zurich, Switzerland. 9. Frangi, A.F., Laclaustra, M., Lamata, P.: A registration-based approach to quantify flow-sequences. IEEE Trans. Med. Imaging 22(11), 1458–1469 (2003). https://doi.org/10.1109/tmi.2003.819278 10. Gao, Y., Sandhu, R., Fichtinger, G., Tannenbaum, A.R.: A coupled global registration and segmentation framework with application to magnetic resonance prostate imagery. IEEE Trans. Med. Imaging 29(10), 1781–1794 (2010). https://doi.org/10.1109/TMI.2010.2052065 Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in pub- lished maps and institutional affiliations. Received: 12 July 2017 Accepted: 5 January 2018 Received: 12 July 2017 Accepted: 5 January 2018 In this work, the technique is used to investigate the occurrence of local deformations in wood provoked by moisture changes. The datasets are acquired with X-ray Tomography, thus leading to little changes in the grey- value intensity within the cell walls. This allows the use of squared sum of intensity differences (SSD) as similarity criterion. However, there are cases, especially in medi- cine, where the two datasets, i.e., reference and moving, are acquired with different configurations leading to con- trast enhancement (i.e., pre- and post-contrast agent with magnetic resonance imaging). In such case, another simi- larity criterion, insensitive to intensity changes, would be more suitable, as already demonstrated in Rueckert et al. [28]. The proposed method is expected to be widely applicable in material science and in medicine for detect- ing locally object deformations. Authors’ contributions DD d JC d h DD and JC conceived the project. AP, DD and JC defined the objectives and methodology. AP, SC and DD acquired the data, with the contribution of MS. AP and SC developed and implemented the code. AP, DD and JC analyzed the results. All authors contributed to the manuscript. All authors read and approved the final manuscript. 7. Dornheim, L., Tönnies, K. D., Dixon, K.: Automatic segmentation of the left ventricle in 3D SPECT data by registration with a dynamic anatomic model. In: Duncan, J. S., Gerig, G. (eds.) Medical image computing and computer-assisted intervention—MICCAI 2005, pp. 335–342. Springer, Berlin (2005). http://link.springer.com/chapter/10.1007/11566465_42 References 1. Abd-Elmoniem, K.Z., Stuber, M., Prince, J.L.: Direct three- dimensional myocardial strain tensor quantification and tracking using zHARP. Med. Image Anal. 12(6), 778–786 (2008). https://doi.org/10.1016/j. media.2008.03.008 2. Brown, L.G.: A survey of image registration techniques. ACM Comput. Surv. 24(4), 325–376 (1992). https://doi.org/10.1145/146370.146374 2. Brown, L.G.: A survey of image registration techniques. ACM Comput. Surv. 24(4), 325–376 (1992). https://doi.org/10.1145/146370.146374 3. Canny, J.: A computational approach to edge detection. IEEE Trans. Pattern Anal. Mach. Intell. PAMI 8(6), 679–698 (1986). https://doi. org/10.1109/tpami.1986.4767851 g p 4. Collins, D.L., Evans, A.C.: Animal: validation and applications of nonlinear registration-based segmentation. Int. J. Pattern Recognit Artif Intell. 11(08), 1271–1294 (1997). https://doi.org/10.1142/S0218001497000597 p g 5. Derome, D., Griffa, M., Koebel, M., Carmeliet, J.: Hysteretic swelling of wood at cellular scale probed by phase-contrast X-ray tomogra- phy. J. Struct. Biol. 173(1), 180–190 (2011). https://doi.org/10.1016/j. jsb.2010.08.011 6. Dierick, M., Van Loo, D., Masschaele, B., Van den Bulcke, J., Van Acker, J., Cnudde, V., Van Hoorebeke, L.: Recent micro-CT scanner developments at UGCT. Nucl. Instrum. Methods Phys. Res. Sect. B 324, 35–40 (2010) 6. Dierick, M., Van Loo, D., Masschaele, B., Van den Bulcke, J., Van Acker, J., Cnudde, V., Van Hoorebeke, L.: Recent micro-CT scanner developments at UGCT. Nucl. Instrum. Methods Phys. Res. Sect. B 324, 35–40 (2010) Ethics approval and consent to participate Does apply to this study. non-rigid deformations in complex biological materi- als, such as wood. The algorithm contains a wide range of tools for image analysis: in particular, morphologi- cal operations, segmentation and linear and non-linear transformations. This work is mainly focused on the implementation of a non-linear transformation based on B-spline functions. The method can be used for study- ing the behaviour of different existing materials detected with several experimental setups, in 2D, and it can be extended to a 3D case study. Does apply to this study. Acknowledgements 11. Gering, D. T., Nabavi, A., Kikinis, R., Grimson, W. E. L., Hata, N., Everett, P., Wells, W. M.: An integrated visualization system for surgical planning and guidance using image fusion and interventional imaging. In: Taylor, C., Colchester, A. (eds.), Medical image computing and computer-assisted intervention—MICCAI’99, pp. 809–819. Springer, Berlin (1999). http://link. springer.com/chapter/10.1007/10704282_88 Dr. Jan Van Den Bulcke and Prof. Dr. Joris Van Acker are greatly acknowledged. The experiments were carried out at the Centre of X-ray Tomography in Ghent University, Belgium. We acknowledge Dr. Ahmad Rafsanjani for his support in the validation of the algorithm with FEM. Dr. Michele Griffa is greatly acknowl- edged for the insightful discussion on the registration method. Ethics approval and consent to participate Ethics approval and consent to participate Does apply to this study. Conclusion In this work, a new algorithm is developed based on the work of Rueckert using B-spline. The main contribution consists in an accurate combination for the specific case study between the concept of feature recognition within specific regions locally distributed in the material with an optimization problem. The work is validated with a syn- thetically deformed dataset in bending and is used for docu- menting local swelling on a complex cellular material, wood, using two states of moisture content at 25 and 85% RH.h The non-rigid registration algorithm introduced in this work is a powerful tool for detecting and quantifying the The results presented in this section illustrate clearly that non-rigid registration is a powerful tool for Page 10 of 11 Patera et al. Adv Struct Chem Imag (2018) 4:1 ac.in/pluginfile.php/13002/mod_resource/content/0/References/har- ris1988.pdf ac.in/pluginfile.php/13002/mod_resource/content/0/References/har- ris1988.pdf 24. Nocedal, J., Wright, S.: Numerical Optimization. Springer, New York (2006) 25. Patera, A., Derome, D., Griffa, M., Carmeliet, J.: Hysteresis in swelling and in sorption of wood tissue. J. Struct. Biol. 182(3), 226–234 (2013). https://doi. org/10.1016/j.jsb.2013.03.003 15. Huang, X., Ren, J., Guiraudon, G., Boughner, D., Peters, T.M.: Rapid dynamic image registration of the beating heart for diagnosis and surgical naviga- tion. IEEE Trans. Med. Imaging 28(11), 1802–1814 (2009). https://doi. org/10.1109/TMI.2009.2024684 26. Patera, A., Van den Bulcke, J., Boone, M., Derome, D., Carmeliet, J.: Swelling interactions of earlywood and latewood across a growth ring: global and local deformations. Wood Sci. Technol. (2017). https://doi.org/10.1007/ s00226-017-0960-3 16. Isgum, I., Staring, M., Rutten, A., Prokop, M., Viergever, M.A., Bv, Ginneken: Multi-atlas-based segmentation with local decision fusion—application to cardiac and aortic segmenta- tion in CT scans. IEEE Trans. Med. Imag- ing 28(7), 1000–1010 (2009) 27. Pluim, J.P.W., Maintz, J.B.A., Viergever, M.A.: Mutual-information-based registration of medical images: a survey. IEEE Trans. Med. Imaging 22(8), 986–1004 (2003). https://doi.org/10.1109/TMI.2003.815867 17. Lavely, W.C., Scarfone, C., Cevikalp, H., Li, R., Byrne, D.W., Cmelak, A.J., Fitzpatrick, J.M.: Phantom validation of coregistration of PET and CT for image-guided radiotherapy. Med. Phys. 31(5), 1083–1092 (2004). https:// doi.org/10.1118/1.1688041 28. Rueckert, D., Sonoda, L.I., Hayes, C., Hill, D.L.G., Leach, M.O., Hawkes, D.J.: Nonrigid registration using free-form deformations: application to breast MR images. IEEE Trans. Med. Imaging 18(8), 712–721 (1999). https://doi. org/10.1109/42.796284 18. Lee, S., Wolberg, G., Shin, S.Y.: Scattered data interpolation with multilevel B- splines. IEEE Trans. Visual Comput. Graphics 3(3), 228–244 (1997). https://doi.org/10.1109/2945.620490 29. Sederberg, T., Parry, S.: Free-form deformation of solid geometric models. Proc. ACM SIGGRAPH 20(4), 151–160 (1986) g 19. Maksimov, D., Hesser, J., Brockmann, C., Jochum, S., Dietz, T., Schnitzer, A., Diehl, S.: Graph-matching based CTA. IEEE Trans. Med. Imaging 28(12), 1940–1954 (2009). https://doi.org/10.1109/TMI.2009.2026370 30. Staring, M., van der Heide, U.A., Klein, S., Viergever, M.A., Pluim, J.: Registration of cervical MRI using multifeature mutual information. IEEE Trans. Med. Imaging 28(9), 1412–1421 (2009). https://doi.org/10.1109/ TMI.2009.2016560 g 20. Martin, S., Daanen, V., Troccaz, J.: Atlas-based prostate segmentation using an hybrid registration. Int. J. Comput. Assist. Radiol. Surg. 3(6), 485–492 (2008). https://doi.org/10.1007/s11548-008-0247-0 31. Szeliski, R., Coughlan, J.: Spline-based image registration. Int. J. Comput. Vision 22(3), 199–218 (1997). https://doi.org/10.1023/A:1007996332012 21. Masschaele, B.C., Cnudde, V., Dierick, M., Jacobs, P., Van Hoorebeke, L., Vlassenbroeck, J.: UGCT: new X-ray radiography and tomography facility. Nucl. Instrum. Methods Phys. Res. Sect. A 580(1), 266–269 (2007). Consent for publication 14. Harris, C., Stephens, M.: A combined corner and edge detector. In: Alvey vision conference, 15.50. Manchester, UK (1988). http://courses.daiict. Does not apply to this submission. Patera et al. Adv Struct Chem Imag (2018) 4:1 Page 11 of 11 Page 11 of 11 ac.in/pluginfile.php/13002/mod_resource/content/0/References/har- ris1988.pdf https:// doi.org/10.1016/j.nima.2007.05.099 32. Wahba, G.: Spline models for observational data. Soc. Ind. Appl. Math (1990). http://epubs.siam.org/doi/book/10.1137/1.9781611970128 33. Wyawahare, M.V., Patil, P.M., Abhyankar, H.K., et al.: Image registration techniques: an overview. Int. J. Signal Process. Image Process. Pattern Recogn. 2(3), 11–28 (2009) 22. Miller, K., Wittek, A., Joldes, G., Horton, A., Dutta-Roy, T., Berger, J., Morriss, L.: Modelling brain deformations for computer-integrated neurosurgery. Int. J. Numer. Methods Biomed. Eng. 26(1), 117–138 (2010). https://doi. org/10.1002/cnm.1260 34. Zhuang, X., Rhode, K.S., Razavi, R.S., Hawkes, D.J., Ourselin, S.: A registration-based propagation framework for automatic whole heart segmentation of cardiac MRI. IEEE Trans. Med. Imaging 29(9), 1612–1625 (2010). https://doi.org/10.1109/TMI.2010.2047112 23. Mises, R.v.: Mechanik der festen Körper im plastisch-deformablen Zustand. Nachrichten von des Gesellschaft des Wissenschaften zu Göt- tingen, Mathematisch-Physicalisch Klasse 1913, 582–592 (1913)
https://openalex.org/W2952802059	https://peerj.com/articles/5862.pdf	English	null	PromoterPredict: sequence-based modelling of<i>Escherichia coli</i>σ<sup>70</sup>promoter strength yields logarithmic dependence between promoter strength and sequence	PeerJ	2,018	cc-by	7,715	How to cite this article Bharanikumar et al. (2018), PromoterPredict: sequence-based modelling of Escherichia coli s70 promoter strength yields logarithmic dependence between promoter strength and sequence. PeerJ 6:e5862; DOI 10.7717/peerj.5862 Ramit Bharanikumar1, Keshav Aditya R. Premkumar2 and Ashok Palaniappan3 1 Biotechnology, Sri Venkateswara College of Engineering (Autonomous), Sriperumbudur, Tamil Nadu, India 2 Computer Science and Engineering, Sri Venkateswara College of Engineering (Autonomous), Sriperumbudur, Tamil Nadu, India 2 Computer Science and Engineering, Sri Venkateswara College of Engineering (Autonomous), Sriperumbudur, Tamil Nadu, India p 3Bioinformatics, School of Chemical and Biotechnology, SASTRA Deemed University, Thanjavur, Tamil Nadu, India 3Bioinformatics, School of Chemical and Biotechnology, SASTRA Deemed University, Thanjavur, Tamil Nadu, India Subjects Bioinformatics, Computational Biology, Genomics, Synthetic Biology, Data Mining and Machine Learning PromoterPredict: sequence-based modelling of Escherichia coli r70 promoter strength yields logarithmic dependence between promoter strength and sequence Ramit Bharanikumar1, Keshav Aditya R. Premkumar2 and Ashok Palaniappan3 Keywords Regression modelling, Promoter sequences, Promoter strength prediction, Sigma70 promoters, Genetic engineering, Weak promoters, PWM construction, Data mining, Software tools INTRODUCTION The primary Escherichia coli promoter-speciﬁcity factor and the one widely used in recombinant DNA technology is the s70 factor. Promoters recognized by s70-containing RNA polymerase are called core promoters and share the following features: two conserved hexamer sequences, separated by a non-speciﬁc spacer of ideally 17 nucleotides. The two hexamers are located ∼35 and ∼10 bp upstream of the transcription start site, and are called the -35 and -10 sequences, respectively (Maquat & Reznikoff, 1978; Bujard, 1980; Paget & Helmann, 2003; Kadonaga, 2012). -35 and -10 sequences matching the consensi motifs (TTGACA and TATAAT, respectively) are known as canonical hexamers (Galas, Eggert & Waterman, 1985; Deuschle et al., 1986; Stormo, 1990). It is known that the conserved hexamer regions are vital for recognizing and optimizing the interactions between DNA and the RNA polymerase (Hawley & McClure, 1983; Knaus & Bujard, 1990; Hook-Barnard, Johnson & Hinton, 2006; Feklistov & Darst, 2011; Basu et al., 2014). Theory has yielded a linear relationship between the total promoter score and the natural log of promoter strength (Berg & Von Hippel, 1987; Li & Zhang, 2014). Nucleotide occurrence frequencies were ﬁrst used by Weller & Recknagel (1994) in promoter strength prediction. Additivity in promoter-polymerase interaction has been afﬁrmed by Benos, Bulyk & Stormo (2002). Patterns in s70 promoters have been quantiﬁed by Huerta & Collado-Vides (2003). Strength of E. coli sE RNA polymerase promoters were studied by Rhodius & Mutalik (2010). The complexity of E. coli s70 promoter sequences has been treated from an information theoretic standpoint by Shultzaberger et al. (2007). More recently, an support vector machines (SVM) model has been successfully applied to predicting the strength of a mutation library of E. coli Trc promoter sequences (Meng et al., 2017). One drawback with an SVM or artiﬁcial neural networks (ANN) machine learning model is the ‘black-box’ approach; that is, the absence of any mechanistic insights that could be gleaned with respect to the relationship between promoter sequence and strength. Such an understanding could be vital in the prediction of promoter strengths in different contexts, as well as the forward design of promoters in ﬁnely-tuned genetic circuits (see Endy, 2005; De Mey et al., 2007; Salis, Mirsky & Voigt, 2009; Li & Zhang, 2014). ABSTRACT We present PromoterPredict, a dynamic multiple regression approach to predict the strength of Escherichia coli promoters binding the s70 factor of RNA polymerase. s70 promoters are ubiquitously used in recombinant DNA technology, but characterizing their strength is demanding in terms of both time and money. We parsed a comprehensive database of bacterial promoters for the -35 and -10 hexamer regions of s70-binding promoters and used these sequences to construct the respective position weight matrices (PWM). Next we used a well-characterized set of promoters to train a multivariate linear regression model and learn the mapping between PWM scores of the -35 and -10 hexamers and the promoter strength. We found that the log of the promoter strength is signiﬁcantly linearly associated with a weighted sum of the -10 and -35 sequence proﬁle scores. We applied our model to 100 sets of 100 randomly generated promoter sequences to generate a sampling distribution of mean strengths of random promoter sequences and obtained a mean of 6E-4 ± 1E-7. Our model was further validated by cross-validation and on independent datasets of characterized promoters. PromoterPredict accepts -10 and -35 hexamer sequences and returns the predicted promoter strength. It is capable of dynamic learning from user-supplied data to reﬁne the model construction and yield more robust estimates of promoter strength. PromoterPredict is available as both a web service (https://promoterpredict.com) and standalone tool (https://github.com/PromoterPredict). Our work presents an intuitive generalization applicable to modelling the strength of other promoter classes. Submitted 3 April 2018 Accepted 3 October 2018 Published 7 November 2018 Corresponding author Ashok Palaniappan, apalania@scbt.sastra.edu Academic editor Yuriy Orlov Additional Information and Declarations can be found on page 13 DOI 10.7717/peerj.5862 Copyright 2018 Bharanikumar et al. Distributed under Creative Commons CC-BY 4.0 Subjects Bioinformatics, Computational Biology, Genomics, Synthetic Biology, Data Mining and Machine Learning d ll h d Keywords Regression modelling, Promoter sequences, Promoter strength prediction, Sigma70 promoters, Genetic engineering, Weak promoters, PWM construction, Data mining, Software tools Distributed under Creative Commons CC-BY 4.0 How to cite this article Bharanikumar et al. (2018), PromoterPredict: sequence-based modelling of Escherichia coli s70 promoter strength yields logarithmic dependence between promoter strength and sequence. PeerJ 6:e5862; DOI 10.7717/peerj.5862 Bharanikumar et al. (2018), PeerJ, DOI 10.7717/peerj.5862 INTRODUCTION Many freely available resources predict the location of promoters in a genomic sequence mainly by identifying the -10 and -35 regulatory sequences (De Jong et al., 2012), but very few tools are available to predict the strength of such sequences. One tool provides qualitative predictions (‘strong’ or not) of promoter strength based on the occurrence of a triad pattern (Dekhtyar, Morin & Sakanyan, 2008), and is available as a macro. Here, we present a two-step approach to the predictive modelling of the strength of s70 core promoters, and a companion web-based platform and Python standalone tool that implement our method along with the option to dynamically include user data into the prediction model. Our implementation is the ﬁrst freely available tool/web-server for the quantitative prediction of promoter strength. Generative model of promoter sequences Generative model of promoter sequences Generative model of promoter sequences A generative model of the -10 and -35 promoter sequences is constructed using two position weight matrices (PWM–10 and PWM–35) in the following manner. Bharanikumar et al. (2018), PeerJ, DOI 10.7717/peerj.5862 2/15 A comprehensive set of s70-binding promoter sequences was extracted from the RegulonDB (Gama-Castro et al., 2016). For each promoter sequence, we extracted a -35 region of 13 nucleotides centred at -35 position, and a -10 region of 13 nucleotides centred at the -10 position, to allow for uncertainties in the precise position of occurrence of the hexamers. For each -35 region, we used FIMO (Grant, Bailey & Noble, 2011) to ﬁnd the best match to the consensus -35 motif, and similarly for the -10 regions, to obtain a dataset of -35 and -10 hexamer sequences. This dataset was then ﬁltered for only signiﬁcant hits to the consensi motifs (p-value < 0.05) and the resulting dataset was used to determine the weights of each nucleotide at each position of the -35 and -10 hexamers. Nucleotide-wise counts at each position of the hexamer motifs were augmented by a pseudo-count prior to correct for E. coli GC content of 50.8% and the resulting frequency matrices were converted into log-odds matrices. Biopython routines (www.biopython.org) were used. A comprehensive set of s70-binding promoter sequences was extracted from the RegulonDB (Gama-Castro et al., 2016). For each promoter sequence, we extracted a -35 region of 13 nucleotides centred at -35 position, and a -10 region of 13 nucleotides centred at the -10 position, to allow for uncertainties in the precise position of occurrence of the hexamers. For each -35 region, we used FIMO (Grant, Bailey & Noble, 2011) to ﬁnd the best match to the consensus -35 motif, and similarly for the -10 regions, to obtain a dataset of -35 and -10 hexamer sequences. This dataset was then ﬁltered for only signiﬁcant hits to the consensi motifs (p-value < 0.05) and the resulting dataset was used to determine the weights of each nucleotide at each position of the -35 and -10 hexamers. Nucleotide-wise counts at each position of the hexamer motifs were augmented by a pseudo-count prior to correct for E. coli GC content of 50.8% and the resulting frequency matrices were converted into log-odds matrices. Biopython routines (www.biopython.org) were used. Linear modelling of promoter strength Following Berg & Von Hippel (1987), we modelled the relationship between the promoter sequences and the ln of the promoter strength using multiple linear regression. The training set of 18 promoters is drawn from the Anderson library of activator- independent plasmid tet promoter variants maintained at the Registry of standard biological parts (http://parts.igem.org/Promoters/Catalog/Anderson). Each promoter sequence is scored with respect to the generative models of the -10 and -35 motifs (i.e. the PWM–10 and PWM–35 matrices) and the two scores obtained formed the feature space of the regression modelling. The regression coefﬁcients to be determined represent the weights of the -10 and -35 regions in the regression analysis. The Anderson library provided promoter strengths spanning two orders of magnitude and normalized in the range 0.00–1.00 with respect to the strongest (i.e. reference) promoter. It was noted that the normalisation step would not affect a linear relationship, altering only the constant of the regression. The normalised strength values were log-transformed to obtain the required response variable values. Since the ln function rapidly descends towards—Inf with decreasing promoter strength, we capped the inﬁmum of promoter strength at 0.0001 prior to log-transformation. The least-squares cost function was minimized using iterative gradient descent. The model parameters were assessed using t-statistics, and the overall model was assessed using F-statistic and the adjusted multiple coefﬁcient of determination given by: Adj: R2¼ 1 1R2 n1 ð Þ= nm1 ð Þ ½ (1) (1) where m is the number of features and n is the number of instances. The adjustment is a penalty for increasing model complexity. where m is the number of features and n is the number of instances. The adjustment is a penalty for increasing model complexity. Bharanikumar et al. (2018), PeerJ, DOI 10.7717/peerj.5862 RESULTS The entire datasets of 1,004 -35 hexamers and 1,046 -10 hexamers parsed out of RegulonDB are available as Supplementary Information. The conservation proﬁles of the extracted -35 and -10 hexamer sequences of the promoters in the RegulonDB were visualized and shown in Fig. 1. Based on these PWMs, the site scores of each promoter sequence in the Anderson library were regressed on the corresponding ln of the promoter strength. A summary of this process with the training data, log-transformation of the promoter strength and predicted response values is presented in Table 1. The modelling process converged within 105 iterations by tuning the gradient descent to a learning rate (a) of 0.015, and the following model was obtained: ln promoter strength ð Þ ¼ 5:1046 þ 0:4271 PWM35 ð Þ þ 0:2726 PWM10 ð Þ (2) (2) We derived an independent solution of the multiple regression using R (www.r-project.org) and obtained a correlation coefﬁcient of 0.998 between the ﬁtt values of the two models. The interval estimates of the coefﬁcients of the regression computed in R using confint (fit, level = 0.95), and obtained the following conﬁdence intervals: We derived an independent solution of the multiple regression using R (www.r-project.org) and obtained a correlation coefﬁcient of 0.998 between th We derived an independent solution of the multiple regression using R (www.r-project.org) and obtained a correlation coefﬁcient of 0.998 between the ﬁtted values of the two models. The interval estimates of the coefﬁcients of the regression were computed in R using confint (fit, level = 0.95), and obtained the following 95% conﬁdence intervals: (www.r-project.org) and obtained a correlation coefﬁcient of 0.998 between the ﬁtted Intercept : (-6.4974449, -3.7118421) PWM_35 : (0.2445358, 0.6095848) PWM_10 : (0.1434939, 0.4017307) Intercept : (-6.4974449, -3.7118421) PWM_35 : (0.2445358, 0.6095848) PWM_10 : (0.1434939, 0.4017307) The interval estimates did not include zero, and this implied that the coefﬁcients were signiﬁcant at the 0.05 level. In fact, all the three estimates were signiﬁcant at a p-value of 1E-3. The F-statistic of the overall regression was signiﬁcant at a p-value of 2E-4 and adj. R2 was ≈0.65. The plane of best ﬁt corresponding to the above model is visualized in Fig. 2. The model was then cross-validated using a 18-fold LOOCV (similar to jack-knife). Cross-validation yielded a correlation coefﬁcient of ∼0.76 (Table 2). Model validation The model of promoter strength was validated in three ways: i) The model was validated using leave-one-out cross-validation (LOOCV). Bharanikumar et al. (2018), PeerJ, DOI 10.7717/peerj.5862 3/15 3/15 ii) We generated 100 sets of 100 randomly generated promoter sequences each, using the sample function in Python. From the obtained sampling distribution of mean strengths of random promoter sequences, we calculated the estimate of the true mean strength of a random promoter sequence, together with its standard error. iii) We further validated our model on independent datasets of characterized promoters available in Davis, Rubin & Sauer (2011), Dekhtyar, Morin & Sakanyan (2008), and Dayton et al. (1984). iii) We further validated our model on independent datasets of characterized promoters available in Davis, Rubin & Sauer (2011), Dekhtyar, Morin & Sakanyan (2008), and Dayton et al. (1984). iii) We further validated our model on independent datasets of characterized promoters available in Davis, Rubin & Sauer (2011), Dekhtyar, Morin & Sakanyan (2008), and Dayton et al. (1984). Bharanikumar et al. (2018), PeerJ, DOI 10.7717/peerj.5862 Bharanikumar et al. (2018), PeerJ, DOI 10.7717/peerj.5862 RESULTS We sought to benchmark our model on a negative test set by generating random -35 and -10 hexamer sequences. To this end, we applied our model to 100 sets of 100 random promoter sequences each (available in Supplementary Information) and estimated the true mean of the sampling distribution as 0.00055. The standard error of the estimate was 1.04E-7. The low predicted strength along with the very small standard error indicated that the Bharanikumar et al. (2018), PeerJ, DOI 10.7717/peerj.5862 4/15 Figure 1 Sequence logos of the -35 and -10 hexamers of the selected RegulonDB promoters. (A) -35 motif; (B) -10 motif. Figure was made using WebLogo (Crooks et al., 2004). Full-size  DOI: 10.7717/peerj.5862/ﬁg-1 Figure 1 Sequence logos of the -35 and -10 hexamers of the selected RegulonDB promoters. (A) -35 motif; (B) -10 motif. Figure was made using WebLogo (Crooks et al., 2004). Full-size  DOI: 10.7717/peerj.5862/ﬁg-1 d l di d h i b i h d i Table 1 Summary of promoter information. Promoter -35 hexamer -10 hexamer Promoter activity ln (Promoter activity) Predicted ln (Promoter activity) BBa_J23100 TTGACG TACAGT 1 0 -1.6336486579 BBa_J23101 TTTACA TATTAT 0.7 -0.35667494 0.0555718065 BBa_J23102 TTGACA TACTGT 0.86 -0.15082289 -1.0957849491 BBa_J23104 TTGACA TATTGT 0.72 -0.32850407 0.1647181133 BBa_J23105 TTTACG TACTAT 0.24 -1.42711636 -2.2871659092 BBa_J23106 TTTACG TATAGT 0.47 -0.75502258 -1.3174788735 BBa_J23107 TTTACG TATTAT 0.36 -1.02165125 -1.0266628468 BBa_J23108 CTGACA TATAAT 0.51 -0.67334455 -0.4282477098 BBa_J23109 TTTACA GACTGT 0.04 -3.21887582 -3.3693144659 BBa_J23110 TTTAGG TACAAT 0.33 -1.10866262 -3.3946866337 BBa_J23111 TTGACG TATAGT 0.58 -0.54472718 -0.3731455955 BBa_J23112 CTGATA GATTAT 0.01 -4.60517019 -3.1533888284 BBa_J23113 CTGATG GATTAT 0.01 -4.60517019 -4.2356234817 BBa_J23114 TTTATG TACAAT 0.1 -2.30258509 -2.5943689001 BBa_J23115 TTTATA TACAAT 0.15 -1.89711998 -1.5121342469 BBa_J23116 TTGACA GACTAT 0.16 -1.83258146 -1.5897942167 BBa_J23117 TTGACA GATTGT 0.06 -2.81341072 -1.1644781255 BBa_J23118 TTGACG TATTGT 0.56 -0.5798185 -0.91751654 Note: The promoter activities (strengths) are seen to span two orders of magnitude in the range (0.0, 1.0). The promoters follow the naming in the Anderson dataset. Note: The promoter activities (strengths) are seen to span two orders of magnitude in the range (0.0, 1.0). The promoters follow the naming in the Anderson dataset. model predicted these instances to be non-promoter sequences with good certainty. Thi fﬁ d th iﬁit f d l f t t This afﬁrmed the speciﬁcity of our model for true promoters. his afﬁrmed the speciﬁcity of our model for true promoters. model predicted these instances to be non-promoter sequences with good certainty. Bharanikumar et al. (2018), PeerJ, DOI 10.7717/peerj.5862 RESULTS The following results were obtained: i) For the 10 promoters discussed by Davis, Rubin & Sauer (2011), we ranked the promoters in Table 1 of the same reference according to their strengths and observed a 1,000-fold span of promoter strengths, 1E-3 to 1 (Table 3). Promoters 2 and 3 were identically strong, hence we took the average of their predicted strengths in ranking the promoters. With this arrangement, we found that the predicted order of promoters in terms of strength exactly reproduced the experimentally characterized order. Despite the fact that Anderson library and these promoters were characterized and normalized using different systems, the model was able to predict surprisingly well across a promoter strength spectrum spanning three orders of magnitude. i) For the 10 promoters discussed by Davis, Rubin & Sauer (2011), we ranked the promoters in Table 1 of the same reference according to their strengths and observed a 1,000-fold span of promoter strengths, 1E-3 to 1 (Table 3). Promoters 2 and 3 were identically strong, hence we took the average of their predicted strengths in ranking the promoters. With this arrangement, we found that the predicted order of promoters in terms of strength exactly reproduced the experimentally characterized order. Despite the fact that Anderson library and these promoters were characterized and normalized using different systems, the model was able to predict surprisingly well across a promoter strength spectrum spanning three orders of magnitude. ii) Next, we applied our model to the set of 13 strong promoter candidates of Thermotoga maritima discussed in Dekhtyar, Morin & Sakanyan (2008). Using the hexamer sequences provided in Fig. 5 of the same reference, we applied our model and obtained quantitative predictions of promoter strengths (Table 4). Almost all the promoters had predicted strengths >0.38 and promoters with canonical hexamers even had strengths >1.00. One promoter (TM0032) was predicted as ‘weak’ with a strength ∼0.056 and seemed to point to an apparent anomaly in the relationship between promoter sequence and strength, possibly highlighting the need for further experimentation on this promoter. Our observations were corroborated by Fig. 4 in the same reference that showed the least and greatly reduced expression from this particular promoter. These results taken in conjunction with the results on random promoter sequences afﬁrmed the ability of our model to discriminate between promoters at opposite ends of the strength spectrum. RESULTS To validate our model further on true promoter sequences and experimentally characterized promoter strengths, we used datasets available in the literature and To validate our model further on true promoter sequences and experimentally characterized promoter strengths, we used datasets available in the literature and Bharanikumar et al. (2018), PeerJ, DOI 10.7717/peerj.5862 Figure 2 The regression surface of the estimated model with the training data points (red). x- and y-axes represent PWM scores and the z-axis (vertical) represents the predicted ln(promoter strength). Full-size  DOI: 10.7717/peerj.5862/ﬁg-2 Figure 2 The regression surface of the estimated model with the training data points (red). x- and y-axes represent PWM scores and the z-axis (vertical) represents the predicted ln(promoter strength). Full-size  DOI: 10.7717/peerj.5862/ﬁg-2 Table 2 Cross-validation results. Table 2 Cross-validation results. Fold PWM_35 PWM_10 Combined logStrength cvpred cvres 1 6.5966 2.398 9 0 -1.757 1.757 2 6.9195 8.089 15.01 -0.357 0.145 -0.50 3 9.1308 0.402 9.53 -0.151 -1.3 1.15 4 9.1308 5.025 14.16 -0.329 0.286 -0.62 5 4.3854 3.465 7.85 -1.427 -2.36 0.93 6 4.3854 7.022 11.41 -0.755 -1.377 0.62 7 4.3854 8.089 12.47 -1.022 -1.027 0.00 8 4.5119 10.086 14.6 -0.673 -0.362 -0.31 9 6.9195 -4.474 2.45 -3.219 -3.463 0.24 10 4.3854 5.462 9.85 -1.109 -1.792 0.68 11 6.5966 7.022 13.62 -0.545 -0.349 -0.20 12 2.5179 3.213 5.73 -4.605 -2.847 -1.76 13 -0.0162 3.213 3.2 -4.605 -3.977 -0.63 14 2.3914 5.462 7.85 -2.303 -2.646 0.34 15 4.9255 5.462 10.39 -1.897 -1.485 -0.41 16 9.1308 -1.411 7.72 -1.833 -1.518 -0.32 17 9.1308 0.15 9.28 -2.813 -0.796 -2.02 18 6.5966 5.025 11.62 -0.58 -0.944 0.36 Note: In each fold of cross-validation, the instance corresponding to the fold was designated as the test instance while the prediction model was built using the rest of the instances. This process was repeated 18 times, once for each test instance and the cross-validation (CV) residuals were obtained. combined, sum of the PWM scores; cvpred, predicted log strength of the test instance; cvres, cross-validation residual. Bharanikumar et al. (2018), PeerJ, DOI 10.7717/peerj.5862 6/15 compared the predicted strength with the experimental results and examined their concordance. The following results were obtained: compared the predicted strength with the experimental results and examined their concordance. RESULTS iii) We also applied our model on the ﬁve promoters discussed in Dayton et al. (1984). Of these, the ﬁrst three are known as ‘major’ promoters that are active even at low concentrations of the polymerase, whereas the last two are ‘minor’, less strong promoters that are only active when the polymerase is present at high concentrations. We applied our model on the promoter sequences found in Fig. 5 of the same reference and found the predictions in line with the nature of these promoters (Table 5). The activity of the least strong ‘major’ promoter is about two times more than the activity of the strongest ‘minor’ promoter. Hence, our modelling approach was able to discriminate between major and minor promoters. Bharanikumar et al. (2018), PeerJ, DOI 10.7717/peerj.5862 DISCUSSION In addition to the independent contributions of -35 and -10 sites to promoter strength, we were interested in exploring if any interactions between them could contribute to promoter strength. To this end, we examined the following model in R: lm(logStrength ∼PWM35 PWM10) lm(logStrength ∼PWM35 PWM10) where PWM35 and PWM10 represent the corresponding site scores. This model resulted in a lower adj. R2-value than that without any interactions. Further, the p-value of the Bharanikumar et al. (2018), PeerJ, DOI 10.7717/peerj.5862 Table 3 Validation results: using data of Davis, Rubin & Sauer (2011). Actual rank Promoter -35 sequence -10 sequence Strength Predicted exp(logStrength) Predicted rank 1 pro1 tttacg gtatct 0.009 0.0079073845 1 2.5 pro2 gcggtg tataat 0.017 0.0306978849 2.5 2.5 pro3 ttgacg gaggat 0.017 0.0306978849 2.5 4 proA tttacg taggct 0.03 0.0482647297 4 5 pro4 tttacg gatgat 0.033 0.0809816409 5 6 pro5 tttacg taggat 0.05 0.0867400443 6 7 proB tttacg taatat 0.119 0.1534857959 7 8 pro6 tttacg taaaat 0.193 0.2645364297 8 9 proC tttacg tatgat 0.278 0.3059490889 9 10 proD tttacg tataat 1 0.6173668247 10 Note: The promoters were ordered based on the rank of their strength, and given as input to our model. The predicted promoter log strengths were then examined for agreement with the actual rank and the ordering obtained matched the original ordering. The individual predicted values for pro2 and pro3 were 0.0024 and 0.059, respectively. Note: The promoters were ordered based on the rank of their strength, and given as input to our model. The predicted promoter log strengths were then examined for agreement with the actual rank and the ordering obtained matched the original ordering. The individual predicted values for pro2 and pro3 were 0.0024 and 0.059, respectively. PWM10 score dropped below signiﬁcance (0.31), and the interaction term turned out to be totally insigniﬁcant (p-value: 0.97), thus discounting any interaction between the sites in the present dataset. On this basis, the null hypothesis of absence of any interaction could not be rejected, and we concluded that there is little evidence for interaction between the -35 and -10 sites in contributing to promoter strength. Our model assumed that both the predictors carried independent information about the promoter strength, and together they are able to provide sufﬁcient information about the strength. The basis of this assumption was probed to determine if both predictors are necessary to the model. Could one predictor provide sufﬁcient information about the promoter strength in the absence of the other? There are at least three angles to address this question, and all of them were considered to interpret the model better. Table 4 Validation with T. maritima strong promoter candidates. Bharanikumar et al. (2018), PeerJ, DOI 10.7717/peerj.5862 lm(logStrength ∼PWM35 PWM10) Promoter -35 sequence -10 sequence Strength Predicted exp(logStrength) Predicted class TM0373 ttgaca tataat Strong 4.6845788997 Strong TM1016 ttgaat tttaat Strong 0.3808572257 Strong TM1272 ttgaca tttaat Strong 1.6386551999 Strong TM1429 ttgaca tataat Strong 4.6845788997 Strong TM1667 ttgaaa tataat Strong 2.5859432664 Strong TM1780 ttcata tataat Strong 0.463878289 Strong Tmt11 ttgaat taaaat Strong 0.4665383797 Strong TM0032 tcgaaa cataat Strong 0.0562167049 Weak TM0477 ttgaat tataat Strong 1.0887926414 Strong TM1067 ttgacc tattat Strong 0.7046782664 Strong TM1271 ttgaca tataat Strong 4.6845788997 Strong Tmt45 ttgaac tataat Strong 0.670434893 Strong TM1490 ttgact taaaat Strong 0.8451600149 Strong PWM10 score dropped below signiﬁcance (0.31), and the interaction term turned out to be totally insigniﬁcant (p value: 0 97) thus discounting any interaction between the Table 4 Validation with T. maritima strong promoter candidates. Promoter -35 sequence -10 sequence Strength Predicted exp(logStrength) Predicted class TM0373 ttgaca tataat Strong 4.6845788997 Strong TM1016 ttgaat tttaat Strong 0.3808572257 Strong TM1272 ttgaca tttaat Strong 1.6386551999 Strong TM1429 ttgaca tataat Strong 4.6845788997 Strong TM1667 ttgaaa tataat Strong 2.5859432664 Strong TM1780 ttcata tataat Strong 0.463878289 Strong Tmt11 ttgaat taaaat Strong 0.4665383797 Strong TM0032 tcgaaa cataat Strong 0.0562167049 Weak TM0477 ttgaat tataat Strong 1.0887926414 Strong TM1067 ttgacc tattat Strong 0.7046782664 Strong TM1271 ttgaca tataat Strong 4.6845788997 Strong Tmt45 ttgaac tataat Strong 0.670434893 Strong TM1490 ttgact taaaat Strong 0.8451600149 Strong Table 4 Validation with T. maritima strong promoter candidates. PWM10 score dropped below signiﬁcance (0.31), and the interaction term turned out to be totally insigniﬁcant (p-value: 0.97), thus discounting any interaction between the sites in the present dataset. On this basis, the null hypothesis of absence of any interaction could not be rejected, and we concluded that there is little evidence for interaction between the -35 and -10 sites in contributing to promoter strength. PWM10 score dropped below signiﬁcance (0.31), and the interaction term turned out to be totally insigniﬁcant (p-value: 0.97), thus discounting any interaction between the sites in the present dataset. On this basis, the null hypothesis of absence of any interaction could not be rejected, and we concluded that there is little evidence for interaction between the -35 and -10 sites in contributing to promoter strength. Our model assumed that both the predictors carried independent information about the promoter strength, and together they are able to provide sufﬁcient information about the strength. The basis of this assumption was probed to determine if both predictors are necessary to the model. Bharanikumar et al. (2018), PeerJ, DOI 10.7717/peerj.5862 lm(logStrength ∼PWM35 PWM10) Further, the highest correlation between the features and response variable was observed between the combined score and log of the promoter strength (∼0.79), but the combined score showed only a moderate correlation with the promoter strength prior to log-transformation (∼0.63). This was in keeping with similar observations for the strength of sE promoters (Rhodius & Mutalik, 2010) and underscored the logarithmic dependence between the promoter strength and sequence. 3. Another way to address the question is to compute the correlation coefﬁcients between all the variables of interest, including a variable with the combined effects of -35 and -10 sites. This is shown in Table 6. Three features were used, namely PWM-10 score, PWM-35 score, and the combined score (i.e. PWM-10 + PWM-35). These feature variables were correlated with two response variables, namely promoter strength and its corresponding log-transformation. It was ﬁrst observed that the PWM-10 and PWM-35 scores were anti-correlated with each other (correlation coefﬁcient = -0.37), thus supporting the hypothesis that they are two independent features that could compensate for each other in determining promoter strength. It was signiﬁcant that the each feature was better correlated with the log of the strength than the strength itself. We tried to regress the strength on the PWM scores, but the model had a very low adj. R2 (≈0.40) and the intercept term was not signiﬁcant at the 0.05 level. Further, the highest correlation between the features and response variable was observed between the combined score and log of the promoter strength (∼0.79), but the combined score showed only a moderate correlation with the promoter strength prior to log-transformation (∼0.63). This was in keeping with similar observations for the strength of sE promoters (Rhodius & Mutalik, 2010) and underscored the logarithmic dependence between the promoter strength and sequence. Finally, the assumptions of linear modelling were investigated with reference to our problem. Model diagnostics of four basic assumptions were plotted (shown in Fig. 4). Speciﬁcally: Plot A: The residuals were plotted against the ﬁtted values. No trend was visible in the plot, indicating the residuals did not increase with the ﬁtted values and followed a random pattern about zero. This validated the assumption that the errors were independent. Plot B: The square root of the relative error (standardized residual) was plotted against the ﬁtted value. An almost ﬂat trend was observed, indicating that the standardized residual did not vary with the ﬁtted value. lm(logStrength ∼PWM35 PWM10) Conﬁdence in the effect of -35 site increases with the score from 0 to about 7, and then is susceptible to edge effects as the score reaches 8. Conﬁdence in the effect of the -10 site increases with the score from -4 to about 5, and then is susceptible to edge effects as the score reaches 10. The results are shown in Fig. 3 where the PWM scores are plotted against the level of conﬁdence in the predicted response. Conﬁdence in the effect of -35 site increases with the score from 0 to about 7, and then is susceptible to edge effects as the score reaches 8. Conﬁdence in the effect of the -10 site increases with the score from -4 to about 5, and then is susceptible to edge effects as the score reaches 10. The results are shown in Fig. 3 where the PWM scores are plotted against the level of conﬁdence in the predicted response. Conﬁdence in the effect of -35 site increases with the score from 0 to about 7, and then is susceptible to edge effects as the score reaches 8. Conﬁdence in the effect of the -10 site increases with the score from -4 to about 5, and then is susceptible to edge effects as the score reaches 10. 3. Another way to address the question is to compute the correlation coefﬁcients between all the variables of interest, including a variable with the combined effects of -35 and -10 sites. This is shown in Table 6. Three features were used, namely PWM-10 score, PWM-35 score, and the combined score (i.e. PWM-10 + PWM-35). These feature variables were correlated with two response variables, namely promoter strength and its corresponding log-transformation. It was ﬁrst observed that the PWM-10 and PWM-35 scores were anti-correlated with each other (correlation coefﬁcient = -0.37), thus supporting the hypothesis that they are two independent features that could compensate for each other in determining promoter strength. It was signiﬁcant that the each feature was better correlated with the log of the strength than the strength itself. We tried to regress the strength on the PWM scores, but the model had a very low adj. R2 (≈0.40) and the intercept term was not signiﬁcant at the 0.05 level. Bharanikumar et al. (2018), PeerJ, DOI 10.7717/peerj.5862 lm(logStrength ∼PWM35 PWM10) Could one predictor provide sufﬁcient information about the promoter strength in the absence of the other? There are at least three angles to address this question, and all of them were considered to interpret the model better. Bharanikumar et al. (2018), PeerJ, DOI 10.7717/peerj.5862 8/15 1. Comparing the raw, unadjusted R2 with the adjusted R2. The corresponding values were: Table 5 Validation with major (A1, A2, A3) and minor (C, D) promoters. Promoter -35 sequence -10 sequence Strength Predicted exp(logStrength) Predicted class A1 ttgact gatact strong 0.2904988307 Medium A2 ttgaca taagat strong 0.9947607331 Strong A3 ttgaca tacgat strong 0.658183377 Strong C ttgacg tagtct minor 0.1452865585 Minor D ttgact taggct minor 0.1541996302 Minor Figure 3 Effects plots of promoter sites on promoter strength. (A) –35 promoter site; and (B) –10 promoter site. Full-size  DOI: 10.7717/peerj.5862/ﬁg-3 Table 5 Validation with major (A1, A2, A3) and minor (C, D) promoters. Figure 3 Effects plots of promoter sites on promoter strength. (A) –35 promoter site; and (B) –10 promoter site. Full-size  DOI: 10.7717/peerj.5862/ﬁg-3 Figure 3 Effects plots of promoter sites on promoter strength. (A) –35 promoter site; and (B) –10 promoter site. Full-size  DOI: 10.7717/peerj.5862/ﬁg-3 1. Comparing the raw, unadjusted R2 with the adjusted R2. The corresponding values were: 1. Comparing the raw, unadjusted R2 with the adjusted R2. The corresponding values were: R2 ≈0.69 Adj. R2 ≈0.65 R2 ≈0.69 Adj. R2 ≈0.65 R2 ≈0.69 Adj. R2 ≈0.65 Since there is not much difference between R2 and adj. R2, we could say that both predictors contribute substantially to the response variable (promoter strength) and account for about 65% of its variance. 2. Since the p-values of both predictors are signiﬁcant, it would be interesting to observe their effect on the response variable in more detail. This was performed using the effects package in R: 2. Since the p-values of both predictors are signiﬁcant, it would be interesting to observe their effect on the response variable in more detail. This was performed using the effects package in R: library(effects) fit = lm(logStrength∼PWM35+ PWM10, data) plot(allEffects(fit)) library(effects) fit = lm(logStrength∼PWM35+ PWM10, data) plot(allEffects(fit)) fit = lm(logStrength∼PWM35+ PWM10, data) fit = lm(logStrength∼PWM35+ PWM10, data) plot(allEffects(fit)) plot(allEffects(fit)) Bharanikumar et al. (2018), PeerJ, DOI 10.7717/peerj.5862 9/15 The results are shown in Fig. 3 where the PWM scores are plotted against the level of conﬁdence in the predicted response. Bharanikumar et al. (2018), PeerJ, DOI 10.7717/peerj.5862 lm(logStrength ∼PWM35 PWM10) This further validated the assumption that the errors were independent. Plot C: To test the assumption that the errors were normally distributed, the standardized residuals were plotted against the theoretical quantiles of a normal distribution. The residual distribution closely followed the theoretical quantiles, except for minor deviations towards the tails of the distribution. Plot D: Since the least-squares cost function is sensitive to outliers, the number of outliers should be kept to a minimum. This was investigated by plotting the standardized residual against the corresponding instance’s model leverage. This plot showed that there were no signiﬁcant outliers in the dataset that could exert an undue inﬂuence on the regression parameters. 10/15 Bharanikumar et al. (2018), PeerJ, DOI 10.7717/peerj.5862 Table 6 Correlation matrix of features and response variables. Correlation coefﬁcient PWM–35 PWM–10 Combined Strength Log-strength PWM–35 1 -0.3715610 0.3401672 0.4558838 0.5153622 PWM–10 -0.3715610 1 0.7466500 0.3025062 0.4115533 Combined 0.3401672 0.7466500 1 0.6330488 0.7861173 Strength 0.4558838 0.3025062 0.6330488 1 0.8665495 Log-strength 0.5153622 0.4115533 0.7861173 0.8665495 1 Table 6 Correlation matrix of features and response variables. Log strength 0.5153622 0.4115533 0.7861173 0.8665495 1 Figure 4 Model diagnostics plots for investigating the assumptions underlying linear modelling. (A) Residuals vs. ﬁtted values; (B) homogeneity of residual variances; (C) normal Q-Q plot; and (D) residuals vs. leverage plot. Full-size  DOI: 10.7717/peerj.5862/ﬁg-4 rJ, DOI 10.7717/peerj.5862 11/ Figure 4 Model diagnostics plots for investigating the assumptions underlying linear modelling. (A) Residuals vs. ﬁtted values; (B) homogeneity of residual variances; (C) normal Q-Q plot; and (D) residuals vs. leverage plot. Full-size  DOI: 10.7717/peerj.5862/ﬁg-4 Bharanikumar et al. (2018), PeerJ, DOI 10.7717/peerj.5862 11/15 Figure 4 Model diagnostics plots for investigating the assumptions underlying linear modelling. (A) Residuals vs. ﬁtted values; (B) homogeneity of residual variances; (C) normal Q-Q plot; and (D) residuals vs. leverage plot. Full-size  DOI: 10.7717/peerj.5862/ﬁg-4 Bharanikumar et al. (2018), PeerJ, DOI 10.7717/peerj.5862 An alternative univariate regression model using only the combined score of the PWMs found the coefﬁcient of regression and the F-statistic signiﬁcant (both p-values ≈10-4). However, the adj. R2 of the model (≈0.59) was much lower than that for Eq. (2), so the original multiple linear regression model was retained for the estimation of the promoter strength. In summary, our model performed equally well on datasets of strong promoter sequences and datasets of weak random promoter sequences. Bharanikumar et al. (2018), PeerJ, DOI 10.7717/peerj.5862 lm(logStrength ∼PWM35 PWM10) Our model was consistent in detecting promoter strengths across a 1,000-fold span of promoter strengths in E. coli as well as the promoter strengths of a different species, T. maritima. The model was further able to discriminate between the major and minor promoters of bacteriophage T7. Based on these results, an open-access open-source web server and standalone tool offering the prediction service have been implemented. Since the linear modelling results are dependent on the dataset, our implementation provides a facility to augment the learning based on user-provided inputs. The web interface is based on Python web module (web.py) and nginx server. The computational layer is based on numpy, Biopython and matplotlib. The user is provided with an option to add any number of promoter instances with -10 and -35 sequences and the corresponding strengths to augment the training data of the supervised model. The measurement of promoter strength could be done in the manner of Kelly et al. (2009), where the GFP (reporter gene) synthesis rate is measured per unit biomass, and this could be normalized relative to the reference promoter. In order to assess the goodness of ﬁt of the updated model, the R2-value is re-computed, along with the 3D plot of the regression surface. This would enable the user to decide whether the data added to the model has improved its performance for further experiments with the software. Based on the trained model, the user could predict the strength of an uncharacterised promoter given its -10 and -35 hexamers. Author Contributions Ramit Bharanikumar performed the experiments, analysed the data, contributed reagents/materials/analysis tools, prepared ﬁgures and/or tables, approved the ﬁnal draft. Keshav Aditya R. Premkumar performed the experiments, analysed the data, contributed reagents/materials/analysis tools, prepared ﬁgures and/or tables, approved the ﬁnal draft. Ashok Palaniappan conceived and designed the experiments, performed the experiments, analysed the data, contributed reagents/materials/analysis tools, prepared ﬁgures and/or tables, authored or reviewed drafts of the paper, approved the ﬁnal draft. CONCLUSION The following important conclusions were drawn from our study. (1) Sequence-based modelling yielded a non-linear, logarithmic dependence between promoter strength and sequence. (2) The model was able to discriminate equally well between strong/major promoters and weak/minor/random promoter sequences, indicating successful learning of the essential features of promoter strength prediction. (3) The combined score (PWM–35 + PWM–10) emerged as the single most important predictor of the promoter strength. Our model yielded robust quantitative prediction across a 1,000-fold span of promoter strengths. It is straightforward to extend our methodology to the study of new promoter classes of other s factors. Our implementation and web service could be useful in characterizing promoters identiﬁed in genome sequencing projects as well in engineering promoters for the design of ﬁnely-tuned genetic circuits in synthetic biology. The dynamic feature of our implementation would enable users to incorporate their own data into the model and obtain more reliable estimates of promoter strength. The service will be periodically updated based on the availability of new training instances, user input data and/or models for promoters of other s factors. Bharanikumar et al. (2018), PeerJ, DOI 10.7717/peerj.5862 Competing Interests The authors declare that they have no competing interests. ACKNOWLEDGEMENTS We would like to thank the reviewers for helping improve an earlier version of the manuscript. We are grateful for computing facilities at SASTRA Deemed University for support. Data Availability The following information was supplied regarding data availability: The following information was supplied regarding data availability: Raw code: https://github.com/PromoterPredict/PromoterStrengthPredictor Palaniappan, Ashok; Aditya, Keshav; Bharanikumar, Ramit (2018): PromoterPredict: sequence-based modelling of promoter strength: supplementary information. ﬁgshare. Fileset. https://doi.org/10.6084/m9.ﬁgshare.6794939.v1 Palaniappan, Ashok; Aditya, Keshav; Bharanikumar, Ramit (2018): PromoterPredict: sequence-based modelling of promoter strength: supplementary information. ﬁgshare. Fileset. https://doi.org/10.6084/m9.ﬁgshare.6794939.v1 Funding The authors received no funding for this work. Bharanikumar et al. (2018), PeerJ, DOI 10.7717/peerj.5862 REFERENCES Basu RS, Warner BA, Molodtsov V, Pupov D, Esyunina D, Fernández-Tornero C, Kulbachinskiy A, Murakami KS. 2014. Structural basis of transcription initiation by bacterial RNA polymerase holoenzyme. Journal of Biological Chemistry 289(35):24549–24559 DOI 10.1074/jbc.m114.584037. Basu RS, Warner BA, Molodtsov V, Pupov D, Esyunina D, Fernández-Tornero C, Kulbachinskiy A, Murakami KS. 2014. Structural basis of transcription initiation by bacterial RNA polymerase holoenzyme. Journal of Biological Chemistry 289(35):24549–24559 DOI 10.1074/jbc.m114.584037. Basu RS, Warner BA, Molodtsov V, Pupov D, Esyunina D, Fernández-Tornero C, Kulbachinskiy A, Murakami KS. 2014. Structural basis of transcription initiation by bacterial RNA polymerase holoenzyme. Journal of Biological Chemistry 289(35):24549–24559 DOI 10.1074/jbc.m114.584037. Benos PV, Bulyk ML, Stormo GD. 2002. Additivity in protein-DNA interactions: how good an approximation is it? Nucleic Acids Research 30(20):4442–4451 DOI 10.1093/nar/gkf578. Benos PV, Bulyk ML, Stormo GD. 2002. Additivity in protein-DNA interactions: how good an approximation is it? Nucleic Acids Research 30(20):4442–4451 DOI 10.1093/nar/gkf578. Berg OG, Von Hippel PH. 1987. Selection of DNA binding sites by regulatory proteins. Statistical-mechanical theory and application to operators and promoters. Journal of Molecular Biology 193(4):723–750. Berg OG, Von Hippel PH. 1987. Selection of DNA binding sites by regulatory proteins. Statistical-mechanical theory and application to operators and promoters. Journal of Molecular Biology 193(4):723–750. Bujard H. 1980. The interaction of E. coli RNA polymerase with promoters. Trends in Biochemical Sciences 5(10):274–278 DOI 10.1016/0968-0004(80)90036-5. Bujard H. 1980. The interaction of E. coli RNA polymerase with promoters. Trends in Biochemical Sciences 5(10):274–278 DOI 10.1016/0968-0004(80)90036-5. Bharanikumar et al. (2018), PeerJ, DOI 10.7717/peerj.5862 13/15 Crooks GE, Hon G, Chandonia JM, Brenner SE. 2004. WebLogo: a sequence logo generator. Genome Research 14(6):1188–1190 DOI 10.1101/gr.849004. Davis JH, Rubin AJ, Sauer RT. 2011. Design, construction and characterization of a set of insulated bacterial promoters. Nucleic Acids Research 39(3):1131–1141 DOI 10.1093/nar/gkq810. Dayton CJ, Prosen DE, Parker KL, Cech CL. 1984. Kinetic measurements of Escherichia coli RNA polymerase association with bacteriophage T7 early promoters. Journal of Biological Chemistry 259:1616. De Jong A, Pietersma H, Cordes M, Kuipers OP, Kok J. 2012. PePPER: a webserver for prediction of prokaryote promoter elements and regulons. BMC Genomics 13(1):299 DOI 10.1186/1471-2164-13-299. De Mey M, Maertens J, Lequeux GJ, Soetaert WK, Vandamme EJ. 2007. Construction and model-based analysis of a promoter library for E. coli: an indispensable tool for metabolic engineering. BMC Biotechnology 7(1):34 DOI 10.1186/1472-6750-7-34. Dekhtyar M, Morin A, Sakanyan V. 2008. Triad pattern algorithm for predicting strong promoter candidates in bacterial genomes. Bharanikumar et al. (2018), PeerJ, DOI 10.7717/peerj.5862 Bharanikumar et al. (2018), PeerJ, DOI 10.7717/peerj.5862 REFERENCES BMC Bioinformatics 9(1):233 DOI 10.1186/1471-2105-9-233. Deuschle U, Kammerer W, Gentz R, Bujard H. 1986. Promoters of Escherichia coli: a hierarchy of in vivo strength indicates alternate structures. EMBO Journal 5(11):2987–2994 DOI 10.1002/j.1460-2075.1986.tb04596.x. Endy D. 2005. Foundations for engineering biology. Nature 438(7067):449–453 DOI 10.1038/nature04342. Feklistov A, Darst SA. 2011. Structural basis for promoter–10 element recognition by the bacterial RNA polymerase s subunit. Cell 147(6):1257–1269 DOI 10.1016/j.cell.2011.10.041. Galas DJ, Eggert M, Waterman MS. 1985. Rigorous pattern-recognition methods for DNA sequences. Analysis of promoter sequences from Escherichia coli. Journal of Molecular Biology 186(1):117–128 DOI 10.1016/0022-2836(85)90262-1. Gama-Castro S, Salgado H, Santos-Zavaleta A, Ledezma-Tejeida D, Muñiz-Rascado L, García-Sotelo JS, Alquicira-Hernández K, Martínez-Flores I, Pannier L, Castro-Mondragón JA, Medina-Rivera A, Solano-Lira H, Bonavides-Martínez C, Pérez-Rueda E, Alquicira-Hernández S, Porrón-Sotelo L, López-Fuentes A, Hernández-Koutoucheva A, Del Moral-Chávez V, Rinaldi F, Collado-Vides J. 2016. RegulonDB version 9.0: high-level integration of gene regulation, coexpression, motif clustering and beyond. Nucleic Acids Research 44(D1):D133–D143 DOI 10.1093/nar/gkv1156. Grant CE, Bailey TL, Noble WS. 2011. FIMO: scanning for occurrences of a given motif. Bioinformatics 27(7):1017–1018 DOI 10.1093/bioinformatics/btr064. Grant CE, Bailey TL, Noble WS. 2011. FIMO: scanning for occurrences of a given motif. Bioinformatics 27(7):1017–1018 DOI 10.1093/bioinformatics/btr064. Hawley DK, McClure WR. 1983. Compilation and analysis of Escherichia coli promoter DNA sequences. Nucleic Acids Research 11(8):2237–2255. Hawley DK, McClure WR. 1983. Compilation and analysis of Escherichia coli promoter DNA sequences. Nucleic Acids Research 11(8):2237–2255. Hook-Barnard I, Johnson XB, Hinton DM. 2006. Escherichia coli RNA polymerase recognition of a s70-dependent promoter requiring a -35 DNA element and an extended -10 TGn motif. Journal of Bacteriology 188(24):8352–8359 DOI 10.1128/jb.00853-06. Huerta AM, Collado-Vides J. 2003. Sigma70 promoters in Escherichia coli: speciﬁc transcription in dense regions of overlapping promoter-like signals. Journal of Molecular Biology 333(2):261–278 DOI 10.1016/j.jmb.2003.07.017. Kadonaga JT. 2012. Perspectives on the RNA polymerase II core promoter. Wiley Interdisciplinary Reviews: Developmental Biology 1(1):40–51 DOI 10.1002/wdev.21. Kelly JR, Rubin AJ, Davis JH, Ajo-Franklin CM, Cumbers J, Czar MJ, De Mora K, Glieberman AL, Monie DD, Endy D. 2009. Measuring the activity of biobrick promoters Bharanikumar et al. (2018), PeerJ, DOI 10.7717/peerj.5862 14/15 using an in vivo reference standard. Journal of Biological Engineering 3(1):4 DOI 10.1186/1754-1611-3-4. Knaus R, Bujard H. 1990. Principles governing the activity of E. coli promoters. In: Eckstein F, Lilley DMJ, eds. Nucleic Acids and Molecular Biology. Vol. 4. Berlin: Springer-Verlag, 110–122. Knaus R, Bujard H. 1990. Principles governing the activity of E. coli promoters. REFERENCES In: Eckstein F, Lilley DMJ, eds. Nucleic Acids and Molecular Biology. Vol. 4. Berlin: Springer-Verlag, 110–122. Li J, Zhang Y. 2014. Relationship between promoter sequence and its strength in gene expression. European Physical Journal E 37(9):44 DOI 10.1140/epje/i2014-14086-1. Li J, Zhang Y. 2014. Relationship between promoter sequence and its strength in gene expression. European Physical Journal E 37(9):44 DOI 10.1140/epje/i2014-14086-1. Maquat LE, Reznikoff WS. 1978. In vitro analysis of the Escherichia coli RNA polymerase interaction with wild-type and mutant lactose promoters. Journal of Molecular Biology 125(4):467–490 DOI 10.1016/0022-2836(78)90311-x. Meng H, Ma Y, Mai G, Wang Y, Liu C. 2017. Construction of precise support vector machine based models for predicting promoter strength. Quantitative Biology 5(1):90–98 DOI 10.1007/s40484-017-0096-3. Paget MS, Helmann JD. 2003. The s70 family of sigma factors. Genome Biology 4(1):203. Rhodius VA, Mutalik VK. 2010. Predicting strength and function for promoters of the Escherichia coli alternate sigma factor, sE. Proceedings of the National Academy of Sciences of the United States of America 107(7):2854–2859 DOI 10.1073/pnas.0915066107. Salis HM, Mirsky EA, Voigt CA. 2009. Automated design of synthetic ribosome binding sites to control protein expression. Nature Biotechnology 27(10):946–950 DOI 10.1038/nbt.1568. Shultzaberger RK, Chen Z, Lewis KA, Schneider TD. 2007. Anatomy of Escherichia coli sigma70 promoters. Nucleic Acids Research 35:771–788. Stormo GD. 1990. Consensus patterns in DNA. In: Doolittle RF, ed. Methods in Enzymology, Vol. 183. Molecular Evolution: Computer Analysis of Protein and Nucleic Acid Sequences. San Diego: Academic Press, 211–221. Weller K, Recknagel RD. 1994. Promoter strength prediction based on occurrence frequencies of consensus patterns. Journal of Theoretical Biology 171(4):355–359 DOI 10.1006/jtbi.1994.1239. 15/15
https://openalex.org/W2123016804	https://diagnosticpathology.biomedcentral.com/counter/pdf/10.1186/1746-1596-9-126	English	null	Heterogenous mismatch-repair status in colorectal cancer	Diagnostic pathology	2,014	cc-by	6,684	Joost et al. Diagnostic Pathology 2014, 9:126 http://www.diagnosticpathology.org/content/9/1/126 Joost et al. Diagnostic Pathology 2014, 9:126 http://www.diagnosticpathology.org/content/9/1/126 * Correspondence: patrick.joost@med.lu.se 1Department of Oncology and Pathology, Institute of Clinical Sciences, Lund University, SE-22381, Lund, Sweden Full list of author information is available at the end of the article © 2014 Joost et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. RESEARCH Open Access Heterogenous mismatch-repair status in colorectal cancer Patrick Joost1, Nynke Veurink1, Susanne Holck2, Louise Klarskov3, Anders Bojesen4, Maria Harbo4, Bo Baldetorp1, Eva Rambech1 and Mef Nilbert1 Abstract Background: Immunohistochemical staining for mismatch repair proteins is efficient and widely used to identify mismatch repair defective tumors. The tumors typically show uniform and widespread loss of MMR protein staining. We identified and characterized colorectal cancers with alternative, heterogenous mismatch repair protein staining in order to delineate expression patterns and underlying mechanisms. Methods: Heterogenous staining patterns that affected at least one of the mismatch repair proteins MLH1, PMS2, MSH2 and MSH6 were identified in 14 colorectal cancers. Based on alternative expression patterns macro-dissected and micro-dissected tumor areas were separately analyzed for microsatellite instability and MLH1 promoter methylation. Results: Heterogenous retained/lost mismatch repair protein expression could be classified as intraglandular (within or in-between glandular formations), clonal (in whole glands or groups of glands) and compartmental (in larger tumor areas/compartments or in between different tumor blocks). These patterns coexisted in 9/14 tumors and in the majority of the tumors correlated with differences in microsatellite instability/MLH1 methylation status. Conclusions: Heterogenous mismatch repair status can be demonstrated in colorectal cancer. Though rare, attention to this phenomenon is recommended since it corresponds to differences in mismatch repair status that are relevant for correct classification. Virtual Slides: The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/ 1771940323126788 Keywords: Mismatch repair, immunohistochemistry, heterogeneity, MLH1, MSH2, MSH6, PMS2 Keywords: Mismatch repair, immunohistochemistry, heterogeneity, MLH1, MSH2, MSH6, PMS2 MMR protein immunostaining S f ll bl k ( Sections from all tumor blocks (n = 4-11) from the 14 cases were subjected to independent MMR protein staining using alternative MMR protein antibodies from other manufac- turers (Table 2). Fresh 4-μm sections from formalin-fixed, paraffin-embedded tumors were mounted on Dako REAL™ Capillary gap microscope slides (Dako, Glostrup, Denmark). The slides were dried overnight at room temperature and thereafter at 60°C for 1–2 hours. The tissue was deparaffinized in xylene for two times 5 min, followed by 5 min each in 99.5% and 95% etha- nol and 5 min in distilled water. Heat-induced epitope retrieval was achieved by pressure boiler-treatment in F: female; M: Male; Adca: adenocarcinoma; MMR: mismatch repair. Background Variable epitope expres- sion may also result in alternative expression patterns, e.g. cytoplasmic staining and perinuclear staining, which are typically present throughout the tumor [10]. MMR protein immunostainings are generally stable and rela- tively easy to interpret; though challenges and pitfalls have been reported with false positive as well as false negative interpretations [10-12]. Most commonly, these observations relate to technical artifacts caused by sub- optimal fixation or paraffin-embedding, necrotic areas, sample storage, antibody specificity, clone selection or staining conditions [13,14]. Also, neoadjuvant chemo- therapy and radiotherapy may influence the results with a particular effect on MSH2/MSH6 staining [15,16]. Heterogenous expression patterns with retained staining in the adenomatous part and loss of staining in a smaller, invasive part of the tumor have been reported but their relevance is uncertain [17]. We systematically collected colorectal cancers with heterogenous MMR protein staining patterns for detailed analysis with correlations e.g. to MSI status and MLH1 promoter methylation. heterodimerizing proteins [8,9]. Variable epitope expres- sion may also result in alternative expression patterns, e.g. cytoplasmic staining and perinuclear staining, which are typically present throughout the tumor [10]. MMR protein immunostainings are generally stable and rela- tively easy to interpret; though challenges and pitfalls have been reported with false positive as well as false negative interpretations [10-12]. Most commonly, these observations relate to technical artifacts caused by sub- optimal fixation or paraffin-embedding, necrotic areas, sample storage, antibody specificity, clone selection or staining conditions [13,14]. Also, neoadjuvant chemo- therapy and radiotherapy may influence the results with a particular effect on MSH2/MSH6 staining [15,16]. Heterogenous expression patterns with retained staining in the adenomatous part and loss of staining in a smaller, invasive part of the tumor have been reported but their relevance is uncertain [17]. We systematically collected colorectal cancers with heterogenous MMR protein staining patterns for detailed analysis with correlations e.g. to MSI status and MLH1 promoter methylation. Background syndrome for further molecular diagnostics and to ob- tain treatment-predictive information linked to somatic methylation of MLH1 [6]. Mismatch repair (MMR) defects characterize 2-4% of colorectal cancers linked to Lynch syndrome and 15% of sporadic colorectal cancers caused by epigenetic MLH1 promoter methylation. Various strategies can be used to preselect colorectal cancers for MMR protein testing, e.g. clinical guidelines for hereditary cancer, MMR pre- diction models that combine clinical and pathological information and potentially novel biomarker-based strategies [1-5]. The monoclonal antibodies used for immunohisto- chemical MMR protein staining generally result in stable and consistent staining patterns with retained staining or loss of staining. Functional interaction between the MLH1/PMS2 and the MSH2/MSH6 proteins implies that the expression pattern of the heterodimerizing pro- tein partner may be used to direct mutation analysis. Aberrant MMR function typically leads to complete loss of nuclear staining in the tumor cells, particularly when linked to MLH1 promoter hypermethylation that leads to complete gene silencing [7]. In Lynch syndrome, the multitude of disease-predisposing mutations may have variable effects on epitope expression, from complete loss to weak or retained expression for one or both Universal assessment of immunohistochemical MMR staining is increasingly applied in colorectal cancer diag- nostics in order to identify cases suspected of Lynch Page 2 of 10 Joost et al. Diagnostic Pathology 2014, 9:126 http://www.diagnosticpathology.org/content/9/1/126 Joost et al. Diagnostic Pathology 2014, 9:126 http://www.diagnosticpathology.org/content/9/1/126 Joost et al. Diagnostic Pathology 2014, 9:126 http://www.diagnosticpathology.org/content/9/1/126 materials consisted of resection specimens from 12 colon cancers and 2 rectal cancers. None of the patients had received neoadjuvant radiotherapy or chemotherapy. All cases were histologically re-evaluated by one pathologist (P.J.). Tumor stage was determined according to the American Joint Cancer Committee/Union Internationale Contre le Cancer (AJCC/UICC) staging system and the grade according to the WHO system. Mucinous cancers were considered poorly differentiated. A tumor was classified as mucinous cancer if more than 50% of the tumor area showed such differentiation [18]. Tumors with mucinous components that encompassed <50% of the area were classified as having a mucinous component, though not fulfilling the criteria for mucinous tumors [19]. Mucinous cancer was observed in 6 cases. MMR gene mu- tation testing had been performed in 8 cases, 4 of which carried disease-predisposing mutations. Ethical approval for the study was granted from the ethical committees at Lund University, Sweden and at the Capital Region, Copenhagen. heterodimerizing proteins [8,9]. Methylation-specific PCR analysis Methylation-specific PCR analysis Extracted DNA was treated with bisulfite using the EZ DNA Methylation-Lightning™Kit (Zymo Research, CA, USA) according to the manufacturer’s instructions. MLH1 promoter methylation status was analyzed by means of a fluorescence-based, real-time methylation- specific PCR assay, as described previously [20]. Two sets of primers and probes, designed specifically for bisulfite-converted DNA, were used: MLH1-M2B for the methylation-specific reaction [21] and ALU-C4 for the methylation independent control reaction used to meas- ure the amount of bisulfite-converted input DNA [22]. Amplification was performed on QuantStudio™12 K Flex Real-Time PCR System (Life Technologies). Samples were run in duplicate, including positive and negative controls. Methods Materials Colorectal cancers with heterogenous MMR protein ex- pression were identified during evaluations at the Depart- ments of Pathology, Helsingborg Hospital, Sweden and Hvidore Hospital, Denmark. Following the first observation of heterogenous MMR protein staining in 2007, two gastro- intestinal pathologists (PJ and SH) collected all such cases identified at these two institutions during 5 years. In total, 14 colorectal cancers with heterogenous MMR protein ex- pression were identified for in-depth analysis (Table 1). The Table 1 Summary of clinical and pathological data Table 1 Summary of clinical and pathological data Case Sex/Age Tumor location Histologic type Differentiation pTNM Stage MMR gene mutation 1 F/57 Cecum Adca Moderate T2N0MX I Not tested 2 F/33 Cecum Mucinous adca Poor T2N0MX I MSH2 4 M/41 Rectum Adca Moderate T3N0MX II MSH6 5 F/63 Transverse colon Mucinous adca Moderate T4N1MX III Not tested 6 F/82 Rectum Mucinous adca Moderate T2N0MX I Not identified 8 M/85 Cecum Adca Poor T4N0MX II Not identified 9 M/71 Ascending colon Mucinous adca Poor T3N0MX II Not tested 10 F/66 Ascending colon Mucinous adca Poor T3N0MX II Not identified 11 F/57 Descending colon Adca Moderate T3N0MX II MSH6 12 F/55 Ascending colon Adca Poor T3N0MX II MLH1 14 M/82 Ascending colon Adca Moderate T3N0MX II Not tested 15 M/83 Ascending colon Adca Moderate T3N0MX II Not tested 16 M/74 Transverse colon Mucinous adca Poor T3N1MX III Not tested 17 F/74 Cecum Adca Poor T3N1MX III Not identified F: female; M: Male; Adca: adenocarcinoma; MMR: mismatch repair. Joost et al. Diagnostic Pathology 2014, 9:126 http://www.diagnosticpathology.org/content/9/1/126 Page 3 of 10 Table 2 Information on the MMR protein antibodies used MMR protein Lab Supplier Clone Dilution Immunogen/epitope MLH1 L BD Pharmingen G168-15 1:100 Full-length C DAKO ES05 RTU Recombinant protein, 210 aa L DAKO ES05 1:100 Recombinant protein, 210 aa PMS2 L BD Pharmingen A16-4 1:300 aa 431–862, C-terminal H Ventana (Cell Marque) EPR3947 RTU 100 aa, C-terminal C Epitomics EPR3947 1:50 100 aa, C-terminal MSH2 L Calbiochem FE11 1:100 C-terminal H Ventana (Cell Marque) G219-1129 RTU Full-length C Novocastra 25D12 1:50 Full-length MSH6 C Epitomics EP49 1:100 Synthetic peptide, N-terminal L Epitomics EP3945 1:100 Synthetic peptide, N-terminal L BD Transduction Lab. 44 1:500 Synthectic peptide aa 225-333 MMR: mismatch repair; L: Lund; H Helsingborg; C: Copenhagen; RTU: ready to use. Table 2 Information on the MMR protein antibodies used dissected tissues or whole tumor sections. Methods Materials MSI ana- lyses were performed using the MSI Analysis System, Version 1.2 (Promega, Madison, WI), PCR products were size separated on a 3130xl Genetic Analyzer (Applied Biosystems, Foster City, CA). The results were evaluated using the GeneMapper® software Version 4.0 (Applied Biosystems, Foster City, CA). The analysis in- cluded the 5 mononucleotide markers BAT-25, BAT-26, NR-21, NR-24, and MONO-27 (Promega, MSI Analysis System, Version 1.2, Madison, WI). Tumors with instability for 1 marker were classified as MSI low, tumors with instability for ≥2 markers were classified as MSI-high (MSI-H), and tumors with stability for all markers were classified as microsatellite stable (MSS). ethylene diamine tetraacetic acid (EDTA)-Tris buffer (1:10 mM, pH 9.0) for 20 min. Hereafter, the slides were cooled for 20 min and rinsed in distilled water. Immunostaining was performed using the Dako Auto- stainer and the EnVision™visualization method (Dako, Glostrup, Denmark). Endogenous peroxidase activity was blocked for 5 min and primary mouse monoclonal IgG antibodies were used (Table 2). Following primary antibody incubation, the slides were incubated with EnVision™/horseradish peroxidase (HRP) rabbit/mouse (Dako) and stained using the EnVision™Detection Sys- tem peroxidase/DAB rabbit/mouse (Dako). The immu- nohistochemical stainings were classified as retained, lost or reduced, i.e. a weaker than expected staining in the tumor cells compared to the stromal cells. The areas of the respective expression patterns were esti- mated in each block end expressed in percentage. Microsatellite instability analysis To assess the impact from heterogenous MMR protein stainings on MMR protein function, the tumors were subjected analysis of microsatellite instability (MSI). De- pending on the extent of the area involved, microdissec- tion or macrodissction was used to obtain material from areas with the respective expression patterns (Table 3). Laser capture micro-dissection was performed using Polyethylene Teraphthalate (PET)-membrane Frame- Slides (Carl Zeiss MicroImaging, Germany). In tumors where larger areas showed variable expression pat- terns, macro-dissection was performed. Tissue from 2–6 tumor areas with the respective MMR protein stained patterns were collected from 10-μm tissue sec- tions. DNA extraction was performed using QIAamp® DNA micro kit (Qiagen) for laser micro-dissected tis- sues and using the QIAcube machine (Qiagen) or the QIAamp® DNA FFPE tissue kit (Qiagen) for macro- Flow cytometry l Flow cytometry Flow cytometric DNA analysis was performed as previ- ously described [23,24]. The separated cells were then treated with ribonuclease (Sigma-Aldrich, Stockholm, Sweden), incubated with trypsin for 48 h (Merck, Joost et al. Diagnostic Pathology 2014, 9:126 http://www.diagnosticpathology.org/content/9/1/126 Page 4 of 10 Table 3 Summary of MMR heterogeneity Case Block no. Flow cytometry l Hetero- geneity Pattern Involved area (%) MMR protein immunostaining Fraction of MSI in markers MLH1 promoter methylation MLH1 PMS2 MSH2 MSH6 1 1A¤/ + CL 15 +/− +/− + + 0/5 and 4/5 +/− 1B + CL 5 - - +/R +/− 1C - H 0 - - + + 1D - H 0 - - + + 1E¤ + CL, IG 70 / 10 +/− +/− +/R +/− 0/3 vs 4/5 2 24 - H 0 + + + + - 25* - H 0 + + + + 0/5 26 - H 0 + + + + 27* - H 0 + + + + 0/5 28 + CL, IG 15 +/− +/− + + 29* + CL, IG 40 +/− +/− + + 5/5 30 + IG 10 +/− +/− + + 31 + IG 5 +/− +/− + + 32 + IG 5 +/− +/− + + 4 2 + IG 97 + + +/− R/- NE 3¤/* + CL, IG 95 + + +/− R/- 3/3 vs 3/5 4 + CL, IG 80 + + +/− R/- 5* + IG 95 + + +/− R/- 5/5 5 20* + CL, IG 50 / 100 + +/− - R/- 5/5 - 21 + IG 100 + + - R/- 23 + IG 100 + + - R/- 24 + IG 100 + + - R/- 25 + COM, IG 100 + - - R/- 26 + COM, IG 100 + - - R/- 27 + COM IG 100 + - - R/- 28 + COM, IG 100 + - - R/- 29 + COM, IG 100 + - - R/- 30 + COM, IG 100 + - - R/- 31 + COM, IG 100 + - - R/- 6 5¤ + IG 100 + R/- + + 0/5 vs 0/4 NE 6* + IG 100 + R/- + + 0/5 7* - H 0 + + + + 0/5 8* + IG 100 + R/- + + 0/5 9* + IG 95 + R/- + + 0/5 10 + IG 95 + R/- + + 11* + IG 100 + R/- + + 0/5 8 5* - H 0 - - + + 5/5 + 6 + CL, IG 60 - - + R/- 7 + CL 20 - - + +/− 8 - H 0 - - + + 9 - H 0 - - + + 10 + CL, IG 20 - - +/R +/− Table 3 Summary of MMR heterogeneity Joost et al. Flow cytometry l Joost et al. Diagnostic Pathology 2014, 9:126 Page 6 of 10 http://www.diagnosticpathology.org/content/9/1/126 Joost et al. Diagnostic Pathology 2014, 9:126 http://www.diagnosticpathology.org/content/9/1/126 Page 6 of 10 Table 3 Summary of MMR heterogeneity (Continued) 19 - H 0 - - + + 20 + CL 8 - - +/− +/− 21 - H 0 - - + + 22 - H 0 - - + + microdissection; : macrodissection; CL: clonal, IG: intraglandular; COM: compartmental; H: homogenous; R: reduced staining; +/-: heterogenous staining; MMR: mismatch repair; MSI: microsatellite instability; NE: not evaluated. Table 3 Summary of MMR heterogeneity (Continued) 19 - H 0 - - + + 20 + CL 8 - - +/− +/− 21 - H 0 - - + + 22 - H 0 - - + + microdissection; : macrodissection; CL: clonal, IG: intraglandular; COM: compartmental; H: homogenous; R: reduced staining; +/-: heterogenous staining; MMR: mismatch repair; MSI: microsatellite instability; NE: not evaluated. Table 3 Summary of MMR heterogeneity (Continued) microdissection; : macrodissection; CL: clonal, IG: intraglandular; COM: compartmental; H: homogenous; R: reduced staining; +/-: heterogenous staining; MMR: mismatch repair; MSI: microsatellite instability; NE: not evaluated. ction; CL: clonal, IG: intraglandular; COM: compartmental; H: homogenous; R: reduced staining; +/-: heterogenous staining; MMR: tellite instability; NE: not evaluated. microdissection; : macrodissection; CL: clonal, IG: intraglandular; COM: compartmental; H: homogenous; R: reduced staining; +/-: heterogenous staining; MMR: mismatch repair; MSI: microsatellite instability; NE: not evaluated. microdissection; : macrodissection; CL: clonal, IG: intraglandular; COM: compartmental; H: homogenous; R: reduced staining; +/-: mismatch repair; MSI: microsatellite instability; NE: not evaluated. well demarcated and appeared in three distinct pat- terns: “intraglandular” (retained/lost staining within or in between glandular formations), “clonal” (retained/ lost staining in whole glands or groups of glands) and “compartmental” (retained/lost staining in larger tumor areas/compartments leading to retained/lost staining in between different tumor blocks) (Figure 1, Table 3). Various heterogenous expression patterns co-existed in 9/14 tu- mors, most commonly as intraglandular and clonal hetero- geneity (figure 1c). The heterogenous staining patterns were present in 3-100% of the examined tumor area. In 4/ 14 cases, all tumor blocks showed heterogeneity, whereas the remaining tumors showed heterogeneity in a variable fraction of the tumor blocks (Table 3). Darmstadt, Germany), and stained with propidium iod- ide (Sigma-Aldrich, Stockholm). Flow cytometric DNA analysis was performed in a FACS Caliber (Becton, Dick- inson, BD Biosciences, USA). Up to 20,000 nuclei were analysed from each sample. Flow cytometry l The DNA histograms ob- tained were automatically processed using Modfit LT 3.3™software. The DNA index (DI) was calculated as the ratio of the respective modal channel values of the non- diploid and the diploid G0/G1 peaks. The S-phase frac- tion (Spf) was estimated assuming that the S-phase compartment constituted a rectangular distribution be- tween the modal values of the G0/G1 and G2 peaks. Flow cytometry l Diagnostic Pathology 2014, 9:126 http://www.diagnosticpathology.org/content/9/1/126 Page 5 of 10 Page 5 of 10 Table 3 Summary of MMR heterogeneity (Continued) 11 - H 0 - - + + 12 + CL 20 - - +/− +/− 9 16 - H 0 + + + + 19 - H 0 + + + + 20 - H 0 + + + + 0/5 21* - COM 100 - - + + 5/5 22 - H 0 + + + + 40 - H 0 + + + + 44 - H 0 + + + + 10 6 + CL 50 - - +/− +/− 7 - H 0 - - + + 8 - H 0 - - + + 9* - H 0 - - + + 5/5 10 - H 0 - - + + 11 - H 0 - - + + 11 3 - H 0 - - + + 4 + CL, IG 5 - - +/− +/− 5 - H 0 - - + + 6* - H 0 - - + + 5/5 7 - H 0 - - + + 8* + CL, IG 30 - - +/R +/− 5/5 12 3* + CL 5 - - +/− +/− 5/5 4 + CL, IG 90 - - +/− +/− 5 + CL, IG 80 - - +/− +/− 6 - H 100 - - - - 7 + CL 60 - - +/− +/− 8 + CL, IG 90 - - +/− +/− 9* + CL, IG 60 - - +/− +/− 5/5 14 1A* - H 0 - - + + 5/5 1B* + CL 5 - - +/R +/− 5/5 1C* + CL, IG 30 - - R/- +/− 5/5 1D* + CL 40 - - +/R +/− 5/5 1E* + CL, IG 40 - - R/- +/− 5/5 15 4A* + IG 100 + + +/R R/- 5/5 4B* + IG 100 + + + R/- 5/5 4C + IG 100 + + +/R R/- 4D + IG 100 + + + R/- 4E + IG 100 + + +/R R/- 16 6A + IG 15 - - R +/− 6B* + CL 3 - - +/R +/− 5/5 6C + CL 10 - - +/R +/− 6D + IG 5 - - R +/− 6E + IG 5 - - +/R +/− 17 18* + CL 55 - - +/− +/− 5/5 Table 3 Summary of MMR heterogeneity (Continued) Table 3 Summary of MMR heterogeneity (Continued) 19 - H 0 - - + + 20 + CL 8 - - +/− +/− 21 - H 0 - - + + 22 - H 0 - - + + microdissection; *: macrodissection; CL: clonal, IG: intraglandular; COM: compartmental; H: homogenous; R: reduced staining; +/-: heterogenous staining; MMR: mismatch repair; MSI: microsatellite instability; NE: not evaluated. Results MSI was demonstrated in 13/14 tumors. Intra-tumor differences in MMR status, i.e.MSI versus MSS, in line with MMR protein staining expression was observed in 3 tumors (Table 3; case 1, 2 and 9). Non-consistent, homogenous MSI status in tumors with heterogenous MMR protein expression was observed in 2 cases (Table 3; cases 4 and 6). MLH1 promoter methylation was demonstrated in all 7 cases with complete (non- Immunohistochemical staining using alternative MMR pro- tein antibodies confirmed heterogenous MMR protein ex- pression in all 14 tumors. Heterogenous expression affected MLH1/PMS2 in 3 tumors, PMS2 in 2 tumors, MSH2/ MSH6 in 10 tumors (of which two also expressed hetero- geneity for MLH1/PMS2) and MSH6 only in 1 tumor (in which one block also expressed heterogeneity for PMS2). Areas with alternative expression patterns were Figure 1 Examples of the different MMR protein staining patterns. A) clonal loss, B) intraglandular loss, C) co-existence of clonal and intraglandular loss and D) compartmental loss with different patterns in two separate tumor blocks. Figure 1 Examples of the different MMR protein staining patterns. A) clonal loss, B) intraglandular loss, C) co-existence of clonal and intraglandular loss and D) compartmental loss with different patterns in two separate tumor blocks. Joost et al. Diagnostic Pathology 2014, 9:126 http://www.diagnosticpathology.org/content/9/1/126 Joost et al. Diagnostic Pathology 2014, 9:126 http://www.diagnosticpathology.org/content/9/1/126 Joost et al. Diagnostic Pathology 2014, 9:126 http://www.diagnosticpathology.org/content/9/1/126 Page 7 of 10 Page 7 of 10 (Figure 2). DNA flow cytometric analysis was performed in one tumor (case 1) and demonstrated differences in DNA content within the heterogenous areas, which had DNA indices of 1.13 and 1.57, respectively (Figure 2). heterogenic) loss of MLH1/PMS2. In 2 cases (Table 3; cases 1 and 9) heterogenous MMR protein staining for MLH1/PMS2 correlated with heterogenous MLH1 pro- moter methylation, i.e. tumor areas with retained MLH1 expression did not show MLH1 methylation, whereas areas with loss of MLH1 expression showed MLH1 methylation. Concordant immunostaining and methyla- tion status suggest functional intratumoral heterogeneity Discussion Heterogeneous MMR protein expression is a rare phenomenon, but corresponds to differences in MMR Figure 2 An adenocarcinoma (case 1) with 4 different expression patterns and various combinations of heterogeneity, loss of MLH1/ PMS2 and heterogeneity/retained expression for MSH2/MSH6. A) clonal loss of MLH1 staining. B) MSI corresponding to loss of staining, C) MSS corresponding to retained MMR protein staining. Methylation analysis revealed D) presence and E) absence, respectively, of MLH1 promoter methylation, which verifies clonal MLH1 methylation status. Flow cytometric analysis showing different DNA indices, i.e. F) 1.13 in the MSI area and G) 1.57 in the MSS area. Figure 2 An adenocarcinoma (case 1) with 4 different expression patterns and various combinations of heterogeneity, loss of MLH1/ PMS2 and heterogeneity/retained expression for MSH2/MSH6. A) clonal loss of MLH1 staining. B) MSI corresponding to loss of staining, C) MSS corresponding to retained MMR protein staining. Methylation analysis revealed D) presence and E) absence, respectively, of MLH1 promoter methylation, which verifies clonal MLH1 methylation status. Flow cytometric analysis showing different DNA indices, i.e. F) 1.13 in the MSI area and G) 1.57 in the MSS area. Joost et al. Diagnostic Pathology 2014, 9:126 http://www.diagnosticpathology.org/content/9/1/126 Page 8 of 10 promoter methylation and DNA content, suggestive of a tumor composed of two distinct clones (case 1, Figure 2). status within the tumor and is therefore important to recognize to prevent false-positive or false-negative eval- uations. We identified three distinct patterns of hetero- geneity, i.e. intraglandular, clonal and compartmental heterogenous MMR protein expression. The different patterns co-existed within the same tumor and the ex- tent of the tumor involved varied. In-depth analysis suggests that multiple causes may apply, e.g. variable epitope expression, expression related to variable differ- entiation, second hit mutations or methylation in se- lected tumor clones and possibly influence from factors linked to the tumor microenvironment such as hypoxia and oxidative stress [25]. Variable MMR status did in some tumors correspond to variable differentiation, e.g. mucinous areas (cases 5 and 15), poor differentiation (cases 16 and 17) or aden- omatous components (case 2, Figure 3). Correlation be- tween MSI status and expression also of other molecular markers has been described [28,29]. Discussion Different, through homogenous, MMR protein expression patterns in dis- tinct tumor compartments were observed in a mucinous adenocarcinoma (case 9) with loss of MLH1/PMS2 expres- sion, MSI and MLH1 methylation in 1/7 tumor blocks that corresponded to an adenomatous tumor component (Figure 1d). Sample mix was excluded through histologic review and penta-D marker fragment analysis (data not shown). Homogenous loss/reduced staining of MSH2/ MSH6 throughout the tumor and additional loss of PMS2 in 7/10 tumor blocks was observed in a mucinous adeno- carcinoma (case 5). This case most likely reflects how the mucinous tumor component progressed in another line than the non-mucinous tumor component. Though com- partmental loss of MMR protein expression is rare, this ob- servation motivates thorough evaluation of different tumor compartments, particularly when areas with variable ex- pression are identified. Intraglandular and/or clonal heterogeneity throughout the tumor, which may be caused by variable epitope ex- pression, was identified in 4 tumors (cases 4, 5, 15 and 16, Table 3). Homogenous loss of MLH1/PMS2 and heterogenous expression of MSH2/MSH6 was identified in 7 tumors that were consistently MSI and showed MLH1 promoter methylation (Table 3). This expression pattern has previously been observed and may relate either to a germline MSH2/MSH6 mutation that allows for partial epitope binding in the presence of somatic MLH1 methylation or to secondary MSH2/MSH6 inacti- vation [25-27]. Heterogenous MLH1 and/or PMS2 expres- sion, suggestive of variable MLH1 methylation/second hit mutations was observed in 2 tumors (case 2 and 6, Table 3). Case 1 showed a more complex pattern of MMR protein expression and intra-tumor differences in MSI, MLH1 Limitations to our study include analysis based on surgi- cal specimens though biopsy material may produce stain- ings of better technical quality [30-33]. At the same time use of biopsy material implies analysis of a restricted tumor Figure 3 Variable MMR protein expression in relation to tumor differentiation. A) case 5 with retained expression for PMS2 in a mucinous tumor component and loss of PMS2 expression in a non-mucinous component. B) case 2 with clonal and intraglandular heterogeneity for MLH1 in the adenomatous component of the tumor, whereas the remaining tumors showed retained expression for MLH1. C-D) case 17 with clonal heterogeneity for MLH6 in a poorly differentiated tumor component and homogenous expression in a well-differentiated tumor component. Figure 3 Variable MMR protein expression in relation to tumor differentiation. Joost et al. Diagnostic Pathology 2014, 9:126 http://www.diagnosticpathology.org/content/9/1/126 Joost et al. Diagnostic Pathology 2014, 9:126 http://www.diagnosticpathology.org/content/9/1/126 Joost et al. Diagnostic Pathology 2014, 9:126 http://www.diagnosticpathology.org/content/9/1/126 Joost et al. Diagnostic Pathology 2014, 9:126 http://www.diagnosticpathology.org/content/9/1/126 area that may not capture areas with alternative expression. Also, information on MMR gene mutation status was not available in all cases. The 4 tumors from Lynch syndrome mutation carriers, however, expressed heterogeneity in be- tween different tumor blocks, which showed homogenous as well as heterogenous loss in clonal and intraglandular patterns. Recognition of so-called “patchy” MMR protein staining has been reported and were also considered herein (Table 3). This phenomenon differs from the heterogenous staining patterns described herein in that it primarily relates to MSH6 stainings, neoadjuvant treatment [15,16] or repre- sents a weak or cytoplasmic staining rather than the distinct and well-demarcated areas of retained staining and loss of staining described herein. predicting mismatch repair gene mutations in Lynch syndrome: a comprehensive analysis of 3,671 families. Int J Cancer 2014, 135:69–77. Umar A, Boland CR, Terdiman JP, Syngal S, de la Chapelle A, Ruschoff J, Fishel R, Lindor NM, Burgart LJ, Hamelin R, Hamilton SR, Hiatt RA, Jass J, Lindblom A, Lynch HT, Peltomaki P, Ramsey SD, Rodriguez-Bigas MA, Vasen HF, Hawk ET, Barrett JC, Freedman AN, Srivastava S: Revised Bethesda Guidelines for hereditary nonpolyposis colorectal cancer (Lynch syndrome) and microsatellite instability. J Natl Cancer Inst 2004, 96:261–268. Vasen HF, Watson P, Mecklin JP, Lynch HT: New clinical criteria for hereditary nonpolyposis colorectal cancer (HNPCC, Lynch syndrome) proposed by the International Collaborative group on HNPCC. Gastroenterology 1999, 116:1453–1456. 6. Moreira L, Balaguer F, Lindor N, de la Chapelle A, Hampel H, Aaltonen LA, Hopper JL, Le Marchand L, Gallinger S, Newcomb PA, Haile R, Thibodeau SN, Gunawardena S, Jenkins MA, Buchanan DD, Potter JD, Baron JA, Ahnen DJ, Andreu M, Ponz de Leon M, Rustgi AK, Castells A: Identification of Lynch syndrome among patients with colorectal cancer. JAMA 2012, 308:1555–1565. Abbreviations Abbreviations MMR: Mismatch repair; MSI: Microsatellite instability; MSI-H: MSI-high; MSS: Microsatellite stable. 11. Klarskov L, Ladelund S, Holck S, Roenlund K, Lindebjerg J, Elebro J, Halvarsson B, von Salome J, Bernstein I, Nilbert M: Interobserver variability in the evaluation of mismatch repair protein immunostaining. Hum Pathol 2010, 41:1387–1396. 11. Klarskov L, Ladelund S, Holck S, Roenlund K, Lindebjerg J, Elebro J, Halvarsson B, von Salome J, Bernstein I, Nilbert M: Interobserver variability in the evaluation of mismatch repair protein immunostaining. Hum Pathol 2010, 41:1387–1396. Received: 12 April 2014 Accepted: 22 June 2014 Published: 26 June 2014 18. Hamilton SR, Bosman FT, Boffetta P, Ilyas M, Morreau H, Nakamura SI, Quirke P, Riboli E, Sobin LH: Carcinoma of the colon and rectum. In WHO Classification of Tumours of the Digestive System. Volume 3. 4th edition. Edited by Bosman FT, Carneiro F, Hruban RH, Theise ND. Lyon, France: IARC Press; 2010:134–146. Competing interests Th h ’ d l h Competing interests The author’s declare that they have no competing interests. Acknowledgments 15. Bao F, Panarelli NC, Rennert H, Sherr DL, Yantiss RK: Neoadjuvant therapy induces loss of MSH6 expression in colorectal carcinoma. Am J Surg Pathol 2010, 34:1798–1804. 15. Bao F, Panarelli NC, Rennert H, Sherr DL, Yantiss RK: Neoadjuvant therapy induces loss of MSH6 expression in colorectal carcinoma. Am J Surg Pathol 2010, 34:1798–1804. Financial support was granted from the Swedish Cancer Society, the Nilsson Cancer Foundation, the Kamprad Cancer Foundation and through an ALF grant from the Lund University Medical Faculty, Sweden. 16. Radu OM, Nikiforova MN, Farkas LM, Krasinskas AM: Challenging cases encountered in colorectal cancer screening for Lynch syndrome reveal novel findings: nucleolar MSH6 staining and impact of prior chemoradiation therapy. Hum Pathol 2011, 42:1247–1258. Authors' contributions 12. Overbeek LI, Ligtenberg MJ, Willems RW, Hermens RP, Blokx WA, Dubois SV, van der Linden H, Meijer JW, Mlynek-Kersjes ML, Hoogerbrugge N, Hebeda KM, van Krieken JH: Interpretation of immunohistochemistry for mismatch repair proteins is only reliable in a specialized setting. Am J Surg Pathol 2008, 32:1246–1251. PJ: corresponding author, study design, review of pathology samples, data analysis and manuscript writing, NV: data collection, macrodissection, MSI analysis, SH: identification of relevant cases, histopathologic review, LK: identification of relevant cases, histopathologic review, AB/MH: MLH1 methylation analysis, BB: DNA ploidy analysis, ER: sample collection, MMR protein immunostaining, MN: study design, data analysis, MMR evaluation, manuscript review. All authors read and approved the final manuscript. 13. Engel KB, Moore HM: Effects of preanalytical variables on the detection of proteins by immunohistochemistry in formalin-fixed, paraffin-embedded tissue. Arch Pathol Lab Med 2011, 135:537–543. 13. Engel KB, Moore HM: Effects of preanalytical variables on the detection of proteins by immunohistochemistry in formalin-fixed, paraffin-embedded tissue. Arch Pathol Lab Med 2011, 135:537–543. 14. Shia J, Ellis NA, Klimstra DS: The utility of immunohistochemical detection of DNA mismatch repair gene proteins. Virchows Arch 2004, 445:431–441. 14. Shia J, Ellis NA, Klimstra DS: The utility of immunohistochemical detection of DNA mismatch repair gene proteins. Virchows Arch 2004, 445:431–441. Conclusions y g p 7. Li X, Yao X, Wang Y, Hu F, Wang F, Jiang L, Liu Y, Wang D, Sun G, Zhao Y: MLH1 promoter methylation frequency in colorectal cancer patients and related clinicopathological and molecular features. PLoS One 2013, 8:e59064. Our study verifies heterogenous MMR status in a subset of colorectal cancer. Heterogenous MMR protein ex- pression appears in three major forms, which frequently co-exist and correlate to differences in MMR status. We suggest that variable MMR protein staining patterns should be considered and when observed linked to ex- tended analysis in order to ensure correct classification of MMR status. 8. Mangold E, Pagenstecher C, Friedl W, Fischer HP, Merkelbach-Bruse S, Ohlendorf M, Friedrichs N, Aretz S, Buettner R, Propping P, Mathiak M: Tumours from MSH2 mutation carriers show loss of MSH2 expression but many tumours from MLH1 mutation carriers exhibit weak positive MLH1 staining. J Pathol 2005, 207:385–395. g 9. Shia J, Klimstra DS, Nafa K, Offit K, Guillem JG, Markowitz AJ, Gerald WL, Ellis NA: Value of immunohistochemical detection of DNA mismatch repair proteins in predicting germline mutation in hereditary colorectal neoplasms. Am J Surg Pathol 2005, 29:96–104. 10. Muller A, Giuffre G, Edmonston TB, Mathiak M, Roggendorf B, Heinmoller E, Brodegger T, Tuccari G, Mangold E, Buettner R, Ruschoff J: Challenges and pitfalls in HNPCC screening by microsatellite analysis and immunohistochemistry. J Mol Diagn 2004, 6:308–315. 10. Muller A, Giuffre G, Edmonston TB, Mathiak M, Roggendorf B, Heinmoller E, Brodegger T, Tuccari G, Mangold E, Buettner R, Ruschoff J: Challenges and pitfalls in HNPCC screening by microsatellite analysis and immunohistochemistry. J Mol Diagn 2004, 6:308–315. Author details 1 1Department of Oncology and Pathology, Institute of Clinical Sciences, Lund University, SE-22381, Lund, Sweden. 2Department of Pathology and Clinical 1Department of Oncology and Pathology, Institute of Clinical Sciences, Lund University, SE-22381, Lund, Sweden. 2Department of Pathology and Clinical Research Centre, University Hospital of Copenhagen, DK-2650, Hvidore, 17. Giuffre G, Muller A, Brodegger T, Bocker-Edmonston T, Gebert J, Kloor M, Dietmaier W, Kullmann F, Buttner R, Tuccari G, Ruschoff J: Microsatellite analysis of hereditary nonpolyposis colorectal cancer-associated colorectal adenomas by laser-assisted microdissection: correlation with mismatch repair protein expression provides new insights in early steps of tumorigenesis. J Mol Diagn 2005, 7:160–170. Denmark. 3Department of Pathology, Herlev Hospital, DK-2730, Herlev, Denmark. 4Department of Clinical Genetics, Vejle Hospital, DK-7100, Vejle, D k Denmark. 4Department of Clinical Genetics, Vejle Hospital, DK-7100, Vejle, Received: 12 April 2014 Accepted: 22 June 2014 Published: 26 June 2014 Discussion A) case 5 with retained expression for PMS2 in a mucinous tumor component and loss of PMS2 expression in a non-mucinous component. B) case 2 with clonal and intraglandular heterogeneity for MLH1 in the adenomatous component of the tumor, whereas the remaining tumors showed retained expression for MLH1. C-D) case 17 with clonal heterogeneity for MLH6 in a poorly differentiated tumor component and homogenous expression in a well-differentiated tumor component. Page 9 of 10 2. Steinke V, Holzapfel S, Loeffler M, Holinski-Feder E, Morak M, Schackert HK, Gorgens H, Pox C, Royer-Pokora B, von Knebel-Doeberitz M, Buttner R, Propping P, Engel C: Evaluating the performance of clinical criteria for 1. Kastrinos F, Balmana J, Syngal S: Prediction models in Lynch syndrome. Fam Cancer 2013, 12:217–228. Joost et al. Diagnostic Pathology 2014, 9:126 http://www.diagnosticpathology.org/content/9/1/126 20. Eads CA, Danenberg KD, Kawakami K, Saltz LB, Blake C, Shibata D, Danenberg PV, Laird PW: MethyLight: a high-throughput assay to measure DNA methylation. Nucleic Acids Res 2000, 28:E32. 21. Fiegl H, Gattringer C, Widschwendter A, Schneitter A, Ramoni A, Sarlay D, Gaugg I, Goebel G, Muller HM, Mueller-Holzner E, Marth C, Widschwendter M: Methylated DNA collected by tampons–a new tool to detect endometrial cancer. Cancer Epidemiol Biomarkers Prev 2004, 13:882–888. 22. Weisenberger DJ, Campan M, Long TI, Kim M, Woods C, Fiala E, Ehrlich M, Laird PW: Analysis of repetitive element DNA methylation by MethyLight Nucleic Acids Res 2005, 33:6823–6836. 23. Baldetorp B, Dalberg M, Holst U, Lindgren G: Statistical evaluation of cell kinetic data from DNA flow cytometry (FCM) by the EM algorithm. Cytometry 1989, 10:695–705. 24. Schutte B, Reynders MM, Bosman FT, Blijham GH: Flow cytometric determination of DNA ploidy level in nuclei isolated from paraffin- embedded tissue. Cytometry 1985, 6:26–30. 25. Shia J: Immunohistochemistry versus microsatellite instability testing for screening colorectal cancer patients at risk for hereditary nonpolyposis colorectal cancer syndrome. Part I. The utility of immunohistochemistry. J Mol Diagn 2008, 10:293–300. 26. Chapusot C, Martin L, Bouvier AM, Bonithon-Kopp C, Ecarnot-Laubriet A, Rageot D, Ponnelle T, Laurent Puig P, Faivre J, Piard F: Microsatellite instability and intratumoural heterogeneity in 100 right-sided sporadic colon carcinomas. Br J Cancer 2002, 87:400–404. 27. Watson N, Grieu F, Morris M, Harvey J, Stewart C, Schofield L, Goldblatt J, Iacopetta B: Heterogeneous staining for mismatch repair proteins during population-based prescreening for hereditary nonpolyposis colorectal cancer. J Mol Diagn 2007, 9:472–478. 28. Nodin B, Johannesson H, Wangefjord S, O’Connor DP, Lindquist KE, Uhlen M, Jirstrom K, Eberhard J: Molecular correlates and prognostic significance of SATB1 expression in colorectal cancer. Diagn Pathol 2012, 7:115. 29. Wangefjord S, Brandstedt J, Lindquist KE, Nodin B, Jirstrom K, Eberhard J: Associations of beta-catenin alterations and MSI screening status with expression of key cell cycle regulating proteins and survival from colorectal cancer. Diagn Pathol 2013, 8:10. 30. Fadhil W, Ilyas M: Immunostaining for mismatch repair (MMR) protein expression in colorectal cancer is better and easier to interpret when performed on diagnostic biopsies. Histopathology 2012, 60:653–655. 31. Kumarasinghe AP, de Boer B, Bateman AC, Kumarasinghe MP: DNA mismatch repair enzyme immunohistochemistry in colorectal cancer: a comparison of biopsy and resection material. Pathology 2010, 42:414–420. 32. References 1. Kastrinos F, Balmana J, Syngal S: Prediction models in Lynch syndrome. Fam Cancer 2013, 12:217–228. 19. Halvarsson B, Anderson H, Domanska K, Lindmark G, Nilbert M: Clinicopathologic factors identify sporadic mismatch repair-defective colon cancers. Am J Clin Pathol 2008, 129:238–244. 2. Steinke V, Holzapfel S, Loeffler M, Holinski-Feder E, Morak M, Schackert HK, Gorgens H, Pox C, Royer-Pokora B, von Knebel-Doeberitz M, Buttner R, Propping P, Engel C: Evaluating the performance of clinical criteria for Page 10 of 10 Joost et al. Diagnostic Pathology 2014, 9:126 http://www.diagnosticpathology.org/content/9/1/126 Shia J, Stadler Z, Weiser MR, Rentz M, Gonen M, Tang LH, Vakiani E, Katabi N, Xiong X, Markowitz AJ, Shike M, Guillem J, Klimstra DS: Immunohistochemical staining for DNA mismatch repair proteins in intestinal tract carcinoma: how reliable are biopsy samples? Am J Surg Pathol 2011, 35:447–454. 33. Warrier SK, Trainer AH, Lynch AC, Mitchell C, Hiscock R, Sawyer S, Boussioutas A, Heriot AG: Preoperative diagnosis of Lynch syndrome with DNA mismatch repair immunohistochemistry on a diagnostic biopsy. Dis Colon Rectum 2011, 54:1480–1487. doi:10.1186/1746-1596-9-126 Cite this article as: Joost et al.: Heterogenous mismatch-repair status in colorectal cancer. Diagnostic Pathology 2014 9:126. 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: • Convenient online submission
https://openalex.org/W3163746043	https://www.researchsquare.com/article/rs-523388/v1.pdf?c=1631881720000	English	null	Non-Transgenic Functional Rescue of Neuropeptides	Research Square (Research Square)	2,021	cc-by	8,681	Non-Transgenic Functional Rescue of Neuropeptides Elizabeth DiLoreto Worcester Polytechnic Institute Douglas Reilly Worcester Polytechnic Institute Jagan Srinivasan (  jsrinivasan@wpi.edu ) Worcester Polytechnic Institute Research Article Research Article Keywords: neuromodulation, neuropeptides, animal behavior Posted Date: May 17th, 2021 DOI: https://doi.org/10.21203/rs.3.rs-523388/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Non-Transgenic Functional Rescue of Neuropeptides Elizabeth M. DiLoreto1†, Douglas K. Reilly1,2†, Jagan Srinivasan1,3* 1 Department of Biology and Biotechnology, Worcester Polytechnic Institute, Worcester, MA, USA 2 Current Position: Department of Biology, Tufts University, Medford, MA, USA 3 Neuroscience Program, Worcester Polytechnic Institute, Worcester, MA, USA † These authors contributed equally to this work. * Corresponding author. email: jsrinivasan@wpi.edu * Corresponding author. email: jsrinivasan@wpi.edu Non-Transgenic Functional Rescue of Neuropeptides Elizabeth M. DiLoreto1†, Douglas K. Reilly1,2†, Jagan Srinivasan1,3* 1 1 Introduction Introduction Abstract Animals constantly respond to changes in their environment and internal states via neuromodulation. Neuropeptide genes modulate neural circuits by encoding either multiple copies of the same neuropeptide or different neuropeptides. This architectural complexity makes it difficult to determine the function of discrete and active neuropeptides. Here, we present a novel genetic tool that facilitates functional analysis of individual peptides. We engineered Escherichia coli bacteria to express active peptides and fed loss-of-function Caenorhabditis elegans to rescue gene activity. Using this approach, we rescued the activity of different neuropeptide genes with varying lengths and functions: trh-1, ins-6, and pdf-1. While some peptides are functionally redundant, others exhibited unique and previously uncharacterized functions. The mechanism of peptide uptake is reminiscent of RNA interference, suggesting convergent mechanisms of gene regulation in organisms. Our rescue-by-feeding paradigm provides a high-throughput screening strategy to elucidate the functional landscape of neuropeptide genes regulating different behavioral and physiological processes. 2 2 Neuropeptide Signaling is Complex When presented with a host of environmental cues, organisms’ sense, interpret, and enact appropriate responses to stimuli 1,2. The interpretation and integration of stimuli is a dynamic process that requires neural circuits to be flexible to elicit the proper behavior. For example, a single stimulus can drive multiple reactions depending on internal or environmental states of the organism 3. In the roundworm Caenorhabditis elegans, when presented with the same small molecular cue (ascr#3), the sexes respond differently: males are attracted while hermaphrodites avoid the cue 4-6. Indole elicits different reactions depending on the amount present in the environment. At low concentrations, indole is attractive with a pleasant, floral aroma, though at high concentrations, it is repulsive, smelling pungent, reminiscent of feces or rot 7,8. To modulate competing behaviors in response to the same cue, for example, different neurons can be involved at the circuit level or changes can occur at the synaptic level. A more dynamic approach for modulating neural responses is peptidergic signaling. Neuropeptides are short amino acid chains that serve to regulate neural circuits, while also functioning as neurotransmitters and neurohormones 9,10. Neuropeptides signal on longer time scales than, though often in concert with neurotransmitters to regulate synaptic activity, typically through G-protein coupled receptor signaling cascades 10,11. Multiple modulators allow neurons to serve unique functions within discrete neural circuits 12. Neuropeptides serve a broad array of functions across the animal kingdom. Oxytocin-like and vasopressin-like peptides are neurohormones that regulate social attachment, lactation, and blood pressure by contracting muscles in mammals, dating back 600-700 million years 13-18. However, in the Echinoderm sea star, Asterias rubens, vasopressin-like and oxytocin-like neuropeptide 3 ortholog asterotocin serves instead to in relax muscles in the cardiac stomach during fictive feeding 18. The C. elegans vasopressin ortholog, nematocin, interacts with serotonin and dopamine signaling to modulate gustatory associative memory and male mating behaviors 13,19. C. elegans are a microscopic nematode that displays robust behaviors driven by just over 300 neurons 20,21. The C. elegans genome encodes three classes of neuropeptides: FMRFamide-like peptides (FLP), insulin-like peptides (INS), and non-FLP/insulin neuropeptide-like peptides (NLP) 22-24, encoding over 300 individual neuropeptides through 131 genes that modulate the functional connectome 9,25,26. The complexity of the neuropeptide genome, combined with extra- synaptic neuropeptides signaling makes elucidating the role of individual peptides difficult, as canonical studies often rely on null mutations and transgenic rescues 27-30. Neuropeptide Signaling is Complex As such, these studies are often incomplete, as full gene rescue restores complete preproproteins and makes discrimination of discrete peptide function impossible 9,31. Here we present a strategy that rescues neuropeptides synthesized endogenously within the worm genome via engineered bacteria expressing individual peptides. Development of Rescue-by-Feeding Paradigm Current methods of genetic rescue in Caenorhabditis elegans are not ideal for understanding the function of individual peptides; whether by cost inhibition or equipment limitations 32,33 or due to whole gene rescue not being ideal for studying discrete peptides 34-37. Feeding of peptides via E. coli has proven successful in manipulating C. elegans biological function. Feeding the scorpion venom protein, mBmKTX, modulates lifespan and egg laying behaviors 38. Neuropeptide rescue-by feeding, outlined here, expands on RNA interference (RNAi) feeding paradigms, which have been successfully used to test gene function 39,40, wherein a plasmid encoding the RNA of interest is driven by isopropyl-β-D-thiogalactoside (IPTG) -induction. 4 Wherein RNAi employs paired T7 promoters facing one another to produce double-stranded RNA, our protocol uses one T7 promoter to generate mRNA encoding the peptide of interest. The use of Gateway Cloning to develop expression vectors allows for the development of high-throughput experiments targeting individual peptides, or combinations thereof 41,42 (Figure 1a). Additionally, this technique is readily accessible compared to traditional transgenic rescue approaches. Wherein RNAi employs paired T7 promoters facing one another to produce double-stranded RNA, our protocol uses one T7 promoter to generate mRNA encoding the peptide of interest. The use of Gateway Cloning to develop expression vectors allows for the development of high-throughput experiments targeting individual peptides, or combinations thereof 41,42 (Figure 1a). Additionally, this technique is readily accessible compared to traditional transgenic rescue approaches. The paradigm rescues individual, processed neuropeptides, leveraging the genetic amenability of the C. elegans food source, Escherichia coli, to circumvent the need for transgenic development and enable high-throughput rescue and elucidation of individual neuropeptide function. We demonstrate the application of this paradigm by rescuing behaviors driven by neuropeptides synthesized from trh-1, ins-6, and pdf-1 (Figure 1b). Results and Discussion Functional Redundancy of Thyrotropin-Releasing Hormone (TRH)-like Peptides in C. elegans Functional Redundancy of Thyrotropin-Releasing Hormone (TRH)-like Peptides in C. elegans Recent work has revealed that C. elegans express homologs of mammalian thyrotropin- releasing hormone (TRH) 43. Expression of the nematode gene, trh-1, in the pharyngeal motor neurons M4 and M5 results in the production of a TRH-like neuropeptide precursor that is processed into two matured peptides: TRH-1A (GRELF-NH2), and TRH-1B (ANELF-NH2) 31,43. Like FLP and other NLP peptides, these peptides are also flanked by di/tribasic residues, allowing them to be processed by EGL-3 44,45. In mammals, TRH is essential for proper metabolism and growth 46, and can even induce metamorphosis in certain amphibians 47. The initial characterization of trh-1 and the cognate receptor gene, trhr-1, revealed a similar role within the C. elegans nervous system 43. Animals 5 expressing a trh-1 pro-peptide that is truncated prior to peptide translation due to an 8-bp indel- frameshift are significantly shorter and thinner than their wild-type counterparts, resulting in a relative body volume defect (Figure 1b, Size Comparisons), 48-hours after larval stage 1 (L1) arrest (Mann-Whitney test, p = 0.0012) (Figure 2a) 43. In-vitro studies involving biochemical activation of the TRHR-1 receptor by either peptide (TRH-1A or TRH-1B) suggests functional redundancy of these peptides in rescuing body volume defect in trh-1 animals 43. We cloned both TRH-1 peptides; TRH-1A and TRH-1B in our expression vector as described in Figure 1. trh-1 loss-of-function (lof) mutant animals fed scramble peptide (SCRAMBLE, NSKLHRGGGRSRTSGSTGSMASHARGSPGLQ-NH2) expressed in E. coli DH5α cells experience a significant increase in their body volume compared to wild-type animals (Kruskal- Wallis followed by Dunn’s multiple comparison test, p = 0.0015) (Figure 2b). However, feeding trh-1 lof animals with E. coli bacteria expressing either TRH-1A, or TRH-1B (trh-1 lof versus TRH-1A or TRH-1B fed, p < 0.0001) or a combination of both TRH-1A and TRH-1B peptides resulted in a complete restoration of wild-type body volume (trh-1 lof versus TRH-1A+B fed, p = 0.0019; wild-type versus TRH-1A+B fed, p > 0.9999) (Figure 2b, Supp. Figure 1a-b, c-h for length, width and area). This suggests that feeding bacteria expressing TRH-1A or TRH-1B, or a combination of the two peptides, to trh-1 mutant animals results in rescue of body volume, suggesting that they are functionally redundant. While transgenic C. elegans expressing the full- length trh-1 gene under its endogenous promoter restores wild-type body volume 43, the role of individual peptides (TRH-1A and TRH-1B) in regulating this phenotype was not addressed. elegans Our rescue-by-feeding strategy offers an easy, high-throughput in vivo biological confirmation of function. 6 6 An interesting observation we noticed is that the quality of food determines the body morphology phenotype in trh-1 mutant worms (Figure 2a, b). trh-1 mutant worms when fed on the standard food source, E.coli OP50, displayed reduced body volume as previously published (Figure 2a) 43. However, trh-1 lof worms reared on E. coli DH5a cells exhibited increased body volume compared to wildtype animals raised under similar conditions (Figure 2b). In addition, we observed that other body morphology characteristics such as relative body length (Supp. Figure 1c, d), width (Supp. Figure 1e, f), and area (Supp. Figure 1g, h) are different in worms fed with E. coli DH5a compared to E. coli OP50 fed animals. E. coli OP50 strain, an uracil auxotroph is the most commonly used laboratory bacterial food source as it grows in thin lawns which allow easier visualization of worms 48. However, studies have shown that C. elegans prefer other more nutritious bacteria such as HB101 or Comamonas sp. for its nutrition 49. We propose that the reduced body volume defect in trh-1 lof worms reared on E. coli OP50 could be a result of an inefficiency of nutrient absorption due to a difference in the nutrient composition of the two E. coli strains DH5a and OP50. Our studies corroborate previous literature, wherein trh-1 mutant worms reared on E. coli HB101 exhibit no defect in relative body volume in compared to E. coli OP50 fed animals 43. ng of INS-6 Peptides Rescues Chemotaxis Defects Exhibited by ins-6 Mutants Insulin and insulin-like peptides serve signaling functions in Drosophila and C. elegans homologous to the human insulin-like growth factor (IGF), which regulates FOXO activity 50. In C. elegans, ins-6 encodes only one processed INS-6 peptide (VPAPGETRACGRKLISLVMAVCGDLCNPQEGKDIATECCGNQCSDDYIRSACCP-NH2) 51,52, though the gene was originally postulated to encode two putative proteins 9,23 . ins-6 functions 7 in dauer formation 51,53 and sensory modulation of large fluctuations in salt concentration, with loss of ins-6 causing dysfunction of NaCl attraction (Figure 1b, Chemotaxis) 54. While wild-type worms were attracted to high concentrations of salt (750 mM), there was a significant decrease in attraction in ins-6 mutant animals, as measured by a Chemotaxis Index (CI) (Paired t-test with samples of equal variance, p = 0.0248) (Figure 3a) 54. Salt attraction was partially rescued with genetic reintroduction of ins-6 under its endogenous promotor (Figure 3a). Similarly, the CI of ins-6 lof animals fed scramble peptide was significantly different from the wild-type animals fed scramble (Figure 3b). Feeding of E. coli expressing INS-6 peptide to ins-6 lof animals resulted in a complete restoration of attraction to high salt concentration (ANOVA, followed by Bonferroni’s Correction, ins-6 lof fed scramble versus INS-6 fed, p = 0.0101) (Figure 3b). Overexpression of INS-6 did not increase chemotaxis towards high salt, as wild-type animals fed INS-6, along with a full ins-6 genetic rescue, exhibited slight defects in CI towards high salt (ANOVA, followed by Bonferroni’s Correction, wild-type versus fed, p = 0.08071; wild-type versus transgenic overexpression, p = 0.2403) (Supp. Figure 2b). Partial genetic rescue of ins-6 limited to the ASI sensory neuron (ins-6;ASI::ins-6) was sufficient to rescue neurophysiological function of AWCON NaCl sensation 54, though it was not sufficient for rescuing behavioral attraction to salt (ANOVA, followed by Bonferroni’s Correction, p = 0.2136) (Supp. Figure 2b). While our rescue-by-feeding paradigm was able to rescue the behavioral phenotypes of aberrant ins-6 signaling, the ability to combine this method with calcium imaging techniques is still in development. 8 8 Differential Functional Activity of Peptides Encoded by Pigment Dispersing Factor (pdf)-1 Wild-type males display a characteristic exploratory behavior when left on a lawn of food, as well-fed males leave food in search of mates 55,56. The pigment dispersing factor pdf-1 neuropeptide plays a significant role in male mate-searching behavior, balancing the neural circuits controlling two predominant male interests: finding food and finding mates 55,57. Like trh-1, the pdf-1 precursor encodes two neuropeptides: PDF-1A (SNAELINGLIGMDLGKLSAVG-NH2) and PDF-1B (SNAELINGLLSMNLNKLSGAG-NH2) 58,59. We quantified the behavioral activity of pdf-1 lof males using a food-leaving behavior as previously described 55,60. Individuals were placed on a small food spot, and track patterns were scored at three different time points (2, 6, 24 hrs) (Figure 4a). ‘‘Never left food’’ indicate the absence of tracks outside the food spot. ‘‘Minor excursion’’ indicates that tracks were observed not beyond 1 cm from the food. ‘‘Major excursion’’ indicates the presence of tracks past the 1 cm boundary (Figure 1b, Excursion Assay). We additionally quantified this food-leaving behavior as a measure of mate-searching behavior. In this behavior well-fed males left food in search of mates and the data is represented as a Probability of Leaving (PL) (Supp. Figure 3a, b) 55,56. pdf-1 lof males did not leave food as readily as wild-type worms suggesting that these worms do not display exploratory behavior, as previously described (Paired t-test with samples of equal variance, p < 0.0001) (Supp. Figure 3b, c) 55. When fed either PDF-1 peptide (or a 1:1 combination of both), pdf-1 lof males had a higher PL compared to scramble fed pdf-1 lof males (ANOVA, followed by Bonferroni’s Correction, p < 0.0001) (Figure 4b). However, PDF-1B and a 1:1 ratio of both peptides resulted in a significantly different PL from pdf-1 males fed PDF-1A alone (ANOVA, followed by Bonferroni’s Correction, p < 0.0001). Rescue-by-feeding resulted in 9 9 a partial rescue: while the PL of peptide-fed males were significantly higher than pdf-1 lof males fed scramble, they were still significantly lower than wild-type males fed scramble peptide (ANOVA, followed by Bonferroni’s Correction, p < 0.0001) (Figure 4b). Future studies employing this paradigm can now focus on teasing apart the difference in PDF-1A and PDF-1B function in mate-finding behavior, as we have successfully employed in dissecting FLP-3 peptide functions 61. We present here a method of neuropeptide rescue that exploits bacterial expression to deliver individual peptides to rescue behavioral and growth-related phenotypes. Differential Functional Activity of Peptides Encoded by Pigment Dispersing Factor (pdf)-1 We show that while some peptides are redundant in function (Figure 2), others rescue phenotypes driven by large peptides (Figure 3), while novel functions are displayed by other peptides over long timescales (Figure 4). Our novel technology to rescue peptide function is advantageous over transgenic studies, as it allows for functional characterization of individual peptides. This genetic tool is built off the principles of RNAi feeding techniques to supply worms with the peptide of interest through their food source as previously shown for the scorpion venom peptide mBmKTX to alter lifespan and egg-laying behavior in C. elegans 38. More recently, we have improved upon this paradigm to characterize the FMRFamide-like peptide gene, flp-3 61. This gene encodes ten different peptides and we leveraged the rescue-by-feeding technology to elucidate that only two of the ten peptides encoded by the precursor are active in controlling the behavioral response of males to a mating pheromone 61. These studies support the assertion that feeding peptides to C. elegans via their E. coli food source is sufficient to rescue mutant phenotypes. Based on our results, we propose that the neuropeptide rescue-by-feeding strategy delivers mRNA ready for translation by the C. elegans cellular machinery, rather than supply C. elegans with fully translated and processed peptides. The plasma membrane of a cell is an intricate complex 10 10 of multiple lipid and protein molecules. Small molecules with moderate polarity diffuse through the cell membrane passively, but most metabolites and short peptides require specialized membrane transporters for translocation 62. Given the large number of neuropeptides encoded in the genome of C. elegans 9, having specialized transporters for peptide transport is not feasible 63. The strongest piece of evidence for this statement lies in that the processed INS-6 peptide is 54 amino acids in length: the C. elegans intestine expresses peptide transporters that only uptake smaller, inactive di- and tri-amino acid peptide chains 63. Thus, rescue of chemotaxis behavior by INS-6 peptide feeding (Figure 3b) suggests that the mechanism of rescue may be similar to double-stranded RNA (dsRNA) uptake, rather than peptidergic transport across the intestinal membrane 64,65. Furthermore, previous studies have shown that dsRNA exhibit transgenerational inheritance 66, despite degradation rates,. Our rescue of the exploratory behavior of pdf-1 lof males even in the 24-hour timescale (Figure 4) suggest that our feeding paradigm exploits similar mechanisms, allowing peptide-encoding mRNAs to remain present throughout the assay. Differential Functional Activity of Peptides Encoded by Pigment Dispersing Factor (pdf)-1 We propose that if peptides were taken up, their degradation rates would likely impede rescue efficiency at later timepoints unlike what we observe in our experiments suggesting that the mechanism is similar to double-stranded RNA (dsRNA) feeding, with mRNA uptake driving rescue rather than peptide uptake. Feeding E. coli DH5α expressing peptides results in altered phenotypes compared to E. coli OP50 (Figure 2, 3) and (Reilly et al., in review) 61. Future studies will focus towards optimizing experimental protocols for feeding. Two areas of optimization we envision involve designing an expression vector strategy that can multiplex multiple neuropeptide genes and finding the “best” food source for the rescue experiments. Given the differences in nutrient composition of the 11 11 different E. coli strains and worms preferring more nutritious bacteria 49, expressing peptides in HB101 or HB115 strains offer viable alternatives. Understanding neuropeptide function is essential both from a perspective of regulation of neuronal (synaptic and non-synaptic) channels of communication, and form a global view of general neuronal functional assignments throughout the brain. Revealing how a particular neuropeptide acts both at the cellular and subcellular peptide receptor level is critical link in understanding the role of neuromodulation of circuits. An enhanced experimental pipeline to investigate peptide function will enable progress toward answering how neural circuit activity within the network in its different states and identification are modulated by neuropeptides, resulting in flexible decision-making during behaviors. 12 C. elegans strains The C. elegans strains N2 and CB4088 (him-5(e1490)) were obtained from the Caenorhabditis Genetics Center. The LSC1118 (trh-1(lst1118)) strain was generously provided by Isabel Beets at KU Leuven for the body morphology tests. The strains used in the Chemotaxis Assay were gifted by Prof. Shreekanth Chalasani, Salk Insitutute (IV302 (ins-6(tm2416); kyEx2595) at the Salk Institute, and Yun Zhang (ZC239 (ins-6(tm2416)II; yxEx175[Pins-6::ins-6; Punc-122::gfp]) at Harvard University. The UR954 (pdf-1(tm1886);him-5(e1490)) strain was provided by Douglas Portman at the University of Rochester. Peptide plasmid design and generation Peptide plasmid design and generation DNA sequences encoding individual peptides were identified via www.wormbase.org. Sequences were flanked with the endogenous cleavage sites for the EGL-3 processing enzyme, which cleaves dibasic resides. Sequences encoding MRFGKR and KRK-STOP codons were therefore placed prior to, and following the peptide codon sequences, respectively. Finally, Gateway Cloning sites attB1 and attB2 sites were attached to the ends of the sequences. These final sequences (comprised of attB1::MRFGKR::peptide::KRK-STOP::attB2) were ordered from IDT (Integrated DNA Technologies) using their DNA Oligo and Ultramer DNA Oligo services, depending on the size of the oligo ordered. Both forward and reverse sequences were ordered (Supp. Table 1a). Lyophilized oligos were prepared following IDT Annealing Oligonucleotides Protocol (https://www.idtdna.com/pages/education/decoded/article/annealing-oligonucleotides). In short, they were resuspended in Duplex Buffer (100 mM Potassium Acetate; 30 mM HEPES, pH 7.5; available from IDT), preheated to 94 °C to a final concentration of 40 μM. Complimentary oligo 13 sequences were then mixed in equimolar ratios, and placed in a thermocycler at 94 °C for 2 minutes, prior to a stepwise cooling to room temperature. Annealed oligos were used to perform a BP reaction with pDONR p1-2 donor vector to generate pENTRY clones (Gateway Cloning, ThermoFisher). Entry clones were then recombined with pDEST-527 (a gift from Dominic Esposito (Addgene plasmid # 11518)) in LR reactions generating expression clones. The scramble control was generated in an identical manner, with the sequence between the cleavage sites being amplified from pL4440 (provided by Victor Ambros, University of Massachusetts Medical School, MA) (Supp. Table 1b). Purified plasmids were stored at -20 °C or -80 °C for long term storage. Peptide Plate Preparation The expression clone for both the peptide of interest and the control was transformed into competent DH5α cells (NEB® [New England Biolabs] 5-alpha Competent E. coli (High Efficiency) Cat# C2987I). Peptide-expressing cultures on LB agar plates (10 g/L NaCl, 10 g/L Tryptone, 5 g/L Yeast Extract, 5 g/L Agar) containing 100 μg/μL ampicillin (AMP), were stored at 4 °C for up to 2 months. From this plate, single colonies were selected for growth in 5 mL of LB media containing ampicillin at 37 °C for 16 hours. The optical density of the grown cultures (OD600) was adjusted to 1.0. 75 μL of 1.0 OD600 peptide-expressing culture was plated onto 60 mm NGM agar plates (50 mM NaCl. 25 mM KPO4 (pH 6.0), 1mM MgSO4, 1mM CaCl2, 5 µg/L cholesterol, 17 g/L agar, 2.5 g/L peptone) containing 100 μg/μL AMP and 1 mM isopropyl-β-D-thiogalactoside (IPTG) at room temperature for at least 8 hours prior to use, for no longer than 1 week. 14 14 All animals were maintained on bacterial lawns of OP50 E. coli, at 20 °C, on NGM agar plates until the start of experiments. Mutant or control animals were transferred onto lawns of DH5α E. coli expressing either scramble peptide or the rescue peptide-of-interest on NGM plates containing 100 μg/μL AMP and 1 mM IPTG. Animals were reared on the peptide-expressing lawns for at least 48 hours at 20 °C before assaying for rescue of mutant phenotype at the appropriate developmental stage. See each section of the behavioral assay methods for the exact peptide feeding parameters. Size Comparison (trh-1) The body morphology different of trh-1 mutants was compared to wild-type worms by body volume measurements. Wild-type (N2) and trh-1 animals were synchronized by embryonic starvation following alkaline hypochlorite protocols 67. Ten gravid hermaphrodites from OP50 E. coli plates were picked into 30 μL drops of alkaline hypochlorite solution (20% sodium hypochlorite, 500 mM KOH). Two drops were placed on either side of an unseeded NGM plate, resulting in 20 animals per plate. Plates were then incubated for 24 hours at 20 °C before L1 larval animals washed with 1 mL M9 (22 mM KH2PO4, 42.25 mM Na2HPO4, 85.5 mM NaCl, 1 mM MgSO4) per unseeded NGM plate. Allow L1 worms to settle into pellet before removing M9 supernatant. Repeat washing step 2 more times. Plate worm pellet onto NGM with 100 μg/μL AMP and 1 mM IPTG plates containing 75 µL peptide lawns. After at 48 hours at 20 °C 43, animals were recorded using a Basler acA2500-14uM camera using WormLab software (WormLab 4.1; MBF Bioscience, 2017) for 65 seconds. Worm length, width, and area were calculated within the WormLab software (Supp. Figure 1 c-h). As the WormLab worm width metric is an average of cross-sections throughout the length of each worm, 15 this was used to calculate the volume of a worm as a cylinder, with the Volume = π * (0.5 * width)2 * length (Figure 2a, b, Supp. Figure 1a, b). Chemotaxis Assay (ins-6) A salt chemotaxis assay was performed as previously described in 68,69, to test the functionality of ins-6 after rescue-by-feeding. Wild-type (N2) and ins-6 worms were fed either scramble or INS-6 peptide passing 4-6 L4 larval onto an NGM plate containing 100 μg/μL AMP and 1 mM IPTG with 75 µL of peptide OD600 = 1.0. These worms were grown at 20 °C for about 4 days until the progeny of the initial L4 worms were young adults. The day prior to testing, a thin, 10 mL layer of agar was added to 10 cm petri dishes to generate a 10 cm Chemotaxis Plates (5 mM KPO4 (pH 6.0), 1 µM MgSO4, 1 µM CaCl2, 20 g/L agar, and 8 g/L Difco Nutrient Broth). To prepare the “high salt” plates, 750 mM NaCl was added prior to plate pouring. The high salt plate were stored at 4 °C for up to one month, while the Chemotaxis Plates were made one day prior to testing. To create the high and low salt plugs, the back end of a Pasteur pipette was used to punch a 5 mm plug out of each plate. These 2 plugs were placed on opposite edges of the plate. A 10 mm radius was be drawn around the location of the plugs (Figure 1 for schematic). Plates were stored, lightly covered, overnight at room temperature (~20 °C, <40% humidity). The day of the assay, young adult worms, either control worms fed OP50 E. coli or peptide- fed worms, were washed off peptide plates with M9. Worms were allowed to settle by gravity for 4 minutes before removing the supernatant, washing the worms. Worms were then washed with Chemotaxis Buffer (5 mM KPO4 (pH 6.0), 1 mM MgSO4, 1 mM CaCl2) three times. To prepare the Chemotaxis plates for the assay, high and low salt plugs were removed with forceps, and the plates spotted with 1 µL of 0.5 M sodium azide (NaN3). Approximately 30 µL of 16 worms in Chemotaxis Buffer were spotted onto the edge of the agar, between the location of the high and low salt gradient (Supp. Figure 2). Worms were left to chemotax for 1 hour before scoring the number of worms within the 10 mm radii of high and low salt areas. Excursion Assay (pdf-1) Excursion Assay (pdf-1) The mate searching behavior of pdf-1 mutants was quantified in a food leaving assay. him-5 animals were used as wild-type control to ensure the presence of males. him-5 and pdf-1 animals were fed OP50, scramble peptide, PDF-1A, PDF-1B, or a 1:1 mixture of PDF-1A and PDF-1B cultures. Four to six larval L4 hermaphrodites were passed onto an NGM plate containing 100 μg/μL AMP and 1 mM IPTG with 75 µL of peptide. These worms were grown at 20 °C for about 4 days until the male progeny of the initial L4 worms were young adults. The day prior to the assay, young adult male pdf-1 or him-5 worms were singled onto 60 mm plates containing lawns of either OP50 (NGM plates) or appropriate peptide cultures (NGM containing 100 μg/μL AMP and 1mM IPTG). To determine the mate searching effects of pdf-1, we performed a food-leaving assay as described previously 55,56,60, with modifications. In this assay, individuals were placed on a small food spot, and track patterns were scored at the indicated times. Assay plates were prepared one day prior to assaying on 100 mm plates with 10 mL of Leaving Assay media (25 mM KPO4 (pH 6.0), 1 mM MgSO4, 1mM CaCl2, 50 mM NaCl, 2.5 g/L Peptone, 17 g/L Agar). On center of plate, 7 µL of OP50 was added. Plates were lightly covered and stored at room temperature overnight (~20⁰C, <40% humidity). On the day of the assay, a single male was plated on the assay plate and placed in a dark, 20°C incubator. At 2, 6, and 24 hours after the worms are plated, plates were scored for male worm tracks. Scored were separated into binned ranges 60. “Never left food” indicates the absence of 17 17 tracks outside the food spot. “Minor excursion” indicates that tracks were observed not beyond 10 mm from the food. “Major excursion” indicates the presence of tracks past the 1 cm boundary. We quantified the food leaving behavior at three different time points (2, 6, and 24 hours) 55. (Figure 4a, Supp. Figure 3b) 55. We additionally quantified the mate searching defect of pdf-1 as a Probability of Leaving per hour (PL) (Figure 4b, Supp Figure. 3a, c) 56. Excursion Assay (pdf-1) In this method, we reduce the analysis of the leaving behavior to a single value, by scoring the worms leaving the food lawn as Leavers (traveling beyond 35 mm from the food source) or Non-Leavers (did not travel further than 35 mm from center of food). # #$%&' () (+ ',-./# $0#$%&' () -$# ',-. .$.,- # #$%&' . At least 10 Chemotaxis Index (CI) was calculated as 𝐶𝐼= # #$%&' () (+ ',-./# $0#$%&' () -$# ',-. .$.,- # #$%&' . At least 10 plates were assayed in each condition: OP50 control, scramble fed, and INS-6 fed. Plates were excluded from final calculations if (1) the total number of worms on the assay plate was <20, or (2) if the plate CI was greater than two standard deviations from the average CI. Conditions were compared using Two-tailed t-tests with samples of equal variable to compare between two conditions with small samples or ANOVA followed by Bonferroni’s multiple comparison for multiple condition comparisons. Chemotaxis Index (CI) was calculated as 𝐶𝐼= # #$%&' () (+ ',-./# $0#$%&' () -$# ',-. .$.,- # #$%&' . At least 10 plates were assayed in each condition: OP50 control, scramble fed, and INS-6 fed. Plates were excluded from final calculations if (1) the total number of worms on the assay plate was <20, or (2) if the plate CI was greater than two standard deviations from the average CI. Conditions were compared using Two-tailed t-tests with samples of equal variable to compare between two conditions with small samples or ANOVA followed by Bonferroni’s multiple comparison for multiple condition comparisons. Excursion Assay The assay was performed across at least two days per condition, with at least n = 50, wherein each assay plate with one male is equal to n = 1. In all experiments, the investigators were blinded to genotype. Worm in the >35 mm from the center of food bin were scored as “leavers” 56. The probability of leaving per hour (Probability of Leaving (PL)) was calculated using an R script developed in 55, first cited in 56, with assistance by (Personal Communications to Barrios, 2020). PL was calculated by the “hazard obtained by fitting an exponential parametric survival model to the censored data using maximum likelihood” (Supp. Figure 3a) 56. The PLh-1 values for all pdf-1 animals fed scramble or peptide rescue were compared to him-5 worms fed scramble using an ANOVA followed by Bonferroni’s multiple comparison (Figure 4b, Supp. Figure 3c). Data Availability The raw data for all figures is available with this manuscript. Additionally, a summary containing the average, n, standard error measure, and associated statistical test is also available. Size Comparisons Two to four plates were recorded per day for at least three days to generate data sets. Control plates of N2 and trh-1 animals raised on the scramble peptide were grown alongside every rescue feeding. Each “worm track” was considered an n = 1. Tracks were excluded if (1) worm tracks less than 10 seconds, or (2) worms were further than two standard deviations from the mean metric. Following tests for normality, comparisons were made using either Mann-Whitney tests for direct comparisons between N2 and trh-1 animals reared on OP50, or Kruskal-Wallis non-parametric ANOVAs and Dunn’s multiple comparisons tests for animals raised on peptide feeding strains. Comparisons were made for metrics: length (Supp. Figure 1c, d), width (Supp. Figure 1e, f), area (Supp. Figure 1g, h), and volume (Figure 2a, b, and Supp. Figure 1a, b). Chemotaxis Assay Young adult hermaphrodites are tested in the Chemotaxis Assay with no more than three replicates per day, over a minimum of three days, with 100-200 worms/assay plate. The 18 Author’s Contribution EMD, DKR and JS designed all experiments, EMD and DKR performed all experiments and statistical analysis. EMD co-wrote the paper with DKR and JS. Acknowledgements Acknowledgements 19 19 We would like to thank Jeffrey Marsh for assistance in running the Chemotaxis Assays. We would like to thank Dr. Arantza Barrios for help in executing the leaving assay analysis in R. We extend our thanks to Dr. Shreekanth Chalasani and Dr. Douglas Portman for their insights and updates to method for testing the phenotypes related to ins-6 and pdf-1, respectively. Thank you to Dr. Isabel Beets for her insights into the trh-1 analysis. This work was funded by NIH R01DC016058 (JS). Ethics Declaration The authors declare no competing financial or non-financial interests as defined by Nature Research. Research. 20 References 1 Ghosh, D. D., Nitabach, M. N., Zhang, Y. & Harris, G. Multisensory integration in C. elegans. Curr Opin Neurobiol 43, 110-118, doi:10.1016/j.conb.2017.01.005 (2017). 1 Ghosh, D. D., Nitabach, M. N., Zhang, Y. & Harris, G. Multisensory integration in C. elegans. Curr Opin Neurobiol 43, 110-118, doi:10.1016/j.conb.2017.01.005 (2017). 2 Mesulam, M. M. From sensation to cognition. Brain 121 ( Pt 6), 1013-1052, doi:10.1093/brain/121.6.1013 (1998). 3 Gonzalez-Suarez, A. D. & Nitabach, M. N. Peptide-Mediated Neurotransmission Takes Center Stage. Trends Neurosci 41, 325-327, doi:10.1016/j.tins.2018.03.013 (2018). 4 Srinivasan, J. et al. A blend of small molecules regulates both mating and development in Caenorhabditis elegans. Nature 454, 1115-1118, doi:10.1038/nature07168 (2008). 5 Jang, H. et al. Neuromodulatory state and sex specify alternative behaviors through antagonistic synaptic pathways in C. elegans. Neuron 75, 585-592, doi:10.1016/j.neuron.2012.06.034 (2012). 6 Macosko, E. Z. et al. A hub-and-spoke circuit drives pheromone attraction and social behaviour in C. elegans. Nature 458, 1171-1175, doi:10.1038/nature07886 (2009). 7 Chacko, R. et al. Data based predictive models for odor perception. Scientific Reports 10, 17136, doi:10.1038/s41598-020-73978-1 (2020). Malnic, B., Hirono, J., Sato, T. & Buck, L. B. Combinatorial receptor codes for odors. Cell 9 23, doi:10.1016/s0092-8674(00)80581-4 (1999). , ( ) ( ) 9 Li, C. & Kim, K. Neuropeptides. WormBook, 1-36, doi:10.1895/wormbook.1.142.1 (2008). 10 Dale Purves, G. J. A., David Fitzpatrick, William C Hall, Anthony-Samuel LaMantia, Richard D Mooney, Michael L Platt, Leonard E White. Neuroscience. 6 edn, (Oxford University Press, 2018). 11 Frooninckx, L. et al. Neuropeptide GPCRs in C. elegans. Frontiers in Endocrinology 3, doi:10.3389/fendo.2012.00167 (2012). 12 Nusbaum, M. P., Blitz, D. M. & Marder, E. Functional consequences of neuropeptide and small- molecule co-transmission. Nat Rev Neurosci 18, 389-403, doi:10.1038/nrn.2017.56 (2017). 13 Beets, I. et al. Vasopressin/oxytocin-related signaling regulates gustatory associative learning in C. elegans. Science 338, 543-545, doi:10.1126/science.1226860 (2012). 14 Gruber, C. W. Physiology of invertebrate oxytocin and vasopressin neuropeptides. Experimental Physiology 99, 55-61, doi:https://doi.org/10.1113/expphysiol.2013.072561 (2014). 15 Insel, T. R. & Young, L. J. The neurobiology of attachment. Nature Reviews Neuroscience 2, 129- 136, doi:10.1038/35053579 (2001). 16 Willets, J. M. et al. Regulation of oxytocin receptor responsiveness by G protein-coupled receptor kinase 6 in human myometrial smooth muscle. Mol Endocrinol 23, 1272-1280, doi:10.1210/me.2009-0047 (2009). 17 Lukas, M. et al. The Neuropeptide Oxytocin Facilitates Pro-Social Behavior and Prevents Social Avoidance in Rats and Mice. Ethics Declaration Neuropsychopharmacology 36, 2159-2168, doi:10.1038/npp.2011.95 (2011). 18 Odekunle, E. A. et al. Ancient role of vasopressin/oxytocin-type neuropeptides as regulators of feeding revealed in an echinoderm. BMC Biology 17, 60, doi:10.1186/s12915-019-0680-2 (2019). i l i / i l d id h i l i d i 18 Odekunle, E. A. et al. Ancient role of vasopressin/oxytocin-type neuropeptides as regulators of feeding revealed in an echinoderm. BMC Biology 17, 60, doi:10.1186/s12915-019-0680-2 (2019). 19 Garrison, J. L. et al. Oxytocin/vasopressin-related peptides have an ancient role in reproductive behavior. Science 338, 540-543, doi:10.1126/science.1226201 (2012). g gy , , ( ) 19 Garrison, J. L. et al. Oxytocin/vasopressin-related peptides have an ancient role in reproductive behavior. Science 338, 540-543, doi:10.1126/science.1226201 (2012). 20 White, J. G., Southgate, E., Thomson, J. N. & Brenner, S. The structure of the nervous system of the nematode Caenorhabditis elegans. Philos Trans R Soc Lond B Biol Sci 314, 1-340 (1986). 21 Sammut, M. et al. Glia-derived neurons are required for sex-specific learning in C. elegans. Nature 526, 385-390, doi:10.1038/nature15700 (2015). 22 Li, C., Kim, K. & Nelson, L. S. FMRFamide-related neuropeptide gene family in Caenorhabditis elegans. Brain Res 848, 26-34, doi:10.1016/s0006-8993(99)01972-1 (1999). 23 Pierce, S. B. et al. Regulation of DAF-2 receptor signaling by human insulin and ins-1, a member of the unusually large and diverse C. elegans insulin gene family. Genes Dev 15, 672-686, doi:10.1101/gad.867301 (2001). 21 24 Nathoo, A. N., Moeller, R. A., Westlund, B. A. & Hart, A. C. Identification of neuropeptide-like protein gene families in Caenorhabditiselegans and other species. Proc Natl Acad Sci U S A 98, 14000-14005, doi:10.1073/pnas.241231298 (2001). 24 25 Van Bael, S. et al. Mass spectrometric evidence for neuropeptide-amidating enzymes in Caenorhabditis elegans. J Biol Chem 293, 6052-6063, doi:10.1074/jbc.RA117.000731 (2018). 26 Hobert, O. in WormBook: The Online Review of C. elegans Biology (Pasadena, CA, 2005-2018). 27 Barrios, A., Ghosh, R., Fang, C., Emmons, S. W. & Barr, M. M. PDF-1 neuropeptide signaling modulates a neural circuit for mate-searching behavior in C. elegans. Nature Neuroscience 15, 1675-1684, doi:10.1038/nn.3253 (2012). 28 Hussey, R. et al. Oxygen-sensing neurons reciprocally regulate peripheral lipid metabolism via neuropeptide signaling in Caenorhabditis elegans. PLoS genetics 14, e1007305-e1007305, doi:10.1371/journal.pgen.1007305 (2018). 29 29 Nath, R. D., Chow, E. S., Wang, H., Schwarz, E. M. & Sternberg, P. W. C. elegans Stress-Induced Sleep Emerges from the Collective Action of Multiple Neuropeptides. Current biology 26, 2446- 2455, doi:10.1016/j.cub.2016.07.048 (2016). Ethics Declaration Deciphering the Role of EGL-3 for Neuropeptides Processing in Caenorhabditis elegans Using High-Resolution Quadrupole-Orbitrap Mass Spectrometry. Neurochem Res 43, 2121-2131, doi:10.1007/s11064-018-2636-2 (2018). 46 Mullur, R., Liu, Y. Y. & Brent, G. A. Thyroid hormone regulation of metabolism. Physiol Rev 94, 355-382, doi:10.1152/physrev.00030.2013 (2014). 47 Galas, L. et al. TRH acts as a multifunctional hypophysiotropic factor in vertebrates. Gen Comp Endocrinol 164, 40-50, doi:10.1016/j.ygcen.2009.05.003 (2009). 48 Zečić, A., Dhondt, I. & Braeckman, B. P. The nutritional requirements of Caenorhabditis elegans. Genes & Nutrition 14, 15, doi:10.1186/s12263-019-0637-7 (2019). 49 Shtonda, B. B. & Avery, L. Dietary choice behavior in Caenorhabditis elegans. J Exp Biol 209, 89-102, doi:10.1242/jeb.01955 (2006). 50 Chen, Z. et al. Two insulin-like peptides antagonistically regulate aversive olfactory learning in C. elegans. Neuron 77, 572-585, doi:10.1016/j.neuron.2012.11.025 (2013). 51 Hung, W. L., Wang, Y., Chitturi, J. & Zhen, M. A Caenorhabditis elegans developmental decision requires insulin signaling-mediated neuron-intestine communication. Development 141, 1767- 1779, doi:10.1242/dev.103846 (2014). 52 Hua, Q.-X. et al. A divergent INS protein in Caenorhabditis elegans structurally resembles human insulin and activates the human insulin receptor. Genes Dev 17, 826-831, doi:10.1101/gad.1058003 (2003). 53 Zheng, S. et al. A functional study of all 40 Caenorhabditis elegans insulin-like peptides. J Biol Chem 293, 16912-16922, doi:10.1074/jbc.RA118.004542 (2018). 54 Leinwand, S. G. & Chalasani, S. H. Neuropeptide signaling remodels chemosensory circuit composition in Caenorhabditis elegans. Nat Neurosci 16, 1461-1467, doi:10.1038/nn.3511 (2013). 55 Barrios, A., Ghosh, R., Fang, C., Emmons, S. W. & Barr, M. M. PDF-1 neuropeptide signaling d l t l i it f t hi b h i i C l N t N i 15 1675 1682 54 Leinwand, S. G. & Chalasani, S. H. Neuropeptide signaling remodels chemosensory circuit composition in Caenorhabditis elegans. Nat Neurosci 16, 1461-1467, doi:10.1038/nn.3511 (2013). 55 Barrios, A., Ghosh, R., Fang, C., Emmons, S. W. & Barr, M. M. PDF-1 neuropeptide signaling modulates a neural circuit for mate-searching behavior in C. elegans. Nat Neurosci 15, 1675-1682, doi:10.1038/nn.3253 (2012). 56 Lipton, J., Kleemann, G., Ghosh, R., Lints, R. & Emmons, S. W. Mate searching in Caenorhabditis elegans: a genetic model for sex drive in a simple invertebrate. The Journal of neuroscience : the official journal of the Society for Neuroscience 24, 7427-7434, doi:10.1523/JNEUROSCI.1746- 04.2004 (2004). 57 Reilly, D. K. Neuromodulation of Sex-Specific Pheromone-Mediated Behaviors PhD thesis, Worcester Polytechnic Institute, (2020). 58 Janssen, T. et al. Ethics Declaration , j ( ) 30 Nelson, M. D. et al. FRPR-4 Is a G-Protein Coupled Neuropeptide Receptor That Regulates Behavioral Quiescence and Posture in Caenorhabditis elegans. PLoS One 10, e0142938, doi:10.1371/journal.pone.0142938 (2015). 31 Van Bael, S., Edwards, S. L., Husson, S. J. & Temmerman, L. Identification of Endogenous Neuropeptides in the Nematode C. elegans Using Mass Spectrometry. Methods Mol Biol 1719, 271-291, doi:10.1007/978-1-4939-7537-2_18 (2018). _ ( ) 32 TC, E. Transformation and microinjection. . WormBook: The Online Review of C. elegans Biology (2006). 33 Rieckher, M. & Tavernarakis, N. Caenorhabditis elegans Microinjection. Bio Protoc 7, e2565, doi:10.21769/BioProtoc.2565 (2017). 34 Conte, D., Jr., MacNeil, L. T., Walhout, A. J. M. & Mello, C. C. RNA Interference in Caenorhabditis elegans. Curr Protoc Mol Biol 109, 26.23.21-26.23.30, doi:10.1002/0471142727.mb2603s109 (2015). 35 Timmons, L., Court, D. L. & Fire, A. Ingestion of bacterially expressed dsRNAs can produce specific and potent genetic interference in Caenorhabditis elegans. Gene 263, 103-112, doi:10.1016/s0378-1119(00)00579-5 (2001). 36 Timmons, L. & Fire, A. Specific interference by ingested dsRNA. Nature 395, 854, doi:10.1038/27579 (1998). 37 Fire, A. et al. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 391, 806-811, doi:10.1038/35888 (1998). 38 Xu, J. et al. Feeding recombinant E. coli with GST-mBmKTX fusion protein increases the fecundity and lifespan of Caenorhabditis elegans. Peptides 89, 1-8, doi:10.1016/j.peptides.2017.01.003 (2017). 39 Fraser, A. G. et al. Functional genomic analysis of C. elegans chromosome I by systematic RNA interference. Nature 408, 325-330, doi:10.1038/35042517 (2000). 40 Kamath, R. S., Martinez-Campos, M., Zipperlen, P., Fraser, A. G. & Ahringer, J. Effectiveness of specific RNA-mediated interference through ingested double-stranded RNA in Caenorhabditis elegans. Genome Biol 2 (2001). g ( ) 41 Reece-Hoyes, J. S. & Walhout, A. J. M. Gateway Recombinational Cloning. Cold Spring Harb Protoc 2018, doi:10.1101/pdb.top094912 (2018). 42 Petersen, L. K. & Stowers, R. S. A Gateway MultiSite recombination cloning toolkit. PLoS One 6, e24531, doi:10.1371/journal.pone.0024531 (2011). 43 Van Sinay, E. et al. Evolutionarily conserved TRH neuropeptide pathway regulates growth in Caenorhabditis elegans. Proc Natl Acad Sci U S A 114, E4065-e4074, doi:10.1073/pnas.1617392114 (2017). 22 44 44 Husson, S. J., Clynen, E., Baggerman, G., Janssen, T. & Schoofs, L. Defective processing of neuropeptide precursors in Caenorhabditis elegans lacking proprotein convertase 2 (KPC-2/EGL- 3): mutant analysis by mass spectrometry. J Neurochem 98, 1999-2012, doi:10.1111/j.1471- 4159.2006.04014.x (2006). 45 45 Salem, J. B., Nkambeu, B., Arvanitis, D. N. & Beaudry, F. ( ) 62 Yang, N. J. & Hinner, M. J. Getting across the cell membrane: an overview for small molecules, peptides, and proteins. Methods Mol Biol 1266, 29-53, doi:10.1007/978-1-4939-2272-7_3 (2015). Ethics Declaration Functional characterization of three G protein-coupled receptors for pigment dispersing factors in Caenorhabditis elegans. J Biol Chem 283, 15241-15249, doi:10.1074/jbc.M709060200 (2008). 59 59 Janssen, T. et al. Discovery and characterization of a conserved pigment dispersing factor-like neuropeptide pathway in Caenorhabditis elegans. J Neurochem 111, 228-241, doi:10.1111/j.1471- 4159.2009.06323.x (2009). 60 60 Ryan, D. A. et al. Sex, age, and hunger regulate behavioral prioritization through dynamic modulation of chemoreceptor expression. Curr Biol 24, 2509-2517, doi:10.1016/j.cub.2014.09.032 (2014). 61 61 Reilly, D. K. et al. Distinct Neuropeptide-Receptor Modules Regulate a Sex-Specific Behavioral Response to a Pheromone. bioRxiv, 2020.2012.2009.417857, doi:10.1101/2020.12.09.417857 (2020). 62 62 Yang, N. J. & Hinner, M. J. Getting across the cell membrane: an overview for small molecules, peptides, and proteins. Methods Mol Biol 1266, 29-53, doi:10.1007/978-1-4939-2272-7_3 (2015). 23 63 63 Meissner, B., Boll, M., Daniel, H. & Baumeister, R. Deletion of the intestinal peptide transporter affects insulin and TOR signaling in Caenorhabditis elegans. J Biol Chem 279, 36739-36745, doi:10.1074/jbc.M403415200 (2004). 64 64 Rao, M. & Sockanathan, S. Molecular mechanisms of RNAi: Implications for development and disease. Birth Defects Research Part C: Embryo Today: Reviews 75, 28-42, doi:https://doi.org/10.1002/bdrc.20030 (2005). 65 65 Bernstein, E., Caudy, A. A., Hammond, S. M. & Hannon, G. J. Role for a bidentate ribonuclease in the initiation step of RNA interference. Nature 409, 363-366, doi:10.1038/35053110 (2001). 66 Seydoux G & Fire A Soma-germline asymmetry in the distributions of embryonic RNAs in 65 Bernstein, E., Caudy, A. A., Hammond, S. M. & Hannon, G. J. Role for a bidentate ribonuclease in the initiation step of RNA interference. Nature 409, 363-366, doi:10.1038/35053110 (2001). 66 Seydoux, G. & Fire, A. Soma-germline asymmetry in the distributions of embryonic RNAs in Caenorhabditis elegans. Development 120, 2823-2834 (1994). p , , ( ) 66 Seydoux, G. & Fire, A. Soma-germline asymmetry in the distributions of embryonic RNAs in Caenorhabditis elegans. Development 120, 2823-2834 (1994). 67 Stiernagle, T. Maintenance of C. elegans. WormBook, 1-11, doi:10.1895/wormbook.1.101.1 (2006). 68 68 Leinwand, S. G. & Chalasani, S. H. Neuropeptide signaling remodels chemosensory circuit composition in Caenorhabditis elegans. Nature neuroscience 16, 1461-1467, doi:10.1038/nn.3511 (2013). 69 69 Bargmann, C. I. & Horvitz, H. R. Chemosensory neurons with overlapping functions direct chemotaxis to multiple chemicals in C. elegans. Neuron 7, 729-742 (1991). 24 Figure 1. Overview of the Rescue-By-Feeding Technique to Rescue Neuropept a) Design of an expression vector containing the peptide sequence of interest. The vector sequence includes Gateway Cloning sites attR1 and attR2, a 6x-histidine tag (6x-His), and MRFGKR and KRK-STOP sequences flanking the peptide sequence of interest. This vector is transformed into E. coli DH5α cells allowing for the expression of peptides. The bacteria expressing the neuropeptides is then fed to neuropeptide loss-of-function worms and assayed for rescue of behavior. b) Different assays used to quantify behavioral activity. We used three different assays for the quantification of rescue; (1) Size Comparison, (2) Chemotaxis and (3) Excursion Assay. For Size Comparison, mutant worms are fed the peptide for 48 hours before assaying for body morphology on an automated worm tracker. In the second assay, Chemotaxis, we used an NaCl gradient chemotaxis assay to salt attraction of worms of rescued neuropeptides. The Excursion Assay quantified the ability for males to leave a food source in search of mates. Minor Excursion denotes worms that did not leave the food source. Major Excursion denotes worms that left the food source in search of mates, indicating rescue (See Methods for details of the assays). Figure 2. Feeding of TRH-1 Peptides Rescues the Body Volume Defects in trh-1 Mutants. a) Figure Captions Figure Captions Figure 3. Chemotaxis Defects of ins-6 Neuropeptide Mutants are Rescued by INS-6 Peptide. a) Chemotaxis Index to 750 mM NaCl of wild-type (WT), ins-6 loss of function, and genetic ins-6 rescue (ins-6;Pins-6;ins-6), animals fed E. coli OP50. Animals with loss-of- function ins-6 gene exhibit significant decreased attraction and genetic rescue of ins-6 results in a partial rescue of chemotaxis. b) Chemotaxis Index of peptide fed worms to 750 mM NaCl. ins-6 lof worms fed scramble display significantly lower chemotaxis compared to scramble-fed wild type animals. Feeding of INS-6 peptide to ins-6 lof worms results in complete rescue of NaCl chemotaxis. Overexpression of INS-6 does not result in changes in attraction towards 750 mM NaCl. n values are denoted in graphs. Error bars denote SEM. One-Way ANOVA, followed by Bonferroni’s Correction * p < 0.05, p < 0.01, * p < 0.001, ** p < 0.0001, comparisons noted with line over bars. Figure 4. pdf-1 lof males fed discrete PDF-1 peptides differentially rescue food-leaving behavior. a) Percent of male worms leaving food across time is used to visualize the location of the males a different time points. him-5 males serve as the wild-type worms in this assay compared to males deficient in pdf-1 fed PDF-1 peptide to rescue behavior. At each time point the percent of worm within each boundary is noted (Major Excursion is beyond 10 mm radius of the food (purple), Minor Excursion is worms who left food but stayed within 10 mm (pink), and those remaining on food were classified as Never Left Food (yellow)). b) Probability of Leaving. To compare the leaving behavior of the worms as a likelihood of the worms seeking to travel beyond 35 mm from the food. n; him-5 males fed scramble = 55, pdf-1 males fed scramble = 53, pdf-1 males fed PDF-1A = 58, pdf-1 males fed PDF-1B = 51, pdf-1 males fed PDF-1A and PDF-1B = Figure 2. Feeding of TRH-1 Peptides Rescues the Body Volume Defects in trh- trh-1 mutant worms exhibit body volume defects. trh-1 mutants show a reduced body volume compared with wild-type worms when fed E. coli OP50 (Mann-Whitney test, p = 0.0012). b) Feeding of TRH-1 peptides results in restoration of body volume in trh-1 lof worms. trh-1 mutant worms fed with scramble peptide, display significant increase in body volume feeding compared to wild-type animals. trh-1 mutants fed either TRH-1A or TRH-1B or even a combination of both exhibit a restoration of wild-type body volume. Error bars denote SEM. n values denoted in graph. Kruskal-Wallis followed by Dunn’s multiple comparisons, */## p < 0.01, #### p < 0.0001, 25 denote mutant worms compared to wild-type control, #denote mutant worm condition compared to mutant worms fed scramble peptide. 51). Error bars denote SEM. ANOVA, followed by Bonferroni’s Correction, **/### p < 0.001, #### p < 0.0001, denote mutant worms compared to wild-type control, #denote mutant worm condition compared to mutant worms fed scramble peptide. Figure 4. pdf-1 lof males fed discrete PDF-1 peptides differentially rescue food-leaving 26 51). Error bars denote SEM. ANOVA, followed by Bonferroni’s Correction, **/### p < 0.001, #### p < 0.0001, denote mutant worms compared to wild-type control, #denote mutant worm condition compared to mutant worms fed scramble peptide. 27 a) Peptide Feeding No peptide Bacterially-expressed peptides Null mutant phenotypes Rescued phenotypes Peptide Expression Chemotaxis Excursion Assay E. coli Neuropeptides Minor excursion Major excursion High NaCl Low NaCl Size Comparisons Figure-1 a) b) Expression Vector Peptide Expression E. coli Neuropeptides Expression Vector E. coli Neuropeptides Peptide Expression Peptide Feeding b) Null mutant phenotypes Null mutant phenotypes Rescued phenotypes Size Comparisons Excursion Assay Minor excursion Major excursion Excursion Assay Major excursion Minor excursion Major excursion Figure-1 Relative Body Volume (% N2) 0 25 50 75 100 125 496 243 WT trh-1 a) b) Relative Body Volume (% N2) 0 25 50 75 100 125 WT trh-1 #### ## #### 221 441 483 559 454 SCRAMBLE TRH-1A TRH-1B + + + + + + - - - - - - - - - Relative Body Volume (% N2) 0 25 50 75 100 125 496 243 WT trh-1 a) b Relative Body Volume (% N2) 0 25 50 75 100 125 WT trh-1 #### ## #### 221 441 483 559 454 SCRAMBLE TRH-1A TRH-1B + + + + + + - - - - - - - - - b) a) Figure-2 Figure-2 Figure-2 a) b) 0 0.5 1.0 10 26 11 WT ins-6 ins-6 pins-6::ins-6 * Chemotaxis Index SCRAMBLE INS-6 + + + - + - - - * Chemotaxis Index 0 0.5 1.0 WT ins-6 ins-6 pins-6::ins-6 11 12 12 15 a) 0 0.5 1.0 10 26 11 WT ins-6 ins-6 pins-6::ins-6 * Chemotaxis Index b) SCRAMBLE INS-6 + + + - + - - - * Chemotaxis Index 0 0.5 1.0 WT ins-6 ins-6 pins-6::ins-6 11 12 12 15 b) Figure-3 Figure-3 Figure-3 a) b) 0 20 40 60 80 100 % Leaving Food Never Left Food Minor Excursion Major Excursion 2 hrs 6 hrs 24 hrs SCRAMBLE PDF-1A PDF-1B + + + + + + - - - - - - - - - + + + + + + - - - - - - - - - + + + + + + - - - - - - - - - pdf-1 him-5 pdf-1 him-5 pdf-1 him-5 WT pdf-1 SCRAMBLE PDF-1A PDF-1B + + + + + + - - - - - - - - - Probability of leaving 0.00 0.02 0.04 0.06 0.08 0.10 (PLh-1) * ### a) b) 0 20 40 60 80 100 % Leaving Food Never Left Food Minor Excursion Major Excursion 2 hrs 6 hrs 24 hrs SCRAMBLE PDF-1A PDF-1B + + + + + + - - - - - - - - - + + + + + + - - - - - - - - - + + + + + + - - - - - - - - - pdf-1 him-5 pdf-1 him-5 pdf-1 him-5 WT pdf-1 SCRAMBLE PDF-1A PDF-1B + + + + + + - - - - - - - - - Probability of leaving 0.00 0.02 0.04 0.06 0.08 0.10 (PLh-1) * ### b) a) Figure-4 Supplementary Files This is a list of supplementary ¦les associated with this preprint. Click to download. SupplementaryInformationFinal.pdf
https://openalex.org/W4391226220	https://jurnalbisnismahasiswa.com/index.php/jurnal/article/download/176/144	Indonesian	null	Analisis Pengaruh Strategi Pemasaran melalui TikTok terhadap Minat Beli Konsumen: Studi Literatur	Jurnal Bisnis Mahasiswa	2,024	cc-by-sa	4,293	DOI DOI 10.60036/jbm.v4i1.art2 Analisis Pengaruh Strategi Pemasaran melalui TikTok terhadap Minat Beli Konsumen: Studi Literatur Analisis Pengaruh Strategi Pemasaran melalui TikTok terhadap Minat Beli Konsumen: Studi Literatur Febri Annisa, Mochammad Reza Fadli, Novia Suherman, Ida Farida Adi Prawira Universitas Pendidikan Indonesia, Jl. Dr. Setiabudi No.229, Bandung, Indonesia. Informasi Artikel Diterima: November 2023 Direvisi: Desember 2023 Disetujui: Desember 2023 Copyright © 2024 Author(s). This is an open-access article distributed under the terms of the Creative Commons Attribution-ShareAlike 4.0 International License (http://creativecommons.org/licences/by-sa/4.0/) Jurnal Bisnis Mahasiswa Penulis Korespondensi febriannisa@upi.edu *Penulis Korespondensi febriannisa@upi.edu Abstrak TikTok, dengan lebih dari 1 miliar pengguna aktif global, telah menjadi platform pilihan untuk pemasaran bisnis, khususnya di Indonesia dengan 22,2 juta pengguna aktif. Bisnis menggunakan TikTok untuk menciptakan konten menarik, berkolaborasi dengan influencer, dan menjalankan iklan berbayar. Strategi pemasaran ini bertujuan untuk meningkatkan kesadaran merek dan mendorong interaksi dengan konsumen. Beberapa penelitian telah menunjukkan kesuksesan kampanye TikTok, seperti kampanye "BFI Smile Ramadhan" oleh BFI Finance. TikTok juga digunakan untuk mempengaruhi keputusan pembelian, seperti dalam studi yang melibatkan salah satu merek yaitu Bittersweet by Najla. Semua ini menegaskan peran penting TikTok dalam pemasaran digital. Penelitian yang dilakukan bertujuan untuk mengumpulkan bukti melalui metode studi literatur berdasarkan penelitian yang telah dilakukan mengenai Pengaruh Pemasaran melalui TikTok terhadap Minat Beli Konsumen. Kata Kunci Citra Merk, Harga, Keputusan Pembelian. PENDAHULUAN Pemasaran merupakan salah satu aspek krusial dalam dunia bisnis. Salah satu tanggung jawabnya adalah mengelola cara produk diperkenalkan dan didistribusikan kepada konsumen. Saat ini, terdapat berbagai metode dan teknik yang dapat digunakan dalam upaya memasarkan produk atau layanan. Namun, dalam mengikuti perkembangan zaman dan untuk dapat bersaing di pasar yang kompetitif, para pemasar perlu menyesuaikan strategi dan taktik pemasaran mereka. Menurut Gumilang, (2019) penting bagi pelaku usaha untuk mengadopsi strategi pemasaran dan media yang tepat guna menarik pasar yang mereka targetkan, sehingga dapat meningkatkan volume penjualan dan profitabilitas. Salah satu solusi yang banyak diminati oleh masyarakat adalah penggunaan Digital Marketing. Perlahan tapi pasti, banyak yang beralih dari metode pemasaran konvensional atau tradisional ke metode pemasaran modern yang disebut digital marketing. Dalam digital marketing, komunikasi dan transaksi dapat terjadi secara real-time, dan potensinya untuk mencapai pasar global sangat besar. Dengan banyaknya pengguna media sosial berbasis pesan instan, peluang ini semakin berkembang, membuka pintu bagi Usaha Kecil dan Menengah (UKM) untuk memperluas jangkauan pasar mereka melalui perangkat smartphone. Digital marketing telah dilakukan melalui berbagai jenis metode dan media, salah satunya melalui TikTok. TikTok telah menjadi salah satu platform pemasaran JBM \| Vol 4 No 1, 2024 JBM \| Vol 4 No 1, 2024 digital yang digunakan oleh pelaku bisnis. Menurut penelitian yang dilakukan oleh (Bulele & Wibowo, 2020), TikTok, sebuah media sosial yang saat ini telah merambah ke Indonesia, banyak dimanfaatkan oleh masyarakat tidak hanya sebagai wadah untuk berkreasi, tetapi juga sebagai sarana untuk berbisnis. TikTok adalah salah satu platform media sosial yang memungkinkan pengguna untuk mengekspresikan diri dan berkreasi melalui video, seperti yang diungkapkan oleh (Arrofi & Hasfi, 2019) Mayoritas pengguna TikTok berasal dari kalangan remaja dan dewasa muda, khususnya dalam rentang usia sekitar 16 hingga 24 tahun dengan jumlah pengguna nya di Indonesia sebanyak 30,7 juta. Mereka merupakan target audiens yang sangat potensial untuk pemasaran di TikTok, sebagaimana yang ditegaskan oleh Helmy Rasyid, (2020) Penggunaan platform media TikTok dapat menjadi bagian integral dari strategi pemasaran digital, mengingat adanya fitur-fitur tambahan yang dapat meningkatkan daya tarik konten yang dihasilkan. Pemasaran digital melalui TikTok dapat dilakukan melalui Live Streaming, pembuatan konten endorsement melalui influencer, dan TikTok Shop akan tetapi TikTok Shop sendiri telah resmi ditutup pada 4 Oktober 2023. Minat dan keputusan seseorang dalam membeli dapat dipengaruhi oleh berbagai faktor salah satunya melalui strategi pemasaran. PENDAHULUAN Keputusan pembelian terhadap suatu produk muncul setelah konsumen melalui serangkaian pertimbangan yang melibatkan persepsi mereka terhadap merek dan faktor-faktor lingkungan yang mempengaruhinya. Ini kemudian membentuk perilaku mereka dalam memilih merek tertentu. Sebelum seseorang akhirnya memutuskan untuk membuat pembelian, minat untuk membeli akan muncul terlebih dahulu dalam pikiran konsumen (Kotler & Armstrong, 2013) Internet menghasilkan banyak sekali media sosial yang dapat digunakan oleh masyarakat luas sebagai alat komunikasi, alat hiburan, alat pemasaran, dan masih banyak lagi. Kehadiran Internet telah memiliki dampak yang signifikan dalam dunia bisnis, terutama dalam pemasaran yang dilakukan melalui media sosial. Istilah yang muncul dari jenis pemasaran ini adalah digital marketing (pemasaran digital). Tujuan dari digital marketing atau pemasaran digital ini sendiri yaitu untuk menghubungkan konsumen dan juga perusahaan yang mana dapat berbagi informasi serta berkomunikasi (Coviello et al., 2001). Kegiatan pemasaran digital memang telah menjadi sebuah trend yang khas pada era saat ini. Terdapat beberapa platform media sosial yang dapat dijadikan sebagai sarana pemasaran produk, dan salah satunya adalah Tiktok. Seiring berjalannya waktu, Tiktok bertransformasi menjadi sebuah media dalam mengembangkan diri dan berfokus pada komersialisasi, memungkinkan penggunanya untuk menghasilkan pendapatan melalui pembelian dalam aplikasi serta reaksi atau hadiah berbayar sebagai respons terhadap video yang mereka bagikan. Media sosial Tiktok ini dapat digunakan untuk sebuah digital marketing karena fitur yang terdapat pada Tiktok sangat membantu untuk membuat konten yang dapat memunculkan niat beli seseorang. Salah satu fitur yang dapat digunakan adalah melakukan pemasaran melalui Live Streaming Tiktok. Kehadiran Live Streaming dapat meningkatkan kepercayaan konsumen saat berbelanja secara online. Upaya membangun kepercayaan ini sangat penting karena media internet tidak dapat 15 JBM \| Vol 4 No 1, 2024 JBM \| Vol 4 No 1, 2024 menciptakan suasana dan produk seperti yang terjadi dalam belanja konvensional. Meskipun demikian, ketersediaan fitur Live Streaming memungkinkan pelaku bisnis untuk berkomunikasi secara detail, melakukan demonstrasi, dan menjawab pertanyaan calon pembeli secara langsung (Saputra & Fadhilah, 2022). Live Streaming telah menjadi bagian dari strategi pemasaran yang bertujuan untuk memperkenalkan suatu produk kepada masyarakat, menjadikannya sebagai bentuk periklanan. Live Streaming didefinisikan sebagai platform yang merupakan bagian dari fitur perdagangan yang mengintegrasikan interaksi sosial secara real-time dalam digital marketing. Cara lain untuk memasarkan produk di Tiktok adalah melalui influencer endorsement. Beberapa faktor dapat memicu minat konsumen, salah satu pemicunya adalah promosi. Promosi bisa dijelaskan sebagai bentuk komunikasi pemasaran yang berfungsi untuk mengajak dan menyampaikan informasi tentang produk atau layanan yang ditawarkan. PENDAHULUAN Kehadiran internet memiliki dampak yang cukup besar pada perkembangan teknik pemasaran baru yang bisa digunakan oleh perusahaan. Disini, Influencer memiliki peran yang signifikan dalam mempengaruhi konsumen karena dengan pemikiran, sikap, dan pandangan mereka, sehingga berdampak besar pada tren permintaan produk tertentu (Zak & Hasprova, 2020). Pengaruh yang diberikan oleh influencer dapat memiliki dampak signifikan pada keputusan konsumen ketika mereka hendak membeli produk atau jasa. Walaupun ada banyak faktor lain yang turut mempengaruhi keputusan pembelian, tetapi tidak dapat diabaikan bahwa pemasaran melalui influencer juga memiliki potensi untuk mempengaruhi keputusan pembelian konsumen (Zak & Hasprova, 2020). Keputusan pembelian didefinisikan sebagai proses yang dilalui oleh konsumen mulai dari mengidentifikasi masalah, mencari informasi tentang produk tertentu, hingga mengevaluasi opsi yang dapat memecahkan masalah tersebut, yang kemudian mempengaruhi keputusan dan minat seseorang dalam membeli suatu produk tertentu. (Kotler & Keller, 2021). Minat beli sendiri menggambarkan keputusan pembelian sebagai serangkaian tahap yang harus dilalui oleh konsumen sebelum mereka membeli produk. Minat beli menurut (Kotler & Keller, 2021), “Minat beli merupakan perilaku yang muncul sebagai respon terhadap objek yang menunjukkan keinginan konsumen untuk melakukan pembelian”. Menurut penelitian yang dilakukan oleh (NST Rizky & Yasin, 2014) minat yang dimiliki oleh calon pembeli sering kali tidak sejalan dengan kondisi keuangan mereka. Minat beli konsumen mencerminkan keinginan yang tersembunyi dalam diri mereka. Minat beli konsumen selalu tersembunyi dalam masing-masing individu dan tidak dapat diketahui oleh orang lain, serta merupakan harapan dari konsumen tersebut. METODE Penelitian ini bertujuan untuk mengidentifikasi dan menjelaskan permasalahan yang terkait dengan pelaksanaan evaluasi pembelajaran dalam mencapai tujuan pendidikan pada era belajar mandiri. Penelitian ini menggunakan metode deskriptif dengan pendekatan kualitatif. Seperti yang dinyatakan oleh Nurdin & Hartati, (2019), penelitian kualitatif adalah jenis penelitian yang berdasarkan pada 16 JBM \| Vol 4 No 1, 2024 data, menggunakan teori yang ada sebagai dasar penjelasan, dan berakhir dengan pembentukan suatu teori. Dalam penelitian ini, metode yang diterapkan adalah studi literatur, yang melibatkan evaluasi dan analisis artikel serta jurnal ilmiah yang relevan dengan masalah penelitian. Fokus utama penelitian adalah untuk mengeksplorasi teori-teori yang terkait dengan dampak pemasaran media sosial pada keputusan pembelian konsumen. Teori-teori ini akan diuraikan secara rinci dalam bagian literatur yang diselidiki, dan akan digunakan sebagai dasar untuk merumuskan permasalahan penelitian. Selain itu, literatur ini akan dijadikan dasar untuk melakukan perbandingan dengan temuan penelitian sebelumnya, dengan tujuan untuk menilai sejauh mana pemasaran media sosial memiliki pengaruh pada minat beli konsumen. HASIL DAN PEMBAHASAN (Geyser, 2022) mendefinisikan TikTok Marketing sebagai salah satu strategi pemasaran yang melibatkan penggunaan platform media sosial TikTok sebagai alat untuk mempromosikan merek, produk, atau layanan. TikTok adalah aplikasi video pendek yang memungkinkan pengguna membuat dan berbagi video, seringkali dengan musik. Dengan lebih dari 1 miliar pengguna aktif global, TikTok telah menjadi pilihan populer bagi bisnis untuk mencapai audiens yang lebih muda dan meningkatkan kesadaran merek. Strategi pemasaran TikTok melibatkan pembuatan konten merek, kerjasama dengan pengaruh, dan pelaksanaan kampanye iklan berbayar. Tujuannya adalah menciptakan konten menarik dan menghibur yang dapat meraih perhatian pengguna, mendorong keterlibatan mereka dengan merek. Perkembangan TikTok sebagai salah satu media pemasaran digital telah berlangsung di Indonesia sejak awal pandemi Covid-19. Perkembangan tersebut sejalan dengan pertumbuhan pesat platform ini dan pangsa pasar yang besar, mencapai 22,2 juta pengguna aktif (Francesca & Utami, 2022). Marketing yang dilakukan dalam media sosial TikTok memanfaatkan kreativitas dalam pengelolaan konten berupa video maupun foto atau melalui kampanye-kampanye yang mengundang ketertarikan konsumen dalam membeli sebuah produk maupun jasa. Dalam beberapa penelitian yang dilakukan, berikut merupakan contoh penerapan strategi pemasaran di media sosial TikTok: p p p g p a. Penelitian yang dilakukan oleh Francesca & Utami (2022) memaparkan bahwa BFI Finance, sebuah perusahaan pembiayaan, meluncurkan kampanye TikTok bernama "BFI Smile Ramadhan" untuk brand awareness. Berdasarkan hasil penelitian, strategi pemasaran melalui TikTok ini berhasil membangun brand awareness dari BFI Finance. b. Penelitian oleh Yulianti (2022) menjelaskan bahwa terdapat hubungan antara content marketing melalui TikTok oleh Bittersweet by Najla dengan keputusan membeli followers yang juga menunjukkan bahwa content marketing yang relevan, informatif, andal, memiliki value, dan keunikan berpengaruh terhadap keputusan pembelian followers nya. c. Penelitian oleh Yunani & Kamilla (2023) yang meneliti mengenai salah satu brand skincare yaitu Somethinc menemukan bahwa pemasaran konten melalui TikTok memiliki dampak yang besar pada minat pembelian dan kesadaran merek pada c. Penelitian oleh Yunani & Kamilla (2023) yang meneliti mengenai salah satu brand skincare yaitu Somethinc menemukan bahwa pemasaran konten melalui TikTok memiliki dampak yang besar pada minat pembelian dan kesadaran merek pada 17 JBM \| Vol 4 No 1, 2024 akun @somethincofficial. Tidak hanya itu, kesadaran merek juga berperan sebagai perantara dalam pengaruh pemasaran konten terhadap minat beli. Pengaruh penggunaan media sosial TikTok melalui berbagai cara seperti penggunaan influencer, pembangunan citra merek, dan pembuatan konten kreatif terhadap minat beli konsumen telah dibuktikan melalui beberapa penelitian. HASIL DAN PEMBAHASAN Hingga saat ini, strategi pemasaran melalui TikTok semakin marak diterapkan karena telah memberikan hasil yang signifikan saat konten yang disajikan dalam pemasaran produk terkait dikelola dengan baik dan sekreatif mungkin. Berikut merupakan matriks dari 5 penelitian relevan yang menunjukkan pengaruh pemasaran melalui TikTok terhadap minat beli konsumen: Judul, Penulis, & Tahun Variabel & Metode Hasil Penelitian Judul: Pengaruh Media Sosial Marketing Tiktok Terhadap Minat Beli Online Di Shopee Penulis: Frida Eka Setianingsih & Fauzan Aziz Tahun: 2022 Variabel: Variabel dependen: minat beli konsumen Variabel independen: Metode Strategi Pemasaran melalui TikTok Metode: Metode penelitian deskriptif dan kausalitas dengan pendekatan kuantitatif. Penelitian menunjukkan bahwa TikTok berpengaruh secara signifikan pada minat beli konsumen, terutama melalui konten dan informasi produk. Kepercayaan konsumen dapat ditingkatkan dengan memastikan privasi dan keamanan di platform media sosial. Judul: The Effect of Social Media Marketing TikTok and Product Quality Towards Purchase Intention Penulis: Tiara Meliawati, Sweety Celendine Gerald, & Akhmad Edhy Aruman Tahun: 2023 Variabel: Variabel dependen: Minat beli Variabel independen: Pemasaran melalui media sosial TikTok, dan kualitas produk. Metode: Metode kuantitatif dengan pendekatan survei eksplanatif asosiatif melalui kuesioner. Pemasaran melalui media sosial TikTok dan kualitas produk memiliki pengaruh signifikan terhadap minat beli. TikTok terbukti menjadi indikator yang paling signifikan dalam variabel pemasaran melalui media sosial, sedangkan penampilan produk menjadi indikator yang paling signifikan dalam variabel kualitas produk. Judul: TikTok as a Platform for Marketing Campaigns: The effect of Brand Awareness Variabel: Variabel dependen: Minat beli Variabel independen: Marketing Campaigns on Pemasaran melalui TikTok mempengaruhi kesadaran merek generasi milenial secara signifikan. Namun, tidak berpengaruh signifikan pada Pemasaran melalui media sosial TikTok dan kualitas produk memiliki pengaruh signifikan terhadap minat beli. TikTok terbukti menjadi indikator yang paling signifikan dalam variabel pemasaran melalui media sosial, sedangkan penampilan produk menjadi indikator yang paling signifikan dalam variabel kualitas produk. 18 JBM \| Vol 4 No 1, 2024 JBM \| Vol 4 No 1, 2024 \| Judul, Penulis, & Tahun Variabel & Metode Hasil Penelitian and Brand Recall on the Purchase Intentions of Millennials Penulis: Mary Aubrey G. Gesmundo, Melvin Dave S. Jordan, Wee Hansei D. Meridor, Dainielle Vien C. Muyot, Mary Caroline N. Castano, & Agnes Jocelyn P. Bandojo Tahun: 2022 TikTok & Brand Awareness, & Brand Recall. Metode: Metode penelitian deskriptif-kuantitatif dengan teknik sampling convenience. Sampel terdiri dari 300 responden milenial berusia 25-40 tahun di Wilayah Ibukota Negara (NCR) Filipina. HASIL DAN PEMBAHASAN Kuesioner deskriptif diadopsi dari penelitian Bilal Ahmed Memon dan dianalisis menggunakan statistik deskriptif, distribusi frekuensi, mean, deviasi standar, serta PLS- SEM (Partial Least Square- Structural Equation Modeling) untuk mengevaluasi hubungan antar variabel yang diteliti. ingatan merek mereka. Meskipun begitu, kesadaran merek tetap berhubungan dengan ingatan merek, dan keduanya berpengaruh terhadap niat pembelian generasi milenial. Judul: Pengaruh Live streaming Sales Tiktok terhadap Minat Beli Konsumen Penulis: Muhammad Ali Fakri & Santi Indra Astuti Tahun: 2023 Variabel: Variabel dependen: Minat beli konsumen Variabel independen: Pengaruh Live streaming Sales Tiktok yang terdiri dari beberapa indikator, yaitu “Persepsi Hiburan", "Persepsi Diskon", "Persepsi Interaktivitas", "Persepsi Profesionalisme", dan "Persepsi Kesamaan" Metode: Metode penelitian yang digunakan dalam studi ini adalah metode kuantitatif Penelitian ini menemukan adanya hubungan yang signifikan antara live streaming penjualan di TikTok dengan minat beli konsumen terhadap merek Screamous yang menandakan bahwa semakin besar pengaruh live streaming penjualan TikTok, semakin tinggi minat beli konsumen. Selain itu, hasil penelitian juga menunjukkan bahwa faktor- faktor seperti persepsi hiburan, persepsi diskon, persepsi interaktivitas, persepsi profesionalisme, dan persepsi kesamaan dalam live Judul, Penulis, & Tahun Variabel & Metode H and Brand Recall on the Purchase Intentions of Millennials Penulis: Mary Aubrey G. Gesmundo, Melvin Dave S. Jordan, Wee Hansei D. Meridor, Dainielle Vien C. Muyot, Mary Caroline N. Castano, & Agnes Jocelyn P. Bandojo Tahun: 2022 TikTok & Brand Awareness, & Brand Recall. Metode: Metode penelitian deskriptif-kuantitatif dengan teknik sampling convenience. Sampel terdiri dari 300 responden milenial berusia 25-40 tahun di Wilayah Ibukota Negara (NCR) Filipina. Kuesioner deskriptif diadopsi dari penelitian Bilal Ahmed Memon dan dianalisis menggunakan statistik deskriptif, distribusi frekuensi, mean, deviasi standar, serta PLS- SEM (Partial Least Square- Structural Equation Modeling) untuk mengevaluasi hubungan antar variabel yang diteliti. ingatan Meskipu merek dengan keduany terhadap generasi Judul: Pengaruh Live streaming Sales Tiktok terhadap Minat Beli Konsumen Penulis: Muhammad Ali Fakri & Santi Indra Astuti Tahun: 2023 Variabel: Variabel dependen: Minat beli konsumen Variabel independen: Pengaruh Live streaming Sales Tiktok yang terdiri dari beberapa indikator, yaitu “Persepsi Hiburan", "Persepsi Diskon", "Persepsi Interaktivitas", "Persepsi Profesionalisme", dan "Persepsi Kesamaan" Metode: Metode penelitian yang digunakan dalam studi ini adalah metode kuantitatif Penelitia adanya signifikan penjuala minat be merek menanda besar pe penjuala tinggi mi Selain itu menunju faktor hiburan, persepsi persepsi persepsi Judul, Penulis, & Tahun Variabel & Metode Hasil Penelitian and Brand Recall on the Purchase Intentions of Millennials Penulis: Mary Aubrey G. Gesmundo, Melvin Dave S. HASIL DAN PEMBAHASAN Jordan, Wee Hansei D. Meridor, Dainielle Vien C. Muyot, Mary Caroline N. Castano, & Agnes Jocelyn P. Bandojo Tahun: 2022 TikTok & Brand Awareness, & Brand Recall. Metode: Metode penelitian deskriptif-kuantitatif dengan teknik sampling convenience. Sampel terdiri dari 300 responden milenial berusia 25-40 tahun di Wilayah Ibukota Negara (NCR) Filipina. Kuesioner deskriptif diadopsi dari penelitian Bilal Ahmed Memon dan dianalisis menggunakan statistik deskriptif, distribusi frekuensi, mean, deviasi standar, serta PLS- SEM (Partial Least Square- Structural Equation Modeling) untuk mengevaluasi hubungan antar variabel yang diteliti. ingatan merek mereka. Meskipun begitu, kesadaran merek tetap berhubungan dengan ingatan merek, dan keduanya berpengaruh terhadap niat pembelian generasi milenial. Penelitian ini menemukan adanya hubungan yang signifikan antara live streaming penjualan di TikTok dengan minat beli konsumen terhadap merek Screamous yang menandakan bahwa semakin besar pengaruh live streaming penjualan TikTok, semakin tinggi minat beli konsumen. gg Selain itu, hasil penelitian juga menunjukkan bahwa faktor- faktor seperti persepsi hiburan, persepsi diskon, persepsi interaktivitas, persepsi profesionalisme, dan persepsi kesamaan dalam live 19 JBM \| Vol 4 No 1, 2024 Judul, Penulis, & Tahun Variabel & Metode Hasil Pe dengan pendekatan korelasional. Data dikumpulkan melalui penggunaan kuesioner & dianalisis menggunakan teknik analisis korelasi Rank Spearman. streaming pe juga memiliki signifikan pad konsumen. menunjukkan variabel mempengaruh konsumen k dalam live strea TikTok dari me Namun, pentin bahwa pengaru penjualan TikT faktor lain yan menjelaskan variasi dalam konsumen. Ma faktor lain d penelitian yan mempengaruh konsumen. Judul: Analisis Pengaruh Marketing Influencer Tiktok Terhadap Keinginan Pembelian Pada Pengguna Aplikasi Tiktok Penulis: Made Putri Ariasih & I Putu Dharmawan Suryagita Susila Putra. Tahun: 2022 Variabel: Variabel dependen: Keinginan pembelian pengguna aplikasi TikTok. Variabel independen: Marketing influencer TikTok Metode: Metode penelitian deskriptif kualitatif Penelitian ini beberapa hal: - Perusahaan influencer memperken dan merang pada pen influencer. - Konsumen cenderung informasi p membeli, melalui ulas influencer. M tanggapan mengenai diminati. - Video konte mampu Hasil Penelitian streaming penjualan TikTok juga memiliki dampak yang signifikan pada minat beli konsumen. Hal ini menunjukkan bahwa variabel- variabel tersebut mempengaruhi minat beli konsumen ketika terlibat dalam live streaming penjualan TikTok dari merek Screamous. Namun, penting untuk diingat bahwa pengaruh live streaming penjualan TikTok dan faktor- faktor lain yang diteliti hanya menjelaskan sekitar 30,36% variasi dalam minat beli konsumen. Masih ada faktor- faktor lain di luar lingkup penelitian yang juga dapat mempengaruhi minat beli konsumen. Simpulan Berdasarkan literatur review yang telah dilakukan, terlihat bahwa pemasaran melalui TikTok memiliki dampak yang signifikan terhadap minat beli konsumen. TikTok, sebagai platform media sosial, telah terbukti menjadi alat yang sangat efektif dalam memperkenalkan merek, produk, atau layanan. Dengan memanfaatkan konten kreatif, kerjasama dengan influencer, dan kampanye iklan berbayar, pemasaran melalui TikTok dapat meningkatkan kesadaran merek, membangun pemahaman merek, dan merangsang minat beli konsumen. Berbagai faktor mempengaruhi keberhasilan pemasaran melalui TikTok, termasuk pemahaman konsumen tentang merek, kualitas konten yang disajikan, penggunaan pemasaran afiliasi, persepsi terhadap citra merek, serta mutu produk dan ulasan dari influencer dan konsumen lainnya. Hasil penelitian menunjukkan bahwa pemasaran melalui TikTok dapat meningkatkan minat beli konsumen terhadap produk tertentu. Namun, penting untuk dicatat bahwa pemasaran melalui TikTok hanya merupakan bagian dari strategi pemasaran yang lebih luas. Keberhasilan pemasaran tetap bergantung pada faktor-faktor lain seperti kualitas produk, kepercayaan konsumen, dan elemen lingkungan yang mempengaruhi keputusan pembelian. Oleh karena itu, perusahaan harus mempertimbangkan semua faktor ini saat merencanakan strategi pemasaran yang efektif melalui TikTok atau platform media sosial lainnya. Keterbatasan Keterbatasan dari penelitian ini adalah bahwa penelitian ini hanya menggunakan metode deskriptif dengan pendekatan kualitatif. Hal ini menunjukan bahwa penelitian ini mungkin tidak dapat memberikan gambaran yang lengkap tentang dampak pemasaran media sosial pada keputusan pembelian konsumen. HASIL DAN PEMBAHASAN Kualitas produk & review atau ulasan dari influencer dan konsumen lain Berdasarkan penelitian oleh (Ariasih & Putra, 2022) menjelaskan bahwa konsumen saat ini lebih suka mencari informasi tentang produk sebelum melakukan pembelian, seringkali melalui ulasan produk yang dibuat oleh influencer. Mereka tertarik untuk mengetahui pandangan influencer terhadap produk yang mereka minati sebelum membuat keputusan pembelian. e. Kualitas produk & review atau ulasan dari influencer dan konsumen lain Berdasarkan penelitian oleh (Ariasih & Putra, 2022) menjelaskan bahwa konsumen saat ini lebih suka mencari informasi tentang produk sebelum melakukan pembelian, seringkali melalui ulasan produk yang dibuat oleh influencer. Mereka tertarik untuk mengetahui pandangan influencer terhadap produk yang mereka minati sebelum membuat keputusan pembelian. HASIL DAN PEMBAHASAN - Konsumen saat ini cenderung mencari informasi produk sebelum membeli, salah satunya melalui ulasan produk oleh influencer. Mereka mencari tanggapan influencer mengenai produk yang diminati. - Video konten dari influencer mampu membangkitkan 20 JBM \| Vol 4 No 1, 2024 Judul, Penulis, & Tahun Variabel & Metode Hasil Penelitian minat beli konsumen terhadap produk yang direkomendasikan. Dengan demikian, dapat disimpulkan bahwa pemasaran melalui influencer TikTok memiliki dampak atau pengaruh pada keinginan beli pengguna TikTok. Berdasarkan matriks yang telah dirumuskan, membuktikan bahwa pemasaran melalui TikTok merupakan pemasaran yang efektif untuk dilakukan pada era digital seperti saat ini dan seluruh penelitian membuktikan bahwa Pemasaran Digital melalui Media Sosial TikTok memiliki pengaruh signifikan terhadap Minat Beli Konsumen pada beberapa produk tertentu. Pemasaran melalui TikTok tentunya melibatkan aspek-aspek lain yang mendukung keberhasilan penguasaan pasar/penarikan minat beli konsumen seperti sebagai berikut: a. Pemahaman/kesadaran tentang merek Pengetahuan yang dimiliki konsumen mengenai suatu merek dapat memiliki dampak yang besar pada keinginan mereka untuk membeli. Hasil penelitian dari (Yunani & Kamilla, 2023) menunjukkan bahwa pengetahuan konsumen tentang merek berperan sebagai perantara dalam hubungan antara pemasaran konten TikTok dan keinginan pembelian mereka. b. Kualitas konten Cara konten pemasaran disajikan di TikTok memiliki potensi mempengaruhi minat pembelian konsumen. Studi menunjukkan bahwa bagaimana pemasaran konten disusun di TikTok dapat berdampak nyata pada keinginan untuk membeli produk atau layanan. (Yunani & Kamilla, 2023) c. Pelibatan affiliate marketing Affiliate marketing didefinisikan sebagai salah satu cara pemasaran digital yang terkenal di Indonesia, melibatkan kolaborasi antara perusahaan dan pembuat konten media sosial untuk mengiklankan produk. Studi oleh (Arafah et al., 2023) menunjukkan bahwa pendekatan ini telah terbukti meningkatkan penjualan produk secara signifikan. d. Persepsi terhadap citra merek Pandangan konsumen terhadap suatu merek memiliki potensi mempengaruhi keinginan mereka untuk membeli produk tersebut. Penelitian oleh Yunani & Kamila (2023) menunjukkan bahwa pandangan ini memiliki peranan krusial dalam membentuk tindakan pembelian konsumen. Selain itu, (Fakri & Indra Astuti, 2023) juga memaparkan persepsi lain seperti “Persepsi Hiburan", "Persepsi Diskon", "Persepsi Interaktivitas", "Persepsi Profesionalisme", dan "Persepsi 21 JBM \| Vol 4 No 1, 2024 JBM \| Vol 4 No 1, 2024 Kesamaan" yang dapat mempengaruhi minat beli konsumen saat melihat konten terkait pemasaran digital yang dilakukan oleh perusahaan dalam media sosial TikTok. Kesamaan" yang dapat mempengaruhi minat beli konsumen saat melihat konten terkait pemasaran digital yang dilakukan oleh perusahaan dalam media sosial TikTok. e. Implikasi Penelitian Implikasi dari penelitian ini adalah bahwa pemasaran melalui TikTok memiliki dampak yang signifikan terhadap minat beli konsumen, terutama dalam mencapai audiens yang lebih muda dan meningkatkan kesadaran merek. Hal ini menunjukkan bahwa bisnis dapat memanfaatkan platform TikTok sebagai bagian dari strategi pemasaran digital mereka untuk mencapai target audiens yang lebih muda dan mempengaruhi keputusan pembelian konsumen. Selain itu, penelitian ini juga 22 JBM \| Vol 4 No 1, 2024 JBM \| Vol 4 No 1, 2024 menunjukkan bahwa faktor-faktor seperti pemahaman konsumen tentang merek, kualitas konten, affiliate marketing, persepsi terhadap citra merek, dan kualitas produk serta ulasan dari influencer dan konsumen lainnya mempengaruhi keberhasilan pemasaran melalui TikTok. Implikasi praktis dari penelitian ini adalah bahwa bisnis dapat mempertimbangkan untuk mengintegrasikan pemasaran melalui TikTok sebagai bagian dari strategi pemasaran mereka, terutama jika target audiens mereka adalah generasi muda. Selain itu, bisnis juga perlu memperhatikan kualitas produk, kepercayaan konsumen, dan elemen lingkungan dalam melaksanakan strategi pemasaran melalui TikTok. Implikasi teoritis dari penelitian ini adalah bahwa penelitian lebih lanjut dapat dilakukan untuk mendalami faktor-faktor yang mempengaruhi keberhasilan pemasaran melalui TikTok, serta untuk memahami lebih dalam dampak pemasaran digital terhadap perilaku konsumen. DAFTAR RUJUKAN Arafah, N., Karunia Pratama, D., Wahyudi, R., Puteri Utami, M., Prehanto, A., & Mariska Purwaamijaya, B. (2023). PERSEPSI PELAKU USAHA FASHION DI KOTA TASIKMALAYA TERHADAP PENERAPAN AFILIASI MARKETING. Jurnal Industri Kreatif Dan Kewirausahaan, 6(1). Ariasih, M. P., & Putra, I. P. D. S. S. (2022). Analisis Pengaruh Marketing Influencer Tiktok Terhadap Keinginan Pembelian Pada Pengguna Aplikasi Tiktok. Jurnal Sutasoma, 1(1), 22–27. https://doi.org/10.58878/sutasoma.v1i1.178 Arrofi, A., & Hasfi, N. (2019). MEMAHAMI PENGALAMAN KOMUNIKASI ORANG TUA- ANAK KETIKA MENYAKSIKAN TAYANGAN ANAK-ANAK DI MEDIA SOSIAL TIK TOK. http://www.fisip.undip.ac.id Bulele, Y. N., & Wibowo, T. (2020). ANALISIS FENOMENA SOSIAL MEDIA DAN KAUM MILENIAL: STUDI KASUS TIKTOK. Conference on Business, Social Sciences and Innovation Technology, 1(1), 565–572. http://journal.uib.ac.id/index.php/cbssit Coviello, N., Milley, R., & Marcolin, B. (2001). Understanding IT-enabled interactivity in contemporary marketing. Journal of Interactive Marketing, 15(4), 18–33. https://doi.org/10.1002/dir.1020 Fakri, M. A., & Indra Astuti, S. (2023). Pengaruh Live Streaming Sales Tiktok terhadap Minat Beli Konsumen. Bandung Conference Series: Journalism, 3(2), 110–116. https://doi.org/10.29313/bcsj.v3i2.7714 Geyser, W. (2022, June 21). TikTok Marketing 101: How to Market Your Brand on One of the Most Popular Social Apps. Influencer Marketing Hub. Gumilang, R. R. (2019). Implementasi Digital MarketingTerhadap Peningkatan Penjualan Hasil Home Industri. Coopetition : Jurnal Ilmiah Manajemen, 10(1), 9– 14. Helmy Rasyid, M. (2020). Pembelajaran Puisi Secara Daring dengan Media Pembelajara Berbasis Aplikasi (Tik Tok) Kelas X SMA Negeri 3 Pati. Kotler, P., & Armstrong, G. (2013). Principle of Marketing (14th ed.). Prentice Hall. Kotler, P., & Armstrong, G. (2013). Principle of Marketing (14th ed.). Prentice Hall. Kotler P & Keller K L (2021) Marketing Management (16th ed ) Pearson Education Kotler, P., & Armstrong, G. (2013). Principle of Marketing (14th ed.). Prentice Hall. Kotler, P., & Keller, K. L. (2021). Marketing Management (16th ed.). Pearson Education. Kotler, P., & Keller, K. L. (2021). Marketing Management (16th ed.). Pearson Education. 23 JBM \| Vol 4 No 1, 2024 NST Rizky, M. F., & Yasin, H. (2014). PENGARUH PROMOSI DAN HARGA TERHADAP MINAT BELI PERUMAHAN OBAMA PT. NAILAH ADI KURNIA SEI MENCIRIM MEDAN. JURNAL MANAJEMEN & BISNIS, 14(2), 135–143. Nurdin, I., & Hartati, S. (2019). METODOLOGI PENELITIAN SOSIAL. Media Sahabat Cendekia. Saputra, G. G., & Fadhilah, F. (2022). Pengaruh Live Streaming Shopping Instagram Terhadap Kepercayaan Konsumen Online dan Dampaknya pada Keputusan Pembelian. Ekonomi, Keuangan, Investasi Dan Syariah (EKUITAS), 4(2), 442–452. https://doi.org/10.47065/ekuitas.v4i2.2353 Yunani, A., & Kamilla, Z. N. (2023). Pengaruh Content Marketing Tiktok terhadap Minat Beli @Somethincofficial Melalui Brand Awareness. DAFTAR RUJUKAN Al-Kharaj : Jurnal Ekonomi, Keuangan & Bisnis Syariah, 6(2), 3809–3825. https://doi.org/10.47467/alkharaj.v6i2.4100 Zak, S., & Hasprova, M. (2020). The role of influencers in the consumer decision- making process. SHS Web of Conferences, 74, 03014. https://doi.org/10.1051/shsconf/20207403014 24 24
https://openalex.org/W4386937827	https://link.springer.com/content/pdf/10.1007/978-3-031-43950-6_6.pdf	English	null	Drug Intervention Follow up with Internet of Things: A Case Study	Lecture notes in computer science	2,023	cc-by	4,431	© The Author(s) 2023 K. Jongbae et al. (Eds.): ICOST 2023, LNCS 14237, pp. 65–75, 2023. https://doi.org/10.1007/978-3-031-43950-6_6 Hassan M. Ahmed1(B), Souhail Maraoui1, Bessam Abdulrazak1, Benoît Cossette2, and F. Guillaume Blanchet3,4,5 Hassan M. Ahmed1(B), Souhail Maraoui1, Bessam Abdulrazak1, Benoît Cossette2, and F. Guillaume Blanchet3,4,5 {hassan.mostafa.ahmed.fahmy,souhail.maraoui, bessam.abdulrazak}@usherbrooke.ca 2 Centre de recherche sur le vieillissement du CIUSSS de l’Estrie – CHUS, Sherbrooke, Canada benoit.cossette@usherbrooke.ca 3 Département de Biologie, Faculté des sciences, Université de Sherbrooke, Sherbrooke, Canad guillaume.blanchet@usherbrooke.ca Département de Mathématiques, Faculté des sciences, Université de Sherbrooke, Sherbrooke Canada Département de Mathématiques, Faculté des sciences, Université de Sherbrooke, Sherbrooke, Canada 5 Canada 5 sciences de la santé communautaire, Faculté de médecine et des sciences de Département des sciences de la santé communautaire, Faculté de médecine et des sciences d la santé, Université de Sherbrooke, Sherbrooke, Canada la santé, Université de Sherbrooke, Sherbrooke, Canada Abstract. Advancements on the Internet of Things (IoT) have enabled the devel- opment of advanced monitoring systems that can track human behavior and vital signs in real-time, which can have a real impact in the way healthcare is provided. This paper presents a system implementation to monitor and analyze a subject’s behavior changes over time using IoT, with the objective of detecting the impact of an inhibitor drug on the subject’s activity levels. In this research we present a case study by which we showed it is possible to follow the effect of an anticholinergic drug by means of an unobtrusive IoT system. We have monitored the physical activity of a subject in his residence for seven consecutive months to study the effect of the inhibiting drug doses introduced at three known speciﬁc timestamps. Following, we compared our detection results for the subject’s physical activ- ity change timestamps with the medical staff medication doses timestamps. Our results show that we can detect the physical activity change at close timestamps compared to those indicated by the medical staff. Keywords: Physical Activity Change Detection · Outliers Detection · Medication Follow-up Keywords: Physical Activity Change Detection · Outliers Detection · Medication Follow-up 1 Introduction The Internet of Things (IoT) is a rapidly growing network of interconnected devices that can share data and communicate with one another, and it has revolutionized a wide range of industries, including healthcare. IoT devices and systems can be used to gather and analyze data on patients, monitor their health conditions, and provide personalized and H. M. Ahmed et al. 66 timely healthcare [1]. One area where IoT is particularly promising is in the development and monitoring of new drugs, such as inhibitor drugs used to slow down or stop speciﬁc biological processes or activities [2]. Monitoring the behavior and activity of subjects under the inﬂuence of such drugs is crucial to address two main issues: the drug effect itself and the pharmaceutical metrics [3, 4]. In this paper, we are presenting a proof of concept based on a case study to show we can follow the effect of drug intervention on a subject using IoT. To this end, we have monitored a subject in his residence where he was given three timestamped doses of anticholinergic drug. We used our IoT framework to monitor his daily physical activity inside the residence and analyzing the captured timeseries afterwards. To achieve such a task, the physical activity is monitored using motion sensors embedded in the ambient environment. Following, we used an outliers’ detection technique applied to the physical activity of the subject that relied on a sliding window applied to the entire timeseries collected. The mean and standard deviation of the subject’s physical activity was quantity within the sliding window. We considered the change in the subject’s physical behavior to be deﬁned as outliers from the calculated mean and standard deviation gathered across the sliding window. This paper is organized as following, Sect. 2 comprises the related work in this particular ﬁeld of research. The methodology is presented in Sect. 3. Results and dis- cussion are presented in Sects. 4 and 5, respectively. Finally, the conclusion is presented in Sect. 6. 2 Related Work For example, clinical researchers are interested in evaluating how deprescribing anticholinergic and sedative medications impact people behavior. Based on a literature review, Reeve et al. [14] deﬁned deprescribing as “the process of withdrawal of an inappropriate medication, supervised by a health care pro- fessional with the goal of managing polypharmacy and improving outcomes.” Although this deﬁnition has not yet been validated, it can be used as a basis for any deprescribing drug, even if further work is needed to get expert consensus to develop an internationally accepted deﬁnition [14]. Considering Reeves et al. [14] deﬁnition, in recent years there are accumulating evi- dence showing health improvement associated with deprescribing anticholinergic and sedative in older adults. For example, Ailabouni N. et al. [15] found that reducing anti- cholinergic and sedative medications in frail older people was feasible and beneﬁcial and resulted in better health outcomes, fewer adverse effects, and improved mood and vigor. They also concluded that deprescribing with a patient-centered approach can be safe and effective. Dearing et al. [16] evaluated the effectiveness of a pharmacist-led medication optimization initiative using an electronic tool called the Drug Burden Index (BDI) Calculator in four hospital sites in Canada. This study aimed to achieve three objectives: First, to determine the impact of integrating an electronic clinical decision support tool, the DBI Calculator, into pharmacist medication optimization activities on medication changes and clinical outcomes; Second, to assess whether the effect of the intervention varies based on participant characteristics such as frailty status and sex, as well as the setting of the intervention, including different ward and hospital characteris- tics; and Third, to explore the implementation of the DBI Calculator into pharmacist-led medication optimization activities during inpatient admissions. The study found signiﬁ- cant changes in DBI scores and clinical outcomes, indicating the potential beneﬁt of such interventions. Similarly, Arvisais et al. [17] conducted a study to assess the efﬁcacy of a pharmacist-physician intervention model in reducing the use of high-risk medications in older adult patients. The study reported that the intervention model was effective in reducing the use of high-risk medications. Also, Cossette et al. [18, 19] investigated the impact of a computerized alert system-based pharmacist-physician intervention model on Potentially Inappropriate Medications (PIM) use. The interventions resulted in a higher number of drug cessation and dosage reductions for targeted PIMs compared to a control group. In a pilot study conducted by Cossette et al. 2 Related Work There has been signiﬁcant interest in IoT research within the healthcare domain, resulting in the development of various applications for different diseases, settings, and popula- tions. A comprehensive review conducted by Lin et al. [5] surveyed representative and active IoT application products and categorized their focuses into three care settings: (i) acute disease care, (ii) chronic disease care, and (iii) self-health management. In acute disease care, it is crucial to collect patients’ vital signs accurately and in a timely manner. For example, Gao et. al. [6] collaborated closely with medical profes- sionals to identify the key needs in health emergencies. They designed and implemented a system that records patients’ vital signs in real-time using wearable sensors, detects anomalies through a vital sign monitoring algorithm, and shares the data with authorized personnel via a web portal. IoT applications in chronic disease management aim to enable remote health moni- toring. For example, Al-Khafajiy et al. [7] presented Wearable Sensors for Smart Health- care Monitoring System (SW-SHMS), a smart healthcare monitoring system that collects physiological data from wearable devices. In their system, the collected data is trans- mitted to a smartphone app via Bluetooth and then to the cloud over the internet. The processed data is displayed on a monitoring platform accessible by doctors. Another example is IMMED (Indexing MultiMEdia data from wearable sensors for diagnostics and treatment of Dementia) was proposed by Mégret et al. [8]. IMMED relies on wear- able cameras to capture video and audio data of older adults’ daily activities to assess cognitive decline. 67 Drug Intervention Follow up with Internet of Things As the importance of health monitoring becomes increasingly recognized, more IT applications are being developed to assist with self-health management. For example, Sadek et al. [9] proposed an unobtrusive Apnea detection using a bed sensor and Sadek et al. [10] proposed a sleep monitoring system using a smart bed. Additionally, smart- watches have been utilized in activity recognition [11] and as a stress detection using heart rate variability [12]. Another technique for monitoring the indoor daily activity was proposed by Ahmed et al. [13] who relied on a thermal sensor array-based (TSA-based) system that can monitor sleeping activity, daily activity, and the no-activity class of a person. Drug intervention management is important for health management and started to attract attention of clinical research. 2 Related Work [20] in a primary care setting, an interdisciplinary pharmacist-physician intervention model based on alerts generated by a computerized alert system was implemented to reduce the use of PIM H. M. Ahmed et al. 68 in community-dwelling older adults. The study found that a majority of the patients included in the study had medication change suggested by the pharmacist, indicating the potential effectiveness of this intervention model. These results can be generalized older adults living in different regions of the world. Studies conducted in New Zealand [21], Taiwan [22], and Texas [23] have highlighted the prevalence of PIM use and associated factors and suggested the need for screening tools such as the Beers criteria to identify PIMs and interventions to optimize or minimize their use. p Additionally, the use of PIMs can increase the likelihood of adverse drug reactions (ADRs), the harmful or unintended reactions to medications. The prevalence of ADRs is yet another signiﬁcant concern for health care. Speciﬁcally, Beijer and de Blaey [24] found that among the older adult population one in six patients are for ADR as opposed to ~ 4% in the younger population [24]. Various studies have highlighted the burden of ADRs in hospital inpatients at large [25]. These studies have identiﬁed risk factors such as increasing age, admission to medical wards, and the number of regular medica- tions. Strategies to minimize the burden of ADRs include computerized prescribing and monitoring systems, pharmacist involvement in ward rounds, improved monitoring, and education on prescribing practices. Harmful events resulting from PIMs also includes adverse drug events (ADEs) [26] that are more likely to occur in older adults with mul- tiple chronic conditions as well as older adults taking speciﬁc medication classes. This highlights the importance of considering the overall health and medication proﬁle of older adults to minimize the risk of ADEs. A study by Bressler et al. [27] emphasized the need for physicians to be continuously aware of potential adverse drug interac- tions in older patients and to consult pharmacists and/or patients to ensure appropriate medication prescribing. As highlighted by numerous studies [18, 17, 27], ensuring appropriate medication prescription and monitoring of older patients is crucial to avoid ADEs and ADRs. This emphasizes the need for continuous monitoring of patients’ behavior and vital signs, a task that can be facilitated using home-based non-intrusive IoT technology. 3 Methodology In this section, we ﬁrst describe our methodology at the system implementation level where we are present our system architecture and our approach to deployment. Following, we present the data analysis algorithm we used in the data collection section. 2 Related Work In this context, our proposed system aims at extending the monitoring of patients’ physical activity levels using IoT technology, with the objective of detecting the impact of an inhibitor drug on the subject’s behavior changes over time. 3.1 System Implementation Architecture Overview: This project relies on the AMI-Platform [28] solution we have developed. Data ﬂows through the platform as follows: A cluster of heterogeneous sensors at the end-user layer collects environmental and biomedical data, such as tem- perature, heart rate, and location. This data is then sent to the physical gateway for pre-processing and transmitted to the cloud layer through a secure channel established Drug Intervention Follow up with Internet of Things 69 by the network layer. At the cloud layer, the data is decoded, processed, and stored in databases. The business layer serves as the user interface, offering dynamic graphs and charts for improved data visualization. Deployment: We setup two gateway nodes. The ﬁrst node handles serial communi- cation with the sleep mat sensor that collects Ballistocardiogram (BCG), from which we can infer on the participant’s breathing rate and heartrate during sleep. The second node handles communication with all other deployed sensors that measures the participant’s indoor activity, including door sensors but also motion sensors that report on luminance, humidity, and temperature. 3.2 Data Collection and Analysis In this study, a subject has been monitored for 7 months, from the end of July 2021 to early January 2022. The objective of this study is to detect changes in the overall behavior of the monitored subject after being given doses of a drug that inhibits physical activity. We have imputed missing activity values using the approach proposed by Ahmed et al. [29]. The overall activity curve for the subject inside his residence during the entire monitoring period is presented by Fig. 1. The timestamps at which these doses are given to the subject are tabulated in Table 1. Table 1. Timestamps at which actions were performed by the team. able 1. Timestamps at which actions were performed by the te Timestamp Action performed 27th July 2021 System installation 3rd August 2021 First visit 26th October 2021 Second visit 6th January 2022 Last visit The detection algorithm measures the mean and standard deviation within a 3-days sliding window of activity calculated along the complete time series for the subject’s physical activity. Box-and-whisker plots were than calculated (Fig. 2) and used to deﬁne outliers for both the averages and standard deviations. Note that in our box-and-whisker plots outliers are calculated as all values above or below 1.5 times the interquartile range (the interquartile range where 50% of the data around the median). We deﬁned outliers as change timestamps for the subject’s behavior. These outlier values are then back projected to the time domain to obtain the corresponding change timestamps. The corresponding change timestamps are presented in Table 2. The mean and standard deviation timestamps by both average and standard deviation at which the behavior changes are plotted with red dots overlapped on the same overall activity curve and presented by Fig. 2. 70 H. M. Ahmed et al. The change detected by the mean sliding window conforms with the expected times- tamps for the inhibition effect more than that detected by the standard deviation sliding window. This is because these timestamps are close enough in temporal domain to those timestamps at which the 3 doses are given to the subject. However, the standard deviation sliding window detects more change timestamps than the average sliding window does. Although the change is detected at the end of the window, the right change timestamp is corrected by the window size, i.e., three days are subtracted from the detected timestamp. 4 Data Analysis Results Ahmed et al. H. M. Ahmed et al. 72 Table 2. Detected changes’ timestamps and their corresponding values and source of detection. Detected Change Timestamp Outlier Value Source of Detection 7/29/2021 6181 Average 7/30/2021 10400 Average Standard Deviation 7/31/2021 3750 Standard Deviation 8/1/2021 4545 Standard Deviation 8/19/2021 4227 Average 10/30/2021 2337 Standard Deviation 11/8/2021 3922 Standard Deviation 11/9/2021 4877 Average 12/4/2021 2834 Standard Deviation 12/7/2021 508 Average Standard Deviation 12/9/2021 3181 Average 12/10/2021 7076 Average Standard Deviation 12/11/2021 5463 Standard Deviation 12/23/2021 7501 Average 12/25/2021 7204 Average Detected changes’ timestamps and their corresponding values and source of detection. 4 Data Analysis Results Daily activity time series for the subject under the effect of an activity inhibiting drug is presented in Fig. 1. There are two behavior states that can be found along the time series, and both have a similar meaning. One is characterized by a relatively low standard devi- ation, we referred to this state as the background activity state. The other is characterized by high standard deviations, we refer to this state as an (low or high) activity induced state. The background activity state is dominant across the monitoring period while the activity induced state occurs for short period in mid-August 2021, at the beginning of November 2021 and during the ﬁrst week of December 2021. The background activity state is characterized by activity values ranging approximately between 5000 to 7000 movements (per day) with a standard deviation ranging between 0 and roughly 1800 (Fig. 3. Subject behavior changes timestamps overlapped on the activity timeseries). While the activity induced state is characterized by activity values higher than 7000 or lower than 4000 movements (per day). Fig. 1. Subject physical activity time series. Fig. 1. Subject physical activity time series. Drug Intervention Follow up with Internet of Things 71 71 Fig. 2. Boxplot and outliers for both average and standard deviation sliding window. Fig. 2. Boxplot and outliers for both average and standard deviation sliding window. In our pilot study, 60% of the detected change (outliers) timestamps are associated with the standard deviation sliding windows’ outliers while the remaining 40% of the detected change timestamps are associated to the mean sliding. Note also that there is a change detection overlap of for 25% of the detected timestamps. There are six groups of change timestamps distributed along the entire daily physical activity timeseries of the subject (Fig. 3. Subject behavior changes timestamps overlapped on the activity timeseries). The 1st group starts in late July and ends in early August 2021, the 2nd group span the second half of August 2021, the 3rd group is in late October 2021, the 4th group is at the beginning of November 2021, the 5th group is at the beginning of December 2021, and the 6th group is in late December 2021. The typical changes’ timestamps are tabulated in Table 2. Fig. 3. Subject behavior changes timestamps overlapped on the activity timeseries. Fig. 3. Subject behavior changes timestamps overlapped on the activity timeseries. H. M. 6 Conclusion In this paper we presented a promising application of IoT to monitor inhibitor drugs. We proposed to monitor the behavior and activity of individuals under the inﬂuence of drugs to assess their effectiveness, identify side effects, and make timely adjustments to drug levels based on patient status. As such, we have presented a novel approach for monitoringandanalyzingthebehaviorofasubjectinanindoorenvironmentoveraperiod of seven months. Our approach relies on the IoT AMI-Platform solution which collected data from a variety of sensors deployed in the participant’s environment. Our method uses slidingwindowstocalculatethemeanandstandarddeviationofthesubject’sactivityover a 3-day period. Following, we rely on box-and-whisker plots to detect outliers in these values. These outliers represent changes in the subject’s behavior, which can be further analyzed to understand the effects of speciﬁc interventions, such as the administration of an inhibitor drug. Our results also show the effectiveness of our approach in detecting changes in behavior over a 7-month monitoring period. Speciﬁcally, we were able to observe the expected decline in the subject’s physical activity following the administration of the inhibitor drug, as well as recovery periods following each dose. Such information is useful to medical professionals to evaluate the effectiveness of the drug and making decisions about its administration in the future. They also highlight our platform as a valuable tool for deploying and analyzing data collected from sensor networks in a variety of applications, including in healthcare as presented in our case study. 5 Discussion For the inhibition drug experiment the average activity level is very high; about 6000 movements on average per day as the subject is a young person. Based on the timeseries, the subject’s activity level declines from late July 2021 to mid-August 2021. This decline is conformed with the expected outcome of the inhibition drug given to the subject as the effect of the drug is to inhibit the physical activity of the subject. This observation is in-line with the timestamp marked by the medical staff as the drug’s 1st dose intake, as reported in Table 1. There is another decline in the subject’s physical activity from October 30th 2021, to November 9th November 2021, which matches the administration of the 2nd dose as reported by the medical staff. A 3rd decline in the subject’s physical activity is observed and detected during the period extending from late December 2021 to early January 2022. In addition, a recovery period representing the subject’s convales- cence from the inhibition drug to his normal overall daily physical activity is observed from mid-August 2021 to early September 2021. A 2nd recovery period is also observed in mid-November 2021. From the subject’s activity timeseries, we can observe that a convalescence period of roughly one week is required for the subject to recover from the effect of the inhibition drug. 73 Drug Intervention Follow up with Internet of Things References 1. Georgios, L., Kerstin, S., Theofylaktos, A.: Internet of things in the context of industry 4.0: An overview (2019) 2. Bolden, J.E., Peart, M.J., Johnstone, R.W.: Anticancer activities of histone deacetylase inhibitors. Nat. Rev. Drug Discov. 5(9), 769–784 (2006) 3. Julien, R.M.: A primer of drug action: A concise nontechnical guide to the actions, uses, and side effects of psychoactive drugs, revised and updated. Holt Paperbacks (2013) 4. Fowler, J.S., Volkow, N.D., Wang, G.-J., Ding, Y.-S., Dewey, S.L.: PET and drug research and development. J. Nucl. Med. 40(7), 1154–1163 (1999) 5. Lin, Q., Zhao, Q.: IoT Applications in Healthcare. Internet of Things: Cases and Studies, pp. 115–133 (2021) 6. Gao, T., Greenspan, D., Welsh, M., Juang, R.R., Alm, A.: “Vital signs monitoring and patient tracking over a wireless network. In: 2005 IEEE Engineering in Medicine and Biology 27th Annual Conference, pp. 102–105 (2006) pp 7. Al-Khafajiy, M., et al.: Remote health monitoring of elderly through wearable sensors. Multimed Tools Appl. 78(17), 24681–24706 (2019) 8. Mégret, R., et al.: The IMMED project: wearable video monitoring of people with age demen- tia. In: Proceedings of the 18th ACM International Conference on Multimedia, pp. 1299–1302 (2010) 9. Sadek, I., Seet, E., Biswas, J., Abdulrazak, B., Mokhtari, M.: Nonintrusive vital signs monitoring for sleep apnea patients: a preliminary study. IEEE Access 6, 2506–2514 (2017) 10. Sun, X., Qiu, L., Wu, Y., Tang, Y., Cao, G.: Sleepmonitor: Monitoring respiratory rate and body position during sleep using smartwatch. Proc. ACM Interact Mob. Wearable Ubiquitous Technol. 1(3), 1–22 (2017) 74 H. M. Ahmed et al. 11. Shahmohammadi, F., Hosseini, A., King, C.E., Sarrafzadeh, M.: Smartwatch based activity recognition using active learning. In: 2017 IEEE/ACM International Conference on Con- nected Health: Applications, Systems and Engineering Technologies (CHASE), pp. 321–329 (2017) 12. Chalmers, T., et al.: Stress watch: The use of heart rate and heart rate variability to detect stress: a pilot study using smart watch wearables. Sensors 22(1) (2022). https://doi.org/10. 3390/s22010151 13. Ahmed, H.M., Abdulrazak, B.: Monitoring indoor activity of daily living using thermal imag- ing: a case study. Inter. J. Adv. Comput. Sci. Appli. 12(9) (2021). https://doi.org/10.14569/ ijacsa.2021.0120902 14. Reeve, E., Gnjidic, D., Long, J., Hilmer, S.: A systematic review of the emerging deﬁnition of ‘deprescribing’with network analysis: implications for future research and clinical practice. Br. J Clin. Pharmacol. 80(6), 1254–1268 (2015) 15. References Ailabouni, N., Mangin, D., Nishtala, P.S.: DEFEAT-polypharmacy: deprescribing anticholin- ergic and sedative medicines feasibility trial in residential aged care facilities. Int. J. Clin. Pharm. 41(1), 167–178 (2019) 16. Dearing, M.E., et al.: Pharmacist-led intervention to improve medication use in older inpa- tients using the Drug Burden Index: a study protocol for a before/after intervention with a retrospective control group and multiple case analysis. BMJ Open 10(2), e035656 (2020) 17. Arvisais, K., et al.: A pharmacist–physician intervention model using a computerized alert system to reduce high-risk medication use in elderly inpatients. Drugs Aging 32, 663–670 (2015) 18. Cossette, B., et al.: Knowledge translation strategy to reduce the use of potentially inap- propriate medications in hospitalized elderly adults. J Am Geriatr Soc 64(12), 2487–2494 (2016) 19. Cossette, B., et al.: Reduction in targeted potentially inappropriate medication use in elderly inpatients: a pragmatic randomized controlled trial. Eur J Clin Pharmacol 73, 1237–1245 (2017) 20. “A pharmacist-physician intervention model using a computerized alert system to reduce high-risk medication use in primary care” 21. Nishtala, P.S., Bagge, M.L., Campbell, A.J., Tordoff, J.M.: Potentially inappropriate medicines in a cohort of community-dwelling older people in N ew Z ealand. Geriatr Gerontol Int 14(1), 89–93 (2014) 22. Iversen, H.H., Bjertnæs, Ø.A., Skudal, K.E.: Patient evaluation of hospital outcomes: An analysis of open-ended comments from extreme clusters in a national survey. BMJ Open 4(5) (2014). https://doi.org/10.1136/bmjopen-2014-004848 23. Holmes, H.M., Luo, R., Kuo, Y.F., Baillargeon, J., Goodwin, J.S.: Association of potentially inappropriate medication use with patient and prescriber characteristics in Medicare Part D. Pharmacoepidemiol Drug Saf 22(7), 728–734 (2013). https://doi.org/10.1002/pds.3431 24. Beijer, H.J.M., de Blaey, C.J.: Hospitalisations caused by adverse drug reactions (ADR): a meta-analysis of observational studies. Pharm. World Sci. 24(2), 46–54 (2002). https://doi. org/10.1023/A:1015570104121 25. Davies, E.C., Green, C.F., Taylor, S., Williamson, P.R., Mottram, D.R., Pirmohamed, M.: Adverse drug reactions in hospital in-patients: a prospective analysis of 3695 patient-episodes. PLoS ONE 4(2), e4439 (2009) 26. Field, T.S., et al.: Risk factors for adverse drug events among older adults in the ambulatory setting. J Am Geriatr Soc 52(8), 1349–1354 (2004). https://doi.org/10.1111/j.1532-5415. 2004.52367.x 27. Bressler, R., Bahl, J.J.: Principles of drug therapy for the elderly patient. In: Mayo Clinic Proceedings, pp. 1564–1577 (2003) Drug Intervention Follow up with Internet of Things 75 28. Abdulrazak, B., Paul, S., Maraoui, S., Rezaei, A., Xiao, T.: IoT Architecture with Plug and Play for fast deployment and system reliability: AMI Platform. References In: International Conference on Smart Homes and Health Telematics, pp. 43–57 (2022) 29. Ahmed, H.M., Abdulrazak, B., Blanchet, F.G., Aloulou, H., Mokhtari, M.: Long gaps missing IoT sensors time series data imputation: a bayesian gaussian approach. 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https://openalex.org/W2976298046	https://europepmc.org/articles/pmc6972717?pdf=render	English	null	A systematic review on the effects of group singing on persistent pain in people with long‐term health conditions	European journal of pain	2,019	cc-by	13,425	R E V I E W A R T I C L E R E V I E W A R T I C L E J. Yoon Irons1,2 \| David Sheffield3 \| Freddie Ballington4 \| Donald E. Stewart5 Group singing may be an effective and safe approach for reducing persistent pain and depression in people with long‐term health conditions. Significance: This systematic review assesses research evidence for the effective- ness of group singing on chronic pain in people with long‐term health conditions. Narrative syntheses revealed that there is partial support for singing effects on some pain outcomes based on the limited available evidence of varied quality. Qualitative data provided additional support of physical, psychological and social benefits. The review highlights implications for practice and future studies. Abstract Background and Objectives: Singing can have a range of health benefits; this paper reviews the evidence of the effects of group singing for chronic pain in people with long‐term health conditions. Database and Data Treatment: We searched for published peer‐reviewed singing studies reporting pain measures (intensity, interference and depression) using major electronic databases (last search date 31 July 2018). After screening 123 full texts, 13 studies met the inclusion criteria: five randomized controlled trials (RCTs), seven non‐RCTs and one qualitative study. Included studies were appraised using Downs and Black and the Critical Appraisals Skills Programme quality assessments. 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. European Journal of Pain published by John Wiley & Sons Ltd on behalf of European Pain Federation ‐ EFIC ® 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. wileyonlinelibrary.com/journal/ejp \| Received: 29 March 2019 \| Revised: 16 September 2019 \| Accepted: 19 September 2019 Received: 29 March 2019 \| Revised: 16 September 2019 \| Accepted: 19 September 2019 Received: 29 March 2019 \| Revised: 16 September 2019 \| Accepted: 19 September 2019 DOI: 10.1002/ejp.1485 J. Yoon Irons1,2 \| David Sheffield3 \| Freddie Ballington4 \| Donald E. Stewart5 1Health and Social Care Research Centre, University of Derby, Derby, UK 2Queensland Conservatorium Research Centre, Griffith University, Brisbane, Australia 3University of Derby Online Learning, Derby, UK 4University of Derby, Derby, UK 5School of Medicine, Griffith University, Brisbane, Australia Correspondence J. Yoon Irons, Health and Social Care Research Centre (N304), University of Derby, Kedleston Road, Derby DE22 1GB, UK. Email: y.irons@derby.ac.uk 1Health and Social Care Research Centre, University of Derby, Derby, UK 2Queensland Conservatorium Research Centre, Griffith University, Brisbane, Australia 3University of Derby Online Learning, Derby, UK 4University of Derby, Derby, UK 5School of Medicine, Griffith University, Brisbane, Australia Correspondence J. Yoon Irons, Health and Social Care Research Centre (N304), University of Derby, Kedleston Road, Derby DE22 1GB, UK. Email: y.irons@derby.ac.uk 1Health and Social Care Research Centre, University of Derby, Derby, UK 2Queensland Conservatorium Research Centre, Griffith University, Brisbane, Australia 3University of Derby Online Learning, Derby, UK 4University of Derby, Derby, UK 5School of Medicine, Griffith University, Brisbane, Australia Abstract Background and Objectives: Singing can have a range of health benefits; this paper reviews the evidence of the effects of group singing for chronic pain in people with long‐term health conditions. Database and Data Treatment: We searched for published peer‐reviewed singing studies reporting pain measures (intensity, interference and depression) using major electronic databases (last search date 31 July 2018). After screening 123 full texts, 13 studies met the inclusion criteria: five randomized controlled trials (RCTs), seven non‐RCTs and one qualitative study. Included studies were appraised using Downs and Black and the Critical Appraisals Skills Programme quality assessments. Results: Included studies reported differences in the type of singing intervention, long‐term condition and pain measures. Due to the high heterogeneity, we conducted a narrative review. Singing interventions were found to reduce pain intensity in most studies, but there was more equivocal support for reducing pain interference and depression. Additionally, qualitative data synthesis identified three key linked and complementary themes: physical, psychological and social benefits. Conclusion: Group singing appears to have the potential to reduce pain intensity, pain interference and depression; however, we conclude that there is only partial support for singing on some pain outcomes based on the limited available evidence of varied quality. Given the positive findings of qualitative studies, this review recommends that practitioners are encouraged to continue this work. More studies of better quality are needed. Future studies should adopt more robust methodology and report their singing intervention in details. 72 \| 1 \| INTRODUCTION IRONS IRONS 72 for one's life (Price, 1999) as manifested by negative emo- tions related to pain (e.g. depression and anxiety). The final component of this model concerns overt behavioural expres- sions of pain (e.g. ability to participate in daily responsibili- ties), which accord with quality of life measures, particularly when they pertain to activities of daily life and interference. Focusing on these different components may aid understand- ing of how singing influences pain processing. Singing requires the active involvement of both vocal appa- ratus and the respiratory system (Irons, Kenny, McElrea, & Chang, 2012; Irons, Kuipers, & Petoz, 2013; Irons, Petocz, Kenny, & Chang, 2019). Singing encourages the active use of diaphragmatic breathing, which promotes deep and slow breathing. This diaphragmatic breathing influences a num- ber of important physiological functions, such as the cardio- vascular system and the autonomic nervous system (Russo, Santarelli, & O’Rourke, 2017). Singing also stimulates both the auditory and sensory‐motor pathways in the brain (Wan, Ruber, Hohmann, & Schlaug, 2010). Recently, increasing number of studies have shown the potential health benefits of group singing programmes for people with long‐term health conditions, such as chronic obstructive pulmonary disease (Bonilha, Onofre, Vieira, Almeida Prado, & Martinez, 2009; Lord et al., 2010, 2012; Morrison et al., 2013; Skingley, Clift, Hurley, Price, & Stephens, 2018) and Parkinson's (Fogg‐ Rogers et al., 2016; Stegemoller, Radig, Hibbing, Wingate, & Sapienza, 2017). However, the role of singing in chronic health conditions with persistent pain is unclear. It is plausible that singing will influence pain as singing increases body relaxation and reduces stress levels (Ma et al., 2017). A recent study found that people who practiced relax- ing deep slow breathing patterns had increased pain thresh- olds and reduced pain sensitivity (Busch et al., 2012). Singing, compared with passively listening to music, also activates the body's own pain relief function (endorphins) and elevates positive mood (Dunbar, Kaskatis, MacDonald, & Barra, 2012). Further, singing might influence pain processing through distraction—focusing attention away from pain may reduce the impact of pain and how it interferes with activities of daily living (Blomqvist & Hallberg, 2002). Correspondence Results: Included studies reported differences in the type of singing intervention, long‐term condition and pain measures. Due to the high heterogeneity, we conducted a narrative review. Singing interventions were found to reduce pain intensity in most studies, but there was more equivocal support for reducing pain interference and depression. Additionally, qualitative data synthesis identified three key linked and complementary themes: physical, psychological and social benefits. Conclusion: Group singing appears to have the potential to reduce pain intensity, pain interference and depression; however, we conclude that there is only partial support for singing on some pain outcomes based on the limited available evidence of varied quality. Given the positive findings of qualitative studies, this review recommends that practitioners are encouraged to continue this work. More studies of better quality are needed. Future studies should adopt more robust methodology and report their singing intervention in details. Group singing may be an effective and safe approach for reducing persistent pain and depression in people with long‐term health conditions. Significance: This systematic review assesses research evidence for the effective- ness of group singing on chronic pain in people with long‐term health conditions. Narrative syntheses revealed that there is partial support for singing effects on some pain outcomes based on the limited available evidence of varied quality. Qualitative data provided additional support of physical, psychological and social benefits. The review highlights implications for practice and future studies. Eur J Pain. 2020;24:71–90. 72 \| 1 \| INTRODUCTION Finally, singing is a group activity that leads to social cohesion and emotional support (Pearce et al., 2016; Tarr, Launay, & Dunbar, 2014; Weinstein, Launay, Pearce, Dunbar, & Stewart, 2016), which has been related to pain (Bernardes, Forgeron, Fournier, & Reszel, 2017; Brown, Sheffield, Leary, & Robinson, 2003; Pearce et al., 2016; Tarr et al., 2014; Weinstein et al., 2016). Using the pain processing model domains, this review aims to answer: To what extent does singing influence pain inten- sity, unpleasantness, interference and depressive symptoms in people with long‐term health conditions? Chronic pain is defined by its persisting for more than 3 months in one or more parts of the body (Treede et al., 2015). Chronic pain is a multi‐dimensional experience, in- volving not only biological but also sensory, cognitive and affective processes, which negatively affects one's physical, emotional and social functioning (Gatchel, Peng, Peters, Fuchs, & Turk, 2007; Moseley & Butler, 2015). A large European survey study found that chronic pain is prevalent in adults: 19% of survey respondents reported to have moderate to severe pain intensity, and to have changed or lost jobs as well as being diagnosed with depression due to pain (Breivik, Collett, Ventafridda, Cohen, & Gallacher, 2006). Persistent pain due to long‐term health conditions (e.g. Parkinson's, stroke and dementia) is common (Achterberg et al., 2013; Hénon, 2006; Skogar & Lokk, 2016). People living with persistent pain also experience depression, anxiety, anger, reduced self‐efficacy and self‐esteem (Burke, Mathias, & Denson, 2015). Pain is usually recognized as multi‐dimen- sional comprising at least sensory and affective compo- nents as well as having consequences on everyday activities (Melzack & Casey, 1968). One approach to understand the ways in which singing might influence individuals is through Price's Pain Processing model (Price, 1999; Riley et al., 2002). This model consists of a number of stages: first, an initial sensory‐discriminative stage, where the major compo- nent is the perceived intensity of the pain sensation; second, a stage of pain processing, unpleasantness, reflects an indi- vidual's immediate affective response and involves limited cognitive processing; third, there is a stage of pain processing that involves longer term reflective or cognitive processes that relate to the meanings or implications that pain holds 2.4 Two review authors (JYI & DS) screened the eligibility of studies for inclusion in the review, based on the inclu- sion criteria. Any disagreements were resolved in consul- tation with a third author (FB); however, there were no discrepancies. p y p Based on the framework of Price's Pain Model (Price, 1999; Riley et al., 2002) and consistent with current re- search in chronic pain (Aaron, Fisher, de la Vega, Lumley, & Palermo, 2019), we chose these pain outcomes. Pain inten- sity is “magnitude of experienced pain” (Cook et al., 2013); unpleasantness is individual's immediate affective response (e.g. how unpleasant/horrible the pain is) (Riley et al., 2002). Pain interference refers to functional limitations, i.e., how much does pain impact on physical function, work, recre- ation, social activities, family roles, activities of daily living and sleep (Cook et al., 2013). Depression commonly coexists with chronic pain; can further aggravate the severity of pain experience as well as depression (Sheng, Liu, Wang, Cui, & Zhang, 2017). Thus, it is vital to assess depression when as- sessing pain (Dansie & Turk, 2013). Two review authors (JYI & DS) independently extracted data from a data collection form, including citation details, trial setting, inclusion and exclusion criteria, participants (e.g. age, gender, health condition, related pain history), intervention de- tails (e.g. intensity and duration of intervention, types of sing- ing, lists of songs, information on facilitators, etc.), outcome measures (e.g. self‐reported pain measures, depression) and results (statistical techniques used, p values and effect sizes). We contacted study authors for any missing data and further information. A third author (FB) independently reviewed the extracted data to ensure accuracy and reliability, with review- ers meeting to confirm agreement of extraction and to estab- lish reliability. Where there were discrepancies, these were resolved by discussion; agreement was high (Kappa = 0.98). Secondary outcomes: number of general practitioner (GP)/ pain specialist's visits. These were assessed by self‐report. Methodological quality of the included trials was as- sessed by two independent reviewers (JYI & FB) and a third author was consulted when there were discrepancies. For both RCTs and non‐RCTs, we have utilized a modi- fied Downs and Black quality assessment checklist to as- sess study qualities (Downs & Black, 1998). We simplified the question No.27 where we scored one for carrying out a power calculation and zero for no power calculation based on previous recommendations (Kennelly, 2011) (Please see Appendix S2). 2.1 \| Inclusion and exclusion criteria Given singing interventions are relatively new in the field of pain, we aimed to quantify the field and identify the research gaps. Hence, we included non‐controlled studies as well as randomized controlled trials (RCTs). We included peer‐re- viewed singing studies with people with long‐term health con- ditions associated with persistent pain: for example, arthritis, cardiovascular diseases, cancers, chronic respiratory diseases, diabetes, fibromyalgia and dementia (Wimo, Jönsson, Bond, Prince, & Winblad, 2013). We also included studies involving cancer patients, as cancer is associated with persistent pain. We excluded long‐term mental health conditions, as a recent systematic review on the effects of singing for mental health service users has been published (Williams, Dingle, & Clift, 2018). We excluded studies of professional singers and stud- ies with children and adolescent samples. We also excluded studies that had very brief singing interventions (<2 weeks), had interventions that were not facilitated by professionals with a relevant qualification (e.g. music therapist, professional 73 IRONS singing teachers, speech therapists, musicians, nurses or occu- pational therapists) or did not collect pre‐ and post‐interven- tion pain data. We did not have language restrictions. and Repository for Arts and Health Resources (www.artsh ealthresources.org.uk/) for relevant studies. We inspected the reference lists of relevant studies and citing articles to capture any articles missed through our database searches. The end date for searches was 31 July 2018. 2.4 Further, a Critical Appraisals Skills Programme (CASP) checklist was employed to assess qual- ities in the studies which reported qualitative data (CASP, 2018). We graded studies' qualities as poor, fair and good according to their scores of either the Downs and Black or CASP checklist. 2.2 Primary outcomes: Pain intensity, unpleasantness, pain inter- ference and depression associated with pain measured by val- idated questionnaires, such as the Brief Pain Inventory (BPI) and Hospital Anxiety Depression Scale (HADS). 2.3 The protocol for this review was registered with the International Prospective Register of Systematic Reviews (PROSPERO) (ID 2016 CRD42016047557). This re- view was conducted in accordance with the Cochrane Systematic Review methods (Higgins & Green, 2011); it is reported using the Preferred Reporting Items for Systematic Reviews and Meta‐Analyses (PRISMA) guidelines (Moher, Liberati, Tetzlaff, & Altman, 2009) and in accordance with A MeaSurement Tool to Assess systematic Reviews criteria (Shea et al., 2007), to allow for evaluation and to reduce the potential for bias in the review. Major database searches were performed by the authors with experience of conducting systematic reviews (JYI, DS and FB), using key words, based on advice from experi- enced librarians, related to singing, chronic health conditions and pain (Please see Appendix S1 for the detailed search terms). Databases searched included Cochrane Database of Systematic Reviews, Cochrane Central Register of controlled trials, MEDLINE (EBSCO platform), PUBMED, CINAHL, WEB OF SCIENCE, PSCYINFO, SCOPUS, CORE* and Google Scholar* (first 20 most relevant studies were in- spected). In addition, we searched clinical trials registry (www.clinicaltrials.gov/), Dimensions (www.dimensions.ai/) 2.5 \| Effect sizes were extracted for each study and, where nec- essary, were calculated using means, standard deviations and sample sizes at baseline and post‐intervention of ex- perimental and control conditions (Decoster & Claypool, 2004). Where such statistics were missing, we used F‐sta- tistics, t‐values and p‐values to calculate effect sizes; we used Cohen’s (1988) suggestions to categorize effect size as small, medium and large. RCT effects were categorized IRONS 74 Morrison et al., 2013; Reagon et al., 2017; Stegemoller et al., 2017) and one qualitative study (Hopper, Curtis, Hodge, & Simm, 2016). All RCTs and non‐RCTs reported the effects of group singing on pain intensity and/or interference. Two studies recruited chronic pain patients (Bradt et al., 2016; Kenny & Faunce, 2004), three studies involved old people with Alzheimer's disease (Clements‐Cortes, 2013, 2015; Pongan et al., 2017), two studies recruited cancer survivors (Gale et al., 2012; Reagon et al., 2017) and two studies were with people with Parkinson's and stroke survivors (Fogg‐ Rogers et al., 2016; Stegemoller et al., 2017). Descriptions of included studies are presented in Table 1. Two Chronic Obstructive Pulmonary Disease (COPD) trials could not be included in the meta‐analysis, as the trials' authors were un- able to provide data on pain interference (SF‐12) (Lord et al., 2010, 2012). One study (Hopper et al., 2016) reported only qualitative findings of group singing interventions for chronic pain patients, while seven studies reported qualita- tive data within a mixed‐methods design (Bradt et al., 2016; Clements‐Cortes, 2013, 2015; Fogg‐Rogers et al., 2016; Gale et al., 2012; Reagon et al., 2017; Tamplin et al., 2013). using “significantly favours intervention,” “trends towards intervention,” “no difference,” “trends towards control” and “significantly favours control” (Cooper, Hedges, & Valentine, 2009) Authors were contacted when relevant data were not reported in the article; the following au- thors provided additional data: Kenny and Faunce (2004); Clements‐Cortes (2013, 2015); Grape, Töres, Britt‐Maj, and Rolf (2009); Morrison et al. (2013); Pongan et al. (2017); Tamplin (2013); Stegemoller (2017) and Reagon (2017). We conducted narrative syntheses based on the framework by Popay and colleagues (Popay et al., 2006). 3.1 \| Description of studies and results of searches The electronic database searches found 575 records. After screening abstracts and full‐text review, 13 studies met the inclusion criteria (Figure 1 for PRISMA flow diagram) in- cluding five randomized controlled trials (RCTs) (Bradt, Norris, Shim, Gracely, & Gerrity, 2016; Grape et al., 2009; Kenny & Faunce, 2004; Pongan et al., 2017; Tamplin et al., 2013); seven non‐randomized controlled trials (non‐ RCTs) (Clements‐Cortes, 2013, 2015; Fogg‐Rogers et al., 2016; Gale, Enright, Reagon, Lewis, & Van Deursen, 2012; Twelve included studies, with a total of 311 participants, evaluated the effects of group singing on pain intensity and/ or pain interference, and seven of those studies also reported the effects of singing on depression; no included study re- ported pain unpleasantness (Tables 2 and 3). All studies were FIGURE 1 PRISMA flow diagram Records idenﬁed through database searching (n = 575) Screening Included Eligibility Idenﬁcaon Addional records idenﬁed through other sources (n = 1) Records aer duplicates removed (n = 443) Records screened (n = 283) Records excluded (n = 160) Full-text arcles assessed for eligibility (n = 123) Full-text arcles excluded, with reasons (n = 110) • No pain assessment = 51 • Intervenon included singing and/or other acvies = 30 • Including children = 9 • Literature review = 8 • Study Protocol = 1 • Thesis = 1 • Non-chronic condions = 8 • Pain data was not available = 2 Studies included in narrave synthesis (n = 13) RCT (n = 5) Mixed-methods (n =7) Qualitave (n =1) Records idenﬁed through database searching (n = 575) Addional records idenﬁed through other sources (n = 1) Records aer duplicates removed (n = 443) Records screened (n = 283) Records excluded (n = 160) Full-text arcles assessed for eligibility (n = 123) IRONS Study ID (1st au- thor, year, Country) Diagnosis, number of Participants at baseline, Mean age Study design, control group condition Singing interven- tion frequency and intensity, total hours of singing Setting, facilitator, repertoires Pain outcome measures, Pain questionnaire Depression measure/ Questionnaire Comments Kenny & Faunce (2004)a, Australia Chronic Pain, N = 77, 40.02 years RCT, Exercises group 0.5‐hr session, three times per week for 3 weeks, 4.5 hr Chronic Pain Management Centre (Royal North Shore Hospital, Sydney), Singing/ piano teacher, Songs with repetitive phrases Pain interfer- ence, (pain disabil- ity questionnaire) Zung depression inventory Grape et al. 3.1 \| Description of studies and results of searches (2009)a, Sweden Irritable Bowel Syndrome, N = 55, NR RCT, Education group 1‐hr session, once weekly for 1 year (46 hr) Not reported, Not reported, Not reported Pain Interference (GSRS‐IBS’s Pain subscale) None Gale et al. (2012)a, UK Cancer, N = 32, 59 years Non‐RCT, No com- parison group 2‐hr session, once weekly for 12 weeks (24 hr) Community, Professional musicians, Arranged music Pain intensity & Pain interference (SF−36 Bodily pain subscale) Hospital Anxiety Depression Scale (HADS) Qualitative analysis was performed on interviews. Tamplin et al. (2013)a, Australia Spinal cord injury, N = 24, 45 years RCT, Music appre- ciation & relaxa- tion group 1‐hr session, three times per week for 12 weeks (36 hr) Not reported, Music therapist, Not reported Pain Intensity (AQoL−4D) Profile of mood States (POMS) Qualitative analysis was performed on post‐intervention interviews. Morrison et al. (2013)a, UK COPD, N = 106, 69.5 years Non‐RCT, No com- parison group 1.5‐hr session, once weekly over 36 weeks (54 hr) Community, Experienced group singing facilitators, Familiar & new songs Pain Interference (SF‐12 Bodily pain subscale) EQ‐5D (depression/ anxiety subscale) Participants' written comments about their experience were presented. Clements‐Cortes (2013)a, Canada Dementia, N = 28, 72.9 years Non‐RCT, No com- parison group 1‐hr session, once weekly for 16 weeks (16 hr) Adult day‐care centre, Music therapist, Familiar & simple songs that participants chosen Pain Intensity & Pain Interference (AQoL) None Qualitative analysis was performed on interview transcripts. Clements‐Cortes (2015)a, Canada mild to moder- ate Alzheimer's Disease, N = 35, range 66–99 years Non‐RCT, (Group 1) older people with AD; (Group 2) significant others/staff/ volunteers 1‐hr session, once weekly for 16 weeks (16 hr) Long‐term care facility, Music therapist, Participants' pre- ferred songs from the 1930s to 1940s Pain Intensity Scale (adopted from FLACC Scale) None Qualitative analysis was performed on interview transcripts. Bradt et al. (2016), USA Chronic Pain, N = 55, 53.75 years RCT, Wait‐list group 1‐hr session, once weekly for 8 weeks (8 hr) Health Centre, Music thera- pist, Inspirational songs cho- sen by participants; Circle songs; Vocal improvisation Pain Interference (Westhaven‐Yale Multidimensional Pain Inventory) Hospital Anxiety Depression Scale (HADS) Qualitative analy- sis was performed on focus group transcripts. 3.1 \| Description of studies and results of searches (Continues) TABLE 1 Description of included studies (k = 13) 76 \| IRONS 76 Study ID (1st au- thor, year, Country) Diagnosis, number of Participants at baseline, Mean age Study design, control group condition Singing interven- tion frequency and intensity, total hours of singing Setting, facilitator, repertoires Pain outcome measures, Pain questionnaire Depression measure/ Questionnaire Comments Fogg‐Rogers et al. (2016)a, New Zealand Parkinson's & Stroke, N = 23 (N = 9 car- ers), 64.4 years Non‐RCT, No com- parison group 1 hr, once weekly for 12 weeks (12 hr) Community, Music therapist, Not reported Pain Interference (WHO‐QOL_Bref) None Qualitative analysis was performed on interview transcripts. Hopper et al. (2016), UK Chronic pain, N = 7, range 44–79 years Non‐RCT, Qualitative study Weekly ongoing choir Community Pain Service Centre, A service‐user (per- son with chronic pain), Not reported None None Qualitative analysis was performed on interview transcripts. Pongan et al. (2017)a, France Alzheimer's Disease, N = 77, 78.8 years (singing group); 80.2 years (control group) RCT, Painting group 2‐hr session, once weekly for 12 weeks (24 hr) Memory clinics located in University Hospitals, Professional choir conductor, Well‐known songs chosen by participants Pain Intensity (EQ−5D); Pain Interference (BPI) Geriatric Depression Scale (GDS) Stegemoller et al. (2017)a, USA Parkinson's, N = 27, 67 years Non‐RCT, (Group 1) high intensity singing; (Group 2) low intensity singing (Group 1) = 1 hr, twice weekly for 8 weeks (16 hr), (Group 2) = 1‐ hr session, once weekly for 8 weeks (8 hr) Community, Music therapist, popular songs Pain Interference (WHO‐QOL_Bref) None Reagon et al. (2017)a, UK Cancer, N = 816 (N = 222 cancer patients, N = 593 carers), 62 years (cancer patients); 56 years (carers) Non‐RCT, No com- parison group 1.5‐hr session, once weekly for 12 weeks (18 hr) Community, Professional musicians, Contemporary & traditional songs Pain intensity & Pain interference (SF‐36 Bodily pain subscale) Hospital Anxiety Depression Scale (HADS) Qualitative analysis was performed on interviews and focus groups data. Abbreviations: AQoL, Assessment of Quality of life; BPI, Brief Pain Inventory; COPD, Chronic Obstructive Pulmonary Disease; EQ‐5D, EuroQoL‐5D; FLACC Scale, Face Legs Activity Cry Consolability Scale; GSRS‐IBS, Gastrointestinal Symptom Rating Scale for IBS; hrs, hours; IBS, Irritable Bowel Syndrome; N, number; NR, Not Reported; RCT, Randomized Controlled Trial; SF‐12, Short Form Health Survey 12; SF‐36, Short Form Health Survey 36; WHO‐QOL_Bref, World Health Organization Quality of Life Scale; yrs, years. 3.1 \| Description of studies and results of searches IRONS IRONS IRONS TABLE 2 Included studies' results on pain intensity, interference and depression measures and quality assessments Study ID (1st Author, Year, Country) Pain questionnaire Pain questions Effect size (g) (95% Confidence Intervals) Participants Number Quality Assessment§ Pain intensity Tamplin et al. (2013)a Australia AQoL−4D (Assessment of Quality of Life−4D) “How much pain or discomfort do I experience?” −0.18 (−0.75–0.39) 13 23 Morrison et al. (2013) UK EQ−5D (EuroQual−5D) “I have no pain/slight pain/moderate pain/severe pain or extreme pain.” −0.33 (−0.61–0.05) 69 11 Clements‐Cortes (2015) Canada Author has used a visual analogue scale adopted from FLACC Scale. Visual Analogue Scale 0 = no pain ‐ 5 = a lot of pain −0.63 (−1.23–−0.03) 14 11 Pongan et al. (2017)a France EQ−5D (EuroQual−5D) “I have no pain/slight pain/moderate pain/severe pain or extreme pain.” −0.47* (−0.89–−0.04) 25 24 Pain interference Kenny and Faunce (2004)a Australia Pain Disability Questionnaire “Does your pain interfere with your normal work/personal care/travelling?” −0.53 (−1.14–0.07) 13 18 Grape et al. (2009)a Sweden GSRS‐IBS's Pain subscale (Gastrointestinal Symptom Rating Scale modified for use in patients with Irritable Bowel Syndrome) “Have you been bothered by abdominal pain during past week?” −0.25 (−0.63–0.13) 28 14 Morrison et al. (2013) UK SF−12 (Short Form−12) “During the past 4 weeks, how much did pain interfere with your normal work (including work outside the home and housework?” −0.19 (−0.41–0.03) 71 11 Fogg‐Rogers et al. (2016) New Zealand WHOQOL_BREF (World Health Organization‐Quality Of Life‐Bref) “To what extent do you feel that physical pain prevents you from doing what you need to do?” 0.38 (−0.21–0.96) 13 9 Bradt et al. (2016)a USA Westhaven‐Yale Multidimensional Pain Inventory “How much does your pain problem interfere with your day to day activities?” −0.65* (−1.13–−0.18) 22 19 Pongan et al. (2017)a France BPI (Brief Pain Inventory) “How, during the past 24 hr, pain has interfered with your general activity/mood/walking ability/normal work/relation- ship with others/sleep?” 0.01 (−0.25–0.27) 31 24 Stegemoller et al. (2017) USA WHOQOL_BREF (World Health Organization‐Quality Of Life‐Bref) “To what extent do you feel that physical pain prevents you from doing what you need to do?” −0.38* (−0.76–0.00) 29 12 results on pain intensity, interference and depression measures and quality assessments IRONS 78 Study ID (1st Author, Year, Country) Pain questionnaire Pain questions Effect size (g) (95% Confidence Intervals) Participants Number Quality Assessment§ Pain intensity + interference Gale et al. 3.1 \| Description of studies and results of searches (2012) UK SF−36 (Short Form−36) “How much bodily pain have you had during the past 4 weeks?” “During the past 4 weeks, how much did pain in- terfere with your normal work (including both work outside of the home and housework)?” −0.36 (−0.83–0.10) 20 11 Clements‐Cortes (2013) Canada AQoL (Assessment of Quality of Life) “How much pain or discomfort do you experience?” “How often does pain interfere with your usual activities?” −0.15 (−0.53–0.23) 28 11 Reagon (2017) UK SF−36 (Short Form−36) “How much bodily pain have you had during the past 4 weeks?” “During the past 4 weeks, how much did pain in- terfere with your normal work (including both work outside of the home and housework)?” −0.23 (−0.40–0.03) 60 11 Depression Kenny and Faunce (2004)a Australia Zung Depression Inventory “I feel down‐hearted and blue–a little of the time/some of the time/good part of the time/most of the time.” −0.61 (−1.25–0.03) 12 18 Gale et al. (2012) UK HAD (Hospital Anxiety Depression) “I feel cheerful–not at all/not often/sometimes/most of the time” −0.22 (−0.68–0.23) 20 11 Tamplin et al. (2013)a Australia POMS (Profile of Mood States) Depression is one of the six domains. “Below is a list of words that describe feelings people have. Please circle the number that best describes how you feel right now.” For example, Feeling unhappy/sad/hopeless/ satisfied/worthless −0.26 (−0.84–0.31) 13 23 Morrison et al. (2013) UK EQ−5D “I am not/slightly/moderately/severely/extremely anxious or depressed” −0.28* (−0.52–−0.04) 71 11 Bradt et al. (2016)a USA HAD (Hospital Anxiety Depression) “I feel cheerful–not at all/not often/sometimes/most of the time” −0.06 (−48–0.37) 22 19 Pongan et al. (2017)a France GDS (Geriatric Depression scale) “Do you feel happy most of the time? – Yes/No” 0.11 (−0.25–0.46) 31 24 Reagon et al. (2017) UK HAD (Hospital Anxiety Depression) “I feel cheerful – not at all/not often/sometimes/most of the time” −0.11 (−0.36–0.15) 60 11 TABLE 2 (Continued) IRONS 79 IRONS carried out in developed countries. Singing programmes were facilitated by music therapists, experienced professional musicians or a service‐user, a person with chronic pain plus musical background (Hopper et al., 2016), and provided at community centres or hospital/health centres. Singing inter- vention length varied between 3 weeks and 1 year. The dura- tion of each session was commonly between 60 and 90 min, although Kenny's programme comprised 30‐min sessions (Kenny & Faunce, 2004). 3.1 \| Description of studies and results of searches All eight studies report- ing qualitative data employed recognized analysis methods, such as thematic analysis, or interpretative phenomenologi- cal analysis. All authors listed representative quotes with the exception of Tamplin (2013) (for further details, please see Appendix S3). All qualitative studies were appraised as mak- ing a valuable contribution to the existing knowledge. 3.3 \| Relationship between singing and chronic pain Overall, all included 12 studies have a great heterogeneity in populations, outcome assessment tools, singing interventions (settings, facilitators, repertoires, length and frequencies of sessions) and study designs. Only five included RCTs were assessed to have low to good quality. Inspection of RCTs re- vealed differences in chronic condition, intervention length and comparator. Due to these differences, it was not possible to con- duct a meta‐analysis. Instead, we present a mixed‐methods re- view, synthesizing current evidence in the effects of singing for persistent pain in people with chronic health conditions (Popay et al., 2006). Tables 2 and 3 present the effect sizes and 95% Confidence Intervals of singing interventions in each study. Only one study (Bradt et al., 2016) reported including a predominantly African‐American ethnic population. None of the included studies reported the number of GP/pain spe- cialist's visits nor pain unpleasantness. We summarized each study's details of pain and depression measures in Table 2 along with each study's effect size and confidence intervals. 3.2 Twelve included studies were quality assessed according to the Downs and Black quality assessment tool (Downs & Black, 1998) (Table 4). Overall, 12 included studies were appraised to have low to high qualities: Fogg‐Rogers et al. (2016) scored lowest nine, while Pongan et al., 2017 scored 24 out of a possible 28. Amongst the RCTs, Pongan et al. (2017) and Tamplin et al. (2013) studies were judged to be of good quality, in part as they attempted to blind the experimen- tal condition (singing) by offering active comparators, namely painting (Pongan et al., 2017) and a music appreciation and relaxation programme (Tamplin et al., 2013); blinding asses- sors was also achieved in both studies. Three RCTs (Bradt et al., 2016; Grape et al., 2009; Kenny & Faunce, 2004) and seven non‐RCTs were unable to blind participants nor asses- sors. In terms of comprehensive attempt to measure adverse events and describing principal confounders (Question No 5), none of the included studies reported any details (Table 4). 3.1 \| Description of studies and results of searches Most interventions were conducted weekly (Bradt et al., 2016; Gale et al., 2012; Grape et al., 2009; Morrison et al., 2013; Pongan et al., 2017; Reagon et al., 2017; Stegemoller et al., 2017); however, one met thrice weekly (Tamplin et al., 2013), while one assembled three or more times weekly (Kenny & Faunce, 2004). The interventions were delivered predominantly by an individual with relevant musical skill, such as music therapists (Bradt et al., 2016; Clements‐Cortes, 2013, 2015; Fogg‐Rogers et al., 2016; Stegemoller et al., 2017; Tamplin et al., 2013) or professional musicians (Gale et al., 2012; Grape et al., 2009; Kenny & Faunce, 2004; Morrison et al., 2013; Pongan et al., 2017; Reagon et al., 2017). The majority of studies provided little details of the singing interventions; two studies provided no information on repertoires (Grape et al., 2009; Hopper et al., 2016) (Table 1). 2015; Fogg‐Rogers et al., 2016; Gale et al., 2012; Reagon et al., 2017; Tamplin et al., 2013) reported qualitative data as part of a mixed‐methods study design, while Hopper et al. (2016) was the only qualitative study (Table 5). We appraised qualitative studies, with the exception of Fogg‐Rogers et al. (2016), as having good qualities: studies included clear state- ments of the aims, appropriate methodologies and recruitment strategies. Clements‐Cortes (2015) and Hopper et al. (2016) considered possible implications of the relationship between researcher and participants within their study settings; other articles did not address this issue. All eight studies report- ing qualitative data employed recognized analysis methods, such as thematic analysis, or interpretative phenomenologi- cal analysis. All authors listed representative quotes with the exception of Tamplin (2013) (for further details, please see Appendix S3). All qualitative studies were appraised as mak- ing a valuable contribution to the existing knowledge. 2015; Fogg‐Rogers et al., 2016; Gale et al., 2012; Reagon et al., 2017; Tamplin et al., 2013) reported qualitative data as part of a mixed‐methods study design, while Hopper et al. (2016) was the only qualitative study (Table 5). We appraised qualitative studies, with the exception of Fogg‐Rogers et al. (2016), as having good qualities: studies included clear state- ments of the aims, appropriate methodologies and recruitment strategies. Clements‐Cortes (2015) and Hopper et al. (2016) considered possible implications of the relationship between researcher and participants within their study settings; other articles did not address this issue. 3.4 Four studies (Clements‐Cortes, 2015; Morrison et al., 2013; Pongan et al., 2017; Tamplin et al., 2013) involv- ing 121 participants, measured pain intensity before and after group singing interventions. Pain intensity questions include “How much pain or discomfort do you experience? None at all/I have moderate pain/I suffer from severe pain/I suffer unbearable pain” (AQoL‐4D). All four stud- ies found reductions in pain intensity from pre‐ to post‐ singing intervention (Clements‐Cortes, 2015; Morrison et al., 2013; Pongan, 2017; Tamplin, 2013). The effect size in studies with people with COPD (Morrison et al., 2013) and dementia (Pongan et al., 2017) was medium, although Morrison et al. (2013) did not have a comparison group and was appraised to be of fair quality. Tamplin's study (2013) of spinal cord injury patients demonstrated a small effect size, whereas Clements‐Cortes (2015) study with Alzheimer's disease showed a large effect size. However, In addition, we assessed studies reporting qualitative data using CASP qualitative checklist (CASP Programme, 2018). Seven studies (Bradt et al., 2016; Clements‐Cortes, 2013, IRONS 80 TABLE 3 Effect size of RCTs of pain intensity, interference and depression and quality assessment Study ID (1st Author, Year) Number of Participants in each group Comparison (Control group) Effect size (g) (95% Confidence Intervals) Comments Quality Assessment§ Pain intensity Tamplin et al. (2013) SGa = 13 Music relaxation/appreciation group (ac- tive comparator) −0.24 (−0.66–0.17) Trend towards intervention 23 CGb = 11 Pongan et al. (2017)c SGa = 31 Painting (active comparator) −0.15 (−0.41–0.11) Trend towards intervention 24 CGb = 28 Pain interference Kenny and Faunce (2004) SGa = 12 Exercise while listening to the sing- ing group singing practice (active comparator) 0.24 (−0.04–0.52) Trend towards control 18 CGb = 39 Grape et al. (2009) SGa = 11 Education group (passive comparator) −0.55 (−0.97–−0.11) Significantly fa- vours intervention 14 CGb = 14 Bradt et al. (2016) SGa = 22 Wait‐list (passive comparator) −0.58 (−0.91–−0.26) Significantly fa- vours intervention 19 CGb = 22 Pongan et al. (2017)c SGa = 31 Painting (active comparator) 0.42 (0.15–0.68) Significantly fa- vours control 24 CGb = 28 Depression Kenny and Faunce (2004) SGa = 12 Exercise while listening to the sing- ing group singing practice (active comparator) 0.09 (−0.18–0.37) No difference 18 CGb = 39 Tamplin et al. (2013) SGa = 13 Music relaxation/appreciation group (ac- tive comparator) −0.18 (−0.23–0.59) No difference 23 CGb =11 Bradt et al. 3.4 (2016) SGa = 22 Wait‐list (passive comparator) −0.27 (−0.54–0.00) Trend towards intervention 19 CGb = 22 Pongan et al. (2017)c SGa = 31 Painting (active comparator) 0.43 (0.16–0.69) Significantly fa- vours control 24 CGb = 28 Note: SGa = Singing Group; CGb = Control Group c = authors reported Intention‐To‐Treat analysis. §Downs & Black quality assessment; possible max. score = 28. TABLE 3 Effect size of RCTs of pain intensity, interference and depression and quality assessment Note: SGa = Singing Group; CGb = Control Group c = authors reported Intention‐To‐Treat analysis. §Downs & Black quality assessment; possible max. score = 28. g g p p §Downs & Black quality assessment; possible max. score = 28. in studies with chronic pain patients (Bradt, 2016), people with Parkinson's (Stegemoeller, 2017), people with COPD (Morrison et al., 2013) and people with irritable bowel syn- drome (IBS) (Grape et al., 2009); however, the decrease was only significant in Bradt et al. (2016). Four RCTs yield mixed results: Bradt et al. (2016) and Grape et al. (2009) studies, rated as of less than good quality, reported findings that “significantly favours intervention,” whereas Pongan et al. (2017) findings “significantly favours control” and Kenny's (2004) had a “trend towards control.” Pongan's study (2017) was appraised to be of good quality, whereas Kenny's study (2004) had a number of weaknesses, such as a high attrition rate (only 31% of singing group participants completed the 3‐week long singing programme) and not reporting missing data. Both Grape (2009) and Bradt (2016) RCTs had pas- sive comparator groups (education group and wait‐list, re- spectively), and had medium intervention effects. In contrast, singing did not result in decreased pain interference com- pared to painting (Pongan, 2017) or exercise and listening Clements‐Cortes (2015) study was uncontrolled and as- sessed to be a fair quality study. Inspection of two good quality RCTs' effect sizes and 95% Confidence Intervals (Pongan, 2017; Tamplin, 2013) revealed that there is “trend towards intervention” in pain intensity outcome measures with small effect sizes in both studies (Table 3). 3.5 \| Pain interference The effects of singing on pain interference were examined in seven quantitative studies with 205 participants (Bradt, 2016; Fogg‐Rogers et al., 2016; Grape et al., 2009; Kenny & Faunce, 2004; Morrison et al., 2013; Pongan et al., 2017; Stegemoller, 2017). These studies utilized pain question- naires, which asked the degrees of pain interference in a nu- merical scale: for example, “Mark the box beside the number that describes how, during the past 24 hr, pain has interfered with your general activity, normal work, etc.,” (BPI). Pain interference decreased from pre‐ to post‐singing intervention IRONS IRONS Downs & Black quality assessment Kenny 2004a Grape 2009a Gale 2012b Morrison 2013b Clement‐Cortes 2013b Tamplin 2013a Clement‐Cortes 2015b Bradt 2016a Fogg‐Rogers 2016b Stegemoeller 2017b Reagon 2017b Pongan 2017a Reporting (0–11c) Q1. Aims 1 1 1 1 1 1 1 1 0 1 1 1 Q2. Outcomes 1 1 1 1 1 1 1 1 0 1 1 1 Q3. Participants 1 1 1 1 1 1 1 1 1 1 1 1 Q4. Intervention 1 0 1 1 1 1 1 1 1 1 1 1 Q5. Confoundersd 0 0 0 0 0 0 0 0 0 0 0 0 Q6. Findings 1 1 1 1 1 1 1 1 1 1 1 1 Q7. Variability 1 0 0 0 0 1 0 1 0 0 1 Q8. Adverse event 0 0 0 0 0 0 0 0 0 0 0 0 Q9. Drop‐outs 0 0 1 1 1 1 1 1 1 1 1 1 Q10. Probability 1 1 1 1 1 1 1 1 1 1 1 1 Reporting sub‐scores 7 5 7 7 7 8 7 8 5 7 7 8 External validity (0–3c) Q11. Participants 1 1 0 0 0 1 0 1 0 0 0 1 Q12. Population 0 0 0 0 0 0 0 0 0 0 0 0 Q13. Treatment 1 0 0 0 0 1 0 1 0 0 0 1 External validity sub‐scores 2 1 0 0 0 2 0 2 0 0 0 2 Internal validity (0–7c) Q14. intervention 0 0 0 0 0 1 0 0 0 0 0 1 Q15. blinding 0 0 0 0 0 1 0 0 0 0 0 1 Q16. results 1 1 1 1 1 1 1 1 1 1 1 1 Q17. follow‐up 1 1 0 0 0 1 0 1 0 0 0 1 Q18. 3.5 \| Pain interference statistics 1 1 1 1 1 1 1 1 1 1 1 1 Q19. compliance 1 1 1 1 1 1 1 1 1 1 1 1 Q20. measures 1 1 1 1 1 1 1 1 1 1 1 1 Internal validity sub‐scores 5 5 4 4 4 7 4 5 4 4 4 7 Selection Bias (confounding) (0–6c) Q21. Participants 1 1 0 0 0 1 0 1 0 0 0 1 Q22. Time 1 1 0 0 0 1 0 1 0 0 0 1 Q y q ( ) Downs & Black quality assessment Kenny 2004a Grape 2009a Gale 2012b Morrison 2013b Clement‐Cortes 2013b Tamplin 2013a Clement‐Cortes 2015b Bradt 2016a Fogg‐Rogers 2016b Stegemoeller 2017b Reagon 2017b Pongan 2017a IRONS IRONS 82 to singing (Kenny, 2004); indeed, painting resulted in larger decreases in interference than singing (Tables 2 and 3). Downs & Black quality assessment Kenny 2004a Grape 2009a Gale 2012b Morrison 2013b Clement‐Cortes 2013b Tamplin 2013a Clement‐Cortes 2015b Bradt 2016a Fogg‐Rogers 2016b Stegemoeller 2017b Reagon 2017b Pongan 2017a Q23. Randomization 1 1 0 0 0 1 0 1 0 0 0 1 Q24. Concealment 0 0 0 0 0 1 0 0 0 0 0 1 Q25. Analysis 0 0 0 0 0 0 0 0 0 1 0 1 Q26. Follow‐up 0 0 0 0 0 1 0 1 0 0 0 1 Selection bias sub‐scores 3 3 0 0 0 5 0 4 0 1 0 6 Powere (0–1c) Q27. Calculation 1 0 0 0 0 1 0 0 0 0 0 1 Overall (0–28c) 18 14 11 11 11 23 11 19 9 12 11 24 Rating fair fair fair fair fair good fair fair poor fair fair good 3.7 \| Depression Four RCTs (Bradt, 2016; Kenny & Faunce, 2004; Pongan, 2017; Tamplin et al., 2013) and two non‐RCTs (Morrison et al., 2013; Reagon et al., 2017) with a total of 229 participants evaluated the effects of singing on depression. Inspection of four RCTs revealed mixed results: Pongon study (2017) in- dicated “significantly favours control,” whereas Kenny and Faunce (2004) and Tamplin et al. (2013) showed “no differ- ence.” Only one RCT (Bradt et al., 2016), rated as of less than good quality, revealed a “trend towards intervention” (Table 2 and 3). 3.6 \| Pain interference and pain intensity Three studies measured aspects of both pain intensity and in- terference simultaneously; both the SF‐36 (Gale et al., 2012; Reagon et al., 2017) and AQoL‐8D (Clements‐Cortes, 2013) have a pain subscale, which includes questions on both pain intensity and pain interference. These two questions' responses are scored together as overall pain scores. Thus, we grouped these studies together. Both cancer studies (Gale, 2012; Reagon et al., 2017) demonstrated reduced pain intensity and inference, although these studies had no comparator groups and were appraised to have fair quality. Clement‐Cortes study with people with Alzheimer's disease (2013) also reported a reduction in this combined pain outcome measure (Table 2). No RCTs included this combined pain measures. 3.8 Three of five RCTs employed active comparators: Kenny and Faunce (2004) compared group singing with an exer- cise and listening to singing condition, while Pongan et al. (2017) had a painting group comparator and Tamplin (2013) had a music appreciation/relaxation comparator group. These three RCTs with the active comparators yield results favour- ing the comparator conditions. In Kenny and Faunce (2004) and Pongan et al. (2017) studies, the control groups demon- strated improvement in pain interference, while the singing groups did not. Tamplin et al. (2013) also found no differ- ence between singing and control group on depression meas- ure. Two other RCTs with passive control groups, namely an education (Grape et al., 2009) and wait‐list (Bradt et al., 2016), found significant reductions in pain interference and depression (only in Bradt et al., 2016) in the singing group. Attending an education group does not require active engage- ment, while painting and exercise groups require physical and mental engagement. Positive trends towards group sing- ing are apparent in the passive group; however, the effects \| 83 IRONS 83 TABLE 5 Quality assessment of qualitative studies (using CASP) (k = 8) CASP* Qualitative Checklist Gale et al. (2012)a Tamplin (2013)a Clements‐ Cortes (2013)a Clements‐ Cortes (2015)a Hopper (2016) Bradt (2016)a Fogg‐Rogers et al. (2016)a Reagon et al. (2017)a Q1. Was there a clear statement of the aims of the research? Yes Yes Yes Yes Yes Yes Yes Yes Q2. Is a qualitative methodology appropriate? Yes Yes Yes Yes Yes Yes Yes Yes Q3. Was the research design appropriate to address the aims of the research? Yes Yes Yes Yes Yes Yes Yes Yes Q4. Was the recruitment strategy appropri- ate to the aims of the research? Yes Yes Yes Yes Yes Yes Yes Yes Q5. Was the data collected in a way that addressed the research issue? Yes Yes Yes Yes Yes Yes Yes Yes Q6. Has the relationship between re- searcher and participants been adequately considered? Can't tell Can't tell Can't tell Yes Yes Can't tell Can't tell Can't tell Q7. Have ethical issues been taken into consideration? Yes Yes Yes Yes Yes Yes Yes Yes Q8. Was the data analysis sufficiently rigorous? Yes Yes Yes Yes Yes Yes Yes Yes Q9. Is there a clear statement of findings? Yes Yes Yes Yes Yes Yes No Yes Q10. How valuable is the research? 3.8 Yes Yes Yes Yes Yes Yes Yes Yes Note: CASP*, Critical Appraisals Skills Programme (downloaded from https://casp-uk.net/wp-content/uploads/2018/03/CASP-Qualitative-Checklist-2018_fillable_form.pdf.) Possible answers are Yes/Can't Tell/No. aQualitative data were part of a quantitative study. IRONS 84 and pain intensity combined with interference measures. However, Pongan's et al. (2017) findings showed signifi- cantly favoured control condition in pain interference and depression, but there was a trend towards intervention for pain intensity. In the third group of patients with impaired lung function (Morrison et al., 2013; Tamplin et al., 2013), the singing groups demonstrated reduced pain intensity with small to medium effect sizes. Lastly, patients with cancer (Gale et al., 2012; Reagon et al., 2017) or with IBS (Grape et al., 2009) also demonstrated reductions in pain: Reagon et al. (2017) and Gale et al. (2012) studies revealed that singing reduced pain intensity and interference with small to medium effect sizes; Grape's study of IBS patients (2009) also yielded reductions in pain interference with a small effect size. Although RCTs and non‐RCTs cannot be directly compared, this synthesis suggests that there is preliminary evidence that group singing is potentially effective in reducing pain in patients with impaired lung function, IBS and cancer. Inconsistent results were found in patients with neurological conditions (Parkinson's, stroke and Alzheimer's disease) and chronic pain. Moreover, there is no evidence of the effects of singing on pain in other long‐term health conditions. of group singing are not greater than, and in some cases is smaller than, those of painting, exercise or music apprecia- tion/relaxation programmes. 3.9 \| Synthesis II: length of singing intervention The shortest singing intervention length was less than 1 month in Kenny's study (Kenny & Faunce, 2004). Five studies adopted 3‐month intervention (Fogg‐Rogers et al., 2016; Gale et al., 2012; Pongan et al., 2017; Reagon et al., 2017; Tamplin et al., 2013), while two studies were 2‐month long (Bradt et al., 2016; Stegemoller et al., 2017). Morrison et al. (2013) evaluated 9‐month singing programme, and Grape study (2009) lasted for 1 year. These two longer term stud- ies (Grape et al., 2009; Morrison et al., 2013) found reduced pain interference with small effect sizes. In singing studies lasting less than 6 months, there are conflicting results: Some reported a positive trend towards intervention in pain inter- ference (Bradt et al., 2016; Stegemoller et al., 2017), others reported a trend towards control (Pongan et al., 2017) and yet others reported no difference (Gale et al., 2012; Reagon et al., 2017). Kenny's study (2004) RCT of 3 weeks dura- tion indicated a “trend towards control” in pain interference. Although RCTs and non‐RCTs cannot be compared directly, this synthesis suggests that there is preliminary evidence that longer term interventions (>6‐month, weekly singing) can have positive impact on reducing pain intensity, pain inter- ference and depression. 3.11 Eight studies explored participants' perspectives of partici- pating in the group singing programme and its impact on their health using a qualitative approach (Bradt et al., 2016; Clements‐Cortes, 2013, 2015; Fogg‐Rogers et al., 2016; Gale et al., 2012; Hopper et al., 2016; Reagon et al., 2017; Tamplin et al., 2013) (see Appendix S3). Hopper et al. (2016) was the only purely qualitative study; the others employed qualitative approach in a mixed‐methods research design. The authors argued that qualitative data would complement the quantita- tive data, as experience of the group singing programme can be individual and often the depth and intensity of such expe- rience cannot be captured appropriately in the quantitative data. Two studies utilized focus group discussions (Bradt et al., 2016; Reagon et al., 2017), while others conducted semi‐ structured interviews with participants (Clements‐Cortes, 2013, 2015; Gale et al., 2012; Hopper et al., 2016; Riley et al., 2002; Tamplin et al., 2013). Overall, singing programmes were well‐received by participants. Through synthesizing qualitative data from eight studies, three key themes were identified: (a) physical benefits; (b) psychological benefits; (c) social benefits. Figure 2 illustrates these key themes and the interactions between them. Participants demonstrated that they experienced positive impacts of singing on the physi- cal, psychological and social aspects and these three domains appear to be interwoven, complementary and correspondent. Increasing positive mood may be due to having friendship/ companionship and support, which also impact on reducing 4 \| DISCUSSION The current narrative review presented new syntheses of re- search evidence about the effects of group singing on pain in- tensity, pain interference and depression in people living with long‐term health conditions. Thirteen studies with 318 par- ticipants were included in the review: five RCTs, seven non‐ RCTs and one qualitative study. Additionally, seven studies (one RCT, six non‐RCTs) collected qualitative data within a mixed‐methods study design. These studies varied in study design, study quality, sample (variety of long‐term health conditions), pain outcomes measured and details of singing interventions (facilitators, settings, intensities and frequen- cies of sessions). Due to the high heterogeneity, conducting a meta‐analysis was not appropriate. Therefore, we presented effect size, quality assessments alongside a narrative review. Based on the limited available evidence of varied quality, we conclude there is partial support for singing on some pain outcomes. Specifically, we found in all the studies that there is a positive trend of singing interventions decreasing pain intensity, while there is more equivocal support for singing influencing pain interference and depression. g p p Analyses of a small subset of five RCTs yielded more mixed effects of singing on pain interference and depres- sion measures. Studies with passive comparator groups (Bradt et al., 2016; Grape et al., 2009) found reductions in pain interference and depression, suggesting that sing- ing may yield benefits compared to education or waiting for the intervention. Studies with active comparator groups (Kenny & Faunce, 2004; Pongan et al., 2017; Tamplin et al., 2013) demonstrated either no difference between singing and control groups, or results favouring control conditions. However, the comparator groups in these studies involved interventions have known positive effects, e.g., exercise (Kenny & Faunce, 2004, or were creative arts interventions likely to activate similar psycho‐social processes as sing- ing, e.g., painting (Pongan et al., 2017). Moreover, there are number of general processes in both active comparative conditions and art making conditions, such as leadership, a focus on meaningful activity and social support from the group (Archer, Buxton, & Sheffield, 2015; Kaptein, Hughes, Murray, & Smyth, 2018). Further, our syntheses suggest that the beneficial effects of group singing on depression appear to be disputable: only one RCT (Bradt et al., 2016) found a trend towards singing inter- vention, while other studies (Kenny & Faunce, 2004; Pongan et al., 2017; Tamplin et al., 2013) did not. 3.10 The pathways by which singing improves pain processing appear to be non‐linear, and interwoven within those phys- ical, psychological and social domains (Figure 3). Potential mechanisms of group singing should be further studied to gain a deeper understanding of how they are activated and complement each other; it would also be informative to examine how they relate to specific elements of group singing programmes (repertoire, being part of a group, fa- cilitator, breathing, etc.) and result in greater benefits in pain patients. people with persistent pain. For example, deep breathing is a central element for singing, which can impact on both physiological (e.g. cardiovascular and autonomic nervous system) and emotional functions (Busch et al., 2012; Ma et al., 2017; Russo et al., 2017). Further, studies found that group singing can reduce pain threshold and pain percep- tion (Dunbar, Baron, et al., 2012; Dunbar, Kaskatis, et al., 2012). Other studies also found that group singing had greater social bonding effects than other social activities such as creative writing or craft activity (Pearce, Launay, & Dunbar, 2015; Pearce, Launay, MacCarron, & Dunbar, 2017). Enhanced social bonding is relevant in the pain context, as there is a positive relationship between social isolation and pain interference (Karayannis, Baumann, Sturgeon, Melloh, & Mackey, 2018). Getting together with people with the same or similar health condition and en- gaging in an enjoyable activity, such as singing, can re- duce social isolation, and increase social support which in turn can reduce pain processing (Brown et al., 2003). We identified that singing interventions were well‐received and participants reported great positivity, which supports quantitative findings of reductions in pain interference (Figure 2). While the quantitative evidence of depression reduction was inconsistent, qualitative evidence highlights that singing greatly improved mood across all eight studies. The pathways by which singing improves pain processing appear to be non‐linear, and interwoven within those phys- ical, psychological and social domains (Figure 3). Potential mechanisms of group singing should be further studied to gain a deeper understanding of how they are activated and complement each other; it would also be informative to examine how they relate to specific elements of group singing programmes (repertoire, being part of a group, fa- cilitator, breathing, etc.) and result in greater benefits in pain patients. stress and anxiety, and increasing relaxation as well as cop- ing with pain. 3.10 Thus, these themes interact with one another and suggest complex pathways linking group singing to pain (Figure 3). 3.10 We also grouped the 12 included quantitative studies ac- cording to participants' diagnosis and/or similar condi- tions. (a) Kenny (2004) and Bradt (2016) recruited patients with a chronic pain diagnosis. (b) Four studies included people with neurological conditions, such as Parkinson's (Stegemoller et al., 2017), Parkinson's and stroke (Fogg‐ Rogers et al., 2016) and Alzheimer's disease (Clements‐ Cortes, 2013, 2015; Pongan et al., 2017). (c) Two studies included patients with impaired lung function (Morrison et al., 2013; Tamplin et al., 2013). (d) Two cancer stud- ies (Gale et al., 2012; Reagon et al., 2017) and one IBS (Grape et al., 2009) study formed one group. In the chronic pain diagnosis group, there are some differences between the two RCTs (Bradt et al., 2016; Kenny & Faunce, 2004): their findings are inconsistent. While Bradt (2016) found reduction in pain interference, Kenny and Faunce (2004) found an increase. In the second group, the neurological conditions, Stegemoeller's study (2017) demonstrated re- duced pain interference with medium effect sizes, whereas Fogg‐Rogers' study (2016) did not. Further, Clement‐ Cortes (2013) indicated improvement in pain intensity, 85 IRONS 85 people with persistent pain. For example, deep breathing is a central element for singing, which can impact on both physiological (e.g. cardiovascular and autonomic nervous system) and emotional functions (Busch et al., 2012; Ma et al., 2017; Russo et al., 2017). Further, studies found that group singing can reduce pain threshold and pain percep- tion (Dunbar, Baron, et al., 2012; Dunbar, Kaskatis, et al., 2012). Other studies also found that group singing had greater social bonding effects than other social activities such as creative writing or craft activity (Pearce, Launay, & Dunbar, 2015; Pearce, Launay, MacCarron, & Dunbar, 2017). Enhanced social bonding is relevant in the pain context, as there is a positive relationship between social isolation and pain interference (Karayannis, Baumann, Sturgeon, Melloh, & Mackey, 2018). Getting together with people with the same or similar health condition and en- gaging in an enjoyable activity, such as singing, can re- duce social isolation, and increase social support which in turn can reduce pain processing (Brown et al., 2003). We identified that singing interventions were well‐received and participants reported great positivity, which supports quantitative findings of reductions in pain interference (Figure 2). While the quantitative evidence of depression reduction was inconsistent, qualitative evidence highlights that singing greatly improved mood across all eight studies. 4 \| DISCUSSION This could be due to small sample size, high attrition rate, short intervention period and active comparator group. In contrast to our findings, a re- cent systematic review on singing for mental health indicated that singing reduced mental ill‐health with moderate to large effect sizes (Williams et al., 2018). A number of previous qual- itative studies also affirm that participating in group singing can enhance mood and promote mental well‐being (Plumb & Stickley, 2017; Skingley, Martin, & Clift, 2015). Further, our qualitative synthesis suggests that patients have reported great psychological and social benefits from group singing. In our review, two qualitative studies (Bradt et al., 2016; Hopper et al., 2016) suggested that group singing interventions can re- duce negative emotions and increase self‐efficacy. There is Qualitative analysis supported and augmented the quan- titative data. The qualitative synthesis identified three key themes: physical, psychological and social benefits. As re- flected in the qualitative themes (Appendix S3 and Figure 2), singing interventions may offer unique advantages for 86 \| IR FIGURE 2 Three key themes from qualitative data Participating in Group Singing has 1. Physical benefits Physical changes; Relaxing; Stress release; Living well with pain 3. Social benefits Togetherness; Friendship, Companionship & Support; Sharing the music and spreading the word Emotional impact; Personal growth; Improved mood & attitude; Singing is uplifting; Increased confidence & self-esteem 2. Psychological benefits FIGURE 3 Pathways by which singing improves pain processing SINGING PAIN Physical benefits Pain intensity Psychological benefits Social benefits Depression Pain interference Confounders Group singing 86 \| FIGURE 2 Three key themes qualitative data Participating in Group Singing has 1. Physical benefits Physical changes; Relaxing; Stress release; Living well with pain 3. Social benefits Togetherness; Friendship, Companionship & Support; Sharing the music and spreading the word Emotional impact; Personal growth; Improved mood & attitude; Singing is uplifting; Increased confidence & self-esteem 2. Psychological benefits IRONS FIGURE 2 Three key themes from qualitative data Emotional impact; Personal growth; Improved mood & attitude; Singing is uplifting; Increased confidence & self-esteem 2. Psychological benefits qualitative data Participating in Group Singing has 3. 4 \| DISCUSSION Social benefits Togetherness; Friendship, Companionship & Support; Sharing the music and spreading the word FIGURE 3 Pathways by which singing improves pain processing Physical benefits Pain intensity Group singing Psychological benefits SINGING PAIN information, such as settings, facilitator's qualifications and repertoires (Table 1); however, more details of the protocol, the delivery of and compliance with their singing intervention were missing. It is important that researchers address inter- nal validity in order to increase certainty that study findings can be attributed to the intervention (Horner, Rew, & Torres, 2006). Moreover, reporting these details assist future studies with designing their singing interventions, so that high‐stan- dard singing interventions can be developed and delivered. Additionally, none of included studies addressed possible confounders, such as previous singing experience and expec- tation. Previous singing experience and expectations may have confounding impacts on the results, as people with previous positive singing experience and those who are aware of group singing benefits from popular media may have positive ex- pectations, which in turn lead to positive outcomes. Another deficient quality issue of the included studies was measuring adverse effects. No study has reported their attempt to mea- sure adverse events, but qualitative studies indicated that sing- ing interventions were well‐received by patients. also evidence that suggests depression would be mediated by the relationship between social isolation and physical health, with higher social isolation scores predicting higher depression scores (Karayannis et al., 2018). As we presented in Figure 3, singing may have a knock‐on effect, such that reduced pain intensity, in turn, reduces pain interference and suffering, and consequently depression. In summary, there is sufficient qual- itative evidence supporting singing intervention for reducing depression; inconsistent evidence from quantitative studies in this review warrants further investigation. Moreover, our evidence syntheses suggest that studies with longer term interventions (>6 months) appear to be effective in reducing pain and depression, as interventions that lasted less than 6 months showed inconsistent findings. Thus, it can be rec- ommended that patients with long‐term health conditions should take part in group singing for months and years. Additionally, future studies may assess high‐intensity (>1 hr weekly) short‐ term intervention (<6 months), as there is currently no available evidence. Further, more studies are needed to establish appro- priate singing intervention length and frequency for people with chronic health conditions in order to manage pain effectively. 4 \| DISCUSSION The included studies have variable study quality with many having a number of weaknesses: in particular, internal valid- ity, confounders and measuring adverse effects were identi- fied as common deficiency. Included studies reported basic REFERENCES Although all possible efforts were made to find relevant stud- ies through major databases and other repository channels and having no language restrictions, the current review only included articles published in English, which may have pre- sented a publication bias. The current review is also limited by the small number of RCTs and the inclusion of uncon- trolled studies, which may produce overestimates of effects (Thornton & Lee, 2000). Aaron, R. V., Fisher, E. A., de la Vega, R., Lumley, M. A., & Palermo, T. M. (2019). Alexithymia in individuals with chronic pain and its rela- tion to pain intensity, physical interference, depression, and anxiety: A systematic review and meta‐analysis. Pain, 160(5), 994–1006. https://doi.org/10.1097/j.pain.0000000000001487 Achterberg, W., Pieper, M. J. C., van Dalen‐Kok, A. H., de Waal, M. W. M., Husebo, B. S., Lautenbacher, S., … Corbett, A. (2013). Pain management in patients with dementia. Clinical Interventions in Aging, 8, 1471–1482. https://doi.org/10.2147/CIA.S36739 ging, 8, 1471–1482. https://doi.org/10.2147/CIA.S36739 Archer, S., Buxton, S., & Sheffield, D. (2015). The effect of creative psychological interventions on psychological outcomes for adult cancer patients: A systematic review of randomised controlled trials. Psychooncology, 24(1), 1–10. https://doi.org/10.1002/ pon.3607 AUTHOR CONTRIBUTIONS JYI: primary investigator involved in study selection, data extraction, analysis and manuscript writing. DS: participated in searches, study selection, data ex- traction as a second reviewer, initial meta‐analysis and man- uscript writing. FB: participated in searches, study selection, quality as- sessments and manuscript writing. DES: contributed to manuscript writing. All authors discussed the results and commented on the manuscript. All authors discussed the results and commented on the manuscript. ACKNOWLEDGEMENTS The authors thank the reviewers for their comments on the manuscript. We also thank Jill Boland, academic librarian at the University of Derby, for her advice on the searches. Finally, we thank authors who shared data with us. The 1st author received funding from Queensland Conservatorium Research centre for the early stage of this review. 4.1 The present review provides an analysis of the current evi- dence of the effects of singing on chronic pain management. IRONS 87 More RCTs with active comparators with larger sample sizes are needed to better understand the potential effects of group singing on chronic pain for people with long‐term health conditions. Further, given the widespread popular- ity of singing and perception of potential health benefits, we advocate measuring participants' expectations prior to the singing intervention. Additionally, researchers need to identify a minimally required number of singing sessions, along with cost‐benefit analysis, so that group singing can be administered in the most effective way, to achieve op- timal pain‐related outcomes for patients. Finally, future studies should have a fuller and more consistent approach to reporting the details of sessions, in order to identify po- tential mediators of the effects. For practice, this review affirms that group singing has a range of potential benefits for people with pain. Based on our synthesis, we recom- mend that at least 6 months of singing is most likely to be effective for reducing pain in people with long‐term health conditions. Group singing facilitators' roles are important, in order to engage participants fully into singing activities, as most people would require encouragement and support. Ongoing training and more resources are also required for group singing facilitators to provide the most effective singing sessions. group singing, practitioners are encouraged to continue this work and consider pain measures in evaluations of it. group singing, practitioners are encouraged to continue this work and consider pain measures in evaluations of it. 5 \| CONCLUSION (2012). A pilot investigation of quality of life and lung func- tion following choral singing in cancer survivors and their carers. Ecancermedicalscience, 6(1), 1–13. https://doi.org/10.3332/ecanc er.2012.261 Brown, J. L., Sheffield, D., Leary, M. R., & Robinson, M. E. (2003). Social support and experimental pain. Psychosomatic Medicine, 65(2), 276–283. https://doi.org/10.1097/01.PSY.00000 30388.62434.46 Gatchel, R. J., Peng, Y. B., Peters, M. L., Fuchs, P. N., & Turk, D. C. (2007). The biopsychosocial approach to chronic pain: Scientific advances and future directions. Psychological Bulletin, 133(4), 581–624. https://doi.org/10.1037/0033-2909.133.4.581 Burke, A. L., Mathias, J. L., & Denson, L. A. (2015). Psychological functioning of people living with chronic pain: A meta‐analytic re- view. British Journal of Clinical Psychology, 54(3), 345–360. https ://doi.org/10.1111/bjc.12078 Grape, C., Töres, T., Britt‐Maj, W., & Rolf, E. (2009). Choir singing and fibrinogen. VEGF, cholecystokinin and motilin in IBS patients. Medical Hypotheses, 72(2), 223–225. https://doi.org/10.1016/j. mehy.2008.09.019 Busch, V., Magerl, W., Kern, U., Haas, J., Hajak, G., & Eichhammer, P. (2012). The effect of deep and slow breathing on pain per- ception, autonomic activity, and mood processing—an ex- perimental study. Pain Medicine, 13(2), 215–228. https://doi. org/10.1111/j.1526-4637.2011.01243.x Hénon, H. (2006). Pain after stroke: A neglected issue. Journal of Neurology, Neurosurgery, and Psychiatry, 77(5), 569–569. https:// doi.org/10.1136/jnnp.2005.085761 Higgins, J. P., & Green, S. (Eds.). (2011). Cochrane Handbook for Systematic Reviews of Interventions Version 5.1. Available from www.cochrane‐handbook.org: The Cochrane Collaboration. Accessed 08/04/2019. CASP Programme. (2018). CASP Qualitative Checklist. [online]. Accessed 15/07/2019 https://casp-uk.net/wp-content/uploa ds/2018/03/CASP-Qualitative-Checklist-2018_fillable_form.pdf Clements‐Cortes, A. (2013). Buddy's glee club: Singing for life. Act Adapt & Aging, 37, 273–290. https://doi.org/10.1080/01924 788.2013.845716 Hopper, M. J., Curtis, S., Hodge, S., & Simm, R. (2016). A qualitative study exploring the effects of attending a community pain service choir on wellbeing in people who experience chronic pain. British Journal of Pain, 10(3), 124–134. https://doi.org/10.1177/20494 63716638368 Clements‐Cortes, A. (2015). Singing for health, connection and care. Muscle and Medical, 7(4), 13–23. Cohen, J. (1988). Statistical power analysis for the behavioral sciences. (2nd ed.). NJ: Erlbaum: Hillsdale. Horner, S., Rew, L., & Torres, R. (2006). Enhancing interven- tion fidelity: A means of strengthening study impact. Journal for Specialists in Pediatric Nursing, 11(2), 80–89. https://doi. org/10.1111/j.1744-6155.2006.00050.x Cook, K. F., Dunn, W., Griffith, J. W., Morrison, M. T., Tanquary, J., Sabata, D., … Gershon, R. C. (2013). Pain assessment using the NIH Toolbox. Neurology, 80(11 Suppl 3), S49–S53. https://doi. org/10.1212/WNL.0b013e3182872e80 Irons, J. Y., Kenny, D. T., McElrea, M., & Chang, A. B. (2012). 5 \| CONCLUSION Based on the research evidence syntheses presented in this review, currently there is limited support for the effects of group singing on chronic pain in people with long‐term health conditions. Group singing appears to have the poten- tial to reduce pain intensity, pain interference and depression based on the limited corpus of studies with variable quality. Qualitative data in this review also highlighted that singing programmes were enthusiastically received by participants and had positive impacts on the physical, psychological and social aspects of participants' lives suggesting a variety of mechanisms. However, additional well‐designed studies are needed to investigate whether singing intervention has greater effects than other non‐pharmacological interventions on pain experience. Finally, given the wide‐ranging health benefits of Based on the research evidence syntheses presented in this review, currently there is limited support for the effects of group singing on chronic pain in people with long‐term health conditions. Group singing appears to have the poten- tial to reduce pain intensity, pain interference and depression based on the limited corpus of studies with variable quality. Bernardes, S. F., Forgeron, P., Fournier, K., & Reszel, J. (2017). Beyond solicitousness: A comprehensive review on informal pain‐related social support. Pain, 158(11), 2066–2076. https://doi.org/10.1097/j. pain.0000000000001033 Blomqvist, K., & Hallberg, I. R. (2002). Managing pain in older persons who receive home‐help for their daily living. Perceptions by older persons and care providers. Scandinavian Journal of Caring Sciences, 16(3), 319–328. https://doi. org/10.1046/j.1471-6712.2002.00087.x Bonilha, A. G., Onofre, F., Vieira, M. L., Almeida Prado, M. Y., & Martinez, J. A. B. (2009). Effects of singing classes on pulmonary function and quality of life of COPD patients. International Journal of Chronic Obstructive Pulmonary Disease, 4(1), 1–8. of Chronic Obstructive Pulmonary Disease, 4(1), 1–8. Bradt, J., Norris, M., Shim, M., Gracely, E. J., & Gerrity, P. (2016). Vocal music therapy for chronic pain management in inner‐city 88 \| African Americans: a mixed methods feasibility study. Journal of Music Therapy, 53(2), 178–206. https://doi.org/10.1093/jmt/thw004 IRONS IRONS 88 following stroke or Parkinsons disease: An exploration of partici- pants experiences. Disability and Rehabilitation, 38(10), 952–962. https://doi.org/10.3109/09638288.2015.1068875 African Americans: a mixed methods feasibility study. Journal of Music Therapy, 53(2), 178–206. https://doi.org/10.1093/jmt/thw004 Breivik, H., Collett, B., Ventafridda, V., Cohen, R., & Gallacher, D. (2006). Survey of chronic pain in Europe: Prevalence, impact on daily life, and treatment. European Journal of Pain, 10(4), 287–287. https://doi.org/10.1016/j.ejpain.2005.06.009 Gale, N. S., Enright, S., Reagon, C., Lewis, I., & Van Deursen, R. 5 \| CONCLUSION Singing therapy for young people with cystic fibrosis a randomized con- trolled pilot study. Music and Medicine, 4, 136–145. https://doi. org/10.1177/1943862112452150 Cooper, H., Hedges, L. V., & Valentine, J. C. (2009). The Handbook of Research Synthesis and Meta‐Analysis. (2nd ed.). New York: Russell Sage Foundation. Dansie, E. J., & Turk, D. C. (2013). Assessment of patients with chronic pain. British Journal of Anaesthesia, 111(1), 19–25. https://doi. org/10.1093/bja/aet124 Irons, J. Y., Kuipers, K., & Petoz, P. (2013). Exploring the health ben- efits of singing for young people with cystic fibrosis, using the in- ternational classification of functioning, disability and health for children and youth (ICF‐CY). International Journal of Therapy and Rehabiliation, 20(3), 144–153. https://doi.org/10.12968/ ijtr.2013.20.3.144 Decoster, J., & Claypool, H. M. (2004). A meta‐analysis of priming ef- fects on impression formation supporting a general model of infor- mational biases. Personality and Social Psychology Review, 8(1), 2–27. https://doi.org/10.1207/s15327957pspr0801_1 Irons, J. Y., Petocz, P., Kenny, D. T., & Chang, A. B. (2019). Singing as an adjunct therapy for children and adults with cystic fibrosis. Cochrane Database of Systematic Reviews, (7, 27). https://doi. org/10.1002/14651858.CD008036.pub5 Downs, S. H., & Black, N. (1998). The feasibility of creating a checklist for the assessment of the methodological quality both of randomized and non‐randomized studies of health care interventions. Journal of Epidemiology and Community Health, 52, 377–384. Kaptein, A. A., Hughes, B. M., Murray, M., & Smyth, J. M. (2018). Start making sense: Art informing health psychology. Health Psychology Open, 5(1), 2055102918760042. https://doi.org/10.1177/20551 02918760042 Dunbar, R. I. M., Baron, R., Frangou, A., Pearce, E., van Leeuwen, E. J. C., Stow, J., … van Vugt, M. (2012). Social laughter is correlated with an elevated pain threshold. Proceedings of the Royal Society of London. Series B: Biological Sciences, 279(1731), 1161–1167. https://doi.org/10.1098/rspb.2011.1373 Karayannis, N. V., Baumann, I., Sturgeon, J. A., Melloh, M., & Mackey, S. C. (2018). The impact of social isolation on pain interference: A longitudinal study. Annals of Behavioral Medicine, 53(1), 65–74. https://doi.org/10.1093/abm/kay017 Dunbar, R. I. M., Kaskatis, K., MacDonald, I., & Barra, V. (2012). Performance of music elevates pain threshold and positive affect: Implications for the evolutionary function of music. Evolutionary Psychology, 10(4), 688–702. https://doi.org/10.1177/1474704912 01000403 Kennelly, J. (2011). Chapter 2. Methodological approach to assessing the evidence. In A. Handler, J. Kennelly, & N. Peacock (Eds.), Reducing racial/ethnic disparities in reproductive and peri- natal outcomes (pp. 7–19). Boston, MA: Springer. https://doi. org/10.1007/978-1-4419-1499-6_2. 5 \| CONCLUSION Kenshalo (Ed.), The skin senses: Proceedings of the first interna- tional symposium on the skin senses. Tallahassee, FL: The Florida State University. Skingley, A., Clift, S., Hurley, S., Price, S., & Stephens, L. (2018). Community singing groups for people with chronic obstructive pul- monary disease: Participant perspectives. Perspect Public Health, 138(1), 66–75. https://doi.org/10.1177/1757913917740930 Moher, D., Liberati, A., Tetzlaff, J., & Altman, D. G. (2009). Preferred reporting items for systematic reviews and meta‐analyses: The PRISMA statement. Annals of Internal Medicine, 151(4), 264–269. https://doi.org/10.7326/0003-4819-151-4-200908180-00135 Skingley, A., Martin, A., & Clift, S. (2015). The contribution of com- munity singing groups to the well‐being of older people: partici- pant perspectives from the United Kingdom. Journal of Applied Gerontology, 35(12), 1302–1324. https://doi.org/10.1177/07334 64815577141 Morrison, I., Clift, S., Page, S., Salisbury, I., Shipton, M., Skingley, A., & Vella‐Burrows, T. (2013). A UK feasibility study on the value of singing for people with Chronic Obstructive Pulmonary Disease (COPD). UNESCO Observatory Multi‐Disciplinary J Arts, 3(3), 1–19. Skogar, O., & Lokk, J. (2016). Pain management in patients with Parkinson's disease: Challenges and solutions. Journal of Multidisciplinary Healthcare, 9, 469–479. https://doi.org/10.2147/ JMDH.S105857 Moseley, L., & Butler, D. (2015). 15 years of explaining pain ‐ The past, present and future (Vol. 16). Pearce, E., Launay, J., & Dunbar, R. I. M. (2015). The ice‐breaker effect: Singing mediates fast social bonding. Royal Society Open Science, 2(10), 150221. https://doi.org/10.1098/rsos.150221. Stegemoller, E. L., Radig, H., Hibbing, P., Wingate, J., & Sapienza, C. (2017). Effects of singing on voice, respiratory control and quality of life in persons with Parkinson's disease. Disability and Rehabilitation, 39(6), 594–600. https://doi.org/10.3109/09638 288.2016.1152610 Pearce, E., Launay, J., MacCarron, P., & Dunbar, R. I. M. (2017). Tuning in to others: Exploring relational and collective bonding in singing and non‐singing groups over time. Psychology of Music, 45(4), 496– 512. https://doi.org/10.1177/0305735616667543 Tamplin, J., Baker, F. A., Grocke, D., Brazzale, D. J., Pretto, J. J., Ruehland, W. R., … Berlowitz, D. J. (2013). Effect of singing on re- spiratory function, voice, and mood after quadriplegia: A randomized controlled trial. Archives of Physical Medicine and Rehabilitation, 94(3), 426–434. https://doi.org/10.1016/j.apmr.2012.10.006 Pearce, E., Launay, J., Van Duijn, M., Rotkirch, A., David‐Barrett, T., & Dunbar, R. I. M. (2016). Singing together or apart: The effect of competitive and cooperative singing on social bonding within and between sub‐groups of a university Fraternity. Psychology of Music, 44(6), 1255–1273. https://doi.org/10.1177/0305735616636208 , 426–434. https://doi.org/10.1016/j.apmr.2012.10.006 Tarr, B., Launay, J., & Dunbar, R. I. M. 5 \| CONCLUSION Fogg‐Rogers, L., Buetow, S., Talmage, A., McCann, C. M., Leão, S. H. S., Tippett, L., … Purdy, S. C. (2016). Choral singing therapy IRONS IRONS 89 Kenny, D. T., & Faunce, G. (2004). The impact of group singing on mood, coping, and perceived pain in chronic pain patients attend- ing a multidisciplinary pain clinic. Journal of Music Therapy, 41(3), 241–258. https://doi.org/10.1093/jmt/41.3.241 Price, D. D. (1999). Psychological mechanisms of pain and analgesia, Vol. 15. Seattle: IASP Press. Reagon, C., Gale, N. S., Dow, R., Lewis, I., & van Deursen, R. (2017). Choir singing and health status in people affected by can- cer. European Journal of Cancer Care, 26(5), e12568. https://doi. org/10.1111/ecc.12568 Lord, V. M., Cave, P., Hume, V. J., Flude, E. J., Evans, A., Kelly, J. L., … Hopkinson, N. S. (2010). Singing teaching as a therapy for chronic respiratory disease – A randomised controlled trial and qualita- tive evaluation. BMC Pulmonary Medicine, 10, 41–48. https://doi. org/10.1186/1471-2466-10-41 Riley, J. L., Wade, J. B., Myers, C. D., Sheffield, D., Papas, R. K., & Price, D. D. (2002). Racial/ethnic differences in the experience of chronic pain. Pain, 100(3), 291–298. https://doi.org/10.1016/ S0304-3959(02)00306-8 Lord, V. M., Hume, V. J., Kelly, J. L., Cave, P., Silver, J., Waldman, M., … Hopkinson, N. S. (2012). Singing classes for chronic obstructive pulmonary disease: A randomized controlled trial. BMC Pulmonary Medicine, 12(1), 69–69. https://doi. org/10.1186/1471-2466-12-69 Russo, M. A., Santarelli, D. M., & O’Rourke, D. (2017). The physio- logical effects of slow breathing in the healthy human. Breathe, 13, 298–309. https://doi.org/10.1183/20734735.009817 Shea, B. J., Grimshaw, J. M., Wells, G. A., Boers, M., Andersson, N., Hamel, C., … Bouter, L. M. (2007). Development of AMSTAR: A measurement tool to assess the methodological quality of systematic reviews. BMC Medical Research Methodology, 7, 10. https://doi. org/10.1186/1471-2288-7-10 Ma, X., Yue, Z.‐Q., Gong, Z.‐Q., Zhang, H., Duan, N.‐Y., Shi, Y.‐T., … Li, Y.‐F. (2017). The effect of diaphragmatic breathing on attention, negative affect and stress in healthy adults. Frontiers in Psychology, 8, 1–12. https://doi.org/10.3389/fpsyg.2017.00874 8, 1–12. https://doi.org/10.3389/fpsyg.2017.00874 Sheng, J., Liu, S., Wang, Y., Cui, R., & Zhang, X. (2017). The link between depression and chronic pain: neural mechanisms in the brain. Neural Plasticity, 2017, 9724371–9724371. https://doi. org/10.1155/2017/9724371 Melzack, R., & Casey, K. L. (1968). Sensory, motivational, and cen- tral control determinants of pain: A new conceptual model. In D. R. 5 \| CONCLUSION (2014). Music and social bonding: “self‐other” merging and neurohormonal mechanisms. Frontiers in Psychology, 5(1096), 1–10. https://doi.org/10.3389/ fpsyg.2014.01096 Plumb, L., & Stickley, T. (2017). Singing to promote mental health and well‐being. Mental Health Practice, 20(8), 31–36. https://doi. org/10.7748/mhp.2017.e1182 Thornton, A., & Lee, P. (2000). Publication bias in meta‐analysis: Its causes and consequences. Journal of Clinical Epidemiology, 53(2), 207–216. https://doi.org/10.1016/S0895-4356(99)00161-4 Pongan, E., Tillmann, B., Leveque, Y., Trombert, B., Getenet, J. C., Auguste, N., … Rouch, I. (2017). Can musical or painting interven- tions improve chronic pain, mood, quality of life, and cognition in patients with mild Alzheimer's disease? Evidence from a random- ized controlled trial. Journal of Alzheimer's Disease, 60(2), 663– 677. https://doi.org/10.3233/JAD-170410 Treede, R.‐D., Rief, W., Barke, A., Aziz, Q., Bennett, M. I., Benoliel, R., … Wang, S.‐J. (2015). A classification of chronic pain for ICD‐11. Pain, 156(6), 1003–1007. https://doi.org/10.1097/j.pain.00000 00000000160 Popay, J., Roberts, H., Sowden, A., Petticrew, M., Arai, L., & Rodgers, M., …Duffy, S. (2006). A product from the ESRC methods pro- gramme: Guidance on the conduct of narrative synthesis in system- atic reviews. Wan, C. Y., Ruber, T., Hohmann, A., & Schlaug, G. (2010). The thera- peutic effects of singing in neurological disorders. Music Perception, 27(4), 287–295. https://doi.org/10.1525/mp.2010.27.4.287 IRONS IRONS 90 SUPPORTING INFORMATION SUPPORTING INFORMATION Weinstein, D., Launay, J., Pearce, E., Dunbar, R. I., & Stewart, L. (2016). Group music performance causes elevated pain thresholds and social bonding in small and large groups of singers. Evolution and Human Behavior, 37(2), 152–158. https://doi.org/10.1016/j. evolhumbehav.2015.10.002 Additional supporting information may be found online in the Supporting Information section at the end of the article. Williams, E., Dingle, G. A., & Clift, S. (2018). A systematic review of mental health and wellbeing outcomes of group singing for adults with a mental health condition. European Journal of Public Health. https://doi.org/10.1093/eurpub/cky115 How to cite this article: Irons JY, Sheffield D, Ballington F, Stewart DE. A systematic review on the effects of group singing on persistent pain in people with long‐term health conditions. Eur J Pain. 2020;24:71–90. https://doi.org/10.1002/ejp.1485 How to cite this article: Irons JY, Sheffield D, Ballington F, Stewart DE. A systematic review on the effects of group singing on persistent pain in people with long‐term health conditions. Eur J Pain. 2020;24:71–90. https://doi.org/10.1002/ejp.1485 Wimo, A., Jönsson, L., Bond, J., Prince, M., & Winblad, B. (2013). The worldwide economic impact of dementia 2010. Alzheimer's & Dementia: the Journal of the Alzheimer's Association, 9(1), 1–11. e13. https://doi.org/10.1016/j.jalz.2012.11.006
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* # # * GFP CEP170-3'UTR Vector miR-137 miR-141 miR-203 0.0 0.2 0.4 0.6 0.8 1.0 1.2 Relative luciferase activity ## ## ## GFP NUCKS1-3'UTR Vector miR-101 miR-125 miR-137 miR-144 miR-200 miR-497 0.0 0.2 0.4 0.6 0.8 1.0 1.2 Relative luciferase activity ## ## ## # ** * # * # GFP ZC3H11A-3'UTR A B C D E miR-135 miR-141 miR-144 miR-200 miR-203 0.0 0.5 1.0 1.5 2.0 2.5 Relative enrichment GFP CEP170-3'UTR * ** * * miR-135 miR-137 miR-203 0 1 2 3 4 Relative enrichment GFP NUCKS1-3'UTR * * miR-101 miR-125 miR-137 miR-144 miR-200 miR-497 0.0 0.5 1.0 1.5 2.0 2.5 Relative enrichment GFP ZC3H11A-3'UTR ** * * * F G H GFP ceRNA 3’UTR Luc MRE i miRNA ii GFP ceRNA 3’UTR biotinylated probes Streptavidin beads miRNAs 105 Pri Met 10-1 100 101 102 103 FPKM Pri Met 10-2 10-1 100 101 102 103 FPKM Pri Met 10-2 10-1 100 101 102 103 FPKM * 102 * 104 106 * 103 Pri Met 102 103 104 105 FPKM Pri Met 10-2 10-1 100 101 102 FPKM miR-101 miR-135 miR-137 miR-141 miR-144 miR-200a miR-200b miR-200c miR-203a miR-497 I miR-135 miR-137 miR-144 miR-203 0.0 0.2 0.4 0.6 0.8 1.0 1.2 miRNA reporter Relative luciferase activity GFP CEP170-3'UTR NUCKS1-3'UTR ZC3H11A-3'UTR Combo-3'UTR * ns ns ns ns * ns * E A GFP ceRNA 3’UTR Luc MRE i miRNA ii GFP ceRNA 3’UTR biotinylated probes Streptavidin beads miRNAs E A miR-135 miR-137 miR-144 miR-203 0.0 0.2 0.4 0.6 0.8 1.0 1.2 miRNA reporter Relative luciferase activity GFP CEP170-3'UTR NUCKS1-3'UTR ZC3H11A-3'UTR Combo-3'UTR * ns ns ns ns * ns * Vector miR-135 miR-141 miR-144 miR-200 miR-203 0.0 0.2 0.4 0.6 0.8 1.0 1.2 Relative luciferase activity * * ** * * # # ## * # # * GFP CEP170-3'UTR Vector miR-137 miR-141 miR-203 0.0 0.2 0.4 0.6 0.8 1.0 1.2 Relative luciferase activity ## ## ## GFP NUCKS1-3'UTR Vector miR-101 miR-125 miR-137 miR-144 miR-200 miR-497 0.0 0.2 0.4 0.6 0.8 1.0 1.2 Relative luciferase activity ## ## ## # ** * # * # GFP ZC3H11A-3'UTR A B C D E GFP ceRNA 3’UTR Luc MRE i miRNA ii GFP ceRNA 3’UTR biotinylated probes Streptavidin beads miRNAs i miR-135 miR-137 miR-144 miR-203 0.0 0.2 0.4 0.6 0.8 miRNA reporter Relative luciferase a GFP CEP170-3'UTR NUCKS1-3'UTR ZC3H11A-3'UTR Combo-3'UTR Vector miR-135 miR-141 miR-144 miR-200 miR-203 0.0 0.2 0.4 0.6 0.8 1.0 1.2 Relative luciferase activity * * ** * * # # ## * # # * GFP CEP170-3'UTR Vector miR-137 miR-141 miR-203 0.0 0.2 0.4 0.6 0.8 1.0 1.2 Relative luciferase activity ## ## ## GFP NUCKS1-3'UTR Vector miR-101 miR-125 miR-137 miR-144 miR-200 miR-497 0.0 0.2 0.4 0.6 0.8 1.0 1.2 Relative luciferase activity ## ## ## # ** * # * # GFP ZC3H11A-3'UTR B C D et al Figure S4 miR-135 miR-141 miR-144 miR-200 miR-203 0.0 0.5 1.0 1.5 2.0 2.5 Relative enrichment GFP CEP170-3'UTR * ** * * miR-135 miR-137 miR-203 0 1 2 3 4 Relative enrichment GFP NUCKS1-3'UTR * * miR-101 miR-125 miR-137 miR-144 miR-200 miR-497 0.0 0.5 1.0 1.5 2.0 2.5 Relative enrichment GFP ZC3H11A-3'UTR ** * * * F G H Luc MRE ii GFP ceRNA 3’UTR biotinylated probes Streptavidin beads miRNAs Pri Met 100 101 102 103 104 105 FPKM * Pri Met 10-1 100 101 102 103 FPKM Pri Met 10-2 10-1 100 101 102 103 FPKM Pri Met 10-2 10-1 100 101 102 103 FPKM * Pri Met 10-2 10-1 100 101 102 FPKM * Pri Met 10-1 100 101 102 103 104 FPKM * Pri Met 100 101 102 103 104 105 106 FPKM * Pri Met 10-1 100 101 102 103 FPKM Pri Met 102 103 104 105 FPKM Pri Met 10-2 10-1 100 101 102 FPKM miR-101 miR-135 miR-137 miR-141 miR-144 miR-200a miR-200b miR-200c miR-203a miR-497 I Vector miR-135 miR-141 miR-144 miR-200 miR-203 0.0 0.2 0.4 0.6 0.8 1.0 1.2 Relative luciferase activity * * ** * * # # ## * # # * GFP CEP170-3'UTR Vector miR-137 miR-141 miR-203 0.0 0.2 0.4 0.6 0.8 1.0 1.2 Relative luciferase activity ## ## ## GFP NUCKS1-3'UTR Vector miR-101 miR-125 miR-137 miR-144 miR-200 miR-497 0.0 0.2 0.4 0.6 0.8 1.0 1.2 Relative luciferase activity ## ## ## # ** * # * # GFP ZC3H11A-3'UTR B C D Vector miR-137 miR-141 miR-203 0.0 0.2 0.4 0.6 0.8 1.0 1.2 Relative luciferase activity ## ## ** ## GFP NUCKS1-3'UTR C Vector miR-135 miR-141 miR-144 miR-200 miR-203 0.0 0.2 0.4 0.6 0.8 1.0 1.2 Relative luciferase activity * * ** * * # # ## * # # * GFP CEP170-3'UTR B C Vector miR-101 miR-125 miR-137 miR-144 miR-200 miR-497 0.0 0.2 0.4 0.6 0.8 1.0 1.2 Relative luciferase activity ## ## ## # * # * # GFP ZC3H11A-3'UTR D D C C B V miR miR miR miR miR V miR miR miR V miR miR miR miR miR miR miR-135 miR-141 miR-144 miR-200 miR-203 0.0 0.5 1.0 1.5 2.0 2.5 Relative enrichment GFP CEP170-3'UTR * ** * * miR-135 miR-137 miR-203 0 1 2 3 4 Relative enrichment GFP NUCKS1-3'UTR * * miR-101 miR-125 miR-137 miR-144 miR-200 miR-497 0.0 0.5 1.0 1.5 2.0 2.5 Relative enrichment GFP ZC3H11A-3'UTR ** * * * F G H m m m miR-135 miR-137 miR-203 0 1 2 3 4 Relative enrichment GFP NUCKS1-3'UTR * * G miR-135 miR-141 miR-144 miR-200 miR-203 0.0 0.5 1.0 1.5 2.0 2.5 Relative enrichment GFP CEP170-3'UTR * ** * * F miR-101 miR-125 miR-137 miR-144 miR-200 miR-497 0.0 0.5 1.0 1.5 2.0 2.5 Relative enrichment GFP ZC3H11A-3'UTR ** * * * H H G F Xu et al., Figure S4 Pri Met 100 101 102 103 104 105 FPKM * Pri Met 10-1 100 101 102 103 FPKM Pri Met 10-2 10-1 100 101 102 103 FPKM Pri Met 10-2 10-1 100 101 102 103 FPKM * Pri Met 10-2 10-1 100 101 102 FPKM * Pri Met 10-1 100 101 102 103 104 FPKM * Pri Met 100 101 102 103 104 105 106 FPKM * Pri Met 10-1 100 101 102 103 FPKM Pri Met 102 103 104 105 FPKM Pri Met 10-2 10-1 100 101 102 FPKM miR-101 miR-135 miR-137 miR-141 miR-144 miR-200a miR-200b miR-200c miR-203a miR-497 I Xu et al., Figure S4 Pri Met 100 101 102 103 104 105 FPKM * Pri Met 10-1 100 101 102 103 FPKM Pri Met 10-2 10-1 100 101 102 103 FPKM Pri Met 10-2 10-1 100 101 102 103 FPKM * Pri Met 10-2 10-1 100 101 102 FPKM * Pri Met 10-1 100 101 102 103 104 FPKM * Pri Met 100 101 102 103 104 105 106 FPKM * Pri Met 10-1 100 101 102 103 FPKM Pri Met 102 103 104 105 FPKM Pri Met 10-2 10-1 100 101 102 FPKM miR-101 miR-135 miR-137 miR-141 miR-144 miR-200a miR-200b miR-200c miR-203a miR-497 I Pri Met 100 101 102 103 104 105 FPKM * Pri Met 10-1 100 101 102 103 Pri Met 10-2 10-1 100 101 102 103 FPKM Pri Met 10-2 10-1 100 101 102 103 FPKM * Pri Met 10-1 100 101 102 103 104 * Pri Met 100 101 102 103 104 105 106 FPKM * 10-1 100 101 102 103 FPKM 10-2 10-1 100 101 102 FPKM miR-135 miR-137 miR-141 miR-200b miR-200c miR-203a Pri Met 100 101 102 103 104 105 FPKM * Pri Met 10-2 10-1 100 101 102 103 FPKM FPKM miR-137 miR-200c Pri Met 100 101 102 103 104 105 FPKM * Pri Met 10-1 100 101 102 103 FPKM Pri Met 10-2 10-1 100 101 102 103 FPKM Pri Met 10-2 10-1 100 101 102 103 FPKM * Pri Met 10-2 10-1 100 101 102 FPKM * Pri Met 10-1 100 101 102 103 104 FPKM * Pri Met 100 101 102 103 104 105 106 FPKM * Pri Met 102 103 104 105 FPKM miR-101 miR-135 miR-137 miR-141 miR-200a miR-200b miR-200c miR-203a I Pri Met 10-1 100 101 102 103 FPKM Pri Met 10-2 10-1 100 101 102 FPKM * Pri Met 10-1 100 101 102 103 104 FPKM * Pri Met 102 103 104 105 FPKM miR-101 miR-135 miR-200a miR-200b I Pri Met 100 101 102 103 104 105 FPKM * Pri Met 0-1 00 01 02 03 Pri Met 10-2 10-1 100 101 102 103 FPKM Pri Met 0-1 00 01 02 03 04 * miR-135 miR-137 miR-200b miR-200c Pri Met 10-2 10-1 100 101 102 103 FPKM * Pri Met 100 101 102 103 104 105 106 FPKM * Pri Met 10-1 100 101 102 103 FPKM Pri Met 10-2 10-1 100 101 102 FPKM miR-141 miR-144 miR-203a miR-497 I I Pri Met 10-1 100 101 102 103 FPKM miR-135 Pri Met 10-2 10-1 100 101 102 FPKM miR-144 Pri Met 10-2 10-1 100 101 102 103 FPKM * miR-141 FPKM FPKM FPKM FPKM FPKM Pri Met 10-1 100 101 102 103 104 FPKM * miR-200b Pri Met 100 101 102 103 104 105 106 FPKM *** FPKM miR-203a Pri Met 10-1 100 101 102 103 FPKM miR-497 FPKM FPKM FPKM FPKM Xu et al., Figure S4 Xu et al., Figure S4
https://openalex.org/W2281967437	http://metsatieteenaikakauskirja.fi/pdf/6251	Finnish	null	Metsäojitusalueen puuston boniteettimalli	Metsätieteen aikakauskirja	1,970	cc-by-sa	2,065	Tausta S uomessa on ojitettuja soita valtakunnan metsien uusimman inventoinnin mukaan 4,7 miljoonaa hehtaaria, mikä vastaa noin 18:aa prosenttia koko metsätalousmaan pinta-alasta. Keskikasvu niillä oji- tusalueilla, joilla kunnostusojitukset on tehty ajois- sa, on ollut melko voimakas jo lähes 40 vuoden ajan. Tästä on seurannut huomattavan suuri metsävarojen lisäys. Suopuustojen tilavuuden yhä voimistuva ke- hitys ei ole siten yhdentekevää. Tarkka kasvupaikan hyvyyden arviointi antaa varman pohjan ojitusaluei- den puustojen tulevalle kasvuennusteelle. Kivennäismailla yleisesti käytetty, valtapituuteen ja ikään perustuva kasvupaikkojen luokittelu ei edellä esitetyn perusteella sovi kovin hyvin ojitus- alueiden puustoille. Puun ikä ja kehitysvaihe eivät ole samalla tavalla sidoksissa toisiinsa niin kuin ki- vennäismailla. Puut voivat olla ojitushetkellä hyvin vanhoja, vaikka ne ovat kooltaan pieniä. Puuston ikää/kehitysvaihetta on vaikea määrittää järkevästi ojitetuilla soilla, minkä vuoksi kovien maiden pi- tuusbonitointimenetelmää ei ole juurikaan sovellet- tu Suomen suometsiin. Arvioinnin perustuessa vain mittaushetken valtapituuteen saadaan virheellisiä tuloksia paikoilla, joilla oli puustoa jo ojitushetkel- lä (vanhoja ojitusalueita). Arvioinnissa pitäisi ottaa huomioon myös puuston kehitys ennen ojitusta. S u u Jo vuosikymmen sitten pidetyssä kasvupaikkase- minaarissa (Reinikainen ja Lehtinen 1994) käytän- nön metsätalouden edustajat ja tutkijat korostivat kasvupaikan luokitustapojen rinnakkaista käyttöä ja edelleen kehittelyä. Tällöin ehdotettiin pituusbo- nitoinnin mahdollista käyttöä myös ojitusalueilla (myös Penttilä 1984). Aluskasvillisuuteen nojau- tuvaa silmävaraista suotyypittelyä voitaisiin näin vahvistaa käyttämällä puustoon perustavaa objek- tiivisempaa menetelmää. Ojituksen jälkeinen valtapituuden kehitys – uuden arviointimallin perusta Ratkaisu uuteen puustobonitointimalliin löydettiin suontutkijoiden jo pitkään käyttämistä tunnuksista: ojituksenjälkeinen valtapuiden kehitys ja aika oji- tuksesta (ojitusikä) (esim. Seppälä 1969, Paarlahti 1988). Ojitusikä on nykyisin tärkeä muuttuja myös ojitusalueiden puuston kasvumalleissa (Mela-mal- lit, Hökkä 1997). Tutkimuksen tuloksena oli uusi pituusboniteettimalli, jossa puiden ikä on korvattu helposti määritettävällä ojitusiällä ja jossa valtapi- tuus mittaushetkellä on korvattu valtapituuden ke- hityksellä ojituksen jälkeen. Laadittu malli perustuu Metsäojitusalueen puuston boniteettimalli Nämä olosuhteet aiheuttavat vaihtelevaa riippuvuut- ta puiden kasvun ja kasvutekijöiden välillä. Nämä olosuhteet aiheuttavat vaihtelevaa riippuvuut- ta puiden kasvun ja kasvutekijöiden välillä. Hans Gustav Gustavsen Metsäojitusalueen puuston boniteettimalli Hans Gustav Gustavsen Metsätieteen aikakauskirja 2/2004 Metsätieteen aikakauskirja 2/2004 Metsätieteen aikakauskirja 2/2004 Tieteen tori Metsäojitusalueiden erikoispiirteet vaikeuttavat arviointia Ojitetuilla soilla kasvupaikka muuttuu jatkuvasti mm. turpeen kuivumisen, painumisen ja minerali- soitumisen seurauksena. Turvemaa voi soistua uu- delleen, jos ojat eivät toimi kunnolla. Vesitalouden lisäksi myös ravinnetalous muuttuu aikaa myöten. 220 Metsätieteen aikakauskirja 2/2004 Tieteen tori Metsäntutkimuslaitoksen yli 45 vuotta seuratuilta kestokoealoilta mitattuun aineistoon Etelä-Suomen metsäojitusalueilta. Bonitointi antaa välituloksena (kuva 1) valtapi- tuuden kasvun (Hdomdr) 40 vuoden ojitusiällä (Tdr) eli ns. pituusboniteetti-indeksin (H40dr). Pituusbo- niteetit on esitetty myös taulukossa 1, josta tarkka boniteetti-indeksi voidaan määrittää interpoloimalla ojituksenjälkeisen valtapituuden ja ojitusiän avul- la. Bonitoinnin lopputuloksena saadaan kasvupaikan ojituksenjälkeinen keskikasvu (MAI40dr, m3/ha/v), joka on laskettu 40 vuoden jaksolle ojituksen jälkeen (ojitusikä). Pituusboniteettiluokkien edustama puun- tuotoskyky (taulukko 2) voidaan laskea malleilla: Kuusivaltaiset metsiköt: MAI40dr = 1,850 + 0,018 × H40dr2 Mäntyvaltaiset metsiköt: MAI40dr = 1,828 + 0,015 × H40dr2 Bonitoinnin varmistamiseksi on kuitenkin hyödyl- listä käyttää sekä puustoon että aluskasvillisuuteen perustuvaa menetelmää Taulukossa 2 on esitetty eri Taulukko 1. Ojituksenjälkeisen valtapituuden (Hdomdr , m) kehitys ojitusiän (Tdr , v) mukaan pituusboniteettiluo- kissa H40dr = 8–22. Tdr,v H40dr 8 10 12 14 16 18 20 22 7 9 11 13 15 17 19 21 23 5 1,0 1,4 1,7 2,1 2,5 2,9 3,4 3,9 4,4 10 2,0 2,6 3,3 4,0 4,8 5,5 6,4 7,2 8,2 15 2,9 3,9 4,8 5,8 6,8 7,9 9,0 10,2 11,4 20 3,8 5,0 6,2 7,5 8,7 10,1 11,4 12,6 14,3 25 4,7 6,1 7,5 9,0 10,5 12,0 13,6 15,2 16,9 30 5,5 7,1 8,8 10,4 12,1 13,8 15,6 17,3 19,1 35 6,3 8,1 9,9 11,8 13,6 15,5 17,0 19,3 21,2 40 7,0 9,0 11,0 13,0 15,0 17,0 19,0 21,0 23,0 45 7,7 9,9 12,0 14,2 16,3 18,4 20,5 22,6 24,7 50 8,4 10,7 13,0 15,3 17,5 19,7 21,9 24,11 26,2 55 9,0 11,5 13,9 16,3 18,7 20,9 23,2 25,4 27,6 60 9,8 12,3 14,8 17,3 19,7 22,1 24,4 26,6 28,8 7 9 11 13 15 17 19 21 23 "ÌÕÃiBii Û>Ì>«ÌÕÕÃÊ``À®]Ê ä]ä Ó]ä {]ä È]ä n]ä £ä]ä £Ó]ä £{]ä £È]ä £n]ä Óä]ä ÓÓ]ä Ó{]ä ÓÈ]ä Ón]ä Îä]ä n £ä £Ó £{ £È £n Óä ÓÓ ä Èä {ä xä Îä Óä £ä "ÌÕÃBÊ/`À®]ÊÛ {ä`À Kuva 1. Etelä-Suomen ojitusalueiden kuusi-, koivu- ja mäntyvaltaisten metsien pituusboniteetit. Hdomdr on mit- taushetken valtapituuden (Hd ) ja ojitushetken valtapi- Taulukko 1. Ojituksenjälkeisen valtapituuden (Hdomdr , m) kehitys ojitusiän (Tdr , v) mukaan pituusboniteettiluo- kissa H40dr = 8–22. Taulukko 1. Metsäojitusalueiden erikoispiirteet vaikeuttavat arviointia Ojituksenjälkeisen valtapituuden (Hdomdr , m) kehitys ojitusiän (Tdr , v) mukaan pituusboniteettiluo- kissa H40dr = 8–22. Tdr,v H40dr 8 10 12 14 16 18 20 22 7 9 11 13 15 17 19 21 23 5 1,0 1,4 1,7 2,1 2,5 2,9 3,4 3,9 4,4 10 2,0 2,6 3,3 4,0 4,8 5,5 6,4 7,2 8,2 15 2,9 3,9 4,8 5,8 6,8 7,9 9,0 10,2 11,4 20 3,8 5,0 6,2 7,5 8,7 10,1 11,4 12,6 14,3 25 4,7 6,1 7,5 9,0 10,5 12,0 13,6 15,2 16,9 30 5,5 7,1 8,8 10,4 12,1 13,8 15,6 17,3 19,1 35 6,3 8,1 9,9 11,8 13,6 15,5 17,0 19,3 21,2 40 7,0 9,0 11,0 13,0 15,0 17,0 19,0 21,0 23,0 45 7,7 9,9 12,0 14,2 16,3 18,4 20,5 22,6 24,7 50 8,4 10,7 13,0 15,3 17,5 19,7 21,9 24,11 26,2 55 9,0 11,5 13,9 16,3 18,7 20,9 23,2 25,4 27,6 60 9,8 12,3 14,8 17,3 19,7 22,1 24,4 26,6 28,8 7 9 11 13 15 17 19 21 23 "ÌÕÃiBii Û>Ì>«ÌÕÕÃÊ``À®]Ê ä]ä Ó]ä {]ä È]ä n]ä £ä]ä £Ó]ä £{]ä £È]ä £n]ä Óä]ä ÓÓ]ä Ó{]ä ÓÈ]ä Ón]ä Îä]ä n £ä £Ó £{ £È £n Óä ÓÓ ä Èä {ä xä Îä Óä £ä "ÌÕÃBÊ/`À®]ÊÛ {ä`À Kuva 1. Etelä-Suomen ojitusalueiden kuusi-, koivu- ja mäntyvaltaisten metsien pituusboniteetit. Hdomdr on mit- taushetken valtapituuden (Hdom) ja ojitushetken valtapi- tuuden (Hdom0) erotus (Gustavsen 1996). "ÌÕÃiBii Û>Ì>«ÌÕÕÃÊ``À®]Ê ä]ä Ó]ä {]ä È]ä n]ä £ä]ä £Ó]ä £{]ä £È]ä £n]ä Óä]ä ÓÓ]ä Ó{]ä ÓÈ]ä Ón]ä Îä]ä n £ä £Ó £{ £È £n Óä ÓÓ ä Èä {ä xä Îä Óä £ä "ÌÕÃBÊ/`À®]ÊÛ {ä`À Metsäntutkimuslaitoksen yli 45 vuotta seuratuilta kestokoealoilta mitattuun aineistoon Etelä-Suomen metsäojitusalueilta. Metsäntutkimuslaitoksen yli 45 vuotta seuratuilta kestokoealoilta mitattuun aineistoon Etelä-Suomen metsäojitusalueilta. Bonitointi antaa välituloksena (kuva 1) valtapi- tuuden kasvun (Hdomdr) 40 vuoden ojitusiällä (Tdr) eli ns. pituusboniteetti-indeksin (H40dr). Pituusbo- niteetit on esitetty myös taulukossa 1, josta tarkka boniteetti-indeksi voidaan määrittää interpoloimalla ojituksenjälkeisen valtapituuden ja ojitusiän avul- la. Bonitoinnin lopputuloksena saadaan kasvupaikan ojituksenjälkeinen keskikasvu (MAI40dr, m3/ha/v), joka on laskettu 40 vuoden jaksolle ojituksen jälkeen (ojitusikä). Metsäojitusalueiden erikoispiirteet vaikeuttavat arviointia Pituusboniteettiluokkien edustama puun- tuotoskyky (taulukko 2) voidaan laskea malleilla: Kuusivaltaiset metsiköt: MAI40dr = 1,850 + 0,018 × H40dr2 Mäntyvaltaiset metsiköt: MAI40dr = 1,828 + 0,015 × H40dr2 Kuusivaltaiset metsiköt: MAI40dr = 1,850 + 0,018 × H40dr2 Mäntyvaltaiset metsiköt: MAI40dr = 1,828 + 0,015 × H40dr2 Kuusivaltaiset metsiköt: MAI40dr = 1,850 + 0,018 × H40dr2 Mäntyvaltaiset metsiköt: MAI40dr = 1,828 + 0,015 × H40dr2 Kuusivaltaiset metsiköt: MAI40dr = 1,850 + 0,018 × H40dr2 Mäntyvaltaiset metsiköt: Bonitoinnin varmistamiseksi on kuitenkin hyödyl- listä käyttää sekä puustoon että aluskasvillisuuteen perustuvaa menetelmää. Taulukossa 2 on esitetty eri luokitusten keskinäinen rinnastus. Kuva 1. Etelä-Suomen ojitusalueiden kuusi-, koivu- ja mäntyvaltaisten metsien pituusboniteetit. Hdomdr on mit- taushetken valtapituuden (Hdom) ja ojitushetken valtapi- tuuden (Hdom0) erotus (Gustavsen 1996). 221 Metsätieteen aikakauskirja 2/2004 Tieteen tori Taulukko 2. Pituusboniteettiluokkien (H40dr) ja suotyyppien sekä ravinteisuus- ja tuotos- luokkien vastaavuus ja puuntuotoskyky (MAI40dr). H40dr MAI40dr Alkuperäinen Ravinteisuusluokka Tuotosluokka suotyyppi (ojitettuna) Heikurainen 1968 Huikari 1952 Gustavsen ym. 1998 19 8,0 LhK,RhK 1–2 I 21–18 9,2–7,3 16 6,0 MK,VSK,PK,KgK, 2–3 II 17,9–15 7,2–5,6 RhSK,VLR,RhSR 13 4,5 PsK,VSR,LkSR,PsR, 3–4 III 14,9–12 5,5–4,0 KR,KgR,VSN,RhSN 10 3,0 IR,TR,RaR,LkN 5–6 IV 11,9–8 3,9–2,1 Taulukko 2. Pituusboniteettiluokkien (H40dr) ja suotyyppien sekä ravinteisuus- ja tuotos- luokkien vastaavuus ja puuntuotoskyky (MAI40dr). Hyvä ojitus edellytyksenä mallin käytölle Ojitushetken valtapituus Hdom0 = 0,7 × Hdom – 0,2 × Tdr Ojitushetken valtapituus Hdom0 = 0,7 × Hdom – 0,2 × Tdr Ojitushetken valtapituus Hdom0 = 0,7 × Hdom – 0,2 × Tdr Mallin tärkein edellytys on, että alueen ojasto on kunnossa ja kuivatus toimii hyvin. Mikäli ojitus on huono, puuston kehitys ei kuvaa kasvupaikan luonnollista ojituksenjälkeistä puuntuotoskykyä. Jos harvennuksessa on poistettu paksuimmat puut tai jos alue on lannoitettu, ei pituusbonitointimalli myöskään toimi luotettavasti. Boniteettimallia testattiin erikseen mänty-, kuusi- ja koivuvaltaisten sekä sekapuustojen (mänty-kuusi koivu) osalta. Yhteiseen aineistoon perustuva valta- pituusmalli yliarvioi lievästi mäntyvaltaisten mutta aliarvioi kuusi- ja koivuvaltaisten metsien valta- pituuksia. Sekametsissä malli oli harhaton. Tämä osoittaa, että mallin tarkkuutta voidaan parantaa tekemällä siitä puulajisuhteet huomioon ottava. Tähän pituusboniteettimallin sovellukseen liit- tyvät samat rajoitukset kuin kangasmetsien ikä- valtapituussysteemiin. Esimerkiksi bonitointitulos on epävarma nuorella oijitusiällä. Ojitusalueiden puustot ovat kangasmetsistä poiketen varsin usein eri-ikäisiä ja erikokoisia. Mittaushetken valtapituus on järkevintä määrittää valitsemalla objektiivises- ti metsikköön sijoitettujen relaskooppikoealojen (kerroin = 1) paksuin puu ja mittaamalla sen pituus. Epätasaisissa turvemaiden metsiköissä joudutaan yleensä mittaamaan useampia koealoja kuin kan- gasmaiden metsiköissä tietyn tarkkuuden saavutta- miseen. Bonitointimallin antaman indeksin ja kasvilli- suuden välistä yhteyttä on myös äskettäin tutkittu riippumattomassa aineistossa (Hotanen 2003). Tu- lokset osoittavat sen, että puustoon ja kasvillisuuteen perustuvat bonitoinnit korreloivat melko voimak- kaasti keskenään, ja ne yhdessä voivat täsmentää ojitusalueen hyvyyden arviointia. Myös taulukossa 2 esitetyt valtapituusindeksit 40 vuoden ojitusiällä ovat likimain samoja kuin ne ojituksenjälkeiset valtapi- tuusarviot, jotka Seppälä (1969) on aiemmin esittänyt valtakoepuille eräiden suotyyppien osalta. Tämän metsikkömallin vertailu Hökän (2001) Pohjois-Suomen rämeille laatimaan puukohtaiseen valtapituusmalliin osoittaa selviä eroja ja puolustaa soiden alueellisten bonitointimallien käyttöä Etelä- ja Pohjois-Suomessa. Hökän malli on laadittu ns. ”sekamallin” periaatteella (kiinteitä ja satunnaispa- rametreja) (Ojansuu 1996), jota nykyisin käytetään Suomessa yksipuolisesti ja joskus kritiikittömästi kasvu- ja tuotosmallien laadinnassa. Malli pyrkii kuvaamaan samanaikaisesti sekä luonnontilaisen Malli on erityisen käyttökelpoinen ojitusalueilla, joilla puusto on syntynyt ojitushetkellä. Tällöin mit- taushetken valtapituus on suoraan ojituksenjälkeinen pituus. Vanhoilla ojitusalueilla, joilla oli puustoa ojitushetkellä, tarvitaan tietoja alkuvaltapituudesta jotta ojituksenjälkeinen valtapituus saadaan laske- tuksi ennen bonitoimista. Jos tietoja ei ole, voidaan alkuvaltapituus karkeasti määrittää mittaushetken valtapituuden ja ojitusiän avulla: 222 Metsätieteen aikakauskirja 2/2004 Tieteen tori Hökkä, H 1997. Models for predicting growth and yield in drained peatland stands in Finland. Metsäntutkimus- laitoksen tiedonantoja 651. 45 + 53 s. että ojitetun suopuuston valtapituuskehitystä in- tegroimalla kasvupaikkamuuttujien valtapituuden ikäriippuvuuteen. Sen tavoite poikkeaa selvästi tässä kirjoituksessa esitetystä metsikkökohtaisesta mallista. Sekamalli edellyttää mm. Hyvä ojitus edellytyksenä mallin käytölle valtapuuston kes- ki-iän määrittämistä johon liittyy epäluotettavuutta, erityisesti käytännön metsän inventoinnissa (Ojan- suu 1996). Iän määrittäminen on lisäksi hidasta ja kallista käytännössä. Yleensä voidaan todeta että sellaisen monimutkaisen valtapituusmallin tulokset ovat käyttäjille vaikeampia arvioida, koska niitä ei esitetä selvästi valtapituusboniteettikäyrästönä, josta käy ilmi boniteetti-indeksit ja kasvukyvyt (m3/ha/v). Tästä johtuen on hyvä, että meillä on tarjolla erilai- siin käyttötarkoituksiin myös traditionaalisia met- sikkömalleja. j — 2001. Puuston valtapituuskehitys rämekasvupaikkojen kuvauksessa. Julkaisussa: Varmola, M. & Tapaninen, S. (toim.). Pohjoisten metsien hoito – 30 vuotta tut- kimuspäiviä Rovaniemellä. Metsäntutkimuslaitoksen tiedonantoja 803: 119–132. Ojansuu, R. 1996. Kangasmaiden kasvupaikan kuvaus MELA-järjestelmässä. Julkaisussa: Hynynen, J. & Ojansuu, R. (toim.). Puuston kehityksen ennusta- minen – MELA ja vaihtoehtoja. Tutkimusseminaari Vantaalla 1996. Metsäntutkimuslaitoksen tiedonantoja 612: 39–56. Paarlahti, K. 1988. Suometsien tuotos vanhoilla kesto- koealoilla. Metsäntutkimuslaitoksen tiedonantoja 308: 180–186. Penttilä, T. 1984. Suometsien puustobonitoinnin mahdol- lisuudet. Metsäntutkimuspäivät Rovaniemellä 1984. Metsäntutkimuslaitoksen tiedonantoja 148: 79–89. ■ MMT Hans Gustav Gustavsen, Metla, Joensuun tutkimus- keskus. Osoite: Vanhatie 73, 78200 Varkaus Huikari, O. 1952. Suotyypin määritys maa- ja metsäta- loudellista käyttöarvoa silmällä pitäen. Summary: On the determination of mire types especially considering their drainage value for agriculture and forestry. Silva Fennica 75: 1–22. Kirjallisuus Reinikainen, A. & Lehtinen, K-M. (toim.). 1994. Kasvu- paikaluokituksen tutkijaseminaari. Vantaa 27.10. 1994. Metsäntutkimuslaitoksen tiedonantoja 531. 116 s. Gustavsen, H.G. 1996. Site index model approach for drained peatland forest stands. Pituusboniteettisovellus ojitusalueiden metsille. Suo 47(2): 37–46. Seppälä, K. 1969. Kuusen ja männyn kasvun kehitys oji- tetuilla turvemailla. Summary: Post-drainage growth rate of Norway spruce and Scots pine on peat. Acta Forestalia Fennica 93. 88 s. — , Heinonen, R., Paavilainen, E. & Reinikainen, A. 1998. Growth and yield models for forest stands on drained peatland sites in southern Finland. Forest Eco- logy and Management 107(1998): 1–17. Heikurainen, L. 1968. Suo-opas. 2. painos. Kirjayhtymä, Helsinki. 40 s. Heikurainen, L. 1968. Suo-opas. 2. painos. Kirjayhtymä, Helsinki. 40 s. Hotanen, J.P. 2003. Multidimensional site description of peatlands drained for forestry. Silva Fennica 37(1): 55–93. Hotanen, J.P. 2003. Multidimensional site description of peatlands drained for forestry. Silva Fennica 37(1): 55–93. Huikari, O. 1952. Suotyypin määritys maa- ja metsäta- loudellista käyttöarvoa silmällä pitäen. Summary: On the determination of mire types especially considering their drainage value for agriculture and forestry. Silva Fennica 75: 1–22. Huikari, O. 1952. Suotyypin määritys maa- ja metsäta- loudellista käyttöarvoa silmällä pitäen. Summary: On the determination of mire types especially considering their drainage value for agriculture and forestry. Silva Fennica 75: 1–22. 223
https://openalex.org/W2917078541	https://www.arpgweb.com/pdf-files/jssr5(2)354-359.pdf	English	null	Action Research in Teaching English for Students of Non-Linguistic Specialties in Higher Schools	The journal of social sciences research	2,019	cc-by	4,345	Abstract This article presents the theoretical and practical part of using the action research in teaching English for students of non-linguistic specialties in higher schools. Action research is a new process of research in Kazakhstan education that presents conducting of research by action researchers who share their findings with others in teaching. Our daily life connects with action research in order to investigate, make analysis and evaluate our work. The term action research has become particularly popular in education, especially in teaching foreign languages. Different approaches can be applied to improve the process of learning in it. Action Research is a great opportunity for creative teachers to develop different skills of students. The main purpose of this paper is to study basic concepts of action research, develop a model of action research process in teaching English, and determine the effectiveness and advantages of action research in teaching English for students of non-language specialties. g g g g g p eywords: Action research; Teaching English;Practical lessons; Representatives; Inspectors;Developers. The Journal of Social Sciences Research ISSN(e): 2411-9458, ISSN(p): 2413-6670 Vol. 5, Issue. 2, pp: 354-359, 2019 URL: https://arpgweb.com/journal/journal/7 DOI: https://doi.org/10.32861/jssr.52.354.359 Academic Research Publishing Group Original Research Open Access Action Research in Teaching English for Students of Non-Linguistic Specialties in Higher Schools M. Zh. Tussupbekova* L.N. Gumilyov Eurasian National UniversityAstana, Kazakhstan M. A. Idrissova L.N. Gumilyov Eurasian National UniversityAstana, Kazakhstan B. G. Smagulova L.N. Gumilyov Eurasian National UniversityAstana, Kazakhstan N. K. Kazhikenova N. K. Kazhikenova L.N. Gumilyov Eurasian National UniversityAstana, Kazakhstan Zh. M. Konyratbaeva L.N. Gumilyov Eurasian National UniversityAstana, Kazakhstan B. Abduali L.N. Gumilyov Eurasian National UniversityAstana, Kazakhstan Corresponding Author The Journal of Social Sciences Research ISSN(e): 2411-9458, ISSN(p): 2413-6670 Vol. 5, Issue. 2, pp: 354-359, 2019 URL: https://arpgweb.com/journal/journal/7 DOI: https://doi.org/10.32861/jssr.52.354.359 Academic Research Publishing Group Original Research Open Access Action Research in Teaching English for Students of Non-Linguistic Specialties in Higher Schools M. Zh. Tussupbekova L.N. Gumilyov Eurasian National UniversityAstana, Kazakhstan M. A. Idrissova L.N. Gumilyov Eurasian National UniversityAstana, Kazakhstan B. G. Smagulova L.N. Gumilyov Eurasian National UniversityAstana, Kazakhstan N. K. Kazhikenova L.N. Gumilyov Eurasian National UniversityAstana, Kazakhstan Zh. M. Konyratbaeva L.N. Gumilyov Eurasian National UniversityAstana, Kazakhstan B. Abduali L.N. Gumilyov Eurasian National UniversityAstana, Kazakhstan Abstract The Journal of Social Sciences Research ISSN(e): 2411-9458, ISSN(p): 2413-6670 Vol. 5, Issue. 2, pp: 354-359, 2019 URL: https://arpgweb.com/journal/journal/7 DOI: https://doi.org/10.32861/jssr.52.354.359 Academic Research Publishing Group Original Research Open Access 1.Introduction Within a historically short period after gaining independence in 1991, Kazakhstan has managed to take a strong position on the international scene. Kazakhstan became a member state of the United Nations and other international organizations. Since then, Kazakhstan has been actively involved in the activities of many UN organizations, such as UNESCO, UNICEF, ECOSOC, UNHCR, and others. Kazakhstan also began to collaborate with a number of major international organizations, including the International Monetary Fund (IMF), the International Bank for Reconstruction and Development (IBRD), the Organization for Security and Cooperation in Europe (OSCE). Kazakhstan is also an initiator of the convening of the Conference on Interaction and Confidence-Building Measures in Asia (CICA). In addition, it is actively involved in the integration processes within the framework of the Economic Cooperation Organization (ECO), the Collective Security Treaty Organization, the Central Asian Economic Union (CAPS), and the Shanghai Cooperation Organization (SCO). In 2010, Kazakhstan became the first Asian country to chair the OSCE. y Thus, in view of the political and economic internationalization, the good command of English has been becoming more and more important. Since Kazakhstan announced its independence, the President of the Republic of Kazakhstan Nursultan Nazarbayev has constantly been advocating a trilingual model. Each citizen of Kazakhstan should strive to gain fluency in English, Russian, and Kazakh. Accordingly, the national project „Trinity of languages‟ determines Kazakh as the state language, Russian as an official language, and English as the language of international communication (Nazarbayev, 2007); (Hilao, 2016); (Charoensuk and Jaipetch, 2017); (Unnanantn, 2017). Simultaneously, this trinity is understood as harmony rather than competition. Therefore, it is important to accompany as well as to support the implementation of the language policy of trilingualism by scientific explorations. It needs to be related to the multicultural policy, which requires the knowledge of the native language and stimulates learning of other languages. Transformation into the new multilevel system of higher education in Kazakhstan has shaken all academic community to enter a modern curriculum, to create methodical techniques, to publish teaching aids, to introduce new *Corresponding Author 354 The Journal of Social Sciences Research technology of education. Modules, a syllabus, criteria of grades, current, intermediate controls, students‟ self-work may be applied in academic terms of teaching. technology of education. Modules, a syllabus, criteria of grades, current, intermediate controls, students‟ self-work may be applied in academic terms of teaching. 1.Introduction The multilevel system of education in Kazakhstan should correspond to the European standards and meet the requirements of «L3» idea (Life Long Learning), which is a visiting card of Bologna transformation. Each level of education provides opportunity for learning foreign languages. The goal and task of teaching English in higher institutions is the practical acquiring of colloquial and professional ways of speaking for active using as in real and professional conversation (Abdul Amir, 2015; Passov, 2002; Pradhan, 2016; Taher, 2016). 2. The Basic Concept of Action Research in Teaching English This study of action research is based on works of Lewin (1946); Masters (1995); McNiff (2002); Kemmis et al. (2004), Reason and Bradbury (2007) and others. The main goal of these works is to identify the meaning and concept of action research from different perspectives. K. Lewin described action research as “a comparative research on the conditions and effects of various forms of social action and research leading to social action.” In other words, each research is done by action. Dewey (1986), completely describes the educational traditional action research in his book and gives real samples from his own experience. He always reminds about the process of action research in teaching (planning, action, observation and reflection). g p g Therefore, action research is a strategy of teachers to investigate a problem or area of specific interest to their professional context. It provides the structure to engage in a planned, systematic and documented process of professional growth. The important aim of action research in teaching is to contribute the experience of teachers in solving of problematic situations, following the plan and targeted goals. It is concurrently collaborative work with students or colleagues.Gilmore et al. (1986), notes, that action research requires the active collaboration of a researcher (or a teacher) and a client (or a student). Scientifically, action research is a systematic inquiry of knowledge in educational practice to understand clearly the situations that are carried out in practice and develop strategies geared towards the problem‟s improvement. It can be focused on the teaching and learning processes to solve a problem. Thus, action research is a practical approach that can be used in any social situations to generalize different ideas of solving research problems in real life. It is believed that action research develops powers of thoughts, discussions, decisions and actions by ordinary people who participate in research on “private troubles” that they have in common (Boonyarattanasoontorn, 2017; Junnak and Veerachaisantikul, 2016; Mills, 2000). In other words, action research refers to the unity of three parts: action, research, and participation. Freire (1970), tries to integrate three main aspects in this issue: participation, action and research. It makes orientation on methodology that enables teachers or researchers to work in partnership in a manner that leads to action for change. 2. The Basic Concept of Action Research in Teaching English p p g The studying of the basic concept of action research helps us to develop our model of action resear teaching English for students of non-linguistic specialties. 3. An Action Research Model Development in Teaching English 3. An Action Research Model Development in Teaching English The plan of the research action Stages Date Events I Friday 20th January Introductory lecture Friday 23rd February Tutorial (a session to allow students to meet with teachers and discuss the following steps of work on the research action) II Friday 21th March Submission of the proposal to Inspectors Friday 18th April Submission of the proposal to Local Authority III Friday 28th April Inquiry meeting Friday 19th May Feedback on ideas (Comments on topic) h th f th h l t d di t th t d t ‟ i lti d th i ll b (T bl 2 The Journal of Social Sciences Research The themes of the research are selected according to the students‟ specialties and their syllabus (Table 2). The themes of the research are selected according to the students‟ specialties and their syllabus (Table 2). Table-2. Research topics Groups Research topics Departments G1 Restoring the building of Cinema City (Imanova Street, Astana) Engineering G2 Retirement center (Borovoe, Kokshetau) Tourism G3 Nursery school (Koktal village, Astana) Philology During the second meeting, teachers discuss the stages of work on the research action. 12 students and 6 teachers take part in this study. Teachers play the role of representatives of the local authority; some students perform the role of inspectors and another group of students are in the role of developers. Students-developers work in pairs on the suggested topic, students-inspectors solve the problem which are presented to the representatives of the local authority (Table 3). Table-3. The working group Groups G1 G2 G3 Students (Developers) Abdualieva Damira Akibaeva Kamila Esenamanova Nargiz Doskaliev Serik Kabylbekov Temirlan Ongarbekova Aisulu Students (Inspectors) Afandiev Rasul Darbosova Kamila Zhuaspaev Timur Zhadiger Madikhan Samizhan Maral Tussunova Aziza Teachers (representatives of the local authority) Tussupbekova Madina Abduali Bekzhan Idrissova Mapruza Smagulova Botagoz N.K. Kazhikenova, Zh.M. Konyratbaeva Table-3. The working group Groups G1 G2 G3 Students (Developers) Abdualieva Damira Akibaeva Kamila Esenamanova Nargiz Doskaliev Serik Kabylbekov Temirlan Ongarbekova Aisulu Students (Inspectors) Afandiev Rasul Darbosova Kamila Zhuaspaev Timur Zhadiger Madikhan Samizhan Maral Tussunova Aziza Teachers (representatives of the local authority) Tussupbekova Madina Abduali Bekzhan Idrissova Mapruza Smagulova Botagoz N.K. Kazhikenova, Zh.M. Konyratbaeva 3.2. The Second Stage: Action g The aim of the stage is to develop students‟ ability to collect and analyze data. Teachers prepare special guidelines for research projects. Each project consists of introduction (a short description of working project); main issues (a short description of the main issues in the project), a proposal (suggestions with a description of all reasons), and a conclusion. The main purpose of collecting data in this action research is to find a decision for the main issues of the chosen topic. Collecting data means a collection of information on the given topic about the location of Cinema City. First of all, it is necessary to get information about the construction of this construction, about the developers, then, make photos and videos, learn all gathered computer information, do mathematical operations to get quantitative information for analyzing data, create a chart or tables, transcribe the contents of photos and video recordings. Analyzing data involves examining all gathered information to make some conclusions to understand better the researched problem and the whole situation. There exist two kinds of data to analyze. Quantitative data include numbers, which can be analyzed mathematically. Qualitative data consist of descriptions, opinions, quotes, interpretations that are more valuable and informative in action research. 3. An Action Research Model Development in Teaching English p g g Developing action research model in teaching English assists to investigate clearly our teaching experience and students‟ learning. This model explains and directs teachers to use action research in practice. If we take into account Freire‟s three main aspects of action research. According to it, we determine the group of students to participate in our experiment. Then, we describe the following action and do research (Figure 1). Make a plan Identif y topics Participat ion Collect data Analyze data Action Plan applicatio n Submit a paper Research Figure-1. Action research process in teaching English 3.1. The First Stage: Participation In this stage, we make a plan of the research and identify topics. Our participants are 2 second year students of non-language departments of L.N. Gumilyov Eurasian National University (engineering, tourism, and philology specialties). The first meeting includes a short description of the research action, discussion of the plan, indication of special dates, and deadline of the whole research (Table 1). Figure-1. Action research process in teaching English Make a plan Identif y topics Participat ion Collect data Analyze data Action Plan applicatio n Submit a paper Research Make l Plan li 3.1. The First Stage: Participation g p In this stage, we make a plan of the research and identify topics. Our participants are 2 second year students of non-language departments of L.N. Gumilyov Eurasian National University (engineering, tourism, and philology specialties). The first meeting includes a short description of the research action, discussion of the plan, indication of special dates, and deadline of the whole research (Table 1). In this stage, we make a plan of the research and identify topics. Our participants are 2 second year students of non-language departments of L.N. Gumilyov Eurasian National University (engineering, tourism, and philology specialties). The first meeting includes a short description of the research action, discussion of the plan, indication of special dates, and deadline of the whole research (Table 1). 355 The Journal of Social Sciences Research Table-1. 3.3. The Third Stage: Research The third stage requires planning an application and submission a paper. Developers prepare a planning application and submit it to inspectors. Inspectors decide whether the proposal should be allowed or dismissed. Therefore, inspectors should submit a planning application to the authority (teachers), backed up with appropriate reports. The planning of application is presented by developers at the Inquiry meeting. After listening to the developers‟ planning applications, inspectors can ask questions to decide whether the proposal should be realized. The representatives of the local authority consider whether the proposal in the planning of application is acceptable, in terms of its compliance with the guidance and decision making. 356 The Journal of Social Sciences Research The Journal of Social Sciences Research The Journal of Social Sciences Research Planning Application G1 - Restoring of the Cinema City Introduction Cinema City is situated in Imanova Street, Astana city. It is located in the centre of the city. Its building is near the main street (Republic Avenue). It was constructed in 2000. It was considered as one of the big and new cinemas during several years. Cinema City provided only one big screen and was closed two years ago, because it was uneconomic to operate. Main issues - Cinema City is adjacent to the city highway and it is used by drivers as a parking place for cars; - The availability of only one big screen in the cinema can significantly reduce the prospects of the development of a new cinema in this part of the city. Proposal The proposal is to increase the number of screens to 5. The largest hall will hold 200 seats. The number of seats in the other halls will be 100. It is planned that the most popular films to be shown in the largest hall and other films in the small ones. Conclusion Today, all new cinemas have multi-screen designs. Screening of a popular film attracts a large audience of film amateurs. The number of filmgoers may be a huge amount in a week and can bring even the annual income for the short period. The new cinema can show at least 4 different movies simultaneously. In this way, it is possible to avoid the uneconomic operations as they are with the old one. From economic point of view, we think that this new cinema may get more financial benefits. 3.3. The Third Stage: Research First, its location in the centre of the city will satisfy the leisure demands of the residents of this area. G 2 – Retirement centre Introduction Retirement centre is in the place called Borovoye. Borovoye is situated over 200 kilometres to the northeast of Astana and almost 100 kilometres to the south of Kokshetau. This place by Kazakh standards makes it very accessible for the inhabitants of this region. All people appreciate this beautiful place in Kazakhstan with picturesque mounting cliffs and lakes with crystal-clear bluish water. There is a very large old building far from Borovoye in the pine forest. This building is proposed to change into a retirement centre for old age people. Main issues - The building is very old, located in a remote area; - There are no public transport services, no cycle lanes or footways leading to and from the site. Proposal The proposal includes the creation of a retirement centre, consisting of 50 beds. Moreover, this centre will provide quiet and safe place, where elderly can take pleasure in this retirement center; the old building will be restored and all living conditions will be created. Conclusion The retirement centre is situated within an area of a beautiful countryside and clean environment especially for elderly people. The centre provides with all internal arrangements and there will be a social club (a gymnasium, game rooms, hairdresser‟s salon, and a medical center), a restaurant, a cafe, and a small supermarket. In spite of remote area, there will be a number of cycling and jogging tracks for pedestrians. G 3 - Nursery school Introduction Children‟s nursery has been operating in a small hall close to the shopping centre. The size of the hall only allows the nursery to accommodate 30 children. Main issues - Nursery school is too small to accommodate more children. This is a popular, well- run nursery and so there is already a waiting list of parents wishing to send their children to the nursery. - Nursery school is likely to increase the number of parking places for private cars Proposal The proposal includes increasing the capacity to at least 80 children and a longer period of free pre-school attendance in this nursery. Moreover, there will be the area outside where parents can park their cars for a short period that is clear because of the highway. 4. Conclusion Action research is concluded in improving English language teaching techniques and in empowering teachers‟ competencies. Following the model of action research, students can work on presented issues. Each research is done by action and action is accompanied by participation. During this action research, students allocate the role of developers and inspectors. Students work in pairs; follow the plan with deadlines and events. Moreover, students are learnt to collect and analyze data to find the right decision, plan an application (introduction, main issues, proposal and conclusion). Students participate in inquiry meeting with all participants of action research (developers, inspectors and authority). It is important to follow the stages in action research process. In planning it is to identify the problem, find the causes of the problem and its solutions, how to implement it and decide whether the action successful or not by using collection and analyzing data. In the second stage it is necessary to take the right decision, in observation is to gather evidence, which you will analyze in order to solve an issue and whether a solution is successful or not. The last stage is reflection where you should know how to analyze issues, before finding the solution to the researched problem, how to give and support ideas, disagree with others, ask research questions, what roles the participants play in groups, how to solve the problems in order to improve practice. Finally, action research is a very beneficial tool, but it needs a lot of time to conduct to be done. y g y g g y g in observation is to gather evidence, which you will analyze in order to solve an issue and whether a solution is successful or not. The last stage is reflection where you should know how to analyze issues, before finding the solution to the researched problem, how to give and support ideas, disagree with others, ask research questions, what roles the participants play in groups, how to solve the problems in order to improve practice. Finally, action research is a very beneficial tool, but it needs a lot of time to conduct to be done. Action research is an operating process of reflection of the most effective learning environment. It is to note that to solve problems, the results are not immediate as one might expect. The Journal of Social Sciences Research The Journal of Social Sciences Research Developers submit their planning applications to inspectors. Developers can submit their applications in written form. Inspectors get acquainted with the planning applications and decide whether it should be allowed or dismissed. After inspectors‟ decisions, the planning applications are submitted to the authorities (teachers). At the end of all processes, the planning applications are presented by developers at the Inquiry meeting. The inquiry process has the following requirements: g q 1) Developers present a brief introduction of their researched topics (1-2 min.); opers prepare a presentation with photos and videos ( ) p p p p p ( ) 3) Inspectors present their solutions about the planning applications (1-2 minutes); 3) Inspectors present their solutions about the planning applications (1- 4) Authority questions to developers and inspectors (5-7 minutes); 5) Cross-examinations (up to 10 minutes): 6) Feedback of authority (up to 10 minutes). Developers (only whose planning applications are accepted) present their research topic with all arguments to inspectors and authority. After listening to the developers‟ proposals, inspectors and authority decide whether the proposed decision should be allowed to go ahead. Inquiry meeting helps to finish researching and shows the real results according to the model of action research (participation of students, action in researching, and doing research work). 4. Conclusion In fact, action research is an essential process of education to develop learning skills of students in English. References Abdul Amir, A. R. Z. (2015). Utilization of request mitigators by Omani learners of english and native speakers, a comparative study. International Journal of Humanities, Arts and Social Sciences, 1(4): 156-72. p y f Boonyarattanasoontorn, P. (2017). An investigation of Thai students‟ english language writing difficul use of writing strategies. Journal of Advanced Research in Social Sciences and Humanities, 2(2 , ( ) g g g g g of writing strategies. Journal of Advanced Research in Social Sciences and Humanities, 2(2): 111-18. g g f ( ) Charoensuk, V. and Jaipetch, D. (2017). Attitudes toward english, a study of first-year students at king mongkut‟s university of technology North Bangkok. Journal of Advances in Humanities and Social Sciences,2017, 3(1): 42-57. Dewey, J. (1986). Experience and education. In The Educational Forum, 50(3): 241-52. Freire, P. (1970). Pedagogy of the oppressed MB ramos, trans.continuum, 2007. New York. Gilmore, T., Krantz, J. and Ramirez, R. (1986). Action based modes of inquiry and the host-researcher Consultation, An International Journal, 5(3): 160-76. ( ) Hilao, M. P. (2016). Creative teaching as perceived by english language teachers in private universities. Journal of Advances in Humanities and Social Sciences, 2(5): 278-86. Junnak, C. and Veerachaisantikul, A. (2016). Reporting verb in research projects of efl english major students. Journal of Advanced Research in Social Sciences and Humanities, 1(1): 41-46. Junnak, C. and Veerachaisantikul, A. (2016). Reporting verb in research projects of efl english Journal of Advanced Research in Social Sciences and Humanities, 1(1): 41-46. Kemmis, S., McTaggart, R. and Retallick, J. (2004). The action research planner. gg p Lewin, K. (1946). Action research and minority problems. Journal Of Social Issues, 2(4): 34-46. Lewin, K. (1946). Action research and minority problems. Journal Of Social Is McNiff, J. (2002). Action research, Principles and practice. Routledge. 69-102. Mills, C. W. (2000). The sociological imagination. Oxford University Press. Nazarbayev, N. A. (2007). Trinity of languages‟—the base for multicultural personality. 54(9): 212-40. Passov, E. I. (2002). Communicative method of teaching foreign language speaking. M. Prosveshchenie: 101-201. P dh S (2016) E li h l t hi t t t i l I t ti l J l f H iti assov, E. I. (2002). Communicative method of teaching foreign language speaking. M. Prosveshchenie: 10 adhan, S. (2016). English language teaching: a next gate to social awareness. International Journal of Hu Passov, E. I. (2002). Communicative method of teaching foreign language speaking. M. Prosveshchenie: 101-201. 3.3. The Third Stage: Research Conclusion This nursery school will accommodate 80 children and has the potential to provide a further increase in capacity The redundant barn has become available and this area has a Planning Application G1 - Restoring of the Cinema City 357 ( ) eason and Bradbury (2007). Handbook of action research. 2nd edn: Sage: London. Taher, M. A., Shrestha, P. N., Rahman, M. M., & Khalid, A. K. M. I. (2016). Curriculum linked video CLV as a tool for english language teaching ELT at secondary school classrooms in Bangladesh. International Journal of Humanities, Arts and Social Sciences, 2(4): 126-32. ( ) Unnanantn, T. (2017). Enhancing Thai undergraduates‟ ability on scholarly English presentation, M based instruction. Journal of Advances in Humanities and Social Sciences, 3(2): 82-94. The Journal of Social Sciences Research Taher, M. A., Shrestha, P. N., Rahman, M. M., & Khalid, A. K. M. I. (2016). Curriculum linked video CLV as a tool for english language teaching ELT at secondary school classrooms in Bangladesh. International Journal of Humanities, Arts and Social Sciences, 2(4): 126-32. Unnanantn, T. (2017). Enhancing Thai undergraduates‟ ability on scholarly English presentation, Miller's model- based instruction Journal of Advances in Humanities and Social Sciences 3(2): 82 94 References Pradhan, S. (2016). English language teaching: a next gate to social awareness. International Journal of Humanities, Arts and Social Sciences, 2(4): 156-58. Pradhan, S. (2016). English language teaching: a next gate to social awareness. International Journal of Humanities, Arts and Social Sciences, 2(4): 156-58. Reason and Bradbury (2007). Handbook of action research. 2nd edn: Sage: London. 358 The Journal of Social Sciences Research Taher, M. A., Shrestha, P. N., Rahman, M. M., & Khalid, A. K. M. I. (2016). Curriculum linked video CLV as a tool for english language teaching ELT at secondary school classrooms in Bangladesh. International Journal of Humanities, Arts and Social Sciences, 2(4): 126-32. 359
https://openalex.org/W4360614683	https://backend.orbit.dtu.dk/ws/files/314999712/microorganisms_11_00819.pdf	English	null	Coxiella burnetii Seroprevalence and Associated Risk Factors in Cattle, Sheep, and Goats in Estonia	Microorganisms	2,023	cc-by	8,680	Citation (APA): Neare, K., Tummeleht, L., Lassen, B., & Viltrop, A. (2023). Coxiella burnetii Seroprevalence and Associated Risk Factors in Cattle, Sheep, and Goats in Estonia. Microorganisms, 11(4), Article 819. https://doi.org/10.3390/microorganisms11040819 Downloaded from orbit.dtu.dk on: Oct 24, 2024 Downloaded from orbit.dtu.dk on: Oct 24, 2024 Downloaded from orbit.dtu.dk on: Oct 24, 2024 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 and 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  You may freely distribute the URL identifying the publication in the public portal If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. Coxiella burnetii Seroprevalence and Associated Risk Factors in Cattle, Sheep, and Goats in Estonia Neare, Kädi; Tummeleht, Lea; Lassen, Brian; Viltrop, Arvo Neare, Kädi; Tummeleht, Lea; Lassen, Brian; Viltrop, Arvo Document Version Publisher's PDF, also known as Version of record 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 and it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights. 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 and 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 Citation: Neare, K.; Tummeleht, L.; Lassen, B.; Viltrop, A. Coxiella burnetii Seroprevalence and Associated Risk Factors in Cattle, Sheep, and Goats in Estonia. Microorganisms 2023, 11, 819. https://doi.org/10.3390/ microorganisms11040819 Kädi Neare 1,* , Lea Tummeleht 1 , Brian Lassen 2,* and Arvo Viltrop 1 1 Chair of Veterinary Biomedicine and Food Hygiene, Institute of Veterinary Medicine and Animal Sciences, Estonian University of Life Sciences, 51006 Tartu, Estonia 2 Research Group for Foodborne Pathogens and Epidemiology, National Food Institute, Technical University of Denmark, 2800 Kongens Lyngby, Denmark * Correspondence: kadi.neare@emu.ee (K.N.); brlas@food.dtu.dk (B.L.) 1 Chair of Veterinary Biomedicine and Food Hygiene, Institute of Veterinary Medicine and Animal Sciences, Estonian University of Life Sciences, 51006 Tartu, Estonia y 2 Research Group for Foodborne Pathogens and Epidemiology, National Food Institute, Technical University of Denmark 2800 Kongens Lyngby Denmark 2 Research Group for Foodborne Pathogens and Epidemiology, National Food In * Correspondence: kadi.neare@emu.ee (K.N.); brlas@food.dtu.dk (B.L.) Abstract: Q fever, a disease caused by Coxiella burnetii (CB), is an emerging zoonotic health prob- lem. The prevalence data from potential sources are valuable for assessing the risk to human and animal health. To estimate the prevalence of CB antibodies in Estonian ruminants, pooled milk and serum samples from cattle (Bos taurus) and pooled serum samples from sheep (Ovis aries) and goats (Capra hircus) were analyzed. Additionally, bulk tank milk samples (BTM; n = 72) were analyzed for the presence of CB DNA. Questionnaires and herd-level datasets were used to identify the risk factors for exposure using binary logistic regression analysis. The prevalence of CB-positive dairy cattle herds (27.16%) was signiﬁcantly higher than that in beef cattle herds (6.67%) and sheep ﬂocks (2.35%). No CB antibodies were detected in the goat ﬂocks. CB DNA was found in 11.36% of the BTM samples. The odds of seropositivity were higher in dairy cattle herds, with an increasing number of cattle in the herd, and with location in southwestern, northeastern and northwestern Estonia. Dairy cattle herds had higher odds of testing positive for CB in BTM if the dairy cows were kept loose and lower odds if the herd was located in northwestern Estonia. Keywords: foodborne zoonotic diseases; infectious disease; one health; public health; vector-borne diseases Keywords: foodborne zoonotic diseases; infectious disease; one health; public health; vector-borne diseases Article Coxiella burnetii Seroprevalence and Associated Risk Factors in Cattle, Sheep, and Goats in Estonia Kädi Neare 1,* , Lea Tummeleht 1 , Brian Lassen 2,* and Arvo Viltrop 1 Link back to DTU Orbit Link back to DTU Orbit Citation (APA): Neare, K., Tummeleht, L., Lassen, B., & Viltrop, A. (2023). Coxiella burnetii Seroprevalence and Associated Risk Factors in Cattle, Sheep, and Goats in Estonia. Microorganisms, 11(4), Article 819. https://doi.org/10.3390/microorganisms11040819 this document breaches copyright please contact us providing details, and we will remove access to the work immediate ur claim. microorganisms 1. Introduction Q fever (QF) is a zoonosis caused by the intracellular bacterium Coxiella burnetii (CB). The bacteria are considered to be globally distributed but may be overlooked in some countries due to the irregular or absence of investigations [1,2]. The main transmission routes of CB are via the inhalation of spore-like stages transported in aerosols or via contact with excretions (urine, feces, vaginal mucus, semen, and milk) from infected animals or contaminated materials [3]. In rare cases, CB is transmitted through tick bites [4]. Humans can acquire infection by consuming unpasteurized milk or dairy products containing viable CB [3]. Academic Editor: Paweł Stefanoff Received: 22 February 2023 Revised: 16 March 2023 Accepted: 20 March 2023 Published: 23 March 2023 In ruminants, cats, and dogs, the course of the infection is commonly asymptomatic but can, in some cases, affect reproduction, manifesting as stillbirths, infertility, retained placenta, abortions, and weak offspring [5]. Domestic ruminants are considered the most important reservoir for human infections [3]. In humans, the average incubation period of CB infection is 2 to 3 weeks, and the symptoms include flu-like illness, pneumonia, and hepatitis. Chronic QF is rare and generally manifests clinically as vascular infection and endocarditis [3]. Copyright: © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). In response to the rising concerns about health risks regarding a QF outbreak in the Netherlands, the European Food Safety Authority (EFSA) assembled a scientiﬁc opinion that called for the need for harmonized passive and active monitoring of QF [1]. Following a Dutch outbreak, human CB infections were generally found near dairy goat farms with abortion waves induced by CB [6]. https://www.mdpi.com/journal/microorganisms Microorganisms 2023, 11, 819. https://doi.org/10.3390/microorganisms11040819 2 of 12 Microorganisms 2023, 11, 819 Herd-level seroprevalence estimates of CB infections in European ruminants range from 0.0% to 81.6% in cattle, 0.0% to 78.6% in sheep and 0.0% to 43.1% in goats [7–10]. In previous studies, herd size, a loose housing system, and location have been identiﬁed as risk factors for herd-level CB seropositivity. Breed, parity, use of windshield and use of artiﬁcial insemination were detected as animal-level risk factors [7,9,11]. In Estonia, there are few data on the presence of CB in domestic animals. Evidence of infection has previously been detected serologically in ﬁve cattle from three differ- ent farms [12]. Information is lacking on the presence of CB in other domestic animals, including sheep, goats, and pets. This study aimed to estimate the exposure to CB in domestic ruminants in Estonia based on seroprevalence and identify potential associated risk factors. 2. Materials and Methods 2. Materials and Methods 2.1. Setting and Sample Size 2.1. Setting and Sample Size 2.1.1. Estimation of CB Herd-Level Seroprevalence in Cattle 2.1.1. Estimation of CB Herd-Level Seroprevalence in Cattle 2.1.1. Estimation of CB Herd-Level Seroprevalence in Cattle The study population included cattle herds sampled for the ofﬁcial surveillance of enzootic bovine leukosis (EBL) in Estonia. Milk or serum samples from all cattle aged ≥24 months were collected from all cattle herds in Estonia by authorized veteri- narians for EBL surveillance in 2012. Available aliquots of these samples were stored at the Estonian National Centre for Laboratory Research and Risk Assessment and tested for CB antibodies at the Estonian University of Life Sciences (EMU). In 2012, 4665 cattle herds were registered in the Estonian national production animal register [13] The herds that kept more dairy breed cattle than beef breeds were classiﬁed as dairy herds, and the remainder were considered as beef herds. Based on this categorization, there were 3417 dairy and 1248 beef herds. Herds with at least ﬁve adult animals were included. Using a random number generator EpiTools® [14], 504 cattle herds (337 dairy and 167 beef herds) were selected from the original list of herds and included in this study. The selection was further stratiﬁed by administrative region (county) in proportion to the number of herds in each herd category (dairy or beef) in each county. Assuming a 20% herd prevalence in the population, the sample size enabled the estimation of the prevalence at the country level in each herd category, with 95% conﬁdence and 5% accepted error. p From each beef herd, 30 serum samples were randomly selected for CB antibody testing, unless ≤30 samples were collected, in which case all the samples were tested. The selected serum samples were pooled from 3 to 6 samples before testing for CB antibodies using 150 µL of each sample. g p Fifty individual milk samples were randomly selected from each dairy herd and pooled for CB antibody testing. If the number of individual samples per herd was ≤50, all the samples were pooled and tested. p p The sampling scheme of included cattle herds is presented in Figure S1. A sample size of 30 animals per herd enabled the detection of a seropositive herd with 95% conﬁdence if the within-herd seroprevalence was 12%, assuming a sensitivity of 82.6% and a speciﬁcity of 100% for testing antibodies in cattle blood serum [15], and a very good match (Kappa = 0.89) between the results of serum and milk samples [16]. 2.1.2. Detection of CB Seropositive Sheep and Goat Flocks 2.1.2. Detection of CB Seropositive Sheep and Goat Flocks 2.1.3. Risk Factor Analyses in Cattle Herds Two studies were performed to identify risk factors for CB infection in Estonian cattle herds. In the ﬁrst study (Risk Factor Study 1), herds and data from the seroprevalence study based on surveillance program samples were included. Data on the herds’ geographical locations, sizes (number of animals), and breeds were retrieved from the National Animal Register database [13]. The second study (Risk Factor Study 2) included dairy cattle herds that volunteered to participate in the study in 2013. One tank milk sample (BTM) was submitted by farmers from each herd to test for CB antibodies and deﬁne the herd’s infection status (positive or negative), as speciﬁed in Figure S1. In addition, information on putative risk factors in the farm was gathered using a structured questionnaire (Table S1). 2.2.1. Antibody Detection An indirect enzyme-linked immunosorbent assay PrioCHECK Ruminant Q Fever Ab Plate Kit (Thermo Fisher Scientiﬁc, Waltham, MA, USA) was used to detect CB antibodies in milk and serum samples. Sample preparation, analyses, and interpretation of the results were performed according to the manufacturer’s instructions [17]. 2.1.2. Detection of CB Seropositive Sheep and Goat Flocks In Estonia, 1949 sheep ﬂocks and 653 goat ﬂocks were registered in the production animal register in 2012 [13]. Flocks involved in the national breeding program and subjected to ofﬁcial surveillance for brucellosis were included. All herds sampled in 2012 and 2013 were included. Leftover serum samples from the Brucella test were used for further investigation of CB antibodies at EMU. Microorganisms 2023, 11, 819 3 of 12 3 of 12 Additional sheep and goat ﬂocks not included in the brucellosis surveillance pro- gram were sampled to increase the herd sample size. Blood samples were collected from 30 randomly selected adult animals from each ﬂock. y The sampling procedure is summarized in Figure S1. y The sampling procedure is summarized in Figure S1. The number of ﬂocks tested enabled the detection of an infected ﬂock with 95% conﬁdence if the herd-level prevalence in the population was 2% in sheep and 20% in goats, assuming herd-level sensitivity and speciﬁcity of 95% and 100%, respectively. The number of samples collected from each ﬂock (1–65) enabled the identiﬁcation of an infected ﬂock with 95% conﬁdence, at least on a 20% prevalence level, assuming 88.8% sensitivity and 98.5% speciﬁcity of the test in sheep, and 91.6% sensitivity and 98.9% speciﬁcity in goats, respectively [15]. 2.2. Sample Analysis Collected samples were stored at −20 ◦C in labelled plastic vials until analysis. All laboratory analyses were performed in 2012–2014. 2.3. Statistical Analysis 2.3. Statistical Analysis 2.2.2. DNA Detection All pooled milk samples that were positive for CB antibodies (n = 88) were tested for the presence of CB DNA using polymerase chain reaction. DNA was puriﬁed from milk samples using Chelex 100 Resin (Bio-Rad Laboratories, Hercules, CA, USA). Trans-1 (5′-TAT GTA TCC ACC GTA GCC AGT C-3′) and Trans-2 (5′-CCC AAC AAC ACC TCC TTA TTC-3′) primers were used to detect the presence of speciﬁc IS1111 repetitive elements in CB DNA. The use of these primers had been described by Berri et al. [18]. Positive control was obtained from Vircell Microbiologists [19] and puriﬁed distilled water was used as no template negative control. Initial denaturation took place at 95 ◦C, the remaining denaturations at 94 ◦C. During ﬁrst ﬁve cycles the annealing was performed at 66–62 ◦C (lowered 1 ◦C with each cycle), and the rest of the annealings were performed at 61 ◦C. Elongation processes were carried out at 72 ◦C and the reaction was terminated at 4 ◦C [20]. 2.3.2. Risk Factor Analysis 2.3.2. Risk Factor Analysis Logistic regression analyses were used to estimate the association between herd CB infection status based on antibodies and potential risk factors. Herd size was evaluated in the models as a continuous variable (number of animals in the herd) and categorical variable (herd size). Prior analyses of cattle herds were categorized according to the number of animals as small (<101 animals), medium-sized (101–300 animals), and large herds (>300 animals). The effects of speciﬁc cattle breeds (Estonian Holstein Friesian, Estonian Red, Blonde d’Aquitaine and Hereford) were evaluated using the number of animals of speciﬁc breeds registered in the herd. Cattle herds were grouped into regions based on Estonian counties (N = 15): southwest (Pärnu, Saare, and Viljandi counties), southeast (Põlva, Tartu, Valga, and Võru counties), northeast (Ida-Viru, Jõgeva, Järva, and Lääne-Viru counties), and northwest (Harju, Hiiu, Lääne, and Rapla counties) (Figure S2). The season of sample collection was determined as astronomical seasons: spring, summer, autumn, and winter, with the respective starting dates. The percentage of registered beef cattle was calculated from the total number of animals registered in the cattle herd. The keeping systems of lactating cows and pregnant heifers were categorized as tied, loose, or mixed (tied and loose). The grazing of adult animals was categorized as ’no grazing’, ‘grazing of dry cows’, or ‘grazing of all animals’. Participation in animal shows, pastures being in contact with the neighboring farms’ pastures, animals drinking from natural waterbodies, and employees keeping production animals at home were evaluated as binary variables (yes/no). Univariable regression analyses were performed to identify possible risk factors for herd CB seropositivity. Variables with p < 0.35 (Table S2) were included in the ﬁrst mul- tivariable model building. A stepwise forward inclusion procedure was used to develop the model by ﬁrst adding the most biologically plausible factors and controlling for con- founders and interaction terms. The ﬁrst regression model was developed to compare the prevalence of CB-seropositive animals in different categories of cattle herds. The interaction terms for production type (beef/dairy) and herd size (number of animals) were added to the model because of the different sizes of the beef and dairy cattle herds and the signiﬁcant effect of herd size on CB infection status of the herd. The second multivariable model was built to identify the risk factors for CB seroposi- tivity status of a herd among dairy cattle herds. 2.3.2. Risk Factor Analysis The inclusion of variables (Table S3) and the process of model building were the same as those described above. The keeping system, number of animals in the herd, and region were included in the ﬁnal model. Akaike information criterion (AIC) was used to monitor the ﬁt of the models during construction. All variables with p-values < 0.05 were considered to have a statistically signiﬁcant impact on the herds’ infection status. The analyses were performed using R version 4.1.0 [21]. R version 4.1.0 [21]. 2.3.1. Prevalence Estimation 2.3.1. Prevalence Estimation Cattle herds were categorized as dairy herds (≥50% dairy breed cattle) and beef herds (<50% dairy breed cattle), and herd-level prevalence estimates were calculated for both subgroups as well as for sheep and goat herds. Wilson’s 95% conﬁdence intervals (CI) Microorganisms 2023, 11, 819 4 of 12 were calculated using the EpiTools® epidemiological calculator ’Conﬁdence limits for a proportion’ [14]. were calculated using the EpiTools® epidemiological calculator ’Conﬁdence limits for a proportion’ [14]. 3. Results 3.1. Seroprevalence Study and Risk Factor Study 1 3.1. Seroprevalence Study and Risk Factor Study 1 The sample included 504 cattle herds, of which 324 were dairy and 180 were beef cattle herds. The average size of the herds was 278 for dairy herds (median 82) and 72 for beef herds (median 45). More than 90% of the dairy cattle were Estonian Holstein Friesian, Estonian Red, and/or Estonian Native cattle breeds. The beef cattle population consisted of Microorganisms 2023, 11, 819 5 of 12 more than 10 different breeds, of which Hereford, Aberdeen-Angus, and Limousine breeds were dominant. more than 10 different breeds, of which Hereford, Aberdeen-Angus, and Limousine breeds were dominant. The small ruminant ﬂocks consisted of 170 sheep and 18 goat ﬂocks. The average ﬂock size was 165 for sheep (median 110) and 52 for goats (median 16). p g The prevalence of cattle herds, sheep, and goat ﬂocks with at least one CB-seropositive test result is presented in Table 1. Table 1. Prevalence of Coxiella burnetii antibody-positive herds among beef and dairy herds and sheep and goat ﬂocks sampled for national surveillance programs in Estonia during 2012–2013. Table 1. Prevalence of Coxiella burnetii antibody-positive herds among beef and dairy herds and sheep and goat ﬂocks sampled for national surveillance programs in Estonia during 2012–2013. Ruminants n/N 1 Prevalence, % (95% CI 2) Beef cattle herds 12/180 6.67 (3.85–11.29) Dairy cattle herds 88/324 27.16 (22.61–32.25) Sheep ﬂocks 4/170 2.35 (0.92–5.89) Goat ﬂocks 0/18 0.00 (0.00–16.82) 1 number of herds (n) with at least one CB-seropositive test result and number of tested herds (N). 2 95% conﬁdence intervals. The apparent prevalence of CB-positive herds was signiﬁcantly higher in dairy cattle herds than in beef cattle herds and sheep ﬂocks. The prevalence of CB-positive beef cattle herds and sheep ﬂocks did not differ signiﬁcantly. p g y DNA of CB was found in 11.36% (6.29–19.67 95% CI) of the CB-seropositive pooled milk samples (n/N = 10/88) from the EBL surveillance program. p ( ) p g The results of the univariable binomial logistic regression analysis for the risk factors for cattle herds are presented in Table S2. Risk factors associated with a cattle herd testing CB-positive in the univariable analysis were production type, region, number of animals, herd size, and season. The production type, number of animals, and region were included in the ﬁnal multivariable model (Table 2). Cattle breeds were excluded from the ﬁnal model because of evidence of collinearity with herd size. 3.2. Risk Factor Study 2 (Dairy Cattle Herds) In total, 72 dairy farms participated in this study, submitted bulk tank milk samples for CB antibody testing, and replied to the questionnaire. The results of the univariable binomial logistic regression analysis of the risk factors for dairy herds testing CB-positive are presented in Table S3. The univariable analysis revealed that herds with all animals grazing, not keeping lactating cows tied, and located in the northeast region compared to those in southwest Estonia had higher odds of testing CB positive. Of the variables that passed the inclusion criteria for the multivariable analysis (Table S3), three remained in the final regression model, as presented in Table 3. The keeping system was added as a fixed variable and the number of animals in the herd and location were kept in the final model as possible confounders. Table 3. Multivariable binomial logistic regression analyses for the risk factors associated with dairy cattle herds testing seropositive for Coxiella burnetii in a bulk tank milk sample in Estonia. Table 3. Multivariable binomial logistic regression analyses for the risk factors associated with dairy cattle herds testing seropositive for Coxiella burnetii in a bulk tank milk sample in Estonia. Variable n/N 7 Odds Ratio (95% CI 8) p-Value ANOVA p-Value Keeping system (lactating cows) Tied 5/36 1 0.087 Mixed 9 3/5 3.54 (0.28–45.64) 0.332 Loose 17/31 5.56 (1.17–26.39) 0.031 No. of animals 10 1.001 (1.000–1.002) 0.359 Region Southwest 8/25 1 0.002 Southeast 3/13 0.50 (0.07–3.30) 0.468 Northeast 13/18 3.29 (0.71–15.17) 0.127 Northwest 1/16 0.09 (0.01–0.92) 0.042 7 number of herds with at least one CB antibody positive result (n) and number of tested herds (N); 8 95% conﬁdence intervals; 9 loose and tied keeping systems used; 10 number of animals in the herd. 7 number of herds with at least one CB antibody positive result (n) and number of tested herds (N); 8 95% conﬁdence intervals; 9 loose and tied keeping systems used; 10 number of animals in the herd. 3.1. Seroprevalence Study and Risk Factor Study 1 Including the interaction term for herd type (dairy or beef) and the total number of animals resulted in a slight decrease in the odds of testing positive, and was decided to retain in the model. Table 2. Binomial logistic regression analyses of risk factors associated with Coxiella burnetii- seropositive cattle herds sampled for national surveillance programs in Estonia during 2012–2013. Table 2. Binomial logistic regression analyses of risk factors associated with Coxiella burnetii- seropositive cattle herds sampled for national surveillance programs in Estonia during 2012–2013. Table 2. Binomial logistic regression analyses of risk factors associated with Coxiella burnetii seropositive cattle herds sampled for national surveillance programs in Estonia during 2012–2013. Variable n/N 3 Odds Ratio (95% CI 4) p-Value ANOVA p-Value Production type Beef cattle 12/180 1 <0.001 Dairy cattle 88/324 5.40 (2.07–14.04) <0.001 No. of animals 5 1.007 (1.001–1.013) 0.016 <0.001 Region Southwest 14/156 1 <0.001 Southeast 25/99 3.80 (1.75–8.22) <0.001 Northeast 40/121 4.60 (2.24–9.41) <0.001 Northwest 21/128 2.46 (1.13–5.39) 0.024 Interaction 6 Beef cattle herds: No. of animals 1 0.114 Dairy cattle herds: No. of animals 0.99 (0.99–1.00) 0.100 3 number of herds (n) with at least one CB antibody positive result and number of tested herds (N); 4 95% conﬁdence intervals (95% CI); 5 total number of animals in the herd (No. of animals); 6 interaction between production type and total number of animals in the herd 3 number of herds (n) with at least one CB antibody positive result and number of tested herds (N); 4 95% conﬁdence intervals (95% CI); 5 total number of animals in the herd (No. of animals); 6 interaction between production type and total number of animals in the herd Microorganisms 2023, 11, 819 6 of 12 The ﬁnal multivariable model based on samples collected via surveillance programs predicted higher odds for detecting CB antibodies in animals in dairy cattle herds, and if the herd was located in regions other than Southwest of Estonia (Figure S2). 3.2. Risk Factor Study 2 (Dairy Cattle Herds) 4.2. Detection of Coxiella Burnetii DNA in Pooled Milk Samples CB DNA has been detected in the feces, vaginal mucus, milk, and abortion material of dairy cattle, indicating an active infection [29,30]. The results of CB DNA-positive pooled milk samples collected for surveillance indicated that more than one out of ten herds in Estonia had at least one animal with active infection at any given time. Older animals are at a higher risk of being antibody positive to CB likely because of a potentially longer exposure time [31]. In 2012, the average number of lactations for dairy cows in Estonian dairy herds was 2.4 [32]. Approximately 60% of cows were in their ﬁrst or second lactation cycles, whereas 22% had been in the herd for more than three lactation cycles [32]. As we could not determine the average age of animals in study herds, and young animals (calves and heifers) were not sampled, the relationship between exposure and age factors potentially inﬂuencing the onset of CB infection could not be determined in Estonian cattle herds. Parturition may or may not be associated with the secretion of CB, and the current state of knowledge suggests that control measures should focus on the whole herd, not only on infected or susceptible animals [29,33]. DNA detection in pooled milk may indicate intermittent or persistent excretion of the pathogen [29,33], which may be caused by intramammary infection [34]. Intramammary infections may result in increased environmental contamination and CB transmission at the conventional milking parlor, which is commonly used in Estonian dairy cattle herds (personal observation). The risk of detecting CB DNA in tank milk has been found to be lower in herds using automatic milking systems [10], which is attributed to differences in management practices, hygiene, behavioral patterns, and interactions between animals [35]. 4.1. Coxiella Burnetii Seroprevalence in Cattle Herds 4.1. Coxiella Burnetii Seroprevalence in Cattle Herds CB antibodies were found in herds from all regions of Estonia, where CB infections may already be endemic. The proportion of Estonian dairy cattle herds that tested positive for CB was lower than that observed in Danish, Dutch, and French dairy cattle herds [10,23,24], but higher than that observed in northern parts of Europe closer to Estonia, such as Norway and Sweden [8,25]. CB infections have spread in different regions of France and Denmark [23,24] and are linked to abortion cases in cattle [24]. This suggests that CB infection is endemic in Western Europe and poses a threat to animal health. The study populations and laboratory methods used in the different studies varied, and comparisons between their results should be made with caution. The presence of CB antibodies found in cattle herds indicates that cattle, as hosts of an active infection, pose a risk of spreading zoonosis to humans, such as farmers and veterinarians [26,27]. This risk has been described in human QF outbreaks associated with dairy cattle farming [28]. Since the stage of the infection has not been clariﬁed and QF has not been diagnosed in Estonian cattle, further studies are needed to detect the dynamics of CB infection in cattle herds and the risk to people handling the animals. 4. Discussion To our knowledge, this was the ﬁrst study in Estonia to estimate the extent of the spread of CB infection in Estonian domestic ruminant herds and to identify possible associated risk factors. The reuse of sera from the EBL surveillance program allowed taking a randomized sample of the Estonian cattle herds balanced according to the regional distribution of herds. Therefore, the obtained sample could be considered relatively well representing the target population and the prevalence estimates valid. However, the small number of small ruminant ﬂocks sampled in Brucella surveillance program did not allow to estimate reliably the herd prevalence in the population and the results reﬂect primarily the situation in ﬂocks involved in breeding program, dairy ﬂocks and larger production ﬂocks. g p The number of samples that could be obtained from goat ﬂocks was particularly limited, and the results accordingly should be interpreted with caution. The number of samples collected from each cattle herd was sufﬁcient to detect a seropositive herd at deﬁned prevalence level (design prevalence). However, the number of samples collected from small ruminant ﬂocks might have been insufﬁcient to detect all CB-seropositive ﬂocks among the sampled ones as the within-herd prevalence may be lower than 20% in some situations [22]. 7 of 12 7 of 12 Microorganisms 2023, 11, 819 4.4. Risk Factor Analysis A higher CB seroprevalence was observed in dairy cattle herds than in beef cattle herds. These results are in accordance with those reported in other countries [24,31]. A similar trend has also been observed between sheep used for dairy and meat production [9,24]. A higher prevalence of CB-positive herds has been associated with larger herds, higher herd density of the area, managemental differences including housing practices and herd size, and outdoor and indoor environmental conditions [9,24]. Ryan et al. [37] identiﬁed dairy cattle breed as a potential animal-level risk factor, but mentioned that breed-speciﬁc effects need to be further studied, as breeds may reﬂect different husbandry and management techniques. In this study, speciﬁc breeds were associated with the production type and herd size and were not included in the ﬁnal model. Although human QF outbreaks are mostly associated with small ruminants [6], out- breaks can also be related with cattle [28]. In Estonia, QF is a notiﬁable disease in ruminants, and animals are tested when there is a clinical suspicion of the disease or due to trade requirements [38]. As this study revealed that CB infection is present in Estonian cattle herds, especially in dairy cattle, it is necessary to raise farmers’ awareness about the possible risks associated with CB infection in humans and animals. Schimmer et al. [39,40] found that working with CB-seropositive goats increased the odds of the farmer being seropositive as well, while no such relation was identiﬁed in dairy cattle farmers. Farmers should also be encouraged to contact veterinary authorities in case of animal health issues possibly related to QF, so that the spread of the infection can be promptly limited. Testing of imported animals originating from QF-endemic areas should be encouraged to restrict the purchase of CB-infected animals. In Estonia, 9.62% of veterinary professionals and 7.73% of dairy cattle farmers tested CB seropositive, which is higher than that in the general population (3.9%) [27]. Farmers and veterinarians thus represent a risk group for infections with CB and should be informed of the risks associated with working or having contact with ruminants of unknown CB status. Larger cattle herds had higher odds of being exposed to CB in Estonia. Similarly, Spanish, Danish, and Irish cattle farms with more animals are more likely to be CB an- tibody positive [11,37,41]. These observations have also been reported in goats in the Netherlands [7] and sheep in Italy [36]. 4.3. Coxiella Burnetii-Seropositivity Detection in Sheep and Goat Flocks The study results demonstrated that the prevalence of CB-seropositive Estonian sheep ﬂocks was relatively low, but it indicated that the infection occasionally occurs. Compared to Western and Central European sheep ﬂocks, the proportion of CB seropositive Estonian sheep ﬂocks was lower, similar to what has been observed in Northern Europe. A lower CB seroprevalence has been reported in sheep ﬂocks in Norway and Sweden [8,25], and higher in the Netherlands, France, and Sicily, Italy [9,24,36]. CB has been detected in different areas of France and the Netherlands [9,24] and clinical signs of QF occur [24], suggesting that CB infection is endemic in sheep in these countries. CB antibodies have been detected in both dairy and meat production systems [9,36]. Kampen et al. [8] suggested that the low prevalence in Norwegian sheep could be explained by the limited import of animals, as domestic ruminant populations are not separated. Estonian cattle and sheep populations are usually separated, preventing spread between the species through direct contact [13]. Microorganisms 2023, 11, 819 8 of 12 Additionally, sheep are kept and managed similarly to beef cattle, which may partially explain the low CB seroprevalence (personal observation). Additionally, sheep are kept and managed similarly to beef cattle, which may partially explain the low CB seroprevalence (personal observation). p p p Because human QF outbreaks are often associated with small ruminants farming or contact with infected animals or their products, even a low CB prevalence in sheep ﬂocks may pose a public health risk [3]. No CB antibody-positive goat ﬂocks were detected in Estonia. This ﬁnding is similar to those reported in Norway and Sweden [8,25]. However, this study found no positive goat ﬂocks, the number of tested goat ﬂocks was small in this study, and the absence of the pathogen in this host could not be veriﬁed. In other parts of Europe, the prevalence is high in goats. For example, 43.1% of goat farms tested positive for CB antibodies in the Netherlands and 61% in France [7,24]. Kampen et al. [8] suggested that the low prevalence may be due to limited imports and combined ruminant farming. Ohlson et al. [25] reported that a low prevalence in goats coincided with a low prevalence in cattle in Sweden, indicating that ruminants may share similar risk factors for transmission. 4.4. Risk Factor Analysis An increased number of interactions between animals may increase the possibility of contact with the pathogen from an infected animal. Differences in herd characteristics, herd management practices, and a larger number of em- ployees and visitors to larger herds could also facilitate the introduction and transmission of infection [7,11,31,37]. Our ﬁndings support some of these ﬁndings. According to our observations, Estonian beef cattle are often raised extensively on grasslands with no or min- imal natural shelter during summer and are housed in winter. Dairy cattle are often kept in intensive production systems that commonly use large indoor living areas, where CB can Microorganisms 2023, 11, 819 9 of 12 easily spread. In these farms, dairy cattle are kept loose and interact more closely with each other than beef cattle on pastures. When using indoor housing, infectious pathogens might move more easily in the environment owing to the accumulation of dust, formation of aerosols, and transmission between animals. Moreover, larger dairy herds have a number of contacts with visitors, including veterinarians, inseminators, hoof trimers, and animal husbandry consultants, who can present a risk of introducing CB to the farm or between animals. However, a negative association in the univariable analysis of veterinary service was detected in Denmark presumably due to adequate personal hygiene measures [11] g y y was detected in Denmark, presumably due to adequate personal hygiene measures [11]. , p y q p yg [ ] Estonian cattle herds were relatively evenly distributed between the counties [13] and CB-seropositive cattle herds were found in all regions. Cattle from herds located in the southwestern part and dairy cattle from northwestern herds were less commonly CB seropositive than those from other regions of Estonia. Regional differences have been observed in other European countries. In Northern Ireland, CB seroprevalence in differ- ent regions ranges from 33% to 63% in cattle herds and from 3% to 10% in individual animals [31]. Signiﬁcant regional differences and clusters have been reported in Swedish dairy cattle herds [25]. Production density, climate, and geographical differences have been suggested as potential factors that can affect the presence of CB in different regions [25,42]. In Estonia, the climate differs distinctively between the eastern and western halves of the country (e.g., the yearly average temperature in the western region is 1 ◦C higher than that in the eastern region). 4.4. Risk Factor Analysis More detailed studies on regional differences in CB-seropositive herds are needed and can be used to conceptualize strategies for eradication programs. p g p g The prevalence was lower in dairy cattle herds where animals were kept tied compared to herds, keeping the animals loose, or combining tied and free-range management in uni- variable analysis. A higher CB seroprevalence has also been found in Danish loose housing systems than in cattle kept tied [11]. Keeping heifers in tie stalls has signiﬁcantly reduced the risk of seroconversion in Belgian dairy cattle herds, although this result was considered to be related to a low exposure level [43]. The protective effect of keeping animals tied may be due to less contact between animals shedding CB bacteria and susceptible animals in the herd. Furthermore, limited movement has been suggested to reduce transmission within farms [11]. However, the tied keeping of cattle is a disappearing keeping system in the European Union according to animal welfare rules for cattle and cannot be considered a measure to control infections. Farmers must ﬁnd other ways to mitigate infection risk. Data Availability Statement: Data are available on request. Acknowledgments: The study group would like to thank the Estonian National Centre for Lab- oratory Research and Risk Assessment for providing the cattle, sheep and goat samples used in prevalence study, Estonian Agricultural Registers and Information Board for providing data on do- mestic ruminant herds in Estonia, Tanel Kaart for advice on statistical analysis, and Annely Aleksejev and Julia Jeremejeva for technical laboratory work. Special gratitude goes to Ants Kuks who helped to collect additional samples from sheep and goat ﬂocks and to all the volunteered farmers participating in this study. Conﬂicts of Interest: The authors declare no conﬂict of interest. References 1. European Food Safety Authority (EFSA). Scientiﬁc Opinion on Q fever. EFSA J. 2010, 8, 1595. [Cros 1. European Food Safety Authority (EFSA). Scientiﬁc Opinion on Q fever. EFSA J. 2010, 8, 1595. [CrossRef] 2. Lau, C.; Musso, D.; Fournier, P.-E.; Parola, P.; Raoult, D.; Weinstein, P. Absence of serological evidence of Rickettsia spp., Bartonella spp., Ehrlichia spp. and Coxiella burnetii infections in American Samoa. Ticks Tick. Borne. Dis. 2016, 7, 703–705. [CrossRef] [PubMed] 1. European Food Safety Authority (EFSA). Scientiﬁc Opinion on Q fever. EFSA J. 2010, 8, 1595. [CrossRef] 2. Lau, C.; Musso, D.; Fournier, P.-E.; Parola, P.; Raoult, D.; Weinstein, P. Absence of serological evidence of Rickettsia spp., Bartonella 1. European Food Safety Authority (EFSA). Scientiﬁc Opinion on Q fever. EFSA J. 2010, 8, 1595. [CrossRef] 3. Eldin, C.; Mélenotte, C.; Mediannikov, O.; Ghigo, E.; Million, M.; Edouard, S.; Mege, J.-L.; Maurin, M.; Raoult, D. From Q Fever to Coxiella burnetii Infection: A Paradigm Change. Clin. Microbiol. Rev. 2017, 30, 115–190. [CrossRef] [PubMed] 4. Duron, O.; Sidi-Boumedine, K.; Rousset, E.; Moutailler, S.; Jourdain, E. The Importance of Ticks in Has (and Has Not) Been Demonstrated? Trends Parasitol. 2015, 31, 536–552. [CrossRef] 5. Hansen, M.S.; Rodolakis, A.; Cochonneau, D.; Agger, J.F.; Christoffersen, A.-B.; Jensen, T.K.; Agerholm, J.S. Coxiella burnetii associated placental lesions and infection level in parturient cows. Vet. J. 2011, 190, e135–e139. [CrossRef] p p 6. Dijkstra, F.; van der Hoek, W.; Wijers, N.; Schimmer, B.; Rietveld, A.; Wijkmans, C.J.; Vellema, P.; Schneeberger, P.M. The 2007–2010 Q fever epidemic in the Netherlands: Characteristics of notiﬁed acute Q fever patients and the association with dairy goat farming. FEMS Immunol. Med. Microbiol. 2012, 64, 3–12. [CrossRef] g 7. Schimmer, B.; Luttikholt, S.; Hautvast, J.L.A.; Graat, E.A.M.; Vellema, P.; van Duynhoven, Y.T.H.P. Serop of Q fever in goats on commercial dairy goat farms in the Netherlands, 2009–2010. BMC Vet. Res. 2011, 7, 7. Schimmer, B.; Luttikholt, S.; Hautvast, J.L.A.; Graat, E.A.M.; Vellema, P.; van Duynhoven, Y.T.H.P. Seroprevalence and risk factors of Q fever in goats on commercial dairy goat farms in the Netherlands, 2009–2010. BMC Vet. Res. 2011, 7, 81. [CrossRef] [PubMed] 8. Kampen, A.H.; Hopp, P.; Grøneng, G.M.; Melkild, I.; Urdahl, A.M.; Karlsson, A.-C.; Tharaldsen, J. No indication of Coxiella burnetii infection in Norwegian farmed ruminants. BMC Vet. Res. 2012, 8, 59. [CrossRef] of Q fever in goats on commercial dairy goat farms in the Netherlands, 2009–2010. BMC Vet. References Res. 2011, 7, 81. [CrossRef] [PubMed] 8. Kampen, A.H.; Hopp, P.; Grøneng, G.M.; Melkild, I.; Urdahl, A.M.; Karlsson, A.-C.; Tharaldsen, J. No indication of Coxiella burnetii infection in Norwegian farmed ruminants. BMC Vet. Res. 2012, 8, 59. [CrossRef] g [ ] 9. Schimmer, B.; de Lange, M.M.A.; Hautvast, J.L.A.; Vellema, P.; van Duynhoven, Y.T.H.P. Coxiella burnetii seroprevalence and risk factors on commercial sheep farms in The Netherlands. Vet. Rec. 2014, 175, 17. [CrossRef] 9. Schimmer, B.; de Lange, M.M.A.; Hautvast, J.L.A.; Vellema, P.; van Duynhoven, Y.T.H.P. Coxiella burnet factors on commercial sheep farms in The Netherlands. Vet. Rec. 2014, 175, 17. [CrossRef] 10. van Engelen, E.; Schotten, N.; Schimmer, B.; Hautvast, J.L.A.; van Schaik, G.; van Duijnhoven, Y.T.H.P. Prevalence and risk factors for Coxiella burnetii (Q fever) in Dutch dairy cattle herds based on bulk tank milk testing. Prev. Vet. Med. 2014, 117, 103–109. [CrossRef] 11. Paul, S.; Agger, J.F.; Markussen, B.; Christoffersen, A.-B.; Agerholm, J.S. Factors associated with Coxiella burnetii antibody positivity in Danish dairy cows. Prev. Vet. Med. 2012, 107, 57–64. [CrossRef] [PubMed] p y y 12. Estonian National Centre for Laboratory Research and Risk Assessment (LABRIS). Annual Repo LABRIS: Tartu, Estonia, 2002. Available online: https://labris.agri.ee/et/loomatervis/aruanded (ac 12. Estonian National Centre for Laboratory Research and Risk Assessment (LABRIS). Annual Report 2001; Part 1 (In Estonian). LABRIS: Tartu, Estonia, 2002. Available online: https://labris.agri.ee/et/loomatervis/aruanded (accessed on 6 September 2018). 13 E t i A i lt l R i t d I f ti B d R i t f A i lt l A i l A il bl li htt // i / 12. Estonian National Centre for Laboratory Research and Risk Assessment (LABRIS). Annual Report 2001; Part 1 (In Estonian). LABRIS: Tartu, Estonia, 2002. Available online: https://labris.agri.ee/et/loomatervis/aruanded (accessed on 6 September 2018). 13. Estonian Agricultural Registers and Information Board. Register of Agricultural Animals. Available online: http://www.pria.ee/ en/ (accessed on 20 March 2012). , , p // g / / / ( p ) 13. Estonian Agricultural Registers and Information Board. Register of Agricultural Animals. Available online: http://www.pria.ee/ en/ (accessed on 20 March 2012). 14. Sergeant, E.S.G. EpiTools Epidemiological Calculators. Available online: http://epitools.ausvet.com.au/ (accessed on 17 March 2021). Tools Epidemiological Calculators. Available online: http://epitools.ausvet.com.au/content.php?page=home ch 2021). 15. Horigan, M.W.; Bell, M.M.; Pollard, T.R.; Sayers, A.R.; Pritchard, G.C. Q fever diagnosis in domestic ruminants: Comparison between complement ﬁxation and commercial enzyme-linked immunosorbent assays. J. Vet. Diagnostic Investig. 5. Conclusions This study established serological evidence of exposure to CB for beef and dairy cattle and sheep, but not for goats in Estonia. Approximately one-tenth of the seropositive samples from cattle also contained CB DNA, indicating that a considerable proportion of exposed cattle may also shed the bacteria, and cattle may pose a risk for human infections. A larger herd size appears to be a risk factor for CB seropositivity in cattle herds, indicating that certain management practices may play a role in CB spread. Regional differences in CB seropositivity in cattle herds may reﬂect the effect of climatic conditions on CB spread in Estonia. The results of this study can be used to inform risk groups for zoonosis and to implement control strategies to limit the spread of CB in ruminant herds. Supplementary Materials: The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/microorganisms11040819/s1, Figure S1: Collection of samples from Estonian cattle, sheep and goats to identify Coxiella burnetii seroprevalence and related risk factors; Table S1: Questionnaire used to clarify possible risk factors for Coxiella burnetii infection in volunteered dairy cattle herds; Figure S2: Study regions according to Estonian counties; Table S2: Univariable regression model of risk factors predicting Coxiella burnetii seroprevalence of 504 Estonian cattle herds based on surveillance data; Table S3: Univariable regression analysis results to predict Coxiella burnetii seroprevalence in bulk tank milk samples from volunteering Estonian dairy cattle herds. 10 of 12 10 of 12 Microorganisms 2023, 11, 819 Author Contributions: Conceptualization, K.N., B.L. and A.V.; data curation, K.N., B.L. and A.V.; formal analysis, K.N. and A.V.; funding acquisition, A.V.; investigation, K.N., L.T., B.L. and A.V.; methodology, A.V. and B.L.; writing—original draft, K.N. and A.V.; writing—review and editing, K.N., L.T., B.L. and A.V. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by the Estonian Research Council health promotion research program TerVE (project No. 3.2.1002.11-0002). Institutional Review Board Statement: This study was approved by the Ministry of Agriculture of the Republic of Estonia (license no. 7.2-11/2, 2 April 2013). Institutional Review Board Statement: This study was approved by the Ministry of Agriculture of the Republic of Estonia (license no. 7.2-11/2, 2 April 2013). Informed Consent Statement: Not applicable. Data Availability Statement: Data are available on request. References 2011, 23, 924–931. [CrossRef] [PubMed] 11 of 12 11 of 12 Microorganisms 2023, 11, 819 16. Guatteo, R.; Beaudeau, F.; Joly, A.; Seegers, H. Performances of an ELISA applied to serum and milk for the detection of antibodies to Coxiella burnetii in dairy cattle. Rev. Med. Vet. 2007, 158, 250–252. y 17. Thermo Fisher Scientiﬁc. Home Page. Available online: https://www.thermoﬁsher.com/ee/en/home.html (accessed on 24 April 2019). 18. Berri, M.; Laroucau, K.; Rodolakis, A. The detection of Coxiella burnetii from ovine genital swabs, milk and fecal samples by the use of a single touchdown polymerase chain reaction. Vet. Microbiol. 2000, 72, 285–293. [CrossRef] rcell Microbiologists. Home Page. Available online: https://en.vircell.com/ (accessed on 20 March 2012). 20. Vaidya, V.M.; Malik, S.V.S.; Kaur, S.; Kumar, S.; Barbuddhe, S.B. Comparison of PCR, immunoﬂuorescence assay, and pathogen isolation for diagnosis of Q fever in humans with spontaneous abortions. J. Clin. Microbiol. 2008, 46, 2038–2044. [CrossRef] 20. Vaidya, V.M.; Malik, S.V.S.; Kaur, S.; Kumar, S.; Barbuddhe, S.B. Comparison of PCR, immunoﬂuorescence assay, and pathogen isolation for diagnosis of Q fever in humans with spontaneous abortions. J. Clin. Microbiol. 2008, 46, 2038–2044. [CrossRef] 21. R Core Team. R: A Language and Environment for Statistical Computing. Available online: https://www.r-project.org/ (accessed on 20 June 2021). isolation for diagnosis of Q fever in humans with spontaneous abortions. J. Clin. Microbiol. 2008, 46, 2038–2044. [CrossRef] 21. R Core Team. R: A Language and Environment for Statistical Computing. Available online: https://www.r-project.org/ (accessed on 20 June 2021). J ) 22. Georgiev, M.; Afonso, A.; Neubauer, H.; Needham, H.; Thiéry, R.; Rodolakis, A.; Roest, H.J.; Stärk, K.D.; Stegeman, J.A.; Vellema, P.; et al. Q fever in humans and farm animals in four European countries, 1982 to 2010. Eurosurveillance 2013, 18, 20407. [CrossRef] 23. Agger, J.F.; Paul, S. Increasing prevalence of Coxiella burnetii seropositive Danish dairy cattle herds. Acta Vet. Scand. 2014, 56, 46. [CrossRef] 24. Gache, K.; Rousset, É.; Perrin, J.B.; De Cremoux, R.; Hosteing, S.; Jourdain, E.; Guatteo, R.; Nicollet, P.; Touratier, A.; Calavas, D.; et al. Estimation of the frequency of Q fever in sheep, goat and cattle herds in France: Results of a 3-year study of the seroprevalence of Q fever and excretion level of Coxiella burnetii in abortive episodes. Epidemiol. Infect. 2017, 145, 3131–3142. [CrossRef] p p f 25. Ohlson, A.; Malmsten, J.; Frössling, J.; Bölske, G.; Aspán, A.; Dalin, A.-M.; Lindberg, A. References Surveys on Coxiella burnetii infections in Swedish cattle, sheep, goats and moose. Acta Vet. Scand. 2014, 56, 39. [CrossRef] 26. Bacci, S.; Villumsen, S.; Valentiner-Branth, P.; Smith, B.; Krogfelt, K.A.; Mølbak, K. Epidemiology and Clinical Features of Human Infection with Coxiella burnetii in Denmark During 2006–2007. Zoonoses Public Health 2011, 59, 61–68. [CrossRef] 27. Neare, K.; Janson, M.; Hütt, P.; Lassen, B.; Viltrop, A. Coxiella burnetii Antibody Prevalence and Risk Factors of Infection in the Human Population of Estonia. Microorganisms 2019, 7, 629. [CrossRef] [PubMed] 27. Neare, K.; Janson, M.; Hütt, P.; Lassen, B.; Viltrop, A. Coxiella burnetii Antibody Prevalence and Risk Human Population of Estonia. Microorganisms 2019, 7, 629. [CrossRef] [PubMed] 28. Chmielewski, T.; Tylewska-Wierzbanowska, S. Q fever outbreaks in Poland during 2005–2011. M 1073–1079. [CrossRef] 28. Chmielewski, T.; Tylewska-Wierzbanowska, S. Q fever outbreaks in Poland during 2005–2011. Med. Sci. Monit. 2013, 19, 1073–1079. [CrossRef] 29 G tt R B d F J l A S H C i ll b tii h ddi b d i V t R 2007 38 849 860 [C R f] 1073–1079. [CrossRef] 29 Guatteo R ; Beaudeau F ; Joly A ; Seegers H Coxiella burnetii shedding by dairy cows Vet Res 2007 38 849–860 [CrossRef] [ ] 29. Guatteo, R.; Beaudeau, F.; Joly, A.; Seegers, H. Coxiella burnetii shedding by dairy cows. Vet. Res. 2007, 38, 849–860. [CrossRef] [PubMed] 29. Guatteo, R.; Beaudeau, F.; Joly, A.; Seegers, H. Coxiella burnetii shedding by dairy cows. Vet. Res. 2007 [PubMed] 30. Clemente, L.; Barahona, M.J.; Andrade, M.F.; Botelho, A. Diagnosis by PCR of Coxiella burnetii in aborted fetuses of domestic ruminants in Portugal. Vet. Rec. 2009, 164, 373–374. [CrossRef] g 31. McCaughey, C.; Murray, L.J.; McKenna, J.P.; Menzies, F.D.; McCullough, S.J.; O’Neill, H.J.; Wyatt, D.E.; Cardwell, C.R.; Coyle, P.V. Coxiella burnetii (Q fever) seroprevalence in cattle. Epidemiol. Infect. 2010, 138, 21–27. [CrossRef] 32. Estonian Livestock Performance Recording Ltd. Home Page. Available online: https://www.epj.ee/jkk/en.html (accessed on 17 April 2021). p 33. Rodolakis, A.; Berri, M.; Héchard, C.; Caudron, C.; Souriau, A.; Bodier, C.C.; Blanchard, B.; Camuset, P.; Devillechaise, P.; Natorp, J.C.; et al. Comparison of Coxiella burnetii shedding in milk of dairy bovine, caprine, and ovine herds. J. Dairy Sci. 2007, 90, 5352–5360. [CrossRef] [PubMed] 34. Barlow, J.; Rauch, B.; Welcome, F.; Kim, S.G.; Dubovi, E.; Schukken, Y. Association between Coxiella burnetii shedding in milk and subclinical mastitis in dairy cattle. Vet. Res. 41. Alvarez, J.; Perez, A.; Mardones, F.O.; Pérez-Sancho, M.; García-Seco, T.; Pagés, E.; Mirat, F.; Díaz, R.; Carpintero, J.; Domínguez, L. Epidemiological factors associated with the exposure of cattle to Coxiella burnetii in the Madrid region of Spain. Vet. J. 2012, 194, 102–107. [CrossRef] [PubMed] 42. Nusinovici, S.; Frössling, J.; Widgren, S.; Beaudeau, F.; Lindberg, A. Q fever infection in dairy cattle herds: Increased risk with high wind speed and low precipitation. Epidemiol. Infect. 2015, 143, 3316–3326. [CrossRef] [PubMed] g p p p p f 43. Czaplicki, G.; Houtain, J.-Y.; Mullender, C.; Porter, S.R.; Humblet, M.-F.; Manteca, C.; Saegerman, C. Apparent prevalence of antibodies to Coxiella burnetii (Q fever) in bulk tank milk from dairy herds in southern Belgium. Vet. J. 2012, 192, 529–531. [CrossRef] 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. References 2008, 39, 23. [CrossRef] 34. Barlow, J.; Rauch, B.; Welcome, F.; Kim, S.G.; Dubovi, E.; Schukken, Y. Association betw and subclinical mastitis in dairy cattle. Vet. Res. 2008, 39, 23. [CrossRef] 35. Jacobs, J.A.; Siegford, J.M. Invited review: The impact of automatic milking systems on dairy cow management, behavior, health, and welfare. J. Dairy Sci. 2012, 95, 2227–2247. [CrossRef] [PubMed] 35. Jacobs, J.A.; Siegford, J.M. Invited review: The impact of automatic m and welfare. J. Dairy Sci. 2012, 95, 2227–2247. [CrossRef] [PubMed] 36. Villari, S.; Galluzzo, P.; Arnone, M.; Alfano, M.; Geraci, F.; Chiarenza, G. Seroprevalence of Coxiella burn sheep farms located in Sicily (Southern Italy) and related risk factors. Small Rumin. Res. 2018, 164, 82–8 p y y 37. Ryan, E.D.; Kirby, M.; Collins, D.M.; Sayers, R.; Mee, J.F.; Clegg, T. Prevalence of Coxiella burnetii (Q fever) antibodies in bovine serum and bulk-milk samples. Epidemiol. Infect. 2011, 139, 1413–1417. [CrossRef] 38. Parliament of the Estonian Republic. Veterinary Act. Available onli 8072022001/consolide (accessed on 1 November 2022). 38. Parliament of the Estonian Republic. Veterinary Act. Available online: https://www.riigiteataja.ee/en/eli/ee/Riigikogu/act/50 8072022001/consolide (accessed on 1 November 2022). 39. Schimmer, B.; Lenferink, A.; Schneeberger, P.; Aangenend, H.; Vellema, P.; Hautvast, J.; van Duynhoven, Y. Seroprevalence and risk factors for Coxiella burnetii (Q fever) seropositivity in dairy goat farmers’ households in the Netherlands, 2009–2010. PLoS ONE 2012, 7, e42364. [CrossRef] [PubMed] 40. Schimmer, B.; Schotten, N.; van Engelen, E.; Hautvast, J.L.A.; Schneeberger, P.M.; van Duijnhoven, Y.T.H.P. Coxiella burnetii Seroprevalence and Risk for Humans on Dairy Cattle Farms, the Netherlands, 2010–2011. Emerg. Infect. Dis. 2014, 20, 417–425. [CrossRef] [PubMed] 41. Alvarez, J.; Perez, A.; Mardones, F.O.; Pérez-Sancho, M.; García-Seco, T.; Pagés, E.; Mirat, F.; Díaz, R.; Carpintero, J.; Domínguez, L. Epidemiological factors associated with the exposure of cattle to Coxiella burnetii in the Madrid region of Spain. Vet. J. 2012, 194, 102–107. [CrossRef] [PubMed] 12 of 12 12 of 12 Microorganisms 2023, 11, 819 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/W2166963738	https://bmcchem.biomedcentral.com/counter/pdf/10.1186/1752-153X-6-148	English	null	Development and immunochemical evaluation of a novel chicken IgY antibody specific for KLK6	Chemistry central journal	2,012	cc-by	6,952	RESEARCH ARTICLE Open Access Sotiropoulou et al. Chemistry Central Journal 2012, 6:148 http://journal.chemistrycentral.com/content/6/1/148 Open Access * Correspondence: gdsotiro@upatras.gr 1Department of Pharmacy, University of Patras, 265 00 Rion-Patras, Greece Full list of author information is available at the end of the article Development and immunochemical evaluation of a novel chicken IgY antibody specific for KLK6 Georgia Sotiropoulou1, Georgios Pampalakis1, Evangelia Prosnikli1, Gregory P Evangelatos2 and Evangelia Livaniou2 © 2012 Sotiropoulou et al.; licensee Chemistry Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Abstract Background: Human kallikrein-related peptidase 6 (KLK6) has been implicated in various types of cancer and in neurodegenerative and demyelinating diseases including multiple sclerosis. Further, anti-KLK6 antibodies attenuated disease manifestations in the mouse model of multiple sclerosis. Availability of specific antibodies against KLK6 is fundamental to the development of improved diagnostic and/or immunotherapeutic applications. Here, we exploited the enhanced immunogenicity of mammalian proteins in avian species to generate a polyclonal antibody against KLK6. Results: Chicken were immunized with recombinant KLK6 and antibodies Y (IgYs) were purified from egg yolk with a simple procedure and evaluated for KLK6 detection by ELISA and Western blot using recombinant proteins and human cell lysates and supernatants. The anti-KLK6 Y polyclonal exhibited high affinity for KLK6 with a detection limit of 30 fmol. On the other hand, the widely used rabbit polyclonal antibody that was raised against the same recombinant KLK6 had a detection limit of 300 fmol. Moreover, the IgYs did not display any crossreactivity with recombinant KLKs or endogenous KLKs and other cellular proteins. Conclusions: Based on its high specificity and sensitivity the developed anti-KLK6 IgY is expected to aid the development of improved diagnostic tools for the detection of KLK6 in biological and clinical samples. Keywords: Kallikrein-related peptidase 6(KLK6), IgY, Immunoassays Production of anti-KLK6 IgYs Chicken were immunized with rKLK6, the laying eggs were collected and IgYs were purified by a modification of the acidified water procedure [28]. The structure of Y antibodies resembles the structure of G antibodies ex- cept for the longer Fc fragment (Figure 1) [2]. A major difference between Y and G is the inability of Y anti- bodies to bind to bacterial Fc receptors such as the staphylococcal Protein A or streptococcal Protein G [1]. The purity of the generated IgYs was very high and no contaminating proteins could be detected by SDS-PAGE resolution of IgYs (data not shown). The two detected bands of about ~67 kDa and 25 kDa correspond to the heavy and light chains of IgYs, respectively. The 35 kDa band of the vitellogenin II precursor observed in Chicken were immunized with rKLK6, the laying eggs were collected and IgYs were purified by a modification of the acidified water procedure [28]. The structure of Y antibodies resembles the structure of G antibodies ex- cept for the longer Fc fragment (Figure 1) [2]. A major difference between Y and G is the inability of Y anti- bodies to bind to bacterial Fc receptors such as the staphylococcal Protein A or streptococcal Protein G [1]. p [ 9] KLK6 has also been suggested as a new biomarker for neurodegenerative diseases. Specifically, plasma levels of KLK6 were found reduced in patients with Alzheimer disease or other forms of dementia compared to age- related healthy individuals [20,21]. Further, KLK6 was found elevated in the serum of multiple sclerosis patients with highest levels being associated with sec- ondary progressive disease [22]. On the other hand, KLK6 can degrade myelin. Consequently, it is considered to play important roles in the physiological demyelin- ation and remyelination process [23,24], while its aber- rant activity has been associated with pathological demyelination typical of multiple sclerosis and other de- myelinating diseases. Importantly, passive immunization through administration of anti-KLK6 antibodies or active immunization through administration of recombinant rat Klk6, to induce the production of anti-KLK6 anti- bodies, delayed significantly the onset and attenuated the symptoms of experimental autoimmune encephalo- myelitis (EAE), the mouse model for multiple sclerosis [25]. Recently, Klk6 neutralizing antibodies slowed dis- ease progression in the TMEV (Theiler’s murine enceph- alomyelitis virus) mouse demyelination model [24]. Also, KLK6 was shown to be new serum prognostic marker for aneurismal subarachnoid hemorrhage. Background these advantages for the generation of a novel polyclonal that recognizes the KLK6 protease with high affinity and specificity. Y antibodies are the predominant serum immunoglobulins in birds, reptiles and amphibians. In the female, transfer of IgYs from serum to egg yolk confers passive immunity to embryos and neonates [1,2] similarly to placental IgG transfer in mammals which also confers passive immunity to the fetus. The enrichment of these immunoglobulins in egg yolk led Leslie and Clem to name this antibody class IgYs [3]. There are several advantages associated with the development and usage of Y antibodies, including better immune responsiveness to mammalian antigens, higher affinity with persistent titer, non-invasive collection, sim- ple and low cost isolation process, large yield and scalable production [1], while enhanced immune response results in antibodies with improved specificity and sensitivity as compared to mammalian IgGs. In this study, we exploited KLK6 was originally identified and cloned based on its aberrant expression in mammary and ovarian cancers and was proposed as a potential diagnostic biomarker [4]. Now it is known that KLK6 has a tissue-wide range of expression, including breast, central nervous system, kidney etc. [5]. It should be emphasized that KLK6 has many transcript and splice variants [6,7]. Transcript var- iants result from alternative promoters usage and encode for the same KLK6 protein, since all mRNA sequence changes occur at the 50-untranslated region, while splice variants result mainly by skipping coding exons and they either encode for small proteins with low identity to KLK6 or to no proteins at all [6,7]. Recently, it was shown that stromal cell-associated ex- pression of KLK6 is an indicator of poor prognosis in ovarian cancer patients [8]. KLK6-positive ovarian can- cer patients show an increased risk of relapse and death Correspondence: gdsotiro@upatras.gr 1Department of Pharmacy, University of Patras, 265 00 Rion-Patras, Greece Full list of author information is available at the end of the article Sotiropoulou et al. Chemistry Central Journal 2012, 6:148 http://journal.chemistrycentral.com/content/6/1/148 Sotiropoulou et al. Chemistry Central Journal 2012, 6:148 http://journal.chemistrycentral.com/content/6/1/148 Page 2 of 8 including KLK6, with proteases of the thrombostasis axis [27]. Potential regulatory interaction between KLK6 and proteases of the thrombostasis axis could have a large impact in various human diseases, including neurode- generation and cancer, as discussed above [27]. Conse- quently, the development of new reagents for the detection of KLK6 with potential diagnostic applications is of great importance. Background Presently, a limited number of rabbit polyclonal or mouse monoclonal antibodies have been generated against KLK6. Often, their specificity (i.e. lack of crossreactivity with other KLKs or non-KLK pro- teins) has been debated. Using IgY technology, we devel- oped a new polyclonal antibody that displayed high specificity and sensitivity for KLK6 in Western blot and ELISA immunoassays. compared to KLK6 negative [9], and the combination of KLK6 and CA-125 enhances the diagnostic power [10]. Additionally, KLK6 has been found up-regulated both in tumor specimens and serum of patients with colon cancer and high KLK6 expression was associated with poor prog- nosis [11]. Elevated expression of KLK6 in gastric cancers as compared to noncancerous tissues was associated with lymphatic invasion [12]. Finally, KLK6 shows strong ex- pression in low grade in contrast to high grade renal cell carcinomas [13] and represents a potential serum bio- marker for uterine serous papillary cancer [14]. The role(s) of KLK6 in the progression of human malig- nancies are not clear and they may vary with the type of cancer and KLK6 levels of expression [15]. KLK6 has been shown to cleave components of the extracellular matrix (ECM), therefore it was concluded that KLK6 promotes cancer invasion and metastasis [16]. In addition, KLK6 was shown to release angiostatin for plasminogen, thus it may have anti-angiogenic potential [17]. In breast cancer expression of KLK6 at physiological levels has tumor- suppressor properties, while over-expression results in tumor promotion [18]. Overexpression of KLK6 in lung cancer is related to increase in cyclin E and increase in cell proliferation [19]. Antibody characterization previous IgY preparations was absent [28]. The yield of IgY production was 100 mg per single yolk, which is substantially higher than yields for IgG production. The generated chicken anti-KLK6 antibody (IgYs) was compared with a rabbit polyclonal against KLK6 that has been widely used for analyses of biological and clinical samples [5,21,29,30]. The sensitivity of IgYs and that of the widely used rabbit polyclonal were compared in par- allel by Western using serial dilutions of rKLK6. As shown in Figure 3A, the detection limit of IgYs was down to one ng or ~30 fmol of KLK6 compared to 300 fmol for the rabbit polyclonal. Production of anti-KLK6 IgYs Specifically, serum KLK6 was decreased in patients relative to adult population and the lowest concentrations were corre- lated with worse outcome [26]. The purity of the generated IgYs was very high and no contaminating proteins could be detected by SDS-PAGE resolution of IgYs (data not shown). The two detected bands of about ~67 kDa and 25 kDa correspond to the heavy and light chains of IgYs, respectively. The 35 kDa band of the vitellogenin II precursor observed in Hinge Antigen binding sites Fab Fc C 2 C 3 C 4 C 1 CH2 CH3 CH1 VH VH VL CL Figure 1 Structure of IgYs. Representation of Y and G antibodies [modified from 2]. Figure 1 Structure of IgYs. Representation of Y and G antibodies [modified from 2]. On the other hand, a large body of emerging evidence suggests that there is a functional interaction of KLKs, Page 3 of 8 Page 3 of 8 Page 3 of 8 Sotiropoulou et al. Chemistry Central Journal 2012, 6:148 http://journal.chemistrycentral.com/content/6/1/148 Titration and displacement ELISA The developed IgYs were initially evaluated by ELISA. As shown in Figure 2A, under the conditions used (details in Materials and Methods section), the titer of the obtained antibody was approximately 0.125 μg/ml (negative controls either no coating or without IgYs gave absorbance values between 0.402 and 0.509). More spe- cifically, the 0.125 μg/ml concentration of the antibody gave absorbance values >1.5-times the negative controls. Figure 2B shows the displacement curves obtained for the antibody. The addition of rKLK6 displaced bound IgYs in a concentration-dependent manner. Then, we tested the potential crossreactivity of the pro- duced IgYs with other recombinant KLKs that we had pro- duced [17]. Recombinant KLK5 and KLK13, which is the most closely related by sequence to KLK6 [31], were tested. As shown in Figure 3B, no crossreactivity could be detected even for high (1–1.5 μg) amounts of recombinant proteins. y = -0,2731x + 1,2498 R2 = 0,9798 0 0,2 0,4 0,6 0,8 1 1,2 1,4 1,6 1,8 2 -2,5 -2 -1,5 -1 -0,5 0 0,5 1 1,5 2 2,5 log Cst A405 0 0,5 1 1,5 2 2,5 3 0 0,2 0,4 0,6 0,8 1 1,2 A405 A B IgY , µg/ml Figure 2 Titration and displacement ELISAs. A, Antibody titer determination by ELISA. B, Displacement ELISA. 0 0,5 1 1,5 2 2,5 3 0 0,2 0,4 0,6 0,8 1 1,2 A405 A IgY µg/ml A y = -0,2731x + 1,2498 R2 = 0,9798 0 0,2 0,4 0,6 0,8 1 1,2 1,4 1,6 1,8 2 -2,5 -2 -1,5 -1 -0,5 0 0,5 1 1,5 2 2,5 log Cst A405 B IgY , µg/ml B y = -0,2731x + 1,2498 R2 = 0,9798 Figure 2 Titration and displacement ELISAs. A, Antibody titer determination by ELISA. B, Displacement ELISA Page 4 of 8 Sotiropoulou et al. Chemistry Central Journal 2012, 6:148 http://journal.chemistrycentral.com/content/6/1/148 IgYs Rabbit poly A C B rKLK6 rKLK5 rKLK13 D 31 kDa rKLK6 5 sec 10 sec 37 kDa 0 0.5 1 3 5 10 20 50 100 ng rKLK6 5 10 20 min 1 2 3 1 2 3 37 kDa Figure 3 Sensitivity and specificity of IgYs. Recombinant and endogenous KLK6 were detected by Western blotting using IgYs at 1:2,500. A, IgYs could detect as low as 1 ng (30 fmol) of rKLK6, while the widely used anti-KLK6 rabbit polyclonal [30] was limited to 10 ng (300 fmol). Titration and displacement ELISA A single band of 37 kDa corresponding to endogenous KLK6 could be detected in MDA-MB-468 and MDA-MB-231 C5WT, a cDNA-transfected clone to stably express KLK6 [18] (lanes 2 and 3 respectively; left). Moreover, lack of crossreactivity of IgYs with other cellular proteins was confirmed by analysis of whole cell lysates (100 μg) isolated from MDA-MB-231 C5WT (lane 1, right) and MDA-MB-468 (lane 3, right). Additionally, IgYs could recognize a mutant form of KLK6 produced by MDA-MB-231 C7MS transfectants (lane 2, right) that lacks enzymatic activity [18]. D, Heat-induced denaturation of rKLK6 increases the sensitivity of IgYs. 5, 10 and 20 min refer to times of incubation at 95°C, while 5 and 10 sec denote the time of exposure; the two bands correspond to glycosylated (31 kDa) and non-glycosylated (25 kDa) rKLK6 produced in Pichia pastoris. Figure 3 Sensitivity and specificity of IgYs. Recombinant and endogenous KLK6 were detected by Western blotting using IgYs at 1:2,500. A, IgYs could detect as low as 1 ng (30 fmol) of rKLK6, while the widely used anti-KLK6 rabbit polyclonal [30] was limited to 10 ng (300 fmol). B, IgYs could recognize rKLK6 (100 ng) but lacked crossreactivity with its most homologous KLK13 (rKLK13, 1.5 μg) but also KLK5 (rKLK5, 1 μg) (Western blot, upper). Integrity of the loaded proteins was confirmed by Coomassie staining (SDS-PAGE gel, lower). C, IgYs lack crossreactivity with any of the secreted proteins as no band could be detected in MDA-MB-231 cells that do not produce KLK6 (lane 1, left). A single band of 37 kDa corresponding to endogenous KLK6 could be detected in MDA-MB-468 and MDA-MB-231 C5WT, a cDNA-transfected clone to stably express KLK6 [18] (lanes 2 and 3 respectively; left). Moreover, lack of crossreactivity of IgYs with other cellular proteins was confirmed by analysis of whole cell lysates (100 μg) isolated from MDA-MB-231 C5WT (lane 1, right) and MDA-MB-468 (lane 3, right). Additionally, IgYs could recognize a mutant form of KLK6 produced by MDA-MB-231 C7MS transfectants (lane 2, right) that lacks enzymatic activity [18]. D, Heat-induced denaturation of rKLK6 increases the sensitivity of IgYs. 5, 10 and 20 min refer to times of incubation at 95°C, while 5 and 10 sec denote the time of exposure; the two bands correspond to glycosylated (31 kDa) and non-glycosylated (25 kDa) rKLK6 produced in Pichia pastoris. Titration and displacement ELISA B, IgYs could recognize rKLK6 (100 ng) but lacked crossreactivity with its most homologous KLK13 (rKLK13, 1.5 μg) but also KLK5 (rKLK5, 1 μg) (Western blot, upper). Integrity of the loaded proteins was confirmed by Coomassie staining (SDS-PAGE gel, lower). C, IgYs lack crossreactivity with any of the secreted proteins as no band could be detected in MDA-MB-231 cells that do not produce KLK6 (lane 1, left). A single band of 37 kDa corresponding to endogenous KLK6 could be detected in MDA-MB-468 and MDA-MB-231 C5WT, a cDNA-transfected clone to stably express KLK6 [18] (lanes 2 and 3 respectively; left). Moreover, lack of crossreactivity of IgYs with other cellular proteins was confirmed by analysis of whole cell lysates (100 μg) isolated from MDA-MB-231 C5WT (lane 1, right) and MDA-MB-468 (lane 3, right). Additionally, IgYs could recognize a mutant form of KLK6 produced by MDA-MB-231 C7MS transfectants (lane 2, right) that lacks enzymatic activity [18]. D, Heat-induced denaturation of rKLK6 increases the sensitivity of IgYs. 5, 10 and 20 min refer to times of incubation at 95°C, while 5 and 10 sec denote the time of exposure; the two bands correspond to glycosylated (31 kDa) and non-glycosylated (25 kDa) rKLK6 produced in Pichia pastoris. B rKLK6 rKLK5 rKLK13 31 kDa IgYs Rabbit poly A 0 0.5 1 3 5 10 20 50 100 ng rKLK6 B A C D 37 kDa 1 2 3 1 2 3 37 kDa 1 2 3 37 kDa C 37 kDa 1 2 3 D rKLK6 5 sec 10 sec 5 10 20 min D C Figure 3 Sensitivity and specificity of IgYs. Recombinant and endogenous KLK6 were detected by Western blotting using IgYs at 1:2,500. A, IgYs could detect as low as 1 ng (30 fmol) of rKLK6, while the widely used anti-KLK6 rabbit polyclonal [30] was limited to 10 ng (300 fmol). B, IgYs could recognize rKLK6 (100 ng) but lacked crossreactivity with its most homologous KLK13 (rKLK13, 1.5 μg) but also KLK5 (rKLK5, 1 μg) (Western blot, upper). Integrity of the loaded proteins was confirmed by Coomassie staining (SDS-PAGE gel, lower). C, IgYs lack crossreactivity with any of the secreted proteins as no band could be detected in MDA-MB-231 cells that do not produce KLK6 (lane 1, left). Titration and displacement ELISA In particular, sensitivity of IgYs for detection of endogenous KLK6 and lack of crossreactivity with other KLKs and unre- lated proteins was assessed by analyzing whole cell lysates and supernatants isolated from MDA-MB-468 breast can- cer cell line that expresses the KLK5, KLK6 and KLK10 proteins. The MDA-MB-231 KLK6-non-expressing cell line was used as a negative control for KLK6 but a positive con- trol for KLK1 [32]. In addition, cDNA-transfected MDA- MB-231 cells with stably reconstituted KLK6 expression were included in the analysis [18]. The IgYs could detect the endogenous (secreted and intracellular) KLK6 protein produced by MDA-MB-468 and MDA-MB-231 cells stably transfected with the KLK6 cDNA (Figure 3C) and no other proteins were detected indicating high specificity of IgYs for detection of endogenous KLK6 by Westerns. On the other hand, complete absence of crossreactivity of the anti- KLK6 IgYs with endogenous KLK5 and KLK10 or any other KLK-unrelated proteins could be demonstrated in lysates and supernatants of MDA-MB-468. Also, lack of crossreactivity with KLK1 was shown in MDA-MB-231 cells (Figure 3C). If KLK10 was detected in MDA-MB-468 it would have given a band that would not overlap with the KLK6 due to difference in molecular weight (30 kDa for KLK10 compared to 37 kDa for glycosylated KLK6). In addition, KLK6 protein was detected in differentiated SH- SY5Y neuroblastoma cells, while other bands derived from non-specific binding of IgYs could not be detected (Vekrellis, Sotiropoulou et al. unpublished data). Finally, we showed that denaturation of the KLK6 protein due to prolonged heating at 95°C enhanced the sensitivity of IgYs against KLK6 (Figure 3D). This obser- vation can be exploited in Westerns to reduce the detec- tion limit of IgYs below the 1 ng (30 fmol) KLK6 threshold. The generated IgYs were shown unable to immunoprecipi- tate KLK6 (not shown), which is common for Y antibodies due to their shorter hinge structure that usually renders them less effective in immune-precipitations as compared to G antibodies [1]. Effect of IgYs on KLK6 activity The activity was measured in the absence (A) and presence of IgYs at increasing molar ratios of IgYs:rKLK6 equal to 1:1, 20:1 and 30:1 (B, C and D, respectively). Then, the trypsin substrate N-benzoyl-L-arginine ethyl ester (BAEE) was added and the change in absorbance at A254 was measured relative to time for 3 min. The corresponding changes in the absorbance at A254 were normalized and expressed % of the rKLK6 without addition of the antibodies. IgYs could inhibit rKLK6 only moderately as 61% of its activity remained at a molar ratio as high as 30:1, while the E24 mouse monoclonal control could extinguish 60% of the activity at 1:1. Rates of hydrolysis of the BAEE substrate by rKLK6 are shown. BAEE was used at 250 μΜ and rKLK6 at 12 nM. specifically KLK5 and KLK13 and endogenous KLK1 and KLK10, but also with any other cellular proteins produced by mammary (and neuronal) cells, likely due to the evolu- tionary distance between avian and human species. Lack of specificity of anti-KLK antibodies, especially their cross- reactivity with multiple KLKs, has been an obstacle to the optimization of immunoassays for clinical applications. Even the extensively used and characterized anti-KLK3 (PSA) antibodies (rabbit polyclonal and mouse monoclo- nal antibodies) were shown to crossreact with KLK2 and KLK1 [34]. Moreover, our IgY production offers the advantages of easy scale-up to gram levels and simple purification process. Generation of 100 mg of IgGs that can be obtained from a single yolk, would require approxi- mately 20 ml of rabbit antiserum [28]. inhibition of KLK6 proteolytic activity was observed at molar ratios IgYs:KLK6 as high as 30:1, indicative of a small fraction of IgYs recognizing three-dimensional epi- topes. Thus, it may still be possible to raise antibodies with enzyme-inhibitory activity in avian species. The well-described E24 mouse monoclonal that blocks the enzymatic activity of KLK6 [16] was used as positive control, which at molar ratio 1:1 caused about 60% in- hibition of KLK6 activity. Allowing KLK6-IgYs complex formation for longer time (10 min) did not increase fur- ther the extent of inhibition (not shown). Effect of IgYs on KLK6 activity Effect of IgYs on KLK6 activity IgYs were tested for inhibitory ability against the enzym- atic activity of KLK6. As shown in Figure 4, partial Sotiropoulou et al. Chemistry Central Journal 2012, 6:148 http://journal.chemistrycentral.com/content/6/1/148 Sotiropoulou et al. Chemistry Central Journal 2012, 6:148 http://journal.chemistrycentral.com/content/6/1/148 Page 5 of 8 100 50 0 0.5 1.0 1.5 2.0 2.5 3.0 A B C E24 D Relative Absorbance Time, min 75 25 0 Figure 4 Inhibition of KLK6 activity by IgYs. The activity was measured in the absence (A) and presence of IgYs at increasing molar ratios of IgYs:rKLK6 equal to 1:1, 20:1 and 30:1 (B, C and D, respectively). Then, the trypsin substrate N-benzoyl-L-arginine ethyl ester (BAEE) was added and the change in absorbance at A254 was measured relative to time for 3 min. The corresponding changes in the absorbance at A254 were normalized and expressed % of the rKLK6 without addition of the antibodies. IgYs could inhibit rKLK6 only moderately as 61% of its activity remained at a molar ratio as high as 30:1, while the E24 mouse monoclonal control could extinguish 60% of the activity at 1:1. Rates of hydrolysis of the BAEE substrate by rKLK6 are shown. BAEE was used at 250 μΜ and rKLK6 at 12 nM. 100 50 0 0.5 1.0 1.5 2.0 2.5 3.0 A B C E24 D Relative Absorbance Time, min 75 25 0 Figure 4 Inhibition of KLK6 activity by IgYs. The activity was measured in the absence (A) and presence of IgYs at increasing molar ratios of IgYs:rKLK6 equal to 1:1, 20:1 and 30:1 (B, C and D, respectively). Then, the trypsin substrate N-benzoyl-L-arginine ethyl ester (BAEE) was added and the change in absorbance at A254 was measured relative to time for 3 min. The corresponding changes in the absorbance at A254 were normalized and expressed % of the rKLK6 without addition of the antibodies. IgYs could inhibit rKLK6 only moderately as 61% of its activity remained at a molar ratio as high as 30:1, while the E24 mouse monoclonal control could extinguish 60% of the activity at 1:1. Rates of hydrolysis of the BAEE substrate by rKLK6 are shown. BAEE was used at 250 μΜ and rKLK6 at 12 nM. Figure 4 Inhibition of KLK6 activity by IgYs. Discussion Despite the fact that IgYs were described about 120 year ago their applications in immunoassays have been lim- ited compared to IgGs [1]. In the recent years however, the development of chicken antibodies is considered an advantageous alternative compared to the classical mam- malian immunization procedures, mainly because mam- malian proteins exhibit enhanced immunogenicity in avian species due to phylogenetic distance. Recently, the so-called IgY technology was suggested as a potential new way for passive immunization to treat human and animal diseases [33]. Recently, an anti-peptide rabbit polyclonal was generated against the KLK6 (109–119) region that lacks homology with other KLKs, thus, the affinity-purified polyclonal dis- played high specificity against KLK6 [8]. To the advantages of our production method, high yield and no need for af- finity purification should be added. Availability of adequate amounts should facilitate pharmacological validation of IgYs for potential therapeutic applications. Indeed, anti- KLK6 therapy was effective in mouse models of multiple sclerosis [23-25]. However, our antibody has certain limita- tions in its applications. Specifically, we demonstrated that the developed antibody cannot be used for immunopreci- pitations which is a more general drawback of Y antibodies. Further, it cannot be used as a potent inhibitor of KLK6 protease activity since it shows moderate inhibition and only in high molecular ratios. We applied IgY technology to high-yield generation of Y antibodies against rKLK6 that were purified to near homo- geneity with a very simple and inexpensive procedure and characterized in established immunochemical methods for future validation in clinical diagnosis. The utility of our anti-KLK6 Y antibodies lies in their absence of immuno- logical crossreactivity with enzymes of the KLK family, Page 6 of 8 Sotiropoulou et al. Chemistry Central Journal 2012, 6:148 http://journal.chemistrycentral.com/content/6/1/148 Sotiropoulou et al. Chemistry Central Journal 2012, 6:148 http://journal.chemistrycentral.com/content/6/1/148 Page 6 of 8 Overall, the IgY technology will help to reduce the cost of clinical or research immunochemical tests. Further it reduces the number of animals used since a single hen can produce eggs having the desired IgYs for at least 10 months leading to the production of very high amounts of antibodies [2]. Finally, it meets the recom- mendations of the European Centre for the Validation of Alternative Methods (ECVAM) that specifies that IgYs are suggested to be used instead of mammalian anti- bodies for animal welfare purposes [35]. Conclusions This study generated a novel chicken polyclonal antibody against KLK6. KLK6 is an emerging new biochemical mar- ker for clinical diagnosis of various forms of cancer, in- cluding ovarian cancer and for neurodegerative disorders. The developed antibody exhibited sensitivity in the subpi- comolar range and very importantly it lacked crossreactiv- ity with other KLK enzymes or cellular proteins. Western blot Samples were resolved on 12% SDS-PAGE and transferred onto PVDF membranes. Membranes were blocked with 5% milk in PBS. IgYs (or rabbit IgGs) was added to 1:2,500 di- lution in 1% milk in PBS containing 0.05% Tween (PBST) for 1 h at room temperature. Then, membranes were washed with PBST and anti-IgY (Sigma) was added to 1:3,000 dilution in 1% milk in PBST for 1 h at room temperature. Specific immunoreactive bands were detected with West Pico ECL (Pierce). For immunoprecipitation, rKLK6 (2 μg of rKLK6 in 800 μl PBS) was pre-incubated with 20 μg of IgYs for 1 h at room temperature, then, a rabbit anti-IgY polyclonal antibody (10 μg) was added and the mixture incubated for another 1 h at room temperature. Finally, 50 μl of protein G beads (1:1) were added and incu- bated for 16 h at 4°C. Beads were recovered by centrifuga- tion, proteins eluted in sample buffer and analyzed by Western. et ods Materials All chemicals used were obtained from Sigma (St. Louis, MO, USA) or Merck (Darmstadt, Germany). Isolation of IgYs IgYs were isolated from egg yolk according to a modified version of the acidified water dilution method [28,38]. Briefly, yolks from 15 eggs were separated from white, washed with distilled water and allowed to drip through pharmaceutical gauze into a beaker. Yolk was diluted ten times (v/v) with acidified water (pH 5.2), the mixture was remained 16 h at 4°C and, then, centrifuged at 8,500xg for 30 min at 4°C. Lipid-containing residue was discarded and supernatant was collected. Na2SO4 was added to the supernatant up to 19% concentration and the mixture remained at 37°C for 3 h and, then, at room temperature for 16 h. Subsequently, the mixture was centrifuged at 8,500xg for 30 min at 25°C and the precipitant containing IgYs (100 mg/egg determined by Bradford assay) was collected, dialyzed against water (72 h, 4°C, MWCO of 12 kDa) and lyophilized (100 mg/ egg determined by Bradford assay). 3 mg of IgYs were dissolved in 1 ml PBS and stored in aliquots at −20°C. Discussion To our know- ledge this is the first study that develops a chicken antibody against KLK6 and in general against a member of the human KLK family of serine proteases. Hen immunization Laying hens (Leghorn hybrids) 3-month old were immu- nized by subcutaneous injections on the neck. The immunogen, rKLK6, was injected (100 μg per injection) as an emulsion (1:1 v/v) in Complete Freund’s Adjuvant. rKLK6 encompasses the aminoacid residues 22–244 of the Uniprot sequence Q92876, thus corresponds to the full-length KLK6 protein. Booster injections were admi- nistered every 2 weeks for a total of 3 months. Care of animals was in accordance with European legislation and our Institution’s Guidelines pertaining to the use of laboratory animals. Production of recombinant KLKs Recombinant KLK6 (rKLK6) and other recombinant KLKs were produced in the methylotrophic yeast Pichia pastoris KM71 strain, purified to homogeneity and activity-tested as described [15,36,37]. The identity of the proteins was verified by N-terminal analysis by Edman degradation and/or MALDI-MS or ESI-MS, while the absence of un- desired mutations was also confirmed by sequencing the cloned KLK cDNA fragment before yeast transformation [17,36,37]. Mature rKLK6 was used for hen immunization. Cell culture and preparation of cell lysates and supernatants All cell lines were grown as described [18]. For prepar- ation of cell lysates, cells were lysed in 50 mM Tris– HCl, pH 8.0, 150 mM NaCl, 1% Igepal CA-630 for 30 min on ice and lysates were clarified by centrifugation at 16,000xg for 10 min and used immediately for ana- lysis. Serum-free conditioned media (SFCM) were col- lected from confluent cultures at 24 h, clarified by centrifugation and concentrated by 10-fold using centri- fugal filter devices (Amicon, MWCO of 10 kDa). Titration ELISA ELISA microtiter plates were coated with rKLK6 (100 ng/ ml, 100 μl/microwell, 16 h at 37°C), then, washed and Page 7 of 8 Page 7 of 8 Sotiropoulou et al. Chemistry Central Journal 2012, 6:148 http://journal.chemistrycentral.com/content/6/1/148 Sotiropoulou et al. Chemistry Central Journal 2012, 6:148 http://journal.chemistrycentral.com/content/6/1/148 Chemistry Laboratory, National Centre for Scientific Research (NCSR) “Demokritos”, 153 10 Aghia Paraskevi-Attiki, Greece. blocked with 2% BSA in PBST (200 μl/well, 1 h at room temperature). Blocking was discarded and wells were washed with PBST and incubated with serial dilutions of IgYs in PBST containing 0.2% BSA (100 μl/well, 2 h at 37°C). Following, wells were washed with PBST and incubated with rabbit anti-IgY secondary antibody coupled to horseradish peroxidase (HRP) at 1:5,000 di- lution in PBST with 0.2% BSA (100 μl/well, 2 h at 37°C). Finally, wells were washed with PBST, incubated with ABTS/H2O2 (100 μl/well, 30 min at room temperature) and the absorbance was measured at 405 nm. Abbreviations 0 13. Gabril M, White NM, Moussa M, Chow TF, Metias SM, Fatoohi E, Yousef GM: Immunohistochemical analysis of kallikrein-related peptidases in the normal kidney and renal tumors: potential clinical implications. Biol Chem 2010, 391:403–409. ABTS: 2,20-azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid) diammonium salt; BAEE: N-benzoyl-L-arginine ethyl ester; BSA: Bovine serum albumin; IgY: Immunoglobulin Y; KLK6: Kallikrein-related peptidase 6; MWCO: Molecular weight - cut off; PBST: 0.01 M phosphate buffered saline, pH 7.4, containing 0.05%, v/v, Tween-20; SFCM: Serum-free conditioned media. 14. Santin AD, Diamandis EP, Bellone S, Soosaipillai A, Cane S, Palmieri M, Burnett A, Roman JJ, Pecorelli S: Human kallikrein 6: a new potential serum biomarker for uterine serous papillary cancer. Clin Cancer Res 2005, 11:3320–3325. References 1. Zhang WW: The use of gene-specific IgY antibodies for drug target discovery. Drug Discov Today 2003, 8:364–371. 2. Dias da Silva W, Tambourgi DV: IgY: a promising antibody for use in immunodiagnostic and immunotherapy. Vet Immunol Immunopathol 2010, 135:173–180. 3. Leslie GA, Clem LW: Phylogeny of immunoglobulin structure and function. 3. Immunoglobulins of the chicken. J Exp Med 1969, 130:1337–1352. 4. Anisowicz A, Sotiropoulou G, Stenman G, Mok SC, Sager R: A novel protease homolog differentially expressed in breast and ovarian cancer. Mol Med 1996, 2:624–636. Displacement ELISA Microtiter plates were coated and blocked as described above. Then, microwells were incubated with varying concentrations of anti-KLK6 IgYs concomitantly with a series of KLK6 standard solutions. After incubation the plates were processed as described for titration ELISA. 5. Petraki CD, Karavana VN, Skoufogiannis PT, Little SP, Howarth DJ, Yousef GM, Diamandis EP: The spectrum of human kallikrein 6 (zyme/protease M/neurosin) expression in human tissues as assessed by immunohistochemistry. J Histochem Cytochem 2001, 49:1431–1441. 5. Petraki CD, Karavana VN, Skoufogiannis PT, Little SP, Howarth DJ, Yousef GM, Diamandis EP: The spectrum of human kallikrein 6 (zyme/protease M/neurosin) expression in human tissues as assessed by immunohistochemistry. J Histochem Cytochem 2001, 49:1431–1441. 6. Pampalakis G, Kurlender L, Diamandis EP, Sotiropoulou G: Cloning and characterization of novel isoforms of the human kallikrein 6 gene. Biochem Biophys Res Commun 2004, 320:54–61. 6. Pampalakis G, Kurlender L, Diamandis EP, Sotiropoulou G: Cloning and characterization of novel isoforms of the human kallikrein 6 gene. Biochem Biophys Res Commun 2004, 320:54–61. Acknowledgements We would like to thank Prof Eleftherios P. Diamandis (Mount Sinai Hospital, and University of Toronto, Toronto, Ontario, Canada) for providing the E24 monoclonal and rabbit polyclonal antibodies, Osahon Obasuyi for performing the Western in Figure 3A, and Georgia Arvaniti for assistance in the designing of the graphical abstract. 18. Pampalakis G, Prosnikli E, Agalioti T, Vlahou A, Zoumpourlis V, Sotiropoulou G: A tumor protective role for human kallikrein-related peptidase 6 in breast cancer mediated by inhibition of epithelial-to-mesenchymal transition. Cancer Res 2009, 69:3779–3787. 19. Nathalie HV, Chris P, Serge G, Catherine C, Benjamine B, Claire B, Christelle P, Briollais L, Pascale R, Marie-Lise J, Yves C: High kallikrein-related peptidase 6 in non-small cell lung cancer cells: an indicator of tumour proliferation and poor prognosis. J Cell Mol Med 2009, 13:4014–4022. Enzyme kinetics p y 7. Yousef GM, Borgoño CA, White NM, Robb JD, Michael IP, Oikonomopoulou K, Khan S, Diamandis EP: In silico analysis of the human kallikrein gene 6. Tumour Biol 2004, 25:282–289. p y 7. Yousef GM, Borgoño CA, White NM, Robb JD, Michael IP, Oikonomopoulou K, Khan S, Diamandis EP: In silico analysis of the human kallikrein gene 6. Tumour Biol 2004, 25:282–289. The enzymatic activity of rKLK6 [37] was measured using the N-benzoyl-L-arginine ethyl ester (BAEE) sub- strate in 67 mM Na2HPO4, pH 7.6. For KLK6 enzyme kinetics, rKLK6 (R80Q) was used, a mutant form of KLK6 with stabilized activity compared to wild-type that is amenable to autocatalytic inactivation via cleavage at R80 [37]. Rates of hydrolysis were measured based on changes in the absorbance at 254 nm monitored by a double beam UV–vis spectrophotometer (Perkin Elmer). As control for specific inhibition of KLK6 activity, the E24 anti-KLK6 blocking mouse monoclonal was used [16]. For inhibition assays, the antibodies were pre-mixed with rKLK6 and incubated for 1 min, then, substrate was added and the rate of hydrolysis was monitored. 8. Seiz L, Dorn J, Kotzsch M, Walch A, Grebenchtchikov NI, Gkazepis A, Schmalfeldt B, Kiechle M, Bayani J, Diamandis EP, Langer R, Sweep FC, Schmitt M, Magdolen V: Stromal cell-associated expression of kallikrein- related peptidase 6 (KLK6) indicates poor prognosis of ovarian cancer patients. Biol Chem 2012, 393:391–401. p 9. Shan SJ, Scorilas A, Katsaros D, Diamandis EP: Transcriptional upregulation of human tissue kallikrein 6 in ovarian cancer: clinical and mechanistic aspects. Br J Cancer 2007, 96:362–372. 10. Rosen DG, Wang L, Atkinson JN, Yu Y, Lu KH, Diamandis EP, Hellstrom I, Mok SC, Liu J, Bast RC: Potential markers that complement expression of CA125 in epithelial ovarian cancer. Gynecol Oncol 2005, 99:267–277. 11. Kim JT, Song EY, Chung KS, Kang MA, Kim JW, Kima SJ, Yeom YI, Kim JH, Kim KH, Lee HG: Up-regulation and clinical significance of serine protease kallikrein 6 in colon cancer. Cancer 2011, 117:2608–2619. 12. Nagahara H, Mimori K, Utsunomiya T, Barnard GF, Ohira M, Hirakawa K, Mori M: Clinicopathologic and biological significance of kallikrein 6 overexpression in human gastric cancer. Clin Cancer Res 2005, 11:6800–6806. Received: 11 October 2012 Accepted: 19 November 2012 Published: 5 December 2012 Received: 11 October 2012 Accepted: 19 November 2012 Published: 5 December 2012 Competing interests The authors declare that they have no competing interests. 15. Sotiropoulou G, Pampalakis G, Diamandis EP: Functional roles of human kallikrein-related peptidass. J Biol Chem 2009, 284:32989–32994. Authors’ contributions 16. Ghosh MC, Grass L, Soosaipillai A, Sotiropoulou G, Diamandis EP: Human kallikrein 6 degrades extracellular matrix proteins and may enhance the metastatic potential of tumour cells. Tumour Biol 2004, 25:193–199. GS conceived, and designed the project, and drafted the manuscript. GP performed experiments, drafting the manuscript. EP, GE, EL performed experiments. All authors have read and approved the final manuscript. 17. Sotiropoulou G, Rogakos V, Tsetsenis T, Pampalakis G, Zafiropoulos N, Simillides G, Yiotakis A, Diamandis EP: Emerging interest in the kallikrein gene family for understanding and diagnosing cancer. Oncol Res 2003, 13:381–391. Sotiropoulou et al. Chemistry Central Journal 2012, 6:148 http://journal.chemistrycentral.com/content/6/1/148 20. Menendez-Gonzalez M, Castro-Santos P, Suarez A, Calatayud MT, Perez- Pinera P, Martinez M, Ribacoba R, Gutierrez C: Value of measuring plasmatic levels of neurosin in the diagnosis of Alzheimer’s disease. J Alzheimers Dis 2008, 14:59–67. 21. Diamandis EP, Yousef GM, Petraki C, Soosaipillai AR: Human kallikrein 6 as a biomarker of alzheimer’s disease. Clin Biochem 2000, 33:663–667. 22. Scarisbrick IA, Linbo R, Vandell AG, Keegan M, Blaber SI, Blaber M, Sneve D, Lucchinetti CF, Rodriguez M, Diamandis EP: Kallikreins are associated with secondary progressive multiple sclerosis and promote neurodegeneration. Biol Chem 2008, 389:739–745. g 23. Scarisbrick IA, Yoon H, Panos M, Larson N, Blaber SI, Blaber M, Rodriguez M: Kallikrein 6 regulates early CNS demyelination in a viral model of multiple sclerosis. Brain Pathol 2012, 22:709–722. 24. Bando Y, Ito S, Nagai Y, Terayama R, Kishibe M, Jiang YP, Mitrovic B, Takahashi T, Yoshida S: Implications of protease M/neurosin in myelination during experimental demyelination and remyelination. Neurosci Lett 2006, 405:175–180. 25. Blaber SI, Ciric B, Christophi GP, Bernett MJ, Blaber M, Rodriguez M, Scarisbrick IA: Targeting kallikrein 6 proteolysis attenuates CNS inflammatory disease. FASEB J 2004, 18:920–922. inflammatory disease. FASEB J 2004, 18:920–922. 26. Martínez-Morillo E, Diamandis A, Romaschin AD, Diamandis EP: Kallikrein 6 as a serum prognostic marker in patients with aneurysmal subarachnoid hemorrhage. PLoS One 2012, 7:e45676. 27. Blaber M, Yoon H, Juliano MA, Scarisbrick IA, Blaber SI: Functional intersection of the kallikrein-related peptidases (KLKs) and thrombostasis axis. Biol Chem 2010, 391:311–320. 28. Klimentzou P, Paravatou-Petsotas M, Zikos C, Beck A, Skopeliti M, Czarnecki 28. Klimentzou P, Paravatou-Petsotas M, Zikos C, Beck A, Skopeliti M, Czarnecki J, Tsitsilonis O: Development and immunochemical evaluation of antibodies Y for the poorly immunogenic polypeptide prothymosin alpha. Peptides 2006, 27:183–193. 29. Strojnik T, Kavalar R, Zajc I, Diamandis EP, Oikonomopoulou K, Lah TT: Prognostic impact of CD68 and kallikrein 6 in human glioma. Anticancer Res 2009, 29:3269–3279. 30. Diamandis EP, Yousef GM, Soosaipillai AR, Grass L, Porter A, Little S, Sotiropoulou G: Immunofluorometric assay of human kallikrein 6 (zyme/ protease M/neurosin) and preliminary clinical applications. Clin Biochem 2000, 33:369–375. 31. Pavlopoulou A, Pampalakis G, Michalopoulos I, Sotiropoulou G: Evolution history of tissue kallikreins. PLoS One 2010, 5:e13781. 32. Wolf WC, Evans DM, Chao L, Chao J: A synthetic tissue kallikrein inhibitor suppresses cancer cell invasiveness. Am J Pathol 2001, 159:1797–1805. 33. Kovacs-Nolan J, Mine Y: Egg yolk antibodies for passive immunity. Author details 1D f 1Department of Pharmacy, University of Patras, 265 00 Rion-Patras, Greece. 2Institute of Radioisotopes & Radiodiagnostic Products, Immunopeptide Page 8 of 8 Page 8 of 8 Sotiropoulou et al. Chemistry Central Journal 2012, 6:148 http://journal.chemistrycentral.com/content/6/1/148 Open access provides opportunities to our colleagues in other parts of the globe, by allowing anyone to view the content free of charge. Publish with ChemistryCentral and every scientist can read your work free of charge W. Jeffery Hurst, The Hershey Company. available free of charge to the entire scientific community peer reviewed and published immediately upon acceptance cited in PubMed and archived on PubMed Central yours you keep the copyright Submit your manuscript here: http://www.chemistrycentral.com/manuscript/ Sotiropoulou et al. Chemistry Central Journal 2012, 6:148 http://journal.chemistrycentral.com/content/6/1/148 Annu Rev Food Sci Technol 2012, 3:163–182. 34. Finlay JA, Day JR, Rittenhouse HG: Polyclonal and monoclonal antibodies to prostate-specific antigen can crossreact with human kallikrein 2 and human kallikrein 1. Urology 1999, 53:746–751. 35. Schade R, Calzado EG, Sarmiento R, Chacana PA, Porankiewicz-Asplund J, Terzolo HR: Chicken egg yold antibodies (IgY-technology). A review of progress in production and use in research and human and veterinary medicine. Altern Lab Anim 2005, 33:129–154. 36. Michael IP, Sotiropoulou G, Pampalakis G, Magklara A, Ghosh M, Wasney G, Diamandis EP: Biochemical and enzymatic characterization of human kallikrein 5 (hK5), a novel serine protease potentially involved in cancer progression. J Biol Chem 2005, 280:14628–14635. 37. Bayés A, Tsetsenis T, Ventura S, Vendrell J, Aviles FX, Sotiropoulou G: Human kallikrein 6 activity is regulated via an autoproteolytic mechanism of activation/inactivation. Biol Chem 2004, 385:517–524. Open access provides opportunities to our colleagues in other parts of the globe, by allowing anyone to view the content free of charge. Publish with ChemistryCentral and every scientist can read your work free of charge W. Jeffery Hurst, The Hershey Company. available free of charge to the entire scientific community peer reviewed and published immediately upon acceptance cited in PubMed and archived on PubMed Central yours you keep the copyright Submit your manuscript here: http://www.chemistrycentral.com/manuscript/ 38. Akita EM, Nakai S: Immunoglobulins form egg yolk: isolation and purification. J Food Sci 1992, 57:629–634. doi:10.1186/1752-153X-6-148 Cite this article as: Sotiropoulou et al.: Development and immunochemical evaluation of a novel chicken IgY antibody specific for KLK6. Chemistry Central Journal 2012 6:148. available free of charge to the entire scientific community peer reviewed and published immediately upon acceptance cited in PubMed and archived on PubMed Central yours you keep the copyright al al
https://openalex.org/W3117374183	https://europepmc.org/articles/pmc7790564?pdf=render	English	null	Comprehensive Management of Ectodermal Dysplasia with Interceptive Orthodontics in a Young Boy Who Was Bullied at School	Case Reports in Dentistry/Case reports in dentistry	2,020	cc-by	3,613	Hindawi Case Reports in Dentistry Volume 2020, Article ID 6691235, 7 pages https://doi.org/10.1155/2020/6691235 Hindawi Case Reports in Dentistry Volume 2020, Article ID 6691235, 7 pages https://doi.org/10.1155/2020/6691235 Hindawi Case Reports in Dentistry Volume 2020, Article ID 6691235, 7 pages https://doi.org/10.1155/2020/6691235 A. A. A. K. Wimalarathna ,1 W. B. M. C. R. D. Weerasekara ,2 and E. M. U. C. K. Herath 2 1Department of Prosthetic Dentistry, Faculty of Dental Sciences, University of Peradeniya, Sri Lanka 2Department of Community Dental Health, Faculty of Dental Sciences, University of Peradeniya, Sri Lanka A. A. A. K. Wimalarathna ,1 W. B. M. C. R. D. Weerasekara ,2 and E. M. U. C. K. Herath 2 1Department of Prosthetic Dentistry, Faculty of Dental Sciences, University of Peradeniya, Sri Lanka 2Department of Community Dental Health, Faculty of Dental Sciences, University of Peradeniya, Sri Lanka Correspondence should be addressed to A. A. A. K. Wimalarathna; arunaw@dental.pdn.ac.lk eceived 4 November 2020; Revised 7 December 2020; Accepted 15 December 2020; Published 31 December 202 Academic Editor: Leandro Napier de Souza Copyright © 2020 A. A. A. K. Wimalarathna 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. Aim. The management of hypohidrotic ectodermal dysplasia with oligodontia in Class-I malocclusion in late mix dentition. Case Report. An 11-year-old boy with ED was treated and managed by means of interceptive orthodontic treatment accompanied by direct and indirect restorative methods in a successful manner. The patient was prepared to receive deﬁnitive oral rehabilitation with dental implants for the missing teeth when the patient reaches a suitable age. The patient was followed for 5 years from the beginning of treatment. Conclusion. Management of the child with ectodermal dysplasia with oligodontia was a real challenge. Early diagnosis, necessary investigation, and providing age-appropriate multidisciplinary treatment were key steps in successful outcomes. The objectives were not only just orthodontic or paedodontics but also prosthetic and psychological. 1. Introduction with an incidence of 1-7 per 100,000 live births, with 28% of mortality rate in males up to 3 years of age [3]. The term ectodermal dysplasia (ED) is described as a rare heterogeneous group of congenital disorders which is charac- terized by defects of two or more ectodermaly derived tissues [1]. These are primarily the nails, skin, hair, sweat glands, and teeth [2]. Though only eight forms of ED were identiﬁed by 1971, currently, it comprises more than 200 diﬀerent types; about 30% have been identiﬁed at the molecular level with identiﬁcation of the causative genes [3]. Patients with ED can easily be recognized from normal individuals by their typical facial and clinical features such as frontal bossing, prominent supraorbital ridges, sunken cheeks, wrinkles and periorbital hyperpigmentation, saddle nose, everted lips, large low-set ears, dysplastic nails, and reduced lower facial height [2]. Occasionally some of them have asthma and eczema [5]. Dental manifestations include multiple congenitally missing teeth, conical- or pegged-shaped anterior teeth, ankylosed primary molars due to missing permanents, delayed eruption of permanent teeth, hypoplastic alveoli, xer- ostomia and thickened frena, macrodontia or microdontia, enamel hypoplasia, and taurodontism [2]. Normally, chil- dren with ED have normal psychological and social develop- ment [6]. According to the mode of inheritance, ED can be classi- ﬁed into autosomal dominant, autosomal recessive, or X- linked. Hypodontia, hypohidrosis, and hypotrichosis are a triad of clinical features characteristic of the hypohidrotic form [4]. Based on the function and number of sweat glands, ED can be broadly divided into two clinically distinguished forms: ﬁrst, autosomal dominant hidrotic type (Clouston’s syndrome) with normal sweat glands, and second, the X- linked recessive anhidrotic or hypohidrotic type (Christ-Sie- mens-Touraine Syndrome) with absent or diminished sweat glands. The X-linked trait is the most common phenotype Considering oral aesthetics, rehabilitation of a patient with ED has a big challenge due to missing teeth. According to the number of missing teeth, it can be classiﬁed into hypo- dontia (missing less than six teeth), oligodontia (six or more 2 Case Reports in Dentistry Figure 1: The genealogy of the presenting patient. missing teeth), and anodontia (completely absence of teeth) [7]. The problems associated with missing teeth are directly proportionate to the number of missing teeth in both decid- uous and permanent dentition. The most rarely missing teeth are maxillary central incisors, maxillary canines, and maxil- lary ﬁrst molars [8]. 1. Introduction Mostly, the presenting teeth are conical in shape, malformed, with restricted alveolar bone develop- ment, and often showing unaesthetic spacing. Figure 1: The genealogy of the presenting patient. A multidisciplinary approach is now recommended for managing ED patients. Early and extensive dental treatment is needed throughout childhood because of the absence of most of the deciduous and permanent teeth, and they are still in the developing stage. Therefore, the management basically consists of prevention of caries, restoration of malformed teeth, replacement of missing teeth, management of infraoc- clusion, and correction of malocclusions [7]. Figure 2: Facial image of the patient. Therefore, in many cases of hypodontia, orthodontic treatment can greatly facilitate any restorative treatment or sometimes even eliminate the need for it [9]. Carrying out orthodontic treatment in order to eliminate or to simplify later orthodontic treatment in young patients is called “inter- ceptive orthodontics” (IO) [10]. The main aims of IO treat- ments in hypodontia are management of available space, uprighting and alignment of teeth, and management of increased overbite, as well as interdisciplinary inputs of hypodontia management [9]. Figure 2: Facial image of the patient. The presenting case shows the total care management of a paediatric patient with ED which is associated with oligo- dontia was managed by the means of interceptive orthodon- tic treatment accompanied by direct and indirect restorative methods in a successful manner. mobility. The lower permanent incisors (32 and 42) were conical in shape (Figure 3). All four deciduous canines were slightly overretained with the canine bulges evident in relation to 13 and 43, whereas 33 had already erupted. There was a restoration on 55 with secondary caries. With mild attrited facets on 85, the occlusal surface showed slight mobility. 65 and 75 had been extracted due to caries, and 37 was erupting (Figures 3). His dmft index was 4. He had a Class-I skeletal pattern, with average “Frankfort mandibular plane angle” associated with incisor Class-I and bilateral molar Class-II 1/2 unit relationship. Lips were competent and everted. There was evidence of normal tongue and adaptive tongue thrust during the activity. 2. Case Report An 11-year-old schoolboy presented to the paedodontic clinic with the main complaint of having abnormal and uneven teeth and requesting treatments. The parents had noticed that the permanent teeth were abnormal in shape and malaligned. Most importantly, when the child was inquired about his concern, he said that his biggest problem was being teased and bullied by his peers at school. There was no history of trauma related to the teeth. The child has had a history of asthma but is otherwise healthy. He had been brushing his teeth with ﬂuoridated toothpaste twice daily, using a toothbrush. The child’s dietary habits did not show a high consumption of sugar. There was no his- tory of parafunctional habits. Parents have no consanguine- ous marriage (Figure 1). The radiographic investigation, sensibility tests, and basic periodontal examination (BPE) were performed to identify missing permanent teeth, pulp status, and periodontal health, respectively. All the teeth gave a vital response, and the pla- que score was 29%. There was no evidence of deep pockets more than 3 mm. The radiological ﬁndings revealed 6 miss- ing teeth: 12, 15, 22, 23, 31, and 41 while 25 was impacted due to space loss. There were anterior conical teeth and over- retained 81 with resorbing root and taurodontic molar teeth (Figure 4). On examination, it was observed that the overall develop- ment was appropriate for his chronological age. On extraoral examination, he was noticed to have ﬁne sparse hair, scanty eyelashes and eyebrows, protruding lips with reduced lower facial height, periorbital pigmentation, and protruding low set of ears (Figure 2). His palms and soles showed hyperkeratosis. With the information gathered from the history, exami- nation, and investigations, this case was diagnosed as “a patient with hypohidrotic-type ectodermal dysplasia with oligodontia in Class-I malocclusion in late mix dentition on skeletal base Class-I.”. Intraoral examination showed normal soft tissues and no signs of periodontal disease. The lower anterior teeth were stained and showed crowding with overretained 81 and 62. There was a superﬁcial dentinal caries on 73 with slight Written consent was obtained from the parents after explaining the proposed treatment plan. At the initial stage, 3 Case Reports in Dentistry 3 (a) (b) (c) (d) (e) Figure 3: Preoperative intraoral images of (a) right buccal, (b) closed mouth, (c) left buccal, (d) lower arch, and (e) upper arch views. 2. Case Report (b) (a) (a) (b) (d) (c) (c) (d) (e) (e) Figure 3: Preoperative intraoral images of (a) right buccal, (b) closed mouth, (c) left buccal, (d) lower arch, a ive intraoral images of (a) right buccal, (b) closed mouth, (c) left buccal, (d) lower arch, and (e) upper arch view Figure 4: Preoperative photoimage of dental panoramic tomograph. Figure 4: Preoperative photoimage of dental panoramic tomograph. the routine preventive measures like the use of ﬂuoridated toothpaste and correct brush instructions were introduced after plaque demonstration. At the second visit, dietary mod- iﬁcation and counselling were done. Fluoride varnish was introduced for professional application. Further CPP-ACP containing GC tooth mousse plus was introduced to use at home on a daily basis. separators to facilitate molar bands and stainless steel crown on 55 in the next visit. At the fourth visit, ﬁssure sealants on permanent molars and cementation of a stainless steel crown for 55 were done using luting GIC. The molar bands were cemented to all ﬁrst molars (Figure 5). During that period, the permanent canines erupted as expected after extraction of deciduous canines; the patient was reassessed at the ortho-paedodo joint clinic, and the fol- lowing management steps were planned: derotation of 13 and 24, surgical exposure of 25, extraction of 81, uprighting and distalization of distally tilted 72, distalization of 26 to During the third visit, the child demonstrated a good relationship with the dental team and seemed to have adapted well to the clinical environment. According to the treatment plan, 53, 73, and 83 were extracted after placing 4 Case Reports in Dentistry Figure 5: Intraoral photograph shows cemented crown and molar bands. Figure 7: Intraoral photograph showing cemented palatal button on rotated 13 and 24. Figure 7: Intraoral photograph showing cemented palatal button on rotated 13 and 24. Figure 5: Intraoral photograph shows cemented crown and molar bands. Figure 6: Lower arch “Kesling diagnostic setup.” Figure 8: Intraoral photograph showing immediately after extraction of 81. Figure 6: Lower arch “Kesling diagnostic setup.” Figure 8: Intraoral photograph showing immediately after extraction of 81. facilitate the eruption of 25, recontouring of upper and lower incisors with composites after completing orthodontic treat- ment, and replacement of missing 31 and 41 spaces with one tooth with a cantilevered resin-bonded bridge (RBB) from 32. and function. 2. Case Report In order to provide aesthetically and function- ally pleasing outcomes, a multidisciplinary management approach may be required. The aims of that approach should include improving aesthetics, maintaining the existing denti- tion, improving speech, enhancing the masticatory eﬃcacy, improving acceptance by family and peers, and promoting psychological wellbeing [7]. Most of these requirements can be achieved by addressing the hypodontia by replacing miss- ing teeth with recommended options such as removable prosthesis, conventional or resin adhesive bridgework, implant-supported prosthesis, or autotransplantation [11]. All the options have a common prerequisite in which they need space management before replacing the missing teeth. Before commencing the second half of the orthodontic treatment, the lower arch “Kesling diagnostic setup” was made (Figure 6). At the next visit, palatal buttons were ﬁxed on rotated 13 and 24 in order to derotate (Figure 7). The overretained 81 was extracted prior to bond up of the lower arch (Figure 8). In parallel to the orthodontic treatment, surgical expo- sure of 25 and distalization of 26 with a coiled spring was done to facilitate the eruption of 25 (Figure 9). Prior to debonding the ﬁxed applications, a “near-end radiograph” was taken to observe the status of root resorption, teeth posi- tion, and the available spaces and root parallelism for future implant placement of the overretained teeth (Figure 10). Therefore, managing the child especially in mixed denti- tion with ED and interceptive orthodontics plays a major role. The patient is awaiting the orthodontic treatments which mean he/she became a candidate for the preventive care like plaque control, ﬁssure sealant, dietary counselling, and ﬂuoride therapy [12]. Therefore, our patient also under- went all the preventive measures. Even though we clinically diagnosed this case as hypohidrotic ED, the ideal way of con- ﬁrming it is a skin biopsy that shows absent or hypoplastic sweat glands. Furthermore, to diagnose the subtypes of EDs, many ways of genetic tests are also available [13]. Immediately after debonding, all the conical incisors and canines were recontoured using composites (Figure 11). The missing upper laterals were managed by right-side canine lat- eralization and left-side overretained deciduous canine recontouring by selective grinding and composite buildups. The missing 41 was replaced by a cantilever RBB (Figure 12). The total care rehabilitation, including aesthetic and pre- ventive measures, seemed to be alright in our patient at the end of the 5-year follow-up period (Figure 13). He is still on review. 2. Case Report Implant-supported crowns for the missing 15, 22, 23, 31, and 41 could be considered when the patient is at the age of 18 years. Currently, there is no causative treatment available [14]. ED-associated hypodontia can be aﬀected by both deciduous and permanent dentition [15]. Most often, the permanent maxillary central incisors, maxillary ﬁrst molars, mandibular ﬁrst molars, and maxillary canines are present in hypohidro- tic ED [16]. The most common concern of the children with ED is about the dental anomalies and facial appearance [17], and it was common for the present case study also. 3. Discussion Ectodermal dysplasia associated with oligodontia is a com- plex dental condition that signiﬁcantly eﬀects on aesthetics Case Reports in Dentistry 5 Case Reports in Dentistry (a) (b) Figure 9: Intraoral photographs of 25 (a) before and (b) after surgical exposure. (a) (b) (b) (a) Figure 9: Intraoral photographs of 25 (a) before and (b) after surgical exposure. Figure 10: The “near-end radiograph” (digital dental panoramic tomograph). Figure 10: The “near-end radiograph” (digital dental panoramic tomograph). Figure 12: RBB was cemented to replace missing lower central incisors. Figure 11: All conical incisors were recontoured with LCC. Figure 11: All conical incisors were recontoured with LCC. Figure 12: RBB was cemented to replace missing lower central incisors. The main demand of these patients is replacing their missing teeth and recontouring of the malformed teeth to improve their aesthetics and functions. But the conventional prosthodontic treatments are considered problematic in severe hypodontic cases [18] that may be associated with underdeveloped alveolar ridges in edentulous areas and mal- formed or overretained deciduous abutment teeth which will lead to poor support, stability, and retention of the prosthesis. implant therapy. Interceptive orthodontic treatments are a method to restore a normal occlusion when a malocclusion has started to occur. Furthermore, the development of the dentoskeletal complex, possible discrepancies, and malposi- tion are identiﬁed and removed throughout the interceptive orthodontic period. Applications undertaken in interceptive orthodontics are serial extraction, correction of developing crossbite, control of abnormal habits, space reclamation and distribution, extraction of supernumerary and retained primary teeth, root parallelism, and correction of possible predictable malocclusions [20, 21]. The main factors guiding the decision towards the orthodontic and prosthetic choice The new trends of managing EDs are moving towards implant-supported rehabilitation [19]. Therefore, the intro- duction of interceptive orthodontics to the ED patients is of utmost importance to prepare them as candidates for future 6 Case Reports in Dentistry 6 (a) (b) Figure 13: Comparison of preoperative and postoperative aesthetic outcomes. (b) (a) (a) (a) (b) Figure 13: Comparison of preoperative and postoperative aesthetic outcomes. Figure 13: Comparison of preoperative and postoperative aesthetic outcomes. [4] I. Tarjan, K. Gabris, and N. Rozsa, “Early prosthetic treatment of patients with ectodermal dysplasia: a clinical report,” The Journal of Prosthetic Dentistry, vol. 93, no. 5, pp. 419–424, 2005. are the presence of posterior natural teeth, facial aesthetics, lip support, number and size of existing natural teeth, and the occlusal vertical dimension. 3. Discussion The successful outcomes of the patient presented in this case report are due to good understanding and the commit- ments of both clinicians and the child’s parents, who always had positive attitudes towards the proposed multidisciplinary treatment plan. [5] K. Salem and F. Moazami, “Dental reconstruction in hypohy- drotic ectodermal dysplasia: case report,” Iranian Journal of Pediatric Dentistry, vol. 11, no. 2, pp. 77–82, 2016. [6] M. A. Pigno, R. B. Blackman, R. J. Cronin Jr., and E. Cavazos, “Prosthodontic management of ectodermal dysplasia: a review of the literature,” The Journal of Prosthetic Dentistry, vol. 76, no. 5, pp. 541–545, 1996. 4. Conclusion [7] J. H. Nunn, N. E. Carter, T. J. Gillgrass et al., “The interdisci- plinary management of hypodontia: background and role of paediatric dentistry,” British Dental Journal, vol. 194, no. 5, pp. 245–251, 2003. Management of a child with ectodermal dysplasia with oligo- dontia was a real challenge. Early diagnosis, necessary inves- tigation, and providing age-appropriate multidisciplinary treatment were key steps in successful outcomes. Further- more, selecting the most suitable treatment option among a verity of modalities is of utmost importance to the long- term sustainability of both provided and future treatments as well. Finally, providing the treatments for ED patients while preparing them for future implant therapy is an added advantage for the patients. Most importantly, upliftment of the psychological wellbeing of the young children provided by proper management of ED is of utmost importance to improve their quality of life. [8] L. Suri, E. Gagari, and H. Vastardis, “Delayed tooth eruption: pathogenesis, diagnosis, and treatment. A literature review,” American Journal of Orthodontics and Dentofacial Orthope- dics, vol. 126, no. 4, pp. 432–445, 2004. [9] N. E. Carter, T. J. Gillgrass, R. S. Hobson et al., “The interdis- ciplinary management of hypodontia: orthodontics,” British Dental Journal, vol. 194, no. 7, pp. 361–366, 2003. [10] J. L. Ackerman and W. R. Proﬃt, “Preventive and interceptive orthodontics: a strong theory proves weak in practice,” The Angle Orthodontist, vol. 50, no. 2, pp. 75–87, 1980. [11] R. Holliday, N. Lush, J. Chapple, F. Nohl, and B. Cole, “Hypo- dontia: aesthetics and function part 2: management,” Dental update, vol. 41, no. 10, pp. 891–898, 2014. Data Availability All the available data which relevant to this case report has presented in the manuscript. [12] R. Hawkins, D. Locker, J. Noble, and E. J. Kay, “Prevention. Part 7: professionally applied topical ﬂuorides for caries pre- vention,” British Dental Journal, vol. 195, no. 6, pp. 313–317, 2003. Conflicts of Interest The authors are declared that there is no conﬂict of interest in any above mentioned products or materials. [13] F. Clauss, M. C. Manière, F. Obry et al., “Dento-craniofacial phenotypes and underlying molecular mechanisms in hypohi- drotic ectodermal dysplasia (HED): a review,” Journal of Den- tal Research, vol. 87, no. 12, pp. 1089–1099, 2008. References [14] D. Celli, A. Manente, C. Grippaudo, and M. Cordaro, “Inter- ceptive treatment in ectodermal dysplasia using an innovative orthodontic/prosthetic modular appliance A case report with 10- year follow-up,” European Journal of Paediatric Dentistry, vol. 19, no. 4, pp. 307–312, 2018. [1] S. Y. Chee, C. H. Wanga, W. D. Lina, and F. J. Tsaia, “Ectoder- mal dysplasia (ED) syndrome,” Biomedicine (Taipei), vol. 4, no. 4, p. 27, 2014. [2] T. Halai and C. Stevens, “Ectodermal dysplasia: a clinical over- view for the dental practitioner,” Dental Update, vol. 42, no. 8, pp. 779–790, 2015. [15] R. S. Hobson, N. E. Carter, T. J. Gillgrass et al., “The interdis- ciplinary management of hypodontia: the relationship between an interdisciplinary team and the general dental prac- titioner,” British Dental Journal, vol. 194, no. 9, pp. 479–482, 2003. [3] G. Rashu and M. Manjul, “Prosthodontic management of chil- dren with ectodermal dysplasia: review of literature,” Den- tistry, vol. 5, no. 11, 2015. 7 Case Reports in Dentistry Case Reports in Dentistry [16] A. D. Guckes, M. W. Roberts, and G. R. McCarthy, “Pattern of permanent teeth present in individuals with ectodermal dys- plasia and severe hypodontia suggests treatment with dental implants,” Pediatric Dentistry, vol. 20, no. 4, pp. 278–280, 1998. [17] M. B. Siegel and W. P. Potsic, “Ectodermal dysplasia: the oto- laryngologic manifestations and management,” International Journal of Pediatric Otorhinolaryngology, vol. 19, no. 3, pp. 265–271, 1990. [18] J. R. Boj, J. Duran von Arx, M. Cortada, A. Jimenez, and J. Golobart, “Dentures for a 3-yr-old child with ectodermal dysplasia: case report,” American Journal of Dentistry, vol. 6, no. 3, pp. 165–167, 1993. [19] I. P. Sweeney, J. W. Ferguson, A. A. Heggie, and J. O. Lucas, “Treatment outcomes for adolescent ectodermal dysplasia patients treated with dental implants,” International Journal of Paediatric Dentistry, vol. 15, no. 4, pp. 241–248, 2005. [20] R. M. Ricketts, “Dr. Robert M. Ricketts on early treatment (part 1),” Journal of Clinical Orthodontics, vol. 13, no. 1, pp. 23–38, 1979. [21] N. Karaiskos, W. A. Wiltshire, O. Odlum, D. Brothwell, and T. H. Hassard, “Preventive and interceptive orthodontic treat- ment needs of an inner-city group of 6- and 9-year-old Cana- dian children,” Journal of the Canadian Dental Association, vol. 71, no. 9, p. 649, 2005.
https://openalex.org/W3217221255	https://www.nature.com/articles/s43247-021-00312-6.pdf	English	null	A Bayesian framework for deriving sector-based methane emissions from top-down fluxes	Communications earth & environment	2,021	cc-by	8,771	1 Arizona Institutes for Resilience, University of Arizona, Tucson, AZ, USA. 2 Jet Propulsion Laboratory, California Institute of Technology, Pasadena, CA, USA. 3 Division of Geological and Planetary Sciences, California Institute of Technology, Pasadena, CA, USA. 4 SRON Netherlands Institute for Space Research, Utrecht, Netherlands. 5 Key Laboratory of Coastal Environment and Resources of Zhejiang Province, School of Engineering, Westlake University, Hangzhou, Zhejiang Province, China. 6 Institute of Advanced Technology, Westlake Institute for Advanced Study, Hangzhou, Zhejiang Province, China. 7 Department of Earth and Planetary Sciences, Harvard University, Cambridge, MA, USA. 8 School of Engineering and Applied Sciences, Harvard University, Cambridge, MA, USA. ✉email: dcusworth@arizona.edu ARTICLE https://doi.org/10.1038/s43247-021-00312-6 OPEN COMMUNICATIONS EARTH & ENVIRONMENT \| (2021) 2:242 \| https://doi.org/10.1038/s43247-021-00312-6 \| www.nature.com/commse A Bayesian framework for deriving sector-based methane emissions from top-down ﬂuxes Daniel H. Cusworth1,2✉, A. Anthony Bloom2, Shuang Ma2, Charles E. Miller 2, Kevin Bowman2, Yi Yin 3, Joannes D. Maasakkers4, Yuzhong Zhang 5,6, Tia R. Scarpelli7, Zhen Qu 8, Daniel J. Jacob 7,8 & John R. Worden 2 A Bayesian framework for deriving sector-based methane emissions from top-down ﬂuxes Daniel H. Cusworth1,2✉, A. Anthony Bloom2, Shuang Ma2, Charles E. Miller 2, Kevin Bowman2, Yi Yin 3, Joannes D. Maasakkers4, Yuzhong Zhang 5,6, Tia R. Scarpelli7, Zhen Qu 8, Daniel J. Jacob 7,8 & John R. Worden 2 Atmospheric methane observations are used to test methane emission inventories as the sum of emissions should correspond to observed methane concentrations. Typically, con- centrations are inversely projected to a net ﬂux through an atmospheric chemistry-transport model. Current methods to partition net ﬂuxes to underlying sector-based emissions often scale ﬂuxes based on the relative weight of sectors in a prior inventory. However, this approach imposes correlation between emission sectors which may not exist. Here we present a Bayesian optimal estimation method that projects inverse methane ﬂuxes directly to emission sectors while accounting uncertainty structure and spatial resolution of prior ﬂuxes and emissions. We apply this method to satellite-derived ﬂuxes over the U.S. and at higher resolution over the Permian Basin to demonstrate that we can characterize a sector- based emission budget. This approach provides more robust comparisons between different top-down estimates, critical for assessing the efﬁcacy of policies intended to reduce emissions. 1 1 ARTICLE COMMUNICATIONS EARTH & ENVIRONMENT \| https://doi.org/10.1038/s43247-021-00312-6 A d A s the second highest greenhouse gas (GHG) contributor to global radiative forcing, understanding the global budget of methane (CH4) is a top climate priority1,2. Methane is emitted from a variety of anthropogenic and natural emission sectors, including oil and gas operations, waste management, coal mining, agriculture, wetlands, and ﬁres among others3. Article 14 of the Paris Agreement4 requires participating countries to report progress towards achieving their climate mitigation goals, or nationally determined contributions. Reporting progress, including any changes in the CH4 budget, necessitates inven- torying all possible emission sources. CH4 emission inventories can be constructed from “bottom-up” or derived from “top- down” observations. Bottom-up accounting relies on a knowledge of activity data and emission factors for anthropogenic sectors and/or detailed processed-based models that predict CH4 emis- sions based on a set of environmental factors for natural emission sectors. A Bayesian framework for deriving sector-based methane emissions from top-down ﬂuxes Daniel H. Cusworth1,2✉, A. Anthony Bloom2, Shuang Ma2, Charles E. Miller 2, Kevin Bowman2, Yi Yin 3, Joannes D. Maasakkers4, Yuzhong Zhang 5,6, Tia R. Scarpelli7, Zhen Qu 8, Daniel J. Jacob 7,8 & John R. Worden 2 The full derivation is documented in the Methods section and Supporting Information (SI) and has been mechanically veriﬁed and tested using simulated emissions, concentrations, and ﬂuxes. This approach has been previously described for atmospheric trace gas retrievals15 but is here modiﬁed and applied for ﬂux comparisons. The full derivation is documented in the Methods section and Supporting Information (SI) and has been mechanically veriﬁed and tested using simulated emissions, concentrations, and ﬂuxes. This approach has been previously described for atmospheric trace gas retrievals15 but is here modiﬁed and applied for ﬂux comparisons. This approach has been previously described for atmospheric trace gas retrievals15 but is here modiﬁed and applied for ﬂux comparisons. Applying Eqs. 1 and 2 allows for the ability to “swap priors.” This means that no matter what prior was used in an initial ﬂux inversion, we can swap it with a different prior emissions vector zA, which can include sector-based information. This a critical component for comparison between two different top-down inventories, as it removes error that may arise from choice of prior. Another advantage of this Bayesian approach is that ﬂuxes are partitioned according to not just the prior emission state zA, but also according prior uncertainties on those emissions (i.e., ZA). For example, if we have evidence that a particular emission sector is well-characterized in bottom-up inventories (i.e., a tight prior uncertainty), Eqs. 1–2 take this knowledge into account when optimizing emissions. In this sense, our approach is similar to updated methods that use prior ratios with prior error variance when computing RWs used for partitioning16. However, any RW- based approach still assumes that correlation exists between emission sectors at the grid-level, creating a relationship that may bias results depending on prior construction. g y ) Atmospheric observations of CH4, combined with an atmo- spheric transport model and a regularizing statistical approach, provide a top-down constraint on the global CH4 budget. Typi- cally referred to as “inversions” or top-down inventories, these methods estimate CH4 ﬂuxes by assimilating tower, aircraft, or satellite-based CH4 measurements8–14. Generally, these top-down methods only estimate total ﬂuxes (i.e., sum of all emission sector contributions) explicitly, and may rely on using prior ratios or relative weights (RWs) between source categories to partition ﬂuxes to speciﬁc source sectors. Top-down inverse models may be driven by different regularization or prior conditions, compli- cating direct comparison between an ensemble of inventories. A Bayesian framework for deriving sector-based methane emissions from top-down ﬂuxes Daniel H. Cusworth1,2✉, A. Anthony Bloom2, Shuang Ma2, Charles E. Miller 2, Kevin Bowman2, Yi Yin 3, Joannes D. Maasakkers4, Yuzhong Zhang 5,6, Tia R. Scarpelli7, Zhen Qu 8, Daniel J. Jacob 7,8 & John R. Worden 2 Therefore, these partitioning approaches are prone to error when comparing with bottom-up inventories if the prior distribution of emissions is biased, or if different sectors have different uncer- tainties. For example, Saunois et al. compared an ensemble of 22 top-down CH4 global inversions to an ensemble of bottom-up inventories, and found the bottom-up estimate to be 30% higher than the top-down ensemble mean. The study attributes much of this total discrepancy to large differences in non-wetland natural sources (e.g., lakes and rivers, oceans seeps, termites, geologic sources, wild animals, etc.). However, when integrating emissions over the whole globe, they ﬁnd that other source categories from bottom-up and top-down approaches are consistent within their reported uncertainties (fossil fuel top-down: 81–131 Tg a−1, bottom-up: 113–154 Tg a−1; agriculture+waste top-down: 207–240 Tg a−1, bottom-up: 191–223 Tg a−1; and biomass +biofuel burning top-down: 22–36 Tg a−1, bottom-up: 26–40 Tg a−1). Reported uncertainties reﬂect the range of estimates among distinct bottom-up and top-down inventories across various emission sectors. Saunois et al. also relied on using RWs to partition top-down ﬂuxes to individual bottom-up sectors. An explicit approach for comparison between top-down and bottom- up inventories, and between independent top-down based inventories, is needed to reduce this uncertainty in the global CH4 budget by sector and by region. The main caveat of this Bayesian approach is that an explicit representation of ^S is needed. For analytical ﬂux inversions, this matrix has a closed-form representation that is computed as part of the inversion. For adjoint-based inversions10,11, a closed-form representation of ^S is not directly computed. Though the error covariance can still be estimated17,18, this is computationally expensive and is generally not done. Analytical frameworks are best suited for direct comparison between inverse products due to explicit error characterization. A Bayesian framework for deriving sector-based methane emissions from top-down ﬂuxes Daniel H. Cusworth1,2✉, A. Anthony Bloom2, Shuang Ma2, Charles E. Miller 2, Kevin Bowman2, Yi Yin 3, Joannes D. Maasakkers4, Yuzhong Zhang 5,6, Tia R. Scarpelli7, Zhen Qu 8, Daniel J. Jacob 7,8 & John R. Worden 2 By aggregating an ensemble of bottom-up inventories and process-models, Saunois et al.5 calculated a global methane budget for 2008–2017 and estimated total emissions of 594–880 TgCH4 a−1, with 113–154 TgCH4 a−1 from fossil fuels, 191–223 TgCH4 a−1 from agriculture and waste, 26–40 TgCH4 a−1 from biomass and biofuel burning, 102–182 TgCH4 a−1 from wetlands, and 143–306 TgCH4 a−1 from other natural sources. Uncertainty and bias in bottom-up CH4 emissions in some geographic regions may be caused by imprecise emission factors and activity data that are not readily available at necessary spatial and temporal scales, or by process-based models that perform poorly due to a host of environmental factors (e.g., wetland models rely on wet- land inundation maps, biogeochemical process parameterizations, and knowledge of carbon availability6,7). Bayesian estimation of emissions from ﬂuxes. We propose a comprehensive Bayesian framework that derives top-down grid- ded CH4 emissions (^z) and their error covariance (^Z) from inverse ﬂuxes (^x) and their error covariance (^S) without reliance on RWs from an inventory. This framework takes the following form: ^z ¼ zA þ ^ZMT^S 1½ðI ^SS1 A ÞðxA MzAÞ þ ð^x xAÞ ð1Þ ð1Þ Where the vector ^x represents inverse CH4 ﬂuxes on a spatial grid with full error characterization given by the covariance matrix ^S, xA is the vector of prior ﬂuxes, I is the identity matrix, SA is the prior error covariance matrix, and M is an aggregation matrix that sums emissions to ﬂuxes (Methods section: Eqs. 8–9). The posterior emission error covariance matrix ^Z is calculated expli- citly given M, SA, ^S, and prior emissions error covariance matrix ZA: Where the vector ^x represents inverse CH4 ﬂuxes on a spatial grid with full error characterization given by the covariance matrix ^S, xA is the vector of prior ﬂuxes, I is the identity matrix, SA is the prior error covariance matrix, and M is an aggregation matrix that sums emissions to ﬂuxes (Methods section: Eqs. 8–9). The posterior emission error covariance matrix ^Z is calculated expli- citly given M, SA, ^S, and prior emissions error covariance matrix ZA: ^Z ¼ ðMTð^S 1 S1 A ÞM þ Z1 A Þ 1 : ð2Þ ð2Þ The full derivation is documented in the Methods section and Supporting Information (SI) and has been mechanically veriﬁed and tested using simulated emissions, concentrations, and ﬂuxes. COMMUNICATIONS EARTH & ENVIRONMENT \| https://doi.org/10.1038/s43247-021-00312-6 ﬂ l h l b l h total CH4 emissions per grid cell over CONUS using 2010–2015 GOSAT inverse ﬂuxes6. Panel a shows CH4 ﬂuxes at 0.5 × 0.625° b and c show optimized emissions and the change from the prior for the gas sector, respectively at 0.1 × 0.1° resolution. Circled boxes in gh outlines of major U.S. gas producing basins: (i) Permian, (ii) Anadarko, (iii) Haynesville, (iv) Eagle Ford, (v) Barnett Shale, (vi) Bakken, and (viii) Niobara. Panels d and e show optimized emissions and the change from the prior for the livestock sector, respectively. d total CH4 emissions per grid cell over CONUS using 2010–2015 GOSAT inverse ﬂuxes6. Panel a shows CH4 ﬂ els b and c show optimized emissions and the change from the prior for the gas sector, respectively at 0.1 × 0.1° resolu Fig. 1 Optimized total CH4 emissions per grid cell over CONUS using 2010–2015 GOSAT inverse ﬂuxes6. Panel a shows CH4 ﬂuxes at 0.5 × 0.625° resolution. Panels b and c show optimized emissions and the change from the prior for the gas sector, respectively at 0.1 × 0.1° resolution. Circled boxes in Panel b show rough outlines of major U.S. gas producing basins: (i) Permian, (ii) Anadarko, (iii) Haynesville, (iv) Eagle Ford, (v) Barnett Shale, (vi) Appalachia, (vii) Bakken, and (viii) Niobara. Panels d and e show optimized emissions and the change from the prior for the livestock sector, respectively. Partitioning CH4 ﬂuxes over CONUS. We apply the partitioning algorithm described by Eqs. 1 and 2 to a 2010–2015 0.50 × 0.625° resolution North American CH4 ﬂux inversion6 performed using Greenhouse Gas Observing Satellite20 (GOSAT) dry air column mixing ratios of CH4. We partition emission to seven distinct sectors at 0.1 × 0.1° resolution: oil, gas, coal, livestock, waste management, wetlands, and other emission sources (soil, ﬁre, etc.). For oil, gas, and coal prior emissions and uncertainties, we use a global inventory for 2016 based on national reports to the United Nations Framework Convention on Climate Change (Scarpelli et al.21). For wetland prior emissions and uncertainties, we take the ensemble average and standard deviation of wetland models that were found to be the highest performing when compared with a global CH4 ﬂux inversion22. Results h In what follows, we show examples of this approach using a previously performed 2010–2015 GOSAT ﬂux inversion of the Continental United States9 (CONUS). We also apply Eqs. 1 and 2 to a previously performed 2018–2019 TROPOMI ﬂux inversion over the Permian Basin in western Texas, southern New Mexico19. We compare with the partitioned results from the 2010–2015 GOSAT inversion to show how projection to a common prior can be used to assess regional emission trends since uncertainties from different priors and different spatial resolutions of the ﬂux inversions are removed. OMMUNICATIONS EARTH & ENVIRONMENT \| (2021) 2:242 \| https://doi.org/10.1038/s43247-021-00312-6 \| www.nature.com/commsenv 2 ARTICLE COMMUNICATIONS EARTH & ENVIRONMENT \| https://doi.org/10.1038/s43247-021-00312-6 COMMUNICATIONS EARTH & ENVIRONMENT \| https://doi.org/10.1038/s43247-021-00312-6 ΔCH4 = 0.00/−0.06 TgCH4 a−1), the small CH4 ﬂux enhancement observed in Fig. 1a is partitioned entirely to the oil sector, but produces emissions lower than the prior, which contrasts with the increasing production reported in the basin24. This emission reduction may be due to an overestimate in the prior inventory or a difﬁculty in GOSAT sampling over that region, factors which could be veriﬁed with additional study. Posterior livestock emissions show increases from the prior that are distributed across the central and eastern United States, and a 0.07 TgCH4 a−1 decrease in emissions over central California. Comparing inverse ﬂuxes in the Permian Basin. As seen from Figs. 1 and S1, the Permian Basin shows large increases in CH4 emissions for both oil and gas sectors compared to the prior inventory. Production data from the Energy Information Administration24 (EIA) indicates that between 2010 and 2019, the Permian increased oil production by 190% and gas produc- tion by 150%. We expect that fugitive emissions from these sec- tors would increase proportionately to the production increases, but the actual posterior estimates do not differentiate between intentional (planned) and unintentional (unplanned) emissions. The sensitivity study in Fig. 2 shows that the application of Eqs. 1–2 can be used to compare distinct inverse ﬂuxes when a common emissions prior is used. We compare the 2010–2015 GOSAT ﬂux product with a Permian 0.25° × 0.325° ﬂux product19 based on May 2018–March 2019 TROPOspheric Monitoring Instrument27 (TROPOMI). We partition ﬂuxes to the same sec- tors as Fig. 1, but at a ﬁner 0.1 × 0.1° grid resolution. A major advantage of using Eqs. 1–2 to estimate emissions from independent inverse ﬂuxes is that any discrepancies in ﬂux priors can be accounted for when partitioning to a common emission prior. We show this through a sensitivity study whose results are summarized in Fig. 2. Here, we use two inverse ﬂux products: (1) the 2010–2015 GOSAT CONUS ﬂux shown in Fig. 1a, and (2) inverse ﬂuxes from 2010–2015 GOSAT recomputed by using the EDGAR v5.0 emission inventory for oil, gas, waste, and livestock sectors for 201525. Differences in these prior inventories are summarized in Table S1 and Figs. S2–S3. Global inventories like EDGAR use consistent bottom-up aggregation methods across countries, which some- times leads to discrepancies when compared with national inventories for particular sectors21. COMMUNICATIONS EARTH & ENVIRONMENT \| https://doi.org/10.1038/s43247-021-00312-6 Fig. 2 Emissions partitioned or optimized from 2010–2015 GOSAT ﬂux inversions. Two different ﬂux products are partitioned using two different partitioning approaches. Blue bars represent ﬂuxes that were derived using the same ﬂux prior as Fig. 1 (Table S1). Red bars represent ﬂuxes that were Fig. 2 Emissions partitioned or optimized from 2010–2015 GOSAT ﬂux inversions. Two different ﬂux products are partitioned using two different partitioning approaches. Blue bars represent ﬂuxes that were derived using the same ﬂux prior as Fig. 1 (Table S1). Red bars represent ﬂuxes that were derived using EDGAR v5.0 for oil, gas, livestock, and waste sectors. Solid bars represent the optimal emission (OE) approach from Eqs. 1–2. Hatched bars represent partitioning done by computing the relative weights (RW) of emission sectors that made up the ﬂux prior. Fig. 2 Emissions partitioned or optimized from 2010–2015 GOSAT ﬂux inversions. Two different ﬂux products are partitioned using two different partitioning approaches. Blue bars represent ﬂuxes that were derived using the same ﬂux prior as Fig. 1 (Table S1). Red bars represent ﬂuxes that were derived using EDGAR v5.0 for oil, gas, livestock, and waste sectors. Solid bars represent the optimal emission (OE) approach from Eqs. 1–2. Hatched bars represent partitioning done by computing the relative weights (RW) of emission sectors that made up the ﬂux prior. RWs (11.3 TgCH4 a−1 and 8.1 TgCH4 a−1, respectively). Similarly, wetland and waste emissions differ depending on which prior are used for RWs. Using prior ratios assumes total correlation between emission sectors in each grid cell as each sector’s RW depends on emissions from other sectors. Therefore, we see that different assumptions on the ﬂux prior can complicate comparison even between inverse ﬂuxes that use the same atmospheric observations and transport model. ΔCH4 = 0.00/−0.06 TgCH4 a−1), the small CH4 ﬂux enhancement observed in Fig. 1a is partitioned entirely to the oil sector, but produces emissions lower than the prior, which contrasts with the increasing production reported in the basin24. This emission reduction may be due to an overestimate in the prior inventory or a difﬁculty in GOSAT sampling over that region, factors which could be veriﬁed with additional study. Posterior livestock emissions show increases from the prior that are distributed across the central and eastern United States, and a 0.07 TgCH4 a−1 decrease in emissions over central California. COMMUNICATIONS EARTH & ENVIRONMENT \| https://doi.org/10.1038/s43247-021-00312-6 For all other emission sectors, we use the 2012 EPA gridded CH4 inventory as the prior estimate23, and assume a one standard deviation uncertainty equal to 50% of the mean value. For illustration and computational tractability, the prior error covariances are repre- sented as diagonal matrices. Figure 1a shows the inverse ﬂuxes ^x over CONUS, optimized emissions ^z for the gas and livestock sectors when applying Eqs. 1–2, and the change in emissions compared to the emission prior ð^z zAÞ. Other optimized emission sectors are shown in Fig. S1. Optimized oil and gas emissions show large changes from the prior at the basin scale in several major producing basins and shale plays: Permian (New Mexico/Texas; ΔCH4: gas/oil = 0.40/ 0.76 TgCH4 a−1), Eagle Ford (southern Texas; ΔCH4 = 0.14/0.11 TgCH4 a−1), Haynesville (Texas/Louisiana; ΔCH4 = 0.18/0.0 TgCH4 a−1), Barnett Shale (Texas; ΔCH4 = 0.21/0.0 TgCH4 a−1), Anadarko (Oklahoma; ΔCH4 = 0.52/0.05 TgCH4 a−1), and the Appalachia Basin (Ohio/Pennsylvania/West Virginia; ΔCH4 = 0.20/0.0 TgCH4 a−1). The Niobrara (Wyoming/Colorado; ΔCH4 = 0.05/0.0 TgCH4 a−1) region shows much less or no change from the prior. For the Bakken (Montana/North Dakota; MMUNICATIONS EARTH & ENVIRONMENT \| (2021) 2:242 \| https://doi.org/10.1038/s43247-021-00312-6 \| www.nature.com/commsenv 3 ARTICLE COMMUNICATIONS EARTH & ENVIRONMENT \| https://doi.org/10.1038/s43247-021-00312-6 We employ two methods for optimizing/partitioning inverse ﬂuxes: (1) using optimal estimation (OE) from this study’s Eqs. 1–2 to optimize the same emissions prior from Fig. 1, and (2) by computing RWs of each emission sector in the ﬂux prior and partitioning the inverse ﬂuxes to emissions using these weights. In Fig. 2 we show that by employing OE on each separate inverse ﬂux, we get the exact same answer for optimized emissions. This is a result of the ðI ^SS1 A Þ term from Eq. 1. Sometimes called the averaging kernel matrix26, this term represents the spatial resolution of the ﬂux estimate, or alternatively, the degree of smoothing of the ﬂux prior to the estimate. Since the averaging kernel is applied to the prior swap term ðxA MzAÞ, we account for discrepancies between ﬂux and emission priors as a condition of emission optimization, so the choice of ﬂux prior is immaterial. However, Fig. 2 shows the result when a relative weighting scheme is used for partitioning. The livestock to gas ratio in EDGAR is higher than in the EPA and Scarpelli et al. inventories. The result is that RW-partitioned livestock emissions are higher and gas emissions are lower when using EDGAR RWs (13.2 TgCH4 a−1 and 5.8 TgCH4 a−1, respectively) than when using EPA-Scarpelli g g Figure 3a, b shows the 2010–2015 GOSAT ﬂux inversion and the 2018–2019 TROPOMI ﬂux inversion over the Permian Basin, respectively. Within the Permian domain (black line in Fig. 3), the GOSAT inverse ﬂux estimate is 2.01 ± 0.01 TgCH4 a−1 and the TROPOMI inverse ﬂux estimate is 2.68 ± 0.5 TgCH4 a−1, though these inversions were performed over different time periods and used different ﬂux priors and prior error covariance matrices to constrain their solutions. The GOSAT and TRO- POMI inverse products show distinct spatial patterns. The TROPOMI inversion shows two main regions of elevated CH4 ﬂux, which correspond to the Delaware Basin on the western side of the Permian (west Texas, southeast New Mexico) and the Midland Basin on the eastern side of the Permian. The GOSAT inversion shows a more distributed region of ﬂux enhancement across the Permian. COMMUNICATIONS EARTH & ENVIRONMENT \| https://doi.org/10.1038/s43247-021-00312-6 The difference in spatial distribution of these CH4 ﬂux maps could be due to the more limited GOSAT spatial observing coverage over the Permian and the coarser spatial resolution over which the 2010–2015 GOSAT ﬂux inversion was MUNICATIONS EARTH & ENVIRONMENT \| (2021) 2:242 \| https://doi.org/10.1038/s43247-021-00312-6 \| www.nature.com/commsenv 4 4 ARTICLE COMMUNICATIONS EARTH & ENVIRONMENT \| https://doi.org/10.1038/s43247-021-00312-6 Fig. 3 Comparison of two satellite inverse ﬂux whose emissions were optimized using the same prior in the Permian Basin (black outline). Panel a shows the 2010–2015 GOSAT inverse ﬂux result over the Permian Basin. Panel b shows the 2018–2019 TROPOMI inverse ﬂux13, with the Delaware and Midland basins circled in red. Panel c is the prior oil and gas inventory used for partitioning15. Panels d and e represent posterior oil and gas emissions, optimized from ﬂuxes in panels a and b, respectively. Fig. 3 Comparison of two satellite inverse ﬂux whose emissions were optimized using the same prior in the Permian Basin (black outline). Panel a shows the 2010–2015 GOSAT inverse ﬂux result over the Permian Basin. Panel b shows the 2018–2019 TROPOMI inverse ﬂux13, with the Delaware and Midland basins circled in red. Panel c is the prior oil and gas inventory used for partitioning15. Panels d and e represent posterior oil and gas emissions, optimized from ﬂuxes in panels a and b, respectively. performed. However, given that the two ﬂux inversions are asynchronous and that oil and gas production increased dramatically between 2015 and 2018, spatial differences in CH4 ﬂuxes could also be the result of changing infrastructure throughout the basin. discrepancy between top-down estimates corresponds explicitly to differences in ﬂux priors used in the original inversions that is now accounted for with this approach. Therefore, we can quantify how much the choice of ﬂux prior directly impacts any quantiﬁed changes between top-down inventories. g Figure 3d, e show the optimized emissions for the oil, gas, and livestock sectors in the Permian, respectively, and compared to the prior inventory (Fig. 3c). Optimized emissions are derived from GOSAT and TROPOMI ﬂuxes (Fig. 3a, b) that were swapped with a consistent prior (Fig. 3c) using Eqs. 1 and 2. The partitioned GOSAT emissions continue to show more distributed oil and gas CH4 emissions across the basin when compared to the partitioned TROPOMI emissions, which are mostly concentrated to the Delaware and Midland Basins. COMMUNICATIONS EARTH & ENVIRONMENT \| https://doi.org/10.1038/s43247-021-00312-6 Figure 4 shows the aggregated top- down oil and gas emissions in the Permian compared with reported trends from EIA production reports. The mean 2010–2015 oil and gas production in the Permian was 1.3 million bbl per day and 5.0 million Mcf per day, respectively. This increased to 3.8 million bbl per day and 12.7 million Mcf per day between May 2018 and March 2019 mean production, respectively24. Though not linear with increased production, comparing partitioned 2010–2015 GOSAT oil and gas emissions and 2018–2019 TROPOMI emissions, we see a 0.52 ± 0.29 TgCH4 a−1 change in CH4 emissions, representing a 29% increase. Optimized emissions summed across all sectors increased by 0.42 ± 0.33 TgCH4 a−1, with the increase driven mostly by gas, some contribution by oil, and offset by a small decrease in livestock emissions. Originally reported posterior ﬂux estimates showed a 0.67 ± 0.5 TgCH4 a−1 difference between TROPOMI and GOSAT inversions, larger than the 0.42 ± 0.33 TgCH4 a−1 difference we quantify here after reprojection to a common prior. This difference Though consistent with the increase in gas production, the quantiﬁed 0.42 TgCH4 a−1 increase in oil and gas emissions from 2010–2015 to 2018–2019 could also be due to observing constraints and/or biases in satellite retrievals. For example, Qu et al.28 performed global 2 × 2.5° CH4 inversions for 2019 using GOSAT and TROPOMI using the same prior and atmospheric chemistry- transport model, and derived Permian net ﬂuxes of 2.36 TgCH4 a−1 and 2.43 TgCH4 a−1, respectively, showing consistency between signals observed by these independent remote sensing platforms for this region. Therefore, we conclude that the trends observed in Fig. 3 are likely due to changes in gas operations, and not bias in observing systems. However, in other global regions where the surface is less bright and homogeneous than the Permian, ﬂux results derived from TROPOMI and GOSAT inversion may not agree28. pðxjyÞ / pðyjxÞpðxÞ Where p (y\|x) is the maximum likelihood given by Eq. 3, and pðxÞ is the prior distribution. If we assume that p (y\|x) and p(x) are Gaussian distributions, and y and xA represent the modes of those respective distributions, then the mode of the posterior distribution ^x has a closed form solution, known as the Maximum A Posteriori (MAP; Rodgers, 2000) solution: ^x ¼ xA þ ^SKTS1 y ðy KxAÞ ð5Þ ^S ¼ ðKTS1 y K þ S1 A Þ 1 ð6Þ ð5Þ ^S ¼ ðKTS1 y K þ S1 A Þ 1 ð6Þ While our estimates account for the spatial resolution and error associated with the inversion of observations to ﬂuxes, we do not explicitly account for error in model transport and chemistry. These errors could be important when comparing emissions between seasons or in regions where transport is poorly modeled such as in the tropics. For example, a study of global carbon monoxide emissions, a trace gas that (like CH4) is affected by reaction with the hydroxl radical (OH) and transport, found that convective mass ﬂux in the tropics is likely responsible for errors in emissions31. While our approach can account for this error if a corresponding posterior covariance is provided, we emphasize that studies that both characterize and mitigate this part of the ﬂux error budget are needed to better use satellite observations of methane and of other trace gases. Ultimately, improved emission and error characterization from top-down information will allow for better updates and comparisons with bottom-up inventories, which can guide progress towards CH4 mitigation. For policy-relevance and CH4 budget quantiﬁcation, we really wish to optimize emissions using atmospheric observations, i.e., we want to compute the explicit posterior representation pðz j yÞ without re-simulation of an atmospheric transport model. pðxjyÞ / pðyjxÞpðxÞ The relationship between z and x is simple aggregation, and can represented by matrix M: x ¼ Mz: ð7Þ x ¼ Mz: ð7Þ If z and x share the same grid resolution (i.e., if z 2 Rm and x 2 Rn and s is the number of emission sectors, then m = ns), the matrix M 2 Rn ´ m is represented with the following terms: mi;j ¼ 1 ði; jÞ pertain to same grid 0 otherwise ð8Þ ð8Þ If z and x pertain to different grids, the relationship M is deﬁned by the geo- graphic area (Ω) and intersections (\) of grid cells: mi;j ¼ Ωxi\zj Ωzj : ð9Þ ð9Þ Since M is simply a summation matrix, we assume there is no noise associated with its application. Using M, we can update Eqs. 5 and 6 to ﬁnd the optimal emission state vector ^z and its posterior error covariance ^Z: ^z ¼ zA þ ^ZðKMÞTS1 y ðy ðKMÞzAÞ ð10Þ ^Z ¼ ððKMÞTS1 y ðKMÞ þ Z1 A Þ 1 ð11Þ ^z ¼ zA þ ^ZðKMÞTS1 y ðy ðKMÞzAÞ ð10Þ ^z ¼ zA þ ^ZðKMÞTS1 y ðy ðKMÞzAÞ COMMUNICATIONS EARTH & ENVIRONMENT \| https://doi.org/10.1038/s43247-021-00312-6 ARTICLE COMMUNICATIONS EARTH & ENVIRONMENT \| https://doi.org/10.1038/s43247-021-00312-6 Fig. 4 Aggregated CH4 emissions and reported production from the Energy Information Administration (EIA) in the Permian Basin. Panel a shows top- down emissions optimized from the independent ﬂux products from Fig. 3 aggregated to the basin. Error bars are an integration of prior and posterior error covariance matrices for the respective emission sectors. Panel b compares mean 2010–2015 and May 2018–March 2019 EIA production data21 for oil and gas sectors to the oil+gas emissions from panel a. Fig. 4 Aggregated CH4 emissions and reported production from the Energy Information Administration (EIA) in the Permian Basin. Panel a shows top- down emissions optimized from the independent ﬂux products from Fig. 3 aggregated to the basin. Error bars are an integration of prior and posterior error covariance matrices for the respective emission sectors. Panel b compares mean 2010–2015 and May 2018–March 2019 EIA production data21 for oil and gas sectors to the oil+gas emissions from panel a. p (x\|y), or ^x. A transformation or Jacobian matrix K can be derived from atmo- spheric transport simulations (e.g., GEOS-Chem), such that we can represent the relationship between ﬂuxes and observations: diagnosing uncertainty in the estimated methane budget. Ulti- mately, this information can be combined to provide better global understanding of methane emissions and ﬁner and more policy- relevant spatial and temporal scales. Likewise, the Bayesian fra- mework we describe in this study can be applied to other atmospheric gas ﬂuxes like carbon dioxide (CO2). Partitioning between biospheric and anthropogenic CO2 emissions remains highly uncertain30, so incorporating this framework that directly optimizes emission sectors could be useful for reconciling the budget. This approach does require a move from traditional ensemble or adjoint-based inversions, created to reduce cost of this computationally expensive problem, to an analytic or optimal estimation inversion as an explicit representation of the posterior covariance is required and this covariance is not easily calculated from ensemble or 4D-var methods. y ¼ Kx þ n ð3Þ ð3Þ Where n represents noise. We apply Bayes Theorem to estimate the posterior distribution p (x\|y): pðxjyÞ / pðyjxÞpðxÞ ð4Þ ð4Þ Discussion Having robust intercomparison methods in place are needed for interpreting the ever increasing number of atmospheric obser- vations of CH4, particularly with regard to the expected launches of several CH4-observing satellites in the 2020s29. As described in this study, inverse ﬂuxes derived from these satellite observations will depend on the observations themselves, the chemical trans- port model, and the prior constraint. Having the ability to directly quantify how these terms inﬂuence emissions is needed for MMUNICATIONS EARTH & ENVIRONMENT \| (2021) 2:242 \| https://doi.org/10.1038/s43247-021-00312-6 \| www.nature.com/commsenv 5 Methods In this section we derive a method to estimate CH4 emissions from atmospheric observations. The SI contains and alternate derivation (Section S1) and conceptual examples to further clarify the mechanics of prior-swapping. Emissions can be represented as a vector i.e., ðz 2 RmÞ that contains both sectoral and spatial information. Atmospheric observations i.e., ðy 2 RpÞ often represent concentra- tions of CH4 observed by surface, satellite, airborne, or some other observing system. Generally, atmospheric inversions do not directly optimize CH4 sectoral emissions from observations, and instead optimize CH4 ﬂuxes i.e., ðx 2 RnÞ, which represent the summation of CH4 emissions within a grid cell. In the ﬂux inversion setup, atmospheric CH4 observations y are used to estimate CH4 ﬂuxes x. We estimate this optimal state by ﬁnding the mode of the posterior ﬂux distribution ^Z ¼ ððKMÞTS1 y ðKMÞ þ Z1 A Þ 1 ð11Þ ^Z ¼ ððKMÞTS1 y ðKMÞ þ Z1 A Þ ð16Þ HðMzÞ ¼ xA þ AðMz xAÞ Code availability And G is the Gain Matrix G ¼ ∂^x ∂y: Code used for this analysis can be found at https://github.com/dcusworth/ partition_ﬂuxes_to_emissions. Code used for this analysis can be found at https://github.com/dcusworth/ partition_ﬂuxes_to_emissions. And x are the true atmospheric ﬂuxes. Therefore, Eq. 12 shows that the optimal solution ^x is a combination of the truth, smoothed by some prior and includes noise. From Eq. 12, we can create a ﬂux to posterior ﬂux operation H, given that our relationship M is known: Received: 19 May 2021; Accepted: 27 October 2021; Received: 19 May 2021; Accepted: 27 October 2021; HðxÞ ¼ xA þ Aðx xAÞ ð15Þ HðMzÞ ¼ xA þ AðMz xAÞ ð16Þ HðxÞ ¼ xA þ Aðx xAÞ ð15Þ ð15Þ Data availability Equation 5 can be shown to have an equivalent form that is often used in atmospheric trace gas retrievals26: y Fossil fuel prior emission inventories are available for download at https://doi.org/ 10.7910/DVN/HH4EUM. Wetland emission prior inventories are available at (https:// doi.org/10.3334/ORNLDAAC/1502). The EPA gridded methane inventory is available for download at https://www.epa.gov/ghgemissions/gridded-2012-methane-emissions. ^x ¼ xA þ Aðx xAÞ þ Gn ð12Þ Where A is the averaging kernel matrix A ¼ ∂^x ∂x: A ¼ I ^SS1 A ð13Þ And G is the Gain Matrix G ¼ ∂^x ∂y: G ¼ SAKTðKSAKT þ SyÞ 1 ð14Þ Where A is the averaging kernel matrix A ¼ ∂^x ∂x: ð13Þ ^Z ¼ ððKMÞTS1 y ðKMÞ þ Z1 A Þ 1 Equations 10 and 11 provide an explicit closed form solution for ^z, which is sufﬁcient for emission optimization without reliance on RWs. Therefore, appli- cation of Eqs. 10 and 11 into existing inverse frameworks would provide posterior emission estimates constrained by atmospheric observations. However, these equations require the computation of the matrix (KM), which can be large in cases where many atmospheric observations exist. An exactly equivalent solution is possible with just the products of the ﬂux inversion, speciﬁcally ^S, ^x, SA, and xA. We show one derivation below and provide an alternative derivation in the SI: OMMUNICATIONS EARTH & ENVIRONMENT \| (2021) 2:242 \| https://doi.org/10.1038/s43247-021-00312-6 \| www.nature.com/commsenv 6 ARTICLE References y Sm ¼ GSyGT: ð18Þ Sm ¼ GSyGT: ð18Þ Sm ¼ GSyGT: ð18Þ 4. UNFCCC. Adoption of the Paris Agreement. Report No. FCCC/CP/2015/L.9/ Rev.1, http://unfccc.int/resource/docs/2015/cop21/eng/l09r01.pdf, 2015 While Ss has the following representation: While Ss has the following representation: Ss ¼ ðI AÞSAðI AÞT ð19Þ 5. Saunois, M. et al. The global methane budget 2000–2017. Earth Syst. Sci. Data 12, 1561–1623 (2020). Ss ¼ ðI AÞSAðI AÞT ð19Þ We can combine Eqs. 17 and 19 to get an alternate form of Sm that does not require Sy and G explicitly: 6. Melton, J. R. et al. Present state of global wetland extent and wetland methane modelling: Conclusions from a model inter-comparison project (WETCHIMP). Biogeosciences 10, 753–788 (2013). Sm ¼ ^S ðI AÞSAðI AÞT ð20Þ Sm ¼ ^S ðI AÞSAðI AÞT ð20Þ 7. Poulter, B. et al. Global wetland contribution to 2000-2012 atmospheric methane growth rate dynamics. Environ. Res. Lett. https://iopscience.iop.org/ article/10.1088/1748-9326/aa8391 (2013). Using Sm for observational error covariance, we can apply the MAP solution to derive ^z and ^Z: ^z ¼ zA þ ^ZMTATS1 m ð^x HðMzAÞÞ ð21Þ ^z ¼ zA þ ^ZMTATS1 m ð^x xA AðMzA xAÞÞ ð22Þ ^Z ¼ ðMTATS1 m AM þ Z1 A Þ 1: ð23Þ ^z ¼ zA þ ^ZMTATS1 m ð^x HðMzAÞÞ 8. Zhang, Y. et al. Attribution of the accelerating increase in atmospheric methane during 2010–2018 by inverse analysis of GOSAT observations. Atmos. Chem. Phys. 21, 3643–3666 (2021). ð21Þ ð22Þ 9. Maasakkers, J. D. et al. 2010–2015 North American methane emissions, sectoral contributions, and trends: a high-resolution inversion of GOSAT satellite observations of atmospheric methane. Atmos. Chem. Phys. 21, 4339–4356 (2021). ^Z ¼ ðMTATS1 m AM þ Z1 A Þ 1: ^ ^Z ¼ ðMTATS1 m AM þ Z1 A Þ 1 108, 4116 https://doi.org/10.1029/ 2002JD002299, D3 (2003). 15. Rodgers, C. D. & Connor, B. J. Intercomparison of remote sounding instruments. J. Geophys. Res. Atmos. 108, 4116 https://doi.org/10.1029/ 2002JD002299, D3 (2003). Taking the inverse of Sm using the Eq. 27, we have: S1 m ¼ ð^SATÞ 1 ð28Þ 16. Shen, L. et al. Unravelling a large methane emission discrepancy in Mexico using satellite observations. Remote Sens. Environ. 260, 112461 (2021). 16. Shen, L. et al. Unravelling a large methane emission discrepancy in Mexico using satellite observations. Remote Sens. Environ. 260, 112461 (2021). S1 m ¼ ðATÞ 1^S 1: ð29Þ S1 m ¼ ðATÞ 1^S 1 17. Meirink, J. F., Bergamaschi, P. & Krol, M. C. Four-dimensional variational data assimilation for inverse modelling of atmospheric methane emissions: method and comparison with synthesis inversion. Atmos. Chem. Phys. 8, 6341–6353 (2008). The algebra for 28 and 29 are only possible if ðATÞ1 exists. For overdetermined systems (i.e., dimension of y » dimension of x), this is generally valid. Multiplying both sides of Eq. 29 by AT ﬁnishes the proof: 18. Bousserez, N. & Henze, D. K. Optimal and scalable methods to approximate the solutions of large-scale Bayesian problems: theory and application to atmospheric inversion and data assimilation. Q. J. R. Meteorol. Soc. 144, 365–390 (2018). ATS1 m ¼ ^S 1: In a similar fashion, we can show that Eqs. 2 and 23 are equivalent if ATS1 m A ¼ ^S 1 S1 A . To do this, we multiply Eq. 30 by A: In a similar fashion, we can show that Eqs. 2 and 23 are equivalent if ATS1 m A ¼ ^S 1 S1 A . To do this, we multiply Eq. 30 by A: 19. Zhang, Y. et al. Quantifying methane emissions from the largest oil-producing basin in the United States from space. Sci. Adv. 6, eaaz5120 (2020). ATS1 m A ¼ ^S 1A ð31Þ ¼ ^S 1ðI ^SS1 A Þ ð32Þ ¼ ^S 1 S1 A : ð33Þ 19. Zhang, Y. et al. Quantifying methane emissions from the largest oil-producing basin in the United States from space. Sci. Adv. 6, eaaz5120 (2020). 20. Kuze, A. et al. Update on GOSAT TANSO-FTS performance, operations, and data products after more than 6 years in space. Atmos. Meas. Tech. 9, 2445–2461 (2016). 21. Scarpelli, T. R. et al. References Þ el, as 7Þ Sm 8Þ 9Þ t 0Þ to 1Þ 2Þ 3Þ f a - s hat 4Þ 5Þ 6Þ 7Þ 8Þ 9Þ ed ng 0Þ 1Þ 2Þ 3Þ References 1. Myhre, G. et al. Anthropogenic and Natural Radiative Forcing Supplementary Material. In: Climate Change 2013: The Physical Science Basis. Contribution of Working Group I to the Fifth Assessment Report of the Intergovernmental Panel on Climate Change Available from www.climatechange2013.org and www.ipcc.ch. (2013). 2. United Nations Environment Programme and Climate and Clean Air Coalition (2021). Global Methane Assessment: Beneﬁts and Costs of Mitigating Methane Emissions. Nairobi: United Nations Environment Programme (2021). 3. Kirschke, S. et al. Three decades of global methane sources and sinks. Nature Geoscience 6, 813–823 (2013). 4. UNFCCC. Adoption of the Paris Agreement. Report No. FCCC/CP/2015/L.9/ Rev.1, http://unfccc.int/resource/docs/2015/cop21/eng/l09r01.pdf, 2015 5. Saunois, M. et al. The global methane budget 2000–2017. Earth Syst. Sci. Data 12, 1561–1623 (2020). 6. Melton, J. R. et al. Present state of global wetland extent and wetland methane modelling: Conclusions from a model inter-comparison project (WETCHIMP). Biogeosciences 10, 753–788 (2013). 7. Poulter, B. et al. Global wetland contribution to 2000-2012 atmospheric methane growth rate dynamics. Environ. Res. Lett. https://iopscience.iop.org/ article/10.1088/1748-9326/aa8391 (2013). 8. Zhang, Y. et al. Attribution of the accelerating increase in atmospheric methane during 2010–2018 by inverse analysis of GOSAT observations. Atmos. Chem. Phys. 21, 3643–3666 (2021). 9. Maasakkers, J. D. et al. 2010–2015 North American methane emissions, sectoral contributions, and trends: a high-resolution inversion of GOSAT satellite observations of atmospheric methane. Atmos. Chem. Phys. 21, 4339–4356 (2021). 10. Bergamaschi, P. et al. Inverse modelling of European CH4 emissions during 2006–2012 using different inverse models and reassessed atmospheric observations. Atmos. Chem. Phys. 18, 901–920 (2018). 11. Alexe, M. et al. Inverse modelling of CH4 emissions for 2010–2011 using different satellite retrieval products from GOSAT and SCIAMACHY. Atmos. Chem. Phys. 15, 113–133 (2015). 12. Yadav, V. et al. Spatio‐temporally resolved methane ﬂuxes from the Los Angeles Megacity. J. Geophys. Res. Atmos. 124, 5131–5148 (2019). 13. Miller, S. M. et al. Anthropogenic emissions of methane in the United States. Proc. Natl Acad. Sci. USA 110, 20018–20022 (2013). 14. Ganesan, A. L. et al. Quantifying methane and nitrous oxide emissions from the UK and Ireland using a national-scale monitoring network. Atmos. Chem. Phys. 15, 6393–6406 (2015). 15. Rodgers, C. D. & Connor, B. J. Intercomparison of remote sounding instruments. J. Geophys. Res. Atmos. NICATIONS EARTH & ENVIRONMENT \| (2021) 2:242 \| https://doi.org/10.1038/s43247-021-00312-6 \| www.nature.com/commsenv References 108, 4116 https://doi.org/10.1029/ 2002JD002299, D3 (2003). 16. Shen, L. et al. Unravelling a large methane emission discrepancy in Mexico using satellite observations. Remote Sens. Environ. 260, 112461 (2021). 17. Meirink, J. F., Bergamaschi, P. & Krol, M. C. Four-dimensional variational data assimilation for inverse modelling of atmospheric methane emissions: method and comparison with synthesis inversion. Atmos. Chem. Phys. 8, 6341–6353 (2008). 18. Bousserez, N. & Henze, D. K. Optimal and scalable methods to approximate the solutions of large-scale Bayesian problems: theory and application to atmospheric inversion and data assimilation. Q. J. R. Meteorol. Soc. 144, 365–390 (2018). 19. Zhang, Y. et al. Quantifying methane emissions from the largest oil-producing basin in the United States from space. Sci. Adv. 6, eaaz5120 (2020). 20. Kuze, A. et al. Update on GOSAT TANSO-FTS performance, operations, and data products after more than 6 years in space. Atmos. Meas. Tech. 9, 2445–2461 (2016). 21. Scarpelli, T. R. et al. A global gridded (0.1× 0.1) inventory of methane emissions from oil, gas, and coal exploitation based on national reports to the 1. Myhre, G. et al. Anthropogenic and Natural Radiative Forcing Supplementary Material. In: Climate Change 2013: The Physical Science Basis. Contribution of Working Group I to the Fifth Assessment Report of the Intergovernmental Panel on Climate Change Available from www.climatechange2013.org and www.ipcc.ch. (2013). The operation H allows for emissions to be smoothed with an averaging kernel, allowing for direct comparison with ^x. With this operator relationship, we treat ^x as an observable. The error covariance ^S includes both smoothing (Ss) and mea- surement error (Sm): ^S ¼ Ss þ Sm ð17Þ 2. United Nations Environment Programme and Climate and Clean Air Coalition (2021). Global Methane Assessment: Beneﬁts and Costs of Mitigating Methane Emissions. Nairobi: United Nations Environment Programme (2021). 2. United Nations Environment Programme and Climate and Clean Air Coalition (2021). Global Methane Assessment: Beneﬁts and Costs of Mitigating Methane Emissions. Nairobi: United Nations Environment Programme (2021). 3. Kirschke, S. et al. Three decades of global methane sources and sinks. Nature Geoscience 6 813 823 (2013) For ﬂux partitioning, we want to isolate the Sm error component ^x, as the H operator already accounts for smoothing via the averaging kernel. The matrix Sm can be represented32 using G and Sy: g 3. Kirschke, S. et al. Three decades of global methane sources and sinks. Nature Geoscience 6, 813–823 (2013). ^Z ¼ ðMTATS1 m AM þ Z1 A Þ 1 ð23Þ Now we have a direct solution for ^z and ^Z derived only from the products of a ﬂux inversion (speciﬁcally, xA; SA, ^x, ^S), an emissions prior (ZA), and an aggre- gation matrix M. Equations 22 and 23 can be shown to be of the same form as Eqs. 1 and 2 by showing that ATS1 m ¼ ^S 1. Decomposing AT and recognizing that ^S and SA are symmetric matrices, we have 10. Bergamaschi, P. et al. Inverse modelling of European CH4 emissions during 2006–2012 using different inverse models and reassessed atmospheric observations. Atmos. Chem. Phys. 18, 901–920 (2018). 11. Alexe, M. et al. Inverse modelling of CH4 emissions for 2010–2011 using different satellite retrieval products from GOSAT and SCIAMACHY. Atmos. Chem. Phys. 15, 113–133 (2015). AT ¼ ðI ^SS1 A Þ T ¼ I ðS1 A Þ T^S T ¼ I S1 A ^S: ð24Þ AT ¼ ðI ^SS1 A Þ T ¼ I ðS1 A Þ T^S T ¼ I S1 A ^S: ð24Þ We ﬁrst expand Eq. 19 for an alternative expression of Sm: y 12. Yadav, V. et al. Spatio‐temporally resolved methane ﬂuxes from the Los Angeles Megacity. J. Geophys. Res. Atmos. 124, 5131–5148 (2019). 12. Yadav, V. et al. Spatio‐temporally resolved methane ﬂuxes from the Los A l M i J G h R A 124 5131 5148 (2019) Sm ¼ ^S ðI AÞSAðI AÞT Sm ¼ ^S ðI AÞSAðI AÞT ¼ ^S ðI I þ ^SS1 A ÞSAðI I þ ^SS1 A Þ T ¼ ^SAT: Sm ¼ ^S ðI AÞSAðI AÞT ð25Þ ¼ ^S ðI I þ ^SS1 A ÞSAðI I þ ^SS1 A Þ T ð26Þ ¼ ^SAT: ð27Þ ð25Þ g g y p y 13. Miller, S. M. et al. Anthropogenic emissions of methane in the United States. Proc. Natl Acad. Sci. USA 110, 20018–20022 (2013). ¼ ^S ðI I þ ^SS1 A ÞSAðI I þ ^SS1 A Þ T ð26Þ 14. Ganesan, A. L. et al. Quantifying methane and nitrous oxide emissions from the UK and Ireland using a national-scale monitoring network. Atmos. Chem. Phys. 15, 6393–6406 (2015). ¼ ^SAT: ð27Þ y 15. Rodgers, C. D. & Connor, B. J. Intercomparison of remote sounding instruments. J. Geophys. Res. Atmos. Reprints and permission information is available at http://www.nature.com/reprints Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional afﬁliations. Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional afﬁliations. 30. Friedlingstein, P. et al. Global carbon budget 2020. Earth Syst. Sci. Data 12, 3269–3340 (2020). 31. Jiang, Z. et al. Impact of model errors in convective transport on CO source estimates inferred from MOPITT CO retrievals. J. Geophys. Res. Atmos. 118, 2073–2083 (2013). Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/ licenses/by/4.0/. 32. Bowman, K. W. et al. Tropospheric emission spectrometer: retrieval method and error analysis. IEEE Trans. Geosci. Remote Sens. 44, 1297–1307 (2006). Author contributions United Nations Framework Convention on Climate Change. Earth System Science. Data 12, 563–575 (2020). D.H.C. and J.R.W. designed the study. D.H.C. performed the analysis and wrote the manuscript. D.H.C., Y.Y., J.R.W., A.A.B., and K.B. developed/derived the algebraic equations. S.M., J.D.M., C.E.M., T.R.S., Z.Q., D.J.J., and Y.Z. provided and guided interpretation and integration of prior and posterior inventories. All authors provided feedback and edits on the manuscript. D.H.C. and J.R.W. designed the study. D.H.C. performed the analysis and wrote the manuscript. D.H.C., Y.Y., J.R.W., A.A.B., and K.B. developed/derived the algebraic equations. S.M., J.D.M., C.E.M., T.R.S., Z.Q., D.J.J., and Y.Z. provided and guided interpretation and integration of prior and posterior inventories. All authors provided feedback and edits on the manuscript. 22. Ma, S. et al. Satellite constraints on the latitudinal distribution and temperature sensitivity of wetland methane emissions. AGU Adv. 2, e2021AV000408 (2021). 23. Maasakkers, J. D. et al. Gridded national inventory of US methane emissions. Environ. Sci. Technol. 50, 13123–13133 (2016). 24. EIA. Drilling Productivity Report, URL https://www.eia.gov/petroleum/ drilling/, Last Accessed 2 Feb 2021 (2020). p g The authors declare no competing interests. g 25. Crippa, M. et al. Fossil CO2 and GHG emissions of all world countries - 2019 Report, EUR 29849 EN, Publications Ofﬁce of the European Union, Luxembourg, ISBN 978-92-76-11100-9, 10.2760/687800, JRC117610. (2019). Additional information Supplementary information The online version contains supplementary material available at https://doi.org/10.1038/s43247-021-00312-6. g 26. Rodgers, C. D. Inverse methods for atmospheric sounding: theory and practice (Vol. 2). World Scientiﬁc (World Scientiﬁc Publishing Co. Pte. Ltd, 2000). 27. Veefkind, J. P. et al. TROPOMI on the ESA Sentinel-5 Precursor: A GMES mission for global observations of the atmospheric composition for climate, air quality and ozone layer applications. Remote Sens. Environ. 120, 70–83 (2012). Correspondence and requests for materials should be addressed to Daniel H. Cusworth. Correspondence and requests for materials should be addressed to Daniel H. Cusworth. Peer review information Communications Earth & Environment thanks Luke Western, Hossein Maazallahi and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Primary Handling Editors: Joshua Dean and Clare Davis. Peer reviewer reports are available. Peer review information Communications Earth & Environment thanks Luke Western, Hossein Maazallahi and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Primary Handling Editors: Joshua Dean and Clare Davis. Peer reviewer reports are available. 28. Qu, Z. et al. Global distribution of methane emissions: a comparative inverse analysis of observations from the TROPOMI and GOSAT satellite instruments. Atmos. Chem. Phys. Discussions 21, 14159–14175 (2021). 29. Crisp, D. et al. A constellation architecture for monitoring carbon dioxide and methane from space. Prepared by the CEOS Atmospheric Constellation Greenhouse Gas Team, Version, 1(8), https://ceos.org/document_management/ Virtual_Constellations/ACC/Documents/CEOS_AC-VC_GHG_White_Paper_ Version_1_20181009.pdf (2018). Reprints and permission information is available at http://www.nature.com/reprints ^Z ¼ ðMTATS1 m AM þ Z1 A Þ 1 A global gridded (0.1× 0.1) inventory of methane emissions from oil, gas, and coal exploitation based on national reports to the ATS1 m A ¼ ^S 1A ¼ ^S 1ðI ^SS1 A Þ ¼ ^S 1 S1 A : ¼ S ðI SSA Þ ð32Þ ¼ ^S 1 S1 A : ð33Þ data products after more than 6 years in space. Atmos. Meas. Tech. 9, 2445–2461 (2016). 21. Scarpelli, T. R. et al. A global gridded (0.1× 0.1) inventory of methane emissions from oil, gas, and coal exploitation based on national reports to the ð33Þ 21. Scarpelli, T. R. et al. A global gridded (0.1× 0.1) inventory of methane emissions from oil, gas, and coal exploitation based on national reports to the ¼ ^S 1 S1 A : ð33Þ 21. Scarpelli, T. R. et al. A global gridded (0.1× 0.1) inventory of methane emissions from oil, gas, and coal exploitation based on national reports to the ¼ ^S 1 S1 A : ð33Þ 21. Scarpelli, T. R. et al. A global gridded (0.1× 0.1) inventory of methane emissions from oil, gas, and coal exploitation based on national reports to the COMMUNICATIONS EARTH & ENVIRONMENT \| (2021) 2:242 \| https://doi.org/10.1038/s43247-021-00312-6 \| www.nature.com/commsenv COMMUNICATIONS EARTH & ENVIRONMENT \| (2021) 2:242 \| https://doi.org/10.1038/s43247-021-00312-6 \| www.nature.com/commse 7 ARTICLE © The Author(s) 2021 Acknowledgements g Portions of this work research was carried out at the Jet Propulsion Laboratory, Cali- fornia Institute of Technology, under a contract with the National Aeronautics and Space Ad i i t ti (80NM0018D0004) S f th k t d b NASA’ C b Portions of this work research was carried out at the Jet Propulsion Laboratory, Cali- fornia Institute of Technology, under a contract with the National Aeronautics and Space Administration (80NM0018D0004). Some of the work was supported by NASA’s Carbon Monitoring System program. Yuzhong Zhang acknowledges funding by NSFC (42007198). Government sponsorship acknowledged. Portions of this work research was carried out at the Jet Propulsion Laboratory, Cali- fornia Institute of Technology, under a contract with the National Aeronautics and Space Administration (80NM0018D0004). Some of the work was supported by NASA’s Carbon Monitoring System program. Yuzhong Zhang acknowledges funding by NSFC (42007198). Government sponsorship acknowledged. © The Author(s) 2021 COMMUNICATIONS EARTH & ENVIRONMENT \| (2021) 2:242 \| https://doi.org/10.1038/s43247-021-00312-6 \| www.nature.com/commsenv OMMUNICATIONS EARTH & ENVIRONMENT \| (2021) 2:242 \| https://doi.org/10.1038/s43247-021-00312-6 \| www.nature.com/commsenv 8 8
https://openalex.org/W2617618349	https://europepmc.org/articles/pmc5444844?pdf=render	English	null	Revisiting the discriminatory accuracy of traditional risk factors in preeclampsia screening	PloS one	2,017	cc-by	11,266	Methods A stratified analysis by parity was conducted using the Swedish Birth Register between 2002–2010 including 626.600 pregnancies. The discriminatory accuracy (DA) of traditional definitions of high-risk women was compared with a new definition based on 1) specific com- binations of individual variables and 2) a centile cut-off of the probability of PE predicted by a multiple logistic regression model. Copyright: © 2017 Rodriguez-Lopez et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Editor: Cassandra Nichole Spracklen, University of North Carolina at Chapel Hill, UNITED STATES Editor: Cassandra Nichole Spracklen, University of North Carolina at Chapel Hill, UNITED STATES Received: January 19, 2017 Accepted: May 15, 2017 Published: May 25, 2017 Copyright: © 2017 Rodriguez-Lopez et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Received: January 19, 2017 Accepted: May 15, 2017 Published: May 25, 2017 Merida Rodriguez-Lopez1,2, Philippe Wagner1, Raquel Perez-Vicente1, Fatima Crispi2, Juan Merlo1 Merida Rodriguez-Lopez1,2, Philippe Wagner1, Raquel Perez-Vicente1, Fatima Crispi2, Juan Merlo1 1 Unit for Social Epidemiology, Faculty of Medicine, Lund University, Malmo¨, Sweden, 2 Fetal i+D Fetal Medicine Research Center, BCNatal—Barcelona Center for Maternal-Fetal and Neonatal Medicine (Hospital Clı´nic and Hospital Sant Joan de Deu), Institut Clı´nic de Ginecologia, Obstetricia i Neonatologia, Institut d’Investigacions Biomèdiques August Pi i Sunyer, Universitat de Barcelona, and Centre for Biomedical Research on Rare Diseases (CIBER-ER), Barcelona, Spain a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 * merida.rdguez@gmail.com Background Preeclampsia (PE) is associated with a high risk of perinatal morbidity and mortality. How- ever, there is no consensus in the definition of high-risk women. Citation: Rodriguez-Lopez M, Wagner P, Perez- Vicente R, Crispi F, Merlo J (2017) Revisiting the discriminatory accuracy of traditional risk factors in preeclampsia screening. PLoS ONE 12(5): e0178528. https://doi.org/10.1371/journal. pone.0178528 RESEARCH ARTICLE * merida.rdguez@gmail.com Aim To question current definition of high PE risk and propose a definition that considers individ- ual heterogeneity to improves risk classification. Introduction Preeclampsia (PE) is defined as the new onset of hypertension and proteinuria after the 20th gestational week. This complication affects 3–10% of all pregnancies [1], and associates a higher risk of perinatal complication such as intrauterine growth restriction, prematurity and maternal mortality[2]. Although the underlying causes of PE are still unknown[3], a number of different risk-factors have been recognized and are currently recommended in screening guidelines for its early prediction and prevention[4–6]. In fact, low-dose aspirin (LDA) has been proved to reduce the risk of PE in selected high-risk patients and it is usually recom- mended for preventing PE. However, there is no consensus among guidelines on how to iden- tify those women at higher risk who might benefit from a close follow-up and prophylactic treatment with LDA. Competing interests: The authors have declared that no competing interests exist. For instance, the NICE guideline classifies women as at high-risk for PE when any major risk factor (i.e. previous PE, chronic kidney disease (CKD), autoimmune disease, diabetes mel- litus (DM), or chronic hypertension (HBP)) or at least two moderate risk factors (i.e., primi- parity, 40 years old, inter-pregnancy interval >10 years, obesity at first visit, family history of PE or multiple pregnancy) are present[6]. The Task Force guidelines[7], however, defines high- risk for screening when at least one of the above risk factors (major or minor) is present, but recommends LDA only in those with more than one previous PE or one previous PE deliver- ing preterm. The World Health Organization guideline[5] define high risk as the existence of at least one major risk factor including multiple pregnancy. Likewise, local guidelines are used in Sweden to define high-risk of PE. For example, in the region of Skåne, Sweden, the definition of high risk includes only major risk factors, and specifies that only those diabetics with vascu- lar damage and those previous PE with severe, early or simultaneous fetal growth restriction should be included[8]. Overall, current screening guidelines are easy to apply in everyday practice as they are not based on complicated risk equations but mainly on the existence of one or two risk factors. The selection of those risk factors is mainly based on the difference in the average risk between exposed and non-exposed women, which is normally appraised by measures of association like the odds ratio (OR). Conclusions Funding: This work was partially supported by the Erasmus + Programme of the European Union (Framework Agreement number: 2013-0040) to MR-L. This publication reflects the views only of the author, and the Commission cannot be held responsible for any use, which may be made of the information contained therein. This work was also supported by the Swedish Research Council (PI: JM, 2013-2484). Additionally, the research leading to these results has received funding from “la Caixa” Foundation; Cerebra Foundation for the Brain Injured Child (Carmarthen, Wales, UK) and AGAUR 2014 SGR grant nº 928. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. In preeclampsia prediction the combination of specific risk factors provided a better discrimi- natory accuracy than traditional single risk approach. Our results contribute to a more per- sonalized risk estimation of preeclampsia. Risk factors in preeclampsia screening Results None of the classical risk-factors alone reached an acceptable DA. In multiparous, any com- bination of a risk-factor with previous PE or HBP reached a +LR>10. The combination of obesity and multiple pregnancy reached a good DA particularly in the presence of previous preeclampsia (positive likelihood ratio (LR+) = 26.5 or chronic hypertension (HBP) LR+ = 40.5. In primiparous, a LR+>15 was observed in multiple pregnancies with the simultaneous presence of obesity and diabetes mellitus or with HBP. Predicted probabilities above 97 centile in multiparous and 99 centile in primiparous provided high (LR+ = 12.5), and moder- ate (LR+ = 5.85), respectively. No one risk factor alone or in combination provided a LR- suf- ficiently low to rule-out the disease. Data Availability Statement: Data are from the Swedish Medical Birth Register, which is available upon request from the Swedish National Board of Health and Welfare, a central government agency, following presentation of an appropriate ethical permit. For information in English please see: http://www.socialstyrelsen.se/register/ halsodataregister/medicinskafodelseregistret/ inenglish. Researchers can contact Sara Segelson (sara.segelson@socialstyrelsen.se) for more information. 1 / 19 PLOS ONE \| https://doi.org/10.1371/journal.pone.0178528 May 25, 2017 Population and study design This study is based on the Swedish Medical Birth Registry (MBR) that includes detailed and standardized information on nearly all pregnancies in Sweden culminating in delivery. Over- all, the quality of the register is rather high as described elsewhere[15,16]. Using the unique Swedish personal identification number, the National Board of Health and Welfare and Statis- tics in Sweden linked the MBR to the Patient register that records all inpatient and outpatient hospital diagnoses. In addition, the longitudinal integration database for health insurance and labour market studies (LISA) records information on socioeconomic factors. Data is codified by a personal identification code without access to the real personal identification number in order to protect the participants’ anonymity. The regional ethical review board in southern Sweden approved the database use and did not required the explicit informed consent from women. We identified all the 938.932 deliveries recorded in the MBR from 1st January 2002 to 31th December 2010. We decided to restrict the analysis to deliveries from Swedish mothers that has been residing in Sweden for more or equal to 5 years in order to improve the homogeneity, completeness and validity of the information. To prevent that the same episode of PE counted twice in multiple pregnancies, only one children were randomly selected among those belong- ing to the same pregnancy. The flow diagram of the study population is shown in Fig 1. The final study sample consisted of 626.600 pregnancies, representing 67% of the initial population. The prevalence of PE was close to 4% in both the final sample and the original population. Characteristics of the included and excluded population is shown in supplemental data (S1 Table). Introduction However, this form of screening could be criticized, as previous meth- odological studies[9–12] have stressed that measures of association alone are inappropriate to discriminate between individuals who will subsequently suffer a disease from those who will not. Actually, what it is often considered as a robust association (e.g., OR5), is related to a rather low discriminatory accuracy due to a high false positive fraction (FPF) and/or false neg- ative fraction (FNF) in the population[9–12]. Therefore, current guidelines may translate in an overdiagnosis (thereby unnecessary treatments, avoidable monitoring, and increase parental anxiety) or, on the contrary, underdiagnosis that may prevent a close follow-up of patients at risk of PE. A suitable approach to improve discriminatory accuracy in clinical decision making could be to adopt the perspective of precision medicine[13] aiming to better understand individual heterogeneity in PE risk and, thereby, achieve a higher predictive accuracy. Therefore, the PLOS ONE \| https://doi.org/10.1371/journal.pone.0178528 May 25, 2017 2 / 19 Risk factors in preeclampsia screening objective of this study was double. Firstly, using measures of discriminatory accuracy, we aimed to question established risk classification guidelines for PE. Subsequently, we aimed to apply two alternative definitions of high-risk. The first was based on specific combinations of risk factors selected by random forest analysis[14] and the second was based on predicted prob- abilities derived from multiple logistic regression models. Assessment of variables The main outcome of our study was PE or eclampsia defined as a diagnosis coded O14 or O15, respectively, according the International Classification of Diseases 10th version (ICD-10). In this manuscript both pathologies are named PE. PE was recoded as positive whether it was present in the MBR or in the Patient register from 20 weeks of gestational age and up to six weeks after delivery. Gestational hypertension (ICD-10 code O13), chronic hypertension with proteinuria (O11) and edema and proteinuria (O12) were included as non-PE. Non-specified hypertensive disorder (O16) was considered as HBP. When the same patient was categorized as Gestational hypertension and PE was included as PE as previous studies with the MBR[17]. All the exposure variables were dichotomized in order to follow a similar approach as in current clinical guidelines and categorized as: maternal age into <40 or 40 years, educational achievement as 11 years or 12 years of formal education obtained the year before the deliv- ery and family situation as cohabiting with the child’s father or not. The presence of disease (ICD-10 codes) was identified at the first antenatal visit, at the hospital discharge after delivery and/or during a five-year period before pregnancy, and was considered as positive whether it was present in the MBR or in the Patient register. The codes obtained were: HBP (I10-I15)), DM (E10, E11-E14)), CKD (N18, N19)), and autoimmune diseases including systemic lupus erythematosus (M32) and rheumatoid arthritis (M05, M06). There were only 26 pregnancies The main outcome of our study was PE or eclampsia defined as a diagnosis coded O14 or O15, respectively, according the International Classification of Diseases 10th version (ICD-10). In this manuscript both pathologies are named PE. PE was recoded as positive whether it was present in the MBR or in the Patient register from 20 weeks of gestational age and up to six weeks after delivery. Gestational hypertension (ICD-10 code O13), chronic hypertension with proteinuria (O11) and edema and proteinuria (O12) were included as non-PE. Non-specified hypertensive disorder (O16) was considered as HBP. When the same patient was categorized as Gestational hypertension and PE was included as PE as previous studies with the MBR[17]. PLOS ONE \| https://doi.org/10.1371/journal.pone.0178528 May 25, 2017 Assessment of variables All the exposure variables were dichotomized in order to follow a similar approach as in current clinical guidelines and categorized as: maternal age into <40 or 40 years, educational achievement as 11 years or 12 years of formal education obtained the year before the deliv- ery and family situation as cohabiting with the child’s father or not. The presence of disease (ICD-10 codes) was identified at the first antenatal visit, at the hospital discharge after delivery and/or during a five-year period before pregnancy, and was considered as positive whether it was present in the MBR or in the Patient register. The codes obtained were: HBP (I10-I15)), DM (E10, E11-E14)), CKD (N18, N19)), and autoimmune diseases including systemic lupus erythematosus (M32) and rheumatoid arthritis (M05, M06). There were only 26 pregnancies PLOS ONE \| https://doi.org/10.1371/journal.pone.0178528 May 25, 2017 3 / 19 Risk factors in preeclampsia screening with antiphospholipid syndrome (D686), therefore it was included together with autoimmune diseases. Obesity was defined as body mass index (BMI) 30kg/m2[17–19]. Information on smoking habits before pregnancy was self-reported and concerned the status up to 3 months Fig 1. Flow diagram showing the selection of study population of deliveries in the Swedish birth register. https://doi.org/10.1371/journal.pone.0178528.g001 Risk factors in preeclampsia screening Fig 1. Flow diagram showing the selection of study population of deliveries in the Swedish birth register. https://doi.org/10.1371/journal.pone.0178528.g001 https://doi.org/10.1371/journal.pone.0178528.g001 with antiphospholipid syndrome (D686), therefore it was included together with autoimmune diseases. Obesity was defined as body mass index (BMI) 30kg/m2[17–19]. Information on smoking habits before pregnancy was self-reported and concerned the status up to 3 months PLOS ONE \| https://doi.org/10.1371/journal.pone.0178528 May 25, 2017 4 / 19 Risk factors in preeclampsia screening before pregnancy, and dichotomized into smoker (including mild: 1–9 and heavy 10 ciga- rettes per day) or non-smoker. Previous PE was defined whether O14-O15 codes were present in the Patient register from 270 days and up to five-year period before birthdate. Parity was dichotomized into primiparous or multiparous. Pregnancy characteristics included multiple pregnancy, conception by assisted reproductive technologies (ART) and gestational diabetes (O24). In the MBR, the data concerning the current pregnancy is collected prospectively, before onset of potential adverse pregnancy outcomes, which prevents recall bias. PE diagnose is noted by the responsible doctor at discharge from hospital. Statistical analysis Stratified analysis by parity. To decide whether to perform the analysis in the overall population or stratified by parity, we explored the interaction effect between each factor and parity by adding one interaction term to the baseline logistic model. Each model was specified as i = 1 if nulliparous and 0 if multiparous, and j = 1 when the risk factor was present and 0 otherwise. Then, OR11, OR10 and OR01 was obtained, OR00 was considered the reference category. Multiplicative interaction was determined by the ratio of Odds ratio (ratio of ORs = OR11 / (OR10 x OR01), directly obtained from the output of multiple logistic models. Additive interaction was determined by the relative excess of risk due to interaction relative to the risk without exposure also called Interaction contrast ratio (ICR = OR11−OR10−OR01+1), using the regression coefficients and covariance matrix obtained from the multiple logistic regres- sions. When the confidence interval did not include the value of one or zero, the interaction was considered statistically significant in the multiplicative or additive scale, respectively. The interaction was classified as positive in the multiplicative/additive scale if ratio of ORs>1/ ICR>0, negative if ratio if ORs<1/ICR<0 or absent if ratio of ORs = 1/ICR = 0. As significant modification was found in both multiplicative and the additive scales, all analyses were strati- fied on parity, also considering that previous PE can only be present in multiparous woman. The prevalence of risk factors in women with and without PE was calculated using point estimations and confidence intervals. Logistic regression analysis was performed to obtain crude and adjusted ORs stratified by parity. Starting from a saturated model containing all the variables, a backward elimination strategy was applied to construct the final multiple regres- sion model. A significance level of 0.1 was defined to exclude variables from the saturated model. When the confidence interval did not contain the null, the difference was considered statistically significant. Since the data was correlated (children from different pregnancies clus- tered within mothers) we obtained robust standard errors and 95%CI. Models fit were com- pared by Akaike information criteria (AIC). Thereafter, a variable based on the number of current risk factors[4–6] (i.e. previous PE, CKD, autoimmune disease, DM, HBP, 40 years old, obesity, multiple pregnancy and including ART and gestational diabetes) was created and then we explored whether their combinations could increase the risk of PE by subgroups of parity. Assessment of variables The information about chronic hypertension and other maternal demographic, clinical and reproductive characteristics are recorded by a midwife during mother´s first visit for antenatal care. Then, the data is for- warded to the MBR where the information is computerized. The records are standardized and are identical throughout the country, which minimizes information bias. Statistical analysis Smoking, cohabiting and education achievement were included as covariates in multi- ple logistic regression models. Current and new definition of high-risk groups for PE. To identify women at the high- est PE risk, we first adopted a similar approach as that used by current guidelines[4–6], and recreated three different classification of high risk of PE as having 1) one risk factor, 2) one 5 / 19 PLOS ONE \| https://doi.org/10.1371/journal.pone.0178528 May 25, 2017 Risk factors in preeclampsia screening major or two moderate and 3) one major including multiple pregnancy as explained in the introduction section. Information on family history of PE and pregnancy intervals was not available in all women. We included instead gestational diabetes and conception by ART as moderate risk factors. Then, random forest (RF)[14] was performed to guide variable selection for specific risk combination. The RF analysis also included smoking habits, cohabiting and education achievement. In short, RF fits many classification trees by randomly selected predic- tors and creating different trees by bootstrapping techniques. For this reason, RF produces more stable and accurate predictions than a single tree analysis. This algorithm allowed to identify the most important variables for splitting the data and therefore were subsequently used to create subgroups. RF are covered in more detail elsewhere[14–20]. Finally, we also cre- ated subgroups of risk based on predicted probabilities derived from multiple logistic regres- sion model. For this purpose, the predicted probabilities were dichotomized using three cut- offs and those women (> 95, >97 and >99 centiles) were considered as higher risk. Discriminatory accuracy of high risk groups. The discriminatory accuracy of the high- risk definition was determined based on a 1) single risk factors, 2) recreating three guidelines approach described above[5–7], 3) subgroups generated using RF and 4) individual risk proba- bilities predicted by multiple regression analysis. As previous PE and HBP are usually well rec- ognized by clinicians as very important risk factors for PE, we additionally evaluate the performance of the selected combination of risk factors among those without these conditions. For the same reason, an additional analysis was performed to identify combinations of risk fac- tors associated with a higher risk of PE among those without major factors. The absolute risk (AR), attributable risk (AF), true positive fraction (TPF), false positive fraction (FPF), and likelihood ratio (LR) were calculated. Statistical analysis Briefly, the LR shows the probability of having or not the risk factor (i.e., positive or negative) in patients with PE and compare to the probability of the same results in patients without PE. The null value is 1. In general, a LR+ >10 is considered high enough to rule-in PE, 5–10 moderate and 2–5 small[21,22]. The estab- lished criteria to rule-in PE is a LR+ > 10 and LR- < 0.2 to rule-out the disease with confi- dence[7]. We also evaluated the area under the ROC curve (AUC) and TPF for a FPF of 10% of the multiple logistic regression models. To consider the assumption of independency for RF, we chose one birth randomly from each multiparous mother. This strategy was also applied to obtained the measures of DA but the results were similar than those obtained keep- ing the clustered data. Therefore, we reported all results keeping the clustered data in multipa- rous. Analyses were performed using STATA 14 (Statacorp, College Station, Texas, US) and the randomForest Package in R version 0.99.893 (R Foundation for Statistical Computing, Vienna, Austria). PLOS ONE \| https://doi.org/10.1371/journal.pone.0178528 May 25, 2017 Heterogeneity of risk factors for preeclampsia and parity Preeclampsia was present in 3.83% of the population. The characteristics of the deliveries in multiparous and primiparous are presented in Table 1. Primiparous women presented a higher incidence of PE, lower education, and higher rate of no cohabiting with children’s father, smoking before pregnancy and conception by ART when compared to multiparous. The rest of the risk factors were higher in multiparous group with the exception of autoim- mune diseases that showed the same prevalence in both groups. In the overall population, DA of primiparity in relation to PE was rather low with a LR+ of 1.48 (1.46–1.49) and a LR- of 0.62 (0.61–0.63). An interaction effect was observed between each traditional risk factors and parity, in one or both scales. The effect of risk factors on PE was lower in nulliparous than in multiparous. PLOS ONE \| https://doi.org/10.1371/journal.pone.0178528 May 25, 2017 6 / 19 ( ) p value <0.001 from cluster-weighted Χ2 test p value 0.001 from cluster weighted Χ test ART: assisted reproductive technology, CKD: Chronic kidney disease, DM: Diabetes Mellitus, HBP: chronic hypert https://doi.org/10.1371/journal.pone.0178528.t001 Accuracy of current definition of high- risk groups Concerning a single factor, a LR+ around 10 was only present in multiparous women with HBP or previous PE. No one of the studied variables showed a LR- lower than 0.2 (Tables 2 and 3). Table 4 shows the DA of the high-risk definition analogous to the current guidelines approach. The higher sensitivity was observed when at least one risk factor was present. The inclusion of multiple pregnancy or two moderate risk factors reached similar DA. In the over- all population without a major risk factor no one combination of moderate risk-factors reached a LR10 neither a LR0.2 (S4 Table). The combination of primiparity and multiple pregnancy showed the higher OR, DA (LR+ = 6.66) and absolute risk or positive predictive value (AR = 18.61%). The adjusted OR associated with unspecific combination of risk factors was greater in multiparous than in primiparous, even when the analysis was restricted to those mothers without previous PE (S3 Table). For illustrative purposes, Fig 2 shows the magnitude of OR (in logarithm scale) with the increment of the number of clinical risk factors stratified by parity. Risk factors in preeclampsia screening Table 1. Sociodemographic, pre-pregnancy and pregnancy characteristics of the 626600 Swedish deliveries recorded between 2002 and 2010 stratified by parity. MultiparousN = 344001 PrimiparousN = 282599 Preeclampsia 8291 (2.41) 15732 (5.57) Sociodemographic Maternal age <40 328384 (95.46) 278016 (98.38) 40 15617 (4.54) 4583 (1.62)* Educational status Medium-High 136942 (39.81) 125662 (44.47) Low 207059 (60.19) 156937 (55.53)* Family situation Cohabiting 33148 (97.14) 265590 (93.98) Non-Cohabiting 9853 (2.86) 17009 (6.02)* Pre-pregnancy DM No 341490 (99.27) 280899 (99.40) Yes 2511 (0.73) 1700 (0.60)* HBP No 341150 (99.17) 280523 (99.27) Yes 2851 (0.83) 2076 (0.73)* Autoinmune disorders No 343230 (99.78) 281984 (99.78) Yes 771 (0.22) 615 (0.22) CKD No 342153 (99.456) 281266 (99.53) Yes 1848 (0.54) 1333 (0.47)* Obesity No 299824 (87.16) 254513 (90.06) Yes 44177 (12.84) 28086 (9.94)* Previous preeclampsia No 330594 (96.10) NA Yes 13407 (3.90) Smoker No 292038 (84.89) 223392 (79.05) Yes 51963 (15.11) 59207 (20.95)* Pregnancy Multiple pregnancy No 338376 (98.36) 279145 (98.78) Yes 5625 (1.64) 3454 (1.22)* Conception by ART No 338838 (98.50) 271486 (96.07) Yes 5163 (1.50) 11113 (3.93)* Gestational diabetes No 341039 (99.14) 280536 (99.27) Yes 2962 (0.86) 2063 (0.73)* Data are N (%). p value <0.001 from cluster-weighted Χ2 test ART: assisted reproductive technology CKD: Chronic kidney disease DM: Diabetes Mellitus HBP: chronic hypertension NA: Non applicable PLOS ONE \| https://doi.org/10.1371/journal.pone.0178528 May 25, 2017 7 / 19 Risk factors in preeclampsia screening The lowest ICR (ICR<2) was observed for the interaction of parity with HBP and with multi- ple pregnancy. An opposite direction of the interaction measures was observed between parity and diabetes and between parity and smoking.(S2 Table). 35.45% of PE cases occurred in preg- nancies without a known risk factor in multiparous and in 64.75% among primiparous. Tables 2 and 3 show the relationship between PE and sociodemographic, pre-pregnancy and preg- nancy characteristics among multiparous and primiparous women, respectively. In multipa- rous, all variables were positively associated with PE, with particularly strong associations for HBP, previous PE and multiple pregnancy. In both groups, the risk of PE was lower in smok- ers, while autoimmune disease was no longer associated with PE after adjustment. Despite the heterogeneity of the effect by parity, the direction of associations observed in multiple model was similar but family situation remained significant only among multiparous. PLOS ONE \| https://doi.org/10.1371/journal.pone.0178528 May 25, 2017 Accuracy of an alternative definition of high risk for preeclampsia in multiparous vs primiparous Fig 3 shows the most relevant variables for classifying women in relation to their PE risk according the results from the random forest analyses. Previous PE was the most relevant vari- able among multiparous while obesity was the most relevant variable in primiparous. In mul- tiparous, a combination of multiple pregnancy and obesity with HBP or previous PE was related to the greatest PE risk with an OR above 50 (Table 5). Any combination of a risk factor with previous PE or HBP also presented a LR+>10 (data no shown). In those without previous PE nor HBP, a LR>10 was also observed when multiple pregnancy and obesity were simulta- neously combined with: DM (n = 9, LR+ = 18(3.5–81.1), ERC (n = 4, LR+ = 19.7(2.05–189), ART (n = 59, LR+ = 30.3(17.6–51.9) or >40 years (n = 41, LR+ = 12.2(5.39–27.4)). Among those without major risk factors, we also observed that only specific combinations of risk fac- tors were associated with a greater risk of PE (S5 Table). Among those that were spontaneously conceived without major risk factors, multiple pregnancy and gestational diabetes was also associated with a higher risk in non-obese mothers (n = 22, LR+18.30(6.75–49). Regarding primiparous, the combination of multiple pregnancy and obesity increased the risk particularly in the presence of DM. The combination of HBP and multiple pregnancy also presented a LR>10. A moderate LR was observed in those with HBP and obesity, DM and obe- sity and DM with multiple pregnancy. There were no patients with these 4 factors neither with the combination of DM, HBP and multiple pregnancy. In the absence of HBP, the combina- tion of multiple pregnancy and obesity with gestational diabetes reached a moderate LR+ PLOS ONE \| https://doi.org/10.1371/journal.pone.0178528 May 25, 2017 8 / 19 Risk factors in preeclampsia screening raphic, pre-pregnancy and pregnancy characteristics and preeclampsia (PE) in the 344001 Swedish deliv- d between 2002 and 2010. Table 2. Relation between sociodemographic, pre-pregnancy and pregnancy characteristics and preeclampsia (PE) in the 344001 Swedish deliv- eries from multiparous woman recorded between 2002 and 2010. Risk Factor AR AF TPF (95% CI) FPF (95% CI) LR+ (95% IC) LR- (95% CI) ORc (95% CI) ORa (95% CI) Maternal age, y <40 2.33 Ref Ref 40 4.04 1.71 7.6 (7–8.2) 4.5 (4.4–4.5) 1.70 (1.58–1.84) 0.97 (0.96–0.97) 1.76 (1.62–1.91) 1.73 (1.58–1.89) Educational status Medium-High 2 Ref Ref Low 2.68 0.68 66.9 (65.9–67.9) 60 (59.2–60.2) 1.12 (1.10–1.13) 0.83 (0.80–0.85) 1.35 (1.29–1.41) 1.20 (1.14–1.26) Family situation Cohabiting 2.39 Ref Ref Non-Cohabiting 3.04 0.65 3.6 (3.2–4) 2.8 (2.8–2.9) 1.27 (1.14–1.42) 0.99 (0.99–1) 1.28 (1.14–1.44) 1.24 (1.09–1.41) Diabetes No 2.35 Ref Ref Yes 10.5 8.16 3.2 (2.8–3.6) 0.7 (0.6–0.7) 4.76 (4.20–5.39) 0.97 (0.97–0.98) 4.88 (4.26–5.57) 2.64 (2.25–3.10) HBP No 2.26 Ref Ref Yes 20.1 17.8 6.9 (6.46–7.5) 0.7 (0.7–0.7) 10.1 (9.30–11.1) 0.94 (0.93–0.94) 10.8 (9.85–11.9) 4.63 (4.07–5.27) Autoinmune No 2.40 Ref Ref Yes 5.58 3.18 0.5 (0.4–0.7) 0.2 (0.2–0.2) 2.39 (1.76–3.25) 1 2.39 (1.74–3.31) 1.54 (1.06–2.24) CKD No 2.38 Ref Ref Yes 7.52 5.14 1.7(1.4–2) 0.1 (0.1–0.1) 3.29 (2.77–3.91) 0.99 (0.99–0.99) 3.33 (2.78–3.99) 2.15 (1.73–2.68) Obesity No 1.91 Ref Ref Yes 5.80 3.89 30.9 (29.9–31.9) 12.4 (12.3–12.5) 2.49 (2.41–2.58) 0.79 (0.78–0.80) 3.16 (3.01–3.32) 2.44 (2.31–2.58) Previous PE No 1.76 Ref Ref Yes 18.4 16.6 19.7 (28.7–30.7) 3.3 (3.2–3.3) 9.10 (8.76–9.45) 0.73 (0.72–0.74) 12.51 (11.9–13.2) 10.6 (10–11.25) Smoker No 2.43 Ref Ref Yes 2.30 -0.13 14.4 (13.7–15.2) 15.1(15–15.2) 0.95 (0.91–1.01) 1.01 (1–1.02) 0.95 (0.89–1) 0.90 (0.84–0.97) Multiple pregnancy No 2.28 Ref Ref Yes 10.1 7.82 6.9 (6.3–7.4) 1.5 (1.5–1.5) 4.55 (4.18–4.95) 0.95 (0.94–0.95) 4.88 (4.40–5.26) 5.42 (4.91–5.99) Conception by ART No 2.37 Ref Ref Yes 5 2.63 3.1 (2.7–3.5) 1.5 (1.4–1.5) 2.13 (1.88–2.41) 0.98 (0.98–0.99) 2.16 (1.90–2.46) 1.71 (1.48–1.96) Gestational diabetes No 2.37 Ref Ref Yes 7.49 5.12 2.7 (2.3–3) 0.8 (0.8–0.8) 3.28 (2.87–3.75) 0.98 (0.98–0.98) 3.34 (2.91–3.84) 1.85 (1.57–2.19) AF: Attributable fraction, AR: Attributable risk, TPF: True positive fraction, ORc: crude Odds ratio, ORa: mutually adjusted Odds ratios, LR+: Positive Likelihood ratio, LR-: Negative Likelihood ratio, ART: assisted reproductive technology, CKD: Chronic kidney disease. https://doi.org/10.1371/journal.pone.0178528.t002 Table 2. Relation between sociodemographic, pre-pregnancy and pregnancy characteristics and preeclam eries from multiparous woman recorded between 2002 and 2010. Risk factors in preeclampsia screening Table 3. Relation between sociodemographic, pre-pregnancy and pregnancy characteristics and risk of preeclampsia (PE) in the 282599 Swedish deliveries from nulliparous woman recorded between 2002 and 2010. Risk Factor AR AF TPF (95% CI) FPF (95% CI) LR+ (95% IC) LR- (95% CI) Orc (95% CI) ORa (95% CI) Maternal age, y <40 5.51 Ref Ref 40 9.14 3.63 2.7 (2.4–2.9) 0.6 (0.5–0.6) 1.71 (1.55–1.88) 0.99 (0.99–0.99) 1.73 (1.56–1.91) 1.49 (1.31–1.62) Educational status Medium-High 5.06 Ref Ref Low 5.97 0.91 59.6 (58.8–60.3) 55.3 (55.1–55.5) 1.08 (1.06–1.09) 0.90 (0.89–0.92) 1.19 (1.15–1.23) 1.13 (1.09–1.17) Family situation Cohabiting 5.58 Ref Ref Non-Cohabiting 5.36 -0.22 5.8 (5.4–6.2) 6 (5.9–6.1) 0.96 (0.90–1.02) 1 0.96 (0.89–1.02) 0.94 (0.88–1.01) Diabetes No 5.46 Ref Ref Yes 22.7 17.3 2.5 (2.2–2.7) 0.5 (0.5–0.5) 4.98 (4.45–5.58) 0.98 (0.98–0.98) 5.08 (4.53–5.70) 4.29 (3.77–4.88) HBP No 5.53 Ref Ref Yes 24.1 18.5 3.2 (2.9–3.5) 0.6 (0.6–0.6) 5.37 (4.86–5.93) 0.97 (0.97–0.98) 5.51 (4.98–6.10) 4.07 (3.63–4.57) Autoimmune No 5.56 Ref Ref Yes 9.27 3.71 0.4 (0.3–0.5) 0.2 (0.2–0.2) 1.73 (1.32–2.27) 1 1.73 (1.32–2.28) 1.12 (0.78–1.60) CKD No 5.55 Ref Ref Yes 10.1 4.50 0.9 (0.7–1) 0.4 (0.4–0.5) 1.90 (1.59–2.26) 1 (0.99–1) 1.90 (1.59–2.28) 1.34 (1.09–1.66) Obesity No 4.84 Ref Ref Yes 12.1 7.28 21.6 (21–22.3) 9.2 (9.1–9.4) 2.34 (2.27–2.42) 0.86 (0.86–0.87) 2.71 (2.60–2.82) 2.56 (2.45–2.66) Smoker No 5.68 Ref Ref Yes 5.13 -0.55 19.3 (18.7–19.9) 21 (20.9–21.2) 0.92 (0.89–0.95) 1.02 (1.01–1.03) 0.89 (0.86–0.93) 0.83 (0.79–0.88) Multiple pregnancy No 5.39 Ref Ref Yes 19.7 14.3 4.3 (4–4.7) 1 (1–1.1) 4.17 (3.84–4.52) 0.97 (0.96–0.97) 4.31 (3.96–4.69) 4.27 (3.90–4.67) Conception by ART No 5.48 Ref Ref Yes 7.59 2.11 5.4 (5–5.7) 3.8 (3.8–3.9) 1.39 (1.30–1.49) 0.98 (0.98–0.99) 1.41 (1.32–1.52) 1.17 (1.08–1.25) Gestational diabetes No 5.51 Ref Ref Yes 13.9 8.40 1.8 (1.6–2) 0.7 (0.6–0.7) 2.74 (2.42–3.10) 0.99 (0.99–0.99) 2.77 (2.44–3.14) 2.04 (1.79–2.33) AF: Attributable fraction, AR: Attributable risk, TPF: True positive fraction, ORc: crude Odds ratio; ORa: mutually adjusted Odds ratios, LR+: Positive Likelihood ratio, LR-: Negative Likelihood ratio, ART: assisted reproductive technology, CKD: Chronic kidney disease, HBP: Chronic hypertension. htt //d i /10 1371/j l 0178528 t003 hic, pre-pregnancy and pregnancy characteristics and risk of preeclampsia (PE) in the 282599 Swedish d between 2002 and 2010. Table 3. Relation between sociodemographic, pre-pregnancy and pregnancy characteristics and risk of pre deliveries from nulliparous woman recorded between 2002 and 2010. AF: Attributable fraction, AR: Attributable risk, TPF: True positive fraction, ORc: crude Odds ratio, ORa: mutually adjusted Odds ratios, LR+: Positive Likelihood ratio, LR-: Negative Likelihood ratio, ART: assisted reproductive technology, CKD: Chronic kidney disease. AF: Attributable fraction, AR: Attributable risk, TPF: True positive fraction, ORc: crude Odds ratio, ORa: mutually ad Likelihood ratio, LR-: Negative Likelihood ratio, ART: assisted reproductive technology, CKD: Chronic kidney disea AF: Attributable fraction, AR: Attributable risk, TPF: True positive fraction, ORc: crude Odds ratio, ORa: mutually adjusted Odds ratios, LR+: Positive Likelihood ratio, LR-: Negative Likelihood ratio, ART: assisted reproductive technology, CKD: Chronic kidney disease. (n = 9, LR+ = 8.71(2.18–34.8) but a predictive value of 33%. In primiparous with no major risk factors, no one combination reached a LR>10 but a moderate LR was observed when multiple pregnancy was present in mother older than 40 or with obesity. PLOS ONE \| https://doi.org/10.1371/journal.pone.0178528 May 25, 2017 9 / 19 The AUC and sensitivity for the model including all variables was higher in multiparous when compared to primiparous(Table 5). Based on multiple regression model, those with a predicted probability>97 centile showed a LR>10 in multiparous (Table 6). In primiparous, those with a predicted probability >99centile showed a moderate LR+. The absence of any sin- gle risk factor, neither specific combination nor lower centiles from multiple regression model, reached a LR-below 0.2 in multiparous and primiparous. A summary of the highest risk group is shown in Table 7. PLOS ONE \| https://doi.org/10.1371/journal.pone.0178528 May 25, 2017 PLOS ONE \| https://doi.org/10.1371/journal.pone.0178528 May 25, 2017 10 / 19 Risk factors in preeclampsia screening Fig 2. Log-odds ratio for preeclampsia according to the number of clinical risk factors in multiparous and primiparous women adjusted by smoking, education and family situation. https://doi.org/10.1371/journal.pone.0178528.g002 Fig 2. Log-odds ratio for preeclampsia according to the number of clinical risk factors in multiparous and primiparous women adjusted by smoking, education and family situation. Fig 2. Log-odds ratio for preeclampsia according to the number of clinical risk factors in multiparous and primiparous women adjusted by smoking, education and family situation. https://doi.org/10.1371/journal.pone.0178528.g002 https://doi.org/10.1371/journal.pone.0178528.g002 https://doi.org/10.1371/journal.pone.0178528.g002 g y data obtained from multiple regression model adjusted by smoke, educational status and family situation AIC: Akaike information criteria, AF: attributable fraction, AR: attributable risk, TPF: true positive fraction, ORa: adjusted Odds ratios by smoker, educational status and family situation. LR+: Positive Likelihood ratio; LR-: Negative Likelihood ratio, CI: conﬁdence interval. Major risk factors included previous preeclampsia, chronic kidney disease, autoimmune disease, diabetes mellitus or chronic hypertension. Moderate risk factors included, maternal age40 years old, obesity at ﬁrst visit, multiple pregnancy; conception by assisted reproductive technologies and multiple pregnancy. https://doi.org/10.1371/journal.pone.0178528.t004 PLOS ONE \| https://doi.org/10.1371/journal.pone.0178528 May 25, 2017 Discussion Our population-based study confirms previous associations between PE and traditional risk factors, alone or in simple combinations. As recently recommended[23], we complement mea- sures of associations with measure of DA. When doing so, we found that neither single risk ditional approach for defining high- risk for preeclampsia in multiparous and in primiparous women. Table 4. Discriminatory accuracy of the traditional approach for defining high- risk for preeclampsia in multiparous and in primiparous women. Criteria N AR AF TPF (95% CI) FPF (95% CI) LR+ (95% CI) LR- (95% CI) ORa (95% CI)* AUC (95% CI) AIC* Multiparous Any Risk 81162 6.59 5.47 64.6 (63.5– 65.6) 22.6 (22.4– 22.7) 2.86 (2.81– 2.91) 0.46 (0.44– 0.47) 6.18 (5.90– 6.47) 72.0 (71.5–72.6) 71652 1 major or 2 moderate 25135 13.8 12.3 41.9 (40.9–43) 6.5 (6.4–6.5) 6.50 (6.35– 6.69) 0.62 (0.61– 0.63) 10.3 (9.87– 10.8) 69.9 (69–70.6) 70031 1major and Multiple pregnancy 25218 13.9 12.5 42.5 (41.4– 43.5) 6.5 (6.4–6.5) 6.57 (6.39– 6.76) 0.62 (0.60– 0.63) 10.6 (10.1– 11.1) 70.6 (69–71.2) 69805 Primiparous Any Risk 48924 11.3 6.97 35.2 (34.5–36) 16.3 (16.1– 16.4) 2.17 (2.12– 2.22) 0.77 (0.76– 0.78) 2.77 (2.69– 2.87) 60.9 (60.4–61.4) 118237 1 major or 2 moderate 9235 17.9 12.7 10.5 (10–11) 2.8 (2.9–2.8) 3.69 (3.51– 3.88) 0.92 (0.92– 0.93) 3.96 (3.75– 4.19) 56.2 (55.8–56.7) 119488 1major and Multiple pregnancy 8769 19.1 14.1 10.6 (10.2– 11.1) 2.7 (2.6–2.7) 4 (3.80–4.21) 0.92 (0.91– 0.92) 4.35 (4.11– 4.60) 56.5 (56.3–57) 119310 *data obtained from multiple regression model adjusted by smoke, educational status and family situation AIC: Akaike information criteria, AF: attributable fraction, AR: attributable risk, TPF: true positive fraction, ORa: adjusted Odds ratios by smoker, educational status and family situation. LR+: Positive Likelihood ratio; LR-: Negative Likelihood ratio, CI: conﬁdence interval. PLOS ONE \| https://doi.org/10.1371/journal.pone.0178528 May 25, 2017 11 / 19 Risk factors in preeclampsia screening factors alone nor their unspecific combination had an acceptable DA. Therefore, their use in clinical practice may lead to both over and under diagnoses and, thereby, unwanted conse- quences like over and under-treatment with LDA. However, by using machine learning tech- niques (i.e., RF) and stratification for parity, we inform the existence of individual heterogeneity in PE and identify specific combinations of specific risk factors with a high LR+ that permit a more assertive PE risk assessment for specialist referral and LDA prescription. Discussion Additionally, we demonstrate that a more extreme cut-off of the individual probabilities predicted by multiple logistic regression analysis is needed in primiparous when compared to multiparous in order to predict PE with confidence. Risk factors Our results are compatible with previous findings reporting a positive association between PE and low education[17], maternal age[24], previous PE, HBP, CKD[25,26–28], ART[7], multiple pregnancy[29–31], DM and obesity [26,27,30] and a negative association with smoking before pregnancy[32]. To our knowledge this is the first study reporting an effect modification of parity by other risk factors which also justify the stratified analysis. The effect of traditional risks on the incidence of PE was lower for nulliparous than for multiparous. HBP and multiple pregnancy showed the highest negative effect on the additive scale, which highlight the importance of these risk factors mainly among multiparous. These results could be explained, at least in part, by the shorter time of exposure or less severity of diseases in nulliparous, which tend to be younger than multiparous. The interpretation of the interaction with opposite direction in the multiplica- tive and additive scale needs caution, and biological plausibility should be taken into consider- ation. For example, in the interaction between diabetes and parity, negative additive interaction seems more biologically plausible than a positive multiplicative interaction. We also found a dose response association between the number of risk factors and PE risk in multiparous, de- scribing almost a linear trend. A non-linear effect was observed in primiparous, suggesting that the combination of clinical risk-factors among multiparous imply a more deleterious effect than in primiparous, most probably explained by the presence of previous PE only among them. https://doi.org/10.1371/journal.pone.0178528.g003 PLOS ONE \| https://doi.org/10.1371/journal.pone.0178528 May 25, 2017 Risk factors in preeclampsia screening Table 5. Discriminatory accuracy for specific combinations of risk factor for preeclampsia based on random forest variable importance. Combination n AR AF TPF (95% CI) FPF (95% CI) LR+ (95% CI) LR- (95% CI) ORa (95% CI) Multiparous Incidence PE 2.41% PP HBP Obese Multiple 283462 1.23 Ref 0 0 0 1 4698 8.56 7.33 4.8 (4.4–5) 1.3 (1.2–1.3) 3.79 (3.43–4.19) 0.96 (0.96–0.97) 7.02 (6.28–7.83) 0 0 1 0 39533 3.79 2.56 18.1 (17.3–18.9) 11.3 (11.2–11.4) 1.60 (1.25–1.62) 0.92 (0.91–0.93) 3.05 (2.86–3.25) 0 0 1 1 708 13 11.8 1.1 (0.9–1.4) 0.2 (0.2–0.2) 6.05 (4.86–7.52) 0.99 (0.99–0.99) 11.1 (8.86–13.8) 0 1 0 0 1453 14.7 13.5 2.6 (2.2–2.9) 0.4 (0.3–0.4) 6.96 (6.02–8.03) 0.98 (0.97–0.98) 11.6 (9.94–13.6) 0 1 0 1 24 16.7 15.5 ~0 ~0 8.10 (2.77–23.7) ~1 14.6 (4.86–43.9) 0 1 1 0 706 19.6 18.4 1.7 (1.4–2) 0.2 (0.1–0.2) 9.84 (8.18–11.8) 0.99 (0.98–0.99) 16.2 (13.2–19.8) 0 1 1 1 10 50 48.8 0.1 (0–0.1) ~0 40.5 (11.7–139) ~1 57.2 (9.99–328) 1 0 0 0 9635 15.1 13.9 17.5 (16.7–18.4) 2.4 (2.4–2.5) 7.20 (6.84–7.58) 0.85 (0.84–0.85) 14 (13.2–15.2) 1 0 0 1 136 34.6 33.4 0.6 (0.4–0.6) ~0 21.4 (15–30.4) 0.99 (0.99–1) 41 (28.7–58.8) 1 0 1 0 2935 24.8 23.6 8.8 (8.2–9.4) 0.3 (0.2–0.3) 13.4 (12.3–14.5) 0.92 (0.91–0.92) 25.8 (23.5–28.3) 1 0 1 1 43 39.5 38.3 0.2 (0.1–0.3) ~0 26.5 (14.4–48.8) ~1 51.9 (28–96.5) 1 1 0 0 410 31.5 30.3 1.6 (1.3–1.8) 0.1 (0.1–0.1) 18.6 (11.6–22.9) 0.99 (0.98–0.99) 32 (26.2–40.6) 1 1 0 1 6 16.7 15.5 ~0 ~0 8.10 (0.95–69.3) ~1 13.9 (1.8–105) 1 1 1 0 242 33.8 32.6 1 (0.8–1.2) ~0 20.8 (15.9–27.1) 0.99 (0.99–0.99) 36.1 (27.6–40.6) Multiple model including single variables AUC:75.35(74.74–75.97) Sensitivity (FP10%):45.01(43.87–46.15) AIC:67934 Multiple model including combination of variables AUC:75.23(74.61–75.85) Sensitivity (FP10%):43.34(42.23–44.44) AIC:67632 Primiparous Incidence PE = 5.57% DM HBP Obese Multiple 248600 4.47 Ref 0 0 0 1 3056 19.1 14.6 3.7 (3.4–4) 0.9 (0.9–1) 4 (3.66–4.37) 0.97 (0.97–0.97) 4.84 (4.14–5.31) 0 0 1 0 26939 11.5 7.03 19.6 (19–20.2) 8.9 (8.8–9) 2.19 (2.12–2.27) 0.88 (0.88–0.89) 2.69 (2.59–2.81) 0 0 1 1 358 23.5 19 0.5 (0.4–0.5) 0.1 (0.1–0.1) 5.20 (4.07–6.64) 1 (0.99–1) 6.10 (4.76–7.84) 0 1 0 0 1418 22.5 18 2 (1.8–2.2) 0.4 (0.4–0.4) 4.88 (4.31–5.53) 0.98 (0.98–0.99) 5.87 (5.16–6.67) 0 1 0 1 17 47.1 42.6 0.1 (0-.01) ~0 15.1 (5.82–39) ~1 17.4 (6.63–45.5) 0 1 1 0 505 29.7 25.2 1 (0.8–1.1) 0.1 (0.1–0.11) 7.17 (5.93–8.67) 0.99 (0.99–0.99) 8.15 (6.70–9.91) 0 1 1 1 6 16.7 12.2 ~0 ~0 3.39 (0.40–29) ~1 3.74 (0.38–36.4) 1 0 0 0 1316 21.7 17.2 1.8 (1.6–2) 0.4 (0.4–0.4) 4.71 (4.14–5.36) 0.99 (0.99–0.99) 5.91 (5.18–6.76) 1 0 0 1 13 23.1 18.6 ~0 ~0 5.09 (1.40–18.5) ~1 6.03 (1.60–22.6) 1 0 1 0 237 30.4 25.9 0.5 (0.4–0.6) 0.1 (0.1–0.1) 7.40 (5.61–9.76) ~1 8.99 (6.80–11.9) 1 0 1 1 4 50 45.5 ~0 ~0 16.96 (2.39–120) ~1 18.9 (2.98–120) 1 1 0 0 93 12.9 8.43 0.1 (0–0.1) ~0 2.51 (1.37–4.61) ~1 2.42 (1.24–4.69) 1 1 1 0 37 29.7 25.2 0.1 (0–0.1) ~0 7.18 (3.55–14.5) ~1 7.84 (3.84–16) Multiple model including single variables AUC: 61.48(60.99–61.96) Sensitivity (FP10%): 24.73(24.04–25.44) AIC:117232 Multiple model including combination of variables AUC: 61.50(61.02–61.98) Sensitivity (FP10%): 24.72(24.05–25.39) AIC:117060 AIC: Akaike information criteria, AF: Attributable fraction, AR: Attributable risk, TPF: True positive fraction, ORa: mutually adjusted Odds ratios. Accuracy of high- risk groups definitions Accuracy of some current definitions for high- risk groups. In everyday clinical practice the definition of high risk women varies across guidelines. In some of them[5,6] the same Fig 3. Criteria for risk factor combination based on variable importance in A) Multiparous B) Primiparous for the prediction of preeclampsia according to Random Forest. https://doi.org/10.1371/journal.pone.0178528.g003 Fig 3. Criteria for risk factor combination based on variable importance in A) Multiparous B) Primiparous for the prediction of preeclampsia according to Random Forest. Fig 3. Criteria for risk factor combination based on variable importance in A) Multiparous B) Primiparous for the prediction of preeclampsia according to Random Forest. https://doi.org/10.1371/journal.pone.0178528.g003 PLOS ONE \| https://doi.org/10.1371/journal.pone.0178528 May 25, 2017 12 / 19 https://doi.org/10.1371/journal.pone.0178528.t005 LR+: Positive Likelihood ratio; LR-: Negative Likelihood ratio, HBP: chronic hypertension; DM: Diabetes Mellitus Table 5. Discriminatory accuracy for specific combinations of risk factor for preeclampsia based on random forest variable importance. Combination n AR AF TPF (95% CI) FPF (95% CI) LR+ (95% CI) LR- (95% CI) ORa (95% CI) Multiparous Incidence PE 2.41% PP HBP Obese Multiple 283462 1.23 Ref 0 0 0 1 4698 8.56 7.33 4.8 (4.4–5) 1.3 (1.2–1.3) 3.79 (3.43–4.19) 0.96 (0.96–0.97) 7.02 (6.28–7.83) 0 0 1 0 39533 3.79 2.56 18.1 (17.3–18.9) 11.3 (11.2–11.4) 1.60 (1.25–1.62) 0.92 (0.91–0.93) 3.05 (2.86–3.25) 0 0 1 1 708 13 11.8 1.1 (0.9–1.4) 0.2 (0.2–0.2) 6.05 (4.86–7.52) 0.99 (0.99–0.99) 11.1 (8.86–13.8) 0 1 0 0 1453 14.7 13.5 2.6 (2.2–2.9) 0.4 (0.3–0.4) 6.96 (6.02–8.03) 0.98 (0.97–0.98) 11.6 (9.94–13.6) 0 1 0 1 24 16.7 15.5 ~0 ~0 8.10 (2.77–23.7) ~1 14.6 (4.86–43.9) 0 1 1 0 706 19.6 18.4 1.7 (1.4–2) 0.2 (0.1–0.2) 9.84 (8.18–11.8) 0.99 (0.98–0.99) 16.2 (13.2–19.8) 0 1 1 1 10 50 48.8 0.1 (0–0.1) ~0 40.5 (11.7–139) ~1 57.2 (9.99–328) 1 0 0 0 9635 15.1 13.9 17.5 (16.7–18.4) 2.4 (2.4–2.5) 7.20 (6.84–7.58) 0.85 (0.84–0.85) 14 (13.2–15.2) 1 0 0 1 136 34.6 33.4 0.6 (0.4–0.6) ~0 21.4 (15–30.4) 0.99 (0.99–1) 41 (28.7–58.8) 1 0 1 0 2935 24.8 23.6 8.8 (8.2–9.4) 0.3 (0.2–0.3) 13.4 (12.3–14.5) 0.92 (0.91–0.92) 25.8 (23.5–28.3) 1 0 1 1 43 39.5 38.3 0.2 (0.1–0.3) ~0 26.5 (14.4–48.8) ~1 51.9 (28–96.5) 1 1 0 0 410 31.5 30.3 1.6 (1.3–1.8) 0.1 (0.1–0.1) 18.6 (11.6–22.9) 0.99 (0.98–0.99) 32 (26.2–40.6) 1 1 0 1 6 16.7 15.5 ~0 ~0 8.10 (0.95–69.3) ~1 13.9 (1.8–105) 1 1 1 0 242 33.8 32.6 1 (0.8–1.2) ~0 20.8 (15.9–27.1) 0.99 (0.99–0.99) 36.1 (27.6–40.6) Multiple model including single variables AUC:75.35(74.74–75.97) Sensitivity (FP10%):45.01(43.87–46.15) AIC:67934 Multiple model including combination of variables AUC:75.23(74.61–75.85) Sensitivity (FP10%):43.34(42.23–44.44) AIC:67632 P i i I id PE 5 57% fic combinations of risk factor for preeclampsia based on random forest variable importance. Table 5. Discriminatory accuracy for specific combinations of risk factor for preeclampsi Multiple model including single variables AIC: Akaike information criteria, AF: Attributable fraction, AR: Attributable risk, TPF: True positive fraction, ORa: m Positive Likelihood ratio; LR-: Negative Likelihood ratio, HBP: chronic hypertension; DM: Diabetes Mellitus AIC: Akaike information criteria, AF: Attributable fraction, AR: Attributable risk, TPF: True positive fraction, ORa: mutually adjusted Odds ratios. PLOS ONE \| https://doi.org/10.1371/journal.pone.0178528 May 25, 2017 Risk factors in preeclampsia screening Table 6. Discriminatory accuracy of the multiple model at different cut-off of the predicted preeclampsia probability in multiparous and in primipa- rous women. Centile(cut-off) n AR AF TPF (95% CI) FPF (95% CI) LR+ (95% CI) LR- (95% CI) ORa (95% CI) Multiparous 95 centile (0.07) 16847 17.5 15.9 35.5 (34.5–36.6) 4.1 (4.1–4.2) 8.58 (8.30–8.87) 0.67 (0.66–0.68) 12.8 (12.2–13.4) 97 centile (0.13) 6237 23.6 21.6 17.8 (17–18.6) 1.4 (1.4–1.5) 12.5 (11.8–13.2) 0.83 (0.83–0.84) 15 (14.1–16) 99 centile (0.25) 3160 27.5 25.3 10.5 (9.8–11.2) 0.7 (0.7–0.7) 15.4 (14.3–16.6) 0.90 (0.89–0.91) 17 (15.71–18) Primiparous 95 centile (0.12) 8884 20.8 15.7 11.7 (11.2–12.3) 2.6 (2.6–2.7) 4.46 (4.24–4.68) 0.91 (0.90–0.91) 4.92 (4.66–5.19) 97 centile (0.13) 7997 21.5 16.5 11 (10.5–11.5) 2.4 (2.3–2.4) 4.66 (4.43–4.90) 0.91 (0.91–0.92) 5.11 (4.83–5.40) 99 centile (0.19) 2392 25.6 20.2 3.9 (3.6–4.2) 0.7 (0.6–0.7) 5.85 (5.34–6.40) 0.97 (0.96–0.97) 6.04 (5.50–6.63) AF: Attributable fraction, AR: Attributable risk, TPF: True positive fraction, ORa: mutually adjusted Odds ratios. LR+: Positive Likelihood ratio; LR-: Negative Likelihood ratio. Table 6. Discriminatory accuracy of the multiple model at different cut-off of the predicted preeclampsia pro rous women. risk, TPF: True positive fraction, ORa: mutually adjusted Odds ratios. LR+: Positive Likelihood ratio; LR-: Negative AF: Attributable fraction, AR: Attributable risk, TPF: True positive fraction, ORa: mutually adjusted Odds ratios. LR+ Likelihood ratio. AF: Attributable fraction, AR: Attributable risk, TPF: True positive fraction, ORa: mutually adjusted Odds ratios. LR+: Positive Likelihood ratio; LR-: Negative Likelihood ratio. however in those with multiple and HBP pregnancies the presence of obesity rises the LR+ from 8 to 40. This assumption of homogeneity is one limitation of current high risk definitions for PE. That is, any single major or any combination of two moderate risk factor carries a simi- lar PE risk and this risk is the same in multiparous and primiparous. Accuracy of a new definition for high- risk groups. Applying different approaches (i.e., stratification, multiple logistic regression and random forest) we identified specific com- bination of risk-factors that provided a higher discriminatory accuracy to rule-in PE with confidence. For instance, among multiparous, any bivariate combination including HBP or previous PE reached a LR>10. There are few studies analyzing the impact of combinations rather than a single factor in PE risk. LR+: Positive Likelihood ratio; LR-: Negative Likelihood ratio, HBP: chronic hypertension; DM: Diabetes Mellitus definition is used interchangeably for further screening tests and for prescription of LDA. definition is used interchangeably for further screening tests and for prescription of LDA. However, we demonstrate that the predictive accuracy of this current practice falls to reach the standard cutoff for acceptable discrimination[7]. For example, in the whole population, primi- parity vs multiparity is currently considered as a moderate risk-factor. However, our analysis indicates that its LR+ is very low (i.e., LR+ = 1.48). Besides, among primiparous, the existence of another independent risk factor did not improve PE prediction which seriously question the use of this condition alone or in unspecific bivariate combination for specialist referral or prescription of LDA. Likewise, obesity provided a LR+~ 2 and OR ~ 3 among multiparous, definition is used interchangeably for further screening tests and for prescription of LDA. However, we demonstrate that the predictive accuracy of this current practice falls to reach the standard cutoff for acceptable discrimination[7]. For example, in the whole population, primi- parity vs multiparity is currently considered as a moderate risk-factor. However, our analysis indicates that its LR+ is very low (i.e., LR+ = 1.48). Besides, among primiparous, the existence of another independent risk factor did not improve PE prediction which seriously question the use of this condition alone or in unspecific bivariate combination for specialist referral or prescription of LDA. Likewise, obesity provided a LR+~ 2 and OR ~ 3 among multiparous, PLOS ONE \| https://doi.org/10.1371/journal.pone.0178528 May 25, 2017 13 / 19 Interesting, despite CKD has been considered a major risk factor for PE, our results are in accordance with a previous study reporting that only those with the simultaneous presence of HBP are at a higher risk of PE[33]. We additionally identi- fied a higher risk in those with CKD, multiple pregnancy and obesity even in the absence of HBP or previous PE. Among those without major risk factors at least three rather than two moderate factors are needed to reach a LR+>10. In primiparous, only “rare” combinations of risk factors achieve a LR>10, particularly mul- tiple pregnancy with HBP or with the simultaneous presence of DM and obesity. However, some combinations with a moderate LR provided an absolute risk above 30%, i.e. obesity with HBP or with DM. As observed in multiparous, the combination of multiple pregnancy and Table 7. Subgroups combinations with the highest positive likelihood ratio to rule-in preeclampsia. Population Multiparous Primiparous All • Any combination of a risk factor with previous PE or HBP • DM +Multiple pregnancy+ obesity • DM +HBP+ obesity • No DM + No obesity +Multiple pregnancy+ HBP • No DM + No Multiple pregnancy +Obesity + HBP • >97 centile of individual risk probabilities • >99 centile of individual risk probabilities Without Previous PE nor HBP • Multiple pregnancy +Obese +(DM or CKD or ART or <40) • Multiple pregnancy + Obese + Gestational diabetes Without major risk factor • >40 +Multiple pregnancy +ART •<40+Multiple pregnancy +ART +Obese • Multiple pregnancy + gestational diabetes +<40 +No obese + no ART • No obese +Multiple pregnancy + ART • Obese +Multiple pregnancy +<40 +no ART ART: assisted reproductive technologies, CKD: chronic kidney disease, DM: diabetes mellitus, HBP: chronic hypertension https://doi org/10 1371/journal pone 0178528 t007 Table 7. Subgroups combinations with the highest positive likelihood ratio to rule-in preeclampsia. https://doi.org/10.1371/journal.pone.0178528.t006 Population Multiparous Primiparous All • Any combination of a risk factor with previous PE or HBP • DM +Multiple pregnancy+ obesity • DM +HBP+ obesity • No DM + No obesity +Multiple pregnancy+ HBP • No DM + No Multiple pregnancy +Obesity + HBP • >97 centile of individual risk probabilities • >99 centile of individual risk probabilities Without Previous PE nor HBP • Multiple pregnancy +Obese +(DM or CKD or ART or <40) • Multiple pregnancy + Obese + Gestational diabetes Without major risk factor • >40 +Multiple pregnancy +ART •<40+Multiple pregnancy +ART +Obese • Multiple pregnancy + gestational diabetes +<40 +No obese + no ART • No obese +Multiple pregnancy + ART • Obese +Multiple pregnancy +<40 +no ART ART: assisted reproductive technologies, CKD: chronic kidney disease, DM: diabetes mellitus, HBP: chronic hypertension roups combinations with the highest positive likelihood ratio to rule-in preeclampsia. 14 / 19 Risk factors in preeclampsia screening obesity was associated with an increased OR and LR+, particularly among those without major risk factors. This finding might suspect the role of volume overload in the pathogenesis of PE as a hypertensive disease[34,35]. Other factors could be needed to improve the prediction in this group. For example, a recent study in primiparous healthy women[36] has pointed out a higher risk among those with systolic blood pressure>120mmHg and maternal low birth- weight, or in those with family history of PE and vaginal bleeding>5 days. We speculate that the increment in the number of risk factors might leads to an increment in preventive treat- ment and self-care, which could simulate the flattening in the risk of PE when three or more factors are present in primiparous.(Fig 2 and Table 6). The contribution of traditional risk-factors to the DA for PE was modest as previously reported[37]. Using different prediction rules, the TPF varies from 18–31% for a FPR of 10% [30,38]. In our study, the AUC was significantly lower in primiparous reinforcing the necessity to identify new risk-factors in this population. These results are in contrast with a recent study reporting a similar AUC in multiparous and primiparous[39]. However, that study was per- formed in a higher risk population and included biomarkers, therefore results are not directly comparable. Our results agrees with Poon et al[24,40] demonstrating a better DA from multi- ple regression models when compared to NICE recommendations, but we additionally identify a different cut-off of the predicted probability in multiparous and primiparous to predict PE with confidence. Despite the improvement in model fit when the subgroups were incorporated in multiple regression models, the sensitivity (TPF) and specificity (TNF) remained constant. This can be explained by the low prevalence of high-risk subgroups in the population. Contrarily to gen- eral belief, the TPF and the FPF depend of the prevalence ratio[41]. That is, the ratio between the prevalence of the risk factor and the prevalence of the disease. For the same prevalence of a disease, a risk factor with a lower prevalence (i.e., combination of risk), is related with a lower TPF and FPF when compared with a factor with a higher prevalence (i.e. single risk), since nei- ther many cases nor many controls can be exposed to such combinations. Then, a large subset of non-exposed women develops PE possibly because of the existence of other factors that were not included in this analysis. Therefore, the DA of rare combination is generally low at the population level but could be high to predict PE at individual level. Some researchers have pointed out that measures of association alone are unsuitable for discriminatory purpose[9,23,42]. OR is obtained by multiplying sensitivity and specificity (TPTN/FNFP). Then, the same OR can be obtained with very different scenarios of sensitiv- ity and specificity. Therefore, the traditional OR approach prevent a more personalized medi- cine. Therefore, we propose the use of measures of DA to disentangle the utility of a risk-factor for screening or treatment purposes. The main advantage of LR versus sensitivity and specific- ity is that clinicians can use them to quantify the probability of a disease for an individual patient. The LR summarizes how many times more (or less) likely patients with the PE are to have that particular risk factor (or combination) than patients without the disease[21,43]. Risk factors in preeclampsia screening possible combinations of variables, we used a machine learning approach by RF algorithm as a guide to identify the most important variables for subsequently generate subgroups at a higher-risk of PE. This study also has potential limitations. First, we have excluded missing data, but we can- not assure that missing values were completely at random even if included and excluded deliv- eries were balanced concerning the prevalence important risk-factors. Second, we had no information on interventions during pregnancy, such as aspirin prophylaxis. At the study interval, there was no preeclampsia screening in place, however, some patients at risk were possibly on LDA treatment, particularly women with prior preterm preeclampsia, i.e., proba- bly around 2%-5% of all preeclampsia cases, which might bias the estimations towards the null in this population. Third, we were not able to evaluate all possible combinations of moderate risk-factors reported in published guidelines, neither include the histories of previous PE from longer pregnancy intervals. Fourth, we have not validated our finding in a separate population or by mean of bootstraps so we cannot rule out that our multiple regression model might be overfitted and the AUCs overestimated. This is particularly important with the results from the smaller subgroups that may be the product of overfitting and may not readily reproduce in other study samples. We have used dichotomous variables in other to adopt a similar approach as that used in current guidelines, however ordinal or continuous variables could produce more precise estimations. Finally, even though the quality of the MBR seems appropriate[15], our results need to be validated in other populations. Conclusions No one risk-factors alone or unspecific combinations reached an acceptable accuracy, and 3 moderate risk combinations are needed in those without major risk-factors to reach a LR+> 10. Consequently, current approach based exclusively in OR, might be associated with inef- ficient specialist referral and unnecessary treatment with LDA. The prediction of PE was improved with a more individualized approach, by identifying specific combinations or by defining a differential cut-off to the distribution of the predicted probability for multiparous and nulliparous obtained by multiple regression analysis. However, the absence of any single neither relevant combinations were enough to rule-out the disease. The identification of such specific subgroups can improve the reliability of LDA prescription, but those with any single risk might need further screening. Our results contribute to a more personalized risk estima- tion of preeclampsia. Strength and limitations To our knowledge, this is the first study explicitly focused on understanding heterogeneity in PE risk in order to identify high-risk subgroups of PE by combining specific risk-factors. While most previous studies have provided risk equations for the whole population of women [24,44] or mainly focused in primiparous[36], we stratified the analysis by parity. We included all pregnancies even in the presence of congenital malformation (ICD-10 codes Q00-Q99) or HBP to extend the results in real clinical settings. Additionally, we included post-partum PE that is usually excluded when data is exclusively based on birth registers. As there are many PLOS ONE \| https://doi.org/10.1371/journal.pone.0178528 May 25, 2017 15 / 19 Acknowledgments The authors would like to thanks Dr. Carlos Campillo for his valuable comments on the final draft and to Dr. Pelle Lindqvist for his valuable inputs about the Swedish medical practice. Conceptualization: MR-L FC JM. Formal analysis: MR-L PW RP-V JM. Investigation: MR-L RP-V JM. Resources: MR-L RP-V JM. Supervision: JM. Visualization: MR-L FC JM. Writing – original draft: MR-L JM. Writing – review & editing: MR-L PW RP-V FC JM. S4 Table. Discriminatory accuracy of specific bivariate combinations of risk factor for pre- eclampsia in the overall population of pregnancies without major risk factors. (DOCX) S4 Table. Discriminatory accuracy of specific bivariate combinations of risk factor for pre- eclampsia in the overall population of pregnancies without major risk factors. (DOCX) S5 Table. Discriminatory accuracy of specific bivariate combinations of risk factor for pre- eclampsia in multiparous and primiparous pregnancies without major risk factors. (DOCX) Supporting information S1 Table. Characteristics of included and missing data among multiparous and primipa- rous women. ( ) S2 Table. Bivariate interaction effect between parity and each risk factors in multiple logis- tic regression models. One interaction term was included in each model. Previous preeclamp- sia was considered a dummy variable with three categories: yes or no in multiparous and no applicable in primiparous. S3 Table. Association between number of clinical risk factor and the risk of PE in both pri- miparous and multiparous adjusted by smoking, education and family situation. (DOCX) S3 Table. Association between number of clinical risk factor and the risk of PE in both pri- miparous and multiparous adjusted by smoking, education and family situation. (DOCX) S3 Table. Association between number of clinical risk factor and the risk of PE in both pri- miparous and multiparous adjusted by smoking, education and family situation. (DOCX) 16 / 19 PLOS ONE \| https://doi.org/10.1371/journal.pone.0178528 May 25, 2017 Risk factors in preeclampsia screening References Applying measures of discriminatory accuracy to revisit traditional risk factors for being small for gestational age in Sweden: a national cross-sectional study. BMJ Open. 2014; 4(7):e005388. https://doi.org/10.1136/bmjopen-2014-005388 PMID: 25079936 12. Wald NJ, Hackshaw AK, Frost CD. When can a risk factor be used as a worthwhile screening test? BMJ. 1999; 319:1562–5. PMID: 10591726 13. Khoury MJ, Iademarco MF, Riley WT. Precision Public Health for the Era of Precision Medicine. Am J Prev Med. 2016; 50(3):398–401. https://doi.org/10.1016/j.amepre.2015.08.031 PMID: 26547538 14. Yoo W, Ference BA, Cote ML, Schwartz A. A Comparison of Logistic Regression, Logic Regression, Classification Tree, and Random Forests to Identify Effective Gene-Gene and Gene-Environmental Interactions. Int J Appl Sci Technol. 2012 Aug; 2(7):268-. PMID: 23795347 15. Petersson K, Persson M, Lindkvist M, Hammarstrom M, Nilses C, Haglund I, et al. Internal validity of the Swedish Maternal Health Care Register. BMC Health Serv Res. 14:364. https://doi.org/10.1186/1472- 6963-14-364 PMID: 25175811 16. Cnattingius S, Ericson A, Gunnarskog J, Kallen B. A quality study of a medical birth registry. Scand J Soc Med. 1990 Jun; 18(2):143–8. PMID: 2367825 17. Wikstrom AK, Stephansson O, Cnattingius S. Tobacco use during pregnancy and preeclampsia risk: effects of cigarette smoking and snuff. Hypertension. 2010 May; 55(5):1254–9. https://doi.org/10.1161/ HYPERTENSIONAHA.109.147082 PMID: 20231527 18. Cnattingius S, Villamor E, Johansson S, Edstedt Bonamy AK, Persson M, Wikstrom AK, et al. Maternal obesity and risk of preterm delivery. JAMA. 2013 Jun 12; 309(22):2362–70. https://doi.org/10.1001/ jama.2013.6295 PMID: 23757084 19. Sohlberg S, Stephansson O, Cnattingius S, Wikstrom AK. Maternal body mass index, height, and risks of preeclampsia. Am J Hypertens. 2012 Jan; 25(1):120–5. https://doi.org/10.1038/ajh.2011.175 PMID: 21976280 20. Breiman L. Random Forests. Machine Learning. 2001; 45(1):5–32. 21. Deeks JJ, Altman DG. Diagnostic tests 4: likelihood ratios. BMJ. 2004 Jul 17; 329(7458):168–9. https:// doi.org/10.1136/bmj.329.7458.168 PMID: 15258077 22. Grimes DA, Schulz KF. Refining clinical diagnosis with likelihood ratios. Lancet. 2005 Apr 23; 365 (9469):1500–5. https://doi.org/10.1016/S0140-6736(05)66422-7 PMID: 15850636 23. Merlo J, Mulinari S. Measures of discriminatory accuracy and categorizations in public health: a response to Allan Krasnik’s editorial. Eur J Public Health. 2015 Dec; 25(6):910. https://doi.org/10.1093/ eurpub/ckv209 PMID: 26604325 24. Poon LC, Kametas NA, Chelemen T, Leal A, Nicolaides KH. Maternal risk factors for hypertensive disor- ders in pregnancy: a multivariate approach. J Hum Hypertens. 2010 Feb; 24(2):104–10. https://doi.org/ 10.1038/jhh.2009.45 PMID: 19516271 25. Bartsch E, Medcalf KE, Park AL, Ray JG. Clinical risk factors for pre-eclampsia determined in early pregnancy: systematic review and meta-analysis of large cohort studies. References 1. Roberts CL, Ford JB, Algert CS, Antonsen S, Chalmers J, Cnattingius S, et al. Population-based trends in pregnancy hypertension and pre-eclampsia: an international comparative study. BMJ Open. 2011 Jan 1; 1(1):e000101. https://doi.org/10.1136/bmjopen-2011-000101 PMID: 22021762 2. Steegers EA, von Dadelszen P, Duvekot JJ, Pijnenborg R. Pre-eclampsia. Lancet. 2010 Aug 21; 376 (9741):631–44. https://doi.org/10.1016/S0140-6736(10)60279-6 PMID: 20598363 3. Craici I, Wagner S, Garovic VD. Preeclampsia and future cardiovascular risk: formal risk factor or failed stress test? Ther Adv Cardiovasc Dis. 2008; 2(4):249–59. https://doi.org/10.1177/1753944708094227 PMID: 19124425 4. Robinson C, Goodnight W. Hypertension-Management of Hypertensive Disorders of Pregnancy According to International Guidelines: A Panel Discussion (Case 1: Role of Proteinuria). Am J Perinatol. 2015 Epub 2015 Apr 17. 5. World Health Organization. WHO recommendations for prevention and treatment of pre-eclampsia and eclampsia Geneva: World Health Organization; 2011 [cited 2016 Dec 27]. Available from: https://www. ncbi.nlm.nih.gov/books/NBK140561/ 6. Redman CW. Hypertension in pregnancy: the NICE guidelines. Heart. Dec; 97(23):1967–9. https://doi. org/10.1136/heartjnl-2011-300949 PMID: 21990386 7. American College of Obstetricians and Gynecologists’ Task Force on Hypertension in Pregnancy. Hypertension in pregnancy. Report of the American College of Obstetricians and Gynecologists’ Task Force on Hypertension in Pregnancy. Obstet Gynecol. 2013; 122(5):1122–31. https://doi.org/10.1097/ 01.AOG.0000437382.03963.88 PMID: 24150027 8. vardgivare.skane.se[Internet], Sweden: Regionala riktlinjer fo¨r hypertoni under graviditet i basmo¨dra- ha¨lsovård. 2014 [cited 2016 October 28]; Available from: http://vardgivare.skane.se/vardriktlinjer/ medicinska-omraden/kvinnosjukvard-graviditet-och-forlossning. 9. Pepe MS, Janes H, Longton G, Leisenring W, Newcomb P. Limitations of the odds ratio in gauging the performance of a diagnostic, prognostic, or screening marker. Am J Epidemiol. 2004 May; 159(9):882– 90. PMID: 15105181 17 / 19 PLOS ONE \| https://doi.org/10.1371/journal.pone.0178528 May 25, 2017 Risk factors in preeclampsia screening 10. Merlo J, Wagner P, Juarez S, Mulinari S, Hedblad B. The tyranny of the averages and the indiscriminate use of risk factors and population attributable fractions in Public Health: the case of coronary heart dis- ease. Working papers at the Unit for Social Epidemiology, Lund University. 2013 Available from: http:// www.med.lu.se/english/klinvetmalmo/unit_for_social_epidemiology/abstracts_c_working_papers_c 10. Merlo J, Wagner P, Juarez S, Mulinari S, Hedblad B. The tyranny of the averages and the indiscriminate use of risk factors and population attributable fractions in Public Health: the case of coronary heart dis- ease. Working papers at the Unit for Social Epidemiology, Lund University. 2013 Available from: http:// www.med.lu.se/english/klinvetmalmo/unit_for_social_epidemiology/abstracts_c_working_papers_c 11. Juarez SP, Wagner P, Merlo J. References BMJ. 2016 Apr 19; 353:i1753. https://doi.org/10.1136/bmj.i1753 PMID: 27094586 26. Li X, Tan H, Huang X, Zhou S, Hu S, Wang X, et al. Similarities and differences between the risk factors for gestational hypertension and preeclampsia: A population based cohort study in south China. Preg- nancy Hypertens. 2016 Jan; 6(1):66–71. https://doi.org/10.1016/j.preghy.2015.11.004 PMID: 26955775 27. Goetzinger KR, Tuuli MG, Cahill AG, Macones GA, Odibo AO. Development and validation of a risk fac- tor scoring system for first-trimester prediction of preeclampsia. Am J Perinatol. 2014 Dec; 31 (12):1049–56. https://doi.org/10.1055/s-0034-1371705 PMID: 24705967 28. Egeland GM, Klungsoyr K, Oyen N, Tell GS, Naess O, Skjaerven R. Preconception Cardiovascular Risk Factor Differences Between Gestational Hypertension and Preeclampsia: Cohort Norway Study. Hypertension. 2016Jun; 67(6):1173–80. https://doi.org/10.1161/HYPERTENSIONAHA.116.07099 PMID: 27113053 29. Ros HS, Cnattingius S, Lipworth L. Comparison of risk factors for preeclampsia and gestational hyper- tension in a population-based cohort study. Am J Epidemiol. 1998 Jun 1; 147(11):1062–70. PMID: 9620050 30. Baschat AA, Magder LS, Doyle LE, Atlas RO, Jenkins CB, Blitzer MG. Prediction of preeclampsia utiliz- ing the first trimester screening examination. Am J Obstet Gynecol. 2014 Nov; 211(5):514 e1–7. PLOS ONE \| https://doi.org/10.1371/journal.pone.0178528 May 25, 2017 18 / 19 Risk factors in preeclampsia screening 31. Sibai B, Dekker G, Kupferminc M. Pre-eclampsia. Lancet. 2005 Feb 26; 365(9461):785–99. https://doi. org/10.1016/S0140-6736(05)17987-2 PMID: 15733721 32. England LJ, Levine RJ, Qian C, Morris CD, Sibai BM, Catalano PM, et al. Smoking before pregnancy and risk of gestational hypertension and preeclampsia. Am J Obstet Gynecol. 2002 May; 186(5):1035– 40. PMID: 12015533 33. Munkhaugen J, Lydersen S, Romundstad PR, Wideroe TE, Vikse BE, Hallan S. Kidney function and future risk for adverse pregnancy outcomes: a population-based study from HUNT II, Norway. Nephrol Dial Transplant. 2009 Dec; 24(12):3744–50. https://doi.org/10.1093/ndt/gfp320 PMID: 19578097 34. Kotsis V, Stabouli S, Papakatsika S, Rizos Z, Parati G. Mechanisms of obesity-induced hypertension. Hypertens Res.2010 May; 33(5):386–93. https://doi.org/10.1038/hr.2010.9 PMID: 20442753 35. Hunter S, Robson SC. Adaptation of the maternal heart in pregnancy. Br Heart J. 1992 Dec; 68(6):540– 3. PMID: 1467047 36. North RA, McCowan LM, Dekker GA, Poston L, Chan EH, Stewart AW, et al. Clinical risk prediction for pre-eclampsia in nulliparous women: development of model in international prospective cohort. BMJ. 2011; 342:d1875. https://doi.org/10.1136/bmj.d1875 PMID: 21474517 37. Milne F, Redman C, Walker J, Baker P, Bradley J, Cooper C, et al. The pre-eclampsia community guideline (PRECOG): how to screen for and detect onset of pre-eclampsia in the community. BMJ. PLOS ONE \| https://doi.org/10.1371/journal.pone.0178528 May 25, 2017 References 2005 Mar 12; 330(7491):576–80. https://doi.org/10.1136/bmj.330.7491.576 PMID: 15760998 38. Oliveira N, Magder LS, Blitzer MG, Baschat AA. First-trimester prediction of pre-eclampsia: external validity of algorithms in a prospectively enrolled cohort. Ultrasound Obstet Gynecol. 2014 Sep; 44 (3):279–85. https://doi.org/10.1002/uog.13435 PMID: 24913190 39. Moon M, Odibo A. First-trimester screening for preeclampsia: impact of maternal parity on modeling and screening effectiveness. J Matern Fetal Neonatal Med. 28(17):2028–33. https://doi.org/10.3109/ 14767058.2014.978758 PMID: 25330843 40. Poon LC, Nicolaides KH. Early prediction of preeclampsia. Obstet Gynecol Int. 2014; 2014:297397. PubMed Central PMCID: PMC4127237 https://doi.org/10.1155/2014/297397 PMID: 25136369 41. Choi BC. Causal modeling to estimate sensitivity and specificity of a test when prevalence changes. Epidemiology. 1997 Jan; 8(1):80–6. PMID: 9116101 42. Merlo J. Multilevel analytical approaches in social epidemiology: measures of health variation compared with traditional measures of association. J Epidemiol Community Health. 2003 Aug; 57(8):550–2. https://doi.org/10.1136/jech.57.8.550 PMID: 12883048 43. McGee S. Simplifying likelihood ratios. J Gen Intern Med. 2002 Aug; 17(8):646–9. PMID: 12213147 44. Wright D, Syngelaki A, Akolekar R, Poon LC, Nicolaides KH. Competing risks model in screening for preeclampsia by maternal characteristics and medical history. Am J Obstet Gynecol. Jul; 213(1):62 e1– 10. 19 / 19
https://openalex.org/W2588456184	https://periodicos.unifesp.br/index.php/olhares/article/download/507/219	Portuguese	null	ANÁLISE DAS CONCEPÇÕES DE PROFESSORES DE BIOLOGIA SOBRE MODELOS CIENTÍFICOS ANTES E APÓS UM CURSO DE FORMAÇÃO CONTINUADA	Olh@res	2,016	cc-by	5,915	Maria Delourdes Maciel Universidade Cruzeiro do Sul RESUMO: Este artigo apresenta parte dos resultados de uma pesquisa de doutorado, em que se investigou as compreensões/concepções de professores de Biologia da rede pública Estadual de Belo Horizonte sobre interações entre Ciência – Tecnologia e Sociedade CTS e Natureza da Ciência e Tecnologia (NdC&T). O objetivo foi respaldar ações em um projeto de extensão com vistas à formação continuada. As compreensões/concepções dos professores foram obtidas por meio de aplicação do Questionário de Opiniões sobre a Ciência, a Tecnologia e a Sociedade (COCTS), antes e após o curso de formação, envolvendo a análise de uma questão, entre as treze respondidas pelos professores, que versava sobre modelos científicos. Buscou-se avaliar as dimensões das mudanças identificadas após o curso. Concluímos que, no geral, o curso promoveu resultados positivos, pois se identificou mudanças nas concepções dos professores acerca dos modelos científicos, além do interesse destes em alterar sua prática a partir dos pressupostos CTS. PALAVRAS-CHAVE: educação continuada, CTS, NdC&T. PALAVRAS-CHAVE: educação continuada, CTS, NdC&T. KEY WORDS: continuing education, CTS, NOS&T ANÁLISE DAS CONCEPÇÕES DE PROFESSORES DE BIOLOGIA SOBRE MODELOS CIENTÍFICOS ANTES E APÓS UM CURSO DE FORMAÇÃO CONTINUADA Rosiane Resende Leite rosianeresende@deii.cefetmg.br Centro Federal de Educação Tecnológica de Minas Gerais Maria Delourdes Maciel maria.maciel@cruzeirodosul.edu.br Universidade Cruzeiro do Sul Maria Delourdes Maciel maria.maciel@cruzeirodosul.edu.br Universidade Cruzeiro do Sul ANALYSIS OF BIOLOGY TEACHERS' CONCEPTIONS ABOUT SCIENTIFIC MODELS BEFORE AND AFTER A CONTINUING EDUCATION COURSE ABSTRACT: This article presents part of the results of a doctoral research in progress, in which they investigated the understandings / conceptions of Biology teachers from Belo Horizonte's state public school system about interactions between Science, Technology and Society/ Nature of Science and Technology (CTS/NOS&T). The objective was to endorse actions in an extension project with a view to continuing education courses. Understandings / teachers' conceptions were obtained by applying Opinions Questionnaire on Science, Technology and Society (COCTS) before and after the training course, involving the analysis of an issue between the thirteen answered by teachers, which was about scientific models. We sought to assess the dimensions of the changes identified after the course. We conclude that, in general, the course promoted positive results, as it was identified changes in teachers' conceptions about scientific models, besides their interest in changing their practice from the CTS assumptions. KEY WORDS: continuing education, CTS, NOS&T Olh@res, Guarulhos, v. 4, n. 2, p. 165-178, novembro 2016. 165 Rosiane Resende Leite; Maria Delourdes Maciel Rosiane Resende Leite; Maria Delourdes Maciel Olh@res, Guarulhos, v. 4, n. 2, p. 165-178, novembro 2016. Introdução Análise das concepções de professores de biologia sobre modelos científicos antes e após um curso d formação continuada ensinadas, além da transposição didática operada pelos professores de Ciências (GALAGOVSKY, L.; ADÚRIZ-BRAVO, 2001). Dessa forma, a compreensão da NdC é altamente relevante para que os professores consigam implementar atividades de cunho CTS em sala de aula, já que a “imersão na cultura científica e tecnológica é fundamental para a formação de cidadãos e cidadãs críticos que, no futuro, participarão na tomada de decisões” (PRAIA et al., 2007, p.152). A implementação de uma abordagem CTS pelo professor, em sala de aula, não é usual, devido a vários obstáculos, entre eles as visões distorcidas que o professor tem sobre a própria Ciência e sobre as relações existentes entre Ciência, Tecnologia e Sociedade. Estas visões normalmente são provenientes do histórico de sua vivência escolar e do modelo de sua formação inicial, ou, até mesmo, das influências recebidas por meio das mídias. Assim, é mister que haja modificação na imagem de NdC que os professores têm e que transmitem aos estudantes (PRAIA et al., 2007). Corroborando com esta visão, Knauss (2005, p. 281) afirma que: “as pesquisas sobre as noções que o professorado detém do conhecimento científico indicam uma defasagem que o aproxima da perspectiva do aluno e do senso comum, ao mesmo tempo que o distancia da ciência”. Parece evidente que o desenvolvimento de práticas de ensino pautadas pelas orientações CTS reportam à necessidade de formação adequada dos professores, pois o ponto chave desta questão é: o que os professores farão em sala de aula? (TENREIRO- VIEIRA e VIEIRA, 2005). Os mesmos autores apontam, ainda, que existem duas razões para se considerar a formação dos professores: “o currículo enunciado exige mudança de mentalidades” e “os próprios professores reconhecem que não sabem como integrar a orientação CTS no ensino de Ciências” (p.192). Embora estas questões refiram-se ao contexto de Portugal, a situação no Brasil é semelhante como evidenciam as pesquisas de Delizoicov, Angotti e Pernambuco (2002); Nardi, Bastos e Diniz (2004); Cachapuz et al (2011); Carvalho e Gil-Pérez (2006), Gatti e Nunes (2009), entre outros, que também discutem esta problemática no contexto brasileiro. Auler e Bazzo (2001) apontam um trabalho de revisão realizado por Auler (1998), que elenca alguns problemas e desafios em relação ao movimento CTS no campo educacional, entre eles, destacam a “compreensão dos professores sobre as interações entre Ciência, Tecnologia e Sociedade”. Introdução Este trabalho configura-se como um recorte de uma tese de doutorado, em andamento, sobre Ensino de Ciências na Escola Básica, onde se investigou as compreensões/concepções de professores de Biologia da rede pública Estadual de Belo Horizonte sobre interações entre Ciência – Tecnologia e Sociedade (CTS) e Natureza da Ciência e Tecnologia (NdC&T), tendo por objetivo analisar as concepções prévias de cinco professores de Biologia acerca da Natureza da Ciência (NdC) e questões sobre a aplicação dos pressupostos que envolvem Ciência – Tecnologia e Sociedade (CTS) em sala de aula, a fim de desenhar um curso de formação continuada para atender as reais necessidades dos docentes. O projeto fez parte de uma colaboração internacional conhecida por Projeto Ibero-Americano de Avaliação de Atitudes Relacionadas com a Ciência, a Tecnologia e a Sociedade (PIEARCTS) desenvolvida por países como Argentina, Brasil, Colômbia, Espanha, México, Portugal e Uruguai, e que tem como objetivo principal avaliar as atitudes e compreensões dos professores quanto à NdC. A seleção dos professores participantes da pesquisa foi aleatória, pois os interessados se inscreveram voluntariamente no curso de extensão ofertado pelo Centro Federal de Educação Tecnológica de Minas Gerais, após a divulgação do mesmo no site do Centro de Referência Virtual do Professor (disponível em: <http://crv.educacao.mg.gov.br/sistema_crv/index2.aspx??id_objeto=23967>, acesso em: 20 jul.2014), sobre o tema em questão, em que se ofertou 20 vagas. A rede pública Estadual de Belo Horizonte conta com aproximadamente 201 professores de Biologia. Embora o número de inscritos no curso não tenha sido expressivo (5), considerando o número de professores da rede (201), prosseguimos com o mesmo por entender que era uma situação importante e relevante. Também porque, à época da divulgação do curso, os professores estavam de férias (adiantadas), devido à copa do mundo, por isso foi feita a divulgação, naquele período, apenas no site da Secretaria de Educação. O aprendizado sobre a NdC é um objetivo de vital importância no currículo de Ciência. A NdC pode ser definida, no contexto do ensino, como o conjunto de conteúdos metacientíficos relevantes para a educação científica, incluindo ideias procedentes de diferentes campos (epistemologia, história e sociologia da ciência) e que precisam ser 166 Análise das concepções de professores de biologia sobre modelos científicos antes e após um curso de formação continuada ensinadas, além da transposição didática operada pelos professores de Ciências (GALAGOVSKY, L.; ADÚRIZ-BRAVO, 2001). Olh@res, Guarulhos, v. 4, n. 2, p. 165-178, novembro 2016. @res, Guarulhos, v. 4, n. 2, p. 165-178, novembro 2016. Introdução Os autores reforçam, ainda, a escassez de publicações sobre “a utilização do enfoque CTS no ensino, no contexto brasileiro” (p.2). 167 Rosiane Resende Leite; Maria Delourdes Maciel Rosiane Resende Leite; Maria Delourdes Maciel Rosiane Resende Leite; Maria Delourdes Maciel Vasconcelos et al., (2012) apresentaram um estudo em que se realizou a caracterização das concepções de professores de Química relacionadas à CTS. Chegaram à conclusão que é de primordial importância compreender as concepções prévias dos professores para que ações sejam adotadas no sentido de corrigir possíveis distorções. Neste sentido, a formação continuada configura-se como um campo desafiador que engloba muitas questões, entre elas a consideração da trajetória do professor, pois sabemos que a atividade profissional destes sujeitos baseia-se em suas práticas, crenças e aprendizagens por eles vivenciadas. Há um movimento contínuo de construção e reconstrução dessas concepções, por parte dos professores, as quais adquirem um formato moldável ao longo de sua trajetória (LOPES e MACEDO, 2002). Compreendendo esta perspectiva e este desafio, buscou-se planejar e desenvolver um curso de formação continuada, direcionando-o a partir da avaliação de crenças e atitudes dos professores de Biologia da rede pública Estadual de Belo Horizonte sobre os temas CTS. Analisa-se, neste trabalho, uma questão do Questionário de Opiniões sobre a Ciência, a Tecnologia e a Sociedade (COCTS) que foi aplicada no início e no final do curso, cujo tema é modelo científico. Para os propósitos deste texto, destacamos a seguinte questão: ocorrem mudanças nas concepções dos professores acerca de modelos científicos, após terem realizado o curso de formação proposto? Olh@res, Guarulhos, v. 4, n. 2, p. 165-178, novembro 2016. Instrumento para recolha de dados O instrumento de coleta de dados utilizado para a realização deste trabalho foi uma questão extraída do COCTS, o qual já foi utilizado e validado por outros estudos realizados no Brasil e em outros países da Europa e América Latina. As questões extraídas do COCTS têm sido consideradas relevantes como instrumentos para a discussão de percepções/concepções dos sujeitos sobre CTS/NdC&T. Por meio de uma análise comparativa das respostas dos sujeitos, obtidas no pré-teste e no pós-teste, é possível verificar quais as mudanças evidenciadas após a realização de um curso de formação. As questões do COCTS seguem um modelo de respostas múltiplas, que permite a análise qualitativa e quantitativa, aplicação de teste estatístico e comparação entre diferentes grupos de sujeitos (grupo controle e grupo experimental). Para a pesquisa desenvolvida na tese foram trabalhadas 13 questões do COCTS, aplicadas aos cinco professores participantes do curso de extensão cuja finalidade foi a 168 Análise das concepções de professores de biologia sobre modelos científicos antes e após um curso de formação continuada Análise das concepções de professores de biologia sobre modelos científicos antes e após um curso d formação continuada formação continuada dos mesmos em relação ao enfoque CTS e NdC&T (Natureza da Ciência e Tecnologia). Cada questão segue um modelo de respostas múltiplas, a partir do qual as respostas são valoradas segundo o grau de concordância para cada uma das opções apresentadas nas questões, numa escala de nove pontos, ou seja, as respostas para cada frase em cada questão variam de 1 a 9, onde 1 indica discordância total e 9 (na outra extremidade) significa total concordância com a frase. Para a análise dos dados obtidos, é utilizada uma métrica que aceita a geração de um índice referente às atitudes, denominado índice atitudinal, que pode variar entre -1 e 1. Independente da categoria da resposta (adequada, plausível ou ingênua), quanto maior a proximidade com o valor 1, maior é a proximidade com as respostas atribuídas pelos juízes peritos (VÁZQUEZ et al., 2011). De acordo com o autor, a equipe de juízes peritos (16 experts no campo CTS/NdC&T: quatro filósofos, quatro pesquisadores em didática de ciências, três professores de ciências e cinco formadores de professores) convidados a avaliar e validar as questões do COCTS e que classificaram e categorizaram as respostas em adequadas, plausíveis e ingênuas. Para este artigo consideramos apenas uma das treze questões utilizadas na tese (Questão 90211). Olh@res, Guarulhos, v. 4, n. 2, p. 165-178, novembro 2016. Instrumento para recolha de dados Ao responder a questão, os sujeitos deveriam classificá-las da seguinte maneira (Figura 1): respostas pontuadas de 1 a 4 (Desacordo), respostas pontuadas com valor 5 (Indecisos/Neutros), respostas pontuadas de 6 a 9 (Acordo), E (Não Entendo) e S (Não Sei o Suficiente para avaliar/responder). Reportamo-nos, aqui, à análise quantitativa e qualitativa da questão 90211 (Figura 1) do COCTS, que trata dos modelos científicos usados nos laboratórios de investigação. A questão apresenta como subtema modelos científicos. 169 Rosiane Resende Leite; Maria Delourdes Maciel Figura 01 – Questão 90211, COCTS (adaptada) DESACORDO INDECISO ACORDO OUTRO QUESTÃO 90211 - Muitos modelos científicos usados nos laboratórios de investigação, tais como o modelo do calor, dos neurônios, do DNA ou do átomo, são cópias da realidade. Os modelos científicos SÃO cópias da realidade: T A M B B M A T NE NS A. Porque os cientistas dizem que são verdadeiros, portanto devem sê- lo. 1 2 3 4 5 6 7 8 9 E S B. Porque há muitas provas científicas que demonstram que são verdadeiros. 1 2 3 4 5 6 7 8 9 E S C. Porque são verdadeiros para a vida. O seu objetivo é mostrar-nos a realidade ou ensinarnos algo sobre ela. 1 2 3 4 5 6 7 8 9 E S D. Os modelos científicos são, muito aproximadamente, cópias da realidade, porque são baseados em observações científicas e em investigação. 1 2 3 4 5 6 7 8 9 E S Os modelos científicos NÃO são cópias da realidade: E. Porque simplesmente são úteis para aprender e explicar, dentro das suas limitações. 1 2 3 4 5 6 7 8 9 E S F. Porque mudam com o tempo e com o estado do conhecimento, como o fazem as teorias 1 2 3 4 5 6 7 8 9 E S G. Porque estes modelos devem ser ideais ou conjecturas bem informadas, já que o objeto real não se pode ver. 1 2 3 4 5 6 7 8 9 E S Fonte: tese de doutorado da primeira autora Figura 01 – Questão 90211, COCTS (adaptada) Fonte: tese de doutorado da primeira autora Na questão 90211, as frases A B e C são consideradas ingênuas, já as frases D e G são consideradas plausíveis e as frases E e F são consideradas adequadas. Instrumento para recolha de dados Esta questão foi escolhida para este trabalho, entre as 13 questões aplicadas, por ter apresentado resultados significativos, tanto no âmbito qualitativo, quanto no quantitativo, mensurado utilizando-se o teste estatístico de Wilcoxon. Olh@res, Guarulhos, v. 4, n. 2, p. 165-178, novembro 2016. Os cursistas De forma simplificada apresentamos o perfil dos participantes do curso de formação no Quadro 1: Olh@res, Guarulhos, v. 4, n. 2, p. 165-178, novembro 2016. 170 Análise das concepções de professores de biologia sobre modelos científicos antes e após um curso de formação continuada Análise das concepções de professores de biologia sobre modelos científicos antes e após um curso d formação continuada Quadro 1 - Perfil dos Profissionais Cursistas (professores) do curso de extensão Fonte: Produção própria Legenda: 1- Nível de formação: graduação(G); Especialização (E) Mestrado(M); 2- gênero: Masculino (M) ou Feminino (F). Anos de experiência idade Sexo2 Menos de 5 anos De 6 a 12 anos Nível de formação1 Nível de formação1 G E M G E M 25 a 33 M P1 F P3 e P4 P2 34 a 42 M F P5 Quadro 1 - Perfil dos Profissionais Cursistas (professores) do curso de extensão Legenda: 1- Nível de formação: graduação(G); Especialização (E) Mestrado(M); 2- gênero: Masculino (M) ou Feminino (F). Olh@res, Guarulhos, v. 4, n. 2, p. 165-178, novembro 2016. A proposta para os cursistas Na elaboração do projeto que foi apresentado e aprovado pelo departamento de Extensão do Centro Federal de Educação Tecnológica de Minas Gerais, os proponentes indicaram os seguintes objetivos do curso: (i) investigar saberes envolvendo Alfabetização Científica (AC) mobilizada pelos professores de Biologia que atuam no Ensino Médio; (ii) identificar quais são as dificuldades, as tensões que os professores de Biologia encontram ou enfrentam durante a implementação de uma proposta didática que envolva a AC; (iii) identificar quais as estratégias didáticas utilizadas pelos professores de Biologia no desenvolvimento dos conteúdos com enfoque CTS; (iv) promover uma reflexão sobre o potencial e as limitações da abordagem CTS no contexto da AC para o desenvolvimento da aprendizagem de Biologia e, finalmente, (v) desenvolver com os professores sequências didáticas na perspectiva CTS. Atendendo aos propósitos do curso de formação e, de acordo com a revisão de literatura efetuada, principalmente após leitura do livro “A Necessária Renovação do Ensino de Ciências” de Cachapuz et al., (2011) vimos que o curso deveria se basear em três aspectos: (i) levantamento das concepções dos professores sobre CTS/NdC&T; (ii) formação didática sobre CTS/NdC&T; (iii) elaboração, pelos professores cursistas, de sequências didáticas no contexto CTS e sua aplicação em sala de aula, se possível. O curso de formação foi ministrado a cinco professores cursistas, identificados como (P1, P2, P3, P4 e P5), durante 9 encontros de 4 horas, cada, divididos em 2 módulos de aulas, com um intervalo entre eles. Assim, no total foram 36 horas de aula de 50 171 Rosiane Resende Leite; Maria Delourdes Maciel minutos. Além das aulas presenciais, o curso também constou de 24 horas de atividades não presenciais, desenvolvidas pelos professores cursistas com suas turmas, em sala de aula; leituras de textos indicados na bibliografia e preparação de seminários, contabilizando um total de 60 horas. A seguir, no Quadro 2, temos a indicação do que foi tratado durante o curso de formação: Quadro 2 – Atividades Desenvolvidas no curso de Formação Quadro 2 – Atividades Desenvolvidas no curso de Formação Primeiro Encontro – 02 de agosto de 2014 8h-10h: Apresentação dos formadores e professores cursistas Apresentação da disciplina, do plano de ensino (Entrega do cronograma de atividades) Relatório individual sobre a experiência profissional de cada participante e levantamento oral sobre o entendimento de CTS pelos professores cursistas. Apresentação ppt pela pesquisadora sobre Conceitos básicos CTS. A proposta para os cursistas 10h – 10:20: Café 10:25 – 11:00: Leitura e Discussão de Artigo: A educação científica sob a Perspectiva da Pedagogia Histórico-Crítica e do Movimento CTS no ensino de Ciências - Paulo Marcelo M.Teixeira.Ciência e Educação, v.9, n.2, p. 177-190, 2003 11:00 – 12:00: Aplicação do questionário (Parte I e II – COCTS). Segundo Encontro – 22 de agosto de 2014 8h-10h – Apresentação e Exploração da Plataforma Ápice (Disponível em :<http://apice.febrace.org.br>.Acesso em 8 ago.2014), do curso: Introdução à Metodologia de Pesquisa. Discussão do módulo 2, 3 e 4 do curso Ápice 10h – 10:20: Café 10:25 – 12:00 – Apresentação PPT (PowerPoint) sobre Metodologia Científica. Leitura e Discussão de Artigo: Alfabetização Científica: Processos de Ensino e Aprendizagem que contribuem para a popularização da Ciência. Autores: Josiane Ladelfo, Flávia Santos da Costa, Maraes Huber. Maiêutica - Curso de Ciências Biológicas, v. 01, n. 01, jul./dez. 2011. Terceiro Encontro - 30 de agosto de 2014 8h-10h: Apresentação do programa Cmaptools (programa disponível gratuitamente na internet). Discussão do módulo 5, 6 e 7 do curso Ápice 10h – 10:20: Café 10:25 – 11:00: Leitura e discussão do capítulo 1 do livro: "CACHAPUZ, A.; GIL- PÉREZ, D.; CARVALHO, A. M. P de; PRAIA, J. e VILCHES, A. (Org.). A necessária renovação do ensino das ciências. 2 ed. São Paulo: Cortez, 2011” e do artigo de Santos (2007). 11:00 – 12:00: Elaboração de um mapa conceitual pelos cursistas, cada um elaborou um mapa referente a um tópico do capítulo do livro e/ou artigo, utilizando o programa Cmaptools. Quarto Encontro - 13 de setembro de 2014 8h-10h – Discussão do módulo 8, 9 e 10 do curso Ápice 10h – 10:20: Café 10:25 – 12:00 – Análise e Discussão do artigo: capítulo 3 do livro A necessária renovação do ensino de Ciências. Quinto Encontro - 27 de setembro de 2014 8h-10h: Leitura e discussão do capítulo 2 do livro A necessária renovação do ensino das Ciências. Discussão em grupo a partir das questões propostas. 10h – 10:20: Café Leitura prévia do texto em espanhol: LA BIBLIOTECA DE BABEL por Jorge Luís Borges.Explicitação do entendimento acerca do texto lido pelos cursistas. 10:25 – 12:00: Palestra sobre Filosofia da Ciência proferida pelo professor Juracy Coelho Ventura Sexto Encontro - 18 de outubro de 2014 8h-10h – Leitura e discussão do artigo de BAZZO et al., 2003. Palestra sobre Sequência Didática ministrada pela professora Sônia Cabral. Olh@res, Guarulhos, v. 4, n. 2, p. 165-178, novembro 2016. A proposta para os cursistas 10h – 10:20: Café 10:25 – 12:00 – Apresentação de uma sequência didática desenvolvida pela professora Sônia Aparecida Cabral e aplicada no ensino fundamental; Olh@res, Guarulhos, v. 4, n. 2, p. 165-178, novembro 2016. 172 172 Análise das concepções de professores de biologia sobre modelos científicos antes e após um curso de formação continuada Análise das concepções de professores de biologia sobre modelos científicos antes e após um curso de formação continuada Atividade sobre Classificação Biológica utilizando botões que foi usada na sequência da professora Sônia. Atividade proposta – Desenvolver uma sequência didática e aplicar em sala de aula se possível. Sétimo Encontro – 8 de novembro de 2014 8h-10h: Leitura e discussão de artigo sobre contextualização (workshop). Discussão do módulo 11 a 14 do curso Ápice 10h – 10:20: Café 10:25 – 12:00: Discussão dos artigos:  VAZQUEZ, A. A Importância da alfabetização científica e do conhecimento acerca da natureza da ciência e da Tecnologia para a formação de um cidadão.  BRECHT, E.; SILVA, M.P. A formação de professores sob o enfoque da Ciência, Tecnologia e Sociedade. Oitavo Encontro – 22 de novembro de 2014 8h-10h – Apresentação das sequências didáticas produzidas pelos professores P1 e P2; Discussão do módulo 11, 12 e 13 do curso Ápice. Apresentação do vídeo: O que é Ciência da profa. Dra. Luciana Massi. (Disponível em: <https://www.youtube.com/watch?v=ZYz0O8gFbyQ>. Acesso em 22 nov. 2014). 10h – 10:20: Café 10:25 – 12:00 – Apresentação da sequência didática produzida pela professora P3 Nono Encontro – 06 de dezembro de 2014 8h-10h – Reaplicação do questionário COCTS Apresentação da Sequência didática dos professores P4 e P5 10h –12:00 – Confraternização e Avaliação do curso Olh@res, Guarulhos, v. 4, n. 2, p. 165-178, novembro 2016. Analisamos, a seguir, cada uma das sete (7) frases da questão 90211 do COCTS. Olh@res, Guarulhos, v. 4, n. 2, p. 165-178, novembro 2016. Resultados e Discussão A questão (90211) do COCTS analisada refere-se aos modelos científicos. Por meio dela é possível inferir a ideia que o respondente tem sobre modelos científicos. Verificou-se que os cinco professores apresentavam uma fragilidade em relação à questão pesquisada, corroborando com algumas pesquisas que apontam esta dificuldade em relação ao ensino de Ciências. Neste sentido, Moreira (2014, p.2) aponta que: As teorias e modelos científicos são ensinados como verdades, como “descobertas geniais”, como definitivos, acabados. Os professores de ciência normalmente aceitam que o aluno é um construtor de seu próprio conhecimento e procuram fazer a mediação necessária para a reconstrução interna de conhecimentos científicos externamente construídos. Mas não apresentam estes conhecimentos como construções científicas. Este é um grande problema de ensino de ciências: ensina-se ciências sem uma concepção do que é ciência. (MOREIRA, 2014). Vê-se que a não apresentação dos conhecimentos como construções científicas talvez seja uma decorrência da falta de compreensão do professor sobre o assunto em questão. Vimos que as ideias ingênuas dos professores foram amenizadas, ou seja, se aproximaram do desejável, que é o valor atitudinal próximo de 1 após o curso de formação. Analisamos, a seguir, cada uma das sete (7) frases da questão 90211 do COCTS Olh@res, Guarulhos, v. 4, n. 2, p. 165-178, novembro 2016. 173 Rosiane Resende Leite; Maria Delourdes Maciel Rosiane Resende Leite; Maria Delourdes Maciel Em relação à frase A considerada pelos juízes como sendo ingênua, é possível inferir que houve uma melhoria no nível de aproximação com o resultado esperado. Apenas um professor (P3) apresentou discordância em relação a esta frase no pré-teste, mas após o curso de formação verificou-se que houve mudança nas compreensões desse mesmo professor. O gráfico 1 apresenta os índices médios obtidos para cada frase da questão 90211, antes (pré-teste) e após (pós-teste) o curso de formação. Resultados e Discussão Gráfico 1 – índices médios entre os professores de Biologia no pré-teste e pós-teste para a questão 90211 PRÉ-TESTE QUESTÃO 90211 frase A frase B frase C frase D frase E frase F frase G -0.6 -0.4 -0.2 -0.0 0.2 0.4 0.6 INDICE ATITUDINAL PÓS-TESTE QUESTÃO 90211 frase A frase B frase C frase D frase E frase F frase G -0.4 -0.2 0.0 0.2 0.4 0.6 0.8 INDICE ATITUDINAL Fonte: dados da pesquisa desenvolvida na tese PÓS-TESTE QUESTÃO 90211 frase A frase B frase C frase D frase E frase F frase G -0.4 -0.2 0.0 0.2 0.4 0.6 0.8 INDICE ATITUDINAL INDICE ATITUDINAL INDICE ATITUDINAL Fonte: dados da pesquisa desenvolvida na tese Fonte: dados da pesquisa desenvolvida na tese Para as frases B e C, que também são ingênuas, é possível verificar que os professores, exceto o professor P5, apresentaram grande discordância com o resultado dos especialistas, ou seja, os professores estão de acordo com estas questões ingênuas. Embora os professores continuassem com a visão ingênua após a aplicação do pós-teste, é possível verificar que para a maioria houve um deslocamento quanto ao nível de concordância, ou seja, as compreensões ainda são ingênuas, mas em um grau de intensidade menor que antes do curso conforme pode ser verificado pela análise do gráfico 1. De acordo com Moreira e Ostermann (1993), a permanente evolução dos modelos científicos caracteriza o conhecimento científico como não definitivo, porém o que em geral acontece em relação ao ensino de ciências é a tendência em reforçar o contrário, ou seja, de tratar o conhecimento científico como algo pronto e imutável. Talvez isso ocorra devido à “representação idiossincrásica que constroem os alunos sobre o mundo natural e as correspondentes representações científicas” (GALAGOVSKY & ADÚRIZ- BRAVO, 2001 p.232), o que é um impeditivo para a produção de uma aprendizagem significativa. Parece evidente a necessidade de um trabalho mais sistemático de desmistificação sobre o fazer científico. Olh@res, Guarulhos, v. 4, n. 2, p. 165-178, novembro 2016. Olh@res, Guarulhos, v. 4, n. 2, p. 165-178, novembro 2016. Resultados e Discussão 174 Análise das concepções de professores de biologia sobre modelos científicos antes e após um curso de formação continuada Análise das concepções de professores de biologia sobre modelos científicos antes e após um curso de formação continuada Em relação às frases D, E, F, G (plausíveis e adequadas), os professores apresentaram grande concordância com os juízes, de modo geral, e após o curso houve uma melhora significativa nas concepções relacionadas com estas frases. Todos os professores que apresentaram discordância com os juízes antes do curso de formação mudaram suas opiniões após o curso. Podemos inferir que, sobre estas questões, o curso obteve razoável efeito sobre as compreensões dos professores, operando de forma positiva na construção de ideias mais plausíveis acerca dos modelos científicos, o que foi possível ser validado com o emprego do teste estatístico wilcoxon (Quadro 3). Usando o programa de estatística Action 2,8, temos que a estatística de teste foi de 0. Este valor corresponde a um valor-p 0,01. Como o valor é maior que o nível de significância de 5%, ou seja, pode-se dizer que houve diferença significativa entre os grupos pesquisados (pré-teste e pós-teste) Quadro 3 - teste estatístico wilcoxon pareado para a questão 90211 Informação Valor V 0 P-valor 0,015625 Hipótese Nula 0 Método Wilcoxon signed rank test (Pseudo) Mediana -0,3625 Intervalo de Confiança 95% Limite Inferior -0,625 Limite Superior -0,2 Fonte: Execução do programa Action 2,8 no Excel Quadro 3 - teste estatístico wilcoxon pareado para a questão 90211 Quadro 3 - teste estatístico wilcoxon pareado para a questão 90211 Informação Valor V 0 P-valor 0,015625 Hipótese Nula 0 Método Wilcoxon signed rank test (Pseudo) Mediana -0,3625 Intervalo de Confiança 95% Limite Inferior -0,625 Limite Superior -0,2 Fonte: Execução do programa Action 2,8 no Excel Olh@res, Guarulhos, v. 4, n. 2, p. 165-178, novembro 2016. Olh@res, Guarulhos, v. 4, n. 2, p. 165-178, novembro 2016. Considerações Finais A avaliação do curso se deu tanto no aspecto quantitativo quanto no qualitativo. Apesar dos professores não conseguirem demonstrar que melhoraram 100% as suas concepções, as respostas mostraram-se satisfatórias, pois se verificou que ocorreu uma significativa mudança das mesmas para melhor, se compararmos as respostas do pré-teste com o pós-teste. Verificou-se um melhor desempenho dos participantes que apresentaram concepções ingênuas no pré-teste e, com isso, foi possível atuar de forma mais sistemática sobre as fragilidades apresentadas pelos professores cursistas, em relação à discussão da questão colocada. Acreditamos que o curso tenha contribuído efetivamente para a mudança das concepções destes professores e pode ser tomado como exemplo por aqueles que desejarem promover mudanças na formação docente, a partir de atividades acessíveis ao 175 Rosiane Resende Leite; Maria Delourdes Maciel professor e que possam ser facilmente aplicadas em situações de sala de aula. Ou seja, visualizar e compreender os pontos frágeis das concepções para, então, atuar sobre elas, reforçando-as de maneira que os docentes possam agir adequadamente em questões relacionadas a CTS/NdC&T, transmitindo aos seus alunos uma visão mais correta sobre a ciência. No curso ofertado tivemos a preocupação não só não de detectar as concepções errôneas dos docentes, mas também de usar estratégias didáticas que possibilitassem aos professores tomar consciência dos seus próprios erros. Para isto, elaboramos uma lista de afirmações falsas e verdadeiras a respeito da Natureza da Ciência (NdC) e sua aprendizagem, baseada em Vazquez e Manassero (2012, p. 8-9) e Pozo e Crespo (2009, p.18). Pedimos aos professores que assinalassem V (verdadeiro) ou F (falso) ao lado de cada frase. Após, indicamos o que era considerado atitude e crença inadequada por estes autores, para que os professores pudessem identificar suas fragilidades em relação à NdC. Ao final do curso reaplicamos o questionário COCTS (pós-teste) e, a seguir, devolvemos para os professores o questionário respondido no início (pré-teste), solicitando que fizessem uma comparação de suas respostas dadas no início e no fim do curso. Além disso, apontamos quais questões eram ingênuas, adequadas e plausíveis. Logo, no início da formação foi detectada a necessidade de desenvolver temas que promovessem a cultura científica dos professores, para que os mesmos conseguissem introduzir a abordagem CTS nas suas aulas. Olh@res, Guarulhos, v. 4, n. 2, p. 165-178, novembro 2016. Considerações Finais Vimos que, em relação à NdC, o entendimento acerca da construção do conhecimento, o passo a passo de uma pesquisa, era o principal a ser trabalhado, já que os professores cursistas relataram no primeiro encontro esta dificuldade, pois na sua formação inicial não tiveram a oportunidade de cursar a disciplina metodologia científica, que não era ofertada na graduação, ou mesmo de realizar uma iniciação científica. Dessa forma, concluímos ser necessário incentivar os professores a trabalhar numa perspectiva CTS; ofertar cursos de formação nesta linha e criar espaços e tempos para a sua formação sobre o fazer ciência. Olh@res, Guarulhos, v. 4, n. 2, p. 165-178, novembro 2016. 176 Olh@res, Guarulhos, v. 4, n. 2, p. 165-178, novembro 2016. REFERÊNCIAS BIBLIOGRÁFICAS ALBRECHT, E.; SILVA, M.P. A formação de professores sob o enfoque da Ciência, Tecnologia e Sociedade. In: MACIEL, M.D.; AMARAL, C.L.C; GUAZZELLI, I.R.B. Ciência Tecnologia & Sociedade. São Paulo: Terracota, 2010. p. 157-164 AULER, D. Movimento Ciência-Tecnologia-Sociedade (CTS): modalidades, problemas e perspectivas em sua Implementação no ensino de física. In: Encontro de Pesquisa em Ensino de Física, 6, Resumos..., Florianópolis, 1998. AULER, D e BAZZO, W. A. Reflexões para a implementação do movimento CTS no contexto educacional brasileiro. Ciência & Educação, Bauru, v. 7, n. 1. p. 1-13, 2001. BAZZO, W. A., VON LINSINGEN, I.; PEREIRA, L.T.V. Introdução aos estudos CTS (Ciência, tecnologia e sociedade). OEI. Organização dos Estados Ibero – americanos.Cadernos de Ibero-América,Madri, Espanha. 2003. Disponivel em:<http://www.oei.es/salactsi/Livro_CTS_OEI.pdf>. Acesso em 12.jan.2013. BORGES, J. L. La Biblioteca de Babel. Disponível em <http://www.literaberinto.com/vueltamundo/bibliotecabor mar.2013. BORGES, J. L. La Biblioteca de Babel. Disponível em <http://www.literaberinto.com/vueltamundo/bibliotecaborges.htm>. Acesso em mar.2013. CACHAPUZ, A.; GIL- PÉREZ, D.; CARVALHO, A. M. P.; PRAIA, J.; VILCHES, A. (Org.). A necessária renovação do ensino das ciências. 2 ed. São Paulo: Cortez, 2011. CARVALHO, A. M. P. de; GIL-PÉREZ, D. Formação de professores de ciências: tendências e inovações. 8 ed. São Paulo: Cortez, 2006. DELIZOICOV, D.; ANGOTTI, J. A.; PERNAMBUCO, M. M. Ensino de Ciências: fundamentos e métodos. São Paulo: Cortez, 2002. GALAGOVSKY, L.; ADÚRIZ-BRAVO, A. Modelos y analogías en la enseñanza de las ciências naturales. El concepto de modelo didáctico analógico. Enseñanza de las ciencias, v.19, n.2, p. 231-242, 2001. GATTI, B.A.; NUNES, M.M.R. Formação de professores para o ensino fundamental: estudo de currículos das licenciaturas em Pedagogia, Língua Portuguesa, Matemática e Ciências Biológicas. São Paulo: FCC/DPE, 2009, v. 29. KNAUSS, P. O desafio da ciência: modelos científicos no ensino de história. Cad. Cedes, Campinas, vol. 25, n. 67, p. 279-295, set./dez. 2005. LADELFO, J.; COSTA, F.S.; HUBER, M. Alfabetização Científica: processos de ensino e aprendizagem que contribuem para a popularização da ciência. Maiêutica - Curso de Ciências Biológicas, v.01, n.01, jul./dez. 2011. LOPES, A.C e MACEDO, E. O pensamento curricular no Brasil, In: Currículo: debates contemporâneos. LOPES E MACEDO (Orgs.), São Paulo, SP, Cortez Editora, 2002. Olh@res, Guarulhos, v. 4, n. 2, p. 165-178, novembro 2016. 177 177 Rosiane Resende Leite; Maria Delourdes Maciel MOREIRA, M.A; OSTERMANN, F. Sobre o ensino do Método Cientifico, cad.ens. Fís, v.10, n.2, p. 108 – 177, ago. 1993. MOREIRA, M.A. Olh@res, Guarulhos, v. 4, n. 2, p. 165-178, novembro 2016. REFERÊNCIAS BIBLIOGRÁFICAS Modelos científicos, modelos mentais, modelagem computacional e modelagem matemática: aspectos epistemológicos e implicações para o ensino. R. B. E. C. T.v. 7, n. 2, mai-Ago, 2014 NARDI, R.; BASTOS, F.; DINIZ, R. E. da S. (Org.) Pesquisas em ensino de Ciências: contribuições para a formação de professores. São Paulo: Escrituras, 2004. POZO, J. I.; CRESPO, M.Á.G. Aprendizagem e o ensino de ciências: do conhecimento cotidiano ao conhecimento científico. Tradução Naila Freitas. 5 ed. Porto Alegre: Artmed, 2009. PRAIA, J; GIL-PEREZ, D.; VILCHES, A. O papel da natureza da Ciência na educação para a cidadania Ciência & Educação, v. 13, n. 2, p. 141-156, 2007. SANTOS, W. L.P. dos. Educação científica na perspectiva de letramento como prática social: funções, princípios e desafios. Revista Brasileira de Educação, Rio de Janeiro, v. 12, n. 36, p.474-550, set./dez. 2007. Disponível em: <http://www.scielo.br/pdf/rbedu/v12n36/a07v1236.pdf>. Acesso em: 22 mai. 2015. TEIXEIRA, P. M. M. A Educação Científica sob a Perspectiva da Pedagogia Histórico- social e do movimento CTS no Ensino de Ciências. Revista Ciências & Educação, Bauru: Unesp v. 9, n. 2, p. 177- 190, 2003. TENREIRO-VIEIRA e VIEIRA. Construção de práticas didático-pedagógicas com orientação CTS: impacto de um programa de formação continuada de professores de Ciências do ensino básico. Ciencia & Educação, v.11, n.2, p.191-211, 2005. VÁZQUEZ, A.V. Importância da Alfabetização Científica e do Conhecimento acerca da Natureza da Ciência e da Tecnologia para a formação de um cidadão. In: MACIEL, M.D.; AMARAL, C.L.C; GUAZZELLI, I.R.B. Ciência Tecnologia & Sociedade. São Paulo: Terracota, 2010. p. 43-70 VÁZQUEZ, A.; MACIEL, M.D.; MANASSERO, M. A., CHRISPINO, A. “A compreensão dos temas de ciência, tecnologia e sociedade no Brasil: análise comparativa com outros países do PIEARCTS. CTS e Educação Científica: desafios, tendências e resultados de pesquisas”. SANTOS, W.L.P.; AULER, D. Editora Universidade de Brasilia. 460p, 2011. VÁZQUEZ, A.; MACIEL, M.D.; MANASSERO, M. A., CHRISPINO, A. “A compreensão dos temas de ciência, tecnologia e sociedade no Brasil: análise comparativa com outros países do PIEARCTS. CTS e Educação Científica: desafios, tendências e resultados de pesquisas”. SANTOS, W.L.P.; AULER, D. Editora Universidade de Brasilia. 460p, 2011. VÁZQUEZ-ALONSO, Á.; MANASSERO-MAS, M. A. La selección de contenidos para enseñar naturaleza de la ciencia y tecnología (parte 1): Una revisión de las aportaciones de la investigación didática. Cádiz, España, Revista Eureka sobre Enseñanza y Divulgación de las Ciencias, v. 9, n. 1, p. 2-31, 2012 Disponível em:<http://www.redalyc.org/pdf/920/92024530002.pdf>. Acesso em fev 2014. REFERÊNCIAS BIBLIOGRÁFICAS Recebido em: 22/02/2016 Aprovado em: 23/08/2016 Recebido em: 22/02/2016 Aprovado em: 23/08/2016 Olh@res, Guarulhos, v. 4, n. 2, p. 165-178, novembro 2016. 178
https://openalex.org/W4362417716	https://figshare.com/articles/journal_contribution/Supplementary_Figure_4_from_Characterization_of_Chemokines_and_Adhesion_Molecules_Associated_with_T_cell_Presence_in_Tertiary_Lymphoid_Structures_in_Human_Lung_Cancer/22388460/1/files/39833946.pdf	English	null	Supplementary Figure 2 from Characterization of Chemokines and Adhesion Molecules Associated with T cell Presence in Tertiary Lymphoid Structures in Human Lung Cancer	null	2,023	cc-by	200	Appendix A6 : Model of a role of Ti-BALT in the establishment of protective anti- tumoral immune responses Appendix A6 : Model of a role of Ti-BALT in the establishment of protective anti- tumoral immune responses Appendix A6 : Model of a role of Ti-BALT in the establishment of protective anti- tumoral immune responses Appendix A6 : Model of a role of Ti-BALT in the establishment of protective anti- tumoral immune responses This model recapitulates our findings and hypothesis on the migration of T cells to and from Ti-BALT, and consequences on the establishment of protective immune responses. A subset f i h l T ll i t t th t i th HEV Thi fi t i t ti i l of peripheral T cells emigrates to the tumor via the HEV. This first interaction involves a unique combination of receptor/ligand pairs, as mentioned in the figure. Specific T cells encounter antigen-loaded DC in the T-cell zone. This interaction induces the differentiation of naïve T cells into effector and central-memory T cells. The central-memory T cells expressing CCR7 may leave the tumor via CCL21+ lymphatic vessels to the draining lymph node, and provide a systemic protection against tumor metastasis.
https://openalex.org/W3123502880	https://dergipark.org.tr/tr/download/article-file/1456947	English	null	Urinary tract stone surgery in patients with urinary diversion and vesicostomy: a single center experience	Journal of health sciences and medicine :	2,021	cc-by	4,870	ABSTRACT Objective: To report our experience in percutaneous nephrolithotomy and endoscopic urinary tract stone surgery in patients with urinary diversion or vesicostomy. Material and Method: Data of 21 patients with urinary diversion or vesicostomy who underwent surgery for urinary tract stones in our clinic between January 2008 and January 2020 were retrospectively analyzed. Eight patients (38%) underwent percutaneous nephrolithotomy, 2 patients (9.5%) underwent antegrade flexible ureteroscopy, 4 patients (19.0%) underwent retrograde semi-rigid or flexible ureteroscopy, 5 patients (23.8%) underwent retrograde pouch lithotripsy and 2 patients (9.5%) underwent percutaneous cystolithotripsy with vesicostomy tract entrance. Preoperative and postoperative data of the patients were evaluated. Results: The male to female ratio was 16/5.The mean age of the patients was 54.6±10.1 years and mean preoperative stone diameter was 2.8±4.5 cm. It was determined that 14 patients (66.6%) had ileal conduit (Bricker anastomosis), 5 patients (23.8%) had ureterocutaneostomy, and 2 patients (9.5%) had vesicostomy. Stone-free rate was 85.7% after single session of treatment. In the postoperative period, febrile urinary tract infection was observed in 4 (19.0%) patients, urinary system obstruction secondary to stone in 3 (14.2%) patients and anastomotic leakage in 1 (4.7%) patient. Conclusion: Percutaneous nephrolithotomy, antegrade ureterorenoscopy, retrograde ureterorenoscopy and vesicostomy entry cystolithotripsy are highly effective and safe methods in patients with urinary diversion and vesicostomy. The most important factors affecting the success are the experience of surgical team that can apply procedural options together with careful preoperative preparation and correct instrumentation. Keywords: Urinary diversion, stone formation, stone surgery Urinary tract stone surgery in patients with urinary diversion and vesicostomy: a single center experience Kubilay Sarıkaya, Çağrı Şenocak, Fahri Erkan Sadioğlu, Mehmet Çiftçi, Ömer Faruk Bozkurt University of Health Sciences, Keçiören Training and Research Hospital, Department of Urology, Ankara, Turkey Cite this article as: Sarıkaya K, Şenocak Ç, Sadioğlu FE, Çiftçi M, Bozkurt ÖF. Urinary tract stone surgery in patients with urinary diversion and vesicostomy: a single center experience. J Health Sci Med 2021; 4(1): 78-83. HEALTH SCIENCES MEDICINE DOI: 10.32322/jhsm.843304 Original Article Original Article J Health Sci Med 2021; 4(1): 78-83 DOI: 10.32322/jhsm.843304 Corresponding Author: Kubilay Sarıkaya, drkubilay.sarikaya76@outlook.com This work is licensed under a Creative Commons Attribution 4.0 International License. Surgical Procedures combined antegrade and retrograde approaches constitute the preferred surgical options (11). Although successful results of extracorporeal shoch wave lithotripsy (ESWL) have been reported in stone patients with urinary diversion in stones smaller than 2 cm, it is known that the success rate of ESWL decreases in larger stones (10). Percutaneus nephrolithotomy (PNL): All patients with renal stones were evaluated primarily in lithotomy position in terms of performing retrograde ureteral catheterization and, if possible, retrograde intrarenal surgery (RIRC). Patients whose ureter orifices cannot be seen and retrograde access was not possible were prepared for percutaneous intervention, a 16-18 fr foley catheter was inserted into the pouch from the stoma, the balloon was inflated with 5-6 cc saline, and the patient was turned to the prone position. After the appropriate position was given, radiopaque liquid diluted with 50% saline was given into the pouch through the foley catheter, and it was tried to pass from the ureter to the kidney to visualize the renal collecting system. In cases which sufficient radiopaque material could not reached into the kidney and the renal collecting system, the ultrasound-guided entry technique was used. An 18-gauge percutaneous needle was used to enter the renal collecting system, and the needle was removed so that the outer sheath remained in the renal collecting system by providing the targeted renal calix entry. Afterwards, radiopaque fluid was injected into the renal collecting system through the outer sheath of the percutaneous needle, and the collecting system was visualized, and the entrance location and anatomical structure were evaluated. In cases where it was seen that proper calix entry could not be achieved, re-entry was made with a second needle and the outer sheath of the first entry needle was not removed and used for radiopaque fluid infusion into the renal collecting system in order to ensure adequate visualization. After proper renal calix insertion was provided, a 0.035-inch hydrophilic guide wire was advanced into the renal collecting system through the outer sheath of the needle, followed by percutaneous dilatation with percutaneous dilatators ranging from 24fr-30fr, allowing percutaneous access to the renal collecting system with rigid nephroscope (Karl- storz®-22 fr). Following the percutaneous entry, the stone was fragmented and removed with use of an ultrasonic lithotripter (EMS®), or pneumatic lithotripter (EMS®). A flexible cystoscope (Olympus®-21 fr) was used in some calix stones that cannot be reached with rigid nephroscope. Surgical Procedures Following the procedure, a 16-fr catheter was placed in all patients as a nephrostomy, and the catheter had placed in the pouch was removed. As the literature data and our clinical experience indicate, urinary system stone stone surgery in patients with urinary diversion and vesicostomy requires preoperative instrumentation preparation and surgical team experience, which allows all options to be applied during the operation. In this study, we aimed to share our experiences in urinary system stone surgeries performed in patients with urinary diversion and vesicostomy in our clinic, which is one of the centers working intensively on urinary system stone surgery. INTRODUCTION Ureterosigmoidostomy procedure was first applied after radical cystectomy by Simon et al. (1) and following this, different types of urinary diversion such as cutaneous conduit, orthotopic neobladder and continent urinary diversion were developed. Stone formation in the upper urinary tract as well as in the reservoir or conduit is one of the most common complications in patients with urinary diversion (2). The incidence of stone formation in patients with ileal conduit was reported to be from 9%to 11%, while it was reported that 17% patients with Koch pouch and from 11% to 12.9% of patients with Indiana pouch developed urinary tract stones in the long term follow-up period (3-5). Similar to patients with urinary diversion, it is known that both bladder stones and upper urinary tract stones are frequently develop in patients with neurogenic bladder developing secondary to spinal cord injury (6). Stone surgery in patients with urinary diversion presents various difficulties for urologists. Difficulties in visualizing the ureter orifices through the pouch and entering the ureter during both imaging and retrograde approach due to the impaired anatomical structure constitute the main problem in this area (7-9). On the other hand, stone recurrence reported in 33% to 63% of patients with urinary diversion within 3-5 years after the first surgical intervention significantly limits the option of the open stone surgery (10). Therefore, in patients with urinary diversion, percutaneous nephrolithotomy (PNL), semi-rigid or flexible antegrade ureteroscopy (URS) performed through percutaneous tract, or Received: 19.12.2020 Accepted: 22.12.2020 This work is licensed under a Creative Commons Attribution 4.0 International License. Sarıkaya et al. Stone surgery in patients with urinary diversion J Health Sci Med 2021; 4(1): 78-83 RESULTS The male to female ratio was 16/5. The mean age of the patients was 54.6±10.1 (16-71) years. The most common comorbidity factor detected in the preoperative period was hypertension which was seen in 14 (66.6%) patients. The mean preoperative creatinine level was 1.1±0.3 (80.7- 2.2) mg/dL. The most common stone localization was kidney in 11 (52.3%) patients, while it was found in the reservoir pouch in 5 (23.8%) patients and in the ureter in 3 (14.2%) patients. Bladder stones were detected in the other 2 (9.5%) patients with vesicostomy included in the study. Preoperative patient characteristics are shown in Table 1. Table 1. Preoperative patient’s characteristics Age, mean±SD (minimum-maximum),years 54.6±10.1 (16-71) Gender (n,%) - Male 16 (76.1%) Female 5 (23.8%) Comorbidities (n,%) - Hypertension 14 (66.6%) Diabetes mellitus 4 (19.0%) Dyslipidemia 5 (23.8%) Chronic obstructive pulmonary disease 2 (9.5%) Rheumatoid arthritis 1 (4.7) Preoperative serum creatinine level (mg/dL) 1.1±0.3 (0.7-2.2) Stone localization (n,%) - Kidney 11 (52.3%) Ureter 3 (14.2%) Reservoirpouch 5 (23.8%) Bladder 2 (9.5%) Stone size (cm) 2.8±4.5 (1.4-8.5) Diversion type (n,%) - İleal conduit (Bricker type) 14 (66.6%) Ureterocutaneostomy 5 (23.8%) Vesicostomy 2 (9.5%) Table 1. Preoperative patient’s characteristics Age, mean±SD (minimum-maximum),years 54.6±10.1 (16-71) Gender (n,%) - Male 16 (76.1%) Female 5 (23.8%) Comorbidities (n,%) - Hypertension 14 (66.6%) Diabetes mellitus 4 (19.0%) Dyslipidemia 5 (23.8%) Chronic obstructive pulmonary disease 2 (9.5%) Rheumatoid arthritis 1 (4.7) Preoperative serum creatinine level (mg/dL) 1.1±0.3 (0.7-2.2) Stone localization (n,%) - Kidney 11 (52.3%) Ureter 3 (14.2%) Reservoirpouch 5 (23.8%) Bladder 2 (9.5%) Stone size (cm) 2.8±4.5 (1.4-8.5) Diversion type (n,%) - İleal conduit (Bricker type) 14 (66.6%) Ureterocutaneostomy 5 (23.8%) Vesicostomy 2 (9.5%) Retrograde pouch lithotripsy with stomal entry: Retrograde pouch lithotripsy with stomal entry was applied in patients with reservoir stones. For this purpose, the pouch was inserted retrograde from the stoma with using a 22 fr nephroscope and the stone was fragmented with a pneumatic or ultrasonic lithotriptor and removed with the aid of a stone basket. During the procedure, the presence of stone fragments between the mucosal folds was checked by fluroscopy, and possible fragmented stones were removed. At the end of the procedure, radiopaque fluid was injected into the pouch and the presence of possible anastomotic leakage was checked, and at the end of the procedure, a 16-18 fr foley catheter was placed in the pouch and the procedure was terminated. MATERIAL AND METHODh The study was carried out with the permission of Keçiören Training and Research Hospital Health Application Research Center Medical Specialty Education Board (Date: 08.12.2020 IRB: 2012-KAEK-15/2202) of our institution, the data of 21 patients with urinary diversion or vesicostomy who underwent surgical intervention for urinary tract stones between January 2008 and January 2020 were retrospectively analyzed. All procedures were performed adhered to the ethical rules and the Helsinki Declaration of Principles. During the preoperative preparation period, all patients were evaluated with routine preoperative blood tests, urine analysis, urine culture, and non-contrast abdominal computed tomography (CT). Intravenous urography (IVU) was performed to evaluate the anatomical structure of kidney. Possible anatomical variations were evaluated according to the type of operation and diversion the patients had, and the possible difficulties to be encountered during the operation were discussed and necessary and sufficient instrumentation was provided according to these possibilities. In the preoperative evaluation, it was determined that 14 (66.6%) patients had Bricker-type ileal conduit diversion, 5 (23.5%) had ureterocutaneostomy performed during radical cystectomy, and 2 (9.5%) patients had vesicostomy due to neurogenic bladder developing secondary to spina bifida. Appropriate prophlylactic and therapeutic antibiotic was administered to patients according to preoperative urine culture results. Intravenous 1 g 3rd generation cephalosporin was administered to all patients for preoperative prophylaxis. The operations included in the study were performed by a total of six surgeons working in the same center. Antegrade URS: Antegrade URS procedure was used in upper ureteral stones where retrograde intervention was not possible and for stones that could not be reached with a rigid or flexible nephroscope. At the entrance, the same procedure was applied as the PNL entry and percutaneous entry to the kidney through the appropriate calix was provided. Then, a 0.035-inch hydrophilic guide wire was advanced antegradely into the ureter and then the stone was reached by entering the ureter with 79 Sarıkaya et al. Stone surgery in patients with urinary diversion J Health Sci Med 2021; 4(1): 78-83 a flexible ureteroscope (Olympus®-9.5 fr) through the nephrostomy tract. The stone was fragmented with using a 200 µm or 500 µm Ho:YAG laser energy (StoneLight®) and removed with a 2.2 fr nitinol stone basket. After the stone was removed, imaging was performed by antegrade ureterography in terms of possible obstruction and anostomosis integrity, and a 5 fr doule-j stent was placed through the nephrostomy tube up to the reservoir pouch. Statistical Analysis All statistical analyses were performed with SPSS 24.0 (IBM Corp., Chicago) for Windows. The mean±standard deviation was used for parametric data and the median and minimum-maximum values were used for nonparametric data. Retrograde URS: Except for patients with vesicostomy, ureteral catheterization for PNL or URS was tried in all 19 patients with urinary diversion, but only in 4 (21.0%) of the patients procedure wassuccessful. Retrograde URS was performed in cases that the ureter orifice can be seen through the diversion pouch. Using a 17 fr rigit cystoscope or 9.5-fr semi-rigid ureterorenoscope (Olymus® 9.5 fr), a 0.035-inch hydrophilic guidewire was advanced to the ureter under direct visualition of the ureter orifice of the stone side. Subsequently, the ureter was entered by using a semi-rigid or flexible ureterorenoscope under the guidance of a hydrophilic guidewire. The stone was fragmented with a 200 µm or 500 µm Ho: YAG laser energy and removed using a 2.2 fr nitinol stone basket. At the end of the procedure, a 5 fr double-j stent was placed over the hydrophilic guidewire into the ureter and the procedure was terminated.The double-j stent was removed on the postoperative day 7 by retrograde route using a rigit or flexible cystoscope. MATERIAL AND METHODh Subsequently, a 16 fr nephrostomy catheter was inserted and the procedure was terminated. The nephrostomy tube was removed on the postoperative day 3, and then on the 7th day, the duoble-j stend was removed through the pouch using a rigit (Karl-Storz®-17 fr) or flexible cystoscope. was entered through the vesicostomy tract with a 22-fr nephroscope, and then the stone was fragmented using an ultrasonic or pneumatic lithotripter and removed using a stone basket. When necessary, 200 µm or 500 µm Ho: YAG laser energy was also used for stone fragmentation. At the end of the procedure, a 16-18 fr foley catheter was inserted through the vesicostomy tract into the bladder and removed on the second postoperative day. RESULTS The foley catheter in the pouch was removed on the second postoperative day. Percutaneous cystolithotripsy with vesicostomy entry: In patients with vesicostomy, which was performed due to neurogenic bladder developing secondary to spina bifida, percutaneous cystolithotripsy with vesicostomy entry was applied for bladder stone. In this procedure, the bladder 80 J Health Sci Med 2021; 4(1): 78-83 Sarıkaya et al. Stone surgery in patients with urinary diversion caused by it, stomal stenosis and urinary retention are also important factors that cause stone formation in the pouch (16). Metabolic changes are characterized by systemic acidosis, hypercalciuria, hyperoxaluria, and hypocitraturia (17). These factors increase the risk of calcium-containing stones (17). However, the most common factor causing stone formation in patients with urinary diversion is the presence of increased chronic infection (18). Therefore, struvite stones are the most common stone formation in patients with urinary diversion. In our study, similar to the literature, struvite stones were found to be the most common stone formation. It was observed that the mean operation time was 74.5±14.6 (40-130) minutes and the most common operationtype was PNLthat was performed in 8 (38.0%) patients. Other procedures performed were antegrade URS in 2 (9.5%) patients, retrograde URS in 4 patients (19.0%), retrograde pouch lithotripsy with stomal entry in 5 patients (23.8%) and percutaneous cystolithotripsy with vesicostomy entry in 2 patients (9.5%). In the early postoperative period, febrile urinary tract infection was developed in 4 (19.0%) patients. In total of 3 (14.2%) patients, 2 (9.5%) who underwent PNL and 1 (4.7%) who underwent antegrade URS, urinary obstruction was developed due to residual stones in the postoperative period and secondary surgical intervention was required. Minimal anastomotic leakage was found at the end of the procedure in 1 (4.7%) of the patients who underwent retrograde URS, and it was observed that the urinary leakage spontaneously recovered by a double-j stent without additional surgical intervention. Postoperative findings of the patients are shown in Table 2. In patients with urinary diversion with asymptomatic small size urinary stone, ESWL is recommended as a non-invasive initial treatment option in order to reduce potential surgical difficulties and complication (19). Ahmed et al. (20) reported that they were performed ESWLin 27 patients with urinary diversion with stones smaller than 1 cm. According to this study, the success rate of upper urinary tract stones in urinary diversion patients with single session ESWL treatment was reported as 81.5% (22/27). RESULTS However, in this study, it was also reported that the stone-free status could not completely reached in 2 (7.4%) patientsand 1 (3.7%) had a significant residual stone, 2 (7.4%) patients developed renal obstruction after ESWL and 5 (18.5%) patients had PNL, antegrade URS or a secondary procedure such as open operation is required. The fact that the stone fragments after ESWL enters into the intestinal folds in the pouch and do not drain spontaneously and cause the formation of large stones again by undergoing reformation is considered as an important factor limiting the effectiveness of this procedure, especially in stones larger than 1 cm (18-20). Table 2. Surgical procedures and postoperative results Surgical procedure (n,%) - Percutaneus nephrolithotomy 8 (38.0%) Antegradeureteroscopy (flexible) 2 (9.5%) Retrogradeureteroscopy (semi-rigid or flexible) 4 (19.0%) Retrograde pouch lithotripsy (transstomal) 5 (23.8%) Percutaneus cystolithotripsy (vesicostomy-entry) 2 (9.5%) Operation time (minutes) 74.5±14.6 (40-130) Postoperative serum creatinine level (mg/dL) 0.8±0.4 (0.7-1.9) Complication (n,%) - Febrile urinary tract infection 4 (19.0%) Urinary obstruction and secondary surgery 3 (14.2%) Anastomotic urine leakage 1 (4.7%) Stone composition (n,%) - Calcium-oxalate 9 (42.8%) Struvite 10 (47.6%) Calcium-phosphate 1 (4.7%) Urine-acite 1 (4.7%) Percutaneous nephrolithotomy is one of the most important options in the surgical treatment of large stones in the upper urinary system in patients with urinary diversion, and its success rate has been reported to be from 60% to 80% (21). In a study conducted by Lindsay et al. (22) involving 77 patients, the effectiveness of PNL, retrograde URS and ESWL in the treatment of upper urinary tract stones in patients with urinary diversion was compared. In this study, it was stated that the average stone size in the PNL group was significantly higher than the URS and ESWL groups (2.1 vs 0.9 and 1.0 cm,respectively, p<0.0001).In this study, although the mean stone size was significantly larger, the total stone-free rate was significantly better in PNL than the URS and ESWL groups (83.3% vs 33.3% and 30%, respectively, p<0.0001). Total complication rates were reported to be similar between the groups (p=0.900). In another similar study by Zhong et al. (23) including 20 patients with urinary diversion, 8 patients underwent PNL, 3 patients had antegrade URS, 6 patients had percutaneous pouch lithotripsy, 2 patients DISCUSSION The most common postoperative complication seen in patients with urinary diversion is urinary stone formation with a prevalence of 2.6% to 15.3% (12,13). The main cause of urinary system stone disease in these patients is the presence of increased chronic infection and metabolic changes, as well as the structural and mechanical differentiation in the urinary system (14). Stasis in the upper urinary system and the presence of hydronephrosis developing secondary to this, together with the change of the anatomical structure as a result of urinary diversion, are seen with a frequency of up to 80% in these patients (15). Mucus production originating from the intestinal mucosa and the foreign body reaction 81 Sarıkaya et al. Stone surgery in patients with urinary diversion J Health Sci Med 2021; 4(1): 78-83 had transurethral neo-bladder lithotripsy, 1 patient had open stone surgery. It was reported that 18 (90%) patients werestone-free at the end of the procedures. According to this study, an insignificant residual stone remained in 1 (5%) patient who underwent PNL and 1 (5%) patient who underwent percutaneous pouch surgery, and only 2 (15%) patients had postoperative fever and 1 (5%) patient had postoperative urinary extravasation. In our study, it was seen that the most common surgical procedure used in patients with urinary diversion was PNL, and similar to the literature, a high rate of stone-free was achieved. In our study, secondary intervention was required due to urinary obstruction in only 1 of the patients who underwent PNL. This result supports the idea that PNL should be the preferred surgical method for the surgical treatment of upper urinary tract stones in patients with urinary diversion due to its high success rate. The most common problem encountered when performing PNL in patients with urinary diversion is that the retrograde catheterization cannot be performed most of the time due to the difficulty in visualizing the ureter orifices through the pouch (17). Ultrasonographic approach can be used in these patients for achieving proper access to the collecting system (24). At this stage, proper preoperative preparation and equipment competence, as well as surgical team experience come to the fore. In our study, there was no access problem in any urinary diversion patient who underwent PNL, and therefore, there was no need to switch to another surgical method. Limitationsh The most important limitation of our study is its retrospective nature. In addition, the fact that urinary system stone surgery is one of the rare cases in patients with urinary diversion and therefore the low number of patients in the study can be considered as another limitation. The fact that no comparison was made due to the absence of a control group in our study can be considered as another limitation. The most important problem with retrograde URS in patients with urinary diversion is the difficulty in identifying the ureteral orifices within the reservoir (3,4,19,20,23). Singla N et al. (25) reported the results of retrograde URS intervention performed in 45 neobladder patients due to several upper urinary tract anomalies. According to this study, it was reported that retrograde URS intervention was successful in 2 of 4 urinary system stone patients. In the study of Delveccio et al. (11) it was reported that retrograde URS can be performed more easily under the guidance of the guidewire advanced into the neo-bladder from antegrade route, but the procedure is very time consuming. Therefore, antegrade URS is recommended for upper ureteral stones or renal calix stones that cannot be reached with PNL in urinary diversion patients, and the procedure can be successfully performed with a semi-rigid or flexible ureterorenoscope (26). In our study, retrograde URS intervention was largely unsuccessful because the ureteral orifices could not be clearly visualized so the procedure changed to antegrade URS or PNL in these patients. This result indicates that the antegrade URS or PNL should be the more preferred choice rather than retrograde URS in patients with urinary diversion with upper urinary tract stones due to the loss of time and effort. DISCUSSION In several studies, it has been reported that stones in the diversion pouch can be successfully treated with transurethral lithotripsy, percutaneous pouch lithotripsy or stomal entry pouch lithotripsy in patients with both orthotopic urinary diversion and cutaneous urinary diversion (7,11,15). The surgical method to be chosen depends on the type of diversion and the size of the stone. In our study, stomal entry pouch lithotripsy was applied to all patients with stones in the reservoir pouch and complete stone-free was achieved in all patients without complications, similar to the literature. Various studies have reported that the risk of bladder stone formation is increased in neurogenic bladder disorder that develops in patients with spinal cord injury (27). Ordet al. (28) reported that the risk of bladder stone formation was significantly increased in patients with spinal cord injury, both in patients who underwent intermittent urethral catheterization, and in patients with vesicostomy and cystostomy. In our study, bladder stones were detected in 2 patients who had vesicostomy due to neurogenic bladder secondary to spina bifida, and complete stone-free was achieved with vesicostomy-entry cystolithotripsy. REFERENCES 22. Hertzig LL, Iwaszko MR, Rangel LJ, Patterson DE, Gettman MT, Krambeck AE. Urolithiasis after ileal conduit urinary diversion: a comparison of minimally invasive therapies. J Urol 2013; 189: 2152-7. . 1. Simon J. Extrophia vesicae (absence of the anterior walls of the bladder and pubic abdominal parietes); operation for directing the orifices of the ureters into the rectum; temporary success; subsequent death; autopsy. Lancet 1852; 2: 568. 23. Zhong W, Yang B, He F, Wang L, Swami S, Zeng G. Surgical management of urolithiasis in patients after urinary diversion. PLoS One 2014; 31: 9. 2. Urh A, Soliman PT, Schmeler KM, et al. Postoperative outcomes after continent versus incontinent urinary diversion at the time of pelvic exenteration for gynecologic malignancies. Gynecol Oncol 2013; 129: 580-5. 24. Desai M. Ultrasonography-guided punctures-with and without puncture guide. J Endourol 2009; 23: 1641–3. 3. Turk TM, Koleski FC, Albala DM. Incidence of urolithiasis in cystectomy patients after intestinal conduit or continent urinary diversion. World J Urol 1999; 17: 305–7. 25. Singla N, Montie JE, Lee CT, Wolf JS Jr, Faerber GJ. Experience with 45 Consecutive Patients with Neobladders Undergoing Retrograde Ureteroscopy for Upper Tract Abnormalities. Urol Pract 2015; 2: 244-9. 4. Ginsberg D, Huffman JL, Lieskovsky G, Boyd S, Skinner DG. Urinary tract stones: a complication of the Kock pouch continent urinary diversion. J Urol 1991; 145: 956–9. 26. Li X, He Z, Wu K, Li SK, Zeng G. Chinese minimally invasive percutaneous nephrolithotomy: the Guangzhou experience. J Endourol 2009; 23: 1693–7. 5. Arai Y, Kawakita M, Terachi T, et al. Long-term follow up of the Kock and Indiana pouch procedures. J Urol 1993; 150: 51–5. 27. Yamamoto M, Kashiwai H, Hirayama A, et al. [Long-term follow- up of female tetraplegic patients with cutaneous vesicostomy]. Hinyokika Kiyo1997; 43: 263-6. 6. Ku JH, Jung TY, Lee JK, Park WH, Shim HB. Risk factors for urinary stone formation in men with spinal cord injury: a 17-year follow-up study. BJU Int 2006; 97: 790-3. 28. Ord J, Lunn D, Reynard J. Bladder management and risk of bladder stone formation in spinal cord injured patients. J Urol 2003; 170: 1734-7. 7. Okhunov Z, Duty B, Smith AD, Okeke Z. Management of urolithiasis in patients after urinary diversions. BJU Int 2011; 108: 330–6. 8. Fernandez A, Foell K, Nott L, Fernandez A. Percutaneous nephrolithotripsy in patients with urinary diversions: A case- control comparison of perioperative outcomes. ETHICAL DECLARATIONS 12. Terai A, Arai Y, Kawakita M et al. Effect of urinary intestinal diversion on urinary risk factors for urolithiasis. J Urol 1995; 153: 37. Ethics Committee Approval: The study was carried out with the permission of Keçiören Training and Research Hospital Health Application Research Center Medical Specialty Education Board (Date: 08.12.2020 IRB: 2012- KAEK-15/2202). 13. Shimko MS, Tollefson MK, Umbreit EC, et al. Long-term complications of conduit urinary diversion. J Urol 2011; 185: 562. 14. Beiko DT and Razvi H: Stones in urinary diversions: update on medical and surgical issues. Curr Opin Urol 2002; 12: 297. 15. el-Nahas AR, Eraky I, el-Assmy AM, et al. Percutaneous treatment of large upper tract stones after urinary diversion. Urology 2006; 68: 500-4. Informed Consent: Because the study was designed retrospectively, no written informed consent form was obtained from patients. 16. Haselhuhn GD, Kropp KA, Keck RW, Selman SH. Photochemical ablation of intestinal mucosa for bladder augmentation. J Urol. 1994; 152: 2267-71. Referee Evaluation Process: Externally peer-reviewed. 17. Cohen TD, Streem SB, and Lammert GK: Selective minimally invasive management of calculi in patients with urinary diversions. J Urol 1994; 152: 1091–4. Conflict of Interest Statement: The authors have no conflicts of interest to declare. 18. Razvi HA, Martin TV, Sosa ER, et al. Endourologic management of complications of urinary intestinal diversion. AUA Update Series 11(lesson 22): 174 –9, 1996. Financial Disclosure: The authors declared that this study has received no financial support. 19. Cass AS, Lee JY, Aliabadi.Extracorporeal shock wave lithotripsy and endoscopic management of renal calculi with urinary diversions. J Urol 1992; 148: 1123–5. Author Contributions: All of the authors declare that they have all participated in the design, execution, and analysis of the paper, and that they have approved the final version. 20. El-Assmy A, El-Nahas AR, Mohsen T, et al. Extracorporeal shock wave lithotripsy of upper urinary tract calculi in patients with cystectomy and urinary diversion. Urology 2005; 66: 510-3. 21. Madbouly K. Large orthotopic reservoir stone burden: Role of open surgery. Urol Ann 2010; 2: 96–9. CONCLUSION Surgical treatment of urinary tract stones in patients with urinary diversion and vesicostomy varies according to the type of diversion, stone location and stone size. In upper urinary tract stones, antegrade URS and PNL should be the primary choice due to their short operation time and effort advantage. Retrograde URS should be preferred in patients where ureter orifices can be clearly visualized through the diversion pouch. Stones in diversion pouch can be easily treated with both stomal entry and percutaneous stomal entry, and high stone-free rate can be achieved. Surgical treatment of bladder stones in patients with vesicostomy can be successfully performed with vesicostomy entry. The most important factors in the success of urinary tract stone surgery in patients with urinary diversion and vesicostomy are the correct management of the preoperative surgical preparation process and instrumentation preparation in which all interventions can be performed, as well as surgical team experience. 82 J Health Sci Med 2021; 4(1): 78-83 Sarıkaya et al. Stone surgery in patients with urinary diversion REFERENCES J Endourol 2011; 25: 1615–8. 9. Franzoni DF, Decter RM. Percutaneous vesicolithotomy: an alternative to open bladder surgery in patients with an impassable or surgically ablated urethra. J Urol 1999; 162: 777–8. 10. Cohen TD, Streem SB, Lammert G. Long-term incidence and risks for recurrent stones following contemporary management of upper tract calculi in patients with a urinary diversion. J Urol1996; 155: 62–5. 11. Delvecchio FC, Kou RL, Iselin CE, et al: Combined antegrade and retrograde endoscopic approach for management of urinary diversion-associated pathology. J Endourol 2000; 14: 251–6. 83
https://openalex.org/W2983172950	https://discovery.ucl.ac.uk/id/eprint/10114324/1/Manganese-Catalyzed%20Electrochemical%20Deconstructive%20Chlorination%20of%20Cycloalkanols%20via%20Alkoxy%20Radicals.pdf	English	null	Manganese-Catalyzed Electrochemical Deconstructive Chlorination of Cycloalkanols via Alkoxy Radicals	Organic letters	2,019	cc-by	5,716	Manganese-Catalyzed Electrochemical Deconstructive Chlorination of Cycloalkanols via Alkoxy Radicals Benjamin D. W. Allen,†,§ Mishra Deepak Hareram,†,§ Alex C. Seastram,† Tom McBride,† Thomas Wirth,‡ Duncan L. Browne,,† and Louis C. Morrill,† †CardiﬀCatalysis Institute, School of Chemistry, CardiﬀUniversity, Main Building, Park Place, CardiﬀCF10 3AT, U.K. ‡School of Chemistry, CardiﬀUniversity, Main Building, Park Place, CardiﬀCF10 3AT, U.K. Letter pubs.acs.org/OrgLett Cite This: Org. Lett. 2019, 21, 9241−9246 This is an open access article published under a Creative Commons Attribution (CC-BY) License, which permits unrestricted use, distribution and reproduction in any medium, provided the author and source are cited. Manganese-Catalyzed Electrochemical Deconstructive Chlorination of Cycloalkanols via Alkoxy Radicals Benjamin D. W. Allen,†,§ Mishra Deepak Hareram,†,§ Alex C. Seastram,† Tom McBride,† Thomas Wirth,‡ Duncan L. Browne,,† and Louis C. Morrill,† †CardiﬀCatalysis Institute, School of Chemistry, CardiﬀUniversity, Main Building, Park Place, CardiﬀCF10 3AT, U.K. ‡School of Chemistry, CardiﬀUniversity, Main Building, Park Place, CardiﬀCF10 3AT, U.K. Letter pubs.acs.org/OrgLett Cite This: Org. Lett. 2019, 21, 9241−9246 This is an open access article published under a Creative Commons Attribution (CC-BY) License, which permits unrestricted use, distribution and reproduction in any medium, provided the author and source are cited. This is an open access article published under a Creative Commons Attribution (CC-BY) License, which permits unrestricted use, distribution and reproduction in any medium, provided the author and source are cited. This is an open access article published under a Creative Commons Attribution (CC-BY) License, which permits unrestricted use, distribution and reproduction in any medium, provided the author and source are cited. Letter pubs.acs.org/OrgLett Cite This: Org. Lett. 2019, 21, 9241−9246 , p , p y , provided the author and source are cited. pubs.acs.org/OrgLett Manganese-Catalyzed Electrochemical Deconstructive Chlorination of Cycloalkanols via Alkoxy Radicals Benjamin D. W. Allen,†,§ Mishra Deepak Hareram,†,§ Alex C. Seastram,† Tom McBride,† Thomas Wirth,‡ Duncan L. Browne,,† and Louis C. Morrill,† †CardiﬀCatalysis Institute, School of Chemistry, CardiﬀUniversity, Main Building, Park Place, CardiﬀCF10 3AT, U.K. ‡School of Chemistry, CardiﬀUniversity, Main Building, Park Place, CardiﬀCF10 3AT, U.K. * S Supporting Information ABSTRACT: A manganese-catalyzed electrochemical deconstructive chlorination of cycloalkanols has been developed. This electrochemical method provides access to alkoxy radicals from alcohols and exhibits a broad substrate scope, with various cyclopropanols and cyclobutanols converted into synthetically useful β- and γ-chlorinated ketones (40 examples). Furthermore, the combination of recirculating ﬂow electrochemistry and continuous inline puriﬁcation was employed to access products on a gram scale. A traditional methods for alkoxy radical generation involve the homolysis of weak oxygen-heteroatom bonds within prefunc- tionalized radical precursors in combination with radical initiators and/or thermal or photochemical activation (Scheme 1B).1,2 Alternatively, transition metal salts can be employed in combination with stoichiometric oxidants (e.g., K2S2O8 or hypervalent iodine reagents) for the generation of alkoxy radicals.4 Recent advances have developed photocatalytic approaches for alkoxy radical generation employing various radical precursors5 including peroxides,6 N-alkoxyphthali- mides,7 N-alkoxypyridiniums,8 N-alkoxybenzimidazoles,9 N- alkoxytriazoliums,10 and unprotected alcohols.11 Despite these important advances, many existing approaches require the use of prefunctionalized substrates, (super)stoichiometric reagents (generating waste/byproducts), and/or precious metal (photo)catalysts. Organic Letters Letter Mn(OTf)2 could be lowered to 2 equiv and 5 mol %, respectively, without signiﬁcant reduction in conversion (entries 14 and 15). A Faradaic eﬃciency of 67% was obtained when 2 F/mol of charge was passed (entry 16), which indicated that most of the electricity passing through the cell is utilized productively. the manganese-catalyzed electrochemical deconstructive chlorination of cycloalkanols via alkoxy radical intermediates,17 the manganese-catalyzed electrochemical deconstructive chlorination of cycloalkanols via alkoxy radical intermediates,17 accessing synthetically useful β- and γ-chlorinated ketones (Scheme 1C). Furthermore, by employing microreactor technology and recirculating ﬂow, the electrochemical method can be performed on gram scale, with continuous inline puriﬁcation incorporated. The full scope of the electrochemical process was explored starting with the deconstructive chlorination of cyclobutanols to form γ-functionalized ketones (Scheme 2A). From the outset, it was found that 1-arylcyclobutan-1-ols containing aromatic systems with electron-releasing groups at the 2- or 4- positions (e.g., 4-tBu) or extended π systems (e.g., 4-Ph) undergo decomposition using the optimized reaction con- ditions (Table 1, entry 11). This instability was attributed to ionization of the C−OH bond in the presence of Brønsted and/or Lewis acids, forming stabilized carbocations that are unproductive for the desired transformation. In such cases, this issue was addressed by employing syringe pump addition of the substrate over 2 h and using TBAOAc as the supporting electrolyte. With a choice of two suitable reaction conditions in hand, a variety of 1-arylcyclobutan-1-ols were converted to the corresponding γ-chlorinated ketone products in good to excellent isolated yields (products 2−20). Within the aryl unit, various alkyl and aryl substitution was tolerated at the 4-, 3-, and 2-positions in addition to halides and electron- withdrawing substituents (e.g., 4-CF3). The electrochemical method exhibits good functional group tolerance as demon- strated by the presence of aldehyde, carboxylic acid, methyl ester, primary amide, nitrile, benzylic primary alcohol, and silyl ether functionalities present within products 14−20. A selection of 1-alkylcyclobutan-1-ols were also converted into the corresponding γ-chlorinated ketones in good isolated yields (products 21−26). Benzo-fused cyclobutanols participated in deconstructive chlorination, giving benzyl chloride products 27−31, including the formation of 7-, 8-, 9-, and 10-membered rings. This strategy was also applied to the formation of disubstituted cycloheptane 32 in 77% isolated yield. Additional substitution at the 2- and 3-positions of the cyclobutanol was tolerated, accessing γ-chlorinated ketones 33−35 in high yields. We also investigated the deconstructive chlorination of cyclopropanols (Scheme 2B). Manganese-Catalyzed Electrochemical Deconstructive Chlorination of Cycloalkanols via Alkoxy Radicals Benjamin D. W. Allen,†,§ Mishra Deepak Hareram,†,§ Alex C. Seastram,† Tom McBride,† Thomas Wirth,‡ Duncan L. Browne,,† and Louis C. Morrill,† †CardiﬀCatalysis Institute, School of Chemistry, CardiﬀUniversity, Main Building, Park Place, CardiﬀCF10 3AT, U.K. ‡School of Chemistry, CardiﬀUniversity, Main Building, Park Place, CardiﬀCF10 3AT, U.K. Letter pubs.acs.org/OrgLett Cite This: Org. Lett. 2019, 21, 9241−9246 This is an open access article published under a Creative Commons Attribution (CC-BY) License, which permits unrestricted use, distribution and reproduction in any medium, provided the author and source are cited. A lkoxy radicals are highly transient species that exhibit diverse reactivity, including hydrogen atom transfer,1 addition to π systems,2 and β-scission processes (Scheme 1A).3 The generation of alkoxy radicals directly from aliphatic alcohols is challenging, partly due to the high dissociation energy of RO−H bonds (∼105 kcal/mol).2b As such, y p The generation of alkoxy radicals directly from aliphatic alcohols is challenging, partly due to the high dissociation energy of RO−H bonds (∼105 kcal/mol).2b As such, Scheme 1. Context and Outline of Electrochemical Strategy © 2019 American Chemical Society 9 Scheme 1. Context and Outline of Electrochemical Strategy Scheme 1. Context and Outline of Electrochemical Strategy Organic electrochemistry represents one of the cleanest possible chemical processing technologies,12 which has recently undergone a renaissance due partly to the increasing availability of standardized batch and ﬂow electrochemical reactors.13 By careful tuning of electrochemical parameters, speciﬁc single electron transfer processes can be targeted, accessing powerful radical intermediates.14 Despite these characteristics, the development of electrochemical methods for the generation of alkoxy radicals from alcohols has received little attention and remains largely limited to the generation of methoxy radicals,15 which requires expensive boron-doped diamond or platinum anodes.16 To this end, herein we report Received: October 16, 2019 Published: November 5, 2019 Received: October 16, 2019 Published: November 5, 2019 © 2019 American Chemical Society DOI: 10.1021/acs.orglett.9b03652 Org. Lett. 2019, 21, 9241−9246 9241 Organic Letters Gratifyingly, it was found that a representative selection of 1-arylcyclopropan-1-ols and 1- alkylcyclopropan-1-ols could be readily converted to the corresponding β-chlorinated ketones in good yields (36−39). Additional substitution is tolerated within the cyclopropanol, giving secondary radical derived product 40 as the major regioisomer. Furthermore, bicyclo[4.1.0]heptan-1-ol was con- verted to 3-chlorocycloheptan-1-one 41 in 48% isolated yield. At the current stage of development, the electrochemical method does not tolerate larger ring sizes with reduced ring strain. For example, despite assessing various reaction conditions, 1-phenylcyclopentan-1-ol underwent decomposi- tion, whereas 1-phenethylcyclopentan-1-ol was unreactive.21 In order to demonstrate scalability the batch process was To commence our studies, 1-phenylcyclobutan-1-ol 1 was selected as the model substrate (Table 1). After extensive Table 1. Optimization of Electrochemical Processa Table 1. Optimization of Electrochemical Processa Table 1. Optimization of Electrochemical Processa entry variation from “standard” conditions yieldb (%) 1 none 82 2 no electricity <2 3 no MnCl2.4H2O <2 4 Ecell = 2.4 V 66 5 i = 12.5 mA, janode = 9.8 mA/cm2 74 6 i = 7.5 mA, janode = 5.9 mA/cm2 80 7 TBAPF6 instead of LiClO4 82 8 Pt foil cathode instead of graphite 82 9 Ni plate cathode instead of graphite 75 10 Mn(OAc)2·4H2O instead of MnCl2·4H2O 82 11 Mn(OTf)2 instead of MnCl2·4H2O 97 (78) 12c LiCl instead of MgCl2 64 13c NaCl instead of MgCl2 <2 14c MgCl2 (2 equiv) 76 15c Mn(OTf)2 (5 mol %) 75 16c,d Q = 2 F/mol 67 aReactions performed with 0.3 mmol of cyclobutanol 1 using the ElectraSyn 2.0 batch electrochemical reactor. [1] = 0.05 M. bYield after 3 h as determined by 1H NMR analysis of the crude reaction mixture with 1,3,5-trimethylbenzene as the internal standard. Isolated yield given in parentheses. cMn(OTf)2 as catalyst. d96 min reaction time. optimization,18 it was found that an electrochemical system composed of MnCl2.4H2O (10 mol %) as catalyst,19 MgCl2 (5 equiv) as chloride source, and LiClO4 as supporting electrolyte in MeCN/AcOH (7:1, [1] = 0.05 M) using galvanostatic conditions (i = 10 mA, janode = 7.8 mA/cm2, Q = 3.73 F/mol) and graphite electrodes at 25 °C for 3 h under N2, enabled the deconstructive chlorination of 1, giving γ-chlorinated ketone 2 in 82% NMR yield (entry 1). No conversion occurs in the absence of electricity or the manganese catalyst (entries 2 and 3). DOI: 10.1021/acs.orglett.9b03652 Org. Lett. 2019, 21, 9241−9246 Organic Letters Employing a constant cell potential (Ecell = 2.4 V) or variation of the current (i = 12.5 mA or 7.5 mA) lowered the NMR yield of 2 (entries 4−6). Employing TBAPF6 as electrolyte (entry 7) or substituting the graphite cathode for Pt foil or Ni plate (entries 8 and 9) each had a negligible impact on conversion. However, upon evaluating alternative Mn(II) salts (entries 10 and 11), it was found that 97% conversion was obtained using Mn(OTf)2 as catalyst, which was adopted for further optimization. Employing LiCl or NaCl as the chloride source was detrimental to conversion, presumably due to decreased solubility in MeCN/AcOH (entries 12 and 13).20 Gratifyingly, the quantities of MgCl2 and In order to demonstrate scalability, the batch process was translated to a ﬂow electrochemical setup.22 By employing the commercially available Ammonite8 ﬂow electroreactor,23 a variety of reaction parameters were evaluated including electrolyte loading, temperature, solvent ratio, residence time, charge, and mixing eﬃciency (Scheme 2C). However, by using MnCl2·4H2O (10 mol %) as catalyst, the conversion to 2 could not be increased beyond 20% using single-pass ﬂow electro- chemistry.18 The yield was increased by applying the optimized reaction parameters to a recirculating ﬂow electrochemical 9242 Letter Letter Organic Letters Letter setup, which provided access to 2 in 84% isolated yield (Scheme 2D).24 Advantageously, due to the decreased distance between the electrodes in ﬂow, a supporting electrolyte was not required. Furthermore, by employing a 6-port 2-position switching valve, the ﬂow could be redirected from recirculation to continuous inline puriﬁcation (Scheme 2E). Once the electrochemical reaction was complete, the valve was switched from position 1 to position 2 to redirect the ﬂow into the path of workup solvents. The ﬂow was passed through a mixing unit before entering a liquid/liquid phase separator containing a hydrophobic membrane that allowed separation of the organic layer, which was subsequently dried over MgSO4, ﬁltered, and concentrated in vacuo to provide 1.2 g of product. This ﬂow setup, which combines recirculating ﬂow electrochemistry and continuous inline puriﬁcation for the ﬁrst time, might be suitable for >1 g scale processing by increasing reactor volume and operation time. [Mn(II)X2Cl]−from Mn(II)X2 and MgCl2, which is oxidized at the anode to form Mn(III)X2Cl (Scheme 3B). This intermediate undergoes ligand exchange with the cycloalkanol to form a Mn(III) alkoxide, with subsequent homolysis generating an alkoxy radical, which can undergo reversible β- scission. Alternatively, the Mn(III) alkoxide may undergo reversible β-scission, rather than a free alkoxy radical intermediate. Trapping of the transient primary carbon- centered radical with the persistent Mn(III)X2Cl species forms a new C−Cl bond.26 Hydrogen gas is generated via proton reduction at the cathode. [Mn(II)X2Cl]−from Mn(II)X2 and MgCl2, which is oxidized at the anode to form Mn(III)X2Cl (Scheme 3B). This intermediate undergoes ligand exchange with the cycloalkanol to form a Mn(III) alkoxide, with subsequent homolysis generating an alkoxy radical, which can undergo reversible β- scission. Alternatively, the Mn(III) alkoxide may undergo reversible β-scission, rather than a free alkoxy radical intermediate. Trapping of the transient primary carbon- centered radical with the persistent Mn(III)X2Cl species forms a new C−Cl bond.26 Hydrogen gas is generated via proton reduction at the cathode. In conclusion, we have developed a new electrochemical method for alkoxy radical generation from alcohols and utilized this for the manganese-catalyzed electrochemical deconstruc- tive chlorination of cycloalkanols. The method is applicable across various cyclopropanols and cyclobutanols, accessing a broad range of synthetically useful β- and γ-chlorinated ketones (40 examples). Furthermore, the combination of recirculating ﬂow electrochemistry and continuous inline puriﬁcation was employed to access products on gram scale. Organic Letters Scheme 2. Substrate Scope: Batch and Flow Electrochemistry* Scheme 2. Substrate Scope: Batch and Flow Electrochemistry* Scheme 2. Substrate Scope: Batch and Flow Electrochemistry* Scheme 2. Substrate Scope: Batch and Flow Electrochemistry* p y Reactions performed with 0.3 mmol of cycloalkanol using the ElectraSyn 2.0 batch electrochemical reactor with isolated yields aft hromatographic puriﬁcation quoted unless stated otherwise. aCycloalkanol was added over 2 h via syringe pump, TBAOAc (0.1 M) as electroly TBAOAc (0.1 M) as electrolyte. cYield as determined by 1H NMR analysis of the crude reaction mixture with 1,3,5-trimethylbenzene as t ternal standard. d6 h. Reactions performed with 0.3 mmol of cycloalkanol using the ElectraSyn 2.0 batch electrochemical reactor with isolated yields after chromatographic puriﬁcation quoted unless stated otherwise. aCycloalkanol was added over 2 h via syringe pump, TBAOAc (0.1 M) as electrolyte. bTBAOAc (0.1 M) as electrolyte. cYield as determined by 1H NMR analysis of the crude reaction mixture with 1,3,5-trimethylbenzene as the internal standard. d6 h. Reactions performed with 0.3 mmol of cycloalkanol using the ElectraSyn 2.0 batch electrochemical reactor with isolated yields after chromatographic puriﬁcation quoted unless stated otherwise. aCycloalkanol was added over 2 h via syringe pump, TBAOAc (0.1 M) as electrolyte. bTBAOAc (0.1 M) as electrolyte. cYield as determined by 1H NMR analysis of the crude reaction mixture with 1,3,5-trimethylbenzene as the internal standard. d6 h. 9243 DOI: 10.1021/acs.orglett.9b03652 Org. Lett. 2019, 21, 9241−9246 * S Supporting Information * S Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.or- glett.9b03652. Optimization data, experimental procedures, character- ization of new compounds and spectral data (PDF) ■ACKNOWLEDGMENTS We gratefully acknowledge the School of Chemistry, Cardiﬀ University, for generous support and the EPSRC for a standard research grant (EP/R006504/1) and doctoral training grants for Ph.D. studentships (A.S., EP/R513003/1; T.M., EP/ N509449/1). Organic Letters Ongoing studies are focused on further applications of earth-abundant transition metals in synthetic organic electrochemistry, and these results will be reported in due course.27 Cyclic voltammetry was employed in order to gain mechanistic insight into the electrochemical process.18 In accordance with the literature,19d the combination of Mn(OTf)2 and MgCl2 produced a new quasi-reversible redox event at ∼0.8 V vs Fc/Fc+,25 which provided evidence for the generation of a Mn(III)X2Cl species from [Mn(II)X2Cl]−. Furthermore, an increase in the oxidation current was observed upon addition of 1-phenylcyclobutan-1-ol 1, which suggested that Mn(III)X2Cl is consumed by 1. When methyl ether cyclobutane 42 was employed as the substrate using the standard electrochemical reaction conditions, no γ-chlorinated ketone 2 was observed, with 82% starting material recovered (Scheme 3A). This indicated that the proposed Mn(III)X2Cl species does not promote cyclobutane ring opening in the absence of a hydroxyl functional group. As such, a plausible reaction mechanism initiates with the formation of Notes The authors declare no competing ﬁnancial interest. Information about the data that underpins the results presented in this article, including how to access them, can be found in the CardiﬀUniversity data catalogue at http://doi. org/10.17035/d.2019.0087324419. ORCID Duncan L. Browne: 0000-0002-8604-229X Louis C. Morrill: 0000-0002-6453-7531 ■AUTHOR INFORMATION Corresponding Authors Scheme 3. Mechanistic Studies aYield as determined by 1H NMR analysis of the crude reaction mixture with 1,3,5-trimethylbenzene as the internal standard. Scheme 3. Mechanistic Studies E-mail: morrilllc@cardiﬀ.ac.uk. E-mail: dlbrowne@cardiﬀ.ac.uk. E-mail: morrilllc@cardiﬀ.ac.uk. E-mail: dlbrowne@cardiﬀ.ac.uk. Author Contributions §B.D.W.A. and M.D.H. contributed equally to this work. Organic Letters Catalytic Ring-Opening of Cyclic Alcohols Enabled by PCET Activation of Strong O-H Bonds. J. Am. Chem. Soc. 2016, 138, 10794−10797. (c) Guo, J.-J.; Hu, A.; Chen, Y.; Sun, J.; Tang, H.; Zuo, Z. Photocatalytic C-C Bond Cleavage and Amination of Cycloalkanols by Cerium(III) Chloride Complex. Angew. Chem., Int. Ed. 2016, 55, 15319−15322. (d) Wu, X.; Wang, M.; Huan, L.; Wang, D.; Wang, J.; Zhu, C. Tertiary-Alcohol- Directed Functionalization of Remote C(sp3)-H Bonds by Sequential Hydrogen Atom and Heteroaryl Migrations. Angew. Chem., Int. Ed. 2018, 57, 1640−1644. (e) Schwarz, J.; König, B. Visible-light mediated C-C bond cleavage of 1,2-diols to carbonyls by cerium- photocatalysis. Chem. Commun. 2019, 55, 486−488. (f) Li, G.-X.; Hu, X.; He, G.; Chen, G. Photoredox-mediated remote C(sp3)-H heteroarylation of free alcohols. Chem. Sci. 2019, 10, 688−693. ( ) ( ) (2) (a) Hartung, J. Stereoselective Construction of the Tetrahy- drofuran Nucleus by Alkoxyl Radical Cyclizations. Eur. J. Org. Chem. 2001, 2001, 619−632. (b) Hartung, J.; Gottwald, T.; Špehar, K. Selectivity in the Chemistry of Oxygen-Centered Radicals −The Formation of Carbon-Oxygen Bonds. Synthesis 2002, 1469−1498. yg y (3) (a) Murakami, M.; Ishida, N. β-Scission of Alkoxy Radicals in Synthetic Transformations. Chem. Lett. 2017, 46, 1692−1700. (b) Wu, X.; Zhu, C. Recent Advances in Radical-Mediated C-C Bond Fragmentation of Non-Strained Molecules. Chin. J. Chem. 2019, 37, 171−182. (4) For selected reviews, see: (a) Ren, R.; Zhu, C. Radical-Mediated Ring-Opening Functionalization of Cyclobutanols: A Shortcut to γ- Substituted Ketones. Synlett 2016, 27, 1139−1144. (b) Wu, X.; Zhu, C. Recent Advances in Ring-Opening Functionalization of Cyclo- alkanols by C-C σ-Bond Cleavage. Chem. Rec. 2018, 18, 587−598. y g (5) (a) Jia, K.; Chen, Y. Visible-light-induced alkoxyl radical generation for inert chemical bond cleavage/functionalization. Chem. Commun. 2018, 54, 6105−6112. (b) Guo, J.-J.; Hu, A.; Zuo, y , , (12) For selected recent reviews, see: (a) Yan, M.; Kawamata, Y.; Baran, P. S. Synthetic Organic Electrochemical Methods Since 2000: On the Verge of a Renaissance. Chem. Rev. 2017, 117, 13230−13319. (b) Wiebe, A.; Gieshoff, T.; Möhle, S.; Rodrigo, E.; Zirbes, M.; Waldvogel, S. R. Electrifying Organic Synthesis. Angew. Chem., Int. Ed. 2018, 57, 5594−5619. (c) Kärkäs, M. D. Electrochemical strategies for C-H functionalization and C-N bond formation. Chem. Soc. Rev. 2018, 47, 5786−5865. (d) Moeller, K. D. Using Physical Organic Chemistry To Shape the Course of Electrochemical Reactions. Chem. Rev. 2018, 118, 4817−4833. (e) Tang, S.; Liu, Y.; Lei, A. ■REFERENCES aYield as determined by 1H NMR analysis of the crude reaction mixture with 1,3,5-trimethylbenzene as the internal standard. (1) (a) C̆eković, Ž. Reactions of δ-carbon radicals generated by 1,5- hydrogen transfer to alkoxyl radicals. Tetrahedron 2003, 59, 8073− DOI: 10.1021/acs.orglett.9b03652 Org. Lett. 2019, 21, 9241−9246 9244 DOI: 10.1021/acs.orglett.9b03652 Org. Lett. 2019, 21, 9241−9246 Organic Letters Letter omethoxylation of arenes and heteroarenes. Chem. Sci. 2019, 10, 3217−3222. 8090. (b) Salamone, M.; Bietti, M. Tuning Reactivity and Selectivity in Hydrogen Atom Transfer from Aliphatic C-H Bonds to Alkoxyl Radicals: Role of Structural and Medium Effects. Acc. Chem. Res. 2015, 48, 2895−2903. omethoxylation of arenes and heteroarenes. Chem. Sci. 2019, 10, 3217−3222. 3217 3222. (11) For selected examples, see: (a) Jia, K.; Zhang, F.; Huang, H.; Chen, Y. Visible-Light-Induced Alkoxyl Radical Generation Enables Selective C(sp3)-C(sp3) Bond Cleavage and Functionalizations. J. Am. Chem. Soc. 2016, 138, 1514−1517. (b) Yayla, H. G.; Wang, H.; Tarantino, K. T.; Orbe, H. S.; Knowles, R. R. Catalytic Ring-Opening of Cyclic Alcohols Enabled by PCET Activation of Strong O-H Bonds. J. Am. Chem. Soc. 2016, 138, 10794−10797. (c) Guo, J.-J.; Hu, A.; Chen, Y.; Sun, J.; Tang, H.; Zuo, Z. Photocatalytic C-C Bond Cleavage and Amination of Cycloalkanols by Cerium(III) Chloride Complex. Angew. Chem., Int. Ed. 2016, 55, 15319−15322. (d) Wu, X.; Wang, M.; Huan, L.; Wang, D.; Wang, J.; Zhu, C. Tertiary-Alcohol- Directed Functionalization of Remote C(sp3)-H Bonds by Sequential Hydrogen Atom and Heteroaryl Migrations. Angew. Chem., Int. Ed. 2018, 57, 1640−1644. (e) Schwarz, J.; König, B. Visible-light mediated C-C bond cleavage of 1,2-diols to carbonyls by cerium- photocatalysis. Chem. Commun. 2019, 55, 486−488. (f) Li, G.-X.; Hu, X.; He, G.; Chen, G. Photoredox-mediated remote C(sp3)-H heteroarylation of free alcohols. Chem. Sci. 2019, 10, 688−693. (12) For selected recent reviews, see: (a) Yan, M.; Kawamata, Y.; Baran, P. S. Synthetic Organic Electrochemical Methods Since 2000: On the Verge of a Renaissance. Chem. Rev. 2017, 117, 13230−13319. (b) Wiebe, A.; Gieshoff, T.; Möhle, S.; Rodrigo, E.; Zirbes, M.; Waldvogel, S. R. Electrifying Organic Synthesis. Angew. Chem., Int. Ed. 2018, 57, 5594−5619. (c) Kärkäs, M. D. Electrochemical strategies for C-H functionalization and C-N bond formation. Chem. Soc. Rev. 2018, 47, 5786−5865. (d) Moeller, K. D. Using Physical Organic Chemistry To Shape the Course of Electrochemical Reactions. Chem. Rev. 2018, 118, 4817−4833. (e) Tang, S.; Liu, Y.; Lei, A. Electrochemical Oxidative Cross-coupling with Hydrogen Evolution: A Green and Sustainable Way for Bond Formation. Chem. 2018, 4, 27−45. (11) For selected examples, see: (a) Jia, K.; Zhang, F.; Huang, H.; Chen, Y. Visible-Light-Induced Alkoxyl Radical Generation Enables Selective C(sp3)-C(sp3) Bond Cleavage and Functionalizations. J. Am. Chem. Soc. 2016, 138, 1514−1517. (b) Yayla, H. G.; Wang, H.; Tarantino, K. T.; Orbe, H. S.; Knowles, R. R. Organic Letters Electrochemical Oxidative Cross-coupling with Hydrogen Evolution: A Green and Sustainable Way for Bond Formation. Chem. 2018, 4, 27−45. Z. Photocatalytic alkoxy radical-mediated transformations. Tetrahe- dron Lett. 2018, 59, 2103−2111. (6) DiRocco, D. A.; Dykstra, K.; Krska, S.; Vachal, P.; Conway, D. V.; Tudge, M. Late-Stage Functionalization of Biologically Active Heterocycles Through Photoredox Catalysis. Angew. Chem., Int. Ed. 2014, 53, 4802−4806. (7) For selected examples, see: (a) Zhang, J.; Li, Y.; Zhang, F.; Hu, C.; Chen, Y. Generation of Alkoxyl Radicals by Photoredox Catalysis Enables Selective C(sp3)-H Functionalization under Mild Reaction Conditions. Angew. Chem., Int. Ed. 2016, 55, 1872−1875. (b) Wang, C.; Harms, K.; Meggers, E. Catalytic Asymmetric C(sp3)-H Functionalization under Photoredox Conditions by Radical Trans- location and Stereocontrolled Alkene Addition. Angew. Chem., Int. Ed. 2016, 55, 13495−13498. (c) Zhang, J.; Li, Y.; Xu, R.; Chen, Y. Donor-Acceptor Complex Enables Alkoxyl Radical Generation for Metal-Free C(sp3)-C(sp3) Cleavage and Allylation/Alkenylation. Angew. Chem., Int. Ed. 2017, 56, 12619−12623. (13) (a) Yan, M.; Kawamata, Y.; Baran, P. S. Synthetic Organic Electrochemistry: Calling All Engineers. Angew. Chem., Int. Ed. 2018, 57, 4149−4155. (b) Gütz, C.; Klöckner, B.; Waldvogel, S. R. Electrochemical Screening for Electroorganic Synthesis. Org. Process Res. Dev. 2016, 20, 26−32. (14) Yan, M.; Lo, J. C.; Edwards, J. T.; Baran, P. S. Radicals: Reactive Intermediates with Translational Potential. J. Am. Chem. Soc. 2016, 138, 12692−12714. g (8) For selected examples, see: (a) Quint, V.; Morlet-Savary, F.; Lohier, J.-F.; Lalevée, J.; Gaumont, A.-C.; Lakhdar, S. Metal-Free, Visible Light-Photocatalyzed Synthesis of Benzo[b]phosphole Oxides: Synthetic and Mechanistic Investigations. J. Am. Chem. Soc. 2016, 138, 7436−7441. (b) Kim, I.; Min, M.; Kang, D.; Kim, K.; Hong, S. Direct Phosphonation of Quinolinones and Coumarins Driven by the Photochemical Activity of Substrates and Products. Org. Lett. 2017, 19, 1394−1397. (c) Jelier, B. J.; Tripet, P. F.; Pietrasiak, E.; Franzoni, I.; Jeschke, G.; Togni, A. Radical Trifluoromethoxylation of Arenes Triggered by a Visible-Light-Mediated N-O Bond Redox Fragmenta- tion. Angew. Chem., Int. Ed. 2018, 57, 13784−13789. (d) Barthelemy, A.-L.; Tuccio, B.; Magnier, E.; Dagousset, G. Alkoxyl Radicals Generated under Photoredox Catalysis: A Strategy for anti- Markovnikov Alkoxylation Reactions. Angew. Chem., Int. Ed. 2018, 57, 13790−13794. (e) Kim, I.; Park, B.; Kang, G.; Kim, J.; Jung, H.; Lee, H.; Baik, M.-H.; Hong, S. Visible-Light-Induced Pyridylation of Remote C(sp3)-H Bonds by Radical Translocation of N-Alkoxypyr- idinium Salts. Angew. Chem., Int. Ed. 2018, 57, 15517−15522. (f) Bao, X.; Wang, Q.; Zhu, J. (9) Zheng, W.; Morales-Rivera, C. A.; Lee, J. W.; Liu, P.; Ngai, M.-Y. Catalytic C-H Trifluoromethoxylation of Arenes and Heteroarenes. Angew. Chem., Int. Ed. 2018, 57, 9645−9649. (10) (a) Zheng, W.; Lee, J. W.; Morales-Rivera, C. A.; Liu, P.; Ngai, M.-Y. Redox-Active Reagents for Photocatalytic Generation of the OCF3 Radical and (Hetero)Aryl C-H Trifluoromethoxylation. Angew. Chem., Int. Ed. 2018, 57, 13795−13799. (b) Lee, J. W.; Zheng, W.; Morales-Rivera, C.; Liu, P.; Ngai, M.-Y. Catalytic radical difluor- DOI: 10.1021/acs.orglett.9b03652 Org. Lett. 2019, 21, 9241−9246 Organic Letters Dual Photoredox/Copper Catalysis for the Remote C(sp3)-H Functionalization of Alcohols and Alkyl Halides by N-Alkoxypyridinium Salts. Angew. Chem., Int. Ed. 2019, 58, 2139− 2143. g (8) For selected examples, see: (a) Quint, V.; Morlet-Savary, F.; Lohier, J.-F.; Lalevée, J.; Gaumont, A.-C.; Lakhdar, S. Metal-Free, Visible Light-Photocatalyzed Synthesis of Benzo[b]phosphole Oxides: Synthetic and Mechanistic Investigations. J. Am. Chem. Soc. 2016, 138, 7436−7441. (b) Kim, I.; Min, M.; Kang, D.; Kim, K.; Hong, S. Direct Phosphonation of Quinolinones and Coumarins Driven by the Photochemical Activity of Substrates and Products. Org. Lett. 2017, 19, 1394−1397. (c) Jelier, B. J.; Tripet, P. F.; Pietrasiak, E.; Franzoni, I.; Jeschke, G.; Togni, A. Radical Trifluoromethoxylation of Arenes Triggered by a Visible-Light-Mediated N-O Bond Redox Fragmenta- tion. Angew. Chem., Int. Ed. 2018, 57, 13784−13789. (d) Barthelemy, A.-L.; Tuccio, B.; Magnier, E.; Dagousset, G. Alkoxyl Radicals Generated under Photoredox Catalysis: A Strategy for anti- Markovnikov Alkoxylation Reactions. Angew. Chem., Int. Ed. 2018, 57, 13790−13794. (e) Kim, I.; Park, B.; Kang, G.; Kim, J.; Jung, H.; Lee, H.; Baik, M.-H.; Hong, S. Visible-Light-Induced Pyridylation of Remote C(sp3)-H Bonds by Radical Translocation of N-Alkoxypyr- idinium Salts. Angew. Chem., Int. Ed. 2018, 57, 15517−15522. (f) Bao, X.; Wang, Q.; Zhu, J. Dual Photoredox/Copper Catalysis for the Remote C(sp3)-H Functionalization of Alcohols and Alkyl Halides by N-Alkoxypyridinium Salts. Angew. Chem., Int. Ed. 2019, 58, 2139− 2143. (15) For selected examples, see: (a) Dolson, M. G.; Swenton, J. S. Product and Mechanistic Studies of the Anodic Oxidation of Methoxylated Naphthalenes. The EECrCp Mechanism. J. Am. Chem. ( ) Soc. 1981, 103, 2361. (b) Carreño, M. C.; Ribagorda, M. Anodic Oxidation of N-Protected 4-Methoxy Anilines: Improved Synthesis of Quinone Imine Acetals. J. Org. Chem. 2000, 65, 1231−1234. (c) Sumi, T.; Saitoh, T.; Natsui, K.; Yamamoto, T.; Atobe, M.; Einaga, Y.; Nishiyama, S. Anodic Oxidation on a Boron-Doped Diamond Electrode Mediated by Methoxy Radicals. Angew. Chem., Int. Ed. 2012, 51, 5443−5446. (d) Yajima, S.; Saitoh, T.; Kawa, K.; Nakamura, K.; Nagase, H.; Einaga, Y.; Nishiyama, S. Asymmetric Induction in cyclohexadienones carrying α-D-glucopyranosyl moiety. Tetrahedron 2016, 72, 8428−8435. (16) For a spiroketal formation, which is proposed to involve anodic oxidation of alkoxides to form alkoxy radicals, see: Markó, I. E. Electrochemical oxidative cyclisation of ω-hydroxy-tetrahydropyrans to spiroketals. Tetrahedron Lett. 2000, 41, 4383−4387. p (17) For a recent review of deconstructive functionalization processes, see: (a) Morcillo, S. P. Organic Letters Letter Manganese-catalyzed ring-opening chlorination of cyclobutanols: regiospecific synthesis of γ-chloroketones. Org. Chem. Front. 2016, 3, 1467−1471. (e) Dauncey, E. M.; Morcillo, S. P.; Douglas, J. J.; Sheikh, N. S.; Leonori, D. Photoinduced Remote Functionalisations by Iminyl Radical Promoted C-C and C-H Bond Cleavage Cascades. Angew. Chem., Int. Ed. 2018, 57, 744−748. (f) Roque, J. B.; Kuroda, Y.; Göttemann, L. T.; Sarpong, R. Deconstructive diversification of cyclic amines. Nature 2018, 564, 244−248. and Applications. J. Am. Chem. Soc. 2019, 141, 6392−6402. (b) Peters, B. K.; Rodriguez, K. X.; Reisberg, S. H.; Beil, S. B.; Hickey, D. P.; Kawamata, Y.; Collins, M.; Starr, J.; Chen, L.; Udyavara, S.; Klunder, K.; Gorey, T. J.; Anderson, S. L.; Neurock, M.; Minteer, S. D.; Baran, P. S. Scalable and safe synthetic organic electroreduction inspired by Li-ion battery chemistry. Science 2019, 363, 838−845. (25) Sandford, C.; Edwards, M. A.; Klunder, K. J.; Hickey, D. P.; Li, M.; Barman, K.; Sigman, M. S.; White, H. S.; Minteer, S. D. A synthetic chemist’s guide to electroanalytical tools for studying reaction mechanisms. Chem. Sci. 2019, 10, 6404−6422. y , , (18) See the Supporting Information for full experimental details. (19) For examples of Mn-mediated electrochemical reactions, see: (a) Shundo, R.; Nishiguchi, I.; Matsubara, Y.; Hirashima, T. Mn3+- Mediated Coupling-Cyclization of 5-Arylpent-1-enes with Active Methylene Compounds by Electrooxidation. Chem. Lett. 1991, 20, 235−236. (b) Shundo, R.; Nishiguchi, I.; Matsubara, Y.; Hirashima, T. Carbon-Carbon Bond Formation Using Manganese(III) Acetate as an Electrochemical Mediator. Tetrahedron 1991, 47, 831−840. (c) Fu, N.; Sauer, G. S.; Saha, A.; Loo, A.; Lin, S. Metal-catalyzed electrochemical diazidation of alkenes. Science 2017, 357, 575−579. (d) Fu, N.; Sauer, G. S.; Lin, S. Electrocatalytic Radical Dichlorination of Alkenes with Nucleophilic Chlorine Sources. J. Am. Chem. Soc. 2017, 139, 15548−15553. (e) Ye, K.-Y.; Pombar, G.; Fu, N.; Sauer, G.; Keresztes, I.; Lin, S. Anodically Coupled Electrolysis for the Heterodifunctionalization of Alkenes. J. Am. Chem. Soc. 2018, 140, 2438−2441. (f) Ye, K.-Y.; Song, Z.; Sauer, G. S.; Harenberg, J. H.; Fu, N.; Lin, S. Synthesis of Chlorotrifluoromethylated Pyrrolidines by Electrocatalytic Radical Ene-Yne Cyclization. Chem. - Eur. J. 2018, 24, 12274−12279. (g) Merchant, R. R.; Oberg, K. M.; Lin, Y.; Novak, A. J. E.; Felding, J.; Baran, P. S. Divergent Synthesis of Pyrone Diterpenes via Radical Cross Coupling. J. Am. Chem. Soc. 2018, 140, 7462−7465. (h) Fu, N.; Sauer, G. S.; Lin, S. Organic Letters Radical-promoted C-C bond cleavage: a deconstructive approach for selective functionalization. Angew. Chem., Int. Ed. 2019, 58, 14044. For examples of deconstructive chlorination, see: (b) Fan, X.; Zhao, H.; Yu, J.; Bao, X.; Zhu, C. Regiospecific synthesis of distally chlorinated ketones via C-C bond cleavage of cycloalkanols. Org. Chem. Front. 2016, 3, 227− 232. (c) Huang, F.-Q.; Xie, J.; Sun, J.-G.; Wang, Y.-W.; Dong, X.; Qi, L.-W.; Zhang, B. Regioselective Synthesis of Carbonyl-Containing Alkyl Chlorides via Silver-Catalyzed Ring-Opening Chlorination of Cycloalkanols. Org. Lett. 2016, 18, 684−687. (d) Huan, L.; Zhu, C. g (10) (a) Zheng, W.; Lee, J. W.; Morales-Rivera, C. A.; Liu, P.; Ngai, M.-Y. Redox-Active Reagents for Photocatalytic Generation of the OCF3 Radical and (Hetero)Aryl C-H Trifluoromethoxylation. Angew. Chem., Int. Ed. 2018, 57, 13795−13799. (b) Lee, J. W.; Zheng, W.; Morales-Rivera, C.; Liu, P.; Ngai, M.-Y. Catalytic radical difluor- (10) (a) Zheng, W.; Lee, J. W.; Morales-Rivera, C. A.; Liu, P.; Ngai, M.-Y. Redox-Active Reagents for Photocatalytic Generation of the OCF3 Radical and (Hetero)Aryl C-H Trifluoromethoxylation. Angew. Chem., Int. Ed. 2018, 57, 13795−13799. (b) Lee, J. W.; Zheng, W.; Morales-Rivera, C.; Liu, P.; Ngai, M.-Y. Catalytic radical difluor- 9245 DOI: 10.1021/acs.orglett.9b03652 Org. Lett. 2019, 21, 9241−9246 DOI: 10.1021/acs.orglett.9b03652 Org. Lett. 2019, 21, 9241−9246 (24) (a) Kawamata, Y.; Vantourout, J. C.; Hickey, D. P.; Bai, P.; Chen, L.; Hou, Q.; Qiao, W.; Barman, K.; Edwards, M. A.; Garrido- Castro, A. F.; deGruyter, J. N.; Nakamura, H.; Knouse, K.; Qin, C.; Clay, K. J.; Bao, D.; Li, C.; Starr, J. T.; Garcia-Irizarry, C.; Sach, N.; White, H. S.; Neurock, M.; Minteer, S. D.; Baran, P. S. Electro- chemically Driven, Ni-Catalyzed Aryl Amination: Scope, Mechanism, Organic Letters A general, electrocatalytic approach to the synthesis of vicinal diamines. Nat. Protoc. 2018, 13, 1725−1743. (i) Fu, N.; Shen, Y.; Allen, A. R.; Song, L.; Ozaki, A.; Lin, S. Mn-Catalyzed Electrochemical Chloroalkylation of Alkenes. ACS Catal. 2019, 9, 746−754. (j) Zhang, Z.; Zhang, L.; Cao, Y.; Li, F.; Bai, G.; Liu, G.; Yang, Y.; Mo, F. Mn-Mediated Electrochemical Trifluoromethylation/C(sp2)-H Functionalization Cascade for the Synthesis of Azaheterocycles. Org. Lett. 2019, 21, 762−766. (k) Lu, L.; Fu, N.; Lin, S. Three-Component Chlorophosphinoylation of Alkenes via Anodically Coupled Electrolysis. Synlett 2019, 30, 1199− 1203. (l) Strehl, J.; Hilt, G. Electrochemical, Mangenese-Assisted Carbon-Carbon Bond Formation between β-Keto Esters and Silyl Enol Ethers. Org. Lett. 2019, 21, 5259−5263. (26) (a) Studer, A. The Persitent Radical Effect in Organic Synthesis. Chem. - Eur. J. 2001, 7, 1159−1164. (b) Fischer, H. The Persistent Radical Effect: A Principle for Selective Radical Reactions and Living Radical Polymerizations. Chem. Rev. 2001, 101, 3581− 3610. (27) A prior version of the present article was deposited as a preprint on ChemRxiv: Allen, B. D. W.; Hareram, M. D.; Seastram, A. C.; McBride, T.; Wirth, T.; Browne, D. L.; Morrill, L. C. Manganese- Catalyzed Electrochemical Deconstructive Chlorination of Cyclo- alkanols via Alkoxy Radicals. ChemRxiv 2019. DOI: 10.26434/ chemrxiv.9275441.v1. (20) The substitution of MgCl2 with other halide salts, namely KBr or NaI, resulted in >90% recovery of cyclobutanol 1. (21) Wilsey, S.; Dowd, P.; Houk, K. N. Effect of Alkyl Substituents and Ring Size on Alkoxy Radical Cleavage Reactions. J. Org. Chem. 1999, 64, 8801−8811. (22) (a) Yoshida, J.-i.; Nagaki, A. Electrochemical Reactions in Microreactors. In Microreactors in Preparative Chemistry; Reschetilowski, W., Ed.; Wiley-VCH: Weinheim, Germany, 2013; pp 231−242. (b) Folgueiras-Amador, A. A.; Wirth, T. Electrosyn- thesis in Continuous Flow. Science of Synthesis: Flow Chemistry in Organic Synthesis 2018, 147−189. (c) Laudadio, G.; de Smet, W.; Struik, L.; Cao, Y.; Noël, T. Design and application of a modular and scalable electrochemical flow microreactor. J. Flow Chem. 2018, 8, 157−165. (d) Pletcher, D.; Green, R. A.; Brown, R. C. D. Flow Electrolysis Cells for the Synthetic Organic Chemistry Laboratory. Chem. Rev. 2018, 118, 4573−4591. (e) Atobe, M.; Tateno, H.; Matsumura, Y. Applications of Flow Microreactors in Electrosynthetic Processes. Chem. Rev. 2018, 118, 4541−4572. (22) (a) Yoshida, J.-i.; Nagaki, A. Electrochemical Reactions in Microreactors. In Microreactors in Preparative Chemistry; Reschetilowski, W., Ed.; Wiley-VCH: Weinheim, Germany, 2013; pp 231−242. (b) Folgueiras-Amador, A. A.; Wirth, T. Organic Letters Electrosyn- thesis in Continuous Flow. Science of Synthesis: Flow Chemistry in Organic Synthesis 2018, 147−189. (c) Laudadio, G.; de Smet, W.; Struik, L.; Cao, Y.; Noël, T. Design and application of a modular and scalable electrochemical flow microreactor. J. Flow Chem. 2018, 8, Matsumura, Y. Applications of Flow Microreactors in Electrosynthetic Processes. Chem. Rev. 2018, 118, 4541−4572. (23) Green, R. A.; Brown, R. C. D.; Pletcher, D.; Harji, B. A. Microflow Electrolysis Cell for Laboratory Synthesis on the Multigram Scale. Org. Process Res. Dev. 2015, 19, 1424−1427. g g (24) (a) Kawamata, Y.; Vantourout, J. C.; Hickey, D. P.; Bai, P.; Chen, L.; Hou, Q.; Qiao, W.; Barman, K.; Edwards, M. A.; Garrido- Castro, A. F.; deGruyter, J. N.; Nakamura, H.; Knouse, K.; Qin, C.; Clay, K. J.; Bao, D.; Li, C.; Starr, J. T.; Garcia-Irizarry, C.; Sach, N.; White, H. S.; Neurock, M.; Minteer, S. D.; Baran, P. S. Electro- chemically Driven, Ni-Catalyzed Aryl Amination: Scope, Mechanism, DOI: 10.1021/acs.orglett.9b03652 Org. Lett. 2019, 21, 9241−9246 9246
https://openalex.org/W2082817342	https://researchonline.lshtm.ac.uk/id/eprint/1635826/1/pone.0092386.pdf	English	null	Can Timely Vector Control Interventions Triggered by Atypical Environmental Conditions Prevent Malaria Epidemics? A Case-Study from Wajir County, Kenya	PloS one	2,014	cc-by	5,800	Peter Maes1, Anthony D. Harries2,3, Rafael Van den Bergh4, Abdisalan Noor5,6, Robert W. Snow5,6, Katherine Tayler-Smith4, Sven Gudmund Hinderaker7, Rony Zachariah4, Richard Allan8 1 Medical Department, Water, Hygiene and Sanitation Unit, Me´decins Sans Frontie`res, Operational Center Brussels, Brussels, Belgium, 2 International Union Against Tuberculosis and Lung Disease (The Union), Paris, France, 3 London School of Hygiene and Tropical Medicine, London, United Kingdom, 4 Medical Department, Operational Research Unit (LuxOR), Operational Center Brussels, Me´decins Sans Frontie`res -Luxembourg, Luxembourg, Luxembourg, 5 Malaria Public Health Department, KEMRI-University of Oxford-Wellcome Trust Collaborative Programme, Nairobi, Kenya, 6 Centre for Tropical Medicine, University of Oxford, Oxford, United Kingdom, 7 Centre for International Health, University of Bergen, Bergen, Norway, 8 The Mentor Initiative, Crawley, United Kingdom Abstract This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits tion, and reproduction in any medium, provided the original author and source are credited. Copyright: 2014 Maes et al. This is an open-access article distributed under the terms of the Creative Commons Attributi unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: Funding for the course was from the innumerable donors who collectively provide in 85% of the total MSF budget; was from the Department for International Development, UK; and was from Me´decins Sans Frontie`res, Luxembourg. AMN is supported by the Wellcome Trust as an Intermediate Fellow (# 095127); RWS is supported by the Wellcome Trust as Principal Research Fellow (# 079080) and both acknowledge the support provided by the Wellcome Trust Major Overseas Programme grant to the KEMRI/Wellcome Trust Research Programme (#092654). 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: Peter.Maes@brussels.msf.org In Kenya, like many other African countries, malaria is a leading cause of morbidity and mortality, especially amongst children. Kenya has four malaria epidemiological zones: an endemic zone; seasonal malaria transmission zones; zones that are prone to malaria epidemics, and a zone of low malaria risk. There are limited data on effectiveness of interventions to prevent or control malaria outbreaks in zones where seasonal transmission or Can Timely Vector Control Interventions Triggered by Atypical Environmental Conditions Prevent Malaria Epidemics? A Case-Study from Wajir County, Kenya Peter Maes1*, Anthony D. Harries2,3, Rafael Van den Bergh4, Abdisalan Noor5,6, Robert W. Snow5,6, Katherine Tayler-Smith4, Sven Gudmund Hinderaker7, Rony Zachariah4, Richard Allan8 Abstract Background: Atypical environmental conditions with drought followed by heavy rainfall and flooding in arid areas in sub- Saharan Africa can lead to explosive epidemics of malaria, which might be prevented through timely vector-control interventions. Objectives: Wajir County in Northeast Kenya is classified as having seasonal malaria transmission. The aim of this study was to describe in Wajir town the environmental conditions, the scope and timing of vector-control interventions and the associated resulting burden of malaria at two time periods (1996–1998 and 2005–2007). Methods: This is a cross-sectional descriptive and ecological study using data collected for routine program monitoring and evaluation. Results: In both time periods, there were atypical environmental conditions with drought and malnutrition followed by massive monthly rainfall resulting in flooding and animal/human Rift Valley Fever. In 1998, this was associated with a large and explosive malaria epidemic (weekly incidence rates peaking at 54/1,000 population/week) with vector-control interventions starting over six months after the massive rainfall and when the malaria epidemic was abating. In 2007, vector- control interventions started sooner within about three months after the massive rainfall and no malaria epidemic was recorded with weekly malaria incidence rates never exceeding 0.5 per 1,000 population per week. Discussion and Conclusion: Did timely vector-control interventions in Wajir town prevent a malaria epidemic? In 2007, the neighboring county of Garissa experienced similar climatic events as Wajir, but vector-control interventions started six months after the heavy un-seasonal rainfall and large scale flooding resulted in a malaria epidemic with monthly incidence rates peaking at 40/1,000 population. In conclusion, this study suggests that atypical environmental conditions can herald a malaria outbreak in certain settings. In turn, this should alert responsible stakeholders about the need to act rapidly and preemptively with appropriate and wide-scale vector-control interventions to mitigate the risk. Citation: Maes P, Harries AD, Van den Bergh R, Noor A, Snow RW, et al. (2014) Can Timely Vector Control Interventions Triggered by Atypical Environmental Conditions Prevent Malaria Epidemics? A Case-Study from Wajir County, Kenya. PLoS ONE 9(4): e92386. doi:10.1371/journal.pone.0092386 Editor: Georges Snounou, Universite´ Pierre et Marie Curie, France Received August 8, 2013; Accepted February 21, 2014; Published April 3, 2014 Received August 8, 2013; Accepted February 21, 2014; Published April 3, 2014 es et al. Citation: Maes P, Harries AD, Van den Bergh R, Noor A, Snow RW, et al. (2014) Can Timely Vector Control Interventions Triggered by Atypical Environmental Conditions Prevent Malaria Epidemics? A Case-Study from Wajir County, Kenya. PLoS ONE 9(4): e92386. doi:10.1371/journal.pone.0092386 Methods This was a cross-sectional descriptive and ecological study using previously collected data. The county experiences the Sahel climate characterized by long dry spells and two rainy seasons each year from September to January and March to May. The mean annual precipitation between 1932 and 1998 was just over 300 mm [3]. These desert fringe conditions create seasonal malaria transmission during, and immediately following, the two rainy seasons. The intensity of the resulting malaria seasonal transmission varies considerably from year to year, depending largely on the climatic conditions. Temperatures are usually high and average rainfall creates surface water pools suitable for vector breeding sites. Extreme climatic conditions with unseasonal heavy rainfall can result in flooding in many parts of this county. Surface flood water, whilst initially washing away most existing mosquito larvae, creates unusually large vector breeding sites as the flood waters gradually recede and Data sources Sources of data for the study included:- i) A Wellcome Trust electronic Excel 2003 database on rainfall, temperature and malaria from 1999 to 2008 [7]; ii) A Kenya Meteorological Department, Wajir station Excel 2003 data base for temperature and rainfall from 1998 to 2006; iii) MSF-Epicentre internal electronic reports on flooding, malnutrition and RVF cases in 1997/1998 and 2007; iv) MSF-Epicentre internal electronic reports on malaria vector control interventions in 1998 and 2007; v) MSF Mobile Clinic registers and Ministry of Health hospital and county reports on malaria cases in 1997/1998 and 2007; vi) The MENTOR Initiative internal electronic reports on a 2007 emergency Malaria Control intervention for the most vulnerable flood affected communities in Kenya; and vii) The Nutrition Information in Crisis Situation electronic database by the United Nations system Standing Committee on Nutrition for nutritional information.. Similar environmental conditions occurred in 2007 with protracted drought resulting in wide-spread under-nutrition, followed by very heavy rainfall and flooding across much of the province, causing mass population displacement and a RVF outbreak in the county. Prompted by these environmental conditions and before malaria cases had increased above normal baseline numbers, Me´decins Sans Frontie`res (MSF) intervened with an emergency approach designed to mitigate the risk of a malaria epidemic occurring. The ongoing surveillance and case management activities in the county were promptly reinforced. Also, within ten days, malaria vector control interventions were launched with the aim of cutting malaria transmission and mitigating against the risk of a large scale malaria epidemic developing, as had occurred in 1997. Setting G General. Kenya is a large country in East Africa with a population of 42 million in 2011, according to the World Bank. It has eight provinces including the North Eastern Province, which was, at the time, divided into four districts, now counties, from north to south; Mandera, Wajir, Garissa and Ijara. Wajir County. The study took place in Wajir County which is the second largest county in Kenya, covering an area of 56,501 km2 [3]. Wajir County ranges from very arid in the north, to semi-arid in the south. In 2007, the county had 13 administrative divisions with a total of 74 locations and 88 sub- locations. Wajir County includes Wajir town, which consists of the centre of the town or Township surrounded by the five major villages of Jog Baru, Wagberi, Hodhan, Alimao and Barwaqo. The population was about 500,000 people with an annual growth of 2.5%, and the majority of the population consisted of nomadic pastoralists. Over 63% of the population depended solely on livestock for their livelihood, 57% lived in absolute poverty (,1 USD/day) and the overall literacy level was 12.5%. [8]. In 2007, the county reportedly had one district hospital in Wajir town, five sub-district hospitals, one health center and more than 30 dispensaries. The aim of this study is to describe certain environmental conditions and the associated resulting burden of malaria in Wajir town at two time periods (1996–1998 and 2005–2007), each characterized by vector control interventions which started at different phases of the malaria transmission process. Timely Vector Control to Prevent Malaria Epidemics epidemics of malaria may occur. Control measures, when taken, are often implemented too late and with minimal coordination and expertise, and often under intense political pressure [2]. larvicides were applied: iii) for malaria:- number of admissions each month in Wajir hospital diagnosed with malaria based on clinical features; number of persons diagnosed with malaria each week in Wajir town per 1000 population (weekly malaria incidence). For the weekly malaria incidence figures, malaria was diagnosed based on clinical features of ‘‘any patient reporting a recent history of fever, in the absence of any other cause’’ confirmed by thin blood smears or malaria rapid diagnostic tests (Paracheck-Pf). Wajir County in Northeast Kenya is usually dry and hot and is a zone classified as having seasonal malaria transmission. When certain environmental conditions occur, epidemics of malaria may ensue. Since 1932, there have been two recorded major malaria epidemics in the county, one in 1961 and one in late 1997. The epidemic in 1997–1998 was preceded by twelve months of high temperatures and drought that led to widespread malnutrition [3]. El Nin˜o then caused virtually uninterrupted rainfall and the worst flooding in the county for over 50 years, resulting in large scale population displacement, and an outbreak of Rift Valley Fever (RVF) in the county. The remaining shallow flood waters covered an extensive area, and provided ideal breeding conditions for Anopheles mosquitoes (malaria vectors). This resulted in an exponential growth of the vector population and an explosive epidemic of malaria [4]. The poor nutritional status of the population, displacement from their homes, and lack of access to treatment services due to flooding and a national health staff strike, made individuals highly susceptible to malaria infection and to developing severe disease. Between February and May 1998, a total of 23,377 malaria cases (a malaria attack rate of 39% in the population) were reported [5]. The average crude mortality was approximately 9 per 10,000 per day and in the under-5 population this rose to approximately 28 per 10,000 per day. Introduction According to the World Health Organization (WHO) 2011 malaria report, there were 216 million cases of malaria globally in 2010, with over 80% occurring in sub-Saharan Africa [1]. There were 655,000 deaths, with 86% of the victims being children under the age of five years and 91% of malaria deaths occurring in Africa. April 2014 \| Volume 9 \| Issue 4 \| e92386 1 April 2014 \| Volume 9 \| Issue 4 \| e92386 PLOS ONE \| www.plosone.org Timely Vector Control to Prevent Malaria Epidemics Environmental conditions and hospital malaria admissions: 1996–1998 Environmental conditions and hospital malaria admissions: 1996–1998 The monthly rainfall, the presence of floods and RVF and the number of malaria admissions to Wajir hospital between January 1996 and December 1998 are shown in Figure 1. The seasonal periods of rainfall followed by drought are shown up to August 1997. From September 1997 to February 1998 there was very heavy monthly rainfall, at its peak reaching nearly 500 mm for the month of November. This resulted in extensive flooding between the months of October 1997 to February 1998 and RVF from November 1997 to February 1998. The number of malaria admissions from January 1996 to December 1997 was relatively stable ranging from 13 to 43 per month (with the exception of December when there was a hospital strike and no admissions). The increase in malaria admissions was first noted in January 1998 with 92 cases for the month and this peaked at 456 in February, before dropping to 427 in March and 69 in April. A RVF outbreak also requires heavy rainfall and extensive flooding of low lying grassland depressions associated with the rapid and mass emergence of Aedes mosquitoes reaching their maximum population about ten days after the flood event [12,13,14]. The breeding sites of the Anopheles mosquitoes that transmit malaria take much longer to recover from flooding compared with those of Aedes, and thus the Anopheles mosquito population reaches a maximum about one to two months after the flood event [15]. Thus, episodes of RVF often herald malaria epidemics in this sort of context. Malaria control. In 1997/1998 MSF carried out water rehabilitation and cholera preparedness projects, and moni- toring of RVF. MSF supported Wajir town hospital during this period, with out-patient and in-patient case management of malaria. Following atypical environmental conditions, an explosive malaria outbreak occurred in Wajir County between February and May 1998. MSF also started an Indoor Residual Spraying campaign in Wajir town about four to five weeks after the peak of the malaria outbreak in close collaboration with the District Public Health Office (DPHO). MERLIN, a UK non-governmental organization, worked in parallel with MSF, providing primary health care centre support, mobile clinics and insecticide treated bed-nets in flood affected areas in the county, but not in Wajir town. Ethics statement This study met the Me´decins Sans Frontie`res’ Ethics Review Board (Geneva, Switzerland) -approved criteria for analysis of routinely-collected program data, and was also approved by the Ethics Advisory Group of the International Union Against Tuberculosis and Lung Disease, Paris, France. Analysis was done on previously collected program data only: patients were not contacted, and no identifying characteristics of patients were collected. As such, informed consent of involved patients was not indicated or sought. Study participants During this three year period the daily recorded temperature never fell below 22uC. The prevalence of malnutrition and severe acute malnutrition in children under the age of five years in the county was 15.6% and 4.1% respectively in May 2006 and 23.0% and 2.8% in April 2007. Study participants included all adult and pediatric (less than 15 years of age) inpatients diagnosed with malaria at Wajir district hospital and all outpatients diagnosed with malaria in Wajir town during the two time periods (1996–1998 and 2005– 2007). Environmental conditions and hospital malaria admissions: 1996–1998 The Catholic mission, located in Wajir town provided additional clinical services, and the Swedish Rotarians had two volunteers providing labora- tory and clinical support at the hospital. MSF left Wajir in 1998. During this three year period the daily temperature never dropped below 21uC. The prevalence of malnutrition and severe acute malnutrition in children under the age of five years in the county and the town during the three year period is shown in Table 1. Data variables The following data variables were collected: g i) for environmental conditions and their impact on the population: -daily temperature (uC); rainfall (mm per month); reported flooding in the county (yes/no); internationally reported cases of animal and/or human RVF in the county (yes/no); and malnutrition and severe acute malnutrition in the under-fives in the county and Wajir town as measured by two stage cluster surveys and defined by z-scores [6]: ii) for malaria vector control interventions:- number of houses that received indoor residual spraying; number of long-lasting impregnated nets (LLIN) distributed; and number of surface flood water bodies where April 2014 \| Volume 9 \| Issue 4 \| e92386 April 2014 \| Volume 9 \| Issue 4 \| e92386 PLOS ONE \| www.plosone.org 2 Timely Vector Control to Prevent Malaria Epidemics become shallow surface water in the following weeks. Mass vector breeding results in very high rates of malaria transmission, and epidemic outbreaks of malaria with high morbidity and mortality rates [9,10,11]. Analysis and statistics Data were entered into an electronic Excel 2003 database and a simple descriptive analysis was carried out. The scope of malaria vector control interventions including the number of houses sprayed, LLINs distributed, modeled LLIN coverage based on the numbers distributed, and shallow pools treated with larvicides in Wajir town in 1998 and 2007 is shown in Table 2. Over 90% of houses were sprayed in the two periods, but in 1998 there were no LLINs distributed in the town, and no shallow pools were treated, in contrast to 2007. Environmental conditions and hospital malaria admissions: 2005–2007 The monthly rainfall, the presence of floods and RVF, and the number of malaria admissions in persons aged 15 years and below to Wajir hospital from January 2005 to December 2007 are shown in Figure 2. The seasonal periods of rainfall followed by drought are shown up to September 2006. At the end of September 2006, very heavy rains began, and in October the monthly rainfall reached almost 250 mm, declining to 95 mm in November and then back to the monthly average. This resulted in widespread flooding between the months of October and December 2006 and a RVF outbreak from November 2006 to January 2007. The number of malaria admissions in persons under the age of 15 years remained relatively stable during the whole three year period and there was no increase in cases during or after the time of rainfall and flooding. Heavy rains and flooding affected Wajir County and Greater Garissa from the end of September 2006, creating conditions very similar to those leading to the large malaria epidemic in 1997/1998. In response, MSF managed an outbreak of RVF in the neighbouring Garissa county and in January 2007 launched integrated malaria vector control activities in Wajir town. Discussion This cross-sectional descriptive and ecological study showed that in 1997/1998 and 2006/2007 Wajir County in north-eastern Kenya experienced atypical environmental conditions with drought and malnutrition, followed by massive monthly rainfall resulting in flooding and animal/human RVF. In 1998, this was associated with a large and explosive epidemic of malaria resulting in large numbers of admissions to Wajir Hospital and a weekly malaria incidence of 40–55 cases per 1000 population per week in all persons and children. Vector control interventions at that time consisted solely of indoor residual spraying of houses, and these started over 6 months after the onset of heavy rains when the malaria epidemic was already abating. In 2007, as a result of similar environmental conditions, MSF instituted a package of vector control interventions that included indoor residual spraying, distribution of LLINs and applying larvicides to shallow pools. These interventions started over three months after the onset of heavy rains and no malaria epidemic was recorded with weekly In 2007, the incidence of malaria per 1000 population per week and the timing of indoor residual spraying and distribution of LLINs are shown in Figure 4. The two malaria vector control interventions illustrated in Figure 3 and the treatment of shallow pools with larvicides started in the third week of January before any increase in malaria incidence was observed. It took ten days between the decision to intervene and the start of activities. Seven weeks after starting activities, 93% of all houses in Wajir town were sprayed and there was 76% household coverage of LLINs meaning that 76% of Wajir town population had received one LLIN per 1.8 persons. The incidence of all malaria cases and malaria in children under the age of five years remained low at less than 0.5 per 1000 population per week between the months of January to March in Wajir Town. The malaria surveillance in the peripheral areas illustrated that the malaria incidence in Wajir Table 1. Documented prevalence of malnutrition in Wajir county and Wajir town in children under the age of five years from 1996–1998. Site Year Month Prevalence of Malnutrition % Prevalence of severe acute malnutrition % Source of data Wajir County Oct 1996 27.9 3.8 OXFAM Feb 1997 25.1 4.2 MERLIN Jul 1997 8.8 1.0 OXFAM/MOH Mar 1998 16.0 2.9 OXFAM Wajir Town Jul 1997 15.0 1.7 OXFAM/MOH1 Mar 1998 25.3 3.7 MSF 1Ministry of Health. Timing of malaria vector control interventions and malaria incidence: 1998 and 2007 In 1998, the incidence of malaria per 1000 population per week and the timing of indoor residual spraying are shown in Figure 3. The incidence of all malaria cases and malaria in children under the age of five years peaked at the end of March 1998 at 54 per PLOS ONE \| www.plosone.org April 2014 \| Volume 9 \| Issue 4 \| e92386 3 Timely Vector Control to Prevent Malaria Epidemics Figure 1. Monthly rainfall, presence of floods and Rift Valley Fever and malaria admissions to Wajir Hospital: January 1996– December 1998. doi:10.1371/journal.pone.0092386.g001 Figure 1. Monthly rainfall, presence of floods and Rift Valley Fever and malaria admissions to Wajir Hospital: January 1996– December 1998. doi:10.1371/journal.pone.0092386.g001 County did not exceed 1 per 1000 population per week between the months of January to March. 1000 population per week and 46 per 1000 per week respectively. Indoor residual spraying started in mid-April reaching full house coverage of Wajir Town after two weeks. However, the start of this intervention occurred at a time when malaria incidence was already decreasing. Discussion doi:10.1371/journal.pone.0092386.t001 PLOS ONE \| www.plosone.org 4 April 2014 \| Volume 9 \| Issue 4 \| e92386 valence of malnutrition in Wajir county and Wajir town in children under the age of five years from Timely Vector Control to Prevent Malaria Epidemics Figure 2. Monthly rainfall, presence of floods and Rift Valley Fever and malaria admissions in persons under 15 years of age to Wajir Hospital: January 2005–December 2007. doi:10.1371/journal.pone.0092386.g002 Figure 2. Monthly rainfall, presence of floods and Rift Valley Fever and malaria admissions in persons under 15 years of age to Wajir Hospital: January 2005–December 2007. doi:10.1371/journal.pone.0092386.g002 malaria incidence consistently below 0.5 per 1000 population per week (100 times less than in 1998). can be said to have experienced similar climatic events as Wajir, malaria control interventions were started much later, and there was a significant increase in malaria cases [16]. The big question is whether these vector control interventions triggered by environmental cues, prevented a malaria epidemic? This is difficult to definitively answer, but the neighboring county of Greater Garissa County provides some sort of control arm. This county has similar characteristics to Wajir (semi arid) and experienced very similar atypical environmental conditions as Wajir County in 2006/2007 with massive rainfall followed by flooding, mass population displacement, and human/animal cases of RVF [16]. Malnutrition rates during the same time period were similar to those experienced in Wajir, reaching a mean of 24% global acute malnutrition in 2006 [17]. In this case, donors were slow to respond and large scale vector control interventions by the MENTOR initiative in Garissa did not start until March 2007, over six months after the start of heavy rains, which is similar to what happened in Wajir in 1997/1998. In Garissa County, there was almost a doubling of reported malaria cases from 7719 in 2006 to 13739 in 2007, and monthly malaria incidence peaked at 42 per 1000 population in January 2007 and continued at high levels of above 30 per 1000 per month for the next three to four months [16]. Thus, while impossible to formally compare these two regions due to differences in data collection systems, Garissa There are several strengths to this study. Weekly malaria incidence figures at the town level in Wajir were based on clinical features observed first-hand by MSF combined with thin blood smears showing malaria parasites or positive malaria rapid diagnostic tests. Discussion There was also a robust multidisciplinary approach with several different and concurring sources of data on the measurement of environmental changes and the impact on the population such as rates of malnutrition and RVF. Limitations relate to the operational nature of the study. First, malaria admission data from Wajir hospital during the two time periods were obtained from different sources, they did not describe the same populations and the diagnosis might have been made without parasite or serological confirmation. Second, the catch- ment area for Wajir Hospital might have been the county as well as the town, and it was principally the town that was targeted for vector control interventions by MSF. Third, we only have second- hand data over a limited period of time, from a control county such as Garissa, collected through separate channels precluding formal comparison - more data would have helped to strengthen the case for linking timely vector control interventions and malaria prevention in Wajir county in 2006/2007. Additionally, there was a report by Epicentre that in the neighboring county of Ijara, no malaria outbreak occurred between 22 January and 11 February 2007. While distribution of insecticide-treated bed nets reportedly also took place in this county, the scope and timing of such interventions could not be discerned, and it is therefore difficult to assess whether Ijara can be regarded as a control county for Wajir [18]. Table 2. Scope of malaria control interventions in 1998 and 2007 in Wajir Town. Table 2. Scope of malaria control interventions in 1998 and 2007 in Wajir Town. Malaria control interventions 1998 2007 Number of houses 9468 28536 Number (%) houses sprayed 9372 (99%) 26544 (93%) Number of LLINs needed for 100% coverage No data 45708 Number (coverage %) LLINs distributed 0 34716 (76%) Shallow pools treated with larvicides 0 5 LLIN = long-lasting insecticide treated nets. doi:10.1371/journal.pone.0092386.t002 The rainfall was truly exceptional in 1996–1998. In 2006 there was massive rainfall, although not to the level or the duration experienced ten years previously. However, major outbreaks of RVF have occurred in this county only during the two study periods. Discussion This phenomenon requires heavy rainfall and extensive flooding of low lying grassland depressions associated with the rapid and mass emergence of Aedes mosquitoes, and such episodes April 2014 \| Volume 9 \| Issue 4 \| e92386 April 2014 \| Volume 9 \| Issue 4 \| e92386 PLOS ONE \| www.plosone.org 5 Timely Vector Control to Prevent Malaria Epidemics Figure 3. Incidence of malaria per 1000 population per week in Wajir town and timing of indoor residual spraying in 1998. doi:10.1371/journal.pone.0092386.g003 malaria per 1000 population per week in Wajir town and timing of indoor residual spraying in 1998. .0092386.g003 Figure 3. Incidence of malaria per 1000 population per week in Wajir town and timing of indoor residual spraying in 1998. doi:10.1371/journal.pone.0092386.g003 of RVF often herald malaria epidemics in this sort of context. Furthermore, in Wajir county the high temperatures which stayed above 22uC favor the development of the malaria parasite in the Anopheles mosquitoes [19]. community level [23,24,25]. However, in an area with seasonal malaria transmission it seems vital to implement these interven- tions in a timely way. For this, there needs to be sufficient information for Ministries of Health and Aid agencies to raise a red early warning flag to prevent a malaria epidemic from occurring [26]. This study suggests that very heavy rainfall in an area like Wajir County that is followed by flooding and reports of RVF should be enough to trigger a wide scale vector control intervention to prevent epidemic malaria. This concurs with previous suggestions [2,27] that area specific monitoring of rainfall is very important, and that this can provide key early epidemic warning indicators to inform responsible stakeholders of the need to mount rapid integrated epidemic prevention responses. Wajir County exemplifies the epidemiology of an unstable malaria zone: this is typified by limited overall rainfall, protracted periods of drought with few infective bites, limited transmission of Plasmodium falciparum and thus low collective immunity of the population for malaria which is then followed by heavy rainfall leading to conditions that are ripe for a true malaria epidemic. Other people living in moderate to high malaria transmission regions of the country experience many infective bites throughout the year, and the children that survive repeated malaria infections in their first five years of life develop partial protective immunity to severe P. falciparum malaria [5,19,20,21]. References 14. Sang R, Kioko E, Lutomiah J, Warigia M, Ochieng C, et al. (2010) Rift Valley Fever Virus epidemic in Kenya, 2006/2007: the entomological investigations. Am J Trop Med Hyg 83: 28–37. 1. World Health Organization (2011) WHO Global Malaria Report 2011. World Health Organization, Geneva, Switzerland. 1. World Health Organization (2011) WHO Global Malaria Report 2011. World Health Organization, Geneva, Switzerland. 2. WHO (2001) Malaria early warning systems. Concepts, indicators and partners. Roll Back Malaria, World Health Organization, Geneva, Switzerland. WHO/ CDS/RBM/2001.32. J p yg 15. Linthicum KJ, Davies FG, Bailey CL (1983) Mosquito species succession in a dambo in an east African forest. Mosquito News 43: 464–470. 16. Allan R (2013) Basic data sheet. Extension and Expansion of Emergency Malaria Control for vulnerable communities in North Eastern and Coastal Province, Kenya. The MENTOR Initiative (contact person richard@mentor- intiative.net). 3. Snow RW, Ikoku A, Omumbo J, Ouma J (1999) The epidemiology, politics and control of malaria epidemics in Kenya:1900–1998. Report for the Roll Back Malaria. Resource network on epidemics, World Health Organization, Geneva, Switzerland. 17. USAID (2006) Understanding nutrition data and the causes of malnutrition in Kenya. A special report by the Famine Early Warning Systems Network (FEWSNET). 4. Allan R, Nam S, Doull L (1998) MERLIN and malaria epidemic in north-east Kenya. Lancet 351: 1966–7. 5. Brown V, Issak MA, Rossi M, Barboza P, Paugam A (1998) Epidemic of malaria in North East Kenya. Lancet 352: 1356–1357. 18. Grandesso F, Wichman Ole. Investigation of a Rift valley Fever outbreak and implementation if a malaria surveillance system. Ijara County, Kenya 2007. Paris, EpiCentre Medicins sans Frontier: 2007. P. 3–4. 6. WHO and UNICEF (2009) WHO child growth standards and the identification of sever acute malnutrition in infants and children. World Health Organization, Geneva, Switzerland. Available: http://www.who.int/nutrition/publications/ severemalnutrition/9789241598163_eng.pdf (accessed 12 June 2013). 19. Craig MH, Snow RW, le Sueur (1999) A climate-based distribution model of malaria transmission in Sub-Saharan Africa. Parasitology Today 15: 105–111. gy y 20. Hay S, Rogers DJ, Shanks GD, Myers MF, Snow RW (2001) Malaria early warning in Kenya. Trends Parasitol 17:95–99. 7. Okiro E A, Alegana V A, Noor A M, Mutheu J J, 1, Elizabeth Juma, et al (2009) Malaria paediatric hospitalization between 1999 and 2008 across Kenya. BMC Medicine 7:75 doi:10.1186/1741-7015-7-75. g y 21. Reiter P, Thomas CJ, Atkinson PM, Hay SI, Randolph SE, et al. (2003). Timely Vector Control to Prevent Malaria Epidemics Timely Vector Control to Prevent Malaria Epidemics International Health, University of Bergen, Norway and the Institute of Tropical Medicine, Antwerp, Belgium. We are grateful to Dr Emelda Okiro for help with the data. be needed that allows resources to be made available to start rapid and widespread scale up of vector control interventions before any significant rise in malaria cases is declared. Acknowledgments Conceived and designed the experiments: PM ADH RVDB KTS SGH RZ RA. Performed the experiments: PM ADH RVDB AMN RWS RA. Analyzed the data: PM ADH RVDB KTS SGH. Contributed reagents/ materials/analysis tools: PM ADH RVDB AMN RWS RZ SGH RA. Wrote the paper: PM ADH RVDB AMN RWS KTS SGH RZ RA. This research was supported through an operational research course, which was jointly developed and run by the Operational Research Unit (LUXOR), Me´decins Sans Frontie`res, Brussels-Luxembourg, The Centre for Operational Research, International Union Against Tuberculosis and Lung Disease, France, and The Union South-East Asia Regional Office. Additional support for running the course was provided by the Center for Discussion Periods of drought also cause widespread malnutrition and there is evidence that malaria prevalence is further increased in such circumstances [22]. In conclusion, this study suggests that atypical environmental conditions with massive rainfall, flooding and RVF following a period of drought can herald a malaria outbreak in semi arid settings such as the North Eastern Province of Kenya, and that this could be prevented by timely, preemptive and rapid delivery of appropriate vector control interventions. A paradigm shift might Vector control interventions, such as the ones used in the study, are well-established methods for controlling malaria at the Figure 4. Incidence of malaria per 1000 population per week in Wajir town and timing of indoor residual spraying and modeled long-lasting insecticidal nets coverage in 2007. doi:10.1371/journal.pone.0092386.g004 Figure 4. Incidence of malaria per 1000 population per week in Wajir town and timing of indoor residual spraying and modeled long-lasting insecticidal nets coverage in 2007. doi:10.1371/journal.pone.0092386.g004 PLOS ONE \| www.plosone.org 6 References Global warming and malaria: a call for accuracy. The lancet Infectious diseases 4:323– 324. 8. Kenya National Bureau of Statistics (KNBS) and ICF Macro (2010) Kenya Demographic and Health Survey 2008–09. Calverton, Meryland: KNBS and ICF Macro. 22. Khogali M, Zachariah R, Keiluhu A, Van den Brande K, Tayler-Smith K, et al. (2011) Detection of malaria in relation to fever and grade of malnutrition amongst malnourished children in Ethiopia. Public Health Action 1: 16–18. 9. Githeko AK, Lindsay SW, Confalonieri UE, Patz JA (2000) Climate change and vector-borne diseases – a regional analysis. Bull World Health Organ 78: 1136– 1147. g p 23. Lengeler C (2009) Insecticide-treated bed nets and curtains for preventing malaria. The Cochrane Library, Issue 2. 10. Zhou G, Minakawa N, Githeko AK, Yan G (2004) Association between climate variability and malaria epidemics in the East African highlands. PNAS 101: 2375–2380 24. Pluess B, Tanser FC, Lengeler C, Sharp BL (2010) Indoor residual spraying for preventing malaria. Cochrane Database Syst Rev 4: CD006657. 25. Alba S, Hetzel MW, Nathan R, Alexander M, Lengeler C (2011) Assessing the impact of malaria interventions on morbidity through a community-based surveillance system. Int J Epidemiol 40: 405–416. 11. Peterson A (2009) Shifting suitability for malaria vectors across Africa with warming climates. BMC Infectious Diseases 9:59 12. Woods C, Karpati A, Grein T, McCarthy N, Gaturuku P, et al. (2002) An outbreak of Rift Valley Fever in Northeastern Kenya, 1997–98. Emerg Infect Dis 8: 138–144 26. Snow B, Hay S, Noor AM (2006) Rapid situation analysis of possible malaria epidemic in Wajir County, North Eastern Kenya and Somalia. Nairobi: Kemri- Wellcome Trust. Report. 13. Nguku PM, Sharif SK, Mutonga D, Amwayi S, Omolo J, et al. (2010) An investigation of a major outbreak of Rift Valley Fever in Kenya, 2006–2007. Am J Trop Med Hyg 83: 5–13. p 27. Hay S, Renshaw M, Ochola S, Noor AM, Snow RW (2003) Performance of forecasting, warning and deterction of malaria epidemics in the highlands of western Kenya. Trends Parasitol 19: 394–399. April 2014 \| Volume 9 \| Issue 4 \| e92386 PLOS ONE \| www.plosone.org 7
https://openalex.org/W2995618033	http://ejournal.uin-suska.ac.id/index.php/juring/article/download/7813/4423	Indonesian	null	Pengaruh Penerapan Strategi Pembelajaran Metakognitif terhadap Kemampuan Koneksi Matematis Berdasarkan Kemandirian Belajar Siswa SMP/MTs	Juring	2,019	cc-by-sa	4,906	Pengaruh Penerapan Strategi Pembelajaran Metakognitif Terhadap Kemampuan Koneksi Matematis Berdasarkan Kemandirian Belajar Siswa SMP/MTs Asmawati1, Risnawati2, dan Ramon Muhandaz3 1 2.3 Program studi pendidikan matematika, Universitas Islam Negeri Sultan Syarif Kasim Riau e mail: ramon muhan@uin suska ac id 1 2.3 Program studi pendidikan matematika, Universitas Islam Negeri Sultan Syarif Kasim Riau e-mail: ramon.muhan@uin-suska.ac.id ABSTRAK. Penelitian ini bertujuan untuk mengetahui pengaruh penerapan strategi pembelajaran metakognitif terhadap kemampuan koneksi matematis berdasarkan kemandirian belajar siswa. Jenis penelitian ini merupakan penelitian Quasi Eksperimental dengan desain The Nonequivalent Posttest Only Control Group Design. Penelitian ini dilakukan di MTsN 1 Pekanbaru. Tenik pengambilan sampel yang digunakan adalah Purposive Sampling. Populasi pada penelitian ini adalah seluruh siswa kelas VII MTsN 1 Pekanbaru dengan sampel yaitu kelas VII.7 sebagai kelas eksperimen dan VII.8 sebagai kelas kontrol. Instrumen penelitian ini berupa soal tes kemampuan koneksi matematis, angket kemandirian belajar dan lembar observasi. Teknik analisis data yang digunakan untuk hipotesis 1 adalah uji-t sedangkan untuk hipotesis 2 dan 3 menggunakan anova dua arah. Berdasarkan penelitian yang dilakukan diperoleh beberapa kesimpulan, yaitu untuk hipotesis pertama disimpulkan bahwa terdapat perbedaan kemampuan koneksi matematis antara siswa yang belajar menggunakan strategi pembelajaran metakognitif dengan siswa yang belajar tanpa menggunakan strategi pembelajaran metakognitif dengan hasil analisis data dengan menggunakan uji-t menunjukkan nilai dan kemampuan koneksi matematis siswa di kelas eksperimen lebih baik dari pada kelas kontrol, dimana nilai rata-rata kelas eksperimen adalah dan nilai rata-rata kelas kontrol adalah Untuk hipotesis kedua dan ketiga dapat disimpulkan dengan hasil analisis data menggunakan anova dua arah yang menunjukkan sehingga disimpulkan terdapat perbedaan kemampuan koneksi matematis antara siswa yang memiliki kemandirian belajar tinggi, sedang, dan rendah serta menunjukkan sehingga disimpulkan bahwa tidak terdapat interaksi antara strategi pembelajaran dengan kemandirian belajar siswa dalam mempengaruhi kemampuan koneksi matematis siswa. Hasil tersebut mengidentifikasi bahwa strategi pembelajaran metakognitif mempengaruhi kemampuan koneksi matematis. Kata kunci: Strategi Pembelajaran Metakognitif, Kemampuan Koneksi Matematis, Kemandirian Belajar Juring (Journal for Research in Mathematics Learning) p-ISSN: 2621-7430 \|e-ISSN: 2621-7422 Vol. 2, No. 3, 1 September 2019, 273– 284 Juring (Journal for Research in Mathematics Learning), Vol. 2, No. 3, 1 September 2019, 273– 284 PENDAHULUAN Matematika merupakan salah satu mata pelajaran wajib yang harus dipelajari siswa tingkat dasar dan menengah, hal ini sesuai dengan Undang-undang tentang sistem pendidikan nasional nomor 20 Tahun 2003 pasal 37 menyatakan bahwa kurikulum pendidikan dasar dan menengah di Indonesia wajib memuat mata pelajaran matematika yang memiliki peranan penting dalam berbagai ilmu untuk memajukan daya pikir manusia (UU No. 20, 2003). Sebagai disiplin ilmu yang memiliki peranan penting dan wajib untuk dipahami oleh setiap siswa, tentunya ada standar kemampuan yang harus dicapai. Menurut NCTM (National Council of Teacher of Mathematics) (Walle, 2006, hal. 4) bahwa standar proses dalam pembelajaran matematika yaitu kemampuan pemecahan masalah (problem solving), kemampuan penalaran (reasoning), kemampuan komunikasi (communication), kemampuan membuat koneksi (connection), dan kemampuan representasi (representation). Pentingnya kemampuan koneksi matematis juga terdapat 273 Juring (Journal for Research in Mathematics Learning), Vol. 2, No. 3, 1 September 2019, 273– 284 Asmawati, Risnawati, dan Ramon Muhandaz pada salah satu tujuan pembelajaran matematika di sekolah. Berdasarkan kurikulum tahun 2013 (Permendikbud, 2014, hal. 325) salah satu tujuan pembelajaran matematika adalah “bahwa siswa memahami konsep matematika, menjelaskan keterkaitan konsep dan menerapkan konsep atau algoritma secara fleksibel, akurat, efisien, dan tepat dalam pemecahan masalah.”. Berdasarkan NCTM dan tujuan pembelajaran matematika yang termuat dalam kurikulum 2013, maka peneliti menyimpulkan bahwa kemampuan koneksi matematis penting dimiliki oleh siswa. Namun pada kenyataan yang ditemukan dari berbagai sumber yang peneliti lihat, kemampuan koneksi matematis ini masih tergolong rendah. Buktinya hasil penelitian yang dilakukan oleh PISA (Programme of International Study Assesment) (2016:44), kemampuan matematika siswa indonesia mendapat skor 386 di bawah rata-rata skor internasional, yakni 490 dan menduduki peringkat ke-63 dari 70 negara yang berpartisipasi. Selanjutnya salah satu hasil penelitian terdahulu yang dilakukan oleh Anandita (2015, hal. 94). Hasil penelitian menunjukkan bahwa dari 37 peserta didik terdapat 18 peserta didik yang masuk pada kategori sangat kurang, 10 peserta didik pada kategori kurang, 6 peserta didik pada kategori cukup, 2 peserta didik pada kategori baik, dan 1 peserta didik pada kategori sangat baik. Permasalahan serupa peneliti temukan di MTsN 1 Pekanbaru. Berdasarkan hasil wawancara yang dilakukan dengan salah satu guru mata pelajaran matematika mengungkapkan bahwa kebanyakan siswa masih kesulitan dalam menyelesaikan masalah matematika. Juring (Journal for Research in Mathematics Learning), Vol. 2, No. 3, 1 September 2019, 273– 284 PENDAHULUAN Sehingga dapat dikatakan bahwa pembelajaran dengan strategi metakognitif mengarahkan perhatian siswa pada apa yang relevan dan membimbing 274 \| Pengaruh Penerapan Strategi Pembelajaran Metakognitif Terhadap Kemampuan Koneksi Matematis Berdasarkan Kemandirian Belajar Siswa SMP/MTs ngaruh Penerapan Strategi Pembelajaran Metakognitif Terhadap Kemampuan Koneksi Matematis Berdasarkan Kemandirian Belajar Siswa SMP/MTs Pengaruh Penerapan Strategi Pembelajaran Metakognitif Terhadap Kemampuan Koneksi Matematis Berdasarkan Kemandirian Belajar Siswa SMP/MTs mereka untuk memilih strategi yang tepat untuk menyelesaikan soal-soal terkait dengan kebutuhan siswa dalam menyelesaikan masalah matematis. y Pada strategi pembelajaran metakognitif memiliki tiga tahapan atau langkah pembelajaran yaitu: perencanaan, pemantauan, dan evaluasi. Pada tahap perencanaan adalah tahap proses mengidentifikasi strategi berpikir dan keterampilan berpikir. Pada tahap pemantauan adalah tahap mampu mendeteksi kemajuan perencanaan dan pelaksanaan proses berfikir. Kemudian tahap evaluasi adalah tahap proses mengoreksi dan menentukan kualitas produk dan proses berpikir yang telah dilalui (Zakaria, dkk., 2007, hal. 135-136). y g ( ) Berdasarkan hasil penelitian yang dilakukan oleh Azma (2017, hal. 102) yang melakukan penelitian tentang Peningkatan Kemampuan Koneksi Matematis dan Self Efficacy Siswa SMP Melalui Pembelajaran Strategi Metakognisi menunjukkan hasil peningkatan kemampuan koneksi matematis siswa yang memperoleh pembelajaran dengan strategi metakognisi lebih baik dari pada siswa yang memperoleh pembelajaran konvensional ditinjau dari keseluruhan siswa dan pengelompokan siswa (tinggi, sedang, dan rendah). Selain itu, sama hal nya dengan penelitian yang telah dilakukan oleh Lestari (2019) yang menerapkan strategi metakognitif dalam pembelajaran matematika dengan melihat salah satu kemampuan matematika yaitu komunikasi matematis yang menghasilkan kesimpulan bahwa kemampuan komunikasi matematis siswa yang belajar dengan menggunakan strategi metakognitif lebih baik dibandingkan dengan kemampuan komunikasi matematis siswa yang belajar dengan strategi yang bukan strategi metakognitif. Dengan demikian pembelajaran dengan strategi metakognitif diharapkan dapat menjadi solusi dalam mempengaruhi kemampuan koneksi matematis. Indikator kemampuan koneksi matematis yang peneliti gunakan adalah mengenali dan menggunakan hubungan antara gagasan/ide matematis; mengenali dan menggunakan hubungan-hubungan antara matematika dengan bidang studi lain; serta mengenali dan menerapkan matematika dalam kehidupan sehari-hari. Selain faktor strategi pembelajaran, terdapat faktor lain yang mempengaruhi hasil belajar siswa yaitu faktor internal yang berasal dari siswa itu sendiri, seperti kemampuan, minat, bakat, motivasi instrinsik dan kemandirian belajar siswa. Faktor yang diambil peneliti dalam mempengaruhi hasil belajar, khususnya koneksi matematis siswa adalah kemandirian belajar. Kemandirian belajar merupakan faktor penting yang mempengaruhi belajar siswa. Hal ini sejalan dengan pendapat Heris dkk. (2017, hal. 227) yang menyatakan bahwa salah satu faktor penting dari keadaan individu yang mempengaruhi belajar adalah kemandirian belajar (Self Regulated Learning). PENDAHULUAN Hal ini tampak ketika siswa kurang tepat dalam menuliskan langkah-langkah penyelesaian dan siswa masih kesulitan dalam menentukan rumus atau konsep apa yang digunakan untuk menyelesaikan masalah karena kurang mampunya siswa dalam mengidentifikasi hubungan konsep dalam matematika baik antar konsep matematika itu sendiri maupun konsep matematika dengan konsep diluar matematika, terkhusus pada soal berbentuk cerita yang berkaitan dengan kehidupan sehari-hari. Berdasarkan hasil wawancara yang dilakukan, peneliti juga melakukan pra riset dengan memberikan beberapa soal koneksi matematis pada sebagian siswa kelas VII, yang mana rata- rata nilai hasil untuk koneksi matematis yang diperoleh siswa masih rendah. Berdasarkan hasil pra riset secara keseluruhan tingkat keberhasilan siswa dalam menyelesaikan soal kemampuan koneksi matematis sebesar , artinya sebanyak dari jawaban siswa belum mampu dalam menyelesaikan soal kemampuan koneksi matematis. p Mengingat betapa pentingnya kemampuan koneksi matematis dan juga menyikapi beberapa permasalahan yang telah dipaparkan sebelumnya harus diperlukan suatu inovasi pembelajaran sebagai upaya untuk meningkatkan kemampuan koneksi matematis. Salah satu faktor yang mempengaruhi berkembangnya kemampuan kognitif siswa adalah strategi pembelajaran. Sebagaimana yang diungkapkan oleh Muhibbin Syah (2004, hal. 144) bahwa salah satu faktor yang mempengaruhi hasil belajar siswa adalah “Faktor pendekatan belajar (approach to learning), yakni jenis upaya belajar siswa yang meliputi model/strategi dan metode yang digunakan siswa untuk melakukan kegiatan pembelajaran materi-materi pelajaran. g p j p j Oleh karena itu, perlu diterapkan suatu strategi pembelajaran yang dapat mempengaruhi kemampuan koneksi matematis. Salah satu strategi pembelajaran yang sesuai dengan permasalahan tersebut adalah strategi metakognitif. Husamah dan Setyaningrum (2013, hal. 178) menyatakan bahwa strategi yang digunakan untuk mengetahui proses kognitif seseorang dan caranya berfikir tentang bagaimana informasi diproses dikenal sebagai strategi metakognitif. Kemudian mereka melanjutkan bahwa strategi metakognitif adalah menghubungkan informasi baru dengan pengetahuan terdahulu (koneksi), memilih strategi berpikir secara sengaja, merencanakan, memantau, dan mengevaluasi proses berpikir. Selanjutnya Chairani (2016, hal. 8) mengungkapkan bahwa bentuk kesadaran seseorang yang terkait dengan kemampuan kognisinya tentang apa yang diketahuinya, dan yang tidak diketahuinya berdasarkan pengetahuan yang sudah dimilikinya, pengalaman, proses, dan kontrol dimana ia sendiri terlibat dalam kegiatan kognisinya sendiri adalah aspek dari aktivitas metakognisi. Juring (Journal for Research in Mathematics Learning), Vol. 2, No. 3, 1 September 2019, 273– 284 PENDAHULUAN Kemandirian belajar diperlukan bagi siswa agar siswa tersebut mempunyai tanggung jawab dalam mengatur dan mendisiplinkan dirinya, menjadikan siswa belajar aktif dalam proses pembelajaran karena siswa memiliki kemauan sendiri dalam belajar. Hal ini sesuai dengan pernyataan Wedemeyer dalam Rusman (2016, hal. 354) yang menyatakan bahwa kemandirian belajar perlu diberikan kepada perserta didik supaya mereka mempunyai tanggung jawab dalam mengatur dan mendisiplinkan dirinya dan dalam mengembangkan kemampuan belajar atas kemauan sendiri. Kemandirian belajar pada penelitian ini menggunakan indikator menurut sumarmo (Hendriana, dkk., 2017, hal. 233) yaitu inisiatif dan motivasi belajar instrinsik; kebiasaan mendiagnosa kebutuhan belajar; menetapkan tujuan/target belajar; memonitor, mengatur dan mengontrol belajar; memandang kesulitan sebagai tantangan; memanfaatkan dan mencari sumber yang relevan; memilih, menerapkan strategi belajar; mengevaluasi proses dan hasil belajar; self efficacy/konsep diri/ kemampuan diri. Penelitian ini berfokus pada strategi pembelajaran metakognitif terhadap kemampuan koneksi matematis berdasarkan kemandirian belajar siswa. Oleh karena itu penelitian ini bertujuan untuk mengetahui apakah terdapat perbedaan kemampuan koneksi matematis antara siswa yang belajar menggunakan strategi pembelajaran metakognitif dengan siswa yang belajar tanpa menggunakan strategi pembelajaran metakognitif, dan untuk mengetahui apakah terdapat perbedaan kemampuan koneksi matematis antara siswa yang memiliki kemandirian belajar tinggi, sedang dan rendah, serta untuk mengetahui apakah terdapat interaksi antara strategi pembelajaran dengan kemandirian belajar siswa dalam mempengaruhi kemampuan koneksi matematis. \| 275 Juring (Journal for Research in Mathematics Learning), Vol. 2, No. 3, 1 September 2019, 273– 284 Asmawati, Risnawati, dan Ramon Muhandaz Juring (Journal for Research in Mathematics Learning), Vol. 2, No. 3, 1 September 2019, 273– 284 METODE 276 Pengaruh Penerapan Strategi Pembelajaran Metakognitif Terhadap Kemampuan Koneksi Matematis Berdasarkan Kemandirian Belajar Siswa SMP/MTs Pengaruh Penerapan Strategi Pembelajaran Metakognitif Terhadap Kemampuan Koneksi Matematis Berdasarkan Kemandirian Belajar Siswa SMP/MTs METODE Penelitian ini menggunakan pendekatan kuantitatif dalam bentuk Quasi Experimental Design. Eksperimen semu memiliki kelompok kontrol, namun tidak dapat berfungsi secara utuh untuk mengontrol variabel-variabel luar yang mempengaruhi hasil eksperimen (Sugiyono, 2014, hal. 144). Penelitian ini menggunakan dua kelas yaitu kelas eksprimen dan kelas kontrol. Kelas eksperimen adalah kelas yang sengaja diberi seperangkat perlakuan yaitu proses pembelajaran yang menggunakan strategi metakognitif sedangkan kelas kontrol proses pembelajaran dengan menggunakan strategi pembelajaran selain metakognitif. Penelitian ini menggunakan rancangan penelitian The Nonequivalent Posttest Only Control Group Design. Rancangan penelitian Nonequivalent Posttest Only Control Group Design dapat dilihat pada Tabel 1 berikut (Lestari & Ridwan, 2017:136). Tabel 1. Rancangan Penelitian X O O Tabel 1. Rancangan Penelitian X O O Tabel 1. Rancangan Penelitian X O O Keterangan : g X : perlakuan/treatment yang diberikan (variabel independen) O : pretest/posttest (variabel dependen yang diobservasi) g X : perlakuan/treatment yang diberikan (variabel independen) O : pretest/posttest (variabel dependen yang diobservasi) X : perlakuan/treatment yang diberikan (variabel independen) p y g ( p ) O : pretest/posttest (variabel dependen yang diobservasi) Untuk kemandirian belajar siswa, digunakan angket diawal pada kelas eksperimen dan kelas kontrol. Skala kemandirian belajar siswa dikelompokkan menjadi tinggi, sedang dan rendah. kriteria pengelompokan kemandirian belajar bisa dilihat pada Tabel 2 berikut ini (Muhandaz, 2015:39). Tabel 2. Kriteria Penilaian Kemandirian Belajar Kriteria Kemandirian Belajar Keterangan Tinggi Sedang Rendah Penelitian ini dilaksanakan di MTsN 1 Pekanbaru pada semester genap tahun ajaran 2018/2019. Populasi pada penelitian ini adalah kelas VII MTsN 1 Pekanbaru. Teknik pengambilan sampel yang digunakan dalam penelitian ini adalah Pusposive Sampling. Sampel yang terpilih adalah kelas VII.7 sebagai kelas eksperimen dan kelas VII.8 sebagai kelas kontrol. Variabel bebas pada penelitian ini adalah strategi pembelajaran metakognitif, sedangkan variabel terikat adalah kemampuan koneksi matematis, serta variabel moderat nya adalah kemandirian belajar siswa. Instrumen penelitian yang digunakan adalah tes kemampuan koneksi matematis, angket kemandirian belajar siswa, dan lembar observasi. Instrumen yang digunakan untuk mengukur harus divalidasi untuk mendapatkan data yang benar-benar valid. Untuk memvalidasi tes kemampuan koneksi matematis yang dilakukan adalah dengan melakukan pengujian validitas dan reliabilitas, serta menganalisis tingkat kesukaran dan menentukan daya beda butir instrumen. Untuk memvalidasi angket kemandirian belajar yang dilakukan adalah dengan melakukan pengujian validitas dan reliabilitas. Teknik analisis data yang digunakan untuk menguji hipotesis 1 adalah uji-t sedangkan untuk hipotesis 2 dan 3 menggunakan anova dua arah. Hasil Uji Normalitas Soal Posttest Kemampuan Koneksi Matematis Tabel 5. Uji Homogenitas Soal Posttest Kemampuan Koneksi Matematis Dari tabel dapat diketahui bahwa data berdistribusi normal dan varians homogen, maka selanjutnya dilakukan uji hipotesis. Untuk hipotesis 1, hasil uji-t yang diperoleh adalah sebagai berikut: Tabel 6. Uji-t Posttest Keterangan Ha diterima Berdasarkan hasil analisis uji-t yang terdapat pada Tabel 6 diperoleh harga , maka ditolak dan diterima artinya hal ini menunjukkan bahwa pembelajaran yang menggunakan strategi pembelajaran metakognitif dengan pembelajaran tanpa menggunakan strategi metakognitif memberikan pengaruh yang berbeda terhadap kemampuan koneksi matematis siswa kelas VII MTsN 1 Pekanbaru. Juring (Journal for Research in Mathematics Learning), Vol. 2, No. 3, 1 September 2019, 273– 284 Hasil Hasil penelitian ini adalah terdapat perbedaan kemampuan koneksi matematis antara siswa yang belajar menggunakan strategi pembelajaran metakognitif dengan siswa yang belajar tanpa menggunakan strategi pembelajaran metakognitif, dan terdapat perbedaan kemampuan koneksi matematis antara siswa yang memiliki kemandirian belajar tinggi, sedang, dan rendah, serta tidak terdapat interaksi antara strategi pembelajaran dengan kemandirian belajar dalam mempengaruhi kemampuan koneksi matematis siswa. p Untuk hasil analisis angket kemandirian belajar siswa, siswa di kelompokkan dengan kriteria tinggi, sedang dan rendah. Berdasarkan hasil perhitungan yang telah dilakukan, diperoleh kriteria pengelompokkan kemandirian belajar siswa pada Tabel 3. Tabel 3. Kriteria Pengelompokan Kemandirian Belajar Kriteria Kemandirian Belajar Keterangan Eksperimen Kontrol Tinggi 5 Orang 4 Orang Sedang 26 Orang 25 Orang Rendah 4 Orang 6 Orang Tabel 3. Kriteria Pengelompokan Kemandirian Belajar Pada Tabel 3 dapat kita lihat bahwa, siswa yang memperoleh skor sama atau lebih dari 74,02, berarti siswa tersebut termasuk ke dalam siswa yang memiliki kemandirian belajar kelompok tinggi. Sedangkan siswa yang skornya antara 59,74 sampai 74,02, termasuk siswa yang memiliki kemandirian belajar kelompok sedang. Apabila siswa memperoleh skor sama atau kurang dari 59,74, maka siswa tersebut termasuk ke dalam siswa yang memilki kemandirian belajar kelompok rendah. Untuk analisis data posttest, secara deskriptif terlihat bahwa hasil Posttest pada kelas eksperimen dengan strategi pembelajaran metakognitif memperoleh rata-rata nilai sebesar . Selanjutnya, hasil Posttest pada kelas kontrol dengan model pembelajaran scientific memperoleh rata- rata nilai sebesar . Perbandingan rata-rata nilai Posttest antara kelas eksperimen dan kontrol dapat dilihat pada gambar 1 berikut. Gambar 1. Perbandingan Rata-Rata Nilai Posttest Kelas Eksperimen Dan Kontrol 77,57 69,71 50 60 70 80 Eksperimen Kontrol Rata-Rata Kelas Gambar 1. Perbandingan Rata-Rata Nilai Posttest Kelas Eksperimen Dan Kontro Selanjutnya, secara inferensial dilakukan uji hipotesis 1 dengan uji-t dan hipotesis 2 dan 3 dengan uji anova dua arah. Sebelum melakukan uji hipotesis terlebih dahulu dilakukan uji normalitas menggunakan rumus Chi Kuadrat dan homogenitas menggunakan rumus Uji F. Hasil uji normalitas dan homogenitas kemampuan koneksi matematis siswa kelas eksperimen dan kontrol MTsN 1 Pekanbaru dapat dilihat pada Tabel 4 dan Tabel 5 berikut. \| 277 Juring (Journal for Research in Mathematics Learning), Vol. 2, No. 3, 1 September 2019, 273– 284 Asmawati, Risnawati, dan Ramon Muhandaz Tabel 4. Uji Normalitas Soal Posttest Kemampuan Koneksi Matematis Kelas Kriteria Eksperimen 11.07 Normal Kontrol 11.07 Normal Tabel 5. Uji Homogenitas Soal Posttest Kemampuan Koneksi Matematis Nilai Varians Sampel Kelas Eskperimen Kontrol N 35 35 Tabel 4. Pembahasan Dikatakan bahwa strategi metakognitif yaitu strategi yang membimbing dan mengarahkan siswa untuk memikirkan strategi yang tepat dalam proses menguasai materi pembelajaran. Strategi ini membantu siswa dalam menjalankan proses berpikir secara bermakna (Zakaria, dkk., 2007:135). Ciri utama dalam pembelajaran metakognitif adalah pertanyaan-pertanyaan metakognitif yang berisi pemahaman masalah, perencanaan penyelesaian masalah dan meriview hasil penyelesaian masalah (Hutahuruk, 2016:180). Melalui pertanyaan-pertanyaan yang dapat mengarahkan dan membimbing siswa pada proses kognitifnya dapat menanamkan kesadaran pada siswa bagaimana untuk terlibat dalam proses berpikir sehingga meningkatkan proses belajar dan memori serta meningkatkan kemampuan siswa dalam menghubungkan atau mengaitkan konteks antar matematika dan matematika dengan konteks diluar matematika. Pada kelas eksperimen, siswa terlibat secara sadar dalam proses belajar yang dilakukannya dengan melalui tahap perencanaan, pemantauan, dan melakukan evaluasi pembelajarannya sendiri. Guru membimbing dan mengarahkan siswa untuk melakukan kegiatan tersebut dengan pertanyaan yang merangsang siswa untuk sadar akan proses kognisi sehingga kemampuan koneksi matematis siswa dapat berkembang lebih baik. Dengan demikian Strategi Pembelajaran Metakognitif memberikan kesempatan siswa untuk mampu melihat keterkaitan-keterkaitan yang terdapat dalam matematika, baik antar konsep matematika, matematika dengan ilmu lainnya, serta matematika dengan kehidupan sehari-hari. Berdasarkan penjelasan tersebut dapat dikatakan bahwa hal-hal tersebutlah yang dapat memicu kemampuan koneksi matematis siswa pada kelas eksperimen lebih tinggi dari pada kemampuan koneksi matematis siswa pada kelas kontrol. Hasil penelitian yang dilakukan oleh Fauzi (2011, hal. 113) dengan judul penelitian Peningkatan Kemampuan Koneksi Matematis Dan Kemandirian Belajar Siswa Dengan Pendekatan Pembelajaran Metakognitif Di Sekolah Menengah Pertama menunjukkan hasil bahwa 278 \| Pengaruh Penerapan Strategi Pembelajaran Metakognitif Terhadap Kemampuan Koneksi Matematis Berdasarkan Kemandirian Belajar Siswa SMP/MTs Pengaruh Penerapan Strategi Pembelajaran Metakognitif Terhadap Kemampuan Koneksi Matematis Berdasarkan Kemandirian B l j Si SMP/MT Pengaruh Penerapan Strategi Pembelajaran Metakognitif Terhadap Kemampuan Koneksi Matematis Berdasarkan Kemandirian Belajar Siswa SMP/MTs secara umum dapat dilihat bahwa siswa yang mendapat pendekatan metakognitif peningkatan kemampuan koneksi matematis siswa yang secara signifikan lebih tinggi dari pada siswa yang mendapat pendekatan konvensional. Begitu juga penelitian yang dilakukan oleh Azma (2017:102) dengan judul Peningkatan Kemampuan Koneksi Matematis dan Self Efficacy Siswa SMP Melalui Pembelajaran Strategi Metakognisi. Hasil penelitian menunjukkan bahwa peningkatan kemampuan koneksi matematis siswa yang memperoleh pembelajaran dengan strategi metakognisi lebih baik dari pada siswa yang memperoleh pembelajaran konvensional. Berdasarkan hal tersebut dapat dikatakan bahwa pembelajaran dengan penerapan strategi metakognitif lebih baik dari pada pembelajaran tanpa menggunakan strategi metakognitif (saintifik), hal ini bisa dilihat dari rata-rata nilai posttest per masing-masing kelas dan hasil analisis posttest yang menggunakan uji-t. Pembahasan Selain dari rata-rata nilai posttest per masing-masing kelas dan hasil uji-t, pengaruh ini dapat dilihat dari rata- rata nilai siswa dari kelas eksperimen dan kelas kontrol ditinjau dari masing-masing soal kemampuan koneksi matematis. Untuk lebih jelasnya, peneliti akan menjabarkan kemampuan koneksi matematis siswa berdasarkan butir-butir soal dan indikator kemampuan koneksi matematis. Soal Kemampuan Koneksi Matematis Nomor Satu Pada butir soal koneksi matematis mengandung indikator yaitu mengenali dan menggunakan hubungan-hubungan antara gagasan/ide matematis. Hubungan yang ditunjukkan pada soal nomor satu yaitu hubungan antara materi persegi panjang dan persegi dengan materi aljabar. Tingkat keberhasilan siswa dikelas eksperimen untuk soal nomor satu sebesar . Jika dibandingkan dengan tingkat keberhasilan di kelas kontrol, keberhasilan siswa dikelas eksperimen lebih unggul karena tingkat keberhasilan di kelas kontrol adalah . Berikut hasil lembar jawaban siswa pada soal nomor 1. Gambar 2. Lembar Jawaban Siswa Kelas Eksperimen Soal No.1 Gambar 2. Lembar Jawaban Siswa Kelas Eksperimen Soal No.1 Gambar 3. Lembar Jawaban Siswa Kelas Kontrol Soal No.1 Gambar 3. Lembar Jawaban Siswa Kelas Kontrol Soal No.1 Juring (Journal for Research in Mathematics Learning), Vol. 2, No. 3, 1 September 2019, 273– 284 \| 279 Asmawati, Risnawati, dan Ramon Muhandaz Soal Kemampuan Koneksi Matematis Nomor Dua Pada butir soal koneksi matematis mengandung indikator yaitu mengenali dan menggunakan hubungan-hubungan antara gagasan/ide matematis. Hubungan/koneksi yang ditunjukkan pada soal nomor dua yaitu hubungan antara materi jajargenjang dengan materi perbandingan dan aljabar. Tingkat keberhasilan siswa dikelas eksperimen untuk soal nomor dua sebesar sedangkan tingkat keberhasilan siswa dikelas kontrol adalah . Berikut hasil lembar jawaban siswa pada soal nomor 2. Gambar 4. Lembar Jawaban Siswa Kelas Eksperimen Soal No.2 Gambar 4. Lembar Jawaban Siswa Kelas Eksperimen Soal No.2 Gambar 5. Lembar Jawaban Siswa Kelas Kontrol Soal No.2 Gambar 5. Lembar Jawaban Siswa Kelas Kontrol Soal No.2 Soal Kemampuan Koneksi Matematis Nomor Empat Pada butir soal koneksi matematis mengandung indikator yang ketiga yaitu mengenali dan menerapkan matematika dalam kehidupan sehari-hari. Tingkat keberhasilan siswa dikelas eksperimen untuk soal nomor empat sebesar . Jika dibandingkan dengan tingkat keberhasilan di kelas kontrol, keberhasilan siswa dikelas eksperimen lebih unggul karena tingkat keberhasilan di kelas kontrol adalah . Berikut hasil lembar jawaban: Gambar 8. Lembar Jawaban Siswa Kelas Eksperiemen Soal No.4 Gambar 8. Lembar Jawaban Siswa Kelas Eksperiemen Soal No.4 Gambar 9. Lembar Jawaban Siswa Kelas Kontrol Soal No.4 Gambar 9. Lembar Jawaban Siswa Kelas Kontrol Soal No.4 Gambar 9. Lembar Jawaban Siswa Kelas Kontrol Soal No.4 Juring (Journal for Research in Mathematics Learning), Vol. 2, No. 3, 1 September 2019, 273– 284 Soal Kemampuan Koneksi Matematis Nomor Tiga Pada butir soal koneksi matematis mengandung indikator yaitu mengenali dan menggunakan hubungan-hubungan antara matematika dengan bidang studi lain. Hubungan/koneksi yang ditunjukkan pada soal nomor tiga yaitu hubungan antara materi belah ketupat dan teorema pythagoras dengan pelajaran fisika yaitu tentang jarak, kecepatan, dan waktu. Tingkat keberhasilan siswa dikelas eksperimen untuk soal nomor tiga sebesar . Hal ini tentu menjadi sebuah catatan bagi peneliti dikarenakan soal ketiga keberhasilan siswa dikelas eksperimen tidak terlalu tinggi. Pada soal ini kebanyakan siswa kurang mampu mengaitkan materi segiempat dengan materi jarak, kecepatan dan waktu. Namun jika dibandingkan dengan tingkat keberhasilan di kelas kontrol, keberhasilan dikelas eksperimen lebih unggul karena tingkat keberhasilan di kelas kontrol adalah . Berikut hasil lembar jawaban siswa pada soal nomor 3. Gambar 6. Lembar Jawaban Siswa Kelas Eksperimen Soal No.3 Gambar 6. Lembar Jawaban Siswa Kelas Eksperimen Soal No.3 280 \| Juring (Journal for Research in Mathematics Learning), Vol. 2, No. 3, 1 September 2019, 273– 284 280 \| Juring (Journal for Research in Mathematics Learning), Vol. 2, No. 3, 1 September 2019, 273– 284 280 Pengaruh Penerapan Strategi Pembelajaran Metakognitif Terhadap Kemampuan Koneksi Matematis Berdasarkan Kemandirian Belajar Siswa SMP/MTs Pengaruh Penerapan Strategi Pembelajaran Metakognitif Terhadap Kemampuan Koneksi Matematis Berdasarkan Kemandirian Belajar Siswa SMP/MTs Pengaruh Penerapan Strategi Pembelajaran Metakognitif Terhadap Kemampuan Koneksi Matematis Berdasarkan Kemandirian Belajar Siswa SMP/MTs Gambar 7. Lembar Jawaban Siswa Kelas Kontrol Soal No.3 Gambar 7. Lembar Jawaban Siswa Kelas Kontrol Soal No.3 Juring (Journal for Research in Mathematics Learning), Vol. 2, No. 3, 1 September 2019, 273– 284 Soal Kemampuan Koneksi Matematis Nomor Lima Pada butir soal koneksi matematis mengandung indikator yang ketiga yaitu mengenali dan menerapkan matematika dalam kehidupan sehari-hari. Tingkat keberhasilan siswa dikelas eksperimen untuk soal nomor lima sebesar sedangkan tingkat keberhasilan di kelas kontrol adalah . Berikut hasil lembar jawaban siswa pada soal nomor 5. \| 281 Juring (Journal for Research in Mathematics Learning), Vol. 2, No. 3, 1 September 2019, 273– 284 Asmawati, Risnawati, dan Ramon Muhandaz Gambar 10. Lembar Jawaban Siswa Kelas Eksperimen Soal No.5 Gambar 10. Lembar Jawaban Siswa Kelas Eksperimen Soal No.5 Gambar 11. Lembar Jawaban Siswa Kelas Kontrol Soal No.5 Gambar 11. Lembar Jawaban Siswa Kelas Kontrol Soal No.5 Untuk hipotesis 2 dan 3, hasil analisis data dengan anova dua arah diperoleh hasil sebagai berikut: Tabel 7. Hasil Uji Anova Dua Arah Dk JK RK Fh Antar baris (Model) A 1 Antar kolom (Kemandirian Belajar) B 2 Interaksi Kemandirian Belajar *Model (A×B) 2 Error 64 Total 69 Tabel 7. Hasil Uji Anova Dua Arah Berdasarkan Tabel 7 diketahui bahwa dan dan dan maka, atau , sehingga diterima dan ditolak. Kesimpulannya adalah pada taraf signifikan 5% terdapat perbedaan kemampuan koneksi matematis antara siswa yang memiliki kemandirian belajar tinggi, sedang, dan rendah. Hasil penelitian ini sejalan dengan penelitian yang dilakukan oleh Sumarni (2016:96) bahwa terdapat pengaruh kemandirian belajar terhadap koneksi matematis. Siswa yang memiliki kemandirian belajar yang baik, pencapaian kemampuan koneksi matematisnya juga baik dan sebaliknya siswa yang dengan kemandirian belajar yang kurang, pencapaian kemampuan koneksi matematisnya juga kurang baik. Kemudian atau , sehingga diterima dan ditolak. Kesimpulannya adalah pada taraf signifikan 5% tidak terdapat interaksi antara strategi pembelajaran dengan kemandirian belajar dalam mempengaruhi kemampuan koneksi matematis siswa. Ini berarti antara antara strategi pembelajaran metakognitif dan kemandirian belajar tidak 82 \| Juring (Journal for Research in Mathematics Learning), Vol. 2, No. 3, 1 September 2019, 273– 284 282 Pengaruh Penerapan Strategi Pembelajaran Metakognitif Terhadap Kemampuan Koneksi Matematis Berdasarkan Kemandirian Belajar Siswa SMP/MTs Pengaruh Penerapan Strategi Pembelajaran Metakognitif Terhadap Kemampuan Koneksi Matematis Berdasarkan Kemandirian Belajar Siswa SMP/MTs Pengaruh Penerapan Strategi Pembelajaran Metakognitif Terhadap Kemampuan Koneksi Matematis Berdasarkan Kemandirian Belajar Siswa SMP/MTs memberikan pengaruh bersama yang signifikan terhadap kemampuan koneksi matematis siswa. Hasil penelitian ini juga relevan dengan penelitian Muhammad Gazali (2015:76) yang menunjukkan bahwa model pembelajaran dan kemandirian belajar tidak berinteraksi secara signifikan dalam prestasi belajar siswa. . Berdasarkan hasil penelitian yang telah dilakukan penulis, disarankan kepada guru bidang studi matematika yang ingin menerapkan strategi pembelajaran metakognitif agar mempersiapkan banyak pertanyaan yang dapat mengarahkan siswa pada kesadaran proses kognitif yang sedang dilakukannya. Penelitian ini masih terbatas pada kemampuan koneksi matematis siswa, diharapkan pada rekan penulis untuk dapat melakukan penelitian lanjutan dalam ruang lingkup yang lebih. Soal Kemampuan Koneksi Matematis Nomor Lima Artinya, tidak adanya interaksi tersebut karena tidak dominannya pengaruh strategi pembelajaran dari kemandirian belajar terhadap kemampuan koneksi matematis, atau pengaruh kemandirian belajar tidak lebih dominan dari strategi pembelajaran terhadap kemampuan koneksi matematis, sehingga melemahkan interaksi yang ada. KESIMPULAN Berdasarkan analisis data dan pengujian hipotesis yang telah dilakukan, maka dapat disimpulkan bahwa: 1) Terdapat perbedaan kemampuan koneksi matematis antara siswa yang belajar menggunakan strategi pembelajaran metakognitif dengan siswa yang belajar tanpa menggunakan strategi pembelajaran metakognitif dengan hasil analisis data menggunakan uji-t menunjukkan nilai dan nilai rata-rata kelas eksperimen adalah dan nilai rata-rata kelas kontrol adalah yang menunjukkan kemampuan koneksi matematis siswa di kelas eksperimen lebih baik dari pada kelas control. 2) Terdapat perbedaan kemampuan koneksi matematis berdasarkan kemandirian belajar tinggi, sedang dan rendah dengan hasil analisis data menggunakan anova dua arah yang menunjukkan . Dan 3)Tidak terdapat interaksi antara strategi pembelajaran dengan kemandirian belajar dalam mempengaruhi kemampuan koneksi matematis siswa dengan hasil analisis data menggunakan anova dua arah yaitu . REFERENSI Amir, Z. (2014). Strategi Metakognitif: Suatu Kajian Penerapannya Dalam Pembelajaran Matematika. Prosiding Seminar Nasional Pendidikan Matematika. Bandung: Universitas Islam Nusantara. 185 Anandita, G. P. (2015). Analisis Kemampuan Koneksi Matematis Siswa SMP kelas VIII Pada Materi Kubus dan Balok. (Thesis, Universitas Negeri Semarang, 2015). Diakses dari https://lib.unnes.ac.id/21529/1/4101411075-S.pdf. Azma, D. N. (2017). Peningkatan Kemampuan Koneksi Matematis dan Self Efficacy Siswa SMP Melalui Pembelajaran Strategi Metakognisi. (Thesis, Universitas Syiah Kuala Darussalam, 2017). Diakses dari http://etd.unsyiah.ac.id/baca/index.php?id=35533&page=1.102 Fauzi, M. A. (2011). Peningkatan Kemampuan Koneksi Matematis dan Kemandirian Belajar Siswa dengan Pendekatan Pembelajaran Metakognitif Di Sekolah Menengah Pertama. Prosiding Seminar Internasional dan Konferensi Nasional Keempat Tentang Pendidikan Matematika. Yogyakarta: Universitas Yogyakarta. 113 ______. (2013). “Kemampuan Koneksi Matematis Siswa dengan Pendekatan Pembelajaran Metakognitif Di Sekolah Menengah Pertama”. Jurnal Pendidikan Matematika PARADIKMA. Vol. 6. No. 1. 50 Fauzi, M. A. (2011). Peningkatan Kemampuan Koneksi Matematis dan Kemandirian Belajar Siswa dengan Pendekatan Pembelajaran Metakognitif Di Sekolah Menengah Pertama. Prosiding Seminar Internasional dan Konferensi Nasional Keempat Tentang Pendidikan Matematika. Yogyakarta: Universitas Yogyakarta. 113 Prosiding Seminar Internasional dan Konferensi Nasional Keempat Tentang Pendidikan Matematika. Yogyakarta: Universitas Yogyakarta. 113 ______. (2013). “Kemampuan Koneksi Matematis Siswa dengan Pendekatan Pembelajaran Metakognitif Di Sekolah Menengah Pertama”. Jurnal Pendidikan Matematika PARADIKMA. Vol. 6. No. 1. 50 ______. (2013). “Kemampuan Koneksi Matematis Siswa dengan Pendekatan Pembelajaran Metakognitif Di Sekolah Menengah Pertama”. Jurnal Pendidikan Matematika PARADIKMA. Vol. 6. No. 1. 50 Gazali, M. (2015). “Eksperimentasi Model Pembelajaran Team Assisted Individualization Ditinjau Dari Kemandirian Belajar”. Jurnal Elemen. Vol 1. No.1. 76 Juring (Journal for Research in Mathematics Learning), Vol. 2, No. 3, 1 September 2019, 273– 284 \| 283 Asmawati, Risnawati, dan Ramon Muhandaz Hendriana, H., Rohaeti, E. E. & Sumarmo, U. (2017). Hard Skill dan Soft Skill Matematik Siswa. Bandung: PT. Refika Aditama. Hutahuruk, A.J.B. (2016). Pendekatan Metakognitif dalam Pembelajaran Matematika. Prosiding Seminar Nasional Matematika dan Pendidikan Matematika. Cirebon: Universitas Swadaya Gunung Jati Cirebon. 180 Lestari, K.A. & Yudhanegara, M. R. (2017). Penelitian Pendidikan Matematika. Bandung: PT. Refika Aditama. Menteri Pendidikan dan Kebudayaan Republik Indonesia. (2014). Peraturan Menteri Pendidikan dan Kebudayaan Republik Indonesia No. 58 Tahun 2014. Tentang Kurikulum 2013 SMP/ MTs. Jakarta: Menteri Pendidikan dan Kebudayaan Republik Indonesia. Muhandaz, R. (2015). “Pengaruh Model Pembelajaran Kooperatif Tipe Investigasi Kelompok Terhadap Pemecahan Masalah Matematis Siswa Kelas VIII MTsN Kota Padang”. Suska Jurnal of Mathematics Education. Vol. 1 No. 1. 39 PISA (Programme of International Study Assesment). (2016). Result: excellent and Equity in Education Volume 1. Paris: OECD Publishing. Juring (Journal for Research in Mathematics Learning), Vol. 2, No. 3, 1 September 2019, 273– 284 REFERENSI Risnawati, Mardianita, W. & Hernety. “Pengembangan Lks Pemecahan Masalah Kaidah Pencacahan Dengan Pendekatan Metakognitif Untuk Sma Kelas Xi”. JPPM. Vol. 9. No. 1. 139 Septiani Putri L, Ramon M, & Risnawati. (2019). “Pengaruh Penerapan Strategi Metakognitif terhadap Kemampuan Komunikasi Matematis Berdasarkan Kemandirian Belajar Siswa Sekolah Menengah Pertama Pekanbaru”. Journal For Research in Matheatics Learning. Vol. 2 No.2 171. Sumarni. (2016). “Tinjauan Korelasi Antara Kemampuan Koneksi Matematis Dan Self-Regulated Learning Matematika Siswa Yang Pembelajarannya Melalui Learning Cycle 5E”. Jurnal JES-MAT. Vol. 2. No. 1. 96 Suprapto, E. (2015). “Pengaruh Model Pembelajaran Kontekstual, Pembelajaran Langsung dan Motivasi Berprestasi Terhadap Hasil Belajar Kognitif”. INVOTEC. Vol. XI. No. 1. 37. Undang-undang Republik Indonesia Nomor 20 Tahun 2003. (2018). Tentang Sistem Pendidikan Nasional di kelembagaan.ristekdikti.go.id/wp- content/uploads/2016/08/UU_no_20_th_2003.pdf. Diakses pada 2 september 2018. g g p ontent/uploads/2016/08/UU_no_20_th_2003.pdf. Diakses pada 2 september 2018. Zakaria, E., Nordin, N.M., & Ahmad, S. (2007). Trend Pengajaran dan Pembelajaran Matematik. Kuala Lumpur: Prin-Ad Sdn. Bhd. 284 \| Juring (Journal for Research in Mathematics Learning), Vol. 2, No. 3, 1 September 2019, 273– 284
https://openalex.org/W3217431468	https://hal.inrae.fr/hal-03481132/file/Abdollahi-GSE-2021.pdf	English	null	Dissecting genetic trends to understand breeding practices in livestock: a maternal pig line example	Genetics selection evolution	2,021	cc-by	7,993	To cite this version: Rostam Abdollahi-Arpanahi, Daniela Lourenco, Andres Legarra, Ignacy Misztal. Dissecting genetic trends to understand breeding practices in livestock: a maternal pig line example. Genetics Selection Evolution, 2021, 53 (1), 10 p. 10.1186/s12711-021-00683-6. hal-03481132 Dissecting genetic trends to understand breeding practices in livestock: a maternal pig line example Rostam Abdollahi-Arpanahi, Daniela Lourenco, Andres Legarra, Ignacy Misztal Distributed under a Creative Commons Attribution 4.0 International License © The Author(s) 2021. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativeco mmons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. HAL Id: hal-03481132 https://hal.inrae.fr/hal-03481132v1 Submitted on 23 Feb 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 Abdollahi‑Arpanahi et al. Genetics Selection Evolution (2021) 53:89 https://doi.org/10.1186/s12711-021-00683-6 Ge n e t i c s Selection Evolution Ge n e t i c s Selection Evolution Open Access Abstract Background: Understanding whether genomic selection has been effective in livestock and when the results of genomic selection became visible are essential questions which we have addressed in this paper. Three criteria were used to identify practices of breeding programs over time: (1) the point of divergence of estimated genetic trends based on pedigree-based best linear unbiased prediction (BLUP) versus single-step genomic BLUP (ssGBLUP), (2) the point of divergence of realized Mendelian sampling (RMS) trends based on BLUP and ssGBLUP, and (3) the partition of genetic trends into that contributed by genotyped and non-genotyped individuals and by males and females. Methods: We used data on 282,035 animals from a commercial maternal line of pigs, of which 32,856 were geno‑ typed for 36,612 single nucleotide polymorphisms (SNPs) after quality control. Phenotypic data included 228,427, 101,225, and 11,444 records for birth weight, average daily gain in the nursery, and feed intake, respectively. Breeding values were predicted in a multiple-trait framework using BLUP and ssGBLUP. Results: The points of divergence of the genetic and RMS trends estimated by BLUP and ssGBLUP indicated that genomic selection effectively started in 2019. Partitioning the overall genetic trends into that for genotyped and non-genotyped individuals revealed that the contribution of genotyped animals to the overall genetic trend increased rapidly from ~ 74% in 2016 to 90% in 2019. The contribution of the female pathway to the genetic trend also increased since genomic selec‑ tion was implemented in this pig population, which reflects the changes in the genotyping strategy in recent years. Conclusions: Our results show that an assessment of breeding program practices can be done based on the point of divergence of genetic and RMS trends between BLUP and ssGBLUP and based on the partitioning of the genetic trend into contributions from different selection pathways. However, it should be noted that genetic trends can diverge before the onset of genomic selection if superior animals are genotyped retroactively. For the pig population example, the results showed that genomic selection was effective in this population. Dissecting genetic trends to understand breeding practices in livestock: a maternal pig line example Rostam Abdollahi‑Arpanahi1* , Daniela Lourenco1, Andres Legarra2 and Ignacy Misztal1 Background breeding industries, such as for pigs, broilers, beef, etc. have also heavily invested in this technology [3]. Hence, understanding whether implementation of genomic selection has been effective in these species and when the results of genomic selection became visible, are essential questions which need to be addressed. Genomic selection has revolutionized the livestock breeding industry and has doubled genetic gain in some species, such as dairy cattle [1, 2]. Other livestock Correspondence: rostam.abdollahi@uga.edu 1 Department of Animal and Dairy Science, University of Georgia, Athens, GA 30602, USA Full list of author information is available at the end of the article The genetic progress of a population is a combination of the progress in different selection pathways [4], which are under different selection pressures and that have GA 30602, USA ull list of author information is available at the end of the article Correspondence: rostam.abdollahi@uga.edu 1 Department of Animal and Dairy Science, University of Georgia, Athens, GA 30602, USA Full list of author information is available at the end of the article © The Author(s) 2021. 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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. Abdollahi‑Arpanahi et al. Genetics Selection Evolution (2021) 53:89 Page 2 of 10 Abdollahi‑Arpanahi et al. Genetics Selection Evolution different accuracies of selection. The impact of the imple- mentation of genomic selection on different selection pathways can be measured by the decomposition of the overall genetic trend into that contributed by each path- way over the course of selection [5]. from 0 would show selective genotyping, and a divergent trend in average estimates of RMS between ssGBLUP and BLUP would display the starting date of genomic selection. García-Cortés et al. [14] introduced a procedure for partitioning the genetic gain into contributions from par- ent averages and RMS and for allocating these contribu- tions into pre-defined “paths” (e.g., by country, gender, line, etc.), summarizing path-specific terms to quan- tify the contributions of different sources to the overall genetic trend. This procedure has been used to quan- tify the contribution of different countries to the overall genetic trend in Brown Swiss bulls [15] and to explore the impact of national selection and importation in Landrace and Large-White pigs in Croatia [16]. When a population is under genomic selection, genetic evaluation based on the conventional pedigree-based method i.e., pedigree-based best linear unbiased pre- diction (PBLUP), is biased because it neither considers genomic information nor takes genomic preselection into account [6–8]. Genomic preselection occurs when only animals that are selected based on genomic esti- mated breeding values (GEBV), and therefore on posi- tive Mendelian sampling terms, are phenotyped [9, 10]. In this case, the breeding values predicted by PBLUP for these animals are, on average, underestimated [11], while single-step genomic BLUP (ssGBLUP) is expected to pro- vide unbiased genetic trends [12, 13] because it combines all the information that has been used for selection deci- sions (pedigree, genomic data, and phenotypic records). Therefore, the point when estimated genetic trends based on PBLUP and ssGBLUP diverge indicates the starting date of effective genomic selection [13]. This is the time- point when young animals are selected based on estimates of their Mendelian sampling terms based on genomics, in addition to parent average and, perhaps, own information. If all genotyped individuals are phenotyped, then the dif- ference between genetic trends by PBLUP and ssGBLUP depends on the corresponding prediction accuracy. In principle, GEBV are more accurate than EBV and result in better genetic progress. However, if multiple-trait selec- tion is practiced, it is important that the genetic trends are estimated using a multiple-trait model. Partitioning the total genetic trend into contributions from genotyped and non-genotyped animals or into con- tributions from males and females can be used to deter- mine the impact of different selection pathways over time. For instance, if genotyped animals have a greater contribution to genetic gain than non-genotyped indi- viduals, it can indicate selective genotyping (elite animals are genotyped) or that genomic selection is effective and most of the parents are selected from the genotyped can- didates. In species such as pigs, the number of progeny is smaller per male and larger per female than in dairy cattle. Therefore, the impact of female paths on genetic progress is potentially higher than that of male paths [17] and it is worth studying this in different species. f In general, PBLUP and ssGBLUP are expected to esti- mate similar genetic trends before the starting date of genomic selection. However, when the elite animals are genotyped retroactively or genotyping is done after selec- tion, PBLUP and ssGBLUP can estimate different trends. The pig population that was used here has experienced both situations for some of the traits. Thus, we used three approaches to identify and investigate changes in breed- ing practices over time, in particular the use of genomic selection, namely: (1) based on differences in genetic trends estimated using PBLUP versus ssGBLUP, (2) based on differences in trends in RMS estimated using PBLUP versus ssGBLUP, and (3) based on partitioning the esti- mated genetic trends into different selection pathways as in [14]. These approaches were applied to a real dataset from a purebred maternal pig line. The average genetic merit of an individual can be decomposed as the sum of the parent average and a Mendelian sampling term that represents the devia- tion of an animal’s breeding value from the average of its parents. Thus, the superiority of the selected candidates over the mean of their parents represents the extra gain compared to the previous generation, by capturing the Mendelian sampling terms. Realized Mendelian sam- pling (RMS) terms have zero expectation when all ani- mals are genotyped or when genotyping is at random. When animals (e.g., young boars that are kept till they reach sexual maturity and reproduce) are selected based on estimated parent average, their RMS is also 0. How- ever, the average RMS will not be 0 when the genotyped animals are selected based on own phenotype or prog- eny performance, i.e. with selective genotyping. Moreo- ver, with selective genotyping, the average RMS will be greater with ssGBLUP than with BLUP because the lat- ter does not account for genomic information and prese- lection. Consequently, a deviation of the average RMS Statistical analysis Analysis of the data was based on a multi-trait mixed linear model of the three traits considered, with the sta- tistical model for each trait described in the following and with fixed effects denoted in uppercase and random effects in lowercase letters. j qf In matrix notation, the general model for each trait can be written as: f For BW, the model was: (1) yijokqn = Si + Pj + YHMo + b(tnbk) + lq + an + mn + eijkoqn, where yt is the vector of observations for trait t ; t refers to BW, ADG, and FEED; bt is the vector of fixed effects; lt , pet , at , and mt are the vectors of random effects for litter, pen, direct additive genetic, and maternal genetic effects, respectively; et is the vector of residuals; and X , W1 , W2 , W3 , and W4 are design matrices for the effects in lt , pet , at , and mt , respectively. (4) yt = Xbt + W1lt + W2pet + W3at + W4mt + et, where yt is the vector of observations for trait t ; t refers to BW, ADG, and FEED; bt is the vector of fixed effects; lt , pet , at , and mt are the vectors of random effects for litter, pen, direct additive genetic, and maternal genetic effects, respectively; et is the vector of residuals; and X , W1 , W2 , W3 , and W4 are design matrices for the effects in lt , pet , at , and mt , respectively. (4) yt = Xbt + W1lt + W2pet + W3at + W4mt + et, where yijokqn denotes the BW record of animal n , Si is the effect of the i-th sex ( i = 1 or 2), Pj is the effect of the j-th parity ( j = 1, …,9), YHMo is the effect of the o-th herd- year-month ( o = 1,…, 173), b is the linear regression of BW on total number of piglets born ( tnbk ), lq is the ran- dom litter effect of sow q ( q = 1,…, 18,394), an is the ran- dom direct genetic effect of animal n ( n = 1, …, 282,035), mn is the maternal genetic effect associated with dam of animal n , and eijkoqn is the residual effect for the BW record. Data structureh The phenotypes of the Landrace pigs used in this study were collected from 2012 to 2021 and included 228,427 records for birth weight (BW), 101,225 records for aver- age daily gain from birth to the end of the nursery period (ADG) at 11 weeks of age, and 11,444 records for average Page 3 of 10 Abdollahi‑Arpanahi et al. Genetics Selection Evolution (2021) 53:89 Abdollahi‑Arpanahi et al. Genetics Selection Evolution (2021) 53:89 Page 3 of 10 For ADG, the model was: daily feed intake during the finishing period (FEED) at 23 weeks of age. FEED was measured on males only. Descriptive statistics for each trait are in Table 1. Pedi- gree information was available for 282,035 animals, of which 32,856 were genotyped for 36,612 single nucleotide polymorphisms (SNPs) after quality control. The animals in the pedigree were the progeny of 809 sires and 14,674 dams. The number of pigs that had either both or one par- ent unknown was 2011 and 835, respectively. The ratio of genotyped pigs to all pigs born in each year ranged from 4% in 2015 to 18% in 2019. Of the genotyped pigs, 92% had own records and 32% of those had offspring. (2) yijqn = Si + YHW j + lq + an + eijqn, (2) where yijqn denotes the ADG record of animal n , Si , is the effect of the i-th sex ( i = 1 or 2), YHW j is the effect of the j-th year-herd-week ( j = 1,…, 1059), lq is the random litter effect of sow q ( q = 1,…, 18,394), an is the random direct genetic effect of animal n ( n = 1, …, 282,035), and eijqn is the residual effect for the ADG record. Data structureh jqf For FEED, the model was: (3) yikqon = Bi + b Agek + lq + po + an + eikqon, (3) where yikqon denotes the FEED record of animal n , Bi is the fixed effect of barn i ( i = 1,…, 512), b is the lin- ear regression of FEED on age of weighing ( Agek ), lq is the random litter effect of sow q ( q = 1,…, 18,394), po is the random pen effect ( o = 1,…, 4197), an is the random direct genetic effect of animal n ( n = 1, …, 282,035), and eijkoqn is the residual effect for the FEED record. Realized Mendelian sampling termsh where σ 2 i is the variance of the i-th random effect; σij denotes the covariance components of the i-th effect for the j-th combination of traits, and R = 3 × 3 matrix with (co)variance between traits; A is the numerator relation- ship matrix constructed based on pedigree information for PBLUP, and I is an identity matrix. For the ssGBLUP analysis, A was replaced by H , with H−1 computed as in Aguilar et al. [8]: where σ 2 i is the variance of the i-th random effect; σij denotes the covariance components of the i-th effect for the j-th combination of traits, and R = 3 × 3 matrix with (co)variance between traits; A is the numerator relation- ship matrix constructed based on pedigree information for PBLUP, and I is an identity matrix. For the ssGBLUP analysis, A was replaced by H , with H−1 computed as in Aguilar et al. [8]: The RMS for individual i for a given trait was estimated as: h as: (6) RMSi = (G)EBV i −PAi, (6) where PA is the parent average (average (G)EBV of the parents) and (G)EBV i denotes the (genomic) estimated breeding value of individual i . More theoretical details about the RMS can be found in Abdollahi-Arpanahi et al. [13]. H−1 = A−1 + 0 0 0 G−1 −A−1 22 , When animals are randomly sampled for genotyping at a young age before any source of information (not even genomics) is available, RMS is 0 on average. However, if the “best” animals based on progeny testing or own per- formance are genotyped (i.e. selective genotyping), then the RMS of the genotyped animals will be nonzero, (i.e. positive if they selected for a higher value and negative if they are selected for a lower value). Since genomic prese- lection has taken place in most of the livestock popula- tions, the divergence in RMS trends obtained based on EBV and GEBV of genotyped animals can also indicate the starting point of genomic selection. The same animals as used to estimate genetic trends were also used to esti- mate RMS trends. where G−1 is the inverse of the genomic relationship matrix and A−1 22 is the inverse of the pedigree relationship matrix for genotyped individuals. Partitioning of genetic trends Predictions of breeding values can be partitioned to quantify the contribution of genotyped versus non-geno- typed or males versus females as follows: (7) ai = 1 2as(i) + 1 2ad(i) + mi, (7) where mi is the estimate of the RMS and a is the (G)EBV; subscripts s and d refer to the sire and dam of animal i , respectively. for founder animals ai = mi . For the whole population Eq. (7) can be written as: Realized Mendelian sampling termsh The genomic relation- ship matrix ( G ) was constructed using the first method of VanRaden [18]: G = ZZ′ 2 pi(1 −pi), where Z is a matrix of genotypes coded as 0, 1, and 2 for AA, AB, and BB, respectively, and then centered by sub- tracting twice the frequency of the major allele of SNP i (pi ) ( i = 1, …, 36,612). To avoid singularity problems, G was blended with A22 as G = 0.95 G + 0.05 A22. Solutions for the multi-trait PBLUP and ssGBLUP were obtained using the preconditioned conjugate gra- dient algorithm with iteration on data, as implemented in the BLUP90IOD2 program [19]. The (co)variance components were the most recent estimates derived using PBLUP. The GEBV from ssGBLUP were set to the same base (i.e., year 2015) as the mean EBV from PBLUP. To facilitate comparisons between traits, G(EBV) were divided by the square root of the additive genetic variance. Statistical analysis where yijokqn denotes the BW record of animal n , Si is the effect of the i-th sex ( i = 1 or 2), Pj is the effect of the j-th parity ( j = 1, …,9), YHMo is the effect of the o-th herd- year-month ( o = 1,…, 173), b is the linear regression of BW on total number of piglets born ( tnbk ), lq is the ran- dom litter effect of sow q ( q = 1,…, 18,394), an is the ran- dom direct genetic effect of animal n ( n = 1, …, 282,035), mn is the maternal genetic effect associated with dam of animal n , and eijkoqn is the residual effect for the BW The assumed (co)variance structure of random effects for the multiple-trait analysis was as follows: record. (5) Var       lt pet at mt et       = Var               lBW lADG lFEED peFEED aBW aADG aFEED mBW e               =                Iσ 2 lBW IσlBW,ADG IσlBW,FEED 0 0 0 0 0 Iσ 2 lADG IσlADG,FEED 0 0 0 0 0 Iσ 2 lFEED 0 0 0 0 0 Iσ 2 peFEED 0 0 0 0 Aσ 2 aBW AσaBW,ADG AσaBW,FEED Aσ aBW,mBW Aσ 2 aADG AσaADG,FEED 0 symmetric Aσ 2 aFEED 0 Aσ 2 mBW R ⊗I                , (5) , Table 1 Descriptive statistics of the Landrace pig breed dataset BW Birth weight, ADG average daily gain through the end of the nursery, FEED feed intake Trait (unit) Number of records Mean SD h2 Minimum Maximum BW (g) 228,427 1281.6 327.8 0.05 45 3992 ADG (g) 101,255 343.5 61.5 0.22 200 600 FEED (g) 11,444 1917.8 301.9 0.26 827 3872 Abdollahi‑Arpanahi et al. Genetics Selection Evolution (2021) 53:89 Abdollahi‑Arpanahi et al. Genetics Selection Evolution (2021) 53:89 Page 4 of 10 Genetic trends Estimated genetic trends for genotyped individuals, in genetic standard deviation units, based on PBLUP and ssGBLUP are presented in Fig. 1. The genetic trends were favorable for all traits, with a faster improvement in recent years. The changes in average EBV from 2015 to 2020 for BW, ADG, and FEED were 0.66, 0.72, and 0.20, respectively based on PBLUP and 0.65, 1.03, and Divergence of genetic trendsh (8) a = T m, a = T m, (8) The point of divergence of the genetic trends obtained by ssGBLUP and PBLUP was used to identify the onset of genomic selection. Details on the theory of predicting breeding values by PBLUP and ssGBLUP, are in Abdol- lahi-Arpanahi et al. [13]. To estimate the genetic trends using PBLUP and ssGBLUP, the (G)EBV for a given trait were averaged by year of birth for animals with both phe- notypes and genotypes. The reason for using only ani- mals with genotypes and phenotypes to estimate genetic trends is that young animals without genotypes and phe- notypes (own and progeny) do not contribute informa- tion to the evaluation and their average EBV is equal to the parent average. where T is a triangular matrix that relates each animal to its parents [20]. Following Eq. (8) and considering that m = T−1a , the vector of (G)EBV for the entire popula- tion (Eq. (8)) can be partitioned into contributions of defined selection pathways [14] as: (9) a = TP1T−1a + TP2T−1a = a1 + a2, (9) where Pi is a diagonal matrix of 1s and 0s to select the corresponding columns of T and is used to allocate the RMS of males versus females or of genotyped versus non- genotyped individuals to the i-th partition of a. Page 5 of 10 Abdollahi‑Arpanahi et al. Genetics Selection Evolution (2021) 53:89 This procedure was implemented using the R package AlphaPart 0.8.1. [21], using the GEBV obtained using ssGBLUP for all animals (i.e., genotyped and non-geno- typed) as the input. The contribution of each pathway for each birth year was expressed as a percentage by dividing the average GEBV of a partition by the average GEBV of the whole population. 0.31 based on ssGBLUP. For ADG and FEED, the genetic trends estimated using PBLUP and ssGBLUP started to diverge in 2018, but for BW, which is not under direct selection, there was no evidence of divergence. In the last year of data (i.e., 2020), the difference between aver- age EBV based on ssGBLUP and PBLUP were − 0.01, 0.31, and 0.11 SD for BW, ADG, and FEED, respectively. The positive genetic trend for BW is due to its indirect response to selection for increasing the maternal effect on BW and for other correlated traits in the selection index. Mendelian sampling trendsh The RMS trends for genotyped individuals, in genetic standard deviation units, estimated using PBLUP and ssGBLUP are shown in Fig. 2. The pattern of changes in RMS trends was the same for the three traits and started BW ADG FEED 2015 2016 2017 2018 2019 2020 2015 2016 2017 2018 2019 2020 2015 2016 2017 2018 2019 2020 0.04 0.24 0.44 0.64 0.84 1.04 0.04 0.24 0.44 0.64 0.84 1.04 0.04 0.24 0.44 0.64 0.84 1.04 Birth Year Mean EBV/SD Method BLUP ssGBLUP Fig. 1 Genetic trends for birth weight (BW), average daily gain through the end of the nursery (ADG), and feed intake (FEED) for genotyped Landrace pigs. Genetic trends are presented on the additive genetic standard deviation scale and the genetic base was adjusted to the 2015 birth year Fig. 1 Genetic trends for birth weight (BW), average daily gain through the end of the nursery (ADG), and feed intake (FEED) for genotyped Landrace pigs. Genetic trends are presented on the additive genetic standard deviation scale and the genetic base was adjusted to the 2015 birth year Abdollahi‑Arpanahi et al. Genetics Selection Evolution (2021) 53:89 Page 6 of 10 BW ADG FEED 2015 2016 2017 2018 2019 2020 2015 2016 2017 2018 2019 2020 2015 2016 2017 2018 2019 2020 0.020 0.058 0.096 0.134 0.172 0.020 0.058 0.096 0.134 0.172 0.020 0.058 0.096 0.134 0.172 Birth Year Mean RMS/SD Method BLUP ssGBLUP Fig. 2 Mendelian sampling trends for birth weight (BW), average daily gain through the end of the nursery (ADG), and feed intake (FEED) for genotyped Landrace pigs. Mendelian sampling trends are presented on the additive genetic standard deviation scale. The solid black lines represent the zero-base and the dotted green vertical lines shows the start date of genomic selection Fig. 2 Mendelian sampling trends for birth weight (BW), average daily gain through the end of the nursery (ADG), and feed intake (FEED) for genotyped Landrace pigs. Mendelian sampling trends are presented on the additive genetic standard deviation scale. The solid black lines represent the zero-base and the dotted green vertical lines shows the start date of genomic selection to non-genotyped individuals before 2016, but from 2016 onwards, genotyped individuals were responsible for the genetic gain. Mendelian sampling trendsh For ADG, non-genotyped individuals had a greater contribution to the genetic trend than genotyped animals until 2016, but from 2017 onwards, the contri- bution of genotyped individuals to genetic gain increased from 74% in 2016 to 94% in 2020. For feed intake, in 2015, all the genetic gain was driven by non-genotyped pigs, but the contribution of genotyped pigs increased rapidly after that, from 76% in 2016 to 97% in 2020. to diverge in 2019. In the last year of data (i.e., 2020), the difference between average estimates of RMS based on PBLUP and ssGBLUP was 0.10, 0.05, and 0.06 SD for BW, ADG, and FEED, respectively. The positive estimated trend in RMS and the considerable difference of RMS from 0 from 2015 to 2017 are due to the genotyping in 2018 of elite culled or active boars that were born before 2018 and were retrieved from stored tissue samples. Contributions of females versus males to genetic trends The decomposition of genetic trends into Mendelian sampling contributions from genotyped and non-gen- otyped individuals is shown in Fig. 3. The percentage of individuals born from 2015 to 2020 that were genotyped ranged from 5 to 21%. All genetic gain in BW was due Contributions of females versus males to genetic trends Figure 4 shows the decomposition of genetic trends into contributions from males and females for all traits. For BW, most of the genetic gain was driven by females from 2019 onwards. For ADG, males and females had similar Abdollahi‑Arpanahi et al. Genetics Selection Evolution (2021) 53:89 Page 7 of 10 BW ADGn FEED 2015 2016 2017 2018 2019 2020 2015 2016 2017 2018 2019 2020 2015 2016 2017 2018 2019 2020 −0.26 0.24 0.74 −0.26 0.24 0.74 −0.26 0.24 0.74 Birth Year Mean EBV/SD variable overall Genotyped Nongenotyped Fig. 3 Decomposition of overall genetic trends into genotyped and non-genotyped animals for birth weight (BW), average daily gain through the end of the nursery (ADG), and average daily feed intake (FEED) for Landrace pigs. Average breeding values are presented on the additive genetic d d d i i l BW ADGn FEED 2015 2016 2017 2018 2019 2020 2015 2016 2017 2018 2019 2020 2015 2016 2017 2018 2019 2020 −0.26 0.24 0.74 −0.26 0.24 0.74 −0.26 0.24 0.74 Birth Year Mean EBV/SD variable overall Genotyped Nongenotyped Fig. 3 Decomposition of overall genetic trends into genotyped and non-genotyped animals for birth weight (BW), average daily gain through the end of the nursery (ADG), and average daily feed intake (FEED) for Landrace pigs. Average breeding values are presented on the additive genetic standard deviation scale Fig. 3 Decomposition of overall genetic trends into genotyped and non-genotyped animals for birth weight (BW), average daily gain through the end of the nursery (ADG), and average daily feed intake (FEED) for Landrace pigs. Average breeding values are presented on the additive genetic standard deviation scale versus females. Quantifying the contribution of geno- typed individuals to the genetic trends revealed the presence of selective genotyping (only elite animals were genotyped) and the impact of genomic selection (faster genetic improvement) in this breeding program. Discussion Genetic trend The effective starting point of genomic selection was found to be in 2019 in the offspring of the first animals selected based on GEBV in 2018. This finding agrees with the history of genomic selection in this pig population. Genotyping started in late 2016, for pigs born between 2015 and 2017 that were active boars or culled boars with stored tissue samples. Consequently, the higher genetic Contributions of females versus males to genetic trends Decomposition of genetic trends into contributions from males versus females allowed the monitoring of the contribution of males versus females to genetic trends after the implementation of genomic selection.hf contributions to the genetic trend up to 2017, but from 2018 onwards, the contribution of females was greater than that of males. The pattern of genetic trend for feed intake was similar to that of males, while the contribu- tion of females to the genetic trend for feed intake was in the undesirable direction. For BW and ADG, the impact of the female pathway increased after implementation of genomic selection. Genetic trends We used the divergence of genetic and RMS trends obtained by PBLUP versus ssGBLUP to determine the effectiveness of genomic selection. We also parti- tioned genetic trends into contributions from geno- typed versus non-genotyped animals and from males Abdollahi‑Arpanahi et al. Genetics Selection Evolution (2021) 53:89 Abdollahi‑Arpanahi et al. Genetics Selection Evolution Page 8 of 10 trends estimated from 2015 to 2018 for ADG using ssGBLUP than using PBLUP can be the result of selec- tive genotyping. The decline in genetic trend for ADG estimated by PBLUP after 2018 is due to genomic prese- lection bias. When only genotyped animals receive phe- notype records, PBLUP do not account for the positive RMS of those young animals. Similar results are reported in the literature [12, 13, 22]. Masuda et al. [12] com- pared genetic trends estimated using PBLUP and ssGB- LUP for milk production traits in US Holstein cattle and found that after the implementation of genomic selec- tion, the genetic trend based on PBLUP was underesti- mated because of genomic preselection. In a simulation study, Jibrila et al. [22] showed that genomic preselec- tion caused bias in estimates of genetic gain based on PBLUB, while the bias was smaller when based on ssG- BLUP. According to Abdollahi-Arpanahi et al. [13], after implementing genomic selection in pig, broiler, and beef cattle populations, the genetic trends obtained by ssG- BLUP accelerated and those estimated using PBLUP decelerated. In the pig population under study, genotyping was done retroactively, which means that for the genotyped animals born from 2015 to 2017 the selection decisions were practiced by another method such as PBLUP, thus even if ssGBLUP during this period results in higher accuracy than PBLUP, we do not expect a higher genetic trend for ssGBLUP. In fact, the accuracy of ssGBLUP will be greater than the accuracy of PBLUP at any point when a sufficient number of animals is genotyped. How- ever, the prediction accuracy at the time of selection is what is reflected in the genetic trend. Genetic trends The reason is that if the company/breeder invested in genotyping but has not used the genomic information in selection decisions, the higher accuracy of evaluation by ssGBLUP compared to PBLUP using accumulated data does not necessarily BW ADGn FEED 2015 2016 2017 2018 2019 2020 2015 2016 2017 2018 2019 2020 2015 2016 2017 2018 2019 2020 −0.26 0.24 0.74 −0.26 0.24 0.74 −0.26 0.24 0.74 Birth Year Mean EBV/SD variable overall male female Fig. 4 Decomposition of overall genetic trends into males and females for birth weight (BW), average daily gain through the end of the nursery (ADG), and feed intake (FEED) for Landrace pigs. Average breeding values are presented on the additive genetic standard deviation scale Fig. 4 Decomposition of overall genetic trends into males and females for birth weight (BW), average daily gain through the end of the nursery (ADG), and feed intake (FEED) for Landrace pigs. Average breeding values are presented on the additive genetic standard deviation scale Abdollahi‑Arpanahi et al. Genetics Selection Evolution (2021) 53:89 Abdollahi‑Arpanahi et al. Genetics Selection Evolution Page 9 of 10 translate into the genetic trend. Overall, the changes of prediction accuracies over time may not follow the genetic trends estimated by PBLUP or ssGBLUP.l greater contribution to genetic gain than non-genotyped individuals. The greater contribution of genotyped indi- viduals to genetic trends does not necessarily depict the effectiveness of genomic selection. For instance, if geno- typed animals are preselected based on PBLUP EBV, we expect genetic trends to be higher for genotyped than for non-genotyped animals, which is the case for the period from 2015 to 2018 before genomic selection started. Although investigating the fluctuations in the genetic trend across time for each trait is beyond the scope of this study, the changes in genetic trends observed are consist- ent with the breeding practices and the periodic modifi- cations of weights in the selection index and the genetic correlations between traits under selection. One example is the estimated increase of 0.6 and 0.7 SD in BW from 2015 to 2020 based on ssGBLUP and PBLUP, respec- tively. Although the direct genetic value of BW has not been selected for in this population, the maternal genetic value for BW has. The genetic correlation between direct and maternal genetic values is about 0.1. Genetic trends However, the genetic correlations of direct BW with growth rate in the nursery, finisher average daily gain, and finisher average daily feed intake are positive and high, e.g., 0.42, 0.31 and 0.25, respectively. Therefore, we believe that the observed genetic trend for BW is due to the correlated responses to selection. A greater impact of females on genetic trends would be because selection decisions in a maternal line are placed more on females than males, and in pigs, each selected female has a larger contribution because it pro- duces more progeny. However, the pig breed analyzed here is a maternal line and 40% of the traits under selec- tion are only measured in females, which results in the females being the main drivers of changes in these traits. Thus, it is expected that females contribute more to the next generation than males in a pig breeding program, although the selection intensity for males is higher than for females. We found that the contribution of females to the genetic trends for BW and ADG was greater than that of males after the implementation of genomic selection. For FEED, while a flat to slightly positive genetic trend was observed for males, the trend for females was posi- tive and unfavorable. Feed intake has a negative eco- nomic value, while ADG has a positive value in the index. Therefore, the breeding objective is to achieve a positive response in growth rate and a flat or slightly positive response in FEED, which, in turn, improves feed effi- ciency. Few studies investigated the contribution of dif- ferent selection paths to genetic trends. For example, García-Ruiz et al. [2] demonstrated that 73 to 90% of the selection differential for milk production traits in US Holstein cattle is due to the sire of the bull and sire of the cow pathways. Mendelian sampling trendsh The RMS trends revealed signatures of selective genotyp- ing for the three traits. All males born from 2017 to 2020 were genotyped, but only a subset of females selected based on phenotypes were genotyped during this period. Hence, the deviations of RMS from 0 for birth years after 2018 are due to the strong selective genotyping of females rather than of males. Moreover, inferior males, e.g., those with a small BW, may be removed from the tested popu- lation before genotyping, which can result in positive RMS. As genotyping becomes less expensive, genotyping more young animals becomes economically justified and we expect a convergence of the RMS trends estimated using PBLUP versus ssGBLUP if phenotypic records are available for all animals.h Conclusions The advantages of ssGBLUP in reducing prediction bias increase when animals have been preselected based on GEBV. Genetic evaluation using PBLUP assumes that RMS average 0, but when genotyped animals with posi- tive or negative RMS receive phenotypes or progeny, the average RMS is no longer 0 [7, 9]. In this regard, a simula- tion study showed that the RMS for bulls clearly deviated from 0 after genomic preselection was implemented in a dairy cattle population [10]. Divergence of genetic trends for genotyped animals esti- mated using PBLUP versus ssGBLUP indicates the pres- ence of genomic selection. This divergence may occur before the onset of genomic selection if superior ani- mals are genotyped retroactively. Presence of nonzero average RMS by ssGBLUP or PBLUP indicates selective genotyping. Selective genotyping can be deliberate, e.g., genotyping of animals with superior genotypes, or inci- dental due to removal of weak/sick/dead animals. Under genomic selection, trends for RMS are higher when esti- mated using ssGBLUP than using PBLUP, with the point of divergence indicating the effective onset of genomic selection. Partitioning of genetic trends into contribu- tions by various classes of animals such as genotyped versus ungenotyped or males versus females allows the Author details 1 ut o deta s 1 Department of Animal and Dairy Science, University of Georgia, Athens, GA 30602, USA. 2 UMR GenPhySE, INRA Toulouse, Castanet Tolosan, France. 1 Department of Animal and Dairy Science, University of Georgia, Athens, GA 30602, USA. 2 UMR GenPhySE, INRA Toulouse, Castanet Tolosan, France. Acknowledgements g We would like to thank the data provider and their comments on the paper. We also greatly acknowledge the very helpful comments and English editing by Jack Dekkers. 12. Masuda Y, VanRaden PM, Misztal I, Lawlor TJ. Differing genetic trend estimates from traditional and genomic evaluations of genotyped animals as evidence of preselection bias in US Holsteins. J Dairy Sci. 2018;101:5194–206.f Availability of data and materials 17. Lourenco DAL, Fragomeni BO, Tsuruta S, Aguilar I, Zumbach B, Hawken RJ, et al. Accuracy of estimated breeding values with genomic information on males, females, or both: an example on broiler chicken. Genet Sel Evol. 2015;47:56. Availability of data and materials Provider of data does not intend to disclose its identity. y Provider of data does not intend to disclose its identity. Decomposition of genetic trends To quantify the contribution of genotyped individuals to genetic trends, we partitioned the genetic trends into the genetic gain derived by genotyped individuals and that achieved by non-genotyped individuals. Regardless of the trait, in recent years, genotyped individuals had a Abdollahi‑Arpanahi et al. Genetics Selection Evolution (2021) 53:89 Page 10 of 10 Page 10 of 10 6. Party C, Ducrocq V. Bias due to genomic selection. Interbull Bull. 2009;39:77–82. determination of the relative impact of genotyping for different groups of animals. In particular, the observation that nearly all the genetic progress is contributed by gen- otyped animals confirms the increasing interest in geno- typing animals, and the observation that a large fraction of the genetic progress is contributed by females validates the importance of the females in the genetic progress of a pig population. In summary, post-processing of EBV and GEBV can help to investigate the effectiveness of genomic selection and assess breeding program practices. 7. Patry C, Ducrocq V. Evidence of biases in genetic evaluations due to genomic preselection in dairy cattle. J Dairy Sci. 2011;94:1011–20. 8. Aguilar I, Misztal I, Johnson DL, Legarra A, Tsuruta S, Lawlor TJ. Hot topic: a unified approach to utilize phenotypic, full pedigree, and genomic information for genetic evaluation of Holstein final score. J Dairy Sci. 2010;93:743–52. 9. Patry C, Ducrocq V. Accounting for genomic pre-selection in national BLUP evaluations in dairy cattle. Genet Sel Evol. 2011;43:30. 10. Tyrisevä AM, Mäntysaari EA, Jakobsen J, Aamand GP, Dürr J, Fikse WF, et al. Detection of evaluation bias caused by genomic preselection. J Dairy Sci. 2018;101:3155–63. 11. Koivula M, Stranden I, Aamand GP, Mantysaari EA. Reducing bias in the dairy cattle single-step genomic evaluation by ignoring bulls without progeny. J Anim Breed Genet. 2018;135:107–15. Competing interests Competing interests The authors declare that they have no competing interests. Competing interests The authors declare that they have no competing interests. 22. Jibrila I, ten Napel J, Vandenplas J, Veerkamp RF, Calus MPL. Investigating the impact of preselection on subsequent single-step genomic BLUP evaluation of preselected animals. Genet Sel Evol. 2020;52:42. 22. Jibrila I, ten Napel J, Vandenplas J, Veerkamp RF, Calus MPL. Investigating the impact of preselection on subsequent single-step genomic BLUP evaluation of preselected animals. Genet Sel Evol. 2020;52:42. Declarations 18. VanRaden PM. Efficient methods to compute genomic predictions. J Dairy Sci. 2008;91:4414–23. Publisher’s Note Received: 4 June 2021 Accepted: 9 November 2021 Springer Nature remains neutral with regard to jurisdictional claims in pub‑ lished maps and institutional affiliations. Springer Nature remains neutral with regard to jurisdictional claims in pub‑ lished maps and institutional affiliations. Springer Nature remains neutral with regard to jurisdictional claims in pub‑ lished maps and institutional affiliations. Authors’ contributions 13. Abdollahi-Arpanahi R, Lourenco D, Misztal I. Detecting effective date of genomic selection by divergent trends from BLUP and ssGBLUP in pigs, beef cattle, and broilers. BioRXiv. 2021;101:5166. IM, DL and RAA conceived and designed the study. RAA analyzed the data and wrote the first draft of the manuscript. IM, DL and AL provided critical insights and revised the manuscript. All authors read and approved the final manuscript. 14. García-Cortés LA, Martínez-Ávila JC, Toro MA. Partition of the genetic trend to validate multiple selection decisions. Animal. 2008;2:821–4. 15. Gorjanc G, Potočnik K, García-Cortés LA, Jakobsen J, Dürr J. Partitioning of international genetic trends by origin in Brown Swiss bulls. Interbull Bull. 2011;44:81–6. References 1. Georges M, Charlier C, Hayes B. Harnessing genomic information for livestock improvement. Nat Rev Genet. 2019;20:135–56. • f • t • r • s • g m • At B Read Read 1. Georges M, Charlier C, Hayes B. Harnessing genomic information for livestock improvement. Nat Rev Genet. 2019;20:135–56. 2. García-Ruiz A, Cole JB, VanRaden PM, Wiggans GR, Ruiz-López FJ, Van Tas‑ sell CP. Changes in genetic selection differentials and generation intervals in US Holstein dairy cattle as a result of genomic selection. Proc Natl Acad Sci USA. 2016;113:E3995-4004. 3. Misztal I, Lourenco D, Legarra A. Current status of genomic evaluation. J Anim Sci. 2020. https://doi.org/10.1093/jas/skaa101. 4. Rendel JM, Robertson A. Estimation of genetic gain in milk yield by selec‑ tion in a closed herd of dairy cattle. J Genet. 1950;50:1–8. 5. Woolliams JA, Bijma P, Villanueva B. Expected genetic contributions and their impact on gene flow and genetic gain. Genetics. 1999;153:1009–20. livestock improvement. Nat Rev Genet. 2019;20:135–56. • fast, convenient online submission • thorough peer review by experienced researchers in your ﬁeld • rapid publication on acceptance • support for research data, including large and complex data types • gold Open Access which fosters wider collaboration and increased citations maximum visibility for your research: over 100M website views per year • At BMC, research is always in progress. Learn more biomedcentral.com/submissions Ready to submit your research Ready to submit your research ? Choose BMC and benefit from: ? Choose BMC and benefit from: • fast, convenient online submission • thorough peer review by experienced researchers in your ﬁeld • rapid publication on acceptance • support for research data, including large and complex data types • gold Open Access which fosters wider collaboration and increased citations maximum visibility for your research: over 100M website views per year • At BMC, research is always in progress. Learn more biomedcentral.com/submissions Ready to submit your research Ready to submit your research ? Choose BMC and benefit from: ? Choose BMC and benefit from: • 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. Consent for publication Not applicable. Consent for publication Not applicable. 20. Quaas RL. Computing the diagonal elements and inverse of a large numerator relationship matrix. Biometrics. 1976;32:949–53. 21. Obšteter J, Holl J, Hickey JM, Gorjanc G. AlphaPart-R implementation of the method for partitioning genetic trends. Genet Sel Evol. 2021;53:30. 21. Obšteter J, Holl J, Hickey JM, Gorjanc G. AlphaPart-R implementation of the method for partitioning genetic trends. Genet Sel Evol. 2021;53:30. Funding g This study was supported by Agriculture and Food Research Initiative Com‑ petitive Grant no. 2020–67015-31030 from the US Department of Agriculture’s National Institute of Food and Agriculture. 16. Škorput D, Gorjanc G, Kasap A, Luković Z. Partition of genetic trends by origin in Landrace and Large-White pigs. Animal. 2015;9:1605–9. Ethics approval and consent to participate Not applicable. 19. Misztal I, Tsuruta S, Lourenco DAL, Masuda Y, Aguilar I, Legarra A, et al. Manual for BLUPF90 family of programs. Athens: University of Georgia; 2014. References Learn more biomedcentral.com/submissions Ready to submit your research Ready to submit your research ? Choose BMC and benefit from: ? Choose BMC and benefit from: 2. García-Ruiz A, Cole JB, VanRaden PM, Wiggans GR, Ruiz-López FJ, Van Tas‑ sell CP. Changes in genetic selection differentials and generation intervals in US Holstein dairy cattle as a result of genomic selection. Proc Natl Acad Sci USA. 2016;113:E3995-4004. 3. Misztal I, Lourenco D, Legarra A. Current status of genomic evaluation. J Anim Sci. 2020. https://doi.org/10.1093/jas/skaa101. 4. Rendel JM, Robertson A. Estimation of genetic gain in milk yield by selec‑ tion in a closed herd of dairy cattle. J Genet. 1950;50:1–8. 5. Woolliams JA, Bijma P, Villanueva B. Expected genetic contributions and their impact on gene flow and genetic gain. Genetics. 1999;153:1009–20.
https://openalex.org/W3177841865	https://ccforum.biomedcentral.com/counter/pdf/10.1186/s13054-021-03658-7	English	null	Diagnostic yield of routine daily blood culture in patients on veno-arterial extracorporeal membrane oxygenation	Critical care	2,021	cc-by	9,264	Abstract Without these three conditions, only two BSIs diagnosed with routine BCs would have been missed by on-demand BCs sampling. Results: On the 150 V-A ECMO included, 2146 BCs were performed (1162 routine and 984 on-demand BCs); 190 (9%) were positive, including 68 contaminants. Fifty-one (4%) routine BCs revealed BSIs; meanwhile, 71 (7%) on-demand BCs revealed BSIs (p = 0.005). Performing routine BCs was negatively associated with BSIs diagnosis (OR 0.55, 95% CI [0.38; 0.81], p = 0.002). However, 16 (31%) BSIs diagnosed by routine BCs would have been missed by on-demand BCs. Independent variables for BSIs diagnosis after routine BCs were: V-A ECMO for cardiac graft failure (OR 2.43, 95% CI [1.20; 4.92], p = 0.013) and sampling with on-going antimicrobial therapy (OR 2.15, 95% CI [1.08; 4.27], p = 0.029) or renal replacement therapy (OR 2.05, 95% CI [1.10; 3.81], p = 0.008). Without these three conditions, only two BSIs diagnosed with routine BCs would have been missed by on-demand BCs sampling. Conclusions: Although routine daily BCs are less effective than on-demand BCs and expose to contamination and inappropriate antimicrobial therapy, a policy restricted to on-demand BCs would omit a significant proportion of BSIs. This argues for a tailored approach to routine daily BCs on V-A ECMO, based on risk factors for positivity. Keywords: Extracorporeal membrane oxygenation, Cardiogenic shock, Blood culture, Bloodstream infection, Contamination © The Author(s) 2021. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativeco mmons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Introduction Veno-arterial extracorporeal membrane oxygenation (V-A ECMO) is increasingly used to support various causes of refractory shock [1]. Although it is life-saving support, it generates new issues and side effects. Emer- gent management, invasive procedures and devices, and intensive care unit (ICU)-acquired immunodepres- sion increase infectious risk. Indeed, half of patients will Correspondence: nicolas.mongardon@aphp.fr 1 Service d’Anesthésie‑Réanimations Chirurgicales, DMU CARE, DHU A‑TVB, Assistance Publique‑Hôpitaux de Paris (AP‑HP), Hôpitaux Universitaires Henri Mondor, 1 rue Gustave Eiffel, 94000 Créteil, France Full list of author information is available at the end of the article Diagnostic yield of routine daily blood culture in patients on veno‑arterial extracorporeal membrane oxygenation Quentin de Roux1,7,8 , Marie Renaudier1, Wulfran Bougouin2, Johanna Boccara1, Vincent Fihman3,7, Raphaël Lepeule4, Chamsedine Cherait1, Antonio Fiore5, François Hemery6, Jean‑Winoc Decousser3,7, Olivier Langeron1,7 and Nicolas Mongardon1,7,8 Abstract Background: Bloodstream infections (BSIs) are frequent on veno-arterial extracorporeal membrane oxygenation (V-A ECMO). Performing routine blood cultures (BCs) may identify early paucisymptomatic BSIs. We investigated the contribution of systematic daily BCs to detect BSIs on V-A ECMO. Methods: This was a retrospective study including all adult patients requiring V-A ECMO and surviving more than 24 h. Our protocol included routine daily BCs, from V-A ECMO insertion up to 5 days after withdrawal; other BCs were performed on-demand. Methods: This was a retrospective study including all adult patients requiring V-A ECMO and surviving more than 24 h. Our protocol included routine daily BCs, from V-A ECMO insertion up to 5 days after withdrawal; other BCs were performed on-demand. Results: On the 150 V-A ECMO included, 2146 BCs were performed (1162 routine and 984 on-demand BCs); 190 (9%) were positive, including 68 contaminants. Fifty-one (4%) routine BCs revealed BSIs; meanwhile, 71 (7%) on-demand BCs revealed BSIs (p = 0.005). Performing routine BCs was negatively associated with BSIs diagnosis (OR 0.55, 95% CI [0.38; 0.81], p = 0.002). However, 16 (31%) BSIs diagnosed by routine BCs would have been missed by on-demand BCs. Independent variables for BSIs diagnosis after routine BCs were: V-A ECMO for cardiac graft failure (OR 2.43, 95% CI [1.20; 4.92], p = 0.013) and sampling with on-going antimicrobial therapy (OR 2.15, 95% CI [1.08; 4.27], p = 0.029) or renal replacement therapy (OR 2.05, 95% CI [1.10; 3.81], p = 0.008). Without these three conditions, only two BSIs diagnosed with routine BCs would have been missed by on-demand BCs sampling. Results: On the 150 V-A ECMO included, 2146 BCs were performed (1162 routine and 984 on-demand BCs); 190 (9%) were positive, including 68 contaminants. Fifty-one (4%) routine BCs revealed BSIs; meanwhile, 71 (7%) on-demand BCs revealed BSIs (p = 0.005). Performing routine BCs was negatively associated with BSIs diagnosis (OR 0.55, 95% CI [0.38; 0.81], p = 0.002). However, 16 (31%) BSIs diagnosed by routine BCs would have been missed by on-demand BCs. Independent variables for BSIs diagnosis after routine BCs were: V-A ECMO for cardiac graft failure (OR 2.43, 95% CI [1.20; 4.92], p = 0.013) and sampling with on-going antimicrobial therapy (OR 2.15, 95% CI [1.08; 4.27], p = 0.029) or renal replacement therapy (OR 2.05, 95% CI [1.10; 3.81], p = 0.008). Correspondence: nicolas.mongardon@aphp.fr 1 Service d’Anesthésie‑Réanimations Chirurgicales, DMU CARE, DHU A‑TVB, Assistance Publique‑Hôpitaux de Paris (AP‑HP), Hôpitaux Universitaires Henri Mondor, 1 rue Gustave Eiffel, 94000 Créteil, France Full list of author information is available at the end of the article de Roux et al. Crit Care (2021) 25:241 https://doi.org/10.1186/s13054-021-03658-7 de Roux et al. Crit Care (2021) 25:241 https://doi.org/10.1186/s13054-021-03658-7 RESEARCH Diagnostic yield of routine daily blood culture in patients on veno‑arterial extracorporeal membrane oxygenation Quentin de Roux1,7,8 , Marie Renaudier1, Wulfran Bougouin2, Johanna Boccara1, Vincent Fihman3,7, Raphaël Lepeule4, Chamsedine Cherait1, Antonio Fiore5, François Hemery6, Jean‑Winoc Decousser3,7, Olivier Langeron1,7 and Nicolas Mongardon1,7,8 Open Access © The Author(s) 2021. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativeco mmons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. de Roux et al. Crit Care (2021) 25:241 Page 2 of 11 further develop sepsis on V-A ECMO support [2]. Infec- tious complications are associated with an around 50% increase of mortality [3].h nor systemic antimicrobial prophylaxis is implemented during ICU stay. If sepsis is suspected, an empiric antimi- crobial therapy is initiated according to our local protocol and secondarily tailored to the laboratory findings. Daily rounds with microbiologists and infectious diseases spe- cialists are carried out [14]. The diagnosis of sepsis in adult population supported by V-A ECMO is challenging. Body temperature can- not be interpreted because of blood exposure to heat exchanger. White blood cell count and common bio- markers lack of specificity in the setting of cardiogenic shock both on medical treatment and on V-A ECMO [4, 5]. To overcome diagnosis, routine blood cultures (BCs) are proposed [6, 7]. In a survey of the Extracorporeal Life Support Organization (ELSO), one-third of the Ameri- can centers performed daily routine BCs [8]. However, this practice is still debated and has never been evaluated on V-A ECMO [9, 10]. According to the low incidence of poorly symptomatic BSIs and the high rate of contami- nants leading to unnecessary antimicrobial therapy, we hypothesized that systematic BCs have a lowest interest than clinically guided BCs. Study design and population All adult patients who underwent peripheral V-A ECMO for medical or surgical refractory cardiogenic shock, or for refractory cardiac arrest, in our 15-bed cardiovascular surgical ICU (Henri Mondor Teaching Hospital, Créteil, France) from January 2013 to January 2017 were retro- spectively included. Patients who died within 24 h after V-A ECMO implantation were excluded. Bloodstream infection definitioni As defined by the Centers for Disease Control and Pre- vention, Corynebacterium spp, Bacillus spp, Cutibac- terium spp, coagulase-negative Staphylococci (CoNS), viridians group Streptococci, Aerococcus spp and Micro- coccus spp were considered as common skin contami- nants unless the same bacterial strain was isolated from two separate BCs within 48 h of each other [18, 19]. All other pathogens were considered as BSIs from the first positive BC. BSI was considered as primary when the microorganism isolated in the BC was not clinically related to an infectious source. Otherwise, if the patho- gen isolated in the BC corresponded to a pathogen iden- tified from another sterile site, BSI was considered as secondary. Several BCs positive with the same pathogen on consecutive samples or days were considered as multi- ple positive BCs but belonging to the same single episode of BSI [20]. In case of refractory cardiac arrest or refractory cardio- genic shock, femoro-femoral V-A ECMO was inserted through surgical approach. Indications for V-A ECMO assistance followed recommendations for management of shock refractory to fluid optimization and inotrope/ vasopressive drugs administration [11]. Withdrawal was performed according to standard recommendations [12]. In case of scheduled cardiac surgery, nasal carriage of Staphylococcus aureus is screened the day before surgery and treated by a five-day course of nasal mupirocin in case of positivity, or otherwise stopped. For body routine care, daily skin washing with chlo- rhexidine 4% (Hibiscrub®, BCM Limited, Nottingham, UK) is performed during the first seven postoperative days in Staphylococcus aureus nasal carriers; other usual hygiene procedures are applied [15]. The sites of can- nulations are cleaned with chlorhexidine 2% in alcohol isopropyl 70% (Bactiseptic®, Vesismin S.L., Barcelona, Spain) and covered with occlusive dressings changed each two days. No antiseptic dressing is used. Care bun- dles for the prevention of ventilator-associated pneumo- nia follow international recommendations [16]. Beyond determining incidence, risk factors, and con- sequences of positive BCs, the aim of this study was to determine the respective contribution of routine versus on-demand BCs in a population of adult patients requir- ing V-A ECMO for refractory cardiogenic shock. Blood culture microbiological samples practices O l l l d d l Our local protocol recommends daily routine BCs, from V-A ECMO implantation up to five days after withdrawal. After decontamination of the skin and lid of bottles with chlorhexidine 2%, 10 mL of blood is sampled in one aero- bic and anaerobic bottle from the arterial line [17]. BCs performed between 4 and 7 a.m. were defined as routine BCs; all other BCs were considered as on-demand BCs. BCs results from the day of V-A ECMO implantation up to five days after withdrawal were included. Other micro- biological samples were left to the intensivist’s discretion. Antimicrobial strategy and hygiene practices Our protocol does not include antimicrobial prophylaxis in case of V-A ECMO implantation in ICU [13]. When the implantation is performed in the operating room in case of immediate cardiopulmonary bypass weaning fail- ure, antibiotic prophylaxis is restricted to the ongoing surgical procedure (repeated dose of cefazolin up to chest skin closure, or a single bolus of vancomycin in case of beta-lactam allergy). Neither digestive decontamination de Roux et al. Crit Care (2021) 25:241 Page 3 of 11 de Roux et al. Crit Care (2021) 25:241 Appropriateness of antimicrobial therapy Mann–Whitney, or Kruskal–Wallis test for continuous variables, as appropriate. Secondly, BCs were compared (BSI vs no BSI) using χ2 test for categorical variables and Student t-test, Mann–Whitney, or Kruskal–Wallis test for continuous variables, considering each BC as an inde- pendent unit. Finally, variables associated with BSI in univariate analysis (with p < 0.15) were included in a mul- tivariable logistic regression to identify factors indepen- dently associated with BSI. After multivariable logistic regression, we assessed the test performance (sensitivity, specificity, positive predictive value, and negative predic- tive value) of risk factors identified in the multivariable model. A sensitivity analysis restricted to routine BCs was performed. As a sensitivity analysis, we performed a multilevel logistic modeling, considering blood samples as level 1 (with “level 1 variables” including collection on V-A ECMO, routine sampling, ongoing microbial ther- apy, and ongoing RRT) and patients as level 2 (with BMI, KDIGO, lactate level, and bilirubin level as “level 2 vari- ables”). All tests were two-sided, with p < 0.05 considered statistically significant. STATA/SE 15.0 software (College Station, TX, USA) was used for statistical analysis. We considered antimicrobial therapy as appropriate (defined as the use of agents with in vitro activity against the etiologic pathogens) when it was administered for relevant BSI and within 24 h after the final antimicrobial report [21, 22]. Antimicrobial therapy was a posteriori deemed inappropriate when beginning for a single BC positive with a contaminant pathogen. Data collection and ethical considerations Data were collected from patient’s medical files and from the microbiological department database. We collected pre-morbid and demographic conditions, characteristics of V-A ECMO support, clinical and biological param- eters, and ICU/hospital course and outcomes. For each BC, body temperature, site of sampling, and sampling conditions were analyzed. According to the French law, patients were informed of the anonymous data extraction and analysis from medi- cal files [23]. This study was approved by the Comité d’Ethique pour la Recherche en Anesthésie-Réanimation (CERAR, IRB 00010254-2019-027). Blood cultures practices and results Overall, 2146 BCs were collected (including 363 BCs up to five days after V-A ECMO withdrawal), correspond- ing to a mean of 1.5 BCs per day. Seventy percent of the 1422 days of V-A ECMO had been subject to one rou- tine BC sampling. Overall, 190 BCs (9%) were positive: 116 with bacteria and 6 with yeasts, i.e., 122 BSIs, after exclusion of 68 contaminants. Forty-five (36%) BSIs were considered as primary. During V-A ECMO course, 49 different episodes of BSIs were observed, i.e., a BSI rate of 34.5 cases/1.000 days of V-A ECMO support. Results Continuous variables were expressed as median and interquartile range [25–75%] or mean (standard devi- ation), as appropriate. Categorical variables were expressed as proportions. Firstly, patients were com- pared according to occurrence of at least one BSI episode using χ2 test for categorical variables and Student t-test, During the 4-year study period, 206 consecutive VA- ECMO were inserted for refractory cardiogenic shock or cardiac arrest. After exclusion of 56 V-A ECMO (9 patients with central V-A ECMO and 47 deaths within 24 h after implantation), 150 V-A ECMO were analyzed (Fig. 1). Patients who received two V-A ECMO during 206 V-A ECMO screened in cardio- vascular surgical ICU from January 2013 to January 2017 150 ECMO analyzed 2146 BC collected 87 positive BC 1162 systematic BC 103 positive BC 51 pathogens 71 pathogens 36 contaminants 32 contaminants 56 V-A ECMO excluded : - 9 central V-A ECMO - 47 deaths within 24 hours after implantation 984 “on-demand” BC Fig. 1 Flow chart 206 V-A ECMO screened in cardio- vascular surgical ICU from January 2013 to January 2017 56 V-A ECMO excluded : - 9 central V-A ECMO - 47 deaths within 24 hours after implantation 150 ECMO analyzed 2146 BC collected 87 positive BC 32 contaminants 71 pathogens 51 pathogens 36 contaminants de Roux et al. Crit Care (2021) 25:241 Page 4 of 11 hospitalization (n = 3) were considered as independent cases. mechanical ventilation support duration for patients with BSIs was 6 days longer (p = 0.02). Overall, ICU mortality reached 56% without significant difference between patients with or without BSIs. ICU and hospital lengths of stay were significantly longer in case of BSIs (Table 3). V-A ECMO, veno-arterial extracorporeal membrane oxygenation; BSI, bloodstream infection; BMI, body mass index; COPD, chronic obstructive pulmonary disease; SAPS II, Simplified Acute Physiology Score 2; SOFA, Sepsis-related Organ Failure Assessment Data are expressed as median (interquartile 25–75) or number (percentage), as appropriate Baseline patients’ characteristics Median age was 58 [48–69] years. Comorbidities are reported in Table 1. V-A ECMO indication was mainly acute medical heart failure (n = 85; including 39 refrac- tory cardiac arrest); post-cardiotomy shock concerned 65 V-A ECMO indications (including 14 primary graft failure after heart transplantation). All patients except one were mechanically ventilated. Baseline patients’ char- acteristics did not differ regarding BSIs onset, except for patients with V-A ECMO for graft failure for whom BSIs were significantly more frequent, and for lactate level at admission, which was higher in patients without BSIs. Clinical characteristics and onset of bloodstream infection during ECMO course 1 1 0 0 Morganella morganii 1 1 0 0 Stenotrophomonas maltophilia 1 1 0 0 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 Days after V-A ECMO implantation 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 1 2 3 4 5 BSI (n) Days after V-A ECMO withdrawal BSI (n) Day of V-A ECMO support 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 Number of V-A ECMO 150 150 145 131 121 101 85 71 63 59 52 45 39 36 29 23 19 18 16 14 11 7 6 6 6 6 5 3 3 2 2 2 2 1 1 1 (A) (B) Fig. 2 Bloodstream infections per day, according to time elapsed after V-A ECMO implantation (panel A) or withdrawal (panel B) 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 Days after V-A ECMO implantation 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 1 2 3 4 5 BSI (n) Days after V-A ECMO withdrawal BSI (n) Day of V-A ECMO support 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 Number of V-A ECMO 150 150 145 131 121 101 85 71 63 59 52 45 39 36 29 23 19 18 16 14 11 7 6 6 6 6 5 3 3 2 2 2 2 1 1 1 (A) (B) Fig. Clinical characteristics and onset of bloodstream infection during ECMO course Crit Care (2021) 25:241 Page 5 of 11 i i l d f i BC ( 5) Blood culture characteristics variables associated 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 Days after V-A ECMO implantation 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 1 2 3 4 5 BSI (n) Days after V-A ECMO withdrawal BSI (n) Day of V-A ECMO support 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 Number of V-A ECMO 150 150 145 131 121 101 85 71 63 59 52 45 39 36 29 23 19 18 16 14 11 7 6 6 6 6 5 3 3 2 2 2 2 1 1 1 (A) (B) Fig. 2 Bloodstream infections per day, according to time elapsed after V-A ECMO implantation (panel A) or withdrawal (panel B) Table 2 Pathogens responsible for bloodstream infection on V-A ECMO Total numbers of pathogens differ from number of BSI due to polymicrobial BSI Pathogen Overall Within day 0–day 7 Within day 7 withdrawal After ECMO withdrawal Klebsiella pneumoniae 24 14 8 2 Enterobacter aerogenes 18 2 3 13 Proteus mirabilis 15 8 1 6 Enterobacter cloacae 13 10 2 1 Escherichia coli 11 6 2 3 Pseudomonas aeruginosa 13 3 0 10 Yeast (including Candida spp.) 6 0 5 1 Enterococcus faecalis 6 5 0 1 Hafnia alvei 5 5 0 0 Bacteroides fragilis 3 1 1 1 Klebsiella oxytoca 3 0 2 1 Pantoea spp. 3 3 0 0 Serratia marcescens 2 0 0 2 Staphylococcus aureus 2 2 0 0 Citrobacter braakii 1 1 0 0 Enterococcus faecium 1 0 0 1 Haemophilus influenzae 1 1 0 0 Lactobacillus casei 1 1 0 0 Neisseria spp. 1 1 0 0 Leuconostoc spp. Clinical characteristics and onset of bloodstream infection during ECMO course Regarding sampling categorization, on 1162 routine BCs performed, 51 were positive for non-contaminants pathogens (4%). Conversely, 984 on-demand BCs were sampled and 71 were positive for non-contaminants pathogens (7%) (p = 0.005). On the 68 total BCs posi- tive for contaminants, 10 (15%) led to an inappropriate antimicrobial therapy; this rate did not differ whether Median duration of V-A ECMO support was 7 [5–13] days, representing a total of 1422 ECMO-days. BSIs occurred in 50 (33%) patients, with increased incidence during the first week and after withdrawal (Fig. 2). Pathogens identified in BSIs were reported in Table 2. V-A ECMO support duration was significantly longer in case of BSIs (11 versus 6 days, p = 0.0002). Similarly, Table 1 Baseline patients characteristics Table 1 Baseline patients characteristics Data are expressed as median (interquartile 25–75) or number (percentage), as appropriate Characteristics All V-A ECMO (n = 150) Patient without BSI (n = 100) Patient with BSI (n = 50) p Age (years) 58 (16) 59 (16) 55 (15) 0.14 Male gender 104 (69) 68 (68) 36 (72) 0.62 BMI (kg/m)2 25.4 (22.8–29.0) 24.7 (22.8–28.7) 27.1 (22.6–29.4) 0.13 Comorbidities COPD 10 (7) 7 (7) 3 (6) 0.82 Cirrhosis 3 (2) 1 (1) 2 (4) 0.22 Long-term corticosteroid 7 (5) 4 (4) 3 (6) 0.58 Diabetes 36 (24) 27 (27) 9 (18) 0.22 V-A ECMO for post-cardiotomy shock 65 (43) 43 (43) 22 (44) 0.91 Including primary graft failure after heart transplantation 14 (21) 5 (11) 9 (41) 0.01 V-A ECMO for acute heart failure 85 (57) 57 (57) 28 (56) 0.91 Including refractory cardiac arrest 39 (26) 12 (24) 27 (27) 0.69 Percutaneous V-A ECMO insertion 9 (6) 8 (8) 1 (2) 0.27 Intra-aortic balloon pump 26 (17) 20 (20) 6 (12) 0.22 Lactate level at day 0 (mmol/L) 5.2 (3.0–9.1) 6.1 (3.7–10.3) 3.8 (2.2–8.3) 0.003 Creatinine level at day 0 (µmol/L) 159 (98) 154 (93) 169 (109) 0.39 SAPS II 54 (38–70) 56 (40–74) 50 (37–66) 0.19 Admission SOFA score 13 (11–15) 14 (12–16) 12 (11–15) 0.10 Vasoactive–inotropic score 70 (34–139) 68 (36–130) 73 (29–141) 0.91 de Roux et al. Clinical characteristics and onset of bloodstream infection during ECMO course 2 Bloodstream infections per day, according to time elapsed after V-A ECMO implantation (panel A) or withdrawal (panel B) (A) Table 2 Pathogens responsible for bloodstream infection on V-A ECMO Total numbers of pathogens differ from number of BSI due to polymicrobial BSI Pathogen Overall Within day 0–day 7 Within day 7 withdrawal After ECMO withdrawal Klebsiella pneumoniae 24 14 8 2 Enterobacter aerogenes 18 2 3 13 Proteus mirabilis 15 8 1 6 Enterobacter cloacae 13 10 2 1 Escherichia coli 11 6 2 3 Pseudomonas aeruginosa 13 3 0 10 Yeast (including Candida spp.) 6 0 5 1 Enterococcus faecalis 6 5 0 1 Hafnia alvei 5 5 0 0 Bacteroides fragilis 3 1 1 1 Klebsiella oxytoca 3 0 2 1 Pantoea spp. 3 3 0 0 Serratia marcescens 2 0 0 2 Staphylococcus aureus 2 2 0 0 Citrobacter braakii 1 1 0 0 Enterococcus faecium 1 0 0 1 Haemophilus influenzae 1 1 0 0 Lactobacillus casei 1 1 0 0 Neisseria spp. 1 1 0 0 Leuconostoc spp. 1 1 0 0 Morganella morganii 1 1 0 0 Stenotrophomonas maltophilia 1 1 0 0 Table 2 Pathogens responsible for bloodstream infection on V-A ECMO Total numbers of pathogens differ from number of BSI due to polymicrobial BSI Total numbers of pathogens differ from number of BSI due to polymicrobial BSI Blood culture characteristics variables associated with bloodstream infection contaminant was isolated from routine BCs (n = 5) or on-demand BCs (n = 5) (13 vs. 14%, p = 0.85). Focusing on routine BCs, 16 (31%) BSIs from routine BCs would not have been diagnosed by on-demand BCs, i.e., patients had no on-demand BCs. contaminant was isolated from routine BCs (n = 5) or on-demand BCs (n = 5) (13 vs. 14%, p = 0.85). Considering each BC as an independent event, clinical and BC characteristics during V-A ECMO course are summarized in Table 4. Body temperature at the time of Considering each BC as an independent event, clinical and BC characteristics during V-A ECMO course are summarized in Table 4. Body temperature at the time of Focusing on routine BCs, 16 (31%) BSIs from routine BCs would not have been diagnosed by on-demand BCs, i.e., patients had no on-demand BCs. de Roux et al. V-A ECMO, veno-arterial extracorporeal membrane oxygenation; BC, blood culture; BMI, body mass index; BSI, bloodstream infection; KDIGO, Kidney Disease Improving Global Outcomes; RRT, renal replacement therapy Data are expressed as median [interquartile 25–75] or number (percentage), as appropriate Discussion In this 4-year series of 150 consecutive V-A ECMO, BSIs were observed in one-third of patients, with a rate of 34.5 BSI/1.000 days of V-A ECMO support. Routine BCs iden- tified significantly less BSIs than on-demand BCs and led to a high proportion of contaminations with subsequent inappropriate antimicrobial therapy. In addition, per- forming routine BCs were negatively associated with BSIs diagnosis. However, one-third of BSIs would have been missed by a policy restricted to on-demand BCs. This argues for better selecting conditions of routine BCs pre- scription on V-A ECMO. Indeed, this study highlighted three independent risk factors for BCs positivity when routine BCs were performed: patient with V-A ECMO for graft failure after heart transplantation, BCs collected with ongoing antimicrobial therapy or renal replacement therapy. BC sampling was not different in case of BSI. Whereas BCs were preferably drawn from arterial line (70%), BSI was more observed when BCs were retrieved from cen- tral venous line or direct venipuncture (p < 0.001). p (p ) In multivariate analysis considering all BCs (Table 5), independent risk factors associated with BSIs were: BMI (OR 1.1, 95% CI [1.0; 1.1], p = 0.007), lactate level at admission (OR 0.90, 95% CI [0.85; 0.95], p < 0.001), bili- rubin level at admission (OR 1.00, 95% CI [1.00; 1.01], p = 0.019), BCs collected on V-A ECMO (OR 0.52, 95% CI [0.34; 0.81], p = 0.004), BCs collected with ongo- ing antimicrobial therapy (OR 1.56, 95% CI [1.03; 2.35], p = 0.037), and BCs collected with ongoing renal replace- ment therapy (OR 2.76, 95% CI [1.86; 4.09], p < 0.001). In addition, performing routine BC was negatively asso- ciated with BSI diagnosis (OR 0.55, 95% CI [0.38; 0.81], p = 0.002). Sensitivity analysis with multilevel model adjusted found consistent results (OR of routine BC for BSI = 0.44, 95% CI [0.28; 0.67], p < 0.001). py A recent study of 220 V-A ECMO reported an inci- dence of nosocomial infections of 64%, with ventilator- associated pneumonia and BSI being the most frequent (55% and 18%, respectively) [2]. The specific burden of infectious complications is hard to evaluate, because the most severe patients die early, while the survivors expe- rience prolonged ECMO support and ICU length of stay. Clinical characteristics and onset of bloodstream infection during ECMO course Crit Care (2021) 25:241 Page 7 of 11 Table 5 Multivariate analysis of independent variables of probability of blood culture to diagnose bloodstream infection V-A ECMO, veno-arterial extracorporeal membrane oxygenation; BSI, bloodstream infection; BMI, body mass index; KDIGO, Kidney Disease Improving Global Outcomes; RRT, renal replacement therapy BSI characteristics OR [95% CI] p BMI (kg/m)2 1.06 [1.01; 1.11] 0.007 KDIGO stage 1 1.95 [1.19; 3.17] 0.007 2 1.64 [0.91; 2.95] 0.101 3 1.36 [0.77; 2.40] 0.285 Lactate level at day 0 (mmol/L) 0.90 [0.85; 0.95] < 0.001 Total bilirubin level at day 0 (µmol/L) 1.00 [1.00; 1.01] 0.019 Collection on V-A ECMO 0.52 [0.34; 0.81] 0.004 Routine sampling 0.55 [0.38; 0.81] 0.002 On-going antimicrobial therapy 1.56 [1.03; 2.35] 0.037 On-going RRT 2.76 [1.86; 4.09] < 0.001 Table 6 Multivariate analysis of independent variables associated with positive routine blood culture BSI, bloodstream infection; RRT, renal replacement therapy BSI characteristics OR [95% CI] p Primary graft failure 2.43 [1.20; 4.92] 0.013 Ongoing antimicrobial therapy 2.15 [1.08; 4.27] 0.029 Ongoing RRT 2.05 [1.10; 3.81] 0.008 Table 6 Multivariate analysis of independent variables associated with positive routine blood culture revealed BSIs in the presence of all the three conditions. Test performance of the routine BCs strategy comparing the existence of at least one risk factor with the absence of risk factor was 96%, 30%, 5%, and 99%, for sensitivity, specificity, positive predictive value, and negative predic- tive value, respectively. V-A ECMO, veno-arterial extracorporeal membrane oxygenation; BSI, bloodstream infection; BMI, body mass index; KDIGO, Kidney Disease Improving Global Outcomes; RRT, renal replacement therapy Clinical characteristics and onset of bloodstream infection during ECMO course Crit Care (2021) 25:241 Page 6 of 11 Table 3 Clinical characteristics during ECMO course Table 3 Clinical characteristics during ECMO course Data are expressed as median (interquartile 25–75) or number (percentage), as appropriate V-A ECMO, veno-arterial extracorporeal membrane oxygenation; BSI, bloodstream infection; ICU, intensive care unit All V-A ECMO (n = 150) Patient without BSI (n = 100) Patient with BSI (n = 50) p ECMO implantation in the operative room 81 (54) 55 (55) 26 (52) 0.73 V-A ECMO support duration (days) 7 (5–13) 6 (5–11) 11 (6–16) 0.0002 Total red blood cell unit transfusion during V-A ECMO support 13 (9) 13 (9) 13 (9) 0.83 Acute mesenteric ischemia during V-A ECMO support 14 (9) 8 (8) 6 (12) 0.43 Total duration of mechanical ventilation (days) 14 (6–27) 12 (5.5–25) 18 (10–30) 0.02 ICU mortality 84 (56) 54 (54) 30 (60) 0.49 ICU length of stay (days) 19 (10–32) 17 (9–26) 23 (14–38) 0.02 Hospital length of stay (days) 24 (14–38) 21 (10–37) 31 (17–41) 0.01 Table 4 Blood culture characteristics Data are expressed as median [interquartile 25–75] or number (percentage), as appropriate V-A ECMO, veno-arterial extracorporeal membrane oxygenation; BC, blood culture; BMI, body mass index; BSI, bloodstream infection; KDIGO, Kidney Disease All BC (n = 2146) No BSI (n = 2024) BSI (n = 122) p Age (years) 57 (16) 57 (16) 54 (16) 0.04 BMI (kg/m)2 26.1 (4.6) 26.1 (4.6) 26.8 (4.6) 0.12 Indications for V-A ECMO < 0.001 Acute heart failure 763 (36) 720 (36) 43 (35) Refractory cardiac arrest 466 (22) 446 (22) 20 (16) Post-cardiotomy shock 722 (34) 691 (34) 31 (25) Primary graft failure 195 (9) 167 (8) 28 (23) Percutaneous V-A ECMO insertion 119 (6) 111 (9) 8 (1) < 0.001 Hemoglobin at day 0 (g/dL) 10.0 (2.4) 9.9 (2.3) 10,2 (2.7) 0.23 White blood cell count at day 0 (G/L) 14.2 (6.8) 14.2 (6.8) 14.5 (7.5) 0.69 Total bilirubin level at day 0 (µmol/L) 33 (46) 32 (43) 46 (85) 0.002 Lactate level at day 0 (mmol/L) 6.0 (4.1) 6.1 (4.2) 4.5 (3.4) < 0.0001 KDIGO stage 0.026 0 813 (38) 781 (39) 32 (26) 1 634 (30) 587 (29) 47 (39) 2 276 (13) 255 (13) 21 (17) 3 423 (20) 401 (20) 22 (18) Timing between V-A ECMO implantation and BC sampling 0.001 < 7 days 1124 (52) 1069 (53) 55 (45) 7–15 days 733 (34) 696 (34) 37 (30) > 15 days 289 (13) 259 (13) 30 (25) BC sampling on V-A ECMO 1783 (83) 1694 (84) 89 (73) 0.002 Routine BC 1162 (54) 1111 (55) 51 (42) 0.005 Body temperature 36.8 (36.4–37.3) 36.8 (36.4–37.3) 36.9 (36.4–37.3) 0.57 ≥ 38.3 °C 156 (7) 150 (7) 6 (5) 0.30 BC sampling site < 0.001 Arterial line 1705 (82) 1629 (83) 76 (66) Central venous catheter 210 (10) 186 (9) 24 (21) Peripheral venipuncture 161 (8) 146 (7) 15 (13) Ongoing antimicrobial therapy 1318 (61) 1231 (61) 87 (72) 0.02 Ongoing RRT 582 (27) 527 (26) 55 (45) < 0.001 de Roux et al. Discussion Regarding our BSIs rate, this large variation from a factor 1 to 10 prevents from drawing an “usual” rate of BSIs on ECMO. Our high rate of BSIs may be explained by (1) our daily routine BCs sampling protocol, (2) the inclusion of post-cardiac arrest patients who develop high rate of infectious complica- tions, and (3) the extent of the study period up to 5 days after V-A ECMO withdrawal. patient, an increase of antibiotic prescription, and length of stay [34, 35]. It has been demonstrated than half of patients with contaminated BCs were inappropriately treated with antibiotics, one-third receiving vancomy- cin [17]. In our unit, antimicrobial therapy was deemed a posteriori inadequate in only 14% of cases, thanks to a rigorous anti-antimicrobial therapy policy and daily rounds with infectious diseases specialists. This may counterbalance our high rate of BSIs and contaminated BCs. Second, collecting 20 ml of blood per BC on the top of others daily blood samples exposes to progressive ane- mia [36]. On the contrary, a policy of reduction of blood laboratory tests reduced red blood cell transfusion, hos- pitalization costs, without impacting ICU outcome [37]. (pediatric for the most of studies, or a mix of adult and children) creates heterogeneity [24–28]. In addition, pro- phylactic anti-infective strategies and BCs contaminants varied between studies. Similarly, while the ELSO Infec- tious Disease Task Force does not recommend antibiotic prophylaxis on ECMO, 74% of centers reported perform- ing it [29, 30]. The risk of BSIs and the well-established consequence of delayed or inappropriate antimicrobial therapy must be balanced with the negative impact of antibiotics on microbiota and subsequent infections [31]. Overall, a recent review (in pediatric and adult popula- tion with V-V and V-A ECMO) found a BSIs prevalence ranging from 3 to 18% and a incidence range from 3 to 31 episodes per 1.000 ECMO-days [3]. Regarding our BSIs rate, this large variation from a factor 1 to 10 prevents from drawing an “usual” rate of BSIs on ECMO. Our high rate of BSIs may be explained by (1) our daily routine BCs sampling protocol, (2) the inclusion of post-cardiac arrest patients who develop high rate of infectious complica- tions, and (3) the extent of the study period up to 5 days after V-A ECMO withdrawal. Our results question the optimal policy of BC prac- tices. Discussion For instance, V-A ECMO support duration, ICU, and hospital length of stay were significantly longer in patients with at least one BSI, but this link reflects a longer vulnerability period, prone to septic complica- tions. This precludes comparison of the mortality of infected versus non-infected patients, in addition to dis- crepancies between definitions and differences of case mix between studies. Focusing on routine BCs, independent risk factors associated with BSIs were: V-A ECMO for graft failure after heart transplantation (OR 2.43, 95% CI [1.20; 4.92], p = 0.013) and BCs performed with ongoing antimicro- bial therapy or renal replacement therapy (OR 2.15, 95% CI [1.08; 4.27], p = 0.029, and OR 2.05, 95% CI [1.10; 3.81], p = 0.008, respectively) (Table 6). Figure 3 represents the occurrence of BSIs when BCs were collected systematically according to the presence of none or at least one of the three independent variables described above. In case of the absence of all these three conditions, only two BSIs from routine BCs were posi- tive and would not have been caught up by on-demand BCs sampling. On the contrary, 15% of routine BCs A few studies focused on the incidence of BSI on ECMO support. Unfortunately, mixture of veno-venous (V-V) and V-A ECMO, various indications of support (from acute respiratory distress syndrome to refrac- tory cardiogenic shock), and various types of population de Roux et al. Crit Care (2021) 25:241 Page 8 of 11 0 2 4 6 8 10 12 14 16 18 None of the three risk factors One risk factor Two risk factors Three risk factors Fig. 3 Occurrence of bloodstream infection diagnosis (%) with routine blood culture depending on risk factors (pediatric for the most of studies, or a mix of adult and children) creates heterogeneity [24–28]. In addition, pro- phylactic anti-infective strategies and BCs contaminants varied between studies. Similarly, while the ELSO Infec- tious Disease Task Force does not recommend antibiotic prophylaxis on ECMO, 74% of centers reported perform- ing it [29, 30]. The risk of BSIs and the well-established consequence of delayed or inappropriate antimicrobial therapy must be balanced with the negative impact of antibiotics on microbiota and subsequent infections [31]. Overall, a recent review (in pediatric and adult popula- tion with V-V and V-A ECMO) found a BSIs prevalence ranging from 3 to 18% and a incidence range from 3 to 31 episodes per 1.000 ECMO-days [3]. Discussion Our results could mean that BSIs are often sympto- matic in this population, with either worsening shock or clinical/radiological signs of sepsis. Alternatively, physi- cians may have not prescribed on-demand BCs, knowing the fact that routine BCs had been sampled a few hours before. This subjective point may have lowered the diag- nosis yield of on-demand BCs. Aiming to identify the clinical settings that increase the putative contribution of systematic BCs, we highlighted three risk factors for BC positivity. ECMO support after cardiac transplanta- tion raises the question of performing systematic daily BC, as immunodepression increases the risk of BSI, and thus, the suitability of routine BC. Similarly, ongoing renal replacement therapy denotes higher severity and thus higher risk of nosocomial infections. Alternatively, the need for intravascular dialysis catheter may be a sup- plementary BSI risk factor. At least, BC sampling dur- ing ongoing antimicrobial therapy may reveal creeping bacteremia that are uncompleted treated by antibiotics. Overall, we propose a mitigated strategy, i.e., performing daily BC in adult patients on V-A ECMO with risk fac- tor. This approach could be an acceptable compromise To deal with the challenging issue of diagnosis of infec- tion on ECMO, routine BCs sampling is sometimes advocated but is still debated [6, 32]. According to ELSO surveys, between 34 and 49% of centers reported per- forming routine BCs, with variable intervals [8, 30]. As far as we are aware, this is the first study focusing on the interest and impact of a policy of routine BCs on V-A ECMO. Multiple and non-clinically oriented routine BCs could increase the probability of diagnosing paucisymptomatic BSIs, but also the risk of positivity with contaminants. Our contamination rate was 2.5%; a rate within previ- ously reported contamination ranges from 0.6 to 6% [33]. This raises several points of concern. First, the isolation of positive BC with potential contaminants generates an additional cost of hospitalization of around $8000 per de Roux et al. Crit Care (2021) 25:241 de Roux et al. Crit Care (2021) 25:241 Page 9 of 11 positive BCs risk factors are present. Further prospec- tive studies should test these criteria. between an aggressive BC collection policy leading to an excess of contaminant identification and blood depletion, and a sparing policy missing clinically relevant BSI. Our work presents limitations. Availability of data and materials Availability of data and materials d l l bl Availability of data and materials Data and materials are available on request to the corresponding author. Discussion First, in the absence of consensual definition of contaminated BCs in the pre- sent particular clinical setting, the CDC definition was applied [18, 38]. Second, our study population mixed patients with post-cardiotomy shock, acute heart failure, and refractory cardiac arrest. The latter is more prone to infectious complications, notably BSI [39]. Third, in case of death on V-A ECMO support, BC may not have been sampled the day of death. Fourth, the retrospective design of this study did not allow us to assess formally that all BCs taken between 4 and 7 a.m. were all routine sampling. Fifth, the reason of performing on-demand BC was not collected. We also acknowledge that provid- ers who know their patients will receive daily BCs in the morning might be less likely to order on-demand cul- tures, or consider that the routine BCs were enough for diagnosing infection, even in the setting of new signs or symptoms. In addition, ongoing antimicrobial therapy was analyzed as a yes/no variable, but it may have dif- ferent impact according to the duration of treatment. Also, we did not extract biological variables at each BC sampling but only those at V-A ECMO implantation, i.e., at day 0. Similarly, location and number of intravas- cular catheters were not collected, whereas they are per se a major risk factor for BSI. Sixth, BC was considered as the gold-standard of BSI diagnostic method. However, BC detect only culturable bacteria. Innovative techniques like bacterial DNA detection in rapid molecular assays such as PCR or more recently metagenomic next-gener- ation sequencing (mNGS) test using cell-free DNA from blood could be promising tools for diagnosis supplemen- tary BSI [40, 41]. Finally, the temporal window of BCs collection is debatable. Indeed, the time frame of ECMO- related BSIs advocated by some authors is from the day of V-A ECMO implantation up to two days after withdrawal [2, 42]. However, risk of delayed V-A ECMO-related BSIs persists several days after withdrawal. Our results con- firm this recent finding, arguing for large BCs sampling after V-A ECMO withdrawal [43]. Author details 1 Service d’Anesthésie‑Réanimations Chirurgicales, DMU CARE, DHU A‑TVB, Assistance Publique‑Hôpitaux de Paris (AP‑HP), Hôpitaux Universitaires Henri Mondor, 1 rue Gustave Eiffel, 94000 Créteil, France. 2 Réanimation Polyvalente, Hôpital Privé Jacques Cartier, 91300 Massy, France. 3 Département de préven‑ tion, diagnostic et traitement des infections, Assistance Publique des Hôpitaux de Paris (APHP), Hôpitaux Universitaires Henri Mondor, 94010 Créteil, France. 4 Unité transversale de traitement des infections, Assistance Publique des Hôpitaux de Paris (APHP), Hôpitaux Universitaires Henri Mondor, 94010 Créteil, France. 5 Service de chirurgie cardiaque, Assistance Publique des Hôpitaux de Paris (APHP), Hôpitaux Universitaires Henri Mondor, 94010 Créteil, France. 6 Département d’information médicale, Assistance Publique des Hôpitaux de Paris (APHP), Hôpitaux Universitaires Henri Mondor, 94010 Créteil, France. 7 Faculté de Santé, Univ Paris Est Créteil, 94010 Créteil, France. 8 U955‑IMRB, Equipe 03 “Pharmacologie et Technologies pour les Maladies Cardiovasculaires (PROTECT)”, Inserm, Univ Paris Est Créteil (UPEC), Ecole Nationale Vétérinaire d’Alfort (EnVA), 94700 Maisons‑Alfort, France. Competing interests l NM serves as a consultant for Amomed. The other authors have not disclosed any potential competing interests. Ethical approval and consent to participate f d f h d pp p p Patients were informed of the anonymous data extraction and analysis from medical files. This study was approved by the Comité d’Ethique pour la Recherche en Anesthésie-Réanimation (CERAR, IRB 00010254-2019-027). Authors’ contributions QdR and NM designed the study and wrote the manuscript. WB, QdR, and NM performed the statistical analyses. QdR, MR, JB, FH, and NM collected study data. All authors participated in writing and revising the manuscript. All authors read and approved the final manuscript. Abbreviations BC: Blood culture; BMI: Body mass index; BSI: Bloodstream infection; CERAR : Comité d’Ethique pour la Recherche en Anesthésie-Réanimation; CoNS: Coagulase-negative staphylococci; COPD: Chronic obstructive pulmonary dis‑ ease; ELSO: Extracorporeal Life Support Organization; ICU: Intensive care unit; KDIGO: Kidney Disease Improving Global Outcomes; RRT: Renal replacement therapy; SAPS II: Simplified Acute Physiology Score 2; SOFA: Sepsis-related organ failure assessment; V-A ECMO: Veno-arterial extracorporeal membrane oxygenation; V-V ECMO: Veno-venous extracorporeal membrane oxygenation. Consent for publication All authors have given their consent for publication. Funding Funding The authors received no financial support for the study or publication. Availability of data and materials Data and materials are available on request to the corresponding author. References The diagnostic yield of routine admission blood cultures in criti‑ cally ill patients. Crit Care Med. 2021;49(1):60–9. 28. Butler DF, Lee B, Molitor-Kirsch E, Newland JG. Extracorporeal mem‑ brane oxygenation-associated bloodstream infections in children. Pediatr Infect Dis J. 2017;36(3):346–7. 8. Glater-Welt LB, Schneider JB, Zinger MM, Rosen L, Sweberg TM. Noso‑ comial bloodstream infections in patients receiving extracorporeal life support: variability in prevention practices: a survey of the extra‑ corporeal life support organization members. J Intensive Care Med. 2016;31(10):654–69. 29. Extracorporeal Life Support Organization-ECMO and ECLS > About Us > Task Forces > Infectious Disease Task Force. 2019. Disponible sur: https://www.elso.org/AboutUs/TaskForces/InfectiousDiseaseTaskForce. aspx. 9. Elerian LF, Sparks JW, Meyer TA, Zwischenberger JB, Doski J, Goretsky MJ, et al. Usefulness of surveillance cultures in neonatal extracorporeal membrane oxygenation. ASAIO J. 2001;47(3):220–3. 30. Kao LS, Fleming GM, Escamilla RJ, Lew DF, Lally KP. Antimicrobial prophy‑ laxis and infection surveillance in extracorporeal membrane oxygena‑ tion patients: a multi-institutional survey of practice patterns. ASAIO J. 2011;57(3):231–8. y 10. Abrams D, Grasselli G, Schmidt M, Mueller T, Brodie D. ECLS-associated infections in adults: what we know and what we don’t yet know. Inten‑ sive Care Med. 2020;46(2):182–91. 11. Le Gall A, Follin A, Cholley B, Mantz J, Aissaoui N, Pirracchio R. Veno- arterial-ECMO in the intensive care unit: From technical aspects to clinical practice. Anaesth Crit Care Pain Med. 2018;37(3):259–68. 31. Bassetti M, Rello J, Blasi F, Goossens H, Sotgiu G, Tavoschi L, et al. System‑ atic review of the impact of appropriate versus inappropriate initial anti‑ biotic therapy on outcomes of patients with severe bacterial infections. Int J Antimicrob Agents. 2020;56(6):106184. 12. Aissaoui N, El-Banayosy A, Combes A. How to wean a patient from veno-arterial extracorporeal membrane oxygenation. Intensive Care Med. 2015;41(5):902–5. 32. Kaczala GW, Paulus SC, Al-Dajani N, Jang W, Blondel-Hill E, Dobson S, et al. Bloodstream infections in pediatric ECLS: usefulness of daily blood culture monitoring and predictive value of biological markers. The British Columbia experience. Pediatr Surg Int. 2009;25(2):169–73. 13. Martin C, Auboyer C, Boisson M, Dupont H, Gauzit R, Kitzis M, et al. Anti‑ bioprophylaxis in surgery and interventional medicine (adult patients). Update 2017. Anaesth Crit Care Pain Med. 2019;38(5):549–62. 33. Hall KK, Lyman JA. Updated review of blood culture contamination. Clin Microbiol Rev. 2006;19(4):788–802. 14. Leone M, Roberts JA, Bassetti M, Bouglé A, Lavigne J-P, Legrand M, et al. References 1. Mongardon N, De Roux Q, Clariot S. Veno-arterial ECMO in criti‑ cally ill patients: the age of maturity? Anaesth Crit Care Pain Med. 2018;37(3):193–4. Infect Dis Off Publ Eur Soc Clin Microbiol. 2015;34(7):1395–40 22. Siempos II, Ioannidou E, Falagas ME. The difference between adequate and appropriate antimicrobial treatment. Clin Infect Dis. 2008;46(4):642–4. 2. Schmidt M, Bréchot N, Hariri S, Guiguet M, Luyt CE, Makri R, et al. Nosocomial infections in adult cardiogenic shock patients supported by venoarterial extracorporeal membrane oxygenation. Clin Infect Dis. 2012;55(12):1633–41. 23. Toulouse E, Lafont B, Granier S, Mcgurk G, Bazin J-E. French legal approach to patient consent in clinical research. Anaesth Crit Care Pain Med. 2020;39(6):883–5. 3. Biffi S, Di Bella S, Scaravilli V, Peri AM, Grasselli G, Alagna L, et al. Infec‑ tions during extracorporeal membrane oxygenation: epidemiology, risk factors, pathogenesis and prevention. Int J Antimicrob Agents. 2017;50(1):9–16. 24. Kim GS, Lee KS, Park CK, Kang SK, Kim DW, Oh SG, et al. Nosocomial infection in adult patients undergoing veno-arterial extracorporeal membrane oxygenation. J Korean Med Sci. 2017;32(4):593–8. 4. Kataja A, Tarvasmäki T, Lassus J, Sionis A, Mebazaa A, Pulkki K, et al. Kinet‑ ics of procalcitonin, C-reactive protein and interleukin-6 in cardiogenic shock—insights from the CardShock study. Int J Cardiol. 2021;322:191–6. 25. Na SJ, Chung CR, Choi HJ, Cho YH, Yang JH, Suh GY, et al. Blood stream infection in patients on venovenous extracorporeal membrane oxygenation for respiratory failure. Infect Control Hosp Epidemiol. 2018;39(7):871–4. 5. Kim DW, Cho HJ, Kim GS, Song SY, Na KJ, Oh SG, et al. Predictive value of procalcitonin for infection and survival in adult cardiogenic shock patients treated with extracorporeal membrane oxygenation. Chonnam Med J. 2018;54(1):48–54. 26. Silvetti S, Ranucci M, Pistuddi V, Isgrò G, Ballotta A, Ferraris L, et al. Bloodstream infections during post-cardiotomy extracorporeal mem‑ brane oxygenation: incidence, risk factors, and outcomes. Int J Artif Organs. 2018;391398818817325. 6. Steiner CK, Stewart DL, Bond SJ, Hornung CA, McKay VJ. Predictors of acquiring a nosocomial bloodstream infection on extracorporeal mem‑ brane oxygenation. J Pediatr Surg. 2001;36(3):487–92. g 27. Kutleša M, Santini M, Krajinović V, Papić N, Novokmet A, Josipović Mraović R, et al. Nosocomial blood stream infections in patients treated with venovenous extracorporeal membrane oxygenation for acute respiratory distress syndrome. Minerva Anestesiol. 2017;83(5):493–501. 7. Verboom DM, van de Groep K, Boel CHE, Haas PJA, Derde LPG, Cremer OL, et al. References Update in antibiotic therapy in intensive care unit: report from the 2019 Nîmes international symposium. Anaesth Crit Care Pain Med. 2019;38(6):647–56. 34. Alahmadi YM, Aldeyab MA, McElnay JC, Scott MG, Darwish Elhajji FW, Magee FA, et al. Clinical and economic impact of contaminated blood cultures within the hospital setting. J Hosp Infect. 2011;77(3):233–6. 35. Bates DW, Goldman L, Lee TH. Contaminant blood cultures and resource utilization: the true consequences of false-positive results. JAMA. 1991;265(3):365–9. 15. Climo MW, Yokoe DS, Warren DK, Perl TM, Bolon M, Herwaldt LA, et al. Effect of daily chlorhexidine bathing on hospital-acquired infection. N Engl J Med. 2013;368(6):533–42. 16. Leone M, Bouadma L, Bouhemad B, Brissaud O, Dauger S, Gibot S, et al. Hospital-acquired pneumonia in ICU. Anaesth Crit Care Pain Med. 2018;37(1):83–98. 36. Lyon AW, Chin AC, Slotsve GA, Lyon ME. Simulation of repetitive diagnos‑ tic blood loss and onset of iatrogenic anemia in critical care patients with a mathematical model. Comput Biol Med. 2013;43(2):84–90. 17. Lamy B, Dargère S, Arendrup MC, Parienti J-J, Tattevin P. How to optimize the use of blood cultures for the diagnosis of bloodstream infections? A State-of-the Art. Front Microbiol. 2016;7:697. 37. Kumwilaisak K, Noto A, Schmidt UH, Beck CI, Crimi C, Lewandrowski K, et al. Effect of laboratory testing guidelines on the utilization of tests and order entries in a surgical intensive care unit. Crit Care Med. 2008;36(11):2993–9. 18. Horan TC, Andrus M, Dudeck MA. CDC/NHSN surveillance definition of health care-associated infection and criteria for specific types of infec‑ tions in the acute care setting. Am J Infect Control. 2008;36(5):309–32. 38. Beekmann SE, Diekema DJ, Doern GV. Determining the clinical signifi‑ cance of coagulase-negative staphylococci isolated from blood cultures. Infect Control Hosp Epidemiol. 2005;26(6):559–66. 19. Surveillance of healthcare-associated infections and prevention indica‑ tors in European intensive care units: HAI-Net ICU protocol, version 2.2. European Centre for Disease Prevention and Control. 2017. Disponible sur: https://www.ecdc.europa.eu/en/publications-data/surveillance- healthcare-associated-infections-and-prevention-indicators-european. 39. Mongardon N, Perbet S, Lemiale V, Dumas F, Poupet H, Charpentier J, et al. Infectious complications in out-of-hospital cardiac arrest patients in the therapeutic hypothermia era. Crit Care Med. 2011;39:1359–64. 40. Bloos F, Hinder F, Becker K, Sachse S, Mekontso Dessap A, Straube E, et al. A multicenter trial to compare blood culture with polymerase chain reac‑ tion in severe human sepsis. Intensive Care Med. 2010;36(2):241–7. 20. Almyroudis NG, Fuller A, Jakubowski A, Sepkowitz K, Jaffe D, Small TN, et al. Conclusion We describe for the first time the consequences of BC practices policy in an adult population supported by V-A ECMO. Whereas routine daily BCs were less clini- cally relevant than on-demand BCs and lead to a sig- nificant proportion of inappropriate antimicrobial therapy, a restricting policy of BCs sampling misses a significant proportion of BSIs. We suggest daily BCs sampling on V-A ECMO support when easy-to-identify Received: 23 March 2021 Accepted: 27 June 2021 de Roux et al. Crit Care (2021) 25:241 Page 10 of 11 Page 10 of 11 21. Park SY, Kwon KH, Chung J-W, Huh HJ, Chae SL. Coagulase-negative staphylococcal bacteremia: risk factors for mortality and impact of ini‑ tial appropriate antimicrobial therapy on outcome. Eur J Clin Microbiol Infect Dis Off Publ Eur Soc Clin Microbiol. 2015;34(7):1395–401.f Page 11 of 11 de Roux et al. Crit Care (2021) 25:241 42. Grasselli G, Scaravilli V, Di Bella S, Biffi S, Bombino M, Patroniti N, et al. Nosocomial infections during extracorporeal membrane oxygenation: incidence, etiology, and impact on patients’ outcome. Crit Care Med. 2017;45(10):1726–33. 43. Messika J, Schmidt M, Aubry A, Combes A, Ricard J-D. Extracorporeal membrane oxygenation-associated infections: carefully consider cannula infections! Crit Care Med. 2018;46(2):e171–2. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in pub‑ lished maps and institutional affiliations. de Roux et al. Crit Care (2021) 25:241 de Roux et al. Crit Care (2021) 25:241 Page 11 of 11 Page 11 of 11 43. Messika J, Schmidt M, Aubry A, Combes A, Ricard J-D. Extracorporeal membrane oxygenation-associated infections: carefully consider cannula infections! Crit Care Med. 2018;46(2):e171–2. 42. Grasselli G, Scaravilli V, Di Bella S, Biffi S, Bombino M, Patroniti N, et al. Nosocomial infections during extracorporeal membrane oxygenation: incidence, etiology, and impact on patients’ outcome. Crit Care Med. 2017;45(10):1726–33. References Pre- and post-engraftment bloodstream infection rates and associated mortality in allogeneic hematopoietic stem cell transplant recipients. Transpl Infect Dis Off J Transplant Soc. 2005;7(1):11–7. 41. Gu W, Deng X, Lee M, Sucu YD, Arevalo S, Stryke D, et al. Rapid pathogen detection by metagenomic next-generation sequencing of infected body fluids. Nat Med. 2021;27(1):115–24. 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 Ready to submit your research ? Choose BMC and benefit from: ? 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https://openalex.org/W113834033	https://inria.hal.science/hal-01483814/file/978-3-642-35764-0_15_Chapter.pdf	English	null	A Networked Evidence Theory Framework for Critical Infrastructure Modeling	IFIP advances in information and communication technology	2,012	cc-by	4,396	To cite this version: Chiara Foglietta, Andrea Gasparri, Stefano Panzieri. A Networked Evidence Theory Framework for Critical Infrastructure Modeling. 6th International Conference on Critical Infrastructure Protection (ICCIP), Mar 2012, Washington, DC, United States. pp.205-215, 10.1007/978-3-642-35764-0_15. hal-01483814 Distributed under a Creative Commons Attribution 4.0 International License Abstract This paper describes a distributed approach for data fusion and informa- tion sharing based on evidence theory and the transferable belief model. Evidence theory aggregates data generated from diﬀerent sources in or- der to better assess an ongoing situation and to aid in the response and decision making processes. In the domain of critical infrastructure protection, researchers are forced to develop distributed approaches for modeling and control with a minimal exchange of data due to the exis- tence of multiple stakeholders and interconnections between infrastruc- ture components. Evidence theory permits the modeling of uncertainty in data fusion, but it is typically applied in a centralized manner. This paper proposes a decentralized extension of the transferable belief model that facilitates the application of evidence theory to data fusion in criti- cal infrastructure applications. A case study is provided to demonstrate the convergence of results similar to the centralized approach, and to show the utility of fusing data in a distributed manner for interdepen- dent critical infrastructure systems. Keywords: Modeling, evidence theory, situational awareness, data fusion Chiara Foglietta, Andrea Gasparri and Stefano Panzieri Chiara Foglietta, Andrea Gasparri and Stefano Panzieri HAL Id: hal-01483814 https://inria.hal.science/hal-01483814v1 Submitted on 6 Mar 2017 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 Keywords: Modeling, evidence theory, situational awareness, data fusion 1. Introduction A nation’s critical infrastructure comprises complex, interdependent sys- tems whose proper operation and interaction are essential to the welfare of society. Modeling the interdependencies between the diﬀerent critical infras- tructure sectors is a complex task, but this is vital to correctly analyze and predict cascading phenomena. Interdependencies, from the viewpoint of data fusion, can be analyzed to dis- cern critical events and failures. Events associated with critical infrastructures typically have low probabilities but high impact. The impact is exacerbated 206 CRITICAL INFRASTRUCTURE PROTECTION VI by eﬀects that are not localized and tend to propagate to interconnected in- frastructures. This implies that a “signature” of the event can be identiﬁed in the interconnected system and, sometimes, in independent agencies such as meteorological services or police departments. Therefore, there is a need to fuse together the available data and derive a common belief of the current sit- uation. Developing a common belief facilitates eﬃcient and eﬀective decisions and plans of action. Since critical infrastructures are inherently distributed [5], the fusion mechanism should also be distributed. This paper provides an extension of the transferable belief model to facilitate the application of evidence theory. The existing transferable belief model pro- vides a centralized technique for fusing data. However, a decentralized approach is required for interdependent critical infrastructures. This paper demonstrates the application of an extended decentralized transferable belief model to the domain of critical infrastructure protection and shows how data fusion can help develop more accurate beliefs about interdependent infrastructures. 2. Evidence theory is a methodology that is commonly applied to data fusion problems. Data fusion seeks to combine data from heterogeneous sources or sensors to provide estimates of ongoing events [3]. Evidence theory stems primarily from the pioneering work of Dempster [4] and Shafer [10] and is often considered in the same light as Bayesian networks. The primary diﬀerence, however, is the ability to deal with uncertainty [9]. The Dempster-Shafer theory [10] explicitly considers uncertainty and examines if the various sources of data are inconsistent or if there is an error in the modeling process. On the other hand, Bayesian networks use the recognition of input values as a likelihood from pre-determined patterns. The application of the two approaches depends on the type of knowledge that has to be represented and fused [8]. Several methods have been proposed for combining and correlating the avail- able data in the context of the Dempster-Shafer framework. This work uses the methodology proposed by Smets [11], which extends the Dempster-Shafer framework by assuming that the correct answer might not be among the con- sidered ones (i.e., it engages the open world assumption). Smets’ approach also allows the computation of the amount of contradictory data in the value of the empty set. p y The major limitation to applying evidence theory in a real context is the number of hypotheses required to model the application of interest. This can be explained by the fact that, from a computational perspective, the power set of the set of hypotheses has to be computed, causing the complexity to grow exponentially with the number of hypotheses. However, evidence theory has been successfully applied to a variety of practical problems using approxima- tions (see, e.g., [7, 13]). Data fusion can also aid in impact assessment. In fact, limiting an impact assessment strictly to measured events can lead to heavy underestimation or in- 207 Foglietta, Gasparri & Panzieri correct estimation. For example, an isolated failure can propagate in a number of ways depending on the cause and the detection methods. Examples include a ﬁre blast detected by a sensor and a computer virus detected by an intru- sion detection system. In the ﬁrst example, the ﬁre blast propagation eﬀects are associated with an interdependency model according to a spatial proximity pattern. In the second example, a computer virus may propagate to similar, and highly dispersed, telecommunication nodes. 3. Evidence Theory 3. 3. The rule strongly emphasizes the agreement between multiple sources and ignores conﬂicting evidence using a normalization CRITICAL INFRASTRUCTURE PROTECTION VI 208 factor as shown in the following equation: factor as shown in the following equation: factor as shown in the following equation: Dempster{mi, mj}(∅) = 0 Dempster{mi, mj}(γa) = ! γb∩γc=γa mi(γb)mj(γc) 1 − ! γb∩γc=∅ mi(γb)mj(γc) ∀γa ∈Γ(Ω). Dempster{mi, mj}(∅) = 0 On the other hand, Smets’ rule of combination [11] provides the ability to explicitly express the contradiction in the transferable belief model by letting m(∅) ̸= 0. This combination rule, unlike Dempster’s rule, avoids normalization while preserving commutativity and associativity: Smets{mi, mj}(γa) = mi(γa) ⊗mj(γa) ∀γa ∈Γ(Ω) where where mi(γa) ⊗mj(γa) = ! γb∩γc=γa mi(γb)mj(γc) ∀γa ∈Γ(Ω). The relation m(∅) > 0 can be explained in two ways: (i) open world as- sumption; and (ii) quantiﬁed conﬂict. The open world assumption, proposed by Dempster, reﬂects the idea that the frame of discernment must contain the true value. If the open world assumption is true, then the set of hypotheses must contain all possibilities. Under this interpretation, if ∅is the complement of Ω, the mass m(∅) > 0 represents the case where the truth is not contained in Ω. Alternatively, the notion of quantiﬁed conﬂict means that there is some underlying conﬂict between the sources that are combined to produce the BBA. Hence, the mass assigned to m(∅) represents the degree of conﬂict. Speciﬁcally, it is computed as: mi(∅) ⊗mj(∅) = 1 − ! γa∈Γ,γa̸=∅ (mi(γa) ⊗mj(γa)) . 3. Evidence theory provides a means to form a consolidated belief by correlating evidence from diﬀerent sources [4, 10, 11]. This section provides an overview of the evidence theory formalisms used in this work. Let ωi represent a cause of system failure and Ω= {ω1, · · · , ωn} be the set of hypotheses containing known possible failures. This set is called the “frame of discernment.” For example, the possible causes of failures of a critical infrastructure asset include sabotage, device failure, fault due to weather and a (cyber) denial-of-service attack. Note that the hypotheses are assumed to be mutually exclusive in evidence theory. The power set of the frame of discernment is expressed as Γ(Ω) = {γ1, · · · , γ2\|Ω\|}, which has cardinality \|Γ(Ω)\| = 2\|Ω\|. The power set contains all possible subsets of Ω, including the empty set γ1 = ∅and the universal set γ2\|Ω\| = Ω. , g p y γ γ2 The transferable belief model [11] is derived from the basic belief mass func- tion m: m : Γ(Ω) →[0, 1]. This function, also called the “basic belief assignment” (BBA), maps each el- ement of the power set to a value between 0 and 1. Each BBA is an atomic element in the transferable belief model. In fact, each sensor, agent or node must be able to assign the BBA values by some subjective assumptions or through algorithms that automatically determine the assignment. The BBA function is constrained by: ! γa⊆Γ(Ω) m(γa) = 1 with m(∅) = 0. The transferable belief model examines propositions accordingly as: “the true value of ωi is in γa” where γa ∈Γ(Ω). For γa ∈Γ(Ω), m(γa) is the conﬁdence that supports exactly γa. This implies that the true value is in the set γa; however, due to the lack of additional data, it is not possible to support any strict subset of γa. In the case of diﬀerent independent data sources, a rule is necessary to aggregate the data. Several rules of combination exist in the literature; the most widely used rules are Dempster’s rule [4] and Smets’ rule [11]. Dempster’s rule of combination [4], which was the ﬁrst to be proposed, is a purely conjunctive operation. 4. Consider a network of multiple agents described by an indirect graph G = {V, E} where V = {vi \| i = 1, · · · , nV } is the set of nodes and E = {eij \| (vi, vj)} is the set of edges that represent a communication channel between the nodes. Edges are indirect and, thus, the existence of an arc eij implies the existence of an edge eji. In this work, we assume that no central unit is available to perform data aggregation. Additionally, communications between nodes are limited to the neighbors of the node under consideration (i.e., nodes that are physically or directly connected to the node under consideration). These assumptions are reasonable for data fusion problems in the area of sensor networks. 209 Foglietta, Gasparri & Panzieri Table 1. BBA assignments for a telecommunications network. Table 1. BBA assignments for a telecommunications network. Set Node 1 Node 2 Node 3 m12 m123 ∅ 0.0 0.0 0.0 0.44 0.770 {a} 0.1 0.5 0.7 0.11 0.095 {b} 0.8 0.4 0.2 0.44 0.134 {a,b} 0.1 0.1 0.1 0.01 0.010 A direct consequence of the assumptions, however, is that Smets’ rule of composition cannot be directly applied. Indeed, applying Smets’ rule multiple times over the same BBAs leads to diﬀerent outputs. Note that this could easily happen in a distributed context where communications are local and limited to the one-hop neighborhood. Consider, for example, a scenario where it is necessary to identify the degra- dation of services in a telecommunications network. In the case of extensive delays in packet transmission, it may be necessary to determine if the situation is a temporary congestion or a persistent malicious attack. Network sensors (e.g., intrusion detection systems) can detect a denial-of-service (DoS) attack. The DoS attack may result in cascading eﬀects within the network that are identiﬁed by other sensors. If the attack is conducted on a suﬃciently large scale, entire regions can be compromised, with diﬀerent sensors providing dif- ferent inputs based on localized knowledge. A general method to deﬁne a BBA allocation is to consider the reliability of the data source. Suppose that the data obtained from the source supports a set of hypotheses in Ω. Then, the subset γa of the power set Γ(Ω), containing the set of hypotheses, receives a mass m(γa) equal to the reliability of the source. m1 ⊗m2 = {0.44, 0.11, 0.44, 0.01}. S is the set of local states of each node in the network. while end condition do while end condition do while end condition do Select an edge eij ∈E(t) according to e; Update the states of the selected nodes by applying the operator R: si(t + 1) = si(t) ⊗sj(t) sj(t + 1) = sj(t) ⊗si(t) Set t = t + 1 end g ij ( ) g ; pdate the states of the selected nodes by applying the operator R: g j ( ) g ; Update the states of the selected nodes by applying the operator R: p si(t + 1) = si(t) ⊗sj(t) sj(t + 1) = sj(t) ⊗si(t) If Node 1 then communicates with Node 3 by exchanging data about the possi- ble cause of the congestion, then the result obtained by centralized aggregation is equal to: m12 ⊗m3 = {0.77, 0.095, 0.134, 0.001}. Next, we consider the communications between Node 1 and Node 3 by ap- plying Smets’ operator. The result, which is diﬀerent from the one obtained in the centralized system, is given by: m123 ⊗m3 = {0.8828, 0.0767, 0.0404, 0.0001}. m123 ⊗m3 = {0.8828, 0.0767, 0.0404, 0.0001}. If a decision on the cause of the fault has to be made, then the latter case results in a change of opinion; according to the latest aggregations, the cause is a denial-of-service attack (hypothesis a) instead of network routing congestion (hypothesis b). ( ) As shown, Smets’ rule of combination cannot be directly used in a distributed data aggregation context. The next section presents a distributed algorithm that can update the knowledge of all the nodes in a network. 4. The remaining mass 1−m(γa) is assigned to the universal set because no other data is available. Table 1 shows the BBA assignments for a cause classiﬁcation problem in a telecommunications network using the centralized approach. The network has three sources (nodes) that relay data to determine the probable causes of a network congestion incident. The table shows the BBA values assigned to the various network nodes. The frame of discernment is Ω= {a, b}, where hypothesis a is a denial-of-service attack (i.e., congestion of one or more net- work nodes that intentionally degrades the telecommunications network), and hypothesis b indicates congestion in the telecommunications network due to routing problems. If we assume that Node 1 is coordinating with Node 2, then upon applying Smets’ operator, the result is: m1 ⊗m2 = {0.44, 0.11, 0.44, 0.01}. CRITICAL INFRASTRUCTURE PROTECTION VI 210 Algorithm 1 : Gossip Algorithm Data: t = 0, si(t = 0) ∀i = 1, · · · , n Results: si(tend) ∀i = 1, · · · , n 5. The algorithm proposed by Gasparri, et al. [6] provides the ability to divide the knowledge of each node into two parts: (i) data shared between two nodes; and (ii) localized data retained by each node. In the decentralized algorithm, the network is described by an indirect graph G = {V, E}. A spanning tree T = {V, !E} is derived, where !E ⊆E is available to all nodes. Note that the nodes must have the capacity to save data. Interested readers are referred to [2] for details about spanning tree construction. Communications between nodes are asynchronous and follow the Gossip Pro- tocol [1]. This protocol is formalized as Algorithm 1. The triplet {S, R, e} speciﬁes the network such that: 211 Foglietta, Gasparri & Panzieri Foglietta, Gasparri & Panzieri R is local interaction rule, i.e., for each pair of nodes (i, j) such that ∈E th f ll i ti h ld R is local interaction rule, i.e., for each pair of nodes (i, j) such that eij ∈E, the following equation holds: R is local interaction rule, i.e., for each pair of nodes (i, j) such that eij ∈E, the following equation holds: R : Rq × Rq →Rq where q is the number of elements called “focal sets.” where q is the number of elements called “focal sets.” e is the process of edge selection for which eij ∈E(t) is the selected edge at time t. To deﬁne the operator R, it is ﬁrst necessary to introduce the operator ⊙. Consider two sets of BBAs: mk = {mk(γa) \| ∀γa ∈Γ(Ω)} and mi = {mi(γa) \| ∀γa ∈Γ(Ω)}, such that mk = mi ⊗mj. The operator ⊙can then be deﬁned as: such that mk = mi ⊗mj. The operator ⊙can then be deﬁned as: mj = mk ⊙mi ≜˜mi k. Starting with the power set element with the highest cardinality and exam- ining elements with decreasing cardinality, the value of a BBA can be computed recursively as follows: mj(γa) = mk(γa) − ! γb∩γc=γa,γa⊂γb mj(γb)mi(γc) ! γa⊆γb mi(γb) . It is now possible to introduce the operator R, along with ⊕, to aggregate BBAs of the nodes: mi(t + 1) = mj(t + 1) = mi(t) ⊕mj(t) = { " ˜mj i(t, γa) ⊗˜mj i(t, γa) # ⊗¯mi,j(t, γa), ∀γa ∈Γ(Ω)}. 6. Application Scenario Applying the transferable belief model involves modeling a problem, specify- ing the frame of discernment and selecting the BBAs. Note that the assignment of BBAs is problem dependent and depends signiﬁcantly on the source of infor- mation and knowledge about the system. In this work, we assume that experts provide advice on assigning the BBAs. Other possible approaches are described in [3, 12], where the reliability of the data sources is considered along with the eﬀect of mass assignment on the compound hypotheses. Our scenario involves a supervisory control and data acquisition (SCADA) system that aggregates data from a large number sensors positioned at remote locations. Operators located at a control center manage operations by acquiring system data and updating system parameters. Note that the remote facilities all have data regarding critical events and that the data is generated from diﬀerent sources. Communications between the control center and the devices in the ﬁeld occur via dedicated telecommunications circuits or shared public media such as the Internet. Note that the same communications channels can support information sharing among critical infrastructures to help discern the possible causes of failures. The concept of information sharing across sectors can help prevent cascading eﬀects or prevent the impact from propagating to interconnected infrastructures that have not yet been aﬀected. y Our scenario considers n = 5 interdependent critical infrastructures. Each infrastructure owner determines the BBAs in his/her infrastructure. To discern the cause of a fault, the BBAs have to be aggregated and shared among the ﬁve infrastructures. The frame of discernment is Ω= {a, b, c}, where hypothesis a represents a cyber attack, hypothesis b represents the failure of an isolated single unit, and hypothesis c represents a natural disaster (e.g., earthquake). Table 2 shows the BBA assignments for the ﬁve infrastructure nodes. First, we evaluate the scenario using the centralized algorithm. The results as shown in Table 3. Each column presents the results obtained using Smets’ operator, with column “NN 12” representing the aggregation of Nodes 1 and 2, column “NN 123” representing the aggregation of Nodes 1, 2 and 3, and so on. The last column “C-TBM” shows the results for the centralized transferable belief model. Next, we evaluate the scenario using the distributed algorithm. Table 4 shows the edge selection data. Table 5 shows the output of the R-operator. 212 Gasparri, result as the distributed a tionally, the of Smets’ op 6. A Applying ing the fram of BBAs is p mation and provide advi in [3, 12], wh eﬀect of mas Our scena system that locations. O system data all have dat diﬀerent sou Communi via dedicate Internet. No sharing amo failures. Th cascading eﬀ 212 CRITICAL INFRAS 212 CRITICAL INFRASTRUCTURE PROTECTION VI Gasparri, et al. [6] have shown that the algorithm converges to the same result as the centralized aggregation algorithm. The convergence time of the distributed algorithm is related to the diameter d of the spanning tree. Addi- tionally, the computational complexity of the R-operator is the same as that of Smets’ operator. 5. Note that the term ˜mj i(t, γa) indicates the innovation of node i with respect to the node j, which can be calculated recursively using the operator S: ˜mj i(t, γa) = mi(t, γa) ⊙¯mi,j(t, γa). The element ¯mi,j(t, γa) express the common knowledge (i.e., knowledge ex- changed between two agents (i, j) after the last aggregation) such that: ¯mi,j(t, γa) = n = {0, 0, · · · , 0, 1}. {0.6334, 0.0451, 0.3070, 0.0125, 0.0014, 0.0, 0.0004, 0.0001} {0.6334, 0.0451, 0.3070, 0.0125, 0.0014, 0.0, 0.0004, 0.0001} and are consistent with the centralized transferable belief model outputs. Note that the algorithm terminates when the nature of the edge selection process is known [6]. The results demonstrate that the decentralized approach (Table 5) yields the same values as the centralized approach (Table 3). 6. Application Scenario Each column corresponds to the aggregation of two nodes as related to the sequential timing. 213 Foglietta, Gasparri & Panzieri Table 2. BBA assignments for the ﬁve nodes. Set Node 1 Node 2 Node 3 Node 4 Node 5 ∅ 0.0 0.0 0.0 0.0 0.0 {a} 0.3 0.0 0.0 0.0 0.2 {b} 0.3 0.4 0.1 0.4 0.4 {c} 0.0 0.4 0.5 0.4 0.1 {a,b} 0.3 0.0 0.0 0.0 0.0 {a,c} 0.0 0.0 0.0 0.0 0.0 {b,c} 0.0 0.0 0.3 0.0 0.0 {a,b,c} 0.1 0.2 0.1 0.2 0.3 Table 2. BBA assignments for the ﬁve nodes. Table 3. Centralized algorithm output with incremental aggregation. Set NN 12 NN 123 NN 1234 NN 12345 C-TBM ∅ 0.36 0.468 0.5304 0.6334 0.6334 {a} 0.18 0.108 0.0648 0.0451 0.0451 {b} 0.34 0.346 0.3676 0.3070 0.3070 {c} 0.04 0.046 0.0308 0.0125 0.0125 {a,b} 0.06 0.024 0.0048 0.0014 0.0014 {a,c} 0.0 0.0 0.0 0.0 0.0 {b,c} 0.0 0.006 0.0012 0.0004 0.0004 {a,b,c} 0.20 0.002 0.0004 0.0001 0.0001 Table 3. Centralized algorithm output with incremental aggregation. Set NN 12 NN 123 NN 1234 NN 12345 C-TBM Table 4. Temporal edge selection. Time t=1 t=2 t=3 t=4 t=5 t=6 t=7 Edge e12 e23 e34 e45 e34 e23 e12 Table 4. Temporal edge selection. After each exchange of knowledge between two nodes, the R-operator reveals that the two nodes have the same knowledge value as the operator output. For example, at t = 5, the nodes have the same value: CRITICAL INFRASTRUCTURE PROTECTION VI 214 Table 5. Distributed algorithm output. Set s1 ⊕s2 s2 ⊕s3 s3 ⊕s4 s4 ⊕s5 s3 ⊕s4 s2 ⊕s3 s1 ⊕s2 ∅ 0.36 0.468 0.5304 0.6334 0.6334 0.6334 0.6334 {a} 0.18 0.108 0.0648 0.0451 0.0451 0.0451 0.0451 {b} 0.34 0.346 0.3676 0.3070 0.3070 0.3070 0.3070 {c} 0.04 0.046 0.0308 0.0125 0.0125 0.0125 0.0125 {a,b} 0.06 0.024 0.0048 0.0014 0.0014 0.0014 0.0014 {a,c} 0.0 0.0 0.0 0.0 0.0 0.0 0.0 {b,c} 0.0 0.006 0.0012 0.0004 0.0004 0.0004 0.0004 {a,b,c} 0.20 0.002 0.0004 0.0001 0.0001 0.0001 0.0001 Table 5. Distributed algorithm output. This is important because centralized computation is ill-suited to critical infras- tructure applications due to the associated interdependencies. The case study shows that the decentralized approach produces the same results as the central- ized approach, and also demonstrates the utility of fusing data in a distributed manner for interdependent critical infrastructures. Integrating situational awareness with distributed monitoring is an appeal- ing concept for networked critical infrastructures. This is important because the ability to leverage and share sensor data can signiﬁcantly enhance system resilience and robustness. For example, in the case of a cyber attack, data from intrusion detection systems coupled with data from standard ﬁeld sensors can be combined to obtain more accurate belief assessments. The decentralized approach oﬀers the same advantages as the traditional transferable belief model in terms of its ability to deal with uncertainty. It is important to note, however, that the same disadvantages exist (e.g., expo- nential growth in the computational complexity with respect to the number of hypotheses). Nevertheless, both approaches are useful for modeling beliefs in interdependent infrastructures for the purpose of enhancing situational aware- ness. Acknowledgement This research was partially supported by the European Commission through the FP7 Cockpit CI Project. 7. The decentralized extension of the transferable belief model enables the ap- plication of evidence theory to data fusion in critical infrastructure applications. CRITICAL INFRASTRUCTURE PROTECTION VI References [1] S. Boyd, A. Ghosh, B. Prabhakar and D. Shah, Randomized gossip algo- rithms, IEEE Transactions on Information Theory, vol. 52(6), pp. 2508– 2530, 2006. [2] Y. Dalal, A distributed algorithm for constructing minimal spanning trees, IEEE Transactions on Software Engineering, vol. 13(3), pp. 398–405, 1987. [2] Y. Dalal, A distributed algorithm for constructing minimal spanning trees, IEEE Transactions on Software Engineering, vol. 13(3), pp. 398–405, 1987. [3] S. Das, High-Level Data Fusion, Artech House, Norwood, Massachusetts, 2008. [3] S. Das, High-Level Data Fusion, Artech House, Norwood, Massachusetts, 2008. 215 Foglietta, Gasparri & Panzieri [4] A. Dempster, Upper and lower probabilities induced by a multivalued map- ping, in Classic Works of the Dempster-Shafer Theory of Belief Functions, Studies in Fuzziness and Soft Computing, R. Yager and L. Liu (Eds.), Springer, Berlin-Heidelberg, Germany, pp. 57–72, 2008. [5] S. De Porcellinis, S. Panzieri and R. Setola, Modeling critical infrastruc- ture via a mixed holistic reductionistic approach, International Journal of Critical Infrastructures, vol. 5(1/2), pp. 86–99, 2009. [6] A. Gasparri, F. Fiorini, M. DiRocco and S. Panzieri, A networked transfer- able belief model approach for distributed data aggregation, IEEE Trans- actions on Systems, Man and Cybernetics, Part B, vol. 42(2), pp. 391–405, 2012. [7] J. Hall and J. Lawry, Generation, combination and extension of random set approximations to coherent lower and upper probabilities, Reliability Engineering and System Safety, vol. 85(1-3), pp. 89–101, 2004. [8] F. Jensen and T. Nielsen, Bayesian Networks and Decision Graphs, Springer, New York, 2007. [9] K. Ng and B. Abramson, Uncertainty management in expert systems, IEEE Expert, vol. 5(2), pp. 29–48, 1990. [10] G. Shafer, A Mathematical Theory of Evidence, Princeton University Press, Princeton, New Jersey, 1976. [11] P. Smets and R. Kennes, The transferable belief network, Artiﬁcial Intel- ligence, vol. 66(2), pp. 191–234, 1994. [12] A. Veremme, D. Dupont, E. Lefevre and D. Mercier, Belief assignment on compound hypotheses within the framework of the transferable belief model, Proceedings of the Twelfth International Conference on Information Fusion, pp. 498–505, 2009. [13] F. Voorbraak, A computationally eﬃcient approximation of Dempster- Shafer Theory, International Journal of Man-Machine Studies, vol. 30(5), pp. 525–536, 1989.
https://openalex.org/W4385497949	https://www.frontiersin.org/articles/10.3389/fenvs.2023.1256044/pdf	English	null	Editorial: Urban nature-based solutions and green infrastructure as strategies for climate change adaptation	Frontiers in environmental science	2,023	cc-by	1,552	TYPE Editorial PUBLISHED 02 August 2023 DOI 10.3389/fenvs.2023.1256044 TYPE Editorial PUBLISHED 02 August 2023 DOI 10.3389/fenvs.2023.1256044 TYPE Editorial PUBLISHED 02 August 2023 DOI 10.3389/fenvs.2023.1256044 KEYWORDS COPYRIGHT © 2023 Matos, Monteiro, Santos, Briga-Sá and Imteaz. 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. nature-based solutions, urban resiliency, environmental sustainability, human wellbeing, climate change OPEN ACCESS EDITED AND REVIEWED BY Riccardo Buccolieri, University of Salento, Italy CORRESPONDENCE Cristina M. Monteiro, cmonteiro@ucp.pt Cristina Matos1,2, Cristina M. Monteiro3, Cristina Santos1,4, Ana Briga-Sá2,5 and Monzur Alam Imteaz6 1CIIMAR – Centro Interdisciplinar de Investigação Marinha e Ambiental, Matosinhos, Portugal, 2Escola de Ciências e Tecnologia- Departamento de Engenharias, UTAD - Universidade de Trás os Montes e Alto Douro, Vila Real, Portugal, 3CBQF - Centro de Biotecnologia e Química Fina, Escola Superior de Biotecnologia - Universidade Católica Portuguesa, Porto, Portugal, 4Departamento de Engenharia Civil, FEUP - Faculdade de Engenharia da Universidade do Porto, Porto, Portugal, 5CQ-VR-Centro Química Vila Real, Vila Real, Portugal, 6Swinburne University of Technology, Hawthorn, VIC, Australia Matos C, Monteiro CM, Santos C, Briga-Sá A and Imteaz MA (2023), Editorial: Urban nature-based solutions and green infrastructure as strategies for climate change adaptation. Front. Environ. Sci. 11:1256044. doi: 10.3389/fenvs.2023.1256044 frontiersin.org Editorial on the Research Topic Editorial on the Research Topic Urban nature-based solutions and green infrastructure as strategies for climate change adaptation The consequences of climate change, such as ﬂooding, urban heat island effects, heat waves, and air pollution, pose serious threats to urban environments, which are exacerbated by the signiﬁcant increase in impervious surfaces. The European Union (EU) is committed to tackling these impacts and aims to become the world’s ﬁrst climate-neutral continent, in line with the key climate and energy targets launched last decade. Nature-based Solutions (NbS) are designed to use our environmental resources without signiﬁcantly altering the natural habitat; this not only reduces our carbon footprint but also renders cost-effective solutions. NbS measures are proven to beneﬁt our environment and are likely to increase urban resiliency to climate change. However, as an emerging concept, they need further investigation. This Research Topic focused on urban NbS research (rain gardens and green infrastructure, such as green roofs and green walls) and aimed to welcome a Research Topic of high-quality research outputs on minimizing climate change impacts. The published contributions will allow the scientiﬁc community to shed light on the technical solutions to improve urban sustainability and resiliency, with a focus on NbS solutions to mitigate the effects of climate change. Applegate and Tilt published an interesting paper on how the concept of urban resiliency is operationalized across spatial scales. This paper is an important contribution to the Research Topic, as much of the work about green cities falls under the concepts of urban resiliency, Green Infrastructure (GI), and Ecosystem Services (ES). This study seeks to understand the criteria considered for the planning, development, implementation, and maintenance of urban resiliency at the city and international levels. The present work, by clarifying wide-ranging terms like resiliency, ecosystem services, and Green Infrastructure, performs a comparative analysis that provides a detailed understanding of the similarities and differences between plans Frontiers in Environmental Science Frontiers in Environmental Science 01 frontiersin.org 10.3389/fenvs.2023.1256044 Matos et al. communities, offering an approach to sustainable urban development and establishing positive human-nature connections. Furthermore, the present paper has an innovative feature with respect to the current literature when trying to address the gap related to the design phase and adoption of biophilic design in high-rise buildings and the possible social or cultural impacts that may be experienced as a result. from a national perspective, along with an analysis of city-to-city comparisons. Editorial on the Research Topic The results suggest that there are differences in the focus on key aspects of resiliency and the strategies suggested for resilient cities. Key differences were found in the importance placed on transportation, the future role of GI, and the deﬁnitions of ES, which may have potential impacts on the outcomes of resiliency project development and maintenance. Another important aspect is that urbanization presents sustainability challenges for the natural environment, resources, and ecological systems, while high levels of pollution and disconnection from the natural environment can adversely impact the health and wellbeing of urban residents. The originality of the work by Sturiale et al. lies in the potential of the approach used to become an effective and widely used tool for planning urban GI’s implementation and involving citizens in GI’s co-management (planning and monetary contribution). It was concluded that the involvement of the population will contribute to the easy acceptance of strategic public investments for the implementation of GI and to changing the attitude of citizens in terms of recognition and awareness of the importance of urban greening and the related beneﬁts on climate change mitigation, which for some citizens are still questionable, conﬁrming that there is still a cultural gap that must be urgently addressed. O’Sullivan et al. examined ﬁndings from community focus groups that explored perceptions of a proposed sustainable urban development in Wales, United Kingdom (Biophilic urbanism and biophilic design) that included the incorporation of nature and green infrastructure within the city in order to positively affect human health and wellbeing, in addition to beneﬁting environmental, social, and economic sustainability. This research looked at how community members understood and negotiated possible development impacts on the social, environmental, and economic landscape of the city by drawing on their own experiences. From a different GI perspective, the value of the study by Khajah et al. lies in the design and development of a multistage vertical ﬂow constructed wetland with a tidal ﬂow strategy coupled with a step- feed approach that was able to efﬁciently treat synthetic domestic wastewater, removing high concentration levels of organic and nitrogen loads, through diverse bacterial metabolic pathways. Also approaching social aspects, Sturiale et al. surveyed the citizens of Catania (Italy) and analyzed the perception of GI in tackling the effects of climate change. Editorial on the Research Topic This study revealed the level of awareness of climate change, the value attributed to GI, and ﬁnally the willingness to pay to contribute to the maintenance of GI in the city. The results showed that the citizens involved perceive GI as strategic elements of urban quality of life, although they are not always aware of their positive impact on climate change. This type of study is important because understanding citizens’ views will be a strategic tool for planning and managing public investments in GI as solutions to improving the quality of life in the urban ecosystem. The different contributions show that NbS is on the agenda and that its beneﬁts should be further developed and researched, taking into account social, economic, and environmental aspects. In general, NbS systems are perceived by stakeholders as a cost–intensive burden, as they require signiﬁcant initial investments. However, a payback period analysis considering all social and environmental beneﬁts often depicts a true cost–beneﬁt picture of such a system. Future studies should focus on case studies of such payback period analysis that translate some intangible environmental beneﬁts. Khajah et al. presented work in a completely different area within the NbS. They analyzed the efﬁciency of a multistage vertical ﬂow mesocosm-constructed wetland system, an NbS, in treating domestic wastewater with a high nitrogen (N) load. This study demonstrated the beneﬁts of step-feeding strategies in tidal ﬂow- constructed wetlands as a cost-effective solution to minimize external carbon input and achieve effective N removal. Author contributions CMa: Writing–original draft. CMo: Writing–review and editing. CS: Writing–review and editing. AB-S: Writing–review and editing. MI: Writing–review and editing. Published papers on this Research Topic add signiﬁcant knowledge regarding the implementation of NbS and GI in urban environments in several aspects. Conﬂict of interest Applegate and Tilt published paper, adds knowledge to the current literature, in the systematization criteria on the several terms used to describe city urban resiliency in all the dimensions (planning, development, implementation and maintenance), showing that differences in the way problems are framed can impact resiliency planning at different scales. Furthermore, the novelty of the presented methodology of analyzing planning documents, lies within its possibility to be employed on other cities, allowing its comparison between different scales, and providing detailed understanding of the similarities and differences in resiliency concepts which may have potential impacts on outcomes for resiliency project development and maintenance. 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. Frontiers in Environmental Science 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. The novelty of the paper published by O’Sullivan et al. is related to biophilic urbanism and design and its aim to be sustained by 02 frontiersin.org
https://openalex.org/W2156821941	https://europepmc.org/articles/pmc4382075?pdf=render	English	null	Cognitive Training for Attention-Deficit/Hyperactivity Disorder: Meta-Analysis of Clinical and Neuropsychological Outcomes From Randomized Controlled Trials	Journal of the American Academy of Child and Adolescent Psychiatry	2,015	cc-by	8,992	Cognitive Training for Attention-Deﬁcit/Hyperactivity Disorder: Meta-Analysis of Clinical and Neuropsychological Outcomes From Randomized Controlled Trials Samuele Cortese, MD, PhD, Maite Ferrin, MD, PhD, Daniel Brandeis, PhD, Jan Buitelaar, MD, PhD, David Daley, PhD, Ralf W. Dittmann, MD, PhD, Martin Holtmann, MD, Paramala Santosh, MD, PhD, Jim Stevenson, PhD, Argyris Stringaris, MD, PhD, MRCPsych, Alessandro Zuddas, MD, Edmund J.S. Sonuga-Barke, PhD, on behalf of the European ADHD Guidelines Group (EAGG) Objective: The authors performed meta-analyses of ran- domized controlled trials to examine the effects of cogni- tive training on attention-deﬁcit/hyperactivity disorder (ADHD) symptoms, neuropsychological deﬁcits, and ac- ademic skills in children/adolescents with ADHD. signiﬁcant. There were signiﬁcant effects on laboratory tests of working memory (verbal: SMD ¼ 0.52, 95% CI ¼ 0.24–0.80; visual: SMD ¼ 0.47, 95% CI ¼ 0.23–0.70) and parent ratings of executive function (SMD ¼ 0.35, 95% CI ¼ 0.08–0.61). Effects on academic performance were not statistically signiﬁcant. There were no effects of working memory training, speciﬁcally on ADHD symptoms. In- terventions targeting multiple neuropsychological deﬁcits had large effects on ADHD symptoms rated by most proximal assessors (SMD ¼ 0.79, 95% CI ¼ 0.46–1.12). Method: The authors searched Pubmed, Ovid, Web of Science, ERIC, and CINAHAL databases through May 18, 2014. Data were aggregated using random-effects models. Studies were evaluated with the Cochrane risk of bias tool. Results: Sixteen of 695 nonduplicate records were analyzed (759 children with ADHD). When all types of training were considered together, there were signiﬁcant effects on total ADHD (standardized mean difference [SMD] ¼ 0.37, 95% CI ¼ 0.09–0.66) and inattentive symptoms (SMD ¼ 0.47, 95% CI ¼ 0.14–0.80) for reports by raters most proximal to the treatment setting (i.e., typically unblinded). These ﬁgures decreased substan- tially when the outcomes were provided by probably blinded raters (ADHD total: SMD ¼ 0.20, 95% CI ¼ 0.01– 0.40; inattention: SMD ¼ 0.32, 95% CI ¼ 0.01 to 0.66). Effects on hyperactivity/impulsivity symptoms were not Conclusion: Despite improving working memory per- formance, cognitive training had limited effects on ADHD symptoms according to assessments based on blinded measures. Approaches targeting multiple neuropsycho- logical processes may optimize the transfer of effects from cognitive deﬁcits to clinical symptoms. Key Words: ADHD, nonpharmacological, working memory, executive functions, evidence-based psychiatry J Am Acad Child Adolesc Psychiatry 2015;54(3):164–174. J Am Acad Child Adolesc Psychiatry 2015;54(3):164–174. A h effects4; uncertainty about long-term costs and beneﬁts5; poor adherence6; and negative medication-related attitudes from patients, parents, or clinicians.7 Psychological treatments such as behavioral parent training are also widely used. JOURNAL OF THE AMERICAN ACADEMY OF CHILD & ADOLESCENT PSYCHIATRY VOLUME 54 NUMBER 3 MARCH 2015 Study Selection Articles’ titles and abstracts were screened independently by the ﬁrst 2 authors. Final inclusion was based on the full text. Trials were blindly double coded for eligibility by the ﬁrst 2 authors (S.C., M.F). Disagreement was resolved by the senior author for 3 trials. Search Strategy 1 Sonuga-Barke et al.14 included studies up to April 3, 2012. Here, using the same search strategy, our ﬁnal search date was May 18, 2014. Supplement 1, available online, reports details about the search strategy and syntax for each database. Parallel searches were conducted separately by the ﬁrst 2 authors. A signiﬁcant number of new RCTs of cognitive training for ADHD, not available for inclusion in these previous 2 meta-analyses,9,14 have been published in the past 2 years, reﬂecting the current interest in cognitive training in this ﬁeld. The greater number of trials now available allows a much more deﬁnitive estimate of the effects of cognitive training to be made. In the present article, we update the ﬁrst EAGG cognitive training meta-analysis to include these new trials, and we extend its focus to cover effects on neuro- psychological processes and academic functioning, which were not addressed in the previous EAGG meta-analysis.14 A signiﬁcant number of new RCTs of cognitive training for ADHD, not available for inclusion in these previous 2 meta-analyses,9,14 have been published in the past 2 years, reﬂecting the current interest in cognitive training in this ﬁeld. The greater number of trials now available allows a much more deﬁnitive estimate of the effects of cognitive training to be made. In the present article, we update the ﬁrst EAGG cognitive training meta-analysis to include these new trials, and we extend its focus to cover effects on neuro- psychological processes and academic functioning, which were not addressed in the previous EAGG meta-analysis.14 The focus on neuropsychological processes is important for 2 reasons. First, neuropsychological deﬁcits are postulated to mediate the pathways between originating causes and dis- order onset: improvements in neuropsychological func- tioning may therefore be a prerequisite for ADHD symptom reduction.15 Second, they are associated with functional impairments in their own right, independent of their asso- ciation with ADHD symptomatology, especially in social and academic contexts.16 A broad range of training ap- proaches have been used with ADHD populations. In the METHOD The EAGG protocol for nonpharmacological interventions for ADHD was registered on the International Prospective Register of Systematic Reviews PROSPERO (http://www.crd.york.ac.uk/ PROSPERO, protocol number: CRD42011001393). The same proto- col was followed here. y g g The efﬁcacy of cognitive training for ADHD was addressed in a meta-analysis of nonpharmacological treatments for ADHD by Sonuga-Barke et al.14 on behalf of the European ADHD Guidelines Group (EAGG). This meta-analysis focused solely on RCTs. Importantly, it addressed the issue of blinding by comparing outcomes rated by individuals most proximal to the therapeutic setting (often unblinded and invested in the patient and/or intervention) and those provided by reporters judged to be probably blinded. Effects of cognitive training on ADHD symptoms calculated using unblinded ratings were highly signiﬁcant (standardized mean difference [SMD]¼ 0.64, 95% CI ¼ 0.33–0.95). These effects dropped substantially (SMD ¼ 0.24) and became statistically nonsigniﬁcant (95% CI ¼ 0.24 to 0.72) when probably blinded measures were used. However, these results should be considered as pre- liminary because only 6 RCTs were included. The authors concluded that more evidence was required, especially from trials in which assessments were effectively blinded, before cognitive training could be supported as an ADHD treatment. A second meta-analysis by Rapport et al.,9 published more recently and exploring a wider range of outcomes, found similar effects. However, compared to Sonuga-Barke et al.,14 this more recent meta-analysis included only 2 additional peer-reviewed RCTs with outcomes related to ADHD core symptoms. Moreover, to increase statistical power, Rapport et al.9 also included non-RCTs and pooled across design types, making effect size estimates of the effects of cognitive training on ADHD core symptoms and related neuropsychological impairment difﬁcult to interpret. Inclusion and Exclusion Criteria Only RCTs including interventions aimed to directly train a cogni- tive function were retained. As reported by the Cochrane group,17 to ensure high levels of methodological adequacy and to avoid the inevitable bias caused by dependence on investigators agreeing to provide data from unpublished studies, only published studies were included. Trials were included if participants had an ADHD diag- nosis (any subtype) or met accepted cut-offs on validated ADHD rating scales and were between 3 and 18 years of age. Trials involving children with ADHD comorbid with rare disorders (e.g., fragile X syndrome) only were excluded. Control conditions allowed were “treatment as usual,” “wait list,” “active/placebo/sham” (i.e., involving other forms of computer-based activity or alternative training regimen). Trials were not excluded if patients received medication as part of normal treatment. In an extension of the EAGG protocol,14 trials could be included in this updated meta- analysis despite not reporting an ADHD outcome if they reported neuropsychological and/or academic outcomes. Outcome Measures For consistency with previous EAGG meta-analyses8 and to provide a robust estimate of effects, outcome domains were analyzed only if 5 or more RCTs were available. The outcomes analyzed were: ADHD symptoms (total ADHD as well as inattention and hyperactivity/ impulsivity symptoms), parent ratings of executive functioning (e.g., Behavior Rating Inventory of Executive Function [BRIEF]), standard- ized measures of reading and arithmetic ability, and laboratory-based measures of verbal and visual working memory, inhibition, and atten- tion. For neuropsychological outcomes, only scores from tasks different from those used for training were included in the analysis. The focus on neuropsychological processes is important for 2 reasons. First, neuropsychological deﬁcits are postulated to mediate the pathways between originating causes and dis- order onset: improvements in neuropsychological func- tioning may therefore be a prerequisite for ADHD symptom reduction.15 Second, they are associated with functional impairments in their own right, independent of their asso- ciation with ADHD symptomatology, especially in social and academic contexts.16 A broad range of training ap- proaches have been used with ADHD populations. In the Cognitive Training for Attention-Deﬁcit/Hyperactivity Disorder: Meta-Analysis of Clinical and Neuropsychological Outcomes From Randomized Controlled Trials However, a recent meta-analysis8 found no effects on ADHD symptoms when only ratings by assessors blind to treatment allocation were considered. A ttention-deﬁcit/hyperactivity disorder (ADHD) is a childhood-onset condition characterized by perva- sive patterns of inattention and/or impulsivity- hyperactivity that often persist into later life.1 Combinations of pharmacological and psychological approaches are rec- ommended for its treatment.2 Although medication is efﬁ- cacious in randomized controlled trials (RCT) in the short/ medium-term and is indicated as the ﬁrst-line treatment (at least for severe cases2), it has a number of potential limitations—each affecting some patients. These include the following: partial response or nonresponse3; possible adverse In recent years, cognitive training has been investigated as a potential ADHD treatment.9 Building on evidence of brain plasticity from rehabilitation science and contempo- rary developmental neuroscience, cognitive training is pre- mised on the notion that key brain networks implicated in ADHD can be strengthened, and the cognitive processes they subserve improved, through controlled exposures to information processing tasks.10 Thus, it is argued that cognitive training can reduce ADHD symptoms and improve functioning by targeting neuropsychological deﬁ- cits thought to mediate ADHD pathophysiology. In keeping This article can be used to obtain continuing medical education (CME) at www.jaacap.org. Supplemental material cited in this article is available online. JOURNAL OF THE AMERICAN ACADEMY OF CHILD & ADOLESCENT PSYCHIATRY VOLUME 54 NUMBER 3 MARCH 2015 164 www.jaacap.org COGNITIVE TRAINING IN ADHD meta-analysis by Sonuga-Barke et al.,14 given the small number of studies available, trials with different techniques had to be pooled to generate an effect size estimate. How- ever, given the increased number of trials now available, our aim was to explore training-type speciﬁc effects through the use of subanalyses where sufﬁcient numbers of trials existed. with the complex nature of ADHD neuropsychology,11 cognitive training approaches have targeted a range of def- icits (e.g., attentional control, working memory, inhibitory control). Currently, such training is typically delivered via computers using adaptive procedures, whereby training task difﬁculty is automatically increased across sessions to continually challenge the patient at the boundaries of his or her competence. This has been shown in neuroimaging studies to be necessary for sustaining neuronal changes.12,13 Risk of Bias Assessment Two authors independently assessed trial risk of bias using 5 do- mains of the Cochrane Collaborations tool17: namely, selection bias, performance bias, detection bias, attrition bias, and other bias. If JOURNAL OF THE AMERICAN ACADEMY OF CHILD & ADOLESCENT PSYCHIATRY VOLUME 54 NUMBER 3 MARCH 2015 165 www.jaacap.org CORTESE et al. FIGURE 1 Preferred Reporting Items in Systematic Reviews and Meta-Analyses (PRISMA) ﬂow diagram of selection of studies (last search updated May 18, 2014). Note: aA total of 259 studies were not on attention-deﬁcit/hyperactivity disorder (ADHD); 342 were not on cognitive training; 7 were not randomized controlled trials (RCTs); 47 were reviews; 3 were studies in adults; and 1 was a study protocol. bReasons for exclusion of each study are reported in Table S2, available online. cEgeland et al.24 and Hovik et al.25 refer to the same study. there was disagreement between the 2 raters, the ﬁnal rating was established through consensus with the involvement of the senior author. This occurred for 4 trials. judged to be blinded, whereas for school-based interventions, par- ents were judged to be blinded except where this could be inferred not to be the case from the text20,21 or from e-mail exchange with the authors. As per protocol, where direct observations were available, we selected these over rating-scale scores. This decision was based on the judgement that direct observations are likely, in general, to be better blinded than parent- or teacher-rated outcomes, even when the latter are made in a setting other than the therapeutic setting. Where multiple measures were available for a single outcome (as was sometimes the case for laboratory tasks), the measure most frequently reported across included trials and/or that which was judged to tap the core of the construct was selected. Sensitivity an- alyses were conducted including only trials meeting the following criteria: use of active/sham control; use of working memory training; use of training targeting more than 1 neuropsychological domain (termed here “multiple process training”); and use of no/ low medication (i.e., with <30% of participants receiving medica- tions). We also performed an additional sensitivity analysis excluding the study by Gray et al.,22 in which all participants had a diagnosis of ADHD plus coexisting intellectual disability. Publica- tion bias was assessed with funnel plots and Egger’s tests. Risk of Bias Assessment Finally, we also conducted a meta-regression analysis, using the metareg command in STATA,23 to assess the relationship between age and SMD for most proximal and probably blinded assessments of ADHD core symptoms. This analysis was conducted to establish JOURNAL OF THE AMERICAN ACADEMY OF CHILD & ADOLESCENT PSYCHIATRY VOLUME 54 NUMBER 3 MARCH 2015 www.jaacap.org Data Extraction and Statistical Analysis Trial information was entered into RevMan 5.0.18 Data extraction was independently performed and cross-checked by the ﬁrst 2 au- thors. SMD was calculated as mean pre- to posttreatment change in the intervention group minus the mean pre- to posttreatment change in the control group divided by the pooled pretest standard devia- tion with a bias adjustment.19 SMDs for each trial were combined using the inverse variance method. Given the inherent heterogeneity of studies, random effects models were used. The I2 statistic was calculated a posteriori to estimate between-trial SMD heterogeneity. For the most proximal analysis, parent ratings, if available, were used for home-based interventions, and teacher ratings were used for school-based interventions, except when it could be inferred from the manuscript’s text that teachers were less blinded than parents for home-based interventions and parents less blinded than teachers for school-based interventions (2 trials20,21). Probably blin- ded assessments were those made by an individual judged likely to be unaware of treatment allocation. In trials in which more than 1 such measure was available, the best-blinded measure was chosen. Data Extraction and Statistical Analysis For home-delivered interventions, teachers’ ratings were usually JOURNAL OF THE AMERICAN ACADEMY OF CHILD & ADOLESCENT PSYCHIATRY VOLUME 54 NUMBER 3 MARCH 2015 166 www.jaacap.org e Meta-Analysis Training Sample Outcomes th ng ys) FU Type Setting n T C Meds T (%) C (%) Age (mo) ADHD M-Prox ADHD P-Blind Included Neuropsychology Outcomes Academic Functioning U: mo WMT RoboMemo43 School/home 26 27 0a 0 116 (mean) Parent Teacher Digit span (verbal WM); span board (visual WM); stroop accuracy (inhibition) N/A FU Attention training Clinic 20 16 0b 0b 72e156 Parent Parent N/A In-house tests FU Inhibitory and WMT Home 20 20 47c 78c 95e149 Parent Parent No go errors % (inhibition) N/A U: hin y Attention training Captain’s Log44 School/home 25 25e 7 NS Teacher N/A N/A Woodcock- Johnson test 0 FU Attention/WMT BrainTrain44 School 13 15 60 148.8 10.8 (mean) Parent Teacher N/A N/A FU Attention training AixTent Welfare service, home or lab 16 16 100 100 124e138 N/A N/A Vigilance omissions (inattention) N/A U: wk Adaptive inhibitory training and WMT Home 22 20 90 95e145 Parent NA Counting span (verbal WM); Go NoGo, RT incongruent stimuli (inhibition); oddball task correct (attention) N/A FU Adaptive WMT RoboMemo43 School 32 20 98 144e204 Teacher N/A Digit span back (verbal WM); CANTAB spatial WM (visual WM); D2 test total (attention) Wide-Range Achieve- ment FU Adaptive WMT RoboMemo43 Home 12 14 67 14 84e168 Parent Parent WISC index (verbal WM) N/A U: wk Adaptive EF training (inhibition, WM, ﬂexibility) Home 18 22 66 96e144 Parent Teacher N/A N/A FU Adaptive attention training Pay Attention! School 45 46 65 73 84e180 Parent Clinician Digit span (verbal WM); D-KEFS scaled score (inhibition), omissions (inhibition) N/A CORTESE et al. ADHD Symptoms ( ADHD Symptoms ADHD symptoms (total score or inattention or hyperactivity/ impulsivity separately) were an outcome in up to 14 trials. Probably blinded measures were available in up to 11 trials (Table 2). ADHD Symptoms ( When most proximal assessments were the outcome, there was a moderate but signiﬁcant effect on total ADHD and in- attentionsymptomsbutnoeffectonhyperactivity/impulsivity TABLE 2 Summary of Results Showing Pooled Standardized Mean Differences (SMD) Between Treatment and Control Arms for E h O t TABLE 2 Summary of Results Showing Pooled Standardized Mean Differences (SMD) Between Treatment and Control Arms for Each Outcome esults Showing Pooled Standardized Mean Differences (SMD) Between Treatment and Control Arms for TABLE 2 Summary of Results Showing Pooled Standardized Mean Differences (SMD) Between Treatm Each Outcome ummary of Results Showing Pooled Standardized Mean Differences (SMD) Between Treatment and Con TABLE 2 Summary of Results Showing Pooled Standardized Mean Differences (SMD) Between Treatment and Control Arms for Each Outcome Outcome Trials Included Measure Study n Effect Heterogeneity SMD 95% CI p I2 p ADHD total All MProx 14 0.37 0.09 to 0.66 .01 71 <.001 PBlind 11 0.20 0.01 to 0.40 .04 30 .16 Active control MProx 7 0.16 0.23 to 0.55 .41 71 <.001 PBlind 6 0.22 0.09 to 0.53 .17 42 .13 WMT MProx 6 0.00 0.31 to 0.31 1.00 56 .05 MPT MProx 5 0.79 0.46 to 1.12 <.001 36 .18 MED MProx 5 0.19 0.16 to 0.54 .30 56 .06 PBlind 5 0.11 0.10 to 0.32 .31 0 .74 Inattention All MProx 11 0.47 0.14 to 0.80 <.01 76 <.001 PBlind 9 0.32 0.01 to 0.66 .06 69 <.001 Active control MProx 5 0.30 0.17 to 0.76 .21 72 <.001 WMT MProx 5 0.22 0.18 to 0.62 .28 66 <.001 MED MProx 5 0.35 0.09 to 0.79 .29 71 .02 Hyper/Imp All MProx 9 0.14 0.07 to 0.35 .18 28 .28 PBlind 8 0.18 0.01 to 0.37 .06 0 .50 Active control MProx 5 0.01 0.25 to 0.22 .91 0 .60 WMT MProx 5 0.02 0.24 to 0.21 .89 0 .68 Executive function rating All MProx 6 0.35 0.08 to 0.61 .01 22 .22 Working memory (visual) All Objective 5 0.47 0.23 to 0.70 <.01 69 <.001 Active control Objective Insufﬁcient trials (n ¼ 4) WMT Objective 5 0.47 0.23 to 0.70 <.01 69 <.001 Working memory (verbal) All Objective 8 0.52 0.24 to 0.80 <.01 48 .06 Active control Objective 5 0.58 0.23 to 0.94 .001 45 .12 WMT Objective 6 0.57 0.29 to 0.82 <.001 32 .19 Inhibition All Objective 6 0.07 0.15 to 0.28 .53 2 .4 Attention All Objective 7 0.14 0.19 to 0.48 .41 58 .03 Reading All Standardized tests 5 0.09 0.09 to 0.27 .33 23 .26 Arithmetic All Standardized tests 5 0.01 0.13 to 0.11 .84 0 .44 Note: Signiﬁcant effects are expressed in boldface. Data Extraction and Statistical Analysis Sample Outcomes Meds T (%) C (%) Age (mo) ADHD M-Prox ADHD P-Blind Included Neuropsychology Outcomes Academic Functioning 27 32 84e132 Parent Teacher AWMA listening (verbal WM); dot matrix (visual WM); CPT commissions (inhibition); omissions (attention) Wide-Range Achieve- ment 68 120e144 Teacher N/A Stroop interference score (inhibition; CPT focus (attention) Logos Test 68 120e144 Teacher N/A Digit span (verbal WM); Leiter visual span (visual WM) N/A 41 55 100.8 14.8 (mean) Parent Direct observa- tion (BOSS) N/A N/A 0 0 71.5e87.6 Investigator Teacher Digit span (verbal WM); Knox Cubes (visual WM); Stroop difference (inhibition); Sustained attention dots: SDRT (attention) N/A able S1, available online; long-term follow-up is listed under “Length of Training” after ﬁrst outcome ADHD ¼ attention-deﬁcit/hyperactivity disorder; AWMA ¼ automated working memory assessment; d Battery; D-KEFS ¼ Delis-Kaplan Executive Function System; EF ¼ executive functions; FU ¼ follow-up; not speciﬁed; PBlind ¼ probably blinded rater; RT ¼ reaction time; SDRT ¼ Spatial Delayed Response ning. n 1 week before the study, and the other participants were stimulant naive. of the study. None were medicated either during the training sessions or during the pre- and posttesting lysis. aining in attention deﬁcit hyperactivity disorder. Aula Abierta 2012;40:55-60. analysis for consistency with interventions included in the other studies retained in the meta-analysis. g Setting MT 43 Home 4 4 MT 43 School 3 3 MT 43 School 3 3 on School 3 3 MT 3) Home, except for 1 participant 2 2 dy reference number, as i T) and control (C) condition ropsychological Test Autom rking memory training; NS WMT ¼ working memory continued stimulant medica mulants throughout the dura fore testing. dered for the present meta- Stasik D, Tucha O. Attentio included in the present me ent outcomes. e W emo W emo W emo atten WM W med emo Type tive oMe tive oMe tive oMe ve a d W tive Cogm oMe wed b eatm ngth of ining ays) d FU 35 o FU A 25 FU: mo A 25 FU: mo A 91 o FU Ad 35 o FU A n and are dividuals in ANTAB ¼ C -WMT ¼ no gence Scale efore the stu ol group rece dication in t truction that omputer-ass layattention a L, Hauser oring” arm, d present an COGNITIVE TRAINING IN ADHD whether the efﬁcacy of cognitive training varied across age, a ﬁnding that could be of clinical signiﬁcance. Data Extraction and Statistical Analysis 1 trial in the clinic, and 1 at the welfare service/children center, home or laboratory. Five trials had no/low medication levels. Figure S1 (available online) depicts the graphic output for the risk of bias assessment. Risk of bias was generally low or unclear. No trials were scored as “high risk” with regard to “random sequence generation,” “allocation concealment,” and “incomplete outcome data,” and only 3 and 4 trials scored high for “blinding of participants/personnel” and “blinding of outcome assessment,” respectively (the rating of each study is available upon request). RESULTS Fifteen trials (reported in 16 papers) met entry criteria (see Table S1, available online). Studies not included in the meta- analysis are listed (with reasons for their exclusion) in Table S2 (available online). Figure 1 reports the trial selection ﬂowchart. Table 1 gives information about retained trials. Results of all analyses are summarized in Table 2. Six trials were on working memory training, 4 on attention training, 2 combined attention and working memory training, 2 inhibi- tion and working memory training, and 1 trial provided a general executive function training covering working mem- ory, inhibition, and cognitive ﬂexibility. All training schedules had an “adaptive” component, that is, task difﬁculty was increased across sessions to track performance improvement. Eight trials had an active control condition. Six trials were implemented at home, 5 at school, 2 at either school or home, Note: Signiﬁcant effects are expressed in boldface. Table reports only measures for which 5 or more trials were available. Where only most proximal rater (MProx) is reported, there were insufﬁcient trials with probably blinded rater (PBlind) measures. Active controls ¼ all trials with an active control arm such as easy or non-adaptive training; ADHD ¼ attention-deﬁcit/hyperactivity disorder; All ¼ all trials meeting inclusion criteria with available measure; MED ¼ trials in which <30% of participants were treated with ADHD medication; MPT ¼ multiple process training; WMT ¼ all trials using just working memory training. ADHD Symptoms ( Table reports only measures for which 5 or more trials were available. Where only most proximal rater (MProx) is reported, there were insufﬁcient trials with probably blinded rater (PBlind) measures. Active controls ¼ all trials with an active control arm such as easy or non-adaptive training; ADHD ¼ attention-deﬁcit/hyperactivity disorder; All ¼ all trials meeting inclusion criteria with available measure; MED ¼ trials in which <30% of participants were treated with ADHD medication; MPT ¼ multiple process training; WMT ¼ all trials using just working memory training. Note: Signiﬁcant effects are expressed in boldface. Table reports only measures for which 5 or more trials were available. Where only most proximal rater (MProx) is reported, there were insufﬁcient trials with probably blinded rater (PBlind) measures. Active controls ¼ all trials with an active control arm such as easy or non-adaptive training; ADHD ¼ attention-deﬁcit/hyperactivity disorder; All ¼ all trials meeting inclusion criteria with available measure; MED ¼ trials in which <30% of participants were treated with ADHD medication; MPT ¼ multiple process training; WMT ¼ all trials using just working memory training. JOURNAL OF THE AMERICAN ACADEMY OF CHILD & ADOLESCENT PSYCHIATRY VOLUME 54 NUMBER 3 MARCH 2015 JOURNAL OF THE AMERICAN ACADEMY OF CHILD & ADOLESCENT PSYCHIATRY VOLUME 54 NUMBER 3 MARCH 2015 169 www.jaacap.org CORTESE et al. and signiﬁcant effect of cognitive training on both compo- nents (Figure 3), which was maintained in sensitivity ana- lyses considering trials with active controls only or working memory training trials only (Figure S5, available online; sensitivity analyses were not performed for visual working memory because of an insufﬁcient number of trials). The number of trials using multi-component training and no/ low medication trials was insufﬁcient to perform sensitivity analyses. There were no signiﬁcant effects of training on laboratory tests of inhibition (6 trials) or attention (7 trials) (Figure 3). Six trials included most proximal ratings of ex- ecutive functioning using the BRIEF rating scale (Figure S6, available online). These demonstrated a small-to-moderate, signiﬁcant SMD. There was an insufﬁcient number of trials with ratings of executive functioning to perform planned sensitivity analyses. (Figure 2; SMD and CI data for all outcomes are presented in Table 2). In sensitivity analyses (Figures S2 and S3, available online), considering only trials with an active control, the effects were no longer statistically signiﬁcant for any ADHD core symptoms outcomes. ADHD Symptoms ( There was no effect of working memory training when implemented on its own (Figures S2 and S3, available online). In contrast, multi-process training approaches (i.e., approaches targeting more than 1 neuropsy- chological domain) gave a large effect size for total ADHD symptoms (Figure S2, available online). Between-study heterogeneity of effect sizes was high and signiﬁcant for total ADHD and inattention symptoms. When analyses were restricted to probably blinded measures (Figure 2), in general, effect sizes were reduced with small and statistically marginal effects for all ADHD outcomes. In a sensitivity analysis (Figure S4, available on- line), effect sizes dropped further to nonsigniﬁcant levels when only trials with an active control arm were included. There were insufﬁcient studies (n < 5) for an analysis of probably blinded measures in multi-component training trials, as well as for a number of other sensitivity analyses. Academic Ability Five trials included standardized measures of reading and 5 of arithmetic. There were no signiﬁcant effects in either domain (Figure 3). There was an insufﬁcient number of trials to perform planned sensitivity analyses. y y When analysis was restricted to no/low medication trials, effects on total ADHD symptoms were not signiﬁcant for either most proximal or, when available, probably blinded assess- ments in any ADHD core symptom–related outcome (Table 2). Publication Bias FIGURE 3 Forest plots for meta-analysis of effects on neuropsychological and academic outcomes. Note: Cogn ¼ cognitive; std ¼ standard. FIGURE 3 Forest plots for meta-analysis of effects on neuropsychological and academic outcomes. Note: Cogn ¼ cognitive; std ¼ standard. FIGURE 3 Forest plots for meta-analysis of effects on neuropsychological and academic outcomes. Note: Cogn ¼ cognitive; std ¼ standard FIGURE 3 Forest plots for meta-analysis of effects on neuropsychological and academic outcomes. Note: Cogn ¼ cognitive; std ¼ standard. blinded raters, the test failed to reach the p < 0.05 level, suggesting no signiﬁcant publication bias. blinded raters, the test failed to reach the p < 0.05 level, suggesting no signiﬁcant publication bias. RCTs compared to the previous study by Sonuga-Barke et al.,14 provided little support for cognitive training as a front-line ADHD treatment. There were statistically signiﬁ- cant effects on ADHD symptoms when considering raters most proximal to treatment delivery, especially for symp- toms of inattention. However, these effects were reduced substantially when analyses were limited to trials with an active control arm or where assessors were probably blind to treatment allocation. The evidence was somewhat stronger for the beneﬁts of cognitive training as an adjunctive treat- ment aimed at reducing neuropsychological impairment. There were large and highly signiﬁcant improvements on objective tests of both visual and verbal working memory, although there were no effects on inhibition or inattention. Furthermore, the effects of cognitive training on working memory did not extend to the academic outcomes explored. Meta-Regression Analysis For most proximal or probably blinded assessments of ADHD core symptoms, there was no signiﬁcant effect of age on SMD (see Supplement 3, available online). Sensitivity Analysis Excluding the Study by Gray et al.22 The main results considering most proximal assessment of ADHD core symptoms were substantially unchanged, as reported in Figure S7 (available online). As this study was not included in “probably blinded” analyses, no sensitivity analysis was conducted considering probably blinded assessment. The substantial drop in SMDs between most proximal and probably blinded analyses for ADHD symptoms is similar to the pattern seen in previous meta-analyses of nonpharmacological treatments using the EAGG protocol (e.g., behavioral intervention;8 neurofeedback14). This is probably caused by the inﬂation of effect size estimates that inevitably occurs when one relies on raters who are both likely to be aware of treatment allocation and heavily invested in the delivery and outcome of treatment. FIGURE 3 Forest plots for meta-analysis of effects on neuropsychological and academic outcomes. Note: Cogn ¼ cognitive; std ¼ standard. It is also possible that probably blinded and most proximal assess- ments differed in some way that reduced the sensitivity of the former to treatment-related change. However, the same measurement approaches were used for each (some parent, JOURNAL OF THE AMERICAN ACADEMY OF CHILD & ADOLESCENT PSYCHIATRY VOLUME 54 NUMBER 3 MARCH 2015 www.jaacap.org Neuropsychological Outcomes Neuropsychological Outcomes Funnel plots and results of Egger’s test are reported in Supplement 2 (available online). For both meta-analyses of ADHD symptoms scored by most proximal and probably Eight trials included laboratory measures of verbal, and 5 trials visual working memory (Table 2). There was a large FIGURE 2 Forest plots for meta-analysis of effects on attention-deﬁcit/hyperactivity disorder (ADHD) core symptoms assessed by the most proximal and probably blinded raters. Note: Cogn ¼ cognitive; std ¼ standard. JOURNAL OF THE AMERICAN ACADEMY OF CHILD & ADOLESCENT PSYCHIATRY 70 www.jaacap.org VOLUME 54 NUMBER 3 MARCH 2015 JOURNAL OF THE AMERICAN ACADEMY OF CHILD & ADOLESCENT PSYCHIATRY VOLUME 54 NUMBER 3 MARCH 2015 JOURNAL OF THE AMERICAN ACADEMY OF CHILD & ADOLESCENT PSYCHIATRY VOLUME 54 NUMBER 3 MARCH 2015 170 www.jaacap.org COGNITIVE TRAINING IN ADHD DISCUSSION In this regard, our ﬁndings suggest that choosing substrates that have emerged from experimental research as treatment targets may not necessarily translate into clinical beneﬁts. This possible dissociation between candidate mechanisms of a disorder and clinical targets is important when adopting pathophysiology-based research approaches such as the Research Domain Criteria (RDoC).40 From a clinical standpoint, developing techniques to extend transfer from the effects on core working memory processes to broader neuropsychological processes and important domains of impairment and/or clinical presentation is the most pressing challenge for the future. The reasons for the lack of effect on inhibitory and attentional control are hard to determine on the basis of the current analysis, given the small number of trials that speciﬁcally targeted these do- mains. Although we might predict that training targeting multiple deﬁcit domains would show effects on these neu- ropsychological processes, there were insufﬁcient trials with multi-component training and measures of inhibition and/ or attention to test this. Approaches focusing on motiva- tional or energetic processes may also be valuable (i.e., training to increase delay of gratiﬁcation).41 The trials included in the meta-analysis used a wide range of training approaches targeting different neuropsy- chological processes. There was a sufﬁcient number of trials to look at 2 classes of intervention individually, which was not possible in the previous meta-analysis by Sonuga-Barke et al.14: namely, training of working memory only, and training focusing on multiple neuropsychological domains. The results for trials implementing working memory training only departed in a striking way from the most proximal/probably blinded pattern described above. Effects on ADHD were negligible even considering most proximal measures. This suggests that this form of training, which has been widely promoted for use with patients with ADHD (as discussed by Rapport et al.,9), has little or no efﬁcacy for core ADHD symptoms. On the other hand, the SMD for most proximal assessment of ADHD symptoms was substantially larger for trials based on training targeting multiple domains than for all studies as a whole. Unfortunately, there was an insufﬁcient number of trials (n ¼ 4) with probably blinded measures to corroborate these effects using independent sources. The superiority of these approaches may be due to the typically greater number of training sessions in multi- compared to single-component approaches (in our analysis, an average of 9 weeks compared to 6 weeks, respectively). DISCUSSION There are 2 perspectives on cognitive training in ADHD. From 1 perspective, cognitive training is a front-line ADHD treatment: this is based on the hypothesis that because causal pathways to disorder are mediated by neuropsychological deﬁcits, strengthening deﬁcient neuropsychological functions should reduce ADHD symptoms and associated impairment. From the second perspective, it is perceived as an adjunctive treatment that reduces impairment associated with neuro- psychological deﬁcits commonly seen in children with ADHD, independent of any effects on core ADHD symptoms itself. The current meta-analysis, including an additional 10 JOURNAL OF THE AMERICAN ACADEMY OF CHILD & ADOLESCENT PSYCHIATRY VOLUME 54 NUMBER 3 MARCH 2015 171 www.jaacap.org CORTESE et al. ﬁnding may be relevant in clinical practice. Indeed, parents may currently favor cognitive training with the hope that they can improve academic performance. Our results show that this is not supported by empirical evidence. some teacher, and some direct observation measures). Another possibility is that most proximal assessments accurately captured treatment effects established in the therapeutic setting but that these effects did not generalize to the settings in which probably blinded assessments were made. However, in a substantial minority of trials (Table 1), especially those with an active control arm, probably blin- ded measures were collected in the treatment setting, and the effects for these trials were no larger than those for trials in which they were collected in a different setting. The success of working memory training in improving working memory performance draws into even sharper re- lief its failure to improve ADHD symptoms, suggesting dissociation between neuropsychological functioning and disorder. There are 4 possible explanations for this: ﬁrst, that working memory deﬁcits do not, in fact, mediate ADHD pathophysiology39; second, that although they do mediate the development of ADHD, they have become entrenched and not susceptible to the type of training implemented in trials conducted to date; third, that training as currently implemented targets types of working memory not funda- mental to the deﬁcits in ADHD9; and fourth, that training produces only peripheral, practice-like effects on working memory, with no profound impact on the brain networks underpinning neuropsychological deﬁcits responsible for ADHD. Whether or not working memory deﬁcits are part of the causal mechanism underpinning ADHD, based on our results, strengthening working memory appears to be neither a necessary nor a sufﬁcient condition for ADHD symptom reduction. JOURNAL OF THE AMERICAN ACADEMY OF CHILD & ADOLESCENT PSYCHIATRY VOLUME 54 NUMBER 3 MARCH 2015 www.jaacap.org DISCUSSION Dittmann has received compensation for serving as a consultant or speaker for, or he or the institution he works for have received research support or royalties from the companies or organizations indicated: the European Union (EU FP7 Pro- gramme), US National Institute of Mental Health (NIMH), German Federal Ministry of Health/Regulatory Agency (BMG/BfArM), German Federal Ministry of Education and Research (BMBF), German Research Foundation (DFG), Volkswagen Foundation, Ferring, Janssen-Cilag, Eli Lilly and Co., Otsuka, Shire, and Theravance. He owns Eli Lilly and Co. stock. Prof. Holtmann has served as an advisor or consultant for Eli Lilly and Co., Novartis, Shire, and Bristol-Myers Squibb and has received conference attendance support or was paid for public speaking by AstraZeneca, Bristol- Myers Squibb, Janssen-Cilag, Eli Lilly and Co., Medice, Neuroconn, Novartis, and Shire. Dr. Santosh has received research funding from the EU (FP7 Programme) and the National Institute for Health Research (NIHR). He is also a director and shareholder of HealthTracker, Ltd., UK, and HighStreet Medical Dental, Gillingham, UK. Dr. Stringaris has received grant or research support from the Wellcome Trust, the NIHR, and the Department of Health UK. He has received royalties from Cambridge University Press for his book The Maudsley Reader in Phenomenological Psychiatry. Prof. Zuddas has received research grants from the EU (FP7 Programme), Italian National Institute of Health, Sardinian Secretary of Public Health, Eli Lilly and Co., Shire, and Vifor and has served as speaker, adviser, or consultant for AstraZeneca, Bristol- Myers Squibb, Eli Lilly and Co., Lundbeck, Schering-Plough, Shire, and Vifor. He has been a member of Data Safety Monitoring Boards for Otsuka and Lundbeck. Prof. Sonuga-Barke has served on the speakers’ board of Shire and Janssen-Cilag; has served as a consultant to Shire and NeuroTechnology Solutions, Ltd.; has received research support from Janssen-Cilag and Shire; has served on the advisory board of Shire and NeuroTechnology Solutions, Ltd.; and has received conference support from Shire. Profs. Brandeis and Stevenson report no biomedical ﬁnancial interests or potential conﬂicts of interest. In summary, the current meta-analysis found limited evidence for the clinical value of cognitive training for chil- dren with ADHD outside of the narrow conﬁnes of speciﬁc targeted neuropsychological processes (i.e., working mem- ory training improved working memory function). Given the evidence for neuropsychological heterogeneity in ADHD, future efforts should be directed at developing protocols to target a broader range of neuropsychological deﬁcits. DISCUSSION However, the ﬁnding opens up the interesting possibility that multi-component training models may be more successful for ADHD, given the complex and heterogeneous nature of the condition. Because children with ADHD differ from one another in their neuropsychological proﬁle, and because children may be affected by more than 1 deﬁcit,37,38 multi- component training may be used to target a series of neuro- psychological domains that may be more important than working memory alone in the pathophysiology of ADHD symptoms. The development and evaluation of multi- component training models should be a future priority. A number of limitations need to be taken into account when interpreting the current analysis. First, there was signiﬁcant SMD heterogeneity for some analyses (most proximal total ADHD, symptoms of inattention, and visual working mem- ory). This leaves open the possibility that cognitive training may be effective under speciﬁc circumstances in individual trials. Given the limited number of trials available, we were unable to identify speciﬁc features of positive trials (apart from therapeutic content, working memory training). Second, only a minority of trials (n ¼ 5) reported using intention-to-treat analyses, a situation that may have inﬂated the effects for some outcomes, as participants who are more difﬁcult to treat or who perceive the treatment as less beneﬁcial may drop out of trials; however, drop-out was relatively low in most trials. The effects on neuropsychological outcomes were restricted to working memory, which were substantial, with no effects on inhibitory or attentional control. There were signiﬁcant effects on parents’ ratings of executive function, but these could not be corroborated by independent blinded evidence. All 6 trials that included a working memory outcome were working memory training trials. Therefore, although these trials produced “near transfer” of training effects to untrained working memory measures, there was no evidence of “far transfer” to other neuropsychological processes. Crucially, there was also no evidence that these effects generalized to important areas of everyday func- tioning, which themselves are inﬂuenced by working memory ability,16 such as reading and arithmetic. This JOURNAL OF THE AMERICAN ACADEMY OF CHILD & ADOLESCENT PSYCHIATRY VOLUME 54 NUMBER 3 MARCH 2015 172 www.jaacap.org COGNITIVE TRAINING IN ADHD Third, despite the recent substantial increase in the number of available cognitive training trials, there was an insufﬁcient number of trials to evaluate training approaches targeting speciﬁc neuropsychological constructs other than working memory training. DISCUSSION Fourth, there were insufﬁcient trials to run analyses for some important outcomes (e.g., functional impairment, IQ), as well as for sensitivity analyses or analyses restricted to probably blinded measures for a number of out- comes. Fifth, too few trials included long-term outcomes (Table 1) toallowanevaluationoftheextenttowhich effects on clinical symptoms grew over time or effects on neuropsycho- logical processes persisted. Sixth, no trials were restricted to individuals with both ADHD and the speciﬁc neuropsycho- logical deﬁcit to be trained. As a consequence, effect sizes for both neuropsychological deﬁcits and ADHD symptoms may have beentruncated:intheformer case becausethere wouldbe little room for improvement where no deﬁcit existed; in the latter case because targeting a neuropsychological deﬁcit that was not causing the condition would be unlikely to reduce symptoms of the core condition. Seventh, in the neuropsy- chologicaldomains,diversemeasuresfromdifferenttasks(still tapping the same domain, however) were combined across studies to allow the calculation of pooled SMD estimates. Eighth, it is important to understand whether initial symptom- related and neuropsychological treatment effects persist over time and generalize to other domains, if they do. There were insufﬁcient trials that examined long-term outcomes to address this issue. Finally, the categorization of studies as “probably blinded,” although carried out according to previ- ously agreed and clear decision rules set out in the protocol, is limited by an inevitable degree of uncertainty because of lim- itations in the information reported in some trials. Mannheim, Heidelberg University, Germany. Prof. Brandeis is also with the University of Zurich, Switzerland. Prof. Buitelaar is with the Donders Institute for Brain, Cognition and Behaviour, Radboud University Medical Centre, Nij- megen, The Netherlands and the Karakter Child and Adolescent Psychiatry University Centre, Nijmegen. Prof. Holtmann is with the LWL-University Hos- pital for Child and Adolescent Psychiatry, Ruhr University, Bochum, Germany. Prof. Zuddas is with the Unit of Child Neuropsychiatry, University of Cagliari, Cagliari, Italy. Mannheim, Heidelberg University, Germany. Prof. Brandeis is also with the University of Zurich, Switzerland. Prof. Buitelaar is with the Donders Institute for Brain, Cognition and Behaviour, Radboud University Medical Centre, Nij- megen, The Netherlands and the Karakter Child and Adolescent Psychiatry University Centre, Nijmegen. Prof. Holtmann is with the LWL-University Hos- pital for Child and Adolescent Psychiatry, Ruhr University, Bochum, Germany. Prof. Zuddas is with the Unit of Child Neuropsychiatry, University of Cagliari, Cagliari, Italy. JOURNAL OF THE AMERICAN ACADEMY OF CHILD & ADOLESCENT PSYCHIATRY VOLUME 54 NUMBER 3 MARCH 2015 Accepted December 24, 2014. Dr. Cortese and Profs. Stevenson and Sonuga-Barke are with the Develop- mental Brain-Behaviour Laboratory, University of Southampton, UK. Dr. Cor- tese is also with the School of Medicine, University of Nottingham, UK and the New York University Child Study Center, New York. Prof. Sonuga-Barke is also with Ghent University, Belgium and Aarhus University, Denmark. Prof. Daley is with the School of Medicine, University of Nottingham, UK and the Centre for ADHD and Neurodevelopmental Disorders Across the Lifespan, Institute of Mental Health, University of Nottingham, UK. Drs. Ferrin, Santosh, and Stringaris are with the King’s College London, Institute of Psychiatry, UK. Dr. Ferrin is also with the Centro de Salud Mental de Estella, Navarra, Spain and the Huntercombe Hospital Maidenhead, UK. Profs. Brandeis and Ditt- mann are with the Central Institute of Mental Health, Medical Faculty DISCUSSION Support for meetings and analyses was received from Brain Products GMBH, Janssen-Cilag, Eli Lilly and Co., Medice, Shire, and Vifor. No honoraria were received, and funders had no input for the review and meta-analysis process or the writing of this article. The European ADHD Guidelines Group (EAGG) is a workgroup of the Euro- pean Network for Hyperkinetic Disorder (EUNETHYDIS) and consists of the following members and associates co-opted to work on this review (listed in alphabetical order): T. Banaschewski, MD, PhD; D. Brandeis, PhD; J. Buite- laar, MD, PhD; D. Coghill, MD; S. Cortese, MD, PhD; D. Daley, PhD; M. Danckaerts, MD, PhD; R.W. Dittmann, MD, PhD; M. D€opfner, PhD; M. Ferrin, MD, PhD; C. Hollis, MD, PhD; M. Holtmann, MD, PhD; E. Konofal, MD, PhD; M. Lecendreux, MD; A. Rothenberger, MD; P. Santosh, MD; J.A. Sergeant, PhD; E. Simonoff, MD; E.J. Sonuga-Barke, PhD; C. Soutullo, MD, PhD; H-Ch. Steinhausen, MD, PhD; J. Stevenson, PhD; A. Stringaris, MD, PhD; E. Taylor, MD; S. van der Oord, PhD; I. Wong, PhD; and A. Zuddas, MD. The authors thank the following individuals for providing additional information on studies and/or advice: Corrado Barbui, MD, PhD, Verona University, Italy; Andrea Cipriani, MD, PhD, Oxford University, UK; Jens Egeland, PhD, Oslo University, Norway; Stuart J. Johnstone, PhD, University of Wollongong, Australia; Marianna Purgato, PhD, Johns Hopkins University, USA; David Rabiner, PhD, Duke University, USA; Lilach Shalev-Mevorah, PhD, Tel-Aviv University, Israel; Leanne Tamm, PhD, Cincinnati Children’s Hospital, USA; Rosemary Tannock, PhD, Hospital for Sick Children, Toronto, Canada; Olivier Tucha, PhD, Groningen University, Netherlands; and Martine van Dongen- Boomsma, MD, Radboud University Nijmegen, Netherlands. Disclosure: Dr. Cortese has received royalties from Argon Healthcare Italia. Dr. Ferrin has received economic support from the Instituto de Salud Carlos III, Consejeria de Salud Junta de Andalucia, Gobierno de Navarra (Beca Jer- onimo Ayanz), and Fundacion Alicia Koplowitz. Prof. Buitelaar has served as a consultant to/advisory board member of/ and/or speaker for Janssen Cilag BV, Eli Lilly and Co., and Servier. Prof. Daley has provided educational talks for Eli Lilly and Co. and Shire; has attended an advisory board for Eli Lilly and Co.; has received support for educational travel from Eli Lilly and Co., Shire, and HP Pharma; and has received funding from Shire. Prof. REFERENCES Training of attention functions in children with attention deﬁcit hyperactivity disorder. Atten Deﬁc Hyperact Disord. 2011;3:271-278. y y 8. Daley D, Van der Oord S, Ferrin M, et al. Behavioral interventions in attention-deﬁcit/hyperactivity disorder: a meta-analysis of randomized controlled trials across multiple outcome domains. J Am Acad Child Adolesc Psychiatry. 2014;53:835-847. yp 31. Johnstone SJ, Roodenrys S, Blackman R, et al. Neurocognitive training for children with and without AD/HD. Atten Deﬁc Hyperact Disord. 2012; 4:11-23. 9. Rapport MD, Orban SA, Koﬂer MJ, Friedman LM. Do programs designed to train working memory, other executive functions, and attention beneﬁt children with ADHD? A meta-analytic review of cognitive, academic, and behavioral outcomes. Clin Psychol Rev. 2013;33:1237-1252. 32. Green CT, Long DL, Green D, et al. Will working memory training generalize to improve off-task behavior in children with attention- deﬁcit/hyperactivity disorder? Neurotherapeutics. 2012;9:639-648. y 10. Vinogradov S, Fisher M, de Villers-Sidani E. Cognitive training for impaired neural systems in neuropsychiatric illness. Neuro- psychopharmacology. 2012;37:43-76. yp y p 33. Van der Oord S, Ponsioen AJ, Geurts HM, Brink EL, Prins PJ. A Pilot Study of the Efﬁcacy of a Computerized Executive Functioning Remediation Training With Game Elements for Children With ADHD in an Outpatient Setting: Outcome on Parent- and Teacher-Rated Executive Functioning and ADHD Behavior. J Atten Disord 2012, in press, http://dx.doi.org/10.1177/1087 054712453167. p y p gy 11. Sonuga-Barke EJ, Coghill D. The foundations of next generation attention-deﬁcit/hyperactivity disorder neuropsychology: building on progress during the last 30 years. J Child Psychol Psychiatry. 2014; 55:e1-e5. 34. Tamm L, Epstein JN, Peugh JL, Nakonezny PA, Hughes CW. Preliminary data suggesting the efﬁcacy of attention training for school-aged children with ADHD. Dev Cogn Neurosci. 2013;4:16-28. 12. Lewis CM, Baldassarre A, Committeri G, Romani GL, Corbetta M. Learning sculpts the spontaneous activity of the resting human brain. Proc Natl Acad Sci U S A. 2009;106:17558-17563. 35. Chacko A, Bedard AC, Marks DJ, et al. A randomized clinical trial of Cogmed Working Memory Training in school-age children with ADHD: a replication in a diverse sample using a control condition. J Child Psy- chol Psychiatry. 2014;55:247-255. 13. Poldrack RA, Gabrieli JD. Characterizing the neural mechanisms of skill learning and repetition priming: evidence from mirror reading. Brain. 2001;124:67-82. 36. van Dongen-Boomsma M, Vollebregt MA, Buitelaar JK, Slaats-Willemse D. Working memory training in young children with ADHD: a randomized placebo-controlled trial. J Child Psychol Psychiatry. 2014;55:886-896. 14. REFERENCES a randomized controlled trial. J Child Psychol Psychiatry. 2012;53: 1277-1284. 1. Ramos-Quiroga JA, Montoya A, Kutzelnigg A, Deberdt W, Sobanski E. Attention deﬁcit hyperactivity disorder in the European adult popula- tion: prevalence, disease awareness, and treatment guidelines. Curr Med Res Opin. 2013;29:1093-1104. 23. StataCorp. Stata Statistical Software: release 13. College Station, TX: StataCorp, 2013. p 24. Egeland J, Aarlien AK, Saunes BK. Few effects of far transfer of working memory training in ADHD: a randomized controlled trial. PLoS One. 2013;e75660. p 2. Taylor E, Dopfner M, Sergeant J, et al. European Clinical Guidelines for Hyperkinetic Disorder—ﬁrst upgrade. Eur Child Adolesc Psychiatry. 2004;13(Suppl 1):I7-I30. pp 3. Faraone SV, Biederman J, Spencer TJ, Aleardi M. Comparing the efﬁcacy of medications for ADHD using meta-analysis. MedGenMed. 2006;8:4. 25. Hovik KT, Saunes BK, Aarlien AK, Egeland J. RCT of working memory training in ADHD: long-term near-transfer effects. PLoS One. 2013;8:e80561. 26. Klingberg T, Fernell E, Olesen PJ, et al. Computerized training of working memory in children with ADHD-a randomized, controlled trial. J Am Acad Child Adolesc Psychiatry. 2005;44:177-186. 4. Cortese S, Holtmann M, Banaschewski T, et al. Practitioner review: cur- rent best practice in the management of adverse events during treatment with ADHD medications in children and adolescents. J Child Psychol Psychiatry. 2013;54:227-246. Acad Child Adolesc Psychiatry. 2005;44:177-186. 27. Shalev L, Tsal Y, Mevorach C. Computerized progressive Attentional (CPAT) Program: effective direct intervention for children with ADHD. Child Neuropsychology. 2006;13:382-388. y y 5. Molina BS, Hinshaw SP, Swanson JM, et al. MTA at 8 Years: Prospective follow-up of children treated for combined-type ADHD in a multisite study. J Am Acad Child Adolesc Psychiatry. 2009;48:484-500. p y gy 28. Johnstone SJ, Roodenrys S, Phillips E, Watt AJ, Mantz S. A pilot study of combined working memory and inhibition training for children with AD/HD. Atten Deﬁc Hyperact Disord. 2010;2:31-42. y y y 6. Adler LD, Nierenberg AA. Review of medication adherence in children and adults with ADHD. Postgrad Med. 2010;122:184-191. 7. Kovshoff H, Williams S, Vrijens M, et al. The Decisions Regarding ADHD Management (DRAMa) study: uncertainties and complexities in assess- ment, diagnosis and treatment, from the clinician’s point of view. Eur Child Adolesc Psychiatry. 2012;21:87-99. 29. Rabiner DL, Murray DW, Skinner AT, Malone PS. A randomized trial of two promising computer-based interventions for students with attention difﬁculties. J Abnorm Child Psychol. 2010;38:131-142. y 30. Tucha O, Tucha L, Kaumann G, et al. 0890-8567/$36.00/ª2015 American Academy of Child and Adolescent Psychiatry Correspondence to Edmund J.S. Sonuga-Barke, PhD, Developmental Brain- Behaviour Laboratory, Psychology Institute for Disorders of Impulse and Attention, University of Southampton, Highﬁeld Campus, SO17 1BJ, UK; e-mail: ejb3@soton.ac.uk http://dx.doi.org/10.1016/j.jaac.2014.12.010 http://dx.doi.org/10.1016/j.jaac.2014.12.010 DISCUSSION Furthermore, therapeutic innovation is required to enhance the “far transfer” of speciﬁc neuropsychological gains to everyday patterns of functional impairment through more ecologically valid training approaches.42 Future trials should more consistently include active control arms, a broader range of functional outcomes, and long-term follow-up. & JOURNAL OF THE AMERICAN ACADEMY OF CHILD & ADOLESCENT PSYCHIATRY VOLUME 54 NUMBER 3 MARCH 2015 173 www.jaacap.org CORTESE et al. 0890-8567/$36.00/ª2015 American Academy of Child and Adolescent Psychiatry REFERENCES Sonuga-Barke EJ, Brandeis D, Cortese S, et al. Nonpharmacological in- terventions for ADHD: systematic review and meta-analyses of ran- domized controlled trials of dietary and psychological treatments. Am J Psychiatry. 2013;170:275-289. 37. Pauli-Pott U, Becker K. Neuropsychological basic deﬁcits in preschoolers at risk for ADHD: a meta-analysis. Clin Psychol Rev. 2011;31:626-637. y y 15. Coghill D, Nigg J, Rothenberger A, Sonuga-Barke E, Tannock R. Whither causal models in the neuroscience of ADHD? Dev Sci. 2005;8:105-114. 38. Frazier TW, Demaree HA, Youngstrom EA. Meta-analysis of intellec- tual and neuropsychological test performance in attention-deﬁcit/ hyperactivity disorder. Neuropsychology. 2004;18:543-555. 16. Holmes J, Gathercole SE, Dunning DL. Poor working memory: impact and interventions. Adv Child Dev Behav. 2010;39:1-43. 39. Coghill DR, Hayward D, Rhodes SM, Grimmer C, Matthews K. A longitudinal examination of neuropsychological and clinical func- tioning in boys with attention deﬁcit hyperactivity disorder (ADHD): improvements in executive functioning do not explain clinical improve- ment. Psychol Med. 2014;44:1087-1099. 17. Cochrane Collaboration. Higgins JPT, Green S, editors. Cochrane Handbook for Systematic Reviews of Interventions, Version 5.1.0 [updated March 2011]. Available at: www.cochrane-handbook.org p g 18. Cochrane Collaboration. RevMan, version 5.1. Copenhagen: Nordic Cochrane Centre; 2011. 19. Morris SB. Estimating effect sizes from pretest-posttest-control group designs. Organ Res Methods. 2008;11:364-386. 40. Sonuga-Barke EJ. Editorial: ’What’s up, (R)DoC?’—can identifying core dimensions of early functioning help us understand, and then reduce, developmental risk for mental disorders? J Child Psychol Psychiatry. 2014;55:849-851. 20. Steiner NJ, Sheldrick RC, Gotthelf D, Perrin EC. Computer-based attention training in the schools for children with attention deﬁcit/hyperactivity disorder: a preliminary trial. Clin Pediatr (Phila). 2011;50:615-622. 41. Sonuga-BarkeEJ.Onthereorganizationofincentivestructuretopromotedelay tolerance: a therapeutic possibility for AD/HD? Neural Plast. 2004;11:23-28. p y ( ) 21. Steiner NJ, Frenette EC, Rene KM, Brennan RT, Perrin EC. Neurofeed- back and cognitive attention training for children with attention-deﬁcit hyperactivity disorder in schools. J Dev Behav Pediatr. 2014;35:18-27. p p y 42. Moreau D, Conway AR. The case for an ecological approach to cognitive training. Trends Cogn Sci. 2014;18:334-336. g g 43. Cognitive Medical Systems AB. RoboMemo. Stockholm: Cognitive Medical Systems AB, 2005. yp y 22. Gray SA, Chaban P, Martinussen R, et al. Effects of a computerized working memory training program on working memory, attention, and academics in adolescents with severe LD and comorbid ADHD: y 44. BrainTrain. Captain’s Log. Richmond, VA: BrainTrain, 2012. JOURNAL OF THE AMERICAN ACADEMY OF CHILD & ADOLESCENT PSYCHIATRY VOLUME 54 NUMBER 3 MARCH 2015 REFERENCES JOURNAL OF THE AMERICAN ACADEMY OF CHILD & ADOLESCENT PSYCHIATRY VOLUME 54 NUMBER 3 MARCH 2015 JOURNAL OF THE AMERICAN ACADEMY OF CHILD & ADOLESCENT PSYCHIATRY VOLUME 54 NUMBER 3 MARCH 2015 JOURNAL OF THE AMERICAN ACADEMY OF CHILD & ADOLESCENT PSYCHIATRY VOLUME 54 NUMBER 3 MARCH 2015 174 www.jaacap.org
https://openalex.org/W4239826138	https://www.qeios.com/read/I9MW78/pdf	English	null	Staphylococcal Nuclease and Tudor Domain Containing 1	Definitions	2,020	cc-by	84	Qeios · Definition, February 8, 2020 Open Peer Review on Qeios Staphylococcal Nuclease and Tudor Domain Containing 1 National Cancer Institute National Cancer Institute Qeios ID: I9MW78 · https://doi.org/10.32388/I9MW78 Source National Cancer Institute. Staphylococcal Nuclease and Tudor Domain Containing 1. NCI Thesaurus. Code C96333. Thesaurus. Code C96333. Staphylococcal nuclease domain-containing protein 1 (910 aa, ~102 kDa) is encoded by the human SND1 gene. This protein plays a role in both RNA interference and the positive regulation of transcription. Qeios ID: I9MW78 · https://doi.org/10.32388/I9MW78 1/1
https://openalex.org/W3094545861	https://hal.archives-ouvertes.fr/hal-02971994/document	English	null	Dynamical well-killing simulation of a vertical H<sub>2</sub>S-containing natural gas well	Oil & gas science and technology	2,020	cc-by	15,604	To cite this version: Liangjie Mao, Mingjie Cai, Qingyou Liu, Guorong Wang. Dynamical well-killing simulation of a vertical H2S-containing natural gas well. Oil & Gas Science and Technology - Revue d’IFP Energies nouvelles, 2020, 75, pp.71. 10.2516/ogst/2020065. hal-02971994 Received: 3 March 2020 / Accepted: 27 August 2020 Abstract. This work aims to explore the dynamical well-killing process of a vertical H2S-containing natural gas well. A dynamical well-killing model considering an H2S solubility was established to simulate the overﬂow and well-killing process of a vertical H2S-containing natural gas well. The mass and momentum equations of the coupled model were solved using ﬁnite difference method, while the transient temperature prediction model was solved using ﬁnite volume method. The coupled model was validated by reproducing experimental data and ﬁeld data of Well Tiandong #5. The effect of H2S content, mud displacement, drilling ﬂuid density, and initial overﬂow volume on the dynamical well-killing process of an H2S-containing natural gas well were obtained and analyzed in this work. Results showed that H2S will gasify near wellhead during well killing when casing pressure decreases. To balance the bottom hole pressure, when H2S releases, the casing pressure increases as H2S content increases. As initial overﬂow volume increases, the annular temperature, annular pressure and the casing pressure increase signiﬁcantly. When H2S gasiﬁes, the casing pressure applied at wellhead should be higher at lower initial overﬂow volume to balance bottom hole pressure. In the well-killing process, the annular pressure and temperature decrease as drilling ﬂuid density increases and a lower casing pressure is needed for balancing bottom hole pressure. The casing pressure is lower at a higher displacement for higher friction resistance. Besides, as well-killing displacement increases H2S will gasify at an earlier time. When drilling for H2S-containing natural gas well, early detection of gas kick should be more frequent to avoid severe overﬂow. Besides, higher displacement and density of drilling ﬂuid should be considered to avoid stratum fracturing and prevent leakage accidents under the premise of meeting drilling requirements. Received: 3 March 2020 / Accepted: 27 August 2020 Nomenclature Qg Gas production QgH2S Released H2S qg Densities of gas ql Density of drilling ﬂuid vg Velocity of gas vl Velocity of drilling ﬂuid Eg Gas void fraction El Liquid holdup A Cross-sectional area g Local acceleration of gravity oP oz fr Friction pressure drop c1 Speciﬁc heat capacity of the drilling ﬂuid in the drilling string T1 Drilling ﬂuid temperature in the drilling string up Velocity in the x direction of the drilling ﬂuid in the drilling string vp Velocity in the y direction of the drilling ﬂuid in the drilling string Cx Heat transfer coefﬁcient of the drilling ﬂuid in the x direction Cy Heat transfer coefﬁcient of the drilling ﬂuid in the y direction q2 Density of the drilling string T2 Drilling string temperature h Well depth Ql Mud displacement V pg Pit gain at shut-in time P0 Wellhead pressure Pp Formation pressure vsg Superﬁcial velocities of gas vsl Superﬁcial velocities of liquid * Correspondence authors: maoliangjie@qq.com; liuqy66@aliyun.com Available online at: ogst.ifpenergiesnouvelles.fr Oil & Gas Science and Technology – Rev. IFP Energies nouvelles 75, 71 (2020) Available online at: L. Mao et al., published by IFP Energies nouvelles, 2020 ogst.ifpenergiesnouvelles.fr https://doi.org/10.2516/ogst/2020065 Oil & Gas Science and Technology – Rev. IFP Energies nouvelles 75, 71 (2020) L. Mao et al., published by IFP Energies nouvelles, 2020 https://doi.org/10.2516/ogst/2020065 Oil & Gas Science and Technology – Rev. IFP Energies nouvelles 75, 71 (2020) L. Mao et al., published by IFP Energies nouvelles, 2020 https://doi.org/10.2516/ogst/2020065 HAL Id: hal-02971994 https://hal.science/hal-02971994v1 Submitted on 20 Oct 2020 L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. Oil & Gas Science and Technology – Rev. IFP Energies nouvelles 75, 71 (2020) L. Mao et al., published by IFP Energies nouvelles, 2020 https://doi.org/10.2516/ogst/2020065 Dynamical well-killing simulation of a vertical H2S-containing natural gas well Liangjie Mao1,2,, Mingjie Cai1,2, Qingyou Liu1,, and Guorong Wang1 1 State Key Laboratory of Oil and Gas Reservoir Geology and Exploitation, Southwest Petroleum University, Sichuan, Chengdu 610500, China 2 Petroleum Engineering School, Southwest Petroleum University, Sichuan, Chengdu 610500, China Liangjie Mao1,2,, Mingjie Cai1,2, Qingyou Liu1,, and Guorong Wang1 Liangjie Mao1,2,, Mingjie Cai1,2, Qingyou Liu1,, and Guorong Wang1 1 State Key Laboratory of Oil and Gas Reservoir Geology and Exploitation, Southwest Petroleum University, Sichuan, Ch d 610500 Chi g , 2 Petroleum Engineering School, Southwest Petroleum University, Sichuan, Chengdu 610500, China Nomenclature (2014) established a transient model to study the changing rule of wellhead parameters during blowout and found that the gas outlet velocity increases as well depth increases. Hou et al. (2019) conducted an experiment to study the heat transfer for gas-liquid two-phase ﬂow in wellbore and devel- oped a new model of convective heat transfer coefﬁcient. Fu et al. (2019) proposed the mathematic model of wellbore annulus transient water hammer with the consideration of transient multi-phase ﬂow characteristics and pointed out that both the additional water hammer pressure and shut- in casing pressure generated by the closure of BOP are likely to cause formation at the shallow casing shoe damage. voo Gas drift velocity r Surface tension k1, k2 Correction coefﬁcients yi Mole fractions of H2S in the gas xi Mole fractions of H2S in the liquid uV i Fugacity coefﬁcients of H2S in the gas ul i Fugacity coefﬁcients of H2S in the liquid kij Interaction coefﬁcient between H2S and hydro- carbon components in natural gas Tci Critical temperature of H2S Pci Critical pressure of H2S Tri Correspondent temperature of H2S w Acentric factor of H2S D Borehole size d Drill pipe diameter vgr Drift velocity C0 Distribution coefﬁcient vm Mixed velocity of gas and liquid Hr Viscous friction head De Equivalent diameter n Flow index of the drilling ﬂuid Re Reynolds number of the mixed ﬂuids ee Equivalent absolute roughness Pb Bottom hole pressure rw Hole radius K Reservoir permeability c System compressibility lg Gas viscosity t Overﬂow time W g Molar mass of natural gas R Molar gas constant Pa Casing pressure Pla Circulating pressure loss Pma Hydrostatic pressure created by the ﬂuid column Re Mean Reynolds number of the mixed ﬂuids uv i Fugacity coefﬁcients of H2S in the gas ul i Fugacity coefﬁcients of H2S in the liquid Z Natural gas compression factor With the increasing demand for natural gas, an increas- ing number of gas reservoirs that contain H2S will be drilled (Guo and Wang, 2016; Yin et al., 2015; Zhang et al., 2020; Zhu et al., 2011). The existing states of H2S are greatly affected by temperature and pressure. The state may trans- form from supercritical to gas in the wellbore as annular pressure and temperature decrease, and this condition inﬂu- ences annular pressure and ﬂow pattern distribution. Nomenclature This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. L. Mao et al.: Oil & Gas Science and Technology – Rev. IFP Energies nouvelles 75, 71 (2020) 2 Gas drift velocity Surface tension k2 Correction coefﬁcients Mole fractions of H2S in the gas Mole fractions of H2S in the liquid Fugacity coefﬁcients of H2S in the gas Fugacity coefﬁcients of H2S in the liquid Interaction coefﬁcient between H2S and hydro- carbon components in natural gas Critical temperature of H2S Critical pressure of H2S Correspondent temperature of H2S Acentric factor of H2S Borehole size Drill pipe diameter Drift velocity Distribution coefﬁcient Mixed velocity of gas and liquid Viscous friction head Equivalent diameter Flow index of the drilling ﬂuid Reynolds number of the mixed ﬂuids Equivalent absolute roughness Bottom hole pressure Hole radius Reservoir permeability System compressibility Gas viscosity Overﬂow time Molar mass of natural gas Molar gas constant Casing pressure Circulating pressure loss Hydrostatic pressure created by the ﬂuid column Mean Reynolds number of the mixed ﬂuids Fugacity coefﬁcients of H2S in the gas Fugacity coefﬁcients of H2S in the liquid Natural gas compression factor conditions. Nickens (1987) proposed a dynamic computer model by a solution of the mass and momentum balance equations for predicting the dynamical two-phase ﬂow behavior and pressure response of the wellbore. Sun and Wang proposed a mathematical model to predict the multi- phase ﬂow in a wellbore for deep water wells (Wang et al., 2016). Their model has also been used to study the phase transition of hydrate during the exploitation of natural gas hydrate (Wang and Sun, 2009, 2014). Xu et al. (2018, 2019) developed a non-isothermal two-phase ﬂow model to investigate the effect of major parameters on the two-phase ﬂow behavior in the wellbore. They found that temperature, pressure, and solubility ﬁelds are mutually inﬂuential. The gas solubility effect and heat transfer effect inﬂuence gas kick characteristics signiﬁcantly. Meng et al. (2015) pro- posed a transient gas–liquid drift ﬂux model based on Shi slip relation for simulating gas kick/injection and solved the model numerically using ﬁnite volume method and advection upstream splitting method V scheme. Xie et al. Nomenclature Therefore, the typical gas–liquid two-phase ﬂow models may be unsuitable for predicting the multiphase ﬂow behavior in H2S-containing natural gas wells. The “1223” Gas Blowout Accident in Kaixian County, Chongqing has elicited scholarly attention on phase transition of H2S in well control. Some scholars have focused on multiphase ﬂow behavior of H2S-containing natural gas wells. Sun and Wang et al. established a multiphase ﬂow model that considers the phase change of the H2S in the drilling ﬂuid; simulation results showed that, as the invasion gas moves up along the wellbore to the critical position of H2S, the released H2S may cause a rapid volume expansion, thereby increasing the blowout risk (Sun et al., 2013, 2018). (1) Overﬂow period ; 4. The inﬂuence of annulus eccentricity is disregarded; 4. The inﬂuence of annulus eccentricity is disregarded; 5. The geothermal gradient is regarded as constant. ( ) p When overﬂow occurs, the wellhead is open and the parameters under normal drilling conditions can be used as the initial conditions of a well kick: y g ; 5. The geothermal gradient is regarded as constant. 5. The geothermal gradient is regarded as constant. Then, the continuity equations of gas and liquid phases are expressed as follows (Wei et al., 2018): P h; 0 ð Þ ¼ Pb Q h; 0 ð Þ ¼ Ql Egðh; 0Þ ¼ 0 Elðh; 0Þ ¼ 1 vlðh; 0Þ ¼ Ql=A : 8 > > > > > < > > > > > : ð8Þ P h; 0 ð Þ ¼ Pb Q h; 0 ð Þ ¼ Ql Egðh; 0Þ ¼ 0 Elðh; 0Þ ¼ 1 vlðh; 0Þ ¼ Ql=A : 8 > > > > > < > > > > > : ð8Þ oðqgvgEgAÞ oz þ oðqgEgAÞ ot ¼ Qg þ QgH2Sðoverflow periodÞ QgH2Sðwell killing periodÞ ( ; ð1Þ oðqlvlElAÞ oz þ oðqlElAÞ ot ¼ 0: ð2Þ oðqgvgEgAÞ oz þ oðqgEgAÞ ot ¼ Qg þ QgH2Sðoverflow periodÞ QgH2Sðwell killing periodÞ ( ; ð8Þ ð1Þ The boundary conditions are shown as follows: The boundary conditions are shown as follows: oðqlvlElAÞ oz þ oðqlElAÞ ot ¼ 0: ð2Þ P 0; t ð Þ ¼ P0 Q h; t ð Þ ¼ Ql þ Qgðh; tÞ Pg ¼ V pg 8 > < > : : ð9Þ ð2Þ ð9Þ On the basis of the law of conservation of momentum, the momentum equation can be obtained as follows: (2) Killing period 2.1 Governing equations for mass and momentum oðq3c3T 3Þ ot þ o ox q3c3uaT 3 ð Þ þ o oy q3c3vaT 3 ð Þ ¼ o ox C3xc3 oT 3 ox þ o oy C3yc3 oT 3 oy þ Sa: ð6Þ During drilling, gas enters the wellbore from the stratum when overﬂow occurs, and gas–liquid two-phase ﬂow appears below the location the gas reaches. Given that two-phase ﬂow exists in overﬂow and well-killing periods, a mathematical model is established in this work to obtain the two-phase ﬂow behavior. (4) Heat transfer model of casing, cement sheath, and formation: To simplify the calculation, the following assumptions are made in this work: oðqiciT iÞ ot ¼ o ox Cixci oT i ox þ o oy Ciyci oT i oy : ð7Þ ð7Þ 1. The gas and drilling ﬂuid ﬂow in a vertical wellbore is considered one dimensional; 2. The compressibility of the drilling ﬂuid is ignored; 2 Mathematical model (3) Heat transfer model in the annular: (3) Heat transfer model in the annular: 1 Introduction Gas kick and well-killing are the main concerns of well con- trol during the drilling of oil and gas ﬁelds. Various multi- phase ﬂow models have been proposed to predict the ﬂow pattern distribution in the wellbore after gas kick (Amaya-Gómez et al., 2019; Ansari et al., 1994; Bilicki and Kestin, 1987; Ebrahimi and Khamehchi, 2015; Feng et al., 2015; Guo et al., 2018; Hasan and Kabir, 1988; Keles- sidis and Dukler, 1989; Li and Mouline, 1997; Zhang et al., 2019); among them, the most widely used in drilling engi- neering are the patterns of bubble, slug, churn, and annular ﬂows (Osman, 2004; Raghavan, 1989; Sun et al., 2017). On the basis of these studies, many researchers have focused on the multiphase ﬂow in the wellbore under different ( ) It can be seen that most of the existing studies are focused on the gas kick simulation of a well and, the well- killing simulation after gas kick is scarce, especially for an H2S-containing natural gas well. By applying casing pres- sure in the wellhead during well killing, the two-phase ﬂow behavior is different from that in the overﬂow (gas kick). Thus, the phase change behavior of H2S may be different from that described in the abovementioned published L. Mao et al.: Oil & Gas Science and Technology – Rev. IFP Energies nouvelles 75, 71 (2020) 3 literature. Thus, it is of signiﬁcance to study the well-killing process of an H2S-containing natural gas well to offer a guidance for exploitation of H2S-containing gas reservoir. 2.2 Transient temperature prediction model To accurately predict the phase change behavior of H2S in wellbore, a transient temperature prediction model is developed (Mao and Zhang, 2018). The unsteady two- dimensional convection–diffusion and unsteady two- dimensional diffusion equations are used to describe the heat transfer models as follows: g g g Thus, the aim of this study is to establish a dynamical well-killing model considering an H2S solubility to simulate the overﬂow and well-killing process of a vertical H2S- containing natural gas well. The mass and momentum equations of the coupled model were solved using ﬁnite difference method, while the transient temperature predic- tion model was solved using ﬁnite volume method. The con- tinuity and momentum equations are decoupled from temperature prediction model. The effect of H2S content, mud displacement, drilling ﬂuid density, and initial over- ﬂow volume on the dynamical well-killing process of an H2S-containing natural gas well were obtained and ana- lyzed in this work. (1) Heat transfer model in the drilling string: oðq1c1T 1Þ ot þ o ox q1c1upT 1 ð Þ þ o oy q1c1vpT 1 ð Þ ¼ o ox C1xc1 oT 1 ox þ o oy C1yc1 oT 1 oy þ Sp: ð4Þ ð4Þ (2) Heat transfer model of drilling string: (2) Heat transfer model of drilling string: oðq2c2T 2Þ ot ¼ o ox C2xc2 oT 2 ox þ o oy C2yc2 oT 2 oy : ð5Þ ð5Þ 2.2 Initial and boundary conditions 3. Gas and liquid phases are continuous in the control unit; (1) Overﬂow period (2) Killing period ( ) After the shut-in operation is performed, the ﬂow pat- tern, gas void fraction, and annular pressure distributions at the shut-in time can be considered the initial conditions of the well-killing period. During the well-killing process, the bottom hole pressure is equal to the formation pressure, and the boundary conditions are shown as follows: o ot ðqlvlEl þ qgvgEgÞ þ o oz ðqlv2 l El þ qgv2 gEgÞ þ oP oz þðqlEl þ qgEgÞg þ oP oz fr ¼ 0: ð3Þ ð3Þ L. Mao et al.: Oil & Gas Science and Technology – Rev. IFP Energies nouvelles 75, 71 (2020) L. Mao et al.: Oil & Gas Science and Technology – Rev. IFP Energies nouvelles 75, 71 (2020) 4 4 Pð0; tÞ ¼ Pp Qlð0; tÞ ¼ Ql Qgð0; tÞ ¼ 0 8 > < > : : ð10Þ ai ¼ 1 þ mð1 T 0:5 ri Þ 2; ð21Þ aj ¼ 0:45724R2T 2 ci Pci ai; ð22Þ ð21Þ ð10Þ ð22Þ and 2.4.1 H2S solubility The solubility of H2S in the drilling ﬂuid can be expressed as (Sun et al., 2018): The solubility of H2S in the drilling ﬂuid can be expressed as (Sun et al., 2018): C 0 ¼ 1: The solubility of H2S in the drilling ﬂuid can be expressed as (Sun et al., 2018): C 0 ¼ 1:182 þ 0:9 d D : ð28Þ C 0 ¼ 1:182 þ 0:9 d D : ð28Þ ð28Þ xi ¼ pyiuv i pul i ; ð16Þ Churn and annular ﬂows: xi ¼ pyiuv i pul i ; ð16Þ Churn and annular ﬂows: ð16Þ P ¼ RT V b a VðV þ bÞ þ bðV bÞ : ð17Þ vgr ¼ 0:35 þ 0:22 D d g Dd ð ÞðqlqgÞ ql 0:5 ; C 0 ¼ 1: ð29Þ ð17Þ ð29Þ With A ¼ ap R2T2, B ¼ bp RT, and Z ¼ PV RT, equation (13) can be written as With A ¼ ap R2T2, B ¼ bp RT, and Z ¼ PV RT, equation (13) can be written as With A ¼ ap R2T2, B ¼ bp RT, and Z ¼ PV RT, equation (13) can be written as Thus, the gas void fraction can be calculated by the follow- ing equations: With A ¼ ap R2T2, B ¼ bp RT, and Z ¼ PV RT, equation (13) can be written as Thus, the gas void fraction can be calculated by the follow- ing equations: Thus, the gas void fraction can be calculated by the follow- ing equations: Eg ¼ vsg C ovm þ vgr ; ð30Þ Z 3 ð1 BÞZ 2 þ ðA 3B2 2BÞZ ðAB B2 B3Þ ¼ 0; ð18Þ ð30Þ ð18Þ vm ¼ vsg þ vsl; ð31Þ ð31Þ a ¼ X i X j xixj ﬃﬃﬃﬃﬃﬃﬃﬃ aiaj p 1 kij ; ð19Þ b ¼ X i xi 0:0778RT ci Pci ; ð20Þ vm 2.4.3 Friction pressure For single phase ﬂow, t obtained using the frictio a ¼ X i X j xixj ﬃﬃﬃﬃﬃﬃﬃﬃ aiaj p 1 kij ; ð19Þ b ¼ X i xi 0:0778RT ci Pci ; ð20Þ vm ¼ vsg þ vsl; ð31Þ 2.4.3 Friction pressure drop For single phase ﬂow, the friction pressure drop can be obtained using the friction factor of the power-law ﬂuid. ð19Þ ð19Þ 2.4.2 Drift ﬂux model vsg < 3:1 gðqlqgÞr q2g h i0:25 qgv2 g > 25:41g qlv2 sl 39 ! if qlv2 sl > 74:4 qgv2 g > 0:0051ðqlv2 slÞ1:7 ! if qlv2 sl 74:4 8 > > > < > > > : : ð14Þ In the drift ﬂux model, the corresponding slip rate of the gas phases and distribution coefﬁcient in case of different ﬂow patterns are shown as follows (Wei et al., 2018): Bubble ﬂow: vgr ¼ 1:53 gðql qgÞr ql2 0:25 ; ð25Þ Annular ﬂow: ð25Þ : vsg > 3:1 gðql qgÞr q2 g " #0:25 : ð15Þ vgr ¼ 1:53 gðql qgÞ ql2 ; ð25Þ C 0 ¼ 1:20 þ 0:371 d D : ð26Þ vsg > 3:1 gðql qgÞr q2 g " #0:25 : ð15Þ : ð15Þ C 0 ¼ 1:20 þ 0:371 d D : ð26Þ ð15Þ C 0 ¼ 1:20 þ 0:371 d D : ð26Þ ð26Þ 2.4 Calculation of key parameters Slug ﬂow: 2.3 Flow pattern discriminant m ¼ 0:37464 þ 1:5226 0:26992w2: ð23Þ ð23Þ The main patterns of a gas–liquid two-phase ﬂow are bub- ble, slug, churn, and annular ﬂows (Hasan and Kabir, 1988). They can be discriminated by the following equations: Thus, the fugacity coefﬁcient of a certain component is expressed as: Thus, the fugacity coefﬁcient of a certain component is expressed as: ln ui ¼ bi b Z 1 ð Þ ln Z b ð Þ a 2 ﬃﬃﬃ 2 p b 2 P j xiaij a bi b 0 @ 1 A ln ui ¼ bi b Z 1 ð Þ ln Z b ð Þ a 2 ﬃﬃﬃ 2 p b 2 P j xiaij a bi b 0 @ 1 A ln Z þ 1 þ ﬃﬃﬃ 2 p b Z þ 12 ﬃﬃﬃ 2 p b " # : ð24Þ Bubble ﬂow: vsg k1 0:429 vsl þ 0:357 voo ð Þ; ð11Þ voo ¼ 1:53 gðql qgÞr ql2 0:25 : ð12Þ ð11Þ ln Z þ 1 þ ﬃﬃﬃ 2 p b Z þ 12 ﬃﬃﬃ 2 p b " # : ð24Þ ð12Þ ð12Þ ð24Þ Slug ﬂow: The solubility of H2S in the liquid can be obtained by combining equation (16) with equation (24). vsg > k1 0:429 vsl þ 0:357 voo ð Þ: ð13Þ ð13Þ ð13Þ Churn ﬂow: 2.4 Calculation of key parameters vgr ¼ 0:35 gDðql qgÞ ql 0:5 ; ð27Þ 2.4 Calculation of key parameters 2.4.1 H2S solubility The solubility of H2S in the drilling ﬂuid can be expressed as (Sun et al., 2018): py uv vgr ¼ 0:35 gDðql qgÞ ql 0:5 ; ð27Þ C 0 ¼ 1:182 þ 0:9 d D : ð28Þ 2.4.1 H2S solubility vgr ¼ 0:35 ð27Þ 3 Model solution Bubble ﬂow: Bubble ﬂow: 2.4.4 Gas production Under the effect of pressure differential between bottom hole and formation pressures, the H2S-containing natural gas enters the wellbore. In this work, the following equation is adopted to calculate gas production (Elsharkawy, 2004): ZZ oX ot þ oY oz dtdz ¼ Z L Xdz Ydt ¼ Z L1 þ Z L2 þ Z L3 þ Z L4 ¼ Z t iþ1 ð Þ t ið Þ Yðt; zðjÞÞdt þ Z z jþ1 ð Þ z jð Þ Xðtði þ 1Þ; zÞdz þ Z t iþ1 ð Þ t ið Þ Yðt; zðj þ 1ÞÞdt Z z jþ1 ð Þ z jð Þ XðtðiÞ; zÞdz ¼ 0: ð Þ Qg ¼ 2:64 1020KhðP2 p P2 bÞ ð0:8 þ IntDÞ ðT 255ÞZlg ; ð40Þ tD ¼ max 10; 1:47 109t rw2 K c/lg : ð41Þ ð40Þ ð41Þ ð45Þ ð45Þ The above-mentioned equation can be converted to the following equation by simpliﬁcation: 2.4.6 Convective heat transfer coefﬁcients Convective heat transfer coefﬁcients can be expressed as follows (Mao and Zhang, 2018): f ¼ 8 v2 l ql 8vl De 3n þ 1 4n n ; if Re 2000 1ﬃﬃﬃf p ¼ 2:1 n0:75 log Re f 4 1n=2 " # 0:2 n1:2 > 2000: 8 > > > > < > > > > : ð33Þ h ¼ Nu k d : ð43Þ h ¼ Nu k d : ð43Þ ð33Þ ð43Þ For laminar ﬂow, Nu = 4.36; for turbulent ﬂow, Nu = 0.023 Re0.8 Pr0.33. For gas–liquid two-phase ﬂow, the friction pressure drop can be calculated using the following equations established by Yin et al. (2017). (2) Model discretization (2) Model discretization Churn and annular ﬂows: Churn and annular ﬂows: ( ) Figure 1b shows the cell grid integration area K. The partial differential equation in the mathematical model can be written as Hr ¼ 2fv2 mqm=ðDeE2 gÞ ð38Þ 0:079 1 þ 75ð1 EgÞ =ðReg0:25Þ ð39Þ ð38Þ ð39Þ oX ot þ oY oz ¼ 0: ð44Þ ð44Þ Then, by integrating this equation into area K, a curvi- linear integral along the boundary of L can be obtained in accordance with Green’s theorem: 2.4.3 Friction pressure drop ð20Þ b ¼ X i xi 0:0778RT ci Pci ; ð20Þ For single phase ﬂow, the friction pressure drop can be obtained using the friction factor of the power-law ﬂuid. ð20Þ For single phase ﬂow, the friction pressure drop can be obtained using the friction factor of the power-law ﬂuid. L. Mao et al.: Oil & Gas Science and Technology – Rev. IFP Energies nouvelles 75, 71 (2020) 5 5 Hr ¼ 2fvl 2ql=De; ð32Þ Hr ¼ 2fvl 2ql=De; ð32Þ 3.1 Solution for mass and momentum governing equations Hr ¼ 2fvm 2qm=De; ð34Þ Hr ¼ 2fvm 2qm=De; ð34Þ 1ﬃﬃﬃf p ¼ 4 log ee 3:71De 5:05 log A Re ; A ¼ m ee 2:56De 1:11 þ 7:149 Re 0:898 : 8 > > > < > > > : ð35Þ ð34Þ The ﬁnite difference method is used in this work to solve the mass and momentum governing equations. Meanwhile, the inﬂuence of the boundary and initial conditions in each control region are considered in the solving model. The solution is completed by three steps: generating discrete grids, constructing discrete equations, and solving these equations. 1ﬃﬃﬃf p ¼ 4 log ee 3:71De 5:05 log A Re ; A ¼ m ee 2:56De 1:11 þ 7:149 Re 0:898 : 8 > > > < > > > : ð35Þ ð35Þ Slug ﬂow: (1) Generating discrete grids (1) Generating discrete grids I ﬁ d ff l Hr ¼ 2ð1 EgÞfvm 2qm=De ð36Þ 1ﬃﬃ f p ¼ 4 log ee 3:71De 5:05 log A Re A ¼ ee 2:56De 1:11 þ 7:149 Re 0:898 8 > > > < > > > : ð37Þ ð36Þ In ﬁnite difference numerical calculation, the spatial domain is the wellbore. For the overﬂow simulation, the time domain is the entire time from the overﬂow to the shut-in. For the well-killing simulation, the time domain is from the shut-in to the time the lower boundary of two-phase reaches wellhead. The schematic of discrete grid nodes of the wellbore is shown in Figure 1a. ð37Þ 3.2 Solution for transient temperature prediction model Figure 2 shows the grid diagram of the wellbore and forma- tion. By using ﬁnite volume method, equations (4)–(7) can be expressed as (Mao and Zhang, 2018): j ¼ 1 aPT P ¼ aNNT NN þ aNT N þ aWT W þ aST S þ aET E þ b; ð52 ð52Þ j ¼ 2 aPT P ¼ aNT N þ aWT W þ aST S þ aET E þ b; ð53Þ ð53Þ Fig. 1. Schematic diagram of solution for the mass and momentum equations: (a) Schematic of discrete grid nodes of the wellbore; (b) Diagram of cell grid integration area K. j ¼ 3 aPT P ¼ aSST SS þ aNT N þ aWT W þ aST S þ aET E þ b; ð54Þ ð54Þ j 4 aPT P ¼ aNT N þ aWT W þ aST S þ aET E þ b: ð55Þ X ¼ qgEg; Y ¼ qgEgvg: ( ð47Þ ð55Þ ð47Þ By using under-relaxation iteration method, discretized scheme of the heat transfer control equations are obtained as follows: Thus, the gas-phase difference equation can be obtained by combining equations (46) and (47): ðqgV gEgÞjþ1 iþ1 ðqgV gEgÞjþ1 i ¼ 0:5z=t h ðqgEgÞj i þ ðqgEgÞjþ1 i ðqgEgÞiþ1 j ðqgEgÞjþ1 iþ1 i þ 0:5z QgH2S jþ1 iþ1 þ QgH2S j iþ1 ð4 See equations (56)–(59) top of next page Figure 3 shows the ﬂow chart of model solution. 2.4.5 Gas density The gas density can be described by introducing a compres- sion factor into the ideal gas equation, as deﬁned in the equation below (Yin et al., 2017): Y jþ1 iþ1 Y j iþ1 ¼ z 2t Xi j þ Xi jþ1 Xiþ1 j X iþ1 jþ1 ; ð46Þ ð46Þ qg ¼ PW g ZRT : ð42Þ (3) Numeralization of the continuity equation F h i i i l (3) Numeralization of the continuity equation ð42Þ For gas-phase continuity equation, we let For gas-phase continuity equation, we let L. Mao et al.: Oil & Gas Science and Technology – Rev. IFP Energies nouvelles 75, 71 (2020) L. Mao et al.: Oil & Gas Science and Technology – Rev. IFP Energies nouvelles 75, 71 (2020) 6 Schematic of discrete grid nodes of the wellbore. Fig. 1. Schematic diagram of solution for the mass and Schematic of discrete grid nodes of the wellbore. Schematic of discrete grid nodes of the wellbore. X ¼ qgEgvg þ qlElvl; Y ¼ qgEgvg2 þ qlElvl2 þ qgEg þ qlElg þ P þ Pfr: ( ð50Þ ð50Þ Thus, the mixed momentum difference equation can be obtained by combining equations (46) and (50): Thus, the mixed momentum difference equation can be obtained by combining equations (46) and (50): Pjþ1 iþ1 Pj iþ1 ¼ z 2t ½ðqlvlEl þ qgvgEgÞj i þ ½ðqlvlEl þqgvgEgÞjþ1 iþ1 ½ðqlvlEl þ qgvgEgÞj iþ1 ½ðqlvlEl þ qgvgEgÞjþ1 iþ1g þ ðqlv2 l El þ qgv2 gEgÞj iþ1 0:5zgðqlEl þ qgEgÞj iþ1 þ ðqlv2 l El þ qgv2 gEgÞjþ1 iþ1 0:5zgðqlEl þ qgEgÞjþ1 iþ1 0:5z oP oz fr " #j iþ1 0:5z oP oz fr " #jþ1 iþ1 : ð Schematic of discrete grid nodes of the wellbore. ð51Þ ( ) q For mixed momentum equation, we let 3.3 Model coupling IFP Energies nouvelles 75, 71 (2020) 7 7 j ¼ 1 T tþt i;1 ¼ T t i;1 þ x aNNtþt i1 T tþt i2;1 þ aNtþt i1 T tþt i1;1 þ aStþt i1 T tþt iþ1;1 þ aEtþt i1 T tþt i;2 þ btþt i1 aPtþt i1 T t i;1 h i aP tþt i1 ; ð56Þ j ¼ 2 T tþt i;2 ¼ T t i;2 þ x aNtþt i2 T tþt i1;2 þ aWtþt i2 T tþt i;1 þ aStþt i2 T tþt iþ1;2 þ aEtþt i2 T tþt i;3 þ btþt i2 aPtþt i2 T t i;2 h i aP tþt i2 ; ð57Þ ð56Þ ð57Þ ð58Þ ð59Þ Determine the well structure and size Divide the grid and determine the steps Reverse circulation Set the direction of the flow item Set the initial and boundary conditions Iterative calculation The physical properties of each unit are calculated based on the initial and iteration temperature Determine laminar flow or turbulence Calculate the friction heat source, convective heat transfer coefficient Calculate the unit temperature at a certain time Calculate the unit temperature at the next moment Output downhole temperature distribution data Normal circulation Laminar flow Turbulence Converge Yes No Fig. 3. Flow chart of the model solution process. Fig. 2. The grid diagram of the wellbore and formation. Determine the well structure and size Divide the grid and determine the steps Reverse circulation Set the direction of the flow item Set the initial and boundary conditions Iterative calculation The physical properties of each unit are calculated based on the initial and iteration temperature Determine laminar flow or turbulence Calculate the friction heat source, convective heat transfer coefficient Calculate the unit temperature at a certain time Calculate the unit temperature at the next moment O t t d h l t t di t ib ti d t Normal circulation Laminar flow Turbulence Converge Yes No Fig. 2. The grid diagram of the wellbore and formation. a certain well depth. And the temperature also has a signiﬁcant effect on the ﬂuid properties (Xu et al., 2019). Therefore, before solving the mass and momentum equations, we must solve the transient temperature predic- tion model. The well-killing simulation is based on the overﬂow con- dition at shut-in time; thus, the entire solution process can be divided into two steps: overﬂow and well-killing periods. 3.3 Model coupling ð48Þ Moreover, the continuity and momentum equations are decoupled from temperature prediction model. Consistent time step can be expressed as the following equation: Similarly, the liquid-phase difference equation can be obtained as follows: ðqlV lElÞjþ1 iþ1 ðqlV lElÞjþ1 i ¼ 0:5z=t h ðqlElÞj i þðqlElÞjþ1 i ðqlElÞiþ1 j qlEl ð Þjþ1 iþ1 i : ð4 t ¼ z vg : ð60Þ ð60Þ ð49Þ When H2S is released from the drilling ﬂuid, the state changes from supercritical to gas, which directly leads to a signiﬁcant increase in gas void fraction. The gasiﬁcation volume of H2S is inﬂuenced greatly by the temperature at When H2S is released from the drilling ﬂuid, the state changes from supercritical to gas, which directly leads to a signiﬁcant increase in gas void fraction. The gasiﬁcation volume of H2S is inﬂuenced greatly by the temperature at (4) Numeralization of the momentum equation For mixed momentum equation, we let (4) Numeralization of the momentum equation F i d i l For mixed momentum equation, we let a certain well depth. And the temperature also has a signiﬁcant effect on the ﬂuid properties (Xu et al., 2019). Therefore, before solving the mass and momentum equations, we must solve the transient temperature predic- tion model. The well-killing simulation is based on the overﬂow con- dition at shut-in time; thus, the entire solution process can be divided into two steps: overﬂow and well-killing periods. 3.3 Model coupling F ﬂ i d th l ti ﬂ h t f ﬂ j ¼ 1 T tþt i;1 ¼ T t i;1 þ x aNNtþt i1 T tþt i2;1 þ aNtþt i1 T tþt i1;1 þ aStþt i1 T tþt iþ1;1 þ aEtþt i1 T tþt i;2 þ btþt i1 aPtþt i1 T t i;1 h i aP tþt i1 ; ð56Þ j ¼ 2 T tþt i;2 ¼ T t i;2 þ x aNtþt i2 T tþt i1;2 þ aWtþt i2 T tþt i;1 þ aStþt i2 T tþt iþ1;2 þ aEtþt i2 T tþt i;3 þ btþt i2 aPtþt i2 T t i;2 h i aP tþt i2 ; ð57Þ j ¼ 3 T tþt i;3 ¼ T t i;3 þ x aSStþt i3 T tþt iþ2;3 þ aNtþt i3 T tþt i1;3 þ aWtþt i3 T tþt i;2 þ aStþt i3 T tþt iþ1;3 þ aEtþt i3 T tþt i;4 þ btþt i3 aPtþt i3 T t i;3 h i aP tþt i3 ; ð58Þ j 4 T tþt i;j ¼ T t i;j þ x aNtþt ij T tþt i1;j þ aWtþt ij T tþt i;j1 þ aStþt ij T tþt iþ1;j þ aEtþt ij T tþt i;jþ1 þ btþt ij aPtþt ij T t i;j h i aP tþt ij : ð59Þ Fig. 2. The grid diagram of the wellbore and formation. Determine the well structure and size Divide the grid and determine the steps Reverse circulation Set the direction of the flow item Set the initial and boundary conditions Iterative calculation The physical properties of each unit are calculated based on the initial and iteration temperature Determine laminar flow or turbulence Calculate the friction heat source, convective heat transfer coefficient Calculate the unit temperature at a certain time Normal circulation Laminar flow Turbulence No L. Mao et al.: Oil & Gas Science and Technology – Rev. IFP Energies nouvelles 75, 71 (2020) 7 L. Mao et al.: Oil & Gas Science and Technology – Rev. IFP Energies nouvelles 75, 71 (2020) L. Mao et al.: Oil & Gas Science and Technology – Rev. 3.3 Model coupling For overﬂow period, the solution ﬂowchart of overﬂow simulation is depicted in Figure 4. For well-killing period, the solution ﬂowchart of well-killing simulation is depicted in Figure 5. As shown in the ﬁgure, depending on whether the gas reaches the wellhead, the solution process can be divided into two parts: gas rising and gas exhausting stages. The boundary positions of two-phase segment are key parameters in distinguishing the killing stage. The two- phase ﬂow module used in the overﬂow period is also called Converge Fig. 3. Flow chart of the model solution process. Fig. 3. Flow chart of the model solution process. 8 L. Mao et al.: Oil & Gas Science and Technology – Rev. IFP Energies nouvelles 75, 71 (2020 8 Fig. 4. Solution ﬂowchart of overﬂow simulation for coupling model. Fig. 4. Solution ﬂowchart of overﬂow simulation for coupling model. 1.65 m3 at 22:14. At 22:44, the wellhead emitted a loud noise, and a violent blowout accident occurred. The pit gain was recorded every 5 min, and the gas well was shut in when the pit gain reached 6 m3 at 22:44. Table 1 shows the basic parameters of Well Tiandong #5 (Blowout accident of TianDong #5 well, 1990). As shown in Figure 6a, the simulation results are in good agreement with the ﬁeld data of Well Tiandong #5. When H2S is released, the pit gain shows a sudden increase in simulation results and ﬁeld data. The slight difference between the two may be due to that the compressibility of the drilling ﬂuid is disregarded. to solve the two-phase ﬂow behavior in the well-killing period. 4 Model validation The basic data of an H2S-containing gas well Tiandong #5 were used to simulate the overﬂow process for verifying the validity of the dynamical well-killing model considering an H2S solubility. The overﬂow was found in Well Tiandong #5 at the drilling depth of 3570 m, and the pit gain was 9 L. Mao et al.: Oil & Gas Science and Technology – Rev. IFP Energies nouvelles 75, 71 (2020) ader conducted a gas kick and well-killing experiment artiﬁcial well L.S.U. 7 and provided the experimental (Rader et al., 1975). The basic data of experimental L.S.U. 7 were used to simulate the killing process, as i T bl 2 f lid i h lidi f h ll pit gain was 1.59 m3, and gas was continued to injec the shut-in operation; (2) injection of gas was stopp the casing pressure reached the desired value; (3) w ing was started. Figure 6b shows the comparison i l i d i l l A h i Fi 5. Solution ﬂowchart of well-killing simulation. Parameter Value Unit Inner diameter of casing 0.1243 m Outer diameter of drilling string 0.073 m Diameter of gas injection pipe 0.0254 m Drilling ﬂuid density 1.03 g/cm3 Well depth 1832.2 m Pumping speed of killing 60 r/min Surface temperature 23.9 C Reservoir thickness 1.9 m Plastic viscosity of drilling ﬂuid 0.0126 Pa s Casing pressure at shut-in time 3.447 MPa Gas void fraction 0.75 Table 3. Major computational parameters of the gas well in Sichuan. Parameter Value Unit Initial gas ﬂux 0.06 m3/s Drilling ﬂuid density 1.2 g/cm3 Weighted drilling ﬂuid 1.4 g/cm3 H2S content 2% Surface temperature 40 C Well depth 3000 m Drilling speed 0.08 m/s Reservoir permeability 40 mD Reservoir thickness 2 m Plastic viscosity of drilling ﬂuid 0.03 Pa s Yield value of drilling ﬂuid 1.5 Pa Mud displacement 20 L/s Threshold value of pit gain 3 m3 Geothermal gradient 0.024 C/m L. Mao et al.: Oil & Gas Science and Technology – Rev. IFP Energies nouvelles 75, 71 (2020) 10 Table 1. Basic parameters of Well Tiandong #5. Parameter Value Unit Initial gas ﬂux 0.054 m3/s Drilling ﬂuid density 1.3 g/cm3 H2S content 10% Surface temperature 10 C Well depth 3570 m Drilling speed 0.05 m/s Reservoir permeability 27 mD Reservoir thickness 1.9 m Plastic viscosity of drilling ﬂuid 0.027 Pa s Yield value of drilling ﬂuid 1.2 Pa Mud displacement 24 L/s Geothermal gradient 0.026 C/m Table 2. Basic data of experimental well L.S.U 7. Table 1. Basic parameters of Well Tiandong #5. Table 3. Major computational parameters of the gas well in Sichuan. 0 250 500 750 1000 1250 1500 1750 2000 0 1 2 3 4 5 6 7 Pit gain (m 3) Time (s) Result of this model Field data of TianDong #5 H2S releases (a) Comparison between simulation and field data of Well Tiandong #5 0 250 500 750 1000 1250 1500 1750 2000 0 1 2 3 4 5 6 7 Pit gain (m 3) Time (s) Result of this model Field data of TianDong #5 H2S releases Table 3. Major computational parameters of the gas well in Sichuan. Parameter Value Unit Initial gas ﬂux 0.06 m3/s Drilling ﬂuid density 1.2 g/cm3 Weighted drilling ﬂuid 1.4 g/cm3 H2S content 2% Surface temperature 40 C Well depth 3000 m Drilling speed 0.08 m/s Reservoir permeability 40 mD Reservoir thickness 2 m Plastic viscosity of drilling ﬂuid 0.03 Pa s Yield value of drilling ﬂuid 1.5 Pa Mud displacement 20 L/s Threshold value of pit gain 3 m3 Geothermal gradient 0.024 C/m (a) Comparison between simulation and field data of Well Tiandong #5 0 200 400 600 800 1000 1200 1400 1600 2 3 4 5 6 7 8 Casing pressure (MPa) Time (s) Model of this work Gas column model Experimental result (b) Comparison between simulation and experimental results Fig. 6. Comparison results: (a) is the comparison between simulation and ﬁeld data of Well Tiandong #5; (b) is the comparison between simulation and experimental results. 0 200 400 600 800 1000 1200 1400 1600 2 3 4 5 6 7 8 Casing pressure (MPa) Time (s) Model of this work Gas column model Experimental result Model of this work Gas column model Experimental result simulation results of our model and traditional “Gas column model” is also shown in Figure 6b. Large error exists in the simulation results of “Gas column model” because the fric- tion and slip between gas and liquid are ignored. Casing pressure (MPa) Fig. 5. Solution ﬂowchart of well-killing simulation. pit gain was 1.59 m3, and gas was continued to inject during the shut-in operation; (2) injection of gas was stopped when the casing pressure reached the desired value; (3) well kill- ing was started. Figure 6b shows the comparison between simulation and experimental results. As shown in Figure 6b, the simulation results of our model are in good agreement with the experimental results. The comparison between Rader conducted a gas kick and well-killing experiment in an artiﬁcial well L.S.U. 7 and provided the experimental data (Rader et al., 1975). The basic data of experimental well L.S.U. 7 were used to simulate the killing process, as shown in Table 2, for validating the validity of the well- killing model. In the experiment, (1) nitrogen was injected into the well bottom hole from the injection pipe until the Mao et al.: Oil & Gas Science and Technology – Rev. IFP Energies nouvelles 75, 71 (2020) 10 simulation results of our model and traditional “Gas column model” is also shown in Figure 6b. Large error exists in the simulation results of “Gas column model” because the fric- tion and slip between gas and liquid are ignored. 5 C d Table 1. Basic parameters of Well Tiandong #5. Parameter Value Unit Initial gas ﬂux 0.054 m3/s Drilling ﬂuid density 1.3 g/cm3 H2S content 10% Surface temperature 10 C Well depth 3570 m Drilling speed 0.05 m/s Reservoir permeability 27 mD Reservoir thickness 1.9 m Plastic viscosity of drilling ﬂuid 0.027 Pa s Yield value of drilling ﬂuid 1.2 Pa Mud displacement 24 L/s Geothermal gradient 0.026 C/m 0 250 500 750 1000 1250 1500 1750 2000 0 1 2 3 4 5 6 7 Pit gain (m 3) Time (s) Result of this model Field data of TianDong #5 H2S releases (a) Comparison between simulation and field data of Well Tiandong #5 5 6 7 8 pressure (MPa) Model of this work Gas column model Experimental result Table 2. Basic data of experimental well L.S.U 7. 5 Case study 0 50 100 150 200 250 3000 2500 2000 1500 1000 500 0 Well depth (m) Solubility of H2S (kg/m 3) -0.1 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 Gas o d act o eg=0.471 Slug flow Bubble flow Single phase flow 0.78 kg/m3 a) Gas void fraction and solubility of H2S 3000 2500 2000 1500 1000 500 0 30 40 50 60 70 80 90 100 110 120 130 Well depth (m) Temperature ( ) Temperature inside the drill string Temperature inside the annulus Geothermal temperature (b) Annular temperature 3000 2500 2000 1500 1000 500 0 Formation Fracture Pressure Well depth (m) Formation Pressure 0 10 20 30 40 50 60 70 Annular Pressure (MPa) (c) Annular pressure Fig. 8. Change in gas void fraction, solubility of H2S, annular temperature and annular pressure with the variation in wel depth at shut-in time: (a) Gas void fraction and solubility of H2S; (b) Annular temperature; (c) Annular pressure. a) Gas void fraction and solubility of H2S a) Gas void fraction and solubility of H2S 3000 2500 2000 1500 1000 500 0 30 40 50 60 70 80 90 100 110 120 130 Well depth (m) Temperature ( ) Temperature inside the drill string Temperature inside the annulus Geothermal temperature Well depth (m) Fig. 7. Wellbore conﬁguration of the well in Sichuan. (b) Annular temperature 3000 2500 2000 1500 1000 500 0 Formation Fracture Pressure Well depth (m) Formation Pressure 0 10 20 30 40 50 60 70 Annular Pressure (MPa) (c) Annular pressure Figure 8a shows the change in gas void fraction and solubility of H2S with the variation in well depth at shut- in time. As shown in the ﬁgure, gas rises to a location that is 1800 m below the wellhead, and the maximal gas void fraction is approximately 0.471. The ﬂow pattern criterion proposed by Hasan and Kabir (1988) indicates that three ﬂow patterns exist in the wellbore: bubble, slug, and single phase ﬂows. Besides, the H2S solubility decreases as the well depth decreases. This phenomenon can be attributed to the decrease in annular temperature and pressure with the well depth, as shown in Figures 8b and 8c. When the solubility of H2S decreases to H2S concentration in the drilling ﬂuid at about 500 m, the phase change of H2S occurs. 5 Case study 0 50 100 150 200 250 3000 2500 2000 1500 1000 500 0 Well depth (m) Solubility of H2S (kg/m 3) -0.1 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 Gas void fraction eg=0.471 Slug flow Bubble flow Single phase flow 0.78 kg/m3 a) Gas void fraction and solubility of H2S 3000 2500 2000 1500 1000 500 0 30 40 50 60 70 80 90 100 110 120 130 Well depth (m) Temperature ( ) Temperature inside the drill string Temperature inside the annulus Geothermal temperature (b) Annular temperature 2500 2000 1500 1000 500 0 Formation Fracture Pressure Well depth (m) Formation Pressure 0 10 20 30 40 50 60 70 Annular Pressure (MPa) and ut- hat oid ion ree ngle well the well lity 0 50 100 150 200 250 3000 2500 2000 1500 1000 500 0 Well depth (m) Solubility of H2S (kg/m 3) -0.1 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 Gas void fraction eg=0.471 Slug flow Bubble flow Single phase flow 0.78 kg/m3 a) Gas void fraction and solubility of H2S 3000 2500 2000 1500 1000 500 0 30 40 50 60 70 80 90 100 110 120 130 Well depth (m) Temperature ( ) Temperature inside the drill string Temperature inside the annulus Geothermal temperature (b) Annular temperature 2000 1500 1000 500 0 Formation Fracture Pressure Well depth (m) Formation Pressure 0 10 20 30 40 50 60 70 Annular Pressure (MPa) 0 50 100 150 200 250 3000 2500 2000 1500 1000 500 0 Well depth (m) Solubility of H2S (kg/m 3) -0.1 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 Gas void fraction eg=0.471 Slug flow Bubble flow Single phase flow 0.78 kg/m3 Fig. 7. Wellbore conﬁguration of the well in Sichuan. 5 Case study As shown in re 8c, the annular pressure below 2000 m is lower than the formation pressure. This phenomenon can be attributed to an invasion of natural gas, which increases the dif- ferential pressure between bottom hole and formation. 7. Wellbore conﬁguration of the well in Sichuan. 0 50 100 150 200 250 3000 2500 2000 1500 1000 500 0 Well depth (m) Solubility of H2S (kg/m 3) -0.1 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 Gas void fraction eg=0.471 Slug flow Bubble flow Single phase flow 0.78 kg/m3 a) Gas void fraction and solubility of H2S 3000 2500 2000 1500 1000 500 0 30 40 50 60 70 80 90 100 110 120 130 Well depth (m) Temperature ( ) Temperature inside the drill string Temperature inside the annulus Geothermal temperature (b) Annular temperature 3000 2500 2000 1500 1000 500 0 Formation Fracture Pressure Well depth (m) Formation Pressure 0 10 20 30 40 50 60 70 Annular Pressure (MPa) (c) Annular pressure Fig. 8. Change in gas void fraction, solubility of H2S, annular temperature and annular pressure with the variation in well depth at shut-in time: (a) Gas void fraction and solubility of H2S; (b) Annular temperature; (c) Annular pressure. L. Mao et al.: Oil & Gas Science and Technology – Rev. IFP Energies nouvelles 75, 71 (2020) 11 L. Mao et al.: Oil & Gas Science and Technology – Rev. IFP Energies nouvelles 75, 71 (2020) 11 in gas void fraction and ion in well depth at shut- gas rises to a location that and the maximal gas void The ﬂow pattern criterion 1988) indicates that three re: bubble, slug, and single ubility decreases as the well on can be attributed to the and pressure with the well nd 8c. When the solubility ation in the drilling ﬂuid at of H2S occurs. ﬂuid temperature distribu- l t h t i ti Th the well in Sichuan. 5 Case study The basic parameters from Table 3 are used to simulate the killing process of an H2S-containing natural gas well in Sichuan. The number of spatial nodes is 60 and number of spatial nodes is 80 in the simulation. The wellbore conﬁg- uration of this well is shown in Figure 7. When overﬂow behavior occurs, the invaded gas will push the drilling ﬂuid with the same volume out of the wellbore, thereby leading to an increase in the pit gain. In drilling engineering, once the pit gain exceeds a threshold value, a shut-in operation must be performed. On the basis of ﬁeld experience, the threshold value of pit gain is set as 3 m3 in this work. (b) Comparison between simulation and experimental results Fig. 6. Comparison results: (a) is the comparison between simulation and ﬁeld data of Well Tiandong #5; (b) is the comparison between simulation and experimental results. igure 8a shows the change in gas void fraction and ility of H2S with the variation in well depth at shut- me. As shown in the ﬁgure, gas rises to a location that 00 m below the wellhead, and the maximal gas void on is approximately 0.471. The ﬂow pattern criterion osed by Hasan and Kabir (1988) indicates that three patterns exist in the wellbore: bubble, slug, and single e ﬂows. Besides, the H2S solubility decreases as the well h decreases. This phenomenon can be attributed to the ase in annular temperature and pressure with the well h, as shown in Figures 8b and 8c. When the solubility S decreases to H2S concentration in the drilling ﬂuid at t 500 m, the phase change of H2S occurs. igure 8b shows the drilling ﬂuid temperature distribu- in the drilling string and annulus at shut-in time. The lar temperature is always higher than that in the ng string because the drilling ﬂuid in the annulus is r to the formation, thus, more heat can be gotten from formation. Moreover, the annular temperature and hermal temperature intersect at about 2250 m, which ates that when the well depth is less than 2250 m, rilling ﬂuid in the annulus can be cooled by the forma- When the well depth is greater than 2250 m, the dril- ﬂuid in the annulus can be heated by the formation. re 8c shows the change in annular pressure with the tion in well depth at shut-in time. 5 Case study The increasing differential pressure will cause an increase in gas ﬂow from formation to the wellbore and result in blow- out accidents easily if not controlled. Time (s) (b) Casing pressure Driller’s method is widely used in well killing in oil and gas ﬁelds (Feng et al., 2016a). Thus, the method is also used in the simulation of this work. Figure 9 shows the physical model of the killing process. During killing, the pressure balance relationship can be expressed as follows (Feng et al., 2016b): Fig. 10. Change in bottom hole pressure and casing pressure the variation in time: (a) Bottom hole pressure; (b) Casing pressure. Fig. 10. Change in bottom hole pressure and casing pressure the variation in time: (a) Bottom hole pressure; (b) Casing pressure. Pp Pb ¼ Pa þ Pla þ Pma: ð61Þ ð61Þ Figure 10a shows the change in bottom hole pressure with the variation in time. As shown in the ﬁgure, the bottom hole pressure ﬁrst decreases during the overﬂow period and then keeps constant (greater or equal to forma- tion pressure) when well killing is conducted. Therefore, no gas ﬂows into the wellbore from the formation during killing. Figure 10a shows the change in bottom hole pressure with the variation in time. As shown in the ﬁgure, the bottom hole pressure ﬁrst decreases during the overﬂow period and then keeps constant (greater or equal to forma- tion pressure) when well killing is conducted. Therefore, no gas ﬂows into the wellbore from the formation during killing. time. Stage 2 indicates that the two-phase section in the wellbore is rising up toward the wellbore prior to reaching the wellhead. At this time, the ﬂow pattern distribution in the wellbore is single phase ﬂow, gas–liquid two-phase ﬂow, and single phase ﬂow in sequence. Moreover, the casing pressure increases with the increase in killing time. This phenomenon is attributed to the slippage and expan- sion of gas. The gas will expand as it rises up along the well- bore because the annular and formation temperatures and depth decrease (Sun et al., 2018). Thus, the hydrostatic pressure decreases accordingly and the casing pressure increases to keep the bottom hole pressure constant. Stage 3 indicates that the gas reaches the wellhead and single phase and gas–liquid two-phase ﬂows exist in the wellbore. The total gas volume reaches a maximum at this point. 5 Case study Formation Pressure Figure 8b shows the drilling ﬂuid temperature distribu- tion in the drilling string and annulus at shut-in time. The annular temperature is always higher than that in the drilling string because the drilling ﬂuid in the annulus is closer to the formation, thus, more heat can be gotten from the formation. Moreover, the annular temperature and geothermal temperature intersect at about 2250 m, which indicates that when the well depth is less than 2250 m, the drilling ﬂuid in the annulus can be cooled by the forma- tion. When the well depth is greater than 2250 m, the dril- ling ﬂuid in the annulus can be heated by the formation. Figure 8c shows the change in annular pressure with the variation in well depth at shut-in time. As shown in Figure 8c, the annular pressure below 2000 m is lower than (c) Annular pressure (c) Annular pressure Fig. 8. Change in gas void fraction, solubility of H2S, annular temperature and annular pressure with the variation in well depth at shut-in time: (a) Gas void fraction and solubility of H2S; (b) Annular temperature; (c) Annular pressure. the formation pressure. This phenomenon can be attributed to an invasion of natural gas, which increases the dif- ferential pressure between bottom hole and formation. L. Mao et al.: Oil & Gas Science and Technology – Rev. IFP Energies nouvelles 75, 71 (2020) 12 Pump Throttle valve Formation Cement Casing Natural gas Supercritical H 2S Acid gas Phase change of H 2S Polluted drilling fluid Well killing fluid Drilling string Drill bit Heat Conduction Heat Convection Fig. 9. Physical model of the killing process. Pump Throttle valve Formation Cement Casing Natural gas Supercritical H 2S Acid gas Phase change of H 2S Polluted drilling fluid Well killing fluid Drilling string Drill bit Heat Conduction Heat Convection Fig. 9. Physical model of the killing process. 0 500 1000 1500 2000 2500 3000 3500 4000 30 31 32 33 34 35 36 37 38 39 Bottom hole pressure (MPa) Time (s) Start killing well Time (s) (a) Bottom hole pressure ( ) p 1 2 3 4 5 6 0 500 1000 1500 2000 2500 3000 3500 4000 4500 0 2 4 6 8 10 12 Casing Pressure (MPa) Time (s) H2S release (b) Casing pressure Casing Pressure (MPa) Fig. 9. Physical model of the killing process. 5.1 Analysis under different H2S contents Considering that the phase change of H2S occurs at low pressure and temperature and the phase change of H2S has a great effect on the pressure distribution (Sun et al., 2013), the diagrams of wellhead ﬂow pattern when gas reaches wellhead during well killing under different working conditions are depicted to study the ﬂows at wellhead. a) Gas kick y Figure 12 shows the diagram of wellhead ﬂow pattern when gas reaches wellhead during well killing with H2S con- tents of 0%, 2%, 4%, and 6%. As shown in the ﬁgure, the gas void fraction increases with the increase in H2S content. This phenomenon is caused by the increase in gasiﬁcation volume of H2S as H2S content increases. When the well depth decreases, the annular pressure and annular temper- ature decrease, as shown in Figure 13. Besides, the higher the H2S content is, the lower the annular temperature will be as shown in Figure 13b. This is because, near the critical point of H2S, the viscosity of supercritical H2S decreases sig- niﬁcantly (Vesovic et al., 1998). Therefore, in the upper well section, the convective heat transfer coefﬁcient between the drilling ﬂuid and the formation increases as viscosity of supercritical H2S decreases. Thus, more heat will be diffused from the drilling ﬂuid in the annulus to the formation. As H2S content increases, this effect will be enhanced. There- fore, in the upper well section which is near the critical point of H2S, the annular temperature is lower at higher H2S con- tent and, as a result, at the same well depth, the H2S solu- bility will be lower and more H2S will gasify. (b) Well killing Fig. 11. Dynamical pressure distribution in the wellbore: (a) during gas kick; (b) during well killing. y g y Additional H2S will gasify at higher H2S content, and this condition changes the wellhead ﬂow pattern. Churn ﬂow appears at the wellhead with H2S content of 0%; how- ever, when H2S is released, annular ﬂow appears and the droplets in the bubble decrease as H2S content increases. Therefore, the hydrostatic pressure decreases with the increase in H2S content, and the casing pressure increases to keep the bottom hole pressure constant. Figure 14 shows that, when H2S is released, the casing pressure increases with H2S content. Thus, the annular pressure of the upper well section increases with the increase in H2S content, as shown in Figure 13a. 5 Case study With the change in hydrostatic pressure created by the ﬂuid column, the casing pressure of the wellhead can be changed by adjusting the throttle opening to maintain the bottom hole pressure constant. Figure 10b shows the change in casing pressure with the variation in time. As shown in the ﬁgure, the entire killing process can be divided into six stages. Stage 1 is the initial stage of the killing pro- cess, which is determined by the ﬂow pattern at shut-in L. Mao et al.: Oil & Gas Science and Technology – Rev. IFP Energies nouvelles 75, 71 (2020) 13 a) Gas kick (b) Well killing Fig. 11. Dynamical pressure distribution in the wellbore: (a) during gas kick; (b) during well killing. a) Gas kick pressure distribution in the wellbore (Fig. 11). As shown in Figure 11, the pressure of each location along the wellbore is higher than that of the overﬂow process during the killing process. Therefore, the H2S solubility of each location in the killing process increases compared with that of shut-in time. Although it reaches the wellhead, the H2S will not release until the H2S solubility is lower than its content in the drilling ﬂuid. Therefore, when the casing pressure decreases, the H2S solubility decreases. As a result, the H2S transforms from a supercritical phase to a gas phase, gas volume expands suddenly, and gas void fraction increases abruptly. Thus, the casing pressure drops slowly. 5.1 Analysis under different H2S contents However, H2S content has a small effect on entire wellbore pressure distribution, as shown in Figure 14. Stage 4 indicates that all the gas has been pushed out of the wellbore and only single phase ﬂow exists in the wellbore. This stage will last for some time until the weighted drilling ﬂuid enters the annulus. Furthermore, the casing pressure keeps constant in Stage 4. Stage 5 indicates that the weighted drilling ﬂuid enters the annulus and the hydro- static pressure increases. Thus, the casing pressure decreases. Stage 6 indicates that the annulus is full of the weighted drilling ﬂuid and the casing pressure decreases to 0. The well-killing operation ends. Figure 10b also shows that, when all the gas is about to be pushed out of the well, H2S is released, and the curve drops slowly. This phenomenon is the result of casing pres- sure applied at the wellhead. Figure 8a shows that, when overﬂow behavior occurs, H2S will release at a location close to the wellhead if shut-in operation is not performed. How- ever, H2S solubility decreases with the increase in pressure, and the casing pressure applied at the wellhead changes the From comparison between Figures 8c and 13a, it can be concluded that well killing can rebuild the pressure distribu- tion in the wellbore and stop the overﬂow behavior caused by the pressure differential between bottom hole and forma- tion. However, for the stratum with low formation fracture L. Mao et al.: Oil & Gas Science and Technology – Rev. IFP Energies nouvelles 75, 71 (2020) 14 Fig. 12. Diagram of wellhead ﬂow pattern when gas reaches wellhead during well killing with H2S contents of 0%, 2%, 4%, and 6%. Fig. 12. Diagram of wellhead ﬂow pattern when gas reaches wellhead during well killing with H2S contents of 0%, 2%, 4%, and 6%. Fig. 12. 5.1 Analysis under different H2S contents Diagram of wellhead ﬂow pattern when gas reaches wellhead during well killing with H2S con 3000 2500 2000 1500 1000 500 0 0 10 20 30 40 50 60 Formation Pressure Well depth (m) 0 % 2 % 4 % 6 % Formation Fracture Pressure Annular pressure (MPa) (a) Annular pressure 3000 2500 2000 1500 1000 500 0 40 50 60 70 80 90 100 110 1000 800 600 400 200 040 45 50 55 60 65 70 Well depth (m) 0% 2% 4% 6% Temperature ( ) Temperature ( ) (b) Annular temperature. Fig. 13. Change in annular pressure and temperature with the variation in well depth at 1300 s during well killing with H2S contents of 0%, 2%, 4%, and 6%: (a) Annular pressure; (b) Annular temperature. 3000 2500 2000 1500 1000 500 0 0 10 20 30 40 50 60 Formation Pressure Well depth (m) 0 % 2 % 4 % 6 % Formation Fracture Pressure Annular pressure (MPa) (a) Annular pressure pressure, the well-killing operation may cause annulus pressure to approach or exceed formation fracture pres- sure, thereby causing well leakage. This scenario should be highly regarded, especially for H2S-containing natural gas wells. Figure 13a shows that, when H2S is released, the annular pressure of the upper well section will exceed the formation fracture pressure. Moreover, a high H2S content indicates serious fracture of the stratum. Therefore, during the late period of well killing in H2S-containing natural gas wells, the casing pressure should be reduced appropriately to avoid stratum fracturing and prevent leak- age accidents. (b) Annular temperature. 5.2 Analysis under different initial overﬂow volumes Figure 15 shows the diagram of wellhead ﬂow pattern when gas reaches wellhead during well killing with initial overﬂow volumes of 2, 4, and 8 m3. As shown in the ﬁgure, the gas void fraction increases with the increase in initial overﬂow volume because a large amount of gas enters the wellbore at high initial overﬂow volume. Moreover, the wellhead ﬂow pattern with initial overﬂow volume of 2 m3 can be re- garded as slug ﬂow, whereas the annular ﬂow appears at high initial overﬂow volumes of 4 and 8 m3. The gas phase becomes continuous at initial overﬂow volumes of 4 and 8 m3. The continuous phase volume increases as the initial overﬂow volume increases. (a) Annular pressure 3000 2500 2000 1500 1000 500 0 40 50 60 70 80 90 100 110 1000 800 600 400 200 040 45 50 55 60 65 70 Well depth (m) 0% 2% 4% 6% Temperature ( ) Temperature ( ) (b) Annular temperature. Well depth (m) A high initial overﬂow volume indicates gas rises to a high location in the wellbore at shut-in time. When initial overﬂow volume reaches 8 m3, the gas enters the wellhead at shut-in time. This conclusion can also be drawn from Figure 16. Figure 16b shows the change in casing pressure with the variation in time and initial overﬂow volumes of 2, 4, and 8 m3. As shown in the ﬁgure, the surface casing pressure increases with the increase in initial overﬂow volume. This phenomenon can be attributed to the increase in hydrostatic pressure loss as gas volume increases. Thus, additional surface casing pressure should be applied to balance the bottom hole pressure at high initial overﬂow volume. Moreover, as shown in Figure 16b, the pressure change in the wellbore intensiﬁes as initial overﬂow volume increases. (b) Annular temperature. (b) Annular temperature. Fig. 13. Change in annular pressure and temperature with the variation in well depth at 1300 s during well killing with H2S contents of 0%, 2%, 4%, and 6%: (a) Annular pressure; (b) Annular temperature. L. Mao et al.: Oil & Gas Science and Technology – Rev. 5.2 Analysis under different initial overﬂow volumes We can see that, the drilling ﬂuid temperature is higher at higher initial overﬂow volume. This is because, as more gas enters the wellbore, more heat from the formation will be carried into the well- bore. Thus, the H2S solubility will be higher at the same well depth and less H2S will gasify. Therefore, when H2S gasiﬁes, the casing pressure applied at wellhead is higher at lower initial overﬂow volume. ﬁgure, the gas void fraction at wellhead increases with the increase in drilling ﬂuid density. This phenomenon can be attributed to the decrease in casing pressure caused by the increase in drilling ﬂuid density. The annular pressure is low at shut-in time with low drilling ﬂuid density because the hydrostatic pressure is proportional to drilling ﬂuid den- sity. Therefore, to maintain the bottom hole pressure, the surface casing pressure should be higher at low drilling ﬂuid density, as shown in Figure 19. During well killing, the annular pressure is high at low drilling ﬂuid density, as shown in Figure 20a. The gas void fraction is high at high drilling ﬂuid density because the gas expands violently at low pressure. Figure 18 shows that the drilling ﬂuid density slightly affects the wellhead ﬂow pattern, and annular ﬂow is the common ﬂow pattern under drilling ﬂuid densities of 1.20, 1.25, and 1.30 g/m3. Figure 17b shows the change in annular pressure with the variation in well depth and initial overﬂow volumes of 2, 4, and 8 m3. As initial overﬂow volume increases, the gas expansion capacity is limited. Thus, pressure changed rapidly from 2 m3 to 4 m3, but slowly from 4 m3 to 8 m3. As shown in the ﬁgure, the annular pressure is high with high initial overﬂow volume. This phenomenon is caused by the increase in surface casing pressure as initial overﬂow volume increases. However, when initial overﬂow volume increases, the annular pressure may approach or even ex- ceed the formation fracture pressure, which may lead to for- mation fracturing. Therefore, careful monitoring of the pit gain is necessary during the exploitation of H2S-containing natural gas wells. Once the pit gain exceeds a threshold va- lue, the shut-in operation should be performed immediately to prevent further overﬂow. 5.2 Analysis under different initial overﬂow volumes IFP Energies nouvelles 75, 71 (2020) 15 (a) Wellbore pressure 0% 2% 4% 6% 0 500 1000 1500 2000 2500 3000 0 2 4 6 8 10 12 14 1100 1150 1200 1250 1300 1350 1400 1450 3.0 3.5 4.0 4.5 5.0 0% 2% 4% 6% Casing pressure (MPa) Time (s) H2S release (b) Casing pressure 14. Change in wellbore pressure and casing pressure with the variation in time and H2S contents of 0%, 2%, 4%, and L. Mao et al.: Oil & Gas Science and Technology Rev. IFP Energies nouvelles 75, 71 (2020) (a) Wellbore pressure 0% 2% 4% 6% 0% 2% 6% (a) Wellbore pressure 4% 6% (a) Wellbore pressure (a) Wellbore pressure 0 500 1000 1500 2000 2500 3000 0 2 4 6 8 10 12 14 1100 1150 1200 1250 1300 1350 1400 1450 3.0 3.5 4.0 4.5 5.0 0% 2% 4% 6% Casing pressure (MPa) Time (s) H2S release (b) Casing pressure Fig. 14. Change in wellbore pressure and casing pressure with the variation in time and H2S contents of 0%, 2%, 4%, and 6%: g. 14. Change in wellbore pressure and casing pressure with the variation in time and H2S contents o re pressure and casing pressure with the variation in time and H2S contents of 0%, 2%, 4%, and 6%: Fig. 14. Change in wellbore pressure and casing pressure with the variation in time and H2S contents of 0%, 2%, 4%, and 6%: L. Mao et al.: Oil & Gas Science and Technology – Rev. IFP Energies nouvelles 75, 71 (2020) 16 Fig. 15. Diagram of wellhead ﬂow pattern when gas reaches wellhead during well killing with initial overﬂow volumes of 2, 4, and 8 m3. Fig. 15. Diagram of wellhead ﬂow pattern when gas reaches wellhead during well killing with initial overﬂow volumes of 2, 4, and 8 m3. Fig. 15. Diagram of wellhead ﬂow pattern when gas reaches wellhead during well killing with initial over Besides, as the initial overﬂow volume increases, the gasiﬁcation starting time of H2S increases and, the casing pressure is lower as shown in Figure 16a. This phenomenon can be attributed to the change of drilling ﬂuid tempera- ture. Figure 17a shows the change in annular temperature with the variation in well depth at 1300 s during well killing with different initial overﬂow volume. 5.2 Analysis under different initial overﬂow volumes Furthermore, during the late period of well killing in H2S-containing natural gas wells with high initial overﬂow volume, the surface casing pres- sure should be decreased appropriately to avoid stratum fracturing and prevent leakage accidents. Early detection of gas kick should be more frequent to avoid severe overﬂow. Figure 20b shows the change in annular temperature with the variation in well depth at 1300 s during well killing with different drilling ﬂuid density. As shown in Figure 20b, for the deeper well section, the drilling ﬂuid temperature increases with the decrease in drilling ﬂuid density. This is because, when the same heat is obtained from the forma- tion, the greater the drilling ﬂuid density, the less the increase in temperature (Gao et al., 2017). For the upper well section, the drilling ﬂuid temperature is closer to the formation temperature, the temperature differences decrease. Considering that H2S only gasiﬁes in the upper well section, the effect of temperature difference caused by the change in drilling ﬂuid density on gasiﬁcation of H2S is small as shown in Figure 19b. Furthermore, annular pressure of the upper well section will exceed the formation fracture pressure, and low drilling ﬂuid density indicates serious fracture of the stratum. Thus, during the well killing in H2S-containing natural gas wells, higher drilling ﬂuid density should be used to avoid stratum fracturing and prevent leakage accidents. 5.4 Analysis under different well-killing displacements Figure 18 shows the diagram of wellhead ﬂow pattern when gas reaches wellhead during well killing with drilling ﬂuid densities of 1.20, 1.25, and 1.30 g/cm3. As shown in the Figure 21 shows the diagram of wellhead ﬂow pattern when gas reaches wellhead during well killing with displacements L. Mao et al.: Oil & Gas Science and Technology – Rev. IFP Energies nouvelles 75, 71 (2020) 17 (a) Wellbore pressure 0 500 1000 1500 2000 2500 3000 0 2 4 6 8 10 Casing pressure (MPa) Time (s) 2 (m 3) 4 (m 3) 8 (m 3) (b) Casing pressure 2m3 4m3 8m3 16. Change in wellbore pressure and casing pressure with the variation in time and initial overﬂow volumes of 2, 4, and 8 m3: Wellbore pressure; (b) Casing pressure. L. Mao et al.: Oil & Gas Science and Technology – Rev. IFP Energies nouvelles 75, 71 (2020) 17 2m3 4m3 4m3 (a) Wellbore pressure 4m3 8m3 4m3 2m3 8m3 (a) Wellbore pressure (a) Wellbore pressure 0 500 1000 1500 2000 2500 3000 0 2 4 6 8 10 Casing pressure (MPa) Time (s) 2 (m 3) 4 (m 3) 8 (m 3) (b) Casing pressure Fig. 16. Change in wellbore pressure and casing pressure with the variation in time and initial overﬂow volumes of 2, 4, and 8 m3: (a) Wellbore pressure; (b) Casing pressure. (a) Wellbore pressure 0 500 1000 1500 2000 2500 3000 0 2 4 6 8 10 Casing pressure (MPa) Time (s) 2 (m 3) 4 (m 3) 8 (m 3) (b) Casing pressure Casing pressure (MPa) Fig. 16. Change in wellbore pressure and casing pressure with the variation in time and initial overﬂow volumes of 2, 4, and 8 m3: (a) Wellbore pressure; (b) Casing pressure. L. Mao et al.: Oil & Gas Science and Technology – Rev. IFP Energies nouvelles 75, 71 (2020) 18 3000 2500 2000 1500 1000 500 0 Formation Fracture Pressure Well depth (m) 2 (m 3) 4 (m 3) 8 (m 3) Formation Pressure 0 10 20 30 40 50 60 70 Annular Pressure (MPa) (b) Annular pressure. 5.4 Analysis under different well-killing displacements 3000 2500 2000 1500 1000 500 0 40 50 60 70 80 90 100 110 120 Well depth (m) 2(m 3) 4(m 3) 8(m 3) (a) Annular temperature 3000 2500 2000 1500 1000 500 0 Formation Fracture Pressure Well depth (m) 2 (m 3) 4 (m 3) 8 (m 3) Formation Pressure 0 10 20 30 40 50 60 70 Annular Pressure (MPa) (b) Annular pressure. Fig. 17. Change in annular temperature and annular pressure with the variation in well depth at 1300 s during well killing with initial overﬂow volumes of 2, 4, and 8 m3: (a) Annular temperature; (b) Annular pressure. 3000 2500 2000 1500 1000 500 0 40 50 60 70 80 90 100 110 120 Well depth (m) 2(m 3) 4(m 3) 8(m 3) (a) Annular temperature (b) Annular pressure. (b) Annular pressure. (a) Annular temperature Fig. 17. Change in annular temperature and annular pressure with the variation in well depth at 1300 s during well killing with initial overﬂow volumes of 2, 4, and 8 m3: (a) Annular temperature; (b) Annular pressure. Fig. 18. Diagram of wellhead ﬂow pattern when gas reaches wellhead during well killing with drilling ﬂuid densities of 1.20, 1.25, and 1.30 g/cm3. Fig. 18. Diagram of wellhead ﬂow pattern when gas reaches wellhead during well killing with drilling ﬂuid densities of 1.20, 1.25, and 1 30 g/cm3 Fig. 18. Diagram of wellhead ﬂow pattern when gas reaches wellhead during well killing with drilling ﬂuid densities of 1.20, 1.25, and 1.30 g/cm3. However, the effect of displacement on temperature distri- bution in annulus is small. of 20, 30, and 40 L/s. As shown in the ﬁgure, the gas void fraction at wellhead increases slightly with the increase in displacement. The annular ﬂow is the common wellhead ﬂow pattern under displacements of 20, 30, and 40 L/s, and the number of droplets in the continuous gas decreases with the increase in displacement. Figure 23 shows the change in wellbore pressure and casing pressure with the variation in time and displace- ments of 20, 30, and 40 L/s. As shown in the ﬁgure, the higher the displacement is, the faster the gas kick leaves wellbore and, the lower the casing pressure will be. Besides, H2S will gasify at an earlier time. This phenomenon can be attributed to the increased friction resistance due to the increase in displacement (Shi et al., 2014). 5.4 Analysis under different well-killing displacements Thus, the bot- tom hole pressure increases as displacement increases and, the casing pressure decreases as a result. Therefore, the annular pressure is lower at higher displacement in the upper well section as shown in Figure 22b. Besides, the annular pressure of the upper well section will exceed the formation fracture pressure, and low displacement indicates serious fracture of the stratum. Thus, during well Figure 22a shows the change in annular temperature with the variation in well depth at 1300 s during well killing with different displacement. The lower the displacement is, the longer time for heat exchange with the formation will be. Thus, for the upper well section, the annular tempera- ture is higher than formation, at higher displacement, the drilling ﬂuid temperature is higher because lesser heat of drilling ﬂuid will loss. For the deeper well section, the annu- lar temperature is lower than formation, at higher displace- ment, the drilling ﬂuid temperature is lower because lesser heat from formation will be exchanged to the drilling ﬂuid. 19 L. Mao et al.: Oil & Gas Science and Technology – Rev. IFP Energies nouvelles 75, 71 (2020) (a) Wellbore pressure 0 500 1000 1500 2000 2500 3000 3500 0 1 2 3 4 5 6 7 8 9 Casing pressure (MPa) Time (s) 1.20 (g/cm 3) 1.25 (g/cm 3) 1.30 (g/cm 3) 1.20g/cm3 1.25g/cm3 1.30g/cm3 (b) Casing pressure Fig. 19. Change in wellbore pressure and casing pressure with the variation in time and drilling ﬂuid densities of 1.20, 1.25, and / 3 ( ) ( ) L. Mao et al.: Oil & Gas Science and Technology – Rev. IFP Energies nouvelles 75, 71 (2020) 19 1.25g/cm3 1.25g/cm3 1.20g/cm3 (a) Wellbore pressure 1.30g/cm3 1.30g/cm3 (a) Wellbore pressure 1.30g/cm3 (a) Wellbore pressure 0 500 1000 1500 2000 2500 3000 3500 0 1 2 3 4 5 6 7 8 9 Casing pressure (MPa) Time (s) 1.20 (g/cm 3) 1.25 (g/cm 3) 1.30 (g/cm 3) (b) Casing pressure Fig. 19. Change in wellbore pressure and casing pressure with the variation in time and drilling ﬂuid densities of 1.20, 1.25, and 1.30 g/cm3: (a) Wellbore pressure; (b) Casing pressure. (a) Wellbore pressure 0 500 1000 1500 2000 2500 3000 3500 0 1 2 3 4 5 6 7 8 9 Casing pressure (MPa) Time (s) 1.20 (g/cm 3) 1.25 (g/cm 3) 1.30 (g/cm 3) (b) Casing pressure (a) Wellbore pressure Fig. 19. 5.4 Analysis under different well-killing displacements Change in wellbore pressure and casing pressure with the variation in time and drilling ﬂuid densities of 1.20, 1.25, and 1.30 g/cm3: (a) Wellbore pressure; (b) Casing pressure. L. Mao et al.: Oil & Gas Science and Technology – Rev. IFP Energies nouvelles 75, 71 (2020) 20 3000 2500 2000 1500 1000 500 0 Formation Fracture Pressure Well depth (m) 1.20 (g/m 3) 1.25 (g/m 3) 1.30 (g/m 3) Formation Pressure 0 10 20 30 40 50 60 70 Annular Pressure (MPa) (a) Annular pressure 3000 2500 2000 1500 1000 500 0 40 50 60 70 80 90 100 110 Well depth(m) 1.20(g/cm 3) 1.25(g/cm 3) 1.30(g/cm 3) Temperature ( ) (b) Annular temperature Fig. 20. Change in annular temperature and annular pressure with the variation in well depth at 1300 s during well killing with drilling ﬂuid densities of 1.20, 1.25, and 1.30 g/cm3: (a) Annular pressure; (b) Annular temperature. 3000 2500 2000 1500 1000 500 0 Formation Fracture Pressure Well depth (m) 1.20 (g/m 3) 1.25 (g/m 3) 1.30 (g/m 3) Formation Pressure 0 10 20 30 40 50 60 70 Annular Pressure (MPa) (a) Annular pressure 3000 2500 2000 1500 1000 500 0 40 50 60 70 80 90 100 110 Well depth(m) 1.20(g/cm 3) 1.25(g/cm 3) 1.30(g/cm 3) Temperature ( ) (b) Annular temperature Annular Pressure (MPa) Fig. 20. Change in annular temperature and annular pressure with the variation in well depth at 1300 s during well killing with drilling ﬂuid densities of 1.20, 1.25, and 1.30 g/cm3: (a) Annular pressure; (b) Annular temperature. Fig. 21. Diagram of wellhead ﬂow pattern when gas reaches wellhead during well killing with displacements of 20, 30, and 40 L/s. Fig. 21. Diagram of wellhead ﬂow pattern when gas reaches wellhead during well killing with displacements of 20, 30, and 40 L/s. Fig. 21. Diagram of wellhead ﬂow pattern when gas reaches wellhead during well killing with displac 3000 2500 2000 1500 1000 500 0 35 40 45 50 55 60 65 70 75 80 85 90 95 100 105 110 Well depth (m) 20 L/s 30 L/s 40 L/s Temperature ( ) (a) Annular temperature 3000 2500 2000 1500 1000 500 0 Formation Fracture Pressure Well depth (m) 20 (L/s) 30 (L/s) 40 (L/s) Formation Pressure 0 10 20 30 40 50 60 70 Annular Pressure (MPa) (b) Annular pressure Fig. 22. 6 Conclusion Ebrahimi A., Khamehchi E. (2015) A robust model for comput- ing pressure drop in vertical multiphase ﬂow, J. Nat. Gas. Sci. Eng. 26, 1306–1316. This work established a dynamical well-killing model con- sidering an H2S solubility to simulate the well-killing pro- cess of a vertical H2S-containing natural gas well. The following important conclusions can be drawn: Elsharkawy A.M. (2004) Efﬁcient methods for calculations of compressibility, density and viscosity of natural gases, Fluid Phase Equilibr. 218, 1, 1–13. Feng J., Fu J., Chen P., Liu Z., Wei H. (2015) Predicting pressure behavior during dynamic kill drilling with a two- phase ﬂow, J. Nat. Gas Sci. Eng. 22, 591–597. 1. H2S will gasify near wellhead during well killing when casing pressure decreases. Near the critical point of H2S, the annular temperature decreases as H2S con- tent increases and, the H2S solubility will be lower and more H2S will gasify. To balance the bottom hole pressure, when H2S releases, the casing pressure increases as H2S content increase. Feng J., Fu J., Chen P., Luo J., Kou B. (2016a) Comparisons of the Driller’s method and the wait and weight method in deep- water well killing operation, Arab. J. Sci. Eng. 41, 7, 2699–2706. Feng J., Fu J., Luo J., Liu Z., Wei H. (2016b) An advanced Driller’s Method simulator for deepwater well control, J. Loss Prevent. Proc. 39, 131–140. 2. As initial overﬂow volume increases, the annular tem- perature, annular pressure and the casing pressure increase signiﬁcantly. When H2S gasiﬁes, the casing pressure applied at wellhead should be higher at lower initial overﬂow volume to balance bottom hole pressure. Fu J., Su Y., Jiang W., Li S., Chen Y. (2019) Wellbore annulus water hammer pressure prediction based on transient multi- phase ﬂow characteristics, Oil Gas Sci. Technol. – Rev. IFP Energies nouvelles 74, 84. Gao Y., Cui Y., Xu B., Sun B., Zhao X., Li H., Chen L. (2017) Two phase ﬂow heat transfer analysis at different ﬂow patterns in the wellbore, Appl. Therm. Eng. 117, 544–552. p 3. In the well-killing process, the annular pressure and temperature decrease as drilling ﬂuid density increases and a lower casing pressure is needed for balancing bottom hole pressure. No signiﬁcant effect of drilling ﬂuid density on the gasiﬁcation of H2S is found. As well-killing displacement increases, for the upper well section, the annular temperature increases while the annular pressure decreases. 5.4 Analysis under different well-killing displacements IFP Energies nouvelles 75, 71 (2020) 22 killing in H2S-containing natural gas wells, higher well- killing displacement should be used to avoid stratum fracturing and prevent leakage accidents. Bilicki Z., Kestin J. (1987) Transition criteria for two-phase ﬂow patterns in vertical upward ﬂow, Int. J. Multiphase Flow. 13, 3, 283–294. Blowout accident of TianDong well #5 (1990) Petrochina Southwest Oil and Gas ﬁeld Company. 5.4 Analysis under different well-killing displacements Change in annular temperature and annular pressure with the variation in well depth at 1300 s during well killing with displacements of 20, 30, and 40 L/s: (a) Annular temperature; (b) Annular pressure. 3000 2500 2000 1500 1000 500 0 Formation Fracture Pressure Well depth (m) 20 (L/s) 30 (L/s) 40 (L/s) Formation Pressure 0 10 20 30 40 50 60 70 Annular Pressure (MPa) (b) Annular pressure 3000 2500 2000 1500 1000 500 0 35 40 45 50 55 60 65 70 75 80 85 90 95 100 105 110 Well depth (m) 20 L/s 30 L/s 40 L/s Temperature ( ) (a) Annular temperature Well depth (m) (b) Annular pressure Fig. 22. Change in annular temperature and annular pressure with the variation in well depth at 1300 s during well killing with displacements of 20, 30, and 40 L/s: (a) Annular temperature; (b) Annular pressure. 21 L. Mao et al.: Oil & Gas Science and Technology – Rev. IFP Energies nouvelles 75, 71 (2020) (a) Wellbore pressure 0 500 1000 1500 2000 2500 3000 0 1 2 3 4 5 6 7 8 9 10 Casing pressure (MPa) Time (s) 20 (L/s) 25 (L/s) 30 (L/s) (b) Casing pressure 20L/s 30L/s 40L/s Fig. 23. Change in wellbore pressure and casing pressure with the variation in time with displacements of 20, 30, and 40 a) Wellbore pressure; (b) Casing pressure. L. Mao et al.: Oil & Gas Science and Technology – Rev. IFP Energies nouvelles 75, 71 (2020) 30L/s 30L/s 20L/s 40L/s (a) Wellbore pressure (a) Wellbore pressure 0 500 1000 1500 2000 2500 3000 0 1 2 3 4 5 6 7 8 9 10 Casing pressure (MPa) Time (s) 20 (L/s) 25 (L/s) 30 (L/s) (b) Casing pressure 0 500 1000 1500 2000 2500 3000 0 1 2 3 4 5 6 7 8 9 10 Casing pressure (MPa) Time (s) 20 (L/s) 25 (L/s) 30 (L/s) (b) Casing pressure Casing pressure (MPa) Fig. 23. Change in wellbore pressure and casing pressure with the variation in time with displacements of 20, 30, and 40 L/s: (a) Wellbore pressure; (b) Casing pressure. L. Mao et al.: Oil & Gas Science and Technology – Rev. IFP Energies nouvelles 75, 71 (2020) L. Mao et al.: Oil & Gas Science and Technology – Rev. 6 Conclusion The casing pressure is lower at a higher displacement for higher friction resis- tance. Besides, H2S will gasify at an earlier time. Guo R., Chen Y., Waltrich P.J., Williams W.C. (2018) An experimental investigation on ﬂow pattern map and drift-ﬂux model for Co-Current upward liquid-gas two-phase ﬂow in narrow annuli, J. Nat. Gas. Sci. Eng. 51, 65–72. Guo X., Wang Q. (2016) A new prediction model of elemental sulfur solubility in sour gas mixtures, J. Nat. Gas Sci. Eng. 31, 98–107. Hasan A.R., Kabir C.S. (1988) A study of multiphase ﬂow behavior in vertical wells, Spede. 3, 2, 263–272. https://doi.org/10.2118/ 15138-PA. 4. When drilling for H2S-containing natural gas well, early detection of gas kick should be more frequent to avoid severe overﬂow. Besides, during well killing, higher displacement and density of drilling ﬂuid should be considered to avoid stratum fracturing and prevent leakage accidents under the premise of meeting drilling requirements. Hou Z., Yan T., Li Z., Feng J., Sun S., Yuan Y. (2019) Temperature prediction of two phase ﬂow in wellbore using modiﬁed heat transfer model: An experimental analysis, Appl Therm. Eng. 149, 54–61. Kelessidis V.C., Dukler A.E. (1989) Modeling ﬂow pattern transitions for upward gas-liquid ﬂow in vertical concentric and eccentric annuli, Int. J. Multiphase Flow. 15, 2, 173–191. Li H.Z., Mouline Y. (1997) Chaotic bubble coalescence in non- newtonian ﬂuids, Int. J. Multiphase Flow. 23, 713–723. Acknowledgments. This work was supported by the National Key Research and Development Program of China (2019YFC0312303) and Sichuan Science and Technology Project (2019YFS0045). Mao L., Zhang Z. (2018) Transient temperature prediction model of horizontal wells during drilling shale gas and geothermal energy, J. Petrol. Sci. Eng. 169, 610–622. Meng Y., Xu C., Na W., Gao L., Hongtao L., Mubai D. (2015) Numerical simulation and experiment of the annular pressure variation caused by gas kick/injection in wells, J Nat Gas Sci Eng. 22, 646–655. References Nickens H.V. (1987) A dynamic computer model of kick Well, SPE Drill. Eng. 2, 2, 158–173. https://doi.org/10.2118/ 14183-PA. Amaya-Gómez R., López J., Pineda H., Urbano-Caguasango D., Pinilla J., Ratkovich N., Muñoz F. (2019) Probabilistic approach of a ﬂow pattern map for horizontal, vertical, and inclined pipes, Oil Gas Sci. Technol. - Rev. IFP Energies nouvelles 74, 67. Osman E.S.A. (2004) Artiﬁcial neural network models for identifying ﬂow regimes and predicting liquid holdup in horizontal multiphase ﬂow, SPE Prod. Facil. 19, 1, 33–40. https://doi org/10 2118/68219 MS Ansari A.M., Sylvester N.D., Sarica C., Shoham O., Brill J.P. (1994) A comprehensive mechanistic model for upward two phase ﬂow in wellbores, SPE Prod. Facil. 5, 143–152. https:// doi.org/10.2523/20630-MS. https://doi.org/10.2118/68219-MS. Rader D.W., Bourgoyne A.T., Ward R.H. (1975, May 1) Factors affecting bubble-rise velocity of gas kicks, Society of Petroleum Engineers. https://doi.org/10.2118/4647-PA. L. Mao et al.: Oil & Gas Science and Technology – Rev. IFP Energies nouvelles 75, 71 (2020) L. Mao et al.: Oil & Gas Science and Technology – Rev. IFP Energies nouvelles 75, 71 (2020) 23 Raghavan R. (1989) Well-test analysis for multiphase ﬂow, SPE Form. Eval. 4, 4, 585–594. https://doi.org/10.2118/14098-PA. Xu Z.M., Song X.Z., Li G.S., Wu K., Pang Z., Zhu Z. (2018) Development of a transient non-isothermal two-phase ﬂow model for gas kick simulation in HTHP deep well drilling, Appl. Therm. Eng. 141, 1055–1069. Shi H., Li G., Huang Z., Shi S. (2014) Properties and testing of a hydraulic pulse jet and its application in offshore drilling, Petrol. Sci. 11, 3, 401–407. Xu Z.M., Song X.Z., Li G.S., Zhu Z., Zhu B. (2019) Gas kick simulation in oil-based drilling ﬂuids with the gas solubility effect during high-temperature and high-pressure well drilling, Appl. Therm. Eng. 149, 1080–1097. Sun B., Gong P.B., Wang Z.Y. (2013) Simulation of gas kick with high H2S content in deep well, J. Hydrodyn. 25, 2, 264–273. Sun B.J., Sun X.H., Wang Z.Y., Chen Y.H. (2017) Effects of phase transition on gas kick migration in deepwater horizontal drilling, J. Nat. Gas Sci. Eng. 46, 710–729. Xie J., Zhang X., Tang Y., Wang Y., Shao Q., Yu B. (2014) Transient simulation of the blowing-out process of the air pockets in vertical wellbore, Appl. Therm. Eng. 72, 97–103. Sun B.J., Guo Y.H., Sun W.T., Gao Y., Hao L., Wang Z., Zhang H. (2018) Multiphase ﬂow behavior for acid-gas mixture and drilling ﬂuid ﬂow in vertical wellbore, J. Petrol. Sci. Eng. References 165, 388–396. Yin H., Liu P., Li Q., Wang Q., Gao D. (2015) A new approach to risk control of gas kick in high-pressure sour gas wells, J. Nat. Gas Sci. Eng. 26, 142–148. Vesovic V., Assael M.J., Gallis Z.A. (1998) Prediction of the viscosity of supercritical ﬂuid mixtures, Int. J. Thermophys. 19, 5, 1297–1313. Yin B., Gang L., Li X. (2017) Multiphase transient ﬂow model in wellbore annuli during gas kick in deepwater drilling based on oil-based mud, Appl. Math. Model. 51, 159–198. Zhang X., Li Q., Zheng L., Li X., Xu L. (2020) Numerical simulation and feasibility assessment of acid gas injection in a carbonate formation of the Tarim Basin, China. Oil Gas, Sci. Technol. 75, 1–17. Wang N., Sun B., Wang Z., Wang J., Yang C. (2016) Numerical simulation of two phase ﬂow in wellbores by means of drift ﬂux model and pressure based method, J. Nat. Gas. Sci. Eng. 36, 811–823. Zhang J., Li X., Tang X., Luo W. (2019) Establishment and analysis of temperature ﬁeld of riserless mud recovery system, Oil Gas Sci. Technol. – Rev. IFP Energies nouvelles 74, 19. Wang Z.Y., Sun B.J. (2014) Deepwater gas kick simulation with consideration of the gas hydrate phase transition, J. Hydro- dyn. 26, 1, 94–103. Zhu G., Zhang S., Huang H., Liang Y., Meng S., Li Y. (2011) Gas genetic type and origin of hydrogen sulﬁde in the Zhongba gas ﬁeld of the western Sichuan Basin, China, Appl. Geochem. 26, 7, 1261–1273. Wang Z., Sun B. (2009) Annular multiphase ﬂow behavior during deep water drilling and the effect of hydrate phase transition, Petrol. Sci. 6, 1, 57–63. Wei N., Xu C.Y., Meng Y.F., Li G. (2018) Numerical simulation of gas-liquid two-phase ﬂow in wellbore based on drift ﬂux model, Appl. Math. Comput. 338, 175–191.
W4386403006.txt	https://zenodo.org/record/8313086/files/NJSTR_2023_08_015.pdf	en		Performance Analysis of Mixed-Mode Solar Dryer for Drying Vegetables	Zenodo (CERN European Organization for Nuclear Research)	2,023	cc-by	2,889	Journal of Science and Technology Research 5(3) 2023 pp. 136-143 ISSN-2682-5821 Performance Analysis of Mixed-Mode Solar Dryer for Drying Vegetables Agbonkhese, K1& Okojie,G2 1,2Department of Mechanical Engineering, National Institute of Construction Technology and Management, Uromi, Edo State, Nigeria. Article Info Abstract Keywords: Mixed-mode, vegetables, solar energy, dryer, performance,analysis This work presents the design, fabrication and performance analysis of mixed-mode solar dryer for vegetables. In the dryer, the heated air from a separate solar collector will pass through a grain bed and at the same time, the drying cabinet absorbs solar energy directly through the transparent walls and roof. The main components of the dryer are the solar collector (air heated), the drying, cabinets, drying trays. The results obtained during the analysis period shown that dryer and the solar collector was higher than the average ambient temperature during most hours of the day light. The rate of drying and efficiency of the system were found to be 0.75kg/h and 62.5% respectively. These, values obtained for the dryer exhibited sufficient ability to dry vegetables products to a safe moisture level and simultaneously ensured a superior quality of dry product. Received 11 May 2023 Revised 29 May 2023 Accepted 20 June 2023 Available online 3 Sept. 2023 https://doi.org/10.5281/zenodo.8313086 ISSN-2682-5821/© 2023 NIPES Pub. All rights reserved. 1.0. Introduction Drying is one of the oldest methods of food preservation [1-9]. Traditionally, drying is carried out openly in the field. Open air drying using direct sunlight has numerous draw backs which include subjection to adverse weather conditions like rain, dusts, wind, insects and sometimes rodents. This method slows down the drying rate, causing mold formation, for several thousand years, people have been preserving apricots, grapes, herbs, potatoes, corn, milk, meat and fish by drying [4,10]. Until canning was developed at the end of the 18th century, drying was virtually the only method of food preservation. Open sun drying has no quality control and also has a risk of contamination, creating a potential health hazard. The product’s quality is seriously degraded, sometimes to the extent that they are inedible [10, 3, 8]. Food deterioration and spoilage is caused by the action of yeasts, bacteria and enzymes. The drying process removes enough moisture from food to prevent the growth and reproduction of microorganisms like bacteria, yeasts and molds causing decay. Food drying can reduce loss of a harvest surplus, allow storage for food shortages, and in some cases facilitate export to high value markets. To guide against the aforementioned demerits and also to speed up the rate of drying the product, control the final moisture content and elimination of microorganisms effect, various types of solar dehydrators can used [11]. Upon the subsequent development in the technical world, efforts are focused towards improve drying and this led to solar drying as an upgrade from sun drying. Solar dryers are special drying enclosure that is used to dry food, produce and has numerous merits which include controlled drying rate, drying of higher temperature, lower relative humidity, and reduced moisture content of dried food [12]. Also it protects the produce against adverse weather 136 Agbonkhese, K & Okojie,G./ Journal of Science and Technology Research 5(3) 2023 pp. 136-143 conditions, pests and rodents [12]. In many parts of the world there is a growing awareness that renewable energy has an important role to play in extending technology to the farmer in development countries to increase their productivity [12]. However, to improve traditional drying, solar dryers which have the potential of substantially reducing the demerits of open-air drying, have received considerable attention over the past 20 years [2, 6, 5, 12, 1, 9]. Nowadays, several solar dryers designs exist in the market, and most of these designs require expensive materials and source of energy, which makes the prototype expensive and difficult to obtain for small scale producer [12, 13]. In this work we designed and fabricate a mixed-mode solar dryer which exhibit a sufficient ability to dry vegetable produce for domestic purpose 2.0. Materials and Methods For this study, the research methodology covered the research design, selection of materials, design of the amount of moisture to be removed, quantity of air required to effect drying, Blower design and capacity, Quantity of heat required to effect drying, thermal efficiency of the dryer and drying rate. 2.1. Materials The selection of materials for this study was guided by the design that was done, work they are expected to perform, the environmental condition in which they would function, their availability in the local market at cheaper costs. The body of the cabinet was made of plywood because it is a poor conductor of heat. Hence, heat loss from the cabinet would be greatly minimized. The inside of the cabinet was lined with aluminum sheet in order to reflect heat back to the cabinet also prevent decaying of the wood due to humid air, galvanized sheet metal also has outstanding advantages of toughness and ability to conduct and radiate heat, burnt bricks was used to build the wall of the heating unit because of its poor conductor of heat. 2.2 Method: i. Collector (Air Heater) A flat plate collector is used since it is easy to fabricate and also economical. The collector was constructed using plywood, galvanized iron sheet, clear glass which were locally available with low cost. The collector is made up of wood. GI sheet of 27mm gauge is used as absorber as it is a good conductor and economical. It is painted black to increase the absorption of heat. The space between the inner box and outer box is filled with foam material of about 40mm thickness and thermal conductivity of 0.043Wm-1K-1. The insulating material was selected to be plywood as it is a good insulator as well as environmentally friendly. It also does not have carcinogenic effects which other popular insulating materials like glass wool have. The collector glazing is a single layer of 4mm thick transparent glass sheet that has a surface area of 720mm by 980mm and of transmittance above 0.7 for the wave lengths in the range of 0.2 – 2.0 m and opaque to wave length greater than 4.5 m . One side of the solar collector has an air inlet vent of area 0.0888m2 which is covered by a galvanized wire mesh to prevent entrance of rodents while the other end opens to the plenum chambers. ii. Drying Cabinets. This is the place or space where drying of vegetable occur. The design of the drying cabinet was done to permit the user to use it as in –bin storage dryer. Since the dryer is a prototype and drying is to be done in racks, five racks were arranged at a distance of 100mm in drying cabinet the dimensions of the racks were taken as 370 x 370 x 100mm. the drying cabinet consisted of a square box dimension: 400 x 400 x 500mm. in order to minimize heat loss by conduction to the ambient 137 Agbonkhese, K & Okojie,G./ Journal of Science and Technology Research 5(3) 2023 pp. 136-143 environment; it was painted black both inside and outside to increase its heat absorption. The drying cabinet had in addition a digital thermometer/hygrometer inserted inside the drying cabinet in order to measure the temperature of the drying air and humidity in the drying cabinet. The mixed mode dryer isometric view is shown in Fig 1and Fig 2. 138 Agbonkhese, K & Okojie,G./ Journal of Science and Technology Research 5(3) 2023 pp. 136-143 iii. Drying Trays The drying trays are contained inside the drying cabinet and were constructed from galvanized wire mesh with a fairly open structure to allow drying air to pass through the vegetable 2.3. Determination of the Tilting Angle for the Solar Collector The tilting angle for the solar collector was determined from [14] (1)  = 100 + Lat  Where,  = Tilting angle Lat  = Latitude angle of the Solar collector location The latitude angle of Uromi where the dryer was designed and fabricated is 7.50N, Substituting into equation (1) gives  = 100 + 7.50 = 17.50 The flat plate solar collector is normally tilted and oriented in such a away that it receives maximum solar radiation in the desired season of use. The best stationary orientation is due South in the Northern hemisphere and due North in Southern hemisphere. The Solar collector for this work was therefore oriented facing the South and tilted at 17.110 to the horizontal. 2.4. Experimental Setup and Approach The test was conducted between the period of 5th of March, 2023 and 13th March, 2023. An initial test was first conducted for six bright days to evaluate the thermal profile of the solar collector from 6.00am to 6.00pm. The ambient air conditions were noted and recorded by the use of a digital thermometer, as well as their corresponding drying cabinet temperature. The vegetables used were bought from a Uromi Market to determine the maximum amount of vegetable that can be handled per batch for optimal drying base on the quality of heat produced as well as the drying time. A maximum of 0.4kg of vegetable were sufficiently dried. At the start of the solar drying test, the fabricated solar dryer was positioned at the front of Mechanical Engineering Department such that a shadow will not be cast in order to prevent shading effect. The dryer was positioned to align with the North-South axis and with solar collector tilted to face the South Pole. The moisture content of the vegetables was determined by weighing the fresh vegetables and drying them until no significant weight loss was observed. The weight was measured and recorded in one hour intervals with the use of an electronic weighing balance. Before the staring of solar drying test, the mass of the vegetables was recorded. Then the weighed tagged vegetables was put into the drying cabinet, the starting time was recorded. The temperature in the solar collector cabined and the ambient air temperature was measured with a digital thermometer and hygrometer respectively. At the end of the test which lasted for 4-6 hours, the moisture content was calculated as a percentage of the initial dried mass of the vegetable and the difference between the initial mass and the hourly measured mass was used to calculate the percentage weight loss for vegetable. 2.5. Performance and Data Analysis 2.5.1. Collector Efficiency Collector efficiency measures the thermal performance, i.e the useful energy gain of the collector. Not all of the solar radiation from the sun incident on the collector surface is converted to heat. Part of the radiation is reflected back to the sky and the other component is absorbed by the glazing. Once the collector absorbs heat and as a result temperature gets higher than the surrounding, there will also be a heat loss to the atmosphere by convection and radiation. 139 Agbonkhese, K & Okojie,G./ Journal of Science and Technology Research 5(3) 2023 pp. 136-143 Collector Efficiency,  c = Ma(To − Ti)Cp IcAc (2) 2.5.2. Moisture Content Moisture content was taken at the beginning and at the end of each drying day drying and calculated using the following equation Mi − Mf Moisture content Mc = (3) x100 Mi 2.5.3. Drying Rate Drying rate is the amount of evaporated moisture over time Mi − Md Drying Rate DR= (4) t 2.5.4. Drying Efficiency Drying efficiency is the ratio of the energy needed to evaporate moisture from the material to the heat supplied to the dryer. This term is used to measure the overall effectiveness of a drying system. But it may not be used for comparing one dryer with another due to different factors such as the particular material being dried, the air temperature and mode of air flow may differ for various dryers. System efficiency can be expressed mathematically as  d = ML It Ac (5) 2.5.5. Amount of Moisture to be removed The formula to calculate the total amount of moisture to be removed (Mw) is given by [14] Mm = Ww (Mi % - Mf%) I - Mf (6) 2.5.6. Amount of heat required to remove moisture content is given by QR = (Mm x hfg ) + (Mm x hf) (7) 3.0. Results and Discussion The day results of hourly variation of the temperatures in the Solar collector and drying cabinet compared to the ambient temperature. The dryer is hottest about 13.00 hour when the sun is usually overhead. The temperatures inside the dryer and solar collector were much higher than the ambient temperature during most hours of the day light. The temperature rise inside drying cabinet was up to 250c (75%) for about three hours immediately 13.00hour and this indicates prospect for performance than open-air sun drying. The variation of the relative humidity of the ambient air and drying chamber is shown in fig 4. Comparison of this figure with fig 3 shows that the drying processes were enhanced by heated air at very low humidity. Fig 5 shows the drying curve for vegetable in mixed mode solar dryer. It was observed that the drying rate increased due increase in temperature between 10.00hours and 14.00hours but decreased thereafter which shows the earlier and faster removal of moisture from dried vegetables. 140 Agbonkhese, K & Okojie,G./ Journal of Science and Technology Research 5(3) 2023 pp. 136-143 141 Agbonkhese, K & Okojie,G./ Journal of Science and Technology Research 5(3) 2023 pp. 136-143 The dryer was able to remove 85.4% of moisture, dry basis, from 7.5kg of vegetable in one day of 10.00 hour drying time, which is about 0.75kg/h drying rate. The collector efficiency of the mixed-mode solar dryer during the test period was found to be 63.2%. 4.0. Conclusion An easy and low cost mixed mode solar dryer was designed and fabricated using locally sourced materials. The hourly variation of the temperature inside the cabinet and air-heater are much higher than the ambient temperature during the most hours of the day light. The temperature rise inside the drying cabinet was up to 25% (75%) for about three hours immediately after 13.00hour. The drying rate, collector efficiency and percentage of moisture removed from drying vegetable were 0.75kgh-1, 63.2% and 85.4% respectively. The dryer exhibited sufficient ability to dry vegetables reasonably rapidly to a safe moisture level and simultaneously it ensures a superior quality of the dried vegetable. Nomenclature T - air Temperature elevation, 0C Ie - insolation on collector surface, W/m2 Ac - Collector Area, m2 ME- Mass of vegetable slides before drying Re - Collector efficiency Mc -Moisture content DR – Drying rate Md- Mass of sample after drying E - drying period  d – System efficiency M -Mass of moisture evaporated per second (Kg/s) L - Latent heat of evaporation of water (KJ/kg) It - Insolation on tilted collector surface (W/m2) Ac - Collector area (m2) Mm – Amount of moisture to be removed Mw -amount of moisture removed 142 Agbonkhese, K & Okojie,G./ Journal of Science and Technology Research 5(3) 2023 pp. 136-143 Ww- initial total weight  - Tilting angle Lat  - Latitude angle of the solar collector location Acknowledgement The authors wish to express their profound gratitude to the Management of National Institute of Construction Technology and Management, Uromi, Edo State and Tertiary Education Trust Fund (TETFund) for the financial support for this study References [1] Abdullahi Y Momoh M, Garba MM, Musa M. Design and Construction of an Adjustable and Collapsible Natural Convection Solar Frod Dryer. International Journal of Computational Engineering Research 2013. 3 (6) 1-8 [2] Bassey MW, Development and Use of Solar drying technologies. Nigerian Journal of Solar Energy 1989, 133164 [3] Diamante LM, Munro PA. Mathematical/Modelling of Thin Layer Solar Drying of Sweet Potatoes, Solar Energy 2004, (51), 176-271 [4] Ekechukwu OV, Norton B. Review of Solar Energy Dry Systems iii: Low Temperature Air-Heating Solar Collector for Drying Application, Energy Conversion and Management 1999, 40, 615-655 [5] Gartea AA. Design, Construction and Performance evaluation of solar maize dryer. Journal of Agricultural Extension and Rural Development 2009, 1(2), 42-49 [6] Irtwange SV, Adebayo S. Development and Performance of a Laboratory-Scale Passive Solar Grain Dryer. Journal of Agricultural Extension and Rural Development 2009, 1(2) 42-49 [7] Nejat TV. Alternative Energy Source viii, Hemisphere Publishing Company 1989, 1. [8] Raju RVS, Reddy RM, Reddy ES. Design and Fabrication of Efficient Solar Dryer, International Journal of Engineering Research and Applications 2013, 3(6) 1445 -1458 [9] Umogbai VI, Iorter HA. Design, Construction and Performance evaluation of passive solar dryer for maize cobs. African Journal of Food Science and Technology 2013; 4(5), 110-115 [10] Whitefield DE. Solar Dryer Systems and Internet, Important, Resources to improve food preparation. Proceedings of International Conference on Solar cooking, Kimberly, South Africa 2000, [11] Yaldiz O, Ertekin C, Uzun HB. Mathematical modeling of thin layer solar drying of sultana grapes. Energy 2001, 26, 457-465 [12] Ikechukwu CU, Ibukun BI, Ogbe EI, 2019. Design and Development of a mixed-mode Domestic Solar Dryer. International Journal of Engineering and Manufacturing, 3, 55-65 [13] Vishnuvardhan R, Mugi PD and Rawakrisma B. 2022. A review of Natural Energy Storage Materials used in Solar Dryers of Food drying Application. Journal of Energy Storage 49, 104198. [14] Gulti B, Kima S and Mustafa BG (2012). Design and Construction of Forced/Natural Convection Solar Vegetable Dryer with Heat Storage.ARPN Journal of Engineering and Applied Sciences, 7(10): 1213-1217 143
https://openalex.org/W4280643646	https://link.springer.com/content/pdf/10.1007/s43979-022-00015-7.pdf	English	null	Role of CCUS in carbon neutral power system	Carbon neutrality	2,022	cc-by	9,096	© 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/. 1 Background information Anthropogenic CO2 emissions, especially the utilization of fossil fuels for power generation, is the leading cause of global warming. Reduction of CO2 emissions is crucial in our efforts towards mitigation global warming. Among all the mitigation strategies, CO2 capture, utilization and storage (CCUS) is an efficient and reliable technology especially for high carbon fuels, while there is no alterna- tive yet existing for mitigating emissions from the contin- ued use of fossil fuel for electricity generation [1–5]. By the end of 2020, most countries in the world have successively proposed carbon-neutral targets. Among Among all the fields, power industry is the key area for carbon emission reduction due to the large amount of Abstract Achieving carbon neutrality by 2060 is an ambitious goal to promote the green transition of economy and society in China. Highly relying on coal and contributing nearly half of CO2 emission, power industry is the key area for reaching carbon-neutral goal. On basis of carbon balance, a criterial equation of carbon neutral for power system is provided. By means of the equation, the different effects of three technical approaches to achieve carbon neutrality, including energy efficiency improvement, shifting energy structure and CO2 capture, utilization and storage (CCUS) technology, had been evaluated. The results indicate that building a carbon-neutral power system requires comprehensive coor- dination between energy efficiency, renewable energy and CCUS technology. In particular, the unique role of CCUS in achieving carbon neutral target was investigated. For any power systems with fossil energy input, CCUS and negative emission technologies is indispensable to reach carbon neutrality. However, rather high energy consumption and costs is the critical gas deterring the large scale deployment of CCUS. Considering the specific conditions of China’s power industry, before the time window between 2030 and 2040 being closed, CCUS would either be ready for large scale deployment by reducing energy consumption and costs, or be phased out along with the most coal power plants. Conclusively, carbon neutral scenario will give CCUS the last chance to decarbonize the fossil fuel, which has great significance for China. Keywords: Carbon neutrality, CCUS, Power system, Carbon emission intensity Keywords: Carbon neutrality, CCUS, Power system, Carbon emission intensity them, the major economies in the world, such as the United States, the European Union, Japan, and Canada, have proposed to achieve carbon-neutral targets by 2050 [6–8]. As the world’s largest carbon emitter, China has a total carbon emissions of more than 11.3 billion tons, accounting for about 30% of global carbon emissions [9]. Therefore, the carbon emission reduction required for China is much higher than that of other economies, and its carbon-neutral tasks are obviously more complex and diversified. Recently (22 September 2020), the Chinese government announced in the 75th session of the UN General Assembly (New York, USA) that China aims to achieve carbon neutrality before 2060. The announce- ment of the carbon neutral target clearly indicated that China needs to carry out a self-revolution to achieve zero CO2 emissions before 2060 [10]. Role of CCUS in carbon neutral power system Yawen Zheng1,2, Lin Gao1, Rui Dong1,3 and Song He1,2 Carbon Neutrality Carbon Neutrality Zheng et al. Carbon Neutrality (2022) 1:19 https://doi.org/10.1007/s43979-022-00015-7 Correspondence: gaolin@iet.cn 1 Laboratory of Integrated Energy System and Renewable Energy, Institute of Engineering Thermophysics, Chinese Academy of Sciences, Beijing 100190, China Full list of author information is available at the end of the article Zheng et al. Carbon Neutrality (2022) 1:19 Page 2 of 12 Page 2 of 12 CO2 emissions [9]. The carbon neutral target challenges traditional energy systems, where energy saving, renew- able energy and carbon removal technologies (CCUS) are three pathways in achieving carbon neutral goal. China is home to over half the world’s existing coal capacity and projects under construction. Whereas the announcement of the carbon neutrality clearly indicated that fossil fuel should be either decarbonized or replaced by renewable energy before 2060. Considering the large amount of and the highly reliance on coal, the role of CCUS has to be shifted or significantly enhanced in the scenario of car- bon neutrality, otherwise the coal has to be phased out to achieve carbon neutral target.h energy such as solar energy and wind energy, which con- tain no carbon elements and does not directly emit CO2 during utilization. Moreover, carbon-neutral energy refers to biomass, whose carbon elements come from the atmosphere. During the short period of growth and utili- zation, the carbon element is absorbed by biomass from the atmosphere and transferred to the atmosphere. Thus, the net CO2 content in the atmosphere is not changed by carbon-neutral energy. Carbonaceous and carbon-neutral energy are the most important carbon sources in the power industry. And the CO2 cycle in the atmosphere will be affected by both of them during the utilization processes. According to characteristics of different energy and diverse energy utilization technologies, the carbon emissions during the utilization process can be obtained by the following equation [13]: Thus, in order to better understand and achieve the goal of carbon neutrality, this paper provided the crite- rial equation of carbon neutrality for power systems with discussion the pathways in achieving carbon neutral tar- get. More seriously, the important role of CCUS was dis- cussed, and correspondingly, the challenges and possible path for the deployment of CCUS were explored. (1) E = 44 12FCO (1) E is the amount of CO2 emissions; F is the total energy of the fuel consumed, depending on the amount of fuel consumed and the heating value of the fuel; C is the car- bon content per unit of fuel energy, depending on the type of fuel; O is oxidant factor, indicating the percentage of carbon in the fuel that is eventually converted to CO2, depending on whether the fuel is burned adequately. Considering the professional technology of boilers and with the advancement of combustion technology, the oxi- dation factor of most energy conversion processes have approached to 1 [14]. Thus, the oxidation factor will be assumed to be 1 in the following discussion [13].h 2.1 Formula derivationh The concept of carbon neutrality is essentially a balance between carbon sources and carbon sinks. The carbon source is the process of releasing CO2 into the atmos- phere, and the carbon sink is the process of absorbing CO2 from the atmosphere. Investigating the atmospheric system within a certain period of time, when the amount of CO2 emitted by all carbon sources into the system is equal to the amount of CO2 absorbed by all carbon sinks from the system, carbon neutrality can be achieved. There are various carbon sources and sinks in nature with complicated relationships. Among them, the most important carbon source related to human activities is energy production. In 2020, China’s power sector emit- ted about 4 billion tons of CO2 per year, which is close to 40% of the total emissions. Thus, the power sector is one of the key areas in emission reduction to achieve the carbon-neutral goal. To this end, this article focuses on the issues of carbon neutrality in the power industry. The net carbon emissions of power system can be expressed as Eq. (2) based on carbon balance: (2) NE = Source + Sink (2) Source refers to the amount of carbon emissions from carbon sources in the system, sink represents the carbon absorption from carbon sinks in the system.i Define K as the carbon recovery ratio, representing the proportion of CO2 emitted by carbon sources that is absorbed by carbon sinks. According to the elemental balance, the carbon-balanced equation for different types of energy can be established as follows. The subscripts C, CN, and CF represent carbonaceous energy, carbon-neu- tral, and carbon free energy, respectively: According to the amount and sources of carbon elements, energy can be divided into three types: Carbonaceous energy [11], Carbon free energy and Car- bon-neutral energy [12]. Carbonaceous energy mainly refers to fossil fuel, which contains carbon elements mined underground (the carbon elements in the ancient atmosphere are transformed into biomass through pho- tosynthesis, and then become fossil fuels through long geological years). And in the process of utilization, the carbon element emitted to the atmosphere in the form of CO2. 2.1 Formula derivationh In addition, carbon free energy refers to renewable As for carbonaceous energy: As for carbonaceous energy: (3) NEC = 44 12FCCC + −44 12FCCCKC (3) The carbon recovery ratio KC < 1 and NCEC > 0 due to the technical limitation; The carbon recovery ratio KC < 1 and NCEC > 0 due to the technical limitation; Zheng et al. Carbon Neutrality (2022) 1:19 Page 3 of 12 Page 3 of 12 Zheng et al. Carbon Neutrality (2022) 1:19 For carbon neutral energy: where Ic is the carbon emission intensity of the power system, which represents the amount of CO2 emitted per unit of power generated by the power system. ηi repre- sents the power supply efficiency, i Fi · ηi is the total power generation of the power system. (4) NECN = 44 12 FCN CCN + ( −44 12 FCN CCN KCN ) + ( −44 12 FCN CCN ) (4) Considering that the amount of CO2 emitted ( FCN · CCN · 44 12 ) and absorbed ( −FCN · CCN · 44 12 ) by car- bon-neutral energy are equal. Thus, Eq. (4) can be simpli- fied as: The carbon emission intensity Ic ≤ 0 must be met by power systems achieving carbon neutrality. Thus, the formula (6) can be further simplified, and the follow- ing carbon-neutral formula can be obtained: (5) NECN = −44 12FCN CCN KCN (5) (11) [R(1 −KC) −(1 −R)KCN] i=C,CN (Fi Ci)/ i=C,CN,CF (Fi ηi) ≤0 when the carbon recovery ratio KCN > 0, the carbon recovery technology is equipped in biomass power plants and the negative emission can be obtained (NECN < 0). (11) For power systems with multiple energy inputs, the above carbon neutrality equation must be satisfied to achieve the goal of carbon neutrality. CN However, for carbon free energy, the net carbon emis- sion NECF always equal to 0 due to its carbon free charac- teristics (CF = 0), as shown in Eq. (6): (6) NECF = 44 12FCF CCF CCF = 0 0 (6) 2.2 The different effects of emission reduction approaches As a major carbon emitter, the power industry will play a key role in achieving carbon neutrality. 2.1 Formula derivationh According to the criterial equation of carbon neutrality derived above, there are three major ways to establish a carbon- neutral power system: (1) Improve energy efficiency thus to save the consumption of carbonaceous energy; (2) Shift the energy structure and reduce the propor- tion of carbonaceous energy (3) Rebuild the balance of sources and sinks by adopting CO2 capture, utilization and storage technology to achieve low-carbon utiliza- tion of carbonaceous energy. Based on Eqs. (3), (5) and (6), the total net carbon emission of the power system composed of fossil energy (carbonaceous energy), carbon free energy (non-biomass renewable energy) and carbon neutral energy (biomass energy) is: (7) NE = i=C,CN,CF NEi = 44 12FC CC + i=C,CN −44 12Fi Ci Ki (7) NE is the net carbon emission of power system. If the dimensionless parameter R is defined as the proportion of carbon contained in carbonaceous energy among the total carbon input of the system: 2.2.1 Energy efficiency improvement ‑ potential A d h ( ) h 2.2.1 Energy efficiency improvement ‑ potential According to the Eq. (10), the consumption of carbo- naceous energy can be reduced by improving the uti- lization efficiency, thereby reducing carbon emissions from the power system. Taking coal power plants as an example, Fig. 1 shows the trend of average stand- ard coal consumption and its corresponding carbon emission intensity in the past 20 years in China. It can be seen from the figure that with the development of power generation technology focusing on thermal cycle upgrading, such as the improvement of initial steam parameters and the introduction of reheating and recuperation, the power generation efficiency of coal power plants has risen from ~ 20% in the middle of the twentieth century to the current ~ 47% power genera- tion efficiency. At the same time, the average standard coal consumption of electricity has been declining year by year, dropping to 304.7 g/kWh by 2020. The reduc- tion in coal consumption has also significantly reduced the carbon emission intensity of coal power plants. As shown in the green curve in Fig. 1, in the past 20 years, (8) R = FC CC FC CC + FCN CCN (8) Equation (7) can be simplified as: Equation (7) can be simplified as: ( NE = 44 12[R(1 −KC) −(1 −R)KCN] i=C,CN (Fi Ci) (9) Obviously, the power system can achieve carbon neutrality when NE ≤ 0. But in fact, the realization of carbon neutrality must meet the demand for energy products. In other words, carbon neutrality is a car- bon balance under the premise of a certain power out- put. Therefore, carbon emission intensity is required to be adopted to characterize the carbon emission of the power system: (10) Ic = NE∕ ∑ i=C,CN,CF (Fi 휂i ) = 44 12 [ R ( 1 −KC ) −(1 −R)KCN ] ∑ i=C,CN (Fi Ci )∕ ∑ i=C,CN,CF (Fi 휂i ) (10) CF Zheng et al. Carbon Neutrality (2022) 1:19 Page 4 of 12 Zheng et al. Carbon Neutrality Fig. 1 The trend of average coal consumption and carbon emission intensity of thermal power plants in China 1 kWh of electricity output by traditional coal power plants, and natural gas power plants adopting combined cycle only emit about 0.35 kg of CO2 to the environment for every 1 kWh of electricity output, which is about 1/3 of that of coal power plants. 2.2.1 Energy efficiency improvement ‑ potential A d h ( ) h Figure 2 shows the carbon emission intensity of different fossil fuels with different power efficiency, and renewable energy. Peat and cok- ing coal represent two typical coals with low and high carbon content per calorific value, respectively. When power generation efficiency (η) equals to 100%, the value of carbon emission intensity equals to the carbon emis- sions per unit of fuel energy (44/12C). It’s naturally that the net carbon emission intensity are both 0 gCO2/kWh for carbon-neutral and carbon free renewable energy. Although biomass will produce a certain amount of CO2 emissions during collection, processing (~ 20 gCO2/kWh emissions) and transportation (about 3 gCO2/kWh emis- sions for 50 km) processes [15], the emission amount is very small and can be ignored. Thus, carbon emission reduction can be achieved by increasing the proportion of renewable energy in the power system. the carbon emission intensity of coal power plants in China has dropped from 1078 gCO2/kWh to 838 gCO2/kWh (the carbon content of coal is assumed to be 75 wt.% [12] and the oxidant factor as 1.0). However, it can also be found that the declining trend of power generation energy consumption has gradually flat- tened. The reason for this trend comes from the follow- ing reality, super-critical or ultra-super-critical power plants had already accounted around 70% of installed capacity, while “650 °C” or “700 °C” technologies are still in research due to material limitation. Thus, the potential for continuing to reduce carbon emissions through energy saving is confined. 2.2.2 Shifting the energy structure ‑ reliability g gy y Coal, natural gas and oil are the most widely used car- bonaceous energy at present. Due to composition and energy characteristics of fossil fuels and technical utili- zation efficiency, different fossil fuels have various CO2 emission characteristics. Among fossil fuels, coal has the highest carbon content ranging from 0.024 kg /MJ to 0.026 kg /MJ [13], which means that 0.08 kg ~ 0.1 kg CO2 will be produced by direct combustion of coal with every 1 MJ energy released. CH4 is the main component of natural gas with the carbon content about 0.015 kg/MJ, which is only half of that of coal. Obviously, natural gas is lower carbon than coal from the perspective of CO2 emission characteristics. On the other hand, the average efficiency of coal-based power generation technology is only about 40%, while combined cycle burning natural gas can reach higher than 60%. Correspondingly, about 1 kg of CO2 will be emitted into the atmosphere for every From 2000 to 2020, the electricity installed capac- ity and generation ratio of renewable energy in China increased from 24.9% and 16.4% to 26.8% and 41.2%, respectively. This change in the energy structure has reduced the carbon emission intensity of the power system by about 30-35%. The temperature control plan of the 13th Five-Year Plan mentioned that CO2 emis- sions intensity need to be controlled within 550 g/ kWh, mainly by increasing the proportion of renewable energy [16]. At present, most strategic studies suggest that in a carbon-neutral scenario by 2060, renewables Zheng et al. Carbon Neutrality (2022) 1:19 Page 5 of 12 Zheng et al. Carbon Neutrality Fig. 2 Carbon emission intensity of different energy resources and technologies will be the main source of electricity accounted for more than 80% electricity generation ratio [17, 18]. carbonaceous energy and storing it in the ground. For carbon-neutral energy, CCUS breaks the original carbon cycle by storing the CO2 absorbed from the atmosphere into the ground, creating a negative emission effect. The more fossil energy power plants or biomass power plants equipped with CCUS technology, the higher the recovery ratio KC or KCN in the carbon neutral formula. For a power plant, KC or KCN represents the CO2 recov- ery rate of the specific power plant, or it represents the proportion of the power plants equipped with CCUS in 2.2.3 CO2 capture, utilization and storage technology – energy penalty CO2 Capture, Utilization and Storage (CCUS) is the only technology that can achieve low-carbon utiliza- tion of high-carbon fuel and realize negative emissions. As shown in Fig. 3, for carbonaceous energy, CCUS constructs a carbon cycle, capturing CO2 released from Fig. 3 The impact of CCUS technology on the carbon cycle Zheng et al. Carbon Neutrality (2022) 1:19 Page 6 of 12 for Ic. Assuming that the capture energy consumption per CO2 (ECCO2) is the same for carbon neutral and car- bonaceous energy, the carbon emission intensity Ic after modifying ηi can be written as follows: a regional energy system after comprehensive considera- tion the CCUS deployment conditions. At present, the average carbon emission intensity of 600 MW newly-built power generation plants are about 770 gCO2/kWh and 350 gCO2/kWh for ultra-supercriti- cal power plants and gas-fired power plants, respectively. The carbon emission intensity is close to or even bet- ter than that in the United States and the United King- dom [19]. Under the carbon neutral scenario, the role of CCUS has to be shifted or significantly enhanced. After installing CCUS, the average carbon emission intensity of coal power plants can be reduced to about 80 gCO2/kWh when the carbon capture rate reaches 90%. However, it’s inevitable that some coal fired power plants are un-retro- fitable, due to practical reasons like lacking of space for CO2 separation unit, or no matched storage sites. So, it will be difficult to install CCUS for all power plants in a specific region, which also means that KC for a regional power system could not reach 100%. For biomass power plants, the average carbon emission intensity is about 1200 gCO2/kWh without considering the carbon source of the biomass itself [20, 21]. If CCUS (90% carbon cap- ture rate) is further introduced, the average carbon emis- sions will drop to about − 1080 gCO2/kWh under 90% capture rate, creating so called negative emission effect. Correspondingly, an capture rate K for an energy system with both KC and KCN can be defined, indicating the ratio of the total amount of CO2 captured of the power system with CCUS to the total CO2 emissions of the power sys- tem without CCUS. Similarly, K can be derived from Eq. 2.2.3 CO2 capture, utilization and storage technology – energy penalty (10) as follows: (14) Ic = 44 12 (R −K) ∑ i=C,CN Fi ⋅Ci ∕ ∑ i=C,CN,CF Fi ⋅휂i −44 12 ∑ i=C,CN,CF Fi ⋅Ci ⋅K ⋅ECCO2 = 44 12 (R −K) ∑ i=C,CN Fi ⋅Ci ∕ ∑ i=C,CN,CF Fi ⋅ 휂i −44 12 Ci ⋅K ⋅ECCO2 = 44 12 (R −K) ∑ i=C,CN Fi ⋅Ci ∕ ∑ i=C,CN,CF Fi ⋅ 휂i −휂i’ (14) 44 12 i=C,CN,CF (Fi · Ci · K · ECCO2) represents the total t g ti f CCUS ’ i th d 44 12 i=C,CN,CF (Fi · Ci · K · ECCO2) represents the total capture energy consumption of CCUS, ηi’ is the reduc- tion in power generation efficiency of the original system caused by the adoption of CCUS. i=C,CN,CF capture energy consumption of CCUS, ηi’ is the reduc- tion in power generation efficiency of the original system caused by the adoption of CCUS. The relationship and trend of Ic and Ic ’ are shown in Fig. 4. Considering the deterioration of system perfor- mance due to CCUS, the carbon emission intensity Ic ’ of the system after the introduction of CCUS will be higher than that under the ideal situation Ic (the impact of CCUS on system performance is not considered). When R = K, the energy system achieves carbon neutrality. As discussed above, the contribution of each technical approach on carbon neutrality can be further evaluated by Eq. (10). From 2000 to 2020, the total CO2 emission intensity of China’s power sector decreased by 317.3 gCO2/kWh (from 878.6 gCO2/kWh to 561.3 gCO2/ kWh), whose calculation is based on generation data of China Electricity Council (CEC) and fuel emission factor data from Intergovernmental Panel on Climate Change (IPCC) [13, 22] with the oxidant factor being assumed to be 1.0. The share of power generation from non-fos- sil fuel had increased from 19% to 32.2%, contributing 157 gCO2/kWh of CO2 emission reduction. Meanwhile, average coal consumption of fossil fuel power plants had (12) K = R −[R(1 −KC) −(1 −R)KCN] (12) By defining K, the overall contribution and the impor- tance of CCUS to carbon neutral system can be reflected. Thus, Eq. (9) can be further written as: Fig. 2.2.3 CO2 capture, utilization and storage technology – energy penalty 4 The relationship between K and Ic (13) Ic = 44 12(R −K) i=C,CN (Fi Ci)/ i=C,CN,CF (Fi ηi) (13) R represents the ratio of system carbon emissions to the total carbon input, and K represents the ratio of cap- tured carbon to input carbon. When R = K, the energy system achieves carbon neutrality. It is worth noting that although increasing capture rate can directly reduce carbon emissions, the capture rate and ηi are not independent of each other. As CO2 capture requires additional energy consumption, the power gen- eration efficiency of power plants equipped with CCUS technology will be significantly reduced, which is cur- rently one of the key obstacles hindering the large-scale promotion of CCUS technology. Therefore, the influence of the capture rate K on ηi should be further considered Fig. 4 The relationship between K and Ic Page 7 of 12 Zheng et al. Carbon Neutrality (2022) 1:19 been decreased from 392 g/kWh to 305 g/kWh with the power generation proportion of that decreased to 67.8%, which leads to 160.3 g CO2/kWh of CO2 mitigation. It can be seen that the increment of renewable share and energy saving of fossil fuel power plants play the similar role (nearly half to half contribution for total CO2 emis- sion reduction) for decarbonization of power sector in China in the past two decades. Due to the immaturity of CCUS technology, the contribution of it is negligible in the absence of domestic CCUS demonstration projects. negative emission technology if more coal fired power plants are deployed. The left side of Fig. 5 indicates the optimum area for CCUS retrofit under a comparatively lower capture rates. However, carbon neutrality is diffi- cult to be achieved even with negative emission technol- ogy when R increases to a certain value (as shown in the shaded areas in Fig. 5), such as R > 0.9 under KC = 0.9 con- dition, due to the unreachable value of KCN (KCN ≥ 0.9). Similarly, when KC increases (less than 0.9), the demand of carbon neutral system for negative emission technol- ogy declines, and a higher proportion of carbonaceous energy can be input into energy system under carbon neutral scenario. In addition, as renewable energy is expected to account for about 80% electricity generation ratio in 2060 [17, 18], which will definitely become the main contribu- tor. 2.3 Indispensable role of CCUS in carbon neutral target 2.3 Indispensable role of CCUS in carbon neutral target Due to the high reliance of carbonaceous energy in China and the instability and unsafety of high portion of renew- able energy, it’s really difficult to imagine an energy sys- tem totally phasing out the utilization of carbonaceous energy over a long period of time, which means R will not be 0. Correspondingly, according to Eq. (13), it’s appar- ently that Ic could not reach 0 when R is not 0, only if K equals R, and naturally, the CCUS technology for both carbonaceous energy and carbon neutral energy is indis- pensable in achieving carbon neutral target. However, KC and KCN play the different role in power system reaching carbon neutral target. The Boundary Dam Carbon Capture, Sequestra- tion and Utilization (CCUS) facility is the world’s first industrial-scale post-combustion CCUS project [26]. It is located outside Estevan, Saskatchewan and com- menced operations in October 2014. Thus, this sec- tion goes to give a detailed analysis of Boundary Dam to help understand the energy consumption and cost of CCUS. In May 2011, SaskPower retrofitted the existing plant and installed CO2 capture facilities in this pro- ject. The total investment of the project is about $1.24 Based on Eq. (11), the relationship of capture rates between fossil fuel (KC) and biomass (KCN) can be derived as follows in the carbon neutral scenario: Fig. 5 The relationship between energy inputs and capture rates of different CO2 capture technologies in the carbon neutral scenario (15) KCN = R 1 −R − R 1 −RKC (15) KC and KCN represents the capture rates of carbona- ceous energy and carbon neutral energy for an area or an energy system. On the basis of Eq. (15), Fig. 5 depicts the relationship between energy inputs and capture rates of different CO2 capture technologies in the carbon neutral scenario. Considering that the typical or the optimized value of KC for fossil fuel power plants is around 0.7 ~ 0.9 [23–25], KC is adopted as a variable parameter with spe- cific value of 0.7, 0.8 and 0.9 in the analysis. As shown in Fig. 5, with the increase of R, the ratio of KCN / KC increases dramatically as its growth rate increases under a specific KC, which implies a much higher demand for Fig. 2.2.3 CO2 capture, utilization and storage technology – energy penalty At the same time, if the average coal consumption of thermal power plant declines to about 200-250 g/kWh in 2060 with around 20% electricity generation ratio, about 10-20% of the carbon emission intensity reduction of power system can be anticipated by improving energy efficiency. Finally, the left part of carbon emissions will be covered by CCUS technology on the scenario of carbon neutrality. 3 Challenge for CCUSh The rather high energy consumption and cost is widely recognized as the leading gap deterring the develop- ment and deployment of CCUS. In order to better understand the compositions of the energy consump- tion or cost of CCUS, the thermodynamic and eco- nomic analysis for the representative demonstration power plant will be conducted in this section.h 2.3 Indispensable role of CCUS in carbon neutral target Fortunately, the considerable ben- efits of EOR (60.8 $/t CO2) have helped the Boundary Dam survive successfully. Among CCUS full chain, the cost of CO2 capture is undoubtedly the leading cause of the dilemma of Boundary Dam, accounting for around 90% of the total cost of CCUS. Thus, cutting down the energy consumption and cost of the CO2 capture process (16) GP = COELC −COEC CEC −CELC (16) where GP refers to Green Premium, COE is the cost of electricity of the specific power generation technology, CE is the amount of CO2 emissions, LC and C represent the low-carbon technology and carbon emission technol- ogy (reference case), respectively. Table 1 Technical and economic analysis of the retrofitted Boundary Dam demonstration project [27] Table 1 Technical and economic analysis of the retrofitted Boundary Dam demonstration project [27] CO2 capture unit Fuel type Saskatchewan brown coal Fuel input, MW 397.1 CO2 capture rate, K ~ 91% (1 million t CO2/y) CO2 capture technology Cansolv amine-based carbon capture process CANSOLV Steam parameters 29 MPa/593 °C/621 °C Net output work without CCS, MW 150 Net output work with CCS, MW 110 System efficiency without CCS, % 37.8 System efficiency with CCS, % 23.9 ~ 27.7 CO2 utilization Weyburn EOR COE, $/kWh 0.252 CO2 capture cost, $/t CO2 100-130 CO2 transport cost, $/t CO2 10 CO2-EOR unit Net profit of CO2-EOR 24.3 $/bbl CO2 cost/bbl 2.5 bbl/t CO2 Net profit of CO2-EOR 60.8$/t CO2 Take the current cost of low-carbon technologies in China as an example. Considering that the coal power plant is the most common and traditional power genera- tion technology with great amount of carbon emissions in China, it is set as the reference case for the calculation of GP. Table 2 shows the costs of diverse power genera- tion technologies in China. The data of COE comes from the report of IEA in 2020 [31], and the median value is adopted. The amount of CO2 emission of coal power plants is around 800 g CO2/kWh in 2020 according to Fig. 1, and 90% CO2 capture rate is assumed for CCS. For solar and wind technologies, due to the large load fluc- tuations, energy storage technology is often needed to ensure its safe and stable operation. 2.3 Indispensable role of CCUS in carbon neutral target 5 The relationship between energy inputs and capture rates of different CO2 capture technologies in the carbon neutral scenario Zheng et al. Carbon Neutrality (2022) 1:19 Page 8 of 12 may be the only way to rescue CCUS from the current dilemma. billion, of which 50% is for capture facilities, 30% for renovation of existing power plants, and about 20% for emission control and other facilities. Although the CO2 capture cost of Boundary Dam is around 130 $/t-CO2, as high as 67% cost reduction will be anticipated in the second demonstration project (Shand Power station) according to the pre-feasibility study of Saskpower with the technology progress [26]. In addi- tion, as indicated by the roadmap issued by China in 2015 [29] and 2019 [28], the avoidance cost of the first- generation CO2 capture technology, mainly post-com- bustion capture, is anticipated to be reduced to around 40 $/t CO2 by 2030, mainly driven by the improvement of separation technologies and accumulation of engineering experience, etc. Table 1 summarizes the thermodynamic and eco- nomic performance of this project. As shown in Table 1, the system power generation efficiency can reach 37.8% after retrofitting without considering CCUS. However, a drop of 10.1-13.9% points of sys- tem efficiency will be caused after the installation of CCUS, decreasing to about 23.9-27.7%. It is obviously that the introduction of CCUS highly deteriorates the thermodynamic performance of the Boundary Dam. As the major energy penalty is caused by CO2 desorption process, nearly accounting for 80% among CO2 capture energy consumption, the large improvement of CO2 separation process may be expected to reduce the effi- ciency penalty of CCUS to 5-8% points when the des- orption energy consumption declines to 2-2.5 GJ/t CO2 [28]. Further, the challenges CCUS faces in achieving carbon neutrality also come from other low-carbon power gener- ation technologies. Green Premium is a crucial indicator for evaluation of the cost of CO2 avoidance of low-carbon technologies [30]. In power industry, the Green Premium for electricity amounts to the additional cost of getting all power in the grid from non-emitting sources like wind, solar, nuclear power, and fossil fuel plants equipped with carbon-capture technology over one that emits a greater amount of greenhouse gases. It can be calculated by the following equation: Similarly, as indicated by Table 1, CO2 capture cost reaches 100-130 $/t CO2, which is unaffordable for the long-time operation. 2.3 Indispensable role of CCUS in carbon neutral target Assuming that when the annual energy storage capacity accounts for ~ 20% of the total electricity production [32–34], the stability of a fully-renewable power supply can be achieved. And the cost of energy storage is assumed to be 100 $/MWh [35]. Based on the above information and assumptions, the GP can be obtained following the Eq. (16), as shown in Page 9 of 12 Zheng et al. Carbon Neutrality (2022) 1:19 Zheng et al. Carbon Neutrality Table 2. The results show that half of show obviously advantage in GP, due even with considering the cost of en nology. CCUS may not be competitiv renewable energy technologies at pres if it has lower GP than solar CSP and imperative to reduce the energy consu CCUS technology under the carbon soon as possible. 4 Time window for CCUS retrofitti With the fast development of renewab of coal is being knocked out, especia oped countries. Figure 6a shows the capacity of coal-fired power plants in Table 2 Comparison of the costs of diver technologies in China (2020) Technology COE [31] $/MWh COE (with energy storage) COE (wit Wind onshore 58 78 – Wind offshore 82 102 – Solar PV 51 71 – Solar CSP 119 139 – Hydro 51 – – Biomass 113 – – Nuclear 66 – – Coal 75 – 105 Fig. 6 The installed capacity and service life of Table 2 Comparison of the costs of diverse power generation technologies in China (2020) Technology COE [31] $/MWh COE (with energy storage) COE (with CCS) GP $/t CO2 (o/w energy storage) Wind onshore 58 78 – −21.25/3.75 Wind offshore 82 102 – 8.75/33.75 Solar PV 51 71 – −30/−5 Solar CSP 119 139 – 55/80 Hydro 51 – – −30 Biomass 113 – – 47.5 Nuclear 66 – – −11.25 Coal 75 – 105 41.7 Table 2 Comparison of the costs of diverse power generation technologies in China (2020) rate of that has decreased from > 10% between 2003 and 2008 to > 5% between 2008 and 2020 mainly due to mar- ket and the energy revolution policy mentioned by the Chinese Government in 2016 with the aims of achieving low-carbon and clean energy structure by adjusting the energy mix, reducing the energy consumption, innovat- ing the energy technologies and strengthening the role of market. 2.3 Indispensable role of CCUS in carbon neutral target More seriously, the carbon neutral target pro- posed recently by Chinese government will undoubtedly accelerate the reduction of living space for coal power plants in the power industry. However, the coal power plants in China are still very young, much shorter than its service life, as shown in Fig. 6b. Currently, more than 80% of 300-400 MW (the largest proportion) coal-fired power plants have been in service for less than 20 years (most of them were put into operation after 2000). For the 500-999 MW units, no more than 5% are in service for more than 20 years. And the maximum service life of 1000 MW units is 12 years at present. In other words, there are 530 million kW units in China that have been in service for less than 10 years, and the proportion of units with service life of more than 30 years is less than 1.1%. By contrast, in the world, more than 24% of the coal power units have been in service for more than 30 years. Among them, the longest service life has reached more than 60 years. In some countries, more than 90% of coal power units have been in service for 30 to 60 years. Therefore, most of the installed capacity of coal power plants in China is still in the “youth” stage. Table 2. The results show that half of renewable energies show obviously advantage in GP, due to the lower COE, even with considering the cost of energy storage tech- nology. CCUS may not be competitive with most of the renewable energy technologies at present in China, even if it has lower GP than solar CSP and biomass. Thus, it is imperative to reduce the energy consumption and cost of CCUS technology under the carbon neutral scenario as soon as possible. Table 2. The results show that half of renewable energies show obviously advantage in GP, due to the lower COE, even with considering the cost of energy storage tech- nology. CCUS may not be competitive with most of the renewable energy technologies at present in China, even if it has lower GP than solar CSP and biomass. Thus, it is imperative to reduce the energy consumption and cost of CCUS technology under the carbon neutral scenario as soon as possible. 5 Conclusionsh This paper introduced the critical equation of carbon neutral for power system based on the carbon balance principle. According to the carbon-neutral equation, three technical approaches to achieve carbon neutral- ity are discussed, including improving energy efficiency, shifting energy structure and adopting CCUS technology. And the role of CCUS in achieving carbon neutral tar- get is focused on in this paper. Some conclusions can be drawn as follows: (1) Building a carbon-neutral power system in the future requires a combination of energy efficiency, renewable energy and CCUS technology, neverthe- less, the role of each approach will be different. For power systems that use fossil energy, CCUS tech- nologies will be indispensable to achieve the goal of carbon neutrality. Considering the limitation of capture ratio, negative emission technology, like BECCS, will play the key role in neutralizing the carbon emission of power system with fossil fuel input. Finally, some possible countermeasures can be antici- pated to support the technology innovation of CCUS. The innovation of CCUS technology can be conducted from two aspects: the improvement of CO2 separa- tion technologies (mainly indicate the innovation of the first-generation of CCUS technology [28, 29]) and the exploration of new CO2 capture technologies (mainly indicate the second-generation of CCUS technology [28, 29]). The rather high energy consumption of CO2 separation technologies, like chemical absorption, caus- ing even more than 10% points efficiency penalty. With the innovation of separation technology, the power sys- tem efficiency penalty is expected to be reduced to 5% points. In addition to the improvement of separation processes, the innovation of energy conversion meth- ods can facilitate CO2 separation or directly realize fully enrichment of CO2 to avoid separation. From the system- atic perspective, this technical direction can be referred to as source control technologies, which possess the fol- lowing technical characteristics: (1) Different from the direct combustion in which 1/3 of fuel chemical exergy will be destructed, source control technology try to find new path to transform the chemical energy of fuel in cascade way. 4 Time window for CCUS retrofitting is closing With the fast development of renewable energy, the role of coal is being knocked out, especially in some devel- oped countries. Figure 6a shows the trend of installed capacity of coal-fired power plants in China. The growth As China’s coal power units are still very young, most of them will still have a large residual value before 2030 ~ 2040, even considering a rather short designed Fig. 6 The installed capacity and service life of coal power plants in China Fig 6 The installed capacity and service life of coal power plants in Ch Fig. 6 The installed capacity and service life of coal power plants in China Zheng et al. Carbon Neutrality (2022) 1:19 Zheng et al. Carbon Neutrality (2022) 1:19 Page 10 of 12 Page 10 of 12 life like 30 years. But facing the goal of carbon neutral- ity, most coal power plants will have to face two choices: (1) Forced to decommission, to be replaced by either renewable power or new coal power plants with CCUS; (2) Low-carbon retrofit. It’s obviously that retrofitting the original power station before decommissioning can extend its service life and save investment, compared with building new low-carbon power stations with lower energy penalty. In other words, almost all existing coal power units in China are bound to face the challenge of low-carbon retrofit, if power industry want to reduce the sunk cost. Under the goal of carbon neutrality, the criti- cal time window for coal power plants to make choices will be around 2030 ~ 2040, which is also the peak for low-carbon retrofitting of coal power plants. Before the “window” closed, it is imperative to make CCUS ready for large scale deployment. If CCUS can be ready with competitive low energy consumption and cost compared with other low-carbon technologies, the coal power plant is anticipated to remain a certain amount of installed capacity with CCUS even until 2060. Otherwise, most of the retrofittable coal power plants in the youth stage will be forced to decommission. chemical looping combustion), instead of being diluted by air. Concentration of CO2 along with the fuel conver- sion will facilitate or even cancel the separation process; (3) and finally, the efficiency of source control system can profit from both efficient utilization of chemical energy of fuel and reduced energy consumption in separation. Finally, zero or even negative penalty for CO2 capture can be expected. 5 Conclusionsh Just like polygeneration system [36–39] or chemical looping combustion [40, 41], the exergy destruction of fuel conversion will be reduced owing to the cascade utilization of chemical energy of fuel, which leads to improvement in efficiency of energy utilization; (2) and meanwhile, the carbon components will be par- tially (in polygeneration system) or fully concentrated (in (2) The coal power plants in China are still “young”, and almost all the existing power plants are bound to face the challenge of CO2 emission reduction. Under the goal of carbon neutrality, the critical time window for coal power plants to make choices will be around 2030 ~ 2040, which is also the peak for low-carbon retrofitting of coal power plants. i (3) Compared with renewable energy, the costs of CCUS have fallen far less than expected. As the fos- sil fuel with CCUS and renewable energy are both the promising technologies in low carbon path- ways, if the CCUS technology cannot make a break- through before the window closing, much more energy, environment and economic penalty will be paid for coping with the climate change. (4) Carbon neutral scenario may be the last chance for CCUS to get a foothold in the future emissions- reduction portfolio. Greatly reducing the energy consumption and costs of CCUS technology by accelerating the innovation of new-generation cap- ture technology may be the breakthrough point for CCUS. Page 11 of 12 Page 11 of 12 Zheng et al. Carbon Neutrality (2022) 1:19 Zheng et al. Carbon Neutrality (2022) 1:19 Zheng et al. Carbon Neutrality Availability of data and materials 15. Uddin SN, Barreto L (2007) Biomass-fired cogeneration systems with CO2 capture and storage. Renew Energy 32:1006–1019 Data sharing not applicable to this article as no datasets were generated or analyzed during the current study. 16. S. Council (2016) Thirteenth five-year plan. In: Work p 16. S. Council (2016) Thirteenth five-year plan. In: Work plan for controlling greenhouse gas emissions. State Council, China greenhouse gas emissions. State Council, China Funding This work is funded by National Science and Technology Major Project (J2019-I-0009-0009). 14. Houghton JT, Meira Filho LG, Lim B, Treanton K, Mamaty I, Bonduki Y et al (1996) Revised 1996 IPCC guidelines for national greenhouse gas inven- tories: workbook. IPCC, UK Subscripts publications 9. IEA (2020) Global energy review: CO2 emissions in 2020. IEA, Paris C: Carbonaceous energy/ Carbon emission technology; CN: Carbon-neutral energy; CF: Carbon free energy; LC: Carbon emission technology. p 9. IEA (2020) Global energy review: CO2 emissions in 2020. IEA, Paris 10. IEA (2021) An energy sector roadmap to carbon neutrality in China. IEA, Paris 11. Piatkowski N, Wieckert C, Weimer AW, Steinfeld A (2011) Solar-driven gasification of carbonaceous feedstock—a review. Energy Environ Sci 4:73–82 η: Power supply efficiency. η: Power supply efficiency. 8. European Commission (2017) Going climate-neutral by 2050. EU publications Abbreviations 5. Mcculloch S, Remme U, Bains P, Baylin-Stern A (2020) Energy technology perspectives 2020-special report on carbon capture utilisation and stor- age, IEA CCUS: CO2 capture, utilization and storage; CEC: China Electricity Council; IPCC: Intergovernmental Panel on Climate Change; E, kg: CO2 emissions; F, kJ: Total energy of the fuel consumed; C, kg C/kJ: Carbon content per unit of fuel energy; O: Oxidant factor; NE, kg: Net carbon emissions; Source, kg: Carbon emissions from carbon sources; Sink, kg: Carbon absorption from carbon sinks; K: Carbon recovery ratio; R: Proportion of carbon contained in carbonaceous energy among the total carbon input of the system; I, kgCO2/kWh: Carbon emission intensity; ECCO2, kWh/kgCO2: Capture energy consumption; GP, $/t CO2: Green Premium; COE, $/MWh: Cost of electricity. 6. Wang F, Harindintwali JD, Yuan Z, Wang M, Wang F, Li S, Yin Z, Huang L, Fu Y, Li L, Chang SX, Zhang L, Rinklebe J, Yuan Z, Zhu Q, Xiang L, Tsang DCW, Xu L, Jiang X, Liu J, Wei N, Kastner M, Zou Y, Ok YS, Shen J, Peng D, Zhang W, Barcelo D, Zhou Y, Bai Z, Li B, Zhang B, Wei K, Cao H, Tan Z, Zhao LB, He X, Zheng J, Bolan N, Liu X, Huang C, Dietmann S, Luo M, Sun N, Gong J, Gong Y, Brahushi F, Zhang T, Xiao C, Li X, Chen W, Jiao N, Lehmann J, Zhu YG, Jin H, Schaffer A, Tiedje JM, Chen JM (2021) Technologies and perspectives for achieving carbon neutrality. Innovation 2:100180 7. Schreyer F, Luderer G, Rodrigues R, Pietzcker RC, Baumstark L, Sugiyama M, Brecha RJ, Ueckerdt F (2020) Common but differentiated leadership: strategies and challenges for carbon neutrality by 2050 across industrial- ized economies. Environ Res Lett 15(11):114016 Consent for publication Not applicable. fi g gy 21. NASA (2011) Carbon emissions from burning biomass for energy. PFPI 22. (2021) China Electricity Council. Power statistics at a glance [DB/ OL]. https://www.cec.org.cn/ 22. (2021) China Electricity Council. Power statistics at a glance [ Competing interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. 23. Brandl P, Bui M, Hallett JP, Mac Dowell N (2021) Beyond 90% capture: pos- sible, but at what cost? Int Greenh Gas Con 105:103239 24. Liu X, Yang S, Hu Z, Qian Y (2015) Simulation and assessment of an inte- grated acid gas removal process with higher CO2 capture rate. Comput Chem Eng 83:48–57 Authors’ contributions YWZ: Methodology, Data curation, Formal analysis, Investigation and Writ- ing–original draft. LG: Conceptualization, Formal analysis, Funding acquisition, Methodology, Supervision, Writing-review & editing. RD: Resources. SH: Writ- ing-review & editing. The author(s) read and approved the final manuscript. 12. Saidur R, Abdelaziz EA, Demirbas A, Hossain MS, Mekhilef S (2011) A review on biomass as a fuel for boilers. Renew Sust Energ Rev 15:2262–2289 13. Eggleston HS, Buendia L, Miwa K, Ngara T, Tanabe K (2006) 2006 IPCC guidelines for national greenhouse gas inventories. IPCC, Japan Author details 1 1 Laboratory of Integrated Energy System and Renewable Energy, Institute of Engineering Thermophysics, Chinese Academy of Sciences, Beijing 100190, China. 2 University of Chinese Academy of Sciences, Beijing 100049, China. 3 North China Electric Power University, Beijing 100096, China. 25. Mletzko J, Ehlers S, Kather A (2016) Comparison of natural gas combined cycle power plants with post combustion and Oxyfuel Technology at Different CO2 capture rates. Energy Procedia 86:2–11 f 2 p gy 26. International CCS Knowledge Center (2018) The Shand CCS feasibility study public report 26. International CCS Knowledge Center (2018) The Shand Received: 19 November 2021 Accepted: 18 April 2022 Received: 19 November 2021 Accepted: 18 April 2022 27. Glennie JM, Sc MBACFA (2015) Full report analysis of the cash and carbon flows of boundary dam coal-fired power station. Saskatchewan com- munity wind, Canada 27. Glennie JM, Sc MBACFA (2015) Full report analysis of the cash and carbon flows of boundary dam coal-fired power station. Saskatchewan com- munity wind, Canada 28. Ministry of Science and Technology (2019) Roadmap for carbon capture, utilization and storage technology in China. China 28. Ministry of Science and Technology (2019) Roadmap for carbon capture, utilization and storage technology in China. China pp p p All included patients gave their oral and written informed consent. All included patients gave their oral and written informed consent. 19. (2020) China’s emissions trading scheme. IEA 20. Fajardy M, Mac Dowell N (2017) Can BECCS deliver sustainable and resource efficient negative emissions? Energy Environ Sci 10:1389–1426 Declarations 17. Mallapaty S (2020) How China could be carbon neutral by mid-century. Nature 586:482–483 18. Jiankun H (2020) Research on China’s long-term low-carbon develop- ment strategy and pathway. Tsinghua University, China Ethics approval and consent to participate All included patients gave their oral and written informed consent. ment strategy and pathway. Tsinghua University, China 1. Metz B, Ogunlade D, de Coninck H, Loos M, Meyer L (2005) IPCC special report on carbon dioxide capture and storage. IPCC 2. Organisation for Economic Co-operation and Development (2008) CO2 capture and storage: a key carbon abatement option. IEA, Paris 3. IEA (2012) Energy technology perspectives 2012—how to secure a clean energy future. IEA, Paris 4. Allen MR, Babiker M, Chen Y et al (2018) IPCC special report on global warming of 1.5 °C. IPCC References Jackson RG (1989) Polygeneration system for power and methanol based on coal gasification. Coal Conversion 3:60–64 i 37. Gao L, Jin H, Liu Z, Zheng D (2004) Exergy analysis of coal-based polygen- eration system for power and chemical production. Energy 29:2359–2371 37. Gao L, Jin H, Liu Z, Zheng D (2004) Exergy analysis of coal-based polygen- eration system for power and chemical production. Energy 29:2359–2371 y y 38. Wu H, Gao L, Jin H, Li S (2017) Low-energy-penalty principles of CO2 capture in polygeneration systems. Appl Energy 203:571–581 38. Wu H, Gao L, Jin H, Li S (2017) Low-energy-penalty principles of CO2 capture in polygeneration systems. Appl Energy 203:571–581 39. Cai R, Jin H, Gao L, Hong H (2010) Development of multifunctional energy systems (MESs). Energy 35:4375–4382 39. Cai R, Jin H, Gao L, Hong H (2010) Development of multifunctional energy systems (MESs). Energy 35:4375–4382 40. Kierzkowska AM, Müller CR (2012) Development of calcium-based, copper-functionalised CO2 sorbents to integrate chemical looping com- bustion into calcium looping. Energy Environ Sci 5 41. Blamey J, Anthony EJ, Wang J, Fennell PS (2010) The calcium looping cycle for large-scale CO2 capture. Prog Energy Combust Sci 36:260–279 41. Blamey J, Anthony EJ, Wang J, Fennell PS (2010) The calcium looping cycle for large-scale CO2 capture. Prog Energy Combust Sci 36:260–279 References 29. (2015) Roadmap for carbon capture and storage demonstration and deployment in the People’s Republic of China. Asian Development Bank 1. Metz B, Ogunlade D, de Coninck H, Loos M, Meyer L (2005) IPCC special report on carbon dioxide capture and storage. IPCC 2. Organisation for Economic Co-operation and Development (2008) CO2 capture and storage: a key carbon abatement option. IEA, Paris 3. IEA (2012) Energy technology perspectives 2012—how to secure a clean energy future. IEA, Paris 4. Allen MR, Babiker M, Chen Y et al (2018) IPCC special report on global warming of 1.5 °C. IPCC 30. Gates B (2021) How to avoid a climate disaster: the solutions we have and the breakthroughs we need. US 31. IEA (2020) Projected costs of generating electricity. IEA, Paris 32. Haas J, Cebulla F, Nowak W, Rahmann C, Palma-Behnke R (2018) A multi-service approach for planning the optimal mix of energy storage technologies in a fully-renewable power supply. Energy Convers Manag 178:355–368 4. Allen MR, Babiker M, Chen Y et al (2018) IPCC special report on global warming of 1.5 °C. IPCC Page 12 of 12 Zheng et al. Carbon Neutrality (2022) 1:19 Zheng et al. Carbon Neutrality Zheng et al. Carbon Neutrality (2022) 1:19 33. Gulagi A, Bogdanov D, Breyer C (2018) The role of storage technologies in energy transition pathways towards achieving a fully sustainable energy system for India. J Energy Storage 17:525–539 33. Gulagi A, Bogdanov D, Breyer C (2018) The role of storage technologies in energy transition pathways towards achieving a fully sustainable energy system for India. J Energy Storage 17:525–539 34. Bussar C, Moos M, Alvarez R, Wolf P, Thien T, Chen H, Cai Z, Leuthold M, Sauer DU, Moser A (2014) Optimal allocation and capacity of energy stor- age systems in a future european power system with 100% renewable energy generation. Energy Procedia 46:40–47 energy generation. Energy Procedia 46:40–47 gy g gy 35. Schmidt O, Hawkes A, Gambhir A, Staffell I (2017) The future cost of electrical energy storage based on experience rates. Nat Energy 2(8):1–8 36. Jackson RG (1989) Polygeneration system for power and methanol based on coal gasification. Coal Conversion 3:60–64 35. Schmidt O, Hawkes A, Gambhir A, Staffell I (2017) The future cost of electrical energy storage based on experience rates. Nat Energy 2(8):1–8 36. Jackson RG (1989) Polygeneration system for power and methanol based on coal gasification. Coal Conversion 3:60–64 36. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in pub- lished maps and institutional affiliations. Springer Nature remains neutral with regard to jurisdictional claims in pub- lished maps and institutional affiliations. Springer Nature remains neutral with regard to jurisdictional claims in pub- lished maps and institutional affiliations.
https://openalex.org/W4390860227	http://joiv.org/index.php/joiv/article/download/1740/797	English	null	Stock Price Time Series Data Forecasting Using the Light Gradient Boosting Machine (LightGBM) Model	JOIV : International Journal on Informatics Visualization	2,023	cc-by-sa	7,930	JOIV : Int. J. Inform. Visualization, 7(4) - December 2023 2270-2279 JOIV : Int. J. Inform. Visualization, 7(4) - December 2023 2270-2279 INTERNATIONAL JOURNAL ON INFORMATICS VISUALIZATION Keywords— Machine learning; prediction; forecasting; time series; LightGBM. Manuscript received 6 Apr. 2023; revised 12 Jun. 2023; accepted 26 Jul. 2023. Date of publication 31 Dec. 2023. International Journal on Informatics Visualization is licensed under a Creative Commons Attribution-Share Alike 4.0 International License. or forecast the future. Forecasting is a technique that can be used to predict future data trends based on existing and past data [7]–[10]. I. INTRODUCTION journal homepage : www.joiv.org/index.php/joiv Stock Price Time Series Data Forecasting Using the Light Gradient Boosting Machine (LightGBM) Model Anggit Dwi Hartanto a,, Yanuar Nur Kholik a, Yoga Pristyanto a a Department of System Information, Universitas Amikom Yogyakarta, Depok, Sleman, 55282, Indonesia Corresponding author: anggit@amikom.ac.id Stock Price Time Series Data Forecasting Using the Light Gradient Boosting Machine (LightGBM) Model Anggit Dwi Hartanto a,, Yanuar Nur Kholik a, Yoga Pristyanto a a Department of System Information, Universitas Amikom Yogyakarta, Depok, Sleman, 55282, Indonesia Corresponding author: anggit@amikom.ac.id Anggit Dwi Hartanto a,, Yanuar Nur Kholik a, Yoga Pristyanto a a Department of System Information, Universitas Amikom Yogyakarta, Depok, Sleman, 55282, Indonesia Corresponding author: anggit@amikom.ac.id Abstract— In the world of stock investment, one of the things that commonly happens is stock price fluctuations or the ups and downs of stock prices. As a result of these fluctuations, many novice investors are afraid to play stocks. However, on the other hand, stocks are a type of investment that can be relied upon during disasters or economic turmoil, such as in 2019, namely the Covid-19 pandemic. For stock price fluctuations to be estimated by investors, it is necessary to carry out a forecasting activity. This study builds stock price forecasting using the Light Gradient Boosting Machine (LightGBM) algorithm, which has high accuracy and efficiency. To forecast stock price time series, the model used is the LightGBM ensemble. At the same time, they were optimizing the determination of hyperparameters using Grid Search Cross Validation (GSCV). This study will also compare the LGBM algorithm with other algorithms to see which model is optimal in forecasting price stock data. In this study, the test used the RMSE metric by comparing the original data (testing data) with the predicted results. The experimental results show that the LightGBM model can compete with and outperform boosting-based forecasting models like XGBoost, AdaBoost, and CatBoost. In comparing forecasting models, the same dataset is used so that the results are accurate, and the comparisons are equivalent. In future research, paying attention to the data during pre-processing is necessary because it has many outliers. In addition, it is necessary to include exogenous variables and external variables, which are determined to involve many parties. Manuscript received 6 Apr. 2023; revised 12 Jun. 2023; accepted 26 Jul. 2023. Date of publication 31 Dec. 2023. International Journal on Informatics Visualization is licensed under a Creative Commons Attribution-Share Alike 4.0 International License. INTERNATIONAL JOURNAL ON INFORMATICS VISUALIZATION journal homepage : www.joiv.org/index.php/joiv I. INTRODUCTION GOSS is a sampling method for GDBT that can balance reducing data instances and maintaining accuracy in the decision tree that has been studied [29]. [ ] A similar study was also carried out by Pokhrel [30] by predicting dominant ocean waves using the Light Gradient Boosting Machine (LightGBM). The data used is The Coastal Data Information Program (CDIP) which has been filtered based on specific parameters to improve data quality. Features based on sea waves, such as wave height, period, kurtosis, and skewness, and then features related to the atmosphere, such as humidity, pressure, and temperature, are extracted from the data set. The model used is a decision tree-based model, Extra Trees (ET), which performs the bagging process, and LightGBM performs the booting process. Both processes use the ensemble method in making predictions or forecasting. Then the model is easy to use and has a high-efficiency level. The study's results by Pokhrel [30] showed that the two forecasting models proposed experienced a decrease in performance from zero to one day (one day range). However, the performance was relatively consistent in the 15-day and 30-day trials. The proposed forecasting model also outperforms comparison data originating from weather forecasting institutions or bodies concerned with marine research, such as the Fleet Numerical Meteorology and Oceanography Center (FNMOC), European Center for Medium-Range Weather Forecasts (ECMWF), National Centers for Environmental Prediction (NCEP) and others. So, the two proposed models show better performance when paired with data spanning 15 or 30 days compared to one day. Of the two proposed models, LightGBM has better results than ET and data from several marine research agencies or institutions. Similar research was also carried out by Li [33] applying LightGBM to predictions of monthly house rental prices. The result is that LightGBM has an RMSE value of 0.1429 and a goodness of fit (R2) value of 96.13 percent. In his research, Gan et al. [29] collected hourly water discharge data from the National Oceanic and Atmospheric Administration (NOAA) by searching data via the Internet. The research methodology conducted by Gan et al. [29] is the same as the studies above, using the Boosting model. The results show that the small learning rate parameters require iteration with high hyperparameter values to get the best RMSE; num_leaves and max_depth are selected by comparing each combination and displayed on a grid graph to calculate the NMSE. I. INTRODUCTION Another advantage of LightGBM is that it can perform sparse optimization, parallel training, multiple loss functions, regularization, bagging, and early stopping [25]. used the Cross Industry Standard Process for Data Mining (CRISP-DM) framework by comparing five different algorithms, namely Linear Regression (LR), Ridge Regression (RR), XGBoost Classifier, LightGBM, and LSTM, which Root Mean Squared Errors (RMSE) then evaluated. RMSE was chosen because it is more concerned with the most significant errors. The result is that LightGBM has the lowest error value compared to other alternative models. used the Cross Industry Standard Process for Data Mining (CRISP-DM) framework by comparing five different algorithms, namely Linear Regression (LR), Ridge Regression (RR), XGBoost Classifier, LightGBM, and LSTM, which Root Mean Squared Errors (RMSE) then evaluated. RMSE was chosen because it is more concerned with the most significant errors. The result is that LightGBM has the lowest error value compared to other alternative models. Forecasting in the economic field is also carried out by Pokhrel [30], predicting stock returns from Nvidia. This study uses regression as the basis for forecasting calculations. Therefore, the data is sorted based on predetermined features. The same thing was done by Chlebus et al. [32] when preparing data before carrying out the training process. However, Pokhrel [30] adds several data sets as exogenous variables or variables that can affect fluctuations in data originating from outside. The data used is from competitor companies or business partners of Nvidia and public or investor sentiment towards Nvidia. In this study, the models whose performance was tested included ARIMA, ARIMAX, K-Nearest Neighbors (KNN), Support Vector Regression (SVR), XGBoost, LightGBM, and LSTM. The results show that SVR and LightGBM have nearly the same performance, but SVR has a better test score than other alternative forecasting methods based on stationary variables. In addition, machine learning forecasting models have better predictive performance than econometric models. The difference between LightGBM and Extreme Gradient Boosting (XGBoost) lies in how it increases Gradient Boosting (GB) by using multiple cores from the Central Processing Unit (CPU) so that the learning process can be carried out in parallel, distributing calculations, cache optimization, and out-of-core processing. Whereas LightGBM has a leaf-wise growth structure, XGBoost has level-wise growth or levels [27], [28]. LightGBM was built using two novel techniques: GOSS and Exclusive Feature Bundling (EFB). I. INTRODUCTION At the end of 2019, the coronavirus disease 2019 (COVID- 19) hit the world. At the beginning of the emergence of COVID-19, Indonesia experienced losses in various sectors. The Ministry of Finance of the Republic of Indonesia stated that COVID-19 caused Indonesia to experience a contraction in economic growth 2020 of -2.07 percent. Investment, according to KBBI, is the investment to gain profit. Investment is needed to face the future if unexpected events such as COVID-19 occur [1]–[3]. By investing, the community can minimize the problems caused, such as the COVID-19 pandemic. However, many people need help to start investing or make beginner mistakes when making decisions due to various psychological factors, namely overconfidence or vice versa [4]–[6]. Stock predictions generally use data in the form of time series or time series data which is a collection of data arranged chronologically and measured over a certain period. Therefore, stock market predictions are critical when making decisions in the financial sector, making it a considerable challenge. The general and popular forecasting models used to make predictions on time series data include Autoregressive Integrated Moving Average (ARIMA) [11], [12], Seasonal Autoregressive Integrated Moving Average (SARIMA) [13], [14], Long Short Term Memory (LSTM) [15]–[17], and Gradient Boosting Decision Tree (GBDT) [18], [19], [20]. Forecasting techniques and models commonly used to predict data [21]–[25]. A study conducted by Ke et al. [25] developed the Light Gradient Boosting Machine (LightGBM) to overcome the shortcomings of GBDT when handling big data by speeding up the training process up to 20 times with nearly the same One form of investment is stocks which have always been a hot topic of conversation both in the financial and technical fields. If we look at the last few decades, the development of computers has reached a stage where computers can forecast 2270 accuracy. In the M5 Forecasting-Accuracy competition, LightGBM got first place and proved that LightGBM is superior to other techniques or models and can be used effectively to process many correlated and exogenous series and minimize the potential for forecasting errors [26]. In addition, one advantage of LightGBM is that LightGBM is compatible with several algorithms, such as Gradient Decision Trees (GDT), Gradient Boosting Decision Trees (GBDT), Gradient-based One-Side Sampling (GOSS), Dropouts meet Multiple Additive Regression Trees (DART), and Random Forests (RF). 1) Data Transformation: One of the elements that can affect the data is the unit root. The unit root is a stochastic (uncertain) attribute that can cause problems in drawing statistical conclusions, especially series data. To eliminate the unit root, data transformation can be performed, or it can also be called the de-trending process so that the data can be stationary before carrying out the data transformation. A unit root test, also known as the unit root process, is carried out to determine the trend of data based on mathematical calculations so that the data distribution can be known with certainty. The following subsections explain each step carried out in the research flow. The following subsections explain each step carried out in the research flow. The Augmented Dickey-Fuller (ADF) [34], [35] and Kwiatkowski–Phillips–Schmidt–Shin (KPSS) [36], [37] tests were carried out. The ADF test was conducted to find out the unit root of the data, while the KPSS test was to find out the stationarity of the data. The parameters used in the ADF test are regression equal to 'ct,' which means constant and trend in ADF and KPSS, and the auto lag parameter is the same as 'Akaike's and Schwarz's Information Criteria' (AIC) in ADF. This is done so that the results obtained are more valid and can also determine the cause of the problem if there is an oddity in the data. II. MATERIAL AND METHODS II. MATERIAL AND METHODS This research was conducted based on several stages illustrated in Figure 1 below. Then outlier handling aims to eliminate outlier data. Outlier data or also known as anomalies in the data can cause forecasting bias. Outlier data is illustrated as data that does not follow the "flow" because it has a difference in value that is too high with the value of the previous index or the value of most indexes. This causes bias because the model learns data patterns to make predictions so that if the model encounters an outlier, it can affect the pattern studied. Therefore, data indicated as an outlier must be removed or replaced, generally replaced with the mean or median. Fig. 1 Research Stages The following subsections explain each step carried out in A. Dataset Acquisition The data used in this study is stock data taken from Yahoo Finance using an application programming interface (API), namely finance. Time series data with the stock code AAPL was withdrawn from 01/01/1992 to 01/01/2022 with a total of 7558. Data pulled from finance was collected in July 2, 2022. The AAPL stock code dataset pulled from finance has six features: open, high, low, close, and volume. For research purposes, the data used is comparative column data because close is the market closing price. At the stock market's closing, the price will no longer change in recording stock transactions. This study divides the data into training data, data validation, and data testing. Data training starts from 02/01/1992 to 11/03/2011, data validation from 14/032011 to 30/12/2015, and data testing from 04/01/2016 to 31/12/2021. It can be seen in Table 1 that there are no signs of stationary data in the raw data column or raw data, namely data that has not gone through any treatment. The raw data has an ADF p- value of 1.0 and a KPSS p-value of 0.01; this means that the data has a trend or is not constant. The results of the ADF and KPSS tests can be interpreted by looking at Table 2, or it can be said that the raw data rejects the 0 KPSS hypothesis because the p-value is less than the alpha value (0.05) and the p-value of the raw data also accepts the 0 ADF hypothesis because the value is more than the alpha (0.05). From the two hypothesis tests, it can be concluded that the raw data is not stationary because it rejects the 0 KPSS hypothesis and accepts the 0 ADF hypothesis. The proof can be seen in Figure 2 above, and stock price data still has a trend in an exponential form that has risen suddenly in the last decade. Data that is not stationary has a mean and median that are not constant because they still have a trend, so a de-trending procedure must be carried out utilizing data transformation. I. INTRODUCTION Evaluation is done by comparing LightGBM with physics-based models, such as non-stationary tidal harmonic analysis (NS_TIDE). The result is that LightGBM can outperform NS_TIDE on RMSE, MAE, non-dimensional skill score (SS), and correlation coefficient (CC) scores. ( ) Most previous studies on forecasting used the Boosting model, but some of the Boosting models experienced overfitting. Therefore, this study proposes the LightGBM method with hyperparameter tuning for forecasting stock market prices. This is done because the LightGBM method has little risk of overfitting. Then the proposed method will be compared with several boosting methods in previous studies, including XGBoost, CatBoost, and AdaBoost. The contributions to this research include the following. First, the proposed method can handle the risk of overfitting in predicting stock market prices. Both results of the proposed method can be used as a technology model to predict stock market prices. The three proposed methods can be a reference for future research related to forecasting in the economic field, especially stock market prices. Forecasting based on machine learning has also been widely used in economics to support decision-making processes. Examples are forecasting sales predictions, stock predictions, sentiment analysis, and others. Research by Husein and Harahap [31] reveals that forecasting product sales time series with the M5 Forecasting dataset. This study 2271 2272 data have higher accuracy after differentiating data sets with the same reaction as the data sets used in the experiment. 2) Outlier Handling transformation; of course, this is remarkably interesting. This is because the differentiation transformation data no longer has a unit root, but the data is still not stationary but stationary differentiation. Data that has been trended out but still needs to be constant based on the KPSS test. Stationary data that should have a deterministic or definite mean becomes a stochastic or uncertain mean, and this is a particular case in this study. One way to prevent biased forecasting results is to reduce or eliminate outlier data using specific techniques or methods. The most manageable technique to detect outliers is by visualizing data using graphs. Boxplots are the easiest way to display the distribution of data which is summarized into five indicators ("minimum," first quartile (Q1), median, third quartile (Q3), and "maximum") as shown in Figure 2. Based on the illustration in Figure 3, outlier data are points outside the box plot's minimum and maximum limits [38], [39]. Because square root data transformation and differentiation transformation cannot turn the data into static data, this study tries to combine the two techniques into differentiation roots. This experiment produced results; this can be seen in Figure 2. The image at the bottom of the graph shows stationary data located not far from the mean or with a deterministic mean. Then the ADF p-value is relatively tiny, but the data meets the stationary requirements, namely rejecting the 0 hypotheses because the value is less than the alpha value (0.05). Then the p-value of KPSS can also fulfill the stationary requirements because the value accepts the 0 hypotheses, or it can be said that the p-value is more than the alpha value (0.05). Fig. 3 Boxplot Visualization Then in Figure 4, the top picture is a box plot visualization of the AAPL stock price data, which has many outliers, while good data is data with no outliers. This can also cause the data distribution to be abnormal, as in the distribution chart in Figure 4 below, which shows the frequency of abnormal data gathered in the median. Fig. 3 Boxplot Visualization Fig. 3 Boxplot Visualization TABLE I ADF AND KPSS TEST Metric ADF p-value KPSS p-value Data 1.0 0.010 Square Root 1.0 0.010 Differentiation 0.000000000000000000003721 0.010 Square Root- Differentiation 0.000000000000000000000601 7 0.051 TABLE II ADF AND KPSS TEST HYPOTHESIS KPSS ADF H0: if the p-value > 0.05 then the data is stationary. B. Data Pre-processing A critical element in forecasting besides data pre- processing refers to manipulating data so that the data becomes of higher quality or maintains the model's performance during training [32]. At this step, data manipulation does not mean altering data with bad intentions but processing data with specific techniques so that data becomes another form that data can display certain information or remove information that is not needed. In this study, data pre-processing was carried out using data transformation techniques and outlier handling. The purpose of data transformation is to make data stationery. In short, stationary data can be described as data that has a constant mean and variance. Chlebus et al. [32] show that selecting models and variables included in the essential category in data processing and feature engineering or feature engineering is vital to get stationary data. They conducted several stock price forecasting experiments, one comparing forecasting using stationary and non-stationary data. The result is that stationary Data transformation can be done in various ways, including using square roots. Then the data that has undergone the procedure is tested again using KPSS and ADF. However, in Table 1, the sqrt column of the transformation results still does not change, so differentiation is made to replace the square root [32]. After differentiating unexpected things, it turns out that the ADF and KPSS tests have different conclusions on the data resulting from the differentiation 2273 C. Forecasting Model Using LGBM In this study, the model used is the Light Gradient Boosting Machine (LGBM). The LightGBM model has many hyperparameters, some of which can be adjusted to improve model performance, while others can shorten training time due to the large volume of data. In short, a hyperparameter is a parameter whose value controls the learning process and determines the value of a model parameter which is eventually learned by the learning algorithm. Therefore, the hyperparameter is one of the most critical variables and can determine the conclusion in machine learning [29], [30], [32], [33]. Outlier handling can be done by removing the outliers based on the index of the captured data and then filling them back in with the mean or median value. Filling in the deleted values is known as imputed. Impute is done so that the information in the data is recovered. However, in Table 5, imputing the transformation data does not affect kurtosis. Then experiment with a scaler or smooth the values so that the outliers are not too extreme. Smoothing the value using a scaler reduces a data value based on a specific range of values, generally zero to one. This process is called data normalization. In short, data normalization occurs when there is a significant difference between the smallest and largest values so that the most significant value is considered an outlier. Usually, the methods that are often used are the robust scaler, min-max scaler, standard scaler, and power scaler methods. However, the scaler method could more successfully overcome abnormal data distribution due to outliers. Then the next experiment removes outlier data based on data captured by Tukey's Method and ThymeBoost. It can be seen in Figure 5 that the distribution graph is normal with kurtosis and skewness values of 0.304 and 0.159, The technique used in determining hyperparameters is grid search cross-validation (GSCV). GSCV is a hyperparameter optimization standard commonly used in machine learning [40], [41], [42], [40]. GSCV has two methods: grid search (GS) and cross-validation (CV). GSCV looks for the best combination of parameter values by mapping the hyperparameters illustrated as the x-axis, then CV is illustrated as the y-axis so that cross-validation and parameter values intersect and when drawn vertical and horizontal straight lines will form a grid (grid) like Figure 6. 2) Outlier Handling H0: if the p-value > 0.05 then the data is not stationary. H1: if the p-value < 0.05 then the data is not stationary. H1: if the p-value < 0.05 then the data is stationary. Fig. 2 Visualization of data conditions Fig. 3 Boxplot Visualization Fig. 3 Boxplot Visualization Then in Figure 4, the top picture is a box plot visualization of the AAPL stock price data, which has many outliers, while good data is data with no outliers. This can also cause the data distribution to be abnormal, as in the distribution chart in Figure 4 below, which shows the frequency of abnormal data gathered in the median. Fig. 4 Data Visualization Before Outlier Handling Fig. 2 Visualization of data conditions Fig. 4 Data Visualization Before Outlier Handling Fig. 4 Data Visualization Before Outlier Handling The abnormal distribution of the data can also be proven by the high kurtosis and skewness values, as shown in Table 3. It turns out that the data distribution becomes abnormal after transforming the data. The kurtosis and skewness values are respectively 7,459 and 2,696, whereas normal kurtosis and skewness values are values greater than equal to negative Fig. 2 Visualization of data conditions 2274 respectively, and also the boxplot shows that the data still has outliers, but these outliers are not too influential. three and less than equal to three. For this reason, it is necessary to detect outliers and then take further action on them so that the distribution becomes normal. TABLE V KURTOSIS AND SKEWNESS VALUES AFTER OUTLIERS HANDLING Kurtosis Skewness Imputer 28.324 -0.249 Scaler 26.667 -0.248 Delete (outlier) 0.304 0.159 Fig. 5 Data Visualization After Outlier Handling C F ti M d l U i LGBM TABLE V KURTOSIS AND SKEWNESS VALUES AFTER OUTLIERS HANDLING TABLE III KURTOSIS AND SKEWNESS VALUES BEFORE OUTLIERS HANDLING Kurtosis Skewness Before Transformation 7.459 2.696 After Transformation 26.668 -0.249 Tukey's method separates outlier probability (possibility) and probability (probability). The outlier probability value is between the inner (inside) and outer (outer) fence, while the outlier probability value is outside the outer fence. In contrast to the whisker, which is shaped like a horizontal line that connects the IQR box with the minimum and maximum limits, the inner fence and outer fence do not appear in the boxplot visualization. 2) Outlier Handling For this reason, the inner fence and outer fence are calculated by entering the IQR into equation (2), and to find the IQR values, it is done as in equation (1). 3 1 (1) 1 1.5 ∗, 3 1.5 ∗] 1 3 ∗, 3 3 ∗ (2) (1) Fig. 5 Data Visualization After Outlier Handling (2) (2) ThymeBoost is a forecasting model like LightGBM, but in this study, ThymeBoost is used because of its ability to detect outliers in data by using the detect outlier function [39]. Then in, Table 4 shows the number of outliers netted by Tukey's Method and ThymeBoost. It can be seen that the Tukey method produces two outputs, as mentioned in the previous presentation, namely, the number of outlier probability values is 424, and the number of outlier probability values is 1038 then the number of ThymeBoost outlier values is 740. Fig. 5 Data Visualization After Outlier Handling TABLE IV NUMBER OF OUTLIERS Outlier Tukey (prob) Tukey (poss) ThymeBoost Amount 424 1038 740 C. Forecasting Model Using LGBM C. Forecasting Model Using LGBM Many of these iterations can be calculated by the number of parameters raised to the power of length from the list of each hyperparameter. The number of candidates hyperparameters is the number of CV folds multiplied by the number of 2275 Rank Index Params Mean Test Score Mean Train Score 364 242 {'bagging_fr action': 0.5, 'bagging_fre q': 20, 'boosting_ty pe': 'rf', 'learning_rat e': 0.001, 'max_depth': 10, 'metric': 'rmse', 'n_estimator s': 1000} 0.52765508 48 0.52785740 62 729 692 {'bagging_fr action': 0.95, 'bagging_fr eq': 20, 'boosting_ty pe': 'gbdt', 'learning_rat e': 0.01, 'max_depth': 10, 'metric': 'rmse', 'n_estimator s': 1000} 0.99985720 55 0.99989368 74 Fig. 6 Grid Search Cross Validation (GSCV) Illustration D. Evaluation In this study, testing was carried out based on the scenario that had been made. The scenarios tested vary according to needs. For example, researchers want to test algorithms compatible with LightGBM for the best results. This study will compare these algorithms with balanced hyperparameter settings in the sense that the parameters of the hyperparameters can work or are compatible with the Rank Index Params Mean Test Score Mean Train Score 364 242 {'bagging_fr action': 0.5, 'bagging_fre q': 20, 'boosting_ty pe': 'rf', 'learning_rat e': 0.001, 'max_depth': 10, 'metric': 'rmse', 'n_estimator s': 1000} 0.52765508 48 0.52785740 62 729 692 {'bagging_fr action': 0.95, 'bagging_fr eq': 20, 'boosting_ty pe': 'gbdt', 'learning_rat e': 0.01, 'max_depth': 10, 'metric': 'rmse', 'n_estimator s': 1000} 0.99985720 55 0.99989368 74 iterations, so there were 729 iterations and 3645 hyperparameter candidates in this study. yp p y In Table 6, the 666th GSCV index has the best mean test score and mean training score because it is calculated based on the RMSE parameter in the model. The max_depth parameter is the depth of the model tree; max_depth can also overcome over-fitting if the training data set is small. Directly, overfitting means 'too fitting'; overfitting is a phenomenon where the training data used is 'too fitting' so that it can reduce accuracy if paired with other data. The max_depth parameter has an integer data type, meaning that the hyperparameter must be filled with integers. Then the training model uses the parameters bagging freq and bagging_fraction because the Random Forest (RF) algorithm requires both of these; however, GOSS cannot use 'bagging' at all. Fig. 6 Grid Search Cross Validation (GSCV) Illustration Fig. 6 Grid Search Cross Validation (GSCV) Illustration TABLE VI SAMPLE OF GRID SEARCH CROSS VALIDATION RESULTS TABLE VI SAMPLE OF GRID SEARCH CROSS VALIDATION RESULTS Rank Index Params Mean Test Score Mean Train Score 1 666 {'bagging_fr action': 0.95, 'bagging_fre q': 20, 'boosting_ty pe': 'dart', 'learning_rat e': 0.001, 'max_depth': 3, 'metric': 'rmse', 'n_estimator s': 100} 0.03069857 617 0.03079684 814 C. Forecasting Model Using LGBM The value of bagging freq must be an integer greater than zero (n>0), and the bagging fraction value must be a decimal number greater than zero and less than one (1.0<n>0.0). "Bagging" means taking random samples without changing values during training. The bagging_freq parameter is equal to 20, and the bagging_fraction parameter is equal to 0.95. This means the model is told to resample without changing values every 20 iterations and sample 95 percent of the training data. However, the GOSS algorithm cannot use bagging freq and bagging fraction. Then n_estimator is an alias of num_iteration, and n_estimator must be assigned an integer value. The n_estimator parameter equals 100, which means the model must boost for 100 iterations. Then the learning_rate parameter or learning rate is a parameter that determines the step size of each iteration during the training process. The learning_rate value must be a decimal number that is greater than zero (n> 0). The smaller the learning_rate value, the higher the accuracy of the learning. The boosting_type parameter is filled with the boosting or algorithm compatible with LightGBM, the boosting type compatible with LGBM, namely GBDT, RF, GOSS, and DART. Then the last one is the objective, which has many parameters, some of which are regression, Huber, Poisson, and others. Then the objective parameter in the model is 'regression_l1', which means regularization or L1 regularization (regularization lasso). Regularization is a form of regression, and the model is given regularization to control over-fitting phenomena. Fig. 6 Grid Search Cross Validation (GSCV) Illustration A. Experimental Given the many challenges in the pre-processing circuit, experiments need to be carried out to get the best results. This step also provides insight or description of data that has undergone previous processes. In the outlier handling step, it is indicated that the data has many outliers, so the data distribution becomes abnormal. This can be seen in Table 3. This is also supported by the large number of outliers netted in Table 4. Because there are many outliers in the data, the data is treated so that it can remove outliers. In Table 4, the outlier detection methods, namely Tukey's Method and ThymeBoost, each provide two outputs and one output. Because each output has different conclusions, a step is needed to determine which output is the most ideal as a basis for conducting model training. Fig. 8 Comparison of LGBM Model Initially, the stock price data that has been transformed is deleted based on the index data, which is indicated as an outlier. Then the data is entered into the training model with default hyperparameter settings. Default settings provide more "natural" results because the model is still not optimal so the data quality will be more visible. Table 7 and Figure 7 shows that the data subject to reduced probability outlier data from Tukey's Method has better RMSE, MAE, and MedAE scores than others. While the data subjected to reduction of Tukey's Method, outlier probability data has the worst score. From this, it can be concluded that the more outlier data that is captured, the data will have a better score. Fig. 8 Comparison of LGBM Model III. RESULTS AND DISCUSSION III. RESULTS AND DISCUSSION TABLE VIII COMPARISON OF LGBM Metric RMSE MAE MedAE Default 0.02483357 0.01986791 0.0167887 GBDT 0.02180185 0.01790202 0.0161667 GOSS 0.02179386 0.01789748 0.0161734 RF 0.02169517 0.01780414 0.0160431 DART 0.02145566 0.01765761 0.0160092 Fig. 8 Comparison of LGBM Model TABLE VIII COMPARISON OF LGBM Metric RMSE MAE MedAE Default 0.02483357 0.01986791 0.0167887 GBDT 0.02180185 0.01790202 0.0161667 GOSS 0.02179386 0.01789748 0.0161734 RF 0.02169517 0.01780414 0.0160431 DART 0.02145566 0.01765761 0.0160092 Fig. 8 Comparison of LGBM Model TABLE VIII COMPARISON OF LGBM D. Evaluation In this study, testing was carried out based on the scenario that had been made. The scenarios tested vary according to needs. For example, researchers want to test algorithms compatible with LightGBM for the best results. This study will compare these algorithms with balanced hyperparameter settings in the sense that the parameters of the hyperparameters can work or are compatible with the algorithm. Then the best model is selected, then the model is compared with other alternative models. In this study, testing uses metrics that are based on the course of research. The metric used is RMSE. RMSE compares the original data (testing data) with the predicted data. RMSE can be interpreted as "how concentrated the data is around the regression line" so the smaller the RMSE value, the better. However, RMSE is quite sensitive to outliers, so researchers use MAE and MedAE because they resist outliers. 2276 max_bin parameter means the data set has a unique maximum value per feature based on the max_bin value. MAE measures the average magnitude of error in the data, while MedAE measures the median value of the error. MAE and MedAE measure data error regardless of the direction of the data (positive/negative). MAE measures the average magnitude of error in the data, while MedAE measures the median value of the error. MAE and MedAE measure data error regardless of the direction of the data (positive/negative). Table 8 and Figure 8 show that DART has the best RMSE, MAE, and MedAE scores. So, in the next stage, the algorithm used in the LightGBM model is DART because it is proven to have the best score compared to other algorithms. B. Evaluation In the model evaluation phase, alternative forecasting models such as XGBoost, Adaptive Boosting (AdaBoost), and CatBoost are used. In testing, the hyperparameter model is adjusted to GSCV, and the training and validation data sets are installed with the max_bin parameter. Table 9 and Figure 9 show that LightGBM has the lowest RMSE, MAE, and MedAE scores of the other alternative forecasting models. In short, LightGBM has the slightest error difference compared to other models. Then CatBoost took second place with a score difference just a short distance from LightGBM. Then the third rank is XGBoost, and the fourth rank is AdaBoost. TABLE VII COMPARISON OF RESULTS BASED ON THE OUTLIER HANDLING METHOD Metric Tukey (poss) Tukey (prob) ThymeBoost RMSE 0.0248335694 0.0402051601 0.0300122047 MAE 0.0198679087 0.0318043206 0.0244017374 MedAE 0.0167887351 0.0265773916 0.0217605065 TABLE VII COMPARISON OF RESULTS BASED ON THE OUTLIER HANDLING METHOD TABLE VII Fig. 7 Outlier Detection Model Comparison TABLE IX LGBM VERSUS ANOTHER FORECASTING MODEL Metric LGBM CatBoost XGBoost AdaBoost RMSE 0.021456 0.021992 0.023581 0.024612 MAE 0.017658 0.017988 0.019075 0.019712 MedAE 0.016009 0.015980 0.016094 0.016481 Fig. 9 LGBM Model versus Another Forecasting Model TABLE IX LGBM VERSUS ANOTHER FORECASTING MODEL Metric LGBM CatBoost XGBoost AdaBoost RMSE 0.021456 0.021992 0.023581 0.024612 MAE 0.017658 0.017988 0.019075 0.019712 MedAE 0.016009 0.015980 0.016094 0.016481 Fig. 9 LGBM Model versus Another Forecasting Model TABLE IX LGBM VERSUS ANOTHER FORECASTING MODEL Metric LGBM CatBoost XGBoost AdaBoost RMSE 0.021456 0.021992 0.023581 0.024612 MAE 0.017658 0.017988 0.019075 0.019712 MedAE 0.016009 0.015980 0.016094 0.016481 Fig. 9 LGBM Model versus Another Forecasting Model Fig. 7 Outlier Detection Model Comparison After finding out a better data set, the next thing that needs to be proven is the effect of hyperparameters and the results of learning each LightGBM algorithm, such as DART, GOSS, RF, and GBDT. Then the hyperparameters are adjusted according to the GSCV results, and the max_bin parameters are added to develop training and validation data sets. The Fig. 9 LGBM Model versus Another Forecasting Model 2277 To find out a model's ability and the data's validity, the researchers tested it by forecasting using data with different timescales. In practice, the hyperparameters are adjusted to the results of the GSCV. The alternative model used is the same as the previous evaluation: CatBoost, XGBoost, and AdaBoost. Based on the tests conducted, LightGBM occupies the top position despite using a different timeframe. B. Evaluation In Table 10, LightGBM and other alternative models will give better results using more data. data accept the stationary hypothesis, while the KPSS states the opposite. For this reason, researchers use square root differentiation to transform the data into stationary. In addition, this study also encountered several obstacles in the pre-processing stage because the stock price data had many outliers after undergoing the data transformation stage. This condition indicates the distribution graph's extreme shape and the high kurtosis value. For this reason, efforts are made to smooth the values or normalize using scalers such as min-max scalers, robust scalers, standard scalers, and power transformers. However, these efforts did not produce results. This is because the outlier deviation is too apparent, so data indicated as an outlier must delete or handled using another method. In future research, paying attention to the data during pre-processing is necessary because it has many outliers. In addition, it is necessary to include exogenous variables and external variables, which are determined to involve many parties. TABLE X FORECASTING RESULTS WITH DIFFERENT TIME RANGES Range Model RMSE MAE MedAE 30 years (1992-2022) LightGBM 0.021456 0.017658 0.016009 CatBoost 0.021992 0.017988 0.015980 XGBoost 0.023581 0.019075 0.016094 AdaBoost 0.024612 0.019712 0.016481 20 years (2002-2022) LightGBM 0.031023 0.025378 0.021841 CatBoost 0.033460 0.027367 0.024269 XGBoost 0.036289 0.029398 0.025747 AdaBoost 0.037814 0.030789 0.026934 15 years (2007-2022) LightGBM 0.042737 0.035547 0.031844 CatBoost 0.046121 0.038053 0.033140 XGBoost 0.051026 0.041805 0.035897 AdaBoost 0.052323 0.042610 0.036483 10 years (2012-2022) LightGBM 0.052208 0.042680 0.038333 CatBoost 0.056176 0.046187 0.040557 XGBoost 0.061559 0.050350 0.042521 AdaBoost 0.063670 0.052129 0.043877 5 years (2017-2022) LightGBM 0.063721 0.051706 0.048172 CatBoost 0.067825 0.055354 0.050920 XGBoost 0.076624 0.061588 0.056169 AdaBoost 0.079039 0.062733 0.052944 1 year (2021-2022) LightGBM 0.095871 0.072390 0.046005 CatBoost 0.101959 0.077168 0.050629 XGBoost 0.118092 0.094113 0.073922 AdaBoost 0.128051 0.102729 0.085067 TABLE X FORECASTING RESULTS WITH DIFFERENT TIME RANGES ACKNOWLEDGMENT The authors thank the Department of Information Systems, Faculty of Computer Science, and Department for Research and Community Service, Universitas Amikom Yogyakarta, for supporting this research. REFERENCES [1] M. M. S. Saragih, T.Nurhaida, S. Sinaga, R. N. Ilham, and Faisal, “The impact of the Covid-19 pandemic on stock performance : Evidence from Indonesia,” Manag. Res. Behav. J., vol. 1, no. 1, pp. 1–6, 2021. [1] M. M. S. Saragih, T.Nurhaida, S. Sinaga, R. N. Ilham, and Faisal, “The impact of the Covid-19 pandemic on stock performance : Evidence from Indonesia,” Manag. Res. Behav. J., vol. 1, no. 1, pp. 1–6, 2021. [1] M. M. S. Saragih, T.Nurhaida, S. Sinaga, R. N. Ilham, and Faisal, “The impact of the Covid-19 pandemic on stock performance : Evidence from Indonesia,” Manag. Res. Behav. J., vol. 1, no. 1, pp. 1–6, 2021. [2] H. Rezaei, H. Faaljou, and G. Mansourfar, “Stock price prediction using deep learning and frequency decomposition,” Expert Systems with Applications, vol. 169, p. 114332, May 2021, doi: 10.1016/j.eswa.2020.114332. j [3] R. Chandra and Y. He, “Bayesian neural networks for stock price forecasting before and during COVID-19 pandemic,” PLOS ONE, vol. 16, no. 7, p. e0253217, Jul. 2021, doi: 10.1371/journal.pone.0253217. [4] W. Lu, J. Li, J. Wang, and L. Qin, “A CNN-BiLSTM-AM method for stock price prediction,” Neural Computing and Applications, vol. 33, no. 10, pp. 4741–4753, Nov. 2020, doi: 10.1007/s00521-020-05532-z. [5] H. T. H. Ton and T. K. Dao, “The Effects of Psychology on Individual Investors’ Behaviors: Evidence from the Vietnam Stock Exchange,” Journal of Management and Sustainability, vol. 4, no. 3, Aug. 2014, doi: 10.5539/jms.v4n3p125. j p [6] E. Mulyani, H. Fitra, and F. F. Honesty, “Investment Decisions: The Effect of Risk Perceptions and Risk Propensity for Beginner Investors in West Sumatra,” Seventh Padang Int. …, vol. 192, no. Piceeba, pp. 49–55, 2021. [7] W. Budiharto, “Data science approach to stock prices forecasting in Indonesia during Covid-19 using Long Short-Term Memory (LSTM),” Journal of Big Data, vol. 8, no. 1, Mar. 2021, doi:10.1186/s40537-021-00430-0. IV. CONCLUSION This study uses stock price time series data derived from the Yahoo Finance and LightGBM ensemble model as a forecasting model. LightGBM's ability to use the DART algorithm is proven superior in obtaining RMSE, MAE, and MedAE scores compared to other alternative forecasting models. In comparing forecasting models, the same dataset is used to make the results accurate and the comparisons equivalent. This is supported by the increased capability of the post-GSCV hyperparameter tuning model compared to the default hyperparameter setting. Then the researcher found an exciting discovery when conducting unit root tests on the data. The conclusions given by the KPSS and ADF tests are contradictory, even though the data has gone through a transformation using square roots. The ADF states that the [8] M. Shahvaroughi Farahani and S. H. Razavi Hajiagha, “Forecasting stock price using integrated artificial neural network and metaheuristic algorithms compared to time series models,” Soft Computing, vol. 25, no. 13, pp. 8483–8513, Apr. 2021, doi: 10.1007/s00500-021-05775-5. [9] D. Cheng, F. Yang, S. Xiang, and J. Liu, “Financial time series forecasting with multi-modality graph neural network,” Pattern Recognition, vol. 121, p. 108218, Jan. 2022, doi:10.1016/j.patcog.2021.108218. [10] E. S. Abdulla, A. Hamdan, and H. Akeel, “The Impact of Artificial Intelligence on Financial Institutes Services During Crisis: A Review of the Literature,” in Digitalisation: Opportunities and Challenges for Business, 2023, pp. 642–655. [11] H. Yu, L. J. Ming, R. Sumei, and Z. Shuping, “A Hybrid Model for Financial Time Series Forecasting—Integration of EWT, ARIMA With The Improved ABC Optimized ELM,” IEEE Access, vol. 8, pp. 84501–84518, 2020, doi: 10.1109/access.2020.2987547. [12] Z. Li, J. Han, and Y. Song, “On the forecasting of high‐frequency financial time series based on ARIMA model improved by deep 2278 learning,” Journal of Forecasting, vol. 39, no. 7, pp. 1081–1097, Mar. 2020, doi: 10.1002/for.2677. [28] A. A. Taha and S. J. Malebary, “An Intelligent Approach to Credit Card Fraud Detection Using an Optimized Light Gradient Boosting Machine,” IEEE Access, vol. 8, pp. 25579–25587, 2020, doi:10.1109/access.2020.2971354. [13] U. M. Sirisha, M. C. Belavagi, and G. Attigeri, “Profit Prediction Using ARIMA, SARIMA and LSTM Models in Time Series Forecasting: A Comparison,” IEEE Access, vol. 10, pp. 124715– 124727, 2022, doi: 10.1109/access.2022.3224938. [29] M. Gan, S. Pan, Y. Chen, C. Cheng, H. Pan, and X. IV. CONCLUSION Zhu, “Application of the Machine Learning LightGBM Model to the Prediction of the Water Levels of the Lower Columbia River,” Journal of Marine Science and Engineering, vol. 9, no. 5, p. 496, May 2021, doi:10.3390/jmse9050496. [14] T. C. Nokeri, “Forecasting Using ARIMA, SARIMA, and the Additive Model,” in Implementing Machine Learning for Finance: A Systematic Approach to Predictive Risk and Performance Analysis for Investment Portfolios, Berkeley, CA: Apress, 2021, pp. 21–50. j [30] P. Pokhrel, “A LightGBM based Forecasting of Dominant Wave Periods in Oceanic Waters,” in Proceedings of ACM Conference on Information and Knowledge Management. (CIKM’21), 2018, vol. 9. [15] Z. Fang, X. Ma, H. Pan, G. Yang, and G. R. Arce, “Movement forecasting of financial time series based on adaptive LSTM-BN network,” Expert Systems with Applications, vol. 213, p. 119207, Mar. 2023, doi: 10.1016/j.eswa.2022.119207. [31] A. M. Husein and M. Harahap, “Pendekatan Data Science untuk Menemukan Churn Pelanggan pada Sector Perbankan dengan Machine Learning,” Data Sciences Indonesia (DSI), vol. 1, no. 1, pp. 8–13, Nov. 2021, doi: 10.47709/dsi.v1i1.1169. [16] A. H. Bukhari, M. A. Z. Raja, M. Sulaiman, S. Islam, M. Shoaib, and P. Kumam, “Fractional Neuro-Sequential ARFIMA-LSTM for Financial Market Forecasting,” IEEE Access, vol. 8, pp. 71326– 71338, 2020, doi: 10.1109/access.2020.2985763. [32] M. Chlebus, M. Dyczko, and M. Woźniak, “Nvidia’s Stock Returns Prediction Using Machine Learning Techniques for Time Series Forecasting Problem,” Central European Economic Journal, vol. 8, no. 55, pp. 44–62, Jan. 2021, doi: 10.2478/ceej-2021-0004. [17] J. Cao, Z. Li, and J. Li, “Financial time series forecasting model based on CEEMDAN and LSTM,” Physica A: Statistical Mechanics and its Applications, vol. 519, pp. 127–139, Apr. 2019, doi:10.1016/j.physa.2018.11.061. [33] J. Li, “Monthly Housing Rent Forecast based on LightGBM (Light Gradient Boosting) Model,” NCCP Int. J. Intell. Inf. Manag. Sci., vol. 7, no. 6, pp. 2307–0692, 2018. [18] Q. Gu, Y. Chang, N. Xiong, and L. Chen, “Forecasting Nickel futures price based on the empirical wavelet transform and gradient boosting decision trees,” Applied Soft Computing, vol. 109, p. 107472, Sep. 2021, doi: 10.1016/j.asoc.2021.107472. [34] J. Wang, T. Ji, and M. Li, “A Combined Short-Term Forecast Model of Wind Power Based on Empirical Mode Decomposition and Augmented Dickey-Fuller Test,” Journal of Physics: Conference Series, vol. 2022, no. 1, p. 012017, Sep. 2021, doi: 10.1088/1742- 6596/2022/1/012017. [19] T. Le, B. Vo, H. Fujita, N.-T. Nguyen, and S. W. IV. CONCLUSION Baik, “A fast and accurate approach for bankruptcy forecasting using squared logistics loss with GPU-based extreme gradient boosting,” Information Sciences, vol. 494, pp. 294–310, Aug. 2019, doi:10.1016/j.ins.2019.04.060. [35] M. Ahmed et al., “Bubble Identification in the Emerging Economy Fuel Price Series: Evidence from Generalized Sup Augmented Dickey–Fuller Test,” Processes, vol. 10, no. 1, p. 65, Dec. 2021, doi:10.3390/pr10010065. [20] F. Zhou, Q. Zhang, D. Sornette, and L. Jiang, “Cascading logistic regression onto gradient boosted decision trees for forecasting and trading stock indices,” Applied Soft Computing, vol. 84, p. 105747, Nov. 2019, doi: 10.1016/j.asoc.2019.105747 [36] A. Kagalwala, “kpsstest: A command that implements the Kwiatkowski, Phillips, Schmidt, and Shin test with sample-specific critical values and reports p-values,” The Stata Journal: Promoting communications on statistics and Stata, vol. 22, no. 2, pp. 269–292, Jun. 2022, doi: 10.1177/1536867x221106371. [21] F. I. Durrah, Y. Yulia, T. P. Parhusip, and A. Rusyana, “Peramalan Jumlah Penumpang Pesawat Di Bandara Sultan Iskandar Muda Dengan Metode SARIMA (Seasonal Autoregressive Integrated Moving Average),” Journal of Data Analysis, vol. 1, no. 1, pp. 1–11, Sep. 2018, doi: 10.24815/jda.v1i1.11847. [37] Marsani, Ani Shabri, Basri Badyalina, Nur Amalina Mat Jan, and Mohd Shareduwan Mohd Kasihmuddin, “Efficient Market Hypothesis for Malaysian Extreme Stock Return: Peaks over a Threshold Method,” Mat. Mjim, vol. 38, no. 2, pp. 141–155, 2022. [22] N. S. Arunraj, D. Ahrens, and M. Fernandes, “Application of SARIMAX Model to Forecast Daily Sales in Food Retail Industry,” International Journal of Operations Research and Information Systems, vol. 7, no. 2, pp. 1–21, Apr. 2016, doi:10.4018/ijoris.2016040101. [38] H. Alimohammadi and S. Nancy Chen, “Performance evaluation of outlier detection techniques in production timeseries: A systematic review and meta-analysis,” Expert Systems with Applications, vol. 191, p. 116371, Apr. 2022, doi: 10.1016/j.eswa.2021.116371. j [23] A. Khumaidi, R. Raafi’udin, and I. P. Solihin, “Pengujian Algoritma Long Short-Term Memory untuk Prediksi Kualitas Udara dan Suhu Kota Bandung,” J. Telemat., vol. 15, no. 1, pp. 13–18, 2020. [39] A. Blázquez-García, A. Conde, U. Mori, and J. A. Lozano, “A Review on Outlier/Anomaly Detection in Time Series Data,” ACM Computing Surveys, vol. 54, no. 3, pp. 1–33, Apr. 2021, doi: 10.1145/3444690. [40] D. M. Belete and M. D. Huchaiah, “Grid search in hyperparameter optimization of machine learning models for prediction of HIV/AIDS test results,” International Journal of Computers and Applications, vol. 44, no. 9, pp. 875–886, Sep. 2021, doi:10.1080/1206212x.2021.1974663. [24] J. H. IV. CONCLUSION Friedman, “Greedy function approximation: A gradient boosting machine.,” The Annals of Statistics, vol. 29, no. 5, Oct. 2001, doi:10.1214/aos/1013203451. [25] G. Ke et al., “LightGBM: A highly efficient gradient boosting decision tree,” in 31st Conference on Neural Information Processing Systems (NIPS 2017), 2017, vol. 2017-Decem, no. Nips, pp. 3147–3155. [41] M. Adnan, A. A. S. Alarood, M. I. Uddin, and I. ur Rehman, “Utilizing grid search cross-validation with adaptive boosting for augmenting performance of machine learning models,” PeerJ Computer Science, vol. 8, p. e803, Feb. 2022, doi: 10.7717/peerj-cs.803. [26] S. Makridakis, E. Spiliotis, and V. Assimakopoulos, “M5 accuracy competition: Results, findings, and conclusions,” International Journal of Forecasting, vol. 38, no. 4, pp. 1346–1364, Oct. 2022, doi: 10.1016/j.ijforecast.2021.11.013. [42] S. Chen, H. Zhu, W. Liang, L. Yuan, and X. Wei, “A Stock Index Prediction Method and Trading Strategy Based on the Combination of Lasso-Grid Search-Random Forest,” in Intelligent Computing and Block Chain, 2021, pp. 431–448. [27] T. Chen et al., “Prediction of Extubation Failure for Intensive Care Unit Patients Using Light Gradient Boosting Machine,” IEEE Access, vol. 7, pp. 150960–150968, 2019, doi: 10.1109/access.2019.2946980. 2279
https://openalex.org/W2058623711	https://discovery.ucl.ac.uk/1429246/1/bdi12203.pdf	English	null	Analysis of <i>ANK3</i> and <i>CACNA1C</i> variants identified in bipolar disorder whole genome sequence data	Bipolar disorders	2,014	cc-by	7,648	doi: 10.1111/bdi.12203 Key words: allelic association study – ankyrin 3 – bipolar disorder – DNA sequencing – genetic – L-type calcium channel Received 19 September 2013, revised and accepted for publication 27 December 2013 Corresponding author: Dr. Andrew McQuillin Molecular Psychiatry Laboratory Division of Psychiatry University College London Rockefeller Building London WC1E 6BT UK Fax: +44-20-3108-2194 E-mail: a.mcquillin@ucl.ac.uk In the authors’ opinion, both of these authors contributed equally to the work and should be considered joint ﬁrst authors. †Professor Gurling is deceased. Key words: allelic association study – ankyrin 3 – bipolar disorder – DNA sequencing – genetic – L-type calcium channel Received 19 September 2013, revised and accepted for publication 27 December 2013 Corresponding author: Dr. Andrew McQuillin Molecular Psychiatry Laboratory Division of Psychiatry University College London Rockefeller Building London WC1E 6BT UK Fax: +44-20-3108-2194 E-mail: a.mcquillin@ucl.ac.uk In the authors’ opinion, both of these authors contributed equally to the work and should be considered joint ﬁrst authors. †Professor Gurling is deceased. Methods: In order to screen both coding and non-coding regions to identify potential aetiological variants, we used whole-genome sequencing in 99 BP cases. Variants with markedly diﬀerent allele frequencies in the BP samples and the 1,000 genomes project European data were genotyped in 1,510 BP cases and 1,095 controls. Results: We found that the CACNA1C intron 3 variant, rs79398153, potentially aﬀecting an ENCyclopedia of DNA Elements (ENCODE)- deﬁned region, showed an association with BP (p = 0.015). We also found the ANK3 BP-associated variant rs139972937, responsible for an asparagine to serine change (p = 0.042). However, a previous study had not found support for an association between rs139972937 and BP. The variants at ANK3 and CACNA1C previously known to be associated with BP were not in linkage disequilibrium with either of the two variants that we identiﬁed and these are therefore independent of the previous haplotypes implicated by genome-wide association. Conclusions: Sequencing in additional BP samples is needed to ﬁnd the molecular pathology that explains the previous association ﬁndings. If changes similar to those we have found can be shown to have an eﬀect on the expression and function of ANK3 and CACNA1C, they might help to explain the so-called ‘missing heritability’ of BP. (3–6), with a ten-fold increase in risk to the relatives of probands with BP (7). Bipolar Disorders 2014: 16: 583–591 BIPOLAR DISORDERS Analysis of ANK3 and CACNA1C variants identiﬁed in bipolar disorder whole genome sequence data Alessia Fiorentinoa,, Niamh Louise O’Briena,, Devin Paul Lockeb, Andrew McQuillina, Alexandra Jarrama, Adebayo Anjorina, Radhika Kandaswamya, David Curtisc, Robert Alan Blizarda and Hugh Malcolm Douglas Gurlinga,† aMolecular Psychiatry Laboratory, Division of Psychiatry, University College London, London, UK, bKnome Inc., Cambridge, MA, USA, cDepartment of Psychological Medicine, Queen Mary University of London, London, UK Fiorentino A, O’Brien NL, Locke DP, McQuillin A, Jarram A, Anjorin A, Kandaswamy R, Curtis D, Blizard RA, Gurling HMD. Analysis of ANK3 and CACNA1C variants identiﬁed in bipolar disorder whole genome sequence data. Bipolar Disord 2014: 16: 583–591. © 2014 The Authors. Bipolar Disorders Published by John Wiley & Sons Ltd. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. Objectives: Genetic markers in the genes encoding ankyrin 3 (ANK3) and the a-calcium channel subunit (CACNA1C) are associated with bipolar disorder (BP). The associated variants in the CACNA1C gene are mainly within intron 3 of the gene. ANK3 BP-associated variants are in two distinct clusters at the ends of the gene, indicating disease allele heterogeneity. doi: 10.1111/bdi.12203 The rs1006737 risk variant has been associated with increased amygdala func- tioning observed by magnetic resonance imaging during emotional processing; the enhancement of activation leads to impaired facial emotion recog- nition in BP patients (20, 23–26). There has been conﬂicting evidence as to whether the presence of the CACNA1C variant results in brain volumetric alteration. Some reports state that this SNP has been associated with brainstem alterations, increased grey matter density, as well as a cortical volume increase (27–29). A conﬂicting study did not report any association between this SNP and brain volumetric alterations (30). Mutations/vari- ants located in intronic regions can also aﬀect the stability of RNA and protein expression, and can have a strong eﬀect on the transcriptional regula- tion of the gene. In ANK3 the strongest evidence for allelic asso- Fiorentino et al. doi: 10.1111/bdi.12203 Many linkage studies of speciﬁc chromosomal regions and whole genomes in multiply aﬀected families support the presence of locus heterogeneity, with multiple sus- ceptibility loci (8–10). About half of the segrega- tion analyses of systematically ascertained families Bipolar disorder (BP) is a common disease with a worldwide average population prevalence of 1.4%, which rises to 2.4% if bipolar spectrum disorders are included (1). Twin and family studies indicate that BP is genetically related to some types of uni- polar aﬀective disorder (2). The genetic heritability of BP is thought to be between 79% and 93% 583 imply that BP has an autosomal dominant mode of inheritance (11). Other models have favoured a single major locus with a polygenic multifactorial background and pure polygenic transmission. Linkage and linkage disequilibrium (LD) analyses demonstrate locus heterogeneity (12, 13). Genome- wide association studies (GWAS), meta-analyses, and replication studies focusing on BP have been carried out on combined cohort sizes of up to 7,481 cases and 9,250 controls (14–17). These and other single-locus case–control association studies have repeatedly implicated the L-type calcium channel a1C subunit (CACNA1C) and ankyrin 3 (ANK3) genes in BP. The strongest allelic associa- tion signal in CACNA1C is localized entirely within intron 3 of the gene with the single nucleo- tide polymorphisms (SNPs) rs1006737 (p = 7.0 9 108) (14, 18, 19), rs4765913 (p = 1.52 9 108) (14), rs4765914 (p = 1.52 9 108) (20), and rs1024 582 (p = 1.7 9 107) (17, 21). GWAS results across ﬁve diﬀerent psychiatric illnesses further implicate rs1024582 in susceptibility to both BP and schizophrenia, assuming that there has not been substantial misdiagnosis, especially where schizoaﬀective BP cases are included in the schizo- phrenia group (20). Intron 3 of CACNA1C con- tains a chromosomal region with high levels of LD, strong mammalian conservation, and multiple sites designated by the ENCyclopedia of DNA Ele- ments (ENCODE) project as being able to aﬀect gene expression. Studies show that that the pres- ence of the rs1006737 CACNA1C BP risk variant may have an impact on certain brain activities. One study showed that the rs1006737 risk variant in healthy males is associated with lower extraver- sion, trait anxiety, paranoid ideation, and higher harm avoidance (22). Fiorentino et al. 107) (31), rs4948418 (p = 8.93 9 109) (15), rs 10994336 (p = 9.1 9 109) (14, 26, 32–34), rs1099 4397 (p = 7.1 9 109) (17) at the 50 end, and the SNP rs9804190 (p = 1.20 9 104) (17, 26, 35) at the 30 end of the longest isoform (NM_001204403) of the gene. These regions are over 340 kb apart and appear to be independently associated with BP, with no signiﬁcant interactions between SNPs from the two regions (32). However, the existing data on ANK3 show that only low-frequency aetio- logical base-pair (bp) changes are present with an odds ratio less than 1.35 for BP (17). The SNP associations are not replicated in every study (36– 39); however, the ANK3 association has been reported in several diﬀerent ancestral populations (36, 40–42). Several novel, rare potential aetiologi- cal bp changes have been identiﬁed by us through sequencing the gene in our samples. These were selected for having haplotypes associated with BP (43). Doyle et al. (44) sequenced the 8 kb brain- expressed exon 48 of ANK3 but could not ﬁnd potential aetiological bp changes that were associ- ated with BP. This exon is of recent evolutionary history, and variation in the exon appears to be tolerated. Sequencing analysis of ANK3 demon- strated the impact of heterogeneity on replication of allelic associations, even within well-deﬁned ancestral populations (43). mRNA analysis has detected diﬀerential regulation of distinct ANK3 transcription start sites and coupling of speciﬁc 50 ends with 30 mRNA splicing events, suggesting that brain-speciﬁc cis-regulatory transcriptional changes might be relevant to BP molecular pathol- ogy (45). Gene network analysis and test of epista- sis have found further support for an association of ANK3 with BP (46, 47). The genetic variants associated with disease have no known biological function. However, one study showed that the presence of the rs10994336 BP risk variant in healthy males might predict lower novelty seeking, lower behavioural activation scores, and high star- tle reactivity (22). In healthy volunteers, rs10994 336 may be associated with reduced white matter integrity in the anterior limb of the internal cap- sule, as well as with altered set-shifting and deci- sion-making (48). These ﬁndings may be consistent with previous diﬀusion tensor imaging studies in patients with BP (49–54) and core phenotypes of BP (55–58). Fiorentino et al. Lithium has been shown to alter Ank3 mRNA levels in the mouse brain (59), and lithium and sodium valproate have been shown to change Ank3 protein amounts in rat neuronal dendritic spines (60). In another animal model, RNA inter- ference of Ank3 in the hippocampus dentate gyrus induced a reduction of anxiety-related behaviours d i d ti it d i th li ht h hi h 107) (31), rs4948418 (p = 8.93 9 109) (15), rs 10994336 (p = 9.1 9 109) (14, 26, 32–34), rs1099 4397 (p = 7.1 9 109) (17) at the 50 end, and the SNP rs9804190 (p = 1.20 9 104) (17, 26, 35) at the 30 end of the longest isoform (NM_001204403) of the gene. These regions are over 340 kb apart and appear to be independently associated with BP, with no signiﬁcant interactions between SNPs from the two regions (32). However, the existing data on ANK3 show that only low-frequency aetio- logical base-pair (bp) changes are present with an odds ratio less than 1.35 for BP (17). The SNP associations are not replicated in every study (36– 39); however, the ANK3 association has been reported in several diﬀerent ancestral populations (36, 40–42). Several novel, rare potential aetiologi- cal bp changes have been identiﬁed by us through sequencing the gene in our samples. These were selected for having haplotypes associated with BP (43). Doyle et al. (44) sequenced the 8 kb brain- expressed exon 48 of ANK3 but could not ﬁnd potential aetiological bp changes that were associ- ated with BP. This exon is of recent evolutionary history, and variation in the exon appears to be tolerated. Sequencing analysis of ANK3 demon- strated the impact of heterogeneity on replication of allelic associations, even within well-deﬁned ancestral populations (43). mRNA analysis has detected diﬀerential regulation of distinct ANK3 transcription start sites and coupling of speciﬁc 50 ends with 30 mRNA splicing events, suggesting that brain-speciﬁc cis-regulatory transcriptional changes might be relevant to BP molecular pathol- ogy (45). Gene network analysis and test of epista- sis have found further support for an association of ANK3 with BP (46, 47). The genetic variants associated with disease have no known biological function. However, one study showed that the presence of the rs10994336 BP risk variant in healthy males might predict lower novelty seeking, lower behavioural activation scores, and high star- tle reactivity (22). Subjects This study included 1,510 aﬀected research subjects with BP. These were sampled in three cohorts. The ﬁrst cohort, UCL1, included 506 research subjects with bipolar I disorder (BP-I), deﬁned by the pres- ence of mania and hospitalization according to Research Diagnostic Criteria (RDC) (62). UCL1 was included in the previously reported mega- analysis by the Psychiatric Genetic Consortium (PGC) BP GWAS (17). The second and third cohorts, UCL2 and UCL3, consisted, respectively, of 593 and 411 subjects with BP-I or bipolar II dis- order (BP-II). Ancestry screening was used as a selection criterion for the inclusion of cases. Sam- ples were included if at least three out of four grandparents were English, Irish, Scottish, or Welsh and if the fourth grandparent was non-Jew- ish European, before the European Union enlarge- ment in 2004. The sample of 1,095 controls comprised 614 screened subjects who had no ﬁrst- degree family or personal history of psychiatric ill- ness and an additional 481 unscreened normal British subjects, obtained from the European Col- lection of Animal Cell Cultures (ECACC). National Health Service (NHS) multicentre research ethics approval was obtained. All partici- pants provided signed consent. Bipolar variants in ANK3 and CACNA1C Bipolar variants in ANK3 and CACNA1C saliva samples for the UCL2 cohort, and a mix- ture of both blood and saliva for the UCL3 sam- ples. DNA from blood samples was extracted using a standard phenol–chloroform method and from saliva samples using the Oragene protocol for DNA extraction (DNA Genotek, Ottawa, ON, Canada). were attenuated by chronic treatment with the mood stabilizer, lithium. Similar behavioural alter- ations of reduced anxiety and increased motivation for reward were also exhibited by Ank3+/ hetero- zygous mice compared with wild-type Ank3+/+ mice (61). Given the typical natural history of BP, which consists of episodes of both mania and depression with complete recovery very often between epi- sodes, it can be argued that genetic susceptibility will involve aetiological bp changes inﬂuencing the control of gene expression and mRNA translation rather than mutations creating structural protein abnormalities. Therefore, we chose whole-genome sequencing (WGS) rather than exome sequencing in order to be able to investigate intronic and non- coding control regions of susceptibility genes along with the exonic coding regions. WGS WGS was performed on 99 of the subjects with BP-I selected from all our cohorts who had a posi- tive family history of BP or bipolar spectrum disor- der and an early age at onset. The genomic DNA was sequenced using 100 bp paired-end reads on a Hi-Seq 1000 (Illumina Inc., San Diego, CA, USA). Sequence data alignment to the National Center for Biotechnology Information human reference genome 37.1 (hg19) and variant calling was performed using the CASAVA 1.8.2 pipeline at Illu- mina (http://res.illumina.com/documents/products/ technotes/technote_snp_caller_sequencing.pdf). The sequence data from these individuals were further analysed and annotated using kGAP (Knome Inc., Boston, MA, USA). Fiorentino et al. In healthy volunteers, rs10994 336 may be associated with reduced white matter integrity in the anterior limb of the internal cap- sule, as well as with altered set-shifting and deci- sion-making (48). These ﬁndings may be consistent with previous diﬀusion tensor imaging studies in patients with BP (49–54) and core phenotypes of BP (55–58). Lithium has been shown to alter Ank3 mRNA levels in the mouse brain (59), and lithium and sodium valproate have been shown to change Ank3 protein amounts in rat neuronal dendritic spines (60). In another animal model, RNA inter- In ANK3, the strongest evidence for allelic asso- ciation comes from SNPs rs10994338 (p = 1.20 9 584 Fiorentino et al. Haplotype analysis (i) Located in the third intron of CACNA1C between ﬂanking high recombination peaks (chr12:2271532-2425994 hg19); Haplotype analysis was performed using Haplo- view (66) to determine the LD between GWAS- associated SNPs and the rare variants reported here. Haplotype blocks were determined using a solid spine of LD (D0 = 1). (ii) Located in a putative functional site deﬁned by being an ENCODE-marked element (65); (ii) Located in a putative functional site deﬁned by being an ENCODE-marked element (65); (iii) Located in a conserved non-coding sequence, determined using the mammalian conservation track on the University of California Santa Cruz (UCSC) genome browser (http://genome.ucsc.edu/) or by their Genomic Evolutionary Rate Proﬁling (GERP) scores; Variant calling and selection (iv) Not in a repeat region. WGS in 99 samples with BP produced a mean depth coverage of 37.0 with 90% of the genome sequenced. A total of 0.12% of bases were hetero- zygous and the transition/transversion (Ti/Tv) ratio was 2.0 (see Supplementary Table 3). Each variant was validated by conﬁrming the bp call conﬁdence using individual Binary Alignment/ Map (BAM) ﬁles before genotyping. Bioinformatic analysis to determine potentially functional SNPs was carried out using the UCSC genome browser, Alibaba2.1 (http://www.gene-regulation.com/pub/ programs/alibaba2/index.html), targetscan 6.2 (http://www.targetscan.org/), miRanda (http:// www.microrna.org/microrna/home.do), PolyPhen-2 (http://genetics.bwh.harvard.edu/pph2/), and Sort- ing Intolerant From Tolerant (SIFT) (http://sift. jcvi.org/). Using the criteria set out above, 82 ANK3 and 43 CACNA1C variants were identiﬁed in the cod- ing exons, 50UTR, 30UTR, splicing sites, and pro- moter region; and a further 108 CACNA1C variants were identiﬁed in the intron 3 region 12:2271532-2425994 (Supplementary Table 1, Sup- plementary Fig. 1, Supplementary Fig. 2). These variants were further ﬁltered using a cut- oﬀthreshold of p < 0.05 using Fisher’s exact test against the 1,000G Project (67) European allele fre- quencies. After ﬁltering, three variants remained in ANK3 and these comprised two known SNPs, rs184389434 and rs139972937, and a previously unreported SNP at position ss825679002; ten CACNA1C variants remained after ﬁltering and these comprised ﬁve known SNPs (rs146482058, rs79398153, rs191953785, rs112312080, and rs113 414207) and four previously unreported variants (ss825679004, ss825679005, ss825679006, and ss825679007 (Table 1, Supplementary Fig. 1, Sup- plementary Fig. 2). Genotyping Genotyping for the selected SNPs in 1,510 BP cases (UCL1, UCL2, and UCL3 samples) and 1,095 ancestrally matched controls was performed in-house with allele-speciﬁc polymerase chain reac- tion (PCR) using KASPar reagents (KBiosciences, Hoddesdon, UK) on a LightCycler 480 (Roche, Burgess Hill, UK) real-time PCR machine. For all SNPs genotyped, 17% of samples were duplicated to detect error and conﬁrm the reproducibility of genotypes. Allele-speciﬁc primers were designed for each of the SNPs using Primer Picker (KBio- sciences), as shown in Supplementary Table 2. All these data were analysed to conﬁrm Hardy–Wein- berg equilibrium (HWE). Allelic associations for SNPs were performed using Fisher’s Exact test. Signiﬁcance values shown for all analyses are uncorrected for multiple testing, and a cutoﬀsig- niﬁcance value of p < 0.05 was used. The ANK3 variant rs184389434 is located in the promoter region of the gene and was pre- dicted to create a binding site for three new tran- scription factors [ETS-related gene (Erg-1), Ultrabithorax (Ubx) and Octamen-1 (Oct-1)]. rs139972937 causes a non-conservative amino acid change from asparagine to serine at position 2,643 (N2643S) in exon 34 of the alternative iso- forms NM_020987.3 and CCDS7258.1. The N2643S amino acid substitution was predicted to be benign with a score of 0 (sensitivity 1, speciﬁc- ity 0) by PolyPhen-2 and tolerated with a score of 0.71 by SIFT. The previously unreported ANK3 variant, ss825679002, was located in the 30UTR of the gene and was found to be in a microRNA binding site. Bioinformatic analysis using target- scan and miRanda predicted no eﬀect on microR- NA binding. Variant selection ANK3 and CACNA1C non-synonymous variants present in the coding exons were identiﬁed using the Knome VARIANTS software (Knome Inc.) (Supplementary Table 1). The same software was used to identify variants in the 50 untranslated region (UTR), 30UTR, splicing sites [donor site consists of 5 bp in the exon and 6 bp in the intron, acceptor site consist of 3 bp in the exon and 20 bp in the intron (64)], promoter region (1,000 bp from the ﬁrst exon of every coding isoform), and the third intron of CACNA1C (Supplementary Table 1, Supplementary Fig. 1, Supplementary Fig. 2). Allele counts for each SNP in the BP samples were compared to those from the 372 European samples in the 1,000 Ge- nomes (1,000G) Project (phase 1, version 3; ftp:// ftp.1000genomes.ebi.ac.uk/vol1/ftp/release/20110521/ ALL.wgs.phase1_release_v3.20101123.snps_indels_ sv.sites.vcf.gz). SNPs for which the variant allele was more common in subjects with BP than in the 1,000G Project data, signiﬁcant at p < 0.05 using Fisher’s exact test, were chosen for geno- typing in the complete UCL case–control sample. Variants which were present in poly-base regions and insertions in repeat regions were excluded from genotyping. Research subjects with BP had been given an NHS clinical diagnosis of ICD-10 BP and then needed to fulﬁl RDC (62) for BP with clinical data collected by the lifetime version of the Schizophrenia and Aﬀective Disorder Schedule (SADS-L) (63). DNA samples were collected from blood samples from the UCL1 cohort, The variants in the third intron of CACNA1C were selected if they met four criteria: 585 Results Variant calling and selection Burden analysis A burden analysis was performed on the data sepa- rately for ANK3 and CACNA1C. A chi-square test was used to compare the numbers of case and con- trol individuals carrying one or more of the variant alleles against the numbers of case and control individuals who were found to be homozygous for the reference alleles at all of the loci tested. 586 Bipolar variants in ANK3 and CACNA1C Table 1. Test of association with ANK3 and CACNA1C variants with bipolar disorder Table 1. Test of association with ANK3 and CACNA1C variants with bipolar disorder Gene SNP Location (hg19) Position in gene Change European 1,000G MAF MLP Number of samples Genotype counts MAF p- valuea ANK3 rs184389434 10: 62493837 Promoter A/T 0 1.39 BP 1,469 0/15/1454 0.0051 0.41 Control 1,031 0/12/1019 0.0058 ANK3 ss825679002 10: 61788626 30UTR C/A 0 1.39 BP 1,467 0/36/1431 0.012 0.35 Control 1,026 0/28/998 0.014 ANK3 rs139972937 10: 61832711 Exon (N2643S) A/G 0 1.39 BP 1,474 0/9/1465 0.0031 0.042 Control 1,030 0/1/1029 0.0005 CACNA1C ss825679004 12: 2161934 Promoter G/A 0 1.39 BP 1,467 0/5/1462 0.0017 0.57 Control 1,031 0/4/1027 0.0019 CACNA1C ss825679005 12: 2694668 Splice site G/A 0 1.39 BP 1,473 0/3/1470 0.0010 0.7 Control 1,032 0/2/1030 0.0010 CACNA1C rs146482058 12: 2403077 Intronb,e –/T 0 10.9 BP 1,486 3/134/1322 0.048 0.31 Control 1,022 4/96/922 0.051 CACNA1C rs79398153 12: 2295156 Intronb,e C/T 0.01 1.57 BP 1,498 3/88/1380 0.032 0.015 Control 1,029 0/44/985 0.021 CACNA1C rs191953785 12: 2425097 Intronb,e C/T 0 2.29 BP 1,475 0/25/1450 0.0085 0.34 Control 1,020 0/20/1005 0.0098 CACNA1C rs112312080 12: 2354510 Intronb,c,e C/T 0.001 2.02 BP 1,501 0/35/1439 0.012 0.30 Control 1,028 0/20/1008 0.0097 CACNA1C rs113414207 12: 2292742- 2292743 Intronb,d,e –/C 0 2.15 BP 1,497 0/76/1394 0.026 0.19 Control 1,026 0/44/982 0.021 CACNA1C ss825679006 12: 2329069 Intronb,d,e G/T 0 2.15 BP 1,498 0/14/1457 0.0048 0.43 Control 1,033 0/8/1024 0.0039 CACNA1C ss825679007 12: 2423175 Intronb,c,d,e G/C 0 2.15 BP 1,502 0/4/1471 0.0014 0.34 Control 1,026 0/1/1025 0.0010 1,000G = 1,000 Genomes; ANK3 = ankyrin 3; BP = bipolar disorder; CACNA1C = L-type calcium channel a1C subunit; MAF = minor allele frequency; SNP = single nucleotide polymorphism; UTR = untranslated region; European 1,000G MAF = MAF for European sam- ples in the 1,000G Project (72); MLP = minus log10 of Fisher’s exact p-value comparing UCL whole-genome sequence allele frequency to the European 1,000G MAF. ap-signiﬁcance value for a Fisher’s exact test. bH3K4Me1, H3K4Me3, and H3K27Ac marks. Burden analysis cDNAse hypersensitivity cluster marks. dHuman mRNA. eConserved region. The distributions of genotype data for all SNPs were in Hardy–Weinberg equilibrium in the cases and controls. 1,000G = 1,000 Genomes; ANK3 = ankyrin 3; BP = bipolar disorder; CACNA1C = L-type calcium channel a1C subunit; MAF = minor allele frequency; SNP = single nucleotide polymorphism; UTR = untranslated region; European 1,000G MAF = MAF for European sam- ples in the 1,000G Project (72); MLP = minus log10 of Fisher’s exact p-value comparing UCL whole-genome sequence allele frequency to the European 1,000G MAF. a p ap-signiﬁcance value for a Fisher’s exact test. b dHuman mRNA. eConserved region. The distributions of genotype data for all SNPs were in Hardy–Weinberg equilibrium in the cases and controls. Human mRNA. eConserved region. The distributions of genotype data for all SNPs were in Hardy–Weinberg equilibrium in the cases and controls. 27 (H3K27ac), and active transcriptional enhanc- ers with distinct chromatin signatures (68). Enrich- ment for H3K4me1 and H3K27ac at a genetic level distinguishes active enhancers from inactive or poised enhancers (69, 70). The presence of H3K4me1- and H3K27ac-marked chromatin, with low levels of H3K4me3 and an absence of another histone marker, H3K27me3, represent putative human embryonic stem cell (hESC) enhancers and have been shown to localize proximally to genes that are expressed during development in hESCs and in epiblast cells (70). Additionally, rs112312080 and ss825679007 were found to be present on DNAse I hypersensitivity sites, as listed by the ENCODE project. One of the novel variants in CACNA1C, ss825679004, was in the promoter region of the gene. Alibaba 2.1 analysis of ss825679004 pre- dicted it to disrupt the binding sites for three tran- scription factors [Activating Enhancer Binding Protein-2alpha (AP-2alph), NF-muE1, Speciﬁcity Protein-1 (Sp1)] and to create a new one for a dif- ferent transcription factor [GC Factor (GCF)]. CACNA1C variant ss825679005 is the sixth base in the intron of the splice donor site for exon 17. This exon is present in all known isoforms of the gene and this variant might alter splicing eﬃciency. The CACNA1C intron 3 variants rs146482058, rs79398153, rs191953785, rs112312080, rs113414 207, ss825679006, and ss825679007 were present in the region of high LD between chr12:2,230,353 and chr12:2,559,413. Each variant was also located in an ENCODE marked region (65). All seven SNP regions are marked by H3 mono-methylation of lysine 4 (H3K4me1), H3 tri-methylation of lysine 4 (H3K4me3), and H3 acetylation of lysine Discussion We have analysed genetic variation by WGS in two of the best-replicated bipolar susceptibility genes, CACNA1C and ANK3. This analysis has identiﬁed novel possible BP susceptibility variants in both of these genes. The CACNA1C intron 3 variant rs79398153 may impact CACNA1C gene expression by virtue of its presence in an ENCODE-marked transcriptional enhancer region. Of the SNPs found in the CACNA1C intronic region, rs79398153 was found to be associated with BP (Fisher’s exact test p = 0.015). We detected 88 heterozygote and three homozygote cases for this variant and 44 heterozygote controls. The excess of homozygotes may possibly represent a recessive eﬀect, as only one homozygote would be expected under HWE, but this excess is not statistically signiﬁcant (71). rs79398153 is located in an ENCODE-marked region for H3K4me1, H3K27ac, and H3Kme3. None of the other intron- ic CACNA1C SNPs were associated with BP. Imputation for rs79398153 in UCL1 using GWAS data showed that it was still signiﬁcantly associated with BP (p = 0.022) (17). Burden analysis showed that, overall, there was no excess of the variants genotyped in cases versus controls for either CAC- NA1C or ANK3. g The ANK3 amino acid changing variant, rs139972937 (N2643S), was associated with BP in our sample (p = 0.042). This rare variant was also found in two of 1,119 cases and one of 1,078 con- trols and in a family containing seven subjects with BP, a father and six oﬀspring, where only the father and two of the oﬀspring possessed the vari- ant (44). These conﬂicting ﬁndings are not uncom- mon in the genetics of complex diseases but this lack of support casts doubt on the true aetiological importance of this variant in BP. It is of note that the allele frequencies of some of the variants selected for genotyping were found to be markedly diﬀerent in our control samples compared to the frequencies in the European 1,000G Project. This underlies the importance of typing variants in matched control samples on the same platform rather than relying on publi- cally available data such as the 1,000G Project in order to mitigate possible spurious association ﬁndings. Fiorentino et al. generated for 12 of these variants and the genotype distributions for each SNP followed HWE in the case and control cohorts. The non-synonymous ANK3 variant rs139972937 was found to be associ- ated with BP (Fisher’s exact test p = 0.042) (Table 1). Nine cases with BP and only one control were found to be heterozygous for this variant. None of the other ANK3 variants were found to be associated with BP, as shown in Table 1. were more common in the control subjects. Thus, these variants could not be accounting for the GWAS signals. The results of the LD analysis are shown in Table 2. Genotyping Assays were designed for 13 SNPs which passed ﬁltering tests for genotyping in the complete UCL BP case–control sample. Genotype data were 587 Acknowledgements unipolar depression. Arch Gen Psychiatry 2003; 60: 497– 502. The UCL clinical and control samples were collected with the support from the Bipolar Organization, the Neuroscience Research Charitable Trust, the Central London NHS Blood Transfusion Service, The Stanley Medical Research Institute, Cheshire and Wirral Partnership NHS Foundation Trust, Cumbria Partnership NHS Foundation Trust, Cambridgeshire and Peterborough NHS Foundation Trust, Suﬀolk Mental Health Partnership NHS Trust, South Essex Partnership Uni- versity NHS Foundation Trust (in services based in Bedford- shire and Luton), West London Mental Health NHS Trust, Camden and Islington NHS Foundation Trust, East London NHS Foundation Trust, North East London Mental Health NHS Trust, Hertfordshire Partnership NHS Foundation Trust, Berkshire Healthcare NHS Foundation Trust, North Essex Partnership NHS Foundation Trust, Oxfordshire and Buckinghamshire Mental Health NHS Foundation Trust, South Essex Partnership University NHS Foundation Trust, South London and Maudsley NHS Foundation Trust, Oxleas NHS Foundation Trust, Surrey and Borders Partnership NHS Foundation Trust, Kent and Medway NHS and Social Care Partnership Trust, South West London and St George’s Men- tal Health NHS Trust, Sussex Partnership Trust, South Essex Partnership University NHS Foundation Trust, Cornwall Partnership NHS Trust, Somerset Partnership NHS Founda- tion Trust, Salisbury NHS Foundation Trust, Central and North West London NHS Foundation Trust, the National Institute for Health Research Mental Health Research Net- work, and the NIHR-supported Primary Care Research Net- work. Genetic analysis of the UCL cohort has been supported by UK Medical Research Council project grants G9623693N, G0500791, G0701007, and G1000708. NLOB is supported by a joint Ph.D. studentship funded by a UCL IMPACT award and Equilibrium, the Bipolar Foundation. RK was funded by a UK government Overseas Research Student award. The Stanley Foundation and the Stanley Psychiatric Research Cen- ter at the Broad Institute, Boston, MA, USA, funded the gen- ome-wide association study. 7. Shih RA, Belmonte PL, Zandi PP. A review of the evi- dence from family, twin and adoption studies for a genetic contribution to adult psychiatric disorders. Int Rev Psychi- atry 2004; 16: 260–283. y 8. Badner JA, Gershon ES. Meta-analysis of whole-genome linkage scans of bipolar disorder and schizophrenia. Mol Psychiatry 2002; 7: 405–411. 9. Segurado R, Detera-Wadleigh SD, Levinson DF et al. Genome scan meta-analysis of schizophrenia and bipolar disorder, part III: bipolar disorder. Am J Hum Genet 2003; 73: 49–62. 10. McQueen MB, Devlin B, Faraone SV et al. Disclosures The authors of this paper do not have any commercial associa- tions that might pose a conﬂict of interest in connection with this manuscript. 18. Sklar P, Smoller JW, Fan J et al. Whole-genome associa- tion study of bipolar disorder. Mol Psychiatry 2008; 13: 558–569. 19. Consortium WTCC. Genome-wide association study of 14,000 cases of seven common diseases and 3,000 shared controls. Nature 2007; 447: 661–678. Acknowledgements Combined analysis from eleven linkage studies of bipolar disorder provides strong evidence of susceptibility loci on chromo- somes 6q and 8q. Am J Hum Genet 2005; 77: 582–595. 11. Gurling H, Rifkin L. Genetic aspects of aﬀective disorders. In: Horton KW, Katona C eds. Biological Aspects of Aﬀective Disorders. London: Academic Press, 1991: 305– 329. 12. Spence MA, Flodman PL, Sadovnick AD et al. Bipolar disorder: evidence for a major locus. Am J Med Genet 1995; 60: 370–376. 13. Craddock N, Khodel V, Van Eerdewegh P, Reich T. Mathematical limits of multilocus models: the genetic transmission of bipolar disorder. Am J Hum Genet 1995; 57: 690–702. 14. Ferreira MA, O’Donovan MC, Meng YA et al. Collabo- rative genome-wide association analysis supports a role for ANK3 and CACNA1C in bipolar disorder. Nat Genet 2008; 40: 1056–1058. 15. Chen DT, Jiang X, Akula N et al. Genome-wide associa- tion study meta-analysis of European and Asian-ancestry samples identiﬁes three novel loci associated with bipolar disorder. Mol Psychiatry 2013; 18: 195–205. 16. Wray NR, Pergadia ML, Blackwood DH et al. Genome- wide association study of major depressive disorder: new results, meta-analysis, and lessons learned. Mol Psychiatry 2012; 17: 36–48. 17. Sklar P, Ripke S, Scott LJ et al. Large-scale genome-wide association analysis of bipolar disorder identiﬁes a new sus- ceptibility locus near ODZ4. Nat Genet 2011; 43: 977–983. Discussion Haplotype and LD analysis at ANK3 and CAC- NA1C was performed separately in Haploview (66) to examine the co-occurrence of the BP GWAS SNP alleles [ANK3 rs10994285, rs2018783, rs10994336, rs10994397, rs10821792, rs1938526 (14, 26, 32–34); CACNA1C rs1006737 (14); rs4765913 (14); rs476590 (31); and rs4765914 (20)] with the BP risk variant alleles (rs139972937, allele G; rs79398153, allele T). For both sets of analyses, the BP risk variant alleles reported here were found to be associated with the GWAS SNP alleles that The variants we have found appear to be acting independently of the allelic and haplotypic associa- tions found in the previous BP allelic association studies. Independent genetic replication and bio- logical validation of intronic potential aetiological bp changes would support the argument for carry- ing out WGS as well as exome sequencing in BP. The biphasic nature of BP makes a compelling argument for the existence of genetically deter- mined pathological switch mechanisms that may manifest themselves in the loss of control of gene expression. Findings such as ours may help to explain the ‘missing heritability’ in this common complex disorder. Further analyses in much larger samples are needed to ﬁnd aetiological bp changes in the ANK3 and CACNA1C genes that are carried by the main haplotypes showing strong association with BP. The outcome could be personalized treat- ment for BP, based on susceptibility genotypes. Table 2. Pairwise linkage disequilibrium analysis between bipolar dis- order-associated SNPs reported here and previous GWAS SNPs Gene Marker 1 Marker 2 D0 r2 LOD ANK3 rs139972937 rs10994285 1 0 0.17 ANK3 rs139972937 rs2018783 1 0.002 0.51 ANK3 rs139972937 rs10994336 1 0 0.03 ANK3 rs139972937 rs10994397 1 0 0.03 ANK3 rs139972937 rs10821792 1 0 0.21 ANK3 rs139972937 rs1938526 1 0 0.26 CACNA1C rs79398153 rs1006737 0.754 0.016 1.06 CACNA1C rs79398153 rs1024582 0.729 0.014 0.87 CACNA1C rs79398153 rs4765913 0.822 0.029 2.12 CACNA1C rs79398153 rs4765914 0.829 0.032 2.31 ANK3 = ankyrin; CACNA1C = L-type calcium channel a1C sub- unit; GWAS = genome-wide association studies; LOD = loga- rithm of the odds; SNPs = single nucleotide polymorphisms. Table 2. Pairwise linkage disequilibrium analysis between bipolar dis- order-associated SNPs reported here and previous GWAS SNPs 588 Fiorentino et al. Genome-wide supported risk variant for bipolar disorder alters anatomical connec- tivity in the human brain. Neuroimage 2012; 59: 3288– 3296. 31. Liu Y, Blackwood DH, Caesar S et al. Meta-analysis of genome-wide association data of bipolar disorder and major depressive disorder. Mol Psychiatry 2011; 16: 2–4. 49. Chaddock CA, Barker GJ, Marshall N et al. White matter microstructural impairments and genetic liability to famil- ial bipolar I disorder. Br J Psychiatry 2009; 194: 527–534. 32. Schulze TG, Detera-Wadleigh SD, Akula N et al. Two variants in Ankyrin 3 (ANK3) are independent genetic risk factors for bipolar disorder. Mol Psychiatry 2009; 14: 487– 491. 50. Sussmann JE, Lymer GK, McKirdy J et al. White matter abnormalities in bipolar disorder and schizophrenia detected using diﬀusion tensor magnetic resonance imag- ing. Bipolar Disord 2009; 11: 11–18. 33. Scott LJ, Muglia P, Kong XQ et al. Genome-wide associa- tion and meta-analysis of bipolar disorder in individuals of European ancestry. Proc Natl Acad Sci U S A 2009; 106: 7501–7506. 51. McIntosh AM, Mu~noz Maniega S, Lymer GK et al. White matter tractography in bipolar disorder and schizophrenia. Biol Psychiatry 2008; 64: 1088–1092. 52. McIntosh AM, Job DE, Moorhead TW et al. White mat- ter density in patients with schizophrenia, bipolar disorder and their unaﬀected relatives. Biol Psychiatry 2005; 58: 254–257. 34. Lett TA, Zai CC, Tiwari AK et al. ANK3, CACNA1C and ZNF804A gene variants in bipolar disorders and psy- chosis subphenotype. World J Biol Psychiatry 2011; 12: 392–397. 53. Lin F, Weng S, Xie B, Wu G, Lei H. Abnormal frontal cortex white matter connections in bipolar disorder: a DTI tractography study. J Aﬀect Disord 2011; 131: 299–306. 35. Smith EN, Bloss CS, Badner JA et al. Genome-wide asso- ciation study of bipolar disorder in European American and African American individuals. Mol Psychiatry 2009; 14: 755–763. 54. Sui J, Adali T, Pearlson GD, Calhoun VD. An ICA-based method for the identiﬁcation of optimal FMRI features and components using combined group-discriminative techniques. Neuroimage 2009; 46: 73–86. 36. Belmonte Mahon P, Pirooznia M, Goes FS et al. Genome- wide association analysis of age at onset and psychotic symptoms in bipolar disorder. Am J Med Genet B Neuro- psychiatr Genet 2011; 156B: 370–378. 55. Adida M, Jollant F, Clark L et al. Trait-related decision- making impairment in the three phases of bipolar disorder. Biol Psychiatry 2011; 70: 357–365. 37. Fiorentino et al. Fiorentino et al. 25. Small SA, Schobel SA, Buxton RB, Witter MP, Barnes CA. A pathophysiological framework of hippocampal dys- function in ageing and disease. Nat Rev Neurosci 2011; 12: 585–601. 42. Gonzalez SD, Xu C, Ramirez ME et al. Family-based association of an ANK3 haplotype with bipolar disorder in Latino populations. Transl Psychiatry 2013; 3: e265. 43. Dedman A, McQuillin A, Kandaswamy R et al. Sequenc- ing of the ANKYRIN 3 gene (ANK3) encoding ankyrin G in bipolar disorder reveals a non-conservative amino acid change in a short isoform of ankyrin G. Am J Med Genet B Neuropsychiatr Genet 2012; 159B: 328–335. 26. Tesli M, Koefoed P, Athanasiu L et al. Association analy- sis of ANK3 gene variants in nordic bipolar disorder and schizophrenia case–control samples. Am J Med Genet B Neuropsychiatr Genet 2011; 156B: 969–974. 44. Doyle GA, Lai AT, Chou AD et al. Re-sequencing of ankyrin 3 exon 48 and case–control association analysis of rare variants in bipolar disorder type I. Bipolar Disord 2012; 14: 809–821. 27. Franke B, Vasquez AA, Veltman JA et al. Genetic vari- ation in CACNA1C, a gene associated with bipolar dis- order, inﬂuences brainstem rather than gray matter volume in healthy individuals. Biol Psychiatry 2010; 68: 586–588. 45. Rueckert EH, Barker D, Ruderfer D et al. Cis-acting regu- lation of brain-speciﬁc ANK3 gene expression by a genetic variant associated with bipolar disorder. Mol Psychiatry 2013; 18: 922–929. 28. Perrier E, Pompei F, Ruberto G et al. Initial evidence for the role of CACNA1C on subcortical brain morphology in patients with bipolar disorder. Eur Psychiatry 2011; 26: 135–137. 46. Pandey A, Davis NA, White BC et al. Epistasis network centrality analysis yields pathway replication across two GWAS cohorts for bipolar disorder. Transl Psychiatry 2012; 2: e154. 29. Kempton MJ, Ruberto G, Vassos E et al. Eﬀects of the CACNA1C risk allele for bipolar disorder on cerebral gray matter volume in healthy individuals. Am J Psychiatry 2009; 166: 1413–1414. 47. Judy JT, Seifuddin F, Pirooznia M et al. Converging evi- dence for epistasis between ANK3 and potassium channel gene KCNQ2 in bipolar disorder. Front Genet 2013; 4: 87. 30. Tesli M, Egeland R, Sønderby IE et al. No evidence for association between bipolar disorder risk gene variants and brain structural phenotypes. J Aﬀect Disord 2013; 151: 291–297. 48. Linke J, Witt SH, King AV et al. References 1. Merikangas KR, Jin R, He JP et al. Prevalence and corre- lates of bipolar spectrum disorder in the world mental health survey initiative. Arch Gen Psychiatry 2011; 68: 241–251. 20. Smoller JW, Craddock N, Kendler K et al. Identiﬁcation of risk loci with shared eﬀects on ﬁve major psychiatric dis- orders: a genome-wide analysis. Lancet 2013; 381: 1371– 1379. 2. Bertelsen A. A Danish twin study of manic-depressive dis- orders. Br J Psychiatry 1987; 130: 330–351. 21. Group PGCBDW. Large-scale genome-wide association analysis of bipolar disorder identiﬁes a new susceptibility locus near ODZ4. Nat Genet 2011; 43: 977–983. 3. Barnett JH, Smoller JW. The genetics of bipolar disorder. Neurosci 2009; 164: 331–343. 22. Roussos P, Giakoumaki SG, Georgakopoulos A, Robakis NK, Bitsios P. The CACNA1C and ANK3 risk alleles impact on aﬀective personality traits and startle reactivity but not on cognition or gating in healthy males. Bipolar Disord 2011; 13: 250–259. 4. Kendler KS, Pedersen NL, Farahmand BY, Persson PG. The treated incidence of psychotic and aﬀective illness in twins compared with population expectation: a study in the Swedish Twin and Psychiatric Registries. Psychol Med 1996; 26: 1135–1144. 23. Jogia J, Ruberto G, Lelli-Chiesa G et al. The impact of the CACNA1C gene polymorphism on frontolimbic function in bipolar disorder. Mol Psychiatry 2011; 16: 1070–1071. 5. Kieseppa T, Partonen T, Haukka J, Kaprio J, Lonnqvist J. High concordance of bipolar I disorder in a nationwide sample of twins. Am J Psychiatry 2004; 161: 1814–1821. 24. Bhat S, Dao DT, Terrillion CE et al. CACNA1C (Cav1.2) in the pathophysiology of psychiatric disease. Prog Neuro- biol 2012; 99: 1–14. 6. McGuﬃn P, Rijsdijk F, Andrew M et al. The heritability of bipolar aﬀective disorder and the genetic relationship to 589 Supporting Information Additional Supporting Information may be found in the online version of this article: Additional Supporting Information may be found in the online version of this article: 64. Stephens RM, Schneider TD. Features of spliceosome evo- lution and function inferred from an analysis of the infor- mation at human splice sites. J Mol Biol 1992; 228: 1124– 1136. Figure S1. The introns and exons of the diﬀerent splice variants of the ANK3 gene are shown, along with the genomic regions of the gene that were analysed for variant selection. The loca- tions of the variants detected by sequencing are shown, as are the variants that were selected for genotyping in the full case control sample. Figure S1. The introns and exons of the diﬀerent splice variants of the ANK3 gene are shown, along with the genomic regions of the gene that were analysed for variant selection. The loca- tions of the variants detected by sequencing are shown, as are the variants that were selected for genotyping in the full case control sample. 65. Consortium TEP. The ENCODE (ENCyclopedia Of DNA Elements) Project. Science 2004; 306: 636–640. 66. Barrett JC, Fry B, Maller J, Daly MJ. Haploview: analysis and visualization of LD and haplotype maps. Bioinformat 2005; 21: 263–265. Figure S2. The introns and exons of the diﬀerent splice variants of the CACNA1C gene are shown, along with the genomic regions of the gene that were analysed for variant selection. The locations of the variants detected by sequencing are shown, as are the variants that were selected for genotyping in the full case–control sample. 67. Jia P, Wang L, Fanous AH et al. A bias-reducing pathway enrichment analysis of genome-wide association data con- ﬁrmed association of the MHC region with schizophrenia. J Med Genet 2012; 49: 96–103. 68. Heintzman ND, Stuart RK, Hon G et al. Distinct and pre- dictive chromatin signatures of transcriptional promoters and enhancers in the human genome. Nat Genet 2007; 39: 311–318. Table S1. Variants identiﬁed using the Knome VARIANTS software (Knome). VAF = variant allele(s) frequency. Table S1. Variants identiﬁed using the Knome VARIANTS software (Knome). VAF = variant allele(s) frequency. Table S2. Allele-speciﬁc primers designed using Primer Picker (KBiosciences) for genotyping. Table S2. Allele-speciﬁc primers designed using Primer Picker (KBiosciences) for genotyping. 69. Creyghton MP, Cheng AW, Welstead GG et al. Histone H3K27ac separates active from poised enhancers and pre- dicts developmental state. Bipolar variants in ANK3 and CACNA1C Bipolar variants in ANK3 and CACNA1C 60. Nanavati D, Austin DR, Catapano LA et al. The eﬀects of chronic treatment with mood stabilizers on the rat hippo- campal post-synaptic density proteome. J Neurochem 2011; 119: 617–629. 70. Rada-Iglesias A, Bajpai R, Swigut T et al. A unique chro- matin signature uncovers early developmental enhancers in humans. Nature 2011; 470: 279–283. 71. Curtis D. Consideration of plausible genetic architectures for schizophrenia and implications for analytic approaches in the era of next generation sequencing. Psychiatr Genet 2013; 23: 1–10. 61. Leussis MP, Berry-Scott EM, Saito M et al. The ANK3 bipolar disorder gene regulates psychiatric-related behav- iors that are modulated by lithium and stress. Biol Psychia- try 2013; 73: 683–690. 72. Clarke L, Zheng-Bradley X, Smith R et al. The 1000 Ge- nomes Project: data management and community access. Nat Methods 2012; 9: 459–462. 62. Spitzer RL, Endicott J, Robins E. Research diagnostic cri- teria: rationale and reliability. Arch Gen Psychiatry 1978; 35: 773–782. 63. Spitzer R, Endicott J. The Schedule for Aﬀective Disorder and Schizophrenia, Lifetime Version. New York, NY: New York State Psychiatric Institute, 1977. Fiorentino et al. Green EK, Hamshere M, Forty L et al. Replication of bipolar disorder susceptibility alleles and identiﬁcation of two novel genome-wide signiﬁcant associations in a new bipolar disorder case–control sample. Mol Psychiatry 2013; 18: 1302–1307. 56. Bora E, Yucel M, Pantelis C. Cognitive endophenotypes of bipolar disorder: a meta-analysis of neuropsychological deﬁcits in euthymic patients and their ﬁrst-degree relatives. J Aﬀect Disord 2009; 113: 1–20. 38. Gella A, Segura M, Durany N et al. Is Ankyrin a genetic risk factor for psychiatric phenotypes? BMC Psychiatry 2011; 11: 103. 57. Chandler RA, Wakeley J, Goodwin GM, Rogers RD. Altered risk-aversion and risk-seeking behavior in bipolar disorder. Biol Psychiatry 2009; 66: 840–846. 39. Kloiber S, Czamara D, Karbalai N et al. ANK3 and CACNA1C–missing genetic link for bipolar disorder and major depressive disorder in two German case–control samples. J Psychiatr Res 2012; 46: 973–979. y y 58. McKirdy J, Sussmann JE, Hall J et al. Set shifting and reversal learning in patients with bipolar disorder or schizophrenia. Psychol Med 2009; 39: 1289–1293. 59. McQuillin A, Rizig M, Gurling HM. A microarray gene expression study of the molecular pharmacology of lithium carbonate on mouse brain mRNA to understand the neu- robiology of mood stabilization and treatment of bipolar aﬀective disorder. Pharmacogenet Genomics 2007; 17: 605–617. 40. Takata A, Kim SH, Ozaki N et al. Association of ANK3 with bipolar disorder conﬁrmed in East Asia. Am J Med Genet B Neuropsychiatr Genet 2011; 156B: 312–315. 41. Lee MT, Chen CH, Lee CS et al. Genome-wide associa- tion study of bipolar I disorder in the Han Chinese popula- tion. Mol Psychiatry 2011; 16: 548–556. 590 Supporting Information Proc Natl Acad Sci U S A 2010; 107: 21931–21936. Table S3. Next Generation Sequencing Control information. Table S3. Next Generation Sequencing Control information. 591 591
https://openalex.org/W4309763475	https://www.repo.uni-hannover.de/bitstream/123456789/14019/1/Effect_of_low-temperature_precipitates.pdf	English	null	Effect of low-temperature precipitates on microstructure and pseudoelasticity of an Fe–Mn–Si-based shape memory alloy	Materials characterization	2,023	cc-by	10,084	Effect of low-temperature precipitates on microstructure and pseudoelasticity of an Fe–Mn–Si-based shape memory alloy Hesamodin Khodaverdi a, Maryam Mohri b,, Amir Sabet Ghorabaei a, Elyas Ghafoori b,c Mahmoud Nili-Ahmadabadi a, a School of Metallurgy and Materials Engineering, College of Engineering, University of Tehran, Tehran, Iran b Empa, Swiss Federal Laboratories for Materials Science and Technology, 8600 Dübendorf, Switzerland c Institute for Steel Construction, Faculty of Civil Engineering and Geodetic Science, Leibniz University Hannover, 30167 Hannover, Germany A R T I C L E I N F O Keywords: Fe–Mn–Si-based shape memory alloy Superelasticity Precipitation Equilibrium phase diagram Transmission electron microscopy Fe–Mn–Si-based shape memory alloys (Fe-SMAs) have attracted much research attention due to their potential applications for vibration mitigation, energy dissipation, and re-centering in the construction sector. Because of the crucial impact of precipitation on the pseudoelasticity (PE) behavior of Fe-SMAs, the equilibrium phase diagram of an Fe–17Mn–5Si–10Cr–4Ni–1(V-C) (wt%) SMA was used in this study to identify a low-temperature precipitate and study its effect on the microstructure and PE of the alloy after a low-temperature aging process. Transmission electron microscopy (TEM) studies revealed that aging at 485 ◦C for 6 h after aging at 750 ◦C for 6 h led to the precipitation of fresh, parallelogram-shaped, (Cr–V–C)-rich precipitates along with elliptical-shaped, V-rich precipitates in the austenite grains of the recrystallized samples. Numerous parallel stacking faults (SFs) were formed due to the presence of the precipitates within the austenite grains. It is postulated that such an arrangement of SFs can further improve the PE by reducing the activation energy for the nucleation of ε-martensite laths and inhibiting them from colliding with each other and consequent formation of α'-martensite, resulting in a residual strain reduction to 2.7% after 4.0% tensile straining. A combination of the reversible motion of Shockley partial disloca tions (SPDs) and back transformation from ε-martensite (with hexagonal close-packed (HCP) lattice structure) to γ-austenite (with face-centered cubic (FCC) lattice structure) in unloading is believed to be the micro structural reason for the PE effect in Fe–Mn–Si-based SMAs [19]. Several attempts have been made to enhance the PE of Fe–Mn–Si-based SMAs, such as controlling the grain size [20,21], texturizing, and precipitation [22] in combination with stacking fault energy (SFE) control [23]. Among the different approaches, SFE control and precipitation have had a greater impact on PE improvement. Precipitates such as Cr23C6 [24], TiC [25], VN [26], VC [27], and NbC [28] are used for PE improvement. * Corresponding authors. E-mail addresses: maryam.mohri@empa.ch (M. Mohri), nili@ut.ac.ir (M. Nili-Ahmadabadi). * Corresponding authors. E-mail addresses: maryam Available online 17 November 2022 1044-5803/© 2022 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). https://doi.org/10.1016/j.matchar.2022.112486 Received 26 August 2022; Received in revised form 10 November 2022; Accepted 12 November 2022 Materials Characterization 195 (2023) 112486 Materials Characterization 195 (2023) 112486 Available online 17 November 2022 1044-5803/© 2022 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). * Corresponding authors. E-mail addresses: maryam.mohri@empa.ch (M. Mohri), nili@ut.ac.ir (M. Nili-Ahmadabadi). https://doi.org/10.1016/j.matchar.2022.112486 Received 26 August 2022; Received in revised form 10 November 2022; Accepted 12 November 2022 1. Introduction Fe–Mn–Si-based shape memory alloys (SMAs), as one of the most practical categories of Fe-based SMAs, have attracted much research attention [1–4]. Due to their low material cost, high Young's modulus and strength, exceptional workability, and high stress and strain re covery [5,6], these alloys have been used for a wide range of applica tions such as coupling devices [7], mechanical tightening, structural elements, active controls, pre-stressing or post-tensioning of structures [8], and damping devices [9]. However, low pseudoelasticity (PE) has prevented them from being used in many applications such as dissipa tive re-centering systems in civil engineering [10]. It has been revealed that NbC carbides increase the strength of the γ-austenite phase and assist in the reversible movement of the γ/ε in terfaces through a specific crystallographic path. The back-stress on the growing ε-martensite lath due to the stress fields induced by the NbC carbides causes this back transformation [28,29]. In addition, VC pre cipitates promote the formation of ε-martensite by reducing the critical resolved shear stress (CRSS) of austenite [19]. Precipitates also increase A new design for Fe-based SMAs, Fe–17Mn–5Si–10Cr–4Ni–1(V-C) (wt%), has been developed in Swiss Federal Laboratories for Materials Science and Technology (Empa) [11–15]. The alloy can be produced by a relatively cheap casting process under atmospheric conditions [16] and has high potential for large-scale repair and strengthening in the construction section [17] and passive vibration damping [18]. Materials Characterization 195 (2023) 112486 H. Khodaverdi et al. was considered to analyze Kα radiation of the alloying elements. The EDS spectra were quantified by the TIA software based on the Cliff- Lorimer method [37] with k-factors calculated with respect to silicon. A maximum relative uncertainty value of ~5% was detected for the reported chemical compositions. Samples for the OM and SEM obser vations were mechanically polished and etched with a solution of H2O2 (35%), HNO3 (65%), and HCl (32%) (7:30:9). The HR-TEM specimens were prepared by standard mechanical thinning followed by electro polishing in a solution of HClO4 and C2H5OH (1:9) at −20 ◦C and 22 V. the number of dislocations and stacking faults (SFs) in the γ-austenite matrix, which act as embryos for ε-martensite nucleation [30–34]. It has recently been found that having a fine-grained austenite matrix along with uniform distribution of precipitates facilitates the γ ⇌ ε trans formation and enhances the PE behavior of the alloy [35]. 1. Introduction In a recent study on the newly designed Fe–Mn–Si-based SMA [36], it has been shown that (Cr–V–C)-rich precipitates uniformly form inside the austenite grains by high-temperature aging at 750 ◦C, resulting in a significant improvement of PE. To recognize any new low-temperature precipitates, it is essential to investigate the equilibrium phase dia gram of the alloy. In the present work, the effect of low-temperature aging at 485 ◦C on the evolution of a new type of precipitate is stud ied, and its combination with the primary high-temperature precipitates is employed to further enhance the PE of the alloy. Low-temperature precipitates, which may improve shape memory and superelasticity properties, are highly desirable because lowering the aging temperature decreases manufacturing costs and associated problems like oxidation and grain growth. It is shown that after 4.0% loading in tension, a re sidual strain of 2.7% can be achieved. The underlying mechanisms of the observed improvement in PE after low-temperature aging at 485 ◦C are studied by detailed microstructural characterizations, which provide new insights into the processing of this alloy for vibration mitigation and seismic damping applications. To evaluate the mechanical properties of the heat-treated samples, monotonic loading–unloading experiments were performed with a Zwick/Roell Z020 tensile test machine. During the loading–unloading tests, the strain evolution was measured with a calibrated sensor-arm extensometer type BTC-EXMULTI.011 made by Zwick/Roell Company. Longitudinal tensile specimens were machined from the samples and prepared in a dog-bone shape with a reduced section length of 32.0 mm and a cross-section of 1.0 × 0.8 mm2 (Fig. 1). Furthermore, to study the microstructural evolution of the samples after loading–unloading tests, sub-size tensile specimens with a gauge length of 25.00 mm and a width of 6.25 mm were prepared according to the ASTM E8M-04 standard. Tensile loading to a maximum strain of 4% was carried out at room temperature with a crosshead speed of 0.5 mm/min followed by unloading with the same crosshead speed until a force of 10 N was attained. 3. Results Rebars with a diameter of 18 mm and a nominal composition of Fe–17Mn–5Si–10Cr–4Ni–1(V-C) (wt%), provided by re-fer AG, Switzerland, were used in this study. The material was produced by hot rolling at 1000 ◦C after casting. The microstructure of the as-received rebars consisted of equiaxed austenite grains and fine precipitates at the grain boundaries [35]. The as-received alloy was cold–caliber-rolled to an octagonal-shaped speciemen with equal sides of 14.2 mm. The cold rolling was performed in 16 consecutive steps to reach an equivalent strain of 0.25 at ambient temperature, using a caliber rolling machine with a roller diameter of 110 mm and a rolling speed of 800 mm/min. The cold-rolled specimens were recrystallized in a vacuum furnace at 925 ◦C for 50 min followed by air cooling to room temperature [35]. High-temperature aging was performed on the recrystallized specimens at 750 ◦C for 6 h in a muffle furnace followed by air cooling to ambient tempearture. Subsequently, a set of the aged specimens was double aged at a relatively lower temperature of 485 ◦C for 6 h and then cooled down to the room temperature under atmospheric conditions. The recrystal lized, single aged, and double aged specimens are hereafter named as Rex, Rex-Aged, and Rex-Double aged, respectively. 3.1. Equilibrium phase diagram of Fe–17Mn–5Si–10Cr–4Ni–1(V-C) (wt %) alloy Fig. 2 shows the equilibrium phase diagram of the studied alloy calculated by using Thermo-Calc software with TCFE7 database [38]. The liquid phase is thermodynamically stable at high temperatures down to approximately 1320 ◦C, where it starts to solidify to form the γ-austenite phase in a temperature range of about 95 ◦C until 1225 ◦C. Further cooling to 1000 ◦C promotes the formation of M7C3 carbide via a precipitation reaction of γ → γ + M7C3. Subsequently, the Sigma1 phase is formed by a precipitation reaction of γ → γ + Sigma1 at 665 ◦C, and its molar fraction reaches a maximum of ~0.15 at 485 ◦C (inset of Fig. 2). Additionally, the thermodynamic calculations predict that the Sigma1 phase transforms into the Sigma2 and A2 phases below 485 ◦C. In the present study, the possible precipitation of the Sigma1 phase and its relation to PE evolution were investigated through double aging of the Rex-Aged samples at 485 ◦C. 3.2. Microstructure development after high- and low-temperature aging Dilatometry analysis with a cooling/heating rate of 1 ◦C/s was conducted on cylindrical samples with a length of 10 mm and a diameter of up to 2 mm in an Adamel DT1000 dilatometer. A schematic view of the heat treatment cycles and the optical microstructures of the as- received material before and after the cold rolling are provided in the Supplementary Material File (see Fig. S1). Figs. 3a–c show the FE-SEM micrographs of the Rex (a), Rex-Aged (b), and Rex-Double aged (c) samples. The microstructure of the Rex sample mainly consisted of recrystallized austenite grains with an average grain size of 5 μm and reversed austenite regions with a diam eter of approximately 1 μm. Since grain boundary junctions act as preferential nucleation sites for the martensitic transformation, most strain-induced martensite forms in these locations [39]. Therefore, the reversed austenite grains usually appeared in the triple junctions of recrystallized austenite grains (Fig. 3a). Such formation of reversed The volume fractions of phase constituents were measured using X- ray diffraction (XRD) analysis with a Cu Kα radiation source (λ = 0.154 nm) at an acceleration voltage of 45 kV and a tube current of 200 mA. The microstructures of the specimens were studied using optical mi croscopy (OM; ZEISS Axioskop 2 MAT), secondary electron analysis by field-emission scanning electron microscopy (FE-SEM; FEI Nova Nano SEM 450), and high-resolution transmission electron microscopy (HR- TEM; FEI Tecnai F20 series). The chemical compositions of precipitates were analyzed using energy-dispersive X-ray spectroscopy (EDS; Oxford X-MaxN 80 T silicon drift detector with a take-off angle of 14.6◦and a resolution of 134 eV FWHM at reference energy of 5.9 keV) by the TEM. Standard samples of pure elements were used for calibrating the TEM imaging and analysis (TIA) interface of the microscope software. Moreover, the multi-polynomial background correction for the X-ray signals along with thin foil thickness correction for the TEM specimens Fig. 1. Geometry of the dog-bone specimens used for the tensile tests (di mensions in mm). Fig. 1. Geometry of the dog-bone specimens used for the tensile tests (di mensions in mm). 2 Materials Characterization 195 (2023) 112486 H. Khodaverdi et al. H. Khodaverdi et al. Fig. 2. Calculated equilibrium phase diagram of the studied Fe–17Mn–5Si–10Cr–4Ni–1(V-C) (wt%) alloy at 1 atm pressure by the Thermo- Calc software. (Fig. 3c). 3.2. Microstructure development after high- and low-temperature aging In addition, the number of (Cr–V–C)-rich precipitates (poly hedral-shaped) was reduced, but some new needle-shaped precipitates formed inside the austenite grains. Similar to the Rex and Rex-Aged samples, the grain size of recrystallized austenite for the Rex-Double aged sample did not change, but the grain size of reversed austenite slightly decreased (Fig. 3c). Fig. 3d illustrates the XRD diffractograms of the specimens from which the volume fractions of α'-martensite and ε-martensite were calculated according to the method provided in Ref. [41]. The Rex sample contained 14 vol% ε-martensite and 7.9 vol% α'-martensite, while the Rex-Aged sample possessed a much higher volume fraction of ε-martensite of about 43% and, interestingly, had almost zero volume fraction of α'-martensite. The disappearance of α'-martensite and the increase in the volume fraction of ε-martensite in the Rex-Aged spec imen could be related to the formation of precipitates, which can change the chemical composition of the austenite matrix and inhibit the for mation of randomly oriented ε-martensite laths inside the austenite grains [42]. Furthermore, the XRD diffractogram of the Rex-Aged sam ple showed peak splitting (indicated by the small arrow in Fig. 3d), which can be related to the presence of precipitates, SFs, and ε-martensite that reduce the symmetry of the microstructure [43]. In comparison to the Rex-Aged sample, the volume fraction of ε-martensite reduced to nearly 16% and the degree of peak splitting decreased for the Rex-Double aged sample. Fig. 2. Calculated equilibrium phase diagram of the studied Fe–17Mn–5Si–10Cr–4Ni–1(V-C) (wt%) alloy at 1 atm pressure by the Thermo- Calc software. austenite at the triple junctions of recrystallized austenite grains during the annealing of cold-rolled austenite has also been reported elsewhere [39,40]. Moreover, there were some constituents with sizes of less than 800 nm located inside the austenite grains and also at the grain boundaries of austenite, which were (Cr–V–C)-rich precipitates ac cording to our previous findings [35]. In comparison to the Rex sample, the Rex-Aged sample showed a sharp increase in the number of pre cipitates with a higher volume fraction inside the austenite grains than at the grain boundaries. The average austenite grain size of the Rex sample remained close to 5 μm after aging (Fig. 3b). After low- temperature aging of the Rex-Aged sample at 485 ◦C for 6 h, the vol ume fraction and continuity of reversed austenite regions decreased 3.4. TEM study on the microstructures after aging at high and low temperatures Figs. 5a–b show TEM images from the substructure of the Rex sam ple. Particles with sizes between 15 and 20 nm were uniformly distrib uted in the austenite matrix (white arrows in Fig. 5a). It has recently been indicated that these particles are rich in vanadium and can be considered as embryos for the V-rich precipitates [35]. Additionally, a number of parallel SFs (blue arrows in Figs. 5a–b) could be observed in the microstructure of the Rex sample. The TEM images of the Rex-Aged sample are shown in Figs. 5c–d, indicating the formation of a higher number of SFs and ε-martensite laths after aging in comparison to the Rex sample. The TEM-EDS analysis showed that the polyhedral-shaped precipitates were rich in Cr, V and C elements (Fig. 5c). These parti cles with approximately similar chemical compositions but with spher ical morphologies and relatively smaller sizes were also present in the substructure of the Rex sample (white arrows in Fig. 5b). The details of TEM-EDS analyses are provided in the Supplementary Material File (Figs. S2–S9). A dark-field TEM image obtained from the [0110] reflection of ε-martensite is shown in the inset of Fig. 5d. It was observed that ε-martensite laths generally grew parallel to each other in separate groups in the samples. Fig. 4. Loading–unloading curves of the As-Received aged [35], Rex, Rex- Aged, and Rex-Double aged samples strained to 4% in tension. Figs. 6a–d illustrate TEM images from the substructure of the Rex- Double aged sample. Fig. 6a shows an austenite grain with a large number of SFs that are all located in the same orientation inside the grain. Such a parallel orientation of SFs, but with a lower degree of orientation, was also detected in the TEM micrographs of the Rex and Rex-Aged samples (Fig. 5). In contrast to the other samples, parallelogram-shaped precipitates were observed in addition to polyhedral-shaped precipitates in the microstructure of the Rex-Double aged sample (Figs. 6b–c). As shown in the inset of Fig. 6c, the parallelogram-shaped precipitates indicated a range of 170–330 nm in length. It seemed that these precipitates were the same as the needle- shaped precipitates observed in the FE-SEM image (Fig. 3c), which a portion of their cross-section could be seen in the TEM images (Figs. 6b–c). Furthermore, ε-martensite laths with a limited number of variants were observed in the Rex-Double aged sample (Fig. 6b). 3.4. TEM study on the microstructures after aging at high and low temperatures An HR- TEM image from a section of one of the ε-martensite laths with a width of nearly 2.2 nm is shown in Fig. 6d. By applying the fast Fourier transform (FFT) and then the inverse FFT (IFFT) to a portion of the image (dashed white square), the displacement of atomic planes for the formation of ε-martensite from γ-austenite was revealed as shown with red lines in the inset of Fig. 6d. calculated by using Hooke's law, the calculated values corresponded to mixed microstructures of austenite, ε-martensite, and α’-martensite with different volume fractions of the constituents. Furthermore, it has been shown that mainly the stress-induced martensitic transformation occurs until the stress level reaches the yielding point of this alloy [44]. Therefore, the mixture of phases is changed by increasing (decreasing) the strain in the elastic part, which justifies the nonlinear deformation behavior during loading (unloading). The PE strain was calculated ac cording to Eq. (1): calculated by using Hooke's law, the calculated values corresponded to mixed microstructures of austenite, ε-martensite, and α’-martensite with different volume fractions of the constituents. Furthermore, it has been shown that mainly the stress-induced martensitic transformation occurs until the stress level reaches the yielding point of this alloy [44]. Therefore, the mixture of phases is changed by increasing (decreasing) the strain in the elastic part, which justifies the nonlinear deformation behavior during loading (unloading). The PE strain was calculated ac cording to Eq. (1): εtotal = εres + εpse + εE = εres + εpse + σ4% Eunloading (1) (1) where εres, εpse, and εE are the residual strain, PE strain, and elastic strain, respectively. σ4% denotes the strength at the peak tensile strain, and Eunloading is the elastic modulus in unloading. g The Rex sample exhibited an elastic modulus of 160 GPa in loading which decreased to 105 and 80 GPa for the Rex-Aged and Rex-Double aged samples, respectively. The value of σY0.1% was 440 MPa for the Rex sample, while it slightly increased and reached 475 MPa for the Rex- Aged sample. After performing the aging heat treatment at 485 ◦C for 6 h, the σY0.1% increased to 525 MPa for the Rex-Double aged sample. Similarly, the σ4% increased from 670 MPa for the Rex sample to 737 and 750 MPa for the Rex-Aged and Rex-Double aged samples, respectively. 3.4. TEM study on the microstructures after aging at high and low temperatures Furthermore, in comparison to the Rex sample with a residual strain of 3.09%, the Rex-Aged and Rex-Double aged samples showed relatively lower residual strains of 2.87% and 2.70%, respectively. Such behavior could be mainly related to the higher σ4% values, lower Young's moduli and higher PE strains of the aged samples compared with those of the Rex sample (Table 1 and Eq. (1)). It is worth mentioning that the ob tained residual strain of 2.70% for the Rex-Double aged samples after 4% loading in tension is the lowest residual strain value reported so far for this particular alloy. Figs. 7a–b show TEM micrographs from the substructure of the Rex- Double aged sample after performing a loading–unloading test to a peak strain of 2% in tension. Unlike the substructures of the non-deformed samples (Figs. 5 and 6), parallel SFs crossed each other after applying the deformation (blue arrows in Fig. 7a). Moreover, the crossing SFs were trapped by the polyhedral-shaped, (Cr–V–C)-rich precipitates, resulting in the formation of highly distorted areas around the pre cipitates in the austenite matrix (yellow arrows in Fig. 7a). Fig. 7b in dicates sparse V-rich precipitates, ranging from 70 to115 nm in size, inside a variant of annealing twins in the austenite phase. No SFs were observed in the twinned crystals that could tangle around the elliptical- shaped, V-rich precipitates, compared with the polyhedral-shaped 3.3. Loading–unloading experiments The engineering stress-strain curves after loading–unloading tests for the As-Received aged [35] and the heat-treated samples are shown in Fig. 4. The values of PE and other tensile properties of the samples inferred from Fig. 4 are summarized in Table 1. For this particular alloy, the 0.1% offset yield stress (σY0.1%) was considered for estimating the onset stress of the martensitic transformation [44]. The elastic modulus of this alloy can vary between 40 and 170 GPa under different micro structural conditions [45]. Since the elastic moduli of the samples were 3 Fig. 3. SEM micrograph of the (a) Rex, (b) Rex-Aged, and (c) Rex-Double aged samples. (d) XRD diffractograms of the specimens. D EM micrograph of the (a) Rex, (b) Rex-Aged, and (c) Rex-Double aged samples. (d) XRD diffractograms of the specimens. Materials Characterization 195 (2023) 112486 H. Khodaverdi et al. Fig. 4. Loading–unloading curves of the As-Received aged [35], Rex, Rex- Aged, and Rex-Double aged samples strained to 4% in tension. Table 1 Table 1 Mechanical characteristics of the As-Received aged [35], Rex, Rex-Aged, and Rex-Double aged samples after loading–unloading experiments with a peak strain of 4% in tension. εres: residual strain, εpse: PE strain, εE: elastic strain, σY0.1%: 0.1% offset yield stress, σ4%: strength at peak tensile strain, Eloading: elastic modulus in loading, E l ti d l i l di Mechanical characteristics of the As-Received aged [35], Rex, Rex-Aged, and Rex-Double aged samples after loading–unloading experiments with a peak strain of 4% in tension. εres: residual strain, εpse: PE strain, εE: elastic strain, σY0.1%: 0.1% offset yield stress, σ4%: strength at peak tensile strain, Eloading: elastic modulus in loading, Eunloading: elastic modulus in unloading. Sample name E loading (GPa) σY0.1% (Mpa) σ4% (Mpa) Eunloading (GPa) εE (%) εpse (%) εres (%) As-Received aged [35] 160 275 600 130 0.46 0.54 3 Rex 160 440 670 135 0.50 0.41 3.09 Rex-Aged 105 475 737 115 0.64 0.49 2.87 Rex-Double aged 80 525 750 140 0.53 0.77 2.70 unloading g Sample name E loading (GPa) σY0.1% (Mpa) σ4% (Mpa) Eunloading (GPa) εE (%) εpse (%) εres (%) As-Received aged [35] 160 275 600 130 0.46 0.54 3 Rex 160 440 670 135 0.50 0.41 3.09 Rex-Aged 105 475 737 115 0.64 0.49 2.87 Rex-Double aged 80 525 750 140 0.53 0.77 2.70 Materials Characterization 195 (2023) 112486 H. Khodaverdi et al. a–b) Bright-field TEM images of the Rex sample illustrating SFs and precipitates in the austenite matrix. (c) Bright-field TEM image of SFs and (Cr–V–C)-rich tes in the Rex-Aged sample. (d) Bright-field and dark-field TEM images from ε-martensite laths in the Rex-Aged sample; the [0110] reflection of ε-martensite d for dark-field imaging as shown in the corresponding selected area electron diffraction pattern. Fig. 5. (a–b) Bright-field TEM images of the Rex sample illustrating SFs and precipitates in the austenite matrix. (c) Bright-field TEM image of SFs and (Cr–V–C)-rich precipitates in the Rex-Aged sample. (d) Bright-field and dark-field TEM images from ε-martensite laths in the Rex-Aged sample; the [0110] reflection of ε-martensite was used for dark-field imaging as shown in the corresponding selected area electron diffraction pattern. accomplished by forming SFs on (111) planes of the FCC crystal in every two layers at pre-existing SFs or as newly developing SFs [47,48]. Applying external stress will generate a dislocation loop two layers away from the existing SFs to produce a four-layer ε-martensite plate. Table 1 When the thickness of the ε-martensite plate reaches a threshold value, a new martensite plate will then be formed at a certain distance to the first martensite plate to relax the lattice strain of the initially formed martensite plate. Therefore, the SFs are considered as embryos for the nucleation of ε-martensite [49,50]. It has also been reported that the essential factor for obtaining good PE behavior in Fe-based SMAs is the propensity to produce very thin ε-martensite plates by applying external stress [51]. Having a high density of SFs in the austenite grains is necessary to form extremely thin and single-variant ε-martensite plates with a uniform distribution within each grain of the austenite phase. Given the fact that α'-martensite, which weakens the PE behavior of Fe- based SMAs, can nucleate at the intersection of two ε-martensite laths [52], the formation of SFs in random orientations should be minimized in the microstructure. precipitates in the non-twinned regions. It was realized that these V-rich precipitates evolved from the fine particles observed in the Rex sample (Fig. 5a) such that their size increased at the expense of the reduction in their volume fraction by double aging the sample (Figs. 6a and 7b). Table 2 lists the reported chemical compositions of observed precipitates in several FeMnSi-SMAs subjected to different heat treatment conditions and the average chemical compositions of observed precipitates in this study. 4. Discussion (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) show elliptical-shaped particles with a size of 15–20 nm in the micro structure of the Rex sample (Fig. 5a). Stanford et al. [53] have shown that fine particles with a size of less than 30 nm can be counterpro ductive to achieving superior PE behavior by pinning stress-induced ε-martensite and inhibiting its reversion to γ-austenite. M7C3 carbides in the microstructure. By comparing these data with the precipitation behavior of the studied alloy at three different tempera tures of 925, 750, and 485 ◦C (Figs. 5 and 6), the polyhedral-shaped and parallelogram-shaped precipitates in the Rex-Double aged sample could be related to M7C3-type and sigma-type precipitates in the phase dia gram, respectively. Although there are some studies that support these arguments [55,56], the crystallography of the parallelogram-shaped precipitates with the average chemical composition of Fe–14Mn–5Si–26Cr–3Ni–6V–12C (wt%) needs to be investigated in detail in the future. The εres value of the Rex-Aged sample (2.87%) shows about 7.1% improvement over that of the Rex sample (3.09%). This enhancement is mainly related to the more formation of (Cr–V–C)-rich precipitates in side the austenite grains with the same austenite grain size (5 μm) compared with the Rex sample. These polyhedral-shaped precipitates, which create lattice strains (Fig. 5c), are uniformly distributed in the austenite matrix (Fig. 3b), leading to the formation of a significant number of SFs accompanied by a high fraction (43 vol%) of ε-martensite (Figs. 5c–d). The high number of SFs not only facilitates the γ → ε martensitic transformation but also results in the significant reduction of Young's modulus compared with the Rex sample (Table 1). The reduc tion in Young's modulus is related to the change in the interatomic bonding configuration at the SFs [54]. The observed improvement of εpse in the Rex-Double aged sample is thought to be partly caused by the formation of the new precipitates, which can induce the development of a higher number of SFs grouped in the same orientation inside the austenite grains compared with the other samples (Figs. 6a–b). The generation of a high number of precipitate/ matrix interfaces in the double-aged sample can play a role in the for mation of new SFs in the microstructure. 4. Discussion Several studies have investigated the mechanism of PE in Fe–Mn–Si- based SMAs. In the first attempts, Sawaguchi et al. [46] justified the PE behavior by the reverse transformation from ε-martensite to γ-austenite in unloading caused by the back-stress experienced by SPDs residing at the top of the ε-martensite plates. Leinenbach et al. [19] later showed that the microstructural reason for PE is a combination of the back transformation from ε-martensite to γ-austenite and reversible motions of SPDs. In low SFE materials, a dislocation typically can dissociate into two extended dislocations, creating an SF bounded by SPDs. Therefore, SFs play an important role in the PE response of the studied Fe–Mn–Si- based SMA with an SFE value of 5.75 mJ/m2 [35]. Although the microstructure of the Rex specimen contains relatively equiaxed austenite grains with a small grain size, the tensile test does not show a significant PE response for this specimen (Figs. 3 and 4). The presence of 7.9 vol% α'-martensite in the microstructure is one of the reasons for this inferior PE behavior. Furthermore, the TEM images Generally, the nucleation of ε-martensite in γ-austenite can be 5 5 Materials Characterization 195 (2023) 112486 H. Khodaverdi et al. Fig. 6. Bright-field TEM images of the Rex-Double aged sample illustrating (a) a high number of parallel SFs in an austenite grain, (b) polyhedral- and parallelogram- shaped precipitates along with parallel SFs and ε-martensite laths in the austenite matrix, and (c) the distribution of parallelogram-shaped precipitates in the austenite matrix. (d) HR-TEM image and corresponding FFT and IFFT images of an ε-martensite lath formed parallel to the {111} planes of austenite. Selected lattice spots for producing the IFFT image are shown by the red circles in the FFT pattern. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) Fig. 6. Bright-field TEM images of the Rex-Double aged sample illustrating (a) a high number of parallel SFs in an austenite grain, (b) polyhedral- and parallelogram- shaped precipitates along with parallel SFs and ε-martensite laths in the austenite matrix, and (c) the distribution of parallelogram-shaped precipitates in the austenite matrix. (d) HR-TEM image and corresponding FFT and IFFT images of an ε-martensite lath formed parallel to the {111} planes of austenite. Selected lattice spots for producing the IFFT image are shown by the red circles in the FFT pattern. Table 2 Fe–30Mn–6Si–5Cr Hot-rolled GB-phase Fe–29.5Mn–5.9Si–6.3Cr [70] Fe–15Mn–7Si–9Cr–5Ni Aged at 900 ◦C for 1 h Intermetallic Fe–17.5Mn–13.5Si–11.2Cr–7.6Ni [71] Fe–15Mn–6Si–9Cr–5Ni–1.5Ti–0.16C Aged at 850 ◦C for 0.5 h Rod-like Fe–16Mn–8.5Si–10Cr–4Ni [72] Fe–(13–27)Mn–5.5Si–8.5Cr–5Ni As-cast Sigma-phase Fe–17.7Mn–8.1Si–15.8Cr–3Ni [73] Chi-phase Fe–31.5Mn–15.8Si–7.8Cr–10.6Ni Lathy-phase Fe–12.5Mn–7Si–12.1Cr–3Ni Fe–17Mn–5Si–10Cr–4Ni–1(V-C) Aged at 750 ◦C for 6 h Polyhedral-shaped Fe–14Mn–6Si–30Cr–2Ni–6V–8C * Fe–17Mn–5Si–10Cr–4Ni–1(V-C) Aged at 485 ◦C for 6 h Parallelogram-shaped Fe–14Mn–5Si–26Cr–3Ni–6V–12C * Fe–17Mn–5Si–10Cr–4Ni–1(V-C) Aged at 485 ◦C for 6 h Elliptical-shaped Fe–7Mn–10Cr–2Ni–50V–0.2Si −0.2C * aging can also stem from the segregation of solute atoms at the newly generated dislocations during aging, resulting in the local lowering of the SFE at the dislocations and causing the dislocations to dissociate to nucleate new SFs [30]. The formation of SFs in one orientation inhibits the development of new SFs in random orientations and reduces the chance of the intersection of SFs with each other. Furthermore, the in crease in the size of the pre-existed V-rich particles from less than 30 nm (15–20 nm) to about 70–115 nm can have a synergistic effect on the PE improvement of the Rex-Double aged sample compared with the Rex and Rex-Aged samples. aging can also stem from the segregation of solute atoms at the newly generated dislocations during aging, resulting in the local lowering of the SFE at the dislocations and causing the dislocations to dissociate to nucleate new SFs [30]. The formation of SFs in one orientation inhibits the development of new SFs in random orientations and reduces the chance of the intersection of SFs with each other. Furthermore, the in crease in the size of the pre-existed V-rich particles from less than 30 nm (15–20 nm) to about 70–115 nm can have a synergistic effect on the PE improvement of the Rex-Double aged sample compared with the Rex and Rex-Aged samples. In the Rex-Aged sample with fewer oriented SFs, in comparison with the Rex-Double aged sample, multiple martensite variants form in different orientations within each austenite grain (Fig. 5d). However, due to the presence of enough large precipitates (70–330 nm) along with the higher number of oriented SFs in the Rex-Double aged sample, ε-martensite laths almost form in one orientation within each austenite grain (Fig. 6b). 4. Discussion It has been shown that dislo cations form at the precipitate/matrix interface to accommodate the elastic strain fields (represented in Figs. 5c and 6b) accompanied by the formation of the precipitate [35,57]. Consequently, the SFs can be formed by the dissociation of the dislocations associated with the pre cipitate/matrix interface [58]. In addition, the formation of SFs after i In comparison to the εpse value of the Rex-Aged sample, an increase of 57% in εpse is observed for the Rex-Double aged sample, resulting in a higher PE strain of 0.77%. According to the equilibrium thermodynamic data at 485 ◦C (Fig. 2), sigma-type precipitates can form along with 6 Materials Characterization 195 (2023) 112486 H. Khodaverdi et al. Fig. 7. Bright-field TEM images of the Rex-Double aged sample after a loading–unloading experiment to a peak strain of 2% in tension. (a) Groups of parallel SFs crossing each other (blue arrows) around a (Cr–V–C)-rich precipitate in non-twinned regions of the austenite matrix, creating distorted zones around the precipitate (yellow arrows). (b) Three elliptical-shaped, V-rich precipitates (inset) in a variant of annealing twins inside the austenite matrix with no observation of SFs around them. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) Fig. 7. Bright-field TEM images of the Rex-Double aged sample after a loading–unloading experiment to a peak strain of 2% in tension. (a) Groups of parallel SFs crossing each other (blue arrows) around a (Cr–V–C)-rich precipitate in non-twinned regions of the austenite matrix, creating distorted zones around the precipitate (yellow arrows). (b) Three elliptical-shaped, V-rich precipitates (inset) in a variant of annealing twins inside the austenite matrix with no observation of SFs around them. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) Table 2 aging can also stem from the segregation of solute atoms at the newly generated dislocations during aging, resulting in the local lowering of the SFE at the dislocations and causing the dislocations to dissociate to nucleate new SFs [30]. The formation of SFs in one orientation inhibits the development of new SFs in random orientations and reduces the chance of the intersection of SFs with each other. Furthermore, the in crease in the size of the pre-existed V-rich particles from less than 30 nm (15–20 nm) to about 70–115 nm can have a synergistic effect on the PE improvement of the Rex-Double aged sample compared with the Rex and Rex-Aged samples. The shape and morphology of the precipitates are determined by the interplay between the elastic strains and interfacial energy minimization during precipitation. The elastic energy is dependent on the elastic stiffness, misfit strain, precipitate size, and applied stress, and the interfacial energy is dependent on imbalanced atomic forces in the precipitate/matrix interface [57,59]. As shown in Fig. 6b, the SFs form around the polyhedral-shaped, M7C3-type precipitate, and after applying tensile strain they are multiplied in different orientations and trapped by the precipitate (Fig. 7a). However, the stress fields around the parallelogram-shaped, sigma-type precipitate can to some extent prevent the SFs from colliding with the precipitate (Fig. 6b), resulting in a more oriented distribution of the SFs in the austenite matrix that may reduce the probability of their trapping by the precipitate during the deformation. It should be noted that possible local chemical fluctuations in the matrix near the precipitates and their effects on local SFE varia tions may also influence the formation of SFs, which require further investigations in the future. In the Rex-Aged sample with fewer oriented SFs, in comparison with the Rex-Double aged sample, multiple martensite variants form in different orientations within each austenite grain (Fig. 5d). However, due to the presence of enough large precipitates (70–330 nm) along with the higher number of oriented SFs in the Rex-Double aged sample, ε-martensite laths almost form in one orientation within each austenite grain (Fig. 6b). As a result, a relatively lower volume fraction of ther mally formed ε-martensite develops in the Rex-Double aged sample (16%) compared with that of the Rex-Aged sample (43%), which may be beneficial for PE improvement because the chance of intersection be tween thermally formed and stress-induced formed ε-martensite is reduced. Table 2 To investigate the role of martensite-start (MS) and austenite-start (AS) temperatures of the γ ⇌ ε phase transformation in the evolution of thermally formed ε-martensite, dilatometry tests were performed on the heat-treated samples. As shown in Fig. 8, the measured MS tem peratures for the Rex, Rex-Aged, and Rex-Double aged samples are 240, 255, and 260 ◦C, respectively, which are well above the room temper ature. The increases in the MS values after aging can be related to the formation of the precipitates and the resultant changes in the chemical composition of the austenite matrix near the precipitates and also to the lattice strains generated during the evolution of the precipitates [60–62]. Moreover, the measured AS temperatures for the Rex, Rex- Aged, and Rex-Double aged samples are 155, 140, and 115 ◦C, respec tively, which are lower than the corresponding MS temperatures. These results can indicate the facilitation of martensite-to-austenite trans formation by precipitation; obstacles such as precipitates can increase Table 2 Chemical compositions of various kinds of precipitates formed under different heat treatment conditions in several FeMnSi-SMAs and average chemical compositions of precipitates observed in this study (). Alloy composition (wt%) Heat treatment Comments Precipitate composition (wt%) Ref. Fe–30Mn–6Si–5Cr Hot-rolled GB-phase Fe–29.5Mn–5.9Si–6.3Cr [70] Fe–15Mn–7Si–9Cr–5Ni Aged at 900 ◦C for 1 h Intermetallic Fe–17.5Mn–13.5Si–11.2Cr–7.6Ni [71] Fe–15Mn–6Si–9Cr–5Ni–1.5Ti–0.16C Aged at 850 ◦C for 0.5 h Rod-like Fe–16Mn–8.5Si–10Cr–4Ni [72] Fe–(13–27)Mn–5.5Si–8.5Cr–5Ni As-cast Sigma-phase Fe–17.7Mn–8.1Si–15.8Cr–3Ni [73] Chi-phase Fe–31.5Mn–15.8Si–7.8Cr–10.6Ni Lathy-phase Fe–12.5Mn–7Si–12.1Cr–3Ni Fe–17Mn–5Si–10Cr–4Ni–1(V-C) Aged at 750 ◦C for 6 h Polyhedral-shaped Fe–14Mn–6Si–30Cr–2Ni–6V–8C Fe–17Mn–5Si–10Cr–4Ni–1(V-C) Aged at 485 ◦C for 6 h Parallelogram-shaped Fe–14Mn–5Si–26Cr–3Ni–6V–12C * Fe–17Mn–5Si–10Cr–4Ni–1(V-C) Aged at 485 ◦C for 6 h Elliptical-shaped Fe–7Mn–10Cr–2Ni–50V–0.2Si −0.2C * nder different heat treatment conditions in several FeMnSi-SMAs and average chemical compositions Chemical compositions of various kinds of precipitates formed under different heat treatment conditions in several FeMnSi-SMAs and average chemical compositions of precipitates observed in this study (*). Alloy composition (wt%) Heat treatment Comments Precipitate composition (wt%) Ref. Table 2 As a result, a relatively lower volume fraction of ther mally formed ε-martensite develops in the Rex-Double aged sample (16%) compared with that of the Rex-Aged sample (43%), which may be beneficial for PE improvement because the chance of intersection be tween thermally formed and stress-induced formed ε-martensite is reduced. The shape and morphology of the precipitates are determined by the interplay between the elastic strains and interfacial energy minimization during precipitation. The elastic energy is dependent on the elastic stiffness, misfit strain, precipitate size, and applied stress, and the interfacial energy is dependent on imbalanced atomic forces in the precipitate/matrix interface [57,59]. As shown in Fig. 6b, the SFs form around the polyhedral-shaped, M7C3-type precipitate, and after applying tensile strain they are multiplied in different orientations and trapped by the precipitate (Fig. 7a). However, the stress fields around the parallelogram-shaped, sigma-type precipitate can to some extent prevent the SFs from colliding with the precipitate (Fig. 6b), resulting in a more oriented distribution of the SFs in the austenite matrix that may reduce the probability of their trapping by the precipitate during the deformation. It should be noted that possible local chemical fluctuations in the matrix near the precipitates and their effects on local SFE varia tions may also influence the formation of SFs, which require further investigations in the future. To investigate the role of martensite-start (MS) and austenite-start (AS) temperatures of the γ ⇌ ε phase transformation in the evolution of thermally formed ε-martensite, dilatometry tests were performed on the heat-treated samples. As shown in Fig. 8, the measured MS tem peratures for the Rex, Rex-Aged, and Rex-Double aged samples are 240, 255, and 260 ◦C, respectively, which are well above the room temper ature. The increases in the MS values after aging can be related to the formation of the precipitates and the resultant changes in the chemical composition of the austenite matrix near the precipitates and also to the lattice strains generated during the evolution of the precipitates [60–62]. Moreover, the measured AS temperatures for the Rex, Rex- Aged, and Rex-Double aged samples are 155, 140, and 115 ◦C, respec tively, which are lower than the corresponding MS temperatures. These results can indicate the facilitation of martensite-to-austenite trans formation by precipitation; obstacles such as precipitates can increase 7 Materials Characterization 195 (2023) 112486 H. Khodaverdi et al. Fig. 8. Table 2 Dilatation curves versus temperature at the cooling/heating rate of 1 ◦C/s for the Rex (C1 and H1), Rex-Aged (C2 and H2), and Rex-Double aged (C3 and H3) samples. The samples were first cooled from the heat treatment temperature and then heated to measure the γ ⇌ ε phase transformation temperatures. can be drawn: 1. The Rex specimen with a γ-austenite grain size of 5 μm has the lowest measured PE strain of 0.41% among the other specimens. Although the microstructure of the Rex specimen contains fine, equiaxed austenite grains, the presence of 7.9 vol% α'-martensite along with small V-rich particles with a size of 15–20 nm reduces the PE effect. 2. A significant improvement of approximately 20% in the PE strain is achieved by aging the Rex specimen at 750 ◦C for 6 h. This enhancement of PE for the Rex-Aged specimen is mainly due to the precipitation of polyhedral-shaped, (Cr–V–C)-rich precipitates (M7C3-type) with a uniform distribution inside the austenite grains, which causes the formation of a large number of stacking faults (SFs) and ε-martensite laths in the microstructure. 3. The highest measured PE strain of 0.77% for this alloy is attained by performing double aging heat treatment on the Rex-Aged specimen at 485 ◦C for 6 h. The formation of fresh, parallelogram-shaped, (Cr–V–C)-rich precipitates (sigma-type) with the average chemical composition of Fe–14Mn–5Si–26Cr–3Ni–6V–12C (wt%) and an in crease in the size of pre-existed V-rich particles to 70–115 nm with the chemical composition of Fe–7Mn–10Cr–2Ni–50V–0.2Si–0.2C (wt %) are believed to be responsible for the PE improvement in the Rex- Double aged specimen. Fig. 8. Dilatation curves versus temperature at the cooling/heating rate of 1 ◦C/s for the Rex (C1 and H1), Rex-Aged (C2 and H2), and Rex-Double aged (C3 and H3) samples. The samples were first cooled from the heat treatment temperature and then heated to measure the γ ⇌ ε phase transformation temperatures. 4. The formation of sigma-type precipitates in the microstructure of the Rex-Double aged specimen causes a large number of SFs to be ori ented in the same direction inside the austenite grains. It is postu lated that the parallel alignment of these SFs may further improve the γ ⇌ ε martensitic phase transformation by reducing the proba bility of the collision of ε-martensite laths with each other and consequent formation of α’-martensite during tensile loa ding–unloading experiments. Table 2 This results in a residual strain of 2.7% after 4% loading in tension for the Rex-Double aged specimen, which shows about 13% improvement compared with that of the Rex sample. 4. The formation of sigma-type precipitates in the microstructure of the Rex-Double aged specimen causes a large number of SFs to be ori ented in the same direction inside the austenite grains. It is postu lated that the parallel alignment of these SFs may further improve the γ ⇌ ε martensitic phase transformation by reducing the proba bility of the collision of ε-martensite laths with each other and consequent formation of α’-martensite during tensile loa ding–unloading experiments. This results in a residual strain of 2.7% after 4% loading in tension for the Rex-Double aged specimen, which shows about 13% improvement compared with that of the Rex sample. the elastic energy storage during the γ → ε phase transformation, assisting the reverse transformation [63–66]. These observations sug gest that the type of precipitates and the orientation of SFs are the decisive factors in determining the volume fraction of thermally formed ε-martensite in the microstructures. Jian et al. [67] have shown that during applying strain, more SFs would form on a specific set of {111} γ planes and combine with the pre- existing SFs on these planes. The accumulation and extension of SFs on the same plane lead to the coalescence of individual SFs, generating longer SFs. The long SFs tend to be spaced regularly with the increase in the applied strain (Fig. 7a). When the spacing between adjacent long SFs reaches twice the spacing between adjacent {111} γ planes, the local regions will transform into ε-martensite. Similar to the role of pre cipitates, having more oriented groups of SFs may also assist in the formation of ε-martensite laths just on a specific set of {111} γ planes, thereby reducing the possibility of the intersection of ε-martensite var iants with each other and consequent formation of α'-martensite, which is detrimental to PE. Additionally, the activation energy required for the γ → ε martensitic transformation is reduced by forming a high number of SFs in the matrix, resulting in better PE behavior [19]. These conditions can be met in the Rex-Double aged sample due to the formation of the sigma-type precipitates alongside the M7C3-type precipitates in the austenite matrix compared with the Rex-Aged sample (Figs. 6 and 7). Acknowledgment The authors acknowledge the support from re-fer AG, Switzerland, for providing the material for this research study. Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Data availability The raw/processed data required to reproduce the findings of this study are available from the corresponding authors upon reasonable request. The acquired EDS spectra used for calculating the chemical compositions of the precipitates are provided in the Supplementary Material File. Another factor that improves the PE of the aged samples is the absence of coarse annealing twins in the microstructures. To obtain a good PE response, the formation of annealing twins should be sup pressed in Fe–Mn–Si-based SMAs [21,68,69]. The interactions between annealing twins and stress-induced ε-martensite not only distort the twin boundaries but also significantly inhibit the γ → ε martensitic transformation. The annealing twins may also prevent the formation of thermally induced ε-martensite because a rise in the fraction of the twins lowers the MS temperature [69]. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi. org/10.1016/j.matchar.2022.112486. [1] H. Koohdar, M. Nili-Ahmadabadi, F. Javadzadeh Kalahroudi, H.R. Jafarian, T. G. Langdon, The effect of high-pressure torsion on the microstructure and outstanding pseudoelasticity of a ternary Fe–Ni–Mn shape memory alloy, Mater. Sci. Eng. A 802 (2021), https://doi.org/10.1016/j.msea.2020.140647. [2] M. Vollmer, C. Segel, P. Krooß, J. Günther, L. Tseng, On the effect of gamma phase formation on the pseudoelastic performance of polycrystalline Fe–Mn–Al–Ni shape [1] H. Koohdar, M. Nili-Ahmadabadi, F. Javadzadeh Kalahroudi, H.R. Jafarian, T. G. Langdon, The effect of high-pressure torsion on the microstructure and outstanding pseudoelasticity of a ternary Fe–Ni–Mn shape memory alloy, Mater. Sci. Eng. A 802 (2021), https://doi.org/10.1016/j.msea.2020.140647. [2] M. Vollmer, C. Segel, P. Krooß, J. Günther, L. Tseng, On the effect of gamma phase formation on the pseudoelastic performance of polycrystalline Fe–Mn–Al–Ni shape References 15 (1967) 157–159, https://doi.org/ 10.1016/0001-6160(67)90170-8. p g j [8] W.J. Lee, B. Weber, C. Leinenbach, Recovery stress formation in a restrained Fe–Mn–Si-based shape memory alloy used for prestressing or mechanical joining, Constr. Build. Mater. 95 (2015) 600–610, https://doi.org/10.1016/J. CONBUILDMAT.2015.07.098. [33] K. Kamei, Y. Maehara, Y. Ohmori, Effect of stacking fault precipitation on hot deformation of austenitic stainless steel, Trans. Iron Steel Inst. Jpn. 26 (1986) 159–166, https://doi.org/10.2355/ISIJINTERNATIONAL1966.26.159. [9] L. Janke, C. Czaderski, M. Motavalli, J. Ruth, Applications of shape memory alloys in civil engineering structures—overview, limits and new ideas, Mater. Struct. 38 (5) (2005) 578–592, https://doi.org/10.1007/BF02479550. p g [34] J.J. Irani, R.T. Weiner, Precipitation of vanadium carbide on stacking faults, Nature 205 (4973) (1965) 795, https://doi.org/10.1038/205795a0. [10] H. Otsuka, K. Nakajima, T. Maruyama, Superelastic behavior of Fe–Mn–Si–Cr shape memory alloy coil, Mater. Trans. 41 (2000) 547–549, https://doi.org/ 10.2320/MATERTRANS1989.41.547. [35] H. Khodaverdi, M. Mohri, E. Ghafoori, A. Sabet Ghorabaei, M. Nili-Ahmadabadi, Enhanced pseudoelasticity of an Fe–Mn–Si-based shape memory alloy by applying microstructural engineering through recrystallization and precipitation, J. Mater. Res. Technol. 21C (2022) 2999–3013, https://doi.org/10.1016/j. jmrt.2022.10.092. [11] Z. Dong, U.E. Klotz, C. Leinenbach, A. Bergamini, C. Czaderski, M. Motavalli, A novel Fe-Mn-Si shape memory alloy with improved shape recovery properties by VC precipitation, Adv. Eng. Mater. 11 (2009) 40–44, https://doi.org/10.1002/ adem.200800312. [36] M. Mohri, I. Ferretto, C. Leinenbach, D. Kim, D.G. Lignos, E. Ghafoori, Effect of thermomechanical treatment and microstructure on pseudo-elastic behavior of Fe–Mn–Si–Cr–Ni-(V, C) shape memory alloy, Mater. Sci. Eng. A 855 (2022), 143917, https://doi.org/10.1016/J.MSEA.2022.143917. [12] W. Wang, A. Hosseini, E. Ghafoori, Experimental study on Fe-SMA-to-steel adhesively bonded interfaces using DIC, Eng. Fract. Mech. 244 (2021), 107553, https://doi.org/10.1016/J.ENGFRACMECH.2021.107553. g [37] G. Cliff, G.W. Lorimer, The quantitative analysis of thin specimens, J. Microsc. 103 (1975) 203–207, https://doi.org/10.1111/j.1365-2818.1975.tb03895.x. p g [13] E. Ghafoori, M. Neuenschwander, M. Shahverdi, C. Czaderski, M. Fontana, Elevated temperature behavior of an iron-based shape memory alloy used for prestressed strengthening of civil structures, Constr. Build. Mater. 211 (2019) 437–452, https://doi.org/10.1016/J.CONBUILDMAT.2019.03.098. [ ] , , [39] T. Song, B.C. de Cooman, Martensite nucleation at grain boundaries containing intrinsic grain boundary dislocations, ISIJ Int. 54 (2014) 2394–2403, https://doi. org/10.2355/ISIJINTERNATIONAL.54.2394. [14] D.I.H. Rosa, A. Hartloper, A. de Castro e Sousa, D.G. Lignos, M. Motavalli, E. Ghafoori, Experimental behavior of iron-based shape memory alloys under cyclic loading histories, Constr. Build. Mater. 272 (2021), 121712, https://doi.org/ 10.1016/J.CONBUILDMAT.2020.121712. [40] M.J. Sohrabi, M. Naghizadeh, H. References The microstructure and pseudoelasticity (PE) of an Fe–17Mn–5Si–10Cr–4Ni–1(V-C) (wt%), Fe-based shape memory alloy have been studied under recrystallized (Rex), recrystallized and aged (Rex-Aged), and recrystallized and double aged (Rex-Double aged) conditions. According to experimental investigations and detailed microstructural and mechanical assessments, the following conclusions 8 H. Khodaverdi et al. Materials Characterization 195 (2023) 112486 memory alloys, Scr. Mater. 108 (2015) 23–26, https://doi.org/10.1016/j. scriptamat.2015.06.013. [27] L. Chengxin, W. Guixin, W. Yandong, L. Qingsuo, Z. Jianjun, Effect of addition of V and C on strain recovery characteristics in Fe–Mn–Si alloy, Mater. Sci. Eng. A 438–440 (2006) 808–811, https://doi.org/10.1016/J.MSEA.2006.01.098. memory alloys, Scr. Mater. 108 (2015) 23–26, https://doi.org/10.1016/j. scriptamat.2015.06.013. [3] A. Bauer, M. Vollmer, T. Niendorf, Effect of crystallographic orientation and grain boundaries on martensitic transformation and Superelastic response of Oligocrystalline Fe–Mn–Al–Ni shape memory alloys, Shape Mem. Superelast. 7 (2021) 373–382, https://doi.org/10.1007/S40830-021-00340-3. [28] S. Kajiwara, D. Liu, T. Kikuchi, N. Shinya, Remarkable improvement of shape memory effect in Fe-Mn-Si based shape memory alloys by producing NbC precipitates, Scr. Mater. 44 (2001) 2809–2814, https://doi.org/10.1016/S1359- 6462(01)00978-2. [4] Z. Zhang, J. Zhang, H. Wu, Y. Ji, D. Kumar, Iron-based shape memory alloys in construction: research, applications and opportunities, Materials. 1723 (2022), https://doi.org/10.3390/ma15051723. [29] A. Baruj, T. Kikuchi, S. Kajiwara, N. Shinya, Improvement of shape memory properties of NbC containing Fe–Mn–Si based shape memory alloys by simple thermomechanical treatments, Mater. Sci. Eng. A 378 (2004) 333–336, https://doi. org/10.1016/j.msea.2003.10.357. p g [5] X.L. Gu, Z.Y. Chen, Q.Q. Yu, E. Ghafoori, Stress recovery behavior of an Fe-Mn-Si shape memory alloy, Eng. Struct. 243 (2021), 112710, https://doi.org/10.1016/J. ENGSTRUCT.2021.112710. [30] M.J. Lai, Y.J. Li, L. Lillpopp, D. Ponge, S. Will, D. Raabe, On the origin of the improvement of shape memory effect by precipitating VC in Fe–Mn–Si-based shape memory alloys, Acta Mater. 155 (2018) 222–235, https://doi.org/10.1016/j. actamat.2018.06.008. [6] H. Peng, G. Wang, S. Wang, J. Chen, I. MacLaren, Y. Wen, Key criterion for achieving giant recovery strains in polycrystalline Fe-Mn-Si based shape memory alloys, Mater. Sci. Eng. A 712 (2018) 37–49, https://doi.org/10.1016/j. msea.2017.11.071. [31] J. van Aswegen, R.D.W. Honeycombe, Precipitation on stacking faults in Cr-Ni austenitic steels, Acta Metall. 12 (1965) 1–13, https://doi.org/10.1016/0001-6160 (64)90048-3. [7] A.V. Druker, A. Perotti, I. Esquivel, J. Malarría, A manufacturing process for shaft and pipe couplings of Fe–Mn–Si–Ni–Cr shape memory alloys, Mater. Des. 56 (2014) 878–888, https://doi.org/10.1016/j.matdes.2013.11.032. [32] F.H. Froes, B.W.K. Honeycombe, D.H. Warrington, Conditions controlling matrix and stacking fault precipitation, Acta Metall. References Mirzadeh, Deformation-induced martensite in austenitic stainless steels: a review, Arch. Civil Mech. Eng. 20 (2020) 1–24, https:// doi.org/10.1007/s43452-020-00130-1. [15] E. Hosseini, E. Ghafoori, C. Leinenbach, M. Motavalli, S.R. Holdsworth, Stress recovery and cyclic behaviour of an Fe–Mn–Si shape memory alloy after multiple thermal activation, Smart Mater. Struct. 27 (2018), 025009, https://doi.org/ 10.1088/1361-665X/AAA2C9. l g [41] J.H. Robertson, IUCr, Elements of X-ray Diffraction by B. D. Cullity, Urn:Issn:0567- 7394 35, 1979, p. 350, https://doi.org/10.1107/S0567739479000917, 1-509. [42] M.M. Pan, X.M. Zhang, D. Zhou, R.D.K. Misra, P. Chen, Fe–Mn–Si–Cr–Ni based shape memory alloy: thermal and stress-induced martensite, Mater. Sci. Eng. A 797 (2020), https://doi.org/10.1016/j.msea.2020.140107. [16] C. Leinenbach, H. Kramer, C. Bernhard, D. Eifler, Thermo-mechanical properties of an Fe–Mn–Si–Cr–Ni–VC shape memory alloy with low transformation temperature, Adv. Eng. Mater. 14 (2012) 62–67, https://doi.org/10.1002/adem.201100129. [43] G. Nolze, PowderCell: A Mixture between Crystal Structure Visualizer, Simulation and Refinement Tool, International School on Powder Diffraction, 2002. [17] J. Vůjtˇech, P. Ryj´aˇcek, J.C. Matos, E. Ghafoori, Iron-based shape memory alloy for strengthening of 113-year bridge, Eng. Struct. 248 (2021), 113231, https://doi. org/10.1016/j.engstruct.2021.113231. i [44] W.J. Lee, B. Weber, G. Feltrin, C. Czaderski, M. Motavalli, C. Leinenbach, Phase transformation behavior under uniaxial deformation of an Fe–Mn–Si–Cr–Ni–VC shape memory alloy, Mater. Sci. Eng. A 581 (2013) 1–7, https://doi.org/10.1016/ j.msea.2013.06.002. g j g [18] R. Fosdick, Y. Ketema, Shape memory alloys for passive vibration damping, J. Intell. Mater. Syst. Struct. 9 (1998) 854–870, https://doi.org/10.1177/ 1045389X9800901009. [45] M. Beßling, C. Czaderski, J. Orlowsky, Prestressing effect of shape memory alloy reinforcements under serviceability tensile loads, Buildings 11 (2021) (2021) 101, https://doi.org/10.3390/buildings11030101. [19] C. Leinenbach, A. Arabi-Hashemi, W.J. Lee, A. Lis, M. Sadegh-Ahmadi, S. van Petegem, T. Panzner, H. van Swygenhoven, Characterization of the deformation and phase transformation behavior of VC-free and VC-containing FeMnSi-based shape memory alloys by in situ neutron diffraction, Mater. Sci. Eng. A 703 (2017) 314–323, https://doi.org/10.1016/j.msea.2017.07.077. p g g [46] T. Sawaguchi, T. Kikuchi, S. Kajiwara, The pseudoelastic behavior of Fe–Mn–Si- based shape memory alloys containing Nb and C, Smart Mater. Struct. 14 (2005) S317, https://doi.org/10.1088/0964-1726/14/5/022. [47] N. Bergeon, G. Guenin, C. Esnouf, Microstructural analysis of the stress-induced ε martensite in a Fe–Mn–Si–Cr–Ni shape memory alloy: part I—calculated description of the microstructure, Mater. Sci. Eng. A 242 (1998) 77–86, https:// doi.org/10.1016/S0921-5093(97)00511-X. p g j [20] T. Omori, M. Okano, R. Kainuma, Effect of grain size on superelasticity in Fe-Mn- Al-Ni shape memory alloy wire, APL Mater. 1 (2013), https://doi.org/10.1063/ 1.4820429. References doi.org/10.1016/S0921-5093(97)00511-X. [21] G. Wang, H. Peng, C. Zhang, S. Wang, Y. Wen, Relationship among grain size, annealing twins and shape memory effect in Fe–Mn–Si based shape memory alloys, Smart Mater. Struct. 25 (2016), 075013, https://doi.org/10.1088/0964-1726/25/ 7/075013. [48] N. Bergeon, G. Guenin, C. Esnouf, Microstructural analysis of the stress-induced ε martensite in a Fe–Mn–Si–Cr–Ni shape memory alloy: part II: transformation reversibility, Mater. Sci. Eng. A 242 (1998) 87–95, https://doi.org/10.1016/ S0921-5093(97)00512-1. [49] E. Gartstein, A. Rabinkin, On the f.c.c. → h.c.p. phase transformation in high manganese-iron alloys, Acta Metall. 27 (1979) 1053–1064, https://doi.org/ 10.1016/0001-6160(79)90193-7. l [22] A. Arabi-Hashemi, W.J. Lee, C. Leinenbach, Recovery stress formation in FeMnSi based shape memory alloys: impact of precipitates, texture and grain size, Mater. Des. 139 (2018) 258–268, https://doi.org/10.1016/j.matdes.2017.11.006. [50] H. Li, D. Dunne, N. Kennon, Factors influencing shape memory effect and phase transformation behaviour of Fe–Mn–Si based shape memory alloys, Mater. Sci. Eng. A 273–275 (1999) 517–523, https://doi.org/10.1016/S0921-5093(99)00391- 3. g j [23] O. Matsumura, T. Sumi, N. Tamura, K. Sakao, T. Furukawa, H. Otsuka, Pseudoelasticity in an Fe–28Mn–6Si–5Cr shape memory alloy, Mater. Sci. Eng. A 279 (2000) 201–206, https://doi.org/10.1016/S0921-5093(99)00644-9. [24] Y.H. Wen, L.R. Xiong, N. Li, W. Zhang, Remarkable improvement of shape memory effect in an Fe–Mn–Si–Cr–Ni–C alloy through controlling precipitation direction of Cr23C6, Mater. Sci. Eng. A 474 (2008) 60–63, https://doi.org/10.1016/J. MSEA.2007.05.043. [51] S. Kajiwara, Characteristic features of shape memory effect and related transformation behavior in Fe-based alloys, Mater. Sci. Eng. A 273 (1999) 67–88, https://doi.org/10.1016/S0921-5093(99)00290-7. [25] N. Stanford, D.P. Dunne, Effect of NbC and TiC precipitation on shape memory in an iron-based alloy, J. Mater. Sci. 41 (2006) 4883–4891, https://doi.org/10.1007/ S10853-006-0050-7. [52] L. Bracke, L. Kestens, J. Penning, Transformation mechanism of α’-martensite in an austenitic Fe–Mn–C–N alloy, Scr. Mater. 57 (2007) 385–388, https://doi.org/ 10.1016/j.scriptamat.2007.05.003. [26] K. Li, Z. Dong, Y. Liu, L. Zhang, A newly developed Fe-based shape memory alloy suitable for smart civil engineering, Smart Mater. Struct. 22 (2013), 045002, https://doi.org/10.1088/0964-1726/22/4/045002. j p [53] N. Stanford, D.P. Dunne, Effect of second-phase particles on shape memory in Fe–Mn–Si-based alloys, Mater. Sci. Eng. A 454 (2007) 407–415, https://doi.org/ 10.1016/j.msea.2006.11.084. 9 9 H. Khodaverdi et al. Materials Characterization 195 (2023) 112486 [64] D.P. Dunne, C.M. Wayman, The effect of austenite ordering on the martensite transformation in Fe-Pt alloys near the composition Fe3Pt: II. Crystallography and general features, Metall. Trans. A. 4 (1) (1973) 147–152, https://doi.org/10.1007/ BF02649613. [54] Y. Chen, T. Burgess, X. An, Y.-W. Mai, H.H. References Tan, J. Zou, S.P. Ringer, C. Jagadish, X. Liao, Effect of a high density of stacking faults on the Young’s modulus of GaAs nanowires, Nano Lett. 16 (2016) 1911–1916, https://doi.org/10.1021/acs. nanolett.5b05095. [65] E. Acar, G.P. Toker, H. Kurkcu, H.E. Karaca, High temperature shape memory behavior of Ni47.3Ti29.7Hf20Pd3 alloys, Intermetallics (Barking) 111 (2019), 106518, https://doi.org/10.1016/J.INTERMET.2019.106518. [55] A.I. Gorunov, Investigation of M7C3, M23C6 and M3C carbides synthesized on austenitic stainless steel and carbon fibers using laser metal deposition, Surf. Coat. Technol. 401 (2020), 126294, https://doi.org/10.1016/J. SURFCOAT 2020 126294 [66] E. Acar, M. Kok, I.N. Qader, Exploring surface oxidation behavior of NiTi–V alloys, Eur. Phys. J. Plus 135 (1) (2020) 1–9, https://doi.org/10.1140/EPJP/S13360-019- 00087-Y. [56] F. Ernst, D. Li, H. Kahn, G.M. Michal, A.H. Heuer, The carbide M7C3 in low- temperature-carburized austenitic stainless steel, Acta Mater. 59 (2011) 2268–2276, https://doi.org/10.1016/J.ACTAMAT.2010.11.058. [67] L. Jian, C.M. Wayman, On the mechanism of the shape memory effect associated with γ(fcc) to ε(hcp) martensitic transformations in Fe-Mn-Si based alloys, Scr. Metall. Mater. 27 (1992) 279–284, https://doi.org/10.1016/0956-716X(92) 90512-D. [57] R. Shi, N. Ma, Y. Wang, Predicting equilibrium shape of precipitates as function of coherency state, Acta Mater. 60 (2012) 4172–4184, https://doi.org/10.1016/J. ACTAMAT.2012.04.019. [58] J.R. Patel, K.A. Jackson, H. Reiss, Oxygen precipitation and stacking-fault formation in dislocation-free silicon, J. Appl. Phys. 48 (2008) 5279, https://doi. org/10.1063/1.323558. [68] H. Peng, G. Wang, Y. Du, S. Wang, J. Chen, Y. Wen, A novel training-free processed Fe-Mn-Si-Cr-Ni shape memory alloy undergoing δ → γ phase transformation, Metall. Mater. Trans. A Phys. Metall. Mater. Sci. 47 (2016) 3277–3283, https://doi. org/10.1007/S11661-016-3521-8. g [59] A.M. Jokisaari, S.S. Naghavi, C. Wolverton, P.W. Voorhees, O.G. Heinonen, Predicting the morphologies of γ’ precipitates in cobalt-based superalloys, Acta Mater. 141 (2017) 273–284, https://doi.org/10.1016/J.ACTAMAT.2017.09.003. l [69] Y.H. Wen, H.B. Peng, D. Raabe, I. Gutierrez-Urrutia, J. Chen, Y.Y. Du, Large recovery strain in Fe-Mn-Si-based shape memory steels obtained by engineering annealing twin boundaries, Nat. Commun. 5 (1) (2014) 1–9, https://doi.org/ 10.1038/ncomms5964. [60] D.L. Rittermann, A. Kyrolainen, P.J. Ferreira, Influence of annealing treatment on the formation of nano/submicron grain size AISI 301 Austenitic stainless steels, Metall. Mater. Trans. A 37 (8) (2006) 2325–2338, https://doi.org/10.1007/ BF02586207. [70] H. Lin, C. Lin, K. Lin, Y. Chuang, An investigation of grain-boundary phase in Fe–30Mn–6Si–5Cr shape memory alloy, J. Alloys Compd. 27 (2001) 279–284. [61] A. Sato, K. Soma, T. Mori, Hardening due to pre-existing ϵ-Martensite in an Fe- 30Mn-1Si alloy single crystal, Acta Metall. References 30 (1982) 1901–1907, https://doi.org/ 10.1016/0001-6160(82)90030-X. [71] B. Maji, M. Krishnan, The effect of microstructure on the shape recovery of a Fe–Mn–Si–Cr–Ni stainless steel shape memory alloy, Scr. Mater. 48 (2003) 71–77, https://doi.org/10.1016/S1359-6462(02)00348-2. l p g [72] D. Bu, H. Peng, Y. Wen, N. Li, Influence of ageing on wear resistance of an Fe–Mn–Si–Cr–Ni–Ti–C shape memory alloy, Mater. Des. 32 (2011) 2969–2973, https://doi.org/10.1016/j.msea.2017.11.071. [62] V. Kokorin, L. Kozlova, A. Titenko, Temperature hysteresis of martensite transformation in aging Cu–Mn–Al alloy, Scr. Mater. 47 (2002) 499–502, https:// doi.org/10.1016/S1359-6462(02)00136-7. [63] D.P. Dunne, C.M. Wayman, The effect of austenite ordering on the martensite transformation in Fe-Pt alloys near the composition Fe3Pt: I. Morphology and transformation characteristics, Metall. Trans. A. 4 (1) (1973) 137–145, https://doi. org/10.1007/BF02649612. [73] H. Peng, Y. Wen, Y. Du, Q. Yu, Q. Yang, Effect of manganese on microstructures and solidification modes of cast Fe-Mn-Si-Cr-Ni shape memory alloys, Metall. Mater. Trans. B Process Metall. Mater. Process. Sci. 44 (2013) 1137–1143, https:// doi.org/10.1007/S11663-013-9880-2. 10
https://openalex.org/W4318671056	https://frontiersinzoology.biomedcentral.com/counter/pdf/10.1186/s12983-023-00486-z	English	null	What coloration brings: Implications of background adaptation to oxidative stress in anurans	Frontiers in zoology	2,023	cc-by	7,899	Frontiers in Zoology Frontiers in Zoology Radovanović et al. Frontiers in Zoology (2023) 20:6 https://doi.org/10.1186/s12983-023-00486-z Open Access Abstract Background Urban development results in habitat destruction, affecting populations of amphibians, the most frag- ile group of vertebrates. With changes in the environment, these animals become more exposed to light and preda- tors. To enhance their chances of survival, they display plasticity of body coloration. Aside from adaptive benefits, animals exhibiting background matching meet the energetic costs and restrictions of changing body tones. To study the physiological consequences of Hyla arborea tadpole adaptation to background color, we followed oxidative stress parameters after rearing larvae on a constant background (black/white) and after changing the background color. Results Larvae cultivated for 20 days on constant substrate color exhibited differences in body coloration but with- out differences in lipid peroxidation (LPO) concentration between dark and pale individuals, suggesting that colora- tion investment during this period did not induce higher oxidative damage in darker tadpoles. Prolonged exposure of larvae (37 days) to a dark habitat increased antioxidative system defense and LPO concentrations, compared to animals reared permanently in the white surroundings. The positive correlation of oxidative damage with color intensity of individuals points to the physiological consequences of higher investment in the number of pigment cells necessary for dark pigmentation. In individuals faced with non-matching background and change in body coloration, defense system declined and LPO occurred relative to individuals cultivated in white habitat. Conclusion Here, we have pointed to consequences related to background matching and stress that amphibians experienced during chromatic adaptations. Background color change causes a complex physiological response affecting the antioxidative defense parameters. This investigation elucidates the accompanying cost of amphibiansʼ adjustment to an altered environment. Keywords Amphibian larvae, Background color change, Adaptive plasticity, Physiological cost, Oxidative damage, Antioxidant system Background Natural habitats are greatly altered by anthropogenic activities, which lead to increased pollution and cli- mate change. Urbanization and industrialization result in accelerated habitat loss. The outcome can be veg- etation fragmentation or the development of gaps in a formerly contiguous habitat. Changes in forest cover near aquatic areas could be quite important for local amphibians. This process of vegetation loss increases Correspondence: Tijana B. Radovanović tijana@ibiss.bg.ac.rs 1 Department of Physiology, Institute for Biological Research “Siniša Stanković”, National Institute of the Republic of Serbia, University of Belgrade, Bulevar despota Stefana 142, Belgrade 11060, Serbia 2 Department of Evolutionary Biology, Institute for Biological Research “Siniša Stanković”, National Institute of the Republic of Serbia, University of Belgrade, Bulevar despota Stefana 142, Belgrade 11060, Serbia © The Author(s) 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativeco mmons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. What coloration brings: Implications of background adaptation to oxidative stress in anurans Tijana B. Radovanović1, Tamara G. Petrović1, Branka R. Gavrilović1, Svetlana G. Despotović1, Jelena P. Gavrić1, Ana Kijanović2, Marko Mirč2, Nataša Tomašević Kolarov2, Tanja Vukov2 and Marko D. Prokić1 The strong preference for certain backgrounds indicates that individuals try to avoid unnecessary color change, consequently reducing the involved energy costs [12, 15]. Depending on the species, alterations in body color can develop in different time ranges, from a couple of seconds to over a month [6, 7]. Physiological color change occurs in a shorter time interval of a few seconds, minutes or hours and is based on the dispersion and clustering of pigment inside cells. Changes that happen over days and weeks, due to the production and reorganization of pig- ment cells in the dermis, are frequently called morpho- logical or quantitative [7, 8] and are usually linked to expected, long-term variations in the environment [6]. According to [9, 10] prolonged background adaptation (lasting for several days) leads to prolonged physiological color change, which precedes the morphological changes of color. Even though changes in body color induced by changes in the environment (background matching) are associ- ated with an increased metabolic rate, oxidative phos- phorylation and, consequently, increased production of reactive oxygen species (ROS) [5], how a change in body color affects the physiology and antioxidant defense in anurans has not been examined. ROS disrupt the struc- ture and function of macromolecules, cause tissue injury and have a detrimental influence on overall fitness. Therefore, the antioxidant protective apparatus must limit and neutralize their prooxidant activity and convert ROS into less harmful molecules [18].h For amphibians, cryptic coloration can be the initial defense line and a form of antipredator adaptation [11]. By adapting to the color of the substrate and their envi- ronment, amphibians become less visible to predators, thereby increasing their fitness and survival chances. The proportion of this adaptive plasticity during ontogenesis is age-related as adult individuals exhibit lower plastic- ity than juveniles [6, 12]. The process of color change in amphibians is enabled by three types of dermal chroma- tophores (xanthophores, iridophores, and melanophores) [13]. When individuals are exposed to a different back- ground color, a reaction-relocation of pigment organelles (melanosomes) in the dermal melanophores occurs. On a light background, pigment aggregation in melanophores results in skin lightening, while a dark background leads to pigment dispersion and darkening of the dorsal skin. Chromatophores modify their dimensions and density when chromatic adjustment lasts a week or more [14]. This color change is a reversible process in amphibians. © The Author(s) 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativeco mmons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Radovanović et al. Frontiers in Zoology (2023) 20:6 Page 2 of 12 Radovanović et al. Frontiers in Zoology be affected by adrenaline, noradrenaline, progesterone and testosterone [16].i the risk of animals being detected or recognized by natural predators [1]. Therefore, animals are faced with new challenges that affect their survival. The ability of some animal species to modify the phenotype, because of changes in environmental conditions, is considered as adaptive plasticity [2–4]. This capacity enables some organisms to change their natural body coloration and pattern (metachrosis) as an adaptation to a non-match- ing background. With adaptive plasticity organisms can improve performance when faced with pressures such as climate change, UV radiation, chemical pollutants and predators [5]. Chromatic adaptation, despite its benefits, also has an inevitable impact on other body processes and energy budgets and is linked to costs and restrictions. Color change through pigment reorganization or de novo syn- thesis involves complex neuroendocrine control of the chromatophores and may be followed by high energetic costs and metabolic activities [15]. These metabolic expenditures are the result of the many physiological reactions required for this adaptive response [17]. In color-changing animals, energy constraints are regulated by incidence and the category of change itself, thus, mod- erate change, based on diet, is less costly than rapid and continuous variations [12]. Amphibians select habitats that match their own coloration to maximize camouflage. The model organism for our experiment was the Hyla arborea tadpole (Fig. 1). Throughout the aquatic stage of life, amphibians encounter a variety of different inverte- brate and vertebrate predators [19, 20]. Amphibian larvae frequently alter their morphology in response to preda- tors, starting with the size, shape and color of their tails. For H. arborea tadpoles, dragonfly nymphs are a serious threat to their survival and can induce a plastic pheno- typic response. Tadpoles developed deeper tails with black pigmented patches and changed their swimming behavior when predatory dragonfly larvae were present [21–23]. Energy investment in color change due to preda- tory risk represents an additional challenge for larvae which are already confronted with serious physiological, morphological and behavioral transformations essential for the crossover to a terrestrial habitat.hi h Intracellular transport of pigments is mediated by dif- ferent hormones. Alpha-melanocyte stimulating hor- mone (α-MSH) induces the dispersion of melanin in melanophores that, then cover other color cells. In con- trast, melanin-concentrating hormone (MCH) leads to the aggregation of pigment that causes lightening of the skin tone [15]. Besides these two primary hormones, pig- ment modifications in some amphibian species can also This study aimed to examine whether there are signifi- cant physiological consequences of changing coloration. We examined the evidence for the possible oxidative cost of adaptation of H. arborea tadpoles to background color. We assumed that: (i) tadpoles will adjust their body pigmentation in response to a change in background Radovanović et al. Frontiers in Zoology (2023) 20:6 Page 3 of 12 Radovanović et al. Frontiers in Zoology Fig. 1 Tadpoles of Hyla arborea developed on a white (A) and black (B) background Fig. 1 Tadpoles of Hyla arborea developed on a white (A) and black (B) background inspected. The well-being of the animals was checked daily. color; (ii) greater investment in pigmentation and its maintenance in darker larvae will result in an oxida- tive cost manifested through changes in oxidative sta- tus and induction of lipid peroxidation (LPO) due to an increase in ROS generated by a higher metabolic rate; (iii) exposure of individuals to a background color that is opposite to their body coloration (inadequate matching) will induce processes of color change, stress response and result in oxidative damage. The oxidative cost was assessed through changes in antioxidative system param- eters and oxidative damage levels. All surplus specimens (30 eggs) were returned to their natural habitat, 50 eggs never developed. Sixty contain- ers had a black paper sleeve and were placed on a black surface (B—black background coloration treatment) and 60 containers had a white paper sleeve and were placed on a white surface (W – white background coloration treatment). On the 20th day of the experiment 20 animals from the black surface (B treatment), and 20 animals from the white surface (W treatment) were randomly chosen and euthanized for analysis of their biochemical parameters of oxidative stress. Specimens were euthanized by sub- merging in liquid nitrogen. From the remaining 80 tad- poles, 20 animals from the B treatment were switched to the white containers (BW treatment) and the same num- ber of animals from the W treatment were switched to the black containers (WB treatment). The remaining 40 animals resumed development under the original con- ditions (now BB and WW treatments, 20 animals each). Thereby four treatment groups were established. At the moment of the switch, the tadpoles were at GS 30 (Fig. 2). Materials and methods Experimental design Two hundred Hyla arborea eggs were collected from Reva pond, located in the vicinity of Belgrade, Serbia, (44° 50′53.8′′ N, 20° 32′07.7′′ E (44.848272, 20.535476)) during April 2021. The sample was transported in 1 L transparent containers (15.5·14.5·6.5 cm) and reared at the Department of Evolutionary Biology, Institute for Biological Research “Siniša Stanković”, University of Bel- grade. The experiment started when tadpoles reached Gosner stage 25 (GS 25) [24]. In total, 120 specimens were randomly chosen for the experiment. Tadpoles were individually placed in plastic 0.5 L containers filled with dechlorinated tap water and reared at a constant room temperature (20 °C) under a natural day and night cycle. Water in the containers was changed every four days, and the tadpoles were fed ad libitum every other day with the same amount of pulverized commercial fish food tablets (Tetra TabiMin®, Tetra GmbH, Melle, Germany). The survival and general health of the tadpoles were visually On the 37th day of the experiment (17 days after the switch), all individuals from the treatment groups were euthanized in the same way as mentioned before, to analyze the parameters of oxidative stress caused by prolonged background adaptation. At this point, the tad- poles were at GS 37. On the 20th and 37th days of the experiment, tadpoles were photographed to obtain length measurements. All photographs contained a ruler to standardize length measurements. Using the tpsDig program [25] we placed Radovanović et al. Frontiers in Zoology (2023) 20:6 Page 4 of 12 Fig. 2 Schematic presentation of the experimental design Fig. 2 Schematic presentation of the experimental design markers on the tip of the snout and the tip of the tail of tadpoles. Two markers 10 mm apart were placed on the ruler. Then, using tmorphgen from the Integrated Mor- phometrics Program (IMP) package [26] we calculated the lengths of the tadpoles. Additionally, on the same days, high-resolution images of the dorsal side of the tadpoles were taken for analysis of the pigmentation. All images were created using a DSLR Nikon D7500 camera, mounted perpendicularly to the animals and at a fixed height. were centrifuged for 10 min at 10,000 × g at 4 °C in 40% trichloroacetic acid (TCA). The resulting supernatant was used LPO determination at 532 nm and was pre- sented in nmol TBARS/mg tissue.h The remaining homogenous tissue mass was used for the analysis of antioxidant parameters. Biochemical analysesh The Lowry method was used for the determination of the total protein concentration at 500 nm [31]. The activity of superoxide dismutase (SOD) was evaluated at 480 nm, by monitoring the autoxidation of adrenaline to adreno- chrome [32]. The Claiborne method [33] that was used for catalase (CAT) activity determination is based on the quantification of hydrogen peroxide decomposition and was carried out at 240 nm. The assays for glutathione peroxidase (GSH-Px) and glutathione reductase (GR) activity were performed according to the methods of [34] and [35], respectively. The [36] procedure was used for the evaluation of glutathione-S-transferase (GST) activ- ity by measuring the interaction of 1-chloro-2,4-dini- trobenzene (CDNB) with the sulfhydryl group of GSH. Materials and methods Experimental design The tissue was homogenized at pH 7.4 in 25 mM sucrose buffer [28] with 10 mM Tris-HCl and 5 mM EDTA [29] using an Ultra-Turrax homogenizer (T-18, IKA-Werk, Germany). Subsequently, using a Sonopuls ultrasonic homogenizer (HD 2070, Bandelin Electronic, Germany) the homoge- nates were sonicated at 20 kHz for 30 s. A part of the sonicate was used for measurement of total glutathione (GSH) concentration, and the remaining sonicate was centrifuged at 100,000×g for 90 min (4 °C) [30]. The obtained supernatant was used for the estimation of additional biochemical parameters. Pigmentation was described as the percentage of dark pixels on a tadpole’s body surface, calculated from pho- tographs in Adobe Photoshop CC 2015 (Adobe System Inc., 2015). Pixels were described as light or dark based on their mean RGB value. First, we defined the RGB threshold values for each group and date, as the mean RGB value calculated from a subsample of specimens from each group and for each date. Pixels with lower RGB values were described as light and those with higher values as dark. Second, from the Adobe Photoshop histo- gram, we read the percentages of light and dark pixels for every tadpole. Statistical analysish y The Grubbs test was applied to check and exclude out- lier data (we did not detect any outlier). The data were verified with the Kolmogorov-Smirnov one-sample test and all investigated parameters had a normal distribu- tion. To test total snout-vent length (SVL), color intensity and differences in oxidative stress components between the studied groups of tadpoles (between the B and W group on the 20th day, and among WW, WB, BW and BB groups on the 37th day), one-way analysis of variance (ANOVA) was implemented with the background color as an independent variable. For parameters that showed significant differences (on the 37th day), we applied the post hoc Fisher least significant difference (LSD) test to determine further differences. The significant level was determined at p < 0.05. Linear regression was used to observe the relationship between the intensity of pigmentation/coloration and oxidative stress damage (LPO) in individuals exposed constantly on a white and black background. We used discriminant analysis (DA) to identify the differences among the examined tadpole groups (WW, WB, BW and BB) on the 37th day based on all measured oxidative stress components. In all fig- ures and tables, the obtained data are presented as the The results for one-way ANOVA are given in Table 2. Only the activity of CAT was significantly reduced in the group of tadpoles that were constantly exposed to the black background (B) with respect to the W group (p = 0.0032). The other examined biochemical param- eters did not differ between these two groups (Table 2; Fig. 3). One-way ANOVA showed significant differences for all oxidative stress parameters among WW, WB, BW and BB groups. Figure 4 presents the results of Fisher LSD post hoc test for oxidative stress parameters after prolonged background adaptation. SOD activity was significantly increased in the WW group compared to the WB (p = 0.0115) and BW groups (p = 0.0006), and also in the Table 1 The total snout-vent length (SVL) and color intensity (% of dark pixels) of Hyla arborea tadpoles in experimental groups (W – white, B – black, WW – white-white, WB – white-black, BW – black-white and BB – black-black) The data are expressed as the mean ± SE. Resultsh The measured values for total snout-vent length (SVL) and color intensity (% of dark pixels) of Hyla arborea tadpoles in the experimental groups are given in Table 1. One-way ANOVA revealed that there were no signifi- cant differences between SVL values in W and B groups on the 20th day of the experiment (p = 0.6455). However, body pigmentation differed significantly between the two groups (p < 0.0001). For prolonged background adapta- tion, after 37 days of the experiment the Fisher LSD test revealed that individuals from the BB group were sta- tistically longer than WB individuals (p = 0.0199). All groups displayed a statistically significant difference in pigmentation among themselves, except groups BB and WB (WW vs. WB–p < 0.0001; WW vs. BW– p = 0.0007; WW vs. BB– p < 0.0001; WB vs. BW– p < 0.0001; BB vs. BW–p < 0.0001).h All investigated parameters were rated at 25 °C using an UV-VIS spectrophotometer (UV-1800, Shimadzu, Japan). Tissue processingh The total body of the tadpoles used in biochemical analy- ses was minced and mixed into one homogenous mass; 0.2 g of the tissue was separated for the determina- tion of thiobarbituric acid-reactive substance (TBARS) concentration, and the remaining homogenous tissue mass was used for antioxidative system (AOS) param- eter assessment. The TBARS method [27] included tis- sue homogenization and sonication in 10 volumes of ice-cold Tris-HCl buffer, pH 7.4. The obtained sonicates Radovanović et al. Frontiers in Zoology (2023) 20:6 Page 5 of 12 Radovanović et al. Frontiers in Zoology The activities of GSH-Px, GR, and GST were monitored at 340 nm. The activities of the enzymes were interpreted in U/mg protein. mean ± standard error (SE). All results were calculated with STATISTICA 8.0. Software, with exception of DA which was performed in XLSTAT, Ver. 2014.5.03. The Griffith method [37] was applied for the analysis of total GSH concentration, based on the oxidation of GSH by 5,50-dithiobis-(2- nitrobenzoic acid (DTNB) and NADPH reduction. Total GSH concentration was meas- ured at 412 nm and expressed as nmol/g tissue. Discussion In many ectotherms, body color is susceptible to change and is fine-tuned at the individual level. Color adapta- tions require the formation, degradation, translocation and maintenance of pigments in chromatophores. These processes are energetically expensive and have significant implications on numerous physiological and biochemi- cal processes. They can affect cortisol and corticosterone level [38, 39], body weight [40], morphology [14], stand- ard metabolic rate and behavior [15]. Even though color change provides a direct benefit for animals in heteroge- neous environments, it can allocate energy from other internal functions (antioxidant protection, the immune system) with effects on overall fitness [41]. It was found that background color affects the growth and survival of Hippocampus kuda juveniles [42] and the body length of Microhylafissipes tadpoles, suggesting that pigmentation is prioritized over somatic development [5].f BB group as compared to group BW (p = 0.0271). In the BW group, CAT activity was significantly reduced com- pared to WB (p = 0.0067) and BB groups (p = 0.0072). In individuals that were continuously exposed to the black background (BB), the activity of GSH-Px was sta- tistically increased in comparison to the other studied groups (BB vs. WW–p < 0.0001; BB vs. WB–p < 0.0001; BB vs. BW–p < 0.0001). The activity of GR was increased in the group of animals constantly subjected to the black background compared to the other groups (BB vs. WW–p = 0.0107; BB vs. WB–p < 0.0001; BB vs. BW–p < 0.0001), as it was in the group of animals con- stantly exposed to the white surroundings compared with groups WB (p = 0.0036) and BW (p = 0.015). The activity of GST was considerably lower in the BW group with respect to other investigated groups (BW vs. WW–p = 0.0004; BW vs. WB–p = 0.0329; BW vs. BB–p = 0.0064). The concentration of GSH in the WW group was lower than in all other groups (WW vs. WB–p = 0.0017; WW vs. BW–p = 0.0462; WW vs. BB–p < 0.0001), but it was enhanced in the BB group with respect to the BW group (p = 0.0218). The concentration of TBARS was the lowest in the WW group in compari- son with other groups (WW vs. WB–p = 0.0135; WW vs. BW–p < 0.0001; WW vs. BB–p < 0.0001), and lower in the WB group in comparison to individuals from the BW group (p = 0.0008). Statistical analysish Data in bold indicate statistical differences (p < 0.05) based on one-way ANOVA with the Fisher LSD post hoc test Groups Mean ± SE W versus B WW versus WB WW versus BW WW versus BB WB versus BW BB versus BW BB versus WB SVL (mm) W B 32.07 ± 0.98 32.58 ± 0.51 p = 0.6455 p = 0.2059 p = 0.9646 p = 0.2870 p = 0.2022 p = 0.3220 p = 0.0199 WW WB BW BB 46.96 ± 0.59 45.61 ± 0.75 47.01 ± 1.06 48.07 ± 0.59 Color (dark pixels %) W B 57.84 ± 2.74 92.29 ± 1.30 p < 0.0001 p < 0.0001 p = 0.0007 p < 0.0001 p < 0.0001 p < 0.0001 p = 0.7011 WW WB BW BB 70.39 ± 2.59 91.41 ± 1.27 59.88 ± 2.19 90.41 ± 1.52 t length (SVL) and color intensity (% of dark pixels) of Hyla arborea tadpoles in experimental groups (W – -white, WB – white-black, BW – black-white and BB – black-black) The data are expressed as the mean ± SE. Data in bold indicate statistical differences (p < 0.05) based on one-way ANOVA with the Fisher LSD post hoc test The data are expressed as the mean ± SE. Data in bold indicate statistical differences (p < 0.05) based on one-way ANOVA with the Fisher LSD post hoc test Page 6 of 12 Radovanović et al. Statistical analysish Frontiers in Zoology (2023) 20:6 Table 2 Results of one-way ANOVA of the comparison among different treatment groups of Hyla arborea tadpoles (W—white and B—black groups on the 20th day; WW—white–white, WB— white–black, BW—black–white and BB—black–black groups on the 37th day) for oxidative stress parameters Data in bold indicate statistical differences (p < 0.05) Variable dF F p 20th day SOD 1 0.01 0.9369 CAT 1 11.56 0.0032 GSH-Px 1 3.35 0.0838 GR 1 3.33 0.0848 GST 1 1.61 0.2202 GSH 1 0.02 0.8820 LPO 1 2.08 0.1681 37th day SOD 3 5.07 0.0042 CAT 3 3.68 0.0189 GSH-Px 3 14.45 < 0.0001 GR 3 16.26 < 0.0001 GST 3 5.22 0.0036 GSH 3 7.26 0.0005 LPO 3 14.16 < 0.0001 Table 2 Results of one-way ANOVA of the comparison among different treatment groups of Hyla arborea tadpoles (W—white and B—black groups on the 20th day; WW—white–white, WB— white–black, BW—black–white and BB—black–black groups on the 37th day) for oxidative stress parameters positive correlation with the intensity of coloration in individuals continuously maintained on dark and white backgrounds (r = 0.57; p = 0.016). g p Based on the measurements of oxidative stress compo- nents, discriminant analysis revealed differences between groups that developed on a background with different colors (WW, WB, BW and BB) after 37 days (Table 3; Fig. 5). The first function (F1) accounted for 59.58% of the total heterogeneity. The BB group was separated along the first function in relation to other groups, and was mostly differentiated regarding to GSH-Px and GR activi- ties. The second function (F2) in the analysis accounted for 33.77% of the total heterogeneity. LPO concentration and SOD activity were the main factors that contributed to the differentiation of the WW group along the second canonical function in relation to the BW group. Discussion TBARS values displayed a significant In the present study, we assessed the effect of back- ground color and its influence on body coloration and oxidative status parameters of anuran tadpoles. Our investigation revealed that tadpoles of H. arborea mani- fest the ability to adjust their body pigmentation to the background. A high-reflecting environment induced lower pigmentation, whereas a black, low-reflecting envi- ronment induced increased pigmentation. Our assumption was that higher investment in pig- mentation will result in an oxidative cost manifested as changes in the oxidative status and greater lipid peroxi- dation. Rearing larvae for 20 days on a dark (B) and light (W) substrate induced differences in body coloration, but did not affect antioxidant components (except CAT activity). The absence of significant differences in the concentration of LPO products indicates that investment in body pigmentation during this period did not cause greater oxidative stress in darker tadpoles of H. arborea. Radovanović et al. Frontiers in Zoology (2023) 20:6 Page 7 of 12 Radovanović et al. Frontiers in Zoology Fig. 3 Antioxidant system (A—superoxide dismutase (SOD); B—catalase (CAT); C—glutathione peroxidase (GSH-Px); D—glutathione reductase (GR); E—glutathione-S-transferase (GST); F—glutathione (GSH)) and oxidative stress (G—lipid peroxidation (LPO)) parameters in two experimental groups (W—white, B—black) of Hyla arborea tadpoles. indicates significant differences between groups (one-way ANOVA, p < 0.05) Fig. 3 Antioxidant system (A—superoxide dismutase (SOD); B—catalase (CAT); C—glutathione peroxidase (GSH-Px); D—glutathione reductase (GR); E—glutathione-S-transferase (GST); F—glutathione (GSH)) and oxidative stress (G—lipid peroxidation (LPO)) parameters in two experimental groups (W—white, B—black) of Hyla arborea tadpoles. indicates significant differences between groups (one-way ANOVA, p < 0.05) Larvae (early stages of development, GS 30) were capa- ble of neutralizing potential variations in ROS formation at different substrate colors and successfully maintain homeostasis. Larvae (early stages of development, GS 30) were capa- ble of neutralizing potential variations in ROS formation at different substrate colors and successfully maintain homeostasis. (LPO). The level of oxidative damage showed a positive correlation with the color intensity of individuals. These results in H. arborea tadpoles could be the physiologi- cal consequences of the increase in the number/density of pigment cells and maintenance of a darker coloration for a prolonged time. Animals required to establish and maintain cryptic behavior in dark surroundings could experience elevated metabolic expenditure as a conse- quence of chromatic adaptation compared to animals in light surroundings. Discussion Previous studies suggested that indi- viduals with higher pigmentations require greater food Nevertheless, prolonged exposure of larvae to a con- stantly dark habitat (for 37 days) led to major changes in the antioxidant status of individuals in the form of increased AOS activity and concentration of investigated parameters (GSH-Px, GR, GSH) as compared to the group kept continuously on a white background. Darker- pigmented larvae also exhibited greater oxidative damage Radovanović et al. Frontiers in Zoology (2023) 20:6 Radovanović et al. Frontiers in Zoology Page 8 of 12 Fig. 4 Antioxidant system (A—superoxide dismutase (SOD); B—catalase (CAT); C—glutathione peroxidase (GSH-Px); D—glutathione reductase (GR); E—glutathione-S-transferase (GST); F—glutathione (GSH)) and oxidative stress (G—lipid peroxidation (LPO)) parameters in four experimental groups (WW—white–white, WB—white–black, BW—black–white and BB—black–black) of Hyla arborea tadpoles after the background switch. indicates significant differences between groups (Fisher LSD, p < 0.05) Fig. 4 Antioxidant system (A—superoxide dismutase (SOD); B—catalase (CAT); C—glutathione peroxidase (GSH-Px); D—glutathione reductase (GR); E—glutathione-S-transferase (GST); F—glutathione (GSH)) and oxidative stress (G—lipid peroxidation (LPO)) parameters in four experimental groups (WW—white–white, WB—white–black, BW—black–white and BB—black–black) of Hyla arborea tadpoles after the background switch. indicates significant differences between groups (Fisher LSD, p < 0.05) Radovanović et al. Frontiers in Zoology (2023) 20:6 Page 9 of 12 Table 3 Standardized discriminant analysis coefficient for oxidative stress parameters in four experimental groups (WW— white–white, WB—white–black, BW—black–white and BB— black–black) of Hyla arborea tadpoles A significant positive correlation between body color and SMR was obtained, and it was concluded that darker lar- vae displayed a higher percentage of oxygen consump- tion. In Microhyla fissipes tadpoles, a darker background caused oxidative phosphorylation and an overturn of protein metabolism, which was identified at the tran- scriptional level [5]. [43] assessed the oxidative status of Lates calcarifer juveniles in different colored tanks and observed that background colors did not induce sig- nificant variations in malondialdehyde, CAT or GST activities; however, SOD and GSH-Px activities were sig- nificantly elevated together with plasma cortisol in fish cultivated in black tanks, confirming stressful conditions. F1 F2 F3 SOD 0.2018 − 0.5924 − 0.1530 CAT 0.2683 − 0.1742 0.7147 GST 0.2208 − 0.5889 0.1794 GSH-Px 0.8097 0.0332 0.1356 GR 0.7603 − 0.3595 − 0.2165 GSH 0.3991 0.4062 0.6577 LPO 0.1646 0.8561 − 0.2060 Eigenvalue 2.9802 1.6892 0.3330 Discrimination (%) 59.5752 33.7674 6.6574 Cumulative % 59.5752 93.3426 100.0000 i Our investigation discloses that skin coloration of H. Competing interests The authors declare no competing interests. Received: 17 August 2022 Accepted: 25 January 2023 Received: 17 August 2022 Accepted: 25 January 2023 Availability of data and materials y The dataset used and analysed during the current study are available from the corresponding author on request. References 1. Skelly DK, Freidenburg LK, Kiesecker JM. Forest canopy and the perfor- mance of larval amphibians. Ecology. 2002;83(4):983–92. 2. Ghalambor CK, McKay JK, Carroll SP, Reznick DN. Adaptive versus non- adaptive phenotypic plasticity and the potential for contemporary adaptation in new environments. Funct Ecol. 2007;21(3):394–407. 3. Kelly PW, Pfennig DW, Pfennig KS. Adaptive plasticity as a fitness benefit of mate choice. Trends Ecol Evol. 2021;36(4):294–307. 4. Brooker R, Brown LK, George TS, Pakeman RJ, Palmer S, Ramsay L, Schöb C, Schurch N, Wilkinson MJ. Active and adaptive plasticity in a changing climate. Trends Plant Sci. 2022;27(7):717–28. Received: 17 August 2022 Accepted: 25 January 2023 Even though body color modification is an effective tactic in many animals, its physiological repercussions have received limited attention. We deduce that altera- tion of background color induces very complex biologi- cal responses. These results indicate that amphibians encounter stress during chromatic adaptations, which eventually affects the components of their antioxidative defense. Author contributions TR, TP and MP conceived the study. AK, MM, NTK, and TV collected the samples and conducted the experiment. TR, TP, BG, JG, SD and MP performed methodology. TR, TP and MP performed the statistical analysis. TR drafted the manuscript. All authors edited the draft and prepared the manuscript for publication. TR and MP oversaw the project. All authors read and approved the final manuscript. Discussion arborea tadpoles is reversible as individuals respond to changes in substrate hue. Exposure to altered substrate color can activate neural-hormonal events that require energy for pigment translocations within chromato- phores. A dark background can enhance the transcrip- tion of proopiomelanocortin (POMC) and the expression of α-melanocyte-stimulating hormone (α-MSH), which controls the production of melanin and dispersion of melanosome in darker individuals [44]. These hormones also have an important role in the control of energy intake and encounter higher metabolic costs. [15] meas- ured the standard metabolic rate (SMR) in newt larvae (Lissotriton boscai) subjected to different background conditions. After 14 days, larvae reared in a dark micro- habitat exhibited a 64% increase in oxygen consumption in comparison with ones cultivated in a lighter substrate. Fig. 5 Discriminant analysis of investigated oxidative stress parameters in four experimental groups (WW—white–white, WB—white–black, BW — black–white and BB—black–black) of Hyla arborea tadpoles on the factor plan Fig. 5 Discriminant analysis of investigated oxidative stress parameters in four experimental groups (WW—white–white, WB—white–black, BW — black–white and BB—black–black) of Hyla arborea tadpoles on the factor plan Radovanović et al. Frontiers in Zoology (2023) 20:6 Radovanović et al. Frontiers in Zoology Page 10 of 12 on the development, fitness and survival of amphibians is of special concern. Examination of the ability of the amphibian population to cope with environmental vari- ations, such as changes in substrate color, could help us understand how amphibians deal with background diver- sity and predict their susceptibility and resistance to these changes. homeostasis [45]. The transfer to non-body-matching surroundings can cause pressure due to greater visibility and predation risk. This state activates CORT levels and the HPI-axis, which can also alter the energy budget and oxidative status. In this study, we report on major changes in antioxi- dant protection parameters followed by variations in skin pigmentation. In individuals that reverse body coloration (BW group), the defense system (SOD, GR, GST) failed and LPO occurred in comparison to individuals reared continuously in the white surroundings. We observed consequences related to background color matching. The presence of oxidative damage in tadpoles that devel- oped on altered substrates and in ones that were continu- ously maintained on a dark background suggests that the change in body coloration carries an oxidative cost simi- lar to the one imposed by maintaining prolonged dark pigmentation. Consent for publication Not applicable. Funding Thi t d This study was funded by the Ministry of Education, Science and Technological Development of the Republic of Serbia, Grant No. 451-03-68/2022-14/200007. Inverted body coloration as a response to a non- matching background can induce oxidative stress. How- ever, color change from dark to lighter carries a higher oxidative cost. These individuals experienced both pro- cesses of melanin synthesis (aggregation) and destruc- tion (dispersion), in contrast to only pigment production (aggregation) during the change from a pale to a darker coloration. Our results revealed that both processes involved in color change include oxidative stress as an associated cost. Acknowledgements f Acknowledgements The authors are grateful to Goran Poznanović for proofreading the manuscript. g The authors are grateful to Goran Poznanović for proofreading the manuscript. Ethics approval and consent to participate The collection of H. arborea eggs from the natural population was approved by the Ministry of Environmental Protection of the Republic of Serbia (permit No. 353-0 L-1613/2021-04). All applicable international, national and institu- tional guidelines for the care and use of animals were followed. All procedures performed involving animals were in accordance with the ethical standards of the Institute for Biological Research “Siniša Stanković”, University of Belgrade (permit No. 01-739). All experimental procedures complied with the European Directive (2010/63/EU) on the protection of animals used in experimental and other scientific purposes. It has been shown that pigments involved in appear- ance modification are also significant for other body functions related to the immunologic response [7, 12] or antioxidant protection [46]. A significant increase in pig- ment melanin in amphibian cells was noted in different stressful conditions such as oxygen and food deprivation or low temperature [47–50]. According to some data, melanin has a significant part in the elimination of ROS and other toxicants in amphibians and other species [50, 51]. Some authors even assume that the antioxidant role of SOD and GSH could be replaced with this pigment in amphibians [52, 53]. 1. Skelly DK, Freidenburg LK, Kiesecker JM. Forest canopy and the perfor- mance of larval amphibians. Ecology. 2002;83(4):983–92. 2. Ghalambor CK, McKay JK, Carroll SP, Reznick DN. Adaptive versus non- adaptive phenotypic plasticity and the potential for contemporary adaptation in new environments. Funct Ecol. 2007;21(3):394–407. 3. Kelly PW, Pfennig DW, Pfennig KS. Adaptive plasticity as a fitness benefit of mate choice. Trends Ecol Evol. 2021;36(4):294–307. 4. Brooker R, Brown LK, George TS, Pakeman RJ, Palmer S, Ramsay L, Schöb C, Schurch N, Wilkinson MJ. Active and adaptive plasticity in a changing climate. Trends Plant Sci. 2022;27(7):717–28. 5. Chang L, Wang B, Zhang M, Liu J, Zhao T, Zhu W, Jiang J. The effects of corticosterone and background colour on tadpole physiological plastic- ity. Comp Biochem Physiol Part D Genomics Proteomics. 2021;39:100872. 6. Caro T, Sherratt TN, Stevens M. The ecology of multiple colour defences. Evol Ecol. 2016;30:797–809. 4. Brooker R, Brown LK, George TS, Pakeman RJ, Palmer S, Ramsay L, Schöb C, Schurch N, Wilkinson MJ. Active and adaptive plasticity in a changing climate. Trends Plant Sci. 2022;27(7):717–28. Conclusionhl 5. Chang L, Wang B, Zhang M, Liu J, Zhao T, Zhu W, Jiang J. The effects of corticosterone and background colour on tadpole physiological plastic- ity. Comp Biochem Physiol Part D Genomics Proteomics. 2021;39:100872. The influence of surrounding color tones on the behav- ior and physiology of animals is a field of science that is expanding and information concerning the effect of color 6. Caro T, Sherratt TN, Stevens M. The ecology of multiple colour defences. Evol Ecol. 2016;30:797–809. Page 11 of 12 Page 11 of 12 Radovanović et al. Frontiers in Zoology (2023) 20:6 Radovanović et al. Frontiers in Zoology Radovanović et al. Frontiers in Zoology (2023) 20:6 7. Stevens M. Color change, phenotypic plasticity, and camouflage. Front Ecol Evol. 2016;4:51. 32. Misra HP, Fridovich I. The role of superoxide anion in the autoxidation of epinephrine and simple assay for superoxide dismutase. J Biol Chem. 1972;247:3170–5. 8. Noor MA, Parnell RS, Grant BS. A reversible color polyphenism in ameri- can peppered moth (Biston betularia cognataria) caterpillars. PLoS ONE. 2008;3(9):e3142. 33. Claiborne A. Catalase activity. In: Greenwald RA, editor. Handbook of methods for Oxygen Radical Research. Boca Raton: CRC Press Inc; 1984. pp. 283–4. 9. Sugimoto M. Morphological color changes in the medaka, Oryzias latipes, after prolonged background adaptation: I. Changes in the population and morphology of melanophores. Comp Biochem Physiol. 1993;104A:513–8. 34. Tamura M, Oshino N, Chance B. Some characteristics of hydrogen- and alkylhydroperoxides metabolizing systems in cardiac tissue. J Biochem. 1982;92:1019–31. 10. Sugimoto M. Morphological color changes in the medaka, Oryzias latipes, after prolonged background adaptation: II. Changes in the responsive- ness of melanophores. Comp Biochem Physiol. 1993;104A:519–23. 35. Glatzle D, Vuilleumier JP, Weber F, Decker K. Glutathione reductase test with whole blood, a convenient procedure for the assessment of the riboflavin status in humans. Experientia. 1974;30:665–7. y 11. Wells KD. The ecology and behavior of amphibians. Chicago: University of Chicago Press; 2007.l l 36. Habig WH, Pabst MJ, Jakoby WB. Glutathione S-transferases. The first enzymaticstep in mercapturic acid formation. J Biol Chem. 1974;249:7130–9. 12. Duarte RC, Flores AAV, Stevens M. Camouflage through colour change: mechanisms, adaptive value and ecological significance. Philos Trans R Soc Lond B Biol Sci. 2017;372(1724):20160342. 37. Griffith OW. Determination of glutathione and glutathione disulfide using glutathione reductase and 2-vinylpyridine. Anal Biochem. 1980;106:207–12. 13. Li Y, Correll N. From natural to artificial camouflage: components and systems. arXiv. 2018;1809.04171 38. Conclusionhl Chovanec A. The influence of tadpole swimming behaviour on predation by dragonfly nymphs. Amphibia-Reptilia. 1992;13(4):341–9. yl y y 22. van Buskirk J, Arioli M. Dosage response of an induced defense: how sensitive are tadpoles to predation risk? Ecology. 2002;83(6):1580–5. 45. Schiöth HB, Haitina T, Ling MK, Ringholm A, Fredriksson R, Cerdá-Reverter JM, Klovins J. Evolutionary conservation of the structural, pharmacologi- cal, and genomic characteristics of the melanocortin receptor subtypes. Peptides. 2005;26(10):1886–900. 23. Lemcke C. Phänotypische Plastizität bei Kaulquappen des Europäischen Laubfrosches, Hyla arborea. PhD Dissertation, LMU München: Faculty of Biology; 2006. 46. Huang S, Pan Y, Gan D, Ouyang X, Tang S, Ekunwe SIN, Wang H. Antioxi- dant activities and UV-protective properties of melanin from the berry of Cinnamomum burmannii and Osmanthus fragrans. Med Chem Res. 2011;20:475–81. 24. Gosner KL. A simplified table for staging anuran embryos and larvae with notes on identification. Herpetologica. 1960;16:183–90. i 25. Rohlf FJ. (2006) tpsDig, version 2.10. Available at http://life.bio.sunysb. edu/morph/index.html. 47. Barni S, Bertone V, Croce AC, Bottiroli G, Bernini F, Gerzeli G. Increase in liver pigmentation during natural hibernation in some amphibians. J Anat. 1999;195:19–25. 26. Sheets HD. (2000) Integrated morphometrics package (IMP) Available at http://www2.canisius.edu/sheets. 48. Frangioni G, Borgioli G, Bianchi S, Pillozzi S. Relationships between hepatic melanogenesis and respiratory conditions in the newt, Triturus carnifex. J Exp Zool. 2000;287:120–7. 27. Rehncrona S, Smith DS, Akesson B, Westerberg E, Siesjö BK. Peroxidative changes in brain cortical fatty acids and phospholipids, as characterized during Fe2+ and ascorbic acid stimulated lipid peroxidation in vitro. J Neurochem. 1980;34:1630–8. 49. Silva JP, da Silva DC, Melo FT, Giese EG, Furtado AP, Santos JN. Liver histopathology in the cane toad, Rhinella marina (Amphibia: Bufonidae), induced by Ortleppascaris sp. larvae (Nematoda: Ascarididae). J Parasitol. 2013;99(2):250–6. 28. Lionetto MG, Caricato R, Giordano ME, Pascariello MF, Marinosci L, Schet- tino T. Integrated use of biomarkers (acetylcholineesterase and antioxi- dant enzyme activities) in Mytilus galloprovincialis and Mullus barbatus in an italian coastal marine area. Mar Pollut Bull. 2003;46:324–30. 50. Bernabò I, Biasone P, Macirella R, Tripepi S, Brunelli E. Liver histology and ultrastructure of the Italian Newt (Lissotriton Italicus): normal structure and modifications after acute exposure to nonylphenol ethoxylates. Exp Toxicol Pathol. 2014;66:455–68. 29. Rossi MA, Cecchini G, Dianzani MM. Glutathione peroxidase, glutathione reductase and glutathione transferase in two different hepatomas and in normal liver. IRCS Med Sci Biochem. 1983;11:805. 30. Takada Y, Noguchi T, Okabe T, Kajiyama M. Conclusionhl Papoutsoglou SE, Mylonakis G, Miliou H, Karakatsouli NP, Chadio S. Effects of background color on growth performance and physiological responses of scaled carp (Cyprinus carpio L.) reared in closed circulated systems. Aquac Eng. 2000;22:309–18. 14. Sugimoto M, Yuki M, Miyakoshi T, Maruko K. The influence of long-term chromatic adaptation on pigment cells and striped pigment patterns in the skin of the zebrafish, Danio rerio. J Exp Zool A Comp Exp Biol. 2005;303(6):430–40. 39. Holmes AM, Emmans CJ, Jones N, Coleman R, Smith TE, Hosie CA. Impact of tank background on the welfare of the African clawed frog, Xenopus laevis (Daudin). Appl Anim Behav Sci. 2016;185:131–6. 15. Polo-Cavia N, Gomez-Mestre I. Pigmentation plasticity enhances crypsis in larval newts: associated metabolic cost and background choice behav- iour. Sci Rep. 2017;7:39739. 40. Nawang S, Ching FF, Senoo S. Comparison on growth performance, body coloration changes and stress response of juvenile river catfish, Pangasius hypophthalmus reared in different tank background colour. Aquac Res. 2019;50:2591–9. 16. Nilsson Sköld H, Aspengren S, Wallin M. Rapid color change in fish and amphibians–function, regulation, and emerging applications. Pigment Cell Melanoma Res. 2013;26(1):29–38. 17. Merilä J, Laurila A, Lindgren B. Variation in the degree and costs of adap- tive phenotypic plasticity among Rana temporaria populations. J Evol Biol. 2004;17(5):1132–40. 41. Alfakih A, Watt PJ, Nadeau NJ. The physiological cost of col- our change: evidence, implications and mitigations. J Exp Biol. 2022;225(10):jeb210401. 18. Halliwell B, Gutteridge JMC. Free radicals in biology and medicine. 5th ed. Oxford: Oxford University Press; 2015. 42. Pawar HB, Sanaye SV, Murugan A, Sreepada RA, Tanu. Effect of back- ground color of tanks on growth and survival of juvenile yellow seahorse, Hippocampus kuda (Bleeker 1852), in the pelagic phase. Isr J Aquac- Bamid. 2011;2011(63):621–6. 19. Wassersug RJ, Sperry DG. The relationship of locomotion to dif- ferential predation on Pseudacris triseriata (Anura: Hylidae). Ecology. 1977;58:830–9. 43. Sotoudeh E, Feizollahi S, Banan A, Morshedi V, Hosseini A. Oxidative status and digestive histology of Barramundi perch in response to different tank colors. J Aquat Anim Health. 2019;31(2):139–46. 20. Vonesh JR. Sequential predation in a complex life-history: interactions among egg, larval, and post-metamorphic predators of the east African treefrog, Hyperolius spinigularis. PhD Dissertation, University of Florida; 2003. 44. Bertolesi GE, McFarlane S. Seeing the light to change colour: an evolu- tionary perspective on the role of melanopsin in neuroendocrine circuits regulating light-mediated skin pigmentation. Pigment Cell Melanoma Res. 2018;31(3):354–73. 21. Conclusionhl Superoxide dismutase in various tissues from rabbits bearing the Vx-2 carcinoma in the maxillary sinus. Cancer Res. 1982;42:4233–5. 51. Loumbourdis NS. Liver histopathologic alterations in the frog Rana ridibunda from a small river of northern Greece. Arch Environ Contam Toxicol. 2007;53:418–25. 31. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951;193:265–75. 52. Sichel G, Corsaro C, Scalia M, Sciuto S, Geremia E. Relationship between melanin content and superoxide dismutase (SOD) activity in the liver of various species of animals. Cell Biochem Funct. 1987;5:123–8. Page 12 of 12 Radovanović et al. Frontiers in Zoology (2023) 20:6 Radovanović et al. Frontiers in Zoology (2023) 20:6 53. Geremia E, Corsaro C, Baratta D, Santoro C, Scalia M, Sichel G. Antioxidant enzymatic system in pigment tissue of amphibia. Pigment Cell Res. 1989;2:208–12. 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https://openalex.org/W2939923122	https://journals.sub.uni-hamburg.de/aethiopica/article/download/1147/1181	English	null	The World’s Oldest Living Proverb Discovered Thriving in Ethiopia	Aethiopica	2,019	cc-by-sa	4,937	Aethiopica 21 (2018) International Journal of Ethiopian and Eritrean Studies PETER UNSETH, Dallas International University, Dallas–SIL International, Dallas * As always, I gratefully acknowledge the patience, editorial insight, and support of my wife Carole Unseth. Also, I am grateful for suggestions from an anonymous referee, especially regarding artistic effects in the Guǧǧi proverb. I am indebted to Mark Har- lan for help with the Arabic form, including keying the proverb in Arabic script. I am very grateful to Ted Kai Gatwich, now of Khartoum, for information about the Nuer proverb form. He told me in 2017 by email that it is a new proverb in Nuer, but did not speculate where it was borrowed from. I am very grateful to Joseph Malual for his help, giving the wordbyword gloss, even though the proverb is from a form of Nuer further east than his own. I am happy to acknowledge Ervin Starwalt for his help with glossing Greek examples. Miscellaneous The World’s Oldest Living Proverb Discovered Thriving in Ethiopia Aethiopica 21 (2018), 226–236 ISSN: 1430-1938 The World’s Oldest Living Proverb Discovered Thriving in Ethiopia Aethiopica 21 (2018), 226–236 ISSN: 1430-1938 Aethiopica 21 (2018), 226–236 ISSN: 1430-1938 Edited in the Asien-Afrika-Institut Hiob-Ludolf-Zentrum für Äthiopistik der Universität Hamburg Abteilung für Afrikanistik und Äthiopistik 2 Alster 1979, 5. p g g p 1 Moran 1978, 17–18. ‘Bitch’ is a technical and somewhat archaic term for ‘female dog’. 2 Alster 1979, 5. Introduction to the Proverb The oldest living proverb in the world is about a dog being hasty and there- fore giving birth to blind puppies. An ancient king of Assyria, ŠamšiAdad, wrote on a clay tablet to YasmahAddu, his son and viceroy in Mari, advis- ing him not to be too hasty in his actions. In his instructions to his son he warned, ‘Heaven forbid that, as in the ancient proverb, kalbatum ina šutebu/puriša ḫuppudūtim ūlid “The bitch by her acting too hastily brought forth the blind,” you now do likewise’.1 Based on this, Alster has described this proverb as having ‘a longer history than any other recorded proverb in the world’, going back to ‘around 1800 BC’.2 Note that even at the time of his writing the father referred to it as an ‘ancient proverb’. It is a wonderful example of a metaphorical proverb, the reader of the letter would clearly have understood that the message had nothing to do with literal dogs giving birth to literal puppies. 1 Moran 1978, 17–18. ‘Bitch’ is a technical and somewhat archaic term for ‘female dog’. 2 Alster 1979, 5. Aethiopica 21 (2018) 4 Erasmus is known for his compilation of a book of proverbs in Latin, but also for producing the first critical edition of the entire New Testament in Greek. I am in- debted to him for both of these, since both have led to my employment and enjoy- ment. 5 Mieder 2014, 13. ; ; g ; 7 All rejoice in the great increase in recent proverb study in Ethiopian languages by Ethiopian scholars (Fekade Azeze 2001). Since Fekade’s article was published, the proverb scholarship by Ethiopians has continued to increase, as seem in the sources cited by Unseth et al. 2017, 23, 24. 6 Alster 1979; Avishur 1981; Bodi 2015; Bonechi 2014; Bremmer 1980; Chavalas 2014, 84, 85; Dalley and Reyes 1997; Gordon 1958a; Gordon 1958b; Hinz 2004; Moran 1978; Moran 2002; Slings 1976; West 1997. 3 Moran 1978, 17. 5 Mieder 2014, 13. 6 3 Moran 1978, 17. 4 Erasmus is known for his compilation of a book of proverbs in Latin, but also for producing the first critical edition of the entire New Testament in Greek. I am in- debted to him for both of these, since both have led to my employment and enjoy- ment. 5 Mieder 2014, 13. 6 Alster 1979; Avishur 1981; Bodi 2015; Bonechi 2014; Bremmer 1980; Chavalas 2014, 84, 85; Dalley and Reyes 1997; Gordon 1958a; Gordon 1958b; Hinz 2004; Moran 1978; Moran 2002; Slings 1976; West 1997. 7 All rejoice in the great increase in recent proverb study in Ethiopian languages by Ethiopian scholars (Fekade Azeze 2001). Since Fekade’s article was published, the proverb scholarship by Ethiopians has continued to increase, as seem in the sources cited by Unseth et al 2017 23 24 Miscellaneous Moran wrote, ‘The proverb about the hasty bitch producing blind whelps needs no introduction to the [European] classicist. Familiar with it perhaps in many other languages (English, German, Italian, Turkish, and so on), he certainly knows the [ancient] Greek version’.3 y Erasmus gave a Latin form of it in his published collection of proverbs, his Adagia: ‘Canis festinans caecos parit catulos’. 4 We can assume that Erasmus’s Latin Adagia (published in 1500 with subsequently enlarged editions until 1536), which spread many other proverbs across Europe,5 was instrumental in spreading this one across Europe where it had not previous- ly been known. Many scholars have written about this proverb, but always in languages from a limited geographical range: Mesopotamia and Europe.6 These lan- guages are all north and west of the earliest Mesopotamian documentation. Now, far from these areas where scholars are familiar with this proverb, this study documents it to the south of Mesopotamia, in contemporary languages of Ethiopia, a land famous for its riches in proverbs.7 At this point in time, we can document that the proverb about the dog giving birth to blind puppies is found far from the regions where it has been previously reported. But we can only speculate about its original creation and the de- tails of its spread between the Middle East and Ethiopia. p p What makes this discovery noteworthy is not merely that yet another proverb is documented as having a wider range than had been known be- fore. Rather, it is noteworthy because this is the world’s oldest living prov- erb and it has been studied by many, but always studied within Europe and the Middle East. Now this paper documents it in Ethiopia. Additionally, this paper explores two variants of this proverb, one with a hasty dog and one with a hasty cat, presenting evidence that the dog version is original. 4 Erasmus is known for his compilation of a book of proverbs in Latin, but also for producing the first critical edition of the entire New Testament in Greek. I am in- debted to him for both of these, since both have led to my employment and enjoy- ment. 7 All rejoice in the great increase in recent proverb study in Ethiopian languages by Ethiopian scholars (Fekade Azeze 2001). Mieder 2014, 13. 6 Alster 1979; Avishur 1981; Bodi 2015; Bonechi 2014; Bremmer 1980; Chavalas 2014, 84, 85; Dalley and Reyes 1997; Gordon 1958a; Gordon 1958b; Hinz 2004; Moran 1978; Moran 2002; Slings 1976; West 1997. 7 All rejoice in the great increase in recent proverb study in Ethiopian languages by Ethiopian scholars (Fekade Azeze 2001). Since Fekade’s article was published, the proverb scholarship by Ethiopians has continued to increase, as seem in the sources cited by Unseth et al. 2017, 23, 24. MidEast The earliest recorded version of this proverb was written by King ŠamšiAdad in Mesopotamia, an area that is now within Iraq. The proverb is still found in Mesopotamia, in modern Iraqi Arabic, one version with a hasty dog and another with a cat: ‘The bitch in her hurry whelps blind pups’ and ‘The cat in her haste kittens blind kittens’.8 عميين ولدها تجيب عجلته من البزونة عميين ولدها تجيب عجلته من البزونة عميين ولدها تجيب عجلته من البزونة blind heroffspring bringsout herhaste from Thecat blind heroffspring bringsout herhaste from Thecat ‘The cat in her haste bears blind kittens.’ What is striking about this proverb from Iraq is that Avishur reported that he found it only in the Iraqi form of Arabic, not in other Arabic speak- ing areas. It is noteworthy that the syntax of the proverb is identical in both Akkadian and Arabic, i.e., the Arabic version is a wordbyword translation of the Akkadian text. These sentences are build [sic] in the same way. The proverb, created by the folk mouth, appears to have been transmitted among the populace of Mesopotamia for genera- tions, and translated by them according to which tongue they spoke: from Akkadian to Aramaic, and from Aramaic to Arabic. This as- sumption is supported by the fact, that in the collections of folk proverbs from Arabic countries (Syria, Lebanon, Palestine, Egypt, Yemen and Bedouins) I have not found this proverb; it is also missing in the corpus of ‘Comparative Proverbs’ published by AlTikriti.9 Thus it appears that this proverb is not an original Arabic text, nor is it borrowed from a European culture.10 10 Avishur 1981, 38. 11 Moran 1978, 18, n. 7. 8 Avishur 1981, 37, 38. , , 9 AbdulRahman AlTikriti (1966–1969) compared the proverbs of twelve Arab na- tions, but found this proverb only in Iraqi Arabic. 10 Avishur 1981, 38. 11 Moran 1978, 18, n. 7. 9 AbdulRahman AlTikriti (1966–1969) compared the proverbs of twelve Arab n tions, but found this proverb only in Iraqi Arabic. 10 Miscellaneous Since Fekade’s article was published, the proverb scholarship by Ethiopians has continued to increase, as seem in the sources cited by Unseth et al. 2017, 23, 24. Aethiopica 21 (2018) 227 Peter Unseth Peter Unseth 14 Bodi 2015, 77. 13 Moran 1978; 2002. 12 Bodi 2015, 78. 13 Moran 1978; 2002. 14 Bodi 2015, 77. 15 The fable is number 223 in Perry’s standard index of Aesop’s fables (Perry 1952). 16 Bodi 2015, 77. 17 Houghton 1915, 28. 18 Moran 2002, 90. 12 Bodi 2015, 78. 16 Bodi 2015, 77. g 18 Moran 2002, 90. Peter Unseth CE.19 Though this was technically on the African continent, the fact that it was written in Greek and was from an era when Egypt was under GrecoRoman domination (both culturally and politically) allows us to conclude that the finding of this proverb was still within a Greek context more than African. Greece Moran believed that the proverb originated in Mesopotamia, ‘[t]his of course would not be the only piece of oriental wisdom to have worked its way west.’11 If we presume that the proverb spread from Mesopotamia to- ward Europe, the earliest European attestations are (not surprisingly) in Aethiopica 21 (2018) 228 Miscellaneous Greek. The proverb may have travelled to Greece via Turkish, but this can- not be proven since the earliest Turkish record of the proverb is from the fifteenth century. Describing the location of the example from Turkey, be- tween Mesopotamia and Greece, Bodi noted it is ‘geographically […] signif- icant’.12 The proverb’s adoption into Greek was early, Archilochus of Paros (sev- enth century BCE) referenced it: ‘I am afraid, lest acting hastily out of eager- ness, I begat like a bitch in the proverb children blind and untimely’.13 [δε]δοιχ όπωσ τυφλα καλιτημερα [σπ]ουδη επειγομενοσ τωσ ʹωςπερ ή fear as blind gooddays hi bi h fear as blind gooddays rushing gavebirth so over theF dog the rushing gavebirth so over theF dog then ‘I fear like the dog rushing, to give birth to blind pups.’ ‘I fear like the dog rushing, to give birth to blind pups.’ Other Greeks used it later, such as Aesop (sixth century BCE) in a fable about a bitch bragging to a sow how fast she gives birth,15 in return the sow taunted the bitch, ‘You give birth to the blind’.16 κύων επισπευδουσα τυφλα γεννα17 dog hurrying blind birth ‘You give birth to the blind.’ ‘You give birth to the blind.’ Aristophanes used the proverb in his play Peace in 421 BCE: ‘The bitch in her haste gave birth to the blind’. ἡ κύων σπεύδουσα τυφλα τίκτει18 the dog rushing blind pups ‘The dog by haste produced blind pups.’ A fragment of Archilochus’s poetry citing this proverb was used as part of the wrapping of an Egyptian mummy from the first or second century 12 Bodi 2015, 78. 13 Moran 1978; 2002. 15 The fable is number 223 in Perry’s standard index of Aesop’s fables (Perry 1952) 16 Bodi 2015, 77. 16 Bodi 2015, 77. 17 Houghton 1915, 28. 18 Moran 2002, 90. Aethiopica 21 (2018) Aethiopica 21 (2018) 229 Peter Unseth Peter Unseth 19 Merkelbach and West 1974. 20 Hinz 2004. 25 For Guǧǧi Oromo see Unseth 2011, 433; for Allaaba see Unseth 2013, 461. 26 Fekede Menuta 2014, 36. 27 Avishur 1981, 38. 28 Personal communication (2017). 29 Tadesse Jaleta 2004, 85 and Tadesse Jaleta Jirata 2009, 50. The symbol <j> is used by multiple authors quoted in this article to represent a voiced affricate [d͡ʒ]. 30 Mieder and Litovkina 2006, 20. Ethiopia Documentation is provided here that this proverb, previously well docu- mented and studied in Mesopotamia and Europe, has now been noted in at least four languages of Ethiopia. In book reviews, I have previously men- tioned the parallel between the Akkadian proverb and proverbs currently found in Guǧǧi Oromo and Allaaba.25 Also, the proverb has been docu- mented in Western Gurage, a form of Gurage, 26 a Semitic language of southcentral Ethiopia. Gurage is contiguous to Allaaba and near Guǧǧi Oromo, all in the southcentral part of Ethiopia, near to each other. Allaaba is Highland East Cushitic, Guǧǧi Oromo is Lowland East Cushitic, and Gurage is Semitic. Speakers of all three of these language communities con- tain large Muslim populations which might suggest a link toward Arabic speaking areas to the north, but Avishur reported finding it only in the Ar- abic of Iraq.27 It might also be suspected that the proverb is found in nearby Somali, a Lowland East Cushitic language like Guǧǧi Oromo. However, Georgi Kapchits, editor of a large collection of Somali proverbs, reported that this proverb is not found in Somali.28 Also, it is not found in Amharic. p Examples of the proverb from each of the Ethiopian languages where it is found are transcribed as in the original sources. ǧǧ 26 Fekede Menuta 2014, 36. 28 Personal communication (2017). 27 Avishur 1981, 38. p q 30 Mieder and Litovkina 2006, 20. Europe In Europe, it seems that the proverb first entered the continent via the Greek language. The Greek scholar and copyist Michael Apostolios (b.1420) transcribed many Greek proverbs, likely the source that brought this prov- erb to the attention of Erasmus.20 Proverbs that refer to a bitch giving birth to blind pups (or a cat giving birth to blind kittens) because of her haste have also been found across Eu- rope all the way to Britain where it has been passed down as ‘The hasty bitch bringeth forth blind whelps’.21 The proverb still lives on today in Modern Greek: ‘Η σκύλα από τη βιάση της τα κάνει στραβά τα κουτάβια της’ (‘The bitch made such haste that she gave birth to blind puppies’).22 It was adopted into German, where it lives on as ‘Die eilende Hündin wirft blinde Junge’. In French, it has come down as ‘La chienne dans sa hâte a mis bas des chiots aveugles’. In Italian, it be- came known in two forms, one with a hasty dog (‘Cagna frettolosa fa catellini ciechi’) and another with a hasty cat (‘Gatta frettolosa fa i gattini ciechi’).23 Similarly, Portuguese has two versions, one with a dog (‘Cadelas apressadas parem cães tortos’) and one with a cat (‘Cachorra apressada pare filhos cegos’). In Spanish, the version with a cat is also found, ‘La gata pre- surosa para los gatitos ciegos’. In Romanian, the cat version of this is also found, ‘Căţeaua de pripă işi naşte căţeii fără ochi’.24 19 Merkelbach and West 1974. 22 Personal communication from Marina Mogli. 23 Taylor 1962, 25; Bodi 2015, 77. y 24 Flonta 2012, 1. 24 Flonta 2012, 1. Aethiopica 21 (2018) 230 230 25 For Guǧǧi Oromo see Unseth 2011, 433; for Allaaba see Unseth 2013, 461. 26 Gurage gɨjǝ mǝt’ jǝft’ǝre barǝm; furt’ ʧ’ǝnǝm33 bitch labour 3SgMfast sayPST blind give.birthPST34 ‘Saying give birth faster; a bitch gave birth to a blind puppy.’ In the Gurage proverb, we find a striking number of ejective t’ and ʧ’, here in bold: ‘gɨjǝ mǝt’ jǝft’ǝre barǝm; furt’ ʧ’ǝnǝm’. Also, note the sequences of vowel<r>: ‘jǝft’ǝre barǝm; furt’’. Also, the Gurage form of the proverb is a couplet, and both halves end with a rhyme of ǝm. Additionally, remembering that Gurage is a Semitic language, where the consonants are psycholinguistical- ly more prominent than the vowels, it is significant that the couplets, when we ignore the vowels, contain the similar consonantal sequences ft’r and frt’. Clearly, this Gurage proverb was formed artistically. Peter Unseth Horn of Africa.31 Additionally, this proverb specifies the number of the blind puppies as ‘nine’, ‘sagal’. In a Guǧǧi riddle game about numbers, the standard answer for ‘eight’ is that it is the number of puppies in a litter, but in this proverb the number of puppies in the litter is larger, nine. This is significant because among the Guǧǧi the ‘birth of a ninth child is believed to bring misfortune.’32 g The Guǧǧi proverb shows clear signs of artistic efforts, including being formed with only twosyllable words, the two words of the quotation allit- eratively beginning with ma, sound similarities being heard in ‘jette’ and ‘deettee’, and sequences of vowel assonance. Guǧǧi Oromo ‘Mali maqnee’, jette sareen jaamaa sagal deettee29 why? evil said dog blind nine gavebirth ‘“What’s our sin?” said a bitch after giving birth to nine blind pups. ‘“What’s our sin?” said a bitch after giving birth to nine blind pups.’ The Guǧǧi Oromo version of the proverb is recognizably similar, but different in some interesting ways. First, the matter of haste is not men- tioned, rather the broader term ‘sin’. Secondly, the proverb is formed as a wellerism, a quotation proverb in which there is a statement, a speaker, and (often) an unusual circumstance,30 a proverb structure very common in the p q 30 Mieder and Litovkina 2006, 20. Aethiopica 21 (2018) 231 Peter Unseth 31 Unseth et al. 2017, 23, 24. 32 Taddesse Berisso 2013, 59. 34 In Semitic languages of Ethiopia, it is common to use the verb ‘say’ to indicate inten- tion. A clearer translation could be ‘Trying to give birth faster, a bitch gave birth to a blind puppy’. 36 Sudan Tribune 2014. Miscellaneous In Allaaba, the last two words have a pattern of alliteration of the ejective velar consonant, here in bold: ‘k’ook’á k’altáa’. Clearly, this is artistically crafted for the ear. In addition to these three neighbouring languages in central Ethiopia— Guǧǧi Oromo, Allaaba, and Gurage—the proverb is also found in the Nuer language, spoken in far western Ethiopia and in South Sudan. ɛ pɛ̈ɛ̈th ɛn min daap kɛ jiok kɛ pääth diɔk kɛ gaat tä ken nyien kien kueth (gaat ti coor) itis hurry SUB that givebirth ? dog with month three with babies that NEG eyes their mature (babies that blind) ‘Because she rushed her pregnancy, that is why the dog gave birth in three months to the puppies whose eyes are not mature (blind babies).’ ɛ pɛ̈ɛ̈th ɛn min daap kɛ jiok kɛ pääth diɔk kɛ gaat tä ken nyien kien kueth (gaat ti coor) gaat tä ken nyien kien kueth (gaat ti coor) itis hurry SUB that givebirth ? dog with month three with babies that NEG eyes their mature (babies that blind) ‘Because she rushed her pregnancy, that is why the dog gave birth in itis hurry SUB that givebirth ? dog with month three with babies that NEG eyes their mature (babies that blind) ‘Because she rushed her pregnancy, that is why the dog gave birth in three months to the puppies whose eyes are not mature (blind babies).’ Looking for evidence of poetic structure in the Nuer form, the eight rep- etitions of the consonant k are striking. Also, the following seems artistic: ‘jiok kɛ […] diɔk kɛ’. This proverb, though reported to be new among the Nuer, has become established enough among them that a government official quoted the prov- erb to underline the need for patience when addressing a group of Nuer: ‘It is lack of patience, just after three months of pregnancy that makes [a] dog give birth to blind puppies.’36 This is a wonderful use of the proverb in a context where proverbs are traditionally frequent, a call for patience and reconciliation. Whether these language communities found in Ethiopia have borrowed the proverb or have created it, all have crafted forms of the proverb that contain verbal art. Allaaba wússhat(i), daʔlansí batiɲɲíih(a) k’ook’á k’altáa35 dogFEM bequick extreme blind gavebirth ‘The shedog, because she is in extreme hurry, gives birth to blind ones.’ wússhat(i), daʔlansí batiɲɲíih(a) k’ook’á k’altáa35 dogFEM bequick extreme blind gavebirth ‘The shedog, because she is in extreme hurry, gives birth to blind ones.’ 31 Unseth et al. 2017, 23, 24. 32 Taddesse Berisso 2013, 59. 33 Fekede Menuta 2014, 36. , 34 In Semitic languages of Ethiopia, it is common to use the verb ‘say’ to indicate inten- tion. A clearer translation could be ‘Trying to give birth faster, a bitch gave birth to a blind puppy’. 34 In Semitic languages of Ethiopia, it is common to use the verb ‘say’ to indicate inten- tion. A clearer translation could be ‘Trying to give birth faster, a bitch gave birth to a blind puppy’. p ppy 35 SchneiderBlum 2009, 95. Aethiopica 21 (2018) 232 Peter Unseth oped as an axiom that older forms are usually preserved at the geographic periphery.37 The dog is the animal found in the proverb in the geographic peripheries: Britain and Ethiopia. The Ethiopian data is important in show- ing that the dog form of the proverb is original. Conclusion The evidence presented here calls attention to the fact that this wellstudied ancient proverb from Mesopotamia and Europe is also found in the Horn of Africa, outside of the previously documented regions in the Middle East and Europe. There have been ancient trade links between these regions, but the specifics of directions and how this proverb has spread are lost. Howev- er, links between Mesopotamia and Ethiopia were not limited to commer- cial trade, as is seen by the borrowing of a word from Mesopotamia into Gǝʿǝz for an astronomical term, a borrowing that was not via Greek.38 This proverb data indicates that scholars should search for traces of this proverb in the regions between Mesopotamia and the Horn of Africa, as well as in additional languages in the Horn. This data from Ethiopia raises many more questions and calls for alertness among proverb scholars as they study proverbs from other languages in the region. 38 Lourié 2012. 37 Hock 1986, 440. 37 Hock 1986, 440. 38 Lourié 2012. Versions with a Hasty Cat instead of Dog Versions with a Hasty Cat instead of Dog The oldest version of this proverb mentions a dog. Later versions of this proverb with a hasty cat instead of a dog are found in a number of places in Europe and also in Iraq. The proverbs from Ethiopia only have a dog, no examples have been found in the Horn of Africa with a cat. This leads to the question of which form of the proverb is original, the dog or the cat? A useful heuristic is taken from historical linguistics, where it has been devel- Aethiopica 21 (2018) Aethiopica 21 (2018) 233 Peter Unseth References Alster, B. 1979. ‘An Akkadian and a Greek Proverb: A Comparative Study’, Die Welt des Orients, 10 (1979), 1–5. Apperson, G. L. 1993. The Wordsworth Dictionary of Proverbs: A lexicon of folklore and traditional wisdom, Wordsworth Reference (Ware: Wordsworth, 1993). Avishur, Y. 1981. ‘Additional Parallels of an Akkadian Proverb Found in the Iraqi Ver- nacular Arabic’, Die Welt des Orients, 12 (1981), 37–38. Bodi, D. 2015. ‘CrossCultural Transformation of Animal Proverbs (Sumer, Mari, Hebrew Bible, Aramaic Ahiqar and Aesop’s Fables)’, in M.S. Ortola and G. AchardBayle, eds, Concepts éthiques et moraux: Approches multiculturelles et inter- disciplinaires, Sémantique des énoncés parémiques, Aliento, Échanges sapientiels en Méditerranée, 6 (Nancy: Edulor–Presses Universitaires de Nancy–Éditions Universi- taires de Lorraine, 2015), 61–112. Bonechi, M. 2014. ‘Materiali per una definizione della più antica sapienza siriana: I pro- verbi di Mari e di Ebla’, Studi Epigrafici e Linguistici, 31 (2014), 81–110. Bremmer, J. 1980. ‘An Akkadian Hasty Bitch and the New Archilochus’, Zeitschrift für Papyrologie und Epigraphik, 39 (1980), 28. 234 Aethiopica 21 (2018) Miscellaneous Chavalas, M. W. 2014. Women in the Ancient Near East: A Sourcebook, ed. M. W. Chavalas, Routledge Sourcebooks for the Ancient World (London: Routledge, 2014). Dalley, S. and A. T. Reyes 1997. ‘Mesopotamian Contact and Influence in the Greek World: 1. To the Persian Conquest, 2: Persia, Alexander, and Rome’, in S. Dalley, ed., The Legacy of Mesopotamia (Oxford: Oxford University Press, 1997), 85–124. Fekade Azeze 2001. ‘The State of Oral Literature Research in Ethiopia: Retrospect and Prospect’, Journal of Ethiopian Studies, 34/1 (2001), 43–85. Fekede Menuta 2014. ‘Discourses of Development in Gurage Proverbs’, Ethiopian Jour- nal of Social Sciences and Language Studies, 1/1 (2014), 25–40. Flonta, T. 2012. A Dictionary of English and Romance Languages Equivalent Proverbs: Over 3,200 English Proverbs and Thousands of Equivalents in French, Italian, Span- ish, Portuguese and Romanian (Hobart, TAS: DeProverbio.com, 2012). Gordon, E. I. 1958a. ‘Sumerian Animal Proverbs and Fables: “Collection Five”’, Journal of Cuneiform Studies, 12/1 (1958), 1–21. — 1958b. ‘Sumerian Animal Proverbs and Fables: “Collection Five” (Conclusion)’, Journal of Cuneiform Studies, 12/2 (1958), 43–75. Hinz, V. 2004. ‘«Die eilende Hündin wirft blinde Junge» und einige andere antike Sprichwörter bei Michael Apostolios und Erasmus’, Antike und Abendland, 50 (2004), 124–148. Hock, H. H. 1986. Principles of Historical Linguistics (Berlin–New York, NY– Amsterdam: Mouton de Gruyter, 1986). Houghton, H. P. 1915. References Moral Significance of Animals as Indicated in Greek Proverbs (Amherst, MA: Carpenter & Morehouse, 1915). Lourié, B. 2012. ‘Kéntros, another Term of Mesopotamian Origin in the Ethiopian As- tronomy’, Aethiopica, 15 (2012), 225–227. Merkelbach, R. and M. L. West 1974. ‘Ein ArchilochosPapyrus’, Zeitschrift für Papyro- logie und Epigraphik, 14 (1974), 97–113. Mieder, W. 2014. Behold the Proverbs of a People: Proverbial Wisdom in Culture, Liter- ature, and Politics (Jackson, MS: University Press of Mississippi, 2014). Mieder, W. and A. T. Litovkina 2006. Old proverbs never die, they just diversify: a col- lection of antiproverbs (Burlington, VT: University of Vermont, Veszprém: Panno- nian University of Veszprém, 2006). Moran, W. L. 1978. ‘An Assyriological Gloss on the New Archilochus Fragment’, Har- vard Studies in Classical Philology, 82 (1978), 17–19. — 2002. ‘Puppies in Proverbs—From ŠamšiAdad to Archilochus?’, in W. L. Moran, ed. R. S. Hendel, The Most Magic Word: Essays on Babylonian and Biblical Literature, The Catholic Biblical Quarterly, Monograph Series, 35 (Washington DC: Catholic Biblical Association of America, 2002), 87–97. Perry, B. E. 1952. Aesopica: A Series of Texts Relating to Aesop or Ascribed to him or Closely Connected with the Literary Tradition that Bears his Name, Collected and Critically Edited, in part Translated from Oriental Languages, with a Commentary and Historical Essay, I: Greek and Latin Texts, ed. B. E. Perry (Urbana, IL: Univer- sity of Illinois Press, 1952). Aethiopica 21 (2018) 235 Peter Unseth SchneiderBlum, G. 2009. Máakut(i) tʾawá shuultáa: Proverbs finish the problems, Say- ings of the Alaaba (Ethiopia), ed. G. SchneiderBlum, Verbal Art and Documentary Literature in African Languages, 28 (Köln: Rüdiger Köppe Verlag, 2009). Slings, S. R. 1976. ‘Archilochus, the Hasty Mind and the Hasty Bitch’, Zeitschrift für Papyrologie und Epigraphik, 21 (1976), 283–288. Sudan Tribune 2014. ‘Jonglei governor calls for peace at grassroots level’, Sudan Tribune (13 October 2014), http://sudantribune.com/spip.php?article52707, accessed on 15 August 2018. Taddesse Berisso 2013. ‘The Riddles of the Number Nine in GujiOromo Culture’, in Bekele Gutema and C. C. Verharen, eds, African Philosophy in Ethiopia, Ethiopian Philosophical Studies II, Cultural Heritage and Contemporary Change, 2, Africa, 15 (Washington DC: The Council for Research in Values and Philosophy, 2013), 53–68. Tadesse Jaleta [Tadesse Jaleta Jirata] 2004. A Contextual Study of GujiOromo Proverbs: functions in Focus, MA Thesis, Addis Ababa University (2004). — 2009. References A Contextual Study of the Social Functions of GujiOromo Proverbs: The Savor and Rhetoric Power of Verbal Arts in Everyday Communications of African Peoples (Saarbrücken: Verlag Dr. Müller, 2009). Taylor, A. 1962. The Proverb and an Index to The Proverb (Hatboro, PA: Folklore Associates, Copenhagen: Rosenkilde and Bagger, 1962). Unseth, P. 2011. ‘Review of Tadesse Jaleta Jirata, A Contextual Study of the Social Func- tions of GujiOromo Proverbs: The Savor and Rhetoric Power of Verbal Arts in Eve- ryday Communications of African Peoples (Saarbrücken: Verlag Dr. Müller, 2009) and Abdullahi A. Shongolo and Günther Schlee, Boran Proverbs in their Cultural Context (Köln: Rüdiger Köppe Verlag, 2007)’, Proverbium, 28 (2011), 427–434. — 2013. ‘Review of Gertrud SchneiderBlum, ed., Máakut(i) tʾawá shuultáa: Proverbs finish the problems, Sayings of the Alaaba (Ethiopia), Verbal Art and Documentary Literature in African Languages, 28 (Köln: Rüdiger Köppe Verlag, 2009)’, Proverbium, 30 (2013), 459–461. Unseth, P., D. Kliemt, L. Morgan, S. Nelson, and E. M. Scherrer 2017. ‘Wellerism prov- erbs: Mapping their distribution’, GIALens, 11/3 (2017), 1–35. West, M. L. 1997. The East Face of Helicon: West Asiatic Elements in Greek Poetry and Myth (Oxford: Clarendon Press, 1997). Summary The world’s oldest living proverb, from around 3,800 years ago, is found on a tablet from the Assyrian empire. The proverb has been documented from later eras, northwest from the Middle East up into Europe, as far northwest as Britain. Evidence is given here now demonstrating that the proverb is also found to the south of the Middle East, in Ethiopia. In some places, a cat is substituted for the dog, but the Ethiopian evidence indicates that the dog version of the proverb is original. Aethiopica 21 (2018) Aethiopica 21 (2018) 236
https://openalex.org/W4312799095	https://journals.kozminski.edu.pl/system/files/Buchalska_0.pdf	English	null	Using a Surname in a Trademark: Has the Messi Case Changed Case-Law?	Krytyka Prawa	2,022	cc-by	9,067	Using a Surname in a Trademark: Has the Messi Case Changed Case-Law? 3 Wpłynął: 3.05.2021. Akceptacja: 23.08.2022 p 3 The research has not been supported financially by any institution. 1 Van Anh Le, PhD – University of Oxford, Faculty of Law (United Kingdom); e-mail: van-anh.le@law. ox.ac.uk; ORCID: 0000-0001-8592-3135. „Krytyka Prawa”, tom 14, nr 3/2022, s. 67–84, ISSN 2080-1084, e-ISSN 2450-7938, © 2022 Authors. This is an open access article distributed under the Creative Commons BY 4.0 license: http://creativecommons.org/licenses/by/4.0 „Krytyka Prawa”, tom 14, nr 3/2022, s. 67–84, ISSN 2080-1084, e-ISSN 2450-7938, © 2022 Authors. This is an open access article distributed under the Creative Commons BY 4.0 license: http://creativecommons.org/licenses/by/4.0 VAN ANH LE1, JOANNA BUCHALSKA2 2 Joanna Buchalska, PhD – Kozminski University, Civil Department (Poland); e-mail: jbuchalska@ kozminski.edu.pl; ORCID: 0000-0001-9012-950X. Abstract Using a surname as a trademark or part of a trademark has been the subject of numerous case-laws and has been widely discussed in the literature. However, it seems that after the Messi case (cases C-449/18 P and C-474/18 P), the Court of Justice of the European Union (CJEU) seems to have departed from their previous approach where it was held that surnames in trademarks should be treated as normal signs. In the Messi case, the CJEU, however, ruled that the reputation of Messi – an internationally famous football player – is so well known that an average consumer, seeing the ‘MESSI’ mark placed on clothing, gymnastic or sporting articles and protective equipment, will establish a link between the mark and the sports personality, despite the similarity between the ‘MESSI’ mark to the ‘MASSI’ brand name, a previously registered trademark. However, this ruling gives rise to the following questions, which this article seeks to address: How can one prove that someone’s surname is globally recognised? Why did the CJEU decide that Messi is better known than, for instance, Picasso who was the subject of the previous case? Who else can be as famous as Messi if Picasso was held not to be? Has Messi changed the CJEU’s approach and opened the floodgates to expand trademark protection for an unlimited number of trademarks? This article attempts to answer these questions. This article attempts to answer these questions. 3 The research has not been supported financially by any institution. VAN ANH LE, JOANNA BUCHALSKA VAN ANH LE, JOANNA BUCHALSKA 4 Badania wykorzystane w artykule nie zostały sfinansowane przez żadną instytucję. Keywords: trademark, surname, reputation. Keywords: trademark, surname, reputation. 1 Van Anh Le, PhD – University of Oxford, Faculty of Law (United Kingdom); e-mail: van-anh.le@law. ox.ac.uk; ORCID: 0000-0001-8592-3135. 2 Joanna Buchalska, PhD – Kozminski University, Civil Department (Poland); e-mail: jbuchalska@ kozminski.edu.pl; ORCID: 0000-0001-9012-950X. DOI: 10.7206/kp.2080-1084.541 Tom 14, nr 3/2022 „Krytyka Prawa”, tom 14, nr 3/2022, s. 67–84, ISSN 2080-1084, e-ISSN 2450-7938, © 2022 Authors. This is an open access article distributed under the Creative Commons BY 4.0 license: http://creativecommons.org/licenses/by/4.0 „Krytyka Prawa”, tom 14, nr 3/2022, s. 67–84, ISSN 2080-1084, e-ISSN 2450-7938, © 2022 Authors. This is an open access article distributed under the Creative Commons BY 4.0 license: http://creativecommons.org/licenses/by/4.0 5 Judgment of the Court (Second Chamber) of 16 September 2004, Nichols plc v. Registrar of Trade Marks, Case C-404/02, Judgment of the General Court (Second Chamber) of 1 March 2005, Vincenzo Fusco v. European Union Intellectual Property Office, Case T-185/03, Judgment of the General Court (Fourth Chamber) of 13 July 2005, Julián Murúa Entrena v. European Union Intellectual Property Office, Case T-40/03. Introduction Using a surname as a trademark or part of a brand has been subject to numerous CJEU cases where surnames have been held to be treated as normal signs.5 How- ever, after the Messi case (cases C‑449/18 P and C‑474/18 P), the Court seems to have changed its previous approach. It held that the reputation of Messi – an internationally well-known football player, is so famous that an average consumer, seeing the ‘MESSI’ mark, cannot ignore the link between the sign and the sports personality, despite its similarity to ‘MASSI’, a previously registered trademark. However, this ruling gives rise to the following questions, which this article seeks to address: 1. How to prove that someone’s surname is globally recognised? 2. Why did the Court decide that Messi is better known than, e.g. Picasso, the subject of the previous case?l 2. Why did the Court decide that Messi is better known than, e.g. Picasso, the subject of the previous case? 3. Has the Messi case changed the Court’s interpretation and opened the flood gates to expand their trademark protection for an unlimited number of trade marks? Who else can be as famous as Messi if Picasso was held not to be? Streszczenie Posługiwanie się nazwiskiem jako znakiem towarowym lub jego częścią było już przedmiotem licznych wypowiedzi przedstawicieli doktryny oraz judykatury. Wydaje się jednak, że po sprawie Messi (sprawy C-449/18 P i C-474/18 P) Trybunał Sprawiedliwości Unii Europejskiej (TSUE) odszedł od swojej wcześniejszej linii orzeczniczej, zgodnie z którą uznawano, że nazwiska zawarte lub będące znakiem towarowym należy traktować tak jak inne oznaczenia (znaki towarowe). W sprawie Posługiwanie się nazwiskiem jako znakiem towarowym lub jego częścią było już przedmiotem licznych wypowiedzi przedstawicieli doktryny oraz judykatury. Wydaje się jednak, że po sprawie Messi (sprawy C-449/18 P i C-474/18 P) Trybunał Sprawiedliwości Unii Europejskiej (TSUE) odszedł od swojej wcześniejszej linii orzeczniczej, zgodnie z którą uznawano, że nazwiska zawarte lub będące znakiem towarowym należy traktować tak jak inne oznaczenia (znaki towarowe). W sprawie Messi Trybunał przyjął, że renoma Messiego – piłkarza o międzynarodowej sławie – jest tak duża, że przeciętny konsument, widząc znak „MESSI” umieszczony na odzieży, artykułach gimnastycznych lub sportowych oraz sprzęcie ochronnym, nie skojarzy go ze znakiem „MASSI”, który został wcześniej zarejestrowanym znakiem towarowym. Messi Trybunał przyjął, że renoma Messiego – piłkarza o międzynarodo – jest tak duża, że przeciętny konsument, widząc znak „MESSI” umieszczony na odzieży, artykułach gimnastycznych lub sportowych oraz sprzęcie ochronnym, nie skojarzy go ze znakiem „MASSI”, który został wcześniej zarejestrowanym znakiem towarowym. – jest tak duża, że przeciętny konsument, widząc znak „MESSI” umieszczony na odzieży, artykułach gimnastycznych lub sportowych oraz sprzęcie ochronnym, nie skojarzy go ze znakiem „MASSI”, który został wcześniej zarejestrowanym znakiem towarowym. Orzeczenie to rodzi następujące pytania, na które postarano się odpowiedzieć w niniejszym artykule: jak udowodnić, że czyjeś nazwisko jest globalnie rozpoznawalne? dlaczego TSUE uznał, że Messi jest bardziej znany niż np. Picasso, którego dotyczyło wcześniejsze orzecznictwo? kto jeszcze mógłby być „tak sławny” jak Messi, aby w inny sposób traktować taki znak towarowy (renomowany)? czy sprawa Messi zmieni podejście TSUE i rozszerzy ochronę znaków towarowych na nieograniczoną liczbę znaków towarowych? Słowa kluczowe: znak towarowy, nazwisko, reputacja. Tom 14, nr 3/2022 Tom 14, nr 3/2022 DOI: 10.7206/kp.2080-1084.541 USING A SURNAME IN A TRADEMARK: HAS THE MESSI CASE CHANGED… 69 6 J. Buchalska, Nazwisko jako przedmiot ochrony w prawie polskim, Warszawa 2016, pp. 15 ff. The Significance of Surnames A surname accompanies a person from birth to death and sometimes ‘lives’ for centuries, occupying a permanent place in a nation’s history, e.g. Napoleon, Picasso, Chopin. The surname remains the crucial element of identification of a person and it is credited with stability and immutability.6 The surname is an element of legal identification, created in the long-term development process, in the course of which the aim was initially only a temporary, current identification, based on Tom 14, nr 3/2022 DOI: 10.7206/kp.2080-1084.541 Van Anh Le, Joanna Buchalska 70 various variable markings.7 It is a source of knowledge about the social and politi cal history of a nation, about the development of its culture and realities of life, and sometimes about certain values. A person’s surname is part of his or her perso nality because in the public space, one is defined precisely by his or her surname and at the same time, it can – and it often should – carry a specific image of the person who uses it.8 However, many characteristics are ascribed to the surname. For instance, it individualises natural persons, which is the basis for assigning them specific rights and obligations. The surname also shapes the principle of separating individual natural persons in the civil law sphere. One can distinguish three basic functions of the surname – identification (ordinal function), the role of a personality symbol (personal right) and the function of family belonging.9 The additional functions of the surname are to define the nationality or ethnic ties; the shape of the surname also allows, in some languages, to determine the gender of the person holding it. The surname also creates a kind of portrait of the person holding it, which allows one to recognise the person and their characteristics.10 For instance, this ‘metaphysical’ portrait11 can frequently be equated with protecting personal rights. This is because many celebrities have associated their brands bearing their surname.12 Naturally, we feel the differences between the surname as part of a personal trademark and the surname as such. The differences between those two rights were limited by creating the right of publicity, which can be transferable as an economic right. However, the right of publicity has a dichotomous aspect – audience perception and a physical human individual (natural person). Moreover, it does not allow the person to control the use of the name and reputation. 12 Compare: https://www.vogue.com/article/catherine-malandrino-new-see-now-buy-now-hsn-collection; http://www.southflorida.com/video/sf-boca-fashion-kate-spade-20160412-story.html (access: 12.11.2021); https://www.wsj.com/articles/when-is-kate-spade-not-kate-spade-when-shes-frances-valen- tine-1471971817 (access: 12.11.2021). 9 P. Lagarde, L’oeuvre de la Commission Internationale de l’Etat Civil en matière de nom des personnes, [in:] H.-P. Mansel, T. Pfeiffer, H. Kronke, Ch. Köhler, R. Hausmann (eds.), Festschrift für Erik Jayme, Band II, Munich 2004, p. 1291; K. Funken, Das Anerkennungsprinzip im internationalen Privatrecht. Perspektiven eines euro- päischen Anerkennungskollisionsrechts für Statusfragen, München 2009, p. 293; P. Meier, E. de Luze, Droit des personnes. Articles 11–89a CC, Schulthess 2014, pp. 120 ff. , p 8 R. Freitag, Subjektive Anknüpfung: Vorstellung des Vorschlags, [in:] A. Dutta, T. Helms, W. Printens (eds.), Ein Name in ganz Europa, Vorschläge für ein Internationales Namensrecht der Europäischen Union, Frankfurt am Main 2016, p. 51. p pp 10 S. Nautré, Le nom en droit compare, Frankfurt am Main 1977, pp. 9 ff.; R. Freitag, Subjektive Anknüpfung…, p. 51. 11 S. Nautré, Le nom…, p. 29. 7 Ibidem, p. 24. 8 R. Freitag, Subjektive Anknüpfung: Vorstellung des Vorschlags, [in:] A. Dutta, T. Helms, W. Printens (eds.), Ein Name in ganz Europa, Vorschläge für ein Internationales Namensrecht der Europäischen Union, Frankfurt am Main 2016, p. 51. 9 P. Lagarde, L’oeuvre de la Commission Internationale de l’Etat Civil en matière de nom des personnes, [in:] H.-P. Mansel, T. Pfeiffer, H. Kronke, Ch. Köhler, R. Hausmann (eds.), Festschrift für Erik Jayme, Band II, Munich 2004, p. 1291; K. Funken, Das Anerkennungsprinzip im internationalen Privatrecht. Perspektiven eines euro- päischen Anerkennungskollisionsrechts für Statusfragen, München 2009, p. 293; P. Meier, E. de Luze, Droit des personnes. Articles 11–89a CC, Schulthess 2014, pp. 120 ff. 10 S. Nautré, Le nom en droit compare, Frankfurt am Main 1977, pp. 9 ff.; R. Freitag, Subjektive Anknüpfung…, p. 51. 11 S. Nautré, Le nom…, p. 29. 12 Compare: https://www.vogue.com/article/catherine-malandrino-new-see-now-buy-now-hsn-collection; http://www.southflorida.com/video/sf-boca-fashion-kate-spade-20160412-story.html (access: 12.11.2021); https://www.wsj.com/articles/when-is-kate-spade-not-kate-spade-when-shes-frances-valen- tine-1471971817 (access: 12.11.2021). 7 Ibidem, p. 24. The Significance of Surnames In those cases, after transferring the name as part of the right of publicity, it is no longer under the control of the surname bearer. It can also be split into two – on the one hand, 12 Compare: https://www.vogue.com/article/catherine-malandrino-new-see-now-buy-now-hsn-collection; http://www.southflorida.com/video/sf-boca-fashion-kate-spade-20160412-story.html (access: 12.11.2021); https://www.wsj.com/articles/when-is-kate-spade-not-kate-spade-when-shes-frances-valen- tine-1471971817 (access: 12.11.2021). DOI: 10.7206/kp.2080-1084.541 Tom 14, nr 3/2022 USING A SURNAME IN A TRADEMARK: HAS THE MESSI CASE CHANGED… 71 representing a product of their actual identity and creativity. On the other hand, a public person is controlled by the assignee using their identity. Many brands also engage celebrities to increase the popularity of products or services they sell.13 Usually, this results in registering their surname as a trademark. However, along with financial problems, a surname – as a trademark – becomes the property of another entity, which uses it.14 On the other hand, according to Article 14(1) of Regulation (EU) 2017/1001,15 an EU trademark (EUTM) owner cannot prohibit the third party from using the name or the address of the third party, where that third party is a natural person.16 14 Compare: https://www.vogue.com/article/catherine-malandrino-new-see-now-buy-now-hsn-collection; http://www.southflorida.com/video/sf-boca-fashion-kate-spade-20160412-story.html (access: 12.11.2021); https://www.wsj.com/articles/when-is-kate-spade-not-kate-spade-when-shes-frances-valen- tine-1471971817 (access: 12.11.2021). 13 Compare: [2013] EWHC 3459 (Ch) http://www.vogue.co.uk/article/jane-birkin-asks-remove-name-her- mes-bag-peta-investigation; http://www.vogue.co.uk/article/tommy-hilfiger-defends-gigi-hadid (access: 12.11.2021). 15 Regulation (EU) 2017/1001 of the European Parliament and of the Council of 14 June 2017 on the Eu- ropean Union trademark. 16 ECJ: Judgment in Céline SARL v. Céline SA C-17/06, EU:C:2007:497. Summary of the Messi Case In August 2011, Lionel Andrés Messi Cuccittini, the famous football player (the Applicant), applied for the registration of a figurative mark ‘MESSI’ with the Euro- pean Union Intellectual Property Office (EUIPO), in Nice Classes 9, 25 and 28 as follows: In November 2011, the owner of Massi – a Spanish company dealing in cycling equipment and clothing – the products registered in Classes 9, 25 and 28 – filed an opposition to Messi’s registration on the ground of the likelihood of confusion. As an aside, the term massi means ‘large rocks’ in Italian. 13 Compare: [2013] EWHC 3459 (Ch) http://www.vogue.co.uk/article/jane-birkin-asks-remove-name-her- mes-bag-peta-investigation; http://www.vogue.co.uk/article/tommy-hilfiger-defends-gigi-hadid (access: 12.11.2021). 14 Compare: https://www.vogue.com/article/catherine-malandrino-new-see-now-buy-now-hsn-collection; http://www.southflorida.com/video/sf-boca-fashion-kate-spade-20160412-story.html (access: 12.11.2021); https://www.wsj.com/articles/when-is-kate-spade-not-kate-spade-when-shes-frances-valen- tine-1471971817 (access: 12.11.2021). 15 Regulation (EU) 2017/1001 of the European Parliament and of the Council of 14 June 2017 on the Eu- ropean Union trademark. 16 ECJ Judgment in Céline SARL Céline SA C 17/06 EU C 2007 497 DOI: 10.7206/kp.2080-1084.541 Tom 14, nr 3/2022 Van Anh Le, Joanna Buchalska 72 In June 2013, the EUIPO Division of Opposition sided with the opponent. Dissatisfied with this decision, Messi appealed to the Board of Appeal, which rejected his request, confirming the conclusion of the Division of Opposition. The Board of Appeal held that their dominant components comprised of the terms ‘MASSI’ and ‘MESSI’ were virtually identical visually and phonetically. Although there is a possible conceptual differentiation, if at all, by a part of the public, such a difference cannot outweigh the phonetic and visual similarities. Moreover, Messi’s goods are almost identical to the goods covered by the earlier registration of ‘Massi’. In July 2014, Lionel Messi filed an appeal before the EU General Court, claim- ing that the Board of Appeal had erred by holding the risk of confusion between the conflicting trademarks. The EU General Court agreed with Lionel Messi. The EUIPO and Massi were displeased with the General Court’s ruling, so they took the case to the CJEU. Firstly, the CJEU found that visually the marks are only similar because consumers are accustomed to graphic elements being pro- moted by trademarks. These are stylised differently in both marks.17 Secondly, although the Court endorsed the position of the Board of Appeal, claiming that the marks are phonetically similar, it took a different view regarding the concep- tual layer of marks. 17 Paragraph 43 et seq. of the justification. 18 Paragraph 50 et seq. of the justification of the judgment. 19 Paragraph 52 of the justification. 20 Paragraph 61 of the judgment. Summary of the Messi Case According to the Court, the Board of Appeal wrongly claimed that Messi’s reputation was recognised only by a part of the public interested in football and sports.18 In the Court’s view, Messi is a public figure known recognised by informed, attentive and competent customers who read the press, watch the news on TV, go to the cinema or listen to the radio. It is possible to see him and where he is mentioned.19 The reputation of Messi as the surname of a world-famous football player and public figure is a well-known fact. Therefore, an average consumer of sporting articles and clothing in many cases would likely associate the term ‘MESSI’ directly with this famous player and distinguish him from another Italian-sounding brand. According to the CJEU, the Board of Appeal had such information and it should have considered the conceptual similarity.20 In the General Court’s view, the con- cept of the mark stemming from the reputation of Messi neutralises the similarity in the phonetic and visual sphere. In the light of all those elements, the degree of similarity between the marks at issue is not sufficiently high to be concluded that the relevant public may believe that the goods in question come from the same undertaking or, as may be the case, from economically related performances. Tom 14, nr 3/2022 DOI: 10.7206/kp.2080-1084.541 USING A SURNAME IN A TRADEMARK: HAS THE MESSI CASE CHANGED… 73 21 J. Buchalska, op. cit., pp. 219 ff. Jurisprudence in Matters Concerning Surnames Placed in Trademarks It is worth comparing the considerations with the position of EU bodies assessing the registration of surnames as trademarks21. Based on the jurisprudence of the General Court (EU Court) and the Court of Justice, the following cases considering the registration of a surname as a trademark can be identified: 1) Nichols plc v. Registrar of Trade Marks22 2) Vincenzo Fusco v. OHIM23 3) Julian Murúa Entrena v. OHIM24 4) Elio Fiorucci v. OHIM,25 and later Edwin Co. Ltd v. OHIM (trademark ‘ELIO FIORUCCI’)26 5) I Marchi Italiani Srl and Antonio Basile v. OHIM (trademark ‘B. Antonio Basile 1952’)27 6) Jackson International Trading Co. Kurt D. Brühl GmbH & Co. KG v. OHIM (trademark ‘ROYAL SHAKESPEARE’)28 p pp 22 Judgment of the Court (Second Chamber) of 16 September 2004, Nichols plc v. Registrar of Trade Marks, Case C-404/02, https://curia.europa.eu/juris/document/document.jsf?text=&docid=49511&pageIn- dex=0&doclang=EN&mode=lst&dir=&occ=first&part=1&cid=4821259 (access: 12.11.2021). 23 Judgment of the General Court (Second Chamber) of 1 March 2005, Vincenzo Fusco v. European Union Intellectual Property Office, Case T-185/03, https://curia.europa.eu/juris/document/document.jsf?tex- t=&docid=55026&pageIndex=0&doclang=EN&mode=lst&dir=&occ=first&part=1&cid=4821259 (access: 12.11.2021). 24 Judgment of the General Court (Fourth Chamber) of 13 July 2005, Julián Murúa Entrena v. European Union Intellectual Property Office, Case T-40/03, https://curia.europa.eu/juris/document/document. jsf?text=&docid=60415&pageIndex=0&doclang=EN&mode=lst&dir=&occ=first&part=1&cid=4821259 (access: 12.11.2021). 24 Judgment of the General Court (Fourth Chamber) of 13 July 2005, Julián Murúa Entrena v. European Union Intellectual Property Office, Case T-40/03, https://curia.europa.eu/juris/document/document. jsf?text=&docid=60415&pageIndex=0&doclang=EN&mode=lst&dir=&occ=first&part=1&cid=4821259 (access: 12.11.2021). 25 Judgment of the Court of First Instance (Fifth Chamber) of 14 May 2009, Elio Fiorucci v. Office for Harmonisation in the Internal Market (Trade Marks and Designs) (OHIM), Case T-165/06, https://eur- lex.europa.eu/legal-content/EN/TXT/HTML/?uri=CELEX:62006TJ0165&qid=1633421485195&from=EN (access: 12.11.2021). 25 Judgment of the Court of First Instance (Fifth Chamber) of 14 May 2009, Elio Fiorucci v. Office for Harmonisation in the Internal Market (Trade Marks and Designs) (OHIM), Case T-165/06, https://eur- lex.europa.eu/legal-content/EN/TXT/HTML/?uri=CELEX:62006TJ0165&qid=1633421485195&from=EN (access: 12.11.2021). 25 Judgment of the Court of First Instance (Fifth Chamber) of 14 May 2009, Elio Fiorucci v. Office for Harmonisation in the Internal Market (Trade Marks and Designs) (OHIM), Case T-165/06, https://eur- lex.europa.eu/legal-content/EN/TXT/HTML/?uri=CELEX:62006TJ0165&qid=1633421485195&from=EN (access: 12.11.2021).i 26 Judgment of the Court (Grand Chamber) of 5 July 2011, Edwin Co. Ltd v. Office for Harmonisation in the Internal Market (Trade Marks and Designs), Case C-263/09 P, https://curia.europa.eu/juris/docu- ment/document.jsf?text=&docid=106861&pageIndex=0&doclang=EN&mode=lst&dir=&occ=- first&part=1&cid=7563152 (access: 12.11.2021). 26 Judgment of the Court (Grand Chamber) of 5 July 2011, Edwin Co. Ltd v. Office for Harmonisation in the Internal Market (Trade Marks and Designs), Case C-263/09 P, https://curia.europa.eu/juris/docu- ment/document.jsf?text=&docid=106861&pageIndex=0&doclang=EN&mode=lst&dir=&occ=- first&part=1&cid=7563152 (access: 12.11.2021). 22 Judgment of the Court (Second Chamber) of 16 September 2004, Nichols plc v. Registrar of Trade Marks, Case C-404/02, https://curia.europa.eu/juris/document/document.jsf?text=&docid=49511&pageIn- dex=0&doclang=EN&mode=lst&dir=&occ=first&part=1&cid=4821259 (access: 12.11.2021). i 23 Judgment of the General Court (Second Chamber) of 1 March 2005, Vincenzo Fusco v. European Union Intellectual Property Office, Case T-185/03, https://curia.europa.eu/juris/document/document.jsf?tex- t=&docid=55026&pageIndex=0&doclang=EN&mode=lst&dir=&occ=first&part=1&cid=4821259 (access: 12.11.2021). 24 Judgment of the General Court (Fourth Chamber) of 13 July 2005, Julián Murúa Entrena v. European Union Intellectual Property Office, Case T-40/03, https://curia.europa.eu/juris/document/document. jsf?text=&docid=60415&pageIndex=0&doclang=EN&mode=lst&dir=&occ=first&part=1&cid=4821259 (access: 12.11.2021). Jurisprudence in Matters Concerning Surnames Placed in Trademarks 27 Judgment of the General Court (Sixth Chamber) of 28 June 2012, I Marchi Italiani Srl and Antonio Basile v. Office for Harmonisation in the Internal Market (Trade Marks and Designs) (OHIM), Case T-133/09, https://eur-lex.europa.eu/legal-content/EN/TXT/HTML/?uri=CELEX:62009TJ0133& qid=1633421686435&from=EN (access: 12.11.2021). 28 Judgment of the General Court (First Chamber) of 6 July 2012, Jackson International Trading Co. Kurt D. Brühl GmbH & Co. KG v. Office for Harmonisation in the Internal Market (Trade Marks and Designs) (OHIM), Case T-60/10, https://curia.europa.eu/juris/document/document.jsf?text=&do- 28 Judgment of the General Court (First Chamber) of 6 July 2012, Jackson International Trading Co. Kurt D. Brühl GmbH & Co. KG v. Office for Harmonisation in the Internal Market (Trade Marks and Designs) (OHIM), Case T-60/10, https://curia.europa.eu/juris/document/document.jsf?text=&do- Tom 14, nr 3/2022 DOI: 10.7206/kp.2080-1084.541 74 Van Anh Le, Joanna Buchalska 74 7) El Corte Inglés, SA v. OHIM (trademark ‘PUCCI’)29 8) Claude Ruiz-Picasso, Paloma Ruiz-Picasso, Maya Widmaier-Picasso, Marina Ruiz-Picasso, Bernard Ruiz-Picasso v. OHIM (trademark ‘Picasso’)30 9) Harman International Industries, Inc. v. OHIM (trademark ‘Barbara Becker’).31 7) El Corte Inglés, SA v. OHIM (trademark ‘PUCCI’)29 7) El Corte Inglés, SA v. OHIM (trademark ‘PUCCI’)29 8) Claude Ruiz-Picasso, Paloma Ruiz-Picasso, Maya Widmaier-Picasso, Marina Ruiz-Picasso, Bernard Ruiz-Picasso v. OHIM (trademark ‘Picasso’)30 9) Harman International Industries, Inc. v. OHIM (trademark ‘Barbara Becker’).31 7) El Corte Inglés, SA v. OHIM (trademark PUCCI ) 8) Claude Ruiz-Picasso, Paloma Ruiz-Picasso, Maya Widmaier-Picasso, Marina Ruiz-Picasso, Bernard Ruiz-Picasso v. OHIM (trademark ‘Picasso’)30 9) H I i l I d i I OHIM ( d k ‘B b B k ’) 31 8) Claude Ruiz-Picasso, Paloma Ruiz-Picasso, Maya Widmaier-Picasso, Marina Ruiz-Picasso, Bernard Ruiz-Picasso v. OHIM (trademark ‘Picasso’)30 9) Harman International Industries, Inc. v. OHIM (trademark ‘Barbara Becker’).31 9) Harman International Industries, Inc. v. OHIM (trademark ‘Barbara Becker’).31 Ad 1) This case is the prototype of issues relating to assessing the registration of a surname as a trademark. It was based on the refusal to register an application for registration by Nichols plc, a company established in the United Kingdom, against the Registrar of Trade Marks (head of the trademark registration office). It concerned the refusal to register a popular surname as a trademark for vending machines and food products, including beverages, typically dispensed through such machines. The Registrar of Trade Marks stated, given the number of entries in the London telephone directory, that the surname ‘Nichols’, including its phonetic equivalent ‘Nicholls’ and its singular form ‘Nichol’, is common in the United Kingdom. 29 Judgment of the General Court (Sixth Chamber) of 27 September 2012, El Corte Inglés, SA v. Office for Harmonisation in the Internal Market (Trade Marks and Designs), Case T‑39/10, https://curia.europa. eu/juris/document/document.jsf?text=&docid=127624&pageIndex=0&doclang=EN&mode=l- st&dir=&occ=first&part=1&cid=4821259 (access: 12.11.2021). 31 Judgment of the Court of First Instance (First Chamber) of 2 December 2008, Harman International Industries, Inc. v. Office for Harmonisation in the Internal Market (Trade Marks and Designs) (OHIM), Case T-212/07, https://curia.europa.eu/juris/document/document.jsf?text=&docid=72680&pageIndex- =0&doclang=EN&mode=lst&dir=&occ=first&part=1&cid=4821259; Judgment of the Court (Fourth Chamber) of 24 June 2010, Barbara Becker v. Harman International Industries Inc., https://curia.europa. eu/juris/document/document.jsf?text=&docid=81387&pageIndex=0&doclang=EN&mode=l- st&dir=&occ=first&part=1&cid=4821259 (access: 12.11.2021). 30 Judgment of the Court (First Chamber) of 12 January 2006, Claude Ruiz-Picasso and Others v. European Union Intellectual Property Office, Case C-361/04 P, https://curia.europa.eu/juris/document/document. jsf?text=&docid=57309&pageIndex=0&doclang=EN&mode=lst&dir=&occ=first&part=1&cid=4821259 (access: 12.11.2021). cid=124801&pageIndex=0&doclang=EN&mode=lst&dir=&occ=first&part=1&cid=416758 (access: 12.11.2021).i cid=124801&pageIndex=0&doclang=EN&mode=lst&dir=&occ=first&part=1&cid=416758 (access: 12.11.2021). 29 Judgment of the General Court (Sixth Chamber) of 27 September 2012, El Corte Inglés, SA v. Office for Harmonisation in the Internal Market (Trade Marks and Designs), Case T‑39/10, https://curia.europa. eu/juris/document/document.jsf?text=&docid=127624&pageIndex=0&doclang=EN&mode=l- st&dir=&occ=first&part=1&cid=4821259 (access: 12.11.2021). 30 Judgment of the Court (First Chamber) of 12 January 2006, Claude Ruiz-Picasso and Others v. European Union Intellectual Property Office, Case C-361/04 P, https://curia.europa.eu/juris/document/document. jsf?text=&docid=57309&pageIndex=0&doclang=EN&mode=lst&dir=&occ=first&part=1&cid=4821259 (access: 12.11.2021). 31 Judgment of the Court of First Instance (First Chamber) of 2 December 2008, Harman International Industries, Inc. v. Office for Harmonisation in the Internal Market (Trade Marks and Designs) (OHIM), Case T-212/07, https://curia.europa.eu/juris/document/document.jsf?text=&docid=72680&pageIndex- =0&doclang=EN&mode=lst&dir=&occ=first&part=1&cid=4821259; Judgment of the Court (Fourth Chamber) of 24 June 2010, Barbara Becker v. Harman International Industries Inc., https://curia.europa. eu/juris/document/document.jsf?text=&docid=81387&pageIndex=0&doclang=EN&mode=l- st&dir=&occ=first&part=1&cid=4821259 (access: 12.11.2021). i p ( ) 30 Judgment of the Court (First Chamber) of 12 January 2006, Claude Ruiz-Picasso and Others v. European Union Intellectual Property Office, Case C-361/04 P, https://curia.europa.eu/juris/document/document. jsf?text=&docid=57309&pageIndex=0&doclang=EN&mode=lst&dir=&occ=first&part=1&cid=4821259 (access: 12.11.2021). 31 Judgment of the Court of First Instance (First Chamber) of 2 December 2008, Harman International Industries, Inc. v. Office for Harmonisation in the Internal Market (Trade Marks and Designs) (OHIM), Case T-212/07, https://curia.europa.eu/juris/document/document.jsf?text=&docid=72680&pageIndex- 0&d l EN& d l t&di & fi t& t 1& id 4821259 J d t f th C t (F th Jurisprudence in Matters Concerning Surnames Placed in Trademarks There- fore, such a surname cannot communicate as a badge of origin. The High Court took a similar view that common surnames should be considered carefully to ensure that an unfair advantage is not given to those names. A question was sub- mitted to the CJEU for a preliminary ruling in those circumstances. The CJEU stated that the criteria to assess the distinctiveness of a surname mark are the same as those applied to other marks. Those criteria are, for instance, a predetermined number of persons with the same surname, above which that name may be regarded as devoid of distinctive character – the number of undertakings providing products or services of the type covered by the application for registration – or the prevalence, or the use of surnames in the relevant trade, cannot be applied DOI: 10.7206/kp.2080-1084.541 Tom 14, nr 3/2022 USING A SURNAME IN A TRADEMARK: HAS THE MESSI CASE CHANGED… 75 to such trademarks. According to the CJEU, a surname, even a popular one, may be registered as a trademark if it is distinctive. Advocate General Dámas Ruiz-Jarabo Colomer expressed a slightly different opinion.32 The potential distinctiveness of a surname depends on the goods or services covered by the marks, the relevant consumers who consider that the mark identifies those of one undertaking rather than those of another. The commonness of the surname is one of the factors but not the decisive one. Ad 2) This dispute involved the refusal to register the word mark – ‘ENZO FUSCO’ by the OHIM, against the registration of which the owner of the trademark ‘ANTONIO FUSCO’ filed an opposition. The Board of Appeal of OHIM upheld the initial decision, stating that the surname ‘Fusco’, which appears in both signs, was neither rare nor particularly common in Italy. The individual character of these signs was given by the names ‘Antonio’ and ‘Enzo’, which are familiar names. Therefore, the presence of the word ‘Fusco’ in both signs was to give rise to a reaso nable likelihood of confusion in the mind of the reference public. One of the grounds for refusal was that the marks were applied to identical goods.i In this case, the court of first instance adopted an interesting position, con ditioning the assessment of a surname trademark through the prism of differentia tion about individual states of the community. 32 https://curia.europa.eu/juris/document/document.jsf?text=&docid=48847&pageIndex=0&do- clang=EN&mode=lst&dir=&occ=first&part=1&cid=414747 (access: 12.11.2021). Jurisprudence in Matters Concerning Surnames Placed in Trademarks According to the court, the percep- tion of signs made up of personal names may vary from country to country within the European Community. In determining whether, in a particular country, the relevant public generally attributes greater distinctiveness to the surname than the forename, the case-law of that country, although not binding on the Commu- nity courts, may provide valuable guidelines. In this case, the court of first instance referred to Italian case-law, which generally considers that the surname constitutes the heart of a sign made up of a forename and a surname. Moreover, both parties agreed that ‘Fusco’ is not one of the most common surnames in Italy. Therefore, the court of first instance decided that the indicated signs are characterised by a certain similarity resulting from their most characteristic feature being the same.i Ad 3) This case concerned the figurative mark ‘Julian Murúa Entrena’, in Nice Class 33 dedicated to wines. The Opposition Division of OHIM refused to register the mark because an earlier trademark, ‘MURÚA’, was registered in the same class and for the same goods. This position was supported by the Board of Appeal, pointing out that the conflict ing trademarks designated identical goods. Moreover, the dominant verbal element Tom 14, nr 3/2022 DOI: 10.7206/kp.2080-1084.541 Van Anh Le, Joanna Buchalska 76 in both marks is the word ‘Murúa’, the first surname in the sign at issue. Such regi stration would be misleading. In this case, the applicant’s position about using a name in trademarks is inter- esting. The applicant stated that the first name ‘Julián’, whose mark was composed, is relatively common. However, the combination of ‘Murúa’ and ‘Entrena’, which is rare, has a unique and distinctive character that is aurally easy to distinguish. According to the applicant, when confronted with a verbal element composed of a first name, in this case, ‘Julián’, and two surnames, in this case, ‘Murúa’ and ‘Entrena’, Spanish consumers tend to disregard the first name and the second surname. On the contrary, in a legal system such as the one existing in Spain, where a person’s civil status is based on two surnames, according to the applicant, the critical factor is the distinctiveness of each of the components of the verbal element in question. 33 OJ L 040, 11/02/1989 P. 0001–0007, hereinafter referred to as ‘Directive 89/104’, no longer in force; currently Directive 2008/95/EC of the European Parliament and of the Council of 22 October 2008 to approximate the laws of the Member States relating to trade marks, OJ L 299, 8 November 2008, pp. 25–33. 34 OJ L 011, 14/01/1994 P. 0001–0036, hereinafter referred to as ‘Regulation 40/94’. USING A SURNAME IN A TRADEMARK: HAS THE MESSI CASE CHANGED… USING A SURNAME IN A TRADEMARK: HAS THE MESSI CASE CHANGED… 77 Ad 4) The facts of the case were as follows: Fiorucci SpA, a company incorpo- rated under Italian law set up by E. Fiorucci, a fashion designer who gained some popularity in Italy in the 1970s, transferred on 21 December 1990 to Edwin Co. Ltd all of its ‘creative assets’, including all trademarks it owned, many of which contained the element ‘FIORUCCI’. On 6 April 1999, upon the request of Edwin Co. Ltd, OHIM registered the word mark ‘ELIO FIORUCCI’ in respect of a series of goods falling within Classes 3, 18 and 25. Four years after the registration, E. Fiorucci applied for revocation and a declaration of the invalidity of that mark. The basis of the request was the premise that as a result of the use by the proprietor of the trade mark or with his consent in respect of the goods or services for which it is registered, the trade mark is liable to mislead the public, particularly as to the nature, quality or geographical origin of those goods or services. The other prem- ise was also Article 52(2) of Regulation 40/94 determining the relative grounds for invalidity, according to which a Community trade mark will also be declared invalid on application to OHIM or based on a counterclaim in infringement pro- ceedings where the use of such trademark may be prohibited under the national law governing the protection of any other earlier right and in particular: a right to a name; a right of personal portrayal; copyright; an industrial property right. By the decision of 23 December 2004, the Cancellation Division of OHIM allowed the application for a declaration of invalidity because it was necessary to receive permission to register his name as a Community trademark. Such permission has never been given. However, the Board of Appeal annulled the decision of the Cancellation Divi- sion, referring to the provisions of national law which prevented the registration of a commonly known surname by a person other than the person bearing this surname. The Board of Appeal held that E. Fiorucci’s case did not fall within the scope of that regulation. Its purpose is to prevent third parties from exploiting for commercial purposes the name of a person famous in sectors other than strictly commercial. Consequently, E. Jurisprudence in Matters Concerning Surnames Placed in Trademarks Moreover, the applicant pointed out that the refusal to register that surname would prevent him from using his patronymic in the trade mark.i The court of first instance pointed out that the Directive 89/104/EEC33 indicates that the trademark does not entitle the proprietor to prohibit a third party from using, in the course of trade: his name or address; indications concerning the kind, quality, quantity, intended purpose, value, geographical origin, the time of produc tion of goods or rendering of the service, or other characteristics of goods or services; the trade mark where it is necessary to indicate the intended purpose of a product or service, in particular as accessories or spare parts, provided he uses them by honest practices in industrial or commercial matters. The court of first instance also stated that based on Council Regulation (EC) No 40/94,34 there are no specific regulations regarding the registration of a surname as a trademark. The criteria for assessing the distinctive character constituted by a personal name are the same as those applicable to the other categories of the trademark. The same must hold for criteria for assessing the likelihood of confusion between a Community trade mark whose registration has been sought and an earlier trademark unless otherwise provided by that regulation. In this case, the court of first instance agreed with the position of the Board of Appeal, pointing to the impossibility of registering the mark in question. The surname ‘Murúa’, shared by the conflicting signs, has the same origin, namely the applicant’s father, who transferred the earlier trademark, registered in Spain, to the proprietor of the ‘Murúa’ mark could potentially mislead consumers. DOI: 10.7206/kp.2080-1084.541 Tom 14, nr 3/2022 Tom 14, nr 3/2022 USING A SURNAME IN A TRADEMARK: HAS THE MESSI CASE CHANGED… Fiorucci cannot rely on the right to a surname under that provision. In its judgment, the court of first instance upheld the position of the Board of Appeal, according to which, the fact that a mark and a patronymic were identical does not mean that the public concerned will think that the person whose surname constitutes the mark designed the goods bearing that mark. According to the Board of Appeal, the use of patronymic marks is a widespread practice in all sectors of com- merce and the public concerned is well aware that, behind every patronymic mark, there is not necessarily a fashion designer of that name. However, the court of first instance pointed out that the renown of a surname does not exclude the possibility Tom 14, nr 3/2022 DOI: 10.7206/kp.2080-1084.541 Van Anh Le, Joanna Buchalska 78 of its protection as a trademark. This protection may be dualistic under national laws concerning the protection of a surname EU and national law about trademarks. of its protection as a trademark. This protection may be dualistic under national laws concerning the protection of a surname EU and national law about trademarks. Subsequently, the subject of the proceedings was the application of Edwin Co. Ltd to set aside the judgment of the Court of First Instance of 14 May 2009 in Case T‑165/06 Fiorucci v. OHIM – Edwin (ELIO FIORUCCI), by which that Court upheld in part the action brought by E. Fiorucci against the decision of the First Board of Appeal of the Office for Harmonisation in the Internal Market (Trade Marks and Designs) (OHIM) of 6 April 2006 (Case R 238/2005‑1) concerning invalidity and revocation proceedings between E. Fiorucci and Edwin, which was submitted to the Court of Justice for consideration. In the opinion of the Court, the content of national law made it possible to prevent the registration of a trademark containing the surname of a well-known person without that person’s consent. However, it is essential to present circumstances that confirm the renown of the surname in question.35i Ad 5) The mark contested in this case was the figurative mark B. Antonio Basile 1952, registered by OHIM assigned to a sole trader under the name B. Antonio Basile 1952. Given the registration of this mark, the company Osra SA applied for a declaration of invalidity referring to its Italian and international trademark BASILE. 35 Advocate General Kokott argued against this position in an opinion delivered on 27 January 2011, https://curia.europa.eu/juris/document/document.jsf?text=&docid=79193&pageIndex=0&do- clang=EN&mode=lst&dir=&occ=first&part=1&cid=7563152 (access: 12.11.2021). 36 Cf. Judgment of the Court (Fourth Chamber) of 24 June 2010, Barbara Becker v, Harman International Industries Inc., Case C-51/09 P, Court Reports I-5805, paragraphs 36 and 37. https://curia.europa.eu/juris/document/document.jsf?text=&docid=79193&pageIndex=0&do- clang=EN&mode=lst&dir=&occ=first&part=1&cid=7563152 (access: 12.11.2021). 36 Cf. Judgment of the Court (Fourth Chamber) of 24 June 2010, Barbara Becker v, Harman International Industries Inc., Case C-51/09 P, Court Reports I-5805, paragraphs 36 and 37. 37 Cf. Judgment of the General Court (Sixth Chamber) of 28 June 2012, Antonio Basile and I Marchi Italiani Srl v. Office for Harmonisation in the Internal Market (Trade Marks and Designs), Case T-134/09, https://curia.europa.eu/juris/document/document.jsf?text=&docid=124470&pageIndex=0&doclang=- FR&mode=lst&dir=&occ=first&part=1&cid=419456 (access: 12.11.2021). USING A SURNAME IN A TRADEMARK: HAS THE MESSI CASE CHANGED… Both marks were registered for the same goods – clothing. What is signi ficant is that the surname Basile was the subject of many registrations made in Italy. Additionally, the mark B. Antonio Basile 1952 is widely known and has a particular reputation. In this case, the EU Court, as in the case of ‘ENZO FUSCO’, stated that the Italian consumer would generally attribute greater distinctiveness to the surname than to the forename in trademarks. In that regard, in particular, the account must be taken that the surname concerned is unusual or, on the contrary, very common, which is likely to affect that distinctive character. Account must also be taken of whether the person who requests that his forename and surname, taken together, be registered as a trademark is well known.36 Because the surname Basile is widely known and even one of the most popular in Italy, despite the combination with the name Antonio, it is an element that makes the trademark more distinctive. In the judgment, the Court compared the marks in question on three levels – visual, phone DOI: 10.7206/kp.2080-1084.541 Tom 14, nr 3/2022 Tom 14, nr 3/2022 USING A SURNAME IN A TRADEMARK: HAS THE MESSI CASE CHANGED… 79 tical, and conceptual, stressing, in line with the previous case-law, that trademarks with an element of surname should be assessed according to the same method.37 Ad 6) The subject of the proceedings, in this case, was the application for the word mark ‘ROYAL SHAKESPEARE’ for beverages, including alcohols. The Royal Shakespeare Company applied for a declaration of invalidity of this registration, indicating that the use of the indicated mark may bring unfair advantage to the applicant, may be detrimental to the distinctive character and reputation of the unre- gistered, reputable trademark RSC, where the abbreviation was described below as ‘The Royal Shakespeare Company’ and was used by The Royal Shakespeare Company. The Court found that the surname of William Shakespeare is well known and, as such, in and of itself, is not highly distinctive for theatre productions. It is also not distinguished by the words ‘royal’ or ‘Shakespeare’. However, combining these two words gives rise to a distinctive mark. It is the expression ‘Royal Shakespeare’, which is at the same time dominant, which was repeated in the mark at issue. USING A SURNAME IN A TRADEMARK: HAS THE MESSI CASE CHANGED… In the opinion of the Court, it is also significant that the mark was used in a way that targeted the public at large, not only a specialised group of consumers. Conse- quently, consumers could be misled by the existence of these two marks simulta- neously on the market. Ad 7) The subject of the dispute was the registration of the word mark ‘PUCCI’ El Corte Inglés, SA, filed a notice of opposition regarding this registration. The opposition was based on the earlier registration of the marks ‘E. TUCCI’ and ‘EMIDO TUCCI’ both as figurative and word marks. The Cancellation Division upheld the registration of the PUCCI mark registered for clothing and footwear, as did the Board of Appeal, arguing that the marks were not similar. The lack of similarity consists of a different surname used in the marks – PUCCI – TUCCI. It is proceeded by an additional element – E. TUCCI or EMIDO TUCCI. The EU Court, in this case, referred to the previous practice of using the criteria of the average consumers about the national law. As in previous cases, the Court argued that a Spanish consumer, like an Italian one, will primarily pay attention to the surname appearing in the mark. Therefore, the surname PUCCI and TUCCI may mislead the average consumer since they only differ by one letter. However, the name EMIDO is not popular in Spain. Thus, the average consumer will consider the trademark as a whole, disregarding the dominant nature of the word TUCCI. Moreover, this form of the trademark will suggest the Italian origin of the goods Tom 14, nr 3/2022 DOI: 10.7206/kp.2080-1084.541 Van Anh Le, Joanna Buchalska 80 covered by it. In the opinion of the EU Court, in the case of trademarks containing a surname, the assessment of misleading an average consumer in this respect should be performed each time, in the same way as in the case of other trademarks. Ad 8) The issue of registering a historical surname was also the subject of the Court’s considerations. In this case, the painter’s heirs opposed using the artist’s name by a car brand. While this judgment does not refer to the issue, particular attention should be paid to the opinion of Advocate General Colomer.38 In this case, the Court had no doubts that the famous artist’s name could be used as the desi gnation of cars. 38 https://curia.europa.eu/juris/document/document.jsf;jsessionid=9ea7d2dc30d60bb9aefc02af49c38984b- 566f8c205ef.e34KaxiLc3qMb40Rch0SaxuOb3r0?text=&docid=59707&pageIndex=0&doclang=EN& mode=lst&dir=&occ=first&part=1&cid=539996#Footref36 (access: 12.11.2021). 39 Cf. Paragraph 69 of the opinion. USING A SURNAME IN A TRADEMARK: HAS THE MESSI CASE CHANGED… The Advocate General pointed out that attention should be paid to two aspects concerning protecting a historical surname. Firstly, when such a surname is allowed to be used in a different context to that in which its reputa- tion was earned, the greater protection which must be given to marks with a highly distinctive character cannot automatically be claimed because it is not certain if the name provides any information about the commercial origin of the goods or services. Secondly, to safeguard their work from trivialisation, there is a general interest in protecting great artists’ surnames, which represents a universal cultural heritage from insatiable commercial greed. It is sad to think that an averagely informed, reasonably aware, and perceptive consumer, who no longer links names such as Opel, Renault, Ford or Porsche with the outstanding engineers whose products were named after them, will, in the not-too-distant future, be subjected to the same process as the surname Picasso.39 Ad 9) The Board of Appeal held that the mark Barbara Becker would be perceived by the relevant public as a person’s name consisting of a first name and a surname, identical to the surname that comprises the earlier mark. It follows that the surname Becker is likely to have attributed to it a stronger distinctive character than the first name Barbara in the mark Barbara Becker. However, the court of first instance stated that the fact that Barbara Becker enjoys celebrity status in Germany as the former wife of Boris Becker does not mean that, conceptually, the marks at issue are not similar. The earlier trademark BECKER and the mark Barbara Becker refer to the same surname Becker. In the present case, it is clear that the component ‘becker’ will be perceived as a surname, which is commonly used to describe a person. It must be held that that component retains a distinctive independent role in the mark Barbara Becker. USING A SURNAME IN A TRADEMARK: HAS THE MESSI CASE CHANGED… However, in the view of Advocate General Villalón, such grounds may lead to the misconception that any surname, similar to an earlier trademark, may constitute an obstacle to the registration of a complex trademark Tom 14, nr 3/2022 DOI: 10.7206/kp.2080-1084.541 USING A SURNAME IN A TRADEMARK: HAS THE MESSI CASE CHANGED… 81 composed of the first and last name in question, due to the existence of a likelihood of confusion within the meaning of Article 8(1)(b) of Regulation No 40/94.40 However, this case was brought to the Court, which pointed to an essential ele- ment in using a surname in trade marks. According to the Court, when assessing the similarity of the marks, account must also be taken of whether the person who requests that his first name and surname, taken together, be registered as a trade- mark is well known, since that factor may influence the perception of the mark by the relevant public. 40 Opinion of Advocate General Cruz Villalón delivered on 25 March 2010, Case C-51/09 P, Barbara Becker, https://curia.europa.eu/juris/document/document.jsf?text=&docid=79651&pageIndex=0&do- clang=EN&mode=lst&dir=&occ=first&part=1&cid=540605 (access: 12.11.2021). 40 Opinion of Advocate General Cruz Villalón delivered on 25 March 2010, Case C-51/09 P, Barbara Becker, https://curia.europa.eu/juris/document/document.jsf?text=&docid=79651&pageIndex=0&do- clang=EN&mode=lst&dir=&occ=first&part=1&cid=540605 (access: 12.11.2021). 41 7.2. point to the Guidelines EUIPO, https://guidelines.euipo.europa.eu/1922895/1788130/trade-mark- guidelines/7-2-1-names (access: 12.11.2021). 42 Com point 7.2.1. 42 Com point 7.2.1. gi p ( ) 41 7.2. point to the Guidelines EUIPO, https://guidelines.euipo.europa.eu/1922895/1788130/trade-mark- guidelines/7-2-1-names (access: 12.11.2021). 42 C i 7 2 1 Comments Following the previous analysis, we can observe some remarks. First of all, accord- ing to the EUIPO Guidelines, ‘there are no specific criteria to be taken into account when the likelihood of confusion between names is assessed. However, because of the very nature of names and surnames, certain aspects come into play that have to be carefully considered and balanced, such as whether a given name and surname is common or not in the relevant territory.’41 Trademarks consisting of names can be perceived differently in different EU countries.42 For instance, the special role of the average consumer – initially Italian and later Spanish. This was the first obvious step, which showed that trademarks could not be limited to trade- mark rights because of their functions. g Based on case-law, trademarks consisting of common surnames are also assessed in certain ways. Renown is of particular importance in assessing the similarity of trademarks. However, renown should be evaluated in relation to the reputation of trademarks but their bearers. A celebrity may also have an impact on the possi bility of registering a trademark containing one’s surname. Turning to trademarks containing historical surnames, it is possible to distinguish the different positions of the Advocate General, who recognised the historical name as an object of parti cular respect. Therefore, it is emphasised in recent judgments that these surnames have characteristic renown identified with the persons who bear them. However, in the Messi case, the CJEU sheds essential light on the likelihood of confusion on test interpretation when assessing the conceptual differences in Tom 14, nr 3/2022 DOI: 10.7206/kp.2080-1084.541 Van Anh Le, Joanna Buchalska 82 determining similar marks. The Court confirmed that even if a sign has a high degree of visual and phonetic similarity, a substantial dissimilarity in concept may be sufficient to prevent confusion. It is indisputable that Messi is a globally famous figure. However, would it be fair to grant Messi broader protection against the previously registered trademark because of his extraordinary publicity? Would the Court have arrived at a different conclusion if the ‘MESSI’ mark owner had been someone else, for instance, a company or a family member? Messi won because he is too famous for being confused with anyone else. However, we should think about human rights (or civil ones), allowing us to identify a person by their name. 43 Decision of the Opposition Division of EUIPO No B 2 166 034. 44 E.g. 16/12/2010, T-345/08 & T-357/08, Botolist / Botocyl, confirmed 10/05/2012, C-100/11 P, 10/01/2011, R 43/2010-4, FFR (fig.) / CONSORZIO VINO CHIANTI CLASSICO (fig.). 45 Com. A. Sztoldman, The Lionel Messi Case: Trade Mark’s Reputation Blurred with Personal Notoriety, “Euro- pean Intellectual Property Review”, 2021, 43(6), pp. 408–411. 46 Com. M. Mancinella, The Visual, the Phonetic and the Famous: Trade Mark Similarity in the Wake of Messi v EUIPO, “European Intellectual Property Review” 2018, 40(10), pp. 665–668. 48 ‘From a trade mark perspective, the ruling implies that opposition against an application consisting of the name of a celebrity is doomed to fail regardless of the level of attention paid by the relevant public, the identity of the goods or services or the visual and phonetic similarity of the signs. This judgment merely confirms the truth of the well-known adage that it is “better to be rich and healthy than poor and sick”’ – S. Martin, Lionel Messi v EUIPO: 2-0. Court of Justice Blows Final Whistle on Opposi tion Proceedings Involving Leo Messi, “Journal of Intellectual Property Law & Practice” 2021, 16(1), p. 11. 47 M. Giannino, Messi Like Picasso? General Court Rejects Opposition Against Registration as an EUTM Featur- ing the Name of Lionel Messi, “Journal of Intellectual Property Law & Practice” 2018, 13(9), p. 686. USING A SURNAME IN A TRADEMARK: HAS THE MESSI CASE CHANGED… USING A SURNAME IN A TRADEMARK: HAS THE MESSI CASE CHANGED… 83 Although the CJEU quickly confirmed that such instances would only arise in unique circumstances, there is a lack of clarity arising from the decision. Although the celebrity of a football star certainly seems obvious in Messi, personal notoriety cannot be supposed. Lionel Messi’s fame among football fans only concerns the public interest in football and sport. This is also hard to agree implausible that average consumers have never heard of Lionel Messi and be incapable of associat ing the MESSI mark to him.47 If personal fame is superior to other factors, where is the limit and what are the criteria for assessing the fame of celebrities in trade- mark matters? Moreover, it is hard to agree that Messi is better known in public than Lady Gaga, who is active in many areas, e.g. as a singer, actress and performer. Addi- tionally, she is one of the most notable performers. Somehow, it brings to a very sad conclusion, pointed out by S. Martine that in trademark cases, when marks consist of surnames or trademarks, it is not necessary to compare trademarks taking into account the relevant public, the identity of the goods or services or the visual and phonetic similarity of the signs. What is essential in those cases is the reputa- tion of the person bearing the name, which, unfortunately, based on this judgment, cannot be evaluated on a transparent basis.48 Comments According to the CJEU’s decision, only one person bearing the name ‘Messi’ is well known, but what about someone else named Messi?i The decision also highlights another significant legal issue. The CJEU held that Messi’s legal team did not need to submit any evidence of his reputation because his world-renowned reputation was already apparent. It poses challenges for practitioners to garner proof to prove the reputation of a celebrity figure. How famous must someone be so that the evidence of their notoriety can be waived? Especially when we consider the previous judgments. The EUIPO’s decision con- cerned an earlier EU trademark LADY GAGA and a later EU trademark GAGA.43 The EUIPO concluded that the opponent (Lady Gaga – the singer) failed to prove that her trademark LADY GAGA has a reputation. The evidence did not provide information on using the LADY GAGA trademark. Moreover, the evidence did not indicate the degree of recognition of the trademark by the relevant public. g g y p Additionally, the evidence proving someone’s reputation must be clear and con- vincing.44 In MESSI, contrary to the previous one, the courts did not judge the reputation based on legal facts. As the doctrine points out, the ECJ clarified that ascertaining a person’s notoriety is a question of fact and therefore is inadmissible in court. Thus, the ECJ accepted a priori the recognition of Messi, which affects the distinctiveness of the goods and eliminates the risk of confusion. This point of view is objectionable, and General Court should also consider the lack of evidence of personal fame.45 Lack of evidence requirements to judge the reputation of the trademark, which is (or includes) famous surnames, can be too unclear and cause the subjective feeling of the judge becomes the basis of the decision.46 Tom 14, nr 3/2022 DOI: 10.7206/kp.2080-1084.541 DOI: 10.7206/kp.2080-1084.541 Sztoldman A., The Lionel Messi Case: Trade Mark’s Reputation Blurred with Personal Notoriety, “European Intellectual Property Review” 2021, 43(6). Lagarde P., L’oeuvre de la Commission Internationale de l’Etat Civil en matière de nom des personnes, [in:] H.- Mansel P., Pfeiffer T., Kronke H., Köhler Ch., Hausmann R. (eds.), Festschrift für Erik Jayme, Band II, Munich 2004. Mancinella M., The Visual, the Phonetic and the Famous: Trade Mark Similarity in the Wake of Messi v EUIPO, “European Intellectual Property Review” 2018, 40(10). utré S., Le nom en droit compare, Frankfurt am Main 1977. Bibliography Buchalska J., Nazwisko jako przedmiot ochrony w prawie polskim, Warszawa 2016. Buchalska J., Nazwisko jako przedmiot ochrony w prawie polskim, Warszawa 2016. Freitag R., Subjektive Anknüpfung: Vorstellung des Vorschlags, [in:] A. Dutta, T. Helms, W. Printens, Ein Name in ganz Europa, Vorschläge für ein Internationales Namensrecht der Europäischen Union, Frankfurt am Main 2016. Funken K., Das Anerkennungsprinzip im internationalen Privatrecht. Perspektiven eines euro- päischen Anerkennungskollisionsrechts für Statusfragen, München 2009. Funken K., Das Anerkennungsprinzip im internationalen Privatrecht. Perspektiven eines euro- päischen Anerkennungskollisionsrechts für Statusfragen, München 2009. Giannino M., Messi Like Picasso? General Court Rejects Opposition Against Registration as an EUTM Featuring the Name of Lionel Messi, “Journal of Intellectual Property Law & Prac- tice” 2018, 13(9). Tom 14, nr 3/2022 DOI: 10.7206/kp.2080-1084.541 Van Anh Le, Joanna Buchalska 84 Lagarde P., L’oeuvre de la Commission Internationale de l’Etat Civil en matière de nom des personnes, [in:] H.- Mansel P., Pfeiffer T., Kronke H., Köhler Ch., Hausmann R. (eds.), Festschrift für Erik Jayme, Band II, Munich 2004. Mancinella M., The Visual, the Phonetic and the Famous: Trade Mark Similarity in the Wake of Messi v EUIPO, “European Intellectual Property Review” 2018, 40(10). Mancinella M., The Visual, the Phonetic and the Famous: Trade Mark Similarity in the Wake of Messi v EUIPO, “European Intellectual Property Review” 2018, 40(10). Martin S., Lionel Messi v EUIPO: 2-0. Court of Justice Blows Final Whistle on Opposition Proceed- ings Involving Leo Messi, “Journal of Intellectual Property Law & Practice” 2021, 16(1). Meier P., de Luze E., Droit des personnes. Articles 11–89a CC, Schulthess 2014. Martin S., Lionel Messi v EUIPO: 2-0. Court of Justice Blows Final Whistle on Opposition Proceed- ings Involving Leo Messi, “Journal of Intellectual Property Law & Practice” 2021, 16(1). Martin S., Lionel Messi v EUIPO: 2-0. Court of Justice Blows Final Whistle on Opposition Proceed- ings Involving Leo Messi, “Journal of Intellectual Property Law & Practice” 2021, 16(1). Meier P., de Luze E., Droit des personnes. Articles 11–89a CC, Schulthess 2014. Nautré S., Le nom en droit compare, Frankfurt am Main 1977. Sztoldman A., The Lionel Messi Case: Trade Mark’s Reputation Blurred with Personal Notoriety, “European Intellectual Property Review” 2021, 43(6). Sztoldman A., The Lionel Messi Case: Trade Mark’s Reputation B “European Intellectual Property Review” 2021, 43(6). DOI: 10.7206/kp.2080-1084.541 Tom 14, nr 3/2022 Tom 14, nr 3/2022
W3025254207.txt	http://bsj.pitt.edu/ojs/index.php/bsj/article/download/212/1396	en		Three Takes on De-Colonizing the State Apparatus in Bolivia	Bolivian studies journal/Bolivian studies	2,020	cc-by	1,596	Three Takes on De-Colonizing the State Apparatus in Bolivia Chuck Sturtevant Davidson College Abstract This response summarizes and compares three scholars’ approaches (Marcelo Bohrt, Robert Albro and Pamela Calla) to the Morales administration’s efforts to decolonize the government of Bolivia. Seeking the common ground among them, I find that all three recognize the importance of symbolic and discursive changes, which have allowed some previously-excluded individuals to access positions of authority within the state apparatus. On the other hand, these changes have been uneven, exposing rifts between indigenous communities, exacerbating existing inequities, and establishing new or renewed hierarchies of subordination. Keywords Bolivia, decolonization, Evo Morales, indigenous people, state apparatus Resumen Este texto resume y compara tres enfoques académicos (Marcelo Bohrt, Robert Albro y Pamela Calla) sobre los esfuerzos de la gestión de Morales para descolonizar el gobierno de Bolivia. Al buscar un terreno común entre Bolivian Studies Journal /Revista de Estudios Bolivianos Vol. 25 • 2019 • doi: 10.5195/bsj.2019.212 • ISSN 1074-2247 (print) https://bsj.pitt.edu • ISSN 2156-5163 (online) Chuck Sturtevant 61 ellos, encuentro que los tres reconocen la importancia de cambios simbólicos y discursivos que han permitido que algunos individuos previamente excluidos accedan a posiciones de autoridad dentro del aparato estatal. Por otro lado, estos cambios han sido desiguales; exponiendo, así, divisiones entre las comunidades indígenas, exacerbando desigualdades existentes y estableciendo jerarquías de subordinación nuevas o renovadas. Palabras claves Aparato estatal, Bolivia, descolonización, Evo Morales, pueblos indígenas From the moment President Evo Morales took office in 2006, his administration set an ambitious agenda. It aimed to decolonize the state, promote indigenous frameworks for environmental sustainability, and combat all forms of racism and discrimination, all while maintaining strict control over natural resources and political processes. The scholarship gathered together here asks how the implementation of that agenda has played out in practice, and what legacies it might leave for the future. Each of these scholars examines the practices and institutions of the state –the state apparatus as Abrams puts it– from different perspectives and drawing on distinct materials. They triangulate on a conclusion that there have been important shifts in the discursive and symbolic expressions of the state apparatus, and that these have real affects in terms of which individuals can access positions within that apparatus. Nevertheless, that access has been uneven, exposing rifts between indigenous communities, exacerbating existing inequities, and establishing new or renewed hierarchies of subordination. Marcelo Bohrt’s analysis of the position of indigenous bureaucrats describes an effort to reframe narratives about progress and development. Previous regimes described progress in terms of resolving “the Indian problem.” From this perspective, heterogeneity of ethnicities and the supposed backwardness of indigenous peoples prevented progress and development. Whiteness, on the other hand, was (implicitly and often explicitly) associated with modernity. Instead, the Morales administration has worked to reframe progress and development in terms of overcoming “the colonial problem.” Bohrt analyses this reframing –from a concern with “the Indian problem” to a concern with the colonial problem– in the administration’s official decolonization discourse, and the 2006-2010 National Development Plan, in particular. In the bureaucracy, this involved an effort to overturn the status quo of a “deeply ingrained, racialized system,” which Bolivian Studies Journal /Revista de Estudios Bolivianos Vol. 25 • 2019 • doi: 10.5195/bsj.2019.212 • ISSN 1074-2247 (print) https://bsj.pitt.edu • ISSN 2156-5163 (online) 62 Three Takes on De-Colonizing the State Apparatus in Bolivia marked those spaces as hostile to indigenous people and reproduced a “boundary between indigeneity and the state.” Bohrt argues that these discursive and symbolic shifts redrew the symbolic boundaries between indigeneity and state and overthrew the hegemonic notion that state authority is embodied in white-mestizo bodies. Indigenous people now work within the bureaucracies and participate in the exercise of statecraft. Now, indigenous people –bureaucrats and petitioners alike– “come in as if they were in their own house,” as one of his interlocutors told him. The fact that indigenous individuals have been able to gain access to positions within the state, clearly shifts the constitution of bureaucratic spaces in ways that make them more accessible to some indigenous people. Yet the state remains a principle venue for enacting the “actualities of social subordination” (Abrams 63), through which various interests, communities, ethnic groups, and social sectors compete to exert power, and state-craft still depends on bureaucracy, resource distribution, and political parties. There may have been –as Bohrt argues– a dramatic shift in the racial or ethnic make-up of the state. The participation of dark-skinned, indigenous-identifying bureaucrats in the “palpable nexus of practice and institutional structure centered in government,” as Abrams (82) puts it, would have been unimaginable just ten years before Morales’ administration. But these transformations have not substantially altered the nexus of practice and institutional structure themselves. Robert Albro argues that, in practice, the structural dynamics reproduced within the state continue to marginalize indigenous peoples as communities, interest groups, or ethnic classifications. Albro identifies a tension between indigenous priorities as the diversity of these interests come into conflict. For example, discourses reproduced in the constitution and other legal frameworks codify indigenous rights, offer stringent critiques of colonialism, and introduce an indigenous framework for environmental sustainability. These framings depend on a collective notion of indigenous subjects, with their own language, historical traditions, cosmovision, and territory. Yet indigeneity means something very different for urban indigenous people, who go largely unrecognized in the constitution and whose interests differ drastically from those of such rural, collectively conceived indigenous communities. Further, as these interests collide, it is these collective indigenous communities that remain marginalized from the exercise of power through state-craft, and who face disproportionately severe consequences from the extractive practices that benefit indigenous majorities in urban areas. The result is that MAS’s political Bolivian Studies Journal /Revista de Estudios Bolivianos Vol. 25 • 2019 • doi: 10.5195/bsj.2019.212 • ISSN 1074-2247 (print) https://bsj.pitt.edu • ISSN 2156-5163 (online) Chuck Sturtevant 63 agenda (as produced in the constitution and, particularly, as projected into international sphere) continues to be informed by key indigenous cosmological concepts and a critique of liberal economic system even as these frameworks are disconnected from specific concerns of particular indigenous groups, and the everyday exercise of power through state-craft. One of the consequences of these tensions has been that efforts at decolonization of the state have stagnated. Pamela Calla has explored the work 1 –or, more pointedly, the scarcity of work– conducted by the ViceMinistry for Decolonization and the National Committee Against Racism and All Forms of Discrimination (Comité Nacional Contra el Racismo y Toda Forma de Discriminación). As Calla frames it, the MAS party hijacked the radical elements of the indigenous movement and coopted them to achieve its own goal: building a one-party government. The push from below by grassroots activists demanding a response to racist and discriminatory scaffolds that are embedded in the state has led to a string of resolutions, the formation of committees, and a string of meetings, but has not led to changes in public policies. Discrimination complaints go unprocessed, regional committees fail to meet, language training for public officials is ineffective, and the Vice-Ministry itself is poorly funded. That is, the state apparatus continues to operate along preexisting trajectories. The emotional bond between the party and the activists has been broken and the activists, particularly indigenous and women activists, who pushed for these changes, have found that they, once again, have to do the work of imagining an alternative. This involves looking beyond the Bolivian state as an emancipatory institution to build international networks. The question that panelists put forward, one form or another, is, “How successful have the Morales administration and the MAS party been in decolonizing the state?” There have certainly been symbolic and discursive changes. Further, these changes in discursive or symbolic registers are important, as Bohrt argues. Individuals who identify as indigenous have access to bureaucratic spaces that were once closed off to them, both as officials and petitioners. If the outlook seems positive from the perspective of Bohrt’s indigenous informants, perhaps that says more about their position working within the state apparatus, as indigenous bureaucrats who have managed to attain a position within the state apparatus. For those outside, the picture is less encouraging. While bureaucracies and political parties may no longer be 1 Pamela Calla’s paper is not collected in this special issue, but its contents and critique can be appreciated throughout this discussion. Bolivian Studies Journal /Revista de Estudios Bolivianos Vol. 25 • 2019 • doi: 10.5195/bsj.2019.212 • ISSN 1074-2247 (print) https://bsj.pitt.edu • ISSN 2156-5163 (online) 64 Three Takes on De-Colonizing the State Apparatus in Bolivia constituted as white spaces from which indigenous people are inherently excluded, they still operate as bureaucracies and political parties that enact the interests of specific actors. The state apparatus still produces a palpable nexus of practices and institutional structure that continue to enact the realities of social subordination. These are not the practices and structures imagined by the grassroots activists, indigenous communities, and other actors who saw the election of an indigenous president as an opportunity to radically reimagine the idea of the state. Works Cited ABRAMS, Philip. 1988. "Notes on the Difficulty of Studying the State (1977)." Journal of Historical Sociology 1(1): 58-89. New articles in this journal are licensed under a Creative Commons Attribution 4.0 United States License. This journal is published by the University Library System of the University of Pittsburgh as part of its D-Scribe Digital Publishing Program, and is cosponsored by the University of Pittsburgh Press. Bolivian Studies Journal /Revista de Estudios Bolivianos Vol. 25 • 2019 • doi: 10.5195/bsj.2019.212 • ISSN 1074-2247 (print) https://bsj.pitt.edu • ISSN 2156-5163 (online)
https://openalex.org/W2060832571	https://europepmc.org/articles/pmc4396555?pdf=render	English	null	Spatial Mutual Information Based Hyperspectral Band Selection for Classification	The scientific world journal/TheScientificWorldjournal	2,015	cc-by	4,179	1. Introduction on the information measures such as mutual information between a pair of vectors. The vectors are then clustered into several groups based on their dissimilarity. In our work, we use hierarchical clustering [7] in the dissimilarity space. In the end, for each of the clusters, a band is selected to represent each cluster. The dissimilarity metric used will influence the shape of the clusters, as some elements may be close to one another according to one distance and farther away according to another.h Hyperspectral imaging consists of a large number of closely spaced bands that range from 0.4 𝜇m to 2.5 𝜇m [1]. The high dimensionality in hyperspectral imagery makes it useful for many applications such as agriculture, medicine, and surveillance. However, the high dimensionality of hyperspec- tral data leads to high computational cost and can contain redundant information. Thus, there is need to select the relevant bands to reduce computational cost and data storage while maintaining accuracy. The maximization of mutual information criterion postu- lates that mutual information is maximal, when image bands are similar. Mutual information has been demonstrated to be a very general and powerful similarity metric, which can be applied automatically and very reliably, without prior preprocessing, on a large variety of applications [8]. Mutual information treats all pixels the same during signal matching regardless of the position and usefulness of the pixel in the image. However, it does not incorporate useful spatial information which is a drawback. Band selection or feature extraction can be used to reduce hyperspectral data. In band selection, a representative subset of the original hyperspectral information is selected [2, 3]. Feature extraction involves the reduction of the original information by transforming the initial information [4, 5]. In hyperspectral imaging band selection is preferred since orig- inal information is preserved, whereas in feature extraction the original and required information may be distorted [6]. In pixel classification a good band selection method can not only reduce computational cost but also improve the classification accuracy. In this work, we propose spatial mutual information which combines mutual information and a weighting func- tion based on absolute difference of corresponding pixels as the dissimilarity metric and hierarchical clustering to select the bands considered most relevant. Hindawi Publishing Corporation e Scientiﬁc World Journal Volume 2015, Article ID 630918, 7 pages http://dx.doi.org/10.1155/2015/630918 Hindawi Publishing Corporation e Scientiﬁc World Journal Volume 2015, Article ID 630918, 7 pages http://dx.doi.org/10.1155/2015/630918 Hindawi Publishing Corporation e Scientiﬁc World Journal Volume 2015, Article ID 630918, 7 pages http://dx.doi.org/10.1155/2015/630918 Anthony Amankwah Computer Science Department, University of Ghana, Legon, Ghana Correspondence should be addressed to Anthony Amankwah; amankwah@ieee.org Received 1 September 2014; Revised 29 November 2014; Accepted 30 November 2014 Academic Editor: Lucile Rossi Copyright © 2015 Anthony Amankwah. 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 amount of information involved in hyperspectral imaging is large. Hyperspectral band selection is a popular method for reducing dimensionality. Several information based measures such as mutual information have been proposed to reduce information redundancy among spectral bands. Unfortunately, mutual information does not take into account the spatial dependency between adjacent pixels in images thus reducing its robustness as a similarity measure. In this paper, we propose a new band selection method based on spatial mutual information. As validation criteria, a supervised classification method using support vector machine (SVM) is used. Experimental results of the classification of hyperspectral datasets show that the proposed method can achieve more accurate results. Research Article Spatial Mutual Information Based Hyperspectral Band Selection for Classification Anthony Amankwah 2. Dissimilarity Measures 2.2. Spatial Mutual Information. We have extended MI to include spatial information. MI is estimated on a pixel to pixel basis, meaning that it takes into account only the relationships between corresponding individual pixels and not those of each pixel in the respective neighbourhood. As a result, much of spatial information inherent in images is not utilized. If an image band is reshuffled it will yield the same MI. Thus, the MI between Figure 2(a) and Figure 2(a) (itself) and the MI between Figure 2(a) and Figure 2(b) are the same. Figure 2(c) is the histogram of image in Figure 2(a) or Figure 2(b). 2.2. Spatial Mutual Information. We have extended MI to include spatial information. MI is estimated on a pixel to pixel basis, meaning that it takes into account only the relationships between corresponding individual pixels and not those of each pixel in the respective neighbourhood. As a result, much of spatial information inherent in images is not utilized. If an image band is reshuffled it will yield the same MI. Thus, the MI between Figure 2(a) and Figure 2(a) (itself) and the MI between Figure 2(a) and Figure 2(b) are the same. Figure 2(c) is the histogram of image in Figure 2(a) or Figure 2(b). The independence of bands is one of the main factors used to select a subset of image bands for pixel classification. The independence of bands is one of the main factors used to select a subset of image bands for pixel classification. Dissimilarity measures are used to quantify the degree of independence of image bands. Information measures such as mutual information are widely used to measure the correlation between information from different sensors. Dissimilarity measures are used to quantify the degree of independence of image bands. Information measures such as mutual information are widely used to measure the correlation between information from different sensors. 2.1. Mutual Information. If 𝑋and 𝑌are two image bands, the mutual information MI can be defined by Our proposed spatial mutual information (SMI) com- bines mutual information with a weighting function based on the absolute difference of corresponding pixel values. The absolute differences provide the spatial information. The sum of absolute difference can be considered as another similarity metric. where 𝑝𝑋(𝑖) is the probability distribution. 𝑝𝑋( ) p y Equation (1) contains the term −𝐻(𝑋, 𝑌), and it means minimizing joint entropy is increasing mutual information. Since generally joint entropy increases with increasing dis- similarity, the mutual information decreases with increasing dissimilarity. In other words, if image bands are similar the amount of mutual information they contain about each other is high. 𝑝𝑋 p y Equation (1) contains the term −𝐻(𝑋, 𝑌), and it means minimizing joint entropy is increasing mutual information. Since generally joint entropy increases with increasing dis- similarity, the mutual information decreases with increasing dissimilarity. In other words, if image bands are similar the amount of mutual information they contain about each other is high. (4) where Diff(𝑋, 𝑌) is the weighting function based on the absolute difference of corresponding pixels. Figure 3 shows the dissimilarity matrix of 220-band AVIRIS Indian Pines image scene using SMI. In our work, the histogram method was used to estimate the MI between image bands; thus, MI = 1 𝐸∑ 𝑥 ∑ 𝑦 Hist𝑥𝑦(𝑋, 𝑌) ∗log ( 𝐸∗Hist𝑥𝑦(𝑋, 𝑌) Hist𝑥(𝑋) ∗Hist𝑦(𝑌)) , (3) 1. Introduction We tested our proposed algorithm on two hyperspectral AVIRIS datasets with 220 Typically, in band selection, the similarity space is defined among hyperspectral bands after converting the image bands into vectors, where a dissimilarity measure is defined based The 50 100 150 200 20 40 60 80 100 120 140 160 180 200 220 Figure 1: Dissimilarity matrix of hyperspectral image with 220 bands using MI. 2 The Scientific World Journal 2 Figure 1: Dissimilarity matrix of hyperspectral image with 220 bands using MI. where 𝐸is the number of entries. Hist𝑥(𝑋) and Hist𝑦(𝑌) are defined as their histograms and Hist𝑥𝑦(𝑋, 𝑌) as joint histogram. and 204 band images, respectively, and their corresponding ground truths. The experimental results show that using our proposed dissimilarity metric provides a more suitable subset of bands for pixel classification. Figure 1 shows the dissimilarity matrix of 220-band AVIRIS Indian Pines image scene using MI. 2. Dissimilarity Measures MI = 𝐻(𝑋) + 𝐻(𝑌) −𝐻(𝑋, 𝑌) , (1) (1) where 𝐻(𝑋) and 𝐻(𝑌) are the Shannon entropies [8] of 𝑋 and 𝑌, respectively, and 𝐻(𝑋, 𝑌) is the Shannon entropy of the joint distribution of 𝑋and 𝑌. 𝐻(𝑋) is defined as If 𝑋and 𝑌are image bands the spatial mutual information is defined by 𝐻(𝑋) = 𝑁 ∑ 𝑖=1 𝑝𝑋(𝑖) log 𝑝𝑋(𝑖) , (2) (2) SMI = 1 𝑀∑ 𝑥 ∑ 𝑦 Diff (𝑋, 𝑌) ∗Hist𝑥𝑦(𝑋, 𝑌) ∗log ( 𝑀∗Hist𝑥𝑦(𝑋, 𝑌) Hist𝑥(𝑋) ∗Hist𝑦(𝑌)) , (4) where 𝑝𝑋(𝑖) is the probability distribution. where 𝑝𝑋(𝑖) is the probability distribution. 4. Experiments and Results In our experiments, datasets are used to evaluate the per- formance of the proposed method. The first dataset is the Airborne Visible/Infrared Imaging Spectrometer (AVIRIS) image taken over northwestern Indiana’s Indian Pine test site, which has been widely used for experiments [11, 12]. The Indian Pine dataset is with the resolution of 145 × 145 pixels and has 220 spectral bands. There are 16 classes in total, ranging in size from 20 to 2455 pixels. The dataset is accom- panied with a reference map, indicating the ground truth. The background class was not considered for classification. The Salinas dataset consists of 204 spectral bands with size of 217 × 512 pixels [13]. There are 16 classes in total ranging from 916 to 11721 pixels. The background area was not used for classification. Indian Pines AVIRIS Ground Truth Classes (1) Background (2) Alfalfa (3) Corn no Till (4) Corn-min Till (5) Corn (6) Grass-pasture (7) Grass-trees (8) Grass/Pasture-mowed (9) Hay-windrowed (10) Oats (11) Soybean no Till (12) Soybean min Till (13) Soybean-clean (14) Wheat (15) Woods (16) Building-Grass Tree-Drives (17) Stone-Steel Towers. i Hierarchical clustering is used in this work. It is normally represented in tree structures with a nested set of partitions. The dissimilarity space is used to obtain a sequence of disjoint partitions. The distance between each pair of groups is used to decide how to link nested clusters in the consecutive levels of the hierarchy. One interesting characteristic of hierarchical methods is the fact that different linkage strategies create different tree structures. We use an agglomerative strategy in this work. That is, it starts with 𝑚initial clusters and, at each step, merges the two most similar groups to form a new cluster. Thus, the number of groups is reduced one by one [10]. In the end, bands are grouped according to the amount of information they share. In a final stage, a band representing each cluster is chosen, in such a way that the band selected will share as much information with respect to the other bands in the cluster. 3. Our Proposed Band Selection Algorithm The goal of our algorithm is to select a subset of image bands that are independent as possible. The independence (3) 3 3 The Scientific World Journal 20 40 60 80 100 120 140 20 40 60 80 100 120 140 (a) 20 40 60 80 100 120 140 20 40 60 80 100 120 140 (b) 1000 1200 1400 1600 1800 2000 2200 2400 0 10 20 30 40 50 60 70 80 90 (c) Figure 2: (a) 144th band of AVIRIS Indian Pines scene. (b) Randomized image in (a). (c) Histogram of images in (a) and (b). 20 40 60 80 100 120 140 20 40 60 80 100 120 140 (a) 20 40 60 80 100 120 140 20 40 60 80 100 120 140 (b) (b) 1000 1200 1400 1600 1800 2000 2200 2400 0 10 20 30 40 50 60 70 80 90 (c) (c) Figure 2: (a) 144th band of AVIRIS Indian Pines scene. (b) Randomized image in (a). (c) Histogram of images in (a) and (b). 50 100 150 200 20 40 60 80 100 120 140 160 180 200 220 Figure 3: Dissimilarity matrix of hyperspectral image with 220 bands using SMI. Figure 3: Dissimilarity matrix of hyperspectral image with 220 bands using SMI. The Scientific World Journal 4 The following lists show the classes of the Indian Pines and Salinas datasets, respectively. of selected bands increases the accuracy of classification of pixels [9]. We use the dissimilarity measure spatial mutual information to define a dissimilarity space as shown in Figure 3. Then, clustering is used to group bands according to the information they share. Finally, a band representing each cluster is selected for classification purposes. Indian Pines AVIRIS Ground Truth Classes Salinas AVIRIS Ground Truth Classes (1) Background (2) Brocoli green weeds 1 (3) Brocoli green weeds 2 (4) Fallow (5) Fallow rough plow (6) Fallow smooth (7) Stubble (8) Celery (9) Grapes untrained (10) Soil vineyard develop (11) Corn senesced green weeds (12) Lettuce romaine 4 wk (13) Lettuce romaine 5 wk (14) Lettuce romaine 6 wk (15) Lettuce romaine 7 wk (16) Vinyard untrained (17) Vinyard vertical trellis. (1) Background (2) Brocoli green weeds 1 (3) Brocoli green weeds 2 (4) Fallow (5) Fallow rough plow (6) Fallow smooth (7) Stubble (8) Celery (9) Grapes untrained (10) Soil vineyard develop (11) Corn senesced green weeds (12) Lettuce romaine 4 wk (13) Lettuce romaine 5 wk (14) Lettuce romaine 6 wk (15) Lettuce romaine 7 wk (16) Vinyard untrained (17) Vinyard vertical trellis. i In this work, use the support vector machine (SVM) for classification. The SVM classifies data into two groups by constructing a hyperplane [14]. Intuitively, a good separation is achieved by the hyperplane that has the largest distance to the nearest training data point of two classes. Generally the larger the margin the lower the generalization error of the classifier. In this work, we use the multiclass SVM scheme, named one-versus-all. The one-versus-all scheme involves the division of an 𝑁number of classes dataset into 𝑁two- class cases. The radial basis function (RBF) is used as the kernel function in this experiment.h The pixels from every 16 classes are randomly sepa- rated into 55% and 45% as the training and testing data, respectively. For our experiment, 5,702 and 61107 pixels form the training data of the Indian Pines and Salinas datasets, respectively. The rest of the pixels for each dataset form the testing data. The ground truths of the Indian Pines and Salinas datasets are shown in Figures 5 and 6, respectively. (17) Vinyard vertical trellis. We evaluated the overall accuracy which is the total number of correctly classified samples versus the number of samples. Salinas AVIRIS Ground Truth Classes Figures 4(a) and 4(b) compare the classification accuracy using our proposed algorithm and one of the popular methods used for band selection [15–17], which has The Scientific World Journal 5 0 10 20 30 40 50 40 50 60 70 80 90 100 Number of selected bands Classification accuracy Indian pines Spatial mutual information Mutual information (a) 0 10 20 30 40 50 50 55 60 65 70 75 80 85 90 95 100 Number of selected bands Classification accuracy Salinas Spatial mutual information Mutual information (b) Figure 4: Classification results between our proposed algorithm and using mutual information to define the dissimilarity space for (a) Indian Pines dataset and (b) Salinas dataset. 0 10 20 30 40 50 50 55 60 65 70 75 80 85 90 95 100 Number of selected bands Classification accuracy Salinas 0 10 20 30 40 50 40 50 60 70 80 90 100 Number of selected bands Classification accuracy Indian pines Classification accuracy Figure 4: Classification results between our proposed algorithm and using mutual information to define the dissimilarity space for (a) Indian Pines dataset and (b) Salinas dataset. 20 40 60 80 100 120 140 160 180 200 50 100 150 200 250 300 350 400 450 500 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 Figure 6: Ground truth for Salinas dataset. 20 40 60 80 100 120 140 20 40 60 80 100 120 140 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 Figure 5: Ground truth for Indian Pines dataset. 20 40 60 80 100 120 140 20 40 60 80 100 120 140 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 Figure 5: Ground truth for Indian Pines dataset. 20 40 60 80 100 120 140 160 180 200 50 100 150 200 250 300 350 400 450 500 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 Figure 6: Ground truth for Salinas dataset. 20 40 60 80 100 120 140 160 180 200 50 100 150 200 250 300 350 400 450 500 20 40 60 80 100 120 140 20 40 60 80 100 120 140 Figure 5: Ground truth for Indian Pines dataset. Figure 6: Ground truth for Salinas dataset. Salinas AVIRIS Ground Truth Classes for our proposed method and using MI is 73% and 67%, respectively. Figures 7 and 8 visualize the classification results of our experiment. The figures show that there is general improvement in classification accuracy with the increasing with number of bands selected. a similar configuration as in our proposed algorithm but MI is used to define the dissimilarity space as shown in Figure 1.hi a similar configuration as in our proposed algorithm but MI is used to define the dissimilarity space as shown in Figure 1. The classification accuracy of our proposed algorithm is generally higher than using MI. For smaller numbers of band selection our proposed method is particularly more robust. The average classification accuracy for the Indian Pines dataset using number of bands selected from 2 to 10 for our proposed method and using MI is 70% and 65%, respectively. The average classification accuracy for the Salinas dataset using the same number of bands range The classification accuracy of our proposed algorithm is generally higher than using MI. For smaller numbers of band selection our proposed method is particularly more robust. The average classification accuracy for the Indian Pines dataset using number of bands selected from 2 to 10 for our proposed method and using MI is 70% and 65%, respectively. The average classification accuracy for the Salinas dataset using the same number of bands range 5. Conclusions In this paper, we propose a new hyperspectral band selection algorithm for pixel classification. The algorithm uses spatial 6 The Scientific World Journal 6 20 40 60 80 100 120 140 20 40 60 80 100 120 140 (53%, 2 bands) 20 40 60 80 100 120 140 20 40 60 80 100 120 140 20 40 60 80 100 120 140 20 40 60 80 100 120 140 (73%, 5 bands) 20 40 60 80 100 120 140 20 40 60 80 100 120 140 20 40 60 80 100 120 140 20 40 60 80 100 120 140 (82%, 10 bands) 20 40 60 80 100 120 140 20 40 60 80 100 120 140 (87%, 15 bands) (91%, 20 bands) (94%, 25 bands) Figure 7: Classification results of Indian Pines dataset using our proposed algorithm. 20 40 60 80 100 120 140 20 40 60 80 100 120 140 (82%, 10 bands) 20 40 60 80 100 120 140 20 40 60 80 100 120 140 (73%, 5 bands) 20 40 60 80 100 120 140 20 40 60 80 100 120 140 (53%, 2 bands) (53%, 2 bands) (82%, 10 bands) (73%, 5 bands) (73%, 5 bands) 20 40 60 80 100 120 140 20 40 60 80 100 120 140 (94%, 25 bands) 20 40 60 80 100 120 140 20 40 60 80 100 120 140 (91%, 20 bands) 20 40 60 80 100 120 140 20 40 60 80 100 120 140 (87%, 15 bands) (87%, 15 bands) (91%, 20 bands) (94%, 25 bands) Figure 7: Classification results of Indian Pines dataset using our proposed algorithm. 10 20 30 40 50 60 70 80 90 100 50 100 150 200 250 10 20 30 40 50 60 70 80 90 100 50 100 150 200 250 10 20 30 40 50 60 70 80 90 100 50 100 150 200 250 10 20 30 40 50 60 70 80 90 100 50 100 150 200 250 10 20 30 40 50 60 70 80 90 100 50 100 150 200 250 10 20 30 40 50 60 70 80 90 100 50 100 150 200 250 (60%, 2 bands) (77%, 5 bands) (82%, 10 bands) (90%, 15 bands) (95%, 20 bands) (96%, 25 bands) Figure 8: Classification results of Salinas dataset using our proposed algorithm. Conflict of Interests The author declares that there is no conflict of interests regarding the publication of this paper. [15] A. Mart´ınez-Us´o, F. Pla, J. M. Sotoca, and P. Garc´ıa-Sevilla, “Clustering-based hyperspectral band selection using informa- tion measures,” IEEE Transactions on Geoscience and Remote Sensing, vol. 45, no. 12, pp. 4158–4171, 2007. Acknowledgment [16] I. S. Dhillon, S. Mallela, and R. Kumar, “A divisive information- theoretic feature clustering algorithm for text classification,” Journal of Machine Learning Research, vol. 3, pp. 1265–1287, 2003. The author would like to thank Professor Turgay Celik for his help in the implementation of the algorithms. The author would like to thank Professor Turgay Celik for his help in the implementation of the algorithms. [17] A. Gersho and R. M. Gray, Vector Quantization and Signal Compression, Kluwer, Norwell, Mass, USA, 1992. 5. Conclusions 10 20 30 40 50 60 70 80 90 100 50 100 150 200 250 (82%, 10 bands) 10 20 30 40 50 60 70 80 90 100 50 100 150 200 250 (77%, 5 bands) 10 20 30 40 50 60 70 80 90 100 50 100 150 200 250 (60%, 2 bands) 10 20 30 40 50 60 70 80 90 100 (82%, 10 bands) 10 20 30 40 50 60 70 80 90 100 50 100 150 200 250 (90%, 15 bands) 10 20 30 40 50 60 70 80 90 100 50 100 150 200 250 (95%, 20 bands) 10 20 30 40 50 60 70 80 90 100 50 100 150 200 250 (96%, 25 bands) 10 20 30 40 50 60 70 80 90 100 (96%, 25 bands) (95%, 20 bands) (95%, 20 bands) Figure 8: Classification results of Salinas dataset using our proposed algorithm. The Scientific World Journal 7 mutual information to calculate the dissimilarity space for band selection. We compare our method to a state-of-the-art method where mutual information is used as the dissimilarity metric. The experiments demonstrate that our proposed method can achieve more accurate pixel classification results than using mutual information. In future, we will apply our proposed method to other large datasets and investigate optimization algorithms to reduce computational cost. [12] G. Camps-Valls, T. V. B. Marsheva, and D. Zhou, “Semi- supervised graph-based hyperspectral image classification,” IEEE Transactions on Geoscience and Remote Sensing, vol. 45, no. 10, pp. 3044–3054, 2007. [13] R. Nakamura, J. Papa, and L. Fonseca, “Hyperspectral band selection through optimum-path forest and evolutionary-based algorithms,” in Proceedings of the IEEE International Geoscience and Remote Sensing Symposium (IGARSS ’12), pp. 3066–3069, 2012. [14] N. Cristianini and J. Shawe-Taylor, An Introduction to Support Vector Machines and Other Kernel-Based Learning Methods, Cambridge University Press, Cambridge, UK, 2000. References [1] D. Landgrebe, “Hyperspectral image data analysis,” IEEE Signal Processing Magazine, vol. 19, no. 1, pp. 17–28, 2002. [2] S. B. Serpico and L. Bruzzone, “A new search algorithm for feature selection in hyperspectral remote sensing images,” IEEE Transactions on Geoscience and Remote Sensing, vol. 39, no. 7, pp. 1360–1367, 2001. [3] L. Bruzzonne, F. Roli, and S. B. Serpico, “An extension to multi- class cases of the Jeffreys-Matusita distance,” IEEE Transactions on Geoscience and Remote Sensing, vol. 33, no. 6, pp. 1318–1321, 1995. [4] L. O. Jim´enez-Rodr´ıguez, E. Arzuaga-Cruz, and M. V´elez- Reyes, “Unsupervised linear feature-extraction methods and their effects in the classification of high-dimensional data,” IEEE Transactions on Geoscience and Remote Sensing, vol. 45, no. 2, pp. 469–483, 2007. [5] J. Wang and C.-I. Chang, “Independent component analysis- based dimensionality reduction with applications in hyper- spectral image analysis,” IEEE Transactions on Geoscience and Remote Sensing, vol. 44, no. 6, pp. 1586–1600, 2006. [6] C.-I. Chang and S. Wang, “Constrained band selection for hyperspectral imagery,” IEEE Transactions on Geoscience and Remote Sensing, vol. 44, no. 6, pp. 1575–1585, 2006. [7] C. Ding and X. He, “Cluster merging and splitting in hierar- chical clustering algorithms,” in Proceedings of the 2nd IEEE International Conference on Data Mining (ICDM ’02), vol. 1, pp. 139–146, December 2002. [8] C. E. Shannon, “A mathematical theory of communication,” The Bell System Technical Journal, vol. 27, pp. 379–423, 1948. [9] S. Kumar, J. Ghosh, and M. M. Crawford, “Best-bases feature extraction algorithms for classification of hyperspectral data,” IEEE Transactions on Geoscience and Remote Sensing, vol. 39, no. 7, pp. 1368–1379, 2001. [10] J. Ward, “Hierarchical grouping to optimize an objective func- tion,” Journal of the American Statistical Association, vol. 58, no. 301, pp. 236–244, 1963. [11] T. V. Bandos, L. Bruzzone, and G. Camps-Valls, “Classification of hyperspectral images with regularized linear discriminant analysis,” IEEE Transactions on Geoscience and Remote Sensing, vol. 47, no. 3, pp. 862–873, 2009.
https://openalex.org/W2769534618	https://uknowledge.uky.edu/cgi/viewcontent.cgi?article=1095&context=microbio_facpub	English	null	Ablation of an Ovarian Tumor Family Deubiquitinase Exposes the Underlying Regulation Governing the Plasticity of Cell Cycle Progression in <i>Toxoplasma gondii</i>	MBio	2,017	cc-by	23,894	University of Kentucky University of Kentucky UKnowledge UKnowledge Microbiology, Immunology, and Molecular Genetics Faculty Publications Microbiology, Immunology, and Molecula Genetics 11-21-2017 Ablation of an Ovarian Tumor Family Deubiquitinase Exposes the Ablation of an Ovarian Tumor Family Deubiquitinase Exposes the Underlying Regulation Governing the Plasticity of Cell Cycle Underlying Regulation Governing the Plasticity of Cell Cycle Progression in Progression in Toxoplasma gondii Animesh Dhara University of Kentucky, animesh.dhara@uky.edu Rodrigo de Paula Baptista University of Georgia Jessica C. Kissinger University of Georgia Ernest Charles Snow University of Kentucky, ecsnow01@uky.edu Anthony P. Sinai University of Kentucky, sinai@uky.edu Follow this and additional works at: https://uknowledge.uky.edu/microbio_facpub Part of the Medical Immunology Commons, Medical Microbiology Commons, and the Molecular Genetics Commons Right click to open a feedback form in a new tab to let us know how this document benefits you. Right click to open a feedback form in a new tab to let us know how this document benefits you. Repository Citation Repository Citation Dhara, Animesh; de Paula Baptista, Rodrigo; Kissinger, Jessica C.; Snow, Ernest Charles; and Sinai, Anthony P., "Ablation of an Ovarian Tumor Family Deubiquitinase Exposes the Underlying Regulation Governing the Plasticity of Cell Cycle Progression in Toxoplasma gondii" (2017). Microbiology, Immunology, and Molecular Genetics Faculty Publications. 96. https://uknowledge.uky.edu/microbio_facpub/96 This Article is brought to you for free and open access by the Microbiology, Immunology, and Molecular Genetics at UKnowledge. It has been accepted for inclusion in Microbiology, Immunology, and Molecular Genetics Faculty Publications by an authorized administrator of UKnowledge. For more information, please contact UKnowledge@lsv.uky.edu. University of Kentucky University of Kentucky UKnowledge UKnowledge Microbiology, Immunology, and Molecular Genetics Faculty Publications Microbiology, Immunology, and Molecular Genetics 11-21-2017 Ablation of an Ovarian Tumor Family Deubiquitinase Exposes the Ablation of an Ovarian Tumor Family Deubiquitinase Exposes the Underlying Regulation Governing the Plasticity of Cell Cycle Underlying Regulation Governing the Plasticity of Cell Cycle Progression in Progression in Toxoplasma gondii Animesh Dhara University of Kentucky, animesh.dhara@uky.edu Rodrigo de Paula Baptista University of Georgia Jessica C. Kissinger University of Georgia Ernest Charles Snow University of Kentucky, ecsnow01@uky.edu Anthony P. Sinai University of Kentucky, sinai@uky.edu Follow this and additional works at: https://uknowledge.uky.edu/microbio_facpub Part of the Medical Immunology Commons, Medical Microbiology Commons, and the Molecular Genetics Commons Right click to open a feedback form in a new tab to let us know how this document benefits you. Right click to open a feedback form in a new tab to let us know how this document benefits you. Repository Citation Repository Citation Dhara, Animesh; de Paula Baptista, Rodrigo; Kissinger, Jessica C.; Snow, Ernest Charles; and Sinai, Anthony P., "Ablation of an Ovarian Tumor Family Deubiquitinase Exposes the Underlying Regulation Governing the Plasticity of Cell Cycle Progression in Toxoplasma gondii" (2017). Microbiology, Immunology, and Molecular Genetics Faculty Publications. 96. https://uknowledge.uky.edu/microbio_facpub/96 This Article is brought to you for free and open access by the Microbiology, Immunology, and Molecular Genetics at UKnowledge. It has been accepted for inclusion in Microbiology, Immunology, and Molecular Genetics Faculty Publications by an authorized administrator of UKnowledge. For more information, please contact UK l d @l k d University of Kentucky University of Kentucky UKnowledge UKnowledge Microbiology, Immunology, and Molecular Genetics Microbiology, Immunology, and Molecular Genetics Faculty Publications Microbiology, Immunology, and Molecular Genetics Faculty Publications Ablation of an Ovarian Tumor Family Deubiquitinase Exposes the Ablation of an Ovarian Tumor Family Deubiquitinase Exposes the Underlying Regulation Governing the Plasticity of Cell Cycle Underlying Regulation Governing the Plasticity of Cell Cycle Progression in Progression in Toxoplasma gondii Animesh Dhara University of Kentucky, animesh.dhara@uky.edu Repository Citation Repository Citation p y p y Dhara, Animesh; de Paula Baptista, Rodrigo; Kissinger, Jessica C.; Snow, Ernest Charles; and Sinai, Anthony P., "Ablation of an Ovarian Tumor Family Deubiquitinase Exposes the Underlying Regulation Governing the Plasticity of Cell Cycle Progression in Toxoplasma gondii" (2017). Microbiology, Immunology, and Molecular Genetics Faculty Publications. 96. https://uknowledge.uky.edu/microbio_facpub/96 This Article is brought to you for free and open access by the Microbiology, Immunology, and Molecular Genetics at UKnowledge. It has been accepted for inclusion in Microbiology, Immunology, and Molecular Genetics Faculty Publications by an authorized administrator of UKnowledge. For more information, please contact UKnowledge@lsv.uky.edu. Ablation of an Ovarian Tumor Family Deubiquitinase Exposes the Underlying Ablation of an Ovarian Tumor Family Deubiquitinase Exposes the Underlying Regulation Governing the Plasticity of Cell Cycle Progression in Regulation Governing the Plasticity of Cell Cycle Progression in Toxoplasma gondii Digital Object Identifier (DOI) https://doi.org/10.1128/mBio.01846-17 Notes/Citation Information Notes/Citation Information Published in mBio, v. 8, issue 6, e01846-17, p. 1-28. Notes/Citation Information Notes/Citation Information Published in mBio, v. 8, issue 6, e01846-17, p. 1-28. Copyright © 2017 Dhara et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license. This article is available at UKnowledge: https://uknowledge.uky.edu/microbio_facpub/96 This article is available at UKnowledge: https://uknowledge.uky.edu/microbio_facpub/96 November/December 2017 Volume 8 Issue 6 e01846-17 ® mbio.asm.org 1 Animesh Dhara,a Rodrigo de Paula Baptista,b Jessica C. Kissinger,b,c,d E. Charles Snow,a Anthony P. Sinaia Animesh Dhara,a Rodrigo de Paula Baptista,b Jessica C. Kissinger,b,c,d E. Charles Snow,a Anthony P. Sinaia Department of Microbiology, Immunology and Molecular Genetics, University of Kentucky College of Medicine, Lexington, Kentucky, USAa; Center for Tropical and Emerging Global Diseases, University of Georgia, Athens, Georgia, USAb; Institute of Bioinformatics, University of Georgia, Athens, Georgia, USAc; Department of Genetics, University of Georgia, Athens, Georgia, USAd Department of Microbiology, Immunology and Molecular Genetics, University of Kentucky College of Medicine, Lexington, Kentucky, USAa; Center for Tropical and Emerging Global Diseases, University of Georgia, Athens, Georgia, USAb; Institute of Bioinformatics, University of Georgia, Athens, Georgia, USAc; Department of Genetics, University of Georgia, Athens, Georgia, USAd ABSTRACT The Toxoplasma genome encodes the capacity for distinct architectures underlying cell cycle progression in a life cycle stage-dependent manner. Replication in intermediate hosts occurs by endodyogeny, whereas a hybrid of schizogony and endopolygeny occurs in the gut of the deﬁnitive feline host. Here, we characterize the consequence of the loss of a cell cycle-regulated ovarian tumor (OTU family) deubiquitinase, OTUD3A of Toxoplasma gondii (TgOTUD3A; TGGT1_258780), in T. gondii tachyzoites. Rather than the mutation being detrimental, mutant parasites ex- hibited a ﬁtness advantage, outcompeting the wild type. This phenotype was due to roughly one-third of TgOTUD3A-knockout (TgOTUD3A-KO) tachyzoites exhibiting de- viations from endodyogeny by employing replication strategies that produced 3, 4, or 5 viable progeny within a gravid mother instead of the usual 2. We established the mechanistic basis underlying these altered replication strategies to be a dysregu- lation of centrosome duplication, causing a transient loss of stoichiometry between the inner and outer cores that resulted in a failure to terminate S phase at the at- tainment of 2N ploidy and/or the decoupling of mitosis and cytokinesis. The result- ing dysregulation manifested as deviations in the normal transitions from S phase to mitosis (S/M) (endopolygeny-like) or M phase to cytokinesis (M/C) (schizogony-like). Notably, these imbalances are corrected prior to cytokinesis, resulting in the genera- tion of normal progeny. Our ﬁndings suggest that decisions regarding the utilization of speciﬁc cell cycle architectures are controlled by a ubiquitin-mediated mechanism that is dependent on the absolute threshold levels of an as-yet-unknown target(s). Analysis of the TgOTUD3A-KO mutant provides new insights into mechanisms un- derlying the plasticity of apicomplexan cell cycle architecture. Received 4 October 2017 Accepted 9 October 2017 Published 21 November 2017 Citation Dhara A, de Paula Baptista R, Kissinger JC, Snow EC, Sinai AP. 2017. Ablation of an ovarian tumor family deubiquitinase exposes the underlying regulation governing the plasticity of cell cycle progression in Toxoplasma gondii. mBio 8:e01846-17. https:// doi.org/10.1128/mBio.01846-17. Editor Louis M. Weiss, Albert Einstein College of Medicine Copyright © 2017 Dhara et al. This is an open- access article distributed under the terms of the Creative Commons Attribution 4.0 International license. Address correspondence to Anthony P. Sinai, sinai@uky.edu. Animesh Dhara,a Rodrigo de Paula Baptista,b Jessica C. Kissinger,b,c,d E. Charles Snow,a Anthony P. Sinaia Apicomplexa exhibit diverse repli- cation strategies that are deﬁned by the organization of their cell cycles, which are more complex than those of higher eukaryotes (3). The apicomplexan cell cycle is deﬁned by two distinct phases: the nuclear cycle (comprising DNA synthesis, mitosis, and karyokinesis) and the budding cycle (comprising cytokinesis) (see Fig. S1 in the supplemental material). This architecture accounts for the capacity of these parasites to uncouple the nuclear cycle from the budding cycle depending on the replication strategy employed (3). P Toxoplasma gondii, an important member of the Apicomplexa, is of particular concern in immunocompromised individuals and as a source of congenitally acquired infections (4). Toxoplasma parasites exhibit the capacity to alter their replication strategy, with tachyzoites (associated with the acute asexual phase) (5) and bradyzoites (associated with the chronic asexual phase) replicating by endodyogeny (6, 7), while the replication of merozoites in the feline intestine occurs by a hybrid of schizogony and endopolygeny (8, 9). As such, the Toxoplasma genome encodes the capacity to repli- cate by all 3 major apicomplexan cell cycle mechanisms. Endodyogeny is an internal budding mechanism by which 2 daughters are formed within the mother (Fig. S1) (5, 7). In the course of endodyogeny, each cycle is associated with a single round of DNA synthesis, progressing to mitosis and karyokinesis, which overlaps with cytokinesis, resulting in the formation of the daughter parasites within the mother cell (3, 10). In contrast, Plasmodium spp., the agents of malaria, replicate by schizogony wherein rounds of DNA synthesis and mitosis/karyokinesis in the absence of cytokinesis result in a multinucleated cell that then undergoes synchronous cytoki- nesis (budding cycle), resulting in an even number of progeny (Fig. S1) (11). The third apicomplexan strategy is endopolygeny, (e.g., Sarcocystis spp.), in which the nuclear cycle is deﬁned by successive rounds of DNA synthesis and what appears to be mitosis (segregation of replicated genomes) without the execution of karyokinesis (nuclear division), resulting in the formation of a polyploid nuclear mass within the corpus of the parasite (3). Upon entry into the budding cycle (cytokinesis), the execution of karyoki- nesis results in the packaging of individual nuclei into the forming daughter scaffolds, which allows the birth of multiple progeny (64 in the case of Sarcocystis neurona) (Fig. S1) (3). Animesh Dhara,a Rodrigo de Paula Baptista,b Jessica C. Kissinger,b,c,d E. Charles Snow,a Anthony P. Sinaia Received 4 October 2017 Accepted 9 October 2017 Published 21 November 2017 Citation Dhara A, de Paula Baptista R, Kissinger JC, Snow EC, Sinai AP. 2017. Ablation of an ovarian tumor family deubiquitinase exposes the underlying regulation governing the plasticity of cell cycle progression in Toxoplasma gondii. mBio 8:e01846-17. https:// doi.org/10.1128/mBio.01846-17. Editor Louis M. Weiss, Albert Einstein College of Medicine Copyright © 2017 Dhara et al. This is an open- access article distributed under the terms of the Creative Commons Attribution 4.0 International license. Address correspondence to Anthony P. Sinai, sinai@uky.edu. Received 4 October 2017 Accepted 9 October 2017 Published 21 November 2017 Citation Dhara A, de Paula Baptista R, Kissinger JC, Snow EC, Sinai AP. 2017. Ablation of an ovarian tumor family deubiquitinase exposes the underlying regulation governing the plasticity of cell cycle progression in Toxoplasma gondii. mBio 8:e01846-17. https:// doi.org/10.1128/mBio.01846-17. Editor Louis M. Weiss, Albert Einstein College of Medicine Copyright © 2017 Dhara et al. This is an open- access article distributed under the terms of the Creative Commons Attribution 4.0 International license. Address correspondence to Anthony P. Sinai, sinai@uky.edu. Address correspondence to Anthony P. Sinai, sinai@uky.edu. Address correspondence to Anthony P. Sinai, sinai@uky.edu. IMPORTANCE Replication by Toxoplasma gondii can occur by 3 distinct cell cycle architectures. Endodyogeny is used by asexual stages, while a hybrid of schizogony and endopolygeny is used by merozoites in the deﬁnitive feline host. Here, we es- tablish that the disruption of an ovarian-tumor (OTU) family deubiquitinase, TgOTUD3A, in tachyzoites results in dysregulation of the mechanism controlling the selection of replication strategy in a subset of parasites. The mechanistic basis for these altered cell cycles lies in the unique biology of the bipartite centrosome that is associated with the transient loss of stoichiometry between the inner and outer centrosome cores in the TgOTUD3A-KO mutant. This highlights the importance of ubiquitin-mediated regulation in the transition from the nuclear to the budding ® mbio.asm.org 1 ® mbio.asm.org 1 November/December 2017 Volume 8 Issue 6 e01846-17 ® Dhara et al. phases of the cell cycle and provides new mechanistic insights into the regulation of the organization of the apicomplexan cell cycle. KEYWORDS deubiquitinase, Toxoplasma gondii, apicomplexan parasites, cell cycle, centrosomes P arasites of the phylum Apicomplexa are responsible for numerous diseases of both human and veterinary importance (1, 2). At the heart of their pathogenesis is their ability to replicate intracellularly within their hosts. November/December 2017 Volume 8 Issue 6 e01846-17 mbio.asm.org 2 Animesh Dhara,a Rodrigo de Paula Baptista,b Jessica C. Kissinger,b,c,d E. Charles Snow,a Anthony P. Sinaia The mechanistic basis underlying the capacity of Toxoplasma to employ each of these three replication strategies in a life cycle stage-dependent context has not been explored. The replication of eukaryotes is a highly regulated unidirectional process that is deﬁned by the cell cycle (12, 13). Tight control is achieved by the spatiotemporal regulation of protein complexes governed by phosphorylation/dephosphorylation (14, 15) and protein stabilization/turnover (15). The latter mechanism is orchestrated pre- dominantly by a balance between ubiquitination and deubiquitination of speciﬁc targets (16), driven by a complex interplay between ubiquitin ligases and deubiquiti- nases (DUBs) (17, 18). The importance of ubiquitin-mediated mechanisms in the regulation of the Toxo- plasma cell cycle can be inferred by the disproportionate number of cell cycle-regulated genes whose products are subject to ubiquitination (19). Ubiquitination itself is a highly versatile posttranslational modiﬁcation based on the covalent linkage of ubiquitin (Ub) via one of 7 possible lysine residues (K6, K11, K27, K29, K33, K48, and K63) to a target protein that deﬁnes the ubiquitin code (20, 21). The resultant combinatorial diversity observed in polyUb structures provides a mechanism to achieve a very high degree of mbio.asm.org 2 ® A Toxoplasma DUB Controls Cell Cycle Architecture functional speciﬁcity (22) that is catalyzed by the activity of speciﬁc Ub ligases (23). On the other side of the equation, the deubiquitinases (DUBs) present a similar range of speciﬁcities regarding the Ub chains that they selectively target (18). This permits an exquisite level of ﬁne control that not only governs the stability/turnover of the target protein but can also have nondegradative signaling and trafﬁcking functions (24–26). The contribution of DUBs in the ﬁne-tuning of the cell cycle in higher eukaryotes is well documented (27–29). How the ubiquitin cycle regulates the diverse replication strate- gies of Apicomplexa remains unexplored. Here, we focus on the role ubiquitination plays as a means for controlling the ﬁdelity of Toxoplasma tachyzoite replication. While several studies have looked into the contribution of ubiquitin ligases in Apicomplexa (30–32), investigations on deubiquiti- nases are limited (30). We reasoned that DUBs that exhibit a strong cell cycle- dependent transcriptional expression proﬁle are more likely to be involved in the regulation of cell cycle progression. One gene, encoding a member of the ovarian tumor (OTU) family of DUBs (33), which we designated TgOTUD3A (T. gondii OTUD3A), exhibits tight cell cycle-dependent gene and protein expression (34). Animesh Dhara,a Rodrigo de Paula Baptista,b Jessica C. Kissinger,b,c,d E. Charles Snow,a Anthony P. Sinaia The functional characterization of recombinant TgOTUD3A conﬁrms its enzymatic activity and speci- ﬁcity (for K48-linked polyUb) (34). Somewhat to our surprise, the genetic ablation of TgOTUD3A failed to generate a gross growth defect despite a clear defect within a subset of parasites in the ﬁdelity of endodyogeny. In these instances, the generation of multiple daughter parasites often exhibited typical features of schizogony and endo- polygeny, suggestive of a partial shift in cell cycle architecture. Characterization of the TgOTUD3A-knockout (TgOTUD3A-KO) mutant exposes an important role for a ubiquitin-mediated process in regulating the selection of the replication strategy. Here, we investigate how the mechanistic basis underlying the selection of the replication strategy is connected to alterations in the dynamics of the bipartite Toxoplasma centrosome (35), particularly as it relates to the control of the transition from the nuclear to the budding phase of the cell cycle. This work establishes a framework for a better understanding of the complexity inherent in the architecture of the apicomplexan cell cycle. November/December 2017 Volume 8 Issue 6 e01846-17 RESULTS (A) Distribution and relative levels of TgOTUD3A-HA as functions of cell cycle progression, visualized using TgIMC3, nuclear morphology, and labeling intensity. TgOTUD3A-HA levels increase with the progression of the cell cycle from G1 through S/M, peaking at the end of mitosis and with the progression of cytokinesis. TgOTUD3A-HA localizes to the cytoplasm and associates with daughter scaffolds when present. The large labeled structure in the top right corner of the DNA panel is the nucleus of the infected host cell. Scale bars 10 m. (B) Location of the site targeted for CRISPR-Cas9-mediated cleavage at the TgOTUD3A genomic locus just downstream from the ATG start codon. The TgOTUD3A-targeting CRISPR–Cas9-GFP construct was cotransfected with a DNA sequence encoding the DHFR cassette, which can integrate into the CRISPR break site in two different orientations. Integration of DHFR into the locus was conﬁrmed using the indicated PCR primers (red, Fwd/R1 and Fwd/R2). (C) Immunoblot analysis of four pyrimethamine-resistant clones (2A1, 2B1, 2C3, and 2H1) with polyclonal TgOTUD3A mouse antibody reveals a loss of TgOTUD3A protein expression to conﬁrm the TgOTUD3A knockout. Independent clones 2C3 and 2H1 were characterized in this study. Protein from wild-type (WT) parasites was run as a positive antibody control. (D) Sequence analysis of theTgOTUD3A-KO DHFR insertion junction conﬁrms that 2C3 and 2H1 are independent clones. Sequence differences are highlighted in yellow. (E) Effect of TgOTUD3A knockout on growth assessed using a plaque assay: the areas of approximately 150 randomly acquired plaques (~50 plaques for 3 different lines each from three independent experiments) generated at 6 days postinfection by wild-type (either the RH or TgOTUD3A-HA-tagged line) and TgOTUD3A-KO parasites were measured and plotted. The mean plaque sizes of wild-type (RH and TgOTUD3A-HA) and TgOTUD3A-KO parasites were not statistically signiﬁcant by one-way ANOVA (F2,150 4.094, P 0.0186). (F) A head-to-head competition assay (see Materials and Methods for details) was performed. Data compiled from 4 independent experiments conﬁrm that there were mean 1.2-fold and 2-fold increases in the DNA content of TgOTUD3A-KO parasites relative to the DNA content of the wild type (TgOTUD3-HA) at 24 h and 48 h postinfection. When analyzed with ANOVA (F3,12 5.085, P 0.0168) and multiple comparisons, the average gDNA copy number increase at 48 h was found to be signiﬁcant, suggesting some ﬁtness advantage of the TgOTUD3A-KO mutant over the wild type. , P 0.01. Error bars represent standard deviations. RESULTS T. gondii with ablation of TgOTUD3A does not exhibit a gross growth defect. The progression of the Toxoplasma gondii tachyzoite cell cycle can be morphologically tracked based on the appearance of the nucleus and the development of the daughter scaffolds, detected using inner membrane complex (IMC) proteins as markers within the mother parasite undergoing endodyogeny. Parasites in G1 possessed a compact nucleus and weak labeling of the maternal inner membrane complex when visualized using the T. gondii IMC3 (TgIMC3) marker (Fig. 1A). Entry and progression into S phase was characterized by increased size and intensity of the parasite nuclei, which toward the end of S phase and the onset of mitosis (S/M) possessed a bilobed, heart-shaped conﬁguration (Fig. 1A, DNA). In light of the overlapping organization of the stages in endodyogeny (10), the early daughter buds were present during the initial phases of mitosis and are seen as the bright TgIMC3-labeled structures (Fig. 1A, IMC3, red). The completion of mitosis was evident from the resolution of the bilobed nuclear mass into two discrete nuclei and the expansion of the daughter buds into mature scaffolds, eventually resulting in the emergence of two new parasites upon the recycling of maternal components (Fig. 1A). TgOTUD3A is a cell cycle-regulated deubiquitinase of the ovarian tumor family (33), the levels of which increased upon entry into S phase, reaching peak levels during cytokinesis, when they appeared to be associated with the developing daughters (Fig. 1A) (34). This pattern of protein expression mirrored that of its gene transcription (36), suggesting a potentially important function in cell cycle progression. We used a clustered regularly interspaced short palindromic repeat– CRISPR-associated protein 9 (CRISPR-Cas9) system that has been adapted to Toxo- plasma gondii (37) to introduce a knock-in mutation into the open reading frame of the TgOTUD3A gene, just downstream from the ATG start codon, using a dihydrofolate mbio.asm.org 3 November/December 2017 Volume 8 Issue 6 e01846-17 mbio.asm.org on February 15, 2018 - Published by mbio.asm.org Downloaded from Dhara et al. mbio.asm.org on February 15, 2018 - Published by mbio.asm.org ded from g y , y g FIG 1 Cell cycle dynamics of T. gondii TgOTUD3A wild type and growth phenotype of TgOTUD3A-KO mutant. (A) Distribution and relative levels of TgOTUD3A-HA as functions of cell cycle progression, visualized using TgIMC3, nuclear morphology, and labeling intensity. RESULTS TgOTUD3A-HA levels increase with the progression of the cell cycle from G1 through S/M, peaking at the end of mitosis and with the progression of cytokinesis. TgOTUD3A-HA localizes to the cytoplasm and associates with daughter scaffolds when present. The large labeled structure in the top right corner of the DNA panel is the nucleus of the infected host cell. Scale bars 10 m. (B) Location of the site targeted for CRISPR-Cas9-mediated cleavage at the TgOTUD3A genomic locus just downstream from the ATG start codon. The TgOTUD3A-targeting CRISPR–Cas9-GFP construct was cotransfected with a DNA sequence encoding the DHFR cassette, which can integrate into the CRISPR break site in two different orientations. Integration of DHFR into the locus was conﬁrmed using the indicated PCR primers (red, Fwd/R1 and Fwd/R2). (C) Immunoblot analysis of four pyrimethamine-resistant clones (2A1, 2B1, 2C3, and 2H1) with polyclonal TgOTUD3A mouse antibody reveals a loss of TgOTUD3A protein expression to conﬁrm the TgOTUD3A knockout. Independent clones 2C3 and 2H1 were characterized in this study. Protein from wild-type (WT) parasites was run as a positive antibody control. (D) Sequence analysis of theTgOTUD3A-KO DHFR insertion junction conﬁrms that 2C3 and 2H1 are independent clones. Sequence differences are highlighted in yellow. (E) Effect of TgOTUD3A knockout on growth assessed using a plaque assay: the areas of approximately 150 randomly acquired plaques (~50 plaques for 3 different lines each from three independent experiments) generated at 6 days postinfection by wild-type (either the RH or TgOTUD3A-HA-tagged line) and TgOTUD3A-KO parasites were measured and plotted. The mean plaque sizes of wild-type (RH and TgOTUD3A-HA) and TgOTUD3A-KO parasites were not statistically signiﬁcant by one-way ANOVA (F2,150 4.094, P 0.0186). (F) A head-to-head competition assay (see Materials and Methods for details) was performed. Data compiled from 4 independent experiments conﬁrm that there were mean 1.2-fold and 2-fold increases in the DNA content of TgOTUD3A-KO parasites relative to the DNA content of the wild type (TgOTUD3-HA) at 24 h and 48 h postinfection. When analyzed with ANOVA (F3,12 5.085, P 0.0168) and multiple comparisons, the average gDNA copy number increase at 48 h was found to be signiﬁcant, suggesting some ﬁtness advantage of the TgOTUD3A-KO mutant over the wild type. , P 0.01. Error bars represent standard deviations. mbio.asm.org 2018 - Published by FIG 1 Cell cycle dynamics of T. gondii TgOTUD3A wild type and growth phenotype of TgOTUD3A-KO mutant. November/December 2017 Volume 8 Issue 6 e01846-17 mbio.asm.org 4 RESULTS 2A, Phase, white number 4) each contained two forming daughters, resulting in eight progeny within the vacuole (Fig. 2A, Phase, red number 8). This normal geometric progression, however, was differentially altered in a subset of TgOTUD3A-KO parasites that exhibited non-2n numbers of parasites per vacuole (Fig. 2C and D, Phase, white numbers). Deviation from a 2n progression of parasites per vacuole could be explained either by the loss of replicative synchrony or the production of more than two daughters per mother parasite per replicative cycle. Such deviation from normal endodyogeny has been reported to occur at a low level in wild-type parasites and to be enhanced under speciﬁc conditions (5, 39, 40), and it also occurs in conditional mutants with mutations affecting the cell cycle (41). We therefore examined the number of daughter scaffolds within gravid mother parasites within vacuoles using TgIMC3. As opposed to the wild-type parasites, where each gravid mother typically contains two daughters, each with an equally divided nucleus (Fig. 2A), a subset of the TgOTUD3A-KO parasites exhibited a diverse range of phenotypes, often within the same vacuoles, as pre- sented in Fig. 2B to D. Examination of the daughter scaffold burden (TgIMC3) revealed the presence of mothers with two (not marked), three, and four daughter scaffolds, respectively. In many instances, the mother parasites containing two scaffolds ap- peared to have progressed further along cytokinesis than those with three and four daughter scaffolds, based on the size of the TgIMC3 scaffolds (Fig. 2B). In other vacuoles, particularly those containing relatively immature scaffolds, the sizes of form- ing daughters with 2, 3, and 4 scaffolds were comparable (Fig. 2B and C). What was uniform, however, was that the size of daughter scaffolds within a given parasite (whether 2, 3, or 4) was always the same, suggesting coordination of the initiation and progression of cytokinesis within a given mother (Fig. 2B and C). In addition, despite the differences in the numbers of daughters within individual members of a vacuole, all of the parasites within a vacuole were gravid, as partial gravidity was rarely observed. Thus, despite individual parasites within a vacuole displaying differing numbers of developing daughters, they appeared to be synchronized with regard to transitioning from the nuclear to the budding cycle (cytokinesis). Insights into the patterns of replication were also provided from the nuclear morphology, best observed in the grayscale rendition of the DNA (Hoechst-stained) images. RESULTS reductase (DHFR) cassette that confers pyrimethamine resistance (Pyrr) (Fig. 1B). We were able to select multiple knockout clones that were conﬁrmed using immunoblot analysis (Fig. 1C), with the insertion and orientation of the drug resistance cassette conﬁrmed by PCR (data not shown), using primers Fwd/R1 and Fwd/R2 (Fig. 1B), and sequencing of the amplicons (Fig. 1D). The recovery of viable TgOTUD3A-KO clones indicated that the gene was nonessential. Further sequencing of the clones conﬁrmed the integration of DHFR, and sequence variation at the integration sites conﬁrmed that 2H1 and 2C3 were independent clonal lines (Fig. 1D). We sought to establish whether the loss of TgOTUD3A had any detrimental effect on parasite infectivity and growth using a plaque assay. Surprisingly, despite its being tightly cell cycle regulated (34) (Fig. 1A), mutants with the ablation of TgOTUD3A had no apparent growth defects relative to the growth of the wild-type (WT) parental (RH) or the hemagglutinin mbio.asm.org 4 November/December 2017 Volume 8 Issue 6 e01846-17 ® A Toxoplasma DUB Controls Cell Cycle Architecture (HA)-tagged line (in an RHΔKu80 background [34]) (Fig. 1E). We next conducted a head-to-head competition assay (see Materials and Methods for details), in which the TgOTUD3A-KO mutants consistently outcompeted the wild-type line, with mean in- creases of 1.2- and 2-fold, respectively, at 24 and 48 h postinfection (Fig. 1F). Of note, this ﬁtness advantage was reported for TgOTUD3A-KO in a recently published genome- wide CRISPR-based competition screen for essentiality (38). This unexpected result led us to perform cell cycle analysis based on DNA content for both wild-type and mutant parasites using ﬂow cytometry. Although small shifts in the proportions of parasites exhibiting 2N ploidy were observed for the TgOTUD3A-KO parasites, their levels were not statistically signiﬁcant (Fig. S2). This indicated that increased ploidy alone was not the sole driver of the higher genome equivalents observed in the mutant. We therefore assessed whether the observed increase in ﬁtness was due to an actual increase in parasite numbers. Increased asynchrony and abnormal endodyogeny in a subset of TgOTUD3A-KO mutants are associated with dysregulation of the nuclear cycle. A feature of tachyzoite replication is the geometric increase in parasite numbers due to a high degree of synchrony, resulting in their following a 2n progression for parasite growth within individual vacuoles. This outcome was seen in the example of the wild type, where four mother parasites (Fig. November/December 2017 Volume 8 Issue 6 e01846-17 RESULTS For mother parasites containing 3 scaffolds (Fig. 2B and C), 3 distinct nuclear states were evident. The ﬁrst contained a single large polyploid nucleus that was denser than the other nuclei within the same vacuole (Fig. 2B, DNA, blue number 1). The second state displayed 2 asymmetrical nuclei within the mother, one small and one large, that differed in both size and density (Fig. 2C, DNA, blue number 2). Finally, a state where the 3 daughter scaffolds were accompanied by 3 equivalently sized mbio.asm.org 5 mbio.asm.org on February 15, 2018 - Published by mbio.asm.org Downloaded from ® Dhara et al. FIG 2 TgOTUD3A-KO parasites exhibit a range of altered/aberrant endodyogeny phenotypes. WT and TgOTUD3A-KO parasites in HFFs were ﬁxed at 24 h postinfection and labeled with rat anti-TgIMC3 and rabbit anti-TgSAG1 antibodies. Nuclear DNA was labeled with Hoechst dye. The DNA images were converted into gray scale for better visualization. Phase images show parasite morphology, with total numbers of parasites (white numbers) and of mature/immature daughter scaffolds (red numbers) per vacuole indicated. (A) Wild-type parasites exhibit synchronous 2n replication as determined by the number of daughter scaffolds (TgIMC3, green) in each replicating parasite (TgSAG1, red), representing normal endodyogeny. (B and C) The replication phenotypes in TgOTUD3A-KO parasites are often altered with regard to the total number (non-2n) of parasites within vacuoles, the numbers of daughter scaffolds within individual mothers, the numbers of nuclei, and the nuclear morphology. In each case presented where daughter scaffolds are present, aberrant and normal endodyogeny are evident within the same vacuole. In the DNA images, symmetrical nuclear division is indicated with green numbers, while polyploid and asymmetrical nuclei within individual parasites are marked with blue numbers. In the Phase and TgSAG1 images, note that mother parasites containing 3 or 4 TgIMC3-positive (yellow numbers in TgIMC3 images) daughter scaffolds tend to be enlarged and morphologically distinct relative to mother parasites that are undergoing normal endodyogeny. In the case of multiple daughters, all the daughter scaffolds within a given mother were found to be of equal size. (D) Evidence of mitosis (nuclei marked with green 2 in DNA image) in the absence of linked cytokinesis suggests a disconnect between nuclear and budding cycles. (A to D) Scale bars 10 m. (E) Quantiﬁcation of aberrant replication. RESULTS Parasitophorous vacuoles (PV) were labeled with the PV marker mouse anti-TgGRA3 antibody, while individual parasites were identiﬁed using the surface marker rabbit anti-TgSAG1 antibody. All slides were coded, and the numbers of parasites per vacuole (2n [1, 2, 4, 8, and 16] versus non-2n) were counted blindly by three individuals. A total of 4 biological replicates with 3 independent samples per replicate were counted. One-way ANOVA indicated that there was a signiﬁcant difference (F11,36 P 0.0001) between the wild-type and TgOTUD3A-KO lines with regard to the incidence of deviation from a 2n progression that manifests following the ﬁrst 2 rounds of replication. A total of 1,649 vacuoles (WT 756 and KO 893) were counted for this analysis. (F) Comparison between numbers of daughter scaffolds (TgIMC3, green) per gravid parasite (TgSAG1, red) counted blindly in both wild-type and TgOTUD3A-KO parasites reveals that a signiﬁcantly higher proportion of gravid mother parasites bear 2 progeny in the TgOTUD3A-KO than in the parental (WT) line. Analysis was restricted to parasites that contained daughter scaffolds (TgIMC3 positive). One-way ANOVA indicated signiﬁcant differences (F9,25 761.8, P 0.0001) between wild-type and KO parasites. (E and F) Tukey’s pairwise multiple-comparison test ( 0.05) was used to compare the level of signiﬁcance for comparison between wild-type and KO parasites for each group analyzed. Signiﬁcant differences (P 0.05) are indicated by asterisks as follows: , P 0.01; , P 0.001; *, P 0.0001. A total of 2,059 (WT 980, KO 1,079) gravid parasites were counted in this analysis. Error bars represent standard deviations of the means. FIG 2 TgOTUD3A-KO parasites exhibit a range of altered/aberrant endodyogeny phenotypes. WT and TgOTUD3A-KO parasites in HFFs were ﬁxed at 24 h postinfection and labeled with rat anti-TgIMC3 and rabbit anti-TgSAG1 antibodies. Nuclear DNA was labeled with Hoechst dye. The DNA images were converted into gray scale for better visualization. Phase images show parasite morphology, with total numbers of parasites (white numbers) and of mature/immature daughter scaffolds (red numbers) per vacuole indicated. (A) Wild-type parasites exhibit synchronous 2n replication as determined by the number of daughter scaffolds (TgIMC3, green) in each replicating parasite (TgSAG1, red), representing normal endodyogeny. RESULTS (B and C) The replication phenotypes in TgOTUD3A-KO parasites are often altered with regard to the total number (non-2n) of parasites within vacuoles, the numbers of daughter scaffolds within individual mothers, the numbers of nuclei, and the nuclear morphology. In each case presented where daughter scaffolds are present, aberrant and normal endodyogeny are evident within the same vacuole. In the DNA images, symmetrical nuclear division is indicated with green numbers, while polyploid and asymmetrical nuclei within individual parasites are marked with blue numbers. In the Phase and TgSAG1 images, note that mother parasites containing 3 or 4 TgIMC3-positive (yellow numbers in TgIMC3 images) daughter scaffolds tend to be enlarged and morphologically distinct relative to mother parasites that are undergoing normal endodyogeny. In the case of multiple daughters, all the daughter scaffolds within a given mother were found to be of equal size. (D) Evidence of mitosis (nuclei marked with green 2 in DNA image) in the absence of linked cytokinesis suggests a disconnect between nuclear and budding cycles. (A to D) Scale bars 10 m. (E) Quantiﬁcation of aberrant replication. Parasitophorous vacuoles (PV) were labeled with the PV marker mouse anti-TgGRA3 antibody, while individual parasites were identiﬁed using the surface marker rabbit anti-TgSAG1 antibody. All slides were coded, and the numbers of parasites per vacuole (2n [1, 2, 4, 8, and 16] versus non-2n) were counted blindly by three individuals. A total of 4 biological replicates with 3 independent samples per replicate were counted. One-way ANOVA indicated that there was a signiﬁcant difference (F11,36 P 0.0001) between the wild-type and TgOTUD3A-KO lines with regard to the incidence of deviation from a 2n progression that manifests following the ﬁrst 2 rounds of replication. A total of 1,649 vacuoles (WT 756 and KO 893) were counted for this analysis. (F) Comparison between numbers of daughter scaffolds (TgIMC3, green) per gravid parasite (TgSAG1, red) counted blindly in both wild-type and TgOTUD3A-KO parasites reveals that a signiﬁcantly higher proportion of gravid mother parasites bear 2 progeny in the TgOTUD3A-KO than in the parental (WT) line. Analysis was restricted to parasites that contained daughter scaffolds (TgIMC3 positive). One-way ANOVA indicated signiﬁcant differences (F9,25 761.8, P 0.0001) between wild-type and KO parasites. November/December 2017 Volume 8 Issue 6 e01846-17 mbio.asm.org 6 RESULTS (E and F) Tukey’s pairwise multiple-comparison test ( 0.05) was used to compare the level of signiﬁcance for comparison between wild-type and KO parasites for each group analyzed. Signiﬁcant differences (P 0.05) are indicated by asterisks as follows: , P 0.01; *, P 0.001; , P 0.0001. A total of 2,059 (WT 980, KO 1,079) gravid parasites were counted in this analysis. Error bars represent standard deviations of the means. postmitotic nuclei (Fig. S3A) suggested the initial dysregulation was/could be corrected to produce multiple progeny from the gravid mother. For mother parasites containing 4 daughter scaffolds, 2 distinct nuclear morphologies were evident. The ﬁrst possessed 2 equivalently sized larger nuclei, with the small immature daughter buds suggesting a pending second round of nuclear division (karyokinesis) (Fig. 2C, DNA, green number 2). The second nuclear morphology exhibited 4 distinct nuclei suggestive of a post- karyokinesis state and associated with well-developed daughter TgIMC3 scaffolds (Fig. 2B, DNA, green number 4). In addition to instances where daughter scaffolds indicative of active cytokinesis were observed (Fig. 2B and C), the detection of karyokinesis (the presence of 2 [or more] discrete nuclei within a single mother parasite) with an absence of any visible daughter scaffolds (Fig. 2D) was indicative of an uncoupling of mitosis and cytokinesis that was typical of a schizogony-like replication strategy (5). Among these examples were instances where we appeared to have captured mitosis as it was occurring, seen as a mbio.asm.org 6 ® A Toxoplasma DUB Controls Cell Cycle Architecture dividing nucleus (Fig. 2D, DNA, green number 2). The unmarked nuclei in Fig. 2D appeared to be in parasites that were not cycling. Finally, the detection of a single immature daughter scaffold (Fig. S3D, TgIMC3, yellow arrowhead) in a mother parasite with completed mitosis within a vacuole where the other parasites had undergone or were in the process of mitosis indicated the potential for the aberrant initiation of cytokinesis at low frequency. The deviations from normal endodyogeny were observed at higher frequencies in TgOTUD3A-KO parasites. We therefore plotted the distribution of parasite numbers within randomly acquired vacuoles counted on blinded samples. The results revealed the expected progression of growth for wild-type parasites (both RH and RH- TgOTUD3A-HA), with a distribution that follows the 2n pattern and a small fraction of the vacuoles deviating from this progression (Fig. 2E). RESULTS The identical analysis performed using TgOTUD3A-KO mutants revealed a signiﬁcant increase (38%) in the proportion of non-2n parasite-containing vacuoles (Fig. 2E). Notably, the proportions of vacuoles harboring 1 to 4 mutant parasites were not statistically different from the results for the wild type, but there was signiﬁcant deviation at the 8-parasite stage (Fig. 2E). This suggested that the phenotype resulting in deviation from the 2n growth progression manifested following the initial rounds of replication. We also counted the numbers of daughters within gravid mother parasites by counting the numbers of daughter scaffolds (detected using TgIMC3 labeling) within them. Consistent with previously published data (39, 40) for actively growing wild-type parasites, 6.6% of mothers contained 3 daughter scaffolds (Fig. 2F). In contrast, the frequency of TgOTUD3A-KO mutant mother parasites with 3 daughters was 14.5% among gravid parasites (Fig. 2F). A similar proportional increase was observed for mothers bearing 4 daughter scaffolds (Fig. 2F), with a small number (1 out of 500 gravid parasites) containing 5 scaffolds (Fig. 2F). This suggested that the partial loss in the ﬁdelity of the counting mechanism to ensure accurate endodyogeny (resulting in 2 progeny) was evident in the TgOTUD3A-KO organisms. Notably, this analysis did not include instances of the schizogony-like phenotype (Fig. 2D) or the presence of polyploid nuclei without daughter scaffolds (see discussion of electron microscopy [EM] results below), thereby underestimating the scope of deviations from endodyogeny within the TgOTUD3A-KO parasites. Notably, the phenotype of the independent clone 2C3 (data not shown) mirrored that of clone 2H1 detailed here. In addition to examining the progression of mitosis and cytokinesis, we also examined the characteristics of the inheritance and formation of key organelles in mutant parasites. Multiple Golgi stacks were found in the mothers producing multiple daughters (Fig. S4), but in general, the numbers of apicoplasts and Golgi stacks matched the inferred numbers of daughter progeny based on the morphologies and intensities of the nuclei, suggesting faithful inheritance (Fig. S4 and S5). In addition, no defects were observed in the population with regard to the organization of the mitochondria, rhoptries, and micronemes (Fig. S6). These data suggested that the progeny resulting from the aberrant endodyogeny were normal and viable. This is consistent with the mutant out-competing the wild-type parasites in a head-to-head competition (Fig. 1F). Electron microscopy conﬁrms the diverse patterns of replication in TgOTUD3A-KO parasites. November/December 2017 Volume 8 Issue 6 e01846-17 RESULTS In light of the signiﬁcant deviations from conventional endodyogeny exhib- ited by the TgOTUD3A-KO, we sought to examine growing parasites by transmission electron microscopy (TEM). In addition to gravid mother parasites undergoing conven- tional endodyogeny, with 2 nuclei and 2 daughter scaffolds evident (Fig. 3A, green block arrows), mother parasites containing various numbers of daughter scaffolds were also observed in the same vacuole (Fig. 3A), consistent with an altered replication strategy that was found at higher frequencies in TgOTUD3A-KO parasites captured by immunoﬂuorescence assay (IFA) (Fig. 2B to D). In the same vacuole, there was a parasite with a single scaffold visible in the section (an additional scaffold is likely in a distinct section) (Fig. 3A). The parasite shown in Fig. 3B represented a clear apparent example of the schizogony-like phenotype that was seen, as 2 distinct nuclei (Fig. 3B, yellow mbio.asm.org 7 November/December 2017 Volume 8 Issue 6 e01846-17 mbio.asm.org on February 15, 2018 - Published by mbio.asm.org Downloaded from ® Dhara et al. FIG 3 Replication patterns deviating from normal endodyogeny revealed by electron microscopy. Actively growing TgOTUD3A-KO parasites were prepared for electron microscopy and viewed with the aim of capturing evidence for deviations from normal endodyogeny. Normal endodyogeny, with 2 nearly mature daughter scaffolds within a TgOTUD3A-KO mother parasite, was detected (A; green block arrows) in the same vacuole shared with a mother having multiple daughter scaffolds (A, C; yellow block arrows) and multiple discrete nuclear proﬁles (B, C, D; yellow asterisks). A schizogony-like phenotype, the completion of mitosis without the initiation of cytokinesis (absence of daughter scaffolds), was also detected in a subset of mutant parasites (B; yellow asterisks). We see instances of what appears to be the reinitiation of a replicative cycle prior to the completion of a round of cytokinesis, presenting as 4 daughter scaffolds with 2 scaffolds in each arm of the aberrant parasite shown (C). Furthermore, the detection of large, likely polyploid nuclear masses without a daughter scaffold (D; yellow asterisk and line) is suggestive of an endopolygeny-like replication strategy. Parasites containing multiple da ghter scaffolds and progressing thro gh c tokinesis appear to possess normal micronemes ( ello line arro ) rhoptries (solid ello FIG 3 Replication patterns deviating from normal endodyogeny revealed by electron microscopy. Actively growing TgOTUD3A-KO parasites were prepared for electron microscopy and viewed with the aim of capturing evidence for deviations from normal endodyogeny. RESULTS Normal endodyogeny, with 2 nearly mature daughter scaffolds within a TgOTUD3A-KO mother parasite, was detected (A; green block arrows) in the same vacuole shared with a mother having multiple daughter scaffolds (A, C; yellow block arrows) and multiple discrete nuclear proﬁles (B, C, D; yellow asterisks). A schizogony-like phenotype, the completion of mitosis without the initiation of cytokinesis (absence of daughter scaffolds), was also detected in a subset of mutant parasites (B; yellow asterisks). We see instances of what appears to be the reinitiation of a replicative cycle prior to the completion of a round of cytokinesis, presenting as 4 daughter scaffolds with 2 scaffolds in each arm of the aberrant parasite shown (C). Furthermore, the detection of large, likely polyploid nuclear masses without a daughter scaffold (D; yellow asterisk and line) is suggestive of an endopolygeny-like replication strategy. Parasites containing multiple daughter scaffolds and progressing through cytokinesis appear to possess normal micronemes (yellow-line arrow), rhoptries (solid yellow arrowhead), and Golgi bodies (open yellow arrowheads), consistent with the formation of these organelles observed by ﬂuorescence microscopy (see Fig. S4 to S6 in the supplemental material). In all instances, the scale bars represent 1 m. FIG 3 Replication patterns deviating from normal endodyogeny revealed by electron microscopy. Actively growing TgOTUD3A-KO parasites were prepared for electron microscopy and viewed with the aim of capturing evidence for deviations from normal endodyogeny. Normal endodyogeny, with 2 nearly mature daughter scaffolds within a TgOTUD3A-KO mother parasite, was detected (A; green block arrows) in the same vacuole shared with a mother having multiple daughter scaffolds (A, C; yellow block arrows) and multiple discrete nuclear proﬁles (B, C, D; yellow asterisks). A schizogony-like phenotype, the completion of mitosis without the initiation of cytokinesis (absence of daughter scaffolds), was also detected in a subset of mutant parasites (B; yellow asterisks). We see instances of what appears to be the reinitiation of a replicative cycle prior to the completion of a round of cytokinesis, presenting as 4 daughter scaffolds with 2 scaffolds in each arm of the aberrant parasite shown (C). Furthermore, the detection of large, likely polyploid nuclear masses without a daughter scaffold (D; yellow asterisk and line) is suggestive of an endopolygeny-like replication strategy. November/December 2017 Volume 8 Issue 6 e01846-17 RESULTS 3C, yellow asterisks) in an overall conﬁguration mbio.asm.org 8 November/December 2017 Volume 8 Issue 6 e01846-17 ® A Toxoplasma DUB Controls Cell Cycle Architecture that appeared to indicate that 2 distinct rounds of mitosis might be occurring simul- taneously or, alternatively, a new entry into cytokinesis was initiated without comple- tion of the prior cycle. Finally, the vacuole shown in Fig. 3D was distinctive, as it contained a parasite containing a very large nucleus (Fig. 3D, yellow asterisk and line), much like an endopolygeny-like polyploid nucleus, as well as the parasite on the left, which exhibited a forked nucleus suggestive of ongoing karyokinesis that was curiously oriented away from the apical end, while its cytoplasm was connected to a fragment of another parasite. Notably, examination at the ultrastructural level conﬁrmed that the developing daughter parasites appeared to have morphologically normal micronemes (Fig. 3A, yellow-line arrow), rhoptries (Fig. 3A, closed yellow arrowhead), and Golgi bodies (Fig. 3C, open yellow arrowheads). In addition, mitochondrion and apicoplast proﬁles were also evident in parasites with aberrant endodyogeny that possessed morpholog- ical features of schizogony-like and endopolygeny-like replication architectures. The stoichiometry of the centrosome cores matches that of the daughter scaffolds. As in higher eukaryotes, the centrosome in Apicomplexa is the main micro- tubule organizing center (MTOC) involved in coordinating mitosis and karyokinesis. A recent study (35) showed that the Toxoplasma centrosome has a unique bipartite structure (comprising outer and inner cores) that critically regulates the ﬁdelity of the parasite cell cycle by regulating and integrating the dynamics of the nuclear and budding cycles. We therefore investigated whether the aberrant replication pheno- types seen in the TgOTUD3A-KO parasites were due to dysregulation of centrosome dynamics. Using an outer core protein (Centrin-1) and an inner core protein tagged with hemagglutinin (CEP250L1-HA) as markers, we examined the numbers and the distribution of centrosomes relative to the numbers of developing daughter scaf- folds in gravid parasites, with a focus on mothers bearing more than two daughters (Fig. 4). The outer core is believed to associate with and regulate the spatial organization of the daughter scaffolds (35). As expected, for wild-type parasites undergoing mitosis (bilobed nuclear mass) with 2 immature daughter buds per mother, 2 Centrin-1 spots were associated with each nucleus/nuclear arm (Fig. 4A, wild type). The same was observed for the TgOTUD3A-KO parasites with 2 daughter scaffolds, whether the nuclei were divided (Fig. RESULTS Parasites containing multiple daughter scaffolds and progressing through cytokinesis appear to possess normal micronemes (yellow-line arrow), rhoptries (solid yellow arrowhead), and Golgi bodies (open yellow arrowheads), consistent with the formation of these organelles observed by ﬂuorescence microscopy (see Fig. S4 to S6 in the supplemental material). In all instances, the scale bars represent 1 m. FIG 3 Replication patterns deviating from normal endodyogeny revealed by electron microscopy. Actively growing TgOTUD3A-KO parasites were prepared for electron microscopy and viewed with the aim of capturing evidence for deviations from normal endodyogeny. Normal endodyogeny, with 2 nearly mature daughter scaffolds within a TgOTUD3A-KO mother parasite, was detected (A; green block arrows) in the same vacuole shared with a mother having multiple daughter scaffolds (A, C; yellow block arrows) and multiple discrete nuclear proﬁles (B, C, D; yellow asterisks). A schizogony-like phenotype, the completion of mitosis without the initiation of cytokinesis (absence of daughter scaffolds), was also detected in a subset of mutant parasites (B; yellow asterisks). We see instances of what appears to be the reinitiation of a replicative cycle prior to the completion of a round of cytokinesis, presenting as 4 daughter scaffolds with 2 scaffolds in each arm of the aberrant parasite shown (C). Furthermore, the detection of large, likely polyploid nuclear masses without a daughter scaffold (D; yellow asterisk and line) is suggestive of an endopolygeny-like replication strategy. Parasites containing multiple daughter scaffolds and progressing through cytokinesis appear to possess normal micronemes (yellow-line arrow), rhoptries (solid yellow arrowhead), and Golgi bodies (open yellow arrowheads), consistent with the formation of these organelles observed by ﬂuorescence microscopy (see Fig. S4 to S6 in the supplemental material). In all instances, the scale bars represent 1 m. asterisks) were evident, indicative of completed karyokinesis with the absence of any cytokinesis (no daughter scaffolds). Although one cannot rule out the presence of scaffolds on a different EM section, it is unlikely due to the fact that we also captured this evidence by IFA (Fig. 2D). We further observed a parasite mass that appeared to contain 4 daughter scaffolds (Fig. 3C, yellow block arrows) and a dividing nucleus segregating between 2 progeny (Fig. November/December 2017 Volume 8 Issue 6 e01846-17 RESULTS (Middle) In the gravid TgOTUD3A-KO parasites, mothers containing 2 daughter scaffolds have matched numbers of Centrin-1 spots (green, not marked). The gravid mothers containing 4 daughter scaffolds each (IMC3, red with white 4’s) are matched with regard to both Centrin-1 (green, marked with polygons) and discrete postmitotic nuclei (DNA and Merge). (Bottom) The parasites containing 2 daughter scaffolds (IMC3, red) each contain a single nucleus with 2 centrosomes (Centrin-1, green). Two larger mother parasites (Phase) with large polyploid nuclei (DNA) each contain 3 daughter scaffolds (IMC3, red with white 3’s), which are matched to 3 centrosomes (Centrin-1, green, circled). (B) (Top) In wild-type parasites containing 2 immature daughter scaffolds (IMC1, green), the numbers of centrosome inner cores (CEP250L1-HA, red) are matched and in close apposition to the intensely labeled undivided nuclei (DNA). (Middle) Mother TgOTUD3A-KO parasites containing the normal 2 daughter scaffolds (IMC1, green, not marked) are matched to the number of CEP20L1-HA (red)-labeled centrosome inner cores. Two additional parasites each contain four daughter scaffolds (IMC1, white 4’s) matched to 4 CEP240L1-HA (red) centrosome inner cores (circled). Each of these mothers possesses 2 equivalently labeled nuclei. (Bottom) Three TgOTUD3A-KO mother parasites containing 4 daughters (IMC1, green with white 4), 3 daughters (IMC1, green with white 3), and a rare failed replication event (IMC1, not numbered). Both the mothers containing 4 (circled) and 3 (marked with yellow triangle) daughters contain matched numbers of CEP250L1-HA-labeled centrosome inner cores. The nuclei in these parasites appear intensely labeled and polyploid (DNA and Merge). The apparently failed replication event contains 4 daughter scaffolds (IMC1) and 4 centrosome inner cores (CEP250L1-HA, red, marked with white triangle) but only 3 nuclei (DNA), one of which (Merge and DNA, red asterisk) is not associated with any daughter scaffold. In addition, two daughter scaffolds (IMC1 and Merge, yellow asterisk) lack a nucleus, while a free centrosome (Merge, open yellow arrowhead) appears to no longer be associated with either a nucleus or a scaffold. Scale bars 10 m. Dhara et al. FIG 4 Centrosome numbers match the numbers of daughter scaffolds in gravid parasites. The bipartite T. gondii centrosome consists of an outer (Centrin-1 marker) and an inner (CEP250L1-HA marker) core. (A) (Top) Wild-type parasites, each with two daughter scaffolds (IMC3, red), possess duplicated centrosomes (Centrin-1, green) adjacent to the dividing nuclei. RESULTS 4A, middle row, 2 scaffolds [IMC3] and 2 discrete nuclei) or undivided polypoid masses (Fig. 4A, bottom row, 2 scaffolds [IMC3] with 1 large nucleus each). In instances where more than 2 daughter scaffolds were detected in the mother, the number of Centrin-1 spots matched the number of scaffolds (Fig. 4A, TgOTUD3A-KO, Centrin-1, polygons and circles). Of note, the number of Centrin-1 spots within parasites containing very large nuclei but lacking any daughter scaffolds was found to be an indicator of the number of progeny that would be expected to develop. The inner centrosome core is known to engage the intranuclear centrocone (which houses the mitotic spindle) (42, 43) to coordinate the outer core and ensure the ﬁdelity of inheritance of the nuclear genome (35). Parasites in the later stages of S phase (inferred from the intensity of DNA staining) (Fig. 4B, Wild type, DNA) or initiating mitosis with the normal 2 immature daughter scaffolds possessed 2 CEP250L1-HA spots (inner core marker) associated with the nucleus (Fig. 4B, Wild type). This relationship was maintained for gravid TgOTUD3A-KO parasites bearing 2 daughter scaffolds both with an undivided nucleus and with already-separated nuclei following karyokinesis (Fig. 4B, TgOTUD3A-KO). As was observed with the outer core marker Centrin-1, the number of CEP250L1-HA-positive spots matched the number of daughter scaffolds whether the mother bore 3 daughter scaffolds (Fig. 4B, CEP250L1-HA, bottom row, bold yellow triangle) or 4 daughter scaffolds (Fig. 4B, CEP250L1-HA, middle and bottom rows, ovals). These data indicated that at the time cytokinesis was initiated (appearance of daughter buds), the numbers of scaffolds, outer cores, and inner cores were in a 1:1:1 ratio. In rare cases, we observed that the progeny of multiple-daughter parasites were present as scaffolds lacking nuclei (Fig. 4B, Merge, bottom row, yellow asterisks). In this instance, a nucleus (Fig. 4B, DNA, bottom row, red asterisk) appeared outside its parent mbio.asm.org 9 November/December 2017 Volume 8 Issue 6 e01846-17 ® Dhara et al. parasite with a rogue centrosome that was no longer associated with it (Fig. 4B, bottom FIG 4 Centrosome numbers match the numbers of daughter scaffolds in gravid parasites. The bipartite T. gondii centrosome consists of an outer (Centrin-1 marker) and an inner (CEP250L1-HA marker) core. (A) (Top) Wild-type parasites, each with two daughter scaffolds (IMC3, red), possess duplicated centrosomes (Centrin-1, green) adjacent to the dividing nuclei. mbio.asm.org 10 RESULTS mbio.asm.org 10 November/December 2017 Volume 8 Issue 6 e01846-17 ® A Toxoplasma DUB Controls Cell Cycle Architecture Transient dysregulation in the stoichiometry of the inner and outer cores of the centrosome drives deviation from endodyogeny. Ploidy within apicomplexan parasites undergoing different modes of replication is controlled at the level of the centrosome (35). Deviations from a ploidy of 2 (endodyogeny) in TgOTUD3A-KO parasites are noted by the centrosome number matching that of the daughter scaffolds as gravid parasites enter cytokinesis (Fig. 4). This suggests that any aberration during centrosome duplication (as would be required for the generation of more than 2 progeny) would have to occur by an aberrant reduplication of one (to result in 3 progeny) or both (resulting in 4 progeny) centrosomes. These changes would likely be reﬂected as transient imbalances in centrosome dynamics during the phase of DNA synthesis, serving to alter S-phase progression while ensuring the completion of the required ploidy to match the eventual number of daughters. A failure to match the ploidy to the number of daughters would result in catastrophic defects that would be inconsistent with the enhanced ﬁtness of TgOTUD3A-KO mutants (Fig. 1) (38). g g Duplication of the bipartite centrosome during endodyogeny followed a prescribed pattern that was initiated with the duplication of the outer (Centrin-1) core prior to that of the inner (CEP250L1-HA) (35) core. The completion of normal centrosome core duplication during endodyogeny resulted in a 1:1 stoichiometry with the cores in close apposition to each other. The association of the inner core with the centrocone (spindle) coupled with the attachment of the DNA constituted the priming for mitosis. Toward establishing whether there were transient defects in the stoichiometry of the centromere cores that were corrected prior to entry into cytokinesis, we examined the stoichiometry of the centrosome cores using the Centrin-1 and CEP250L1-HA signals. These patterns were correlated to the size, shape, and intensity (a measure of total DNA content) of the relevant nuclei (Fig. 5). Accordingly, the wild-type parasites (Fig. 5, Wild type) appeared to be in the midst of mitosis, typiﬁed by a larger nucleus, likely at the end of S phase based on the high concentrations of DNA and the distribution of inner and outer cores (Fig. 5). RESULTS (Middle) In the gravid TgOTUD3A-KO parasites, mothers containing 2 daughter scaffolds have matched numbers of Centrin-1 spots (green, not marked). The gravid mothers containing 4 daughter scaffolds each (IMC3, red with white 4’s) are matched with regard to both Centrin-1 (green, marked with polygons) and discrete postmitotic nuclei (DNA and Merge). (Bottom) The parasites containing 2 daughter scaffolds (IMC3, red) each contain a single nucleus with 2 centrosomes (Centrin-1, green). Two larger mother parasites (Phase) with large polyploid nuclei (DNA) each contain 3 daughter scaffolds (IMC3, red with white 3’s), which are matched to 3 centrosomes (Centrin-1, green, circled). (B) (Top) In wild-type parasites containing 2 immature daughter scaffolds (IMC1, green), the numbers of centrosome inner cores (CEP250L1-HA, red) are matched and in close apposition to the intensely labeled undivided nuclei (DNA). (Middle) Mother TgOTUD3A-KO parasites containing the normal 2 daughter scaffolds (IMC1, green, not marked) are matched to the number of CEP20L1-HA (red)-labeled centrosome inner cores. Two additional parasites each contain four daughter scaffolds (IMC1, white 4’s) matched to 4 CEP240L1-HA (red) centrosome inner cores (circled). Each of these mothers possesses 2 equivalently labeled nuclei. (Bottom) Three TgOTUD3A-KO mother parasites containing 4 daughters (IMC1, green with white 4), 3 daughters (IMC1, green with white 3), and a rare failed replication event (IMC1, not numbered). Both the mothers containing 4 (circled) and 3 (marked with yellow triangle) daughters contain matched numbers of CEP250L1-HA-labeled centrosome inner cores. The nuclei in these parasites appear intensely labeled and polyploid (DNA and Merge). The apparently failed replication event contains 4 daughter scaffolds (IMC1) and 4 centrosome inner cores (CEP250L1-HA, red, marked with white triangle) but only 3 nuclei (DNA), one of which (Merge and DNA, red asterisk) is not associated with any daughter scaffold. In addition, two daughter scaffolds (IMC1 and Merge, yellow asterisk) lack a nucleus, while a free centrosome (Merge, open yellow arrowhead) appears to no longer be associated with either a nucleus or a scaffold. Scale bars 10 m. parasite with a rogue centrosome that was no longer associated with it (Fig. 4B, bottom row, white triangle and open yellow arrowhead). These data clearly show that parasites gravid with multiple daughters, captured early in the budding cycle (based on the small immature daughter buds) at the onset of cytokinesis, are competent to produce viable progeny. November/December 2017 Volume 8 Issue 6 e01846-17 mbio.asm.org 11 RESULTS For these 4 wild-type parasites, there was a 1:1 stoichiometry between the outer core (Centrin-1) and inner core (CEP250L1-HA), with the 2 signals found to be associated with the nucleus and in close apposition to each other (Fig. 5, Wild type, Merge). When examining the TgOTUD3A-KO mutant parasites, we often detected 3 or more centrosomal cores (outer and/or inner) in a subset of parasites (Fig. 5, TgOTUD3A-KO parasites circled with bold yellow dotted lines) within a vacuole, indicating a deviation from normal endodyogeny. Those circled with white dotted lines in Fig. 5 appeared to be executing conventional endodyogeny. Notably, as seen with the daughter scaffolds (Fig. 2), parasites within the same vacuole (phase image, not shown) exhibit features of classical endodyogeny and signiﬁcant variation from this replication strategy. More often we saw instances where the outer core number exceeded the inner core number (Fig. 5, TgOTUD3A-KO, top row, 4 o’clock, 9 o’clock, and 11 o’clock positions, white open arrowheads). A lower frequency was observed for instances where there were more inner cores than outer cores. An example of this phenotype was evident within a vacuole where one parasite possessed 3 outer cores and 5 inner cores (Fig. 5, TgOTUD3A-KO, middle row, 2 o’clock position). Besides observing the imbalance, we also captured the instances where 3 or more outer or inner cores were in perfect 1:1 stoichiometry and sitting in close appositions (Fig. 5, TgOTUD3A-KO, middle row, 7 o’clock and 5 o’clock positions, and bottom row, 10 o’clock and 3 o’clock positions). This suggested that 1:1 stoichiometry could be or is achieved in these TgOTUD3A-KO parasites even though there is a transient loss of core stoichiometry. Upon the restoration of inner and outer core stoichiometry, we expected that entry into the budding cycle (cytokinesis) was now permissible. Next, we investigated whether this transient asymmetry in the centrosome cores drives aberrant DNA replication (more than 2N ploidy), thereby altering the nuclear cycle in the TgOTUD3A-KO parasites. The grayscale images of nuclei in the TgOTUD3A-KO parasites containing multiple inner or outer core spots revealed that a prominent imbalance in the stoichiometry or the presence of more than 2 centrosome cores was often associated with abnormal nuclear morphologies, seen as signiﬁcantly mbio.asm.org 11 ® Dhara et al. FIG 5 Reduplication and transient dysregulation in the stoichiometry of the inner and outer cores drive the plasticity of the replication mode. RESULTS The 1st row displays wild-type parasites both primed for mitosis (based on nuclear size and intensity) and undergoing mitosis, possessing matched outer (Centrin-1, green) and inner (CEP250L1-HA, red) cores in close apposition to each other and the nucleus (Merge). The 2nd and 3rd rows show the diverse phenotypes associated with the TgOTUD3A-KO mutant. These include instances of two matched inner and outer centrosome cores (circled in white dashed lines), similar to the phenotype of wild-type parasites. TgOTUD3A-KO parasites with more than 2 inner or outer cores are circled in yellow dashed lines. These include parasites within which the number of outer cores (Centrin-1) exceeds the number of inner cores (CEP250L1-HA); the extra cores are marked with open white arrowheads. The 1st row for the TgOTUD3A-KO line displays three parasites containing mismatched outer and inner cores. These parasites display aberrant nuclear morphologies (red asterisks), with two nuclei presenting as larger masses (9 o’clock and 11 o’clock) relative to the sizes of the mitotically resolved nuclei within the normal parasite (circled in white dashed lines). The third parasite (5 o’clock) appears to have undergone one round of mitosis but displays unevenly sized nuclei. The 2nd row for the TgOTUD3A-KO line displays a parasite with both higher numbers and mismatch (in terms of their relative positioning) of both outer and inner cores (Centrin-1, 7 o’clock) that possesses 2 intensely labeled nuclei of equivalent size. In addition, this vacuole contains a parasite with additional inner cores (CEP250L1-HA, 2 o’clock). In this case, the weak signal of an outer core (Centrin-1, white asterisk) and some inner cores (CEP250L1-HA, yellow asterisk) may suggest an immature centrosome or breakdown products of a centrosome. Of note, the parasite in the center (5 o’clock) possesses 4 matched Centrin-1- and CEP250L1-HA-labeled centrosomes and appears, based on the intensity of the DNA synthesis, closer to the end of replication and poised for the second round of symmetrical mitosis and cytokinesis. Of the two TgOTUD3A-KO parasites displayed in the 3rd row with more than 2 centrosome cores, one (10 o’clock) possesses matched though spatially disorganized centro- somes together with 2 largely matched nuclei. The second aberrant parasite (3 o’clock) exhibits an intensely labeled apparently polyploid nucleus within which the stoichiometry between the inner and outer cores appears to have been resolved. RESULTS Of the two TgOTUD3A-KO parasites displayed in the 3rd row with more than 2 centrosome cores, one (10 o’clock) possesses matched though spatially disorganized centro- somes together with 2 largely matched nuclei. The second aberrant parasite (3 o’clock) exhibits an intensely labeled apparently polyploid nucleus within which the stoichiometry between the inner and outer cores appears to have been resolved. The resolution of transient imbalances in favor of either the inner or outer core appears to be linked to an aberrant S phase, promoting either a schizogony-like or endopolygeny-like nuclear cycle. In all instances, the scale bars represent 10 m. FIG 5 Reduplication and transient dysregulation in the stoichiometry of the inner and outer cores drive the plasticity of the replication mode. The 1st row displays wild-type parasites both primed for mitosis (based on nuclear size and intensity) and undergoing mitosis, possessing matched outer (Centrin-1, green) and inner (CEP250L1-HA, red) cores in close apposition to each other and the nucleus (Merge). The 2nd and 3rd rows show the diverse phenotypes associated with the TgOTUD3A-KO mutant. These include instances of two matched inner and outer centrosome cores (circled in white dashed lines), similar to the phenotype of wild-type parasites. TgOTUD3A-KO parasites with more than 2 inner or outer cores are circled in yellow dashed lines. These include parasites within which the number of outer cores (Centrin-1) exceeds the number of inner cores (CEP250L1-HA); the extra cores are marked with open white arrowheads. The 1st row for the TgOTUD3A-KO line displays three parasites containing mismatched outer and inner cores. These parasites display aberrant nuclear morphologies (red asterisks), with two nuclei presenting as larger masses (9 o’clock and 11 o’clock) relative to the sizes of the mitotically resolved nuclei within the normal parasite (circled in white dashed lines). The third parasite (5 o’clock) appears to have undergone one round of mitosis but displays unevenly sized nuclei. The 2nd row for the TgOTUD3A-KO line displays a parasite with both higher numbers and mismatch (in terms of their relative positioning) of both outer and inner cores (Centrin-1, 7 o’clock) that possesses 2 intensely labeled nuclei of equivalent size. In addition, this vacuole contains a parasite with additional inner cores (CEP250L1-HA, 2 o’clock). In this case, the weak signal of an outer core (Centrin-1, white asterisk) and some inner cores (CEP250L1-HA, yellow asterisk) may suggest an immature centrosome or breakdown products of a centrosome. November/December 2017 Volume 8 Issue 6 e01846-17 mbio.asm.org 12 RESULTS The resolution of transient imbalances in favor of either the inner or outer core appears to be linked to an aberrant S phase, promoting either a schizogony-like or endopolygeny-like nuclear cycle. In all instances, the scale bars represent 10 m. FIG 5 Reduplication and transient dysregulation in the stoichiometry of the inner and outer cores drive the plasticity of the replication mode. The 1st row displays wild-type parasites both primed for mitosis (based on nuclear size and intensity) and undergoing mitosis, possessing matched outer (Centrin-1, green) and inner (CEP250L1-HA, red) cores in close apposition to each other and the nucleus (Merge). The 2nd and 3rd rows show the diverse phenotypes associated with the TgOTUD3A-KO mutant. These include instances of two matched inner and outer centrosome cores (circled in white dashed lines), similar to the phenotype of wild-type parasites. TgOTUD3A-KO parasites with more than 2 inner or outer cores are circled in yellow dashed lines. These include parasites within which the number of outer cores (Centrin-1) exceeds the number of inner cores (CEP250L1-HA); the extra cores are marked with open white arrowheads. The 1st row for the TgOTUD3A-KO line displays three parasites containing mismatched outer and inner cores. These parasites display aberrant nuclear morphologies (red asterisks), with two nuclei presenting as larger masses (9 o’clock and 11 o’clock) relative to the sizes of the mitotically resolved nuclei within the normal parasite (circled in white dashed lines). The third parasite (5 o’clock) appears to have undergone one round of mitosis but displays unevenly sized nuclei. The 2nd row for the TgOTUD3A-KO line displays a parasite with both higher numbers and mismatch (in terms of their relative positioning) of both outer and inner cores (Centrin-1, 7 o’clock) that possesses 2 intensely labeled nuclei of equivalent size. In addition, this vacuole contains a parasite with additional inner cores (CEP250L1-HA, 2 o’clock). In this case, the weak signal of an outer core (Centrin-1, white asterisk) and some inner cores (CEP250L1-HA, yellow asterisk) may suggest an immature centrosome or breakdown products of a centrosome. Of note, the parasite in the center (5 o’clock) possesses 4 matched Centrin-1- and CEP250L1-HA-labeled centrosomes and appears, based on the intensity of the DNA synthesis, closer to the end of replication and poised for the second round of symmetrical mitosis and cytokinesis. mbio.asm.org 12 RESULTS Of note, the parasite in the center (5 o’clock) possesses 4 matched Centrin-1- and CEP250L1-HA-labeled centrosomes and appears, based on the intensity of the DNA synthesis, closer to the end of replication and poised for the second round of symmetrical mitosis and cytokinesis. Of the two TgOTUD3A-KO parasites displayed in the 3rd row with more than 2 centrosome cores, one (10 o’clock) possesses matched though spatially disorganized centro- somes together with 2 largely matched nuclei. The second aberrant parasite (3 o’clock) exhibits an intensely labeled apparently polyploid nucleus within which the stoichiometry between the inner and outer cores appears to have been resolved. The resolution of transient imbalances in favor of either the inner or outer core appears to be linked to an aberrant S phase, promoting either a schizogony-like or endopolygeny-like nuclear cycle. In all instances, the scale bars represent 10 m. larger nuclei with more than 2N nuclear mass (Fig. 5, TgOTUD3A-KO, DNA, red aster- isks), suggesting that there was failure to terminate S phase at 2N ploidy and the excess centrosome drove the DNA replication to continue until the correct ploidy was achieved for successful entry into the budding cycle. These ﬁndings provide a mech- anistic basis underlying the multiple-daughter phenotype and the potential for schizogony-like and endopolygeny-like nuclear cycles evidenced by IFA (Fig. 2 and 4) and electron microscopy (Fig. 3). Incomplete centrosome engagement with a partner centrocone (spindle) pro- motes an altered progression of the nuclear cycle. The most critical function of the mbio.asm.org 12 ® A Toxoplasma DUB Controls Cell Cycle Architecture centrosome is to anchor the mitotic spindle at the kinetochore to drive mitosis and karyokinesis. In light of mitosis in Toxoplasma being closed (the nuclear envelope does not disintegrate as the chromosomes separate), a specialized structure termed the centrocone (42–44) is required to establish access for the cytoplasmically located centrosomes to the chromosomes in the nucleus. The presence, therefore, of multi- ple centrosomes in multiply gravid TgOTUD3A-KO parasites would necessitate the formation of matched centrocones to ensure the correct engagement of the respective genome equivalents to the centrosomes, ensuring the ﬁdelity of inheritance. This issue is particularly relevant in instances where a single polyploid (2N ploidy) nuclear mass is present. To test this, we used an antibody against membrane occupation and recognition nexus protein 1 (MORN1), a protein marker of the centrocone (Fig. RESULTS 6, yellow open arrowheads and closed white arrowheads) (42, 43), in the CEP250L1-HA-tagged TgOTUD3A-KO line to establish the relationship between the centrosome and centro- cone. Importantly, the MORN1 protein is found not only in the centrocone (Fig. 6, yellow arrowhead, and Fig. 7) but also at the apical (not shown) and basal ends of the parasite (Fig. 6A, white closed arrowhead, and Fig. 7, Normal endodyogeny) (42, 43). As the parasite completes G1 and enters S phase, DNA synthesis proceeds along with the duplication of the centrosomal cores and the centrocone also duplicates, establishing its association with the centrosome inner core (Fig. 6A and 7, Normal endodyogeny). When the ploidy reaches 1.8N, the newly duplicated chromosomes associate with the centrosome through the centrocone, which functions as the conduit (Fig. 7, Normal endodyogeny). This effectively engages the spindle checkpoint to terminate DNA synthesis (S phase), initiating mitosis and karyokinesis. This proceeds with the increase in the distance between centrosome pairs as the nucleus divides (Fig. 6A, bottom row, open yellow arrowheads, and Fig. 7, Normal endodyogeny) (3). Given the overlapping organization of the cell cycle, the process of formation of daughter buds is initiated before mitosis is complete (Fig. 7) (5, 10). As mitosis proceeds, the daughter scaffolds (IMC signal) extend (Fig. 7, Normal endodyogeny) and connect at their leading edges to MORN1, which will form the eventual basal complex in the daughter parasites (Fig. 6A, closed white arrowheads, and Fig. 7, Normal endodyogeny) (5, 10, 42). The altered progression of endodyogeny resulting in the generation of 3 or 4 (or more) daughter parasites appeared to be associated with two different mechanisms revealed by the TgOTUD3A-KO mutant parasites. The ﬁrst is uncoupling of the nuclear and budding cycles due to reduplication of one or both of the centrosomes associated with the nuclei that have completed the ﬁrst nuclear cycle (schizogony-like) (Fig. 6B and C and 7A and D). Instead of moving into the budding cycle, one or both individual nuclei (Fig. 6B and C, red-line arrows) undergo further duplication to match the centrosome number, resulting in 3 and 4 progeny, respectively. In each of these instances, the nuclei that are undergoing a second round of division are either larger or denser than the 1N nucleus (Fig. 6B and C, red-line arrows, and Fig. 7A and D). November/December 2017 Volume 8 Issue 6 e01846-17 RESULTS (C) Two perpendicularly arranged nuclei exhibiting the reduplication of the centrosome inner core (CEP250L1-HA) appear matched with the centrocone marker MORN1 (open yellow arrowheads), suggesting that they are capable of mitosis. Note the intensity of the nuclear signal, suggestive of more DNA content than in 1N ploidy. (D) The parasite in the center of the ﬁeld exhibits two nuclear MORN1-labeled centrocones (open yellow arrowheads) but 3 distinct centrosome inner cores (CEDP250L1-HA). Notably, the orphan centrosome (open white arrowhead) renders it incompetent for the engagement of the spindle checkpoint. The nucleus in the parasite (red-line arrow) appears to be a large and polyploid nuclear mass. (E) Experimental evidence also demonstrates that one gravid parasite that has four inner core spots (CEP250L1-HA, red, circled in white) and four matching centrocone spots (MORN1, green) contains a very large polypoid nucleus (DNA, red arrow), suggesting that they are primed for making 4 progenies. (D and E) Both patterns of labeling are suggestive of an endopolygeny-like replication event in process, as in both cases, mitosis may have been completed even though karyokinesis is yet to happen. Scale bars 10 m. FIG 6 Centrocone (MORN1) and centrosome dynamics deﬁne the mechanistic basis for the production of multiple progeny. MORN1 is a marker of the nuclear centrocone (open yellow arrowheads), as well as the basal complex (closed white arrowheads). (A) In the wild-type parasites, the centrocone is in close apposition to the centrosome inner core (CEP250L1-HA). The images in the top and bottom rows exhibit the association of the centrocone with the centrosome in early and late S phase, respectively, which facilitates engagement of the genome with the centrosome. A balanced stoichiometry between the centrocones and centrosomes facilitates mitosis and karyo- kinesis. (B) While 4 of the parasites in the vacuole possess correctly organized centrosome-centrocone pairs, the ﬁfth organism appears to have reduplicated one centrosome (CEP250L1-HA, DNA, red-line arrow) and the other remains poised for division. (A and B) In both panels, second reduplications of one or both centrocone-centrosome pairs appear to be happening in the nuclei that have undergone ﬁrst-round karyokinesis, similar to a schizogony- like replication strategy. (C) Two perpendicularly arranged nuclei exhibiting the reduplication of the centrosome inner core (CEP250L1-HA) appear matched with the centrocone marker MORN1 (open yellow arrowheads), suggesting that they are capable of mitosis. Note the intensity of the nuclear signal, suggestive of more DNA content than in 1N ploidy. RESULTS The second mechanism is attributable to the failure of S phase to terminate upon reaching 2N ploidy due to the reduplication of one or both centrosomes, resulting in a 3N or 4N polypoid nucleus (endopolygeny-like) that ﬁnally resolves into 3 or 4 progeny (Fig. 6D and E and 7B and C). We were able to capture the orphan centrosome (Fig. 6D, white arrowhead, and Fig. 7B and C, purple) waiting to receive its partner centrocone. This centrosome is expected to receive its partner centrocone (MORN1) upon completion of 3N ploidy. The absence of a MORN1 signal suggests that the spindle checkpoint cannot be engaged at this orphan centrosome. This absence of MORN1 attachment and the larger nuclear mor- phology and higher nuclear intensity (compared to that of adjacent parasite with 2 centrosomes) of these nuclei (Fig. 6D and E, red-line arrows, and Fig. 7B and C) suggest that these nuclei are still undergoing S phase. Together, these data support the idea that the centrosome dynamics and its association with spindle checkpoints drive the generation of multiple-daughter phenotypes in TgOTUD3A-KO parasites. Failure of complementation upon restoration of TgOTUD3A to the KO mutant. In order to conﬁrm that the disruption of TgOTUD3A was responsible for the increased mbio.asm.org 13 November/December 2017 Volume 8 Issue 6 e01846-17 ® Dhara et al. FIG 6 Centrocone (MORN1) and centrosome dynamics deﬁne the mechanistic basis for the production of multiple progeny. MORN1 is a marker of the nuclear centrocone (open yellow arrowheads), as well as the basal complex (closed white arrowheads). (A) In the wild-type parasites, the centrocone is in close apposition to the centrosome inner core (CEP250L1-HA). The images in the top and bottom rows exhibit the association of the centrocone with the centrosome in early and late S phase, respectively, which facilitates engagement of the genome with the centrosome. A balanced stoichiometry between the centrocones and centrosomes facilitates mitosis and karyo- kinesis. (B) While 4 of the parasites in the vacuole possess correctly organized centrosome-centrocone pairs, the ﬁfth organism appears to have reduplicated one centrosome (CEP250L1-HA, DNA, red-line arrow) and the other remains poised for division. (A and B) In both panels, second reduplications of one or both centrocone-centrosome pairs appear to be happening in the nuclei that have undergone ﬁrst-round karyokinesis, similar to a schizogony- like replication strategy. November/December 2017 Volume 8 Issue 6 e01846-17 mbio.asm.org 14 RESULTS (D) The parasite in the center of the ﬁeld exhibits two nuclear MORN1-labeled centrocones (open yellow arrowheads) but 3 distinct centrosome inner cores (CEDP250L1-HA). Notably, the orphan centrosome (open white arrowhead) renders it incompetent for the engagement of the spindle checkpoint. The nucleus in the parasite (red-line arrow) appears to be a large and polyploid nuclear mass. (E) Experimental evidence also demonstrates that one gravid parasite that has four inner core spots (CEP250L1-HA, red, circled in white) and four matching centrocone spots (MORN1, green) contains a very large polypoid nucleus (DNA, red arrow), suggesting that they are primed for making 4 progenies. (D and E) Both patterns of labeling are suggestive of an endopolygeny-like replication event in process, as in both cases, mitosis may have been completed even though karyokinesis is yet to happen. Scale bars 10 m. frequency of the multiple-daughter phenotype, we sought to restore the wild-type gene under the control of its native promoter at its original chromosomal location. The approach relied on the use of 2 CRISPR sites to drop out the mutagenizing (DHFR- containing) cassette and replace it with a C-terminally HA-tagged complementing cassette that included a hypoxanthine-guanine phosphoribosyltransferase (HXGPRT) frequency of the multiple-daughter phenotype, we sought to restore the wild-type gene under the control of its native promoter at its original chromosomal location. The approach relied on the use of 2 CRISPR sites to drop out the mutagenizing (DHFR- containing) cassette and replace it with a C-terminally HA-tagged complementing cassette that included a hypoxanthine-guanine phosphoribosyltransferase (HXGPRT) mbio.asm.org 14 November/December 2017 Volume 8 Issue 6 e01846-17 mbio.asm.org on February 15, 2018 - Published by mbio.asm.org Downloaded from ® A Toxoplasma DUB Controls Cell Cycle Architecture FIG 7 Schematic models for the generation of multiple progeny in the TgOTUD3A-KO line. (Middle) Schematic displays the process of normal endodyogeny, highlighting key milestones with regard to both the nuclear and budding cycles. With conventional endodyogeny, an integrated nuclear (DNA synthesis and mitosis, karyokinesis) and budding (formation of daughter scaffold) cycle progresses to generate 2 daughters. (A, B) Deviations from endodyogeny in TgOTUD3A-KO parasites resulting in the formation of three daughter progeny by both an endopolygeny-like and a schizogony-like mechanism due to an asymmetrical division of the genome(s) during the nuclear cycle. (C, D) Generation of four daughter parasites using both a schizogony-like and an endopolygeny-like mechanism, driven by the symmetrical reduplication of the genome. November/December 2017 Volume 8 Issue 6 e01846-17 mbio.asm.org 15 RESULTS These deviations from conventional endodyogeny are driven by aberrant reduplication of the centrosome and the capacity of the new centrosomes to engage partner centrocones. Incomplete centrosome-centrocone pairing prevents the arrest of DNA synthesis, as the spindle checkpoint cannot be engaged (see text for more details). FIG 7 Schematic models for the generation of multiple progeny in the TgOTUD3A-KO line. (Middle) Schematic displays the process of normal endodyogeny, highlighting key milestones with regard to both the nuclear and budding cycles. With conventional endodyogeny, an integrated nuclear (DNA synthesis and mitosis, karyokinesis) and budding (formation of daughter scaffold) cycle progresses to generate 2 daughters. (A, B) Deviations from endodyogeny in TgOTUD3A-KO parasites resulting in the formation of three daughter progeny by both an endopolygeny-like and a schizogony-like mechanism due to an asymmetrical division of the genome(s) during the nuclear cycle. (C, D) Generation of four daughter parasites using both a schizogony-like and an endopolygeny-like mechanism, driven by the symmetrical reduplication of the genome. These deviations from conventional endodyogeny are driven by aberrant reduplication of the centrosome and the capacity of the new centrosomes to engage partner centrocones. Incomplete centrosome-centrocone pairing prevents the arrest of DNA synthesis, as the spindle checkpoint cannot be engaged (see text for more details). gene to afford mycophenolic acid/xanthine (MPA/Xan) resistance in the ΔHXGPRT background of the strain (Fig. 8A). The resulting complemented clones were selected for MPA/Xan resistance and further screened for resistance or sensitivity for pyrimeth- amine to establish the retention or replacement of the original DHFR/thymidylate synthase (TS) resistance cassette. Two complemented clones of the 2H1 line, D5 (retaining both the mutagenized gene and a single copy of the complementing cassette encoding MPA/Xanr and Pyrr; also designated Compl-2H1 below) and C4 (containing a tandem insertion of the complementing cassette and eliminating the mutagenized gene encoding MPA/Xanr and Pyrs) were chosen for further analysis mbio.asm.org 15 November/December 2017 Volume 8 Issue 6 e01846-17 mbio.asm.org on February 15, 2018 - Published by mbio.asm.org Downloaded from FIG 8 Complementation of TgOTUD3A-KO parasites does not appear to restore wild-type phenotypes. (A) Schematic shows the complementation strategy using the CRISPR-Cas9 gene-editing approach. Two CRISPR sites were designed to ﬂank the 5= and 3= UTRs of the TgOTUD3A locus in the KO-2H1 strain, which has a knock-in mutated DHFR cassette. RESULTS The complementing gene copy has its own endogenous promoter and C-terminal 3HA tag and a functional HXGPRT cassette to provide mycophenolic acid/xanthine resistance (MPA/Xanr). Two complemented clones, which were selected based on resistance to MPA/Xan (clone D5 retains pyrimethamine resistance [Pyrr], and clone C4 is sensitive to pyrimethamine [Pyrs]), were used for further experiments. (B) The presence or absence of the DHFR cassette in those complemented lines was further conﬁrmed by PCR (primer location is indicated in the schematic in panel A). (C) Immunoﬂuorescence analysis with anti-HA antibody exhibits a higher level of TgOTUD3A expression in C4. Scale bars 10 m. (D) Immunoblotting with anti-TgOTUD3A and anti-HA antibodies conﬁrms TgOTUD3A expression in the complemented lines and also at higher levels in C4 than in D5. (E) Comparison of multiple-daughter phenotypes between the WT, TgOTUD3A-KO, and 2 complemented lines (D5 and C4). Parasites were stained with anti-TgIMC3 antibody (to visualize daughter scaffolds) in gravid parasites (SAG1 labels individual parasites) and were blindly counted by two individuals (A.D. and A.P.S.). One-way ANOVA was done (F22 63 Dhara et al. ® Dhara et al. FIG 8 Complementation of TgOTUD3A-KO parasites does not appear to restore wild-type phenotypes. (A) Schematic shows the complementation strategy using the CRISPR-Cas9 gene-editing approach. Two CRISPR sites were designed to ﬂank the 5= and 3= UTRs of the TgOTUD3A locus in the KO-2H1 strain, which has a knock-in mutated DHFR cassette. The complementing gene copy has its own endogenous promoter and C-terminal 3HA tag and a functional HXGPRT cassette to provide mycophenolic acid/xanthine resistance (MPA/Xanr). Two complemented clones, which were selected based on resistance to MPA/Xan (clone D5 retains pyrimethamine resistance [Pyrr], and clone C4 is sensitive to pyrimethamine [Pyrs]), were used for further experiments. (B) The presence or absence of the DHFR cassette in those complemented lines was further conﬁrmed by PCR (primer location is indicated in the schematic in panel A). (C) Immunoﬂuorescence analysis with anti-HA antibody exhibits a higher level of TgOTUD3A expression in C4. Scale bars 10 m. (D) Immunoblotting with anti-TgOTUD3A and anti-HA antibodies conﬁrms TgOTUD3A expression in the complemented lines and also at higher levels in C4 than in D5. (E) Comparison of multiple-daughter phenotypes between the WT, TgOTUD3A-KO, and 2 complemented lines (D5 and C4). RESULTS Parasites were stained with anti-TgIMC3 antibody (to visualize daughter scaffolds) in gravid parasites (SAG1 labels individual parasites) and were blindly counted by two individuals (A.D. and A.P.S.). One-way ANOVA was done (F22,63 3.874, P 0.0001), and the number of daughters per gravid parasite compared between any two groups using Tukey’s pairwise multiple-comparison test ( 0.05). Signiﬁcant differences (P 0.05) between groups are marked by asterisks as follows: , P 0.01; *, P 0.001; , P 0.0001. The data analyzed were from 4 biological replicate experiments. In total, 3,819 wild-type, 2,319 KO, 1,841 D5, and 1,664 C4 parasites were counted. Error bars represent standard deviations. FIG 8 Complementation of TgOTUD3A-KO parasites does not appear to restore wild-type phenotypes. (A) Schematic shows the complementation strategy using the CRISPR-Cas9 gene-editing approach. Two CRISPR sites were designed to ﬂank the 5= and 3= UTRs of the TgOTUD3A locus in the KO-2H1 strain, which has a knock-in mutated DHFR cassette. The complementing gene copy has its own endogenous promoter and C-terminal 3HA tag and a functional HXGPRT cassette to provide mycophenolic acid/xanthine resistance (MPA/Xanr). Two complemented clones, which were selected based on resistance to MPA/Xan (clone D5 retains pyrimethamine resistance [Pyrr], and clone C4 is sensitive to pyrimethamine [Pyrs]), were used for further experiments. (B) The presence or absence of the DHFR cassette in those complemented lines was further conﬁrmed by PCR (primer location is indicated in the schematic in panel A). (C) Immunoﬂuorescence analysis with anti-HA antibody exhibits a higher level of TgOTUD3A expression in C4. Scale bars 10 m. (D) Immunoblotting with anti-TgOTUD3A and anti-HA antibodies conﬁrms TgOTUD3A expression in the complemented lines and also at higher levels in C4 than in D5. (E) Comparison of multiple-daughter phenotypes between the WT, TgOTUD3A-KO, and 2 complemented lines (D5 and C4). Parasites were stained with anti-TgIMC3 antibody (to visualize daughter scaffolds) in gravid parasites (SAG1 labels individual parasites) and were blindly counted by two individuals (A.D. and A.P.S.). One-way ANOVA was done (F22,63 3.874, P 0.0001), and the number of daughters per gravid parasite compared between any two groups using Tukey’s pairwise multiple-comparison test ( 0.05). Signiﬁcant differences (P 0.05) between groups are marked by asterisks as follows: , P 0.01; *, P 0.001; **, P 0.0001. The data analyzed were from 4 biological replicate experiments. November/December 2017 Volume 8 Issue 6 e01846-17 RESULTS In total, 3,819 wild-type, 2,319 KO, 1,841 D5, and 1,664 C4 parasites were counted. Error bars represent standard deviations. mbio.asm.org 16 November/December 2017 Volume 8 Issue 6 e01846-17 ® A Toxoplasma DUB Controls Cell Cycle Architecture (Fig. 8A). The retention of the DHFR/TS cassette in the D5 clone was conﬁrmed by PCR (Fig. 8B), and the restoration of the functional TgOTUD3A gene in both D5 and C4 was validated using immunoblotting and IFA in addition (Fig. 8C and D). Consistent with the presence of an additional copy of TgOTUD3A, complemented line C4 expressed higher levels of the protein than D5, as captured by both immunoﬂuorescence (Fig. 8C) and immunoblot (Fig. 8D) analysis. The increase in the size of the complementing gene was due to the presence of the 3HA epitope tag (Fig. 8D). The ability to restore TgOTUD3A to the KO mutant was assessed using the multiple- daughter progeny phenotype counted on double-blinded samples. Remarkably, de- spite the expression of nearly wild-type levels (clone D5) or overexpression (clone C4) of TgOTUD3A, restoration of the wild-type phenotype was not achieved (Fig. 8E). Rather, the frequencies of multiply gravid mother parasites within the complemented lines were at the levels seen in the TgOTUD3A-KO parasites. In addition, the comple- mented line behaved exactly like the TgOTUD3A-KO line in the plaque assay (data not shown). At face value, this suggested that the multiple-daughter phenotype was due to an off-target mutation of a gene(s) other than TgOTUD3A. Alternatively, the loss of TgOTUD3A established an irreversible change as a compensatory mechanism, whereby the restoration of the gene had no effect on what was now the “new normal.” Whole-genome sequencing fails to identify a credible second site mutation responsible for the phenotype. The inability to complement the defect in the TgOTUD3A-KO line (clone 2H1) suggested that a second site mutation potentially caused by the mistargeting of CRISPR-Cas9 or other means was responsible for the observed multiple-daughter phenotype. We reasoned that if this were the case, the potential off-target mutation would also be found in the independently isolated clone 2C3 and would be shared with the complemented 2H1 line (Compl-2H1 [clone D5]). In order to directly assess this, we performed whole-genome Illumina sequencing on a parental line (WT, RHΔHXGPRT), KO clones 2C3 and 2H1, and the Compl-2H1 line (D5). Details regarding the assembly and analysis of the NGS data are described in Text S1A in the supplemental material. November/December 2017 Volume 8 Issue 6 e01846-17 RESULTS Genomic DNA libraries from 4 clonal lines were made using the TruSeq DNA PCR-free method and sequenced by Illumina HiSeq 2500 rapid-run sequencing, whereby 100- to 125-nucleotide (nt)-long fragments were sequenced. We obtained excellent average coverage of the genomes (WT, 53; 2H1, 43; 2C3, 40; and Compl-2H1 [D5], 46). This depth of coverage was evident for the number of reads at the DHFR/TS locus, which was doubled for the exons (as the mutagenizing cassette encodes the cDNA and not the genomic copy of the gene) of DHFR/TS in the 2 KO clones, resulting in a series of peaks for those strains (Fig. 9A, red peaks). The peak height for the DHFR/TS exons in the complemented line was double that of the wild type but two-thirds of that for the 2 KO lines, indicating that one of the 2 DHFR/TS cassettes was removed during complementation. In the Compl-2H1 (D5) strain, one of the two DHFR/TS cassettes was replaced with the epitope-tagged TgOTUD3A-HA and the HXGPRT selection marker. The higher red and blue peaks at the 5= and 3= ends for the complemented line, at levels similar to those for the KO lines, reﬂected that the same 5= and 3= untranslated regions (UTRs) of DHFR were used in the HXGPRT gene cassette as in the ﬂanking sequences (Fig. 9A). Because more than one DHFR/TS cassette got inserted into 2 independent KO lines, 2C3 and H1, we investigated whether those extra copies of the cassette were inserted in tandem at the TgOTUD3A locus or in a different locus other than the CRISPR-Cas9 target site (in this case TgOTUD3A), thereby resulting in an off-target insertional mutation. The recovery of DNA sequences ﬂanking the mutagenesis cassette ﬂanking region revealed that all 7 nodes captured by homology to the ﬂanking 50 nt on both ends of the DHFR/TS cassette matched either the TgOTUD3A gene or the DHFR cassette itself, consistent with the tandem insertion of the DHFR cassettes at the TgOTUD3A locus (Fig. 9B). We next sought to establish all the differences identiﬁed on our sequenced clones relative to the recently published Toxoplasma gondii RH reference genome (45). The RH mbio.asm.org 17 November/December 2017 Volume 8 Issue 6 e01846-17 mbio.asm.org on February 15, 2018 - Published by mbio.asm.org Downloaded from ® Dhara et al. FIG 9 Whole-genome next-generation sequencing of 4 parasite lines (WT, 2 KOs, and a complemented line). November/December 2017 Volume 8 Issue 6 e01846-17 RESULTS (A) Each chart shows the read depth coverage of a portion of the T. gondii chromosome XIIb, where the red region represents the DHFR/TS locus, showing 3 copies of DHFR in the TgOTUD3A-KO samples (clones 2C3 and 2H1) and 2 copies of DHFR in the complemented 2H1 (Compl-2H1 [clone D5]) samples, compared to 1 in the wild type. (B) Analysis of the (Continued on next page) November/December 2017 Volume 8 Issue 6 e01846-17 mbio asm org 18 FIG 9 Whole-genome next-generation sequencing of 4 parasite lines (WT, 2 KOs, and a complemented line). (A) Each chart shows the read depth coverage of a portion of the T. gondii chromosome XIIb, where the red region represents the DHFR/TS locus, showing 3 copies of DHFR in the TgOTUD3A-KO samples (clones 2C3 and 2H1) and 2 copies of DHFR in the complemented 2H1 (Compl-2H1 [clone D5]) samples, compared to 1 in the wild type. (B) Analysis of the (Continued on next page) FIG 9 Whole-genome next-generation sequencing of 4 parasite lines (WT, 2 KOs, and a complemented line). (A) Each chart shows the read depth coverage of a portion of the T. gondii chromosome XIIb, where the red region represents the DHFR/TS locus, showing 3 copies of DHFR in the TgOTUD3A-KO samples (clones 2C3 and 2H1) and 2 copies of DHFR in the complemented 2H1 (Compl-2H1 [clone D5]) samples, compared to 1 in the wild type. (B) Analysis of the (Continued on next page) November/December 2017 Volume 8 Issue 6 e01846-17 ® A Toxoplasma DUB Controls Cell Cycle Architecture genome showed excellent concordance (98% identical) with the reference type I GT1 sequence (http://www.toxodb.org) (Fig. S7). Using the published Toxoplasma RH ge- nome as the reference, we identiﬁed between 6,122 and 6,346 variations in the wild-type (WT), mutant (2C3 and 2H1), and complemented (Compl-2H1) lines (Fig. 9C). Given the size of the published genome, this corresponded to an averaged frequency of a mutation from the reference genome roughly every 11,000 bases. The Poisson distribution P value for each of these samples, in the range of 0.00476 to 0.00010, served as a measure of a random occurrence not driven by a speciﬁc factor(s) (Fig. 9C). We also tested the signiﬁcance of the observed variance between strains by single- factor analysis of variance (ANOVA) (Text S1B). Neither statistical analysis identiﬁed these variations as any different from random variations. RESULTS Not surprisingly, the vast majority of these deviations from the reference genome (GT1; 4,305 differences) were shared among all the sequenced strains (Fig. 9D). We next established the frequency of shared deviations from the reference genome in the 2 mutants and the complemented line (2C3, 2H1, and Compl-2H1 [D5]) that were not present in the parental parasites (WT). This population had 260 deviations from the reference genome (Fig. 9D, red circle), which were manually curated to eliminate deviations that were present in intragenic regions and within predicted introns. The survivors of this analysis comprised shared deviations within predicted open reading frames and were found to comprise insertions, deletions, and single-nucleotide poly- morphisms (SNPs) (Fig. 7E; Text S1C). We sought therefore to assess the impact of loss-of-function mutations of these genes by exploiting the results from a recent whole-genome CRISPR knockout analysis (38). In this competition-based study, a score of 0 indicates no impact on growth, a negative score indicates a ﬁtness defect, and a positive score is indicative of a growth advantage as a result of a loss of the targeted activity. Notably, these essentiality scores were based on the relative depletion or enrichment of CRISPR-disrupted genes over 3 lytic cycles, representing a relatively short time frame. As these TgOTUD3A-KO or complemented lines have been passaged for multiple lytic cycles and did not show any gross detrimental growth abnormality, these loci can be excluded as being the basis of the phenotype. Thus, any ﬁtness defect or advantage would be expected to be exaggerated further with increasing lytic/passage cycles if these phenotypes were inherited. Each integer (positive or negative) reﬂects a 2-fold difference (log2) in growth. Of the genes for which essentiality scores were available, 8 resulted in signiﬁcant growth defects (essentiality scores of 3.53 to 4.89) and 2 had a roughly 2-fold effect on growth (essentiality scores of 1.01 and 1.06) (Fig. 9E). This predicted negative impact on growth would exclude these genes as being responsible for the defect attributed to the TgOTUD3A-KO line, where the mutant parasites have a growth advantage (Fig. 1E). A single predicted gene presented a growth advantage (essentiality score of 1.56), indicating a roughly 3-fold increase in representation within the population over the 7-day-infection protocol used in the genome-wide KO study. g ( ) locations of the extra DHFR copies in the TgOTUD3A-KO and complemented lines. Flanking sequences of the drug cassettes in the top panel of the genome browser (derived from ToxoDB) output represent the TgOTUD3A locus, where 3 of the 7 hits match, and the bottom panel shows the DHFR locus, where the remaining 4 of the 7 hits aligned, indicating that the drug cassettes integrated in tandem in the TgOTUD3A locus without random insertion in the genome. (C) Statistical analysis shows that the average frequencies of mutation (based on the total number of variations and RH genome size) found in each sample and between samples are random and not caused by CRISPR editing. (D) Venn diagram comparing all variants called by our analysis, showing that 260 of them (circled in red) were shared between the mutants and not with the wild type. (E) Further analysis determined that out of 260 variants, only 15 were found inside the coding regions; these are listed in the table. Since there is no RH annotation available in the database, we used the GT1 gene identiﬁcation number (ID) as the reference gene. Position, type of mutation, and essentiality score based on a recent genome-wide CRISPR study (38) are provided. November/December 2017 Volume 8 Issue 6 e01846-17 RESULTS We could safely eliminate this hypothetical gene from con- sideration, as extrapolation of the 2-fold difference in head-to-head competition be- tween the wild-type and the TgOTUD3A-KO mutant at 48 h (Fig. 1E) would outstrip a 3-fold increase if extended to 7 days. Finally, the 3 targets that were not included in the whole-genome CRISPR screen all had SNPs predicted to result in amino acid substitutions (Fig. S6D). The predicted phenylalanine-4-hydroxylase (TGGT1_411100), involved in the production of tyrosine FIG 9 Legend (Continued) (B) Phylogenetic analysis revealed that the two signiﬁcantly upregulated D1 TgOTUs (red box) are phylogenetically among the closest homologs of TgOTUD3A (green box). The position of TgOTU7 in the phylogenetic tree is highlighted by the blue box. Scale bars 10 M. Error bars represent standard deviations. from phenylalanine, presented with a substitution of R for L at position 772 (L772R). A recent study showed that ME49 tachyzoites with this gene homolog knocked out had no signiﬁcant growth defect (46). And last, the substitutions in 2 hypothetical open reading frames (ORFs) TGGT1_217961 (L110I) and TGGT1_318880 (E668Q) were more conservative and unlikely to result in a loss of function. In addition, the nature of the phenotype was stochastic and it only appeared in some parasites within a given a vacuole, which indicated that a single amino acid substitution was not responsible. Taken together, we were unable to identify any credible shared mutation that could provide the basis of an off-target effect at the level of the genome that accounted for the phenotype exposed by the loss of TgOTUD3A. We therefore sought to establish whether the loss of TgOTUD3A triggered a compensatory change that irreversibly altered the expression landscape, thereby precluding the complementation of the lesion. Selective overexpression of closely related TgOTU family members in the TgOTUD3A-KO line. In order to establish whether the loss of TgOTUD3A resulted in compensatory changes in the expression levels of other TgOTU family members, we performed quantitative real-time PCR (qRT-PCR) on the wild type, the TgOTUD3A-KO mutant 2H1, and the complemented 2H1 line D5. Of the 6 OTU family members tested, 3 exhibited signiﬁcant upregulation in their steady-state mRNA levels (Fig. 10A). Based on our previously published analysis, TgOTUD1B and TgOTUD1C, which are most closely related to TgOTUD3A (Fig. 10B) (34), were induced roughly 10-fold in the TgOTUD3A-KO line. Unlike TgOTUD3A, which was tightly cell cycle regulated, neither TgOTUD1B nor TgOTUD1C was cell cycle regulated (36) (http://www.toxodb.org). In contrast, of the 2 cell cycle-regulated TgOTU members (34, 36) (http://www.toxodb .org), TgOTU7 was also upregulated roughly 3-fold, while TgOTU5 was not (Fig. 10A). While these changes occurred in response to the loss of TgOTUD3A (in either clone 2H1 or 2C3), they were not restored to wild-type levels upon complementation (Fig. 10A), providing credence to the notion that a fundamental and irreversible change in the expression landscape had occurred within the KO parasites. FIG 9 Legend (Continued) mbio.asm.org 19 mbio.asm.org 19 ® Dhara et al. FIG 10 Members of the TgOTU family are selectively upregulated in TgOTUD3A-KO parasites, and complementation did not restore wild-type expression. (A) Knockout of TgOTUD3A results in the selective upregulation of speciﬁc TgOTUs. Steady-state mRNA expression of unsynchronized wild-type and TgOTUD3A-KO parasites infected for 24 h was quantiﬁed using real-time quantitative PCR. Data from quadruplicate samples from 3 independent experiments were analyzed by the ΔΔCT method after being normalized against TgSAG1 expression (template control). The mRNA expression of TgOTU7, one of the other 2 cell cycle-associated TgOTUs (TgOTU5 and TgOTU7), was increased by more than 3-fold. Signiﬁ- cantly, the expression of two members of clade D1 TgOTUs (TgOTUD1B and TgOTUD1C) was upregulated 10- to 12-fold. Complemented line D5 (which has a single functional copy of the TgOTUD3A gene) showed an expression proﬁle similar to that of the KO parasites, suggesting a failure of functional complementation even though the complemented gene copy was expressed in a dynamic pattern similar to the expression in the wild type. (B) Phylogenetic analysis revealed that the two signiﬁcantly upregulated D1 TgOTUs (red box) are phylogenetically among the closest homologs of TgOTUD3A (green box). The position of TgOTU7 in the phylogenetic tree is highlighted by the blue box. Scale bars 10 M. Error bars represent standard deviations. FIG 10 Members of the TgOTU family are selectively upregulated in TgOTUD3A-KO parasites, and complementation did not restore wild-type expression. (A) Knockout of TgOTUD3A results in the selective upregulation of speciﬁc TgOTUs. Steady-state mRNA expression of unsynchronized wild-type and TgOTUD3A-KO parasites infected for 24 h was quantiﬁed using real-time quantitative PCR. Data from quadruplicate samples from 3 independent experiments were analyzed by the ΔΔCT method after being normalized against TgSAG1 expression (template control). The mRNA expression of TgOTU7, one of the other 2 cell cycle-associated TgOTUs (TgOTU5 and TgOTU7), was increased by more than 3-fold. Signiﬁ- cantly, the expression of two members of clade D1 TgOTUs (TgOTUD1B and TgOTUD1C) was upregulated 10- to 12-fold. Complemented line D5 (which has a single functional copy of the TgOTUD3A gene) showed an expression proﬁle similar to that of the KO parasites, suggesting a failure of functional complementation even though the complemented gene copy was expressed in a dynamic pattern similar to the expression in the wild type. November/December 2017 Volume 8 Issue 6 e01846-17 mbio.asm.org 20 mbio.asm.org 20 DISCUSSION Unlike conventional eukaryotic cell cycles, which are deﬁned by discrete steps and are unidirectional, the replication cycles of Apicomplexa tend to possess overlapping stages that can contain phases that are repetitive and thereby no longer unidirectional (see Fig. S1 in the supplemental material) (3). Functionally, the apicomplexan cell cycle can be broadly classiﬁed into the nuclear and budding cycles. The variations in the organization of the nuclear cycle and the patterns by which it transitions into the budding cycle deﬁne the three major replication strategies (endodyogeny, schizogony, and endopolygeny), each of which has different capacities with regard to the number of progeny generated during the replicative cycle (35, 47). While endodyogeny pro- duces 2 daughters per cycle, schizogony and endopolygeny each produce multiple progeny. Toxoplasma genomes encode the capacity to execute all three replication mechanisms in a life cycle stage-dependent manner. The mechanistic basis restricting the parasites to a speciﬁc mechanism in a particular life cycle stage has remained unknown. Recent studies show that the bipartite centrosome in T. gondii plays a major regulatory role in determining the ploidy and, by extension, the number of progeny (35, 48). Cell cycle control is mediated to a signiﬁcant degree by the activity of atypical cyclins and cyclin-dependent kinase (Cdk)-related kinases (Crks), serving as checkpoints in the progression of the T. gondii cell cycle (44, 49). p g g y The TgOTUD3A-KO mutant is unique in that it appears to have partially altered the regulatory landscape, which manifests as T. gondii tachyzoites deploying the replication strategies that are typically used in the feline gut at the onset of the sexual cycle. Despite being genetically clonal, TgOTUD3A-KO parasites can propagate by multiple replication strategies even within the same vacuole (Fig. 2 and 3). This suggests a defect that is not entirely penetrant and likely related to a not-yet-determined factor(s), the absolute levels of which deﬁne the thresholds and, thereby, the phenotypic outcomes related to the speciﬁc choice of replication strategy. As such, the differential phenotypic outcomes for the TgOTUD3A-KO parasites must be determined at the level of individual parasites and governed at a posttranslational level consistent with the dysregulation of ubiquitination/deubiquitination mechanisms to afford a highly sensitive but plastic means of control affecting cell cycle strategy and progression. The understanding of the role of ubiquitin-mediated mechanisms in the regulation of the cell cycle of Toxoplasma and other Apicomplexa is in its infancy. FIG 9 Legend (Continued) The irreversibility of this change and the alterations in the patterns of replication resulting in the increase in the mbio.asm.org 20 ® A Toxoplasma DUB Controls Cell Cycle Architecture multiple-daughter phenotype could suggest a shift in the cell cycle architecture within the TgOTUD3A-KO (and complemented) parasites. November/December 2017 Volume 8 Issue 6 e01846-17 mbio.asm.org 21 DISCUSSION Our recent work established that the progression of endodyogeny is associated with dynamic changes in the relative levels and spatial distribution of K48-, K11-, and K63-linked polyubiquitin chains (34). The complexity of the phenotypes associated with the TgOTUD3A-KO parasites described in the present study matches the conﬁrmed speciﬁcity of TgOTUD3A for the K48 linkage (34), a modiﬁcation associated with directing ubiquitin- mediated protein turnover (20). Notably, the levels of K48-linked polyubiquitin are highest at the S/M transition and the early phases of cytokinesis, coinciding with the transcript and protein expression peaks of TgOTUD3A expression (Fig. 1A) (34). This spatiotemporal expression proﬁle suggests that functions of TgOTUD3A at the S/M transition and entry into cytokinesis are needed to achieve the absolute levels of the critical target(s) to allow for the progression of endodyogeny. This is entirely consistent with a threshold-driven mechanism underlying the selection of the replication strategy. A recently completed proteomic analysis of the Toxoplasma (tachyzoite) ubiquitome established that 35% of ubiquitin-modiﬁed proteins are themselves transcriptionally regulated in a cell cycle-dependent manner (19). In that study, the ﬁnding that a cohort of ubiquitin ligases and deubiquitinases are transcriptionally controlled in a cell cycle- dependent manner suggests that the establishment of a dynamic balance of key factors by ubiquitin-mediated turnover is critical to ensure the ﬁdelity of cell cycle transitions. Among ubiquitinated targets, histones, histone-modifying enzymes, DNA licensing factors, and enzymes regulating epigenetic processes point to the centrality mbio.asm.org 21 November/December 2017 Volume 8 Issue 6 e01846-17 ® Dhara et al. of ubiquitin-mediated regulation in ensuring the ﬁdelity of replication (19). A recent functional study further shows that one Crk family protein, Crk5, and its interacting partner ECR1 (essential for chromosome replication 1), regulating DNA licensing at the origin of replication, are regulated by ubiquitin-mediated mechanisms (44). Identifying the relevant target of TgOTUD3A responsible for the observed cell cycle phenotypes is a challenging task for multiple reasons. As an exodeubiquitinase, TgOTUD3A recognizes the K48 polyubiquitin chain (34) rather than the modiﬁed target protein, with the K48 linkage representing the most abundant polyubiquitin species in the parasite (34). In addition, the loss of TgOTUD3A is compensated for by the overexpression of three related TgOTUs (TgOTUD1B, TgOTUD1C, and TgOTU7) (Fig. 10), of which only TgOTU7 is cell cycle regulated at the transcriptional level (34). DISCUSSION This complicates the identiﬁcation of the target using comparative proteomics, as one cannot distinguish whether the putative target is destabilized (by the loss of the TgOTUD3A) or stabilized (by the overexpression of one or more of the related TgOTUs). Toward establishing the mechanistic basis for the multiple-daughter phenotype, our attention was directed at the Toxoplasma centrosome. Unlike the centrosome of higher eukaryotes, the Toxoplasma centrosome is a bipartite structure, comprised of compo- sitionally distinct inner and outer cores (35, 50). Within this framework, the inner core of the centrosome (CEP250L1-HA was used as a marker) correlates with the control of the nuclear cycle, while the outer core (Centrin-1 was used as a marker) is linked to ensuring the ﬁdelity of cytokinesis (35). Notably, during the nuclear cycle, when progression to cytokinesis is blocked, the outer core has limited function (35). This could be a potential regulatory mechanism to ensure the repetition of the nuclear cycle, as observed in schizogony and endopolygeny. Full outer core functionality is restored during the ﬁnal nuclear cycle and upon entry into the budding cycle, when DNA synthesis proceeds to mitosis/karyokinesis and on to cytokinesis with the forma- tion of multiple daughter buds (35). In Toxoplasma tachyzoite endodyogeny, the norm is a single nuclear cycle that is immediately coupled with an overlapping budding cycle (5). This restriction is partially lost in the TgOTUD3A-KO parasites, as we clearly see the uncoupling of the nuclear and budding cycles (within a subset of parasites), either because of extended DNA replication (beyond 2N), resulting in a large polyploid nuclear mass (3N or 4N or more) (endopolygeny-like), or reinitiation of DNA replication after the ﬁrst round of karyokinesis without entering into the budding cycle (schizogony-like) (Fig. 2 and 6). The synchronized entry into the budding cycle, regard- less of the number of daughter parasites within individual parasites and within a vacuole (Fig. 2B to D), suggests that the defect associated with the multiple-daughter phenotype precedes cytokinesis and is corrected prior to the initiation of the budding cycle. This transient defect has been determined to be a high frequency of aberrant centrosome duplication following the ﬁrst duplication. These deviations manifest in two different ways. One is an imbalanced reduplication (1 of the 2 centrosomes reduplicating) that results in progression to a 3N or greater odd-number ploidy. November/December 2017 Volume 8 Issue 6 e01846-17 mbio.asm.org 22 mbio.asm.org 22 DISCUSSION One obvious question now arises, as to how a cytoplasmic factor like TgOTUD3A (34) can regulate the nuclear cycle by controlling centrosome duplication, as all Apicom- plexa, including T. gondii, undergo closed mitosis, wherein the nuclear envelope does not completely break down. Evidence from the ﬁssion yeast, Schizosaccharomyces pombe, which also undergoes closed mitosis, reveals that there is a transient localized breakdown of the nuclear envelope that allows localized contact between the cyto- plasm and nucleoplasm surrounding the area where the centrosome is tethered to the nuclear envelope (55). Such a function may be associated with centrocone dynamics (44, 51) or could provide an alternative mechanism to engage the spindle checkpoint, arresting further DNA synthesis for the speciﬁc genome complement associated with a given centrosome. There is ample evidence from literature on mammals that ubiquiti- nation controls the duplication dynamics of centrosomes (56–58). The overexpression of a cell cycle-dependent E2 Ub ligase (which is functionally equivalent to the knock- down or knockout of a cell cycle-regulated DUB), UbcH10, results in the generation of multiple centrosomes, which despite being in clonal lines, occurs in 28 to 33% of the cells (56), a very similar phenotype to that observed in TgOTUD3A-KO parasites. The multiple-daughter and/or aberrant endodyogeny phenotypes have been re- ported previously in other T. gondii mutants with mutations affecting lipid export (NPC) (59) and protein sorting through the Golgi complex (Rab6) (60). Although these mutants are viable (at least in the short term for the Rab6 mutant [60]), none of these studies dissected these phenotypes from the perspective of the cell cycle and centro- some dynamics. Interestingly, the nuclear phenotypes in those mutant parasites are found to be similar to that of TgOTUD3A-KO parasites, suggesting that perturbation of the functions of these genes impacts the cell by mediating the dysregulation of cell cycle checkpoints that govern which replication strategy to adopt. In contrast, most of the conditional (ts) mutant parasites that uncouple the nuclear and budding cycles produce multiple progeny that are not viable (41). However, the TgOTUD3A-KO mu- tants, which appear to transiently uncouple the nuclear and budding cycles, are viable, as shown by plaque assay, competition assay (Fig. 1C and D), and morphology (Fig. 2 and 3). The facts that this multiple-daughter phenotype is not fully penetrant and is not reversed by complementation (Fig. DISCUSSION Given that the centromeres of the chromosomes are sequestered to the centrocone through- out the cell cycle (51), this imbalanced reduplication happens in two different formats, either after the ﬁrst round of karyokinesis is complete (schizogony-like) (Fig. 7A and D) or before the karyokinesis occurs (endopolygeny-like) (Fig. 7B and C). In the latter case, even though the karyokinesis is not complete, presumably the mitosis is complete in the polyploid mass, as described for Sarcocystis endopolygeny (52), and most likely, differential signaling events through one of the centrosomes causes this aberrant reduplication. The orphan centrosome resulting from this imbalanced reduplication, which is lacking the partner centrocone (Fig. 6D, MORN1), indicates that the connection to the centrosome cannot have been established, probably because the synthesis of the third genome equivalent was underway and it was therefore not ready for mitosis. The other deviation from the norm is the balanced reduplication wherein both the centrosomes reduplicate either before or after the ﬁrst round of karyokinesis is com- plete and that can be explained by signaling events similar to those described above. Of interest is a recent ﬁnding of a role for the ECR1/TgCrk complex in centrocone mbio.asm.org 22 November/December 2017 Volume 8 Issue 6 e01846-17 ® A Toxoplasma DUB Controls Cell Cycle Architecture duplication and the ubiquitin-mediated turnover of ECR1, which is related to the geminins in higher eukaryotes (44). Geminins play a critical role in preventing relicens- ing of DNA synthesis, chromosome duplication, and spindle formation (53, 54). It is possible that a geminin-like molecule could be a target of TgOTUD3A in regulating the centrosome duplication and maintaining the ﬁdelity of replication. The drivers of aberrant reduplication may be related to the degree of centrosome maturity following the initial duplication, as suggested by differences in the intensities of the centrosome signals (size) within individual parasites (Fig. 4 and 5). It is unclear, however, whether aberrant reduplication is exhibited by the mature mother or puta- tively immature daughter centrosome, as speciﬁc markers for centrosome maturation are not known. Of note, however, a study examining the role of centriolar plaques in asymmetrical mitotic division (1 nucleus divides while another does not) in Plasmodium suggests that it is the mother centrosome which initiates reduplication in the second S phase (11). November/December 2017 Volume 8 Issue 6 e01846-17 MATERIALS AND METHODS Parasite culture and maintenance. Type I RHΔHXGPRT and RHΔHXGPRTΔku80, HA-tagged TgOTUD3A and TgOTUD3A-KO, and complemented Toxoplasma gondii lines were cultured in primary human foreskin ﬁbroblast (HFF; ATCC) cells in Minimal Essential Medium with alpha modiﬁcation (-MEM) supplemented with 7% heat-inactivated fetal bovine serum (FBS; Gemini Bio-Products, West Sacramento, CA), 100 U/ml penicillin, 100 mg/ml streptomycin, and 2 mM L-glutamine (Gibco BRL, Rockville, MD) at 37°C and 5% CO2 in a humidiﬁed incubator. Modiﬁed parasite lines selected on the basis of drug resistance were grown on medium containing appropriate drugs. Epitope tagging of TgOTUD3A. The TgOTUD3A gene (TGGT1_258780) was tagged at the C terminus with a triple-HA tag by using the pHA3xLIC tagging plasmid (kind gift from Peter Bradley, UCLA) as described elsewhere (34). Both the wild-type and TgOTUD3A-KO lines were tagged using a fosmid containing a tagged copy of CEP250L1-HA and the chloramphenicol resistance gene (kind gift from Michael White, USF), and clonal lines were isolated for further analysis. Epitope tagging of TgOTUD3A. The TgOTUD3A gene (TGGT1_258780) was tagged at the C terminus with a triple-HA tag by using the pHA3xLIC tagging plasmid (kind gift from Peter Bradley, UCLA) as described elsewhere (34). Both the wild-type and TgOTUD3A-KO lines were tagged using a fosmid containing a tagged copy of CEP250L1-HA and the chloramphenicol resistance gene (kind gift from Michael White, USF), and clonal lines were isolated for further analysis. TgOTUD3A knockout and complementation by CRISPR-Cas9 gene editing. The TgOTUD3A gene open reading frame was disrupted by using clustered regularly interspaced short palindromic repeat (CRISPR)–CRISPR-associated gene 9 (Cas9)-mediated gene editing technology (62). The base CRISPR-Cas9 plasmid (37) was a kind gift from L. D. Sibley, Washington University, St. Louis, MO. TgOTUD3A gene (TGGT1_258780)-speciﬁc guide RNAs (gRNAs) were designed using the online E-CRISP portal (http:// www.e-crisp.org/E-CRISP/designcrispr.html). Based on the ranking, 4 gRNA sequences were selected to replace the endogenously encoded anti-TgUPRT gRNA, using the Q5 mutagenesis kit (New England BioLogicals) as previously described (37). For selection based on pyrimethamine resistance, a mutated version of the dihydrofolate reductase (DHFR) cassette (63) was ampliﬁed from the pBioID-HA3x-DHFR plasmid (Peter Bradley, UCLA) by using a primer pair attached to the 20-nt sequence ﬂanking the CRISPR target sequence. All the primers used to make the TgOTUD3A gene knockout mutant by CRISPR are listed in Table S1 in the supplemental material. Dhara et al. Dhara et al. In summary, the analysis of the TgOTUD3A-KO mutant reinforces a role for ubi- quitination-mediated mechanisms in the sophisticated decisions underlying the pro- gression and organization of the apicomplexan cell cycle. It further demonstrates that ubiquitin-mediated mechanisms likely control key switches governing the mechanisms that typically restrict the selection of cell cycle architecture of Toxoplasma in a life cycle-determined manner. By loosening the controls that restrict tachyzoites to endodyogeny, a subset of TgOTUD3A-KO parasites follow cell cycle progression strat- egies while retaining the capacity to undergo endodyogeny. The basis of this plasticity appears to be linked to the centrosome dynamics, which reinforces earlier studies (35). The analysis further presents a potential mechanistic foundation to dissect all three replication strategies displayed by Apicomplexa. These insights reveal how Toxoplasma executes three distinct replication strategies across its life cycle, ensuring the ﬁdelity of cell cycle progression potentially tuned to the requirements of the speciﬁc life cycle stage. November/December 2017 Volume 8 Issue 6 e01846-17 DISCUSSION 8) suggest some fundamental changes in the physiological landscape, deﬁning what is now a new normal in both the mutant and complemented lines (Fig. 8). The absence of credible off-target mutations (Fig. 9) predicts compensation by other factors. Such compensatory changes may be mediated by the upregulation of other closely related TgOTUs (Fig. 10), allowing adaptation to this new normal state. Interestingly, the Plasmodium ortholog (PF3D7_0923100) of TgOTUD3A is only expressed at the schizont stages in a genome-wide microarray analysis (PlasmoDB) (61). In addition, an anti-TgOTUD3A antibody cross-reacts with the orthologous protein only from puriﬁed schizonts and not from other erythrocytic stages (data not shown), suggesting potentially conserved cell cycle-speciﬁc roles in Apicomplexa, governing the transition from the nuclear to the budding cycle. mbio.asm.org 23 November/December 2017 Volume 8 Issue 6 e01846-17 ® mbio.asm.org 24 MATERIALS AND METHODS Both the PCR-ampliﬁed DHFR expression cassette and the CRISPR-Cas9 plasmid containing TgOTUD3A-speciﬁc gRNA were transfected into Toxoplasma gondii strain RH. At 24 h posttransfection, the parasites were harvested and syringe passaged. The syringe- passaged material was ﬁltered using membrane ﬁlters (CellTrics; Partec, GmbH) with a 50-micron cutoff prior to sorting for Cas9-green ﬂuorescent protein (GFP) on a ﬂow cytometer (Sony SY3200, installed in a biosafety level II cabinet). Sorted parasites were used to infect HFF cells in a 6-well plate. Following 2 passages in the pyrimethamine-containing medium, the parasites were cloned by limiting dilution. Each line was grown, and the parasite lysates were tested for immunoreactivity by Western blot analysis using the mouse polyclonal anti-TgOTUD3A antibody (34). Genomic DNA (gDNA) was isolated from the lines that showed no TgOTUD3A protein expression. The integration of the DHFR cassette into the double- stranded DNA break site was conﬁrmed by PCR, using the gDNA as the template. The PCR products were sequenced to conﬁrm that the knock-in mutation disrupted the open reading frame and thereby caused the loss of protein expression. Complementation was done by using the same CRISPR-mediated gene-editing approach. Two CRISPR gRNA-containing plasmids targeting the ﬂanking region of the DHFR cassette in the TgOTUD3A locus, along with an HA-tagged copy of functional full-length TgOTUD3A DNA (with its endogenous promoter), were transfected into the TgOTUD3A-KO 2H1 line, and clones were selected based on their resistance or sensitivity to mycophenolic acid (MPA)/xanthine and pyrimethamine. The expression of the complemented TgOTUD3A gene was tested by immunoﬂuorescence and immunoblot analyses and further veriﬁed by whole-genome sequencing. Plaque assay. HFF monolayers were infected with a small number (150 parasites/well in 6-well plates) of parasites of the wild-type RH, TgOTUD3A-HA, and TgOTUD3A-KO lines. The plaque sizes were monitored on a daily basis. On day 6 postinfection, the wells were washed with 1 phosphate-buffered saline (PBS), ﬁxed with ice-cold methanol (MeOH), and stained with crystal violet for visualization of the plaques. Images of a total of 150 plaques were randomly acquired (using a 10 objective) from all the parasite lines from three independent experiments. The areas of the plaques in arbitrary units were determined by measuring the plaques using NIH ImageJ software. mbio.asm.org 24 ® A Toxoplasma DUB Controls Cell Cycle Architecture Competition assay. The TgOTUD3A-HA (wild-type) and TgOTUD3A-KO lines were used for a head-to-head competition assay. MATERIALS AND METHODS Based on the sequence variation at the TgOTUD3A locus due to the introduction of the HA-DHFR drug cassette at the 3= UTR in the wild-type HA-tagged line and the DHFR drug cassette near the ATG start codon, we designed speciﬁc primer sets that will only amplify the parasite line-speciﬁc genomic DNA. The assay was standardized and validated by mixing different ratios of both of the genomic DNAs and using a single primer set for both combinations. The real-time data (not shown) for standardization faithfully represented the real combinatorial ratios used for speciﬁc reaction mixtures. For the actual experiment, HFF monolayers in each well of a 6-well plate were infected with equal numbers (2 105 and 5 103 for 24-h and 48-h time points, respectively) of each parasite line. Parasites were harvested after 24 and 48 h postinfection with equal numbers of wild-type (TgOTUD3A-HA) and TgOTUD3A-KO parasites, and total gDNA was collected and treated with RNase to remove RNA contamination. In each real-time PCR, 100 ng of genomic DNA was used, along with a speciﬁc set of primers (either wild type or TgOTUD3A-KO speciﬁc). The amount of template was normalized using TgSAG1 expression. Real-time PCR data were obtained using a Bio-Rad CFX instrument and analyzed by the cycle threshold (ΔΔCT) method. Cell cycle (DNA content) analysis. To measure the parasite DNA content, a previously described protocol (64) was followed. In brief, HFF monolayers were infected with both WT and TgOTUD3A-KO parasites. At 24 h and 36 h postinfection, parasites were harvested, syringe passaged, and ﬁltered through 10-m ﬁlters (CellTrics; Partec, GmbH). For a negative control, host cells only were harvested and treated the same way. Parasites ﬁxed with ﬁltered ethyl alcohol (EtOH; overnight ﬁxation at 20°C) were treated with RNase, stained with the DNA dye SYTOX green (Invitrogen), and analyzed with a ﬂow cytometer (Sony SY3200) by collecting 50,000 events for each parasite line. Data were analyzed using WinList 8.0 (Verity Software House). Immunoﬂuorescence assay. HFF monolayers were grown in conﬂuent monolayers on coverslips in 24-well plates and infected (1 104 per well) with either wild-type (RH) or TgOTUD3A-KO parasites. Around 16 to 24 h postinfection, infected cell monolayers were ﬁxed as previously described (65). MATERIALS AND METHODS The following primary antibodies were used at the indicated dilutions: rabbit anti-HA antibodies (from Covance, 1:250, and from Cell Signaling, 1:100), rat anti-TgIMC3 antibody (1:1,000) (66), mouse anti- TgIMC1 antibody (1:1,000) (kind gift from Gary Ward, University of Vermont), rat anti-MORN1 antibody (1:10) (43) (kind gift from Ke Hu, Indiana University), rabbit anti-SAG1 antibody (1:30,000) (kind gift from John Boothryod, Stanford University), mouse anti-Chlamydomonas centrin monoclonal antibody (20H5) (EMD Millipore), mouse anti-TgAtrx1 antibody (apicoplast; 1:1,000) (67), mouse anti-TgMIC3 antibody (microneme; 1:1,000) (68), mouse anti-TgROP7 antibody (rhoptry, 1:1,000) (69), mouse anti-TgF1 antibody (mitochondrion; 1:1,000) (65) (gift from Peter Bradley), mouse anti-TgGRA3 antibody (parasi- tophorous vacuole membrane; 1:1,000) (70), and rabbit anti-TgSAG1 antibody (surface antigen; 1:3,000) (71). Slides were visualized using a Zeiss AxioVision stand with a 100 1.4 numeric aperture (NA) oil immersion objective and images acquired using a high-resolution grayscale Zeiss AxioCam MRM digital camera. Where relevant, Z stack images were acquired and were deconvoluted using an iterative algorithm (AxioVision Deconvolution Suite; Zeiss). Grayscale images were pseudocolored to reﬂect the emission of the speciﬁc ﬂuorophore, brightness and contrast were adjusted, and the merged images were generated using Adobe Photoshop CS6. All manipulations (brightness/contrast) of the images acquired were applied uniformly to the images presented. qRT-PCR assay. Total RNA was extracted from 1 108 RH and TgOTUD3A-KO parasites using RNeasy columns (Qiagen) in which on-column DNase digestion was done to degrade all the genomic DNA. Total RNA was quantiﬁed on a NanoDrop 1000 instrument (Thermo Scientiﬁc). Equal amounts (maximum 1 g) of total RNA were used for cDNA synthesis (Promega reverse transcription system). Brieﬂy, PCRs using 20 l each of cDNA synthesis reaction mixture [4 l MgCl2 (25 mM), 2 l 10 reverse transcriptase buffer, 2 l deoxynucleoside triphosphate (dNTP) mixture, 0.5 g oligo(dT) primer, 0.5 l RNase inhibitor, and 1 l AMV reverse transcriptase] were run under the following PCR cycling conditions: 42°C for 60 min, 95°C for 5 min, and 4°C for 5 min. For each TgOTU, a primer pair capable of amplifying a 200-bp PCR product was designed (see Table S1 for primer sequences) and as a housekeeping internal control, a SAG1 (TGGT1_233460) primer pair was used to amplify the SAG1 transcript to normalize the initial transcript copy number. Primers that only ampliﬁed products of the expected size (single band on agarose gel) were used for this assay. November/December 2017 Volume 8 Issue 6 e01846-17 mbio.asm.org 25 mbio.asm.org 25 MATERIALS AND METHODS The quantitative real-time PCR (qRT-PCR) mixture (10 l), containing iTaq Universal SYBR green supermix (BioRad), template cDNA (100 ng/reaction mixture volume), and speciﬁc primer pairs, was run on a CFX Connect real-time PCR detection system (Bio-Rad) for 40 cycles under the following conditions: ampliﬁcation reaction at 95°C for 3 min, 95°C for 15 s, and 60°C for 30 s, and melt curve analysis from 65°C to 95°C in a 0.5°C increment every 5 s. Real-time PCR data were analyzed by the ΔΔCT method. y y T Statistical analysis. Based on the data types and distribution, one-way analysis of variance (ANOVA) tests were done to determine the levels of signiﬁcance for the mean differences between wild-type and TgOTUD3A-KO parasites for a number of variable traits that were looked at, such as plaque size, parasite number per vacuole, number of daughter scaffolds per gravid parasite, etc. The P value was determined by the Brown-Forsythe test and corrected Bartlett’s test (corrected for any deviation from Gaussian distribution). The difference between any two group means (for the speciﬁc test metric) was analyzed using Tukey’s multiple-comparison test with a 95% conﬁdence interval. The details of each test metric and level of signiﬁcance are mentioned in Results and in individual ﬁgure legends GraphPad Prism version 6.0 (La Jolla, CA) was used to generate all the bar graphs and to perform all statistical analysis. Next-generation whole-genome sequencing and analysis. Genomic DNA was isolated from 4 Next-generation whole-genome sequencing and analysis. Genomic DNA was isolated from 4 clonal lines (WT, KO mutant 2C3, KO mutant 2H1, and Compl-2H1 [D5]), and sequencing libraries were mbio.asm.org 25 ® Dhara et al. made using the TruSeq DNA PCR-free method, run on lanes of split ﬂow cells, and sequenced by Illumina HiSeq 2500 rapid run sequencing at the University of Kentucky Genomics Core Laboratory. The output paired-end 100- to 125-nucleotide reads were mapped against the RH reference genome. Mapped reads were analyzed through a pipeline of programs/tools to ﬁnd unique and shared mutations. A detailed description of the analysis is available in Text S1. SUPPLEMENTAL MATERIAL Supplemental material for this article may be found at https://doi.org/10.1128/mBio .01846-17. Supplemental material for this article may be found at https://doi.org/10.1128/mBio .01846-17. TEXT S1, PDF ﬁle, 0.2 MB. TEXT S1, PDF ﬁle, 0.2 MB. FIG S1, TIF ﬁle, 0.2 MB. FIG S2, TIF ﬁle, 0.4 MB. FIG S3, TIF ﬁle, 0.3 MB. FIG S4, TIF ﬁle, 0.4 MB. FIG S5, TIF ﬁle, 0.3 MB. FIG S6, TIF ﬁle, 0.6 MB. FIG S7, TIF ﬁle, 13 MB. TABLE S1, PDF ﬁle, 0.1 MB FIG S1, TIF ﬁle, 0.2 MB. FIG S2, TIF ﬁle, 0.4 MB. FIG S3, TIF ﬁle, 0.3 MB. FIG S4, TIF ﬁle, 0.4 MB. FIG S5, TIF ﬁle, 0.3 MB. FIG S6, TIF ﬁle, 0.6 MB. FIG S7, TIF ﬁle, 13 MB. TABLE S1, PDF ﬁle, 0.1 MB. REFERENCES Mol Biochem Parasitol 115:165–175. https://doi.org/10.1016/S0166-6851 (01)00284-5. Mol Biochem Parasitol 115:165–175. https://doi.org/10.1016/S0166-6851 (01)00284-5. 1. Hill DE, Chirukandoth S, Dubey JP. 2005. Biology and epidemiology of Toxoplasma gondii in man and animals. Anim Health Res Rev 6:41–61. https://doi.org/10.1079/AHR2005100. 11. Arnot DE, Ronander E, Bengtsson DC. 2011. The progression of the intra- erythrocytic cell cycle of Plasmodium falciparum and the role of the cen- triolar plaques in asynchronous mitotic division during schizogony. Int J Parasitol 41:71–80. https://doi.org/10.1016/j.ijpara.2010.07.012. 2. Dubey JP, Jones JL. 2008. Toxoplasma gondii infection in humans and animals in the United States. Int J Parasitol 38:1257–1278. https://doi .org/10.1016/j.ijpara.2008.03.007. 2. Dubey JP, Jones JL. 2008. Toxoplasma gondii infection in humans and animals in the United States. Int J Parasitol 38:1257–1278. https://doi .org/10.1016/j.ijpara.2008.03.007. 3. Francia ME, Striepen B. 2014. Cell division in apicomplexan parasites. Nat Rev Microbiol 12:125–136. https://doi.org/10.1038/nrmicro3184. 3. Francia ME, Striepen B. 2014. Cell division in apicomplexan parasites. Nat Rev Microbiol 12:125–136. https://doi.org/10.1038/nrmicro3184. 12. Hartwell LH, Culotti J, Pringle JR, Reid BJ. 1974. Genetic control of the cell division cycle in yeast. Science 183:46–51. https://doi.org/10.1126/ science.183.4120.46. 4. Hill D, Dubey JP. 2002. Toxoplasma gondii: transmission, diagnosis and prevention. Clin Microbiol Infect 8:634–640. https://doi.org/10.1046/j .1469-0691.2002.00485.x. 4. Hill D, Dubey JP. 2002. Toxoplasma gondii: transmission, diagnosis and prevention. Clin Microbiol Infect 8:634–640. https://doi.org/10.1046/j .1469-0691.2002.00485.x. 13. Hartwell LH, Weinert TA. 1989. Checkpoints: controls that ensure the order of cell cycle events. Science 246:629–634. https://doi.org/10.1126/ science.2683079. 5. Gubbels MJ, White M, Szatanek T. 2008. The cell cycle and Toxoplasma gondii cell division: tightly knit or loosely stitched? Int J Parasitol 38: 1343–1358. https://doi.org/10.1016/j.ijpara.2008.06.004. 5. Gubbels MJ, White M, Szatanek T. 2008. The cell cycle and Toxoplasma gondii cell division: tightly knit or loosely stitched? Int J Parasitol 38: 1343–1358. https://doi.org/10.1016/j.ijpara.2008.06.004. 14. Fisher D, Krasinska L, Coudreuse D, Novák B. 2012. Phosphorylation network dynamics in the control of cell cycle transitions. J Cell Sci 125:4703–4711. https://doi.org/10.1242/jcs.106351. 6. Ferguson DJ, Hutchison WM. 1987. An ultrastructural study of the early development and tissue cyst formation of Toxoplasma gondii in the brains of mice. Parasitol Res 73:483–491. https://doi.org/10.1007/BF00535321. 6. Ferguson DJ, Hutchison WM. 1987. An ultrastructural study of the early development and tissue cyst formation of Toxoplasma gondii in the brains of mice. Parasitol Res 73:483–491. https://doi.org/10.1007/BF00535321. 15. Nurse PM. 2002. Nobel lecture. Cyclin dependent kinases and cell cycle control. Biosci Rep 22:487–499. https://doi.org/10.1023/A:10220177 01871. 7. Watts E, Zhao Y, Dhara A, Eller B, Patwardhan A, Sinai AP. 2015. ACKNOWLEDGMENTS We acknowledge Michael White (University of South Florida), Marc-Jan Gubbels (Boston College), Daniel Howe (University of Kentucky), and Gary Ward (University of Vermont) for stimulating discussions that played an important role in the evolution of this study. We also acknowledge Michael White, Marc-Jan Gubbels, Gary Ward, John Boothroyd (Stanford University), Peter Bradley (UCLA), Ke Hu (Indiana University), and L. David Sibley (Washington University) for sharing critical reagents without which this work would not have been possible. This work was supported by Kentucky Science and Engineering Foundation grant number KSEF-2624-RDE-015 and National Institutes of Health grant number NIH R21 AI122894 (both awarded to A.P.S.). November/December 2017 Volume 8 Issue 6 e01846-17 REFERENCES Novel approaches reveal that Toxoplasma gondii bradyzoites within tissue cysts are dynamic and replicating entities in vivo. mBio 6:e01155-15. https://doi.org/10.1128/mBio.01155-15. 7. Watts E, Zhao Y, Dhara A, Eller B, Patwardhan A, Sinai AP. 2015. Novel approaches reveal that Toxoplasma gondii bradyzoites within tissue cysts are dynamic and replicating entities in vivo. mBio 6:e01155-15. https://doi.org/10.1128/mBio.01155-15. 16. Mocciaro A, Rape M. 2012. Emerging regulatory mechanisms in ubiquitin-dependent cell cycle control. J Cell Sci 125:255–263. https:// doi.org/10.1242/jcs.091199. 8. Speer CA, Dubey JP. 2005. Ultrastructural differentiation of Toxoplasma gondii schizonts (types B to E) and gamonts in the intestines of cats fed bradyzoites. Int J Parasitol 35:193–206. https://doi.org/10.1016/j.ijpara .2004.11.005. 8. Speer CA, Dubey JP. 2005. Ultrastructural differentiation of Toxoplasma gondii schizonts (types B to E) and gamonts in the intestines of cats fed bradyzoites. Int J Parasitol 35:193–206. https://doi.org/10.1016/j.ijpara .2004.11.005. 17. Sivakumar S, Gorbsky GJ. 2015. Spatiotemporal regulation of the anaphase-promoting complex in mitosis. Nat Rev Mol Cell Biol 16:82–94. https://doi.org/10.1038/nrm3934. 18. Eletr ZM, Wilkinson KD. 2014. Regulation of proteolysis by human deu- biquitinating enzymes. Biochim Biophys Acta 1843:114–128. https://doi .org/10.1016/j.bbamcr.2013.06.027. 9. Ferguson DJ, Sahoo N, Pinches RA, Bumstead JM, Tomley FM, Gubbels MJ. 2008. MORN1 has a conserved role in asexual and sexual develop- ment across the Apicomplexa. Eukaryot Cell 7:698–711. https://doi.org/ 10.1128/EC.00021-08. 19. Silmon de Monerri NC, Yakubu RR, Chen AL, Bradley PJ, Nieves E, Weiss LM, Kim K. 2015. The ubiquitin proteome of Toxoplasma gondii reveals roles for protein ubiquitination in cell cycle transitions. Cell Host Microbe 18:621–633. https://doi.org/10.1016/j.chom.2015.10.014. 10. Radke JR, Striepen B, Guerini MN, Jerome ME, Roos DS, White MW. 2001. Deﬁning the cell cycle for the tachyzoite stage of Toxoplasma gondii. November/December 2017 Volume 8 Issue 6 e01846-17 mbio.asm.org 26 mbio.asm.org on February 15, 2018 - Published by mbio.asm.org Downloaded from ® A Toxoplasma DUB Controls Cell Cycle Architecture 20. Trempe JF. 2011. Reading the ubiquitin postal code. Curr Opin Struct Biol 21:792–801. https://doi.org/10.1016/j.sbi.2011.09.009. Flynn J, Parrot B, Radke J, Striepen B, White MW. 2008. Forward genetic analysis of the apicomplexan cell division cycle in Toxoplasma gondii. PLoS Pathog 4:e36. https://doi.org/10.1371/journal.ppat.0040036. Flynn J, Parrot B, Radke J, Striepen B, White MW. 2008. Forward genetic analysis of the apicomplexan cell division cycle in Toxoplasma gondii. PLoS Pathog 4:e36. https://doi.org/10.1371/journal.ppat.0040036. 21. Perica T, Chothia C. 2010. Ubiquitin—molecular mechanisms for recog- nition of different structures. Curr Opin Struct Biol 20:367–376. https:// doi.org/10.1016/j.sbi.2010.03.007. Pathog 4:e36. https://doi.org/10.1371/journal.ppat.0040036. 42. REFERENCES Gubbels MJ, Vaishnava S, Boot N, Dubremetz JF, Striepen B. 2006. A MORN-repeat protein is a dynamic component of the Toxoplasma gon- dii cell division apparatus. J Cell Sci 119:2236–2245. https://doi.org/10 .1242/jcs.02949. g j 22. Ikeda F, Crosetto N, Dikic I. 2010. What determines the speciﬁcity and outcomes of ubiquitin signaling? Cell 143:677–681. https://doi.org/10 .1016/j.cell.2010.10.026. 43. Hu K. 2008. Organizational changes of the daughter basal complex during the parasite replication of Toxoplasma gondii. PLoS Pathog 4:e10. https://doi.org/10.1371/journal.ppat.0040010. j 23. Kuhlbrodt K, Mouysset J, Hoppe T. 2005. Orchestra for assembly and fate of polyubiquitin chains. Essays Biochem 41:1–14. https://doi.org/10 .1042/EB0410001. 44. Naumov A, Kratzer S, Ting LM, Kim K, Suvorova ES, White MW. 2017. The toxoplasma centrocone houses cell cycle regulatory factors. mBio 8:e00579-17. https://doi.org/10.1128/mBio.00579-17. 24. Chen AL, Kim EW, Toh JY, Vashisht AA, Rashoff AQ, Van C, Huang AS, Moon AS, Bell HN, Bentolila LA, Wohlschlegel JA, Bradley PJ. 2015. Novel components of the Toxoplasma inner membrane complex revealed by BioID. mBio 6:e02357-14. https://doi.org/10.1128/mBio.02357-14. 45. Lau YL, Lee WC, Gudimella R, Zhang G, Ching XT, Razali R, Aziz F, Anwar A, Fong MY. 2016. Deciphering the draft genome of Toxoplasma gondii RH strain. PLoS One 11:e0157901. https://doi.org/10.1371/journal.pone .0157901. 25. Kayagaki N, Phung Q, Chan S, Chaudhari R, Quan C, O’Rourke KM, Eby M, Pietras E, Cheng G, Bazan JF, Zhang Z, Arnott D, Dixit VM. 2007. DUBA: a deubiquitinase that regulates type I interferon production. Science 318:1628–1632. https://doi.org/10.1126/science.1145918. 46. Wang ZT, Verma SK, Dubey JP, Sibley LD. 2017. The aromatic amino acid hydroxylase genes AAH1 and AAH2 in Toxoplasma gondii contribute to transmission in the cat. PLoS Pathog 13:e1006272. https://doi.org/10 .1371/journal.ppat.1006272. 26. Kulathu Y, Komander D. 2012. Atypical ubiquitylation—the unexplored world of polyubiquitin beyond Lys48 and Lys63 linkages. Nat Rev Mol Cell Biol 13:508–523. https://doi.org/10.1038/nrm3394. 47. Francia ME, Jordan CN, Patel JD, Sheiner L, Demerly JL, Fellows JD, de Leon JC, Morrissette NS, Dubremetz JF, Striepen B. 2012. Cell division in apicomplexan parasites is organized by a homolog of the striated rootlet ﬁber of algal ﬂagella. PLoS Biol 10:e1001444. https://doi.org/10.1371/ journal.pbio.1001444. 27. Komander D, Clague MJ, Urbé S. 2009. Breaking the chains: structure and function of the deubiquitinases. Nat Rev Mol Cell Biol 10:550–563. https://doi.org/10.1038/nrm2731. 28. Huang X, Summers MK, Pham V, Lill JR, Liu J, Lee G, Kirkpatrick DS, Jackson PK, Fang G, Dixit VM. 2011. REFERENCES Deubiquitinase USP37 is activated by CDK2 to antagonize APC(CDH1) and promote S phase entry. Mol Cell 42:511–523. https://doi.org/10.1016/j.molcel.2011.03.027. 48. Chen CT, Gubbels MJ. 2015. Apicomplexan cell cycle ﬂexibility: centro- some controls the clutch. Trends Parasitol 31:229–230. https://doi.org/ 10.1016/j.pt.2015.04.003. 29. Song L, Rape M. 2008. Reverse the curse—the role of deubiquitination in cell cycle control. Curr Opin Cell Biol 20:156–163. https://doi.org/10 .1016/j.ceb.2008.01.012. 49. Alvarez CA, Suvorova ES. 2017. Checkpoints of apicomplexan cell divi- sion identiﬁed in Toxoplasma gondii. PLoS Pathog 13:e1006483. https:// doi.org/10.1371/journal.ppat.1006483. 30. Ponts N, Yang J, Chung DW, Prudhomme J, Girke T, Horrocks P, Le Roch KG. 2008. Deciphering the ubiquitin-mediated pathway in apicomplexan parasites: a potential strategy to interfere with parasite virulence. PLoS One 3:e2386. https://doi.org/10.1371/journal.pone.0002386. 50. Morlon-Guyot J, Francia ME, Dubremetcz JF, Daher W. 2016. Towards a molecular architecture of the centrosome in Toxoplasma gondii. Cyto- skeleton 74:55–71. https://doi.org/10.1002/cm.21353. 51. Brooks CF, Francia ME, Gissot M, Croken MM, Kim K, Striepen B. 2011. Toxoplasma gondii sequesters centromeres to a speciﬁc nuclear region throughout the cell cycle. Proc Natl Acad Sci U S A 108:3767–3772. https://doi.org/10.1073/pnas.1006741108. 31. Frickel EM, Quesada V, Muething L, Gubbels MJ, Spooner E, Ploegh H, Artavanis-Tsakonas K. 2007. Apicomplexan UCHL3 retains dual speciﬁc- ity for ubiquitin and Nedd8 throughout evolution. Cell Microbiol 9:1601–1610. https://doi.org/10.1111/j.1462-5822.2007.00896.x. 52. Vaishnava S, Morrison DP, Gaji RY, Murray JM, Entzeroth R, Howe DK, Striepen B. 2005. Plastid segregation and cell division in the apicompl- exan parasite Sarcocystis neurona. J Cell Sci 118:3397–3407. https://doi .org/10.1242/jcs.02458. 32. Agrawal S, Chung DW, Ponts N, van Dooren GG, Prudhomme J, Brooks CF, Rodrigues EM, Tan JC, Ferdig MT, Striepen B, Le Roch KG. 2013. An apicoplast localized ubiquitylation system is required for the import of nuclear-encoded plastid proteins. PLoS Pathog 9:e1003426. https://doi .org/10.1371/journal.ppat.1003426. 53. Li A, Blow JJ. 2005. Cdt1 downregulation by proteolysis and geminin inhibition prevents DNA re-replication in Xenopus. EMBO J 24:395–404. https://doi.org/10.1038/sj.emboj.7600520. 33. Mevissen TE, Hospenthal MK, Geurink PP, Elliott PR, Akutsu M, Arnaudo N, Ekkebus R, Kulathu Y, Wauer T, El Oualid F, Freund SM, Ovaa H, Komander D. 2013. OTU deubiquitinases reveal mechanisms of linkage speciﬁcity and enable ubiquitin chain restriction analysis. Cell 154: 169–184. https://doi.org/10.1016/j.cell.2013.05.046. 54. Tachibana KE, Gonzalez MA, Guarguaglini G, Nigg EA, Laskey RA. 2005. Depletion of licensing inhibitor geminin causes centrosome overdupli- cation and mitotic defects. EMBO Rep 6:1052–1057. https://doi.org/10 .1038/sj.embor.7400527. 34. Dhara A, Sinai AP. 2016. November/December 2017 Volume 8 Issue 6 e01846-17 REFERENCES A cell cycle-regulated toxoplasma deubiquiti- nase, TgOTUD3A, targets polyubiquitins with speciﬁc lysine linkages. mSphere 1:e00085-16. https://doi.org/10.1128/mSphere.00085-16. 55. Fernández-Álvarez A, Bez C, O’Toole ET, Morphew M, Cooper JP. 2016. Mitotic nuclear envelope breakdown and spindle nucleation are con- trolled by interphase contacts between centromeres and the nuclear envelope. Dev Cell 39:544–559. https://doi.org/10.1016/j.devcel.2016.10 .021. 35. Suvorova ES, Francia M, Striepen B, White MW. 2015. A novel bipartite centrosome coordinates the apicomplexan cell cycle. PLoS Biol 13: e1002093. https://doi.org/10.1371/journal.pbio.1002093. 56. van Ree JH, Jeganathan KB, Malureanu L, van Deursen JM. 2010. Over- expression of the E2 ubiquitin-conjugating enzyme UbcH10 causes chromosome missegregation and tumor formation. J Cell Biol 188: 83–100. https://doi.org/10.1083/jcb.200906147. 36. Behnke MS, Wootton JC, Lehmann MM, Radke JB, Lucas O, Nawas J, Sibley LD, White MW. 2010. Coordinated progression through two sub- transcriptomes underlies the tachyzoite cycle of Toxoplasma gondii. PLoS One 5:e12354. https://doi.org/10.1371/journal.pone.0012354. p g j 57. Cˇajánek L, Glatter T, Nigg EA. 2015. The E3 ubiquitin ligase Mib1 regu- lates Plk4 and centriole biogenesis. J Cell Sci 128:1674–1682. https://doi .org/10.1242/jcs.166496. 37. Shen B, Brown KM, Lee TD, Sibley LD. 2014. Efﬁcient gene disruption in diverse strains of Toxoplasma gondii using CRISPR/CAS9. mBio 5:e01114 -14. https://doi.org/10.1128/mBio.01114-14. 58. Li J, D’Angiolella V, Seeley ES, Kim S, Kobayashi T, Fu W, Campos EI, Pagano M, Dynlacht BD. 2013. USP33 regulates centrosome biogenesis via deubiquitination of the centriolar protein CP110. Nature 495: 255–259. https://doi.org/10.1038/nature11941. 38. Sidik SM, Huet D, Ganesan SM, Huynh MH, Wang T, Nasamu AS, Thiru P, Saeij JP, Carruthers VB, Niles JC, Lourido S. 2016. A genome-wide CRISPR screen in toxoplasma identiﬁes essential apicomplexan genes. Cell 166: 1423–1435.e12. https://doi.org/10.1016/j.cell.2016.08.019. 59. Lige B, Romano JD, Bandaru VV, Ehrenman K, Levitskaya J, Sampels V, Haughey NJ, Coppens I. 2011. Deﬁciency of a Niemann-Pick, type C1- related protein in toxoplasma is associated with multiple lipidoses and increased pathogenicity. PLoS Pathog 7:e1002410. https://doi.org/10 .1371/journal.ppat.1002410. g j 39. Hu K, Mann T, Striepen B, Beckers CJ, Roos DS, Murray JM. 2002. Daughter cell assembly in the protozoan parasite Toxoplasma gondii. Mol Biol Cell 13:593–606. https://doi.org/10.1091/mbc.01-06-0309. 40. Hu K, Roos DS, Angel SO, Murray JM. 2004. Variability and heritability of cell division pathways in Toxoplasma gondii. J Cell Sci 117:5697–5705. https://doi.org/10.1242/jcs.01494. 60. Stedman TT, Sussmann AR, Joiner KA. 2003. Toxoplasma gondii Rab6 mediates a retrograde pathway for sorting of constitutively secreted 41. Gubbels MJ, Lehmann M, Muthalagi M, Jerome ME, Brooks CF, Szatanek T, 41. November/December 2017 Volume 8 Issue 6 e01846-17 REFERENCES Gubbels MJ, Lehmann M, Muthalagi M, Jerome ME, Brooks CF, Szatanek T, mbio.asm.org on February 15, 2018 - Published by mbio.asm.org Downloaded from ® Dhara et al. proteins to the Golgi complex. J Biol Chem 278:5433–5443. https://doi .org/10.1074/jbc.M209390200. CJ, Ferguson DJ, Sahoo N, Gubbels MJ. 2011. A family of intermediate ﬁlament-like proteins is sequentially assembled into the cytoskeleton of Toxoplasma gondii. Cell Microbiol 13:18–31. https://doi.org/10.1111/j .1462-5822.2010.01514.x. 61. Shock JL, Fischer KF, DeRisi JL. 2007. Whole-genome analysis of mRNA decay in Plasmodium falciparum reveals a global lengthening of mRNA half-life during the intra-erythrocytic development cycle. Genome Biol 8:R134. https://doi.org/10.1186/gb-2007-8-7-r134. 67. 67. Karnataki A, DeRocher AE, Feagin JE, Parsons M. 2009. Sequential process- ing of the Toxoplasma apicoplast membrane protein FtsH1 in topologically distinct domains during intracellular trafﬁcking. Mol Biochem Parasitol 166: 126–133. https://doi.org/10.1016/j.molbiopara.2009.03.004. 62. Ran FA, Hsu PD, Wright J, Agarwala V, Scott DA, Zhang F. 2013. Genome engineering using the CRISPR-Cas9 system. Nat Protoc 8:2281–2308. https://doi.org/10.1038/nprot.2013.143. 126–133. https://doi.org/10.1016/j.molbiopara.2009.03.004. 68. Carruthers VB, Tomley FM. 2008. Microneme proteins in apicomplex- ans. Subcell Biochem 47:33–45. https://doi.org/10.1007/978-0-387 -78267-6_2. 63. Donald RG, Roos DS. 1994. Homologous recombination and gene re- placement at the dihydrofolate reductase-thymidylate synthase locus in Toxoplasma gondii. Mol Biochem Parasitol 63:243–253. https://doi.org/ 10.1016/0166-6851(94)90060-4. 69. Rome ME, Beck JR, Turetzky JM, Webster P, Bradley PJ. 2008. Intervacu- olar transport and unique topology of GRA14, a novel dense granule protein in Toxoplasma gondii. Infect Immun 76:4865–4875. https://doi .org/10.1128/IAI.00782-08. 64. Jammallo L, Eidell K, Davis PH, Dufort FJ, Cronin C, Thirugnanam S, Chiles TC, Roos DS, Gubbels MJ. 2011. An insertional trap for conditional gene expression in Toxoplasma gondii: identiﬁcation of TAF250 as an essen- tial gene. Mol Biochem Parasitol 175:133–143. https://doi.org/10.1016/j .molbiopara.2010.10.007. 70. Bermudes D, Dubremetz JF, Joiner KA. 1994. Molecular characterization of the dense granule protein GRA3 from Toxoplasma gondii. Mol Biochem Parasit 68:247–257. 65. Ghosh D, Walton JL, Roepe PD, Sinai AP. 2012. Autophagy is a cell death mechanism in Toxoplasma gondii. Cell Microbiol 14:589–607. https:// doi.org/10.1111/j.1462-5822.2011.01745.x. 71. Manger ID, Hehl AB, Boothroyd JC. 1998. The surface of Toxoplasma tachyzoites is dominated by a family of glycosylphosphatidylinositol- anchored antigens related to SAG1. Infect Immun 66:2237–2244. 66. Anderson-White BR, Ivey FD, Cheng K, Szatanek T, Lorestani A, Beckers mbio.asm.org 28
https://openalex.org/W2597468530	https://zenodo.org/record/3731880/files/yakpo_2017_language-contact-guianas-ams.pdf	English	null	Language contact and change in the multilingual ecologies of the Guianas	De Gruyter eBooks	2,017	cc-by	7,411	Yakpo, Kofi & Pieter Muysken. 2017. Language contact and change in the multilingual ecologies of the Guianas. In Kofi Yakpo & Pieter Muysken (eds.), Boundaries and bridges: Language contact in multilingual ecologies (Language Contact and Bilingualism (LCB) 14), 3–19. Berlin: De Gruyter Mouton. doi:10.1515/9781614514886-001. AUTHOR MANUSCRIPT VERSION Yakpo, Kofi & Pieter Muysken. 2017. Language contact and change in the multilingual ecologies of the Guianas. In Kofi Yakpo & Pieter Muysken (eds.), Boundaries and bridges: Language contact in multilingual ecologies (Language Contact and Bilingualism (LCB) 14), 3–19. Berlin: De Gruyter Mouton. doi:10.1515/9781614514886-001. AUTHOR MANUSCRIPT VERSION Yakpo, Kofi & Pieter Muysken. 2017. Language contact and change in the multilingual ecologies of the Guianas. In Kofi Yakpo & Pieter Muysken (eds.), Boundaries and bridges: Language contact in multilingual ecologies (Language Contact and Bilingualism (LCB) 14), 3–19. Berlin: De Gruyter Mouton. doi:10.1515/9781614514886-001. AUTHOR MANUSCRIPT VERSION Yakpo, Kofi & Pieter Muysken. 2017. Language contact and change in the multilingual ecologies of the Guianas. In Kofi Yakpo & Pieter Muysken (eds.), Boundaries and bridges: Language contact in multilingual ecologies (Language Contact and Bilingualism (LCB) 14), 3–19. Berlin: De Gruyter Mouton. doi:10.1515/9781614514886-001. AUTHOR MANUSCRIPT VERSION 1 Introduction This book deals with multilingualism, language contact, language change and convergence in the Guianas of South America, with a focus on Suriname. The Guianas are a very complex region. The national identity of the countries in the Guianas involves both a sense of common destiny and of multiple ethnic affilia- tions. In this sense it presents a condensed microcosm of the Latin American and Caribbean quest for identity that we find in works as apart geographically, if not intellectually, as José Vasconcelos’ La Raza Cósmica in Mexico (1925), José Carlos Mariátegui’s 7 Ensayos de Interpretación de la Realidad Peruana in Peru (1928), or Jean Price-Mars’ Ainsi parla l’oncle in Haiti (1928) (see also the overview of essays on ethnicity and boundaries gathered in Oostindie 1996). We have named our volume Boundaries and bridges because it reflects at the same time the maintenance of ethnic and linguistic boundaries, through the languages involved, but also the numerous instances of cross-linguistic influ- ence across these boundaries. It illustrates the point that in the complex multi- lingual and multi-ethnic area of the Guianas, the languages spoken have been part of an effort of groups to keep themselves apart, as boundaries, but have also undergone numerous changes in the presence of other languages, and thus form bridges. The Guianas, or any part of them, do not form a single language community, but rather a chain of interacting and intersecting communities, which have very diverse and complex relations among themselves. Hence the term multilingual ecologies in our subtitle. However, these cases of cross-linguistic influence are very diverse in nature, and involve many parts of language. They result from different contact scenarios and include maintenance, shift, and creation. Our main focus in this book will be the Republic of Suriname, but it is useful to consider Suriname for a moment in the more global context of the Guianas. The term Guianas refers to a region bordering on the Amazon basin but straddled along the Caribbean coast of northern South America. The tropical coastal areas were discovered to be suitable for sugar plantations, as we will discuss below, and the inlands are mostly tropical rainforest, which are currently being exploited. The southern part of the Guianas is a plateau, separating the coastal plains from the Amazon basin proper. 4 Kofi Yakpo and Pieter Muysken Different parts of the Guianas were occupied by five European colonial powers: from west to east these were Spain, England, the Netherlands, France, and Portugal. This has resulted in the fact that nowadays, they correspond to five different political entities: the provinces Amazonas, Bolivar, and Delta Amacuro of Venezuela, the Republic of Guyana, the Republic of Suriname, the French colony Guyane, and the state of Amapá in Brazil. The histories of these five entities are necessarily quite different, but there are a number of common threads as well: the continued presence of different Amerindian peoples particularly belonging to the Cariban and Arawakan language families, the deportation from Africa and exploi- tation of enslaved Africans in the seventeenth through the nineteenth century, struggles for independence as well as internal strife and some border disputes. In addition to the Amerindians, people of European, and African origin, there is also the presence of Asian-descended populations. Their immigration started around the middle of the nineteenth century. Thus, the region is thoroughly Tab. 1: Overview of the five Guianas in terms of their history and ethnic composition Venezuelan Guayana Guyana Suriname Guyane Amapá Status Three states in Venezuela Republic (since 1970, indepen- dence 1966) Former British colony Republic (independence in 1975) Former Dutch colony French colony State of Brazilian Federal Republic Official language Spanish English Dutch French Portuguese Creole languages Guyanese English Creole, Berbice Dutch, Skepi Dutch Sranantongo, Maroon Creole languages, Haitian, Guyanese English Creole Guyanese French Creole, Haitian, Maroon Creole languages Guyanese French Creole (Lanc-Patuá), Karipuna French Creole Amerindian languages Arawakan, Warao Arawakan, Warao Arawakan, Cariban, (Warao) Arawakan, Cariban, Tupí-Guaraní Arawakan (Palikúr), Tupí-Guaraní (Karipuna, Wayampí), Galibi (Cariban), Iapamá (isolate) Asian languages Chinese Guyanese Bhojpuri Sarnami, Javanese, Kejia (Hakka) Kejia (Hakka), Hmong, Vietnamese Other languages English Portuguese, English Portuguese, Spanish, Dutch Tab. 1: Overview of the five Guianas in terms of their history and ethnic composition Language contact and change in the multilingual ecologies of the Guianas 5 5 multi-ethnic and multi-lingual. In Tab. 1 above, we give a broad overview of the five Guianas in terms of their history and ethnic composition. Suriname, the focus of the present book, is linguistically and ethnically the most diverse of the five Guianas, as we will illustrate in the following sections. Through processes of con- vergence, it has also become a linguistic area in its own right. 6 Kofi Yakpo and Pieter Muysken 6 Kofi Yakpo and Pieter Muysken 6 Kofi Yakpo and Pieter Muysken This book attempts to draw the outlines of language contact and change in mul- tilingual ecologies more clearly, and it proposes a framework for comparable studies. The case studies are centred on the Guianas in South America, and Suriname in particular. In this region, we can see the formation and transformation of a lin- guistic area unfolding before our eyes. The area has been recognized by anthro- pologists, historians, and linguists alike as an object of intense study for several reasons: – The socio-history, culture, and demographic development of this region, within a diachronic depth of about five centuries, are exceptionally well documented; – The Guianas, and Suriname in particular, boast a higher number of “new” languages and cultures that have arisen through language contact, than many other regions of the world; these have been the subject of very central theore- tical debate. But many aspects of these languages still remain to be studied; – Currently more than twenty genetically diverse languages are spoken in the region; – There is an astounding typological spread with representatives of the fol- lowing major linguistic groupings: Afro-Caribbean English-lexifier Creoles, Indo-Aryan, Indo-Germanic, Austronesian, Sinitic, Arawakan, Cariban; – Language contact is multidirectional and minimally involves two main donor or target languages, namely a dominant Creole language and European colonial languages; – The impact of the main target languages is temporally layered, as well as sociolinguistically diverse with correspondingly differentiated impacts on the domains of phonology, lexicon and morphosyntax, as well as style and register; – We have identified various hotspots of contact and structural convergence between the languages studied and we draw numerous comparisons with other contact scenarios in the wider region and other parts of the world. – We have identified various hotspots of contact and structural convergence between the languages studied and we draw numerous comparisons with other contact scenarios in the wider region and other parts of the world. 2 Linguistic areas and contact linguistics in the Guianas This collective volume looks at the dynamics of a specific type of linguistic area from a fresh perspective. Based on case studies, it strives to integrate common concepts in contact linguistics such as borrowing, contact-induced change, lan- guage maintenance and shift, creolization, koineization, genealogical differenti- ation, typological change, and areal convergence. The most common contact situations described in the linguistic literature involve (1) synchronic and diachronic studies of language contact with one lan- guage uni-directionally exerting influence on another (e.g. the studies in Thomason and Kaufmann 1988, Thomason 2001, Matras and Sakel 2007a, 2007b); (2) studies in areal linguistics addressing the outcomes of contact between more than two languages. Here the time-depth and lack of documentation often make it difficult to describe with more accuracy the processes leading to contact-induced changes and their directionality (e.g. Aikhenvald and Dixon 2007; Nicolai and Zima 2002). This study adds to the types of studies listed above in looking at the situation of multiple language contact, characterized by multilevel interactions between more than two languages, involving Sranantongo, Dutch, and multiple other langua- ges, and simultaneous multidirectional change. Furthermore, it studies contact- induced change in a selection of languages that are typologically and genetically highly diverse, and describes the emergence of complex linguistic areas over a five hundred year period, taking into account both synchronic variation, and changes at various time depths, involving shifting sociolinguistic configurations. The volume offers detailed information on different grammatical domains (among which tense, mood, aspect and argument realization) across various case studies, and in doing so, allows a thorough reassessment of the two important notions of borrowability and stability of linguistic elements and structures in language contact. The studies and findings of this volume have a high potential for generalization. Multiple language contact and change involving more than two languages is little described. However, it probably represents the most common type of contact ecology in most parts of the (non-Western) world, where fast demographic and socio-eco- nomic change, and multilingualism have led to equally rapid linguistic change. 3 Multilingualism and language contact in Suriname Suriname has been the scene of complex and overlapping population movements throughout its history. In this section, we give a brief overview of how these move- ments have driven the development of multilingualism in the recent history of Suriname and in present times. Patterns of community-wide multilingualism have probably characterized the societies of Suriname from well before colonial conquest. Linguistic diversity in Language contact and change in the multilingual ecologies of the Guianas 7 2: Some key events in the history of Suriname and their sociolinguistic significance (taken from Yakpo and Muysken 2014) 8 Kofi Yakpo and Pieter Muysken Date Event and its demographic significance Contact-related aspects 1876 Dutch introduced into schools as only medium of instruction and part of universal education The beginnings of urban Dutch-Sranan multilingualism in a slightly larger population; begin of prestige loss of Sranan. The adoption of Dutch as an L2 by increasing numbers of Surinamese causes the creation of a profoundly Sranan-influenced Surinamese Dutch. 1873–1916 Arrival of Indian indentured laborers Varieties of north Indian languages brought to Suriname, such as Bhojpuri, Magahi and Maithili 1890–1939 Arrival of Javanese indentured laborers Varieties of Javanese brought to Suriname 1975 Independence of the Republic of Suriname Symbolic break with the former colonial power, possibility for autonomous developments in Surinamese Dutch, stronger Dutch influence on Sranan due to circular migration Netherlands-Suriname 1990s Economic development Gradual influx of Haitians, Brazilians, Chinese 3.1 Pre-colonial contact and creolization in the colonial period Tab. 2 (continued) Symbolic break with the former colonial power, possibility for autonomous developments in Surinamese Dutch, stronger Dutch influence on Sranan due to circular migration Netherlands-Suriname Gradual influx of Haitians, Brazilians, Chinese Language contact and change in the multilingual ecologies of the Guianas 7 Language contact and change in the multilingual ecologies of the Guianas 7 7 Suriname has increased significantly since the beginning of the colonial period, reaching a peak in contemporary Suriname and ushering in the type of extensive language contact that characterizes the country today. For detailed overviews of multilingual Suriname see Charry, Koefoed and Muysken 1983; Carlin and Arends 2002 and the chapter by Borges in this volume, which also includes a historical overview. Some key events in the history of Suriname with sociolinguistic significance are presented in Tab. 2. Tab. 2: Some key events in the history of Suriname and their sociolinguistic significance (taken from Yakpo and Muysken 2014) Date Event and its demographic significance Contact-related aspects 1200–1500s Migratory movements in the Guianas Extensive contact between Warao, Cariban and Arawak languages 1650 Establishment of an English colony in Suriname Varieties of English brought to Suriname 1652– Beginning of the deportation and enslavement of West Africans in Suriname Gradual creation of an English lexicon coastal Creole language which would develop into Sranan in the latter part of the 17th century 1665 Arrival of the Portuguese Jewish planters from Brazil, possibly with some enslaved Africans Varieties of Portuguese and quite possibly Portuguese-based Creole brought to Suriname 1667 Suriname becomes a Dutch colonial possession Varieties of Dutch brought to Suriname as an elite language; speedy end to the presence of English 1685 Emergence of the Saramaccans as a separate ethnic group Creation of the Saramaccan (or Saamaka) language out of West-African languages, a Portuguese and the English lexicon pidgin/Creole 1730 Emergence of the Ndyuka as a separate ethnic group Creation of Ndyuka out of West-African languages and the English lexicon Creole 1804–1816 English occupation English superstrate influence on Sranan lexicon limited to few words 1844–1854 Enslaved Africans were allowed to learn how to read and write Sranan texts created; consolidation of a written register of the language 1863 Emancipation of the enslaved rural population and dismantlement of the traditional plantation system Increased presence of Sranan speakers in the urban centre Paramaribo 1853 First arrival of Chinese indentured laborers and traders Hakka and other Chinese languages brought to Suriname Tab. 3.1 Pre-colonial contact and creolization in the colonial period Amongst these, Sranantongo has spread beyond the coastal belt into the interior to become the most-widely spoken Creole of the country. The Creole languages of Suriname, however, thrived and have differentiated into the three distinct clusters of Sranantongo, Western and Eastern Maroon Creole (Smith 1987; Smith 2002). Amongst these, Sranantongo has spread beyond the coastal belt into the interior to become the most-widely spoken Creole of the country. The indigenous languages of Suriname have undergone quite fundamental contact-induced changes since colonization as well, both through contact with each other (Carlin 2006, this volume) as well as with Sranantongo and Dutch (Rybka, this volume). The indigenous languages of Suriname have undergone quite fundamental contact-induced changes since colonization as well, both through contact with each other (Carlin 2006, this volume) as well as with Sranantongo and Dutch (Rybka, this volume). 3.1 Pre-colonial contact and creolization in the colonial period Taking pre- and early colonial times as a starting point, there were originally three indigenous language families represented on the territory of present-day Suriname, namely Warao, Carib, and Arawak (cf. Hoff 1995). Particularly striking is the partly convergent development within the Arawak and Cariban languages, including the creation of a sixteenth century Carib Coastal Pidgin (Taylor and Hoff 1980). Convergence must have been the consequence of multilingualism in a situation of language maintenance, probably with both Carib and Arawak enjoy- ing similar degrees of prestige. With the beginning of the European colonization of Suriname in the seven- teenth century, the linguistic situation becomes more complex. The Netherlands ends up being the sole colonial power in 1667. The establishment of a plantation economy leads to the deportation from the western seaboard of Africa and ens- lavement of an estimated total of approximately 350,000 Africans by the Dutch between 1675 and 1803 (Postma 1990). Various interlocking linguistic processes played a role in the emergence of the Creole languages of Suriname, among them the present-day lingua franca Sranan- tongo. Language creation led to the rise of early Creole varieties largely drawing Language contact and change in the multilingual ecologies of the Guianas 9 9 on first Portuguese, then English superstrate lexicon, and as well as grammati- cal features from African substrate languages (cf. e.g. Huttar 1983; Huttar, Esseg- bey and Ameka 2007; Winford and Migge 2007). High mortality rates under the brutal laboring conditions on Dutch-owned plantations made it impossible for the enslaved African population to replenish itself through natural growth (Arends 1995). Therefore most sources agree that creolization in Suriname must have been gradual, involving a long period of multilingualism in the emerging Creole, and African and European languages (Selbach, Cardoso and Van den Berg 2009). Lan- guage creation must therefore have been accompanied both by gradual language shift (to the Creole and for some Dutch) by Suriname-born Africans as well as maintenance of African languages among African-born Africans and Suriname- born children. African languages have only survived into the present in a fossi- lized form in the ritual languages Kumanti, Ampuku and Papa (Thoden van Velzen and van Wetering 1988; Thoden van Velzen and van Wetering 2004). The Creole languages of Suriname, however, thrived and have differentiated into the three distinct clusters of Sranantongo, Western and Eastern Maroon Creole (Smith 1987; Smith 2002). 3.2 The abolition of slavery and the Asian languages of Suriname This is probably also due to the fact that a small but not insi- gnificant part of the “Javanese” population of Suriname had its origins elsewhere in the Indonesian Archipelago than Java (Gobardhan-Rambocus and Sarmo 1993). Contrary to Sarnami, there are indications that Javanese is not as vital anymore as it still was in the second half of the twentieth century and that there is an ongoing language shift, particularly by speakers below twenty to Sranantongo and Dutch. The language of the Chinese community was, for a long time, chiefly Hakka (also called “Kejia”). But Cantonese and more recently Mandarin have played important roles as prestige languages within the community and there is an ongoing language shift to Sranantongo and Dutch (Tjon Sie Fat 2002). Besides change due to contact with Sranantongo and Dutch, some degree of koineization also affected the Javanese language since it arrived in Suriname (cf. Vruggink 1987). This is probably also due to the fact that a small but not insi- gnificant part of the “Javanese” population of Suriname had its origins elsewhere in the Indonesian Archipelago than Java (Gobardhan-Rambocus and Sarmo 1993). Contrary to Sarnami, there are indications that Javanese is not as vital anymore as it still was in the second half of the twentieth century and that there is an ongoing language shift, particularly by speakers below twenty to Sranantongo and Dutch. The language of the Chinese community was, for a long time, chiefly Hakka (also called “Kejia”). But Cantonese and more recently Mandarin have played important roles as prestige languages within the community and there is an ongoing language shift to Sranantongo and Dutch (Tjon Sie Fat 2002). 3.2 The abolition of slavery and the Asian languages of Suriname The full abolition of slavery in 1873 after a transitional period of ten years of forced labor prompted the Dutch colonial regime to “import” indentured laborers from Asia, as in other plantation economies throughout the Caribbean and elsewhere in the colonial world in order to substitute for slave labor (Saunders 1984; Kale 1998). Through these arrangements, a total of about 30,000 (male and female) labo- rers were transshipped to Suriname from northern India between 1873 and 1916 (Damsteegt 1988: 95). A total of about 30,000 laborers arrived from Java (Indonesia) between 1890 and 1939 (Bersselaar, Ketelaars and Dalhuisen 1991). A third, much smaller wave of migrants arrived from Guangdong province of southern China from the 1850s onwards as laborers and traders, numbering only about two thousand but constituting an important community in economic terms (Fat 2009: 52). These migratory movements brought about a fundamental transformation of the previously established demographic constellation in Suriname. A country Kofi Yakpo and Pieter Muysken 10 with a largely African-descended population with relatively small Indigenous American and European components in the mid-nineteenth century had acquired an Asian-descended population numbering nearly half the size of the population by the turn of the twenty-first century. Hence in the 2004 national census about 27% of the total Surinamese population of half a million self-identifies as “Hindoe- staans” (Indian-descended) and 15% as “Javaans” (Javanese-descended) while the category “others” of 6% subsumes amongst others the Chinese-descended population and the Indigenous peoples of Suriname. Self-identified “Kreolen” and “Marrons” (both African-descended) Surinamese make up 18% and 15% respec- tively of the population. The substantial number of Surinamese who self-classify themselves as “mixed” (12%) or leave their ethnicity unreported (6%) is indicative of a growing proportion of Surinamese either claiming a mixed heritage of various constellations or rejecting ethnic labelling altogether. The migratory mass movements of the indenture period have been equally transformative for the linguistic situation in Suriname as they have been for the demography of the country. Various northern Indian language varieties merged to form the koiné Sarnami, the community language of the Indian-descended population of Suriname (Damsteegt 1988, Yakpo, this volume). Besides change due to contact with Sranantongo and Dutch, some degree of koineization also affected the Javanese language since it arrived in Suriname (cf. Vruggink 1987). 3.3 Sranantongo and Dutch as lingua francas Sranantongo and Dutch play a special role in Suriname: they are the only langua- ges extensively used outside of their traditional speaker communities (principally the Afro-Surinamese population of the coastal belt). Within the four hundred years or so since its creation by enslaved Africans on the European plantations of Suriname, Sranantongo has evolved into a multi-ethnic dia-system used as a Language contact and change in the multilingual ecologies of the Guianas 11 lingua franca by the ethnically diverse population of the coast. The language has also made inroads into the interior where it shares a common space with various Maroon Creoles (Migge 2007; Migge and Léglise 2011, 2013) and Indigenous languages. Sranantongo served as the primary donor of lexical material to the Asian languages of Suriname during the indenture period, when knowledge of Dutch was not yet as widespread within these communities as it now is. Nowa- days Sranantongo plays the role of a donor language together with Dutch. Sranan- tongo is the only language of Suriname that virtually every Surinamese has at least some knowledge of, however in growing competition with Dutch. It should be pointed out that the expansion of Sranantongo is solely a consequence of an incremental growth because the language has not benefited from state support of any kind whatsoever since it was abolished as a language of instruction in 1876 (cf. Tab. 2). This stands in stark contrast to the development of Dutch, which has also witnessed a considerable growth in speaker numbers throughout the 20th century due to sustained institutional and elite support. Since colonial times, Dutch has been the sole language of government business and parliamentary affairs, and the de facto language of education at the primary, secondary and tertiary levels. It has remained the language of upward social mobility and high prestige and is extensively used by officialdom and by coastal Surinamese in a variety of registers. One of the consequences of this disposition is that Dutch has witnessed a fundamental transformation within the last hundred years or so. From being a language of the colonial administration and a relatively small Dutch-educa- ted elite, it has been appropriated by larger sections of Surinamese society. In the process, Dutch has engaged on a trajectory of its own and today plays an important role as a donor language to Sranantongo and other languages of Suri- name. 3.3 Sranantongo and Dutch as lingua francas At the same time, Dutch has itself become a recipient language for lexical (cf. De Bies, Martin and Smedts 2009) and structural borrowing from Sranantongo (De Kleine 1999). Our sociolinguistic interviews show widespread competence in (varieties of) spoken Dutch with Surinamese of diverse class backgrounds hence beyond the traditional patterns of upper and middle class use of Dutch inherited from the colonial period. Together with Sranantongo, Dutch is also a target for language shift from traditional community languages such as Javanese, Sarnami and Hakka. The hierarchical superposition of Dutch to Sranantongo and the other lan- guages of Suriname is being driven by a similar set of ideological, political and economic factors as in other postcolonial societies (cf. Omondi and Sure 1997; Heine 1990; Veiga 1999 for the status quo of colonial and African languages in African nations). The widespread assumption and acceptance of the “superior” status of Dutch in Suriname is reflected in often negative and self-denigrating 12 Kofi Yakpo and Pieter Muysken 12 attitudes of speakers towards the non-European languages they speak and in the corresponding language practices. However, the social and functional division of labor between Dutch and Sranantongo outlined above has also led to Sranantongo enjoying a large amount of covert prestige. In many contexts, using Sranantongo is an act of identity asser- tion, defiance and resistance against norms transmitted through Dutch, with all its problematic associations with elitism, the colonial past and a post-colonial present. 4 Data collection methods in the present volume The studies reported on this book rely for the largest part on field data collected in Suriname in 2011–12 as part of the ERC project “Traces of Contact” at the Centre for Language Studies at Radboud University Nijmegen. The corpus contains recordings in eight Surinamese languages: The Creole languages Sranantongo, Ndyuka, Kwinti and Saramaccan, as well as Sarnami, Surinamese Javanese, Surinamese Hakka and Suri- namese Dutch. Comparative data has been collected in India, the Netherlands, West Africa and Mauritius. The corpus consists of a total of about hundred and fifty hours of data, of which the recordings of Sarnami and its control groups in India (Awadhi, Bhojpuri, Maithili, Magahi) and Mauritius (Mauritian Bhojpuri) make up about thirty hours. All unreferenced examples in this paper stem from our own field data. The chapters of Carlin, Rybka, and Migge rely on data collected in the course of many years of field research in the Guianas. The data was collected according to a unified methodology in order to allow comparison across varieties and languages. Data collection methods involved the use of broad (story-based) and narrow (video clip-based) visual stimuli on the one hand and (semi-)structured interviews on specific topics on the other. Elicitation was complemented by recordings of natural discourse. In Suriname, we also conducted about fifty sociolinguistic interviews in Sranantongo on the backgrounds of speakers and their attitudes vis-à-vis the languages they speak. We are much in favor of approaches employing quantitative analyses based on large diachronic and synchronic corpora in order to differentiate between codeswitching and borrowing, as well as between “normal” variability and contact-induced change (e.g., Van Hout and Muysken 1994; Poplack, Zentz and Dion 2012). However, when working with less documented languages, as in the case of Suriname, one is in a less fortunate position. There is a lack of sizeable corpora of diachronic data for all languages but Sranantongo (cf. http: //suca.ruhosting.nl), and the collection and handling of even modest corpora of synchronic data invol- ves considerable efforts. It seems then that only a mixed strategy is feasible. This involves quantitative investigations based on smaller corpora and extrapolation based on in-depth morpho-syntactic investigations of particular structural areas. 3.4 Contemporary data on multilingualism in Suriname Determining the size of speaker communities in present-day Suriname is not easy in the absence of a comprehensive linguistic survey. Chapter 2 by Borges provides a detailed overview of the highly complex and still rapidly changing current situation. Sranantongo and Dutch constitute the two main axes of multilingualism. These two languages show the highest total percentages of self-reported “most often” and “second language” uses. At the same time they manifest the largest differences between “most often” and “second language” uses. The differences in social function between these two most widely spoken languages of Suriname transpire in the significant differences in percentage of “most spoken”. The percentage of 9% for Sranantongo for “language spoken most often” is surprisingly low, particularly in comparison to an equally surpri- singly high score of 46.6% for Dutch. We attribute these percentages to prevai- ling language attitudes in Suriname that result from the functional and prestige differences between these languages referred to in the preceding section. Hence the high prestige of Dutch leads to over-reporting of use as “language spoken most often”, while the, the low prestige of Sranantongo leads to underreporting of use as a primary language. As for the other languages listed in Tab. 2, the lower percentages in the “second language” column seem to point to these languages largely functioning as in-group “ethnic” languages. For Sarnami for example, the relation of “most spoken” (about 75% of the total) and “second language” (about 25% of the total) may well be indicative of a partial language shift to Dutch and Sranantongo, or at least a certain decline in use. The same holds for Javanese. We have seen that language creation has been of primordial historical impor- tance for the rise of linguistic diversity in the country. In the present context, we find the maintenance of community languages alongside language shift to the two dominant languages, Sranantongo and Dutch. Language contact and change in the multilingual ecologies of the Guianas 13 5 Theoretical background Language contact and mutual borrowing of lexical items and structures in the langu- ages of Suriname is a consequence of widespread multilingualism. We will therefore first review some of the concepts related to language contact and multilingualism 14 Kofi Yakpo and Pieter Muysken 14 that we will be referring to. The typology of language contact that Thomason and Kaufman (1988) propose provides for three principal contact scenarios. We define scenario as the organized fashion in which multilingual speakers, in certain social settings, deal with the various languages in their repertoire. In the maintenance scenario the language that borrows (henceforth the recipient language), from another language (henceforth the donor language) continues to be spoken by its speaker community, i.e. it is maintained. The literature shows that there is a large range of variation in maintenance scena- rios. In some cases of maintenance, the recipient language may undergo more moderate lexical and structural transfer from a donor language. Other cases of maintenance show extensive transfers of phonological features, lexical mate- rial and structural patterns (e.g. Hainan Cham, whose Austronesian typological profile has been significantly altered due to contact with Sinitic, cf. Thurgood and Li 2003). The classification of a scenario as involving maintenance may also be theory-dependent. For example, a strong position on relexification – i.e. the mapping of one language’s semantic and morphosyntactic properties onto another’s phonological shapes – may in fact be seen as an extreme case of maintenance of the language providing the semantic and morphosyntactic content. Such a position is implicit in Lefebvre’s (1993, 1998) interpretation of the rise of Haitian Creole. In the second scenario suggested by Thomason and Kaufman (1988), shift, a community leaves behind its traditional language and shifts to another lan- guage, usually due to the socio-economic and/or political dominance of the community speaking the language shifted to. Contact effects in shift scenarios may be very similar to those encountered in maintenance scenarios. Studies have shown that intermediary stages of language shift and obsolescence (cf. e.g. the case study in Aikhenvald 2012) show the same kind of heavy structural and lexical borrowing that may characterize maintenance scenarios in which a recipient language is not threatened by language loss (for an illustrative example, cf. Gómez-Rendón 2007). 5 Theoretical background A principal difference between shift and maintenance is pointed out by Van Coetsem (2000): Language shift involves a change in directionality of borro- wing (termed “agentivity” by Van Coetsem) between a recipient language and a source language. Hence during a shift, contact effects chiefly manifest themsel- ves through structural rather than lexical influence from a shifting community’s traditional language which is usually still spoken alongside the dominant lan- guage by some proportion of the shifting community. In a maintenance scena- rio, however, the traditional language of the community remains the dominant language and lexical borrowing is usually far more common, than or at least as common as structural borrowing from the donor language. The distinction is Language contact and change in the multilingual ecologies of the Guianas 15 also relevant for Suriname, which features a range of maintenance scenarios of varying depth or extensiveness of contact. The third major scenario proposed by Thomason and Kaufman (1988) involves the creation of new linguistic systems composed of elements of contributing languages. Creolization as one type of language creation is particu- larly important in the linguistic trajectory of Suriname. In the Surinamese cre- olization scenarios, European superstrate languages (English and Portuguese) provided most of the lexicon while several African substrate languages provi- ded some lexicon and substantial parts of the grammatical and phonological systems. Next to genetic inheritance from contributing languages, creolization in Suriname also seems to have involved various degrees of restructuring of the input languages driven by linguistic-cognitive factors – the respective contribu- tion ascribed to either of the two factors being subject to theoretical preferences (Alleyne 1980; Lefebvre 1998; McWhorter 2005; Bickerton 2009). However, in the context of Suriname, other scenarios play a role as well. Language creation in Suriname concerns not only creolization but also koineiza- tion as diachronic and synchronic processes. We understand koineization as a less pervasive type of language creation in that there is less restructuring of the input languages involved in the creation of the koiné, as has been amply observed in cases of dialect contact (cf. e.g. Auer 1998b and the classic study of the rise of the Indic koiné of Fiji by Siegel 1985, 1987). The literature suggests that typological proximity and mutual intelligibility are the chief linguistic reasons responsible for the more modest restructuring of an interlanguage or koiné with respect to its input languages (cf. e.g. 5 Theoretical background the studies in Braunmüller 2009; Kühl and Petersen 2009). Stable multilingualism over some generations, as in Suriname, can lead to structural convergence between the various languages spoken in the same geo- graphical space (Winford 2003). In the process, the languages in contact may become more similar by mutual accommodation, i.e. bidirectional change, for example by adopting a compromise on the basis of already existing common structures. In this paper, we employ “convergence” in a broader sense, however, as a cover term for the multiple contact scenario characteristic for Suriname. Here, borrowed structures may stem from the two dominant donor languages Dutch and Sranantongo simultaneously, and these two languages may interact in their influence on a recipient language. Due to this circumstance, it is often dif- ficult to attribute instances of contact-induced change in a language like Sarnami to a single source. In our classification of contact phenomena in the Surinamese languages we rely on models that differentiate between the borrowing of a specific form or matter (morphemes and their phonological shapes) and structure or pattern (morphosyntactic and semantic structures without the corresponding forms). Kofi Yakpo and Pieter Muysken 16 The latter phenomenon has been also been referred to in the literature (with varying degrees of overlap in meaning) by terms like “calquing” (Haugen 1950), “metatypy” (Ross 1996), “grammatical replication” (Heine and Kuteva 2003; Heine and Kuteva 2010), “pattern replication” (Matras and Sakel 2007; Sakel 2007), “rule borrowing” (Boretzky 1985) and last but not least “relexification” (e.g. Muysken 1981; Lefebvre 1993). We also refer to the code-copying model pro- posed by Johanson (1992). Such approach not only allow operationalizing these two fundamental types of borrowing, it also allows yet finer distinctions of structural borrowing. Borrowing of patterns allows us to differentiate for example, between the repli- cation of lexical versus grammatical structures. It may also encompass cases of partial replication in which a donor language pattern undergoes adaptation, i.e. is grammaticalized to fulfill functions in the recipient language that differ to some degree from those attested in the donor language (Heine and Kuteva 2003; Meyerhoff 2009). The differentiation between the borrowings of forms (matter) and structure (pattern) also leaves room for identifying combinations of matter and pattern borrowing, in which a form and its morphosyntactic and semantic specifications are carried over into another language. 5 Theoretical background As we move on, we will see that both types of borrowing and combinations between them can be found in our Surinamese corpus. Before moving on to the next section with the chapters in this book, we wish to point out that we share the general understanding that the outcomes of multi- lingualism and language contact are of course not solely determined by linguistic factors. Socio-economic, political, cultural and demographic factors, the time- depth of cultural and linguistic contact between communities and so forth, are at least as important in fashioning the processes and outcomes of contact between languages (Myers-Scotton 1993; Roberts 2005; Gómez-Rendón 2008). Our focus in this paper is on the linguistic as much as the extra-linguistic factors of contact- induced language change in Suriname. 6 Chapters in the present volume In The people and languages of Suriname, Robert Borges reviews the various contact processes in the multilingual Guianas. Five processes are considered: (1) the partly convergent development within the Arawakan and Cariban languages (2) the introduction of the languages of three competing colonial powers: English, Dutch, French, and Portuguese; (3) the introduction of West African Gbe langua- ges and languages of the Central African Kikongo cluster with enslaved Africans. Language contact and change in the multilingual ecologies of the Guianas 17 17 This led to the emergence of the various Creoles; (4) after the abolition of slavery indentured laborers were brought in, speaking northern Indian languages, Kejia, Cantonese, and Javanese; these languages underwent leveling; (5) with increasing regional migration in recent times speakers of Guyanese Creole, Haitian Creole, Brazilian Portuguese, and Mandarin have come to Suriname. Currently Surinamese Dutch is the dominant language, transformed from a metropolitan standard lan- guage to a local interethnic urban variety. Kofi Yakpo presents a systematic overview, in Creole in transition: Contact with Dutch and typological change in Sranantongo, of how the expression of spatial relations and grammatical relations in Sranantongo has undergone typo- logical change from a more West African typology inherited from the substrates to a more Dutch-like system. Sranantongo is the only language of Suriname spoken to some degree by the vast majority of the Surinamese population, irrespective of their class, ethno-linguistic and regional background. Robert Borges, in The Maroon Creoles of the Guianas: Expansion, contact, and hybridization provides an account of the historical developments, up to the present, of the Maroons and Maroon languages in Suriname and French Guiana. Following the arrival of enslaved Africans, groups of individuals fled their capti- vity, taking creole varieties with them as they established independent commu- nities outside the plantation area. The relative isolation of the Maroon groups provided a setting in which the Maroon languages diverged substantially from the Creole varieties associated with the plantation area. This isolation came to a gradual end, leading to increased contact between Maroon varieties on the one hand, and Sranan and Dutch, on the other, and to the leveling of Maroon varieties. 6 Chapters in the present volume In Out of India: Language contact and change in Sarnami (Caribbean Hindustani), Kofi Yakpo describes how Sarnami of Suriname, the only Indo- Aryan koiné of the Caribbean that still enjoys a stable speaker community, has diverged from its north Indian contributing languages due to contact with Sranan and Dutch. The study shows that head-initial order has increased or supplan- ted head-final order inherited from Indo-Aryan in some domains (i.e. SVO, AuxV, NRel), while other domains have remained head-final (e.g. NAdp, AdjN). Sophie Villerius, in Developments in Surinamese Javanese, shows how Java- nese was brought to Suriname during the colonial labor trade within roughly the same period as Sarnami and still serves as a community language to a sizeable portion of the Javanese-descended population of Suriname. This chapter addres- ses effects of contact with Dutch and Sranan that have so far remained unstudied. In an exploratory paper Luis Miguel Rojas-Berscia with Jia Shi in an explo- ratory paper entitled Hakka as spoken in Suriname, analyze recordings of Hakka or Kejia Chinese. An ethnic Chinese population began constituting itself in Kofi Yakpo and Pieter Muysken 18 Suriname in the mid nineteenth century, when the Dutch colonial regime started importing Asian indentured labor as a substitute for African slave labor. This first cohort of Chinese migrants largely stemmed from Hakka/Kejia-speaking villages in the Fuidung’on Region. In the 1960s, a second wave consisting of acculturated Fuidung’on Hakka chain migrants joined the Chinese population already present in Suriname via Hong Kong. The third and latest migratory wave began in the 1990s after the People’s Republic of China (PRC) eased restrictions on emigration. The paper Cariban in contact: New perspectives on Trio-Ndyuka pidgin by Sergio Meira and Pieter Muysken, analyse the Trio-Ndyuka pidgin of the interior of Suriname, and situate the pidgin in the context of Carib/non-Carib contacts during the last five or six centuries. In earlier important work by Huttar and Venantie (1997) the pidgin was looked at from the Ndyuka perspective. Here the Carib perspective is focused upon. In Language contact in Southern Suriname: The case of Trio and Wayana, Eithne Carlin presents an in-depth overview of different types of language contact phenomena involving the Amerindians of southern Suriname and non- Amerindian groups, over a period of approximately 400 years. Overall, most of the language contact has resulted in lexical borrowing, but this process has been quite complicated. 6 Chapters in the present volume Konrad Rybka, in Contact-induced phenomena in Lokono (Arawakan) shows that a number of contact-induced phenomena have affected a variety of Lokono spoken in Suriname. Setting out from an account of the different layers of old nominal borrowings, it moves on to focus on synchronic examples involving hybrid verbal paradigms. This category shift is a sign of a new contact situation in the history of Lokono – the widespread multilingualism in Lokono, Sranantongo and Dutch makes it possible for the languages to exert influence on one another far beyond simple lexical borrowing. Pieter Muysken, in The transformation of a colonial language: Surinamese Dutch analyzes the structural evolution of Dutch in Suriname since the mid seventeenth century. Historical records show that the language was used by Dutch colonists, and albeit fewer, free and enslaved Africans alike from the very beginning of its implantation in Suriname and the Dutch Antilles. In the course of this development, Surinamese Dutch has also undergone dramatic changes due to substratal influence, mainly from Sranantongo. Robert Borges, Pieter Muysken, Sophie Villerius and Kofi Yakpo describe in The tense-mood-aspect systems of the languages of Suriname how the expres- sion of Tense, Mood, and Aspect (TMA) has received a great deal of scholarly attention by contact linguists, in particularly by those interested in pidgins and creoles. Thus there is a wealth of data on the expression of TMA in monolingual varieties of these languages, and on how these expressions emerged, but we know Language contact and change in the multilingual ecologies of the Guianas 19 very little about their behavior in multilingual practices. This chapter compares the use of TMA markers in several types of contact languages (creoles, koinés, Surinamese Dutch) in their respective monolingual and multilingual settings. Bettina Migge in From grammar to meaning: Towards a framework for studying synchronic language contact, explores the dynamic nature of language contact. Taking a language or system-based perspective, research on language contact tends to focus on describing the structural effects of language contact on particular structural sub-domains. Contact mechanisms and processes are inferred from macro-social data and structural linguistic comparative data alone. Such an approach creates homogenizing and one-dimensional contact scenarios. 6 Chapters in the present volume This chapter critically examines the viability of this approach for understanding synchronic contact settings, and drawing on contact between Maroon Creoles, Srananatongo, Dutch and other languages, shows how people make use of lan- guage contact to negotiate different social and interactional meanings. In the final chapter Multilingual ecologies in the Guianas: Overview, typology, prospects, Kofi Yakpo and Pieter Muysken discuss to what extent and in which way the grammatical systems of the various languages covered in this volume have converged. Are the changes these languages have undergone the result of contact or are they motivated by other factors? How stable are specific structural features and areas in this particular contact setting? The crucial aspects of bor- rowability and stability of linguistic features during contact are also addressed here.
https://openalex.org/W2262521939	http://www.scielo.br/pdf/tce/v24n3/0104-0707-tce-24-03-00818.pdf	English	null	STRATEGIES DEVELOPED BY COMMUNITY-DWELLING ELDERLY PEOPLE TO LIVE ALONE	Texto & contexto enfermagem	2,015	cc-by	5,578	STRATEGIES DEVELOPED BY COMMUNITY-DWELLING ELDERLY PEOPLE TO LIVE ALONE1 Francine Melo da Costa2, Priscila Tadei Nakata3, Eliane Pinheiro de Morais4 1 Extract from the thesis - Strategies developed by the elderly to dwell alone, presented to the Programa de Pós-Graduação em Enfermagem (PPGENF) of the Universidade Federal do Rio Grande do Sul (UFRGS), in 2013. 1 Extract from the thesis - Strategies developed by the elderly to dwell alone, presented to the Programa de Pós-Graduação em Enfermagem (PPGENF) of the Universidade Federal do Rio Grande do Sul (UFRGS), in 2013. 2 h l d lí d l l d d l l l f ) ( ) g. Nurse at the Hospital de Clínicas de Porto Alegre. Porto Alegre, Rio Grande do Sul, Brazil. E-mail: mcfrancine f g ( ) ( ) 2 M.Sc. in Nursing. Nurse at the Hospital de Clínicas de Porto Alegre. Porto Alegre, Rio Grande do Sul, Brazil. E gmail.com f g ( ) ( ) 2 M.Sc. in Nursing. Nurse at the Hospital de Clínicas de Porto Alegre. Porto Alegre, Rio Grande do Sul, Braz gmail.com g 3 Nurse of the Unidade de Saúde da Família Campo Novo. Porto Alegre, Rio Grande do Sul, Brazil. E-mail: priscilanakata@gmail. com g 3 Nurse of the Unidade de Saúde da Família Campo Novo. Porto Alegre, Rio Grande do Sul, Brazil. E-mail: priscilanakata@gmail. comi 4 Ph.D. in Fundamentals of Nursing. Adjunct Professor of the Departamento de Assistência e Orientação Profissional da Escola de Enfermagem and the PPGENF/UFRGS. Porto Alegre, Rio Grande do Sul, Brazil. E-Mail: epmorais@hotmail.com 4 Ph.D. in Fundamentals of Nursing. Adjunct Professor of the Departamento de Assistência e Orientação Profissional da Escola de Enfermagem and the PPGENF/UFRGS. Porto Alegre, Rio Grande do Sul, Brazil. E-Mail: epmorais@hotmail.com ABSTRACT: The aim of this study was to analyze strategies developed by the elderly in order to live alone. A qualitative, exploratory research was conducted with 14 elderly individuals, who lived in a community that belongs to the area assisted by a basic health unit in a city in southern Brazil. Data were collected through interviews and analyzed by means of the thematic content analysis technique. In order to analyze the strategies, three categories were constructed, according to the identified behaviors. Strategy 1: behaviors in search of social support; strategy 2: behaviors in search of keeping active; strategy 3: behaviors in search of religiosity. ESTRATÉGIAS DESENVOLVIDAS PELOS IDOSOS RESIDENTES NA COMUNIDADE PARA MORAREM SOZINHOS RESUMO: Este estudo objetivou analisar as estratégias desenvolvidas pelos idosos para morarem sozinhos. Trata-se de pesquisa qualitativa do tipo exploratória. Participaram 14 idosos residentes na comunidade pertencente à área de abrangência de uma unidade básica de saúde de um município do Sul do País. As informações foram coletadas por meio de entrevistas e analisadas pela técnica de análise de conteúdo temática. Para a análise das estratégias, foram constituídas três categorias, de acordo com os comportamentos identificados. Estratégia 1: comportamentos em busca de apoio social; estratégia 2: comportamentos em busca de manter-se ativo; estratégia 3: comportamentos em busca de religiosidade. A análise das estratégias desenvolvidas pelos idosos possibilitou compreender de que forma utilizam os recursos disponíveis para lidar com as dificuldades inerentes ao processo de envelhecimento. DESCRITORES: Idoso. Comportamento. Habitação. - 818 - Original Article - 818 - Original Article http://dx.doi.org/10.1590/0104-07072015002730014 STRATEGIES DEVELOPED BY COMMUNITY-DWELLING ELDERLY PEOPLE TO LIVE ALONE1 The analysis of the strategies developed by the elderly enabled us to understand how they use the available resources to handle difficulties that are inherent to the process of aging. DESCRIPTORS: Aged. Behavior. Housing. DESCRIPTORS: Aged. Behavior. Housing. METHODS An exploratory study, with a qualitative ap- proach,8 was conducted with participants selected from the cohort study Porto Alegre Longitudinal Aging (PALA)9 and from the records of the Basic Health Unit (BHU) of a teaching hospital in the state of Rio Grande do Sul, which is a reference unit for the neighborhoods where the elderly from the PALA live. This service, as well as the researched area, belong to the city’s sanitary district with the highest proportion of elderly individuals.10 National studies on this subject are scarce. A study researched the diverse conditional aspects of the lives of elderly individuals who live alone. It called attention to the fact that spirituality and social activity were strategies employed by them to handle loneliness. The elderly individuals received help in everyday practical situations, especially from their family members and, in lower numbers, from friends and neighbors. There was no mention of help offered by organs of the state.3 The researchers decided to select participants from the cohort and users of the BHU for the inves- tigation on the recommendation of a study3 already conducted in the Brazilian scenario with elderly individuals who live alone. This study calls atten- tion to the difficulty of access to this population because of their fear of sharing information and the feeling of vulnerability they report. Therefore, we intended to have easier access to the participants, since they were already linked to a study/refer- ence service they trusted. In the international scenario, many studies have been conducted regarding this theme and they claim that living alone, for elderly individu- als, can be a risk factor for morbidity, mortality and lower quality of life, generating high demand for healthcare services.4-5 This happens due to the aging process, whose set of changes results in increased dependency in terms of needing help when conducting activities of daily living; thus, living alone can be considered a complex circumstance. The participants were selected by intentional sampling.8 Elderly individuals from different age groups participated, with the aim of contemplat- ing younger and older elderly individuals from both genders. The inclusion criteria were: being a subject from the PALA cohort and/or living in the area assisted by the aforementioned BHU; be- ing registered in the unit and who self-reported as living alone for more than six months. ESTRATÉGIAS DESARROLLADAS POR LOS ANCIANOS RESIDENTES EN LA COMUNIDAD PARA VIVIER SOLOS RESUMEN: Este estudio objetivó hacer un análisis de las estrategias desarrolladas por los ancianos para que vivan solos. Se trata de una investigación cualitativa de tipo exploratoria. Participaron 14 ancianos residentes en la comunidad perteneciente al área de alcance de una unidad básica de salud de un municipio del sur del País. Las informaciones fueron recolectadas a través de entrevistas y analizadas por medio del contenido temático. Para el análisis de las estrategias se constituyeron tres categorías de acuerdo con los comportamientos identificados. Estrategia 1: comportamientos en búsqueda de apoyo social; estrategia 2: comportamientos en búsqueda de mantenerse activo; estrategia 3: comportamientos en búsqueda de religiosidad. El análisis de las estrategias desarrolladas por los ancianos posibilita comprender la forma en que utilizan los recursos disponibles para lidiar con las dificultades inherentes al proceso de envejecimiento. DESCRIPTORES: Anciano. Conducta. Vivienda. Text Context Nursing, 2015 Jul-Sep; 24(3): 818-25. Strategies developed by community-dwelling elderly people... - 819 - INTRODUCTION health problems that possibly arise from the aging process? To answer this question, the aim of this study was to analyze strategies developed by the elderly in order to live alone. There is a worldwide tendency for the el- derly to live alone, especially women who are older, widowed and poor.1 In Brazil, among people aged 60 or older, 13.8% live in one-person house- holds – those composed of a single person. There is evidence of a positive correlation between the proportion of elderly individuals and the propor- tion of one-person household units.2 Text Context Nursing, 2015 Jul-Sep; 24(3): 818-25. METHODS Exclu- sion criteria were: failing to be located after three attempts at different times and days of the week, including Saturdays and Sundays, by telephone or in home visits. Facing the complex factors involved in this situation, it is necessary to know which strategies were developed by elderly individuals to live alone, be it an option or a life circumstance. We consider strategies “[...] the behaviors, manifested or not, aiming at minimizing the action of envi- ronmental conditions that cause harm and losses to the individual and, at the same time, increase their chances of recovery and well-being”.6:1351 The developed strategies become behaviors,6 which are understood as an individual’s set of reactions to stimuli that are objectively observable. The adop- tion of a behavior involves many factors that are individual and collective, which vary from person to person; and the maintenance of these behaviors is linked to the prospect of success.7 From the PALA cohort, we identified eight elderly individuals who lived alone, of which four met the inclusion criteria. For gathering at the BHU, we located 10 elderly individuals who met the inclusion criteria after recommendations from the healthcare professionals. The sample included a sufficient number of elderly individuals for the theoretical saturation of information,8 totaling 14 participants. In view of the evident increase in the num- ber of elderly individuals who live alone in Brazil and this being a situation that needs to be inves- tigated nationally, we ask: what are the strategies developed by elderly individuals so they can live alone facing physical, economical, emotional and Information were collected between April and September 2012, through structured inter- - 820 - Costa FM, Nakata PT, Morais EP views with open-ended and close-ended ques- tions. From the 14 interviews, five were conducted in the presence of a companion, observing the elderly individuals’ preferences. and another for the individuals from the BHU. The participants were identified by codes to have their anonymity preserved. RESULTS AND DISCUSSION Be a source of support for the family. Live close to those who provide support. Keep close affective relationships. Strategy 2 Behaviors in search of remaining active Practice leisure activities. Participate in activities of the community. Strategy 3 Behaviors in search of religiosity Pray for health. Pray to avoid loneliness. of strategies developed by the elderly to live alone according to the identified re, Rio Grande do Sul, Brazil, 2013 Text Context Nursing, 2015 Jul-Sep; 24(3): 818-25. RESULTS AND DISCUSSION We used thematic analysis,11 with support from the software Qualitative Solutions Research NVivo (QSR NVivo) 8.0. This content analysis technique is carried out in three stages: 1) pre-anal- ysis, 2) exploration of the subject and 3) treatment and interpretation of results.11 The data regarding the participants’ characteristics were analyzed by means of descriptive statistics. Of the 14 elderly interviewees, 11 were female. The interviewees’ ages varied between 60 and 91 years, with a mean of 79.3 (standard deviation [SD]±9.2). The mean length of educa- tion was 8.6 years (SD±4.8). Most of them were widowed and lived with up to two minimum wages. The mean time they had been living alone was 19 years (SD±14.2), varying between three and 60 years. As for the reasons for living alone, most (12) interviewees said it was because of the death of those who lived with them. The research proposal was approved by the Research Ethics Committee of the institution under protocol number 120058/2012 and met the terms of resolution number 196/1996.12 We obtained authorization from the coordinators of the PALA study for use of information. Participants signed a free and informed consent form, with a written version for the elderly individuals from the PALA Analysis of the produced material allowed to group the interviewees’ speeches under two empirical categories, according to the themes that emerged, as presented in Table 1. Table 1 – Description of strategies developed by the elderly to live alone according to the identified behaviors. Porto Alegre, Rio Grande do Sul, Brazil, 2013 Strategies Behaviors Findings Strategy 1 Behaviors in search of social support Look for support from family members, friends and neighbors. Be a source of support for the family. Live close to those who provide support. Keep close affective relationships. Strategy 2 Behaviors in search of remaining active Practice leisure activities. Participate in activities of the community. Strategy 3 Behaviors in search of religiosity Pray for health. Pray to avoid loneliness. Table 1 – Description of strategies developed by the elderly to live alone according to the identified behaviors. Porto Alegre, Rio Grande do Sul, Brazil, 2013 Table 1 – Description of strategies developed by the elderly to live alone according to the identified behaviors. Porto Alegre, Rio Grande do Sul, Brazil, 2013 Strategies Behaviors Findings Strategy 1 Behaviors in search of social support Look for support from family members, friends and neighbors. Strategy 1 – Behaviors in search of social support Therefore, more balanced ex- change relationships cause higher physical and psychological benefits for them,14 which may be a possible explanation for their satisfaction in helping family members and friends, highlight- ing said help. providing and complementing resources and handling new situations.14 A social support network refers to the organization of relationships among people. A formal support network consists of public policies directed at the elderly population in general, gath- ering healthcare services. The informal support network has characteristics such as spontaneity and reciprocity, which help the elderly keep re- lationships and provide well-being. It consists of family, community, friends and neighbors.14 In Brazil, as shown in this study, the family usually provides care for the elderly and, in their absence, friends and neighbors do so. There are no formal state programs effectively applied to provide care for the elderly who are not helped by their families and for whom institutionalization is still the main alternative.3 Most of the elderly interviewees also referred to institutionalization as the first choice when they cannot live alone anymore, even among those who have children: I will have to surrender myself to a geriatric house or some such thing (I6). We identified in the elderly individuals’ speeches that living close to support providers made it easier to receive it: live close to people, ‘cause nobody lives alone. I can live alone, but there should be somebody to the right or to the left to ask for help. Of course! (I12). The elderly individuals who had more than one child lived close to the one who provided the most support. The findings show that proximity to the children’s home can be understood as a behavior both from the elderly individuals and from the family in order to sup- port them in their activities of daily living and to be readily available in case of need. The speeches from the I3 interviewee and her daughter (present at the time of the interview) exemplify this finding: I am always observing her [the elderly individual]. In the morning, I put the paper under the door, with a small part sticking out, so that I can observe it. If she gets the paper, it’s a sign that she already got up, that she is fine, get it? (I3’s daughter). Strategy 1 – Behaviors in search of social support should do (I10). These difficulties were associated to physical limitations, such as the weight of grocer- ies and fear of falling; or issues of security, such as going to the bank. This confirms the findings of a study in which one of the biggest difficulties for elderly individuals who lived alone was the need for physical security.13 Analysis of the findings indicates that the elderly interviewees, even when living alone, presented limitations to perform some activities of daily living and, thus, searched for support from family members, friends and neighbors, especially daughters, to fulfill their needs, as observed in the following speech: to organize the house, my daughter comes and does it [...] I have lunch with her [daughter], too. [Does she cook for you?] She does (I13). Social support is fundamental in order to handle specific issues of old age. Social support is defined as the total resources offered to other people, forming mutual exchanges in which both those who receive and those who offer are ben- efited. Its function is to provide the emotional and practical support that individuals need. It is considered one of the most important predictors for physical health and well-being, consisting of a process that involves individuals and their social networks with the aim of satisfying their needs, Most elderly interviewees did not report needing for help in self-care. However, they frequently reported difficulties with activities in the community, such as shopping and going to the bank: if I need to go out, I call my son-in-law or my daughter. Because walking alone is showboating. Because I know I am 90 and I know I must know what I - 821 - Strategies developed by community-dwelling elderly people... for the family, friends, neighbors and, especially, children and grandchildren. The main form of support is financial: now my grandson is unemployed. He had not finished college and I am paying it for him [...] I feel happy about it (I4). The importance that the elderly individuals give to the fact that they are also support providers calls attention. A study showed that the elderly individuals’ self-esteem may be shaken by the perception of dependency, lack of autonomy and impossibility to return the help they receive. Text Context Nursing, 2015 Jul-Sep; 24(3): 818-25. Strategy 1 – Behaviors in search of social support Moreover, it also enables the elderly individuals to have someone to ask for help in case they need, or even to help in activities of daily living, including the mainte- The elderly individuals called attention, with no questioning regarding the subject, to the fact that they commonly were the providers of support - 822 - Costa FM, Nakata PT, Morais EP Another identified behavior that seems to have a positive effect on the elderly is participating in community activities. From the 14 interviewees, nine participated in groups linked to the univer- sity, to health care services, to social clubs, to volunteer or religious institutions: try to have some activity, participate, because it’s useless to live alone and stay boxed in at home with no one to talk with. My group is very fun and the people there are very nice! We have many activities there. For example, next week we will cook carreteiro rice. On the 29th, we will throw a June party [...] and we have a protest scheduled for September [...] you can exchange telephone numbers, e-mails and everything with the people you like the most (I14). nance of their security.3 Elderly individuals who never married or did not have children develop, throughout the years, an extrafamilial lifestyle, enhancing their relationships with friends and guaranteeing the maintenance of an independent life in old age.15 The strategy that includes behaviors of search for social support - especially informal support, provided by family members, friends and neigh- bors - is also used. Our findings show that, despite their independent lifestyle, the elderly interviewees presented limitations for some activities of daily living. It is essential for them to receive support so they can perform these activities and, at the same time, remain alone and safe at their homes. Other studies rarely report this population’s participation in groups, which disagrees with this investigation. Strategy 1 – Behaviors in search of social support An alarming aspect related to the capacity to perform activities of daily living and self-care was the significant number of reports of falling. Among the interviewees, ten reported at least one fall at home or on the street in the last year: a while ago, I fell near the front door, then I couldn’t get up and I couldn’t even reach the phone. When I fall, I can’t get up. [...] Then, I crawled like a child to the sofa (I1). An investigation shows that elderly individuals who live alone tend to fall more frequently than those who live with other people.4 Falls usually happen at home. The conse- quences of lesions on older people are more seri- ous than those on younger people. In the case of lesions of the same severity, elderly individuals suffer more incapacity, longer hospital stay and rehabilitation, higher dependency later on and even risk of death.1 In addition to geographical proximity, close- ness in affective relationships with friends and neighbors was also mentioned as an important aspect in the search for social support, especially among those who did not live close to their chil- dren or those who did not have them: I also left my key [from the house] at the bazaar [close to the elderly individual’s home]. They are trustworthy people. I told them that, if one day I lose my key, or if I need something, they have it. They are very caring, they are very good to me (I8). The traditional perception of falls as “ac- cidents” resulted in a historic negligence of this public health area. Most falls can be prevented. Buildings must consider the health and security needs of the elderly, as well as physical obstacles in houses. An assessment by healthcare professionals through home visits is an alternative, where they can suggest or help in adapting the house.1 This closeness and/or good relationship with friends and neighbors is essential for maintaining friendship and companionship. Strategy 1 – Behaviors in search of social support Activity groups have an important role for elderly individuals who live alone, how- ever this kind of initiative is still incipient and does not meet the demand created by those who do not adapt to defined activities.3,16 These are important actions, given that feeling socially included is a considerable benefit for this group by decreasing monotony and sadness and by reaffirming the pos- sibility of being active.7 Furthermore, relationship with friends resulting from group participation is a protection against functional losses and it also enables relationships of cooperation and interac- tivity, demonstrating the relevance of social and affective relationships.17 Strategy 2 – Behaviors in search of remaining active The findings show that despite the limita- tions that are inherent to old age, the elderly indi- viduals try to keep active: I go out a lot during the day. During the day you cannot find me at home because I have many activities [...] I do gymnastics, exercises, knitting, handicraft pieces, I do a lot of things. I cross stitch. It’s very nice, it’s a very good thing. It is good entertainment (I5). These activities received great attention in the speeches of the participants because they represent a way to put body and mind to work, keeping busy and productive. This fact contradicts studies conducted with this population,3,16 which do not present in their results aspects related to leisure activities. We infer that higher education and socio-economic levels where the elderly indi- viduals live may have influenced the adoption of such activities. Behaviors in search of remaining active through leisure activities and participation in com- munity activities are part of a strategy that enables higher self-esteem for the elderly, avoids idleness, has a protective effect against functional capacity losses, helps the maintenance or the creation of friendship relationships and offers possible sources and/or exchanges of support. Higher availability of activity groups is among the alternatives to in- crease the use of this strategy, with higher diversity of activities, contemplating the many preferences of elderly individuals and the availability of these groups in isolated communities. This facilitates the movement of people living in these places and, consequently, increases their participation. A study with elderly individuals from the state of São Paulo found that leisure activities, such as watching television and doing handicrafts, can have a protective effect on functional capacity, since they involve learning, cognitive stimulus and compensatory mechanisms from the social support network.17 The awareness that certain behaviors bring benefits for the individuals acts as a motivational factor for their maintenance,7 something com- monly observed in relation to this category, as shown in the following speech: I do extremely hard crosswords. So I have a very good memory. I don’t need to write stuff down [...] the elderly sometimes have prob- lems, but that’s because they don’t do crosswords (I4). Text Context Nursing, 2015 Jul-Sep; 24(3): 818-25. Strategy 3 – Behaviors in search of religiosity In this study, we opted for the term “religi- osity”, because all elderly individuals mentioned specific religions, with their practices and rituals. Religion may be understood as an organized Text Context Nursing, 2015 Jul-Sep; 24(3): 818-25. - 823 - Strategies developed by community-dwelling elderly people... nisms related to religiosity.3,16 In the international context, a review of almost 850 studies examined the relationship between religiosity and mental health, reinforcing the association of religious involvement with higher levels of life satisfaction, well-being, hope and optimism.23 system of beliefs, practices, rituals and symbols destined to facilitate the individuals proximity to the sacred and transcendent.18 Religious practices were highlighted as important aspects for most elderly individuals in this research, considering that religious be- haviors are very frequent in old age.19 The most frequently mentioned activities were individual practices, such as prayer. Group practices, such as religious rituals, were also mentioned, but not as frequently. A study that investigated religiosity among elderly individuals admitted to a geriatric ward reported that physical limitations, such as locomotion problems, fear of falling and fear of going out unaccompanied, make them prefer individual practices.20 The strategy that involves behaviors in search of religiosity has positive repercussions on the physical and mental health of elderly indi- viduals, since according to what is highlighted in literature, it offers specific benefits for those who live alone, especially in relation to compensatory mechanisms for avoiding loneliness. However, it is necessary to analyze this practice. A study warns that religious belief is loaded with disbelief in public healthcare services. When the elderly pray to solve their health problems, incapacities or loneliness, they transfer to God the responsibility for coping or solving their situation. Such behavior generates passivity and conformism, which tend to naturalize the process of ageing with incapacities. This is a characteristic of the Brazilian culture that collaborates for minimizing social and governmen- tal responsibility.21 Therefore, one of the identified behaviors was prayer for health, as shown in the following speeches: I was hospitalized for 40 days and the doctors said I wouldn’t walk anymore, but through my prayer I walked again (I12). I thank God everyday [...] Jesus blesses me so I don’t have to buy medicines (I9). Strategy 3 – Behaviors in search of religiosity For the elderly individuals, remaining independent and autonomous is usually more associated with health than with the lack of illnesses, a fact that is clear in the following speech: I care for myself and every day I ask God to help me stay that way (I3). Text Context Nursing, 2015 Jul-Sep; 24(3): 818-25. CONCLUSIONS The analysis of strategies developed by the elderly to live alone helps the broadening of knowledge regarding their routines and the un- derstanding of how they handle the difficulties they face daily and the available resources for that. Studies show the implications that religios- ity may have on phenomena related to health and disease, with an emphasis on its role as a coping strategy when experiencing the health-disease pro- cess.19,21-22 A study found that religious coping has the function of regulating the emotional response caused by the bodily experience of incapacity, since, in the perception of the elderly individuals in this study, the realities of functional incapacity were difficult and painful.21 A study reaching a more representative sample of the elderly population who live alone can paint a more comprehensive picture of this population, investigating, among other aspects, the assessment of functional capacity and the support networks of these elderly individuals. We suggest the analysis of groups from different socio- economic levels and cultural contexts, since there may be considerable differences in developed strategies and their characteristics. For some elderly individuals in this study, in addition to repercussions on physical health, the religious practice of praying helps in avoiding the loneliness implied in the situation of living alone: some days I wake up sad, I don’t know. Then I argue with myself, I say: ‘wake up! Go to work!’. Some days ago my sister visited me, she went to the kitchen and said: ‘who are you talking to?’ God is listening to me! He does not leave me alone! (I11). For these individu- als, “the presence of God” represents company that alleviates loneliness, compensating the need for another human being.21 There are clear nursing problems regarding elderly individuals who live alone, such as limita- tions for performing some everyday activities and the high number of falls. The roles of nurses and healthcare teams as important intervention devices in these problems may be based on higher incen- tives for adopting the behaviors identified in this study and in the adequate assessment of elderly individuals who are capable or not capable of liv- ing alone, in addition to meeting their families. National studies show that, in order to handle loneliness, female elderly individuals who lived alone adopted compensatory mecha- Costa FM, Nakata PT, Morais EP - 824 - 10. Instituto Brasileiro de Geografia e Estatística. REFERENCES 12. Ministério da Saúde (BR). Conselho Nacional de Saúde, Comissão Nacional de Ética em Pesquisa. Resolução No 196 de 10 de outubro de 1996: diretrizes e normas regulamentadoras de pesquisa envolvendo seres humanos. Brasília (DF): MS; 1996. 1. Organização Mundial da Saúde. Envelhecimento ativo: uma política de saúde. Brasília (DF): Organização Pan-Americana de Saúde; 2005. 2. Instituto Brasileiro de Geografia e Estatística (IBGE). Censo demográfico 2010: famílias e domicílios. Rio de Janeiro (RJ) [online]. 2010. [acesso 2013 Mai 20]. Disponível em: ftp://ftp.ibge.gov.br/Censos/ Censo_Demografico_2010/Familias_e_Domicilios/ censo_fam_dom.pdf 13. Ramos JL, Ramos C, Menezes MR, Meira EC. Idosos que moram sozinhos: desafios e potencialidades do cotidiano. Rev Baiana Enferm. 2010 Dez-Jan; 24(1-3):43-54. 14. Rosa TEC. Redes de apoio social. In: Litvoc J, Brito FC, organizadores. Envelhecimento: prevenção e promoção da saúde. São Paulo (SP): Atheneu; 2004. p. 203-17. 3. Camargos MCS. Enfim só: um olhar sobre o universo de pessoas idosas que moram sozinhas no município de Belo Horizonte (MG) [tese]. Belo Horizonte (MG): Universidade Federal de Minas Gerais. Centro de Desenvolvimento e Planejamento Regional; 2008. 15. Larsson K, Silverstein M. The effects of marital and parental status on informal support and service utilization: a study of older swedes living alone. J Aging Stud. 2004 Mai; 18(2):231-44. 4. Sok SR, Yun EK. A comparison of physical health status, self-esteem, family support and health- promoting behaviours between aged living alone and living with family in Korea. J Clin Nurs. 2011 Jun; 20(11-12):1606-12. 16. Capitanini MES. Sentimentos de solidão, bem-estar subjetivo e relações sociais em idosas vivendo sós [dissertação]. Campinas (SP): Universidade Estadual de Campinas. Faculdade de Educação; 2000. 5. Kandler U, Meisinger C, Baumert J, Löwel H. Living alone is a risk factor or mortality in men but not women from the general population: a prospective cohort study. BMC Public Health [online]. 2007 [acesso 2013 Jul 31]; 7(335). Disponível em: http://www.ncbi.nlm.nih.gov/pmc/articles/ PMC2225416/pdf/1471-2458-7-335.pdf 17. Orsi E, Xavier AJ, Ramos LR. Trabalho, suporte social e lazer protegem idosos da perda funcional: estudo epidoso. Rev Saúde Pública. 2011; 45(4):685- 92. 18. Koenig HG, Mccullough ME, Larson DB. Handbook of religion and health. New York (US): Oxford University; 2001. 6. Tavares JSC, Trad LAB. Estratégias de enfrentamento do câncer de mama: um estudo de caso com famílias de mulheres mastectomizadas. Ciênc Saúde Coletiva. 2010; 15(1):349-58. 19. Teixeira JJV, Lefèvre F. Significado da intervenção médica e da fé religiosa para o paciente idoso com câncer. Ciênc Saúde Coletiva. CONCLUSIONS Síntese dos indicadores sociais: uma análise das condições de vida da população brasileira [online]. Rio de Janeiro (RJ): IBGE; 2010 [acesso 2013 Mai 20]. Disponível em: http://www.ibge.gov.br/ home/estatistica/populacao/condicaodevida/ indicadoresminimos/sinteseindicsociais2010/ SIS_2010.pdf Therefore, more alternatives for formal support would be developed, qualifying care practices and enabling the planning of health actions focused on these individuals, in a perspec- tive of individual assessment and social support. Such measure would encourage promoting and protective behaviors both for health and for well- being, helping elderly individuals in the “task” of living alone. 11. Minayo MCS. O desafio do conhecimento: pesquisa qualitativa em saúde. 8ª ed. São Paulo (SP): Hucitec; 2004. Text Context Nursing, 2015 Jul-Sep; 24(3): 818-25. Strategies developed by community-dwelling elderly people... - 825 - Enferm [online]; 2014 Jul-Set [acesso 2015 Fev 08]; 23(3):648-55. Disponível em: http://www.scielo.br/ pdf/tce/v23n3/pt_0104-0707-tce-23-03-00648.pdf 23. Koenig HG, Larson DB, Larson SS. Religion and coping with serious medical illness. Ann Pharmacother. 2001 Mar; 35(3):352-9. Received: September 06, 2014 Approved: May 12, 2015 REFERENCES 2008 Jul-Ago; 13(4):1247-56. 7. Silva MCS, Lautert L. O senso de auto-eficácia na manutenção de comportamentos promotores de saúde de idosos. Rev Esc Enferm USP [online]. 2010 [acesso 2013 Mai 20]; 44(1). Disponível em: http:// www.scielo.br/scielo.php?script=sci_arttext&pid= S008062342010000100009&lng=en&nrm=iso 20. Duarte FM, Wanderley KS. Religião e espiritualidade de idosos internados em uma enfermaria geriátrica. Psicol Teor Pesqui. 2011 Jan-Mar; 27(1):49-53. 21. Santos WJ, Giacomin KC, Pereira JK, Firmo JOA. Enfrentamento da incapacidade funcional por idosos por meio de crenças religiosas. Ciênc Saúde Coletiva [online]; 2013 [acesso 2014 Ago 06]; 18(8):2319-28. Disponível em: http://www.scielo. br/pdf/csc/v18n8/16.pdf 8. Polit DF, Beck CT, Hungler BP. Fundamentos de pesquisa em enfermagem: métodos, avaliação e utilização. 5ª ed. Porto Alegre (RS): Artmed; 2004. 9. Rinaldi J. Relação da satisfação de vida e aspectos biopsicossociais no estudo Porto Alegre Longitudinal Aging (PALA) [tese]. Porto Alegre (RS): Universidade Federal do Rio Grande do Sul. Faculdade de Medicina; 2010. 22. Chaves ECL, Paulino CFP, Souza VHS, Mesquita AC, Carvalho FSC, Nogueira DA. Qualidade de vida, sintomas depressivos e religiosidade em idosos: um estudo transversal. Texto Contexto Text Context Nursing, 2015 Jul-Sep; 24(3): 818-25. - 825 - Text Context Nursing, 2015 Jul-Sep; 24(3): 818-25. Correspondence: Francine Melo da Costa Rua Praça Jayme Telles, 40, compl 03 90650-100 - Santana, Porto Alegre, RS, Brasil E-mail: mcfrancine@gmail.com Received: September 06, 2014 Approved: May 12, 2015 Text Context Nursing, 2015 Jul-Sep; 24(3): 818-25.
W4200147169.txt	https://amt.copernicus.org/articles/14/7681/2021/amt-14-7681-2021.pdf	en		Idealized simulation study of the relationship of disdrometer sampling statistics with the precision of precipitation rate measurement	Atmospheric measurement techniques	2,021	cc-by	6,848	Atmos. Meas. Tech., 14, 7681–7691, 2021 https://doi.org/10.5194/amt-14-7681-2021 © Author(s) 2021. This work is distributed under the Creative Commons Attribution 4.0 License. Idealized simulation study of the relationship of disdrometer sampling statistics with the precision of precipitation rate measurement Karlie N. Rees and Timothy J. Garrett Department of Atmospheric Sciences, University of Utah, Salt Lake City, UT, USA Correspondence: Timothy J. Garrett (tim.garrett@utah.edu) Received: 29 September 2020 – Discussion started: 17 October 2020 Revised: 26 October 2021 – Accepted: 2 November 2021 – Published: 8 December 2021 Abstract. Due to the discretized nature of rain, the measurement of a continuous precipitation rate by disdrometers is subject to statistical sampling errors. Here, Monte Carlo simulations are employed to obtain the precision of rain detection and rate as a function of disdrometer collection area and compared with World Meteorological Organization guidelines for a 1 min sample interval and 95 % probability. To meet these requirements, simulations suggest that measurements of light rain with rain rates R ≤ 0.50 mm h−1 require a collection area of at least 6 cm × 6 cm, and for R = 1 mm h−1 , the minimum collection area is 13 cm × 13 cm. For R = 0.01 mm h−1 , a collection area of 2 cm × 2 cm is sufficient to detect a single drop. Simulations are compared with field measurements using a new hotplate device, the Differential Emissivity Imaging Disdrometer. The field results suggest an even larger plate may be required to meet the stated accuracy, likely in part due to non-Poissonian hydrometeor clustering. 1 Introduction Ground-based precipitation sensors are commonly used to validate remotely sensed precipitation measurement systems, including satellite (TRMM), WSR-88D radar measurements (Kummerow et al., 2000; Fulton et al., 1998), and numerical weather prediction models (Colle et al., 2005) aimed at hydrology, agriculture, transportation, and recreation applications (WMO, 2018; Estévez et al., 2011; Campbell and Langevin, 1995; Brun et al., 1992). The ability of an automated weather station to detect the presence of light pre- cipitation can be crucial for weather forecasting in remote locations where a human observer is not available to verify the presence of rainfall (Horel et al., 2002; Miller and Barth, 2003). Light rain or drizzle can severely impede road safety (Andrey and Yagar, 1993; Andrey and Mills, 2003; BergelHayat et al., 2013; Theofilatos and Yannis, 2014). Disdrometers measure particle drop size distributions and provide calculated precipitation rate from the integrated mass flux. Among available disdrometers are the mechanical Joss– Waldvogel (JW) disdrometer (Joss and Waldvogel, 1967), laser or optical sensors such as the OTT Parsivel2 (Tokay et al., 2013; Bartholomew, 2014), and video disdrometers such as the 2DVD (Kruger and Krajewski, 2002; Thurai et al., 2011; Brandes et al., 2007). In instrument development, striking a balance between sampling area and accurate fine-scale precipitation detection is a well-known problem. Among other instrument-specific considerations, disdrometer accuracy depends on the sampling area and time interval. Gultepe (2008) highlights a universal difficulty in accurately measuring very light precipitation. In their study, they compared different co-located precipitation sensors and found large absolute and relative errors (32 %–44 %) for all instruments when precipitation rate R < 0.3 mm h−1 , but the relative errors were approximately 10 % when R > 1.5 mm h−1 . Despite these observations, they found that optical and hotplate disdrometers can more accurately detect light precipitation compared to weighing gauges, despite having comparatively small sampling areas, typically 50 cm2 , where the VRG101 weighing gauge sampling area is 400 cm2 . Here, we focus solely on the effect of disdrometer sampling area and time interval on the precision of precipitation rate measure- Published by Copernicus Publications on behalf of the European Geosciences Union. 7682 K. N. Rees and T. J. Garrett: Disdrometer precision and first drop detection ment distinct from any other uncertainties associated with the instruments themselves. The World Meteorological Organization (WMO) recommends that a disdrometer measure precipitation with an output averaging time of 1 min (WMO, 2018). Measurement uncertainty is defined as “the uncertainty of the reported value with respect to the true value and indicates the interval in which the true value lies within a stated probability” specified to be 95 %. For liquid precipitation intensity, the uncertainty requirements for rates of 0.2 to 2 mm h−1 are ±0.1 mm h−1 , and for precipitation rates > 2 mm h−1 they are 5 % (see Table 1). For < 0.2 mm h−1 , the requirement is only detection. Most commercial instruments do not reach such strict standards. Table 1 provides the reported uncertainties for several precipitation instruments. The instrumentation used by Automated Surface Observing Systems (ASOS) weather stations located at major airports utilizes a heated tipping bucket (HTB) for precipitation accumulation and a light-emitting diode weather identifier (LEDWI) for precipitation type and intensity. The LEDWI uses signal power return to determine the drop size distribution of rain or snow and then classifies the precipitation intensity based on the size distribution (Nadolski, 1998). A significant limitation of the ASOS system is that it cannot discriminate drizzle from light rain, and it qualitatively expresses small amounts as a trace (Wade, 2003; Nadolski, 1998). Vaisala manufactures a range of optical precipitation sensors that detect and categorize precipitation from the forward scattering of a light beam, including the PWD12, PWD22, PWD52, and FD71P. The PWD12 detects rain, snow, unknown precipitation, drizzle, fog, and haze. The PWD22 has the same resolution and accuracy as the PWD12, but it also detects freezing drizzle, freezing rain, and ice pellets (Vaisala, 2019b). The PWD52 has an increased observation range of 50 km compared to 20 km for the PWD22 (Vaisala, 2018a). All three PWD instruments have a precipitation intensity resolution of 0.05 mm h−1 for a 10 min sampling interval at 10 % uncertainty (Vaisala, 2019b). The PWD22 is included with tactical weather instrumentation intended for US military and aviation operations (TACMET) and reports precipitation type in WMO METAR code format (Vaisala, 2018b). The FD71P has a higher stated sampling frequency and resolution than the PWD instruments of 0.01 mm h−1 with 2.2 % uncertainty in a 5 s measurement cycle (Vaisala, 2019a), although there has yet to be independent scientific evaluation of the device. Hotplate disdrometers offer an alternative with the advantage of requiring fewer assumptions as mass is inferred from the energy required for evaporation. The Yankee Environmental Systems TPS-3100 determines the liquid water precipitation rate of rain or snow by taking the power difference between upward- and downward-facing hotplates as a measure of the latent heat energy required to evaporate precipitation (Rasmussen et al., 2010). The technology is currently marketed as the Pond Engineering Laboratories K63 HotAtmos. Meas. Tech., 14, 7681–7691, 2021 plate Total Precipitation Gauge (Pond Engineering, 2020). The hotplate is 5 in. in diameter, or 126.7 cm2 , which is equivalent to a square with a width of 11.26 cm. It measures precipitation rate with a resolution of 0.10 ± 0.5 mm h−1 and can detect the onset of light snow within 1 min (Yankee Environmental Systems, 2011; Pond Engineering, 2020). A newer hotplate disdrometer, yet to be commercialized, is the Differential Emissivity Imaging Disdrometer (DEID) developed at the University of Utah. The DEID measures the mass of individual hydrometeors using a hotplate and a thermal camera, which provide accurate, fine-scale measurements. A larger hotplate sampling area increases the operating cost through higher power consumption. The work here was originally motivated by a desire to minimize DEID power and maximize measurement precision, although the calculations are applicable more generally to other disdrometers such as those described. We employ a Monte Carlo approach (Liu et al., 2012, 2018; Jameson and Kostinski, 1999, 2001a, 2002) to stochastically generate raindrops based on canonical size distributions aimed at determining the minimum required disdrometer collection area and sampling frequency for precise measurement of precipitation rates between 0.01 and 10 mm h−1 . We consider the precipitation rate uncertainty relative to WMO standards. Inherent precipitation measurement uncertainties associated with the instrument mechanism are not addressed here. Where Joss and Waldvogel (1969) approached the problem analytically by assuming the interarrival times of droplets up to 6 mm in diameter are distributed according to a Poisson distribution, here we approach the problem numerically by employing a Monte Carlo approach. In principle, the results should converge, although the Monte Carlo approach also facilitates the calculation here of the time required to measure the “first drop” in a precipitation event. Joss and Waldvogel (1969) defined a sample size as the product of an area and a sample time. Under the assumption that raindrop size follows an exponential distribution, to measure a precipitation rate of R = 1 mm h−1 to a precision of 10 % within 95 % confidence bounds, the required sample size is 1.5 m2 s, corresponding to a cross-sectional sampling area of A = 250 cm2 with a square sampling area width W = 15.8 cm for a nominal 60 s collection interval. The required square width W found in this work for the same parameters is 13 cm. 2 Differential Emissivity Imaging Disdrometer principle The DEID obtains the mass of individual precipitation particles by assuming conservation of energy during heat transfer from a square plate to a melting hydrometeor. To determine particle size during evaporation, a thermal camera is directed at a heated aluminum sheet. Since aluminum is a thermal reflector (thermal emissivity ≈ 0.03), whereas water is not ( ≈ 0.96), particles have high brightness temperahttps://doi.org/10.5194/amt-14-7681-2021 K. N. Rees and T. J. Garrett: Disdrometer precision and first drop detection 7683 Table 1. Precipitation measurement instrument specifications. Model Type Vaisala PWD12/22/52 Vaisala FD71P ASOS HTB ASOS LEDWI YES TPS-3100/Pond K63 Optical Optical Tipping bucket Optical Hotplate Resolution (mm h−1 ) WMO Uncertainty Time (s) 0.05 0.01 0.25 0.25 0.10 10 % 2.2 % 0.5 mm or 4 %∗ > 4 mm h−1 0.5 mm h−1 0.10 0.1 mm h−1 or 5 %* 600 5 60 60 60 Meets WMO requirement N Y N N N 60 ∗ Whichever is greater. ture and appear as white regions on a low brightness temperature, black background. From the measured cross-sectional surface area, temperature, and evaporation time of each hydrometeor, the effective diameter and volume and the mass of each particle can be calculated with high precision (Singh et al., 2021). A piece of polyimide tape with ≈ 0.95 is placed on the side of the sampling area as a reference for the differential emissivity calculation and determination of the camera’s pixel resolution. For the study described here, the DEID’s aluminum plate had an area of 15.24 cm × 15.24 cm, and the camera pixel resolution was 0.2 mm. Polyimide tape applied to the surface restricted the collection area to A ≈ 7 cm × 5 cm, equivalent to a square width of W = 5.8 cm. Thermal camera imagery with a sampling frequency between 2 and 60 Hz was used to determine the cross-sectional surface area, temperature, and evaporation time of each hydrometeor (Singh et al., 2021). From these parameters, individual hydrometeor mass is calculated from conservation of energy, whereby the heat gained by the hydrometeor is equal to the heat lost by the hotplate when evaporating water through Z cp 1T Zt Z dm + Leqv dm = K A(t)(Tp − Tw (t))dt, (1) H Figure 1. Photograph of the DEID during the Red Butte field experiment in Salt Lake City, Utah, with the thermal camera pointed at the hotplate surface. 0 where cp is the specific heat capacity of water at constant pressure, 1T is the difference in temperature between 0 and time t, m is the mass of the hydrometeor, and Leqv is the equivalent latent heat required for the conversion of the hydrometeor to gas. For liquid precipitation Leqv = Lv , where Lv is the latent heat of vaporization of water. K is the thermal conductivity of the plate, H is hotplate thickness, A(t) is the area of the water droplet at time t, Tp is the temperature of the hotplate, and Tw (t) is the temperature of the water at time t. When combining the constants into a single value Kd , Eq. (1) simplifies to Zt Z dm = Kd A(t)(Tp − Tw (t))dt, 0 https://doi.org/10.5194/amt-14-7681-2021 (2) where Kd is determined experimentally. The precipitation rate RDEID is calculated from the total mass of hydrometeors evaporated on the hotplate during a given sample time interval. For each frame of the hotplate captured by the thermal camera, RDEID = βfs Kd Aevap Imean , ρw Ahot (3) where β = 3.6×106 mm s m−1 h−1 , fs is the camera resolution (frame s−1 ), Aevap is the total area of water on the sampling area (m2 ), Imean is the pixel intensity related to the temperature difference between the plate and water through Tp −Tw (t) ≈ (255−Imean )/256×Tp , ρw is the density of water (1000 kg m−3 ), and Ahot is the hotplate area (m2 ). Atmos. Meas. Tech., 14, 7681–7691, 2021 7684 K. N. Rees and T. J. Garrett: Disdrometer precision and first drop detection Table 2. Minimum collection area required to meet WMO precision criteria for various precipitation rates and time intervals evaluated within 1 cm intervals. 1t = 10 s Rate (mm h−1 ) WMO precision 0.01 0.10 0.20 0.50 1.00 2.00 10.00 First drop (µ = 2) First drop (µ = 2) ±0.1 mm h−1 ±0.1 mm h−1 ±0.1 mm h−1 ±0.1 mm h−1 & ±5 % ±5 % 1t = 60 s 1t = 300 s 1t = 600 s Width (cm) 5 <1 6 14 25 40 30 2 1 1 6 13 20 15 <1 <1 1 3 5 8 6 <1 <1 <1 3 4 5 4 Figure 2. Precipitation rate Rcalc calculated 1000 times for each W with a uniformly distributed random selection of particle sizes from the Marshall–Palmer distribution with diameters up to 6 mm for 1t = 60 s. Vertical curves represent a probability density function (PDF) of calculated Rcalc with the widths scaled according to the widest curve; 95th and 5th percentile bounds are smoothed and shown in red, dotted lines. WMO standards (±5 %, dashed, and ±0.1 mm h−1 , dot-dashed) are shown as horizontal lines. The intersection of these lines indicates the required square disdrometer sampling area width to determine R according to WMO standards. Hydrometeor catch inefficiency is a large contributor to precipitation rate measurement error, especially in buckettype precipitation gauges (Pollock et al., 2018). Rasmussen et al. (2010) found that the Yankee hotplate had a catch efficiency of only 50 % with a wind speed of 5 m s−1 and 35 % with a wind speed of 8 m s−1 . The DEID underwent a series of calibration wind tunnel tests to determine the effect of wind on mass measurements. During these experiments, mass measurements remained approximately constant, revealing that catch inefficiency is not a contributor to the precipitation rate measurement (Singh et al., 2021). The high catch efficiency from the DEID was demonstrated during a storm that took place at Alta, UT, on 16 April 2020 between 00:00 and 16:00 UTC (Fig. 10 of Singh et al., 2021). The colocated weighing gauge was located inside of a wind fence, while the DEID was not. Despite wind speeds during the storm ranging from 4 to 13 m s−1 sustained with 8–19 m s−1 gusts, DEID precipitation measurements were within 6 % of the co-located weighing gauge. For this reason, precipitation rate as a function of catch efficiency is not explored in this work. Atmos. Meas. Tech., 14, 7681–7691, 2021 3 3.1 Monte Carlo simulations Size distribution generation For a range of collection areas A and time intervals 1t, a raindrop distribution is generated stochastically and compared with the assumed rate R. Initially, we adopt the Marshall–Palmer (Marshall and Palmer, 1948) drop size distribution n(D) = n0 e−3D , (4) where n0 = 8000 m−3 mm−1 and 3 = 4.1R −0.21 mm−1 . D ranges between 0 and Dmax in linear bins evenly spaced by 1D. We establish Dmax = 6 mm to account for the expected breakup of large raindrops (Villermaux and Bossa, 2009) and 1D = 0.25 mm for 50 bins. The value of 1D is arbitrary but was chosen to approximate the spatial measurement resolution of the prototype DEID. For an assumed value of R, the array of drops is stochastically generated according to P Eq. (4), where NMP = n(D)1D is the total number of drops generated from each bin. Each drop in the bin is assigned a diameter of Dmean = D + 1D/2. For each value of https://doi.org/10.5194/amt-14-7681-2021 K. N. Rees and T. J. Garrett: Disdrometer precision and first drop detection 7685 Figure 3. Comparison of R = 0.10, 1.00, and 10.00 mm h−1 for 1t = 10 s, 5 min, and 10 min. The applicable WMO standards for each rate (±5 %, dashed, and ±0.1 mm h−1 , dot-dashed) are shown as horizontal lines. 3.2 Dmean , the fall speed is b v = aDmean , (5) where the coefficient a and the exponent b are determined for the Stokes regime for Dmean ≤ 0.08 mm, the intermediate regime for 0.08 mm ≤ Dmean < 1.2 mm, and the turbulent regime for Dmean ≥ 1.2 mm following Lamb and Verlinde (2011). The maximum value of v is used to determine the sample volume of the generated Marshall–Palmer distribution of drops vmax A1t m3 . The calculated size distribution of drops incident on the collection area during sampling time 1t is then N(D)coll = n(D)Av1t, (6) with units of mm−1 . The total calculated number of drops Ncoll incident on the collection area is a summation of Eq. (6) over the range 0 to Dmax . https://doi.org/10.5194/amt-14-7681-2021 Calculated precipitation rate Ncoll drops are randomly sampled assuming the Marshall– Palmer distribution. From the drops that impact the collection area, the calculated precipitation rate Rcalc is (cf. Lane et al., 2009) 50 P π Rcalc = α 6 i=1 Ni Di3 A1t , (7) where α = 3.6 ×10−3 m2 s mm−2 h−1 and Ni is the number of drops with diameter Di , with i corresponding to bin number; 100 simulations of Rcalc are performed √ for each value of equivalent sampling area width W = A, each evenly spaced by 1 cm. The 95th and 5th percentile bounds of Rcalc are specified as the upper and lower bounds of sampling uncertainty (Rcalc /R − 1). Atmos. Meas. Tech., 14, 7681–7691, 2021 7686 K. N. Rees and T. J. Garrett: Disdrometer precision and first drop detection Figure 4. Generated size distributions (a, d, g), first drop simulation (b, e, h), and 100th drop simulation (c, f, i) for µ = 0, 1, and 2 (top, middle, and bottom). 3.3 First and hundredth drops To determine the sampling time required to detect the onset of precipitation, 100 simulations were performed for R = 0.01, 0.1, and 1 mm h−1 and for 100 evenly spaced width bins W between 1 and 20 cm. For each width bin, the number size distribution was calculated from n(D) for a 1 m3 volume directly above the collection area with height h = 1/A. A sample of drops is generated from Eq. (6). To ensure that the drop with the smallest size Dmean could feasibly fall from the top to the bottom of the sample volume, 1t = h/v is maximized using the fall speed of the minimum value of Dmean . In general, small drops contribute negligibly to calculations of precipitation rate (Smith et al., 2003), but due to their higher concentrations they may nonetheless be the first detected. Accordingly, the functional form of n(D) is adjusted from the simpler exponential form described by Eq. (4) to a gamma distribution n(D) = n0 D µ e−3D . (8) So that small particles with D < 1 mm are not overrepresented (Ulbrich and Atlas, 1984), the drop size distribution is modified by the shape parameter µ and generated according to Eq. (6). Each drop is assigned a random height 1z above the collection area within a distance h above the plate, and the time elapsed for the plate to detect a drop is Atmos. Meas. Tech., 14, 7681–7691, 2021 tp = 1z/v. The shortest of these times is the first drop detection time t1 . Following the collection approach taken by Marshall et al. (1947), reproduction of the Marshall–Palmer size distribution is assumed to require collection of 100 drops. The time elapsed for the calculated incidence of 100 drops is t100 . If fewer than 100 drops were obtained in Ncoll , a new sample of drops is obtained from Eq. (6) with an increased value of 1t. 4 4.1 Results Monte Carlo simulations The sampling uncertainty in the precipitation rate is illustrated in Fig. 2. Precipitation rates of R = 0.02, 0.20, and 2.00 mm h−1 were analyzed for a standard sampling time of 60 s. Collection areas smaller than approximately 6 cm × 6 cm meet WMO standards for R ≤ 0.50 mm h−1 , but a collection area of over 10 cm × 10 cm is required for R > 1 mm h−1 . To illustrate the relationship between accuracy and sampling time, Fig. 3 compares R = 0.10, 1.00, and 10.00 mm h−1 with sampling times of 10 s, 5 min, and 10 min. For heavy rain rates of 10 mm h−1 , for a time interval of 5 min, a disdrometer sampling area width of 4 cm yields measured rain rates with a precision of ±5 % error https://doi.org/10.5194/amt-14-7681-2021 K. N. Rees and T. J. Garrett: Disdrometer precision and first drop detection 7687 Figure 5. Recalculation of Rcalc (Eq. 7) from 1000 uniformly distributed, randomly sampled iterations of a 1 min DEID dataset from 8 March 2020 at 14:43 MST with rain rate RDEID = 4.2 mm h−1 (a). Rcalc was recalculated using uniformly distributed random segments of time (c) and hotplate sampling area width (d). As t → 60 and W → WDEID , Rcalc approaches RDEID . for 95 % of the measurements. The intersection between 95th and 5th percentile bounds and WMO accuracy criteria occurs in larger collection areas as R increases. First drop simulation results are shown in Fig. 4. Three simulations were performed using three values of µ, where µ = 0 represents the Marshall–Palmer exponential distribution and closely represents the distributions generated by the uncertainty simulations. Following Ulbrich and Atlas (1984), DEID measurements show values of µ between 1 and 2 best represent the distribution of drops that arrive on the hotplate (Figs. 5b and 6b). Monte Carlo calculations of the size distribution Ncoll are shown in Fig. 4. Note that the size distribution for N (D)coll does not converge to N (D)MP for long sampling times because smaller drops fall slowly. Rather, the distribution more closely resembles a gamma distribution (Ulbrich and Atlas, 1984). Nevertheless, the contribution of small drops to ground-based measurements of precipitation tends to be small as they are comparatively less massive. Also, precipitation particles form primarily from droplet collisions in the updrafts within clouds and so must attain the size that they fall sufficiently quickly to leave cloud base and fall to the ground where they can be sampled by https://doi.org/10.5194/amt-14-7681-2021 ground-based instruments (Garrett, 2019). For µ = 2, few of the smallest drops with D < 1 mm are incident on the collection area. A square collection sampling area width of 2 cm × 2 cm is sufficient to detect the onset of light precipitation with a rate of R = 0.01 mm h−1 within 1 min. 4.2 Application to DEID measurements During a field campaign that took place at the University of Utah between April 2019 and March 2020, the DEID recorded the mass and density of individual hydrometeors, along with the 1 min-averaged precipitation rate RDEID for six rain events and five snow events with data spanning 1185 total minutes. DEID particle mass distributions for two contrasting 1 min samples of convective moderate (RDEID = 4.2 mm h−1 ) and light (RDEID = 0.1 mm h−1 ) rain are shown in Figs. 5 and 6. These samples were selected from rain-only events with measured RDEID values closest to the values of R that were analyzed in Sect. 4.1. These disdrometer data are used here in place of an assumed size distribution to calculate Rcalc (Eq. 7) in 100 iterations, each taken from data randomly sampled over time interval segments of Atmos. Meas. Tech., 14, 7681–7691, 2021 7688 K. N. Rees and T. J. Garrett: Disdrometer precision and first drop detection Figure 6. Recalculation of Rcalc from 1000 iterations of a 1 min DEID dataset from 8 March 2020 at 15:14 MST with rain rate RDEID = 0.1 mm h−1 (a). Rcalc was calculated using uniformly distributed random segments of time (c) and hotplate sampling area width (d). As t → 60 and W → WDEID , Rcalc approaches RDEID . 1t = 10, 20, 30, 40, and 50 s (Figs. 5c and 6c) and plate area segments of W = 1, 2, 3, 4, and 5 cm (Figs. 5d and 6d). Segments of 1t include all particles within the DEID collection area, and segments in W encompass the entire 60 s time interval. In a three-dimensional space of plate cross-sectional area and sampling time, the associated number concentration of drops for each case is shown in Figs. 5a–b and 6a–b. The version of the DEID used in this study had a maximum collection area width of W = 5.8 cm. For moderate rain where R = 1 mm h−1 , the derived minimum required sampling area width to meet WMO requirements is 13 cm. That is, the size of plate used was insufficient for the measurement of rain this intense. For light rain, the statistical uncertainty bounds at 95 % confidence converge to within ±0.1 mm h−1 of the DEID 1 min measured rate for a plate collection area width of W ∼ 3 cm. This value is larger than the 2 cm suggested by the Monte Carlo calculation shown in Table 2. One possibility is that the raindrop interarrival time and spacing were not in fact Poissonian (Jameson and Kostinski, 2001b). In the event of clustering, a larger plate would be required to provide an accurate assessment of the average rain rate during a given time interval. Atmos. Meas. Tech., 14, 7681–7691, 2021 To assess whether the presence of non-Poissonian clustering is the case, two-point correlation functions η were calculated following Shaw et al. (2002). A value of unity indicates interarrival times that are Poissonian and values greater than unity the presence of non-random clustering. Based on the location of hydrometeor centroids as they arrive on the DEID plate, storm-averaged values of η were found to be equal to 1.01 for rain falling under light winds on 8 March 2020, between 13:38 and 15:49 MST, and equal to 1.10 for the period between 05:00 and 07:34 MST earlier that day when high winds were present. For contrast, the value of η was 1.55 for a snow event with large aggregate snowflakes that took place on 14 January 2020 at 12:43–14:06 MST. While more extensive analysis is required, the implication is that non-Poissonian clustering can occur. 5 Conclusions A Monte Carlo approach was used to determine the minimum required cross-sectional collection area for a disdrometer to measure a given precipitation rate with a WMO target https://doi.org/10.5194/amt-14-7681-2021 K. N. Rees and T. J. Garrett: Disdrometer precision and first drop detection precision at 95 % probability for a 1 min collection period. Intrinsic instrument uncertainties were not considered, only those associated with statistical sampling errors associated with the raindrop size distribution. Following these criteria, a square collection area of 6 cm × 6 cm is sufficient to detect the onset of light rain with R ≤ 0.50 mm h−1 . For R > 1 mm h−1 , a sample area of over 10 cm × 10 cm is required, although a smaller collection area may achieve the required accuracy by increasing the sampling time. For example, in 10 min, a 4 cm × 4 cm collection area can measure 10 mm h−1 precipitation rates to within the WMO required precision 95 % of the time. A collection area as small as 2 cm × 2 cm may detect the onset of light drizzle with R = 0.01 mm h−1 within 1 min, even in instances where small particles in the drop size distribution fall too slowly to intercept the collection area. Theoretical results obtained from Monte Carlo simulations were compared with observed field measurements from a new precipitation sensor, the Differential Emissivity Imaging Disdrometer, for both light and moderate rain. Randomly selected segments of decreasing sampling time and area from the DEID were used to recalculate the precipitation rate. The results suggest a larger plate may be required to meet a specified precision than those indicated by the Monte Carlo simulations that were performed. A possible explanation is the presence of non-Poissonian clustering that was revealed by two-point correlation function calculations, particularly during high wind and snow events. The results presented here have general implications for the sampling limitations of other widely used particle-by-particle disdrometers such as the PARSIVEL with a sample area of 48.6 cm2 or effective W of 7 cm (Battaglia et al., 2010) and the 2DVD (Kruger and Krajewski, 2002) with a W of 10 cm. Despite their sizable collection areas, like the Differential Emissivity Imaging Disdrometer, they may nonetheless fail to meet WMO standards if operated at a nominal 1 min sampling interval. NMP R Rcalc RDEID Tw Tp t1 t100 v vmax W z η µ Parameters Ahot cp Dmax Dmean fs H K Kd Leqv Lv WDEID α β Appendix A: Nomenclature Variables A Aw Aevap D h Imean m Ncoll Sampling area (m2 ) Cross-sectional area of a hydrometeor as it appears on the hotplate (m2 ) Total cross-sectional area of evaporated water on the hotplate (m2 ) Spherical diameter of a raindrop (mm) Height above hotplate with sampling area A required to create a 1 m3 sample volume (m) Mean pixel intensity of each hydrometeor Mass (kg) Number of drops simulated to fall on the hotplate during a given time interval (mm−1 ) https://doi.org/10.5194/amt-14-7681-2021 3 ρw 7689 Number of drops associated with the Marshall–Palmer distribution (mm−1 ) True precipitation rate (mm h−1 ) Calculated precipitation rate (mm h−1 ) Precipitation rate measured by the DEID (mm h−1 ) Water temperature (◦ C or K) Hotplate temperature (◦ C or K) Time to detect the first drop (s) Time to detect the 100th drop (s) Hydrometeor fall speed (m s−1 ) Maximum fall speed associated with the smallest hydrometeor in the sample drop size distribution (m s−1 ) √ Square sampling area width = A (cm) Randomly assigned raindrop height above hotplate (m) Two-point correlation function (Shaw et al., 2002) Shape parameter: controls number of small particles in size distribution Hotplate surface area (m2 ) Specific heat capacity of water at constant pressure = 4.28 × 103 J K−1 kg−1 Maximum generated drop diameter = 6 mm Mean diameter value in each bin (mm) Camera resolution (frame s−1 ) Hotplate thickness = 0.0508 m Thermal conductivity of Al = 205 W m−1 K−1 Experimentally derived constant = 1.54 ×10−3 kg s−1 K−1 m−2 Equivalent latent heat required for the conversion of the hydrometeor to gas Latent heat of vaporization of water = 2.26 ×106 J kg−1 Square sampling area width of the DEID (cm) Conversion factor = 3.6 × 10−3 m2 s mm−2 h−1 Conversion factor from m s−1 to 6 −1 −1 −1 mm h = 3.6 × 10 mm s m h Thermal emissivity Slope parameter = 4.1R −0.21 for the Marshall–Palmer distribution Density of liquid water = 1000 kg m−3 Atmos. Meas. Tech., 14, 7681–7691, 2021 7690 K. N. Rees and T. J. Garrett: Disdrometer precision and first drop detection Data availability. The dataset used in this work can be accessed at https://doi.org/10.7278/S50D-SPT1-FNHH (Rees et al., 2021). Author contributions. KNR led collection and analysis of the data. KNR and TJG contributed equally to the writing of the manuscript. Competing interests. The DEID intellectual property is protected through patent US20210172855A1 and is commercially available through Particle Flux Analytics, Inc. Karlie N. Rees and Timothy J. Garrett are co-authors on the DEID patent. Timothy J. Garrett is a co-owner of Particle Flux Analytics, Inc., which has a licence from the University of Utah to commercialize the DEID. Disclaimer. Publisher’s note: Copernicus Publications remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Acknowledgements. We are grateful to Dhiraj Singh, Eric Pardyjak, Spencer Donovan, and Allan Reaburn and colleagues at Particle Flux Analytics, Inc. for their contributions to the field program and the development of the DEID. We thank Darrel Baumgardner and the three anonymous reviewers for their constructive comments. Financial support. This research has been supported by the US Department of Energy Atmospheric System Research program (award no. DE-SC0016282) and the National Science Foundation (grant no. 1841870). Review statement. This paper was edited by Alexis Berne and reviewed by Darrel Baumgardner and four anonymous referees. References Andrey, J. and Yagar, S.: A temporal analysis of rainrelated crash risk, Accident Anal. Prev., 25, 465–472, https://doi.org/10.1016/0001-4575(93)90076-9, 1993. Andrey, J. C. and Mills, B. E.: Collisions, Casualties, and Costs: Weathering the Elements on Canadian Roads, Institute for Catastrophic Loss Reduction Canada, 2003. Bartholomew, M. J.: Laser Disdrometer Instrument Handbook, Tech. rep., Department of Energy Atmospheric Radiation Measurement Program, available at: https://www.arm.gov/ publications/tech_reports/handbooks/\\ldis_handbook.pdf (last access: 3 March 2020), 2014. Battaglia, A., Rustemeier, E., Tokay, A., Blahak, U., and Simmer, C.: PARSIVEL snow observations: A critical assessment, J. Atmos. Ocean. Tech., 27, 333–344, https://doi.org/10.1175/2009JTECHA1332.1, 2010. Bergel-Hayat, R., Debbarh, M., Antoniou, C., and Yannis, G.: Explaining the road accident risk: Weather effects, Accident Anal. Atmos. Meas. Tech., 14, 7681–7691, 2021 Prev., 60, 456–465, https://doi.org/10.1016/j.aap.2013.03.006, 2013. Brandes, E. A., Ikeda, K., Zhang, G., Schönhuber, M., and Rasmussen, R. M.: A Statistical and Physical Description of Hydrometeor Distributions in Colorado Snowstorms Using a Video Disdrometer, J. Appl. Meteorol. Clim., 46, 634–650, https://doi.org/10.1175/JAM2489.1, 2007. Brun, E., David, P., Sudul, M., and Brunot, G.: A numerical model to simulate snow-cover stratigraphy for operational avalanche forecasting, J. Glaciol., 38, 13–22, https://doi.org/10.3189/S0022143000009552, 1992. Campbell, J. F. and Langevin, A.: Operations management for urban snow removal and disposal, Transportation Res. A-Pol., 29, 359– 370, https://doi.org/10.1016/0965-8564(95)00002-6, 1995. Colle, B. A., Garvert, M. F., Wolfe, J. B., Mass, C. F., and Woods, C. P.: The 13–14 December 2001 IMPROVE-2 Event. Part III: Simulated Microphysical Budgets and Sensitivity Studies, J. Atmos. Sci., 62, 3535–3558, 2005. Estévez, J., Gavilán, P., and García-Marín, A. P.: Data validation procedures in agricultural meteorology – a prerequisite for their use, Adv. Sci. Res., 6, 141–146, https://doi.org/10.5194/asr-6141-2011, 2011. Fulton, R. A., Breidenbach, J. P., Seo, D.-J., Miller, D. A., and O’Bannon, T.: The WSR-88D Rainfall Algorithm, Weather Forecast., 13, 377–395, 1998. Garrett, T. J.: Analytical solutions for precipitation size distributions at steady-state, J. Atmos. Sci., 76, 1031–1037, https://doi.org/10.1175/JAS-D-18-0309.1, 2019. Gultepe, I.: Measurements of light rain, drizzle and heavy fog, in: Precipitation: Advances in measurement, estimation and prediction, Springer, 59–82, 2008. Horel, J., Splitt, M., Dunn, L., Pechmann, J., White, B., Ciliberti, C., Lazarus, S., Slemmer, J., Zaff, D., and Burks, J.: Mesowest: Cooperative Mesonets in the Western United States, B. Am. Meteorol. Soc., 83, 211–226, 2002. Jameson, A. R. and Kostinski, A. B.: Fluctuation Properties of Precipitation. Part V: Distribution of Rain Rates–Theory and Observations in Clustered Rain, J. Atmos. Sci., 56, 3920–3932, 1999. Jameson, A. R. and Kostinski, A. B.: Reconsideration of the physical and empirical origins of Z–R relations in radar meteorology, Q. J. Roy. Meteor. Soc., 127, 517–538, 2001a. Jameson, A. R. and Kostinski, A. B.: What is a Raindrop Size Distribution?, B. Am. Meteorol. Soc., 82, 1169–1178, 2001b. Jameson, A. R. and Kostinski, A. B.: Spurious power-law relations among rainfall and radar parameters, Q. J. Roy. Meteor. Soc., 128, 2045–2058, 2002. Joss, J. and Waldvogel, A.: Ein spektrograph für niederschlagstropfen mit automatischer auswertung, Pure Appl. Geophys., 68, 240–246, 1967. Joss, J. and Waldvogel, A.: Raindrop Size Distribution and Sampling Size Errors, J. Atmos. Sci., 26, 566–569, 1969. Kruger, A. and Krajewski, W. F.: Two-Dimensional Video Disdrometer: A Description, J. Atmos. Ocean. Tech., 19, 602–617, 2002. Kummerow, C., Simpson, J., Thiele, O., Barnes, W., Chang, A. T. C., Stocker, E., Adler, R. F., Hou, A., Kakar, R., Wentz, F., Ashcroft, P., Kozu, T., Hong, Y., Okamoto, K., Iguchi, T., Kuroiwa, H., Im, E., Haddad, Z., Huffman, G., Ferrier, B., Olson, W. S., Zipser, E., Smith, E. A., Wilheit, T. T., North, G., Krishnamurti, T., and Nakamura, K.: The Status of the Tropical https://doi.org/10.5194/amt-14-7681-2021 K. N. Rees and T. J. Garrett: Disdrometer precision and first drop detection Rainfall Measuring Mission (TRMM) after Two Years in Orbit, J. Appl. Meteorol., 39, 1965–1982, 2000. Lamb, D. and Verlinde, J.: Physics and Chemistry of Clouds, Cambridge University Press, 2011. Lane, J. E., Kasparis, T., Metzger, P. T., and Jones, W. L.: Spatial and Temporal Extrapolation of Disdrometer Size Distributions Based on a Lagrangian Trajectory Model of Falling Rain, Open Atmos. Sci. J., 3, 172–186, https://doi.org/10.2174/1874282300903010172, 2009. Liu, X., Gao, T., Hu, Y., and Shu, X.: Measuring hydrometeors using a precipitation microphysical characteristics sensor: Sampling effect of different bin sizes on drop size distribution parameters, Adv. Meteorol., 2018, 1–15, https://doi.org/10.1155/2018/9727345, 2018. Liu, X. C., Gao, T. C., and Liu, L.: Effect of sampling variation on error of rainfall variables measured by optical disdrometer, Atmos. Meas. Tech. Discuss., 5, 8895–8924, https://doi.org/10.5194/amtd-5-8895-2012, 2012. Marshall, J. S. and Palmer, W.: The distribution of raindrops with size, J. Meteorol., 5, 165–166, 1948. Marshall, J. S., Langille, R. C., and Palmer, W. M. K.: Measurement of Rainfall by Radar, J. Meteorol., 4, 186–192, 1947. Miller, P. A. and Barth, M. F.: Ingest, integration, quality control, and distribution of observations from state transportation departments using MADIS, in: 19th International Conference on Interactive Information and Processing Systems, 2003. Nadolski, V.: Automated surface observing system (ASOS) user’s guide, Tech. rep., available at: https://www.weather.gov/media/ asos/aum-toc.pdf (last access: 23 May 2020), 1998. Pollock, M. D., O’Donnell, G., Quinn, P., Dutton, M., Black, A., Wilkinson, M. E., Colli, M., Stagnaro, M., Lanza, L. G., Lewis, E., Kilsby, C. G., and O’Connell, P. E.: Quantifying and Mitigating Wind-Induced Undercatch in Rainfall Measurements, Water Resour. Res., 54, 3863–3875, https://doi.org/10.1029/2017WR022421, 2018. Pond Engineering: Model K63 Hotplate Total Precipitation Gauge Operation & Maintenance Manual, available at: http: //www.pondengineering.com/s/K63_man_Mar2020.pdf, last access: 7 July 2020. Rasmussen, R. M., Hallett, J., Purcell, R., Landolt, S. D., and Cole, J.: The Hotplate Precipitation Gauge, J. Atmos. Ocean. Tech., 28, 148–164, https://doi.org/10.1175/2010JTECHA1375.1, 2010. Rees, K. N., Singh, D. K., Pardyjak, E. R., and Garrett, T. J.: Mass and density of individual frozen hydrometeors and A differential emissivity imaging technique for measuring hydrometeors, The Hive: University of Utah Research Data Repository [data set], https://doi.org/10.7278/S50D-SPT1-FNHH, 2021. Shaw, R. A., Kostinski, A. B., and Lanterman, D. D.: Superexponential extinction of radiation in a negatively correlated random medium, J. Quant. Spectrosc. Ra., 75, 13–20, 2002 https://doi.org/10.5194/amt-14-7681-2021 7691 Singh, D. K., Donovan, S., Pardyjak, E. R., and Garrett, T. J.: A differential emissivity imaging technique for measuring hydrometeor mass and type, Atmos. Meas. Tech., 14, 6973–6990, https://doi.org/10.5194/amt-14-6973-2021, 2021. Smith, R. B., Jiang, Q., Fearon, M. G., Tabary, P., Dorninger, M., Doyle, J. D., and Benoit, R.: Orographic precipitation and air mass transformation: An Alpine example, Q. J. Roy. Meteor. Soc., 129, 433–454, https://doi.org/10.1256/qj.01.212, 2003. Theofilatos, A. and Yannis, G.: A review of the effect of traffic and weather characteristics on road safety, Accident Anal. Prev., 72, 244–256, https://doi.org/10.1016/j.aap.2014.06.017, 2014. Thurai, M., Petersen, W. A., Tokay, A., Schultz, C., and Gatlin, P.: Drop size distribution comparisons between Parsivel and 2-D video disdrometers, Adv. Geosci., 30, 3–9, https://doi.org/10.5194/adgeo-30-3-2011, 2011. Tokay, A., Petersen, W. A., Gatlin, P., and Wingo, M.: Comparison of Raindrop Size Distribution Measurements by Collocated Disdrometers, J. Atmos. Ocean. Tech., 30, 1672–1690, https://doi.org/10.1175/JTECH-D-12-00163.1, 2013. Ulbrich, C. W. and Atlas, D.: Assessment of the contribution of differential polarization to improved rainfall measurements, Radio Science, 19, 49–57, https://doi.org/10.1029/RS019i001p00049, 1984. Vaisala: Present Weather Detector PWD52 B211065EN-C, datasheet, available at: https://www.vaisala.com/sites/default/ files/documents/\\PWD52-Datasheet-B211065EN.pdf (last access: 3 March 2020), 2018a. Vaisala: MAWS201M Datasheet B210730EN-F, datasheet, available at: https://www.vaisala.com/sites/default/files/documents/ MAWS201-Datasheet-B211006EN-D.pdf (last access: 28 November 2021), 2018b. Vaisala: Forward Scatter FD70 Series B211744EN-C, datasheet, available at: https://www.vaisala.com/sites/default/files/ documents/\\FD70-Series-Datasheet-B211744EN.pdf (last access: 3 March 2020), 2019a. Vaisala: Present Weather and Visibility Sensors PWD10, PWD12, PWD20, and PWD22 B210385EN-F, datasheet, available at: https://www.vaisala.com/sites/default/files/documents/\\ PWD-Series-Datasheet-B210385EN.pdf (last access: 3 March 2020), 2019b. Villermaux, E. and Bossa, B.: Single-drop fragmentation determines size distribution of raindrops, Nature Phys., 5, 697–702, 2009. Wade, C. G.: A Multisensor Approach to Detecting Drizzle on ASOS, J. Atmos. Ocean. Tech., 20, 820–832, 2003. WMO: Guide to meteorological instruments and methods of observation (WMO-No. 8), World Meteorological Organization, available at: https://library.wmo.int/doc_num.php?explnum_id=3148 (last access: 28 November 2021), 2018. Yankee Environmental Systems, I.: TPS-3100 Total Precipitation Sensor, Installation and User Guide, Version 2.0, Rev M, 2011. Atmos. Meas. Tech., 14, 7681–7691, 2021
https://openalex.org/W4235154200	https://www.researchsquare.com/article/rs-119282/v1.pdf?c=1631881874000	English	null	Psychomotor Skills in Root Canal Treatment: Influence of Preclinical Training Time on Clinical Performance and Confidence Level of Undergraduate Dental Students	Research Square (Research Square)	2,020	cc-by	7,163	Psychomotor Skills in Root Canal Treatment: Influence of Preclinical Training Time on Clinical Performance and Confidence Level of Undergraduate Dental Students Clinical Dental Sciences Department, College of Dentistry, Princess Nourah Bint Abdulrahman University, Riyadh Rahaf Almohareb (  almohareb.r@hotmail.com ) Clinical Dental Sciences Department, College of Dentistry, Princess Nourah Bint Abdulrahman University, Riyadh Hanan Balto Department of Restorative Dental Sciences, College of Dentistry, King Saud University, Ri Research Article Keywords: Confidence, Endodontic, Preclinical, Technical Quality, Undergraduate Posted Date: December 16th, 2020 DOI: https://doi.org/10.21203/rs.3.rs-119282/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Page 1/13 Page 1/13 Abstract Background: Performing root canal treatment is complex and requires the development of psychomotor skills adapted to working without the advantage of vision. Students have allocated special importance to preclinical training in helping them acquire these skills. The aim of this prospective study was to investigate the influence of exposure to additional preclinical training on undergraduate students’ confidence level and clinical performance defined by technical quality and quantity of root canal treatment. Methods: Clinical root canal treatment performed by a cohort of fifth-year undergraduate dental students was followed after half of them attended an additional (elective) endodontic preclinical course the year before. Root canal treatment was radiographically evaluated according to root canal filling length, density and presence of procedural errors. Technical quality and quantity of root canal treatment performed by students who had attended the elective course (attendees) and non- attendees, were compared. All students were also invited to participate in a survey to rate their undergraduate endodontic training and confidence levels performing root canal treatment. Statistical analysis of data was performed using Person chi- square test, Fisher Freeman Halton exact test, and T-test. A p-value <0.05 was considered statistically significant. Results: No significant difference between the two groups in overall root canal treatment quality (p=0.619) was found. Although elective attendees performed significantly less procedural errors (p=0.004), non-attendees completed more root canal treatments (p=0.012). Despite, no significant difference in the reported level of confidence between the attendees and the non- attendees, significantly more elective attendees rated their undergraduate endodontic training as adequate (p=0.002). Conclusion: While there was no significant difference in overall technical quality of root canal treatment, nor confidence levels, between both groups, undergraduate students who attended additional preclinical training performed significantly fewer procedural errors. Furthermore, students who attended additional preclinical training were more satisfied with their undergraduate endodontic education. Background The dental pulp - a richly neuro vascularized connective tissue located in a complex canal system enclosed within the hard tissues of the tooth crown and root [1]- is subject to painful infection due to dental caries or trauma [2]. This cascades into the periapical tissues, resulting in periapical disease with possible systemic disease associations [3]. Success of treating of pulpal and periapical disease depends on performing non-surgical root canal (endodontic) treatment which aims to remove the entire infected tissue from the root canal system, shaping then sealing it with a three-dimensional filling, that promotes healing of the periapical tissue [4]. Performing root canal treatment is complex and requires the development of cognitive-perceptual ability integrated with fine motor skills otherwise known as psychomotor skills [5], especially since much of the procedure is performed deep within narrow root canals without the advantage of vision. The desired outcome of an undergraduate dental curriculum is for graduating students to achieve clinical competence in performing root canal treatment of uncomplicated anterior and posterior teeth [6]. More than any other subject, teaching endodontics has challenged those involved in delivering the required knowledge and skills [7]. Most students view this discipline as complex and difficult to master, which in turn reflects on how confident they feel when it comes to performing endodontic treatment, especially on molar teeth [8]. This has been echoed by a high frequency of inadequate root canal fillings and procedural errors [9–12]. Students have allocated special importance to preclinical training in helping them acquire the necessary skills [8]. Limited preclinical and clinical training time were cited as major roadblocks to attaining competency and achieving self-confidence in performing root canal treatment [7, 8, 13, 14]. Despite the expert guidelines specifying the scope of endodontic education [15], preclinical and clinical teaching methodologies on endodontic education, the minimum number of cases required to achieve Page 2/13 Page 2/13 competency remain vague [8, 16]. Little is known about the influence of preclinical training time on improving clinical performance of complex psychomotor tasks as that required in root canal treatment. In addition to how this would reflect on students’ confidence level. Methods This prospective cohort study involved all registered fifth-year (final) undergraduate students (n=46) at PNU, College of Dentistry, during the academic year 2019-2020. Half of these student (n=23) attended an elective endodontic preclinical course during the previous year. The study was exempt from ethical approval by the institutional review board at PNU (IRB#19-0165). The undergraduate dental curriculum at PNU, College of Dentistry is a five-year program, composed of a mixture of the block (modules) and stream (longitudinal) courses. Undergraduate endodontic teaching starts with a preclinical block course in the third year composed of a theoretical component and hands-on preclinical sessions that run for seven weeks, culminating in a total of ninety contact hours. All preclinical training takes place in a simulated setting, on mannequin heads, on which students are expected to complete root canal treatment for plastic and natural anterior and posterior teeth. Upon passing this preclinical course, students can commence performing non-surgical root canal treatment on patients under close supervision as part of stream comprehensive clinical courses. An elective advanced preclinical endodontic block course is also offered in the fourth year. This course runs for five weeks (a total of forty-five contact hours) and offers advanced theoretical knowledge along with simulated preclinical training sessions focused on molar teeth. Background The undergraduate curriculum at College of Dentistry, Princess Nourah Bint Abdulrahman University (PNU), Kingdom of Saudi Arabia which offered an extra elective endodontic preclinical course provided a unique opportunity to investigate the influence of exposure to additional preclinical training time on clinical performance, by comparing the technical quality and quantity of root canal treatment performed clinically by fifth-year undergraduate students, who attended and who did not attend the elective course. Their confidence level in performing root canal treatment was also compared. The alternative hypothesis was that students exposed to additional preclinical training would demonstrate higher confidence levels and improved clinical performance. Case selection All completed non-surgical root canal treatment cases performed by the fifth-year students during the academic year were evaluated. Students performed root canal treatment at the college of dentistry’s dental clinics under the supervision of endodontic specialists, with an average student to staff ratio of 1:7. An aseptic technique with rubber dam isolation was applied in all cases. Working length was established using electronic apex locator and periapical radiographs. Canal cleaning and shaping was done either using manual 0.02mm taper stainless-steel k-files (Medin, A.S. Czech Republic) with step-back technique or rotary instrumentation using nickel-titanium Protaper Universal files (Dentsply Maillefer, Ballaigues, Switzerland). Canals were irrigated with Sodium hypochlorite 2.25% for chemical disinfection, dried with paper points then filled with gutta- percha cones and AH plus sealer (Dentsply-Sirona, United States) using a lateral condensation technique. On completion, coronal restoration was performed, and a postoperative periapical radiograph was exposed using the paralleling technique. The academic dental software system, AxiUm (Exan, BC, Canada) - employed at the dental clinics - was used to specify all non- surgical root canal treatment cases completed by fifth-year students from the start of the academic year on the first of September 2019 until work at the dental clinics was abruptly terminated on the eighth of March 2020 due to the COVID 19 pandemic. Retreatment cases were excluded. During evaluation, records with poor quality or missing periapical radiographs were also excluded. The cases were divided into two groups: Group 1; (attendees), teeth treated by students who had attended both the preclinical endodontic block course in the third-year and the elective preclinical course in the fourth-year. Group 2; (non-attendees), teeth treated by students who had not attended the elective preclinical course. Page 3/13 Page 3/13 Assessment of clinical performance Root canal treatment quality and procedural errors Clinical performance of the students was assessed by evaluating the technical quality of the performed root canal treatment and the presence of procedural errors. Technical quality was determined based on criteria for adequate root canal filling: distance between the end of the root canal filling and the radiographic apex, filling density and the detection of procedural errors, which had been adopted by Barriesh-Nusaair et al. [10] and Balto et al. [9]. and are summarized in Table 1 and Table 2. The tooth was considered as one unit, scored according to the presence of errors, and/or defects in obturation length and density in any of its canals, for clinical failure of one root will eventually lead to failure of the tooth [9]. Table 1: Criteria for evaluation of technical quality of root canal treatment Criteria Definition Length of root canal filling Adequate End of root canal filling is either at or ≤ 2mm from radiographic apex. Inadequate Root canal filling is either short more than 2mm from the radiographic apex or extends beyond radiographic apex. Density of root canal filling Adequate No voids visible within the root canal filling or between the filling and the canal walls. Inadequate Voids are visible within the root canal filling or between the filling and the canal walls. Presence of procedural errors Present One or more procedural errors are detected. Non present No procedural errors detected. Table 2: Criteria for detection of procedural errors. Procedural Error Criteria of Diagnosis Ledge formation The obturation was at least 1 mm shorter than the working length and deviated from the original canal shape in teeth where root canal curvature occurred. Apical transportation In the apical third, the obturation was located on the outside curve of the canal. Apical perforation The apical termination of the filled canal was different from the original canal terminus or when the obturation extruded through the apical foramen. Root perforation Extrusion of canal obturation in any other area of a root except the furcation area and the inner wall of the root Strip perforation Extrusion of canal obturation in the lateral (inner) wall of the root canal Presence of fractured instrument A fractured instrument was detected inside a root canal or with its tip extending into the periapical area Furcation perforation Extrusion of filling material through the furcation area in multi-rooted teeth Table 2: Criteria for detection of procedural errors. Root canal treatment quality and procedural errors Procedural Error Criteria of Diagnosis Ledge formation The obturation was at least 1 mm shorter than the working length and deviated from the original canal shape in teeth where root canal curvature occurred. Apical transportation In the apical third, the obturation was located on the outside curve of the canal. Apical perforation The apical termination of the filled canal was different from the original canal terminus or when the obturation extruded through the apical foramen. Root perforation Extrusion of canal obturation in any other area of a root except the furcation area and the inner wall of the root Strip perforation Extrusion of canal obturation in the lateral (inner) wall of the root canal Presence of fractured instrument A fractured instrument was detected inside a root canal or with its tip extending into the periapical area Furcation perforation Extrusion of filling material through the furcation area in multi-rooted teeth Table 2: Criteria for detection of procedural errors. Page 4/13 Number of cases performed by each student was also recorded. Two endodontists with a minimum experience of three years, Page 4/13 Number of cases performed by each student was also recorded. Two endodontists with a minimum experience of three years, blinded to the treating students, evaluated independently, the technical quality of canal obturations, and presence of procedural errors by studying the preoperative, working length, and postoperative radiographs. Mesial and distal angulated radiographs were included for multi-rooted teeth. The periapical radiographic images were retrieved from the digital archives, viewed, and analyzed using Mipax (Microtek, Taiwan). All radiographs were examined at 1.5 magnification on the same 21-inch LCD monitor resolution (1920 × 1200 at 60 Hz) in a darkened room, and the same ambient conditions were sustained during all the radiographic evaluation. Each original digital image was manipulated by the investigator to enhance the contrast and brightness of the image to give the subjectively clearest image of the root canal and radiographic apex as recommended by Akdeniz and Soǧuon [17]. Prior to the actual study, intra- and inter-examiner reliability was determined by evaluating eighteen endodontically treated teeth randomly selected from AxiUm records, these were not included in the study. These cases were evaluated twice by the same examiners, four weeks apart. Inter- and intra- examiner agreement were measured by Cohen’s kappa (k). Undergraduate students’ confidence survey All students completing the five-year dental program in 2019-2020 were invited to participate in an online questionnaire survey at the beginning of their internship year in July 2020. Prior to completing the questionnaire, the students were made aware of the objectives of the survey and how the results will be used. They were informed that participation is voluntary, their answers will remain entirely anonymous, and they can withdraw at any time during or after completing the questionnaire. The questionnaire adapted from Davey et al. [14], and Murray and Chandler [22] contained thirteen multiple-choice format questions and three open-ended questions. The questionnaire indicated students’ attendance of the endodontic elective course, assessed their experiences, perceived competence performing root canal treatments, along with their self-rated levels of the confidence in carrying out various endodontic tasks and their views on their undergraduate endodontic training. Participants were asked to classify their perceived level of confidence over a five-point scale: very confident, confident, neutral, low confidence, or extremely low confidence. Statistical analysis Statistical analysis was performed using the Statistical Package for Social Sciences software (SPSS version 27.0 IBM Inc., Chicago; IL, USA), Descriptive data analysis was carried out and Person chi-square test, Fisher Freeman Halton exact test were conducted to analyze the categorical data: root canal filling quality, confidence level and satisfaction. Independent samples T- test was used to compare the number of teeth completed students in each group. A p-value <0.05 was considered statistically significant. Root canal treatment quality and procedural errors Values for inter- examiner agreement for obturation length, density, and procedural errors were k=0.702, k=0.667, and k=0.824 respectively, while intra-examiner reliability ranged from (k=0.77 -k=1). This indicated good to excellent agreement [18]. Therefore, as reported by previous studies [19–21], it seemed acceptable to randomly allocate the sample radiographs equally between both endodontists. Assessment of endodontic technical quality and procedural errors One hundred and ninety teeth were evaluated through the AxiUm software system. Thirty-one teeth were excluded either due to missing or unclear radiographs, or records revealing that the treatment was incomplete. Of the remaining one hundred and fifty- nine treated teeth, the majority (95.6%) were posterior teeth: molars (n=69) and premolars (n=83). Root canal fillings were regarded as adequate in 60.38% (n=96) of these cases. Procedural errors were recorded in only thirty teeth (18.8%), of which apical perforation and instrument separation were the most frequent: 50% and 26.6%, respectively. On assessing root canal treatment quality, 63.5% (n=40) of the teeth were treated by elective course attendees, and 58.33% (n=56) of non-attendee cases were regarded as adequate quality. Table 3 shows distribution of root canal treatment quality Page 5/13 Page 5/13 according to tooth type among students in both groups. Although significantly more teeth treated by attendees were of adequate filling length (p=0.015) and lacked procedural errors (p=0.004). There was no significant difference between the two groups in overall quality (p=0.619). The mean number of teeth treated by students who had attended the elective course (2.74) was significantly lower than that of non-attendees (4.17); (p=0.012). according to tooth type among students in both groups. Although significantly more teeth treated by attendees were of adequate filling length (p=0.015) and lacked procedural errors (p=0.004). There was no significant difference between the two groups in overall quality (p=0.619). The mean number of teeth treated by students who had attended the elective course (2.74) was significantly lower than that of non-attendees (4.17); (p=0.012). Table 3: Obturation quality of treated teeth according to tooth type among students who attended and did not attend the endodontic elective course. treated teeth according to tooth type among students who attended and did not attend the Table 3: Obturation quality of treated teeth according to tooth type among students who attended and did not attend the endodontic elective course. endodontic elective course. Assessment of endodontic technical quality and procedural errors Tooth type Molars N=69 Premolars N=83 Anteriors N=7 Attend Elective Yes N=27 No N=42 Yes N=35 No N=48 Yes N=1 No N=6 Technical Quality of Obturation N % N % N % N % N % N % Obturation length Adequate 26 96.29 25 59.52 33 94.28 43 89.58 1 100 4 66.66 Overfill 0 0 8 19.04 1 2.85 2 4.16 0 0 2 33.33 Short 1 3.70 6 14.28 1 2.85 1 2.08 0 0 0 0 Obturation density Adequate 15 55.55 24 57.14 27 77.14 39 81.25 0 0 6 100 Inadequate 12 44.44 15 35.71 8 22.85 8 16.66 1 100 0 0 Procedural errors No errors 24 88.88 24 57.14 33 94.28 41 85.41 1 100 5 83.33 Errors 3 11.11 18 42.85 2 5.71 7 14.58 0 0 1 16.66 Overall obturation quality Adequate 14 51.85 15 35.71 26 74.28 37 77.08 0 0 4 66.66 Inadequate 13 48.14 27 64.28 9 25.71 11 22.91 1 100 2 33.33 Undergraduate students’ confidence survey The questionnaire response rate was very high 97.8%. All students who had attended the elective course participated. When asked about their reason for attending the course, twelve students (52.1%) stated the desire to improve their hand skills and performance, while the remaining eleven students (47.9%) were motivated by their interest in endodontics. The questionnaire response rate was very high 97.8%. All students who had attended the elective course participated. When asked about their reason for attending the course, twelve students (52.1%) stated the desire to improve their hand skills and performance, while the remaining eleven students (47.9%) were motivated by their interest in endodontics. In regards to students’ confidence level in performing different stages of root canal treatment, there was no significant difference in the level of confidence between the attendees and the non-attendees (Table 4). Although more students who attended the elective course perceived themselves competent in treating multi-rooted teeth (Table 5), this was not statistically significant (p=0.069). Attending the elective course was also not associated with a significant increase in the perception of competence in treating single-rooted teeth (p=1.00). However, the number of treated premolars was significantly associated with a perception of competence in treating multi-rooted teeth (p= 0.043). Table 4: Self-evaluated level of confidence in performing different stages of root canal treatment among elective course attendees (A) and non-attendees (NA). Undergraduate students’ confidence survey Page 7/13 Page 7/13 Page 7/13 Perceived Competence in Root Canal Treatment Lacking Neutral Competent Tooth type Single- rooted Multi-rooted Single- rooted Multi-rooted Single-rooted Multi-rooted Attended elective Yes No Yes No Yes No Yes No Yes No Yes No % 0% 0% 8.7% 13.6% 4.3% 4.5% 13% 40.9% 95.6% 95.4% 78.3% 45.4% On asking students about their perceived quality of undergraduate education, elective course attendees were more likely to describe all aspects of their undergraduate endodontic training as adequate (Table 6). This proved significant concerning the overall undergraduate training and theory content (p=0.002), (p=0.006) respectively. Another element associated with having an adequate opinion of undergraduate endodontic training was the student’s perception of being competent in performing endodontic treatment on both single and multi-rooted teeth (p= 0.021) (p=0.038). Students’ perception regarding the quality of their undergraduate endodontic education. regarding the quality of their undergraduate endodontic education. Perceived quality of undergraduate education Attended elective endodontic course Yes (N=23) N (%) No (N=22) N (%) Training time Lacking 6 (26.1%) 10 (45.5%) Neutral 4 (17.4%) 4 (18.2%) Adequate 13 (56.5%) 8 (36.4%) Lectures Lacking 0 (0%) 5 (22.7%) Neutral 1(4.3%) 4 (18.2%) Adequate 22 (95.7%) 13 (59.1%) Lab Lacking 6 (26.1%) 6 (27.3%) Neutral 1 (4.3%) 4 (18.2%) Adequate 16 (69.6%) 12 (54.5%) Overall training Lacking 3(13%) 4 (18.2%) Neutral 0 (0%) 8 (36.4%) Adequate 20 (87%) 10 (45.4%) In regards to the open-ended questions targeting student opinion and feedback on their undergraduate endodontic training: only nineteen students (42%) completed these questions. Students clearly expressed the desire to increase clinical and preclinical endodontic training hours, with a special focus on the use of rotary instrumentation and managing endodontic procedural errors. In regards to the open-ended questions targeting student opinion and feedback on their undergraduate endodontic training: only nineteen students (42%) completed these questions. Students clearly expressed the desire to increase clinical and preclinical endodontic training hours, with a special focus on the use of rotary instrumentation and managing endodontic procedural errors. Undergraduate students’ confidence survey Page 6/13 Perceived level of confidence Very confident Confident Neutral Little confidence Very little confidence p- value A NA A NA A NA A NA A NA Endodontic diagnosis 60.8% 63.6% 34.7% 36.3% 4.5% 0% 4.3% 0% 0% 0% 1.00 Patient referral for complicated cases 39.1% 50% 43.4% 45.4% 17.3% 0% 4.3% 4.5% 0% 0% 0.166 Providing adequate anesthesia 39.1% 45.4% 34.7% 45.4% 21.7% 4.5% 0% 4.5% 0% 0% 0.413 Rubber dam placement 60.8% 63.6% 34.7% 31.8% 0% 4.5% 4.3% 0% 0% 0% 1.00 Access cavity preparation 30.4% 45.4% 43.4% 31.8% 17.3% 22.7% 0% 0% 0% 0% 0.627 Finding all canals in multi-rooted teeth 0% 4.5% 47.8% 36.3% 34.7% 22.7% 4.3% 36.3% 0% 0% 0.301 Determining working length 30.4% 27.2% 65.2% 54.5% 4.5% 18.1% 17.3% 0% 0% 0% 0.423 Cleaning and shaping root canal system 26% 50% 56.5% 39.1% 17.3% 9% 0% 0% 0% 0% 0.275 Selecting proper irrigant and irrigation technique 43.4% 45.4% 43.4% 45.4% 13% 9% 8.6% 0% 0% 0% 1.00 Placing an inter- appointment dressing 34.7% 54.5% 52.1% 22.7% 8.6% 13.6% 0% 9% 0% 0% 0.242 Inter-appointment flare- ups management 4.3% 18.1% 39.1% 30.4% 39.1% 27.2% 4.3% 22.7% 0% 0% 0.489 Root canal system obturation 13% 36.3% 73.9% 40.9% 13% 18.1% 17.3% 4.5% 0% 0% 0.094 Taking radiographs 52.1% 59% 34.7% 27.2% 13% 9% 4.3% 4.5% 0% 0% 0.870 Interpreting radiographs 47.8% 54.5% 43.4% 31.8% 8.6% 13.6% 0% 0% 0% 0% 0.693 Giving post-operative instructions 52.1% 72.7% 43.4% 13.6% 4.3% 13.6% 0% 0% 0% 0% 0.066 Assessing quality of a root filling post- operatively 60.8% 68.1% 34.7% 18.1% 4.3% 13.6% 0% 0% 0% 0% 0.299 Determining correct recall Period for patient 39.1% 50% 39.1% 31.8% 13% 13.6% 0% 4.5% 0% 0% 0.903 Performing non- surgical root canal retreatment 39.1% 36.3% 47.8% 40.9% 8.6% 13.6% 0% 4.5% 0% 4.5% 0.935 Table 5: Perceived competence in performing root canal treatment according to tooth type and elective course attendance. Table 5: Perceived competence in performing root canal treatment according to tooth type and elective course attendance. Discussion Overall, the frequency of procedural errors (18.8%) was fairly low which is in accordance with other studies [20, 21, 29, 30] . Adequate root canal treatment was seen in 60.3% of the cases performed by the undergraduate students, in spite of the fact that the majority of the treated teeth were posterior teeth (premolars and molars) which are usually associated with lower frequencies of adequate treatment when performed by undergraduate dental students (49.5% in premolars and 26.3% in molars) [31]. An explanation may have been that the students were constantly supervised by endodontists with a low student to instructor ratio [16, 21]. Another explanation may be the exclusion of root canal filling taper as an evaluation criterion of root canal filling quality in this study [25, 32]. Studies considering taper had reported lower frequencies of adequate root canal treatment [9, 10, 30]. Taper is a subjective matter that cannot be adequately reflected in a two-dimensional radiograph, moreover, it may be influenced by the canal’s original anatomy giving the impression of a large taper irrespective of actual instrumentation [33]. Accordingly, it was excluded as an evaluation criterion in this study [25, 32]. The mean number of teeth completed by non-attendees was significantly higher compared to attendees. One possible explanation is that non-attendees sought out more opportunities to practice through clinical experience. Other factors that could lend an explanation to this result is the fact that the academic year was cut short due to the COVID-19 pandemic, and that retreatment cases performed by the students were not accounted for in the study [34]. Although more attendees 78.3% perceived themselves competent in treating multi-rooted teeth compared to non-attendees 45.4%, survey results showed no significant difference between the two groups in confidence levels whether in performing a particular stage of endodontic treatment or in their self-perception of competence in treating single or multi-rooted teeth. These findings are in line with other studies exploring what was termed self-efficacy - a combination of competence and confidence- of students [32, 34], which found no difference between students who attended different theoretical and clinical modules. Confidence levels in this study were also in close proximity to those reported by similar studies despite the difficulty in drawing conclusions from other studies due to variations in methodologies [14, 22, 35, 36]. The difference between attendees and non-attendees lay in their satisfaction with the quality of their endodontic undergraduate education. Discussion The issue of time in education is a matter of concern as it incurs greater costs and effort [23]. The undergraduate students in this study were supervised and taught by the same instructors, in relatively equal clinical training conditions [24, 25] only half of them had undergone the extended preclinical training. This permitted investigating the effect of extending preclinical training Page 8/13 Page 8/13 Page 8/13 time on undergraduate students’ confidence levels and clinical performance of root canal treatment [24, 25]. Previous studies have examined the effect of certain modifications in preclinical endodontic teaching; for example, training on artificial instead of natural teeth and using the electronic apex locator, by evaluating the technical quality of root canal treatment performed by the undergraduate students in a similar manner [19, 26]. time on undergraduate students’ confidence levels and clinical performance of root canal treatment [24, 25]. Previous studies have examined the effect of certain modifications in preclinical endodontic teaching; for example, training on artificial instead of natural teeth and using the electronic apex locator, by evaluating the technical quality of root canal treatment performed by the undergraduate students in a similar manner [19, 26]. The present findings showed no significant difference between the teeth performed by students who had attended the elective course and those who had not, as far as the overall technical quality of root canal treatment is concerned. At first glance this suggests that preclinical training time did not influence the undergraduate students’ clinical performance of root canal treatment. This can be explained by what Chambers et al. [27] described as the “learning curve” relationship between practice and competence, where a certain point is reached beyond which extra practice no longer lends to the acquisition of skill. A study also looking into the acquisition of medical clinical skills through practice concluded that while the amount of practice time played a role in achieving an “initial mastery” of basic skills, progress beyond that was not achieved through repetition, but by performing tasks that specifically target the areas of performance deficiency [28]. However, students who did not attend the elective preclinical course performed significantly more procedural errors than those who attended. In fact, cases with procedural errors (instrument fracture, ledging, perforation) that required either referral to an endodontic specialist or board residents to complete the treatment or tooth extraction due to non-restorability were by non- attendee students. PNU PNU Princess Nourah Bint Abdulrahman University. Princess Nourah Bint Abdulrahman University. Discussion Case difficulty which has a major part to play in undergraduate root canal treatment performance [29], was not taken into consideration in this study. It was assumed that endodontists supervising the students assess the case and refer moderate to high difficulty cases to post-graduate students or endodontic specialists within the Dental College’s clinics. Although the overall technical quality of root canal treatment performed by undergraduate students and their confidence levels were not affected by their preclinical training time. Students who attended the elective course performed significantly fewer procedural errors and were more satisfied with their undergraduate endodontic education. In spite of students’ perspective regarding lack of training time, more focus should be placed on how preclinical endodontics is taught, how to facilitate deliberate practice and not how much, i.e., the quality not the quantity [37]. Further research is required on pinpointing the challenges of acquiring of psychomotor skills required in root canal treatment and introducing novel teaching strategies targeting these unique skills. Discussion When asked about their perception of the overall training quality of endodontic education, 87% (n = 20) of elective course attendees reported adequate training, in contrast to only 45.4% (n = 10) of non-attendees. Students attribute great importance to preclinical training in helping them acquire the necessary skills in root canal treatment [8], which would explain this significant difference in the level of satisfaction. Sukotjo et al. [37] found that shortening preclinical hours in the undergraduate dental curriculum affected student anxiety and stress levels especially those measured prior to entering the clinics. The replies to the open-ended questions at the end of the questionnaire echoed this desire for more Page 9/13 training time (clinical and preclinical), similar to what has been reported by previous studies when sounding out student perspectives on their undergraduate education [7, 8]. A recent study by Baaij et al. [32] concluded that undergraduate students’ feelings of competence and confidence were influenced by clinical experiences in endodontics, specifically, successful ones. In accordance with this, the findings of the present study show that students’ self-perception of competence was associated with the number of teeth they reportedly treated, in particular: premolars. A reason for that maybe that students were required to pass an endodontic competency assessment, which involved completing root canal treatment on a two-canal upper premolar. Certain limitations to this study should be kept in mind: a relatively small sample size. Students from only a single academic year were included, as the elective course was unavailable to students from the previous year, and the elective course became mandatory for subsequent fourth-year students. The attendees’ reasons for taking the course were mainly either a desire to increase skill or having an interest in endodontics. However, the reason why non-attendees did not choose to take the elective course was not explored. This could have shed more light on specific characteristics of that group. Students’ innate cognitive, perceptual and psychomotor abilities related to acquiring competence in psychomotor skills were also not investigated [38]. Studies have shown that these abilities may play a role during the initial stages of skill acquisition and their influence tends to diminish with increased training [38]. However, innate spatial abilities remained of influence regardless of practice, for tasks of an important spatial nature, which could very well apply to procedures in root canal treatment [39]. Conclusion Within the limitations of this study, no significant difference was found in overall quality of clinical root canal treatment performed by undergraduate students who attended extended preclinical training compared to those who did not, nor were their confidence levels affected. However, students who attended additional preclinical training performed significantly fewer procedural errors and were more satisfied with their undergraduate endodontic education. Ethics approval and consent to participate Page 10/13 This study was exempt from requiring ethical approval by the institutional review board at Princess Nourah Bint Abdulrahman University (IRB#19-0165). All methods were performed in accordance to the proposal submitted to the institutional review board Page 10/13 at Princess Nourah, to which the exemption was granted. All participants gave verbal informed consent to participate upon which the survey link was e-mailed to them. Author details 1Clinical Dental Sciences Department, College of Dentistry, Princess Nourah Bint Abdulrahman University, Riyadh, Saudi Arabia. 2Department of Restorative Dental Sciences, College of Dentistry, King Saud University, Riyadh, Saudi Arabia. Acknowledgement This research was funded by the Deanship of Scientific Research at Princess Nourah Bint Abdulrahman University through the Fast-track Research Funding Program. Authors’ contributions R.B. and R.A. collected the data, R.B. conducted the statistical analysis. All authors interpreted the results, drafted the manuscript and read and approved the final manuscript. Competing interests The authors declare that they have no competing interests. This project received no funding. Consent for publication Not applicable The datasets supporting the findings of this article are available from the corresponding author upon reasonable request. Competing interests References Determinants of individual differences during skill acquisition: Cognitive abilities and information processing. J Exp Psychol Gen. 1988;117:288–318. doi:10.1037/0096-3445.117.3.288. 11. Ackerman PL. Determinants of individual differences during skill acquisition: Cognitive abilities and information processing. J Exp Psychol Gen. 1988;117:288–318. doi:10.1037/0096-3445.117.3.288. 12. Al-Anesi MS, AlKhawlani MM, Alkheraif AA, Al-Basmi AA, Alhajj MN. An audit of root canal filling quality performed by undergraduate pre-clinical dental students, Yemen. BMC Med Educ. 2019;19:350. doi:10.1186/s12909-019-1798-1. 12. Al-Anesi MS, AlKhawlani MM, Alkheraif AA, Al-Basmi AA, Alhajj MN. An audit of root canal filling quality performed by undergraduate pre-clinical dental students, Yemen. BMC Med Educ. 2019;19:350. doi:10.1186/s12909-019-1798-1. 13. Luz LB, Grock CH, Oliveira VF, Bizarro L, Ardenghi TM, Ferreira MBC, et al. Self-reported confidence and anxiety over endodontic procedures in undergraduate students—Quantitative and qualitative study. Eur J Dent Educ. 2019;23:482–90. doi:10.1111/eje.12456. 13. Luz LB, Grock CH, Oliveira VF, Bizarro L, Ardenghi TM, Ferreira MBC, et al. Self-reported confidence and anxiety over endodontic procedures in undergraduate students—Quantitative and qualitative study. Eur J Dent Educ. 2019;23:482–90. doi:10.1111/eje.12456. 14. Davey J, Bryant ST, Dummer PMH. The confidence of undergraduate dental students when performing root canal treatment and their perception of the quality of endodontic education. Eur J Dent Educ. 2015;19:229–34. doi:10.1111/eje.12130. 14. Davey J, Bryant ST, Dummer PMH. The confidence of undergraduate dental students when performing root canal treatment and their perception of the quality of endodontic education. Eur J Dent Educ. 2015;19:229–34. doi:10.1111/eje.12130. 15. De Moor R, Hülsmann M, Kirkevang L-L, Tanalp J, Whitworth J. Undergraduate Curriculum Guidelines for Endodontology. Int Endod J. 2013;46:1105–14. doi:10.1111/iej.12186. 16. Al Raisi H, Dummer PMH, Vianna ME. How is Endodontics taught? A survey to evaluate undergraduate endodontic teaching in dental schools within the United Kingdom. Int Endod J. 2019;52:1077–85. doi:10.1111/iej.13089. 16. Al Raisi H, Dummer PMH, Vianna ME. How is Endodontics taught? A survey to evaluate undergraduate endodontic teaching in dental schools within the United Kingdom. Int Endod J. 2019;52:1077–85. doi:10.1111/iej.13089. 17. Akdeniz BG, Sogur E. An ex vivo comparison of conventional and digital radiography for perceived image quality of root fillings. Int Endod J. 2005;38:397–401. doi:10.1111/j.1365-2591.2005.00958.x. 17. Akdeniz BG, Sogur E. An ex vivo comparison of conventional and digital radiography for perceived image quality of root fillings. Int Endod J. 2005;38:397–401. doi:10.1111/j.1365-2591.2005.00958.x. 18. Valachovic RW, Douglass CW, Berkey CS, McNeil BJ, Chauncey HH. Examiner Reliability in Dental Radiography. J Dent Res. 1986;65:432–6. doi:10.1177/00220345860650031201. 19. References 1. Demarco FF, Conde MCM, Cavalcanti BN, Casagrande L, Sakai VT, Nör JE. Dental pulp tissue engineering. Braz Dent J. 2011;22:3–13. doi:10.1590/S0103-64402011000100001. 1. Demarco FF, Conde MCM, Cavalcanti BN, Casagrande L, Sakai VT, Nör JE. Dental pulp tissue engineering. Braz Dent J. 2011;22:3–13. doi:10.1590/S0103-64402011000100001. 2. Pak JG, White SN. Pain Prevalence and Severity before, during, and after Root Canal Treatment: A Systematic Review. J Endod. 2011;37:429–38. doi:10.1016/j.joen.2010.12.016. 2. Pak JG, White SN. Pain Prevalence and Severity before, during, and after Root Canal Treatment: A Systematic Review. J Endod. 2011;37:429–38. doi:10.1016/j.joen.2010.12.016. 3. Khalighinejad N, Aminoshariae MR, Aminoshariae A, Kulild JC, Mickel A, Fouad AF. Association between Systemic Diseases and Apical Periodontitis. J Endod. 2016;42:1427–34. doi:10.1016/j.joen.2016.07.007. 3. Khalighinejad N, Aminoshariae MR, Aminoshariae A, Kulild JC, Mickel A, Fouad AF. Association between Systemic Diseases and Apical Periodontitis. J Endod. 2016;42:1427–34. doi:10.1016/j.joen.2016.07.007. 4. Estrela C, Holland R, Estrela CR de A, Alencar AHG, Sousa-Neto MD, Pécora JD. Characterization of Successful Root Canal Treatment. Braz Dent J. 2014;25:3–11. doi:10.1590/0103-6440201302356. 4. Estrela C, Holland R, Estrela CR de A, Alencar AHG, Sousa-Neto MD, Pécora JD. Characterization of Successful Root Canal Treatment. Braz Dent J. 2014;25:3–11. doi:10.1590/0103-6440201302356. 5. El-Kishawi M. Improving students’ learning and performance in pre-clinical endodontics. University of Adelaide, School of Dentistry; 2016. http://hdl.handle.net/2440/100211. 6. Qualtrough AJE, Whitworth JM, Dummer PMH. Preclinical endodontology: an international comparison. Int Endod J. 1999;32:406–14. doi:10.1046/j.1365-2591.1999.00253.x. 6. Qualtrough AJE, Whitworth JM, Dummer PMH. Preclinical endodontology: an international comparison. Int Endod J. 1999;32:406–14. doi:10.1046/j.1365-2591.1999.00253.x. 7. Qualtrough AJE. Undergraduate endodontic education: what are the challenges? Br Dent J. 2014;216:361–4. doi:10.1038/sj.bdj.2014.227. 7. Qualtrough AJE. Undergraduate endodontic education: what are the challenges? Br Dent J. 2014;216:361–4. doi:10.1038/sj.bdj.2014.227. Page 11/13 8. Seijo MOS, Ferreira EF, Ribeiro Sobrinho AP, Paiva SM, Martins RC. Learning Experience in Endodontics: Brazilian Students’ Perceptions. J Dent Educ. 2013;77:648–55. doi:10.1002/j.0022-0337.2013.77.5.tb05515.x. 9. Balto H, Al Khalifah S, Al Mugairin S, Al Deeb M, Al-Madi E. Technical quality of root fillings performed by undergraduate students in Saudi Arabia. Int Endod J. 2010;43:292–300. doi:10.1111/j.1365-2591.2009.01679.x. 10. Barrieshi-Nusair K., Al-Omari M., Al-Hiyasat A. Radiographic technical quality of root canal treatment performed by dental students at the Dental Teaching Center in Jordan. J Dent. 2004;32:301–7. doi:10.1016/j.jdent.2004.01.002. 10. Barrieshi-Nusair K., Al-Omari M., Al-Hiyasat A. Radiographic technical quality of root canal treatment performed by dental students at the Dental Teaching Center in Jordan. J Dent. 2004;32:301–7. doi:10.1016/j.jdent.2004.01.002. 11. Ackerman PL. References Tchorz JP, Brandl M, Ganter PA, Karygianni L, Polydorou O, Vach K, et al. Pre-clinical endodontic training with artificial instead of extracted human teeth: does the type of exercise have an influence on clinical endodontic outcomes? Int Endod J. 2015;48:888–93. doi:10.1111/iej.12385. 20. Alsulaimani RS, Al-Manei KK, A Alsubait S, AlAqeely RS, A M Al-Shehri S, M Al-Madi E. Effects of Clinical Training and Case Difficulty on the Radiographic Quality of Root Canal Fillings Performed by Dental Students in Saudi Arabia. Iran Endod J. 2015;10:268–73. doi: 10.7508/iej.2015.04.012. 21. Zambon da Silva P, Carlos Ribeiro F, Machado Barroso Xavier J, Pratte-Santos R, Demuner C. Radiographic evaluation of root canal treatment performed by undergraduate students, part I; iatrogenic errors. Iran Endod J. 2018;13:30–6. doi: 10.22037/iej.v13i1.16800. 22. Murray CM, Chandler NP. Undergraduate endodontic teaching in New Zealand: Students’ experience, perceptions and self- confidence levels. Aust Endod J. 2014;40:116–22. doi:10.1111/aej.12084. 22. Murray CM, Chandler NP. Undergraduate endodontic teaching in New Zealand: Students’ experience, perceptions and self- confidence levels. Aust Endod J. 2014;40:116–22. doi:10.1111/aej.12084. 23. Jones RF, Korn D. On the cost of educating a medical student. Acad Med. 1997;72:200–10. doi:10.1097/00001888- 199703000-00015. 23. Jones RF, Korn D. On the cost of educating a medical student. Acad Med. 1997;72:200–10. doi:10.1097/00001888- 199703000-00015. 24. Ng Y-L, Mann V, Rahbaran S, Lewsey J, Gulabivala K. Outcome of primary root canal treatment: systematic review of the literature – Part 2. Influence of clinical factors. Int Endod J. 2007;41:6–31. doi:10.1111/j.1365-2591.2007.01323.x. 24. Ng Y-L, Mann V, Rahbaran S, Lewsey J, Gulabivala K. Outcome of primary root canal treatment: systematic review of the literature – Part 2. Influence of clinical factors. Int Endod J. 2007;41:6–31. doi:10.1111/j.1365-2591.2007.01323.x. 25. Burke Fm, Lynch Cd, Ní Ríordáin R, Hannigan A. Technical quality of root canal fillings performed in a dental school and the associated retention of root-filled teeth: a clinical follow-up study over a 5-year period. J Oral Rehabil. 2009;36:508–15. doi:10.1111/j.1365-2842.2009.01966.x. 25. Burke Fm, Lynch Cd, Ní Ríordáin R, Hannigan A. Technical quality of root canal fillings performed in a dental school and the associated retention of root-filled teeth: a clinical follow-up study over a 5-year period. J Oral Rehabil. 2009;36:508–15. doi:10.1111/j.1365-2842.2009.01966.x. 26. Tchorz JP, Ganter PA, Woelber JP, Stampf S, Hellwig E, Altenburger MJ. Evaluation of an improved endodontic teaching model: do preclinical exercises have an influence on the technical quality of root canal treatments? Int Endod J. 2014;47:410–5. doi:10.1111/iej.12162. Page 12/13 Page 12/13 27. Chambers D. References Learning Curves: What Do Dental Students Learn from Repeated Practice of Clinical Procedures? J Dent Educ. 2012;76:291–302. doi:10.1002/j.0022-0337.2012.76.3.tb05258.x. 28. Duvivier RJ, van Dalen J, Muijtjens AM, Moulaert VR, van der Vleuten CP, Scherpbier AJ. The role of deliberate practice in the acquisition of clinical skills. BMC Med Educ. 2011;11:101. doi:10.1186/1472-6920-11-101. 29. Haug SR, Solfjeld AF, Ranheim LE, Bårdsen A. Impact of Case Difficulty on Endodontic Mishaps in an Undergraduate Student Clinic. J Endod. 2018;44:1088–95. doi:10.1016/j.joen.2018.03.012. 30. AlRahabi MK. Evaluation of complications of root canal treatment performed by undergraduate dental students. Libyan J Med. 2017;12. doi:10.1080/19932820.2017.1345582. 31. Ribeiro DM, Réus JC, Felippe WT, Pacheco-Pereira C, Dutra KL, Santos JN, et al. Technical quality of root canal treatment performed by undergraduate students using hand instrumentation: a meta-analysis. Int Endod J. 2018;51:269–83. doi:10.1111/iej.12853. 32. Baaij A, Özok AR. Method of teaching undergraduate students to perform root canal treatment: It’s influence on the quality of root fillings. Eur J Dent Educ. 2018;22:e221–7. doi:10.1111/eje.12275. 33. Vantorre T, Bécavin T, Deveaux E, Marchandise P, Chai F, Robberecht L. Are the evaluation criteria used in preclinical endodontic training courses relevant? A preliminary study. Aust Endod J. 2020;:aej.12417. doi:10.1111/aej.12417. 34. Baaij A, Özok AR, Væth M, Musaeus P, Kirkevang L -L. Self‐efficacy of undergraduate dental students in Endodontics within Aarhus and Amsterdam. Int Endod J. 2020;53:276–84. doi:10.1111/iej.13218. 35. Mirza MB. Difficulties Encountered during Transition from Preclinical to Clinical Endodontics among Salman bin Abdul Aziz University Dental Students. J Int oral Heal JIOH. 2015;7 Suppl 1:22–7. http://www.ncbi.nlm.nih.gov/pubmed/26225100%0Ahttp://www.pubmedcentral.nih.gov/articlerender.fcgi? artid=PMC4516078. 36. Alqisi AY, Alsalhi IYA, Hussain W, Alwadei A, Alsafi ZA, Alshahrani EA. Confidence Levels of Intern Doctors during Root Canal Treatment Procedures in Saudi Arabia. Egypt J Hosp Med. 2018;71:3148–52. doi: 10.12816/0046617. 36. Alqisi AY, Alsalhi IYA, Hussain W, Alwadei A, Alsafi ZA, Alshahrani EA. Confidence Levels of Intern Doctors during Root Canal Treatment Procedures in Saudi Arabia. Egypt J Hosp Med. 2018;71:3148–52. doi: 10.12816/0046617. 37. Sukotjo C, Thammasitboon K, Howell H, Karimbux N. The Impact of Targeted Shortened Preclinical Exercises on Student Perceptions and Outcomes. J Dent Educ. 2007;71:1070–9. doi:10.1002/j.0022-0337.2007.71.8.tb04373.x. 37. Sukotjo C, Thammasitboon K, Howell H, Karimbux N. The Impact of Targeted Shortened Preclinical Exercises on Student Perceptions and Outcomes. J Dent Educ. 2007;71:1070–9. doi:10.1002/j.0022-0337.2007.71.8.tb04373.x. 38. Suksudaj N, Townsend GC, Kaidonis J, Lekkas D, Winning TA. Acquiring psychomotor skills in operative dentistry: do innate ability and motivation matter? Eur J Dent Educ. 2012;16:e187–94. doi:10.1111/j.1600-0579.2011.00696.x. 38. References Suksudaj N, Townsend GC, Kaidonis J, Lekkas D, Winning TA. Acquiring psychomotor skills in operative dentistry: do innate ability and motivation matter? Eur J Dent Educ. 2012;16:e187–94. doi:10.1111/j.1600-0579.2011.00696.x. 39. Keehner M, Lippa Y, Montello DR, Tendick F, Hegarty M. Learning a spatial skill for surgery: how the contributions of abilities change with practice. Appl Cogn Psychol. 2006;20:487–503. doi:10.1002/acp.1198. 39. Keehner M, Lippa Y, Montello DR, Tendick F, Hegarty M. Learning a spatial skill for surgery: how the contributions of abilities change with practice. Appl Cogn Psychol. 2006;20:487–503. doi:10.1002/acp.1198. Page 13/13
https://openalex.org/W4206999677	https://link.springer.com/content/pdf/10.1007/JHEP01(2022)109.pdf	English	null	Q-monopole-ball: a topological and nontopological soliton	The Journal of high energy physics/The journal of high energy physics	2,022	cc-by	16,220	Published for SISSA by Springer Received: December 7, 2021 Accepted: January 10, 2022 Published: January 20, 2022 Received: December 7, 2021 Accepted: January 10, 2022 Published: January 20, 2022 Open Access, c⃝The Authors. Article funded by SCOAP3. Q-monopole-ball: a topological and nontopological soliton JHEP01(2022)109 Yang Bai,a Sida Lub and Nicholas Orlofskyc https://doi.org/10.1007/JHEP01(2022)109 Contents 1 Introduction 1 2 QMBs with unit magnetic charge (q = 2) 3 2.1 Classical equations of motion 5 2.2 Large Q-ball charge 7 2.3 Small Q-ball charge 9 2.4 Numerical results 11 3 Multiply magnetically charged QMBs (q > 2) 14 4 Early-universe formation of QMBs 17 5 Implications for phenomenology 20 6 Conclusions 22 Contents 1 Introduction 1 2 QMBs with unit magnetic charge (q = 2) 3 2.1 Classical equations of motion 5 2.2 Large Q-ball charge 7 2.3 Small Q-ball charge 9 2.4 Numerical results 11 3 Multiply magnetically charged QMBs (q > 2) 14 4 Early-universe formation of QMBs 17 5 Implications for phenomenology 20 6 Conclusions 22 Contents 1 Introduction 1 2 QMBs with unit magnetic charge (q = 2) 3 2.1 Classical equations of motion 5 2.2 Large Q-ball charge 7 2.3 Small Q-ball charge 9 2.4 Numerical results 11 3 Multiply magnetically charged QMBs (q > 2) 14 4 Early-universe formation of QMBs 17 5 Implications for phenomenology 20 6 Conclusions 22 1 3 5 7 9 11 14 17 20 22 JHEP01(2022)109 6 Conclusions Yang Bai,a Sida Lub and Nicholas Orlofskyc Yang Bai,a Sida Lub and Nicholas Orlofskyc aDepartment of Physics, University of Wisconsin-Madison, Madison, WI 53706, U.S.A. bSchool of Physics and Astronomy, Tel Aviv University, Tel-Aviv 69978, Israel cDepartment of Physics, Carleton University, Ottawa, ON K1S 5B6, Canada E-mail: yangbai@physics.wisc.edu, sidalu@mail.tau.ac.il, norlofsky@physics.carleton.ca Yang Bai,a Sida Lub and Nicholas Orlofskyc aDepartment of Physics, University of Wisconsin-Madison, Madison, WI 53706, U.S.A. bSchool of Physics and Astronomy, Tel Aviv University, Tel-Aviv 69978, Israel cDepartment of Physics, Carleton University, Ottawa, ON K1S 5B6, Canada E-mail: yangbai@physics.wisc.edu, sidalu@mail.tau.ac.il, norlofsky@physics.carleton.ca Abstract: Magnetic monopoles and Q-balls are examples of topological and nontopolog- ical solitons, respectively. A new soliton state with both topological and nontopological charges is shown to also exist, given a monopole sector with a portal coupling to an addi- tional scalar ﬁeld S with a global U(1) symmetry. This new state, the Q-monopole-ball, is more stable than an isolated Q-ball made of only S particles, and it could be stable against ﬁssioning into monopoles and free S particles. Stable Q-monopole-balls can contain large magnetic charges, providing a novel nongravitational mechanism for binding like-charged monopoles together. They could be produced from a phase transition in the early universe and account for all dark matter. Keywords: Beyond Standard Model, Solitons Monopoles and Instantons Keywords: Beyond Standard Model, Solitons Monopoles and Instantons ArXiv ePrint: 2111.10360 Open Access, c⃝The Authors. Article funded by SCOAP3. Open Access, c⃝The Authors. Article funded by SCOAP3. 1 Introduction Quantum ﬁeld theory oﬀers a rich variety of possibilities for still-undiscovered soliton states beyond the Standard Model. Many of these states can be cosmologically long-lived relics if formed in the early universe. Among these are nontopological solitons such as Q-balls. For a scalar ﬁeld with nonlinear interactions and charged under a global symmetry, it has long been pointed out [1–3] that there may exist a classical solution where the scalar ﬁeld condenses and forms macroscopic states with large global charge (see [4, 5] for reviews). Such conﬁgurations are energetically preferred against decaying into free particles, and hence their stabilities are ensured by global charge conservation. Examples of such con- ﬁgurations exists, for example, in the Minimal Supersymmetric Standard Model (MSSM) where the squarks or sleptons have nonlinear interactions [6] and carry baryon or lepton number. Also, in a Higgs-portal dark matter scenario where the dark sector contains a complex scalar ﬁeld, dark matter may live as solitons in which the electroweak symmetry is restored [7]. Another possible solitonic state is the magnetic monopole, which carries topological charge. After being proposed by Dirac [8] to explain the observed quantization of electric charge, monopoles have drawn the attentions of physicists from both theoretical and ex- perimental directions. On the theory side, the simplest model of monopoles was realized by ’t Hooft and Polyakov [9, 10] in a spontaneously broken SU(2) gauge theory, and has been applied to the Grand Uniﬁcation Theory [11]. In electroweak theory, a diﬀerent monopole topology has also been pointed out [12]. On the experimental side, monopoles have been searched for in quantum interference devices [13] through detecting the quantized jump of – 1 – magnetic ﬂux, in detectors or materials that record the tracks of monopoles [14–16], and at the Large Hadron Collider [17, 18] through direct production from ion collisions. For a recent review, see [19]. In this work we propose a novel soliton state, the Q-monopole-ball (QMB), which in- herits the features of both the two aforementioned examples. In a theory with an unbroken global symmetry and a spontaneously broken gauge symmetry that admits monopoles, it is possible to obtain a macroscopic state that is charged both topologically and nontopo- logically. This is achieved by introducing a quartic scalar interaction between the scalar ﬁeld of the global symmetry and the scalar ﬁeld breaking the gauge symmetry. 1 Introduction From a bottom-up perspective, the model is fairly minimal, invoking only a gauged scalar to provide for the existence of a monopole (which is well motivated by charge quantization) and one new gauge-singlet scalar, then including a renormalizable scalar portal coupling between these two scalars, which should exist in the absence of any symmetry forbidding it. From a top-down perspective, such a setup can be naturally realized by a symmetry- breaking pattern of G/H, where G contains both gauge symmetries and a global U(1) symmetry, and H contains the unbroken U(1)em electromagnetic gauge symmetry and the global U(1) symmetry. Note, this setup diﬀers from “gauged Q-balls” [20–23], where the global nontopological charge is gauged and anyways only carries the electric-charge analog of its gauge group. JHEP01(2022)109 Depending on the size of its global charge Q, a QMB may behave mainly like either a Q-ball or a monopole, which can be interpreted as one or more monopoles bound inside a large Q-ball or a small Q-ball bound inside a monopole, respectively. The existence and stability of such bound states can be understood qualitatively. Both an isolated Q-ball and a monopole tend to have the spontaneously broken gauge symmetry restored in their interiors, which costs vacuum energy. Having the Q-ball and monopole “overlap” to form a bound state reduces the volume of space in the false vacuum, reducing the overall energy of the system. Additionally, for a monopole bound inside a larger-radius Q-ball, the magnetic charge is free to “spread out” over a larger volume, reducing the energy contained in the magnetic ﬁeld. For monopoles with magnetic charge q = 2, analytic expressions for the spherically symmetric ﬁeld conﬁgurations only exist in the Bogomol’nyi-Prasad-Sommerﬁeld (BPS) limit [24, 25], when the self interaction of the gauged scalar ﬁeld vanishes. Away from this limit, spherical solutions can be readily obtained numerically. In this work, we apply a similar numerical treatment to QMBs, which are also spherically symmetric when q = 2. We show that QMBs with q = 2 are always more stable than Q-balls of the same global charge Q. Additionally, smaller-Q solutions exist for QMBs than for isolated Q-balls. It is known that a monopole can have multiple magnetic charges, i.e. q > 2, although such monopole conﬁgurations cannot be spherically symmetric [26]. 1 Introduction It has been proposed that a large number of BPS monopoles may cluster into a spherical bag structure [27], where the monopoles reside on a quasi-regular lattice on the spherical surface [28] (see also [29–34]). Such states exist only in the BPS limit, where repulsive magnetic forces and attractive Yukawa forces (mediated by the massless symmetry-breaking scalar ﬁeld) be- tween monopoles cancel. However, gravitational forces could stabilize bound states of – 2 – non-BPS monopoles, allowing for gravitational magnetic bag and magnetic black hole states [35–37]. Nambu’s dumbbell construction [38] and cosmic necklaces [39–41] oﬀer other bound state alternatives where non-BPS monopoles can be bound by string(s), al- though their geometry diﬀers from the compact, approximately spherical conﬁguration of interest here. A two-monopole (q = 4) nonspherical conﬁguration bound by a Skyrmion ﬁeld was considered in [42]. Our work proposes a novel nongravitational way to stabilize large-magnetic-charge con- ﬁgurations away from the BPS limit by having them bound in QMBs, without attempting to decipher the nonspherical ﬁeld conﬁgurations of QMBs with q > 2. As long as the magnetic ﬁeld energy is not too large, it can be energetically favorable for many monopoles to be bound in a larger Q-ball, according to the energetic arguments presented above. The typical size of the magnetic charge in a QMB, on the other hand, relies on the details of the QMB formation and evolution, which we brieﬂy discuss when examining the relic abundance of QMBs. JHEP01(2022)109 The organization of this paper is as follows. We ﬁrst focus on QMBs with unit magnetic charge q = 2 in section 2. We work out the equations of motion for a QMB and the expressions for its mass M and global charge Q, discuss its properties in both the large and small Q limit, and present the computed parameter space where QMBs are stable. Then, in section 3, we discuss QMBs with q > 2. We provide an argument why it could be energetically preferred for a QMB to have more than unit magnetic charge, and provide the corresponding expressions for QMBs’ masses and global charges at q > 2. Based on this, we presented the parameter region where the QMBs are cosmologically stable. In section 4 we brieﬂy discuss the formation of QMBs from cosmic phase transitions and the allowed parameter space for QMBs to make up all the cosmic dark matter. 1 Introduction Finally in section 5 we discuss the phenomenological consequences and the detection of QMBs, before concluding in section 6. 2 QMBs with unit magnetic charge (q = 2) A QMB is charged under both the U(1)em electromagnetic gauge symmetry and a U(1)S global symmetry. One could treat it as a composite state that is made of an isolated Q-ball and an isolated magnetic monopole. In our study, we use a simple toy model containing a magnetic monopole, but the qualitative results can be applied to a realistic model containing a magnetic monopole. Consider a theory with a complex gauge-singlet scalar S and a gauged SU(2) triplet scalar φa with a = 1, 2, 3 the gauge index. The most general renormalizable Lagrangian invariant under the SU(2) gauge symmetry and U(1)S global symmetry is L = \|∂µS\|2 + 1 2(Dµφa)2 −1 4F a µνF aµν −V (S, φ) , (2.1) (S, φ) = 1 8λφ(φaφa −v2)2 + 1 2λφS\|S\|2(φaφa) + λS\|S\|4 + m2 S,0\|S\|2 , (2.2) (2.1) (2.2) where Dµφa = ∂µφa + eϵabcAb µφc, F a µν = ∂µAa ν −∂νAa µ + eϵabcAb µAc ν, Aa µ is the SU(2) gauge ﬁeld, and e is the SU(2) gauge coupling. Here, we take all model parameters – 3 – λφ, λφS, λS, m2 S,0, v2 ≥0, so that the vacuum expectation values (VEVs) for the scalar ﬁelds are ⟨φaφa⟩= v2 and ⟨\|S\|2⟩= 0.1 This theory admits several stable nonvacuum ﬁeld conﬁgurations. One, the ’t Hooft- Polyakov monopole [9, 10], is a topological ﬁeld conﬁguration resulting from the residual U(1)em symmetry from the spontaneous SU(2) breaking (whose homotopy group satisﬁes π2[G/H] = π2[SU(2)/U(1)] = Z). It involves only the φa and Aa µ ﬁelds, with \|S\|2 = 0 being irrelevant. Indeed, λφS could be zero, or equivalently S need not be present in the theory for this solution. Alternatively, for diﬀerent sign choices in the Lagrangian parameters, the S VEV could be modiﬁed inside the monopole [45]. Another conﬁguration, the nontopological soliton [2] or Q-ball [3], allows for a large global “charge” of S owing to the global U(1)S symmetry in the theory and a nontrivial interplay with the φa ﬁeld [7]. This soliton solution has no interplay with the gauge ﬁeld Aa µ and exists even in the limit e = 0. JHEP01(2022)109 Both of the prior two solutions have been explored extensively in the literature. 1A somewhat similar theory containing a gauged spontaneously broken U(1) and a global U(1) was considered in [43, 44]. That theory results in cosmic strings rather than monopoles. In those works, the equivalent Lagrangian parameters are not all positive, so the global U(1) symmetry is spontaneously broken inside the string. One could also explore the similar parameter space in the Lagrangian considered here. 2 QMBs with unit magnetic charge (q = 2) Here, we point out another novel solution to the classical equations of motion of ﬁelds in (2.1) and a new object: the Q-Monopole-Ball, which is charged under both the U(1)em gauge and U(1)S global symmetry. By allowing all three ﬁelds to interplay with one another, it is possible to form a mixed state containing both topological and nontopological charges labeled by q and Q, respectively. In certain limits, this mixed state could be approximately interpreted as a monopole trapped inside a Q-ball or several S quanta trapped inside a monopole. We demonstrate this solution and its various limits in the following subsections. In this section, we focus on unit monopole charges, which admit spherical solutions. By unit charge, we mean the magnetic charge is qmag = 2πq/e with q = 2. The Dirac quantization of electric and magnetic charge is satisﬁed for integer q with e qmag = 2πq, and the spherical ’t Hooft-Polyakov monopole solution corresponds to q = 2. Higher charges q > 2 can only be achieved with nonspherical solutions [26], making it analytically intractable to determine their ﬁeld conﬁgurations. We return to these in section 3. We additionally look for solutions that are stable against decaying to other states. For convenience, we label states according to their magnetic and U(1)S charges by the notation (q, Q). In this notation, an ordinary monopole without S ﬁeld is (2, 0), a free S particle is (0,1), an ordinary Q-ball without gauge ﬁelds is (0, Q), and a QMB can have both entries nonzero. The most important channels through which a QMB with unit magnetic charge q = 2 can decay are Q-particle decay : (2, Q) →Q × (0, 1) + (2, 0) , (2.3) Q-ball decay : (2, Q) →(0, Q) + (2, 0) , (2.4) 1-particle decay : (2, Q) →(2, Q −1) + (0, 1) . (2.5) (2.3) (2.3) (2.4) (2.5) (2.4) (2.5) (2.5) We will see later that the ﬁrst process (2.3) is the most important, so we will focus on this one when discussing analytic approximations. The process (2.4) is disfavored be- We will see later that the ﬁrst process (2.3) is the most important, so we will focus on this one when discussing analytic approximations. The process (2.4) is disfavored be- 1A somewhat similar theory containing a gauged spontaneously broken U(1) and a global U(1) was considered in [43, 44]. 2 QMBs with unit magnetic charge (q = 2) That theory results in cosmic strings rather than monopoles. In those works, the equivalent Lagrangian parameters are not all positive, so the global U(1) symmetry is spontaneously broken inside the string. One could also explore the similar parameter space in the Lagrangian considered here. – 4 – cause Q-balls are more stable when mixed with monopoles. Additionally, Q-ball and QMB solutions generally only exist if emitting a single free S particle like in process (2.5) is energetically impossible (we show this in the following subsection). 2.1 Classical equations of motion To write the equations of motion, we parametrize the ﬁelds by the following spherically symmetric ansatz depending on radius r: φa = ˆra v f(r) , S = e−iωt v √ 2 s(r) , A0 = 0 , Aa i = ϵaij ˆrj e r a(r) , (2.6) (2.6) JHEP01(2022)109 where ˆra = ra/r. With a rescaling of the coordinate r = v r and parameters Ω= ω/v and µ0 = mS,0/v, the equations of motion can be written in terms of dimensionless quantities: a′′ −1 r2 a(1 −a)(2 −a) + e2 (1 −a) f2 = 0 , (2.7) f′′ + 2 r f′ −2 r2 (1 −a)2 f −1 2 λφ f (f2 −1) −1 2 λφS s2f = 0 , (2.8) s′′ + 2 r s′ + Ω2 s −1 2 λφS f2 s −λS s3 −µ2 0 s = 0 , (2.9) (2.7) (2.8) (2.9) where primes denote derivatives with respect to r. where primes denote derivatives with respect to r. For the QMB, the boundary conditions at the origin are determined by demanding that the equations of motion not diverge. At inﬁnity, the scalar ﬁelds must take their VEVs and the gauge ﬁeld must behave as a monopole. Thus, the boundary conditions are f(0) = 0 , f(∞) = 1 , s′(0) = 0 , s(∞) = 0 , a(0) = 0 , a(∞) = 1 . (2.10) (2.10) Notice the Neumann boundary condition for s at the origin. For convenience, we deﬁne s(0) = s0, which will depend on Ω. These boundary conditions could be compared to those of a pure monopole, which has s = 0 identically but the same conditions on f and a as in (2.10). They could also be compared to those of a pure nontopological soliton, which has a = 0 identically and a Neumann boundary condition f′(0) = 0 replacing the Dirichlet condition on f(0) in (2.10).2 Notice the Neumann boundary condition for s at the origin. For convenience, we deﬁne s(0) = s0, which will depend on Ω. These boundary conditions could be compared to those of a pure monopole, which has s = 0 identically but the same conditions on f and a as in (2.10). 2.1 Classical equations of motion (2.12) (2.12) As \|Ω\| approaches \|Ωc\| from above, the U(1)S charge will increase. One can think of the s ﬁeld taking a long time to roll to the origin as Ωsaturates this limit, resulting in a larger radius and charge for the Q-ball. Note that the combination of (2.11) and (2.12) implies As \|Ω\| approaches \|Ωc\| from above, the U(1)S charge will increase. One can think of the s ﬁeld taking a long time to roll to the origin as Ωsaturates this limit, resulting in a larger radius and charge for the Q-ball. Note that the combination of (2.11) and (2.12) implies JHEP01(2022)109 λφS > q 2 λS λφ . (2.13) (2.13) Otherwise, no Q-ball or QMB solution exists. Otherwise, no Q-ball or QMB solution exists. Once a solution is determined, the global U(1)S charge Q of the solution is Once a solution is determined, the global U(1)S charge Q of the solution is Q = i Z d3x S† ∂t S −S ∂t S† = 4π Ω Z ∞ 0 dr r2s2 . (2.14) (2.14) Classically, any irrational value of Q is allowed by the equations of motion. However, because the S particles are quantized, Q can only take on integer values in practice. For clarity, we will limit ourselves to Ω> 0 in future discussions. A sign ﬂip leads to otherwise identical solutions with opposite Q. The solution’s mass is M = 4πv Z ∞ 0 dr 1 2e2 2a′2 + 1 r2 2a −a22 + (1 −a)2 f2 + r2 1 2f′2 + 1 8λφ(f2 −1)2 + r2 1 2s′2 + 1 2Ω2s2 + 1 4λφSf2s2 + 1 4λSs4 + 1 2µ2 0s2 . (2.15) (2.15) (2.15) That the 1-particle decay channel (2.5) is stable can be understood from these equa- tions. Note that ∂M/∂Ω= Ωv(∂Q/∂Ω). The change in the QMB energy for a change in its charge ∆Q = 1 is thus ∆M ≈∆Q(∂M/∂Q) = Ωv < q m2 S,0 + λφSv2/2, using (2.11) in the last inequality. Thus, there is more rest mass energy in a free particle than energy contained in the bound particle. This argument is strictly true in the classical theory where ∆Q →0 is allowed, although it need not necessarily hold for quantized ∆Q = 1 (numerically, we found no counterexamples in our scans). 2.1 Classical equations of motion They could also be compared to those of a pure nontopological soliton, which has a = 0 identically and a Neumann boundary condition f′(0) = 0 replacing the Dirichlet condition on f(0) in (2.10).2 The solution for s can be thought of as a particle starting at rest at some large value s0 at r = 0, then rolling towards a stationary point at the origin, coming to rest there as r →∞[3]. Its eﬀective potential is Ueﬀ= Ω2s2/2 −V (s, f), with the last term given by plugging appropriate expressions from (2.6) into (2.2). To have a nontrivial solution with s0 ̸= 0 that rolls monotonically to s = 0, s must have a negative mass term around s = 0 (when f = 1) in Ueﬀ. Thus, Ωmust satisfy Ω2 ≡Ω2 −µ2 0 < λφS 2 . (2.11) (2.11) 2The pure nontopological soliton uses the gauge choice φb = δ3bvf(r), which does not contain a topo- logical charge when a = 0, instead of the expression in (2.6). So, the third term ∝r−2 in (2.8) is absent, enabling the Neumann boundary condition without introducing a singularity in the equations of motion. Note that using the ansatz in (2.6) and without the gauge ﬁeld, one could have a soliton state with global topological and global nontopological charges, which we do not explore here. 2The pure nontopological soliton uses the gauge choice φb = δ3bvf(r), which does not contain a topo- logical charge when a = 0, instead of the expression in (2.6). So, the third term ∝r−2 in (2.8) is absent, enabling the Neumann boundary condition without introducing a singularity in the equations of motion. Note that using the ansatz in (2.6) and without the gauge ﬁeld, one could have a soliton state with global topological and global nontopological charges, which we do not explore here. – 5 – Additionally, there is a minimum imposed on \|Ω\| by requiring Ueﬀ> 0 for some value s > 0. If \|Ω\| is too small, the quartic λφ term dominates Ueﬀand makes it negative, such that there is no Q-ball solution for which the ﬁeld rolls towards the origin.3 This condition is derived by setting f = 0 and ﬁnding a positive solution smax for ∂Ueﬀ/∂s = 0, then demanding Ueﬀ(s = smax) > 0. The result is Ω4 > Ω4 c ≡1 2 λS λφ . 3See the left panel of ﬁgure 4 in ref. [7]. 2.1 Classical equations of motion This is because the extra mass contribution from µ0 in (2.16) goes to zero as Q →0. For the remainder of this paper, we will take µ0 = 0 for convenience. The mass, charge, and stability for otherwise identical QMBs with µ0 > 0 can be determined using these relationships. It is easy to show using (2.11) that λφS ≥λφS > 0, where the ﬁrst inequality is only equal when µ0 = 0, and that λφS is a monotonically decreasing function of µ0. In short, increasing µ0 leads to decreased binding energy, making QMBs (or Q-balls) less stable. This can increase the minimum stable charge satisfying ∆M > 0. However, if a QMB is stable for any Q > 0 when µ0 = 0, a similar QMB with µ0 > 0 and all other Lagrangian parameters ﬁxed should also be stable. This is because the extra mass contribution from µ0 in (2.16) goes to zero as Q →0. For the remainder of this paper, we will take µ0 = 0 for convenience. The mass, charge, and stability for otherwise identical QMBs with µ0 > 0 can be determined using these relationships. An interesting relation for the mass can be derived for the s-dependent terms in M. By substituting the equation of motion (2.9) to eliminate λφS then integrating by parts, the s-dependent part of the mass [all terms in the second line of (2.15)] can be written as MS = Q Ωv −4πv Z ∞ 0 dr r2 1 4 λS s4 . (2.19) (2.19) Note that Ωdepends on Q and s(r) via (2.14). Nevertheless, this equation gives the qualitatively correct behavior for a mass of a Q-ball with large charge: M ∝Qv [note that the λS term in (2.19) is cancelled by the λφ term in the ﬁrst line of (2.15), at least in the step-function approximation in section 2.2 using (2.20) and (2.27)]. Indeed, we will see in the following subsection that as Q increases, Ωsaturates to its lower limit (2.12) and can be treated as approximately constant. 2.1 Classical equations of motion Indeed, this does not guarantee stability in the Q-particle decay channel (2.3), where ∆Q is too large for the approximation of constant partial derivative to hold. Let us brieﬂy comment on the bare Lagrangian mass µ0. The solutions for the ﬁeld proﬁles a(r), f(r), s(r) for a ﬁxed value of Ωwith µ0 > 0 are identical to the ﬁeld proﬁle solutions when µ0 = 0 and Ω= Ω. However, the mass and charge for these two cases diﬀer. Deﬁning M0(Ω) ≡M\|µ0=0, Ω=Ωin (2.15), the mass for arbitrary µ0 can be written in terms of M0 and Q as M = M0(ΩQ) + µ2 0 q Ω2 Q + µ2 0 Q v . (2.16) (2.16) – 6 – This will have an eﬀect on the stability of the QMB (or Q-ball). Considering the most relevant decay channel (2.3), the binding energies for the cases µ0 = 0 and µ0 > 0 are This will have an eﬀect on the stability of the QMB (or Q-ball). Considering the most relevant decay channel (2.3), the binding energies for the cases µ0 = 0 and µ0 > 0 are ∆M =                M0(ΩQ) − s λφS 2 Q v −M(2,0) , µ0 = 0 M0(ΩQ) −   s λφS 2 + µ2 0 − µ2 0 q Ω2 Q + µ2 0  Q v −M(2,0) , µ0 > 0 . (2.17) (2.17) Here, M(2,0) is the isolated monopole mass. Thus, for the purposes of binding energy and stability in this channel, the results when µ0 = 0 can be translated to the results when µ0 > 0 by replacing λφS with λφS satisfying JHEP01(2022)109 s λφS 2 = s λφS 2 + µ2 0 − µ2 0 q Ω2 Q + µ2 0 . (2.18) (2.18) It is easy to show using (2.11) that λφS ≥λφS > 0, where the ﬁrst inequality is only equal when µ0 = 0, and that λφS is a monotonically decreasing function of µ0. In short, increasing µ0 leads to decreased binding energy, making QMBs (or Q-balls) less stable. This can increase the minimum stable charge satisfying ∆M > 0. However, if a QMB is stable for any Q > 0 when µ0 = 0, a similar QMB with µ0 > 0 and all other Lagrangian parameters ﬁxed should also be stable. 2.2 Large Q-ball charge It is useful to obtain analytic estimates for how the mass and charge depend on the pa- rameters in the equations of motion. One estimate is obtained in the large Q limit by assuming the ﬁelds f(r) and s(r) behave like step functions and the a(r) ﬁeld behaves like a power-law function: f(r) ≈ ( 0 , r < rb 1 , r > rb , s(r) ≈ ( s0 , r < rb 0 , r > rb , a(r) ≈ ( r2/r2 b , r < rb 1 , r > rb . (2.20) (2.20) – 7 – Here, the r2 power for a(r) is derived from (2.7) for f(r) = 0 and in the limit of a(r) ≪1. Then, the S charge is 4 Then, the S charge is Q ≈4π 3 r3 b Ωs2 0 . (2.21) (2.21) For simplicity, we will take µ0 = 0 in the following. Then, the mass is (ignoring the gradient energies for f(r) and s(r), which are only important near the boundary rb and thus small in the large Q limit) For simplicity, we will take µ0 = 0 in the following. Then, the mass is (ignoring the gradient energies for f(r) and s(r), which are only important near the boundary rb and thus small in the large Q limit) M(2,Q) ≈304 π v 35 e2 rb + 4π 3 r3 bv 1 4λSs4 0 + 1 8λφ + 1 2Ω2s2 0 . (2.22) (2.22) The ﬁrst term of (2.22) can be thought of as the energy contained in the magnetic ﬁeld B generated by the bound monopoles, or roughly R d3xB2/2. The other terms are the volume contributions to the vacuum energy from S and φa diﬀering from their VEVs. After substituting (2.21) to eliminate Ωin favor of Q and s0, the expression for M is minimized (taking ∂M/∂s0 = ∂M/∂rb = 0) by The ﬁrst term of (2.22) can be thought of as the energy contained in the magnetic ﬁeld B generated by the bound monopoles, or roughly R d3xB2/2. The other terms are the volume contributions to the vacuum energy from S and φa diﬀering from their VEVs. 2.2 Large Q-ball charge The second term ∝Q−1/3 is the mass energy contained in the gauge ﬁeld a coming from the ﬁrst term in (2.22). The leading mass term in (2.26) is canceled by the subtraction. Thus, the mass of the combined QMB system can always be smaller than the sum of the masses of an isolated monopole and a Q-ball for suﬃciently large Q. q Additionally, if (M(2,Q) −M(2,0))/Q < mS = v q λφS/2, where mS is the mass of the S particles in the true vacuum, such QMBs are stable against decays to free S particles. Using (2.26) in the limit M(2,Q) ≫M(2,0), this stability condition is equivalent to the existence condition in (2.13). This implies that there always exists a suﬃciently large Q such that QMB (and Q-ball) solutions are stable if such solutions exist. JHEP01(2022)109 Beyond the step-function approximation, the frequency Ωasymptotes to Ωc in the large Q limit. The radius depends on Ωas rb = c1/(Ω−Ωc), with c1 > 0 as a numerical coeﬃcient [7]. Ignoring the magnetic ﬁeld energy, the Q-ball mass is M(2,Q) ≈M(0,Q) ≈Q v Ω≈Q v Ωc + c2 Q−1/3 , (2.30) (2.30) with c2 > 0 related to c1 and other couplings. The subleading term ∝Q2/3 ∝r2 b is the surface energy contribution to the mass. We conﬁrm this behavior numerically for the QMB. The above Q-dependence for mass means that two Q-balls with charge Q1 and Q2 have a binding energy of ∆M = MQ1 + MQ2 −MQ1+Q2 = c2 v h Q2/3 1 + Q2/3 2 −(Q1 + Q2)2/3i . (2.31) (2.31) This expression is always positive, so a Q-ball or QMB is stable against ﬁssioning to multiple Q-balls of smaller charge. This expression is always positive, so a Q-ball or QMB is stable against ﬁssioning to multiple Q b ll f ll h Q-balls of smaller charge. 2.2 Large Q-ball charge After substituting (2.21) to eliminate Ωin favor of Q and s0, the expression for M is minimized (taking ∂M/∂s0 = ∂M/∂rb = 0) by JHEP01(2022)109 s0 ≈1 rb 9 Q2 16 π2 λS !1/6 , (2.23) (2.23) rb ≈ 2432 π + 105 (6/π)1/3 λ1/3 S e2 Q4/3 140 π e2 λφ !1/4 . (2.24) (2.24) It is useful to go to the limit λ1/3 S e2Q4/3 ≫60 because the step function approximation would not be expected to hold in the opposite limit anyways (for the case λS = 0, a diﬀerent ansatz is necessary, cf. [7]). This is equivalent to neglecting the ﬁrst term in (2.22), meaning that the magnetic ﬁeld energy is negligible. Then, It is useful to go to the limit λ1/3 S e2Q4/3 ≫60 because the step function approximation would not be expected to hold in the opposite limit anyways (for the case λS = 0, a diﬀerent ansatz is necessary, cf. [7]). This is equivalent to neglecting the ﬁrst term in (2.22), meaning that the magnetic ﬁeld energy is negligible. Then, rb ≈(3/π)1/3 25/12 λ1/12 S λ1/4 φ Q1/3 , (2.25) (2.25) M(2,Q) ≈M(0,Q) ≈Q Ωc v , (2.26) s0 ≈ λφ 2 λS 1/4 , (2.27) (2.27) Ω≈Ωc . (2.28) (2.28) Note, Ωsaturates the bound in (2.12) in the large Q limit, and M ∝r3 b ∝Q so the QMB has a ﬁxed energy density. In this large Q limit, the system can be thought of as a small-radius monopole trapped in a large-radius Q-ball. There always exists a suﬃciently large Q such that the QMB conﬁguration is stable against decay to an isolated Q-ball and monopole. As Q and rb increase, the ﬁrst term in (2.22) corresponding to the magnetic ﬁeld energy becomes less relevant, and the mass approaches that of a Q-ball without a magnetic charge as in (2.26). Including this magnetic ﬁeld energy term in M(2,Q), the binding energy for a monopole and Q-ball is ∆M = M(2,0) + M(0,Q) −M(2,Q) ≈4πv e Y −304 × 25/12 π4/3 35 × 31/3 λ1/4 φ e2 λ1/12 S Q−1/3 v . (2.29) (2.29) – 8 – The ﬁrst term is the mass of an isolated monopole M(2,0) = 4πvY/e. Here, Y ≡Y (λφ/e2) is monotonic with Y (0) = 1 corresponding to the BPS limit and Y (∞) ≈1.787. 2.3 Small Q-ball charge In the opposite limit of small Q, the S ﬁeld only causes a small perturbation to the monopole ﬁeld conﬁguration. Thus, it is energetically preferred for a small number of S particles to be trapped in a monopole. Provided they cluster near the center of the monopole where f ∼0, the S particles themselves have smaller mass inside (where mS ∼mS,0) than outside (where mS = q m2 S,0 + λφSv2/2). q , To get a sense of the small Q limit, we make the following approximations. First, take µ0 = λS = 0 and approximate µ0 = λS = 0 and approximate s(r) ≈      s0 sin(Ωr) Ωr , r < rb = π Ω 0 , r > rb . (2.32) (2.32) ≈   0 , r > rb This is the analytic solution for s when f = 0 in (2.9). We will further simplify by considering the BPS monopole [24, 25] (the limit λφ/e2 →0), for which an analytic solution is known: fBPS(r) = coth(er) −1/(er). This is bounded above by fBPS(r) ≤er/3 (to good approximation, one could replace e with e + p2λφ in this equation and throughout the – 9 – rest of this paragraph in the non-BPS limit). We assume that the small perturbation in f from the λφS term is unimportant. Plugging this upper limit on fBPS and (2.32) into the second line of (2.15)—what we called MS above — then minimizing with respect to Ω, rest of this paragraph in the non-BPS limit). We assume that the small perturbation in f from the λφS term is unimportant. Plugging this upper limit on fBPS and (2.32) into the second line of (2.15)—what we called MS above — then minimizing with respect to Ω, MS ≲2(4π2 −6)1/4 3 √ 3 Q e1/2 λ1/4 φS v . (2.33) (2.33) The numerical prefactor is ≈0.93. Comparing to the mass of Q free S particles Q(λφS/2)1/2v, we see that the Q-ball should be stable unless the gauge coupling is large: e ≳λ1/2 φS . While we have assumed λS = 0, we observe a posteriori that the λS term can gen- erally be neglected compared to the Ω2 term in MS at small Q: λSs4 0/(Ω2s2 0) = λSQ/(2π2), which for small enough Q and perturbative λS should always be less than unity. 2.3 Small Q-ball charge JHEP01(2022)109 In the regime of large gauge coupling, e ≳λ1/2 φS , the prior approximation that f = fBPS is not valid because the Q-ball radius is comparable to the monopole radius. The eﬀects of the S ﬁeld on the φ VEV must be included. To see this, we ﬁrst simplify the ansatz in (2.32) to make it more analytically tractable: s(r) ∼      s0 , r < rb = π Ω 0 , r > rb . (2.34) (2.34) The scalar potential (2.2) can be rewritten to reﬂect the dependence of the φ VEV on S: 1 8λφ h φaφa −(v2 −2λφSλ−1 φ \|S\|2) i2 + 1 8λφ h v4 −(v2 −2λφSλ−1 φ \|S\|2)2i + m2 S,0\|S\|2 + λS\|S\|4 . (2.35) (2.35) Deﬁning the VEV inside the monopole for suﬃciently small s0 as v2 ≡v2 1 −λφSλ−1 φ s2 0 , (2.36) (2.36) and assuming the monopole and Q-ball radii are comparable, the mass is given approxi- mately by [using (2.21)] M(2,Q) = 4πv e Y + " π 2 + λS − λ2 φS 2λφ ! 3 16π2 Q # Qv rb + λφS 4π + µ2 0 2π ! Q v rb . (2.37) (2.37) The ﬁrst term is M(2,0) with v set to v. Taking µ0 = 0 and neglecting higher powers of Q, the mass M is minimized by rb ≃π q 2/λφS. To justify the assumption of comparable monopole and Q-ball radii so that v can be replaced by v, we require rb ≳1/e, or λφS/e2 ≲2π2. Indeed, this is exactly where the prior approximation (f = fBPS) appeared to give unstable Q-balls, revealing that approximation was invalid in this limit. With this value for rb, we obtain the mass at leading order in Q after a binomial expansion on ¯v: M(2,Q) ≃4πv e Y + Qv   s λφS 2 − 3 λ2 φS 4π3 e λφ Y  . (2.38) (2.38) – 10 – Figure 1. Example QMB ﬁeld conﬁgurations for small (left) and large (right) S charge Q. Both panels take mS,0 = 0 and q = 2. 2.3 Small Q-ball charge In the left panel, the dashed blue and green lines indicate the Q = 0 solution for an isolated monopole with the same λφ and e, which is barely distinguishable from the Q = 2 ﬁeld proﬁles (solid) for a and f. The dotted yellow line shows s0 sin(Ωr)/(Ωr), which well approximates the small-radius solution for s in the small Q regime. In the right panel, two diﬀerent choices for large Q are shown in solid and dashed lines. JHEP01(2022)109 Figure 1. Example QMB ﬁeld conﬁgurations for small (left) and large (right) S charge Q. Both panels take mS,0 = 0 and q = 2. In the left panel, the dashed blue and green lines indicate the Q = 0 solution for an isolated monopole with the same λφ and e, which is barely distinguishable from the Q = 2 ﬁeld proﬁles (solid) for a and f. The dotted yellow line shows s0 sin(Ωr)/(Ωr), which well approximates the small-radius solution for s in the small Q regime. In the right panel, two diﬀerent choices for large Q are shown in solid and dashed lines. This indicates that the mixed QMB is stable in this limit because the free monopole mass is 4πvY/e and the free S particle mass is q λφS/2 v. q The above arguments cover both the cases when the Q-ball radius is either larger or smaller than the monopole radius and indicate that the QMB is stable in both cases. However, they assume that the monopole ﬁelds are relatively unchanged by the presence of the s ﬁeld—f ≈fBPS for the ﬁrst case and v > 0 in (2.36) for the second case. When these assumptions are violated, on the other hand, analytic approximation is intractable, and we must resort to qualitative explanations to explain the stability of small-Q QMBs. First, consider the BPS limit λφ = 0. When S particles are added to the monopole starting from Q = 0, the region where φ = 0 is expanded because the λφS term contributes a positive mass-square to φ. There is no vacuum energy cost to expanding the φ = 0 region in this limit. Thus, all charges Q are stable in that they decrease the overall energy of the system by decreasing the mass energy of S particles. 2.3 Small Q-ball charge This explains why the analytic approximation above where we took f = fBPS breaks down in the large e limit, where the isolated monopole radius ∝(ev)−1 is small. Going away from the BPS limit so that λφ > 0, there is now a vacuum energy cost to expanding the radius of the region with φ = 0. As λφ increases, the radius where f ∼0 (which goes as r ∼min[e−1, λ−1/2 φ ] for an isolated monopole) shrinks, and it also costs more vacuum energy to increase its radius. Thus, at suﬃciently large λφ, the region with f ∼0 has too narrow of a radius to admit bound states of S particles. Only at suﬃciently large Q, where there is an additional energy saving from having many S particles together in a Q-ball, do bound states exist. 2.4 Numerical results To be more quantitative, we solve the system numerically using the ﬁnite diﬀerence method. We use the rescaling in [23] to recast the semi-inﬁnite boundary at r = ∞into a ﬁnite – 11 – - - - - Figure 2. Diﬀerence between the initial and ﬁnal masses Mi and Mf (normalized to v) for the Q-particle (2.3), Q-ball (2.4), and 1-particle (2.5) decay channels shown solid, dashed, and dotted, respectively. Diﬀerent colors denote diﬀerent choices of λφS; the other parameters are ﬁxed, in- cluding mS,0 = 0. Values greater than zero are unstable; the charge at which the Q-particle decay crosses zero is denoted Qs in the text. When curves are cut oﬀon the left, no solution exists for the QMB (solid and dotted curves) or for the isolated Q-ball (dashed curve), denoted Qc in the text. Two diﬀerent-mass and same-charge solutions exist; part of the higher-energy solution for the QMB is shown in the solid curves. The Q-ball decay channel (dashed) has been multiplied by 10−2 on the y-axis to make it visible in the plot range. - - - - JHEP01(2022)109 Figure 2. Diﬀerence between the initial and ﬁnal masses Mi and Mf (normalized to v) for the Q-particle (2.3), Q-ball (2.4), and 1-particle (2.5) decay channels shown solid, dashed, and dotted, respectively. Diﬀerent colors denote diﬀerent choices of λφS; the other parameters are ﬁxed, in- cluding mS,0 = 0. Values greater than zero are unstable; the charge at which the Q-particle decay crosses zero is denoted Qs in the text. When curves are cut oﬀon the left, no solution exists for the QMB (solid and dotted curves) or for the isolated Q-ball (dashed curve), denoted Qc in the text. Two diﬀerent-mass and same-charge solutions exist; part of the higher-energy solution for the QMB is shown in the solid curves. The Q-ball decay channel (dashed) has been multiplied by 10−2 on the y-axis to make it visible in the plot range. range, taking y = r/(1 + r/a) as the dependent variable with a an arbitrary positive constant and y ∈[0, a]. We scan for solutions over the parameters λφS, λS, λφ, e, and Ω. 2.4 Numerical results When scanning along a given parameter, we take small steps in that parameter and use the previous solution as the initial guess for the next solution. This recursive approach allows us to scan to points near the limits in (2.11)–(2.13). We focus on the case µ0 = 0 in the plots presented here, but we will later comment on the changes when µ0 > 0. Example numerical solutions are shown in ﬁgure 1, focusing on the small and large Q limits. At small Q (left panel), the f and a ﬁelds for the QMB are barely distinguishable from those of an isolated monopole when Q = 0 (the dashed lines in the left panel). The s ﬁeld is well-approximated by (2.32), shown dotted. When Q is large (right panel), the radius where f and a diﬀer from unity is signiﬁcantly expanded to be of comparable radius to the s proﬁle, agreeing with the approximation in (2.20). Increasing Q in this limit causes the radius to increase but has almost no eﬀect on s0, matching the analytic estimates in (2.25) and (2.27). That s0 ≈1 in the right panel is a numerical accident — other choices of λφ and λS would result in diﬀerent s0 at large Q according to (2.27). With these solutions, we can numerically compute their masses using (2.15), and com- pare to the masses of other states to numerically assess their stability. The diﬀerence of the initial and ﬁnal state masses for each of the possible decay channels in (2.3)–(2.5) are com- pared in ﬁgure 2 for a few choices of parameters as a function of Q. When all three channels have mass diﬀerences less than zero for a given charge, the QMB is stable. The largest S charge Q for which any of these channels’ mass diﬀerences crosses zero is the minimum sta- – 12 – Figure 3. Contours of the minimum stable S charge Qs for QMBs with magnetic charge q = 2. When a Lagrangian parameter is not varied on the x- or y-axis, it is ﬁxed to λφ = 0.5, e2 = 0.5, λS = 0.3, λφS = 1, and mS,0 = 0. 2.4 Numerical results In the dark shaded regions, no Q-ball state exists of any kind, mixed QMB or isolated Q-ball. In the light shaded regions, Q > Qs is required for a stable solution. For Q < Qs, the states decay to Q free S particles and an isolated monopole. The contours in the left and right panels should extend all the way to the dark shaded regions but are truncated due to limited numerical precision. In the white unshaded regions, all S charges Q down to unity are stable. JHEP01(2022)109 Figure 3. Contours of the minimum stable S charge Qs for QMBs with magnetic charge q = 2. When a Lagrangian parameter is not varied on the x- or y-axis, it is ﬁxed to λφ = 0.5, e2 = 0.5, λS = 0.3, λφS = 1, and mS,0 = 0. In the dark shaded regions, no Q-ball state exists of any kind, mixed QMB or isolated Q-ball. In the light shaded regions, Q > Qs is required for a stable solution. For Q < Qs, the states decay to Q free S particles and an isolated monopole. The contours in the left and right panels should extend all the way to the dark shaded regions but are truncated due to limited numerical precision. In the white unshaded regions, all S charges Q down to unity are stable. ble charge Qs. In all cases in our full parameter scan, the decay to Q free S particles (2.3) is the most important channel for determining Qs. The green lines (λφS = 1.1) in ﬁgure 2 show an example with Qs = 0, so QMBs with any charge Q are stable. There is also a cutoﬀcharge Qc below which no QMB or isolated Q-ball solutions (stable or unstable) exist. Recall that decreasing Q corresponds to increasing Ω. However, when Ωis increased suﬃciently, the charge begins to increase again from Qc, but the solutions are at a higher energy [2]. A portion of these higher-energy solutions for the QMBs are shown in the solid curves of ﬁgure 2, above the kinks. 2.4 Numerical results Ωis increasing from right to left along the lower branch, then continues from left to right along the upper branch as Ωapproaches q λφS/2. The higher-energy solutions are unstable, so we will only focus on the lower-energy branch. A similar eﬀect occurs for isolated Q-balls [7], but for the decays to isolated Q-balls we have cut oﬀthe corresponding dashed lines at Qc for the isolated Q-balls, omitting the higher-energy branch. In Q-ball literature, these higher- energy solutions are referred to as “Q-clouds.” Notice that Qc and Qs for the QMB are always smaller than Qc for the isolated Q-ball for ﬁxed choice of Lagrangian parameters. This is because there is an additional energy savings from having the Q-ball and monopole overlap. The tunneling rate for quantum-mechanically unstable Q-balls with Qc < Q < Qs in the lower energy branch to decay to free particles was considered in [46]. We show contours of Qs for example parameter choices in ﬁgures 3. In the white regions, all values for Q down to Q = 0 are stable (though in the quantized theory only integer Q are allowed). As explained in section 2.3, our numerical scans conﬁrm that when λφ = 0, all charges Q provide stable mixed states, i.e., Qs = 0. However, for λφ > 0, there is a minimum stable U(1)S charge Qs below which mixed QMBs are unstable to decays to Q free S particles. In the darkest shaded region no QMB or Q-ball solution is present. This region is only present when λSλφ > 0 due to eq. (2.13), otherwise λφS can – 13 – be arbitrarily small. In the intermediate lightly shaded region, QMBs are only stable if Q > Qs. It is interesting to note that these stability threshold values are always smaller than the threshold Qs for an isolated Q-ball. For example, for the benchmark values in ﬁgure 2, λφ = 0.5, e2 = 0.55, λS = 0.3, λφS = 1, and mS,0 = 0, Qs ≈55 for a QMB with q = 2 while Qs ≈345 for a Q-ball with q = 0. Other patterns are also evident. Decreasing e leads to increased stability for small Q because the monopole radius is increased. The S bound state energy is reduced because it is in a larger-radius potential well. 2.4 Numerical results Increasing λφS also leads to increased stability for small Q because a smaller s0 is required to restore the non-Abelian gauge symmetry associated with φ, which requires λφSs2 0 > λφv2/2. In the middle panel, the boundary between the white and light shaded regions is vertical because the eﬀect of λS on the QMB mass is negligible in the small-Q limit, as discussed in section 2.3. JHEP01(2022)109 Let us brieﬂy discuss the case mS,0 > 0 in relation to these numerical results, which all took mS,0 = 0. In ﬁgure 3, the boundaries between the dark shaded, light shaded, and white regions do not change when µ0 is varied. The boundary for the dark shaded regions is given by (2.13), whose derivation held for arbitrary µ0. The boundary between the white and light shaded regions is determined in the Q →0 limit. In this limit, the mass contribution from nonzero µ0 in (2.16) goes to zero proportional to Q. Thus, if a stable solution exists when µ0 = 0 for arbitrarily small Q, a stable Q →0 solution also exists for µ0 > 0 when holding all other Lagrangian parameters ﬁxed. Therefore, only the contours of Qs > 0 shown in the light shaded regions of ﬁgure 3 are modiﬁed by varying µ0. How these Qs contours vary with µ0 can be described by replacing λφS with λφS in (2.18). Because λφS is a monotonically decreasing function of µ0, the value for Qs increases as µ0 increases; i.e., the QMBs become less stable as µ0 increases. 4The condition that the ﬁrst term in (3.1) is negligible is q ≪λ1/6 S e Q2/3, less important than the condition in (3.2) for large Q. 3 Multiply magnetically charged QMBs (q > 2) We can extend the conclusions from the analysis of unit magnetically charged Q-balls to those with higher magnetic charges. It is well-known that spherical solutions for monopoles do not exist above unit monopole charge [26]. Thus, we cannot easily apply the numerical methods of section 2 to this case. Nevertheless, we can make similar analytic approxima- tions to argue for the stability of certain multiply charged conﬁgurations. The case that most easily admits higher monopole charges is the case where the Q-ball radius is much larger than that of an isolated monopole. Then, as described in section 2.2, the QMB state can be thought of as a monopole bound inside a Q-ball. This modiﬁes the energy in two ways. First, there is a vacuum energy savings because the interiors of both the monopole and Q-ball have φ = 0, which contributes to the vacuum energy in each system (when λφ > 0). The volume of the QMB is smaller than the sum of the volumes of the isolated monopole and Q-ball, so the total vacuum energy is less for a QMB. We expect that adding additional monopoles to a QMB also has a negligible eﬀect on the QMB radius, as long as q is not too large compared to Q that it signiﬁcantly backreacts on the s ﬁeld in the equations of motion. Second, there is a change to the energy contained in the magnetic ﬁeld. An isolated monopole’s magnetic ﬁeld energy is proportional to v/e. – 14 – Once a monopole is bound inside a Q-ball, the magnetic charge spreads out throughout the QMB’s volume (see the proﬁle for a in ﬁgure 1 right panel). Thus, the energy contained in the ﬁeld is proportional to v/(e2rb) as in (2.22). As a result, there is a reduction of the magnetic ﬁeld energy when a single monopole (with characteristic radius r ∼e−1 for a(r)) is trapped in a larger-radius Q-ball with radius rb > e−1. This argument remains true as the monopole charge q of the Q-ball increases, with the energy in the magnetic ﬁeld going proportional to q2v/(e2rb). Only once q is very large can the magnetic ﬁeld energy of the (q, Q) state be larger than the magnetic ﬁeld energy for separated (2, 0)+ (q −2, Q) states. Thus, q-charged QMBs should be stable up until some maximum value for q. 3 Multiply magnetically charged QMBs (q > 2) To be a bit more quantitative, we can generalize the approximation for the QMB mass in (2.22) to arbitrary magnetic charge q as JHEP01(2022)109 M(q,Q) ∼304πv(q/2)2 35e2rb + 4π 3 r3 bv 1 4λSs4 0 + 1 8λφ + 1 2Ω2s2 0 . (3.1) (3.1) While this does not capture the full nonspherical structure of the solution, it should give a reasonable approximation in the Q, rb ≫1 limit. In this limit, the ﬁrst term is negligible compared to the second term as we noted previously, and the other terms set rb, s0, and M as a function of Q as in (2.25)–(2.27). Using the notation from eqs. (2.3)–(2.5), in order for the bound (q, Q) state to be stable against decay to (q, Q) →(q −2, Q) + (2, 0), there is an upper bound on q as4 q ≲1 + 35 76 e rb Y ≈35(3/π)1/3 76 × 25/12 e Y λ1/12 S λ1/4 φ Q1/3 , (3.2) (3.2) where in the last step we have substituted (2.25), which is only valid when both λφ, λS > 0. The other possible decay channel to (q/2)×(2, 0)+(0, Q) is less important, having a larger upper bound on q by a factor of 2 than the one above. The binding energy to add an isolated (2, 0) monopole to an existing (q, Q) state to form a (q + 2, Q) state is the same as (2.29) but with the last term on the right-hand side multiplied by (q + 1). In the limit of (q + 1) ≪e λ1/12 S λ−1/4 φ Q1/3, the binding energy saturates the isolated q = 2 monopole mass ∆M ≈M(2,0). This approximation supports our claim that mixed (q, Q) solutions exist and are sta- ble for a suﬃciently large hierarchy between q and Q. While more detailed numerical work is largely intractable, the conclusion remains that large-charged monopoles may exist in nature. QMBs thus oﬀer a wider variety of charges and masses for magnetically-charged objects than other systems. For an ordinary isolated monopole, the mass and charge are ﬁxed to M = 4πvY/e and q = 2, respectively. On the other hand, a QMB will have mass given approximately by (2.26) and magnetic charge satisfying (3.2). Thus, M(q,Q) ≳21 λφ e3Y 3 q3v . 3 Multiply magnetically charged QMBs (q > 2) (3.3) (3.3) 4The condition that the ﬁrst term in (3.1) is negligible is q ≪λ1/6 S e Q2/3, less important than the condition in (3.2) for large Q. – 15 – Figure 4. Allowed mass-to-charge ratio M/q and magnetic charge q for various cosmologically stable magnetically charged states. Orange and red regions: QMBs with λφ = 0.5, λS > 0 (in- sensitive to exact value), e2 = 4π/137 the SM value, and v = 246 (1016) GeV for orange (red). The bottom-left boundaries of each region with small q and Q are not reliable because the large Q limit is assumed. Hashed regions show where the Schwarzschild radius exceeds the QMB radius, so gravitational eﬀects (ignored here) should be taken into account. Blue vertical line: isolated monopoles with e the SM value and v varying from 246 GeV to Mpl, using the same λφ and e as the QMB. Black: magnetically charged black holes, which exist as cosmologically stable relics today either on the horizontal black line if they are extremal or in the shaded region if they are not extremal (in between, they evaporate to the extremal line with lifetime τ ≪t0 the age of the universe). Lilac horizontal dashed lines indicate the bound on the relic abundance as a fraction of the dark matter density fDM from the Parker bound applied to M31 (assuming all relics have the same charge and mass). Above the line labeled “fDM < 1,” magnetically charged states could explain all of dark matter (though other model-dependent bounds exist). Gray dotted lines show contours of constant mass. JHEP01(2022)109 Figure 4. Allowed mass-to-charge ratio M/q and magnetic charge q for various cosmologically stable magnetically charged states. Orange and red regions: QMBs with λφ = 0.5, λS > 0 (in- sensitive to exact value), e2 = 4π/137 the SM value, and v = 246 (1016) GeV for orange (red). The bottom-left boundaries of each region with small q and Q are not reliable because the large Q limit is assumed. Hashed regions show where the Schwarzschild radius exceeds the QMB radius, so gravitational eﬀects (ignored here) should be taken into account. Blue vertical line: isolated monopoles with e the SM value and v varying from 246 GeV to Mpl, using the same λφ and e as the QMB. 5If the magnetic charge q is suﬃciently small — much less than extremal, so that they do not have an enhanced Hawking evaporation rate — magnetic BHs down to about 1015 g are cosmologically stable. However, evaporation products of PBHs below 1017 g set limits on their abundance (for review, see [50]). Thus, while the “BH, τ > t0” line in ﬁgure 4 would curve slightly downwards as it approaches smaller q, we neglect this because such BHs could only be a small fraction of DM. 3 Multiply magnetically charged QMBs (q > 2) Black: magnetically charged black holes, which exist as cosmologically stable relics today either on the horizontal black line if they are extremal or in the shaded region if they are not extremal (in between, they evaporate to the extremal line with lifetime τ ≪t0 the age of the universe). Lilac horizontal dashed lines indicate the bound on the relic abundance as a fraction of the dark matter density fDM from the Parker bound applied to M31 (assuming all relics have the same charge and mass). Above the line labeled “fDM < 1,” magnetically charged states could explain all of dark matter (though other model-dependent bounds exist). Gray dotted lines show contours of constant mass. Again, this is only valid when Q ≫1 and both λφ, λS > 0. This could also be contrasted with magnetically charged black holes (BH) [36, 37, 47, 48], another example of a bound state admitting larger q > 2. If lighter than about 1017 g, they evaporate to extremal very quickly with lifetime τ ≪t0 the age of the universe, at which point they become cosmologically stable [49].5 Extremal black holes have a ﬁxed mass-to-charge ratio M/q = cos θW √πe−1Mpl ≈5.2 Mpl with Mpl the Planck mass and θW the weak mixing angle. Their masses also cannot be smaller than the Planck scale. The possible masses and magnetic charges for various magnetically charged states are compared in ﬁgure 4. Isolated monopoles (blue line) can only have charge q = 2 and their – 16 – mass depends on v. QMBs can have larger charge and mass than isolated monopoles; their mass is always larger than that of isolated monopoles. The boundary from (3.3) is derived in the large Q limit and thus should not be trusted in the small q and Q limit (bottom left of both the orange and red regions). Magnetic black holes may either be near- extremal (along the horizontal black line) or suﬃciently massive to have not evaporated to near-extremal (shaded region). In comparison, QMBs allow a larger variety of masses for a given magnetic charge. QMBs can have large magnetic charges with masses below the Planck scale, not allowed for black holes. QMBs can also exist in the parameter region where magnetic BHs would have evaporated to extremal (see ﬁgure 4, between the black horizontal line and black shaded region). 3 Multiply magnetically charged QMBs (q > 2) JHEP01(2022)109 All of our derivations so far have neglected gravitational eﬀects. However, once the Schwarzschild radius rs = 2M/M2 pl is larger than the QMB radius, we expect such eﬀects to be important, potentially leading to a soliton star or black hole [4, 51, 52]. Comparing rs to (2.25), gravitational eﬀects are important when M(q,Q) ≳ r 3 16π λ−1/2 φ Mpl v 2 Mpl . (3.4) (3.4) This is denoted by the hatched region in ﬁgure 4. Larger v leads to more compact QMBs, so gravitational eﬀects are more important. 4 Early-universe formation of QMBs The QMB could be formed during a phase transition in the early universe at a temperature T ∼v. For example, if the phase transition is ﬁrst order, the true-vacuum bubbles could “snowplow” the global charge into a small pocket and form the Q-balls [7, 53]. The average global charge for the Q-balls depends on the symmetry breaking scale v, the rate of bubble nucleation for the phase transition, and the potentially nonzero initial matter-antimatter asymmetry for the S particles ηQ = \|nS −nS†\|/(nS +nS†) where nS(†) is the S (anti)particle number density. The QMB’s acquisition of magnetic charge is trickier and relies on the topological structure of the ﬁeld conﬁguration. The Kibble mechanism is usually adopted to estimate the formation abundance for a unit magnetic charge with q = 2. The probability to form a large winding number is usually suppressed, which will be also discussed later. Let us consider a ﬁrst order phase transition based on the ﬁnite-temperature potential of φa. We begin with the nontopological global charge, then later discuss the topological magnetic charge. The global charge of QMBs and Q-balls relies on the total number of S particles within one Hubble patch during the phase transition and the number of nucleation sites at the end of the phase transition. The total number of S particles (including both S and S†) within one Hubble volume at the temperature Tf when the phase transition ends is NHubble S ∼nS d3 H ∼(1 × 1046) v−3 3 , (4.1) (4.1) where v3 ≡v/(103 GeV), 1/dH = H(Tf) = p π2g∗/90 T 2 f /Mpl, and nS = (2ζ(3)/π2)T 3 f the (relativistic) S number density with g∗≈100 and Tf ∼v. The number of bubble where v3 ≡v/(103 GeV), 1/dH = H(Tf) = p π2g∗/90 T 2 f /Mpl, and nS = (2ζ(3)/π2)T 3 f the (relativistic) S number density with g∗≈100 and Tf ∼v. The number of bubble – 17 – nucleation sites per Hubble patch is [7] nucleation sites per Hubble patch is [7] nucleation sites per Hubble patch is [7] NHubble QMB ∼(1 × 1013) × λφS 3 −14 , (4.2) (4.2) where the large power 14 comes from a numerical ﬁt. Note that for large enough λφS ≳2, the phase transition is strongly ﬁrst order [7, 54, 55]. 4 Early-universe formation of QMBs So, the average number of both S and S† particles in one QMB is NQMB S ∼(1 × 1033) v−3 3 λφS 3 14 , (4.3) (4.3) JHEP01(2022)109 assuming most or all of the S particles remain in the false vacuum. Taking the particle- antiparticle annihilations into account, the typical Q charge for each QMB is (taking ηQ ≪2) ⟨QQMB⟩∼max (3 × 1016) v−3/2 3 λφS 3 7 , ηQ (1 × 1033) v−3 3 λφS 3 14 . (4.4) (4.4) The second term comes from if the asymmetric component dominates. Otherwise, the ﬁrst term gives the typical net charge from the symmetric component Q ∼(NQMB S )1/2. Since the net charge in this equation is typically large, the QMB’s mass is dominated by the Q-ball part, and the average mass of QMBs at their formation is ⟨MQMB⟩≈⟨QQMB⟩Ωcv. The second term comes from if the asymmetric component dominates. Otherwise, the ﬁrst term gives the typical net charge from the symmetric component Q ∼(NQMB S )1/2. Since the net charge in this equation is typically large, the QMB’s mass is dominated by the Q-ball part, and the average mass of QMBs at their formation is ⟨MQMB⟩≈⟨QQMB⟩Ωcv. If QMB annihilations are not important, the yield of QMBs YQMB ∼NHubble QMB d−3 H s−1, with s = (2π2/45)g∗ST 3 f and g∗S ≈100, is constant. It can be compared to the observed dark matter abundance ΩDMh2 ≈0.12 [56], which corresponds to YDM ≈ 3.6 × 10−10(1 GeV/MDM). Thus, QMBs and Q-balls would make up a fraction fDM of dark matter If QMB annihilations are not important, the yield of QMBs YQMB ∼NHubble QMB d−3 H s−1, with s = (2π2/45)g∗ST 3 f and g∗S ≈100, is constant. It can be compared to the observed dark matter abundance ΩDMh2 ≈0.12 [56], which corresponds to YDM ≈ 3.6 × 10−10(1 GeV/MDM). Thus, QMBs and Q-balls would make up a fraction fDM of dark matter fDM ∼max (4 × 10−7)v5/2 3 3 λφS !7 , ηQ(2 × 1010)v3 Ωc . 4 Early-universe formation of QMBs (4.5) (4.5) For QMBs and Q-balls to account for all the dark matter abundance    asymmetric component dominates:                        asymmetric component dominates: ηQ ∼(6 × 10−11v−1 3 )Ω−1 c and v ≲(3 × 105 GeV) λφS 3 14/5 Ω−2/5 c symmetric component dominates: v ∼(3 × 105 GeV) λφS 3 14/5 Ω−2/5 c and ηQ ≲(2 × 10−13)Ω−1 c 3 λφS !14/5 .(4.6) ymmetric component dominates: ηQ ∼(6 × 10−11v−1 3 )Ω−1 c and v ≲(3 × 105 GeV) λφS 3 14/5 Ω−2/5 c .(4.6)     symmetric component dominates: mmetric component dominates: v ∼(3 × 105 GeV) λφS 3 14/5 Ω−2/5 c and ηQ ≲(2 × 10−13)Ω−1 c 3 λφS !14/5 The upper line corresponds to the asymmetric component dominating ⟨QQMB⟩, while the lower corresponds to statistical ﬂuctuations in the symmetric component dominating (the second and ﬁrst terms, respectively, of the max functions in (4.4)–(4.5)). Note that ηQ may be similar to the baryon asymmetry ηB ≈6 × 10−11, suggesting a potential shared asymmetry formation mechanism for both baryons and S (to which we remain agnostic – 18 – here). Thus, the typical QMB and Q-ball mass if they explain all of dark matter (fDM = 1) is ⟨MQMB⟩∼(8×1025 GeV) v−3 3 λφS 3 14 for v ≲(3×105 GeV) λφS 3 14/5 Ω−2/5 c . (4 The upper limit on v corresponds to a lower limit ⟨MQMB⟩≳(2×1018 GeV)(λφS/3)28/5Ω6/5 c pp p ⟨ QMB⟩≳( )( φS/ ) Topological defects form where multiple bubbles with diﬀerent symmetry-breaking phases meet. In a ﬁrst order phase transition, these topological defect formation sites coincide with where Q-balls form due to the snowplow mechanism — both form where multiple bubbles meet. Thus, where a monopole is formed, it is almost guaranteed to be already inside a Q-ball. This is true as long as the typical charge ⟨QQMB⟩≫Qs, which should generally be the case for the large charges in (4.4). Conversely, not every site where a Q-ball is formed will have a monopole. Most QMBs formed in such a manner will have initial magnetic charge q = 2. The probability for a larger winding number q > 2 for QMBs is suppressed for geometric reasons. 4 Early-universe formation of QMBs The number of true vacuum bubbles surrounding the pocket of false vacuum which forms a QMB determines the probability of the topological winding number. As a simple example, in two spatial dimensions, a vortex is formed with probability 1/4 when three bubbles meet. Three bubbles cannot form a vortex with larger than unit winding number — at least ﬁve bubbles must meet to form winding number two, and so forth for higher windings. Even if ﬁve bubbles meet, the probability to form winding number two is only ∼0.005. Similarly, for three spatial dimensions, the probability for four bubbles (in a tetrahedron shape) to form a unit winding number is 1/8, and higher monopole charges require the conﬂuence of more bubbles [57–60]. The probability to form higher monopole charges q > 2 is thus suppressed by both the probability for many (> 4) bubbles to collide at approximately the same point and the probability for those bubbles to produce a higher winding number. For simplicity, we parameterize the probability of Q-balls to carry a unit magnetic charge to be p2 between about 10−1 and 10−2. So, at the end of the phase transition, there are a fractional abundance of 1 −p2 Q-ball states and p2 QMB states with magnetic charge q = 2. The fraction of QMBs carrying a larger q and the fraction of isolated monopoles without a global charge are suppressed. JHEP01(2022)109 After the formation of Q-balls and QMBs, the charge distributions could continue to evolve via the so-called “solitosynthesis” [61]. This can proceed in two ways: (a) Q- balls and QMBs can merge if they have same-sign global charge or (partially) annihilate if opposite-sign, and (b) Q-balls and QMBs can absorb unbound S particles. For a suﬃciently strong ﬁrst order phase transition corresponding to larger λφS, (b) can be approximately neglected because almost all S particles will be bound inside Q-balls or QMBs [7]. Further, a simple estimate reveals (a) to be unimportant. Assuming for simplicity that all Q-balls and QMBs have initial nontopological charge Q, the rate for two such objects to merge or annihilate is Γ = σ vrelnQ with σ ∼πR2 Q and RQ ∼Q1/3 v−1. 4 Early-universe formation of QMBs The relative speed is vrel ∼ q T/MQ, and the number density is nQ ∼TeqT 3/MQ with Teq ≈1 eV as the temperature of matter-radiation equality and assuming Q-balls and QMBs account for all dark matter or fDM = 1. Comparing Γ to the Hubble rate H(T) ∼T 2/Mpl, the interaction – 19 – rate is important when Q < 6×104 v−12/5 3 with T ∼v. Comparing this to the typical charge in (4.4), the additional charge evolution eﬀects (in both q and Q) are not important in this formation mechanism. Here, we have taken the geometric cross section of the QMBs for σ. Note that the cross section for magnetic monopole interactions is generally much larger and plays a role in annihilations of isolated monopoles [62]. However, we have veriﬁed that this eﬀect is unimportant for QMBs carrying small q ∼2 and large mass M(q,Q) ≫M(2,0). (q,Q) ( , ) If the phase transition is second order, Q-balls can form in ﬁnite regions of false vac- uum. The probability for a region to remain in the false vacuum is determined by the Boltzmann distribution comparing the region’s free energy to the Ginzburg temperature (the temperature at which thermal ﬂuctuations between the false and true vacua freeze out) [63–65]. Then, Q-balls form in these false vacuum regions either due to an initial asymmetry ηQ or statistical ﬂuctuations in the diﬀerence of S and S† particles, similar to the ﬁrst order phase transition. Meanwhile, monopoles can form isolated from Q-balls via the Kibble-Zurek mechanism [66, 67]. Unlike the ﬁrst order phase transition, the S particles are not snowplowed to the boundaries between diﬀerent coherent regions of symmetry-breaking phases, so Q-balls and monopoles need not initially overlap. Addi- tionally, it cannot be assumed that few free S particles remain unbound after the phase transition. Thus, following the phase transition, the charges of the states may evolve. If Qs is suﬃciently small, the monopoles could form bound states with free S particles, building up their U(1)S charge Q either due to an asymmetry in S particles or due to ﬂuctuations in a 1-dimensional random walk in Q. Importantly, because Qs can be arbitrarily small for QMBs (contrary to Q-balls), it is easier to build QMBs from fusion of free S particles than it is to build Q-balls, the latter of which requires many S particles (> Qs) to meet simul- taneously [61, 65]. 4 Early-universe formation of QMBs Thus, solitosynthesis can more easily produce a population of QMBs. Additionally, such monopoles or QMBs could encounter isolated Q-balls and become bound by them. Q-balls and QMBs could also be destroyed by free (anti)particles if their charge is annihilated below Qs [61]. The details of the many types of interactions between free particles and bound states require tracking of interactions of the many diﬀerent possible states, which is beyond the scope of this work. JHEP01(2022)109 5 Implications for phenomenology The Parker bound [68, 69] states that coherent magnetic ﬁelds cannot be drained of energy by accelerated monopoles, otherwise we would not observe them. When a monopole passes through a coherent magnetic ﬁeld, it is accelerated to a speed vmag ≃min  1, s 2B(2πq/e)ℓc M  ≃min 1, 4 × 10−5 r q M 5.2Mplℓ21B3 , (5.1) (5.1) where ℓ21 = ℓc/(1021 cm) is the coherent length of the magnetic ﬁeld with strength B3 = B/(3 µG). The Parker bound was initially derived for Milky Way coherent ﬁelds. It was recently extended to M31 [47], which has larger-scale approximately axisymmetric magnetic ﬁelds in its disc with ℓ21 ∼30 [70] and a corresponding regeneration time treg ∼10 Gyr [71]. – 20 – Its strength is B3 ∼5/3 [70], and its local dark matter (DM) density and escape velocity are similar to the Milky Way: ρ0.4 = ρ/(0.4 GeV cm−3) [72, 73] and v ∼10−3. The M31 Parker bound on the DM energy density fraction of QMBs fDM = ρQMB/ρDM thus takes the form Its strength is B3 ∼5/3 [70], and its local dark matter (DM) density and escape velocity are similar to the Milky Way: ρ0.4 = ρ/(0.4 GeV cm−3) [72, 73] and v ∼10−3. The M31 Parker bound on the DM energy density fraction of QMBs fDM = ρQMB/ρDM thus takes the form fDM ≲                6 × 10−3 M 5.2 q Mpl !2 v−3 ρ0.4(ℓ21/30)(t15/300) , vmag ≲10−3 102 M 5.2 q Mpl ! B3/(5/3) v−3(ρ0.4/10−6)(t15/300) , vmag ≳10−3 . (5.2) (5.2) JHEP01(2022)109 Here, v−3 = v/10−3 is the velocity of the QMBs. If vmag ≲10−3, the QMBs can be bound in the galaxy with v−3 ∼1 and could explain DM. Otherwise, they cannot be bound and must have v−3 ≳1; in that case, they cannot explain all of DM. If they are bound, fDM is compared to the local DM density ρ0.4 ∼1; otherwise, if they are unbound fDM is compared to the average DM density ρ0.4 ∼10−6. These diﬀerences in ρ are reﬂected in the top and bottom lines of (5.2). The parameter t15 = treg/(1015 s). This bound is represented by lilac dashed lines in ﬁgure 4. 5 Implications for phenomenology The most constraining direct search for q ≥2 monopoles comes from searches for tracks in ancient mica [15], which limits fDM < (M/1023 GeV) (about an order of magnitude stronger than the strongest “laboratory” experiment MACRO [14]). Note, we expect the sensitivity of these searches is roughly insensitive to the magnetic charge q. They are slightly more constraining than the Parker bound for small q but subdominant at large q. Direct detection constraints could also apply independent of the magnetic charge, especially if the symmetry breaking in the full theory makes the QMB interior electroweak symmetric [7, 45, 74, 75]. These are most stringent for smaller v (near the electroweak scale) because the QMB radius goes as 1/v in the large-Q limit. They can be completely alleviated at larger v, reducing the eﬀective cross section. Other model-dependent bounds on magnetically charged objects can be found in refs. [19, 47, 76–78], though things like stopping power in materials may need to be re- calculated to apply some of these bounds to QMBs (especially if the QMB radius is large). Additionally, magnetic monopoles could be detected via their eﬀects as they pass through the atmosphere. Previous atmospheric studies have focused on states with dipole rather than monopole magnetic ﬁelds [79, 80] or with a ﬁxed geometric scattering cross section [81] and would need to be reinterpreted accordingly. We brieﬂy mention a novel proposal for a detection strategy, although it is unlikely to compete against other established bounds unless undertaken at great scale. Magnetic anomaly detectors (MADs) [82] oﬀer another interesting method to search for passing magnetically charged particles like QMBs that has not been previously considered in lit- erature to our knowledge. The advantage of such detectors is that they are commercially available, compact, and work at room temperature. They have typical sensitivities of order SMAD ∼1 to 103 fT/ √ Hz (see, e.g., [83–86]). A passing QMB, which generates a monopole magnetic ﬁeld Bm = (10−6 fT)(q/2)(km/r)2 at a distance r from its center, could generate a signal in a MAD. If bound in the galaxy, its velocity is v ∼10−3, so the passing time for – 21 – it to be within a distance r from a MAD is tpass ∼(3×10−3 s)(r/km)(v−3)−1. To be above the MAD threshold, Bm √tpass > SMAD is required. 5 Implications for phenomenology Using the local density of dark matter, the number of QMBs that pass within a distance r of a MAD during an exposure time T is N ∼fDM(T/s)(1017 GeV/M)(r/km)2v−3 ρ0.4. Allowing there to be NMAD diﬀerent MAD detectors in an experiment (spaced suﬃciently far apart that their eﬀective sensitive areas do not overlap), the number of detected QMBs Ndet above the MAD threshold sensitivity is thus Ndet ∼7 × 10−7 NMAD fDM q4/3 1017 GeV M ! fT/ √ Hz SMAD !4/3 T yr (v−3)1/3 . (5.3) (5.3) JHEP01(2022)109 Additionally, MADs may lose sensitivity at small frequencies, for example of order Hz for SQUID detectors [87]. If we conservatively require tpass < tpass,max = 1 s, this limits the eﬀective distance of closest approach to r < 300 km (tpass,max/s). Then, there is a bound on Ndet going as Ndet ≲4 × 1012 NMAD fDM 1017 GeV M ! T yr tpass,max s 2 . (5.4) (5.4) Unlike (5.3), this is independent of q, so it sets an upper mass reach. A search could be sensitive when Ndet ≳3, assuming no backgrounds. To reduce backgrounds, a lattice conﬁguration of MADs could be used to look for correlated signals of a passing QMB. A less sensitive network of over 100 detectors, accurate to the nT level, is already in place for monitoring the geomagnetic ﬁeld [88]. Compared to other direct searches, this search strategy has the advantage that one detector can cover a large cross sectional area, espe- cially for larger q. However, barring a very large number NMAD or very sensitive detectors with much smaller SMAD, the Parker bound above tends to give a stronger constraint on fDM. Note, these sensitivities do not account for the possibility that large-q and small-M QMBs could be stopped in the atmosphere. 6 Conclusions In this work, we have established the existence of a new physical state carrying both topological and nontopological charge. In the analytically tractable case of unit magnetic charge q = 2, we have solved the spherically symmetric equations of motion for the ﬁelds to explicitly demonstrate the QMB properties and stability. We have further argued that states with q > 2 should also be stable. This is the ﬁrst such example of stable q > 2 monopoles away from the BPS limit that are bound nongravitationally. QMBs with large magnetic charge could exist with masses either well above or below the Planck scale. The theoretical structure required for such states to exist is surprisingly minimal. If a theory contains a magnetic monopole (already favored as an explanation for the quantization of electric charge), then QMB states can form given only one new gauge-singlet complex scalar ﬁeld with a good U(1) global symmetry and a renormalizable portal coupling to the gauged scalar of the monopole sector. – 22 – QMBs can form in a phase transition in the early universe. According to our estimates, QMBs formed in such a manner may have masses around or in excess of the Planck scale, as well as large radii far in excess of their intrinsic scale 1/v. A mixture of QMBs and Q-balls may even make up all of dark matter. We have provided an analytic estimate for the abundance and average charge of QMBs following a phase transition. The existence of QMBs allows for new and interesting phenomena in their formation and evolution. First, Q-balls can aggregate monopoles as they cross through the interior of Q-balls and form possibly multiply magnetically charged bound states. Second, monopoles can act as seeds of Q-ball formation following a phase transition. Because the minimum stable global charge Qs is smaller for a QMB than for a Q-ball, sometimes as small as unity, it is easier to form QMBs from free S particles and isolated monopoles than it is to form Q-balls from S particle fusion [61]. Detailed numerical work similar to solitosynthesis on this formation mechanism, though beyond the scope of this work, would be of interest to verify our estimates. As an extension to this work, it would be interesting to examine the likelihood of forming magnetic black holes through this approach. 6 Conclusions If possible, the QMBs may serve as a novel formation mechanism for magnetic black holes in addition to the collapse of primordial density perturbations. JHEP01(2022)109 As the existence of monopole bound states could be a generic feature of high energy the- ories containing global charge, QMBs should be considered when devising and interpreting monopole searches. We have discussed how the Parker bound and direct monopole searches apply to QMBs. However, many more monopole search strategies exist [19, 47, 76–78] or could be recast from similar searches [79–81]. Several of these depend on the stopping power of monopoles in various materials (the Sun, the Earth, interstellar gases, etc.). QMBs may have electroweak or Grand Uniﬁed Theory symmetry restoration in their (potentially large radii) cores, giving them a potentially large geometric cross section with Standard Model matter or the ability to mediate baryon number violation. By establishing the existence of QMB states, we have shown that magnetically charged objects can have a larger variety of mass and magnetic charge than previously envisioned. Thus, an interesting topic of future study, beyond the scope of this work, would be to recast existing monopole bounds and to propose new search strategies for arbitrary values of mass, radius, and magnetic charge. Acknowledgments The work of YB is supported by the U.S. Department of Energy under the contract DE- SC-0017647. The work of SL is supported in part by Israel Science Foundation under Grant No. 1302/19. The work of NO is supported by the Arthur B. McDonald Canadian Astroparticle Physics Research Institute. Open Access. This article is distributed under the terms of the Creative Commons Attribution License (CC-BY 4.0), which permits any use, distribution and reproduction in any medium, provided the original author(s) and source are credited. – 23 – References [1] G. Rosen, Particlelike solutions to nonlinear complex scalar ﬁeld theories with positive-deﬁnite energy densities, J. Math. Phys. 9 (1968) 996 [INSPIRE]. [2] R. Friedberg, T.D. Lee and A. Sirlin, A class of scalar-ﬁeld soliton solutions in three space dimensions, Phys. Rev. D 13 (1976) 2739 [INSPIRE]. [3] S.R. Coleman, Q-balls, Nucl. Phys. B 262 (1985) 263 [Addendum ibid. 269 (1986) 744] [INSPIRE]. [4] T.D. Lee and Y. Pang, Nontopological solitons, Phys. Rept. 221 (1992) 251 [INSPIRE]. [5] E.Y. Nugaev and A.V. Shkerin, Review of nontopological solitons in theories with U(1)-symmetry, J. Exp. Theor. Phys. 130 (2020) 301 [arXiv:1905.05146] [INSPIRE]. JHEP01(2022)109 JHEP01(2022)109 [6] A. Kusenko and M.E. Shaposhnikov, Supersymmetric Q balls as dark matter, Phys. Lett. B 418 (1998) 46 [hep-ph/9709492] [INSPIRE]. [7] E. Pontón, Y. Bai and B. Jain, Electroweak symmetric dark matter balls, JHEP 09 (2019) 011 [arXiv:1906.10739] [INSPIRE]. [8] P.A.M. Dirac, Quantised singularities in the electromagnetic ﬁeld, Proc. Roy. Soc. Lond. A 133 (1931) 60 [INSPIRE]. [9] G. ’t Hooft, Magnetic monopoles in uniﬁed gauge theories, Nucl. Phys. B 79 (1974) 276 [INSPIRE]. [10] A.M. Polyakov, Particle spectrum in quantum ﬁeld theory, JETP Lett. 20 (1974) 194 [Pisma Zh. Eksp. Teor. Fiz. 20 (1974) 430] [INSPIRE]. [11] H. Georgi and S.L. Glashow, Unity of all elementary particle forces, Phys. Rev. Lett. 32 (1974) 438 [INSPIRE]. [12] Y.M. Cho and D. Maison, Monopoles in Weinberg-Salam model, Phys. Lett. B 391 (1997) 360 [hep-th/9601028] [INSPIRE]. [13] B. Cabrera, First results from a superconductive detector for moving magnetic monopoles, Phys. Rev. Lett. 48 (1982) 1378 [INSPIRE]. [14] MACRO collaboration, Final results of magnetic monopole searches with the MACRO experiment, Eur. Phys. J. C 25 (2002) 511 [hep-ex/0207020] [INSPIRE]. [15] D. Ghosh and S. Chatterjea, Supermassive magnetic monopoles ﬂux from the oldest mica samples, Europhys. Lett. 12 (1990) 25 [INSPIRE]. [15] D. Ghosh and S. Chatterjea, Supermassive magnetic monopoles ﬂux from the oldest mica samples, Europhys. Lett. 12 (1990) 25 [INSPIRE]. [16] IceCube collaboration, Searches for relativistic magnetic monopoles in IceCube, Eur. Phys. J. C 76 (2016) 133 [arXiv:1511.01350] [INSPIRE]. [16] IceCube collaboration, Searches for relativistic magnetic monopoles in IceCube, Eur. Phys. J. C 76 (2016) 133 [arXiv:1511.01350] [INSPIRE]. 7] MoEDAL collaboration, The physics programme Of The MoEDAL experiment At The LHC, Int. J. Mod. Phys. A 29 (2014) 1430050 [arXiv:1405.7662] [INSPIRE]. [17] MoEDAL collaboration, The physics programme Of The MoEDAL experiment At The LHC, Int. J. Mod. Phys. References A 29 (2014) 1430050 [arXiv:1405.7662] [INSPIRE]. [18] O. Gould and A. Rajantie, Magnetic monopole mass bounds from heavy ion collisions and neutron stars, Phys. Rev. Lett. 119 (2017) 241601 [arXiv:1705.07052] [INSPIRE]. [19] N.E. Mavromatos and V.A. Mitsou, Magnetic monopoles revisited: Models and searches at colliders and in the Cosmos, Int. J. Mod. Phys. A 35 (2020) 2030012 [arXiv:2005.05100] [INSPIRE]. [20] K.-M. Lee, J.A. Stein-Schabes, R. Watkins and L.M. Widrow, Gauged Q balls, Phys. Rev. D 39 (1989) 1665 [INSPIRE]. – 24 – [21] I.E. Gulamov, E.Y. Nugaev, A.G. Panin and M.N. Smolyakov, Some properties of U(1) gauged Q-balls, Phys. Rev. D 92 (2015) 045011 [arXiv:1506.05786] [INSPIRE]. [22] Y. Brihaye, A. Cisterna, B. Hartmann and G. Luchini, From topological to nontopological solitons: kinks, domain walls, and Q-balls in a scalar ﬁeld model with a nontrivial vacuum manifold, Phys. Rev. D 92 (2015) 124061 [arXiv:1511.02757] [INSPIRE]. [23] J. Heeck, A. Rajaraman, R. Riley and C.B. Verhaaren, Mapping gauged Q-balls, Phys. Rev. D 103 (2021) 116004 [arXiv:2103.06905] [INSPIRE]. [24] E.B. Bogomolny, Stability of classical solutions, Sov. J. Nucl. Phys. 24 (1976) 449 [Yad. Fiz. 24 (1976) 861] [INSPIRE]. JHEP01(2022)109 JHEP01(2022)109 [25] M.K. Prasad and C.M. Sommerﬁeld, An exact classical solution for the ’t Hooft monopole and the Julia-Zee dyon, Phys. Rev. Lett. 35 (1975) 760 [INSPIRE]. [26] E.J. Weinberg and A.H. Guth, Nonexistence of spherically symmetric monopoles with multiple magnetic charge, Phys. Rev. D 14 (1976) 1660 [INSPIRE]. [27] S. Bolognesi, Multi-monopoles and magnetic bags, Nucl. Phys. B 752 (2006) 93 [hep-th/0512133] [INSPIRE]. [28] K.-M. Lee and E.J. Weinberg, BPS magnetic monopole bags, Phys. Rev. D 79 (2009) 025013 [arXiv:0810.4962] [INSPIRE]. [29] N.J. Hitchin, N.S. Manton and M.K. Murray, Symmetric monopoles, Nonlinearity 8 (1995) 661 [dg-ga/9503016] [INSPIRE]. [30] C.J. Houghton and P.M. Sutcliﬀe, Tetrahedral and cubic monopoles, Commun. Math. Phys. 180 (1996) 343 [hep-th/9601146] [INSPIRE]. [31] C.J. Houghton and P.M. Sutcliﬀe, Octahedral and dodecahedral monopoles, Nonlinearity 9 (1996) 385 [hep-th/9601147] [INSPIRE]. [32] N.S. Manton, Monopole planets and galaxies, Phys. Rev. D 85 (2012) 045022 [arXiv:1111.2934] [INSPIRE]. [33] J. Evslin and S.B. Gudnason, High Q BPS monopole bags are urchins, Int. J. Mod. Phys. A 29 (2014) 1450004 [arXiv:1111.3891] [INSPIRE]. [34] J. Evslin and S.B. Gudnason, Dwarf galaxy sized monopoles as dark matter?, arXiv:1202.0560 [INSPIRE]. [35] S. Bolognesi, Magnetic bags and black holes, Nucl. Phys. B 845 (2011) 324 [arXiv:1005.4642] [INSPIRE]. [36] K.-M. Lee and E.J. References Weinberg, Nontopological magnetic monopoles and new magnetically charged black holes, Phys. Rev. Lett. 73 (1994) 1203 [hep-th/9406021] [INSPIRE]. [37] J. Maldacena, Comments on magnetic black holes, JHEP 04 (2021) 079 [arXiv:2004.0608 [INSPIRE]. [38] Y. Nambu, String-like conﬁgurations in the Weinberg-Salam theory, Nucl. Phys. B 130 (1977) 505 [INSPIRE]. [39] M. Hindmarsh and T.W.B. Kibble, Beads on strings, Phys. Rev. Lett. 55 (1985) 2398 [INSPIRE]. [40] V. Berezinsky and A. Vilenkin, Cosmic necklaces and ultrahigh-energy cosmic rays, Phys. Rev. Lett. 79 (1997) 5202 [astro-ph/9704257] [INSPIRE]. – 25 – [41] M. Hindmarsh, K. Rummukainen and D.J. Weir, Numerical simulations of necklaces in SU(2) gauge-Higgs ﬁeld theory, Phys. Rev. D 95 (2017) 063520 [arXiv:1611.08456] [INSPIRE]. [42] S.B. Gudnason and J. Evslin, Global monopoles of charge 2, Phys. Rev. D 92 (2015) 045044 [arXiv:1507.03400] [INSPIRE]. [43] P. Peter, Superconducting cosmic string: Equation of state for space-like and time - like current in the neutral limit, Phys. Rev. D 45 (1992) 1091 [INSPIRE]. [44] R.A. Battye and S.J. Cotterill, Stable cosmic vortons in bosonic ﬁeld theory, Phys. Rev. Lett. 127 (2021) 241601 [arXiv:2111.07822] [INSPIRE]. [45] Y. Bai, M. Korwar and N. Orlofsky, Electroweak-symmetric dark monopoles from preheating, JHEP 07 (2020) 167 [arXiv:2005.00503] [INSPIRE]. JHEP01(2022)109 JHEP01(2022)109 [46] D. Levkov, E. Nugaev and A. Popescu, The fate of small classically stable Q-balls, JHEP 12 (2017) 131 [arXiv:1711.05279] [INSPIRE]. [47] Y. Bai, J. Berger, M. Korwar and N. Orlofsky, Phenomenology of magnetic black holes with electroweak-symmetric coronas, JHEP 10 (2020) 210 [arXiv:2007.03703] [INSPIRE]. [48] Y. Bai and M. Korwar, Hairy magnetic and dyonic black holes in the standard model, JHEP 04 (2021) 119 [arXiv:2012.15430] [INSPIRE]. [49] Y. Bai and N. Orlofsky, Primordial extremal black holes as dark matter, Phys. Rev. D 101 (2020) 055006 [arXiv:1906.04858] [INSPIRE]. [50] B. Carr, K. Kohri, Y. Sendouda and J. Yokoyama, Constraints on primordial black holes, Rept. Prog. Phys. 84 (2021) 116902 [arXiv:2002.12778] [INSPIRE]. [51] T.D. Lee, Soliton stars and the critical masses of black holes, Phys. Rev. D 35 (1987) 3637 [INSPIRE]. [52] R. Friedberg, T.D. Lee and Y. Pang, Scalar soliton stars and black holes, Phys. Rev. D 35 (1987) 3658 [INSPIRE]. [53] E. Witten, Cosmic separation of phases, Phys. Rev. D 30 (1984) 272 [INSPIRE]. [54] A. Katz and M. Perelstein, Higgs couplings and electroweak phase transition, JHEP 07 (2014) 108 [arXiv:1401.1827] [INSPIRE]. [55] B. Jain, S.J. Lee and M. References Son, Validity of the eﬀective potential and the precision of Higgs ﬁeld self-couplings, Phys. Rev. D 98 (2018) 075002 [arXiv:1709.03232] [INSPIRE]. [56] Planck collaboration, Planck 2018 results. VI. Cosmological parameters, Astron. Astrophys. 641 (2020) A6 [Erratum ibid. 652 (2021) C4] [arXiv:1807.06209] [INSPIRE]. [57] T. Prokopec, Formation of topological and nontopological defects in the early universe, Phys. Lett. B 262 (1991) 215 [INSPIRE]. [58] R. Leese and T. Prokopec, Clustering of cosmological defects at the time of formation, Phys. Lett. B 260 (1991) 27 [INSPIRE]. [59] R.A. Leese and T. Prokopec, Monte Carlo simulation of texture formation, Phys. Rev. D 44 (1991) 3749 [INSPIRE]. [60] T. Okabe and M. Nagasawa, Formation of multiple winding topological defects in the early universe, Phys. Lett. B 461 (1999) 49 [hep-ph/9905401] [INSPIRE]. [61] K. Griest and E.W. Kolb, Solitosynthesis: cosmological evolution of nontopological solitons, Phys. Rev. D 40 (1989) 3231 [INSPIRE]. – 26 – [62] J. Preskill, Cosmological production of superheavy magnetic monopoles, Phys. Rev. Lett. 43 (1979) 1365 [INSPIRE]. [63] J.A. Frieman, G.B. Gelmini, M. Gleiser and E.W. Kolb, Solitogenesis: primordial origin of nontopological solitons, Phys. Rev. Lett. 60 (1988) 2101 [INSPIRE]. [64] K. Griest, E.W. Kolb and A. Massarotti, Statistical ﬂuctuations as the origin of nontopological solitons, Phys. Rev. D 40 (1989) 3529 [INSPIRE]. [65] J.A. Frieman, A.V. Olinto, M. Gleiser and C. Alcock, Cosmic evolution of nontopological solitons. 1, Phys. Rev. D 40 (1989) 3241 [INSPIRE]. [66] W.H. Zurek, Cosmological experiments in superﬂuid helium?, Nature 317 (1985) 505 [INSPIRE]. JHEP01(2022)109 JHEP01(2022)109 [67] H. Murayama and J. Shu, Topological dark matter, Phys. Lett. B 686 (2010) 162 [arXiv:0905.1720] [INSPIRE]. [68] E.N. Parker, The origin of magnetic ﬁelds, Astrophys. J. 160 (1970) 383 [INSPIRE]. [69] M.S. Turner, E.N. Parker and T.J. Bogdan, Magnetic monopoles and the survival of galactic magnetic ﬁelds, Phys. Rev. D 26 (1982) 1296 [INSPIRE]. [70] A. Fletcher, E.M. Berkhuijsen, R. Beck and A. Shukurov, The magnetic ﬁeld of M31 from multi-wavelength radio polarization observations, Astron. Astrophys. 414 (2004) 53 [astro-ph/0310258] [INSPIRE]. [71] T.G. Arshakian, R. Beck, M. Krause and D. Sokoloﬀ, Evolution of magnetic ﬁelds in galaxies and future observational tests with the Square Kilometre Array, Astron. Astrophys. 494 (2009) 21 [arXiv:0810.3114] [INSPIRE]. [72] A. Klypin, H. Zhao and R.S. Somerville, Lambda CDM-based models for the Milky Way and M31 I: dynamical models, Astrophys. J. 573 (2002) 597 [astro-ph/0110390] [INSPIRE]. [73] A. Tamm, E. Tempel, P. Tenjes, O. References Tihhonova and T. Tuvikene, Stellar mass map and dark matter distribution in M31, Astron. Astrophys. 546 (2012) A4 [arXiv:1208.5712] [INSPIRE]. [74] Y. Bai and J. Berger, Nucleus capture by macroscopic dark matter, JHEP 05 (2020) 160 [arXiv:1912.02813] [INSPIRE]. [75] P. Adhikari et al., First direct detection constraints on Planck-scale mass dark matter with multiple-scatter signatures using the DEAP-3600 detector, Phys. Rev. Lett. 128 (2022) 011801 [arXiv:2108.09405] [INSPIRE]. [76] D. Ghosh, A. Thalapillil and F. Ullah, Astrophysical hints for magnetic black holes, Phys. Rev. D 103 (2021) 023006 [arXiv:2009.03363] [INSPIRE]. [77] M.D. Diamond and D.E. Kaplan, Constraints on relic magnetic black holes, arXiv:2103.01850 [INSPIRE]. [78] Y. Bai, S. Lu and N. Orlofsky, Searching for magnetic monopoles with the Earth’s magnetic ﬁeld, Phys. Rev. Lett. 127 (2021) 101801 [arXiv:2103.06286] [INSPIRE]. [79] J.P. VanDevender et al., Detection of magnetized quark-nuggets, a candidate for dark matter, Sci. Rep. 7 (2017) 8758 [arXiv:1708.07490] [INSPIRE]. [80] T. Sloan et al., Magnetised quark nuggets in the atmosphere, Sci. Rep. 11 (2021) 22432 [arXiv:2109.14480] [INSPIRE]. – 27 – [81] J.S. Sidhu and G. Starkman, Macroscopic dark matter constraints from bolide camera networks, Phys. Rev. D 100 (2019) 123008 [arXiv:1908.00557] [INSPIRE]. [82] Y. Zhao et al., A brief review of magnetic anomaly detection, Meas. Sci. Techol. 32 (2020) 042002. [83] I. Kominis, T. Kornack, J. Allred and M. Romalis, A subfemtotesla multichannel atomic magnetometer, Nature 422 (2003) 596. [84] H. B. Dang, A. C. Maloof and M.V. Romalis, Ultrahigh sensitivity magnetic ﬁeld and magnetization measurements with an atomic magnetometer, Appl. Phys. Lett. 97 (2010) 151110. JHEP01(2022)109 [85] D. A. Keder, D. W. Prescott, A. W. Conovaloﬀand K.L. Sauer, An unshielded radio-frequency atomic magnetometer with sub-femtoTesla sensitivity, AIP Adv. 4 (2014) 127159. radio-frequency atomic magnetometer with sub-femtoTesla sensitivity, AIP Adv. 4 (2014) 127159. [86] R. Li et al., A dual-axis, high-sensitivity atomic magnetometer, Chin. Phys. B 26 (2017) 120702. [87] P Ripka Magnetic sensors and magnetometers Artech house London U K (2021) [86] R. Li et al., A dual-axis, high-sensitivity atomic magnetometer, Chin. Phys. B 26 (2017) 120702. [87] P. Ripka, Magnetic sensors and magnetometers, Artech house, London U.K. (2021). [87] P. Ripka, Magnetic sensors and magnetometers, Artech house, London U.K. (2021). [88] J.J. Love, Magnetic monitoring of earth and space, Phys. Today 61 (2008) 31. [88] J.J. Love, Magnetic monitoring of earth and space, Phys. Today 61 (2008) 31. – 28 –
https://openalex.org/W2080721256	https://zenodo.org/records/1996196/files/article.pdf	English	null	THE INCREASE OF INSANITY.	Lancet	1,907	public-domain	1,864	To the Editors of THE LANCET. To the Editors of THE LANCET. SiBS,—My attention having been called to a letter by my friend, Dr. M. S. Paterson, of the Frimley Sanatorium, in your issue of March 23rd, apropos of an article by Mr. C. 1-1. Garland and myself under the above title, may I be allowed to correct what seems to be a misconception when he infers that our views were based on our visits to Frimley ? Our scheme was framed originally in 1903 and 1904 and in the inclosed copy of my interim report as chairman of the Sitep and Buildings Subcommittee (p. 7, Fourthly) printed for March llth, 1904, you will find that our advocacy of the adoption of the system of employing selected patients in saratorilims-which, by the way, is no new thing-antedated not only our visits to the Brompton Hospital Sanatorium by a few years but also, I believe, the erection and opening and Dr. Paterson’s appointment thereto. With regard to the infantile death-rate, it should be con- sidered that the wealthy class in Paignton are largely people who come here after retiring from the army, navy, or business, and are therefore for the most part past middle life. It follows that by far the larger number of births are among the artisans and labouring classes amongst whom the rate is always high I am Sirs yours faithfully appointment The scheme of the National Association for Establishing and Maintaining Sanatoria, for which I have had the honour and pleasure of working continuously since its inception, has been developed almost without modification on the lines laid down in the report quoted. The other medical members referred to therein were Dr. A. P. Hillier and Mrs. Percy Alden, who were both unfortunately obliged to give up this work by other public calls upon them, but not before giving very substantial assistance. Dr. Paterson has achieved a striking piece of organisation and administration, and no one engaged on the development of similar methods on a large scalE in England can afford to ignore his demonstration of the possi- bilities of this work. I wish to take this opportunity, in case our article did not convey our gratitude as sufficiently as it might have done, of thanking Dr. Paterson and his colleagues for enabling Mr. Garland and myself, as well as Dr. L. 70 the Editors of THE LANCET. SIRS,-L have seen in THE LANCET of March 23rd an article on the subject of the forthcoming International Red Cross Conference. The statements it contains require correction. In regard to no official intimation of the conference having been given, an official notice was sent to you and was published in THE LANCET on Feb. 16th. It was also extensively circulated throughout the press of the country. I inclose a fresh copy of this notice which fully states who are responsible for the arrangements and where the meetings will be held. Further than this, you had on Feb. 2nd already published an article giving the same information, and in regard to the exhibition we advertised it in THE LANCET of, I believe, Feb. 9th. , g y I am, Sirs, yours faithfully, To the Editors of THE LANCET. SIRS,-I greatly regret that I should have caused annoyance to Dr. Marr by my suggestion that the difference between the increase among general paralytics in Glasgow and in Edinburgh might be the result of better clinical observation in the latter. For this suggestion I have no ground, but still it is a very difficult thing to believe that it is a fact that the disease increases more in Edinburgh than in Glasgow, and I ought to have put it that it probably was a question of difference of diagnosis. Any wav I am certain that the careful examination of patients at Woodilee leaves nothing to be desired. Yours truly, be desired. ou s truly, -. Vevonshire-place, W., April 9th, 1907. GEO. H. SAVAGE. u s truly, -. GEO. H. SAVAGE. right I consider the statement of your local critic as to illness in the town over-coloured and made upon hearsay evidence. My own son is one of those who are at present suffering from scarlet fever, and the most careful research fails to show how it was contracted or any connexion with the few other cases in Paignton. The question of an addi- tional ward at the hospital should be considered to overcome any future difficulty caused by the wards being occupied by enteric cases, but I believe the fact that the hospital is empty for months at a time could easily be proved. Your correspondent has omitted to point out that last summer a large sum was spent on the outfall of the sewer, that a break was located and mended, and the sewer was carried 250 feet farther out to sea. With regard to the water, I can only say that I have never had to boil it and my Pasteur filter shows no trace or indication of any organic matter being contained in the water. truly, -. Vevonshire-place, W., April 9th, 1907. GEO. H. SAVAGE. Vevonshire-place, W., April 9th, 1907. Vevonshire-place, W., April 9th, 1907. To the Editors of THE LANCET. Crossley, the medical superintendent of our first sana- torium at Benenden, to see what was being done at Frimley. I Sirs faithfully always high. am, Sirs, yours faithfully, Paignton, April 4th, 1907. W. PIERPOINT ROBERTS. always high. a , S s, yours faithfully, Paignton, April 4th, 1907. W. PIERPOINT ROBERTS. 1041 1041 1041 A.C. under "normal " do not pretend to represent accurate measurements of B.C. and A.C. the road to be made 24 feet wide to form a footpath with curb and channel if the council will lay the sewer and ballast and form the new piece of road. The question whether this was a new road within the meaning of the Private Street Works Act was raised at the Local Government Board inquiry and disregarded by the inspector, and the sanction for the loan has been received. The new water scheme referred to by you is an auxiliary scheme for distributing the water to the higher levels of the town. The main water scheme is finished, and the water which has been brought 18 miles from the centre of the moor is now running into the home reservoirs, and the formal opening is fixed for June. The total cost of this portion of the scheme is £120,000, and both Teignmouth and Brixham will be supplied from Paignton. The dispute as to the relative values of the three schemes submitted to the council is purely an engineering question and cannot affect the health of the town in any shape or form, but the fact that the council has decided to leave it in the hands of the engineer, who is responsible for and has carried out the main scheme, would seem to be the right course to have taken. A.C. under "normal " do not pretend to represent accurate measurements of B.C. and A.C. asurements of B.C. and A.C. I am, Sirs, yours faithfully, asurements B.C. I am, Sirs, yours faithfully, rements .C. I am, Sirs, yours faithfully, Sirs, yours faithfully, - - - -.. D. MATHESON MACKAY, M.D. Edin., Sirs, yours faithfully, - - - -.. D. MATHESON MACKAY, M.D. Edin., Sirs, yours faithfully, - - - -.. D. MATHESON MACKAY, M.D. Edin., , , Late Senior Clinical Assistant, Hospital for Diseases of the Throat, Golden-square, London, W. Hull, March 22nd, 1907. y y Believe me, yours faithfully. J a,. Signed) J. DANVERS POWER, Chairman, Conference Committee. British Red Cross Society, 9. Victoria-street, London, S.W., March 27th. 1907. March 27th, 1907. - * We regret to have been misled with regard to the steps taken by the Red Cross Society for giving publicity to the Conference of 1907. We refer our readers to an editorial article in THE LANCET of Feb. 2nd, p. 311.-ED. L. SANITARY ADMINISTRATION IN PAIGNTON. of, believe, I may add that all the Central Red Cross Societies who are entitled to send delegates to the Conference have been made fully acquainted with the manner in which it has to be conducted, that having been prescribed at the last conference at St. Petersburg in 1902. EIGHTH INTERNATIONAL CONFERENCE OF BED CROSS SOCIETIES. 70 the Editors of THE LANCET. To the Editors of THE LANCET. To the Editors of THE LANCET. SIRS,-I have had a copy of your editorial note under the above heading sent to me, and as a resident in Paignton for the past 18 years and recently a member of the urban district council I wish to reply to some points upon which I venture to think you have been misled by the report of the medical officer of health, who has evidently overlooked the fact that the report may come into the hands of those who have no local knowledge. Petersburg In reference to your general remarks about this society perhaps you will allow me to send the inclosed pamphlet which explains its scope and origin. I shall have much pleasure at any time in providing you with the fullest information should you give me the opportunity, and I think you will find that the Conference will be conducted in every way in a manner worthy of the occasion. way worthy Believe me, yours faithfully. J a,. Signed) J. DANVERS POWER, Chairman, Conference Committee. British Red Cross Society, 9. Victoria-street, London, S.W., March 27th. 1907. March 27th, 1907. - knowledge. I will take the case of the supposed disregard of, the by-laws, where it is said that the council is permitting a road 600 feet long to be only 24 feet wide. The report does not state that this is a parish road leading from Paignton to Marldon and having an average width of 13 feet. All the council could compel the adjoining owners to do in the event of houses being erected would be to keep the frontages back 18 feet from the centre of the road. An owner has offered to give a strip of land which would enable worthy Believe me, yours faithfully. J a,. way worthy Believe me, yours faithfully. J a,. Signed) J. DANVERS POWER, Chairman, Conference Committee. British Red Cross Society, 9. Victoria-street, London, S.W., March 27th. 1907. March 27th, 1907. - * We regret to have been misled with regard to the steps taken by the Red Cross Society for giving publicity to the Conference of 1907. We refer our readers to an editorial article in THE LANCET of Feb. 2nd, p. 311.-ED. L. , * We regret to have been misled with regard to the steps taken by the Red Cross Society for giving publicity to the Conference of 1907. , * We regret to have been misled with regard to the steps taken by the Red Cross Society for giving publicity to the Conference of 1907. We refer our readers to an editorial article in THE LANCET of Feb. 2nd, p. 311.-ED. L. To the Editors of THE LANCET. We refer our readers to an editorial article in THE LANCET of Feb. 2nd, p. 311.-ED. L.
https://openalex.org/W4386109783	https://journal.uokufa.edu.iq/index.php/kufa_arts/article/download/692/635	Chinese	null	المستوي التركيبي في الدعاء القرآني	Ādāb al-Kūfaẗ/Ādāb al-kūfaẗ	2,021	cc-by	6,482	) ٥١١ ( ) ٥١١ ( ﺍﳌﺴﺘﻮﻱ ﺍﻟﱰﻛﻴﺒﻲ ﰲ ﺍﻟﺪﻋﺎء ﺍﻟﻘﺮﺁﻧﻲ ﺍﳌﻠﺨﺺ     آمء اا ه ّا ل و ا و أ   ا  ا ف. ا  أل دراا ي ا    ي اآمء ا ا   دورا ّ   ءا و   م ا  ا ا ا  و   م  ا ا ّ أنت اا   ءا    م ا ااا وا  و ظ    اا وا و  ّع ء وع ا     و و ا .و اار ظا  و ا   اأ د   ام  ت  و ّة  ا  ا أنّ و ا ا  ظ   ا و م   رة وا . ﻜﺍﻟ ﻠﻤﺎﺕ ﺍﻟﺮﺋ ﺔﻴﺴﻴ اء،ا ،اآما ي ا ،ا  ، ا .او ام ١ . ﺍﳌﻘﺪﻣﺔ ١.٢ ﻣﻨﻬﺞ ﺍﻟﺒﺤﺚ  ان ط  عا  ا ّد ا اا إ ط  ام ، و  ذ ما     ا - ا يا    تآ ءا و ا  ا  و  ا ّن  ءا اآم . ﺍﳌﺴﺘﻮﻱ ﺍﻟﱰﻛﻴﺒﻲ ﰲ ﺍﻟﺪﻋﺎء ﺍﻟﻘﺮﺁﻧﻲ ل  با     را ي ا وظ  ا  ا . « ) ّا ، ٧٩٩١  :٣٩ (    ت  ة  ا ي ا   و ا و ا و ا و      ا  ا ي ا    ءا آما يا ول درا ا  و ا ا    ءا   و ا و ا و درا ا و اام   ا و ا و .ا ا ضا » ا  ا ، اذ ا   ط ا و ا    ا  .ول ا دة  ه ا   ط،ّ ،وا    و  ا ط    .و ا ء ا رة،اا ا ي    ا ،أي ا ي ا   ت ا و ام  . « )ا ،٤٠٠٢  :٥٩١  ( و  ا م ة اّف ا   ا       تا اا ا ا ي و  ا و اا و   ا سا  ا ا   : ا ا و ا ا.     درا ا و اام   ا و ا و .ا ا ضا » ا  ا ، اذ ا   ط ا و ا    ا  .ول ا دة  ه ا   ط،ّ ،وا    و  ا ط    .و ا ء ا رة،اا ا ي    ا ،أي ا ي ا   ت ا و ام  . « )ا ،٤٠٠٢  :٥٩١  ( و  ا م ة اّف ا   ا       تا اا ا ا ي و  ا و اا و   ا سا  ا ا   : ا ا و ا ا. ١.١ ﺃﻫﺪﺍﻑ ﺍﻟﺒﺤﺚ ١.  و    ا  ا      ا  ا  ءا آما ب يا و با ما و ضا  اإ . ١.  و    ا  ا      ا  ا  ءا آما ب يا و با ما و ضا  اإ . ٢.  و    ا ا و ا و ضا  اا    . ٣.ا   ا   ا و ا و م ءا . ١ . ﺍﳌﻘﺪﻣﺔ ّ ءا  زأ أ آن اا و د   د زهإ ا ، آن ان ا م   إذ ت ه و       و ر ءت  ،  و ظأ وار ار ا . ا »    را را دا  درا   .و     يا ص ، وا     ا،  » درا ا ا  ا ) ٦١١ ( ﺍﳌﺴﺘﻮﻱ ﺍﻟﱰﻛﻴﺒﻲ ﰲ ﺍﻟﺪﻋﺎء ﺍﻟﻘﺮﺁﻧﻲ ﺍﳌﺴﺘﻮﻱ ﺍﻟﱰﻛﻴﺒﻲ ﰲ ﺍﻟﺪﻋﺎء ﺍﻟﻘﺮﺁﻧﻲ ١.٥ ﺔﻴﺧﻠﻔ ﺍﻟﺒﺤﺚ  ت درا    ءا و،آما  ام ّ  م   درا  را  ان : »ه ي   د در آن    «   ر م و ا  ي ر   م م     ،نإ ٦٨٣١ ش ، دُرس ها ا   ءا آما و زا ا  إ ر ظا و ما و اعأم ا ا  ءا . و  و ع     اّ    ما ر   ان : » ءا أ ه و ارهأ « ،  أ يا ١٠٠٢  أو،   ءا و  و ار اا .  ط وداد اّ  ر  ان : » ءد ءما  انا ا « ،  حا طا  ،م  ٠١٠٢  و  ر ءا و  درا  .   ّ  راه ا     ّ ّ  راا . ١.٣ ﺍﺳﺌﻠﺔ ﺍﻟﺒﺤﺚ ١اع ا ام  .  و ا ا  يا ا  ءا ؟آما ٢ .  اعام ا ا    ءا ؟آما ﺍﳌﺴﺘﻮﻱ ﺍﻟﱰﻛﻴﺒﻲ ﰲ ﺍﻟﺪﻋﺎء ﺍﻟﻘﺮﺁﻧﻲ ........................................................... .... ) ٧١١ ( ٣ .    و ا اا  ء؟ا ١.٤ ﻓﺮﺿ ﺎﺕﻴ ﺍﻟﺒﺤﺚ ١-  ع  ءا    و ا و اا    ورو   ا و    ه يا . ٢-  ءا ا م ب ا ي اض   ؤلا   با أو رظ صا  عو أو     . ٣ ا اا ا ض.ات،ا ا و ارا ا و ا  ار ديا . ً ﺍﻟﺪﻋﺎء ﻟﻐﺔ ، د ر ءا »     ا ط و ِ«.ه ) ا ،ة دة، (و.ع.د ) ٨١١ ( ﺍﺮﺁﻲ ﻮﻱﺍﱰﻴﺒﻲﰲﺍ ﺍﳌ ) ( » و تُ ا "د ر ء ا انا" ء هُأد ًءد ، اأ را  ا : ُل ُ ًءد   ل ُ  . « ) ا،ي دة د.ع.و(،  ذ و  ءا ا م ُ: »  إا ا   « ،ر .)ا ٨٩٩١ دة  :  (د.ع.و و ذ  ان  ي ء ؛ » ة إا ا : ، ةوا  ا :    ... ودا ا   ك  عا   ه و ا ان وذ اذا  ٌوا و آ ُه    ولا د م . « ،رس )ا ٢٢٤١ ق،ص ٩٧٢- ٠٨٢ ( ﺍﻟﺪﻋﺎء ﺍﺻﻄﻼﺣﺎً  ء ت  ة  ا :  ح »  إا ا دةوا .« ءا)ا ، ٢٨٩١ ، ص ٧٤٤ ( ء  ان ا و »  إم  دال   ا  ع و    ا . « م)ا ، ي ٨١٤١ ه ، ص ، ( ٢٤١  اّ م زيا » دم اط  ا    اء.« ز ا)ا ي ، ٤٠٤١ ه ، ٢٩٢ ، ( م  اّ  »  ا رء اا و ادا اه ،م و    ءا رإظ را اءةوا  لا ةوا ا ، و   دا ، روا ا ا ، و   ءا   ا « ،) ا ٢٩٩١ ،ص (٤ ﺍﳌﺴﺘﻮﻱ ﺍﻟﱰﻛﻴﺒﻲ ﰲ ﺍﻟﺪﻋﺎء ﺍﻟﻘﺮﺁﻧﻲ . ل آات ءا  »با « ا ي  ،صا دون  ا )ا ( ا ي    ا ، ا  ا  ) ي- ( م دون اا ف اءا ) (  : M § ¨ © ª « ¬ L ) ٥٢ : ط أو ( : M ½ ¾ ¿ À Á Â Ã Ä Å Æ Ç È É Ê Ë Ì Í L ) آل ان :( ٨ أُ ي  ّا  ف )ء ( اء؟ا اب وا !»  ء ااط  ّناد  آا اء ان م ف اءا »«  ) ّرب ( ،ا و ّام   ّ آنا و ّا ا  ذ ، ّنا ) (اءا  اء ا و ا   بأ ِه   را  ن   ا  . « ) ا ،٥١٠٢ ،ص٢٥ (، M » ¼ ½ ¾ ¿ À Á Â Ã Ä Å Æ Ç È É Ê Ë Ì Í Î L : ه)ا ٦٨١ ( » ذ  و   ا  ر اا   ّر و ءا ب و  با  ة و ر   ) (     M § ¶ ¸ ¹ º » ّاا ا    : M Ì Í Î Ï Ð Ñ Ò Ó Ô Õ Ö × Ø Ù Ú Û L )فا ٩٨ : -٨٨ (  ا ا ةا  ) ّبا ( ف اءا )( ، و   ذ اء  م ّ ّأ ل و  َغأ ه  ةد ِ وارإم   د ِا د     ط ّا   ِاده ط ة   ّم  َ  ّبا  ِم و ه أن ّ إ ،نا   ف اءا ز دة  إا ا  ا و با ُهر « . وي،)٥٠٠٢ ،ص٩٦١ ( ﺍﳋﺼﺎﺋﺺ ﺍﻟﻠﻔﻈ ﰲ ﺔﻴ ﺍﻟﺪﻋﺎء ﺍﻟﻘﺮﺁﻧﻲ إذا أ ةم ّ    ءا ،آما    ا و ام ، م ّما َدُ ،ا    ،ا ل ا ،  اءا  ا ،     ،ا وا  ا ا و ا ادا .    ء ا  ،آما م وا ا ، و   ظ ود،ا   ،ا    ،رةا    ،ا م ا ،ن مو ي  إ و  ا ،ا و  اإ و ٍ. أي و  رةوا ،آمء ا ا        و   ا   ،   طا    ا  و ُ  رب ذانا و نا .   نا ،ظأ ،وا  و    ّ ٍو ور و ا   ّَ   ا. رةوا ،آمء ا ا        و   ا   ،   طا    ا  و ُ  رب ذانا و نا .   نا ،ظأ ،وا  و    ّ ٍو ور و ا   ّَ   ا. ﺍﳌﺴﺘﻮﻱ ﺍﻟﱰﻛﻴﺒﻲ ﰲ ﺍﻟﺪﻋﺎء ﺍﻟﻘﺮﺁﻧﻲ . ) ٩١١ ( ﺍﳌﺴﺘﻮ ﺍﻟﱰ ﻱ ﻛﻴ ﰲ ﺒﻲ ﺍﻟﺪﻋﺎء ﺍﻟﻘﺮﺁﻧﻲ ّع ا يا   ءا آما و ّم    و أ ، او    م ،    ورو   ه و    ه يا .   ءا  آنا م     : ﺍﳌﺴﺘﻮﻱ ﺍﻟﱰﻛﻴﺒﻲ ﰲ ﺍﻟﺪﻋﺎء ﺍﻟﻘﺮﺁﻧﻲ . ﺍﳌﺴﺘﻮﻱ ﺍﻟﱰﻛﻴﺒﻲ ﰲ ﺍﻟﺪﻋﺎء ﺍﻟﻘﺮﺁﻧﻲ . ) ٠٢١ ( ﺍﳌﺴﺘﻮﻱ ﺍﻟﱰﻛﻴﺒﻲ ﰲ ﺍﻟﺪﻋﺎء ﺍﻟﻘﺮﺁﻧﻲ ١- وء اماع ا أم أ :ب اااد ، ط ل ا  طا    ء و اوج ا  ه ا إ ن   ف  اق و ا الا .و      ا  وج ا إ  ء،ءا  » ا   و ّعا و عا  : M Ó Ô Õ Ö × Ø Ù Ú Û Ü Ý Þ ß à á â L )حم : ٨٢ ( و  ن  دما ا ا  . « و  أ    ءا ورودا  ا طا   وزن )إ (         ن اا : M ! " # $ % & ' ( ) * + , - L )اا ٥٨ :-٤٨ و (   ن    ا   ) إ و (   وزن )ّ و ّ    (  : M µ ¶ ¸ ¹ º » ¼ ½ ¾ ¿ À L ) آل ان :٣٩١ ( ا  ما ا ء   ا  را ا   ا    ءت ة ةوا     ):ام  ّر و إ  ا (أي إ ، )ةا ٥٨٢ ( . ٢- :ا » ا ط   ا ّ  ء  و ء،ا و ،ا نا ا  ، ن ًءا  دما ، ًءود  ا و ا  ا . ﺍﳌﺴﺘﻮﻱ ﺍﻟﱰﻛﻴﺒﻲ ﰲ ﺍﻟﺪﻋﺎء ﺍﻟﻘﺮﺁﻧﻲ . ﺍﳌﺴﺘﻮﻱ ﺍﻟﱰﻛﻴﺒﻲ ﰲ ﺍﻟﺪﻋﺎء ﺍﻟﻘﺮﺁﻧﻲ . ﺮﻲ ﱰﻴﺒﻲﰲ ﻮﻱ ) ( ٣- ي اب ا ءء ا :يب ا ءا  ءم ا ّ اض   :ؤلا   با او     او رظ صا  و     : M h i j k l m n o p q r s t u L ) ما:٧٨ (. )   ا اام (  ًءد ان و     اظ و ا     ءهد    : M v w x y z { \| } ~ L ) ما:٨٨ (. و    ء أدب ()     : M / 0 1 2 3 4 5 6 7 8 9 : L ) ماء :٣٨ » (  ب  ع ا ا  ض   أدب و أط    ء  ا ض و ّ   ط ر ءا ) «. ا ،م١٠٠٢ ،ص ( ١٤ ٤ -  :ب ا ءاج ا  ه ا إ ن     اق و  ء       ن   () : M « ¬ ® ¯ ° ± ² ³ ´ µ ¶ ¸ ¹ º » ¼ ½ ¾ ¿ À Á Â Ã Ä Å Æ Ç È É Ê Ë Ì Í Î Ï Ð Ñ Ò Ó Ô Õ Ö × Ø Ù Ú Û L أي   ، »  ا إ     ءدا ّ     فا و  « ،م)ا ٢٩٩١ ،٩٨١ ( و       : )  م  إ وج     (أي "أ " ) :١١ و (  ءا آما ا  »ذأ ، َذ ا «     : M ~ ¡ ¢ £ ¤ ¥ ¦ L )ها : ٧٦ ا و (.   ] : َل َذ ا ُ ّ اِمّر َأ ا[ ي )  : «( ٣٢ إنّ أا م   أ   ءا آما ب ا   ا  ا و ا ب ا . ﺍﳌﺴﺘﻮ ﺍﻟﱰ ﻱ ﻛﻴ ﰲ ﺒﻲ ﺍﻟﺪﻋﺎء ﺍﻟﻘﺮﺁﻧﻲ « ،)ا ٣٤٩١ ،ص ( ٤١١  وج ا  ُه ا  إ ن ا ء  ا زا     : M ¿ À Á Â Ã Ä Å Æ Ç L )حم ( ٦٢ :  ا ون  ) ا واب   آن ٍوا (  : M § ¨ © ª « ¬ ® ¯ ° ± ² ³ ´ µ ¶ ¸ ¹ º » ¼ ½ ¾ ¿ À Á Â Ã Ä Å Æ Ç È É Ê Ë Ì Í Î Ï Ð Ñ Ò Ó Ô Õ Ö × Ø Ù Ú Û Ü Ý Þ ß à L )ةا : ٦٨٢ ( ٢- :ا » ا ط   ا ّ  ء  و ء،ا و ،ا نا ا  ، ن ًءا  دما ، ًءود  ا و ا  ا . « ،)ا ٣٤٩١ ،ص ( ٤١١ ) ١٢١ ( ﺍﳌﺴﺘﻮﻱ ﺍﻟﱰﻛﻴﺒﻲ ﰲ ﺍﻟﺪﻋﺎء ﺍﻟﻘﺮﺁﻧﻲ . ﺍﳌﺴﺘﻮﻱ ﺍﻟﱰﻛﻴﺒﻲ ﰲ ﺍﻟﺪﻋﺎء ﺍﻟﻘﺮﺁﻧﻲ . ٣- ي اب ا ءء ا :يب ا ءا  ءم ا ّ اض   :ؤلا   با او     او رظ صا  و     : M h i j k l m n o p q r s t u L ) ما:٧٨ (. )   ا اام (  ًءد ان و     اظ و ا     ءهد    : M v w x y z { \| } ~ L ) ما:٨٨ (. ﺍﳌﺴﺘﻮﻱ ﺍﻟﱰﻛﻴﺒﻲ ﰲ ﺍﻟﺪﻋﺎء ﺍﻟﻘﺮﺁﻧﻲ . ﺍﳌﺴﺘﻮﻱ ﺍﻟﱰﻛﻴﺒﻲ ﰲ ﺍﻟﺪﻋﺎء ﺍﻟﻘﺮﺁﻧﻲ ) ٢٢١ (  ك ء ا ع آم آنا ا   و و ء ا    و و  ا و      آن،وا   ظ    ا   ):و ٌ   ّ ٍه ٍه )(ها و ( ١: : M ¦ § ¨ L ) ا :( ١و   ءا ّرا ر با     : M ° ± ² ³ ´ µ ¶ L :)٨( ،أي )   (و َل   : ¡ ¢ £ ¤ L )د :و ، ( ٨٦M » ¼ ½ ¾ ¿ À Á L )د :، ( ٨٦: ا ُوا .ك و    ء إد  : M × Ø Ù Ú Û Ü L )٩٧ :ص (.......)  ّ ا ا دك  ا ( M è é ê ë ì í î ï ð ñ L )٣٨ :ص (  د و   ّ و ٍ و  ول  ا عا  ءا م   أ و ءا ا و  ا و ّعا . – ﺍﳉﻤﻞ ﺍﻷﲰ ﻴﺔ ﻭ ﺍﳉﻤﻞ ﺍﻟﻔﻌﻠ ﻴﺔ: انّا   ا و ا وب ا  ءا اما  ا ا   اد تا ا و ارا ، و  ا ا   اد   ا  ّد .  ا  ا ن ر ا   أوُو   ر أو ا  أو ا ر ف   ، ّا ا ا  ا ر   أوم . ) ٣٢١ ( ﺍﳌﺴﺘﻮﻱ ﺍﻟﱰﻛﻴﺒﻲ ﰲ ﺍﻟﺪﻋﺎء ﺍﻟﻘﺮﺁﻧﻲ . و  ا ا ا ا    : M S T U V W X Y Z [ \ ] ^ L ا)ا : ( ٧٣ ءت ا ا ( َ رّاِم ُا )  ل أا ا ي ل   با و  ا إ ) ( ام ز ه ا أي  و ر  ّرب آ  ن ا  ا أا   واراد و   ا ا  اا و  أ ا ا     ر ذا M 4 5 6 7 8 L : )ا ( ١M P Q R S T L :س)ا (١ و   ا ا ) ذأ ، ( ارا ّديا ن  ر و ا  أ ا ا    M 7 8 9 : L ) ا ( و ٦: ضا  ذ ط واا و ارا ا  . ﺍﳌﺴﺘﻮﻱ ﺍﻟﱰﻛﻴﺒﻲ ﰲ ﺍﻟﺪﻋﺎء ﺍﻟﻘﺮﺁﻧﻲ . ﺍﳌﺴﺘﻮﻱ ﺍﻟﱰﻛﻴﺒﻲ ﰲ ﺍﻟﺪﻋﺎء ﺍﻟﻘﺮﺁﻧﻲ . ) ٤٢١ ( ٢- وا  ر  ا ط : واء اد »    ن  رإظ دا عوا ّوا موار أي  ن  ط دا ،  أوا و اهٍم و  إ ابا .« ،صر م)ا ( ١٦  أد  ا  ي   ن   () ل ر : ]رب حا   ر و( ٥٢ )ي    ا ٦٢ )ي ( وا م ِ ة  )٧٢ ( ا   )٨٢ ( وا   وزا  أ )٩٢ ( رون أ )٠٣ ( دا  ازر ١٣ ) ي و ( أ   أ ٢٣ )ي (.[) رة ،آ ط ٥٢- ( ٢٣ »   د ا ن   ) ا و ّا (   ا ءد دة و  ن  ) ّبا (  ءد  و ط نِ  ء ا . «،صر م)ا ( ٢٦ -     ن ا    ءد دة،ا   ،   ل اا أوا أو  ،  ءد حم () : M < = > ? @ A B C D E F G H I J K L M N O P L )د : ( ٧٤  اأ   ءد ا ءت  وا    ) إ أو  ( : M ¶ ¸ ¹ º » ¼ ½ ¾ ¿ À Á Â Ã Ä Å Æ Ç È É Ê Ë Ì Í Î Ï Ð Ñ Ò Ó Ô Õ Ö × Ø Ù Ú Û Ü Ý Þ ß à L )ةا : ٦٨٢ (     ءد دةا إ ءا أو  ا أ  ءد ا    ا  ا . ﺃﺳﺎﻟ ﻴﺐ ﺩﻋﺎء ﺍﻷﻧﺒ ﰲ ﺎءﻴ ﺍﻟﻘﺮﺁﻥ ﺍﻟ ﻢﻳﺮﻜ ا اترزةاءدماء آنآا  فإأ  ءا ﺃﻧﻮﺍﻉ ﺍﻟﺪﻋﺎء ﰲ ﺍﻟﻘﺮﺁﻥ ﺍﻟ ﻜﺮﻳﻢ ﰲ ﺃﻮﺍﻉﺍ ﺍﺮﺁﻥﺍ ﺮﻳﻢ  ّ ءا  آنا ا   ا   : ١- ءدة واء اد ٢-  واء اد ١- :ءدة واء اد »    ن  الا ات ،ا  ا أو  و   ارإ    .و  رة  ا ءوا   ا و  ن  و ط و ًءر و       اعام دةا ةا طوا  الا لوا وات ووا ك ا  با  ا و  « ،)ط ٠١٠٢ ،ص ١٦ (، و آ ت ءد دةا    ] :  ا   ا   ا  ءَ و ُعَ ا  ء و   ء و ل  ء ك ا وأم      ء   )[. آل ان :٦٢ ( آت ءد دةا ا وردت      ا وا وا ءوا . ﺃﺳﺎﻟ ﻴﺐ ﺩﻋﺎء ﺍﻷﻧﺒ ﰲ ﺎءﻴ ﺍﻟﻘﺮﺁﻥ ﺍﻟ ﻢﻳﺮﻜ  ا ات رزةا  ءد ما ء  آنآ ا    فإ أ   ءا إ ،ا  رة ً ون ا ن    ورة ن ا ن لا    ط  ا دوا ،    را اا ا  : م  ر؛   ﺍﳌﺴﺘﻮﻱ ﺍﻟﱰﻛﻴﺒﻲ ﰲ ﺍﻟﺪﻋﺎء ﺍﻟﻘﺮﺁﻧﻲ ........................................................... .... ) ٥٢١ ( ١- :لن ا با »  دا ا ،  ب لا ،   ّ ءد ر  ما ،ء  ءإم    با ن لا .« ،)ط ٠١٠٢ ،ص٣٢١ ( »   ءا ا با ط ن و    ،  اب  ن    » إ «ال ا   ا ءدو () : M ! " # $ % & ' L )اءا : ( ٨٤  و  ن   )  ( ، أي   ،عا  ا ل حم () : M ½ ¾ ¿ À Á Â Ã Ä Å Æ Ç L )حم ( ٦٢ :  ا ون  ) ا واب   آن ٍوا (  ] : َر مِاُ إنم وأ مأ ... ُوا ّ ِوا َوَار ... [ ) ةا : ٦٨٢ ( ٢- :لن ا بأ »   ءا ا با دة نا وا  ن م ر وا م «،صر م)ا ٤٢١ (  ءل دن ا ءال إ و أ ، و  ،م و  ا اَُ ،     ،أ  ام د  ي ا   . « ،ام)ا ،ص (٩ »  م ا ا ء د ن  أ ، وا أم   ا روا  ه و   ا ا  و ور  م   ،    أم ءر ه و ر ا  ر    ا  . ا  با ا ن   و ل اا  ّ أم     را و ت ،ة و  ث فاو . ﺃﺳﺎﻟ ﻴﺐ ﺩﻋﺎء ﺍﻷﻧﺒ ﰲ ﺎءﻴ ﺍﻟﻘﺮﺁﻥ ﺍﻟ ﻢﻳﺮﻜ ١-  ؛ل ا و ده ا   را ما ء إ ا    ه    ورد   ن ز  () : M / 0 1 2 3 4 5 6 7 8 9 : ; < = L )   ، (٤ ﺍﳌﺴﺘﻮﻱ ﺍﻟﱰﻛﻴﺒﻲ ﰲ ﺍﻟﺪﻋﺎء ﺍﻟﻘﺮﺁﻧﻲ . ﺍﳌﺴﺘﻮﻱ ﺍﻟﱰﻛﻴﺒﻲ ﰲ ﺍﻟﺪﻋﺎء ﺍﻟﻘﺮﺁﻧﻲ . ) ٥٢١ ( ١- :لن ا با »  دا ا ،  ب لا ،   ّ ءد ر  ما ،ء  ءإم    با ن لا .« ،)ط ٠١٠٢ ،ص٣٢١ ( »   ءا ا با ط ن و    ،  اب  ن    » إ «ال ا   ا ءدو () : M ! " # $ % & ' L )اءا : ( ٨٤  و  ن   )  ( ، أي   ،عا  ا ل حم () : M ½ ¾ ¿ À Á Â Ã Ä Å Æ Ç L )حم ( ٦٢ :  ا ون  ) ا واب   آن ٍوا (  ] : َر مِاُ إنم وأ مأ ... ُوا ّ ِوا َوَار ... [ ) ةا : ٦٨٢ ( ٢- :لن ا بأ »   ءا ا با دة نا وا  ن م ر وا م «،صر م)ا ٤٢١ (  ءل دن ا ءال إ و أ ، و  ،م و  ا اَُ ،     ،أ  ام د  ي ا   . « ،ام)ا ،ص (٩ »  م ا ا ء د ن  أ ، وا أم   ا روا  ه و   ا ا  و ور  م   ،    أم ءر ه و ر ا  ر    ا  . ا  با ا ن   و ل اا  ّ أم     را و ت ،ة و  ث فاو . ﺍﳌﺴﺘﻮﻱ ﺍﻟﱰﻛﻴﺒﻲ ﰲ ﺍﻟﺪﻋﺎء ﺍﻟﻘﺮﺁﻧﻲ . ﺍﳌﺴﺘﻮﻱ ﺍﻟﱰﻛﻴﺒﻲ ﰲ ﺍﻟﺪﻋﺎء ﺍﻟﻘﺮﺁﻧﻲ ........................................................... .... ) ٦٢١ ( » أورد و () را أ د   ا ،وا ؛  اظ و ط ا ا طا  ظ  ا وا ا يا    وا ء ا    أسا اوا  ادا ،. )ط ٠١٠٢ ،ص ٦٢١ ( ٢- :ولل ا و » ب ا ا    ا   ا    ا  .  ءوا   و ن  ا   ِده   م و  ذ ل   () : M ¯ ° ± ² ³ ´ µ ¶ ¸ ¹ º » ¼ ½ ¾ ¿ À Á Â Ã Ä Å Æ Ç È L )  : ( ١٠١      ()  ا   ُم هآ  ا و    ؤا  و  اتا    ل  ا و  آ وع ا  .ء «،صر م)ا ٤٢١ ( ٣- :ول وال ا و » أ له ا ءان ا  ت ا   أ ماء م     ر و ة د  و اا  و   ّ رّ أم درا فا دا   . « ،صر م)ا ٦٢١ (  ذ و ء أدب () : M / 0 1 2 3 4 5 6 7 8 9 : L ) ماء : ( ٣٨ ل ا  ، » ّأ أنب () ، و ّر    ال ر    ،ه و        الا ، و ا  ب  دبا  الا ءوا .« ،صر م)ا ٦٢١ ( ﺍﻟﻨﺘﺎﺋﺞ » أورد و () را أ د   ا ،وا ؛  اظ و ط ا ا طا  ظ  ا وا ا يا    وا ء ا    أسا اوا  ادا ،. )ط ٠١٠٢ ،ص ٦٢١ ( ٢- :ولل ا و » ب ا ا    ا   ا    ا  . ﺍﳌﺴﺘﻮﻱ ﺍﻟﱰﻛﻴﺒﻲ ﰲ ﺍﻟﺪﻋﺎء ﺍﻟﻘﺮﺁﻧﻲ . ١-  ؛ل ا و ده ا   را ما ء إ ا    ه    ورد   ن ز  () : M / 0 1 2 3 4 5 6 7 8 9 : ; < = L )   ، (٤ ١- :لن ا با »  دا ا ،  ب لا ،   ّ ءد ر  ما ،ء  ءإم    با ن لا .« ،)ط ٠١٠٢ ،ص٣٢١ ( ر  ما ،ء  ءإم    با ن لا .« ،)ط ٠١٠٢ ،ص٣٢١ ( »   ءا ا با ط ن و    ،  اب  ن    » إ «ال ا   ا ءدو () : M ! " # $ % & ' L )اءا : ( ٨٤  و  ن   )  ( ، أي   ،عا  ا ل حم () : M ½ ¾ ¿ À Á Â Ã Ä Å Æ Ç L )حم ( ٦٢ :  ا ون  ) ا واب   آن ٍوا (  ] : َر مِاُ إنم وأ مأ ... ُوا ّ ِوا َوَار ... [ ) ةا : ٦٨٢ ( ٢- :لن ا بأ »   ءا ا با دة نا وا  ن م ر وا م «،صر م)ا ٤٢١ (  ءل دن ا ءال إ و أ ، و  ،م و  ا اَُ ،     ،أ  ام د  ي ا   . « ،ام)ا ،ص(٩    ا  با ا ن   و ل اا  ّ أم     را و ت ،ة و  ث فاو . ) ٦٢١ ( ﺍﳌﺴﺘﻮﻱ ﺍﻟﱰﻛﻴﺒﻲ ﰲ ﺍﻟﺪﻋﺎء ﺍﻟﻘﺮﺁﻧﻲ . ﺍﳌﺴﺘﻮﻱ ﺍﻟﱰﻛﻴﺒﻲ ﰲ ﺍﻟﺪﻋﺎء ﺍﻟﻘﺮﺁﻧﻲ .  ءوا   و ن  ا   ِده   م و  ذ ل   () : M ¯ ° ± ² ³ ´ µ ¶ ¸ ¹ º » ¼ ½ ¾ ¿ À Á Â Ã Ä Å Æ Ç È L ) ( ١ ١ ﺍﳌﺴﺘﻮﻱ ﺍﻟﱰﻛﻴﺒﻲ ﰲ ﺍﻟﺪﻋﺎء ﺍﻟﻘﺮﺁﻧﻲ . ﺍﳌﺴﺘﻮﻱ ﺍﻟﱰﻛﻴﺒﻲ ﰲ ﺍﻟﺪﻋﺎء ﺍﻟﻘﺮﺁﻧﻲ . ﺮﻲ ﱰﻴﺒﻲﰲ ﻮﻱ ٢- ا ا  ن  ر و اع و اء ا ا  اا   ًءد ء ا : M × Ø Ù Ú Û Ü Ý L )٩٧ :ص (....... M è é ê ë ì í î ï ð ñ L ) ( ٣٨ :ص ٣-  ا اءة ا  ) ّرب - ّر (و   ا ) ّا (   ا  م ا و ّا و ءا إ ر ا أ  ا . ٤ - ل آا ت ا  ء »با « ا ي  ،صا دون  ا )ا ( يا  ،ا  ا  ا ي)- ( م دون اا ف اءا ) (و ذ ا ر اا   ّبا . ٥-  د ود و ّ  آمء اأنّ ا و م م  ا . ٦-  انّ ا ا و ا وب ا  ءا ،آما ا ا  اد تا ا و ارا  ): ا رب ا ،و ( ا ا  اد  ا  ّد  ) : مإ اا ا (. ٢- ا ا  ن  ر و اع و اء ا ا  اا   ًءد ء ا : M × Ø Ù Ú Û Ü Ý L )٩٧ :ص (....... M è é ê ë ì í î ï ð ñ L ) ( ٣٨ :ص ٣-  ا اءة ا  ) ّرب - ّر (و   ا ) ّا (   ا  م ا و ّا و ءا إ ر ا أ  ا .      إ   ٤ - ل آا ت ا  ء »با « ا ي  ،صا دون  ا )ا ( يا  ،ا  ا  ا ي)- ( م دون اا ف اءا ) (و ذ ا ر اا   ّبا . ٥-  د ود و ّ  آمء اأنّ ا و م م  ا . ٦-  انّ ا ا و ا وب ا  ءا ،آما ا ا  اد تا ا و ارا  ): ا رب ا ،و ( ا ا  اد  ا  ّد  ) : مإ اا ا (. ﺍﻟﻨﺘﺎﺋﺞ  ، م ا  ا          ؛ ١- مء اد ء  آنا ا  و   دا  ا و اام و  . ) ٧٢١ ( ﺍﳌﺴﺘﻮﻱ ﺍﻟﱰﻛﻴﺒﻲ ﰲ ﺍﻟﺪﻋﺎء ﺍﻟﻘﺮﺁﻧﻲ .  ٧-  ا ،  ا ، ّدة ا آمء ا ا رةا ،  اءا  ا  ٨ - ع ا  ءا و ّن   ا   ورو  ا و  ه يا . ٩ -  ءا ما ب يا اض ّ  ،ب ا  ؤل ا  أو و صر اظ أو  . ٠١-  ا ءا ورودا  آنا ،ا ءا ب ا   ا  ا و ا ب ،ا ورد  ا  و م و  ة و  ا أر ة ة و ء با ا ا ة ة و ّر أ ب ا  ا را با  ورد ة ةوا  .  ١١-  انّ ا ا و ا وب ا  ءا ،آما ا ا  اد تا ا و ارا  ): ا رب ا ،و ( ) ٨٢١ ( ﺍﳌﺴﺘﻮﻱ ﺍﻟﱰﻛﻴﺒﻲ ﰲ ﺍﻟﺪﻋﺎء ﺍﻟﻘﺮﺁﻧﻲ .. Abstract Quranic pray method is distincted by art statement and firm style and strong and unparatleled tissue. this is essay is investgating stylish compound level in Quranic pray. and has an important role in showing the structural elements of Quranic pray. the conclusion of this discussion shows that consists of compound dimension in Quranic pray like originative and predicative. noun and verb clauses are used there alternately that is suitable for the kinds of pray then it shows kinds of formulas that are used in Quranic pray like imperative and negative interrogation. choosing they styles and imperative sentences is not random but is has the most care lessness and influence on the reader and harmony is seen clearly between the sentences and their meaning. Kay words : stylistic , Quranic pray , compound level , vocabulary , sentences kinds. ﻗﺎﺋﻤﺔ ﺍﳌﺼﺎﺩﺭ ﻭ ﺍﳌﺮﺍﺟﻊ ﻗﺎﺋﻤﺔ ﺍﳌﺼﺎﺩﺭ ﻭ ﺍﳌﺮﺍﺟﻊ  ىءم و آن اﻠﻜر . ١- ال ا ،زيا ين ا حا ،ا  ام اظا   ها و ا، ،وت ،ا ٤٠٤١ ه. ٢-  ا ، ا  ،اوع اي د، ا ،٤٠٠٢ . ٣-  ا ه، اا   ،ا ،ّا وت ،دارا   . ٤- ،وت،رون ا :، ا  ،رس، ا ا ٢٢٤١ ق. ٥-  ،ار ا ا   ل اﻛ ،ر وت، دار  ، ب ن ،در٨٩٩١ . ٦- ي، اء ا اا ب  ، ا،ت  تا و وقا ،ا  ن دروم  و  ،ي،دا ١٨٩١-٢٨٩١  . ٧- ، اطم اما ن،  اا ،، ،وت،دارا٢٩٩١ . ﻗﺎﺋﻤﺔ ﺍﳌﺼﺎﺩﺭ ﻭ ﺍﳌﺮﺍﺟﻊ  ىءم و آن اﻠﻜر . ١- ال ا ،زيا ين ا حا ،ا  ام اظا   ها و ا، ،وت ،ا ٤٠٤١ ه. ٢-  ا ، ا  ،اوع اي د، ا ،٤٠٠٢ . ٣-  ا ه، اا   ،ا ،ّا وت ،دارا   . ٤- ،وت،رون ا :، ا  ،رس، ا ا ٢٢٤١ ق. ٥-  ،ار ا ا   ل اﻛ ،ر وت، دار  ، ب ن ،در٨٩٩١ . ٦- ي، اء ا اا ب  ، ا،ت  تا و وقا ،ا  ن دروم  و  ،ي،دا ١٨٩١-٢٨٩١  . ٧- ، اطم اما ن،  اا ،، ،وت،دارا٢٩٩١ . ٦- ي، اء ا اا ب  ، ا،ت  تا و وقا ،ا  ن دروم  و  ،ي،دا ١٨٩١-٢٨٩١  . ٧- ، اطم اما ن،  اا ،، ،وت،دارا٢٩٩١ . ) ٩٢١ ﺍﳌﺴﺘﻮﻱ ﺍﻟﱰﻛﻴﺒﻲ ﰲ ﺍﻟﺪﻋﺎء ﺍﻟﻘﺮﺁﻧﻲ . ٨- ،وي آن ا ا ،ا ، ،ه، ا م ٥٠٠٢ . ٩- ،نت اف ا،  ،يما وت،دار ا ،ا ٨١٤١ ه. ٨- ،وي آن ا ا ،ا ، ،ه، ا م ٥٠٠٢ . ٩- ،نت اف ا،  ،يما وت،دار ا ،ا ٨١٤١ ه. ٠١- ا،را   ، ا ا  ا ي،ا ١، ٠١- ا،را   ، ا ا  ا ي،ا ، ١ ،ن،دارا٤٠٠٢ . ٠١- ا،را   ، ا ا  ا ي،ا ، ١ ،ن،دارا٤٠٠٢ . ١١- ، اا،ن  ،  اء، ان ا   ،قا ،ذاراد ،ا ٢٩٩١ . ٢١-  اء ازاق،اا د ،اما ا  با و ا ، ة،ا . ٢١-  اء ازاق،اا د ،اما ا  با و ا ، ة،ا . ٣١- ا ي،  ، ، ،س اا  وسج ا ءوت،دارإ اثا ،ا  . ٣١- ا ي،  ، ، ،س اا  وسج ا ءوت،دارإ اثا ،ا  . ٤١- و، ارا،ما ، ،داراب،ا ا٧٩٩١ . ٤١- و، ارا،ما ، ،داراب،ا ا٧٩٩١ . ٥١- ا ،  ،اا آن ا ا   ،زا  ،ردنا دم ،٥١٠٢  . ٥١- ا ،  ،اا آن ا ا   ،زا  ،ردنا دم ،٥١٠٢  . ٦١- آن ا ا ءمء ا،د م ، ودادط ﻟﻜر ح  ا :،م ،طا ٠١٠٢ . ٧١- ا ،م   ، ا آن ا ا ء  و ه و ارها  ا، ا ي، ١٠٠٢ . ٨١- ، ارف ات وم، ،اا.،، اا ٣٤٩١ ﺍﳌﺴﺘﻮﻱ ﺍﻟﱰﻛﻴﺒﻲ ﰲ ﺍﻟﺪﻋﺎء ﺍﻟﻘﺮﺁﻧﻲ ........................................................... .... ) ٠٣١ ( ﺍﳌﻠﺤﻘﺎﺕ اول اا   ءا ﺍﳌﺴﺘﻮﻱ ﺍﻟﱰﻛﻴﺒﻲ ﰲ ﺍﻟﺪﻋﺎء ﺍﻟﻘﺮﺁﻧﻲ ﺴﺘﻮﻱ ﺍﻟﱰﻛﻴﺒﻲ ﰲ ﺍﻟﺪﻋﺎء ﺍﻟﻘﺮﺁﻧﻲ ........................................................... .... ) ٠٣١ ( ﺍﳌﻠﺤﻘﺎﺕ اول اا   ءا
https://openalex.org/W4223902728	https://bmcmededuc.biomedcentral.com/track/pdf/10.1186/s12909-022-03331-9	English	null	Assessment of an intensive education program for pharmacists on treatment of tobacco use disorder using an objective structured clinical examination: a randomized controlled trial	BMC medical education	2,022	cc-by	8,308	El Hajj et al. BMC Medical Education (2022) 22:289 https://doi.org/10.1186/s12909-022-03331-9 El Hajj et al. BMC Medical Education (2022) 22:289 https://doi.org/10.1186/s12909-022-03331-9 © The Author(s) 2022. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativeco mmons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Abstract Background: Tobacco use is one of the major public health threats globally. Community pharmacists are uniquely positioned to offer tobacco cessation services owing to their easy accessibility by the public. To prepare Qatar com- munity pharmacists to develop the competencies and skills required to offer smoking cessation services, an intensive tobacco control education program was designed and implemented. The study aimed to assess the impact of the tobacco education program on the pharmacists’ skills and competence. Methods: A random sample of community pharmacists in Qatar was chosen for participation in the program. Consenting participants were randomly assigned to either intervention or control groups. The intervention group received an intensive education program on treatment of tobacco-use disorder, while a short didactic session on a non-tobacco-related topic was delivered to the control group. The pharmacists’ tobacco cessation skills and compe- tencies were assessed using an Objective Structured Clinical Examination (OSCE). Results: A total of 54 and 32 community pharmacists in the intervention group and the control group, respectively, completed the OSCE. The intensive tobacco education group achieved significantly higher total scores than the control group in all the OSCE cases. Specifically, the mean total scores for the intervention group were 15.2, 15.3, 14.2, 14.6, 16.3, and 15.2 compared to 8.8, 6.2, 7.7, 9.2, 8.3, and 11.3 for the control group (p < 0.001) for cases one to six respectively. Conclusion: The study demonstrated that an intensive tobacco cessation education program can improve pharma- cists’ tobacco cessation skills and increase their tobacco cessation counseling abilities. Trial registration: Clinical Trials NCT03518476 (https://clinicaltrials.gov/ct2/show/NCT03518476) Registration date: May 8, 2018. Keywords: Qatar, Education program, Tobacco control, Smoking cessation, Pharmacist, OSCE Assessment of an intensive education program for pharmacists on treatment of tobacco use disorder using an objective structured clinical examination: a randomized controlled trial Maguy Saffouh El Hajj1, Ahmed Awaisu1, Mohamad Haniki Nik Mohamed2, Rana Ahmed Saleh1, Noora Mohammed Al Hamad3, Nadir Kheir4 and Ziyad R. Mahfoud5,6 Maguy Saffouh El Hajj1, Ahmed Awaisu1, Mohamad Haniki Nik Mohamed2, Rana Ahmed Saleh1, Noora Mohammed Al Hamad3, Nadir Kheir4 and Ziyad R. Mahfoud5,6 Background Around 942 million men and 175 million women aged 15 or older currently smoke cigarettes globally [1]. About three-quarters of male daily smokers reside in countries Correspondence: maguyh@qu.edu.qa 1 College of Pharmacy, QU Health, Qatar University, 2713 Doha, Qatar 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. El Hajj et al. BMC Medical Education (2022) 22:289 Page 2 of 12 Page 2 of 12 patients with specific medical conditions or problems, or health professionals with certain requests or tasks). A candidate at an interactive station is then assessed using a standardized rubric by an assessor. Non-interac- tive stations can be for preparation purposes or for the candidate to resolve a given problem without any direct observation or assessment at that point of time [6–10]. with a medium or high human development index (HDI), while half of female daily smokers live in very high- HDI countries [1]. In Qatar, the prevalence of tobacco smoking among adults is reported as 18.2% [2]. Despite that fewer men smoke typically in Qatar than on average in very high-HDI countries, there are over 260,000 men who smoke cigarettes daily, which makes tobacco use a public health problem in the country [2]. Hence, it is imperative for all health professionals in Qatar to offer tobacco cessation services to increase quit rates and to prevent non-smokers from starting. Performance of candidates being assessed via OSCE focuses on cognitive skills, including critical thinking, problem-solving, communication and interpersonal skills. In addition, elements of ethical and professional judgment can be assessed using a well-designed OSCE [6–10]. In this paper, we report the results of an OSCE used for the purpose of assessing the effectiveness of an intensive tobacco education program targeting commu- nity pharmacists in Qatar. Study designh The study was a randomized controlled trial (RCT) aimed to evaluate the effectiveness of a tobacco-cessation education program for community pharmacists using an OSCE. Community pharmacists were randomly assigned to either an intensive education program on treatment of tobacco-use disorder group (intervention group) or didactic sessions on non-tobacco-related topics (control group). The tobacco dependence treatment skills of par- ticipants in both groups were assessed using a six-station OSCE, targeting evidence-based tobacco cessation core competencies. The study methodology is available in details in the published study protocol [11]. The trial is registered in Clinical Trials. NCT03518476 (https://clini caltrials.gov/ct2/show/NCT03518476) with the registra- tion date: May 8, 2018. Therefore, in an effort to prepare Qatar community pharmacists to have the required knowledge and skills to offer tobacco cessation services, formal tobacco edu- cation program is required. The aim of this randomized controlled study is to design, implement, and evaluate an intensive education program on tobacco-use treatment for pharmacists provided by a team of health professional educators in Qatar. The effectiveness of the program was assessed using a multiple-choice-based evaluation instru- ment and an Objective Structured Clinical Examination (OSCE). Community pharmacists are uniquely positioned healthcare professionals to offer tobacco cessation ser- vices using the 5A’s (ask, advise, assess, assist and arrange follow-up) approach, owing to the ease of their accessi- bility by the public. Evidence has shown that quit rates achieved when pharmacists recommend smoking ces- sation medications and provide counseling are similar to those achieved by other healthcare professionals [3]. Additionally, community pharmacists may also provide cost-effective access to smoking cessation therapy [4]. However, a study conducted among community phar- macists in Qatar confirmed their low involvement in tobacco cessation activities. Moreover, over 80% of com- munity pharmacists never received any formal education or training about tobacco cessation in the past [5].hf Intervention group A f d A six-station OSCE, which targeted the core competen- cies and skills covered in the program, was completed by participants in both groups in the same location of the training program. OSCE cases were developed and vali- dated for content and face validity, through several meet- ings, by a group of educators and researchers experienced in the field of tobacco cessation. The first case targeted tobacco use in a generally healthy female adult patient, the second included a pregnant woman contemplating to stop smoking, the third involved a teenager who smokes, the fourth targeted relapse prevention in a patient who recently quitted and experiencing withdrawal symptoms, the fifth case involved a smoker with cardiovascular dis- ease, and the last case involved a smoker in the precon- templation stage of behavior change. During the OSCE, each participant was allocated 10 min for each case to interact with a standardized patient (SP) in a private counseling room. Each SP was extensively trained by the research team using a validated script for the corre- sponding case. Performance of participants was assessed by trained assessors (faculty members from the College of Pharmacy and advanced pharmacy practitioners) using assessment checklists designed and validated by the same group of experts. The checklists included both analytical and global assessment sections. The analytical assessment section evaluated the participants’ ability to establish rapport, gather relevant information, provide appropriate recommendations and follow-up evaluation for the patient. On the other hand, the global assess- ment part evaluated the participants’ communication skills in general, confidence, and body language using a 5-point rating scale. The number of items in the ana- lytical checklist differed between one case and another. Given the complexity of cases, items were rated on two or three or four level scale (1 to 4 points). Assessors in A team of educators, researchers, and clinicians with expertise in tobacco control and tobacco dependence treatment delivered an intensive tobacco education program to participants in the intervention group. The training program was conducted at Qatar University over four days with an average of eight contact hours per day (i.e., approximately 32 contact hours). The program materials were developed de novo and targeted the Core Competencies for Evidence-based Treatment of Tobacco Dependence by the Association for the Treatment of Tobacco Use and Dependence (ATTUD) [12]. Eligibility of participants and recruitment Any licensed pharmacist who practiced in the commu- nity pharmacy setting in Qatar was eligible to participate in the study. Pharmacists in training or interns or unli- censed pharmacists were excluded. OSCE is a performance-based assessment commonly used and described in the literature to assess clinical skills in health professional education programs [6–10]. Many of such programs have introduced OSCE in their undergraduate education curricula, while in some coun- tries it is used for professional licensure purposes. An OSCE consists of a series of stations, which each exam- inee needs to go through in a rotational manner over a specific time. At each station, the candidate is given a simulated scenario or task, which requires performance of particular activities, such as medication history tak- ing, medication counselling, or responding to drug infor- mation inquiry [6–10]. An OSCE station can be active (interactive or non-interactive) or inactive (e.g., rest sta- tion). Interactive stations usually involve the use of stand- ardized patients (i.e., people who are trained to act as A random sample of 529 community pharmacists was chosen for recruitment from among a total list of 1000 pharmacists, provided by the Health Practitioners Reg- istration and Licensing Section of the Qatar Ministry of Public Health. Consented pharmacists were randomly allocated to the intervention or control groups using permuted block ran- domization with blocks of size 2, 4, and 6. Randomization was made by the study statistician who was not involved in the recruitment process and was concealed from the research assistants recruiting the participants. Given the nature of the intervention, blinding of the study partici- pants about their assigned groups was not feasible. No El Hajj et al. BMC Medical Education (2022) 22:289 Page 3 of 12 incentives were offered to participants in both groups other than the CE units. patients, self and peer debriefing and performance feed- back activities. Outcome measuresh The main outcome measure was the post-intervention tobacco-cessation skills, assessed through OSCE. Sample size determinationh A didactic lecture on women health and contracep- tion was delivered to pharmacists in the control group. This was to prevent contaminating participants’ knowl- edge and skills with tobacco-related elements that could subsequently change their behavior to another level not representative of their tobacco-cessation-related “cur- rent practice” or “usual care.” Pharmacists in the control group received three CE units accredited by QCHP. The sample size estimates were for 54 participants in each group with a power of 80% to detect an effect size of 0.60 between the study groups for the numeric primary outcomes (skills scales) using the independent t test with a significance level of 2.5%. In addition, with 54 partic- ipants per group, the study will be able to detect a dif- ference of at least 27.5% between the two study groups for any dichotomous outcome using the chi-square test with 80% power and a significance level of 5%. With a 10% loss-to-follow-up rate assumed, 60 pharmacists were required per group, resulting in 120 pharmacists to be randomized into groups. Intervention group A f d The train- ing program was accredited with 26.5 CE units by the Qatar Council for Healthcare Practitioners (QCHP) of Qatar Ministry of Public Health.h The training curriculum covered the following topics: (1) Tobacco-use epidemiology in Qatar and globally, (2) Risks and consequences of tobacco use, (3) Benefits of quitting tobacco use, (4) principles of nicotine addiction, 5) Non-pharmacological treatment of tobacco use and dependence 6) Pharmacological treatment of tobacco use and dependence 7) Alternative therapies 8) Coun- seling and communication skills 9) Relapse prevention 10) Treatment of tobacco use and dependence in special populations 11) Treatment of waterpipe dependence 12) Role of pharmacist in tobacco cessation 13) Establishing a tobacco cessation service 14) Tobacco cessation related professional development 15) Qatar law and ethics. The learning outcomes for each topic were mapped against ATTUD Core Competencies.h The program was delivered through a combination of didactic lectures and active learning strategies including problem-based learning (PBL) exercises using case sce- narios, group discussions, games, role plays, videos, sim- ulated practical applications with peers and standardized El Hajj et al. BMC Medical Education (2022) 22:289 El Hajj et al. BMC Medical Education (2022) 22:289 Page 4 of 12 statistical significance was set at 2.5% for the total OSCE score and 5% for all other comparisons. statistical significance was set at 2.5% for the total OSCE score and 5% for all other comparisons. every station were trained on how to use the checklists and were blinded regarding the participants’ groups. The standardized patient was not involved in rating or assess- ing the study participants. Statistical analysis y IBM SPSS software (IBM SPSS® for Windows, Version 24.0; IBM Corp, Armonk, NY, USA) was used for data analyses. The Consolidated Standards of Reporting Trials (CONSORT) guidelines were followed when analyzing the study data [13]. Demographic and pharmacist edu- cation and practice-related questions were summarized using means and standard deviations for numeric vari- ables and frequency (percentage) distributions for cat- egorical variables. These characteristics were compared between the study groups and differences using the inde- pendent t-test or the Chi-square test depending on the scale of measurement of the variable. The primary out- come analyses included comparing the between-group post-training scores on the OSCE using independent t-test and univariate linear regression. Secondary analy- ses included using linear regression to adjust the main analyses to any potential confounders or differences in the demographic or work-related participant’s character- istics found between the two study groups. The level of Ethical considerationsh The study protocol and all related instruments and forms were reviewed and granted ethical approval by the Qatar University Institutional Review Board (QU-IRB) (approval number: QU-IRB 906-E/18). Recruitment Between July and September 2018, 1000 pharmacists were assessed for eligibility and 529 pharmacists were randomly selected and invited through the phone to par- ticipate in the study. A total of 164 pharmacists accepted to participate and were randomly allocated to the inter- vention (n = 77, 46.9%) and control (n = 87,53.1%) groups (Fig. 1). Fifty-seven participants (74.0%) received the intensive education on tobacco dependence treatment (the intervention group), while 37 (42.5%) received the education on contraception (the control group). Fifty-four participants in the intervention group (94.7%) and 32 participants (86.4%) in the control group completed the OSCE. The OSCE was conducted in the same week after the end of both the tobacco education and contraception training groups. Fig. 1 Participant flow chart Page 5 of 12 El Hajj et al. BMC Medical Education (2022) 22:289 Sociodemographic, practice and tobacco related characteristics of the participants The majority of the participants in the intervention group were male (56.1%), while most participants in the con- trol group were female (55.9%) (Table 1). a Standard Deviation Follow up/monitoring scores Pharmacists in the intervention group offered appropri- ate follow-up/monitoring plans with significantly higher mean scores than those in the control group. The mean scores range for follow-up for the intervention group were 0.4 to 0.8 compared to the control group range of mean scores of 0.1–0.4 for cases one, two, three, five and six. There was no follow-up/monitoring section for case four. Global assessment scores per case Table 3 summarizes the results for the OSCE cases for both intervention and control groups. In addition, the global assessment scores were signifi- cantly higher for the intervention group than the con- trol group in all the cases except cases three and six. The mean global assessment scores for cases one, two, four and five ranged from 3.2 to 3.7 versus 2.1 to 2.6 for the intervention group compared to the control group. Total analytical and total OSCE scores Total analytical scores (i.e. combined scores for estab- lishing rapport, data gathering, management strategies, and follow-up) were significantly greater in the group of pharmacists who received the intensive tobacco-related education in all the OSCE cases (Table 4).h Recruitment BMC Medical Education (2022) 22:289 El Hajj et al. BMC Medical Education (2022) 22:289 El Hajj et al. BMC Medical Education (2022) 22:289 Page 6 of 12 Management strategies scores groups was around 36 years and 35 years, respectively. The highest pharmacy academic qualification for par- ticipants in both the intervention and the control groups was B.Pharm/BSc Pharm with proportions of 85.7% and 76.4%, respectively. There were no statistically sig- nificant differences between the two groups in terms of participants’ baseline characteristics. In addition, there were no significant differences between the two groups in relation to their tobacco use status and prior training received on tobacco use management (Table 2). Majority (> 80%) of participants in both groups were non-smokers. Among the current smokers, those in the intervention group were smoking more cigarettes per day than those in the control group; 12.5 vs. 3, respectively. Almost all participants reported not having any previous training on tobacco use management. groups was around 36 years and 35 years, respectively. The highest pharmacy academic qualification for par- ticipants in both the intervention and the control groups was B.Pharm/BSc Pharm with proportions of 85.7% and 76.4%, respectively. There were no statistically sig- nificant differences between the two groups in terms of participants’ baseline characteristics. In addition, there were no significant differences between the two groups in relation to their tobacco use status and prior training received on tobacco use management (Table 2). Majority (> 80%) of participants in both groups were non-smokers. Among the current smokers, those in the intervention group were smoking more cigarettes per day than those in the control group; 12.5 vs. 3, respectively. Almost all participants reported not having any previous training on tobacco use management. With respect to management strategies, the case-specific OSCE scores ranged from 2.1 to 3.5 in the intervention group and 0.4 to 2.2 in the control group. For cases one to six, pharmacists in the intervention group achieved sig- nificantly higher total scores than did those in the control group. Developing rapport scores Pharmacists in the intervention group achieved signifi- cantly higher total scores for establishing rapport with patients than those in the control group across all the OSCE cases. For example, the mean scores for establish- ing rapport in the intervention group ranged from 2.5 to 3.2 compared to 0.5 to 1.3 in the control group for cases one to six. Recruitment The mean age of the participants in the intervention and the control Table 1 Participant socio-demographic and professional characteristics a Standard Deviation Intervention group n = 57 Control group n = 37 p-value N (%) N (%) Gender Male 32 (56.1) 15 (44.1) 0.267 Female 25 (43.9) 19 (55.9) Mean Age (SDa) 35.9 years (6.5) 34.9 years (6.0) 0.482 Country of origin Egypt 23 (40.3) 13 (39.4) 0.276 India 13 (22.8) 14 (42.4) Palestine 0 (0) 1 (3.1) Philippines 10 (17.5) 4 (12.1) Sudan 5 (8.8) 1 (3) Syria 1 (1.8) 0 (0) Nepal 2 (3.5%) 0 (0) Pakistan 3 (5.3%) 0 (0) Highest pharmacy academic qualification B.Pharm/BSc Pha 48 (85.7) 26 (76.4) 0.606 MPharm 5 (8.9) 4 (11.8) MSc/MPhil/PharmD 2 (3.6) 2 (5.9) Ph.D 1 (1.8) 2 (5.9) Country from which the highest pharmacy degree was obtained Qatar 0 (0) 1 (2.9) 0.224 Egypt 23 (40.3) 12 (34.2) India 13 (22.8) 14 (40) Jordan 1 (1.8) 1 (2.9) Philippines 10 (17.5) 5 (14.2) Sudan 5 (8.8) 1 (2.9) Syria 1 (1.8) 0 (0) Pakistan 4 (7) 0 (0) Australia 0 (0) 1 (2.9) Countries where the pharmacists practiced before moving to Qatar Egypt 23 (40.4) 14 (37.8) 0.808 India 13 (22.8) 14 (37.8) 0.116 Philippines 10 (17.5) 5 (13.5) 0.602 Sudan 6 (10.5) 1 (2.7) 0.239 Saudi Arabia 5 (8.8) 2 (5.4) 0.7 United Arab Emirates (UAE) 1 (1.8) 1 (2.7) 1 No previous practice 0 (0) 2 (5.4%) 0.152 Pakistan 3 (5.3) 0 (0) 0.276 Nepal 2 (3.5) 0 (0) 0.518 Years of practicing as a pharmacist in Qatar Less than 5 years 27 (48.2) 18 (51.4) 0.724 5–10 years 24 (42.9) 13 (37.1) 11–15 years 5 (8.9) 3 (8.6) More than 16 years 0 (0) 1 (2.9) Pharmacists’ position in the pharmacy Pharmacist in training 1 (1.8) 1 (2.9) 0.226 Staff pharmacist 36 (63.2) 16 (45.7) Pharmacy supervisor 9 (15.8) 5 (14.3) Pharmacy manager 11 (19.3) 12 (34.2) Senior pharmacist 0 (0) 1 (2.9) Page 5 of 12 El Hajj et al. BMC Medical Education (2022) 22:289 were male (56.1%), while most participants in the con- trol group were female (55.9%) (Table 1). The mean age of the participants in the intervention and the control Countries where the pharmacists practiced before moving to Qatar Years of practicing as a pharmacist in Qatar Pharmacists’ position in the pharmacy El Hajj et al. Data gathering scores The intensive tobacco education group achieved signif- icantly higher overall total score than the control group for all the OSCE cases. Specifically, the mean total scores for the intervention group ranged from 14.2 to 16.3 com- pared to 6.2 to 11.3 for the control group for cases one to six (Table 4). Pharmacists who received the intensive tobacco-related education performed better in data gathering than those who received the contraception training. The mean scores for data gathering were significantly higher in the intervention group than the control group across all the OSCE cases except for case number six. For cases one to five, the mean scores for the intervention group ranged from 5.0 to 6.3 versus 2.5 to 4.6 in the control group. After adjustment for potential confounders: age, gen- der, years of practicing as pharmacist in Qatar, smoking Table 2 Tobacco-related characteristics of participants a Standard Deviation Intervention group Control group P-value N (%) N (%) Smoking status Ex-tobacco user 6 (10.7) 2 (5.9) 0.895 Current tobacco user 3 (5.4) 2 (5.9) Non-tobacco user 47 (83.9) 30 (88.2) Number of cigarettes per day for current smoker Mean (SDa) 12.5 (3.5) 3 (2.8) 0.097 Previous training on tobacco use and treatment Yes 2 (3.8) 5 (15.6) 0.100 No 50 (96.2) 27 (84.4) Table 2 Tobacco-related characteristics of participants El Hajj et al. Mean (SDb) total score for global assessment Case 1: ‘Healthy’ adult smoker indicated that smoking cessation rates and the possibil- ity to quit smoking were significantly higher for patients who were counseled by trained pharmacists compared to those counseled by non-trained pharmacists [17]. Given the accessibility of community pharmacists to the gen- eral population and since most community pharmacists in Qatar are interested in offering smoking cessation counseling, [5] extensive training programs on tobacco cessation should be part of a continuous professional development (CPD) for pharmacists in Qatar. These pro- grams can equip pharmacists with the needed skills and knowledge to overcome the burden of tobacco use in Qatar. status and previous training in relation to tobacco use and treatment, the intensive tobacco education group achieved, on average, significantly higher adjusted over- all total analytical and total OSCE scores than the control group for all the OSCE cases. (Table 4). Data gathering scores BMC Medical Education (2022) 22:289 Page 7 of 12 Page 7 of 12 Table 3 Summary of OSCEa results Table 3 Summary of OSCEa results Intervention group n = 54 Control group n = 32 p-value N (%) N (%) Mean (SDb) total score for Developing rapport Case 1: ‘Healthy’ adult smoker Possible score range: 0–5 2.8 (0.9) 1.0 (0.7) < 0.001 Case 2: Pregnant smoker Possible score range: 0–5 2.7 (1.1) 0.8 (0.8) < 0.001* Case 3: Teenager smoker Possible score range: 0–5 2.8 (1.1) 0.5 (0.8) < 0.001* Case 4: Relapse prevention in a patient who recently quit smoking and has withdrawal symptoms Possible score range: 0–5 2.5 (1.0) 0.9 (0.9) < 0.001* Case 5: Smoker with cardiovascular diseases Possible score range: 0–5 2.6 (1.0) 1.1 (0.7) < 0.001* Case 6: Smoker in precontemplation stage of change Possible score range: 0–4 3.2 (1.1) 1.3 (0.8) < 0.001* Mean (SDb) total score for Gathering information Case 1: ‘Healthy’ adult smoker Possible score range: 0–11 5.0 (1.6) 2.8 (1.5) < 0.001* Case 2: Pregnant smoker Possible score range: 0–12 6.2 (2.2) 2.5 (1.6) < 0.001* Case 3: Teenager smoker Possible score range: 0–9 5.2 (1.6) 3.0 (1.6) < 0.001* Case 4: Relapse prevention in a patient who recently quit smoking and has withdrawal symptoms Possible score range: 0–13 6.0 (2.3) 4.6 (2.1) 0.005* Case 5: Smoker with cardiovascular diseases Possible score range: 0–12 6.3 (2.1) 3.1 (1.6) < 0.001* Case 6: Smoker in precontemplation stage of change Possible score range: 0–11 5.0 (2.0) 4.4 (2.4) 0.184 Mean (SDb) total score for Management Strategies Case 1: ‘Healthy’ adult smoker Possible score range: 0–10 3.5 (1.7) 2.2 (1.2) < 0.001* Case 2: Pregnant smoker Possible score range: 0–6 2.2 (1.4) 0.4 (0.7) < 0.001* Case 3: Teenager smoker Possible score range: 0–7 2.1 (1.6) 1.1 (1.1) 0.002* Case 4: Relapse prevention in a patient who recently quit smoking and has withdrawal symptoms Possible score range: 0–9 2.8 (1.3) 1.7 (1.3) < 0.001* Case 5: Smoker with cardiovascular diseases Possible score range: 0–10 3.0 (1.9) 1.3 (1.3) < 0.001* Case 6: Smoker in precontemplation stage of change Possible score range: 0–7 3.0 (1.5) 2.2 (1.7) 0.025* Mean (SDb) total score for Monitoring/follow-up Case 1: ‘Healthy’ adult smoker Possible score range: 0–1 0.8 (0.4) 0.4 (0.5) < 0.001* Case 2: Pregnant smoker Possible score range: 0–2 0.8 (0.7) 0.1 (0.3) < 0.001* Case 3: Teenager smoker Possible score range: 0–1 0.7 (0.4) 0.2 (0.4) < 0.001* Case 4: Relapse prevention in a patient who recently quit smoking and has withdrawal symptoms Possible score range: - - - - Case 5: Smoker with cardiovascular diseases Possible score range: 0–1 0.7 (0.4) 0.2 (0.4) < 0.001* Case 6: Smoker in precontemplation stage of change Possible score range: 0–1 0.4 (0.4) 0.1 (0.3) 0.001* group n = 54 g p n = 32 p N (%) N (%) Mean (SDb) total score for Developing rapport Case 1: ‘Healthy’ adult smoker Possible score range: 0–5 2.8 (0.9) 1.0 (0.7) < 0.001* Case 2: Pregnant smoker Possible score range: 0–5 2.7 (1.1) 0.8 (0.8) < 0.001* Case 3: Teenager smoker Possible score range: 0–5 2.8 (1.1) 0.5 (0.8) < 0.001* Case 4: Relapse prevention in a patient who recently quit smoking and has withdrawal symptoms Possible score range: 0–5 2.5 (1.0) 0.9 (0.9) < 0.001* Case 5: Smoker with cardiovascular diseases Possible score range: 0–5 2.6 (1.0) 1.1 (0.7) < 0.001* Case 6: Smoker in precontemplation stage of change Possible score range: 0–4 3.2 (1.1) 1.3 (0.8) < 0.001* Mean (SDb) total score for Gathering information Case 1: ‘Healthy’ adult smoker Possible score range: 0–11 5.0 (1.6) 2.8 (1.5) < 0.001* Case 2: Pregnant smoker Possible score range: 0–12 6.2 (2.2) 2.5 (1.6) < 0.001* Case 3: Teenager smoker Possible score range: 0–9 5.2 (1.6) 3.0 (1.6) < 0.001* Case 4: Relapse prevention in a patient who recently quit smoking and has withdrawal symptoms Possible score range: 0–13 6.0 (2.3) 4.6 (2.1) 0.005* Case 5: Smoker with cardiovascular diseases Possible score range: 0–12 6.3 (2.1) 3.1 (1.6) < 0.001* Case 6: Smoker in precontemplation stage of change Possible score range: 0–11 5.0 (2.0) 4.4 (2.4) 0.184 Mean (SDb) total score for Management Strategies Case 1: ‘Healthy’ adult smoker Possible score range: 0–10 3.5 (1.7) 2.2 (1.2) < 0.001* Case 2: Pregnant smoker Possible score range: 0–6 2.2 (1.4) 0.4 (0.7) < 0.001* Case 3: Teenager smoker Possible score range: 0–7 2.1 (1.6) 1.1 (1.1) 0.002* Case 4: Relapse prevention in a patient who recently quit smoking and has withdrawal symptoms Possible score range: 0–9 2.8 (1.3) 1.7 (1.3) < 0.001* Case 5: Smoker with cardiovascular diseases Possible score range: 0–10 3.0 (1.9) 1.3 (1.3) < 0.001* Case 6: Smoker in precontemplation stage of change Possible score range: 0–7 3.0 (1.5) 2.2 (1.7) 0.025* Mean (SDb) total score for Monitoring/follow-up Case 1: ‘Healthy’ adult smoker Possible score range: 0–1 0.8 (0.4) 0.4 (0.5) < 0.001* Case 2: Pregnant smoker Possible score range: 0–2 0.8 (0.7) 0.1 (0.3) < 0.001* Case 3: Teenager smoker Possible score range: 0–1 0.7 (0.4) 0.2 (0.4) < 0.001* Case 4: Relapse prevention in a patient who recently quit smoking and has withdrawal symptoms Possible score range: - - - - Case 5: Smoker with cardiovascular diseases Possible score range: 0–1 0.7 (0.4) 0.2 (0.4) < 0.001* Case 6: Smoker in precontemplation stage of change Possible score range: 0–1 0.4 (0.4) 0.1 (0.3) 0.001* Mean (SDb) total score for Developing rapport El Hajj et al. Data gathering scores BMC Medical Education (2022) 22:289 Page 8 of 12 Table 3 (continued) Table 3 (continued) Table 3 (continued) a Objective Structured Clinical Examination b Standard Deviation Table 3 (continued) Intervention group n = 54 Control group n = 32 p-value N (%) N (%) Mean (SDb) total score for global assessment Case 1: ‘Healthy’ adult smoker Possible score range: 0–5 3.2 (1.4) 2.4 (1.1) 0.01* Case 2: Pregnant smoker Possible score range: 0–5 3.6 (1.0) 2.4 (1.3) < 0.001* Case 3: Teenager smoker Possible score range: 0–5 3.5 (1.4) 3.0 (1.6) 0.145 Case 4: Relapse prevention in a patient who recently quit smoking and has withdrawal symptoms Possible score range: 0–5 3.4 (1.2) 2.1 (1.2) < 0.001* Case 5: Smoker with cardiovascular diseases Possible score range: 0–5 3.7 (1.1) 2.6 (1.6) < 0.001* Case 6: Smoker in precontemplation stage of change Possible score range: 0–5 3.6 (1.1) 3.3 (1.2) 0.256 Discussion To our knowledge, this is the first randomized con- trolled trial (RCT) conducted in Qatar and the Middle East that involved designing, implementing, and evaluat- ing an intensive education program on tobacco use and dependence disorder treatment for community pharma- cists using an Objective Structured Clinical Examination (OSCE). Overall, the outcomes of this study showed that an intensive tobacco education program significantly enhances community pharmacists’ skills and competen- cies on tobacco cessation. Generally, the pharmacists who received the tobacco cessation training achieved higher scores in developing rapport, data gathering, dis- ease management, and patient follow up/monitoring compared to those who did not. These results are com- parable to the findings of other studies, which involved pharmacists or pharmacy students elsewhere [14, 15].h Different interventions and training programs related to tobacco cessation have been implemented and assessed for effectiveness among healthcare students and professionals using OSCE [19–26]. For instance, OSCE was utilized in Denver and Minneapolis, USA to assess the performance of primary care clinicians who were randomized to receive either moderate or high intensity training for motivational interview- ing (MI) to address tobacco use [19]. Clinicians in the high intensity training had significantly higher scores during the OSCE as compared to those in the moder- ate intensity group for three of six global Motivational Interviewing Treatment Integrity scale scores [19]. The practical skills of third year medical students at a German medical school, who were randomly allocated to either an online course or an attendance course on smoking cessation, were also measured through an OSCE [20]. Overall, median OSCE scores were higher in the attendance group (70.8% vs. 62.8%; p = 0.087), but a statistical significance was only found in one The current study demonstrated the feasibility and the benefits of implementing an educational training on tobacco cessation for community pharmacists in Qatar. Evidence supports the real-life benefits of smoking ces- sation-training programs on pharmacists’ ability to offer smoking cessation [16–18]. One study showed that phar- macists are more likely to counsel patients on stopping smoking and to offer advice on the appropriate use of smoking cessation products when they are provided with training on smoking cessation [16]. In addition, a RCT Page 9 of 12 El Hajj et al. Discussion A prospective intervention study evalu- ated the effects of a multimodal and interactive teach- ing module on smoking cessation for medical students in Germany on their knowledge, skills, attitudes, and self-reported practice using written examinations and OSCE. Scores in the OSCE were significantly higher in the intervention than those in the control group (71.5% vs. 60.5%; p < 0.001) [24]. Moreover, OSCE utilizing standardized patients was used for assessing tobacco dependence education of first-year dental students in one U.S. dental school. The investigators concluded that preparing for and participating in the OSCE appeared to contribute to an increase in student tobacco-related knowledge and facilitated their learning [25]. Moreo- ver, another study implemented a teaching intervention on smoking cessation for fourth-year dental students in Germany and assessed its effectiveness on knowl- edge, communication skills and attitudes using written examinations and OSCE. Students in the intervention group had higher scores in OSCE as compared to the control group (74.9% vs 44.7%; p < 0.001; d = 2.3) [26]. The results of these programs are promising; however, extrapolating these findings to the Qatari context is not plausible especially that community pharmacy prac- tice is different and unique in Qatar. Hence, design- ing, implementing and assessing the effectiveness of an intensive tobacco cessation education program tar- geting Qatar community pharmacists’ tobacco cessa- tion skills using OSCE was warranted. The strengths of our program are that the program was designed and aligned according to the Core Competencies for Evidence-based Treatment of Tobacco Dependence by the Association for the Treatment of Tobacco use and Dependence (ATTUD) and the executed OSCE covered the improvement of knowledge and skills of pharma- cists across six different tobacco-related case scenarios directed to patients with different levels of readiness to quit. Another strength of the study as compared to the previous studies was the prospective setup of the study and the inclusion of a control group taking the same assessments. It is noteworthy to mention that the estimated sample size was achieved for the intervention group, but not for the control group. A possible reason for the low enroll- ment rate in the control group could be the topic itself. Pharmacists may not have been interested in the “women health and contraception” topic versus the “ tobacco ces- sation” topic. p This study has some limitations. Discussion BMC Medical Education (2022) 22:289 Table 4 Adjusted and unadjusted total and analytical OSCE† results a Adjusted for age, gender, years of practicing as pharmacist in Qatar, smoking status and previous training in relation to tobacco use and treatment * statistically significant Smoking training group Contraception training group Unadjusted mean difference Adjusteda mean difference Mean Standard Deviation Mean Standard Deviation Mean Standard Error p-value R2 Mean Standard Error p-value R2—adjusted Case 1: ‘Healthy’ adult smoker Total Analytical Score (# of items:26) 12.1 3.0 6.4 2.4 5.7 0.6 < 0.001* 49.7% 6.3 0.60 < 0.001* 63.3% Final Score 15.2 4.0 8.8 3.1 6.4 0.8 < 0.001* 41.8% 7.2 0.80 < 0.00* 59.2% Case 2: Pregnant smoker Total Analytical Score (# of items:24) 11.8 3.8 3.8 2.2 8.0 0.7 < 0.001* 58.9% 8.4 0.84 < 0.001* 59.7% Final Score 15.3 4.5 6.2 3.0 9.1 0.9 < 0.001* 55.3% 9.6 1.01 < 0.001* 58.1% Case 3: Smoking teenager Total Analytical Score (# of items:20) 10.7 3.4 4.7 2.3 6.0 0.7 < 0.001* 48.7% 6.3 0.75 < 0.001* 50.7% Final Score 14.2 4.3 7.7 3.4 6.5 0.9 < 0.001* 39.1% 7.1 0.98 < 0.001* 43.0% Case 4: Relapse prevention in a patient who recently quit smoking and has withdrawal symptoms Total Analytical Score (# of items:26) 11.2 3.4 7.1 3.4 4.1 0.8 < 0.001* 26.1% 3.6 0.86 < 0.001* 19.6% Final Score 14.6 4.2 9.2 4.4 5.4 1.0 < 0.001* 27.7% 4.9 1.10 < 0.001* 21.7% Case 5: Smoker with cardiovascular diseases Total Analytical Score (# of items:27) 12.6 3.9 5.7 2.7 7.0 0.8 < 0.001* 48.6% 6.9 0.83 < 0.001* 53.1% Final Score 16.3 4.7 8.3 3.6 8.0 1.0 < 0.001* 45.4% 8.2 1.05 < 0.001* 50.1% Case 6: Smoker in precontemplation stage of change Total Analytical Score (# of items:23) 11.7 3.6 8.1 4.0 3.6 0.9 < 0.001* 18.5% 4.3 0.90 < 0.001* 28.4% Final Score 15.2 4.2 11.3 4.9 3.9 1.0 < 0.001* 15.7% 4.5 1.05 < 0.001* 28.8% El Hajj et al. BMC Medical Education (2022) 22:289 Page 10 of 12 Page 10 of 12 scenarios that target unmotivated patients in the train- ing program. single counselling sequence ("Assist": 66.7% vs. 51.4%; p = 0.049) [20]. Conclusionh This study has demonstrated that an intensive tobacco cessation education program can improve pharma- cists’ objectively measured tobacco cessation skills and increase their abilities of tobacco cessation counseling. Any improvements in pharmacists’ tobacco cessation related skills could ultimately decrease tobacco use and dependence among their patients. We believe that the tobacco cessation program that we designed and imple- mented can be incorporated into a wider and larger scale CPD program in Qatar to develop a critical mass of knowledgeable pharmacists with effective tobacco cessa- tion intervention skills. It is worthwhile to note that no significant improve- ment was observed for the intervention group for the last OSCE case, which is about a patient who was not motivated in quitting smoking (i.e., in the pre-contem- plation stage of the transtheoretical model of behavior change). Initiating discussions and establishing rap- port with unmotivated smokers is essential for assisting patients to quit. Hence, future training efforts should emphasize more on how to deal with these patients and encourage them to stop smoking. This might be achieved by having more practical cases and simulated Discussion First, allocation con- cealment or blinding of participants was not possible due to the nature of the study design and program. This might have resulted in some bias as the intervention group might have better prepared for the OSCE. Sec- ond, the skills of the participants in both groups were not assessed at baseline before receiving the training programs. Third, the predetermined sample size was not achieved especially in the control group due to difficul- ties in recruiting participants for both the training and the OSCE as most community pharmacists in Qatar do not have flexible working schedules and many have very long working hours. Moreover, in this study we were not able to examine if the study results represent real differ- ences in the pharmacists’ tobacco cessation activities in practice. Despite these limitations, this study showed the usefulness of an intensive tobacco cessation educational program on the skills of community pharmacists. Future studies are required to examine if pharmacists’ OSCE performances are translated into real-life smoking ces- sation counseling interventions in their practice settings. This can be assessed through actual unannounced visits to community pharmacies using standardized patients or simulated clients to evaluate if there are any changes in pharmacists’ actual competencies in their clinical set- tings. Moreover, additional research is warranted to assess the impact of the program on smokers’ quitting rates in Qatar. Availability of data and materials The raw data and anonymized/pseudonymized data are available on request from the corresponding author, if required. The data are not publicly available due to ethical restrictions. 12. Association for the Treatment of Tobacco use and Dependence: Core Competencies For Evidence-based Treatment of Tobacco Dependence [Internet]. 2005. [Accessed 12 May 2018]. Available from: https://attud. org/pdf/Standards.pdf. Declarations 13. Schulz KF, Altman DG, Moher D. CONSORT 2010 Statement: updated guidelines for reporting parallel group randomized trials. BMJ. 2010;1(2):100–7. https://doi.org/10.1136/bmj.c332. Acknowledgements ld l k h k We would like to thank the pharmacists who participated in both groups. Page 11 of 12 Page 11 of 12 El Hajj et al. BMC Medical Education (2022) 22:289 El Hajj et al. BMC Medical Education (2022) 22:289 El Hajj et al. BMC Medical Education (2022) 22:289 Received: 17 October 2021 Accepted: 24 March 2022 Received: 17 October 2021 Accepted: 24 March 2022 19. Fu SS, Roth C, Battaglia CT, et al. Training primary care clinicians in motivational interviewing: A comparison of two models. Patient Educ Couns. 2015;98(1):61–8. https://doi.org/10.1016/j.pec.2014.10.007. 20. Lauerer E, Tiedemann E, Polak T, et al. Can smoking cessation be taught online? A prospective study comparing e-learning and role-playing in medical education. Int J Med Educ. 2021;12:12–21. https://doi.org/10. 5116/ijme.5ff9.bccc (PMID: 33507877). Funding h k 10. Croft H, Gilligan C, Rasiah R, et al. Current Trends and Opportunities for Competency Assessment in Pharmacy Education-A Literature Review. Pharmacy (Basel). 2019;7(2):67. https://doi.org/10.3390/pharmacy70 20067. 10. Croft H, Gilligan C, Rasiah R, et al. Current Trends and Opportunities for Competency Assessment in Pharmacy Education-A Literature Review. Pharmacy (Basel). 2019;7(2):67. https://doi.org/10.3390/pharmacy70 20067. This work was supported by a collaborative grant from Qatar University Office of Research and Graduate Studies [QUCG-CPH-2018\2019–3]. Its contents are solely the responsibility of the authors and do not necessarily represent the official views of Qatar University. 11. El Hajj MS, Awaisu A, Kheir N, et al. Evaluation of an intensive education program on the treatment of tobacco-use disorder for pharmacists: a study protocol for a randomized controlled trial. Trials. 2019;20(1):25. https://doi.org/10.1186/s13063-018-3068-7. Consent for publication formed consents for publication were obtained from the participan 16. Sinclair H, Bond C, Lennox A, et al. Training pharmacists and pharmacy assistants in the stage-of-change model of smoking cessation: a randomised controlled trial in Scotland. Tob Control. 1998;7(3):253–61. https://doi.org/10.1136/tc.7.3.253. Competing interests The authors declare that they have no competing interests. The authors declare that they have no competing interests. Authors’ contributions 7. Khan KZ, Gaunt K, Ramachandran S, Pushkar P. The Objective Struc- tured Clinical Examination (OSCE): AMEE Guide No. 81. Part II: organisa- tion & administration. Med Teach. 2013;35(9):1447–63. https://doi.org/ 10.3109/0142159X.2013.818635. 7. Khan KZ, Gaunt K, Ramachandran S, Pushkar P. The Objective Struc- tured Clinical Examination (OSCE): AMEE Guide No. 81. Part II: organisa- tion & administration. Med Teach. 2013;35(9):1447–63. https://doi.org/ 10.3109/0142159X.2013.818635. MH is the lead principal investigator of the study leading its conception, design and execution. She wrote the study manuscript. AA, MOH and NK contributed in discussions related to the design of the research project, and review of all the drafts throughout the project. RS and NH helped in data collection and recruitment of participants. ZM assisted in data analysis and contributed in writing the results section of the manuscript. All authors read and approved the final manuscript. 8. Patrício MF, Julião M, Fareleira F, et al. Is the OSCE a feasible tool to assess competencies in undergraduate medical education? Med Teach. 2013;35(6):503–14. https://doi.org/10.3109/0142159X.2013.774330. 8. Patrício MF, Julião M, Fareleira F, et al. Is the OSCE a feasible tool to assess competencies in undergraduate medical education? Med Teach. 2013;35(6):503–14. https://doi.org/10.3109/0142159X.2013.774330. 9. Rushforth HE. Objective structured clinical examination (OSCE): review of literature and implications for nursing education. Nurse Educ Today. 2007;27(5):481–90. https://doi.org/10.1016/j.nedt.2006.08.009. 9. Rushforth HE. Objective structured clinical examination (OSCE): review of literature and implications for nursing education. Nurse Educ Today. 2007;27(5):481–90. https://doi.org/10.1016/j.nedt.2006.08.009. Ethics approval and consent to participate The study protocol and all related instruments and forms were reviewed and granted ethical approval by the Qatar University Institutional Review Board (QU-IRB) (approval number: QU-IRB 906-E/18). All methods were performed in accordance with the relevant guidelines and regulations. Informed consent was obtained from all participants. 14. Simansalam S, Brewster JM, Nik Mohamed MH. Training Malaysian Pharmacy Undergraduates with Knowledge and Skills on Smoking Cessation. Am J Pharm Educ. 2015;79(5):71. https://doi.org/10.5688/ ajpe79571. 14. Simansalam S, Brewster JM, Nik Mohamed MH. Training Malaysian Pharmacy Undergraduates with Knowledge and Skills on Smoking Cessation. Am J Pharm Educ. 2015;79(5):71. https://doi.org/10.5688/ ajpe79571. 15. Martin BA, Chewning BA. Evaluating pharmacists’ ability to counsel on tobacco cessation using two standardized patient scenarios. Patient Educ Couns. 2011;83(3):319–24. https://doi.org/10.1016/j.pec.2010.12.010. 15. Martin BA, Chewning BA. Evaluating pharmacists’ ability to counsel on tobacco cessation using two standardized patient scenarios. Patient Educ Couns. 2011;83(3):319–24. https://doi.org/10.1016/j.pec.2010.12.010. Author details 1 17. Caponnetto P, DiPiazza J, Aiello M, et al. Training pharmacists in the stage-of-change model of smoking cessation and motivational interviewing: A randomized controlled trial. Health Psychol Open. 2017;4(2):2055102917736429. https://doi.org/10.1177/2055102917 736429. 1 College of Pharmacy, QU Health, Qatar University, 2713 Doha, Qatar. 2 Kul- liyyah of Pharmacy, International Islamic University Malaysia, 25200 Kuantan, Pahang, Malaysia. 3 Sidra Medicine, Education City, Al Rayyan Municipality, Qatar. 4 College of Pharmacy and Health Sciences, Ajman University, Ajman, UAE. 5 Medical Education, Weill Cornell Medicine-Qatar, P.O. Box 24144, Doha, Qatar. 6 Division of Epidemiology, Department of Population Health Sciences, Weill Cornell Medicine, New York, NY, USA. 18. Baliunas D, Ivanova A, Tanzini E, et al. Impact of comprehensive smoking cessation training of practitioners on patients’ 6-month quit outcome. Can J Public Health. 2020;111(5):766–74. https://doi.org/10. 17269/s41997-020-00318-1. Received: 17 October 2021 Accepted: 24 March 2022 References 1. Prevalence \| Tobacco Atlas. Tobacco Atlas. https://tobaccoatlas.org/ topic/prevalence/#. Accessed 28 Jan 2021 21. Ockene JK, Hayes RB, Churchill LC, et al. Teaching Medical Students to Help Patients Quit Smoking: Outcomes of a 10-School Randomized Controlled Trial. J Gen Intern Med. 2016;31(2):172–81. https://doi.org/ 10.1007/s11606-015-3508-y. 2. Qatar \| Tobacco Atlas. Tobacco Atlas. https://tobaccoatlas.org/country/ qatar/. Accessed 28 Jan 2021 2. Qatar \| Tobacco Atlas. Tobacco Atlas. https://tobaccoatlas.org/country/ qatar/. Accessed 28 Jan 2021 3. Adams AJ, Hudmon KS. Pharmacist prescriptive authority for smok- ing cessation medications in the United States. J Am Pharm Assoc. 2018;58(3):253–7. https://doi.org/10.1016/j.japh.2017.12.015. 22. Fernandez K, Pandve HT, Debnath DJ. Use of interactive teaching methods in tobacco cessation program and examine it by using objec- tive structured clinical exam. J Educ Health Promot. 2013;2:28. https:// doi.org/10.4103/2277-9531.114210. 4. Csikar JI, Douglas GV, Pavitt S, et al. The cost-effectiveness of smoking cessation services provided by general dental practice, general medi- cal practice, pharmacy and NHS Stop Smoking Services in the North of England. Community Dent Oral Epidemiol. 2016;44(2):119–27. https:// doi.org/10.1111/cdoe.12195. 4. Csikar JI, Douglas GV, Pavitt S, et al. The cost-effectiveness of smoking cessation services provided by general dental practice, general medi- cal practice, pharmacy and NHS Stop Smoking Services in the North of England. Community Dent Oral Epidemiol. 2016;44(2):119–27. https:// doi.org/10.1111/cdoe.12195. 23. Park KY, Park HK, Hwang HS. Group randomized trial of teaching tobacco-cessation counseling to senior medical students: a peer role- play module versus a standardized patient module. BMC Med Educ. 2019;19(1):231. https://doi.org/10.1186/s12909-019-1668-x. 5. El Hajj M, Al Nakeeb R, Al-Qudah R. Smoking cessation counseling in Qatar: community pharmacists’ attitudes, role perceptions and practices. Int J Clin Pharm. 2012;34(4):667–76. https://doi.org/10.1007/ s11096-012-9663-x. 24. Herold R, Schiekirka S, Brown J, et al. Structured Smoking Cessation Training for Medical Students: A Prospective Study. Nicotine Tob Res. 2016;18(12):2209–15. https://doi.org/10.1093/ntr/ntw191. 6. Khan KZ, Ramachandran S, Gaunt K, et al. The Objective Structured Clinical Examination (OSCE): AMEE Guide No. 81. Part I: an historical and theoretical perspective. Med Teach. 2013;35(9):1437–46. https:// doi.org/10.3109/0142159X.2013.818634. 25. Romito L, Schrader S, Zahl D. Using experiential learning and OSCEs to teach and assess tobacco dependence education with first-year dental students. J Dent Educ. 2014;78(5):703–13 (PMID: 24789830). Page 12 of 12 El Hajj et al. BMC Medical Education (2022) 22:289 El Hajj et al. BMC Medical Education (2022) 22:289 26. Vollath SE, Bobak A, Jackson S, et al. References Effectiveness of an innovative and interactive smoking cessation training module for dental students: A prospective study. Eur J Dent Educ. 2020;24(2):361–9. https://doi.org/ 10.1111/eje.12507. 26. Vollath SE, Bobak A, Jackson S, et al. Effectiveness of an innovative and interactive smoking cessation training module for dental students: A prospective study. Eur J Dent Educ. 2020;24(2):361–9. https://doi.org/ 10.1111/eje.12507. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in pub- lished maps and institutional affiliations. • fast, convenient online submission • thorough peer review by experienced researchers in your ﬁeld • rapid publication on acceptance • support for research data, including large and complex data types • gold Open Access which fosters wider collaboration and increased citations maximum visibility for your research: over 100M website views per year • At BMC, research is always in progress. 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https://openalex.org/W2571767533	https://europepmc.org/articles/pmc5251250?pdf=render	English	null	How useful is the EQ-5D in assessing the impact of caring for people with Alzheimer’s disease?	Health and quality of life outcomes	2,017	cc-by	7,826	© The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Abstract Background: The impact on informal caregivers of caring for people with Alzheimer's disease (AD) dementia can be substantial, but it remains unclear which measures(s) best assess such impact. Our objective was to use data from the GERAS study to assess the ability of the EuroQol 5-dimension questionnaire (EQ-5D) to measure the impact on caregivers of caring for people with AD dementia and to examine correlations between EQ-5D and caregiver burden. Methods: GERAS was a prospective, non-interventional cohort study in community-dwelling patients with AD dementia and their informal caregivers. The EQ-5D and Zarit Burden Interview (ZBI) were used to measure health- related quality of life and caregiver burden, respectively. Resource-use data collected included caregiver time spent with the patient on activities of daily living (ADL). Spearman correlations were computed between EQ-5D scores, ZBI scores, and time spent on instrumental ADL (T-IADL) at baseline, 18 months, and for 18-month change scores. T-IADL and ZBI change scores were summarized by EQ-5D domain change category (better/stable/worse). Results: At baseline, 1495 caregivers had mean EQ-5D index scores of 0.86, 0.85, and 0.82, and ZBI total scores of 24.6, 29.4, and 34.1 for patients with mild, moderate, and moderately severe/severe AD dementia, respectively. Change in T-IADL showed a stronger correlation with change in ZBI (0.12; P < 0.001) than with change in EQ-5D index score (0.02; P = 0.546) although both correlations were very weak. Worsening within EQ-5D domains was associated with increases in ZBI scores, although 68%–90% of caregivers remained stable within each EQ-5D domain. There was no clear pattern for change in T-IADL by change in EQ-5D domain. Conclusions: EQ-5D may not be the optimum measure of the impact of caring for people with AD dementia due to its focus on physical health. Alternative measures need further investigation. Keywords: Alzheimer’s disease, Caregiver burden, Europe, Health-related quality of life, Informal care, Observational study and psychiatric medication [2]. Caring for a person with AD dementia can also have a social and financial impact, and the overall effect on caregivers is thought to be reflected in deterioration in their health-related quality of life (HRQoL) as patients become increasingly unable to care for themselves and perform their usual activities [3]. Thus, HRQoL is a construct that evaluates the impact of physical and mental disorders and disability on an individ- ual’s general well-being and can be measured using generic or disease-specific measures [4]. How useful is the EQ-5D in assessing the impact of caring for people with Alzheimer’s disease? Catherine Reed1, Annabel Barrett1, Jeremie Lebrec2, Richard Dodel3, Roy W. Jones4, Bruno Vellas5, Anders Wimo6, Josep Maria Argimon7, Giuseppe Bruno8 and Josep Maria Haro9 Reed et al. Health and Quality of Life Outcomes (2017) 15:16 DOI 10.1186/s12955-017-0591-2 Reed et al. Health and Quality of Life Outcomes (2017) 15:16 DOI 10.1186/s12955-017-0591-2 Correspondence: reed_catherine@lilly.com 1Eli Lilly and Company Limited, Erl Wood Manor, Sunninghill Road, Windlesham, Surrey GU20 6PH, UK Full list of author information is available at the end of the article Background The impact of caring for people with Alzheimer’s disease (AD) dementia falls heavily on informal caregivers who are increasingly required to assist with activities of daily living (ADL) and make decisions on behalf of patients as the disease progresses. The health impact on caregivers can be both physical and emotional [1]; nearly half of the caregivers in some studies meet formal diagnostic criteria for depression and show increased use of health services Caregiver burden is an important multidimensional con- struct that describes the objective and subjective responses to the physical, psychological, social and financial demands * Correspondence: reed_catherine@lilly.com 1Eli Lilly and Company Limited, Erl Wood Manor, Sunninghill Road, Windlesham, Surrey GU20 6PH, UK Full list of author information is available at the end of the article Reed et al. Health and Quality of Life Outcomes (2017) 15:16 Page 2 of 9 Page 2 of 9 of caring [5]. Caregiver burden has been associated with AD dementia severity: caregivers feel more burdened as AD dementia severity increases [6], although there are some differences between spousal and adult-child care- givers in perceived burden measured using the Zarit Burden Interview (ZBI) [7]. the primary caregivers; baseline characteristics and per- ceived burden have been reported previously for the overall caregiver and patient cohorts [6] and by caregiver relation- ship to the patient (e.g., adult-child, spouse) [7]. Community-dwelling patients (aged ≥55 years) with probable AD dementia, who presented within the nor- mal course of care and had a Mini-Mental State Examin- ation (MMSE) score of ≤26, were enrolled between October 2010 and September 2011, mostly at specialist secondary care clinics (memory clinics). Patients were also required to have a primary caregiver (responsible for the patient for at least 6 months per year) who was willing to participate in the study. Poorer caregiver HRQoL has not only been associated with increasing patient dependency [3], but also with greater caregiver perceived burden and increased time spent caring [8, 9]. However, two European studies found poor associations between caregiver and patient HRQoL [10, 11] as assessed using the EuroQol-5 dimension ques- tionnaire (EQ-5D), a recognized generic instrument for measuring HRQoL which elicits a utility value (index score) [4]. Patient EQ-5D scores decreased with increasing AD dementia severity, but caregiver EQ-5D scores did not vary by patient disease severity [10, 11]. Data collected Data were collected for patients and caregivers at the baseline visit and at 6, 12, and 18 months during routine care visits. Full details of the baseline patient and caregiver demographics and characteristics, including comorbidities and medications used, have been reported previously [12]. Caregiver burden was assessed at every visit using the 22-item version of the original 29-item ZBI [15]. The ZBI is a self-report inventory where responses to each item are recorded on a 5-point scale (0 = never to 4 = nearly always) and used to derive a ZBI total score ranging from 0 to 88, with higher scores indicating greater burden. The questions focus on the caregiver’s health, psychological well-being, finances, social life, and relationship with the patient. The ZBI is a valid, reliable, and widely recognized measure of subjective caregiver burden [6, 16]. Since the results of studies investigating caregiver HRQoL and burden are inconsistent, further clarification is needed of the role of caregiver EQ-5D scores in asses- sing the impact of caring for patients with AD dementia and the relationship between EQ-5D and alternative caregiver outcomes. The objective of the present study was to assess the ability of caregiver EQ-5D to measure such impact by exploring EQ-5D index scores and their correlations with ZBI total scores and T-IADL within the caregiver population in the GERAS study. In particu- lar, we used longitudinal data to examine change scores over 18 months and summarized ZBI and T-IADL change scores by EQ-5D domain change category. j g HRQoL was self-assessed by caregivers at the baseline and 18-month visits using the EQ-5D [17]. Caregivers scored their current health state in each of five domains (pain/discomfort, anxiety/depression, mobility, usual activ- ities, and self-care) using a 3-point scale (1 = no problems, 2 = moderate problems, 3 = extreme problems). From the health-state profile obtained, a scoring algorithm using country-specific preference weights [18] was used to calcu- late a total utility score (EQ-5D index score) between 0 (represents death) and 1.0 (represents perfect health). Caregivers also rated their current health status on the day of assessment using the EQ-5D visual analog scale (EQ-5D VAS), which ranges from 0 (worst imaginable health) to 100 (best imaginable health). Background These findings contradict those from other studies (e.g., [3]) and raise the question of whether caregiver EQ-5D is able to accurately reflect utility/disutility in AD dementia. The study aimed to recruit equal numbers of people in each of three disease severity groups based on MMSE criteria: mild AD dementia (MMSE 21–26 points), moder- ate AD dementia (MMSE 15–20 points), or moderately severe/severe (MS/S) AD dementia (MMSE ≤14 points). Ethical review board approval was obtained in each coun- try and all participants provided written informed consent before enrollment. Caregiver time spent on instrumental ADL (T-IADL) is an alternative measure of caregiver impact that can be assessed across the full spectrum of disease severity and contributes to informal care costs when cost-of-illness studies take a societal perspective [12]. Time spent on basic ADL such as eating, dressing, and toileting is a less useful measure of caregiver burden in a community-based AD dementia population because basic ADL do not usually become impaired until the moderate-to-severe stages of AD dementia [13]. Caregiver time increases with increasing severity of patient AD dementia [6, 14] Caregiver and patient characteristics The baseline sociodemographic and clinical characteristics of the caregivers and patients are summarized in Table 1. Caregivers had a mean (SD) age of 67.3 (12.0) years and most were female (64.2%), the spouse of the patient (65.9%), and living with the patient (76.0%). The majority of caregivers reported medical conditions (58.6%; mean of 1.1 medical conditions), most commonly hypertension (36.8%), hypercholesterolemia (23.5%), and depression (10.1%). Approximately one-quarter of the caregivers (23.8%) reported working for pay. The patients had a mean (SD) age of 77.6 (7.7) years, 54.8% were female, mean (SD) time since diagnosis was 2.2 (2.2) years, and 73.6% had comorbidities (mean of 1.4 comorbidities). Statistical analysis Baseline characteristics of caregivers and patients were summarized based on non-missing observations. Data are presented as means (standard deviations [SDs], medians, and interquartile range [Q1, Q3]) or as numbers and percentages of caregivers/patients. Comparisons between AD dementia severity groups for caregiver mean EQ-5D, ZBI, and T-IADL used analysis of variance (ANOVA), with country and baseline MMSE sever- ity as independent factors. Although these variables do not follow a normal distribution, the central limit theorem en- sures the validity of the ANOVA to compare the means, which are based on a large sample size. Also, because the ef- fect of country was adjusted for in the ANOVA, it allows the combination of country-specific derived EQ-5D index scores. Spearman correlation coefficients examined the within- subject associations between continuous variables such as caregiver ZBI total scores, EQ-5D index scores, EQ-5D VAS scores, and T-IADL, at baseline, at 18 months, and for the change from baseline to 18 months. We inter- preted correlation coefficients of 0–0.19 as very weak and 0.20–0.39 as weak. Comparisons between AD dementia severity groups for caregiver mean EQ-5D, ZBI, and T-IADL used analysis of variance (ANOVA), with country and baseline MMSE sever- ity as independent factors. Although these variables do not follow a normal distribution, the central limit theorem en- sures the validity of the ANOVA to compare the means, which are based on a large sample size. Also, because the ef- fect of country was adjusted for in the ANOVA, it allows the combination of country-specific derived EQ-5D index scores. Caregiver HRQoL The caregivers’ overall mean (SD) EQ-5D index score at baseline was 0.84 (0.20), and was 0.86 (0.18), 0.85 (0.19), and 0.82 (0.23) for the mild, moderate, and MS/S AD de- mentia groups, respectively (ANOVA P = 0.043), indicating Table 1 Patient and caregiver characteristicsa at baseline Table 1 Patient and caregiver characteristicsa at baseline Characteristic Mean (SD) or n (%) Caregiver, n 1495 Sex, n (%) female 958 (64.2) Age, mean (SD) 67.3 (12.0) Relationship to patient, n (%) Spouse 984 (65.9) Child 405 (27.1) Other 104 (6.9) Lives with patient, n (%) 1135 (76.0) Working for pay, n (%) 355 (23.8) Caregivers with medical conditions, n (%) 875 (58.6) Number of medical conditions, mean (SD) 1.1 (1.2) Patient, n 1495 Sex, n (%) female 819 (54.8) Age, mean (SD) 77.6 (7.7) Time since diagnosis of AD, years, mean (SD) 2.2 (2.2) MMSE score, mean (SD) 17.4 (6.3) Patients with comorbidities, n (%) 1101 (73.6) Number of comorbidities, mean (SD) 1.4 (1.2) AD Alzheimer’s disease; MMSE Mini-Mental State Examination; SD standard deviation aAll data are based on patients/caregivers with non-missing data Spearman correlation coefficients examined the within- subject associations between continuous variables such as caregiver ZBI total scores, EQ-5D index scores, EQ-5D VAS scores, and T-IADL, at baseline, at 18 months, and for the change from baseline to 18 months. We inter- preted correlation coefficients of 0–0.19 as very weak and 0.20–0.39 as weak. The change in caregiver ZBI total score or T-IADL over 18 months was examined for the total population accord- ing to the change in each of the five EQ-5D domains, which was categorized as better, stable, or worse based on the numerical changes in each 3-point scale. All analyses were performed using Statistical Analysis Software (SAS) version 9.2 (SAS Institute, Cary, NC, USA). Study design and participants GERAS was an 18-month, prospective, multicenter, natur- alistic, observational cohort study conducted in France, Germany, and the UK, designed to evaluate the costs and resource use associated with AD for community-dwelling patients and their caregivers [12]. The study design, patient characteristics, and baseline costs and resource-use data have been reported [12]. The present analysis focuses on Caregiver time spent looking after the patient was assessed using the Resource Utilization in Dementia Reed et al. Health and Quality of Life Outcomes (2017) 15:16 Page 3 of 9 Page 3 of 9 119), or the caregiver/patient was lost to follow-up (n = 26). The number of caregivers with available data at the 18-month visit was: ZBI, n = 932; EQ-5D, n = 934; T- IADL, n = 983. The number of caregivers with available data for the change from baseline to 18 months was: ZBI, n = 931; EQ-5D, n = 933; and T-IADL, n = 982. (RUD) instrument [19] (version RUD Complete 3.1), by interview with the caregiver. This is a widely used, standardized instrument for collecting resource-use data in dementia and has been validated for use in different care settings, including in community-dwelling patients [20, 21]. Time (in the month preceding both the baseline visit and the 18-month visit) was recorded as the total number of caregiving hours, including the number of hours spent on basic ADL (e.g., standard self-care tasks such as eating, getting dressed), instrumental ADL (e.g., cooking, shopping), and patient supervision (i.e., watching the patient to prevent dangerous events). 119), or the caregiver/patient was lost to follow-up (n = 26). The number of caregivers with available data at the 18-month visit was: ZBI, n = 932; EQ-5D, n = 934; T- IADL, n = 983. The number of caregivers with available data for the change from baseline to 18 months was: ZBI, n = 931; EQ-5D, n = 933; and T-IADL, n = 982. Caregiver T-IADL Median caregiver T-IADL during the month before baseline was 60.0 (Q1, Q3: 20.0, 120.0) hours (Table 2). This time increased for the patient groups with greater disease severity and was 36.0 (Q1, Q3: 8.0, 90.0), 60.0 (Q1, Q3: 24.0, 120.0), and 90.0 (Q1, Q3: 40.0, 120.8) hours for the month before baseline in the mild, moderate, and MS/ S AD dementia groups, respectively (ANOVA P < 0.001; see Table 2). The median change from baseline to 18 months in T-IADL was 10.0 (Q1, Q3: −15.0, 45.0) hours/month for the overall cohort; the median changes from baseline in the mild, moderate, and MS/S AD dementia groups were 13.0 (Q1, Q3: −5.0, 52.0), 10.0 (Q1, Q3: −15.0, 46.5), and 0.0 (Q1, Q3: −30.0, 45.0) hours/ month, respectively (ANOVA P = 0.151). Examination of the EQ-5D domain scores (Fig. 1) showed that very few caregivers had extreme problems (<5% for any domain) and the levels of problems were similar at baseline and at 18 months. At both time points, more caregivers had some problems in the domains of pain/discomfort (baseline: 42.8%; 18 months: 46.8%) and anxiety/depression (baseline: 30.3%; 18 months: 33.9%) than in the other three domains. Results At baseline, the study cohort analyzed comprised 1495 patients with mild AD dementia (n = 566), moderate AD dementia (n = 472), or MS/S AD dementia (n = 457), and their caregivers (n = 1495). The number of caregivers and patients from each country was as follows: France, n = 419; Germany, n = 550; UK, n = 526. A total of 1040 caregivers attended the 18-month visit (69.6%). The main reasons for discontinuation from the study were patient institutionalization (n = 214), the patient had died (n = 92), the caregiver/patient had decided to leave the study (n = Page 4 of 9 Reed et al. Health and Quality of Life Outcomes (2017) 15:16 worse disease severity: 24.6 (14.2), 29.4 (14.8), and 34.1 (14.8) for the mild, moderate, and MS/S AD dementia groups, respectively (ANOVA P < 0.001) (Table 2). The mean (SD) change in ZBI total score from baseline to 18 months was 4.9 (12.6) for the overall cohort of caregivers, and differed significantly according to patient disease severity at baseline: 5.4 (11.8), 5.9 (13.7), and 3.0 (12.4) for the mild, moderate, and MS/S AD dementia groups, respectively (ANOVA P = 0.028). a very slightly lower caregiver HRQoL for patients with MS/S AD dementia (Table 2). The overall caregiver mean (SD) EQ-5D VAS score at baseline was 75.1 (17.5), and by patient AD severity was 75.8 (16.6), 76.3 (16.5), and 72.9 (19.2) for the mild, moderate, and MS/S AD dementia groups, respectively (ANOVA P = 0.013). The mean (SD) change from baseline to 18 months was −0.02 (0.21) for the caregiver EQ-5D index score and −3.0 (18.5) for the caregiver EQ-5D VAS score. For both caregiver EQ-5D measures, the change from baseline to 18 months in the overall sample was not significant and did not differ significantly across the AD dementia severity groups (data not shown). Missing data (overall): EQ-5D, n = 12; ZBI, n = 10; T-IADL, n = 2 aComparisons between AD severity groups used analysis of variance (ANOVA) with country and baseline Mini-Mental State Examination (MMSE) severity as i d d t f t AD Alzheimer’s disease; EQ-5D EuroQol-5 dimension questionnaire; HRQoL health-related quality of life; MS/S moderately severe/severe; Q1, Q3 interquartile range; SD standard deviation; T-IADL time spent on instrumental activities of daily living; VAS visual analog scale; ZBI Zarit Burden Interview eimer’s disease; EQ-5D EuroQol-5 dimension questionnaire; HRQoL health-related quality of life; MS/S moderately severe/severe; Q1, Q3 dard deviation; T-IADL time spent on instrumental activities of daily living; VAS visual analog scale; ZBI Zarit Burden Interview d t ( ll) EQ 5D 12 ZBI 10 T IADL 2 Caregiver burden ZBI total scores at baseline ranged from 0 to 80 and the mean (SD) score was 29.0 (15.1). The mean (SD) ZBI total score showed a greater burden for the patient groups with Table 2 Caregiver HRQoL (EQ-5D) and burden scores at baseline Measure n Mean (SD) Median (Q1, Q3) P valuea EQ-5D index scoreb 1483 0.84 (0.20) 0.89 (0.79, 1.00) Mild AD dementia 560 0.86 (0.18) 0.89 (0.79, 1.00) 0.043 Moderate AD dementia 469 0.85 (0.19) 0.89 (0.79, 1.00) MS/S AD dementia 454 0.82 (0.23) 0.89 (0.73, 1.00) EQ-5D VAS scorec 1483 75.1 (17.5) 80.0 (65.0, 90.0) Mild AD dementia 560 75.8 (16.6) 80.0 (69.0, 89.0) 0.013 Moderate AD dementia 469 76.3 (16.5) 80.0 (69.0, 90.0) MS/S AD dementia 454 72.9 (19.2) 79.0 (60.0, 89.0) ZBI total scored 1485 29.0 (15.1) 28.0 (17.0, 40.0) Mild AD dementia 560 24.6 (14.2) 22.0 (14.0, 33.0) <0.001 Moderate AD dementia 471 29.4 (14.8) 29.0 (18.0, 39.0) MS/S AD dementia 454 34.1 (14.8) 33.0 (22.0, 45.0) T-IADL (hours/month) 1493 79.3 (89.5) 60.0 (20.0, 120.0) Mild AD dementia 565 61.0 (83.1) 36.0 (8.0, 90.0) <0.001 Moderate AD dementia 472 77.5 (79.2) 60.0 (24.0, 120.0) MS/S AD dementia 456 103.8 (101.1) 90.0 (40.0, 120.8) AD Alzheimer’s disease; EQ-5D EuroQol-5 dimension questionnaire; HRQoL health-related quality of life; MS/S moderately severe/severe; Q1, Q3 interquartile range; SD standard deviation; T-IADL time spent on instrumental activities of daily living; VAS visual analog scale; ZBI Zarit Burden Interview Missing data (overall): EQ-5D, n = 12; ZBI, n = 10; T-IADL, n = 2 aComparisons between AD severity groups used analysis of variance (ANOVA) with country and baseline Mini-Mental State Examination (MMSE) severity as independent factors bCountry-specific health status index score; range 0 to 1.0, higher scores indicate better health-related quality of life cEQ-5D VAS score range = 0–100, higher scores indicate better health-related quality of life dZBI total score range = 0–88, higher scores indicate greater burden Table 2 Caregiver HRQoL (EQ-5D) and burden scores at baseline bCountry-specific health status index score; range 0 to 1.0, higher scores indicate better health-related quality of life cEQ-5D VAS score range = 0–100, higher scores indicate better health-related quality of life d BI total score range = 0–88, higher scores indicate greater burden Reed et al. Health and Quality of Life Outcomes (2017) 15:16 Page 5 of 9 Fig. 1 Problems in caregiver EQ-5D domains at baseline and 18 months. ZBI Scores by EQ-5D Domain Correlations Between EQ-5D, ZBI, and T-IADL Correlations were weak or very weak between EQ-5D (index and VAS scores) and ZBI or T-IADL at each time point and for the change scores (Table 3). For the ZBI, the 18-month change score correlation was −0.16 (95% confidence interval, CI: −0.22, −0.09) for the EQ-5D VAS score and −0.09 (95% CI: −0.15, −0.03) for the EQ- 5D index score. Caregiver T-IADL showed weak correl- ation with ZBI scores, and the 18-month change score correlation was 0.12 (95% CI: 0.05, 0.18). However, there was no significant correlation between the 18- month change scores for T-IADL and the EQ-5D index score (Table 3). The mean ZBI total scores at baseline and at 18 months by EQ-5D domain showed that burden tended to be greater (higher mean ZBI total score) among caregivers with greater problem severity in each EQ-5D domain (data not shown). From baseline to 18 months, the HRQoL of the majority of caregivers (68–90%) was stable in each EQ-5D domain, though some caregivers (3–14%) showed better EQ-5D scores in some domains than at baseline. HRQoL worsened from baseline to 18 months in 7% of caregivers within the self-care domain, 10% within the mobility domain, 13% in the usual activities domain, 17% in anxiety/depression domain, and 18% in the pain/discomfort domain (Fig. 2). Caregiver burden Baseline: n = 1483 (data missing for 12 caregivers); 18 months: n = 934 (data missing for 113 caregivers). EQ-5D, EuroQol-5 dimension questionnaire Fig. 1 Problems in caregiver EQ-5D domains at baseline and 18 months. Baseline: n = 1483 (data missing for 12 caregivers); 18 months: n = 934 (data missing for 113 caregivers). EQ-5D, EuroQol-5 dimension questionnaire ; EQ-5D EuroQol-5 dimension questionnaire; T-IADL time spent on instrumental activities of daily living (hours/month); VAS visual analo n Interview ZBI Scores by EQ-5D Domain Table 3 Correlationsa between caregiver EQ-5D, T-IADL, and ZBI scores Variable Time point EQ-5D VAS score EQ-5D index score T-IADL n Coefficient 95% CI P value n Coefficient 95% CI P value n Coefficient 95% CI P value ZBI total score Baseline vs baseline 1483 −0.21 −0.26, −0.16 <0.001 1483 −0.21 −0.25, −0.16 <0.001 1484 0.30 0.25, 0.34 <0.001 18 months vs 18 months 929 −0.19 −0.26, −0.13 <0.001 929 −0.21 −0.27, −0.15 <0.001 928 0.22 0.15, 0.28 <0.001 Change score vs change scoreb 928 −0.16 −0.22, −0.09 <0.001 928 −0.09 −0.15, −0.03 0.006 926 0.12 0.05, 0.18 <0.001 T-IADL Baseline vs baseline 1482 −0.15 −0.19, −0.10 <0.001 1482 −0.14 −0.19, −0.09 <0.001 18 months vs 18 months 930 −0.08 −0.15, −0.02 0.010 930 −0.10 −0.16, −0.03 0.004 Change score vs change scoreb 928 −0.07 −0.13, −0.01 0.030 928 0.02 −0.04, 0.08 0.546 CI confidence interval; EQ-5D EuroQol-5 dimension questionnaire; T-IADL time spent on instrumental activities of daily living (hours/month); VAS visual analog scale; ZBI Zarit Burden Interview aSpearman correlation coefficients, 95% CIs, and P values bChange in score from baseline to 18 months Reed et al. Health and Quality of Life Outcomes (2017) 15:16 Page 6 of 9 Fig. 2 Change in caregiver ZBI total score by EQ-5D domain change category from baseline to 18 months (n = 931). Change in EQ-5D domain data were missing for three of these caregivers. EQ-5D, EuroQol-5 dimension questionnaire; ZBI, Zarit Burden Interview As evidenced by Fig. 2, which shows the mean change in ZBI total score by EQ-5D domain change category over 18 months, there was a trend for caregivers who experienced a worsening in EQ-5D domain score over 18 months to have the largest increases in ZBI total score (i.e., the greatest increase in caregiver burden). score over 18 months had a greater increase in mean ZBI total score than caregivers whose HRQoL domain score remained stable or improved. However, this trend was not observed when the EQ-5D domains were combined into the single EQ-5D index score (Table 3 shows a very weak correlation between the change scores). g The longitudinal GERAS study included a large sample of community-dwelling patients with a wide range of AD dementia severity who participated with their care- givers, with assessments being made in a naturalistic set- ting. ZBI Scores by EQ-5D Domain The caregivers participating in the GERAS study had a high mean EQ-5D index score (0.84) at baseline, show- ing that they had relatively good HRQoL, similar to that of the general population of people aged 65–74 years in each country (France: 0.81; Germany: 0.89; and UK: 0.78 from Table 3.6 in Szende et al. [18]). The EQ-5D index score from our study is also consistent with a previous study showing little difference in utility values in a UK general population sample (n = 77) and caregivers of people with mild dementia (n = 71), where the mean (SD) EQ-5D scores were 0.79 (0.25) for the general population sample and 0.78 (0.19) for the caregivers [23]. However, these caregivers of people with dementia gave lower utility values for dementia health states than members of the general population; this could impact on the results of cost–utility analyses, which generally use general popula- tion values [23]. T-IADL by EQ-5D Domain Figure 3 summarizes the median change in caregiver T- IADL by EQ-5D domain change category over 18 months. Of the 982 caregivers with available data on the change in T- IADL, the EQ-5D domain change category was missing for 54 (5%). Fig. 3 shows that there was no clear pattern for me- dian change in T-IADL by EQ-5D domain change category. Discussion This is consistent with previous reports that a high proportion of informal caregivers of patients with AD de- mentia report problems in the EQ-5D domains of pain/dis- comfort and anxiety/depression [3, 9]. As the GERAS study required caregivers to be the established caregiver, the im- pact on pain/discomfort and anxiety/depression scores may have been captured at baseline with low caregiver expect- ation of much change over time. These findings suggest that, because some of the EQ-5D domains are at ceiling scores (with few caregivers remaining in the study expected to decline much in the physical or emotional domains), caregiver EQ-5D is not a particularly informative or sensitive measure of the impact of caring for patients with AD de- mentia. It is possible that most caregivers who remained in the study were physically healthy and that patients discon- tinuing the study (for reasons including institutionalization or death) were those experiencing more acute problems. The discontinuation of caregivers of these patients may thereby reduce the likelihood of observing caregivers with extreme problems. during this stage of the disease; we believe that by the time the patient has progressed to more severe AD dementia, caregivers are likely to have adapted their life and reached a peak in their perceived burden, and patients may be receiv- ing more formal care. There was a tendency for the in- crease in mean ZBI total score to be greatest among caregivers who showed a worsening in each of the EQ-5D domains, especially the anxiety/depression domain. Little published information exists on the relationship between caregiver burden and HRQoL over time in dementia. However, our finding that EQ-5D is a relatively insensitive measure of the impact of caring is consistent with a previous longitudinal study where the mean ZBI score was higher for caregivers of home-living patients with dementia (32.4) than for caregivers of patients with dementia in long-term care (24.9), while the mean EQ-5D index score was similar for both types of caregiver (0.76 and 0.78, respectively) [24]. Furthermore, for a subgroup of caregivers where the patient transitioned from home to institutional care during the 3-month follow-up period, the mean ZBI score decreased from 35.4 to 22.4, while the caregiver EQ-5D index score remained stable at 0.77 [24]. Caregiver T-IADL is sensitive to change across the full range of AD dementia severity and is a common out- come measure in clinical trials. Discussion The impact of AD dementia on caregivers is substantial and should be considered when evaluating the societal im- pact of this disease. Although caregiver outcomes are not typically included in economic evaluations, a preference- based measure such as the EQ-5D can be used to inform economic evaluations [7, 22]. However, the results of our analysis suggest that the EQ-5D is not particularly effect- ive for capturing the true impact on caregivers of caring for people with AD dementia: the EQ-5D index score had a low sensitivity to change over an 18-month period and was not clearly differentiated by patient AD dementia se- verity. EQ-5D index and VAS scores had weak or very weak correlations with other potential measures of impact, perceived caregiver burden (ZBI) and a measure of care- giver time (caregiver T-IADL), both assessed using stan- dardized instruments. There were minimal changes in caregiver EQ-5D scores over the 18-month follow-up period. However, as the EQ- 5D is a brief generic measure of health status rather than an AD-specific HRQoL scale, it may miss both disease-specific and caregiver-specific impacts [4]. The majority of caregivers reported no problems in the EQ-5D domains of mobility, self-care, and usual activities, which reflect physical health, When the EQ-5D was analyzed at the domain level, care- givers who experienced a worsening in HRQoL domain Reed et al. Health and Quality of Life Outcomes (2017) 15:16 Page 7 of 9 Fig. 3 Change in caregiver T-IADL by EQ-5D domain change category from baseline to 18 months (n = 982). Change in EQ-5D domain data were missing for 54 of these caregivers. 95% confidence intervals are bootstrap-based. EQ-5D, EuroQol-5 dimension questionnaire; T-IADL, time spent on instrumental activities of daily living (hours/month) Fig. 3 Change in caregiver T-IADL by EQ-5D domain change category from baseline to 18 months (n = 982). Change in EQ-5D domain data were missing for 54 of these caregivers. 95% confidence intervals are bootstrap-based. EQ-5D, EuroQol-5 dimension questionnaire; T-IADL, time spent on instrumental activities of daily living (hours/month) implying that these caregivers generally had the good physical health to be able to perform their role as caregiver. Approximately half of the caregivers reported some or ex- treme problems in the pain/discomfort domain and about one-third of caregivers had some or extreme problems in the anxiety/depression domain, reflecting emotional/mental health. Discussion Our analyses showed that caregiver T-IADL had very weak correlations with caregiver EQ-5D scores (index and VAS scores), and that there was no clear pattern for the change in caregiver T- IADL by change in EQ-5D domains, reinforcing our conclusion that EQ-5D is not a sensitive measure of the impact of caring for people with AD dementia. In our study, the increase in ZBI total score over time dif- fered according to patient AD dementia severity at baseline and was highest for those caring for patients with moderate AD dementia and lowest for caregivers of patients with MS/S AD dementia. The greatest change in caregiver bur- den may be expected to occur in moderate AD dementia because patients often develop more behavioral problems The relationship between caregiver HRQoL and T-IADL needs further investigation. A recent systematic review on Reed et al. Health and Quality of Life Outcomes (2017) 15:16 Page 8 of 9 Page 8 of 9 18 months only. In addition, we used pooled data from three countries and it would be interesting to examine whether there are country-level effects. As part of our descriptive summary of longitudinal GERAS study results, we investigated the EQ-5D index scores from all countries based on the UK population values and have seen little difference from those generated using country-specific values (data available on request). We found no evidence to suggest that the relationship between EQ-5D and the other measures would differ by country. Fifth, changes in ZBI and T-IADL cannot be compared in the same way as EQ- 5D domain changes, as they are continuous scores. Assess- ment of T-IADL by the caregiver may be subject to recall bias although an electronic diary to record time spent giving care was provided for the purposes of this study. Finally, caregiver HRQoL and burden may change after the patient with AD dementia has been institutionalized [33, 34]. We will report caregiver EQ-5D and ZBI after pa- tient institutionalization or death in a future publication. the relationship between caregiver quality of life (QoL, cov- ering the broader concept of complete physical, mental, and social well-being) and the level/quality of care provided to people with AD included only one study, QoL was mea- sured as overall well-being using the Perceived Change Index, and the evidence was equivocal [25]. Funding The GERAS study was funded by Eli Lilly and Company. Acknowledgments The authors would like to acknowledge Deirdre Elmhirst, Karen Goa, and Gillian Gummer (Rx Communications, Mold, UK) for medical writing assistance with the preparation of this article, funded by Eli Lilly and Company. Abbreviations AD Al h i ’ d AD: Alzheimer’s disease; ADL: Activities of daily living; ANOVA: Analysis of variance; CarerQoL: Care-related Quality of Life instrument; CES: Caregiver Experience Scale; EQ-5D: EuroQol-5 dimension questionnaire; HRQoL: Health-related quality of life; MMSE: Mini-Mental State Examination; MS/S: Moderately severe/severe; Q1, Q3: Interquartile range; QoL: Quality of life; RUD: Resource Utilization in Dementia; SAS: Statistical Analysis Software; SD: Standard deviations; T-IADL: Time spent on instrumental ADL; VAS: Visual analog scale; ZBI: Zarit Burden Interview Discussion An increase in caregiver QoL over six months was associated with an in- crease in T-IADL, although there was a decrease in total caregiving hours [25]. This suggests that caregivers with a better HRQoL are able to spend more time addressing spe- cific aspects of care related to instrumental ADL, such as housework, financial management, and correct use of med- ications. Thus, we can speculate that interventions which improve caregiver HRQoL may improve the level of care that caregivers provide to people with AD dementia. Taken together, our results imply that the EQ-5D may not be the most appropriate measure of the impact of caring for people with AD dementia given the structure of its domains. This suggests that, although the EQ-5D is commonly used in health economics and outcomes research involving caregivers of people with dementia [26], it may not be sensitive enough to measure changes in caregiver HRQoL. Thus, the EQ-5D may underesti- mate the impact of an intervention on the caregiver and, therefore, its cost-effectiveness. Conclusions Our findings indicate that EQ-5D may not be the best meas- ure of the impact on caregivers of caring for people with AD dementia because it mainly focuses on physical health. Alter- native measures, including measures of caregiver burden assessed using the ZBI or caregiver time, may provide a more accurate picture of the impact of caring for a person with AD dementia, and require further investigation. This approach of assessing caregiver burden is perhaps more rele- vant than looking at HRQoL, as the current emphasis is on developing interventions aimed at relieving caregiver burden. Other instruments have been developed that measure the impact of caregiving on informal caregivers, which can be used in economic evaluations; these include the Care- related Quality of Life instrument (CarerQoL) [27, 28] and the Caregiver Experience Scale (CES) [29, 30]. However, these instruments are still undergoing validation. g g There are several limitations to our study. First, the GERAS study included only community-dwelling patients at enrollment who had primary caregivers willing to partici- pate in the study. Thus, our sample of caregivers may not be representative of all caregivers for AD, as it includes only those able/willing to participate. This is reflected in the high proportion of caregivers who had no problems in the EQ- 5D domains of mobility, self-care, and usual activities, although we were able to examine changes in EQ-5D over time. Second, as HRQoL (measured using EQ-5D) is a different construct from caregiver burden and time spent on ADL, this has imposed some limitation on our analysis. However, our aim was to understand how these commonly reported measures improve our understanding of the im- pact of AD for the caregiver and enhance understanding of how the measures best align with the decline in patients’ AD severity. Third, other factors that may influence caregiver HRQoL, burden and time spent caring, such as depression in the caregiver or neuropsychiatric symptoms in the person with AD dementia [2, 6, 31, 32], were not in- cluded within the current objective of our analyses. Fourth, our analysis is based only on those caregivers with available data at 18 months, and evaluated score data at baseline and Authors’ contributions CR, JL, BV, AW and JMA conceived the study and participated in the study design. CR, AB, JL, RD, RWJ, AW, GB and JMH were involved with acquisition, analysis or interpretation of data for the work. All authors assisted in the drafting of the manuscript. CR, RWJ, BV, JMA and GB were involved in the supervision of the project and JL performed the statistical analyses. All authors read and approved the final manuscript. References 2014;18:677–84. 6. Haro JM, Kahle-Wrobleski K, Bruno G, et al. Analysis of burden in caregivers of people with Alzheimer’s disease using self-report and supervision hours. J Nutr Health Aging. 2014;18:677–84. 30. Al-Janabi H, Flynn TN, Coast J. Estimation of a preference-based carer experience scale. Med Decis Making. 2011;31:458–68. 7. Reed C, Belger M, Dell’Agnello G, et al. Caregiver burden in Alzheimer’s disease: differential associations in adult-child and spousal caregivers in the GERAS observational study. Dement Geriatr Cogn Dis Extra. 2014;4:51–64. 31. Dauphinot V, Delphin-Combe F, Mouchoux C, Dorey A, Bathsavanis A, Makaroff Z, Rouch I, Krolak-Salmon P. Risk factors of caregiver burden among patients with Alzheimer's disease or related disorders: a cross- sectional study. J Alzheimers Dis. 2015;44:907–16. 8. Dixon S, Walker M, Salek S. Incorporating carer effects into economic evaluation. Pharmacoeconomics. 2006;24:43–53. 9. Andrén S, Elmståhl S. The relationship between caregiver burden, caregivers’ perceived health and their sense of coherence in caring for elders with dementia. J Clin Nurs. 2008;17:790–9. 32. Feast A, Moniz-Cook E, Stoner C, Charlesworth G, Orrell M. A systematic review of the relationship between behavioral and psychological symptoms (BPSD) and caregiver well-being. Int Psychogeriatr. 2016;27:1–14. 10. Lopez-Bastida J, Serrano-Aguilar P, Perestelo-Perez L, Oliva-Moreno J. Social- economic costs and quality of life of Alzheimer disease in the Canary Islands, Spain. Neurology. 2006;67:2186–91. 33. Gaugler JE, Roth DL, Haley WE, Mittelman MS. Can counseling and support reduce burden and depressive symptoms in caregivers of people with Alzheimer’s disease during the transition to institutionalization? Results from the New York University caregiver intervention study. J Am Geriatr Soc. 2008;56:421–8. 11. Mesterton J, Wimo A, By A, et al. Cross sectional observational study on the societal costs of Alzheimer’s disease. Curr Alzheimer Res. 2010;7:358–67. 12. Wimo A, Reed CC, Dodel R, et al. The GERAS study: a prospective observational study of costs and resource use in community dwellers with Alzheimer’s disease in three European countries – study design and baseline findings. J Alzheimers Dis. 2013;36:385–99. 34. Schultz R, Belle SH, Czaja SJ, McGinnis KA, Stevens A, Zhang S. Long-term care placement of dementia patients and caregiver health and well-being. JAMA. 2004;292:961–7. 13. Marshall GA, Amariglio RE, Sperling RA, Rentz DM. Activities of daily living: where do they fit in the diagnosis of Alzheimer’s disease? Neurodegener Dis Manag. 2012;2:483–91. 14. Bell CM, Araki SS, Neumann PJ. The association between caregiver burden and caregiver health-related quality of life in Alzheimer disease. Author details 1 21. Wimo A, Jonsson L, Zbrozek A. The Resource Utilization in Dementia (RUD) instrument is valid for assessing informal care time in community-living patients with dementia. J Nutr Health Aging. 2010;14:685–90. 1Eli Lilly and Company Limited, Erl Wood Manor, Sunninghill Road, Windlesham, Surrey GU20 6PH, UK. 2Lilly Deutschland GmbH, Bad Homburg, Germany. 3Philipps-University, Marburg, Germany. 4RICE (The Research Institute for the Care of Older People), Royal United Hospital, Bath, UK. 5Toulouse University Hospital, Toulouse, France. 6Karolinska Institute, 7 8 1Eli Lilly and Company Limited, Erl Wood Manor, Sunninghill Road, Windlesham, Surrey GU20 6PH, UK. 2Lilly Deutschland GmbH, Bad Homburg, Germany. 3Philipps-University, Marburg, Germany. 4RICE (The Research Institute for the Care of Older People), Royal United Hospital, Bath, UK. 5Toulouse University Hospital, Toulouse, France. 6Karolinska Institute, Stockholm, Sweden. 7Servei Català de la Salut, Barcelona, Spain. 8Sapienza University of Rome, Rome, Italy. 9Parc Sanitari Sant Joan de Déu, CIBERSAM, Universitat de Barcelona, Barcelona, Spain. 22. Shearer J, Green C, Ritchie CW, Zajicek JP. Health state values for use in the economic evaluation of treatments for Alzheimer’s disease. Drugs Aging. 2012;29:31–43. 23. Rowen D, Mulhern B, Banerjee S, et al. Comparison of general population, patient, and carer utility values for dementia health states. Med Decis Making. 2015;35:68–80. University of Rome, Rome, Italy. 9Parc Sanitari Sant Joan de Déu, CIBERSAM, Universitat de Barcelona, Barcelona, Spain. 24. Bleijlevens MH, Stolt M, Stephan A, et al. Changes in caregiver burden and health-related quality of life of informal caregivers of older people with dementia: evidence from the European RightTimePlaceCare prospective cohort study. J Adv Nurs. 2015;71:1378–91. Received: 22 July 2016 Accepted: 14 January 2017 Received: 22 July 2016 Accepted: 14 January 2017 Availability of data and materials h d ll b h d h The data will not be shared as they are proprietary information. The data will not be shared as they are proprietary information. Page 9 of 9 Page 9 of 9 Page 9 of 9 Reed et al. Health and Quality of Life Outcomes (2017) 15:16 Consent for publication Not applicable. 19. Wimo A, Wetterholm A-L, Mastey V, Winblad B. Evaluation of the healthcare resource utilization and caregiver time in anti-dementia drug trials. In: Wimo A, Jönsson B, Karlsson G, Winblad B, editors. Health Economics of Dementia. London: Wiley; 1998. p. 465–99. Ethics approval and consent to participate Ethical review board approval of the GERAS study was obtained in each country according to individual country regulations. The patient (or their legal representative) and caregiver were both required to provide written informed consent prior to enrolment. 20. Wimo A, Nordberg G. Validity and reliability of assessments of time. Comparisons of direct observations and estimates of time by the use of the resource utilization in dementia (RUD)-instrument. Arch Gerontol Geriatr. 2007;44:71–81. References 25. Hazzan AA, Shannon H, Ploeg J, et al. Association between caregiver quality of life and the care provided to persons with Alzheimer’s disease: systematic review. Adv Alzheimer Dis. 2014;3:44–53. doi:10.4236/aad.2014. 31006. Available from: www.scirp.org/Journal/PaperDownload. aspx?paperID=44096. [Accessed January 21, 2016]. 1. Sörensen S, Conwell Y. Issues in dementia caregiving: effects on mental and physical health, intervention strategies, and research needs. Am J Geriatr Psychiatry. 2011;19:491–6. 2. Mohamed S, Rosenheck R, Lyketsos CG, Schneider LS. Caregiver burden in Alzheimer disease: cross-sectional and longitudinal patient correlates. Am J Geriatr Psychiatry. 2010;18:917–27. 26. Jones C, Edwards RT, Hounsome B. Health economics research into supporting carers of people with dementia: a systematic review of outcome measures. Health Qual Life Outcomes. 2012;10:142. 3. Serrano-Aguilar PG, Lopez-Bastida J, Yanes-Lopez V. Impact on health- related quality of life and perceived burden of informal caregivers of individuals with Alzheimer’s disease. Neuroepidemiology. 2006;27:136–42. 3. Serrano-Aguilar PG, Lopez-Bastida J, Yanes-Lopez V. Impact on health- related quality of life and perceived burden of informal caregivers of individuals with Alzheimer’s disease. Neuroepidemiology. 2006;27:136–42. 27. Hoefman RJ, van Exel J, Brouwer WB. Measuring the impact of caregiving on informal carers: a construct validation study of the CarerQol instrument. Health Qual Life Outcomes. 2013;11:173. 4. Hounsome N, Orrell M, Edwards RT. EQ-5D as a quality of life measure in people with dementia and their carers: evidence and key issues. Value Health. 2011;14:390–9. 4. Hounsome N, Orrell M, Edwards RT. EQ-5D as a quality of life measure in people with dementia and their carers: evidence and key issues. Value Health. 2011;14:390–9. 28. Lutomski JE, van Exel NJ, Kempen GI, et al. Validation of the Care-Related Quality of Life Instrument in different study settings: findings from The Older Persons and Informal Caregivers Survey Minimum DataSet (TOPICS- MDS). Qual Life Res. 2015;24:1281–93. 5. Park M, Sung M, Kim SK, Kim S, Lee DY. Multidimensional determinants of family caregiver burden in Alzheimer's disease. Int Psychogeriatr. 2015;27: 1355–64. 5. Park M, Sung M, Kim SK, Kim S, Lee DY. Multidimensional determinants of family caregiver burden in Alzheimer's disease. Int Psychogeriatr. 2015;27: 1355–64. 29. Al-Janabi H, Coast J, Flynn TN. What do people value when they provide unpaid care for an older person? A meta-ethnography with interview follow-up. Soc Sci Med. 2008;67:111–21. 6. Haro JM, Kahle-Wrobleski K, Bruno G, et al. Analysis of burden in caregivers of people with Alzheimer’s disease using self-report and supervision hours. J Nutr Health Aging. Competing interests 15. Zarit SH, Reever KE, Bach-Peterson J. Relatives of the impaired elderly: correlates of feelings of burden. Gerontologist. 1980;20:649–55. 5. Zarit SH, Reever KE, Bach-Peterson J. Relatives of the impaired eld C Reed and A Barrett are employees of Eli Lilly and Company. J Lebrec is a full-time contractor for Eli Lilly and Company. R Dodel, R W Jones, B Vellas, A Wimo, J M Argimon, G Bruno and JM Haro have received financial C Reed and A Barrett are employees of Eli Lilly and Company. J Lebrec is a full-time contractor for Eli Lilly and Company. R Dodel, R W Jones, B Vellas, A Wimo, J M Argimon, G Bruno and JM Haro have received financial compensation from Eli Lilly for participation on the GERAS Advisory Board. This study was supported by Eli Lilly and Company. 16. Bachner YG, O’Rourke N. Reliability generalization of responses by care providers to the Zarit Burden Interview. Aging Ment Health. 2007;11:678–85 6. Bachner YG, O’Rourke N. Reliability generalization of responses by providers to the Zarit Burden Interview. Aging Ment Health. 2007 17. Group EQ. Euro-Qol – a new facility for the measurement of health-related quality of life. Health Policy. 1990;16:199–208. compensation from Eli Lilly for participation on the GERAS Advisory Board. This study was supported by Eli Lilly and Company. 18. Szende A, Janssen B, Cabasés J, eds. Self-Reported Population Health: An International Perspective based on EQ-5D. SpringerLink: Springer Netherlands; 2014. References Alz Dis Assoc Disord. 2001;15:129–36.
https://openalex.org/W2223939602	https://europepmc.org/articles/pmc4701502?pdf=render	English	null	Aflatoxin Contamination Detected in Nutrient and Anti-Oxidant Rich Edible Stink Bug Stored in Recycled Grain Containers	PloS one	2,016	cc-by	8,640	Data Availability Statement: All relevant data are within the paper. Data Availability Statement: All relevant data are within the paper. Funding: German Academic Exchange Service (DAAD) under Postdoctoral Fellowships in SUb- Saharan Africa at DAAD supported Centres of Excellence Grant A/1394098. Abstract Recently, there has been multi-agency promotion of entomophagy as an environmentally- friendly source of food for the ever increasing human population especially in the develop- ing countries. However, food quality and safety concerns must first be addressed in this context. We addressed these concerns in the present study using the edible stink bug Encosternum delegorguei, which is widely consumed in southern Africa. We analysed for mycotoxins, and health beneficials including antioxidants, amino acids and essential fatty acids using liquid chromatography coupled to quadrupole time of flight mass spectrometry (LC-Qtof-MS) and coupled gas chromatography (GC)-MS. We also performed proximate analysis to determine nutritional components. We identified the human carcinogen myco- toxin (aflatoxin B1) at low levels in edible stink bugs that were stored in traditonally woven wooden dung smeared baskets and gunny bags previously used to store cereals. However, it was absent in insects stored in clean zip lock bags. On the other hand, we identified 10 fatty acids, of which 7 are considered essential fatty acids for human nutrition and health; 4 flavonoids and 12 amino acids of which two are considered the most limiting amino acids in cereal based diets. The edible stink bug also contained high crude protein and fats but was a poor source of minerals, except for phosphorus which was found in relatively high levels. Our results show that the edible stink bug is a nutrient- and antioxidant-rich source of food and health benefits for human consumption. As such, use of better handling and storage methods can help eliminate contamination of the edible stink bug with the carcinogen afla- toxin and ensure its safety as human food. Aflatoxin Contamination Detected in Nutrient and Anti-Oxidant Rich Edible Stink Bug Stored in Recycled Grain Containers Robert Musundire1,2, Isaac M. Osuga1,3, Xavier Cheseto1, Janet Irungu1, Baldwyn Torto1* 1 International Centre of Insect Physiology and Ecology (icipe), Behavioural and Chemical Ecology Department, P.O. Box 30772–00100, Nairobi, Kenya, 2 Department of Crop Science and Postharvest Technology, Chinhoyi University of Technology, Off Chirundu Road, Bag 7724, Chinhoyi, Zimbabwe, 3 Department of Agricultural Resources Management, Kenyatta University, P.O. Box 43844–00100, Nairobi, Kenya * btorto@icipe.org a1111 RESEARCH ARTICLE OPEN ACCESS Citation: Musundire R, Osuga IM, Cheseto X, Irungu J, Torto B (2016) Aflatoxin Contamination Detected in Nutrient and Anti-Oxidant Rich Edible Stink Bug Stored in Recycled Grain Containers. PLoS ONE 11(1): e0145914. doi:10.1371/journal.pone.0145914 Editor: Joseph Clifton Dickens, United States Department of Agriculture, Beltsville Agricultural Research Center, UNITED STATES Received: July 29, 2015 Accepted: December 10, 2015 Published: January 5, 2016 Copyright: © 2016 Musundire et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Editor: Joseph Clifton Dickens, United States Department of Agriculture, Beltsville Agricultural Research Center, UNITED STATES Received: July 29, 2015 Accepted: December 10, 2015 Published: January 5, 2016 Copyright: © 2016 Musundire et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Editor: Joseph Clifton Dickens, United States Department of Agriculture, Beltsville Agricultural Research Center, UNITED STATES Received: July 29, 2015 Accepted: December 10, 2015 Published: January 5, 2016 Copyright: © 2016 Musundire et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Editor: Joseph Clifton Dickens, United States Department of Agriculture, Beltsville Agricultural Research Center, UNITED STATES Introduction To meet the growing needs of the world population, currently at 2.3% per year in Sub-Saharan Africa [1], there is need to re-evaluate the source of food and how it is produced. The Food and Competing Interests: The authors have declared that no competing interests exist. PLOS ONE \| DOI:10.1371/journal.pone.0145914 January 5, 2016 1 / 16 Traditional Storage Introduces Aflatoxin in Edible Stink Bug Agricultural Organization of the United Nations (FAO) proposed a global initiative to increase use of insects as food and feed in order to augment the current efforts to ensure future food security [2]. This initiative was based on the fact that insects are consumed as food (Entomoph- agy) by various ethnic groups in several countries in Central and South America, Oceania, Asia and Africa [2]. They have been shown to have vital nutritional components such as proteins, amino acids, fats, carbohydrates, vitamins and trace elements [2,3]. Additionally, they are also reported to contain important phytosterols for human health such as β-sitosterol, campesterol and stigmasterol [4]. Agricultural Organization of the United Nations (FAO) proposed a global initiative to increase use of insects as food and feed in order to augment the current efforts to ensure future food security [2]. This initiative was based on the fact that insects are consumed as food (Entomoph- agy) by various ethnic groups in several countries in Central and South America, Oceania, Asia and Africa [2]. They have been shown to have vital nutritional components such as proteins, amino acids, fats, carbohydrates, vitamins and trace elements [2,3]. Additionally, they are also reported to contain important phytosterols for human health such as β-sitosterol, campesterol and stigmasterol [4]. The edible stink bug, Encosternum delegorguei Spinola (Hemiptera: Tessaratomidae) is widely distributed in subtropical woodland and bushveld in Zimbabwe and Northern prov- inces of South Africa [5, 6]. This bug plays an important role towards attainment of food and nutritional security as well as a source of income to rural communities [7, 8,9]. It is a phytopha- gus insect and feeds on sub-tropical savannah plants such as Uapaca kirkiana Müll. Arg. (Phyl- lanthaceae) and Oxyanthus speciosus DC (Rubiaceae) [6], Dodonaea viscosa Jacq. var. angustifolia (L.f.) Benth. (Sapindaceae), Diospyros mespiliformis Hochst. ex A.DC (Ebenaceae) [2,10], Vangueria apiculata (Verdc.) Lantz (Rubiaceae) among others. Its egg laying sites include grass stems and twigs of Combretum imberbe Wawra (Combretaceae) and Combretum apiculatum Sond. (Combretaceae) [11]. Introduction Host plant associations have potential implications on the chemical composition of the insects and consequently on their nutritional status. In Zimbabwe, harvesting of E. delegorguei is previously described [11] however, with modi- fications. Briefly, insects are collected from tree branches in the early hours of the day using the knockdown approach with the aid of long hooks or by climbing trees and vigorously shaking off insects perched on branches and leaves. The collected insects are then processed for con- sumption using a warm water killing and heating procedure before being stored in traditionally woven wooden baskets or in used grain bags (Fig 1) for later consumption or sale. These han- dling and storage practices are likely to have an impact on the nutritional status and food safety quality attributes of the bugs. The relationships between traditional harvesting; processing practices and nutrition; incidences of mycotoxins contamination in the edible stink bugs have not been studied. We hypothesised that traditional harvesting and storage practices of E. delegorguei favour the occurrence of mycotoxins. Our study therefore sought to (i) investigate the effect of tradi- tional handling and storage of the edible stink bug on mycotoxin contaminations with empha- sis on aflatoxins, and (ii) profile the nutritional and phytochemical composition of the edible stink bugs in relation to processing methods used in South-eastern districts of Zimbabwe. PLOS ONE \| DOI:10.1371/journal.pone.0145914 January 5, 2016 E. delegorguei Samples of E. delegorguei were collected from Jiri Forest (approximately 20°2' 55" S 31° 43' 36" E) in Nerumedzo area of Bikita district, South-eastern Zimbabwe during the peak harvesting period (June, 2014) as this time of the year (May-August) consides with highest abdominal fat composition in E. delegorguei in South Africa Dzerefos et al. [11]. The forest is managed by the traditional customs under a village head who oversees forest conservation and harvesting activ- ities of the edible stink bug. Harvesting of the bugs for this study was therefore done with the permission of the village head. The edible stink bug is utilized by the local community from the forest and therefore, this study did not involve endangered or protected species or endanger other wildlife in the forest. Four harvesting quadrants (100 m2) were demarcated equidistant from each other and ensuring coverage of the entire forest (approximately 8 km2). Harvesting in each quadrant was 2 / 16 PLOS ONE \| DOI:10.1371/journal.pone.0145914 January 5, 2016 Traditional Storage Introduces Aflatoxin in Edible Stink Bug Fig 1. Method of storage of processed insects a) traditionally wooven wooden baskets, b) used grain bags and c) clean zip lock bags. doi:10 1371/journal pone 0145914 g001 Traditional Storage Introduces Aflatoxin in Edible Stink Bug Fig 1. Method of storage of processed insects a) traditionally wooven wooden baskets, b) used grain bags and c) clean zip lock bags. doi:10.1371/journal.pone.0145914.g001 Fig 1. Method of storage of processed insects a) traditionally wooven wooden baskets, b) used grain bags and c) clean zip lock bags. doi:10.1371/journal.pone.0145914.g001 carried out according to two procedures from branches of 10 randomly selected trees. In one approach, 5 kg of the insects were collected from each quadrant into wooden baskets that were sourced locally and then transfered into perforated re-used polypropylene grain bags (tradi- tional practice) [11,12]. The other procedure involved harvesting insects directly into 3 / 16 PLOS ONE \| DOI:10.1371/journal.pone.0145914 January 5, 2016 Traditional Storage Introduces Aflatoxin in Edible Stink Bug perforated clean zip lock bags. In both instances, perforation of bags allowed air circulation thus keeping the insects alive as per traditional practices until processing. The harvested insects were then pooled to give one composite sample (20 kg) for each respective sampling procedure. Analysis of mycotoxins Mycotoxin analysis was carried out on traditionally processed and unprocessed stink bugs as well as samples collected in clean zip lock bags according to a modified method in reference [13]. The stink bugs (10 g) from each fraction were snap frozen in liquid nitrogen, crushed into fine powder and extracted in 40 mL acetonitrile-water (86: 16, v/v) for 30 min while sonicating. Each mixture was allowed to settle for 30 min and then 6 mL of each sample was filtered through a solid phase extraction (SPE) cartridge Multisep1228AflaPat multifunctional col- umns (Romer Labs, MO, USA). An aliquot (4 mL) of each cleaned extract was evaporated to dryness in a stream of nitrogen gas. The dried samples were re-dissolved in 400 μL methanol- water (20: 80, v/v), vortexed for 1 min and then centrifuged at 14, 000 rpm for 5 min prior to analysis using liquid chromatography coupled to quadruple time of flight mass spectrometry (LC-QtoF-MS). Samples derived from different harvesting, storage and processing procedures were analysed in five replicates. Chemicals Aflatoxins B1, B2, G1 and G2 were purchased from Supelco (Bellefonte, Pennsylvania, USA), apigenin ( 99%), luteolin ( 97%), rutin hydrate ( 94%) quercetin dehydrate ( 98%), octa- decanoic acid ( 98.5%), glutamic acid, myristoleic acid, tetradecanoic acid, hexadecanoic acid, (Z)-9-hexadecenoic acid, linoleic acid, (Z)-9-octadecenoic acid, octadecanoic acid, (Z, Z)- 9,12-octadecadienoic acid, oleic acid, eicosanoic acid and amino acid standard solution (AAS 18) were purchased from Sigma-Aldrich (Chemie GmbH, Munich, Germany). E. delegorguei This was based on the observation that traditional harvesting practices allow unrestricted harvesting of the edible stink bugs from as many parts of the forest in any single harvesting expedition. From the composite harvested insect sample, four samples (5 kg each) were prepared by transferring the bugs into clean zip lock bags. One fraction was frozen (Treatment A- clean method- unprocessed) (Fig 1c) and the other fraction, was killed by lukewarm water and heat dried (Treatment B: clean method- processed). Both treatments A and B were stored in the freezer (-80°C) until analysis for detection of aflatoxins. Traditional preparation of insects. A sub-sample of the harvested insects (5 kg) was transferred into re-used grain bags and divided into two fractions (2.5 kg each). One of these fractions was kept in a traditional woven basket (Treatment C: traditional-unprocessed). The other fraction was killed using 5 L of lukewarm water (~37°C), then sieved and cooked by heat- ing on a traditionally prepared clay pot. A colour change from green to golden brown which took approximately 3 min to develop indicated that the insects were well cooked. Dried insects were kept in traditional woven baskets (Treatment D: traditional-processed) at ambient tem- peratures until use for experiments (Fig 1a and 1b) Treatments A and B were used in the afla- toxin analyses only which showed negative results. Treatments C and D were used for aflatoxin, nutritional and secondary metabolite analyses. Analyses of flavonoids Three different samples; processed, unprocessed stink bugs and the leaves of Vangueria apicu- lata, the host plant from which stink bugs were harvested, were analysed for flavonoids. The samples were separately crushed into fine powder in liquid nitrogen. For each sample, 2.5 g were separately extracted in 50 mL methanol-water (80:20 v/v) by ultrasonication in a sonica- tion bath (Branson 2510, Danbury, CT, USA) for 1 hr followed by filtration through a What- man filter paper No. 32. The remaining residue was re-extracted twice, and the filtrate pooled separately. The extracting solvent was removed under reduced pressure at 40°C using a rotary evaporator (Laborata 4000; Heidolph Instruments GmbH & Co. KG, Germany) to give 60, 80 and 160 mg for unprocessed, processed and host plant leaves respectively. The extracts (5 mg) from each of the samples were re-dissolved in 1 mL water-methanol (95: 5 v/v), centrifuged at 14,000 rpm for 5 min and the supernatant analysed using LC-Qtof-MS. Five replicates were carried out with each replicate done on a different harvested and processed sample. Analysis of fatty acids Stink bug samples (traditionally processed and unprocessed) were snap frozen in liquid nitro- gen, and then crushed into a fine powder. A methyl esterification reaction was then performed on 5 g of each sample according to a protocol adapted from [14]. A solution of sodium 4 / 16 PLOS ONE \| DOI:10.1371/journal.pone.0145914 January 5, 2016 Traditional Storage Introduces Aflatoxin in Edible Stink Bug methoxide in methanol was prepared to give a concentration of 15 mg/mL. An aliquot of the soultion (500 μL) was added to each ground insect sample, vortexed for 1 min and then soni- cated for 5 min. The reaction mixture was incubated at 60°C for 1 hr, thereafter quenched by adding 100 μL deionized water followed by vortexing for another 1 min. The resulting methyl esters were extracted using GC-grade hexane (Sigma–Aldrich, St. Louis, USA) and then centri- fuged at 14, 000 rpm for 5 min. The supernatant was dried over anhydrous Na2SO4 and then analysed using gas chromatography coupled to mass spectrometry (GC/MS). Fatty acids were identified as their methyl esters by comparison of gas chromatographic retention times and fragmentation patterns with those of authentic standards and reference spectra publishedby library–MS databases: National Institute of Standards and Technology (NIST) 05, 08, and 11. Serial dilutions of the authentic standard octadecanoic acid (0.2–125 ng/μl) were analyzed by GC/MS in full scan mode to generate a linear calibration curve (peak area vs. concentration) with the following equation [y = 7E+06x −4E+07(R2 = 0.9757)], which was used for the exter- nal quantification of the different fatty acids. Instrument conditions A continuous lock spray reference compound (leucine enkephalin; [M+H] + = 556.2766) was sampled at 10 s intervals for centroid data mass correction. The mass spectrometer was cal- ibrated across the 50–1,200 Da mass range using a 0.5 mM sodium formate solution prepared in 90:10 2-propanol/water (v/v). MassLynx version 4.1 SCN 712 (Waters Corporation, Maple Street, MA) was used for data acquisition and processing. The elemental composition was generated for every analyte. Poten- tial assignments were calculated using mono-isotopic masses with a tolerance of 10 ppm devia- tion and both odd- and even-electron states possible. The number and types of expected atoms was set as follows: carbon 100; hydrogen 100; oxygen 50; nitrogen 6; sulfur 6 [15]. The empirical formula generated was used to predict structures which were proposed based on the online database, fragmentation pattern, literature and confirmed using authentic standards. Serial dilutions of authentic standards of aflatoxin B1 (0.01–20 ng/μl); rutin, quercetin, luteolin, apigenin (1.8–181 ng/μl); and glutamic acid (0.01–10 ng/μl) were also analyzed by LC-Qtof-MS in MSE mode to generate linear calibration curves (peak area vs. concentration) with the following linear equations: aflatoxin B1 [y = 13738x + 6611.5 (R2 = 0.9571)], rutin [y = 5578.4x −39094 (R2 = 0.9960)], quercetin [y = 4372.4x + 79607 (R2 = 0.9854)], luteolin [y = 13433x −23256 (R2 = 0.9994)] apigenin [y = 10288x −11117 (R2 = 0.9995)] and glutamic acid [y = 40137x −1353.1 (R2 = 0.9999)] which served as the basis for the external quantifica- tion of the aflatoxin, flavonoids and amino acid. GC/MS. Fatty acid methyl esters (FAMEs) were analyzed by GC/MS on a 7890A gas chro- matograph (Agilent Technologies, Inc., Santa Clara, CA, USA) linked to a 5975 C mass selec- tive detector (Agilent Technologies, Inc., Santa Clara, CA, USA) by using the following conditions: inlet temperature 270°C, transfer line temperature of 280°C, and column oven tem- perature programmed from 35 to 285°C with the initial temperature maintained for 5 min then 10°Cmin−1 to 280°C, held at this temperature for 20.4 min. The GC was fitted with a HP- 5 MS low bleed capillary column (30 m × 0.25 mm i.d., 0.25 μm) (J&W, Folsom, CA, USA). Helium at a flow rate of 1.25 ml min−1 served as the carrier gas. The mass selective detector was maintained at ion source temperature of 230°C and a quadrapole temperature of 180°C. Instrument conditions LC-Qtof-Ms. The chromatographic separation was achieved on a Waters ACQUITY UPLC (ultra-performance liquid chromatography) I-class system (Waters Corporation, Mil- ford, MA, USA). For amino acid analysis, the UPLC was fitted with an ACE C18 column (250 mm x 4.6 mm, 5μm (Aberdeen, Scotland) with a heater turned off and an autosampler tray cooled to 5°C. Mobile phases of water (A) and acetonitrile (B) each containing 0.01% formic acid was employed. The following gradient was used: 0 min, 5% B; 0–3 min, 5–30% B; 3–6 min, 30% B; 6–7.5 min, 30–80% B; 7.5–10.5 min, 80% B; 10.5–13.0, 80–100% B, 13–18 min, 100% B; 18–20 min, 100–5% B; 20–22 min, 5% B. The flow rate was held constant at 0.7 ml min−1. The injection volume was 1 μL. For analyses of flavonoids and aflatoxin, a UPLC was fitted to a Waters ACQUITY UPLC BEH C18 column (2.1mm × 50 mm, 1.7 μm particle size; Waters Corporation, Dublin, Ireland) heated to 40°C and an auto sampler tray cooled to 15°C. Mobile phases of water (A) and meth- anol (B), each with 0.01% formic acid were employed. The following gradient was used for i) flavonoids: 0–0.2 min, 10% B; 0.2–3 min, 10–60% B; 3–5 min, 60–80% B; 6–8 min, 80% B; 8–9 min, 100% B; 9–10 min, 100% B; 10–10.5 min 100–10% B; 10.5–12 min 10% B, ii) aflatoxin: 0–0.2 min, 10% B; 0.2–3 min, 10–90% B; 3–5 min, 90% B; 5–6 min, 90–10% B; 6–7 min, 10% B. The flow rate was held constant at 0.4 ml min−1 for both analyses. 5 / 16 PLOS ONE \| DOI:10.1371/journal.pone.0145914 January 5, 2016 Traditional Storage Introduces Aflatoxin in Edible Stink Bug The UPLC system was interfaced with electrospray ionization (ESI) to a Waters Xevo QToF-MS operated in full scan MSE in positive mode. Data were acquired in resolution mode over the m/z range 100–1200 for flavonoids and aflatoxin: m/z 100–700 for amino acid analysis with a scan time of 1 s using a capillary voltage of 0.5 kV, sampling cone voltage of 40 V, source temperature 100°C and desolvation temperature of 350°C. The nitrogen desolvation flow rate was 500 L/h. For the high-energy scan function, a collision energy ramp of 25–45 eV was applied in the T-wave collision cell using ultrahigh purity argon (99.999%) as the collision gas. Instrument conditions Electron impact (EI) mass spectra were obtained at the acceleration energy of 70 eV. A 1.0 μl aliquot of sample was injected in the splitless mode using an auto sampler 7683 (Agilent Tech- nologies, Inc., Beijing, China). Fragment ions were analyzed over 40–550 m/z mass range in the full scan mode. The filament delay time was set at 3.3 min. Nutritional analysis Proximate analysis: Crushed stink bugs were analysed based on procedures employed by the Association of Analytical Chemists [16]. The ash content was determined by heating at 550°C overnight. The organic matter (OM) was then determined by subtracting ash content from 100. Velp solvent extractor (SER 148/6) was used to determine fat content with ethyl ether as extractant. Crude protein (CP) was determined using the Kjeldahl method by determining the nitrogen content (%) and multiplying by the factor 6.25. The neutral detergent fibre (NDF) 6 / 16 PLOS ONE \| DOI:10.1371/journal.pone.0145914 January 5, 2016 Traditional Storage Introduces Aflatoxin in Edible Stink Bug and acid detergent fibre (ADF) were analysed with the Velp fibre analyzer (FIWE 6) (VELP Scientifica, Usmate Velate, Italy) using reagents described [17]. Amino acids: The method for protein extraction was adopted from [18]. Processed and unprocessed insect samples were separately snap-frozen in liquid nitrogen and crushed into fine powder. The samples (2 g each) were extracted for 1hr in ice cold 5 v/w 100 mM 4-(2-hydro- xyethyl)-1-piperazineethanesulfonic acid (HEPES) pH 7.2, 2 mM dithiothreitol (DTT), 2.5% Polyvinylpyrrolidone (PVP), 0.5 mM Ethylenediaminetetraacetic acid (EDTA), 1 mM benzami- dine 0.1 mM phenylmethanesulfonylfluoride (PMSF) in a magnetic stirrer. The samples were filtered through KERLIX™Gauze Bandage Rolls Sterile Soft Pouch 5.7 cm × 2.7 m centrifuged at 8000 rpm for 30 min at 4°C to remove solid debris. Protein was precipitated between 45% and 80% (NH4)2SO4 and the pellet recovered by centrifugation at 21,000 rpm for 30 min at 4°C. The protein pellets were desalted in 20 mM HEPES–NaOH pH 8 containing 2 mM DTT using Sephadex G-25 gel filtration chromatography (PD-10 columns, GE Healthcare) to give 80.2 mg and 77.9 mg of proteins from processed and unprocessed insect samples respectively. 10 mg from each of the samples were separately transferred into a 5 ml micro- reaction vial into which 2ml of 6N HCl were added and closed after careful introduction of nitrogen gas. The samples were hydrolyzed for 24 h at 110°C. For tryptophan analysis, 10 mg from each of the samples were separately transferred into a 5 ml micro-reaction vial into which 2 ml of 6N NaOH were added and then capped after careful introduction of nitrogen gas. The samples were hydrolyzed for 24 h at 110°C. After the hydrolysis, the mixtures were evaporated to dry- ness under vacuum. Nutritional analysis The hydrolysates were reconstituted in 1 ml 90:10 water: acetonitrile, vor- texed for 30 s, sonicated for 30 min, and then centrifuged at 14,000 rpm and the supernatant analysed using LC-Qtof-MS. The analysis was replicated three times. Mineral analysis: The crushed insects were ashed and digested in 6N HCl and the content of the various minerals were analysed on an Atomic Absorption Spectrophotometer (Model— AA6300, Shimadzu, Japan). Statistical analysis Statistical analysis for ofaflatoxin, flavonoid and fatty acids contents and nutritional data were done using SAS statistical software [19]. The results were expressed as mean ± standard error. The means were separated using paired t-test for pair-wise comparisons and Tukey’s multiple comparison tests at P < 0.05. Analysis of aflatoxins LC-Qtof-MS analysis for mycotoxins identified only aflatoxin B1 in traditionally harvested unprocessed (0.50±0.17 ng/g) and processed (0.59±0.18 ng/g) insects (Fig 2). There were no significant (P>0.05) differences in the levels of aflatoxin B1 detected in the two different treat- ments. No aflatoxins were detected in insects that were harvested into and stored in clean zip lock bags. Fatty acid analysis A total of 10 fatty acid methyl esters (FAMEs) were identified from the unprocessed and the traditionally processed stink bug samples. Of these, seven were unsaturated fatty acid deriva- tives (methyl oleate, methyl (Z)-9-hexadecenoate, methyl linoleate, methyl (Z)-9-octadeceno- ate, methyl octadecanoate, methyl (Z,Z)-9,12-octadecadienoate and methyl myristoleate), while the remaining three were saturated fatty acid derivatives (Fig 3; Table 1). 7 / 16 PLOS ONE \| DOI:10.1371/journal.pone.0145914 January 5, 2016 Traditional Storage Introduces Aflatoxin in Edible Stink Bug Table 1. Concentration of fatty acid methyl esters (mg/g) in unprocessed and processed E. delegorguei. Peak no RT (min) Fatty acid methyl esters Unprocessed Processed 1 21.17 Methyl myristoleate 0.40±0.05(a) 0.37±0.12(a) 2 21.29 Methyl tetradecanoate 0.6±0.17(a) 0.70±0.11(b) 3 23.15 Methyl (Z)-9-hexadecenoate 10.87±3.32(b) 10.17±3.42(ab) 4 23.35 Methyl hexadecanoate 12.53±3.75(b) 10.46±3.78(b) 5 24.96 Methyl linoleate 4.92±0.08(ab) 5.04±0.18(ab) 6 25.03 Methyl (Z)-9-octadecenoate 6.98±1.51(ab) 7.40±1.95(ab) 7 25.25 Methyl octadecanoate 5.22±0.64(ab) 5.05±0.17(ab) 8 25.47 Methyl (Z,Z)- 9,12-octadecadienoate 6.24±1.95(ab) 8.23±2.54(ab) 9 25.63 Methyl oleoate 0.36±0.07(a) 0.33±0.01(a) 10 27.00 Methyl eicosanoate 0.8±0.05(a) 0.37±0.02(a) Retention time (RT). Concentrations of FAMEs bearing the same letter in either processed or unprocessed (along the column) are not signiﬁcantly different (P0.05, Tukey’s HSD test). ble 1. Concentration of fatty acid methyl esters (mg/g) in unprocessed and processed E. delegorguei. Table 1. Concentration of fatty acid methyl esters (mg/g) in unprocessed and process Retention time (RT). Concentrations of FAMEs bearing the same letter in either processed or unprocessed (along the column) are not signiﬁcantly different (P0.05, Tukey’s HSD test). doi:10 1371/journal pone 0145914 t001 The unsaturated fatty acids include some of the most essential fatty acids (linolenic acid and linoleic acid) for human nutrition. Except for eicosanoic acid, processing of the stink bugs did not significantly (P>0.05) affect the concentration of the fatty acids. Analysis of flavonoids LC-Qtof-MS analysis showed the presence of four flavonoids in the three different treatments (unprocessed, processed and host plant V. apiculata leaves) (Fig 4; Table 2). Generally, apeginin was the most abundant flavonoid while rutin was the least in all the samples analysed. All the four flavonoids were over 40-fold more abundant in the leaves than in the insect samples. Processing did not significantly (P>0.05) affect the flavonoid content. Traditional Storage Introduces Aflatoxin in Edible Stink Bug Traditional Storage Introduces Aflatoxin in Edible Stink Bug Fig 2. Representative Extracted ion chromatograms for m/z 313.0737. The retention time of aflatoxin B1 is 2.20 min. doi:10.1371/journal.pone.0145914.g002 Fig 2. Representative Extracted ion chromatograms for m/z 313.0737. The retention time of aflatoxin B1 is 2.20 min. doi:10.1371/journal.pone.0145914.g002 Fig 3. Representative total ion chromatogram showing fatty acid methyl esters (FAMEs) detected in processed and unprocessed E. delegorguei. Peaks 1–10 indicate FAMEs shown in Table 1. ogram showing fatty acid methyl esters (FAMEs) detected in processed and unprocessed E. delegorguei. ble 1 Fig 3. Representative total ion chromatogram showing fatty acid methyl esters (FAMEs) detected in processed and unprocessed E. delegorguei. Peaks 1–10 indicate FAMEs shown in Table 1. Fig 3. Representative total ion chromatogram showing fatty acid methyl esters (FAMEs) detected in proc Peaks 1–10 indicate FAMEs shown in Table 1. doi:10.1371/journal.pone.0145914.g003 doi:10.1371/journal.pone.0145914.g003 doi:10.1371/journal.pone.0145914.g003 PLOS ONE \| DOI:10.1371/journal.pone.0145914 January 5, 2016 PLOS ONE \| DOI:10.1371/journal.pone.0145914 January 5, 2016 8 / 16 PLOS ONE \| DOI:10.1371/journal.pone.0145914 January 5, 2016 Nutritional Analyses The proximate composition of stink bugs are presented in Table 3. The processed stink bugs had significantly (P<0.05) higher levels of CP and ADF contents than the unprocessed ones. However, the unprocessed stink bugs had significantly (P<0.05) higher levels of fat and NDF than those that were processed. The mineral content of the insects are presented in Table 4. Most of the minerals analysed were detected in E. delegorguei except manganese and sele- nium. The mineral content was generally low except phosphorus. There were minimal differ- ences in the levels of various minerals between processed and unprocessed stink bugs except for zinc and phosphorus. The amino acid profile of E. delegorguei is presented in Fig 5; Table 5. Nine essential amino acids were detected including the most limiting (lysine, tryptophan and methionine) in cereal/legume based diets. Three non-essential amino acids were also detected. The concentrations of the amino acids ranged from 0.86 mg/g (lysine)– 7.44 mg/g (leucine) for processed and 0.88 mg/g (lysine)– 7.11 mg/g (leucine) for unprocessed insects. However, processing did not significantly (P>0.05) affect the amino acid content. Tryptophan was identified in both the processed and unprocessed stink bug based on accu- rate mass determination and literature data as follows: The LC-Qtof-MS peak at retention time 2.33 min had a molecular ion peak [M+H]+ at m/z 205.0974 and fragment ions m/z 91.0557, PLOS ONE \| DOI:10.1371/journal.pone.0145914 January 5, 2016 9 / 16 Traditional Storage Introduces Aflatoxin in Edible Stink Bug Fig 4. Representative total ion chromatogram showing flavonoids detected in samples of V. apiculata leaves and of E. delegorguei. Peaks 1–4 indicate the flavonoids shown in Table 2. Traditional Storage Introduces Aflatoxin in Edible Stink Bug Fig 4. Representative total ion chromatogram showing flavonoids detected in samples of V. apiculata leaves and of E. delegorguei. Peaks 1–4 indicate the flavonoids shown in Table 2. Fig 4. Representative total ion chromatogram showing flavonoids detected in samples of V. apiculata le indicate the flavonoids shown in Table 2. doi:10.1371/journal.pone.0145914.g004 Table 2. Concentrations (ng/g) of flavonoids in unprocessed and processed E. delegorguei and V. apiculata leaves. RT (min) Flavanoid Unprocessed Processed V. apiculata leaves 1.95 Rutin 2.89±0.54 2.89±0.43 75.76±21.53 2.39 Quercitin 4.33±1.57 4.18±1.15 231.02±61.55 2.46 Luteolin 3.25±1.05 3.29±0.81 146.48±41.36 2.66 Apigenin 37.13±12.59 34.52±10.02 1780.94±540.45 doi:10.1371/journal.pone.0145914.t002 Table 3. Proximate composition (%DM) of unprocessed and processed E. delegorguei. Nutritional Analyses Parameter Unprocessed Processed Signiﬁcance (p<0.05) Organic matter 98.4±0.08 98.2±0.07 NS Crude protein 33.2±0.22 37.7±0.05 * Fat 62.4±0.34 57.7±0.38 ** Neutral detergent ﬁbre 37.3±0.78 32.6±1.13 * Acid detergent ﬁbre 17.5±0.18 19.0±0.20 * indicates signiﬁcance at P<0.05 indicates signiﬁcance at P<0.01 NSindicates no signiﬁcance at P>0.05 doi:10.1371/journal.pone.0145914.t003 PLOS ONE \| DOI:10.1371/journal.pone.0145914 January 5, 2016 10 / 16 Table 2. Concentrations (ng/g) of flavonoids in unprocessed and processed E. delegorguei and V. apiculata leaves. RT (min) Flavanoid Unprocessed Processed V. apiculata leaves 1.95 Rutin 2.89±0.54 2.89±0.43 75.76±21.53 2.39 Quercitin 4.33±1.57 4.18±1.15 231.02±61.55 2.46 Luteolin 3.25±1.05 3.29±0.81 146.48±41.36 2.66 Apigenin 37.13±12.59 34.52±10.02 1780.94±540.45 doi:10.1371/journal.pone.0145914.t002 Table 2. Concentrations (ng/g) of flavonoids in unprocessed and processed E. delegorgu . Concentrations (ng/g) of flavonoids in unprocessed and processed E. delegorguei and V. apiculata leaves Table 3. Proximate composition (%DM) of unprocessed and processed E. delegorguei. Parameter Unprocessed Processed Signiﬁcance (p<0.05) Organic matter 98.4±0.08 98.2±0.07 NS Crude protein 33.2±0.22 37.7±0.05 Fat 62.4±0.34 57.7±0.38 ** Neutral detergent ﬁbre 37.3±0.78 32.6±1.13 * Acid detergent ﬁbre 17.5±0.18 19.0±0.20 * indicates signiﬁcance at P<0.05 indicates signiﬁcance at P<0.01 NSindicates no signiﬁcance at P>0.05 doi:10.1371/journal.pone.0145914.t003 PLOS ONE \| DOI:10.1371/journal.pone.0145914 January 5, 2016 10 / 16 Table 3. Proximate composition (%DM) of unprocessed and processed E. delegorguei. 10 / 16 Traditional Storage Introduces Aflatoxin in Edible Stink Bug Table 5. Concentrations (mg/g) of amino acids in unprocessed and processed E. delegorguei. Amino acid Unprocessed Processed Tryptophan 3.39±0.04 3.32±0.13 Arginine 3.03±0.06 3.16±0.08 Isoleucine 3.18±0.27 3.39±0.56 Leucine 7.11±0.18 7.44±0.71 Proline 2.44±0.16 2.42±0.14 Valine 0.97±0.17 0.98±0.15 Methionine 1.08±0.08 1.30±0.24 Hydroxyproline 2.57±0.08 3.06±0.07 Tyrosine 6.37±0.10 6.97±0.41 Threonine 0.42±0.02 0.55±0.08 Lysine 0.88±0.02 0.86±0.05 Phenylalanine 2.10±0.06 2.37±0.09 doi:10.1371/journal.pone.0145914.t005 118.0658, 132.07427, 146.0623 and 188.0244, elemental composition of C11H13N2O2, 1.5 ppm error to theoretical value and a fit confidence of 99.9% consistent with tryptophan [20]. Table 4. Mineral content (% DM) of unprocessed and processed E. delegorguei. Element Unprocessed Processed Signiﬁcance (p<0.05) Zinc 0.02 0.01 Iron 0.07 0.08 NS Copper 0.02 0.02 NS Potassium 0.79 0.85 NS Sodium 0.25 0.27 NS Calcium 0.56 0.58 NS Magnesium 0.16 0.17 NS Phosphorus 1.39 1.46 * Manganese ND ND Selenium ND ND indicates signiﬁcance at P<0.05 NSindicates no signiﬁcance at P>0.05 NDindicates not detected doi:10.1371/journal.pone.0145914.t004 Fig 5. Representative total ion chromatogram of the amino acid profile of unprocessed and processed insects. Peaks 1–10 indicate amino acids shown in Table 5. doi:10.1371/journal.pone.0145914.g005 Traditional Storage Introduces Aflatoxin in Edible Stink Bug Traditional Storage Introduces Aflatoxin in Edible Stink Bug Table 4. Mineral content (% DM) of unprocessed and processed E. delegorguei. Element Unprocessed Processed Signiﬁcance (p<0.05) Zinc 0.02 0.01 Iron 0.07 0.08 NS Copper 0.02 0.02 NS Potassium 0.79 0.85 NS Sodium 0.25 0.27 NS Calcium 0.56 0.58 NS Magnesium 0.16 0.17 NS Phosphorus 1.39 1.46 * Manganese ND ND Selenium ND ND *indicates signiﬁcance at P<0.05 NSindicates no signiﬁcance at P>0.05 NDindicates not detected doi:10.1371/journal.pone.0145914.t004 Table 4. Mineral content (% DM) of unprocessed and processed E. delegorguei. Fig 5. Representative total ion chromatogram of the amino acid profile of unprocessed and processed insects. Peaks 1–10 indicate amino acids shown in Table 5. matogram of the amino acid profile of unprocessed and processed insects. Peaks 1–10 indicate amino acids Fig 5. Representative total ion chromatogram of the amino acid profile of unprocessed and processed insects. Peaks 1–10 indicate amino acids shown in Table 5. tive total ion chromatogram of the amino acid profile of unprocessed and processed insects. Peaks 1–10 ind Fig 5. Representative total ion chromatogram of the amino acid profile of unprocessed and processed in shown in Table 5. doi:10.1371/journal.pone.0145914.g005 doi:10.1371/journal.pone.0145914.g005 PLOS ONE \| DOI:10.1371/journal.pone.0145914 January 5, 2016 11 / 16 PLOS ONE \| DOI:10.1371/journal.pone.0145914 January 5, 2016 Discussion Therefore, using the cur- rent harvesting and traditional handling and storage practices in the South-eastern districts of Zimbabwe poses a great risk to consumers from mycotoxins contamination. However, use of alternative clean plastic bags demonstrated that the risk can be reduced/eliminated. Analyses of fatty acids revealed in general a composition of mostly unsaturated fatty acids (seven out of detected ten fatty acids) in processed and unprocessed insects. Unsaturated fatty acids play important functions to insects such as regulating fluidity of membranes and as pre- cursors of many pathways for the synthesis of substances such as waxes, pheromones, eicosa- noids and other defense secretions [24]. For humans, unsaturated fatty acids are generally con- sidered healthy because they help in reducing cholesterol levels which is often associated with heart diseases [25]. Specifically the detection of methyl palmetoleate and methyl oleate which confirmed the presence of the monounsaturated fatty acids palmetoic and oleic acids in E. dele- gorguei is of great benefit to consumers. Studies have shown that these two fatty acids or their derivatives promote cell viability and mitogenesis and they help to protect β-cells from glucose and palmatic acid-induced apoptosis [26, 27]. On the other hand, the saturated fatty acids pal- mitic acid and stearic acid identified in this study have been shown to enhance flavour and hav- ing hypocholesterolemic effects [28]. Linoleic acid was the only essential polyunsaturated fatty acid found in unprocessed and tra- ditionally processed stink bugs. The edible stink bug therefore is an important source of this vital fatty acid which has to be obtained from dietary sources. Several health and nutritional benefits of this fatty acid in diet include hypocholesterolemic [29], anticoronary [30] and anti- arthritic [31]. Linoleic acid identified in the edible stink bug is expected to lessen deficiencies to consumers in communities where vegetable and animal sources may be limited. Four flavonoids including rutin, quercitin, luteolin and apeginin, were detected in unpro- cessed and processed stink bugs in similar amounts, and these metabolites were traced back to their feed source V. apiculata leaves, which contained relatively high levels of these com- pounds. Flavonoids influence selection of host plants by the phytophagous insects such as the edible stink bug because they may serve a defence function in insects against natural enemies [32,33]. Discussion Aflatoxins are a group of toxic compounds belonging to the difuranocoumarins and produced by Aspergillus flavus and Aspergillus parasiticus [21] in/on cereals, oils seeds, spices, chillies and milk [22]. Growth and development of fungi producing these toxins is favoured by warm temperatures and high humidity [22]. The presence of aflatoxins in human and animal food is undesirable. In humans, a condition called aflatoxicosis, is a primary hepatic disease associated with aflatoxin B1 [23]. In carrying out our study, we observed and hypothesised that traditional harvesting and storage practices of E. delegorguei favour the occurrence of mycotoxins as polypropylene bags and wooden baskets are alternatively used to store cereal grain and legumes which are often associated with mycotoxin contamination [23]. This hypothesis was proven correct by absence of aflatoxins in insects collected and handled using clean and non-contaminated bags. Our study confirmed the occurrence of aflatoxin B1 (AFB1) in traditionally collected and stored processed and unprocessed insect samples. AFB1 is one of the most potent naturally occurring mutagens and carcinogens which cause significant threats to the food industry and animal production [23]. Although the levels detected in the insect sample (0.50 ng/g and 0.59 ng/g for unprocessed and processed samples respectively) were below the maximum limits rec- ommendations (20 ng/g;World Health Organization), concern still remains for the possible adverse effects resulting from long-term exposure to low levels of aflatoxins in the use of the edible stink bug as food for human consumption. Our study was able to associate aflatoxin contamination of edible stink bugs with traditional harvesting and stored practices of insects into wooden baskets and old polypropylene grain bags. However, a follow up study could involve assessing the levels of contamination of these containers before using them for harvesting and storage of edible stink bugs. The nature of the wooden baskets with interwoven strings smeared with cow dung can potentially result in sur- face moisture retention which can promote growth of moulds. On the other hand, harvested and processed insects (usually with moisture content of 33–45%) can get cross-contaminated by the toxin causing fungi or their spores from storage in the old polypropylene bags. In gen- eral, environmental humidity of 70–90%, storage temperature of 20 to 40°C and substrate PLOS ONE \| DOI:10.1371/journal.pone.0145914 January 5, 2016 12 / 16 Traditional Storage Introduces Aflatoxin in Edible Stink Bug humidity of 10–20% are known to favour aflatoxins production [24]. PLOS ONE \| DOI:10.1371/journal.pone.0145914 January 5, 2016 Traditional Storage Introduces Aflatoxin in Edible Stink Bug nutrients. However, during the processing of the bugs, some of the fats appeared to have been lost due to heating. The fat content in insects is an indication of the amount of energy reserves found in the insect body and has an inverse relationship with the length of the diapausing period [9]. The fat content of E. delegorguei was higher than the values previously reported [10]. These differ- ences could be due to the different analytical and sensitivity of the methods used in the two studies. Our method was according to Randall technique (using hot solvent) while [10] used the Soxhlet technique (using cold solvent). Fats are essential in daily human diets as they increase the palatability of foods by absorbing and retaining their flavours [39]. They are also vital in the structural and biological functioning of cells and help in the transport of nutrition- ally essential fat-soluble vitamins [40]. The fat content of E. delegorguei therefore represents a good source of energy. This result is consistent with the results of [10] for E. delegorguei and for other edible insects [41]. The organic matter of the E. delegorguei was very high (98.2% and 98.4%), which implies low ash content. The low ash content was also reported [10] for E. delegorguei, which is con- sistent with the results of this investigation. The low ash content of E. delegorguei implies low mineral content. This was confirmed by low contents of the various minerals analysed. However, E. delegorguei will supply appreciable quantities of phosphorus. The mineral con- tent obtained in this study was lower than that reported [10]. Mineral content in organisms vary depending on the location and type of feed consumed by the organism. The fibre con- tent of insects is mainly influenced by the insect exoskeleton and structure mainly chitin. The fibre content of the E. delegorguei was moderate. The consumption of fibre helps in digestion of foods by enhancing peristaltic movements of the digestive system as well as pro- viding bulk to food and therefore gives a sense of satiety even when small amount of food is eaten [41]. The edible stink bug has both essential and non-essential amino acids. While [10] reported 8 essential amino acids, our investigation revealed 9 essential amino acids, which indicates the high quality of proteins from E. delegorguei. All the three most limiting amino acids in plant based diets (lysine, methionine and tryptophan) were detected. This shows the importance of E. delegorguei as a source of high quality proteins to supplement plant based diets which is the common scenario in the developing countries. The values of the various amino acids in our study were lower than the values reported by [10]. This variation could be to due differences in the hostplants fed upon by the bug before collection, which in turn might be influenced by the season and environmental conditions at the sites in the two studies. Since there is scarcity of information on the influence of host plants and environmental conditions on the nutritional composition of edible insects, more studies are needed to address this gap. PLOS ONE \| DOI:10.1371/journal.pone.0145914 January 5, 2016 Discussion It is most likely that the insects acquired these compounds during herbivory by the actively feeding nymphs which then stored/ sequestered them throughout to the adult diapaus- ing stages. This suggestion however requires further investigation. The four flavonoids reported here are all aglycones found in many plants [33,34]. The edible stink bug would therefore, be a source of these flavonoids, which are normally found in vegetables and fruits in human diets. Studies have shown that these compounds have health-promoting properties due to their high anti-oxidant capacity [35]. Overall, flavonoids have so far been found to exhibit a wide spec- trum of pharmacological properties, including antioxidative, antiallergic, antiinflammatory, antidiabetic, hepato- and gastro-protective, antiviral, and antineoplastic activities [35,36]. Insects contain proteins where they perform various structural and physiological roles vital for their survival. Edible insects have been shown to have higher protein content, on a mass basis, than other animal and plant foods such as beef, chicken, fish, soybeans, and maize [10]. Ramos [37] reported the nutritional value of 78 species of edible insects in Mexico, with protein values ranging from 15–81%, and calorie content ranging from 293–762 kcal/100 g. The pro- tein content (37.7% and 33.2%) in the current study was similar to that reported from South Africa (35.2%) [10]. Teffo et al. [38] suggested that processing of the insects would have an effect on the nutritional quality of the insects. Our study also found significant effect on the nutritional quality of processing the edible stink bug. The crude protein was increased with processing while the fat content also significantly reduced. The increase in crude protein con- tent is because of the reduction in water content due to cooking which concentrates the 13 / 16 PLOS ONE \| DOI:10.1371/journal.pone.0145914 January 5, 2016 References 1. United Nations. World Population Prospects: The 2012 Revision, Highlights and Advance Tables. 2013. Report No.: Working paper no. ESA/P/WP.228. Available: http://esa.un.org/unpd/wpp/Excel- Data/population.htm. 1. United Nations. World Population Prospects: The 2012 Revision, Highlights and Advance Tables. 2013. Report No.: Working paper no. ESA/P/WP.228. Available: http://esa.un.org/unpd/wpp/Excel- Data/population.htm. 2. Van Huis A, Itterbeek JV, Klunder H, Mertens E, Halloran A, Muir G V P. Edible insects. Future pros- pects for food and feed security. FAO. 2013 pp. 1–34. 3. Xiaoming C, Ying F H Z. Review of the nutrition value of edible insects. In: Durst Patrick B., Johnson Dennis V. L RN and S K, editor. Forest insects as food: humans bite back. Chiang-Mai, Thailand: FAO. 2010 pp. 85–92. 4. Cheseto X, Kuate SP, Tchouassi DP, Ndung’u M, Teal PE a., Torto B. Potential of the Desert Locust Schistocerca gregaria (Orthoptera: Acrididae) as an unconventional source of dietary and therapeutic sterols. PLoS One. 2015; 10(5): e0127171. doi: 10.1371/journal.pone.0127171 PMID: 25970517 5. Dzerefos CM, Witkowski ETF, Toms R. Life-history traits of the edible stinkbug, Encosternum delegor- guei (Hem., Tessaratomidae), a traditional food in southern Africa. J Appl Entomol. 2009; 133 (9–10): 749–759. 6. Picker M, Griffiths C, Weaving A. Field guide to insects of South Africa. African Zoology. Struik; 2002 pp. 440. 7. Chavunduka DM. Insects as a source of protein to the African. Rhod Sci News. 1975; (9: ): 217–220. 8. Kwashirai VC. Indigenous management of teak woodland in Zimbabwe, 1850–1900. J Hist Geogr. 2007; 33 (4): 816–832. 9. Dzerefos CM. The life history, use and socio-economics of the edible stinkbug Encosternum delegor- guei (Hemiptera: Tessaratomidae) in South Africa. PhD. Thesis, University of the Witwatersrand Johan- nesburg. 2014. Available: http://wiredspace.wits.ac.za/bitstream/handle/10539/15143/Thesis-C% 20Dzerefos-Encosternum.pdf?sequence=2&isAllowed = y. 10. Teffo LS, Toms RB E J. Preliminary data on the nutritional composition of the composition of the edible stink-bug, Encosternum delegorguei Spinola, consumed in Limpopo privince, South Africa. S Afr J Sci. 2007; 103: 434–436. 11. Dzerefos CM, Witkowski ETF, Toms R. Comparative ethnoentomology of edible stinkbugs in southern Africa and sustainable management considerations. J Ethnobiol Ethnomed. 2013; 9(1): 20. 12. Mawere M. Forest insects, personhood and the Environment:Harurwa (edible stinkbugs) and conserva- tion in south-eastern Zimbabwe.PhD.Thesis, University of Cape Town. 2014. Available: https://open. uct.ac.za/bitstream/handle/11427/12842/thesis_hum_2014_mawere_m.pdf?sequence=1. 13. Cheng Y, Cappozzo J. Sensitive Femtogram determination of aflatoxins B1, B2, G1 and G2 in food matrices using Triple Quadrapole LC/MS. Chromatogr today. 2008; 3–4. 14. Christie WW. Conclusions Aflatoxin B1 was found in both traditionally collected and stored unprocessed and processed insect samples but was not detected in samples harvested in clean zip-lock bags. Proper storage and handling of Encosternum delegorguei which is a good food source for human consumption are therefore important to avoid introducing aflatoxin contamination. Use of alternative cheap materials such as plastic–lined gunny bags which are easy to clean would help to reduce afla- toxin contamination. The presence of flavonoids which were also detected in the host plant would confer health benefits of flavonoids to humans upon consumption. We conclude that the edible stink bug is a nutrient- and antioxidant- rich food source for humans especially in cereal based diets since it contains high amounts of proteins, fats and phosphorus. The fats also contain higher proportions of unsaturated than the saturated fatty acids. PLOS ONE \| DOI:10.1371/journal.pone.0145914 January 5, 2016 14 / 16 Traditional Storage Introduces Aflatoxin in Edible Stink Bug Acknowledgments We thank members of our laboratory for stimulating discussions. Special thanks are extended to Onesmus Wanyama for his technical support and Mr. Philemon Kufandikamwe for his sup- port during sample collection. Author Contributions Conceived and designed the experiments: RM BT. Performed the experiments: RM IMO XC JI. Analyzed the data: RM IMO XC JI BT. Contributed reagents/materials/analysis tools: BT. Wrote the paper: RM IMO XC JI BT. Conceived and designed the experiments: RM BT. Performed the experiments: RM IMO XC JI. Analyzed the data: RM IMO XC JI BT. Contributed reagents/materials/analysis tools: BT. Wrote the paper: RM IMO XC JI BT. Analyzed the data: RM IMO XC JI BT. Contributed reagents/materials/analysis tools: BT. Wrote the paper: RM IMO XC JI BT. PLOS ONE \| DOI:10.1371/journal.pone.0145914 January 5, 2016 References Preparation of Ester Derivatives of Fatty Acids for Chromatographic Analysis. Adv Lipid Methodol. 1993; 69–111. 15. Jared JJ, Murungi LK, Wesonga J, Torto B. Steroidal glycoalkaloids: chemical defence of edible African nightshades against the tomato red spider mite, Tetranychus evansi (Acari: Tetranychidae). Pest Manag Sci. 2015 Available: 10.1002/ps.4100. 16. AOAC. Official methods of Analysis of the AOAC. 15th ed. Helrich KC, editor. USA: Association of Official Analytical Chemists Inc; 1990. pp. 750–800. 17. Van Soest PJ, Robertson JB, Lewis BA. Methods for Dietary Fiber, Neutral Detergent Fiber, and Non- starch Polysaccharides in Relation to Animal Nutrition. J Dairy Sci. 2015; 74 (10): 3583–3597. 18. Hamilton ML, Kuate SP, Brazier-Hicks M, Caulfield JC, Rose R, Edwards R, et al. Elucidation of the bio- synthesis of the di-C-glycosylflavone isoschaftoside, an allelopathic component from Desmodium spp. PLOS ONE \| DOI:10.1371/journal.pone.0145914 January 5, 2016 15 / 16 Traditional Storage Introduces Aflatoxin in Edible Stink Bug that inhibits Striga spp. development. Phytochemistry. 2012; 84: 169–176. doi: 10.1016/j.phytochem 2012.08.005 PMID: 22959223 that inhibits Striga spp. development. Phytochemistry. 2012; 84: 169–176. doi: 10.1016/j.phytochem. 2012.08.005 PMID: 22959223 19. Inc. S institute. SAS/STAT Software. Cary, NC: SAS Institute; 2008. 20. Alberice JV, Amaral AFS, Armitage EG, Lorente JA, Algaba F, Carrilho E, et al. Searching for urine bio- markers of bladder cancer recurrence using a liquid chromatography-mass spectrometry and capillary electrophoresis-mass spectrometry metabolomics approach. J Chromatogr A. 2013; 1318: 163–170. doi: 10.1016/j.chroma.2013.10.002 PMID: 24139504 21. Reddy SV WF. Properties of aflatoxin and its producing fungi. 2015. Available: http://www.icrisat.org/ aflatoxin/aflatoxin.asp. 22. Azziz-Baumgartner E, Lindblade K, Gieseker K, Rogers HS, Kieszak S, Njapau H, et al. Case-Control Study of an Acute Aflatoxicosis Outbreak, Kenya, 2004. Environ Health Perspect. The National Institute of Environmental Health Sciences (NIEHS); 2005; 113 (12): 1779–1783. 23. Dhanasekaran D, Shanmugapriya S, Thajuddin N P A. Aflatoxins and aflatoxicosis in human and ani- mals. In: Guevara-González RG, editor. Aflatoxins—Biochemistry and Molecular Biology. Rijeka, Cro- atia: Intech; 2011. pp. 221–254. 24. Cotty PJ, Jaime-Garcia R. Influences of climate on aflatoxin producing fungi and aflatoxin contamina- tion. Int J Food Microbiol. 2007; 119 (1–2): 109–115. PMID: 17881074 25. Grundy SM. Monounsaturated fatty acids and cholesterol metabolism: implications for dietary recom- mendations. J Nutr. 1989; 119 (4): 529–533. PMID: 2649645 26. Diakogiannaki E, Dhayal S, Childs CE, Calder PC, Welters HJ, Morgan NG. Mechanisms involved in the cytotoxic and cytoprotective actions of saturated versus monounsaturated long-chain fatty acids in pancreatic β-cells. References J Endocrinol. 2007; 194 (2): 283–291. PMID: 17641278 27. Maedler K, Oberholzer J, Bucher P, Spinas GA, Donath MY. Monounsaturated fatty acids prevent the deleterious effects of palmitate and high glucose on human pancreatic β-cell turnover and function. Dia- betes. 2003; 52 (3):726–733. PMID: 12606514 28. Kumar RN, Reddy JS, Gopikrishna G A S. GC-MS determination of bio-active constituents of Cycas- beddomei cones. Int J Pharma Bio Sci. 2012; 3 (3): 344–350. 29. Spiller GA. Handbook of lipids in human nutrition. CRC Press; 1995. pp. 22. 30. Asfaw N, Demissew S. Phytochemical dictionary: A handbook of bioactive compounds from plants. Econ Bot. 1994; 48 (3): 258–258. 31. Abdel-Moein NM, Abdel-Moniem EA, Mohamed DA H E. Evaluación de los efectos anti-inflamatorio y anti-artrítico de algunos extractos de plantas. 2011; 62 (4): 365–374. 32. Simmonds MS. Importance of flavonoids in insect-plant interactions: feeding and oviposition. Phyto- chemistry.001; 56 (3): 245–252. PMID: 11243451 33. Oberdorster E, Clay MA, Cottam DM, Wilmot FA, McLachlan JA, Milner MJ. Common phytochemicals are ecdysteroid agonists and antagonists: a possible evolutionary link between vertebrate and inverte- brate steroid hormones. J Steroid Biochem Mol Biol. 2001; 77 (4–5): 229–238. PMID: 11457661 34. Miean KH, Mohamed S. Flavonoid (Myricetin, Quercetin, Kaempferol, Luteolin, and Apigenin) content of edible tropical plants. J Agric Food Chem. 2001; 49 (6): 3106–3112. PMID: 11410016 35. Yao LH, Jiang YM, Shi J, Tomas-Barberan FA, Datta N, Singanusong R, et al. Flavonoids in food and their health benefits. Plant Foods Hum Nutr. 2004; 59 (3): 113–122. PMID: 15678717 36. Rosenberg Zand RS, Jenkins DJ a, Diamandis EP. Flavonoids and steroid hormone-dependent can- cers. J Chromatogr B Anal Technol Biomed Life Sci. 2002; 777 (1–2): 219–32. 37. Ramos Eldorduy J. Insects: A sustainable source of food? Ecol Food Nutr. 1997; 36 (2–4): 247–276. 38. Teffo LS, Aderogba M a., Eloff JN. Antibacterial and antioxidant activities of four kaempferol methyl ethers isolated from Dodonaea viscosa Jacq. var. angustifolia leaf extracts. South African J Bot. 2009; 76 (1): 25–29. 39. Aiyesanmi AF, Oguntokun MO. Nutrient composition of Dioclea reflexa seed: An underutilized edible legume. Riv Ital delle Sostanze Grasse. Arti Grafiche Stephano Pinelli. 2015; 73 (11): 521–523. 40. Omotoso OT. Nutritional quality, functional properties and anti-nutrient compositions of the larva of Cir- ina forda (Westwood) (Lepidoptera: Saturniidae). J Zhejiang Univ Sci B. Hangzhou: Zhejiang University Press; 2006; 7 (1): 51–55. PMID: 16365926 41. Bengal W. PLOS ONE \| DOI:10.1371/journal.pone.0145914 January 5, 2016 References Assessment of nutritional quality and anti-nutrient composition of two edible grasshoppers (Orthoptera: Acrididae) —a Search for New Food Alternative. IJMPS. 2013; 3 (5): 31–48. 16 / 16 PLOS ONE \| DOI:10.1371/journal.pone.0145914 January 5, 2016
https://openalex.org/W4362628618	https://figshare.com/articles/journal_contribution/Supplemental_Table_S4_from_Effect_of_Green_Tea_Supplements_on_Liver_Enzyme_Elevation_Results_from_a_Randomized_Intervention_Study_in_the_United_States/22534912/1/files/39998185.pdf	English	null	Supplemental Table S2 from Effect of Green Tea Supplements on Liver Enzyme Elevation: Results from a Randomized Intervention Study in the United States	null	2,023	cc-by	380	Supplemental Table S4. The grading of alanine aminotransferase (ALT) during the 12-month treatment period by the level of baseline liver function tests (LFTs) and the interaction with treatment assignment (GTE or Placebo), the Minnesota Green Tea Trial, 2009-2015 Placebo GTE Pa Pinta Participants with all LFTs below ULN at baseline 487 490 0.41 Highest level of ALT during the intervention period Normal, Grade 0 (<75 U/L) 480 451 <0.0001 Abnormal (≥75U/L) Grade 1 (75-150 U/L) 7 23 Grade 2 or above (>150 U/L) 0 16 Participants with any LFT above ULNb at baseline 21 23 Highest level of ALT during the intervention period Normal, Grade 0 (<75 U/L) 19 18 0.27 Abnormal (≥75U/L) Grade 1 (75-150 U/L) 2 4 Grade 2 or above (>150-300 U/L) 0 1 a 2-sided P values were derived from unconditional logistic regression model. ULN, upper limit of normal. b ULN for all 4 liver function tests (LFTs) including alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphate (AKP), and total bilirubin, were defined in Supplemental Table S1. Supplemental Table S4. The grading of alanine aminotransferase (ALT) during the 12-month treatment period by the level of baseline liver function tests (LFTs) and the interaction with treatment assignment (GTE or Placebo), the Minnesota Green Tea Trial, 2009-2015 Placebo GTE Pa Pinta Participants with all LFTs below ULN at baseline 487 490 0.41 Highest level of ALT during the intervention period Normal, Grade 0 (<75 U/L) 480 451 <0.0001 Abnormal (≥75U/L) Grade 1 (75-150 U/L) 7 23 Grade 2 or above (>150 U/L) 0 16 Participants with any LFT above ULNb at baseline 21 23 Highest level of ALT during the intervention period Normal, Grade 0 (<75 U/L) 19 18 0.27 Abnormal (≥75U/L) Grade 1 (75-150 U/L) 2 4 Grade 2 or above (>150-300 U/L) 0 1 a 2-sided P values were derived from unconditional logistic regression model. ULN, upper limit of normal. b ULN for all 4 liver function tests (LFTs) including alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphate (AKP), and total bilirubin, were defined in Supplemental Table S1. Supplemental Table S4. The grading of alanine aminotransferase (ALT) during the 12-month treatment period by the level of baseline liver function tests (LFTs) and the interaction with treatment assignment (GTE or Placebo), the Minnesota Green Tea Trial, 2009-2015
https://openalex.org/W3081985676	https://www.granthaalayahpublication.org/journals/index.php/granthaalayah/article/download/IJRG19_A01_2021/908	English	null	RESEARCH OF PHYSICAL AND MECHANICAL PROPERTIES OF BLENDED BRICKS WİTH FLY ASH BASED, BLAST FURNACE SLAG ADDITION	International journal of research - granthaalayah	2,019	cc-by	5,252	Keywords: Flay Ash; Blast Furnace Slag; Brick; Physical Property; Mechanical Property. Keywords: Flay Ash; Blast Furnace Slag; Brick; Physical Property; Mechanical Property. ords: Flay Ash; Blast Furnace Slag; Brick; Physical Property; Mechanical Property Cite This Article: Hakan Çağlar, and Arzu Çağlar. (2019). “RESEARCH OF PHYSICAL AND MECHANICAL PROPERTIES OF BLENDED BRICKS WİTH FLY ASH BASED, BLAST FURNACE SLAG ADDITION.” International Journal of Research - Granthaalayah, 7(1), 126-136. https://doi.org/10.29121/granthaalayah.v7.i1.2019.1041. Cite This Article: Hakan Çağlar, and Arzu Çağlar. (2019). “RESEARCH OF PHYSICAL AND MECHANICAL PROPERTIES OF BLENDED BRICKS WİTH FLY ASH BASED, BLAST FURNACE SLAG ADDITION.” International Journal of Research - Granthaalayah, 7(1), 126-136. https://doi.org/10.29121/granthaalayah.v7.i1.2019.1041. RESEARCH OF PHYSICAL AND MECHANICAL PROPERTIES OF BLENDED BRICKS WİTH FLY ASH BASED, BLAST FURNACE SLAG ADDITION Hakan Çağlar 1, Arzu Çağlar 2 1 Civil Engineering, Kastamonu University, Turkey 2 Building Control, Kastamonu University, Turkey ISSN- 2350-0530(O), ISSN- 2394-3629(P) DOI: https://doi.org/10.29121/granthaalayah.v7.i1.2019.1041 ISSN- 2350-0530(O), ISSN- 2394-3629(P) DOI: https://doi.org/10.29121/granthaalayah.v7.i1.2019.1041 [Çağlar et. al., Vol.7 (Iss.1): January 2019] Science Abstract In this study, it is aimed to make improvements on blended brick (1) which is the first building material has a history of at least 10,000 years. To the blended brick which is a traditional material was kept constant at 5% the addition of fly ash which is industrial waste. It was aim of determine of the effect on the physical and mechanical properties of the blended brick using different ratios (5%, 10%, 15% and 20%) blast furnace slag. In the first stage, the production of fly ash-based blast furnace slag doped sample of blended brick was performed. In the second stage, a variety of experiments were applied to determine the physical and mechanical properties of the blended brick sample. As a result; It has been determined that unit volume weight and compressive strength decreases with the use of industrial wastes in blended brick production. They have occured an increase in porosity and capillary water absorption values. The use of industrial wastes in the production of blended bricks will contribute both improve the properties of the bricks and the reduction of wastes left to the environment. 1. Introduction The brick one of the most important elements in the construction industry (2) and its manufacturing is one of the oldest industries from 8000 BC to this day (3). Brick is a burnt block obtained after the burning of clay in a furnace (4). It is a large-scale building material, which is generally used in the construction of exterior and interior walls at building (5). One of the important factor determinants of brick quality is the property of clay. The clay used in the production must be made of very fine granules and when mixed with water, it must become the plastic dough which can be given the desired shape (6). [126] Http://www.granthaalayah.com ©International Journal of Research - GRANTHAALAYAH ISSN- 2350-0530(O), ISSN- 2394-3629(P) DOI: 10.5281/zenodo.2550164 [Çağlar et. al., Vol.7 (Iss.1): January 2019] One of the biggest disadvantages of traditional brick production is the rapid consumption of fertile soils in brick production. China is top of the largest brick producing countries worldwide (7). India is the second largest country with annual production of 180 billion tons of bricks (8). 300 million tons of fertile soil is consumed per day for the production of bricks in India (9). The production of bricks in Ontario (Canada) is about 700 million per year (10). Therefore, to overcome this problem, some countries such as China limit the use of clay for brick production (11). Blended brick has lower strenght than factory bricks. In addition, in terms of strength and durability, it was observed that these bricks were weaker than cement blocks (7). To overcome these deficiencies, different pozzolanic materials is added to the blend bricks (12). Various researchers have investigated the use of different waste materials in bricks. Waste glasses can be used as an additive in brick. Using waste glass, bricks that is increased compressive strength and water absorption property were produced (13). In clay bricks, agricultural wastes such as sugarcane pouch ash and rice husk ash can be used (14,15). In addition to agricultural wastes, industrial by- products such as fly ash, blast furnace slag, silica fume and boron waste are also used as additives in brick production (16). In this study, fly ash that is one of the industrial wastes was used. Fly ash that is one of the industrial by-products and which is formed during the burn of coal for energy production, causes environment pollution. 1. Introduction For this reason, the recycling of fly ash as raw waste material for the construction sector is a solution process very useful aspect economical and environmental (17). The properties of fly ash, are vary depend on the type of burned coal, the type of combustion equipment used and the fly ash collection mechanism used (18). The fly ash is a waste that is physically very fine, dusty, predominantly silica-containing, spherically shaped particles and has an excellent pozzolanic property (19). Its color can vary according to the combustion properties of coal. Unburned carbon gives a black color to the fly ash when the combustion process is not sufficient. The color of the fly ash which is formed as a result of complete combustion is lighter than the other (20). The amount of fly ash emerging in the world is about 600 million tons per year (21). In addition, clay which is the main raw material of the bricks, has been understood to develop the properties of the bricks produced by mixing with the fly ash at certain rates (22). Various research was conducted on the use of fly ash as an additive in brick production (24, 24). Fly ash added bricks compared to clay bricks, fly ash added bricks have been seen to obtained 10% lighter bricks (8). Also, fly ash is increased strength and reduced water absorption (5). Leiva et al. (25) found that reduction of compressive strength was obtained by firing the bricks below 1000 ° C and increasing the amount of fly ash additive. Çiçek and Çinçin (26) reported the superior thermal conductivity of fly ash bricks compared to traditional clay bricks. Fly ash added bricks generally have smooth edges compared to clay bricks (8). In many parts of the world, fly ash is used instead of cement (27). Moreover, the production cost of fly ash bricks is 2% lower compared to clay bricks (28). Therefore, the use of fly ash in blended brick production is important in terms of cost effectiveness, strength and durability of bricks. All of these studies showed that fly ash bricks have higher water adsorption, low resistance towards abrasion, low strenght toward fire and high porosity (9). Another waste used in the study is blast furnace slag. 1. Introduction Blast furnace slag which is contained a large amount of silica and alumina and having an amorphous structure is showed pozzolanic properties [127] Http://www.granthaalayah.com ©International Journal of Research - GRANTHAALAYAH ISSN- 2350-0530(O), ISSN- 2394-3629(P) DOI: 10.5281/zenodo.2550164 ISSN- 2350-0530(O), ISSN- 2394-3629(P) DOI: 10.5281/zenodo.2550164 [Çağlar et. al., Vol.7 (Iss.1): January 2019] if they are ground to very fine grains (29). Milled blast furnace slags have different usage styles such as serve as binders (30). Blast furnace slag is generally used as a suitable material for the construction industry. It is used as an auxiliary material to strength construction materials, increase their durability and achieve high performance. It also provides some economic and environmental benefits, such as energy and waste material savings (31). In this study, in the blended brick, which is a traditional material, the fly ash which is industrial waste has kept constant at a rate of 5%. It was aim to determine the effect on physical and mechanical properties of blended brick produced using different ratios (5%, 10%, 15% and 20%) of blast furnace slag. Firstly, fly ash-based blast furnace slag doped sample had produced. Then, various experiments had performed to determine the physical and mechanical properties to the sample of the blended brick produced. 2.1. Materials Http://www.granthaalayah.com ©International Journal of Research - GRANTHAALAYAH [128] ISSN- 2350-0530(O), ISSN- 2394-3629(P) DOI: 10.5281/zenodo.2550164 [Çağlar et. al., Vol.7 (Iss.1): January 2019] 2.1. Materials Fly Ash (FA) is an ash which the remaining a result of the burning of coal in steam power plants. It has cement fineness and pozzolanic properties (32). The fly ash used in the study and the chemical composition given in Table 1; It was obtained from the Seyitömer Thermal Power Plant. In the experiments, F-type fly ash was used which had a bulk density of 0.88 g/cm3, a specific gravity of 1.58 g/cm3, a specific surface area of 0.125 m2/g, and a pH of 8.3, which was lighter than the other fly ash. Table 1: Chemical Analysis of Seyitömer Fly Ash (33) Compound SiO2 CaO MgO Fe2O3 Al2O3 Na2O K2O SO3 Na2O (equ.) Indep. CaO (%) 52.,4 7,47 5,75 9,30 18,91 0,88 2,17 2,25 2,31 0,20 Blast furnace slag (BFS), iron ores are subjected to melt and heat up to high temperatures in blast furnaces. Coking coal is used as fuel. In these furnaces, the carbon of the coking coal with the effect of temperature is create carbon monoxide and carbon dioxide gases to united with the oxygen in the iron oxide of ore. It leaves the material called slag together with the molten iron when these gases leave the furnace. The melt materials are collected from the lower end of the furnace. Since the densities are different, the iron the lower part of the melt materials, the slag the upper part has formed. When the hot liquid slag comes into contact with water, it turns into particles of 1-10 mm grain size. The slag is a by-product which is granulated by sudden cooling with water. This material containing aluminum, silica and lime may be mixed with very little lime or cement. Because this situation gives the property of binding, it is accepted a pozzolan (34). The blast furnace slag used as additive material was obtained from the Karabük Iron and Steel Factory. Clay soil; the clay soil used as the main material was obtained from the clayey soil pile within the boundaries of Taşköprü district of Kastamonu province. It was seen that silicon (Si) element is the most common element in the blended brick which is investigated at Kastamonu University Center Research Laboratory Application and Research Center (Table 2). In addition, aluminum (Al), calcium (Ca), oxygen (O), iron (Fe) and magnesium (Mg) elements are located in clay structure. Capillary Water Absorption The samples were dried until the constant weight in the drying oven. The samples removed from the drying oven were kept in the laboratory for 1 hour and brought to room temperature. The dry weight of the cooled samples was weighed with the help of precision scales (mfirst). Because they must not come into contact with water, all sides of blended brick samples which are bottom surface dimensions 4x4 cm are covered with paraffin in 1 cm height. The bottom surfaces of the coated samples were placed in water-filled vessel which was 1 cm high which touched the surface of the water and with grid. At certain times of the samples (60, 120 and 180 min) weight measurements were made. After 180 minutes, the samples were weighed (mend). The values found were replaced in (1). Thus, capillary water absorption amount was calculated (36). (1) Weight Per Unit of Volume The samples of doped blended brick produced in 4x4x16 cm dimensions were placed in the oven which had a temperature of 105 ± 1 ° C. The samples were dried to until constant mass for 12 hours. The samples dried in this way were cooled until room temperature and then the size was 0.01 mm sensitivity was measured by caliper. The masses of the samples were measured with precision scales and the weight per unit of volume was determined (35). 2.2.1. Production of Fly Ash-Based, Blast Furnace Slag Additive Blended Brick Samples al., Vol.7 (Iss.1): January 2019] ISSN- 2350-0530(O), ISSN- 2394-3629(P) DOI: 10.5281/zenodo.2550164 Figure 1: Cooling of samples 2.2.2. Physical Experiments Applied to Samples Weight Per Unit of Volume The samples of doped blended brick produced in 4x4x16 cm dimensions were placed in the oven which had a temperature of 105 ± 1 ° C. The samples were dried to until constant mass for 12 Çağlar et. al., Vol.7 (Iss.1): January 2019] ISSN- 2350-0530(O), ISSN- 239 DOI: 10.5281/zenod Figure 1: Cooling of samples 2.2.2. Physical Experiments Applied to Samples [Çağlar et. al., Vol.7 (Iss.1): January 2019] ISSN- 2350-0530(O), ISSN- 2394-3629(P) DOI: 10.5281/zenodo.2550164 Figure 1: Cooling of samples 2 2 2 Ph i l E i t A li d t S l D Figure 1: Cooling of samples 2.2.2. Physical Experiments Applied to Samples Figure 1: Cooling of samples Figure 1: Cooling of samples 2.2.2. Physical Experiments Applied to Samples 2.2.1. Production of Fly Ash-Based, Blast Furnace Slag Additive Blended Brick Samples The production of blended brick samples has made in the Emek Brick Factory in Boyabat district of Sinop province. The study started with the removal of the main material from the clayey soil piles in the district of Taşköprü in Kastamonu province. The clayey soil sample taken by the quartering method was ground in the laboratory type roller hammer and then 1 mm sieve material was obtained. The fly ash and blast furnace slag to be used as additive in the experiment were subjected to the same treatments. The recipe for the mixture prepared for brick dough is given in Table 3. The kneading water was added to each mixture in 20% of the total weight of the material. In the study, reference (REF), 5%FA and 5%BFS doped blended brick sample (5%FA+5%BFS), 5% FA and 10% BFS doped blended brick sample (5%FA+10% BFS), 5%FA and 5% BFS doped blended brick sample (5%FA +15%BFS) and 5% FA and %20BFS doped blended brick sample (5%FA+20%BFS). Table 3: Mixing prescription Prescription Clay soil (%) BFS (%) FA (%) Water (%) REF 100 - - 20 %5FA +%5BFS 90 5 5 20 %5FA +%10BFS 85 10 5 20 %5FA +%15BFS 80 15 5 20 %5FA +%20BFS 75 20 5 20 Table 3: Mixing prescription Firstly, fly ash, blast furnace slag and clay soil samples has been turned into furnace dry in the furnace. Dough to be created mixture was weighed in the proportions given in the prescription with the precision scale and taken into the mixing bowl. All materials were mixed with water after the dry mixture was made. The obtained brick dough was left to rest for 24 hours in a way that would not lose its moisture. After resting, it was mixed until no air bubbles were left in mixer (5 minutes). After the kneading process is finished, the mixture prepared in plastic consistency was poured into steel molds. After they were waited 24 hours in normal weather conditions, they were removed from the molds. The extracted brick samples were allowed to dry in a half-open area for 7 days. At the end of the 7 days, they were fired by increasing the temperature gradually in time adjustable electric ash furnace at 900 ° C. Samples of brick removed from the furnace were brought to room temperature (Figure 1). [129] Http://www.granthaalayah.com ©International Journal of Research - GRANTHAALAYAH [Çağlar et. Porosity In order to determine the porosity values of the doped samples, they were boiled in a container for 3 hours and bring to waterlogged situation. Samples were taken from the container and their hanging weights in water were measured (P3). After measurement, the samples were wiped dry with a dry cloth and was bring to dry-surface situation. Then, the waterlogged surface dry weights were measured (P2). The samples were dried in the drying oven at +105 oC for 24 hours and weighed again after they were constant-weight (P1). The values found were replaced in (2) and the porosity values were calculated (35). (2) Porosity (%) = ((P2-P1) / (P2-P3)) x 100 [130] Http://www.granthaalayah.com ©International Journal of Research - GRANTHAALAYAH [Çağlar et. al., Vol.7 (Iss.1): January 2019] ISSN- 2350-0530(O), ISSN- 2394-3629(P) DOI: 10.5281/zenodo.2550164 Compressive Strength The samples were dried at +105 0C in the dry oven until constant-weight. The compressive strength value of the dried samples was calculated by dividing the breaking load to the surface area (4x4 cm) (37). 3. Results and Discussions The data obtained by physical and mechanical experiments of the blended brick samples shows in Table 5. When the table is examined; the REF sample were seen the highest weight per unit of volume with 1.91 g/cm3, while 5%FA + 20%BFS added blended brick samples had the lowest weight per unit of volume with 1, 85 g/cm3. According to the capillary water absorption test data, the REF sample has the lowest value with 99,1 g capillary water absorption amount, the 5%FA + 5%BFS added blended brick sample has the highest value with 102,5 g capillary water absorption amount. The REF sample had the lowest porosity with a ratio of 21,1%, while the 5%FA+20%BFS sample had the highest porosity with a rate of 26,2%. As a result of the compressive strength test to determine the mechanical properties, It was determined that the 5%FA+ 20%BFS added blended brick was the lowest compressive strength with 2,98 MPa, the REF sample has the highest compressive strength with 4,45 MPa. Table 4: Physical and mechanical values of blended bricks Physical Values Mechanical Values Weight Per Unit of Volume (g/cm3) Capillary water absorption (g) Porosity (%) Compressive Strength (MPa) REF 1,91 99,1 21,1 4,45 %5FA +%5BFS 1,90 99,8 22,3 4,02 %5FA +%10BFS 1,88 100,6 23,4 3,87 %5FA +%15BFS 1,87 101,4 25,5 3,40 %5FA +%20BFS 1,85 102,5 26,2 2,98 3.1. Weight Per Unit of Volume The sample values obtained as a result of the weight per unit of volume test which is one of the physical properties are given in Figure 2. When the figure is examined; it was determined that the amount of fly ash was kept constant and with the increase amount of blast furnace slag the weight per unit of volume values of the samples were decreases. The reason of the decrease can be explained as the density of the fly ash is greater than the density of the clay. There is no limitation for the weight per unit of volume of the brick in TS 705 (38), which is related to blended brick. However, the use of bricks which was low weight per unit of volume in building structure will help to reduce the overall weight of the building. This situation will help to increase the performance of buildings in earthquake zones. [131] Http://www.granthaalayah.com ©International Journal of Research - GRANTHAALAYAH [Çağlar et. 3.2. Capillary Water Absorption The rate of capillary water absorption of the materials is associated with clearance ratio in their structures, the volume, size and inter-pore bonds of the open pores related to the material surface (39). The capillary water absorption test data, which is one of the experiments to determine the physical properties of the blended brick samples, is presented in Figure 3. According to the result of the experiment, it was seen that the amount of capillary water absorption increased with the increase of the amount of additive. This situation can be explained by the high-water absorption capacity of fly ash (40, 41). An increase in the amount of capillary water absorption of the blended brick that is used as an external wall filler is created an undesirable situation for the structure. It was thought that the reason of the high of amount of capillary water absorption is that the samples of blended brick, fired at 900 0C, cannot be reached to sufficient sintering (42). Figure 3: Capillary water absorption values of brick samples 96 98 100 102 104 REF %5FA +%5BFS %5FA +%10BFS %5FA +%15BFS %5FA +%20BFS 99.1 99.8 100.6 101.4 102.5 Capillary water absorption (g) Figure 3: Capillary water absorption values of brick samples 3. Results and Discussions al., Vol.7 (Iss.1): January 2019] ISSN- 2350-0530(O), ISSN- 2394-3629(P) DOI: 10.5281/zenodo.2550164 Figure 2: Weight per unit of volume values of brick samples 1.8 1.85 1.9 1.95 REF %5FA +%5BFS %5FA +%10BFS %5FA +%15BFS %5FA +%20BFS 1.91 1.9 1.88 1.87 1.85 Weight Per Unit Of Volume (g/cm3) Weight Per Unit Of Volume (g/cm3) Figure 2: Weight per unit of volume values of brick samples 3.2. Capillary Water Absorption 3.3. Porosity The values of porosity, which is known as the void ratio, are given in Figure 4. When the graph is examined; With the increase in the amount of BFS porosity values are seen to increase. Increasing [132] Http://www.granthaalayah.com ©International Journal of Research - GRANTHAALAYAH ISSN- 2350-0530(O), ISSN- 2394-3629(P) DOI: 10.5281/zenodo.2550164 ISSN- 2350-0530(O), ISSN- 2394-3629(P) DOI: 10.5281/zenodo.2550164 [Çağlar et. al., Vol.7 (Iss.1): January 2019] the amount of porosity is a positive situation for the weight per unit of volume value. However, capillary water is a negative situation for the amount of absorption. In other words, the increase of porosity will lead to increase the amount of capillary water absorption despite the fact that it creates a light construction material. Figure 4: Porosity values of brick samples 0 10 20 30 REF %5FA +%5BFS %5FA +%10BFS %5FA +%15BFS %5FA +%20BFS 21.1 22.3 23.4 25.5 26.2 Porosity (%) Figure 4: Porosity values of brick samples 3.4. Compressive Strength Compressive strength is one of the most important parameters used to ensure engineering quality in construction materials (42). As a result of the experiments, the FA was kept constant and It has been seen that by increasing the blast furnace slag, the compressive strength of the samples decreased (Figure 5). Although BFS increases the compressive strength of the brick sample (42) it needs a high calcination temperature of 1050 °C in order to provide higher strength of blended brick that is included fly ash (43). The best result in the study was obtained from 5%FA+5%BFS added blended brick. Figure 5: Compressive strength values of brick samples 0 1 2 3 4 5 REF %5FA +%5BFS %5FA +%10BFS %5FA +%15BFS %5FA +%20BFS 4.45 4.02 3.87 3.4 2.98 Compressive strength (MPa) Compressive strength (MPa) Compressive strength (MPa) Compressive strength (MPa) Figure 5: Compressive strength values of brick samples Http://www.granthaalayah.com ©International Journal of Research - GRANTHAALAYAH [133] [Çağlar et. al., Vol.7 (Iss.1): January 2019] ISSN- 2350-0530(O), ISSN- 2394-3629(P) DOI: 10.5281/zenodo.2550164 4. Conclusions and Recommendations In this study, physical and mechanical properties of blended bricks containing fly ash-based blast furnace slag were investigated. As a result of the experiments; With the increase in the of amount of blast furnace slag, in the weight per unit of volume values reduction were occurred. This reduction in the unit weight of the blend bricks will increase the performance of the structures in the earthquake affected areas and will help to reduce the total weight of the structures. In the study, it was seen that by increase of the amount of additive was increased porosity and the capillary water absorption values. When the compressive strength that is one of the mechanical properties is examined, it has been determined that there is a decrease in the compressive strength by increase of amount of waste. The use of fly ash and blast furnace slag in the production of blended brick samples is thought to be an effective way to disposal these wastes. The use of waste materials in threshing brick production is provided benefits the protection of natural resources such as fertile soil and the improvement of the properties of the blended bricks. References al., Vol.7 (Iss.1): January 2019] ISSN- 2350-0530(O), ISSN- 2394-3629(P) DOI: 10.5281/zenodo.2550164 [15] Kazmi, SMS., Abbas, S., Munir, MJ., Khitab, A. Exploratory study on the eﬀect of waste rice husk and sugarcane bagasse ashes in burnt clay bricks, Journal of Building Engineering 7, 2016, 372– 378. [16] Çağlar, H., Çağlar, A., Korkmaz, SZ., Demirel, B., Bayraktar, OY. Comparison Of The Physical And Mechanical Properties Of Manually Manufactured And Factory Production Blended Bricks Used In Build Of Traditional Kastamonu Houses, Fırat University Journal of Engineering Sciences, 30 (2), 2018, 39-48. [17] Tokyay, M., Çetin, B. Strength and water absorption properties of volatile culvert bricks applied with pressed steam cure, Technical Journal, TMMOB Construction Engineering, Chamber Publishing, Skin 2, Istanbul, Turkey, 1991, 385–394. [18] Tütünlü, F., Atalay, Ü. Utilization of ﬂy ash in manufacturing of building bricks, International Ash Utilization Symposium, Center for Applied Energy Research, University of Kentucky, Lexington, Kentucky, USA, 2011. [19] Özdemir, M., Öztürk, NU. Utilization of Clay Wastes Containing Boron as Cement Additives, Cement and Concrete Research, 33(10), 2003, 1659-1661. [20] Güler, G, İpekoğlu, Ü., Mordoğan, H. Using Areas and Properties of Fly Ashes, Turkey 19th International Mining Congress and Exhibition, İzmir, Turkey, 2007, 419-420. [21] Murathan, A., Asan, A., Abdulkerem, TA. Evaluation of environmental wastes on building materials, Gazi University. Journal of Engineering Architecture, 28 (2), 2013, 409–416. [22] Xu, L., Gou, W., Wang, T., Yang, N. Study on fired bricks with replacing clay by fly ash in high volume ratio, Construction Building Material 19, 2005, 243–247. [23] Aydın, O. Investigation of the Use of Thermal Power Plant Ashes in Building Brick Production, Master Thesis, Afyon Kocatepe University, Afyonkarahisar, 2007. [24] Abbas, S., Saleem, MA., Kazmi, SMS. ve Munir, MJ. Production of Sustainable Clay Bricks Using Waste Fly Ash: Mechanical and Durability Properties, Journal of Building Engineering, 14, 2017, 7-14. [25] Leiva, C., Arenas, C., Alonso-Farinas B., Vilches, L.F., Peceno, B., Rodriguez-galan M. Characteristics of fired bricks with co-combustion fly ashes, Journal of Building Engineerig 5, 2006, 114–118. [26] Çiçek, T., Çinçin, Y. Use of fly ash in production of light-weight building bricks, Construction Building Material 94, 2015, 521–527. [27] Bouzoubaa, N., Lachemi, M. Self-compacting concrete incorporating high volumes of class fly ash: preliminary results, Cement and Concrete Research 31 (3), 2001, 413–420. [28] Li, Y., Lin, Y. Compacting Solid Waste Materials Generated in Missouri to Form New Products. Final Technical Report to the Solid Waste Management Program. References [1] İspir, M. A Comprehensive Experimental Research On The Behavior Of Historical Brick Masonry Walls Of 19th Century Buildings, Doktora Tezi, Istanbul Technical University, Institute of Science and Technology İstanbul, 2010, 47-48. gy [2] Rahman, ME., Ong, PJ., Nabinejad, O., Islam, S., Khandoker NAN., Pakrashi, V. and Shorowordi, KM. Utilization of Blended Waste Materials in Bricks, Technologies, 6 (20),2018, 1-12. 3] Houben, H., Guillaud, H. Earth construction: A Comprehensive Guide, London, 1994 [4] Niroumand, H., Zain, MFM., Alhosseini, SN. The influence of nano-clays on compressive strength of earth bricks as sustainable materials, Procedia – Social and Behavioral Sciences (89), 2013, 862– 865. [5] Shakir, AA., Naganathan, SK., Mustapha, KNB. Development Of Bricks From Waste Material: A Review Paper. Australian Journal of Basic and Applied Sciences, 7(8), 2013, 812-818. [6] Çelebi, GÜ. Usage Properties for Building Materials and Construction, Gazi University, Faculty of Engineering and Architecture, Department of Architecture, Lecture Notes, Ankara, 2007. [7] More, A., Tarade, A., Anant, A. Assessment Of Suitability Of Fly Ash And Rice Husk Ash Burnt Clay Bricks, International Journal of Sciences. Research Publishing, 4 (7), 2014, 1–6. [8] Pawar, A., Garud, D. Engineering properties of clay bricks with use of fly ash, International Journal of Research in Engineering and Technology, 3 (9), 2014, 75–80. [9] Kumar, R., Hooda, N. An experimental study on properties of fly ash bricks, International Journal of Mechanical Engineering and Robotics Research, 2 (9), 2014, 56–67. [10] Chidiac, SE., Federico, LM. Eﬀects of waste glass additions on the properties and durability of fired clay brick, Canadian Journal of Civil Engineering, 34 (11), 2007, 1458–1466. [11] Chen, Y., Zhan,g Y., Chen, T., Zhao, Y., Bao, S. Preparation of eco-friendly construction bricks from hematite tailings, Construction and Building Material, 25 (4), 2011, 2107–2111. [12] Munoz, VP., Morales, OMP., Mendívil, GMA., Munoz, VL. Fired clay bricks manufactured by adding wastes as sustainable construction material – a review, Construction and Building Material 63, 2014, 97–107. [13] Djangang, C., Kamseu, E., Elimbi, A., Lecomte, G., Blanchart, P., Net-shape clay ceramics with glass waste additive, Materials Sciences and Applications 5, 2014, 592–602. [14] Kazmi, SMS., Abbas, S., Saleem MA., Munir, MJ., Khitab, A. Manufacturing of sustainable clay bricks: utilization of waste sugarcane bagasse and rice husk ashes, Construction and Building Material 120, 2016, 29–41, Http://www.granthaalayah.com ©International Journal of Research - GRANTHAALAYAH [134] [Çağlar et. References Missouri Department of Natural Resources (MDNR) (Contact no. MDNR 00038-1), Capsule Pipeline Research Center, University of Missouri-Columbia, USA, 2002. [29] Öner, M. Determination of the Effects of Grinding Parameters on the Use of Blast Furnace Slag in Cement Production. Hacettepe University Earth Sciences Application and Research Center Bulletin, Ankara, (23), 2001, 61–69. [30] Binici, H., Sevinç, AH., Durgun, MY. Resistance of pumice, barite, colemanite and blast furnace slag additive mortars and sulphate resistance, Electrical Journal of Construction Technology, 7 (1), 2011, 39-51 [31] Erdoğan, TY. Concrete, METU Development Foundation and Publishing, Ankara, 2003. [32] Böke, H., Akkurt, S., Ipekoğlu, B., Uğurlu, E. Characteristics of brick used as aggregate in historic brick-lime mortars and plasters, Cement Concrete Research, 36(6), 2006, 1115-1122. p ( ) [33] Bayat, O. Characterisation of Turkish fly ashes, Fuel, 77(9/10), 1998, 1059-1066. y y [34] Güvercin, T. The Investigation of the effect of the silica fume, fly ash and blast furnace slag on the cement properties, Master Thesis, Atatürk University, Erzurum, 2002. [35] TS EN 772-1, Methods of test for masonry units- Part 1: Determination of compressive strength, Turkish Standards Institute, Ankara, 2012. Http://www.granthaalayah.com ©International Journal of Research - GRANTHAALAYAH [135] [Çağlar et. al., Vol.7 (Iss.1): January 2019] ISSN- 2350-0530(O), ISSN- 2394-3629(P) DOI: 10.5281/zenodo.2550164 [36] TS EN 772-4, Methods of test for masonry units - Part 4: Determination of real and bulk density and of total and open porosity for natural stone masonry units, Ankara, Turkish Standards Institute, 2000. [36] TS EN 772-4, Methods of test for masonry units - Part 4: Determination of real and bulk density and of total and open porosity for natural stone masonry units, Ankara, Turkish Standards Institute, 2000. [37] TS EN 772-11, Methods of test for masonry units Part 11: Determination of water absorption of aggregate concrete manufactured stone and natural stone masonry units due to capillary action on the initial rate of water absorption of clay masonry units, Turkish Standards Institute, Ankara, 2012. p y y [38] TS 705, Solid Bricks and Vertically Perforated Bricks, Turkish Standards Institute, [39] Çağlar, A. Experimental research on improvement with boron waste additive of properties of blend brick used in traditional Kastamonu houses, PhD Thesis, Selçuk University, Konya, 2018. ç y y [40] Fetra, VR., Ismail, A.R., Ahmad, MAZ. Preliminary study of compressed stabilized earth brick, Australian Journal of Basic and Applied Sciences, 5 (9), 2011, 6–12. [40] Fetra, VR., Ismail, A.R., Ahmad, MAZ. Corresponding author. E-mail address: hcaglar@ kastamonu.edu.tr Corresponding author. References Preliminary study of compressed stabilized earth brick, Australian Journal of Basic and Applied Sciences, 5 (9), 2011, 6–12. [41] Freidin, K., Erell, E. Bricks made of coal fly-ash and slag, cured in the open air, Cement and Concrete Composites, 17 (4), 1995, 289–300. [41] Freidin, K., Erell, E. Bricks made of coal fly-ash and slag, cured in the open air, Cement and Concrete Composites, 17 (4), 1995, 289–300. [42] Sürül, O. Investigation of the use of blast furnace slag and fly ash as additives in brick production, Master Thesis, Bülent Ecevit University, Zonguldak, Turkey, 2015, 6-8. [42] Sürül, O. Investigation of the use of blast furnace slag and fly ash as additives in brick production, Master Thesis, Bülent Ecevit University, Zonguldak, Turkey, 2015, 6-8. [43] Fernando, PT., Said, J. Eco-eﬃcient, Construction Building Materials, Springer, Verlag London, 2011. *Corresponding author. E-mail address: hcaglar@ kastamonu.edu.tr [136] Http://www.granthaalayah.com ©International Journal of Research - GRANTHAALAYAH
https://openalex.org/W4378744894	https://www.researchsquare.com/article/rs-2459757/latest.pdf	English	null	Attentional Ptycho-Tomography (APT) for three-dimensional nanoscale X-ray imaging with minimal data acquisition and computation time	Light, science & applications/Light: Science & Applications	2,023	cc-by	9,997	Iksung Kang (  fresneltransform@gmail.com ) Yi Jiang (  yjiang@anl.gov ) Argonne National Laboratory Yudong Yao (  yaoyud1990@gmail.com ) Argonne National Laboratory ABSTRACT Noninvasive X-ray imaging of nanoscale three-dimensional objects, such as integrated circuits (ICs), generally requires two types of scanning: ptychographic, which is translational and returns estimates of the complex electromagnetic ﬁeld through the IC; combined with a tomographic scan, which collects these complex ﬁeld projections from multiple angles. Here, we present Attentional Ptycho-Tomography (APT), an approach to drastically reduce the amount of angular scanning, and thus the total acquisition time. APT is machine learning- based, utilizing axial self-Attention for Ptycho-Tomographic reconstruction. APT is trained to obtain accurate reconstructions of the ICs, despite the incompleteness of the measurements. The training process includes regularizing priors in the form of typical patterns found in IC interiors, and the physics of X-ray propagation through the IC. We show that APT with ×12 reduced angles achieves ﬁdelity comparable to the gold standard Simultaneous Algebraic Reconstruction Technique (SART) with the original set of angles. When using the same set of reduced angles, then APT also outperforms Filtered Back Projection (FBP), Simultaneous Iterative Reconstruction Technique (SIRT) and SART. The time needed to compute the reconstruction is also reduced, because the trained neural network is a forward operation, unlike the iterative nature of these alternatives. Our experiments show that, without loss in quality, for a 4.48×93.2×3.92 µm3 IC (≃6×108 voxels), APT reduces the total data acquisition and computation time from 67.96 hours to 38 minutes. We expect our physics-assisted and attention-utilizing machine learning framework to be applicable to other branches of nanoscale imaging, such as materials science for crystal defects, biological imaging for viruses, etc. Iksung Kang1,‡,†, Ziling Wu2,¶,†, Yi Jiang3, Yudong Yao3,§, Junjing Deng3, Jeffrey Klug3, Stefan Vogt3, and George Barbastathis2,4 1Department of Electrical Engineering and Computer Science, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA 2Department of Mechanical Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA 2Department of Mechanical Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA 3Argonne National Laboratory, Lemont, Illinois 60439, USA g y, , , 4Singapore-MIT Alliance for Research and Technology (SMART) Centre, 1 CREATE Way, Singapore 138602 ‡Present address: Department of Molecular and Cell Biology, University of California, Berkeley, California 94720, USA g y, , , 4Singapore-MIT Alliance for Research and Technology (SMART) Centre, 1 CREATE Way, Singapore 138602 ‡Present address: Department of Molecular and Cell Biology, University of California, Berkeley, California 94720, USA 4Singapore-MIT Alliance for Research and Technology (SMART) Centre, 1 CREATE Way, Singapore 138602 ‡Present address: Department of Molecular and Cell Biology, University of California, Berkeley, California 94720, USA ¶Present address: Singapore-MIT Alliance for Research and Technology (SMART) Centre, 1 CREATE Way, Singapore 138602 ¶Present address: Singapore-MIT Alliance for Research and Technology (SMART) Centre, 1 CREATE Way, Singapore 138602 ¶Present address: Singapore-MIT Alliance for Research and Technology (SMART) Centre, 1 CREATE Way, Singapore 138602 g p §Present address: Center for Transformative Science, ShanghaiTech University, Shanghai 201210, China †These authors contributed equally to this work. s: Center for Transformative Science, ShanghaiTech University, Shanghai 201210, China contributed equally to this work §Present address: Center for Transformative Science, ShanghaiTech University, Shanghai 201210, China †These authors contributed equally to this work. , g y, g , †These authors contributed equally to this work. To whom correspondence should be addressed; E-mail: gbarb@mit.edu. Keywords: DOI: https://doi.org/ License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Additional Declarations: (Not answered) Introduction Three-dimensional X-ray imaging enables noninvasive monitoring of objects’ interiors with nanoscale resolution. Integrated circuits (IC) are especially interesting for this operation, for two reasons: ﬁrst, noninvasive inspection of ICs is important for verifying manufacturing integrity. Second, ICs follow speciﬁc design rules, which makes their geometries highly regular and yet highly diverse. The geometrical properties are then useful as prior knowledge, enabling vast improvements in practical aspects of the imaging process, such as acquisition time as we show here. Prior works have typically used two types of scanning: translational and rotational. The translational scan (ptycho) is inspired by ptychography, i.e. a scanning-based coherent diffraction imaging method for phase retrieval. Ptychography was originally proposed by W. Hoppe1 to solve the phase problem in Scanning Transmission Electron Microscopy (STEM), where a moving aperture resolves the ambiguity in phase based on translational invariance. The term “ptychography” was coined in the following year2. Nellist et al.3 demonstrated resolution improvement in STEM by a factor of 2.5 over the limit imposed by partial coherence, exploiting the redundancy in the ptychographic measurements. As an alternative that does not even require careful aberration correction in the optics, Gerchberg and Saxton4 introduced a lensless iterative phase retrieval algorithm, now referred to as GS after them. This work was extended to lensless ptychography for extended objects by Faulkner5. Subsequently, Rodenburg6 introduced yet another iterative phase retrieval algorithm called Ptychographical Iterative Engine (PIE) that simultaneously retrieves both the object and the probe function. Thus, the requirement of a high-quality lens for imaging is fundamentally lifted. Further advances by Thibault et al.7 and Thibault and Guizar-Sicairos8 led to the Difference Map (DM) algorithm and Maximum Likelihood algorithm, respectively, for iterative ptychographic reconstruction. After the ptychographic reconstruction step, the second angular scan (tomo) operation is required to retrieve the object’s interior, as in tomography. For parallel beam illumination and under the weak scattering approximation, the measurements are interpreted simply as projections through the object, i.e. the measurements implement the object’s Radon transform9,10. The inverse Radon transform is typically implemented as a version of the Filtered Back- Projection (FBP) algorithm, ﬁrst proposed by Bracewell and Riddle11,12. Gordon, Bender, and Herman13 proposed an alternative iterative tomography algorithm called Algebraic Reconstruction Technique (ART) that iteratively, which applies also to non-parallel illumination beams and works by updating the object estimate to sequentially bring each reconstructed projection into agreement with the corresponding measured projection. Introduction Then 2/18 we start reducing the total angular range, meaning that the sampling now becomes denser. The machine-learning regularizer again manages to maintain approximately even ﬁdelity down to a minimum range θ ∗. This is our optimal operating point (N∗,θ ∗). The strategy is depicted in Fig. 1b. In principle, this procedure can be repeated to ﬁnd even tighter operating conditions, but we did not carry that out as we would expect any further gains to be minimal. That machine learning is suitable for achieving even ﬁdelity while the amount of sampled data is decreasing is not entirely surprising by now. The key is the ability of deep neural networks to very effectively capture regularizing priors, especially sparsity, in both supervised mode as we do here and in untrained mode21,22. Previous demonstrations of supervised learning have been carried out for Fourier ptychography23,24 and two-dimensional ptychography25. The reason we chose the supervised learning mode is because we had ample data available from the gold standard ptycho-tomo approach. APT is described schematically in Fig. 1c. We ﬁrst invert the far-ﬁeld diffraction intensities (or ptycho-scans) with an approximate inversion operator. This yields to get an approximate volumetric estimate of the interior of an IC chip, which we dub the “Approximant”26. This step utilizes prior knowledge on underlying imaging physics and pre-processes the input with the physics prior. The Approximant as a result of this pre-processing step (or physics-informing step) is defective in terms that layers are not well separated because of the approximate inversion from diffraction intensities and only a small fraction of tomo-scans used for the computation. During training, the neural network’s weights are optimized based on the Approximant as input. Upon the completion, the trained neural network gives a reﬁned volumetric reconstruction of ICs. The proposed neural network is based on a 3D U-net architecture27,28 and augmented with the multi-head axial self-attention29 to address lack of spatial resolution in the Approximant by taking advantage of its global-range interactions to retrieve information from all layers to resolve each layer’s structure. We choose this multi-head axial self-attention over multi-head self-attention30 to alleviate computational burden. We demonstrate that the present method is capable of providing reliable reconstructions of ICs even when both the number and the total angular range of tomo-scans are largely decreased to N∗∼29 and θ ∗∼±17◦ representing an improvement of ×12 and ×4.2, respectively. Introduction Subsequent improvements of this original iterative method were the Simultaneous Iterative Reconstruction Technique (SIRT)14 and the Simultaneous Algebraic Reconstruction Technique (SART)15, which consider all projections simultaneously and thus drastically reduce the number of iterations for the reconstruction process. Maximum Likelihood methods have also been popular for tomography, with the Bouman-Sauer algorithm16 as one of the most prominent. For X-rays, the high penetration depth facilitates recovery of information deep inside the sample in the angular sampling scheme. Combining this property with translational scanning for lensless high spatial resolution, Dierolf et al.17 proposed the Ptychographic X-ray Computed Tomography (PXCT) scheme to determine the volumetric interior of biological specimens with nanoscale details. Using this technique, Holler et al.18 experimentally demonstrated noninvasive imaging of ICs produced with 22nm technology at 14.6nm resolution. These techniques are limited by the requirement for two types of scanning, angular and translational, and scale badly scales with object volume. A novel X-ray microscope called Velociprobe19 utilizes ﬂy-scan ptychography20 to signiﬁcantly reduce the data acquisition time. Still, total data acquisition and reconstruction time for a typical 100×100×5 µm3 IC (≃2×1010 voxels) is estimated to be in excess of two months. Here, we propose a machine learning framework to reduce data acquisition and computation time for IC reconstruction under the X-ray ptycho-tomography geometry. The reduction in data acquisition is compensated by explicit use of prior knowledge of the typical objects being imaged, and of the optical physics of the imaging system. The length of the acquisition and computation time scale as the number N of tomo-scans. The total angular range θ determines the size of the missing wedge in the Fourier domain and, therefore, is commensurate with loss of ﬁdelity. Our “gold standard” is a ptycho-tomo reconstruction by SART with N = 349 and θ = ±70.4◦. This maximum angle is determined by practical considerations, such as the sample geometry. More details about the gold standard geometry and our approach are available in Methods. To search this two-dimensional space (N,θ), our strategy is as follows: we start with the gold standard nominal values of N and θ. If we reduced N while using a standard reconstruction algorithm, like FBP, SIRT, etc. mentioned earlier, performance would decrease immediately. With machine learning, we ﬁnd that it is possible to regularize for the loss of angular sampling density and still maintain reconstruction ﬁdelity, down to a minimum number N∗. Introduction For the reconstruction of an IC chip over the test volume (4.48×93.2×3.92µm3), 0.63 hours (or 38 minutes) is sufﬁcient for both data acquisition and reconstruction with our machine learning framework. The improvements work out to an approximate overall ×108 reduction in total (acquisition plus computation) time compared to the current up-to-date iterative reconstruction method. Regularization and imaging system physics The reported improvements suggest that the APT algorithm is particularly effective at learning regularizing priors to compensate for the missing information. Fig. 6a shows the power spectral densities of the gold standard, the APT reconstruction, and the baseline tomographic reconstruction methods FBP11, SIRT14, and SART15, all obtained at N∗= 29 and θ ∗= ±16.8◦. The missing wedge is evident in the latter three. The qualitative cross-sections in Fig. 6b conﬁrm that the missing wedge effect leads to severe artifacts in the baseline methods, but not in APT. APT also relies on its input, the Approximant, having carefully taken into account the physics of the imaging system. Unlike earlier works where the illumination on the sample was coherent31,32, the synchrotron may be considered as temporally coherent but is rather less coherent spatially. The mutual intensity is expressed as a linear combination of mutually incoherent states, also known as coherent modes36. Accounting correctly for the synchrotron X-ray’s coherence state has been shown to improve spatial resolution and phase contrast in standard ptychography for thin samples37. For samples thicker than the depth of focus of the probe, multi-slice reconstruction from simple ptychography has been demonstrated with visible light38, X-rays39 and electrons40. This is the starting point for our Approximant as shown Fig. 1c, and please ﬁnd more information on why multi-slice ptychography was used instead of 2D ptychograpy in Gradient calculation in Methods. We form the cost function Ln = Jn ∑ j=1∑ q M ∑ m=1 Pd n P(n)[L] j,r,m O(n)[L] r o 2 − q I(n) j,q !2 (n = 1,2,··· ,N), (1) (1) where N is the number of given tomo-scans; Jn the number of ptycho-scans associated with the n-th tomo-scan; M the number of coherent modes; L the number of slices for our given depth of focus works out to equal to 5; q denotes the coordinates in the reciprocal space; and P(n)[L] j,r,m , I(n) j,q indicate the waveﬁeld before the L-th slice from the m-th coherent mode and experimental diffraction intensity at the j-th ptycho- and the n-th tomo-scans, respectively. We run two iterations of a gradient scheme on Eq. 1 and obtain the argument ∠O(n)[l] r at each one among the l = 1,··· , L slices38,40. We rotate the result back to the original coordinate system, and average the estimates from all tomo-scan steps to yield the ﬁnal Approximant. and quantitatively. Next we ﬁx N = 29 and perform a parameter sweep over θ. Qualitative results are shown in Fig. 4, while the quantitative evaluation according to the same four metrics of the previous section is in Fig. 5. Both analyses lead to θ ∗∼±17◦as the approximate lower bound before drastic degradation occurs. The savings in data acquisition and computation times are ×12 and ×105, respectively, and total time savings (acquisition plus computation) of ×108. Regularization and imaging system physics More details can be found in Methods. The Approximant computation step is the slowest in the pipeline; in our computing hardware (see Methods), it takes 36 minutes when θ = ±70.4◦and 26 minutes when θ ∼±17◦. In addition to the computation time, the spacing between slices in the Approximant is nominally limited by the depth of focus, and that is why we only reconstruct L = 5 of them. The number of desired reconstruction slices is much larger, i.e. 280, so we simply dilate the Approximant slices to match it. As a result, the input to the neural network is poor (more in Supplementary Materials). Nevertheless, the subsequent APT architecture learns how to use the multi-slices as input and, as long as N > N∗and θ > θ ∗, produce a high-ﬁdelity ﬁnal reconstruction with much ﬁner slice spacing. Reducing acquisition and scanning time The synchrotron beam is delivered on the sample, and a full lateral scan is carried out to obtain the ptychographic information for each angular orientation of the sample. Repeating for N angles collects tomographic information for the interior’s reconstruction. The raw intensities past the sample are recorded by a digital camera detector at each scan position. The details of the experimental collection system are in Methods. The collected raw intensities are then processed in two steps: the ﬁrst step embeds the physics of X-ray propagation through an Approximant operator31,32, while the second step consists of the APT network delivering the ﬁnal reconstruction, as described earlier. The details of training and operating this computational pipeline are in Methods. As discussed earlier, our approach is to ﬁrst reduce scanning time by ﬁnding the minimum N∗and then reduce computation time by ﬁnding the minimum θ ∗. A parameter sweep over N is shown qualitatively in Fig. 2. Four quantitative performance comparisons are shown in Fig. 3, in terms of the following metrics: Pearson correlation coefﬁcient (PCC)33, multi-scale structural similarity index metric (MS-SSIM)34, Dice Similarity coefﬁcient (DSC)35, and Bit-Error Rate (BER, more details available in Methods). Both analyses indicate that N∗∼29, representing a reduction of more than ×12 over the gold standard of N = 349. Reducing N signiﬁcantly below this value results in noticeable degradation, both qualitatively 3/18 Discussion APT is trained using the gold standard reconstructions of randomly selected segments from a single IC specimen, which was made available for our experiments. This prompts us to address two related concerns: (1) what can we guarantee about ﬁdelity of the gold standard and, hence, our reconstructions vis-à-vis the ground truth, i.e. the physical specimen? and (2) is our APT overtrained to this speciﬁc IC? 4/18 The ﬁrst concern was partially addressed by Refs.31,32, where the design ﬁles of the geometrical features were treated as ground truth. (That method was still bound by the assumption that the physical specimen matched the design ﬁles; but that was less of a concern, given the size scales involved.) Neither of these algorithms would have worked in the case reported here, because of the great range of feature sizes in the specimen and because the synchrotron X-ray is not spatially coherent. Moreover, the design ﬁles for the specimen are not available to us. On the other hand, the gold standard was obtained quite thoroughly with 95% spatial overlap factor in the ptycho-scan and N = 349 angles in the tomo-scan. Besides, there are no discernible visual artifacts in the gold standard reconstructions. These facts provide us with reasonable assurance about the fairness of our comparisons in Figs. 2-6. Regarding the second concern: if new structures are given where the priors are signiﬁcantly different than the priors learnt here (e.g. features oriented at 45◦) then APT would have to be retrained. This is a necessary limitation of our supervised learning approach. The same holds for non-IC objects such as viruses. If, moreover, not enough physical specimens are available for supervised training, then it is possible to train by rigorously simulating the forward propagation of X-rays through the specimen (as Refs.31,32 did for visible light) or use “untrained” methods, such as deep image prior21,41. The reported best values of N∗∼29 and θ ⋆∼±17◦are not fundamental, but indicative of the effectiveness of IC geometries acting as regularizing prior. Less complex geometries, smooth and with less content at high frequencies in the missing wedge, could achieve even better reductions, whereas complex structures with smaller features and higher refractive index contrast would be more limited. A full theoretical analysis of how N∗and θ ∗depend on the complexity of the prior is beyond the scope of this work. Discussion Lastly, regarding ICs in particular and planar samples more generally, the total attenuation of the X-rays increases at large angles, which leads to artifacts. It may be compensated computationally, or by scanning the illumination wavevectors on a conical surface. The latter scheme is referred to as laminography42,43. It is beyond the scope of our present work, but it would be interesting to investigate if approaches similar to the one reported here are applicable. Gradient calculation Considering the mixed-state (spatially partially coherent) nature of synchrotron X-rays and multi-slice structure of the IC sample, a forward model can be formulated as Considering the mixed-state (spatially partially coherent) nature of synchrotron X-rays and multi-slice structure of the IC sample, a forward model can be formulated as ψ(n)[L] j,r,m = O(n)[L] r P∆z h O(n)[L−1] r P∆z h ···P∆z h Pr-rj,mO(n)[1] r iii , (2) I(n) j,q = M ∑ m=1 ˜ψ(n)[L] j,q,m 2 = M ∑ m=1 F h ψ(n)[L] j,r,m i 2 , (3) (2) (3) where - n: the index of tomo-scans (n = 1,2,··· ,N) - j: the index of ptycho-scans ( j = 1,2,··· ,Jn) - l: the index of multi-slices (l = 1,2,··· ,L) - m: the index of mixed-states (m = 1,2,··· ,M) - n: the index of tomo-scans (n = 1,2,··· ,N) - j: the index of ptycho-scans ( j = 1,2,··· ,Jn) - l: the index of multi-slices (l = 1,2,··· ,L) - m: the index of mixed-states (m = 1,2,··· ,M) - n: the index of tomo-scans (n = 1,2,··· ,N) - j: the index of ptycho-scans ( j = 1,2,··· ,Jn) - l: the index of multi-slices (l = 1,2,··· ,L) - m: the index of mixed-states (m = 1,2,··· ,M) - n: the index of tomo-scans (n = 1,2,··· ,N) - n: the index of tomo-scans (n = 1,2,··· ,N) - j: the index of ptycho-scans ( j = 1,2,··· ,Jn) - j: the index of ptycho-scans ( j = 1,2,··· ,Jn) - j: the index of ptycho-scans ( j = 1,2,··· ,Jn) - l: the index of multi-slices (l = 1,2,··· ,L) - l: the index of multi-slices (l = 1,2,··· ,L) - m: the index of mixed-states (m = 1,2,··· ,M) - m: the index of mixed-states (m = 1,2,··· ,M) - r: the spatial domain coordinates - q: the reciprocal domain coordinates - q: the reciprocal domain coordinates - Pr,m: the m-th coherent mode of the synchrotron X-ray probe - O(n)[l] r : the l-th slice of the object viewed at the n-th tomo-scan. - O(n)[l] r : the l-th slice of the object viewed at the n-th tomo-scan. - O(n)[l] r : the l-th slice of the object viewed at the n-th tomo-scan. The following describes the gradient computation of the loss function in Eq. Experiment and the gold standard preparation Integrated circuits produced with 16-nm technology of size 25.1 × 93.2 × 3.92 µm3 were laterally scanned with synchrotron X-rays of 8.8keV for each tomo scan with Velociprobe19 at the Advanced Photon Source (APS) of the Argonne National Laboratory (ANL). 12 coherent modes of the synchrotron X-ray were used for the experiment. Tomo-scans were carried out from -70.4◦to 70.4◦with angular increment of 0.4◦, and for each tomo-scan, ptycho- scans were recorded on-the-ﬂy at ∼60k lateral positions with Dectris Eiger X 500K area detector (pixel size: 75 µm, sample-to-detector distance: 1.92m) at a frame rate of 500Hz. Elapsed time of this whole data acquisition process (translational and angular) was 12.51hrs, or 129 seconds per tomo-scan. As a ﬁrst step to obtain the gold standard reconstruction, a two-dimensional projection was reconstructed for each tomo-scan with 600 iterations of the least-square maximum likelihood ptychographic algorithm44 as implemented in PtychoShelves45. The ptychographic reconstruction for all 349 tomo-scans was processed with 8 Tesla V100 GPUs in parallel to expedite the process, thus taking 362.09hrs for this step. Then, the projections were aligned to a tomographic rotation axis with an additional correction in the form of a phase ramp removal process. Then, a deep neural network pre-trained on similar images of integrated circuits was applied to the aligned projections for upsampling by ×246,47. The elapsed time of this step was approximately 5hrs. Then, the projections were aligned to a tomographic rotation axis with an additional correction in the form of a phase ramp removal process. Then, a deep neural network pre-trained on similar images of integrated circuits was applied to the aligned projections for upsampling by ×246,47. The elapsed time of this step was approximately 5hrs. Lastly, the ﬁnal tomographic reconstruction was performed using 349 upsampled projections with 10 iterations of SART to generate a ﬁnally three-dimensional reconstruction of the IC sample with the isotropic 14-nm voxel size, which took 1hr with 8 Tesla V100 GPUs. Lastly, the ﬁnal tomographic reconstruction was performed using 349 upsampled projections with 10 iterations of SART to generate a ﬁnally three-dimensional reconstruction of the IC sample with the isotropic 14-nm voxel size, which took 1hr with 8 Tesla V100 GPUs. 5/18 Gradient calculation 1 based on the forward model, which was done automatically with Ptychoshelves45. The gradients of the loss function with respect to the waveﬁeld and the complex object are ∂L ∂P(n)[l] j,r,m =        O(n)[l] r P−∆z ( ∂L ∂P(n)[l+1]∗ j,r,m )!∗ for 1 ≤l < L (4a) O(n)[L] r χ(n)[L]∗ j,r,m for l = L, (4b) and ∂L ∂O(n)[l] r =              Jn ∑ j=1 M ∑ m=1 P(n)[l] j,r,m P−∆z ( ∂L ∂P(n)[l+1]∗ j,r,m )!∗ for 1 ≤l < L (5a) Jn ∑ j=1 M ∑ m=1 P(n)[L] j,r,m χ(n)[L]∗ j,r,m for l = L, (5b) ∂L ∂P(n)[l] j,r,m =        O(n)[l] r P−∆z ( ∂L ∂P(n)[l+1]∗ j,r,m )!∗ for 1 ≤l < L (4a) O(n)[L] r χ(n)[L]∗ j,r,m for l = L, (4b) (4a) (4b) and ∂L ∂O(n)[l] r =              Jn ∑ j=1 M ∑ m=1 P(n)[l] j,r,m P−∆z ( ∂L ∂P(n)[l+1]∗ j,r,m )!∗ for 1 ≤l < L (5a) Jn ∑ j=1 M ∑ m=1 P(n)[L] j,r,m χ(n)[L]∗ j,r,m for l = L, (5b) n)[l] ,r,m P−∆z ( ∂L ∂P(n)[l+1]∗ j,r,m )!∗ for 1 ≤l < L (5a) (5a) ∂O(n)[l] r        Jn ∑ j=1 M ∑ m=1 P(n)[L] j,r,m χ(n)[L]∗ j,r,m for l = L, (5b) (5b) where P(n)[l] j,r,m = P∆z h O(n)[l−1] r P(n)[l−1] j,r,m i , (6) ∂L ∂Pr,m = N ∑ n=1 Jn ∑ j=1 ∂L ∂P(n)[1] j,r+rj,m , (7) χ(n)[L] j,r,m = F −1     1− q I(n) j,q ˜ψ(n)[L] j,q,m  ˜ψ(n)[L] j,q,m   . (8) (6) (8) With two iterations of gradient descent on the loss function in Eq. 1, we obtain the multi-slice estimate O(n)[l] r \|Niter=2 for each tomo-scan and subsequently its argument at each one of the L = 5 slices. For the ﬁnal Approximant, we rotate the results back to the original coordinate system, and average N estimations from all N tomo-scans. Please see Supplementary Materials for visualization. More details on the gradient calculation can be found in Refs.38,40. Machine learning framework Our neural network architecture is based on a 3D U-net structure27,28 augmented with multi-head axial self-attention (“axial self-attention” in short)29. The U-net directly transfers multi-scale encoded features to its decoder arm to preserve spatial information, and the axial attention augments the features with its global-range self-interactions. The U-net backbone encoder design was inﬂuenced by the well-established architecture ResNet5048 with some modiﬁcations so that it can accommodate 3D instead of 2D data. The architecture’s decoder then upsamples the features to result in isotropic voxels of linear size 14nm. More details can be found in Supplementary Materials. The encoder’s low-dimensional manifolds are further enhanced by the axial self-attention which was proposed in order to reduce the computational complexity of multi-head self-attention (“self-attention” in short)30. The axial self-attention factorizes 3D self-attention into three 1D axial self-attention modules, thus reducing the complexity from O(N3) to O(3N). Each axial self-attention attends to voxels along one of x,y,z axes. Fig. 7 visualizes learned attention weights pi j that quantiﬁes normalized “contribution” of other layers sj (j = 1,··· ,N) to the layer si. We assume that the information of layer si is spread along the layers sj ( j = 1,··· ,N) due to lack of spatial resolution; therefore, the axial self-attention gathers the scattered information from the layers to resolve layer si with global- range interactions. Note that in this paper, we used Pytorch instead of the original Tensorﬂow implementation29, and our code should be publicly available in https://github.com/iksungk/APT. Gradient calculation The data were pre-processed with multi-slice ptychography in order to properly address the sample at larger angles, where 2D ptychography may not be suitable since the effective optical path length becomes thicker than the depth of focus of the probe. The depth of focus of the probe is approximately λ 2(NA)2 = 2.82 µm. When the sample is rotated by the largest angle for tomographic scanning, i.e. 70.4◦, the optical path length increases up to 3.92 cos(70.4◦) = 11.7 µm ≃4.46DOF, so 5 slices should be sufﬁcient to address the rotated sample. Quantitative metrics Because each voxel on an IC is occupied by a single material, even if ICs are printed with various materials such as copper, aluminum, and tungsten, ICs can be comfortably classiﬁed into M-ary labels irrespective of the printing material. To further simplify, we binarize the gold standard by thresholding according to the presence of a metal or silicon within each voxel. The gold standard reconstruction, however, may still be ambiguous especially for longitudinal features due to the missing wedge in the Fourier domain as it still does not cover the entire angular range, i.e. ±90◦, due to the tomographic scheme. Since the gold standard also suffers from extensive errors in these ambiguous layers, we exclude them from our quantitative evaluations as well. More details can be found in Supplementary Materials. The quantitative comparisons in Figs. 3 and 5 use four different quantitative metrics to illustrate different aspects of the reconstructions. The ﬁrst two are correlative metrics: the PCC49 and the MS-SSIM with the same weights as in the original reference34. The remaining two metrics are the DSC35 and the BER. The former is a widely accepted similarity measure in image segmentation to compare an algorithm output against its reference in medical applications53,54. The BER measures the ratio of erroneously classiﬁed voxels over the total voxels, and it is allowable because of our binarization approach. Both of these metrics are probabilistic in the sense that they involve the estimation of probability density functions. They are obtained as DSC = 2·TP 2·TP+FN+FP, (10) BER = FP+FN TP+TN+FP+FN, (11) (10) and (11) where TP, TN, FP, and FN indicate the number of true positives, true negatives, false positives, and false negatives, respectively. For the gold standard, the binary thresholds and prior probabilities p(0), p(1) required for these quantities were estimated by an Expectation Maximization (EM) procedure. For the tests, we used Bayes’ rule p(x\|0)p(0) = p(x\|1)p(1) with p(0), p(1) same as for the gold standard. where TP, TN, FP, and FN indicate the number of true positives, true negatives, false positives, and false negatives, respectively. For the gold standard, the binary thresholds and prior probabilities p(0), p(1) required for these quantities were estimated by an Expectation Maximization (EM) procedure. For the tests, we used Bayes’ rule p(x\|0)p(0) = p(x\|1)p(1) with p(0), p(1) same as for the gold standard. Acknowledgments We are grateful to Jung Ki Song, Mo Deng, Baoliang Ge, William Harrod, Ed Cole, Zachary Levine, Bradley Alpert, Nina Weisse-Bernstein, Lee Oesterling and Antonio Orozco for helpful discussions and comments. Funding from the Intelligence Advanced Research Projects Activity, Ofﬁce of the Director of National Intelligence (IARPA-ODNI), contract FA8650-17-C-9113 is gratefully acknowledged. The MIT SuperCloud and Lincoln Laboratory Supercom- puting Center provided resources (high performance computing, database, consultation) that have contributed to the research results reported within this paper. I. Kang acknowledges support from Korea Foundation for Advanced Studies (KFAS). This research used resources of the Advanced Photon Source, a U.S. Department of Energy (DOE) Ofﬁce of Science User Facility, operated for the DOE Ofﬁce of Science by Argonne National Laboratory under Contract No. DE-AC02-06CH11357. The views and conclusions contained herein are those of the authors and should not be interpreted as necessarily representing the ofﬁcial policies or endorsements, either expressed or implied, of the ODNI, IARPA or the US Government. Training and testing environments To prepare a paired dataset for training and testing, both of the Approximant and the gold standard are divided into smaller volumes of 1.792×1.792×3.92 µm3 with 50% lateral overlap. Then, we split the paired dataset into two non-overlapping sub-datasets. One set is reserved for training, and the other for testing. The training and test samples were drawn so as to not be correlated accidentally by spatial overlap during the ptycho- and tomo-scan operations. For training, we use negative Pearson Correlation coefﬁcient (NPCC) as the training loss function31,32,49 and the Adam optimizer for stochastic gradient descent optimization50 with initial learning rate of 2×10−4, β1 = 0.9, β2 = 0.999, and without weight decay. We also update the learning rate schedule according to a polynomial rule51 as lr(epoch) = lr(0)× 1−epoch T p , (9) (9) where T = 200 and p = 0.9. We run the training process for 150 epochs and stabilize it by a mini-batch learning strategy52 with batch size equal to 4. Upon completion of the training process, the network is loaded and ﬁxed with the optimal weights, and used to reconstruct the test volume (4.48×93.2×3.92 µm3), as shown in Figs. 2, 4 and 6. For all computational procedures, i.e. pre-processing and training & testing processes, we used the MIT Supercloud with a Intel Xeon Gold 6248 CPU with 384 GB RAM and dual NVIDIA Volta V100 GPUs with 32 GB 7/18 VRAM. Once the network was trained, it took 45 seconds to generate the reconstruction over the test volume. References 1. Hoppe, W. Beugung im inhomogenen primärstrahlwellenfeld. i. prinzip einer phasenmessung von elektronen- beungungsinterferenzen. Acta Crystallogr. Sect. A: Cryst. Physics, Diffraction, Theor. Gen. Crystallogr. 25, 495–501 (1969). 2. Hegerl, R. & Hoppe, W. Dynamische Theorie der Kristallstrukturanalyse durch Elektronenbeugung im inhomogenen Primärstrahlwellenfeld. Berichte der Bunsengesellschaft für physikalische Chemie 74, 1148–1154 (1970). 3. Nellist, P., McCallum, B. & Rodenburg, J. M. Resolution beyond the information limit in transmission electron microscopy. Nature 374, 630–632 (1995). 4. Gerchberg, R. W. A practical algorithm for the determination of phase from image and diffraction plane pictures. Optik 35, 237–246 (1972). 5. Faulkner, H. M. L. & Rodenburg, J. Movable aperture lensless transmission microscopy: a novel phase retrieval algorithm. Phys. review letters 93, 023903 (2004). 6. Rodenburg, J. M. & Faulkner, H. M. A phase retrieval algorithm for shifting illumination. Appl. Phys. Lett. 85, 4795–4797 (2004). et al. High-resolution scanning X-ray diffraction microscopy. Science 321, 379–382 (2008 7. Thibault, P. et al. High-resolution scanning X-ray diffraction microscopy. Science 321, 8. Thibault, P. & Guizar-Sicairos, M. Maximum-likelihood reﬁnement for coherent diffractive imaging. New J. Phys. 14, 063004 (2012). 9. Radon, J. On the determination of functions from their integrals along certain manifolds. Ber. Verh, Sachs Akad Wiss. 69, 262–277 (1917). 10. Radon, J. On the determination of functions from their integral values along certain manifolds. IEEE transactions on medical imaging 5, 170–176 (1986). 11. Bracewell, R. N. Strip integration in radio astronomy. Aust. J. Phys. 9, 198–217 (1956). 12. Bracewell, R. N. & Riddle, A. Inversion of fan-beam scans in radio astronomy. The Astrophys. J. 150, 427 (1967). 13. Gordon, R., Bender, R. & Herman, G. T. Algebraic reconstruction techniques (ART) for three-dimensional electron microscopy and x-ray photography. J. Theor. Biol. 29, 471–481 (1970). 14. Gilbert, P. Iterative methods for the three-dimensional reconstruction of an object from projections. J. theoretical biology 36, 105–117 (1972). 15. Andersen, A. H. & Kak, A. C. Simultaneous algebraic reconstruction technique (SART): a superior implemen- tation of the art algorithm. Ultrason. Imaging 6, 81–94 (1984). 16. Bouman, C. & Sauer, K. A generalized gaussian image model for edge-preserving map estimation. IEEE Transactions on image processing 2, 296–310 (1993). 7. Dierolf, M. et al. Ptychographic X-ray computed tomography at the nanoscale. Nature 17. Dierolf, M. et al. Ptychographic X-ray computed tomography at the nanoscale. Nature 467, 436–439 (2010). 18. Holler, M. et al. High-resolution non-destructive three-dimensional imaging of integrated circuits. Competing interests The authors declare no competing interests. 8/18 References Nature 543, 402–406 (2017). 19. Deng, J. et al. The velociprobe: An ultrafast hard X-ray nanoprobe for high-resolution ptychographic imaging. Rev. Sci. Instruments 90, 083701 (2019). 20. Huang, X. et al. Fly-scan ptychography. Sci. Reports 5, 1–5 (2015). 21. Ulyanov, D., Vedaldi, A. & Lempitsky, V. Deep image prior. In Proceedings of the IEEE conference on computer vision and pattern recognition, 9446–9454 (2018). 9/18 22. Mataev, G., Milanfar, P. & Elad, M. Deepred: Deep image prior powered by red. In Proceedings of the IEEE/CVF International Conference on Computer Vision Workshops, 0–0 (2019). 23. Kappeler, A., Ghosh, S., Holloway, J., Cossairt, O. & Katsaggelos, A. Ptychnet: CNN based fourier ptychogra- phy. In 2017 IEEE International Conference on Image Processing (ICIP), 1712–1716 (IEEE, 2017). 24. Nguyen, T., Xue, Y., Li, Y., Tian, L. & Nehmetallah, G. Deep learning approach for fourier ptychography microscopy. Opt. express 26, 26470–26484 (2018). 25. Cherukara, M. J. et al. Ai-enabled high-resolution scanning coherent diffraction imaging. Appl. Phys. Lett. 117, 044103 (2020). 26. Goy, A., Arthur, K., Li, S. & Barbastathis, G. Low photon count phase retrieval using deep learning. Phys. Rev. Lett. 121, 243902 (2018). 27. Ronneberger, O., Fischer, P. & Brox, T. U-net: Convolutional networks for biomedical image segmentation. In International Conference on Medical Image Computing and Computer-Assisted Intervention, 234–241 (Springer, 2015). 28. Çiçek, Ö., Abdulkadir, A., Lienkamp, S. S., Brox, T. & Ronneberger, O. 3D U-Net: learning dense volumetric segmentation from sparse annotation. In International Conference on Medical Image Computing and Computer- Assisted Intervention, 424–432 (Springer, 2016). 29. Wang, H. et al. Axial-deeplab: Stand-alone axial-attention for panoptic segmentation. In European Conference on Computer Vision, 108–126 (Springer, 2020). 30. Vaswani, A. et al. Attention is all you need. arXiv preprint arXiv:1706.03762 (2017). 31. Goy, A. et al. High-resolution limited-angle phase tomography of dense layered objects using deep neural networks. Proc. Natl. Acad. Sci. 116, 19848–19856 (2019). 32. Kang, I., Goy, A. & Barbastathis, G. Dynamical machine learning volumetric reconstruction of objects’ interiors from limited angular views. Light. Sci. & Appl. 10 (2021). 33. Benesty, J., Chen, J., Huang, Y. & Cohen, I. Pearson correlation coefﬁcient. In Noise Reduction in Speech Processing, 1–4 (Springer, 2009). 34. Wang, Z., Simoncelli, E. P. & Bovik, A. C. Multiscale structural similarity for image quality assessment. In The Thrity-Seventh Asilomar Conference on Signals, Systems & Computers, 2003, vol. 2, 1398–1402 (Ieee, 2003). 5. Dice, L. R. References Measures of the amount of ecologic association between species. Ecology 2 36. Wolf, E. New theory of partial coherence in the space–frequency domain. part i: spectra and cross spectra of steady-state sources. JOSA 72, 343–351 (1982). 37. Thibault, P. & Menzel, A. Reconstructing state mixtures from diffraction measurements. Nature 494, 68–71 (2013). 38. Tsai, E. H., Usov, I., Diaz, A., Menzel, A. & Guizar-Sicairos, M. X-ray ptychography with extended depth of ﬁeld. Opt. Express 24, 29089–29108 (2016). 39. Suzuki, A. et al. High-resolution multislice X-ray ptychography of extended thick objects. Phys. Rev. Lett. 112, 053903 (2014). 40. Chen, Z. et al. Electron ptychography achieves atomic-resolution limits set by lattice vibrations. Science 372, 826–831, DOI: 10.1126/science.abg2533 (2021). https://science.sciencemag.org/content/372/6544/826.full.pdf. 41. Baguer, D. O., Leuschner, J. & Schmidt, M. Computed tomography reconstruction using deep image prior and learned reconstruction methods. Inverse Probl. 36, 094004 (2020). 42. Helfen, L. et al. High-resolution three-dimensional imaging of ﬂat objects by synchrotron-radiation computed laminography. Appl. Phys. Lett. 86, 071915 (2005). 10/18 43. Holler, M. et al. Three-dimensional imaging of integrated circuits with macro-to nanoscale zoom. Nat. Electron. 2, 464–470 (2019). 44. Odstrˇcil, M., Menzel, A. & Guizar-Sicairos, M. Iterative least-squares solver for generalized maximum- likelihood ptychography. Opt. Express 26, 3108–3123 (2018). 45. Wakonig, K. et al. Ptychoshelves, a versatile high-level framework for high-performance analysis of ptycho- graphic data. J. Appl. Crystallogr. 53 (2020). 46. Dong, C., Loy, C. C., He, K. & Tang, X. Learning a deep convolutional network for image super-resolution. In European conference on computer vision, 184–199 (Springer, 2014). 47. Dong, C., Loy, C. C., He, K. & Tang, X. Image super-resolution using deep convolutional networks. IEEE transactions on pattern analysis machine intelligence 38, 295–307 (2015). 48. He, K., Zhang, X., Ren, S. & Sun, J. Deep residual learning for image recognition. In Proceedings of the IEEE Conference on Computer Vision and Pattern Recognition, 770–778 (2016). 49. Li, S., Deng, M., Lee, J., Sinha, A. & Barbastathis, G. Imaging through glass diffusers using densely connected convolutional networks. Optica 5, 803–813 (2018). 0. Kingma, D. P. & Ba, J. Adam: A method for stochastic optimization. arXiv preprint arX 50. Kingma, D. P. & Ba, J. Adam: A method for stochastic optimization. arXiv preprint arXiv:1412.6980 (2014). 51. Cheng, B. et al. Panoptic-deeplab: A simple, strong, and fast baseline for bottom-up panoptic segmentation. In Proceedings of the IEEE/CVF Conference on Computer Vision and Pattern Recognition, 12475–12485 (2020). 52. 54. Zou, K. H. et al. Statistical validation of image segmentation quality based on a spatial overlap index1: scientiﬁc reports. Acad. Radiol. 11, 178–189 (2004). References Li, M., Zhang, T., Chen, Y. & Smola, A. J. Efﬁcient mini-batch training for stochastic optimization. In Proceedings of the 20th ACM SIGKDD international conference on Knowledge discovery and data mining, 661–670 (2014). 53. Milletari, F., Navab, N. & Ahmadi, S.-A. V-net: Fully convolutional neural networks for volumetric medical image segmentation. In 2016 fourth International Conference on 3D Vision (3DV), 565–571 (IEEE, 2016). 54. Zou, K. H. et al. Statistical validation of image segmentation quality based on a spatial overlap index1: scientiﬁc reports. Acad. Radiol. 11, 178–189 (2004). 11/18 Figures a c ptycho scans tomo scans Raw image (far-field) b Synchrotron X-rays Gold standard (Iterative baseline) ML framework (APT) Full-number, full-range tomo-scans (1) reduce number of tomo-scans (2) limit the range of tomo-scans Optimized tomo-scans Training Convolutional encoder Convolutional decoder Axial self-attention APT Prediction Ground truth Training loss function: Update weights Validation / Testing Convolutional encoder Convolutional decoder Axial self-attention APT (fixed weights) Load & fix best trained weights ML reconstruction Assemble outputs Pre-processing Approximant tomo-scan #1 tomo-scan #N ... ... ... (J1 ptycho-scans) Mixed-state, multi-slice ptychography (L slices) Mixed-state, multi-slice ptychography (L slices) N x Rotate & Sum ... (JN ptycho-scans) Approximate inverse operator LNPCC N θ 349 N = 28.89 Gold standard Optimized tomo-scans θ* = ± 16.93∘ ±70.4∘ (1) (2) Figure 1. X-ray ptycho-tomography and the implementation of APT. (a) Brief schematic of X-ray ptycho-tomography geometry with translational scanning of synchrotron X-rays (ptycho-scans) and symmetric angular scanning of the IC sample with uniform angular increment (tomo-scans). (b) Gold standard uses 349 tomo-scans within the angular range of ±70.4◦, but our machine learning framework (APT) uses fewer tomo-scans optimized through two steps. (c) Diffraction intensities are pre-processed with an approximate inverse operator to generate the Approximant (and more details can be found in Methods and Supplementary Materials.) One of two non-overlapping portions of the Approximant is used for training with a negative Pearson correlation coefﬁcient (NPCC) as the training loss function, where network weights are updated over several training epochs. For testing, best trained weights are loaded and ﬁxed to generate outputs over the test volume (4.48×93.2×3.92 µm3). a c ptycho scans tomo scans Raw image (far-field) b Synchrotron X-rays Gold standard (Iterative baseline) ML framework (APT) Full-number, full-range tomo-scans (1) reduce number of tomo-scans (2) limit the range of tomo-scans Optimized tomo-scans Pre-processing Approximant tomo-scan #1 tomo-scan #N ... ... ... References (J1 ptycho-scans) Mixed-state, multi-slice ptychography (L slices) Mixed-state, multi-slice ptychography (L slices) N x Rotate & Sum ... (JN ptycho-scans) Approximate inverse operator N θ 349 N* = 28.89 Gold standard Optimized tomo-scans θ* = ± 16.93∘ ±70.4∘ (1) (2) a ptycho scans tomo scans Raw image (far-field) Synchrotron X-rays Synchrotron X-rays c Pre-processing tomo-scan #1 tomo-scan #N ... ... ... (J1 ptycho-scans) Mixed-state, multi-slice ptychography (L slices) Mixed-state, multi-slice ptychography (L slices) N x Rotate & Sum ... (JN ptycho-scans) Approximate inverse operator c Pre-processing c Pre-processing Approximant Training Convolutional encoder Convolutional decoder Axial self-attention APT Prediction Ground truth Training loss function: Update weights Validation / Testing Convolutional encoder Convolutional decoder Axial self-attention APT (fixed weights) Load & fix best trained weights ML reconstruction Assemble outputs LNPCC Training Training Ground truth Prediction Ground truth Validation / Testing Convolutional encoder Convolutional decoder Axial self-attention APT (fixed weights) ML reconstruction Assemble outputs ML reconstruction ML reconstruction cho-tomography and the implementation of APT. (a) Brief schematic of X-ray Figure 1. X-ray ptycho-tomography and the implementation of APT. (a) Brief schem Figure 1. X-ray ptycho-tomography and the implementation of APT. (a) Brief schematic of X-ray ptycho-tomography geometry with translational scanning of synchrotron X-rays (ptycho-scans) and symmetric angular scanning of the IC sample with uniform angular increment (tomo-scans). (b) Gold standard uses 349 tomo-scans within the angular range of ±70.4◦, but our machine learning framework (APT) uses fewer tomo-scans optimized through two steps. (c) Diffraction intensities are pre-processed with an approximate inverse operator to generate the Approximant (and more details can be found in Methods and Supplementary Materials.) One of two non-overlapping portions of the Approximant is used for training with a negative Pearson correlation coefﬁcient (NPCC) as the training loss function, where network weights are updated over several training epochs. For testing, best trained weights are loaded and ﬁxed to generate outputs over the test volume (4.48×93.2×3.92 µm3). 12/18 Gold standard N = 5 N = 11 N = 21 N = 43 N = 86 N = 174 N = 349 Number of tomo-scans (N) ML (APT) reconstruction Zoom Gold standard N = 5 N = 11 N = 21 N = 43 N = 86 N = 174 N = 349 a b 1 µm 1 µm y x z x Number of tomo-scans (N) Figure 2. Optimizing the number of tomo-scans - qualitative view. References Qualitative comparison from a parameter sweep over the number of tomo-scans (N) at two different depths: (a) z = 0.364 µm and (b) y = 0.532 µm. The ﬁgure shows APT reconstructions with different N over the test volume (4.48×93.2×3.92 µm3). Gold standard N = 5 N = 11 N = 21 N = 43 N = 86 N = 174 N = 349 Number of tomo-scans (N) ML (APT) reconstruction Zoom a 1 µm y x ML (APT) reconstruction ML (APT) reconstruction a N = 86 Numbe Figure 2. Optimizing the number of tomo-scans - qualitative view. Qualitative comparison from a parameter sweep over the number of tomo-scans (N) at two different depths: (a) z = 0.364 µm and (b) y = 0.532 µm. The ﬁgure shows APT reconstructions with different N over the test volume (4.48×93.2×3.92 µm3). 13/18 a PCC MS-SSIM DSC 1 - BER 1 0.99 0.98 0.96 0.95 0.97 0.94 0.9 0.88 0.92 0.97 0.96 0.94 0.92 t = 0.8136 hrs, N* = 29.54 PCC Fit t = 0.7952 hrs, N* = 28.86 DSC Fit t = 0.7654 hrs, N* = 27.76 MS-SSIM Fit t = 0.8100 hrs, N* = 29.41 1 - BER Fit Data acquisition + computation time (hrs) 0 2 4 6 8 0 2 4 6 8 0 2 4 6 8 0 2 4 6 8 Data acquisition + computation time (hrs) Data acquisition + computation time (hrs) Data acquisition + computation time (hrs) a N = 86 N = 43 N = 349 N = 174 N = 21 N = 11 N = 5 Number of tomo-scans (N) b 1 0.98 0.96 0.94 0.92 0.9 0.96 0.94 0.9 0.88 0.92 topt 0.88 0.86 0.95 0.93 Figure 3. Optimizing the number of tomo-scans - quantitative view. (a) Quantitative comparison from a parameter sweep over the number of tomo-scans (N) with four different quantitative metrics. (b) The number of tomo-scans that optimally compromise the performance (N∗) is 28.89 in average, where APT reduces the data acquisition and computation time by a factor of 85. References PCC MS-SSIM DSC 1 - BER a N = 86 N = 43 N = 349 N = 174 N = 21 N = 11 N = 5 Number of tomo-scans (N) 1 0.98 0.96 0.94 0.92 0.9 0.88 0.86 1 0.99 0.98 0.96 0.95 0.97 0.94 0.9 0.88 0.92 t = 0.8136 hrs, N* = 29.54 PCC Fit t = 0.7952 hrs, N* = 28.86 DSC Fit Data acquisition + computation time (hrs) 0 2 4 6 8 0 2 4 6 8 Data acquisition + computation time (hrs) b topt b 0.97 0.96 0.94 0.92 t = 0.7654 hrs, N* = 27.76 MS-SSIM Fit t = 0.8100 hrs, N* = 29.41 1 - BER Fit 0 2 4 6 8 0 2 4 6 8 Data acquisition + computation time (hrs) Data acquisition + computation time (hrs) 0.96 0.94 0.9 0.88 0.92 0.95 0.93 Figure 3. Optimizing the number of tomo-scans - quantitative view. (a) Quantitative comparison from a parameter sweep over the number of tomo-scans (N) with four different quantitative metrics. (b) The number of tomo-scans that optimally compromise the performance (N∗) is 28.89 in average, where APT reduces the data acquisition and computation time by a factor of 85. 14/18 Gold standard ±5.6 o ±11.2 o ±16.8 o ±22.4 o ±33.6 o ±44.8 o ±56 o Angular range (θ) ML (APT) reconstruction Zoom a 1 µm y x a ±56 o 1 µm y x ±5.6 o ±11.2 o ±16.8 o ±22.4 o ±33.6 o ±44.8 o ±56 o Angular range (θ) Gold standard b 1 µm z x Figure 4. Optimizing the number of angular range - qualitative view. Qualitative comparison from a parameter sweep over the angular scanning range at two different depths: (a) z = 1.092 µm and (b) y = 4.186 µm. The ﬁgure shows APT reconstructions over the test volume (4.48×93.2×3.92 µm3). Gold standard b ±5.6 o ±11.2 o ±16.8 o e (θ) ±22.4 o gular range ±33.6 o Ang ±33.6 o Ang ±44.8 o ±44.8 o ±56 o 1 µm z x Figure 4. Optimizing the number of angular range - qualitative view. Qualitative comparison from a parameter sweep over the angular scanning range at two different depths: (a) z = 1.092 µm and (b) y = 4.186 µm. The ﬁgure shows APT reconstructions over the test volume (4.48×93.2×3.92 µm3). Figure 4. References Optimizing the number of angular range - qualitative view. Qualitative comparison from a parameter sweep over the angular scanning range at two different depths: (a) z = 1.092 µm and (b) y = 4.186 µm. The ﬁgure shows APT reconstructions over the test volume (4.48×93.2×3.92 µm3). 15/18 PCC MS-SSIM DSC 1 - BER 1 0.99 0.98 0.96 0.95 0.97 0.94 0.9 0.88 0.92 0.97 0.96 0.94 0.92 t = 0.6360 hrs, θ* = 17.24 o PCC Fit t = 0.6332 hrs, θ* = 16.32 o DSC Fit t = 0.6396 hrs, θ* = 18.42 o MS-SSIM Fit t = 0.6313 hrs, θ* = 15.72 o 1 - BER Fit Data acquisition + computation time (hrs) 0.6 0.62 0.64 0.7 0.72 0.68 0.66 0.74 0.76 0.6 0.62 0.64 0.7 0.72 0.68 0.66 0.74 0.76 Data acquisition + computation time (hrs) Data acquisition + computation time (hrs) Data acquisition + computation time (hrs) a b 0.98 1 0.96 0.94 0.92 0.9 0.96 0.94 0.9 0.88 0.92 topt 0.88 0.95 0.93 ±16.8 o ±11.2 o ±22.4 o ±33.6 o ±44.8 o ±56 o ±5.6 o Angular range (θ) 0.6 0.62 0.64 0.7 0.72 0.68 0.66 0.74 0.76 0.6 0.62 0.64 0.7 0.72 0.68 0.66 0.74 0.76 Figure 5. Optimizing number of angular range - quantitative view. (a) Quantitative comparison from a parameter sweep over the angular scanning range at N = N∗(= 29). (b) The total angular range that optimally compromise the performance (θ ∗) is θ ∗= ±16.93◦in average. APT decreases the time for whole process by 108 times. PCC MS-SSIM DSC 1 - BER a 0.98 1 0.96 0.94 0.92 0.9 0.88 ±16.8 o ±11.2 o ±22.4 o ±33.6 o ±44.8 o ±56 o ±5.6 o Angular range (θ) ±56 o b 0.97 0.96 0.94 0.92 t = 0.6396 hrs, θ* = 18.42 o MS-SSIM Fit t = 0.6313 hrs, θ* = 15.72 o 1 - BER Fit 0.6 0.62 0.64 0.7 0.72 0.68 0.66 0.74 0.76 Data acquisition + computation time (hrs) Data acquisition + computation time (hrs) 0.96 0.94 0.9 0.88 0.92 0.95 0.93 0.6 0.62 0.64 0.7 0.72 0.68 0.66 0.74 0.76 Figure 5. Optimizing number of angular range - quantitative view. (a) Quantitative comparison from a parameter sweep over the angular scanning range at N = N∗(= 29). (b) The total angular range that optimally compromise the performance (θ ∗) is θ ∗= ±16.93◦in average. References APT decreases the time for whole process by 108 times. 16/18 0 Normalized frequency (kz) -0.5 0.5 Normalized frequency (kx) 0 -0.5 0.5 0 -0.5 0.5 0 -0.5 0.5 0 -0.5 0.5 0 -0.5 0.5 0 -0.5 0.5 0 -0.5 0.5 0 -0.5 0.5 0 -0.5 0.5 kz ky kx a b 0 -2 -4 -6 -8 -10 -12 Power spectral density (log scale, normalized) Gold standard ML (APT) FBP SIRT SART Gold standard FBP SIRT SART ML (APT) Figure 6. Reconstruction performance comparison with baseline methods. (a) The ﬁgure qualitatively compares 3D power spectral densities of reconstructions of gold standard and baseline reconstruction methods. Both APT and baseline reconstruction methods (FBP, SIRT, SART) use the optimal tomo-scans with N∗= 29 and θ ∗= ±16.8◦. Only APT has effectively ﬁlled up the missing wedges. (b) Reconstructions of gold standard and baseline methods are shown and compared along xy, xz, and yz planes. 0 Normalized frequency (kz) -0.5 0.5 Normalized frequency (kx) 0 -0.5 0.5 0 -0.5 0.5 0 -0.5 0.5 0 -0.5 0.5 0 -0.5 0.5 0 -0.5 0.5 0 -0.5 0.5 0 -0.5 0.5 0 -0.5 0.5 kz ky kx a 0 -2 -4 -6 -8 -10 -12 Power spectral density (log scale, normalized) Gold standard ML (APT) FBP SIRT SART SIRT SART Figure 6. Reconstruction performance comparison with baseline methods. (a) The ﬁgure qualitatively compares 3D power spectral densities of reconstructions of gold standard and baseline reconstruction methods. Both APT and baseline reconstruction methods (FBP, SIRT, SART) use the optimal tomo-scans with N∗= 29 and θ ∗= ±16.8◦. Only APT has effectively ﬁlled up the missing wedges. (b) Reconstructions of gold standard and baseline methods are shown and compared along xy, xz, and yz planes. References 17/18 x axis y axis z axis (6th head, x = 0.784 um) (8th head, x = 1.344 um) (4th head, x = 1.792 um) (4th head, y = 0.448 um) (4th head, y = 1.456 um) (5th head, y = 1.680 um) (2nd head, z = 1.232 um) (6th head, z = 2.912 um) (6th head, z = 3.360 um) 0 0.16 0.08 Attention weight 0 0.3 0.2 0.1 Attention weight 0 0.2 0.1 Attention weight 0 1 0.5 Attention weight 0 1 0.5 Attention weight 0 1 0.5 Attention weight 0 1 0.5 Attention weight 0 1 0.5 Attention weight 0 1 0.5 Attention weight Axial self-attention along Figure 7. Learned attention weight visualization. References (8th head, x = 1.344 um) 0 1 0.5 Attention weight x axis (6th head, x = 0.784 um) 0 1 0.5 Attention weight (4th head, x = 1.792 um) 0 1 0.5 Attention weight (4th head, x = 1.792 um) (8th head, x = 1.344 um) (6th head, x = 0.784 um) y axis (4th head, y = 0.448 um) 0 1 0.5 Attention weight Axial self-attention along (5th head, y = 1.680 um) 0 1 0.5 Attention weight (4th head, y = 1.456 um) 0 1 0.5 Attention weight Axial self-attention along (4th head, y = 1.456 um) (4th head, y = 0.448 um) (5th head, y = 1.680 um) z axis (2nd head, z = 1.232 um) 0 0.3 0.2 0.1 Attention weight (6th head, z = 3.360 um) 0 0.16 0.08 Attention weight (6th head, z = 2.912 um) 0 0.2 0.1 Attention weight z axis (2nd head, z = 1.232 um) (6th head, z = 2.912 um) (6th head, z = 3.360 um) 0 0.16 0.08 Attention weight 0 0.3 0.2 0.1 Attention weight 0 0.2 0.1 Attention weight (6th head, z = 2.912 um) (2nd head, z = 1.232 um) (6th head, z = 3.360 um) Figure 7. References Learned attention weights of multi-head axial self-attentions along each x,y,z axis. Parentheses contain information on the selected attention head and the position of the layer of interest (blue, si) that attends to all layers (sj ( j = 1,2,··· ,N)) with attention weights (red, αk i j), showing importance of sj to si. Figures Figure 1 Figure 5 Figure 2 Figure 3 Figure 3 Figure 1 Figure 4 Figure 7 Figure 5 Figure 4 Figure 6 Figure 2 Figure 7 Figure 6 Supplem This is a lis References Learned attention weight visualization. Learned attention weights of multi-head axial self-attentions along each x,y,z axis. Parentheses contain information on the selected attention head and the position of the layer of interest (blue, si) that attends to all layers (sj ( j = 1,2,··· ,N)) with attention weights (red, αk i j), showing importance of sj to si. 18/18 Supplementary Files This is a list of supplementary ¦les associated with this preprint. Click to download. supplementary.pdf supplementary.pdf supplementary.pdf
https://openalex.org/W2055930365	https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0094202&type=printable	English	null	Fusion Transcript Discovery in Formalin-Fixed Paraffin-Embedded Human Breast Cancer Tissues Reveals a Link to Tumor Progression	PloS one	2,014	cc-by	11,891	Abstract The identification of gene fusions promises to play an important role in personalized cancer treatment decisions. Many rare gene fusion events have been identified in fresh frozen solid tumors from common cancers employing next-generation sequencing technology. However the ability to detect transcripts from gene fusions in RNA isolated from formalin-fixed paraffin-embedded (FFPE) tumor tissues, which exist in very large sample repositories for which disease outcome is known, is still limited due to the low complexity of FFPE libraries and the lack of appropriate bioinformatics methods. We sought to develop a bioinformatics method, named gFuse, to detect fusion transcripts in FFPE tumor tissues. An integrated, cohort based strategy has been used in gFuse to examine single-end 50 base pair (bp) reads generated from FFPE RNA-Sequencing (RNA-Seq) datasets employing two breast cancer cohorts of 136 and 76 patients. In total, 118 fusion events were detected transcriptome-wide at base-pair resolution across the 212 samples. We selected 77 candidate fusions based on their biological relevance to cancer and supported 61% of these using TaqMan assays. Direct sequencing of 19 of the fusion sequences identified by TaqMan confirmed them. Three unique fused gene pairs were recurrent across the 212 patients with 6, 3, 2 individuals harboring these fusions respectively. We show here that a high frequency of fusion transcripts detected at the whole transcriptome level correlates with poor outcome (P,0.0005) in human breast cancer patients. This study demonstrates the ability to detect fusion transcripts as biomarkers from archival FFPE tissues, and the potential prognostic value of the fusion transcripts detected. Citation: Ma Y, Ambannavar R, Stephans J, Jeong J, Dei Rossi A, et al. (2014) Fusion Transcript Discovery in Formalin-Fixed Paraffin-Embedded Human Breast Cancer Tissues Reveals a Link to Tumor Progression. PLoS ONE 9(4): e94202. doi:10.1371/journal.pone.0094202 Editor: Shannon M. Hawkins, Baylor College of Medicine, United States of America Editor: Shannon M. Hawkins, Baylor College of Medicine, United States of America Received June 12, 2013; Accepted March 12, 2014; Published April 11, 2014 Copyright: 2014 Ma 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. Fusion Transcript Discovery in Formalin-Fixed Paraffin- Embedded Human Breast Cancer Tissues Reveals a Link to Tumor Progression Yan Ma, Ranjana Ambannavar, James Stephans, Jennie Jeong, Andrew Dei Rossi, Mei-Lan Liu, Adam J. Friedman, Jason J. Londry, Richard Abramson, Ellen M. Beasley, Joffre Baker, Samuel Levy, Kunbin Qu* Genomic Health Inc., Redwood City, California, United States of America Genomic Health Inc., Redwood City, California, United States of America Abstract Funding: The funder Genomic Health Inc provided support in the form of salaries for authors, YM, RA, JS, JJ, ADR, ML, AJF, JJL, RA, EMB, JB, SL, KQ, but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the ‘author contributions’ section. Competing Interests: All authors are salaried employees of Genomic Health, Inc., which funded this work. All have been awarded stock in the company. This does not alter the authors’ adherence to all the PLOS ONE policies on sharing data and materials. * E-mail: kqu@genomichealth.com April 2014 \| Volume 9 \| Issue 4 \| e94202 Breast cancer patients and RNA-Seq dataset Breast cancer patients and RNA Seq dataset One hundred thirty-six primary breast cancer FFPE tumor specimens with clinical outcomes were provided by Providence St. Joseph Medical Center (Burbank, CA), with institutional review board approval [22]. The clinical characteristics, RNA-Seq sample preparation and sequencing of the Providence cohort of 136 primary breast cancer FFPE tumor specimens were described earlier [17]. Briefly, total RNA was isolated from three 10-mm FFPE tissue sections per patient using Epicentre’s MasterPure Purification Kit (Epicenter Biotechnologies, Madison, WI). Paraf- fin was first removed by xylene extraction followed by ethanol wash. A DNase I treatment step was included to remove DNA from total nucleic acids. The same procedure was employed for RNA isolation from a second breast cancer study cohort from Rush University Medical Center. Seventy-eight primary breast cancer FFPE tumor specimens with clinical outcomes were provided by Rush University Medical Center (Chicago, IL), with institutional review board approval [21]. The same method of sample preparation [21] and sequencing [17] was applied to 76 of 78 Rush samples. Two remaining Rush samples did not yield enough RNA for sequencing. Directional RNA-Seq libraries were prepared using ScriptSeq RNA-Seq Library Preparation Kit (Epicenter Biotechnologies, Madison, WI) as described previously [17]. The quality of the RNA-Seq libraries was assessed using Agilent DNA Kits on a 2100 Bioanalyzer instrument (Santa Clara, CA). Sequence reads of 50 bp in length were processed by CASAVA, the standard Illumina package, and data quality assessment was described earlier [17]. The definition of clinical recurrence in these patients was determined as in the original study plans [21]. p In addition to sequence information, expression profiles have been used to provide supporting evidence for fusion transcripts. The utilization of expression data for fusion transcript detection is a feature of the COPA (Cancer Outlier Profiling Analysis) method that was devised for analysis of microarray databases [18]. Cancer- related genes identified as expression outliers in microarray experiments led to the discovery of TMPRSS2 fused to ETS transcription factors, the first known recurrent gene fusions in common solid carcinomas. Fusion RNAs are expected to exhibit a marked expression discontinuity between the preserved side and discarded side of a given fusion junction, compared to expression of these genes in samples without the fusion transcript. Recently published fusions detected using RNA-Seq data have displayed this discrete expression pattern at acceptor fusion junction sites [8,9]. Introduction ALK fusion [4,5]. Recently, the advent of next-generation sequencing technology has enabled detection of a number of rare recurrent gene fusion events that have potential therapeutic relevance to common solid tumors, including KIF5B-RET, which occurs in about 1% lung adenocarcinomas [6–9]. Oncogenesis is understood to be driven by ten distinctive and interactive capabilities that enable tumor growth and metastasis [1]. One of the underlying hallmarks of cancer cells is genome instability, which fosters random mutations and chromosomal rearrangements. These genomic aberrations, which include translocations, deletions and inversions, can produce oncogenic gene fusions that can be exploited pharmacologically. A classic example of oncogenic fusions is BCR-ABL1 in chronic myelog- enous leukemia, which is generated by a translocation between chromosomes 9 and 22 [2], and exhibits constitutive ABL1 tyrosine kinase activity. This discovery led to the development of the targeted tyrosine kinase inhibitor Imatinib approved in 2001 [3]. With advances of modern technology in medicine, the turnover time from discovery of a molecular biomarker to drug approval has been reduced to a period as brief as four years, as demonstrated by the development of Crizotinib treatment for the 2–7% of non-small lung cancer patients possessing the EML4- The detection of functional gene fusion events generated by chromosomal translocations has been facilitated by the application of RNA-Seq technologies. Numerous bioinformatics methods are available to detect fusion transcripts from RNA-Seq paired-end read data (ChimeranScan [10], SnowShoes-FTD [11], GSTRUCT-fusions [12] and GFP [9]) or single-end read (TopHat-Fusion [13], FusionMap [14] and FusionFinder [15]). All fusion transcript detection methods utilize split reads, in which a single-end read or one read from the pair-end read is mapped to each end of two fused genes exactly at the fusion junction site. In addition to split reads, paired-end approaches take advantage of bridging reads in which each read is mapped to each of the fused genes independently, thus providing extra evidence for the existence of a fusion junction than split reads alone. Most of these April 2014 \| Volume 9 \| Issue 4 \| e94202 PLOS ONE \| www.plosone.org 1 April 2014 \| Volume 9 \| Issue 4 \| e94202 Detect Fusion Transcripts in FFPE for Tumor Progression demonstrated that the technology used is sensitive and specific [17]. Here, we apply these single-end 50 bp RNA-Seq data to identify fusion transcripts and relate them to breast cancer prognosis. Introduction published methods evaluate RNA prepared from cell lines or fresh frozen tumor tissue from biopsy or resection. RNA from these sources is generally relatively intact and produces longer insert size libraries for sequencing, which greatly facilitates the detection of fusion transcripts. The standard clinical practice of creating FFPE tissue specimens from biopsies and surgical resections has generated very large numbers of FFPE tissue blocks in pathology archives that have associated, metadata-rich, long term clinical records. Therefore, the detection of fusion transcripts in FFPE tissues may reveal fusion transcripts of clinical relevance. Any attempts to detect fusion transcripts from FFPE tissues must address the extensive RNA fragmentation that occurs during storage of FFPE blocks and continues as block archival age increases [16], and also the substantial amounts of precursor RNAs detected in this tissue source [17]. As a result, FFPE RNA-Seq libraries have short insert sizes, low complexity (i.e., many short sequence segments with identical nucleotide composition) and a large amount of intronic sequence [17]. Difficulties accurately trimming the sequencing adaptor at the 39-end of reads from FFPE samples as well as the chemical modifications of RNA during formalin treatment can also decrease mapping quality such that the mapping rates from FFPE RNA-Seq libraries are lower than those from fresh frozen tissues. As a result of RNA fragmentation in FFPE tissue, whereby a median RNA fragment size of 100 bp is found, we reasoned that 50 bp single-end reads would provide a robust cost-effective sampling methodology for our study. We describe here the development and application of a bioinformatics method, gFuse, for the detection of fusion transcripts in RNA-Seq data from archival FFPE samples. This method addresses the challenges outlined and employs short sequence single-end reads (50 bp) enabling a cost effective method of analyzing large numbers of FFPE samples. Breast cancer patients and RNA-Seq dataset Multiple bioinformatics approaches including FusionSeq [19], deFuse [20] and TopHat-Fusion [13] have used expression data in their pipelines and all these methods rely on the analysis of an individual subject. The cohort-based approach described here compares expression levels across a cohort of subjects, combined with exon/intron level expression interruption, to identify putative fusion transcripts. Due to the large proportion of sequences (65% of uniquely mapped reads) that map to introns in FFPE RNA-Seq data [17], we included reads mapped to the introns to comprehensively measure expression of each gene. Fusion transcript detection pipeline gFuse We define a fusion junction as a unique pair of donor and acceptor genomic positions such as ‘‘+chr17:5250220-.+ chr17:11532734’’, and a fusion or fusion event as an occurrence of a particular fusion junction within a patient sample. The definition of symbols used to define each junction is: ‘‘-.’’ indicates the splicing direction from donor to acceptor, ‘‘+’’ indicates the transcription direction on the top of chromosome strand, and ‘‘-’’ indicates the transcription direction on the bottom of the chromosome strand. The donor genomic position is the last base of the preserved side of the donor and the acceptor genomic position is the first base of the preserved side of the acceptor. The fusion transcript detection pipeline gFuse consists of two strategies, a sample-based strategy and a cohort-based strategy (Figure 1A). The sample-based strategy interrogates each RNA- Seq sample individually and nominates candidate fusion junctions. The cohort-based strategy has two features that take advantage of the cohort-based information. The first feature is to combine the candidate fusion junctions in the beginning step of the cohort based analysis, which increases the chance of identifying recurrent fusion transcripts across the two cohorts studied here. The second feature is to confirm the presence of each fusion candidate in each individual sample across the whole cohort by examining read alignment and expression profiling evidence. The pipeline was developed in Linux Shell, Perl and R languages, and the data In this study, we detected fusion transcripts in two breast cancer cohorts, the Providence cohort of 136 patients and the Rush cohort of 76 patients with average FFPE block archive ages of 8.5 years and 13.4 years respectively [17,21]. These two cohorts have been previously used in the development of a 21-gene qRT-PCR breast cancer recurrence risk assay [21,22]. Recently, the whole transcriptome RNA-Seq analysis of the Providence cohort has April 2014 \| Volume 9 \| Issue 4 \| e94202 April 2014 \| Volume 9 \| Issue 4 \| e94202 PLOS ONE \| www.plosone.org 2 Detect Fusion Transcripts in FFPE for Tumor Progression Figure 1. The schema and workflow of our fusion detection pipeline gFuse, illustrated for two breast cancer cohorts. A. The sample and cohort based strategies are integrated in RNA-Seq fusion transcript detection. Each step of the pipeline is numbered in shade, and explained in Materials and Methods. The percentages show the fusion junctions retained after each step in all Providence samples. B. Detect Fusion Transcripts in FFPE for Tumor Progression Donor and acceptor mRNA or pre-mRNA containing template sequences were used as controls. Since the DNA breakpoints were unknown in RNA-Seq data, a fusion pre-mRNA template could not be created. The genomic sequences were used to generate the pre-mRNA template sequences, and RefSeq sequences were used to generate mRNA template sequences. The sequence of each template in the five template set was retrieved and annotated for each candidate fusion transcript. Candidate fusion junctions were removed if any of their 100 bp templates had the identical sequence with any other template set. BLAST (http://blast.ncbi. nlm.nih.gov/Blast.cgi, version 2.2.25) was used to investigate the homology of the remaining candidate fusions. A separate collection of 300 bp template set was built for each of the fusion junction candidates with the same strategy as described above to provide sequence input to probe designs for qRT-PCR experi- ments. Homologies between the 300 bp donor template and the 300 bp acceptor template, as well as homologies between the 300 bp donor pre-mRNA template and the 300 bp acceptor pre- mRNA template were evaluated. Any candidate fusion satisfying the following criteria was removed from further analysis: (1) sequence identity of more than 14 bp (empirically determined to effectively remove homologous genes) of 300 bp of the donor template and acceptor template; (2) sequence identity of more than 14 bp of 300 bp of the donor genomic template and acceptor genomic template; and (3) less than 50 bp exonic sequence on either side of fusion, donor, or acceptor template sequences. The 100 bp five template sets for each of the remaining candidate fusions were used to create a template index using a tool from the GSNAP package. Donor and acceptor mRNA or pre-mRNA containing template sequences were used as controls. Since the DNA breakpoints were unknown in RNA-Seq data, a fusion pre-mRNA template could not be created. The genomic sequences were used to generate the pre-mRNA template sequences, and RefSeq sequences were used to generate mRNA template sequences. The sequence of each template in the five template set was retrieved and annotated for each candidate fusion transcript. Candidate fusion junctions were removed if any of their 100 bp templates had the identical sequence with any other template set. BLAST (http://blast.ncbi. nlm.nih.gov/Blast.cgi, version 2.2.25) was used to investigate the homology of the remaining candidate fusions. Detect Fusion Transcripts in FFPE for Tumor Progression The remaining reads were grouped according to the distant splicing junction sites, and each junction site was annotated based on the University of California, Santa Cruz RefSeq sequence annotation (ftp:// hgdownload.cse.ucsc.edu/goldenPath/hg19/database/refGene. txt.gz). During this annotation step, any junctions mapped to pseudo-genes, un-annotated gene regions, or multiply mapped RefSeq genes were removed. Also, gene rearrangements within the same gene or potential transcript read-throughs were also eliminated. At this stage, candidate fusions met at least one of the following criteria: (1) they mapped to different chromosomes, (2) they mapped to different RefSeq genes, (3) they were in opposite directions on same chromosome, or (4) they were at least 1 MB apart if on the same chromosome. Step 3: Extract Fusions. The resulting distant splicing junctions were then annotated and candidate fusion transcripts were selected. Specifically, the alignments of reads that passed Step 2 were examined, and reads with any mismatches within 5 bp of the distant splicing junction site or mapped to the anti-sense strand of annotated genes were removed. Anti-sense reads were removed in this step since directional RNA-Seq libraries were constructed in the two cohorts analyzed here. The remaining reads were grouped according to the distant splicing junction sites, and each junction site was annotated based on the University of California, Santa Cruz RefSeq sequence annotation (ftp:// hgdownload.cse.ucsc.edu/goldenPath/hg19/database/refGene. txt.gz). During this annotation step, any junctions mapped to pseudo-genes, un-annotated gene regions, or multiply mapped RefSeq genes were removed. Also, gene rearrangements within the same gene or potential transcript read-throughs were also eliminated. At this stage, candidate fusions met at least one of the following criteria: (1) they mapped to different chromosomes, (2) they mapped to different RefSeq genes, (3) they were in opposite directions on same chromosome, or (4) they were at least 1 MB apart if on the same chromosome. from the GSNAP package. Step 5: Retrieve Reads. In order to increase the sensitivity and to determine the final supporting reads for each candidate junctions, all reads mapped near any junction site based on the genomic location of all candidate fusion template sets and reads not mapped in the original GSNAP BAM file for each RNA-Seq library were selected. The selected reads were re-mapped into the five template set index with GSNAP with the splicing detection parameter turned off. Only good quality reads uniquely mapped to the fusion template were kept. Step 4: Build Templates. Detect Fusion Transcripts in FFPE for Tumor Progression At this stage, fusion junctions from both the Providence and Rush cohorts were combined (Figure 1B). In order to remove false positives introduced by homologous sequences around candidate fusion junctions and to enable accurate mapping of supporting reads, a five template set was created for each candidate fusion. The features of the five template set are depicted in Figure 2A. The set included the following individual templates, each of which included 100 bp of sequence. The set templates were 100 bp, with 50 bp on either side of the candidate junction for fusion templates, because our read length is 50 bp. Step 6: Profile Expression. In order to assess the expression of each fusion transcript with a cohort, both exons and introns present in candidate fusions that had at least one read across the fusion junction site were assessed for each donor and acceptor. The gene table including exons and introns derived from Step 1 was normalized by library size factors as described by R package DEseq [25]. The intron immediately before the splicing site on the acceptor gene or the intron immediately after the splicing site on the donor gene were excluded from expression analyses due to uncertainty of the breaking point in the intron (Figure 2A). Exons or introns having counts below 5 reads were padded to 5 reads. Detect Fusion Transcripts in FFPE for Tumor Progression A separate collection of 300 bp template set was built for each of the fusion junction candidates with the same strategy as described above to provide sequence input to probe designs for qRT-PCR experi- ments. Homologies between the 300 bp donor template and the 300 bp acceptor template, as well as homologies between the 300 bp donor pre-mRNA template and the 300 bp acceptor pre- mRNA template were evaluated. Any candidate fusion satisfying the following criteria was removed from further analysis: (1) sequence identity of more than 14 bp (empirically determined to effectively remove homologous genes) of 300 bp of the donor template and acceptor template; (2) sequence identity of more than 14 bp of 300 bp of the donor genomic template and acceptor genomic template; and (3) less than 50 bp exonic sequence on either side of fusion, donor, or acceptor template sequences. The 100 bp five template sets for each of the remaining candidate fusions were used to create a template index using a tool from the GSNAP package. passing both filtering thresholds and uniquely mapped to human genome were retained to calculate the gene feature counts that provide expression values for exonic and intronic regions. The gene feature count is the number of aligned bases from reads mapped within the feature region. These gene feature counts are referred to as ‘‘gene tables’’ in Figure 1A. Step 2: Retest by GSNAP. In order to remove false positives, potential distant spliced reads in Step 1 were re-tested using GSNAP parameters that favor local alignment. Each alignment from the GSNAP re-run was examined, any reads meeting all of the following criteria were considered false positive distant splicing reads in the original GSNAP output, and removed from further analyses: the total matched length in the local alignment was at least 44 bp with a gap alignment tolerance of 1 bp. Reads that successfully passed through this step were considered to include a distant spliced junction. Step 3: Extract Fusions. The resulting distant splicing junctions were then annotated and candidate fusion transcripts were selected. Specifically, the alignments of reads that passed Step 2 were examined, and reads with any mismatches within 5 bp of the distant splicing junction site or mapped to the anti-sense strand of annotated genes were removed. Anti-sense reads were removed in this step since directional RNA-Seq libraries were constructed in the two cohorts analyzed here. Fusion transcript detection pipeline gFuse Dataflow and main results of fusion events detected in Providence and Rush are shown side-by-side with each step corresponding to the numbered step in Figure 1A. The numbers of fusion events selected for TaqMan assays and the TaqMan supported ones are in parentheses. doi:10.1371/journal.pone.0094202.g001 Figure 1. The schema and workflow of our fusion detection pipeline gFuse, illustrated for two breast cancer cohorts. A. The sample and cohort based strategies are integrated in RNA-Seq fusion transcript detection. Each step of the pipeline is numbered in shade, and explained in Materials and Methods. The percentages show the fusion junctions retained after each step in all Providence samples. B. Dataflow and main results of fusion events detected in Providence and Rush are shown side-by-side with each step corresponding to the numbered step in Figure 1A. The numbers of fusion events selected for TaqMan assays and the TaqMan supported ones are in parentheses. doi:10.1371/journal.pone.0094202.g001 processing was on a Linux cluster. The detailed steps of gFuse (Figure 1A) are described below. from splicing events between different genes or chromosomes. Distant splicing events can also include splicing events occurring within the same gene, but in the opposite transcription direction [24]. Step 1: Map by GSNAP. Raw FASTQ sequencing data from the Providence and Rush cohorts were generated using CASAVA software. The FASTQ files were mapped to the human genome (version GHCh37/hg19) along with RefSeq splicing sites and dbSNP database (version 135) using the RNA-Seq aligner GSNAP [23]. An important feature of GSNAP is its ability to detect a distant splice junction within a single read. Local splice junctions derive from splicing events within a single gene in a consistent transcription direction, whereas distant spliced junctions derive Two filters were installed to remove low quality and unwanted reads. The quality filter retained reads with a minimum 15 bases at any position with a base quality score of 20 or above. To filter out the un-wanted reads, a number of abundant sequences including biological sequences (e.g., ribosomal RNA and mitochondrial sequences), and sequences introduced during library prep (e.g., PhiX) were removed from alignment (BAM) files. Only reads April 2014 \| Volume 9 \| Issue 4 \| e94202 PLOS ONE \| www.plosone.org 3 Detect Fusion Transcripts in FFPE for Tumor Progression Detect Fusion Transcripts in FFPE for Tumor Progression April 2014 \| Volume 9 \| Issue 4 \| e94202 Fusion confirmation by Personal Genome Machine (PGM, Life Technologies) N For fusions with only one non-redundant read, expression profiling evidence was reviewed to select candidates with favorable expression evidence, and ranked as Tier-2; N Fusions were classified as Tier-3 if they were predicted without any read evidence, but sharing a similar expression profiling with a TaqMan supported fusion after Step 8 described below; N Multiple samples sharing the same fusion junctions, but without good expression evidence were removed. A total of 100 unique fusion junctions with 118 fusion events were identified, the full list is available in Table S1. Step 8: TaqMan assay. Quantitative RT-PCR analysis using TaqMan RT-PCR was used to investigate the selected 60 fusion junctions. Reverse transcription was carried out using the Omniscript RT Kit (Qiagen) by incubating amplified RNA with random hexamers and gene-specific primers at 37uC for 1 hour. Primer, probe, and amplicon sequences are shown in Table S2. Fluorogenic probes were dual-labeled with 59-FAM as a reporter and 39-BHQ-2 as a quencher. Primers and probes were designed using the Primer3 program restricting amplicon sizes to 65-85 bps (http://frodo.wi.mit.edu/). When Primer3 failed, primer and probe sequences were optimized manually to ensure optimal performance of the TaqMan assay design for the chimeric transcripts. Reverse transcription reaction in the absence of RNA template (i.e., water) was always used as a negative control in all assays. The samples that were previously identified as positive or negative for a particular fusion junction served as controls when needed. Since the RT reaction was multiplexed by using a pooled gene specific primer set, the cDNA derived from a RNA sample was tested with all fusion gene qPCR assays within an assayed gene set. All RNA samples were assayed in triplicate qPCR reactions with 10 ml per well. Thermal cycling conditions were standard for all assays (A heat activation step of 95uC for 10 minutes followed by 40 cycles of 95uC for 20 seconds and 60uC for 45 seconds). All TaqMan assay results including primer and probe sequences are listed in Table S2. Ion Torrent Suite software was used to generate FASTQ files in which the barcode adaptors and 39 end low quality sequences were removed as recommended. To recover read sequences longer than the desired 100 bp in a case of an expected amplicon of 126 bp, the 39 end quality trimming was turned off for this design. Detect Fusion Transcripts in FFPE for Tumor Progression The expression Interrupt Ratios (IR) of normalized counts between preserved and discarded sides were calculated for donor and acceptor genes for each sample according to the following formula: N Fusion template: The 50 bp exonic sequence of the preserved region of donor gene plus 50 bp exonic sequence of the preserved region of acceptor gene, N Fusion template: The 50 bp exonic sequence of the preserved region of donor gene plus 50 bp exonic sequence of the preserved region of acceptor gene, N Donor template: The 50 bp exonic sequence of the preserved region of donor gene plus 50 bp exonic sequence of the discarded region of donor gene, IR~ ( counts length )preserved ( counts length )discarded IR~ ( counts length )preserved ( counts length )discarded N Acceptor template: The 50 bp exonic sequence of the discarded region of acceptor gene plus 50 bp exonic sequence of the preserved region of acceptor gene, N Donor pre-mRNA template: The 50 bp upstream genomic sequence of donor splicing site plus 50 bp downstream genomic sequence of donor splicing site, The normalized expression counts of exons and introns in each fusion transcript across samples in a cohort were ordered according to IR values, and a heatmap representing the gene features of the predicted fusion transcript within the cohort was N Acceptor pre-mRNA template: The 50 bp upstream genomic sequence of acceptor splicing site plus 50 bp downstream genomic sequence of acceptor splicing site. PLOS ONE \| www.plosone.org April 2014 \| Volume 9 \| Issue 4 \| e94202 April 2014 \| Volume 9 \| Issue 4 \| e94202 4 Detect Fusion Transcripts in FFPE for Tumor Progression PLOS ONE \| www.plosone.org 5 April 2014 \| Volume 9 \| Issue 4 \| e94 PLOS ONE \| www.plosone.org 5 April 2014 \| Volume 9 \| Issue 4 \| e94202 April 2014 \| Volume 9 \| Issue 4 \| e94202 PLOS ONE \| www.plosone.org Detect Fusion Transcripts in FFPE for Tumor Progression Detect Fusion Transcripts in FFPE for Tumor Progression Figure 2. The utilization of a five template set and expression profiling for fusion transcript detection. A. The concept of five template set is illustrated with six RNA transcripts for a fusion transcript in a FFPE RNA sample. Each template is numbered under lines around the corresponding RNA sequence. Fusion confirmation by Personal Genome Machine (PGM, Life Technologies) generated. Expression profiling results for an example candidate fusions are shown in Figure 2D of the Results section. Step 7: Review Evidence. Fusions were classified into these three tiers based on evidence (Figure 1B). Data were manually reviewed to classify candidate fusions. The following rules were then applied to rank candidate fusions into three tiers: Nineteen qPCR supported fusion transcripts were selected to be sequenced on the semi-conductor based Ion Torrent Personal Genome machine (PGM) to confirm the results from qPCR. The selection priority was given to those either recurred in multiple patients or appeared within a single patient as one of the multiple fusion transcripts. N Fusions with a minimum of two non-redundant reads spanning fusion junctions and were kept as Tier-1 fusions regardless of the expression profiling (Figure 1B); Eight replicate wells of PCR products were generated for each of the fusion targets (19 in total) in 12 Providence/RUSH amplified RNA samples in order to prepare enough PCR product for PGM sequencing of the selected gene fusion candidates. Quantitative RT-PCR analysis using TaqMan RT PCR was used to confirm the presence of PCR product before proceeding to PGM sequencing. Reverse transcription was carried out as described in Step 8. The eight replicate wells of PCR product were pooled for each fusion target. Each PCR product was then purified using 1.86 volume of Agencourt AMPure XP beads (Beckman Coulter), and quality checked and quantitated using the Agilent High Sensitivity DNA Kit (Agilent Technologies). Fusion PCR products from the same patient samples were then pooled together. The Fusion PCR products were then prepared for sequencing using the Ion Plus Fragment Library Kit (Life Technologies) and barcoded using the Ion Xpress Barcode Adapters 1–16 (Life Technologies). The library was amplified with 7 cycles after adapter ligation and cleanup, as required by the protocol. The libraries were individually quantitated using the Agilent High Sensitivity DNA Kit (Agilent Technologies) and diluted to a target concentration of 26 pM. The libraries were pooled in equi-molar quantities prior to emulsion PCR on the Ion OneTouch 2 System (Life Technologies) and subsequently sequenced on a PGM 314 Chip Kit v2 (Life Technologies) using 260 flows. Detect Fusion Transcripts in FFPE for Tumor Progression The preserved and discarded sides of donor or acceptor are indicated by arrowed lines indicating transcription directions above each pre-mRNA. The red blocks are DNA breakpoints. The interrupt ratio (IR) is calculated by using the * marked preserved and discarded sides accordingly for donor or acceptor fusion genes. B. All supporting RNA-Seq split reads are aligned to five templates of fusion RABEP1-.DNAH9 in the Providence sample CSG. Each template is numbered according to Figure 1A. The vertical line indicates the junction site. C. Two samples are shown as outliers (solid red dots) when the gene expression levels of donor RABEP1 are plotted against acceptor DNAH9 in the Providence cohort. The expression levels are log2 base counts normalized by library size factors. TaqMan tested negative samples are labeled as solid black dots. D. Exon and intron expression levels of acceptor DNAH9 in the Providence cohort show the interrupted expression pattern in samples CSG and ECI at the predicted fusion junction site (orange line). The base counts of each exon and intron are normalized by library size, then center-scaled across the Providence cohort. The vertical arrow indicates RNA samples from low to high IR values of DNAH9. The exons (black ticks) and introns of DNAH9 are ordered according to the transcription direction (horizontal arrow), with the intron harboring DNA breakpoint omitted in Figure 2D and 2E. E. The base counts of exons and introns of acceptor DNAH9 in two samples show interrupted expression patterns at the fusion junction site. The base counts are normalized by library size then divided by length of each exon or intron. doi:10.1371/journal.pone.0094202.g002 Fusion transcripts were detected by gFuse, an integrated cohort-based approach All short reads mapping near any junction sites in the template index as well as reads not mapped in Step 1 were aligned to the template index for each RNA-Seq library. Fusion templates with at least one supporting short read were selected for further cohort based analysis. There are 3 tiers of candidate fusion transcripts generated by gFuse, Tier-1, Tier-2 and Tier-3. The supporting evidence for Tier-1 transcripts is strong while Tier-3 transcripts have weak evidence. Any fusions with at least 2 non-redundant reads across the fusion junctions are defined as Tier-1. Both Tier-2 and Tier-3 were selected based on the expression profiling described below; Tier-2 consists of fusions with a single non-redundant read across the fusion junction and Tier-3 represents predicted recurrent fusions with no read across the putative fusion junction. The expression profiling step can nominate candidate fusions despite the existence of very limited reads. In fact, here we used the expression profile data to predict known fusions in samples having no detected fusion sequences as illustrated by the example fusion RABEP1-.DNAH9 (Figure 2). This fusion junction was initially found in a single Providence sample (CSG) as a Tier-1 fusion with 2 split reads (Figure 2B). In this Tier-1 fusion, there are a total 17 reads across the donor RABEP1 mRNA and pre-mRNA template junctions, and 1 read across the acceptor DNAH9 mRNA and pre-mRNA template junctions. This evidence suggests that the strong donor promoter drives the expression of fusion transcripts. Consistent with the read coverage around junction sites, this fusion also appears as one of two expression outliers in the Providence cohort (Figure 2C). A second patient (ECI) is the only other patient that appears to have the same discrete expression pattern which exists in the sample CSG as evidenced by examination of the exon/intron expression levels of acceptor DNAH9 across the Providence cohort (Figure 2D). The samples in the cohort were ordered by IR (defined in the Materials and Methods section) to facilitate the expression outlier identification. The individual exon/intron expression levels of DNAH9 also show The underlying fusion transcript method is based on the detection of distant splicing within a single read feature as detected by the RNA-Seq aligner GSNAP [24]. The utility of GSNAP for fusion transcript detection has been demonstrated in fusion transcript detection methods such as GSTRUCT-fusions and GFP [9,12]. Fusion transcripts were detected by gFuse, an integrated cohort-based approach pp Overall, 118 fusion events, representing 100 unique fusion junctions, were identified in the two cohorts (Table S1). Forty three of the fusion junctions are predicted to produce in-frame chimeric proteins. Based on gene associations with cancer, we selected a total of 60 fusion junction candidates, and designed qRT-PCR assays for these fusion transcripts. Some of the candidate fusions selected for TaqMan assay were observed in 2 or more samples. Therefore by using 60 designs, we tested 77 candidate fusion events by quantitative RT-PCR in amplified RNA from selected patients harboring the corresponding candi- date fusions (Table S2). A total of 47 of the 77 fusion events (61%) were supported by TaqMan across the two cohorts irrespective of the sequence evidence. The Tier-1 category of candidate fusions (see Materials and Methods for definitions of Tiers), which have the strongest sequence evidence have the highest support frequency rate (89%). Tier-2 candidates, selected based on the combination of sequence (single read coverage only) and expression profiling, have a 45% support frequency rate. Tier-3 candidates, purely predicted from gene expression patterns, have the lowest support frequency at 23% (Figures 1B). Thus, the TaqMan results are consistent with the level of evidence observed for the three different tiers of fusion candidates. To further confirm fusion junction identified by TaqMan assays, a total of 19 fusion events identified by TaqMan were selected for PGM sequencing. Fusion junctions were amplified by using TaqMan primers, and PCR products containing fusion amplicons were sequenced on the PGM. In all 19 PCR reactions, the PCR amplicons matched the predicted fusion junction sequences (Table 1 and Table S3). In 7 PGM libraries in which a single barcode was used for a single PCR reaction, the amplicon reads represent the most prevalent clonal population in each library indicating that the PCR reactions are specific for these fusion junctions (Table 1). Next, candidate fusion junctions having at least one supporting read were combined from the two cohorts and further tested using the cohort based strategy. The donor and acceptor mRNA or pre- mRNA template sequences were used as controls for the sequence homology search and to generate read alignments in the cohort based approach. This step removed 27% of potential false positive fusion junctions from Step 3. The remaining five template sets were combined and constructed into a single template index. Data access The read alignments which support the fusion transcripts for Providence and Rush cohorts are deposited into Dryad Digital Repository (http://doi.org/10.5061/dryad.98m0m). Briefly, 50 bp single end reads were mapped to the human reference genome to provide candidate reads splitting across potential fusion junctions similar to GSTRUCT-fusion and GFP (Figure 1A). The candidate fusion split reads were re-mapped against the human reference genome under the GSNAP parameters favoring local alignments. Any reads that aligned locally, and were therefore not split across the fusion junction, were discarded. This alignment re-testing step eliminated 28% of distant spliced junctions identified in Step 1. The RefSeq annotation file was used to annotate these distant spliced junctions. Only junctions mapping to two different annotated genes were kept, and 80% of distant spliced junctions identified in Step 2 were eliminated during the annotation step. Survival analysis Patients were stratified into different categories based on the fusion number detected. The time to disease recurrence as defined in the original studies [21,22] was used to generate the Kaplan- Meier plot using the R package Survival. Detect Fusion Transcripts in FFPE for Tumor Progression Detect Fusion Transcripts in FFPE for Tumor Progression sample-based strategy were combined in the beginning step of the cohort based analysis. However, in recognition of inter-cohort differences in block archive ages and library quality, the expression profiling step was carried out separately within each cohort (Figure 1B). The average insert size and complexity of the Providence cohort libraries are higher than those of the Rush cohort libraries. Here we describe results from the Providence RNA-Seq dataset [17] to illustrate the performance of the cohort- based computational approach. Fusion confirmation by Personal Genome Machine (PGM, Life Technologies) All reads were mapped to the 5 template set sequence database containing the fusion templates. For each of expected fusion amplicons in a given sample, the most abundant reads mapped to the fusion template was selected as the PCR amplicon. The sequence of this read was compared to the sequence of the expected amplicon. If the PCR amplicon matches the expected fusion amplicon, the fusion junction sequence is considered as confirmed. April 2014 \| Volume 9 \| Issue 4 \| e94202 PLOS ONE \| www.plosone.org PLOS ONE \| www.plosone.org 6 Fusion transcripts were detected by gFuse, an integrated cohort-based approach The fused AKAP12 protein might have different cellular localization and thus possess modified functions from the wild type AKAP12. In addition, both fusion protein isoforms may cause constitutive ligand-independent signaling. As a result, the patients harboring ESR1-.AKAP12 fusion may exhibit different responses to breast cancer hormone therapy. interact with another AKAP family member AKAP13 [29]. AKAP12 is a scaffold protein in signal transduction with tumor suppressor activities, and present in the plasma membrane, cytosol or endoplasmic reticulum [30]. The function of AKAP12 to organize the protein kinase A and C at these biological relevant locations might be disrupted if its location is changed. The fused AKAP12 protein might have different cellular localization and thus possess modified functions from the wild type AKAP12. In addition, both fusion protein isoforms may cause constitutive ligand-independent signaling. As a result, the patients harboring ESR1-.AKAP12 fusion may exhibit different responses to breast cancer hormone therapy. the discrete expression patterns around fusion junction site in two Providence samples (Figure 2E). Therefore, we assigned the sample ECI as a Tier-3 candidate for fusion of RABEP1-. DNAH9, even in the absence of reads across fusion junction in ECI. Both fusion events were supported by TaqMan with an average CT of 30.11 (CSG) and 34.86 (ECI) respectively, while other 39 samples tested were negative in the assay (Figure 2C). Therefore we conclude that there are two fusions or recurrent fusion events associated with a particular fusion junction ‘‘+ chr17:5250220 -. +chr17:11532734’’ in the Providence cohort (Figures 2C, D and E). the discrete expression patterns around fusion junction site in two Providence samples (Figure 2E). Therefore, we assigned the sample ECI as a Tier-3 candidate for fusion of RABEP1-. DNAH9, even in the absence of reads across fusion junction in ECI. Both fusion events were supported by TaqMan with an average CT of 30.11 (CSG) and 34.86 (ECI) respectively, while other 39 samples tested were negative in the assay (Figure 2C). Therefore we conclude that there are two fusions or recurrent fusion events associated with a particular fusion junction ‘‘+ chr17:5250220 -. +chr17:11532734’’ in the Providence cohort (Figures 2C, D and E). On the other hand, in certain fusion cases we identified varied junctions between two identical fused partners within a single patient. Fusion transcripts were detected by gFuse, an integrated cohort-based approach Both of these methods depend on GSNAP to provide fusion read candidates, and then apply a set of filtering modules to remove false positives in paired-end RNA-Seq datasets. To compensate for the short FFPE RNA length, we leveraged data from the two patient cohorts as shown in Figure 1A. The sample- based strategy interrogates each RNA-Seq sample individually and nominates candidate fusion junctions for the following cohort- based analysis, which confirms the presence of each fusion candidate in each individual sample across the whole cohort by examining read alignment and expression profiling evidence. To increase the chance of identifying recurrent fusion transcripts across the cohorts, fusion candidate templates provided by the April 2014 \| Volume 9 \| Issue 4 \| e94202 PLOS ONE \| www.plosone.org 7 Detect Fusion Transcripts in FFPE for Tumor Progression Table 1. Fusion junctions are confirmed by PGM sequencing of PCR amplicons. Sample Fusion Junction Tier Number of amplicon reads HM1 ESR1-.AKAP12 +chr6:152265643-.+chr6:151669846 Tier-1 252 HM1 ESR1-.C6orf211 +chr6:152129499-.+chr6:151785588 Tier-2 9702 MJG TFG-.GPR128 +chr3:100438902-.+chr3:100348442 Tier-1 5009 MJG SEMA4C-.BRE -chr2:97527316-.+chr2:28561317 Tier-1 4853 ECI TFG-.GPR128 +chr3:100438902-.+chr3:100348442 Tier-3 2297 ECI RABEP1-.DNAH9 +chr17:5250220-.+chr17:11532734 Tier-3 1927 ECI ERBB2-.IKZF3 +chr17:37868701-.-chr17:37949186 Tier-1 2218 CSG RABEP1-.DNAH9 +chr17:5250220-.+chr17:11532734 Tier-1 7161# D87 TFG-.GPR128 +chr3:100438902-.+chr3:100348442 Tier-2 6187# II6 ESR1-.AKAP12 +chr6:152201906-.+chr6:151669846 Tier-2 6880# IYM ESR1-.AKAP12 +chr6:152201906-.+chr6:151669846 Tier-1 9916# JGV TFG-.GPR128 +chr3:100438902-.+chr3:100348442 Tier-3 6108# L43 TFG-.GPR128 +chr3:100438902-.+chr3:100348442 Tier-2 5979# MGM TFG-.GPR128 +chr3:100438902-.+chr3:100348442 Tier-2 7499# DAP RIMS2-.DPYS +chr8:104709524-.-chr8:105436617 Tier-1 5440 DAP PREX1-.SLC9A8 -chr20:47324798-.+chr20:48431545 Tier-1 5809 GQW TANC2-.RDM1 +chr17:61086987-.-chr17:34247276 Tier-1 1000 GQW DDX5-.IQCG -chr17:62496667-.-chr3:197640913 Tier-1 1919 GQW EIF4A3-.TSPEAR -chr17:78120592-.-chr21:45953806 Tier-2 1296 #In these 7 PGM libraries containing a single PCR reaction with an unique PGM barcode, the fusion amplicons identify the most prevalent clonal population in the library. The detailed experimental results including amplicon sequences are in Table S3. doi:10.1371/journal.pone.0094202.t001 #In these 7 PGM libraries containing a single PCR reaction with an unique PGM barcode, the fusion amplicons identify the most prevalent clonal population in the library. The detailed experimental results including amplicon sequences are in Table S3. doi:10 1371/journal pone 0094202 t001 interact with another AKAP family member AKAP13 [29]. AKAP12 is a scaffold protein in signal transduction with tumor suppressor activities, and present in the plasma membrane, cytosol or endoplasmic reticulum [30]. The function of AKAP12 to organize the protein kinase A and C at these biological relevant locations might be disrupted if its location is changed. Fusion transcripts were detected by gFuse, an integrated cohort-based approach One patient in the Providence cohort has three different ERBB2-.IKZF3 junctions that only differ at the donor junction site, and one patient in the Rush cohort has two different TRIM37-.BCAS3 junctions that only differ at the donor junction site (Table S1). In these two cases qRT-PCR assays were designed to the junction sequences with the greatest number of RNA-Seq reads, and the dominant fusion junctions were supported by TaqMan in each case. Also, multiple recurrent partners fused to different gene partners were identified in our dataset, and supported by TaqMan assay: one tumor harboring ESR1-. AKAP12, another with the fusion gene ESR1-.C6orf211; LRP5 fused to different acceptors KAT6A and SLC22A24 in the same tumor; ADK as an acceptor in the fusion DLG5-.ADK in one patient, and as a donor in the fusion ADK-.C10orf11 in another patient; similarly, ACACA as the donor of ACACA-.MSI2 in one patient, and as the acceptor of UTP18-.ACACA in another patient. Fusion partners are cancer related genes The majority of fusion junctions are intra-chromosomal genomic rearrangements (69 out of total 100 fusion junctions), consistent with findings of others [10,26]. Of the 100 unique fusion junctions, only TFG-.GPR128 had been described previously [11,27,28]. It is noteworthy that a few of these fusion junctions are detected in both of the examined patient cohorts. Three recurrent fusion transcripts including TFG-.GPR128, ESR1-.AKAP12 and RABEP1-.DNAH9 were supported by TaqMan assays using amplified RNA from 6, 3 and 2 patients respectively, in the two cohorts of 212 total patients. Interestingly, among three ESR1-. AKAP12 fusion events in three different patients, there are two unique fusion junctions sharing the same acceptor junction site but differing at the donor junction sites by one exon. Since both these ESR1-.AKAP12 fusion junctions are in frame and the differing ESR1 exon doesn’t harbor any known functional domains (Figure S1A), these two fusion transcripts may possess the same biological function. Both fusion protein isoforms replace the ESR1 ligand binding site with functional domains from AKAP12 (Figures S1B and S1C). The lost ligand binding site of ESR1 is known to April 2014 \| Volume 9 \| Issue 4 \| e94202 PLOS ONE \| www.plosone.org 8 Detect Fusion Transcripts in FFPE for Tumor Progression Figure 3. Breast cancers with high fusion frequency have poor prognosis. A. The distributions of block age, clinical recurrence and ER status are shown according to fusion number categories in Providence and Rush cohorts. The archived block age is plotted as mean and standard deviation for each category. ER status was assessed by immunohistochemistry. The patient number for each category is labeled accordingly. B. Kaplan-Meier plots of each fusion number category show Providence patients with multiple fusions had poor prognosis, and a similar trend exists for Rush patients. The log-rank p-values are indicated in Kaplan-Meier plots. C. Kaplan-Meier plot with the 36 TaqMan supported fusion transcripts in the Providence cohort. There are another 11 TaqMan supported fusion transcripts from the Rush cohort but they are too few to generate a meaningful Kaplan-Meier plot. doi:10 1371/journal pone 0094202 g003 Figure 3. Breast cancers with high fusion frequency have poor prognosis. A. The distributions of block age, clinical recurrence and ER status are shown according to fusion number categories in Providence and Rush cohorts. The archived block age is plotted as mean and standard deviation for each category. ER status was assessed by immunohistochemistry. Fusion partners are cancer related genes The patient number for each category is labeled accordingly. B. Kaplan-Meier plots of each fusion number category show Providence patients with multiple fusions had poor prognosis, and a similar trend exists for Rush patients. The log-rank p-values are indicated in Kaplan-Meier plots. C. Kaplan-Meier plot with the 36 TaqMan supported fusion transcripts in the Providence cohort. There are another 11 TaqMan supported fusion transcripts from the Rush cohort but they are too few to generate a meaningful Kaplan-Meier plot. doi:10 1371/journal pone 0094202 g003 doi:10.1371/journal.pone.0094202.g003 doi:10.1371/journal.pone.0094202.g003 Higher numbers of fusion events are associated with poor tumor prognosis We searched the Mitelman fusion database with all 184 unique fusion partners including donors and acceptors from the final 118 fusion list, and 29 partners were found fused to various different partners in that database [28]. The statistically significant enrichment of Mitelman fusion genes (Fisher’s test P = 76e28) is 3.5 fold compared to all known RefSeq genes. Among them, ACACA, BCAS3, DDX5, FBXL20, IKZF3, RAF1, TFG and TRPS1 were fused to more than one partner in the database. These observations suggest fusion events are unlikely to be random although they appear to be rare in solid tumors. The average number of fusion events detected per patient across Providence and Rush cohorts is 0.63 and 0.29, respectively, far less than the average of 4.2 fusions reported in fresh frozen breast cancer biopsies [10,11]. This difference can reasonably be attributed to the poor quality of FFPE RNA, and a resulting limit on our ability to comprehensively detect fusion events in these samples. Between the Providence and Rush data sets, the latter has older archival ages, poorer quality RNA, and yields far fewer identified fusion transcripts (Figure 1B). The identified fusion partners tend to be cancer-related: 82% of the total 83 fusion junctions (96 fusion events in Figure 1B) identified from the Providence cohort have at least one partner in COSMIC database, which contains sequences of many genes frequently altered in cancers. This is consistent with other evidence for frequently mutated genes prone to genomic rearrangements in the cancer genomes [9]. The discovery of fusion transcripts containing partners that regulate repair of DNA double-strand breaks and homologous recombination, such as RAD21, RDM1, BRCA2 and SHFM1, is consistent with abundant evidence for aberrant regulation of DNA replication in cancer. Within each patient cohort we stratified patients according to the number of fusion events (Figure 3A) to determine whether the number of fusion events detected within individual tumors related to the likelihood of disease recurrence. Because not all candidate fusions were tested by TaqMan assay, all fusion events from Tier- 1, Tier-2 as well as TaqMan supported Tier-3 from the final candidate fusion list (Table S1) were used in stratification regardless of TaqMan results. In view of the limited number of fusions detected in the Rush dataset we evaluated just 2 categories: fusion detected or not detected, whereas in the Providence dataset we evaluated four abundance categories. Discussion We present here novel evidence that increasing frequency of fusion transcripts is associated with poor prognosis. This study also adds to the molecular knowledge of breast cancer complexity by identifying 118 candidate fusion transcripts and many TaqMan supported fusion transcripts, all of which are novel except TFG-. GPR128. Moreover, these fusions could be detected in single-end RNA-Seq data from aged FFPE tumor tissues by applying gFuse, a cohort based bioinformatics method. Among the total 118 candidate fusion transcripts identified, 3 unique fused gene pairs were recurrent and supported by TaqMan in the two cohorts of 212 total patients. The rate at which recurrent fusions were observed and the general novelty of the observed fusion transcripts in this study are in line with the previous publications about the very low recurrence of fusions in solid tumors such as 2–7% EML4-ALK in non-small lung cancer patients [4,5]. It is notable that the recent TCGA (The Cancer Genome Atlas) consortium efforts with large patient cohorts and fresh frozen samples assisted with whole genome sequencing identified primarily private (found in one sample only) fusion transcripts [27,31–33]. In 416 clear cell renal carcinoma patients, 70 out of 83 fusion transcripts are private [27]. In 322 endometrial carcinoma patients, 47 out of 49 fusion transcripts are non-recurrent [31]. In 97 colorectal cancer patients, 35 out of 38 fusion transcripts predicted from DNA translocations only exist in one patient [32]. Our method also addresses intronic RNA sequences, in recognition of the large amount of intronic sequence information present in FFPE RNA. Both donor and acceptor pre-mRNAs are built into 5 template sets to filter out reads mapped to mRNA precursors. On the other hand, the introns are selectively included in the expression profiling analysis to take advantage of abundant intronic sequence information. The two different remapping steps by GSNAP (Step 2 and Step 5 in Figure 1A) were designed to improve the mapping accuracy given the short inset size of FFPE. The success of these FFPE RNA-targeted designs is reflected by the high frequency of TaqMan support rates in the Tier-1 category (Figure 1B). Although the cohort based strategy described here was developed with and applied to FFPE tissue and single end RNA- Seq datasets, it is also relevant to fusion transcript detection in cell lines and fresh frozen samples. Detect Fusion Transcripts in FFPE for Tumor Progression than two fusions (subsequently referred to as multiple fusions) in Providence exhibited a statistically significant increased recurrence risk compared to patients from the three other groups having fewer detected fusion genes (Figure 3B). In the Rush dataset disease recurred at an increased rate among patients with detected fusions, although this relationship does not achieve statistical significance, possibly due to the limited fusion number detected from the low quality FFPE samples. Recognizing that including predicted fusion transcripts in this analysis necessarily reduces confidence in it, we also evaluated only the 36 TaqMan supported fusion transcripts from the Providence cohort. Figure 3C shows that a similar trend is still observed. The Rush data set yields only 11 TaqMan supported fusion transcripts which are too few to generate a meaningful Kaplan-Meier plot. To check whether minimizing the FFPE block age effect alters this relationship, we grouped patients into either upper or lower quartiles of the block age (binning patients with comparable block age or adjusting fusion numbers by RNA-Seq quality was not meaningful given the small numbers of patients and limited fusion numbers in these cohorts). The correction by sub-setting strengthens the association between fusion number and recurrence risk for both cohorts (Figure S2). (mostly acceptor junctions), consistent with the oncogenic gene fusion model. It is also possible that the gene expression filter removes a percentage of true fusion transcripts. When we performed TaqMan assays on a few fusion candidates that had single non-redundant reads without interrupted expression patterns, only one (ESR1-.C6orf21) was supported by TaqMan. It is likely that in many cases fusion gene candidates removed by the gene expression filter that represent true fusion events are expressed at low levels. While it seems plausible that such fusion genes have little or no influence on tumor behavior, in fact their contribution is unknown. To tailor this method to the short insert size and low complexity of FFPE RNA-Seq data, the candidate fusion templates are extended across a cohort or from one cohort to another to maximize the probability of identifying recurrent fusions. The potential of the cohort-based approach was demonstrated by our identification of a total of 6 recurrent TFG-.GPR128 fusions across two cohorts, which include 1 Tier-1 fusion, 3 Tier-2 fusions, and 2 Tier-3 fusions (Table S1). Detect Fusion Transcripts in FFPE for Tumor Progression The Tier-1 fusion was initially identified in a Rush sample, and extension of the Rush fusion templates to the Providence cohort allowed us to identify one Providence Tier-2 fusion, in which a single unique read split across the fusion junction with only 10 bp aligned to its acceptor gene. Sequence alignment tools cannot positively align a 10 bp sequence to its correct position in a whole genome, but this targeted exploration of candidate fusion sites allowed us to recognize recurrent events that were missed in the individual sample analysis. Further, assisted by the expression profiling analysis, another Tier-3 fusion was predicted in the Providence cohort. Higher numbers of fusion events are associated with poor tumor prognosis The 8 patients with more April 2014 \| Volume 9 \| Issue 4 \| e94202 PLOS ONE \| www.plosone.org 9 Detect Fusion Transcripts in FFPE for Tumor Progression Detect Fusion Transcripts in FFPE for Tumor Progression All Tier- 1 and Tier-2 fusions from Figure 2B are included regardless of TaqMan status. The splicing consensus sequences are included for47 TaqMan supported fusion transcripts, which all contain the splicing tag GU-AG. Here we have observed a substantially higher percentage of intronic reads (,60%) than what have been reported in many studies using fresh tissue RNA [37]. We believe this is explained by an intron sequence enrichment that occurs as a result of formalin fixation of RNA. We note that in another study using FFPE tissues more than 50% of the reads are intronic [38]. We have excluded the possibility of genomic DNA (gDNA) contamination in our FFPE RNA preparations by use of criteria: DNAase I treatment, and confirmation by TaqMan assays for gDNA (Table S4). The increased proportion of intronic reads from FFPE specimens may reflect selective degradation of cytoplasmic RNA (i.e., non-intronic RNA) by RNase during formalin fixation [39]. Table S2 An excel file contains all TaqMan results. (XLSX) Table S3 An excel file contains the complete information of 19 fusion junction sequences confirmed by PGM. Table S3 An excel file contains the complete information of 19 fusion junction sequences confirmed by PGM. (XLSX) Table S4 TaqMan assays for examination of residual gDNA contamination of all Providence and Rush cohorts using beta- actin. Each sample from Providence has 6 TaqMan replicates, and each sample from Rush has 3 TaqMan replicates. Each TaqMan plate has Human Genomic DNA (Promega Corporation, Madison, WI) triplicates as positive control, and no template triplicates as negative control. A second DNase I treatment was repeated on 2 Providence RNA samples which didn’t pass the first residual gDNA contamination assays. (XLSX) This study demonstrates the technical feasibility and potential biomedical value of being able to detect fusion transcripts in archival tumor specimens having attached clinical records. Although the average frequency of detected fusion transcripts is relatively low per patient, plausibly attributable to the low quality of FFPE RNA-Seq libraries, the frequency of fusion events found in our cohort nevertheless appears to have prognostic significance. Many of the identified fusion partner genes belong to the kinase, phosphatase and ubiquitin ligase families, which are attractive pharmaceutical targets in oncology. Both fusion frequency and tumor prognosis may be linked to cancer genome instability, which can generate chromosome rearrangements and fusion transcripts. Detect Fusion Transcripts in FFPE for Tumor Progression identified fusions have canonical splicing tags while non-canonical splicing is characteristic of RT-derived trans-splicing [36]. Further evidence against RT based trans-splicing artifacts in this study comes from our TaqMan assay results. TaqMan assays were run against amplified RNA samples that shared the same source RNA as the RNA-Seq libraries but were prepared independently. Systematic RT errors would generate dis-concordance between the fusion calls made by the RNA-Seq fusion detection pipeline and TaqMan assays, but fusion transcripts identified by our pipeline and by the TaqMan assays are completely concordant (Table S2). Taken together, these data suggest that the fusion events we identified are unlikely to be due to artifactual trans- splicing events during RNA-Seq library preparation and thus represent bona-fide fusions of genomic or transcriptomic origin. We do acknowledge that the TaqMan assays can tolerate a few single nucleotide variants within the assayed amplicons and, while we think it is unlikely, it is conceivable that some of the identified fusion transcripts are not accurate. vertical line indicates the fusion position on the corresponding protein. The amino acid length and amino acid positions of each fusion position are labeled on the top of each protein. A. The protein domains of ESR1 protein P03372 (UniProt ID). B. The protein domains of AKAP12 protein Q02952 (UniProt ID). C. The protein domains of two predicted fusion protein isoforms ESR1-.AKAP12. The one amino acid insertion generated from the fusion event is labeled on each fusion protein. (EPS) vertical line indicates the fusion position on the corresponding protein. The amino acid length and amino acid positions of each fusion position are labeled on the top of each protein. A. The protein domains of ESR1 protein P03372 (UniProt ID). B. The protein domains of AKAP12 protein Q02952 (UniProt ID). C. The protein domains of two predicted fusion protein isoforms ESR1-.AKAP12. The one amino acid insertion generated from the fusion event is labeled on each fusion protein. (EPS) Figure S2 Kaplan-Meier plots of patient subsets of Providence or Rush patients as a function of fusion numbers, segregated by block age. Either upper quartile or lower quartile based on block age is selected to examine the effect of the block ages on the disease outcome. The log-rank p-values are displayed. (EPS) Table S1 An excel file contains the complete information of all 118 fusion transcripts from Providence and Rush cohorts. Acknowledgments We thank Thomas Wu from Genentech for technical discussion on using GSNAP. We also thank Chris Baker and Iltcho Kerelsky from Genomic Health for providing IT support for this work. Detect Fusion Transcripts in FFPE for Tumor Progression In conclusion, this study significantly enriches the current understanding of breast tumor complexity by discovering a large number of novel fusion transcripts. It confirms one of the challenges of cancer therapeutics, namely that each cancer is different and personalized treatment is needed. In parallel we demonstrate a unique approach that reveals the genetic compo- sitions of individual cancers employing short read sequencing methods and bioinformatics analysis adapted for FFPE tumor tissues. Author Contributions Conceived and designed the experiments: YM JS ML KQ EMB SL R. Ambannavar R. Abramson. Performed the experiments: YM JS JJ AJF JJL R. Ambannavar. Analyzed the data: YM JS ADR KQ R. Ambannavar. Contributed reagents/materials/analysis tools: YM JJ JS SL R. Amban- navar R. Abramson. Wrote the paper: YM JB SL EMB KQ. Supporting Information Figure S1 Protein domains of fusion ESR1-.AKAP12 are illustrated based on UniProt database (www.uniprot.org). The red Philadelphia Chromosome. New England Journal of Medicine 344: 1038–1042. doi:10.1056/NEJM200104053441402 4. Soda M, Choi YL, Enomoto M, Takada S, Yamashita Y, et al. (2007) Identification of the transforming EML4–ALK fusion gene in non-small-cell lung cancer. Nature 448: 561–566. doi:10.1038/nature05945 3. Druker BJ, Sawyers CL, Kantarjian H, Resta DJ, Reese SF, et al. (2001) Activity of a Specific Inhibitor of the BCR-ABL Tyrosine Kinase in the Blast Crisis of Chronic Myeloid Leukemia and Acute Lymphoblastic Leukemia with the 2. Nowell PC, Hungerford DA (1960) A minute chromosome in chronic granulocytic leukemia. Science 132: 1497–1501. Discussion Single molecule sequencing and other long read approaches aimed at increasing read length are expected to generally improve detection of genomic rearrange- ments, but the benefit of these improvements for FFPE specimens will be limited due to the short RNA fragments isolated from archived FFPE samples. Rapidly decreasing sequencing costs will enable data collection on more archived FFPE samples, therefore we anticipate that the method presented here will continue to facilitate fusion transcript detection and biomarker discovery in FFPE RNA. An important feature of the fusion transcript detection pipeline described here is the use of expression profiling to nominate candidate fusion transcripts from RNA-Seq data that has sparse coverage of fusion junctions. With the dataset analyzed here, this step (Step 7) retains 8% of fusion candidates (Figure 1A). Generally, pathologically important gene fusions in cancer are characterized by one gene that is expressed at relatively high levels in non-fused state fused to another gene that is expressed at relatively low levels in non-fused state, the strong promoter of the 59 gene up-regulates expression of an oncogenic 39 gene (‘‘oncogenic gene fusion model’’) [34]. This predicts discontinuous expression patterns could be observed at either 59 donor or 39 acceptor fusion junction sites. Among 31 TaqMan supported high confidence Tier-1 fusions identified here (Figure 1B), 77% of them exhibit such interrupted expression patterns at fusion junctions Fusion transcripts may result from genuine genomic rearrange- ments or transcript level rearrangements such as trans-splicing [35]. One type of widely occurring, but biologically irrelevant trans-splicing, is a reverse transcriptase (RT) artifact derived from sequence homology [36]. Although our method doesn’t distinguish genuine genomic rearrangement-derived gene fusions from trans- splicing derived fusions, there is no evidence of RT derived fusion artifacts in our study. First, our method searches for template sequence homologies to effectively remove false positive fusions generated by mapping algorithm or RT errors. Second, the April 2014 \| Volume 9 \| Issue 4 \| e94202 PLOS ONE \| www.plosone.org 10 Detect Fusion Transcripts in FFPE for Tumor Progression 1. Hanahan D, Weinberg RA (2011) Hallmarks of Cancer: The Next Generation. Cell 144: 646–674. doi:10.1016/j.cell.2011.02.013 2. Nowell PC, Hungerford DA (1960) A minute chromosome in chronic granulocytic leukemia. Science 132: 1497–1501. 3. Druker BJ, Sawyers CL, Kantarjian H, Resta DJ, Reese SF, et al. (2001) Activity of a Specific Inhibitor of the BCR-ABL Tyrosine Kinase in the Blast Crisis of Chronic Myeloid Leukemia and Acute Lymphoblastic Leukemia with the 1. Hanahan D, Weinberg RA (2011) Hallmarks of Cancer: The Next Generation. Cell 144: 646–674. doi:10.1016/j.cell.2011.02.013 of a Specific Inhibitor of the BCR-ABL Tyrosine Kinase in the Blast Crisis of Chronic Myeloid Leukemia and Acute Lymphoblastic Leukemia with the Detect Fusion Transcripts in FFPE for Tumor Progression Detect Fusion Transcripts in FFPE for Tumor Progression 5. Kwak EL, Bang Y-J, Camidge DR, Shaw AT, Solomon B, et al. (2010) Anaplastic Lymphoma Kinase Inhibition in Non–Small-Cell Lung Cancer. New England Journal of Medicine 363: 1693–1703. doi:10.1056/NEJMoa1006448 21. Cobleigh MA, Tabesh B, Bitterman P, Baker J, Cronin M, et al. (2005) Tumor Gene Expression and Prognosis in Breast Cancer Patients with 10 or More Positive Lymph Nodes. Clin Cancer Res 11: 8623–8631. doi:10.1158/1078- 0432.CCR-05-0735 g 6. Kohno T, Ichikawa H, Totoki Y, Yasuda K, Hiramoto M, et al. (2012) KIF5B- RET fusions in lung adenocarcinoma. Nature Medicine 18: 375–377. doi:10.1038/nm.2644 22. Paik S, Shak S, Tang G, Kim C, Baker J, et al. (2004) A Multigene Assay to Predict Recurrence of Tamoxifen-Treated, Node-Negative Breast Cancer. New England Journal of Medicine 351: 2817–2826. doi:10.1056/NEJMoa041588 7. Takeuchi K, Soda M, Togashi Y, Suzuki R, Sakata S, et al. (2012) RET, ROS1 and ALK fusions in lung cancer. Nature Medicine 18: 378–381. doi:10.1038/ nm.2658 g 23. Wu G, Feng X, Stein L (2010) A human functional protein interaction network and its application to cancer data analysis. Genome Biology 11: R53. doi:10.1186/gb-2010-11-5-r53 8. Lipson D, Capelletti M, Yelensky R, Otto G, Parker A, et al. (2012) Identification of new ALK and RET gene fusions from colorectal and lung cancer biopsies. Nature Medicine 18: 382–384. doi:10.1038/nm.2673 g 24. Wu TD, Nacu S (2010) Fast and SNP-tolerant detection of complex variants and splicing in short reads. Bioinformatics 26: 873–881. doi:10.1093/bioinformatics/ btq057 9. Ju YS, Lee W-C, Shin J-Y, Lee S, Bleazard T, et al. (2012) A transforming KIF5B and RET gene fusion in lung adenocarcinoma revealed from whole- genome and transcriptome sequencing. Genome Res 22: 436–445. doi:10.1101/ gr.133645.111 q 25. Anders S, Huber W (2010) Differential expression analysis for sequence count data. Genome Biol 11: R106. doi:10.1186/gb-2010-11-10-r106 26. Edgren H, Murumagi A, Kangaspeska S, Nicorici D, Hongisto V, et al. (2011) Identification of fusion genes in breast cancer by paired-end RNA-sequencing. Genome Biol 12: R6. doi:10.1186/gb-2011-12-1-r6 10. Robinson DR, Kalyana-Sundaram S, Wu Y-M, Shankar S, Cao X, et al. (2011) Functionally recurrent rearrangements of the MAST kinase and Notch gene families in breast cancer. Nature Medicine 17: 1646–1651. doi:10.1038/ nm.2580 g 27. The Cancer Genome Atlas Research Network (2013) Comprehensive molecular characterization of clear cell renal cell carcinoma. Nature 499: 43–49. doi:10.1038/nature12222 11. Asmann YW, Necela BM, Kalari KR, Hossain A, Baker TR, et al. Detect Fusion Transcripts in FFPE for Tumor Progression (2012) Detection of Redundant Fusion Transcripts as Biomarkers or Disease-Specific Therapeutic Targets in Breast Cancer. Cancer Res 72: 1921–1928. doi:10.1158/0008-5472.CAN-11-3142 28. Mitelman F, Johansson B, Mertens F (2012) Mitelman Database of Chromosome Aberrations and Gene Fusions in Cancer. Available: http:// cgap.nci.nih.gov/Chromosomes/Mitelman. Accessed 2012 October 24 29. Rubino D, Driggers P, Arbit D, Kemp L, Miller B, et al. (1998) Characterization of Brx, a novel Dbl family member that modulates estrogen receptor action. Oncogene 16: 2513–2526. doi:10.1038/sj.onc.1201783 12. Seshagiri S, Stawiski EW, Durinck S, Modrusan Z, Storm EE, et al. (2012) Recurrent R-spondin fusions in colon cancer. Nature 488: 660–664. doi:10.1038/nature11282 30. Streb JW, Kitchen CM, Gelman IH, Miano JM (2004) Multiple Promoters Direct Expression of Three AKAP12 Isoforms with Distinct Subcellular and Tissue Distribution Profiles. J Biol Chem 279: 56014–56023. doi:10.1074/ jbc.M408828200 13. Kim D, Salzberg SL (2011) TopHat-Fusion: an algorithm for discovery of novel fusion transcripts. Genome Biol 12: R72. doi:10.1186/gb-2011-12-8-r72 14. Ge H, Liu K, Juan T, Fang F, Newman M, et al. (2011) FusionMap: detecting fusion genes from next-generation sequencing data at base-pair resolution. Bioinformatics 27: 1922–1928. doi:10.1093/bioinformatics/btr310 31. Network TCGAR (2013) Integrated genomic characterization of endometrial carcinoma. Nature 497: 67–73. doi:10.1038/nature12113 15. Francis RW, Thompson-Wicking K, Carter KW, Anderson D, Kees UR, et al. (2012) FusionFinder: A Software Tool to Identify Expressed Gene Fusion Candidates from RNA-Seq Data. PLoS ONE 7: e39987. doi:10.1371/journal.- pone.0039987 32. Network TCGA (2012) Comprehensive molecular characterization of human colon and rectal cancer. Nature 487: 330–337. doi:10.1038/nature11252 33. Network TCGAR (2012) Comprehensive genomic characterization o cell lung cancers. Nature 489: 519–525. doi:10.1038/nature11404 16. Cronin M, Pho M, Dutta D, Stephans JC, Shak S, et al. (2004) Measurement of Gene Expression in Archival Paraffin-Embedded Tissues: Development and Performance of a 92-Gene Reverse Transcriptase-Polymerase Chain Reaction Assay. The American Journal of Pathology 164: 35–42. doi:10.1016/S0002- 9440(10)63093-3 34. Mitelman F, Johansson B, Mertens F (2007) The impact of translocations and gene fusions on cancer causation. Nature Reviews Cancer 7: 233–245. doi:10.1038/nrc2091 35. Li H, Wang J, Mor G, Sklar J (2008) A Neoplastic Gene Fusion Mimics Trans- Splicing of RNAs in Normal Human Cells. Science 321: 1357–1361. doi:10.1126/science.1156725 17. Sinicropi D, Qu K, Collin F, Crager M, Liu M-L, et al. (2012) Whole Transcriptome RNA-Seq Analysis of Breast Cancer Recurrence Risk Using Formalin-Fixed Paraffin-Embedded Tumor Tissue. PLoS ONE 7: e40092. doi:10.1371/journal.pone.0040092 36. References April 2014 \| Volume 9 \| Issue 4 \| e94202 April 2014 \| Volume 9 \| Issue 4 \| e94202 PLOS ONE \| www.plosone.org 11 Detect Fusion Transcripts in FFPE for Tumor Progression Houseley J, Tollervey D (2010) Apparent Non-Canonical Trans-Splicing Is Generated by Reverse Transcriptase In Vitro. PLoS ONE 5: e12271. doi:10.1371/journal.pone.0012271 18. Tomlins SA, Rhodes DR, Perner S, Dhanasekaran SM, Mehra R, et al. (2005) Recurrent Fusion of TMPRSS2 and ETS Transcription Factor Genes in Prostate Cancer. Science 310: 644–648. doi:10.1126/science.1117679 37. Sharon D, Tilgner H, Grubert F, Snyder M (2013) A single-molecule long-read survey of the human transcriptome. Nat Biotech advance online publication. Available: http://www.nature.com/nbt/journal/vaop/ncurrent/full/nbt.2705. html. Accessed 2013 November 2. 19. Sboner A, Habegger L, Pflueger D, Terry S, Chen DZ, et al. (2010) FusionSeq: a modular framework for finding gene fusions by analyzing paired-end RNA- sequencing data. Genome Biol 11: R104. doi:10.1186/gb-2010-11-10-r104 38. Adiconis X, Borges-Rivera D, Satija R, DeLuca DS, Busby MA, et al. (2013) Comparative analysis of RNA sequencing methods for degraded or low-input samples. Nat Meth 10: 623–629. doi:10.1038/nmeth.2483 20. McPherson A, Hormozdiari F, Zayed A, Giuliany R, Ha G, et al. (2011) deFuse: An Algorithm for Gene Fusion Discovery in Tumor RNA-Seq Data. PLoS Comput Biol 7. Available: http://www.ncbi.nlm.nih.gov/pmc/articles/ PMC3098195/. Accessed 2012 September 11. p 39. Morlan JD, Qu K, Sinicropi DV (2012) Selective Depletion of rRNA Enables Whole Transcriptome Profiling of Archival Fixed Tissue. PLoS ONE 7: e42882. doi:10.1371/journal.pone.0042882 PLOS ONE \| www.plosone.org April 2014 \| Volume 9 \| Issue 4 \| e94202 12
https://openalex.org/W3085580155	https://link.springer.com/content/pdf/10.1007/s10814-020-09149-7.pdf	English	null	Pleistocene Water Crossings and Adaptive Flexibility Within the Homo Genus	Journal of archaeological research	2,020	cc-by	37,830	Journal of Archaeological Research (2021) 29:255–326 https://doi.org/10.1007/s10814-020-09149-7 Journal of Archaeological Research (2021) 29:255–326 https://doi.org/10.1007/s10814-020-09149-7 * Dylan Gaffney dacg2@cam.ac.uk Pleistocene Water Crossings and Adaptive Flexibility Within the Homo Genus Dylan Gaffney1 Published online: 14 September 2020 © The Author(s) 2020 1 Department of Archaeology, University of Cambridge, Downing Street, Cambridge CB2 3DZ, UK Abstract Pleistocene water crossings, long thought to be an innovation of Homo sapiens, may extend beyond our species to encompass Middle and Early Pleistocene Homo. How- ever, it remains unclear how water crossings differed among hominin populations, the extent to which Homo sapiens are uniquely flexible in these adaptive behaviors, and how the tempo and scale of water crossings played out in different regions. I apply the adaptive flexibility hypothesis, derived from cognitive ecology, to model the global data and address these questions. Water-crossing behaviors appear to have emerged among different regional hominin populations in similar ecologies, initially representing nonstrategic range expansion. However, an increasing readiness to form connections with novel environments allowed some H. sapiens populations to even- tually push water crossings to new extremes, moving out of sight of land, making return crossings to maintain social ties and build viable founder populations, and dramatically shifting subsistence and lithic provisioning strategies to meet the chal- lenges of variable ecological settings. Keywords Pleistocene seafaring · Island colonization · Maritime technology · Migration · Hominin behavior · Adaptive flexibility Introduction Crossing substantial bodies of water—lakes, straits, seas, and oceans—to arrive at new landmasses has previously been seen as a unique innovation within our spe- cies, Homo sapiens. These adaptive capacities were thought to be first acquired as Electronic supplementary material The online version of this article (https://doi.org/10.1007/s1081 4-020-09149-7) contains supplementary material, which is available to authorized users. 456789) 3 Journal of Archaeological Research (2021) 29:255–326 256 a population of anatomically and behaviorally “modern” H. sapiens moved out of Africa and skirted the coast of southern Eurasia before entering Wallacea (Island Southeast Asia) and Sahul (Australia and New Guinea), sometime after 60,000 years ago. Over a quarter century ago, Davidson and Noble (1992) described this maritime colonization as the earliest global evidence for modern human behavior, because the deliberate water crossings through island Wallacea to continental Sahul were associated with systems of symbolic communication and shared meaning (i.e., language) to produce effective seagoing vessels. It also required forward planning and technological provisioning to envisage potential futures, to predict currents and weather conditions, and to arrange regular return trips to recruit new individuals for establishing a viable founder population. More recently, several crucial (and controversial) sites around the world have sparked debate and have been cited as evidence that the first water crossings were made by earlier hominin lineages, potentially extending the global history of seafar- ing into the Early and Middle Pleistocene. At the same time, following the discovery of numerous Middle Pleistocene H. sapiens sites across eastern Eurasia, unilinear models for the dispersal of coastally adapted Late Pleistocene H. sapiens along the southern coast of Eurasia are now untenable (Dennell and Petraglia 2012; Rabett 2018). Rather, there seems to have been a series of complex dispersal processes and population interactions predating 70,000 years ago (Martinón-Torres et al. 2017). Moreover, the antiquity of pigment use, engraving, and personal decoration, con- ventionally used to mark the emergence of modern human behavior in H. sapiens, has been pushed back in time and attributed to Homo erectus (Joordens et al. 2015), Homo neanderthalensis (Hoffmann et al. 2018), and possibly Homo heidelbergensis (Burdukiewicz 2014). Thus, the concept of “modernity”—what it is to be a behav- iorally modern and distinct species—and how this interrelates to water-crossing behaviors needs to be reexamined. In this paper, I wade into the debate about Pleistocene water crossings, island colonization, and what this means for understanding behavioral variability within our genus. 1 3 Introduction This builds on pivotal discussions by Keegan and Diamond (1987), Bednarik (2003), Leppard (2015b), and Erlandson (2017), who have investigated the same topic from archaeological and biogeographic perspectives. In particular, I pose three questions: how did water crossings vary across different hominin popu- lations, to what extent did H. sapiens uniquely express adaptive flexibility during water crossings and the colonization of novel environments, and how can this help us understand the rate and scale of hominin adaptive flexibility in different regions? The Pleistocene is a useful temporal focus for this discussion, marking the emer- gence of the Homo genus, and our own species H. sapiens, alongside glacial and interglacial transgressions prior to the establishment of approximately modern sea levels during the Holocene.i The paper first summarizes recent cognitive ecological literature and proposes the concept of adaptive flexibility as a useful work-around to move us past the relatively polarized debate about whether or not different hominin species strategically crossed water gaps. The mechanisms of adaptive flexibility can be tested through two mod- els that describe the rate and scale of behavioral transformations, as set within the framework of established analytical terminology and typology for the study of 1 3 Journal of Archaeological Research (2021) 29:255–326 257 water-crossing behaviors and island colonization processes. Next, paleoenviron- mental information highlights the diachronic rates of global changes in sea level, vegetation, and animal resources during the Pleistocene. Global archaeological data are then presented by region to emphasize temporal and spatial variation present within the clade and to tease apart the variable emergence rates for these behaviors in different parts of the world. My primary aim is to use these data to rethink the nature of hominin adaptive flexibility and how it is tethered to local ecologies and long-term behavioral trajectories in different regional populations. In the discussion section, I look into the behavioral overlaps and contrasts between different regional populations, and examine how this played out among H. sapiens specifically. I argue that ecological contingencies crafted long-term behavioral trajectories in different regional populations, rather than inherent capacities for water crossings existing only within H. sapiens. However, the increasing readiness to form new connections with novel environments seems to have led our species to push water-crossing behaviors, and their adaptive flexibility, into new frontiers. “Modernity” and Adaptive Flexibility Archaeologists usually look for the origins of modern human behavior by identi- fying evidence for symbolism, technological complexity, abstract thinking, and planning depth (McBrearty and Brooks 2000). There remains intense debate as to whether these behaviors emerged within only our species, or within multiple homi- nin species (d’Errico 2003; Klein 2008; Mellars 2005). However, recent studies pre- sent a substantial record for H. neanderthalensis symbolism in the form of pigment use, cave painting, and personal adornment. For instance, on the Iberian Peninsula red pigment hand stencils and rectilinear forms have been directly dated to at least 65,000 years ago, prior to the movement of H. sapiens into the area (Hoffmann et al. 2018). There is also some evidence for primordial H. erectus symbolism, in the form of engraved shell in Java (Joordens et al. 2015). Modernity as a single-species behavioral package begins to breakdown further when we turn to eastern Eurasia and the Pacific (Habgood and Franklin 2008), where there is increasing chronological overlap between late surviving or “pro- gressive” H. erectus with large cranial capacities, Denisovan hominins, archaic H. sapiens or even H. heidelbergensis, and early modern H. sapiens dating to the Mid- dle–Late Pleistocene (Kaifu 2017). In the east, the primary evidence used to distin- guish behavioral modernity among Pleistocene H. sapiens has long been the mari- time technology required to navigate from Sunda to Sahul.i As such, it is clear that the modernity classification itself is regionally varied and problematic in that it creates a false dichotomy between the “moderns” and the “archaics” (Roberts 2015). Shea (2011) suggests that “behavioral variability” is a preferable, scalar description for the broadening of cognitive horizons, which emerged gradually in the Middle and Late Pleistocene. It is then not necessary for specific populations to fulfill a checklist of modern behaviors because these inno- vations, such as pigment use, technological provisioning, and maritime technology, 3 3 Journal of Archaeological Research (2021) 29:255–326 258 arose at different times, in different areas, at different scales, and among different morphological species. Although substantial behavioral variability is evident among Late Pleistocene H. sapiens, the same scale of variability was not innate among all hominin populations; rather it emerged over time, following nondirectional behavioral trajectories. Rob- erts and Stewart’s (2018) reconceptualization of H. sapiens as “generalist special- ists” perfectly illustrates this. Present-day H. “Modernity” and Adaptive Flexibility sapiens have the cognitive potential to adapt to the same range of resources selected by generalist species, such as broad- spectrum scavengers, but individual populations and social units tend to specialize in specific resource webs, often engaging intergenerational knowledge acquired over hundreds or thousands of years. These specializations are not static and H. sapiens are adept at forming new connectivities to novel environments, reorienting our own behavioral trajectories to address challenges, to the extent that we could describe the species as “hyper-adaptable” (Hiscock 2015), “infinitely adaptable” (Veth 2005), or “behaviorally plastic” (Roberts and Amano 2019). Crossing water gaps to explore new lands is one activity that often encourages the expression of this plasticity and experimentation with new adaptive behaviors, as it is in other species pushed to the edge of their ecological range (Kozlovsky et al. 2015; Liebl and Martin 2014; Webb et al. 2014).l Drawing on cognitive ecology literature, I adopt the term “adaptive flexibility” as a way to describe the reorientation of behavioral trajectories for adaptive success when humans move into new environments or at times of perceptible environmen- tal change. Adaptive flexibility is an important response in many species that enter novel environments (Reader and Laland 2002). It allows colonizing populations to exploit novel food sources and habitats as they create new ecological niches for themselves (Price et al. 2008). Substantial adaptive flexibility is identifiable among recent human groups, with proxies coming from changing resource selection, for- aging strategies, shelter selection, group size, and risk reduction strategies. Cross- generational cultural niche construction has been central to hominin evolution, as we are able to alter our behaviors in response to the environments that we ourselves engineer (Boivin et al. 2016). There are inevitably temporal lags in adaptations to novel environments (Laland and Brown 2006). These adaptations are expressed on the individual level, where new behaviors are innovated, and on the social level, where innovations are shared within and between groups. It is one form of rapid response that can operate on the scale of an individual’s lifetime, which may be interrelated with multigenerational genetic and physiological changes (Dukas 1998; West-Eberhard 2003). This type of innovation paired with social learning can be advantageous when environments change, or new environments are explored, but it can be disadvantageous when envi- ronments change so rapidly that prior learning is outdated (Dukas 1998).l The adaptive flexibility hypothesis (Wright et al. 1 3 “Modernity” and Adaptive Flexibility 2010) states that the number of behavioral variants will be high during the introduction stage of colonization due to adaptive innovations and then will gradually decline as successful behaviors are shared within the group during the establishment period of population growth (Fig. 1). This happens because sharing successful behaviors between individuals is favored over further adaptive innovations and results in a consolidation of, and 1 3 Journal of Archaeological Research (2021) 29:255–326 259 2 1 3 urnal of Archaeological Research (2021) 29:255 326 g. 1 The adaptive flexibility hypothesis (adapted from Wright et al. 2010) Fig. 1 The adaptive flexibility hypothesis (adapted from Wright et al. 2010) Fig. 1 The adaptive flexibility hypothesis (adapted from Wright et al. 2010) 3 Journal of Archaeological Research (2021) 29:255–326 260 decline in, in-group behavioral variability. It is possible that adaptively redundant or “nonrational” behaviors also may appear and be shared within groups without compromising that founder population. Later, when the founder population grows large enough, it enters an expansion stage (cf. Wright et al. 2010 “invasion” stage), whereby daughter populations are spawned, encouraging further behavioral varia- tions. This model emphasizes the historical and ecological contingencies of adap- tation, modified by individual and shared experiences in the landscape (social and environmental). It also emphasizes that, contrasting with many models of modern human behavior, adaptive flexibility is not intrinsic to human nature: it can be vari- able over a lifetime, across generations, and between populations. This fits well with current trends in anthropology, which suggest behavior is not hard-wired and bio- logically constant within species but is a dynamic transformation of continuous cultural, material, and environmental engagements (Gosden and Malafouris 2015; Ingold 2003; Malafouris 2013). It also provides a diachronic explanation for how the “generalist specializations” of H. sapiens can emerge, with the capacity for distinct behavioral change in the face of new ecological surroundings, followed by periods of increasing specialization that becomes part of that ecology.l There are several conceptual alternatives to the adaptive flexibility hypothesis (Fig. 1, from Wright et al. 2010). In Alternative 1, some populations might con- sistently operate at high levels of flexibility or inflexibility regardless of context; in Alternative 2, others might display initial innovation, but not share these innovations within the group; or in Alternative 3, some populations might innovate and share behavioral variations but not exhibit increased diversity within daughter populations. “Modernity” and Adaptive Flexibility Coastal and maritime subsistence strategies and making water crossings to tar- get resources previously out of reach are expressions of adaptive flexibility in novel environs. However, is this adaptive flexibility unique to H. sapiens? To what extent did other species also make deliberate water crossings, adapt to island life, and incorporate marine resources into their subsistence strategies? As Leppard and Run- nels (2017) state, this topic is of profound importance for how we understand homi- nin cognitive evolution. Typologizing Water‑Crossing Behaviors The history of thought on hominin capacities for water crossings has ebbed and flowed over the past century (see Erlandson 2001). Fundamentally, water cross- ings do pose distinct challenges for human mobility, which often promote cultural and demographic isolation (Fitzpatrick and Anderson 2008). In the wake of early island biogeographic approaches (e.g., Cherry 1981), Leppard (2015a, b) synthe- sizes this standard model, positing that bodies of water formed important barriers to hominin expansion prior to the Late Pleistocene and that even early water crossings were structured on a global level, with larger continental islands being colonized first, as they present more resource abundance and lower economic risk for incom- ing colonists. This model suggests that intentional seagoing arose solely among H. sapiens (or possibly H. neanderthalensis), following the emergence of “modern cognitive architecture” (Leppard 2015b). According to Leppard (2015a), intentional 1 3 Journal of Archaeological Research (2021) 29:255–326 261 dispersal required projection of future action, group planning, and composite tech- nology. Projection of future action, or thinking ahead about possible realities, is always informed by previous experience and is a core feature of present-day H. sapi- ens water crossings. As Leppard notes, it is a great leap from standing on the shore and wondering about what lies over the horizon, to actually mobilizing oneself with various tools to get there. Group planning not only involves social living and resid- ing in groups large enough to form viable founder populations but also the ability to express complex ideas of abstraction (i.e., possible realities) and to convince oth- ers to achieve mutual goals. Composite technology involves combining component parts that by themselves are insufficient for the intended function, which implies something about the cognitive plasticity of the makers: the ability to project future action or to think ahead of the material (Leppard 2015b). Many nonhuman animals raft or swim to new landmasses (Fig. 2). In this sense water crossings are not exceptional to hominins, rather it is the nature, scale, and deliberateness of the crossings that sets our species apart (Leppard 2015a). A central distinction here is whether dispersals to new pieces of land were intentional. Fol- lowing biogeographic theory, Leppard (2015a) divides maritime dispersal into “pas- sive” and “strategic” (cf. Ruxton and Wilkinson 2012, “accidental” vs. “planned”). Typologizing Water‑Crossing Behaviors Passive dispersals involve no specific cognitive processes about the dispersal itself, while strategic dispersals are self-reflexive, involving cost-benefit projections about the water crossing and subsequent access to new resources. However, it is unclear if a group of hominins raft or swim to new land, forming a viable founder population, Fig. 2 Swimming ranges of human and nonhuman animals (based on data in Bender 2015; Schoener and Schoener 1984) Fig. 2 Swimming ranges of human and nonhuman animals (based on data in Bender 2015; Schoener and Schoener 1984) 1 3 3 Journal of Archaeological Research (2021) 29:255–326 262 whether they chose to do so in the same way that H. sapiens make decisions, and whether this would be considered passive or strategic. In contrast, in a multispecies model proposed by Runnels (2014) in the Medi- terranean and Bednarik (2003) in Wallacea, strategic, long-distance water cross- ings are viewed as distinctly within the capacity of not only H. sapiens but also H. neanderthalensis, H. heidelbergensis, H. erectus, and the ancestors of H. floresien- sis. Erlandson (2001) demonstrates that aquatic resources, including fresh water, shellfish, fish, reptiles, and amphibians, have been important to our genus since the Plio-Pleistocene, and that hominins would have experimented with water crossings (even short river crossings) during any major dispersal around the planet. From this revisionist perspective (see Leppard 2015b), archipelagos are described as express highways rather than barriers. These models are ultimately borne from paradigmatic shifts in the late 20th century, which emphasized coastal and island adaptations in human migration narratives, especially as they pertain to the “southern disper- sal route,” the rapid colonization of Sahul by H. sapiens, coastal occupation by H. neanderthalensis, and the peopling of the Americas (Bailey and Milner 2002). I contend that in this debate—the archaeological questions about which popu- lations made deliberate water crossings and which species succeeded with strate- gic oceanic colonizations—two behavioral processes have been conflated. We can tease apart these questions by examining water crossings as mobility behaviors and island habitation as colonization processes. Most of the Pleistocene record is devoid of direct evidence for maritime mobility behaviors (i.e., boats), and we rely on indirect site distributions, artifact sourcing, and isotopic data relative to Pleis- tocene sea levels. Typologizing Water‑Crossing Behaviors To characterize these behaviors, Broodbank (2006) puts forward a scalar typology: “seagoing” indicates the simplest sea-crossing techniques, “sea- faring” indicates more proficient mid-range technologies, and “voyaging” indicates highly skilled, intentional, and often long-range navigation. I also incorporate the term “water crossing” where there is insufficient evidence to describe the scale and complexity of how a water gap was crossed. These mobility behaviors often cor- respond with, but are not necessarily causally linked to, the scale and success of colonization, for which we have distinct evidence in the form of paleoenvironmental change, faunal extinctions, tool modification, ancient genetics, settlement scale and longevity, and lithic landscape learning. Using such datasets, we can start to address the nature, timing, and scale of water crossings made by different regional populations throughout the Pleisto- cene and how they articulate with colonization processes in new environs. Using the adaptive flexibility hypothesis, we can posit that hominins, whether they crossed water gaps intentionally or not, would have displayed wider behavioral variability during introduction phases of the colonization process, perhaps sam- pling from varied and novel food webs and experimenting with new raw mate- rials to overcome technological challenges such as constructing watercraft to further expand resource patches. Perhaps counterintuitively, these innovations would come at the time when a founder population was small and most sensitive to local extinction (Leppard 2015c, 2016). The variability and effectiveness of these innovations, including additional water crossings, is reflective of the scope of adaptive flexibility expressed within each population and the tempo of these 1 3 Journal of Archaeological Research (2021) 29:255–326 263 adaptive changes. I put forward two testable models (Fig. 3). The “rapid flex- ibility hypothesis” stresses hominins, particularly H. sapiens, are highly adept at changing to new environments. They are able to alter subsistence and techno- logical behaviors so rapidly (within a few generations or several hundred years) that the switch is archaeologically undetectable. This results in hominins arriving in coastal areas, monopolizing aquatic resources, and developing water-crossing abilities very quickly. A second, alternative model is the “gradual modification hypothesis,” which stresses that hominins, including H. sapiens, are adaptively flexible, but major subsistence and technological changes such as water crossings and maritime lifeways derive from long-term behavioral trajectories and can take several millennia, or tens of millennia to emerge, especially if there is no immedi- ate incentive for such behavioral change. 1 3 Fig. Typologizing Water‑Crossing Behaviors 3 Two models for the tempo of adaptive flexibility Fig 3 Two models for the tempo of adaptive flexibility Fig. 3 Two models for the tempo of adaptive flexibility 3 Journal of Archaeological Research (2021) 29:255–326 264 Global Environmental Changes To understand the rates of adaptive flexibility within different regional populations as it pertains to water crossings, it is crucial to understand the rate of environmen- tal change through archaeological time (Fig. 4). This has direct implications for the distance required to get from one landmass to another and the ecologies encountered on arrival. Since the beginning of the Middle Pleistocene, global sea levels have fluctuated over 130 m approximately every 100,000 years during glacial/interglacial transitions (Bintanja et al. 2005). It is likely that Marine Isotope Stage (MIS) 2 (the Last Glacial Maximum, LGM), MIS-6, MIS-12, and MIS-16 were the most extreme glacial maxima, which resulted in the lowest eustatic sea levels as well as ice sheet corridors in the Northern Hemisphere, while MIS-5e, MIS-9, and MIS-11 were the warmest interglacials with high sea levels (Murray-Wallace and Woodroffe 2014, p, 262). Although many MIS-3 and MIS-4 paleobeaches are now submerged, limiting Fig. 4 A timeline of global temperate and sea-level change from the Middle Pleistocene to Holocene. Climate curve data derive from Lowe and Walker (2014) and elevation maps derive from Zong (2015), which show land exposed by glacial cycles in red Fig. 4 A timeline of global temperate and sea-level change from the Middle Pleistocene to Holocene. Climate curve data derive from Lowe and Walker (2014) and elevation maps derive from Zong (2015), which show land exposed by glacial cycles in red 1 3 Journal of Archaeological Research (2021) 29:255–326 265 our ability to understand major dispersals out of Africa (Bailey and Milner 2002), the shorelines of MIS-5e, MIS-13, and MIS-15 would be similar to today, while oth- ers such as MIS-11 would be over 10 m higher than present and some way inland (Bowen 2003). Regionally, there also was substantial variation in relative sea levels due to local emergence rates and more dramatic tectonically induced uplift and sub- sidence events (Murray-Wallace and Woodroffe 2014, p. 278). f In Asia, the Sunda shelf, forming an extension to mainland Southeast Asia, has been repeatedly submerged and exposed during marine transgressions. This turned the islands of Borneo, Java, Sumatra, Taiwan, and the seabed of western Indone- sia into a large continental peninsula the size of present-day Europe, although the Philippines always remained insular (Sathiamurthy and Voris 2006). Global Environmental Changes To the east, the islands of New Guinea and Tasmania were attached to Australia during low stands, forming the continent of Sahul, with the Gulf of Carpentaria becoming a large pale- olake (Summerhayes and Ford 2014). Located between Sunda and Sahul, Wallacea has always consisted of islands such as Sulawesi, Flores, Timor, Seram, Halmahera, and parts of the Raja Ampat group, separated by deep sea trenches. Off Sahul’s northeast coast, the Bismarck Archipelago and Solomon Island chain also were always separated from the mainland. Farther north, Japan was part of a larger land- mass and variously connected to continental Eurasia, although the Ryukyu Islands between Kyusu and Taiwan were predominantly insular (Takamiya et al. in press), while the Beringia land bridge accompanied by ice sheets connected eastern Eura- sia to the Americas during glacial periods; the two continents were separated most recently by approximately 15,000 years ago (Hopkins 1982). In the Mediterranean, following the Messinian Salinity Crisis, the Zanclean Del- uge refilled the basin with water from the Atlantic, forming many of the islands in the region about 5.33 million years ago (Blanc 2002). Although some islands such as Crete, Gavdos, and Cyprus were clearly separated from mainland Greece throughout the Pleistocene, it is uncertain if other islands were due to variable rates of tectonism (Benjamin et al. 2017). Lykousis (2009) suggests that the margins of the Aegean have been subsiding for the past 400,000 years ago at rates of 0.7–1.9 m per 1000 years, particularly during MIS-12 and MIS-10–MIS-8. During these Mid- dle Pleistocene glacial periods, 50–60% of the present-day Aegean would have been attached to the mainland, part of a basin with extensive drainage systems, lakes, and river deltas. Sea-level change and global climate shifts also were core mechanisms that shaped biogeography during the Pleistocene (Hanebuth et al. 2011). During glacial periods, lowered sea levels broadly correspond with aridity and slow sedimentation rates, especially in Sahul (Vannieuwenhuyse 2016) and savannah grasslands in Sunda and highland New Guinea (Louys and Turner 2012). Conversely, during the climatic ameliorations of interglacial and interstadial periods, stable increases in sea level, particularly in equatorial zones, led to expansions of coral reefs, swamps, forests, and wetlands rich in littoral resources (Chappell 1993) and massively reconfigured river systems and estuarine environments (Voris 2000). These periods also were associated with increased humidity, weathering, and pedogenesis (Burckle 1993). Getting Our Feet Wet in Africa, Arabia, and Southern Eurasia Africa was the evolutionary hub for a number of different Homo species, where riv- erine and lakeside aquatic resources, including drinking water and shellfish, were opportunistically targeted since the Early and Middle Pleistocene (Erlandson 2001). Excavations at Blombos Cave in southern Africa show that Middle Stone Age homi- nins were beginning to adapt to the coastal zone, which included diving for marine shells, catching large littoral fish, hunting seals, and probably scavenging beached dolphins, in addition to targeting a wide array of terrestrial animals (Henshilwood et al. 2001). At the Pinnacle Point site, also in southern Africa, the move to the coast occurred 164,000 years ago as a response to harsh aridity, although the change to targeting marine resources, including shellfish and perhaps even processing beached whales, developed over time, becoming prominent 110,000 years ago (Marean et al. 2007; see also Marean 2014).i A change to coastal subsistence, especially collecting shellfish and scavenging large marine mammals, has important implications for adaptive flexibility, with a shift to fixed resources enabling short-term sedentism. Additionally, reliable protein sources, and the fatty acids common to shellfish in particular, may have profoundly affected the evolution of the brain (Kyriacou et al. 2016). It remains unclear if this change at the southern African sites represents the same continuous lineage of hom- inins or marks a distinct replacement by separate, coastally adapted groups. Recent excavations on the north coast, along with genetic work, have pushed back our species, H. sapiens, to over 300,000 years ago (Hublin et al. 2017; Schlebusch et al. 2017), and there is substantial evidence throughout the continent for subse- quent innovations such as pigment use (Watts et al. 2016), blade production (Wilkins and Chazan 2012), future planning (Texier et al. 2010), and marine shell ornamenta- tion (d’Errico et al. 2009) thought to mark a stretching of behavioral plasticity.f As different African lineages moved north through the Levant, they maintained physical and symbolic connections to the sea. There is evidence for perforated gas- tropod shells from Skhul in the Levant and shell beads from Oued Djebbana in northern Africa 100,000–135,000 years ago (Vanhaeren et al. 2006). Later shell tools at Ksâr ‘Akil dated to 50,000 years ago, signaling a pulse of Homo sapiens into northwest Eurasia through the Levantine corridor (Bosch et al. 2015). Global Environmental Changes Warming from about 14,500 years ago was typically unstable and, in the tropics, was associated with severe monsoon and drought conditions, with modern Holocene 1 3 3 Journal of Archaeological Research (2021) 29:255–326 266 sea levels established approximately 8,000 years ago. These variable and changing Pleistocene environments formed the backdrop for the very first water crossings within our genus. Getting Our Feet Wet in Africa, Arabia, and Southern Eurasia There is no direct evidence for hominins making water crossings on their way out of Africa, but the Bab el Mandab route through the Red Sea remains a viable possibility (Armitage et al. 2011). The Red Sea has been open to the Gulf of Aden since at least MIS-12, 440,000 years ago (Lambeck et al. 2011), but during low stands in MIS-5–MIS-6, crossings of about 4 km were possible. At Abdur, along the Eritrea coast of the Red Sea, early Middle Stone Age artifacts in an emergent reef terrace are dated by U-Th 1 3 Journal of Archaeological Research (2021) 29:255–326 267 dating to about 125,000 years ago, and faunal remains demonstrate these people were coastally adapted (Walter et al. 2000), in accord with evidence for climatically induced movements in southern Africa, due to fluctuating glacial cycles. Some out- of-Africa expansions may reflect pull factors into new coastal areas, with competi- tion driven by increased population sizes and sedentism related to coastal adaptation (Walter et al. 2000).f It is now thought that there were a number of dispersals out of Africa by different hominin populations, both terrestrially and coastally. The Sahel vegetation corridor linking Africa, Arabia, and South Asia may have formed one route following abun- dant coastal resources into Southeast Asia. This route was reliable on a latitudinal basis, but over the long term this dispersal would have been punctuated by envi- ronmental variation, particularly glacial cycles (Timmermann and Friedrich 2016). Hypothetically, such environmental instability alongside a diet rich in omega acids may have encouraged ongoing adaptive flexibility in a coastal context. However, no direct evidence exists for maritime technology along this coastal route, and a swathe of new sites in southern and eastern Eurasia now challenge the importance of the coastal model (e.g., Bae et al. 2014; Li et al. 2018; Liu et al. 2015; Shen et al. 2013). The sites, dating to c. 130,000–70,000 years ago, indicate there was an earlier exo- dus of H. sapiens from Africa, perhaps primarily terrestrial, with migrations along southern Eurasia representing a relatively recent dispersal within our genus. The only tentative evidence for strategic Pleistocene seafaring in the region comes from Madagascar, where Hansford et al. (2018) claim elephant bird (Aepyor- nis) long bones dated to >10,500 years ago (Table 1) display peri-mortem butchery marks, potentially demonstrating H. Getting Our Feet Wet in Africa, Arabia, and Southern Eurasia sapiens crossed the Mozambique Channel to Madagascar around the Terminal Pleistocene and Early Holocene. The evidence is still contentious, however, and Anderson et al. (2018) present counter-arguments for an initial Late Holocene colonization from Island Southeast Asia. A critical review of Madagascar’s radiocarbon chronology finds that it was settled by at least the Late Holocene, but Early Holocene occupation is also likely (Douglass et al. in press). Further paleoenvironmental and zooarchaeological analyses are needed to clarify this important point, which has major implications for how we understand the tempo of island megafaunal extinctions (Douglass et al. 2018), as well as whether humans around Africa could navigate substantial distances and out of sight of land (Table 2). The Madagascan data are outliers, and most evidence for open water crossings around Africa itself is not present until well into the Holocene (Mitchell 2004). In future, it will be crucially important to establish if marine lifeways, and perhaps sea- faring, emerged gradually in Africa and along the southern dispersal route during the Pleistocene, or if they appeared suddenly in the east, as a response to humans encountering large archipelagos (Fig. 5). Drifting and Voyaging Through Southeast Asia The earliest secure global evidence for open water crossings comes from Island Southeast Asia. Because of the region’s steep geomorphology, records of Pleisto- cene island and coastal occupation survive, where in other parts of the world it is 3 3 Journal of Archaeological Research (2021) 29:255–326 268 Table 1 Key island sites directly dated to the Pleistocene showing earliest dated material in association with human occupation. Where potentially problematic dates (freshwater shell, bone apatite) occur, they have been included alongside more secure dates from the same site. Radiocarbon determinations were calibrated using Oxcal 4.3 (IntCal 13), but there was no attempt to correct for inbuilt reservoir effects in marine/riverine shell due to high variability in the global data. Only sites from excavated contexts are included Region, island Site Method Lab code Earliest date (BP) Cal. BP (2σ) Material Reference Africa Madagascar Ilaka (Christmas River) Radiocarbon Hela-1774 9,535±70 11,136–10,654 (94.5%) 10,619–10,605 (0.9%) Bone (Elephant bird) Hansford et al. (2018) Wallacea Flores Wolo Sege Ar-Ar QL-OSU-17 1,010,000±20,000 – Ignimbrite Brumm et al. (2010) Flores Mata Menge Fission-track ‘MM1’ 880,000±70,000 – Sediment Morwood et al. (1998) Flores Mata Menge U-series ‘3543B-14’ 546,500±91,700 – Sediment Brumm et al. (2016) Flores Boa Lesa Fission-track – 840,000±70,000 – Sediment Morwood et al. (1999) Flores Kobatuwa Fission-track – 700,000±60,000 – Sediment Morwood et al. (1999) Flores Liang Bua U-series ‘LB1/52’ 86,900±7,900 – Bone (H. floresien- sis) Sutikna et al. (2016) Flores Liang Bua U-series ‘U-s-06/LB/ XI/52/04’ 80,600±11,300 – Bone (Stegodon) Sutikna et al. (2016) Sulawesi Talepu U-series ANU-2942 >600,000 – Tooth enamel (cel- ebochoerus) Van den Bergh et al. (2016a) Sulawesi Leang Burung 2 U-series 3011A 96,000±2,700 – Suid molar (den- tine) Brumm et al. (2018) Sulawesi Leang Burung 2 Red TL SLBRG2-3 82,000±21,000 – Sediment Brumm et al. (2018) Sulawesi Leang Burung 2 U-series GrN-8649 31,260±330 – Riverine shell Glover (1981) Sulawesi Leang Burung 2 Radiocarbon Wk-22791 30,597±323 35,161–33,973 Shell Brumm et al. (2018) 1 3 Journal of Archaeological Research (2021) 29:255–326 269 1 3 Table 1 (continued) Region, island Site Method Lab code Earliest date (BP) Cal. BP (2σ) Material Reference Sulawesi Leang Barugayya 2 U-series ‘LB4.2’ 44,000+910/-830 – Coralloid speleo- them Aubert et al. (2014) Sulawesi Leang Timpuseng U-series ‘LT2.3’ 40,700+870/-840 – Coralloid speleo- them Aubert et al. (2014) Sulawesi Leang Jarie U-series ‘LJ1’ 39,670+320/-320 – Coralloid speleo- them Aubert et al. Drifting and Voyaging Through Southeast Asia (2014) Sulawesi Leang Sampeang U-series ‘LS1.2’ 32,600+760/-760 – Coralloid speleo- them Aubert et al. (2014) Sulawesi Gua Jing U-series ‘GJ1.3’ 30,900+1,700/- 1,800 – Coralloid speleo- them Aubert et al. (2014) Sulawesi Leang Lompoa U-series ‘LL2.2’ 29,300+1,200/- 1,100 – Coralloid speleo- them Aubert et al. (2014) Sulawesi Leang Barugayya 1 U-series ‘LB1.2’ 29,100+320/-310 – Coralloid speleo- them Aubert et al. (2014) Sulawesi Leang Sakapao 1 Radiocarbon Wk-3821 31,280±570 36,405–34,157 Freshwater shell Bulbeck et al. (2004) Sulawesi Leang Sakapao 1 Radiocarbon Wk-3822 25,390±310 30,388–28,790 Marine shell Bulbeck et al. (2004) East Timor Laili Radiocarbon D-AMS 007344 40,417±332 44,668–43,314 Charcoal Hawkins et al. (2017a) East Timor Jerimalai Radiocarbon Wk-17831 38,255±596 43,286–41,606 Marine shell O’Connor (2007) East Timor Lene Hara Radiocarbon Wk-26405 38,207±610 43,279–41,552 Marine shell O’Connor et al. (2010a) East Timor Matja Kuru 2 Radiocarbon OZF785 32,200±300 36,836–35,401 Marine shell O’Connor et al. (2014) East Timor Bui Ceri Uato Radiocarbon ANU-11738 26,520±340 31,210–29,947 Marine shell Selimiotis (2006) East Timor Uai Bobo 2 Radiocarbon ANU-238 13,400±520 17,680–14,470 Charcoal/bone (bulk) Glover (1969) 3 Journal of Archaeological Research (2021) 29:255–326 270 1 3 Table 1 (continued) Region, island Site Method Lab code Earliest date (BP) Cal. BP (2σ) Material Reference Gebe Golo Cave Radiocarbon Wk-4629 32,210±320 36,935–35,350 Marine shell Bellwood et al. (1998) Salibabu, Talaud Leang Sarru Radiocarbon ANU-10499 30,850±340 35,506–34,142 Marine shell Tanudirjo 2001 Rote Lua Meko Radiocarbon ANU-10908 24,420±250 28,958–27,893 Marine shell Mahirta (2003) Rote Lua Manggetek Radiocarbon ANU-10915 13,390±430 17,494–14,850 Marine shell Mahirta (2003) Alor Tron Bon Lei Radiocarbon B-58 17,630±70 21,584–21,029 Marine shell Samper Carro et al. (2016) Morotai Daeo 2 Radiocarbon ANU-9450 13,930±140 17,355–16,430 Marine shell Bellwood et al. (1998) Kisar Here Sorot Entapa Radiocarbon Wk-43368 13,395±33 16,271–15,953 Marine shell O’Connor et al. (2018) Philippines Luzon Kalinga ESR/U-series GCY1501 709,000±68,000 – Tooth enamel (rhino) Ingicco et al. (2018) Luzon Callao Cave ESR/U-series ‘Callao 1’ 66,700+11,000/- 9,000 – Tooth enamel (cervid) Mijares et al. (2010) Palawan Tabon Caves U-series ‘IV-2000-T-197’ 47,000+11,000/- 10,000 – Human tibia Dizon 2003 Palawan Tabon Caves U-series ‘PXIII T436-Sg19’ 31,000+8,000/- 7,000 – Human mandible Dizon (2003) Palawan Pilanduk Cave Radiocarbon – “26,000” – Unknown Kress (1978) Palawan Ille Cave Radiocarbon OxA16666 12,120±60 14,143–13782 Charcoal Lewis et al. (2008) Bismarck Arch. New Ireland Buang Merabak Radiocarbon ANU-15809 40,090±550 44,764–42,860 Marine shell Leavesley (2004) New Ireland Matenkupkum Radiocarbon ANU-5070 32,700±1,550 41,061–34,181 Marine shell Allen et al. (1988) New Ireland Matenbek Radiocarbon Beta-29007 20,430±180 21,152–24,119 Marine shell Allen et al. Drifting and Voyaging Through Southeast Asia (1989) Journal of Archaeological Research (2021) 29:255–326 271 1 3 ab e (co t ued) Region, island Site Method Lab code Earliest date (BP) Cal. BP (2σ) Material Reference New Ireland Panakiwuk Radiocarbon RIDDL-531 15,140±160 18,734–18,003 Charcoal Marshall and Allen (1991) New Ireland Balof 2 Radiocarbon ANU-4848 14,240±400 18,319–16,239 Charcoal White et al. (1991) New Britain Kupona na Dari Fission-track OxL-1426 39,800±5,200 – Obsidian Torrence et al. (2004) New Britain Yombon Radiocarbon Beta-62319 35,570±480 41,274–39,119 Charcoal Pavlides 1999 New Britain Misisil Radiocarbon Beta-62318 14,310±100 17,715–17,108 Charcoal Pavlides (2004) Manus Pamwak Radiocarbon – “20,900” – – Spriggs (2001, p. 367) Solomon Islands Buka Kilu Radiocarbon ANU-5990 28,340±280 33090–31,475 Marine shell Wickler (2001) Palaeo-Honshu Honshu Itaiteragatani Radiocarbon KSU-1142 34,300±730 40,569–36,834 Charcoal Izuho and Kaifu (2015) Honshu Kannoki location 2 Radiocarbon Beta-82580 33,070±540 38,634–36,042 Charcoal Izuho and Kaifu (2015) Honshu Hinatabayashi B Radiocarbon Beta-120859 31,420±280 35,950–34,749 Charcoal Izuho and Kaifu (2015) Honshu Donoshita Radiocarbon IAAA-70603 31,350±210 35,726–34,766 Charcoal Izuho and Kaifu (2015) Honshu MKK Radiocarbon Beta-156135 30,380±400 35,110–33,748 Charcoal Izuho and Kaifu (2015) Honshu Hatsunegahara Radiocarbon Beta-104086 29,750±210 34,263–33,537 Charcoal Izuho and Kaifu (2015) Honshu Owatari II Radiocarbon Gak-17726 27,780±1,060 34,350–30,030 Charcoal Izuho and Kaifu (2015) Honshu Nojiri-ko Radiocarbon – – ‘~42,000-35,000’ Charcoal Ono et al. (2002) 3 Journal of Archaeological Research (2021) 29:255–326 272 1 3 ( ) Region, island Site Method Lab code Earliest date (BP) Cal. BP (2σ) Material Reference Kyushu Ishinomoto 8 Radiocarbon Beta-84290 33,720±430 39,114–36,750 Charcoal Izuho and Kaifu (2015) Kyushu Ishinomoto 54 Radiocarbon Beta-117662 32,650±430 38,128–35,734 Charcoal Izuho and Kaifu (2015) Kyushu Ushiromuta loca- tion 3 Radiocarbon Beta-142854 30,290±200 34,692–33,932 Charcoal Izuho and Kaifu (2015) Ryukyu Okinawa Sakitari Radiocarbon PLD-16469 32,650±130 36,976–36,186 Charcoal Fujita et al. (2016) Okinawa Yamashita-cho I Radiocarbon TK-78 32,100±1000 38,816–34,292 Charcoal Kobayashi et al. (1971) Okinawa Minatogawa Radiocarbon TK-99 18,250±650 23,701–20,545 Charcoal Kobayashi et al. (1974) Miyako Pinza-Abu Cave Radiocarbon – 26,800±1300 34,091–28,552 Charcoal Hamada (1985) Ishigaki Shiraho-Saonetab- aru Cave Radiocarbon MTC-14197 24,556±205 29,046–28,091 Human bone Shinoda and Adachi (2017) Ishigaki Shiraho-Saonetab- aru Cave Radiocarbon MTC-12820 20,416±113 24,995–24,206 Human bone Nakagawa et al. Drifting and Voyaging Through Southeast Asia (2010) Kume Shimojibaru Cave Radiocarbon – 15,200±100 18,709–18,201 Crab Matsu’ura (1999) California Channel Santarosae CA-SMI-678 Radiocarbon OS-80129 10,950±45 12,950–12,711 Shell Erlandson et al (2011) Santarosae CA-SMI-679 Radiocarbon OS-80171 10,800±40 12,754–12,672 Shell Erlandson et al (2011) Santarosae CA-SRI-512W Radiocarbon OS-75147 10,200±45 12,095–11,748 (94.5%) 11,735–11719 (0.9%) Charcoal Erlandson et al (2011) Mediterranean Journal of Archaeological Research (2021) 29:255–326 273 Table 1 (continued) Region, island Site Method Lab code Earliest date (BP) Cal. BP (2σ) Material Reference Crete Preveli 7 OSL LS1368 113,600±10,300 — Sediment Runnels et al. (2014a) Crete Preveli 7 OSL LS1369 93,800±8,900 — Sediment Runnels et al. (2014a) Corsica (Cor- sardinia) A Teppa di U Lupinu Radiocarbon Lyon-3985 15,800±60 19,229–18,890 Bone (owl) Salotti et al. (2008) Sardinia (Cor- sardinia) Grotta di Corbeddu Radiocarbon GR N-11435 13,590±140 16,871–15,994 Bone (deer) Sondaar et al. (1986) Sicily Grotta dell’Acqua Fituasa Radiocarbon F-26 13,760±130 17,041–16,237 Charcoal Bianchini and Gam- bassini (1973) Sicily Riparo del Castello Radiocarbon OxA-10040 13,485±80 16,521–15,979 Charcoal Skeates (2001) Sicily Addaura 1 Radiocarbon KIA-36055 12,890±60 15,639–15,176 Charcoal Mannino et al. (2011) Sicily Grotta Giovanna Radiocarbon R-484 12,840±100 15,705–15,040 Charcoal Cardini (1971) Sicily Grotta di San Teodoro Radiocarbon ETH-34451 12,580±130 15,272–14,234 Charcoal Mannino et al. (2011) Sicily Grotta Niscemi Marine shell OxA-13823 12,090±50 14,096–13,776 Marine shell Mannino and Thomas (2007) Sicily Grotta Perciata Radiocarbon F-27 11,960±330 15,010–13,161 Marine shell Azzi et al. (1973) Sicily Grotta di Cala dei Genovesi Radiocarbon F-19 11,710±295 14,347–12,865 Shell Azzi et al. (1973) Sicily Grotta di Levanzo Radiocarbon F-20 11,700±300 14,331–12,831 – Bianchini and Gam- bassini (1973) Sicily Grotta dell’Uzzo Radiocarbon P-2736 10,050±100 11,976–11,259 – Piperno et al. (1980) Cyprus Akrotiri Aetokrem- nos Radiocarbon Beta-40380 11,720±240 14,140–13,081 Charcoal Simmons (1991) 1 Journal of Archaeological Research (2021) 29:255–326 274 1 3 Table 2 Distances for known or inferred Pleistocene water crossings. Likely crossing windows are estimated based on the earliest known dates for occupation on the respective islands/landmasses. Refer to Figure 4 for information on mean sea levels during marine isotope stages Crossing from Crossing to Route Likely crossing window Present crossing distances ~Minimal distances during crossing window* H. Drifting and Voyaging Through Southeast Asia erectus or similar Island Southeast Asia Sunda (Java/Bali) Sumbawa Selat Strait and Lombok Strait via Lombok >MIS-28 15–35 km 30 km Sumbawa Flores Sape Strait via Komodo and Rinca >MIS-28 1–20 km 10 km Sulawesi Flores Flores Sea via Selayar Islands >MIS-28 1–90 km 85 km Sunda (Borneo) Palawan Balabac Strait via Banggi and Balabac Islands >MIS-17 10–30 km 10 km Palawan Luzon Mindoro Strait via Culion, Busuanga, and Mindoro Islands >MIS-17 1–70 km 15 km Sunda (Borneo) Sulawesi Makassar Strait >MIS-7 –<MIS-19 117 km 30 km H. neanderthalensis Mediterranean Peloponnese (Antikythera) Crete Aegean Sea via Kithera and Antikithera MIS-5b-d 1–32 km 20–25 km Crete Gavdos Libyan Sea MIS-4 to MIS-8? 56 km 25 km Peloponnese Melos Aegean Sea via Cycladic Islands MIS-4 to MIS-8? 1–15 km 0–5 km Peloponnese Naxos Aegean Sea via Cycladic Islands MIS-4 to MIS-8? 1–17 km 0–10 km Peloponnese Kefallinia Ionian Sea via Ithaka MIS-4 to MIS-8? 3–6 km 5 km Peloponnese Zakynthos Ionian Sea MIS-4 to MIS-8? 17 km 5–10 km Homo sapiens Africa Northeast Africa Arabia Bab el Mandab Strait MIS-5? 3–20 km 5 km Southeast Africa Madagascar Mozambique Channel via Comoro Islands MIS-2? 38–300 km 290 km Journal of Archaeological Research (2021) 29:255–326 275 ( ) Crossing from Crossing to Route Likely crossing window Present crossing distances ~Minimal distances during crossing window* sland Southeast Asia unda (Borneo) Palawan Balabac Strait via Banggi and Balabac Islands MIS-3 10–30 km 10 km alawan Luzon Mindoro Strait via Culion, Busuanga, and Mindoro Islands MIS-3 1–70 km 15 km unda (Borneo) Sulawesi Makassar Strait MIS-4? 117 km 30 km ulawesi Talaud Celebes Sea via Sangihe Islands MIS-3 4–111 km 105 km Mindanao Talaud Celebes Sea MIS-3 10–170 km 170 km ulawesi Obi Lifamatola Strait via Sula MIS-4? 1–103 km 100 km Obi Halmahera Obi Strait via Bisa MIS-4? 5–38 km 45 km ulawesi Buru Seram Sea via Sula MIS-4? 1–67 km 65 km Buru Seram Manipa Strait MIS-4? 1–26 km 20 km Halmahera Gebe Halmahera Sea MIS-4? 35 km 35 km Gebe Waigeo Halmahera Sea MIS-4? 70 km 60 km Waigeo Sahul (northwest New Guinea) Sagawin Strait via Batanta MIS-4? 4–22 km 5 km eram Sahul (northwest New Guinea) Seram Sea MIS-4? 82 km 80 km eram Kai Islands Banda Sea via Watubela Archipelago MIS-4? 1–50 km 50 km Kai Islands Sahul (Aru) Arafura Sea MIS-4? Drifting and Voyaging Through Southeast Asia 122 km 80 km unda (Java/Bali) Sumbawa Selat Strait via Lombok MIS-4? 15–35 km 30 km umbawa Flores Sape Strait via Komodo and Rinca MIS-4? 1–20 km 8 km lores Alor Alor Strait via Lambata MIS-4? 3–12 km 5 km Alor Timor Ombai Strait MIS-4? 30 km 15 km Timor Sahul (northwest Australia) Timor Sea MIS-4 450 km 80 km 1 3 Table 2 (continued) Crossing from Crossing to Route Likely crossing window Present crossing distances ~Minimal distances during crossing window* Island Southeast Asia Sunda (Borneo) Palawan Balabac Strait via Banggi and Balabac Islands MIS-3 10–30 km 10 km Palawan Luzon Mindoro Strait via Culion, Busuanga, and Mindoro Islands MIS-3 1–70 km 15 km Sunda (Borneo) Sulawesi Makassar Strait MIS-4? 117 km 30 km Sulawesi Talaud Celebes Sea via Sangihe Islands MIS-3 4–111 km 105 km Mindanao Talaud Celebes Sea MIS-3 10–170 km 170 km Sulawesi Obi Lifamatola Strait via Sula MIS-4? 1–103 km 100 km Obi Halmahera Obi Strait via Bisa MIS-4? 5–38 km 45 km Sulawesi Buru Seram Sea via Sula MIS-4? 1–67 km 65 km Buru Seram Manipa Strait MIS-4? 1–26 km 20 km Halmahera Gebe Halmahera Sea MIS-4? 35 km 35 km Gebe Waigeo Halmahera Sea MIS-4? 70 km 60 km Waigeo Sahul (northwest New Guinea) Sagawin Strait via Batanta MIS-4? 4–22 km 5 km Seram Sahul (northwest New Guinea) Seram Sea MIS-4? 82 km 80 km Seram Kai Islands Banda Sea via Watubela Archipelago MIS-4? 1–50 km 50 km Kai Islands Sahul (Aru) Arafura Sea MIS-4? 122 km 80 km Sunda (Java/Bali) Sumbawa Selat Strait via Lombok MIS-4? 15–35 km 30 km Sumbawa Flores Sape Strait via Komodo and Rinca MIS-4? 1–20 km 8 km Flores Alor Alor Strait via Lambata MIS-4? 3–12 km 5 km Alor Timor Ombai Strait MIS-4? 30 km 15 km Timor Sahul (northwest Australia) Timor Sea MIS-4 450 km 80 km 3 Journal of Archaeological Research (2021) 29:255–326 276 able 2 (continued) Crossing from Crossing to Route Likely crossing window Present crossing distances ~Minimal distances during crossing window* Timor Rote Rote Strait MIS-2 11 km 5 km Timor Kisar Wetar Strait MIS-2 26 km 25 km Pacific ahul (Huon Peninsula) New Britain Vitiaz Strait via Umboi Island MIS-3 25–50 km 40 km New Britain New Ireland St. Drifting and Voyaging Through Southeast Asia George’s Channel via Duke of York Islands MIS-3 10–20 km 20 km New Ireland Solomons Pacific Ocean via Ambitle and Nissan Islands MIS-3 55–65 km 60 km ahul (northeast New Guinea) Manus Pacific Ocean MIS-2 270 km 260 km ahul (southeast New Guinea Rossel Solomon Sea ? 0.5–35 km 10–20 km apan and Ryukyu Eurasia (Korean Peninsula) Palaeo-Honshu Korea Strait and Tsushima MIS-3 25–55 km 40 km Eurasia (Hokkaido) Palaeo-Honshu Tsugaru Strait MIS-3 18 km 5–15 km alaeo-Honshu Kozushima Pacific Ocean via Izu Island chain MIS-3 3–20 km 1–15 km Eurasia (Taiwan) Yonaguni Yonaguni Strait MIS-3 110 km 100 km alaeo-Honshu Tanegashima Osumi Strait MIS-3 35 km 10 km Miyako Okinawa Miyako Strait MIS-3 270 km 220 km Tanegashima Amami-Oshima Tokara Strait MIS-3 230 km 195 km Tanegashima Amami-Oshima Tokara Strait via Tokara Is. chain MIS-3 10–60 km 50 km America California Santarosae Santa Barbara Channel MIS-1 20 km 10 km Journal of Archaeological Research (2021) 29:255–326 277 able 2 (continued) Crossing from Crossing to Route Likely crossing window Present crossing distances ~Minimal distances during crossing window* Mediterranean Eurasia (Italian Peninsula) Corsardinia Tyrrhenian Sea via Isola d’Elba Island MIS-2 10–50 km 15 km Peloponnese Melos Aegean Sea via Cycladic Islands MIS-2 1–15 km 5 km Eurasia (Anatolia) Cyprus Mediterranean Sea MIS-1 75 km 60 km Rounded to nearest 5 km due to poor data resolution in many areas. Calculated with reference to Kealy et al. 2018; Lykousis 2009; Norman et al. 2018; Sahultime.com; Tourloukis and Karkanas 2012 Table 2 (continued) Crossing from Crossing to Route Likely crossing window Present crossing distances ~Minimal distances during crossing window* Mediterranean Eurasia (Italian Peninsula) Corsardinia Tyrrhenian Sea via Isola d’Elba Island MIS-2 10–50 km 15 km Peloponnese Melos Aegean Sea via Cycladic Islands MIS-2 1–15 km 5 km Eurasia (Anatolia) Cyprus Mediterranean Sea MIS-1 75 km 60 km Rounded to nearest 5 km due to poor data resolution in many areas. Calculated with reference to Kealy et al. 2018; Lykousis 2009; Norman et al. Drifting and Voyaging Through Southeast Asia 2018; Sahultime.com; Tourloukis and Karkanas 2012 Table 2 (continued) Crossing from Crossing to Route Likely crossing window Present crossing distances ~Minimal distances during crossing window Mediterranean Eurasia (Italian Peninsula) Corsardinia Tyrrhenian Sea via Isola d’Elba Island MIS-2 10–50 km 15 km Peloponnese Melos Aegean Sea via Cycladic Islands MIS-2 1–15 km 5 km Eurasia (Anatolia) Cyprus Mediterranean Sea MIS-1 75 km 60 km *Rounded to nearest 5 km due to poor data resolution in many areas. Calculated with reference to Kealy et al. 2018; Lykousis 2009; Norman et al. 2018; Sahultime.com; Tourloukis and Karkanas 2012 3 Journal of Archaeological Research (2021) 29:255–326 278 Fig. 5 The Indian Ocean region showing key sites associated with marine resources and major coastal dispersal routes out of Africa Fig. 5 The Indian Ocean region showing key sites associated with marine resources and major coastal dispersal routes out of Africa now submerged. As such, Wallacea allows us to think about how different hominins adapted to specific insular biogeographic zones and how these changing environs impacted long-term behavioral trajectories and water-crossing routes (Fig. 6). H. erectus were present around Sunda since the Middle Pleistocene, probably arriving during one or more sea-level low stands, 1.8–1.74 million years ago. The paleoenvironmental record covering the time span of H. erectus in Southeast Asia (on Java 1.51 million years ago to 117,000–108,000 years ago, see Hyodo et al. 2011; Rizal et al. 2020) is poorly understood. However, there is growing evidence for occupation along lakeshores and in marshy areas within broader mosaic grass- land and forest landscapes, particularly later in the record (Rabett 2017). Specifi- cally, the Trinil and Sangiran H. erectus had access to near-coastal rivers, swamp forests, lagoons, lakes, and marshes with minor marine influences (Bettis et al. 2009). In these environments a wide array of easily caught near-shore fish species were present, and accumulations of Pseudodon and Elongaria shell may indicate predation by H. erectus (Joordens et al. 2009). Moreover, during times of insulariza- tion and increased precipitation (which typified MIS-60, MIS-40, MIS-22, MIS-12, and MIS-6), highly mobile hominins may have found themselves increasingly enter- ing novel environments, even when revisiting known resource patches, with selec- tive pressures favoring groups who proved adaptively flexible. Such environmental variation could have promoted expansions toward the coast to make use of littoral resources. This is supported by findings at both Sangiran and Trinil that suggest H. Drifting and Voyaging Through Southeast Asia erectus were using shell tools and engraving shell by about 500,000 years ago (Choi 1 3 Journal of Archaeological Research (2021) 29:255–326 279 and Driwantoro 2007; Joordens et al. 2015). Along with implications for incipient aquatic adaptation, this has important ramifications for the symbolic and cognitive dimensions of Southeast Asian H. erectus. and Driwantoro 2007; Joordens et al. 2015). Along with implications for incipient aquatic adaptation, this has important ramifications for the symbolic and cognitive dimensions of Southeast Asian H. erectus. As Rabett (2017) points out, it could be this ecological diversity that led to the long survival of these hominins. I would add that it might have also encouraged adaptive flexibility in new environments. This flexibility perhaps led to H. erectus making the first open water crossings in history and adapting to islands. Early and Middle Pleistocene hominins crossed water gaps of at least 20–40 km to reach three islands: Flores, Luzon, and Sulawesi. The earliest known crossings reached Flores in the Lesser Sunda Chain, with evidence for island occupation 1 million years ago at Wolo Sege (Brumm et al. 2010) and 880,000 years ago at the Mata Menge site (Morwood et al. 1998). It is likely that these hominins—H. erectus or an unknown species—moved west to east across several straits into Sumbawa and then Flores, or north to south through Sulawesi (Bellwood 2017, p. 35). Dennell et al. (2014) argue that this dispersal onto Flores followed the latter route via Sulawesi (or perhaps the Sunda mainland around modern Borneo), passively following strong southerly currents. If this were true, we might also expect to see hominins on Sumba Island, just south of Flores, but as yet there is no evidence for this (Turvey et al. 2017). These islands would have been even larger targets with much shorter crossing distances during the Pleistocene (Kealy et al. 2017). Bell- wood (2017) suggests a move from Sulawesi did not require a single catastrophic passive dispersal but a series of small 10–20 km jumps from Sulawesi to Salayar and small reef islands including Taka Bone Rate along with now submerged paleo- islands, followed by a single larger crossing of around 85 km to reach Flores. As Morwood (2014, p. Drifting and Voyaging Through Southeast Asia (2018); c) present-day sea level. Major biogeographic transitions are marked, including the Wallace, Huxley, Weber, Lydekker, and Zollinger Lines. For color legend please refer to online version of the article ▸ make up only 0.23% of the assemblage. This contrasts to later Neolithic H. sapiens levels (Unit 8A-C), which contains 13% aquatic invertebrates, perhaps brought to the site as adornments and tools. Although taphonomic studies are required to con- firm that these changes are behaviorally induced, it tentatively suggests the utiliza- tion of shellfish by recent populations was not mirrored in H. floresiensis or Pleis- tocene H. sapiens, who are first detectable in a shift to lithic raw material selection at c. 46,000 years ago (Sutikna et al 2018). This is important because it shows that variable subsistence and technological strategies were successful on Flores and that long-term behavioral trajectories seem to have informed similar adaptations among multiple species in the same environment. Middle Pleistocene hominins also made crossings into the Philippines, which would have formed several larger landmasses separated from Sunda by the Luzon and Mindoro Straits. At the Kalinga site in Luzon’s Cagayan Valley, Rhinoc- eros philippinensis remains with clear evidence of butchery in association with 57 lithics were found in bone beds dated to 777,000–631,000 years ago (Ingicco et al. 2018). No hominin remains were found during excavations, but the results suggest that H. erectus or an unknown hominin species crossed to the Philippines during a sea-level low stand in MIS-20 or earlier. Support for these finds comes from the Arubo open sites in central Luzon, where bifacial handaxes and horse- hoof cores resemble the Pajitanian industry of H. erectus in Java (Pawlik 2004), and from the Haluga open sites on Mindanao, which contain large choppers and picks (Tiauzon et al. in press). At Callao Cave in northern Luzon, a metatarsal was dated using U-series to sometime before 67,000 years ago (Mijares et al. 2010). Recently, the recovery of additional remains, including teeth and postcranial elements, has led research- ers to suggest they derive from a previously unrecognized species: Homo luzon- ensis (Détroit et al. 2019), which was short in stature and shares morphological characteristics with H. floresiensis, H. erectus, H. sapiens, and the australopiths. Although no lithics were found in association with the bones, many of the cervid remains from the same deposit display cutmarks, akin to that produced with bam- boo knives (Manalo 2011). Drifting and Voyaging Through Southeast Asia 112) notes, even if these hominins passively dispersed to Flores through inclement weather events, it is likely that they were doing things around the coast, for instance, exploiting near-shore resources with natural rafts that increased their chances of being swept out to sea. Whatever arrived on Flores evolved into the small-bodied, small-brained hominin Homo floresiensis, with type fossils discovered at Liang Bua (Fig. 7; Brown et al. 2004; Morwood et al. 2004). These hominins occupied the island until 60,000 or possibly 50,000 years ago (see Table 1; Sutikna et al. 2016), and despite intense initial debate as to whether they represented a diminutive or pathological form of H. sapiens (Argue et al. 2006; Henneberg et al. 2014; Martin et al. 2006), consen- sus now points to H. floresiensis being a separate morpho-species (Aiello 2010). Recent fossil evidence excavated from Mata Menge, dating to about 700,000 years ago, shows that the ancestors to the Liang Bua H. floresiensis were even smaller in stature (Brumm et al. 2016; van den Bergh et al. 2016b), suggesting they very rap- idly dwarfed on Flores or that they existed in a diminutive form prior to arriving on the island, perhaps in Sulawesi. Ultimately, it is unclear if these small-bodied homi- nins derived from H. erectus as most researchers have previously suggested, or an as yet unidentified hominin representing an extremely early pulse out of Africa, given strong morphological similarities with Homo habilis (Argue et al. 2017). The zooarchaeological assemblage at Liang Bua shows remarkable change through time, with Unit IB (120,000–60,000 years ago), which contains H. floresien- sis, being dominated by terrestrial faunas (Table S1). Aquatic invertebrates and fish 1 3 3 3 Journal of Archaeological Research (2021) 29:255–326 280 Fig. 6 Southeast Asia, showing relative sea level during major marine transgressions: a) 120 m below present-day sea level. Sea levels would have been this low during several glacial periods including MIS- 6, MIS-12, and MIS-2 (LGM). Locations of a possible savannah corridor through Sunda and the coastal corridor along Sumatra and Java are indicated; b) 50 m below present-day sea level. This is approxi- mately representative of 65,000 years ago (see Kealy et al. 2017), when H. sapiens may have been enter- ing Wallacea for the first time. Hypothetical dispersals are indicated, following Birdsell’s northern and southern routes (1977), Morwood and Van Oosterzee (2007), Sondaar (1989), and Kealy et al. 1 3 Drifting and Voyaging Through Southeast Asia It is likely that there was substantial morphological variability among homi- nins living in the insular tropics, mimicking what we see in non-Homo primate species (Foley 1991). However, the Callao remains pose an important question: how do we confidently define new species with limited skeletal evidence? Do the finds convincingly indicate allopatric speciation following water-crossing events, or a distinct subgrouping within a broader population of small-bodied Wallacean 1 3 1 3 Journal of Archaeological Research (2021) 29:255–326 281 g ( ) 1 3 Journal of Archaeological Research (2021) 29:255–326 282 . 7 Above: known archaeological sites in Island Southeast Asia relating to Early and Middle Pl ne water crossings, including H. erectus, H. floresiensis, H. luzonensis, and unknown Homo spe ow: archaeological sites relating to the movement of H. sapiens into Wallacea. For color legend p er to online version of the article Fig. 7 Above: known archaeological sites in Island Southeast Asia relating to Early and Middle Pl cene water crossings, including H. erectus, H. floresiensis, H. luzonensis, and unknown Homo spe b l h l i l it l ti t th t f H i i t W ll F l l d Fig. 7 Above: known archaeological sites in Island Southeast Asia relating to Early and Middle Pleisto- cene water crossings, including H. erectus, H. floresiensis, H. luzonensis, and unknown Homo species; below: archaeological sites relating to the movement of H. sapiens into Wallacea. For color legend please refer to online version of the article 1 3 Journal of Archaeological Research (2021) 29:255–326 283 hominins, given the notable decoupling of morphological and molecular genetic variability within the genus (Curnoe 2003). hominins, given the notable decoupling of morphological and molecular genetic variability within the genus (Curnoe 2003). Thirdly, there was a dispersal of unknown hominins into Sulawesi by the end of the Middle Pleistocene at least 200,000 years ago and possibly up to 780,000 years ago (van den Bergh et al. 2016a). Sulawesi was always separated from Sunda by short water crossings (Table 2), but it is not known if these popula- tions spread eastward from what is now Borneo or from the north through the Philippines. Drifting and Voyaging Through Southeast Asia The string of islands north of the northern arm of Sulawesi prob- ably facilitated water crossings between Wallacea and the Philippines, along with routes from Sunda through Palawan, and across the Tapiantana Channel and Basi- lan Strait via the Sulu Archipelago. At Telepu in the Walanae Basin in southwest Sulawesi, hominins discarded 311 stone artifacts in association with megafaunal remains of bovids, stegodons, and suids. The lithics are expedient and show a preference for sharp edges over formal morphology (van den Bergh et al. 2016a), something that is shared among all hominins in the region, along with modern ethnographic toolmakers (White and Thomas 1972), and suggests convergent adaptations within similar ecological spaces. This may relate to the presence of other organic materials that can better facilitate specific tasks such as butchery and plant processing. It also may indicate that, with high levels of mobility and an abundance of raw materials, hominins were able to rapidly find and exploit high-quality lithics and so did not practice lithic conservation for risk reduction as they did not anticipate moving into areas poor in lithics.i The first record of H. sapiens in the region occurs several millennia after these initial dispersals. In Sunda, there is substantial evidence for H. sapiens at Song Terus and Tabuhan Cave in eastern Java by 45,000 years ago (Sémah and Sémah 2013, 2013) and 60,000–70,000 years ago (Westaway et al. 2017), or more debat- ably by <128,000–>118,000 years ago (Westaway et al. 2007). It is unclear if the H. sapiens dispersing into Sunda were already adept sea-goers who entered coast- ally via the southern dispersal route, suggestive of gradual modification (Fig. 6a), or via a savannah corridor bordered by coastal rainforests (Heaney 1991), leading to a major biogeographic leap when the species entered Wallacea for the first time, sug- gestive of rapid flexibility (see Fig. 6a). l The route into Sahul has also long been debated (Fig. 6b), especially whether H. sapiens took Birdsell’s northern route through Sulawesi and Maluku, eventually arriving in today’s Raja Ampat Islands and the Bird’s Head of New Guinea, or Bird- sell’s southern route, through Timor and into Kimberly country in northwest Aus- tralia (Fig. 6b; Birdsell 1977). Although some dispute pre-55,000 dates (O’Connell et al. 2018), there is increasing acceptance of a pre-60,000 date for the arrival of H. sapiens in Sahul (Clarkson et al. 2017). Drifting and Voyaging Through Southeast Asia Recent modeling studies using up-to-date bathymetry are at odds. Kealy et al. (2018) suggest that a northern route into Sahul, through a number of short water crossings, is most likely (see also O’Connell and Allen 2012), although there is a lack of recent research along the northern route. In contrast, Norman et al. (2018) assert that a pre-LGM low stand occurred 60,000–70,000 years ago, in line with the earliest secure dates from northwest Sahul at Madjedbebe in northwest Australia 1 3 Journal of Archaeological Research (2021) 29:255–326 284 (Clarkson et al. 2017), supporting a more easily achieved southerly route, or at least a dual route. Evidence for Birdsell’s southern route comes primarily from Timor. At Laili on East Timor, preserving perhaps the earliest direct evidence of H. sapiens in Wal- lacea at 44,600 years ago, hunters and gatherers exploited local cherts to produce expedient flakes, with minimal evidence for raw material conservation, and targeted a wide array of terrestrial and marine animals (Hawkins et al. 2017b). At Lene Hara, a transit camp between the coast and inland (O’Connor et al. 2002), similar flakes and marine shell date to 42,000 years ago (O’Connor et al. 2010a). Symbolic behavior in the form of anthropomorphic petroglyphs and painted faces at the caves dates maximally to c. 37,000 years ago and continued into the Terminal Pleistocene (Aubert et al. 2007; O’Connor et al, 2010b). Also on Timor, the earliest known occupants of Asitau Kuru (formerly Jeremalai) primarily relied on marine foods (Roberts et al. 2020) and may have been capable of catching a diverse variety of pelagic fish species, along with littoral resources by 42,000 years ago (O’Connor 2015). About one-third of the initial Pleistocene fish assemblage is made up of scombrids, which are known to be fast swimming and hard-to-catch deep-water species, perhaps caught with boat technology (although see critiques from Anderson 2013, who suggests the fish could have been collected near the shore). In support of O’Connor (2015), there is later evidence for line fishing in a Trochus shell bait hook dated to 16,000–23,000 years ago. Interestingly, in the mid-Holocene layers, inshore fishing increased while pelagic fishing decreased, sug- gesting that maritime mobility actually decreased after initial settlement (O’Connor et al. Drifting and Voyaging Through Southeast Asia 2011b), in line with the establishment stage of adaptive flexibility.i l Asitau Kuru also contains shell tools that were modified by drilling, pressure flaking, and grinding and Nautilus shells with pigment staining that were used to process ochre 42,000–38,000 years ago, perhaps indicative of personal adornment and ceremonial behaviors (Langley et al. 2016). Additional evidence is present at Tron Bon Lei rockshelter on Alor Island, where circular rotating fishhooks were found as grave goods in an adult female burial from the Terminal Pleistocene, c. 12,000 years ago (O’Connor et al. 2017b). Obsidian characterization studies from Tron Bon Lei show that stones were imported from at least three different sources around the Sunda Arc, with maritime exchange between Alor and Timor beginning in the Terminal Pleistocene (Reep- meyer et al. 2016). This obsidian exchange, fostering ongoing inter-island support and intermarriage, may have facilitated the initial peopling of even the tiniest of Wallacean islands. One such island was Kisar, where Here Sorot Entapa rockshelter, dating to c. 15,500 years ago, was probably used as a fishing camp where people produced and discarded one-piece fishhooks, caught a variety of fish and turtle spe- cies, collected shellfish, and hunted for locally available bats (O’Connor et al. 2018). Although computer modeling indicates the shortest achievable water crossings existed along Birdsell’s northern route (Kealy et al. 2018), there is sparse archaeo- logical evidence, which mostly postdates the early sites on Timor. Rock art from several cave sites in the Maros Karst area of Sulawesi, dated by U-series to at least c. 40,000 years ago, suggests H. sapiens were present on the island by that time (Aubert et al. 2014, 2019). This represents both the oldest directly dated hand Obsidian characterization studies from Tron Bon Lei show that stones were imported from at least three different sources around the Sunda Arc, with maritime exchange between Alor and Timor beginning in the Terminal Pleistocene (Reep- meyer et al. 2016). This obsidian exchange, fostering ongoing inter-island support and intermarriage, may have facilitated the initial peopling of even the tiniest of Wallacean islands. One such island was Kisar, where Here Sorot Entapa rockshelter, dating to c. 15,500 years ago, was probably used as a fishing camp where people produced and discarded one-piece fishhooks, caught a variety of fish and turtle spe- cies, collected shellfish, and hunted for locally available bats (O’Connor et al. 2018). Drifting and Voyaging Through Southeast Asia i Although computer modeling indicates the shortest achievable water crossings existed along Birdsell’s northern route (Kealy et al. 2018), there is sparse archaeo- logical evidence, which mostly postdates the early sites on Timor. Rock art from several cave sites in the Maros Karst area of Sulawesi, dated by U-series to at least c. 40,000 years ago, suggests H. sapiens were present on the island by that time (Aubert et al. 2014, 2019). This represents both the oldest directly dated hand 1 3 Journal of Archaeological Research (2021) 29:255–326 285 stencils in the world and the earliest evidence of figurative art. Lithics from these areas have previously been dated to 30,000–19,000 years ago (Bulbeck et al. 2004; Glover 1981). Slightly earlier rock art from the east coast of Borneo, at the edge of Sunda, dates to 40,000–52,000 years ago (Aubert et al. 2018), perhaps representing directly ancestral groups to those along the northern route of Wallacea. stencils in the world and the earliest evidence of figurative art. Lithics from these areas have previously been dated to 30,000–19,000 years ago (Bulbeck et al. 2004; Glover 1981). Slightly earlier rock art from the east coast of Borneo, at the edge of Sunda, dates to 40,000–52,000 years ago (Aubert et al. 2018), perhaps representing directly ancestral groups to those along the northern route of Wallacea. Farther east, at Golo cave on Gebe Island, between Halmahera and New Guinea, there is evidence for shell tool production by 35,000–30,000 years ago and an emphasis on marine subsistence (Szabó et al. 2007). However, lithic artifacts from Golo and nearby Wetef cave dated to 25,000 years ago were produced by smash- ing cobbles on an anvil and are highly amorphous, with stronger affinities to north- ern Sahul assemblages than to those from Sulawesi (Tanudirjo 2001, p. 335). These similarities in stone flaking led Tanudirjo (2001, p. 401) to suggest the area may have been occupied first from Sahul, rather than from the northern route. This sce- nario has not been widely addressed in recent literature (except peripherally by Kealy et al. 2017), but it is worth revisiting. Ongoing research by several field teams in Seram, northern Maluku, and the Raja Ampat Islands should help clarify this. At Daeo 2 and Tanjung Pinang on nearby Morotai Island, occupation began after the LGM 16,000 years ago, with evidence for more intensive occupation than on Gebe. Drifting and Voyaging Through Southeast Asia Pebble tools, bone points, unretouched flakes, canarium nut anvils, manu- ports, cooking stones, and ochre are all present, and subsistence strategies involved the collecting of marine shellfish and landsnails and the hunting of rats and cuscus (Tanudirjo 2001, p. 402). The Talaud Island group between northern Sulawesi and the Philippines was occupied by 30,000 years ago, representing inter-island crossings of over 100 km and a possible southerly corridor into Wallacea from the Philippines (Ono et al. 2015). At Leang Sarru on Salibabu Island in the Talaud group, shell data indicate a change to coastal resource use through the LGM and Terminal Pleistocene when seawater temperatures increased. As on Kisar Island, terrestrial resources were dep- auperate, and marine resources played a major role in the diet.i H. sapiens also island-hopped into the Philippines, first into Palawan and then across the Mindoro Strait into Luzon (Mijares 2015). This required crossing Hux- ley’s Line (Fig. 6c), a major biogeographic divide that separates continental Sunda- land faunas from an eclectic mix of insular animals. There is direct evidence of our species at Tabon Cave on Palawan, including crania and mandibles dating to 16,500 years ago and a tibia and mandible dating to 47,000–30,000 years ago (Détroit et al. 2004; Dizon 2003; Dizon et al. 2002). The Tabon lithic sequence resembles that from Callao 28,000 years ago (Mijares 2006), which implies the earliest representa- tives of our species learned common manufacturing techniques. In both industries, a variety of hard hammer flakes were made from siliceous cherts, including some elongate flakes, while blades were not systematically made from these materials (Fox 1970; Mijares 2007). Most of the lithics were applied to hard materials and exhibited a silica gloss with minimal retouch (Xhauflair and Pawlik 2010). l The “bamboo hypothesis” is that these assemblages are technologically simple because they were used to produce composite organic tools from bamboo (Brumm 2010). Usewear and residue studies from the island group confirm that many lithics were used in processing bamboo or possibly other silica-rich plants such as palms 1 3 3 Journal of Archaeological Research (2021) 29:255–326 286 and grasses (Xhauflair et al. 2016); ethnoarchaeological studies from Palawan indi- cate that a huge variety of local plants can be used for technological purposes as well as for medicine and ornaments (Xhauflair et al. 2017), demonstrating sophisti- cated adaptations to rainforest resources. Drifting and Voyaging Through Southeast Asia There seem to have been shared adaptations on the small islands of Wallacea and the Philippines during the Late Pleistocene, including high mobility, the production of expedient tools with some platform preparation, a reliance on marine and coastal subsistence alongside hunting small but reliable game, developments in fishing tech- nology, and early evidence for visually symbolic and ceremonial behaviors. There was a lack of both lithic provisioning and evidence for inter-island exchange until after the LGM. The First Pacific Islanders The possibility that multiple hominin species crossed into northern Sahul and the Pacific has, so far, been overlooked. However, genetic research reveals that the high- est frequency of Denisovan admixture with H. sapiens occurred among the ancestors of Australian Aboriginals and New Guineans, leading Cooper and Stringer (2013) to ponder whether these hominins also crossed into Near Oceania. Jacobs et al. (2019) speculate there was introgression from one lineage of Denisovans east of the Wal- lace Line, which continued into the Terminal Pleistocene. The archaeological and paleoanthropological evidence, however, is nonexistent. With the crossing to continental Sahul, the biogeographic constraints of living on islands were suddenly lifted, and there is substantial evidence that intrepid humans made extensive use of a wide range of challenging environments such as rainfor- ests, arid zones, and mountains. However, coastal and island adaptations contin- ued to facilitate part of the rapid dispersal of H. sapiens along the coast of northern Sahul and beyond (Allen 2000; Bowdler 1990). Although the move into Sahul, and again into the Bismarck Archipelago, would have presented variable floral and fau- nal diversity, the Malesian and Papuasian regions (New Guinea, Indonesia, Malay- sia, and the Philippines) share enough similarities in resources (Paijmans 1976) that technological and subsistence practices could be maintained or gradually modified during the initial phase of movement through relatively similar latitudes (see Florin et al. 2020). Even throughout MIS-3 and MIS-2, climatic conditions around the coast and on low-lying islands would have been relatively consistent and similar to today (Farrera et al. 1999, p. 841). The broader coastal environment was dominated by lowland equatorial rainforests and some savannah, while gradually rising and lowering sea levels produced swamps of brackish water, estuaries, and coral reefs rich in littoral resources (Chappell 1993; Swadling and Hope 1992).i During the first 30,000 years of human activity, occupation was small scale, and the impact on faunal and floral resources seems to have been low (Summerhayes 2007). Between Wallacea and the Bismarcks, a number of Late Pleistocene sites such as Toe, Lachitu, and Wantiglo (Fig. 8) reflect the movement of people along northern Sahul, who made forays into the interior to hunt small game and probably 1 3 1 3 Journal of Archaeological Research (2021) 29:255–326 287 Fig. 8 The western Pacific islands and northern Sahul region, showing known Pleistocene sites and routes of water crossings. The Sahul coastline at c. The First Pacific Islanders 65,000 years ago and at the LGM are marked based on Kealy et al. (2018) and sahultime.com. For color legend please refer to online version of the article Fig. 8 The western Pacific islands and northern Sahul region, showing known Pleistocene sites and routes of water crossings. The Sahul coastline at c. 65,000 years ago and at the LGM are marked based on Kealy et al. (2018) and sahultime.com. For color legend please refer to online version of the article megafauna and to collect highland resources such as Pandanus (Gorecki et al 1991; O’Connor et al. 2011a; Pasveer 2004). At Bobongara Hill, formed of massive uplifted terraces in northeast New Guinea (Groube et al. 1986), numerous “waisted” stone tools dating to ~40,000 years ago may have been hafted and used for ring barking and root clearance for forest man- agement (Groube 1989). The first water crossings into the Bismarck Archipelago were probably made from this area. This journey relied on relatively sophisticated seafaring abilities, requiring, minimally, one crossing of 40 km across the Vitiaz Strait and another of 25 km across the Dampier Strait (Summerhayes et al. 2017). These straits can be treacherous at certain times of year, and so established knowl- edge for predicting and understanding weather patterns would have been essential. Surface finds of robust waisted tools along a 1.8 km stretch of coast on Rossel Island (Shaw 2017) provides further evidence for early seafaring from the tip of south- eastern Papua into the Massim region. Although the tools remain undated, the size and nature of flaking is suggestive of Late Pleistocene lithic traditions on the Sahul mainland. The small size of Rossel, even during the LGM, indicates that humans continued to frequent small offshore islands in the Pacific as they did in Wallacea. fi In the Bismarcks, occupation at Kupona na Dari, on the Willaumez Peninsula of New Britain, dates to 45,000–35,000 years ago (Bonetti et al. 1998; Torrence et al. 2004). Geochemical analyses of obsidian artifacts at the site reveal the import of lithic material from long distances, including local Baki and Gulu material from the peninsula itself and obsidian from the Mopir source in the rainforested interior of New Britain (Torrence 2004). During this time, the landscape would have been exposed to a series of major volcanic events (Neall et al. The First Pacific Islanders 2008), but the archaeo- logical sequences suggest humans only temporarily abandoned the area, engaging flexible and mobile settlement strategies, with a strong social memory that allowed them to resettle former habitation sites (Torrence 2016). 1 3 Journal of Archaeological Research (2021) 29:255–326 288 Yombon in the interior of New Britain, dating to 35,000 years ago, provides further evidence for human occupation in insular rainforests (Pavlides and Gosden 1994). Paleoenvironmental records suggest anthropogenic forest burning was delib- erately used to open gaps for hunting; however, no faunal remains exist to test this (Lentfer et al. 2010). The material from Yombon is almost exclusively local chert that was flaked in an expedient manner characteristic of early northern Sahul assem- blages. These data have two broad implications for human adaptation: the first islanders did not simply hug the coast but ventured into unexplored dense rainforest (for supporting evidence from the mainland of Sahul, see Summerhayes et al. 2010), and they were doing this systematically enough to come across good-quality lithic deposits in the forest. The absence of obsidian at Yombon may relate to the difficulty of accessing the resource at the time, or simply because these flows did not exist yet (see Bonetti et al. 1998). Misissil, a cave close to Yombon, was first occupied in the Terminal Pleistocene, 13,000 years ago (Pavlides 2004; Specht et al. 1981). Obsid- ian was present while local chert was less common, showing that by this point peo- ple had exploited and redistributed higher-quality obsidian around the island. Getting from New Britain to New Ireland required additional water crossings through the dangerous currents of the St. Georges Channel (Summerhayes and Allen 1993). At Buang Merabek on the east coast of New Ireland, occupation stretches back 45,000–43,000 years (Leavesley and Allen 1998), while Matenkupkum dates to about 41,000–30,000 years ago (Allen et al. 1988). The faunal records for the initial period of settlement indicates the caves were used as nonintensive stopover camps by mobile hunter-gatherers, ranging both inland and around the caves for bats and reptiles and on the coast for shellfish and fish (Leavesley 2004; Smith and Allen 1999). In an environment without large, high-rank game such as megafauna on the Sahul mainland, predictable terrestrial resources such as large quantities of fruitbats would have been invaluable. This reflects a tendency for early seafarers to return to reliable resource patches in novel environments. 1 3 The First Pacific Islanders 18,000 years ago (Marshall and Allen 1991); however, in contrast to earlier sites, the diet was terrestrial, with a sparse fish assemblage suggestive of reduced reliance on coastal and marine resources.i Human occupation on Manus Island provides definite evidence for water cross- ings that required seafarers to move out of sight of land. This signals a major shift in technological abilities, especially involving techniques of environmental navi- gation and wayfinding, and suggests that the voyagers were highly confident, even by today’s standards. At Pamwak, dated to c. 21,000 years ago (Spriggs 2001) and probably accessible following a river 10 km inland (Frederickson et al. 1993), the majority of occupation occurred during the LGM and in the Terminal Pleistocene. Obsidian was collected locally from the Admiralty sources by 14,000 years ago (Frederickson 1997), while cuscus (Spilocuscus kraemeri), bandicoot (Echymipera kalubu), and canarium nuts represent separate translocations from mainland Sahul, 230 km away (Kennedy 2002). From northeast New Britain or southeast New Ireland, it would have required a series of island hops of about 60 km through the Feni Islands and the Green Islands to reach the northern Solomons. Kilu cave, first occupied c. 33,000 years ago, is the only known Pleistocene site in the Solomon Island chain, which would have formed one larger island during eustatically lowered sea levels. As at many other island sites in the Pacific, people hunted bats and other terrestrial fauna, collected shellfish, and caught fish (Wickler 2001, pp. 218–220). The fish assemblage may indicate similar deep-water fishing strategies as evidenced in Wallacea at a similar time, and crucial stone-tool residues indicate people were processing wild root vegetables such as taro (Colocasia and Alocasia) by 29,000 years ago (Loy et al. 1992).i After Kilu was first occupied, the material evidence, including the absence of imported obsidian and a dearth of marsupial translocations, suggests that contact with the Bismarcks ceased (Wickler and Spriggs 1988). The Solomons were likely occupied more widely during the Pleistocene, but due to raised sea levels and a lack of uplifted coastal zones (such as on the north coast of New Guinea), attempts to locate similar occupation have failed. The next oldest site is Vatuluma Posovi, with initial occupation beginning in the Early–Mid Holocene (Roe 1993). The First Pacific Islanders There is also evidence for shark hunting at Buang Merabek, where a drilled shark tooth ornament may have had rit- ual or prestige connotations (Leavesley 2007).i As Wallacean and Pacific food sources such as plants, mollusks, and arboreal mammals were relatively sedentary, their local depletion would necessitate frequent relocation. In response, a focus on boat technology for residential mobility would reduce stress on the elderly, pregnant women and children (Torrence 2012). Mari- time activities such as inshore and possibly offshore fishing can then be seen as by- products of this movement through the islands; evidence for this on New Ireland includes shell fishhooks dating to 18,000–20,000 years ago (Smith and Allen 1999). i Later in the Pleistocene, during MIS-2, occupation at Matenbek on New Ire- land began ~24,000–23,000 years ago (Allen et al. 1989). Mopir obsidian was present from the earliest occupation and indicates the deliberate and repeated transport of high-quality flaking material over 350 km, as the crow flies (Sum- merhayes and Allen 1993). This demonstrates provisioning strategies and prior resource knowledge. It may also represent exchange across social boundaries; Summerhayes and Allen (1993) argue that semi-sedentary populations practiced down-the-line exchange to distribute this material. In support, the Matenbek mid- den contains reef fish and shellfish, but cuscus (Phalanger orientalis) bones in the 1 3 Journal of Archaeological Research (2021) 29:255–326 289 initial layers provide the earliest global evidence for animal translocations across bodies of water (Summerhayes et al. 2017). These animals were absent from pre- LGM layers at Buang Merabek and Metenkupkum, but there was a boom in hunt- ing these arboreal mammals following the LGM (Flannery and White 1991). initial layers provide the earliest global evidence for animal translocations across bodies of water (Summerhayes et al. 2017). These animals were absent from pre- LGM layers at Buang Merabek and Metenkupkum, but there was a boom in hunt- ing these arboreal mammals following the LGM (Flannery and White 1991). Residue analysis from the Balof site shows that wild taro and yams also were processed during the Terminal Pleistocene (Barton and White 1993). At Paniki- wuk, farther north, occupation began in the LGM c. The First Pacific Islanders People did not make the next push into Remote Oceania, crossing the threshold of 300 km voyages, until after 3,000 years ago, and into eastern Polynesia later still (Wilmshurst et al. 2011).i In the Pleistocene Pacific we see a continuation of the behavioral trajectories observed in Wallacea and the Philippines, with H. sapiens making use of large con- tinental islands and very small landmasses with high marine ecodiversity and dep- auperate terrestrial resources. In these places, people collected extensively from the littoral and possibly pelagic zone, translocated animals to improve terrestrial protein 1 3 3 Journal of Archaeological Research (2021) 29:255–326 290 availability, and redistributed high-quality lithic material across water gaps. We also see humans pushing the boundaries of these trajectories and expanding into islands that were completely out of relative and absolute inter-visibility, which would have required meticulously planned, multiday voyages through high-risk seas. Insular East Asia Similar evidence demonstrates that hominins expanded into the continental and oceanic islands off the coast of eastern Eurasia (Fig. 9). It is currently debated whether non-sapiens occupied the Japanese islands; however, from what we know about Wallacea it would not be impossible. Matsufuji (2011) makes the case that Fig. 9 Eastern Eurasia and the Americas showing major water crossings into Paleo-Honshu, the Ryukyu Islands, and the California Channel Islands, along with likely coastal dispersals through the Americas Fig. 9 Eastern Eurasia and the Americas showing major water crossings into Paleo-Honshu, the Ryukyu Islands and the California Channel Islands along with likely coastal dispersals through the Americas Fig. 9 Eastern Eurasia and the Americas showing major water crossings into Paleo-Honshu, the Ryukyu Islands, and the California Channel Islands, along with likely coastal dispersals through the Americas 3 Journal of Archaeological Research (2021) 29:255–326 291 Middle Pleistocene hominins entered Japan by MIS-6, a glacial period when sea lev- els were lowered. Possible lithic artifacts from Kanedori are associated with tephra dated to 115,000–84,000 years ago; however, Norton et al. (2010) question whether these remains are indeed artifactual and stress current evidence that H. sapiens first entered the Japanese islands in MIS-3. At that time, the northern island of Hokkaido was connected to Sakhalin and the northeast Eurasian mainland as a peninsula, due to lowered sea levels. Honshu, Shikoku, and Kyushu, on the other hand, formed one larger island called Paleo-Honshu, which was separated from Eurasia by the Tsugaru Strait to the north, the Korea Strait to the west, and the Tokara Strait to the south. There is substantial evidence for H. sapiens crossing one or more of these straits, with over 500 Early Upper Paleolithic sites recorded on Paleo-Honshu, mainly located around river terraces and open-air foothills (Izuho and Kaifu 2015). These sites emerged between 38,000 and 30,000 years ago and are associated with stand- ardized lithic tools and long-distance raw material transport. During initial occupa- tion, the island was characterized by a variety of temperate forests (Takahara and Hayashi 2015), while tropical broadleaf forests were limited to southern Paleo-Hon- shu (Iwase et al 2015). There was also a focus on hunting large terrestrial game and evidence for complex strategizing in the form of pit traps (Sato 2012). However, impacts on megafauna seem to have been relatively small for many millennia (Iwase et al. Insular East Asia This implies there was initially high maritime mobil- ity, exploration, landscape learning, and resource exploitation, followed by a turn inland, perhaps related to the gradual focus on Paleo-Honshu stone resources and the Paleoloxodon-Sinomegaceroides megafaunal complex.f To the south, the offshore Ryukyu Islands preserve evidence for long-distance voyaging of a similar magnitude required to reach Talaud, the Solomons, and Manus (Kaifu et al. 2015). A substantial number of cave and rockshelter sites in karstic formations indicate that the entire island chain was colonized by MIS- 3, and as early as 36,000 years ago. These movements were less extensive than later Holocene settlement efforts such as the Neolithic dispersal into Taiwan from Fujian, but they probably required large founding groups or a period of back-and- forth interaction to establish viable populations on the islands. Kaifu et al. (2015) hypothesize that the initial settlement of the Ryukyus involved multiple migrations from different directions. On Ishigaki in the south- ern Ryukyus, human remains directly date to the LGM 19,000–29,000 years ago (Nakagawa et al. 2010), while similar H. sapiens remains excavated at Yamashita- cho Cave I and Minatogawa on Okinawa Island and Shimojibaru Cave on Kume Island in the central Ryukyus also were dated by association with wood charcoal to a similar time period, 15,000–32,000 years ago (Kobayashi et al. 1974; Suzuki and Hanihara 1982). Mitochondrial DNA analysis successfully extracted from five of the Ishigaki individuals suggests a strong connection with haplogroups common in coastal Mainland Southeast Asia and Taiwan (Shinoda and Adachi 2017), and it is probable that these lineages spread into the Ryukyu chain through what is now Taiwan, with the genesis of the M7a haplogroup arising in the south- ern Ryukyus during the LGM and later spreading into the northern Ryukyus and Paleo-Honshu.i The earliest global evidence for fishhooks comes from Sakitari on Okinawa, dating to c. 20,000–23,000 years ago, along with early shell tools dating to 23,500–25,500 years ago (Fujita et al. 2016). These fishhooks are one piece, made from Trochus shell, and are typologically similar to those from Wallacea and the Pacific. The common innovation of shell technology on small islands with poor lith- ics shows the flexibility of these maritime people to new environments, translating established forms to new materials (Fujita et al. 2016). Insular East Asia 2015), which may be in line with evidence from Madagascar and Sahul. On Paleo-Honshu the Paleoloxodon-Sinomegaceroides megafaunal complex included Naumann’s elephant, Yabe’s giant deer, Sika deer, brown bears, martens, least wea- sels, Eurasian badgers, racoon dogs, monkeys, wolves, and foxes. These became extinct during the LGM or at the Terminal Pleistocene (Iwase et al. 2012), indicat- ing that climatic and vegetational changes, rather than sudden human overhunting, were key factors in their extinction. Lithics that were made on this large island using good-quality cryptocrystalline materials such as obsidians, sanukite, and hard shale include trapezoids, pointed backed blades (also known as knife-shaped tools), and edge-ground axes (Iwase et al. 2015). There seems to have been island-wide technological changes through the Pleistocene, which indicates strong levels of interaction and mobility between Japan and mainland Eurasia. Several artifact types such as the Hakuhen-Sentoki points common around Kyushu (central Paleo-Honshu) and the Korean Peninsula suggest ongoing interaction (whether group or individual movements) across the strait in the Upper Paleolithic (Chang 2013). Morisaki (2015) interprets this as evi- dence for a second small-scale human migration into Paleo-Honshu through the Korean Strait, following the Aira-Tn eruption c. 30,000 years ago. Further evidence for more strategic episodes of seafaring comes from the pro- curement and transport of stone raw material from islands 40–45 km offshore, as early as 38,000 years ago. Geochemical studies demonstrate that obsidian artifacts were obtained from Kozushima (a small island in the Pacific Ocean), transported along the Izu Island chain to Paleo-Honshu, and then discarded at various sites around Mt. Ashitaka (Ikawa-Smith 2008). Ikeya (2015) suggests this may have been achieved through the use of leather-clad boats formed of a light- weight wooden frame made from branches rather than heavier dug-out canoes, which are sometimes found in archaeological sites from the later Jomon period, 1 3 3 Journal of Archaeological Research (2021) 29:255–326 292 and facilitated by changes to axe technology that would have allowed tree fell- ing and hulling. Importantly, the earliest dated sites around Mt. Ashitaka contain abundant Kozushima obsidian, but later sites during the LGM and the Terminal Pleistocene contain much reduced quantities until the Jomon period in the Early Holocene (Tsutsumi 2010). Insular East Asia The faunal remains from Sakitari include a variety of freshwater species (crabs and mollusks), along with eels, frogs, marine fish, small birds, deer, and small mam- mals, which show signs of charring on a fire. Perhaps more importantly, these fauna were being exploited on a seasonal basis, which implies the cave occupants returned to collect marine resources at times of high abundance (Fujita et al. 2016). This indicates that people did not necessarily pass quickly through oceanic islands en route to larger terrestrial sources but adapted to inland, littoral, and marine zones as well. Other sites such as Yamashita-cho I and Minatogawa provided evidence for H. sapiens in association with several species of deer along with wild boar (Table S1), 1 3 Journal of Archaeological Research (2021) 29:255–326 293 suggesting a mixed economy focus on island mammals along with the marine envi- ronment. Kaifu et al. (2015) argue there was an anthropogenic translocation of wild boar into the Ryukyus from mainland Eurasia or Paleo-Honshu to maintain a supply of mid-sized protein sources. suggesting a mixed economy focus on island mammals along with the marine envi- ronment. Kaifu et al. (2015) argue there was an anthropogenic translocation of wild boar into the Ryukyus from mainland Eurasia or Paleo-Honshu to maintain a supply of mid-sized protein sources. In terms of lithic technology, the Pleistocene Ryukyuans used edge-ground axes (Takashi 2012), and surface finds in the Yaeyamas of the southern Ryukyus indi- cate waisted implements that are typologically similar to those found around north- ern Sahul also were used (Anderson and Summerhayes 2008). These likely repre- sent convergent technological innovations in hafting, as waisted implements do not occur in Wallacea or the divide between Japan and Sahul. In contrast to Wallacea and Sahul, a microblade toolkit also developed in the Ryukyus by 20,000 years ago (Takashi 2012). These initial dispersals into Paleo-Honshu and Ryukyu required similarly advanced maritime technologies as in Wallacea and the Pacific, and the mariners sometimes voyaged long distances, out of sight of land. This occurred concurrently with settlement of the Bismarcks, the Talaud group, and many of the islands of Wal- lacea, which demonstrates maritime H. sapiens populations were thriving around the insular tropics and subtropics at a time when mainland Eurasian populations were severely reduced (Kuzmin and Keates 2014). Coastal Highways to the New World Understanding the water-crossing behaviors of eastern Eurasian H. sapiens is cen- tral to debates about Pleistocene seafaring in the Americas. Early evidence from the Bluefish Caves in the Yukon (Fig. 9) suggests that terrestrially adapted hunter- gatherers were active in eastern Beringia at least 24,000 years ago (Bourgeon et al. 2017). This area may have fostered a relatively isolated population that then dis- persed throughout the Americas following climatic amelioration, involving one or two major human lineages that carried multiple language families (Fagundes et al. 2008). This dispersal from Beringia to the tail end of the continent was incredibly rapid. Early coastal sites in South America include Quebrada Jaguay, Tacahuay, Santa Julia, and Huentelauquén, all dating to around 13,000 years ago, as well as Monte Verde dating to at least 14,000 and perhaps up to 18,000 years ago (Dillehay et al. 2015). This is supported by genetic evidence for north-south dispersals over a period of about 1500 years (Brandini et al. 2017). To account for this rapidity, Erlandson proposed the “kelp highway hypothesis”: deglaciation of the Pacific Northwest created a coastal dispersal corridor rich in marine and littoral resources, which allowed migration down the western fringe of the continents (Erlandson et al. 2007, 2008; see also Fladmark 1979). This scenario has major implications for understanding H. sapiens capacities for water crossings in temperate and subarctic zones and for understanding the dispersal of different lineages across the landscape. Unfortunately, although a handful of sites preserve marine fauna that demonstrate foraging activities along the littoral zone (Dillehay 3 Journal of Archaeological Research (2021) 29:255–326 294 et al. 2017), finding evidence for initial coastal and island settlements in this area remains difficult due to postglacial sea-level rise. Some underwater survey has been undertaken on submerged sites off the Californian northern Channel Islands (Watts et al., 2011), but most of the evidence remains inundated.fi Three sites in the northern Channel Islands off California’s Pacific Coast pro- vide the earliest evidence for Pleistocene seafaring in the Americas. These islands (forming one larger island named Santrosae) were separated from the mainland by at least 10 km of open water during the LGM, and the evidence indicates that coastally adapted people intensively hunted a number of marine mammal and seabird species and collected locally abundant shellfish by 12,000–11,000 years ago (Erlandson et al 2011; Hofman et al. 2018). Coastal Highways to the New World These sites are contemporaneous with Clovis and Fol- som sites, which are often situated around lakes in the interior, such as the Western Pluvial Lakes Tradition in the far west (Amick 1993). On Santarosae, there are numerous Channel Island barbed stone points and cres- cents along with red ochre, bone tools, and manufacturing debris at the CA-SRI- 512W site, which is interpreted to be a temporary winter hunting camp that facili- tated easy access to local birdlife and marine mammals that had become abundant on the island due to a lack of terrestrial predators. Midden remains are dominated by sea birds along with some marine mammal bones and smaller numbers of fish (Erlandson et al. 2011); obsidian from the site has been sourced to a flow in eastern California, suggesting trade links with the interior, or residential mobility stretch- ing over 300 km to the Coso Volcanic Field and beyond (Gill et al. 2019). At two nearby lithic scatters (CA-SMI-678 and CA-SMI-679) dating to the same period, people produced numerous bifaces, stemmed points, and crescents from local chert outcrops and consumed abundant local marine shellfish and crabs (Erlandson et al. 2011). Farther south on the arid island of Cedros, formerly connected to the Baja Cali- fornia Peninsula, the Pleistocene occupants hunted marine mammals, sea turtles and birds, collected shellfish and crustaceans, and produced unifacially retouched tools (Des Lauriers 2011). Crucially, oceanic deep-water fish also were caught, probably with hook and line technologies as one-piece shell hooks have been dated to at least 11,300 years ago at the Cerro Pedregoso and Richard’s Ridge sites (Des Lauriers et al. 2017). This provides additional strong evidence that boats were being used along America’s coasts. It remains unclear if the Cedros Island hooks represent remote technological decent from earlier eastern Eurasian examples or a distinct New World innovation; however, leaf-shaped bifaces at the site and other coastal locations stretching from Japan to South America may signify a pre-Clovis coastal dispersal from Eurasia to America (Erlandson and Braje 2011). Finally, recent controversial claims that hominins reached the Americas in MIS-5 are still being scrutinized, but they have important implications for global human migration and water-crossing behaviors (Braje et al. 2017). Holen et al. (2017) assert that hammerstones and stone anvils found at the Cerutti site in southern California are associated with mastodon bones, dated by Th/U to c. 130,000 years ago. 1 3 Coastal Highways to the New World Some of these bones are spirally fractured, perhaps indicating that hominins deliberately broke fresh long bones to extract marrow. These breakage patterns closely resemble experimental breakage from using hafted and unhafted cobble hammers on stone 1 3 Journal of Archaeological Research (2021) 29:255–326 295 anvils. They do not resemble carnivore breakage, but it is unclear if this represents active hunting or indirect scavenging of carcasses. Beringia was submerged during that time period (Hu et al. 2010), and it was not until ~110,000 years ago that the land bridge fleetingly formed, facilitating terrestrial migrations of megafauna (Tim- mermann and Friedrich 2016). If reliable, the most likely time of terrestrial dispersal would have been during MIS-6. anvils. They do not resemble carnivore breakage, but it is unclear if this represents active hunting or indirect scavenging of carcasses. Beringia was submerged during that time period (Hu et al. 2010), and it was not until ~110,000 years ago that the land bridge fleetingly formed, facilitating terrestrial migrations of megafauna (Tim- mermann and Friedrich 2016). If reliable, the most likely time of terrestrial dispersal would have been during MIS-6. From the perspective of Wallacea, it is not unfeasible that hominins, whether early H. sapiens or another population such as northern Denisovans, could have crossed open water gaps to reach North America. However, I would stress that there are key environmental differences in water temperature, marine biodiversity, open-sea distances, prey abundance, and availability of raw materials (see Anderson 2018), which would likely discount the possibility of hominins venturing through northern Pacific waters prior to Late Pleistocene H. sapiens. i Due to these ecological factors, the Americas’ evidence provides an intriguing comparison to the Indo-Pacific records of seafaring and voyaging, which primarily occurred in tropical and subtropical bodies of water. By contrast, the coastal dis- persal of our species down the western fringe of the Americas seems to have been associated with regular use of maritime technologies and the long-distance transfer of lithic materials, but water crossings themselves were restricted to short distances of under 10 km and, when undertaken, targeted known locations of rich offshore resources. The Mediterranean: An Express Route or Major Hurdle? Alongside the seafaring “nurseries” of Wallacea and the Bismarcks (see Irwin 1994, p. 31), the Mediterranean is the only other topographically similar hub of early water-crossing activity in the world (Broodbank 2006). Like Wallacea, the archi- pelagos of the eastern Mediterranean provide a change in geography; instead of the coast and offshore islands following a center-periphery model and providing a corridor for migration, or acting as steppingstones between continents, these areas challenged hominins to think differently about space and their environment. Here, different species were presented with large groups of islands with variable resource abundance that could, potentially, facilitate complex movements and interactions in their own right. The question has resurfaced: did Lower–Middle Paleolithic hominins cross into the Mediterranean islands during the Middle and Late Pleistocene? That time frame would make H. antecessor, H. heidelbergensis, H. neanderthalensis, and H. sapiens all contenders for the earliest water crossings into the islands (see Bartsiokas et al. 2017; Harvati et al. 2009; Martinez and Arsuaga 1997). Runnels (2014) argues that the Mediterranean may have been a Pleistocene express route for different hominin species moving from Africa into the European peninsula rather than a biogeographic barrier, due to similarities between material culture traditions around North Africa, Greece, and on some of the Mediterranean islands (e.g., Galanidou et al. 2016; Tourloukis and Karkanas 2012). To test this, Howitt-Marshall and Runnels (2016) formulate four working hypotheses: that “archaic” hominins were present on the 1 3 Journal of Archaeological Research (2021) 29:255–326 296 islands; that the islands were separated from the mainland during occupation; that cognitive and technological abilities were sufficient to produce watercraft; and that these abilities included wayfinding skills for open-sea crossings. If this multispecies model is demonstrated to be correct with future research, it will drastically revise how we understand the peopling of western Eurasia, potential “backwash” migra- tions into Africa, and long-term exchanges of materials, genes, and ideas between the two. The most convincing support for the multispecies model comes from the Plakias region of southern Crete (Fig. 10), where “Acheulean” lithics, including bifaces and large cutting tools typologically assigned to the Lower Paleolithic, have been found in association with paleosols and uplifted sedimentary terraces (Strasser et al. 2010). The Preveli 2 site lies on marine terraces dated by OSL to c. 45,000–50,000 years ago, providing a minimum possible age for occupation (Strasser et al. The Mediterranean: An Express Route or Major Hurdle? 2011), while sediments at Preveli 7, bracketing artifact-bearing layers, have been dated to c. 100,000–130,000 years ago (Runnels et al. 2014a). Farther west at Loutro, handaxes and other tools made on black flint and limestone have been assigned typologically to the late Lower Paleolithic and early Middle Paleolithic, but they lack direct dat- ing (Mortensen 2008). Similarly, at Mochlos in northeast Crete, an undated qua- ternary alluvial fan is alleged to contain quartz bifaces with affinities to the Lower and Middle Paleolithic on mainland Greece (Runnels et al. 2014b). As Crete was always separated from both Europe and Africa by large channels (Table 2), Pleis- tocene occupation on the island would have required sophisticated water crossings, probably facilitated by rafts or watercraft. On Gavdos Island, separated from Crete by the Libyan Sea, a large proportion of surface finds indicate occupation during the Middle and Late Pleistocene (Kopaka and Matzanas 2009). From the site of Ayios Pavlos-Fetifes, a number of “Paleolithic tools” have been used to construct a tentative temporal sequence stretching from the Fig. 10 Map of archaeological evidence for Pleistocene water crossings in the Mediterranean. Artifacts in association with direct dating marked as filled circles; sporadic undated surface finds with purported Paleolithic and Mesolithic attributes marked as hollow circles. For color legend please refer to online version of the article Fig. 10 Map of archaeological evidence for Pleistocene water crossings in the Mediterranean. Artifacts in association with direct dating marked as filled circles; sporadic undated surface finds with purported Paleolithic and Mesolithic attributes marked as hollow circles. For color legend please refer to online version of the article 3 3 1 Journal of Archaeological Research (2021) 29:255–326 297 Lower Paleolithic through to the Mesolithic (Table S2; Kopaka and Matzanas 2009). This includes chert and black flint implements possibly deriving from Crete. Melian obsidian in the form of a large blade is also present and thought to indicate long- distance maritime contacts by the Mesolithic. There also are claims for Middle Pleistocene occupation in the Cyclades. Typo- logically, chert and sandstone artifacts at Stélida on Naxos are suggested to be both Lower Mesolithic and early Middle Paleolithic (Carter et al. 2014), corresponding to 250,000 and 9,000 years ago. Lykousis (2009) suggests that Naxos was possi- bly connected to mainland Greece and Anatolia during the corresponding glacial periods MIS-6, MIS-8, and MIS-10–MIS-12, although the island’s endemic dwarfed fauna does not support this. In MIS-2 and perhaps MIS-3 corresponding to the Upper Paleolithic and Mesolithic, Naxos was part of the larger island of Cycladia but separated from Euboea by a single short water crossing (Table 2). Chert sources have been geochemically characterized, which will allow for future sourcing studies to assign excavated lithics on other islands to the Stélida outcrops (Skarpelis et al. 2017). Given the relatively extensive exploitation of the outcrop, and the long-range mobility behaviors of hunter-gathering hominins, we would expect this material to appear elsewhere around the islands and on the mainland, which would shed addi- tional light on the question of inter-island travel. Ongoing excavations at Stélida aim to establish artifacts in stratigraphic position (Carter, personal communication 2019).i On Kefallinia and Zakynthos, in the Ionian Islands, surface finds suggest that occupation by H. neanderthalensis began in the Middle Paleolithic (Table S2), per- haps 110,000–35,000 years ago (Ferentinos et al. 2012). This would have required crossing straits that were 5–12 km wide; Ferentinos et al. (2012) posit that the favorable microclimatic conditions in the area played an important role in allowing safe experimentation with water crossings.i Additional possible Lower–Middle Paleolithic surface finds derive from Melos, Alonnisos, Cyprus, and Corsardinia, which were all variably insular during the Pleistocene (Chelidonio 2001; Efstratiou et al. 2014; Panagopoulou et al. 2001; Sondaar et al. 1986; Strasser et al. 2016). To summarize, these tentatively assigned and predominantly undated “Paleolithic tools” are at the center of the debate, but they lack standardized technological analysis, interlaboratory scrutiny, and system- atic reporting. 3 sapiens through continental Europe and the coast of the Mediterranean basin from 45,000 to 40,000 years ago, small numbers of humans seem to have entered the islands of the central and eastern Mediterranean. The first confirmed evidence for Pleistocene seagoing is on Sardinia, which would have formed one larger island with Corsica, named Corsardinia. The fauna of the island is endemic, indicating the island was cut off from continental Europe throughout the Pleistocene. At the cave site of Corbeddu, bone artifacts have been radiocarbon dated to c. 15,000 years ago (Sondaar et al. 1986). The site is situated in a valley system perforated with a number of caves that could have served as a thoroughfare for migrating H. sapiens and also large game (Skeates 2012). Arriving on the island the colonists were confronted with a hugely reduced faunal diversity, but they seem to have rapidly adapted, specializing on large deer (Megaceros) hunting supple- mented by small mammals (ochotonids). Some of the faunal remains are burnt and show clear cutmarks (Skeates 2012, p. 20).l The authors argue that hominins also arrived in the Middle Pleistocene, reflected by a sudden and dramatic change in the extended faunal sequence, from a record dominated by an extinct goat, rodents, and ochotonids to one of deer, voles, and the Sardinian pika (Skeates 2012). This seems plausible; however, the arrival of the canid Cynotherium sardous, which specialized in hunting small, fast game, may also account for such a change. Lithic artifacts are found only throughout Layers 2 and 3, dating to 13,000 years ago, and technological changes are thought to reflect adapta- tion to different functional activities on the island (Hofmeijer et al. 1989). Human remains from the cave suggest H. sapiens, rather than a Middle Pleistocene hominin, were the hunters (Spoor 1999). Bone accumulations at A Teppa di U Lupinu and Coscia caves in Corsica, dat- ing to the LGM and Terminal Pleistocene, are possibly anthropogenic, with bones reworked in the Holocene (Salotti et al. 2008). However, the bones are not associated with any artifacts or patterns of breakage that would characterize butchery or split- ting of green bone to extract marrow. Further, the range of species, mostly compris- ing small mammals, amphibians, and birds, although not outside the range of Pleis- tocene human behavior in Wallacea or the Pacific, is inconsistent with assemblages formed on the adjacent mainland both by H. neanderthalensis and H. 3 A recent review of the lithic evidence from all of the above-men- tioned sites supports assigning some of the tools to the Middle Paleolithic but not the Lower Paleolithic (Papoulia 2017). This suggests that H. neanderthalensis were the first to enter the Mediterranean islands, followed by H. sapiens, strengthening interpretations by Broodbank (2006) and Leppard (2014), although this remains open to future revision given strategic fieldwork efforts to examine the Middle–Late Pleistocene record on the Greek islands that began only a decade ago (Runnels, per- sonal communication 2019). It is not certain if hominins made water crossings exclusively in the eastern Medi- terranean or if the Strait of Gibraltar also could have been a viable passage (Rolland 2013). Based on biogeographic, genetic, and paleontological data, it is an unlikely route (O’Regan et al. 2006), and to date, evidence for Pleistocene water crossings is absent in the western Mediterranean (Ramis and Alcover 2001). However, at 1 1 3 3 Journal of Archaeological Research (2021) 29:255–326 298 Vanguard and Gorham’s Caves in Gibraltar, Neanderthals commonly used coastal resources including marine shell, seals, dolphins, and fish, perhaps as part of broader residential mobility patterns (Stringer et al. 2008). This also occurred in other parts of the Iberian Peninsula and North Africa (Bicho and Haws 2008; Ramos et al. 2008), and there was shell tool manufacture at several sites on mainland Greece and Italy (Douka and Spinapolice 2012). The reliability of coastal environments is evi- dent by the fact these areas were some of the last places that H. neanderthalensis survived (Finlayson 2008). Vanguard and Gorham’s Caves in Gibraltar, Neanderthals commonly used coastal resources including marine shell, seals, dolphins, and fish, perhaps as part of broader residential mobility patterns (Stringer et al. 2008). This also occurred in other parts of the Iberian Peninsula and North Africa (Bicho and Haws 2008; Ramos et al. 2008), and there was shell tool manufacture at several sites on mainland Greece and Italy (Douka and Spinapolice 2012). The reliability of coastal environments is evi- dent by the fact these areas were some of the last places that H. neanderthalensis survived (Finlayson 2008). The record for Upper Paleolithic island occupation is sparse but more secure. Fol- lowing an advance of H. 3 sapiens (Stiner and Kuhn 1992; Vacca 2012) and on Corsardina itself (Sondaar et al. 1986). The hunting of deer on the island seems like a more plausible scenario, and we would expect to see abundant deer remains at the site. Rather, the assemblage closely resembles raptor-genic assemblages from the island (Robert and Vigne 2002). Accu- mulations by a variety of raptor species at the site are a plausible explanation; this 1 3 Journal of Archaeological Research (2021) 29:255–326 299 problem has recently been stressed in cave sites in Wallacea (Hawkins et al. 2018). Another option to account for the large assemblage of small mammals would be the canid Cynotherium sardous, like at Corbeddu (Lyras et al. 2006). However, as noted by Salotti et al. (2008), the absence of digestive marks usually seen on faunal assemblages produced by owls or carnivore gnawing is striking, so the possibility of human accumulation cannot be totally discounted. The timing of the deposits would be well within the timespan of H. sapiens and relatively unsurprising. Evidence for an antler manuport directly dated to c. 8,000–9,000 years ago gives the latest pos- sible date that people could have occupied the cave. problem has recently been stressed in cave sites in Wallacea (Hawkins et al. 2018). Another option to account for the large assemblage of small mammals would be the canid Cynotherium sardous, like at Corbeddu (Lyras et al. 2006). However, as noted by Salotti et al. (2008), the absence of digestive marks usually seen on faunal assemblages produced by owls or carnivore gnawing is striking, so the possibility of human accumulation cannot be totally discounted. The timing of the deposits would be well within the timespan of H. sapiens and relatively unsurprising. Evidence for an antler manuport directly dated to c. 8,000–9,000 years ago gives the latest pos- sible date that people could have occupied the cave. Farther south, there are traces of the Upper Paleolithic on Sicily, at cave sites including Acqua Fituasa, Giovanna, Perciata, Levanzo, Uzzo, San Teodoro, and Addaura, which were all initially occupied c. 17,000–12,000 years ago (Table 1). Genetic and faunal evidence from Grotta d’Oritene suggest H. sapiens crossed into Sicily via a land bridge that formed during the LGM (Mannino et al. 2012). 3 At that time the island was connected to peninsular Italy, and hunter-gathering groups using Epigravettian tools targeted familiar terrestrial food sources such as red deer, aurochs, and boar (Bietti 1990). However, during climatic amelioration and gradual insularization in the Terminal Pleistocene, there are indications that humans were gradually adapting their subsistence to an island lifestyle, including the presence of some sea birds at Grotta di Levanzo (Vigliardi 1982), fish at Grotta dell’Uzzo (Piperno et al. 1980), and marine shell at Addaura (Mannino and Thomas 2007), along with isotopic signatures that suggest a minor shift toward marine foods that were common around the Mediterranean coast (Mannino et al. 2011). However, predominantly terrestrial subsistence strategies persevered, perhaps because of the island’s large size with open environments that supported large mammals. It is unclear if Sicilian hunter-gatherers developed the use of boat technology to return to the mainland. Additionally, at Fontana Nuova cave in southern Sicily, a large assemblage of stone tools, faunal remains, and human bone was excavated unsystematically in the early 20th century and typologically assigned to the Aurignacian (Chilardi et al. 1996), which would implicate pre-LGM water crossings by H. sapiens perhaps 30,000 years ago. However, direct dating on human and animal bone collagen dem- onstrates the site is securely associated with the Early Holocene (Di Maida et al. 2019).f Evidence for more extensive reconnaissance and resource acquisition on off- shore islands comes indirectly from lithic sourcing. Obsidian artifacts excavated at Franchthi Cave in the northeast Peloponnese and dated to c. 15,000 years ago (Laskaris et al. 2011) have been sourced using fission track to Melos (Durrani et al. 1971). Melos was at all times separated from the mainland and Cycladia, but it lacks evidence for Upper Paleolithic settlement, demonstrating the direct procurement and transport of lithic material by mainland groups using watercraft (Renfrew and Aspi- nall 1990). More regular return trips to distant Mediterranean islands began in the Terminal Pleistocene, during a period of rapid cold reversal that resulted in harsh environ- mental conditions on the mainland (Broodbank 2006). H. sapiens visited Akrotiri Aetokremnos on Cyprus by c. 13,000 years ago (Simmons 1991), which was always 1 1 3 3 Journal of Archaeological Research (2021) 29:255–326 300 cut off from Eurasia by substantial water gaps of c. 60 km and provides a difficult target. 3 This suggests the first arrivals, likely coming from southern Anatolia, were supported by relatively sophisticated seafaring technologies (Bar-Yosef Mayer et al. 2015).l Abundant stone tools at the site, comprising flakes, thumbnail scrapers, burins, and a substantial blade component, are distinct from later Neolithic tool kits, and the associated faunal assemblage is dominated by pygmy hippopotamus, an island endemic that may have been overhunted to extinction (Simmons 1988). However, recent taphonomic analyses suggest that the pygmy hippopotami remains represent the reuse of fossil bone for fires rather than direct predation (Zazzo et al. 2015), although either explanation would account for the human translocation of wild boar to the island to provide a reliable protein resource (Vigne et al. 2009). There is little indication that these early Cypriots caught fish (only one fish bone is present in an Early Holocene layer), but they seem to have extensively collected shellfish from the littoral zone, some of which were later used as beads (Simmons 1999, p. 188). Unlike in the east, hominins around the Mediterranean basin may have been “reluctant sea-goers” until the very end of the Pleistocene following the LGM (Broodbank 2013, pp. 148–155). As Will et al. (2019) find, in studying the use of coastal resources by different Mediterranean populations, there were many similari- ties between H. sapiens, H. neanderthalensis, and other Middle Pleistocene homi- nins. The differences are revealed in the scale and diversity of coastally adaptive behaviors, with our species more intensively using a wider diversity of marine resources. In a similar way, the Mediterranean record of Pleistocene water crossings attests to early experimentation and visitation by at least H. neanderthalensis and Late Pleistocene H. sapiens, but the scale and intensity of seafaring by post-LGM H. sapiens sets these groups apart. In the following section I synthesize the global data, first examining the overlaps and contrasts between distinct regional Homo popula- tions and then the nature of adaptive flexibility within our species in particular. Water‑Crossing Behaviors Within the Genus Homo The global evidence shows that a range of Homo morpho-species indisputably crossed water gaps in the Pleistocene. These likely included H. erectus (or a related species including the ancestor of H. floresiensis), H. neanderthalensis, various unknown Homo such as Denisovans, and H. sapiens. The question is not whether these species made water crossings but how we characterize them and understand the implications for adaptive flexibility. Recent reviews (e.g., Dennell et al. 2014; Leppard 2015b) have cautiously accepted that extinct groups crossed water gaps but have stressed that these dispersals were passive. This contrasts with Runnels (2014) and Bednarik (2003) who assert that many of these species were able to deliberately, and perhaps strategically, make water crossings. As Phoca-Cosmetatou and Rabett (2014) keenly point out, the major disjuncture in this debate relates to a priori expectations of hominin cognitive abilities. Instead of comparing hominin decision Journal of Archaeological Research (2021) 29:255–326 301 making against strict criteria of our own abilities for controlled and strategic action, we can use the global data to rethink each regional population and associated water- crossing behaviors in their own right. I posit that some water crossings may have been intentional but fail to meet all of the requirements of strategic dispersal, as we see throughout much of the H. sapiens archaeological and ethnographic records. making against strict criteria of our own abilities for controlled and strategic action, we can use the global data to rethink each regional population and associated water- crossing behaviors in their own right. I posit that some water crossings may have been intentional but fail to meet all of the requirements of strategic dispersal, as we see throughout much of the H. sapiens archaeological and ethnographic records. Biomechanically, it would be possible for many species in our genus to deliber- ately cross water gaps, either by swimming or rafting (see Anderson 2018). Larson et al. (2007) state that changes to the shoulder in H. floresiensis, part of a broader complex of postcranial changes associated with H. erectus but much improved in H. sapiens, may be related to throwing. Interlinked with improvements in throw- ing were improvements in swimming and paddling (see Perry 1983). In support of this, many monkeys and some great apes in states of excitement have recently been observed swimming and diving (see Bender and Bender 2013, which includes video supplementary information). Water‑Crossing Behaviors Within the Genus Homo 1 3 3 302 Journal of Archaeological Research (2021) 29:255–326 This in-between can be illustrated in the difference between proboscids intentionally migrating across deep channels in Indian and Indonesian waters (deliberate, non- strategic) and a rodent drifting on a piece of vegetation after a storm (nondeliberate, nonstrategic). This in-between can be illustrated in the difference between proboscids intentionally migrating across deep channels in Indian and Indonesian waters (deliberate, non- strategic) and a rodent drifting on a piece of vegetation after a storm (nondeliberate, nonstrategic). Thirdly, it remains speculative whether producing and using simple watercraft is more cognitively challenging than producing and hafting a core tool, or building shelter, which have been recorded archaeologically among H. heidelbergensis, H. neanderthalensis, and H. sapiens, or whether watercraft were even needed to cross short water gaps, especially in warm equatorial waters (Anderson 2018). As sum- marized in Table 2, almost all of the water crossings made by hominins prior to MIS-4 took place in these conditions, and likely across water gaps of 30 km or less. Bowdler (1995) summarizes ethnographic and archaeological data from Australia to suggest that rafting with small bark canoes or swimming logs was usually restricted to less than 10 km, although they could be expanded to 30 km, suggesting that early seaward dispersals by other Homo could have been facilitated by simple flotation devices. The range of behaviors expressed by eastern Eurasian H. erectus, proposed to have made jumps into the Philippines, Sulawesi, and Flores, include megafaunal hunting (organized group activity, Storm 2012), engraving (design abstraction, Joordens et al. 2015), and standardized stone-tool production (vertical learning pro- cesses, Miller-Antonio et al. 2004). We also know that these H. erectus had likely evolved relative decentration (the ability to divorce action from concepts of the self and understand lasting consequences) and to coordinate a larger number of concepts, allowing for more complex technological and organizational schemes (Wynn 1993). Analysis of H. floresiensis brain capacity (Falk et al. 2005) shows that, like H. erec- tus, it had derived frontal and temporal lobes and a lunate sulcus in a derived posi- tion. As such, it was capable of higher cognitive processing, although it is unclear if there was a trade-off in intellect, with implications for social organization and motor skills (Kubo et al. 2013). Water‑Crossing Behaviors Within the Genus Homo As Bender (2015) notes, hominin abilities to swim were probably not limited by biomechanics but by cognitive abilities to experiment and learn. If we revisit Leppard’s (2015b) three cognitive requirements for deliberate inter- island travel, Pleistocene groups needed to premeditate future action, organize them- selves anticipating a future unseen goal, and design composite technology. Several comments are relevant. Firstly, the ethnographic analogs for strategic inter-island travel using composite technologies, which fundamentally shape our archaeologi- cal imagination, derive from H. sapiens populations already engaged with muti- generational water-crossing traditions. I contend that it is essential to incorporate the diachronic, incremental emergence of hominin innovation into our models. For instance, over the course of a few millennia or more of living coastally, with children growing up as hydrophilic rather than hydrophobic, perhaps experimenting with noncomposite organic floatation devices and paddles, it is not a big leap to see some adventurous individuals daring to make it over to the piece of land they can see in the distance, perhaps fortified by accidental crossings and attempted returns. In many sheltered archipelagos, such as Wallacea and the Mediterranean, these islands might only be several hundred meters or a few kilometers distant, representing very little risk for experimenting hominins. Secondly, group action among H. sapiens and other species is not always premed- itated and planned but often involves emulation and imitation without relying on language or a theory of mind (Call and Carpenter 2002; Carter and Charles 2013), and some great apes are even capable of spontaneous collaboration (Suchak et al. 2014). Numerous behavioral experiments involving extant hominines, with limited neocortical capacities compared to all fossil Homo, indicate they are capable of “mental time travel,” or episodic future thinking—the ability to project oneself into past and future events—for instance, in anticipating what tools are needed ahead of different activities (Kano and Hirata 2015; Osvath and Osvath 2008). Not only that, but Pan troglodytes at least appear to be able to project the future actions of other individuals within their social group (Osvath and Karvonen 2012). This type of cognition can be differentiated from group-level collective agency required for strategic colonization seen in H. sapiens. There is then likely to be a form of water crossings in the “in-between”: between passive dispersal and strategic colonization. 1 3 Water‑Crossing Behaviors Within the Genus Homo The question is, were these populations experimenting with coastal and marine environments, and, if so, were their brains working in a way to induce deliberate water-crossing behaviors? I suggest the Wallacean Early–Mid- dle Pleistocene record, characterized by short water crossings with little evidence for regular return crossings or adaptive changes to subsistence, represents regional behavioral responses of H. erectus or related species being confronted with equa- torial archipelagos; it is best understood as deliberate logistical range/procurement expansion rather than self-reflexive attempts at colonization. l Although experimental studies have shown that present-day humans with access to Paleolithic technology can deliberately navigate all of the passages crossed by H. erectus, or a similar hominin (Bednarik 1998), this does not shed light on whether H. erectus could have done the same. Such experiments have actually done a disser- vice for the case of deliberate H. erectus water crossings in preparing highly com- plex, multipart rafts made from bamboo. This is unnecessary, as H. erectus could have deliberately used bundles of mangrove vegetation or bamboo to cross small straits. This seems plausible, given we think most H. erectus material culture was noncomposite and did not require extended technological processes to arrive at a useful end result. Over several millennia, such rafts could have been incorporated 1 3 Journal of Archaeological Research (2021) 29:255–326 303 into this regional population’s spatial cognition as mobility extenders, just as chim- panzee cognition is extended when extractive technologies are used. into this regional population’s spatial cognition as mobility extenders, just as chim- panzee cognition is extended when extractive technologies are used. The absence of widespread, sustained occupation by H. erectus or other non- sapiens in the smaller islands of Wallacea, however, does suggest that these cross- ings were not strategic in the way we would understand H. sapiens dispersals into identical islands, and the crossings were likely compounded by accidental drifting and inclement weather. It also demonstrates comparative adaptive inflexibility when islands with dramatic biogeographic differences are reached, such as an absence of open woodlands and lakes, a lack of large terrestrial game, or the presence of dense rainforests. Although Sundaland H. erectus utilized aquatic resources and hunted large grassland animals, zooarchaeological assemblages in Wallacea are domi- nated by terrestrial faunas in contrast to specialized utilization of coastal and deep marine foodwebs or rainforest zones seen in Pleistocene H. sapiens (Roberts and Amano 2019). As O’Connor et al. Water‑Crossing Behaviors Within the Genus Homo (2017a) note, the inability of this specific line- age to quickly adapt their subsistence system may have prevented further expansion eastward into the much smaller and biologically depauperate islands of eastern Wal- lacea. Moreover, the absence of convincing evidence for H. erectus island hopping outside the warm seas of equatorial Wallacea, where water temperatures are fre- quently around 20OC and rafting material is abundant, supports that ecological con- tingency encouraged water crossings only within some regional populations of this species (Anderson 2018). Certainly, the Southeast Asian population of H. erectus (or another related hominin) must have been behaving differently around the coast compared to western H. erectus populations, leading them to reach some islands of Wallacea.l The through-flow currents that pass north–south between the straits separating Bali, Lombok, Komodo, and Flores seem to have undermined other fauna reach- ing the islands (Sprintall et al. 2014), and it is curious that rodents, proboscids, and hominins were the only notable mammalian fauna to cross the gap from Sunda into the Lesser Sunda Island chain (van den Bergh et al. 2001). Proboscids are excellent swimmers up to c. 50 km (Johnson 1980; Fig. 2), while rodents are known to drift on bundles of vegetation. It is notable that in all places where water crossing by non- sapiens have been proposed, proboscids also moved there and experienced insular dwarfism (van der Geer et al. 2016), perhaps reflective of highly mobile hunting par- ties following the migration routes of large game. What is crucial, though, is that the common dwarfing of body size in H. floresiensis and H. luzonensis would suggest there was allopatric speciation and restricted genetic exchange due to limited return trips to Sunda, indicating that initial crossings were outliers of normal behaviors (Leppard 2015a). At the other end of Eurasia, we can split the Mediterranean record into two thresholds: short jumps needed to reach offshore islands (Cycladia and the Ionian Islands) and slightly larger hurdles needed to get to Crete and Gavdos. Given the ranging abilities of all known hominin hunter-gatherers, it seems likely that the ear- liest visits to the Mediterranean islands, probably by H. neanderthalensis or pos- sibly by H. heidelbergensis, involved seasonal or occasional resource acquisition, with return trips to the mainland—and hence no reason to assume colonizing-scale demographics (see Leppard 2015a). Water‑Crossing Behaviors Within the Genus Homo 1 3 304 Journal of Archaeological Research (2021) 29:255–326 The lack of natural floating vegetation in the Mediterranean has been cited as one possible reason that African and western Eurasian H. ergaster/erectus/antecessor did not cross into the Mediterranean islands or Arabia by sea. However, there is evi- dence for H. neanderthalensis using hafted stone tools in Germany by 400,000 years ago (Thieme 2005), tar as a bonding agent in Italy by 200,000 years ago (Mazza et al. 2006), and three-ply fiber technology in France by 40,000 years ago (Hardy et al. 2020). Tar manufacture probably involved extended technological sequences of birch bark processing (Kozowyk et al. 2017), while twisted cords imply a com- prehension of multicomponent parts and mathematical sets (Hardy et al. 2020). It is plausible that similar adhesives and fibers were used for raft construction to get from Anatolia and peninsular Europe to the Mediterranean islands. Moreover, unlike east- ern H. erectus and H. floresiensis, there is substantially more evidence for H. nean- derthalensis being coastally adapted, or at least incorporating coastal resources and locations into wider foraging strategies. Additionally, H. neanderthalensis must have learned by teaching and sharing to produce levallois flakes (Morgan et al 2015). Whether verbal instruction or mostly gestural observation and imitation, the technical knowledge needed to produce a tool blank and the foresight understood in the resultant shape and what it would be used for is not too dissimilar to the level of technical knowledge needed to turn a piece of wood into a flotation device, or to string hides and branches together into a boat to make it to the next island within visible eyesight. Furthermore, the cognitive and technological abilities needed to produce the simplest of watercraft (e.g., some bark canoes or simple flotation devices) have been overstated. It does not require that much abstract thought, especially to get to a nearby island that is visible. It seems entirely plausible that H. neanderthalensis made short jumps into the Mediter- ranean, but these should be understood as adventurous range expansion by small groups of coastally accustomed foragers, rather than strategic colonization or inten- sive adaptation to marine environs (Runnels in press). The longer crossings to Crete and Gavdos, on the other hand, do imply a level of strategic and planned action not previously ascribed to H. neanderthalensis. Water‑Crossing Behaviors Within the Genus Homo This may indicate relatively sophisticated seagoing technologies using rafts and, minimally, a hydrophilic population comfortable with the deep sea. Despite this, on Crete itself there is, as yet, no evidence for dramatic, rapid behavioral shifts to adapt to island life during the colonization process, hinting at relatively low adaptive flex- ibility and experimentation. As Davidson (2001) notes, such short water crossings are remarkable, but they are not in the same order of magnitude as later settlement by H. sapiens. Homo sapiens and Adaptive Flexibility By comparison, H. sapiens generally made longer distance and more frequent water crossings and expressed substantially greater adaptive flexibility during the coloni- zation process, pushing the boundaries of highly diverse ecotones and using islands and marine environments with greater intensity and regularity than other species. For instance, H. sapiens provisioned themselves far more than other species. This 1 3 1 3 Journal of Archaeological Research (2021) 29:255–326 305 is seen in the transport of high-quality obsidian from New Britain to New Ireland, from Melos to mainland Greece, from Kozushima to Honshu, and from the Califor- nian mainland to the Channel Islands. The redistribution of obsidian fulfilled utili- tarian needs but also conveyed aesthetic value and created meaning during recip- rocal exchanges due to its scarcity and fixed origins (Torrence 2005). In a similar way, wild animals and plants were translocated from northern Sahul to the Bismarck Archipelago and Wallacea, from western Eurasia to Cyprus, and possibly from east- ern Eurasia to the Ryukyus. These Pleistocene translocations demonstrate a level of forward planning that is not seen in other species (Hofman and Rick 2018); they are precursors to the fully fledged “transported landscapes” of Holocene maritime societies (see Kirch 2017). Despite these few intraspecies commonalities, there is substantial regional variation in how Pleistocene H. sapiens utilized islands and how they incorporated water crossings into their everyday life.i Among Asian and Pacific H. sapiens, there is clear evidence for seafaring between landmasses and occasional voyaging to islands like Okinawa, Talaud, and Manus. We also see diversification and specialization of subsistence, with increased reliance on marine, littoral, and island resources (Roberts et al. 2020). The earliest faunal assemblages at these sites suggest colonists were extremely broad spectrum in foraging strategies, and in many places, it is likely that there was a seasonal or sporadic interplay between the coast and inland zones, with groups navigating along the shore but also pushing into the interior via river corridors. The earliest dated H. sapiens in Wallacea and Sahul demonstrate long-range mobility, with people mov- ing quickly into a myriad of different ecological zones. The Madjedbebe humans had already moved hundreds of kilometers inland from the northwest Sahul coast where the first settlers would have landed (Clarkson et al. 2017), and in the Ivane Valley of montane northeast Sahul, hunter-gatherers moved between coastal and montane environments from the outset (Summerhayes et al. Homo sapiens and Adaptive Flexibility 2010), in a manner that continued into the Terminal Pleistocene (Gaffney et al. 2015). This process is analo- gous to movements of H. sapiens between the coast and rainforest interiors in East Africa (Shipton et al. 2018). Although the initial colonizing populations of Sahul, Europe, and America may have preferred continental occupation, Late Pleistocene populations in Wallacea, the Ryukyus, the Bismarck Archipelago, and the Massim had a great propensity to inhabit tiny islands with small tracts of forest and depauperate terrestrial faunas. Small islands may pose a risk to terrestrially adapted groups, but those populations with a long-term history of exploiting the littoral and pelagic zones would find such islands ideal, for instance in the expansion of Pleistocene groups onto Kisar, Talaud, Gebe, and Okinawa. It is in these areas, particularly in eastern Wallacean islands like the Raja Ampat group, that we find the highest marine ecodiversity on the planet, with an abundance of marine and coastal resources. And it is the flexibility to shape subsistence strategies in new directions that enabled H. sapiens to monopolize these smaller islands in the first place. In a similar way, Late Holocene populations again showed increasing ingenuity in pushing the use of islands to the limits as they colonized tiny coral islands and reefs, dependent on marine resources and extensive long-distance social networks for survival. 1 3 306 Journal of Archaeological Research (2021) 29:255–326 Burke (2012) argues that H. sapiens evolved a distinct spatial cognition, related to frequent long-range mobility and the maintenance of expansive social and material networks, which would have given them an adaptive edge over H. nean- derthalensis. Similar spatial cognitive advantages probably played an important role in how some lineages of H. sapiens populated the islands of Wallacea, East Asia, the Pacific, and perhaps the Americas. We can visualize the wider social and physical islandscape, rich in resource opportunities and featuring a surround- ing safety net of related social groups, which would have encouraged much more regular return water crossings during the introduction stage of colonization.l Based on the archaeological data reviewed here, the adaptive flexibility hypothesis appears to hold, as a general rule, for Pleistocene H. sapiens in Wal- lacea, the Pacific, and the Ryukyus. This includes initial stages of landscape learning and adaptation to island and marine environments. 1 3 Measuring the Rate of Hominin Adaptive Flexibility The underlying mechanisms of human adaptive flexibility are expressed by the rhythm and tempo of innovative behavioral transformations as hominins crossed into novel environments for the first time. This tests the “rapid flexibility” and “gradual modification” hypotheses against the global data to distinguish if changes took place rapidly, over one or two generations, or if they were slower to unfold, with behaviors maintained over thousands of years by intergenerational cultural traditions. For most areas of the world, we are lacking sites related to the introduction phase of colonization. This can be mitigated by the strength of the empirical evidence, but for now it does not permit either hypothesis to be accepted or rejected. For instance, there is evidence to support rapid flexibility, particularly in lithic landscape learn- ing that globally characterizes Late Pleistocene H. sapiens moving into new islands and scouting out high-quality obsidian, and in the dramatic shifts in subsistence pre- sented in Table S2, which we see among the initial Wallacean, Ryukuan, Bismarck, Santarosae, and Cypriot H. sapiens, who shifted away from hunting large mainland animals to make extensive use of the littoral zone and small, hard-to-catch game.i g Conversely, in support of the gradual modification hypothesis, the earliest evi- dence for H. sapiens occupying the Indo-Pacific islands occurs in the Bismarck Archipelago and Timor, c. 45,000 years ago. Until recently, this was in line with the earliest dates for the colonization of mainland Sahul. However, in light of new dates of >70,000–60,000 years ago from northwest Australia (Clarkson et al. 2017), we may need to rethink the offshore connect. Although there is clearly evidence for complex maritime technology during the Late Pleistocene, no longer do these sites fit into the initial pulse of migration and perhaps represent a buffering lag of 30,000 years, which allowed coastally adapting people to build confidence in watercraft before they decided to settle islands, whereby pelagic fishing, animal translocation, and marine symbolism subsequently developed over tens of millennia in close prox- imity to the sea.l Although this leaves the mechanisms of adaptive flexibility unresolved, the two models offer a way forward to test future archaeological evidence. Further research will clearly be needed to shed light on the rate and scale of Pleistocene Homo adap- tive flexibility. In particular, research projects in several key areas would allow us to fill in the geographically and temporally expansive gaps. Homo sapiens and Adaptive Flexibility The Late Pleistocene archaeological signature in fact points to the opposite: either no occupation or very occasional visits from the 1 3 Journal of Archaeological Research (2021) 29:255–326 307 mainland by terrestrially focused groups. That is, Mediterranean islands were not incorporated into H. sapiens lifeways as extensively as they were in the east, not because European Pleistocene populations lacked some necessary cognitive ability to colonize the islands, but that their long-term behavioral trajectories did not push them toward the coast or to experiment with watercraft as extensively as in the east. Homo sapiens and Adaptive Flexibility I would stress that these innovations are tethered to long-term behavioral trajectories and scaffolded learning strategies in a myriad of ecological niches, which saw our species mov- ing in and out of novel and challenging environments. It was this physical move- ment, part of long-range residential foraging strategies in a number of different and changing ecotones, rather than stasis along the coast, that probably led these groups to experiment with watercraft in the first place. It remains unclear if these first experiments occurred in Wallacea or along the coastal fringes of southern Eurasia, Arabia, and Africa. In contrast, the situation is less convincing in the Mediterranean, and only after the LGM do some groups appear to be regularly seagoing, perhaps seafaring to places like Cyprus. In these islands, there was variable environmental attractiveness so that larger, ecologically stable and more readily accessible islands, such as Cor- sardinia and Sicily, and smaller islands with high-value resources, such as Melos, were preferred over resource-poor islands. The larger continental islands would have contained similar species to those on the mainland, although with less diver- sity, while the smaller oceanic islands contained many endemics but low species diversity, especially in higher taxonomic levels (Alcover et al. 1999). There is lit- tle evidence that islanders dramatically shifted subsistence strategies in the face of Mediterranean island environments, but one initial attraction to these spaces may have been the lack of predators, making prey species docile and easy to catch; after the disappearance of reliable native fauna, humans engineered their islands, such as Cyprus, to mimic the original ecology by introducing mainland animals. Local ecologies and long-term behavioral trajectories may explain variations in the Asian and Mediterranean datasets. Although each archipelago is topographi- cally similar, these H. sapiens populations at opposite ends of Eurasia seem to have approached islands and water crossings differently. As Cherry and Leppard (2018) note, although all H. sapiens were cognitively “capable” of strategic water cross- ings, the insular Mediterranean with reduced terrestrial resource abundance did not appeal on a mass scale to continental foragers. The signature in Wallacea and particularly the western Pacific, which probably hosted a number of strand-looping groups with inter-island voyaging abilities and social networks, seems to be mostly lacking in the Mediterranean. Measuring the Rate of Hominin Adaptive Flexibility At reputedly Lower, Middle, and Upper Palaeolithic sites on Mediterranean islands known to be insular throughout the Middle and Late Pleistocene, investigations could clarify how extensively these hominins made water crossings and how they adapted to island life, in particular by increasing the sample of sites that document subsistence activi- ties. We also require finer resolution in our bathymetric and geomorphological data, particularly in the Mediterranean, so that we can better model Pleistocene sea levels relative to tectonic uplift and subsidence (see recent successful approaches by Kealy et al. 2018; Norman et al. 2018). As we face a climate of increasing sea-level rise, targeting these areas, many of which are already at risk of erosion or inundation, will be paramount in the years ahead. Measuring the Rate of Hominin Adaptive Flexibility Systematic survey and excavation along stretches of the East African, Arabian, and southern Eurasian coasts with high relief that would have been close to the continental shelf edge prior to MIS-3 (see also Will et al. 2019) could establish if there is evidence for behaviors such as offshore fishing that could implicate watercraft, suggesting that H. sapiens were already adept at rudimentary seagoing before they entered island Wallacea, the 1 3 3 Journal of Archaeological Research (2021) 29:255–326 308 Ryukyus, and the Pacific. Such investigations on islands along the northern route through Wallacea could examine if there was a contemporary northerly dispersal into northwest Sahul 70–60,000 years ago, along with the nature of their adapta- tion to the islands, whether rapid or gradual. Research on the Kuril Islands in north- east Eurasia and steep sections of the western coast of the Americas could exam- ine if there is an unbroken link in water-crossing behaviors between what we know occurred in the Japanese islands with that evidenced in California. At reputedly Lower, Middle, and Upper Palaeolithic sites on Mediterranean islands known to be insular throughout the Middle and Late Pleistocene, investigations could clarify how extensively these hominins made water crossings and how they adapted to island life, in particular by increasing the sample of sites that document subsistence activi- ties. We also require finer resolution in our bathymetric and geomorphological data, particularly in the Mediterranean, so that we can better model Pleistocene sea levels relative to tectonic uplift and subsidence (see recent successful approaches by Kealy et al. 2018; Norman et al. 2018). As we face a climate of increasing sea-level rise, targeting these areas, many of which are already at risk of erosion or inundation, will be paramount in the years ahead. Ryukyus, and the Pacific. Such investigations on islands along the northern route through Wallacea could examine if there was a contemporary northerly dispersal into northwest Sahul 70–60,000 years ago, along with the nature of their adapta- tion to the islands, whether rapid or gradual. Research on the Kuril Islands in north- east Eurasia and steep sections of the western coast of the Americas could exam- ine if there is an unbroken link in water-crossing behaviors between what we know occurred in the Japanese islands with that evidenced in California. 1 3 Conclusion sapiens also started to use islands strategically for spe- cific resource acquisition, as people reshaped archipelagos into inter-island exchange networks and manipulated some new environments to better suit human needs by introducing reliable food sources. long, and, upon reaching new landmasses, shifted their behaviors to adapt to chal- lenges in insular environments. In particular, H. sapiens are the only Pleistocene hominins to utilize very small islands with tight biogeographic constraints in Wal- lacea, the Ryukyus, the Pacific, and the Americas. Living on these islands required drastic transformations from the adaptive strategies practiced on continental shelves and larger islands. These H. sapiens also started to use islands strategically for spe- cific resource acquisition, as people reshaped archipelagos into inter-island exchange networks and manipulated some new environments to better suit human needs by introducing reliable food sources. Thirdly, upon moving to the coast of mainland Eurasia, it remains unclear if H. sapiens required thousands of years to gradually innovate boat technology and water-crossing behaviors or if these transformations occurred rapidly, over such a short space of time that they have become archaeologically imperceptible. These two scenarios can be tested with ongoing and future field research across the globe, and they have important implications for how we understand the rate and scale of human adaptive flexibility. Understanding these mechanisms, especially in the face of new and changing environments, is a central concern if 21st century hominins are to effectively adapt to our changing environments. Acknowledgments Incisive review comments from Gary Feinman, Tom Leppard, Curtis Runnels, Glenn Summerhayes, Linda Nicholas, and four anonymous reviewers vastly improved the final manuscript. I also thank Cyprian Broodbank and Graeme Barker for encouraging me to tackle the topic and comment- ing on drafts, as well as Kristina Douglass, Tristan Carter, Ben Shaw, and Curtis Runnels for providing advice, key references, and in-press manuscripts. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Com- mons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. Conclusion Twenty years ago, the temporal and spatial range of Pleistocene water crossings was clear cut. We knew that humans had made it across the Wallace Line as far east as the Solomon Islands and that this was a skill reserved for so-called behav- iorally modern H. sapiens, who had dispersed out of Africa along the southern dis- persal route. This paradigm has recently been challenged by a number of discover- ies around the world, showing that water crossings also took place in the Early and Middle Pleistocene. The swift accumulation of this evidence called for a review to refine how these water crossings reshape our understanding of hominin behavior. i The emergence of water-crossing behaviors and aquatic lifeways occurred asym- metrically across the globe, which emphasizes that ecological and deep historical contingencies shaped water-crossing behaviors among multiple populations in simi- lar environments rather than inherent capacities for water crossings existing innately within specific species. In particular, the Island Southeast Asia region, with its warm equatorial waters and numerous sheltered archipelagos, was a hub of early expan- sions across water gaps by H. erectus, the ancestors of H. floresiensis and H. luzon- ensis, and perhaps one species of Denisovan hominins. These crossings were likely aided by experimentations in swimming and rafting behaviors. In the Mediterra- nean, there were similar expansions into islands by H. neanderthalensis; however, the presence of earlier hominin groups remains to be empirically demonstrated. All of these Early and Middle Pleistocene crossings represent small-scale seagoing, likely less than 30 km, and intentional range expansions to relatively familiar envi- ronments rather than strategic attempts to people new islands and landmasses.ii Secondly, our species, specifically populations in the Indo-Pacific, pushed water- crossing behaviors to new extremes, breaking the 30 km threshold already achieved by other species to move out of sight of land, sometimes on voyages over 200 km 1 3 Journal of Archaeological Research (2021) 29:255–326 309 long, and, upon reaching new landmasses, shifted their behaviors to adapt to chal- lenges in insular environments. In particular, H. sapiens are the only Pleistocene hominins to utilize very small islands with tight biogeographic constraints in Wal- lacea, the Ryukyus, the Pacific, and the Americas. Living on these islands required drastic transformations from the adaptive strategies practiced on continental shelves and larger islands. These H. Conclusion If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licen ses/by/4.0/. References Cited Aiello, L. C. (2010). Five years of Homo floresiensis. American Journal of Physical Anthropology 142: 167–179. Alcover, J. A., Seguí, B., and Bover, P. (1999). Extinctions and local disappearances of vertebrates in the western Mediterranean Islands. In MacPhee, R. D. E., and Sues, H.-D. (eds.), Extinctions in Near Time, Springer, Boston, pp. 165–188. g Allen, J. (2000). From beach to beach: The development of maritime economies in prehistoric Melanesia. Modern Quaternary Research in Southeast Asia 16: 137–176. uaternary Research in Southeast Asia 16: 137–176. Allen, J. Gosden, C., Jones, R., and White, J. P. (1988). Pleistocene dates for the human occupation N I l d h M l i N 331 707 709 Allen, J. Gosden, C., Jones, R., and White, J. P. (1988). Pleistocene da New Ireland, northern Melanesia. Nature 331: 707–709. Allen, J. Gosden, C., Jones, R., and White, J. P. (1988). Pleistocene dates for the human occupation of New Ireland, northern Melanesia. Nature 331: 707–709. Allen, J. Gosden, C., Jones, R., and White, J. P. (1988). Pleistoc New Ireland, northern Melanesia. Nature 331: 707–709. Allen, J. Gosden, C., Jones, R., and White, J. P. (1988). Pleistocene dates for the human occupation New Ireland, northern Melanesia. Nature 331: 707–709. Allen, J., Gosden, C., and White, J. P. (1989). Human Pleistocene adaptations in the tropical islan Pacific: Recent evidence from New Ireland, a Greater Australian outlier. Antiquity 63: 548–561. i Amick, D. S. (1993). Toolstone use and distribution patterns among Western Pluvial Lakes Traditio points from southern Nevada. Current Research in the Pleistocene 10: 49–51. 1 3 Journal of Archaeological Research (2021) 29:255–326 310 Anderson, A. (2013). The antiquity of sustained offshore fishing. Antiquity 87: 879–885. fi Anderson, A. (2018). Ecological contingency accounts for earliest seagoing in the Western Pacific Ocean. The Journal of Island and Coastal Archaeology 13: 224–234.i Anderson, A., and Summerhayes, G. (2008). Edge-ground and waisted axes in the Western Pacific Islands: Implications for an example from the Yaeyama Islands, southernmost Japan. Asian Per- spectives 47: 45–58. Anderson, A., Clark, G., Haberle, S., Higham, T., Nowak-Kemp, M., Prendergast, A., et al. (2018). New evidence of megafaunal bone damage indicates late colonization of Madagascar. PLOS One 13: p.e0204368.l Argue, D., Donlon, D., Groves, C., and Wright, R. (2006). Homo floresiensis: Microcephalic, pygmoid, Australopithecus, or Homo? Journal of Human Evolution 51: 360–374.fil Argue, D., Groves, C. P., Lee, M. S., and Jungers, W. L. (2017). References Cited The affinities of Homo floresiensis based on phylogenetic analyses of cranial, dental, and postcranial characters. Journal of Human Evolu- tion 107: 107–133. Armitage, S. J., Jasim, S. A., Marks, A. E., Parker, A. G., Usik, V. I., and Uerpmann, H.-P. (2011). The southern route “out of Africa”: Evidence for an early expansion of modern humans into Arabia. Science 331: 453–456. Aubert, M., O’Connor, S., McCulloch, M., Mortimer, G., Watchman, A., and Richer-LaFlèche, M. (2007). Uranium-series dating rock art in East Timor. Journal of Archaeological Science 34: 991–996. Aubert, M., Brumm, A., Ramli, M., Sutikna, T., Saptomo, E. W., Hakim, B., et al. (2014). Pleistocen cave art from Sulawesi, Indonesia. Nature 514: 223–227. Aubert, M., Setiawan, P., Oktaviana, A. A., Brumm, A., Sulistyarto, P. H., Saptomo, E. W., et al. (2018 Palaeolithic cave art in Borneo. Nature 564: 254–257. Aubert, M., Lebe, R., Oktaviana, A. A., Tang, M., Burhan, B., Jusdi, A., et al. (2019). Earliest hunting scene in prehistoric art. Nature 576: 442–445. Azzi, C. M., Bigliocca, L., and Piovan, E. (1973). Florence radiocarbon dates, I. Radiocarbon 1 479–487. Bae, C. J., Wang, W., Zhao, J., Huang, S., Tian, F., and Shen, G. (2014). Modern human teeth from la Pleistocene Luna cave (Guangxi, China). Quaternary International 354: 169–183. Bailey, G., and Milner, N. (2002). Coastal hunter-gatherers and social evolution: Marginal or central? Before Farming 3–4: 1–15. Bar-Yosef Mayer, D. E., Kahanov, Y., Roskin, J., and Gildor, H. (2015). Neolithic voyages to Cyprus: Wind patterns, routes, and mechanisms. The Journal of Island and Coastal Archaeology 10: 412–435. Barton, H., and White, J. P. (1993). Use of stone and shell artefacts at Balof 2, New Ireland, Papua Ne Guinea. Asian Perspectives 32: 169–181.i Bartsiokas, A., Arsuaga, J. L., Aubert, M., and Grün, R. (2017). U-series dating and classification of the Apidima 2 hominin from Mani Peninsula, Southern Greece. Journal of Human Evolution 109: 22–29. Bednarik, R. G. (1998). An experiment in Pleistocene seafaring. The International Journal of Nautic Archaeology 27: 139–149. Bednarik, R. G. (2003). Seafaring in the Pleistocene. Cambridge Archaeological Journal 13: 41–66. Bellwood, P. (2017). First Islanders: Prehistory and Human Migration in Island Southeast Asia. John Wiley and Sons, Hoboken. y Bellwood, P., Nitihaminoto, G., Irwin, G. Gunadi, A. W., and Tanudirjo, D. (1998). 35,000 years of pre- history in the northern Moluccas. In Bartstra, G.-J. (ed.), Bird’s Head Approaches: Irian Jaya Studies—A Programme for Interdisciplinary Research, A. A. References Cited Balkema, Rotterdam, pp. 233–275. Bender, P. R. (2015). The Use of Convergence as a Tool in the Reconstruction of Human Past, with Spe- cial Focus on Water Use in Hominin Evolution, Ph.D. thesis, Faculty of Science, University of Witwatersrand, Johannesburg. g Bender, R., and Bender, N. (2013). Swimming and diving behavior in apes (Pan troglodytes and Pongo pygmaeus): First documented report. American Journal of Physical Anthropology 152: 156–162. Benjamin, J., Rovere, A., Fontana, A., Furlani, S., Vacchi, M., Inglis, R. H., et al. (2017). Late Quater- nary sea-level changes and early human societies in the central and eastern Mediterranean Basin: An interdisciplinary review. Quaternary International 449: 29–57. 1 3 Journal of Archaeological Research (2021) 29:255–326 311 Bettis, E. A., Milius, A. K., Carpenter, S. J., Larick, R., Zaim, Y., Rizal, Y., et al. (2009). Way out of Africa: Early Pleistocene paleoenvironments inhabited by Homo erectus in Sangiran, Java. Journal of Human Evolution 56: 11–24. Bianchini, G., and Gambassini, P. (1973). La grotta dell’Acqua Fitusa (Agrigento): I. gli scavi e l’industria litica. Rivista di Scienze Preistoriche 28: 3–55.l Bicho, N., and Haws, J. (2008). At the land’s end: Marine resources and the importance of fluctuations in the coastline in the prehistoric hunter–gatherer economy of Portugal. Quaternary Science Reviews 27: 2166–2175. Bietti, A. (1990). The late Upper Paleolithic in Italy: an overview. Journal of World Prehistory 4: 95–15 Bintanja, R., van de Wal, R. S., and Oerlemans, J. (2005). Modelled atmospheric temperatures and global sea levels over the past million years. Nature 437: 125–128.i Birdsell, J. B. (1977). The recalibration of a paradigm for the first peopling of Greater Australia. In Allen, J., Golson, J., and Jones, R. (eds.), Sunda and Sahul: Prehistoric Studies in Southeast Asia, Mela- nesia and Australia. Academic Press, London, pp. 113–167. Blanc, P.-L. (2002). The opening of the Plio-Quaternary Gibraltar Strait: Assessing the size of a cat clysm. Geodinamica Acta 15: 303–317. Boivin, N. L., Zeder, M. A., Fuller, D. Q., Crowther, A., Larson, G., Erlandson, J. M., Denham, T., and Petraglia, M. D. (2016). Ecological consequences of human niche construction: Examining long- term anthropogenic shaping of global species distributions. Proceedings of the National Academy of Sciences 113: 6388–6396. Bonetti, R., Di Cesare, P., Guglielmetti, A., Malerba, F., Migliorini, E., Oddone, M., et al. (1998). Fission track dating of obsidian source samples from the Willaumez Peninsula, Papua New Guinea and eastern Australia. References Cited Records of the Australian Museum 50: 277–284. Bosch, M. D., Mannino, M. A., Prendergast, A. L., O’Connell, T. C., Demarchi, B., Taylor, S. M., et al. (2015). New chronology for Ksâr ‘Akil (Lebanon) supports Levantine route of modern human dis- persal into Europe. Proceedings of the National Academy of Sciences 112: 7683–7688. Bourgeon, L., Burke, A., and Higham, T. (2017). Earliest human presence in North America dated to the last glacial maximum: new radiocarbon dates from Bluefish Caves, Canada. PLOS One 12: p.e0169486. Bowdler, S. (1990). Peopling Australasia: The ‘coastal colonization’ hypothesis re-examined. In Mellars, P. (ed.), The Emergence of Modern Humans: An Archaeological Perspective, Edinburgh University Press, Edinburgh, pp. 327–346.f g pp Bowdler, S. (1995). Offshore islands and maritime explorations in Australian prehistory. Antiquity 69: 945–958. Bowen, D. Q. (2003). Uncertainty in oxygen isotope stage 11 sea-level: An estimate of ~13±2 m from Great Britain. In Droxier, A. W., Poore, R.Z., and Burckle, L. H. (eds.), Earth’s Climate and Orbital Eccentricity: The Marine Isotope Stage 11 Question, Geophysical Monograph Series 137, American Geophysical Union, Washington, DC, pp. 131–144. Braje, T. J., Dillehay, T. D., Erlandson, J. M., Fitzpatrick, S. M., Grayson, D. K., Holliday, V. T., et al. (2017). Were hominins in California ∼130,000 years ago? PaleoAmerica 3: 200–202. Brandini, S., Bergamaschi, P., Cerna, M. F., Gandini, F., Bastaroli, F., Bertolini, E., et al. (2017). The Paleo-Indian entry into South America according to mitogenomes. Molecular Biology and Evolu- tion 35: 299–311. Broodbank, C. (2006). The origins and early development of Mediterranean maritime activity. Journal of Mediterranean Archaeology 19: 199–230. gy Broodbank, C. (2013). The Making of the Middle Sea: A History of the Mediterranean from the Begin- ning to the Emergence of the Classical World, Thames and Hudson, London, pp. 286–360. Brown, P., Sutikna, T., Morwood, M. J., Soejono, R. P., Saptomo, E. W., and Due, R.A. (2004). A new Brown, P., Sutikna, T., Morwood, M. J., Soejono, R. P., Saptomo, E. W., and Due, R.A. (2004). A n small-bodied hominin from the Late Pleistocene of Flores, Indonesia. Nature 431: 1055–1061. Brumm, A. (2010). The Movius Line and the bamboo hypothesis: Early hominin stone technology Southeast Asia. Lithic Technology 35: 7–24. gy Brumm, A., Jensen, G. M., van den Bergh, G. D., Morwood, M. J., Kurniawan, I., Aziz, F., and Storey, M. (2010). Hominins on Flores, Indonesia, by one million years ago. Nature 464: 748–752. Brumm, A., Bergh, G. References Cited D. van den, Storey, M., Kurniawan, I., Alloway, B. V., Setiawan, R., et al. (2016). Age and context of the oldest known hominin fossils from Flores. Nature 534: 249–253. Brumm, A., Hakim, B., Ramli, M., Aubert, M., van den Bergh, G. D., Li, B., et al. (2018). A reassess- ment of the early archaeological record at Leang Burung 2, a Late Pleistocene rock-shelter site on the Indonesian island of Sulawesi. PLOS One 13: e0193025. 1 3 Journal of Archaeological Research (2021) 29:255–326 312 Bulbeck, F. D., Sumantri, I., and Hiscock, P. (2004). Leang Sakapao 1, a second dated Pleistocene si from South Sulawesi, Indonesia. Modern Quaternary Research in Southeast Asia 18: 111–128. Burckle, L. H. (1993). Late Quaternary interglacial stages warmer than present. Quaternary Science Reviews 12: 825–831. Burdukiewicz, J. M. (2014). The origin of symbolic behavior of Middle Palaeolithic humans: Recent con- troversies. Quaternary International 326: 398–405. Burke, A. (2012). Spatial abilities, cognition and the pattern of Neanderthal and modern human disper- sals. Quaternary International 247: 230–235. Call, J., and Carpenter, M. (2002). Three sources of information in social learning. In Dautenhahn, K., and Nehaniv, C. L. (eds.), Complex Adaptive Systems: Imitation in Animals and Artifacts, MIT Press, Cambridge, MA. pp. 211–228. Cardini, L. (1971). Rinvenimenti paleolitici nella Grotta Giovanna (Siracusa). Atti della XIII Riun, Scient. dell’Istituto ltaliano di Preist e Protostoria 1: 29–35. Carter, B., and Charles, N. (2013). Animals, agency and resistance. Journal for Theory of Social Beha ior 43: 322–340. Carter, T., Contreras, D., Doyle, S., Mihailovic, D., Moutsiou, T., and Skarpelis, N. (2014). The Stél- ida Naxos Archaeological Project: New data on the Middle Palaeolithic and Mesolithic Cyclades. Antiquity Project Gallery 88: 341. Chang, Y. (2013). Human activity and lithic technology between Korea and Japan from MIS 3 to MIS in the Late Paleolithic period. Quaternary International 308: 13–26. Chappell, J. (1993). Late Pleistocene coasts and human migrations in the Austral region. In Spriggs, M., Yen, D., Ambrose, W., Jones, R., Thorne, A., and Andrews, A. (eds.), A Community of Culture, Department of Prehistory, Australian National University, Canberra, pp. 43–48. Chelidonio, G. (2001). Manufatti litici su ciottolo da Milos (isole Cicladi). Pegaso: Rivista di Cultura Mediterranea 1: 117–144. Cherry, J. F. (1981). Pattern and process in the earliest colonization of the Mediterranean islands. Pr ceedings of the Prehistoric Society 47: 41–68. Cherry, J. F., and Leppard, T. P. (2018). References Cited Patterning and its causation in the pre-Neolithic colonization of the Mediterranean islands (Late Pleistocene to Early Holocene). The Journal of Island and Coastal Archaeology 13: 191–205. Chilardi, S., Frayer, D. W., Gioia, P., Macchiarelli, R., and Mussi, M. (1996). Fontana Nuova di Ragusa (Sicily, Italy): Southernmost Aurignacian site in Europe. Antiquity 70: 553–563. ( y y) g p q y Choi, K., and Driwantoro, D. (2007). Shell tool use by early members of Homo erectus in Sa tral Java, Indonesia: Cut mark evidence. Journal of Archaeological Science 34: 48–58. Choi, K., and Driwantoro, D. (2007). Shell tool use by early members of Homo erectus in Sangiran, ce l d i C k id l f A h l l 34 48 58 Choi, K., and Driwantoro, D. (2007). Shell tool use by early members of Homo erectus in Sang tral Java, Indonesia: Cut mark evidence. Journal of Archaeological Science 34: 48–58. tral Java, Indonesia: Cut mark evidence. Journal of Archaeological Science 34: 48–58. Clarkson, C., Jacobs, Z., Marwick, B., Fullagar, R., Wallis, L., Smith, M., et al. (2017). Human occup tion of northern Australia by 65,000 years ago. Nature 547: 306–310. , , , , , , g , , , , , , tion of northern Australia by 65,000 years ago. Nature 547: 306–310. Cooper, A., and Stringer, C. B. (2013). Did the Denisovans cross Wallace’s line? Science 342: 321–323. Curnoe, D. (2003). Problems with the use of cladistic analysis in palaeoanthropology. Homo 53: 225–234. Davidson, I. (2001). The requirements for human colonisation of Australia. In Metcalfe, I., Smith, J. M. B., Morwood, M., and Davidson, I. (eds.), Faunal and Floral Migration and Evolution in SE Asia- A li S d Zi li Li 399 408 Curnoe, D. (2003). Problems with the use of cladistic analysis in palaeoanthropology. Homo 53: 225 234. Davidson, I. (2001). The requirements for human colonisation of Australia. In Metcalfe, I., Smith, J. M. B M d M d D id I ( d ) F l d Fl l Mi i d E l i i SE A i Davidson, I. (2001). The requirements for human colonisation of Australia. In Metcalfe, I., Smith, J. M. B., Morwood, M., and Davidson, I. (eds.), Faunal and Floral Migration and Evolution in SE Asia- Australia, Swets and Zietlinger, Lisse, pp. 399–408.i Davidson, I., and Noble, W. (1992). References Cited Why the first colonisation of the Australian region is the earliest ev dence of modern human behaviour. Archaeology in Oceania 27: 135–142. Dennell, R., and Petraglia, M. D. (2012). The dispersal of Homo sapiens across southern Asia: How early, how often, how complex? Quaternary Science Reviews 47: 15–22. Dennell, R. W., Louys, J., O’Regan, H. J., and Wilkinson, D. M. (2014). The origins and persistence of Homo floresiensis on Flores: Biogeographical and ecological perspectives. Quaternary Science Reviews 96: 98–107. d’Errico, F. (2003). The invisible frontier: A multiple species model for the origin of behavioral mode nity. Evolutionary Anthropology 12: 188–202. d’Errico, F., Vanhaeren, M., Barton, N., Bouzouggar, A., Mienis, H., Richter, D., et al. (2009). Additional evidence on the use of personal ornaments in the Middle Paleolithic of North Africa. Proceedings of the National Academy of Sciences 106: 16051–16056. Des Lauriers, M. R. (2011). Of clams and Clovis: Isla Cedros, Baja California, Mexico. In Bicho, N. F., Haws, J. A., and Davis, L. G. (eds.), Trekking the Shore, Springer, New York pp. 161–177. 1 3 Journal of Archaeological Research (2021) 29:255–326 313 Des Lauriers, M. R., Davis, L. G., Turnbull, J., Southon, J. R., and Taylor, R. E. (2017). The earliest shell fishhooks from the Americas reveal fishing technology of Pleistocene maritime foragers. American Antiquity 82: 498–516. Détroit, F., Dizon, E., Falguères, C., Hameau, S., Ronquillo, W., and Sémah, F. (2004). Upper Pleistocene Homo sapiens from the Tabon cave (Palawan, The Philippines): Description and dating of new discoveries. Comptes Rendus Palevol 3: 705–712. p Détroit, F., Mijares, A. S., Corny, J., Daver, G., Zanolli, C., Dizon, E., et al. (2019). A new species of Homo from the Late Pleistocene of the Philippines. Nature 568: 181–186. Di Maida, G., Mannino, M. A., Krause-Kyora, B., Jensen, T. Z. T., and Talamo, S. (2019). Radiocarbon dating and isotope analysis on the purported Aurignacian skeletal remains from Fontana Nuova (Ragusa, Italy). PLOS One 14: p.e0213173. Dillehay, T. D., Ocampo, C., Saavedra, J., Sawakuchi, A. O., Vega, R. M., Pino, M., et al. (2015). New archaeological evidence for an early human presence at Monte Verde, Chile. PLOS One 10: p.e0141923. Dillehay, T. D., Goodbred, S., Pino, M., Sánchez, V. F. V., Tham, T. R., Adovasio, J., et al. (2017). Simple technologies and diverse food strategies of the Late Pleistocene and Early Holocene at Huaca Pri- eta, coastal Peru. Science Advances 3: p.e1602778. Dizon, E. Z. References Cited (2003). New direct dating of the human fossils from Tabon Cave, Palawan, Philippines. Pro- ceedings of the Society of Philippine Archaeologists 1: 63–67. Dizon, E., Détroit, F., Sémah, F., Falguères, C., Hameau, S., Ronquillo, W., and Cabanis, E. (2002). Notes on the morphology and age of the Tabon Cave fossil Homo sapiens. Current Anthropology 43: 660–666. Douglass, K., Antonites, A. R., Morales, E. M. Q., Grealy, A., Bunce, M., Bruwer, C., and Gough, C. (2018). Multi-analytical approach to zooarchaeological assemblages elucidates Late Holocene coastal lifeways in southwest Madagascar. Quaternary International 471: 111–131. Douglass, K., Hixton, S., Wright, H. T., Godfrey, L. R., Crowley, B. E., Manjakahery, B., et al. (in press). A critical review of radiocarbon dates clarifies the human settlement of Madagascar. Quaternary Science Reviews. Douka, K., and Spinapolice, E. E. (2012). Neanderthal shell tool production: Evidence from Middle Pa aeolithic Italy and Greece. Journal of World Prehistory 25: 45–79. Dukas, R. (1998). Evolutionary ecology of learning. In Dukas, R. (ed.), Cognitive Ecology: The Evolu- tionary Ecology of Information Processing and Decision-making, Chicago University Press, Chi- cago, pp. 129–174.ii g pp Durrani, S. A., Khan, H. A., Taj, M., and Renfrew, C. (1971). Obsidian source identification by fissio track analysis. Nature 233: 242–245. Efstratiou, N., Biagi, P., and Starnini, E. (2014). The Epipalaeolithic site of Ouriakos on the island of Lemnos and its place in the Late Pleistocene peopling of the east Mediterranean region. Adalya 17: 1–23. Erlandson, J. M. (2001). The archaeology of aquatic adaptations: Paradigms for a new millennium. Jour- nal of Archaeological Research 9: 287–350. Erlandson, J. M. (2017). Coastlines, marine ecology, and maritime dispersals in human history. In Boiven, N., Crassard, J., and Petraglia, M. (eds.), Human Dispersal and Species Movement: From Prehistory to Present, Cambridge University Press, Cambridge, pp. 147–163. Erlandson, J. M., and Braje, T. J. (2011). From Asia to the Americas by boat? Paleogeography, paleoecol- ogy, and stemmed points of the northwest Pacific. Quaternary International 239: 28–37. i Erlandson, J. M., Graham, M. H., Bourque, B. J., Corbett, D., Estes, J. A., and Steneck, R. S. (2007). The kelp highway hypothesis: Marine ecology, the coastal migration theory, and the peopling of the Americas. The Journal of Island and Coastal Archaeology 2: 161–174. f gy Erlandson, J. M., Moss, M. L., and Des Lauriers, M. (2008). Life on the edge: Early maritime cultures of the Pacific Coast of North America. References Cited Quaternary Science Reviews 27: 2232–2245. i Erlandson, J. M., Rick, T. C., Braje, T. J., Casperson, M., Culleton, B., Fulfrost, B., et al. (2011). Paleoin- dian seafaring, maritime technologies, and coastal foraging on California’s Channel Islands. Sci- ence 331: 1181–1185. Fagundes, N. J. R., Kanitz, R., Eckert, R., Valls, A. C. S., Bogo, M. R., Salzano, F. M., et al. (2008). Mitochondrial population genomics supports a single pre-Clovis origin with a coastal route for the peopling of the Americas. American Journal of Human Genetics 82: 583–592. p p g f Falk, D., Hildebolt, C., Smith, K., Morwood, M. J., Sutikna, T., Brown, P., et al. (2005). The brain of LB1, Homo floresiensis. Science 308: 242–245. 1 3 Journal of Archaeological Research (2021) 29:255–326 314 Farrera, I., Harrison, S. P., Prentice, I. C., Ramstein, G., Guiot, J., Bartlein, P. J., et al. (1999). Tropical climates at the Last Glacial Maximum: A new synthesis of terrestrial palaeoclimate data. I. Vegeta- tion, lake-levels and geochemistry. Climate Dynamics 15: 823–856. Ferentinos, G., Gkioni, M., Geraga, M., and Papatheodorou, G. (2012). Early seafaring activity in the southern Ionian Islands, Mediterranean Sea. Journal of Archaeological Science 39: 2167–2176. Finlayson, C. (2008). On the importance of coastal areas in the survival of Neanderthal populations du ing the Late Pleistocene. Quaternary Science Reviews 27: 2246–2252. Fitzpatrick, S. M., and Anderson, A. (2008). Islands of isolation: Archaeology and the power of aquat perimeters. The Journal of Island and Coastal Archaeology 3: 4–16. Fladmark, K. (1979). Routes: Alternate migration corridors for early man in North America. American Antiquity 44: 55–69. Flannery, T. F., and White, J. P. (1991). Animal translocations. National Geographic Research and Exploration 7: 96–113. Florin, S. A., Fairbairn, A. S., Nango, M., Djandjomerr, D., Marwick, B., Fullagar, R., et al. (2020). The first Australian plant foods at Madjedbebe, 65,000–53,000 years ago. Nature Communications 11: 924. Foley, R. A. (1991). How many species of hominid should there be? Journal of Human Evolution 20: 413–427. Fox, R. B. (1970). The Tabon Caves: Archaeological Explorations and Excavations on Palawan Island, Philippines, National Museum of the Philippines, Manilla. Frederickson, C. (1997). Changes in Admiralty Islands obsidian source use: The view from Pamwak. Archaeology in Oceania 32: 68–73. Frederickson, C., Spriggs, M., and Ambrose, W. (1993). Pamwak rockshelter: A Pleistocene rockshel- ter on Manus Island. In Smith, M. A., Spriggs, M., and Fankhauser, B. References Cited (eds.), Sahul in Review: Pleistocene Archaeology in Australia and Island Melanesia, Department of Prehistory, Australian National University, Canberra, pp. 144–152. ational University, Canberra, pp. 144–152. Fujita, M., Yamasaki, S., Katagiri, C., Oshiro, I., Sano, K., Kurozumi, T., et al. (2016). Advanced mari- time adaptation in the western Pacific coastal region extends back to 35,000–30,000 years before present. Proceedings of the National Academy of Sciences 113: 11184–11189.f Gaffney, D., Ford, A., and Summerhayes, G. R. (2015). Crossing the Pleistocene-Holocene transition in the New Guinea Highlands: Evidence from the lithic assemblage of Kiowa rockshelter. Journal of Anthropological Archaeology 39: 223–246. Galanidou, N., Athanassas, C., Cole, J., Iliopoulos, G., Katerinopoulos, A., Magganas, A., and McNabb, J. (2016). The Acheulian site at Rodafnidia, Lisvori, on Lesbos, Greece: 2010–2012. In Harvati, K., and Roksandic, M. (eds.), Paleoanthropology of the Balkans and Anatolia, Springer, Dordrecht, pp. 119–138. Gill, K. M., Erlandson, J. M., Hughes, R. E., Origer, T., Rogers, A. K., and Vellanoweth, R. L. (2019). Material conveyance in the Southern California Bight: Obsidian on Alta California’s Chan- nel Islands. The Journal of Island and Coastal Archaeology https://doi.org/10.1080/15564 894.2019.1570988. Glover, I. (1969). Radiocarbon dates from Portuguese Timor. Archaeology and Physical Anthropology Oceania 4: 107–112. Glover, I. (1981). Leang Burung 2: An Upper Palaeolithic rock shelter in South Sulawesi, Indonesi Modern Quaternary Research in Southeast Asia 6: 1–38. y Gorecki, P., Mabin, M., and Campbell, J. (1991). Archaeology and geomorphology of the Vanimo coast, Papua New Guinea: Preliminary results. Archaeology in Oceania 26: 119–122. p y Gosden, C., and Malafouris, L. (2015). Process archaeology (p-arch). World Archaeology 47: 701–717. Gosden, C., and Malafouris, L. (2015). Process archaeology (p-arch). World Archaeology 47: 701–717. Groube, L. (1989). The taming of the rain forests: A model for Late Pleistocene forest exploitation in New Guinea. In Harris, D. R., and Hillman, G. C. (eds.), Foraging and Farming, Unwin Hyman, Gosden, C., and Malafouris, L. (2015). Process archaeology (p-arch). World Archaeology 47: 701–717. Groube L (1989) The taming of the rain forests: A model for Late Pleistocene forest exploitation in Gosden, C., and Malafouris, L. (2015). Process archaeology (p-arch). World Archaeology 47: 701–717. Groube, L. (1989). The taming of the rain forests: A model for Late Pleistocene forest exploitation in New Guinea. In Harris, D. R., and Hillman, G. C. (eds.), Foraging and Farming, Unwin Hyman, London, pp. 292–304. Groube, L., Chappell, J., Muke, J., and Price, D. (1986). References Cited A 40,000-year-old occupation site at Huon Pen- insula, Papua New Guinea. Nature 324: 453–455. Habgood, P. J., and Franklin, N. R. (2008). The revolution that didn’t arrive: A review of Pleistocene Sahul. Journal of Human Evolution 55: 187–222. Hamada, T. (1985). 14C Age of Charcoal from Pinza-Abu Cave Deposits, Miyako Island, Okinawa, Japan: Reports on Excavation of the Pinza-Abu Cave, Department of Education, Okinawa Prefec- ture Government, Naha. 1 3 Journal of Archaeological Research (2021) 29:255–326 315 Hanebuth, T. J., Voris, H. K., Yokoyama, Y., Saito, Y., and Okuno, J. I. (2011). Formation and fate of sedimentary depocentres on Southeast Asia’s Sunda Shelf over the past sea-level cycle and biogeo- graphic implications. Earth-Science Reviews 104: 92–110. Hansford, J., Wright, P. C., Rasoamiaramanana, A., Pérez, V. R., Godfrey, L. R., Errickson, D., Thomp- son, T., and Turvey, S. T. (2018). Early Holocene human presence in Madagascar evidenced by exploitation of avian megafauna. Science Advances 4: eaat6925. p g Hardy, B. L., Moncel, M. H., Kerfant, C., Lebon, M., Bellot-Gurlet, L., and Mélard, N. (2020). Direct evidence of Neanderthal fibre technology and its cognitive and behavioral implications. Scientific Reports 10: 4889. Harvati, K., Panagopoulou, E., and Runnels, C. (2009). The paleoanthropology of Greece. Evolutionary Anthropology 18: 131–143. Hawkins, S., O’Connor, S., and Louys, J. (2017a). Taphonomy of bird (Aves) remains at Laili Cave, Timor-Leste, and implications for human-bird interactions during the Pleistocene. Archaeological and Anthropological Sciences, https://doi.org/10.1007/s12520-017-0568-4. Hawkins, S., O’Connor, S., Maloney, T. R., Litster, M., Kealy, S., Fenner, J. N., et al. (2017b). Old- est human occupation of Wallacea at Laili Cave, Timor-Leste, shows broad-spectrum foraging responses to Late Pleistocene environments. Quaternary Science Reviews 171: 58–72. p y Hawkins, S., Carro, S. C. S., Louys, J., Aplin, K., O’Connor, S., and Mahirta (2018). Human palaeoeco- logical interactions and owl roosting at Tron Bon Lei, Alor Island, Eastern Indonesia. The Journal of Island and Coastal Archaeology 13: 371–387. f gy Heaney, L. R. (1991). A synopsis of climatic and vegetational change in Southeast Asia. In Myers, N. (ed.), Tropical Forests and Climate, Springer, Dordrecht, pp. 53–61. Henneberg, M., Eckhardt, R. B., Chavanaves, S., and Hsü, K. J. (2014). Evolved developmental homeo- stasis disturbed in LB1 from Flores, Indonesia, denotes Down Syndrome and not diagnostic traits of the invalid species Homo floresiensis. Proceedings of the National Academy of Sciences 111: 11967–11972. Henshilwood, C. S., Sealy, J. References Cited C., Yates, R., Cruz-Uribe, K., Goldberg, P., Grine, F. E., et al. (2001). Blombos Cave, Southern Cape, South Africa: Preliminary report on the 1992–1999 excavations of the Middle Stone Age levels. Journal of Archaeological Science 28: 421–448.i Hiscock, P. (2015). Cultural diversification and the global dispersion of Homo sapiens: Lessons from Australia. In Kaifu, Y., Izuho, M., Goebel, T., Sato, H., and Ono, A. (eds.), Emergence and Diver- sity of Modern Human Behaviour in Paleolithic Asia, Texas A&M University Press, College Sta- tion, pp. 225–236. pp Hoffmann, D. L., Angelucci, D. E., Villaverde, V., Zapata, J., and Zilhão, J. (2018). Symbolic use of marine shells and mineral pigments by Iberian Neandertals 115,000 years ago. Science Advances 4: p.eaar5255. Hofman, C. A., and Rick, T. C. (2018). Ancient biological invasions and island ecosystems: Tracking translocations of wild plants and animals. Journal of Archaeological Research 26: 65–115. Hofman, C. A., Rick, T. C., Erlandson, J. M., Reeder-Myers, L., Welch, A. J., and Buckley, M. (2018). Collagen fingerprinting and the earliest marine mammal hunting in North America. Scientific Reports 8: 10014. p Hofmeijer, G. K., Alderliesten, C., Borg, K. V. D., Houston, C. M., Jong, A. F. M., Martini, F., et al (1989). Dating of the Upper Pleistocene lithic industry of Sardinia. Radiocarbon 31: 986–991.f Holen, S. R., Deméré, T. A., Fisher, D. C., Fullagar, R., Paces, J. B., Jefferson, G. T. et al. (2017). 130,000-year-old archaeological site in southern California, USA. Nature 544: 479–483. Hopkins, D. M. (1982). Aspects of the paleogeography of Beringia during the Late Pleistocene. In Hop- kins, D. M., Matthews, J. V., Schweger, C. E., and Young, S. B. (eds.), Paleoecology of Beringia, Academic Press, New York, pp. 3–28. pp Howitt-Marshall, D., and Runnels, C. (2016). Middle Pleistocene sea-crossings in the eastern Mediter- ranean? Journal of Anthropological Archaeology 42: 140–153.l Hu, A., Meehl, G. A., Otto-Bliesner, B. L., Waelbroeck, C., Han, W., Loutre, M. F., et al. (2010). Influ- ence of Bering Strait flow and North Atlantic circulation on glacial sea-level changes. Nature Geo- science 3: 118–121. Hublin, J.-J., Ben-Ncer, A., Bailey, S. E., Freidline, S. E., Neubauer, S., Skinner, M. M., et al. (2017). New fossils from Jebel Irhoud, Morocco and the pan-African origin of Homo sapiens. Nature 546: 289. Hyodo, M., Matsu’ura, S., Kamishima, Y., Kondo, M., Takeshita, Y., Kitaba, I., et al. (2011). References Cited High- resolution record of the Matuyama–Brunhes transition constrains the age of Javanese Homo 1 3 Journal of Archaeological Research (2021) 29:255–326 316 erectus in the Sangiran dome, Indonesia. Proceedings of the National Academy of Sciences 108: 19563–19568. erectus in the Sangiran dome, Indonesia. Proceedings of the National Academy of Sciences 108: 19563–19568. Ikawa-Smith, F. (2008). Living on the edge of the continent: The Japanese Archipelago 30,000–8,000 cal BC. North Pacific Prehistory 2: 45–64. i Ikeya, N. (2015). Maritime transport of obsidian in Japan during the Upper Palaeolithic. In Kaifu, Y., Izuho, M., Goebel, T., Sato, H., and Ono, A. (eds.), Emergence and Diversity of Modern Human Behaviour in Paleolithic Asia, Texas A&M University Press, College Station, pp. 362–375. Ingicco, T., Bergh, G. D., Jago-On, C., Bahain, J. J., Chacón, M. G., Amano, N., et al. (2018). Earlie known hominin activity in the Philippines by 709 thousand years ago. Nature 557: 233–237. Ingold, T. (2003). Three in one: How an ecological approach can obviate the distinctions between body, mind and culture. In Roepstorff, A., Bubandt, N., and Kull, K. (eds.), Imagining Nature: Practices of Cosmology and Identity, Aarhus University Press, Aarhus, pp. 40–55.i Irwin, G. (1994). The Prehistoric Exploration and Colonisation of the Pacific, Cambridge Universi Press, Cambridge. Iwase, A., Hashizume, J., Izuho, M., Takahashi, K., and Sato, H. (2012). Timing of megafaunal extinctio in the late Late Pleistocene on the Japanese Archipelago. Quaternary International 255: 114–124 Iwase, A., Takahashi, K., and and Izuho, M. (2015). Further study on the Late Pleistocene megafaunal extinction in the Japanese Archipelago. In Kaifu, Y., Izuho, M., Goebel, T., Sato, H., and Ono, A. (eds.), Emergence and Diversity of Modern Human Behaviour in Paleolithic Asia, Texas A&M University Press, College Station, pp. 325–344. Izuho, M., and Kaifu, Y. (2015). The appearance and characteristics of the early Upper Paleolithic in the Japanese Archipelago. In Kaifu, Y., Izuho, M., Goebel, T., Sato, H., and Ono, A. (eds.), Emergence and Diversity of Modern Human Behaviour in Paleolithic Asia, Texas A&M University Press, Col- lege Station, pp. 289–313. Jacobs, G. S., Hudjashov, G., Saag, L., Kusuma, P., Darusallam, C. C., Lawson, D. J., et al. (2019). Multi- ple deeply divergent Denisovan ancestries in Papuans. Cell 177: 1010–1021.e32. Johnson, D. L. (1980). Problems in the land vertebrate zoogeography of certain islands and the swim ming powers of elephants. Journal of Biogeography 7: 383–398. Johnson, D. L. (1980). References Cited Problems in the land vertebrate zoogeography of ming powers of elephants. Journal of Biogeography 7: 383–398. Joordens, J. C. A., Wesselingh, F. P., de Vos, J., Vonhof, H. B., and Kroon, D. (2009). Relevance of aquatic environments for hominins: A case study from Trinil (Java, Indonesia). Journal of Human Evolution 57: 656–671. Joordens, J. C. A., d’Errico, F., Wesselingh, F. P., Munro, S., Vos, J. de, Wallinga, J., et al. (2015). Homo erectus at Trinil on Java used shells for tool production and engraving. Nature 518: 228–231. Kaifu, Y. (2017). Archaic hominin populations in Asia before the arrival of modern humans: Their ph logeny and implications for the “southern Denisovans.” Current Anthropology 58: S418–S433. Kaifu, Y., Fujita, M., Yoneda, M., and Yamasaki, S. (2015). Pleistocene seafaring and colonization of the Ryukyu Islands, southwestern Japan. In Kaifu, Y., Izuho, M., Goebel, T., Sato, H., and Ono, A. (eds.), Emergence and Diversity of Modern Human Behaviour in Paleolithic Asia, Texas A&M University Press, College Station, pp. 345–361. y g pp Kano, F., and Hirata, S. (2015). Great apes make anticipatory looks based on long-term memory of single events. Current Biology 25: 2513–2517. Kealy, S., Louys, J., and O’Connor, S. (2017). Reconstructing palaeogeography and inter-island visibility in the Wallacean Archipelago during the likely period of Sahul colonization, 65–45 000 years ago. Archaeological Prospection 24: 259–72. g p Kealy, S., Louys, J., and O’Connor, S. (2018). Least-cost pathway models indicate northern human dis- persal from Sunda to Sahul. Journal of Human Evolution 125: 59–70. Keegan, W. F., and Diamond, J. M. (1987). Colonization of islands by humans: A biogeographical per- spective. Advances in Archaeological Method and Theory 10: 49–92. Kennedy, J. (2002). Manus from the beginning: An archaeological overview. In Kaufmann, C., Kocher- Schmid, C., and Ohnemus, S. (eds.), Admiralty Art: Art from the South Seas, Museum Rietberg, Zurich, pp. 17–28.i pp Kirch, P. V. (2017). On the Road of the Winds: An Archaeological History of the Pacific Islands Before European Contact, University of California Press, Berkeley. Klein, R. G. (2008). Out of Africa and the evolution of human behavior. Evolutionary Anthropology 17: 267–281. Kobayashi, H., Matsui, Y., and Suzuki, H. (1971). University of Tokyo radiocarbon measurements IV. Radiocarbon 13: 97–102. 1 3 Journal of Archaeological Research (2021) 29:255–326 317 Kobayashi, H., Hirose, T., Sugino, M., and Watanabe, N. (1974). University of Tokyo radiocarbon meas- urements V. Radiocarbon 16: 381–387. References Cited Kopaka, K., and Matzanas, C. (2009). Palaeolithic industries from the island of Gavdos, near neighbour to Crete in Greece. Antiquity Project Gallery 83: 321. Kozlovsky, D. Y., Branch, C. L., and Pravosudov, V. V. (2015). Problem-solving ability and response to novelty in mountain chickadees (Poecile gambeli) from different elevations. Behavioral Ecology and Sociobiology 69: 635–643. Kozowyk, P. R. B., Soressi, M., Pomstra, D., and Langejans, G. H. J. (2017). Experimental methods for the Palaeolithic dry distillation of birch bark: Implications for the origin and development of Nean- dertal adhesive technology. Scientific Reports 7: 8033. i Kress, J. H., (1978). The ceramics from Pilanduk Cave and Sa’gung Rockshelter, Quezon Municipality, Palawan Island, the Philippines. Asian Perspectives 21: 58–85.l n Island, the Philippines. Asian Perspectives 21: 58– Kubo, D., Kono, R. T., and Kaifu, Y. (2013). Brain size of Homo floresiensis and its evolutionary impli- cations. Proceedings of the Royal Society of London B 280: 20130338. Kuzmin, Y. V., and Keates, S. G. (2014). Direct radiocarbon dating of Late Pleistocene hominids in Eur sia: Current status, problems, and perspectives. Radiocarbon 56: 753–766. Kyriacou, K., Blackhurst, D. M., Parkington, J. E., and Marais, A. D. (2016). Marine and terrestrial foods as a source of brain-selective nutrients for early modern humans in the southwestern Cape, South Africa. Journal of Human Evolution 97: 86–96. Laland, K. N., and Brown, G. R. (2006). Niche construction, human behavior, and the adaptive‐la hypothesis. Evolutionary Anthropology 15: 95–104. Lambeck, K., Purcell, A., Flemming, N. C., Vita-Finzi, C., Alsharekh, A. M., and Bailey, G. N. (2011). Sea level and shoreline reconstructions for the Red Sea: Isostatic and tectonic considerations and implications for hominin migration out of Africa. Quaternary Science Reviews 30: 3542–3574. Langley, M. C., O’Connor, S., and Piotto, E. (2016). 42,000-year-old worked and pigment-stained Nauti- lus shell from Jerimalai (Timor-Leste): Evidence for an early coastal adaptation in ISEA. Journal of Human Evolution 97: 1–16. Larson, S. G., Jungers, W. L., Morwood, M. J., Sutikna, T., Jatmiko, Saptomo, E. W., Due, R. A., and Djubiantono, T. (2007). Homo floresiensis and the evolution of the hominin shoulder. Journal of Human Evolution 53: 718–731. Laskaris, N., Sampson, A., Mavridis, F., and Liritzis, I. (2011). Late Pleistocene/Early Holocene seafar- ing in the Aegean: New obsidian hydration dates with the SIMS-SS method. Journal of Archaeo- logical Science 38: 2475–2479. Leavesley, M. G. (2004). References Cited Environment, preferred habitats and potential refugia for Pleistocene Homo in Southeast Asia. Comptes Rendus Palevol 11: 203–211. Lowe, J. J., and Walker, M. J. (2014). Reconstructing Quaternary Environments, Routledge, London. Loy, T., Spriggs, M., and Wickler, S. (1992). Direct evidence for human use of plants 28,000 years ago: Starch residues on stone artefacts from the northern Solomon Islands. Antiquity 66: 898–912. Lykousis, V. (2009). Sea-level changes and shelf break prograding sequences during the last 400ka in the Aegean margins: Subsidence rates and palaeogeographic implications. Continental Shelf Research 29: 2037–2044. Lyras, G. A., Geer, A. A. E., Dermitzakis, M. D., and Vos, J. D. (2006). Cynotherium sardous, an insular canid (Mammalia: Carnivora) from the Pleistocene of Sardinia (Italy), and its origin. Journal of Vertebrate Paleontology 26: 735–745. Mahirta (2003). Human Occupation on Roti and Sawu Islands, Nusa Tenggara Timur, Ph.D. thesis, Department of Prehistory, Australian National University, Canberra. Malafouris, L. (2013). How Things Shape the Mind, MIT Press, Cambridge, MA.i Manalo, K. (2011). Preliminary identification of cutmarks morphology on animal bones: Methods and applications. MA thesis, Department of Archaeology, University of the Philippines, Quezon City, Philippines. pp Mannino, M. A., and Thomas, K. D. (2007). New radiocarbon dates for hunter-gatherers and early farm- ers in Sicily. Accordia Research Papers 10: 13–34. Mannino, M. A., Di Salvo, R., Schimmenti, V., Di Patti, C., Incarbona, A., Sineo, L., and Richards, M. P. (2011). Upper Palaeolithic hunter-gatherer subsistence in Mediterranean coastal environments: An isotopic study of the diets of the earliest directly-dated humans from Sicily. Journal of Archaeo- logical Science 38: 3094–3100. g Mannino, M. A., Catalano, G., Talamo, S., Mannino, G., Salvo, R. D., Schimmenti, V., et al. (2012). Ori- gin and diet of the prehistoric hunter-gatherers on the Mediterranean island of Favignana (Ègadi Islands, Sicily). PLOS One 7: e49802.i Marean, C. W. (2014). The origins and significance of coastal resource use in Africa and western Eurasi Journal of Human Evolution 77: 17–40. Marean, C. W., Bar-Matthews, M., Bernatchez, J., Fisher, E., Goldberg, P., Herries, A. I. R., et al. (2007). Early human use of marine resources and pigment in South Africa during the Middle Pleistocene. Nature 449: 905–908. Marshall, B., and Allen, J. (1991). Excavations at Panakiwuk Cave, New Ireland. In Allen, J., and Gos- den, C. (eds.), Report of the Lapita Homeland Project, Department of Prehistory, Australian National University, Canberra, pp. 59–91. y pp Martin, R. D., MacLarnon, A. References Cited Trees to the Sky: Prehistoric Hunting in New Ireland, Papua New Guinea, Ph.D thesis, School of Archaeology and Anthropology, Australian National University, Canberra. eavesley, M. G. (2004). Trees to the Sky: Prehistoric Hunting in New Ireland, Papua New Guinea, Ph.D thesis, School of Archaeology and Anthropology, Australian National University, Canberra. Leavesley, M. G. (2007). A shark-tooth ornament from Pleistocene Sahul. Antiquity 81: 308–315. 7). A shark-tooth ornament from Pleistocene Sahul. A Leavesley, M., and Allen, J. (1998). Dates, disturbance and artefact distributions: Another analysis of Buang Merabak, a Pleistocene site on New Ireland, Papua New Guinea. Archaeology in Oceania 33: 63–82. Lentfer, C., Pavlides, C., and Specht, J. (2010). Natural and human impacts in a 35,000-year vegetation history in central New Britain, Papua New Guinea. Quaternary Science Reviews 29: 3750–3767. Leppard, T. P. (2014). Modeling the impacts of Mediterranean island colonization by archaic homi- nins: The likelihood of an insular Lower Palaeolithic. Journal of Mediterranean Archaeology 27: 231–254. Leppard, T. P. (2015a). Passive dispersal versus strategic dispersal in island colonization by hominins. Current Anthropology 56: 590–595. Leppard, T. P. (2015b). The evolution of modern behaviour and its implications for maritime dispers during the Palaeolithic. Cambridge Archaeological Journal 25: 829–846.f Leppard, T. P. (2015c). Adaptive responses to demographic fragility: Mitigating stochastic effects in ear island colonization. Human Ecology 43: 721–734.i Leppard, T. P. (2016). Between deterministic and random process in prehistoric Pacific Island abandon- ment. Journal of Pacific Archaeology 7: 20–25. i Leppard, T. P., and Runnels, C. (2017). Maritime hominin dispersals in the Pleistocene: Advancing the debate. Antiquity 91: 510–519. Lewis, H., Paz, V., Lara, M., Barton, H., Piper, P., Ochoa, J., et al. (2008). Terminal Pleistocene to mid- Holocene occupation and an early cremation burial at Ille Cave, Palawan, Philippines. Antiquity 82: 318–335. 1 3 Journal of Archaeological Research (2021) 29:255–326 318 , F., Kuhn, S. L., Chen, F., Wang, Y., Southon, J., Peng, F., et al. (2018). The easternmost Middle Pale- olithic (Mousterian) from Jinsitai Cave, North China. Journal of Human Evolution 114: 76–84. Liebl, A. L., and Martin, L. B. (2014). Living on the edge: Range edge birds consume novel foods sooner than established ones. Behavioral Ecology 25: 1089–1096. gy Liu, W., Martinón-Torres, M., Cai, Y. J., Xing, S., Tong, H. W., Pei, S. W., et al. (2015). The earliest unequivocally modern humans in southern China. Nature 526: 696–699. Louys, J., and Turner, A. (2012). References Cited M., Phillips, J. L., and Dobyns, W. B. (2006). Flores hominid: New species or microcephalic dwarf? The Anatomical Record Part A: Discoveries in Molecular, Cellular, and Evolutionary Biology 288: 1123–1145. Martinez, I., and Arsuaga, J. L. (1997). The temporal bones from Sima de los Huesos Middle Pleisto- cene site (Sierra de Atapuerca, Spain): A phylogenetic approach. Journal of Human Evolution 33: 283–318. Martinón-Torres, M., Wu, X., Bermúdez de Castro, J. M., Xing, S., and Liu, W. (2017). Homo sapiens the eastern Asian Late Pleistocene. Current Anthropology 58: S434–S448. Matsufuji K., (2011). When were the earliest hominin migrations to the Japanese islands? In Norto J., and Braun, D. R. (eds.), Asian Palaeoanthropology, Springer, Dordrecht, pp. 191–200. Matsu’ura, S. (1999). A chronological review of Pleistocene human remains from the Japanese Archi- pelago. In Omoto, K. (ed.), Interdisciplinary Perspectives on the Origins of the Japanese, Interna- tional Research Center for Japanese Studies, Kyoto, pp. 181–197. Mazza, P. P. A., Martini, F., Sala, B., Magi, M., Colombini, M. P., Giachi, G., et al. (2006). A new Palae- olithic discovery: Tar-hafted stone tools in a European mid-Pleistocene bone-bearing bed. Journal of Archaeological Science 33: 1310–1318. f g McBrearty, S., and Brooks, A. S. (2000). The revolution that wasn’t: A new interpretation of the origin of modern human behavior. Journal of Human Evolution 39: 453–563. 1 3 Journal of Archaeological Research (2021) 29:255–326 319 Mellars, P. (2005). The impossible coincidence: A single-species model for the origins of modern huma behavior in Europe. Evolutionary Anthropology 14: 12–27. Mijares, A. (2006). Tracking the early human migration into Island Southeast Asia. Hukay 10: 3–24. Mijares, A. S. (2007). Unearthing Prehistory: The Archaeology of Northeastern Luzon, Philippine Islands, British Archaeological Reports, Oxford. Mijares, A. S. (2015). Human emergence and adaptation to an island environment in the Philippine Pale- olithic. In Kaifu, Y., Izuho, M., Goebel, T., Sato, H., and Ono, A. (eds.), Emergence and Diversity of Modern Human Behaviour in Paleolithic Asia, Texas A&M University Press, College Station, pp. 171–181. Mijares, A. S., Détroit, F., Piper, P., Grün, R., Bellwood, P., Aubert, M., et al. (2010). New evidence for a 67,000-year-old human presence at Callao Cave, Luzon, Philippines. Journal of Human Evolution 59: 123–132. Miller-Antonio, S., Schepartz, L.A., Karkanas, P., Yamei, H., Weiwen, H., and Bekken, D. (2004). Lithic raw material use at the late Middle Pleistocene site of Panxian Dadong. Asian Perspectives 43: 314–332. Mitchell, P. (2004). References Cited Towards a comparative archaeology of Africa’s islands. Journal of African Archaeol- ogy 2: 229–250. Morgan, T. J., Uomini, N. T., Rendell, L. E., Chouinard-Thuly, L., Street, S. E., Lewis, H. M., et al. (2015). Experimental evidence for the co-evolution of hominin tool-making teaching and language. Nature Communications 6: 6029. Morisaki, K. (2015). Appearance of Hakuhen-Sentoki (HS points) and second modern human migration into Kyushu, Japan. In Kaifu, Y., Izuho, M., Goebel, T., Sato, H., and Ono, A. (eds.), Emergence and Diversity of Modern Human Behaviour in Paleolithic Asia, Texas A&M University Press, Col- lege Station, pp. 376–388. Mortensen, P. (2008). Lower to Middle Palaeolithic artefacts from Loutró on the south coast of Crete. Antiquity Project Gallery 82: 317. Morwood, M. J. (2014). Faunal biogeography in Island Southeast Asia. In Dennell, R., and Porr, M. (eds.), Southern Asia, Australia, and the Search for Human Origins, Cambridge University Press, Cambridge, pp. 108–117. Morwood, M. J., and van Oosterzee, P. (2007). A New Human: The Startling Discovery and Strange Story of the “Hobbits” of Flores, Indonesia, Left Coast Press, Walnut Creek, CA. Morwood, M. J., O’Sullivan, P. B., Aziz, F., and Raza, A. (1998). Fission-track ages of stone tools and fossils on the east Indonesian island of Flores. Nature 392: 173–176. Morwood, M. J., Aziz, F., O’Sullivan, P., Hobbs, D. R., and Raza, A. (1999). Archaeological and palae- ontological research in central Flores, east Indonesia: Results of fieldwork 1997–98. Antiquity 73: 273–286. Morwood, M. J., Soejono, R. P., Roberts, R. G., Sutikna, T., Turney, C. S., Westaway, K. E., et al. (2004). Archaeology and age of a new hominin from Flores in eastern Indonesia. Nature 431: 1087–1091.f Murray-Wallace, C. V., and Woodroffe, C. D. (2014). Quaternary Sea-level Changes: A Global Perspec- tive, Cambridge University Press, Cambridge. Nakagawa, R., Doi, N., Nishioka, Y., Nunami, S., Yamauchi, H., Fujita, M., et al. (2010). Pleistocene human remains from Shiraho-Saonetabaru Cave on Ishigaki Island, Okinawa, Japan, and their radi- ocarbon dating. Anthropological Science 118: 173–183. g p g Neall, V. E., Wallace, R. C., and Torrence, R. (2008). The volcanic environment for 40,000 years of human occupation on the Willaumez Isthmus, West New Britain, Papua New Guinea. Journal of Volcanology and Geothermal Research 176: 330–343. Norman, K., Inglis, J., Clarkson, C., Faith, J. T., Shulmeister, J., and Harris, D. (2018). An early coloni- sation pathway into northwest Australia 70–60,000 years ago. Quaternary Science Reviews 180: 229–239. References Cited Norton, C. J., Kondo, Y., Ono, A., Zhang, Y. and Diab, M. C. (2010). The nature of megafaunal extinc- tions during the MIS 3–2 transition in Japan. Quaternary International 211: 113–122. O’Connell, J. F., and Allen, J. (2012). The restaurant at the end of the universe: Modelling the colonisa- tion of Sahul. Australian Archaeology 74: 5–31. O’Connell, J. F., and Allen, J. (2012). The restaurant at th tion of Sahul. Australian Archaeology 74: 5–31. O’Connell, J. F., Allen, J., Williams, M. A., Williams, A. N., Turney, C. S., Spooner, N. A., et al. (2018). When did Homo sapiens first reach Southeast Asia and Sahul? Proceedings of the National Acad- emy of Sciences 115: 8482–8490. y f O’Connor, S. (2007). New evidence from East Timor contributes to our understanding of earliest modern human colonisation east of the Sunda Shelf. Antiquity 81: 523–535. 1 3 Journal of Archaeological Research (2021) 29:255–326 320 O’Connor, S. (2015). Crossing the Wallace Line: The maritime skills of the earliest colonists in the Wal- lacean Archipelago. In Kaifu, Y., Izuho, M., Goebel, T., Sato, H., and Ono, A. (eds.), Emergence and Diversity of Modern Human Behaviour in Paleolithic Asia, Texas A&M University Press, Col- lege Station, pp. 214–224. O’Connor, S. Spriggs, M., and Veth, P. (2002). Excavation at Lene Hara Cave establishes occupation in East Timor at least 30,000–35,000 years ago. Antiquity 76: 45–49. O’Connor, S., Barham, A., Spriggs, M., Veth, P., Aplin, K., and Pierre, E. S. (2010a). Cave archaeology and sampling issues in the tropics: A case study from Lene Hara cave, a 42,000 year old occupation site in East Timor, Island Southeast Asia. Australian Archaeology 71: 29–40. O’Connor, S., Aplin, K., Pierre, E. S., and Feng, Y. (2010b). Faces of the ancestors revealed: Discovery and dating of a Pleistocene-age petroglyph in Lene Hara cave, East Timor. Antiquity 84: 649–665. O’Connor, S., Barham, A., Aplin, K., Dobney, K., Fairbairn, A., and Richards, M. (2011a). The power of paradigms: Examining the evidential basis for Early to mid-Holocene pigs and pottery in Melane- sia. Journal of Pacific Archaeology 2: 1–25.i i O’Connor, S., Ono, R., and Clarkson, C. (2011b). Pelagic fishing at 42,000 years before the present an the maritime skills of modern humans. Science 334: 1117–1121. O’Connor, S., Robertson, G., and Aplin, K. P. (2014). Are osseous artefacts a window to perishable mate- rial culture? References Cited Implications of an unusually complex bone tool from the Late Pleistocene of East Timor. Journal of Human Evolution 67: 108–119. O’Connor, S., Louys, J., Kealy, S. and Samper Carro, S. C. (2017a). Hominin dispersal and settlement east of Huxley’s Line: The role of sea level changes, island size, and subsistence behavior. Current Anthropology 58: S567–S582. O’Connor, S. Mahirta, Carro, S. C. S., Hawkins, S., Kealy, S., Louys, J., and Wood, R. (2017b). Fishing in life and death: Pleistocene fish-hooks from a burial context on Alor Island, Indonesia. Antiquity 91: 1451–1468. O’Connor, S., Mahirta, Kealy, S., Boulanger, C., Maloney, T., Hawkins, S., et al. (2018). Kisar and the archaeology of small islands in the Wallacean Archipelago. The Journal of Island and Coastal Archaeology, https://doi.org/10.1080/15564894.2018.1443171. Ono, A., Sato, H., Tsutsumi, T., and Kudo, Y. (2002). Radiocarbon dates and archaeology of the La Pleistocene in the Japanese islands. Radiocarbon 44: 477–494. Ono, R., Nakajima, N., Nishizawa, H., Oda, S., and Soegondho, S. (2015). Maritime migration and lithic assemblage on the Talaud Islands in northern Wallacea during the Late Pleistocene to the Early Holocene. In Kaifu, Y., Izuho, M., Goebel, T., Sato, H., and Ono, A. (eds.), Emergence and Diver- sity of Modern Human Behaviour in Paleolithic Asia, Texas A&M University Press, College Sta- tion, pp. 201–213. O’Regan, H., Bishop, L., Elton, S., Lamb, A., and Turner, A. (2006). Afro-Eurasian mammalian dispersal routes of the Late Pliocene and Early Pleistocene, and their bearing on earliest hominin move- ments. Courier 256: 305–314. Osvath, M., and Karvonen, E. (2012). Spontaneous innovation for future deception in a male chimpanzee. PLOS One 7: e36782. Osvath, M., and Osvath, H. (2008). Chimpanzee (Pan troglodytes) and orangutan (Pongo abelii) fore- thought: Self-control and pre-experience in the face of future tool use. Animal Cognition 11: 661–674. Paijmans, K. (1976). New Guinea Vegetation, Australian National University Press, Canberra. Panagopoulou, E., Kotjaboulou, E., and Karkanas, P. (2001). Geoarchaeological research in Alonnis- sos: New evidence for the Palaeolithic and Mesolithic in the Aegean region. In Sampson, A. (ed.), Archaeological Investigations in the Northern Sporades, Community of Alonnisos, Alonissos, pp. 121–151. Papoulia, C. (2017). Seaward dispersals to the NE Mediterranean islands in the Pleistocene: The lith evidence in retrospect. Quaternary International 431: 64–87. Pasveer, J. (2004). The Djief Hunters: 26,000 Years of Rainforest Exploitation on the Bird’s Head of Papua, Indonesia, A. A. Balkema Publishers, Leiden. Pavlides, C. (1999). References Cited The Story of Imlo: The Organisation of Flaked Stone Technologies from the Lowland Tropical Rainforest of West New Britain, Papua New Guinea, Ph.D. thesis, Department of Archae- ology, La Trobe University, Melbourne. gy, y, Pavlides, C. (2004). From Misisil Cave to Eliva Hamlet: Rediscovering the Pleistocene in interior West New Britain. Records of the Australian Museum S29: 97–108. 1 3 Journal of Archaeological Research (2021) 29:255–326 321 Pavlides, C., and Gosden, C. (1994). 35,000-year-old sites in the rainforests of West New Britain. Antiq- uity 68: 604–610. Pavlides, C., and Gosden, C. (1994). 35,000-year-old sites in the rainforests of West New Britain. Antiq- uity 68: 604–610. Pawlik, A. F. (2004). The Palaeolithic site of Arubo 1 in central Luzon, Philippines. Bulletin of the Indo- Pacific Prehistory Association 24: 3–12. i Perry, J. (1983). Anatomy and biomechanics of the shoulder in throwing, swimming, gymnastics, and tennis. Clinical Sports Medicine 2: 247–270.l i Perry, J. (1983). Anatomy and biomechanics of the tennis. Clinical Sports Medicine 2: 247–270. tennis. Clinical Sports Medicine 2: 247–270. Phoca-Cosmetatou, N., and Rabett, R. (2014). Discussion and debate: Reflections on Pleistocene island occupation. Journal of Mediterranean Archaeology 27: 255–278. Piperno, M., Scali, S., and Tagliacozzo, A. (1980). Mesolitico e Neolitico alla Grotta deli’Uzzo (Tra- pani): Primi dati per un’interpretazione paleoeconomica. Quaternaria 22: 275–300. Price, T., Yeh, P. J., and Harr, B. (2008). Phenotypic plasticity and the evolution of a socially selected trait following colonization of a novel environment. The American Naturalist 172: S49–S62. Price, T., Yeh, P. J., and Harr, B. (2008). Phenotypic plasticity and the evolution of a socially selected trait following colonization of a novel environment. The American Naturalist 172: S49–S62. Rabett, R. J. (2017). The early human occupation of East and Southeast Asia. In Habu, J., Lape, P., and Olsen, J. (eds.), Handbook of East and Southeast Asian Archaeology, Springer, New York, pp. 159–193. Rabett, R. J. (2018). The success of failed Homo sapiens dispersals out of Africa and into Asia. Nature Ecology & Evolution 2: 212–219. Ramis, D., and Alcover, J. A. (2001). Revisiting the earliest human presence in Mallorca, western Medi- terranean. Proceedings of the Prehistoric Society 67: 261–269. Ramos, J., Bernal, D., Domínguez-Bella, S., Calado, D., Ruiz, B., Gil, M. J., et al. (2008). The Benzú rockshelter: A Middle Palaeolithic site on the North African coast. Quaternary Science Reviews 27: 2210–2218. Reader, S. M., and Laland, K. N. (2002). References Cited W., Gallen, S. F., Strasser, T. F., and Panagopoulou, E. (2014a). Lower Palaeolithic artifacts from Plakias, Crete: Implications for hominin dispersals. Eur- asian Prehistory 11: 129–152. Runnels, C., McCoy, F., Bauslaugh, R., and Murray, P. (2014b). Palaeolithic research at Mochlos, Crete: New evidence for Pleistocene maritime activity in the Aegean. Antiquity Project Gallery 88: 342. Ruxton, G. D., and Wilkinson, D. M. (2012). Population trajectories for accidental versus planned colon sation of islands. Journal of Human Evolution 63: 507–511. Salotti, M., Louchart, A., Bailon, S., Lorenzo, S., Oberlin, C., Ottaviani-Spella, M.-M., Pereira, E., and Tramoni, P. (2008). A Teppa di U Lupinu Cave (Corsica, France)—Human presence since 8500 years BC, and the enigmatic origin of the earlier, Late Pleistocene accumulation. Acta Zoologica Cracoviensia - Series A: Vertebrata 51: 15–34. Samper Carro, S.C., O’Connor, S., Louys, J., Hawkins, S., and Mahirta (2016). Human maritime subsist- ence strategies in the Lesser Sunda Islands during the Terminal Pleistocene–Early Holocene: New evidence from Alor, Indonesia. Quaternary International 416: 64–79. Sathiamurthy, E., and Voris, H. K. (2006). Maps of Holocene sea level transgression and submerge lakes on the Sunda Shelf. Tropical Natural History 2: 1–44. Sato, H. (2012). Late Pleistocene trap-pit hunting in the Japanese Archipelago. Quaternary International 248: 43–55. Schlebusch, C. M., Malmström, H., Günther, T., Sjödin, P., Coutinho, A., Edlund, H., et al. (2017). Southern African ancient genomes estimate modern human divergence to 350,000 to 260,000 years ago. Science 358: 652–655.l Schoener, A., and Schoener, T. W. (1984). Experiments on dispersal: Short-term floatation of insul anoles, with a review of similar abilities in other terrestrial animals. Oecologia 63: 289–294. Selimiotis, H. (2006). The Core of the Matter: Core Reduction in Prehistoric East Timor. M.A. thesis, Department of Archaeology, Australian National University, Canberra. Sémah, F., and Sémah, A. M. (2013). Pleistocene migrations in the Southeast Asian archipelagos. In Bell- wood, P. (ed.), The Global Prehistory of Human Migration, Wiley-Blackwell, Oxford, pp. 49–54. Shaw, B. (2017). Late Pleistocene colonisation of the eastern New Guinea islands? The potential implica- tions of robust waisted stone tool finds from Rossel Island on the long term settlement dynamics in the Massim region. Journal of Pacific Archaeology 8: 1–16. g f fi gy Shea, J. J. (2011). Homo sapiens is as Homo sapiens was: Behavioral variability versus “behavioral modernity” in Paleolithic archaeology. Current Anthropology 52: 1–35. Shen, G., Wu, X., Wang, Q., Tu, H., Feng, Y. X., and Zhao, J. References Cited Social intelligence, innovation, and enhanced brain size in pri- mates. Proceedings of the National Academy of Sciences 99: 4436–4441. Reepmeyer, C., O’Connor, S., Mahirta, Maloney, T., and Kealy, S. (2016). Late Pleistocene/Early Holo- cene maritime interaction in southeastern Indonesia – Timor Leste. Journal of Archaeological Sci- ence 76: 21–30. Renfrew, C., and Aspinall, A. (1990). Aegean obsidian and Franchthi cave. In Perles, C. (ed.), Les industries lithiques taillées de Franchthi (Argolide, Grèce): Les industries du Mésolithique et du Néolithique initial, Excavations at Franchthi Cave 5, Indiana University Press, Bloomington, pp. 257–270. Rizal, Y., Westaway, K. E., Zaim, Y., van den Bergh, G. D., Bettis, E. A., Morwood, M. J., et al. (2020). Last appearance of Homo erectus at Ngandong, Java, 117,000–108,000 years ago. Nature 577: 381–385. Robert, I., and Vigne, J.-D. (2002). The bearded vulture (Gypaetus barbatus) as an accumulator of archaeological bones: Late glacial assemblages and present-day reference data in Corsica (western Mediterranean). Journal of Archaeological Science 29: 763–777. Roberts, P. (2015). “We have never been behaviourally modern”: The implications of material engage- ment theory and metaplasticity for understanding the Late Pleistocene record of human behaviour. Quaternary International 30: 1–13. Roberts, P., and Amano, N. (2019). Plastic pioneers: Hominin biogeography east of the Movius Line du ing the Pleistocene. Archaeological Research in Asia 17: 181–192.i Roberts, P., and Stewart, B. A. (2018). Defining the “generalist specialist” niche for Pleistocene Hom sapiens. Nature Human Behaviour 2: 542–550. Roberts, P., Louys, J., Zech, J., Shipton, C., Kealy, S., Carro, S. S., et al. (2020). Isotopic evidence for initial coastal colonization and subsequent diversification in the human occupation of Wallacea. Nature Communications 11: 2068. Roe, D. (1993). Prehistory without Pots: Prehistoric Settlement and Economy of North-west Gaudal- canal, Solomon Islands, Ph.D. thesis, Department of Prehistory, Australian National University, Canberra. Rolland, N. (2013). The Early Pleistocene human dispersals in the Circum-Mediterranean Basin and ini- tial peopling of Europe: Single or multiple pathways? Quaternary International 316: 59–72. Runnels, C. (2014). Early Palaeolithic on the Greek islands? Journal of Mediterranean Archaeology 27: 211–230. Runnels, C. (in press). The Paleolithic exploration of the Greek islands and Middle Pleistocene hominin dispersals: The case for behavioral variability over behavioral modernity. In Napolitano, M. (ed.), The Archaeology of Island Colonization: Global Approaches to Initial Human Settlement, Univer- sity of Florida Press, Gainesville. 1 3 Journal of Archaeological Research (2021) 29:255–326 322 Runnels, C., DiGregorio, C., Wegmann, K. References Cited X. (2013). Mass spectrometric U-series dating of Huanglong Cave in Hubei Province, central China: Evidence for early presence of mod- ern humans in eastern Asia. Journal of Human Evolution 65: 162–167. Shinoda, K., and Adachi, N. (2017). Ancient DNA analysis of Palaeolithic Ryukyu islanders. In Piper, P., Matsumura, H., and Bulbeck, D. (eds.), New Perspectives in Southeast Asian and Pacific Prehis- tory, Australian National University ePress, Canberra, pp. 51–59. , , , ( ) y y y p , , Matsumura, H., and Bulbeck, D. (eds.), New Perspectives in Southeast Asian and Pacific Prehis- tory, Australian National University ePress, Canberra, pp. 51–59. y y pp Shipton, C., Roberts, P., Archer, W., Armitage, S. J., Bita, C., Blinkhorn, J., et al. (2018). 78,000-year-old record of Middle and Later Stone Age innovation in an East African tropical forest. Nature Com- munications 9: 1832. Simmons, A. H. (1988). Extinct pygmy hippopotamus and early man in Cyprus. Nature 333: 554–557. Simmons, A. H. (1991). Humans, island colonization and Pleistocene extinctions in the Mediterranea The view from Akrotiri Aetokremnos, Cyprus. Antiquity 65: 857–869. Simmons, A. H. (1999). Faunal Extinction in an Island Society: Pygmy Hippopotamus Hunters of Cyprus, Kluwer Academic Publishers, New York. Skarpelis, N., Carter, T., Contreras, D. A., and Mihailović, D. D. (2017). Characterization of the siliceous rocks at Stélida, an early prehistoric lithic quarry (northwest Naxos, Greece), by petrography and geochemistry: A first step towards chert sourcing. Journal of Archaeological Science: Reports 12: 819–833. Skeates, R. (2001). New radiocarbon dates for prehistoric Italy and Malta. Accordia Research Papers 8: 165–186. Skeates, R. (2012). Caves in need of context: Prehistoric Sardinia. In Bergsvik, K.A., and Skeates, R. (ed.), Caves in Context: The Cultural Significance of Caves and Rockshelters in Europe, Oxbow Books, Oxford, pp. 166–187. pp Smith, A., and Allen, J. (1999). Pleistocene shell technologies: Evidence from island Melanesia. In Hall, J., and McNiven, I. (eds.), Australian Coastal Archaeology, Department of Archaeology and Natu- ral History, Australian National University, pp. 291–297. 1 3 Journal of Archaeological Research (2021) 29:255–326 323 Sondaar, P. Y. (1989). Did man reach Australia via the giant rat and dingo route? Publication of the Geo- logical Research and Development Center, Paleontology Series 5: 76–83. Sondaar, P. Y., Sanges, M., Kotsakis, T., and de Boer, P. L. (1986). The Pleistocene deer hunter of Sar- dinia. Geobios 19: 17–31. Specht, J., Lilley, J., and Normu, J. (1981). References Cited Radiocarbon dates from West New Britain, Papua New Guinea. Australian Archaeology 12: 13–16. Spoor, F. (1999). The human fossils from Corbeddu Cave, Sardinia: A reappraisal. Deinsea 7: 297–302 Spriggs, M. (2001). How AMS dating changed my life. In Jones, M., and Sheppard, P. (eds.), Australa- sian Connections and New Directions, Department of Anthropology, University of Auckland, Auckland, pp. 365–374.f pp Sprintall, J., Gordon, A. L., Koch-Larrouy, A., Lee, T., Potemra, J. T., Pujiana, K., and Wijffels, S. E. (2014). The Indonesian seas and their role in the coupled ocean–climate system. Nature Geosci- ence 7: 487–492. Stiner, M. C., and Kuhn, S. L. (1992). Subsistence, technology, and adaptive variation in Middle Pale lithic Italy. American Anthropologist 94: 306–339. Storm, P. (2012). A carnivorous niche for Java Man? A preliminary consideration of the abundance fossils in Middle Pleistocene Java. Comptes Rendus Palevol 11: 191–202. Strasser, T. F., Panagopoulou, E., Runnels, C. N., Murray, P. M., Thompson, N., Karkanas, P., McCoy, F. W., and Wegmann, K. W. (2010). Stone Age seafaring in the Mediterranean: Evidence from the Strasser, T. F., Panagopoulou, E., Runnels, C. N., Murray, P. M., Thompson, N., Karkanas, P., McCoy, F. W., and Wegmann, K. W. (2010). Stone Age seafaring in the Mediterranean: Evidence from the Plakias region for Lower Palaeolithic and Mesolithic habitation of Crete. Hesperia 79: 145–190. Strasser, T. F., Runnels, C., Wegmann, K., Panagopoulou, E., Mccoy, F., Digregorio, C., Karkanas, P., and Thompson, N. (2011). Dating Palaeolithic sites in southwestern Crete, Greece. Journal of Q S 26 553 560 Strasser, T. F., Runnels, C., Wegmann, K., Panagopoulou, E., Mccoy, F., Digregorio, C., Karkanas, P., and Thompson, N. (2011). Dating Palaeolithic sites in southwestern Crete, Greece. Journal of Quaternary Science 26: 553–560. Strasser, T. F., Runnels, C., and Vita-Finzi, C. (2016). A possible Palaeolithic handaxe from Cyprus. Antiquity Project Gallery 90: 350. Stringer, C. B., Finlayson, J. C., Barton, R. N. E., Fernández-Jalvo, Y., Cáceres, I., Sabin, R. C., et al. (2008). Neanderthal exploitation of marine mammals in Gibraltar. Proceedings of the National Academy of Sciences 105: 14319–14324. Suchak, M., Eppley, T. M., Campbell, M. W., and de Waal, F. B. (2014). Ape duos and trios: Spontaneou cooperation with free partner choice in chimpanzees. PeerJ 2: e417. Summerhayes, G. R. (2007). Island Melanesian pasts: A view from archaeology. In Friedlaender, J. (ed.), Genes, Language and Culture History in the Southwest Pacific, Oxford University Press, Oxford, pp. 10–35. References Cited MIS3 edge-ground axes and the arrival of the first Homo sapiens in the Japanese Archipelago. Quaternary International 248: 70–78. p g anudirjo, D. A. (2001). Islands in Between: Prehistory of the Northeastern Indonesian Archipelago, Ph D thesis School of Archaeology and Anthropology Australian National University Canberra Tanudirjo, D. A. (2001). Islands in Between: Prehistory of the Northeastern Indonesian Archipelag Ph.D. thesis, School of Archaeology and Anthropology, Australian National University, Canberra j , ( ) y f p g , Ph.D. thesis, School of Archaeology and Anthropology, Australian National University, Canberra. Texier, P. J., Porraz, G., Parkington, J., Rigaud, J. P., Poggenpoel, C., Miller, C., et al. (2010). A Howie- sons Poort tradition of engraving ostrich eggshell containers dated to 60,000 years ago at Diepk- loof rock shelter, South Africa. Proceedings of the National Academy of Sciences 107: 6180–6185. Thieme, H. (2005). The Lower Palaeolithic art of hunting: The case of Schöningen 13 II-4, Lower Saxony, Germany. In Gamble, C., and Porr, M. (eds.), The Hominid Individual in Context: Archaeological Investigations of Lower and Middle Palaeolithic Landscapes, Locales and Arte- facts, Routledge, London, pp. 115–132. Tiauzon, A., Peterson, J., Robles, E. C., Neri, L. A., Forestier, H., Titton, S., et al. (in press). Unfold- ing the technological production strategies of the large toolkits across Philippine Paleolithic sites with specific reference to northern Mindanao. Quaternary International https://doi. org/10.1016/j.quaint.2018.10.037. g j q Timmermann, A., and Friedrich, T. (2016). Late Pleistocene climate drivers of early human migr tion. Nature 538: 92–95. Torrence, R. (2004). Now you see it, now you don’t: Changing obsidian source use in the Willaumez Peninsula, Papua New Guinea. In Cherry, J., Scarre, C., and Shennan, S. (eds.), Explaining Social Change: Studies in Honour of Colin Renfrew, McDonald Institute of Archaeolgoical Research, Cambridge, pp. 115–125. Torrence, R. (2005). Valued stone—How so. In Macfarlane, I., Mountain, M.-J., and Paton, R. (eds.), Many Exchanges: Archaeology, History, Community and the Work of Isabel McBryde, Aborigi- nal History Inc., Canberra, pp. 357–372. Torrence, R. (2012). Boats, stones, and risk: Response to O’Connell and Allen. Australian Archaeol- ogy 74: 26–27. Torrence, R. (2016). Social resilience and long-term adaptation to volcanic disasters: The archaeol- ogy of continuity and innovation in the Willaumez Peninsula, Papua New Guinea. Quaternary international 394: 6–16. Torrence, R., Neall, V., Doelman, T., Rhodes, E., McKee, C., Davies, H., et al. (2004). Pleistocene colonisation of the Bismarck Archipelago: New evidence from West New Britain. References Cited pp Summerhayes, G. R., and Allen, J. (1993). The transport of Mopir obsidian to Late Pleistocene New Ir land. Archaeology in Oceania 28: 144–148. Summerhayes, G. R., and Ford, A. (2014). Late Pleistocene colonisation and adaptation in New Guinea: Implications for modeling modern human behaviour. In Dennell, R., and Porr, M. (eds.), Southern Asia, Australia and the Search for Human Origins, Cambridge University Press, Cambridge, pp. 213–227. Summerhayes, G. R., Leavesley, M., Fairbairn, A., Mandui, H., Field, J., Ford, A., and Fullagar, R. (2010). Human adaptation and use of plants in highland New Guinea 49,000–44,000 years ago. Science 330: 78–81. Summerhayes, G. R., Field, J. H., Shaw, B., and Gaffney, D. (2017). The archaeology of forest exploita- tion and change in the tropics during the Pleistocene: The case of northern Sahul (Pleistocene New Guinea). Quaternary International 448: 14–30. y Sutikna, T., Tocheri, M. W., Morwood, M. J., Saptomo, E. W., Jatmiko, Awe, R. D., et al. (2016). Revised stratigraphy and chronology for Homo floresiensis at Liang Bua in Indonesia. Nature 532: 366. Sutikna, T., Tocheri, M. W., Faith, J. T., Awe, R. D., Meijer, H. J., Saptomo, E. W., and Roberts, R. G. (2018). The spatio-temporal distribution of archaeological and faunal finds at Liang Bua (Flores, Indonesia) in light of the revised chronology for Homo floresiensis. Journal of Human Evolution 124: 52–74. Suzuki, H., and Hanihara, K. (eds.) (1982). The Minatogawa Man: The Upper Pleistocene Man from the Island of Okinawa, University of Tokyo Press, Tokyo. Swadling, P., and Hope, G. (1992). Environmental change in New Guinea since human settlement. In Dodson, J. (ed.), The Naive Lands: Prehistory and Environmental Change in Australia and the Southwest Pacific, Longmans Cheshire, Melbourne, pp. 13–42. fi g pp Szabó, K., Brumm, A., and Bellwood, P. (2007). Shell artefact production at 32,000–28,000 BP in Island Southeast Asia: Thinking across media? Current Anthropology 48: 701–723. 3 Journal of Archaeological Research (2021) 29:255–326 324 Takahara, H., and Hayashi, R. (2015). Paleovegetation during Marine Isotope Stage 3 in East Asia. In Kaifu, Y., Izuho, M., Goebel, T., Sato, H., and Ono, A. (eds.), Emergence and Diversity of Modern Human Behaviour in Paleolithic Asia, Texas A&M University Press, College Station, pp. 314–324. Takamiya, H., Katagiri, C., Yamasaki, S., and Fujita, M. (in press). Human colonization of the cen- tral Ryukyus (Amami and Okinawa archipelagos), Japan. The Journal of Island and Coastal Archaeology.i Takashi, T. (2012). References Cited Archaeology in Oceania 39: 101–130. Tourloukis, V., and Karkanas, P. (2012). The Middle Pleistocene archaeological record of Greece and the role of the Aegean in hominin dispersals: New data and interpretations. Quaternary Science Reviews 43: 1–15. Tsutsumi, T. (2010). Prehistoric procurement of obsidian from sources on Honshu Island (Japan). In Kuzmin, Y. V., and Glascock, M. D. (eds.), Crossing the Straits: Prehistoric Obsidian Source Exploitation in the North Pacific Rim, Archaeopress, Oxford, pp. 27–55.f i Turvey, S. T., Crees, J. J., Hansford, J., Jeffree, T. E., Crumpton, N., Kurniawan, I., et al. (2017). Qua- ternary vertebrate faunas from Sumba, Indonesia: Implications for Wallacean biogeography and evolution. Proceedings of the Royal Society B 284: 20171278. Vacca, B. B. (2012). The hunting of large mammals in the Upper Palaeolithic of southern Italy: A di chronic case study from Grotta del Romito. Quaternary International 252: 155–164. Vacca, B. B. (2012). The hunting of large mammals in the Upper Palaeolithic of southern Italy: A dia- chronic case study from Grotta del Romito. Quaternary International 252: 155–164. van den Bergh, G. D., de Vos, J., and Sondaar, P. Y. (2001). The Late Quaternary palaeogeography of mammal evolution in the Indonesian Archipelago. Palaeogeography, Palaeoclimatology, Pal- aeoecology 171: 385–408. chronic case study from Grotta del Romito. Quaternary International 252: 155 164. van den Bergh, G. D., de Vos, J., and Sondaar, P. Y. (2001). The Late Quaternary palaeogeography of mammal evolution in the Indonesian Archipelago. Palaeogeography, Palaeoclimatology, Pal- aeoecology 171: 385–408. van den Bergh, G. D, Kaifu, Y., Kurniawan, I., Kono, R. T., Brumm, A., Setiyabudi, E., Aziz, F., and Morwood, M. J. (2016a). Homo floresiensis-like fossils from the early Middle Pleistocene of Flores. Nature 534: 245–248. van den Bergh, G. D., Li, B., Brumm, A., Grün, R., Yurnaldi, D., Moore, M. W., et al. (2016b). Earli- est hominin occupation of Sulawesi, Indonesia. Nature 529: 208–211. 1 3 Journal of Archaeological Research (2021) 29:255–326 325 van der Geer, A. A. E., van den Bergh, G. D., Lyras, G. A., Prasetyo, U. W., Due, R. A., Setiyabudi, E., and Drinia, H. (2016). The effect of area and isolation on insular dwarf proboscideans. Jour- nal of Biogeography 43: 1656–1666. Vanhaeren, M., d’Errico, F., Stringer, C., James, S. L., Todd, J. A., and Mienis, H. K. (2006). Middle Paleolithic shell beads in Israel and Algeria. Science 312: 1785–1788. Vannieuwenhuyse, D. (2016). References Cited Mind the Gap: Geoarchaeology and Micromorphology of Cave and Rockshelter Sequences from the Kimberley, North-west Australia, Ph.D. thesis, School of Social Science, University of Western Australia, Crawley. Veth, P. (2005). Between the desert and the sea: Archaeologies of the Western Desert and Pilbara regions, Australia. In Smith, M. A., and Hesse, P. (eds.), 23°S: Archaeology and Environmental History of the Southern Deserts, National Museum of Australia, Canberra, pp.132–141. y f pp Vigliardi, A. (1982). Gli strati paleo-mesolitici della Grotta di Levanzo. Rivista di Scienze Preistoriche 37: 3–58. Vigliardi, A. (1982). Gli strati paleo-mesolitici della Grotta di L Ri i t di S i P i t i h 37 3 58 Vigne, J. D., Zazzo, A., Saliège, J. F., Poplin, F., Guilaine, J. and Simmons, A. (2009). Pre-Neolithic wild boar management and introduction to Cyprus more than 11,400 years ago. Proceedings of the National Academy of Sciences 106: 16135–16138. Voris, H. K. (2000). Maps of Pleistocene sea levels in Southeast Asia: Shorelines, river systems and time durations. Journal of Biogeography 27: 1153–1167.fl Walter, R. C., Buffler, R. T., Bruggemann, J. H., Guillaume, M. M. M., Berhe, S. M., Negassi, B., et al. (2000). Early human occupation of the Red Sea coast of Eritrea during the last interglacial. Nature 405: 65–69. Watts, I., Chazan, M., and Wilkins, J. (2016). Early evidence for brilliant ritualized display: Specularite use in the Northern Cape (South Africa) between ∼500 and ∼300 Ka. Current Anthropology 57: 287–310. Watts, J., Fulfrost, B., and Erlandson, J. (2011). Searching for Santarosae: Surveying submerged land- scapes for evidence of Paleocoastal habitation off California’s northern Channel Islands. In Ford, B. L. (ed.), The Archaeology of Maritime Landscapes, Springer, New York, pp. 11–26.l Webb, J. K., Letnic, M., Jessop, T. S., and Dempster, T. (2014). Behavioural flexibility allows an invasi vertebrate to survive in a semi-arid environment. Biology Letters 10: 20131014. West-Eberhard, M. J. (2003). Developmental Plasticity and Evolution, Oxford University Press, Oxford , ( ) p y , y , Westaway, K. E., Morwood, M. J., Roberts, R. G., Rokus, A. D., Zhao, J. X., Storm, P., et al. (2007). Age and biostratigraphic significance of the Punung rainforest fauna, East Java, Indonesia, and implica- tions for Pongo and Homo. Journal of Human Evolution 53: 709–717. g f Westaway, K. E., Louys, J., Awe, R. D., Morwood, M. J., Price, G. J., Zhao, J. X., et al. (2017). References Cited An early modern human presence in Sumatra 73,000–63,000 years ago. Nature 548: 322–325. White, J. P., and Thomas, D. H. (1972). What mean these stones? Ethnotaxonomic models and archaeo- logical interpretations in the New Guinea Highlands. In Clarke, D. (ed.), Models in Archaeology, Methuen, London, pp. 275–308. pp White, J. P., Flannery, T. F., O’Brien, R., Hancock, R. V., and Pavlish, L. (1991). The Balof shelters, New Ireland. In Allen, J., and Gosden, C. (eds.), Report of the Lapita Homeland Project, Department of Prehistory, Australian National University, Canberra, pp. 46–58. Wickler, S. (2001). The Prehistory of Buka: A Stepping Stone Island in the Northern Solomons, Terra Australis 16, Department of Archaeology and Natural History, Australian National University, Canberra. Wickler, S., and Spriggs, M. (1988). Pleistocene human occupation of the Solomon Islands, Melanesia. Antiquity 62: 703–706. Wilkins, J., and Chazan, M. (2012). Blade production ∼500 thousand years ago at Kathu Pan 1, South Africa: Support for a multiple origins hypothesis for early Middle Pleistocene blade technologies. Journal of Archaeological Science 39: 1883–1900. f g Will, M., Kandel, A. W., and Conard, N. J. (2019). Midden or molehill: The role of coastal adaptations in human evolution and dispersal. Journal of World Prehistory 32: 33–72. Wilmshurst, J. M., Hunt, T. L., Lipo, C. P., and Anderson, A. J. (2011). High-precision radiocarbon dating shows recent and rapid initial human colonization of East Polynesia. Proceedings of the National Academy of Sciences 108: 1815–1820.l Wright, T. F., Eberhard, J. R., Hobson, E. A., Avery, M. L., and Russello, M. A. (2010). Behavioral flex- ibility and species invasions: The adaptive flexibility hypothesis. Ethology Ecology & Evolution 22: 393–404. 1 3 Journal of Archaeological Research (2021) 29:255–326 326 Wynn, T. (1993). Two developments in the mind of early Homo. Journal of Anthropological Archaeology 12: 299–322.l Xhauflair, H., and Pawlik, A. (2010). Usewear and residue analysis: Contribution to the study of the lithic industry from Tabon Cave, Palawan, Philippines. Sezione di Museologia Scientifica e Naturalistica 6: 147–154.l Xhauflair, H., Pawlik, A., Gaillard, C., Forestier, H., Vitales, T. J., Callado, J. R., et al. (2016). Characteri- sation of the use-wear resulting from bamboo working and its importance to address the hypothesis of the existence of a bamboo industry in prehistoric Southeast Asia. Quaternary International 416: 95–125. Xhauflair, H., Revel, N., Vitales, T. J., Callado, J. R., Tandang, D., Gaillard, C., et al. (2017). References Cited What plants might potentially have been used in the forests of prehistoric Southeast Asia? An insight from the resources used nowadays by local communities in the forested highlands of Palawan Island. Qua- ternary International 448: 169–189. Zazzo, A., Lebon, M., Quiles, A., Reiche, I. and Vigne, J. D. (2015). Direct dating and physico-chemical analyses cast doubts on the coexistence of humans and dwarf hippos in Cyprus. PLOS ONE 10: p.e0134429. p Zong, C. (2015). Late Pleistocene Sea Levels and Resulting Changes in Global Land Distributions, Ph.D. dissertation, Department of Geography, University of Kansas, Lawrence. Bibliography of Recent Literature Anderson, A., and Boyle, K. V. (eds.) (2010). The Global Origins and Development of Seafaring, McDonald Institute for Archaeological Research, Cambridge. Bellwood, P. (ed.) (2019). The Spice Islands in Prehistory: Archaeology in the Northern Moluccas, Ind Bellwood, P. (ed.) (2019). The Spice Islands in Prehistory: Archaeology in the Northern Moluccas, Indo nesia, Terra Australis 50, Australian National University Press, Canberra. p y gy nesia, Terra Australis 50, Australian National University Press, Canberra. Dawson, H. (2013). Mediterranean Voyage Left Coast Press, Walnut Creek, CA. Dawson, H. (2013). Mediterranean Voyages: The Archaeology of Island Colonisation and Abandonment, Left Coast Press, Walnut Creek, CA. Left Coast Press, Walnut Creek, CA. Dennell, R., and Porr, M. (eds.) (2014). Southern Asia, Australia, and the Search for Human Origin Cambridge University Press, Cambridge. Fagan, B. (2012). Beyond the Blue Horizon: How the Earliest Mariners Unlocked the Secrets of the Oceans, Bloomsbury Publishing, London. y g Kaifu, Y., Izuho, M., Goebel, T., Sato, H., and Ono, A. (eds.) (2015). Emergence and Diversity of Modern H B h i i P l li hi A i T A&M U i i P C ll S i Kaifu, Y., Izuho, M., Goebel, T., Sato, H., and Ono, A. (eds.) (2015). Emergence and Diversity of Modern Human Behavior in Paleolithic Asia, Texas A&M University Press, College Station. Knapp, A. B. (2013). The Archaeology of Cyprus, Cambridge University Press, Cambridge. O’Connor, S., Bulbeck, D., and Meyer, J. (eds.) (2018). The Archaeology of Sulawesi: Current Research on the Pleistocene to the Historic Period, Terra Australis 48, Australian National University Press, Canberra. Pearson, R. J. (2013). Ancient Ryukyu, University of Hawaii Press, Honolulu. Phoca-Cosmetatou, N. (ed.) (2011). The First Mediterranean Islanders: Initial Occupation and Survival Strategies, Oxbow Books, Oxford. Pydyn, A. (2015). Argonauts of the Stone Age: Early Maritime Activity from the First Migrations fro Africa to the End of the Neolithic, Archaeopress, Oxford. Simmons, A. H. (2014). Stone Age Sailors: Paleolithic Seafaring in the Mediterranean, Routledge, London. Walter, R., and Sheppard, P. (2017). Archaeology of the Solomon Islands, University of Otago Press, Dunedin. Webb, S. G. (2006). The First Boat People, Cambridge University Press, Cambridge. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. 1 3
https://openalex.org/W3011007315	https://www.research-collection.ethz.ch/bitstream/20.500.11850/412425/3/advs.201903697.pdf	English	null	Engineered Polymersomes for the Treatment of Fish Odor Syndrome: A First Randomized Double Blind Olfactory Study	Advanced science	2,020	cc-by	9,098	ETH Library Author(s): ( ) Schmidt, Aaron C.; Hebels, Erik R.; Weitzel, Charlotte; Kletzmayr, Anna; Bao, Yinyin; Steuer, Christian; Leroux, Jean-Christophe Aaron C. Schmidt, Erik R. Hebels, Charlotte Weitzel, Anna Kletzmayr, Yinyin Bao, Christian Steuer, and Jean-Christophe Leroux* This simple ple suffering The smell of TMA, a highly volatile tertiary amine, is readily detectable by humans at levels as low as the µg L−1 range.[4] It is primarily produced by the intestinal breakdown of dietary precur- sors by colonic bacteria, the vast majority being choline, carnitine, and TMA N-oxide (TMAO).[5] After entering systemic cir- culation, TMA is oxidized in the liver by the enzyme flavin-containing monooxy- genase 3 (FMO3) to the nonodorous N-oxide, which is subsequently excreted in the urine. TMAU is caused by an inherited deficiency secondary to muta- tions in this enzyme, yielding a dysfunc- tional metabolism of TMA. So far more than 30 sequence variants of the FMO3 gene have been reported to cause the dis- ease.[6] Deficient oxidation of the metabo- lite results in increased systemic levels of reted through sweat, breath, urine, and other esulting in an offensive fish-like body odor. Trimethylaminuria (TMAU), also known as the “fish odor syn- drome,” is a genetic disorder related to the excretion of elevated levels of the foul-smelling odorant trimethylamine (TMA), making sufferers secrete an odor resembling that of rotten fish. While hundreds of cases have been reported in literature in the past decades, the number of affected individuals is largely unknown since this condition is considered widely undiag- nosed.[1,2] Incidence rates of heterozygous carriers are sug- gested to be in the order of 1% in the white British population, Trimethylaminuria (TMAU), also known as the “fish odor syn- drome,” is a genetic disorder related to the excretion of elevated levels of the foul-smelling odorant trimethylamine (TMA), making sufferers secrete an odor resembling that of rotten fish. While hundreds of cases have been reported in literature in the past decades, the number of affected individuals is largely unknown since this condition is considered widely undiag- nosed.[1,2] Incidence rates of heterozygous carriers are sug- gested to be in the order of 1% in the white British population, Although the condition appears to be of little medical con- cern, it can be devastating from a psychosocial perspective. Communication www.advancedscience.com Aaron C. Schmidt, Erik R. Hebels, Charlotte Weitzel, Anna Kletzmayr, Yinyin Bao, Christian Steuer, and Jean-Christophe Leroux* and are assumed to be higher in other ethnic groups.[3] Trimethylamine (TMA) is a metabolite overtly present in patients suffering from trimethylaminuria (TMAU), a rare genetic disorder characterized by a strong “fishy” body odor. To date, no approved pharmacological treatment to sequester excess TMA on the skin of patients exists. Here, transmembrane pH gradient poly(isoprene)-block-poly(ethylene glycol) (PI-b-PEG) polym- ersomes are investigated for the topical removal of TMA. PI-b-PEG amphi- philes of varying chain length are synthesized and evaluated for their ability to form vesicular structures in aqueous media. The optimization of the PI/ PEG ratio of transmembrane pH gradient polymersomes allows for the rapid and efficient capture of TMA both in solution and after incorporation into a topical hydrogel matrix at the pH of the skin. A subsequent double blind olfactory study reveals a significant decrease in perceived odor intensity after application of the polymersome-based formulation on artificial skin substrates that has been incubated in TMA-containing medium. This simple and novel approach has the potential to ease the burden of people suffering from TMAU. ethnic groups.[3] The smell of TMA, a highly volatile tertiary amine, is readily detectable by humans at levels as low as the µg L−1 range.[4] It is primarily produced by the intestinal breakdown of dietary precur- sors by colonic bacteria, the vast majority being choline, carnitine, and TMA N-oxide (TMAO).[5] After entering systemic cir- culation, TMA is oxidized in the liver by the enzyme flavin-containing monooxy- genase 3 (FMO3) to the nonodorous N-oxide, which is subsequently excreted in the urine. TMAU is caused by an inherited deficiency secondary to muta- tions in this enzyme, yielding a dysfunc- tional metabolism of TMA. So far more than 30 sequence variants of the FMO3 gene have been reported to cause the dis- ease.[6] Deficient oxidation of the metabo- lite results in increased systemic levels of TMA, which is excreted through sweat, breath, urine, and other odily secretions, resulting in an offensive fish-like body odor. s suffering erized by a treatment to smembrane G) polym- EG amphi- their ability n of the PI/ for the rapid ation into ouble blind ntensity ficial skin . Engineered Polymersomes for the Treatment of Fish Odor Syndrome: A First Randomized Double Blind Olfactory Study Aaron C. Schmidt, Erik R. Hebels, Charlotte Weitzel, Anna Kletzmayr Christian Steuer, and Jean-Christophe Leroux* Aaron C. Schmidt, Erik R. Hebels, Charlotte Weitzel, Anna Kletzmayr, Yinyin Bao, Christian Steuer, and Jean-Christophe Leroux* Aaron C. Schmidt, Erik R. Hebels, Charlotte Weitzel, Anna Kletzmayr, Yinyin Bao, Christian Steuer, and Jean-Christophe Leroux* Lit- erature refers to a range of psychological responses, including signs of mental depression and even suicidal tendencies in some cases.[7,8] Currently there is no treatment for TMAU, but only preventive measures, such as dietary restrictions of TMA precursors and frequent washing with acidic soap.[9,10] Dietary restrictions can be problematic especially since choline is vital in the formation of essential membrane phospholipids in humans.[11] There are various reports studying the use of antibiotics to deplete the microorganisms responsible for TMA generation in the large intestine, but most are inconclusive and only refer to small numbers of patients.[12,13] In addition, chronic antibiotic therapy might have a negative impact on the patients’ gut microbiota and contribute to antibiotic resist- ance.[14,15] To address this unmet medical need and improve the patients’ quality of life, the development of novel treatment approaches is of high urgency. The ORCID identification number(s) for the author(s) of this article can be found under https://doi.org/10.1002/advs.201903697. © 2020 The Authors. Published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and re- production in any medium, provided the original work is properly cited. One approach, that has so far not been tested for the treat- ment of TMAU, encompasses the sequestration of TMA into vesicular structures. The low molecular weight and basic char- acter (pKa = 9.80)[16] of TMA make this metabolite suitable for DOI: 10.1002/advs.201903697 © 2020 The Authors. Published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim Adv. Sci. 2020, 7, 1903697 Originally published in: This page was generated automatically upon download from the ETH Zurich Research Collection. For more information, please consult the Terms of use. www.advancedscience.com DOI: 10.1002/advs.201903697 1903697 (1 of 8) © 2020 The Authors. Published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim Adv. Sci. 2020, 7, 1903697 Adv. Sci. 2020, 7, 1903697 www.advancedsciencenews.com www.advancedscience.com uptake into transmembrane pH gradient vesicles (Figure 1a). The underlying principle is the rapid diffusion of the nonproto- nated amine species across the membrane and its subsequent protonation inside an acidic core, trapping it within the vesicle lumen. Our group has previously shown that liposomal carriers bearing a pH gradient could efficiently[17] and in a relatively Figure 1. Capture of trimethylamine (TMA) via transmembrane pH gradient polymersomes (top), chemical structure of PI-b-PEG (bottom) (a). Outline of the screening process of PI-b-PEG polymersomes by light microscopy (top) and of the subsequent in human olfactory testing with PI-b-PEG polymer- some hydrogel formulation (bottom) (b). Representative images of different morphological data recorded with a light microscope (DIC channel, scale bar 50 µm) (c). Fluorescence microscopy images of the encapsulation of pyranine in PI-b-PEG polymersomes (DIC and corresponding fluorescence channel, scale bar 50 µm) (d). Pyranine particle count after encapsulation into PI-b-PEG polymersomes using varying sonication amplitudes as well as sonication times (PI/PEG 1.99), mean + SD (n = 3 images per condition) (e). Cryo-TEM images (top, scale bar: 200 nm) and cryo-SEM images (bottom, scale bar: 1 µm) of PI-b-PEG vesicles (PI/PEG 1.99) (f). www.advancedscience.com Figure 1. Capture of trimethylamine (TMA) via transmembrane pH gradient polymersomes (top), chemical structure of PI-b-PEG (bottom) (a). Outline of the screening process of PI-b-PEG polymersomes by light microscopy (top) and of the subsequent in human olfactory testing with PI-b-PEG polymer- some hydrogel formulation (bottom) (b). Representative images of different morphological data recorded with a light microscope (DIC channel, scale bar 50 µm) (c). Fluorescence microscopy images of the encapsulation of pyranine in PI-b-PEG polymersomes (DIC and corresponding fluorescence channel, scale bar 50 µm) (d). Pyranine particle count after encapsulation into PI-b-PEG polymersomes using varying sonication amplitudes as well as sonication times (PI/PEG 1.99), mean + SD (n = 3 images per condition) (e). Cryo-TEM images (top, scale bar: 200 nm) and cryo-SEM images (bottom, scale bar: 1 µm) of PI-b-PEG vesicles (PI/PEG 1.99) (f). uptake into transmembrane pH gradient vesicles (Figure 1a). The underlying principle is the rapid diffusion of the nonproto- nated amine species across the membrane and its subsequent protonation inside an acidic core, trapping it within the vesicle lumen. 1903697 (2 of 8) Adv. Sci. 2020, 7, 1903697 www.advancedscience.com Most vesicles had a diameter in the lower micrometer range, which was desirable to prevent any penetration of polymersomes across the skin or mucosa. Size, morphology, and vesicular structure were analyzed by electron microscopy (EM) measure- ments (Figure 1f). Whilst confirming the vesicular structure, cryogenic transmission EM (cryo-TEM) as well as cryogenic scanning EM (cryo-SEM) experiments revealed the majority of polymersomes to exhibit smaller sizes (100–600 nm), which is possibly due to overestimation of larger structures by LD (volume distribution) and the fact that the LD analysis does not allow for a differentiation between individual and aggregated vesicles. Further, we evaluated the stability of the ester bond connecting the two polymer blocks towards hydrolysis by SEC, after one month of storage in citric acid buffer (pH = 2.0) at 4 °C (Figure S3, Supporting Information). Only a slight change at In the last decades, polymeric vesicles (i.e., polymersomes) have emerged as synthetic analogs of liposomes, drawing considerable attention in the scientific community.[21] Being made up of synthetic amphiphiles, these vesicular systems are often claimed superior to liposomes both in tunability and stability.[22,23] Polymersomes are investigated in a variety of applications such as drug delivery,[24,25] imaging,[26] and as nanoreactors[27] amongst others. In a recent study, polymeric vesicles comprising the amphiphilic diblock poly(styrene)- block-poly(ethylene glycol) (PS-b-PEG) were shown to effectively capture ammonia in solutions simulating the intestinal fluids, whereas liposomes were destabilized under these conditions.[20] However, due to the high glass transition temperature of the PS block (Tg = 378–382 K),[28] this polymersome system was charac- terized by rather slow uptake kinetics for larger NH3 analogs.[29] Restricted diffusion across the glassy polymersome membrane might play a critical role in these observations. To address this issue, we hypothesized that a polymersome system, consisting of an amphiphile with a hydrophobic block of rather low Tg might accelerate and conclusively increase the diffusion of TMA across the membrane, allowing its application for the symptomatic treatment of TMAU. In order to verify this hypothesis, we selected poly(isoprene)-block-PEG (PI-b-PEG) as amphiphile (Tg,PI = 204–209 K)[30] and synthesized polymers with PI/PEG (w/w) ratios varying between 1 and 4, and a PEG fragment of 2000 g mol−1, via nitroxide-mediated polymeriza- tion (NMP)[31] (Table 1). www.advancedscience.com www.advancedscience.com selective fashion[18] capture the smaller metabolite ammonia in the peritoneal space. This system lowered systemic ammonia and brain edema when applied via peritoneal dialysis in bile- duct ligated rats, a model of hyperammonemia.[19] However, in more hostile environments, such as the intestine, liposomal formulations can be readily destabilized and release their content.[20] emulsification by sonication, nanoprecipitation, and film rehy- dration (Figure 1b). In a first step, the amphiphiles’ ability to form round-shaped structures was investigated under various conditions in order to exclude nonspherical structures, such as irregular aggregates (e.g., pointed with arrows for PI/PEG 3.62 (w/w) in Figure 1c). Subsequently, conditions leading to spher- ical shaped microparticles were tested for their capacity to trap and retain the hydrophilic dye pyranine. Owing to its negative charge, pyranine does not adsorb to hydrophobic matrices and can therefore be used to indirectly monitor polymersome forma- tion.[38] Encapsulation of pyranine varied between samples, but increasing the PI/PEG weight ratio (and therefore the length of the hydrophobic block) from 1.32 to 1.99 improved entrapment (Figure 1d). By increasing the PI/PEG ratio even further, encap- sulation decreased to a point when no dye could be encapsulated (PI/PEG 3.62). The uptake of pyranine was lower in case of film rehydration method compared to the nanoprecipitation and emulsification/sonication procedure (Table S1 and Figure S1, Supporting Information). Furthermore, with the emulsifica- tion/sonication procedure, it was possible to obtain a higher yield of polymersomes (Table S1, Supporting Information). Hence, we selected and further optimized this method for sub- sequent experiments, using the polymer showing the highest encapsulation efficiency (PI/PEG 1.99) (Figure 1e; Figure S2, Supporting Information). This way, it was found that the condi- tions resulting in a high polymersome yield were those of low energy input, i.e., short sonication time (1 min) under a mild amplitude (5%). These conditions were then employed to pre- pare polymersomes with all synthesized amphiphiles. The poly- mersomes were characterized by dynamic light scattering (DLS) as well as laser diffraction (LD) measurements (Table 1). Most vesicles had a diameter in the lower micrometer range, which was desirable to prevent any penetration of polymersomes across the skin or mucosa. Size, morphology, and vesicular structure were analyzed by electron microscopy (EM) measure- ments (Figure 1f). DOI: 10.1002/advs.201903697 Our group has previously shown that liposomal carriers bearing a pH gradient could efficiently[17] and in a relatively 1903697 (2 of 8) Adv. Sci. 2020, 7, 1903697 1903697 (2 of 8) © 2020 The Authors. Published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim © 2020 The Authors. Published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim Adv. Sci. 2020, 7, 1903697 1903697 (3 of 8) © 2020 The Authors. Published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim Adv. Sci. 2020, 7, 1903697 www.advancedscience.com Whilst confirming the vesicular structure, cryogenic transmission EM (cryo-TEM) as well as cryogenic scanning EM (cryo-SEM) experiments revealed the majority of polymersomes to exhibit smaller sizes (100–600 nm), which is possibly due to overestimation of larger structures by LD (volume distribution) and the fact that the LD analysis does not allow for a differentiation between individual and aggregated vesicles. Further, we evaluated the stability of the ester bond connecting the two polymer blocks towards hydrolysis by SEC, after one month of storage in citric acid buffer (pH = 2.0) at 4 °C (Figure S3, Supporting Information). Only a slight change at emulsification by sonication, nanoprecipitation, and film rehy- dration (Figure 1b). In a first step, the amphiphiles’ ability to form round-shaped structures was investigated under various conditions in order to exclude nonspherical structures, such as irregular aggregates (e.g., pointed with arrows for PI/PEG 3.62 (w/w) in Figure 1c). Subsequently, conditions leading to spher- ical shaped microparticles were tested for their capacity to trap and retain the hydrophilic dye pyranine. Owing to its negative charge, pyranine does not adsorb to hydrophobic matrices and can therefore be used to indirectly monitor polymersome forma- tion.[38] Encapsulation of pyranine varied between samples, but increasing the PI/PEG weight ratio (and therefore the length of the hydrophobic block) from 1.32 to 1.99 improved entrapment (Figure 1d). By increasing the PI/PEG ratio even further, encap- sulation decreased to a point when no dye could be encapsulated (PI/PEG 3.62). The uptake of pyranine was lower in case of film rehydration method compared to the nanoprecipitation and emulsification/sonication procedure (Table S1 and Figure S1, Supporting Information). Furthermore, with the emulsifica- tion/sonication procedure, it was possible to obtain a higher yield of polymersomes (Table S1, Supporting Information). Hence, we selected and further optimized this method for sub- sequent experiments, using the polymer showing the highest encapsulation efficiency (PI/PEG 1.99) (Figure 1e; Figure S2, Supporting Information). This way, it was found that the condi- tions resulting in a high polymersome yield were those of low energy input, i.e., short sonication time (1 min) under a mild amplitude (5%). These conditions were then employed to pre- pare polymersomes with all synthesized amphiphiles. The poly- mersomes were characterized by dynamic light scattering (DLS) as well as laser diffraction (LD) measurements (Table 1). www.advancedsciencenews.com www.advancedsciencenews.com www.advancedscience.com A HEC content of 2% was selected, resulting in optimal texture and spreadability characteris- tics for topical administration. The formulations comprising a higher mount of HEC (4% and 8%; Figure S6, Supporting Information) proved noticeably more viscous and difficult to handle. Stability of the vesicular structures in the gel matrix was confirmed by freeze fracture replica TEM, showing struc- tures similar to those obtained in solution (Figure 2g). Vesicles seemed to form aggregates in the gel matrix, which was also confirmed by cryo-SEM measurements (Figure S7, Supporting Information), possibly as a result of the manual mixing pro- cess when producing the formulation. However, the incor- poration of the polymersomes in the gel at a concentration of 7.21 mg mL−1 did not have a major impact on its rheological behavior, with only a slight increase in viscosity (Figure S8, Supporting Information). Since a major part of TMA in patients is secreted in the form of sweat, a topical application of a hydrogel containing PI-b-PEG polymersomes with a pH close to that of human skin (pH 5.8)[40] was considered as a potentially suitable option for the symptomatic treatment of TMAU. We therefore con- ducted additional uptake experiments with polymersome suspensions at pH 5.8 (Figure 2a). Under these conditions, the overall capture of TMA was 50% lower than at pH 6.8, likely due to the reduction of the pH gradient. Nonetheless, the uptake kinetics remained fast, showing an initial slope of 120 µmol g−1 h−1 versus 230 µmol g−1 h−1 within the first 2 h, followed by a plateau. y At pH 5.8, PI-b-PEG polymersomes with a PI/PEG ratio of 1.99 exhibited the highest TMA capture (392 µmol g−1 after 24 h, corresponding to 31% of total TMA) as compared to all other PI/PEG ratios tested (Figure S4, Supporting Informa- tion), confirming the observations of the pyranine experiments. This is also seen in Figure 2b when plotting the TMA capture capacity at various time points as a function of the PI/PEG ratio. Considering that for a given polymer mass, only the polymer present in the form of polymersomes can contribute to TMA capturing, these results indicate that the fraction of polymer in the form of polymersomes increases with the PI block length for PI/PEG ratios up to 1.99 and decreases again for higher ratios, most likely due to more pronounced polymer aggrega- tion in the latter case (Figure 1c). www.advancedscience.com www.advancedscience.com higher retention volumes could be observed, which might how- ever be related to the presence of residual citric acid. higher retention volumes could be observed, which might how- ever be related to the presence of residual citric acid. (Table S3, Supporting Information), indicating miscibility of the two components.[41] For ammonia the difference in solubility parameters was of 13.9 (MJ m−3)½, largely above 5 (MJ m−3)½, suggesting unfavorable interactions. We then investigated the ability of the polymersome sus- pensions to sequester TMA in vitro using side-by-side diffu- sion cells.[18] The experiments were initially performed using previously reported conditions[29] at pH 6.8 to confirm that the uptake kinetics of PI-b-PEG polymersomes (inner pH 2.0) would be improved versus PS-b-PEG. Both polymers had equivalent PEG (2000 g mol−1) and similar hydrophobic/ hydrophilic weight ratios (PI-b-PEG 2.30 and PS-b-PEG 2.15). Polymersomes comprised of PI-b-PEG did indeed exhibit faster TMA uptake kinetics (initial slope of 230 µmol g−1 h−1 vs 60 µmol g−1 h−1 in the first 2 h) and higher encapsulation effi- ciency (530 µmol g−1 vs 240 µmol g−1 after 24 h) as compared to those made of PS-b-PEG (Figure 2a). The enhanced perfor- mance of PI-b-PEG vesicles might be related to the lower Tg of the PI block, allowing for faster diffusion of TMA across the bilayer membrane. In both cases, a slight decrease in cap- ture capacity after 24 h could be observed, likely related to the leakage of TMA following an increase of the inner core osmolarity.[39] As the formulation is intended to be applied on the skin, where ammonia is also present,[42] we performed a competi- tive uptake experiment in buffer containing both ammonia and TMA (10:1 molar ratio). An uptake kinetic in favor of ammonia (which is more abundant than TMA) would bear the risk of rap- idly exhausting the gradient, thereby impairing the capture of TMA despite the higher pKa value of the latter (pKa 9.8 vs 9.25 for ammonia).[43] As shown in Figure 2d, although ammonia was preferentially taken up, TMA capture reached 60% of the level achieved without ammonia. Following the TMA capture experiments for polymersome suspensions in aqueous buffer, we moved to the incorpora- tion of polymersomes in hydrogel formulations. To obtain a hydrogel, hydroxyethyl cellulose (HEC) was used as a thick- ening agent, given its presence in several commercial phar- maceutical dosage forms. www.advancedscience.com We investigated these polymers for their ability to form giant polymersomes (in the low micrometer range, to prevent any permeation of polymersomes through the skin) at concen- trations that would make them compatible with pharmaceu- tical applications. While the self-assembly of amphiphiles has been extensively studied and various methods for the prepara- tion of polymersomes have been reported,[32,33] there are only scarce reports describing supramolecular structures formed by PI-b-PEG in aqueous media, focusing mainly on micellar and hybrid vesicular structures.[31,34–37] We screened the ability of PI-b-PEG to form polymersomes via light/fluorescence microscopy assay using three preparation methods, namely Table 1. Characteristics of PI-b-PEG amphiphiles and polymersomes prepared by the emulsification/sonication method under optimized conditions. PI/PEG [w/w] Mn [g mol−1]a) Mn [g mol−1]b) Ðb) d [nm]c) PDIc) d [µm]d) Spand) 1.32 4600 11 100 1.25 240 0.26 n.d. 1.99 6000 13 700 1.28 * 7.33 1.63 2.30 6600 14 400 1.30 * 5.10 1.11 2.90 7800 16 700 1.25 * 8.52 2.43 3.62 9200 19 600 1.27 * 2.79 0.86 a)Calculated by 1H NMR spectroscopy; b)Measured by SEC in THF; c)Obtained by DLS, polydispersity index (PDI); d)Obtained by LD; *diameter >800 nm; n.d.: not detectable. hiphiles and polymersomes prepared by the emulsification/sonication method under optimized conditions. Table 1. Characteristics of PI-b-PEG amphiphiles and polymersomes prepared by the emulsification/sonication 1903697 (3 of 8) © 2020 The Authors. Published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim Adv. Sci. 2020, 7, 1903697 Adv. Sci. 2020, 7, 1903697 © 2020 The Authors. Published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.advancedsciencenews.com www.advancedscience.com a change in pH (Figure S10, Supporting Information). In case of the hydrogel, the ratio remained constant over 4 h at 37 °C, Following the promising in vitro data, we conducted a double blind olfactory study using an artificial skin substrate Figure 2. TMA capture over time for transmembrane pH gradient PS-b-PEG (PS/PEG 2.15) and PI-b-PEG (PI/PEG 2.30) polymersomes (a). Uptake capacity measured over 24 h incubation as a function of PI/PEG weight ratio (b). TMA capture over time for PI-b-PEG (PI/PEG 1.99) in the pres- ence and absence of a pH gradient (c). TMA capture over time in the presence of tenfold excess NH3 (d). TMA capture of PI-b-PEG HEC hydrogel (Gel-pH-Ves) and HEC hydrogel (Gel) at pH 5.8 (e). Fluorescence intensity ratio I455/I416 (λem 515 nm) of pyranine-containing pH gradient polym- ersomes (PI/PEG 1.99) in HEC hydrogel over time and free pyranine at indicated pH values, mimicking the incubation conditions used in (e) (f). Freeze fracture replica TEM of PI-b-PEG polymersomes (PI/PEG 1.99) in the hydrogel matrix (top) and of the vesicle-free HEC gel (bottom), scale bar is set to 500 nm (g). Results of the in-human olfactory study. The mean difference for 4 comparisons against the shared control “Pos.ctrl” are shown in the above Cumming estimation plot. The raw data are plotted on the upper axes with the line break denoting the mean value of each group and the lines indicating the standard deviation. On the lower axes, mean differences are plotted as bootstrap sampling distributions. Each mean difference is depicted as a dot. Each 95% confidence interval is indicated by the ends of the vertical error bars (Gel: HEC gel; Gel-Ves: HEC gel containing polymersomes without pH gradient; Gel-pH-Ves: HEC gel containing polymersomes with transmembrane pH gradient) (n = 15) (h). For panels (a,) (c), (d), and (e) data are represented as means ± SD (n = 3). For panels (a), (c), and (e) statistics were performed on the AUC0-4h (Table S2, Supporting Information). www.advancedscience.com www.advancedscience.com Figure 2. TMA capture over time for transmembrane pH gradient PS-b-PEG (PS/PEG 2.15) and PI-b-PEG (PI/PEG 2.30) polymersomes (a). Uptake capacity measured over 24 h incubation as a function of PI/PEG weight ratio (b). TMA capture over time for PI-b-PEG (PI/PEG 1.99) in the pres- ence and absence of a pH gradient (c). TMA capture over time in the presence of tenfold excess NH3 (d). TMA capture of PI-b-PEG HEC hydrogel (Gel-pH-Ves) and HEC hydrogel (Gel) at pH 5.8 (e). Fluorescence intensity ratio I455/I416 (λem 515 nm) of pyranine-containing pH gradient polym- ersomes (PI/PEG 1.99) in HEC hydrogel over time and free pyranine at indicated pH values, mimicking the incubation conditions used in (e) (f). Freeze fracture replica TEM of PI-b-PEG polymersomes (PI/PEG 1.99) in the hydrogel matrix (top) and of the vesicle-free HEC gel (bottom), scale bar is set to 500 nm (g). Results of the in-human olfactory study. The mean difference for 4 comparisons against the shared control “Pos.ctrl” are shown in the above Cumming estimation plot. The raw data are plotted on the upper axes with the line break denoting the mean value of each group and the lines indicating the standard deviation. On the lower axes, mean differences are plotted as bootstrap sampling distributions. Each mean difference is depicted as a dot. Each 95% confidence interval is indicated by the ends of the vertical error bars (Gel: HEC gel; Gel-Ves: HEC gel containing polymersomes without pH gradient; Gel-pH-Ves: HEC gel containing polymersomes with transmembrane pH gradient) (n = 15) (h). For panels (a,) (c), (d), and (e) data are represented as means ± SD (n = 3). For panels (a), (c), and (e) statistics were performed on the AUC0-4h (Table S2, Supporting Information). Figure 2. TMA capture over time for transmembrane pH gradient PS-b-PEG (PS/PEG 2.15) and PI-b-PEG (PI/PEG 2.30) polymersomes (a). Uptake capacity measured over 24 h incubation as a function of PI/PEG weight ratio (b). TMA capture over time for PI-b-PEG (PI/PEG 1.99) in the pres- ence and absence of a pH gradient (c). TMA capture over time in the presence of tenfold excess NH3 (d). TMA capture of PI-b-PEG HEC hydrogel (Gel-pH-Ves) and HEC hydrogel (Gel) at pH 5.8 (e). Fluorescence intensity ratio I455/I416 (λem 515 nm) of pyranine-containing pH gradient polym- ersomes (PI/PEG 1.99) in HEC hydrogel over time and free pyranine at indicated pH values, mimicking the incubation conditions used in (e) (f). Freeze fracture replica TEM of PI-b-PEG polymersomes (PI/PEG 1.99) in the hydrogel matrix (top) and of the vesicle-free HEC gel (bottom), scale bar is set to 500 nm (g). Results of the in-human olfactory study. The mean difference for 4 comparisons against the shared control “Pos.ctrl” are shown in the above Cumming estimation plot. www.advancedscience.com Due to its superior TMA cap- ture capacity, all subsequent experiments were carried out using the polymer with a PI/PEG ratio of 1.99. To ensure that TMA capture was indeed driven via the transmembrane pH gradient, we performed a control experiment with polymersomes having the internal pH adjusted to 5.8 (no gradient) (Figure 2c). Sur- prisingly, the TMA capture of these polymersomes still reached approximately 40% of that of the pH gradient system. Such an effect was not observed with ammonia (Figure S5, Supporting Information). We suspect that this may be related to the com- patibility of unprotonated TMA with the PI membrane. In fact, calculation of solubility parameters for TMA and PI provided almost identical values with a difference of only 0.6 (MJ m−3)½ In order to mimic the in vivo conditions following a topical administration of the gel containing polymersomes, we assessed TMA capture with Franz diffusion cells (Figure 2e). The control formulation containing no polymersomes decreased the TMA concentration in the donor compartment due to the diffusion of TMA into the hydrogel. However, the TMA uptake was sig- nificantly higher and faster with the polymersome-containing gel, capturing the majority of TMA within the first 30 min. At 4 h, a capture capacity of 200 ± 50 µmol TMA g−1 polymer, upon correction for TMA diffusion, was measured (Figure S9, Supporting Information) (2.2-fold lower than the results obtained for the polymersome suspensions in side-by-side dif- fusion cells; Figure S4, Supporting Information). This reduced uptake might be attributed to the fact that in Franz cells, the receptor compartment is not stirred and thereby the diffusion of TMA is slower. To evaluate stability of the pH gradient in the gel matrix, we prepared a gel (pHout = 7.0) with polymer- somes containing pyranine in their acidic lumen (pHin = 2.0). The fluorescence spectrum of pyranine is known to be pH dependent, wherefore stability of the gradient can be moni- tored by following the fluorescence excitation spectrum over time. Comparison of the ratio of the wavelengths at maximal pH dependency (455 nm) and the isosbestic point (416 nm) to those of the free dye at set pH values can be used to determine 1903697 (4 of 8) 1903697 (4 of 8) © 2020 The Authors. Published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim Adv. Sci. 2020, 7, 1903697 © 2020 The Authors. Published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.advancedscience.com Nanoprecipitation: Polymers were dissolved in THF (4 mg in 333 µL) and added dropwise to sodium chloride-containing citrate buffer (1 mL, 250 × 10−3 m citric acid, pH = 2, 300 mOsmol kg−1), whilst stirring at room temperature for 5 min. Excess solvent was removed in vacuum (1 h, 40 °C, 15 kPa). Emulsification: Polymers (PI-b-PEG or PSn-b-PEG) were dissolved in DCM (30 mg in 100 µL) and added to sodium chloride-containing citrate buffer (1 mL, 250 × 10−3 m citric acid, pH = 2, 300 mOsmol kg−1) dropwise, whilst sonicating using a FB-705 sonic dismembrator (Thermo Fisher Scientific, Waltham, MA) (Optimized conditions: sonication for 1 min at an amplitude of 5). The sample was cooled with an ice bath during this process. Excess solvent was removed in vacuum (7 min, 40 °C, 65 kPa). Encapsulation of Pyranine: In the case of pyranine containing samples, vesicles were prepared as mentioned above, exchanging the citrate buffer to miliQ water containing 10 × 10−3 m of pyranine. Free pyranine was removed using PD Miditrap G-25 size exclusion columns (GE healthcare, Uppsala, Sweden), applying the following protocol: after conditioning the column with water, the sample (200 µL) was applied. Water (800 µL), followed by another fraction of water (600 µL) were added, of which the latter was collected. Polymersome samples were stored for up to 4 days post preparation at 4 °C. Polymersome Characterization: Polymer concentration: Polymer concentrations in the polymersome suspensions were determined by lyophilization of polymersome-containing samples (75 µL), subsequent dissolution in THF (1 mL), and measurement by SEC. Peak integrals were compared to those of a subset of standards of the polymer used to obtain the polymersomes (0.1, 0.2, 0.5, 1, and 2 mg mL−1). In conclusion, a series of PI-b-PEG polymers with varying chain length were synthesized and formulated into polymer- somes. For the first time, synthetic polymersomes were inves- tigated for the palliative treatment of TMAU. The approach is based on the incorporation of transmembrane pH gradient PI- b-PEG polymersomes in a topical hydrogel formulation. Owing to their fluid membrane at body temperature, these polymer- somes were able to efficiently and rapidly capture TMA in vitro and, as a result, reduced perceived odor intensity when applied on a skin surrogate. www.advancedscience.com It is to note that roughly 7% of the population is anosmic to the smell of TMA.[44] In our case, one of the volunteers was found anosmic to TMA and was therefore excluded from the study. Thresholds of 62.5 ± 33.1 µmol L−1 for the detection and 188 ± 137 µmol L−1 for the recognition of TMA were established (Table S4, Sup- porting Information), which is in accordance with previous findings employing a similar setup.[45] Polymersome Preparation Film Rehydration: Polymers were dissolved in dichloromethane (DCM) (3 mg in 200 µL), added into a 4 mL glass vial and subsequently dried to obtain a thin polymer film. The film was then rehydrated using sodium chloride-containing citrate buffer (1 mL, 250 × 10−3 m citric acid, pH = 2, 300 mOsmol kg−1) and stirred for 3 days at room temperature. i g p y g p In the second part of the study, each individual received a total of 15 samples to assess the TMA odor, comprising three gel formulations (no polymersomes, with polymersomes—no pH gradient, with polymersomes—with pH gradient), as well as positive and negative (no TMA) buffer controls. After 3 h of incubation in a 1 × 10−3 m TMA-containing buffer, the skin substrate was treated with the gel and incubated for 30 min at 37 °C. The herein used TMA concentrations exceeded those usually encountered in healthy humans (≈5 µm in human plasma),[46] to ensure recognition of the distinctive TMA smell by the participants. Perceived odor intensity was rated on a ten-point odor detection scale, ranging from 0, which equaled the negative control, up to 10, equaling the positive control (Figure 1b). Due to the subjective nature of rating the per- ceived odor, an expected variability of the results was observed. Nevertheless, a clear trend was detected when comparing the bootstrap sample distributions of the three HEC formulations (Figure 2h). The control gel devoid of polymersomes did not decrease the perceived odor intensity. While the formulation containing the polymersomes without pH gradient exhibited a slight decrease in TMA odor intensity (as would be expected from the uptake study in Figure 2c), only the formulation pre- pared with the pH gradient polymersomes was significantly dif- ferent from the positive control and the control gel (Table S5, Supporting Information). www.advancedscience.com This study further highlights the potential of polymersomes in detoxification applications and provides a compelling example for the use of microvesicles to sequester target molecules in addition to their conventional use in drug delivery. Size Measurements: Hydrodynamic diameters were determined by dynamic light scattering (DLS) on a DelsaNano (Beckman Coulter, Indianapolis, IN) using the cumulant method. For samples exceeding a diameter of 800 nm, additional measurements using laser diffraction (LD) were performed on a Malvern MasterSizer2000 (Malvern Instruments, Malvern, UK), reporting results as volume distribution. Optical Microscopy: Microscopy images were obtained on a Leica DMI6000B inverted epifluorescence microscope (Leica Microsystems, Wetzlar, Germany) at room temperature. Images were analyzed using ImageJ and particle counting was performed in MatlabR2018b using the Image Processing and Computer Vision toolbox. Code is available upon request. Electron Microscopy: Samples were analyzed by cryo-TEM, cryo-SEM, and freeze fracture replica TEM (see the Supporting Information). In Vitro Uptake Assays-TMA/Ammonia Uptake in Phosphate Buffer: Uptake experiments of PI-b-PEG (or PS-b-PEG) polymersomes were conducted in side-by-side diffusion cells (PermeGear, Hellertown, PA) as described elsewhere.[18] Briefly, two 3.4 mL chambers, separated by a 0.1 µm polycarbonate membrane filter, were prepared by mixing sodium chloride-containing phosphate buffer at chosen pH (5.8 or 6.8) (2.8 mL, 60 × 10−3 m in phosphate, 300 mOsmol kg−1) and 2 M NaOH (125 µL for pH 5.8, 145 µL for pH 6.8) in each chamber, to neutralize the citrate buffer www.advancedscience.com www.advancedscience.com www.advancedscience.com spectra were recorded on an AV-400 400 MHz spectrometer (Bruker, Billerica, MA) at room temperature, using CDCl3 or acetone-d6 as solvent. Chemical shifts are reported in parts per million (ppm) and were adjusted to the corresponding solvent peak. Size exclusion chromatograms were obtained on a Viscothek TDAmax system (Viskotek, Houston, TX) equipped with a differential refractive index detector (TDA 302, Viskotek) and two ViscoGEL columns (GMHHR-M, poly(styrene-co-divinylbenzene)), using tetrahydrofurane (THF) as organic phase. Samples were dissolved in THF and measured at a flow rate of 0.5 mL min−1. Results were obtained by comparison to a poly(methyl methacrylate) standard curve (2500–89 300 g mol−1) (PSS polymer, Mainz, Germany). spectra were recorded on an AV-400 400 MHz spectrometer (Bruker, Billerica, MA) at room temperature, using CDCl3 or acetone-d6 as solvent. Chemical shifts are reported in parts per million (ppm) and were adjusted to the corresponding solvent peak. Size exclusion chromatograms were obtained on a Viscothek TDAmax system (Viskotek, Houston, TX) equipped with a differential refractive index detector (TDA 302, Viskotek) and two ViscoGEL columns (GMHHR-M, poly(styrene-co-divinylbenzene)), using tetrahydrofurane (THF) as organic phase. Samples were dissolved in THF and measured at a flow rate of 0.5 mL min−1. Results were obtained by comparison to a poly(methyl methacrylate) standard curve (2500–89 300 g mol−1) (PSS polymer, Mainz, Germany). hydrogel. Sixteen healthy volunteers were asked to assess the odor of three different formulations in comparison to two con- trols. In a first step, detection and recognition thresholds were defined by asking the volunteers to smell a range of samples with increasing TMA concentrations. It is to note that roughly 7% of the population is anosmic to the smell of TMA.[44] In our case, one of the volunteers was found anosmic to TMA and was therefore excluded from the study. Thresholds of 62.5 ± 33.1 µmol L−1 for the detection and 188 ± 137 µmol L−1 for the recognition of TMA were established (Table S4, Sup- porting Information), which is in accordance with previous findings employing a similar setup.[45] hydrogel. Sixteen healthy volunteers were asked to assess the odor of three different formulations in comparison to two con- trols. In a first step, detection and recognition thresholds were defined by asking the volunteers to smell a range of samples with increasing TMA concentrations. The raw data are plotted on the upper axes with the line break denoting the mean value of each group and the lines indicating the standard deviation. On the lower axes, mean differences are plotted as bootstrap sampling distributions. Each mean difference is depicted as a dot. Each 95% confidence interval is indicated by the ends of the vertical error bars (Gel: HEC gel; Gel-Ves: HEC gel containing polymersomes without pH gradient; Gel-pH-Ves: HEC gel containing polymersomes with transmembrane pH gradient) (n = 15) (h). For panels (a,) (c), (d), and (e) data are represented as means ± SD (n = 3). For panels (a), (c), and (e) statistics were performed on the AUC0-4h (Table S2, Supporting Information). Following the promising in vitro data, we conducted a double blind olfactory study using an artificial skin substrate to assess the formulations’ ability to lower the characteristic TMA smell upon application of the polymersome-loaded a change in pH (Figure S10, Supporting Information). In case of the hydrogel, the ratio remained constant over 4 h at 37 °C, at a value equivalent to the inner pH of the vesicles, indicating stability of the pH gradient (Figure 2f). a change in pH (Figure S10, Supporting Information). In case of the hydrogel, the ratio remained constant over 4 h at 37 °C, at a value equivalent to the inner pH of the vesicles, indicating stability of the pH gradient (Figure 2f). 1903697 (5 of 8) 1903697 (5 of 8) © 2020 The Authors. Published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim Adv. Sci. 2020, 7, 1903697 © 2020 The Authors. Published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim Adv. Sci. 2020, 7, 1903697 www.advancedscience.com www.advancedscience.com subsequently placed on a glass slide, treated with the HEC formulation (≈200 mg), enclosed in a 50 mL falcon tube, and incubated at 37 °C for 30 min. Positive and negative controls, were incubated in the absence of HEC formulation. Samples were rated on a scale of 1 to 10, increasing in TMA smell. Each individual received a total of 15 samples (3 times each: positive control, negative control, PI-b-PEG HEC gel with pH gradient – Gel-pH-Ves, PI-b-PEG HEC gel without pH gradient – Gel-Ves, and pure HEC gel – Gel) in a randomized fashion. Time between samples was 10 min to ensure recovery of the olfactory receptors. that was added in the subsequent step and ensure a final pHout of 5.8 or 6.8). Polymersome suspension in citrate buffer (400 µL, 10.2 mg mL−1) and sodium chloride-containing citrate buffer (400 µL, 250 × 10−3 m citric acid, pH = 2, 300 mOsmol kg−1) were added to the receptor and donor cells, respectively. Water was added to obtain the final volume of 3.4 mL in each cell. After incubation at 37 °C for 15 min, a 100 × 10−3 m TMA (or NH4Cl) solution (25.5 µL) was added to both chambers to start the capture experiment. Samples (50 µL) were drawn at regular time intervals from the donor cell. The TMA content was determined as described previously with minor modifications.[46] In short, calibrator (50 µL) or TMA samples were mixed with internal standard (50 µL) and liberation solution (900 µL, 2 m NaOH/ 0.5 m KCl) in a 20 mL headspace vial and capped directly. Calibration was performed using single point calibration at 500 × 10−6 m. Ammonia concentration of samples was determined with the Berthelot assay.[20] subsequently placed on a glass slide, treated with the HEC formulation (≈200 mg), enclosed in a 50 mL falcon tube, and incubated at 37 °C for 30 min. Positive and negative controls, were incubated in the absence of HEC formulation. Samples were rated on a scale of 1 to 10, increasing in TMA smell. Each individual received a total of 15 samples (3 times each: positive control, negative control, PI-b-PEG HEC gel with pH gradient – Gel-pH-Ves, PI-b-PEG HEC gel without pH gradient – Gel-Ves, and pure HEC gel – Gel) in a randomized fashion. Time between samples was 10 min to ensure recovery of the olfactory receptors. www.advancedscience.com Statistical Analysis: Statistical analysis was carried out using GraphPad Prism (version 8.2.0). In the case of the olfactory study, the groups were compared using a one-way ANOVA (Tukey’s test, paired), assuming normal distribution of the data. Estimation graphics were plotted using the DABEST Python package in Python 3.6.[47] In case of TMA and ammonia in vitro capture assays, statistical analysis was performed on the area under the uptake versus time curve for the first 4 h (AUC0-4h). Multiple groups were compared using a one-way ANOVA (Tukey’s test, unpaired), whereas groups of two were compared using an unpaired t test. Ammonia and TMA Competition Experiment: The competition experiment was conducted in the same fashion as the TMA capture experiment, adding a ten-time excess of NH4Cl (7.5 × 10−3 m) with respect to TMA. Hydrogel Formulation and TMA Capture: HEC (4.0 g) was weighed into a 50 mL cream container and sodium chloride-containing phosphate buffer mixture was added (46 g, 60 × 10−3 m, pH = 5.8, 300 mOsmol kg−1). The mixture was blended using an Unguator Q device (Gako International, Schesslitz, Germany). Immediately after mixing, polymersome suspension (9 g, 16 mg mL−1) in citrate buffer were added to 10 g of the freshly prepared gel. NaOH 10 m was added to reach a final pH of 5.8, and then milliQ water was added to reach a total weight of 20 g. The ingredients were manually mixed and stored at 4 °C for 2 days to obtain the final hydrogel formulation. Polymersome- free gel was prepared the same way, employing citrate buffer without the polymersomes. TMA capture of PI-b-PEG hydrogels was investigated in Franz cells (PermeGear), separating buffer and sample chamber using a 0.1 µm polycarbonate membrane filter. Approximately 1 g of gel formulation was added to the sample chamber. After addition of sodium chloride-containing phosphate buffer (60 × 10−3 m in phosphate, pH = 5.8, 300 mOsmol kg−1), 0.75 × 10−3 m in TMA, capture was evaluated at 37 °C, drawing samples (50 µL) at regular time intervals. Supporting Information Supporting Information is available from the Wiley Online Library or from the author. Acknowledgements The authors gratefully acknowledge funding from the Swiss National Science Foundation (2-77082-16). The authors further thank Britta Hettich for her input on the figures and Anastasia Spyrogianni Roveri for proofreading the manuscript. Frank Steiniger (Electron Microscopy Center of the University Hospital Jena, Germany) and Stephan Handschin (ScopeM, ETH Zurich, Switzerland) are acknowledged for performing the EM measurements. Stability Assessment of pH Gradient in HEC gel: The stability of the pH gradient of the polymersomes in the hydrogel matrix was assessed by encapsulation of pyranine into the vesicles as described previously, using sodium chloride-containing citrate buffer (250 × 10−3 m citric acid, pH = 2, 300 mOsmol kg−1) instead of milliQ water, and subsequent preparation of the vesicle containing hydrogel (pHinside = 2.0, pHoutside = 7.0). Fluorescence excitation spectra (λem 515 nm) were recorded and subsequent comparison of the intensity ratio between λexc 455 nm (maximum pH dependency) and λexc 416 nm (isosbestic point) to those of a series of pyranine solutions (20 × 10−6 m) at set pH values was performed over time. Conflict of Interest The authors declare no conflict of interest. Experimental Section Polymers: PI-b-PEG and PS-b-PEG polymers were synthesized via NMP and atom transfer radical polymerization (ATRP), respectively (see the Supporting Information),[20,31] and characterized by 1H NMR spectroscopy (Figure S11, Supporting Information) and size exclusion chromatography (SEC; Figure S12, Supporting Information). 1H NMR 1903697 (6 of 8) © 2020 The Authors. Published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim Adv. Sci. 2020, 7, 1903697 www.advancedsciencenews.com www.advancedsciencenews.com [1] R. J. Mackay, C. J. McEntyre, C. Henderson, M. Lever, P. M. George, Clin. Biochem. Rev. 2011, 32, 33. [2] J. Messenger, S. Clark, S. Massick, M. Bechtel, J. Clin. Aesthetic Der- matol. 2013, 6, 45. [3] S. C. Mitchell, Oral Dis. 2005, 11, 10. [4] C. van Thriel, M. Schäper, E. Kiesswetter, S. Kleinbeck, S. Juran, M. Blaszkewicz, H. Fricke, L. Altmann, H. Berresheim, T. Brüning, Int. Arch. Occup. Environ. Health 2006, 79, 308. [5] A. Q. Zhang, S. C. Mitchell, R. L. Smith, Food Chem. Toxicol. 1999, 37, 515. [6] E. A. Shephard, E. P. Treacy, I. R. Phillips, Eur. J. Hum. Genet. 2012, 20, 4. [7] S. A. Khan, K. Shagufta, Indian J. Psychiatry 2014, 56, 185. Adv. Sci. 2020, 7, 1903697 [5] A. Q. Zhang, S. C. Mitchell, R. L. Smith, Food Chem. Toxicol. 1999, 37, 515. [6] E. A. Shephard, E. P. Treacy, I. R. Phillips, Eur. J. Hum. Genet. 2012, 20, 4. [1] R. J. Mackay, C. J. McEntyre, C. Henderson, M. Lever, P. M. George, Clin. Biochem. Rev. 2011, 32, 33. [2] J. Messenger, S. Clark, S. Massick, M. Bechtel, J. Clin. Aesthetic Der- matol. 2013, 6, 45. [3] S. C. Mitchell, Oral Dis. 2005, 11, 10. [4] C. van Thriel, M. Schäper, E. Kiesswetter, S. Kleinbeck, S. Juran, M. Blaszkewicz, H. Fricke, L. Altmann, H. Berresheim, T. Brüning, Int. Arch. Occup. Environ. Health 2006, 79, 308. [5] A. Q. Zhang, S. C. Mitchell, R. L. Smith, Food Chem. Toxicol. 1999, [7] S. A. Khan, K. Shagufta, Indian J. Psychiatry 2014, 56, 185. [1] R. J. Mackay, C. J. McEntyre, C. Henderson, M. Lever, P. M. George, Clin. Biochem. Rev. 2011, 32, 33. © 2020 The Authors. Published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim [3] S. C. Mitchell, Oral Dis. 2005, 11, 10. [2] J. Messenger, S. Clark, S. Massick, M. Bechtel, J. Clin. Aesthetic Der- matol. 2013, 6, 45. www.advancedsciencenews.com www.advancedscience.com [8] W. A. Todd, J. Pediatr. 1979, 94, 936. [27] K. T. Kim, J. J. L. M. Cornelissen, R. J. M. Nolte, J. C. M. Van Hest, Adv. Mater. 2009, 21, 2787. [9] M. G. Busby, L. Fischer, K. Costa, D. Thompson, M. Mar, S. H. Zeisel, J. Am. Diet. Assoc. 2004, 104, 1836. [28] J. Rieger, J. Therm. Anal. 1996, 46, 965. [ ] J g , J , , [29] J.-C. Leroux, S. Matoori, A. Schmidt, WO2018033856A1, 2017. [10] B. Wilcken, BMJ 1993, 307, 1497. 29] J.-C. Leroux, S. Matoori, A. Schmidt, WO2018033856A1 [30] J. M. Widmaier, G. C. Meyer, Macromolecules 1981, 14, 450. [11] S. H. Zeisel, K. A. Da Costa, P. D. Franklin, E. A. Alexander, J. T. Lamont, N. F. Sheard, A. Beiser, FASEB J. 1991, 5, 2093. [31] J. K. Wegrzyn, T. Stephan, R. Lau, R. B. Grubbs, J. Polym. Sci., Part A: Polym. Chem. 2005, 43, 2977. [12] R. A. Chalmers, M. D. Bain, H. Michelakakis, J. Zschocke, R. A. Iles, h i d b i 2006 29 62 [12] R. A. Chalmers, M. D. Bain, H. Michelakakis, J. Zschocke, R. A. Iles, J. Inherited Metab. Dis. 2006, 29, 162. y [32] C. Lo Presti, H. Lomas, M. Massignani, T. Smart, G. Battaglia, J. Mater. Chem. 2009, 19, 3576. J. Inherited Metab. Dis. 2006, 29, 162. J [13] E. Treacy, D. Johnson, J. J. Pitt, D. M. Danks, J. Inherited Metab. Dis. 1995, 18, 306. [33] H. N. Yow, A. F. Routh, Soft Matter 2006, 2, 940. [14] M. J. Blaser, Science 2016, 352, 544. [34] S. Pispas, E. Sarantopoulou, Langmuir 2007, 23, 7484. [15] A. J. Alanis, Arch. Med. Res. 2005, 36, 697. [35] M. Krack, H. Hohenberg, A. Kornowski, P. Lindner, H. Weller, S. Förster, J. Am. Chem. Soc. 2008, 130, 7315. [ ] [16] G. W. Chang, W. L. Chang, K. B. K. Lew, J. Food Sci. 1976, 41, 723. [36] J. W. Bartels, S. I. Cauët, P. L. Billings, L. Y. Lin, J. Zhu, C. Fidge, D. J. Pochan, K. L. Wooley, Macromolecules 2010, 43, 7128. [17] V. Forster, R. D. Signorell, M. Roveri, J.-C. Leroux, Sci. Transl. Med. 2014, 6, 258ra141. [18] G. Giacalone, S. Matoori, V. Agostoni, V. Forster, M. Kabbaj, S. Eggenschwiler, M. Lussi, A. De Gottardi, N. Zamboni, J.-C. Leroux, J. Controlled Release 2018, 278, 57. [37] F. Zhao, D. Xie, G. Zhang, S. www.advancedsciencenews.com Pispas, J. Phys. Chem. B 2008, 112, 6358. [38] N. R. Clement, J. M. Gould, Biochemistry 1981, 20, 1534. [19] V. Agostoni, S. H. Lee, V. Forster, M. Kabbaj, C. R. Bosoi, M. Tremblay, M. Zadory, C. F. Rose, J.-C. Leroux, Adv. Funct. Mater. 2016, 26, 8382. [39] N. Bertrand, C. Bouvet, P. Moreau, J. C. Leroux, ACS Nano 2010, 4, 7552. [20] S. Matoori, Y. Bao, A. Schmidt, E. J. Fischer, R. Ochoa-Sanchez, M. Tremblay, M. Oliveira, C. F. Rose, J.-C. Leroux, Small 2019, 15, 1902347. [40] H. C. Korting, K. Hübner, K. Greiner, G. Hamm, O. Braun-Falco, Acta Derm.-Venereol. 1990, 70, 429. [41] D. van Krevelen, K. Nijenhuis, Properties of Polymers, Elsevier, Amsterdam 2009. [21] B. M. Discher, Y. Y. Won, D. S. Ege, J. C. M. Lee, F. S. Bates, D. E. Discher, D. A. Hammer, Science 1999, 284, 1143. 21] B. M. Discher, Y. Y. Won, D. S. Ege, J. C. M. Lee, [42] F. M. Schmidt, O. Vaittinen, M. Metsälä, M. Lehto, C. Forsblom, P.-H. Groop, L. Halonen, J. Breath Res. 2013, 7, 017109. D. E. Discher, D. A. Hammer, Science 1999, 284, 1143. [22] J. Siepmann, A. Faham, S. D. Clas, B. J. Boyd, V. Jannin, [22] J. Siepmann, A. Faham, S. D. Clas, B. J. Boyd, V. Jannin, A. Bernkop-Schnürch, H. Zhao, S. Lecommandoux, J. C. Evans, C. Allen, O. M. Merkel, G. Costabile, M. R. Alexander, R. D. Wildman, C. J. Roberts, J. C. Leroux, Int. J. Pharm. 2019, 558, 128. [43] K. Emerson, R. Russo, R. Lund, R. Thurston, J. Fish. Res. Board Can. 1975, 32, 2379. [44] J. E. Amoore, L. J. Forrester, J. Chem. Ecol. 1976, 2, 49. [23] D. E. Discher, F. Ahmed, Annu. Rev. Biomed. Eng. 2006, 8, 323. [45] P. Garg, K. Carpenter, S. Chong, J. Christodoulou, JIMD Rep. 2013, 8, 11. [24] F. Meng, Z. Zhong, J. Feijen, Biomacromolecules 2009, 10, 197. [25] Y. Zhu, B. Yang, S. Chen, J. Du, Prog. Polym. Sci. 2017, 64, 1. [46] P. Neyer, L. Bernasconi, J. A. Fuchs, M. D. Allenspach, C. Steuer, J. Clin. Lab. Anal. 2020, 34, e23062. [26] P. P. Ghoroghchian, P. R. Frail, K. Susumu, D. Blessington, A. K. Brannan, F. S. Bates, B. Chance, D. A. Hammer, M. J. Therien, Proc. Natl. Acad. Sci. USA 2005, 102, 2922. J [47] J. Ho, T. Tumkaya, S. Aryal, H. Choi, A. Claridge-Chang, Nat. Keywords biodetoxification, engineered polymersomes, fish odor syndrome, trimethylamine, trimethylaminuria In-Human Study: The protocol for the in human study was approved by the Research Ethics Committee of ETH Zurich (EK 2018-N-74). Informed consent of all participating subjects was obtained. A more detailed version of the protocol can be found in the supplementary information. Received: December 19, 2019 Revised: January 31, 2020 Published online: March 9, 2020 Received: December 19, 2019 Revised: January 31, 2020 Published online: March 9, 2020 Threshold Testing: Volunteers were asked to smell and evaluate a range of increasing concentrations of TMA in sodium chloride- containing phosphate buffer (60 × 10−3 m in phosphate, pH = 5.8, 300 mOsmol kg−1). Solutions were prepared in sealable glass containers of 50 mL and incubated at 37 °C for 30 min. Starting from a concentration of 15.6 × 10−6 m in TMA (≈1 mg L−1), concentration was doubled up to the point that the subject was able to detect (detection threshold) and subsequently recognize (recognition threshold) the distinct smell of TMA. [1] R. J. Mackay, C. J. McEntyre, C. Henderson, M. Lever, P. M. George, Clin. Biochem. Rev. 2011, 32, 33. [2] J. Messenger, S. Clark, S. Massick, M. Bechtel, J. Clin. Aesthetic Der- matol. 2013, 6, 45. [4] C. van Thriel, M. Schäper, E. Kiesswetter, S. Kleinbeck, S. Juran, M. Blaszkewicz, H. Fricke, L. Altmann, H. Berresheim, T. Brüning, Int. Arch. Occup. Environ. Health 2006, 79, 308. In-Human Evaluation of PI-b-PEG Formulation: The olfactory evaluation of the hydrogel formulation was performed in a double blind fashion. Artificial skin substrate (VitroSkin, IMS, Bunnell, FL) was incubated in sodium chloride-containing phosphate buffer (60 × 10−3 m, pH = 5.8, 300 mOsmol kg−1), 1 × 10−3 m in TMA, for 3 h (in case of negative controls phosphate buffer without TMA was used). The substrate was [5] A. Q. Zhang, S. C. Mitchell, R. L. Smith, Food Chem. Toxicol. 1999, 37, 515. [7] S. A. Khan, K. Shagufta, Indian J. Psychiatry 2014, 56, 185. 1903697 (7 of 8) © 2020 The Authors. Published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.advancedscience.com www.advancedsciencenews.com www.advancedsciencenews.com Methods 2019, 16, 565. 1903697 (8 of 8) 1903697 (8 of 8) Adv. Sci. 2020, 7, 1903697 © 2020 The Authors. Published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
https://openalex.org/W2047195417	https://ascpjournal.biomedcentral.com/counter/pdf/10.1186/1940-0640-8-S1-A53	English	null	Developing skills to deliver alcohol screening and brief intervention (SBI) in community pharmacy	Addiction science & clinical practice	2,013	cc-by	558	* Correspondence: p.penson@ljmu.ac.uk 1School of Pharmacy and Biomolecular Sciences, Liverpool John Moores University, Liverpool, UK Full list of author information is available at the end of the article Penson et al. Addiction Science & Clinical Practice 2013, 8(Suppl 1):A53 http://www.ascpjournal.org/content/8/S1/A53 Penson et al. Addiction Science & Clinical Practice 2013, 8(Suppl 1):A53 http://www.ascpjournal.org/content/8/S1/A53 Open Access Authors’ details 1 Authors’ details 1School of Pharmacy and Biomolecular Sciences, Liverpool John Moores University, Liverpool, UK. 2Medway School of Pharmacy, The Universities of Greenwich and Kent at Medway, Chatham, Kent, UK. Authors’ details 1School of Pharmacy and Biomolecular Sciences, Liverpool John Moores University, Liverpool, UK. 2Medway School of Pharmacy, The Universities of Greenwich and Kent at Medway, Chatham, Kent, UK. Aim This workshop aims to share and explore learning around developing skills to deliver SBI interventions among the community pharmacy team. © 2013 Penson 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. Question to be addressed How can undergraduate pharmacy students be trained to delivery lifestyle interventions? Developing skills to deliver alcohol screening and brief intervention (SBI) in community pharmacy From International Network on Brief Interventions for Alcohol and Other Drugs (INEBRIA) Meeting 2013 Rome, Italy. 18-20 September 2013 Background services. Role-play was used to consolidate learning, where students were given the role of ‘pharmacist’ ‘potential service user’ or ‘observer’ and given instruc- tions relating to the role they were to play. ‘Pharmacists’ were expected to make an approach the ‘potential service users’ and carry out an alcohol SBI service according to a given protocol. Whole group feedback and discussion fol- lowed small group role-play and participants shared their good and bad experiences with the whole group. Staff working in community pharmacies, where appoint- ments are not needed and environments are more infor- mal, face particular challenges in delivering alcohol SBI services around identifying appropriate individuals, offering the service and engaging service users. Conclusions Feedback from students suggested that the workshop was both enjoyable and perceived to be useful to future involvement in lifestyle intervention services. Presentation We will share our experience of delivering a training package for undergraduate pharmacy students around lifestyle interventions in practice, using alcohol SBI as an exemplar. This training has been run with 170 Level 6 (third year) students studying for the Master of Pharmacy (MPharm) degree at Liverpool John Moores University. The workshop was delivered on three occasions, each with with 3 facilitators and up to 60 students. Students were seated in groups of 4 and undertook structured discussion under the themes ‘Difficult Conversations’, Identifying Opportunities’, ‘Making the approach’ and ‘Making it work’. Facilitators encouraged students to reflect on and share their own relevant experiences from within and outside the pharmacy environment as both a service user and provider, and to consider how best to approach and counsel patients in the context of lifestyle Published: 4 September 2013 doi:10.1186/1940-0640-8-S1-A53 Cite this article as: Penson et al.: Developing skills to deliver alcohol screening and brief intervention (SBI) in community pharmacy. Addiction Science & Clinical Practice 2013 8(Suppl 1):A53. * Correspondence: p.penson@ljmu.ac.uk 1School of Pharmacy and Biomolecular Sciences, Liverpool John Moores University, Liverpool, UK Full list of author information is available at the end of the article
https://openalex.org/W4243860150	https://www.biorxiv.org/content/biorxiv/early/2018/02/13/264929.full.pdf	English	null	The Impact on Authors and Editors of Introducing Data Availability Statements at Nature Journals	International journal of digital curation	2,018	cc-by	4,582	Introduction In September 2016, it was announced that all research papers accepted for publication in Nature, and the life science journals in the Nature family, would be required to include information on whether the data underpinning their study were made available, and how others can access it. This followed a successful 2-month pilot implementation of the policy at five Nature journals between March 2016 and May 2016: Nature Neuroscience, Nature Physics, Nature Communications, Nature Cell Biology and Nature Geoscience. The introduction of data availability statements (DAS) by Nature journals aligned Nature's policies with a standardised Springer Nature research data policy framework, which was introduced earlier in 2016 (Hrynaszkiewicz et al, 2017). DASs are written by authors to provide information on where the data supporting the results reported in their article can be found, and if and how they can be obtained. Although there is no mandated format for these statements, template examples are provided to Nature authors, including: • The datasets generated during and/or analysed during the current study are available in the [NAME] repository, [PERSISTENT WEB LINK TO DATASETS]. • The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request. • The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request. • All data generated or analysed during this study are included in this published article (and its supplementary information files). • All data generated or analysed during this study are included in this published article (and its supplementary information files). Abstract This paper describes the adoption of a standard policy for the inclusion of data availability statements in all research articles published at the Nature family of journals, and the subsequent research which assessed the impacts that these policies had on authors, editors, and the availability of datasets. The key findings of this research project include the determination of average and median times required to add a data availability statement to an article; and a correlation between the way researchers make their data available, and the time required to add a data availability statement. This paper will be presented at the International Digital Curation Conference 2018, and has been submitted to the International Journal of Digital curation. Rebecca Grant and Iain Hrynaszkiewicz Correspondence should be addressed to Rebecca Grant, Springer Nature, The Campus, Trematon Walk, Wharfdale Road, London N1 9FN. Email: rebecca.grant@springernature.com . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted February 13, 2018. ; https://doi.org/10.1101/264929 doi: bioRxiv preprint . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted February 13, 2018. ; https://doi.org/10.1101/264929 doi: bioRxiv preprint . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted February 13, 2018. ; https://doi.org/10.1101/264929 doi: bioRxiv preprint . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted February 13, 2018. ; https://doi.org/10.1101/264929 doi: bioRxiv preprint Analysing Data Availability Statements at Nature Journals Two phases of research were undertaken to analyse the impact of this new policy. The aim of this research was to assess the ways by which researchers chose to make their data available, and to measure the additional time required by editors and production staff to add data availability statements to manuscripts (which gives an indication of cost to the publisher). . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted February 13, 2018. ; https://doi.org/10.1101/264929 doi: bioRxiv preprint . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted February 13, 2018. ; https://doi.org/10.1101/264929 doi: bioRxiv preprint For the five pilot journals, editors were asked to self-report the number of additional minutes it took to process a manuscript to ensure an appropriate DAS was provided. For Nature Communications – one of the five pilot journals, and which publishes a large number of articles – editors self-reported an overall average time for all papers they were responsible for over the pilot period, rather than reporting the time required for every manuscript. As the data were collected with different methodology and with lower precision they are not included in this analysis. For all other journals, editors recorded a time (in minutes) to add a DAS for every paper that they were responsible for in the pilot period. Copy editors and production staff were also asked to provide an estimated average additional manuscript processing time for all papers they handled in this initial period. Editors, production and copy editing staff were also invited to provide comments on the process of providing the DAS. Once the papers were accepted for publication, the text of each DAS was read and categorised into one of four different types according to a coding mechanism created for the project, by Iain Hrynaszkiewicz (Head of Data Publishing, Springer Nature). This was done for four journals (Nature Neuroscience, Nature Physics, Nature Cell Biology and Nature Geoscience), including 82 papers, by assigning the author’s description of their data availability to one of the four categories/codes. Analysing Data Availability Statements at Nature Journals The code that best matched the main message of the DAS or was applicable to the majority of data referred to in the DAS was used. The codes used were the following: • Type 1 stated that the data is available from the author on request. • Type 1 stated that the data is available from the author on request • Type 2 stated that the data had been included in the manuscript or its supplementary material. • Type 2 stated that the data had been included in the manuscript or its supplementary material. • Type 3 stated that some or all of the data is publicly available, for example in a repository. • Type 3 stated that some or all of the data is publicly available, for example in a repository. • Type 4 stated that figure source data was included with the manuscript. This is a method of data sharing used by some authors in a subset of Nature journals that publish life sciences research. Some journals encourage authors to provide the source data behind their figures/plots as spreadsheets. It is specific to the Nature journals and is not mandated and, as such, is relatively uncommon, but was important to capture in this analysis for internal purposes. • Type 4 stated that figure source data was included with the manuscript. This is a method of data sharing used by some authors in a subset of Nature journals that publish life sciences research. Some journals encourage authors to provide the source data behind their figures/plots as spreadsheets. It is specific to the Nature journals and is not mandated and, as such, is relatively uncommon, but was important to capture in this analysis for internal purposes. Univariate and bivariate analysis was then used to interpret the coded data. For the same four journals, it was found that it took 10 minutes extra editorial time on average, or a median time of 8 minutes per paper, to add a DAS (Figure 1). Additionally, 5 minutes extra copy editing time was required. For Nature Communications, 90% of editors reported 15 minutes or less to add a DAS on average. Overall, the addition of the DAS had an impact of approximately 15-20 minutes editorial and production time per accepted paper across all five journals in the 2-month pilot. . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted February 13, 2018. ; https://doi.org/10.1101/264929 doi: bioRxiv preprint Analysing Data Availability Statements at Nature Journals It is made The copyright holder for this preprint (which this version posted February 13, 2018. ; https://doi.org/10.1101/264929 doi: bioRxiv preprint . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted February 13, 2018. ; https://doi.org/10.1101/264929 doi: bioRxiv preprint . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted February 13, 2018. ; https://doi.org/10.1101/264929 doi: bioRxiv preprint manuscripts for which time data were missing (n=112). However, coding of the text of the data availability statements, by Hrynaszkiewicz and Rebecca Grant (Research Data Manager, Springer Nature), for all accepted manuscripts in the study period was carried out. Figure 3. Average time by statement type in minutes (n=329). 3.4 3.7 6.2 12.5 0 2 4 6 8 10 12 14 1 2 3 4 Total Figure 3. Average time by statement type in minutes (n=329). In this larger sample of papers, average editor time to add a DAS decreased for all types of statement compared to the pilot journals (which was 10 minutes on average). In this second analysis (Figure 3), which pooled all data from the initial pilot journals and the additional 20 journals, the type 1 statement remained the fastest (3.4 minutes on average) to add to a paper and the type 3 statement remained slower (average 6.2 minutes) than type 1 and type 2 (3.7 minutes average). The type 4 statement took the longest to implement in the second analysis. It decreased only marginally (average 12.5 minutes) for the type 4 statement but there were the fewest (10) of these statements and papers recorded. As several journals included in the analysis published low numbers of papers requiring a DAS in the 2-month period after implementation of the policy, and to provide a more useful analysis of statement types for 25 journals, statement type data are reported by the journals’ major subject discipline rather than by journal (Figure 4). Analysing Data Availability Statements at Nature Journals When the average time to add a DAS is presented by type of code statement, rather than by journal, the type 1 statement, where data are available on request, was the fastest, on average (5.9 minutes) to add to a paper. The type 3 statement, where some or all data are publicly available, took the most amount of additional time (18.2 minutes), in the four pilot journals included in this analysis. . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted February 13, 2018. ; https://doi.org/10.1101/264929 doi: bioRxiv preprint . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted February 13, 2018. ; https://doi.org/10.1101/264929 doi: bioRxiv preprint Figure 1. Median and average editor time to add DAS by journal (minutes). Figure 1. Median and average editor time to add DAS by journal (minutes). Figure 2. Average time by statement type in minutes (n=82). 5.9 14.5 18.2 12.6 0 2 4 6 8 10 12 14 16 18 20 1 2 3 4 Figure 2. Average time by statement type in minutes (n=82). 5.9 14.5 18.2 12.6 0 2 4 6 8 10 12 14 16 18 20 1 2 3 4 Figure 2. Average time by statement type in minutes (n=82). The second phase of this project gathered data using the same methodology from an additional 20 journals. These were from the biological and physical sciences, which introduced the same DAS policy as the previous journals, from September 2016. These journals provided the same information as the previous five, including the DAS, and time required to add the DAS. Data were gathered by each journal for 2 months after implementation of the policy, between September 2016 and February 2017. Because the time data relied on editor self-reporting, there are a proportion of . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. Limitations Data relating to time was self-reported by around 50 editors and support staff and so may not always be reported consistently, and is subject to individual biases and interpretations of their time. Where no time-related data were provided, those papers were removed from the analysis. Where an editor reported zero minutes or “negligible” these were recorded as 1 minute. The coding method was developed by Hrynaszkiewicz and coding carried out by Hrynaszkiewicz and Grant. Where there was disagreement in coding, this was resolved by consensus. Analysing Data Availability Statements at Nature Journals The classification of journals and papers into four broad subject groups provides some indication of different data sharing practices in different disciplines, For example, in life sciences there was the highest proportion of papers that made supporting data available in a repository (86/151 papers; 57%). In physical sciences the proportion of papers making data available in a repository was the lowest (35/179 papers; 20%). . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted February 13, 2018. ; https://doi.org/10.1101/264929 doi: bioRxiv preprint . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted February 13, 2018. ; https://doi.org/10.1101/264929 doi: bioRxiv preprint Figure 4. Distribution of statement types by journal’s major discipline (n=441). 21 46 7 116 23 13 2 28 31 86 23 35 6 4 0 20 40 60 80 100 120 140 160 180 200 Chemistry and applied Life sciences Multidisciplinary Physical sciences 1 2 3 4 Figure 4. Distribution of statement types by journal’s major discipline (n=441). Discussion and Conclusion As well as understanding the benefits of increasing accessibility to research data is also important to understand costs – particularly for publishers, funding agencies and policy makers. Information from this analysis has informed the selection of data policies by other Springer Nature journals. It has also informed the development of in-house administrative support for academic editors, so that journals without professional editors can also introduce DASs consistently. Simple, practical information, such as additional minutes to process manuscripts, is valuable for editors and support staff in understanding the impacts of editorial policy changes. The disciplinary differences in data sharing practices indicated in Figure 4 are likely due to larger numbers of community repositories being available in life sciences, combined with long standing data sharing mandates for life sciences communities , which are enforced in the editorial process at the Nature journals. In physical sciences, there are fewer mandates and the analysis also includes Nature Physics, where, in high energy physics for example, data produced and analysed can be very large – produced at large central facilities – making sharing of data online challenging. In such cases “data available on reasonable request” can be a pragmatic choice. Although there is an increase in the time required by editorial staff to process articles, the introduction of DAS will also have repercussions for other data curation stakeholders. The incorporation of standardised DASs is likely to become more widespread across journal publishers. Since Springer Nature began introducing standardised data policies, similar initiatives have been introduced by other large publishers such as Elsevier and Wiley . The standardisation of research data policies for journal publishing is being progressed by initiatives such as the Research Data Alliance’s Data Policy Standardisation and Implementation Interest Group . There are also discipline- specific initiatives to standardise and harmonise journal and publisher research data policy, in chemistry and high energy physics and medicine (Taichman et al, 2017). There is growing attention on the provision of mandatory DASs from publishers, institutions and funding agencies, particularly in the UK where DASs are a requirement of the Research Councils UK (RCUK) Common Principles on Data Policy . DASs are simple and interoperable - between stakeholders, publishing platforms and research disciplines - mechanism for communicating the availability of supporting data which can aid monitoring of compliance with data policies of funders, publishers and research communities. Discussion and Conclusion Adding mandatory DASs to all accepted articles in journals operated by professional editors increases manuscript processing time. The publisher deemed the time added to be reasonable in the context of total manuscript submission to publication time given the importance and benefits of including a DAS. Indeed, the additional time did not limit implementation of mandatory DASs at all Nature journals in 2016, after implementation at the five pilot journals. The type 3 DAS, where data are made publicly available, took longer to introduce to papers than statements where data were available on request or declared as being available with the supplementary information files. The type 3 statements include the most non-standard text, as well as links to be verified, which likely caused the longer processing time. This type of statement – making data available with, and linked to published articles – is anticipated to provide the most benefit to authors and readers, such as the potential for greater numbers of citations to those papers (Dorch at al, 2015, Piwowar et al, 2013, Sears, 2014). . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted February 13, 2018. ; https://doi.org/10.1101/264929 doi: bioRxiv preprint . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted February 13, 2018. ; https://doi.org/10.1101/264929 doi: bioRxiv preprint The second, larger, group of journals to introduce mandatory DASs reported that fewer additional minutes were added to the editorial time needed to process a manuscript. Possible reasons for this include greater editor and author awareness of the policy and supporting documents; improved internal communication and editor training after the pilot; greater attention being needed on the pilot journals, which informed, and made more rapid, editor training on handling future DAS for manuscripts in their discipline. The findings of the study demonstrate the impact of data availability statements on editorial staff and the journal publication workflow, and the need to consider increased manuscript processing requirements when mandatory DAS are introduced. Data availability A partially anonymised dataset that supports the figures and graphs in this paper is available in figshare with the identifier: https://doi.org/10.6084/m9.figshare.5809617. In this shared dataset some data columns have been removed, such as personal comments made by authors and editors during correspondence about inserting a DAS, as we do not have consent to publish them. Internal manuscript identifiers have been replaced with ascending numbers. Requests for additional data or for support with reusing the data should be emailed to the authors, or to researchdata@springernature.com Acknowledgements The authors thank Christos Petrou and Graham Smith at Springer Nature for support with data analysis; Sowmya Swaminathan, Head of Editorial Policy at Nature Research for input into policy implementation at the Nature journals; and all editors and administrative staff at the Nature journals who contributed to data acquisition. Discussion and Conclusion They can also support funder policies which require data publication by providing evidence of compliance through descriptions of publicly accessible datasets. The increase of prevalence of DASs by journal publishers necessitates support and training for researchers and editors to enable researchers to share and cite their data wherever possible. . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted February 13, 2018. ; https://doi.org/10.1101/264929 doi: bioRxiv preprint . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted February 13, 2018. ; https://doi.org/10.1101/264929 doi: bioRxiv preprint . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted February 13, 2018. ; https://doi.org/10.1101/264929 doi: bioRxiv preprint Increased prevalence of DASs will also enable further research, using machine-driven approaches (such as with natural language processing, text and data mining), across multiple journals and publishers, to analyse the types of DASs provided – and types of data sharing practiced – by researchers in different disciplines and journals. Further research would also be welcome on associations, if any, between the provision of particular types of data availability statement and research visibility and impact as studies have tended to be limited to specific disciplines and journals (Rowhani-Farid and Barnett, 2016). Increased prevalence of DASs will also enable further research, using machine-driven approaches (such as with natural language processing, text and data mining), across multiple journals and publishers, to analyse the types of DASs provided – and types of data sharing practiced – by researchers in different disciplines and journals. Further research would also be welcome on associations, if any, between the provision of particular types of data availability statement and research visibility and impact as studies have tended to be limited to specific disciplines and journals (Rowhani-Farid and Barnett, 2016). References [preprint] Dorch, S.B.F., Drachen, T. M., Ellengaard, O. (2015) The data sharing advantage in astrophysics. Retrieved from: https://arxiv.org/abs/1511.02512 [preprint] Dorch, S.B.F., Drachen, T. M., Ellengaard, O. (2015) The data sharing advantage in astrophysics. Retrieved from: https://arxiv.org/abs/1511.02512 [preprint] Dorch, S.B.F., Drachen, T. M., Ellengaard, O. (2015) The data sharing advantage in astrophysics. Retrieved from: https://arxiv.org/abs/1511.02512 [journal article] Hrynaszkiewicz, I., Birukou, A., Astell, M., Swaminathan, S., Kenall, A., Khodiyar, V. (2017). Standardising and Harmonising Research Data Policy in Scholarly Publishing. International Journal of Digital Curation 12, Number 1. https://doi.org/10.2218/ijdc.v12i1.531 [data set] Hrynaszkiewicz, I., Grant, R. (2018) The impact on authors and editors of introducing Data Availability Statements at Nature journals [Data set]. Figshare. https://doi.org/10.6084/m9.figshare.5809617 [preprint] Piwowar, H.A., Vision, T.J. (2013) Data Reuse and the open citation advantage. Retrieved from: https://doi.org/10.7717/peerj.175 [journal article] Rowhani-Farid, A., Barnett, A. G. (2016). Has open data arrived at the British Medical Journal (BMJ)? An observational study. BMJ Open, 6(10). https://doi.org/10.1136/bmjopen-2016- 011784 [data set] Sears, J. (2014) Data Sharing Effect on Article Citation Rate in Paleoceanography [Data set]. Figshare. https://doi.org/10.6084/m9.figshare.1222998.v1 [journal article] Hrynaszkiewicz, I., Birukou, A., Astell, M., Swaminathan, S., Kenall, A., Khodiyar, V. (2017). Standardising and Harmonising Research Data Policy in Scholarly Publishing. International Journal of Digital Curation 12, Number 1. https://doi.org/10.2218/ijdc.v12i1.531 [data set] Hrynaszkiewicz, I., Grant, R. (2018) The impact on authors and editors of introducing Data Availability Statements at Nature journals [Data set]. Figshare. https://doi.org/10.6084/m9.figshare.5809617 [journal article] Rowhani-Farid, A., Barnett, A. G. (2016). Has open data arrived at the British Medical Journal (BMJ)? An observational study. BMJ Open, 6(10). https://doi.org/10.1136/bmjopen-2016- 011784 [journal article] Rowhani-Farid, A., Barnett, A. G. (2016). Has open data arrived at the British Medical Journal (BMJ)? An observational study. BMJ Open, 6(10). https://doi.org/10.1136/bmjopen-2016- 011784 [journal article] Rowhani-Farid, A., Barnett, A. G. (2016). Has open data arrived at the British Medical Journal (BMJ)? An observational study. BMJ Open, 6(10). https://doi.org/10.1136/bmjopen-2016- 011784 [data set] Sears, J. (2014) Data Sharing Effect on Article Citation Rate in Paleoceanography [Data set]. Figshare. https://doi.org/10.6084/m9.figshare.1222998.v1 . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted February 13, 2018. ; https://doi.org/10.1101/264929 doi: bioRxiv preprint . References CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted February 13, 2018. ; https://doi.org/10.1101/264929 doi: bioRxiv preprint [journal article] Taichman, D. B., Sahni, P., Pinborg, A., Peiperl, L., Laine, C., James, A., Backus, J. . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted February 13, 2018. ; https://doi.org/10.1101/264929 doi: bioRxiv preprint [journal article] Taichman, D. B., Sahni, P., Pinborg, A., Peiperl, L., Laine, C., James, A., Backus, J. (2017). Data Sharing Statements for Clinical Trials: A Requirement of the International Committee of Medical Journal Editors. PLOS Medicine, 14(6). https://doi.org/10.1371/journal.pmed.1002315 . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted February 13, 2018. ; https://doi.org/10.1101/264929 doi: bioRxiv preprint [journal article] Taichman, D. B., Sahni, P., Pinborg, A., Peiperl, L., Laine, C., James, A., Backus, J. (2017). Data Sharing Statements for Clinical Trials: A Requirement of the International Committee of Medical Journal Editors. PLOS Medicine, 14(6). https://doi.org/10.1371/journal.pmed.1002315 . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted February 13, 2018. ; https://doi.org/10.1101/264929 doi: bioRxiv preprint [journal article] Taichman, D. B., Sahni, P., Pinborg, A., Peiperl, L., Laine, C., James, A., Backus, J. (2017). Data Sharing Statements for Clinical Trials: A Requirement of the International Committee of Medical Journal Editors. PLOS Medicine, 14(6). https://doi.org/10.1371/journal.pmed.1002315 [journal article] Taichman, D. B., Sahni, P., Pinborg, A., Peiperl, L., Laine, C., James, A., Backus, J. (2017). Data Sharing Statements for Clinical Trials: A Requirement of the International Committee of Medical Journal Editors. PLOS Medicine, 14(6). https://doi.org/10.1371/journal.pmed.1002315
https://openalex.org/W1985926961	https://ccsenet.org/journal/index.php/eer/article/download/31987/18605	English	null	Carbon Based Electrochemical Double Layer Capacitors of Low Internal Resistance	Energy and environment research	2,013	cc-by	5,198	Abstract Based on nanoporous carbon electrodes electrochemical double layer capacitors (EDLC), otherwise known as supercapacitors or ultracapacitors, are currently widely used in various energy storage technologies, wherein the EDLC low internal resistance and long cycle life are at an advantage. It is still a good challenge to further reduce the internal resistance of EDLC since this can result in higher power density and higher efficiency of these promising power supply units. In this work it has been found that the EDLC internal resistance depends strongly on the electrolyte diffusion in the carbon electrode nanopores, and two techniques to measure the in-pore diffusion coefficients, namely, those based on spin-echo NMR or cyclic voltammetry with the use of porous rotating disc electrode are described. Cyclic voltammetry, impedance spectroscopy and transmission electron microscopy have also been used to select the best EDLC components. As a result, EDLC devices of very low internal resistance and high power density have been developed. Keywords: double layer capacitor, in-pore electrolyte diffusion, low resistance Energy and Environment Research; Vol. 3, No. 2; 2013 ISSN 1927-0569 E-ISSN 1927-0577 Published by Canadian Center of Science and Education Energy and Environment Research; Vol. 3, No. 2; 2013 ISSN 1927-0569 E-ISSN 1927-0577 Published by Canadian Center of Science and Education Received: September 16, 2013 Accepted: October 25, 2013 Online Published: November 15, 2013 doi:10.5539/eer.v3n2p156 URL: http://dx.doi.org/10.5539/eer.v3n2p156 Received: September 16, 2013 Accepted: October 25, 2013 Online Published: November 15, 2013 doi:10.5539/eer.v3n2p156 URL: http://dx.doi.org/10.5539/eer.v3n2p156 1.1 Why It Is of Interest There is an obvious increasing interest in electrochemical double layer capacitor (EDLC) technology and application all over the world, in particular, in renewable energy and hybrid vehicle applications. However, the EDLC market growth is still rather modest. In our opinion, the main reason for a certain skepticism from automakers side is, on the one hand, a fast progress in Li-ion technology over the past two decades, while, on the other hand, a rather slow progress in EDLC technology. As pure “physical” devices, which do not involve any chemical or electrochemical transformations, any charge or mass transfer through the electrode-electrolyte interface, EDLC’s must demonstrate much faster charge/discharge operations and longer cycle life than any “chemical” batteries (Conway, 1999). Given this, EDLC devices can provide the key to a number of efficient power solutions that are mainly related with various backup systems to compensate short-term voltage surges or drops or with load leveling the batteries in various combined power sources. Low internal resistance can be one of key advantages of EDLC over all other types of energy storage devices since the round trip efficiency and power capability of the devices are inversely proportional to their internal resistance. Besides, low internal resistance predetermines the effectiveness of EDLC application in combined power supply units, wherein an EDLC device is connected with a battery either in parallel or in series. Additionally, high efficiency implies low heat generation and, hence, improved safety. This, accompanied by EDLC long life cycle and wide operation temperature range, can help them to clear their way to the market. To realize it, EDLC devices must clearly demonstrate much higher power capability than Li-ion or any other type of batteries, but this is not always the case. What are the reasons? 1.2 What Are the Hurdles to Overcome Carbon Based Electrochemical Double Layer Capacitors of Low Internal Resistance Yurii Maletin1,2, Volodymyr Strelko2, Natalia Stryzhakova1,2, Sergey Zelinsky1,2, Alexander B. Rozhenko1, Denis Gromadsky1, Vitaliy Volkov3, Sergey Tychina1,2, Oleg Gozhenko1,2 & Dmitry Drobny1,2 1 YUNASKO-Ukraine, Kiev, Ukraine 2 Institute for Sorption and Problems of Endoecology, National Academy of Science of Ukraine, Kiev, Ukraine 3 Institute of Chemical Physics, Russian Academy of Science, Chernogolovka, Moscow Region, Russia Correspondence: Yurii Maletin, YUNASKO-Ukraine, Kiev, Ukraine. Tel: 380-44-450-4043. E-mail: ymaletin@yunasko.com Yurii Maletin1,2, Volodymyr Strelko2, Natalia Stryzhakova1,2, Sergey Zelinsky1,2, Alexander B. Rozhenko1, Denis Gromadsky1, Vitaliy Volkov3, Sergey Tychina1,2, Oleg Gozhenko1,2 & Dmitry Drobny1,2 1 YUNASKO-Ukraine, Kiev, Ukraine 1.3 Our Approach to Solve the Problem According to well-known equations (Bard & Faulkner, 2001, p.137) the electrolyte conductivity is proportional to mobility or to diffusion coefficients of the corresponding ions. On the other hand, there is no potential gradient in narrow pores (if the pore width is close to the Debye length, or to 1–2 nm in concentrated electrolytes, which are normally used in EDLC technology), and therefore, diffusion is the only driving force for charge-discharge processes in nanoporous EDLC electrodes (Maletin et al., 2006). Bearing that in mind, in the present study two known independent experimental techniques based on NMR or electrochemical measurements have been modified and used to measure the diffusion coefficients of the electrolyte inside the carbon nanopores. Since cost is another important issue that hinders the EDLC way to the market, in the present study we are mostly focused on the low cost nanoporous carbons based on natural carbonaceous materials, e.g., coconut shell. In some cases the surface of initial carbons was doped with N-heteroatoms as was offered by Strelko, Stryzhakova, Gozhenko, and Maletin (2009). 1.2 What Are the Hurdles to Overcome The most substantial contributors to the EDLC internal resistance are (see a simplified equivalent circuit in 156 Energy and Environment Research Vol. 3, No. 2; 2013 Energy and Environment Research Vol. 3, No. 2; 2013 Energy and Environment Research Vol. 3, No. 2; 2013 www.ccsenet.org/eer sketch (1) below): contact resistance between Al current collector and active carbon electrode (RAl-C); ohmic resistance of active carbon electrode layer (RC); electrolyte resistance in the electrode nanopores (REl-in-pores); and electrolyte resistance in the bulk solution including electrode and separator macropores (REl-in-bulk). (1) High contact resistance, RAl-C, can result from the native oxide film on the aluminum surface and its effect on EDLC performance is thoroughly discussed in our previous work (Maletin et al., 2008) and also in Section 4 below. (1) High contact resistance, RAl-C, can result from the native oxide film on the aluminum surface and its effect on EDLC performance is thoroughly discussed in our previous work (Maletin et al., 2008) and also in Section 4 below. Ohmic resistance of the active electrode layer, RC, can substantially be reduced by adding a small amount (2–5% wt.) of conductive additives such as carbon black or graphite. On the other hand, it has been found (Maletin et al., 2008; Maletin et al., 2012) that the electrolyte conductivity, though being high enough in the bulk solution, can significantly be reduced in the electrode nanopores, and this can result in the lion’s share of an unexpectedly high internal resistance of EDLC devices. Experimental study of this phenomenon is the main subject of the present paper. 2.3 Diffusion Coefficient Measurements For NMR diffusion measurements the carbon powders listed above were impregnated with ethyl trimethyl ammonium tetrafluoroborate (EtMe3NBF4) dissolved in either acetonitrile (CH3CN) or acetonitrile-d3 (CD3CN) followed by placing each powder into a 5 mm standard NMR tube. All 1H NMR diffusion measurements have been carried out using a Bruker AVANCE 400 spectrometer equipped with the wide-bore magnet. A 5 mm “diff60” diffusion probe head (Bruker) with gradient coils in Z direction was used to generate magnetic field gradient. All experiments were performed at 25 °C. The 90° pulse lengths (14.5–18.5 s) were determined for every sample using the standard routine. The T1 relaxation times were measured by standard inversion-recovery. The T2 relaxation times were determined with the Carr-Purcell-Meiboom-Gill (CPMG) standard sequence. The PGSTE “Pulse Gradient Stimulated Echo” method (DifSte Bruker standard pulse sequence) described by Tanner (1970) and by Cohen, Avram, & Frish (2005) was used for the diffusion measurements. The “diff” automated routine was employed to prepare all the parameters for the diffusion experiments. The square field gradient pulses (δ) and delay between two first radiofrequency pulses () were chosen short enough (0.4 ms and 1.1 ms, respectively) in order to measure the diffusion coefficients for samples with very short (< 2 ms) T2 relaxation times. 32 intensity points were acquired and number of transitions was between 8 and 64, depending on the signal intensity and resulting signal-to-noise ratio. The data were processed in the standard way using the T1/T2 routine and approximating the resulting curve on two different values of diffusion coefficients. The resulting values of self-diffusion coefficients were averaged over three measurements. The 19F NMR diffusion measurements have been carried out with the use of AVANCE-III-400 spectrometer following the same procedure as described above for 1H self-diffusion experiments. As an alternative independent method for diffusion coefficient measurements, a version of the well-known rotating disc electrode (RDE) method (Bard & Faulkner, 2001, p.335) has also been used. The disc electrode (BASi RDE-1) was made of a graphite rod of 3 mm in diameter and covered with a nanoporous carbon layer of 40 micron thick. Rotation rates of 500, 750, 1000, 1250 and 1500 rpm were chosen for measurements. As a reversible redox-pair for RDE measurements, the ferrocene molecule/ferrocenium cation (Fc/Fc+) pair has been chosen since the Fc+ cation is similar by its size and insignificant solvation effect to tetraalkylammonium cations used in EDLC technology. 2.1 Design of EDLC Prototypes and Their Performance Measurements Two-electrode capacitor prototypes were used for performance measurements of various nanoporous carbons and EDLC design solutions. The electrodes were typically prepared by mixing the nanoporous carbon powder with PTFE suspension in water (the latter was used as a binder) until a homogeneous mixture was obtained. No conductive additives were normally added since the carbons selected in this work provided fairly low ohmic resistance, RC. The mixture was rolled to form sheets of 40–100 micron thick followed by cutting off the separate carbon electrodes. The active carbon electrodes thus obtained had their geometric surface area of 15 cm2 each and contained 93% of carbon and 7% of PTFE binder. They were then applied onto electric-spark treated aluminum foil (Maletin et al., 2008) used as a current collector of 15 or 20 micron thick and dried at 220 C under vacuum for 6 hours. A couple of electrodes were then interleaved with a porous insulating sheet (separator) and placed into laminated aluminum shell. The prototypes thus fabricated were filled with 1.3 M Et3MeN (TEMA) BF4 in acetonitrile and sealed. All the assembly operations were carried out in a dry box. Larger EDLC devices comprising a number of positive and negative electrodes connected in parallel and forming a stack with the capacitance between 400 and 1500 F were assembled at Yunasko Pilot Plant according to the same technology. Cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) measurements were carried out with the help of Voltalab-80 PGZ-402 unit. Galvanostatic charge-discharge cycling with the help of Arbin BT-2000 testing unit was also used to measure the capacitance and internal resistance of large EDLC prototypes. The CV measurements were mostly carried out within the voltage range of 0–3 V with the scan rate of 10 mV.s-1. In some cases the voltage range was extended to 3.5 V. Also in some cases three-electrode CV measurements were used to study the behavior of various EDLC components in either positive or negative voltage range. The current loads between 0.1 and 1.0 A.F-1 were used for galvanostatic charge-discharge cycling of the prototypes of different size/capacitance with the sampling rate of 10 ms. All the measurements were carried out at 25 C except special life cycle tests that were carried out at 60 C. 157 Energy and Environment Research Vol. 3, No. 2; 2013 Vol. 3, No. 2.3 Diffusion Coefficient Measurements For RDE diffusion current measurements an Fc sample of 4 mmol.dm-3 was dissolved in 1.3 M Et3MeNBF4 solution in acetonitrile. The potential scan rate in CV measurements was 0.5 mV.s-1. The Ag,AgCl/KCl (1 M) reference electrode was connected with the working electrolyte through a salt bridge. 2.1 Design of EDLC Prototypes and Their Performance Measurements 2; 2013 www.ccsenet.org/eer The internal resistance (Rin) and capacitance (C) values were calculated from galvanostatic cycling results in accordance with the FreedomCAR Ultracapacitor Test Manual (2004). 2.2 Study of Nanoporous Carbon Structure and Surface Chemistry The following nanoporous carbon powders have been chosen for this study: A. ZL-302 (Huzhou Sensheng Activated Carbon Co., Ltd); B. ZL-302-N (ZL-302 carbon powder thermally treated with melamine and containing 15 atomic % of nitrogen on its surface); B. ZL-302-N (ZL-302 carbon powder thermally treated with melamine and containing 15 atomic % of nitrogen on its surface); C. NY1151 (Kuraray Chemical Co.,Ltd); C. NY1151 (Kuraray Chemical Co.,Ltd); D. YP80F (Kuraray Chemical Co.,Ltd); E. YP50F (Kuraray Chemical Co.,Ltd); F. HDLC 20B STUW (Haycarb PLC); G. NC2-1E (EnerG2 Technologies Inc.); H. P2-15 (EnerG2 Technologies Inc.); I. Y-Carbon (Y-Carbon Inc.). The porous structure of carbon materials has been studied with the help of transmission electron microscopy (TEM) with the use of Jeol JEM-2100F, and also from nitrogen adsorption/desorption isotherms at 77 K using a Nova 2200e Surface Area & Pore Size Analyser (Quantachrome Instruments). The concentration of nitrogen heteroatoms on carbon surface has been measured with the help of XPS with the use of KRATOS-800XPS, energy resolution of 1.2 eV, Κα (Al), hν=1486.6 eV, spectral data being treated with the help of XPSPeak 4.0. 3.1 Study of the Carbon Pore Structure 3.2 Electrochemical Measurements Further, the carbon materials listed in Section 2.2 were studied with the use of CV method. A special reference electrode was developed to be used in organic electrolytes, the electrode potential being stable over practically unlimited time and giving a chance to register CV and charge-discharge curves in both positive and negative ranges from the equilibrium potential of carbon material. For the sake of generality, we use the Li/Li+ potential as “zero point” in our scale of potentials. A typical example of CV measurements with the use of three-electrode cell is illustrated in Figure 3-a, wherein a good behaviour of carbon materials selected for positive or negative electrode can be seen in a wide potential range. It should also be noted that a carbon material selected for the positive electrode may be different from that selected for the negative electrode to keep the electrode potential within the certain range when assembling the EDLC. As a result, a CV curve of an EDLC prototype assembled with the use of thus selected electrodes is presented in Figure 3-b. Figure 3-a. Cyclic voltammery curves in positive (right) and negative (left) ranges from the equilibrium potential of nanoporous carbon as a working electrode in a three-electrode cell (room temperature; electrolyte: 1.3 M TEMA BF4 in acetonitrile; scan rate: 10 mV.s-1) Figure 3-a. Cyclic voltammery curves in positive (right) and negative (left) ranges from the equilibrium potential of nanoporous carbon as a working electrode in a three-electrode cell (room temperature; electrolyte: 1.3 M TEMA BF4 in acetonitrile; scan rate: 10 mV.s-1) Figure 3-b. Cyclic voltammery curve of an EDLC prototype with positive and negative electrodes as in Figure 3-a, each electrode area being of 15 cm2 (room temperature; electrolyte: 1.3 M TEMA BF4 in acetonitrile; scan rate: 10 mV.s-1) Figure 3-a. Cyclic voltammery curves in positive (right) and negative (left) ranges from the equilibrium potential of nanoporous carbon as a working electrode in a three-electrode cell (room temperature; electrolyte: 1.3 M TEMA BF4 in acetonitrile; scan rate: 10 mV.s-1) Figure 3-b. Cyclic voltammery curve of an EDLC prototype with positive and negative electrodes as in Figure 3-a, each electrode area being of 15 cm2 (room temperature; electrolyte: 1.3 M TEMA BF4 in acetonitrile; scan rate: 10 mV.s-1) Figure 3-b. 3. Results Most of the carbon materials listed in Section 2.2 have been selected so that they have their pore size in the range 158 Energy and Environment Research Vol. 3, No. 2; 2013 www.ccsenet.org/eer between 1 and 10 nm with a large portion concentrated between 1 and 3 nm. Some examples can be seen in Figure 1 wherein the pore size distribution for three promising carbons is illustrated. One more criterion for preliminary carbon material selection in this work is the presence of mostly shallow slit-shaped pores or just shear cracks of graphene layers in the carbon matrix – see, e.g., in Figure 2, which presents the TEM image for HDLC 20B STUW (F) carbon powder. Figure 1. Pore size distribution obtained from nitrogen adsorption/desorption isotherms with the use of DFT calculations for some of the tested carbon powders (as listed in Section 2.2) Figure 1. Pore size distribution obtained from nitrogen adsorption/desorption isotherms with the use of DFT calculations for some of the tested carbon powders (as listed in Section 2.2) 159 159 Energy and Environment Research Vol. 3, No. 2; 2013 www.ccsenet.org/eer 3.2 Electrochemical Measurements Cyclic voltammery curve of an EDLC prototype with positive and negative electrodes as in Figure 3-a, each electrode area being of 15 cm2 (room temperature; electrolyte: 1.3 M TEMA BF4 in acetonitrile; scan rate: 10 mV.s-1) Figure 4 illustrates the EIS results (Nyquist plots) for three different designs of EDLC devices. Curve 1 illustrates the case of poor electrical contact between the aluminium current collector and active electrode layer resulting in high contact resistance. Curve 2 reflects the design with low contact resistance though with the electrochemical system (i.e. nanoporous carbon electrodes and organic electrolyte) non-optimized properly. The optimization can be achieved due to CV measurements like those presented in Figure 3 above and also due to diffusion coefficient measurements described below. The Nyquist plot for the optimized design is presented by Curve 3 in Figure 4. Figure 4 illustrates the EIS results (Nyquist plots) for three different designs of EDLC devices. Curve 1 illustrates the case of poor electrical contact between the aluminium current collector and active electrode layer resulting in high contact resistance. Curve 2 reflects the design with low contact resistance though with the electrochemical system (i.e. nanoporous carbon electrodes and organic electrolyte) non-optimized properly. The optimization can be achieved due to CV measurements like those presented in Figure 3 above and also due to diffusion coefficient measurements described below. The Nyquist plot for the optimized design is presented by Curve 3 in Figure 4. 160 Energy and Environment Research Vol. 3, No. 2; 2013 www.ccsenet.org/eer Figure 4. Nyquist plots for EDLC prototypes: 1 - with poor contact between the current collector and active carbon layer (high contact resistance); 2 - with improved contact resistance but non-optimized electrochemical system; 3 - with fully optimized design Figure 4. Nyquist plots for EDLC prototypes: 1 - with poor contact between the current collector and active carbon layer (high contact resistance); 2 - with improved contact resistance but non-optimized electrochemical system; 3 - with fully optimized design 3.3 In-Pore Diffusion Coefficient Measurements After impregnating carbon powders with electrolyte, the electrolyte translational self-diffusion coefficients have been calculated from the attenuation of the NMR signals due to the application of the gradient pulses of the constant lengths but various strengths using Equation (2) (Price, 1997): 0 0 2 2 2 3 I I exp D g                     (2) (2) where I and I0 are the NMR signal intensities in the presence and in the absence of the gradient respectively, γ is the gyromagnetic ratio of the nucleus under observation, δ and g are the duration and the strength of the applied gradient pulse, respectively, D0 is the self-diffusion coefficient, and Δ is the time interval between the two successive gradient pulses. It has been found that the theoretical curve practically coincides with experimental values if the attenuation of NMR signals is expressed as a sum of two Equations (2) with different D0 values, a quickly diffusing one (~ 10-9 m2 s-1) and slowly diffusing one (~ 10-10 m2 s-1). This can reflect the fast diffusion in bulk solution or in macropores and slow diffusion in nanopores. The resulting values of thus found effective diffusion coefficients have been averaged over three measurements and are plotted in Figures 5 and 6 (for BF4 - anions and EtMe3N+ cations, respectively) for various nanoporous carbons vs. the internal resistance of EDLC prototypes comprising the same carbons in electrodes. As can be seen from these figures, there is a fairly good correlation between diffusion coefficients of electrolyte ions in carbon nanopores and the internal resistance of the corresponding EDLC prototypes. However, it should be noted that some carbons cannot be used for NMR measurements because of the very short correlation time of electrolytes in their pores resulting in unreliable diffusion coefficient evaluations. Therefore, we have also developed another method to measure the diffusion coefficients of electrolyte ions in carbon nanopores, namely, a version of the well-known RDE method-see Section 2.3 for details. The behaviour of porous rotating disc electrode (PRDE) has recently been studied by Bonnecaze, Mano, Nam, and Heller (2007). To evaluate the diffusion coefficients from PRDE measurements we have used the Levich equation: (3) 161 Energy and Environment Research Vol. 3, No. 3.3 In-Pore Diffusion Coefficient Measurements 2; 2013 www.ccsenet.org/eer where i is the limiting diffusion current value, n is the number of electrons in the electrode semi-reaction, С0 is the concentration of the reacting species, F is the Faraday constant, D is the diffusion coefficient, ν is the kinematic viscosity, and ω is the rotation rate. Figure 5. Internal resistance of EDLC prototypes comprising electrodes of different nanoporous carbons, as listed in Section 2.2, vs. the diffusion coefficients of BF4 - anions in those carbons (19F NMR measurements) Figure 5. Internal resistance of EDLC prototypes comprising electrodes of different nanoporous carbons, as listed in Section 2.2, vs. the diffusion coefficients of BF4 - anions in those carbons (19F NMR measurements) Figure 6. Internal resistance of EDLC prototypes comprising electrodes of different nanoporous carbons, as listed in Section 2.2, vs. the diffusion coefficients of EtMe3N+ cations in those carbons (1H NMR measurements) The results of PRDE measurements of the electrolyte diffusion in various nanoporous carbons, which are listed in Section 2.2, are plotted in Figure 7 vs. the internal resistance of EDLC prototypes comprising the same Figure 6. Internal resistance of EDLC prototypes comprising electrodes of different nanoporous carbons, as listed in Section 2.2, vs. the diffusion coefficients of EtMe3N+ cations in those carbons (1H NMR measurements) The results of PRDE measurements of the electrolyte diffusion in various nanoporous carbons, which are listed in Section 2.2, are plotted in Figure 7 vs. the internal resistance of EDLC prototypes comprising the same The results of PRDE measurements of the electrolyte diffusion in various nanoporous carbons, which are listed in Section 2.2, are plotted in Figure 7 vs. the internal resistance of EDLC prototypes comprising the same 162 Energy and Environment Research Vol. 3, No. 2; 2013 www.ccsenet.org/eer carbons in electrodes. Both NMR and PRDE methods, as illustrated in Figures 5–7, have been employed to select the low resistance electrodes for EDLC manufacture, and the results for large EDLC prototypes are briefly listed in Table 1 below. Table 1. Performance of Yunasko EDLC devices (rated voltage 2.7 V) Capacitance, F Internal resistance, mOhm Time constant (RC), s Spec. energy (CU2/2), W.h kg-1 Spec. power (@95% eff.), kW kg-1 Max. spec. 3.3 In-Pore Diffusion Coefficient Measurements power, kW kg-1 480a 1200a,b 1500b 0.20 0.10 0.09 0.10 0.12 0.14 4.9 5.3 6.1 10.2 8.9 9.1 91 79 81 a) Also tested at the Institute of Transportation Studies, Davis, CA; b) Also tested at JME Inc., Cleveland, OH. Table 1. Performance of Yunasko EDLC devices (rated voltage 2.7 V) Figure 7. Correlation between EDLC internal resistance and diffusion coefficients of ferrocenium-cation in pores of various carbons listed in Section 2.2 and used in electrodes (PRDE measurements) Figure 7. Correlation between EDLC internal resistance and diffusion coefficients of ferrocenium-cation in pores of various carbons listed in Section 2.2 and used in electrodes (PRDE measurements) The cells listed in Table 1 were also tested for their life cycle according to IEC 62391 endurance test procedure, and after holding the cells for at least 2000 hours at 60 C and 2.7 V they demonstrated the capacitance retention within 70% and an increase in internal resistance not exceeding 100%. The cells listed in Table 1 were also tested for their life cycle according to IEC 62391 endurance test procedure, and after holding the cells for at least 2000 hours at 60 C and 2.7 V they demonstrated the capacitance retention within 70% and an increase in internal resistance not exceeding 100%. 4. Discussion Though being beyond the scope of this paper, some methods to reduce the RAl-C contact resistance (see sketch (1)) have recently been disclosed by Maletin et al. (2008). The electrical contact between the active electrode layer and aluminum current collector can be significantly improved due to conductive carbon particles to be locally and individually fused into the current collector surface. New versions of those methods, in particular, the electric spark treatment with the use of graphite electrode are effectively employed in our current technology resulting in a very low contact resistance value, normally not exceeding 10 mOhm *cm2. As regards the choice of nanoporous carbon materials to be used in EDLC electrodes, the materials having shallow slit-shaped pores with their width between 1 and 3 nm (see examples in Figures 1 and 2) look preferable for EDLC electrode application. From our experience the carbons comprising larger mesopores have significantly lower surface area while those with pores predominantly below 1 nm demonstrate too high resistance to be effectively used in EDLC devices. 163 Energy and Environment Research Vol. 3, No. 2; 2013 www.ccsenet.org/eer CV measurements with the use of a three-electrode cell and EIS measurements give a chance to optimize the electrochemical system design in order to improve the EDLC performance. Similar CV and dilatometric studies for different carbon materials and electrolytes were carried out by Hahn, Barbieri, Gallay, & Kötz (2006), and as have now been shown in our work the optimized EDLC prototypes demonstrate a fairly good rectangular shape of CV curves with no visible faradaic processes within a voltage range as wide as 2.9 V and also the lowest resistance and close to ideal vertical line in Nyquist plots at low frequencies– see Figures 3 and 4. As was found in our NMR measurements, the T1 and T2 relaxation times of various nuclei of the electrolyte species (1H, 11B, 19F) change significantly when the electrolyte enters the carbon pores, and this can reflect a strong interaction of electrolyte species with the carbon matrix – see also a similar conclusion made by Price (1997). This phenomenon and some other NMR results will be discussed in more detail in our forthcoming publications, while here we would like to focus on the diffusion coefficient values since they illustrate the electrolyte dynamics in carbon nanopores. 4. Discussion Of course, the D0 values measured for the fluid phase in nanopores are not the true self-diffusion coefficients, but rather the effective molecular diffusivities Deff (Price, 1997). Nevertheless, as can be seen from Figures 5–7, there is a remarkable correlation between the internal resistance of EDLC device and ion diffusion in pores of the corresponding carbon electrode material. Absolute values of diffusion coefficients of EtMe3N+ and Fc+ cations, though being measured by different methods, are close (cf. Figures 6 and 7), and the difference can be accounted for slightly different size of these cations. It is also worth noting that the diffusion coefficient of Fc+ cations, when being measured with the use of a flat graphite electrode, is 1.01×10-9 m2.s-1, which significantly exceeds the values in carbon nanopores presented in Figure 7. It should also be noted that the positive and negative electrodes are typically different to best match the different mobility of anions and cations in their pores. Besides, it has been found that the diffusion coefficients of electrolytes can be increased and, correspondingly, the pore resistance can be reduced due to doping the carbon surface with nitrogen atoms (Strelko et al., 2009). This effect can be accounted for reducing the interaction between the carbon matrix and electrolyte ions. The test results listed in Table 1 and also verified by independent experts clearly show a very low internal resistance of EDLC devices based on optimized electrochemical systems with their RC-constant values being between 0.1 and 0.2 s and maximum power density reaching 80–90 kW/kg. These RC-constant values are lower by a factor of 2–3, and correspondingly, power capabilities of EDLC devices presented in Table 1 are by far higher than those of the best competing devices (Miller, Butler, Ryan, & McNeal, 2013). Very low internal resistance of EDLC devices thus fabricated makes it possible to avoid significant heating during the operation, even under high load conditions. As an example, a module of 16 V comprising 6 single cells of 1200 F each connected in series has the internal resistance below 1 mOhm and after continuous charge/discharge cycling with the current value of 200 A running over 8 hours demonstrates an increase in temperature of about 10–12 °C only. References Bard, A. J., & Faulkner, L. R. (2001). Electrochemical Methods. Fundamentals and Applications (2nd ed). New York: Wiley. Bonnecaze, R. T., Mano, N., Nam, B., & Heller, A. (2007). On the behavior of the porous rotating disk electrode. Journal of The Electrochemical Society, 154(2), F44-F47. http://dx.doi.org/10.1149/1.2403082 Cohen, Y., Avram, L., & Frish, L. (2005). Diffusion NMR spectroscopy in supramolecular and combinatorial chemistry: an old parameter—new insights. Angewandte Chemie International Edition, 44(4), 520-554. http://dx.doi.org/10.1002/anie.200300637 Conway, B. E. (1999). Electrochemical Supercapacitors: Scientific Fundamentals and Technological Applications. New-York: Kluwer-Plenum Press. http://dx.doi.org/10.1007/978-1-4757-3058-6 FreedomCAR Ultracapacitor Test Manual. (2004). Idaho National Engineering Laboratory Report. DOE/NE-ID-11173. Hahn, M., Barbieri, O., Gallay, R., & Kötz, R. (2006). A dilatometric study of the voltage limitation of carbonaceous electrodes in aprotic EDLC type electrolytes by charge-induced strain. Carbon, 44(12), 2523-2533. http://dx.doi.org/10.1016/j.carbon.2006.05.002 Maletin, Y., Novak, P., Shembel, E., Izotov, V., Strizhakova, N., Mironova, A., ... & Podmogilny, S. (2006). Matching the nanoporous carbon electrodes and organic electrolytes in double layer capacitors. Applied Physics A, 82(4), 653-657. http://dx.doi.org/10.1007/s00339-005-3416-9 164 Energy and Environment Research Vol. 3, No. 2; 2013 www.ccsenet.org/eer Maletin, Y., Podmogilny, S., Stryzhakova, N., Mironova, A., Danilin, V., & Maletin, A. (2008). Electrochemical double layer capacitor. US Patent Appl No. 20080151472. Maletin, Y., Stryzhakova, N., Zelinsky, S., Gromadsky, D., Tychina, S., & Drobny, D. (2012). Can the best performance and improved design open the door to EDLC market? Proc. 22nd Internat. Seminar on Double Layer Capacitor and Hybrid Energy Storage Devices. Deerfield Beach, FL, 180-185. Miller, J. R., Butler, S. M., Ryan, D. M., & McNeal, S. (2013). Property, performance, and life of today's large-format electrochemical capacitors. Proc. 3rd European Advanced Automotive Battery Conference, Strasbourg. Retrieved from http://us1.campaign-archive1.com/?u=84cc935cd75c22a368d1cd12e&id=31a3699821&e=193f657ac6 us1.campaign-archive1.com/?u=84cc935cd75c22a368d1cd12e&id=31a3699821&e=193f657ac6 Price, W. S. (1997). Pulsed‐field gradient nuclear magnetic resonance as a tool for studying translational diffusion: Part 1. Basic theory. Concepts in magnetic resonance, 9(5), 299-336. http://dx.doi.org/10.1002/(SICI)1099-0534(1997)9:5<299::AID-CMR2>3.0.CO;2-U Strelko, V., Stryzhakova, N., Gozhenko, O., & Maletin, Y. (2009). N- and P-doped carbons as electrode materials. Proc. 19th Internat. Seminar on Double Layer Capacitor and Hybrid Energy Storage Devices. Deerfield Beach, FL, 115-123. Tanner, J. E. (1970). Use of the stimulated echo in NMR diffusion studies. The Journal of Chemical Physics, 52, 2523. http://dx.doi.org/10.1063/1.1673336 Copyrights Copyright for this article is retained by the author(s), with first publication rights granted to the journa Copyright for this article is retained by the author(s), with first publication rights granted to the journal. This is an open-access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/). 165

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