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https://openalex.org/W1515466761	https://genomebiology.biomedcentral.com/counter/pdf/10.1186/gb-2006-7-12-r120	English	null	Gene function and expression level influence the insertion/fixation dynamics of distinct transposon families in mammalian introns.	GenomeBiology.com	2,006	cc-by	13,022	Correspondence: Uberto Pozzoli. Email: uberto.pozzoli@bp.lnf.it Received: 31 July 2006 Revised: 25 October 2006 Accepted: 20 December 2006 Published: 20 December 2006 The electronic version of this article is the complete one and can be found online at http://genomebiology.com/2006/7/12/R120 © 2006 Sironi 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. D namics of mammalian transposable elements <p>An anal sis of h mans and mo se genomes indicates that gene f nction e pression le el and seq ence conser ation infl ence trans posable elements insertion/fi ation in mammalian introns </p> ; 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. Dynamics of mammalian transposable elements <p>An analysis of humans and mouse genomes indicates that gene function expression level and sequence conservation influence trans posable elements insertion/fixation in mammalian introns </p> Open Access 2006 Sironi et al. Volume 7, Issue 12, Article R120 Research Gene function and expression level influence the insertion/fixation dynamics of distinct transposon families in mammalian introns Manuela Sironi, Giorgia Menozzi, Giacomo P Comi†, Matteo Cereda, Rachele Cagliani, Nereo Bresolin† and Uberto Pozzoli Open Access Addresses: Scientific Institute IRCCS E Medea, Bioinformatic Lab, Via don L Monza, 23842 Bosisio Parini (LC), Italy. †Dino Ferrari Centre, Department of Neurological Sciences, University of Milan, IRCCS Ospedale Maggiore Policlinico, Mangiagalli and Regina Elena Foundation, 20100 Milan, Italy. Correspondence: Uberto Pozzoli. Email: uberto.pozzoli@bp.lnf.it Abstract Background: Transposable elements (TEs) represent more than 45% of the human and mouse genomes. Both parasitic and mutualistic features have been shown to apply to the host-TE relationship but a comprehensive scenario of the forces driving TE fixation within mammalian genes is still missing. Results: We show that intronic multispecies conserved sequences (MCSs) have been affecting TE integration frequency over time. We verify that a selective economizing pressure has been acting on TEs to decrease their frequency in highly expressed genes. After correcting for GC content, MCS density and intron size, we identified TE-enriched and TE-depleted gene categories. In addition to developmental regulators and transcription factors, TE-depleted regions encompass loci that might require subtle regulation of transcript levels or precise activation timing, such as growth factors, cytokines, hormones, and genes involved in the immune response. The latter, despite having reduced frequencies of most TE types, are significantly enriched in mammalian-wide interspersed repeats (MIRs). Analysis of orthologous genes indicated that MIR over-representation also occurs in dog and opossum immune response genes, suggesting, given the partially independent origin of MIR sequences in eutheria and metatheria, the evolutionary conservation of a specific function for MIRs located in these loci. Consistently, the core MIR sequence is over-represented in defense response genes compared to the background intronic frequency. Conclusion: Our data indicate that gene function, expression level, and sequence conservation influence TE insertion/fixation in mammalian introns. Moreover, we provide the first report showing that a specific TE family is evolutionarily associated with a gene function category. Background ments. These sequences have been estimated to represent more than 50% of the human genome [1]. In particular, the great majority of human interspersed repeats derive from ac g ou d It is widely recognized that a large fraction of mammalian genomic DNA is accounted for by interspersed repeated ele- Genome Biology 2006, 7:R120 Genome Biology 2006, 7:R120 R120.2 Genome Biology 2006, Volume 7, Issue 12, Article R120 Sironi et al. http://genomebiology.com/2006/7/12/R120 transposable elements (TEs). Four major classes of mamma- lian TEs have been identified in mammals: long interspersed elements (LINEs), short interspersed elements (SINEs), LTR retrotrasposons and DNA transposons. sequence conservation, function and expression level shape TE representation in human genes. Interestingly, we found evidence that a subset of loci involved in immune responses are enriched with MIR sequences; analysis of opossum orthologous genes, as well as of MIR frequency profiles, indi- cated that these TEs might serve a specific function in these loci. Overall, TEs cover more than 45% of the human genome [1] but, most probably, another huge portion of human DNA is accounted for by ancient transposons that have diverged too far to be recognized as such. Indeed, different TE subtypes have been active over different evolutionary periods [2], implying that multiple copies of propagating elements accu- mulated over discrete time periods depending on the pres- ence of an active source. The result of this age-dependent accumulation is that many TEs are restricted to closely related species: about a half of human repeats cannot be iden- tified in genomes of other than primate origin [3]; similarly, most repeats that can be detected in mouse DNA are specific to rodents. Nonetheless, repeated sequences that are com- mon to all mammalian genomes exist as they probably ampli- fied before the mammalian radiation [3]. Results TE distribution varies with gene class or function We wished to verify whether different TE types might be dif- ferentially represented depending on gene function. TE fre- quency varies with intron length [24] and GC percentage [1]. Moreover, in line with previous findings [24], we show that, although differences exist depending on MCS and TE age, conserved sequences have an overall negative effect on TE fix- ation frequency (Additional data file 1). For each TE type we therefore performed multiple regression analysis on TE number using intronic GC percentage, intron length and con- served sequence length as independent variables. The fitted values were then used to predict the expected TE number per intron (nTEiexp). For each gene, the TE normalized abun- dance (Tena) was calculated as follows: Once considered as merely junk DNA, it is now widely recog- nized that interspersed repeats have been playing a major role in genome structure evolution as well as having an impact on increased protein variability [2,4-8] and gene regulation [9]. Also, recent evidence has suggested that LINE elements have been influencing genome-wide regulation of gene expression [10] and possibly imprinting [11], while several reports [12- 16] showed that specific TEs in noncoding DNA regions have been actively preserved among multiple species during evolu- tion. Still, these observations do not contradict the 'selfish DNA' concept, regarding TEs as parasitic elements that rely more on their replication efficiency than on providing selec- tive advantage to their host [17-19]; rather, evidence of selec- tive benefits offered by TEs indicate that these elements have, in some instances, been 'domesticated' [20] or recruited to serve their host, a process also referred to as exaptation [21]. Several studies have suggested that TE integrations have been subjected to purifying selection to limit the genetic load imposed on their host. For example, genetic damage caused by LINE retrotransposition and ectopic recombination has been hypothesized to be responsible for selection against these elements within human loci [22]. Also, LINE and LTR elements have been reported to be underrepresented in prox- imity to and within genes [23], probably as a cause of their interference with regulatory processes. where nTEiobs is the observed number of TEs per intron. These calculations were performed for all TE families in both human and mouse. (Mean igTEna in contributing genes - mean igTEna in all genes)/\|mean igTEna in all genes\| Results nTEi nTEi nTEi nTEi obs i gene i gene obs i gene ∈ ∈ ∈ ∑ ∑ ∑ − ⎛ ⎝ ⎜ ⎜ ⎞ ⎠ ⎟ ⎟ − exp exp i gene ∈∑ ⎛ ⎝ ⎜ ⎜ ⎞ ⎠ ⎟ ⎟ where nTEiobs is the observed number of TEs per intron. These calculations were performed for all TE families in both human and mouse. where nTEiobs is the observed number of TEs per intron. These calculations were performed for all TE families in both human and mouse. For each TE family, genes displaying three times more or less TE than expected (TEna > 0.5 or TEna < -0.5) were classified as TE-rich or TE-poor, respectively. We next used GeneMerge [25] to retrieve significant associa- tions; database annotations for the three categories desig- nated by the Gene Ontology (GO) Consortium (molecular function, biological process and cellular component) were employed. Correction for multiple tests was applied to all sta- tistical analyses. For each significant GO term retrieved, genes that are present in the study set and associate (there- fore contribute) to the term are designated as 'contributing genes'. We also calculated MCS density and intergenic TE fre- quency of contributing genes. In particular, for intergenic sequences, TEna (igTEna) was calculated as described for introns; for contributing gene sets the fractional igTEna devi- ation was then calculated as: In mammals the great majority of genes are interrupted by introns that usually outsize coding sequences by several fold. Similar to TEs, intervening regions were initially regarded as scrap DNA before being recognized as fundamental elements in the evolution of living organisms. TEs are abundant within intronic regions as well as in 5' and 3' intergenic spacers; yet, a comprehensive analysis of the forces driving TE insertion, fixation and maintenance within mammalian genes has still not been carried out. Here we show that gene features such as Genome Biology 2006, 7:R120 Genome Biology 2006, Volume 7, Issue 12, Article R120 Sironi et al. R120.3 http://genomebiology.com/2006/7/12/R120 Similarly, fractional MCS density deviation was calculated for contributing gene sets. mammals, MIR density in these genes is not accounted for by fewer integrations of younger TEs since MIR frequency remains significantly higher than the average when calculated on TE-free (unique) intron size. Results The second functional category is represented by genes cod- ing for cytokines/growth factors/hormones and, more gener- ally, receptor ligands: these genes do not have, as a whole, higher than average intron conservation and, with the excep- tion of LTR-poor genes, tend to have low igTEna. The last cat- egory (not present among Alu-poor genes) is accounted for by structural molecules, mainly represented by ribosomal pro- teins. These genes have extremely low MCS densities and igTEna. These same associations were retrieved for mouse genes (supplementary Table 1 in Additional data file 2), although no GO term was significantly associated with L1- depleted mouse genes. We next wished to verify whether these genes also had higher frequencies of other ancestral TEs, namely L2s and DNA transposons. The frequencies of these elements were calcu- lated on TE-free intron size and no significant differences were identified in either human or mouse when MIR-rich genes involved in immune responses were compared to all genes (not shown); this finding suggests that relaxation of selective constraints allowing accumulation of ancestral TE insertions is not responsible for MIR over-representation in these genes. Conversely, MIR-rich ion channel introns also displayed significantly higher frequencies of both DNA trans- posons and L2s, indicating, therefore, that the relative enrich- ment in old TEs is not specific to MIRs. Significant associations were also identified with biological process GO terms. As expected [1,26] genes involved in mor- phogenesis/development were over-represented in most TE- poor groups and displayed extremely conserved intronic regions as well as few intergenic TEs (except for LTRs). Also, loci involved in immune defense/response to stimulus were found to be over-represented among TE-poor genes. These loci also have less TEs in their flanking regions and, on aver- age, low MCS densities. Consistently with molecular function GO term retrieval, genes involved in biological processes such as transcription and metabolism were found to be overrepre- sented among TE-poor groups. Again, similar findings were obtained when mouse genes (supplementary Table 1 in Addi- tional data file 2) were analyzed, although no biological proc- ess GO term was significantly over-represented among genes displaying low LINE or DNA transposon frequencies. We therefore wished to verify whether high MIR frequency in immune response genes also occurs in mammalian species other than human and mouse. We therefore analyzed MIR frequency in dog, as well as in our most distant extant mam- malian ancestors, namely metatherian. Results To this aim we searched both Canis familiaris and Monodelphis domestica (gray short-tailed opossum) annotation tables and retrieved dog/opossum genomic positions corresponding to human transcripts in our dataset. A total of 5,476 human genes could be located on the Monodelphis sequence (7,454 on the dog sequence) and, out of 85 MIR-rich immune response genes, 77 were identified in opossum (79 in dog). We then calculated the frequency of mammalian-wide MIRs within dog and opossum genes: in both species (Figure 1) immune response loci displayed significantly higher frequencies compared to the remaining genes (Wilcoxon rank sum test, p < 10-15 and 0.022 for dog and opossum, respectively). Interestingly, in addition to mammalian-wide MIR sequences, metatherian/ monotremata-specific MIR-related TEs are interspersed in the opossum genome. These latter are mainly accounted for by MON1 and MAR1 [3], and show 90% identity with the MIR core sequence [27]. Opossum immune response loci also Moreover, a relatively small set of genes involved in sexual reproduction/spermatogenesis were found to display lower than expected MIR frequencies (both in introns and inter- genic sequences) in humans but not in rodents. Results To gain further insight into this issue, we singled out all genes contributing to at least one GO term in Table 2 (85 genes) and searched for a murine ortholog in our mouse gene dataset; 61 best unique reciprocal orthologs were identified and their MIR density (calculated on unique intron sequence) was significantly higher (Wil- coxon rank sum test, p < 10-14) than the average (calculated on all murine genes in our dataset). The same procedure was applied to mouse MIR-rich genes contributing to GO terms in Table 3; again, human genes displayed significantly higher intronic MIR densities (Wilcoxon rank sum test, p < 10-14). The difference between human and mouse in GO terms asso- ciated with MIR-rich genes, therefore, results from the cut-off we used (TEna > 0.5, corresponding to three times more than expected) to define MIR-rich genes. Data concerning significant (Bonferroni-corrected p value < 0.01) GO associations are summarized in Table 1. Three main molecular function categories were found to be associated with genes displaying low TEna (for more than one TE family). The first one is accounted for by genes involved in nucleic acid binding and transcription; these loci have, on average, high intronic MCS densities and few TEs in their flanking regions. The second functional category is represented by genes cod- ing for cytokines/growth factors/hormones and, more gener- ally, receptor ligands: these genes do not have, as a whole, higher than average intron conservation and, with the excep- tion of LTR-poor genes, tend to have low igTEna. The last cat- egory (not present among Alu-poor genes) is accounted for by structural molecules, mainly represented by ribosomal pro- teins. These genes have extremely low MCS densities and igTEna. These same associations were retrieved for mouse genes (supplementary Table 1 in Additional data file 2), although no GO term was significantly associated with L1- depleted mouse genes. Data concerning significant (Bonferroni-corrected p value < 0.01) GO associations are summarized in Table 1. Three main molecular function categories were found to be associated with genes displaying low TEna (for more than one TE family). The first one is accounted for by genes involved in nucleic acid binding and transcription; these loci have, on average, high intronic MCS densities and few TEs in their flanking regions. TE-rich gene categories Genes displaying higher than expected TE frequencies were also identified for all repeat families, although they were less numerous than TE-poor genes. GO analysis retrieved signifi- cant associations (Bonferroni-corrected p value < 0.01) only for MIR-rich human genes (Table 2). GO terms associated with high MIR density differed between human (Table 2) and mouse (Table 3); in particular, MIR-rich genes belong to the immune response pathway in humans, while they mainly code for ion channels in mice. In both Genome Biology 2006, 7:R120 R120.4 Genome Biology 2006, Volume 7, Issue 12, Article R120 Sironi et al. http://genomebiology.com/2006/7/12/R120 Table 1 GO terms associated with TE-poor genes Under-represented TE type GO term Description Alu L1 L2 LTR DNA transp. Genome Biology 2006, 7:R120 TE-rich gene categories R120.5 http://genomebiology.com/2006/7/12/R120 GO:0007267 Cell-cell signaling 137 0.71 -0.27 162 0.69* 0.03 - - - - - - - - - - - - GO:0007166 Cell surface receptor linked signal transduction 161 0.29* -0.45* - - - - - - - - - - - - - - - GO:0007186 G-protein coupled receptor protein signaling pathway 93 0.17 -0.51* - - - - - - - - - - - - - - - GO:0006952 Defense response 172 0.13* -0.75* 217 -0.08* -0.16 202 -0.11* -0.19 259 0* 0.01 219 -0.04* -0.2 - - - GO:0006955 Immune response 155 0.17* -0.7* 201 -0.08* -0.19 - - - - - - 202 -0.05* -0.17 - - - GO:0050896 Response to stimulus 268 0.13 -0.61* - - - - - - - - - - - - - - - GO:0009607 Response to biotic stimulus 187 0.1* -0.69* 240 -0.1* -0.21 222 -0.14* -0.16 290 -0.05* 0 235 -0.07* -0.25 - - - GO:0009613 Response to pest, pathogen or parasite 99 -0.02* -0.8* - - - - - - - - - 127 -0.33* -0.13 - - - GO:0043207 Response to external biotic stimulus 106 -0.09* -0.86* - - - - - - - - - 134 -0.36* -0.17 - - - GO:0006817 Phosphate transport 27 -0.05 -0.39 - - - - - - - - - - - - - - - GO:0006820 Anion transport 41 0.03 -0.47 - - - - - - - - - - - - - - - GO:0015698 Inorganic anion transport 38 0.03 -0.49 - - - - - - - - - - - - - - - GO:0006350 Transcription - - - - - - - - - 386 1.22* -0.16 - - - 211 1.43* -0.43* GO:0045449 Regulation of transcription - - - - - - - - - 365 1.31* -0.15 - - - 198 1.53* -0.45* GO:0006351 Transcription, DNA- dependent - - - - - - - - - 369 1.25* -0.16 - - - 203 1.48* -0.45* GO:0006355 Regulation of transcription, DNA- dependent - - - - - - 267 1.38* -0.23 355 1.31* -0.16 - - - 196 1.53* -0.46* GO:0006139 Nucleobase, nucleoside, nucleotide and nucleic acid metabolism - - - - - - - - - - - - - - - 301 1.08 -0.23 GO:0019219 Regulation of nucleobase, nucleoside, nucleotide and nucleic acid metabolism - - - - - - - - - 371 1.29* -0.16 - - - 202 1.51* -0.46* GO:0019222 Regulation of metabolism - - - - - - 303 1.32* -0.2 409 1.24* -0.16 - - - 217 1.54* -0.38* GO:0006412 Protein biosynthesis - - - 144 -0.14 -0.34 - - - 179 -0.1* -0.48* - - - - - - GO:0050876 Reproductive physiological process 18 1.19 -0.76 - - - - - - - - - - - - - - - GO:0000003 Reproduction - - - - - - - - - - - - - - - 44 0.09* -0.38 GO:0019953 Sexual reproduction - - - - - - - - - - - - - - - 43 0.06* -0.38 GO:0007276 Gametogenesis - - - - - - - - - - - - - - - 39 0.14* -0.39 GO:0048232 Male gamete generation - - - - - - - - - - - - - - - 33 0.07* -0.05 GO:0007283 Spermatogenesis - - - - - - - - - - - - - - - 33 0.07* -0.05 Significant differences are marked with an asterisk. TE-rich gene categories MIR Molecular function N MCS IG N MCS IG N MCS IG N MCS IG N MCS IG N MCS IG GO:0003676 Nucleic acid binding - - - - - - 468 0.88* -0.44* 598 0.86* -0.27* - - - 327 1.07 -0.29* GO:0003677 DNA binding - - - - - - - - - 394 1.27* -0.17 - - - 219 1.6* -0.34* GO:0003723 RNA binding - - - - - - 131 0.08 -0.49* 153 0.13 -0.42* - - - 91 0.03 -0.12 GO:0003700 Transcription factor activity 138 2.45* -0.63* 171 1.9* -0.51* 160 2.1* -0.41* 220 1.82* -0.09 165 2.18* -0.65* 125 2.23* -0.76* GO:0030528 Transcription regulator activity 159 2.35* -0.59* - - - - - - 279 1.57* -0.1 - - - 152 2.04* -0.67* GO:0004871 Signal transducer activity 348 0.32 -0.45* - - - - - - - - - - - - - - - GO:0004888 Transmembrane receptor activity 138 0.23 -0.31 - - - - - - - - - - - - - - - GO:0005102 Receptor binding 137 0.5 -0.57* 170 0.29 0.03 149 0.24 0.14 192 0.33 0.2 155 0.32 -0.02 - - - GO:0001664 G-protein-coupled receptor binding - - - 25 -0.14 -0.23 - - - - - - 26 -0.16 -0.1 - - - GO:0008083 Growth factor activity 47 0.98 -0.16 - - - - - - 64 0.73 0.45* - - - - - - GO:0005125 Cytokine activity 69 0.59 -0.71* 84 0.29 -0.36 - - - 91 0.44 0.48* 76 0.42 0.24 - - - GO:0008009 Chemokine activity - - - 25 -0.14 -0.23 - - - - - - 26 -0.16 -0.1 - - - GO:0042379 Chemokine receptor binding - - - 25 -0.14 -0.23 - - - - - - 26 -0.16 -0.1 - - - GO:0005179 Hormone activity 33 0.49 -0.71 - - - 41 0.11* -0.44 - - - 34 0.19* -0.47 27 0.49 -0.64 GO:0005184 Neuropeptide hormone activity 10 -0.12 0.27 - - - 11 0.01 0.68 - - - - - - - - - GO:0004252 Serine-type endopeptidase activity - - - 50 -0.34* -0.01 - - - - - - - - - - - - GO:0004263 Chymotrypsin activity - - - 38 -0.45* -0.1 - - - - - - - - - - - - GO:0004295 Trypsin activity - - - 39 -0.45* -0.21 - - - - - - - - - - - - GO:0003735 Structural constituent of ribosome - - - 100 -0.34* -0.25 89 -0.41* -0.72* 116 -0.37* -0.58* 79 -0.35* -0.5 63 -0.33* -0.47 GO:0005198 Structural molecule activity - - - 212 -0.04 -0.4* 192 -0.11* -0.43* 260 -0.07 -0.2 - - - - - - Biological process GO:0007275 Development 335 1.41* -0.55* 410 1.13* -0.45* 386 1.19* -0.23 512 1.09* 0.1 384 1.32* -0.45* 258 1.58* -0.48* GO:0009653 Morphogenesis 222 1.24* -0.48* - - - - - - 334 0.94* 0.21* - - - - - - GO:0009887 Organogenesis 186 1.03* -0.46* - - - - - - 270 0.8* 0.22* - - - - - - GO:0009888 Histogenesis - - - - - - - - - 47 0.49 0.46 - - - - - - GO:0008544 Epidermis development 24 -0.27 -1.4* - - - - - - - - - - - - - - - GO:0001501 Skeletal development 36 1.4* -0.23 - - - - - - - - - - - - - - - GO terms associated with TE-poor genes Genome Biology 2006, 7:R120 Genome Biology 2006, 7:R120 Genome Biology 2006, Volume 7, Issue 12, Article R120 Sironi et al. Significant differences are marked with an asterisk. DNA transp., DNA transposon; N, number of contributing genes; MCS, fractional intronic MCS density deviation (see text); IG, fractional igTEna deviation (see text). TE-rich gene categories DNA transp., DNA transposon; N, number of contributing genes; MCS, fractional intronic MCS Table 1 (Continued) GO terms associated with TE-poor genes Table 1 (Continued) GO terms associated with TE-poor genes Genome Biology 2006, 7:R120 R120.6 Genome Biology 2006, Volume 7, Issue 12, Article R120 Sironi et al. http://genomebiology.com/2006/7/12/R120 Table 2 GO terms associated with TE-rich human genes Over-represented TE types GO term Description Alu L1 L2 LTR DNA transp. TE-rich gene categories DNA transp., DNA transposon; N, number of contributing genes; MCS, fractional intronic MCS al igTEna deviation (see text) Significant differences are marked with an asterisk. DNA transp., DNA transposon; N, number of contributing genes; MCS, fractional intronic MCS density deviation (see text); IG, fractional igTEna deviation (see text). Significant differences are marked with an asterisk. DNA transp., DNA transposon; N, number of contributing genes; MCS, fractional intronic MCS density deviation (see text); IG, fractional igTEna deviation (see text). of 5' or 3' gene boundaries. We next used the different MIR subtype reference sequences [3] to align all instances in immune response gene introns or intergenic spacers sepa- rately. To verify whether any MIR region was over- or under- represented in these genes, we compared the average relative frequency at each position with frequencies derived from 100 samples of an equal number of MIR sequences randomly selected from either introns or intergenic spacers. The mean, as well as the 1st and 99th percentiles in random sample fre- quency distributions were then calculated at each position; they are plotted in Figure 2a together with average frequen- cies of MIRs located in immune response genes. This calcula- tion was not performed for MIRm sequences because of their paucity (47 instances in immune genes). The frequency pro- file of MIR, MIR3 and MIRb sequences located in immune response gene introns indicates that the central core region is over-represented (beyond the 99th percentile) compared to display higher metatherian/monotremata-specific MIR fre- quencies compared to the remaining genes (Wilcoxon rank sum test, p = 0.0023) (Figure 1). display higher metatherian/monotremata-specific MIR fre- quencies compared to the remaining genes (Wilcoxon rank sum test, p = 0.0023) (Figure 1). TE-rich gene categories MIR Molecular function N MCS IG GO:0008009 Chemokine activity - - - - - 9 -0.91* -0.66 GO:0005125 Cytokine activity - - - - - 24 -0.42 -0.13 GO:0001584 Rhodopsin-like receptor activity - - - - - 19 -0.44 0.31 GO:0042379 Chemokine receptor binding - - - - - 9 -0.91* -0.66 GO:0005102 Receptor binding - - - - - 38 -0.45 -0.03 GO:0001664 G-protein-coupled receptor binding - - - - - 9 -0.91* -0.66 Biological process GO:0050874 Organismal physiological process - - - - - 89 -0.57* 0.01 GO:0009607 Response to biotic stimulus - - - - - 70 -0.69* 0.36 GO:0006955 Immune response - - - - - 60 -0.67* 0.23 GO:0009611 Response to wounding - - - - - 31 -0.73* 0.11 GO:0006954 Inflammatory response - - - - - 24 -0.79* 0.06 GO:0006952 Defense response - - - - - 66 -0.7* 0.3 GO:0045087 Innate immune response - - - - - 26 -0.78* 0.07 GO:0016064 Humoral defense mechanism - - - - - 14 -0.65 0.24 GO:0009617 Response to bacteria - - - - - 13 -0.83* 0.34 GO:0009613 Response to pest, pathogen or parasite - - - - - 47 -0.72* 0.21 GO:0043207 Response to external biotic stimulus - - - - - 51 -0.74* 0.16 GO:0006950 Response to stress - - - - - 53 -0.72* 0.16 GO:0042742 Defense response to bacteria - - - - - 9 -0.98* 0.36 GO:0009605 Response to external stimulus - - - - - 65 -0.76* 0.19 GO:0009620 Response to fungi - - - - - 6 -1* 0.91 GO:0009628 Response to abiotic stimulus - - - - - 28 -0.83* 0.55 GO:0042221 Response to chemical substance - - - - - 27 -0.83* 0.7 GO:0050896 Response to stimulus - - - - - 85 -0.71* 0.31 GO:0006968 Cellular defense response - - - - - 14 -0.64 -0.14 GO:0007267 Cell-cell signaling - - - - - 37 -0.26 -0.32 GO:0042330 Taxis - - - - - 17 -0.78* -0.1 GO:0006935 Chemotaxis - - - - - 17 -0.78* -0.1 GO:0030574 Collagen catabolism - - - - - 7 -0.69 -0.77 Significant differences are marked with an asterisk. DNA transp., DNA transposon; N, number of contributing genes; MCS, fractional intronic MCS density deviation (see text); IG, fractional igTEna deviation (see text). GO terms associated with TE-rich human genes n asterisk. Characterization of MIR sequences associated with immune response genes We next wished to verify whether MIR sequences in immune response genes have some feature distinguishing them from MIRs in other genomic locations. Four highly related MIR subtypes (MIR, MIR3, MIRb and MIRm) have been identified in the murine and human genomes [3]; the four subtypes dis- play a central, almost identical 70 base-pair (bp) core region [28]. To verify whether any MIR region has been preferen- tially retained in MIR-rich immune response genes, we retrieved all MIR elements located in the intronic regions of these genes or in their flanking intergenic spacers. In the lat- ter case, we restricted the analysis to TEs located within 15 kb Genome Biology 2006, 7:R120 Genome Biology 2006, Volume 7, Issue 12, Article R120 Sironi et al. R120.7 http://genomebiology.com/2006/7/12/R120 Table 3 GO terms associated with TE-rich mouse genes Table 3 GO terms associated with TE-rich mouse genes Over-represented TE types GO term Description B1 L1 LTR L2 MIR B2/ID/B4 Molecular function N MCS IG N MCS IG N MCS IG GO:0005215 Transporter activity - - - 64 -0.24 0.33 - - - - - - GO:0005216 Ion channel activity - - - - - - 28 0.2 -0.05 - - - GO:0015268 Alpha-type channel activity - - - - - - 33 0.13 0.12 - - - GO:0015267 Channel or pore class transporter activity - - - - - - 33 0.13 0.12 - - - GO:0005261 Cation channel activity - - - - - - 23 0.37* -0.08 - - - GO:0005244 Voltage-gated ion channel activity - - - - - - 19 0.34 -0.1 - - - Biological process GO:0030001 Metal ion transport - - - - - - 26 0.31* -0.06 - - - GO:0007264 Small GTPase mediated signal transduction - - - - - - - - - 14 -0.14 0.79 Significant differences are marked with an asterisk. N, number of contributing genes; MCS, fractional intronic MCS density deviation (see text); IG, fractional igTEna deviation (see text). GO terms associated with TE-rich mouse genes Significant differences are marked with an asterisk. N, number of contributing genes; MCS, fractional intronic MCS density deviation (see text); IG, fractional igTEna deviation (see text). rked with an asterisk. N, number of contributing genes; MCS, fractional intronic MCS density deviation (see text); IG, the background intronic frequency. (TEfintron/meanTEfintron) - (TEfinter/meanTEfinter) where TEfintron is the average TE frequency for all introns in the same gene, meanTEfintron is the average TE frequency for all introns in all genes, TEfinter is the TE frequency averaged for 5' and 3' regions flanking each gene and meanTEfinter is the average TE frequency for all intergenic spacers. Again lowess curves were obtained, as well as empirical probability inter- vals derived from 100 random permutations; as shown in Fig- ure 4b, for highly expressed genes and for all TE types, a significant decreasing trend is observed when frequency dif- ferences are plotted against gene expression. The same obser- vations were confirmed using expression data derived from SAGE experiments and they also apply to mouse genes (sup- plementary Figures 3 to 5 in Additional data file 2). It is worth noting that very similar results were also obtained when the same calculations were performed using 8 kb sequences flanking each gene (4 kb each side) instead of entire inter- genic regions (supplementary Figure 6a,b in Additional data file 2 for human genes and data obtained with either microar- ray or SAGE, respectively). For the latter analyses only genes where TEfintron is the average TE frequency for all introns in the same gene, meanTEfintron is the average TE frequency for all introns in all genes, TEfinter is the TE frequency averaged for 5' and 3' regions flanking each gene and meanTEfinter is the average TE frequency for all intergenic spacers. Again lowess curves were obtained, as well as empirical probability inter- vals derived from 100 random permutations; as shown in Fig- ure 4b, for highly expressed genes and for all TE types, a significant decreasing trend is observed when frequency dif- ferences are plotted against gene expression. The same obser- vations were confirmed using expression data derived from SAGE experiments and they also apply to mouse genes (sup- plementary Figures 3 to 5 in Additional data file 2). It is worth noting that very similar results were also obtained when the same calculations were performed using 8 kb sequences flanking each gene (4 kb each side) instead of entire inter- genic regions (supplementary Figure 6a,b in Additional data file 2 for human genes and data obtained with either microar- ray or SAGE, respectively). For the latter analyses only genes Characterization of MIR sequences associated with immune response genes These same findings did not apply to MIRb and MIR3 sequences in intergenic regions flanking immune response genes (Figure 2b). Similar results (supplemental Figure 2 in Additional data file 2) were obtained for mouse MIR sequences located in immune response genes. gene expression percentile. Results obtained from SAGE expression data, as well as for murine genes, gave similar results and are available in Additional data file 2. To gain further insight, we wished to compare intronic with intergenic TE frequecies (TE number/sequence length). In fact, intergenic and intronic regions belong to the same isochore (that is, they display a similar CG percentage) and their lengths are correlated [31], as well as their MCS density (Spearman rho = 0.37, p < 10-16); therefore, TE density can be directly compared. Thus, for each gene we calculated the rel- ative frequency difference as: We therefore analyzed the human/mouse co-conservation profile (that is, the frequency of bases that, in both human and mouse, are equal to the MIR consensus sequence) of human/mouse orthologous MIR instances. No significant dif- ference was observed (Figure 3a-c) between MIRs located in immune response introns and random MIR samples. Yet, as is evident from Figure 3d, the central portion of intronic MIR sequences, either located in defence response genes or not, is more frequently co-conserved compared to 5' and 3' flanking regions. Analysis o Figure 1 Analysis of MIR frequency in dog and opossum immune defense genes Figure 1 Analysis of MIR frequency in dog and opossum immune defense genes. MIR sequences were divided into mammalian-wide and metatherian/monotremata- specific. Immune response genes displayed significantly higher frequencies of both MIR types compared to the remaining genes. Box height represents sample interquartile range and the bold line depicts the median position. The whiskers extend to the most extreme data point, which is no more than 1.5 times the interquartile range from the box. displaying both 3' and 5' intergenic regions longer than 10 kb were selected (n = 3,477). with different measures of noncoding sequence conservation [24,33,34]. We confirm here (see Additional data file 1) that these observations are explained by the intrinsic mutagenic potential of transposition and the necessity of preserving multispecies conserved sequences from disruption. In fact, TE insertion is counterselected at different degrees depend- ing on the relative timing of MCS fixation and TE activity. Given this premise and considering insertion to be mutagenic irrespective of TE family or type, we analyzed the distribution of different TEs in human introns after correcting for the known parameters affecting either integration frequency or fixation probability, namely GC content [1,35], intron size [24,34] and MCS density (this study and [24]). All analyses have been carried out in parallel on human and mouse genes. Such a procedure strengthens the ensuing conclusions since the majority of TEs are specific to either species [3] and the maintenance of ancestral TEs also differs between primates and rodents due to the higher mutation rate of the latter [34]. Also, we analyzed intronic TE distribution in association with Repeat content as a function of expression level Repeat content as a function of expression level Different TE types have been reported to differentially associ- ate with gene regions depending on expression levels [29]. To get further insight into this issue, we calculated expression level (averaged over all tissues) for human and mouse genes in our dataset. Since different experimental methods for measuring gene expression have been shown to yield differ- ent results [30], we used expression data derived from two different experimental methods, namely microarray and serial analysis of gene expression (SAGE). For each family, TEna was then plotted against expression level and lowess curves calculated (see Materials and methods for details). To address the significance of the observed trends, 100 lowess smooths were calculated after random data permutations and empirical probability intervals were calculated (see Materials and methods). As is evident from Figure 4a, a marked decrease in TEna is observed for genes above the 70th to 80th Genome Biology 2006, 7:R120 R120.8 Genome Biology 2006, Volume 7, Issue 12, Article R120 Sironi et al. http://genomebiology.com/2006/7/12/R120 Analysis of MIR frequency in dog and opossum immune defense genes Figure 1 Analysis of MIR frequency in dog and opossum immune defense genes. MIR sequences were divided into mammalian-wide and metatherian/monotremata- specific. Immune response genes displayed significantly higher frequencies of both MIR types compared to the remaining genes. Box height represents sample interquartile range and the bold line depicts the median position. The whiskers extend to the most extreme data point, which is no more than 1.5 times the interquartile range from the box. Immune response Other 0.0000 0.0005 0.0010 0.0015 Dog mammalian−wide Frequency Immune response Other 0.0000 0.0005 0.0010 0.0015 Opossum mammalian−wide Frequency Immune response Other 0.0000 0.0005 0.0010 0.0015 Opossum metatheria/monotremata−specific Frequency 0.0010 Frequency Frequency Frequency 0.0005 0.0005 Discussion TE di ib i TE distribution in mammalian genomes has been addressed in numerous studies. Yet, many questions concerning the nature of the host-element relationship still remain unan- swered and a comprehensive scenario of the selective forces affecting TE fixation in mammalian genomes is still missing. In particular, genome-wide analyses of TE type distribution within and in proximity to human genes have often neglected relevant features, such as sequence conservation, gene func- tion and expression level. Since the precise removal of an inserted transposon is a rare event [32], present day TE distribution is the result of inser- tion frequency and fixation probability over time. Previous work had indicated that TE frequency inversely correlates Genome Biology 2006, 7:R120 Genome Biology 2006, Volume 7, Issue 12, Article R120 Sironi et al. R120.9 http://genomebiology.com/2006/7/12/R120 0 50 100 150 200 0.000 0.004 0.008 MIR3 Relative frequency 0 50 100 150 200 0.000 0.004 0.008 0 50 100 150 200 0.000 0.004 0.008 Analysis of human MIR sequences associated with immune response genes Figure 2 Analysis of human MIR sequences associated with immune response genes. (a) Relative frequency at each position of MIR (n = 277), MIRb (n = 382) and MIR3 (n = 104) consensus sequences in immune response gene introns (red lines). Mean profiles and intervals corresponding to the 1st and 99th percentiles in 100 random sample frequency distributions are represented by black lines and grey areas, respectively. (b) The same as in (a) for MIRs located in intergenic regions. MIR, n = 239; MIRb, n = 345; MIR3, n = 97. Hatched lines delimit the MIR CORE region. Discussion TE di ib i 0 50 100 150 200 250 0.000 0.004 0.008 MIR Position (bp) Relative frequency (a) 0 50 100 150 200 250 0.000 0.004 0.008 0 50 100 150 200 250 0.000 0.004 0.008 0 50 100 150 200 250 0.000 0.004 0.008 MIRb Position (bp) Relative frequency 0 50 100 150 200 250 0.000 0.004 0.008 0 50 100 150 200 250 0.000 0.004 0.008 0 50 100 150 200 0.000 0.004 0.008 MIR3 Position (bp) Relative frequency 0 50 100 150 200 0.000 0.004 0.008 0 50 100 150 200 0.000 0.004 0.008 0 50 100 150 200 250 0.000 0.004 0.008 MIR Position (bp) Relative frequency (b) 0 50 100 150 200 250 0.000 0.004 0.008 0 50 100 150 200 250 0.000 0.004 0.008 0 50 100 150 200 250 0.000 0.004 0.008 MIRb Position (bp) Relative frequency 0 50 100 150 200 250 0.000 0.004 0.008 0 50 100 150 200 250 0.000 0.004 0.008 0 50 100 150 200 0.000 0.004 0.008 MIR3 Position (bp) Relative frequency 0 50 100 150 200 0.000 0.004 0.008 0 50 100 150 200 0.000 0.004 0.008 0 50 100 150 200 250 0.000 0.004 0.008 MIR Position (bp) Relative frequency (a) 0 50 100 150 200 250 0.000 0.004 0.008 0 50 100 150 200 250 0.000 0.004 0.008 0 50 100 150 200 250 0.000 0.004 0.008 MIRb Position (bp) Relative frequency 0 50 100 150 200 250 0.000 0.004 0.008 0 50 100 150 200 250 0.000 0.004 0.008 0 50 100 150 200 0.000 0.004 0.008 MIR3 Position (bp) Relative frequency 0 50 100 150 200 0.000 0.004 0.008 0 50 100 150 200 0.000 0.004 0.008 0 50 100 150 200 250 0.000 0.004 0.008 MIR Position (bp) Relative frequency (a) 0 50 100 150 200 250 0.000 0.004 0.008 0 50 100 150 200 250 0.000 0.004 0.008 0 50 100 150 200 250 0.000 0.004 0.008 MIRb Position (bp) Relative frequency 0 50 100 150 200 250 0.000 0.004 0.008 0 50 100 150 200 250 0.000 0.004 0.008 (a) Relative frequency Position (bp) 0 50 100 150 200 250 0.000 0.004 0.008 MIR Position (bp) Relative frequency (b) 0 50 100 150 200 250 0.000 0.004 0.008 0 50 100 150 200 250 0.000 0.004 0.008 0 50 100 150 200 0.000 0.004 0.008 MIR3 Position (bp) Relative frequency 0 50 100 150 200 0.000 0.004 0.008 0 50 100 150 200 0.000 0.004 0.008 0 50 100 150 200 250 0.000 0.004 0.008 MIRb Position (bp) Relative frequency 0 50 100 150 200 250 0.000 0.004 0.008 0 50 100 150 200 250 0.000 0.004 0.008 (b) Relative frequency Analysis of human MIR sequences associated with immune response genes Figure 2 Analysis of human MIR sequences associated with immune response genes. Discussion TE di ib i (a) Relative frequency at each position of MIR (n = 277), MIRb (n = 382) and MIR3 (n = 104) consensus sequences in immune response gene introns (red lines). Mean profiles and intervals corresponding to the 1st and 99th percentiles in 100 random sample frequency distributions are represented by black lines and grey areas, respectively. (b) The same as in (a) for MIRs located in intergenic regions. MIR, n = 239; MIRb, n = 345; MIR3, n = 97. Hatched lines delimit the MIR CORE region. Our GO data indicate that functional classes associated with TE-poor genes extend well beyond highly conserved gene cat- egories such as developmental regulators and transcription factors. In fact, some MCS-poor gene function categories also display lower than expected TEs; genes coding for structural molecules and ribosomal proteins are deprived of most TE families in both introns and intergenic spacers. These loci are mainly accounted for by housekeeping genes; if low TE repre- sentation in intronic regions might be explained by the need to reduce transcriptional costs (in agreement with TE paucity in introns of highly expressed genes, as discussed below), the reason why TEs are also excluded from intergenic spacers is more difficult to explain. One possibility is that extensive methylation of repetitive elements might exert a negative reg- ulation on nearby gene expression with detrimental conse- quences for housekeeping genes. Indeed, several reports [37- 40] have suggested the existence of specific methylation pat- terns in TEs (probably representing a cellular defence mech- anism against transposition) and methylation has been shown to spread in cis from TEs to flanking cellular sequences Our GO data indicate that functional classes associated with TE-poor genes extend well beyond highly conserved gene cat- egories such as developmental regulators and transcription factors. In fact, some MCS-poor gene function categories also display lower than expected TEs; genes coding for structural molecules and ribosomal proteins are deprived of most TE families in both introns and intergenic spacers. These loci are mainly accounted for by housekeeping genes; if low TE repre- sentation in intronic regions might be explained by the need to reduce transcriptional costs (in agreement with TE paucity in introns of highly expressed genes, as discussed below), the reason why TEs are also excluded from intergenic spacers is more difficult to explain. Discussion TE di ib i One possibility is that extensive methylation of repetitive elements might exert a negative reg- ulation on nearby gene expression with detrimental conse- quences for housekeeping genes. Indeed, several reports [37- 40] have suggested the existence of specific methylation pat- terns in TEs (probably representing a cellular defence mech- anism against transposition) and methylation has been shown to spread in cis from TEs to flanking cellular sequences both MCS content and TE abundance in intergenic regions. In fact, although we corrected for MCS presence in multiple regression fitting, MCS content represents an indication of gene complexity and regulatory accuracy [36]. On the other hand, TE representation in intergenic spacers might highlight differences in TE effect depending on location; this is espe- cially relevant for TE families that have been previously reported to be preferentially abundant in intergenic versus intronic regions or vice versa [23]. both MCS content and TE abundance in intergenic regions. In fact, although we corrected for MCS presence in multiple regression fitting, MCS content represents an indication of gene complexity and regulatory accuracy [36]. On the other hand, TE representation in intergenic spacers might highlight differences in TE effect depending on location; this is espe- cially relevant for TE families that have been previously reported to be preferentially abundant in intergenic versus intronic regions or vice versa [23]. The initial analysis of the human genome sequence [1] had indicated that the HOX gene cluster is virtually deprived of TEs; the same result was obtained upon analysis of the mouse genome and interpreted in terms of TEs disturbing fine tuned regulation of developmental genes. A more recent study indi- cated that TE-free regions are significantly associated with genes coding for developmental regulators or transcription factors [26]. Genome Biology 2006, 7:R120 R120.10 Genome Biology 2006, Volume 7, Issue 12, Article R120 Sironi et al. http://genomebiology.com/2006/7/12/R120 in plants and yeast [41,42]. In this respect, it is intriguing that Alus, which show lower methylation levels [40], possibly due to their association with a 'protective' sperm protein [43], are not preferentially excluded from these same housekeeping gene sets (Table 1). Similar considerations might be applied to genes coding for cytokines, growth factors, and hormones as well as genes involved in immune responses, all of which display few intronic and intergenic TEs. Still, these genes are not housekeeping genes or highly expressed and they also dis- play lower than expected Alu frequencies. Discussion TE di ib i We speculate that these gene categories might require extremely subtle regula- tion of transcript levels (especially in the case of secreted pro- teins) or precise timing of activation (for example, in response to a stimulus). Indeed, altered hormone or cytokine levels have been associated with human disease and cancer (reviewed in [44,45]), while the effects of immune response gene misregulation are easily envisaged. As mentioned above, TEs can influence gene expression by both altering the epige- netic state of TE-carrying alleles [46,47] and providing pro- moters and transcription factor binding sites (either enhancers or suppressors (reviewed in [48,49]) to the genes neighboring their integration sites. In particular, Alus have been shown to potentially carry functional sites for different transcription factors as well as for both steroid-hormone and retinoic acid receptors (reviewed in [48]); these observations have led to the speculation that Alu integration might cause a genetic disease not through gene coding sequence disruption but rather through alteration of gene expression patterns [50]. Indeed, several gene categories displaying lower than expected intronic Alu frequencies also show significantly fewer Alus in flanking intrergenic spacers. [28]. Previous studies noted a higher representation in mam- malian genomes of MIR core regions compared to flanking 3' and 5' sequences [12,28]; our data indicate that the core sequence is both more frequent and more conserved in the human genome, as assessed by co-conservation profiles. Since MIRs are thought to be long time fossils [28], this observation suggests that the core might serve some general function in mammalian genomes. Indeed, upon analysis of aligned human-mouse intergenic sequences, Silva et al. [12] suggested that the core region is more often present in align- ing orthologous regions than expected on the basis of back- ground genome frequency. Our data indicate that this observation also applies to MIR sequences located in immune response gene introns. To our knowledge, this is the first report showing that a specific TE family is evolutionarily associated with a gene function category. Whether MIRs located in defense response genes serve a specific function or they share a common role with the other core sequences in the genome remains to be elucidated. Recent works indicated that two ancient SINE families have been extensively exapted in the human genome and copies of these TEs have been recruited to serve distinct functions in different genomic loca- tions [14,16]. Co conservation profile of MIR sequences Figure 3 (see following page) Co-conservation profile of MIR sequences. Co-conservation frequency at each position of (a) MIR (n = 277), (b) MIRb (n = 382) and (c) MIR3 (n = 104) consensus sequences in immune response gene introns (red lines). Frequency intervals corresponding to the 1st and 99th percentiles in 100 random sample frequency distributions are represented by the black lines. (d) Co-conservation profiles of MIR sequences located in human introns; in this case, positions correspond to the alignment of the three MIR subtypes: MIR (black), MIRb (red) and MIR3 (blue). Discussion TE di ib i This might also be the case for MIRs; alterna- tively, these sequences might all share a general role in the human genome that is particularly important in immune defense loci. The last part of our work is devoted to studying the influence of gene expression level on TE distribution. In fact, despite the small population size, it has been reported that human genes show signatures consistent with selection mediated by expression levels [56]. In particular, selective pressure aimed at reducing transcriptional cost has been proposed to act on highly expressed human genes and TEs had been suggested as possible targets for selection to act upon [57]. Our findings strongly support this view: all TE families are under-repre- sented in highly expressed genes. While the ability of LINE L1s to affect mRNA transcription/processing efficiency [10] might explain their exclusion from highly expressed introns, Alus have been reported to associate with highly expressed gene regions [29] and no direct effect on transcription or processing has ever been described for ancestral TE families. Therefore, the expression-dependent exclusion of all TE fam- ilies from intronic regions is strongly consistent with the need to reduce the transcription energetic costs. The issue had also been raised as to whether a selective pressure is still acting on highly expressed genes or if we merely witness the remnants of a previous action of selection (still not at equilibrium) [56]. It is interesting to notice that genes involved in immune response, which display extremely low conservation in both coding [51-53] and non-coding sequences [36], as well as a higher content of TEs in their untranslated sequences [54], are deprived of most TE types but enriched in MIR sequences in three eutherian species (human, mouse and dog). Given the partially independent origin of MIR sequences in eutheria and metatheria, it is important to notice that analysis of orthologous genes indicated that MIR over-representation also occurs in opossum immune response genes, suggesting the evolutionary conservation of a specific function for MIRs located in these loci. MIRs belong to a large TE superfamily referred to as CORE- SINE [53]; all CORE-SINE TEs share a common 65 bp central region that was proposed to be either relevant for retrotrans- positional activity [27,55] or functional in the host genome Genome Biology 2006, 7:R120 http://genomebiology.com/2006/7/12/R120 Genome Biology 2006, Volume 7, Issu Genome Biology 2006, Volume 7, Issue 12, Article R120 Sironi et al. Genome Biology 2006, Volume 7, Issue 12, Article R120 Sironi et al. R120.11 http://genomebiology.com/2006/7/12/R120 Genome Biology 2006, Volume 7, Issue 12, Article R120 Sironi et al. R120.11 Discussion TE di ib i http://genomebiology.com/2006/7/12/R120 Gene-expression dependent variation in TE intronic abundance Figure 4 Gene-expression dependent variation in TE intronic abundance. Gene expression levels were derived from microarray data. (a) Lowess fit (solid line) and probability intervals (hatched lines) of TEna versus gene expression level (log transformed values) for the six TE families. (b) Lowess fit (solid line) and probability intervals (hatched lines) of intronic to intergenic relative TE frequency difference (see text) versus gene expression level (log transformed values). Discussion TE di ib i R120.11 http://genomebiology.com/2006/7/12/R120 gure 3 (see legend on previous page) 0 50 100 150 200 250 0.0 0.1 0.2 0.3 0.4 0.5 0.6 Position (bp) Relative frequency 0 50 100 150 200 250 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0 50 100 150 200 250 0.0 0.1 0.2 0.3 0.4 0.5 0.6 (a) 0 50 100 150 200 250 0.0 0.2 0.4 0.6 Position (bp) Relative frequency 0 50 100 150 200 250 0.0 0.2 0.4 0.6 0 50 100 150 200 250 0.0 0.2 0.4 0.6 (b) 0 50 100 150 200 0.0 0.2 0.4 0.6 0.8 1.0 Position (bp) Relative frequency 0 50 100 150 200 0.0 0.2 0.4 0.6 0.8 1.0 0 50 100 150 200 0.0 0.2 0.4 0.6 0.8 1.0 (c) 0 50 100 150 200 250 0.0 0.1 0.2 0.3 0.4 Position (bp) Relative frequency (d) 0 50 100 150 200 250 0.0 0.1 0.2 0.3 0.4 0 50 100 150 200 250 0.0 0.1 0.2 0.3 0.4 0 50 100 150 200 250 0.0 0.1 0.2 0.3 0.4 0.5 0.6 Relative frequency 0 50 100 150 200 250 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0 50 100 150 200 250 0.0 0.1 0.2 0.3 0.4 0.5 0.6 (a) 0 50 100 150 200 250 0.0 0.1 0.2 0.3 0.4 0.5 0.6 Position (bp) Relative frequency 0 50 100 150 200 250 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0 50 100 150 200 250 0.0 0.1 0.2 0.3 0.4 0.5 0.6 (a) 0 50 100 150 200 250 0.0 0.2 0.4 0.6 Position (bp) Relative frequency 0 50 100 150 200 250 0.0 0.2 0.4 0.6 0 50 100 150 200 250 0.0 0.2 0.4 0.6 (b) 0 50 100 150 200 250 0.0 0.2 0.4 0.6 Relative frequency 0 50 100 150 200 250 0.0 0.2 0.4 0.6 0 50 100 150 200 250 0.0 0.2 0.4 0.6 (b) (b) Relative frequency 0 50 100 150 200 250 0.0 0.1 0.2 0.3 0.4 Position (bp) Relative frequency (d) 0 50 100 150 200 250 0.0 0.1 0.2 0.3 0.4 0 50 100 150 200 250 0.0 0.1 0.2 0.3 0.4 0 50 100 150 200 0.0 0.2 0.4 0.6 0.8 1.0 Position (bp) Relative frequency 0 50 100 150 200 0.0 0.2 0.4 0.6 0.8 1.0 0 50 100 150 200 0.0 0.2 0.4 0.6 0.8 1.0 (c) 0 50 100 150 200 250 0.0 0.1 0.2 0.3 0.4 Position (bp) Relative frequency (d) 0 50 100 150 200 250 0.0 0.1 0.2 0.3 0.4 0 50 100 150 200 250 0.0 0.1 0.2 0.3 0.4 0 50 100 150 200 0.0 0.2 0.4 0.6 0.8 1.0 Position (bp) Relative frequency 0 50 100 150 200 0.0 0.2 0.4 0.6 0.8 1.0 0 50 100 150 200 0.0 0.2 0.4 0.6 0.8 1.0 (c) (c) (d) Relative frequency Relative frequency Genome Biology 2006, 7:R120 R120.12 Genome Biology 2006, Volume 7, Issue 12, Article R120 Sironi et al. Discussion TE di ib i 1 2 3 4 5 −1.0 −0.5 0.0 0.5 log10(gene expression) TEna ALU 1 2 3 4 5 −1.0 −0.5 0.0 0.5 log10(gene expression) TEna MIR 1 2 3 4 5 −1.0 −0.5 0.0 0.5 log10(gene expression) TEna L1 1 2 3 4 5 −1.0 −0.5 0.0 0.5 log10(gene expression) TEna L2 1 2 3 4 5 −1.0 −0.5 0.0 0.5 log10(gene expression) TEna LTR 1 2 3 4 5 −1.0 −0.5 0.0 0.5 log10(gene expression) TEna DNA 1 2 3 4 5 −1.0 −0.5 0.0 0.5 log10(gene expression) relative frequency difference 1 2 3 4 5 −1.0 −0.5 0.0 0.5 log10(gene expression) relative frequency difference 1 2 3 4 5 −1.0 −0.5 0.0 0.5 log10(gene expression) relative frequency difference 1 2 3 4 5 −1.0 −0.5 0.0 0.5 log10(gene expression) relative frequency difference 1 2 3 4 5 −1.0 −0.5 0.0 0.5 log10(gene expression) relative frequency difference 1 2 3 4 5 −1.0 −0.5 0.0 0.5 log10(gene expression) relative frequency difference (a) (b) 1 2 3 4 5 −1.0 −0.5 0.0 0.5 log10(gene expression) TEna ALU 1 2 3 4 5 −1.0 −0.5 0.0 0.5 log10(gene expression) TEna MIR 1 2 3 4 5 −1.0 −0.5 0.0 0.5 log10(gene expression) TEna L1 1 2 3 4 5 −1.0 −0.5 0.0 0.5 log10(gene expression) TEna L2 1 2 3 4 5 −1.0 −0.5 0.0 0.5 log10(gene expression) TEna LTR 1 2 3 4 5 −1.0 −0.5 0.0 0.5 log10(gene expression) TEna DNA (a) 1 2 3 4 5 −1.0 −0.5 0.0 0.5 log10(gene expression) relative frequency difference 1 2 3 4 5 −1.0 −0.5 0.0 0.5 log10(gene expression) relative frequency difference 1 2 3 4 5 −1.0 −0.5 0.0 0.5 log10(gene expression) relative frequency difference 1 2 3 4 5 −1.0 −0.5 0.0 0.5 log10(gene expression) relative frequency difference 1 2 3 4 5 −1.0 −0.5 0.0 0.5 log10(gene expression) relative frequency difference 1 2 3 4 5 −1.0 −0.5 0.0 0.5 log10(gene expression) relative frequency difference (b) (b) relative frequency difference Gene expression dependent variation in TE intronic abundance Figure 4 Gene-expression dependent variation in TE intronic abundance. Gene expression levels were derived from microarray data. (a) Lowess fit (solid line) and probability intervals (hatched lines) of TEna versus gene expression level (log transformed values) for the six TE families. Discussion TE di ib i (b) Lowess fit (solid line) and probability intervals (hatched lines) of intronic to intergenic relative TE frequency difference (see text) versus gene expression level (log transformed values). Our data support the first hypothesis: Alus, which represent relatively young TEs are under-represented in highly expressed introns and, in both human and mouse, separation in TE divergence classes did not reveal any different expres- sion-dependent association with TE age (not shown). genes, accounting for 81,599 and 55,553 introns, respectively. For each gene, the closest 5' and 3' known genes were identi- fied (using the UCSC knownGene table [58]); intergenic regions were defined as the genomic portions extending upstream and downstream of the transcribed region to the closest gene. Transposable elements were identified and categorized using the UCSC annotation tables that rely on RepeatMasker. MCS were obtained using phastCons predictions [13,59], which are based on a phylogenetic hidden Markov model and are avail- able through the UCSC database (phastConsElements Table [58]). MCSs were derived from human/chimpanzee/mouse/ rat/dog/chicken/pufferfish/zebrafish multiz alignments [58]. Statistical analysis All statistical analyses were performed using R [68]. Locally weighted scatter plot smoothing was performed using lowess curves [69]. These curves are produced by weighted least- square linear fitting within a window sliding through the data. The size of the window (span) controls the degree of smooth- ing and the curves are made robust by iterating the fit within each window discarding outliers. In all cases 5 robustifying iterations were performed and a span of 0.5 was used. To allow empirical p value calculations, we performed 100 inde- pendent random data permutations of the variable on the y axis. Indeed, computing lowess smooths after random per- mutations of the data can be used as a reference to gauge the significance of the pattern observed on the actual data [70]. For calculation of MIR co-conservation profiles, we used the liftOver utility at the UCSC genome browser [58] to obtain human/mouse orthologous MIR instances. MIRs were then aligned to the reference sequence using ClustalW. Microin- sertions in human instances were ignored. For each MIR position in human instances we calculated the frequency of co-conservation (that is, the frequency of bases that, in both human and mouse, are equal to the MIR consensus sequence). This procedure was applied to both MIRs located in immune response introns and to 100 randomly selected MIR samples of equal size and located within intronic regions. The co-conservation profile was then calculated using a smoothing spline with a span of ten bases over non- CpG positions. Probability interval limits were chosen, for each x value, as corresponding to p = 0.005 and p = 0.995 in the distribution of the 100 permutated y values considered as a Gaussian. Gene classification Gene associations with GO terms and their descriptions were performed by cross-referencing the UCSC hg17 kgXref table [57] with the GO database [61]. Association and description files were then created and significant associations between gene groups and GO terms were identified using GeneMerge [25]. MIR sequence analysis MIR consensus sequences were derived from the Genetic Information Research Institute (RepeatMasker database, release 20060314) [62]. Finally, we added all counts for libraries representing the same tissue type and converted absolute tag counts to relative tag counts (counts per million). For calculation of the MIR relative frequency profile, human or mouse MIR instances were aligned to the consensus sequence using SWAT [63]. Microinsertions in human and mouse instances were ignored. The relative frequency profile at each position of the consensus sequence was calculated as the number of instances covering the position divided by the total number of bases in instances. Expression data Microarray expression data for human and mouse genes were derived from previous high-throughoutput gene expression studies [64,65]; they are publicly accessible through the UCSC database (tables 'gnfHumanAtlas2median' and 'gnfHumanAtlas2medianExps', and 'gnfMouseAtlas2median' and 'gnfMouseAtlas2medianExps') [58]. We only considered probes corresponding to genes that had been included in our database; signals from duplicated probes on the same chip were averaged as well as replicates from the same tissue. A gene was considered to be expressed in a given tissue if its sig- nal level was higher or equal to 200 arbitrary units, as previ- ously recommended [64]. Data derived from tumor tissues were discarded. In the case of SAGE data, for each transcript entry in our databases we extracted a SAGE tag (10 bp down- stream of the most 3' NlaIII site). For both human and mouse, tags were then matched to all RefSeq mRNAs and purged if they corresponded to more than one transcript. For the identification of human-mouse orthologous pairs, the EnsMart database [60] was interrogated and only entries rep- resenting unique best reciprocal hits were selected. Retrieval of opossum and dog genes and annotations was per- formed using UCSC tables [58] referring to assembly monDom1 and canFam2. In particular, opossum/dog mRNA accession numbers were identified by cross-referencing tables 'geneName' and 'gbCdnaInfo' [58]. Genomic locations were next retrieved through tables xenoMrna or blastHg17KG. Monodelphis and dog TE annotations were directly obtained from UCSC [58] and MIR number per gene was calculated as the number of distinct elements fully con- tained between gene boundaries. MIR frequency was calcu- lated as MIR number/gene length. SAGE libraries were obtained from the SAGE Genie website [66]; for both organisms, libraries containing less than 20,000 tags, corresponding to tumor tissues, uncharacter- ized tissues, pharmacological treatments and mutated sam- ples were discarded. As previously suggested [67], libraries with mean tag GC content >0.5 were also removed. We retained 81 human libraries (both long and short tags), accounting for 21 tissues; for mouse, we retained 98 libraries accounting for 41 tissues. Sequence retrieval and analysis For creation of the intron database, human genes that had been annotated in the NCBI Reference Sequence (RefSeq) collection were selected (reviewed or validated entries only); for mouse genes 'Provisional' entries were also included. Genomic sequences, intron/exon boundaries and intergenic regions were derived from the UCSC genome annotation database [58] (assembly hg17 for human and mm5 for mouse). Intronless genes were discarded and, for each gene, the transcript corresponding to the longest genomic sequence and containing the highest number of exons was selected. The datasets are composed of 7,614 human and 5,550 mouse Only purely noncoding phastCons elements were selected (that is, MCSs partially overlapping with exons were dis- carded); a total of 238,005 and 596,018 human MCSs were retrieved in introns and intergenic sequences, respectively. In Only purely noncoding phastCons elements were selected (that is, MCSs partially overlapping with exons were dis- carded); a total of 238,005 and 596,018 human MCSs were retrieved in introns and intergenic sequences, respectively. In Genome Biology 2006, 7:R120 Genome Biology 2006, Volume 7, Issue 12, Article R120 Sironi et al. R120.13 http://genomebiology.com/2006/7/12/R120 mouse, 133,458 intronic and 312,752 intergenic MCSs were identified. mouse, 133,458 intronic and 312,752 intergenic MCSs were identified. 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Mol Biol Evol 2003, 20:1556-1563. 32. van de Lagemaat LN, Gagnier L, Medstrand P, Mager DL: Genomic deletions and precise removal of transposable elements mediated by short identical DNA segments in primates. Genome Res 2005, 15:1243-1249. 33. Chiaromonte F, Yang S, Elnitski L, Yap VB, Miller W, Hardison RC: Association between divergence and interspersed repeats in mammalian noncoding genomic DNA. Proc Natl Acad Sci USA 2001, 98:14503-14508. 9. Jordan IK, Rogozin IB, Glazko GV, Koonin EV: Origin of a substantial fraction of human regulatory sequences from transposable elements. Trends Genet 2003, 19:68-72. p 10. Han JS, Szak ST, Boeke JD: Transcriptional disruption by the L1 retrotransposon and implications for mammalian transcriptomes. Nature 2004, 429:268-274. 34. Pozzoli U, Menozzi G, Comi GP, Cagliani R, Bresolin N, Sironi M: Intron size in mammals: complexity comes to terms with economy. Trends Genet in press. p 11. Allen E, Horvath S, Tong F, Kraft P, Spiteri E, Riggs AD, Marahrens Y: High concentrations of long interspersed nuclear element sequence distinguish monoallelically expressed genes. Proc Natl Acad Sci USA 2003, 100:9940-9945. 35. Waterston RH, Lindblad-Toh K, Birney E, Rogers J, Abril JF, Agarwal P, Agarwala R, Ainscough R, Alexandersson M, An P, et al.: Initial sequencing and comparative analysis of the mouse genome. Nature 2002, 420:520-562. 12. Silva JC, Shabalina SA, Harris DG, Spouge JL, Kondrashovi AS: Con- served fragments of transposable elements in intergenic regions: evidence for widespread recruitment of MIR- and L2-derived sequences within the mouse and human genomes. Genet Res 2003, 82:1-18. 36. References 25. 25. Castillo-Davis CI, Hartl DL: GeneMerge-post-genomic analysis, data mining, and hypothesis testing. Bioinformatics 2003, 19:891-892. 1. 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Versteeg R, van Schaik BD, van Batenburg MF, Roos M, Monajemi R, Caron H, Bussemaker HJ, van Kampen AH: The human transcrip- tome map reveals extremes in gene density, intron length, GC content, and repeat pattern for domains of highly and weakly expressed genes. Genome Res 2003, 13:1998-2004. 5. Nekrutenko A, Li WH: Transposable elements are found in a large number of human protein-coding genes. Trends Genet 2001, 17:619-621. y p g , 30. Additional data files The following additional data are available with the online version of this paper. Additional data file 1 contains supple- Genome Biology 2006, 7:R120 R120.14 Genome Biology 2006, Volume 7, Issue 12, Article R120 Sironi et http://genomebiology.com/2006/7/12/R120 http://genomebiology.com/2006/7/12/R120 mentary text presenting analysis of MCS density and TE inte- gration frequency over evolutionary time. A supplementary figure describing the results is also provided (supplementary Figure 1) together with its legend. Additional data file 2 con- tains supplementary Table 1, and supplementary Figures 2 to 6 and their legends: supplementary Table 1 lists GO terms associated with mouse TE-poor genes; supplementary Figure 2 shows analysis of murine MIR sequences associated with immune response genes; supplementary Figure 3 shows gene-expression dependent variation in TE intronic abun- dance for human genes (SAGE data); supplementary Figure 4 shows gene-expression dependent variation in TE intronic abundance for mouse genes (microarray data); supplemen- tary Figure 5 shows gene-expression dependent variation in TE intronic abundance for mouse genes (SAGE data); supple- mentary Figure 6 shows intronic to intergenic relative fre- quency difference (calculated on gene flanks rather than entire intergenic regions). K, Clawson H, Spieth J, Hillier LW, Richards S, et al.: Evolutionarily conserved elements in vertebrate, insect, worm, and yeast genomes. Genome Res 2005, 15:1034-1050. 14. g Bejerano G, Lowe CB, Ahituv N, King B, Siepel A, Salama SR, Rubin EM, Kent WJ, Haussler D: A distal enhancer and an ultracon- served exon are derived from a novel retroposon. Nature 2006, 441:87-90. 15. 15. Kamal M, Xie X, Lander ES: A large family of ancient repeat ele- ments in the human genome is under strong selection. Proc Natl Acad Sci USA 2006, 103:2740-2745. 16. Nishihara H, Smit AF, Okada N: Functional noncoding sequences derived from SINEs in the mammalian genome. Genome Res in press. p 17. Doolittle WF, Sapienza C: Selfish genes, the phenotype para- digm and genome evolution. Nature 1980, 284:601-603. 18. Orgel LE, Crick FH: Selfish DNA: the ultimate parasite. Nature 1980, 284:604-607. 19. Yoder JA, Walsh CP, Bestor TH: Cytosine methylation and the ecology of intragenomic parasites. Trends Genet 1997, 13:335-340. 20. Miller WJ, McDonald JF, Nouaud D, Anxolabehere D: Molecular domestication - more than a sporadic episode in evolution. Genetica 1999, 107:197-207. 21. Gould SJ, Vrba ES: Exaptation: A missing term in the science of form. Paleobiology 1982, 8:4-15. gy 22. Additional data files Boissinot S, Entezam A, Furano AV: Selection against deleterious LINE-1-containing loci in the human lineage. Mol Biol Evol 2001, 18:926-935. Acknowledgements 23. 23. Medstrand P, van de Lagemaat LN, Mager DL: Retroelement distri- butions in the human genome: variations associated with age and proximity to genes. Genome Res 2002, 12:1483-1495. We are grateful to Dr Roberto Giorda and Matteo Fumagalli for useful dis- cussion about the manuscript. p y g 24. Sironi M, Menozzi G, Comi GP, Bresolin N, Cagliani R, Pozzoli U: Fix- ation of conserved sequences shapes human intron size and influences transposon insertion dynamics. Trends Genet 2005, 21:484-488. 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https://openalex.org/W184052233	https://zenodo.org/records/6516897/files/191-194.pdf	English	null	Spectrophotometric Determination of Uranium and Thorium Using Norwogonin	Zenodo (CERN European Organization for Nuclear Research)	1,963	cc-by	1,481	Mohan Katral and B. P. Singh runium(\'"1) and thorinm(IV) form oranp;e-co)ollftfl eomplexea witb nurwngooin (5,7,8-trihyduq:y- davour:. 'l'hl" complt"xes n.re II(J)ubl~ in aqut'IIIJUB ethanol. The UKO oF thia rpagent for llllpect.rophotometrie deter- mination ofnriLnium and thorium ha• "'-"'n inveotiga"'-"<1 . .ltePr'ol•,.· iH obeye<l ove1 the raDge cf 4 to 20 p.p. m. of mete! ion. The thorium compl•x is •l.abl., h..t,.·een pH :!.7 and 3.11 and that of nr&nium, between pH 2.7 and 5.0. Kulut.ionli cf the tt ( rium t"Omplex become opoiPMrC'nt nt or abov6 pH 3.6 on keeping for a few h~urs. The molar compo•ition• of the compl<•xuo formed by uranium and thorium have been found to be 1:2 and 1:1 tea~tivt•ly. 5-~:::~yfto.vones o.re g>lod chcla.ting agents and mo.ny of them, such aa, marin'•" quercetin', o.nd 1:utin', ho.vo a.lroo.dy been investigated for spectrophotometric estimation of thorium. :\lorin5•6 and quercetin' have also been used in the spectrophotometric deter- mination of uranium. Gslangin8, 3-methylgalangin9, and 5-hydroxy-7,8-dimethoxyflav- one'• ho.Ye been used by the autltors for spectrophotometric determine.tion of uranium and chrysiu" and g<\la.ngin'" for thorium. In the present communication, the suitability of norw,pgonin (5,7 ,8-trihydroxyH.avone) for spectrophotometric determination of the tv.·o metal ions has been investi!;!o.ted. The orange-coloured complexes of uranium (VI) and .thorium (IV) with norwogonin have been studied in 40% ethanolic medium. The reagent possesses the ad\'antages of high sensitivity and availability in a pure form; the complexes o.re stable aver a reasonably wide acidity range. [Joar· IDdiu Chem. Soc., Vol. 40, No. 3, 1963] [Joar· IDdiu Chem. Soc., Vol. 40, No. 3, 1963] [Joar· IDdiu Chem. Soc., Vol. 40, No. 3, 1963] EXPEI\ll[E~TAL The absorption spectra in the UV region were taken, using Perkin-Elmer spectra- cord and for other spectrophotometric measurements, a Coleman spectrophotometer, model No_ 14, was used. pH measurements were taken with a. Metrohm pH meter, type 1. lo"'.etcher &nd llilkey, Scie~~ee, 1954, 119, 445. 2. Idem. AntJ!. Cllem., 1958, 28, 1402. 3. Alimarin el sl., ByuU. Na.tu:l>. TetA. In.form., 1955, No. 7. 61; Vlum. AO.., 11180,54 4. Dev: o.nd Jain, J. I.e.• Common. Mdai.B, 196:!, t, 2!16. 5. Beck &nd Hantoa, Acla 0/aim. Aoad. Bci. Hu•g., 1055, 8, 233. 8. A1mo.ary .t 11!., 0Mm. AbB., 1955, 49, 9438•. 7. Kom...,da, Cl>em. Lioly, 1953, 47, 531. 8. K.o.tyal and Pingh, thi• JoKrntJI, 11182, 39, Ide~, 9. Ide~, ibid .• communlceted. 10- Idem, ibid., 1963, to, 117. , , , , ll. Idem, Proc.l'llll. Acad. Bci., 1962, 56, 12G. , , 12. Idem. tb.ia Jour....&, 1883. 40,121. ll. KATYAL A..'fD R. P. SINGH IIIli E-300. Ura.uyl nitrate and thorium nitrate wero used for preparing standard solutiollB. The solutions were standardised gravimetrically· by ox,Ute methQd. The reagent (nor,.·ogonin) wa.•JJrepared by the method of Saetri and Seeh.a.dri' 3 • A 1 X lO"M s::.lution of the reagent was prepared for the investigations. Oomplez Formation.-Cra.nyl and thorium ions reacted with ljJr.;"ila.vone with im- mediate formation of orange complexes. Complex formation w~stantia.ted by cha.nse in colour and fall in pH. Abwrp&wn Spectra.-1\:Li.x.imum a!J110rption by the flavone was observed at 280 m!L iu tho U\' region and at 370 m!L in tile visible (Fig. I, curve A). Tho absorption spectra ofthe complexes were Htudicd at different pH values (2.7 to 5.0 for uranium complex and 2.7 to3.!J for that oftborium). The absorption maximum in the case of both the oomp~exes was found to lie at 420 m!L (Fig. I, curves B & C). This wave length was consequent~lo -=hoHOn for further ill\"elltigationR. Molar ratio of flo.vone to uranium or thorium was main- tained at IIJ during spectral studios. One mi. of ~lfj I ,00(1 solution ofuranyi or thoriwn salt was mixed with J(J ml (MfJOOO) of the flavone solution and the mixture dilnted to 25.0 ml, maintaining 411% ctbanolic modi urn. The fi11vone solntion was used as blank and these conditions were kciJt coiiiit&nt in subsequent studies. t 1::1 Q BIC 0.~ Q4 Q3 0.2 0.1 A 14 12 1.0 OB 0.6 Q4 0.2 3~0 390 430 470 Waw lenglh (m!L) + FIG. I. Absorption spectra. 13. p...,,lnd. Acad. Sci., 11146, 24A, 251. EXPEI\ll[E~TAL A: Norwogouin ulliog a.q. EtCH u bl&Dk :0: Uru.nyl-nonmgooin complu uaing nor..-ogonin 110 bl.&n.k. C: Thorium-norwogoniD complo.11 uaing norwogonin o.s blank; pH 3.9. ProperlieB of ehe Oomplezu.-The complexes are soluble in aqueous ethanol and aro not extractable in usual organic solvents. No chango in colour was IJ.oticcd on keep- ing the solutions for 11 few hours. 13. p...,,lnd. Acad. Sci., 11146, 24A, 251. t 1::1 Q BIC 0.~ Q4 Q3 0.2 0.1 A 14 12 1.0 OB 0.6 Q4 0.2 3~0 390 430 470 Waw lenglh (m!L) + Waw lenglh (m!L) + FIG. I. Absorption spectra. Waw lenglh (m!L) + FIG. I. Absorption spectra. p p A: Norwogouin ulliog a.q. EtCH u bl&Dk :0: Uru nyl nonmgooin complu uaing nor ogonin 110 bl &n A: Norwogouin ulliog a.q. EtCH u bl&Dk :0: Uru.nyl-nonmgooin complu uaing nor..-ogonin 110 bl.&n.k. C Th i iD l 11 uaing i bl k :0: Uru.nyl-nonmgooin complu uaing nor..-ogonin 110 bl.&n.k. C: Thorium-norwogoniD complo.11 uaing norwogonin o.s blank; pH 3.9. ProperlieB of ehe Oomplezu.-The complexes are soluble in aqueous ethanol and aro not extractable in usual organic solvents. No chango in colour was IJ.oticcd on keep- ing the solutions for 11 few hours. ProperlieB of ehe Oomplezu.-The complexes are soluble in aqueous ethanol and aro not extractable in usual organic solvents. No chango in colour was IJ.oticcd on keep- ing the solutions for 11 few hours. 13. p...,,lnd. Acad. Sci., 11146, 24A, 251. 13. p...,,lnd. Acad. Sci., 11146, 24A, 251. 13. p...,,lnd. Acad. Sci., 11146, 24A, 251. RPECTROPHOTI:'METRI(l DETERl\UNATION OF URANU:)! A!IIO THORIUM 1113 Effect of pH.-The offo~t of pH on thorium complex waR Rtudiod botween pH 2.7 and 3.9. Bt•yoncl pH 3.6, precipijjltt.!_on, h ·nvovor, 11tarts on keeping the Nolution fora few hours. During tlto invostigations carrichut at pH !}.9, readings were taken immediately after preps~ tbo S:llutions of the complex. Uranyl-norwogonin complex was found t.o be quite stable fro H 2.7 to 5.fl. Beyond pH 5.0, the reference solution it..self acquired orangt> rolonr aimit .. "''. that of tho complox. For pH adjustments, dilute solutions of 11odium hydroxi1le and hyu.. , , ic acid wore used. Conformity to Beer'BLaw.-In an attompt to draw the calibration curveR for the two complexes, it wos found that both the complexes 1ulhere<l to Beor's law botween the con- centration range of 4 and 20 p.p.m. of moto.l ion. 06 0.5 -~ :! .;! 14 •. Job, An•. rJoim., 1928, z, 9, 113. 1~. Vooburgh RIOd Cooper, J. A mer. Chem. Soc., 1941, 63, 437. UJtt•AKTM•JfT OJ' CniCHJHTKY'. trwrv&.t~.NITY OF IJJn.ur_. ll~UI1·0, EXPEI\ll[E~TAL ] Q4 ~ QJ 02 3 4 s 6 7 8 9 Fla110ne (ml). FIG. 2. Job'a method. Oompoaition of Ike Oomplezes.-The method of t>nntinue!l ve.rie.tion'4• 1 ~ \'I"BS used for determining the composition of the twn complexes. Optical densitios were det.erminod in 40% ethanolic medium at wave lengths ve.rying from 415 ID(L to 430m(L, employing norwogonin solution of the corresponding strength BoB reforence. The results are shown in Fig. 2. From tho pea.ks in the curvos, tho molo.r r&tio of norwogonin to uranyl ion comes to 2:1 an{l in the case of the thorium complex, this ratio is 1:1. 06 0.5 -~ :! .;! ] Q4 ~ QJ 02 3 4 s 6 7 8 9 Fla110ne (ml) Fla110ne (ml). FIG. 2. Job'a method. Oompoaition of Ike Oomplezes.-The method of t>nntinue!l ve.rie.tion'4• 1 ~ \'I"BS used for determining the composition of the twn complexes. Optical densitios were det.erminod in 40% ethanolic medium at wave lengths ve.rying from 415 ID(L to 430m(L, employing norwogonin solution of the corresponding strength BoB reforence. The results are shown in Fig. 2. From tho pea.ks in the curvos, tho molo.r r&tio of norwogonin to uranyl ion comes to 2:1 an{l in the case of the thorium complex, this ratio is 1:1. ~I, I( ,\'r\' J\1, AN II ft. 1'. fHNOH ,"ffru,tur'-~ r•J 14• f -'MnpiP~nt.· -K.,<•ping in view tiLe previous work!-< and the composi- lir.n o£ tf111 llfiiii,VI· ··nnrwuguuln ("OIIIJ'JnJt, the following te~tative structure is assigned. t.n th1• t•nmplt•ll'. ~ - fn I'AHO nf tlt•>rium l't>lllf'II'X, th(l A.Vailahlc cvidcnee is inijufficient to enao.le UR to lllllliJIII a <lnfinit•• •Lrm•tnrl". }]//f-<1 nf /Ji.,erllt. /tm8.-Thn t•ffoct of anions like oxalate, tartrate, thiosulphate, and phnNphat.ll WRK Hturlic<l on lmth tho "omJ>lexcs. These were found to interfere even at •mall <•ont•cntmtionll (7 ttJ H J>. p.m.), uranium or th'lriurn concentratij!,n_ - of.JW!- IIAIIIn rlf<lf!r a• that uf till' ion added. Thu Author•' thankH arc rlne to Prof. T. R. Seshadri, F. R. 8., Hea.d of the Chemistry Urop~<rtrnont. fur his int<•rtoHt anrl cncoura.gernent ancl to Shri D. K. Bhardwaj for his help in tho •.vntht•ais of tho flo. von.,_ Re<•oved .N ov•mhtr 10, 1118~ .. UJtt•AKTM•JfT OJ' CniCHJHTKY'. trwrv&.t~.NITY OF IJJn.ur_. ll~UI1·0, In U>r l'rh, ; .. ,.,., "" I'· Ullin ltJ,. II ""''lllJ>· oondno X 105 mhOH for Sp. conduo, X 10"'!1 mhoa.
W2984583829.txt	https://escholarship.org/content/qt6k11h74h/qt6k11h74h.pdf?t=q5hn78	en		Outcomes and prognostic factors in parotid gland malignancies: A 10‐year single center experience	Laryngoscope investigative otolaryngology	2,019	cc-by	4,759	UC Irvine UC Irvine Previously Published Works Title Outcomes and prognostic factors in parotid gland malignancies: A 10‐year single center experience Permalink https://escholarship.org/uc/item/6k11h74h Journal Laryngoscope Investigative Otolaryngology, 4(6) ISSN 2378-8038 Authors Parikh, Anuraag S Khawaja, Ayaz Puram, Sidharth V et al. Publication Date 2019-12-01 DOI 10.1002/lio2.326 Copyright Information This work is made available under the terms of a Creative Commons Attribution License, availalbe at https://creativecommons.org/licenses/by/4.0/ Peer reviewed eScholarship.org Powered by the California Digital Library University of California Received: 9 July 2019 Revised: 10 October 2019 Accepted: 25 October 2019 DOI: 10.1002/lio2.326 ORIGINAL RESEARCH Outcomes and prognostic factors in parotid gland malignancies: A 10-year single center experience Anuraag S. Parikh MD1,2 \| Ayaz Khawaja MD1 \| Sidharth V. Puram MD, PhD3 Priya Srikanth MD, PhD1 \| Tjoson Tjoa MD4 \| Hang Lee PhD5 \| Rosh K. V. Sethi MD, MPH1,2 James W. Rocco MD, PhD6 \| \| Mustafa Bulbul MD1 \| Mark A. Varvares MD1,2 Kevin S. Emerick MD1,2 \| Daniel G. Deschler MD1,2 \| \| \| Derrick T. Lin MD1,2 1 Department of Otolaryngology, Massachusetts Eye and Ear Infirmary, Boston, Massachusetts 2 Department of Otology and Laryngology, Harvard Medical School, Boston, Massachusetts 3 Department of Otolaryngology–Head and Neck Surgery and Department of Genetics, Washington University School of Medicine, St. Louis, Missouri 4 Department of Otolaryngology, UC Irvine, Orange, California 5 MGH Biostatistics Center, Massachusetts General Hospital, Boston, Massachusetts 6 Department of Otolaryngology, The Ohio State University, Columbus, Ohio Correspondence Anuraag S. Parikh, Department of Otolaryngology, Massachusetts Eye and Ear Infirmary, 243 Charles Street, Boston, MA 02114. Email: anuraag_parikh@meei.harvard.edu Abstract Objectives: To describe a 10-year single center experience with parotid gland malignancies and to determine factors affecting outcomes. Study Design: Retrospective review. Methods: The institutional cancer registry was used to identify patients treated surgically for malignancies of the parotid gland between January 2005 and December 2014. Clinical and pathologic data were collected retrospectively from patient charts and analyzed for their association with overall survival (OS) and disease-free survival (DFS). Results: Two hundred patients were identified. Mean age at surgery was 57.8 years, and mean follow-up time was 52 months. One hundred two patients underwent total parotidectomy, while 77 underwent superficial parotidectomy, and 21 underwent deep lobe resection. Seventy patients (35%) required facial nerve (FN) sacrifice. Acinic cell carcinoma was the most common histologic type (22%), followed by mucoepidermoid carcinoma (21.5%) and adenoid cystic carcinoma (12.5%). Twentynine patients (14.5%) experienced recurrences, with mean time to recurrence of 23.6 months (range: 1-82 months). Five- and 10-year OS were 81% and 73%, respectively. Five- and 10-year DFS were 80% and 73%, respectively. In univariate analyses, age > 60, histologic type, positive margins, high grade, T-stage, node positivity, Presented as poster presentation at: Triological Society 2017 Combined Sections Meeting, 19 to 21 January 2017, New Orleans, LA. This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made. © 2019 The Authors. Laryngoscope Investigative Otolaryngology published by Wiley Periodicals, Inc. on behalf of The Triological Society. 632 wileyonlinelibrary.com/journal/lio2 Laryngoscope Investigative Otolaryngology. 2019;4:632–639. 633 PARIKH ET AL. perineural invasion, and FN involvement were predictors of OS and DFS. In the multivariate analysis, histology, positive margins, node positivity, and FN involvement were independent predictors of OS and DFS. Conclusions: Our single-center experience of 200 patients suggests that histology, positive margins, node positivity, and FN involvement are independently associated with outcomes in parotid malignancies. Level of Evidence: 4 KEYWORDS acinic cell carcinoma, adenoid cystic carcinoma, mucoepidermoid carcinoma, parotid carcinoma, parotid gland, salivary glands 1 I N T RO DU CT I O N \| underwent surgery for primary malignancy of the parotid gland at MEEI, a tertiary referral center in Boston, Massachusetts, between Neoplasms of the salivary glands comprise a diverse group of at least January 2005 and December 2014. Patient charts were reviewed, and 20 histologically distinct disease entities and frequently pose diagnostic relevant demographic, clinical, and pathologic data were recorded. and management challenges.1 This may be further complicated by histo- Overall survival (OS) was defined as the time elapsed between logic diversity within the same surgical specimen and co-occurrence with the date of surgery and the date of last documented communication benign entities (eg, carcinoma ex pleomorphic adenoma).2 Together, sali- with the patient. Disease-free survival (DFS) was calculated as the vary gland neoplasms account for approximately 6% of all head and neck time elapsed between the date of surgery and the date of last docu- neoplasms,3 with an annual incidence of 0.5 to 2.0 per 100 000 in the mented note from an oncologic provider (surgeon, medical oncologist, general population.4 Approximately 80% of these tumors involve the or radiation oncologist). For patients who had a documented recur- parotid gland. An estimated 20% to 25% are malignant in nature, with rence, DFS was calculated as the time elapsed between the date of mucoepidermoid carcinoma described as the most common histology. surgery and the date of documented recurrence. There are differences in incidence between males and females, with For survival analyses, all patients were assumed to be censored mucoepidermoid carcinomas more common in males and acinic cell and unless documented as deceased (for analyses of OS) or having a recur- adenoid cystic carcinomas found more commonly among females.5 rence (for analyses of DFS). For Cox proportional hazards models, all Surgical resection is the primary treatment for salivary malig- predictor variables were simplified to binary values as follows: age nancy. Prognosis varies widely by histologic subtype, with survival classified as >60 or ≤60, T-stage classified as low (T1 or T2) or high ranging from 95% to 100% for polymorphous low-grade adenocarci- (T3 or T4), N-stage classified as negative (N0) or positive (N1, N2, or noma6 to 23% to 50% in cases of high-grade mucoepidermoid carci- N3), overall stage classified as early (stage I or II) or advanced (stage III 7,8 A variety of factors have been evaluated for prognostic or IV), grade classified as low (grade 1) or high (grade 2 or 3), and mar- significance, including histological subtype, tumor grade and stage, gin status classified as negative or positive (microscopically or grossly cervical lymphadenopathy, facial nerve (FN) involvement, perineural positive). JMP-Pro version 13 (SAS) was used to generate Kaplan- invasion (PNI), and positive surgical margins.9-12 Molecular markers Meier survival curves and for univariate and multivariate Cox propor- have also been discovered that are associated with prognosis and tional hazards models. noma. survival,13,14 but none are widely used in clinical practice. In addition, detailed single institution reviews of parotid malignancies over a substantial time period remain limited. We therefore sought to utilize our 3 \| RE SU LT S own experience to provide insight into demographics and clinical features of parotid gland malignancies over a 10-year period, and thereby Two hundred patients (104 men, 96 women, with average age of determine clinical and pathologic factors affecting survival and 57.8 years [range: 10-96 years]) were treated surgically for primary recurrence. malignancies of the parotid gland at MEEI between January 2005 and December 2014. The most common histologies were acinic cell carcinoma (N = 44), mucoepidermoid carcinoma (N = 43), adenoid cystic 2 \| MATERIALS AND METHODS carcinoma (N = 25), salivary duct carcinoma (N = 18), and carcinoma ex pleomorphic adenoma (N = 15). See Table 1 for a summary of his- This study was reviewed by the Massachusetts Eye and Ear Infirmary tologic subtypes. For Kaplan-Meier curves and Cox proportional haz- (MEEI) institutional review board and deemed to be of minimal risk. ards models, all histologic types other than these top five were The institutional cancer registry was used to identify all patients who grouped under the category “Other Histologies.” 634 PARIKH ET AL. TABLE 1 Summary of histologic types Histology 15.5%), and T4 (n = 46, 23%). One hundred fifty-seven patients N (78.5%) were N0, with 31 being pN0 and 126 being cN0. The remain- % der were N1 (n = 15, 7.5%), N2 (n = 27, 13.5%), and N3 (n = 1, 0.5%). Acinic cell carcinoma 44 22 Mucoepidermoid carcinoma 43 21.5 Adenoid cystic carcinoma 25 12.5 Salivary duct carcinoma 18 9 Carcinoma ex pleomorphic adenoma 15 7.5 reporting was incomplete, with 82 patients (41%) having no grade Squamous cell carcinoma 14 7 reported. All 18 cases of salivary duct carcinoma were high grade, and Histologic grade was determined at the time of original pathologic diagnosis and was classified as low grade (grade 1) or high grade (grade 2 or 3). Grade 2 tumors were classified as high grade to keep approximately equal numbers of high and low grade tumors. Grade Adenocarcinoma NOS 5 2.5 of the remaining cases, 55 (27.5% of total) were high grade, while Cystadenocarcinoma 5 2.5 45 (22.5% of total) were low grade. FN involvement was seen in Epithelial-myoepithelial carcinoma 4 2 44 patients (22%), PNI in 51 (25.5%), LVI in 26 (13%), and ECE in Basal cell adenocarcinoma 3 1.5 5 (2.5% overall, 10% of node-positive). Sixty-eight patients (34%) had Lymphoepithelial carcinoma 3 1.5 microscopic or grossly positive margins, while 132 (66%) had negative Mammary analog secretory carcinoma 3 1.5 margins. Associations between histologic type and clinicopathologic Neuroendocrine carcinoma 3 1.5 features are shown in Table 2. Basaloid carcinoma 2 1 Carcinoma NOS 2 1 Merkel cell carcinoma 2 1 Myoepithelial carcinoma 2 1 Adenocarcinoma, ductal type 1 0.5 mented as deceased over the follow-up period. There were 29 docu- Adenosquamous carcinoma 1 0.5 mented recurrences (14.5% of cases), with a mean time to recurrence Clear cell carcinoma 1 0.5 of 23.6 months. Distant recurrence (18/29 cases, 62%) was more Myofibroblastic sarcoma 1 0.5 common than local (11/29 cases, 38%) or regional (1/29 cases, 3%) Rhabdomyosarcoma 1 0.5 recurrence, with the most common sites for metastasis being the lung Sebaceous adenocarcinoma 1 0.5 (14/18 cases), brain, bone, mediastinum, liver, and peritoneum. Cases Spindle cell carcinoma 1 0.5 of local recurrence were found in the parotid bed (5/11 cases), tempo- Total 200 100 3.3 \| Follow-up and recurrence Mean follow-up time was 4.1 years, and 36 patients (18%) were docu- ral bone, external auditory canal, and infratemporal fossa. One patient experienced both local and regional recurrence, while one patient Abbreviation: NOS, not otherwise specified. experienced both local recurrence and distant metastasis. Four of 11 patients with local recurrence underwent salvage surgery. 3.1 \| Treatment regimens Of 200 patients, 77 (33.5%) underwent superficial parotidectomy, 3.4 \| Survival analysis 21 (10.5%) underwent deep lobe resection, 14 (7%) underwent subtotal parotidectomy, and 88 (44%) underwent total parotidectomy. Total OS (Figure 1A) was 87%, 81%, and 73%, while total DFS Seventy patients (35%) underwent sacrifice of one or more branches (Figure 1B) was 84%, 80%, and 73% at 2 years, 5 years, and 10 years, of the FN. Seventy-four patients (37%) underwent cervical respectively. There was a significant impact of histology on OS and lymphadenectomy. Seventy-seven patients (38.5%) underwent sur- DFS, with salivary duct carcinoma and other histologies having the gery alone, while the remainder received adjuvant therapy, with poorest OS and DFS at 2 and 5 years and adenoid cystic carcinoma, 81 (41%) receiving adjuvant radiation therapy (XRT) alone, 2 (1%) with a significant number of late recurrences and late deaths, having receiving adjuvant chemotherapy alone, and 39 (19.5%) receiving the poorest OS and DFS at 10 years (P = .0016 and .0012 for OS and adjuvant chemotherapy and XRT. DFS, respectively). Accordingly, adenoid cystic carcinoma had a significantly higher average time to recurrence than all other histologic types (3.92 vs 1.29 years, P = .0017). 3.2 \| Pathologic characteristics Patient charts were retrospectively reviewed for data regarding 3.5 \| Predictors of survival AJCC tumor-node-metastasis (TNM) staging, margin status, grade, FN involvement, PNI, lymphovascular invasion (LVI), and extracapsular In the univariate Cox proportional hazards model (n = 200 unless oth- extension (ECE). Patients were distributed across all T stages, includ- erwise specified), predictors of OS and DFS included age > 60 (hazard ing Tis (n = 6, 3%), T1 (n = 66, 33%), T2 (n = 51, 25.5%), T3 (n = 31, ratio, HR [OS] = 4.1, P = .0001; HR [DFS] = 3.5, P < .0001), “other” 635 PARIKH ET AL. TABLE 2 Breakdown of histopathologic characteristics by tumor histology Carcinoma ex pleo Other histology P value (chi-square) 5 (28) 13 (87) 38 (69) <.0001 13 (72) 2 (13) 17 (31) 22 (88) 6 (33) 12 (80) 39 (71) 3 (12) 12 (67) 3 (20) 16 (29) 28 (65) 10 (59) 0 (0) 0 (0) 3 (10) 4 (57) 15 (35) 7 (41) 18 (100) Neg 34 (77) 33 (77) 6 (24) 10 (56) 12 (80) 37 (67) Pos 10 (23) 10 (23) 19 (76) 8 (44) 3 (20) 18 (33) No 42 (96) 42 (98) 25 (100) 10 (56) 12 (80) 43 (78) Yes 2 (4) 1 (2) 0 (0) 8 (44) 3 (20) 12 (22) 36 (82) 30 (70) 12 (48) 3 (17) 9 (60) 17 (31) 8 (18) 13 (30) 13 (52) 15 (83) 6 (40) 38 (69) 43 25 18 Acinic cell Mucoepidermoid Adenoid cystic No 42 (95) 39 (91) 12 (48) Yes 2 (5) 4 (9) 13 (52) N0 40 (91) 38 (88) N+ 4 (9) 5 (12) Low 3 (43) High Salivary duct PNI N stage <.0001 Grade 2 (100) <.0001 28 (90) Margin .0001 LVI <.0001 Stage Early Adv Total N 44 15 <.0001 55 Note: There were significant associations of tumor histology with all histopathologic characteristics. Percentages are shown in parentheses. Abbreviations: Adv, advanced; LVI, lymphovascular invasion; Neg, negative; Pleo, pleomorphic; PNI, perineural invasion; Pos, positive. histology (HR [OS] = 3.2, P = .0008; HR [DFS] = 3.0, P = .0004), posi- Our total OS and DFS are consistent with prior reports in the lit- tive margins (HR [OS] = 3.0, P = .001; HR [DFS] = 3.3, P < .0001), high erature of good prognosis with surgically treated disease.6,12,15-20 In a grade (n = 118; HR [OS] > 100, P < .0001; HR [DFS] > 100, report on 2062 patients with parotid carcinomas from the Swedish P < .0001), advanced T-stage (HR [OS] = 4.5, P < .0001; HR cancer registry, the 10 year survival was 71.6% and the figure did not [DFS] = 5.2, P < .0001), node positivity (HR [OS] = 3.8, P = .0002, HR change significantly from 1960 to 1995.12 Consistent with prior stud- [DFS] = 4.0, P < .0001), PNI (HR [OS] = 3.8, P < .0001; HR [DFS] = 3.8, ies, we found a significant impact of histologic subtype on OS and P < .0001), and FN involvement (HR [OS] = 4.7, P < .0001; HR DFS. At 2 and 5 years, salivary ductal carcinoma had the poorest OS [DFS] = 4.8, P < .0001). ECE (n = 50, HR [OS] = 2.9, P = .21; HR of 77% and 68%, respectively, consistent with prior reports of poor [DFS] = 4.3, P < .05) was associated with DFS but not OS, and LVI survival in patients with this malignancy but higher than previously (HR [OS] = 1.6, P = .42; HR [DFS] = 2.1, P = .06) was not associated quoted rates of only 20% to 35%.21 At 10 years, adenoid cystic carci- with either outcome measure. noma had the poorest OS at 53%, consistent with a propensity for late All predictors that showed significance in the univariate model were recurrence, PNI, and distant metastasis. Prior reports of 5-year sur- tested in the multivariate model, except for grade which was incom- vival ranged from 35% to 70%.22,23 Acinic cell and mucoepidermoid pletely reported and PNI, which was redundant with FN involvement. In carcinomas had the best OS at 2, 5, and 10 years. It is notable that in the multivariate model, histology “other,” positive margins, node positiv- our study, survival was defined by last known communication, which ity, and FN involvement were found to be significant predictors of OS may underestimate potential survival since loss to follow-up or trans- and DFS, and age > 60 showed a trend in this direction (Table 3). fer of care to alternate providers with incomplete communication is Kaplan-Meier survival curves for OS and DFS stratified by margin sta- common in a tertiary referral center. tus, node positivity, and FN involvement are shown in Figure 2. In our cohort, the recurrence rate was 14.5% and the mean time to recurrence was 2 years, consistent with reported recurrence rates of 18.4% to 24.1%.15,17,18 In our cohort, distant metastasis, which was 4 \| DISCUSSION most common in salivary ductal carcinoma (6/18 cases, 33%), was more common than locoregional recurrence, consistent with prior Here, we report on 200 consecutive cases of parotid malignancies at studies.15,18 Salivary ductal carcinomas had the highest rates of early our institution over a 10-year period. We found that histologic sub- recurrence, while adenoid cystic carcinoma had the longest time to type, positive margins, node positivity, and FN involvement were all recurrence (3.29 years) and the greatest rate for recurrence by independent predictors of poor prognosis. 10 years, with an overall recurrence rate of 24%. This propensity for 636 PARIKH ET AL. recurrence at 10 years with good DFS at 2 and 5 years underscores growth the importance of continued surveillance of patients with adenoid manifested by sudden rapid growth, local invasiveness, and distant quiescence, followed by aggressive transformation cystic carcinoma well beyond the 5-year time point, which is gener- metastatic spread.23 To our knowledge, the molecular correlates of ally considered to be curative for other malignancies. Biologically, such an aggressive transformation have not been demonstrated and this late recurrence has been attributed to an initial period of relative warrant further investigation in an effort to guide therapies targeting these lesions. Several factors were noted to impact survival rates. OS and DFS were significantly impacted by FN involvement (Figure 2E,F) and Nstage (Figure 2C,D), and age > 60 showed a trend (Table 3). Many other studies have similarly demonstrated that older age at presentation is associated with worse survival outcomes,15,17 perhaps reflecting the more aggressive nature of late-presenting disease, as well as the fact that older individuals are likely to have more comorbidities, poorer functional status, or weakened immunity.6,17,19 FN involvement, which was seen in 44 patients (22%) in our cohort, has previously been demonstrated to be independently associated with survival outcomes,17,19 though it remains unclear whether this represents a manifestation of more locally aggressive tumors with skip lesions that are difficult to completely resect or a propensity for distant metastasis, as is seen in adenoid cystic carcinoma, which is most classically associated with perineural spread. Nodal involvement was associated with significant reduction in survival. Consistent with prior studies,17,19 5-year OS and DFS, which were 56% and 46%, respectively, in node-positive patients and 88% and 83%, respectively, in node-negative patients (Figure 2C,D). The overall rate of node positivity was 21.5%, which is similar to previous reports of 18% to 28%.16,17,24 However, in our cohort, this was significantly associated with intrinsic factors related to the disease entity, as less than 20% of acinic cell, mucoepidermoid, and adenoid cystic carcinomas demonstrated node positivity, while 66% of salivary ductal carcinomas had nodal metastasis at the time of presentation. These findings suggest that patients with salivary ductal carcinomas should uniformly receive cervical lymphadenectomy at the time of primary tumor removal, while those with other histologic types may be considered on an individualized basis depending on other clinical and pathologic features of the primary tumor. T-stage, tumor grade, and margin status were also found to be F I G U R E 1 Kaplan-Meier curves of A, overall survival and B, disease-free survival by histologic type show a highly significant impact of histology on survival Predictor HR (OS) [95% CI] P value associated OS and DFS in the univariate analysis, with margin status not surprisingly retaining significance on multivariate analysis (Table 3, Figure 2A,B). Due to incomplete reporting, tumor grade was removed HR (DFS) [95% CI] P value Age > 60 2.17 [0.99-5.27] .05 1.81 [0.93-3.76] .08 Histology “Other” 3.23 [1.54-6.81] .002 3.08 [1.59-5.99] .001 Positive margin 2.05 [1.01-4.25] <.05 2.29 [1.21-4.38] .01 T stage (1/2 vs 3/4) 1.52 [0.66-3.67] .33 1.83 [0.87-4.02] .11 N stage (N0 vs N+) 2.05 [1.02-4.06] .04 1.98 [1.06-3.68] .03 FN involvement 2.62 [1.17-5.80] .02 2.32 [1.15-4.66] .02 Note: Although it was significant in the univariate model, grade was eliminated from the multivariate model due to incomplete reporting. Abbreviations: CI, confidence interval; DFS, disease-free survival; FN, facial nerve; HR, hazard ratio; OS, overall survival. T A B L E 3 Multivariate Cox proportional hazards model for predictors of OS and DFS PARIKH ET AL. 637 F I G U R E 2 Kaplan-Meier curves of overall survival and disease-free survival by A and B, margin status, C and D, N stage, and E and F, facial nerve involvement show highly significant impacts of each of these variables on survival from the multivariate analysis, though we anticipate that it remains a Limitations of our study include its retrospective nature, reliance significant factor in determining prognosis. In mucoepidermoid and on the documentation in the electronic medical record, and approxi- acinic cell carcinomas, for example, prior studies have shown 5-year mation of survival by last known communication with the patient. We OS to be as high as 100% in low-grade tumors, decreasing to 50% for recognize that this approach to determining survival may underesti- higher grade tumors.25,26 At minimum, we conclude that more uni- mate true survival, given lack of follow-up, or transfer of care from a form reporting and additional study of the prognostic impact of tumor tertiary referral center to alternate providers with no documented grade in salivary gland malignancies is warranted. T-stage lost signifi- communication. We also acknowledge that it may be biased by pro- cance in the multivariate analysis, and we hypothesize that this finding vider determined need for follow-up and may particularly affect strati- suggests that other clinicopathologic measures better captured the fication by histology. However, our results of survival, both overall biologic impact of larger tumors. and stratified by histology, are consistent with prior reports in the 638 PARIKH ET AL. literature, thus supporting the validity of our data. The diverse group of histopathologic subtypes makes it challenging to comment upon how the specific histologic subtype may affect long-term outcomes, 6. particularly for less common malignancies. Moreover, we recognize that classification of parotid malignancies has evolved in the past 7. decade, leading to potential underreporting of secretory carcinomas and insufficient classification of carcinoma ex pleomorphic adenomas. 8. In addition, incomplete reporting limited our comment on the impact of tumor grade and highlight the importance of histopathologic assessment of this metric, as it is likely that grade does have an impact on outcomes. However, we hope that our relatively large, single insti- 9. tution cohort of parotid malignancies can contribute to an understanding of the relative demographics and differences in prognosis of the variety of salivary gland malignancies commonly treated by the 10. head and neck surgeon. 11. 5 \| C O N CL U S I O N S We report outcomes on 200 consecutive patients with malignancies 12. of the parotid gland managed surgically at a single institution. In our cohort, we found that histologic subtype, N-stage, and FN involve- 13. ment were independent predictors of both OS and DFS. Tumors such as salivary ductal carcinoma were associated with early recurrence, whereas adenoid cystic carcinoma was associated with late 14. recurrence. CONF LICT OF IN TE RE ST 15. The authors have no financial disclosures or conflict of interest. ORCID Anuraag S. Parikh https://orcid.org/0000-0003-0968-1028 Sidharth V. Puram https://orcid.org/0000-0003-3543-889X Rosh K. V. Sethi Mark A. Varvares 16. https://orcid.org/0000-0002-1508-1363 https://orcid.org/0000-0002-6570-5672 ENDNOTE 17. 18. HR > 100 in this case represents the infinite solution resulting from the fact that there were no events in the low-grade group, thus making this a highly significant effect. 19. RE FE R ENC E S 20. 1. Barnes LEJ, Reichart P, Sidransky D. World Health Organization Classification of Tumours: Pathology and Genetics of Head and Neck Tumours. Lyon, France: IARC Press; 2005. 2. Speight PM, Barrett AW. Salivary gland tumours. Oral Dis. 2002;8(5): 229-240. 3. Carvalho AL, Nishimoto IN, Califano JA, Kowalski LP. Trends in incidence and prognosis for head and neck cancer in the United States: a site-specific analysis of the SEER database. Int J Cancer. 2005;114(5): 806-816. 4. Parkin DM, Ferlay J, Curado MP, et al. Fifty years of cancer incidence: CI5 I-IX. Int J Cancer. 2010;127(12):2918-2927. 5. Boukheris H, Curtis RE, Land CE, Dores GM. Incidence of carcinoma of the major salivary glands according to the WHO classification, 21. 22. 23. 1992 to 2006: a population-based study in the United States. Cancer Epidemiol Biomarkers Prev. 2009;18(11):2899-2906. Castle JT, Thompson LD, Frommelt RA, Wenig BM, Kessler HP. Polymorphous low grade adenocarcinoma: a clinicopathologic study of 164 cases. Cancer. 1999;86(2):207-219. Clode AL, Fonseca I, Santos JR, Soares J. Mucoepidermoid carcinoma of the salivary glands: a reappraisal of the influence of tumor differentiation on prognosis. J Surg Oncol. 1991;46(2):100-106. Guzzo M, Andreola S, Sirizzotti G, Cantu G. Mucoepidermoid carcinoma of the salivary glands: clinicopathologic review of 108 patients treated at the National Cancer Institute of Milan. Ann Surg Oncol. 2002;9(7):688-695. Carrillo JF, Vazquez R, Ramirez-Ortega MC, Cano A, OchoaCarrillo FJ, Onate-Ocana LF. Multivariate prediction of the probability of recurrence in patients with carcinoma of the parotid gland. Cancer. 2007;109(10):2043-2051. Cederblad L, Johansson S, Enblad G, Engstrom M, Blomquist E. Cancer of the parotid gland; long-term follow-up. A single centre experience on recurrence and survival. Acta Oncol. 2009;48(4):549-555. Vander Poorten VL, Hart AA, van der Laan BF, et al. Prognostic index for patients with parotid carcinoma: external validation using the nationwide 1985-1994 Dutch Head and Neck Oncology Cooperative Group database. Cancer. 2003;97(6):1453-1463. Wahlberg P, Anderson H, Biorklund A, Moller T, Perfekt R. Carcinoma of the parotid and submandibular glands—a study of survival in 2465 patients. Oral Oncol. 2002;38(7):706-713. Miyabe S, Okabe M, Nagatsuka H, et al. Prognostic significance of p27Kip1, Ki-67, and CRTC1-MAML2 fusion transcript in mucoepidermoid carcinoma: a molecular and clinicopathologic study of 101 cases. J Oral Maxillofac Surg. 2009;67(7):1432-1441. Rao PH, Roberts D, Zhao YJ, et al. Deletion of 1p32-p36 is the most frequent genetic change and poor prognostic marker in adenoid cystic carcinoma of the salivary glands. Clin Cancer Res. 2008;14(16):51815187. Al-Mamgani A, van Rooij P, Verduijn GM, Meeuwis CA, Levendag PC. Long-term outcomes and quality of life of 186 patients with primary parotid carcinoma treated with surgery and radiotherapy at the Daniel den Hoed Cancer Center. Int J Radiat Oncol Biol Phys. 2012;84(1): 189-195. Calearo C, Pastore A, Storchi OF, Polli G. Parotid gland carcinoma: analysis of prognostic factors. Ann Otol Rhinol Laryngol. 1998;107 (11 pt 1):969-973. Chang JW, Hong HJ, Ban MJ, et al. Prognostic factors and treatment outcomes of parotid gland cancer: a 10-year single-center experience. Otolaryngol Head Neck Surg. 2015;153(6):981-989. Erovic BM, Shah MD, Bruch G, et al. Outcome analysis of 215 patients with parotid gland tumors: a retrospective cohort analysis. J Otolaryngol Head Neck Surg. 2015;44:43. Kopec T, Mikaszewski B, Jackowska J, Wasniewska-Okupniak E, Szyfter W, Wierzbicka M. Treatment of parotid malignancies10 years of experience. J Oral Maxillofac Surg. 2015;73(7):1397-1402. Lima RA, Tavares MR, Dias FL, et al. Clinical prognostic factors in malignant parotid gland tumors. Otolaryngol Head Neck Surg. 2005; 133(5):702-708. Barnes L, Rao U, Krause J, Contis L, Schwartz A, Scalamogna P. Salivary duct carcinoma. Part I. A clinicopathologic evaluation and DNA image analysis of 13 cases with review of the literature. Oral Surg Oral Med Oral Pathol. 1994;78(1):64-73. Khafif A, Anavi Y, Haviv J, Fienmesser R, Calderon S, Marshak G. Adenoid cystic carcinoma of the salivary glands: a 20-year review with long-term follow-up. Ear Nose Throat J. 2005;84(10):662, 664-667. Ko YH, Lee MA, Hong YS, et al. Prognostic factors affecting the clinical outcome of adenoid cystic carcinoma of the head and neck. Jpn J Clin Oncol. 2007;37(11):805-811. 639 PARIKH ET AL. 24. Zbaren P, Schupbach J, Nuyens M, Stauffer E, Greiner R, Hausler R. Carcinoma of the parotid gland. Am J Surg. 2003;186(1):57-62. 25. Gomez DR, Katabi N, Zhung J, et al. Clinical and pathologic prognostic features in acinic cell carcinoma of the parotid gland. Cancer. 2009; 115(10):2128-2137. 26. Nance MA, Seethala RR, Wang Y, et al. Treatment and survival outcomes based on histologic grading in patients with head and neck mucoepidermoid carcinoma. Cancer. 2008;113(8):2082-2089. How to cite this article: Parikh AS, Khawaja A, Puram SV, et al. Outcomes and prognostic factors in parotid gland malignancies: A 10-year single center experience. Laryngoscope Investigative Otolaryngology. 2019;4:632–639. https://doi.org/10.1002/lio2.326
https://openalex.org/W3093608596	https://refubium.fu-berlin.de/bitstream/fub188/29320/1/fnbeh-14-590999.pdf	English	null	Mushroom Body Extrinsic Neurons in Walking Bumblebees Correlate With Behavioral States but Not With Spatial Parameters During Exploratory Behavior	Frontiers in behavioral neuroscience	2,020	cc-by	10,676	ORIGINAL RESEARCH published: 20 October 2020 doi: 10.3389/fnbeh.2020.590999 Citation: Jin N, Paffhausen BH, Duer A and Menzel R (2020) Mushroom Body Extrinsic Neurons in Walking Bumblebees Correlate With Behavioral States but Not With Spatial Parameters During Exploratory Behavior. Keywords: bumblebee, mushroom body extrinsic neurons, behavioral states, exploratory behavior, artiﬁcial arena environment Mushroom Body Extrinsic Neurons in Walking Bumblebees Correlate With Behavioral States but Not With Spatial Parameters During Exploratory Behavior Nanxiang Jin†, Benjamin H. Paffhausen, Aron Duer and Randolf Menzel Institut für Biologie – Neurobiologie, Freie Universität Berlin, Berlin, Germany Nanxiang Jin†, Benjamin H. Paffhausen, Aron Duer and Randolf Menzel Institut für Biologie – Neurobiologie, Freie Universität Berlin, Berlin, Germany Central place foraging insects like honeybees and bumblebees learn to navigate efﬁciently between nest and feeding site. Essential components of this behavior can be moved to the laboratory. A major component of navigational learning is the active exploration of the test arena. These conditions have been used here to search for neural correlates of exploratory walking in the central arena (ground), and thigmotactic walking in the periphery (slope). We chose mushroom body extrinsic neurons (MBENs) because of their learning-related plasticity and their multi-modal sensitivities that may code relevant parameters in a brain state-dependent way. Our aim was to test whether MBENs code space-related components or are more involved in state-dependent processes characterizing exploration and thigmotaxis. MBENs did not respond selectively to body directions or locations. Their spiking activity differently correlated with walking speed depending on the animals’ locations: on the ground, reﬂecting exploration, or on the slope, reﬂecting thigmotaxis. This effect depended on walking speed in different ways for different animals. We then asked whether these effects depended on spatial parameters or on the two states, exploration and thigmotaxis. Signiﬁcant epochs of stable changes in spiking did not correlate with restricted locations in the arena, body direction, or walking transitions between ground and slope. We thus conclude that the walking speed dependencies are caused by the two states, exploration and thigmotaxis, rather than by spatial parameters. Edited by: Etsuro Ito, Waseda University, Japan Waseda University, Japan Reviewed by: Takayuki Watanabe, Hokkaido University, Japan Shigehiro Namiki, The University of Tokyo, Japan Correspondence: Nanxiang Jin neil.jnx@163.com †Present address: Nanxiang Jin, A.I. Virtanen Institute for Molecular Sciences, University of Eastern Finland, Kuopio, Finland Reviewed by: Takayuki Watanabe, Hokkaido University, Japan Shigehiro Namiki, The University of Tokyo, Japan Correspondence: Nanxiang Jin neil.jnx@163.com †Present address: Nanxiang Jin, †Present address: Nanxiang Jin, A.I. Virtanen Institute for Molecular Sciences, University of Eastern Finland, Kuopio, Finland Specialty section: This article was submitted to Learning and Memory, a section of the journal Frontiers in Behavioral Neuroscience Received: 03 August 2020 Accepted: 16 September 2020 Published: 20 October 2020 Behavioral Apparatus The apparatus (arena environment, AE) was modiﬁed for our needs based on a previous description (Duer et al., 2015; Figure 1) with the aim of exposing the searching bumble bee to two diﬀerent areas, the open ﬁeld (termed “ground,” including a local cue at a feeding place) and the surrounding slippery slope area that signaled to the animal the border of the arena. In brief, a crimson ceramic plate (rim diameter 35 cm, bottom diameter 24 cm, height of rim 4.5 cm) was placed in the center of a platform (24.5 cm from above the room ground). A round piece of white cardboard paper (diameter 22 cm) was ﬁxed to the center of the ceramic plate surrounded by a crimson-white color contrast boundary (“boundary”). A round piece of blue plastic cardboard (3.5 cm diameter) carrying sucrose solution was placed on the white paper at any location between the center and the edge of ground paper. This blue disk functioned as a local cue to be learned by the bee as a feeding place. The surrounding surface of the plate slope (“slope”) was sprayed with Teﬂon (dry PTFE Spray, Ballistol, Aham, Germany) such that walking bees could not climb over the rim of the plate. Several attempts have been made in the past to include operant behavior in the search for high order integration processes in the honeybee. Neural correlates were found for social interactions and active exploration-like behavior (Duer et al., 2015; Paﬀhausen et al., 2020). Active visual learning could be transferred at least partially to a virtual reality condition in which the bee performed stationary walking on an air-supported ball and controlled the visual environment (Buatois et al., 2018). Mushroom body extrinsic neurons (MBENs) were recorded under these conditions (Zwaka et al., 2019) and neural correlates of operant forms of visual learning could be demonstrated. Most MBENs form dense banding patterns in restricted zones in the vertical lobe and project into the calyx, pedunculus, β-lobe, and the protocerebral lobe (Rybak and Menzel, 1993). MBENs are known to respond to multiple sensory conditions in a integrative or combinatorial way and they are sensitive to the context in which stimuli appear (Filla and Menzel, 2015; Zwaka et al., 2018). We thus expected neural correlates for exploration in MBENs. INTRODUCTION Animals exposed to a novel environment perform typical searching movements during which they explore the environment. Exploration is an active form of learning about the features of the environment that calibrates the reference systems for moving throughout space and time. Social animals are particularly dependent on exploratory learning since they need to return reliably and safely to their home sites. Social bees like honeybees and bumble bees exhibit multiple Front. Behav. Neurosci. 14:590999. doi: 10.3389/fnbeh.2020.590999 October 2020 \| Volume 14 \| Article 590999 Frontiers in Behavioral Neuroscience \| www.frontiersin.org Jin et al. Jin et al. Bumblebee MBENs Reﬂect Behavioral States active exploration or with the behavioral states of exploration and thigmotactic escape. forms of exploratory learning both for local navigation and for way-ﬁnding over greater distances (Srinivasan et al., 2004; Collett et al., 2006; Menzel, 2017). Landmarks need to be distinguished and used for navigation with respect to their spatial properties and identities. MATERIALS AND METHODS Neural systems for navigation are well studied in mammals (Rowland et al., 2016), but little is known in insects. The central complex has been related to the sun compass (Homberg, 2004; Collett, 2019; Currier and Nagel, 2020), and the mushroom body (MB) to multiple forms of learning about object identities (Menzel, 2014). In most of these studies the animals were not able to actively explore the environment, rather they were restricted to experimental conditions that allowed researchers to combine neural recordings with tests of innate control mechanisms and Pavlovian forms of associative learning (Menzel et al., 2007). Active exploration, however, is a major component in navigation and needs to be included in the search for neural correlates as it is the case in studies with rats (Moser et al., 2008), bats (Ulanovsky and Moss, 2007), and cockroaches (Mizunami et al., 1998b). Spatial restriction of the usually small test area often leads to switches between exploration of the open space and escape behavior as indicated by thigmotactic runs along the conﬁning walls. It is, therefore, essential to separate neural correlates of spatial coding from state-dependent behavioral states that cause the animal to switch between exploration and thigmotaxis. Animals Bumblebee (Bombus terrestris) colonies (Schneckenproﬁ, Hennstedt, Germany) were kept in a glasshouse under controlled temperature ranging from 21 to 27◦C. Fresh sucrose solution (30% in volume) and pollen powder were provided daily ad libitum. Forager bumblebees were caught at the feeder, and then their right wings were cut so that they could only walk on the bottom of the ceramic plate (Figures 1A–C). Behavioral Apparatus A wooden dome with a grounded metal mesh hung over the arena and could be lowered down to cover the ceramic plate (Figures 1B,C). In this position it acted as a Faraday cage together with the metal mesh below the ceramic ground plate. The dome was composed of 4 duplications of equal sized trapezoids (short edge 42 cm, long edge 60 cm, height 79 cm). Simple patterns in diﬀerent colors were pasted on inner walls, serving as external cues (panorama). White and infrared LED matrices were symmetrically arranged on the ceiling of the dome. A web camera (Pro 9000, Logitech, Apples, Switzerland) was ﬁxed on top of the ceiling to record movement of the bumblebee through a small hole in the ceiling. Videos were taken with a frame rate of 10fps and a resolution of 1600 by 1200 pixels. After each recording (ended when units stopped ﬁring), non-paper materials were cleaned with 70% ethanol, the ground paper was exchanged, and Teﬂon was re-sprayed and dried at room temperature. p p Bumble bees are more cooperative than honeybees in experimental setups that aim to move essential components of navigation into the laboratory (Jin et al., 2014). Most interestingly they guided their walks with respect to only the panorama. As expected, they also showed thigmotactic escape behavior during which they did not guide their walking trajectories to local or panorama cues. Similarly, in the present study bumblebees performed exploratory runs in a small round arena experiencing for the ﬁrst time a novel environment consisting of an unstructured ground plate. Bumblebees walked actively on the arena ground covering most of the space in multiple trajectories and ran along the slope when trying to escape. This arrangement allowed us to address the question whether the neural activity of the recorded MBENs correlated with spatial experience during Frontiers in Behavioral Neuroscience \| www.frontiersin.org Analysis of Neural Activity The neural activity of single units was manually sorted from multi-units data on the basis of peak-to-trough amplitude and wave shape, and were further conﬁrmed with PCA clustering and event interval analysis, using Spike 2 software (version 7, Cambridge Electronic Design Limited, Cambridge, United Kingdom). Units were included in the analysis if their spike widths (from peak to peak) were longer than 0.6 ms and shorter than 1.2 ms. Recordings longer than 2 min from non-paralyzed bees were included into the database. Customized Matlab (version 2016b, MathWorks, Natick, MA, United States) scripts were written for tracking bee locations from video frames, analyzing neural recordings and statistics. The raw neural data were ﬁrst organized in a time resolution of 100 ms, and then instant animal locations and neural activity were synchronized by time stamps. Next, bee trajectories were smoothed with three ﬁlters: ﬁrst, single “sharp turns” were corrected. Most tracking errors were only one separated point within a sequence of correct tracking. When the trajectory showed a bee turning left or right >135◦, this turning start point (1st point) and the point after turning (3rd point) were directly connected with a line segment, and the 2nd point was re-located as the middle point on this line segment. Second, “jump and stay” were corrected. In rare cases, tracking points jumped to a wrong location and lasted 200–400 ms (2–4 points); therefore, we compared every point with the , , y) The surgery and search for MBENs was performed on a closely attached table outside of the pyramid under an anatomical lens. Single bumblebees were ﬁxed in a metal tube after hypothermic anesthesia, the head was immobilized by clamping the mandibles with forceps, and the antennae were carefully immobilized with Plasticine R⃝(Harbutt, United Kingdom) to reduce disturbance to surgery. A small window was opened in the head capsule with a broken razor blade to expose the position of the right vertical lobe of the MB. The trachea sacks were removed to facilitate the insertion of recording electrodes. The silver electrode was inserted into left ocellus to about 100 µm depth. The diode was slowly inserted into the β-exit region of the vertical lobe (Rybak and Menzel, 1993; Figure 2) with the assistance of a microdrive. The insertion depth was carefully adjusted to 150 µm until stable neural activity was found. Electrodes and Surgery The recording diode was constructed according to previous reports (Mizunami et al., 1998a; Okada et al., 1999, 2007). In brief, two 15 µm diameter copper wires (P155, Elektrisola, Hamburg, October 2020 \| Volume 14 \| Article 590999 2 Jin et al. Bumblebee MBENs Reﬂect Behavioral States FIGURE 1 \| Experimental set-up. (A) Recording set-up. A sketch of the whole apparatus modiﬁed from Duer et al., 2015. The set-up was composed of an artiﬁcial arena environment for behavioral and neural recordings (left half) close to a neural set-up for dissecting the brain, inserting the electrodes, testing for unit activities and for storing neural data (right half). The surgery was done on the table to the right under binoculars. The pyramid hanging over the left part was designed as a Faraday cage and could be lowered down to the ground forming an enclosed space and well shielded. Neural signals were picked up by two twisted 15 µm diameter copper wires and a silver 50 µm diameter ground wire and transmitted to two ampliﬁers (red lines), an oscilloscope, a pair of loud speakers, an analog-digital converter, and ﬁnally stored in a computer. The dashed red line shows the electrode after releasing the bee on ceramic plate, while the black dashed line marks the position when the hanging pyramid was lowered to the ground. (B) The bird’s eye view of the arena environment (AE) with round ground paper (when the pyramid containing panorama patterns was on the ground). A video camera mounted on the ceiling recorded the movements of the bee. Simple patterns were pasted on the inner white walls as panorama. A red ceramic plate was placed in the center of the ground. A piece of round white cardboard paper (diameter 22 cm) was ﬁxed to center of the plate; the edge of the white paper was 1 cm away from the lower end of the slope (black dashed line). Fresh sucrose solution (50% in volume) was supplied on a tiny blue feeding disk. The location of the feeding disk was changed from bee to bee, but was always between the center and the edge of ground paper. (C) The bird’s eye view of the AE with squared ground paper (diagonal 22 cm). This experimental condition only applied to bee # 150512. FIGURE 1 \| Experimental set-up. (A) Recording set-up. A sketch of the whole apparatus modiﬁed from Duer et al., 2015. Electrodes and Surgery The electric resistance of the copper wires was controlled to lower than 50 k by gold plating (Ferguson et al., 2009) as monitored by a digital impedance meter (nanoZTM, White Matter, WA, United States). The diodes were connected to extracellular ampliﬁers (npi electronic GmbH, Tamm, Germany). Germany) were twisted and glued to each other as single- ended electrodes. A 50 µm diameter silver wire (AG548323, Advent Research Materials, Oxford, United Kingdom) was used as reference electrode. The electric resistance of the copper wires was controlled to lower than 50 k by gold plating (Ferguson et al., 2009) as monitored by a digital impedance meter (nanoZTM, White Matter, WA, United States). The diodes were connected to extracellular ampliﬁers (npi electronic GmbH, Tamm, Germany). Electrodes and Surgery The set-up was composed of an artiﬁcial arena environment for behavioral and neural recordings (left half) close to a neural set-up for dissecting the brain, inserting the electrodes, testing for unit activities and for storing neural data (right half). The surgery was done on the table to the right under binoculars. The pyramid hanging over the left part was designed as a Faraday cage and could be lowered down to the ground forming an enclosed space and well shielded. Neural signals were picked up by two twisted 15 µm diameter copper wires and a silver 50 µm diameter ground wire and transmitted to two ampliﬁers (red lines), an oscilloscope, a pair of loud speakers, an analog-digital converter, and ﬁnally stored in a computer. The dashed red line shows the electrode after releasing the bee on ceramic plate, while the black dashed line marks the position when the hanging pyramid was lowered to the ground. (B) The bird’s eye view of the arena environment (AE) with round ground paper (when the pyramid containing panorama patterns was on the ground). A video camera mounted on the ceiling recorded the movements of the bee. Simple patterns were pasted on the inner white walls as panorama. A red ceramic plate was placed in the center of the ground. A piece of round white cardboard paper (diameter 22 cm) was ﬁxed to center of the plate; the edge of the white paper was 1 cm away from the lower end of the slope (black dashed line). Fresh sucrose solution (50% in volume) was supplied on a tiny blue feeding disk. The location of the feeding disk was changed from bee to bee, but was always between the center and the edge of ground paper. (C) The bird’s eye view of the AE with squared ground paper (diagonal 22 cm). This experimental condition only applied to bee # 150512. red light, and then the dome structure was lowered down to cover the ceramic plate. The recordings in white light were started manually after the bee had been left in darkness for 10 min to recover from surgery. Germany) were twisted and glued to each other as single- ended electrodes. A 50 µm diameter silver wire (AG548323, Advent Research Materials, Oxford, United Kingdom) was used as reference electrode. Frontiers in Behavioral Neuroscience \| www.frontiersin.org RESULTS Spatial information content (IC) was calculated according to well-established methods (Markus et al., 1994), which is used to estimate to what extent the spatial distribution of spike frequency predicts the location or direction of an animal. The IC of the location was calculated based on 19 mm × 19 mm squared bins, while the IC of the angle was based on 6◦/sector bins. ICs of three types of angles were tested in every unit: 1. walking direction, 2. target orientation from instant bee location, and 3. target orientation relative to walking direction (i.e., angle 1 – angle 2). We recorded extracellular neuro-activity of MBENs from 5 bees and sorted out a total of 19 units (see Table 1). Recording durations varied from about 3 min to 2 h, and each bee yielded 1–7 units. During the recording, 4 out of 5 bees walked properly and explored the whole arena. One bee (# 140508) circled counter-clockwise throughout the recording time (2 min 54 s), was only occasionally interrupted by immobility. We include the data of this bee although its behavior was rather unnatural since the corresponding spike activities indicated a behaviorally relevant pattern (Figure 3 and Supplementary Video S1). Interestingly, spike activity increased initially when the animal reached the slope, but this eﬀect disappeared over time. Notice that the spatial components (body direction, location) between ground and slope are correlated with the shift between the two behavioral states, exploration on the ground and escape on the slope. None of the bees visited the feeding place frequently enough to allow a relevant analysis (Figure 4), possibly because they were not hungry enough. We collected the bees from the colony or at the feeder. It is also possible that they were not well adapted to the experimental conditions because they were exposed to the 1https://www.mathworks.com/matlabcentral/ﬁleexchange/725-smoothn Analysis of Neural Activity To stabilize the position of electrodes, a tiny amount of two-components silicon Kwik-Kast R⃝(World Precision Instruments Company, Sarasota, FL, United States) was ﬁlled through the window into the space inside the head capsule. Beeswax was additionally applied to ﬁx the electrodes to the outer surface of head capsule. The experimental bumble bee carrying the recording wires was carefully transferred to the arena, released from the metal tube onto the ceramic plate under October 2020 \| Volume 14 \| Article 590999 Frontiers in Behavioral Neuroscience \| www.frontiersin.org 3 Jin et al. Bumblebee MBENs Reﬂect Behavioral States FIGURE 2 \| An optical slice of bumblebee brain. The subﬁgure shows the zoom-in of MB vertical lobe region, and the red dashed-line circle marks the region (β-exit of the MB vertical lobe) where copper electrodes were inserted for neural recordings (courtesy Jürgen Rybak). FIGURE 2 \| An optical slice of bumblebee brain. The subﬁgure shows the zoom-in of MB vertical lobe region, and the red dashed-line circle marks the region (β-exit of the MB vertical lobe) where copper electrodes were inserted for neural recordings (courtesy Jürgen Rybak). frequency was compared between slope and ground within same speed group in the same epoch. Some data sets included sample numbers <30 samples that might lead to less reliable statistic results and thus were excluded from our statistics. Data pairs with big sample numbers (≥30) were compared in independent-samples t-tests and p values were corrected with Bonferroni correction. following 5 points and took the point nearest to the 1st point as the real bee location, the points in between the two real points are evenly re-located between them. In a last step, we applied robust smoothing with a well-established Matlab function SMOOTHN1 to polish up the trajectory curves. Furthermore, every bee location was assigned to a certain region (slope, feeding place or ground without feeding plate). The feeding place did not attract enough visiting times; therefore, it was excluded from all analyses. Frontiers in Behavioral Neuroscience \| www.frontiersin.org Experimental Design and Statistical Analyses To look into the correlation between walking speed and neuro- activity, we ﬁrst pooled spike numbers in the entire arena by every speed segment of 0.5 cm/s from 0 to 8 cm/s and ran a one-way ANOVA to check the overall spiking diﬀerence between speed groups. Next, we compared neural activity on slope and ground separately within each speed group with Wilcoxon rank sum tests followed by Bonferroni correction that controlled type I error from multiple comparisons. To examine the stability of neuro-activity over time, each recording time was arbitrarily cut into 5-min epochs. Spike October 2020 \| Volume 14 \| Article 590999 Frontiers in Behavioral Neuroscience \| www.frontiersin.org 4 Jin et al. Bumblebee MBENs Reﬂect Behavioral States FIGURE 3 \| Bee # 140508 running in a circle. (A) 2D bee trajectory from bird’s eye view; (B) same trajectory in 3D, time goes from down to up. In both sub-ﬁgures, the gray line shows the trajectory, each colorful dot on the trajectory shows a down-sampled bee location (per 0.5 s) and spike activity of that moment. The three red open circles from big to small mark the borders of the plate rim, ground paper and feeding disk, respectively. FIGURE 3 \| Bee # 140508 running in a circle. (A) 2D bee trajectory from bird’s eye view; (B) same trajectory in 3D, time goes from down to up. In both sub-ﬁgures, the gray line shows the trajectory, each colorful dot on the trajectory shows a down-sampled bee location (per 0.5 s) and spike activity of that moment. The three red open circles from big to small mark the borders of the plate rim, ground paper and feeding disk, respectively. test arena to the ﬁrst time. This may have led to stronger escape behavior. referring to the method in rodents place cell analysis (Markus et al., 1994). It turned out that our IC values varied from 0.009 to 0.3, lower than the criterion of 1 as a sign of place cell, indicating that our recorded units did not contain place cell-like properties (Supplementary Figure S1). Similarly, no head direction cell-like properties were found, with IC values ranging from −0.0002 to 0.1; target orientation IC ranged from −0.27 to 0.6; heading direction away from target (i.e., head direction- target orientation) IC varied from −0.03 to 0.12 (Supplementary Figure S2). First, we focused on the neural activity during walking on the ground. Experimental Design and Statistical Analyses We checked if the neuron ﬁring patterns can stably predict location (unit examples in Figure 5) or head direction (unit examples in Figure 6). Therefore, we calculated the so called “information content (IC)” of our recorded units TABLE 1 \| All recorded units. Bee ID Neuron ID Record length Spiking freq. (Hz) SEM Ground Slope Ground Slope 140409 1 28 min 26.10 30.95 0.17 0.22 2 28 min 13.89 22.99 0.23 0.30 3 28 min 9.62 12.30 0.13 0.19 4 28 min 11.56 13.09 0.11 0.15 5 28 min 8.02 10.38 0.12 0.18 6 28 min 1.30 2.10 0.04 0.06 7 28 min 0.53 0.99 0.03 0.04 140508 1 2 min 54 s 93.24 107.27 0.91 3.88 141204 1 59 min 57 s 15.10 17.71 0.07 0.13 2 59 min 57 s 14.16 12.18 0.06 0.12 3 59 min 57 s 13.53 11.30 0.06 0.09 4 59 min 57 s 11.02 16.15 0.09 0.20 5 59 min 57 s 5.44 8.10 0.04 0.09 6 60 min 1.70 1.47 0.02 0.03 7 60 min 7.91 6.14 0.06 0.08 141217 1 120 min 1 s 4.42 7.13 0.04 0.09 2 120 min 1 s 14.34 23.51 0.12 0.18 3 120 min 1 s 12.99 10.80 0.09 0.12 150512 1 59 min 55 s 6.35 4.98 0.07 0.04 In total 19 units were sorted out from 5 bumblebees; each bee yielded 1–7 units and each unit was recorded for ∼3 min – 2 h. Mean spiking frequency and SEM of whole recordings on ground and slope are listed separately. TABLE 1 \| All recorded units. Frontiers in Behavioral Neuroscience \| www.frontiersin.org Correlation Between Walking Speed and Neural Activity y Next, we tested the overall correlation between walking speed and neural activity. One-way ANOVA showed statistically signiﬁcant diﬀerences in every recorded unit (all p values < 0.001), indicating that neural activity between at least two speed groups in each unit was signiﬁcantly diﬀerent. However, for individual units, the highest neuronal activity could appear at any speed (see examples in Figure 7 left panels and Supplementary Figure S3). These results do not indicate that the recorded units were directly controlling walking speed because the sensory and central processing components (both internal and external conditions) leading ﬁnally to changed walking speed may be the parameters inﬂuencing the units’ activities. Therefore, we looked into parameters related to the parameters of the arena that the bees experienced during changes in spike activity. In our ﬁrst approach we asked whether the units were diﬀerently active on ground and slope, since the two areas had diﬀerent textures (paper vs. ceramics), colors (while vs. magenta) and features related to gravity, and they were obviously diﬀerently related to two behavioral states, exploration and thigmotactic escape. To this end, we further divided the spike data between ground and slope, and compared them with Wilcoxon rank sum tests. Because the walking speed distributions on the two October 2020 \| Volume 14 \| Article 590999 5 Jin et al. Bumblebee MBENs Reﬂect Behavioral States FIGURE 4 \| Typical trajectory of a walking bumble bee (#140409) in the arena. Gray lines show the trajectory of a full recording (28 min). The three red open circles from big to small mark the borders of the plate rim, ground paper and feeding disk, respectively. The black dashed-line circle marks the invisible lower end of slope. The bee fully explored the ground area, tried to escape from the slope during thigmotactic walking and visited the feeding place occasionally. in diﬀerent ways for diﬀerent animals (Figure 7 right panels and Supplementary Figure S3). These results clearly show local eﬀects on neural activity possibly related either to area within the whole arena or the two behavioral states related to these two areas (exploration and thigmotaxis). Mushroom body extrinsic neurons are known to respond to multiple sensory conditions in a combinatorial way and they are sensitive to the context in which stimuli appear (Filla and Menzel, 2015; Zwaka et al., 2018). This latter eﬀect could possibly reﬂect state-dependent activities. Correlation Between Walking Speed and Neural Activity Therefore, we went on to test whether the eﬀects we found for the region-speed dependence reﬂected a consistent property or varied over time. The latter was found. The whole recording time of each unit was arbitrarily binned to 5-min epochs. All recorded units (Table 1) that showed diﬀerent spike activities between slope and ground at the same walking speed changed these properties over time, either within each region (Figures 8A,B) or with diﬀerences of neural activity between slope and ground (Figure 8C). For other units, see Supplementary Figure S5. Taken together with the results in Figure 7, the recorded MBENs appeared to combine several conditions the actively exploring animal ﬁnds itself in. The two areas, ground and slope, diﬀered with respect to their spatial arrangement and their meaning to the animal, open area for exploration and boundary of a restricted area that the animal may want to leave (thigmotactic escape). The two areas were well characterized by diﬀerent walking trajectories, ﬂexibly changing walking directions on the ground and stereotypical trajectories when on the slope (Figure 4). The next step will be to analyze repetitive behavioral elements that are consistently related to neural activity changes. Are these epochs similarly or diﬀerently related to spike activity changes in the ground and slope area? FIGURE 4 \| Typical trajectory of a walking bumble bee (#140409) in the arena. Gray lines show the trajectory of a full recording (28 min). The three red open circles from big to small mark the borders of the plate rim, ground paper and feeding disk, respectively. The black dashed-line circle marks the invisible lower end of slope. The bee fully explored the ground area, tried to escape from the slope during thigmotactic walking and visited the feeding place occasionally. structures were signiﬁcantly diﬀerent in every recording of natural walk (Supplementary Figure S4), we ﬁrst normalized the neuro-activity in each narrow speed bin (step = 0.5 cm/s) to the same time unit (spikes/s). As in the initial phase of the circling bee # 141508 (Figure 3), all units were activated diﬀerently when they were on ground and slope. This eﬀect depended on walking speed p y g g p Next we examined the epochs where neural activity was signiﬁcantly diﬀerent between slope and ground explorations at a particular walking speed (mentioned as “sig. epochs” below, marked with ∗in Figure 8C). Correlation Between Walking Speed and Neural Activity Three hypotheses were tested: (1) FIGURE 5 \| Spatial distribution of neural activity of two units in two different animals. The whole arena was divided into squared bins (each bin 19 mm × 19 mm). False colors represent neuro-activity within each spatial bin, which is the mean spike frequency (Hz) at all tracking points in each bin. Spatial information content (IC) was calculated based on well-established methods (Markus et al., 1994). The ICs of unit 1 in bee # 140409 and unit 1 in bee # 141204 were 0.009 and 0.003, indicating no location-predictive (or so-called “place cell”) property in these units. Data of other units are in Supplementary Figure S1. FIGURE 5 \| Spatial distribution of neural activity of two units in two different animals. The whole arena was divided into squared bins (each bin 19 mm × 19 mm). False colors represent neuro-activity within each spatial bin, which is the mean spike frequency (Hz) at all tracking points in each bin. Spatial information content (IC) was calculated based on well-established methods (Markus et al., 1994). The ICs of unit 1 in bee # 140409 and unit 1 in bee # 141204 were 0.009 and 0.003, indicating no location-predictive (or so-called “place cell”) property in these units. Data of other units are in Supplementary Figure S1. October 2020 \| Volume 14 \| Article 590999 Frontiers in Behavioral Neuroscience \| www.frontiersin.org 6 Jin et al. Bumblebee MBENs Reﬂect Behavioral States FIGURE 6 \| Heading direction related neural activity in two neurons. The spiking frequency in 360◦of arena is binned into 60 sectors (i.e., 6◦/sector) and then averaged. Data of other units are in Supplementary Figure S2. The directional ICs of unit 1 in bee # 140409 and unit 1 in bee # 141204 are 0.0004 and 0.001, indicating no direction-predictive (or so-called “head direction cell”) property in these neurons. FIGURE 6 \| Heading direction related neural activity in two neurons. The spiking frequency in 360◦of arena is binned into 60 sectors (i.e., 6◦/sector) and then averaged. Data of other units are in Supplementary Figure S2. The directional ICs of unit 1 in bee # 140409 and unit 1 in bee # 141204 are 0.0004 and 0.001, indicating no direction-predictive (or so-called “head direction cell”) property in these neurons. the feeding site (Jin et al., 2014). Correlation Between Walking Speed and Neural Activity The animals in these experiments were fasted overnight before every training and test, therefore, explored actively the environment and searched for the location of a feeding place both in relation to its local cue and the panorama. The bumble bees relied on spatial relations of the visual features within the test arena as shown by the eﬀects caused by rotating the panorama and displacing the local cue. Other than in these former studies the bees in the experiments reported here were brought to the arena directly from the colony or a feeding place without overnight fasting, and thus they might have been less motivated to feed. They did not suck sucrose solution at the feeder and did not return multiple times to it although they explored the whole arena extensively. We were not concerned about the lack of motivation to feed in the arena because our aim was to search for neural correlates at the level of the MBENs during exploratory behavior as an essential requirement for navigational performance. Animals learn during exploration (Tolman, 1948), a form of learning that has been documented for honeybees in the context of navigation under natural conditions (Degen et al., 2015, 2016). Was the bee at the same location in diﬀerent sig. epochs? (2) Was the bee facing the same direction in diﬀerent sig. epochs? (3) Within each sig. epoch, was the neuro-activity on slope constantly higher or lower than on ground? We found that the same unit in diﬀerent sig. epochs could be activated at diﬀerent locations (one example in Figure 9) and had no preferred heading direction in any sig. epoch (one example in Figure 10), meaning that the inconsecutive sig. epochs resulted from varying neural activity over time rather than returning to a certain spatial location or heading direction. Furthermore, within any sig. epoch the neural activity was not always higher on the slope than on the ground (or the other way round) over time (examples see Figure 11), altogether indicating that the diﬀerent neural activity of MBENs between slope and ground in sig. epochs was a combinatorial and accumulative eﬀect, rather than a single-property or stably spiking in small time scales under the same conditions. Correlation Between Walking Speed and Neural Activity To conclude, the neural activity of all recorded MBENs did not correlate with spatial parameters (location, direction), neither on a macro time scale (up to ∼2 h of whole recording) nor on a micro scale (a few seconds in sig. epochs); instead, they reﬂected in a walking tempo-dynamic way diﬀerent behavioral states when the bees were actively exploring on ground or thigmotactically walking on slope. We aimed to record from MBENs because these neurons read out the high order neural processing in the MB, a structure of the insect brain well known for its convergence of highly processed sensory information and its role in memory formation (Heisenberg, 2003; Menzel, 2012). The MB consists of a large number (∼150,000) of densely packed small neurons (Kenyon cells, KC) that converge on a small number (a few hundred) ENs (Rybak and Menzel, 1993). Other than KC, ENs respond to stimuli of multiple sensory modalities indicating a diﬀerent coding scheme than the highly speciﬁc combinatorial sensory code at the input of the MB (Menzel, 2012). All MBENs so far recorded change their response properties during associative learning. For example, a large EN, the peduncle extrinsic neuron #1 (PE1), was found to reduce Frontiers in Behavioral Neuroscience \| www.frontiersin.org DISCUSSION The search for neural correlates of active exploration in small animals like the bumble bee imposes inevitable compromises with respect to the kind of movement (walking vs. ﬂying), the size of the test arena, the motivation of the animal and the conﬁnement imposed on the animal by the border of the arena. Our emphasis was to replicate as much as possible behavioral studies that documented operant learning of a local color cue at a feeding site and the visual pattern of the panorama characterizing October 2020 \| Volume 14 \| Article 590999 Frontiers in Behavioral Neuroscience \| www.frontiersin.org 7 Jin et al. Bumblebee MBENs Reﬂect Behavioral States FIGURE 7 \| Four examples of neural activity at different walking speeds in the entire arena and in the two different compartments of the arena (ground and slope Spike numbers were pooled by every speed segment of 0.5 cm/s from 0 to 8 cm/s and in each speed group normalized to 1 s. Left panels: entire arena. One-w ANOVA showed statistically signiﬁcant difference in every recorded neuronal unit (all p values < 0.001), indicating that in each neuron at least two speeds were correlated with different neural activity. Right panels: neural activity separately calculated for different walking speeds on the slope and the ground. Units were differently active when the bee walked on the two compartments of the arena at some speeds but not all. p < 0.05 in Wilcoxon rank sum tests with Bonferroni correction. FIGURE 7 \| Four examples of neural activity at different walking speeds in the entire arena and in the two different compartments of the arena (ground and slope). Spike numbers were pooled by every speed segment of 0.5 cm/s from 0 to 8 cm/s and in each speed group normalized to 1 s. Left panels: entire arena. One-way ANOVA showed statistically signiﬁcant difference in every recorded neuronal unit (all p values < 0.001), indicating that in each neuron at least two speeds were correlated with different neural activity. Right panels: neural activity separately calculated for different walking speeds on the slope and the ground. Units were differently active when the bee walked on the two compartments of the arena at some speeds but not all. p < 0.05 in Wilcoxon rank sum tests with Bonferroni correction. DISCUSSION FIGURE 7 \| Four examples of neural activity at different walking speeds in the entire arena and in the two different compartments of the arena (ground and slope). Spike numbers were pooled by every speed segment of 0.5 cm/s from 0 to 8 cm/s and in each speed group normalized to 1 s. Left panels: entire arena. One-way ANOVA showed statistically signiﬁcant difference in every recorded neuronal unit (all p values < 0.001), indicating that in each neuron at least two speeds were correlated with different neural activity. Right panels: neural activity separately calculated for different walking speeds on the slope and the ground. Units were differently active when the bee walked on the two compartments of the arena at some speeds but not all. p < 0.05 in Wilcoxon rank sum tests with Bonferroni correction October 2020 \| Volume 14 \| Article 590999 Frontiers in Behavioral Neuroscience \| www.frontiersin.org 8 Jin et al. Bumblebee MBENs Reﬂect Behavioral States FIGURE 8 \| Differences in neuro-activity between slope and ground at a certain speed were not constant over time. The example shown here stems from Bee 140409, unit 1. Other examples are shown in Supplementary Figure S5. The whole recording time was cut into 5-min epochs. (A) Neuro-activity along time (abscissa) when the bee was on the slope walked at different speed (ordinate). Spike activity is expressed in false color as indicated in the right upper corner. (B) The same graph for the bee walking on the ground. (C) Comparison of time dependence of spike activity between slope and ground. Higher spike activity on the slope is expressed in yellow, and higher spike activity on the ground in blue. marks signiﬁcant differences between slope and ground (p < 0.05 in independent-samples t-tests after Bonferroni correction). × marks low sample numbers (<30) that lead to less reliable statistic results and are thus excluded from our statistics. FIGURE 9 \| Bee 140409 locations in * marked epochs of Figure 8. In each subﬁgure, blue points highlight the bee locations on the slope in that epoch under certain speed, while orange points highlight locations on the ground in the same epoch and same speed. Gray points show other locations of the same epoch in all speeds. Three red open circles, from small to big, mark the feeding place, edge of ground paper and rim of arena. DISCUSSION Higher spike activity on the slope is expressed in yellow, and higher spike activity on the ground in blue. * marks signiﬁcant differences between slope and ground (p < 0.05 in independent-samples t-tests after Bonferroni correction). × marks low sample numbers (<30) that lead to less reliable statistic results and are thus excluded from our statistics. FIGURE 9 \| Bee 140409 locations in * marked epochs of Figure 8. In each subﬁgure, blue points highlight the bee locations on the slope in that epoch under certain speed, while orange points highlight locations on the ground in the same epoch and same speed. Gray points show other locations of the same epoch in all speeds. Three red open circles, from small to big, mark the feeding place, edge of ground paper and rim of arena. The comparison of mean spike numbers between blue and orange points is displayed in the up-right corner of each subﬁgure. Note that in the area marked with a green dashed circle, the unit was not signiﬁcantly more active in epoch 2 on either slope or ground, however, it was more active on the slope when the bee revisited this area in epoch 4. Different signiﬁcant locations in different epochs indicate that in Figure 8 the inconstant difference over time is caused by time instead of revisiting of the same location. More examples of other units are in Supplementary Figure S6. FIGURE 9 \| Bee 140409 locations in * marked epochs of Figure 8. In each subﬁgure, blue points highlight the bee locations on the slope in that epoch under certain speed, while orange points highlight locations on the ground in the same epoch and same speed. Gray points show other locations of the same epoch in all speeds. Three red open circles, from small to big, mark the feeding place, edge of ground paper and rim of arena. The comparison of mean spike numbers between blue and orange points is displayed in the up-right corner of each subﬁgure. Note that in the area marked with a green dashed circle, the unit was not signiﬁcantly more active in epoch 2 on either slope or ground, however, it was more active on the slope when the bee revisited this area in epoch 4. DISCUSSION The comparison of mean spike numbers between blue and orange points is displayed in the up-right corner of each subﬁgure. Note that in the area marked with a green dashed circle, the unit was not signiﬁcantly more active in epoch 2 on either slope or ground, however, it was more active on the slope when the bee revisited this area in epoch 4. Different signiﬁcant locations in different epochs indicate that in Figure 8 the inconstant difference over time is caused by time instead of revisiting of the same location. More examples of other units are in Supplementary Figure S6. its responses to the learned odor (Mauelshagen and Greggers, 1993 Ok d l 2007) b h i i l learned stimulus. These results indicated that PE1 encodes d diﬀ l ibl i l Bumblebee MBENs Reﬂect Behavioral States Jin et al. FIGURE 8 \| Differences in neuro-activity between slope and ground at a certain speed were not constant over time. The example shown here stems from Bee 140409, unit 1. Other examples are shown in Supplementary Figure S5. The whole recording time was cut into 5-min epochs. (A) Neuro-activity along time (abscissa) when the bee was on the slope walked at different speed (ordinate). Spike activity is expressed in false color as indicated in the right upper corner. (B) The same graph for the bee walking on the ground. (C) Comparison of time dependence of spike activity between slope and ground. Higher spike activity on the slope is expressed in yellow, and higher spike activity on the ground in blue. * marks signiﬁcant differences between slope and ground (p < 0.05 in independent-samples t-tests after Bonferroni correction). × marks low sample numbers (<30) that lead to less reliable statistic results and are thus excluded from our statistics. FIGURE 8 \| Differences in neuro-activity between slope and ground at a certain speed were not constant over time. The example shown here stems from Bee 140409, unit 1. Other examples are shown in Supplementary Figure S5. The whole recording time was cut into 5-min epochs. (A) Neuro-activity along time (abscissa) when the bee was on the slope walked at different speed (ordinate). Spike activity is expressed in false color as indicated in the right upper corner. (B) The same graph for the bee walking on the ground. (C) Comparison of time dependence of spike activity between slope and ground. DISCUSSION Different signiﬁcant locations in different epochs indicate that in Figure 8 the inconstant difference over time is caused by time instead of revisiting of the same location. More examples of other units are in Supplementary Figure S6. its responses to the learned odor (Mauelshagen and Greggers, 1993; Okada et al., 2007) but enhances its responses to a visual context stimulus that signaled to the animal the upcoming learned stimulus. These results indicated that PE1 encodes cues and contexts diﬀerently, possibly representing a general property of ENs. October 2020 \| Volume 14 \| Article 590999 Frontiers in Behavioral Neuroscience \| www.frontiersin.org 9 Jin et al. Bumblebee MBENs Reﬂect Behavioral States Jin et al. FIGURE 10 \| Bee 140409 walking directions and neuro-activity in * marked epochs of Figure 8. The 360◦is binned into 12 sectors (i.e., 30◦/sector). Blue sectors: slope, red: ground. Filled sectors: spikes/second; unﬁlled: accumulated walking time. Data of other units are in Supplementary Figure S7. FIGURE 10 \| Bee 140409 walking directions and neuro-activity in * marked epochs of Figure 8. The 360◦is binned into 12 sectors (i.e., 30◦/sector). Blue sectors: slope, red: ground. Filled sectors: spikes/second; unﬁlled: accumulated walking time. Data of other units are in Supplementary Figure S7. 0 \| Bee 140409 walking directions and neuro-activity in * marked epochs of Figure 8. The 360◦is binned into 12 sectors (i.e., 30◦/ ground. Filled sectors: spikes/second; unﬁlled: accumulated walking time. Data of other units are in Supplementary Figure S7. stimuli or actively exploring them. Indeed, MBENs were found to change their responses to visual and olfactory stimuli under operant learning conditions in a virtual reality environment (Zwaka et al., 2018). Furthermore, MBENs were found to predict responses in active social contexts that allowed the recorded animal to move freely within the community of a small colony (Duer et al., 2015; Paﬀhausen et al., 2020). Thus the MB as a whole appears to act as a re-coding device, converting sensory information to value-based information that provides the information for active behavioral control (Menzel, 2014). In this respect the mushroom body shares properties with the mammalian hippocampus (Johnson et al., 2007; Yanike et al., 2009; van der Meer et al., 2010) and prefrontal cortex (Histed et al., 2009; Mansouri et al., 2009). Frontiers in Behavioral Neuroscience \| www.frontiersin.org DISCUSSION Right panels: mean spike numbers on slope and ground during region shifts in the same sig. epoch as left panels. S: slope; G: ground. FIGURE 11 \| Neural activity of MBENs accumulated for each signiﬁcant epoch of walking between slope and ground. Each row shows one signiﬁcant epochs from two bees. Left panels: examples for parts of the arena that included frequent region shifts of the bee between slope and ground. Bee locations in a signiﬁcant epoch are highlighted with color dots; neural activity (spikes/s) from low to high is represented from light blue to magenta (color bar). Other bee locations are marked with open circles. Black bars point to heading direction at that tracking point. Right panels: mean spike numbers on slope and ground during region shifts in the same sig. epoch as left panels. S: slope; G: ground. FIGURE 11 \| Neural activity of MBENs accumulated for each signiﬁcant epoch of walking between slope and ground. Each row shows one signiﬁcant epochs from two bees. Left panels: examples for parts of the arena that included frequent region shifts of the bee between slope and ground. Bee locations in a signiﬁcant epoch are highlighted with color dots; neural activity (spikes/s) from low to high is represented from light blue to magenta (color bar). Other bee locations are marked with open circles. Black bars point to heading direction at that tracking point. Right panels: mean spike numbers on slope and ground during region shifts in the same sig. epoch as left panels. S: slope; G: ground. modulatory systems during recordings of processing neurons is mostly unknown. Hippocampal place cells are not only selectively activated by the local cues of the experienced environment (Moser et al., 2008) but also by the value and meaning of localized olfactory stimuli (Manns and Eichenbaum, 2009), but again it is not known how the selective read- out of the multiple features are controlled. Correlating the activity of single cortical neurons in perceptual or motor tasks is notoriously diﬃcult because of the multiple recurrent networks. What appears as noise may even be the signs of information processing (Engel and Singer, 2001; Salinas et al., 2001; Davis et al., 2019). From a more global perspective, the brain may not require deﬁned external conditions for selecting behavioral acts but rather generates the conditions internally (Gallistel, 2013). neural activity patterns of particular neurons and behavioral acts. DISCUSSION As in any other neural system we do not yet understand the logic of the multiple faceted coding and storing schemes composing higher order neural integration but the limited number and the unique structures of ENs in the bee brain oﬀers an opportunity to gain insight. The MB is characterized by prominent recurrent neurons (Zwaka et al., 2019) whose neural activity relates to novelty detection, context dependence, and expectation (Filla and Menzel, 2015). ENs increase their responses to the learned stimulus, decrease them or do not change. In most cases the range of multi-sensory responses characteristic for the respective neuron is broad and rather unspeciﬁc before learning and becomes more speciﬁc after learning (Strube-Bloss et al., 2011, 2016). For example, half of a subset of ENs (the A1, A2 neurons) change their responses to the reinforced stimuli, the other half continued with stable responses. Most of the plastic neurons change their responses not during the acquisition process but after a consolidation phase of a few hours. Two kinds of changes were observed, qualitative changes (switching) and quantitative changes (modulating). Switching neurons dropped responses and/or developed new responses to one or several of the tested odors. All switches observed with respect to the CS+ odor were recruitments; those to the CS−could have been either recruitment or loss of response. Modulating neurons, however, increased and/or decreased their response rates to diﬀerent odors. One reason for our ignorance about high order integration processes is the lack of information about the internal states of the brain. Animal behavior is highly inﬂuenced by internal states and thus may lead to changing correlations between Most likely, response properties of high order interneurons depend on whether the animals are passively exposed to the October 2020 \| Volume 14 \| Article 590999 10 Jin et al. Bumblebee MBENs Reﬂect Behavioral States FIGURE 11 \| Neural activity of MBENs accumulated for each signiﬁcant epoch of walking between slope and ground. Each row shows one signiﬁcant epochs from two bees. Left panels: examples for parts of the arena that included frequent region shifts of the bee between slope and ground. Bee locations in a signiﬁcant epoch are highlighted with color dots; neural activity (spikes/s) from low to high is represented from light blue to magenta (color bar). Other bee locations are marked with open circles. Black bars point to heading direction at that tracking point. Frontiers in Behavioral Neuroscience \| www.frontiersin.org REFERENCES Filla, I., and Menzel, R. (2015). 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DATA AVAILABILITY STATEMENT The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fnbeh. 2020.590999/full#supplementary-material All datasets presented in this study are included in the article/Supplementary Material. ACKNOWLEDGMENTS We thank Anne Carney for correcting the English. We are grateful to Open Access Funding provided by the Freie Universität Berlin. AUTHOR CONTRIBUTIONS ongoing or future behavioral acts may fail to be uncovered. Here we applied a rigid selection scheme in our search for correlations. The criterion was that the correlation should stay stable over the whole recording time. This is indeed a sharp tool. No selective place-dependent or body direction-dependent spiking changes were found. Walking speed dependence diﬀered in the two main areas of the arena, ground and slope. Walking along the slope is likely related to thigmotaxis/escape, whereas walking on the ground reﬂects exploratory behavior. Thus remaining in one of the two areas may relate more to behavioral states than to locations. In such a case the units‘ diﬀerences in their walking speed dependence indicates behavioral states rather than location dependence, an interpretation supported by the ﬁnding that combinations with other spatial parameters (restricted location, body direction) did not lead to any signiﬁcant eﬀects. RM designed the experiment. BP built the setups and did spike- sorting. NJ did bee surgeries, ran experiments, analyzed data, and prepared all table and ﬁgures. AD and NJ wrote codes for video tracking of bee locations. RM and NJ wrote the manuscript. All authors contributed to the article and approved the submitted version. DISCUSSION Even well-characterized and identiﬁed neurons in rather small nervous systems like that of snails, annelids and arthropods change their spiking activities under state-dependent conditions of the brain or lead to diﬀerent behaviors when selectively stimulated in diﬀerent contexts. For example, a command-like neuron in the leech that releases swimming in water when intracellularly stimulated induces crawling when the leech is placed on solid ground (Esch et al., 2002). The feeding network in Aplysia undergoes network-speciﬁc changes depending on its history of activity in diﬀerent contexts indicating a form of task-speciﬁc inertia (Proekt et al., 2004). Stimulation of a ﬂight-inducing command neuron in insects will lead to ﬂight only in air-suspended animals but not in animals placed on the ground, e.g., in crickets (Nolen and Hoy, 1984). In some cases state-dependence of the brain can be traced to neuro- modulatory circuits controlled by the environment, e.g., in insects (Pﬂüger, 1999; Cohn et al., 2015) but the activity of Given these limitations of an electrophysiological approach to high-order brain functions we may not be surprised that correlations between the current stimulus conditions and Frontiers in Behavioral Neuroscience \| www.frontiersin.org October 2020 \| Volume 14 \| Article 590999 11 Jin et al. Bumblebee MBENs Reﬂect Behavioral States REFERENCES Conﬂict-induced behavioural adjustment: a clue to the executive functions of the prefrontal cortex. Nat. Rev. Neurosci. 10, 141–152. doi: 10.1038/nrn2538 Degen, J., Kirbach, A., Reiter, L., Lehmann, K., Norton, P., Storms, M., et al. (2015). Exploratory behaviour of honeybees during orientation ﬂights. Anim. Behav. 102, 45–57. doi: 10.1016/j.anbehav.2014.12.030 Markus, E. J., Barnes, C. A., McNaughton, B. L., Gladden, V. L., and Skaggs, W. E. (1994). 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https://openalex.org/W4307183693	https://acp.copernicus.org/preprints/acp-2022-648/acp-2022-648.pdf	English	null	Comment on acp-2022-648	null	2,022	cc-by	11,123	Removing the NOx-dependence of SOA formation 32 yields resulted in higher fractional error and fractional bias at both CSN and IMPROVE sites in 33 both summer periods, demonstrating the efficacy of the current formulation of SOA yields. Our 34 Predicted and Observed Changes in Summertime Biogenic and Total Organic 1 Aerosol in the Southeast United States from 2001 to 2010 2 Brian T. Dinkelacker1, Pablo Garcia Rivera1, Ksakousti Skyllakou2, Peter J. Adams3,4, Spyros N. 3 Pandis2,5,* 4 5 1Department of Chemical Engineering, Carnegie Mellon University, Pittsburgh, PA, 15213 6 2Institute of Chemical Engineering Sciences (FORTH/ICE-HT), 26504, Patras, Greece 7 3Department of Civil and Environmental Engineering, Carnegie Mellon University, Pittsburgh, 8 PA, 15213 9 4Department of Engineering and Public Policy, Carnegie Mellon University, Pittsburgh, PA, 10 15213 11 5Department of Chemical Engineering, University of Patras, 26500, Patras, Greece 12 13 Correspondence to: Spyros N. Pandis (spyros@chemeng.upatras.gr) 14 Biogenic secondary organic aerosol (bSOA) is a major component of atmospheric particulate 16 matter (PM2.5) in the southeast United States especially during the summer, when emissions of 17 biogenic volatile organic compound (VOCs) are high and emissions from anthropogenic sources 18 enhance the formation of secondary particulate matter. We evaluate the performance of PM2.5 19 organic aerosol predictions by a chemical transport model (PMCAMx) in response to significant 20 changes in anthropogenic emissions during the summers of 2001 and 2010. Average predicted 21 bSOA concentrations in the southeast US did not change appreciably from the summer of 2001 to 22 the summer of 2010, while the anthropogenic SOA decreased by 45%. As a result, the biogenic 23 fraction of total OA increased from 0.46 in 2001 to 0.63 in 2010. Partitioning effects due to reduced 24 anthropogenic OA from 2001 resulted in 0.4 µg m-3 less biogenic OA on average in the southeast 25 US in the summer of 2010. This was offset by biogenic SOA increases due to higher biogenic 26 vapor emissions in the warmer 2010 summer. Little noticeable difference was observed in OA 27 prediction performance in the southeast US between the two summer simulation periods. The 28 fractional error of OA predictions remained practically the same (0.41 and 0.44 at CSN sites and 29 0.40 to 0.41 at IMPROVE sites in the summers of 2001 and 2010 respectively). https://doi.org/10.5194/acp-2022-648 Preprint. Discussion started: 19 September 2022 c⃝Author(s) 2022. CC BY 4.0 License. Predicted and Observed Changes in Summertime Biogenic and Total Organic 1 Aerosol in the Southeast United States from 2001 to 2010 2 Brian T. Dinkelacker1, Pablo Garcia Rivera1, Ksakousti Skyllakou2, Peter J. Adams3,4, Spyros N. 3 Pandis2,5, 4 5 1Department of Chemical Engineering, Carnegie Mellon University, Pittsburgh, PA, 15213 6 2Institute of Chemical Engineering Sciences (FORTH/ICE-HT), 26504, Patras, Greece 7 3Department of Civil and Environmental Engineering, Carnegie Mellon University, Pittsburgh, 8 PA, 15213 9 4Department of Engineering and Public Policy, Carnegie Mellon University, Pittsburgh, PA, 10 15213 11 5Department of Chemical Engineering, University of Patras, 26500, Patras, Greece 12 13 *Correspondence to: Spyros N. Pandis (spyros@chemeng.upatras.gr) 14 Abstract 15 Biogenic secondary organic aerosol (bSOA) is a major component of atmospheric particulate 16 matter (PM2.5) in the southeast United States especially during the summer, when emissions of 17 biogenic volatile organic compound (VOCs) are high and emissions from anthropogenic sources 18 enhance the formation of secondary particulate matter. We evaluate the performance of PM2.5 19 organic aerosol predictions by a chemical transport model (PMCAMx) in response to significant 20 changes in anthropogenic emissions during the summers of 2001 and 2010. Average predicted 21 bSOA concentrations in the southeast US did not change appreciably from the summer of 2001 to 22 the summer of 2010, while the anthropogenic SOA decreased by 45%. As a result, the biogenic 23 fraction of total OA increased from 0.46 in 2001 to 0.63 in 2010. Partitioning effects due to reduced 24 anthropogenic OA from 2001 resulted in 0.4 µg m-3 less biogenic OA on average in the southeast 25 US in the summer of 2010. This was offset by biogenic SOA increases due to higher biogenic 26 vapor emissions in the warmer 2010 summer. Little noticeable difference was observed in OA 27 prediction performance in the southeast US between the two summer simulation periods. The 28 fractional error of OA predictions remained practically the same (0.41 and 0.44 at CSN sites and 29 0.40 to 0.41 at IMPROVE sites in the summers of 2001 and 2010 respectively). The fractional bias 30 of OA predictions increased from 0.10 to 0.22 at CSN sites and decreased from 0 to -0.09 at 31 IMPROVE sites between the two periods. The fractional bias 30 of OA predictions increased from 0.10 to 0.22 at CSN sites and decreased from 0 to -0.09 at 31 IMPROVE sites between the two periods. Removing the NOx-dependence of SOA formation 32 yields resulted in higher fractional error and fractional bias at both CSN and IMPROVE sites in 33 both summer periods, demonstrating the efficacy of the current formulation of SOA yields. Our 34 1 https://doi.org/10.5194/acp-2022-648 Preprint. Discussion started: 19 September 2022 c⃝Author(s) 2022. CC BY 4.0 License. analysis suggests that the changes in biogenic OA in this forested relatively polluted region appear 35 to be dominated by the partitioning effects and the NOx effects on SOA yields. 36 37 analysis suggests that the changes in biogenic OA in this forested relatively polluted region appear 35 to be dominated by the partitioning effects and the NOx effects on SOA yields. 36 37 1 Introduction 38 Discussion started: 19 September 2022 c⃝Author(s) 2022. CC BY 4.0 License. found to reduce SOA formation from monoterpenes (McFiggans et al., 2019), which have 66 significantly higher SOA yields than that of isoprene. 67 found to reduce SOA formation from monoterpenes (McFiggans et al., 2019), which have 66 significantly higher SOA yields than that of isoprene. 67 Reductions in anthropogenic emissions of NOx have been a consequence of regulatory 68 efforts to reduce tropospheric ozone (Simon et al., 2015). The NOx level affects SOA formation 69 by determining chemical pathways through which SOA is formed from various VOCs, including 70 those from natural sources (Ziemann and Atkinson, 2012). Typically, SOA formation via oxidation 71 of a VOC is measured under both low-NOx and high-NOx conditions (Ng et al., 2007; Lee et al., 72 2011) and these yields are used to implement NOx-dependent SOA formation schemes in models 73 for individual precursors, ultimately determined by the ratio of the rates of organo-peroxy radical 74 reactions with NO (Lane et al., 2008). Impacts of NOx emissions on SOA formation are further 75 complicated, as reductions in NOx have been linked to increases in ozone and OH radical 76 concentrations in urban areas in the southern United States (Tsimpidi et al., 2008). In NOx-limited 77 (rural) areas, however, lower NOx emissions decrease the amount of available oxidants for SOA 78 formation. These complex interactions between various pollutants encourage the implementation 79 of NOx-dependence in air quality models to capture SOA concentration responses to changes in 80 NOx emissions in varied locales. 81 Another important mechanism for anthropogenic influence on bSOA formation is through 82 gas-to-particle partitioning effects. The existence of already formed particle-phase SOA 83 encourages the particle-phase partitioning of additional SOA mass (Liang et al., 1997; Leach et 84 al., 1999; Kroll et al., 2007). This means that the partitioning of bSOA components to the particle 85 phase is facilitated by the presence of anthropogenic secondary aerosol. Implementation of 86 semivolatile SOA partitioning schemes typically follows an approach where the products of SOA 87 forming reactions are lumped to surrogate species according to their volatility such as the Odum 88 2-product model (Odum et al., 1996) or the volatility basis set approach (Donahue et al., 2006; 89 Lane et al., 2008b) which allows for an arbitrary number of volatility bins. Carlton et al. 1 Introduction 38 Many anthropogenic pollutants contribute to the formation of organic aerosol (OA), a 39 major component of atmospheric particulate matter (PM2.5). This material can be directly emitted 40 in the particle phase as primary OA (POA) or formed during the oxidation of volatile organic 41 compounds (VOCs) and condensation or nucleation of their low volatility or semi-volatile 42 oxidation products forming secondary OA (SOA). Emissions from natural sources of VOCs, 43 including isoprene, monoterpenes, and sesquiterpenes have been found to be significant 44 contributors to PM2.5 mass in the form of biogenic SOA (bSOA) (Murphy and Pandis, 2010). 45 Significant efforts have been made to study bSOA formation in the southeast United States during 46 the summer due to a combination of high emissions of biogenic VOCs (Sindelarova et al., 2014) 47 along with the potential for significant anthropogenic influence on the corresponding processes 48 (Carlton et al., 2010). 49 Enforcement of federal air quality regulations such as the Clean Air Act and its 1990 50 amendments, has led to significant reductions in the emissions of anthropogenic pollutants known 51 to impact bSOA formation. US emissions of SO2 decreased by over 50% between 1970 and 2000 52 (Smith et al., 2011) and these reductions continued between 2000 and 2010 (Klimont et al., 2013). 53 Emissions of SO2 contribute inorganic PM2.5 mass in the form of sulfates, often the main acidic 54 component of atmospheric aerosols (Weber et al., 2016). Pye et al. (2013) simulated the effects of 55 acidity on SOA production during the oxidation of isoprene in the Community Multiscale Air 56 Quality model (CMAQv5.0.1). They estimated that with their acid-dependent isoprene-SOA 57 scheme, a 25% reduction in sulfur oxide emissions resulted in a 35-40% reduction in isoprene- 58 derived SOA. A 25% reduction in nitrogen oxides (NOx) was also tested, producing varied results 59 via different pathways due to differences in NOx-dependent SOA parameterizations. The authors 60 observed modest reductions in underpredictions of total organic carbon (OC) from both 61 IMPROVE and Chemical Speciation Network (CSN) sites using the most aggressive uptake 62 scenario. On the other hand, Takahama et al. (2006) found very small, if any statistical correlation 63 between observed OC and particle acidity during the Pittsburgh Air Quality Study. Plenty of 64 uncertainty still exists regarding the role of isoprene in SOA formation. Isoprene itself has been 65 2 https://doi.org/10.5194/acp-2022-648 Preprint. 1 Introduction 38 (2018) 90 found that up to 67% of bSOA mass in the southeast US could be attributed to semivolatile 91 partitioning during July 2013. With anthropogenic emissions of VOCs decreasing from 22 Tg 92 yr-1 to 14 Tg yr-1 between 2000 and 2010 in the United States (Xing et al., 2013), it is likely that 93 there have been changes in bSOA concentrations due to partitioning effects especially in areas 94 such as the southeast US. Relative humidity (RH) is believed to have varied effects on the SOA 95 yields of biogenic precursors. Isoprene SOA yields have been found to be suppressed at higher RH 96 3 3 https://doi.org/10.5194/acp-2022-648 Preprint. Discussion started: 19 September 2022 c⃝Author(s) 2022. CC BY 4.0 License. under high-NOx conditions (Nguyen et al., 2011; Zhang et al., 2011). However, RH has also been 97 found to enhance SOA formation from aromatic compounds, such as toluene (Kamens et al., 2011; 98 Jia and Xu, 2018). Under low-NOx conditions, toluene SOA yields have followed the opposite 99 trend with increasing RH (Cao and Jang, 2010; Hinks et al., 2018). 100 under high-NOx conditions (Nguyen et al., 2011; Zhang et al., 2011). However, RH has also been 97 found to enhance SOA formation from aromatic compounds, such as toluene (Kamens et al., 2011; 98 Jia and Xu, 2018). Under low-NOx conditions, toluene SOA yields have followed the opposite 99 trend with increasing RH (Cao and Jang, 2010; Hinks et al., 2018). 100 The southeast US has been a focus for research on SOA formation in the past due to a high 101 level of biogenic emissions and nearby anthropogenic pollution sources. Lu et al. (2014) leveraged 102 data obtained from the Southern Oxidant and Aerosol Study (SOAS) in 2013 to provide evidence 103 of anthropogenically enhanced biogenic SOA formation. They found that anthropogenic sulfate 104 and NOx in the area impacts 43-70% of measured organic aerosol during the summer. It remains 105 to be seen which of these effects, if any, are the most significant regarding the ability of chemical 106 transport models to reproduce the observed OA concentrations. 107 In this work, we investigate the ability of the Particulate Matter Comprehensive Air Quality 108 Model with Extensions, PMCAMx, relying largely upon NOx-dependent SOA yields and 109 equilibrium semivolatile partitioning, to reproduce changes in predicted bSOA between the 110 summers of 2001 and 2010. Skyllakou et al. 1 Introduction 38 (2021) evaluated the ability of PMCAMx to link 111 changes in emissions to predicted PM2.5 concentrations in 1990, 2001, and 2010 for the purpose 112 of investigating health outcomes. The authors found that while anthropogenic emissions decreased 113 over this period, biogenic OA was predicted to increase from 2001 to 2010 in the southeast US 114 mainly due to higher temperatures in the latter period. Here, we investigate this further by 115 implementing improved sesquiterpene SOA chemistry in PMCAMx, evaluating OA prediction 116 performance, and determining the effect of NOx-dependence and partitioning on predicted bSOA 117 with the goal of identifying the need for additional SOA parameterizations in the model. 118 119 2 Model Description 120 PMCAMx (Karydis, et al., 2010; Murphy and Pandis, 2010; Tsimpidi et al., 2010) is a 121 state-of-the-art chemical transport model, using the framework of CAMx, the Comprehensive Air 122 Quality Model with Extensions (Environ, 2006) that is used to simulate the advection, diffusion, 123 dry and wet deposition, gas and aqueous-phase chemistry, and atmospheric aerosol physical and 124 chemical processes. A 10-size (30 nm to 40 µm) aerosol sectional approach (Gaydos et al., 2003) 125 is used to track the dynamic evolution of aerosol mass and composition. This model simulates the 126 concentrations of aerosol sulfate, nitrate, ammonium, sodium, chloride, water, dust, elemental 127 4 https://doi.org/10.5194/acp-2022-648 Preprint. Discussion started: 19 September 2022 c⃝Author(s) 2022. CC BY 4.0 License. carbon, and both primary and secondary organics (represented as a series of lumped species in the 128 Volatility Basis Set). The ISORROPIA aerosol thermodynamics model (Nenes et al., 1998) is used 129 in this application to calculate the gas/particle partitioning of semi-volatile inorganic species. The 130 condensation and evaporation of inorganic species are simulated in this application assuming 131 equilibrium between the bulk inorganic gas and aerosol phases. The distribution of mass 132 partitioned in each phase to the various aerosol size sections is determined using weighting factors 133 calculated based on the effective surface area in each section (Pandis et al., 1993). 134 Organic aerosol evolution is modeled using the Volatility Basis Set approach (Donahue et 135 al., 2006; Lane et al., 2008b). SOA is split into anthropogenic (aSOA) and biogenic (bSOA) 136 components formed from a variety of SOA-forming VOCs from both human and natural sources. 137 Biogenic SOA formation from isoprene, monoterpenes, and sesquiterpenes is modeled. The latter 138 two are modeled as lumped chemical species meant to represent the entire monoterpene and 139 sesquiterpene families of compounds, respectively. Both aSOA and bSOA are simulated using 4 140 volatility bins ranging from saturation concentration of 100 to 103 µg m-3 at 298 K and use NOx- 141 dependent SOA formation yields (Lane et al., 2008a; Day, 2014). Improved SOA formation yields 142 from sesquiterpenes have been included for the purposes of this study (Sippial et al., 2022). 143 Primary organic aerosol (POA) is simulated using 8 volatility bins ranging from saturation 144 concentration of 10-1 to 106 µg m-3 at 298 K. Gas-phase chemistry is described using the Carbon 145 Bond 05 (CB5) mechanism (Yarwood et al., 2005). 2 Model Description 120 Water vapor influences indirectly the SOA 146 formation in PMCAMx, through its involvement in the gas-phase chemistry, e.g., leading to the 147 production of OH radicals. The mechanism used here includes 193 reactions of 79 gas-phase 148 chemical species. Aqueous-phase chemistry is simulated using the Variable Size Resolution Model 149 of Fahey and Pandis (2001). Notably, particle acidity effects are not included in the model. This is 150 a candidate process for model improvement if inconsistencies arise between the changes in 151 modeled and observed OA. 152 pp PMCAMx was applied with a simulation domain covering the contiguous United States 155 using a grid of 132 by 82 cells with horizontal dimension of 36 x 36 km (total area of 4752 x 2952 156 km2), and 14 layers of varying height up to maximum altitude around 13 km. The domain is 157 equivalent to that of Skyllakou et al. (2021). We apply this simulation approach for the summer 158 5 https://doi.org/10.5194/acp-2022-648 Preprint. Discussion started: 19 September 2022 c⃝Author(s) 2022. CC BY 4.0 License. https://doi.org/10.5194/acp-2022-648 Preprint. Discussion started: 19 September 2022 c⃝Author(s) 2022. CC BY 4.0 License. months (June, July, and August) of 2001 and 2010. Predictions for the summer of 1990 are not 159 used in our analysis due to the lack of available measurements of organic carbon in the southeast 160 United States during that period. The first two days of simulation output from each period were 161 removed from the analysis to account for model spin-up. 162 Meteorological fields were simulated using the Weather Research and Forecasting model 163 (WRFv3.6.1) to provide PMCAMx with wind components, diffusivity, temperature, pressure, 164 humidity, clouds, and precipitation inputs. The initial and boundary conditions for WRF 165 simulations were generated using the ERA-Interim global climate re-analysis database, along with 166 land-use information, soil categories, and terrain height retrieved from the United States 167 Geological Survey. WRF was configured in the manner recommended for air quality simulations 168 (Gilliam and Pleim, 2010; Rogers et al., 2013; Hogrefe et al., 2015). 169 Anthropogenic emissions for the simulations were obtained from the historical emissions 170 inventories of Xing et al. (2013) and include both gaseous and primary particulate emissions. 171 Emissions in this inventory are resolved by source category, however this level of detailed 172 information was not pertinent to the analysis in this work. 2 Model Description 120 A detailed description of these emissions 173 is provided by Skyllakou et al. (2021). The Model of Emissions of Gases and Aerosols from Nature 174 version 3 (MEGAN3) (Guenther et al., 2018) was used to calculate biogenic emissions for all 175 simulations. 176 The subdomain of focus for this study is the southeast United States, as defined by the 177 boundary in Figure 1. Selected changes in emissions used for the two summer simulation periods 178 in the southeast United States are summarized in Figure 2. Significant reductions in key 179 anthropogenic emissions affecting SOA formation have taken place. SO2 emissions in the US fell 180 by 47% between the summers of 2001 and 2010 from an average of 348 g h-1 km-2 to 184 g h-1 181 km-2. Over the same period, NOx emissions were reduced by 42% from 245 g h-1 km-2 to 141 g 182 h-1 km-2. NOx emissions changes can have competing effects in terms of SOA formation depending 183 on the levels of other pollutants. 184 Anthropogenic VOC emissions decreased by 31% between the summer of 2001 and the 185 summer of 2010 from 898 to 623 g h-1 km-2. This reduction is expected to have led to a reduction 186 of anthropogenic OA between the two periods, which should cause a decrease in biogenic OA due 187 to partitioning effects. The magnitude of this effect is quantified in a subsequent section. Increases 188 in the emissions of all biogenic VOCs were estimated between the summer of 2001 and the summer 189 6 https://doi.org/10.5194/acp-2022-648 Preprint. Discussion started: 19 September 2022 c⃝Author(s) 2022. CC BY 4.0 License. of 2010 due to higher temperatures in the second period. Isoprene, monoterpene, and sesquiterpene 190 emissions increased by 27%, 18%, and 21%, respectively as predicted by MEGAN3. This of 191 course would tend to increase bSOA concentrations between the two periods all other factors (NOx, 192 partitioning) being equal. The temperature increase is depicted in Figure S1. 193 A sensitivity test removing the NOx-level dependency of SOA formation yields is 194 considered in this work to determine the efficacy of these parameterizations in capturing the 195 various types of SOA concentration changes that would result from reductions in NOx emissions. 196 197 4 Results 198 4.1 Organic aerosol predictions 199 Average predicted bSOA for the summers of 2001 and 2010 is shown in Figure 3. 2 Model Description 120 A small 200 increase of around 5% (2.19 µg m-3 to 2.35 µg m-3) in the average bSOA concentration in the 201 southeast United States is predicted from 2001 to 2010. This change is much less than the increase 202 (18-27%) in biogenic VOC emissions in the same area. 203 The biogenic fraction of predicted total OA is shown for both simulation periods in Figure 204 4. A notable increase was observed between the summers of 2001 and 2010 in the southeast from 205 46 to 63%. This result points to competing effects producing the changes observed in predicted 206 OA. The small increase in bSOA concentration, especially when compared to the larger increases 207 in biogenic VOC emissions, points to other processes partially offsetting the increases in bSOA. 208 Anthropogenic SOA decreased significantly between the two summers from 2.5 µg m-3 in 2001 to 209 1.3 µg m-3 in 2010. This accounts for most of the increase in the biogenic fraction. Primary organic 210 aerosol (POA) also decreased between the two periods, but only by a small amount (0.1 µg m-3). 211 A breakdown of individual bSOA precursor contributions to total predicted average bSOA 212 concentrations in the southeast United States is provided in Figure S2. Relative contributions from 213 the individual VOC precursors did not change appreciably between the two simulation periods. 214 Between the summer of 2001 and the summer of 2010, contributions to bSOA increased slightly 215 for isoprene (21% to 23%) and sesquiterpenes (35% to 37%) while the contribution from 216 monoterpenes decreased slightly (44% to 40%). 217 Predictions of OA concentrations were compared to available measurements of OA in the 218 southeast United States during the two simulation periods. Measurements from both the primarily 219 urban CSN network (US EPA 2002) as well as the primarily rural IMPROVE network 220 of 2010 due to higher temperatures in the second period. Isoprene, monoterpene, and sesquiterpene 190 emissions increased by 27%, 18%, and 21%, respectively as predicted by MEGAN3. This of 191 course would tend to increase bSOA concentrations between the two periods all other factors (NOx, 192 partitioning) being equal. The temperature increase is depicted in Figure S1. 2 Model Description 120 193 A sensitivity test removing the NOx-level dependency of SOA formation yields is 194 considered in this work to determine the efficacy of these parameterizations in capturing the 195 various types of SOA concentration changes that would result from reductions in NOx emissions. 196 197 4.1 Organic aerosol predictions 199 Metrics for model prediction evaluation here include 222 the fractional error and fractional bias given by: 223 Fractional Error = 2 𝑁∑ \|𝑃𝑖−𝑂𝑖\| 𝑃𝑖+ 𝑂𝑖 𝑁 𝑖=1 (1) 224 Fractional Bias = 2 𝑁∑𝑃𝑖−𝑂𝑖 𝑃𝑖+ 𝑂𝑖 𝑁 𝑖=1 (2) 225 Fractional Error = 2 𝑁∑ \|𝑃𝑖−𝑂𝑖\| 𝑃𝑖+ 𝑂𝑖 𝑁 𝑖=1 (1) 224 Fractional Bias = 2 𝑁∑𝑃𝑖−𝑂𝑖 𝑃𝑖+ 𝑂𝑖 𝑁 𝑖=1 (2) 225 (1) (2) where N is the number of valid measurements, Oi is a single daily measurement, and Pi is a single 226 daily average predicted concentration in the simulation cell where measurement i was taken. Daily 227 average OA concentration measurements from CSN (2001: 7 sites; 2010: 36 sites) and IMPROVE 228 (2001: 13 sites; 2010: 15 sites) sites in the southeastern US (Figure 1) are included in the evaluation 229 dataset. 230 Scatter plots illustrating model OA prediction performance in the southeast United States 231 for summer 2001 and summer 2010 are shown in Figure 5. The corresponding performance metrics 232 for the two simulation periods and measurement networks are provided in Figure 6. In terms of 233 fractional error, OA prediction performance is consistent across the two time periods and site types 234 with the fractional error varying between 0.4 and 0.44. Fractional bias shows greater variability 235 between time periods and networks, with a minimum of -0.09 for rural sites in 2010 and a 236 maximum of +0.22 for urban sites in 2010. While the variability in bias is higher, it never exceeds 237 the bias goals of chemical transport model predictions of ±0.30 (Boylan and Russell, 2006). The 238 consistent performance suggests that the model adequately reproduces OA concentrations in the 239 two time periods. Also, the significant change in anthropogenic emissions does not appear to affect 240 the model error, while the small tendency for overprediction becomes a small tendency towards 241 underprediction. 242 8 The predicted changes at individual sites that were online in both years are directly 243 compared to corresponding observed changes between the two time periods in Figure 7. The 244 changes in OA concentrations are aggregated monthly for each monitor, to reduce the amount of 245 noise in analyzing the changes. The ability of PMCAMx to reproduce the observed changes in 246 each monitoring site is encouraging; large changes in observed concentrations generally 247 correspond to large changes in predicted concentrations. 4.1 Organic aerosol predictions 199 Average predicted bSOA for the summers of 2001 and 2010 is shown in Figure 3. A small 200 increase of around 5% (2.19 µg m-3 to 2.35 µg m-3) in the average bSOA concentration in the 201 southeast United States is predicted from 2001 to 2010. This change is much less than the increase 202 (18-27%) in biogenic VOC emissions in the same area. 203 The biogenic fraction of predicted total OA is shown for both simulation periods in Figure 204 4. A notable increase was observed between the summers of 2001 and 2010 in the southeast from 205 46 to 63%. This result points to competing effects producing the changes observed in predicted 206 OA. The small increase in bSOA concentration, especially when compared to the larger increases 207 in biogenic VOC emissions, points to other processes partially offsetting the increases in bSOA. 208 Anthropogenic SOA decreased significantly between the two summers from 2.5 µg m-3 in 2001 to 209 1.3 µg m-3 in 2010. This accounts for most of the increase in the biogenic fraction. Primary organic 210 aerosol (POA) also decreased between the two periods, but only by a small amount (0.1 µg m-3). 211 A breakdown of individual bSOA precursor contributions to total predicted average bSOA 212 concentrations in the southeast United States is provided in Figure S2. Relative contributions from 213 the individual VOC precursors did not change appreciably between the two simulation periods. 214 Between the summer of 2001 and the summer of 2010, contributions to bSOA increased slightly 215 for isoprene (21% to 23%) and sesquiterpenes (35% to 37%) while the contribution from 216 monoterpenes decreased slightly (44% to 40%). 217 Predictions of OA concentrations were compared to available measurements of OA in the 218 southeast United States during the two simulation periods. Measurements from both the primarily 219 urban CSN network (US EPA, 2002) as well as the primarily rural IMPROVE network 220 7 https://doi.org/10.5194/acp-2022-648 Preprint. Discussion started: 19 September 2022 c⃝Author(s) 2022. CC BY 4.0 License. https://doi.org/10.5194/acp-2022-648 Preprint. Discussion started: 19 September 2022 c⃝Author(s) 2022. CC BY 4.0 License. (IMPROVE, 1995) were used to consider various site locations which could impact the level of 221 anthropogenic influence on SOA formation. 4.1 Organic aerosol predictions 199 Most inconsistencies in the direction of 248 the change between predictions and measurements occur when the changes are small in magnitude 249 8 https://doi.org/10.5194/acp-2022-648 Preprint. Discussion started: 19 September 2022 c⃝Author(s) 2022. CC BY 4.0 License. https://doi.org/10.5194/acp-2022-648 Preprint. Discussion started: 19 September 2022 c⃝Author(s) 2022. CC BY 4.0 License. with two noteworthy exceptions. One of these exceptions is for the month of June at the 250 Okefenokee National Wildlife Refuge (Georgia, IMPROVE) site. Here, measurements of 10, 11, 251 and 23 µg m-3 were recorded on June 3, 9, and 12, respectively. This is due to two reported fires 252 in the refuge that are not in the emissions inventory used with PMCAMx. The abnormally high 253 measured PM2.5 concentrations on these three days result in a much higher predicted decrease in 254 measured PM2.5 at this location between 2001 and 2010 (-4.7 µg m-3) than the model is able to 255 reproduce (+1.4 µg m-3). Removing these three days from the analysis reduces the change in 256 measured PM2.5 to -1.7 µg m-3. The changes in measured and predicted PM2.5 concentrations at the 257 Okefenokee NWR site are in better agreement for the other two months. Measured PM2.5 decreased 258 by 1.6 µg m-3 while predicted PM2.5 decreased by 0.3 µg m-3 in the month of July. In August, 259 measured PM2.5 decreased by 2.3 µg m-3 and predicted PM2.5 decreased by 1.7 µg m-3 between 260 2001 and 2010. The other outlier is for the month of July at the Shining Rock Wilderness (North 261 Carolina, IMPROVE). At this location, only one daily measurement was available during July 262 2001. This means that the entire monthly average of July 2001 (10 measurements) was only 263 compared to one measurement, resulting in a problematic comparison. This datapoint has been 264 excluded from Figure 7 as well as the formal analysis. 265 266 4.2 Effect of semi-volatile partitioning 267 Changes in biogenic emissions between 2001 and 2010 are larger than the change in bSOA 268 concentration predicted by the model pointing to offsetting factors that we explore here. 269 Differences in semivolatile partitioning between the two periods are a potential competing effect 270 that could explain the small change in bSOA concentrations. The large decreases in anthropogenic 271 emissions result in less SOA formed via the oxidation of anthropogenic SOA precursors. Less 272 existing particle phase OA will then result in smaller fractions of biogenic SOA compounds 273 partitioning into the particle phase. This is consistent with the significant predicted increase in the 274 biogenic fraction of total OA with only a small increase in bSOA concentration. 275 To examine the impact of changes in semivolatile partitioning on predicted bSOA 276 concentrations, we estimated the fraction of the total (gas and particulate) biogenic SOA 277 components present in 2010 that would partition to the particle phase if the particle phase 278 anthropogenic SOA remained constant from 2001 to 2010 (2.51 µg m-3), rather than decreasing to 279 1.37 µg m-3. We assume that the total bSOA material concentration (sum of gas and particle phase) 280 9 https://doi.org/10.5194/acp-2022-648 Preprint. Discussion started: 19 September 2022 c⃝Author(s) 2022. CC BY 4.0 License. https://doi.org/10.5194/acp-2022-648 Preprint. Discussion started: 19 September 2022 c⃝Author(s) 2022. CC BY 4.0 License. in 2010 does not change with this hypothetical increase in anthropogenic SOA. The difference 281 between this estimated bSOA concentration (2.7 µg m-3) in this scenario and the predicted biogenic 282 SOA concentration during the summer of 2010 (2.3 µg m-3) is a good estimate of the effect of 283 semivolatile partitioning on predicted biogenic SOA (-0.4 µg m-3). These results are illustrated in 284 Figure 8. If anthropogenic OA remained the same from 2001 to 2010, the biogenic SOA 285 concentration in 2010 would be about 15% higher. In that case the bSOA would increase by 24% 286 from 2001 to 2010, a change that is consistent with the increases in biogenic VOC emissions 287 between the two periods (27%, 18%, 21% for isoprene, monoterpenes, and sesquiterpenes, 288 respectively). Therefore, differences in semivolatile partitioning are predicted to have offset to 289 large extent the increased bSOA due to the higher biogenic emissions in this warmer period. 290 291 4.3 Effect of NOx-dependence 292 We investigated also the effects of the NOx-dependencies of the biogenic SOA yields on 293 both simulation periods and site location types. As a sensitivity test, the same simulations (summer 294 2001 and summer 2010) were performed with NOx-independent SOA formation yields for 295 biogenic SOA. This was done by setting the NOx-level branching ratio to zero, effectively setting 296 the biogenic SOA formation yields equal to their low-NOx level in all conditions. In most cases 297 for bSOA, this represents a higher-yield scenario when compared to the NOx-dependent base case 298 (Lane et al., 2008a). 299 The average bSOA concentrations for these two additional simulations are shown in Figure 300 9. For both simulation periods, the average bSOA concentration in the southeast United States 301 increased when compared to the simulations with NOx-dependent yields. The average predicted 302 bSOA concentration in the southeast United States for the summer of 2001 in the sensitivity test 303 became 2.97 µg m-3 (previously 2.19 µg m-3) and 2.84 µg m-3 (previously 2.35 µg m-3) for 2010. 304 This increase is consistent with expectations based on the assumed NOx dependence of the 305 biogenic SOA yields. 306 The increase in bSOA due to the use of the low-NOx yields was 35% in the summer of 307 2001 and 22% in the summer of 2010, therefore the reduction in NOx emissions resulted in a 308 change of the NOx effect on SOA by approximately 13% of the total biogenic SOA. The biogenic 309 fractions of total OA also increased due to the use of low-NOx yields compared to the NOx- 310 dependent simulations (Figure 10). For summer 2001 the biogenic fraction was 0.50 (previously 311 10 https://doi.org/10.5194/acp-2022-648 Preprint. Discussion started: 19 September 2022 c⃝Author(s) 2022. CC BY 4.0 License. https://doi.org/10.5194/acp-2022-648 Preprint. Discussion started: 19 September 2022 c⃝Author(s) 2022. CC BY 4.0 License. 0.46) and for summer 2010 the biogenic fraction was 0.65 (previously 0.63). These results suggest 312 that the reductions of NOx emissions during the decade caused an increase of the bSOA by roughly 313 10% based on the PMCAMx predictions. 314 0.46) and for summer 2010 the biogenic fraction was 0.65 (previously 0.63). These results suggest 312 that the reductions of NOx emissions during the decade caused an increase of the bSOA by roughly 313 10% based on the PMCAMx predictions. 4.3 Effect of NOx-dependence 292 314 OA prediction performance scatter plots for the NOx-independent bSOA yield simulations 315 are shown in Figure 11 and performance metrics are shown in Figure 12. This sensitivity test 316 produced weaker performance for OA. The fractional error increased in both simulation periods 317 and for both networks when compared to the results of the base case simulation with the NOx- 318 dependent yields. The minimum fractional error for the low-NOx scenario was 0.43 (IMPROVE, 319 summer 2010) and the maximum was 0.53 (CSN, summer 2010). All fractional biases increased 320 by around 0.2, ranging from +0.10 (IMPROVE, summer 2010) to +0.42 (CSN, summer 2010). 321 Performance in this sensitivity test is notably worse in the summer of 2010, indicating that the 322 NOx-dependent bSOA yields implemented in PMCAMx are important at capturing the dynamics 323 of SOA formation with significant concurrent changes in anthropogenic and biogenic emissions. 324 325 5 Conclusions 326 Emissions of most anthropogenic pollutants influencing SOA formation decreased 327 significantly from 2001 to 2010 in the southeast United States. NOx emissions were reduced by 328 42%, impacting NOx-dependent SOA formation yields as well as oxidant levels and the 329 corresponding oxidation pathways. Anthropogenic VOC emissions were reduced by 31%, in turn 330 reducing anthropogenic OA and impacting the gas-to-particle partitioning of semi-volatile 331 biogenic SOA components. SO2 emissions fell by 47% over the same period which could impact 332 any particle acidity effects on SOA formation although this potential effect was not considered 333 here. PMCAMx simulations were performed covering two summer simulation periods of June, 334 July, and August of 2001 and 2010 in order to evaluate the effectiveness of the model at 335 reproducing changes in biogenic SOA between the two summers. This version of PMCAMx 336 simulates SOA formation through semi-volatile partitioning of SOA species and NOx-dependent 337 SOA formation yields neglecting acidity effects and aqueous-phase processes. 338 The average predicted bSOA concentration in the southeast US increased from 1.86 µg 339 m-3 to 1.89 µg m-3 from summer 2001 to summer 2010. The biogenic fraction of total OA based 340 on PMCAMx increased from 46% to 63% over the same period, suggesting significant reductions 341 in anthropogenic contributions. Anthropogenic OA levels were reduced significantly (45%), and 342 11 https://doi.org/10.5194/acp-2022-648 Preprint. Discussion started: 19 September 2022 c⃝Author(s) 2022. CC BY 4.0 License. this resulted in around 0.4 µg m-3 less predicted biogenic SOA through gas-to-particle partitioning 343 effects. 344 With this reduced bSOA in 2010 due to partitioning effects and the NOx-dependent SOA 345 formation chemistry in this formulation of PMCAMx OA observations from IMPROVE and CSN 346 monitoring sites are reproduced reasonably well and consistently in both summers (fractional error 347 of 0.40 and 0.44; fractional bias of -0.09 and 0.22). Removing the NOx-dependence of the SOA 348 formation yields had negative impacts on model performance, increasing fractional error and bias 349 and at both time periods and at both types of monitoring sites. 5 Conclusions 326 The results of this study suggest 350 that a chemical transport model with NOx-dependent yields for SOA precursors, including the 351 explicit treatment of sesquiterpene SOA formation, and semivolatile partitioning of SOA mass 352 using a method such as the volatility basis set has a reasonable performance in reproducing 353 summertime atmospheric OA levels and their changes in response to significant changes in both 354 anthropogenic and biogenic emissions. 355 356 this resulted in around 0.4 µg m-3 less predicted biogenic SOA through gas-to-particle partitioning 343 effects. 344 With this reduced bSOA in 2010 due to partitioning effects and the NOx-dependent SOA 345 formation chemistry in this formulation of PMCAMx OA observations from IMPROVE and CSN 346 monitoring sites are reproduced reasonably well and consistently in both summers (fractional error 347 of 0.40 and 0.44; fractional bias of -0.09 and 0.22). Removing the NOx-dependence of the SOA 348 formation yields had negative impacts on model performance, increasing fractional error and bias 349 and at both time periods and at both types of monitoring sites. The results of this study suggest 350 that a chemical transport model with NOx-dependent yields for SOA precursors, including the 351 explicit treatment of sesquiterpene SOA formation, and semivolatile partitioning of SOA mass 352 using a method such as the volatility basis set has a reasonable performance in reproducing 353 summertime atmospheric OA levels and their changes in response to significant changes in both 354 anthropogenic and biogenic emissions. 355 356 Code and Data Availability. The model code and data used in this study are available from the 357 authors upon request (spyros@chemeng.upatras.gr). 358 359 Code and Data Availability. The model code and data used in this study are available from the 357 authors upon request (spyros@chemeng.upatras.gr). 358 359 Code and Data Availability. The model code and data used in this study are available from the 357 authors upon request (spyros@chemeng.upatras.gr). 358 359 Author contributions. BTD performed the MEGAN and PMCAMx simulations, analyzed the 360 results, and wrote the manuscript. PGR prepared the anthropogenic emissions and other inputs for 361 the PMCAMx simulations. KS performed preliminary PMCAMx simulations and prepared model 362 inputs. SNP and PJA designed and coordinated the study and helped in the writing of the paper. 363 All authors reviewed and commented on the manuscript. 364 Competing Interests. The authors declare that they have no conflict of interest. 366 367 Financial support. 5 Conclusions 326 395 Gaydos, T.M., Koo, B., Pandis, S.N., Chock, D.P.: Development and application of an efficient 396 moving sectional approach for the solution of the atmospheric aerosol 397 condensation/evaporation equations, Atmos. Environ., 37, 3303-3316, doi:10.1016/S1352- 398 2310(03)00267-X, 2003. 399 Gilliam, R.C., Pleim, J.E.: Performance assessment of new land surface and planetary boundary 400 layer physics in the WRF-ARW, J. Appl. Meteorol. Clim., 49, 760-774, 401 References 374 Boylan, J.W., Russell, AG.: PM and light extinction model performance metrics, goals, and criteria 375 for three-dimensional air quality models, Atmos. Environ., 40, 4946-4959, doi: 376 10.1016/j.atmosenv.2005.09.087, 2006. 377 Cao, G., Jang, M.: An SOA model for toluene oxidation in the presence of inorganic aerosols, 378 Environ. Sci. Technol., 44, 727-733, https://doi.org/10.1021/es901682r, 2010. 379 Carlton, A.G., Pinter, R.W., Bhave, P.V., Pouliot, G.A.: To what extent can biogenic SOA be 380 controlled?, Environ. Sci. Technol., 44, 3376-3380. doi:10.1021/es903506b, 2010. 381 Carlton, A.G., Pye, H.O.T., Baker, K.R., Hennigan, C.J.: Additional benefits of federal air-quality 382 rules: model estimates of controllable biogenic secondary organic aerosol, Environ. Sci. 383 Tech., 52, 9254-9265, doi:10.1021/acs.est.8b01869, 2018. 384 Day, M.: Atmospheric organic aerosol and climate change [Doctoral dissertation, Carnegie Mellon 385 University], Proquest Dissertations and Theses Global, 2014. 386 Donahue, N.M., Robinson, A.L., Stanier, C.O., Pandis, S.N.: Coupled partitioning, dilution, and 387 chemical aging of semivolatile organics, Environ. Sci. Tech., 40, 2635-2643, 388 doi:10 1021/es052297c 2006 389 Boylan, J.W., Russell, AG.: PM and light extinction model performance metrics, goals, and criteria 375 for three-dimensional air quality models, Atmos. Environ., 40, 4946-4959, doi: 376 10.1016/j.atmosenv.2005.09.087, 2006. 377 Cao, G., Jang, M.: An SOA model for toluene oxidation in the presence of inorganic aerosols, 378 Environ. Sci. Technol., 44, 727-733, https://doi.org/10.1021/es901682r, 2010. 379 , , g, p g Environ. Sci. Technol., 44, 727-733, https://doi.org/10.1021/es901682r, 2010. 379 Carlton, A.G., Pinter, R.W., Bhave, P.V., Pouliot, G.A.: To what extent can biogenic SOA be 380 controlled?, Environ. Sci. Technol., 44, 3376-3380. doi:10.1021/es903506b, 2010. 381 Carlton, A.G., Pye, H.O.T., Baker, K.R., Hennigan, C.J.: Additional benefits of federal air-quality 382 rules: model estimates of controllable biogenic secondary organic aerosol, Environ. Sci. 383 Tech., 52, 9254-9265, doi:10.1021/acs.est.8b01869, 2018. 384 Day, M.: Atmospheric organic aerosol and climate change [Doctoral dissertation, Carnegie Mellon 385 University], Proquest Dissertations and Theses Global, 2014. 386 y, p g g [ , g University], Proquest Dissertations and Theses Global, 2014. 386 University], Proquest Dissertations and Theses Global, 2014. 5 Conclusions 326 This work was supported by the Center for Air, Climate, and Energy Solutions 368 (CACES) which was supported under Assistance Agreement No. R835873 awarded by the U.S. 369 Environmental Protection Agency and the Horizon-2020 Project FORCeS of the European Union 370 under grant agreement No 821205. 371 367 Financial support. This work was supported by the Center for Air, Climate, and Energy Solutions 368 (CACES) which was supported under Assistance Agreement No. R835873 awarded by the U.S. 369 Environmental Protection Agency and the Horizon-2020 Project FORCeS of the European Union 370 under grant agreement No 821205. 371 367 Financial support. This work was supported by the Center for Air, Climate, and Energy Solutions 368 (CACES) which was supported under Assistance Agreement No. R835873 awarded by the U.S. 369 Environmental Protection Agency and the Horizon-2020 Project FORCeS of the European Union 370 under grant agreement No 821205. 371 3 2 12 12 https://doi.org/10.5194/acp-2022-648 Preprint. Discussion started: 19 September 2022 c⃝Author(s) 2022. CC BY 4.0 License. References 374 Boylan, J.W., Russell, AG.: PM and light extinction model performance metrics, goals, and criteria 375 for three-dimensional air quality models, Atmos. 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Technol., 47, 483 11056-11064, doi:10.1021/es402106h, 2013. 5 Conclusions 326 484 Rogers, R.E., Deng, A., Stauffer, D.R., Gaudet, B.J., Jia, Y., Soong, S.T., Tanrikulu, S.: 485 Application of the weather research and forecasting model for air quality modeling in the 486 San Francisco bay area, J. Appl. Meteorol. Clim., 52, 1953-1973, doi:10.1175/JAMC-D- 487 12-0280.1, 2013. 488 Simon, H., Reff, A., Wells, B., Xing, J., Frank, N.: Ozone trends across the United States over a 489 period of decreasing NOx and VOC emissions, Environ. Sci. Technol., 49, 186-195, 490 doi:10.1021/es504514z, 2015. 491 Sindelarova, K., Granier, C., Bouarar, I., Guenther, A., Tilmes, S., Stavrakou, T., Müller, J.F., 492 Kuhn, U., Stefani, P., Knorr, W.: Global data set of biogenic VOC emissions calculated by 493 the MEGAN model over the last 30 years, Atmos. Chem. Phys., 14, 9317-9341, 494 doi:10.5194/acp-14-9317-2014, 2014. 495 16 https://doi.org/10.5194/acp-2022-648 Preprint. Discussion started: 19 September 2022 c⃝Author(s) 2022. CC BY 4.0 License. https://doi.org/10.5194/acp-2022-648 Preprint. Discussion started: 19 September 2022 c⃝Author(s) 2022. CC BY 4.0 License. Sippial, D., Uruci, P., Kostenidou, E., Pandis, S.N.: Formation of secondary organic aerosol during 496 the dark-ozonolysis of a-humulene, submitted for publication. 497 Skyllakou, K., Garcia Rivera, P., Dinkelacker, B., Karnezi, E., Kioutsioukis, I., Hernandez, C., 498 Adams, P.J., Pandis, S.N.: Changes in PM2.5 concentrations and their sources in the US 499 from 1990 to 2010, Atmos. Chem. Phys., 21, 17115-17132, doi:10.5194/acp-21-17115- 500 2021, 2021. 501 Smith, S.J., van Aardenne, J., Klimont, Z., Andres, R.J., Volke, A., Delgado Arias, S.: 502 Anthropogenic sulfur dioxide emissions: 1850-2005, Atmos. Chem. Phys., 11, 1101-1116, 503 doi:10.5194/acp-11-1101-2011, 2011. 504 Strader, R., Lurmann, F., Pandis, S.N.: Evaluation of secondary organic aerosol formation in 505 winter, Atmos. Environ., 33, 4849-4863, doi:10.1016/S1352-2310(99)00310-6, 1999. 506 Takahama, S., Davidson, C.I., Pandis, S.N.: Semicontinuous measurements of organic carbon and 507 acidity during the Pittsburgh Air Quality Study: Implications for acid-catalyzed organic 508 aerosol formation, Environ. Sci. Technol., 40, 2191-2199, doi:10.1021/ es050856+, 2006. 509 Tsimpidi, A.P., Karydis, V.A., Pandis, S.N.: Response of fine particulate matter to emission 510 changes of NOx and anthropogenic VOCs in the eastern US, J. Air Waste Manage. Assoc., 511 58, 1463-1473, doi:10.3155/1047-3289.58.11.1463, 2008. 512 Tsimpidi, A.P., Karydis, V.A., Zavala, M., Lei, W., Molina, L.T., Ulbrich, I.M., Jimenez, J.L., 513 Pandis, S.N.: Evaluation of the volatility basis-set approach for the simulation of organic 514 aerosol formation in the Mexico City metropolitan area, Atmos. Chem. Phys., 10, 525-546, 515 doi:10.5194/acp-10-525-2010, 2010. 5 Conclusions 326 516 US EPA (United States Environmental Protection Agency): User Guide: Air Quality System, 517 Report, Research Triangle Park, N.C., available at: https://www.epa.gov/ttn/airs/airsaqs/ 518 manuals/AQSUserGuide.pdf (last access: January 2022), 2002. 519 Weber, R.J., Guo, H., Russell, A.G., Nenes, A.: High aerosol acidity despite declining atmospheric 520 sulfate concentrations over the past 15 years, Nat. Geosci., 9, 282-285, 521 doi:10.1038/ngeo2665, 2016. 522 Xing, J., Pleim, J., Mathur, R., Pouliot, G., Hogrefe, C., Gan, C.-M., Wei, C.: Historical gaseous 523 and primary aerosol emissions in the United States from 1990 to 2010, Atmos. Chem. 524 Phys., 13, 7531-7549, doi:10.5194/acp-13-7531-2013, 2013. 525 17 https://doi.org/10.5194/acp-2022-648 Preprint. Discussion started: 19 September 2022 c⃝Author(s) 2022. CC BY 4.0 License. https://doi.org/10.5194/acp-2022-648 Preprint. Discussion started: 19 September 2022 c⃝Author(s) 2022. CC BY 4.0 License. https://doi.org/10.5194/acp-2022-648 Preprint. Discussion started: 19 September 2022 c⃝Author(s) 2022. CC BY 4.0 License. Yarwood, G., Rao, S., Yocke, M., Whitten, G.Z.: Updates to the carbon bond chemical 526 mechanism: CB05, Final Report to the US EPA, RT-0400675, 2005. 527 Zhang, H., Surratt, J.D., Lin, Y.H., Bapat, J., Kamens, R.M.: Effect of relative humidity on SOA 528 formation from isoprene/NO photooxidation: enhancement of 2-methylglyceric acid and 529 its corresponding oligoesters under dry conditions, Atmos. Chem. Phys., 11, 6411-6424, 530 https://doi.org/10.5194/acp-11-6411-2011, 2011. 531 Ziemann, P.J., Atkinson, R.: Kinetics, products, and mechanisms of secondary organic aerosol 532 formation, Chem. Soc. Rev., 41, 6582-6605, https://doi.org/10.1039/C2CS35122F, 2012. 533 534 Yarwood, G., Rao, S., Yocke, M., Whitten, G.Z.: Updates to the carbon bond chemical 526 mechanism: CB05, Final Report to the US EPA, RT-0400675, 2005. 527 Zhang, H., Surratt, J.D., Lin, Y.H., Bapat, J., Kamens, R.M.: Effect of relative humidity on SOA 528 formation from isoprene/NO photooxidation: enhancement of 2-methylglyceric acid and 529 its corresponding oligoesters under dry conditions, Atmos. Chem. Phys., 11, 6411-6424, 530 https://doi.org/10.5194/acp-11-6411-2011, 2011. 531 Yarwood, G., Rao, S., Yocke, M., Whitten, G.Z.: Updates to the carbon bond chemical 526 mechanism: CB05, Final Report to the US EPA, RT-0400675, 2005. 527 Zhang, H., Surratt, J.D., Lin, Y.H., Bapat, J., Kamens, R.M.: Effect of relative humidity on SOA 528 formation from isoprene/NO photooxidation: enhancement of 2-methylglyceric acid and 529 its corresponding oligoesters under dry conditions, Atmos. Chem. Phys., 11, 6411-6424, 530 https://doi.org/10.5194/acp-11-6411-2011, 2011. 531 Yarwood, G., Rao, S., Yocke, M., Whitten, G.Z.: Updates to the carbon bond chemical 526 mechanism: CB05, Final Report to the US EPA, RT-0400675, 2005. 5 Conclusions 326 527 Zhang, H., Surratt, J.D., Lin, Y.H., Bapat, J., Kamens, R.M.: Effect of relative humidity on SOA 528 formation from isoprene/NO photooxidation: enhancement of 2-methylglyceric acid and 529 its corresponding oligoesters under dry conditions, Atmos. Chem. Phys., 11, 6411-6424, 530 https://doi.org/10.5194/acp-11-6411-2011, 2011. 531 Ziemann, P.J., Atkinson, R.: Kinetics, products, and mechanisms of secondary organic aerosol 532 formation, Chem. Soc. Rev., 41, 6582-6605, https://doi.org/10.1039/C2CS35122F, 2012. 533 Ziemann, P.J., Atkinson, R.: Kinetics, products, and mechanisms of secondary organic aerosol 532 formation, Chem. Soc. Rev., 41, 6582-6605, https://doi.org/10.1039/C2CS35122F, 2012. 533 18 535 536 Figure 1: Contiguous United States simulation domain. The boundary of the southeast United 537 States, for the purposes of the analysis in this work is highlighted in red. 538 539 https://doi.org/10.5194/acp-2022-648 Preprint. Discussion started: 19 September 2022 c⃝Author(s) 2022. CC BY 4.0 License. https://doi.org/10.5194/acp-2022-648 Preprint. Discussion started: 19 September 2022 c⃝Author(s) 2022. CC BY 4.0 License. 535 536 Figure 1: Contiguous United States simulation domain. The boundary of the southeast United 537 States, for the purposes of the analysis in this work is highlighted in red. 538 539 535 Figure 1: Contiguous United States simulation domain. The boundary of the southeast United 537 States, for the purposes of the analysis in this work is highlighted in red. 538 Figure 1: Contiguous United States simulation domain. The boundary of the southeast United 537 States, for the purposes of the analysis in this work is highlighted in red. 538 States, for the purposes of the analysis in this work is highlighted in red. 538 19 https://doi.org/10.5194/acp-2022-648 Preprint. Discussion started: 19 September 2022 c⃝Author(s) 2022. CC BY 4.0 License. 540 540 541 542 Figure 2: Average summer emissions of (a) sulfur dioxide (SO2), (b) nitrogen oxides (NOx), (c) 543 non-methane volatile organic compounds (NMVOCs), (d) isoprene, (e) monoterpenes, (f) 544 sesquiterpenes in the southeast United States. 545 546 Figure 2: Average summer emissions of (a) sulfur dioxide (SO2), (b) nitrogen oxides (NOx), (c) 543 non-methane volatile organic compounds (NMVOCs), (d) isoprene, (e) monoterpenes, (f) 544 sesquiterpenes in the southeast United States. 545 20 https://doi.org/10.5194/acp-2022-648 Preprint. Discussion started: 19 September 2022 c⃝Author(s) 2022. CC BY 4.0 License. https://doi.org/10.5194/acp-2022-648 Preprint. Discussion started: 19 September 2022 c⃝Author(s) 2022. CC BY 4.0 License. 547 548 Figure 3: Predicted average summer bSOA concentration for (a) 2001 and (b) 2010. The average 549 value for the southeast US region is provided. 550 551 547 548 Figure 3: Predicted average summer bSOA concentration for (a) 2001 and (b) 2010 The average 549 Figure 3: Predicted average summer bSOA concentration for (a) 2001 and (b) 2010. The average 549 value for the southeast US region is provided. 550 21 21 553 554 Figure 4: Average fraction of predicted organic aerosol that is from biogenic sources for (a) 2001 555 and (b) 2010. 556 557 https://doi.org/10.5194/acp-2022-648 Preprint. Discussion started: 19 September 2022 c⃝Author(s) 2022. CC BY 4.0 License. 553 554 Figure 4: Average fraction of predicted organic aerosol that is from biogenic sources for (a) 2001 555 and (b) 2010. 556 557 https://doi.org/10.5194/acp-2022-648 Preprint. Discussion started: 19 September 2022 c⃝Author(s) 2022. CC BY 4.0 License. https://doi.org/10.5194/acp-2022-648 Preprint. Discussion started: 19 September 2022 c⃝Author(s) 2022. CC BY 4.0 License. 553 554 Figure 4: Average fraction of predicted organic aerosol that is from biogenic sources for (a) 2001 555 and (b) 2010. 556 557 Figure 4: Average fraction of predicted organic aerosol that is from biogenic sources for (a) 2001 555 and (b) 2010. 556 557 22 58 Figure 5: Comparison of predicted daily average OA concentrations with available daily OA 59 measurements in the southeast US from CSN (red) and IMPROVE (blue) sites for the summers 60 of (a) 2001 and (b) 2010. 61 //doi.org/10.5194/acp-2022-648 int. Discussion started: 19 September 2022 uthor(s) 2022. CC BY 4.0 License. 558 Figure 5: Comparison of predicted daily average OA concentrations with available daily OA 559 measurements in the southeast US from CSN (red) and IMPROVE (blue) sites for the summers 560 of (a) 2001 and (b) 2010. 561 562 58 Figure 5: Comparison of predicted daily average OA concentrations with available daily OA 559 measurements in the southeast US from CSN (red) and IMPROVE (blue) sites for the summers 560 of (a) 2001 and (b) 2010. 561 562 Figure 5: Comparison of predicted daily average OA concentrations with available daily OA 559 measurements in the southeast US from CSN (red) and IMPROVE (blue) sites for the summers 560 of (a) 2001 and (b) 2010. https://doi.org/10.5194/acp-2022-648 Preprint. Discussion started: 19 September 2022 c⃝Author(s) 2022. CC BY 4.0 License. 561 562 23 563 564 565 Figure 6: Summary of OA prediction performance in the southeast US based on the fractional 566 error and fractional bias of daily average OA predictions when compared to daily measurements 567 from CSN and IMPROVE sites for the summers of 2001 and 2010. 568 569 https://doi.org/10.5194/acp-2022-648 Preprint. Discussion started: 19 September 2022 c⃝Author(s) 2022. CC BY 4.0 License. 563 564 563 564 565 Figure 6: Summary of OA prediction performance in the southeast US based on the fractional 566 error and fractional bias of daily average OA predictions when compared to daily measurements 567 from CSN and IMPROVE sites for the summers of 2001 and 2010. 568 569 Figure 6: Summary of OA prediction performance in the southeast US based on the fractional 566 error and fractional bias of daily average OA predictions when compared to daily measurements 567 from CSN and IMPROVE sites for the summers of 2001 and 2010. 568 569 570 24 https://doi.org/10.5194/acp-2022-648 Preprint. Discussion started: 19 September 2022 c⃝Author(s) 2022. CC BY 4.0 License. https://doi.org/10.5194/acp-2022-648 Preprint. Discussion started: 19 September 2022 c⃝Author(s) 2022. CC BY 4.0 License. 571 572 573 Figure 7: Change in predicted and measured OA concentrations from summer 2001 to summer 574 2010 using monthly average predicted OA concentrations and monthly averaged measurements 575 from CSN and IMPROVE sites. 576 577 572 Figure 7: Change in predicted and measured OA concentrations from summer 2001 to summer 574 2010 using monthly average predicted OA concentrations and monthly averaged measurements 575 from CSN and IMPROVE sites. 576 577 Figure 7: Change in predicted and measured OA concentrations from summer 2001 to summer 574 2010 using monthly average predicted OA concentrations and monthly averaged measurements 575 from CSN and IMPROVE sites. 576 25 579 580 581 Figure 8: Predicted average summer OA concentration in the southeast US, resolved by 582 anthropogenic or biogenic source for the 2001 and 2010 summer periods, as well as the 2010 583 summer period with 2001 particle phase anthropogenic OA to test the partitioning effect on 584 biogenic OA in 2010. 585 586 2.51 1.37 2.51 2.19 2.35 2.7 0 2 4 6 2001 2010 2010 + 2001 particle phase aOA OA Concentration (mg m−3) Anthropogenic Biogenic 2.51 1.37 2.51 2.19 2.35 2.7 0 2 4 6 2001 2010 2010 + 2001 particle phase aOA OA Concentration (mg m−3) Anthropogenic Biogenic 2 4 OA Concentration (mg m−3) Figure 8: Predicted average summer OA concentration in the southeast US, resolved by 582 anthropogenic or biogenic source for the 2001 and 2010 summer periods, as well as the 2010 583 summer period with 2001 particle phase anthropogenic OA to test the partitioning effect on 584 biogenic OA in 2010. 585 586 26 https://doi.org/10.5194/acp-2022-648 Preprint. Discussion started: 19 September 2022 c⃝Author(s) 2022. CC BY 4.0 License. https://doi.org/10.5194/acp-2022-648 Preprint. Discussion started: 19 September 2022 c⃝Author(s) 2022. CC BY 4.0 License. 587 587 588 589 590 Figure 9: Predicted average summer bSOA concentration for (a) 2001 and (b) 2010 with NOx- 591 independent (low-NOx) yields. The average value for the southeast US region is provided. 592 593 587 588 589 590 Figure 9: Predicted average summer bSOA concentration for (a) 2001 and (b) 2010 with NOx- 591 independent (low-NOx) yields. The average value for the southeast US region is provided. 592 593 Figure 9: Predicted average summer bSOA concentration for (a) 2001 and (b) 2010 with NOx- 591 independent (low-NOx) yields. The average value for the southeast US region is provided. 592 593 Figure 9: Predicted average summer bSOA concentration for (a) 2001 and (b) 2010 with NOx- 591 independent (low-NOx) yields. The average value for the southeast US region is provided. 592 593 27 594 595 Figure 10: Average fraction of predicted organic aerosol that is from biogenic sources for (a) 596 2001 and (b) 2010 with NOx-independent (low-NOx) yields. The average value for the southeast 597 US region is provided 598 599 https://doi.org/10.5194/acp-2022-648 Preprint. Discussion started: 19 September 2022 c⃝Author(s) 2022. CC BY 4.0 License. https://doi.org/10.5194/acp-2022-648 Preprint. Discussion started: 19 September 2022 c⃝Author(s) 2022. CC BY 4.0 License. 594 595 Figure 10: Average fraction of predicted organic aerosol that is from biogenic sources for (a) 596 2001 and (b) 2010 with NOx-independent (low-NOx) yields. The average value for the southeast 597 US region is provided 598 599 Figure 10: Average fraction of predicted organic aerosol that is from biogenic sources for (a) 596 2001 and (b) 2010 with NOx-independent (low-NOx) yields. The average value for the southeast 597 US region is provided 598 28 602 603 Figure 11: Comparison of predicted daily average OA concentrations using NOx-independent 604 (low-NOx) yields with available daily OA measurements in the southeast US from CSN (red) and 605 IMPROVE (blue) sites for the summers of (a) 2001 and (b) 2010. 606 607 //doi.org/10.5194/acp-2022-648 int. Discussion started: 19 September 2022 uthor(s) 2022. CC BY 4.0 License. 602 603 Figure 11: Comparison of predicted daily average OA concentrations using NOx-independent 604 (low-NOx) yields with available daily OA measurements in the southeast US from CSN (red) and 605 IMPROVE (blue) sites for the summers of (a) 2001 and (b) 2010. https://doi.org/10.5194/acp-2022-648 Preprint. Discussion started: 19 September 2022 c⃝Author(s) 2022. CC BY 4.0 License. 606 607 Figure 11: Comparison of predicted daily average OA concentrations using NOx-independent 604 (low-NOx) yields with available daily OA measurements in the southeast US from CSN (red) and 605 IMPROVE (blue) sites for the summers of (a) 2001 and (b) 2010. 606 29 609 610 611 Figure 12: Summary of OA prediction performance in the southeast US based on the fractional 612 error and fractional bias of daily average OA predictions using NOx-independent (low-NOx) yields 613 when compared to daily measurements from CSN and IMPROVE sites for the summers of 2001 614 and 2010. 615 //doi.org/10.5194/acp-2022-648 int. Discussion started: 19 September 2022 uthor(s) 2022. CC BY 4.0 License. 609 609 610 611 609 610 611 Figure 12: Summary of OA prediction performance in the southeast US based on the fractional 612 error and fractional bias of daily average OA predictions using NOx-independent (low-NOx) yields 613 when compared to daily measurements from CSN and IMPROVE sites for the summers of 2001 614 and 2010. 615 616 Figure 12: Summary of OA prediction performance in the southeast US based on the fractional 612 error and fractional bias of daily average OA predictions using NOx-independent (low-NOx) yields 613 when compared to daily measurements from CSN and IMPROVE sites for the summers of 2001 614 and 2010. 615 617 30
https://openalex.org/W2566844572	https://iris.unipa.it/bitstream/10447/226899/1/Chiodini%20et%20al%202016%20ncomms13712-1.pdf	English	null	Magmas near the critical degassing pressure drive volcanic unrest towards a critical state	Nature communications	2,016	cc-by	11,738	ARTICLE Received 17 Mar 2016 \| Accepted 27 Oct 2016 \| Published 20 Dec 2016 Received 17 Mar 2016 \| Accepted 27 Oct 2016 \| Published 20 Dec 2016 1 Istituto Nazionale di Geoﬁsica e Vulcanologia, sezione di Bologna, via D. Creti 12, 40128 Bologna, Italy. 2 Istituto Nazionale di Geoﬁsica e Vulcanologia, sezione di Palermo, via U. La Malfa 153, 90146 Palermo, Italy. 3 DiSTeM, Universita` di Palermo, via Archiraﬁ36, 90123 Palermo, Italy. 4 Istituto Nazionale di Geoﬁsica e Vulcanologia, sezione di Napoli Osservatorio Vesuviano, via Diocleziano 328, 80124 Napoli, Italy. 5 Dipartimento di Scienze, Universita` Roma Tre, Largo San Leonardo Murialdo, 1, 00146 Roma, Italy. 6 Universite´ de Savoie Mont Blanc, Chambe´ry 73000, France. 7 CNRS, ISTerre, F-73376 Le Bourget du Lac, France. Correspondence and requests for materials should be addressed to G.C. (email: giovanni.chiodini@ingv.it). Magmas near the critical degassing pressure drive volcanic unrest towards a critical state Giovanni Chiodini1, Antonio Paonita2, Alessandro Aiuppa2,3, Antonio Costa1, Stef Prospero De Martino4, Valerio Acocella5 & Jean Vandemeulebrouck6,7 Giovanni Chiodini1, Antonio Paonita2, Alessandro Aiuppa2,3, Antonio Costa1, Stefano Caliro4, Prospero De Martino4, Valerio Acocella5 & Jean Vandemeulebrouck6,7 Results The critical degassing pressure during magma decompression. The decompression of fresh magma results in the selective release of dissolved volatiles depending on their solubilities28. This means that while barely soluble CO2 dominates deep degassing3,29,30, more-soluble H2O prevails at shallower depths31. Given this selective release of volatiles from magma and the different capacities of these two species to carry thermal energy, the pattern of heat transfer to overlying rocks and hydrothermal systems will be complex and will vary as the unrest progresses. Several saturation models have been reported in the literature25,28,32–36 that can be used to investigate magma degassing in an H2O–CO2 system. Whereas most of them predict the H2O and CO2 solubilities over narrow ranges of silicate melt composition, one of the models25 can be extended to any silicate melt composition. Here we use that model to predict H2O–CO2 partitioning during magma decompression; however, the use of alternative models would lead to similar results and conclusions. Such magma–hydrothermal interactions are particularly complex and unpredictable at active calderas, where the hydrothermal circulation is particularly intense at the subsurface due to major structural control16,17. This is especially true for Campi Flegrei caldera (CFc), a long-lived resurgent caldera in the metropolitan area of Naples that was formed by the 39-ka Campanian Ignimbrite supereruption, which was the largest in Europe during the past 200 ka (ref. 18). Since the 1950s, CFc has been showing clear signs of potential reawaking, as indicated by frequent episodes of ground uplift (with a total of 43 m of permanent cumulative inﬂation at the caldera centre19), shallow seismicity20, and a visible increase in hydrothermal degassing14. After a period of major unrest in 1983–1984 characterized by thousands of earthquakes and a rapid uplift (B1.8 m over 2 years19), CFc subsided until 2005, when a new inﬂation started, resulting in a minor (B0.4 m over 10 years) but temporally accelerating uplift. The involvement of magma as a causal factor of the current CFc unrest is strongly supported by the composition of volcanic gas21 and deformation changes22. However, it is not clear whether this unrest will culminate in an eruption and, if it does, over what timescale this will occur. Results The presence of more than half a million people living in the proximity of the caldera makes this situation particularly challenging for local authorities and other decision-makers, and highlights the urgency of obtaining a better understanding of interactions between the magma driving the unrest and its overlying hydrothermal system. Model calculations were initialized at conditions relevant to CFc, where the marked variations in the compositions of fumarole emissions37 observed over the last 30 years have been attributed14,21 to decompression-driven open-system degassing of trachybasalt magma. A good match21 between model-calculated solubilities25 and experimentally derived solubilities for CFc or CFc-like magmas has also been demonstrated. Figure 1a shows the total quantities of H2O and CO2 released at equilibrium conditions by 1 kg of magma during open-system depressurization. We assumed isothermal condi- tions (temperature ¼ 1,425 K) and that the CO2–H2O mixture is initially saturated at 200 MPa. This pressure corresponds to a depth of B8 km, where a large magma reservoir below CFc has been detected by seismic tomography38. y g p y A range of total volatile contents was explored, but all of the simulations converged to indicate different degassing behaviours of CO2 and H2O. Figure 1a shows that, during deep (pressure 4150 MPa) degassing stages, less than 0.001 moles per kilogram of magma of CO2-dominated magmatic gas are separated for each 1-MPa decrease in pressure. H2O degassing becomes effective only at lower pressures (Fig. 1a,c), and when this happens there is a narrow pressure interval over which the total amount of separated ﬂuid increases steeply (by more than one order of magnitude). This stage is marked by abrupt variations (Fig. 1a) and it leads to the complete exhaustion of CO2 in the magma, leaving only H2O available for subsequent low-pressure degassing (Fig. 1c). A particularly important feature at this stage is that each model curve shows an inﬂection point (Fig. 1a) at a speciﬁc pressure, which we refer to as the CDP. In each of the model curves of Figs 1 and 2, we set the CDP as the pressure value at which the second derivative of separated gas content with respect to pressure reaches its maximum. This condition marks an abrupt increase in the amount of thermal energy released through ﬂuid expulsion, from o50 J to 41,000 J per kilogram of magma and for each 1-MPa decrease in pressure (Fig. 1b). ARTICLE ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms13712 V V olcanic eruptions1,2 are the surface manifestations of the ﬁnal stages of crustal emplacement of mantle-sourced magmas. Understanding the transition of a volcano from quiescence to eruption is relatively straightforward at the frequently active maﬁc volcanoes, where the rates of magma ascent and the separation of magmatic volatiles drive pressurization of the magmatic systems and ﬁnally eruption3–7. In contrast, interpreting volcanic unrest is difﬁcult at silicic volcanoes, since they commonly develop pervasive hydrothermal systems during their long repose periods8,9. The complex magma–hydrothermal interactions that result as magma ﬁnally makes its way to the surface during the reawaking of a volcano will modulate the physical and chemical signals recorded at the surface10–14, and determine whether the magma will ultimately erupt15. near a bifurcation at which the evolution of the system can either culminate in an eruption or change trend and cool down27. NATURE COMMUNICATIONS \| 7:13712 \| DOI: 10.1038/ncomms13712 \| www.nature.com/naturecommunications Giovanni Chiodini1, Antonio Paonita2, Alessandro Aiuppa2,3, Antonio Costa1, Stefano Caliro4, Prospero De Martino4, Valerio Acocella5 & Jean Vandemeulebrouck6,7 During the reawaking of a volcano, magmas migrating through the shallow crust have to pass through hydrothermal ﬂuids and rocks. The resulting magma–hydrothermal interactions are still poorly understood, which impairs the ability to interpret volcano monitoring signals and perform hazard assessments. Here we use the results of physical and volatile saturation models to demonstrate that magmatic volatiles released by decompressing magmas at a critical degassing pressure (CDP) can drive volcanic unrest towards a critical state. We show that, at the CDP, the abrupt and voluminous release of H2O-rich magmatic gases can heat hydrothermal ﬂuids and rocks, triggering an accelerating deformation that can ultimately culminate in rock failure and eruption. We propose that magma could be approaching the CDP at Campi Flegrei, a volcano in the metropolitan area of Naples, one of the most densely inhabited areas in the world, and where accelerating deformation and heating are currently being observed. 1 Istituto Nazionale di Geoﬁsica e Vulcanologia, sezione di Bologna, via D. Creti 12, 40128 Bologna, Italy. 2 Istituto Nazionale di Geoﬁsica e Vulcanologia, sezione di Palermo, via U. La Malfa 153, 90146 Palermo, Italy. 3 DiSTeM, Universita` di Palermo, via Archiraﬁ36, 90123 Palermo, Italy. 4 Istituto Nazionale di Geoﬁsica e Vulcanologia, sezione di Napoli Osservatorio Vesuviano, via Diocleziano 328, 80124 Napoli, Italy. 5 Dipartimento di Scienze, Universita` Roma Tre, Largo San Leonardo Murialdo, 1, 00146 Roma, Italy. 6 Universite´ de Savoie Mont Blanc, Chambe´ry 73000, France. 7 CNRS, ISTerre, F-73376 Le Bourget du Lac, France. Correspondence and requests for materials should be addressed to G.C. (email: giovanni.chiodini@ingv.it). 1 NATURE COMMUNICATIONS \| 7:13712 \| DOI: 10.1038/ncomms13712 \| www.nature.com/naturecommunications NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms13712 1,000 2,000 0 0 50 100 200 Pressure (MPa) mol kg–1 MPa–1 J kg–1 MPa–1 % of original Separated H2O+CO2 Energy released Original H2O and CO2 (wt%) of magma at 200 MPa 5.39, 0.025 2.86, 0.105 2.02, 0.123 4.89, 0.046 3.89, 0.079 3.38, 0.093 Inflection point Simulation 1 Simulation 1 Simulation 2 Simulation 2 20 40 60 80 Residual gas Separated gas CDP = 160 MPa CDP = 134 MPa CDP = 92 MPa CDP = 75 MPa CDP = 58 MPa 35 MPa H2O CO2 H2O saturation H2O saturation 0.5 1.0 1.5 CO2 / H2O (molar) 0.01 0.02 0.03 a b c d 1,000 2,000 0 0 50 100 200 Pressure (MPa) mol kg–1 MPa–1 J kg–1 MPa–1 % of original Separated H2O+CO2 Energy released Original H2O and CO2 (wt%) of magma at 200 MPa 5.39, 0.025 2.86, 0.105 2.02, 0.123 4.89, 0.046 3.89, 0.079 3.38, 0.093 Inflection point Simulation 1 Simulation 1 Simulation 2 Simulation 2 20 40 60 80 Residual gas Separated gas CDP = 160 MPa CDP = 134 MPa CDP = 92 MPa CDP = 75 MPa CDP = 58 MPa 35 MPa H2O CO2 H2O saturation H2O saturation 0.5 1.0 1.5 CO2 / H2O (molar) 0.01 0.02 0.03 a b c d Figure 1 \| Open-system magma-degassing models for CFc. The different curves refer to different initial H2O and CO2 contents and describe the evolution of the ﬂuids released during magma depressurization in an open-system Rayleigh-type degassing process (where at each inﬁnitesimal decompression step, an inﬁnitesimal parcel of gas phase in excess of the permissible saturation is distilled from the well-mixed magma). Theoretical degassing curves were calculated for the most-primitive magma compositions of CFc (trachybasalt) and show the pressure dependence of (a) the moles of H2O and CO2 released by one kilogram of magma for each 1-MPa decrease in pressure; (b) the energy associated with ﬂuid release (enthalpy of the separated H2O–CO2 mixture at the speciﬁc temperature and pressure, computed using MUFITS63); (c) the residual gas in the melt, as a percentage of the original content; and (d) the CO2/H2O ratio of the released ﬂuids. The blue circle and orange line indicate the conditions used in the corresponding TOUGH2 (ref. 26) simulations. b Figure 1 \| Open-system magma-degassing models for CFc. NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms13712 Our calculations were performed under open-system conditions since the long-lasting variations in the fumarole-gas composition at CFc cannot be reproduced in a closed system, instead requiring efﬁcient separation of gas from the magma21. The large amount of magmatic ﬂuids released by CFc manifestations40 also supports an ongoing open (rather than closed) magma-degassing behaviour. Our open-system Rayleigh-type degassing model assumes that volatiles are continuously separated from magma at each decompression step. However, the release of magmatic ﬂuid from CFc surface manifestations actually shows a pulsed (noncontinuous) behaviour (Supplementary Figs 1 and 2), which suggests a mechanism in which periods of closed-system decompression alternate with episodes of system opening and gas release (that is, a multistep degassing process) over timescales of years. The thermal regime of hydrothermal systems can be strongly impacted if the underlying magma approaches the CDP. Within the typical temperature and pressure hydrothermal range, CO2 behaves as an incondensable species, while H2O can condense and therefore heat the rocks very efﬁciently. Our models of ﬂuid ﬂow in porous media26 that describe the injection of ﬂuids enriched in either CO2 or H2O into a virtual hydrothermal system conﬁrm the different heating capacities of the two volatiles. The ability of ascending magmas to heat any overlying hydrothermal system will therefore be greatly enhanced as the CDP is approached. We use below the CFc example to further illustrate this aspect. Tests show that such multistep degassing can be adequately reproduced as an open-system degassing process provided that there are numerous and recurrent system-opening events, as is likely to be the case. The case of Campi Flegrei caldera. Of the several quiescent calderas worldwide, CFc has recently shown among the clearest signs of unrest. At CFc, several ktons of hydrothermal ﬂuids are emitted daily by the Solfatara-Pisciarelli fumarolic ﬁeld40 (Fig. 3a,b). Stable isotopes of fumarolic steam concur to indicate that such ﬂuids are, at least partially, sourced by magma degassing50. Open-system, unsteady degassing is not only observed at CFc41,42. During extrusive volcanic eruptions, pulsed degassing behaviour can occur even at shorter timescales, and is thought to derive from multiphase ﬂow dynamics within the conduit43. At CFc, we argue that such degassing behaviour can ultimately result from the complex geometry of crustal volcano plumbing systems, whose intricate networks of fractures, dikes, sills and small reservoirs44–47 facilitate the segregation of gas from melt, and the loss of volatiles from a foam layer48. NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms13712 The different curves refer to different initial H2O and CO2 contents and describe the evolution of the ﬂuids released during magma depressurization in an open-system Rayleigh-type degassing process (where at each inﬁnitesimal decompression step, an inﬁnitesimal parcel of gas phase in excess of the permissible saturation is distilled from the well-mixed magma). Theoretical degassing curves were calculated for the most-primitive magma compositions of CFc (trachybasalt) and show the pressure dependence of (a) the moles of H2O and CO2 released by one kilogram of magma for each 1-MPa decrease in pressure; (b) the energy associated with ﬂuid release (enthalpy of the separated H2O–CO2 mixture at the speciﬁc temperature and pressure, computed using MUFITS63); (c) the residual gas in the melt, as a percentage of the original content; and (d) the CO2/H2O ratio of the released ﬂuids. The blue circle and orange line indicate the conditions used in the corresponding TOUGH2 (ref. 26) simulations. further subterraneous gas–magma separation, heating, and (eventually) sustaining an eruption39. l l f d d mass of released volatiles varying less markedly than in open- system conditions. We conclude that the concept of the CDP applies over a wide range of magmatic conditions. We also ﬁnd that CDP conditions are reached independently on the solubility model used, for example, VolatileCalc36 (Supplementary Fig. 3). The thermal regime of hydrothermal systems can be strongly impacted if the underlying magma approaches the CDP. Within the typical temperature and pressure hydrothermal range, CO2 behaves as an incondensable species, while H2O can condense and therefore heat the rocks very efﬁciently. Our models of ﬂuid ﬂow in porous media26 that describe the injection of ﬂuids enriched in either CO2 or H2O into a virtual hydrothermal system conﬁrm the different heating capacities of the two volatiles. The ability of ascending magmas to heat any overlying hydrothermal system will therefore be greatly enhanced as the CDP is approached. We use below the CFc example to further illustrate this aspect. mass of released volatiles varying less markedly than in open- system conditions. We conclude that the concept of the CDP applies over a wide range of magmatic conditions. We also ﬁnd that CDP conditions are reached independently on the solubility model used, for example, VolatileCalc36 (Supplementary Fig. 3). Results While it is universally accepted that the injection of new magma is a common mechanism that drives hydrothermal systems towards the critical state23,24, the mechanisms and timescales of magma–hydrothermal interactions during unrest remain poorly understood and difﬁcult to forecast16. One key aspect that has received little attention is the role that magmatic gases may play in heating the hydrothermal system, and ultimately in driving the unrest. The present study linked magma degassing at depth with the resulting perturbation in the overlying hydrothermal system. Here we initially use the results of volatile saturation25 models to demonstrate that decompressing magmas can reach a critical condition, which we refer as a critical degassing pressure (CDP), at which their ability to release water and convectively transport heat is increased by a least an order of magnitude. We then use physical models26 to show that magmatic volatiles released at the CDP, when injected into an overlying hydrothermal system, lead to extensive heating and expansion, and cause temporally accelerating ground deformation. Finally, we examine ground deformation time series from CFc and some other restless calderas, which identiﬁes consistent accelerating ground uplift trends that are reminiscent of those predicted by our model. We conclude that magmas at the CDP can be recurrent causal factors in driving volcanic unrest towards a critical state; that is, a state We found similar nonlinear degassing behaviour over a range of magma compositions and volatile contents. For any magma type (for example, rhyolitic magma; Fig. 2a), complete CO2 exhaustion in the melt marks a critical condition at which the amount of separated ﬂuids and the energy transfer to the hosting rocks both increase dramatically. It is important to stress that most of the H2O (B95% of the original content) is still dissolved in the magma at the CDP (Fig. 1c) and is therefore available for NATURE COMMUNICATIONS \| 7:13712 \| DOI: 10.1038/ncomms13712 \| www.nature.com/naturecommunications 2 ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms13712 NATURE COMMUNICATIONS \| 7:13712 \| DOI: 10.1038/ncomms13712 \| www.nature.com/naturecommunications NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms13712 Foam growth in low-viscosity maﬁc melts takes place over timescales of months to a few years48,49, which is faster than the observed decennial trends in gas composition. While we therefore favour an open- system scenario, we also show examples of model degassing simulations in closed-system conditions (Fig. 2b) to demonstrate that a CDP can be reached even in that type of system, despite the The large variations in the fumarole emissions of N2–He–CO2–Ar (ref. 21), including the 25-year-long decreasing trend of the N2/He fumarole ratio (Fig. 3c), fully support the idea that a primitive magma degassing in open-system conditions at increasingly lower pressures is sustaining the unrest. A particu- larly important observation is that the ground deﬂation and N2/He gas ratios followed exponential-like trends from 1985 to 2005, with very similar characteristic times, implying common source processes14. The presence of magma depressurization is also supported by modelling of the ground uplift in 2012–2013, which has been interpreted as the effect of a magma intrusion at a 3 NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms13712 ARTICLE Pressure (MPa) 1.45, 0.0397 2.43, 0.0288 3.45, 0.0134 3.78, 0.0071 mol kg–1 MPa–1 mol kg–1 MPa–1 0 25 50 100 0 0.01 0.02 0.03 0 0.01 0.02 0.03 Rhyolitic magma CDP 0 50 100 200 Pressure (MPa) 5.39, 0.025 2.86, 0.105 2.02, 0.123 4.89, 0.046 3.89, 0.079 Open Closed Trachybasaltic magmas CDP Original H2O and CO2 (wt%) of magma at 100 MPa a b Figure 2 \| Results of magma degassing models. The different curves refer to different initial H2O and CO2 contents, reported as couple of values on each and describe the evolution of the ﬂuids released during magma depressurization in various conditions. (a) Theoretical degassing curves were calculated for rhyolitic magma compositions and show the pressure dependence of the moles of H2O and CO2 released during open-system degassing by one kilogram of magma for each 1-MPa pressure decrease. (b) Theoretical degassing curves were calculated for the most-primitive magma compositions of CFc and refer to open and closed degassing (trachybasalt). NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms13712 mol kg–1 MPa–1 0 0.01 0.02 0.03 0 50 100 200 Pressure (MPa) 5.39, 0.025 2.86, 0.105 2.02, 0.123 4.89, 0.046 3.89, 0.079 Open Closed Trachybasaltic magmas CDP b Pressure (MPa) 1.45, 0.0397 2.43, 0.0288 3.45, 0.0134 3.78, 0.0071 mol kg–1 MPa–1 0 25 50 100 0 0.01 0.02 0.03 Rhyolitic magma CDP Original H2O and CO2 (wt%) of magma at 100 MPa a depth of 3 km (ref. 22). At the same time, a generalized heating up of the CFc hydrothermal system is indicated by the 15-year-long exponential increase in CO emissions from the fumaroles (Fig. 3d); note that CO is the fumarole gas most sensitive to temperature changes51. b a Based on these observations, we argue that the CFc magmatic system may be approaching the CDP; that is, that depressurizing magma may release ﬂuids progressively richer in H2O so as to affect the thermal structure of the hydrothermal system. We tested this hypothesis by using TOUGH2 (ref. 26; see Methods) to model the injection of magmatic ﬂuids (IMF) into a hydrothermal system under physical conditions appropriate for CFc13 (Fig. 4). 13 g Our new model simulations reﬁne previous ones13 that ﬁrst identiﬁed the magmatic gas trigger of the unrest. The model involves injecting H2O–CO2 magmatic gas mixtures into a virtual hydrothermal system at subcritical temperature and pressure conditions. The composition (CO2/H2O ratio) of the injected magmatic gas phase is based on the results of our magma-degassing models (see Methods and Fig. 1d). We highlight that these modelled magmatic CO2/H2O ratios can only approximate the composition of ﬂuids entering the real hydrothermal system, since the model does not account for secondary processes potentially occurring along the magma-to-hydrothermal gas cooling path. Figure 2 \| Results of magma degassing models. The different curves refer to different initial H2O and CO2 contents, reported as couple of values on each and describe the evolution of the ﬂuids released during magma depressurization in various conditions. (a) Theoretical degassing curves were calculated for rhyolitic magma compositions and show the pressure dependence of the moles of H2O and CO2 released during open-system degassing by one kilogram of magma for each 1-MPa pressure decrease. (b) Theoretical degassing curves were calculated for the most-primitive magma compositions of CFc and refer to open and closed degassing (trachybasalt). NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms13712 g y g g p We simulated 14 IMF events occurring between 1983 and 2014, whose timing and intensity were constrained based on Depressurization of the 'magmatic' gas reservoir (increase in H2O/CO2) Modelled temperature (simulation 2) Solfatara Meteoric liquid Condensed steam 1 km BRITTLE DUCTILE Magmatic gas Hydrothermal reservoir Solfatara gas plume SW NE CO2 ﬂux ~ 2000 t d–1 1 2 3 Depth (km) Pulsed episodes of magmatic gas emission 200 400 600 800 1982 1986 1990 1994 1998 2002 2006 2010 2014 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 Napoli POZZUOLI Solfatara RITE Pisciarelli Main fumaroles 0 2 km b a CGPS Study area 230 °C 250 °C 270 °C 290 °C 310 °C Temperature increase of the 'hydrothermal reservoir' N2/He CO (p.p.m.) Gas ﬂow Italy c d Figure 3 \| The hydrothermal system of CFc and its signals. (a) Locations of CFc and the main hydrothermal sites: Solfatara and Pisciarelli. (b) Conceptual model of the hydrothermal system feeding the two manifestations: a 4-km-deep zone of magmatic gas accumulation that supplies ﬂuids t a shallower part where they vaporize liquid of meteoric origin to form a 2-km-deep vertical plume of gas14. Previous geochemical investigations based o the stable isotopes of water revealed the presence of typical magmatic waters in the Solfatara fumarole vents50. (c) Temporal evolution of the N2/He rat at the Solfatara fumaroles. (d) Time series of the CO content in the Solfatara fumaroles. The increasing trend indicates heating of the system, and matche the TOUGH2 (ref. 26) model-derived temperatures (magenta line) exceptionally well. Depressurization of the 'magmatic' gas reservoir (increase in H2O/CO2) Modelled temperature (simulation 2) 200 400 600 800 1982 1986 1990 1994 1998 2002 2006 2010 2014 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 230 °C 250 °C 270 °C 290 °C 310 °C Temperature increase of the 'hydrothermal reservoir' N2/He CO (p.p.m.) c d c a Napoli POZZUOLI Solfatara RITE Pisciarelli Main fumaroles 0 2 km a CGPS Study area Italy Solfatara Meteoric liquid Condensed steam 1 km BRITTLE DUCTILE Magmatic gas Hydrothermal reservoir Solfatara gas plume SW NE CO2 ﬂux ~ 2000 t d–1 1 2 3 Depth (km) Pulsed episodes of magmatic gas emission b Gas ﬂow b ystem of CFc and its signals. (a) Locations of CFc and the main hydrothermal sites: Solfatara and Pisciarelli. Figure 3 \| The hydrothermal system of CFc and its signals. NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms13712 Ground deformation pulses and clustered earthquakes support the timing of the IMF events, which were independently ﬁxed based on geochemical anomalies (Supplementary Fig. 2). Previous simulations13 considered injections of hot ﬂuids with a constant CO2/H2O ratio. Here we update these previous calculations to the current CFc state (Simulation 1), but also consider a new scenario (Simulation 2) in which magmatic ﬂuids that are increasingly rich in H2O are injected. The ﬁrst scenario corresponds to degassing of a stationary source at 200 MPa and with original H2O and CO2 contents of 3.89 wt% and 0.079 wt%, respectively (blue circle in Fig. 1a,d). The second scenario describes the depressurization of the same source down to 130 MPa (orange line in Fig. 1a,d). The CO2/H2O ratio used in each IMF event was inferred from the measured N2/He ratios and the results obtained in simulations of open-system magma-degassing models (Supplementary Figs 4 and 5). with obvious implications for volume expansion of the rocks (Fig. 5c). measured geochemical anomalies only (see Methods and Supplementary Fig. 1a,b). Ground deformation pulses and clustered earthquakes support the timing of the IMF events, which were independently ﬁxed based on geochemical anomalies (Supplementary Fig. 2). Previous simulations13 considered injections of hot ﬂuids with a constant CO2/H2O ratio. Here we update these previous calculations to the current CFc state (Simulation 1), but also consider a new scenario (Simulation 2) in which magmatic ﬂuids that are increasingly rich in H2O are injected. The ﬁrst scenario corresponds to degassing of a stationary source at 200 MPa and with original H2O and CO2 contents of 3.89 wt% and 0.079 wt%, respectively (blue circle in Fig. 1a,d). The second scenario describes the depressurization of the same source down to 130 MPa (orange line in Fig. 1a,d). The CO2/H2O ratio used in each IMF event was inferred from the measured N2/He ratios and the results obtained in simulations of open-system magma-degassing models (Supplementary Figs 4 and 5). f d h h d ll d d l h Independent observations support these model predictions of the thermal evolution of the CFc hydrothermal system. The modelled pattern of temperature increase (Fig. 5a) mimics the observed increasing trends in CO content of the fumaroles (Fig. 3d) and temperature estimations obtained from the CO–CO2 geothermometer14 (Fig. 5b). NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms13712 NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms13712 Impermeable and adiabatic Fluid source 300 °C 300 °C 250 °C 200 °C 150 °C 50 °C 100 °C 250 °C 200 °C 150°C 50 °C 100 °C 0 0.2 0.4 0.6 0.8 1.0 Xg 2,000 m 5,000 m Temperature box Checkpoint for gas composition Fixed atmospheric temperature, pressure, and PCO2 Impermeable and adiabatic Fluid source 300 °C 300 °C 250 °C 200 °C 150 °C 50 °C 100 °C 250 °C 200 °C 150°C 50 °C 100 °C 0 0.2 0.4 0.6 0.8 1.0 Xg 2,000 m 5,000 m Temperature box Checkpoint for gas composition Fixed atmospheric temperature, pressure, and PCO2 Figure 4 \| Computational domain of the TOUGH2 simulations. The rock physical properties were homogeneous (porosity ¼ 0.2; thermal capacity ¼ 1,000 J kg 1 C 1; density ¼ 2,000 kg m 3; horizontal permeability ¼ 10 14 m2; vertical permeability ¼ 1.5 10 14 m2; and thermal conductivity ¼ 2.8 Wm 1 C 1). The temperature and the volumetric gas fraction Xg (different shades of gray) refer to steady-state conditions. The ‘checkpoint for gas composition’ is the zone where the simulated CO2/H2O is compared with the measured one (see Methods). The ‘Temperature box’ (yellow rectangle above the injection zone) is the region where the average temperature is calculated during the simulations (see Figs 5 and 6 and Supplementary Fig. 1c). Fixed atmospheric temperature, pressure, and PCO2 Fixed atmospheric temperature, pressure, and PCO2 Impermeable and adiabatic Figure 4 \| Computational domain of the TOUGH2 simulations. The rock physical properties were homogeneous (porosity ¼ 0.2; thermal capacity ¼ 1,000 J kg 1 C 1; density ¼ 2,000 kg m 3; horizontal permeability ¼ 10 14 m2; vertical permeability ¼ 1.5 10 14 m2; and thermal conductivity ¼ 2.8 Wm 1 C 1). The temperature and the volumetric gas fraction Xg (different shades of gray) refer to steady-state conditions. The ‘checkpoint for gas composition’ is the zone where the simulated CO2/H2O is compared with the measured one (see Methods). The ‘Temperature box’ (yellow rectangle above the injection zone) is the region where the average temperature is calculated during the simulations (see Figs 5 and 6 and Supplementary Fig. 1c). measured geochemical anomalies only (see Methods and Supplementary Fig. 1a,b). NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms13712 (a) Locations of CFc and the main hydrothermal sites: S Figure 3 \| The hydrothermal system of CFc and its signals. (a) Locations of CFc and the main hydrothermal sites: Solfatara and Pisciarelli. (b) Conceptual model of the hydrothermal system feeding the two manifestations: a 4-km-deep zone of magmatic gas accumulation that supplies ﬂuids to a shallower part where they vaporize liquid of meteoric origin to form a 2-km-deep vertical plume of gas14. Previous geochemical investigations based on the stable isotopes of water revealed the presence of typical magmatic waters in the Solfatara fumarole vents50. (c) Temporal evolution of the N2/He ratio at the Solfatara fumaroles. (d) Time series of the CO content in the Solfatara fumaroles. The increasing trend indicates heating of the system, and matches the TOUGH2 (ref. 26) model-derived temperatures (magenta line) exceptionally well. gu e 3 \| e yd ot e a syste o C c a d ts s g a s (a) ocat o s o C c a d t e a yd ot e a s tes So ata a a d sc a e (b) Conceptual model of the hydrothermal system feeding the two manifestations: a 4-km-deep zone of magmatic gas accumulation that supplies ﬂuids to a shallower part where they vaporize liquid of meteoric origin to form a 2-km-deep vertical plume of gas14. Previous geochemical investigations based on the stable isotopes of water revealed the presence of typical magmatic waters in the Solfatara fumarole vents50. (c) Temporal evolution of the N2/He ratio at the Solfatara fumaroles. (d) Time series of the CO content in the Solfatara fumaroles. The increasing trend indicates heating of the system, and matches the TOUGH2 (ref. 26) model-derived temperatures (magenta line) exceptionally well. NATURE COMMUNICATIONS \| 7:13712 \| DOI: 10.1038/ncomms13712 \| www.nature.com/naturecommunications 4 ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms13712 Concurrently with the heating onset predicted by the models, the Pisciarelli fumaroles located on the eastern slope of Solfatara visibly increased their ﬂow rates and temperatures in 2005–2006, with recurrent episodes of mud emissions, the formation of new vents, and localized seismic activity14 (Fig. 6). Furthermore, comparison of the locations of earthquakes in 1983–1984 and 2005–2014 highlights the recent disappearance of shallow seismicity in the Solfatara area, suggesting a transition from an elastic-like to a plastic-like behaviour, which is probably due to heating of the rocks52. ) We found that each modelled IMF episode involves the injection of 0.1–25 Mt of magmatic ﬂuids, which is within the range of the gas mass associated with small to moderate-size volcanic eruptions13. The modelled cumulative trends of injected magmatic ﬂuid masses exhibit clear exponential acceleration since the 2000s (Supplementary Fig. 1d). The acceleration trend is steeper in Simulation 2 (in which the gas compositions varied during the simulation) than in Simulation 1. Simulation 2 also predicts an average temperature increase of 60 C in the deep- central part of the hydrothermal system (Figs 3d,5 and 6). One interesting outcome of Simulation 2 is that, while the CO2/H2O ratio of the injected magmatic ﬂuids decreases over time, the simulated gas composition at the ‘checkpoint for gas composition’ (Fig. 4) becomes increasingly rich in CO2 (Supplementary Fig. 1b). This apparent paradox results from H2O condensation in the hydrothermal system, which is the same process heating the rocks. Condensation of a mixed magmatic-meteoric vapor, followed by H2O–CO2 oxygen isotope exchange in the fumaroles’ feeding conduits50, also well account for the observed hydrogen and oxygen isotope composition of fumarolic steam (Supplementary Fig. 6). NATURE COMMUNICATIONS \| 7:13712 \| DOI: 10.1038/ncomms13712 \| www.nature.com/naturecommunications Discussion d We demonstrated, for the ﬁrst time, a discontinuity in the degassing pathway of decompressing magmas. Attainment of this critical condition is purely controlled by the differential solubility of magmatic volatiles. Magmas approaching the CDP abruptly increase their H2O release efﬁciency, and as such enhance their ability to convey heat to the overlying hydrothermal system. Steam-heated host rocks surrounding magma may, in turn, alter their physical properties, lowering their mechanical resistance. Steam injection, which is a technique used in the oil industry for extracting heavy oil53, demonstrates that heating can affect ground deformation to a much larger extent than that expected from pure thermal expansion. Steam injection and the associated latent heat release deform the media also via thermally induced shear dilation54 and enhancing ﬂuid-ﬂow permeability. We conclude, therefore, that the CDP concept may contribute to explain acceleration in signals observed during volcanoes unrests. We propose the CDP can help interpreting the current evolution of the CFc unrest. Escalation in hydrothermal activity, and increasing concentrations of fumarolic species more sensitive to temperature, point to an ongoing heating process of CFc. Our thermo-ﬂuid-dynamic models here suggest that injection of magma-derived H2O, becoming more voluminous and frequent Our model predicts that the injection of increasingly H2O-rich volatiles released by magma approaching the CDP leads to signiﬁcant heating of the hydrothermal system (Fig. 5a) 5 ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms13712 240 260 280 300 2000 2004 2008 2012 2016 240 230 220 Episodes of IMF 30 20 10 0 CO–CO2 temperature Modelled temperature °C °C Vertical displacement and modelled volume increase (thermal expansion) cm 0 m3 2×106 1×106 a b c Figure 5 \| Observations and modelled data for the current period of unrest of CFc. (a) Average temperature obtained by the model (in Simulation 2) for the central deeper zone of the computational domain. (b) Temperatures computed using the CO-CO2 geothermometer at Solfatara fumaroles14 compared with modelled temperatures (gray line). The modelled temperatures, which refer to the central deeper zone of the computational domain (‘Temperature box’ in Fig. 4), have the same temporal evolution but are systematically higher than the CO–CO2 temperatures, which reﬂect the thermal state of the upper part of the hydrothermal system14. (c) Vertical displacements measured at the RITE CGPS station (black line) and modelled volume increases in the computational domain due to thermal expansion (magenta line). Methods D t t Data on the numbers and magnitudes of earthquakes were obtained from the literature14,20 and from INGV Osservatorio Vesuviano. The vertical displacements at Rabaul, Sierra Negra, and Yellowstone were also taken from the literature16,57,61. Data set. Data on the compositions of fumarole emissions as well as the analytical methods used in this study and their uncertainties are available in the literature14. The used data set was updated to December 2015 (Supplementary Data 1). Fifteen continuous-monitoring GPS stations (CGPS) operate at CFc. A full description of the GPS network and the complete data set of the displacement time series is available elsewhere62. Here we used the vertical displacement time series of the RITE CGPS station, which is located in the Pozzuoli area (Fig. 3a) where the uplift has been greatest over the past 15 years. Data on the numbers and magnitudes of earthquakes were obtained from the literature14,20 and from INGV Osservatorio Vesuviano. The vertical displacements at Rabaul, Sierra Negra, and Yellowstone were also taken from the literature16,57,61. Physical modelling of magmatic gas injection into the hydrothermal system. Our physical model aims at assessing the thermal effects caused by the IMF into the CFc hydrothermal system. Among the numerous possible scenarios, we select two reference cases. in time, may well be controlling such heating. We caution that, since our model does not account for rheological properties and heterogeneities of the system, it cannot be used to predict mechanical evolution, for example, caldera deformation. We yet note a similarity between the temporal evolution of caldera uplift and our modelled hydrothermal temperature increase (Fig. 5c). We conclude, therefore, that magmas approaching the CDP may be contributing to accelerating caldera inﬂation, observed since 2005. At CFc, thermal effects can strongly be enhanced by speciﬁc physical characteristics of the rock matrix (Neapolitan Yellow Tuff), which is particularly sensitive to thermal alterations due to the presence of thermally unstable zeolites55 (for example, the tensile strength of these rocks can halve when the temperature increases from 100 to 300 C (ref. 56)). In the ﬁrst case (Simulation 1) we considered an input magmatic ﬂuid of ﬁxed (that is, time invariant) composition, with a CO2/H2O molar ratio of 0.67. This composition corresponds to that of a gas in equilibrium at 200 MPa with a trachybasalt magma (H2O ¼ 3.39 wt%, CO2 ¼ 0.079 wt%; blue circle in Fig. 1). Discussion d We used a coefﬁcient of volumetric thermal expansion of 3 10 5 C 1. 240 260 280 300 240 230 220 Episodes of IMF CO–CO2 temperature Modelled temperature °C °C a b eruptions at Rabaul, Papua New Guinea16 and Sierra Negra, Galapagos57. Like at CFc, the accelerating deformations at these systems can be empirically described by both power-law and exponential growth curves (Fig. 7). Power-law acceleration of the strain rate, earthquakes and other precursors have been widely reported before material-failure phenomena, including volcanic eruptions58–60. The characteristic failure time, tf, for the power- law relationship is B3,900 days for Rabaul and B1,240 days for Sierra Negra (where the volcanoes erupted 3,100 and 950 days after the beginning of the anomaly, respectively). For CFc, similar calculations yield a failure time of 5,670±735 days from the beginning of the anomaly (that is, 2005). Characteristic times, t, of the exponential curve of 240, 850 and 1,600 days are obtained for Sierra Negra, Rabaul and CFc, respectively. The failure time cannot be deﬁned in the exponential model59, but the Rabaul and Sierra Negra examples show that eruptions occurred at 3–4 t (Fig. 7). Episodes of IMF ( g ) However, the temporal evolution of volcanic unrests often exhibits complex, nonlinear behaviour, such as in the case of the Yellowstone caldera, where an initial exponential/power-law acceleration of ground uplift during 2004–2008 was followed by a deceleration61 (Fig. 7). A pausing of ground uplift occurred at CFc between 1982–1984 and 2005. Caution is therefore prudent when forecasting the future mid- to short-term evolution of any period of unrest. Even if the magma underneath CFc is likely to be approaching the CDP, the possible future scenarios can be complicated by additional processes that have not been considered here. For example, increases in the melt liquidus due to H2O release and consequent magma crystallization could increase the melt viscosity, and therefore act against further magma migration. Additional careful scrutiny of monitoring data in the coming months and years is key to interpreting whether hydrothermal heating or magma quenching will prevail. 2000 2004 2008 2012 2016 30 20 10 0 Vertical displacement and modelled volume increase (thermal expansion) cm 0 m3 2×106 1×106 c 2004 NATURE COMMUNICATIONS \| 7:13712 \| DOI: 10.1038/ncomms13712 \| www.nature.com/naturecommunications Methods D t t Figure 5 \| Observations and modelled data for the current period of Figure 5 \| Observations and modelled data for the current period of unrest of CFc. (a) Average temperature obtained by the model (in Simulation 2) for the central deeper zone of the computational domain. (b) Temperatures computed using the CO-CO2 geothermometer at Solfatara fumaroles14 compared with modelled temperatures (gray line). The modelled temperatures, which refer to the central deeper zone of the computational domain (‘Temperature box’ in Fig. 4), have the same temporal evolution but are systematically higher than the CO–CO2 temperatures, which reﬂect the thermal state of the upper part of the hydrothermal system14. (c) Vertical displacements measured at the RITE CGPS station (black line) and modelled volume increases in the computational domain due to thermal expansion (magenta line). We used a coefﬁcient of volumetric thermal expansion of 3 10 5 C 1. Figure 5 \| Observations and modelled data for the current period of unrest of CFc. (a) Average temperature obtained by the model (in Simulation 2) for the central deeper zone of the computational domain. (b) Temperatures computed using the CO-CO2 geothermometer at Solfatara fumaroles14 compared with modelled temperatures (gray line). The modelled temperatures, which refer to the central deeper zone of the computational domain (‘Temperature box’ in Fig. 4), have the same temporal evolution but are systematically higher than the CO–CO2 temperatures, which reﬂect the thermal state of the upper part of the hydrothermal system14. (c) Vertical displacements measured at the RITE CGPS station (black line) and modelled volume increases in the computational domain due to thermal expansion (magenta line). We used a coefﬁcient of volumetric thermal expansion of 3 10 5 C 1. Data set. Data on the compositions of fumarole emissions as well as the analytical methods used in this study and their uncertainties are available in the literature14. The used data set was updated to December 2015 (Supplementary Data 1). Fifteen continuous-monitoring GPS stations (CGPS) operate at CFc. A full description of the GPS network and the complete data set of the displacement time series is available elsewhere62. Here we used the vertical displacement time series of the RITE CGPS station, which is located in the Pozzuoli area (Fig. 3a) where the uplift has been greatest over the past 15 years. NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms13712 NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms13712 Fumarole temperature (°C) Modelled temperature (°C) Boiling temperature of pisciarelli fumarole 90 95 100 105 110 115 120 1999 2003 2007 2011 2015 240 260 280 300 Mud emission Opening of boiling pool Opening of new vent Localized seismic crisis Fumarole temperature (°C) Modelled temperature (°C) Boiling temperature of pisciarelli fumarole 90 95 100 105 110 115 120 1999 2003 2007 2011 2015 240 260 280 300 Mud emission Opening of boiling pool Opening of new vent Localized seismic crisis Figure 6 \| Discharge temperature at the Pisciarelli fumarole compared with the modelled temperature. The temperature at the Pisciarelli fumarole (gray dots) increased from the boiling temperature (95 C) in 2005–2006 to 115–120 C in 2015. During the same time interval, temperature increased of only 3–4 C at the highest temperature fumarole BG, implying clustering of hydrothermal inﬂux on the eastern outer slope of Solfatara crater, where Pisciarelli is sited (Fig. 3). At Pisciarelli, localized low-magnitude seismic swarms20, a weak phreatic activity (mud emission, opening of boiling pools and new vents), and a strong increase in the fumarole ﬂow rate accompanied the temperature increase. The variation occurs concurrently with the increasing temperature of the CFc hydrothermal system modelled in Simulation 2 (magenta line; that is, average temperature inside the yellow box in Fig. 4). ture at the Pisciarelli fumarole compared with the modelle Figure 6 \| Discharge temperature at the Pisciarelli fumarole compared with the modelled temperature. The temperature at the Pisciarelli fumarole (gray dots) increased from the boiling temperature (95 C) in 2005–2006 to 115–120 C in 2015. During the same time interval, temperature increased of only 3–4 C at the highest temperature fumarole BG, implying clustering of hydrothermal inﬂux on the eastern outer slope of Solfatara crater, where Pisciarelli is sited (Fig. 3). At Pisciarelli, localized low-magnitude seismic swarms20, a weak phreatic activity (mud emission, opening of boiling pools and new vents), and a strong increase in the fumarole ﬂow rate accompanied the temperature increase. The variation occurs concurrently with the increasing temperature of the CFc hydrothermal system modelled in Simulation 2 (magenta line; that is, average temperature inside the yellow box in Fig. 4). NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms13712 The degassing decompression trend (that is, from 200 to 130 MPa) of this model is schematically illustrated by the orange lines in Fig. 1a,d. The energy transported by volatiles while they are degassed from magma multiphase (gas/liquid) ﬂuid and heat through a porous medium. The TOUGH2 version used here takes into account the contemporaneous presence of two ﬂuid components (H2O and CO2) at subcritical temperature conditions26. More-recent geothermal simulators65 can also work at supercritical temperature conditions, but we used TOUGH2 to allow comparisons with previous work at CFc13,66,67. The model includes heat ﬂow occurring by both conduction and convection, with the latter including both sensible and latent heat effects26. The model does not account for either the presence of other gas components, solute transport, and chemical reactions, or for deformation or fracturing of the porous rock. model relationship in Supplementary Fig. 4a. The obtained time dependence of the CO2/H2O ratio (see red line in Supplementary Fig. 5b) was sampled at speciﬁc time intervals to infer the magmatic gas CO2/H2O ratio composition of each IMF event (Supplementary Table 1). The degassing decompression trend (that is, from 200 to 130 MPa) of this model is schematically illustrated by the orange lines in Fig. 1a,d. The energy transported by volatiles while they are degassed from magma (Fig. 1b) was calculated from the enthalpy of the separated H2O–CO2 mixture at the magma temperature and pressure. The method63,64 we used can simulate properties of a binary CO2–H2O mixture up to supercritical conditions. However, we injected magmatic ﬂuids into the virtual hydrothermal system at 350 C in both Simulations 1 and 2. This is because the geothermal simulator that we used (TOUGH2 (ref. 26)) can only work below the critical-point temperature of water. Enthalpies of the magmatic gas phase at 350 C were calculated using the EOS2 module26. This yielded that the enthalpies of the injected magmatic gases were far lower than those of magma-released supercritical ﬂuids (Fig. 1b). This difference accounts for the energy lost by ﬂuids as they pass thorough the plastic zone, between the magma and the overlying hydrothermal system. TOUGH2 i i l i l t f th l t th t i f ll The energy transported by volatiles while they are degassed from magma (Fig. 1b) was calculated from the enthalpy of the separated H2O–CO2 mixture at the magma temperature and pressure. NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms13712 6 \| Discharge temperature at the Pisciarelli fumarole com 0 10 20 30 40 50 0 2 0 2 4 6 8 0 Rabaul (September 1994) Rabaul and Sierra Negra eruptions Sierra Negra (October 2005) Exponential growth Power-law growth 0 1 2 3 4 5 6 0 0.2 0.4 0.6 0.8 n =1.7 n = 1.3 n =1 Sierra Negra 241 Rabaul 855 CFc 1,588 33.4 Yellowstone 374 t(days) z(cm) Sierra Negra 1,237 Rabaul 3,893 CFc 5,672 Yellowstone 1,300 tf (days) ζe ζ 36.0 11.9 16.2 1 2 3 4 e 4 3 1 ζp p 1 a b Figure 7 \| Examples of several-years-long accelerating deformation patterns at selected key volcanoes. The curves indicate the vertical ground displacement observed at the different volcanoes (Sierra Negra magenta, Rabaul blue, CFc cyan, Yellowstone gray; see Methods). The patterns can be ﬁtted with either exponential (a) or power-law (b) growth curves. (a) The dimensionless time (te) and vertical displacement (ze) are given by te ¼ t/t* and ze ¼ z/z, respectively, where t and z* are the characteristic time and vertical displacement, respectively, of the exponential curve: z(t) ¼ z* [exp(t/t) 1]. (b) The dimensionless time (tp) and vertical displacement (zp) are given by tp ¼ t/tf and zp ¼ z= C=tn f , respectively, where tf C, and n the adjustable parameters of the power-law curve: z tð Þ ¼ C= tf t ð Þn C=tn f . The initial time for each case refers to the ﬁrst evidence of uplift. Different curve parameters are reported in the ﬁgure legends; the values of C (104 cm daysn) are 43.0, 33.8, 12.0, 67.2 for Sierra Negra, Rabaul, CFc, and Yellowstone, respectively. Methods D t t This CO2-rich composition (CO2 molar fraction ¼ 0.4) was inferred based on volcanic gas evidence50 and has been used in previous CFc simulations13. In the second scenario (Simulation 2) we injected the virtual hydrothermal system with a magmatic gas that has a composition that becomes richer in H2O over time (Supplementary Table 1). This evolving gas composition is based on the output of open-system magma-degassing models of CFc magmas. A comparison of measured fumarole emissions and the model degassing trends21 provides support for open-system magma degassing being an ongoing process at CFc. The results of model simulations21 (Supplementary Fig. 4a) demonstrate that gas observations (in the N2–He–CO2 gas system) can only be quantitatively reproduced if open-system decompression is assumed to be present. We consider the case of a trachybasalt magma decompressing from 200 to 130 MPa, initially coexisting with a gas phase having N2/He ¼ 900 and N2/CO2 ¼ 0.0047 (Supplementary Fig. 4a). We reconstruct the temporal evolution of the CO2/H2O ratio of the magmatic gas feeding the CFc hydrothermal system (Supplementary Fig. 5b) from that of the N2/He ratio measured in CFc fumaroles (Supplementary Fig. 5a). Fumarole N2/He ratios are converted into CO2/H2O ratios in the source magmatic gas using the CFc is unlikely to be an isolated case. A similar, several-year- long period of accelerating inﬂation possibly driven by magma depressurization and heating processes was observed before NATURE COMMUNICATIONS \| 7:13712 \| DOI: 10.1038/ncomms13712 \| www.nature.com/naturecommunications 6 ARTICLE NATURE COMMUNICATIONS \| 7:13712 \| DOI: 10.1038/ncomms13712 \| www.nature.com/naturecommunications NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms13712 0 10 20 30 40 50 0 2 0 2 4 6 8 0 Rabaul (September 1994) Sierra Negra (October 2005) Exponential growth Sierra Negra 241 Rabaul 855 CFc 1,588 33.4 Yellowstone 374 t(days) z(cm) ζe ζ 36.0 11.9 16.2 1 2 3 4 e 4 3 1 a Rabaul and Sierra Negra eruptions Power-law growth 0 1 2 3 4 5 6 0 0.2 0.4 0.6 0.8 n =1.7 n = 1.3 n =1 Sierra Negra 1,237 Rabaul 3,893 CFc 5,672 Yellowstone 1,300 tf (days) ζp p 1 b Rabaul and Sierra Negra eruptions Power-law growth 0 1 2 3 4 5 6 0 0.2 0.4 0.6 0.8 n =1.7 n = 1.3 n =1 Sierra Negra 1,237 Rabaul 3,893 CFc 5,672 Yellowstone 1,300 tf (days) ζp p 1 b b a a Figure 7 \| Examples of several-years-long accelerating deformation patterns at selected key volcanoes. The curve Figure 7 \| Examples of several-years-long accelerating deformation patterns at selected key volcanoes. The curves indicate the vertical ground displacement observed at the different volcanoes (Sierra Negra magenta, Rabaul blue, CFc cyan, Yellowstone gray; see Methods). The patterns can be ﬁtted with either exponential (a) or power-law (b) growth curves. (a) The dimensionless time (te) and vertical displacement (ze) are given by te ¼ t/t and ze ¼ z/z, respectively, where t and z* are the characteristic time and vertical displacement, respectively, of the exponential curve: z(t) ¼ z* [exp(t/t*) 1]. (b) The dimensionless time (tp) and vertical displacement (zp) are given by tp ¼ t/tf and zp ¼ z= C=tn f , respectively, where tf C, and n the adjustable parameters of the power-law curve: z tð Þ ¼ C= tf t ð Þn C=tn f . The initial time for each case refers to the ﬁrst evidence of uplift. Different curve parameters are reported in the ﬁgure legends; the values of C (104 cm daysn) are 43.0, 33.8, 12.0, 67.2 for Sierra Negra, Rabaul, CFc, and Yellowstone, respectively. model relationship in Supplementary Fig. 4a. The obtained time dependence of the CO2/H2O ratio (see red line in Supplementary Fig. 5b) was sampled at speciﬁc time intervals to infer the magmatic gas CO2/H2O ratio composition of each IMF event (Supplementary Table 1). ARTICLE The ﬁrst three events occurred during the major bradyseismic crisis in 1983–1984, and two minor uplift episodes occurred in 1989 and 1994 (ref. 66). Peaks in the CO2/H2O ratio at Solfatara fumaroles (Supplementary Fig. 1b) marked each event. Successive peaks (from 2000 to 2015) in CH4-based geoindicators (that is, the fumarole ratios of CO2/CH4 and He/CH4 (refs 14,70)) are taken as evidence for the occurrence of 11 IMF episodes (Supplementary Figs 1a and 2b). These events typically caused both pulsed inﬂations of the terrain13,14 (Supplementary Fig. 2c) and localized seismic activity (Supplementary Figs 1f and 2d,e). 21. Caliro, S., Chiodini, G. & Paonita, A. Geochemical evidences of magma dynamics at Campi Flegrei (Italy). Geochim. Cosmochim. Acta 132, 1–15 (2014). 22. D’Auria, L. et al. Magma injection beneath the urban area of Naples: a new mechanism for the 2012–2013 volcanic unrest at Campi Flegrei caldera. Sci. Rep. 5, 13100 (2015). 23. Linde, A. T. & Sacks, I. S. Triggering of volcanic eruptions. Nature 395, 888–890 (1998). 24. Pallister, J. S., Hoblitt, R. P. & Reyes, A. G. A basalt trigger for the 1991 eruptions of Pinatubo volcano? Nature 356, 426–428 (1992). Finally, the temporal evolution of the fumarole emissions of H2O–CO2 (Supplementary Fig. 1b) can be used to conﬁne the total amount of ﬂuid injected in each of the 14 IMF events (Supplementary Table 1) using a trial-and-error method. The total mass of each IMF is adjusted until the modelled compositions in the ‘checkpoint for gas composition’ ﬁt the observed fumarole CO2/H2O ratio reasonably well (Supplementary Fig. 1b). As explained above, the CO2/H2O molar ratio of the injected ﬂuids was assumed to be constant ( ¼ 0.67) in Simulation 1, while it decreased from 0.67 to 0.22 in Simulation 2, as in an open-system magma- degassing process (Supplementary Fig. 5b; Supplementary Table 1). 25. Papale, P., Moretti, R. & Barbato, D. The compositional dependence of the saturation surface of H2O þ CO2 ﬂuids in silicate melts. Chem. Geol. 229, 78–95 (2006). 26. Pruess, K. TOUGH2—A General Purpose Numerical Simulator for Multiphase Fluid and Heat Flow 102 (Lawrence Berkeley Lab. Report LBL, 1991). 27. Tilling, R. I. The critical role of volcano monitoring in risk reduction. Adv. Geosci. 14, 3–11 (2008). 28. Holloway, J. R. & Blank, J. G. in Volatiles in Magmas (eds Carroll, M. R & Holloway, J. R.) 185–230 (Mineralogical Society of America, 1994). ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms13712 As in previous work13, the rock physical properties are homogeneous. The ﬂuid source is located at the base of the domain (Fig. 4). 15. Sheridan, M. F. & Wohletz, K. H. Hydrovolcanic explosions: the systematics of water-pyroclast equilibration. Science 212, 1387–1389 (1981). The second step consisted in achieving steady-state conditions. Initially the domain contains liquid water with a low PCO2 (CO2 partial pressure) and a temperature that varies with depth according to the thermal gradient measured in CFc geothermal wells67. This condition is perturbed with a 2000-year-long injection of a 350 C gas mixture at 39 kg s 1, with a CO2/H2O molar ratio of 0.17. The ﬂux and composition reﬂect measurements performed at Solfatara50,67. The system reaches a steady-state condition after 2000 years of injection, which represents time 0 of Simulations 1 and 2. Ideally this initial condition represents the state of the system before 30 October 1983, which is the date on which magmatic gas was ﬁrst injected into the base of the system (Supplementary Fig. 1). Under steady-state conditions, the model predicts the formation of a gas zone (Xg ¼ 1) at 200–220 C at the top of the domain, which is aligned with the injection zone and just below the Solfatara fumarole ﬁeld (‘checkpoint for gas composition’ in Fig. 4). 16. Acocella, V., Di Lorenzo, R., Newhall, C. & Scandone, R. An overview of recent (1988 to 2014) caldera unrest: knowledge and perspectives. Rev. Geophys. 53, 896–955 (2015). 17. Newhall, C. G. & Dzurisin, D. Historical Unrest at Large Calderas of the World 1108 (US Geological Survey Bulletin, 1988). 18. Costa, A. et al. Quantifying volcanic ash dispersal and impact of the Campanian Ignimbrite super-eruption. Geophys. Res. Lett. 39, L10310 (2012). 19. Del Gaudio, C., Aquino, I., Ricciardi, G. P., Ricco, C. & Scandone, R. Unrest episodes at Campi Flegrei: a reconstruction of vertical ground movements during 1905–2009. J. Volcanol. Geotherm. Res. 195, 48–56 (2010). 20. D’Auria, L. et al. Repeated ﬂuid-transfer episodes as a mechanism for the recent dynamics of Campi Flegrei Caldera (1989-2010). J. Geophys Res. 116, B04313 (2011). In the third step, we simulated 14 IMF events. Their timing (Supplementary Table 1) was inferred based on compositional anomalies that have been determined for Solfatara fumaroles. References 34. Iacono Marziano, G., Morizet, Y., Le Trong, E. & Gaillard, F. New experimental data and semi-empirical parameterization of H2O-CO2 solubility in maﬁc melts. Geochim. Cosmochim. Acta 97, 1–23 (2012). 1. Siebert, L., Cottrell, E., Venzke, E. & Andrews, B. in Encyclopedia of Volcanoes 2nd edn (ed. Sigurdsson, H.) 249–261 (2015). 35. Liu, Y., Zhang, Y. & Behrens, H. Solubility of H2O in rhyolitic melts at low pressures and a new empirical model for mixed H2O-CO2 solubility in rhyolitic melts. J. Volcanol. Geotherm. Res. 143, 219–235 (2005). 2. Siebert, L., Simkin, T. & Kimberley, P. Volcanoes of the World (University of California Press, 2010). 3. Aiuppa, A. et al. Forecasting Etna eruptions by real-time observation of volcanic gas composition. Geology 35, 1115–1118 (2007). J , ( ) 36. Newman, S. & Lowenstern, J. B. VolatileCalc: a silicate melt-H2O-CO2 36. Newman, S. & Lowenstern, J. B. VolatileCalc: a silicate melt-H2O-CO2 solution model written in Visual Basic for excel. Comput. Geosci. 28, 597–604 (2002). solution model written in Visual Basic for excel. Comput. Geosci. 28, 597–604 (2002). 4. Patane`, D., De Gori, P., Chiarabba, C. & Bonaccorso, A. Magma ascent and the pressurization of Mount Etna’s volcanic system. Science 299, 2061–2063 (2003). 37. Chiodini, G. et al. Long-term variations of the Campi Flegrei, Italy, volcanic system as revealed by the monitoring of hydrothermal activity. J. Geophys. Res 115, B03205 (2010). 5. Poland, M. P., Miklius, A., Jeff Sutton, A. & Thornber, C. R. A mantle-driven surge in magma supply to Kı%lauea Volcano during 2003-2007. Nat. Geosci. 5, 5. Poland, M. P., Miklius, A., Jeff Sutton, A. & Thornber, C. R. A mantle-driven surge in magma supply to Kı%lauea Volcano during 2003-2007. Nat. Geosci. 5, 295–300 (2012). 5. Poland, M. P., Miklius, A., Jeff Sutton, A. & Thornber, C. R. A mantle-driven i l K%l V l d i N G i surge in magma supply to Kı%lauea Volcano during 2003-2007. Nat. Geosci. 5, 295–300 (2012). 38. Zollo, A. et al. Seismic reﬂections reveal a massive melt layer feeding Campi Flegrei caldera. Geophys. Res. Lett. 35, L12306 (2008). 6. Sigmundsson, F. et al. Segmented lateral dyke growth in a rifting event at Bar[eth]arbunga volcanic system, Iceland. Nature 517, 191 (2015). igmundsson, F. et al. Segmented lateral dyke growth in a rifting 39. Stock, M. J., Humphreys, M. C. S., Smith, V. C., Isaia, R. & Pyle, D. M. ARTICLE The simulation outputs relevant to this study are the simulated values of the total pressure, temperature, Xg (volumetric gas fraction), PCO2, and XCO2 (CO2 mass fraction) in each of the 850 cells. A key model output is the temporal evolution of the system temperature during the injection of the magmatic ﬂuids (Fig. 5a). This corresponds to the volume-weighted average temperature of the cells in a cylinder with a diameter of 0.95 km and a height of 1 km (that is, volume of 0.707 km3) sited above the injection zone (‘temperature box’ in Fig. 4). 29. Blundy, J., Cashman, K. V., Rust, A. & Witham, F. A case for CO2-rich arc magmas. Earth Planet. Sci. Lett. 290, 289–301 (2010). 30. Hurwitz, S. & Lowenstern, J. B. Dynamics of the Yellowstone hydrothermal system. Rev. Geophys. 51, 1–37 (2014). 31. Wallace, P. J., Plank, T., Edmonds, M. & Hauri, E. H. in Encyclopedia of Volcanoes 2nd edn (ed. Sigurdsson, H.) 163–183 (2015). 32. Burgisser, A., Alletti, M. & Scaillet, B. Simulating the behavior of volatiles belonging to the C-O-H-S system in silicate melts under magmatic conditions with the software D-Compress. Comput. Geosci. 79, 1–14 (2015). Data availability. All relevant data are available from the authors. Dixon, J. E. Degassing of alkalic basalts. Am. Mineral. 82, 368–3 NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms13712 The method63,64 we used can simulate properties of a binary CO2–H2O mixture up to supercritical conditions. However, we injected magmatic ﬂuids into the virtual hydrothermal system at 350 C in both Simulations 1 and 2. This is because the geothermal simulator that we used (TOUGH2 (ref. 26)) can only work below the critical-point temperature of water. Enthalpies of the magmatic gas phase at 350 C were calculated using the EOS2 module26. This yielded that the enthalpies of the injected magmatic gases were far lower than those of magma-released supercritical ﬂuids (Fig. 1b). This difference accounts for the energy lost by ﬂuids as they pass thorough the plastic zone, between the magma and the overlying hydrothermal system. The physical model calculations included deﬁning the computational domain, achieving steady-state conditions before simulating IMF events, selecting the number and timing of IMF events, and calculating the mass of ﬂuids injected in each IMF event. These steps are described in detail below. The computational domain consists of 850 cells distributed in a two- dimensional radial space of 2,000 m 2,500 m (Fig. 4). This domain is similar to (but 500 m deeper than) that used in previous simulations66–69 of the CFc hydrothermal system13. The top boundary is at ﬁxed (atmospheric) temperature and pressure conditions, while other boundaries are impermeable and adiabatic. TOUGH2 is a numerical simulator for geothermal systems that is fully described elsewhere26. Brieﬂy, TOUGH2 includes the coupled transport of a 7 NATURE COMMUNICATIONS \| 7:13712 \| DOI: 10.1038/ncomms13712 \| www.nature.com/naturecommunications References Late-stage volatile saturation as a potential trigger for explosive volcanic eruptions. Nat. Geosci. 9, 249 (2016). 7. Sigmundsson, F. et al. Intrusion triggering of the 2010 Eyjafjallajo¨kull explosive eruption. Nature 468, 426–432 (2010). 8. Fischer, T. P. & Chiodini, G. in Encyclopedia of Volcanoes 2nd edn (ed. Sigurdsson, H.) 779–796, (2015). p 40. Aiuppa, A. et al. First observations of the fumarolic gas output from a restless caldera: Implications for the current period of unrest (2005-2013) at Campi Flegrei. Geochem. Geophys. Geosyst. 14, 4153–4169 (2013). g 9. Goff, F. & Janik, C. J. in Encyclopedia of Volcanoes (ed. Sigurdsson, H., Houghton, B., McNutt, S., Rymer, H. & Stix, J.) 817–834 (Academic Press, 2000). 41. Paonita, A., Caracausi, A., Iacono-Marziano, G., Martelli, M. & Rizzo, A. Geochemical evidence for mixing between ﬂuids exsolved at different depths in the magmatic system of Mt Etna (Italy). Geochim. Cosmochim. Acta 84, 380–394 (2012). 10. Sparks, R. S. J. Forecasting volcanic eruptions. Earth Planet. Sci. Lett. 210, 1–15 (2003). 11. Tilling, R. I. Volcanic hazards and their mitigation: progress and problems. Rev. Geophys. 27, 237–269 (1989). 42. Tassi, F. et al. Geochemistry of ﬂuid discharges from Peteroa volcano (Argentina-Chile) in 2010-2015: insights into compositional changes related to the ﬂuid source region(s). Chem. Geol. 432, 41–53 (2016). p y 12. Voight, B. R. & Cornelius, R. R. Prospects for eruption prediction in near real- time. Nature 350, 695–698 (1991). 43. Nicholson, E. J., Mather, T. A., Pyle, D. M., Odbert, H. M. & Christopher, T. Cyclical patterns in volcanic degassing revealed by SO2 ﬂux timeseries analysis: an application to Soufriere Hills Volcano, Montserrat. Earth Planet. Sci. Lett. 375, 209–221 (2013). 13. Chiodini, G., Caliro, S., De Martino, P., Avino, R. & Gherardi, F. Early signals of new volcanic unrest at Campi Flegrei caldera? Insights from geochemical data and physical simulations. Geology 40, 943–946 (2012). 14. Chiodini, G. et al. Evidence of thermal driven processes triggering the 2005-2014 unrest at Campi Flegrei caldera. Earth Planet. Sci. Lett. 414, 58–67 (2015). 44. Cartwright, J. & Hansen, D. M. Magma transport through the crust via interconnected sill complexes. Geology 34, 929–932 (2006). 8 NATURE COMMUNICATIONS \| 7:13712 \| DOI: 10.1038/ncomms13712 \| www.nature.com/naturecommunications ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms13712 45. Dawson, P., Whilldin, D. & Chouet, B. Application of near real-time radial semblance to locate the shallow magmatic conduit at Kilauea Volcano, Hawaii. Geophys. Res. Lett. 31, L21606 (2004). 67. References Todesco, M., Chiodini, G. & Macedonio, G. Monitoring and modelling hydrothermal ﬂuid emission at La Solfatara (Phlegrean Fields, Italy); an interdisciplinary approach to the study of diffuse degassing. J. Volcanol. Geotherm. Res. 125, 57–79 (2003). 46. Paulatto, M., Minshull, T. A. & Henstock, T. J. Constraints on an intrusive system beneath the Soufrie´re Hills Volcano, Montserrat, from ﬁnite difference modelling of a controlled source seismic experiment. Geophys. Res. Lett. 37, L00E01 (2010). 68. Todesco, M. Signals from the Campi Flegrei hydrothermal system: role of a ‘magmatic’ source of ﬂuids. J. Geophys. Res. 114, B05201 (2009). 69. Todesco, M., Rutqvist, J., Chiodini, G., Pruess, K. & Oldenburg, C. M. Modeling of recent volcanic episodes at Phlegrean Fields (Italy); geochemical variations and ground deformation. Geothermics 33, 531–547 (2004). 47. Preston, R. J. Composite minor intrusions as windows into subvolcanic magma reservoir processes: mineralogical and geochemical evidence for complex magmatic plumbing systems in the British Tertiary Igneous Province. J. Geol. Soc. 158, 47–58 (2001). 70. Chiodini, G. CO2/CH4 ratio in fumaroles a powerful tool to detect magma degassing episodes at quiescent volcanoes. Geophys. Res. Lett. 36, L02302 (2009). 48. Menand, T. & Phillips, J. C. Gas segregation in dykes and sills. J. Volcanol. Geotherm. Res. 159, 393–408 (2007). 49. Woods, A. W. & Cardoso, S. S. S. Triggering basaltic volcanic eruptions by bubble-melt separation. Nature 385, 518–520 (1997). Acknowledgements This work received funding from the INGV-DPC Research Agreement 2012–2015, the EU-FP7 Project MED-SUV (grant agreement 308665), the ERC Project ‘Bridge’ (grant agreement 305377), the INGV project COHESO, and DECADE (an initiative from the Deep Carbon Observatory). Comments from V.C. Smith improved the quality of the manuscript. 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To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/ 64. Afanasyev, A. A. Simulation of the properties of a binary carbon dioxide-water mixture under sub-and supercritical conditions. High Temp. Author contributions 54. Dusseault, M. B. Geomechanical challenges in petroleum reservoir exploitation. KSCE J. Civ. Eng. 15, 669–678 (2011). G.C. conceived the initial idea of the study, with all of the coauthors deﬁning the methodology and strategy. G.C., S.C. and P.D.M. acquired geochemical and geodetic data. G.C. ran the simulations, with contributions from A.P. and A.C. G.C., A.P., A.A., A.C., J.V. and V.A. wrote the manuscript with input from all of the coauthors. 55. Heap, M. J., Baud, P., Meredith, P. G., Vinciguerra, S. & Reuschle´, T. The permeability and elastic moduli of tuff from Campi Flegrei, Italy: implications for ground deformation modelling. Solid Earth 5, 1–20 (2014). 56. Heap, M. J. et al. How tough is tuff in the event of ﬁre? 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Lett. 30, 1434 (2003). r The Author(s) 2016 r The Author(s) 2016 NATURE COMMUNICATIONS \| 7:13712 \| DOI: 10.1038/ncomms13712 \| www.nature.com/naturecommunications 9 9 NATURE COMMUNICATIONS \| 7:13712 \| DOI: 10.1038/ncomms13712 \| www.nature.com/naturecommunications
https://openalex.org/W2883456891	https://www.intechopen.com/citation-pdf-url/57382	English	null	Calorimetry Characterization of Carbonaceous Materials for Energy Applications: Review	InTech eBooks	2,018	cc-by	8,556	Abstract Carbonaceous materials are of great interest for several applications in adsorption, cataly- sis, gases storage, and electrochemical energy storage devices because of the ability to modify their pore texture, specific surface area, and surface chemistry. Some of the most used precursors are carbon gels, biomass, carbon nanotubes, and coal. These materials can be doped or functionalized to modify their surface. Immersion calorimetry is one of the techniques used to determine the textural and chemical characterization of solids like carbonaceous materials. Immersion calorimetry provides information about the interac- tions that occur between solids and different immersion liquids. The measurement of heats of immersion into liquids with different molecular sizes allows for the assessment of their pore size distribution. When polar surfaces are analyzed, both the surface accessibil- ity of the immersion liquid and the specific interactions between the solid surface and the liquid’s molecules account for the total value of the heat of immersion. Zapata-Benabithe et al., Castillejos et al., Chen et al., and Centeno et al. prepared different materials and used immersion calorimetry into benzene, toluene, and/or water to correlate the external surface area of microporous solids with energy parameters such as specific capacitance or chemical surface (oxygen content, acid groups, or basic groups). This chapter will be compiling a review of the results founded about the calorimetry characterization of car- bonaceous materials for energy area applications. Keywords: immersion calorimetry, carbonaceous materials, energy applications Additional information is available at the end of the chapter http://dx.doi.org/10.5772/intechopen.71310 http://dx.doi.org/10.5772/intechopen.71310 Selection of our books indexed in the Book Citation Index in Web of Science™ Core Collection (BKCI) Interested in publishing with us? Contact book.department@intechopen.com Numbers displayed above are based on latest data collected. For more information visit www.intechopen.com Open access books available Countries delivered to Contributors from top 500 universities International authors and editors Our authors are among the most cited scientists Downloads We are IntechOpen, the world’s leading publisher of Open Access books Built by scientists, for scientists 14% 191,000 210M TOP 1% 154 7,200 Chapter 4 Calorimetry Characterization of Carbonaceous Materials for Energy Applications: Review Zulamita Zapata Benabithe Additional information is available at t Zulamita Zapata Benabithe Additional information is available at th Additional information is available at the end of the chapter 1. Introduction Nowadays, the demand for electricity worldwide is supplied mainly by conventional energy sources (oil, natural gas, and coal); however, supply from renewable energy sources such as solar, wind, and others has been growing quickly since the end of the 2000’s, from 18% (2000) © 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. ( ) p ttribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, nd reproduction in any medium, provided the original work is properly cited. © 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, di ib i d d i i di id d h i i l k i l i d Calorimetry - Design, Theory and Applications in Porous Solids 78 to 26% (2016) [1]. Although the consumption of these renewable energies has increased dur- ing the last decade, one of the main drawbacks with this type of energy is the reliability and assurance in the energy supply to the consumers, because the energy production fluctuates with the climatological conditions. For this reason, it is necessary to consider energy storage systems (ESSs) that attenuate fluctuations in power generation and can respond to variations in the demand for energy by consumers, facilitating the supply of power to the grid. The production of electrical energy from unconventional sources coupled to an energy stor- age system can be more efficient because they contribute to the reduction of the environmen- tal impact, reducing the carbon footprint and global warming and allow converting back into electrical energy when needed in different periods of time [2]. ESS can be categorized into mechanical (pumped hydroelectric storage, compressed air energy storage, and flywheels), electrochemical (conventional rechargeable batteries and flow batteries), electrical (capacitors, supercapacitors, and superconducting magnetic energy stor- age), thermochemical (solar fuels), chemical (hydrogen storage with fuel cells), and thermal energy storage (sensible heat storage and latent heat storage) [2]. 1. Introduction These differ from each other on their properties, such as the type of primary energy they store, energy density and power density ranges, life cycle, application sector, and the cost of production [3]. In renewable energy–generating devices, such as wind turbines and solar panels, super- capacitors can be used for storing energy to accelerate the turbine after a period with little wind and prevent electrical dropouts in the solar panels. In the case of the transportation sector, batteries, hybrids, and hydrogen are considered as alternatives instead of fossil fuels. Supercapacitors are commonly used in cell phones and computers with backup power for the memory; besides, supercapacitors may also replace the battery in vehicles driven by internal combustion engines [4]. These devices are mainly composed by two electrodes (anode and cathode) and an electro- lyte. Carbonaceous materials (carbon gels, biomass, carbon nanotubes, coal, etc.) are one of the most common materials used as electrodes due to their low cost and high superficial area (400–2000 m2/g), low density (0.3–1 g/cm3), and good conductivity (5–50 S/cm). The energy storage occurs on the surface of the electrodes, and it could be doped or functionalized to modify their superficial chemistry to improve the energy storage [5]. Nitrogen gas adsorption/desorption at 77 K, thermogravimetric analysis (TGA), differential scanning calorimetry (DSC), immersion calorimetry, X-ray diffraction (XRD), X-ray photo- electron spectra (XPS), scanning electron microscope (SEM), and Fourier-transform infrared (FT-IR) spectroscopy are the most techniques used to characterize porous materials, because they give information about textural, porous and morphology surface, and physical-chemical composition. Immersion calorimetry is one of the techniques used to determine the textural and chemical characterization of solids like carbonaceous materials. This technique provides information about the interactions that occur between solids and different immersion liquids [6]. The mea- surement of heats of immersion into liquids with different molecular sizes allows for the assess- ment of their pore size distribution by measuring the enthalpy of immersion of the samples Calorimetry Characterization of Carbonaceous Materials for Energy Applications: Review http://dx.doi.org/10.5772/intechopen.71310 79 79 into liquids of different critical dimensions. When polar surfaces are analyzed, both the surface accessibility of the immersion liquid and the specific interactions between the solid surface and the molecules of the liquid account for the total value of the heat of immersion [7]. 1. Introduction Otherwise, DSC is a thermo-analytical technique in which the difference between the amount of heat required to increase the temperature of a sample and a reference is measured as a func- tion of temperature [8]. DSC profiles provide information about thermal stability and thermal phase transition (transition temperature (Tg), crystalline melting temperature (Tm), and crys- tallization temperature (Tc) from heating curves (enthalpy; ΔH)). DSC analysis can be used to understand the solid–liquid phase transition of room temperature ionic liquids (RTILs) used in energy storage devices. In this chapter, the research results founded that the immersion calorimetry and differential scanning calorimetry as characterization techniques of carbonaceous materials for ESS such as supercapacitors will be compiling. 2. Immersion calorimetry of different liquid technique in supercapacitors application The calorimetric technique is one of the most used techniques to perform the characterization of systems that generate or absorb thermal energy. Isothermal calorimeters exhibit a large heat exchange between the system and the environment. The system in this case is consid- ered as a steel cell in which the liquid adsorbate and the porous material are introduced. Immersion enthalpy is a measure of the amount of heat released when a known mass of a degassed solid is completely immersed in a given liquid; the magnitude of enthalpy depends on the nature of liquid-solid interactions and the extent of available surface. Alonso et al. [9] activated from the pitch was pyrolysis with KOH at different amount of activating agent. The activated carbons obtained were used as electrodes of supercapacitors. The activated carbons were mainly microporous, while the mesopores increased at higher amount of activating agent. The microporosity of the activated carbons was characterized by measuring the enthalpy of immersion of the samples into liquids with different size. Measurements were carried out at 20°C using dichloromethane (CH2Cl2, L = 0.33 nm), ben- zene (C6H6, L = 0.41 nm), cyclohexane (C6H12), carbon tetrachloride (CCl4, L = 0.63 nm), and tri- 2,4-xylylphosphate (TXP, L = 1.5 nm). The increase of the amount of activating agent caused the immersion enthalpy in the liquid of the smallest size (CH2Cl2) to increase from 140 to 281 J/g, which is in agreement with the higher surface developed in the carbons, according to N2 adsorption isotherm data. In the case of the sample with the lowest activating agent ratio (1/1), the immersion enthalpy in TXP were significantly lower than that obtained for the other liquids (8 J/g, respect to 109–177 J/g), indicating that the TXP molecule is not accessible to the pores developed in this sample (dp < 1.5 nm). In the case of the sample with activating agent/ carbon ratio of 2/1, the enthalpy of immersion in TXP was also lower than the rest of the liq- uids. This indicated that only a small proportion of the pores present in this sample are larger than 1.5 nm. Samples activated with a higher proportion of KOH (3/1 and 5/1) showed very Calorimetry - Design, Theory and Applications in Porous Solids 80 high values of immersion enthalpy into TXP, which indicated that most of the pores present in these samples are larger than 1.5 nm. 2. Immersion calorimetry of different liquid technique in supercapacitors application The sample activated at activating agent/carbon ratio of 3/1 showed the highest specific capacitances (300–425 F/g) at different current densities (<1–90 mA/cm2) in H2SO4 2 M. This behavior could be associated with the easy diffusion of the electrolyte due to a heterogeneous porous distribution, in accordance with the highest immer- sion enthalpies values into liquids of different sizes. Mora et al. [10] obtained activated carbons from mesophase pitch with KOH using different proportions of the activating agent (1:1 to 5:1 KOH to carbon mass ratio) and activation tem- peratures (600 and 800°C) to study the effect on the textural characteristics of the resultant activated carbons and how these characteristics influenced their behavior as electrodes in supercapacitors. The textural properties of the activated carbons were studied by gas adsorp- tion of N2 at 77 K and CO2 at 273 K and immersion calorimetry. Enthalpy of immersion of the samples into liquids of different critical dimensions was used to characterize the micro- porosity of the activated carbons. Immersion calorimetry measurements were carried out at 20°C using dichloromethane (CH2Cl2, L = 0.33 nm), benzene (C6H6, L = 0.41 nm), carbon tetrachloride (CCl4, L = 0.63 nm), tetraisopropyl-o-titanate (TIPOT, L = 1.05 nm), and tetra- butyl-o-titanate (TBOT, L = 1.3 nm). Immersion calorimetry into water was used to estimate the number of hydrophilic sites ([O + HCl]∆iH) of the carbon surface according to Eq. (1) [11]: [O + HCl] ∆ i H = [0.21 ∆ i H C 6 H 6 − ∆ i H H 2 O ] / 10 J / mmol (1) (1) The oxygen content is linked with hydrophilic character; samples with high oxygen content showed an increase of hydrophilic sites. Samples were mainly microporous (pore size ~0.9 nm). These samples showed the highest capacitances (200–400 F/g) at low current densities (0.75 mA/ cm2) in 2-M H2SO4. However, the sample obtained at 3 of KOH:mesofase ratio and 600°C showed the highest total oxygen content (13.94 wt.%), but it did not show the highest capacity due to an increase of the equivalent series resistance (ESR). The sample activated at 5 of KOH:mesofase ratio and 700°C showed the highest capacitance at low and high current (0.75 and 75 mA/cm2, respectively). This sample also showed high values of immersion enthalpies into liquids with different size, which suggests the easy access of the electrolyte into the micropores. 2. Immersion calorimetry of different liquid technique in supercapacitors application S av = ( S comp + S phenol + S benzene + S DR ) / 4 (3) (3) S av = ( S comp + S phenol + S benzene + S DR ) / 4 The total surface area can be as high as 1500–1600 m2/g. The relatively low amount of surface oxygen in the present TMCs, as opposed to activated carbons, reduces the contribution of pseudo-capacitance effects and limits the gravimetric capacitance to 200–220 F/g for aqueous electrolytes. In the case of nonaqueous electrolyte, it rarely exceeds 100 F/g. The ionic mobility did not improved due to the mesoporous presence of these TMCs compared with activated carbons of pore widths above 1.0–1.3 nm. Fernández et al. [14] obtained mesoporous materials from mixtures of poly(vinyl alcohol) with magnesium citrate carbonized and evaluated their performance as electrodes in supercapaci- tors. The highest specific capacitance (Co) at low current density (1 mA/cm2) values was 180 F/g in 2 M H2SO4 electrolyte and around 100 F/g in 1 M (C2H5)4NBF4 in acetonitrile. The specific surface area (Sav) was calculated as an average of the several specific surface area values; they were estimated by employing different procedures such as comparison plot (Scomp) and based on the enthalpy of immersion into phenol (Sphenol) and benzene (Sbenzene), according to Eq. (4). S av = ( S comp + S phenol + S benzene ) / 3 (4) (4) The addition up to approximately 40% of MgO to the raw mixture gradually increased the average specific surface area of the resulting carbons up to approximately 1300 m2/g. Co of different mesoporous carbons obtained in aqueous 2-M H2SO4 solution and aprotic electrolyte 1-M (C2H5)4NBF4/CH3CN were correlated with Sav. The relationship of Co vs. Sav showed a lin- ear increase of the limit specified both in an acid medium and aprotic. The lines through the origin correspond to 0.14 F/m2 in aqueous solution and of 0.07 F/m2 in the aprotic electrolyte. Ruiz et al. [15] prepared carbonaceous materials from naphthalene-derived mesophase pitch. These were chemically activated using (3:1) KOH to carbon mass ratio at 700°C for 1 h under nitrogen flow. The activated carbon was thermally treated at 600 and 1000°C under nitrogen flow. The microporosity of the electrodes was characterized by measuring the enthalpy of immersion of the samples into liquids of different critical dimensions. 2. Immersion calorimetry of different liquid technique in supercapacitors application The oxygen content is linked with hydrophilic character; samples with high oxygen content showed an increase of hydrophilic sites. Samples were mainly microporous (pore size ~0.9 nm). These samples showed the highest capacitances (200–400 F/g) at low current densities (0.75 mA/ cm2) in 2-M H2SO4. However, the sample obtained at 3 of KOH:mesofase ratio and 600°C showed the highest total oxygen content (13.94 wt.%), but it did not show the highest capacity due to an increase of the equivalent series resistance (ESR). The sample activated at 5 of KOH:mesofase ratio and 700°C showed the highest capacitance at low and high current (0.75 and 75 mA/cm2, respectively). This sample also showed high values of immersion enthalpies into liquids with different size, which suggests the easy access of the electrolyte into the micropores. Centeno et al. [12] characterized 12 activated carbons with different superficial (378–1270 m2/g) and porous characteristics (micropore widths between 0.7 and 2 nm). The highest specific capacitance value obtained was 320 F/g using 2-M H2SO4 as electrolyte. Eq. (2) shows an empir- ical correlation obtained between Co, the capacitance C (F/g) at 1 mA/cm2, and the enthalpy of immersion ∆iHC6H6 (J/g) at 293 K for 20 microporous carbons. C o = − k ∆ i H C 6 H 6 (2) (2) The deviation of the correlation, with k = 1.15 ± 0.1 (F/J), could be related with specific chemical reactions of the acid with surface groups and to the relatively strong physical interaction between water and the surface oxygen atoms. The enthalpy of immersion of benzene also depends on the Calorimetry Characterization of Carbonaceous Materials for Energy Applications: Review http://dx.doi.org/10.5772/intechopen.71310 81 structural parameters such as microporous (Smi) and external (Se) surface areas and volume (W). Eq. (2) can be used to evaluate empirically the performance of a given carbon as a capacitor. Sevilla et al. [13] prepared templated mesoporous carbons (TMCs) to be used as supercapaci- tors. The double layer capacity formed on their surface corresponds to 0.14 F/m2 in aque- ous electrolytes, such as H2SO4 and KOH, and 0.06 F/m2 for the aprotic medium (C2H5)4NBF4/ CH3CN. The effective surface area was determined by independent techniques from Eq. (3): analysis of the nitrogen isotherms by the comparison plot (Scomp) and DFT (SDFT) and based on the enthalpy of immersion into dilute aqueous solution of phenol (Sphenol) and benzene (Sbenzene). 2. Immersion calorimetry of different liquid technique in supercapacitors application The comparison of the immersion enthalpies into water ΔiH(H2O) and into benzene ΔiH(C6H6) suggested that the sur- face oxygen density for cherry stones–based materials varied between 1.2 and 3.0 μmol/m2. The specific enthalpy of immersion into water, hi[H2O] Eq. (5), was around −0.04 to −0.06 J/m2. h i [ H 2 O] = ∆ i H [ H 2 O] _______ TSA [=] J / g ____ m 2 / g [=] J ___ m 2 (5) (5) (5) The specific capacitances values (C) at low current density as high as 230 F/g in aqueous elec- trolyte 2-M H2SO4 and 120 F/g in the aprotic medium 1-M (C2H5)4NBF4/acetonitrile. The corre- lation between C and TSA showed a specific surface-related capacitance [C (F/g)/TSA (m2/g)] around 0.17 F/m2 in H2SO4 electrolyte and 0.09 F/m2 in (C2H5)4NBF4/acetonitrile medium. The highest value in the acidic electrolyte showed an extra contribution from certain functional surface complexes (containing mainly oxygen and nitrogen) in the form of quick oxidation/ reduction reactions that promoted the pseudo-capacitance effects to be added to the purely double layer capacitance associated with the surface area. In the case of the aprotic electrolyte, the contribution did practically not depend on the chemistry of the carbon surface. The specific capacitances values (C) at low current density as high as 230 F/g in aqueous elec- trolyte 2-M H2SO4 and 120 F/g in the aprotic medium 1-M (C2H5)4NBF4/acetonitrile. The corre- lation between C and TSA showed a specific surface-related capacitance [C (F/g)/TSA (m2/g)] around 0.17 F/m2 in H2SO4 electrolyte and 0.09 F/m2 in (C2H5)4NBF4/acetonitrile medium. The highest value in the acidic electrolyte showed an extra contribution from certain functional surface complexes (containing mainly oxygen and nitrogen) in the form of quick oxidation/ reduction reactions that promoted the pseudo-capacitance effects to be added to the purely double layer capacitance associated with the surface area. In the case of the aprotic electrolyte, the contribution did practically not depend on the chemistry of the carbon surface. Garcia-Gomez et al. [17] prepared cylindrical carbon monoliths, and they studied their behav- ior as electrodes for supercapacitors. The porosity of the carbon monoliths was character- ized by N2 adsorption at 77 K and by immersion calorimetry at 293 K. The total surface area was calculated from the average values of comparison method (Scomp), immersion calorimetry into aqueous solution of phenol (Sphenol), and the Dubinin-Radushkevich approach (SDR), from Eq. 2. Immersion calorimetry of different liquid technique in supercapacitors application Measurements were carried out at 20°C using dichloromethane (0.33 nm) and tri-2,4-xylylphosphate, TXP (1.5 nm). In the thermal treatment at 600°C, a slight reduction in the capacity to adsorb the nitrogen was showed. The total pore volume was reduced from 0.85 to 0.80 cm3/g, and microporous surface Calorimetry - Design, Theory and Applications in Porous Solids 82 area was reduced, Smic, from 1531 to 1407 m2/g (8% reduction compared to original activated carbon). The thermal treatment at 1000°C generated a decrease of the total pore volume up to 0.66 cm3/g, and the Smic decreased up to 1318 m2/g (14%). The average pore size reduced considerably. After thermal treatment, the heat of immersion obtained for CH2Cl2 and TXP diminished respect to the original activated carbon for both temperatures, at 600°C from 197 to 206 J/g with CH2Cl2 and at 1000°C from 82 to 43 J/g with TXP. These could be related with the presence of constrictions and secondly due to the reduction in the average pore size. The specific capacitance of the original activated carbon was 309 F/g in 1 M H2SO4, while the spe- cific capacitance for the activated carbons treated thermally diminished up to 85 F/g for 600°C and 196 F/g for 1000°C. The reason could be the formation of physical constrictions at the entrance of the porous network which makes it more difficult for the electrolyte to gain access. Olivares-Marín et al. [16] produced activated carbon with KOH from cherry stones wastes for electrode material in supercapacitors. The chemical activation of cherry stones was carried out by different agents such as H3PO4, ZnCl2, and KOH. The activated carbons prepared with KOH showed the highest total specific surface area TSA (Smi + Se) (1100–1300 m2/g) and also the con- ductivities 1 and 2 S/cm. The materials obtained by carbonization of a mixture of KOH and cherry stones with a ratio 3:1 at 800 and 900°C (carbons K3–800 and K3–900) consists mainly of micropores (width < 2 nm). Their surface areas are respectively 1244 m2/g and 1039 m2/g. The carbonaceous material obtained with KOH/cherry stones ratio of 1:1 and 3:1 at 800°C (K1–800 and K3–800) showed similar porosity. However, immersion calorimetry with different molecular probes indicated significant differences in the micropore size distribution. 2. Immersion calorimetry of different liquid technique in supercapacitors application (6) instead of surface area estimated from the BET equation (SBET). Calorimetry Characterization of Carbonaceous Materials for Energy Applications: Review http://dx.doi.org/10.5772/intechopen.71310 83 Calorimetry Characterization of Carbonaceous Materials for Energy Applications: Review http://dx.doi.org/10.5772/intechopen.71310 83 S av = ( S comp + S phenol + S DR ) / 3 (6) (6) SBET underestimates the total surface area for carbons with average micropore size below 0.8–0.9 nm. The interfacial capacitance was 14 μF/cm2, obtained in KOH electrolyte, for a specific capacitance of 150 F/g at low current densities (1 mA/cm2 or 10 mA/g) and the specific surface area, Stotal = 1086 m2/g. According to Ref. [18], the double layer capacitance per unit of micropore surface area was found to be very close to that of carbon basal plane (about 15–20 μF/cm2). Moreover, the chemical nature of carbon surface could be estimated from the spe- cific enthalpy of immersion into water −ΔiHH2O (J/g). The low value of the −ΔiHH2O/Stotal ratio (−0.023 J/m2) suggests that the monolith surface has nonoxygenated functionalities. Sánchez-González et al. [19] selected a commercial activated carbon (Norit® C-Granular) and treated it under N2 at 700, 800, and 900°C during 2 h. The activated carbons were evalu- ated electrochemically as electrodes of supercapacitors in aqueous (2 M H2SO4) and organic (1 M (C2H5)4NBF4/CH3CN) electrolytes. The microporosity characterization was based on Dubinin’s theory, micropore volume (Wo), the average width (Lo) of the locally slit-shaped micropores and the surface area of the micropore walls (Smi), the total pore volume (Vp), and the external surface (Se) from N2 adsorption at 77 K isotherm. Other methods such as the com- parison plot (Scomp) and the density functional theory (SDFT) were used. The average surface area was calculated from Eq. (7). S av = ( S totDR + S comp + S DFT ) / 3 (7) (7) The density of surface oxygenated functionalities was estimated by the enthalpies of immersion into water and into benzene at 293 K. After heat treatment did not change significantly the pore structure of carbons, the surface oxygen density, presence of carboxylic acid groups, dimin- ished (9.6 to 4.5–5.6 μmol/m2). The commercial activated carbon showed relatively high surface area (727 m2/g) but a poor electrochemical performance in both aqueous and aprotic media. The cyclic voltammograms based on carbons C700–C900 showed a regular box-like behavior of an ideal capacitor. 2. Immersion calorimetry of different liquid technique in supercapacitors application The rectangular shape was well preserved over a wide range of scan rates (1–50 mV/s), which indicates a quick charge propagation. High gravimetric capacitances were obtained from galvanostatic charge–discharge cycling at 1 mA/cm2 from 124 to 173 F/g in the aqueous H2SO4 electrolyte and around 74–80 F/g in the aprotic medium. The density of surface oxygenated functionalities was estimated by the enthalpies of immersion into water and into benzene at 293 K. After heat treatment did not change significantly the pore structure of carbons, the surface oxygen density, presence of carboxylic acid groups, dimin- ished (9.6 to 4.5–5.6 μmol/m2). The commercial activated carbon showed relatively high surface area (727 m2/g) but a poor electrochemical performance in both aqueous and aprotic media. The cyclic voltammograms based on carbons C700–C900 showed a regular box-like behavior of an ideal capacitor. The rectangular shape was well preserved over a wide range of scan rates Figure 1 shows a relationship between the surface-related capacitances (Co/Sav) with electric conductivity (S/m). The sample C900 showed the lowest Co/Sav with the highest electric con- ductivity, and this behavior could be related to the enhancement in the structural order by thermal annealing of the pseudographitic carbonaceous layers. In aqueous and aprotic elec- trolytes, the sample C900 showed a limited effect on the capacitor capacity for energy storage, but results in power density was almost four times higher than C700. Zapata-Benabithe et al. [20] obtained carbon aerogels by carbonizing organic aerogels pre- pared by polycondensation reaction of resorcinol or pyrocatechol with formaldehyde. They are KOH activated at two KOH/carbon ratios to increase pore volume and surface area, and selected samples were surface treated to introduce oxygen and nitrogen functionalities. Calorimetry - Design, Theory and Applications in Porous Solids 84 Figure 1. Relationship between the surface-related capacitances and electric conductivity. Figure 1. Relationship between the surface-related capacitances and electric conductivity. The samples were evaluated as electrodes for supercapacitors in 1-M H2SO4. The samples were characterized by N2 and CO2 adsorption at −196 and 0°C, respectively, immersion calorimetry, temperature-programmed desorption, and X-ray photoelectron spectroscopy in order to determine their surface area, porosity, and surface chemistry. Two series of sam- ples were obtained: one micro-mesoporous and another basically microporous. After KOH activation, the specific surface area (from BET equation) showed values up to 1935 m2/g. 2. Immersion calorimetry of different liquid technique in supercapacitors application Immersion enthalpies into benzene, −ΔHbenz, water, and −ΔHwater, were used to determine the hydrophobicity (HF) of the samples according to Eq. (8). HF = 1 − (∆ H water / ∆ H benz ) (8) (8) The hydrophobicity factor varied between −0.12 and 0.75 for the activated and functionalized carbon aerogels. The relationship between the hydrophobicity and the surface (OXPS) and total (OTPD) oxygen content of the samples diminished linearly with an increase in the oxygen con- tent. The increase of the oxygen content improved the wettability of the carbon surface by the electrolyte, facilitating the EDL formation. However, this advantage can be offset by the binding of oxygen polar groups (mainly carboxyl groups) with water molecules, hindering and retard- ing the migration of the electrolyte into the pores and thereby increasing the ohmic resistance. One of the samples with the highest gravimetric capacitance in 1-M H2SO4, 221 F/g at 0.125 A/g, was obtained with micro-mesoporous characteristics and the highest oxygen functionalities. Elmouwahidi et al. [21] prepared activated carbons by KOH activation of argan seed shells (ASS), and the activated carbon with the largest surface area was superficially treated to intro- duce oxygen and nitrogen functionalities. Samples were characterized by N2 and CO2 adsorp- tion at −196 and 0°C and immersion calorimetry into benzene and water. Immersion enthalpies into benzene, ΔHbenz, were used to determine the surface area of the activated carbons, Sbenz. Benzene molecule has no specific interactions with surface groups, considering the immer- sion enthalpy into benzene of a nonporous graphitized carbon black to be 0.114 J/m2 [22]. The hydrophobicity of samples was determined from Eq. (8). The Dubinin-Radushkevich (DR) Calorimetry Characterization of Carbonaceous Materials for Energy Applications: Review http://dx.doi.org/10.5772/intechopen.71310 85 equation was applied to the N2 and CO2 isotherms to obtain the micropore volume, W0(N2) and W0(CO2), respectively. All samples showed W0(N2) > W0(CO2), indicating an absence of constrictions at micropore entrances and hence complete accessibility to N2 molecules at −196°C. The apparent surface area (SBET) and Sbenz values were similar, due to the dimensions of N2 (0.36 nm) and benzene (0.37 nm) are almost identical, and the micropore width allowed the accommodation of one N2 monolayer on each micropore wall. The hydrophobicity varied between −0.21 and 0.53 and decreased as a consequence of the fixation of oxygen functional- ities with large polarity like carboxyl groups. 2. Immersion calorimetry of different liquid technique in supercapacitors application Activated carbon aerogel superficially treated with oxygen functional groups showed the lowest specific capacitance at 0.125 and 1 A/g and 259 and 135 F/g, respectively, in 1-M H2SO4. This behavior could be expressed by the presence of surface carboxyl groups hindering electrolyte diffusion into the highly polar pores. Zapata-Benabithe et al. [23] studied the effect of the Boron dopant (boric and phenyl boronic acids) and drying method (supercritical, freeze, microwave oven, and vacuum drying) on the surface physics and chemistry of B-doped resorcinol-formaldehyde gels and their electro- chemical behavior. The surface characteristics were studied by N2 and CO2 adsorption at −196 and 0°C, respectively, and immersion calorimetry into benzene and water. The N2 adsorp- tion–desorption isotherms at −196°C were type IV and showed a type H2 hysteresis cycle for all B-doped carbon gels obtained. The micropore volume (W0(N2)) and specific surface area (SBET) values were similar for all samples (~0.23 cm3/g and 560–590 m2/g, respectively), except the samples with phenyl boronic acids dried in freeze and vacuum drying oven. This behavior suggests that the drying method has practically no influence on porous characteristics. The surface area from enthalpy of immersion into benzene was determined (Simm) from Eq. (9) and compared with SBET values. S C 6 H 6 = ∆ H i, C 6 H 6 _______ ∆ h C 6 H 6 (9) (9) The results showed that the Simm > SBET in all samples. The SBET can underestimate with respect to the Simm, because of the restricted diffusion of N2 at −196°C in very narrow micropores or in those with constrictions at their entrance, whereas benzene molecules can access all micro- pores because of the much higher temperature (30°C) at which immersion took place. The gravimetric capacitances (CCP) from chronopotentiometry technique were obtained in aqueous media 1-M H2SO4. The interfacial or areal capacitance, ICCP, was calculated from CCP and Simm values. The selection of Simm was due to it gave a more realistic value of the sur- face area of the B-doped carbon gels. Most of these values are in fairly good agreement with the interfacial capacitance of a clean graphite surface, 20 μF/cm2, and with the value range between 20 and 30 μF/cm2 reported for different carbons, indicating a good accessibility to the microporosity of the carbon gels. The relationship between ICCP and Simm for B-doped carbon gels showed a good linear agreement (correlation coefficient of 0.927). 3. Differential scanning calorimetry technique in supercapacitors application Electrochemical energy storage devices operate at room temperature and environment condi- tions; therefore, differential scanning calorimetry has used to characterize the thermal decom- position behavior of solid polymer electrolytes and electrodes (metal oxides, polymer and carbon) used in supercapacitors applications. Ghaemi et al. [25] prepared MnO2 materials (γ and layered types) by a novel ultrasonic aided procedure and studied the charge storage mechanism of the prepared samples as a function of the physisorbed water. The water content of manganese dioxide is considered as one of the key factors in the electrochemical performance of MnO2. The hydrous regions in the elec- trode provide the kinetically facile sites needed for the charge-transfer reaction and cation diffusion. To prepare hydrous manganese oxide with different amount of water contents, the samples were thermal treated at 70, 100, and 150°C for 2 h in air. Thermal gravimetric analysis (TGA) and differential scanning calorimetry (DSC) were employed to characterize the water content of the samples. TGA and DSC plots were carried out in air atmosphere with a heating rate of 10°C/min. The DSC analysis showed a wide and steep endothermic peak around 100°C for γ25. The peak was stronger for L25 than γ25 and also shifts toward higher temperature (~125°C) which indicated that the physically adsorbed water is strongly bonded to the porous surface of L25. The heat-treatment temperature decreases the physisorbed water. The cyclic voltammograms (CV) in aqueous 0.5-M Na2SO4 electrolyte within a potential window of 0.0 to +1.0 V versus Ag/AgCl, for both samples, showed almost rectangular and symmetric shape characteristics of a supercapacitor. The specific capacitances values from CV, at a scan rate of 5 mV/s in 0.5 M Na2SO4 at pH 3.3 and 6, were ranged between 100 and 350 F/g. The specific capacitances values decayed gradually through both increasing solution pH and heat-treat- ment temperatures. The pseudocapacitance diminished due to a reduction of the amount of physisorbed water, which is associated with a decline of electrochemical active sites within the electrode. The L25 series showed higher specific capacitances values in comparison with γ25, which could be related to the higher amount of the physisorbed water. Zeng et al. [26] prepared a sheet of Vanadium oxides (V6O13) from NH4VO3 powders to further use it as electrodes of supercapacitors. Vanadium oxides have been widely used as cathode materials for lithium ion battery because of their high-specific capacitance and good cyclabil- ity. 2. Immersion calorimetry of different liquid technique in supercapacitors application The decrease in ICCP with a larger surface area could be explained by the lower EDL capacitance on graphite basal planes versus edges [24]. A rise in the Simm increases the proportion of surface sites on basal planes on the walls Calorimetry - Design, Theory and Applications in Porous Solids 86 of the slit-shaped micropores versus edge sites mainly on the external surface, reducing the interfacial capacitance. ICCP tends to increase with a higher areal oxygen concentration (OXPS), because of the increase in pseudocapacitance effects produced by the surface oxy- gen functionalities, enhancing the total capacitance. 3. Differential scanning calorimetry technique in supercapacitors application V6O13 has a blended valence of V(IV) and V(IV) which is favorable for increasing the electronic conductivity of the material and a promising material in supercapacitors because of its lower cost compared to RuO2. Thermogravimetric and differential scanning calorim- eter (TGA–DSC) were used to study the thermal behavior of NH4VO3 powders. From TGA Calorimetry Characterization of Carbonaceous Materials for Energy Applications: Review http://dx.doi.org/10.5772/intechopen.71310 87 Calorimetry Characterization of Carbonaceous Materials for Energy Applications: Review http://dx.doi.org/10.5772/intechopen.71310 87 curve, there were two weight losses in the ranges of 185–235°C and 290–320°C, which corre- sponding to endothermic peaks showed on the DSC curve. An extra endothermic peak cen- tered at 670°C was observed on DSC curve. The melt temperature for V6O13 was considered at 800°C. Electrochemical properties of the prepared samples were determined by cyclic voltam- metry (CV) and charge–discharge tests in aqueous electrolyte (1 M NaNO3, KNO3, Na2SO4, and LiNO3). The CV curves showed a relatively like rectangular and symmetrical shape thus indicating ideal capacitive property for V6O13. The NaNO3 electrolyte exhibited better specific capacitance, 285 F/g (50 mA/g) and 215 F/g (200 mA/g). Fan et al. [27] developed a novel hierarchical porous carbon membranes using as the source of carbon polyacrylonitrile (PAN), polyvinylpyrrolidone (PVP) as an additive, and N,N-dimethyl- formamide (DMF) as a solvent. The membranes were prepared with the casting solutions by spin coating coupled with a liquid–liquid phase separation technique at room temperature. The morphology and nanostructure of the membranes were tuned by adjusting the addi- tive concentrations in the casting solutions (0–5 wt.%). Later, the membranes were pre-oxi- dized, carbonized, and finally modified with nitric acid. Thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC) of the samples were performed in a nitrogen atmosphere with a heating rate of 10°C/min in the temperature range of 25–900°C. The DSC data showed that there were two broad exothermic peaks in ∼270 and ∼700°C. The signifi- cant weight loss stage below ∼270°C is mainly due to the loss of crystal water and partial dehydrogenation and cross-linking. The weight loss in the temperature range of 270–470°C can be attributed to the decomposition of PVP in the membrane. At temperature exceeding ∼470°C, the weight loss can be assigned to the carbonization of PAN accompanying with further dehydrogenation and partial denitrogenation. 3. Differential scanning calorimetry technique in supercapacitors application The solid polymer PVA–Mix has been used as an electrolyte with RuO2/TiO2 electrodes [29], due to its very good proton conductivity (0.013 S/cm) and stability at environment temperature and relative humidity, forming a solid cell with a thickness of 0.2 mm. At a voltage scan rate of 500 mV/s, the CV profiles were still quite rectangular and showed a capacitance of 50 mF/cm2 in the cell, which suggests that the electrolyte is viable for high rate capacitive devices. The polymer electrolyte not only acted as proton conductor but also facilitated the oxidation and reduction reactions of the electrodes. 84°C. At higher temperatures, there was one endothermic peak for PWA but a split peak for SiWA. In the case of PVA–Mix, two clear endothermic peaks were observed. The water con- tent decreased in the early phase of the temperature scan for all samples. The endothermic peaks could be interpreted as a phase transition or as the escape of certain form of water. The crystallized water in the PVA matrix is more stable than PWA or SiWA, due to the complete release of crystallized protonated water required a higher temperature (122°C for PVA–Mix, respect to 78°C for PVA and 106°C for SiWA). The solid polymer PVA–Mix has been used as an electrolyte with RuO2/TiO2 electrodes [29], due to its very good proton conductivity (0.013 S/cm) and stability at environment temperature and relative humidity, forming a solid cell with a thickness of 0.2 mm. At a voltage scan rate of 500 mV/s, the CV profiles were still quite rectangular and showed a capacitance of 50 mF/cm2 in the cell, which suggests that the electrolyte is viable for high rate capacitive devices. The polymer electrolyte not only acted as proton conductor but also facilitated the oxidation and reduction reactions of the electrodes. Liew et al. [30] investigated the effect of ionic liquid on the PVA-CH3COONH4 polymer electrolytes in supercapacitor application. Ionic liquid-based poly(vinyl alcohol) polymer electrolytes were prepared by means of solution casting. PVA was initially dissolved in dis- tilled water. The weight ratio of PVA:CH3COONH4 was kept at 70:30, and different weight ratio of BmImCl (0–60 wt.%) was thus added into the PVA-CH3COONH4 mixture to prepare ionic liquid-based polymer electrolyte. The increment of BmImCl enhances the ionic con- ductivity, due to strong plasticizing effect of ionic liquid. The glass transition temperature (Tg) of the electrolytes was determined from DSC analysis. 3. Differential scanning calorimetry technique in supercapacitors application The sample prepared with 0.3 wt.% of PVP showed the most reasonable hierarchical pore structure (2–5, 5–50, and >100 nm), high BET surface area (332.9 m2/g), big total pore volume (0.233 m3/g), and the best electrochemical performance in 2-M KOH aqueous solution. The specific capacitance was 278 and 206 F/g at 5 and 50 mA/cm2, respectively, indicating the suitability of the material as electrode materials for supercapacitors. The desirable properties of polymer electrolytes are high ionic conductivity, good tempera- ture, and environmental stability, as well as thin film processability. However, its conductiv- ity is lower than liquid electrolytes and high sensitivity to water are limitation to become viable materials for electrochemical energy conversion and storage devices [28]. Gao and Lian [28] characterized the structural and thermal behavior of solid polymer elec- trolyte using poly(vinyl alcohol) (PVA) and studied the factors contributing to the proton conductivity. Two solid polymer electrolytes were prepared mixing a 15 wt.% PVA solution with a heteropoly acid solution at 32.5 wt.%, one with H4SiW12O40·xH2O (PVA-SWA) and the other one with H3PW12O40·xH2O (PVA-PWA) and 66 wt.% de-ionized water. The PVA-PWA and PVA-SiWA precursors were combined in equal volumes for a mixed polymer electrolyte (PVA–Mix). Differential scanning calorimetry (DSC) analyses were performed with a scan rate of 10°C/min in nitrogen purged cell over a temperature range from 10 to 150°C. The DSC thermograms for PVA–Mix as well as for its individual components (PVA, PWA, and SiWA) showed that the glass transition temperature (Tg) of pure PVA was found around Calorimetry - Design, Theory and Applications in Porous Solids 88 84°C. At higher temperatures, there was one endothermic peak for PWA but a split peak for SiWA. In the case of PVA–Mix, two clear endothermic peaks were observed. The water con- tent decreased in the early phase of the temperature scan for all samples. The endothermic peaks could be interpreted as a phase transition or as the escape of certain form of water. The crystallized water in the PVA matrix is more stable than PWA or SiWA, due to the complete release of crystallized protonated water required a higher temperature (122°C for PVA–Mix, respect to 78°C for PVA and 106°C for SiWA). 3. Differential scanning calorimetry technique in supercapacitors application This study indicated the phase transition of a polymer matrix in the amorphous region, from a hard glassy phase into a flex- ible and soft rubbery characteristic. The Tg decreased further with addition of ionic liquid. This behavior denoted that the plasticizing effect of CH3COONH4 dominates the temporary interactive coordination. This plasticizing effect softens the polymer backbone and thus pro- duces flexible polymer backbone. Polymer electrolyte containing 50 wt.% of BmImCl offered the maximum ionic conductivity of (7.31 ± 0.01) mS/cm at 120°C. The EDLC containing the most conducting polymer electrolyte was assembled and could be charged up to 4.8 V. The specific capacitance of 28.36 F/g was achieved with better electrochemical characteristic in cyclic voltammogram. The higher ion concentration favors the ion migration within the polymer electrolyte (known as separator in EDLC) and promotes the charge accumulation at the electrolyte-electrode boundary. The inclusion of ionic liquid not only improved the interfacial contact between electrode and electrolyte but also increases the electrochemical property of supercapacitors. Yang et al. [31] obtained a promising ionic liquid-gelled polymer electrolyte (GPE) based on semi-crystal polyvinylidene fluoride (PVDF), amorphous polyvinyl acetate (PVAc), and ionic conductive 1-butyl-3-methylimidazolium tetrafluoroborate (BMIMBF4) via solution- casting method. The thermal stability of the GPEs was measured by thermogravimetric/ differential scanning calorimetry (TG/DSC). The PVDF/PVAc/IL (IL, 50 wt.%) GPE film pres- ents good thermal stability (~300°C), wide electrochemical window (>4.0 V), and acceptable ionic conductivity (2.42 × 10−3 S/cm at room temperature) as well. The electrodes were pre- pared from commercial-activated carbon blended with acetylene black and PTFE at the mass Calorimetry Characterization of Carbonaceous Materials for Energy Applications: Review http://dx.doi.org/10.5772/intechopen.71310 89 89 ratio of 85:10:5 wt.%. The solid-state capacitor was assembled with one piece of electrolyte film was placed on one activated carbon electrode surface, and the other symmetrical elec- trode was placed over the gel film to form a “Sandwich Structure”, subsequently sealed into a commercial CR1016 coin cell mold. A 3.0-V C/C solid-state capacitor cell using this GPE film showed a specific capacitance of 93.3 F/g at the current density of 200 mA/g and could retain more than 90% of the initial capacitance after 5000 charge–discharge cycles. Peng et al. [32] prepared gel electrolytes from zwitterionic nature of poly (propylsulfonate dimethylammonium propylmethacrylamide) (PPDP) for solid-state supercapacitors. 3. Differential scanning calorimetry technique in supercapacitors application An ideal gel electrolyte should allow a high ion migration rate, reasonable mechanical strength, and robust water retention ability at the solid state for ensuring excellent work durability. The differ- ential scanning calorimetry (DSC) showed PPDP has high water retention ability. No endother- mic peak could be observed in the thermogram during the heating of PPDP without water and samples with mole ratio of H2O to PDP of 6:1 and 7:1 from −35 to 60°C, suggesting that the polyz- witterion itself does not contribute to the thermal transition behavior. However, an endothermic peak is observed as the mole ratio of H2O to PDP increases to 8:1, which means that the freezable water can be detected in the system when all binding sites of the polyzwitterion are saturated by water molecules. The zwitterionic gel electrolyte were assembled with graphene-based solid- state supercapacitor and reached a volume capacitance of 300.8 F/cm3 at 0.8 A/cm3 with a rate capacity of only 14.9% capacitance loss as the current density increases from 0.8 to 20 A/cm3. Author details Zulamita Zapata Benabithe Address all correspondence to: zulamita.zapata@upb.edu.co Grupo de Energía y Termodinámica, Facultad de Ingeniería Química, Escuela de Ingeniería, Universidad Pontificia Bolivariana, Antioquia, Colombia References [1] Enerdata, Global Energy Statistical Yearbook 2017, France, 2011. https://yearbook.enerd- ata.net/renewables/renewable-in-electricity-production-share.html [1] Enerdata, Global Energy Statistical Yearbook 2017, France, 2011. https://yearbook.enerd- ata.net/renewables/renewable-in-electricity-production-share.html [2] Luo X, Wang J, Dooner M, Clarke J. Overview of current development in electrical energy storage technologies and the application potential in power system operation. Applied Energy. 2015;137:511-536. DOI: 10.1016/j.apenergy.2014.09.081 [3] Kötz R, Hahn M, Gallay R. Temperature behavior and impedance fundamentals of superca- pacitors. Journal of Power Sources. 2006;154:550-555. 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https://openalex.org/W2794072624	https://www.e3s-conferences.org/articles/e3sconf/pdf/2018/08/e3sconf_hrc2018_02071.pdf	English	null	On certain development aspects of an ipsas-based system-target approach to evaluation of net asset sustainability level projects in high-rise construction	E3S web of conferences	2,018	cc-by	8,041	https://doi.org/10.1051/e3sconf/20183302071 https://doi.org/10.1051/e3sconf/20183302071 E3S Web of Conferences 33, 02071 (2018) HRC 2017 © The Authors, published by EDP Sciences. This is an open access article distributed under the terms of the Creative Commons Attribution License 4.0 (http://creativecommons.org/licenses/by/4.0/). On certain development aspects of an ipsas- based system-target approach to evaluation of net asset sustainability level projects in high-rise construction Ruben Kazaryan1,* 1Moscow State University of Civil Engineering, 26 Yaroslavskoe shosse, Moscow, 129337, Russia Ruben Kazaryan1,* 1Moscow State University of Civil Engineering, 26 Yaroslavskoe shosse, Moscow, 129337, Russia Abstract. Problems of accounting and reporting of net assets and the procedure of their formation taking into account the specifics of the economic and legal status of property of a non-commercial autonomous institution are some of the most controversial in the accounting for entities of the public sector. The study focuses on justification of accounting rules for net assets of public sector entities. The methods used in the study are as follows: comparison, synthesis, analysis, logical approach, and system approach. The article examines legal aspects and specifics of recognition of assets of public sector entities in accordance with IPSAS standards (International Public Sector Accounting Standards are a set of accounting standards issued by IPSASB (Council for International Financial Reporting Standards for Public Sector Organizations) used by state-owned enterprises worldwide in preparation of financial statements as of the 31st of August, 2015. The most crucial factor in the modeling of key performance indicators of the system-target approach to estimation of the sustainability level of net assets on the basis of IPSAS is a multicriterial evaluation of the basic management strategy for quality system elements used in operational and strategic planning projects operations in high-rise construction. We offer an alternative evaluation of assets due to be returned to the right holder (the state controller) in the event of liquidation of a public sector entity. Corresponding author: r.kazarian@mail.ru 1 Introduction The concept of the public sector efficiency, forming the basis of public administration reforms in this area, faces a number of outstanding methodological and organizational problems. In particular, such problems include the absence of a generally accepted notion of public sector’s economic efficiency in scientific literature and practice; lack of established economic efficiency criteria and indicators. Methodological approaches and principles of the economic efficiency determination in public and commercial sectors must be uniform. We believe that this approach is untenable in view of fundamental differences of the principles and objectives of the non-profit sector, and its deep-rooted economic and social distinctions, and requires some additional research in the field of financial accounting and reporting. Corresponding author: r.kazarian@mail.ru https://doi.org/10.1051/e3sconf/20183302071 E3S Web of Conferences 33, 02071 (2018) HRC 2017 Acritical indicator of economic efficiency is “net assets”. The key purposes of assessment of a non-profit organization should include not only the achievement of maximum social impact, but also the optimization of budget resources and economic performance. In this regard, special significance is acquired by the “net assets” category, which allows, on the one hand, a review of the investment process in the public sector, and, on the other hand, an estimation of increase in the total value of a non-profit enterprise. This indicator can also be used traditionally as a measure of growth of retained earnings from chargeable operations of a non-profit organization. In the conditions of risk and uncertainty, there is a need for continuous management of economic processes in the public sector. Changes of the last decades have shifted the focus financial and managerial accounting from cost management and financial flows onto economic processes’ management (financial standing, risks, backup enterprise system, reorganization processes, value added control ), based on the use of accounting engineering tools (monitoring, financial, hedging, or other derivative reports). The net assets indicator, in conjunction with net liabilities, represents one of the most important measures of assessing economic processes, efficiency, and sustainable development of a nonprofit enterprise. However, as concerns businesses that are not focused on profit-making or satisfaction of public demand for services as the outcome of investment, this indicator is difficult to determine. Therefore, a lot of proven models and methods of accounting for net assets cannot be directly applied in this area. 1 Introduction This situation leads to a mismatch between the urgent need for scientific methodology in the field of information and analytical support of management and evaluation of the efficiency by using the net assets indicator. A significant enhancement of the financial information quality and the formation of adequate information support for control and management processes to a wider audience, improving the decision-making process regarding the allocation of resources, ensuring greater transparency and accountability of decision-makers (Bellanca&Vandernoot, 2014). Application of these standards allows an improved control and supervision of the budget as well as development of communication tools for promoting dialogue and synchronizing the work of state institutions of different countries. (European Commission, 2012b; European Commission, 2012a). IPSAS have actually become international benchmarks for evaluation of accounting practices in the public sector throughout the world. For these reasons, IPSAS deserves attention of accounting policy makers, as well as practitioners and researchers (KPMG, 2013). Today, the development of national financial accounting and reporting standards is based exactly on this dynamic group of international standards applied worldwide [1-5]. This gives rise to a problem associated with the need to develop new national and international accounting models and tools for net assets in the non-profit sector, and to adapt the existing ones, as well as to develop effective methodologies based thereon. E3S Web of Conferences 33, 02071 (2018) HRC 2017 E3S Web of Conferences 33, 02071 (2018) HRC 2017 https://doi.org/10.1051/e3sconf/20183302071 The synthesis method is used to categorize positive and negative consequences of the IPSAS introduction as the basis of Russian accounting standards in the public sector. The analysis is based on expert examination of publications and documentary materials of the main regulatory authorities (reports, regulations, conference proceedings and discussions). 3.1 The influence of theoretical and legal features of operations of public sector entities on interpretation of the “Net assets” category Public sector entities are characterized by features that pre-determine the peculiar characteristics of the composition and disclosure of information about the accounting items. As defined in IFRS (International Financial Reporting Standards) 22 “Disclosure of Financial Information about the General Government Sector”, a non-profit organization is a legal entity or an organization of other forms, which that are created with the purpose of production or distribution of goods and services, but which do not generate financial benefits for regulatory bodies [2-3]. Thus, their distinctive feature is also the complete absence, or limitation, of the right of ownership to property and other resources administered by a non-profit institution, which are publicly owned and controlled by the state authorities. Distinctive features of non-profit entities include: Distinctive features of non profit entities include: - lack of generation of economic benefits as the main goal of activities. - lack of generation of economic benefits as the main goal of activities. - Predominance of a non-market way of organizing activities. - Production, distribution, and consumption of public goods. - Lack/restriction of the ownership right to property and other resources administered b non-profit institution, which are publicly owned and controlled by the state authorities. - Ensuring an economic equilibrium between demand and supply of public goods throu state mechanisms (social institutions, infrastructure, and resources). - The ability, within acceptable limits, to carry out activities aimed at deriving additional economic benefit while maintaining the objective functions of the public goods distribution and achievement of goals of the state in meeting social needs. - The ability, within acceptable limits, to carry out activities aimed at deriving additional economic benefit while maintaining the objective functions of the public goods distribution and achievement of goals of the state in meeting social needs. Table 1 summarizes key distinctive features of non-profit institutions and determines the influence on recognition of and accounting for net assets/equity of such institutions. Table 1 summarizes key distinctive features of non-profit institutions and determines the influence on recognition of and accounting for net assets/equity of such institutions. The particular features of interpretation of net assets of a non-profit institution shown in the table are approached differently in the methodology of national and international financial reporting standards. 2 Methods The study uses logical system, synthesis, analysis, and analogy methods, as well as the comparative analysis method. The purpose of analysis is to identify differences between IPSAS and the public sector standards published by the Ministry of Finance of Russia. The purpose also includes a system review of draft standards and draft resolutions on the basis of published information and opinions, surveys of the Ministry of Finance and the parties concerned (public sector executives, accounting and state control personnel). The surveys have been conducted in the form of a questionnaire. Of special interest are the respondents’ views on the expedience of the use of IPSAS in the Russian Federation. We appreciate the loyalty of respondents to the prospects of using IPSAS as the IPSAS basis. We also research in the possibility of the accounting tools’ synthesis based on IPSAS and of organization of state and public supervision, including that based on the “Electronic Budget” technology. 2 3.1 The influence of theoretical and legal features of operations of public sector entities on interpretation of the “Net assets” category Description Distinctive features of recognition and accounting in Capital/Net Assets categories Lack of generation of economic benefits as the main goal of activities Eliminates the possibility of using the term “Capital” for financial evaluation of investment into the operational and financial cycle for the purpose of deriving economic benefits Predominance of a non-market way of organizing activities,ensuring an economic equilibrium between demand and supply of public goods through state mechanisms (social institutions, infrastructure, and resources) There is no traditional profit-making and accumulation mechanism characteristic of commercial organizations, with no accumulation of net assets over time Lack/restriction of the ownership right to property and other resources, administered by a non-profit institution, which are publicly owned and controlled by the state power authorities Eliminates the recognition of assets and capital in terms of control over economic benefits/ opportunities of beneficial use. The ability to, within acceptable limits, carry out activities aimed at deriving additional economic benefit while maintaining the objective functions of the public goods distribution and achievement of goals of the state in meeting social needs Creates a traditional business model of the resources’ flow, introduces the concepts of “capital”, “revenue”, “profit”, thus determining a mixed character of the net assets category (containing both a non-commercial component, a financial assessment of the resource transferred by the control entity (the state), and own assets recognized in terms of control of future economic benefits and derived profit) There is no traditional profit-making and accumulation mechanism characteristic of commercial organizations, with no accumulation of net assets over time Eliminates the recognition of assets and capital in terms of control over economic benefits/ opportunities of beneficial use. Creates a traditional business model of the resources’ flow, introduces the concepts of “capital”, “revenue”, “profit”, thus determining a mixed character of the net assets category (containing both a non-commercial component, a financial assessment of the resource transferred by the control entity (the state), and own assets recognized in terms of control of future economic benefits and derived profit) The ability to, within acceptable limits, carry out activities aimed at deriving additional economic benefit while maintaining the objective functions of the public goods distribution and achievement of goals of the state in meeting social needs Pursuant to paragraph 17 of IFRS OS 1 “Presentation of financial statements”, statement of financial position should as a minimum disclose the value of net assets/equity. 3.1 The influence of theoretical and legal features of operations of public sector entities on interpretation of the “Net assets” category Application of international standards and accounting principles is based on the following groups of documents: The particular features of interpretation of net assets of a non-profit institution shown in the table are approached differently in the methodology of national and international financial reporting standards. Application of international standards and accounting principles is based on the following groups of documents: - For commercial organizations – International Accounting Standards (IAS) and International Financial Reporting Standards (IFRS) developed by the Council for IASB (IASB). - For commercial organizations – International Accounting Standards (IAS) and International Financial Reporting Standards (IFRS) developed by the Council for IASB (IASB). - For public sector – International Public Sector Accounting Standards (IPSAS) developed by the Council for IASB OS (IPSASB) under the International Federation of Accountants (IFAC). Definition of the targets and application of both groups of standards is based on the principle of prevalence of economic substance over legal form. This allows qualification of a commercial entity for the purpose of applying the IFRS OS solely on the basis of the objectives of the enterprise and its functions to be implemented in the economic and social environment, regardless of the legal form of ownership and the structure of investment by the state. Therefore, recognition of assets and funding sources in the paradigm of IFRS is based solely on the criteria of their use for achievement of the main goals of the enterprise [2-5] 3 https://doi.org/10.1051/e3sconf/20183302071 E3S Web of Conferences 33, 02071 (2018) HRC 2017 Table 1. Influence of distinctive features of non-profit institutions on recognition of and accounting for net assets. 3.1 The influence of theoretical and legal features of operations of public sector entities on interpretation of the “Net assets” category Pursuant to paragraph 95 of IFRS OS, if an organization does not have any share capital, it should disclose its net assets/ equity either in the financial statements or in the Notes, with breakdown for: - Contributed capital meaning the aggregate amount of contributions by owners less payments to owners as of the reporting date, - Contributed capital meaning the aggregate amount of contributions by owners less payments to owners as of the reporting date, - Accumulated surplus or deficit. - Reserves with description of the nature and purpose of each reserve in the assets/ equity. Minority interest - Reserves with description of the nature and purpose of each reserve in the assets/ equity. Mi i i eserves with description of the nature and purpose of each reserve in the assets/ equity. Minority interest. - Reserves with description of the nature and purpose of each reserve in the assets/ equity - Minority interest. - Minority interest. Review of the net assets of a non-profit institution should take into account particular features of functioning of these enterprises. For instance, net assets of a non-profit enterprise should be understood as a difference between its assets and liabilities. However, this category is not homogeneous from the point of view of its economic and legal content. The proposed composition of the items does not reflect all the specifics of a non-profit organization’s activity; thus, this grouping as implemented within the concept of international accounting standards does not allow for a full analysis or presentation of reliable and relevant information about net assets to the users. 3.2 The problem of recognizing the property and assets of public entities controlled by the state. “Absence/restriction of the right of ownership to the property and other resources administered by a non-profit institution that are publicly owned property belonging to state 4 4 E3S Web of Conferences 33, 02071 (2018) HRC 2017 https://doi.org/10.1051/e3sconf/20183302071 authorities”, identified earlier deserves special attention. In connection with this restriction, there is a problem of IFRS OS application for recognition of the property of a public entity as assets and for reflection thereof within its net assets and sources. There is a problem of recognition of such property within assets, its share in the value of net assets of the company, as well as the order of recognition of settlements with the founders as sources of funding as the organization’s capital or other type of liability. Legal and scientific literature offers different approaches to solving this problem. However, in the light of the IFRS OS positions and reforms of accounting on the basis laid down in the specified standards, these issues remain unsolved and are of a disputable nature. authorities”, identified earlier deserves special attention. In connection with this restriction, there is a problem of IFRS OS application for recognition of the property of a public entity as assets and for reflection thereof within its net assets and sources. There is a problem of recognition of such property within assets, its share in the value of net assets of the company, as well as the order of recognition of settlements with the founders as sources of funding as the organization’s capital or other type of liability. Legal and scientific literature offers different approaches to solving this problem. However, in the light of the IFRS OS positions and reforms of accounting on the basis laid down in the specified standards, these issues remain unsolved and are of a disputable nature. 3.3 Categories of the assets of a public entity in terms of “Control of assets by the state” - Accounting for the aforesaid types of property in the assessment of the net assets of a non- profit autonomous institution from the point of view of serving the different groups of the users’ interests (paying capacity, liquidity, investment attractiveness, sustainable development, etc.). Table 2.Categories of the assets of a public entity in terms of “Control of assets by the state”. Type of asset Subtype of asset Conditions of the asset’s return to the state controller The property of the founder as the holder of the right of ownership and other similar types of property The right vested in the public entity Acquired at the expense of the funds allocated by the subject as the holder of the right of property Demand of return to the founder or another owner in case of liquidation/ termination Most valuable property as reflected by separate accounting. Can be withdrawn as redundant or used other than as intended. The property created and controlled by a non-profit organization at the expense of its own and third-party private sources, which may not be foreclosed upon liquidation of the legal entity, etc. Other state-owned property that may be administered independently May foreclosed upon liquidation of the legal entity. The property acquired at the expense of income-generating activities, the property received gratuitously from the third parties and other similar types of property The property created and controlled by a non-profit organization at the expense of its own and third-party private sources, which may not be foreclosed upon liquidation of the legal entity, etc. The following problems of accounting for and assessing the value of net assets of a non- profit organization are of special urgency: - The conditions and criteria for recognizing the aforesaid property as assets on the IFRS OS basis, including the concepts of control, risks, and benefits. - The conditions and criteria for recognizing settlements with the owners and property controllers, as well as proceeds thereof within the liability side of the balance-sheet (Capital, Liability). - The conditions and criteria for recognizing settlements with the owners and property controllers, as well as proceeds thereof within the liability side of the balance-sheet (Capital, Liability). 3.3 Categories of the assets of a public entity in terms of “Control of assets by the state” We believe that property and its sources should be categorized as follows: We believe that property and its sources should be categorized as follows: - The property of the founder as the holder of the right of ownership and other types of property, which may not be foreclosed upon liquidation of the legal entity. - The property of the founder as the holder of the right of ownership and other types of property, which may not be foreclosed upon liquidation of the legal entity. - The property created and controlled by the non-profit organization at the expense of its own and third-party private sources, which may be foreclosed upon liquidation of the legal entity. We propose the following categories of property for subsequent recognition within the assets (a share in the net assets) (Table 2). Table 2.Categories of the assets of a public entity in terms of “Control of assets by the state”. Type of asset Subtype of asset Conditions of the asset’s return to the state controller The property of the founder as the holder of the right of ownership and other similar types of property The right vested in the public entity Acquired at the expense of the funds allocated by the subject as the holder of the right of property Demand of return to the founder or another owner in case of liquidation/ termination Most valuable property as reflected by separate accounting. Can be withdrawn as redundant or used other than as intended. The property created and controlled by a non-profit organization at the expense of its own and third-party private sources, which may not be foreclosed upon liquidation of the legal entity, etc. Other state-owned property that may be administered independently May foreclosed upon liquidation of the legal entity. The property acquired at the expense of income-generating activities, the property received gratuitously from the third parties and other similar types of property The following problems of accounting for and assessing the value of net assets of a non- profit organization are of special urgency: - The conditions and criteria for recognizing the aforesaid property as assets on the IFRS OS basis, including the concepts of control, risks, and benefits. - The conditions and criteria for recognizing settlements with the owners and property controllers, as well as proceeds thereof within the liability side of the balance-sheet (Capital, Liability). 3.4 The problems of recognizing the value of assets to be returned to the right holder (state controller) upon liquidation of a public sector entity. Evaluation of net assets There is an out standing problem of the conditions and criteria of recognition of settlements with owners and property control entities in the liability side of the balance-sheet (Capital, Liability). Delineation between the funding sources in the liability side of the statement of financial position and their qualification as capital or revenue is contained in IFRS OS 23 “Revenue from non-exchange transactions”. It provides for distinction between contributions of the owner to the institution’s net assets and valuables received in non-exchange transactions (i.e. for no equal valuable consideration) [3,4]. The owner’s contribution is qualified according to IFRS OS 1 and means future economic benefits (including the possibility of beneficial use) from third parties giving no rise to liabilities for the organization and forming a share in the organization’s net assets /capital which: - Gives the right to payments allocable by the organization, both as 1) the future economic benefits or a possibility of beneficial use for the period of its existence at the discretion of the owners or their representatives, and 2) any excess of assets over liabilities in the event of the organization’s liquidation. - Gives the right to payments allocable by the organization, both as 1) the future economic benefits or a possibility of beneficial use for the period of its existence at the discretion of the owners or their representatives, and 2) any excess of assets over liabilities in the event of the organization’s liquidation. g q May be sold, exchanged, transferred, or redeemed. - May be sold, exchanged, transferred, or redeemed. - May be sold, exchanged, transferred, or redeemed. Considering the correspondence of the highlighted assets in the composition of the organization with the specified definitions, we should put an increased focus on the property assigned to the institution and purchased at the expense of the funds that have been allocated by the subject who is the holder of an ownership right [4,5]. - May be sold, exchanged, transferred, or redeemed. Considering the correspondence of the highlighted assets in the composition of the organization with the specified definitions, we should put an increased focus on the property assigned to the institution and purchased at the expense of the funds that have been allocated by the subject who is the holder of an ownership right [4,5]. 3.3 Categories of the assets of a public entity in terms of “Control of assets by the state” - Accounting for the aforesaid types of property in the assessment of the net assets of a non- profit autonomous institution from the point of view of serving the different groups of the users’ interests (paying capacity, liquidity, investment attractiveness, sustainable development, etc.). 5 https://doi.org/10.1051/e3sconf/20183302071 E3S Web of Conferences 33, 02071 (2018) HRC 2017 - The criteria for recognizing the revenue and the accrued financial result. According to IPSAS, a public sector entity has control of its assets, if it can use them for receiving future economic benefits or useful potential in the achievement of its goals and can exclude or otherwise regulate the access of other entities to such economic benefits or the useful potential [1-3]. The useful potential contained in the assets is their ability, independently or jointly with other assets, to be used for providing the state (municipal) services (paid services, works, manufacture of products) and/or performance of the state (municipal) and other functions in compliance with the entity’s purposes, without generating any cash flow (cash equivalents) for the entity [1-3]. In other words, the benefits generated by the asset cannon be evaluated in cash terms, but do exist and have a social or another other useful potential for the society, a feature quite typical for non-profit organizations and their non-market functioning principles. Pursuant to para 7 (IPSAS) 23 “Income from non-exchangeable operations (taxes and transfers)”, control of an asset arises when the organization can use the asset or otherwise derive benefit therefrom, pursuing its goals, as well as close or otherwise regulate the access to this benefit for other parties. The said criterion of the entity’s control over its economic benefits is strictly observed, and all property mentioned in Table 2 can be recognized as assets of a non-profit organization in accordance with IPSAS. In this regard, the right of ownership to the transferred property should be neglected in compliance with the principle of priority of the economic content before the legal form. The property is fully reflected in the assets of the balance sheet / statement of financial position. 3.4 The problems of recognizing the value of assets to be returned to the right holder (state controller) upon liquidation of a public sector entity. Evaluation of net assets For example, when creating a new organization, the budgetary authority at the Department of Finance may perceive the initial transfer of resources to the organization as a share in form of the net assets/ capital, but not as providing funds to meet current needs, • By a formal agreement in respect of the resources’ transfer, creating a new share or increasing the existing one in the net assets/ capital of the organization that may be sold, transferred, or redeemed. In the scientific literature, there is the authors’ position that the settlements with the founding shareholders represent in their initial assessment a current long-term debt of the company, which is based on the approach that the institution must return the received assets during liquidation. As criteria of recognition of the current liability, the authors cite such arguments as occurrence of the event in the past (transfer of property) and the eventual outflow of future economic benefits (in the event of liquidation of the organization). We believe that each approach has its advantages and disadvantages, but does not fully solve the problem of preparation of information on investments of the owner in a part of the secured property and the property created in the process of the enterprise’s functioning, as well as recognition in the financial statements. The net assets of a non-profit institution are formed as an increase of economic benefits arising in the operation process, of the result of changes in the value of assets. However, disputable remains the question of recognition of value of the investments of the owner directly in the composition of net assets, which may be seized without the consent of the institution at the time of liquidation of the organization or other cases stipulated by the law. 3.4 The problems of recognizing the value of assets to be returned to the right holder (state controller) upon liquidation of a public sector entity. Evaluation of net assets There arises a contradiction between the economic and legal substance of this indicator and the definition of the contribution of the owner, forming the capital/net assets of the institution. Since the requirement of the ability to distribute any excess of the assets over liabilities in the event of liquidation of the organization is not implemented to the full extent. Analysis of IPSAS 1 and IPSAS 23 shows that IPSAS does not contain any detailed 6 6 E3S Web of Conferences 33, 02071 (2018) HRC 2017 https://doi.org/10.1051/e3sconf/20183302071 instructions on how to reflect the specificity of the settlements accounting with state property owners. The definition of “the owner’s contribution” is contrary to the economic and legal content of the category of settlements with the founders of a non-profit institution, as it does not take into account the need for the return of property in case of liquidation. According to IPSAS, the paid-up capital in the public sector can be confirmed by the transfer of resources between the parties[3,4,5].The issue of equities during the transfer of resources is not a necessary condition for matching the transfer to the definition of payables from owners. The transfer of resources which rewards a share in the net assets/ capital of an organization is distinguished from the transfer of other resources if it can be confirmed by the following: • By an official confirmation of the contractual parties on the transfer of resources (or on a type of such transfer) as an integral part of the net assets/ capital, either before or during the implementation of the contribution. For example, when creating a new organization, the budgetary authority at the Department of Finance may perceive the initial transfer of resources to the organization as a share in form of the net assets/ capital, but not as providing funds to meet current needs, • By an official confirmation of the contractual parties on the transfer of resources (or on a type of such transfer) as an integral part of the net assets/ capital, either before or during the implementation of the contribution. 3.5 Estimation of the net assets on the basis of value adjustment of the assets subject to return to the right holder (state supervisor) during the liquidation of the public sector entity The composition and value of the net assets represents significant information for the users of external reporting of a non-profit organization. The non-profit organization submits public accountability paperwork based on the IPSAS principles, primarily with the goal of providing users with information about the financial standing both to ensure control and to attract alternative sources of funding. Quite relevant for such institutions there seems to be the investment attractiveness, in connection with additional features of such organizations to provide services and perform projects and works for third parties on a commercial basis. The amount of net assets under this approach characterizes both the financial security, development stability, and the investment component of non-profit institution activities (both from the part of the owner of state property, and directly by the non-profit institution itself). At the same time, the net assets of the company traditionally serve as a tool for assessing solvency and resource endowment from the point of view of their possible distribution among the creditors in case of liquidation of the enterprise, as well as in its current activity. In this 7 E3S Web of Conferences 33, 02071 (2018) HRC 2017 https://doi.org/10.1051/e3sconf/20183302071 case, the principle of conservatism requires a precautionary approach to the recognition of formation sources of assets directly in the capital funds, as well as the inclusion of the assets within the net assets taking into account their feasibility and use to settle claims of the contractors. Despite the fact that the resources transfers can be executed by means of confirmation or official agreement, the organization determines the nature of such transfers on the basis of their economic substance and not merely their legal form[6,7,8]. To achieve the aforesaid purposes of providing the financial statements’ users with the relevant and reliable information, the current IPSAS approach described above is not able to provide any universal method for the formation of the net assets of a nonprofit organization. 4.1 The methodology for the recognition and qualification of assets subject to return to the right holder (the state supervisor) during the liquidation of the public sector entity Assets subject to return to the right holder (the state supervisor) during the liquidation of the public sector entity may be perceived as the initial transfer of resources to the organization as a share in net assets/ capital, and not as provision of funds to meet current needs. However, based on the definition of the owner’s contribution into IFRS OS 1 “Submission of financial statements”, there is no fulfillment of the requirement on possibility of distribution of any excessive assets over liabilities in the event of liquidation of the organization. As already noted, this property may be withdrawn and will not increase the assets in case of liquidation of the organization. We believe that classifying such investments of founders as “the owner’s contribution” in the statement of financial standing is not appropriate, and does not meet the requirements of IFRS OS. The property to be transferred can be fully depleted in the course of the institution activity throughout the useful economic life and physical ability to participate in the operational and financial cycle of the organization. Loss, damage, the asset’s loss of its physical and moral characteristics due to the use does not entail the obligation to repay to the owner the original value of the property or to represent property, similar in its characteristics. The return of the asset to the right holder (the state supervisor) during the liquidation of the public sector entity involves the return of a physical object, not a money equivalent. Thus, no requirements arise in respect of the compulsory equivalent value of the founder’s investments, subject to returned at the time of liquidation and equal to the value of the initial gross proceeds. The transfer of such asset is the symptom of a non-exchange transaction [6-10].Therefore: - The property is settled on a non-profit organization for the implementation of its statutory activities (extraction of economic benefits or opportunities for beneficial use) with the priority objective to fully utilize throughout the useful economic life/ tangible existence. - The property shall be returned in the event of liquidation of the organization as a physical object (right) on the date of liquidation and not as a money equivalent. - The property shall be returned in the event of liquidation of the organization as a physical object (right) on the date of liquidation and not as a money equivalent. 4.2 Alternative evaluation of the assets subject to return to the right holder (the state supervisor) during the liquidation of the public sector entity Based on the above analysis, we can conclude that the asset to be returned to the right holder (the state supervisor) during the liquidation of the public sector entity corresponds to the two sources of formation. The property on the date of investments is the gross inflow of economic benefits in their fair assessment and reflects the investment of the state property owner that implements its objectives through a non-profit institution. At the same time, it is impossible to ignore the fact of the pre-emptive right for the withdrawal of such property during the liquidation of the organization in physical terms and as of the date of the institution’s liquidation. The pre-emptive right of liquidation, of course, creates a present obligation for the enterprise, since the following requirements are met at the same time: - The property shall be returned in the event of liquidation of the organization as a physical object (right) on the date of liquidation and not as a money equivalent. - The property shall be returned in the event of liquidation of the organization as a physical object (right) on the date of liquidation and not as a money equivalent. - The property shall be returned in the same condition and with the same physical and moral characteristics, which it has had on the date of liquidation of the organization. - The property shall be returned in the same condition and with the same physical and moral characteristics, which it has had on the date of liquidation of the organization. q g However, as noted above, the obligation arises only in relation to a physical object in the volume and condition of wear on the liquidation date. The amounts of accumulated depreciation and residual value of this object does not, in our opinion, reflect the true assessment of the property. As depreciation, although they may be correlated with a tendency of loss of physical and moral characteristics by the object in some cases, as a whole, are predominantly a tool to compare the income and expenses of the organization and the resulting residual value cannot give a reliable valuation of the asset at the current date [9-13]. E3S Web of Conferences 33, 02071 (2018) HRC 2017 The requirement of IPSAS to reflect the value of such property at fair value, i.e. the amount by which it is possible to exchange an asset or settle a liability, when a transaction is being made between the well-informed parties, wishing to make such a transaction, and independent from each other. In our opinion, it is necessary to use the rating recommended by IPSAS, as it most accurately reflects the value of the property as contributions of the founder to the organization, provides the required control of evaluation objectivity and allows a more reliable estimation of net asset amount[8-12]. The fair value is, in fact, is an alternative rating of the governmental investments to creating an institution, as a means of implementing the functions of distribution and redistribution of public goods, which is the “effect” of such investments. Thus, when evaluating gross revenue in the part of the fixed assets, we should, as far as possible, be guided by the approach of market evaluation. 4.1 The methodology for the recognition and qualification of assets subject to return to the right holder (the state supervisor) during the liquidation of the public sector entity - The property shall be returned in the same condition and with the same physical and moral characteristics, which it has had on the date of liquidation of the organization. - The property shall be returned in the same condition and with the same physical and moral characteristics, which it has had on the date of liquidation of the organization. That is, there are two features of the property return in liquidation: - The property will be returned not in full (only that property, which exists physically on the date of the liquidation). - The property will be returned not in full (only that property, which exists physically on the date of the liquidation). - The property returns in terms of moral and physical wear and tear, damage, and partial loss of properties. - The property returns in terms of moral and physical wear and tear, damage, and partial loss of properties. - The property returns after an indeterminate period of time, which affects its cost measurements. 8 E3S Web of Conferences 33, 02071 (2018) HRC 2017 https://doi.org/10.1051/e3sconf/20183302071 4.2 Alternative evaluation of the assets subject to return to the right holder (the state supervisor) during the liquidation of the public sector entity g g In its economic content, the property object assigned to the institution represents potential investments of the owner into alternative projects at the time of return (it can be transferred to public auctions and privatization, in front of the other institution, used in private activities, etc.). From this point of view, the cost of the property return is a recoverable amount. The most accurate assessment is, in our opinion, the liquidation value of the asset. According to article 13 IFRS OS 17 “fixed assets”, liquidation value is the estimated amount that the organization would receive currently from the disposal of the asset after deducting the expected costs of disposal, as if the asset was already of age and in the condition expected at the end of the beneficial use [10-14]. Thus, the author of the dissertation research suggests recognizing the obligation of the pre-emptive right for the recovery of property assigned to a public non-profit institution from the part of the owner in the amount of the asset’s liquidation value, and to exclude the specified amount from the composition of the net assets of the organization. The scheme of the liquidation value calculation is as follows: - Corrected (revaluated) cost of the asset. - Corrected (revaluated) cost of the asset. 9 9 https://doi.org/10.1051/e3sconf/20183302071 E3S Web of Conferences 33, 02071 (2018) HRC 2017 - Sum of current costs associated with the liquidation (the costs of maintaining assets prior to their sale, management costs, etc.). - Sum of current costs associated with the liquidation (the costs of maintaining assets prior to their sale, management costs, etc.). - The value of all obligations associated with the liquidation. At the same time, on the liabilities side of the statement of financial standing there must be shown the gross inflows of economic benefits at the fair value of the property assigned to a state non-profit institution from the part of the owner that will result in an increase of the net assets. In our opinion, when calculating the net assets from the gross revenue of economic benefits in fair evaluation on the date of receipt, we must exclude the amount of the liabilities, calculated based on the liquidation value of the assets subject to exemption on the date of liquidation. 4.2 Alternative evaluation of the assets subject to return to the right holder (the state supervisor) during the liquidation of the public sector entity Thus, the cost excess of the gross income from the founder over the amount of the specified liabilities will amount to the proportion of the net assets that are “owned” by the institution and implemented in the process of activity throughout its existence. Also, it is proposed to eliminate the excess of liabilities amount over the initial carrying value of assets in terms of property assigned to the institution. In this case, when evaluating the liability, the author of the dissertation research recommends to use the lower of the two ratings: - The liquidation value of the property intended to be seized. - The liquidation value of the property intended to be seized. q p p y - The carrying value of the property intended for seizure, recognized at the time of investment by the owner. - The carrying value of the property intended for seizure, recognized at the time of investment by the owner. This is due to the principle of interaction between the property owner and the public non- profit institution, according to which the investments sent by the state property owner do not have either the purpose of extracting economic benefits from the use of the transferred assets, or otherwise gaining additional economic benefits. Thus, the owner may not recover economic benefits more than he has invested during the formation and functioning of the non-profit institution in accordance with the principle of non-market interaction [12-20]. 5 Conclusions According to the recommended method, at the time of formation or subsequent investment by the founder it is reflected as the gross inflows of economic benefits/opportunities for beneficial use directly in an increase of the net assets of the organization (see the Section “Net assets/Capital” in accordance with IFRS OS 1 or the Section “Other liabilities” in accordance with Federal Accounting Standards in the public administration sector “Presentation of financial statements”). On the subsequent reporting date of the financial statements, it is recommended to reclassify the above economic category within the liabilities, thus ensuring the exclusion of the property value subject to seizure from the net asset This will ensure adequate assessment and information disclosure: - On the obligations in front of the founder for the property, on which there is a preferential right of withdrawal upon liquidation in the most reliable (liquidation) assessment, reflecting the actual characteristics of the property on the date of withdrawal. - On the share of the gross income from the founder forming the net assets of an institution and “functioning” in the organization. 2. S. Bellanca, &J. Vandernoot, Journal of Modern Accounting and Auditing International Public Sector Accounting Standards (IPSAS) Implementation in the European Union (EU) Member States, 3, 257-269 (2014) 1. M. Bisogno, S. Santis, A. Tommasetti, Int. 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https://openalex.org/W4362579282	https://www.researchsquare.com/article/rs-2732954/latest.pdf	English	null	Application of an improved wide-narrow-band hybrid ANC algorithm in large commercial vehicle cab	Research Square (Research Square)	2,023	cc-by	15,217	Application of an improved wide-narrow-band hybrid ANC algorithm in large commercial vehicle cab Jintao Su Hubei University of Arts and Science jinquan nie Hubei University of Arts and Science Shuming Chen Jilin University Version of Record: A version of this preprint was published at Scienti¦c Reports on May 7th, 2024. See the published version at https://doi.org/10.1038/s41598-024-60979-7. Article License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Additional Declarations: No competing interests reported. Version of Record: A version of this preprint was published at Scienti¦c Reports on May 7th, 2024. See the published version at https://doi.org/10.1038/s41598-024-60979-7. Jintao Su1,2,3* , Jinquan Nie1,2 , Shuming Chen3 Jintao Su1,2,3* , Jinquan Nie1,2 , Shuming Chen3 1 Hubei Key Laboratory of Power System Design and Test for Electrical Vehicle，Hubei University of Arts and Science, Xiangyang 441053 2 School of Automotive and Traffic Engineering,Hubei University of Arts and Science, Xiangyang 441053 3 Jilin University, Changchun, 130022 * Correspondence: nvh2012@163.com Jintao Su1,2,3* , Jinquan Nie1,2 , Shuming Chen3 Jintao Su1,2,3* , Jinquan Nie1,2 , Shuming Chen3 1 Hubei Key Laboratory of Power System Design and Test for Electrical Vehicle，Hubei University of Arts and Science, Xiangyang 441053 2 School of Automotive and Traffic Engineering,Hubei University of Arts and Science, Xiangyang 441053 3 Jilin University, Changchun, 130022 * Correspondence: nvh2012@163.com 1 Hubei Key Laboratory of Power System Design and Test for Electrical Vehicle，Hubei University of Arts and Science, Xiangyang 441053 2 School of Automotive and Traffic Engineering,Hubei University of Arts and Science, Xiangyang 441053 3 Jilin University, Changchun, 130022 * Correspondence: nvh2012@163.com * Correspondence: nvh2012@163.com Abstract:In recent years, active noise reduction technology has become a research hotspot. However, most active noise reduction technologies are developed based on passenger vehicles and are not implemented in large commercial vehicles. The large commercial vehicle sound field space is large, the traditional active noise reduction method is difficult to implement. In order to explore this problem, this paper proposes an improved active noise control method for commercial vehicles :(1) based on the traditional notch filter, a notch FxLMS algorithm based on speed smoothing is proposed; (2) On the basis of the traditional wire-narrowband hybrid ANC algorithm, the reference signal weighting technology is introduced into the wide-band subsystem, and the notch FxLMS algorithm based on speed smoothing is used as the narrowband subsystem, so as to propose an improved wire-narrowband hybrid ANC algorithm. With the help of MATLAB, the simulation model of the designed algorithm is established, and the collected commercial vehicle test noise data is used as the reference signal to simulate and verify the proposed algorithm. The results show that the proposed method has certain practicability. Key words: Noise control; Notch FxLMS algorithm; Wide-narrowband hybrid ANC 1.Introduction At the same time, the control system can be designed according to the characteristics of noise control, so that the noise control is targeted. Therefore, it is very suitable for controlling low frequency noise. In 1933, Paul Lueg proposed the concept of active silencing. Subsequently, a design patent of feed-forward active muffler system was applied for based on the concept of active muffler [1-3]. Limited by the hardware equipment and other objective factors, the system failed to achieve the function of noise cancellation. However, the basic principle of the proposed active noise suppression system lays a foundation for the follow-up research of active noise control technology. In 1953, Harry F.Olson proposed an active noise reduction system [4] named "Electronic sound absorber". Different from Paul Lueg's feed-forward active sound cancellation system, Harry's "electronic sound absorber" consists of an error microphone, a speaker and a controller. The active noise reduction device mainly consists of the sound signal collected by the error microphone and the sound signal emitted by the loudspeaker to construct the reference signal. The electronic sound absorber is regarded as the earliest feedback active noise control system. In 1955, Elvin D.Simshauser and Mones E.Hawley[5] proposed the application of ANC technology to earphones for the first time, which was the first exploration of ANC technology in engineering application. In the same year, William B.Conover and Robert J. Reinglee tried to use ANC technology to control the radiated noise of transformers [6]. This experiment was the first time that active noise control technology was applied in practical situations. During this period, active noise control technology has developed from an idea to the practical application of engineering, and achieved a certain effect of noise reduction. However, the control system in this period can only be applied to specific objects, and the function is single, and the noise control effect can not adapt to the complex requirements of the actual environment. With the rapid development of electronic technology and the deepening of active noise control technology, active noise control technology has entered a period of rapid development. The development of digital signal processor makes it possible to use adaptive control theory to develop With the rapid development of electronic technology and the deepening of active noise control technology, active noise control technology has entered a period of rapid development. 1.Introduction Compared with passive Noise Control technology, Active Noise Control technology (Active Noise Control, ANC) has the advantages of prominent low-frequency Noise Control effect, small volume and lightweight. At the same time, the control system can be designed according to the characteristics of noise control, so that the noise control is targeted. Therefore, it is very suitable for controlling low frequency noise. In 1933, Paul Lueg proposed the concept of active silencing. Subsequently, a design patent of feed-forward active muffler system was applied for based on the concept of active muffler [1-3]. Limited by the hardware equipment and other objective factors, the system failed to achieve the function of noise cancellation. However, the basic principle of the proposed active noise suppression system lays a foundation for the follow-up research of active noise control technology. In 1953, Harry F.Olson proposed an active noise reduction system [4] named "Electronic sound absorber". Different from Paul Lueg's feed-forward active sound cancellation system, Harry's "electronic sound absorber" consists of an error microphone, a speaker and a controller. The active noise reduction device mainly consists of the sound signal collected by the error microphone and the sound signal emitted by the loudspeaker to construct the reference signal. The electronic sound absorber is regarded as the earliest feedback active noise control system. In 1955, Elvin D.Simshauser and Mones E.Hawley[5] proposed the application of ANC technology to earphones for the first time, which was the first exploration of ANC technology in engineering application. In the same year, William B.Conover and Robert J. Reinglee tried to use ANC technology to control the radiated noise of transformers [6]. This experiment was the first time that active noise control technology was applied in practical situations. During this period, active noise control technology has developed from an idea to the practical application of engineering, and achieved a certain effect of noise reduction. However, the control system in this period can only be applied to specific objects, and the function is single, and the noise control effect can not adapt to the complex requirements of the actual environment. Wi h h id d l f l i h l d h d i f i i l Compared with passive Noise Control technology, Active Noise Control technology (Active Noise Control, ANC) has the advantages of prominent low-frequency Noise Control effect, small volume and lightweight. 1.Introduction The development of digital signal processor makes it possible to use adaptive control theory to develop active noise controller. Morgan and Burgress[7] proposed an adaptive active noise control system based on Filtered-x Least Mean Square (FxLMS) algorithm for the first time. The algorithm is studied by computer simulation aiming at pipe noise control. At the same time, the delay of the secondary path of the active noise control system is considered to enhance the system stability. Due to these advantages, FxLMS algorithm has become the most widely used algorithm in ANC system. Oswald[8] developed a single-channel adaptive active noise control system in the car to control the noise caused by diesel engine. The system uses the engine speed signal to calculate the engine noise frequency, which is used to construct a reference noise signal related to the engine speed signal. The results show that the system has a good control effect for engine noise below 200Hz, which is the first successful application of active noise control technology in the car. Subsequently, Lotus Engineering Company of the United Kingdom cooperated with the Institute of Acoustic Vibration of University of Southampton [9] to develop the vehicle adaptive noise active control system for passenger cars. Nissan of Japan applied ANC system to bluebird model and achieved a noise reduction effect of 5.6dB [10]. Honda [11] adopts the method of feedback control to design the active noise control system on Accord. The system integrates in-car audio into an active noise control system without the use of additional speakers. Active noise control technology is gradually applied in practice, and has a certain effect on reducing the sound pressure level of noise. However, the control effect of active noise control technology only stops at the level of reducing noise pressure level. With the introduction of the concept of sound quality, the focus of researchers on noise research has shifted from objective sound pressure values to sound quality indicators, and improving sound quality has become the key direction of active noise control research. Muller-bbm Acoustic Vibration System Co., Ltd. of the United States studied active noise control technology on sound quality earlier [12]. By comparing the test before and after active noise control, they proved that active noise control technology has a broad application prospect in improving sound quality. 1.Introduction Scholars from Nanyang University of Science and Technology in Singapore [13] proposed a new adaptive active noise control method based on equal loudness compensation, which can enhance the perception of the real change of noise level. In 2003, Spanish scholars analyzed sound quality through psycho-acoustic parameter prediction and subjective score, and evaluated the ability of multi-channel active noise control system to obtain more pleasant sounds [14]. In the research of active noise control system, adaptive filter and adaptive algorithm are the core [15]. At present, the research of active noise control algorithm is the focus of active noise control technology. Researchers hope to develop an efficient and stable d i fil i l i h L i Vi [16] i d h F LMS l i h b i h limit of the maximum stride length. The simulation results show that the convergence speed and stability of the control algorithm are improved. Longchen Li[17] proposed a control strategy optimization method based on Genetic Algorithm (Genetic Algorithm-GA), using Genetic Algorithm as a strategy optimization tool for vehicle ANC simulation model optimization and control strategy development. Mohamded Djendi[18] proposed a new fast transverse filtering algorithm with low complexity and good convergence speed based on the dual-Kalman filtering algorithm, and applied it to the field of stereo echo cancellation. Li Tan[19] proposed simplified diagonal structure bilinear filter-X least mean square algorithm and diagonal structure bilinear filter-X least mean square algorithm with channel reduction. Compared with diagonal bilinear filtering - X least mean square algorithm, the computational complexity is reduced. Basant Kumar Mohanty[20] proposed a Delayed Filtered-x Least Mean Square - DFxLMS (Delayed Filtered-x Least Mean Square—DFxLMS) algorithm, which takes into account the Delayed air-electrical connection of active noise controller. Experimental results show that DFxLMS is better than FxLMS(Filter-x Least Mean Square -FxLMS) when the delay is large. Radik Srazhidinov[21] extended the tangential hyperbolic function nonlinear filter-x mean square algorithm(Tangential Hyperbolic Function- Nonlinear Filtered-x Least Mean Square — THF-NLFxLMS) to wiener-Hammerstein system and proposed the Wiener-Hammerstein THF-NLFXLMS algorithm. Simulation results show that wiener-Hammerstein THF-NLFXLMS algorithm has better nonlinear noise reduction effect with the increase of system nonlinearity. M. Ferrer[22] applies the incremental cooperation strategy in the network to the Multiple error filtered-x least mean square -MEFxLMS (Multiple Error Filtered-x Least Mean Square-MEFxLMS), and develops an active noise control system for adaptive distributed networks. 1.Introduction Simulation results show that the proposed distributed algorithm can achieve good performance when appropriate parameters are selected. Mahdi Akraminia[23] proposed an adaptive feedback active noise control system based on wavelet algorithm, and introduced adaptive learning to maintain the stability and convergence speed of the control algorithm. The proposed method is compared with FxLMS and ANC algorithm based on neural network by simulation in typical linear/nonlinear cases. The results show that the proposed method has excellent performance in terms of convergence speed and noise attenuation when the secondary path filter is inaccurate. Jordan Cheer[24] applied the adaptive filtering technology to the multiple-input multiple-output （Multiple Input Multiple Output—MIMO） active control problem, proposed the MIMO FxLMS algorithm of subband adaptive filtering, and then compared the MIMO FxLMS algorithm of self-adaptive filtering with the standard full-band algorithm. The results show that, compared with the full-band implementation, the computational requirements of the adaptive filtering algorithm are significantly reduced with the increase of the number of subbands used. Ho-wuk Kim[25] improved the FuLMS algorithm and applied the ultra-stable adaptive recursive filter into the variable-step LMS algorithm, which improved the stability of the control algorithm without reducing the convergence speed of the algorithm. On the basis of FxLMS algorithm, many scholars normalized the reference signal, thus proposed the normalized least mean square algorithm（Normalized Least Mean Square—NLMS）, and improved NLMS algorithm [26-29], which improved the convergence speed of NLMS algorithm compared with FxLMS algorithm. Xu [30] studied the algorithm of adaptive filtering recursive least square method（Recursive Least Square—RLS）and input the algorithm into the signal processor. By comparing the experimental results with the simulation results, the effectiveness of using RLS algorithm for active noise control was obtained. Shi [31] studied the noise cancellation system of RLS algorithm and confirmed the effect of communication speech signal processing through simulation. Ayesha Zeb[32] proposed several active control algorithms based on FxRLS(Filtered-x Recursive Least Square-FxRLS) to actively control impulse noise, one of which is to ensure the stability of the control algorithm by setting a threshold for the reference noise signal and error signal. Simulation results show that the improved control algorithm can solve the problem of poor stability of FxRLS algorithm when dealing with impulse noise. Lu[33] proposed an active noise control method based on the FxRMC (Filtered-x Recursive Maximum Correntropy - FxRMC), which does not require any prior information of noise and has strong interference to impulse noise. 2 Classical ANC theory and methods There are many classification methods for active noise control systems, which can be divided into finite impulse response (FIR) filters and infinite impulse response (IIR) filters according to the different filter structures of the controllers. According to the reference noise signal can be divided into feed-forward structure and feedback structure. According to the number of secondary sound sources and error microphones, it can be divided into single-channel ANC system and multi-channel ANC system. According to the spectral characteristics of the processed noise, it can be divided into wide-band ANC system, narrowband ANC system, and wide-band narrowband hybrid ANC system. 2.1 ANC controller filter structure 1.Introduction Simulation results and experimental results show that this method has better stability and noise reduction effect. M. Akraminia applies wavelet algorithm to active noise control [34-36]. Simulation results show that this method has faster convergence speed and better noise reduction effect when it is used for active noise control of nonlinear systems. In order to improve the convergence speed of the active control algorithm and reduce the computation amount of the single iteration process of the control algorithm, the two-dimensional coordinate descent method and affine projection method are also proposed a new method to suppress the spike noise based on it. Lifu Wu[42] replaced the scalar leakage factor of FxLMS algorithm with a symmetric Toeplitz matrix, and proposed an improved leakage FxLMS algorithm to solve the "waterbed effect" of ANC feedback system. 2.1 ANC controller filter structure The design of filter is the key to the design of active noise control system. It is very important to choose the appropriate filter structure to ensure the performance of ANC system. Digital filters can be divided into finite impulse response filters and infinite impulse response filters according to whether there is feedback from the output to the input. The IIR filter is recursive. Because of the feedback link, the output of the system is not only related to the input but also affected by the previous output, which leads to the impulse response ( ) h n of the system as an infinite sequence. Figure 1 shows the structure of the direct type I IIR filter, which is obtained directly from the difference equation. It can be seen from the figure that the structure has a two-pole network, with the first level realizing zero and the second level realizing pole. … x(n) y(n) Z-1 b0 b1 x(n-1) Z-1 b2 x(n-2) … bM-1 bM-2 bM-3 x(n-M+3) x(n-M+2) x(n-M+1) Z-1 Z-1 Z-1 … … a1 a2 aN-2 aN-1 aN y(n-1) y(n-2) y(n-N+2) y(n-N+1) y(n-N) Z-1 Z-1 Z-1 Z-1 Z-1 FIG. 1 Flow diagram of direct type I IIR filter FIG. 1 Flow diagram of direct type I IIR filter At time n , the filter output is： At time n , the filter output is： -1 1 0 ( ) ( ) ( - ) ( ) ( - ) N M i i i i y n a n y n i b n x n i = = = +   Where, ( ) ia n and ( ) ib n are the weight coefficients of the filter, M and N are the respective orders of the two-stage filters, ( - ) x n i is the input signal at time - n i , and ( - ) y n i is the output signal at time - n i . The system transfer function is expressed as[4]： Where, ( ) ia n and ( ) ib n are the weight coefficients of the filter, M and N are the respective orders of the two-stage filters, ( - ) x n i is the input signal at time - n i , and ( - ) y n i is the output signal at time - n i . 2.1 ANC controller filter structure For the FIR filter of order N, the output at time n can be expressed as: FIR filter of order N, the output at time n can be expressed as: -1 0 ( ) ( ) ( ) ( ) ( - ) N T i i y n n n w n x n i = = = W X -1 0 ( ) ( ) ( ) ( ) ( - ) N T i i y n n n w n x n i = = = W X (3) The system transfer function can be expressed as[2-5]: The system transfer function can be expressed as[2-5]: 1 0 ( ) ( ) ( ) ( ) N i i i Y Z H Z w n Z X Z − − = = = (4) By comparing equations (2) and (4), it can be seen that, compared with IIR filter, all FIR filter poles are located at z=0, while IIR filter poles are distributed outside the stable region in some cases, which results in poor stability of IIR filter. Under the condition of pursuing the stability of ANC system, FIR filter is preferred as the filter structure of the controller. In addition, compared with IIR filter, FIR filter has better linear phase characteristics. Therefore, the FIR filter structure is selected as the filter structure of ANC controller in this paper. By comparing equations (2) and (4), it can be seen that, compared with IIR filter, all FIR filter poles are located at z=0, while IIR filter poles are distributed outside the stable region in some cases, which results in poor stability of IIR filter. Under the condition of pursuing the stability of ANC system, FIR filter is preferred as the filter structure of the controller. In addition, compared with IIR filter, FIR filter has better linear phase characteristics. Therefore, the FIR filter structure is selected as the filter structure of ANC controller in this paper. 2.2 Classical adaptive active noise control algorithm 2.2 Classical adaptive active noise control algorithm 2.2 Classical adaptive active noise control algorithm 2.1 ANC controller filter structure The system transfer function is expressed as[4]： 1 0 1 ( ) ( ) ( ) ( ) 1 ( ) M i i i N i i i b n Z Y Z H Z X Z a n Z − − = − = = = −   1 0 1 ( ) ( ) ( ) ( ) 1 ( ) M i i i N i i i b n Z Y Z H Z X Z a n Z − − = − = = = −   Different from IIR filter, FIR filter has no feedback link, and the system output is only related to the input, so the impulse response decays to 0 after N sampling periods, so it is called finite impulse response filter. FIG. 2 shows the structure of the direct FIR filter, and the flow diagram is directly obtained from the difference equation. … … x(n) w0(n) w1(n) w2(n) wN-3(n) wN-2(n) wN-1(n) y(n) Z-1 Z-1 Z-1 Z-1 FIG. 2 Flow diagram structure of direct FIR filter x(n) FIG. 2 Flow diagram structure of direct FIR filter FIG. 2 shows that the impulse response of FIR filter is composed of finite sample values. For the FIG. 2 shows that the impulse response of FIR filter is composed of finite sample values. 2.2.1 FxLMS algorithm At present, the most widely used adaptive active noise control algorithm is FxLMS algorithm, which is based on LMS algorithm and considers the influence of secondary sound channel. Figure 3 is the block diagram of FxLMS algorithm, d(n) represents the primary noise signal, x(n)represents the reference noise signal, P(z) represents the transfer function of the primary sound channel, e(n) represents the error signal, y(n) represents the output signal of the secondary loudspeaker, S(z) represents the transfer function of the secondary sound channel, represents the estimation of the transfer function of the secondary sound channel. W(z) represents the transfer function of the adaptive filter. represents the estimation of the transfer function of the secondary sound channel. W(z) represents the transfer function of the adaptive filter. FIG. 3 Block diagram of FxLMS control algorithm FIG. 2.2.1 FxLMS algorithm 3 Block diagram of FxLMS control algorithm Assuming that the order of ANC controller filter is L, then the filter coefficient at time n can be expressed as[3-6]：   1 2 3 ( ) ( ), ( ), ( ), , ( ) T L n w n w n w n w n = W The controller input signal can be expressed as：   ( ) ( ), ( -1), ( -2), , ( - 1) T n x n x n x n x n L = + X The controller output signal can be expressed as： The controller output signal can be expressed as： 1 ( ) ( ) ( ) ( ) ( - 1) L T i i y n n n w n x n i = = = +  X W Assuming that the secondary acoustic channel function is estimated by a filter of order M, the filter coefficient at time n can be expressed as[4-6]: Assuming that the secondary acoustic channel function is estimated by a filter of order M, the filter coefficient at time n can be expressed as[4-6]:   1 2 3 ( ) ( ), ( ), ( ), , ( ) T M n s n s n s n s n = S (8) The error noise signal can be expressed as: The error noise signal can be expressed as: The error noise signal can be expressed as: 1 1 ( ) ( ) ( ) ( - 1) ( ) ( ) ( - 1) ( - 1) ( ) ( ) ( ) M m m M T m m T f e n d n s n y n m d n s n n m n m d n n n = = = + + = + + + = +   X W X W (9) Where, ( ) f n X is the reference noise signal processed by the secondary acoustic channel filtering： Where, ( ) f n X is the reference noise signal processed by the secondary acoustic channel filtering： ( ) ( ), ( -1), ( -2), , ( - 1) T f f f f f n x n x n x n x n L   = +   X (10) 1 ( ) ( ) ( - 1) M f m m x n s n x n m = = +  1 ( ) ( ) ( - 1) M f m m x n s n x n m = = +  FxLMS algorithm follows the minimum mean square error criterion, taking the mean square error of the error noise signal value as the objective function, and its expression is： 2 ( ) ( ) J n E e n   =   Where,  E • represents time averaging of independent variables： Where,  E • represents time averaging of independent variables： Where,  E • represents time averaging of independent variables： Substitute equation (9) into equation (12) to obtain： Substitute equation (9) into equation (12) to obtain： q ( ) q ( ) 2 2 ( )= ( ) ( ) ( ) ( ) ( ) 2 ( ) ( ) ( ) ( ) ( ) ( ) 2 ( ) T T T T f f f T T J n E d n n E n n n n E d n n E d n n n n       + +         = + +   W X X W W X W RW W P (13) ( ) ( ) T f f E n n   =   R X X ( ) ( ) T f E d n n   =   P X 2 2 )= ( ) ( ) ( ) ( ) ( ) 2 ( ) ( ) ( ) ( ) ( ) ( ) 2 ( ) T T T T f f f T T n E d n n E n n n n E d n n E d n n n n       + +         = + +   W X X W W X W RW W P (13) (13) ( ) ( ) T f f E n n   =   R X X ( ) ( ) T f E d n n   =   P X (14) According to Equation (13), J(n) is a quadratic function of the weight vector W(n), while R is a positive definite quadratic matrix. 2.2.1 FxLMS algorithm Therefore: ' ( 1) ( ) ( ) ( ) f n n e n n  + = − W W X ' ' ' ' ' ( ) ( ), ( -1), ( -2), , ( - 1) T f f f f f n x n x n x n x n L   = +   X ' ' ' 1 ( ) ( ) ( ) ( ) ( - 1) M T f m m x n n n s n x n m = = = +  X S ' ' ' ' ' ( ) ( ), ( -1), ( -2), , ( - 1) T f f f f f n x n x n x n x n L   = +   X ' ' ' 1 ( ) ( ) ( ) ( ) ( - 1) M T f m m x n n n s n x n m = = = +  X S In order to ensure the gradual convergence of the algorithm in the iterative process, it is necessary to restrict the convergence factor. The range of convergence factor is： In order to ensure the gradual convergence of the algorithm in the iterative process, it is necessary to restrict the convergence factor. 2.2.1 FxLMS algorithm Therefore, there is a unique minimum point. When J(n) achieves the minimum value, the optimal value Wo of weight coefficient satisfies: According to Equation (13), J(n) is a quadratic function of the weight vector W(n), while R is a positive definite quadratic matrix. Therefore, there is a unique minimum point. When J(n) achieves the minimum value, the optimal value Wo of weight coefficient satisfies: 1 o − = − W R P 1 o − = − W R P 1 o − = − W R P It is too expensive to directly solve the filter weight coefficient of ANC controller according to equation (16). In order to avoid matrix operation, a recursive estimation algorithm is introduced to calculate the filter weights iteratively. According to the steepest descent method, the recursive relation of filter weight coefficients can be obtained as follows: ( 1) ( ) ( ) 2 n n n  + = −  W W In Equation (17), μ represents the convergence factor, which is used to regulate the convergence speed and stability of the algorithm. ( ) n  is the gradient vector and its expression is： In Equation (17), μ represents the convergence factor, which is used to regulate the convergence speed and stability of the algorithm. 2.2.1 FxLMS algorithm The range of convergence factor is： In order to ensure the gradual convergence of the algorithm in the iterative process, it is necessary 1 0 f tr        R 1 0 f tr        R 1 0 f tr        R (23) Where,  tr • represents matrix trace operation, and the specific calculation formula can be expressed as: Where,  tr • represents matrix trace operation, and the specific calculation formula can be expressed as: ( ) ( ) 2 ' 1 1 M f f j tr x n j =  = − +   R ( ) ( ) 2 ' 1 1 M f f j tr x n j =  = − +   R (24) Combined with Equations (23) and (24), with the increase of the order of the adaptive filter, the value range of the convergence factor gradually decreases, and the higher order adaptive filter of the algorithm is more likely to diverge. 2.2.1 FxLMS algorithm ( ) n  is the gradient vector and its expression is： 2 ( ) ( ) ( ) 2 ( ) ( ) ( ) ( ) f J n e n n e n n n n    = = =   X W W ( 2 ( ) ( ) ( ) 2 ( ) ( ) ( ) ( ) f J n e n n e n n n n    = = =   X W W (18) Therefore, the iterative formula of filter weight coefficient of FxLMS algorithm is expressed as： ( 1) ( ) ( ) ( ) f n n e n n  + = − W W X (13) ( 1) ( ) ( ) ( ) f n n e n n  + = − W W X ( 1) ( ) ( ) ( ) f n n e n n  + = − W W X In practice, the value of ( ) f n X cannot be obtained directly and is usually represented by its estimator ' ( ) f n X . ' ( ) f n X is obtained by using the secondary acoustic channel estimation function to process the reference noise signal, and the secondary acoustic channel transfer function needs to be identified and replaced before it is obtained. 3 Design of improved active noise control algorithm The noise in the cab of commercial vehicle includes narrowband noise such as engine order noise and wide-band noise such as road noise and wind noise. In order to achieve good active noise control effect, an improved active noise control algorithm is proposed in this paper. One is based on the traditional notch filter, the speed signal smoothing module is introduced, and the speed smoothing based notch wave FxLMS algorithm is proposed to achieve a good control of the second order noise of commercial vehicle engine. The other is an improved hybrid ANC algorithm. The wide-band subsystem uses FxLMS/F algorithm based on reference model weighting, while the narrowband subsystem uses the notch FxLMS algorithm based on speed smoothing to effectively control the narrowband noise such as engine order noise and the wide-band noise such as road noise and wind noise. In this chapter, the theoretical derivation of the two improved algorithms is given. 3.1 Design of notch FxLMS algorithm based on speed smoothing Based on the theory of adaptive filtering algorithm and the traditional notch algorithm, a notch algorithm FxLMS based on the speed smoothing is proposed by introducing the speed smoothing technique. FIG. 4 shows the block diagram of notch FxLMS algorithm based on speed smoothing. FIG. 4 Notch FxLMS algorithm based on speed smoothing FIG. 4 Notch FxLMS algorithm based on speed smoothing In FIG. 4,x1(n) and x2(n) respectively represent two reference signals constructed according to the engine speed. xf1(n) and xf2(n) respectively represent the two reference signals filtered by the secondary pathway. w1(n) and w2(n) represent the sine and cosine weights of notch filter, respectively. d(n) represents the noise signal transmitted to the error microphone, which is called the expected noise; y(n) represents the output signal from the controller to the loudspeaker. S(z) represents the transfer function of the secondary pathway, ˆ( ) S z represents the estimation of S(z), ys(n) represents the loudspeaker output signal transmitted to the error microphone, and e(n) represents the signal collected by the error microphone, which is the superposition signal of d(n) and ys(n). The engine order noise of commercial vehicles usually satisfies the following relation: i (25) 60 i ni f   = 60 i ni f   = Where, i is the number of engine cylinders, n is the engine speed, τ is the number of engine strokes, and η is the harmonic order. According to the above equation, the engine order frequency corresponding to a specific speed can be calculated, and the reference signal can be constructed accordingly. The traditional ANC algorithm based on notch filter directly uses the collected speed signal to construct the reference signal. However, when the engine speed fluctuates greatly, the variable speed signal will produce the variable reference signal. However, the change of reference signal will adversely affect the noise reduction effect and stability of ANC system. 3.1 Design of notch FxLMS algorithm based on speed smoothing According to the least mean square error criterion, the updated formula of sine component weight coefficient and cosine component weight coefficient can be obtained: 1 1 1 2 2 2 ( 1) ( ) 2 ( ) ( ) ( 1) ( ) 2 ( ) ( ) f f w n w n e n x n w n w n e n x n   + = +  + = +  (30) Where,  is the step size of the algorithm. Filtered reference signals xf1(n) and xf2(n) are calculated as follows： 1 1 2 2 ˆ ( ) ˆ ( ) ( ) ( ) ( ) ( ) T f T f n n x n n x n n  =  =  S S x x (31) Where， 1 1 1 1 ( ) [ ( ) ( 1) ( 1)]T M n x n x n x n = − − + x is the reference signal sequence of sine component， 2 2 2 2 ( ) [ ( ) ( 1) ( 1)]T M n x n x n x n = − − + x is the reference signal sequence of Where， 1 1 1 1 ( ) [ ( ) ( 1) ( 1)]T M n x n x n x n = − − + x is the reference signal sequence of sine component， 2 2 2 2 ( ) [ ( ) ( 1) ( 1)]T M n x n x n x n = − − + x is the reference signal sequence of Where， 1 1 1 1 ( ) [ ( ) ( 1) ( 1)]T M n x n x n x n = − − + x is the reference signal sequence of sine component， 2 2 2 2 ( ) [ ( ) ( 1) ( 1)]T M n x n x n x n = − − + x is the reference signal sequence of cosine component. M is the filter length used to estimate the filter of the secondary pathway. Table 1 shows the calculation flow of notch FxLMS algorithm based on speed smoothing. osine component. M is the filter length used to estimate the filter of the secondary pathway. Table cosine component. M is the filter length used to estimate the filter of the secondary pathway. 3.1 Design of notch FxLMS algorithm based on speed smoothing To solve this problem, equation (26) is used to smooth the speed signal: ( ) ( 1) (1 ) ( ) R n R n r n   = − + − (26) ( ) ( 1) (1 ) ( ) R n R n r n   = − + − Where, R(n) is the smooth speed value at time n, R(n-1) is the smooth speed value at the previous time, and r(n) is the original speed value of the engine at the current time.  is the forgetting factor, and its value is close to 1. FIG. 5 shows the effect of engine speed smoothing in idle driving condition with different values of . FIG. 5 Smoothing effect of engine speed FIG. 5 Smoothing effect of engine speed It can be seen from FIG. 5 that the forgetting factor  controls the smoothness of engine speed. If  is close to 1, the better the smoothness is. But a larger  also means it is harder to track changes in the true engine speed. 3.1 Design of notch FxLMS algorithm based on speed smoothing After calculating the smoothed speed R(n), the reference frequency signal at the nth time is: ( ) ( ) 60 R R n i f n   = ( ) ( ) 60 R R n i f n   = (27) The controller reference signal constructed at time n is: 1 2 ( ) sin(2 ( ) ) ( ) cos(2 ( ) ) R R x n A f n t x n A f n t   =   =  (28) The output signal of the controller at time n can be expressed as： 1 1 2 2 ( ) ( ) ( ) ( ) ( ) y n w n x n w n x n = + The output signal of the controller at time n can be expressed as： The output signal of the controller at time n can be expressed as： The output signal of the controller at time n can be expressed as： 1 1 2 2 ( ) ( ) ( ) ( ) ( ) y n w n x n w n x n = + The output signal of the controller at time n can be expressed as： 1 1 2 2 ( ) ( ) ( ) ( ) ( ) y n w n x n w n x n = + p g p 1 1 2 2 ( ) ( ) ( ) ( ) ( ) y n w n x n w n x n = + 1 1 2 2 ( ) ( ) ( ) ( ) ( ) y n w n x n w n x n = + (29) Where, the notch filter weight coefficients w1(n) and w2(n) are updated based on LMS algorithm. In notch FxLMS algorithm, sine component weight filter and cosine component weight filter usually adopt transverse FIR filter with order 1. 3.1 Design of notch FxLMS algorithm based on speed smoothing ( ) ( 1) (1 ) ( ) R n R n r n   = − + − ( ) ( ) 60 R n i f n   = 1 2 ( ) sin(2 ( ) ) ( ) cos(2 ( ) ) x n A f n t x n A f n t   =   =  ( ) ( 1) (1 ) ( ) R n R n r n   = − + − ( ) ( ) 60 R n i f n   = 1 2 ( ) sin(2 ( ) ) ( ) cos(2 ( ) ) x n A f n t x n A f n t   =   =  1 1 2 2 ( ) ( ) ( ) ( ) ( ) y n w n x n w n x n = + 1 1 2 2 ( ) ( ) ( ) ( ) ( ) y n w n x n w n x n = + 1 1 2 2 ˆ ( ) ˆ ( ) ( ) ( ) ( ) ( ) T f T f n n x n n x n n  =  =  S S x x 1 1 1 2 2 2 ( 1) ( ) 2 ( ) ( ) ( 1) ( ) 2 ( ) ( ) f f w n w n e n x n w n w n e n x n   + = +  + = +  1 1 2 2 ˆ ( ) ˆ ( ) ( ) ( ) ( ) ( ) T f T f n n x n n x n n  =  =  S S x x 1 1 1 2 2 2 ( 1) ( ) 2 ( ) ( ) ( 1) ( ) 2 ( ) ( ) f f w n w n e n x n w n w n e n x n   + = +  + = +  1 1 2 2 ˆ ( ) ˆ ( ) ( ) ( ) ( ) ( ) T f T f n n x n n x n n  =  =  S S x x 3.2 Design of improved active noise control algorithm for wideband and narrowband hybrid In commercial vehicle driving, there will be not only engine order noise, but also road noise, wind noise and other broadband noise. 3.1 Design of notch FxLMS algorithm based on speed smoothing Table 1 shows the calculation flow of notch FxLMS algorithm based on speed smoothing. 1 shows the calculation flow of notch FxLMS algorithm based on speed smoothing. 1 shows the calculation flow of notch FxLMS algorithm based on speed smoothing. Table 1 Calculation flow of notch FxLMS algorithm based on speed smoothing Initialization w1(0)=w2(0)=0, x1(0)=x2(0)=0, xf1(n)=xf2(n)=0, R(0)=r(0) Calculation process For n = 0, 1, 2, … Revolving speed signal r(n), and error microphone signal e(n) at the nth moment are collected. process Revolving speed signal r(n), and error microphone signal e(n) at the nth moment are collected. 3.1 Design of notch FxLMS algorithm based on speed smoothing Therefore, the wideband noise must be controlled. In this section, an improved wide-narrow-band mixed ANC algorithm is proposed. The algorithm consists of wideband subsystem, narrowband subsystem and signal separation subsystem. Compared with the traditional width-narrowband hybrid algorithm, the improved width-narrowband hybrid algorithm introduces speed smoothing module in the narrowband subsystem, and FxLMS/F algorithm based on reference signal weighting is introduced in the wideband subsystem. The block diagram of the improved wideband and narrowband hybrid ANC algorithm is shown in FIG. 6. The detailed introduction of each subsystem in the improved hybrid algorithm is as follows. 3.2.1 Signal separation (SNC) subsystem In the wideband and narrowband ANC algorithm, the function of the signal separation subsystem is to remove the narrowband component in the noise signal collected by the reference microphone, so as to realize the effective separation of the wideband and narrowband components in the wideband narrowband mixed noise, and improve the denoising effect of the wideband subsystem.In the improved wideband hybrid ANC algorithm, the SNC subsystem is used to separate the second-order noise from the cab noise, and the remaining noise signals are processed by the wideband subsystem. Figure 6 Improved wide-narrowband mixed ANC algorithm Figure 6 Improved wide-narrowband mixed ANC algorithm As shown in Figure 6, the input signal of the SNC subsystem is solved by the engine speed. Assuming the engine speed is r(n), the corresponding reference frequency signal is expressed as: ( ) ( ) 60 r n i f n   = (32) The constructed reference signal of SNC subsystem is expressed as： sin(2 ( ) ) cos(2 ( ) ) aS bS x f n n x f n n   =   =  sin(2 ( ) ) cos(2 ( ) ) aS bS x f n n x f n n   =   =  sin(2 ( ) ) cos(2 ( ) ) aS bS x f n n x f n n   =   =  (33) The SNC subsystem uses LMS algorithm to update the filter weights. The output signal of SNC subsystem can be expressed as[7-12]: The SNC subsystem uses LMS algorithm to update the filter weights. 3.1 Design of notch FxLMS algorithm based on speed smoothing The output signal of SNC subsystem can be expressed as[7-12]: ˆ ˆ ( ) ( ) ( ) ( ) ( ) SNC S aS S bS y n a n x n b n x n = + ˆ ˆ ( ) ( ) ( ) ( ) ( ) SNC S aS S bS y n a n x n b n x n = + Where，ˆ ( ) S a n and ˆ ( ) Sb n represent the filter weight coefficients of sine and cosine components Where，ˆ ( ) S a n and ˆ ( ) Sb n represent the filter weight coefficients of sine and cosine components respectively, and their updates follow the following formula： ectively, and their updates follow the following formula： ˆ ˆ ( 1) ( ) ( ) ( ) ˆ ˆ ( 1) ( ) ( ) ( ) S S S S aS S S S S bS a n a n e n x n b n b n e n x n   + = +  + = +  (35) ˆ ˆ ( 1) ( ) ( ) ( ) ˆ ˆ ( 1) ( ) ( ) ( ) S S S S aS S S S S bS a n a n e n x n b n b n e n x n   + = +  + = +  ˆ ˆ ( 1) ( ) ( ) ( ) ˆ ˆ ( 1) ( ) ( ) ( ) S S S S aS S S S S bS a n a n e n x n b n b n e n x n   + = +  + = +  (35) Where， s  is the step size of the SNC subsystem, ( ) se n is the separation error of the SNC subsystem, and the input reference signal ( ) Bx n of the wideband subsystem is expressed as： (36) ˆ ˆ ( ) ( ) ( ) ( ) ( ) ( ) ( ) B S R S aS S bS x n e n x n a n x n b n x n = = − − ˆ ˆ ( ) ( ) ( ) ( ) ( ) ( ) ( ) B S R S aS S bS x n e n x n a n x n b n x n = = − − With the continuous iteration of the filter weight coefficient, the engine order noise component in the vehicle noise is gradually separated, and the separation error ( ) se n gradually becomes the wideband component of the vehicle noise. 3.1 Design of notch FxLMS algorithm based on speed smoothing 3.2.2 Narrowband (NANC) subsystem based on speed smooth notch FxLMS algorithm In the wide-narrow-band hybrid ANC algorithm, the narrow-band subsystem controls the engine order noise in the cab, and its reference signal is obtained from the engine speed signal after smoothing[10-14]： In the wide-narrow-band hybrid ANC algorithm, the narrow-band subsystem controls the engine order noise in the cab, and its reference signal is obtained from the engine speed signal after smoothing[10-14]： ( ) sin(2 ( ) ) ( ) cos(2 ( ) ) a R b R x n A f n t x n A f n t   =   =  ( ) sin(2 ( ) ) ( ) cos(2 ( ) ) a R b R x n A f n t x n A f n t   =   =  (37) The output of the NANC subsystem is expressed as： The output of the NANC subsystem is expressed as： The output of the NANC subsystem is expressed as： ˆ ˆ ( ) ( ) ( ) ( ) ( ) N N a N b y n a n x n b n x n = + ˆ ˆ ( ) ( ) ( ) ( ) ( ) N N a N b y n a n x n b n x n = + (38) The weight update formula of NANC subsystem is expressed as： ˆ ˆ ˆ ( 1) ( ) 2 ( ) ( ) ˆ ˆ ˆ ( 1) ( ) 2 ( ) ( ) N N N a N N N b a n a n e n x n b n b n e n x n   + = +  + = +  (39) (39) The two filtered reference signals in the formula are calculated by the following formula： The two filtered reference signals in the formula are calculated by the following formula： ˆ ( ) ˆ ( ) ˆ ( ) ( ) ˆ ( ) ( ) T a a T b b n n x n n x n n  =  =  S S x x ˆ ( ) ˆ ( ) ˆ ( ) ( ) ˆ ( ) ( ) T a a T b b n n x n n x n n  =  =  S S x x (40) 式中， ( ) [ ( ) ( 1) ( 1)]T a a a a M n x n x n x n = − − + x ， ( ) [ ( ) ( 1) ( 1)] T b b b b M n x n x n x n = − − + x 。式中， ( ) [ ( ) ( 1) ( 1)]T a a a a M n x n x n x n = − − + x ， ( ) [ ( ) ( 1) ( 1)] T b b b b M n x n x n x n = − − + x 。 3.2.3 Wideband ANC subsystem based on FxLMS/F algorithm with reference signal weighting 3.2.3 Wideband ANC subsystem based on FxLMS/F algorithm with reference signal weighting The wideband subsystem of traditional wide-narrowband hybrid ANC algorithm adopts FxLMS algorithm. However, when the algorithm step size is large, the steady state noise reduction will be reduced, and when the step size is small, the convergence speed will be slow. To solve this problem, variable step size strategy is a better solution.The Filtered-x Least Mean Square/Fourth (Filtered-x Least Mean Square/Fourth-FxLMS/F) algorithm adopts threshold parameters to adjust the step size, so that the algorithm takes a larger step size in convergence and a smaller step size in steady-state. Thus, faster convergence speed and better steady-state noise reduction can be achieved. Firstly, the FxLMS/F algorithm is derived. The cost function of FxLMS/F algorithm is defined as follows： 2 2 1 1 ( ) ( ) ln( ( ) ) 2 2 J n e n e n   = − + 2 2 1 1 ( ) ( ) ln( ( ) ) 2 2 J n e n e n   = − + (41) Where,  is the threshold parameter, and e(n) is the error signal. The value of  determines the convergence ability and steady-state noise reduction ability of the algorithm. According to the above cost function, the filter weight updating formula of FxLMS/F algorithm can be derived by using the steepest gradient descent method： 2 2 ( ) ( 1) ( ) ( ) ( ) ( ) e n n n e n n e n    + = + + w w x (42) Where, ( ) n w is the filter weight coefficient vector, and ( ) n x is the filter reference signal. Where, ( ) n w is the filter weight coefficient vector, and ( ) n x is the filter reference signal. For FxLMS and FxLMS/F algorithm, in the process of filter weight coefficient iteration, the reference signal sampling at each time has the same effect. However, in the process of commercial vehicle driving, the road noise and wind noise have strong random characteristics. 3.2.3 Wideband ANC subsystem based on FxLMS/F algorithm with reference signal weighting Therefore, at any time in the process of ANC control, the reference signal sampling closer to the time can better reflect the real-time variation trend of noise, so more weight is given.。Based on this consideration, an FxLMS/F algorithm based on reference signal weighting is used in the wideband subsystem in this study to effectively control the wideband noise in the cab of commercial vehicles. The output signal of the wideband subsystem is calculated by the following formula: ( ) ( ) ( ) B B y n n n = w x ( ) ( ) ( ) B B y n n n = w x (43) Where,   1 0 ( ) ( ) L j j n w n − = = w is the weight coefficient vector of the wideband subsystem filter, ( ) [ ( ) ( 1) ( 1)] B B B B T L n x n x n x n = − − + x is the reference signal vector of the wideband subsystem. The weights of the wideband subsystem filters are updated by the FxLMS/F algorithm weighted by the reference signal, which is expressed as follows: Where,   1 0 ( ) ( ) L j j n w n − = = w is the weight coefficient vector of the wideband subsystem filter, ( ) [ ( ) ( 1) ( 1)] B B B B T L n x n x n x n = − − + x is the reference signal vector of the wideband subsystem. 3.2.3 Wideband ANC subsystem based on FxLMS/F algorithm with reference signal weighting The weights of the wideband subsystem filters are updated by the FxLMS/F algorithm weighted by the reference signal, which is expressed as follows: 2 2 ( ) ˆ ( 1) ( ) ( ) ( ) ( ) QB e n n n e n n e n   + = + + w w x 2 2 ( ) ˆ ( 1) ( ) ( ) ( ) ( ) QB e n n n e n n e n   + = + + w w x ˆ ˆ ( ) ( ) QB B n n = x V x (45) ˆ ˆ ( ) ( ) QB B n n = x V x Where,“ ”is the Hadamard product, ˆ ˆ ˆ ˆ ( ) [ ( ) ( 1) ( 1)] B B B B T L n x n x n x n = − − + x is the filtering reference signal vector，Where, ˆ ( ) ˆ ( ) ( ) T B B n x n n = S x ， V is the weighted vector of the filtered Where,“ ”is the Hadamard product, ˆ ˆ ˆ ˆ ( ) [ ( ) ( 1) ( 1)] B B B B T L n x n x n x n = − − + x is the filtering reference signal vector，Where, ˆ ( ) ˆ ( ) ( ) T B B n x n n = S x ， V is the weighted vector of the filtered ( ) [ ( ) ( ) ( )] B B B B reference signal vector，Where, ˆ ( ) ˆ ( ) ( ) T B B n x n n = S x ， V is the weighted vector of the filtered reference signal vector，Where, ˆ ( ) ˆ ( ) ( ) T B B n x n n = S x ， V is the weighted vector of the filtered reference signal, which can be expressed as the following vector： 0 0 [1,1, , , ] b b L L L = − V 0 0 [1,1, , , ] b b L L L = − V (46) 0 0 [1,1, , , ] b b L L L = − V Where, b is a constant less than 1, and L0 is an integer between 0 and the order L of the wideband filter. 3.2.3 Wideband ANC subsystem based on FxLMS/F algorithm with reference signal weighting To solve the output of the narrowband subsystem and the wideband subsystem, the output of the whole wide-narrowband hybrid ANC algorithm can be expressed as： Where, b is a constant less than 1, and L0 is an integer between 0 and the order L of the wideband filter. To solve the output of the narrowband subsystem and the wideband subsystem, the output of the whole wide-narrowband hybrid ANC algorithm can be expressed as： ( ) ( )+ ( ) N B y n y n y n = ( ) ( )+ ( ) N B y n y n y n = (47) The calculation flow of the proposed improved wire-narrowband hybrid ANC algorithm is shown in Table 2： Table 2 Calculation process of improved broadband and narrowband hybrid ANC algorithm Initializatio n ( ) ( ) ( ) ( ) ˆ ˆ ˆ ˆ 0 0 0 0 0, (0) (0) (0) (0) , (0) (0) S S N N a b B a b a b R r = = = = = = = = = 0 w x x x Table 2 Calculation process of improved broadband and narrowband hybrid ANC algorithm Initializatio n ( ) ( ) ( ) ( ) ˆ ˆ ˆ ˆ 0 0 0 0 0, (0) (0) (0) (0) , (0) (0) S S N N a b B a b a b R r = = = = = = = = = 0 w x x x Calculation For n = 0, 1, 2, … Calculation For n = 0, 1, 2, … Calculation For n = 0, 1, 2, … process Revolving speed signal r(n), reference microphone signal xR(n) and error microphone signal e(n) at the nth moment are collected. 3.2.3 Wideband ANC subsystem based on FxLMS/F algorithm with reference signal weighting ( ) ( 1) (1 ) ( ) R n R n r n   = − + − ( ) ( 1) (1 ) ( ) R n R n r n   = − + − ( ) ( 1) (1 ) ( ) R n R n r n   = − + − ( ) ( ) 60 r n i f n   = ( ) ( ) 60 R R n i f n   = ( ) ( ) 60 r n i f n   = ( ) ( ) 60 R R n i f n   = sin(2 ( ) ) cos(2 ( ) ) aS bS x f n n x f n n   =   =  ˆ ˆ ( ) ( ) ( ) ( ) ( ) SNC S aS S bS y n a n x n b n x n = + ˆ ˆ ( ) ( ) ( )- ( ) ( )- ( ) ( ) B S R S aS S bS x n e n x n a n x n b n x n = = ˆ ˆ ( 1) ( ) ( ) ( ) ˆ ˆ ( 1) ( ) ( ) ( ) S S S S aS S S S S bS a n a n e n x n b n b n e n x n   + = +  + = +  ˆ ˆ ( ) ( ) ( ) ( ) ( ) SNC S aS S bS y n a n x n b n x n = + ˆ ˆ ( ) ( ) ( )- ( ) ( )- ( ) ( ) B S R S aS S bS x n e n x n a n x n b n x n = = ˆ ˆ ( ) ( ) ( )- ( ) ( )- ( ) ( ) B S R S aS S bS x n e n x n a n x n b n x n = = ˆ ˆ ( 1) ( ) ( ) ( ) ˆ ˆ ( 1) ( ) ( ) ( ) S S S S aS S S S S bS a n a n e n x n b n b n e n x n   + = +  + = +  ( ) sin(2 ( ) ) ( ) cos(2 ( ) ) a R b R x n A f n t x n A f n t   =   =  ˆ ˆ ( ) ( ) ( ) ( ) ( ) N N a N b y n a n x n b n x n = + ˆ ( ) ˆ ( ) ˆ ( ) ( ) ˆ ( ) ( ) T a a T b b n n x n n x n n  =  =  S S x x ˆ ˆ ˆ ( 1) ( ) 2 ( ) ( ) ˆ ˆ ˆ ( 1) ( ) 2 ( ) ( ) N N N a N N N b a n a n e n x n b n b n e n x n   + = +  + = +  ( ) ( ) ( ) B B y n n n = w x ˆ ( ) ˆ ( ) ( ) T B B n x n n = S x ˆ ˆ ( ) ( ) QB B n n = x V x ˆ ˆ ˆ ( 1) ( ) 2 ( ) ( ) ˆ ˆ ˆ ( 1) ( ) 2 ( ) ( ) N N N a N N N b a n a n e n x n b n b n e n x n   + = +  + = +  2 2 ( ) ˆ ( 1) ( ) ( ) ( ) ( ) QB e n n n e n n e n   + = + + w w x Output control signal y(n) End 4. Verification of improved active noise control algorithm In this chapter, the simulation model of the algorithm is established based on MATLAB software, and the collected noise data of the commercial vehicle cab is used as the noise signal to be controlled. The notch FxLMS algorithm based on speed smoothing and the improved wide-narrowband hybrid ANC algorithm are simulated. In order to verify the validity of FxLMS algorithm based on speed smoothing and improved hybrid ANC algorithm. Firstly, the simulation model of FxLMS algorithm based on speed smoothing and the improved wide-narrowband hybrid algorithm is established by MATLAB software. According to the calculation process of notch FxLMS algorithm based on speed smoothing and improved wide-narrow-band hybrid ANC algorithm, the corresponding algorithm script file is written by using m language of MATLAB software. Load test noise data, primary acoustic path coefficient and secondary acoustic path coefficient in MATLAB software workspace. 4.1 Idle speed condition verification A commercial vehicle was used as a test vehicle, which was powered by a six-cylinder four-stroke diesel engine. A standard microphone was arranged in the headrest on the right side of the driver to collect the noise data of the test vehicle in the cab at idle speed. The sensor arrangement is shown in FIG. 7 (a). ANC control, loudspeaker, ANC microphone and reference point microphone need to be arranged in the cab. Meanwhile, the engine speed is monitored and recorded in real time. During the test, the sampling frequency of data was 51200 Hz. The proposed active noise control algorithm takes engine speed signal as reference. Therefore, the engine speed signal of the test vehicle was collected in the same period, and its sampling frequency was 200 Hz. The spectrum of acquired noise is shown in Figure 7(b), and the engine speed signal collected is shown in Figure 8. (a) ANC test sensor layout (b) Frequency domain curve of interior noise at idle speed FIG. 7 Sensor placement and test results (a) ANC test sensor layout (a) ANC test sensor layout (b) Frequency domain curve of interior noise at idle speed FIG. 7 Sensor placement and test results (b) Frequency domain curve of interior noise at idle speed FIG. 7 Sensor placement and test results FIG. 7 Sensor placement and test results FIG. 8 Engine speed curve at idle speed FIG 8 Engine speed curve at idle speed FIG. 8 Engine speed curve at idle speed FIG. 8 Engine speed curve at idle speed As can be seen from FIG. 7(b), the noise frequency at idle speed is mainly the engine's order noise and the harmonic frequency excited by it. It can be seen from FIG. 8 that the engine speed of the test vehicle at idle speed is 600 r/min, and the calculated frequency of second-order engine noise is 30 Hz, which is consistent with the result in FIG. 7(b). In the MATLAB simulation model based on the notch FxLMS algorithm of speed smoothing and the improved wide-narrow-band hybrid ANC algorithm, the active noise control simulation of the collected noise signal is carried out. Considering that the ANC system is limited by the computing capacity of the digital signal processor in practical application, its sampling frequency should not be too high. The sampling frequency used in the simulation is set as 8000 Hz. 4.1 Idle speed condition verification The selected algorithm parameters are shown in Table 3. The time-domain noise reduction effect is shown in Figure 9, and the frequency domain noise reduction effect is shown in Figure 10 and Figure 11. Table 3 Algorithm parameter values in idle speed condition Table 3 Algorithm parameter values in idle speed condition Algorithm parameters Notch FxLMS algorithm based on speed smoothing 0.001, 0.9999   = = Improved wide and narrow band hybrid algorithm 0 0.0001, 0.001, 0.001, 0.001, 0.03, 3, 192 S N B b L L     = = = = = = = Table 3 Algorithm parameter values in idle speed condition Algorithm parameters Notch FxLMS algorithm based on speed smoothing 0.001, 0.9999   = = Improved wide and narrow band hybrid algorithm 0 0.0001, 0.001, 0.001, 0.001, 0.03, 3, 192 S N B b L L     = = = = = = = 0.001, 0.9999   = = FIG. 9 Time-domain noise reduction effect diagram at idle speed FIG. 9 Time-domain noise reduction effect diagram at idle speed FIG. 9 Time-domain noise reduction effect diagram at idle speed It can be seen from FIG. 9 that both algorithms can quickly converge to the steady state, and the amplitude of sound pressure in the steady state is significantly reduced compared with that before noise reduction. Relatively speaking, the notch FxLMS algorithm based on speed smoothing converges faster than the improved hybrid ANC algorithm. This is because in the broadband and narrowband hybrid algorithm, ANC subsystem needs a convergence process for the separation of broadband signal and narrowband signal, thus reducing the convergence speed of the whole broadband and narrowband hybrid algorithm. From the steady-state point of view, the improved hybrid ANC algorithm has greater noise reduction than the FxLMS algorithm based on speed smoothing. FIG. 10 shows that notch FxLMS algorithm based on speed smoothing achieves large noise reduction at the second-order frequency of the engine (30 Hz), and the sound pressure level at 30 Hz drops from 89 dB before noise reduction to 52 dB. As can be seen from Figure 11, the sound pressure level of the improved broadband-narrowband hybrid ANC algorithm at 30 Hz drops from 89 dB before noise reduction to 48.3dB, a decrease of 40.7dB. 4.1 Idle speed condition verification The improved wide-and-narrow-band hybrid ANC algorithm not only has a good control effect on the second-order noise of the engine at 30 Hz, but also achieves a good noise reduction effect at the fourth-order frequency of the engine (60 Hz) and a band around it. Among them, the sound pressure level at 60 Hz dropped from 73.6dB to 59.5dB, a decrease of 14.1dB. FIG. 10 Effect of noise reduction in frequency domain at idle speed (Notch FxLMS algorithm based on speed smoothing) FIG. 10 Effect of noise reduction in frequency domain at idle speed (Notch FxLMS algorithm based on speed smoothing) FIG. 11 Effect diagram of noise reduction in frequency domain under idling conditions (improved wide-narrow-band hybrid ANC algorithm) FIG. 11 Effect diagram of noise reduction in frequency domain under idling conditions (improved wide-narrow-band hybrid ANC algorithm) 4.2 Uniform driving condition verification 4.2 Uniform driving condition verification The noise and speed data of the test vehicle at 50 km/h constant speed were collected. The spectrum of collected noise is shown in Figure 12, and the engine speed signal is shown in Figure 13. As can be seen from FIG. 12, noise at 50 km/h uniform speed includes engine order noise, road noise, wind noise and other components. As can be seen from Figure 13, the engine speed of the test vehicle fluctuates around 1190 r/min at a constant speed of 50 km/h, and the fluctuation range is about 1184 r/min to 1195 r/min. The calculated frequency of second-order noise of the engine is about 59.5Hz, which is consistent with the result in FIG. 12. FIG. 12 Frequency domain curve of interior noise at 50 km/h constant speed FIG. 13 Engine speed curve at 50 km/h constant speed engine is about 59.5Hz, which is consistent with the result in FIG. 12. engine is about 59.5Hz, which is consistent with the result in FIG. 12. FIG. 12 Frequency domain curve of interior noise at 50 km/h constant speed FIG. 12 Frequency domain curve of interior noise at 50 km/h constant speed FIG. 13 Engine speed curve at 50 km/h constant speed In the MATLAB simulation model based on the notch FxLMS algorithm of speed smoothing and the improved wide-narrow-band hybrid ANC algorithm, the active noise control simulation of the collected noise signal is carried out. The selected algorithm parameters are shown in Table 4. The time domain noise reduction effect is shown in Figure 14, and the frequency domain noise reduction effect is shown in Figure 15-17. 4.2 Uniform driving condition verification Table 4 Parameter values of the algorithm under 50 km/h uniform driving condition Algorithm Parameters Notch FxLMS algorithm based 0.001, 0.9999   = = Table 4 Parameter values of the algorithm under 50 km/h uniform driving condition Table 4 Parameter values of the algorithm under 50 km/h uniform driving condition Algorithm Parameters Notch FxLMS algorithm based 0.001, 0.9999   = = Parameters 0.001, 0.9999   = = 0.001, 0.9999   = = on speed smoothing Improved wide and narrow band hybrid algorithm 0 0.001, 0.0001, 0.001, 0.001, 0.03, 3, 192 S N B b L L     = = = = = = = As can be seen from Figure 14, the convergence speed of FxLMS algorithm based on speed smoothing is faster than that of the improved wideband and narrowband hybrid ANC algorithm. The improved hybrid ANC algorithm has a higher steady noise reduction than the FxLMS algorithm based on speed smoothing. As can be seen from FIG. 15, notch FxLMS algorithm based on speed smoothing achieves a large noise reduction at the second-order frequency of the engine (59.5Hz), and the sound pressure level drops from 80.4dB to 41.6dB, a decrease of 38.8dB. It can be seen from FIG. 16 that the improved wide-narrow-band hybrid ANC algorithm has a better control effect on second-order noise of engine at 59.5Hz. Moreover, a better noise reduction effect is achieved at the fourth order frequency of the engine (119 Hz). The sound pressure level at 59.5Hz decreased from 80.4dB to 38.0dB, decreasing by 42.4dB. The sound pressure level at 119 Hz decreased from 69.4dB to 58.6dB, decreasing by 10.8dB. FIG. 17 shows the noise reduction effect of 1/3 octave-frequency of the improved wideband and narrowband hybrid algorithm. As can be seen from the figure, sound pressure level at 80 Hz frequency increased by about 2.3 dB, and other frequency ranges achieved good noise reduction effects. The noise reduction of 14.3dB and 8.3dB is achieved for the engine order frequency, and the noise reduction of 1-8dB is achieved for the other frequency range. FIG. 14 Noise reduction effect in time domain under uniform driving condition FIG. 15 Effect of noise reduction in frequency domain under uniform speed condition (notch FxLMS algorithm based on speed smoothing) FIG. 14 Noise reduction effect in time domain under uniform driving condition FIG. 4.2 Uniform driving condition verification 15 Effect of noise reduction in frequency domain under uniform speed condition (notch FxLMS algorithm based on speed smoothing) FIG. 14 Noise reduction effect in time domain under uniform driving condition FIG. 15 Effect of noise reduction in frequency domain under uniform speed condition (notch FxLMS algorithm based on speed smoothing) FIG. 16 Noise reduction effect in frequency domain under uniform driving condition (Improved wide-narrow-band hybrid ANC algorithm) FIG. 17 Effect diagram of 1/3 octave-frequency noise reduction under uniform driving speed (Improved wide-narrow-band hybrid ANC algorithm) 4.3 Acceleration condition verification IG. 16 Noise reduction effect in frequency domain under uniform driving condition (Improved wide-narrow-band FIG. 16 Noise reduction effect in frequency domain under uniform driving condition (Improved wide-narrow-band hybrid ANC algorithm) FIG. 17 Effect diagram of 1/3 octave-frequency noise reduction under uniform driving speed (Improved wide-narrow-band hybrid ANC algorithm) hybrid ANC algorithm) hybrid ANC algorithm) FIG. 17 Effect diagram of 1/3 octave-frequency noise reduction under uniform driving speed (Improved wide-narrow-band hybrid ANC algorithm) 4.3 Acceleration condition verification 4.3 Acceleration condition verification Under the same conditions, the noise and speed data of acceleration from 45 km/h to 90 km/h were collected. The time-frequency diagram of noise is shown in Figure 18, and the engine speed signal collected is shown in Figure 19. As can be seen from FIG. 18, the second-order frequency value of the engine gradually increases as the engine speed continues to rise during acceleration. FIG. 19 shows that the engine speed of the test vehicle increases from 740 r/min to 1400 r/min under accelerated driving conditions. In the MATLAB simulation model based on the notch FxLMS algorithm of speed smoothing and the improved wide-narrowband hybrid ANC algorithm, active noise control simulation is carried out for the noise signals collected above. The selected algorithm parameters are shown in Table 5, time-domain noise reduction effect is shown in Figure 20, time-frequency noise reduction effect is shown in Figure 21. FIG. 18 Time-frequency curve of interior noise under acceleration condition FIG. 18 Time-frequency curve of interior noise under acceleration condition FIG. 4.2 Uniform driving condition verification 19 Engine speed curve under acceleration condition Table 5 Algorithm parameter values in acceleration conditions Algorithm Parameters Notch FxLMS algorithm based on speed smoothing 0.001, 0.9999   = = Improved wide and narrow band hybrid algorithm 0 0.001, 0.0001, 0.001, 0.001, 0.03, 3, 192 S N B b L L     = = = = = = = FIG. 19 Engine speed curve under acceleration condition 0.001, 0.9999   = = 0.001, 0.9999   = = 0.001, 0.9999   = = 0.001, 0.9999   = = As can be seen from FIG. 20, notch FxLMS algorithm based on speed smoothing achieves good time-domain noise reduction in the first half of the acceleration process. However, the noise reduction effect of the second half is relatively poor. This is mainly because the latter half of the vehicle speed is higher, road noise, wind noise and other broadband components accounted for a larger proportion, so the overall noise reduction effect becomes worse. Comparatively, the improved hybrid ANC algorithm achieves high time-domain noise reduction in the whole acceleration process. This benefits from the control of broadband noise. It can be seen from FIG. 21 that, compared with the interior noise before noise reduction, notch FxLMS algorithm based on speed smoothing can produce a larger noise reduction for the second-order frequency noise of the engine during the whole acceleration process. However, there is no noise reduction effect for other orders of noise. Compared with FxLMS algorithm based on speed smoothing, the improved wideband and narrowband hybrid ANC algorithm has better control effect on engine second-order noise. At the same time, the noise of other order of engine (20~200 Hz) can be effectively controlled. FIG. 20 Time-domain noise reduction effect diagram of acceleration condition (a) Time-frequency diagram of interior noise before noise reduction FIG. 20 Time-domain noise reduction effect diagram of acceleration condition FIG. 20 Time-domain noise reduction effect diagram of acceleration condition (a) Time-frequency diagram of interior noise before noise reduction (b) Interior noise based on notch FxLMS algorithm for speed smoothing (c) Improved wide and narrow band hybrid ANC algorithm for interior noise FIG. 4.2 Uniform driving condition verification 21 Time-frequency noise reduction effect diagram under acceleration condition (b) Interior noise based on notch FxLMS algorithm for speed smoothing (b) Interior noise based on notch FxLMS algorithm for speed smoothing (c) Improved wide and narrow band hybrid ANC algorithm for interior noise FIG. 21 Time-frequency noise reduction effect diagram under acceleration condition FIG. 21 Time-frequency noise reduction effect diagram under acceleration condition 5 .Conclusion Aiming at the problem of active noise control in commercial vehicle cab, an improved wide-narrow-band hybrid ANC algorithm was proposed. At the same time, the control effect of the proposed algorithm is analyzed and verified. The main research contents and conclusions are as follows: 1、On the basis of the existing active noise control algorithm, the notch FxLMS algorithm based on speed smoothing is proposed. By smoothing the speed signal, the adverse effect of engine speed fluctuation on the active noise control system is avoided. The method is combined with notch filter and FxLMS algorithm is used to update the weight coefficient of filter. 2、On the basis of FxLMS algorithm, an improved hybrid ANC algorithm with wide and narrow band is proposed. The algorithm consists of narrowband subsystem, wideband subsystem and signal separation subsystem. Narrow band subsystem adopts notch FxLMS algorithm with smooth speed. In wideband subsystem, FxLMS/F algorithm based on signal weighting is adopted to increase the weight of reference signal sampling, so as to enhance the control ability of active noise control system to wideband noise. 3、MATLAB models of FxLMS algorithm based on speed smoothing and ANC algorithm based on improved width and narrow band are established. The simulation results show that the FxLMS algorithm based on speed smoothing can effectively control the second-order engine noise in the cab of commercial vehicle. At idle speed, the sound pressure level of the FxLMS algorithm based on speed smoothing at the second order frequency (30 Hz) of the engine decreases from 89 dB before noise reduction to 52 dB, which decreases by 37 dB. Under uniform driving condition, the SPL at the second order frequency (59.5Hz) of the engine decreases from 80.4dB before noise reduction to 41.6dB, decreasing by 38.8dB. Under the acceleration condition, the SPL at the second order frequency of the engine decreases obviously during the whole acceleration process. on”. on”. Author contributions J. S.: Conceptualization, methodology, data collection, writing—original draf preparation, supervision, and investigations. J. N.: Data collection and investigations. S. C.: Data collection and investigations. Additional information Correspondence and requests for materials should be addressed to J.S. Correspondence and requests for materials should be addressed to J.S. Correspondence and requests for materials should be addressed to J.S. Reprints and permissions information is available at www.nature.com/reprints Reprints and permissions information is available at www.nature.com/reprints Competing interests Te authors declare no competing interests. Acknowledgements the by 6 .Reference 6 .Reference [1] Paul Lueg. Verfahren zur Dämpfung von Schallschwingungen[P]. AT Patent No 141998, 1935. [2] Paul Lueg. 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https://openalex.org/W2922656763	https://europepmc.org/articles/pmc6650761?pdf=render	English	null	Evidence‐based practice integration in hospital wards—The complexities and challenges in achieving evidence‐based practice in clinical nursing	Nursing open	2,019	cc-by	8,113	R E S E A R C H A R T I C L E R E S E A R C H A R T I C L E Åste Renolen1,2 \| Esther Hjälmhult3 \| Sevald Høye4 \| Lars Johan Danbolt5,6 \| Marit Kirkevold1 Åste Renolen1,2 \| Esther Hjälmhult3 \| Sevald Høye4 \| Lars Johan Danbolt5,6 \| Marit Kirkevold1 Abstract Aim: Exploring the processes involved in two different strategies to integrate evi‐ dence‐based practice into nursing practice. Design: Classical grounded theory methodology was used. Methods: Data were collected through 90 hr of observation and 4 focus groups among clinical nurses in two different hospital wards. Results: We identified a multidimensional evidence‐based practice integration frame‐ work that illuminates the complexities involved in the integration process. The di‐ mensions were approaches to evidence‐based practice, positions of evidence‐based practice and levels of evidence‐based practice. The interactions between the dimen‐ sions gave five combinations; an explicit evidence‐based practice performed as a par‐ allel to daily work at the systems level, an implicit evidence‐based practice integrated into daily work at the systems level, an explicit evidence‐based practice integrated into daily work at the individual level, an explicit evidence‐based practice integrated into daily work at the systems level and an implicit evidence‐based practice inte‐ grated into daily work at the individual level. 1Institute of Health and Society, University of Oslo, Oslo, Norway 2Department of Medicine, Innlandet Hospital Trust, Lillehammer, Norway 3Centre for Evidence‐Based Practice, Western Norway University of Applied Sciences, Bergen, Norway 4Faculty of Public Health, Inland Norway University of Applied Sciences, Elverum, Norway 5Centre of Psychology of Religion, Innlandet Hospital Trust, Ottestad, Norway 6Norwegian School of Theology, Oslo, Norway Correspondence Åste Renolen, Department of Medicine, Innlandet Hospital Trust, Lillehammer, Norway. Email: aste.renolen@sykehuset-innlandet.no Funding information Innlandet Hospital Trust, Norway funded the study. Abstract Aim: Exploring the processes involved in two different strategies to integrate evi‐ dence‐based practice into nursing practice. Design: Classical grounded theory methodology was used. Methods: Data were collected through 90 hr of observation and 4 focus groups among clinical nurses in two different hospital wards. Results: We identified a multidimensional evidence‐based practice integration frame‐ work that illuminates the complexities involved in the integration process. The di‐ mensions were approaches to evidence‐based practice, positions of evidence‐based practice and levels of evidence‐based practice. 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. Åste Renolen1,2 \| Esther Hjälmhult3 \| Sevald Høye4 \| Lars Johan Danbolt5,6 \| Marit Kirkevold1 The interactions between the dimen‐ sions gave five combinations; an explicit evidence‐based practice performed as a par‐ allel to daily work at the systems level, an implicit evidence‐based practice integrated into daily work at the systems level, an explicit evidence‐based practice integrated into daily work at the individual level, an explicit evidence‐based practice integrated into daily work at the systems level and an implicit evidence‐based practice inte‐ grated into daily work at the individual level. © 2019 The Authors. Nursing Open published by John Wiley & Sons Ltd. Received: 27 October 2018 \| Revised: 20 December 2018 \| Accepted: 30 January 2019 Received: 27 October 2018 \| Revised: 20 December 2018 \| Accepted: 30 January 2019 Received: 27 October 2018 \| Revised: 20 December 2018 \| Accepted: 30 January 2019 DOI: 10.1002/nop2.259 Abstract Abstract Aim: Exploring the processes involved in two different strategies to integrate evi‐ dence‐based practice into nursing practice. Design: Classical grounded theory methodology was used. Methods: Data were collected through 90 hr of observation and 4 focus groups among clinical nurses in two different hospital wards. Results: We identified a multidimensional evidence‐based practice integration frame‐ work that illuminates the complexities involved in the integration process. The di‐ mensions were approaches to evidence‐based practice, positions of evidence‐based practice and levels of evidence‐based practice. The interactions between the dimen‐ sions gave five combinations; an explicit evidence‐based practice performed as a par‐ allel to daily work at the systems level, an implicit evidence‐based practice integrated into daily work at the systems level, an explicit evidence‐based practice integrated into daily work at the individual level, an explicit evidence‐based practice integrated into daily work at the systems level and an implicit evidence‐based practice inte‐ grated into daily work at the individual level. Correspondence Åste Renolen, Department of Medicine, Innlandet Hospital Trust, Lillehammer, Norway. Email: aste.renolen@sykehuset-innlandet.no Funding information Innlandet Hospital Trust, Norway funded the study. K E Y W O R D S clinical practice guidelines, evidence‐based practice, hospital, huddle board, implementation, nurses, research utilization, whiteboard 1.1 \| Background EBP implies the integration of clinical expertise with systematically obtained research evidence, considering resources available and patient preferences in each patient situation (DiCenso, Guyatt, & Ciliska, 2005; Polit & Beck, 2016; Sackett, Rosenberg, Gray, Haynes, & Richardson, 1996). It may be regarded as a strategy or a general way of thinking aimed at achieving the best treatment and care in each individual patient situation. Furthermore, EBP also involves or‐ ganizational activities such as integrating research evidence through the development of evidence‐based (EB) guidelines (Polit & Beck, 2016). The potential for achieving practice changes through adopting EBP depends on the interaction between the characteristics of the evidence, the clinicians and the context of practice in the health‐ care setting (Greenhalgh, Robert, Macfarlane, Bate, & Kyriakidou, 2004). It occurs as a complex process where people—often through dialogue with others—are active participants in innovations and which research must address (Greenhalgh, 2018; Greenhalgh et al., 2004). The implementation of research evidence has been challenging in nursing practice, and we need more knowledge regarding how to translate research into daily health and nursing care (Kajermo et al., 2010; Mallion & Brooke, 2016; Squires et al., 2011). Clinical nurses seem to value personal experience together with informa‐ tion learned in nursing school and information from colleagues as their most important source of knowledge, rather than basing practice on current research evidence (Adib‐Hajbaghery, 2007; Bischoff & Hinojosa, 2013; Renolen & Hjälmhult, 2015; Yoder et al., 2014). An association between higher reported levels of emotional exhaustion and lower reported levels of research use has been affirmed (Estabrooks, Midodzi, Cummings, & Wallin, 2007). As well, a more favourable context related to culture, good leadership and recognition for a job well done has resulted in higher research use (Estabrooks et al., 2007). In each culture, particular ideas or activities may be more valued than others (Scott‐Findlay & Golden‐Biddle, 2005). In a ward culture charac‐ terized by engagement in EBP and quality improvement, leader‐ ship and clinicians may to a greater extent succeed in changing In this study, we investigated the integration of EBP in clinical practice in hospital wards by studying in depth two different meth‐ ods applied by clinicians. One method involved nurses working with an EBP project to develop local clinical guidelines. The other method included integrating EBP/EB guidelines through an interdisciplinary use of huddle board sessions. practice (Saunders & Vehviläinen‐Julkunen, 2017). A ward culture characterized by rigid completion of practical tasks rather than engagement in EBP may not easily facilitate opportunities for re‐ search use or for changing practice (Henderson, Cooke, Creedy, & Walker, 2012; Ryan, 2016). Furthermore, promoting research use in an environment characterized by work overload among nurses and lack of teamwork structure that facilitate research use, may be demanding (Solomons & Spross, 2011). Studies have indicated that healthcare workers describe a change in practice as hard work and that continuing with the existing practice in daily work with an already huge workload is less demanding (Asadoorian, Hearson, Satyanarayana, & Ursel, 2010; Fink, Thompson, & Bonnes, 2005). EBP in organizational units, while integration refers to the routiniz‐ ing and sustaining of new practices. 1 \| INTRODUCTION Scott‐Findlay, O'Leary, & Gushta, 2003; Funk, Champagne, Wiese, & Tornquist, 1991; Sadeghi‐Bazargani, Tabrizi, & Azami‐Aghdash, 2014; Solomons & Spross, 2011). However, few studies have inves‐ tigated the actual processes of attempting to integrate evidence‐ based practice (EBP) into daily practice, which was the purpose of this study. In the research literature, there has been an inconsistent use of terminologies regarding implementation of new practices (Damschroder et al., 2009; May & Finch, 2009). In this paper, we use the concept of implementation to mean organizing the adoption of 1 Huge amounts of relevant research evidence exist in health and nursing sciences, which is not integrated into clinical practice due to translation and implementation challenges (Greenhalgh, 2018; Grimshaw, Eccles, Lavis, Hill, & Squires, 2012; Song et al., 2010). A large number of the studies have aimed to identify factors that facilitate or hinder the integration of new research evidence into the nursing practice (Cochrane et al., 2007; Estabrooks, Floyd, 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. Nursing Open published by John Wiley & Sons Ltd. 815 \| wileyonlinelibrary.com/journal/nop2 Nursing Open. 2019;6:815–823. RENOLEN et al. 816 2.2.1 \| Setting and participants The study was conducted in a Norwegian hospital trust consisting of six somatic hospitals scattered over a wide geographical area. Data were collected in two medical wards treating patients with differ‐ ent diagnoses in two different geographical locations eight to nine years after the hospital trust introduced EBP with the purpose of 2.1 \| Design The data used in this study were collected and analysed through classical grounded theory methodology (Glaser, 1978, 1998; Glaser & Strauss, 1967). In grounded theory, the researcher initially has an open, inductive approach to data by systematically collecting the data from practice. As codes and categories emerge, one introduces a more focused approach to explore relationships between differ‐ ent properties in codes and categories, based on hypotheses formu‐ lated from the data analysis in the initial phase (Glaser, 1978, 1998). Ward B was using a huddle board to improve clinical practice and reduce patient harm in clinical practice. Huddles are short structural meetings among interdisciplinary healthcare workers (Glymph et al., 2015). Huddle board is a whiteboard used in a huddle as a visual pa‐ tient risk assessment tool (Figure 1) introducing EB guidelines in daily work. Further information about Ward A and Ward B is outlined in Boxes. 2 \| THE STUDY enhancing competence among health professionals (Vandvik & Eiring, 2011). According to grounded theory, wards, research meth‐ ods, participants and situations were selected through theoretical sampling (Glaser & Strauss, 1967). Ward A was chosen based on the ward's engagement in an EBP project, initially guided by a general perspective and problem area. Ward B was included as it was as‐ sumed to be able to contribute information to fortify the emerg‐ ing codes and categories in the theory development (Glaser, 1978; Glaser & Strauss, 1967). The participating wards and nurses are pre‐ sented in Table 1. enhancing competence among health professionals (Vandvik & Eiring, 2011). According to grounded theory, wards, research meth‐ ods, participants and situations were selected through theoretical sampling (Glaser & Strauss, 1967). Ward A was chosen based on the ward's engagement in an EBP project, initially guided by a general perspective and problem area. Ward B was included as it was as‐ sumed to be able to contribute information to fortify the emerg‐ ing codes and categories in the theory development (Glaser, 1978; Glaser & Strauss, 1967). The participating wards and nurses are pre‐ sented in Table 1. 1.2 \| Aim The aim of this study was to explore the processes involved in two different strategies applied to integrate EBP to understand the com‐ plexities and challenges in clinical nurses’ daily work better when they attempt to integrate EBP. TA B LE 1 The participating wards and nurses Ward A Ward B Total Number of beds 18 patient beds 38 patient beds Working groups 2 working groups 4 working groups, of whom 2 groups were participating Staff 33 nurses 63 nurses 96 nurses 3 assistants 5 assistants 8 assistants Hours of observations 36 hr 54 hr 90 hr Number of observed nursesa 28 nurses 35 nurses 63 nurses Focus groups 2 2 4 Nurses participating in focus groups (from the population of observed nurses) 10 nurses 8 nurses 18 nurses aThe nurses (N = 63): 39 registered nurses with a bachelor's degree awarded after 3 years of univer‐ sity‐level education, 9 assistant nurses with two years of upper secondary education. Of the remain‐ ing 15 nurses, two had a master's degree and 13 had twelve‐ to eighteen‐month specializations after their bachelor's degree. The types of specialization were relevant for the wards (here without a further specification to ensure anonymity). Total aThe nurses (N = 63): 39 registered nurses with a bachelor's degree awarded after 3 years of univer‐ sity‐level education, 9 assistant nurses with two years of upper secondary education. Of the remain‐ ing 15 nurses, two had a master's degree and 13 had twelve‐ to eighteen‐month specializations after their bachelor's degree. The types of specialization were relevant for the wards (here without a further specification to ensure anonymity). aThe nurses (N = 63): 39 registered nurses with a bachelor's degree awarded after 3 years of univer‐ sity‐level education, 9 assistant nurses with two years of upper secondary education. Of the remain‐ ing 15 nurses, two had a master's degree and 13 had twelve‐ to eighteen‐month specializations after their bachelor's degree. The types of specialization were relevant for the wards (here without a further specification to ensure anonymity). RENOLEN et al. 817 FI G U R E 1 Example of a risk assessment huddle board 2.2.2 \| Data collection Data were collected between March 2014 and November 2015. The lead researcher was a nurse employed at one of the hospitals 818 RENOLEN et al. where the study was conducted. The researcher therefore knew the organization, general routines, quality improvement meas‐ ures and the system of clinical guidelines. However, at the time of the study, she was acting in a researcher role. The researcher mapped out the EBP activities in the relevant hospital wards, ex‐ cluding wards well known to her. The data collection began with participant observation in Ward A, providing the opportunity to study the nurses’ behaviour in relation to their attempts to inte‐ grate EBP while continuing to conduct their daily work in the ward (Creswell, 2013; Polit & Beck, 2016). The researcher wrote de‐ scriptive and reflective field notes during the observations and di‐ rectly afterwards (Creswell, 2013). On finishing the observations and its analyses in Ward A, two focus groups were held to give the observed nurses an opportunity to discuss their concerns and to bring up questions that had emerged from the collected data (Polit & Beck, 2016). A thematic interview guide was used, starting with an open question about the nurses’ experiences with EBP. In line with grounded theory methodology, we stayed open and let the participants talk about their concerns (Glaser, 2013). Afterwards, data were collected in the same way in Ward B. Based on emerging codes and categories, ward B was chosen because they attempted to integrate EBP into their daily work. The participating nurses in observations and focus groups were chosen to give rich informa‐ tion regarding emerging codes and categories, for instance task accomplishment and adjusting knowledge to practice. All focus groups were conducted at the nurses’ workplaces and consisted of four to five participants. The focus groups were moderated by ÅR and co‐moderated by SH. They lasted between 55–65 min and were audiotaped and transcribed. The data collection and analysis continued until no new categories emerged, and we determined that theoretical saturation was achieved (Glaser, 1978). where the study was conducted. The researcher therefore knew the organization, general routines, quality improvement meas‐ ures and the system of clinical guidelines. However, at the time of the study, she was acting in a researcher role. The researcher mapped out the EBP activities in the relevant hospital wards, ex‐ cluding wards well known to her. 2.4 \| Rigour The use of focus group interviews in grounded theory is less common than the use of individual interviews (Hernandez, 2011). However, data with variety and rich information are recommended in grounded theory (Glaser, 1978, 1998). We consider it a strength that we collected Box 1 Ward A—Participating in an EBP project In Ward A, most nurses participated in an EBP project that had been ongoing for approximately two years. They were working in groups to find new evidence and to develop and im‐ plement clinical EB guidelines with the purpose of improving patient treatment and care. The project manager together with a teaching nurse allocated funds from the hospital to enable the nurses to participate in groups by obtaining dedicated time for this work. The nurses participated voluntarily in four different groups that worked one at a time, each with a self‐determined theme. To a various degree, the nurses were knowledgeable regarding asking and formulating questions, literature search, critical appraisal, applying new knowledge and evaluation. The groups worked to summarize the literature/work and planned to write up the process and results on internal teaching days and when they could find time for it. two wards in relation to each other to explore the challenges in integrating EBP in clinical practice. Box 2 Ward B—Integrating a patient safety huddle board programme The employees in Ward B had a daily focus on quality im‐ provement and had participated in different small EBP projects. When data collection started, the ward was in an early phase of integrating a huddle board programme initiated by the hospital leadership aiming to improve clinical practice and reduce patient harm. The initiative was anchored in the Norwegian Patient Safety Programme, where a group of healthcare experts identified sev‐ eral target areas with recommendations and measures based on the current available evidence, such as systematic reviews and national clinical practice guidelines (Norwegian Ministry of Health & Care Services, 2015). Locally, each ward was assigned target areas determined by the hospital leadership, with some also chosen by the physicians and nurses in the ward. A project manager in the hospital leadership decided which guidelines to locally tailor and implement in each working team through in‐ terdisciplinary daily meetings (i.e. “huddles”). The clinicians were supposed to use the EB guidelines together with their expertise, available resources and patient preferences in EBP performance. A template for checking off and scoring the patients informed by the actual guideline for each target area was used. 2.2.2 \| Data collection The data collection began with participant observation in Ward A, providing the opportunity to study the nurses’ behaviour in relation to their attempts to inte‐ grate EBP while continuing to conduct their daily work in the ward (Creswell, 2013; Polit & Beck, 2016). The researcher wrote de‐ scriptive and reflective field notes during the observations and di‐ rectly afterwards (Creswell, 2013). On finishing the observations and its analyses in Ward A, two focus groups were held to give the observed nurses an opportunity to discuss their concerns and to bring up questions that had emerged from the collected data (Polit & Beck, 2016). A thematic interview guide was used, starting with an open question about the nurses’ experiences with EBP. In line with grounded theory methodology, we stayed open and let the participants talk about their concerns (Glaser, 2013). Afterwards, data were collected in the same way in Ward B. Based on emerging codes and categories, ward B was chosen because they attempted to integrate EBP into their daily work. The participating nurses in observations and focus groups were chosen to give rich informa‐ tion regarding emerging codes and categories, for instance task accomplishment and adjusting knowledge to practice. All focus groups were conducted at the nurses’ workplaces and consisted of four to five participants. The focus groups were moderated by ÅR and co‐moderated by SH. They lasted between 55–65 min and were audiotaped and transcribed. The data collection and analysis continued until no new categories emerged, and we determined that theoretical saturation was achieved (Glaser, 1978). 2.5 \| Ethics Approval for the study was requested from a Regional Committee for Medical and Health Research Ethics, but the study did not require approval (Reference number 2014/35A). The Data Protection Officer for Research and Quality approved the study (Reference number 2013/17344). The hospital where the study was performed also permitted the study (reference number 201200448‐27). The participants were recruited on a voluntary basis, based on information about the study from their leader and oral and written information from the researcher during the obser‐ vation period. When the researcher observed the nurse working with the patient, the nurse first informed the patient and obtained oral consent. The researcher recruited the participants to the focus groups in cooperation with the ward leaders, and written consent was obtained. The multidimensional EBP integration framework visualizes five combinations that give meaning based on data in this study. In the next sections, we explore the five observed patterns of EBP integra‐ tion in further detail. when nurses attempt to integrate EBP in their daily practice. The dimensions are as follows: approach to EBP, position of EBP in daily work and organisational level of EBP. By approach, we mean the way of enacting EBP. Two approaches to EBP were identified; explicit EBP (visible and emphasized in the ward) and implicit EBP (invisible and hidden in the background in the daily work in the ward). We also identified two positions of EBP in daily work. With position, we mean how EBP was related to the daily work in the wards. EBP could either be integrated into the daily workflow or it could be performed as a parallel activity to daily work. Finally, we identified two organisational levels of EBP; the systems level and the individual practitioner level. With organisational level, we mean how EBP was integrated into the work at the wards. It could be built into the general routines of the ward, or it could be con‐ sidered the responsibility of the individual healthcare worker to use EB knowledge when caring for individual patients. The core concept “multidimensional EBP integration” embraces the interac‐ tions between these dimensions (Figure 2). data through both observations and focus groups, endeavouring to perform the data collection and analysis in a manner congruent with grounded theory (Hernandez, 2011). To understand what was hap‐ pening in the investigated fields, we have endeavoured to stay open in the data analysis and refrain from using preconceived ideas or con‐ cepts (Glaser, 2013; Glaser & Strauss, 1967). Throughout the study, we have focused on conceptualizing emerging categories and to be aware of the relationships between the categories. The awareness of these relationships is essential in theoretical sensitivity, which is important in grounded theory (Gibson & Hartmann, 2014; Glaser, 1978). when nurses attempt to integrate EBP in their daily practice. The dimensions are as follows: approach to EBP, position of EBP in daily work and organisational level of EBP. By approach, we mean the way of enacting EBP. Two approaches to EBP were identified; explicit EBP (visible and emphasized in the ward) and implicit EBP (invisible and hidden in the background in the daily work in the ward). We also identified two positions of EBP in daily work. With position, we mean how EBP was related to the daily work in the wards. EBP could either be integrated into the daily workflow or it could be performed as a parallel activity to daily work. Finally, we identified two organisational levels of EBP; the systems level and the individual practitioner level. With organisational level, we mean how EBP was integrated into the work at the wards. It could be built into the general routines of the ward, or it could be con‐ sidered the responsibility of the individual healthcare worker to use EB knowledge when caring for individual patients. The core concept “multidimensional EBP integration” embraces the interac‐ tions between these dimensions (Figure 2). 3.1 \| An explicit EBP as a parallel to daily work at the systems level The EBP project in Ward A represented the dimensions of an explicit EBP performed as a parallel to daily work at the systems level (i.e. alternative 1, Figure 2). Here, the EBP was visible and articulated. All nurses were involved in discussions regarding EBP and the appropri‐ ate knowledge to be used in actual situations, indicating that their attitudes had been influenced and that they were more aware of the knowledge source: 2.3 \| Data analysis We performed an open analysis of the data from the observations and focus groups in the same analysis, concurrently with the data collection, according to the principles of classical grounded theory using the constant comparative method (Glaser, 1978; Glaser & Strauss, 1967). During the analysis, we could see that one of the clinical nurses’ concerns was related to their striving to do the best for the patients based on EBP. We then analysed in depth the data related to the nurses’ challenges in EBP integration. The lead researcher wrote memos, which were assumptions about re‐ lations between the data, articulated as hypotheses that could be tested in the data (Glaser, 1978). As such this was both an induc‐ tive and a deductive approach to the data. In the first step of the analysis, the lead researcher systematically identified the relevant emerged codes from the observations and focus groups using the data from Ward A. Next, the researcher identified the emerged codes from Ward B in the same way. The rest of the research team read transcriptions and field notes as well and the whole group of authors discussed the codes. After finishing the separate coding for the two wards, we analysed the codes and categories for the 819 RENOLEN et al. 3 \| FINDINGS This study revealed three significant and interacting dimensions of EBP integration that may help explain the complexities involved FI G U R E 2 Multidimensional EBP integration framework 820 \| I think that our focus on EBP contributes to a greater awareness of what may be the right thing to do. Not just to find an answer, but to find the right answer for the treatment and for the follow‐up. (Focus group I, SN 4) RENOLEN et al. and were striving to realize it. Nevertheless, the findings indicated a gap between the ideal and the actual performance of individual‐ ized patient care. This gap was related to the challenges of get‐ ting new research evidence to be used and the strong emphasis on standardized routines. Due to the latter, the nurses’ pattern of behaviour was dominated by filling out checklists, whereas their focus on the needs of each individual patient receded into the background. For instance, the nurses in Ward B referred to the whiteboard as a visual checklist, which they appreciated because of better safeguarding of the risk areas. Simultaneously, they ex‐ pressed scepticism of the use of checklists because it was chal‐ lenging to strike the right balance between the risk assessment “check‐offs” and other patient needs for nursing care: This activity running parallel to the nurses’ daily work in the ward could be conflicting for the nurses. On the one hand, the nurses ap‐ preciated the opportunity to work with EBP and quality improvement on a relevant theme, free from daily duties and together with their col‐ leagues. On the other hand, the nurses encountered difficulty in re‐ lating this work to their daily patient work. When the groups finished their project periods, they struggled to put the new evidence to use in the daily work. Even if the project motivated the nurses, they felt that they did not have the power to change practice with a new guideline or just with new evidence. The nurses experienced a strong dependence on the managers and physicians who had to formally approve the new clinical guideline and to accept the new knowledge to be used. The nurses were looking for systems and structures to get new evidence more easily and rapidly incorporated into daily routines. Preventing falls, which is a theme in the huddle board, is part of basic nursing care. 3 \| FINDINGS Holistic nursing care dis‐ appears when filling out the forms. When you have been working for a while, you know what you need to do to prevent falls. I think this [fragmented and task oriented practice] is scary. (Focus group III. AN 6) 3.4 \| An explicit EBP integrated into daily work at the systems level The huddle board programme in Ward B represented the dimensions of an implicit EBP integrated into daily work at the systems level (i.e. alternative 2, Figure 2). The EBP was implied in standardized recom‐ mendations and measures integrated directly into daily routines as a part of the nurses’ daily tasks. This integration made the nurses comply with the request to use the EB recommendations and meas‐ ures. However, the research evidence tied to the huddle board tar‐ get areas was not highlighted in daily work: We could not see an extensive use of an explicit approach to EBP integrated into daily work at the systems level in this study (i.e. alter‐ native 4, Figure 2). Even if some nurses demonstrated their aware‐ ness of the knowledge they used, they seldom could refer to where they had gained it: I am very focused on clinical issues and feel that I up‐ date myself reading every new procedure coming in the ward. But there is a lot of information. We mix it with information about the patient and all the things you should remember during the day. You do not think that “this knowledge” I derived from “there”. You use knowledge without knowing exactly where you got it. (Focus group III, RN 2) I feel that the huddle board in a way has become a visual systematization of things we did already. Everything gets very visible, everyone sees it and it is more organized. We did exactly the same things earlier too, but now it is made visible. (Focus group IV, SN 8) The individual nurses did what the organization expected them to do to promote patient safety and quality improvement, but they did not consciously relate to the evidence or seem to understand their use of knowledge as EBP. The leaders and teaching nurses in the ward did organize reflection groups for the nurses once a week, discussing pro‐ fessional challenges and clinical problems. As such, they stimulated the nurses’ critical thinking and inquiry. Nevertheless, this was not visibly linked to the huddle board target areas. 3.5 \| An implicit EBP integrated into daily work at the individual level The combination of the dimensions of an implicit EBP integrated into daily work at the individual level was difficult for the re‐ searcher to observe in practice and would be difficult for the nurses to put into words because of its implicitness (i.e. alterna‐ tive 5, Figure 2). What we could observe was the nurses provid‐ ing care according to prevailing clinical guidelines at the wards, which indicates integration of EB knowledge. Furthermore, their explicit recognition of the fact that they provided care based on many different sources of knowledge, including new guidelines 4.1 \| EBP as a parallel to daily work The findings showed that clinical nurses who applied the explicit ap‐ proach to EBP as a parallel to daily work increased their awareness of evidence and what might be the right things to do. They wanted to apply new evidence, but at the systems level they did not have the authority to integrate the new knowledge on their own and they lacked an efficient mechanism for ensuring timely integration into their daily work in the ward. This perspective demonstrates chal‐ lenges well known from the literature; clinical nurses striving to learn EBP and develop EB guidelines but failing to integrate the new evi‐ dence (Adib‐Hajbaghery, 2007; Aitken et al., 2011; Pitkänen, Alanen, Rantanen, Kaunonen, & Aalto, 2015; Solomons & Spross, 2011). The lack of organizational structures for adopting new guidelines may be related to an organization's limited capacity for change, which is still a highlighted barrier to EBP integration (Flodgren, Rojas‐Reyes, Cole, & Foxcroft, 2012; Sadeghi‐Bazargani et al., 2014; Solomons & Spross, 2011; Williams, Perillo, & Brown, 2015). We argue that lack of organi‐ zational support must be solved by organizational initiatives to cre‐ ate a structure for integration of new EB guidelines. Otherwise, these organizational barriers will impede healthcare professionals’ ability to increase and maintain their use of EBP, even if they are motivated and have knowledge about the application of EBP (Williams et al., 2015). A movement from the systems level to the individual level entails moving from a structured approach, where EBP is integrated and EB guidelines are applied in daily work at the ward level, to individual‐ ized patient‐tailored care informed by relevant evidence. We argue that this movement could be supported by making EBP explicit and visible at the systems level. This could be achieved by stimulating the clinical nurses’ awareness through systematic reflection and discus‐ sion about the relevance of risk assessment for the individual patients and by making explicit the research evidence underpinning the EB guidelines. Leaders might gradually integrate research activities into the nurses’ everyday routines to change the focus towards valuing re‐ search evidence as a way of providing high‐quality treatment and care for individual patients (Scott‐Findlay & Golden‐Biddle, 2005). This implies discussing the relevance of general guidelines for the individ‐ ual patient. Unless consciously addressed, individualized care could be ousted by EB standardized programmes (Norlyk, Haahr, Dreyer, & Martinsen, 2017). 4 \| DISCUSSION This study revealed three interacting dimensions of EBP integra‐ tion that may explain the complexities and challenges when nurses attempt to integrate EBP in hospital wards. We identified two ap‐ proaches (explicit EBP and implicit EBP), two positions (EBP inte‐ grated into daily work and as a parallel to daily work) and two levels of EBP (the systems level and the individual level). The interactions between the dimensions gave five meaningful combinations in this study. In the following subsections, we have organized the discus‐ sion according to the most central findings; challenges regarding EBP as a parallel to daily work, use of standardization and routiniza‐ tion to promote EBP at the systems level and the movement from the systems level to the individual level. 4.1 \| EBP as a parallel to daily work Patient centeredness and individualized care are necessary to achieve EBP in specific clinical situations (Brown, 2014; Melnyk & Fineout‐Overholt, 2015). A tool such as the huddle board sessions could be a stepping stone to focusing on individual patient situations through combining the standardized risk assessments for individual patients with the integration of patient preferences in clinical problem solving. Leadership may contribute to increased pa‐ tient‐centred care by being close to care delivery, by teaching and su‐ pervising clinicians and by addressing how quality improvement and EBP relate to the care of individual patients (Lalleman, Smid, Dikken, Lagerwey, & Schuurmans, 2017). Giving the clinical nurses and their ward leaders the opportunity to discuss and integrate research evi‐ dence into the nurses’ everyday routines and into the care of indi‐ vidual patients may stimulate the nurses to value and probably use the research findings (Scott‐Findlay & Golden‐Biddle, 2005). being introduced, support the idea of an implicit EBP integrated into daily work at the individual level. or normalization increases clinicians’ use of guidelines and stimulate guideline sustainability (Fleiszer, Semenic, Ritchie, Richer, & Denis, 2015; May, Sibley, & Hunt, 2014). However, the implicit approach to EBP represented a challenge because the nurses lacked awareness about the underlying evi‐ dence and focused rather on the tool and the standardized obser‐ vations, registrations and measures. Thus, the nurses used evidence without being conscious of it. This could constitute a possible risk, as excessive routinization may impede a person's ability to detect, interpret and handle contextual changes, thereby sustaining exist‐ ing patterns of behaviour when change is needed (Ellström, 2006). Furthermore, standardization and routinization could lead to indi‐ vidual patient needs being disregarded. Our findings visualize that a way to succeed in integrating EBP into daily work could be to estab‐ lish measures at the systems level before one can expect EBP to be established at the individual level. A tool, such as the huddle board sessions combined with measures to make and keep the underlying evidence explicit, may make this possible. We turn to this issue next. 3.3 \| An explicit EBP integrated into daily work at the individual level Based on the definition of EBP, the ideal is an explicit EBP inte‐ grated into daily work for each individual patient (i.e. alternative 3, Figure 2). In this study, the clinical nurses recognized this ideal 821 RENOLEN et al. 5 \| CONCLUSIONS AND IMPLICATIONS Asadoorian, J., Hearson, B., Satyanarayana, S., & Ursel, J. (2010). Evidence‐ based practice in healthcare: An exploratory cross‐discipline com‐ parison of enhancers and barriers. Journal for Healthcare Quality, 32, 15–22. https://doi.org/10.1111/j.1945-1474.2010.00081.x This study revealed a multidimensional EBP integration framework. The framework visualizes the complexity in clinical nurses’ daily work and the efforts that need to be put in to achieve EBP integration. Bischoff, W. R., & Hinojosa, R. H. (2013). A descriptive study of point‐ of‐care reference resource use by advanced practice RNs in Texas. Computers, Informatics, Nursing, 31, 530–538. https://doi. org/10.1097/CIN.0000000000000006 This new perspective on the dimensions of EBP integration may have implications for clinical practice and probably could also be a guide for further research. The first objective could be to establish a structure to support EBP with an appropriate tool at the systems level. In such structures, EB guidelines developed by nurses as a parallel to daily work may be easier to apply. Furthermore, organizational and individual initiatives are important steps towards making the evidence in the EB guidelines visible to the nurses in clinical patient situations. Brown, S. J. (2014). Evidence‐based nursing: The research‐practice connec‐ tion, 3rd ed. Burlington: Jones & Bartlett Learning. Cochrane, L. J., Olson, C. A., Murray, S., Dupuis, M., Tooman, T., & Hayes, S. (2007). Gaps between knowing and doing: Understanding and assessing the barriers to optimal health care. Journal of Continuing Education in the Health Professions, 27, 94–102. https://doi. org/10.1002/chp.106 Creswell, J. W. (2013). Qualitative inquiry and research design: Choosing among five approaches, 3rd ed. Thousand Oakes, CA: SAGE Publications Inc. For further research and development of the multidimensional EBP integration framework, we recommend studying more hospital wards in the clinical nurses’ daily work. As shown in this study, re‐ search use through EB guidelines in the implicit approach to EBP in‐ tegrated into daily work might contribute to improved sustainability of guidelines. This could be appropriate for further research using a tool such as a huddle board and conducting a study of participants primarily using an explicit approach to EBP integrated into daily work at the systems level to integrate EB guidelines in clinical practice. Damschroder, L. J., Aron, D. C., Keith, R. E., Kirsh, S. R., Alexander, J. A., & Lowery, J. C. (2009). Fostering implementation of health ser‐ vices research findings into practice: A consolidated framework for advancing implementation science. Implementation Science, 4(1), 50. ACKNOWLEDGEMENTS We would like to thank the participants in this study and their col‐ leagues as well for cooperation doing it possible to perform this study. Estabrooks, C. A., Midodzi, W. K., Cummings, G. G., & Wallin, L. (2007). Predicting research use in nursing organizations: A multilevel anal‐ ysis. Nursing Research, 56, S7–S23. https://doi.org/10.1097/01. nnr.0000280647.18806.98 5 \| CONCLUSIONS AND IMPLICATIONS https://doi.org/10.1186/1748-5908-4-50 DiCenso, A., Guyatt, G., & Ciliska, D. (2005). Evidence‐based nursing: A guide to clinical practice. St. Louis: Elsevier Mosby. Ellström, P.‐E. (2006). The meaning and role of reflection in infor‐ mal learning at work. In D. Boud, P. Cressey, & P. Docherty (Eds.), Productive reflection at work: Learning for changing organisations (pp. 1–10). London, UK: Routledge. Estabrooks, C. A., Floyd, J. A., Scott‐Findlay, S., O'Leary, K. A., & Gushta, M. (2003). Individual determinants of research utilization: A system‐ atic review. Journal of Advanced Nursing, 43, 506–520. https://doi. org/10.1046/j.1365-2648.2003.02748.x CONFLICT OF INTEREST Fink, R., Thompson, C. J., & Bonnes, D. (2005). Overcoming barriers and promoting the use of research in practice. Journal of Nursing Administration, 35, 121–129. https://doi. org/10.1097/00005110-200503000-00005 The authors have declared no conflict of interest. The authors have declared no conflict of interest. \| of data; (b) drafting the article or revising it critically for important intellectual content. of data; (b) drafting the article or revising it critically for important intellectual content. REFERENCES Adib‐Hajbaghery, M. (2007). Factors facilitating and inhibiting evidence‐ based nursing in Iran. 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Nurses’ evidence‐based practice beliefs and the role of evidence‐based practice mentors at university hospitals in Finland. AUTHOR CONTRIBUTIONS Fleiszer, A. R., Semenic, S. E., Ritchie, J. A., Richer, M. C., & Denis, J. L. (2015). The sustainability of healthcare innovations: A concept analysis. Journal of Advanced Nursing, 71, 1484–1498. https://doi. org/10.1111/jan.12633 All authors have agreed on the final version and meet at least one of the following criteria [recommended by the ICMJE (https://www. icmje.org/recommendations/)]: (a) substantial contributions to con‐ ception and design, acquisition of data or analysis and interpretation Flodgren, G., Rojas‐Reyes, M. X., Cole, N., & Foxcroft, D. R. (2012). Effectiveness of organisational infrastructures to promote RENOLEN et al. 823 How to cite this article: Renolen Å, Hjälmhult E, Høye S, Danbolt LJ, Kirkevold M. Evidence‐based practice integration in hospital wards—The complexities and challenges in achieving evidence‐based practice in clinical nursing. Nursing Open. 2019;6:815–823. https://doi.org/10.1002/nop2.259 Implementing, embedding and integrating practices: An outline of normalization process theory. Sociology, 43, 535–554. https://doi.org/10.1177/0038038509103208 Yoder, L. H., Kirkley, D., McFall, D. C., Kirksey, K. M., StalBaum, A. L., & Sellers, D. (2014). Staff nurses’ use of research to facilitate evidence‐ based practice. American Journal of Nursing, 114(9), 26–37. https:// doi.org/10.1097/01.NAJ.0000453753.00894.29 May, C., Sibley, A., & Hunt, K. (2014). The nursing work of hospital‐ based clinical practice guideline implementation: An explanatory systematic review using Normalisation Process Theory. 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https://openalex.org/W3047445549	https://cyberleninka.ru/article/n/application-efficiency-of-bifidobacterium-bifidum-1-and-lactobacillus-reuteri-dsm-17938-cell-free-extracts-in-vivo/pdf	Ukrainian	null	APPLICATION EFFICIENCY OF BIFIDOBACTERIUM BIFIDUM 1 AND LACTOBACILLUS REUTERI DSM 17938 CELL-FREE EXTRACTS IN VIVO	Science Review	2,020	cc-by	3,532	ISSN 2544-9346 ISSN 2544-9346 Science Review ABSTRACT ARTICLE INFO Received 21 April 2020 Accepted 08 June 2020 Published 30 June 2020 Insufficient efficiency and safety of cellular probiotics encourages the search for new effective means of correction of microecological disorders. Most of the beneficial effects of probiotics are due to the biological activity of their structural components and metabolites. Recently, great hope is pinned on postbiotic products as a means of restoring the balance of intestinal microbial populations. The data obtained in this experimental study demonstrate the ability of cell-free extracts from Bifidobacterium bifidum 1 and Lactobacillus reuteri DSM 17938 cultures, cultivated in their own disintegrates supplemented with ascorbic acid, to provide anti-infection protection and correct microecological disturbances at modeling an infectious process against a background of antibiotic-induced dysbiosis in mice. The beneficial effects of cell-free extracts showed up in the acceleration of the pathogen elimination and an increase in the number of representatives of the positive intestinal microbiota. The results of the study justify the need for further clinical trials to determine the therapeutic efficacy of cell-free extracts when included in the protocols of dysbiosis treatment. Pogorila Marina, Senior researcher, Mechnikov Institute of Microbiology and Immunology of the National Academy of Medical Sciences of Ukraine, laboratory and clinical department of molecular immunopharmacology, Ukraine, Kharkiv, ORCID ID: https://orcid.org/0000-0002-1783-9772 Polianska Valentina, Assistant Professor, Ukrainian Medical Stomatological Academy, microbiology, virology and immunology department, Ukraine, Poltava, ORCID ID: https://orcid.org/0000-0002-8727-9029 Zachepylo Svitlana, py Assistant Professor, Ukrainian Medical Stomatological Academy, otorhinolaryngology with ophthalmology department, Ukraine, Poltava, ORCID ID: https://orcid.org/0000-0002-2194-0611 Knysh Oksana, y Senior researcher, Mechnikov Institute of Microbiology and Immunology of the National Academy of Medical Sciences of Ukraine, laboratory of respiratory infections prevention, Ukraine, Kharkiv, ORCID ID: https://orcid.org/0000-0002-4105-1299 P il M i KEYWORDS probiotic cell-free extracts, Bifidobacterium bifidum 1, Lactobacillus reuteri DSM 17938, antibiotic-induced dysbiosis, murine model of intestinal staphylococcal infection. Citation: Knysh Oksana, Pogorila Marina, Polianska Valentina, Zachepylo Svitlana. (2020) Application Efficiency of Bifidobacterium Bifidum 1 and Lactobacillus Reuteri DSM 17938 Cell-Free Extracts in Vivo. Science Review. 5(32). doi: 10.31435/rsglobal_sr/30062020/7137 Copyright: © 2020 Knysh Oksana, Pogorila Marina, Polianska Valentina, Zachepylo Svitlana. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. Робота є фрагментом НДР «Мікробіологічна характеристика нових структурно- метаболітних комплексів лакто- та біфідо-пробіотиків» 0119U100686. Робота є фрагментом НДР «Мікробіологічна характеристика нових структурно- метаболітних комплексів лакто- та біфідо-пробіотиків» 0119U100686. Вступ. Поширеність дисбактеріозів серед населення Східної Європи сягає 90% [1]. До найбільш частих причин розвитку кишкового дисбіозу належать інфекції, вживання ліків та незбалансована дієта [2, 3]. Особливе значення має нераціональне застосування антибіотиків, яке сприяє не лише поширенню антибіотик-асоційованих дисбактеріозів серед населення, але й 5(32), June 2020 RS Global Science Review ISSN 2544-9346 формуванню у мікроорганізмів стійкості до антибіотиків [4]. Збільшення кількості резистентних до антимікробних засобів штамів призводить до зростання захворюваності та смертності від інфекційних захворювань [5]. Основні мікробіологічні ознаки дисбіозу: зменшення кількості корисних бактерій (Bacteroides, Bifidobacterium, Lactobacillus та ін.), експансія патобіонтів (Enterobacteriaceae, Pseudomonadaceae, Staphylococcus та ін.), зменшення мікробного різноманіття та порушення мікробного метаболізму [3, 6]. Мікроекологічний дисбаланс супроводжується порушеннями механізмів вродженого та адаптивного імунного захисту, розвитком запалення в слизовій оболонці кишечника з активацією процесів оксидації, ослабленням і підвищеною проникністю кишкового бар’єру для продуктів запалення, мікробних токсинів, підвищенням ризику мікробної транслокації та метаболічними змінами запального характеру [6]. Зазначені події є важливою патогенетичною ланкою розвитку не лише запальних захворювань кишечника, а й метаболічних (діабету та ожиріння), алергічних, автоімунних, серцево-судинних, нейродегенеративних, ракових та інших захворювань [7]. Недостатня ефективність, безпечність та складність існуючих методів корекції мікроекологічних порушень робить необхідним продовження пошуку нових ефективних засобів. Більшість сприятливих ефектів пробіотиків обумовлені біологічною активністю їх структурних компонентів та продуктів обміну [8, 9]. Тому в останні роки великі сподівання покладаються на постбіотичні продукти як засоби відновлення балансу мікробних популяцій кишечника, запобігання і лікування інфекцій шляхом збільшення колонізації слизових оболонок корисними для здоров’я коменсальними видами бактерій та посилення захисних властивостей імунної системи. KEYWORDS А пробіотичні бактерії розглядають як цінне джерело біологічно активних дериватів [10, 11]. Для налагодження промислового виробництва постбіотичних засобів необхідними є науково і економічно обґрунтований спосіб отримання пробіотичних похідних та достатня доказова база їх біологічної активності, лікувальної ефективності та безпеки. Нами були розроблені оригінальні способи одержання дериватів пробіотичних штамів Bifidobacterium bifidum 1 і Lactobacillus reuteri DSM 17938 та в експериментах in vitro отримані дані, що демонструють високу інгібіторну щодо патобіонтів, стимуляторну щодо пробіотичних бактерій та імуномодуляторну активність дериват-вмісних безклітинних екстрактів (БКЕ) [12, 13, 14, 15]. Зважаючи на те, що активність in vitro не можна ототожнювати з активністю in vivo, перед нами постало завдання дослідити ефективність застосування БКЕ при інфекційному процесі та дисбіозі у лабораторних тварин. Мета дослідження. Встановити здатність БКЕ з культур B. bifidum 1 та L. reuteri DSM 17938 з високою інгібіторною активністю щодо патобіонтів здійснювати протиінфекційний захист та корегувати мікроекологічні порушення in vivo при експериментальному моделюванні кишкової інфекції у мишей на тлі антибіотик-асоційованого дисбіозу. Матеріали і методи. Дослідження з використанням лабораторних тварин були проведені на базі віварію ДУ «ІМІ НАМН» у відповідності до положень вітчизняних і міжнародних біоетичних документів: IV «Європейської конвенції про захист хребетних тварин, які використовуються в експериментальних та інших наукових цілях» (Страсбург, ETS 123, 1986), законодавчих документів України з проведення експериментів на тваринах: «Загальні етичні принципи експериментів на тваринах», ухвалені Першим національним конгресом з біоетики (20.09.2001), методичні рекомендації [16] та були ухвалені Комітетом з біоетики Інституту. Тварини утримувалися у стандартних умовах віварію. В дослідженні були задіяні 44 миші обох статей, віком 2 місяці, вагою 22,0 ± 2,0 г, розподілені на 4 групи: 2 дослідні і 2 контрольні (позитивна і негативна), по 11 тварин у кожній. У тварин позитивної контрольної та дослідних груп було відтворено дисбактеріоз. ру у р р Для відтворення антибіотик-асоційованого дисбактеріозу кишечника у мишей в експерименті було обрано спосіб, описаний і запатентований Дармовим І. В. зі співавторами [17]. Спосіб моделювання дисбактеріозу передбачав щоденне пероральне одноразове введення тваринам впродовж 5 діб 0,1 мл розчину антибіотика з групи аміноглікозидів – гентаміцину у дозі, що перевищувала добову терапевтичну дозу аміноглікозидів для мишей при парентеральному введенні більш ніж у 4,8 рази і становила 2,9 мг (29 мг гентаміцину на 1 мл ізотонічного розчину натрію хлориду). Гентаміцин при пероральному введенні практично не всмоктується в шлунково- кишковому тракті і викликає мікроекологічні порушення в кишечнику. KEYWORDS Згідно з літературними 10 5(32), June 2020 10 RS Global Science Review ISSN 2544-9346 даними, після попередньої індукції антибіотиками дисбіозу пероральне введення мишам будь-якого штаму Staphylococcus aureus у дозі 107 КУО викликає інфекційний процес з підгострим перебігом [18]. Дизайн нашого експерименту передбачав інфікування мишей двох дослідних та позитивної контрольної групи пероральним введенням 0,5 мл суспензії S. aureus AТСС 25923 (3×108 КУО/мл) через дві доби після закінчення введення антибіотика. Миші двох дослідних груп щоденно отримували перорально БКЕ МВА та MLA у дозі 100 мкг. БКЕ МВА був отриманий з культури B. bifidum 1, а БКЕ MLA з культури L. reuteri DSM 17938, що культивувалися у власних дезінтегратах з додаванням аскорбінової кислоти (20 мг/мл) методом, описаним раніше [12]. У дослідних та позитивній контрольній групах тварин реєстрували клінічний перебіг захворювання. Критеріями оцінки перебігу інфекційного процесу були: зміни поведінки та втрата маси тіла тварин, зменшення обʼєму спожитої їжі, ознаки дисфункції кишечника (діарея або помʼякшення фекалій). Неінфікованих та інфікованих мишей зважували перед інфікуванням та кожні 24 години впродовж 7 днів після інфікування. Розраховували показник маси тіла за формулою: М/Мп × 100 %, де М – маса тварин в день зважування, Мп – маса тварин в день інфікування. р ф у Зміни складу кишкової мікрофлори у мишей, спричинених дисбіозом та інфекційним процесом, зокрема, за умови введення БКЕ, досліджували за допомогою бактеріологічного методу. Бактеріологічне дослідження фекалій здійснювали перед відтворенням антибіотик- асоційованого дисбактеріозу, через 2 доби після закінчення введення антибіотика, на 2-у, 3-ю, 5-у, 7-у добу перебігу змодельованого інфекційного процесу на тлі антибіотик-асоційованого дисбактеріозу. Визначали загальну кількість бактерій та кількість окремих представників кишкової мікрофлори: біфідо-, лактобактерій і стафілококів у фекаліях. Відібрані від кожної тварини фекалії зважували та суспендували у стерильному ізотонічному розчині натрію хлориду (1г : 10 мл). Готували ряд десятикратних розведень суспензії фекалій та здійснювали висів у поживні середовища: тіогліколеве середовище, середовище Mанна-Рогоза-Шарпа (Biolife; Італія); біфідум-середовище («Фармактив», Україна) і жовтково-сольовий агар. Посіви інкубували впродовж 24-48 годин за температури 37°С, після чого проводили підрахунок колоній, що виросли. З урахуванням маси фекалій від кожної тварини та числа колоній, що виросли, розраховували кількість бактерій на 1 г фекалій (КУО/г). Отримані показники в таблицях і на рисунках представлені як середнє значення зі стандартним відхиленням (x ± SD), n – відповідає кількості дослідних тварин. Результати аналізували за допомогою однофакторного дисперсійного аналізу ANOVA з подальшим застосуванням t-критерію Стьюдента з корекцією Бонферроні. Значення р < 0,05 вважали статистично значущим. у Результати досліджень. Результати досліджень. KEYWORDS Отримані на 2-у добу після закінчення введення гентаміцину результати бактеріологічного дослідження фекалій свідчили про розвиток дисбактеріозу кишечника у мишей (табл. 1). В середньому на 4 порядки зменшилася загальна кількість бактерій (до ~ 105 КУО/г фекалій), кількість біфідобактерій (до ~ 102 КУО/г фекалій), лактобактерій (до ~ 104 КУО/г фекалій) та спостерігалася поява стафілококів (до ~ 102 КУО/г фекалій). Таблиця 1. Кількість життєздатних бактерій у фекаліях здорових мишей та у мишей з дисбіозом на 2-у добу після закінчення введення антибіотика, (x ± SD), n=11 дисбіозом на 2-у добу після закінчення введення антибіотика, (x ± SD), n=11 Кишкова мікрофлора Групи тварин здорові з дисбіозом Заг. кількість бактерій, КУО/г фекалій 5,82 ± 0,51 × 109 2,3 ± 0,21 × 105* Біфідобактерії, КУО/г фекалій 6,19 ± 0,54 × 106 2,2 ± 0,17 × 102* Лактобактерії, КУО/г фекалій 2,30 ± 0,18 × 108 1,62 ± 0,09 × 104* Стафілококи, КУО/г фекалій – 1,0±0,12 × 102 Примітка. * – відмінності статистично значущі порівняно з показниками у групі здорових тварин. Як відомо, клінічні прояви дисбіозу можуть значно варіювати. В нашому експерименті ознаки дисфункції кишечка або зміни поведінки тварин не спостерігалися. Негативним наслідком дисбактеріозу є формування сприятливого підґрунтя для розвитку інфекційного процесу в 5(32), June 2020 11 RS Global ISSN 2544-9346 Science Review кишечнику. Наступним етапом експерименту було моделювання у мишей інфекційного процесу в кишечнику на тлі антибіотик-асоційованого дисбіозу. Після інфікування S. aureus у мишей групи позитивного контролю спостерігалося значне збільшення виділення стафілококів з фекаліями (рис. 1). На 2-у добу бактеріовиділення становило ~ 108 КУО/г фекалій. Інтенсивність виділення стафілококів з часом зменшувалася, досягаючи на 7-у добу ~ 103 КУО/г фекалій. За умови застосування БКЕ MBА та MLА показники бактеріовиділення були на 2-3 порядки нижчими порівняно з відповідними показниками у групі позитивного контролю протягом усього періоду спостереження. На відміну від групи позитивного контролю, в обох дослідних групах тварин на 7-у добу виділення стафілококів з фекаліями було відсутнім. Рис. 1. Вплив БКЕ на динаміку виділення стафілококів з фекаліями у тварин, інфікованих культурою S. aureus на тлі антибіотик-асоційованого дисбіозу, (x ± SD), n=11. Примітка. ПК – група тварин позитивного контролю; МBA, MLA – дослідні групи тварин, яким вводили БКЕ; відмінності статистично значущі порівняно з: * – показниками у групі ПК; # – показниками у дослідній групі тварин, яким вводили БКЕ МВА. культурою S. aureus на тлі антибіотик асоційованого дисбіозу, (x ± SD), n 11. Примітка. KEYWORDS ПК – група тварин позитивного контролю; МBA, MLA – дослідні групи тварин, яким вводили БКЕ; відмінності статистично значущі порівняно з: * – показниками у групі ПК; # – показниками у дослідній групі тварин, яким вводили БКЕ МВА. у ур ц у, ( ), Примітка. ПК – група тварин позитивного контролю; МBA, MLA – дослідні групи тварин, яким вводили БКЕ; відмінності статистично значущі порівняно з: * – показниками у групі ПК; # – показниками у дослідній групі тварин, яким вводили БКЕ МВА. Загальна кількість бактерій у фекаліях мишей позитивної контрольної групи впродовж всього періоду від інфікування до 7-ї доби зменшувалася з ~ 108 КУО/г фекалій до ~ 105 КУО/г. Кількість біфідо- та лактобактерій у фекаліях мишей групи позитивного контролю впродовж всього періоду спостереження була стабільною і не перевищувала ~ 102 та ~ 104 КУО/г фекалій, відповідно. Введення дослідних екстрактів інфікованим S. aureus мишам із антибіотик- асоційованим дисбіозом сприяло підвищенню загальної кількості бактерій: до ~ 107 КУО/г фекалій за умови застосування БКЕ MBА та до ~ 108 КУО/г фекалій за умови застосування БКЕ MLА. У разі введення БКЕ МВА кількість біфідобактерій зросла з ~ 102 до ~ 104 КУО/г фекалій, а кількість лактобактерій з ~ 104 до ~ 106 КУО/г фекалій. За умови застосування БКЕ МLА кількість біфідо- і лактобактерій до 7-ї доби збільшилася на 3 порядки, досягаючи кількості ~ 105 та ~ 107 КУО/г фекалій, але не досягаючи їх кількості у здорових тварин. Очевидно, для повного відновлення кількості біфідо- і лактобактерій у мишей необхідним було більш тривале введення БКЕ в експерименті. Але тенденція до відновлення нормального мікроекологічного балансу у кишковому біотопі дослідних тварин була достатньо вираженою. При порівнянні ефективності застосування БКЕ з B. bifidum та і L. reuteri за кількісними показниками бактеріовиділення та відновлення вмісту позитивної мікрофлори виявлені очевидні переваги БКЕ MLA перед БКЕ МВА. За умови застосування БКЕ MLA спостерігалися: на порядок нижчий ступінь бактеріовиділення, вищий ступінь відновлення загальної кількості бактерій, зокрема, лакто- і біфідобактерій (табл. 2). Данний ефект можна пояснити більш високим інгібіторним потенціалом БКЕ MLA по відношенню до S. aureus та більш потужною здатністю стимулювати ріст пробіотичної мікрофлори. 12 5(32), June 2020 12 RS Global ISSN 2544-9346 Science Review Таблиця 2. KEYWORDS Кількість життєздатних бактерій у фекаліях різних груп мишей на 7-у добу стафілококової інфекції на тлі антибіотик-асоційованого дисбіозу (x ± SD), n=11 Групи тварин Кількість бактерій, КУО/г- фекалій загальна біфідобактерій лактобактерій Інтактні (НК) 4,77 ± 0,38 × 109 6,54 ± 0,67 × 106 3,40 ± 0,2 × 108 Дисбіоз + інфекція (ПК) 1,33 ± 0,11 × 105# 2,9 ± 0,22 × 102# 2,17 ± 0,16 × 104# Дисбіоз + інфекція + БКЕ МВА 3,9 ± 0,17 × 107#* 3,4 ± 0,25 × 104#* 5,71 ± 0,43 × 106#* Дисбіоз + інфекція + БКЕ МLА 6,8 ± 0,38 × 108#* 2,2 ± 0,17 × 105#* 4,62 ± 0,09 × 107#* Примітка. Відмінності статистично значущі порівняно з: # – показниками у групі тварин НК, * – показниками у групі тварин ПК. Таблиця 2. Кількість життєздатних бактерій у фекаліях різних груп мишей на 7-у добу кокової інфекції на тлі антибіотик-асоційованого дисбіозу (x ± SD), n=11 Примітка. Відмінності статистично значущі порівняно з: # – показниками у групі тварин НК, * – показниками у групі тварин ПК. В ході дослідження у тварин позитивної контрольної групи спостерігалося статистично значуще зниження ваги з 1-ї по 5-у добу після інфікування, із нормалізацією даного показника тяжкості перебігу інфекційного процесу на 7-у добу спостереження (рис. 2). В 1-у та 2-гу добу у тварин цієї групи спостерігали рідкі випорожнення, на 5-у добу – пом’якшення фекалій. В 1-у добу після інфікування реєструвалося деяке зниження об’єму споживання їжі. Порівняно з групою позитивного контролю у тварин дослідних груп, які перорально отримували БКЕ МВА та MLA, спостерігалося достовірно менше зниження маси тіла в критичні перші дні інфекційного процесу, не спостерігалося змін поведінки та консистенції фекальних мас. Рис. 2. Вплив БКЕ МВА та МLА на динаміку маси тіла мишей, інфікованих S. aureus на тлі антибіотик-асоційованого дисбіозу, (x ± SD), n=11. Рис. 2. Вплив БКЕ МВА та МLА на динаміку маси тіла мишей, інфікованих S. aureus на тлі антибіотик-асоційованого дисбіозу, (x ± SD), n=11. Обговорення результатів. Таким чином, результати експериментального дослідження дозволяють стверджувати, що БКЕ з культур B. bifidum та L. reuteri, отриманих при культивуванні у власних дезінтегратах з додаванням аскорбінової кислоти, є перспективними засобами нормалізації складу кишкової мікрофлори. Про це свідчить виявлена здатність БКЕ прискорювати елімінацію збудника інфекційного процесу, сприяти відновленню кількісного вмісту представників позитивної мікрофлори кишечника та полегшувати перебіг інфекційного процесу. ЛІТЕРАТУРА 1. Дуда, О. К., Бойко, В. О., Коцюбайло, Л. П., & Голуб, А. П. (2017). Дисбиоз кишечника и его коррекция в практике врача-инфекциониста. Семейная медицина, 3 (71), 32-36. doi:10.30841/2307- 5112.3(71).2017.115931 2. Thursby, E., & Juge, N. (2017). Introduction to the human gut microbiota. Biochemical Journal, 474(11), 1823–1836. doi:10.1042/bcj20160510 j 3. Walker, W. A. (2017). Dysbiosis. The microbiota in gastrointestinal pathophysiology, 227–232. doi:10.1016/b978-0-12-804024-9.00025-2 4. Becattini, S., Taur, Y., & Pamer, E. G. (2016). Antibiotic-induced changes in the intestinal microbiota and disease. Trends in Molecular Medicine, 22(6), 458–478. doi:10.1016/j.molmed.2016.04.003 j 5. De Kraker, M. E. A., Stewardson, A. J., & Harbarth, S. (2016). Will 10 million people die a year due to antimicrobial resistance by 2050? PLOS Medicine, 13(11), e1002184. doi:10.1371/journal.pmed.1002184 6. Iacob, S., & Iacob, D. G. (2019). Infectious threats, the intestinal barrier, and its Trojan Horse: dysbiosis. Frontiers in Microbiology, 10. doi:10.3389/fmicb.2019.01676 7. Wilkins, L. J., Monga, M., & Miller, A. W. (2019). Defining dysbiosis for a cluster of chronic diseases. Scientific Reports, 9(1). doi:10.1038/s41598-019-49452-y p , ( ) y 8. Shenderov, B. A. (2013). Metabiotics: novel idea or natural development of probiotic conception. Microbial Ecology in Health & Disease 24(0) doi:10 3402/mehd v24i0 20399 p , ( ) y Shenderov, B. A. (2013). Metabiotics: novel idea or natural development of probiotic conceptio Microbial Ecology in Health & Disease, 24(0). doi:10.3402/mehd.v24i0.20399 9. Singh, A., Vishwakarma, V., & Singhal, B. (2018). Metabiotics: the functional metabolic signatures of probiotics: current state-of-art and future research priorities—metabiotics: probiotics effector molecules. Advances in Bioscience and Biotechnology, 09(04), 147–189. doi:10.4236/abb.2018.94012 10. Richards, J. L., Yap, Y. A., McLeod, K. H., Mackay, C. R., & Mariño, E. (2016). Dietary metabolites and the gut microbiota: An alternative approach to control inflammatory and autoimmune diseases. Clinical and Translational Immunology, 5(5), e82. 11. Gagliardi, A., Totino, V., Cacciotti, F., Iebba, V., Neroni, B., Bonfiglio, G., Trancassini, M., Passariello, C., Pantanella, F, & Schippa, S. (2018). Rebuilding the gut microbiota ecosystem. International Journal of Environmental Research and Public Health, 15(8), 1679. 12. Knysh, O. V., & Martynov, A. V. (2020). Potentiation of the antimicrobial effect of Lactobacillus reuteri DSM 17938 cell-free extracts by ascorbic acid. Medicni perspektivi, 25(1), 17-24. doi: 10.26641/2307- 0404.2020.1.200393 13. Knysh, O. V., Pogorila, M. S., & Voyda, Y. V. (2020). In vitro immunomodulatory effect of Bifidobacterium bifidum and Lactobacillus reuteri cell free extracts. Regulatory Mechanisms in Biosystems, 11(1), 93–97. doi:10.15421/022013 14. Knysh, O. V. (2019). KEYWORDS Одержані в даному дослідженні ефекти добре узгоджуються з отриманими нами раніше результатами вивчення бактеріотропних та імунотропних властивостей БКЕ і, очевидно, обумовлені здатністю БКЕ стимулювати проліферацію та біоплівкоутворення власної «корисної» індигенної мікрофлори, сприяти її приживленню у біоплівках, пригнічувати проліферацію умовно-патогенних бактерій та їх здатність до колонізації, а також справляти антиоксидантний вплив та чинити імуномолуляторну дію на клітини вродженого імунітету. Результати нашого дослідження підтверджують той факт, що для отримання пробіотичних ефектів не обов’язковим є збереження цілісності і життєздатності пробіотичних бактерій. Така 5(32), June 2020 13 RS Global ISSN 2544-9346 Science Review думка вже висловлювалася іншими авторами і була не безпідставною [19, 20]. Низка дослідників при застосуванні термічно інактивованих пробіотичних бактерій, супернатантів та бактеріальних екстрактів отримали пробіотичні ефекти на рівні кишечника [21, 22]. Очевидно, що ці ефекти були обумовлені біологічною активністю (імуномодуляторною, протизапальною та інгібіторною щодо патогенів) структурних компонентів та метаболітів, що вивільнялися зі зруйнованих клітин під час дезінтеграції або продукувалися бактеріями під час культивування. Висновки. Безклітинні екстракти з культур B. bifidum 1 та L. reuteri DSM 17938, одержані з додаванням аскорбінової кислоти на етапі культивування, які в попередніх експериментах in vitro виявили високу інгібіторну активність щодо патобіонтів, в даному дослідженні продемонстрували здатність здійснювати протиінфекційний захист та корегувати мікроекологічні порушення in vivo при експериментальному моделюванні кишкової інфекції на тлі антибіотик-асоційованого дисбіозу у мишей. Корисні ефекти безклітинних екстрактів полягали в прискоренні елімінації збудника інфекції та збільшенні чисельності представників позитивної мікробіоти кишкового біотопу. Результати дослідження обґрунтовують необхідність проведення подальших клінічних випробувань для визначення ефективності безклітинних екстрактів при включенні їх у протоколи лікування дисбактеріозу. К ф і і і і ій ЛІТЕРАТУРА Bifidogenic properties of cell-free extracts derived from probiotic strains of Bifidobacterium bifidum and Lactobacillus reuteri. Regulatory Mechanisms in Biosystems, 10(1), 124– 128. doi:10.15421/021919 15. Knysh, O. V. (2019). The effects of cell-free extracts derived from probiotic strains Bifidobacterium bifidum and Lactobacillus reuteri on the proliferation and biofilm formation by Lactobacillus reuteri in vitro. Zaporozhye Medical Journal, 0(6). doi:10.14739/2310-1210.2019.6.186711 14 5(32), June 2020 5(32), June 2020 14 RS Global ISSN 2544-9346 Science Review 16. Резніков, О. Г., Соловйов, А. I., Добреля, Н. В., & Стефанов, О. В. (2006). Біоетична експертиза доклінічних та інших наукових досліджень, що виконуються на тваринах: метод. рекомендації. Вісник фармакології та фармації, (7), 47-61. 17. Дармов, И. В., Чичерин, И. Ю., Ердякова, А. С., Лундовских, И. А., & Погорельский, И. П. Способ моделирования дисбактериоза кишечника у лабораторных животных. Патент № 2477894. Российская Федерация, опубл. 20.03. 2013. Бюл, (8). р у ( ) 18. Larcombe, S., Jiang, J.-H., Hutton, M. L., Abud, H. E., Peleg, A. Y., & Lyras, D. (2020). A mouse model of Staphylococcus aureus small intestinal infection. Journal of Medical Microbiology, 69(2), 290–297. doi:10.1099/jmm.0.001163 j 19. Piqué, N., Berlanga, M., & Miñana-Galbis, D. (2019). Health benefits of heat-killed (tyndallized) probiotics: An overview. International Journal of Molecular Sciences, 20(10), 2534. doi:10.3390/ijms20102534 j 20. Lopetuso, L., Graziani, C., Guarino, A., Lamborghini, A., Masi, S., & Stanghellini, V. (2017). Gelatin tannate and tyndallized probiotics: a novel approach for treatment of diarrhea. Eur Rev Med Pharmacol Sci, 21(4), 873-883. 21. Taverniti, V., & Guglielmetti, S. (2011). The immunomodulatory properties of probiotic microorganisms beyond their viability (ghost probiotics: proposal of paraprobiotic concept). Genes & Nutrition, 6(3), 261–274. doi:10.1007/s12263-011-0218-x 22. Canducci F., Armuzzi A., Cremonini F., Cammarota G., Bartolozzi F., Pola P., Gasbarrini G., Gasbarrini A. (2000). A lyophilized and inactivated culture of Lactobacillus acidophilus increases Helicobacter pylori eradication rates. Alimentary Pharmacology and Therapeutics, 14(12), 1625–1629. doi:10.1046/j.1365-2036.2000.00885.x 15 5(32), June 2020 RS Global 15
https://openalex.org/W2887590359	https://access.archive-ouverte.unige.ch/access/metadata/cfecd9b7-5be0-4a96-b414-f84f9c9f10ae/download	English	null	Search for long-lived particles in final states with displaced dimuon vertices in <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" display="inline"><mml:mi>p</mml:mi><mml:mi>p</mml:mi></mml:math> collisions at <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" display="inline"><mml:msqrt><mml:mi>s</mml:mi></mml:msqrt><mml:mo>=</mml:mo><mml:mn>13</mml:mn><mml:mtext> </mml:mtext><mml:mtext> </mml:mtext><mml:mi>TeV</mml:mi></mml:math> with the ATLAS detector	Physical review. D/Physical review. D.	2,019	cc-by	35,331	Archive ouverte UNIGE https://archive-ouverte.unige.ch Article scientifique Article 2019 Published version Open Access This is the published version of the publication, made available in accordance with the publisher’s policy. Search for long-lived particles in final states with displaced dimuon vertices in pp collisions at √s = 13 TeV with the ATLAS detector Collaborators: Akilli, Ece; Amrouche, Cherifa Sabrina; Ancu, Lucian Stefan; Benoit, Mathieu; Calace, Noemi; Clark, Allan Geoffrey; Della Volpe, Domenico; Di Bello, Francesco Armando; Dubreuil, Arnaud; Fawcett, William; Ferrere, Didier; Gadatsch, Stefan Golling, Tobias [and 19 more] How to cite ATLAS Collaboration. Search for long-lived particles in final states with displaced dimuon vertices in pp collisions at √s = 13 TeV with the ATLAS detector. In: Physical Review. D, 2019, vol. 99, n° 1. doi: Archive ouverte UNIGE https://archive-ouverte.unige.ch Article scientifique Article 2019 Published version Open Access This is the published version of the publication, made available in accordance with the publisher’s policy. Search for long-lived particles in final states with displaced dimuon vertices in pp collisions at √s = 13 TeV with the ATLAS detector Collaborators: Akilli, Ece; Amrouche, Cherifa Sabrina; Ancu, Lucian Stefan; Benoit, Mathieu; Calace, Noemi; Clark, Allan Geoffrey; Della Volpe, Domenico; Di Bello, Francesco Armando; Dubreuil, Arnaud; Fawcett, William; Ferrere, Didier; Gadatsch, Stefan Golling, Tobias [and 19 more] How to cite ATLAS Collaboration. Search for long-lived particles in final states with displaced dimuon vertices in pp collisions at √s = 13 TeV with the ATLAS detector. In: Physical Review. D, 2019, vol. 99, n° 1. doi: Archive ouverte UNIGE Article scientifique Article 2019 published version of the publication, made available in accordance with the publisher’s policy. Search for long-lived particles in final states with displaced dimuon vertices in pp collisions at √s = 13 TeV with the ATLAS detector Collaborators: Akilli, Ece; Amrouche, Cherifa Sabrina; Ancu, Lucian Stefan; Benoit, Mathieu; Calace, Noemi; Clark, Allan Geoffrey; Della Volpe, Domenico; Di Bello, Francesco Armando; Dubreuil, Arnaud; Collaborators: Akilli, Ece; Amrouche, Cherifa Sabrina; Ancu, Lucian Stefan; Benoit, Mathieu; Calace, Noemi; Clark, Allan Geoffrey; Della Volpe, Domenico; Di Bello, Francesco Armando; Dubreuil, Arnaud; Fawcett, William; Ferrere, Didier; Gadatsch, Stefan Golling, Tobias [and 19 more] I. INTRODUCTION decay to the scale of the neutron lifetime. There are a number of BSM models where long-lived particles (LLPs) arise naturally [2,3]. Supersymmetry (SUSY) [4–9] with R-parity violation [10,11], general gauge-mediated (GGM) supersymmetry breaking [12–14], and split supersymmetry [15,16] are examples where small couplings, mass scales associated with the BSM physics, or heavy mediator particles, respectively, lead to high-mass (greater than a few hundred GeV) LLPs. Scenarios with low-mass LLPs include hidden-valley models [17], stealth supersymmetry [18], and dark-sector gauge bosons [3,19]. The ATLAS and CMS experiments at the Large Hadron Collider (LHC) were conceived to address a variety of questions not fully explained within the Standard Model (SM) of particle physics. The data collected by the LHC experiments have not yet revealed evidence of physics beyond the Standard Model (BSM). As a result, there is an increased emphasis on the exploration of unusual final-state signatures that would elude the searches based on exper- imental methods aimed at prompt signatures. In many models of BSM physics there are free parameters that influence the lifetimes of the new particle states, with no strong motivation for assuming that all the particles decay promptly1 and thus give final states investigated with standard analysis techniques. Nor are there any strong demands that these are stable on the detector scale and only weakly interacting, leading to missing transverse momen- tum signatures. Particle lifetimes in the SM, for instance, span roughly 28 orders of magnitude [1], from the strong g g Events with long-lived particles may feature vertices that are significantly displaced from the proton-proton (pp) interaction point (IP). This article presents the results of a search for displaced vertices (DVs) formed by a pair of muons of opposite-sign electric charge, denoted “OS” muons. The search is designed to be sensitive to decays of LLPs with masses between 20 and 1100 GeVand DVs at distances ranging from a few centimeters to a few meters from the IP. The data sample consists of pp collisions at ﬃﬃﬃs p ¼ 13 TeV and an integrated luminosity of 32.9 fb−1 collected with the ATLAS detector at the LHC. Published by the American Physical Society under the terms of the Creative Commons Attribution 4.0 International license. Further distribution of this work must maintain attribution to the author(s) and the published article’s title, journal citation, and DOI. Funded by SCOAP3. How to cite ATLAS Collaboration. Search for long-lived particles in final states with displaced dimuon vertices in pp collisions at √s = 13 TeV with the ATLAS detector. In: Physical Review. D, 2019, vol. 99, n° 1. doi: 10.1103/PhysRevD.99.012001 © The author(s). This work is licensed under a Creative Commons Attribution (CC BY) https://creativecommons.org/licenses/by/4.0 PHYSICAL REVIEW D 99, 012001 (2019) (Received 10 August 2018; published 3 January 2019) (Received 10 August 2018; published 3 January 2019) A search is performed for a long-lived particle decaying into a final state that includes a pair of muons of opposite-sign electric charge, using proton-proton collision data collected at ﬃﬃﬃs p ¼ 13 TeV by the ATLAS detector at the Large Hadron Collider corresponding to an integrated luminosity of 32.9 fb−1. No significant excess over the Standard Model expectation is observed. Limits at 95% confidence level on the lifetime of the long-lived particle are presented in models of new phenomena including gauge-mediated supersymmetry or decay of the Higgs boson, H, to a pair of dark photons, ZD. Lifetimes in the range cτ ¼ 1–2400 cm are excluded, depending on the parameters of the model. In the supersymmetric model, the lightest neutralino is the next-to-lightest supersymmetric particle, with a relatively long lifetime due to its weak coupling to the gravitino, the lightest supersymmetric particle. The lifetime limits are determined for very light gravitino mass and various assumptions for the neutralino mass in the range 300–1000 GeV. In the dark photon model, the lifetime limits are interpreted as exclusion contours in the plane of the coupling between the ZD and the Standard Model Z boson versus the ZD mass (in the range 20–60 GeV), for various assumptions for the H →ZDZD branching fraction. DOI: 10.1103/PhysRevD.99.012001 DOI: 10.1103/PhysRevD.99.012001 Full author list given at the end of the article. 1For the purposes of this analysis, a promptly decaying particle is one with a lifetime no larger than a few tens of picoseconds. Search for long-lived particles in final states with displaced dimuon vertices in pp collisions at ﬃﬃs p = 13 TeV with the ATLAS detector M. Aaboud et al. (ATLAS Collaboration) *Full author list given at the end of the article. 1 © 2019 CERN, for the ATLAS Collaboration 1For the purposes of this analysis, a promptly decaying particle is one with a lifetime no larger than a few tens of picoseconds. g Published by the American Physical Society under the terms of 1For the purposes of this analysis, a promptly decaying particle is one with a lifetime no larger than a few tens of picoseconds. 2ATLAS uses a right-handed coordinate system with its origin at the nominal interaction point in the center of the detector and the z axis along the beam pipe. The x axis points from the IP to the center of the LHC ring, and the y axis points upwards. Cylindrical coordinates ðr; ϕÞ are used in the transverse plane, ϕ being the azimuthal angle around the z axis. The pseudorapidity is defined in terms of the polar angle θ as η ¼ −ln tan ðθ=2Þ. Angular distances are measured in units of ΔR ≡ ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ ðΔηÞ2 þ ðΔϕÞ2 p . II. ATLAS DETECTOR j j Resistive-plate chambers in the barrel and thin-gap chambers in the end cap regions provide triggering capability to the detector as well as ðη; ϕÞ position measurements with a typical spatial resolution of 5– 10 mm. A precise momentum measurement is provided by three layers of monitored drift-tube chambers (MDT), with each chamber providing six to eight η measurements along the muon trajectory. For jηj > 2, the inner layer is instrumented with a quadruplet of cathode-strip chambers (CSC) instead of MDTs. The single-hit resolution in the bending plane for the MDT and the CSC is about 80 and 60 μm, respectively. The muon chambers are aligned with a precision between 30 and 60 μm. The material between the IP and the MS ranges from approximately 100 to 190 radi- ation lengths, depending on η, and consists mostly of the calorimeters. The ATLAS detector [51,52] at the LHC covers nearly the entire solid angle around the collision point.2 It consists of an inner tracking detector surrounded by a thin superconducting solenoid, electromagnetic and hadronic calorimeters, and a muon spectrometer incorporating super- conducting toroidal magnets. The inner detector (ID) is immersed in a 2 T axial magnetic field and provides charged-particle tracking in the range jηj < 2.5. A high-granularity silicon pixel detector covers the vertex region and typically provides four measurements per track, the first hit being normally in the innermost layer. It is followed by a silicon microstrip tracker, which usually provides four two-dimensional measurement points per track. These silicon detectors are complemented by a transition radiation tracker, which enables radially extended track reconstruction up to jηj ¼ 2.0. The transition radiation tracker also provides electron identification information based on the fraction of hits (typically 30 in total) above a higher energy-deposit threshold corresponding to transition radiation. Online event selection is performed with a two-level trigger system [53]. A hardware-based level-1 trigger which uses information from the MS trigger chambers and the calorimeters is followed by a software-based trigger. The calorimeter system covers the pseudorapidity range jηj < 4.9. Within the region jηj < 3.2, electromagnetic calorimetry is provided by barrel and end cap high- granularity lead/liquid-argon (LAr) sampling calorimeters, with an additional thin LAr presampler covering jηj < 1.8 to correct for energy loss in material upstream of the calorimeters. I. INTRODUCTION Although SM decay products typically consist primarily of hadrons, due to the relatively large number of color degrees of freedom for quarks, there are notable advantages to searching for DVs using only tracks of identified muons: the design of the ATLAS muon spectrometer allows detection of dimuon DVs within an unusually large decay volume, free from backgrounds associated with 012001-1 © 2019 CERN, for the ATLAS Collaboration 2470-0010=2019=99(1)=012001(32) PHYS. REV. D 99, 012001 (2019) M. AABOUD et al. calorimeter modules optimized for electromagnetic and hadronic measurements respectively. vertices produced in interactions of hadrons with detector material [20,21]. The muon spectrometer (MS) comprises separate trigger and high-precision tracking chambers measuring the deflection of muons in a magnetic field generated by three superconducting air-core toroidal magnets, each with eight coils. The field integral of the toroids ranges between 2.0 and 6.0 T m across most of the detector. The MS is designed to detect muons in the region jηj < 2.7 and to provide momentum measurements with a relative resolu- tion better than 3% over a wide transverse momentum (pT) range and up to 10% at pT ∼1 TeV. It consists of a barrel (jηj < 1.05), with an inner radius of about 500 cm, and two end cap sections (1.05 < jηj < 2.7). Previous searches by the ATLAS Collaboration for high- mass LLPs that decay within the inner detector to give displaced dilepton vertices excluded LLP lifetimes of cτ ¼ 0.1–100 cm [22]. ATLAS has also searched for very low mass LLPs (< 10 GeV) by considering pairs of highly collimated leptons [23], with sensitivity to LLP lifetimes of cτ ¼ 0.1–20 cm. Several other LLP searches targeting a wide range of lifetimes and signatures have been conducted by the ATLAS [24–33], CMS [34–40], LHCb [41–44], CDF [45], D0 [46,47], BABAR [48], Belle [49], and ALEPH [50] collaborations. II. ATLAS DETECTOR Hadronic calorimetry is provided by a steel/ scintillator-tile calorimeter, segmented into three barrel structures within jηj < 1.7, and two copper/LAr hadronic end cap calorimeters. The solid-angle coverage is com- pleted with forward copper/LAr and tungsten/LAr A. BSM signal samples fraction for the decay ˜χ0 1 →Z ˜G can be Oð1Þ. The lifetime of the ˜χ0 1 is determined by F0 [or, alternatively, by m ˜G, according to Eq. (1)] and its mass m˜χ0 1, Monte Carlo simulated samples from two different BSM physics models are used to tune selection criteria and to evaluate signal efficiencies for use in converting signal yields into cross sections. The chosen models, a general gauge-mediated supersymmetry and dark-sector gauge boson model, represent a variety of BSM physics possibil- ities, as well as final-state topologies and kinematics, to which the analysis may be sensitive. The two processes are illustrated in Fig. 1. Samples for both models were generated with MADGRAPH5_AMC@NLO [59] using the NNPDF23LO PDF set [60] and PYTHIA8 for parton showering and hadronization. The matrix elements were calculated to next-to-leading order in the strong coupling constant. The EVTGEN generator [61] was used for weak decays of heavy-flavor hadrons. The hadronization and underlying-event parameters were set according to the A14 tune [57]. cτ˜χ0 1 ∝16πF2 0 m5 ˜χ0 1 ≈ 100 GeV m˜χ0 1 5 ﬃﬃﬃﬃﬃﬃ F0 p 300 TeV 4 × 1 cm; and hence ˜χ0 1 is long-lived (i.e., nonprompt) for ﬃﬃﬃﬃﬃﬃ F0 p ¼ 103 TeV to 104 TeV. In the GGM model, a pp interaction creates a pair of gluinos, followed by a cascade of decays leading to ˜χ0 1 →Z ˜G. A simplified model is used whereby the cascade of decays of SUSY particles is reduced to a single vertex: ˜g →qq˜χ0 1, where q represents any of the quarks lighter than the top quark, with equal probability for each. Six signal samples were generated with m˜g ¼ 1.1 TeV and ˜χ0 1 masses and lifetimes given in Table I. The value of 1.1 TeV for the gluino mass was chosen to be consistent with the value used in Ref. [22], the previous search for DVs with a GGM interpretation. The signal cross sections are calculated to next-to-leading order in the strong coupling constant, adding the resummation of soft gluon emission at next- to-leading-logarithm accuracy (NLO þ NLL) [63–67]. The nominal cross sections and their uncertainties are taken In R-parity-conserving (RPC) SUSY models where gauge interactions mediate the breaking of the supersym- metry, the gravitino ˜G acquires its mass from a “super- Higgs” mechanism and may be very light: m ˜G ¼ OðkeVÞ. III. DATA AND SIMULATED SAMPLES Proton-proton collision data, collected at the LHC during 2016, with a center-of-mass energy ﬃﬃﬃs p ¼ 13 TeV, are analyzed. After application of detector and data-quality requirements, the integrated luminosity of the data sample is 32.9 fb−1. Samples of Monte Carlo (MC) simulated events are used for studies of both the LLP signal and background processes. The detector response was simulated with GEANT4 [54,55], and the events were processed with the same reconstruction software as used for the data. The distribution of the number of additional pp collisions in the same or neighboring bunch crossings (“pileup”) is accounted for by overlaying minimum-bias events simu- lated with PYTHIA8 [56] using the A2 set of tuned parameters (tune) [57] and MSTW2008LO parton distri- bution function (PDF) set [58]. The pileup profile in the MC samples is reweighted to match the distribution observed in the data. 012001-2 PHYS. REV. D 99, 012001 (2019) SEARCH FOR LONG-LIVED PARTICLES IN FINAL … (a) (b) FIG. 1. Diagrams representing BSM processes considered signals in this article: (a) long-lived neutralino ˜χ0 1 decay in a GGM scenario, and (b) long-lived dark photons ZD produced from Higgs boson decay. The quarks, q, may have different flavors (excluding the top quark). The symbol f represents fermions lighter than half the mass of the Z boson. (a) (b) (b) (a) FIG. 1. Diagrams representing BSM processes considered signals in this article: (a) long-lived neutralino ˜χ0 1 decay in a GGM scenario, and (b) long-lived dark photons ZD produced from Higgs boson decay. The quarks, q, may have different flavors (excluding the top quark). The symbol f represents fermions lighter than half the mass of the Z boson. A. BSM signal samples mZD [GeV] cτZD [cm] BðZD →μþμ−Þ 20 50 0.1475 40 50 0.1370 40 500 0.1370 60 50 0.1066 60 500 0.1066 number of DVs across the full fiducial decay volume of the search: approximately 0 < rvtx < 400 cm. To obtain dis- tributions corresponding to a different lifetime, cτnew, each event is given a weight. The weight wi assigned to each LLP i is computed as The BSM terms in the Lagrangian density include both a hypercharge portal and a Higgs portal, providing kinetic Z-ZD mixing [i.e., mixing between Uð1ÞY and Uð1ÞD] and H-HD mixing, regulated by the small coupling parame- ters ϵ and ζ, respectively. There are two vector-boson mass eigenstates, one that is mostly ZD and another that is mostly SM Z, as well as two scalar mass eigenstates, one that is mostly HD and another that is mostly H. For simplicity, the physical (mass) states are denoted by H, HD, Z, and ZD. wiðtiÞ ¼ τgen e−ti=τgen · e−ti=τnew τnew ; where the first factor reweights the exponential decay to a constant distribution and the second factor reweights to the desired lifetime. The quantity ti is the proper decay time of the LLP and cτgen is the lifetime assumed in generating the sample. The event-level weight is the product of the weights for the two LLPs in each event. The event-level signal efficiency is then the sum of weights for all events for which at least one reconstructed dimuon vertex satisfies the selection criteria, divided by the total number of events generated. This scheme ensures that any dependence of the efficiency on the decay time of both LLPs in the event, and not just the one decaying to a dimuon final state, is properly taken into account for each choice of cτnew. In the scenario where the singlet scalar HD is heavier than the SM H boson, which means that the process H → HDHD is kinematically forbidden, and ZD is lighter than half the H mass, events with a displaced dimuon vertex signature would be observable in experiments at the LHC. The ZD bosons are produced on-shell in Higgs boson decays and decay to SM fermions due to their induced couplings to the electroweak current. A small value of ϵ (≲10−5) results in a long-lived ZD state: cτZD ∝1=ϵ2. A. BSM signal samples The mass is given by m ˜G ¼ F0 ﬃﬃﬃ 3 p MPl ¼ ﬃﬃﬃﬃﬃﬃ F0 p 100 TeV 2 × 2.4 eV; ð1Þ ð1Þ TABLE I. MC signal samples for the GGM SUSYinterpretation. For a given m˜χ0 1, the gravitino mass is chosen to give the desired lifetime. For all samples, m˜g ¼ 1100 GeV, σðpp →˜g ˜gÞ ¼ 163.5 fb, Bð˜χ0 1 →Z ˜GÞ ¼ 1.0, and BðZ →μþμ−Þ ¼ 0.03366. where ﬃﬃﬃﬃﬃﬃ F0 p is the fundamental scale of supersymmetry breaking, typically ≳100 TeV, and MPl is the Planck scale. Hence, the gravitino is the lightest supersymmetric particle (LSP). All heavier supersymmetric particles decay promptly through cascades leading to the next-to-lightest supersymmetric particle (NLSP), which then decays into the LSP gravitino via an interaction with a 1=F0 suppres- sion. The NLSP, depending on model choices, is either the lightest slepton or lightest neutralino, ˜χ0 1. For the latter case, chosen for this search and described in Ref. [62], if ˜χ0 1 has a significant wino or higgsino component the branching m˜χ0 1 [GeV] cτ˜χ0 1 [cm] 300 100 300 500 700 100 700 500 1000 100 1000 500 012001-3 PHYS. REV. D 99, 012001 (2019) M. AABOUD et al. from an envelope of cross-section predictions using differ- ent PDF sets and factorization and renormalization scales, as described in Ref. [68]. TABLE II. MC signal samples for the dark-sector interpreta- tion. For all samples, mH ¼ 125 GeV, mHD ¼ 300 GeV, σðpp →HÞ ¼ 44.1 pb (via the gluon-gluon fusion production process) and BðH →ZDZDÞ ¼ 0.10. A number of BSM theories feature a “hidden” or “dark” sector of matter that does not interact directly with SM particles but may nevertheless interact weakly with SM matter via coupling to the Higgs field. These are “Higgs portal” models that address the dark-matter problem and electroweak baryogenesis. The model considered for this search is one in which there exists a Uð1ÞD symmetry in the dark sector, and the dark vector gauge boson ZD, often called a “dark photon,” is given mass via a singlet scalar field HD that breaks the symmetry and is analogous to the Higgs field H in the visible SM sector [3,69]. A. BSM signal samples The branching fraction for H →ZDZD is determined by the value of ζ and the masses of the scalar singlets: The lifetime reweighting technique is validated by using a signal sample of a given cτgen to predict the efficiency for a different lifetime and comparing with the value directly obtained from a sample generated with that lifetime. BðH →ZDZDÞ ∝ζ m2 H jm2 HD −m2 Hj ; where values as large as 25% have not yet been ruled out by constraints from Higgs coupling fits [70,71]. For ϵ ≪1, the ZD branching fraction to muons, BðZD →μþμ−Þ, is inde- pendent of ϵ but varies with mZD [69]: from a value of 0.1475 for mZD ¼ 20 GeV to a value of 0.1066 for mZD ¼ 60 GeV. Five signal samples were generated with ZD masses and lifetimes given in Table II. The Higgs boson is produced via the gluon-gluon fusion process, assuming a cross section of 44.1 pb, calculated at next-to-next-to- leading order in the strong coupling constant, adding the resummation of soft gluon emission at next-to-next-to- leading-logarithmic accuracy [72]. The inclusion of other production processes was found to have a negligible impact on the analysis. IV. EVENT SELECTION, SIGNAL EFFICIENCIES, AND BACKGROUND ESTIMATE model). All three of the muon triggers use only measure- ments in the MS to identify muons. The thresholds for the Emiss T and collimated-dimuon triggers changed during the course of 2016 data taking. To account for these changes, the highest available thresh- old for each trigger is used and offline requirements are imposed corresponding to the thresholds listed in the table. Moreover, additional stricter requirements are imposed on the corresponding offline quantity in order to ensure that the trigger efficiency falls on the efficiency plateau. Candidate signal events are selected by identifying μþμ− pairs consistent with having been produced in a vertex displaced at least several centimeters from the IP.3 The selection criteria are designed to strongly suppress back- ground from SM processes that produce muons near the IP while efficiently accepting signal events over a wide range of LLP masses, lifetimes and velocities. To retain the greatest possible model independence, minimal require- ments are placed on other aspects of the event. For signal events with displaced dimuon vertices, the single-muon trigger efficiency falls off approximately linearly with jd0j, from a maximum of about 70% at 0 cm to approximately 10% at the fiducial limit of 400 cm, due to requirements that favor muon candidates that originate close to the IP. The calorimeter-based Emiss T trigger is employed to recover some signal efficiency. As muons leave little energy in the calorimeter and the Emiss T at the trigger level is computed only using the calorimeter signals, the Emiss T trigger is an effective muon trigger. The initial event selection is performed with a combi- nation of triggers that require either the presence of a muon candidate or large missing transverse momentum, whose magnitude is denoted Emiss T . Next, offline selection criteria are used to first identify suitable muon candidates, and then pairs of muons of opposite charge consistent with a DV. The backgrounds from all SM beam-collision and non- beam-collision processes (cosmic-ray muons or beam-halo particles) are estimated directly from the data. Finally, the number of vertices expected from background processes is compared with the observed number of vertices in data in two signal regions, distinguished by the dimuon invari- ant mass. T The collimated-dimuon trigger is based on reconstruction of muon tracks with low pT thresholds. SEARCH FOR LONG-LIVED PARTICLES IN FINAL … SEARCH FOR LONG-LIVED PARTICLES IN FINAL … PHYS. REV. D 99, 012001 (2019) TABLE III. The MC generators, hadronization, and showering software package, underlying-event simulation and PDF sets used for the simulated background events. The mass range of the low-mass Drell-Yan sample is restricted to 6 < mμμ < 60 GeV. TABLE III. The MC generators, hadronization, and showering software package, underlying-event simulation and PDF sets used for the simulated background events. The mass range of the low-mass Drell-Yan sample is restricted to 6 < mμμ < 60 GeV. TABLE III. The MC generators, hadronization, and showering software package, underlying-event simulation and PDF sets used for the simulated background events. The mass range of the low-mass Drell-Yan sample is restricted to 6 < mμμ < 60 GeV. the simulated background events. The mass range of the low-mass Drell-Yan sample is restricted to 6 < mμμ < 60 GeV. Sample MC generator Hard-process PDF Hadronization and showering Nonperturbative tune and parton-shower PDF Z þ jets POWHEG [78,79] CT10 [80] PYTHIA8 [56]+EVTGEN [61] AZNLO+CTEQ6L1 [81] Low-mass Drell-Yan POWHEG PYTHIA8+EVTGEN AZNLO+CTEQ6L1 t¯t POWHEG CT10 PYTHIA6 [82]+EVTGEN P2012 [83]+CTEQ6L1 W þ jets POWHEG CT10 PYTHIA8 AZNLO+CTEQ6L1 ZZ POWHEG-BOX v2 [84] CT10nlo PYTHIA8 AZNLO+CTEQ6L1 WW POWHEG-BOX v2 CT10nlo PYTHIA8 AZNLO+CTEQ6L1 WZ POWHEG-BOX v2 CT10nlo PYTHIA8 AZNLO+CTEQ6L1 Single top POWHEG [85,86] CT10 PYTHIA6 P2012+CTEQ6L1 3The RMS spread of the z distribution of the pp interaction vertices is 47.7 mm and the spreads in the x and y directions are less than 0.01 mm. A. Trigger requirements Events must satisfy the requirements of at least one of four different triggers in order to achieve the best possible efficiency for a wide variety of signal topologies and kinematics. The triggers used and their descriptions are given in Table IV. The first two triggers are highly efficient for signals with high-mass states that feature muons with large transverse momentum and large transverse impact parameters, d0, such as the GGM model, while the final two allow efficient selection of signals featuring low-mass states, and therefore lower-pT muons (e.g., the dark-sector IV. EVENT SELECTION, SIGNAL EFFICIENCIES, AND BACKGROUND ESTIMATE The large rates associated with the low pT thresholds are offset by requiring two muons in the MS that are within a cone of size ΔR ¼ 0.5. The efficiency of this trigger for a given signal model is strongly dependent on the magnitude of the boost of the particle decaying to the dimuon final state, as this determines the likelihood of the two muons being found within a cone of size ΔR ¼ 0.5. The trimuon trigger increases the efficiency for selecting events with particles that have a relatively large branching fraction to muons, as is the case of the ZD in the signal model explored in this article. B. SM background samples The MC simulations of background processes are used only as a guide for some of the selection criteria and for categorization of the types of background, while the background yield itself is predicted from techniques that use solely the data. The MC generators, hadronization, and showering software packages, underlying-event sim- ulation and choice of parton distribution functions are summarized in Table III. Further details about the gen- erator settings used for these processes can also be found in Refs. [73–77]. Each of the simulated background samples is scaled to correspond to an integrated luminosity of 32.9 fb−1, the size of the data sample. The signal samples were generated with values of the LLP lifetime that were chosen to provide sufficiently large 012001-4 B. Offline reconstruction and preselection All MS track parameters are expressed at the point of closest approach to the IP and their uncertainties reflect the effects of multiple Coulomb scattering in the detector material. Although the highest track reconstruction efficiency is obtained for muons originating near the IP, appreciable efficiency is obtained for muons with transverse impact parameters as large as 200 cm. In order to optimize the resolution of the track parameters, the following criteria are imposed. The MS tracks are required to have transverse momentum greater than 10 GeV, jηj < 2.5, measurements in each of the three layers of both the precision and trigger chambers, an uncertainty in the d0 measurement less than 20 cm and to not traverse regions of the MS that are poorly aligned. Muon candidates that trigger in a small set of resistive- plate chambers that can have timing jitter are rejected. This amounts to no more than 0.3% of selected muon candidates, which has a negligible effect on the signal acceptance. Interactions between beam protons and beam collimators upstream of the IP are a source of high-momentum muons, denoted beam-induced-background (BIB) muons, that can enter the ATLAS detector nearly parallel to the beam axis. Most MS tracks generated by this process are identified and rejected with the method described in Ref. [91] and results in a negligible reduction in signal efficiency. g g g p To distinguish between muon candidates that originate from prompt and nonprompt decays, the following classi- fication of MS tracks is used. Those for which a successful ID-MS combination has been made, defined by the require- ment that the angular distance between the MS track and nearest combined-muon track is less than 0.1, are referred to as “MScomb” muon candidates and the rest are referred to as “MSonly” muon candidates, as summarized in Table V. The large majority of MS tracks are MScomb, which reflects the fact that most muons are produced very close to the IP. Track reconstruction is performed independently in the ID, and an attempt is made to match each MS track with an ID track. The two matched tracks are then used as input to a combined fit that takes into account all of the ID and MS measurements, the energy loss in the calorimeter and multiple-scattering effects. During the fit, additional MS measurements may be added to or removed from the track to improve the fit quality. B. Offline reconstruction and preselection Interaction vertices from the pp collisions are recon- structed from at least two tracks with pT larger than 400 MeV that are consistent with originating from the beam-collision region in the x-y plane. Selected events are required to have at least one reconstructed interaction vertex. 3The RMS spread of the z distribution of the pp interaction vertices is 47.7 mm and the spreads in the x and y directions are less than 0.01 mm. 012001-5 PHYS. REV. D 99, 012001 (2019) M. AABOUD et al. TABLE IV. Description of triggers used to select events. The quantity ΔRμμ is the angular distance between the two muons in the collimated-dimuon trigger. Signal type Trigger Description Thresholds High mass Emiss T Missing transverse momentum Emiss T > 110 GeV Single muon Single muon restricted to the barrel region Muon jηj < 1.05 and pT > 60 GeV Low mass Collimated dimuon Two muons with small angular separation pT of muons > 15 and 20 GeV and ΔRμμ < 0.5 Trimuon Three muons pT > 6 GeV for all three muons Jet candidates are reconstructed from topological clusters [87], built from energy deposits in the calorimeters calibrated to the electromagnetic scale, using the anti-kt algorithm [88] with radius parameter R ¼ 0.4. The reconstructed jets are calibrated to the hadronic energy scale by scaling their four- momenta to the particle level [89]. The jets are required to have pT > 20 GeV and jηj < 4.4. If a jet in an event fails the “loose” jet-quality requirements of Ref. [90], the event is vetoed in order to suppress detector noise and noncollision backgrounds [90,91]. To reduce the contamination due to jets originating from pileup interactions, an additional requirement on the jet vertex tagger [92] output is made for jets with pT < 60 GeV and jηj < 2.4. momentum greater than 400 MeV, to have a minimum number of hits in each ID subsystem and to have jd0j < 1 cm. Hence, these combined-muon candidates correspond to muons produced within ∼1 cm of the x-y position of the IP. To suppress background from misidentified jets as well as from hadron decays to muons inside jets, all muon candidates are required to have at least a minimum angular separation from all jets (muon-jet overlap removal) and to satisfy track-based isolation criteria. B. Offline reconstruction and preselection The ID track is required to be within the ID acceptance, jηj < 2.5, to have transverse B. Offline reconstruction and preselection Muon-jet overlap removal is accomplished by requiring that ΔRμ−jet > min ð0.4; 0.04 þ 10 GeV=pμ TÞ for all jets in the event, where ΔRμ−jet is the angular separation between the muon candidate and the jet in consideration. The track-based isolation quantity IID ΔR¼0.4 is defined as the ratio of the scalar sum of pT of all ID tracks matched to the primary vertex, and with pT > 0.5 GeV within a cone of size ΔR ¼ 0.4 around the muon candidate, to the muon pT. To remove the contribution of the ID track forming the muon candidate (if it exists), the ID track that is nearest to and within ΔR ¼ 0.1 of the muon candidate and has a pT within 10% of the MS-track pT is not used in the sum. Muon candidates are required to have IID ΔR¼0.4 < 0.05. The muon-jet overlap and isolation requirements are removed in defining control regions used to study backgrounds described in Sec. IV F. The muon reconstruction algorithm [93] starts by finding tracks in the MS, denoted MS tracks, and extrapolating their trajectories towards the IP. All MS track parameters are expressed at the point of closest approach to the IP and their uncertainties reflect the effects of multiple Coulomb scattering in the detector material. Although the highest track reconstruction efficiency is obtained for muons originating near the IP, appreciable efficiency is obtained for muons with transverse impact parameters as large as 200 cm. In order to optimize the resolution of the track parameters, the following criteria are imposed. The MS tracks are required to have transverse momentum greater than 10 GeV, jηj < 2.5, measurements in each of the three layers of both the precision and trigger chambers, an uncertainty in the d0 measurement less than 20 cm and to not traverse regions of the MS that are poorly aligned. Interactions between beam protons and beam collimators upstream of the IP are a source of high-momentum muons, denoted beam-induced-background (BIB) muons, that can enter the ATLAS detector nearly parallel to the beam axis. Most MS tracks generated by this process are identified and rejected with the method described in Ref. [91] and results in a negligible reduction in signal efficiency. The muon reconstruction algorithm [93] starts by finding tracks in the MS, denoted MS tracks, and extrapolating their trajectories towards the IP. SEARCH FOR LONG-LIVED PARTICLES IN FINAL … TABLE V. Definition of categories of muon candidates. Tracks in the ID are reconstructed with maximum jd0j of 1 cm. Muon candidate Definition MScomb Successful ID-MS combination MSonly Standalone MS (no match with ID track) the dimuon invariant mass, mμμ, be larger than 15 GeV. The ability to determine the spatial location of the vertex varies with the pT of the muons in the vertex and the opening angle between them. The average resolutions of rvtx and zvtx are in the range of 2–3 cm. Cosmic-ray muons that pass through the detector in time with a pp collision are sometimes reconstructed as two separate MS tracks that have an opening angle of π: Δϕ ¼ π and Ση ¼ 0, where Δϕ is the difference in ϕ between the two MS tracks and Ση is the sum of their η values. Vertices formed by such MS tracks are effectively eliminated by requiring ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ ðΣηÞ2 þ ðπ −ΔϕÞ2 p > 0.1. additional criteria are applied to form signal regions (SRs) in which data are compared to background estimates, and control regions (CRs) which are used to provide those background estimates. g Within each event, all possible pairs of muon candidates, both MScomb and MSonly, are formed. For each pair, the muon candidate with the largest pT is designated the “leading” muon, while the other is designated the “sub- leading” muon. An algorithm which assumes a straight-line extrapolation of the muon trajectory from the MS inner surface towards the IP is used to determine whether or not the two muons are consistent with originating from a common vertex. The midpoint between the points of closest approach along the trajectories of the two muon candidates is taken to be the three-dimensional location of the vertex. This simple approach is sufficient for the purposes of this analysis, as the location of the putative dimuon vertex is only used in defining the geometrical acceptance of the analysis. The decay length Lvtx and projections onto the x-y plane and z axis, rvtx and zvtx respectively, are measured relative to the IP. It is convenient to sign the vertex radius rvtx according to the following definition. C. Selection of dimuon vertices The selection criteria described below are used to define a sample of dimuon vertices (preselection) to which 012001-6 PHYS. REV. D 99, 012001 (2019) SEARCH FOR LONG-LIVED PARTICLES IN FINAL … SEARCH FOR LONG-LIVED PARTICLES IN FINAL … If the angle between the projections in the x-y plane of the vertex momentum vector (the dimuon momentum vector) and the “flight direction” (the vector connecting the IP with the displaced dimuon vertex) is less than π=2 then it is assigned a positive value, otherwise it is assigned a negative value. When the LLP decays exclusively into a pair of muons or there is a small mass difference between the LLP and the dimuon state, the two vectors are typically closely aligned and the signed rvtx more often has a positive value. Examples are the dark-sector model and the GGM model for cases where there is a relatively small mass difference between the ˜χ0 1 and the Z boson. In all cases, LLPs are distinguished by relatively large values of the magnitude of signed rvtx. Backgrounds that contribute to the preselection sample include SM proton-proton collision processes as well as events with muons that are not associated with the pp collision (e.g., cosmic-ray muons). The dominant contri- butions to the former are low-mass Drell-Yan and Z þ jets processes, collectively referred to simply as DY. At small values of mμμ, dimuon vertices from multijet processes are also substantial. Dimuon vertices reconstructed in t¯t and single-top events make small contributions, while W þ jets and diboson processes are found to be negligibly small backgrounds. Distributions of mμμ and signed rvtx for opposite-charge and same-charge (SS) dimuon vertices satisfying the preselection criteria are shown in Fig. 2. Also shown are the expected contributions from the background processes discussed above. Due to the limited number of simulated multijet events, this source of background is not included in the MC distributions. Its relative contribution is expected to be dominant for SS pairs and most pronounced for OS ones at small values of mμμ, as determined from studies of events where the muon-jet overlap and muon isolation require- ments are inverted, and this is the dominant source of difference between the data and MC distributions in those regions of Fig. 2. The fraction of events in the data with multiple dimuon vertices passing the preselection criteria is 0.065%. When the LLP decays exclusively into a pair of muons or there is a small mass difference between the LLP and the dimuon state, the two vectors are typically closely aligned and the signed rvtx more often has a positive value. SEARCH FOR LONG-LIVED PARTICLES IN FINAL … Examples are the dark-sector model and the GGM model for cases where there is a relatively small mass difference between the ˜χ0 1 and the Z boson. In all cases, LLPs are distinguished by relatively large values of the magnitude of signed rvtx. The preselected dimuon vertices are divided into two regions to be used in searches for low- and high-mass signal models, which are summarized in Table VI. To further suppress the DY background in the high-mass region, where Z þ jets production dominates, and improve the search sensitivity, the transverse boost of the dimuon pair, defined as the ratio of the transverse momentum of the dimuon system to its invariant mass, is required to be larger than 2. This reduces the DY background by approximately a factor of 20, with a small reduction in the signal efficiencies, where the decay of a heavy BSM particle produces the dimuon state (a Z boson in the GGM model) with a relatively large boost. Vertices are selected as follows. To reduce combinatorial background from random track crossings, the distance of closest approach between the two straight-line extrapola- tions is required to be less than 20 cm. As the vertex position is poorly measured for tracks that are nearly parallel to each other, vertices for which the opening angle of the muon pair is less than 0.1 are rejected. Vertices are required to be within the cylindrical fiducial volume jrvtxj < 400 cm and jzvtxj < 600 cm. Background from muons with relatively low momentum in multijet events, as well as ϒ decays to dimuons, is reduced by requiring that The next sections describe the SR and CR selection criteria based on the designation of muon candidates as MScomb or MSonly. 012001-7 PHYS. REV. D 99, 012001 (2019) M. AABOUD et al. SEARCH FOR LONG-LIVED PARTICLES IN FINAL … 20 40 60 80 100 120 140 160 180 200 [GeV] μ μ m 10 2 10 3 10 4 10 5 10 6 10 7 10 Vertices / 2 GeV ATLAS = 13 TeV s -1 32.9 fb − μ + μ Drell-Yan tt Single top quark Data (a) 400 − 300 − 200 − 100 − 0 100 200 300 400 [cm] vtx Signed r 1 10 2 10 3 10 4 10 5 10 6 10 7 10 Vertices / 10 cm ATLAS = 13 TeV s -1 32.9 fb − μ + μ Drell-Yan tt Single top quark Data (b) 20 40 60 80 100 120 140 160 180 200 [GeV] μ μ m 1 10 2 10 3 10 Vertices / 2 GeV ATLAS = 13 TeV s -1 32.9 fb - μ - μ , + μ + μ Drell-Yan tt Single top quark Data (c) 400 − 300 − 200 − 100 − 0 100 200 300 400 [cm] vtx Signed r 1 10 2 10 3 10 4 10 Vertices / 10 cm ATLAS = 13 TeV s -1 32.9 fb - μ - μ , + μ + μ Drell-Yan tt Single top quark Data (d) FIG. 2. Distributions of (a) dimuon invariant mass mμμ and (b) signed vertex radius rvtx for opposite-charge dimuon vertices satisfying the preselection requirements described in the text; (c) mμμ and (d) signed rvtx for same-charge dimuon vertices satisfying the preselection requirements described in the text. The stacked histograms represent the expected contributions from various SM background processes and are derived from MC simulations scaled to an integrated luminosity of 32.9 fb−1. Multijet processes are not included in the background due to the limited number of simulated events. The contributions from these processes are most substantial at small values of mμμ. The shaded bands represent the statistical uncertainties in the simulated background. The observed distributions for data are given by the points with error bars. SEARCH FOR LONG-LIVED PARTICLES IN FINAL … 20 40 60 80 100 120 140 160 180 200 [GeV] μ μ m 10 2 10 3 10 4 10 5 10 6 10 7 10 Vertices / 2 GeV ATLAS = 13 TeV s -1 32.9 fb − μ + μ Drell-Yan tt Single top quark Data (a) 400 − 300 − 200 − 100 − 0 100 200 300 400 [cm] vtx Signed r 1 10 2 10 3 10 4 10 5 10 6 10 7 10 Vertices / 10 cm ATLAS = 13 TeV s -1 32.9 fb − μ + μ Drell-Yan tt Single top quark Data (b) Vertices / 10 cm (b) (a) 20 40 60 80 100 120 140 160 180 200 [GeV] μ μ m 1 10 2 10 3 10 Vertices / 2 GeV ATLAS = 13 TeV s -1 32.9 fb - μ - μ , + μ + μ Drell-Yan tt Single top quark Data (c) 400 − 300 − 200 − 100 − 0 100 200 300 400 [cm] vtx Signed r 1 10 2 10 3 10 4 10 Vertices / 10 cm ATLAS = 13 TeV s -1 32.9 fb - μ - μ , + μ + μ Drell-Yan tt Single top quark Data (d) Vertices / 10 cm (d) (c) FIG. 2. Distributions of (a) dimuon invariant mass mμμ and (b) signed vertex radius rvtx for opposite-charge dimuon vertices satisfying the preselection requirements described in the text; (c) mμμ and (d) signed rvtx for same-charge dimuon vertices satisfying the preselection requirements described in the text. The stacked histograms represent the expected contributions from various SM background processes and are derived from MC simulations scaled to an integrated luminosity of 32.9 fb−1. Multijet processes are not included in the background due to the limited number of simulated events. The contributions from these processes are most substantial at small values of mμμ. The shaded bands represent the statistical uncertainties in the simulated background. The observed distributions for data are given by the points with error bars. SEARCH FOR LONG-LIVED PARTICLES IN FINAL … 0 100 200 300 400 500 600 [cm] vtx L 0 0.05 0.1 0.15 Vertex efficiency = 300 GeV 1 0 χ∼ m = 700 GeV 1 0 χ∼ m = 1000 GeV 1 0 χ∼ m ATLAS Simulation = 13 TeV s high SR (a) 0 50 100 150 200 250 [cm] 0 Leading muon d 0 0.05 0.1 0.15 Vertex efficiency = 300 GeV 1 0 χ∼ m = 700 GeV 1 0 χ∼ m = 1000 GeV 1 0 χ∼ m ATLAS Simulation = 13 TeV s high SR (b) 0 100 200 300 400 500 600 [cm] vtx L 0 0.05 0.1 Vertex efficiency = 20 GeV D Z m = 40 GeV D Z m = 60 GeV D Z m ATLAS Simulation = 13 TeV s low SR (c) 0 50 100 150 200 250 0 Leading muon [cm] d 0 0.05 0.1 Vertex efficiency = 20 GeV D Z m = 40 GeV D Z m = 60 GeV D Z m ATLAS Simulation = 13 TeV s low SR (d) FIG. 3. The efficiency for selecting a displaced dimuon vertex that satisfies the requirements of SRhigh and SRlow as function of (a) and (c) generated decay length Lvtx, and (b) and (d) generated transverse impact parameter d0 of the leading muon. These efficiencies are calculated relative to all generated signal vertices and include geometrical acceptance and reconstruction effects. The distributions in (a) and (b) are derived from signal events with a long-lived neutralino, ˜χ0 1, decaying to a Z boson (with Z →μþμ−) and a gravitino. The distributions in (c) and (d) are derived from signal events with a long-lived dark photon, ZD, that decays to μþμ−. The shaded bands represent the statistical uncertainties in the efficiencies. SEARCH FOR LONG-LIVED PARTICLES IN FINAL … 0 50 100 150 200 250 [cm] 0 Leading muon d 0 0.05 0.1 0.15 Vertex efficiency = 300 GeV 1 0 χ∼ m = 700 GeV 1 0 χ∼ m = 1000 GeV 1 0 χ∼ m ATLAS Simulation = 13 TeV s high SR (b) 0 100 200 300 400 500 600 [cm] vtx L 0 0.05 0.1 0.15 Vertex efficiency = 300 GeV 1 0 χ∼ m = 700 GeV 1 0 χ∼ m = 1000 GeV 1 0 χ∼ m ATLAS Simulation = 13 TeV s high SR (a) Vertex efficiency (a) (b) 0 100 200 300 400 500 600 [cm] vtx L 0 0.05 0.1 Vertex efficiency = 20 GeV D Z m = 40 GeV D Z m = 60 GeV D Z m ATLAS Simulation = 13 TeV s low SR (c) 0 50 100 150 200 250 0 Leading muon [cm] d 0 0.05 0.1 Vertex efficiency = 20 GeV D Z m = 40 GeV D Z m = 60 GeV D Z m ATLAS Simulation = 13 TeV s low SR (d) Vertex efficiency Vertex efficiency Vertex efficiency (c) (d) FIG. 3. The efficiency for selecting a displaced dimuon vertex that satisfies the requirements of SRhigh and SRlow as function of (a) and (c) generated decay length Lvtx, and (b) and (d) generated transverse impact parameter d0 of the leading muon. These efficiencies are calculated relative to all generated signal vertices and include geometrical acceptance and reconstruction effects. The distributions in (a) and (b) are derived from signal events with a long-lived neutralino, ˜χ0 1, decaying to a Z boson (with Z →μþμ−) and a gravitino. The distributions in (c) and (d) are derived from signal events with a long-lived dark photon, ZD, that decays to μþμ−. The shaded bands represent the statistical uncertainties in the efficiencies. parameters of the reconstructed MS tracks and the distri- butions are normalized to the expected yields in the signal regions. muons, while the loss at large values reflects the lower MS reconstruction efficiency for tracks with trajectories that do not extrapolate back to a region close to the IP. The value of Lvtx where maximum efficiency is achieved is different for each choice of ZD mass due to the large differences in boost. D. Signal regions and signal efficiency Signal is characterized by vertices where both muon candidates are MSonly. This requirement unavoidably leads to a reduction in efficiency for decays close to the IP. Displaced-vertex analyses that make use of ID tracks [22] effectively recover such signal events. Two orthogonal signal regions are used to increase the sensitivity to low- and high-mass signal models, SRlow and SRhigh, respec- tively. The two regions are summarized in Table VI. For both SRs, the muons are required to have opposite charge. TABLE VI. Selection criteria for low- and high-mass regions, in addition to the preselection requirements described in the text. The definitions of the low- and high-mass signal regions are also given. given. Selection Low mass High mass pμ T [GeV] > 10 > 20 mμμ [GeV] 15–60 > 60 Dimuon transverse boost > 2 SRlow SRhigh Muon candidates Both MSonly Both MSonly Muon candidate charge Opposite charge Opposite charge The product of acceptance and reconstruction efficiency determined from simulated signal events is shown in Fig. 3 as a function of generated Lvtx and leading muon d0, for the GGM model and for the dark-sector model. The lower efficiency observed for small Lvtx or small jd0j (more apparent in the ZD models) is due to the veto on MScomb 012001-8 SEARCH FOR LONG-LIVED PARTICLES IN FINAL … PHYS. REV. D 99, 012001 (2019) E. Control regions and background estimation 1 − 10 1 10 2 10 3 10 4 10 5 10 6 10 7 10 [cm] τ c 0 0.05 0.1 Total efficiency = 300 GeV 1 0 χ∼ m = 700 GeV 1 0 χ∼ m = 1000 GeV 1 0 χ∼ m ATLAS Simulation = 13 TeV s high SR (a) 1 − 10 1 10 2 10 3 10 4 10 5 10 6 10 7 10 [cm] τ c 0 0.05 Total efficiency = 20 GeV D Z m = 40 GeV D Z m = 60 GeV D Z m ATLAS Simulation = 13 TeV s low SR (b) Total efficiency (b) (a) FIG. 4. Overall event-level efficiencies after the signal-region selections (combining trigger and offline selection), as a function of the lifetime of the long-lived BSM particle, for (a) the GGM model and (b) the dark-sector model. The shaded bands represent the statistical uncertainties only. E. Control regions and background estimation 20 40 60 80 100 120 140 160 180 200 [GeV] μ μ m 0 1 2 3 4 5 6 7 Vertices / 2 GeV ATLAS Simulation = 13 TeV s -1 32.9 fb high SR = 1 m τ = 300 GeV, c 1 0 χ∼ m = 1 m τ = 700 GeV, c 1 0 χ∼ m = 1 m τ = 1000 GeV, c 1 0 χ∼ m (a) 400 − 300 − 200 − 100 − 0 100 200 300 400 [cm] vtx Signed r 1 − 10 1 10 2 10 Vertices / 10 cm ATLAS Simulation = 13 TeV s -1 32.9 fb high SR = 1 m τ = 300 GeV, c 1 0 χ∼ m = 1 m τ = 700 GeV, c 1 0 χ∼ m = 1 m τ = 1000 GeV, c 1 0 χ∼ m (b) 20 30 40 50 60 70 80 [GeV] μ μ m 0 200 400 600 800 1000 1200 1400 Vertices / 2 GeV ATLAS Simulation = 13 TeV s -1 32.9 fb low SR = 0.5 m τ = 20 GeV, c D Z m = 0.5 m τ = 40 GeV, c D Z m = 0.5 m τ = 60 GeV, c D Z m (c) 400 − 300 − 200 − 100 − 0 100 200 300 400 [cm] vtx Signed r 1 10 2 10 3 10 4 10 Vertices / 10 cm ATLAS Simulation = 13 TeV s -1 32.9 fb low SR = 0.5 m τ = 20 GeV, c D Z m = 0.5 m τ = 40 GeV, c D Z m = 0.5 m τ = 60 GeV, c D Z m (d) FIG. 5. Distributions derived from MC simulations of (a) dimuon invariant mass mμμ and (b) vertex radius rvtx for signal vertices in SRhigh with a long-lived neutralino, ˜χ0 1 (m˜χ0 1 ¼ 300, 700, and 1000 GeV and cτ˜χ0 1 ¼ 100 cm) decaying to a Z boson (with Z →μþμ−) and a gravitino; (c) mμμ and (d) rvtx for signal vertices in SRlow with a long-lived dark photon, ZD (mZD ¼ 20, 40, and 60 GeV; and cτZD ¼ 50 cm), that decays to μþμ−. The shaded bands represent the statistical uncertainties. E. Control regions and background estimation The distributions are normalized to the expected yields in the signal regions for m˜g ¼ 1100 GeV, σðpp →˜g ˜gÞ ¼ 0.1635 pb, Bð˜χ0 1 →Z ˜GÞ ¼ 1.0, and BðZ →μþμ−Þ ¼ 0.03366; and mH ¼ 125 GeV, mHD ¼ 300 GeV, σðpp →HÞ ¼ 44.1 pb, BðH →ZDZDÞ ¼ 100%, and the value of BðZD →μþμ−Þ varying between 0.1475 and 0.1066 for the range mZD ¼ 20–60 GeV. 400 − 300 − 200 − 100 − 0 100 200 300 400 [cm] vtx Signed r 1 − 10 1 10 2 10 Vertices / 10 cm ATLAS Simulation = 13 TeV s -1 32.9 fb high SR = 1 m τ = 300 GeV, c 1 0 χ∼ m = 1 m τ = 700 GeV, c 1 0 χ∼ m = 1 m τ = 1000 GeV, c 1 0 χ∼ m (b) 20 40 60 80 100 120 140 160 180 200 [GeV] μ μ m 0 1 2 3 4 5 6 7 Vertices / 2 GeV ATLAS Simulation = 13 TeV s -1 32.9 fb high SR = 1 m τ = 300 GeV, c 1 0 χ∼ m = 1 m τ = 700 GeV, c 1 0 χ∼ m = 1 m τ = 1000 GeV, c 1 0 χ∼ m (a) (b) (a) 20 30 40 50 60 70 80 [GeV] μ μ m 0 200 400 600 800 1000 1200 1400 Vertices / 2 GeV ATLAS Simulation = 13 TeV s -1 32.9 fb low SR = 0.5 m τ = 20 GeV, c D Z m = 0.5 m τ = 40 GeV, c D Z m = 0.5 m τ = 60 GeV, c D Z m (c) 400 − 300 − 200 − 100 − 0 100 200 300 400 [cm] vtx Signed r 1 10 2 10 3 10 4 10 Vertices / 10 cm ATLAS Simulation = 13 TeV s -1 32.9 fb low SR = 0.5 m τ = 20 GeV, c D Z m = 0.5 m τ = 40 GeV, c D Z m = 0.5 m τ = 60 GeV, c D Z m (d) Vertices / 10 cm (c) (d) FIG. 5. E. Control regions and background estimation Dimuon vertices are categorized as described in Table VII. The observed yields of same-charge dimuon vertices in all four regions A, B, C, and D are used to estimate the background yields in the SRs due to muons produced more than about a centimeter from the IP, referred to as nonprompt muons. The observed yields in the opposite-charge B, C, and D CRs are used to predict the background yield from SM processes that produce prompt muons (those produced within about a centimeter of the IP) in the SRs (opposite- charge dimuon vertices in region A). Muons from decays of hadrons containing b and c quarks are, within the context of this analysis, considered to be prompt muons. The total event-level efficiencies, including trigger and offline selection criteria, as functions of the lifetime of the LLP, are shown in Fig. 4 and maximum values are in the cτ region 20–50 cm. The reweighted samples, as described in Sec. III, are used to estimate the efficiencies for values of the lifetime which were not used in generating the simulated samples. This event-level efficiency is defined as the fraction of generated events that are selected and have at least one dimuon DV. Distributions of mμμ and signed rvtx for signal vertices in simulated events, for both SRhigh and SRlow, are displayed in Fig. 5. The vertex properties are computed using the 012001-9 PHYS. REV. D 99, 012001 (2019) M. AABOUD et al. 1 − 10 1 10 2 10 3 10 4 10 5 10 6 10 7 10 [cm] τ c 0 0.05 0.1 Total efficiency = 300 GeV 1 0 χ∼ m = 700 GeV 1 0 χ∼ m = 1000 GeV 1 0 χ∼ m ATLAS Simulation = 13 TeV s high SR (a) 1 − 10 1 10 2 10 3 10 4 10 5 10 6 10 7 10 [cm] τ c 0 0.05 Total efficiency = 20 GeV D Z m = 40 GeV D Z m = 60 GeV D Z m ATLAS Simulation = 13 TeV s low SR (b) FIG. 4. Overall event-level efficiencies after the signal-region selections (combining trigger and offline selection), as a function of the lifetime of the long-lived BSM particle, for (a) the GGM model and (b) the dark-sector model. The shaded bands represent the statistical uncertainties only. F. Nonprompt muon vertices Nonprompt muons are those for which no matching ID track is expected. Examples of such sources of background nonprompt muons are cosmic-ray muons, BIB muons, fake MS tracks generated from random hit combinations, and those arising from pion or kaon decay. depending on the region (A, B, C, or D). As no statistical difference in Rπ=K q is observed for the low- and high-mass regions, a single value is used for both regions. For dimuon vertices composed of BIB or cosmic-ray muons, the muon charges are assumed to be entirely uncorrelated: Rcos =BIB q ¼ 1.0. Cosmic-ray and BIB muons will usually not produce ID tracks, as they rarely pass close enough to the IP to produce an ID track that satisfies the track reconstruction criteria, in particular the jd0j < 1 cm requirement. As a result, they produce mostly MSonly muon candidates. As described in Sec. IV B, dimuon vertices reconstructed from a single cosmic-ray or BIB muon that generates two MS tracks are effectively eliminated in the preselection by taking advan- tage of the fact that the angle between the two tracks will be nearly 180°. On the other hand, vertices formed when a single MS track from a cosmic-ray or BIB muon is paired with an unrelated muon candidate produced from the pp collision will satisfy these selection criteria and more readily contribute to the background. Since the relative composition of the nonprompt dimuon background is unknown, the average of Rπ=K q and Rcos =BIB q is assumed as the nominal value, Rq, with an uncertainty that is half the difference between the two values; this is shown in Table VIII (Rq ¼ 1.24 0.24 for the SRs). The numbers of OS nonprompt vertices in the regions A, B, C, and D are predicted using the number of SS vertices in each region and the appropriate Rq, as described above: Nnonprompt i ¼ Rq;iNSS i , where NSS i is the number of SS vertices observed in region i (i ¼ A, B, C, or D) and Rq;i is the charge ratio for region i. q The predicted yields of nonprompt dimuon vertices for both SRs are given in Table VIII, where the uncertainty in Rq is treated as a systematic uncertainty added in quad- rature to the statistical uncertainties. F. Nonprompt muon vertices Pions and kaons have relatively large lifetimes and feature large branching fractions to final states with one muon. Such decays often result in either no ID track being reconstructed, due to the requirement of at least a minimum number of ID hits, or the ID track of the pion/kaon failing to be matched to the muon MS track. In both of these two cases, a MSonly muon candidate will be produced. SEARCH FOR LONG-LIVED PARTICLES IN FINAL … TABLE VIII. Observed number of SS vertices NSS, charge ratio Rq;SR and predicted yields of nonprompt dimuon vertices Nnonprompt SR in each signal region. The uncertainty in Nnonprompt SR combines in quadrature the statistical uncertainty, derived from the observed number of SS vertices, and the uncertainty in the charge ratio. The statistical uncertainty on NSS for the case where the central value is measured to be zero is taken to be the 68% one-sided Poisson confidence-level interval: þ1.14. TABLE VIII. Observed number of SS vertices NSS, charge ratio Rq;SR and predicted yields of nonprompt dimuon vertices Nnonprompt SR in each signal region. The uncertainty in Nnonprompt SR combines in quadrature the statistical uncertainty, derived from the observed number of SS vertices, and the uncertainty in the charge ratio. The statistical uncertainty on NSS for the case where the central value is measured to be zero is taken to be the 68% one-sided Poisson confidence-level interval: þ1 14 TABLE VII. Description of four regions used in estimating background yields. The ordering of the muon candidates in the description of the dimuon vertex is leading muon first, then subleading muon. Region name Muon candidates in vertex A MSonly-MSonly B MSonly-MScomb C MScomb-MSonly D MScomb-MScomb Region NSS Rq;SR Nnonprompt SR SRlow 11 1.24 0.24 13.6 4.9 SRhigh 0 1.24 0.24 0.0þ1.4 −0.0 E. Control regions and background estimation Distributions derived from MC simulations of (a) dimuon invariant mass mμμ and (b) vertex radius rvtx for signal vertices in SRhigh with a long-lived neutralino, ˜χ0 1 (m˜χ0 1 ¼ 300, 700, and 1000 GeV and cτ˜χ0 1 ¼ 100 cm) decaying to a Z boson (with Z →μþμ−) and a gravitino; (c) mμμ and (d) rvtx for signal vertices in SRlow with a long-lived dark photon, ZD (mZD ¼ 20, 40, and 60 GeV; and cτZD ¼ 50 cm), that decays to μþμ−. The shaded bands represent the statistical uncertainties. The distributions are normalized to the expected yields in the signal regions for m˜g ¼ 1100 GeV, σðpp →˜g ˜gÞ ¼ 0.1635 pb, Bð˜χ0 1 →Z ˜GÞ ¼ 1.0, and BðZ →μþμ−Þ ¼ 0.03366; and mH ¼ 125 GeV, mHD ¼ 300 GeV, σðpp →HÞ ¼ 44.1 pb, BðH →ZDZDÞ ¼ 100%, and the value of BðZD →μþμ−Þ varying between 0.1475 and 0.1066 for the range mZD ¼ 20–60 GeV. 012001-10 012001-10 PHYS. REV. D 99, 012001 (2019) G. Prompt muon vertices The number of dimuon vertices in each of the SRs arising from prompt muon processes is estimated from the observed yields in the OS low-mass and high-mass B, C, and D control regions. Sources of such background in the SRs include SM processes that produce prompt muons that are reconstructed as MSonly due to detector or reconstruction effects, such as ID inefficiencies, or poorly reconstructed combined muons, collectively described as failed combined muon reconstruction. Vertices that contain one or more nonprompt muons are referred to as “nonprompt vertices." If the vertex contains a cosmic-ray or BIB muon paired with an unrelated muon candidate, the charges of the two MS tracks will be largely uncorrelated. However, some charge correlation is expected in vertices containing muons from pion/kaon decay, because the pion/kaon is produced from the same pp collision that produces the other muon in the vertex. For the latter, the charge correlation is studied by measuring the ratio of OS to SS dimuon vertices, Rπ=K q , in the data. As muons from pion/kaon decay are not expected to be isolated from jets, the quantity Rπ=K q is measured in a sample of dimuon vertices where the selection criteria are those of the preselection, except that the jet-muon overlap and isolation requirements are removed for both muons. The value ranges from 1.39 0.09 to 1.55 0.03, To avoid double-counting of dimuons from nonprompt processes, the estimated number of nonprompt OS vertices in each region is subtracted: Nprompt i ¼ NOS i −Nnonprompt i ; i ¼ B; C; D; where NOS i is the number of OS vertices in region i, Nnonprompt i is the number of OS nonprompt vertices in region where NOS i is the number of OS vertices in region i, Nnonprompt i is the number of OS nonprompt vertices in region 012001-11 PHYS. REV. D 99, 012001 (2019) M. AABOUD et al. i (described in Sec. IV F) and Nprompt i is the estimated number of opposite-charge vertices from prompt processes in region i. The quantity Nprompt B (Nprompt C ) is the estimated number of OS vertices from prompt processes with leading (subleading) muons that fail the combined reconstruction and are identified as MSonly, while the other muon candidate is identified as being MScomb. H. Total background The predicted number of nonprompt muon vertices is summed with the predicted number of prompt muon vertices from SM background processes to give the predicted total number of background vertices in each of the SRs: 13.8 4.9 and 0.50þ1.41 −0.07 for SRlow and SRhigh, respectively, where the uncertainties include the statistical components and the systematic uncertainty in Rq. fL ¼ Nprompt B =Nprompt D ; fS ¼ Nprompt C =Nprompt D : fL ¼ Nprompt B =Nprompt D ; fS ¼ Nprompt C =Nprompt D : q The reliability of the background estimation method is validated by applying it to both the sum of the simulated background samples and to a high-mass validation region in the data. The predicted number of dimuon vertices in the simulated sample agrees with the number of observed vertices, to within the statistical precision, in both the low- and high-mass signal regions. As the simulated samples do not include multijet processes or cosmic muon back- grounds, this is primarily a validation of the technique to estimate the background from prompt dimuon vertices. The validation region in data comprises dimuon vertices that satisfy all of the selection criteria of the high-mass region, with the exception that the requirement on the transverse boost of the dimuon system is inverted: it is required to have a value less than two, which ensures that there is negligible contribution from signal processes. The results are given in Table X. These two studies validate the method within the statistical precision. The leading transfer factor multiplied by Nprompt C , or, alternatively, the subleading transfer factor multiplied by Nprompt B , thus gives for prompt muon processes the predicted number of OS vertices in region A, the SRs in this case: Nprompt A ¼ fL · Nprompt C ¼ fS · Nprompt B : The yields in the various regions are summarized in Table IX. The vertices in all CRs are used to verify that the designation of one of the MS tracks in the vertex as MScomb or MSonly is independent of the designation of the other as MScomb or MSonly (the measured correlation is negligibly small, < 0.0015). The predicted number of vertices from prompt background processes in the low- and high-mass SRs are 0.14 0.22 and 0.504 0.070, respec- tively, where the uncertainties are statistical only. G. Prompt muon vertices The quantity Nprompt D is the estimated number of OS vertices from prompt processes with muon candidates that pass the combined reconstruction and are both identified as being MScomb. With these definitions, the leading and subleading “transfer factors” are defined as follows: i (described in Sec. IV F) and Nprompt i is the estimated number of opposite-charge vertices from prompt processes in region i. The quantity Nprompt B (Nprompt C ) is the estimated number of OS vertices from prompt processes with leading (subleading) muons that fail the combined reconstruction and are identified as MSonly, while the other muon candidate is identified as being MScomb. The quantity Nprompt D is the estimated number of OS vertices from prompt processes with muon candidates that pass the combined reconstruction and are both identified as being MScomb. With these definitions, the leading and subleading “transfer factors” are defined as follows: and the transfer factors and predictions of Nprompt in the SRs are recomputed. For both the low-mass and high-mass selection, the sum over the predicted prompt background yields in each bin is consistent with the nominal value. VI. RESULTS The predicted number of nonprompt muon vertices is summed with the predicted number of prompt muon vertices from SM background processes to give the predicted total number of background vertices, Nbkgd, in each SR. The predicted background yields, along with the number of observed vertices in the data, are summarized in Table XI. The distributions of mμμ and rvtx are shown in Fig. 6 for the observed vertices in the two signal regions. Each dimuon vertex is in a separate event, and therefore the number of events observed is equivalent to the number of vertices. The dimuon vertex with the highest mass has mμμ ¼ 381 GeV, rvtx ¼ −220 cm, and zvtx ¼ 99 cm. Close inspection of the event reveals characteristics of being cosmic in origin. The observation of one such dimuon vertex in SRhigh is consistent, within the uncer- tainties, with the nonprompt background estimate of Nnonprompt ¼ 0.0þ1.4 −0.0. The other vertex in SRhigh has a mass compatible with the decay of the SM Z. The dimuon vertex with the largest value of rvtx is in SRlow and has mμμ ¼ 46 GeV, rvtx ¼ 223 cm, and zvtx ¼ 56 cm. The vertex is formed by an MS track passing through the top of the detector combined with another MS track passing through the bottom of the detector, with an angle of nearly 180° between them. This vertex is likely a cosmic- ray muon that narrowly survived the cosmic-ray veto criteria described in Sec. IV C. The systematic uncertainty from pileup effects is deter- mined by varying the pileup reweighting of simulated signal events in a manner that spans the expected uncer- tainty. This results in a systematic uncertainty of 0.2% in the signal efficiency. As no significant excess of vertices over the SM back- ground expectation is observed, 95% confidence-level (C.L.) upper limits on the signal event yields and produc- tion cross sections are calculated for various values of the proper decay distance cτ of the long-lived particle in each of the two BSM scenarios considered.4 The limits are calculated using the CLS prescription [96] with a Poisson likelihood used as the test statistic. Uncertainties in the signal efficiency and background expectation are included g y The methods used to estimate the background are entirely data-driven, with statistical uncertainties arising from the numbers of events in the CRs. 4For events that are selected exclusively by the trimuon trigger the observed signal yield will have a quadratic dependence on BðZD →μþμ−Þ. The collimated-dimuon trigger efficiency domi- nates over the trimuon trigger efficiency for the values of mZD considered in this paper. SEARCH FOR LONG-LIVED PARTICLES IN FINAL … TABLE XI. Predicted nonprompt Nnonprompt, prompt Nprompt, and total Nbkgd background yields and number of observed vertices Nobs in data in SRlow and SRhigh. The uncertainties in the predicted background yields are statistical uncertainties and systematic uncertainties added in quadrature. The uncertainty in the 2016 integrated luminosity is 2.2%. It is derived, following a methodology similar to that detailed in Ref. [94], and using the LUCID-2 detector for the baseline luminosity measurements [95], from calibra- tion of the luminosity scale using x-y beam-separation scans. Yield SRlow SRhigh Nnonprompt 13.6 4.9 0.0þ1.4 −0.0 Nprompt 0.1þ0.2 −0.1 0.50 0.07 Nbkgd 13.8 4.9 0.50þ1.42 −0.07 Nobs 15 2 Sources of systematic uncertainties in the signal effi- ciencies include possible mismodeling of the trigger and MS efficiencies and pileup effects in the MC simulation. For the high-mass SR, the uncertainty associated with trigger and MS track reconstruction efficiency is deter- mined by comparing the observed yields in the data with MC simulation of Z þ jets events, using the selection criteria of the OS B, C, and D control regions and the additional requirement 70 < mμμ < 110 GeV. The differ- ence between the yields in data and the simulated back- ground samples is used to assign a systematic uncertainty of 1% to the combined trigger and MS track-reconstruction efficiency. For the low-mass SR, the efficiency of the trigger and MS track reconstruction is compared between MC simulation and data for J=ψ →μμ events, using a tag- and-probe technique. The efficiency is measured as a function of the angular separation between the two muons, and a maximum deviation of 6% is observed. This differ- ence is taken as an uncertainty in the signal efficiency. The agreement between data and MC simulation for the reconstruction efficiency for MS tracks with large impact parameters was studied by comparing a cosmic-ray muon simulation to cosmic-ray muon candidates in data [22]. Comparing the ratio of the muon candidate d0 distributions in the two samples yields a d0-dependent efficiency correction that is between 1% and 2.5%, with an average value of 1.5%. The systematic uncertainty on MS track reconstruction associated with this procedure is taken from the statistical uncertainty, and is 2% per track in the vertices. V. SYSTEMATIC UNCERTAINTIES As a cross-check, the B, C, and D control regions are subdivided into bins of either muon pT or muon η and ϕ, The systematic uncertainties are described in detail below. They include those in the integrated luminosity, used in converting signal yields to cross sections; the background estimate, derived entirely from the data; and the signal efficiency, determined from MC simulations. All systematic uncertainties are treated as uncorrelated. TABLE IX. The number of opposite-charge vertices, NOS, the number of same-charge vertices, NSS, the estimated ratio of opposite-charge to same-charge nonprompt dimuon vertices, Rq and the estimated number of prompt dimuon vertices Nprompt in each of the control regions B, C, and D. The values of Nprompt are obtained by subtracting the product of NSS and Rq from NOS. The quoted uncertainties in Nprompt include the statistical component and the systematic uncertainty in R TABLE X. Predicted nonprompt Nnonprompt VR , prompt Nprompt VR , and total Nbkgd VR background yields and number of observed vertices Nobs VR in data in the high-mass validation region. The uncertainty in Nprompt VR includes the statistical component and the systematic uncertainty in Rq. Yield High-mass validation region Nnonprompt VR 2.5þ2.3 −1.6 Nprompt VR 7.20 0.25 Nbkgd VR 9.7þ2.3 −1.6 Nobs VR 13 TABLE X. Predicted nonprompt Nnonprompt VR , prompt Nprompt VR , and total Nbkgd VR background yields and number of observed vertices Nobs VR in data in the high-mass validation region. The uncertainty in Nprompt VR includes the statistical component and the systematic uncertainty in Rq. and the systematic uncertainty in Rq. Region NOS NSS Rq Nprompt Low-mass region B 124 63 1.28 0.28 43 23 C 451 335 1.20 0.20 49þ74 −49 D 19599 3220 1.20 0.20 15700 660 High-mass region B 120 0 1.28 0.28 120 11 C 92 0 1.20 0.20 92 10 D 21940 24 1.20 0.20 21900 150 012001-12 PHYS. REV. D 99, 012001 (2019) VI. RESULTS 1 − 10 1 10 2 10 3 10 4 10 5 10 6 10 7 10 [cm] τ c 4 − 10 3 − 10 2 − 10 1 − 10 1 10 2 10 [pb] B x σ 95% CL upper limit (1100 GeV) = 0.163 pb g~ σ ) = 1 G~ Z → 1 0χ∼ ( B = 700 GeV 1 0 χ∼ m Observed Expected σ 1 ± σ 2 ± ATLAS = 13 TeV s -1 32.9 fb 1 − 10 1 10 2 10 3 10 4 10 5 10 6 10 7 10 [cm] τ c 4 − 10 3 − 10 2 − 10 1 − 10 1 10 2 10 [pb] B x σ 95% CL upper limit (1100 GeV) = 0.163 pb g~ σ ) = 1 G~ Z → 1 0χ∼ ( B = 300 GeV 1 0 χ∼ m Observed Expected σ 1 ± σ 2 ± ATLAS = 13 TeV s -1 32.9 fb 1 − 10 1 10 2 10 3 10 4 10 5 10 6 10 7 10 [cm] τ c 4 − 10 3 − 10 2 − 10 1 − 10 1 10 2 10 [pb] B x σ 95% CL upper limit (1100 GeV) = 0.163 pb g~ σ ) = 1 G~ Z → 1 0χ∼ ( B = 700 GeV 1 0 χ∼ m Observed Expected σ 1 ± σ 2 ± ATLAS = 13 TeV s -1 32.9 fb 1 − 10 1 10 2 10 3 10 4 10 5 10 6 10 7 10 [cm] τ c 4 − 10 3 − 10 2 − 10 1 − 10 1 10 2 10 [pb] B x σ 95% CL upper limit (1100 GeV) = 0.163 pb g~ σ ) = 1 G~ Z → 1 0χ∼ ( B = 300 GeV 1 0 χ∼ m Observed Expected σ 1 ± σ 2 ± ATLAS = 13 TeV s -1 32.9 fb (a) 1 − 10 1 10 2 10 3 10 4 10 5 10 6 10 7 10 [cm] τ c 4 − 10 3 − 10 2 − 10 1 − 10 1 10 2 10 [pb] B x σ 95% CL upper limit (1100 GeV) = 0.163 pb g~ σ ) = 1 G~ Z → 1 0χ∼ ( B = 1000 GeV 1 0 χ∼ m Observed Expected σ 1 ± σ 2 ± ATLAS = 13 TeV s -1 32.9 fb (c) (b) (a) (c) FIG. VI. RESULTS The nonprompt- vertex background estimate for both signal regions has a systematic uncertainty of 19% associated with knowledge of the charge correlation Rq, as described in Sec. IV F. Systematic uncertainties in the estimate of the prompt background are determined by varying the quantity that distinguishes MScomb from MSonly muons, the angular distance between the MS track and nearest combined-muon track, by 50% and repeating the ABCD technique described in Sec. IV G. A 9% difference in the prompt background estimate is observed, and this is taken as a systematic uncertainty. 012001-13 PHYS. REV. D 99, 012001 (2019) M. AABOUD et al. 50 100 150 200 250 300 350 400 [GeV] μ μ m 0 1 2 3 4 5 6 7 Vertices / 5 GeV ATLAS -1 32.9 fb = 13 TeV s low data, SR high data, SR (a) 300 − 200 − 100 − 0 100 200 300 [cm] vtx Signed r 0 1 2 3 4 5 6 7 8 Vertices / 10 cm ATLAS -1 32.9 fb = 13 TeV s low data, SR high data, SR (b) FIG. 6. Distributions of (a) dimuon invariant mass mμμ and (b) vertex radius rvtx for displaced dimuon vertices in the low-mass (black circles) and high-mass (red squares) signal regions. 300 − 200 − 100 − 0 100 200 300 [cm] vtx Signed r 0 1 2 3 4 5 6 7 8 Vertices / 10 cm ATLAS -1 32.9 fb = 13 TeV s low data, SR high data, SR (b) 50 100 150 200 250 300 350 400 [GeV] μ μ m 0 1 2 3 4 5 6 7 Vertices / 5 GeV ATLAS -1 32.9 fb = 13 TeV s low data, SR high data, SR (a) (a) (b) FIG. 6. Distributions of (a) dimuon invariant mass mμμ and (b) vertex radius rvtx for displaced dimuon vertices in the low-mass (black circles) and high-mass (red squares) signal regions. VI. RESULTS 1 − 10 1 10 2 10 3 10 4 10 5 10 6 10 7 10 [cm] τ c 4 − 10 3 − 10 2 − 10 1 − 10 1 10 2 10 [pb] B x σ 95% CL upper limit (1100 GeV) = 0.163 pb g~ σ ) = 1 G~ Z → 1 0χ∼ ( B = 300 GeV 1 0 χ∼ m Observed Expected σ 1 ± σ 2 ± ATLAS = 13 TeV s -1 32.9 fb (a) 1 − 10 1 10 2 10 3 10 4 10 5 10 6 10 7 10 [cm] τ c 4 − 10 3 − 10 2 − 10 1 − 10 1 10 2 10 [pb] B x σ 95% CL upper limit (1100 GeV) = 0.163 pb g~ σ ) = 1 G~ Z → 1 0χ∼ ( B = 700 GeV 1 0 χ∼ m Observed Expected σ 1 ± σ 2 ± ATLAS = 13 TeV s -1 32.9 fb (b) 1 − 10 1 10 2 10 3 10 4 10 5 10 6 10 7 10 [cm] τ c 4 − 10 3 − 10 2 − 10 1 − 10 1 10 2 10 [pb] B x σ 95% CL upper limit (1100 GeV) = 0.163 pb g~ σ ) = 1 G~ Z → 1 0χ∼ ( B = 1000 GeV 1 0 χ∼ m Observed Expected σ 1 ± σ 2 ± ATLAS = 13 TeV s -1 32.9 fb (c) FIG. 7. The observed and expected 95% C.L. upper limits on the product of cross section and branching ratios for pair production of gluinos, leading to a final state of μþμ−þ X, in the GGM model, as a function of the ˜χ0 1 lifetime, for m˜g ¼ 1100 GeV and three different choices of m˜χ0 1: (a) 300 GeV, (b) 700 GeV, and (c) 1000 GeV. The shaded green (yellow) bands represent the 1σ (2σ) uncertainties in the expected limits. The dashed horizontal line represents the value of the cross section times branching fractions predicted from simulation, with m˜g ¼ 1100 GeV, σðpp →˜g ˜gÞ ¼ 0.1635 pb, Bð˜χ0 1 →Z ˜GÞ ¼ 1.0, and BðZ →μþμ−Þ ¼ 0.03366. SEARCH FOR LONG-LIVED PARTICLES IN FINAL … 4 1 − 10 1 10 2 10 3 10 4 10 5 10 6 10 7 10 [cm] τ c 3 − 10 2 − 10 1 − 10 1 10 2 10 3 10 [pb] B x σ 95% CL upper limit ) = 0.1 D Z D Z → (H B ) = 0.01 D Z D Z → (H B ) = 0.148 μ μ → D (Z B = 20 GeV D Z m Observed Expected σ 1 ± σ 2 ± ATLAS = 13 TeV s -1 32.9 fb 1 − 10 1 10 2 10 3 10 4 10 5 10 6 10 7 10 [cm] τ c 3 − 10 2 − 10 1 − 10 1 10 2 10 3 10 [pb] B x σ 95% CL upper limit ) = 0.1 D Z D Z → (H B ) = 0.01 D Z D Z → (H B ) = 0.148 μ μ → D (Z B = 20 GeV D Z m Observed Expected σ 1 ± σ 2 ± ATLAS = 13 TeV s -1 32.9 fb 1 − 10 1 10 2 10 3 10 4 10 5 10 6 10 7 10 [cm] τ c 3 − 10 2 − 10 1 − 10 1 10 2 10 3 10 [pb] B x σ 95% CL upper limit ) = 0.1 D Z D Z → (H B ) = 0.01 D Z D Z → (H B ) = 0.137 μ μ → D (Z B = 40 GeV D Z m Observed Expected σ 1 ± σ 2 ± ATLAS = 13 TeV s -1 32.9 fb 1 − 10 1 10 2 10 3 10 4 10 5 10 6 10 7 10 [cm] τ c 3 − 10 2 − 10 1 − 10 1 10 2 10 3 10 [pb] B x σ 95% CL upper limit ) = 0.1 D Z D Z → (H B ) = 0.01 D Z D Z → (H B ) = 0.137 μ μ → D (Z B = 40 GeV D Z m Observed Expected σ 1 ± σ 2 ± ATLAS = 13 TeV s -1 32.9 fb (a) 1 − 10 1 10 2 10 3 10 4 10 5 10 6 10 7 10 [cm] τ c 3 − 10 2 − 10 1 − 10 1 10 2 10 3 10 [pb] B x σ 95% CL upper limit ) = 0.1 D Z D Z → (H B ) = 0.01 D Z D Z → (H B ) = 0.107 μ μ → D (Z B = 60 GeV D Z m Observed Expected σ 1 ± σ 2 ± ATLAS = 13 TeV s -1 32.9 fb (c) (a) (b) (c) FIG. SEARCH FOR LONG-LIVED PARTICLES IN FINAL … PHYS. REV. D 99, 012001 (2019) 1 − 10 1 10 2 10 3 10 4 10 5 10 6 10 7 10 [cm] τ c 3 − 10 2 − 10 1 − 10 1 10 2 10 3 10 [pb] B x σ 95% CL upper limit ) = 0.1 D Z D Z → (H B ) = 0.01 D Z D Z → (H B ) = 0.148 μ μ → D (Z B = 20 GeV D Z m Observed Expected σ 1 ± σ 2 ± ATLAS = 13 TeV s -1 32.9 fb (a) 1 − 10 1 10 2 10 3 10 4 10 5 10 6 10 7 10 [cm] τ c 3 − 10 2 − 10 1 − 10 1 10 2 10 3 10 [pb] B x σ 95% CL upper limit ) = 0.1 D Z D Z → (H B ) = 0.01 D Z D Z → (H B ) = 0.137 μ μ → D (Z B = 40 GeV D Z m Observed Expected σ 1 ± σ 2 ± ATLAS = 13 TeV s -1 32.9 fb (b) 1 − 10 1 10 2 10 3 10 4 10 5 10 6 10 7 10 [cm] τ c 3 − 10 2 − 10 1 − 10 1 10 2 10 3 10 [pb] B x σ 95% CL upper limit ) = 0.1 D Z D Z → (H B ) = 0.01 D Z D Z → (H B ) = 0.107 μ μ → D (Z B = 60 GeV D Z m Observed Expected σ 1 ± σ 2 ± ATLAS = 13 TeV s -1 32.9 fb (c) FIG. 8. The observed and expected 95% C.L. upper limits on the product of cross section and branching ratios, σ × B ¼ σðpp →HÞ × BðH →ZDZDÞ × BðZD →μþμ−Þ, in the dark-sector model, as a function of the ZD lifetime, for three different choices of mZD: (a) 20 GeV, (b) 40 GeV, and (c) 60 GeV. The shaded green (yellow) bands represent the 1σ (2σ) uncertainties in the expected limits. SEARCH FOR LONG-LIVED PARTICLES IN FINAL … 8. The observed and expected 95% C.L. upper limits on the product of cross section and branching ratios, σ × B ¼ σðpp →HÞ × BðH →ZDZDÞ × BðZD →μþμ−Þ, in the dark-sector model, as a function of the ZD lifetime, for three different choices of mZD: (a) 20 GeV, (b) 40 GeV, and (c) 60 GeV. The shaded green (yellow) bands represent the 1σ (2σ) uncertainties in the expected limits. The dashed horizontal lines represent the values of the cross section times branching fractions predicted by simulation, with mH ¼ 125 GeV, mHD ¼ 300 GeV, σðpp →HÞ ¼ 44.1 pb and assuming BðH →ZDZDÞ ¼ 10% or 1%. The value of BðZD →μþμ−Þ varies between 0.1475 and 0.1066 for the range mZD ¼ 20–60 GeV. 20 25 30 35 40 45 50 55 60 [GeV] D Z m 11 − 10 10 − 10 9 − 10 8 − 10 7 − 10 6 − 10 5 − 10 4 − 10 ∈ ) = 10% D Z D Z → (H B Excluded at 95% CL, ) = 1% D Z D Z → (H B Excluded at 95% CL, ATLAS = 13 TeV s -1 32.9 fb FIG. 9. The observed 95% C.L. excluded regions in the plane of ZD-Z kinetic mixing parameter, ϵ, versus ZD mass, for values of BðH →ZDZDÞ ¼ 1% or 10%, and mHD ¼ 300 GeV. The value of BðZD →μþμ−Þ varies between 0.1475 and 0.1066 for the range mZD ¼ 20–60 GeV. 20 25 30 35 40 45 50 55 60 [GeV] D Z m 11 − 10 10 − 10 9 − 10 8 − 10 7 − 10 6 − 10 5 − 10 4 − 10 ∈ ) = 10% D Z D Z → (H B Excluded at 95% CL, ) = 1% D Z D Z → (H B Excluded at 95% CL, ATLAS = 13 TeV s -1 32.9 fb as nuisance parameters, and the CLS values are calculated by generating ensembles of pseudoexperiments corre- sponding to the background-only and signal-plus-back- ground hypotheses. Both the expected and observed limits are shown in Fig. 7 for the GGM model, and in Fig. 8 for the dark-sector model, where SRhigh is used for the GGM model and SRlow is used for the dark-sector model. SEARCH FOR LONG-LIVED PARTICLES IN FINAL … The dashed horizontal lines represent the values of the cross section times branching fractions predicted by simulation, with mH ¼ 125 GeV, mHD ¼ 300 GeV, σðpp →HÞ ¼ 44.1 pb and assuming BðH →ZDZDÞ ¼ 10% or 1%. The value of BðZD →μþμ−Þ varies between 0.1475 and 0.1066 for the range mZD ¼ 20–60 GeV. VI. RESULTS 7. The observed and expected 95% C.L. upper limits on the product of cross section and branching ratios for pair production of gluinos, leading to a final state of μþμ−þ X, in the GGM model, as a function of the ˜χ0 1 lifetime, for m˜g ¼ 1100 GeV and three different choices of m˜χ0 1: (a) 300 GeV, (b) 700 GeV, and (c) 1000 GeV. The shaded green (yellow) bands represent the 1σ (2σ) uncertainties in the expected limits. The dashed horizontal line represents the value of the cross section times branching fractions predicted from simulation, with m˜g ¼ 1100 GeV, σðpp →˜g ˜gÞ ¼ 0.1635 pb, Bð˜χ0 1 →Z ˜GÞ ¼ 1.0, and BðZ →μþμ−Þ ¼ 0.03366. 012001-14 012001-14 SEARCH FOR LONG-LIVED PARTICLES IN FINAL … SEARCH FOR LONG-LIVED PARTICLES IN FINAL … In the GGM model with a gluino mass of 1100 GeV and ˜χ0 1 masses of 300, 700, and 1000 GeV, cτ˜χ0 1 values are excluded in the ranges 3.1–1000 cm, 2.6–1500 cm, and 2.9–1800 cm, respectively. The observed limits are about 1.5σ weaker than the expected limits because of the small excess of events observed in SRhigh. In the dark-sector model with a dark-Higgs-boson mass of 300 GeV, BðH →ZDZDÞ ¼ 10% and ZD masses of 20, 40, and 60 GeV, cτZD values are excluded in the ranges 0.3– 2000 cm, 0.9–2400 cm, and 2.1–1100 cm, respectively. These limits are translated into 95% exclusion contours in the plane of the ZD-Z kinetic mixing parameter, ϵ, and the FIG. 9. The observed 95% C.L. excluded regions in the plane of ZD-Z kinetic mixing parameter, ϵ, versus ZD mass, for values of BðH →ZDZDÞ ¼ 1% or 10%, and mHD ¼ 300 GeV. The value of BðZD →μþμ−Þ varies between 0.1475 and 0.1066 for the range mZD ¼ 20–60 GeV. 012001-15 PHYS. REV. D 99, 012001 (2019) M. AABOUD et al. Colombia; MSMT CR, MPO CR and VSC CR, Czech Republic; DNRF and DNSRC, Denmark; IN2P3-CNRS, CEA-DRF/IRFU, France; SRNSFG, Georgia; BMBF, HGF, and MPG, Germany; GSRT, Greece; RGC, Hong Kong SAR, China; ISF, I-CORE and Benoziyo Center, Israel; INFN, Italy; MEXT and JSPS, Japan; CNRST, Morocco; NWO, Netherlands; RCN, Norway; MNiSW and NCN, Poland; FCT, Portugal; MNE/IFA, Romania; MES of Russia and NRC KI, Russian Federation; JINR; MESTD, Serbia; MSSR, Slovakia; ARRS and MIZŠ, Slovenia; DST/NRF, South Africa; MINECO, Spain; SRC and Wallenberg Foundation, Sweden; SERI, SNSF and Cantons of Bern and Geneva, Switzerland; MOST, Taiwan; TAEK, Turkey; STFC, United Kingdom; DOE and NSF, United States of America. In addition, individual groups and members have received support from BCKDF, the Canada Council, CANARIE, CRC, Compute Canada, FQRNT, and the Ontario Innovation Trust, Canada; EPLANET, ERC, ERDF, FP7, Horizon 2020 and Marie Skłodowska-Curie Actions, European Union; Investissements d’Avenir Labex and Idex, ANR, R´egion Auvergne and Fondation Partager le Savoir, France; DFG and AvH Foundation, Germany; Herakleitos, Thales and Aristeia programmes co-financed by EU-ESF and the Greek NSRF; BSF, GIF and Minerva, Israel; BRF, Norway; CERCA Programme Generalitat de Catalunya, Generalitat Valenciana, Spain; the Royal Society and Leverhulme Trust, United Kingdom. SEARCH FOR LONG-LIVED PARTICLES IN FINAL … The crucial com- puting support from all WLCG partners is acknowledged gratefully, in particular from CERN, the ATLAS Tier-1 facilities at TRIUMF (Canada), NDGF (Denmark, Norway, Sweden), CC-IN2P3 (France), KIT/GridKA (Germany), INFN-CNAF (Italy), NL-T1 (Netherlands), PIC (Spain), ASGC (Taiwan), RAL (UK) and BNL (USA), the Tier-2 facilities worldwide and large non-WLCG resource provid- ers. Major contributors of computing resources are listed in Ref. [97]. ZD mass, and are shown in Fig. 9. Values of ϵ of the order 10−8 are excluded for 20 < mZD < 60 GeV. ZD mass, and are shown in Fig. 9. Values of ϵ of the order 10−8 are excluded for 20 < mZD < 60 GeV. VII. CONCLUSION This article reports on a search for BSM long-lived particles decaying into two muons of opposite-sign electric charge in a sample of pp collisions recorded by the ATLAS detector at the LHC with a center-of-mass energy of ﬃﬃﬃs p ¼ 13 TeV and an integrated luminosity of 32.9 fb−1. The search is performed by identifying dimuon vertices with displacements from the pp interaction point in the range of 1–400 cm and having invariant mass mμμ within one of two signal regions: 20–60 GeV or > 60 GeV. In neither signal region is a significant excess observed in the number of vertices relative to the predicted background. 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Carminati,66a,66b R. M. D. Carney,43a,43b S. Caron,117 E. Carquin,144b S. Carrá,66a,66b 35 32b 14 j 164 14 168 G. D. Carrillo-Montoya,35 D. Casadei,32b M. P. Casado,14,j A. F. Casha,164 M. Casolino,14 D. W G. D. Carrillo-Montoya,35 D. Casadei,32b M. P. Casado,14,j A. F. Casha,164 M. Casolino,14 D. W. Casper,168 R. Castelijn,118 F L C till 171 V C till Gi 171 N F C t 136a 136e A C ti i 35 J R C t 130 A C tt i 35 J C d 24 F. L. Castillo,171 V. Castillo Gimenez,171 N. F. Castro,136a,136e A. Catinaccio,35 J. R. Catmore,13 F. L. Castillo,171 V. Castillo Gimenez,171 N. F. Castro,136a,136e A. Catinaccio,35 J. R. Catmore,130 A. Cattai,35 J. Caudron,24 V. Cavaliere,29 E. Cavallaro,14 D. Cavalli,66a M. Cavalli-Sforza,14 V. Cavasinni,69a,69b E. Celebi,12b F. Ceradini,72a,72b L. Cerda Alberich,171 A. S. Cerqueira,78a A. Cerri,153 L. Cerrito,71a,71b F. Cerutti,18 A. Cervelli,23b,23a S. A. Cetin,12b V. Cavaliere,29 E. Cavallaro,14 D. Cavalli,66a M. Cavalli-Sforza,14 V. Cavasinni,69a,69b E. Celebi,12b F. Ceradini,72a,72b L. Cerda Alberich,171 A. S. Cerqueira,78a A. Cerri,153 L. Cerrito,71a,71b F. Cerutti,18 A. Cervelli,23b,23a S. A. Cetin,12b 34 119 57 118 61 170 31 21 V. Cavaliere,29 E. Cavallaro,14 D. Cavalli,66a M. Cavalli-Sforza,14 V. Cavasinni,69a,69b E. Celebi,12b F. Ceradini,72a,72b L. Cerda Alberich,171 A. S. Cerqueira,78a A. Cerri,153 L. Cerrito,71a,71b F. Cerutti,18 A. Cervelli,23b,23a S. A. Cetin,12b A. Chafaq,34a D Chakraborty,119 S. K. Chan,57 W. S. Chan,118 Y. L. Chan,61a P. Chang,170 J. D. Chapman,31 D. G. Charlton,21 A. Chafaq,34a D Chakraborty,119 S. K. Chan,57 W. S. Chan,118 Y. L. Chan,61a P. Chang,170 J. D. Chapman,31 D. G. Charlton,21 C. C. Chau, C. A. Chavez Barajas, S. Che, A. Chegwidden, S. Chekanov, S. V. Chekulaev, G. A. Chelkov, M. A. Chelstowska,35 C. Chen,58a C. H. Chen,76 H. Chen,29 J. Chen,58a J. Chen,38 S. Chen,133 S. J. Chen,15c X. ACKNOWLEDGMENTS Ali,138 G. Alimonti,66a J. Alison,36 S. P. Alkire,145 C. Allaire,128 B. M. M. Allbrooke,153 B. W. Allen,127 P. P. Allport,21 A. Aloisio,67a,67b A. Alonso,39 F. Alonso,86 C. Alpigiani,145 A. A. Alshehri,55 M. I. Alstaty,99 B. Alvarez Gonzalez,35 012001-19 PHYS. REV. D 99, 012001 (2019) M. AABOUD et al. dyrev,111 A. E. Bolz,59b M. Bomben,132 M. Bona,90 J. S. Bonilla,127 M. Boonekamp,142 A. Bor A. S. Boldyrev,111 A. E. Bolz,59b M. Bomben,132 M. Bona,90 J. S. Bonilla,127 M. Boonekamp,142 A. Borisov,140 G. Borissov,87 J. Bortfeldt,35 D. Bortoletto,131 V. Bortolotto,71a,61b,61c,71b D. Boscherini,23b M. Bosman,14 J. D. Bossio Sola,30 A. S. Boldyrev,111 A. E. Bolz,59b M. Bomben,132 M. Bona,90 J. S. Bonilla,127 M. Boonekamp,142 A. Borisov,140 G. Borissov,87 J. Bortfeldt,35 D. Bortoletto,131 V. Bortolotto,71a,61b,61c,71b D. Boscherini,23b M. Bosman,14 J. D. Bossio Sola,30 y p G. Borissov,87 J. Bortfeldt,35 D. Bortoletto,131 V. Bortolotto,71a,61b,61c,71b D. Boscherini,23b M. Bosman,14 J. D. Bossio Sola,30 ,34a J. Boudreau,135 E. V. Bouhova-Thacker,87 D. Boumediene,37 C. Bourdarios,128 S. K. Bout K. Bouaouda,34a J. Boudreau,135 E. V. Bouhova-Thacker,87 D. Boumediene,37 C. Bourdarios,128 S. K. Boutle,55 A. Boveia,122 J. Boyd,35 I. R. Boyko,77 A. J. Bozson,91 J. Bracinik,21 N. Brahimi,99 A. Brandt,8 G. Brandt,179 O. Brandt,59a A. Boveia,122 J. Boyd,35 I. R. Boyko,77 A. J. Bozson,91 J. Bracinik,21 N. Brahimi,99 A. Brandt,8 G. Brandt,179 O. Brandt,59a F. Braren,44 U. Bratzler,161 B. Brau,100 J. E. Brau,127 W. D. Breaden Madden,55 K. Brendlinge 44 169 177 97 21 55 L. Brenner,44 R. Brenner,169 S. Bressler,177 B. Brickwedde,97 D. L. Briglin,21 D. Britton,55 D. Britzger,59b I. Brock,24 R. Brock,104 G. Brooijmans,38 T. Brooks,91 W. K. Brooks,144b E. Brost,119 J. H. Broughton,21 P. A. Bruckman de Renstrom,82 L. Brenner,44 R. Brenner,169 S. Bressler,177 B. Brickwedde,97 D. L. Briglin,21 D. Britton,55 D. Britzger,59b I. Brock,24 R B k 104 G B ij 38 T B k 91 W K B k 144b E B 119 J H B h 21 P A B k d R 82 4 G. Brooijmans,38 T. Brooks,91 W. K. Brooks,144b E. Brost,119 J. H. Broughton,21 P. A. Bruckman D. Bruncko,28b A. Bruni,23b G. Bruni,23b L. S. Bruni,118 S. Bruno,71a,71b B. H. Brunt,31 M. Bruschi,23b N. Bruscino,135 P. Bryant,36 L. Bryngemark,44 T. Buanes,17 Q. Buat,35 P. Buchholz,148 A. G. Buckley,55 I. A 130 110 8 119 88 118 M. K. Bugge,130 O. Bulekov,110 D. Bullock,8 T. J. Burch,119 S. Burdin,88 C. D. Burgard,118 A. M. Burger,5 B. ACKNOWLEDGMENTS Burghgrave,119 82 141 45 131 50 97 51 55 M. K. Bugge,130 O. Bulekov,110 D. Bullock,8 T. J. Burch,119 S. Burdin,88 C. D. Burgard,118 A. M. Burger,5 B. Burghgrave,119 K. Burka,82 S. Burke,141 I. Burmeister,45 J. T. P. Burr,131 D. Büscher,50 V. Büscher,97 E. Buschmann,51 P. Bussey,55 M. K. Bugge,130 O. Bulekov,110 D. Bullock,8 T. J. Burch,119 S. Burdin,88 C. D. Burgard,118 A. M. Burger,5 B. Burghgrave,119 K. Burka,82 S. Burke,141 I. Burmeister,45 J. T. P. Burr,131 D. Büscher,50 V. Büscher,97 E. Buschmann,51 P. Bussey,55 K. Burka, S. Burke, I. Burmeister, J. T. P. Burr, D. Büscher, V. Büscher, E. Buschmann, P. Bussey, J. M. Butler,25 C. M. Buttar,55 J. M. Butterworth,92 P. Butti,35 W. Buttinger,35 A. Buzatu,155 A. R. Buzykaev,120b,120a G Cabras 23b,23a S Cabrera Urbán 171 D Caforio 138 H Cai 170 V M M Cairo 2 O Cakir 4a N Calace 52 P Calafiura 18 y J. M. Butler,25 C. M. Buttar,55 J. M. Butterworth,92 P. Butti,35 W. Buttinger,35 A. Buzatu,155 A. R. Buzykaev,120b,120a G. Cabras,23b,23a S. Cabrera Urbán,171 D. Caforio,138 H. Cai,170 V. M. M. Cairo,2 O. Cakir,4a N. Calace,52 P. Calafiura,18 J. M. Butler,25 C. M. Buttar,55 J. M. Butterworth,92 P. Butti,35 W. Buttinger,35 A. Buzatu,155 A. R. Buzykaev,120b,120a G. Cabras,23b,23a S. Cabrera Urbán,171 D. Caforio,138 H. Cai,170 V. M. M. Cairo,2 O. Cakir,4a N. Calace,52 P. Calafiura,18 99 132 63 40b 40 78b 96 37 37 A. Calandri,99 G. Calderini,132 P. Calfayan,63 G. Callea,40b,40a L. P. Caloba,78b S. Calvente Lopez,96 D. Calvet,37 S. Calvet,37 T P Calvet 152 M Calvetti 69a,69b R Camacho Toro 132 S Camarda 35 P Camarri 71a,71b D Cameron 130 A. Calandri,99 G. Calderini,132 P. Calfayan,63 G. Callea,40b,40a L. P. Caloba,78b S. Calvente Lopez,96 D. Calvet,37 S. Calvet,37 T. P. Calvet,152 M. Calvetti,69a,69b R. Camacho Toro,132 S. Camarda,35 P. Camarri,71a,71b D. Cameron,130 R. Caminal Armadans,100 C. Camincher,35 S. Campana,35 M. Campanelli,92 A. Camplani,39 A. Campoverde,148 V. Canale,67a,67b M. Cano Bret,58c J. Cantero,125 T. Cao,158 Y. Cao,170 M. D. M. Capeans Garrido,35 I. Caprini,27b M. Caprini,27b M. Capua,40b,40a R. M. Carbone,38 R. Cardarelli,71a F. C. Cardillo,50 I. Carli,139 T. Carli,35 G. Carlino,67a B T Carlson 135 L Carminati 66a,66b R M D Carney 43a,43b S Caron 117 E Carquin 144b S Carrá 66a,66b , , , , , , R. Caminal Armadans,100 C. Camincher,35 S. Campana,35 M. Campanelli,92 A. Camplani,39 A. Campoverde,148 V. Canale,67a,67b M. Cano Bret,58c J. Cantero,125 T. ACKNOWLEDGMENTS Chen,15b,l 80 44 103 15d 77 140 34 5 C. Chen,58a C. H. Chen,76 H. Chen,29 J. Chen,58a J. Chen,38 S. Chen,133 S. J. Chen,15c X. Che . Chen,44 H. C. Cheng,103 H. J. Cheng,15d A. Cheplakov,77 E. Cheremushkina,140 R. Cherkaoui El Y. Chen,80 Y-H. Chen,44 H. C. Cheng,103 H. J. Cheng,15d A. Cheplakov,77 E. Cheremushkina,140 E. Cheu,7 K. Cheung,62 L. Chevalier,142 V. Chiarella,49 G. Chiarelli,69a G. Chiodini,65a A. S. Chisholm,35 A. Chitan,27b I. Chiu,160 Y. H. Chiu,173 M. V. Chizhov,77 K. Choi,63 A. R. Chomont,128 S. Chouridou,159 Y. S. Chow,118 V. Christodoulou,92 M. C. Chu,61a J. Chudoba,137 A. J. Chuinard,101 J. J. Chwastowski,82 L. Chytka,126 D. Cinca,45 E. Cheu, K. Cheung, L. Chevalier, V. Chiarella, G. Chiarelli, G. Chiodini, A. S. Chisholm, A. Chitan, I. Chiu,160 Y. H. Chiu,173 M. V. Chizhov,77 K. Choi,63 A. R. Chomont,128 S. Chouridou,159 Y. S. Chow,118 V Ch i t d l 92 M C Ch 61a J Ch d b 137 A J Ch i d 101 J J Ch t ki 82 L Ch tk 126 D Ci 45 ou,92 M. C. Chu,61a J. Chudoba,137 A. J. Chuinard,101 J. J. Chwastowski,82 L. Chytka,126 D. Ci V. Cindro,89 I. A. Cioară,24 A. Ciocio,18 F. Cirotto,67a,67b Z. H. Citron,177 M. Citterio,66a A. Clark,52 M. R. Clark,38 8 C. Clement,43a,43b Y. Coadou,99 M. Cobal,64a,64c A. Coccaro,53b,53a J. Cochran,76 A. E. C. Co 5 5 P. J. Clark,48 C. Clement,43a,43b Y. Coadou,99 M. Cobal,64a,64c A. Coccaro,53b,53a J. Cochran P. J. Clark, C. Clement, Y. Coadou, M. Cobal, A. Coccaro, J. Cochran, A. E. C. Coimbra, L. Colasurdo,117 B. Cole,38 A. P. Colijn,118 J. Collot,56 P. Conde Muiño,136a,136b E. Coniavitis,50 S. H. Connell,32b L. Colasurdo,117 B. Cole,38 A. P. Colijn,118 J. Collot,56 P. Conde Muiño,136a,136b E. Coniavitis,50 S. H. Connell,32b 98 27b 67 131 172 164 L. Colasurdo,117 B. Cole,38 A. P. Colijn,118 J. Collot,56 P. Conde Muiño,136a,136b E. Coniav 17 B. Cole,38 A. P. Colijn,118 J. Collot,56 P. Conde Muiño,136a,136b E. Coniavitis,50 S. H. Conn I. A. Connelly,98 S. Constantinescu,27b F. Conventi,67a,m A. M. Cooper-Sarkar,131 F. Cormier,172 K. J. R. Cormier,164 M. Corradi,70a,70b E. E. Corrigan,94 F. Corriveau,101,n A. Cortes-Gonzalez,35 M. J. Costa,171 D. Costanzo,146 G. Cottin,31 I. A. Connelly, S. Constantinescu, F. Conventi, A. M. Cooper Sarkar, F. Cormier, K. J. R. Cormier, M. Corradi,70a,70b E. E. Corrigan,94 F. Corriveau,101,n A. Cortes-Gonzalez,35 M. J. Costa,171 D. Costanzo,146 G. Cottin,31 g B. E. Cox,98 J. M. Benoit,52 J. R. Bensinger,26 S. Bentvelsen,118 L. Beresford,131 M. Beretta,49 D. Berge,44 E. Bergeaas Kuutmann,169 N. Berger,5 L. J. Bergsten,26 J. Beringer,18 S. Berlendis,7 N. R. Bernard,100 G. Bernardi,132 C. Bernius,150 F. U. Bernlochner,24 T. Berry,91 P. Berta,97 C. Bertella,15a G. Bertoli,43a,43b I. A. Bertram,87 G. J. Besjes,39 ACKNOWLEDGMENTS Crane,98 K. Cranmer,121 S. J. Crawley,55 R. A. Creager,133 G. Cree,33 S. Cr´ep´e-R G. Cowan,91 B. E. Cox,98 J. Crane,98 K. Cranmer,121 S. J. Crawley,55 R. A. Creager,133 G. Cree,33 S. Cr´ep´e-Renaudin,56 F. Crescioli,132 M. Cristinziani,24 V. Croft,121 G. Crosetti,40b,40a A. Cueto,96 T. Cuhadar Donszelmann,146 F. Crescioli,132 M. Cristinziani,24 V. Croft,121 G. Crosetti,40b,40a A. Cueto,96 T. Cuhadar Donszelmann,146 150 M. Curatolo,49 J. Cúth,97 S. Czekierda,82 P. Czodrowski,35 M. J. Da Cunha Sargedas De Sou A. R. Cukierman,150 M. Curatolo,49 J. Cúth,97 S. Czekierda,82 P. Czodrowski,35 M. J. Da Cunha Sargedas De Sousa,58b,136b C. Da Via,98 W. Dabrowski,81a T. Dado,28a,i S. Dahbi,34e T. Dai,103 F. Dallaire,107 C. Dallapiccola,100 M. Dam,39 G. D’amen,23b,23a J. Damp,97 J. R. Dandoy,133 M. F. Daneri,30 N. P. Dang,178,f N.D Dann,98 M. Danninger,172 V. Dao,35 , , , , , g , C. Da Via,98 W. Dabrowski,81a T. Dado,28a,i S. Dahbi,34e T. Dai,103 F. Dallaire,107 C. Dallapiccola,100 M. Dam,39 G D’amen 23b,23a J Damp 97 J R Dandoy 133 M F Daneri 30 N P Dang 178,f N D Dann 98 M Danninger 172 V Dao 35 , , , , , g , C. Da Via,98 W. Dabrowski,81a T. Dado,28a,i S. Dahbi,34e T. Dai,103 F. Dallaire,107 C. Dallapiccola,100 M. Dam,39 012001-20 PHYS. REV. D 99, 012001 (2019) SEARCH FOR LONG-LIVED PARTICLES IN FINAL … SEARCH FOR LONG-LIVED PARTICLES IN FINAL … G. Darbo,53b S. Darmora,8 O. Dartsi,5 A. Dattagupta,127 T. Daubney,44 S. D’Auria,55 W. Davey,24 C. David,44 T. Davidek,139 D. R. Davis,47 E. Dawe,102 I. Dawson,146 K. De,8 R. De Asmundis,67a A. De Benedetti,124 S. De Castro,23b,23a G. Darbo,53b S. Darmora,8 O. Dartsi,5 A. Dattagupta,127 T. Daubney,44 S. D’Auria,55 W. Davey,24 C. David,44 T. Davidek,139 D. R. Davis,47 E. Dawe,102 I. Dawson,146 K. De,8 R. De Asmundis,67a A. De Benedetti,124 S. De Castro,23b,23a S. De Cecco,70a,70b N. De Groot,117 P. de Jong,118 H. De la Torre,104 F. De Lorenzi,76 A. De Maria,51,o D. De Pedis,70a A. De Salvo,70a U. De Sanctis,71a,71b A. De Santo,153 K. De Vasconcelos Corga,99 J. B. De Vivie De Regie,128 S. De Cecco,70a,70b N. De Groot,117 P. de Jong,118 H. De la Torre,104 F. De Lorenzi,76 A. De Maria,51,o D. De Pedis,70a A. De Salvo,70a U. De Sanctis,71a,71b A. De Santo,153 K. De Vasconcelos Corga,99 J. B. De Vivie De Regie,128 g g C. M. Delitzsch,7 M. Della Pietra,67a,67b D. Della Volpe,52 A. Dell’Acqua,35 L. Dell’Asta,25 M. Delmastro,5 C. Delporte,128 P A D l 56 D A D M 164 S D 180 M D i h 77 S P D i 140 D D i k 118 L D’E 132 A. Di Simone,50 R. Di Sipio,164 D. Di Valentino,33 C. Diaconu,99 M. Diamond,164 F. A. Dias,39 T. Dias Do Vale,136a 5 M. A. Diaz,144a J. Dickinson,18 E. B. Diehl,103 J. Dietrich,19 S. Díez Cornell,44 A. Dimitrievska,18 J. Dingfelder,24 F. Dittus,35 F. Djama,99 T. Djobava,156b J. I. Djuvsland,59a M. A. B. Do Vale,78c M. Dobre,27b D. Dodsworth,26 C. Doglioni,94 j j j J. Dolejsi,139 Z. Dolezal,139 M. Donadelli,78d J. Donini,37 A. D’onofrio,90 M. D’Onofrio,88 J. D 86 55 51 149 51 10 58b M. T. Dova,86 A. T. Doyle,55 E. Drechsler,51 E. Dreyer,149 T. Dreyer,51 M. Dris,10 Y. Du,58b J. Duarte-Campderros,158 F Dubinin 108 M Dubovsky 28a A Dubreuil 52 E Duchovni 177 G Duckeck 112 A Ducourthial 132 O A Ducu 107,p D. Duda,113 A. Dudarev,35 A. C. Dudder,97 E. M. Duffield,18 L. Duflot,128 M. Dührssen,35 C. Dülsen,179 M. Dumancic,177 A. E. Dumitriu,27b,q A. K. Duncan,55 M. Dunford,59a A. Duperrin,99 H. Duran Yildiz,4a M. Düren,54 A. Durglishvili,156b D. Duschinger,46 B. Dutta,44 D. Duvnjak,1 M. Dyndal,44 S. Dysch,98 B. S. Dziedzic,82 C. Eckardt,44 K. M. Ecker,113 C. García,171 J. E. García Navarro,171 J. A. García Pascual,15a M. Garcia-Sciveres,18 R. W. Gardner,36 N. Garelli,150 V. Garonne,130 K. SEARCH FOR LONG-LIVED PARTICLES IN FINAL … Ha 112 14 48 142 20 104 J. D. Hansen, M. C. Hansen, P. H. Hansen, K. Hara, A. S. Hard, T. Harenberg, S. Harkusha, P. F. Harrison, N. M. Hartmann,112 Y. Hasegawa,147 A. Hasib,48 S. Hassani,142 S. Haug,20 R. Hauser,104 L. Hauswald,46 L. B. Havener,38 138 21 35 104 152 131 90 N. M. Hartmann,112 Y. Hasegawa,147 A. Hasib,48 S. Hassani,142 S. Haug,20 R. Hauser,104 L. Hauswald,46 L. B. Havener,38 M. Havranek,138 C. M. Hawkes,21 R. J. Hawkings,35 D. Hayden,104 C. Hayes,152 C. P. Hays,131 J. M. Hays,90 H. S. Hayward,88 S. J. Haywood,141 M. P. Heath,48 V. Hedberg,94 L. Heelan,8 S. Heer,24 K. K. Heidegger,50 J. Heilman,33 S. Heim,44 T. Heim,18 B. Heinemann,44,aa J. J. Heinrich,112 L. Heinrich,121 C. Heinz,54 J. Hejbal,137 L. Helary,35 A. Held,172 S. Hellesund,130 S. Hellman,43a,43b C. Helsens,35 R. C. W. Henderson,87 Y. Heng,178 S. Henkelmann,172 5 5 S. Hellesund,130 S. Hellman,43a,43b C. Helsens,35 R. C. W. Henderson,87 Y. Heng,178 S. Henkelmann,172 A. M. Henriques Correia,35 G. H. Herbert,19 H. Herde,26 V. Herget,174 Y. Hernández Jim´enez 112 35 133 92 165 A. M. Henriques Correia, G. H. Herbert, H. Herde, V. Herget, Y. Hernández Jimenez, H. Herr, G. Herten, R. Hertenberger,112 L. Hervas,35 T. C. Herwig,133 G. G. Hesketh,92 N. P. Hessey,165a J. W. Hetherly,41 S. Higashino,79 q g R. Hertenberger,112 L. Hervas,35 T. C. Herwig,133 G. G. Hesketh,92 N. P. Hessey,165a J. W. Hetherly,41 S. Higashino,79 g g y E. Higón-Rodriguez,171 K. Hildebrand,36 E. Hill,173 J. C. Hill,31 K. K. Hill,29 K. H. Hiller,44 E. Higón-Rodriguez,171 K. Hildebrand,36 E. Hill,173 J. C. Hill,31 K. K. Hill,29 K. H. Hiller,44 S. J. Hillier,21 M. Hils,46 I. Hinchliffe,18 M. Hirose,129 D. Hirschbuehl,179 B. Hiti,89 O. Hladik,137 D. R. Hlaluku,32c X. Hoad,48 J. Hobbs,152 N. Hod,165a M. C. Hodgkinson,146 A. Hoecker,35 M. R. Hoeferkamp,116 F. Hoenig,112 D. Hohn,24 D. Hohov,128 I. Hinchliffe,18 M. Hirose,129 D. Hirschbuehl,179 B. Hiti,89 O. Hladik,137 D. R. Hlaluku,32c X. Hoad,48 J. Hobbs,152 N. Hod,165a M. C. Hodgkinson,146 A. Hoecker,35 M. R. Hoeferkamp,116 F. Hoenig,112 D. Hohn,24 D. Hohov,128 T. R. Holmes,36 M. Holzbock,112 M. Homann,45 S. Honda,166 T. Honda,79 T. M. Hong,135 A. Hönle,113 B. H. Hooberman,170 W. H. Hopkins,127 Y. Horii,115 P. Horn,46 A. J. Horton,149 L. A. Horyn,36 J-Y. Hostachy,56 A. Hostiuc,145 S. Hou,155 A. Hoummada,34a J. Howarth,98 J. Hoya,86 M. Hrabovsky,126 J. Hrdinka,35 I. Hristova,19 J. Hrivnac,128 A. Hrynevich,106 T. Hryn’ova,5 P. J. Hsu,62 S.-C. Hsu,145 Q. D. Goujdami,34c A. G. Goussiou,145 N. Govender,32b,u C. Goy,5 E. Gozani,157 I. Grabowska-Bold,81a P. O. J. Gradin,169 E. C. Graham,88 J. Gramling,168 E. Gramstad,130 S. Grancagnolo,19 V. Gratchev,134 P. M. Gravila,27f C. Gray,55 H. M. Gray,18 Z. D. Greenwood,93,v C. Grefe,24 K. Gregersen,92 I. M. Gregor,44 P. Grenier,150 K. Grevtsov,44 J. Griffiths,8 A. A. Grillo,143 K. Grimm,150 S. Grinstein,14,w Ph. Gris,37 J.-F. Grivaz,128 S. Groh,97 E. Gross,177 J. Grosse-Knetter,51 G. C. Grossi,93 Z. J. Grout,92 C. Grud,103 A. Grummer,116 L. Guan,103 W. Guan,178 J. Guenther,35 A. Guerguichon,128 F. Guescini,165a D. Guest,168 R. Gugel,50 B. Gui,122 T. Guillemin,5 S. Guindon,35 U. Gul,55 C. Gumpert,35 J. Guo,58c W. Guo,103 Y. Guo,58a,x Z. Guo,99 R. Gupta,41 S. Gurbuz,12c G. Gustavino,124 B. J. Gutelman,157 P. Gutierrez,124 C. Gutschow,92 C. Guyot,142 M. P. Guzik,81a C. Gwenlan,131 C. B. Gwilliam,88 A. Haas,121 C. Haber,18 H. K. Hadavand,8 N. Haddad,34e A. Hadef,58a SEARCH FOR LONG-LIVED PARTICLES IN FINAL … Gasnikova,44 A. Gaudiello,53b,53a G. Gaudio,68a I. L. Gavrilenko,108 A. Gavrilyuk,109 C. Gay,172 G. Gaycken,24 E. N. Gazis,10 C. N. P. Gee,141 J. Geisen,51 M. Geisen,97 M. P. Geisler,59a K. Gellerstedt,43a,43b C. Gemme,53b M. H. Genest,56 C. Geng,103 S. Gentile,70a,70b C. Gentsos,159 S. George,91 D. Gerbaudo,14 G. Gessner,45 S. Ghasemi,148 M. Ghasemi Bostanabad,173 M. Ghneimat,24 B. Giacobbe,23b S. Giagu,70a,70b N. Giangiacomi,23b,23a P. Giannetti,69a S. M. Gibson,91 M. Gignac,143 D. Gillberg,33 G. Gilles,179 D. M. Gingrich,3,e M. P. Giordani,64a,64c F. M. Giorgi,23b P. F. Giraud,142 P. Giromini,57 G. Giugliarelli,64a,64c D. Giugni,66a F. Giuli,131 M. Giulini,59b S. Gkaitatzis,159 I. Gkialas,9,t E. L. Gkougkousis,14 P. Gkountoumis,10 L. K. Gladilin,111 C. Glasman,96 J. Glatzer,14 P. C. F. Glaysher,44 A. Glazov,44 M. Goblirsch-Kolb,26 J. Godlewski,82 S. Goldfarb,102 T. Golling,52 D. Golubkov,140 A. Gomes,136a,136b,136d R. Goncalves Gama,78a R. Gonçalo,136a G. Gonella,50 L. Gonella,21 A. Gongadze,77 F. Gonnella,21 J. L. Gonski,57 E. L. Gkougkousis, P. Gkountoumis, L. K. Gladilin, C. Glasman, J. Glatzer, P. C. F. Glaysher, A. Glazov, M. Goblirsch-Kolb,26 J. Godlewski,82 S. Goldfarb,102 T. Golling,52 D. Golubkov,140 A. Gomes,136a,136b,136d R G l G 78a R G l 136a G G ll 50 L G ll 21 A G d 77 F G ll 21 J L G ki 57 ç g S. González de la Hoz,171 S. Gonzalez-Sevilla,52 L. Goossens,35 P. A. Gorbounov,109 H. A. Gordon,29 B. Gorini,35 E. Gorini,65a,65b A. Gorišek,89 A. T. Goshaw,47 C. Gössling,45 M. I. Gostkin,77 C. A. Gottardo,24 C. R. Goudet,128 S. González de la Hoz,171 S. Gonzalez-Sevilla,52 L. Goossens,35 P. A. Gorbounov,109 H. A. Gordon,29 B. Gorini,35 E G i i 65a,65b A G iš k 89 A T G h 47 C Gö li 45 M I G tki 77 C A G tt d 24 C R G d t 128 012001-21 PHYS. REV. D 99, 012001 (2019) M. AABOUD et al. , , , , , , , , S. Hageböck,24 M. Hagihara,166 H. Hakobyan,181,a M. Haleem,174 J. Haley,125 G. Halladjian,104 G. D. Hallewell,99 K. Hamacher,179 P. Hamal,126 K. Hamano,173 A. Hamilton,32a G. N. Hamity,146 K. Han,58a,y L. Han,58a S. Han,15d K. Hamacher,179 P. Hamal,126 K. Hamano,173 A. Hamilton,32a G. N. Hamity,146 K. Han,58a,y L y K. Hanagaki,79,z M. Hance,143 D. M. Handl,112 B. Haney,133 R. Hankache,132 P. Hanke,59a E. Hansen,94 J. B. Hansen,39 J. D. Hansen,39 M. C. Hansen,24 P. H. Hansen,39 K. Hara,166 A. S. Hard,178 T. Harenberg,179 S. SEARCH FOR LONG-LIVED PARTICLES IN FINAL … Jakobs,50 S. Jakobsen,74 T. Jakoubek,137 D. O. Jamin,125 D. K. Jana,93 R. Jansky,52 J. Janssen,24 M. Janus,51 P. A. Janus,81a G. Jarlskog,94 N. Javadov,77,d T. Javůrek,50 M. Javurkova,50 F. Jeanneau,142 L. Jeanty,18 J. Jejelava,156a,dd A. Jelinskas,175 P. Jenni,50,ee J. Jeong,44 C. Jeske,175 S J´ ´ l 5 H Ji 178 J Ji 152 H Ji 76 Y Ji 58a Z Ji 150 ff S Ji i 50 F A Ji M l 37 J Ji P 171 S. Jin,15c A. Jinaru,27b O. Jinnouchi,162 H. Jivan,32c P. Johansson,146 K. A. Johns,7 C. A. Johnson,63 W. J. Johnson,145 K. Jon-And,43a,43b R. W. L. Jones,87 S. D. Jones,153 S. Jones,7 T. J. Jones,88 J. Jongmanns,59a P. M. Jorge,136a,136b J. Jovicevic,165a X. Ju,178 J. J. Junggeburth,113 A. Juste Rozas,14,w A. Kaczmarska,82 M. Kado,128 H. Kagan,122 M. Kagan,150 T. Kaji,176 E. Kajomovitz,157 C. W. Kalderon,94 A. Kaluza,97 S. Kama,41 A. Kamenshchikov,140 L. Kanjir,89 Y. Kano,160 110 79 121 178 32 165b 10 77 T. Kaji,176 E. Kajomovitz,157 C. W. Kalderon,94 A. Kaluza,97 S. Kama,41 A. Kamenshchikov,140 L. Kanjir,89 Y. Kano,160 V. A. Kantserov,110 J. Kanzaki,79 B. Kaplan,121 L. S. Kaplan,178 D. Kar,32c M. J. Kareem,165b E. Karentzos,10 S. N. Karpov,77 Z. M. Karpova,77 V. Kartvelishvili,87 A. N. Karyukhin,140 K. Kasahara,166 L. Kashif,178 R. D. Kass,122 A. Kastanas,151 Y. Kataoka,160 C. Kato,160 J. Katzy,44 K. Kawade,80 K. Kawagoe,85 T. Kawamoto,160 G. Kawamura,51 E. F. Kay,88 V. F. Kazanin,120b,120a R. Keeler,173 R. Kehoe,41 J. S. Keller,33 E. Kellermann,94 J. J. Kempster,21 J. Kendrick,21 O. Kepka,137 S Kersten 179 B P Kerševan 89 R A Keyes 101 M Khader 170 F Khalil Zada 13 A Khanov 125 A G Kharlamov 120b,120a V. A. Kantserov,110 J. Kanzaki,79 B. Kaplan,121 L. S. Kaplan,178 D. Kar,32c M. J. Kareem,165b E. K Z. M. Karpova,77 V. Kartvelishvili,87 A. N. Karyukhin,140 K. Kasahara,166 L. Kashif,178 R. D. Kass,122 A. Kastanas,151 Y. Kataoka,160 C. Kato,160 J. Katzy,44 K. Kawade,80 K. Kawagoe,85 T. Kawamoto,160 G. Kawamura,51 E. F. Kay,88 V. F. Kazanin,120b,120a R. Keeler,173 R. Kehoe,41 J. S. Keller,33 E. Kellermann,94 J. J. Kempster,21 J. Kendrick,21 O. Kepka,137 S. Kersten,179 B. P. Kerševan,89 R. A. Keyes,101 M. Khader,170 F. Khalil-Zada,13 A. Khanov,125 A. G. Kharlamov,120b,120a T. Kharlamova,120b,120a A. Khodinov,163 T. J. Khoo,52 E. Khramov,77 J. Khubua,156b S. Kido,80 M. Kiehn,52 C. R. Kilby,91 S. H. Kim,166 Y. K. Kim,36 N. Kimura,64a,64c O. M. Kind,19 B. T. King,88 D. Kirchmeier,46 J. Kirk,141 A. E. Kiryunin,113 T. SEARCH FOR LONG-LIVED PARTICLES IN FINAL … Hu,29 S. Hu,58c Y. Huang,15a Z. Hubacek,138 F. Hubaut,99 M. Huebner,24 F. Huegging,24 T. B. Huffman,131 E. W. Hughes,38 M. Huhtinen,35 R. F. H. Hunter,33 P. Huo,152 A. M. Hupe,33 N. Huseynov,77,d J. Huston,104 J. Huth,57 R. Hyneman,103 G. Iacobucci,52 G. Iakovidis,29 I. Ibragimov,148 L. Iconomidou-Fayard,128 Z. Idrissi,34e P. Iengo,35 R. Ignazzi,39 O. Igonkina,118,bb R. Iguchi,160 T. Iizawa,52 Y. Ikegami,79 M. Ikeno,79 D. Iliadis,159 N. Ilic,150 F. Iltzsche,46 G. Introzzi,68a,68b M. Iodice,72a K. Iordanidou,38 V. Ippolito,70a,70b M. F. Isacson,169 N. Ishijima,129 M. Ishino,160 M. Ishitsuka,162 W. Islam,125 C. Issever,131 S. Istin,12c,cc F. Ito,166 , j , , , , , , , J. M. Iturbe Ponce,61a R. Iuppa,73a,73b A. Ivina,177 H. Iwasaki,79 J. M. Izen,42 V. Izzo,67a S. Jabbar,3 P. Jacka,137 P. Jackson,1 R. M. Jacobs,24 V. Jain,2 G. Jäkel,179 K. B. Jakobi,97 K. Jakobs,50 S. Jakobsen,74 T. Jakoubek,137 D. O. Jamin,125 D. K. Jana,93 R. Jansky,52 J. Janssen,24 M. Janus,51 P. A. Janus,81a G. Jarlskog,94 N. Javadov,77,d T. Javůrek,50 M. Javurkova,50 F. Jeanneau,142 L. Jeanty,18 J. Jejelava,156a,dd A. Jelinskas,175 P. Jenni,50,ee J. Jeong,44 C. Jeske,175 S. J´ez´equel,5 H. Ji,178 J. Jia,152 H. Jiang,76 Y. Jiang,58a Z. Jiang,150,ff S. Jiggins,50 F. A. Jimenez Morales,37 J. Jimenez Pena,171 S Ji 15c A Ji 27b O Ji hi 162 H Ji 32c P J h 146 K A J h 7 C A J h 63 W J J h 145 J. M. Iturbe Ponce,61a R. Iuppa,73a,73b A. Ivina,177 H. Iwasaki,79 J. M. Izen,42 V. Izzo,67a S. Jabbar,3 P. Jacka,137 P. Jackson,1 R. M. Jacobs,24 V. Jain,2 G. Jäkel,179 K. B. Jakobi,97 K. Jakobs,50 S. Jakobsen,74 T. Jakoubek,137 D. O. Jamin,125 D. K. Jana,93 R. Jansky,52 J. Janssen,24 M. Janus,51 P. A. Janus,81a G. Jarlskog,94 N. Javadov,77,d T. Javůrek,50 M. Javurkova,50 F. Jeanneau,142 L. Jeanty,18 J. Jejelava,156a,dd A. Jelinskas,175 P. Jenni,50,ee J. Jeong,44 C. Jeske,175 S. J´ez´equel,5 H. Ji,178 J. Jia,152 H. Jiang,76 Y. Jiang,58a Z. Jiang,150,ff S. Jiggins,50 F. A. Jimenez Morales,37 J. Jimenez Pena,171 S. Jin,15c A. Jinaru,27b O. Jinnouchi,162 H. Jivan,32c P. Johansson,146 K. A. Johns,7 C. A. Johnson,63 W. J. Johnson,145 K. Jon-And,43a,43b R. W. L. Jones,87 S. D. Jones,153 S. Jones,7 T. J. Jones,88 J. Jongmanns,59a P. M. Jorge,136a,136b J. Jovicevic,165a X. Ju,178 J. J. Junggeburth,113 A. Juste Rozas,14,w A. Kaczmarska,82 M. Kado,128 H. Kagan,122 M. Kagan,150 176 157 94 97 41 140 89 160 R. M. Jacobs,24 V. Jain,2 G. Jäkel,179 K. B. Jakobi,97 K. SEARCH FOR LONG-LIVED PARTICLES IN FINAL … Kishimoto,160 D. Kisielewska,81a V. Kitali,44 O. Kivernyk,5 E. Kladiva,28b T. Klapdor-Kleingrothaus,50 M. H. Klein,103 012001-22 012001-22 PHYS. REV. D 99, 012001 (2019) SEARCH FOR LONG-LIVED PARTICLES IN FINAL … SEARCH FOR LONG-LIVED PARTICLES IN FINAL … C. Leggett,18 N. Lehmann,179 G. Lehmann Miotto,35 W. A. Leight,44 A. Leisos,159,hh M. A. L. Leite,78d R. Leitner,139 177 51 92 24 35 7 69 48 G. Lerner,153 C. Leroy,107 R. Les,164 A. A. J. Lesage,142 C. G. Lester,31 M. Levchenko,134 J. Levêque,5 D. Levin,103 L. J. Levinson,177 D. Lewis,90 B. Li,103 C-Q. Li,58a H. Li,58b L. Li,58c Q. Li,15d Q. Y. Li,58a S. Li,58d,58c X. Li,58c Y. Li,148 Z. Liang,15a B. Liberti,71a A. Liblong,164 K. Lie,61c S. Liem,118 A. Limosani,154 C. Y. Lin,31 K. Lin,104 T. H. Lin,97 R. A. Linck,63 B. E. Lindquist,152 A. L. Lionti,52 E. Lipeles,133 A. Lipniacka,17 M. Lisovyi,59b T. M. Liss,170,ii A. Lister,172 A. M. Litke,143 J. D. Little,8 B. Liu,76 B.L Liu,6 H. B. Liu,29 H. Liu,103 J. B. Liu,58a J. K. K. Liu,131 K. Liu,132 M. Liu,58a A. M. Litke,143 J. D. Little,8 B. Liu,76 B.L Liu,6 H. B. Liu,29 H. Liu,103 J. B. Liu,58a J. K. K. Liu,131 K. Liu,132 M. Liu,58a . , . , . . , . W. , . v , . , J. , S. . y , C. . , F. Lo Sterzo,41 E. M. Lobodzinska,44 P. Loch,7 F. K. Loebinger,98 A. Loesle,50 K. M. Loew,26 T. Lohse,19 K. Lohwasser,146 M L k ji k 137 B A L 25 J D L 170 R E L 87 L L 65a 65b K A L 122 J A L 144b I L P 14 F. Lo Sterzo,41 E. M. Lobodzinska,44 P. Loch,7 F. K. Loebinger,98 A. Loesle,50 K. M. Loew,26 T. Lohse,19 K. Lohwasser,146 137 25 170 87 65a 65b 122 144b 14 j , g, g, g, g , p , p , p , A. Lopez Solis,146 J. Lorenz,112 N. Lorenzo Martinez,5 M. Losada,22 P. J. Lösel,112 X. Lou,44 X. Lou,15a A. Lounis,128 J Love 6 P A Love 87 J J Lozano Bahilo 171 H Lu 61a M Lu 58a N Lu 103 Y J Lu 62 H J Lubatti 145 C Luci 70a,70b A. Lopez Solis,146 J. Lorenz,112 N. Lorenzo Martinez,5 M. Losada,22 P. J. Lösel,112 X. Lou,44 X. Lou,15a A. Lounis,128 A. Lopez Solis,146 J. Lorenz,112 N. Lorenzo Martinez,5 M. Losada,22 P. J. Lösel,112 X. Lou,44 X. Lou,15a A. Lounis,128 6 87 171 61 58 103 62 145 70 70b A. Lucotte,56 C. Luedtke,50 F. Luehring,63 I. Luise,132 W. Lukas,74 L. Luminari,70a B. T. Klioutchnikova,35 F. F. Klitzner,112 P. Kluit,118 S. Kluth,113 E. Kneringer,74 E. B. F. G. Knoops,99 A. Knue,50 A. Kobayashi,160 D. Kobayashi,85 T. Kobayashi,160 M. Kobel,46 M. Kocian,150 P. Kodys,139 T. Koffas,33 E. Koffeman,118 N. M. Köhler,113 T. Koi,150 M. Kolb,59b I. Koletsou,5 T. Kondo,79 N. Kondrashova,58c K. Köneke,50 A. C. König,117 T. Kono,79 R. Konoplich,121,gg V. Konstantinides,92 N. Konstantinidis,92 B. Konya,94 R. Kopeliansky,63 S. Koperny,81a K. Korcyl,82 K. Kordas,159 A. Korn,92 I. Korolkov,14 E. V. Korolkova,146 O. Kortner,113 S. Kortner,113 T. Kosek,139 V. V. Kostyukhin,24 A. Kotwal,47 A. Koulouris,10 A. Kourkoumeli-Charalampidi,68a,68b C. Kourkoumelis,9 E. Kourlitis,146 V. Kouskoura,29 A. B. Kowalewska,82 R. Kowalewski,173 T. Z. Kowalski,81a C. Kozakai,160 W. Kozanecki,142 A. S. Kozhin,140 V. A. Kramarenko,111 G. Kramberger,89 D. Krasnopevtsev,110 M. W. Krasny,132 A. Krasznahorkay,35 D. Krauss,113 J. A. Kremer,81a J. Kretzschmar,88 P. Krieger,164 K. Krizka,18 K. Kroeninger,45 H. Kroha,113 J. Kroll,137 J. Kroll,133 J. Krstic,16 U. Kruchonak,77 H. Krüger,24 N. Krumnack,76 M. C. Kruse,47 T. Kubota,102 S. Kuday,4b J. T. Kuechler,179 S. Kuehn,35 A. Kugel,59a F. Kuger,174 T. Kuhl,44 V. Kukhtin,77 R. Kukla,99 Y. Kulchitsky,105 S. Kuleshov,144b Y. P. Kulinich,170 M. Kuna,56 T. Kunigo,83 A. Kupco,137 T. Kupfer,45 O. Kuprash,158 H. Kurashige,80 L. L. Kurchaninov,165a Y. A. Kurochkin,105 M. G. Kurth,15d E. S. Kuwertz,173 M. Kuze,162 J. Kvita,126 T. Kwan,101 A. La Rosa,113 J. L. La Rosa Navarro,78d L. La Rotonda,40b,40a F. La Ruffa,40b,40a C. Lacasta,171 F. Lacava,70a,70b J. Lacey,44 D. P. J. Lack,98 H. Lacker,19 D. Lacour,132 E. Ladygin,77 R. Lafaye,5 B. Laforge,132 T. Lagouri,32c S. Lai,51 S. Lammers,63 W. Lampl,7 E. Lançon,29 U. Landgraf,50 M. P. J. Landon,90 M. C. Lanfermann,52 V. S. Lang,44 J. C. Lange,14 R. J. Langenberg,35 A. J. Lankford,168 F. Lanni,29 K. Lantzsch,24 A. Lanza,68a A. Lapertosa,53b,53a S. Laplace,132 J. F. Laporte,142 T. Lari,66a F. Lasagni Manghi,23b,23a M. Lassnig,35 T. S. Lau,61a A. Laudrain,128 A. T. Law,143 P. Laycock,88 M. Lazzaroni,66a,66b B. Le,102 O. Le Dortz,132 E. Le Guirriec,99 E. P. Le Quilleuc,142 M. LeBlanc,7 T. LeCompte,6 F. Ledroit-Guillon,56 C. A. Lee,29 G. R. Lee,144a L. Lee,57 S. C. Lee,155 B. Lefebvre,101 M. Lefebvre,173 F. Legger,112 C. Leggett,18 N. Lehmann,179 G. Lehmann Miotto,35 W. A. Leight,44 A. Leisos,159,hh M. A. L. Leite,78d R. Leitner,139 D. Lellouch,177 B. Lemmer,51 K. J. C. Leney,92 T. Lenz,24 B. Lenzi,35 R. Leone,7 S. Leone,69a C. Leonidopoulos,48 G. Lerner,153 C. Leroy,107 R. Les,164 A. A. J. Lesage,142 C. G. Lester,31 M. Levchenko,134 J. Levêque,5 D. Levin,103 L. J. Levinson,177 D. Lewis,90 B. Li,103 C-Q. Li,58a H. Li,58b L. Li,58c Q. Li,15d Q. Y. Li,58a S. Li,58d,58c X. Li,58c Y. Li,148 Z. Liang,15a B. Liberti,71a A. Liblong,164 K. Lie,61c S. Liem,118 A. Limosani,154 C. Y. Lin,31 K. Lin,104 T. H. Lin,97 R. A. Linck,63 B. E. Lindquist,152 A. L. Lionti,52 E. Lipeles,133 A. Lipniacka,17 M. Lisovyi,59b T. M. Liss,170,ii A. Lister,172 A. M. Litke,143 J. D. Little,8 B. Liu,76 B.L Liu,6 H. B. Liu,29 H. Liu,103 J. B. Liu,58a J. K. K. Liu,131 K. Liu,132 M. Liu,58a P. Liu,18 Y. Liu,15a Y. L. Liu,58a Y. W. Liu,58a M. Livan,68a,68b A. Lleres,56 J. Llorente Merino,15a S. L. Lloyd,90 C. Y. Lo,61b F. Lo Sterzo,41 E. M. Lobodzinska,44 P. Loch,7 F. K. Loebinger,98 A. Loesle,50 K. M. Loew,26 T. Lohse,19 K. Lohwasser,146 M Lokajicek 137 B A Long 25 J D Long 170 R E Long 87 L Longo 65a,65b K A Looper 122 J A Lopez 144b I Lopez Paz 14 SEARCH FOR LONG-LIVED PARTICLES IN FINAL … Lund-Jensen,151 M. S. Lutz,100 132 29 137 94 15 77 29 58b 58b A. Lucotte,56 C. Luedtke,50 F. Luehring,63 I. Luise,132 W. Lukas,74 L. Luminari,70a B. Lund-Jensen,151 M. S. Lutz,100 P M Luzi 132 D Lynn 29 R Lysak 137 E Lytken 94 F Lyu 15a V Lyubushkin 77 H Ma 29 L L Ma 58b Y Ma 58b ynn,29 R. Lysak,137 E. Lytken,94 F. Lyu,15a V. Lyubushkin,77 H. Ma,29 L. L. Ma,58b Y. Ma,58b Macchiolo,113 C. M. Macdonald,146 J. Machado Miguens,133,136b D. Madaffari,171 R. Madar,37 er,46 A. Madsen,44 N. Madysa,46 J. Maeda,80 K. Maekawa,160 S. Maeland,17 T. Maeno,29 A. S W. F. Mader,46 A. Madsen,44 N. Madysa,46 J. Maeda,80 K. Maekawa,160 S. Maeland,17 T. Maeno,29 A. S. Maevskiy,111 V. Magerl,50 C. Maidantchik,78b T. Maier,112 A. Maio,136a,136b,136d O. Majersky,28a S. Majewski,127 Y. Makida,79 N. Makovec,128 B. Malaescu,132 Pa. Malecki,82 V. P. Maleev,134 F. Malek,56 U. Mallik,75 D. Malon,6 C. Malone,31 S Maltezos 10 S Malyukov 35 J Mamuzic 171 G Mancini 49 I Mandić 89 J Maneira 136a L Manhaes de Andrade Filho 78a J. Manjarres Ramos,46 K. H. Mankinen,94 A. Mann,112 A. Manousos,74 B. Mansoulie,142 J. D. Mansour,15a M. Mantoani,51 S Manzoni 66a,66b G Marceca 30 L March 52 L Marchese 131 G Marchiori 132 M Marcisovsky 137 C A Marin Tobon 35 J. Manjarres Ramos,46 K. H. Mankinen,94 A. Mann,112 A. Manousos,74 B. Mansoulie,142 J. D. Mansour,15a M. Mantoani,51 66 66b 30 52 131 132 137 35 S. Manzoni,66a,66b G. Marceca,30 L. March,52 L. Marchese,131 G. Marchiori,132 M. Marcisovsky,137 C. A. Marin Tobon,35 M. Marjanovic,37 D. E. Marley,103 F. Marroquim,78b Z. Marshall,18 M. U. F. Martensson,169 S. Marti-Garcia,171 tin,175 V. J. Martin,48 B. Martin dit Latour,17 M. Martinez,14,w V. I. Martinez Outschoorn,100 S. Martin-Haugh,141 V. S. Martoiu,27b A. C. Martyniuk,92 A. Marzin,35 L. Masetti,97 T. Mashimo,160 R. Mashinistov,108 J. Masik,98 A. L. Maslennikov,120b,120a L. H. Mason,102 L. Massa,71a,71b P. Mastrandrea,5 A. Mastroberardino,40b,40a T. Masubuchi,160 P. Mättig,179 J. Maurer,27b B. Maček,89 S. J. Maxfield,88 D. A. Maximov,120b,120a R. Mazini,155 I. Maznas,159 S. M. Mazza,143 N. C. Mc Fadden,116 G. Mc Goldrick,164 S. P. Mc Kee,103 A. McCarn,103 T. G. McCarthy,113 T. Masubuchi,160 P. Mättig,179 J. Maurer,27b B. Maček,89 S. J. Maxfield,88 D. A. Maximov,120b,120a R. Mazini,155 I. Maznas,159 S. M. Mazza,143 N. C. Mc Fadden,116 G. Mc Goldrick,164 S. P. Mc Kee,103 A. McCarn,103 T. G. McCarthy,113 L. I. McClymont,92 E. F. McDonald,102 J. A. Mcfayden,35 G. Mchedlidze,51 M. S. Meehan,145 T. M. Megy,50 S. Mehlhase,112 A. Mehta,88 T. Meideck,56 B. Meirose,42 D. Melini,171,jj B. R. Mellado Garcia,32c J. D. Mellenthin,51 M. Melo,28a F. Meloni,20 A. Melzer,24 S. B. Menary,98 E. D. Mendes Gouveia,136a L. Meng,88 X. T. Meng,103 A. Mengarelli,23b,23a S. Menke,113 E. Meoni,40b,40a S. Mergelmeyer,19 C. Merlassino,20 P. Mermod,52 L. Merola,67a,67b C. Meroni,66a F. S. Merritt,36 A. Messina,70a,70b J. Metcalfe,6 A. S. Mete,168 C. Meyer,133 J. Meyer,157 J-P. Meyer,142 H. Meyer Zu Theenhausen,59a F. Miano,153 R. P. Middleton,141 L. Mijović,48 G. Mikenberg,177 M. Mikestikova,137 M. Mikuž,89 M. Milesi,102 A. Milic,164 D. A. Millar,90 D. W. Miller,36 A. Milov,177 D. A. Milstead,43a,43b A. A. Minaenko,140 M. Miñano Moya,171 I. A. Minashvili,156b A. I. Mincer,121 B. Mindur,81a M. Mineev,77 Y. Minegishi,160 Y. Ming,178 L. M. Mir,14 A. Mirto,65a,65b K. P. Mistry,133 T. Mitani,176 J. Mitrevski,112 V. A. Mitsou,171 A. Miucci,20 P. S. Miyagawa,146 A. Mizukami,79 J. U. Mjörnmark,94 T. Mkrtchyan,181 M. Mlynarikova,139 T. Moa,43a,43b K. Mochizuki,107 P. Mogg,50 S. Mohapatra,38 S. Molander,43a,43b R. Moles-Valls,24 M. C. Mondragon,104 K. Mönig,44 J. Monk,39 E. Monnier,99 A. Montalbano,149 J. Montejo Berlingen,35 F. Monticelli,86 S. Monzani,66a R. W. Moore,3 N. Morange,128 D. Moreno,22 M. Moreno Llácer,35 P. Morettini,53b M. Morgenstern,118 S. Morgenstern,35 D. Mori,149 T. Mori,160 M. Morii,57 M. Morinaga,176 V. Morisbak,130 A. K. Morley,35 G. Mornacchi,35 A. P. Morris,92 J. D. Morris,90 L. Morvaj,152 P. Moschovakos,10 M. Mosidze,156b H. J. Moss,146 J. Moss,150,kk K. Motohashi,162 R. Mount,150 E. Mountricha,35 E. J. W. Moyse,100 S. Muanza,99 F. Mueller,113 J. Mueller,135 R. S. P. Mueller,112 D. Muenstermann,87 P. Mullen,55 G. A. Mullier,20 F. J. Munoz Sanchez,98 P. Murin,28b W. J. Murray,175,141 A. Murrone,66a,66b M. Muškinja,89 C. Mwewa,32a A. G. Myagkov,140,ll J. Myers,127 M. Myska,138 B. P. Nachman,18 O. Nackenhorst,45 K. Nagai,131 K. Nagano,79 Y. Nagasaka,60 K. Nagata,166 M. Nagel,50 E. Nagy,99 A. M. Nairz,35 Y. Nakahama,115 K. Nakamura,79 T. Nakamura,160 I. Nakano,123 H. Nanjo,129 F. Napolitano,59a R. F. Naranjo Garcia,44 R. Narayan,11 D. I. Narrias Villar,59a I. Naryshkin,134 T. Naumann,44 G. Navarro,22 R. Nayyar,7 H. A. Neal,103 P. Y. Nechaeva,108 T. J. Neep,142 A. Negri,68a,68b M. Negrini,23b S. Nektarijevic,117 C. Nellist,51 M. E. Nelson,131 S. Nemecek,137 P. Nemethy,121 M. Nessi,35,mm M. S. Neubauer,170 M. Neumann,179 P. R. Newman,21 T. Y. Ng,61c Y. S. Ng,19 H. D. N. Nguyen,99 T. Nguyen Manh,107 E. Nibigira,37 R. B. Nickerson,131 R. Nicolaidou,142 J. Nielsen,143 N. Nikiforou,11 V. Nikolaenko,140,ll I. Nikolic-Audit,132 K. Nikolopoulos,21 P. Nilsson,29 Y. Ninomiya,79 A. Nisati,70a N. Nishu,58c R. Nisius,113 I. Nitsche,45 T. Nitta,176 T. Nobe,160 Y. Noguchi,83 M. Nomachi,129 I. Nomidis,132 M. A. Nomura,29 T. Nooney,90 M. Nordberg,35 N. Norjoharuddeen,131 T. Novak,89 O. Novgorodova,46 R. Novotny,138 M. Nozaki,79 L. Nozka,126 K. Ntekas,168 E. Nurse,92 F. Nuti,102 F. G. Oakham,33,e H. Oberlack,113 T. Obermann,24 J. Ocariz,132 A. Ochi,80 I. Ochoa,38 J. P. Ochoa-Ricoux,144a K. O’Connor,26 S. Oda,85 S. Odaka,79 A. Oh,98 S. H. Oh,47 C. C. Ohm,151 H. Oide,53b,53a H. Okawa,166 Y. Okazaki,83 Y. Okumura,160 T. Okuyama,79 A. Olariu,27b L. F. Oleiro Seabra,136a S. A. Olivares Pino,144a D. Oliveira Damazio,29 SEARCH FOR LONG-LIVED PARTICLES IN FINAL … A. McKay,41 K. D. McLean,173 S. J. McMahon,141 P. C. McNamara,102 C. J. McNicol,175 R. A. McPherson,173,n J. E. Mdhluli,32c Z. A. Meadows,100 , , , , , y, L. I. McClymont,92 E. F. McDonald,102 J. A. Mcfayden,35 G. Mchedlidze,51 M. A. McKay,41 K. D. McLean,173 012001-23 012001-23 M. AABOUD et al. PHYS. REV. D 99, 012001 (2019) , , gg, p , , , g , K. Mönig,44 J. Monk,39 E. Monnier,99 A. Montalbano,149 J. Montejo Berlingen,35 F. Monticelli,86 S. Monzani,66a R. W. Moore,3 N. Morange,128 D. Moreno,22 M. Moreno Llácer,35 P. Morettini,53b M. Morgen D. Mori,149 T. Mori,160 M. Morii,57 M. Morinaga,176 V. Morisbak,130 A. K. Morley,35 G. Mornacchi,35 A. P. Morris,92 E. Mountricha,35 E. J. W. Moyse,100 S. Muanza,99 F. Mueller,113 J. Mueller,135 R. S. P. Mueller,112 D. Muenstermann,87 55 20 98 28b 175 141 66 66b 89 P. Mullen,55 G. A. Mullier,20 F. J. Munoz Sanchez,98 P. Murin,28b W. J. Murray,175,141 A. Murrone,66a,66b M. Muškinja,89 C. Mwewa,32a A. G. Myagkov,140,ll J. Myers,127 M. Myska,138 B. P. Nachman,18 O. Nackenhorst,45 K. Nagai,131 P. Mullen, G. A. Mullier, F. J. Munoz Sanchez, P. Murin, W. J. Murray, A. Murrone, M. Muškinja, C. Mwewa,32a A. G. Myagkov,140,ll J. Myers,127 M. Myska,138 B. P. Nachman,18 O. Nackenhorst,45 K. Nagai,131 K. Nagano,79 Y. Nagasaka,60 K. Nagata,166 M. Nagel,50 E. Nagy,99 A. M. Nairz,35 Y. Naka T. Nakamura,160 I. Nakano,123 H. Nanjo,129 F. Napolitano,59a R. F. Naranjo Garcia,44 R. Narayan,11 D. I. Narrias Villar,59a T. Nakamura,160 I. Nakano,123 H. Nanjo,129 F. Napolitano,59a R. F. Naranjo Garcia,44 R. Na I. Naryshkin,134 T. Naumann,44 G. Navarro,22 R. Nayyar,7 H. A. Neal,103 P. Y. Nechaeva,108 T. J. Neep,142 A. Negri,68a,68b M. Negrini,23b S. Nektarijevic,117 C. Nellist,51 M. E. Nelson,131 S. Nemecek,137 P. Nemethy,121 M. Nessi,35,mm y yy p g M. Negrini,23b S. Nektarijevic,117 C. Nellist,51 M. E. Nelson,131 S. Nemecek,137 P. Nemethy,121 M. Nessi,35,mm E. Nibigira,37 R. B. Nickerson,131 R. Nicolaidou,142 J. Nielsen,143 N. Nikiforou,11 V. Nikolaenko,140,ll I. Nikolic-Audit,132 K. Nikolopoulos,21 P. Nilsson,29 Y. Ninomiya,79 A. Nisati,70a N. Nishu,58c R. Nisius,113 I. Nitsch p y Y. Noguchi,83 M. Nomachi,129 I. Nomidis,132 M. A. Nomura,29 T. Nooney,90 M. Nordberg,35 N. Norjoharuddeen,131 T. Novak,89 O. Novgorodova,46 R. Novotny,138 M. Nozaki,79 L. Nozka,126 K. Ntekas,168 E. Nurse,92 F. Nuti,102 F. G. Oakham,33,e H. Oberlack,113 T. Obermann,24 J. Ocariz,132 A. Ochi,80 I. Ochoa,38 J. P. Ochoa-Ricoux,144a Y. Noguchi,83 M. Nomachi,129 I. Nomidis,132 M. A. Nomura,29 T. Nooney,90 M. SEARCH FOR LONG-LIVED PARTICLES IN FINAL … Nordberg, K. O’Connor,26 S. Oda,85 S. Odaka,79 A. Oh,98 S. H. Oh,47 C. C. Ohm,151 H. Oide,53b,53a H Y. Okumura,160 T. Okuyama,79 A. Olariu,27b L. F. Oleiro Seabra,136a S. A. Olivares Pino,144a D. Oliveira Damazio,29 J. L. Oliver,1 M. J. R. Olsson,36 A. Olszewski,82 J. Olszowska,82 D. C. O’Neil,149 A. Onofre,136a,136e K. Onogi,115 P. U. E. Onyisi,11 H. Oppen,130 M. J. Oreglia,36 Y. Oren,158 D. Orestano,72a,72b E. C. Orgill,98 N. Orlando,61b 44 164 53b 53 55 58 30 85 A. A. O’Rourke,44 R. S. Orr,164 B. Osculati,53b,53a,a V. O’Shea,55 R. Ospanov,58a G. Otero M. Ouchrif,34d F. Ould-Saada,130 A. Ouraou,142 Q. Ouyang,15a M. Owen,55 R. E. Owen,21 V. E. Ozcan,12c N. Ozturk,8 J. Pacalt,126 H. A. Pacey,31 K. Pachal,149 A. Pacheco Pages,14 L. Pacheco Rodriguez,142 C. Padilla Aranda,14 M. Ouchrif,34d F. Ould-Saada,130 A. Ouraou,142 Q. Ouyang,15a M. Owen,55 R. E. Owen,21 V J. Pacalt,126 H. A. Pacey,31 K. Pachal,149 A. Pacheco Pages,14 L. Pacheco Rodriguez,142 S. Pagan Griso,18 M. Paganini,180 G. Palacino,63 S. Palazzo,40b,40a S. Palestini,35 M. Palka,81b D. Pallin,37 I. Panagoulias,10 C. E. Pandini,35 J. G. Panduro Vazquez,91 P. Pani,35 G. Panizzo,64a,64c L. Paolozzi,52 T. D. Papadopoulou,10 K. Papageorgiou,9,t A. Paramonov,6 D. Paredes Hernandez,61b S. R. Paredes Saenz,131 B. Parida,58c A. J. Parker,87 K. A. Parker,44 M. A. Parker,31 F. Parodi,53b,53a J. A. Parsons,38 U. Parzefall,50 V. R. Pascuzzi,164 J. M. P. Pasner,143 E. Pasqualucci,70a S. Passaggio,53b F. Pastore,91 P. Pasuwan,43a,43b S. Pataraia,97 J. R. Pater,98 A. Pathak,178,f T. Pauly,35 B. Pearson,113 M. Pedersen,130 L. Pedraza Diaz,117 S. Pedraza Lopez,171 R. Pedro,136a,136b S. V. Peleganchuk,120b,120a O. Penc,137 C. Peng,15d H. Peng,58a B. S. Peralva,78a M. M. Perego,142 A. P. Pereira Peixoto,136a D. V. Perepelitsa,29 F. Peri,19 L. Perini,66a,66b H. Pernegger,35 S. Perrella,67a,67b V. D. Peshekhonov,77,a K. Peters,44 R. F. Y. Peters,98 B. A. Petersen,35 T. C. Petersen,39 E. Petit,56 A. Petridis,1 C. Petridou,159 P. Petroff,128 E. Petrolo,70a M. Petrov,131 F. Petrucci,72a,72b M. Pettee,180 N. E. Pettersson,100 A. Peyaud,142 R. Pezoa,144b T. Pham,102 F. H. Phillips,104 P. W. Phillips,141 G. Piacquadio,152 E. Pianori,18 A. Picazio,100 M. A. Pickering,131 R. Piegaia,30 J. E. Pilcher,36 A. D. Pilkington,98 M. Pinamonti,71a,71b J. L. Pinfold,3 M. Pitt,177 M-A. Pleier,29 V. Pleskot,139 E. Plotnikova,77 D. Pluth,76 P. Podberezko,120b,120a R. Poettgen,94 R. Poggi,52 L. Poggioli,128 I. Pogrebnyak,104 D. Pohl,24 I. Pokharel,51 G. Polesello,68a A. Poley,44 A. Policicchio,40b,40a R. Polifka,35 A. Polini,23b C. S. Pollard,44 V. Polychronakos,29 D. Ponomarenko,110 L. Pontecorvo,70a G. A. Popeneciu,27d D. M. SEARCH FOR LONG-LIVED PARTICLES IN FINAL … Portillo Quintero,132 S. Pospisil,138 K. Potamianos,44 I. N. Potrap,77 C. J. Potter,31 H. Potti,11 S. Pagan Griso,18 M. Paganini,180 G. Palacino,63 S. Palazzo,40b,40a S. Palestini,35 M. Palka,81b D. Pallin,37 I. Panagoulias,10 C. E. Pandini,35 J. G. Panduro Vazquez,91 P. Pani,35 G. Panizzo,64a,64c L. Paolozzi,52 T. D. Papadopoulou,10 K. Papageorgiou,9,t A. Paramonov,6 D. Paredes Hernandez,61b S. R. Paredes Saenz,131 B. Parida,58c A. J. Parker,87 K. A. Parker,44 M. A. Parker,31 F. Parodi,53b,53a J. A. Parsons,38 U. Parzefall,50 V. R. Pascuzzi,164 J. M. P. Pasner,143 E. Pasqualucci,70a S. Passaggio,53b F. Pastore,91 P. Pasuwan,43a,43b S. Pataraia,97 J. R. Pater,98 A. Pathak,178,f T. Pauly,35 B. Pearson,113 M. Pedersen,130 L. Pedraza Diaz,117 S. Pedraza Lopez,171 R. Pedro,136a,136b S. V. Peleganchuk,120b,120a O. Penc,137 C. Peng,15d H. Peng,58a B. S. Peralva,78a M. M. Perego,142 A. P. Pereira Peixoto,136a D. V. Perepelitsa,29 F. Peri,19 L. Perini,66a,66b H. Pernegger,35 S. Perrella,67a,67b V. D. Peshekhonov,77,a K. Peters,44 R. F. Y. Peters,98 B. A. Petersen,35 T. C. Petersen,39 E. Petit,56 A. Petridis,1 C. Petridou,159 P. Petroff,128 E. Petrolo,70a M. Petrov,131 F. Petrucci,72a,72b M. Pettee,180 N. E. Pettersson,100 A. Peyaud,142 R. Pezoa,144b T. Pham,102 F. H. Phillips,104 P. W. Phillips,141 G. Piacquadio,152 E. Pianori,18 A. Picazio,100 M. A. Pickering,131 R. Piegaia,30 J. E. Pilcher,36 A. D. Pilkington,98 M. Pinamonti,71a,71b J. L. Pinfold,3 M. Pitt,177 M-A. Pleier,29 V. Pleskot,139 E. Plotnikova,77 D. Pluth,76 P. Podberezko,120b,120a R. Poettgen,94 R. Poggi,52 L. Poggioli,128 I. Pogrebnyak,104 D. Pohl,24 I. Pokharel,51 G. Polesello,68a A. Poley,44 A. Policicchio,40b,40a R. Polifka,35 A. Polini,23b C. S. Pollard,44 V. Polychronakos,29 D. Ponomarenko,110 L. Pontecorvo,70a G. A. Popeneciu,27d D. M. Portillo Quintero,132 S. Pospisil,138 K. Potamianos,44 I. N. Potrap,77 C. J. Potter,31 H. Potti,11 K. A. Parker,44 M. A. Parker,31 F. Parodi,53b,53a J. A. Parsons,38 U. Parzefall,50 V. R. Pascuzzi,164 J. M. P. Pasner,143 E. Pasqualucci,70a S. Passaggio,53b F. Pastore,91 P. Pasuwan,43a,43b S. Pataraia,97 J. R. Pater,98 A. Pathak,178,f T. Pauly,35 B. Pearson,113 M. Pedersen,130 L. Pedraza Diaz,117 S. Pedraza Lopez,171 R. Pedro,136a,136b S. V. Peleganchuk,120b,120a O. Penc,137 C. Peng,15d H. Peng,58a B. S. Peralva,78a M. M. Perego,142 A. P. Pereira Peixoto,136a D. V. Perepelitsa,29 F. Peri,19 L. Perini,66a,66b H. Pernegger,35 S. Perrella,67a,67b V. D. Peshekhonov,77,a K. Peters,44 R. F. Y. Peters,98 B. A. Petersen,35 T. C. Petersen,39 E. Petit,56 A. Petridis,1 C. Petridou,159 P. Petroff,128 E. Petrolo,70a M. Petrov,131 F. Petrucci,72a,72b M. Pettee,180 N. E. Pettersson,100 A. Peyaud,142 R. Pezoa,144b T. Pham,102 F. H. Phillips,104 P. W. Phillips,141 G. Piacquadio,152 E. SEARCH FOR LONG-LIVED PARTICLES IN FINAL … Richter,113 S. Rich g E. D. Resseguie,133 S. Rettie,172 E. Reynolds,21 O. L. Rezanova,120b,120a P. Reznicek,139 R. Ri E. Richter-Was,81b O. Ricken,24 M. Ridel,132 P. Rieck,113 C. J. Riegel,179 O. Rifki,44 M. Rijssenbeek,152 A. Rimoldi,68a,68b L. Rinaldi,23b G. Ripellino,151 B. Ristić,87 E. Ritsch,35 I. Riu,14 J. C. Rivera Vergara,144a F. Riz M. Rimoldi,20 L. Rinaldi,23b G. Ripellino,151 B. Ristić,87 E. Ritsch,35 I. Riu,14 J. C. Rivera Vergara,144a F. Rizatdinova,125 E. Rizvi,90 C. Rizzi,14 R. T. Roberts,98 S. H. Robertson,101,n A. Robichaud-Veronneau,101 D. Robinson,31 , , p , , , , g , , E. Rizvi,90 C. Rizzi,14 R. T. Roberts,98 S. H. Robertson,101,n A. Robichaud-Veronneau,101 D. Robinson,31 vi,90 C. Rizzi,14 R. T. Roberts,98 S. H. Robertson,101,n A. Robichaud-Veronneau,101 D. Robinso son,44 A. Robson,55 E. Rocco,97 C. Roda,69a,69b Y. Rodina,99 S. Rodriguez Bosca,171 A. Rodrigu J. E. M. Robinson,44 A. Robson,55 E. Rocco,97 C. Roda,69a,69b Y. Rodina,99 S. Rodriguez Bosca,171 A. Rodriguez Perez,14 D. Rodriguez Rodriguez,171 A. M. Rodríguez Vera,165b S. Roe,35 C. S. Rogan,57 O. Røhne,130 R. Röhrig,113 D. Rodriguez Rodriguez,171 A. M. Rodríguez Vera,165b S. Roe,35 C. S. Rogan,57 O. Røhne,130 R. Röhrig,113 d,63 J. Roloff,57 A. Romaniouk,110 M. Romano,23b,23a N. Rompotis,88 M. Ronzani,121 L. Roos,132 S C. P. A. Roland,63 J. Roloff,57 A. Romaniouk,110 M. Romano,23b,23a N. Rompotis,88 M. Ronzani K. Rosbach,50 P. Rose,143 N-A. Rosien,51 E. Rossi,67a,67b L. P. Rossi,53b L. Rossini,66a,66b J. H. N. Rosten,31 R. Rosten,14 J. Rothberg,145 D. Rousseau,128 D. Roy,32c A. Rozanov,99 Y. Rozen,157 X. Ruan,32c F. Rubbo,15 M. Rotaru,27b J. Rothberg,145 D. Rousseau,128 D. Roy,32c A. Rozanov,99 Y. Rozen,157 X. Ruan,32c F. Rubbo,150 F. Rühr,50 A. Ruiz-Martinez,33 Z. Rurikova,50 N. A. Rusakovich,77 H. L. Russell,101 J. P. Rutherfoord,7 N. Ruthmann,35 A. Ruiz-Martinez,33 Z. Rurikova,50 N. A. Rusakovich,77 H. L. Russell,101 J. P. Rutherfoord,7 N. Ruthmann,35 E. M. Rüttinger,44,nn Y. F. Ryabov,134 M. Rybar,170 G. Rybkin,128 S. Ryu,6 A. Ryzhov,140 G. F. Rzehorz,51 P. Sabatini,51 G. Sabato,118 S. Sacerdoti,128 H. F-W. Sadrozinski,143 R. Sadykov,77 F. Safai Tehrani,70a P. Saha,119 M. Sahinsoy,59a g y y y y y G. Sabato,118 S. Sacerdoti,128 H. F-W. Sadrozinski,143 R. Sadykov,77 F. Safai Tehrani,70a P. Saha,119 M. Sahinsoy,59a y A. Sahu,179 M. Saimpert,44 M. Saito,160 T. Saito,160 H. Sakamoto,160 A. Sakharov,121,gg D. Salam J. E. Salazar Loyola,144b D. Salek,118 P. H. Sales De Bruin,169 D. Salihagic,113 A. Salnikov,150 J. Salt,171 D. Salvatore,40b,40a F. Salvatore,153 A. Salvucci,61a,61b,61c A. Salzburger,35 D. Sammel,50 D. Sampsonidis,159 D. Sampsonidou,159 J. Sánchez,171 A. SEARCH FOR LONG-LIVED PARTICLES IN FINAL … S g , g, g y, , , , C. Schiavi,53b,53a S. Schier,143 L. K. Schildgen,24 Z. M. Schillaci,26 E. J. Schioppa,35 M. Schioppa,40b,40a K. E. Schleicher,50 35 113 35 97 44 97 50 C. Schiavi,53b,53a S. Schier,143 L. K. Schildgen,24 Z. M. Schillaci,26 E. J. Schioppa,35 M. Schioppa,40b,40a K. E. Schleicher,50 S S hl k 35 K R S h idt S f ld 113 K S h i d 35 C S h itt 97 S S h itt 44 S S h it 97 U S h 50 Schiavi,53b,53a S. Schier,143 L. K. Schildgen,24 Z. M. Schillaci,26 E. J. Schioppa,35 M. Schioppa,4 L. Schoeffel,142 A. Schoening,59b E. Schopf,24 M. Schott,97 J. F. P. Schouwenberg,117 J. Schovancova,35 S. Schramm,52 A. Schulte,97 H-C. Schultz-Coulon,59a M. Schumacher,50 B. A. Schumm,143 Ph. Schune,142 A. Schwartzman,150 T. A. Schwarz,103 H. Schweiger,98 Ph. Schwemling,142 R. Schwienhorst,104 A. Sciandra,24 G. Sciolla,26 M. Scornajenghi,40b,40a F. Scuri,69a F. Scutti,102 L. M. Scyboz,113 J. Searcy,103 C. D. Sebastiani,70a,70b P. Seema,24 S. C. Seidel,116 A. Seiden,143 T. Seiss,36 J. M. Seixas,78b G. Sekhniaidze,67a K. Sekhon,103 S. J. Sekula,41 N. Semprini-Cesari,23b,23a S. Sen,47 S. Senkin,37 C. Serfon,130 L. Serin,128 L. Serkin,64a,64b M. esari,23b,23a S. Sen,47 S. Senkin,37 C. Serfon,130 L. Serin,128 L. Serkin,64a,64b M. Sessa,72a,72b H. p T. Šfiligoj,89 F. Sforza,167 A. Sfyrla,52 E. Shabalina,51 J. D. Shahinian,143 N. W. Shaikh,43a,43b L. Y. Shan,15a R. Shang,170 25 18 1 131 109 153 98 134 J. T. Shank,25 M. Shapiro,18 A. S. Sharma,1 A. Sharma,131 P. B. Shatalov,109 K. Shaw,153 S. M. Shaw,98 A. Shcherbakova,134 Y Sh 124 N Sh f ti 33 A D Sh 25 P Sh d 92 L Shi 155,oo S Shi i 80 C O Shi i 180 M Shi ji 114 J. T. Shank,25 M. Shapiro,18 A. S. Sharma,1 A. Sharma,131 P. B. Shatalov,109 K. Shaw,153 S. M. S 124 33 25 92 155 80 , , , , , , , j , I. P. J. Shipsey,131 S. Shirabe,85 M. Shiyakova,77 J. Shlomi,177 A. Shmeleva,108 D. Shoaleh Saadi,107 M. J. Shochet,36 S. Shojaii,102 D. R. Shope,124 S. Shrestha,122 E. Shulga,110 P. Sicho,137 A. M. Sickles,170 P. E. Sidebo,151 E. Sideras Haddad,32c O. Sidiropoulou,174 A. Sidoti,23b,23a F. Siegert,46 Dj. Sijacki,16 J. Silva,136a M. Silva Jr.,178 M. V. Silva Oliveira,78a S. B. Silverstein,43a L. Simic,77 S. Simion,128 E. Simioni,97 M. Simon,97 P. Sinervo,164 N. B. Sinev,127 M. Sioli,23b,23a G. Siragusa,174 I. Siral,103 S.Yu. Sivoklokov,111 J. SEARCH FOR LONG-LIVED PARTICLES IN FINAL … Sjölin,43a,43b M. B. Skinner,87 P. Skubic,124 M. Slater,21 T. Slavicek,138 M. Slawinska,82 K. Sliwa,167 R. Slovak,139 V. Smakhtin,177 B. H. Smart,5 J. Smiesko,28a N. Smirnov,110 S.Yu. Smirnov,110 Y. Smirnov,110 L. N. Smirnova,111 O. Smirnova,94 J. W. Smith,51 M. N. K. Smith,38 R. W. Smith,38 j I. P. J. Shipsey,131 S. Shirabe,85 M. Shiyakova,77 J. Shlomi,177 A. Shmeleva,108 D. Shoaleh Saadi,107 M. J. Shochet,36 S. Shojaii,102 D. R. Shope,124 S. Shrestha,122 E. Shulga,110 P. Sicho,137 A. M. Sickles,170 P. E. Sidebo,151 E. Sideras Haddad,32c O. Sidiropoulou,174 A. Sidoti,23b,23a F. Siegert,46 Dj. Sijacki,16 J. Silva,136a M. Silva Jr.,178 E. Sideras Haddad,32c O. Sidiropoulou,174 A. Sidoti,23b,23a F. Siegert,46 Dj. Sijacki,16 J. Silva,136a M. Silva Jr.,178 M. V. Silva Oliveira,78a S. B. Silverstein,43a L. Simic,77 S. Simion,128 E. Simioni,97 M. Simon,97 P. Sinervo,164 N. B. Sinev,127 M. Sioli,23b,23a G. Siragusa,174 I. Siral,103 S.Yu. Sivoklokov,111 J. Sjölin,43a,43b M. B. Skinner,87 P. Skubic,124 M. Slater,21 T. Slavicek,138 M. Slawinska,82 K. Sliwa,167 R. Slovak,139 V. Smakhtin,177 B. H. Smart,5 J. Smiesko,28a N. Smirnov,110 S.Yu. Smirnov,110 Y. Smirnov,110 L. N. Smirnova,111 O. Smirnova,94 J. W. Smith,51 M. N. K. Smith,38 R. W. Smith,38 M. Smizanska,87 K. Smolek,138 A. A. Snesarev,108 I. M. Snyder,127 S. Snyder,29 R. Sobie,173,n A. M. Soffa,168 A. Soffer,158 A. Søgaard,48 D. A. Soh,155 G. Sokhrannyi,89 C. A. Solans Sanchez,35 M. Solar,138 E. Yu. Soldatov,110 U. Soldevila,171 A. A. Solodkov,140 A. Soloshenko,77 O. V. Solovyanov,140 V. Solovyev,134 P. Sommer,146 H. Son,167 W. Song,141 M. V. Silva Oliveira, S. B. Silverstein, L. Simic, S. Simion, E. Simioni, M. Simon, P. Sinervo, N. B. Sinev, M. Sioli,23b,23a G. Siragusa,174 I. Siral,103 S.Yu. Sivoklokov,111 J. Sjölin,43a,43b M. B. Skinner,87 P. Skubic,124 M. Slater,21 T. Slavicek,138 M. Slawinska,82 K. Sliwa,167 R. Slovak,139 V. Smakhtin,177 B. H. Smart,5 J. Smiesko,28a N. Smirnov,110 S.Yu. Smirnov,110 Y. Smirnov,110 L. N. Smirnova,111 O. Smirnova,94 J. W. Smith,51 M. N. K. Smith,38 R. W. Smith,38 M. Sioli,23b,23a G. Siragusa,174 I. Siral,103 S.Yu. Sivoklokov,111 J. Sjölin,43a,43b M. B. Skinner,87 P. Skubic,124 M. Slater,21 T. Slavicek,138 M. Slawinska,82 K. Sliwa,167 R. Slovak,139 V. Smakhtin,177 B. H. Smart,5 J. Smiesko,28a N. Smirnov,110 S.Yu. Smirnov,110 Y. Smirnov,110 L. N. Smirnova,111 O. Smirnova,94 J. W. Smith,51 M. N. K. Smith,38 R. W. Smith,38 87 138 108 127 29 173 168 158 A. Søgaard,48 D. A. Soh,155 G. Sokhrannyi,89 C. A. Solans Sanchez,35 M. Solar,138 E. Yu. Soldatov,110 U. Soldevila,171 A. A. Solodkov,140 A. Soloshenko,77 O. V. Solovyanov,140 V. SEARCH FOR LONG-LIVED PARTICLES IN FINAL … Pianori,18 A. Picazio,100 M. A. Pickering,131 R. Piegaia,30 J. E. Pilcher,36 A. D. Pilkington,98 M Pinamonti 71a,71b J L Pinfold 3 M Pitt 177 M A Pleier 29 V Pleskot 139 E Plotnikova 77 D Pluth 76 P Podberezko 120b,120a E. Pasqualucci,70a S. Passaggio,53b F. Pastore,91 P. Pasuwan,43a,43b S. Pataraia,97 J. R. Pater,98 A. Pathak,178,f T. Pauly,35 B. Pearson,113 M. Pedersen,130 L. Pedraza Diaz,117 S. Pedraza Lopez,171 R. Pedro,136a,136b S. V. Peleganchuk,120b,120a O. Penc,137 C. Peng,15d H. Peng,58a B. S. Peralva,78a M. M. Perego,142 A. P. Pereira Peixoto,136a D. V. Perepelitsa,29 F. Peri,19 L. Perini,66a,66b H. Pernegger,35 S. Perrella,67a,67b V. D. Peshekhonov,77,a K. Peters,44 R. F. Y. Peters,98 B. A. Petersen,35 T. C. Petersen,39 E. Petit,56 A. Petridis,1 C. Petridou,159 P. Petroff,128 E. Petrolo,70a M. Petrov,131 F. Petrucci,72a,72b 180 100 142 144b 102 104 141 T. C. Petersen,39 E. Petit,56 A. Petridis,1 C. Petridou,159 P. Petroff,128 E. Petrolo,70a M. Petrov,131 F. Petrucci,72a,72b M. Pettee,180 N. E. Pettersson,100 A. Peyaud,142 R. Pezoa,144b T. Pham,102 F. H. Phillips,104 P. W. Phillips,141 . A. Popeneciu,27d D. M. Portillo Quintero,132 S. Pospisil,138 K. Potamianos,44 I. N. Potrap,77 C 012001-24 012001-24 PHYS. REV. D 99, 012001 (2019) SEARCH FOR LONG-LIVED PARTICLES IN FINAL … j g p p D. M. Rauch,44 F. Rauscher,112 S. Rave,97 B. Ravina,146 I. Ravinovich,177 J. H. Rawling,98 M. , , , , , g, y , , N. P. Readioff,56 M. Reale,65a,65b D. M. Rebuzzi,68a,68b A. Redelbach,174 G. Redlinger,29 R. Reece,143 R. G. Reed,32c K. Reeves,42 L. Rehnisch,19 J. Reichert,133 A. Reiss,97 C. Rembser,35 H. Ren,15d M. Rescigno,70a S. Resconi,66a E. D. Resseguie,133 S. Rettie,172 E. Reynolds,21 O. L. Rezanova,120b,120a P. Reznicek,139 R. Richter,113 S. Richter,92 N. P. Readioff, M. Reale, D. M. Rebuzzi, A. Redelbach, G. Redlinger, R. Reece, R. G. Reed, K. Reeves,42 L. Rehnisch,19 J. Reichert,133 A. Reiss,97 C. Rembser,35 H. Ren,15d M. Rescigno,70a S. Resconi,66a E. D. Resseguie,133 S. Rettie,172 E. Reynolds,21 O. L. Rezanova,120b,120a P. Reznicek,139 R. Richter,113 S. Richter,92 g L. Rehnisch,19 J. Reichert,133 A. Reiss,97 C. Rembser,35 H. Ren,15d M. Rescigno,70a S. Rescon K. Reeves, L. Rehnisch, J. Reichert, A. Reiss, C. Rembser, H. Ren, M. Rescigno, S. Resconi, E. D. Resseguie,133 S. Rettie,172 E. Reynolds,21 O. L. Rezanova,120b,120a P. Reznicek,139 R. Richter,113 S. Richter,92 81b 24 132 113 179 44 152 68 68b , , , , , g , e,133 S. Rettie,172 E. Reynolds,21 O. L. Rezanova,120b,120a P. Reznicek,139 R. SEARCH FOR LONG-LIVED PARTICLES IN FINAL … Sanchez Pineda,64a,64c H. Sandaker,130 C. O. Sander,44 M. Sandhoff,179 C. Sandoval,22 D. P. C. Sankey,141 M. Sannino,53b,53a Y. Sano,115 A. Sansoni,49 C. Santoni,37 H. Santos,136a I. Santoyo Castillo,153 A. Sapronov,77 J. G. Saraiva,136a,136d O. Sasaki,79 K. Sato,166 E. Sauvan,5 P. Savard,164,e N. Savic,113 R. Sawada,160 C. Sawyer,141 L. Sawyer,93,v C. Sbarra,23b A. Sbrizzi,23b,23a T. Scanlon,92 J. Schaarschmidt,145 P. Schacht,113 B. M. Schachtner,112 D. Schaefer,36 L. Schaefer,133 J. Schaeffer,97 S. Schaepe,35 U. Schäfer,97 A. C. Schaffer,128 D. Schaile,112 R. D. Schamberger,152 N. Scharmberg,98 V. A. Schegelsky,134 D. Scheirich,139 F. Schenck,19 M. Schernau,168 53b 53 143 24 26 35 40b 40 50 F. Salvatore,153 A. Salvucci,61a,61b,61c A. Salzburger,35 D. Sammel,50 D. Sampsonidis,159 D. Sampsonidou,159 J. Sánchez,171 A. Sanchez Pineda,64a,64c H. Sandaker,130 C. O. Sander,44 M. Sandhoff,179 C. Sandoval,22 D. P. C. Sankey,141 M. Sannino,53b,53a Y. Sano,115 A. Sansoni,49 C. Santoni,37 H. Santos,136a I. Santoyo Castillo,153 A. Sapronov,77 J. G. Saraiva,136a,136d O. Sasaki,79 K. Sato,166 E. Sauvan,5 P. Savard,164,e N. Savic,113 R. Sawada,160 C. Sawyer,141 L. Sawyer,93,v C. Sbarra,23b A. Sbrizzi,23b,23a T. Scanlon,92 J. Schaarschmidt,145 P. Schacht,113 B. M. Schachtner,112 F. Salvatore, A. Salvucci, A. Salzburger, D. Sammel, D. Sampsonidis, D. Sampsonidou, J. Sánchez, A. Sanchez Pineda,64a,64c H. Sandaker,130 C. O. Sander,44 M. Sandhoff,179 C. Sandoval,22 D. P. C. Sankey,141 M. Sannino,53b,53a Y. Sano,115 A. Sansoni,49 C. Santoni,37 H. Santos,136a I. Santoyo Castillo,153 A. Sapronov,77 J G Saraiva 136a,136d O Sasaki 79 K Sato 166 E Sauvan 5 P Savard 164,e N Savic 113 R Sawada 160 C Sawyer 141 , , g , , p , p , , A. Sanchez Pineda,64a,64c H. Sandaker,130 C. O. Sander,44 M. Sandhoff,179 C. Sandoval,22 D. P. C. Sankey,141 , , g , , p , p , , A. Sanchez Pineda,64a,64c H. Sandaker,130 C. O. Sander,44 M. Sandhoff,179 C. Sandoval,22 D. P. C. Sankey,141 M Sannino 53b,53a Y Sano 115 A Sansoni 49 C Santoni 37 H Santos 136a I Santoyo Castillo 153 A Sapronov 77 M. Sannino,53b,53a Y. Sano,115 A. Sansoni,49 C. Santoni,37 H. Santos,136a I. Santoyo Castillo,153 A. Sapronov,77 J. G. Saraiva,136a,136d O. Sasaki,79 K. Sato,166 E. Sauvan,5 P. Savard,164,e N. Savic,113 R. Sawada,160 C. Sawyer,141 L. Sawyer,93,v C. Sbarra,23b A. Sbrizzi,23b,23a T. Scanlon,92 J. Schaarschmidt,145 P. Schacht,113 y D. Schaefer,36 L. Schaefer,133 J. Schaeffer,97 S. Schaepe,35 U. Schäfer,97 A. C. Schaffer,128 D. Schaile,112 p D. Schamberger,152 N. Scharmberg,98 V. A. Schegelsky,134 D. Scheirich,139 F. Schenck,19 M. T. Poulsen,94 J. Poveda,35 T. D. Powell,146 M. E. Pozo Astigarraga,35 P. Pralavorio,99 S. Prell,76 D. Price,98 M. Primavera,65a S. Prince,101 N. Proklova,110 K. Prokofiev,61c F. Prokoshin,144b S. Protopopescu,29 J. Proudfoot,6 M. Przybycien,81a A. Puri,170 P. Puzo,128 J. Qian,103 Y. Qin,98 A. Quadt,51 M. Queitsch-Maitland,44 A. Qureshi,1 P. Rados,102 F. Ragusa,66a,66b G R h l 95 J A R i 98 S R j l 29 A R i M l 90 T R hid 128 S R 5 M G R tti 66a,66b SEARCH FOR LONG-LIVED PARTICLES IN FINAL … Solovyev,134 P. Sommer,146 H. Son,167 W. Song,141 012001-25 012001-25 PHYS. REV. D 99, 012001 (2019) M. AABOUD et al. PHYS. REV. D 99, 012001 (2019) A. Strubig,48 S. A. Stucci,29 B. Stugu,17 J. Stupak,124 N. A. Styles,44 D. Su,150 J. Su,135 S. Suchek,59a Y. Sugaya,129 M. Suk,138 V. V. Sulin,108 D. M. S. Sultan,52 S. Sultansoy,4c T. Sumida,83 S. Sun,103 X. Sun,3 K. Suruliz,153 C. J. E. Suster,154 M. R. Sutton,153 S. Suzuki,79 M. Svatos,137 M. Swiatlowski,36 S. P. Swift,2 A. Sydorenko,97 I. Sykora,28a T. Sykora,139 D. Ta,97 K. Tackmann,44,qq J. Taenzer,158 A. Taffard,168 R. Tafirout,165a E. Tahirovic,90 N. Taiblum,158 H. Takai,29 R. Takashima,84 E. H. Takasugi,113 K. Takeda,80 T. Takeshita,147 Y. Takubo,79 M. Talby,99 A. A. Talyshev,120b,120a J. Tanaka,160 M. Tanaka,162 R. Tanaka,128 R. Tanioka,80 B. B. Tannenwald,122 S. Tapia Araya,144b S. Tapprogge,97 A. Tarek Abouelfadl Mohamed,132 S. Tarem,157 G. Tarna,27b,q G. F. Tartarelli,66a P. Tas,139 M. Tasevsky,137 T. Tashiro,83 40b 40 136 136b 34 116 48 102 102 E. Tassi,40b,40a A. Tavares Delgado,136a,136b Y. Tayalati,34e A. C. Taylor,116 A. J. Taylor,48 G. N. Taylor,102 P. T. E. Taylor,102 W. Taylor,165b A. S. Tee,87 P. Teixeira-Dias,91 H. Ten Kate,35 P. K. Teng,155 J. J. Teoh,129 F. Tepel,179 S. Terada,79 E. Tassi, A. Tavares Delgado, Y. Tayalati, A. C. Taylor, A. J. Taylor, G. N. Taylor, P. T. E. Taylor, W. Taylor,165b A. S. Tee,87 P. Teixeira-Dias,91 H. Ten Kate,35 P. K. Teng,155 J. J. Teoh,129 F. Tepel,179 S. Terada,79 K. Terashi,160 J. Terron,96 S. Terzo,14 M. Testa,49 R. J. Teuscher,164,n S. J. Thais,180 T. Theve J. P. Thomas,21 A. S. Thompson,55 P. D. Thompson,21 L. A. Thomsen,180 E. Thomson,133 Y. Tian,38 R. E. Ticse Torres,51 V. O. Tikhomirov,108,rr Yu.A. Tikhonov,120b,120a S. Timoshenko,110 P. Tipton,180 S. Tisserant,99 K. Todome,162 J. P. Thomas,21 A. S. Thompson,55 P. D. Thompson,21 L. A. Thomsen,180 E. Thomson,133 Y. Tian,38 R. E. Ticse Torres,51 V. O. Tikhomirov,108,rr Yu.A. Tikhonov,120b,120a S. Timoshenko,110 P. Tipton,180 S. Tisserant,99 K. Todome,162 S. Todorova-Nova,5 S. Todt,46 J. Tojo,85 S. Tokár,28a K. Tokushuku,79 E. Tolley,122 K. G. Tomiwa,32c M. Tomoto,115 L. Tompkins,150,ff K. Toms,116 B. Tong,57 P. Tornambe,50 E. Torrence,127 H. Torres,46 E. Torró Pastor,145 C. Tosciri,131 J. Toth,99,ss F. Touchard,99 D. R. Tovey,146 C. J. Treado,121 T. Trefzger,174 F. Tresoldi,153 A. Tricoli,29 I. M. Trigger,165a 132 14 164 56 128 66a 173 F. Trovato,153 L. Truong,32b M. Trzebinski,82 A. Trzupek,82 F. Tsai,44 J.C-L. Tseng,131 P. V. Tsiareshka,105 N. Tsirintanis,9 V. Tsiskaridze,152 E. G. Tskhadadze,156a I. I. Tsukerman,109 V. Tsulaia,18 S. Tsuno,79 D. Tsybychev,152 Y. Tu,61b A. Tudorache,27b V. Tudorache,27b T. T. Tulbure,27a A. N. PHYS. REV. D 99, 012001 (2019) Vercesi,68a M V d i 72a 72b C M V l I f 76 W V k k 118 A T V l 118 J C V l 118 M C V li 149 e N. Viaux Maira,144b O. Viazlo,94 I. Vichou,170,a T. Vickey,146 O. E. Vickey Boeriu,146 G. H. A. Viehhauser,131 S. Viel,18 L. Vigani,131 M. Villa,23b,23a M. Villaplana Perez,66a,66b E. Vilucchi,49 M. G. Vincter,33 V. B. Vinogradov,77 A. Vishwakarma,44 C. Vittori,23b,23a I. Vivarelli,153 S. Vlachos,10 M. Vogel,179 P. Vokac,138 G. Volpi,14 32 24 139 110 171 88 16 S. E. von Buddenbrock,32c E. Von Toerne,24 V. Vorobel,139 K. Vorobev,110 M. Vos,171 J. H. V S. E. von Buddenbrock, E. Von Toerne, V. Vorobel, K. Vorobev, M. Vos, J. H. Vossebeld, N. Vranjes, M. Vranjes Milosavljevic,16 V. Vrba,138 M. Vreeswijk,118 R. Vuillermet,35 I. Vukotic,36 P. Wagner,24 W. Wagner,179 M. Vranjes Milosavljevic,16 V. Vrba,138 M. Vreeswijk,118 R. Vuillermet,35 I. Vukotic,36 P. Wagner,24 W. Wagner,179 J. Wagner-Kuhr, H. Wahlberg, S. Wahrmund, K. Wakamiya, V. M. Walbrecht, J. Walder, R. Walker, W. Walkowiak,148 V. Wallangen,43a,43b A. M. Wang,57 C. Wang,58b,q F. Wang,178 H. Wang,18 H. Wang,3 J. Wang,154 J. Wang,59b P. Wang,41 Q. Wang,124 R.-J. Wang,132 R. Wang,58a R. Wang,6 S. M. Wang,155 W. T. Wang,58a W. Wang,155,uu W. X. Wang,58a,vv Y. Wang,58a Z. Wang,58c C. Wanotayaroj,44 A. Warburton,101 C. P. Ward,31 D. R. Wardrope,92 A. Washbrook,48 P. M. Watkins,21 A. T. Watson,21 M. F. Watson,21 G. Watts,145 S. Watts,98 B. M. Waugh,92 A. F. Webb,11 S. Webb,97 C. Weber,180 M. S. Weber,20 S. A. Weber,33 S. M. Weber,59a J. S. Webster,6 A. R. Weidberg,131 B. Weinert,63 J W i t 51 M W i i h 97 C W i 50 P S W ll 35 T W 29 T W l 35 S W i 35 N W 24 J. Wagner-Kuhr, H. Wahlberg, S. Wahrmund, K. Wakamiya, V. M. Walbrecht, J. Walder, R. Walker, W. Walkowiak,148 V. Wallangen,43a,43b A. M. Wang,57 C. Wang,58b,q F. Wang,178 H. Wang,18 H. Wang,3 J. Wang,154 J. Wang,59b P. Wang,41 Q. Wang,124 R.-J. Wang,132 R. Wang,58a R. Wang,6 S. M. Wang,155 W. T. Wang,58a W. Wang,155,uu W. X. Wang,58a,vv Y. Wang,58a Z. Wang,58c C. Wanotayaroj,44 A. Warburton,101 C. P. Ward,31 D. R. Wardrope,92 S. Webb,97 C. Weber,180 M. S. Weber,20 S. A. Weber,33 S. M. Weber,59a J. S. Webster,6 A. R. Weidberg,131 B. Weinert,63 J. Weingarten,51 M. Weirich,97 C. Weiser,50 P. S. Wells,35 T. A. Sopczak,138 F. Sopkova,28b D. Sosa,59b C. L. Sotiropoulou,69a,69b S. Sottocornola,68a,68b R. Soualah,64a,64c,pp A. M. Soukharev,120b,120a D. South,44 B. C. Sowden,91 S. Spagnolo,65a,65b M. Spalla,113 M. Spangenberg,175 F. Spanò,91 D. Sperlich,19 F. Spettel,113 T. M. Spieker,59a R. Spighi,23b G. Spigo,35 L. A. Spiller,102 D. P. Spiteri,55 M. Spousta,139 A. Stabile,66a,66b R. Stamen,59a S. Stamm,19 E. Stanecka,82 R. W. Stanek,6 C. Stanescu,72a B. Stanislaus,131 M. M. Stanitzki,44 B. Stapf,118 S. Stapnes,130 E. A. Starchenko,140 G. H. Stark,36 J. Stark,56 S.H Stark,39 P. Staroba,137 P. Starovoitov,59a S. Stärz,35 R. Staszewski,82 M. Stegler,44 P. Steinberg,29 B. Stelzer,149 H. J. Stelzer,35 O. Stelzer-Chilton,165a H. Stenzel,54 T. J. Stevenson,90 G. A. Stewart,55 M. C. Stockton,127 G. Stoicea,27b P. Stolte,51 S. Stonjek,113 A. Straessner,46 J. Strandberg,151 S. Strandberg,43a,43b M. Strauss,124 P. Strizenec,28b R. Ströhmer,174 D. M. Strom,127 R. Stroynowski,41 A. Strubig,48 S. A. Stucci,29 B. Stugu,17 J. Stupak,124 N. A. Styles,44 D. Su,150 J. Su,135 S. Suchek,59a Y. Sugaya,129 M. Suk,138 V. V. Sulin,108 D. M. S. Sultan,52 S. Sultansoy,4c T. Sumida,83 S. Sun,103 X. Sun,3 K. Suruliz,153 C. J. E. Suster,154 M. R. Sutton,153 S. Suzuki,79 M. Svatos,137 M. Swiatlowski,36 S. P. Swift,2 A. Sydorenko,97 I. Sykora,28a T. Sykora,139 D. Ta,97 K. Tackmann,44,qq J. Taenzer,158 A. Taffard,168 R. Tafirout,165a E. Tahirovic,90 N. Taiblum,158 H. Takai,29 R. Takashima,84 E. H. Takasugi,113 K. Takeda,80 T. Takeshita,147 Y. Takubo,79 M. Talby,99 A. A. Talyshev,120b,120a J. Tanaka,160 M. Tanaka,162 R. Tanaka,128 R. Tanioka,80 B. B. Tannenwald,122 S. Tapia Araya,144b S. Tapprogge,97 A. Tarek Abouelfadl Mohamed,132 S. Tarem,157 G. Tarna,27b,q G. F. Tartarelli,66a P. Tas,139 M. Tasevsky,137 T. Tashiro,83 E. Tassi,40b,40a A. Tavares Delgado,136a,136b Y. Tayalati,34e A. C. Taylor,116 A. J. Taylor,48 G. N. Taylor,102 P. T. E. Taylor,102 W. Taylor,165b A. S. Tee,87 P. Teixeira-Dias,91 H. Ten Kate,35 P. K. Teng,155 J. J. Teoh,129 F. Tepel,179 S. Terada,79 K. Terashi,160 J. Terron,96 S. Terzo,14 M. Testa,49 R. J. Teuscher,164,n S. J. Thais,180 T. Theveneaux-Pelzer,44 F. Thiele,39 J. P. Thomas,21 A. S. Thompson,55 P. D. Thompson,21 L. A. Thomsen,180 E. Thomson,133 Y. Tian,38 R. E. Ticse Torres,51 V. O. Tikhomirov,108,rr Yu.A. Tikhonov,120b,120a S. Timoshenko,110 P. Tipton,180 S. Tisserant,99 K. Todome,162 S. Todorova-Nova,5 S. Todt,46 J. Tojo,85 S. Tokár,28a K. Tokushuku,79 E. Tolley,122 K. G. Tomiwa,32c M. Tomoto,115 L. Tompkins,150,ff K. Toms,116 B. Tong,57 P. Tornambe,50 E. Torrence,127 H. Torres,46 E. Torró Pastor,145 C. Tosciri,131 J. Toth,99,ss F. Touchard,99 D. R. Tovey,146 C. J. Treado,121 T. Trefzger,174 F. Tresoldi,153 A. Tricoli,29 I. M. Trigger,165a PHYS. REV. D 99, 012001 (2019) Tuna,57 S. Turchikhin,77 D. Turgeman,177 I. Turk Cakir,4b,tt R Turra 66a P M Tuts 38 E Tzovara 97 G Ucchielli 23b,23a I Ueda 79 M Ughetto 43a,43b F Ukegawa 166 G Unal 35 A. Tudorache, V. Tudorache, T. T. Tulbure, A. N. Tuna, S. Turchikhin, D. Turgeman, I. Turk Cakir, R. Turra,66a P. M. Tuts,38 E. Tzovara,97 G. Ucchielli,23b,23a I. Ueda,79 M. Ughetto,43a,43b F. Ukegawa,166 G. Unal,35 A. Undrus,29 G. Unel,168 F. C. Ungaro,102 Y. Unno,79 K. Uno,160 J. Urban,28b P. Urquijo,102 P. Urrejola,97 G. Usai,8 J. Usui,79 L. Vacavant,99 V. Vacek,138 B. Vachon,101 K. O. H. Vadla,130 A. Vaidya,92 C. Valderanis,112 E. Valdes Santurio,43a,43b M. Valente,52 S. Valentinetti,23b,23a A. Valero,171 L. Val´ery,44 R. A. Vallance,21 A. Vallier,5 J. A. Valls Ferrer,171 T R V D l 14 W V D W ll b 118 H V d G f 118 P V G 6 J V Ni k 149 L. Vacavant,99 V. Vacek,138 B. Vachon,101 K. O. H. Vadla,130 A. Vaidya,92 C. Valderanis,112 E. Valdes Santurio,43a,43b M. Valente,52 S. Valentinetti,23b,23a A. Valero,171 L. Val´ery,44 R. A. Vallance,21 A. Vallier,5 J. A. Valls Ferrer,171 T R Van Daalen 14 W Van Den Wollenberg 118 H Van der Graaf 118 P Van Gemmeren 6 J Van Nieuwkoop 149 L. Vacavant, V. Vacek, B. Vachon, K. O. H. Vadla, A. Vaidya, C. Valderanis, E. Valdes Santurio, M. Valente,52 S. Valentinetti,23b,23a A. Valero,171 L. Val´ery,44 R. A. Vallance,21 A. Vallier,5 J. A. Valls Ferrer,171 T R V D l 14 W V D W ll b 118 H V d G f 118 P V G 6 J V Ni k 149 I. Van Vulpen,118 M. C. van Woerden,118 M. Vanadia,71a,71b W. Vandelli,35 A. Vaniachine,163 P. Vankov,118 R. Vari,70a E. W. Varnes,7 C. Varni,53b,53a T. Varol,41 D. Varouchas,128 A. Vartapetian,8 K. E. Varvell,154 G. A. Vasquez,144b J. G. Vasquez,180 F. Vazeille,37 D. Vazquez Furelos,14 T. Vazquez Schroeder,101 J. Veatch,51 V. Vecchio,72a,72b L. M. Veloce,164 F. Veloso,136a,136c S. Veneziano,70a A. Ventura,65a,65b M. Venturi,173 N. Venturi,35 V. Vercesi,68a E. W. Varnes, C. Varni, T. Varol, D. Varouchas, A. Vartapetian, K. E. Varvell, G. A. Vasquez, J. G. Vasquez,180 F. Vazeille,37 D. Vazquez Furelos,14 T. Vazquez Schroeder,101 J. Veatch,51 V. Vecchio,72a,72b L. M. Veloce,164 F. Veloso,136a,136c S. Veneziano,70a A. Ventura,65a,65b M. Venturi,173 N. Venturi,35 V. SEARCH FOR LONG-LIVED PARTICLES IN FINAL … S. Young,35 C. Young,150 J. Yu,8 J. Yu,76 X. Yue,59a S. P. Y. Yuen,24 I. Yusuff,31,ww B. Zabinski,82 G. Zacharis,10 E. Zaffaroni,52 R. Zaidan,14 A. M. Zaitsev,140,ll N. Zakharchuk,44 J. Zalieckas,17 S. Zambito,57 D. Zanzi,35 D. R. Zaripovas,55 Y. Zhou,7 C. G. Zhu,58b H. L. Zhu,58a H. Zhu,15a J. Zhu,103 Y. Zhu,58a X. Zhuang,15a K. Z 5 A. Zibell,174 D. Zieminska,63 N. I. Zimine,77 S. Zimmermann,50 Z. Zinonos,113 M. Zinser,97 M. Ziolkowski,148 L. Živković,16 G. Zobernig,178 A. Zoccoli,23b,23a K. Zoch,51 T. G. Zorbas,146 R. Zou,36 M. Zur Nedden,19 and L. Zwalinski35 PHYS. REV. D 99, 012001 (2019) Wenaus,29 T. Wengler,35 S. Wenig,35 N. Wermes,24 012001-26 PHYS. REV. D 99, 012001 (2019) M. D. Werner,76 P. Werner,35 M. Wessels,59a T. D. Weston,20 K. Whalen,127 N. L. Whallon,145 A. M. Wharton,87 A. S. White,103 A. White,8 M. J. White,1 R. White,144b D. Whiteson,168 B. W. Whitmore,87 F. J. Wickens,141 W. Wiedenmann,178 M. Wielers,141 C. Wiglesworth,39 L. A. M. Wiik-Fuchs,50 A. Wildauer,113 F. Wilk,98 H. G. Wilkens,35 L. J. Wilkins,91 H. H. Williams,133 S. Williams,31 C. Willis,104 S. Willocq,100 J. A. Wilson,21 I. Wingerter-Seez,5 E. Winkels,153 F. Winklmeier,127 O. J. Winston,153 B. T. Winter,24 M. Wittgen,150 M. Wobisch,93 A. Wolf,97 T. M. H. Wolf,118 R. Wolff,99 M. W. Wolter,82 H. Wolters,136a,136c V. W. S. Wong,172 N. L. Woods,143 S. D. Worm,21 B. K. Wosiek,82 K. W. Woźniak,82 K. Wraight,55 M. Wu,36 S. L. Wu,178 X. Wu,52 Y. Wu,58a T. R. Wyatt,98 B. M. Wynne,48 S. Xella,39 Z. Xi,103 L. Xia,175 D. Xu,15a H. Xu,58a L. Xu,29 T. Xu,142 W. Xu,103 B. Yabsley,154 S. Yacoob,32a K. Yajima,129 D. P. Yallup,92 D. Yamaguchi,162 Y. Yamaguchi,162 A. Yamamoto,79 T. Yamanaka,160 F. Yamane,80 M. Yamatani,160 T. Yamazaki,160 Y. Yamazaki,80 Z. Yan,25 H. J. Yang,58c,58d H. T. Yang,18 S. Yang,75 Y. Yang,160 Z. Yang,17 W-M. Yao,18 Y. C. Yap,44 Y. Yasu,79 E. Yatsenko,58c,58d J. Ye,41 S. Ye,29 I. Yeletskikh,77 E. Yigitbasi,25 E. Yildirim,97 K. Yorita,176 K. Yoshihara,133 C. J. S. Young,35 C. Young,150 J. Yu,8 J. Yu,76 X. Yue,59a S. P. Y. Yuen,24 I. Yusuff,31,ww B. Zabinski,82 G. Zacharis,10 E. Zaffaroni,52 R. Zaidan,14 A. M. Zaitsev,140,ll N. Zakharchuk,44 J. Zalieckas,17 S. Zambito,57 D. Zanzi,35 D. R. Zaripovas,55 S. V. Zeißner,45 C. Zeitnitz,179 G. Zemaityte,131 J. C. Zeng,170 Q. Zeng,150 O. Zenin,140 T. Ženiš,28a D. Zerwas,128 M. Zgubič,131 D. F. Zhang,58b D. Zhang,103 F. Zhang,178 G. Zhang,58a,vv H. Zhang,15c J. Zhang,6 L. Zhang,50 L. Zhang,58a M. Zhang,170 P. Zhang,15c R. Zhang,58a,q R. Zhang,24 X. Zhang,58b Y. Zhang,15d Z. Zhang,128 P. Zhao,47 X. Zhao,41 Y. Zhao,58b,128,y Z. Zhao,58a A. Zhemchugov,77 B. Zhou,103 C. Zhou,178 L. Zhou,41 M. S. Zhou,15d M. Zhou,152 N. Zhou,58c Y. Zhou,7 C. G. Zhu,58b H. L. Zhu,58a H. Zhu,15a J. Zhu,103 Y. Zhu,58a X. Zhuang,15a K. Zhukov,108 V. Zhulanov,120b,120a A. Zibell,174 D. Zieminska,63 N. I. Zimine,77 S. Zimmermann,50 Z. Zinonos,113 M. Zinser,97 M. Ziolkowski,148 L. Živković,16 G. Zobernig,178 A. Zoccoli,23b,23a K. Zoch,51 T. G. Zorbas,146 R. Zou,36 M. Zur Nedden,19 and L. Zwalinski35 Barcelona, Spain 17Department for Physics and Technology, University of Bergen, Bergen, Norway 18Physics Division, Lawrence Berkeley National Laboratory and University of California, (ATLAS Collaboration) 1Department of Physics, University of Adelaide, Adelaide, Australia 2 2Physics Department, SUNY Albany, Albany, New York, USA 3Department of Physics, University of Alberta, Edmonton, Alberta, Canada 4 4aDepartment of Physics, Ankara University, Ankara, Turkey 4b 4bIstanbul Aydin University, Istanbul, Turkey Istanbul Aydin University, Istanbul, Turkey 4cDivision of Physics, TOBB University of Economics and Technology, Ankara, Turkey 5LAPP, Universit´e Grenoble Alpes, Universit´e Savoie Mont Blanc, CNRS/IN2P3, Annecy, France 6High Energy Physics Division, Argonne National Laboratory, Argonne, Illinois, USA 7Department of Physics, University of Arizona, Tucson, Arizona, USA 8Department of Physics, University of Texas at Arlington, Arlington, Texas, USA 9Physics Department, National and Kapodistrian University of Athens, Athens, Greece 10Physics Department, National Technical University of Athens, Zografou, Greece 11Department of Physics, University of Texas at Austin, Austin, Texas, USA 12aBahcesehir University, Faculty of Engineering and Natural Sciences, Istanbul, Turkey 12bIstanbul Bilgi University, Faculty of Engineering and Natural Sciences, Istanbul, Turkey 12cDepartment of Physics, Bogazici University, Istanbul, Turkey 12dDepartment of Physics Engineering, Gaziantep University, Gaziantep, Turkey 13Institute of Physics, Azerbaijan Academy of Sciences, Baku, Azerbaijan 14Institut de Física d’Altes Energies (IFAE), Barcelona Institute of Science and Technology, B l S i Berkeley, California, USA 19Institut für Physik, Humboldt Universität zu Berlin, Berlin, Germany 19Institut für Physik, Humboldt Universität zu Berlin, Berlin, Germany SEARCH FOR LONG-LIVED PARTICLES IN FINAL … SEARCH FOR LONG-LIVED PARTICLES IN FINAL … M. D. Werner,76 P. Werner,35 M. Wessels,59a T. D. Weston,20 K. Whalen,127 N. L. Whallon,145 A. M. Wharton,87 A. S. White,103 A. White,8 M. J. White,1 R. White,144b D. Whiteson,168 B. W. Whitmore,87 F. J. Wickens,141 W. Wiedenmann,178 M. Wielers,141 C. Wiglesworth,39 L. A. M. Wiik-Fuchs,50 A. Wildauer,113 F. Wilk,98 H. G. Wilkens,35 L. J. Wilkins,91 H. H. Williams,133 S. Williams,31 C. Willis,104 S. Willocq,100 J. A. Wilson,21 I. Wingerter-Seez,5 E. Winkels,153 F. Winklmeier,127 O. J. Winston,153 B. T. Winter,24 M. Wittgen,150 M. Wobisch,93 A. Wolf,97 T. M. H. Wolf,118 R. Wolff,99 M. W. Wolter,82 H. Wolters,136a,136c V. W. S. Wong,172 N. L. Woods,143 S. D. Worm,21 B. K. Wosiek,82 K. W. Woźniak,82 K. Wraight,55 M. Wu,36 S. L. Wu,178 X. Wu,52 Y. Wu,58a T. R. Wyatt,98 B. M. Wynne,48 S. Xella,39 Z. Xi,103 L. Xia,175 D. Xu,15a H. Xu,58a L. Xu,29 T. Xu,142 W. Xu,103 B. Yabsley,154 S. Yacoob,32a K. Yajima,129 D. P. Yallup,92 D. Yamaguchi,162 Y. Yamaguchi,162 A. Yamamoto,79 T. Yamanaka,160 F. Yamane,80 M. Yamatani,160 T. Yamazaki,160 Y. Yamazaki,80 Z. Yan,25 H. J. Yang,58c,58d H. T. Yang,18 S. Yang,75 Y. Yang,160 Z. Yang,17 W-M. Yao,18 Y. C. Yap,44 Y. Yasu,79 E. Yatsenko,58c,58d J. Ye,41 S. Ye,29 I. Yeletskikh,77 E. Yigitbasi,25 E. Yildirim,97 K. Yorita,176 K. Yoshihara,133 C. J. S. Young,35 C. Young,150 J. Yu,8 J. Yu,76 X. Yue,59a S. P. Y. Yuen,24 I. Yusuff,31,ww B. Zabinski,82 G. Zacharis,10 E. Zaffaroni,52 R. Zaidan,14 A. M. Zaitsev,140,ll N. Zakharchuk,44 J. Zalieckas,17 S. Zambito,57 D. Zanzi,35 D. R. Zaripovas,55 S. V. Zeißner,45 C. Zeitnitz,179 G. Zemaityte,131 J. C. Zeng,170 Q. Zeng,150 O. Zenin,140 T. Ženiš,28a D. Zerwas,128 M. Zgubič,131 D. F. Zhang,58b D. Zhang,103 F. Zhang,178 G. Zhang,58a,vv H. Zhang,15c J. Zhang,6 L. Zhang,50 L. Zhang,58a M. Zhang,170 P. Zhang,15c R. Zhang,58a,q R. Zhang,24 X. Zhang,58b Y. Zhang,15d Z. Zhang,128 P. Zhao,47 X. Zhao,41 Y. Zhao,58b,128,y Z. Zhao,58a A. Zhemchugov,77 B. Zhou,103 C. Zhou,178 L. Zhou,41 M. S. Zhou,15d M. Zhou,152 N. Zhou,58c Y. Zhou,7 C. G. Zhu,58b H. L. Zhu,58a H. Zhu,15a J. Zhu,103 Y. Zhu,58a X. Zhuang,15a K. Zhukov,108 V. Zhulanov,120b,120a A. Zibell,174 D. Zieminska,63 N. I. Zimine,77 S. Zimmermann,50 Z. Zinonos,113 M. Zinser,97 M. Ziolkowski,148 L. Živković,16 G. Zobernig,178 A. Zoccoli,23b,23a K. Zoch,51 T. G. Zorbas,146 R. Zou,36 M. Zur Nedden,19 and L. Zwalinski35 M. D. Werner,76 P. Werner,35 M. Wessels,59a T. D. Weston,20 K. Whalen,127 N. L. Whallon,145 A. M. SEARCH FOR LONG-LIVED PARTICLES IN FINAL … Wharton,87 A. S. White,103 A. White,8 M. J. White,1 R. White,144b D. Whiteson,168 B. W. Whitmore,87 F. J. Wickens,141 W. Wiedenmann,178 M. Wielers,141 C. Wiglesworth,39 L. A. M. Wiik-Fuchs,50 A. Wildauer,113 F. Wilk,98 H. G. Wilkens,35 L. J. Wilkins,91 H. H. Williams,133 S. Williams,31 C. Willis,104 S. Willocq,100 J. A. Wilson,21 I. Wingerter-Seez,5 E. Winkels,153 F. Winklmeier,127 O. J. Winston,153 B. T. Winter,24 M. Wittgen,150 M. Wobisch,93 A. Wolf,97 T. M. H. Wolf,118 R. Wolff,99 M. W. Wolter,82 H. Wolters,136a,136c V. W. S. Wong,172 N. L. Woods,143 S. D. Worm,21 B. K. Wosiek,82 K. W. Woźniak,82 K. Wraight,55 M. Wu,36 S. L. Wu,178 X. Wu,52 Y. Wu,58a T. R. Wyatt,98 B. M. Wynne,48 S. Xella,39 Z. Xi,103 L. Xia,175 D. Xu,15a H. Xu,58a L. Xu,29 T. Xu,142 W. Xu,103 B. Yabsley,154 S. Yacoob,32a K. Yajima,129 D. P. Yallup,92 D. Yamaguchi,162 Y. Yamaguchi,162 A. Yamamoto,79 T. Yamanaka,160 F. Yamane,80 M. Yamatani,160 T. Yamazaki,160 Y. Yamazaki,80 Z. Yan,25 H. J. Yang,58c,58d H. T. Yang,18 S. Yang,75 Y. Yang,160 Z. Yang,17 W-M. Yao,18 Y. C. Yap,44 Y. Yasu,79 E. Yatsenko,58c,58d J. Ye,41 S. Ye,29 I. Yeletskikh,77 E. Yigitbasi,25 E. Yildirim,97 K. Yorita,176 K. Yoshihara,133 C. J. S. Young,35 C. Young,150 J. Yu,8 J. Yu,76 X. Yue,59a S. P. Y. Yuen,24 I. Yusuff,31,ww B. Zabinski,82 G. Zacharis,10 E. Zaffaroni,52 R. Zaidan,14 A. M. Zaitsev,140,ll N. Zakharchuk,44 J. Zalieckas,17 S. Zambito,57 D. Zanzi,35 D. R. Zaripovas,55 S. V. Zeißner,45 C. Zeitnitz,179 G. Zemaityte,131 J. C. Zeng,170 Q. Zeng,150 O. Zenin,140 T. Ženiš,28a D. Zerwas,128 M. Zgubič,131 D. F. Zhang,58b D. Zhang,103 F. Zhang,178 G. Zhang,58a,vv H. Zhang,15c J. Zhang,6 L. Zhang,50 L. Zhang,58a M. Zhang,170 P. Zhang,15c R. Zhang,58a,q R. Zhang,24 X. Zhang,58b Y. Zhang,15d Z. Zhang,128 P. Zhao,47 X. Zhao,41 Y. Zhao,58b,128,y Z. Zhao,58a A. Zhemchugov,77 B. Zhou,103 C. Zhou,178 L. Zhou,41 M. S. Zhou,15d M. Zhou,152 N. Zhou,58c Y. Zhou,7 C. G. Zhu,58b H. L. Zhu,58a H. Zhu,15a J. Zhu,103 Y. Zhu,58a X. Zhuang,15a K. Zhukov,108 V. Zhulanov,120b,120a A. Zibell,174 D. Zieminska,63 N. I. Zimine,77 S. Zimmermann,50 Z. Zinonos,113 M. Zinser,97 M. Ziolkowski,148 L. Živković,16 G. Zobernig,178 A. Zoccoli,23b,23a K. Zoch,51 T. G. Zorbas,146 R. Zou,36 M. Zur Nedden,19 and L. Zwalinski35 Y. Yamazaki,80 Z. Yan,25 H. J. Yang,58c,58d H. T. Yang,18 S. Yang,75 Y. Yang,160 Z. Yang,17 W-M. Yao,18 Y. C. Yap,44 Y. Yasu,79 E. Yatsenko,58c,58d J. Ye,41 S. Ye,29 I. Yeletskikh,77 E. Yigitbasi,25 E. Yildirim,97 K. Yorita,176 K. Yoshihara,133 C. J. 35CERN, Geneva, Switzerland 51II. Physikalisches Institut, Georg-August-Universität Göttingen, Göttingen, Germany ´ t t d Ph i N l´ i t C l i U i it´ d G ` G ` S it II. Physikalisches Institut, Georg August Universität Göttingen, Göttingen, Germ 2D´epartement de Physique Nucl´eaire et Corpusculaire, Universit´e de Gen`eve, Gen`eve, S 53aDipartimento di Fisica, Universit`a di Genova, Genova, Italy 53b University of Bern, Bern, Switzerland 21School of Physics and Astronomy, University of Birmingham, Birmingham, United Kingdom 012001-27 M. AABOUD et al. PHYS. REV. University of Bern, Bern, Switzerland D 99, 012001 (2019) 22Centro de Investigaciónes, Universidad Antonio Nariño, Bogota, Colombia 23aDipartimento di Fisica e Astronomia, Universit`a di Bologna, Bologna, Italy 23bINFN Sezione di Bologna, Italy 24Physikalisches Institut, Universität Bonn, Bonn, Germany 25Department of Physics, Boston University, Boston, Massachusetts, USA 26Department of Physics, Brandeis University, Waltham, Massachusetts, USA 27aTransilvania University of Brasov, Brasov, Romania 27bHoria Hulubei National Institute of Physics and Nuclear Engineering, Bucharest, Romania 27cDepartment of Physics, Alexandru Ioan Cuza University of Iasi, Iasi, Romania 27dNational Institute for Research and Development of Isotopic and Molecular Technologies, Physics Department, Cluj-Napoca, Romania 27eUniversity Politehnica Bucharest, Bucharest, Romania 27fWest University in Timisoara, Timisoara, Romania 28aFaculty of Mathematics, Physics and Informatics, Comenius University, Bratislava, Slovak Republic 28bDepartment of Subnuclear Physics, Institute of Experimental Physics of the Slovak Academy of Sciences, Kosice, Slovak Republic 29Physics Department, Brookhaven National Laboratory, Upton, New York, USA 30Departamento de Física, Universidad de Buenos Aires, Buenos Aires, Argentina 31Cavendish Laboratory, University of Cambridge, Cambridge, United Kingdom 32aDepartment of Physics, University of Cape Town, Cape Town, South Africa 32bDepartment of Mechanical Engineering Science, University of Johannesburg, Johannesburg, South Africa 32cSchool of Physics, University of the Witwatersrand, Johannesburg, South Africa 33Department of Physics, Carleton University, Ottawa, Ontario, Canada 34aFacult´e des Sciences Ain Chock, R´eseau Universitaire de Physique des Hautes Energies - Universit´e Hassan II, Casablanca, Morocco 34bCentre National de l’Energie des Sciences Techniques Nucleaires (CNESTEN), Rabat, Morocco 34cFacult´e des Sciences Semlalia, Universit´e Cadi Ayyad, LPHEA-Marrakech, Morocco 34dFacult´e des Sciences, Universit´e Mohamed Premier and LPTPM, Oujda, Morocco 34eFacult´e des sciences, Universit´e Mohammed V, Rabat, Morocco 35CERN, Geneva, Switzerland 36Enrico Fermi Institute, University of Chicago, Chicago, Illinois, USA 37LPC, Universit´e Clermont Auvergne, CNRS/IN2P3, Clermont-Ferrand, France 38Nevis Laboratory, Columbia University, Irvington, New York, USA 39Niels Bohr Institute, University of Copenhagen, Copenhagen, Denmark 40aDipartimento di Fisica, Universit`a della Calabria, Rende, Italy 40bINFN Gruppo Collegato di Cosenza, Laboratori Nazionali di Frascati, Italy 41Physics Department, Southern Methodist University, Dallas, Texas, USA 42Physics Department, University of Texas at Dallas, Richardson, Texas, USA 43aDepartment of Physics, Stockholm University, Sweden 43bOskar Klein Centre, Stockholm, Sweden 44Deutsches Elektronen-Synchrotron DESY, Hamburg and Zeuthen, Germany 45Lehrstuhl für Experimentelle Physik IV, Technische Universität Dortmund, Dortmund, Germany 46Institut für Kern- und Teilchenphysik, Technische Universität Dresden, Dresden, Germany 47Department of Physics, Duke University, Durham, North Carolina, USA 48SUPA - School of Physics and Astronomy, University of Edinburgh, Edinburgh, United Kingdom 49INFN e Laboratori Nazionali di Frascati, Frascati, Italy 50Physikalisches Institut, Albert-Ludwigs-Universität Freiburg, Freiburg, Germany 51II. 53bINFN Sezione di Genova, Italy 54II. Physikalisches Institut, Justus-Liebig-Universität Giessen, Giessen, Germany 55 55SUPA - School of Physics and Astronomy, University of Glasgow, Glasgow, United Kingd 56LPSC U i it´ G bl Al CNRS/IN2P3 G bl INP G bl F 57Laboratory for Particle Physics and Cosmology, Harvard University, Cambridge, Massachusetts, USA 58aDepartment of Modern Physics and State Key Laboratory of Particle Detection and Electronics, University of Science and Technology of China, Hefei, China b University of Science and Technology of China, Hefei, China 58bInstitute of Frontier and Interdisciplinary Science and Key Laboratory of Particle P Irradiation (MOE), Shandong University, Qingdao, China y f gy f f 58bInstitute of Frontier and Interdisciplinary Science and Key Laboratory of Particle Physics and Particle Irradiation (MOE), Shandong University, Qingdao, China Institute of Frontier and Interdisciplinary Science and Key Laboratory of Particle P Irradiation (MOE), Shandong University, Qingdao, China University of Bern, Bern, Switzerland Physikalisches Institut, Georg-August-Universität Göttingen, Göttingen, Germany 52D´epartement de Physique Nucl´eaire et Corpusculaire, Universit´e de Gen`eve, Gen`eve, Switzerland 53aDipartimento di Fisica, Universit`a di Genova, Genova, Italy 53bINFN Sezione di Genova, Italy 54II. Physikalisches Institut, Justus-Liebig-Universität Giessen, Giessen, Germany 55SUPA - School of Physics and Astronomy, University of Glasgow, Glasgow, United Kingdom 56LPSC, Universit´e Grenoble Alpes, CNRS/IN2P3, Grenoble INP, Grenoble, France 57Laboratory for Particle Physics and Cosmology, Harvard University, Cambridge, Massachusetts, USA 58aDepartment of Modern Physics and State Key Laboratory of Particle Detection and Electronics, University of Science and Technology of China, Hefei, China 58bInstitute of Frontier and Interdisciplinary Science and Key Laboratory of Particle Physics and Particle I di i (MOE) Sh d U i i Qi d Chi 22Centro de Investigaciónes, Universidad Antonio Nariño, Bogota, Colombia 23 24Physikalisches Institut, Universität Bonn, Bonn, Germany 25Department of Physics, Boston University, Boston, Massachusetts, USA 6 26Department of Physics, Brandeis University, Waltham, Massachusetts, USA 27 27eUniversity Politehnica Bucharest, Bucharest, Romania 27f Krakow, Poland 81bMarian Smoluchowski Institute of Physics, Jagiellonian University, Krakow, Poland 82 82Institute of Nuclear Physics Polish Academy of Sciences, Krakow, Poland 83 83Faculty of Science, Kyoto University, Kyoto, Japan 84 84Kyoto University of Education, Kyoto, Japan 84Kyoto University of Education, Kyoto, Japan 85 85Research Center for Advanced Particle Physics and Department of Physics, Kyushu University, 012001-28 012001-28 PHYS. REV. D 99, 012001 (2019) 73aINFN-TIFPA, Italy y 73bUniversit`a degli Studi di Trento, Trento, Italy 74Institut für Astro- und Teilchenphysik, Leopold-Franzens-Universität, Innsbruck, Austria 75University of Iowa Iowa City Iowa USA f p y p 75University of Iowa, Iowa City, Iowa, USA 76 76Department of Physics and Astronomy, Iowa State University, Ames, Iowa, USA 77 77Joint Institute for Nuclear Research, Dubna, Russia f 78aDepartamento de Engenharia El´etrica, Universidade Federal de Juiz de Fora (UFJF), Juiz de Fora Brazil 78aDepartamento de Engenharia El´etrica, Universidade Federal de Juiz de Fora (UFJF), Fukuoka, Japan 86Instituto de Física La Plata, Universidad Nacional de La Plata and CONICET, La Plata, Argentin 87 87Physics Department, Lancaster University, Lancaster, United Kingdom 88 88Oliver Lodge Laboratory, University of Liverpool, Liverpool, United Kingdom 89Department of Experimental Particle Physics, Jožef Stefan Institute and Department of Physics, University of Ljubljana, Ljubljana, Slovenia 90 90School of Physics and Astronomy, Queen Mary University of London, London, United Kingdom 91Department of Physics, Royal Holloway University of London, Egham, United Kingdom 92 90School of Physics and Astronomy, Queen Mary University of London, London, United Kingdom 91Department of Physics, Royal Holloway University of London, Egham, United Kingdom 92Department of Physics and Astronomy, University College London, London, United Kingdom 92Department of Physics and Astronomy, University College London, London, United Kingdom g 64bICTP, Trieste, Italy 65bDipartimento di Matematica e Fisica, Universit`a del Salento, Lecce, Italy 66 66aINFN Sezione di Milano, Italy 66bDipartimento di Fisica, Universit`a di Milano, Milano, Italy 67 67bDipartimento di Fisica, Universit`a di Napoli, Napoli, Italy 68 67bDipartimento di Fisica, Universit`a di Napoli, Napoli, Italy 68 68aINFN Sezione di Pavia, Italy 68bDipartimento di Fisica, Universit`a di Pavia, Pavia, Italy 69 68bDipartimento di Fisica, Universit`a di Pavia, Pavia, Italy 69 69aINFN Sezione di Pisa, Italy 69bDipartimento di Fisica E. Fermi, Universit`a di Pisa, Pisa, Italy 70 69bDipartimento di Fisica E. Fermi, Universit`a di Pisa, Pisa, Italy 70 70bDipartimento di Fisica, Sapienza Universit`a di Roma, Roma, Italy 71 70bDipartimento di Fisica, Sapienza Universit`a di Roma, Roma, Italy 71 71aINFN Sezione di Roma Tor Vergata, Italy 71aINFN Sezione di Roma Tor Vergata, Italy 71bDipartimento di Fisica, Universit`a di Roma Tor Vergata, Roma, Italy 72a 71bDipartimento di Fisica, Universit`a di Roma Tor Vergata, Roma, Italy 72 72aINFN Sezione di Roma Tre, Italy 72bDipartimento di Matematica e Fisica, Universit`a Roma Tre, Roma, Italy 73aINFN-TIFPA, Italy Juiz de Fora, Brazil 78bUniversidade Federal do Rio De Janeiro COPPE/EE/IF, Rio de Janeiro, Brazil 78c d d d l d d l ( ) d l l 79KEK, High Energy Accelerator Research Organization, Tsukuba, Japan 80 te School of Science, Kobe University, Kobe, Japan 81aAGH University of Science and Technology, Faculty of Physics and Applied Computer Science, Krakow Poland SEARCH FOR LONG-LIVED PARTICLES IN FINAL … SEARCH FOR LONG-LIVED PARTICLES IN FINAL … 58cSchool of Physics and Astronomy, Shanghai Jiao Tong University, KLPPAC-MoE, SKLPPC, Shanghai, China 58dTsung-Dao Lee Institute, Shanghai, China 012001-29 PHYS. REV. D 99, 012001 (2019) M. AABOUD et al. 93Louisiana Tech University, Ruston, Louisiana, USA 94Fysiska institutionen, Lunds universitet, Lund, Sweden 95Centre de Calcul de l’Institut National de Physique Nucl´eaire et de Physique des Particules (IN2P3), Villeurbanne, France 96Departamento de Física Teorica C-15 and CIAFF, Universidad Autónoma de Madrid, Madrid, Spain 97Institut für Physik, Universität Mainz, Mainz, Germany 98School of Physics and Astronomy, University of Manchester, Manchester, United Kingdom 99CPPM, Aix-Marseille Universit´e, CNRS/IN2P3, Marseille, France 100Department of Physics, University of Massachusetts, Amherst, Massachusetts, USA 101Department of Physics, McGill University, Montreal, Quebec, Canada 102School of Physics, University of Melbourne, Victoria, Australia 103Department of Physics, University of Michigan, Ann Arbor, Michigan, USA 104Department of Physics and Astronomy, Michigan State University, East Lansing, Michigan, USA 105B.I. Stepanov Institute of Physics, National Academy of Sciences of Belarus, Minsk, Belarus 106Research Institute for Nuclear Problems of Byelorussian State University, Minsk, Belarus 107Group of Particle Physics, University of Montreal, Montreal, Quebec, Canada 108P.N. Lebedev Physical Institute of the Russian Academy of Sciences, Moscow, Russia 109Institute for Theoretical and Experimental Physics (ITEP), Moscow, Russia 110National Research Nuclear University MEPhI, Moscow, Russia 111D.V. Skobeltsyn Institute of Nuclear Physics, M.V. Lomonosov Moscow State University, Moscow, Russia 112Fakultät für Physik, Ludwig-Maximilians-Universität München, München, Germany 113Max-Planck-Institut für Physik (Werner-Heisenberg-Institut), München, Germany 114Nagasaki Institute of Applied Science, Nagasaki, Japan 115 Norman, Oklahoma, USA 125Department of Physics, Oklahoma State University, Stillwater, Oklahoma, USA 126Palacký University, RCPTM, Joint Laboratory of Optics, Olomouc, Czech Republic 127 125Department of Physics, Oklahoma State University, Stillwater, Oklahoma, USA 126P l ký U i i RCPTM J i L b f O i Ol C h R bli 127Center for High Energy Physics, University of Oregon, Eugene, Oregon, USA 128 128LAL, Universit´e Paris-Sud, CNRS/IN2P3, Universit´e Paris-Saclay, Orsay, France 129 129Graduate School of Science, Osaka University, Osaka, Japan 130 ment of Physics, Oxford University, Oxford, United p f y , f y, f , g 132LPNHE, Sorbonne Universit´e, Paris Diderot Sorbonne Paris Cit´e, CNRS/IN2P3, Paris, France 133Department of Physics, University of Pennsylvania, Philadelphia, Pennsylvania, USA 134Konstantinov Nuclear Physics Institute of National Research Centre “Kurchatov Institute”, PNPI St Petersburg Russia NHE, Sorbonne Universit´e, Paris Diderot Sorbonne Paris Cit´e, CNRS/IN2P3, Paris, France 133Department of Physics, University of Pennsylvania, Philadelphia, Pennsylvania, USA Department of Physics, University of Pennsylvania, Philadelphia, Pennsylvania, USA onstantinov Nuclear Physics Institute of National Research Centre “Kurchatov Institute”, PNPI, St. Petersburg, Russia 135Department of Physics and Astronomy, University of Pittsburgh, Pittsburgh, Pennsylvania, USA 136aLaboratório de Instrumentação e Física Experimental de Partículas - LIP, Portugal 6 ç p g 136bDepartamento de Física, Faculdade de Ciências, Universidade de Lisboa, Lisboa, Portugal 136cDepartamento de Física, Universidade de Coimbra, Coimbra, Portugal 136dCentro de Física Nuclear da Universidade de Lisboa, Lisboa, Portugal 136eDepartamento de Física, Universidade do Minho, Braga, Portugal 36fDepartamento de Física Teorica y del Cosmos, Universidad de Granada, Granada (Spain), Spain 6gDep Física and CEFITEC of Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, 136fDepartamento de Física Teorica y del Cosmos, Universidad de Granada, Granada (Spain), Spain 136gDep Física and CEFITEC of Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, Caparica, Portugal y Moscow, Russia 112Fakultät für Physik, Ludwig-Maximilians-Universität München, München, Germany 113 113Max-Planck-Institut für Physik (Werner-Heisenberg-Institut), München, Germany 114 114Nagasaki Institute of Applied Science, Nagasaki, Japan 115Graduate School of Science and Kobayashi-Maskawa Institute, Nagoya University, Nagoya, Japan 116Department of Physics and Astronomy, University of New Mexico, Albuquerque, New Mexico, USA 117Institute for Mathematics, Astrophysics and Particle Physics, Radboud University Nijmegen/Nikhef, Nijmegen, Netherlands 116Department of Physics and Astronomy, University of New Mexico, Albuquerque, New Mexico, USA 117Institute for Mathematics, Astrophysics and Particle Physics, Radboud University Nijmegen/Nikhef, 116Department of Physics and Astronomy, University of New Mexico, Albuquerque, New Mexico, USA 117 f h h d l h db d kh f Nijmegen, Netherlands hef National Institute for Subatomic Physics and University of Amsterdam, Amsterdam, Netherlands 119 119Department of Physics, Northern Illinois University, DeKalb, Illinois, USA 120 20aBudker Institute of Nuclear Physics, SB RAS, Novosibirsk, Russia 120b 0aBudker Institute of Nuclear Physics, SB RAS, Novosibirsk, Russia 120bN ibi k St t U i it N ibi k R i 20bNovosibirsk State University Novosibirsk, Russia 121Department of Physics, New York University, New York, New York, USA 122 nt of Physics, New York University, New York, New 122The Ohio State University, Columbus, Ohio, USA 23 123Faculty of Science, Okayama University, Okayama, Japan 124Homer L. Dodge Department of Physics and Astronomy, University of Oklahoma, Norman, Oklahoma, USA 125 Caparica, Portugal 137Institute of Physics, Academy of Sciences of the Czech Republic, Prague, Czech Republic 138Czech Technical University in Prague, Prague, Czech Republic 139 139Charles University, Faculty of Mathematics and Physics, Prague, Czech Republic 139Charles University, Faculty of Mathematics and Physics, Prague, Czech Republic 93Louisiana Tech University, Ruston, Louisiana, USA 94 Tokyo, Japan 161Graduate School of Science and Technology, Tokyo Metropolitan University, Tokyo, Japan 163Tomsk State University, Tomsk, Russia 164Department of Physics, University of Toronto, Toronto, Ontario, Canada 165 165aTRIUMF, Vancouver, British Columbia, Canada 165bDepartment of Physics and Astronomy, York University, Toronto, Ontario, Canada 166Di i i f Ph i d T C f h Hi f h U i 165bDepartment of Physics and Astronomy, York University, Toronto, Ontario, Canada 166 Faculty of Pure and Applied Sciences, University of Tsukuba, Tsukuba, Japan 167Department of Physics and Astronomy, Tufts University, Medford, Massachusetts, USA 68Department of Physics and Astronomy, University of California Irvine, Irvine, California, USA 169 Bergische Universität Wuppertal, Wuppertal, Germany 180Department of Physics, Yale University, New Haven, Connecticut, USA 181 Yale University, New Haven, Connecticut, USA of Physics, Yale University, New Haven, Connect 181 181Yerevan Physics Institute, Yerevan, Armenia aDeceased. bAlso at Department of Physics, King’s College London, London, United Kingdom. cAlso at Istanbul University, Department of Physics, Istanbul, Turkey. dAlso at Institute of Physics, Azerbaijan Academy of Sciences, Baku, Azerbaijan. eAlso at TRIUMF, Vancouver, British Columbia, Canada. fAlso at Department of Physics and Astronomy, University of Louisville, Louisville, Kentucky, US gAlso at Department of Physics, California State University, Fresno, Stanford, California, USA. hAlso at Department of Physics, University of Fribourg, Fribourg, Switzerland. iAlso at II. Physikalisches Institut, Georg-August-Universität Göttingen, Göttingen, Germany. jAlso at Departament de Fisica de la Universitat Autonoma de Barcelona, Barcelona, Spain. aDeceased. b bAlso at Department of Physics, King’s College London, London, United Kingdom. bAlso at Department of Physics, King’s College London, London, United Kingdom. cAlso at Istanbul University, Department of Physics, Istanbul, Turkey. d dAlso at Institute of Physics, Azerbaijan Academy of Sciences, Baku, Azerbaijan eAlso at TRIUMF, Vancouver, British Columbia, Canada. f eAlso at TRIUMF, Vancouver, British Columbia, Canada. f p y y y y gAlso at Department of Physics, California State University, Fresno, Stanford, California, USA. h p y y y y gAlso at Department of Physics, California State University, Fresno, Stanford, California, USA. h hAlso at Department of Physics, University of Fribourg, Fribourg, Switzerland. i hAlso at Department of Physics, University of Fribourg, Fribourg, Switzerland. i iAlso at II. Physikalisches Institut, Georg-August-Universität Göttingen, Göttingen, Germany. j jAlso at Departament de Fisica de la Universitat Autonoma de Barcelona, Barcelona, Spain. 012001-30 PHYS. REV. D 99, 012001 (2019) SEARCH FOR LONG-LIVED PARTICLES IN FINAL … SEARCH FOR LONG-LIVED PARTICLES IN FINAL … 140State Research Center Institute for High Energy Physics, NRC KI, Protvino, Russia 141Particle Physics Department, Rutherford Appleton Laboratory, Didcot, United Kingdom 142 140State Research Center Institute for High Energy Physics, NRC KI, Protvino, Russia 141Particle Physics Department Rutherford Appleton Laboratory Didcot United Kingdom y p f pp y 142IRFU, CEA, Universit´e Paris-Saclay, Gif-sur-Yvette, France 43 y f 143Santa Cruz Institute for Particle Physics, University of California Santa Cruz, 143Santa Cruz Institute for Particle Physics, University of California Santa Cruz, Santa Cruz, California, USA 144aDepartamento de Física, Pontificia Universidad Católica de Chile, Santiago, Chile 144b artment Physik, Universität Siegen, Siegen, German 149Department of Physics, Simon Fraser University, Burnaby, British Columbia, Canada 150 y f f 151Physics Department, Royal Institute of Technology, Stockholm, Sweden tment, Royal Institute of Technology, Stockholm, Sw y p y f gy 152Departments of Physics and Astronomy, Stony Brook University, Stony Brook, New York, USA 153 p f y y y y y 153Department of Physics and Astronomy, University of Sussex, Brighton, United Kingdom 154 154School of Physics, University of Sydney, Sydney, Australia 155 156aE. Andronikashvili Institute of Physics, Iv. Javakhishvili Tbilisi State University, Tbilisi, Georgia 156bHigh Energy Physics Institute, Tbilisi State University, Tbilisi, Georgia 157 157Department of Physics, Technion, Israel Institute of Technology, Haifa, Israel 158Raymond and Beverly Sackler School of Physics and Astronomy, Tel Aviv University, Tel Aviv, Israe 159Department of Physics, Aristotle University of Thessaloniki, Thessaloniki, Greece 012001-31 M. AABOUD et al. PHYS. REV. D 99, 012001 (2019) kAlso at Tomsk State University, Tomsk, and Moscow Institute of Physics and Technology State University, Dolgoprudny, Russia. lAlso at The Collaborative Innovation Center of Quantum Matter (CICQM), Beijing, China. Also at Tomsk State University, Tomsk, and Moscow Institute of Physics and Technology State University, Dolgoprudny, Russia. lAlso at The Collaborative Innovation Center of Quantum Matter (CICQM), Beijing, China. y, , y gy y, g p y, lAlso at The Collaborative Innovation Center of Quantum Matter (CICQM), Beijing, China. y, , y lAlso at The Collaborative Innovation Center of Quantum Matter (CIC mAlso at Universita di Napoli Parthenope, Napoli, Italy. mAlso at Universita di Napoli Parthenope, Napoli, Italy. nAlso at Institute of Particle Physics (IPP), Canada. nAlso at Institute of Particle Physics (IPP), Canada. y ( ) oAlso at Dipartimento di Fisica E. Fermi, Universit`a di Pisa, Pisa, Italy. oAlso at Dipartimento di Fisica E. Fermi, Universit`a di Pisa, Pisa, Italy. Also at Dipartimento di Fisica E. Fermi, Universit`a p y pAlso at Horia Hulubei National Institute of Physics and Nuclear Engineering, Bucharest, Romania. pAlso at Horia Hulubei National Institute of Physics and Nuclear Engine qAlso at CPPM, Aix-Marseille Universit´e, CNRS/IN2P3, Marseille, France. qAlso at CPPM, Aix-Marseille Universit´e, CNRS/IN2P3, Marseille, France. qAlso at CPPM, Aix-Marseille Universit´e, CNRS/IN2P3, Marseille, France. sAlso at Borough of Manhattan Community College, City University of New York, New York, USA. t sAlso at Borough of Manhattan Community College, City University of New York, New York, USA. tAlso at Department of Financial and Management Engineering, University of the Aege at Department of Financial and Management Engineering, University of the Aegean, Chios, Greece uAlso at Centre for High Performance Computing, CSIR Campus, Rosebank, Cape Tow at Centre for High Performance Computing, CSIR Campus, Rosebank, Cape Town, South Africa. g p g p vAlso at Louisiana Tech University, Ruston, Louisiana, USA. vAlso at Louisiana Tech University, Ruston, Louisiana, USA. y wAlso at Institucio Catalana de Recerca i Estudis Avancats, ICREA, Barcelona, Spain. wAlso at Institucio Catalana de Recerca i Estudis Avancats, ICREA, Barcelona, Spai p xAlso at Department of Physics, University of Michigan, Ann Arbor, Michigan, USA. xAlso at Department of Physics, University of Michigan, Ann Arbor, Michigan, USA. yAlso at LAL, Universit´e Paris-Sud, CNRS/IN2P3, Universit´e Paris-Saclay, Orsay, France. yAlso at LAL, Universit´e Paris-Sud, CNRS/IN2P3, Universit´e Paris-Sacla zAlso at Graduate School of Science, Osaka University, Osaka, Japan. 012001-31 zAlso at Graduate School of Science, Osaka University, Osaka, Japan. aaAlso at Physikalisches Institut, Albert-Ludwigs-Universität Freiburg, Freiburg, Germany. bb aaAlso at Physikalisches Institut, Albert-Ludwigs-Universität Freiburg, Freib bb bbAlso at Institute for Mathematics, Astrophysics and Particle Physics, Radboud University Nijmegen/Nikhef, Nijmegen, Netherlands. bbAlso at Institute for Mathematics, Astrophysics and Particle Physics, Radboud University Nijmegen/Nikhef, Nijmegen, Netherlands. st University, Nicosia, North Cyprus, Mersin, Turke ddAlso at Institute of Theoretical Physics, Ilia State University, Tbilisi, G ddAlso at Institute of Theoretical Physics, Ilia State University, Tbilisi, Georgia. eeAlso at CERN, Geneva, Switzerland. ff ffAlso at Department of Physics, Stanford University, USA. ffAlso at Department of Physics, Stanford University, USA. ggAlso at Manhattan College, New York, New York, USA. hh ggAlso at Manhattan College, New York, New York, USA. hh hhAlso at Hellenic Open University, Patras, Greece. ii p y iiAlso at The City College of New York, New York, New York, USA. jj iiAlso at The City College of New York, New York jj iiAlso at The City College of New York, New York, New York, USA. jj y g Departamento de Física Teorica y del Cosmos, Universidad de Granada, Granada (Spain), Spain. jjAlso at Departamento de Física Teorica y del Cosmos, Universidad de kk kkAlso at Department of Physics, California State University, Sacramento, Stanford, California, ll llAlso at Moscow Institute of Physics and Technology State University, Dolgoprudny, Russia. mmAlso at D´epartement de Physique Nucl´eaire et Corpusculaire, Universit´e de Gen`eve, Gen`eve, nnAlso at Department of Physics and Astronomy, University of Sheffield, Sheffield, United Kingdom. nnAlso at Department of Physics and Astronomy, University of Sheffield, Sheffield, United Kingdom. l of Physics, Sun Yat-sen University, Guangzhou, C y y g ppAlso at Department of Applied Physics and Astronomy, University of Sharjah, Sharjah, United Arab Emirates. qq ppAlso at Department of Applied Physics and Astronomy, University of Sharjah, Sharjah, United Arab Emirates ppAlso at Department of Applied Physics and Astronomy, University of S qqAlso at Institut für Experimentalphysik, Universität Hamburg, Hamburg, Germany. qqAlso at Institut für Experimentalphysik, Universität Hamburg, Hamburg, Germany. rrAlso at National Research Nuclear University MEPhI, Moscow, Russia. rrAlso at National Research Nuclear University MEPhI, Moscow, Russia. y ssAlso at Institute for Particle and Nuclear Physics, Wigner Research Centre for Physics, Budapest, Hungary. 012001-31 tt l i i i l f i i i k ssAlso at Institute for Particle and Nuclear Physics, Wigner Research Centre for Physics, Budapest, H tt ssAlso at Institute for Particle and Nuclear Physics, Wigner Research Cen ttAlso at Giresun University, Faculty of Engineering, Giresun, Turkey. ttAlso at Giresun University, Faculty of Engineering, Giresun, Turkey. y y g g y uuAlso at Department of Physics, Nanjing University, Nanjing, China. uuAlso at Department of Physics, Nanjing University, Nanjing, China. vvAlso at Institute of Physics, Academia Sinica, Taipei, Taiwan. vvAlso at Institute of Physics, Academia Sinica, Taipei, Taiwan. wwAlso at Department of Physics, University of Malaya, Kuala Lumpur, Malaysia. 012001-32 012001-32
https://openalex.org/W3215922476	https://aip.scitation.org/doi/pdf/10.1063/5.0067920	English	null	Perovskite luminescent solar concentrators for photovoltaics	APL photonics	2,021	cc-by	13,991	Perovskite luminescent solar concentrators for photovoltaics APL Photonics 6, 120901 (2021) https://doi.org/10.1063/5.0067920 APL Photonics 6, 120901 (2021) https://doi.org/10.1063/5.0067920 AFFILIATIONS 1 Advanced Photonics Center, School of Electronic Science and Engineering, Southeast University, Nanjing 210096, People’s Republic of China People s Republic of China 2Department of Electrical and Computer Engineering, Waterloo Institute for Nanotechnology, University of Waterloo, Waterloo, Ontario N2L 3G1, Canada Note: This paper is part of the APL Photonics Special Topic on Perovskite Photonics. a)Authors to whom correspondence should be addressed: wangchl@seu.edu.cn and dban@uwaterloo.ca ABSTRACT As large-area photon collection devices designed for photovoltaics, luminescent solar concentrators (LSCs) have been proposed for more than 40 years. In recent years, the perovskite-based LSCs have received much interest for the convenient preparation process and low cost along with high quantum yields of perovskite luminophores. However, optical losses, such as non-radiative recombination loss and reab- sorption loss, seriously impair the performance of LSCs and further impede the commercialization of such promising photovoltaic devices. Various strategies, such as increasing the Stokes shift and defect passivation, have been implemented to enhance the optical performance in perovskite-based LSCs. Here, we appraise the applications of perovskite luminophores in LSCs and review the typical preparation method of perovskite-based LSCs. The state-of-the-art solutions are presented to address the optical losses, leading to the demonstration of enabling high-performance perovskite-based LSCs. 24 October 2024 05:44:56 © 2021 Author(s). All article content, except where otherwise noted, is licensed under a Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/). https://doi.org/10.1063/5.0067920 Articles You May Be Interested In 24 October 2024 05:44:56 APL Photonics PERSPECTIVE scitation.org/journal/app Perovskite luminescent solar concentrators for photovoltaics Cite as: APL Photon. 6, 120901 (2021); doi: 10.1063/5.0067920 Submitted: 20 August 2021 • Accepted: 22 November 2021 • Published Online: 20 December 2021 II. PEROVSKITE LUMINESCENT SOLAR CONCENTRATORS Perovskite-based LSCs have no essential difference in structure and working mechanism in comparison to traditional LSCs. Such devices can be simply recognized as photon concentration devices, where photons are absorbed from the top surface with a large area, down-converted into low-energy photons, and then converged to the edge side with a small area.48 As perovskite materials attract increasing attention, research on perovskite-based LSCs is growing quickly in recent years (Fig. 2).i The optical efficiency (ηopt), an important parameter to evalu- ate the performance of LSCs,9,18 can be calculated as ηopt = (1 −R)ηTIR ⋅ηabs ⋅ηPLQY ⋅ηheat ⋅ηhost ⋅ηreflection ⋅ηreabsorption, (1)l FIG. 1. Schematic diagram of a typical LSC structure. [1] Non-radiative recombi- nation loss, [2] scattering loss, [3] transmission loss, [4] escape cone loss, and [5] reabsorption loss. on (1) where R is the reflectance at the top surface, ηTIR is the total inter- nal reflection efficiency, ηabs is the fraction of sunlight absorbed by luminophores, ηPLQY is the photoluminescence quantum yield (PLQY) of the luminophores, ηheat is the energy lost due to the heat generation, ηhost is the transport efficiency of the waveguided photons through the matrix, ηreflection is the reflection efficiency of the waveguide, which is determined by the smoothness of the LSCs surface, and ηreabsorption is the transport efficiency of the down- converted photons related to reabsorption. Such a material with excellent optical or electrical properties has been widely used in solar cells, light-emitting diodes, and detectors due to its good solution processing characteristics, and high defect tolerance.25,31–38 We believe that perovskite materials are excellent candidates for LSC device applications because of the following rea- sons: (1) Perovskites can have a high PLQY through ligand mod- ification and additive passivation. (2) The emission of perovskites can be tuned by changing its halide composition, which can match the commercial Si-PV very well. (3) Although perovskites them- selves exhibit relatively high moisture and oxygen and UV sensitivity due to their ionic nature, the stability can be significantly improved after being embedded in transparent matrices, such as glass or poly- mer. II. PEROVSKITE LUMINESCENT SOLAR CONCENTRATORS (4) The adjustable composition and dimensions of perovskites make it feasible to substantially increase its Stokes shift by sim- ply doping impurities of transition metals or introducing multi- ple quantum wells (MQWs), which is therefore very advantageous over organic dyes, particularly those that may have severe reabsorp- tion loss.39–46 (5) The solution-based processing of perovskites does not need high energy consumption equipment, significantly low- ering its synthesis costs. In summary, we believe that the applica- tion of perovskites to the luminophore of LSCs shows great advan- tages to promote the development of high-efficiency and low-cost LSCs.47 A common and convenient way to measure ηopt is to couple a solar cell at the edge of the LSC,49 and ηopt can be calculated as (2) ηopt = (ILSC × SPV)/(ISC × SLSC) = ILSC/(ISC × G), (2) where ILSC is the short circuit current generated by the solar cell coupled to the LSC, ISC is the short circuit current of the same solar cell under direct illumination, SPV is the area of the edge of the LSC, SLSC is the area of the top of the LSC, and G is the geo- metric factor, which is defined as the ratio of the top area to the edge area. Table I shows the performance of 17 LSCs based on dif- ferent luminophores, such as CDs, dyes, and CuInS2/ZnS. In view of the low cost and convenient preparation process of perovskite luminophores, the perovskite-based LSCs show potential advantages for practical applications. As a key parameter for evaluating the performance of LSCs,18 the total solar concentration ratio (C) is defined as C = G × ∫ λmax λmin NPOUT(λ)EQE(λ)d(λ) ∫ λmax λmin NPIN(λ)EQE(λ)d(λ) , (3) In this Perspective, we summarize the recent developments of perovskite luminophores, including zero-dimensional perovskite quantum dots (PQDs)/nanocrystals (PNCs), two-dimensional per- ovskite nanoplates (PNPLs)/nanosheets (PNSs), and other per- ovskite luminophores with doping impurities of transition metal, together with the typical preparation methods of perovskite LSCs. The state-of-the-art solutions to reducing optical losses, such as non-radiative recombination loss, scattering loss, transmission loss, escape cone loss, and reabsorption loss, in perovskite-based LSCs are analyzed in detail. Through this Perspective, we hope to promote the applications of perovskite luminophores in LSCs, as well as pro- viding new ideas for the optimization strategy of optical losses to obtain potential technology advancement in photovoltaic, which will bring us closer to high-performance, commercial perovskite-based LSCs. I. INTRODUCTION photovoltaic devices attached around the LSCs and converted to electricity.7–16 Renewable clean energy, especially solar energy, is attracting great attention due to the emerging depletion of fossil energy.1–5 Meanwhile, climate change becomes prominent as a result of exces- sive consumption of fossil energy. In view of the necessity of sustainable development and the threat of climate change, more countries are increasingly turning to renewable energy technolo- gies and launching their own carbon neutralization policies. As large-area photon harvesting devices, luminescent solar concen- trators (LSCs) are an important supplement to the existing pho- tovoltaic systems, and the cost of LSCs is much lower than that of monocrystalline silicon solar cells of the same size.6 Such low-priced sunlight collection devices are usually composed of a transparent matrix and luminophores embedded in the waveguide with cheap transparent matrices, such as polymethyl methacry- late (PMMA), polystyrene (PS), and glass (Fig. 1), which could absorb high-energy photons in incident sunlight and re-emit a low- energy counterpart. These down-converted photons are fed to the The luminophore, one of the key components of LSCs, directly determines the performance of LSCs. The luminophore candi- dates for high-performance LSC devices should meet the following requirements: (1) high photoluminescence quantum yield (PLQY) to efficiently down-convert the incident high-energy photons, (2) pertinent emission wavelength to match the absorption of the solar cell coupled to the LSCs, (3) good moisture and UV stability to adapt to long-term sunlight irradiation and harsh weather conditions, (4) large Stokes shift or small absorption-emission overlap to minimize reabsorption loss, and (5) low manufacture cost and convenient synthesis to reduce the cost of photovoltaic systems. In the past 40 years, various luminophores have been intro- duced into LSCs such as carbon dots (CDs),17 organic dyes,18,19 nanocrystals (NCs),20,21 and rare earth ions.22,23 In recent years, lead halide perovskites have attracted substantial attention due to its low processing cost and high photoluminescence quantum yield.24–30 APL Photon. 6, 120901 (2021); doi: 10.1063/5.0067920 © Author(s) 2021 6, 120901-1 APL Photon. 6, 120901 (2021); doi: 10.1063/5.0067920 APL Photonics PERSPECTIVE scitation.org/journal/app FIG. 1. Schematic diagram of a typical LSC structure. [1] Non-radiative recombi- nation loss, [2] scattering loss, [3] transmission loss, [4] escape cone loss, and [5] reabsorption loss. II. PEROVSKITE LUMINESCENT SOLAR CONCENTRATORS It is more reasonable to treat the LSC-PV system as an integrated photovoltaic device even though the PV cell is edge-mounted onto where Jsc is the short-circuit current density of the PV cell, Voc is the open-circuit voltage, Isc is the short-circuit current, FF is the fill factor, Pin is the total flux density of the incident solar radiation, and ALSC is the front surface area of the waveguide. g Perovskites are excellent candidates for luminophores in LSCs because of their excellent optical properties. Traditional three- dimensional perovskites with a structural formula of ABX3 (A = methylammonium, formamidinium, Cs; B = Pb, Sn, and X = Cl, Br, I.) have been widely used in solar cells.1,51,63–65 However, in the light-emitting applications such as perovskite light-emitting diodes and perovskite LSCs, where carrier recombination is required to Perovskites are excellent candidates for luminophores in LSCs because of their excellent optical properties. Traditional three- dimensional perovskites with a structural formula of ABX3 (A = methylammonium, formamidinium, Cs; B = Pb, Sn, and X = Cl, Br, I.) have been widely used in solar cells.1,51,63–65 However, in the light-emitting applications such as perovskite light-emitting diodes and perovskite LSCs, where carrier recombination is required to TABLE I. Optical performance of reported LSCs based on different luminophores. PVDF: polyvinylidene fluoride, LR305: Lumogen F Red 305 (dye), and TADF: thermally activated delayed fluorescence. Material PL peak (nm) PLQY (%) Absorption range (nm) LSC dimensions (cm3) G factor Stability ηopt (%) CdSe/CdS11 640 ∼42 300–650 21.5 × 1.3 × 0.5 N.A. N.A. 0.6 Si12 830 ∼46 300–800 12 × 12 × 0.26 11.5 N.A. 2.85 CuInSeS/ZnS13 960 40 300–1000 12 × 12 × 0.3 10 N.A. 3.27 C dots17 540 ∼30 300–650 10 × 10 × 0.2 10 12 h (UV) 1.2 CsPb(IxBr1-x)350 ∼620 ∼60 300–625 9 × 1.3 × 0.2 45 >14 h (UV) 2 CdSe@ZnS/ZnS51 513 ∼65 300–550 10 × 9 × 0.3 7.9 14 h (UV) 1.2 PEA2MA2Pb3I10-PVDF52 618 45.8 300–750 6.5 × 2 × 0.4 5 1440 h (air) 2.8 MAPbBr353 532 75 300–620 5 × 3 × 0.5 10 24 h (UV) 1.57 NaI:Tm2+54 1137 33.2 300–780 N.A. N.A. N.A. 0.35 CsPbI355 698 97.8 300–800 N.A. 10 >720 h (water) 3.1 Mn:CsPbCl356 590 ∼10 300–420 25 × 20 × 0.5 50 N.A. 0.5 LR305-silica NPs57 ∼620 95 300–625 N.A. N.A. N.A. 1.287 CuInS2/ZnS58 862 66 300–826 10 × 10 × 0.14 N.A. N.A. II. PEROVSKITE LUMINESCENT SOLAR CONCENTRATORS (3) (3) where EQE(λ) is the external quantum efficiency of the PV solar cell and NPOUT(λ) and NPIN(λ) are the number of output and input pho- tons at a certain wavelength, respectively. G is the geometric gain, which is defined as the ratio of the top surface aperture area to the total edge area of the LSC. It is worth noting that the calculation method of power conver- sion efficiency (PCE) of LSCs may need to be modified. In the past few years, the PCE of LSCs was usually calculated as follows: PCE = Voc × Jsc × FF Pin = Voc × Isc × FF Aedge × Pin , (4) (4) APL Photon. 6, 120901 (2021); doi: 10.1063/5.0067920 © Author(s) 2021 APL Photon. 6, 120901 (2021); doi: 10.1063/5.0067920 6, 120901-2 APL Photonics APL Photonics PERSPECTIVE scitation.org/journal/app FIG. 2. Number of papers per year pub- lished with the topic “luminescent solar concentrators” and “perovskite lumines- cent solar concentrators” in web of sci- ence database as accessed in July 2021. FIG. 2. Number of papers per year pub- lished with the topic “luminescent solar concentrators” and “perovskite lumines- cent solar concentrators” in web of sci- ence database as accessed in July 2021. an LSC. Therefore, the PCE calculation of LSCs should be revised as follows: where Jsc is the short-circuit current density of PV cell, Voc is the open-circuit voltage, Isc is the short-circuit current, FF is the fill fac- tor, Pin is total flux density of incident solar radiation, and Aedge is the edge area of LSC covered by PV solar cells. PCE = Voc × Jsc × FF Pin = Voc × Isc × FF ALSC × Pin , (5) (5) Very recently, Yang and Lunt pointed out the limitations of this calculation method.10 In the hypothetical LSCs without any non- radiative recombination loss or reabsorption loss, as-generated pho- tocurrent typically increases with the collection area (the waveguide front surface, ALSC). If the side area Aedge is used to calculate Jsc such that Jsc = Isc/Aedge, the calculated Jsc could be diverging to infinity when the thickness of the waveguide (and thus Aedge) approaches to zero, making the calculated PCE value exceed the Shockley–Queisser limit and thereby overestimating the performance of LSCs. II. PEROVSKITE LUMINESCENT SOLAR CONCENTRATORS 8.1 PbS/CdS59 885 ∼50 300–800 10 × 1.5 × 0.2 50 4 h (UV) 1.1 C dots/CsPb(Br0.2I0.8)360 ∼650 N.A. 300–650 10 × 10 × 0.2 N.A. 70 h (UV) 3 CdSe/CdPbS61 620 40 300–650 7 × 1.5 × 0.3 23 N.A. 1.4 TADF dye62 557 N.A. 300–550 12.5 × 3 × 0.3 N.A. >5 h (UV) 2.2 TABLE I. Optical performance of reported LSCs based on different luminophores. PVDF: polyvinylidene fluoride, LR305: Lumogen F Red 305 (dye), and TADF: thermally activated delayed fluorescence. TABLE I. Optical performance of reported LSCs based on different luminophores. PVDF: polyvinylidene fluoride, LR305: Lumogen activated delayed fluorescence. rted LSCs based on different luminophores. PVDF: polyvinylidene fluoride, LR305: Lumogen F Red 305 (dye), and TADF: thermally APL Photon. 6, 120901 (2021); doi: 10.1063/5.0067920 © Author(s) 2021 6, 120901-3 APL Photonics PERSPECTIVE scitation.org/journal/app suppressed reabsorption loss. More specific details will be dis- cussed later in Sec. IV. The 1D perovskite nanowires can be pre- pared through solution processing, which is similar to the syn- thesis of perovskite NCs.47 It is worth mentioning that perovskite nanofibers can be synthesized using electrospinning, directly con- verting the perovskite–polymer slurry to luminescent nanofibers and thus simplifying the fabrication of luminescent films.26 As the surface of the perovskite nanofibers is covered by a polymer, as- prepared perovskites are less sensitive to moisture and become more stable. dominate, luminescent performance is significantly limited by facile dissociation of excitons in perovskites due to their long exciton dif- fusion length and small exciton binding energy.66–69 Various strate- gies have been proposed to reduce the grain size of perovskites or adjust the grain dimensions since the smaller grains can spatially limit the diffusion length of excitons or charge carriers and reduce the possibility of exciton dissociation into carriers.66,70,71 Therefore, in order to obtain a high PLQY and thus high-performance LSCs, the perovskites used as luminophores are usually nanocrystals and nanoplates with reduced dimensions.24,26,27,67,68i The traditional preparation method of LSCs can be divided into three steps: (1) pre-synthesis of luminophores, (2) mixing with a polymer or monomer, and (3) polymerization or coating. Specifi- cally, it can be divided into two categories according to the forming method: one is the overall curing type, and the other is the coating forming type. A. Perovskite nanocrystals for LSCs A typical preparation method of LSCs is to synthesize per- ovskite quantum dots/nanocrystals in advance and then immerse these synthesized luminophores in a transparent matrix, which is denoted as the overall curing type. Zhao et al. synthesized mixed- halide perovskite CsPb(BrxI1−x)3 Nano-crystals (NCs) with opti- mized optical performance.50 As shown in Fig. 3(a), these mixed- halide perovskite NCs show a typical cubic/cuboidal shape with size between 11 and 15 nm and the lattice spacing is ∼0.597 nm. These mixed-halide PNCs shows a higher PLQY compared to pristine NCs, especially when x is over 10%, the PLQY of the mixed-halide PNCs exceeds 60%. Here, crosslinked poly(lauryl methacrylate) (PLMA) is used as the transparent matrix. The specific and typical preparation method of LSCs is mixing the monomer, UV initiator, and mixed- halide PNCs, and then the mixture should be injected into a mold and kept under UV illumination for curing. Photographs of an LSC comprising CsPb(Br0.2I0.8)3 NCs are shown in Figs. 3(b)–3(d). Such a kind of mixed-halide PNC-based LSCs show the optical efficiency of 2% (G = 45) with reduced overlap of absorption and emission spectra compared to the pristine LSCs. The 0D perovskite nanocrystals are commonly used candi- dates for efficient luminophores in LSCs. In NCs, the fraction of surface atoms can be higher than 30% as the NC size decreases to a few nanometers. The surface atoms are responsible for sub- stantial non-radiative recombination observed in II–VI semicon- ductor NCs, thus significantly lowering the corresponding PLQY. As such, specially designed core–shell structures (typically obtained through complicated synthesis procedures) are needed to obtain a high PLQY. Fortunately, benefiting from the high defect toler- ance of perovskite NCs, a high PLQY up to ∼100% can be obtained conveniently through ligand modification or defect passivation in perovskite NCs without a core–shell structure.66–69 Nevertheless, 0D NCs usually show a small Stokes shift, which may cause high reabsorption loss and need further optimization before being used in high-performance LSCs. In contrast to the 0D perovskite NCs, 2D perovskite materials typically exhibit a lower PLQY. However, the Stokes shift can be engineered through dimensional control in 2D perovskite materials,68 making them more advantageous in suppressing reabsorption loss. The coating forming type is different from the above. As shown in Fig. 3(e), Tong et al. prepared LSCs by using the doctor blade method. II. PEROVSKITE LUMINESCENT SOLAR CONCENTRATORS For the overall curing type, the first step is to pre- synthesize the perovskite luminophores, then the luminophores are mixed with the monomer, and finally, the slurry is poured into a mold to polymerize and form the luminescent plate. For the coat- ing forming type, pre-synthesis of the luminophores is still required. As-synthesized luminophores are then mixed with the polymer and finally blade coated on glass or polymer substrates. In this section, several typical works will be reviewed to specifically and intuitively introduce the preparation method of perovskite LSCs, and the appli- cation of different perovskite materials in LSCs will be covered and summarized. Halide perovskites can conventionally be classified into dif- ferent dimensionalities in terms of morphology (size and shape), which is the so-called geometric dimensionality. Zero dimensional (0D) perovskites include quantum dots and nanocrystals, one dimensional (1D) perovskites include nanowires and nanorods, two dimensional (2D) perovskites include nanosheets and nanoplates, and three dimensional (3D) perovskites are bulk materials.47 The geometric dimensionality defines the quantum confinement of the charge carriers—the charge carries are allowed to move in two, one, or zero dimensions in the 2D, 1D, or 0D perovskites, respec- tively. Such spatial confinement of the charge carriers results in novel electronic and optoelectronic properties for useful device applications.i Halide perovskites can also be classified into different structural dimensionalities based on the connectivity of the corner-sharing [BX6]4−octahedral. A general formula of AnBX2+n is used to repre- sent perovskite materials with different structural dimensionalities. The ABX3 structure with n = 1 is referred to as 3D perovskites,7 the A2BX4 with n = 2 as 2D perovskites, the A3BX5 with n = 3 as 1D per- ovskites, and the A4BX6 with n = 4 as 0D perovskites.16 It is worth noting that the structural dimensionality is distinct from the geo- metric dimensionality. The geometric dimensionality is often used to refer conventional nanostructures based on II–VI, II–VI, and III–V semiconductors, and thereafter, the dimensionality of halide perovskites in the following discussion is referred to as geometric dimensionality. B. LSCs based on two-dimensional perovskite nanoplates Very recently, Xia et al. proposed a new LSC preparation strat- egy based on in situ prepared perovskite-polyvinylidene fluoride (PVDF) composite films, which can be ascribed as in situ prepara- tion type.52 Different from the traditional LSCs preparation methods mentioned above, such an in situ preparation strategy avoids the pre- synthesis of perovskite luminophores. The perovskite precursor salts are mixed with PVDF in N, N-Dimethylformamide (DMF) to form a viscous slurry, which can be directly cured into luminescent films by doctor-blade deposition [Fig. 5(a)]. By integrating a phenylethy- lammonium (PEA)-assisted perovskite-PVDF composite film with a polymer anti-reflection/barrier layer, as-prepared composite films show significant improvement in PLQY and optical transmittance [Figs. 5(b) and 5(c)]. As shown in Figs. 5(d) and 5(e), LSCs can be prepared by simply pasting two pieces of glass covered with lumi- nescent films (or replacing one of them with ordinary glass). Such LSCs with a sandwich structure demonstrate a maximum optical efficiency of 2.8% (G = 5) and show good environmental stability. It is worth noting that compared with the traditional pre-synthesis- redispersion strategy, in situ prepared luminescent films are still not widely used in LSCs, although some applications have been reported in backlight display.77 Moreover, the existing research shows that the as-prepared perovskite–polymer composite films exhibit rough surface morphology and high sensitivity to UV curing adhesives, which may increase surface scattering loss and damage device Two-dimensional PNPLs are generally considered to be the preferred material for preparing LSCs, especially when various lay- ers of PNPLs coexist in the system forming the multiple quan- tum wells (MQWs), as-generated energy funneling can effectively increase the Stokes shift, thereby reducing the energy loss caused by reabsorption.69,73–75 As shown in Fig. 4(a), Li et al. modified the solvent-Pb2+ coordination via controlling the polarity and Lewis basicity of the precursor solution so that the synthesized PNPLs showed a uniform MQW distribution, which further improves energy funneling.76 The PNPL-based LSCs can also be prepared by using the doctor blade method. Here, PMMA and glass are used as the slurry matrix and substrate, respectively [Fig. 4(b)]. The energy transfer (ET) between different components can be investigated from the time evolution of various bleach peaks in transient absorp- tion (TA) spectra for chlorobenzene-introduced MQWs, as shown in Fig. 4(c). A. Perovskite nanocrystals for LSCs Dicarboxylic acids are used as ligands to improve the PLQY (up to 90% ± 5%), chemical yield, and stability of FAPbBr3 NCs.72 Then these optimized nanocrystals are mixed with polystyrene (PS) to form a slurry, which will be deposited onto PMMA slabs. It is worth mentioning that the polymer is coated around the perovskite grains, which effectively isolates the direct contact between moisture and the perovskite so that the perovskite-based LSCs exhibits good environmental stability. Although the LSCs based on 1D perovskite nanowires have yet to be reported, patterned/arrayed perovskite nanowires hold potential in the application of LSC luminophores because of their APL Photon. 6, 120901 (2021); doi: 10.1063/5.0067920 © Author(s) 2021 6, 120901-4 6, 120901-4 APL Photonics scitation.org/journal/app FIG. 3. (a) TEM image of CsPb(Br0.4I0.6)3 NCs. Photographs of the LSC comprising CsPb(Br0.2I0.8)3 NCs under (b) ambient, (c) one sun (100 mW/cm2), and (d) UV light illumination.50 Reproduced with permission from Zhao et al., Nano Energy 37, 214–223 (2017). Copyright 2017 Elsevier. (e) Schematic illustration of the synthetic approach for FAPbBr3 NCs and the fabrication procedure of LSCs.72 Reproduced with permission from Tong et al., J. Mater. Chem. A 7, 4872 (2019). Copyright 2019 the Royal Society of Chemistry. 24 October 2024 05:44:56 24 October 2024 05:44:56 FIG. 3. (a) TEM image of CsPb(Br0.4I0.6)3 NCs. Photographs of the LSC comprising CsPb(Br0.2I0.8)3 NCs under (b) ambient, (c) one sun (100 mW/cm2), and (d) UV light illumination.50 Reproduced with permission from Zhao et al., Nano Energy 37, 214–223 (2017). Copyright 2017 Elsevier. (e) Schematic illustration of the synthetic approach for FAPbBr3 NCs and the fabrication procedure of LSCs.72 Reproduced with permission from Tong et al., J. Mater. Chem. A 7, 4872 (2019). Copyright 2019 the Royal Society of Chemistry. B. LSCs based on two-dimensional perovskite nanoplates shallow traps represented by surface vacancies dominate the non- radiative recombination rate, which directly affects the PLQY of the luminophores.78–81 Considering the ionic nature of perovskites, surface passivation via cationic/anionic ligands or additives is one of the most effective ways to improve the PLQY of the per- ovskite luminophore, and the A- and X-site vacancies widely dis- tributed on the grain surface can be effectively filled.48,82 In the past five years, a variety of defect-passivation strategies have been proposed to improve the PLQY and long-term stability of per- ovskite luminophores, and some of these strategies are still appli- cable in perovskite LSCs.47,48 Table II summarizes 18 effective and l l d h dd h shallow traps represented by surface vacancies dominate the non- radiative recombination rate, which directly affects the PLQY of the luminophores.78–81 Considering the ionic nature of perovskites, surface passivation via cationic/anionic ligands or additives is one of the most effective ways to improve the PLQY of the per- ovskite luminophore, and the A- and X-site vacancies widely dis- tributed on the grain surface can be effectively filled.48,82 In the past five years, a variety of defect-passivation strategies have been proposed to improve the PLQY and long-term stability of per- ovskite luminophores, and some of these strategies are still appli- cable in perovskite LSCs.47,48 Table II summarizes 18 effective and typical ligand-attachment strategies or additives with passivation, which are applied to improve the PLQY and stability of perovskite luminophores. It is worth mentioning that halide perovskites, espe- cially their nanocrystals, are usually easy to obtain high PLQY due to their defect-tolerant nature. However, the difficulty lies in how to maintain its high PLQY for a long time, that is, how to improve its stability, which attracts more research interest.48 Benefiting from the novel triphenylphosphine (TPP) treatment, the PLQY and stabil- ity of CsPbI3 nanocrystals have been significantly improved.55 Such molecules with strong coordination and deoxidation could effec- tively protect the perovskite luminophores from decomposition and regrowth during photopolymerization. Note that in addition to the protection of perovskite nanocrystals, TPP can also be used as a UV initiator due to the lone pairs of electrons on the phosphorus III. OPTICAL LOSSES IN LSC AND CORRESPONDING OPTIMIZATION SCHEMES The optical losses, especially non-radiative recombination loss, scattering loss, transmission loss, escape cone loss and reabsorption loss, are the main constraints that limit the improvement of LSC per- formance, thereby delaying its commercialization process.9 In this section, we review the effective and representative strategies that have been proposed for mitigating these losses. In addition, common loss suppression methods for LSCs based on different luminophores will be introduced, and the discussion will not be limited to the scope of perovskite-based LSCs. typical ligand-attachment strategies or additives with passivation, which are applied to improve the PLQY and stability of perovskite luminophores. It is worth mentioning that halide perovskites, espe- cially their nanocrystals, are usually easy to obtain high PLQY due to their defect-tolerant nature. However, the difficulty lies in how to maintain its high PLQY for a long time, that is, how to improve its stability, which attracts more research interest.48 Benefiting from the novel triphenylphosphine (TPP) treatment, the PLQY and stabil- ity of CsPbI3 nanocrystals have been significantly improved.55 Such molecules with strong coordination and deoxidation could effec- tively protect the perovskite luminophores from decomposition and regrowth during photopolymerization. Note that in addition to the protection of perovskite nanocrystals, TPP can also be used as a UV initiator due to the lone pairs of electrons on the phosphorus B. LSCs based on two-dimensional perovskite nanoplates A significant rise in the bleach peak corresponding to the nanosheets with n = 7–10 can be observed over the first 100 ps (n is the number of PbI6 monolayer sheets within a layer), accompanied by a significant fall in the n = 4 sample while the n = 5 and 6 sample signal remains roughly constant. These results indicate the energy transfer from nanosheets with fewer layers to the counterparts with more layers. APL Photon. 6, 120901 (2021); doi: 10.1063/5.0067920 © Author(s) 2021 6, 120901-5 APL Photon. 6, 120901 (2021); doi: 10.1063/5.0067920 © Author(s) 2021 APL Photonics scitation.org/journal/app FIG. 4. (a) Schematic diagram of the LSC structure with energy transfer. (b) Photograph of a PNPL-based LSC. (c) Transient Absorption spectra for the 50% CB sample at various TA bleach peaks as a function of delay time.76 Reproduced with permission from Li et al., Joule 4, 631–643 (2020). Copyright 2020 Elsevier. 24 October 2024 05:44:56 FIG. 4. (a) Schematic diagram of the LSC structure with energy transfer. (b) Photograph of a PNPL-based LSC. (c) Transient Absorption spectra for the 50% CB sample at various TA bleach peaks as a function of delay time.76 Reproduced with permission from Li et al., Joule 4, 631–643 (2020). Copyright 2020 Elsevier. stability. Therefore, more research needs to be done to explore mor- phology control, UV stability, and polymer matrix adaptation in in-situ-prepared perovskite–polymer composite films. The in situ preparation method has potential advantages in preparing lumines- cent films with a high PLQY and simplifying the preparation process of LSCs. A. Non-radiative recombination loss The non-radiative recombination loss is caused by the low PLQY and aging of the luminophore itself, which is an impor- tant indicator for evaluating the performance of LSCs. Usually, APL Photon. 6, 120901 (2021); doi: 10.1063/5.0067920 © Author(s) 2021 6, 120901-6 APL Photonics FIG. 5. (a) Illustration of the preparation process of PEA-assisted perovskite-PVDF composite films and the polymer anti-reflection/barrier approach. Photographs of ⟨n⟩ = 3 perovskite-PVDF composite films (b) with and (c) without the PS anti-reflection/barrier layer on glass substrates. The symbol ⟨n⟩represents the specific precursor salt molar ratio in PEA2MAn-1PbnI3n+1 of the samples. Photographs of ⟨n⟩= 3 perovskite-PVDF composite films with the PS anti-reflection/barrier layer attached to ordinary glass under (d) ambient and (e) UV light.52 Reproduced with permission from Xia et al., Sol. RRL 5, 2100491 (2021). Copyright 2021 Wiley. 24 October 2024 05:44:56 24 October 2024 05:44:56 FIG. 5. (a) Illustration of the preparation process of PEA-assisted perovskite-PVDF composite films and the polymer anti-reflection/barrier approach. Photographs of ⟨n⟩ = 3 perovskite-PVDF composite films (b) with and (c) without the PS anti-reflection/barrier layer on glass substrates. The symbol ⟨n⟩represents the specific precursor salt molar ratio in PEA2MAn-1PbnI3n+1 of the samples. Photographs of ⟨n⟩= 3 perovskite-PVDF composite films with the PS anti-reflection/barrier layer attached to ordinary glass under (d) ambient and (e) UV light.52 Reproduced with permission from Xia et al., Sol. RRL 5, 2100491 (2021). Copyright 2021 Wiley. atoms, which shows better performance than commercial counter- parts. As-prepared perovskite LSCs achieve a high PLQY of 99.4% ± 0.4% [Fig. 6(a)], and the optical conversion efficiency reaches 3.1% (G = 10), accompanied by significant improvement both in environ- mental and UV stability [Fig. 6(b)]. These molecules that can donate electrons to the metal of the nanocrystal surface can effectively bond to the surface of the perovskite grains to reduce surface defects, thereby improving the PLQY and stability of the luminescent film and suppressing the non-radiative recombination loss. and the “light” state for transparency [Fig. 7(c)]. The emission direc- tion can be directly controlled by applied voltages, thereby adjusting the transmittance of LSCs and the scattering of luminophores. Such interesting phenomena in these liquid crystal-based switchable LSCs indicate that it is feasible to suppress the internal waveguide scatter- ing loss by adjusting the emission dipole of the luminophores. B. Scattering loss The sources of scattering loss can be divided into two types: internal waveguide and surface scattering, which are rarely studied in the past few years.9 The surface scattering loss is almost inevitable during the actual installation and application of LSCs due to the change in the angle of the incident sunlight from sunrise until sun- set. In view of this, it is more practical to focus on the scattering loss caused by the internal waveguide.93 Dye embedded supertwist liquid crystals are used to prepare LSCs, which can be switched electrically between three states by applying different voltages: the “dark” state for increasing light absorption and electrical generation [Fig. 7(a)], the “scattering” state for enhancing haziness [Fig. 7(b)], A. Non-radiative recombination loss In addition, it has been reported that ferrimagnetic inorganic nanorods have been used to adjust the optical properties of liquid crystals in 2014.94 Optical tuning can be achieved through manipulating the nanorod orientation using considerably weak external mag- netic fields of 1 mT. Such magnetically responsive liquid crystals show application prospects in controlling the emission dipole of the luminophores and suppressing internal waveguide scattering loss in LSCs because of contactless nature and remote implementation. C. Transmission loss Transmission loss originates from the fact that part of the inci- dent sunlight is not absorbed by the luminophores and directly pen- etrates the LSCs, which results in insufficient absorption of incident photons. Therefore, tandem LSCs are an effective way to increase the utilization rate of incident sunlight. Zhao et al. reported a large- area of 100 cm2 tandem LSC based on CDs and perovskite NCs APL Photon. 6, 120901 (2021); doi: 10.1063/5.0067920 APL Photon. 6, 120901 (2021); doi: 10.1063/5.0067920 © Author(s) 2021 6, 120901-7 6, 120901-7 APL Photonics TABLE II. Representative of a ligand or additive utilized for improving the PLQY and stability of perovskite. RH: relative humidity. Types Additives/ligands Molecular structure PL peak (nm) Enhancement of emission Enhancement of stability Octylammonium63 N.A. N.A. ∼794% (from ∼85 to 760 h) in ambient air at 85 ○Ca A-site vacancy Diethylenetriamine64 ∼763 N.A. >600% (from ∼50 to >350 h) in 50% RH aira 2-Pyridylthiourea65 ∼775 N.A. >100% (from 15 to >30 days) in 30% RH aira Benzylammonium83 ∼810 N.A. ∼400% (from 100 to 500 h) in 80% RH aira Phenylethanaminium52,84 ∼62052 ∼1017% (from 4.1% to 45.8%, PLQY)52 N. A.52 ∼73884 N. A.84 ∼1050% (from four to 46 h) in 52% RH airb 84 Guanidinium85 ∼770 ∼400% (PL intensity) ∼38.5% (from 195 to 270 h) in aira Ethylenediammonium86 ∼705 N.A. ∼600% (from ∼1 to 7 days) in dark dry box at 100 ○Cb 3-Phenyl-2-propen- 1-amine87 ∼528 N.A. ∼253% (from 19 to 67 h)c 2,2 -lminodibenzoic acid47 680 ∼19% (from 80% to 95%, PLQY) >2900% (from ∼0.5 to >15 days)c X-site vacancy Triphenylphosphine55 695 ∼93% (from 52% to 99%, PLQY) >4900% (from ∼2 to >100 h) under 500 W mercury lampc Trioctylphosphine oxide67 532 ∼29% (from 57.3% to 73.8%, PLQY) N.A. BLE II. Representative of a ligand or additive utilized for improving the PLQY and stability of perovskite. RH: relative humidity. ypes Additives/ligands Molecular structure PL peak (nm) Enhancement of emission Enhancement of stability Octylammonium63 N.A. N.A. ∼794% (from ∼85 to 760 h) in ambient air at 85 ○Ca site vacancy Diethylenetriamine64 ∼763 N.A. >600% (from ∼50 to >350 h) in 50% RH aira 2-Pyridylthiourea65 ∼775 N.A. >100% (from 15 to >30 days) in 30% RH aira Benzylammonium83 ∼810 N.A. ∼400% (from 100 to 500 h) in 80% RH aira Phenylethanaminium52,84 ∼62052 ∼1017% (from 4.1% to 45.8%, PLQY)52 N. A.52 ∼73884 N. C. Transmission loss A.84 ∼1050% (from four to 46 h) in 52% RH airb 84 Guanidinium85 ∼770 ∼400% (PL intensity) ∼38.5% (from 195 to 270 h) in aira Ethylenediammonium86 ∼705 N.A. ∼600% (from ∼1 to 7 days) in dark dry box at 100 ○Cb 3-Phenyl-2-propen- 1-amine87 ∼528 N.A. ∼253% (from 19 to 67 h)c 2,2 -lminodibenzoic acid47 680 ∼19% (from 80% to 95%, PLQY) >2900% (from ∼0.5 to >15 days)c site vacancy Triphenylphosphine55 695 ∼93% (from 52% to 99%, PLQY) >4900% (from ∼2 to >100 h) under 500 W mercury lampc Trioctylphosphine oxide67 532 ∼29% (from 57.3% to 73.8%, PLQY) N.A. ative of a ligand or additive utilized for improving the PLQY and stability of perovskite. RH: relative humidity. 24 October 2024 05:44:56 TABLE II. Representative of a ligand or additive utilized for improving the PLQY and stability of perovskite. Types APL Photon. 6, 120901 (2021); doi: 10.1063/5.0067920 © Author(s) 2021 6, 120901-8 © Author(s) 2021 APL Photonics scitation.org/journal/app II. (Continued.) Additives/ligands Molecular structure PL peak (nm) Enhancement of emission Enhancement of stability Dodecylbenzenesulfonic acid79 ∼513 Improve the PLQY to >90% >1233% (from ∼300 to >4000 h)d Ammonium thio- cyanate88 N.A. N.A. >2900% (from ∼2 to >60 days) under continuous light illumina- tion in 30%–40% RH aira Polyethylene glycol89 ∼770 ∼100% (PL intensity) >100% (from ∼7.5 to >15 days) under xenon-lamp illumination in 50% RH aira Thiophene90 ∼775 ∼38% (from 13% to 18%, PLQY) N.A. Iodopentafluorobenzene91 N.A. N.A. N.A. Phosphonic acids92 499 Improve the PLQY to 96.8% N.A. e for the power conversion efficiency of the solar cells decreasing to 80% of the initial value BLE II. (Continued.) ypes Additives/ligands Molecular structure PL peak (nm) Enhancement of emission Enhancement of stability Dodecylbenzenesulfonic acid79 ∼513 Improve the PLQY to >90% >1233% (from ∼300 to >4000 h)d Ammonium thio- cyanate88 N.A. N.A. >2900% (from ∼2 to >60 days) under continuous light illumina- tion in 30%–40% RH aira Polyethylene glycol89 ∼770 ∼100% (PL intensity) >100% (from ∼7.5 to >15 days) under xenon-lamp illumination in 50% RH aira Thiophene90 ∼775 ∼38% (from 13% to 18%, PLQY) N.A. Iodopentafluorobenzene91 N.A. N.A. N.A. Phosphonic acids92 499 Improve the PLQY to 96.8% N.A. fetime for the power conversion efficiency of the solar cells decreasing to 80% of the initial value. ime for the characteristic peaks of precursor materials appearing in the XRD spectra of perovskite films after aging. C. Transmission loss aLifetime for the power conversion efficiency of the solar cells decreasing to 80% of the initial value. bTime for the characteristic peaks of precursor materials appearing in the XRD spectra of perovskite films after aging. cLifetime for the PL intensity decreasing to 80% of the initial value. d aLifetime for the power conversion efficiency of the solar cells decreasing to 80% of the initial value. bTime for the characteristic peaks of precursor materials appearing in the XRD spectra of perovskite films after aging. cLifetime for the PL intensity decreasing to 80% of the initial value. dLifetime for the PLQY decreasing to 80% of the initial value. APL Photon. 6, 120901 (2021); doi: 10.1063/5.0067920 © Author(s) 2021 6, 120901-9 APL Photonics PERSPECTIVE scitation.org/journal/app FIG. 6. (a) PL emission spectra of blank and TPP-CsPbI3 used for measuring the PLQYs. Inset: photograph of a TPP-CsPbI3 NC film in a mounting boat. (b) Photostability of the untreated, the ZnI2 treated, and the TPP treated NC-polymer composite films.55 Reproduced with permission from Wu et al., Angew. Chem., Int. Ed. 59, 7738–7742 (2020). Copyright 2020 Wiley. APL Photonics FIG. 6. (a) PL emission spectra of blank and TPP-CsPbI3 used for measuring the PLQYs. Inset: photograph of a TPP-CsPbI3 NC film in a mounting boat. (b) Photostability of the untreated, the ZnI2 treated, and the TPP treated NC-polymer composite films.55 Reproduced with permission from Wu et al., Angew. Chem., Int. Ed. 59, 7738–7742 (2020). Copyright 2020 Wiley. FIG. 6. (a) PL emission spectra of blank and TPP-CsPbI3 used for measuring the PLQYs. Inset: photograph of a TPP-CsPbI3 NC film in a mounting boat. (b) Photostability of the untreated, the ZnI2 treated, and the TPP treated NC-polymer composite films.55 Reproduced with permission from Wu et al., Angew. Chem., Int. Ed. 59, 7738–7742 (2020). Copyright 2020 Wiley. [Fig. 8(a)].60 In such a tandem system, in addition to the large Stokes shift, the PL spectrum of such carbon dots-based LSC (from 400 to 550 nm) matches the absorption range of the underlying perovskite- based counterparts very well [Fig. 8(b)]. The down-converted pho- tons escaping from the carbon dots-based LSC can be reabsorbed by the perovskite-based ones, thereby improving the utilization rate of incident sunlight and overall external optical efficiency (defined as the ratio of the optical power of re-emitted photons reaching the edges of an LSC to the optical power of incident photons) of the tandem LSC. C. Transmission loss Such semi-transparent LSC exhibits an external G. 7. Photographs of the Yell25c cell at (a) 0 VRMS, (b) 10 VRMS, and (c) 28 VRMS and the corresponding schematic representation of the alignment of the LCs, dyes, and e emission direction. VRMS: voltage root mean square.93 Reproduced with permission from Sol et al., Adv. Energy Mater. 8, 1702922 (2018). Copyright 2018 Wiley. 24 October 2024 05:44:56 24 October 2024 05:44:56 FIG. 7. Photographs of the Yell25c cell at (a) 0 VRMS, (b) 10 VRMS, and (c) 28 VRMS and the corresponding schematic representation of the alignment of the LCs, dyes, and the emission direction. VRMS: voltage root mean square.93 Reproduced with permission from Sol et al., Adv. Energy Mater. 8, 1702922 (2018). Copyright 2018 Wiley. FIG. 7. Photographs of the Yell25c cell at (a) 0 VRMS, (b) 10 VRMS, and (c) 28 VRMS and the corresponding schematic representation of the alignment of the LCs, dyes, and the emission direction. VRMS: voltage root mean square.93 Reproduced with permission from Sol et al., Adv. Energy Mater. 8, 1702922 (2018). Copyright 2018 Wiley. APL Photon. 6, 120901 (2021); doi: 10.1063/5.0067920 APL Photon. 6, 120901 (2021); doi: 10.1063/5.0067920 © Author(s) 2021 6, 120901-10 6, 120901-10 © Author(s) 2021 APL Photonics FIG. 8. (a) Scheme of a typical tandem LSC composed of carbon dot (C-dot), CsPb(Br1−xClx)3, and CsPb(BrxI1−x)3 based LSCs. (b) Absorption and normalized PL spectra of LSCs based on C-dots/PVP on a glass substrate, CsPb(Br0.8Cl0.2)3 QDs, and CsPb(Br0.2I0.8)3 QDs incorporated into a PLMA polymer matrix.60 Reproduced with permission from Zhao et al., Nano Energy 50, 756–765 (2018). Copyright 2018 Elsevier. FIG. 8. (a) Scheme of a typical tandem LSC composed of carbon dot (C-dot), CsPb(Br1−xClx)3, and CsPb(BrxI1−x)3 based LSCs. (b) Absorption and normalized PL spectra of LSCs based on C-dots/PVP on a glass substrate, CsPb(Br0.8Cl0.2)3 QDs, and CsPb(Br0.2I0.8)3 QDs incorporated into a PLMA polymer matrix.60 Reproduced with permission from Zhao et al., Nano Energy 50, 756–765 (2018). Copyright 2018 Elsevier. optical efficiency of ∼3%, which shows a 27% and 117% enhance- ment in efficiency compared to CsPb(BrxI1−x)3 and CsPb(ClxBr1−x)3 LSCs, respectively. structure with toluene solution of the two mixed monomers and the CdSe–CdS QDs before photopolymerization. (4) Immersing the sandwich structure into 2% hydrofluoric acid for 20 s to etch away the silica spheres. The cross section scanning electron microscopy (SEM) image in Fig. 9(c) shows a uniform thickness of the PC layer (∼3.0 μm, ∼12 monolayers of macropores) with an average pore size of ∼375 nm. When photons reach the top and bottom surfaces of the LSC within the escape cone, most of them can be recycled by the PC reflector and trapped, as shown in Figs. 9(c)–9(f). As a result, 95.3% of the total collected photons were contributed from the four edges, leaving only 4.7% emitted from the top/bottom surfaces [Fig. 9(g)], which effectively suppresses the escape cone loss. 24 October 2024 05:44:56 D. Escape cone loss Escape cone loss, one of the factors that significantly inhibit the performance of LSCs, refers to the loss of photons emitted by per- ovskite luminophores in the escape cone through the surface. In view of the planar waveguide structure of LSCs, it can be easily under- stood that the part of the emitted light trapped in the waveguide is determined by the refractive index of the waveguide. According to Snell’s law, all photons approaching an interface between the matrix and air at an angle greater than the critical angle will be totally reflected. This critical angle is defined as Another similar approach to suppress the escape cone loss is using the Distributed Bragg Reflectors (DBRs), which is one of the applications of wavelength selective mirror technology and has been applied in dye-based LSCs, as shown in Fig. 9(h).96 The reflection of fluorophore emission can be achieved by adjusting the DBR thick- ness. Compared with the bare LSC, such a flexible LSC–mirror–DBR system increases the external optical efficiency from 5.0% to 5.7% and 4.5%–5.6% when flat and curved, respectively. When the opti- mized LSCs are coupled to solar cells, the PCE is up to 3.2% with 8.1 mA/cm2 for Jsc and 0.5 V for Voc. The significantly improved performance shows that the DBR strategy effectively inhibits the escape cone loss, which provides a feasible strategy for the subse- quent optimization of perovskite-based LSCs. θc = arcsin(1/n), (6) (6) where n is the refractive index of the waveguide. Herein, for the isotropic luminophores such as perovskite NCs embedded in a wave- guide with a refractive index of 1.5–1.6, ∼25% of the emitted photons will escape from the surface. Two typical strategies for suppressing escape cone loss have been applied in the LSCs based on CdSe–CdS core–shell QDs and dyes, which can also be used as a reference for perovskite-based LSCs. Wang et al. introduced a facile and low-cost approach for the fabrication of a three-dimensional macroporous photonic crys- tal (PC) filter as an efficient photon reflector, which can be coated onto QD-based LSCs, as shown in Fig. 9(a).95 In this work, the preparation of LSCs with reflective coating is mainly divided into four steps [Fig. 9(b)]: (1) Fabricating the PC filter on a glass sub- strate by crystallizing colloidal silica spheres through a convective self-assembly process. (2) Establishing the sandwich structure from two pieces of PC-coated glass substrates. (3) Filling the sandwich E. Reabsorption loss The reabsorption loss is caused by the fact that photons emitted by luminophores are absorbed by other ones, resulting in a decrease of photons that are coupled to the sides of the waveguide. The reab- sorption loss depends on the concentration and Stokes shift of fluo- rophore. The entropy of the LSC system is affected by the absorption APL Photon. 6, 120901 (2021); doi: 10.1063/5.0067920 © Author(s) 2021 APL Photon. 6, 120901 (2021); doi: 10.1063/5.0067920 6, 120901-11 6, 120901-11 APL Photonics scitation.org/journal/app a) Schematic illustration of the top PC surface and the associated photon trapping mechanism. (b) Schematics of fabrication process for the macropo D-based LSC copolymer slab. (c) Cross-sectional SEM image of a PC-LSC device. Inset: top-view of the macroporous structure. Photographs of a PC-LS ed under (d) room light and (e) UV light at 365 nm. (f) Reflection spectrum of the PC reflector as compared with the absorption and emission spectra of Cd ll QDs. (g) PL spectra of the total PC-LSC, edge-covered top/bottom, and the edge emission.95 Reproduced with permission from Wang et al., Nano E 2020). Copyright 2020 Elsevier. (h) Diagram of device configuration (from top to bottom): wavelength selective mirror, dye-doped PDMS, and silver mir phs of two fabricated flexible wavelength-selective mirrors.96 Reproduced with permission from Portnoi et al., Nano Energy 70, 104507 (2020). Copyr 24 October 2024 05:44:56 FIG. 9. (a) Schematic illustration of the top PC surface and the associated photon trapping mechanism. (b) Schematics of fabrication process for the macroporous PC coated QD-based LSC copolymer slab. (c) Cross-sectional SEM image of a PC-LSC device. Inset: top-view of the macroporous structure. Photographs of a PC-LSC device illuminated under (d) room light and (e) UV light at 365 nm. (f) Reflection spectrum of the PC reflector as compared with the absorption and emission spectra of CdSe–CdS core–shell QDs. (g) PL spectra of the total PC-LSC, edge-covered top/bottom, and the edge emission.95 Reproduced with permission from Wang et al., Nano Energy 67, 104217 (2020). Copyright 2020 Elsevier. (h) Diagram of device configuration (from top to bottom): wavelength selective mirror, dye-doped PDMS, and silver mirror (top). Photographs of two fabricated flexible wavelength-selective mirrors.96 Reproduced with permission from Portnoi et al., Nano Energy 70, 104507 (2020). Copyright 2020 Elsevier. FIG. 9. (a) Schematic illustration of the top PC surface and the associated photon trapping mechanism. E. Reabsorption loss 6, 120901 (2021); doi: 10.1063/5.0067920 APL Photonics PERSPECTIVE scitation.org/journal/app and re-emission events of the luminophore embedded in the matrix. According to the reported work, the maximum concentration can be approximated by donor and receptor layers are intimate stacked. Furthermore, when the ensembles of perovskite domains are blocked by the polymer matrix with a long distance, such an ultrafast energy transfer process will not exist. The PLQY, ηedge (defined as the ratio of the number of photons emitted from the side to the total number of photons emit- ted by the LSCs) and the overlap of absorption and emission spectra can be optimized by appropriately changing the acceptor concentra- tion [Figs. 10(d) and 10(e)]. As-prepared devices achieve an optical quantum efficiency (defined as the ratio of the number of photons emitted from the panel edges to the total number of absorbed pho- tons) of 26% and an internal concentration factor of 3.3 for LSCs with an area of 10 × 10 cm2. C ≈(e 3 2 /e 3 1 ) exp[(e1 −e2)/kT0], (7) (7) where e2 and e1 are the photon energies of the emitted and absorbed photons, respectively, k is Boltzmann’s constant, and T0 is ambient temperature.9 In recent years, many strategies have been proposed to increase the Stokes shift or reduce the overlap of absorption and emis- sion spectra. Three typical strategies for inhibiting reabsorption loss based on different luminescence mechanisms have been proposed and demonstrated, which are discussed in the following. The second strategy to suppress reabsorption loss is proposed by Zhao et al.6 Here, Cs4PbBr6 perovskite NCs are used to fab- ricate semi-transparent large-area LSCs. As shown in Fig. 11(a), these Cs4PbBr6 perovskite NCs exhibit a nonspherical shape with an average size of 120 ± 37 nm. Compared to the CsPbBr3 NCs, the Cs4PbBr6 NCs have a large Stokes shift up to 196 nm [Fig. 11(b)], which can be attributed to different luminescence mechanisms. Specifically, in CsPbBr3 NCs, the emission originates from the band energy emission, thereby leading to a very small Stokes shift. However, in Cs4PbBr6 NCs, Br-poor vacancy states are supposed to give rise to the defect-emission, so the emission of Cs4PbBr6 NCs can be considered to come from the mid-band states, which Wei et al. employ layered hybrid impurity halide perovskites to suppress reabsorption loss and improve PLQY.97 Specifically, the ensembles of two-dimensional perovskite domains are used as luminophores in low-loss large-area LSCs [Fig. E. Reabsorption loss (b) Schematics of fabrication process for the macroporous PC coated QD-based LSC copolymer slab. (c) Cross-sectional SEM image of a PC-LSC device. Inset: top-view of the macroporous structure. Photographs of a PC-LSC device illuminated under (d) room light and (e) UV light at 365 nm. (f) Reflection spectrum of the PC reflector as compared with the absorption and emission spectra of CdSe–CdS core–shell QDs. (g) PL spectra of the total PC-LSC, edge-covered top/bottom, and the edge emission.95 Reproduced with permission from Wang et al., Nano Energy 67, 104217 (2020). Copyright 2020 Elsevier. (h) Diagram of device configuration (from top to bottom): wavelength selective mirror, dye-doped PDMS, and silver mirror (top). Photographs of two fabricated flexible wavelength-selective mirrors.96 Reproduced with permission from Portnoi et al., Nano Energy 70, 104507 (2020). Copyright 2020 Elsevier. APL Photon. 6, 120901 (2021); doi: 10.1063/5.0067920 APL Photon. 6, 120901 (2021); doi: 10.1063/5.0067920 © Author(s) 2021 6, 120901-12 6, 120901-12 © Author(s) 2021 APL Photonics ) Schematic diagram of the LSC structure and the energy transfer process within layered perovskite platelets composed of donor phases (n1 and n2) and a . Transient absorption spectral dynamics for (b) the donor and (c) acceptor GSB traces. OD represents optical density. (d) PLQY (purple dots) and OQ s a function of stoichiometry. (e) The absorption–emission overlap coefficient (S), defined as the overlapping area of the absorption spectra and photolumine d relative presence of the final acceptor as the stoichiometry changes. Inset: photograph of a 10 × 10 cm2 PNPL-based LSC.97 Reproduced with permissi Nat. Energy 4, 197–205 (2019). Copyright 2019 Springer Nature. 24 October 2024 05:44:56 FIG. 10. (a) Schematic diagram of the LSC structure and the energy transfer process within layered perovskite platelets composed of donor phases (n1 and n2) and acceptor phase (n3). Transient absorption spectral dynamics for (b) the donor and (c) acceptor GSB traces. OD represents optical density. (d) PLQY (purple dots) and OQE (blue triangles) as a function of stoichiometry. (e) The absorption–emission overlap coefficient (S), defined as the overlapping area of the absorption spectra and photoluminescence spectra, and relative presence of the final acceptor as the stoichiometry changes. Inset: photograph of a 10 × 10 cm2 PNPL-based LSC.97 Reproduced with permission from Wei et al., Nat. Energy 4, 197–205 (2019). Copyright 2019 Springer Nature. APL Photon. 6, 120901 (2021); doi: 10.1063/5.0067920 © Author(s) 2021 APL Photon. E. Reabsorption loss 10(a)], which are carefully adjusted to achieve efficient energy transfer from the donor (n1 and n2) to the acceptor (n3). As shown in Figs. 10(b) and 10(c), the accelerated decrease in the donor ground-state bleaching (GSB) and the increase in the acceptor GSB with increasing acceptor inclu- sions indicate the realization of efficient energy transfer. It should be noted that the ultrafast energy transfer process only exists when the FIG. 11. (a) TEM images of Cs4PbBr6 NCs. (b) Absorption and PL spectra of CsPbBr3 and Cs4PbBr6 NCs in the solution or in the PS thin film. The energy diagram and exciton recombination mechanism in CsPbBr3 and Cs4PbBr6 NCs. (c) Photographs of the Cs4PbBr6 perovskite NCs-based LSC under ambient (left) and one sun (right, 100 mW/cm2) illumination. (d) Integrated PL intensity as a function of detection distance.6 Reproduced with permission from Zhao et al., Adv. Funct. Mater. 29, 1902262 (2019). Copyright 2019 Wiley. 24 October 2024 05:44:56 FIG. 11. (a) TEM images of Cs4PbBr6 NCs. (b) Absorption and PL spectra of CsPbBr3 and Cs4PbBr6 NCs in the solution or in the PS thin film. The energy diagram and exciton recombination mechanism in CsPbBr3 and Cs4PbBr6 NCs. (c) Photographs of the Cs4PbBr6 perovskite NCs-based LSC under ambient (left) and one sun (right, 100 mW/cm2) illumination. (d) Integrated PL intensity as a function of detection distance.6 Reproduced with permission from Zhao et al., Adv. Funct. Mater. 29, 1902262 (2019). Copyright 2019 Wiley. APL Photon. 6, 120901 (2021); doi: 10.1063/5.0067920 © Author(s) 2021 6, 120901-14 APL Photon. 6, 120901 (2021); doi: 10.1063/5.0067920 6, 120901-14 © Author(s) 2021 APL Photonics FIG. 12. (a) Absorption (black line) and PL spectra of Mn2+-doped CsPbCl3 NCs (4% doping level) under 390 nm excitation in toluene solution.56 Reproduced with permission from Meinardi et al., ACS Energy Lett. 2, 2368–2377 (2017). Copyright 2017 American Chemical Society. (b) Absorption (purple) and PL (blue, orange, and red) spectra of the undoped and Mn2+/Yb3+ codoped CsPbCl3 perovskite NCs. Insets: schematics of the perovskite crystal structures and photographs of the samples under UV illumination (365 nm) taken by a visible-camera and a NIR camera with an 800 nm long-path filter. (c) Schematic of the proposed energy transfer (ET) processes in the Mn2+/Yb3+ codoped CsPbCl3 NCs.100 Cai et al., Adv. Sci. 7, 2001317 (2020). Copyright 2020 Author(s), licensed under a Creative Commons Attribution 4.0 License. E. Reabsorption loss (2) The coating forming type, where the pre-synthesized perovskite luminophores are mixed with the polymer and coated on the polymer substrate and perovskite luminophores are embedded into the substrate by swelling. (3) The in situ preparation type, where perovskite precur- sor salts and the polymer are directly mixed in the solvent to form slurry, which can be directly transformed into luminescent films after coating. The third effective way to suppress reabsorption is dop- ing impurities of transition or lanthanide metals in perovskite luminophores.98,99 Here, we review three typical studies based on metal ion doping to increase Stokes shift to show the advantages of the doping strategy in restraining reabsorption loss. Mn2+-doped, Mn2+/Yb3+-codoped, and Yb3+-doped CsPbCl3 NCs are used as luminophores in these highlighted works mentioned below. Meinardi et al. introduced manganese-doped CsPbCl3 NCs as reabsorption-free emitters for large-area LSCs.56 In such Mn2+- doped NCs, the 6A1 singlet ground state and the 4T1 excited triplet state of Mn2+ cations are positioned within the forbidden energy gap of the CsPbCl3 NC host. Figure 12(a) shows the absorption and PL spectrum of these Mn-doped CsPbCl3 NCs, where 80% of emitted photons are due to the 4T1 →6A1 optical transition of the Mn2+ dopants (∼590 nm) accompanied by a large Stokes shift up to 200 nm. In addition to the advances in the preparation process of per- ovskite LSCs mentioned above, the improved stability of perovskite luminophores is also particularly important. Since the vast majority of LSCs require the luminophores to operate in solid or film con- ditions, the importance of the solid or film stability of perovskite, including phase, moisture, temperature, and UV stability cannot be overemphasized, and more efforts to improve optical stability of perovskite films need to be put into practice. The synthesis of Mn2+/Yb3+ codoped CsPbCl3 NCs was real- ized by Cai et al. through the hot-injection technique.100 Compared with pure Mn2+ doped CsPbCl3 NCs, these Mn2+/Yb3+ codoped CsPbCl3 NCs show triple-wavelength emission from the CsPbCl3 host and Mn2+/Yb3+ dopant ions [Fig. 12(b)], and achieve a total PLQY of ∼125.3% due to quantum cutting effects. The energy trans- fer mechanism is shown in Fig. 12(c). Specifically, the emission form CsPbCl3 host (abbreviated as BG-PL) is originated from partial exci- ton recombination at the band edge. E. Reabsorption loss (g) PL spectra (solid lines with shading) of undoped (purple) and Yb3+-doped (dark red) CsPbCl3 NCs excited with a 365 nm light source. The inset shows their absorption spectra.101 Reproduced with permission from Luo et al., Nano Lett. 19, 338–341 (2019). Copyright 2018 American Chemical Society. 24 October 2024 05:44:56 FIG. 12. (a) Absorption (black line) and PL spectra of Mn2+-doped CsPbCl3 NCs (4% doping level) under 390 nm excitation in toluene solution.56 Reproduced with permission from Meinardi et al., ACS Energy Lett. 2, 2368–2377 (2017). Copyright 2017 American Chemical Society. (b) Absorption (purple) and PL (blue, orange, and red) spectra of the undoped and Mn2+/Yb3+ codoped CsPbCl3 perovskite NCs. Insets: schematics of the perovskite crystal structures and photographs of the samples under UV illumination (365 nm) taken by a visible-camera and a NIR camera with an 800 nm long-path filter. (c) Schematic of the proposed energy transfer (ET) processes in the Mn2+/Yb3+ codoped CsPbCl3 NCs.100 Cai et al., Adv. Sci. 7, 2001317 (2020). Copyright 2020 Author(s), licensed under a Creative Commons Attribution 4.0 License. (g) PL spectra (solid lines with shading) of undoped (purple) and Yb3+-doped (dark red) CsPbCl3 NCs excited with a 365 nm light source. The inset shows their absorption spectra.101 Reproduced with permission from Luo et al., Nano Lett. 19, 338–341 (2019). Copyright 2018 American Chemical Society. APL Photon. 6, 120901 (2021); doi: 10.1063/5.0067920 APL Photon. 6, 120901 (2021); doi: 10.1063/5.0067920 © Author(s) 2021 6, 120901-15 6, 120901-15 APL Photonics PERSPECTIVE scitation.org/journal/app alleviate climate problems caused by excessive consumption of fos- sil energy and plays an important role in self-powered buildings. In view of the excellent solution processing properties of perovskite luminophores, perovskite-based LSCs can be processed with simple facilities at low cost. results in the large Stokes shift. Figure 11(c) shows the photographs of the Cs4PbBr6 NC-based LSCs under ambient and one sun (100 mW/cm2) illumination. Such LSCs with large Stokes shift exhibit significantly suppressed reabsorption loss compared with traditional CsPbBr3 NCs-based devices, and significantly reduced PL loss can be observed at different optical distances [Fig. 11(d)]. We review the application of perovskite materials in LSCs and introduce the preparation methods of perovskite-based LSCs. Three commonly used methods for preparing perovskite-based LSCs are introduced: (1) The overall curing type, where the pre-synthesized perovskite luminophores are mixed with monomers and a UV initia- tor and then cured after being irradiated by UV light. 24 October 2024 05:44:56 l As for the pure Yb3+ doped CsPbCl3 NCs, LSCs based on such NCs exhibit an ultra-high PLQY of 164% ± 7% due to quantum cutting and zero self-absorption loss.101 As shown in Fig. 12(g), for the doped sample, due to the energy transfer from the CsPbCl3 host to Yb3+-dopants, the emission of the CsPbCl3 host is strongly quenched while the strong 2F5/2 →2F7/2 f−f emission originated from Yb3+ can be observed. Such Yb3+-doped CsPbCl3 NCs achieved an internal optical efficiency of 118.1% ± 6.7% which shows application prospects in terms of suppressing reabsorption and non-radiative recombination loss. These latest achievements in preparation methods and suppres- sion of optical loss have promoted the development of LSCs and pro- vide new ideas for the preparation of high-performance LSCs, which reduces the obstacles to the commercialization of LSCs and quickly emerges as a promising technology to support future sustainable development. Perovskite-based LSCs hold great potential for the application of energy harvesting systems for building applications due to their low manufacturing and installation costs and good urban environ- mental compatibility. They are complementary to existing photo- voltaic facilities. There are challenges remaining to be addressed and new opportunities are emerging for the research, application, and commercialization of perovskite LSCs. E. Reabsorption loss For the emission of Mn2+, excited electrons can transfer the energy to the excited state (4T1g) of the Mn2+ dopants through the Dexter-type energy transfer process. For the mission of Yb3+, energy transfer first occurs from excitons of the CsPbCl3 host to the localized defects and then to the neighboring Yb3+ ions, followed by a quantum cutting process. The direct energy transfer from 4T1g of Mn2+ to 2F5/2 of Yb3+ may also contribute to the Yb3+ emission. Figures 12(d) and 12(e) show the photographs of the LSC device under UV illumination (365 nm) taken by a visible camera and a near-infrared (NIR)-camera with an 800 nm long-path filter, and Fig. 12(f) shows the bent LSC based on these Mn2+/Yb3+ codoped CsPbCl3 NCs, which shows potential advantages in highly flexible smart windows. i We also review several optical loss suppression schemes in order to provide ideas for the preparation of high-performance LSCs. For the non-radiative recombination loss, surface passiva- tion via cationic/anionic ligands or additives with specific functional groups, such as –NH2 and P = O, is one of the most effective ways to improve the PLQY and stability of the perovskite luminophores. For the scattering loss, it is feasible to suppress the internal waveguide scattering loss by adjusting the emission dipole of the luminophores. For the transmission loss, a tandem LSC is an effective way to increase the utilization rate of incident sunlight. For the escape cone loss, both the three-dimensional macroporous photonic crystal and distributed Bragg reflector can effectively reflect the emission of luminophores within the specific wavelength range so as to sup- press the escape cone loss. For the reabsorption loss, ensembles of two-dimensional perovskite domains with ultrafast energy transfer process are used as the luminophores to suppress the escape cone loss. 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CONCLUSIONS AND PERSPECTIVES As an important supplement to photovoltaic devices, LSC with low cost and convenient preparation is one of the effective ways to APL Photon. 6, 120901 (2021); doi: 10.1063/5.0067920 © Author(s) 2021 APL Photon. 6, 120901 (2021); doi: 10.1063/5.0067920 6, 120901-16 APL Photonics PERSPECTIVE scitation.org/journal/app First, the toxicity of lead halide perovskites needs to be addressed. The heavy metal ions in lead halide perovskites may severely pollute the environment and/or threat human health. Ade- quate and feasible recycling and/or encapsulation strategies are urgently needed to be developed before large scale deployment of lead halide perovskite materials and LSCs. 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https://openalex.org/W4307080405	https://escholarship.org/content/qt9jc673zv/qt9jc673zv.pdf?t=o4e8fj	English	null	Search for direct production of charginos and neutralinos in events with three leptons and missing transverse momentum in <math xmlns="http://www.w3.org/1998/Math/MathML"> <msqrt> <mi>s</mi> </msqrt> <mo>=</mo> </math> 8TeV <math xmlns="http://www.w3.org/1998/Math/MathML"> <mi>p</mi> <mi>p</mi> </math> collisions with the ATLAS detector	OSTI OAI (U.S. Department of Energy Office of Scientific and Technical Information)	2,014	cc-by	23,570	Title Title Search for direct production of charginos and neutralinos in events with three leptons and missing transverse momentum in s=7 TeV pp collisions with the ATLAS detector Permalink Permalink https://escholarship.org/uc/item/9jc673zv Journal Physics Letters B, 718(3) ISSN 0370-2693 Authors Collaboration, ATLAS Aad, G Abajyan, T et al. Publication Date 2013 DOI 10.1016/j.physletb.2012.11.039 Copyright Information This work is made available under the terms of a Creative Commons Attribution License, availalbe at https://creativecommons.org/licenses/by/4.0/ Peer reviewed https://escholarship.org/uc/item/9jc673zv Journal Physics Letters B, 718(3) ISSN 0370-2693 Authors Collaboration, ATLAS Aad, G Abajyan, T et al. Publication Date 2013 DOI 10.1016/j.physletb.2012.11.039 Copyright Information This work is made available under the terms of a Creative Commons Attribution License, availalbe at https://creativecommons.org/licenses/by/4.0/ Copyright Information This work is made available under the terms of a Creative Commons Attribution License, availalbe at https://creativecommons.org/licenses/by/4.0/ UC Santa Cruz UC Santa Cruz UC Santa Cruz Previously Published Works Title Search for direct production of charginos and neutralinos in events with three leptons and missing transverse momentum in s=7 TeV pp collisions with the ATLAS detector 1 ATLAS uses a right-handed coordinate system with its origin at the nominal interaction point (IP) in the centre of the detector and the z-axis along the beam pipe. The x-axis points from the IP to the centre of the LHC ring, and the y-axis points upward. Cylindrical coordinates (r,φ) are used in the transverse plane, φ being the azimuthal angle around the beam pipe. The pseudorapidity is deﬁned in terms of the polar angle θ as η = −ln tan(θ/2). a b s t r a c t Article history: Received 15 August 2012 Received in revised form 5 November 2012 Accepted 13 November 2012 Available online 19 November 2012 Editor: H. Weerts A search for the direct production of charginos and neutralinos in ﬁnal states with three electrons or muons and missing transverse momentum is presented. The analysis is based on 4.7 fb−1 of √ s = 7 TeV proton–proton collision data delivered by the Large Hadron Collider and recorded with the ATLAS detector. Observations are consistent with Standard Model expectations in three signal regions that are either depleted or enriched in Z-boson decays. Upper limits at 95% conﬁdence level are set in R-parity conserving phenomenological minimal supersymmetric models and in simpliﬁed models, signiﬁcantly extending previous results. © 2012 CERN. Published by Elsevier B.V. Open access under CC BY-NC-ND license. 2. Detector description ATLAS [22] is a multipurpose particle detector with forward- backward symmetric cylindrical geometry. It includes an inner tracker (ID) immersed in a 2 T magnetic ﬁeld providing preci- sion tracking of charged particles for pseudorapidities \|η\| < 2.5.1 Calorimeter systems with either liquid argon or scintillating tiles as the active media provide energy measurements over the range \|η\| < 4.9. The muon detectors are positioned outside the calorime- ters and are contained in an air-core toroidal magnetic ﬁeld pro- duced by superconducting magnets with ﬁeld integrals varying from 1 T m to 8 T m. They provide trigger and high-precision track- ing capabilities for \|η\| < 2.4 and \|η\| < 2.7, respectively. Search for direct production of charginos and neutralinos in events with three leptons and missing transverse momentum in √ s = 7 TeV pp collisions with the ATLAS detector ✩ Search for direct production of charginos and neutralinos in events with three leptons and missing transverse momentum in √ s = 7 TeV pp collisions with the ATLAS detector ✩ .ATLAS Collaboration ⋆ 1. Introduction search described here signiﬁcantly extends the current mass limits on charginos and neutralinos set by ATLAS [17,18]. Similar searches have been conducted at the Tevatron [19,20] and LEP [21], where a model-independent lower limit of 103.5 GeV was set at 95% con- ﬁdence level (CL) on the mass of promptly decaying charginos. Supersymmetry (SUSY) [1–9] postulates the existence of SUSY particles, or “sparticles”, with spin differing by one-half unit with respect to that of their Standard Model (SM) partner. If R- parity [10–14] is conserved, the lightest SUSY particle (LSP) is stable and sparticles can only be pair-produced and decay into ﬁ- nal states with SM particles and LSPs. Charginos ( ˜χ± i , i = 1, 2) and neutralinos ( ˜χ 0 j , j = 1, 2, 3, 4) are the mass eigenstates formed from the linear superposition of the SUSY partners of the Higgs and electroweak gauge bosons. These are the Higgsinos, and the winos, zino, and bino, collectively known as gauginos. Natural- ness requires ˜χ± i and ˜χ 0 j (and third-generation sparticles) to have masses in the hundreds of GeV range [15,16]. In scenarios where squark and gluino masses are larger than a few TeV, the direct production of gauginos may be the dominant SUSY process at the Large Hadron Collider (LHC). Charginos can decay into leptonic ﬁ- nal states via sneutrinos (˜νℓ), sleptons ( ˜ℓν) or W bosons (W ˜χ 0 1 ), while unstable neutralinos can decay via sleptons (ℓ˜ℓ) or Z bosons (Z ˜χ 0 1 ). ⋆E-mail address: atlas.publications@cern.ch. be ter 0370-2693/ © 2012 CERN. Published by Elsevier B.V. http://dx.doi.org/10.1016/j.physletb.2012.11.039 Open access under CC BY-NC-ND license. Contents lists available at SciVerse ScienceDirect Contents lists available at SciVerse ScienceDirect Powered by the California Digital Library University of California eScholarship.org Physics Letters B 718 (2013) 841–859 Contents lists available at SciVerse ScienceDirect 0370-2693/ © 2012 CERN. Published by Elsevier B.V. http://dx.doi.org/10.1016/j.physletb.2012.11.039 Open access under CC BY-NC-ND license. 4. Monte Carlo simulation Several Monte Carlo (MC) generators are used to simulate SM processes and new physics signals relevant for this analy- sis. SHERPA [25] is used to simulate diboson processes W Z and Z Z. These include all diagrams leading to three leptons and one neutrino, and to four leptons, respectively, including internal con- versions (virtual photons converting into lepton pairs). HERWIG [26] is used for W W , while MadGraph [27] is used for the t¯tW , t¯tW W , t¯t Z, Wγ and Zγ processes. MC@NLO [28] is cho- sen for the simulation of single- and pair-production of top- quarks, and ALPGEN [29] is used to simulate W /Z + jets. Ex- pected diboson yields are normalised using next-to-leading-order (NLO) QCD predictions obtained with MCFM [30,31]. The top-quark pair-production contribution is normalised to approximate next-to- next-to-leading-order calculations (NNLO) [32] and the t¯tW (W )/Z contributions are normalised to NLO [33,34]. The Wγ and Zγ yields are normalised to be consistent with the ATLAS cross-section measurement [35]. The QCD NNLO FEWZ [36,37] cross-sections are used for normalisation of the inclusive W + light-ﬂavour jets and Z + light-ﬂavour jets. The ratio of the NNLO to LO cross-section is used to rescale the W + heavy-ﬂavour jets and Z + heavy-ﬂavour jets LO cross-sections. Electrons must satisfy “tight” identiﬁcation criteria [47] and ful- ﬁl \|η\| < 2.47 and ET > 10 GeV, where ET and \|η\| are determined from the calibrated clustered energy deposits in the electromag- netic calorimeter and the matched ID track respectively. Muons are reconstructed by combining tracks in the ID and tracks in the muon spectrometer [48]. Reconstructed muons are considered as candidates if they have transverse momentum pT > 10 GeV and \|η\| < 2.4. In this analysis “tagged” leptons are deﬁned for evaluating the background, as described below in Section 7.1. Tagged leptons are leptons separated from each other and from candidate jets as described below. If two candidate electrons are reconstructed with R ≡ (φ)2 + (η)2 < 0.1, the lower energy one is dis- carded. Candidate jets within R = 0.2 of an electron candidate are rejected. To suppress leptons originating from semi-leptonic decays of c- and b-quarks, all lepton candidates are required to be separated from candidate jets by R > 0.4. Muons undergoing bremsstrahlung can be reconstructed with an overlapping electron. 5. Event reconstruction and preselection The data sample was collected with an inclusive selection of single-lepton and double-lepton triggers. If the event is selected by the single-lepton triggers, at least one reconstructed muon (electron) is requested to have transverse momentum pμ T (trans- verse energy Ee T) above 20 GeV (25 GeV). For di-lepton triggers, at least two leptons are required to be present in the event with transverse energy or momentum above threshold. The two muons are required to have pμ T > 12 GeV for di-muon triggers, and the two electrons to have Ee T > 17 GeV for di-electron triggers, while the thresholds for electron–muon triggers are Ee T > 15 GeV and pμ T > 10 GeV. These thresholds on the reconstructed transverse momenta of leptons are higher than those applied by the online trigger selection, and are chosen such that the trigger eﬃciency is high, typically between 90 and 99%, and independent of the trans- verse momentum of the triggerable objects within uncertainties. In the simpliﬁed models considered, the masses of the relevant particles ( ˜χ± 1 , ˜χ 0 2 , ˜χ 0 1 , ˜ν, ˜ℓL) are the only free parameters. The charginos and heavy neutralinos are set to be wino-like and mass degenerate, and the lightest neutralino is set to be bino-like. Two different scenarios are considered. In the ﬁrst case, the ˜χ± 1 and ˜χ 0 2 are pair-produced and decay via left-handed sleptons, including staus, and sneutrinos of mass m˜ν = m ˜ℓL = (m ˜χ 0 1 + m ˜χ± 1 )/2 with a branching ratio of 50% each. In the second scenario, the ˜χ± 1 and ˜χ 0 2 decay via W and Z bosons. Events recorded during normal running conditions are analysed if the primary vertex has ﬁve or more tracks associated to it. The primary vertex of an event is identiﬁed as the vertex with the highest Σ p2 T of associated tracks. 3. New physics scenarios 1 This Letter presents a search with the ATLAS detector for the direct production of charginos and neutralinos decaying to a ﬁ- nal state with three leptons (electrons or muons) and missing transverse momentum, the latter originating from the two unde- tected LSPs and the neutrinos. The analysis is based on 4.7 fb−1 of proton–proton collision data delivered by the LHC at a centre- of-mass energy √ s = 7 TeV between March and October 2011. The In this analysis, results are interpreted in the phenomenolog- ical minimal supersymmetric SM (pMSSM [23]) and in simpliﬁed models [24]. ATLAS Collaboration / Physics Letters B 718 (2013) 841–859 842 In the pMSSM the mixing for the ˜χ± i and ˜χ 0 j depends on the gaugino masses M1 and M2, the Higgs mass parameter μ, and tanβ, the ratio of the expectation values of the two Higgs doublets. The dominant mode for gaugino production leading to three-lepton ﬁnal states is ˜χ± 1 ˜χ 0 2 production via the s-channel ex- change of a virtual gauge boson. Other ˜χ± i ˜χ 0 j processes contribute a maximum of 20% to three-lepton ﬁnal states depending on the values of the mass parameters. The right-handed sleptons (includ- ing third-generation sleptons) are assumed to be degenerate and have a mass m ˜ℓR = (m ˜χ 0 2 + m ˜χ 0 1 )/2, set via the right-handed SUSY- breaking slepton mass parameter at the electroweak scale. In these scenarios, decays to sleptons are favoured. The parameter tanβ is set to 6, yielding comparable branching ratios into each slepton generation. The masses of the gluinos, squarks and left-handed sleptons are chosen to be larger than 2 TeV. In order to achieve maximum mixing in the top-squark sector the corresponding tri- linear couplings are set to non-zero values, while all other trilinear couplings are set to zero. interfaced to HERWIG for simulating the underlying event. For all MC samples, the propagation of particles through the ATLAS de- tector is modelled using GEANT4 [45,46]. The effect of multiple proton–proton collisions from the same or different bunch cross- ings is incorporated into the simulation by overlaying additional minimum bias events onto hard-scatter events using PYTHIA. Simulated events are weighted to match the distribution of the number of interactions per bunch crossing observed in data (pile- up). 6. Signal region selection Selected events must contain exactly three signal leptons. As R-parity conserving leptonic decays of ˜χ 0 j yield same-ﬂavour opposite-sign (SFOS) lepton pairs, the presence of at least one such pair is required. The invariant mass of any SFOS lepton pair must be above 20 GeV to suppress background from low-mass resonances and the missing transverse momentum must satisfy Emiss T > 75 GeV. p The identiﬁcation eﬃciency is measured in data using lepton candidates from Z →ℓℓdecays. Misidentiﬁcation probabilities for each relevant fake type (heavy ﬂavour or conversion) and for each reducible background process, parameterised with the lepton pT and η, are obtained using simulated events with one signal and two tagged leptons. These misidentiﬁcation probabilities are then corrected using the ratio (fake scale factor) of the misidentiﬁcation probability in data to that in simulation obtained from dedicated control samples. For heavy-ﬂavour fakes, the correction factor is measured in a b¯b-dominated control sample. This is deﬁned by se- lecting events with only one b-tagged jet (containing a muon) and a tagged lepton, for which the fake rate is measured. The non- b¯b background includes top-quark pair-production and W bosons produced in association with a b-quark. An Emiss T requirement of less than 40 GeV suppresses both the t¯t and the W contami- nation, while requiring mT < 40 GeV reduces the W background. The remaining (small) background is subtracted from data using MC predictions. The fake scale factor for the conversion candi- dates is determined in a sample of photons radiated from a muon in Z →μμ decays. These are selected by requiring mμμe to lie within 10 GeV of the nominal Z-boson mass value. A weighted av- erage misidentiﬁcation probability is then calculated by weighting the corrected type- and process-dependent misidentiﬁcation prob- abilities according to the relative contributions in a given signal or validation region, deﬁned below. T Three signal regions are then deﬁned: two “Z-depleted” re- gions (SR1a and SR1b), with no SFOS pairs having invariant mass within 10 GeV of the nominal Z-boson mass; and a “Z-enriched” one (SR2), where at least one SFOS pair has an invariant mass within 10 GeV of the Z-boson mass. Events in SR1a and SR1b are further required to contain no b-tagged jets to suppress con- tributions from b-jet-rich background processes, where a lepton could originate from the decay of a heavy-ﬂavor quark. Table 1 Table 1 The selection requirements for the three signal regions. The Z-veto (Z-requirement) rejects (selects) events with mSFOS within 10 GeV of the Z mass (91.2 GeV). The mT is calculated from the Emiss T and the lepton not forming the best Z candidate. Jets are reconstructed using the anti-kt algorithm [49] with a radius parameter of R = 0.4 using clustered energy deposits cal- ibrated at the electromagnetic scale. The jet energy is corrected to account for pile-up and for the non-compensating nature of the calorimeter using correction factors parameterised as a func- tion of the jet ET and η [50]. The correction factors applied to jets have been obtained from simulation and have been tuned and validated using data. Jets considered in this analysis have ET > 20 GeV, \|η\| < 2.5 and a fraction of the jet’s track transverse mo- menta that can be associated with the primary vertex greater than 0.75. Events containing jets failing the quality criteria described in Ref. [50] are rejected to suppress both SM and beam-induced back- ground. Jets are identiﬁed as containing b-hadron decays, and thus called “b-tagged”, using a multivariate technique based on quan- tities such as the impact parameters of the tracks associated to a reconstructed secondary vertex. The b-tagging algorithm [51] cor- rectly identiﬁes b-quark jets in simulated top-quark decays with an eﬃciency of 60% and misidentiﬁes jets containing light-ﬂavour quarks and gluons with a rate of < 1%, for jets with \|η\| < 2.5 and jet ET > 20 GeV. Selection SR1a SR1b SR2 Targeted intermediate decay ˜l(∗) or Z∗ on-shell Z N leptons (e, μ) Exactly 3 Lepton charge, ﬂavour At least one SFOS pair with mℓℓ> 20 GeV Emiss T > 75 GeV mSFOS Z-veto Z-veto Z-requirement N b-jets 0 0 any mT any > 90 GeV > 90 GeV pT all ℓ > 10 GeV > 30 GeV > 10 GeV or an electron from an isolated photon conversion. The contribu- tion from misidentiﬁed light-ﬂavour quark or gluon jets is negligi- ble in the signal regions. The reducible background includes single- and pair-production of top-quarks and W W or W /Z produced in association with jets or photons. The dominant component is the production of top-quarks, with a contribution of 1% or less from Z + jets. The reducible background is estimated using a “matrix method” similar to that described in Ref. [53]. Table 1 In this implementation of the matrix method, the signal lepton with the highest pT or ET is taken to be real, which is a valid assumption in 99% of the cases, based on simulation. The number of observed events with one or two fakes is then extracted from a system of linear equations relating the number of events with two additional signal or tagged candidates to the number of events with two additional candidates that are either real or fake. The coeﬃcients of the linear equations are functions of the real-lepton identiﬁcation eﬃciencies and of the fake-object misidentiﬁcation probabilities. The missing transverse momentum, Emiss T , is the magnitude of the vector sum of the transverse momentum or transverse energy of all pT > 10 GeV muons, ET > 20 GeV electrons, ET > 20 GeV jets, and calibrated calorimeter clusters with \|η\| < 4.9 not associ- ated to these objects [52]. 6. Signal region selection SR1b is designed to increase sensitivity to scenarios characterised by large mass splittings between the heavy gauginos and the LSP by requir- ing all three leptons to have pT > 30 GeV. In both SR1b and SR2, the transverse mass variable mT must take values greater than 90 GeV, where mT is constructed using the Emiss T and the lepton not included in the lepton pair with invariant mass closest to the nom- inal Z-boson mass. The mT requirement is introduced to suppress background from W Z events. The SR1a/b regions target neutralino decays via intermediate sleptons or via off-shell Z bosons while SR2 targets decays via an on-shell Z boson. Table 1 summarises the selection requirements for the three signal regions. 4. Monte Carlo simulation To reject these, tagged electrons and muons separated from jets and reconstructed within R = 0.1 of each other are both dis- carded. Events containing one or more tagged muons that have transverse impact parameter with respect to the primary vertex \|d0\| > 0.2 mm or longitudinal impact parameter with respect to the primary vertex \|z0\| > 1 mm are rejected to suppress cosmic muon background. The choice of the parton distribution functions (PDFs) depends on the generator. The CTEQ6L1 [38] PDFs are used with Mad- Graph and ALPGEN, and the CT10 [39] PDFs with MC@NLO and SHERPA. The MRTSmcal PDF set [40] is used for HERWIG. The pMSSM samples are produced with HERWIG and the sim- pliﬁed model samples with Herwig++ [41]. The yields of the SUSY samples are normalised to the NLO cross-sections obtained from PROSPINO [42] using the PDF set CTEQ6.6 with the renor- malisation/factorisation scales set to the average of the relevant gaugino masses. “Signal leptons” are tagged leptons for which the scalar sum of the transverse momenta of tracks within a cone of R ≡ (φ)2 + (η)2 = 0.2 around the lepton candidate, and exclud- ing the lepton candidate track itself, is less than 10% of the lepton ET for electrons and less than 1.8 GeV for muons. Tracks selected for the electron and muon isolation requirement, deﬁned above, have pT > 1 GeV and are associated to the primary vertex of the event. To suppress leptons originating from secondary vertices, the distance of closest approach of the lepton track to the primary Fragmentation and hadronisation for the ALPGEN and MC@NLO (MadGraph) samples are performed with HERWIG (PYTHIA [43]), while for SHERPA, these are performed internally. JIMMY [44] is ATLAS Collaboration / Physics Letters B 718 (2013) 841–859 843 vertex normalised to its uncertainty is required to be small, with \|d0\|/σ(d0) < 6(3) for electrons (muons). 7.2. Irreducible background processes 7.1. Reducible background processes 9. Systematic uncertainties Several sources of systematic uncertainty are considered in the signal, control and validation regions. The systematic uncertain- ties affecting the simulation-based estimates (the yield of the ir- reducible background, the cross-section weighted misidentiﬁcation probabilities, the signal yield) include the theoretical cross-section uncertainties due to renormalisation and factorisation scale and PDFs, the acceptance uncertainty due to PDFs, the uncertainty on the luminosity, the uncertainty due to the jet energy scale, jet energy resolution, lepton energy scale, lepton energy resolution, lepton eﬃciency, b-tagging eﬃciency, mistag probability, and the choice of MC generator. In SR1a, the total uncertainty on the irre- ducible background is 24%. This is dominated by the uncertainty In all of the above, the value used for the uncertainty on the luminosity is 3.9% [57,58]. Correlations of systematic uncertainties between processes and regions are accounted for. 8. Background model validation The background predictions have been tested in various vali- dation regions. A region (VR1) dominated by Drell–Yan and W Z events is selected by requiring three signal leptons, at least one SFOS lepton pair, 30 GeV < Emiss T < 75 GeV, and a Z-boson veto. A reducible-background dominated region (VR2, where top-quark pair-production and decay to two real and one fake lepton is the main contribution) is built by requiring three signal leptons, Emiss T > 50 GeV and by vetoing SFOS lepton pairs. Finally, a W Z- dominated region (VR3) is deﬁned by selecting events with three signal leptons, at least one SFOS lepton pair, a Z candidate, and 50 GeV < Emiss T < 75 GeV. The data and predictions are in agree- ment within the quoted statistical and systematic uncertainties as shown in Table 2. The total uncertainties on the signal yields are 10–20%, where the largest contribution is from the uncertainty on the cross- sections (7%). Signal cross-sections are calculated to NLO in the strong coupling constant using PROSPINO. An envelope of cross- section predictions is deﬁned using the 68% CL ranges of the CTEQ6.6 [54] (including the αS uncertainty) and the MSTW [55] PDF sets, together with variations of the factorisation and renor- malisation scales by factors of two or one half. The nominal cross- section value is taken to be the midpoint of the envelope and the uncertainty assigned is half the full width of the envelope, follow- ing the PDF4LHC recommendations [56]. Table 2 on the eﬃciency of the signal region selection for the W Z gen- erator, determined by comparing the nominal yield with that ob- tained with the HERWIG generator and found to be 20%. The next largest uncertainties are the uncertainty due to the MC generator (16%) and that on the cross-sections (9%) of the non-W Z back- ground. The MC generator uncertainty partially accounts for the cross-section uncertainty, leading to a slight overestimate of the overall uncertainty. All the remaining uncertainties on the irre- ducible background in this signal region range between 0.5 and 5%. The total uncertainty on the irreducible background in SR1b is slightly larger, at 25%, due to the limited number of simulated events. In SR2, the uncertainty on the irreducible background is 24%, with increased contributions from the jet energy scale and resolution and cross-section uncertainties. The uncertainty on the reducible background includes the MC uncertainty on the weights for the misidentiﬁcation probabilities from the sources listed above (up to 10%) and the uncertainty due to the dependence of the misidentiﬁcation probability on Emiss T (0.6–15%). Also included in the uncertainty on the reducible back- ground is the uncertainty on the fake scale factors (10–34%), and that due to the limited number of data events with three tagged leptons, of which at least one is a signal lepton (19–130%). The lat- ter uncertainty is highest in SR2 where the reducible background is very low. Table 2 Table 2 Expected numbers of events from SM backgrounds and observed numbers of events in data, for 4.7 fb−1, in validation regions VR1, VR2 and VR3. Both statistical and systematic uncertainties are included. Expected numbers of events from SM backgrounds and observed numbers of events in data, for 4.7 fb−1, in validation regions VR1, VR2 and VR3. Both statistical and systematic uncertainties are included. Expected numbers of events from SM backgrounds and observed numbers of events in data, for 4.7 fb−1, in signal regions SR1a, SR1b and SR2. The yield for two of the simpliﬁed model scenarios, “SUSY ref. point 1” with intermediate sleptons, (m ˜χ± 1 ,m ˜χ 0 2 ,m ˜ℓL , m ˜χ 0 1 = 425, 425, 250, 75 GeV) and “SUSY ref. point 2” with no in- termediate sleptons, (m ˜χ± 1 , m ˜χ 0 2 , m ˜χ 0 1 = 150, 150, 0 GeV) are also presented. Both statistical and systematic uncertainties are included. Upper limits on the observed and expected visible production cross-section at 95% CL are also shown. Selection VR1 VR2 VR3 t¯t Z 0.17 ± 0.14 0.12 ± 0.10 1.1 ± 0.9 t¯tW 0.6 ± 0.5 0.7 ± 0.5 0.10 ± 0.08 t¯tW W 0.017 ± 0.014 0.022 ± 0.017 0.0023 ± 0.0019 Z Z 17 ± 15 0.10 ± 0.05 3.9 ± 0.6 W Z 46 ± 8 0.93 ± 0.29 98 ± 12 Reducible Bkg. 50 ± 28 13 ± 7 3.1+4.7 −3.1 Total Bkg. 114 ± 32 15 ± 7 106 ± 13 Data 126 18 109 Selection SR1a SR1b SR2 t¯t Z 0.06 ± 0.05 0.025 ± 0.023 0.6 ± 0.5 t¯tW 0.36 ± 0.29 0.10 ± 0.08 0.09 ± 0.08 t¯tW W 0.010 ± 0.008 0.0023 ± 0.0019 0.004 ± 0.004 Z Z 0.67 ± 0.21 0.09 ± 0.08 0.34 ± 0.17 W Z 13.5 ± 2.9 1.05 ± 0.28 9.3 ± 2.1 Reducible Bkg. 10 ± 5 0.35 ± 0.34 0.5+1.0 −0.5 Total Bkg. 25 ± 6 1.6 ± 0.5 10.9 ± 2.4 Data 24 0 11 SUSY ref. point 1 8.0 ± 0.8 6.5 ± 0.6 0.46 ± 0.05 SUSY ref. 7.1. Reducible background processes A background process is considered “irreducible” if it leads to events with three real and isolated leptons, referred to as “real” leptons below. Such processes include diboson (W Z and Z Z) and t¯tW /Z production, where the gauge boson may be produced off- mass-shell. The Z Z and t¯tW /Z contribution is determined using Several SM processes contribute to the background in the signal regions. A “reducible” process has at least one “fake” object, that is either a lepton from a semileptonic decay of a heavy-ﬂavour quark ATLAS Collaboration / Physics Letters B 718 (2013) 841–859 844 Table 2 point 2 1.03 ± 0.19 0.21 ± 0.09 10.9 ± 1.0 Visible σ (exp) < 3.0 fb < 0.8 fb < 2.0 fb Visible σ (obs) < 3.0 fb < 0.7 fb < 2.0 fb the corresponding MC samples, for which lepton and jet selection eﬃciencies are corrected to account for differences with respect to data. The largest irreducible background, W Z, is determined using a semi-data-driven approach. The W Z background is ﬁt to data in a control region including events with exactly three leptons, one SFOS lepton pair, a Z candidate, Emiss T < 50 GeV, a b-veto, and mT > 40 GeV. The W Z purity in the control region is ∼80%. Non-W Z backgrounds, both irreducible and reducible, are deter- mined based on simulation or by using the matrix method and subtracted. A W Z normalisation factor 1.25 ± 0.12 is obtained in the control region under a background-only hypothesis and used to estimate the W Z background in the validation regions. To obtain the model-independent 95% CL upper limit on the new phenom- ena cross-section, a ﬁt is performed simultaneously in the W Z control region and in the signal region, with ﬂoating W Z normal- isation factor and a non-negative signal in the signal region only. This allows the propagation of the uncertainties on the normalisa- tion factor. When setting limits on speciﬁc new physics scenarios, the potential signal contamination in the W Z control region is ac- counted for in the simultaneous ﬁt. on the eﬃciency of the signal region selection for the W Z gen- erator, determined by comparing the nominal yield with that ob- tained with the HERWIG generator and found to be 20%. The next largest uncertainties are the uncertainty due to the MC generator (16%) and that on the cross-sections (9%) of the non-W Z back- ground. The MC generator uncertainty partially accounts for the cross-section uncertainty, leading to a slight overestimate of the overall uncertainty. All the remaining uncertainties on the irre- ducible background in this signal region range between 0.5 and 5%. The total uncertainty on the irreducible background in SR1b is slightly larger, at 25%, due to the limited number of simulated events. In SR2, the uncertainty on the irreducible background is 24%, with increased contributions from the jet energy scale and resolution and cross-section uncertainties. 10. Results and interpretation The yields for two of the simpliﬁed model scenarios are also shown for illustration purposes: one with intermediate sleptons “SUSY ref. point 1” (m ˜χ± 1 , m ˜χ 0 2 , m ˜ℓL , m ˜χ 0 1 = 425, 425, 250, 75 GeV) and a second with no sleptons “SUSY ref. point 2” (m ˜χ± 1 , m ˜χ 0 2 , m ˜χ 0 1 = 150, 150, 0 GeV). The signal distribution is not stacked on top of the expected background. No signiﬁcant excess of events is found in any of the three sig- nal regions. Upper limits on the visible cross-section, deﬁned as Fig. 1. Emiss T distributions for events in signal regions SR1a (a) and SR2 (b). The uncertainty band includes both statistical and systematic uncertainty, while the un- certainties on the data points are statistical only. The yields for two of the simpliﬁed model scenarios are also shown for illustration purposes: one with intermediate sleptons “SUSY ref. point 1” (m ˜χ± 1 , m ˜χ 0 2 , m ˜ℓL , m ˜χ 0 1 = 425, 425, 250, 75 GeV) and a second with no sleptons “SUSY ref. point 2” (m ˜χ± 1 , m ˜χ 0 2 , m ˜χ 0 1 = 150, 150, 0 GeV). The signal distribution is not stacked on top of the expected background. No signiﬁcant excess of events is found in any of the three sig- nal regions. Upper limits on the visible cross-section, deﬁned as the production cross-section times acceptance times eﬃciency, of 3.0 fb in SR1a, 0.7 fb in SR1b and 2.0 fb in SR2 are placed at 95% CL with the modiﬁed frequentist CLs prescription [59]. All systematic uncertainties and their correlations are taken into account via nui- sance parameters in a proﬁle likelihood ﬁt [60]. The corresponding expected limits are 3.0 fb, 0.8 fb and 2.0 fb, respectively. p p y SR1a and SR1b provide the best sensitivity for the pMSSM scenarios; in particular SR1a (SR1b) targets scenarios with small (large) mass splitting between the heavy gauginos and the LSP. The limits are calculated using the signal region providing the best ex- pected limit for each of the model points. The uncertainties on the signal cross-section are not included in the limit calculation but their impact on the observed limit is shown. 10. Results and interpretation The numbers of observed events and the prediction for SM backgrounds in SR1a, SR1b and SR2 are given in Table 3. Distri- butions of the Emiss T in SR1a and SR2 are presented in Fig. 1. B 718 (2013) 841–859 8 2. Observed and expected 95% CL limit contours for chargino and neutrali uction in the pMSSM for M1 = 100 GeV (a), M1 = 140 GeV (b) and M1 GeV (c). The regions with low values of M2 and μ are the excluded ones for es of M1. The expected and observed limits are calculated without signal cro on uncertainty taken into account. The yellow band is the ±1σ experimen ertainty on the expected limit (black dashed line). The red dotted band is t signal theory uncertainty on the observed limit (red solid line). The LEP2 lim he ﬁgure corresponds to the limit on the ˜χ± 1 mass in [21] as transposed to t SM plane. Linear interpolation is used to account for the discreteness of t al grids. The exclusion contours are optimised by applying in each signal g t the CL values from the most sensitive signal region (lowest expected CL) = 100 GeV and 140 GeV, whereas signal region SR1a is used for M1 = 250 Ge ATLAS Collaboration / Physics Letters B 718 (2013) 841–859 845 Fig. 1. Emiss T distributions for events in signal regions SR1a (a) and SR2 (b). The uncertainty band includes both statistical and systematic uncertainty, while the un- certainties on the data points are statistical only. The yields for two of the simpliﬁed model scenarios are also shown for illustration purposes: one with intermediate sleptons “SUSY ref. point 1” (m ˜χ± 1 , m ˜χ 0 2 , m ˜ℓL , m ˜χ 0 1 = 425, 425, 250, 75 GeV) and a second with no sleptons “SUSY ref. point 2” (m ˜χ± 1 , m ˜χ 0 2 , m ˜χ 0 1 = 150, 150, 0 GeV). The signal distribution is not stacked on top of the expected background. No signiﬁcant excess of events is found in any of the three sig- nal regions. Upper limits on the visible cross-section, deﬁned as Fig. 1. Emiss T distributions for events in signal regions SR1a (a) and SR2 (b). The uncertainty band includes both statistical and systematic uncertainty, while the un- certainties on the data points are statistical only. 10. Results and interpretation The differen between expected and observed limits seen in the upper right c ner of the M1 = 100 GeV exclusion plot, where SR1b has the b sensitivity, is explained by the observed under-ﬂuctuation in d with respect to SM predictions. The value of tanβ does not ha a signiﬁcant impact on σ(pp →˜χ± i ˜χ 0 j ) × BR( ˜χ± i ˜χ 0 j →ℓν ˜χ 0 1 ℓℓ˜χ / y ( ) Fig. 4. Observed and expected 95% CL limit contours for chargino and neutralino production in the simpliﬁed model scenario with intermediate slepton decay (a) and intermediate gauge boson decay (b). The colour coding is the same as that in Fig. 2. For scenarios with intermediate slepton decay (with no intermediate slepton decay) the reference point is “SUSY ref. point 1” (“SUSY ref. point 2”). The “ATLAS 2.06 fb−1 3 leptons” contour corresponds to the result of the ATLAS search docu- mented in [18]. Fig. 4. Observed and expected 95% CL limit contours for chargino and neutralino production in the simpliﬁed model scenario with intermediate slepton decay (a) and intermediate gauge boson decay (b). The colour coding is the same as that in Fig. 2. For scenarios with intermediate slepton decay (with no intermediate slepton decay) the reference point is “SUSY ref. point 1” (“SUSY ref. point 2”). The “ATLAS 2.06 fb−1 3 leptons” contour corresponds to the result of the ATLAS search docu- mented in [18]. of the plane shown for M1 = 250 GeV (Fig. 2(c)), the condition that μ should be greater than M1 is not fulﬁlled and the resulting limits on the same plane become less stringent. Additionally, the reduced reach at high M2 and low μ for M1 = 140 GeV can be ex- plained in terms of smaller cross-section values and smaller mass splittings in that section of the parameter space. The difference between expected and observed limits seen in the upper right cor- ner of the M1 = 100 GeV exclusion plot, where SR1b has the best sensitivity, is explained by the observed under-ﬂuctuation in data with respect to SM predictions. The value of tanβ does not have a signiﬁcant impact on σ(pp →˜χ± i ˜χ 0 j ) × BR( ˜χ± i ˜χ 0 j →ℓν ˜χ 0 1 ℓℓ˜χ 0 1 ), which decreases by 10% if tanβ is raised from 6 to 10. Fig. 3. 10. Results and interpretation The exclusion limits for the pMSSM are shown in Fig. 2 as a function of the three pa- rameters M1, M2 and μ, where the regions with low values of M2 and μ are the excluded ones for all values of M1. In these plots, the main features can be explained in broad terms as follows. For a given value of M1, for example M1 = 100 GeV in Fig. 2(a), the production cross-section decreases as M2 and μ increase, which explains why limits become less stringent when both M2 and μ take high values. In general, the sensitivity is reduced in the region at low M2 and high μ, due to the small mass splitting between Fig. 2. Observed and expected 95% CL limit contours for chargino and neutralino production in the pMSSM for M1 = 100 GeV (a), M1 = 140 GeV (b) and M1 = 250 GeV (c). The regions with low values of M2 and μ are the excluded ones for all values of M1. The expected and observed limits are calculated without signal cross- section uncertainty taken into account. The yellow band is the ±1σ experimental uncertainty on the expected limit (black dashed line). The red dotted band is the ±1σ signal theory uncertainty on the observed limit (red solid line). The LEP2 limit in the ﬁgure corresponds to the limit on the ˜χ± 1 mass in [21] as transposed to this pMSSM plane. Linear interpolation is used to account for the discreteness of the signal grids. The exclusion contours are optimised by applying in each signal grid point the CL values from the most sensitive signal region (lowest expected CL) for M1 = 100 GeV and 140 GeV, whereas signal region SR1a is used for M1 = 250 GeV. (For interpretation of the references to colour in this ﬁgure legend, the reader is referred to the web version of this Letter.) 6 ATLAS Collaboration / g. 3. Observed and expected 95% CL limit contours for chargino and neutr oduction in the pMSSM for M1 = 100 GeV (a), M1 = 140 GeV (b) and M 0 GeV (c). Contours from the combination of the results from this search ose of the two-lepton ATLAS search in [61]. The various limits are as describ g. 2. The colour coding is the same as that in Fig. 2. 10. Results and interpretation e ˜χ 0 2 and the ˜χ 0 1 . When μ is greater than M1 and M2, whic ue for example in the rightmost part of the exclusion plots ATLAS Collaboration / Physics Letters B 718 (2013) 841–859 846 etters B 718 (2013) 841–859 Fig. 4. Observed and expected 95% CL limit contours for chargino and neutralino production in the simpliﬁed model scenario with intermediate slepton decay (a) and intermediate gauge boson decay (b). The colour coding is the same as that in Fig. 2. For scenarios with intermediate slepton decay (with no intermediate slepton decay) the reference point is “SUSY ref. point 1” (“SUSY ref. point 2”). The “ATLAS 2.06 fb−1 3 leptons” contour corresponds to the result of the ATLAS search docu- mented in [18]. 846 ATLAS Collaboration / Physics Letters B 718 (2013) 841 859 Fig. 3. Observed and expected 95% CL limit contours for chargino and neutralino production in the pMSSM for M1 = 100 GeV (a), M1 = 140 GeV (b) and M1 = 250 GeV (c). Contours from the combination of the results from this search with those of the two-lepton ATLAS search in [61]. The various limits are as described in Fig. 2. The colour coding is the same as that in Fig. 2. the ˜χ 0 2 and the ˜χ 0 1 . 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Jovicevic 147, T. Jovin 13b, X. Ju 173, C.A. Jung 43, R.M. Jungst 30, V. Juranek 125, P. Jussel 61, A. Juste Rozas 12, S. Kabana 17, M. Kaci 167, A. Kaczmarska 39, P. Kadlecik 36, M. Kado 115, H. Kagan 109, M. Kagan 57, E. Kajomovitz 152, S. Kalinin 175, L.V. Kalinovskaya 64, S. Kama 40, N. Kanaya 155, M. Kaneda 30, S. Kaneti 28, T. Kanno 157, V.A. Kantserov 96, J. Kanzaki 65, B. Kaplan 108, A. Kapliy 31, J. Kaplon 30, D. Kar 53, M. Karagounis 21, K. Karakostas 10, M. Karnevskiy 42, V. Kartvelishvili 71, A.N. Karyukhin 128, L. Kashif 173, G. Kasieczka 58b, R.D. Kass 109, A. Kastanas 14, M. Kataoka 5, Y. Kataoka 155, E. Katsouﬁs 10, J. Katzy 42, V. Kaushik 7, K. Kawagoe 69, T. Kawamoto 155, G. Kawamura 81, M.S. Kayl 105, S. Kazama 155, V.A. Kazanin 107, M.Y. Kazarinov 64, R. Keeler 169, R. Kehoe 40, M. Keil 54, G.D. Kekelidze 64, J.S. Keller 138, M. Kenyon 53, O. Kepka 125, N. Kerschen 30, B.P. Kerševan 74, S. Kersten 175, K. Kessoku 155, J. Keung 158, F. Khalil-zada 11, H. Khandanyan 146a,146b, A. Khanov 112, D. Kharchenko 64, A. Khodinov 96, A. Khomich 58a, T.J. Khoo 28, G. Khoriauli 21, A. Khoroshilov 175, V. Khovanskiy 95, E. Khramov 64, J. Khubua 51b, H. Kim 146a,146b, S.H. Kim 160, N. Kimura 171, O. Kind 16, B.T. King 73, M. King 66, R.S.B. King 118, S.B. King 168, J. Kirk 129, A.E. Kiryunin 99 ATLAS Collaboration / Physics Letters B 718 (2013) 841–859 851 M.I. Gostkin 64, I. Gough Eschrich 163, M. Gouighri 135a, D. Goujdami 135c, M.P. Goulette 49, A.G. Goussiou 138, C. Goy 5, S. Gozpinar 23, I. Grabowska-Bold 38, P. Grafström 20a,20b, K.-J. Grahn 42, F. Grancagnolo 72a, S. Grancagnolo 16, V. Grassi 148, V. Gratchev 121, N. Grau 35, H.M. Gray 30, J.A. Gray 148, E. Graziani 134a, O.G. Grebenyuk 121, T. Greenshaw 73, Z.D. Greenwood 25,m, K. Gregersen 36, I.M. Gregor 42 P. Grenier 143, J. Griﬃths 8, N. Grigalashvili 64, A.A. Grillo 137, S. Grinstein 12, Ph. Gris 34, Y.V. Grishkevich 97, J.-F. Grivaz 115, E. Gross 172, J. Grosse-Knetter 54, J. 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Hartjes 105, T. Haruyama 65, A. Harvey 56, S. Hasegawa 101, Y. Hasegawa 140, S. Hassani 136 S. Haug 17, M. Hauschild 30, R. Hauser 88, M. Havranek 21, C.M. Hawkes 18, R.J. Hawkings 30, A.D. Hawkins 79, T. Hayakawa 66, T. Hayashi 160, D. Hayden 76, C.P. Hays 118, H.S. Hayward 73, S.J. Haywood 129, S.J. Head 18, V. Hedberg 79, L. Heelan 8, S. Heim 88, B. Heinemann 15, S. Heisterkamp 36, L. Helary 22, C. Heller 98, M. Heller 30, S. Hellman 146a,146b, D. Hellmich 21, C. Helsens 12, R.C.W. Henderson 71, M. Henke 58a, A. Henrichs 54, A.M. Henriques Correia 30, S. Henrot-Versille 115, C. Hensel 54, T. ATLAS Collaboration Henß 175, C.M. Hernandez 8, Y. Hernández Jiménez 167, R. Herrberg 16, G. Herten 48, R. Hertenberger 98, L. Hervas 30, G.G. Hesketh 77, N.P. Hessey 105, E. Higón-Rodriguez 167, J.C. Hill 28, K.H. Hiller 42, S. Hillert 21, S.J. Hillier 18, I. Hinchliffe 15, E. Hines 120, M. Hirose 116, F. Hirsch 43, D. Hirschbuehl 175, J. Hobbs 148, N. Hod 153, M.C. Hodgkinson 139, P. 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Istin 19a, A.V. Ivashin 128, W. Iwanski 39, H. Iwasaki 65, J.M. Izen 41, V. Izzo 102a, B. Jackson 120, J.N. Jackson 73, P. Jackson 1, M.R. Jaekel 30, V. Jain 60, K. Jakobs 48, S. Jakobsen 36, T. Jakoubek 125, J. Jakubek 127, D.K. Jana 111, E. Jansen 77, H. Jansen 30, A. Jantsch 99, M. Janus 48, G. Jarlskog 79, L. Jeanty 57, I. Jen-La Plante 31, D. Jennens 86, P. Jenni 30, A.E. Loevschall-Jensen 36, P. Jež 36 S. Jézéquel 5, M.K. Jha 20a, H. Ji 173, W. Ji 81, J. Jia 148, Y. Jiang 33b, M. Jimenez Belenguer 42, S. Jin 33a, O. Jinnouchi 157, M.D. Joergensen 36, D. Joffe 40, M. Johansen 146a,146b, K.E. Johansson 146a, P. Johansson 13 S. Johnert 42, K.A. Johns 7, K. Jon-And 146a,146b, G. Jones 170, R.W.L. Jones 71, T.J. Jones 73, C. Joram 30, P.M. ATLAS Collaboration Kehoe 40, M. Keil 54, G.D. Kekelidze 64, J.S. Keller 138, M. Kenyon 53, O. Kepka 125, N. 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Shulga 96, M.A. Shupe 7, 125 132a 48 13a 172 124a 153 a , A. Shmeleva , M.J. Shochet , D. Short , S. Shrestha , E. Shulga , M.A. A. Sidoti 132a, F. Siegert 48, Dj. Sijacki 13a, O. Silbert 172, J. Silva 124a, Y. Silver 153, P. Sicho , A. Sidoti , F. Siegert , Dj. Sijacki , O. Silbert , J. Silva , Y. Silver , D. Silverstein 143, S.B. Silverstein 146a, V. Simak 127, O. Simard 136, Lj. Simic 13a, S. Simion 115, E. Simioni 81, , , g , j j , , J , , D. Silverstein 143, S.B. Silverstein 146a, V. Simak 127, O. Simard 136, Lj. Simic 13a, S. Simion 115, E. Simioni 81, 77 89 89b 36 158 114 141 , , y , , , p , G. Siragusa 174, A. Sircar 25, A.N. Sisakyan 64,∗, S.Yu. Sivoklokov 97, J. Sjölin 146a,146b, T.B. Sjursen 14, p V. Smakhtin 172, B.H. Smart 46, S.L. Smestad 117, S.Yu. Smirnov 96, Y. Smirnov 96, L.N. Smirnova 97, A.A. Snesarev 94, S.W. Snow 82, J. Snow 111, S. Snyder 25, R. Sobie 169,k, J. Sodomka 127, A. Soffer 153, J A.A. Solodkov 128, O.V. Solovyanov 128, V. Solovyev 121, N. Soni 1, V. Sopko 127, B. Sopko 127, M. ATLAS Collaboration Tic 125, V.O. Tikhomirov 94, Y.A. Tikhonov 107,f , S. Timoshenko 96 P. Tipton 176, S. Tisserant 83, T. Todorov 5, S. Todorova-Nova 161, B. Toggerson 163, J. Tojo 69, S. Tokár 144a, K. Tokushuku 65, K. Tollefson 88, M. Tomoto 101, L. Tompkins 31, K. Toms 103, A. Tonoyan 14, C. Topfel 17, N.D. Topilin 64, I. Torchiani 30, E. Torrence 114, H. Torres 78, E. Torró Pastor 167, J. Toth 83,ad, F. Touchard 83, D.R. Tovey 139, T. Trefzger 174, L. Tremblet 30, A. Tricoli 30, I.M. Trigger 159a, S. Trincaz-Duvoid 78, M.F. Tripiana 70, N. Triplett 25, W. Trischuk 158, B. Trocmé 55, C. Troncon 89a, M. Trottier-McDonald 142, M. Trzebinski 39, A. Trzupek 39, C. Tsarouchas 30, J.C-L. Tseng 118, M. Tsiakiris 105, P.V. Tsiareshka 90, D. Tsionou 5,ai, G. Tsipolitis 10, S. Tsiskaridze 12, V. Tsiskaridze 48, E.G. Tskhadadze 51a, I.I. Tsukerman 95, V. Tsulaia 15, J.-W. Tsung 21, S. Tsuno 65, D. Tsybychev 148, A. Tua 139, A. Tudorache 26a, V. Tudorache 26a, J.M. Tuggle 31, M. Turala 39, D. Turecek 127, I. Turk Cakir 4e, E. Turlay 105, R. Turra 89a,89b, P.M. Tuts 35, A. Tykhonov 74, M. Tylmad 146a,146b, M. Tyndel 129, G. Tzanakos 9, K. Uchida 21, I. Ueda 155, R. Ueno 29, M. Ugland 14, M. Uhlenbrock 21, M. Uhrmacher 54, F. Ukegawa 160, G. Unal 30, A. Undrus 25, G. Unel 163, Y. Unno 65, D. Urbaniec 35, P. Urquijo 21, G. Usai 8, M. Uslenghi 119a,119b, L. Vacavant 83, V. Vacek 127, ATLAS Collaboration / Physics Letters B 718 (2013) 841–859 856 B. Vachon 85, S. Vahsen 15, J. Valenta 125, S. Valentinetti 20a,20b, A. Valero 167, S. Valkar 126, E. Valladolid Gallego 167, S. Vallecorsa 152, J.A. Valls Ferrer 167, P.C. Van Der Deijl 105, R. van der Geer 105, H. van der Graaf 105, R. Van Der Leeuw 105, E. van der Poel 105, D. van der Ster 30, N. van Eldik 30, P. van Gemmeren 6, I. van Vulpen 105, M. Vanadia 99, W. Vandelli 30, A. Vaniachine 6, P. Vankov 42, F. Vannucci 78, R. Vari 132a, T. Varol 84, D. Varouchas 15, A. Vartapetian 8, K.E. 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Yang 146a,146b, S. Yanush 91, L. Yao 33a, Y. Yao 15, Y. Yasu 65, G.V. Ybeles Smit 130, J. Ye 40, S. Ye 25, M. Yilmaz 4c, R. Yoosoofmiya 123, K. Yorita 171, R. Yoshida 6, C. Young 143, C.J. Young 118, S. Youssef 22, D. Yu 25, J. Yu 8, J. Yu 112, L. Yuan 66, A. Yurkewicz 106, B. Zabinski 39, R. Zaidan 62, A.M. Zaitsev 128, Z. Zajacova 30, L. Zanello 132a,132b, D. Zanzi 99, A. Zaytsev 25, C. Zeitnitz 175, M. Zeman 125, A. Zemla 39, C. Zendler 21, O. Zenin 128, T. Ženiš 144a, Z. Zinonos 122a,122b, S. Zenz 15, D. Zerwas 115, G. Zevi della Porta 57, Z. Zhan 33d, D. Zhang 33b,ak, H. Zhang 88, J. Zhang 6, X. Zhang 33d, Z. Zhang 115, L. Zhao 108, T. Zhao 138, Z. Zhao 33b, A. Zhemchugov 64, J. Zhong 118, B. Zhou 87, N. Zhou 163, Y. Zhou 151, C.G. Zhu 33d, H. Zhu 42, J. Zhu 87, Y. Zhu 33b, X. Zhuang 98, V. Zhuravlov 99, D. Zieminska 60, N.I. Zimin 64, R. Zimmermann 21, S. Zimmermann 21, S. Zimmermann 48, M. Ziolkowski 141, R. Zitoun 5, L. Živkovi´c 35, V.V. Zmouchko 128,∗, G. Zobernig 173, A. Zoccoli 20a,20b, M. zur Nedden 16, V. Zutshi 106, L. a, Edmonton, AB, Canada y, Ankara; (b)Department of Physics, Dumlupinar University, Kutahya; (c)Department of Physics, Gazi University, Ankara; (d)Division of Physics, y, Ankara; (e)Turkish Atomic Energy Authority, Ankara, Turkey Annecy le Vieux France ATLAS Collaboration Zwalinski 30 1 School of Chemistry and Physics, University of Adelaide, Adelaide, Australia 2 Physics Department, SUNY Albany, Albany, NY, United States 3 Department of Physics, University of Alberta, Edmonton, AB, Canada 4 (a)Department of Physics, Ankara University, Ankara; (b)Department of Physics, Dumlupinar University, Kutahya; (c)Department of Physics, Gazi University, Ankara; (d)Division of Physics, TOBB University of Economics and Technology, Ankara; (e)Turkish Atomic Energy Authority, Ankara, Turkey B. Vachon 85, S. Vahsen 15, J. Valenta 125, S. Valentinetti 20a,20b, A. Valero 167, S. Valkar 126, p nucci 78, R. Vari 132a, T. Varol 84, D. Varouchas 15, A. Vartapetian 8, K.E. 30 CERN, Geneva, Switzerland 31 Enrico Fermi Institute, University of Chicago, Chicago, IL, United States 32 ( ) 31 Enrico Fermi Institute, University of Chicago, Chicago, IL, United States , y f g , g , , 32 (a) Departamento de Física, Pontiﬁcia Universidad Católica de Chile, Santiago; (b) Departamento de Física, Universidad Técnica Federico Santa María, Valparaíso, Chile 33 (a) Institute of High Energy Physics, Chinese Academy of Sciences, Beijing; (b) Department of Modern Physics, University of Science and Technology of China, Anhui; (c) Department of Physics, Nanjing University, Jiangsu; (d) School of Physics, Shandong University, Shandong, China 32 (a) Departamento de Física, Pontiﬁcia Universidad Católica de Chile, Santiago; (b) Departamento de Física, Universidad Técnica Federico Santa María, Valparaíso, Chile 33 (a) Institute of High Energy Physics, Chinese Academy of Sciences, Beijing; (b) Department of Modern Physics, University of Science and Technology of China, Anhui; (c) Department of (d) 32 (a) Departamento de Física, Pontiﬁcia Universidad Católica de Chile, Santiago; (b) Departamento de Física, Universidad Técnica Federico Santa María, Valparaíso, Chile 33 (a) I i f Hi h E Ph i Chi A d f S i B iji (b) D f M d Ph i U i i f S i d T h l f Chi A h i(c) D f 32 (a) Departamento de Física, Pontiﬁcia Universidad Católica de Chile, Santiago; (b) Departamento de Física, Universidad Técnica Federic 33 (a) Institute of High Energy Physics, Chinese Academy of Sciences, Beijing; (b) Department of Modern Physics, University of Science and d p ﬁ g p 33 (a) Institute of High Energy Physics, Chinese Academy of Sciences, Beijing; (b) Department of Modern Physics, Univ f g gy y y f j g p f Physics, Nanjing University, Jiangsu; (d) School of Physics, Shandong University, Shandong, China Physics, Nanjing University, Jiangsu; (d) School of Physics, Shandong University, Shandong, China 34 Laboratoire de Physique Corpusculaire, Clermont Université and Université Bla 34 Laboratoire de Physique Corpusculaire, Clermont Université and Université Blaise Pascal and CNRS/IN2P3, Clermont-Ferrand, France 34 Laboratoire de Physique Corpusculaire, Clermont Université and Université Blaise Pascal and CNRS/IN2P3, Clermont-Ferrand, France 34 Laboratoire de Physique Corpusculaire, Clermont Université and Un 35 Nevis Laboratory, Columbia University, Irvington, NY, United States 35 Nevis Laboratory, Columbia University, Irvington, NY, United States 36 Niels Bohr Institute, University of Copenhagen, Kobenhavn, Denmark 36 Niels Bohr Institute, University of Copenhagen, Kobenhavn, Denmark 37 (a) INFN Gruppo Collegato di Cosenza; (b) Dipartimento di Fisica, Università della Calabria, Ar 37 (a) INFN Gruppo Collegato di Cosenza; (b) Dipartimento di Fisica, Uni 37 (a) INFN Gruppo Collegato di Cosenza; (b) Dipartimento di Fisica, Università della Calabria, Arcavata di Rende, Ita 38 38 AGH University of Science and Technology, Faculty of Physics and Applied Computer Science, Krakow 38 AGH University of Science and Technology, Faculty of Physics and Applied Computer Science, Krakow, Poland 39 38 AGH University of Science and Technology, Faculty of Physics and App 39 The Henryk Niewodniczanski Institute of Nuclear Physics, Polish Academy of Sciences, Krakow, Poland 40 39 The Henryk Niewodniczanski Institute of Nuclear Physics, Polish Acad 40 Physics Department, Southern Methodist University, Dallas, TX, Unite 40 Physics Department, Southern Methodist University, Dallas, TX, United States 41 Physics Department, University of Texas at Dallas, Richardson, TX, United States 41 Physics Department, University of Texas at Dallas, Richardson, TX, United States 42 DESY, Hamburg and Zeuthen, Germany g y 43 Institut für Experimentelle Physik IV, Technische Universität Dortmund, Dortmund, Germany 43 Institut für Experimentelle Physik IV, Technische Universität Dortmund, Dortmund, Germany 43 Institut für Experimentelle Physik IV, Technische Universität Dortm f p y 44 Institut für Kern- und Teilchenphysik, Technical University Dresden, Dresden, Germany 44 Institut für Kern- und Teilchenphysik, Technical University Dresden, Dresden, Germany 45 Department of Physics, Duke University, Durham, NC, United States p f y , y, , , 46 SUPA – School of Physics and Astronomy, University of Edinburgh, Edinburgh, United Kingdom 46 SUPA – School of Physics and Astronomy, University of Edinburgh, Edinburgh, United Kingdom 46 SUPA – School of Physics and Astronomy, University of Edinbur 47 INFN Laboratori Nazionali di Frascati, Frascati, Italy 48 Fakultät für Mathematik und Physik, Albert-Ludwigs-Universität, 49 Section de Physique, Université de Genève, Geneva, Switzerland 49 Section de Physique, Université de Genève, Geneva, Switzerland y q 50 (a) INFN Sezione di Genova; (b) Dipartimento di Fisica, Università di Genova, Genova, Italy 50 (a) INFN Sezione di Genova; (b) Dipartimento di Fisica, Università di Genova, Genova, Italy 50 (a) INFN Sezione di Genova; (b) Dipartimento di Fisica, Universit 51 (a) E. ATLAS Collaboration Varvell 150, chool of Chemistry and Physics, University of Adelaide, Adelaide, Australi 2 Physics Department, SUNY Albany, Albany, NY, United States ( )Department of Physics, Ankara University, Ankara; ( )Department of Physics, Dumlupinar University, Kutahya; ( )Department of Physic TOBB University of Economics and Technology, Ankara; (e)Turkish Atomic Energy Authority, Ankara, Turkey 7 Department of Physics, University of Arizona, Tucson, AZ, United States 8 Department of Physics, The University of Texas at Arlington, Arlington, TX, United States 10 Physics Department, National Technical University of Athens, Zografou, Greece 11 Institute of Physics, Azerbaijan Academy of Sciences, Baku, Azerbaijan 12 857 ATLAS Collaboration / Physics Letters B 718 (2013) 841–859 13 (a) Institute of Physics, University of Belgrade, Belgrade; (b) Vinca Institute of Nuclear Sciences, University of Belgrade, Belgrade, Serbia 14 a) Institute of Physics, University of Belgrade, Belgrade; (b) Vinca Institute of Nuclear Sciences, University of Belgrade, Belgrade, Serbia 13 (a) Institute of Physics, University of Belgrade, Belgrade; (b) Vinca Institute of Nuclear Sciences, University of Belgrade, Belgrade, Serbia 13 (a) Institute of Physics, University of Belgrade, Belgrade; (b) Vinca Institute of Nuclear Sciences, University of Be (a) Institute of Physics, University of Belgrade, Belgrade; (b) Vinca Institu 14 Department for Physics and Technology, University of Bergen, Bergen p f y gy y f g g y 15 Physics Division, Lawrence Berkeley National Laboratory and University of California, Berkeley, CA, United States p f y gy y f g g y 15 Physics Division, Lawrence Berkeley National Laboratory and University of California, Berkeley, CA, Unit 15 Physics Division, Lawrence Berkeley National Laboratory and U 16 Department of Physics, Humboldt University, Berlin, Germany p f y y y 17 Albert Einstein Center for Fundamental Physics and Laboratory f y y f g gy y 18 School of Physics and Astronomy, University of Birmingham, Birmingham, United Kingdom f y y f g gy y 18 School of Physics and Astronomy, University of Birmingham, Birmingham, United Kingdom b 18 School of Physics and Astronomy, University of Birmingham, Birming 19 (a) Department of Physics, Bogazici University, Istanbul; (b) Division of Physics, Dogus University, Istanbul; (c) Department of Physics E d 19 (a) Department of Physics, Bogazici University, Istanbul; (b) Division of Physics, Dogus Univers 19 (a) Department of Physics, Bogazici University, Istanbul; (b) Division o (d) Department of Physics, Istanbul Technical University, Istanbul, Turkey p f y y y 20 (a) INFN Sezione di Bologna; (b) Dipartimento di Fisica, Università di Bologna, Bologna, Italy 21 20 (a) INFN Sezione di Bologna; (b) Dipartimento di Fisica, Università di Bologna, Bologna, Italy 20 (a) INFN Sezione di Bologna; (b) Dipartimento di Fisica, Univer 21 Physikalisches Institut, University of Bonn, Bonn, Germany 22 Department of Physics, Boston University, Boston, MA, United States p f y y 23 Department of Physics, Brandeis University, Waltham, MA, United States 23 Department of Physics, Brandeis University, Waltham, MA, United States p f y , y, , , 24 (a) Universidade Federal do Rio De Janeiro COPPE/EE/IF, Rio de Janeiro; (b) Federal University of Juiz de Fora (UFJF), Juiz de Fora; (c) Federal University of Sao Joao del Rei (UFSJ), Sao Joao del Rei; (d) Instituto de Fisica Universidade de Sao Paulo Sao Paulo Brazil 24 (a) Universidade Federal do Rio De Janeiro COPPE/EE/IF, Rio de Janeiro 24 (a) Universidade Federal do Rio De Janeiro COPPE/EE/IF, Rio de Janeiro; (b) Federal University of Juiz de Fora (UFJF), Juiz de Fora; (c) Federal U S J d l R i(d) I i d Fi i U i id d d S P l S P l B il 24 (a) Universidade Federal do Rio De Janeiro COPPE/EE/IF, Rio de Janeiro; (b) Federal University of Juiz de Fora (UFJF), Juiz de Fora; (c) Federal University of Sao Joao del Rei (UFSJ), (d) o Joao del Rei; (d) Instituto de Fisica, Universidade de Sao Paulo, Sao Pau 25 Physics Department, Brookhaven National Laboratory, Upton, NY, United States 25 Physics Department, Brookhaven National Laboratory, Upton, NY, United States y p , y, p , , 26 (a) National Institute of Physics and Nuclear Engineering, Bucharest; (b) University Politehnica Bucharest, Bucharest; (c) West University in Timisoara, Timisoara, Romania 27 Departamento de Física Universidad de Buenos Aires Buenos Aires Argentina 26 (a) National Institute of Physics and Nuclear Engineering, Bucharest; (b) U 2 26 (a) National Institute of Physics and Nuclear Engineering, Bucharest; (b) University Politehnica Bucharest, Bucharest; (c) West University in Timisoara, Timisoara, Romania 27 (a) National Institute of Physics and Nuclear Engineering, Bucharest; (b) 27 Departamento de Física, Universidad de Buenos Aires, Buenos Aires, Argentina 27 Departamento de Física, Universidad de Buenos Aires, Buenos Aires, Argentina 28 Cavendish Laboratory, University of Cambridge, Cambridge, United Kingdom 28 Cavendish Laboratory, University of Cambridge, Cambridge, United Kingdom 29 Department of Physics, Carleton University, Ottawa, ON, Canada 29 Department of Physics, Carleton University, Ottawa, ON, Canada 30 CERN, Geneva, Switzerland Granada, Spain 25 Institute of Physics, Academy of Sciences of the Czech Republic, Praha, f y y f f p p 126 Faculty of Mathematics and Physics, Charles University in Prague, Praha, Czech Republic 2 126 Faculty of Mathematics and Physics, Charles University in Prague, Praha, Czech Republic 126 Faculty of Mathematics and Physics, Charles University in Prague, 127 Czech Technical University in Prague, Praha, Czech Republic 128 State Research Center Institute for High Energy Physics, Protvino, R 129 Particle Physics Department, Rutherford Appleton Laboratory, Didcot, United Kingdom 129 Particle Physics Department, Rutherford Appleton Laboratory, Didcot, United Kingdom 130 129 Particle Physics Department, Rutherford Appleton Laboratory, Di 130 Physics Department, University of Regina, Regina, SK, Canada 131 Ritsumeikan University, Kusatsu, Shiga, Japan 132 (a) INFN Sezione di Roma I; (b) Dipartimento di Fisica, Università La Sapienza, Roma, Italy 132 (a) INFN Sezione di Roma I; (b) Dipartimento di 132 (a) INFN Sezione di Roma I; (b) Dipartimento di Fisica, Università La Sapienza, Roma, Italy 133 (a) INFN Sezione di Roma Tor Vergata; (b) Dipartimento di Fisica, Università di Roma Tor V 133 (a) INFN Sezione di Roma Tor Vergata; (b) Dipartimento di Fisica, Università di Roma Tor Vergata, Roma, Italy 133 (a) INFN Sezione di Roma Tor Vergata; (b) Dipartimento di Fisica, Università di Roma Tor Ver 33 (a) INFN Sezione di Roma Tor Vergata; (b) Dipartimento di Fisica, Univ g p g 134 (a) INFN Sezione di Roma Tre; (b) Dipartimento di Fisica, Università Roma Tre, Roma, Italy g p 134 (a) INFN Sezione di Roma Tre; (b) Dipartimento di Fisica, Università Roma Tre, Roma, Italy 135 (a) Faculté des Sciences Ain Chock, Réseau Universitaire de Physique des Hautes Energies – Université Hassan II, Casablanca; (b) Centre N (c) é é (d) é é 135 (a) Faculté des Sciences Ain Chock, Réseau Universitaire de Physique des Hautes Energies – Université Hassan II, Casablanca; (b) Centre National de l’Energie des Sciences Techniques Nucleaires, Rabat; (c) Faculté des Sciences Semlalia, Université Cadi Ayyad, LPHEA, Marrakech; (d) Faculté des Sciences, Université Mohamed Premier and LPTPM, Oujda; (e) Faculté des Sciences Université Mohammed V Agdal Rabat Morocco 135 (a) Faculté des Sciences Ain Chock, Réseau Universitaire de Physi 135 (a) Faculté des Sciences Ain Chock, Réseau Universitaire de Physique des Hautes Energies – U 135 (a) Faculté des Sciences Ain Chock, Réseau Universitaire de Physique des Hautes Energies – Université Hassan II, Casablanca; (b) Centre National de l Energie des Sciences Techniques Nucleaires, Rabat; (c) Faculté des Sciences Semlalia, Université Cadi Ayyad, LPHEA, Marrakech; (d) Faculté des Sciences, Université Mohamed Premier and LPTPM, Oujda; (e) Faculté des y q g g q Nucleaires, Rabat; (c) Faculté des Sciences Semlalia, Université Cadi Ayyad, LPHEA, Marrakech; (d) Faculté des Sciences, Université Mohamed Premier and LPTPM, Oujda; (e) Faculté des Nucleaires, Rabat; (c) Faculté des Sciences Semlalia, Université Sciences, Université Mohammed V – Agdal, Rabat, Morocco g 136 DSM/IRFU (Institut de Recherches sur les Lois Fondamentales de l’Univers), CEA Saclay (Commissariat a l’Energie Atomique), Gif-sur-Yvett 137 136 DSM/IRFU (Institut de Recherches sur les Lois Fondamentales de l’Univers), CEA Saclay (Commissariat a l’Energie Atomique) 136 DSM/IRFU (Institut de Recherches sur les Lois Fondamenta 136 DSM/IRFU (Institut de Recherches sur les Lois Fondamentales de l’Univers), CEA Saclay (Comm / ( ) y ( g q ) f 137 Santa Cruz Institute for Particle Physics, University of California Santa Cruz, Santa Cruz, CA, United States / ( ) y ( 137 Santa Cruz Institute for Particle Physics, University of California Santa Cruz, Santa Cruz, CA, Unite 37 Santa Cruz Institute for Particle Physics, University of California Santa 138 Department of Physics, University of Washington, Seattle, WA, United States 138 Department of Physics, University of Washington, Seattle, WA, United States p f y y f g 139 Department of Physics and Astronomy, University of Sheﬃeld, Sheﬃeld, United Kingdom 139 Department of Physics and Astronomy, University of Sheﬃeld, Sheﬃeld, United Kingdom 139 Department of Physics and Astronomy, University of Sheﬃeld 140 Department of Physics, Shinshu University, Nagano, Japan 141 Fachbereich Physik, Universität Siegen, Siegen, Germany 142 Department of Physics, Simon Fraser University, Burnaby, BC, Canada 143 SLAC National Accelerator Laboratory, Stanford, CA, United States y f 144 (a) Faculty of Mathematics, Physics & Informatics, Comenius University, Bratislava; (b) Department of Subnuclear Physics, Institute of Exp of Sciences, Kosice, Slovak Republic 144 (a) Faculty of Mathematics, Physics & Informatics, Comenius University, Bratislava; (b) Departme 144 (a) Faculty of Mathematics, Physics & Informatics, Comenius Uni 30 CERN, Geneva, Switzerland Lebedev Institute of Physics, Academy of Sciences, Moscow, Russia 5 Institute for Theoretical and Experimental Physics (ITEP), Moscow, Rus 96 Moscow Engineering and Physics Institute (MEPhI), Moscow, Russia 97 Skobeltsyn Institute of Nuclear Physics, Lomonosov Moscow State U 98 Fakultät für Physik, Ludwig-Maximilians-Universität München, Mü 99 Max-Planck-Institut für Physik (Werner-Heisenberg-Institut), München, Germany 100 Nagasaki Institute of Applied Science, Nagasaki, Japan 101 Graduate School of Science and Kobayashi–Maskawa Institute, Nagoya University, N 101 Graduate School of Science and Kobayashi–Maskawa Institute, Na 102 (a) INFN Sezione di Napoli; (b) Dipartimento di Scienze Fisiche, Università di Napoli, Napoli, Italy p p p 103 Department of Physics and Astronomy, University of New Mexico, Albuquerque, NM, Un 104 Institute for Mathematics, Astrophysics and Particle Physics, Radboud University Nijmegen/Nikhef, 105 Nikhef National Institute for Subatomic Physics and University of Amsterdam, Amsterdam, Netherla 05 Nikhef National Institute for Subatomic Physics and University of Ams f f y y f 106 Department of Physics, Northern Illinois University, DeKalb, IL, United State 106 Department of Physics, Northern Illinois University, DeKalb, IL 106 Department of Physics, Northern Illinois University, DeKalb, IL, U 107 Budker Institute of Nuclear Physics, SB RAS, Novosibirsk, Russia 108 Department of Physics, New York University, New York, NY, United States 108 Department of Physics, New York University, New Yo p f y y 109 Ohio State University, Columbus, OH, United States 109 Ohio State University, Columbus, OH, United States 110 Faculty of Science, Okayama University, Okayama, Jap y f y y y J p 111 Homer L. Dodge Department of Physics and Astronomy, University of Oklahoma, Norman, OK, United States 112 111 Homer L. Dodge Department of Physics and Astronomy, University of Oklahoma, Norman, OK, United States 111 Homer L. 30 CERN, Geneva, Switzerland Dodge Department of Physics and Astronomy, Un g p f y y y f 112 Department of Physics, Oklahoma State University, Stillwater, OK, United States 112 Department of Physics, Oklahoma State University, Stillwater, OK, United States 112 Department of Physics, Oklahoma State University, Stillwater, 113 Palacký University, RCPTM, Olomouc, Czech Republic 114 Center for High Energy Physics, University of Oregon, Eugene, OR, United States 114 Center for High Energy Physics, University of Oregon, Eugene, OR, United States 115 LAL, Université Paris-Sud and CNRS/IN2P3, Orsay, France 116 Graduate School of Science, Osaka University, Osaka, Japan 117 Department of Physics, University of Oslo, Oslo, Norway 118 Department of Physics, Oxford University, Oxford, United Kingdom 119 (a) INFN Sezione di Pavia; (b) Dipartimento di Fisica, Università di Pavia, Pavia, Italy 119 (a) INFN Sezione di Pavia; (b) Dipartimento di Fisica, Università di Pavia, Pavia, Italy 120 Department of Physics, University of Pennsylvania, Phi 121 Petersburg Nuclear Physics Institute, Gatchina, Russia 122 (a) INFN Sezione di Pisa; (b) Dipartimento di Fisica E. Fermi, Università di Pisa, Pisa, Italy 122 (a) INFN Sezione di Pisa; (b) Dipartimento di Fisica E. Fermi, Università di Pisa, Pisa, Italy 122 (a) INFN Sezione di Pisa; (b) Dipartimento di Fisica E. Fe 23 Department of Physics and Astronomy, University of Pittsburgh, Pittsb 123 Department of Physics and Astronomy, University of Pittsburgh, Pittsburgh, PA, United States 124 (a) Laboratorio de Instrumentacao e Fisica Experimental de Particulas – LIP, Lisboa, Portugal; (b) Departamento de Fisica Teorica y del Cosmos and CAFPE, Universidad de Granada, Granada Spain p f y y y f g g 124 (a) Laboratorio de Instrumentacao e Fisica Experimental de Particulas – LIP, Lisboa, Portugal; (b) Departamento de Fisica Teorica y del Cos Granada, Spain 124 (a) Laboratorio de Instrumentacao e Fisica Experimental de Particulas – LIP, Lisboa, Portugal; (b) Departamento de Fisica Teorica y del Cosmos and CAFPE, Universidad de Granada, Granada Spain 124 (a) Laboratorio de Instrumentacao e Fisica Experimental de Particulas – LIP, Lisboa, Portugal; (b) Departamento de Fisica Teorica y del Cosmos and CAFPE, Universidad de Granada, Granada, Spain 30 CERN, Geneva, Switzerland Stepanov Institute of Physics, National Academy of Sciences of Belarus, Minsk, Belarus 91 National Scientiﬁc and Educational Centre for Particle and High Energy Physics, Minsk, Belarus 92 Department of Physics, Massachusetts Institute of Technology, Cambridge, MA, United States 93 Group of Particle Physics, University of Montreal, Montreal, QC, Canada 94 P.N. Lebedev Institute of Physics, Academy of Sciences, Moscow, Russia 95 Institute for Theoretical and Experimental Physics (ITEP), Moscow, Russia 96 Moscow Engineering and Physics Institute (MEPhI), Moscow, Russia 97 Skobeltsyn Institute of Nuclear Physics, Lomonosov Moscow State University, Moscow, Russia 98 Fakultät für Physik, Ludwig-Maximilians-Universität München, München, Germany 99 Max-Planck-Institut für Physik (Werner-Heisenberg-Institut), München, Germany 100 Nagasaki Institute of Applied Science, Nagasaki, Japan 101 Graduate School of Science and Kobayashi–Maskawa Institute, Nagoya University, Nagoya, Japan 102 (a) INFN Sezione di Napoli; (b) Dipartimento di Scienze Fisiche, Università di Napoli, Napoli, Italy 103 Department of Physics and Astronomy, University of New Mexico, Albuquerque, NM, United States 104 Institute for Mathematics, Astrophysics and Particle Physics, Radboud University Nijmegen/Nikhef, Nijmegen, Netherlands 105 Nikhef National Institute for Subatomic Physics and University of Amsterdam, Amsterdam, Netherlands 90 B.I. Stepanov Institute of Physics, National Academy of Sciences of Belarus, Minsk, Belarus 91 National Scientiﬁc and Educational Centre for Particle and High Energy Physics, Minsk, Belarus Department of Physics, Massachusetts Institute of Technology, Cambridge 93 Group of Particle Physics, University of Montreal, Montreal, QC, Canada 4 P.N. 30 CERN, Geneva, Switzerland Andronikashvili Institute of Physics, Tbilisi State University, T 52 II Physikalisches Institut, Justus-Liebig-Universität Giessen, Giessen, Germany 52 II Physikalisches Institut, Justus-Liebig-Universität Giessen, Giessen, Germany 53 SUPA – School of Physics and Astronomy, University of Glasgow, Glasgow, United Kingdom 53 SUPA – School of Physics and Astronomy, University of Glasgow, Glasgow, Un II Physikalisches Institut, Georg-August-Universität, Göttingen, Germany II Physikalisches Institut, Georg August Universität, Göttingen, Germany 55 Laboratoire de Physique Subatomique et de Cosmologie, Université Joseph Fourier and CNRS/IN2P3 and Institut National Polytechnique de Grenoble, Grenoble, France 56 Department of Physics Hampton University Hampton VA United States , g g , g , y que Subatomique et de Cosmologie, Université Joseph Fourier and CNRS/IN2P3 and Institut National Polytechnique de Grenoble, Grenoble, Franc i i i i d 55 Laboratoire de Physique Subatomique et de Cosmologie, Université Joseph Fourier and CNRS/IN2P3 and Institut National Polytechnique de G 56 55 Laboratoire de Physique Subatomique et de Cosmologie, Université Joseph Fourier and CNR 56 Department of Physics, Hampton University, Hampton, VA, United States 56 Department of Physics, Hampton University, Hampton, VA, United Sta 57 Laboratory for Particle Physics and Cosmology, Harvard University, Ca 58 (a) Kirchhoff-Institut für Physik, Ruprecht-Karls-Universität Heidelberg, Heidelberg; (b) Physik 58 (a) Kirchhoff-Institut für Physik, Ruprecht-Karls-Universität Heidelberg, Heidelberg; Technische Informatik, Ruprecht-Karls-Universität Heidelberg, Mannheim, German 59 Faculty of Applied Information Science, Hiroshima Institute of Technology, Hiroshima, Ja 59 Faculty of Applied Information Science, Hiroshima Institute of Technology, Hiroshima, Japan 59 Faculty of Applied Information Science, Hiroshima Institute of Techno 60 Department of Physics, Indiana University, Bloomington, IN, United States 60 Department of Physics, Indiana University, Bloomington, IN, United States 61 Institut für Astro- und Teilchenphysik, Leopold-Franzens-Universität, Innsbruck 62 61 Institut für Astro- und Teilchenphysik, Leopold-Fr 62 University of Iowa, Iowa City, IA, United States y f y 63 Department of Physics and Astronomy, Iowa State University, Ames, IA, United States 63 Department of Physics and Astronomy, Iowa State University, Ames, IA, United St 63 Department of Physics and Astronomy, Iowa Sta 64 Joint Institute for Nuclear Research, JINR Dubna, Dubna, Russia 64 Joint Institute for Nuclear Research, JINR Dubna, Dubna, Russia 65 KEK, High Energy Accelerator Research Organization, Tsukuba, Japan 65 KEK, High Energy Accelerator Research Organization, Tsukuba, Japan 66 Graduate School of Science, Kobe University, Kobe, Japan 66 Graduate School of Science, Kobe University, Kobe, Japan 67 Faculty of Science, Kyoto University, Kyoto, Japan 67 Faculty of Science, Kyoto University, Kyoto, Japan 68 Kyoto University of Education, Kyoto, Japan 68 Kyoto University of Education, Kyoto, Japan 69 Department of Physics, Kyushu University, Fukuoka, Japan 69 Department of Physics, Kyushu University, Fukuoka, Japan 70 Instituto de Física La Plata, Universidad Nacional de La Plata and 70 Instituto de Física La Plata, Universidad Nacional de La Plata and CONICET, La P 71 Physics Department, Lancaster University, Lancaster, United Kingdom 71 Physics Department, Lancaster University, Lancaster, United Kingdom ; p , 73 Oliver Lodge Laboratory, University of Liverpool, Liverpool, United Kingdom Oliver Lodge Laboratory, University of Liverpool, Liverpool, United Kingd 73 Oliver Lodge Laboratory, University of Liverpool, Liverpool, United Kingdom 74 Department of Physics, Jožef Stefan Institute and University of Ljubljana, Ljubljana 74 Department of Physics, Jožef Stefan Institute and University of Ljubljana, Ljubljana, Slovenia 76 Department of Physics, Royal Holloway University of London, Surrey, United Kingdom 76 Department of Physics, Royal Holloway University of London, Surrey, United Kingdom 77 Department of Physics and Astronomy, University College London, London, United Kingdom 77 Department of Physics and Astronomy, University College London, London, United Kingdom y q g 79 Fysiska institutionen, Lunds universitet, Lund, Sweden 79 Fysiska institutionen, Lunds universitet, Lund, Sweden 80 Departamento de Fisica Teorica C-15, Universidad Autonoma de Madrid, Madrid, Spain 80 Departamento de Fisica Teorica C-15, Universidad Autonoma de Madrid, Madrid, Spain 80 Departamento de Fisica Teorica C-15, Universidad Aut 81 Institut für Physik, Universität Mainz, Mainz, Germany 82 School of Physics and Astronomy, University of Manchester, Manchester, United Kingdom 82 School of Physics and Astronomy, University of Manchester, Manchester, United Kingdom 83 CPPM, Aix-Marseille Université and CNRS/IN2P3, Marseille, France 83 CPPM, Aix-Marseille Université and CNRS/IN2P3, Marseille, France 84 Department of Physics, University of Massachusetts, Amherst, MA, 85 Department of Physics, McGill University, Montreal, QC, Canada 85 Department of Physics, McGill University, Montreal, QC, Canada 86 School of Physics, University of Melbourne, Victoria, Australia 86 School of Physics, University of Melbourne, Victoria, Australia 87 Department of Physics, The University of Michigan, Ann Arbor, MI, 858 ATLAS Collaboration / Physics Letters B 718 (2013) 841–859 88 Department of Physics and Astronomy, Michigan State University, East Lansing, MI, United States 89 ( ) (b) 88 Department of Physics and Astronomy, Michigan State University, East Lansing, MI, United States 89 (a) INFN Sezione di Milano; (b) Dipartimento di Fisica, Università di Milano, Milano, Italy 90 B.I. ATLAS Collaboration / Physics Letters B 718 (2013) 841–859 859 163 Department of Physics and Astronomy, University of California Irvine, Irvine, CA, United States 164 (a) (b) (c) 163 Department of Physics and Astronomy, University of California Irvine, Irvine, CA, United States pp g p 165 Department of Physics, University of Illinois, Urbana, IL, United States pp g p 165 Department of Physics, University of Illinois, Urbana, IL, United States 166 Department of Physics and Astronomy, University of Uppsala, Uppsala, Sweden 166 Department of Physics and Astronomy, University of Uppsala, Uppsala, Sweden 167 Instituto de Física Corpuscular (IFIC) and Departamento de Física At 167 Instituto de Física Corpuscular (IFIC) and Departamento de Física Atómica de Barcelona (IMB-CNM), University of Valencia and CSIC, Valencia, Spain p ( ) p de Barcelona (IMB-CNM), University of Valencia and CSIC, Valencia, Spain p ( ) p de Barcelona (IMB-CNM), University of Valencia and CSIC, Valencia, Spain 168 Department of Physics, University of British Columbia, Vancouver, BC, Canada 168 Department of Physics, University of British Columbia, Vancouver, BC, Canada Department of Physics and Astronomy, University of Victoria, Victoria, BC 170 Department of Physics, University of Warwick, Coventry, United Kingdom 170 Department of Physics, University of Warwick, Coventry, United Kingdom 171 Waseda University, Tokyo, Japan y, y , J p 172 Department of Particle Physics, The Weizmann Institute of Science, Rehovot, Israel ment of Particle Physics, The Weizmann Institute of Science, Rehovot, Isra 173 Department of Physics, University of Wisconsin, Madison, WI, United States 173 Department of Physics, University of Wisconsin, Madison, WI, United States 174 Fakultät für Physik und Astronomie, Julius-Maximilians-Universität, Würzburg, Ge 175 174 Fakultät für Physik und Astronomie, Julius-Maximilians-Universität, Würzburg, Germany 175 F hb i h C Ph ik B i h U i i ä W l W l G Fakultät für Physik und Astronomie, Julius Maximilians Universität, Würzburg, Germany 175 Fachbereich C Physik, Bergische Universität Wuppertal, Wuppertal, Germany f y , J , g, y 175 Fachbereich C Physik, Bergische Universität Wuppertal, Wuppertal, Germany 5 Fachbereich C Physik, Bergische Universität Wuppertal, Wuppertal, Ge 176 Department of Physics, Yale University, New Haven, CT, United States 176 Department of Physics, Yale University, New Haven, CT, United States 177 Yerevan Physics Institute, Yerevan, Armenia 177 Yerevan Physics Institute, Yerevan, Armenia y 178 Centre de Calcul de l’Institut National de Physique Nucléaire et de Physique des Particules (IN2P3), Villeurbanne, France a Also at Laboratorio de Instrumentacao e Fisica Experimental de Particulas – LIP, Lisboa, Portugal. a Also at Laboratorio de Instrumentacao e Fisica Experimental de Particulas – LIP, Lisboa, Portugal. b Also at Faculdade de Ciencias and CFNUL, Universidade de Lisboa, Lisboa, Portugal. c Also at Particle Physics Department, Rutherford Appleton Laborat c Also at Particle Physics Department, Rutherford Appleton Laboratory, Didcot, United d Also at TRIUMF, Vancouver, BC, Canada. d Also at TRIUMF, Vancouver, BC, Canada. e Also at Department of Physics, California State University, Fresno, CA, United States. e Also at Department of Physics, California State University, Fresno, CA, United States. e Also at Department of Physics, California State University, Fres f Also at Novosibirsk State University, Novosibirsk, Russia. g Also at Fermilab, Batavia, IL, United States. g Also at Fermilab, Batavia, IL, United States. h Also at Department of Physics, University of Coimbra, Coimbra, Portugal. h Also at Department of Physics, University of Coimbra, Coimbra, Portugal. i Also at Department of Physics, UASLP, San Luis Potosi, Mexico. i Also at Department of Physics, UASLP, San Luis Potosi, Mexico. j Also at Università di Napoli Parthenope, Napoli, Italy. j Also at Università di Napoli Parthenope, Napoli, Ital k Also at Institute of Particle Physics (IPP), Canada. k Also at Institute of Particle Physics (IPP), Canada. l Also at Department of Physics, Middle East Technical University, Ankara, Turkey. l Also at Department of Physics, Middle East Techn l Also at Department of Physics, Middle East Technical University, Ankara, Turkey. m Also at Louisiana Tech University, Ruston, LA, United States. so at Louisiana Tech University, Ruston, LA, United States y, , , n Also at Departamento de Fisica and CEFITEC of Faculdade de Ciencias e Tecnologia, Universidade Nova de Lisboa, Caparica, Portugal. y, , , n Also at Departamento de Fisica and CEFITEC of Faculdade de Ciencias e Tecnologia, Universidade Nova de Lisboa, Caparica, Port y n Also at Departamento de Fisica and CEFITEC of Faculdade de Cienc o Also at Department of Physics and Astronomy, University College London, London, United Kingdom. o Also at Department of Physics and Astronomy, University College London, London, q Also at Department of Physics, University of Cape Town, Cape Town, South Africa q Also at Department of Physics, University of Cape Town, Cape Town, South Africa. r Also at Institute of Physics, Azerbaijan Academy of Sciences, Baku, Azerbaijan. r Also at Institute of Physics, Azerbaijan Academy of Sciences, Baku, Azerbaijan. s Also at Institut für Experimentalphysik, Universität Hamb t Also at Manhattan College, New York, NY, United States. of Sciences, Kosice, Slovak Republic of Sciences, Kosice, Slovak Republic f , , p 145 (a) Department of Physics, University of Johannesburg, Johannesburg; (b) School of Physics, University of the Witwatersrand, Johannesburg, South Africa 146 (a) (b) f , , p 145 (a) Department of Physics, University of Johannesburg, Johannesburg; (b) School of Physics, University of the Witwatersrand, Johannesburg, South Africa b 145 (a) Department of Physics, University of Johannesburg, Johannesburg; (b) School of Physics, University of the Witwatersra Department of Physics, University of Johannesburg, Johannesburg; (b) S (a) Department of Physics, Stockholm University; (b) The Oskar Klein Cen 146 (a) Department of Physics, Stockholm University; (b) The Oskar Klein Centre, Stockholm, Sweden 147 146 (a) Department of Physics, Stockholm University; (b) The Oskar Klein Centre, Stockholm, Sweden 47 Physics Department, Royal Institute of Technology, Stockholm, Sweden 148 Departments of Physics & Astronomy and Chemistry, Stony Brook University, Stony Brook, NY, United States 148 Departments of Physics & Astronomy and Chemistry, Stony Brook University, Stony Brook, NY, United S 148 Departments of Physics & Astronomy and Chemistry, Stony Brook U 149 Department of Physics and Astronomy, University of Sussex, Brighton, United Kingdom 149 Department of Physics and Astronomy, University of Sussex, 149 Department of Physics and Astronomy, University of Sussex, Brighton, United Kingdom 150 School of Physics, University of Sydney, Sydney, Australia 151 Institute of Physics, Academia Sinica, Taipei, Taiwan 152 Department of Physics, Technion: Israel Institute of Technology, Haifa, Israel 152 Department of Physics, Technion: Israel Institute of Technology, Haifa, Israel 152 Department of Physics, Technion: Israel Institute of 53 Raymond and Beverly Sackler School of Physics and Astronomy, Tel Av 154 Department of Physics, Aristotle University of Thessaloniki, Thessaloniki, Greece 154 Department of Physics, Aristotle University of Thessaloniki, Thessaloniki, Greece 155 International Center for Elementary Particle Physics and Department of Physics, The University of Tokyo, Tokyo, Japan 155 International Center for Elementary Particle Physics and Department of Physics, The University of Tokyo, Tokyo, 55 International Center for Elementary Particle Physics and Department 56 Graduate School of Science and Technology, Tokyo Metropolitan Univ 157 Department of Physics, Tokyo Institute of Technology, Tokyo, Japan 158 Department of Physics, University of Toronto, Toronto, ON, Canada 158 Department of Physics, University of Toronto, Toronto, ON, Canada 159 (a) TRIUMF, Vancouver, BC; (b) Department of Physics and Astronom 160 Institute of Pure and Applied Sciences, University of Tsukuba, Tsukuba, Japan 160 Institute of Pure and Applied Sciences, University of Tsukuba, Tsukuba, Japan 161 Science and Technology Center, Tufts University, Medford, MA, United States 161 Science and Technology Center, Tufts University, Medford, MA, United States 62 Centro de Investigaciones, Universidad Antonio Narino, Bogota, Colom ATLAS Collaboration / Physics Letters B 718 (2013) 841–859 a Also at Laboratorio de Instrumentacao e Fisica Experimental de Particulas – LIP, Lisboa, Portugal. t Also at Manhattan College, New York, NY, United States. u Also at School of Physics, Shandong University, Shandong, China u Also at School of Physics, Shandong University, Shandong, China. y g y g v Also at CPPM, Aix-Marseille Université and CNRS/IN2P3, Marseill w Also at School of Physics and Engineering, Sun Yat-sen University, Guanzhou, China. w Also at School of Physics and Engineering, Sun Yat-sen University, Guanzhou, China. x Also at Academia Sinica Grid Computing, Institute of Physics, Academia Sinica, Taipei, Taiwan. x Also at Academia Sinica Grid Computing, Institute of Physics, Academia Sinica, Taipei, Taiwan y Also at Dipartimento di Fisica, Università La Sapienza, Roma, Ital Also at Dipartimento di Fisica, Università La Sapienza, Roma, Italy. z Also at DSM/IRFU (Institut de Recherches sur les Lois Fondamentales de l’Univers), CEA Saclay (Commissariat a l’Energie Atomique), Gif-sur-Yvette, France. aa Also at Section de Physique, Université de Genève, Geneva, Switzerland. p p y z Also at DSM/IRFU (Institut de Recherches sur les Lois Fondamentales de l’Univers), CEA Saclay (Commissariat a l’Energie Atomiq z Also at DSM/IRFU (Institut de Recherches sur les Lois Fondamentales de l’Univers), CEA Saclay (Com z Also at DSM/IRFU (Institut de Recherches sur les Lois Fondament z Also at DSM/IRFU (Institut de Recherches sur les Lois Fondamentales de l’Univers), C / ( ), y ( g q ), , . aa Also at Section de Physique, Université de Genève, Geneva, Switzerland. aa Also at Section de Physique, Université de Genève, Geneva, Switzerland. aa Also at Section de Physique, Université de Genève, Geneva, Switzerland. so at Departamento de Fisica, Universidade de Minho, Braga, Portug ac Also at Department of Physics and Astronomy, University of South Carolina, Columbia, SC, United States. ad Also at Institute for Particle and Nuclear Physics, Wigner Research Centre for Physics, Budapest, Hungary. ad Also at Institute for Particle and Nuclear Physics, Wigner Research Centre for Physics, Budapest, Hung d Also at Institute for Particle and Nuclear Physics, Wigner Researc e Also at California Institute of Technology, Pasadena, CA, United S af Also at Institute of Physics, Jagiellonian University, Krakow, Poland. af Also at Institute of Physics, Jagiellonian University, Krakow, Poland. ag Also at LAL, Université Paris-Sud and CNRS/IN2P3, Orsay, France. ag Also at LAL, Université Paris-Sud and CNRS/IN2P3, Orsay, France. ah Also at Nevis Laboratory, Columbia University, Irvington, NY, United States. ah Also at Nevis Laboratory, Columbia University, Irvington, NY, United States. a Also at Laboratorio de Instrumentacao e Fisica Experimental de Particulas – LIP, Lisboa, Portugal. Also at Department of Physics and Astronomy, University of Sheﬃe ai Also at Department of Physics and Astronomy, University of Sheﬃeld, Sheﬃeld, United King aj Also at Department of Physics, Oxford University, Oxford, United Kingdom. aj Also at Department of Physics, Oxford University, Oxford, United Kingdom. ak Also at Institute of Physics, Academia Sinica, Taipei, Taiwan. ak Also at Institute of Physics, Academia Sinica, Taipei, Taiwan. al Also at Department of Physics, The University of Michigan, Ann Arbor, MI, United States. al Also at Department of Physics, The University of Michigan, Ann Arbor, MI, United ∗Deceased.
https://openalex.org/W2965990483	https://www.nature.com/articles/s41598-019-47393-0.pdf	English	null	Magnetic nanocomposites decorated on multiwalled carbon nanotube for removal of Maxilon Blue 5G using the sono-Fenton method	Scientific reports	2,019	cc-by	8,836	Magnetic nanocomposites decorated on multiwalled carbon nanotube for removal of Maxilon Blue 5G using the sono-Fenton method Mehmet Salih Nas1, Esra Kuyuldar2, Buse Demirkan2, Mehmet Harbi Calimli3, Ozkan Demirbaş4 & Fatih Sen 2 Received: 8 May 2019 Accepted: 12 July 2019 Published: xx xx xxxx Herein, multiwalled carbon nanotube-based Fe3O4 nano-adsorbents (Fe3O4@MWCNT) were synthesized by ultrasonic reduction method. The synthesized nano-adsorbent (Fe3O4@MWCNT) exhibited efficient sonocatalytic activity to remove Maxilon Blue 5G, a textile dye, and present in a cationic form, in aqueous solution under ultrasonic irradiation. The magnetic nano-adsorbent particles were characterized by high-resolution transmission electron microscopy (HR-TEM), transmission electron microscopy (TEM), Raman spectroscopy and X-ray diffraction (XRD). Some important parameters such as nano-adsorbent dosage, solution pH, initial dye and H2O2 concentration, reaction time, ultrasonic power and temperature were tested to determine the optimum conditions for the elimination of Maxilon Blue 5G dye. The reusability results showed that Fe3O4@MWCNT nano- adsorbent has a decrease of about 32.15% in the removal efficiency of Maxilon Blue 5G under ultrasonic irradiation after six times reuse. Additionally, in order to reveal the sufficient kinetic explanation, various experiments were performed at different temperatures and testing three kinetic models like the pseudo-first-order, pseudo-second-order and intraparticle diffusion for removal adsorption process of Maxilon Blue 5G using Fe3O4@MWCNT nano-adsorbent. The experimental kinetic results revealed that the adsorption process of Maxilon Blue 5G in the aquatic mediums using sono-Fenton method was found to be compatible with the intraparticle diffusion. Using kinetic models and studies, some activation parameters like enthalpy, entropy and Gibbs free energy for the adsorption process were calculated. The activation parameters indicated that Fe3O4@MWCNT nano-adsorbent could be used as an effective adsorbent for the removal of Maxilon Blue 5G as a textile dye and the adsorption process of Maxilon Blue 5G with Fe3O4@MWCNT nano-adsorbent is spontaneous. The dyes are one of the classes of chemical compounds that present as the severe hazards in industrial waste- water1. The water pollution caused by dyes poses severe threats to human health. Some diseases such as allergy, dermatitis, skin irritation, cancer, and mutation occur related to dye polluted waters1–3. Most of the industrial pro- duction facilities, including the textile industry, produce a lot of colored effluents which poses a severe threat to water resources4,5. The removal of synthetic wastes from water sources poses a serious threat due to the high dye content and low biodegradability compared to other dyestuffs6,7. It is essential to remove organic substances from water sources for a sustainable environment8. Many studies have been performed to develop effective solutions for the removal of toxic chemical substances from organic dyes9–12. www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports Experimental i l d p Materials and methods. FeCl2.4H2O, FeCl3.6H2O, Potassium permanganate (KMnO4), dimethylfor- mamide, ethanol, hydrogen peroxide (H2O2), NaOH, sulfuric acid (H2SO4), sodium nitrate (NaNO3), 1,2-tetra- decanediol, acetone, and hexanes were purchased from Sigma-Aldrich. Additionally, the natural carbon nanotube chips were supplied from Alfa-Aesar@ company. Experimental studies were carried out with ultrasonic tip soni- cator (Bandelin, 40 kHz, 650 W). Synthesis of Fe3O4@MWCNT nano-adsorbents. The synthesis of Fe3O4@MWCNT nano-adsorbents was achieved by ultrasonic reduction method. Typically, 0.02 g of FeCl2.4H2O and 0.06 g of FeCl3.6H2O were dissolved in 200 mL inert gases purged deonized water. Then 100 mL of 1 M NaOH solution was added into the mixture FeCl2.4H2O and 0.06 g of FeCl3.6H2O solutions and they were stirred vigorously until obtaining of the black colloidal suspension of Fe3O4 nano-adsorbents. The obtained nano-adsorbents solution was mixed with 0.00025 g/mL of MWCNT under ultrasonication. The mixture was continued to stirring for 2 days at room tem- perature. After the obtaing of Fe3O4@MWCNT, they were separated by a magnet, and then washed at least 3 times and dried under inert atmosphere. Experimental adsorption procedure of Maxilon Blue 5G using Fe3O4@MWCNT nano-adsorbents under sono-Fenton waves. The samples for the characterization process were pre- pared by taking a 5 mL solution containing Fe3O4 and MWCNT. This solution was divided into 8 tubes with 5 × 100 volumes and then centrifuged. The formed precipitates were filtered and dried under inert medium, and then stored for further analysis. The X-ray diffraction (XRD) analysis of Fe3O4@MWCNT nano-adsorbent was performed using an analytical Empyrean diffractometer capable of X-ray diffraction (Cu K, λ = 1.54056 Å, at 45 kV and 40 Ma). Transmission electron microscopy (TEM) analysis was conducted with a JEOL 200 kV instru- ment. To take TEM analysis various sample was taken to prepare colloidal slurry and the resulting mixtures were dropped on Cu-TEM grid comprised of carbon. The mean particles size of Fe3O4@MWCNT nano-adsorbent were calculated counting diameter of regions present in TEM patterns. The resulting solution was mixed using Maxilon Blue 5G and Fe3O4@MWCNT in the dark condition to ensure adsorption-desorption balance for 15 min. The desired temperature, pH, Maxilon Blue 5G concentration at a given initial concentration of H2O2 (2 mM) and ultrasonic power were adjusted. 4 ml samples were also taken at regular intervals from the reaction solution medium. Then the wastewater is separated by centrifugation (Sigma 3–30 KS) at 15000 rpm and 10 minutes. Magnetic nanocomposites decorated on multiwalled carbon nanotube for removal of Maxilon Blue 5G using the sono-Fenton method Mehmet Salih Nas1, Esra Kuyuldar2, Buse Demirkan2, Mehmet Harbi Calimli3, Ozkan Demirbaş4 & Fatih Sen 2 Recently, researchers have effectively utilized different methods based on oxidation techniques to remove toxic organic pollutants13,14. Efforts are being made 1Department of Environmental, Faculty of Engineering, University of Igdir, Igdir, Turkey. 2Sen Research Group, Department of Biochemistry, Faculty of Arts and Science, Dumlupınar University, Evliya Çelebi Campus, 43100, Kütahya, Turkey. 3Tuzluca Vocational High School, Igdir University, Igdir, Turkey. 4Department of Chemistry, Faculty of Science and Literature, University of Balikesir, Balikesir, Turkey. Correspondence and requests for materials should be addressed to M.S.N. (email: fatih.sen@dpu.edu.tr) or F.S. (email: mehmet.salih.nas@igdir.edu.tr) Scientific Reports \| (2019) 9:10850 \| https://doi.org/10.1038/s41598-019-47393-0 Scientific Reports \| (2019) 9:10850 \| https://doi.org/10.1038/s41598-019-47393-0 1 1 www.nature.com/scientificreports/ intensively on suitable and efficient technological techniques for the removal of pollutants. One of these methods is the ultrasonic and the Fenton process, which contains an oxidation process15. In heterogeneous Fenton-like processes, OH− radicals are formed as Fe3+ ions and are converted into Fe2+ ions (Reactions 1, 2). Another way to obtain OH− radicals is a fracturing water molecule by sending ultrasound waves (eq. (3)) through cavitation phenomena processes15. Hydrogen peroxide could be released by OH− radicals under the influence of ultrasonic radiation in a solution medium (eq. (4))13–17. (1) (2) (3) (4) (1) (3) (4) There are some cavitation phenomena such as microbubble formation, precipitation, as well as pressure due to the temperature factor under the ultrasonic wave in the solution environment18,19. Furthermore, removal of organic materials by ultrasonic wave method is limited; because a long reaction process is required20. Generally, iron-containing nano-adsorbent can be exceeded by integrating the Fenton-like application process to eliminate this obstacle15. There are also some disadvantages due to the problems such as the removal of the nano-adsorbent from the wastewater and the accumulation of Fe+3 ions in the environment. To overcome this problem, the Fenton processes can be addressed by resorting to heterogeneous catalytic applications21,22. Recently, research- ers have been interested in the use of particles as catalysts23–32. Especially, magnetic nanoparticles provides an opportunity to remove dyes from water sources using nano-adsorbents which have an external magnetic field in heterogeneous Fenton systems33,34. This will allow for quick, efficient and easy separation of the magnetic nano- particles from the water sources33. For this purpose, in this study, Fe3O4@MWCNT were synthesized and used as a nano-adsorbent and not only exhibited a high sonocatalytic activity but also high stability, reusability, and easy application for the removal of Maxilon Blue 5G in aquatic mediums. Magnetic nanocomposites decorated on multiwalled carbon nanotube for removal of Maxilon Blue 5G using the sono-Fenton method Mehmet Salih Nas1, Esra Kuyuldar2, Buse Demirkan2, Mehmet Harbi Calimli3, Ozkan Demirbaş4 & Fatih Sen 2 Fe3O4@MWCNT nano-adsorbent com- bined with sono Fenton technique is a non-toxic, cheap and effective solution for the removal of Maxilon Blue 5G from the solution medium. Fe3O4@MWCNT is an effective example of a new magnetic nano-adsorbent in the sonocatalytic removal of the dye material by the Fenton-like process method. In this study, various parameters such as initial dye concentration, efficient of absorbent, pH, H2O2 concentration and ultrasonic power (US) were investigated under specific standard parameters. Moreover, the reaction mechanism and the parameters of the thermodynamic function were also studied. Scientific Reports \| (2019) 9:10850 \| https://doi.org/10.1038/s41598-019-47393-0 Experimental i l d The measurements were taken by a UV-Vis spectrometer (Perkin Elmer Lambda 750) to determine the concentration of the Maxilon Blue 5G at 410 nm wavelength. The removal efficiency of the dye material was determined from the equation given below. Scientific Reports \| (2019) 9:10850 \| https://doi.org/10.1038/s41598-019-47393-0 2 www.nature.com/scientificreports/ Figure 1. XRD image of MWCNT, Fe3O4 and Fe3O4@MWCNT nano-adsorbents. Figure 1. XRD image of MWCNT, Fe3O4 and Fe3O4@MWCNT nano-adsorbents. Figure 2. Transmission electron microscopy image, high-resolution transmission electron microscopy image, particle size histogram of magnetic Fe3O4@MWCNT nano-adsorbent. Figure 2. Transmission electron microscopy image, high-resolution transmission electron microscopy image, particle size histogram of magnetic Fe3O4@MWCNT nano-adsorbent. = − . q (C C ) V/m t 0 t where, C0 and Ct (mg/L) are the dye concentration for the initial and specific times at equilibrium, respectively. The reusability tests also conducted for the Fe3O4@MWCNT nano-adsorbents. where, C0 and Ct (mg/L) are the dye concentration for the initial and specific times at equilibrium, respectively. The reusability tests also conducted for the Fe3O4@MWCNT nano-adsorbents. Results and Discussion The vibrations created by carbon on the basal plane and the E2g mode form the G band. D and G bands are formed due to the Raman mechanism in double resonance struc- ture. These bands are directly related to lattice structure and particle size [26, 27]. The ratio obtained from the bands D and G (ID/IG) is inversely proportional to the size of the crystalline structure of carbon. The ID/IG ratios of the Fe3O4 nanoparticles supported by MWCNT are very high. Raman spectrum of Fe3O4 is also given in Fig. S2. The effect of experimental conditions on the removal of Maxilon Blue 5G using Fe3O4@ MWCNT nano-adsorbents under ultrasonic waves. In order to compare the effects of experimental conditions on the removal of Maxilon Blue 5G by Fe3O4@MWCNT nano-adsorbent, various conditions such as different nano-adsorbent and dye concentrations, ultrasonic wavelength, H2O2 concentrations, temperatures, and pH were examined and the experimental results achieved at different conditions are given in Fig. 4. The effects of Fe3O4@MWCNT nano-adsorbent concentrations on the removal efficiency. One of the most effective parameters for removing of Maxilon Blue 5G is the amount of nano-adsorbent concentrations. The nano-adsorbent effects were analyzed at pH of 9 for 120 minutes in solutions containing 2 mM H2O2. As shown in Fig. 4(a), the increase in the dosage of Fe3O4@MWCNT magnetic nano-adsorbent was found to be effective for removing Maxilon Blue 5G. As shown in Fig. 4(a), the extraction yield of Maxilon Blue 5G was found to be the highest for the 0.0024 g L−1 Fe3O4@MWCNT magnetic nano-adsorbent dose (about with %98 yield). It can be related to an increase in the number of active catalytic sites due to the increase in the amount of magnetic nano-adsorbent Fe3O4@MWCNT, and therefore, more reactive radicals can be produced. Furthermore, a larger increase in the amount of magnetic nano-adsorbent particles results in a decrease in the efficiency of Maxilon Blue 5G removals in sono adsorption systems. In this case, the addition of the nano-adsorbent may have a clean- ing effect on the %OH− radicals, resulting in a reduction of the Maxilon Blue 5G removal efficiency in the solu- tion medium15. Another reason is that in sonocatalytic heterogeneous systems, excessive screening quantification of ultrasonic waves by the magnetic nano-adsorbent particle prevents the same amount of ultrasonic energy from being absorbed39. Results and Discussion The chemical and morphological analysis of Fe3O4@MWCNT nano-adsorbent. In order to reveal the crystalline structure of the prepared Fe3O4@MWCNT nano-adsorbent, XRD analysis has been con- ducted. The XRD results for Fe3O4@MWCNT nano-adsorbent are given in Fig. 1 and some distinct peaks at about 2θ = 26.3°, 30.1°, 35.4°, 57,1°, and 62.6° are attributed to 220, 311, 400, 511 crystal plane, respectively. Some evident peak which are main peaks of are intensified at 2θ = 35.4°. The crystalline structure of Fe3O4@MWCNT nano-adsorbent is found to be a cubic system. Further, the crystalline size of the prepared nano-adsorbent was calculated as 3.57 nm which is very close to the value found in TEM analysis. XRD analysis also indicated that no other impurities were observed except Fe3O4 present in MWCNT (002, 2θ = 26.3°) samples. These result showed that the structure of Fe3O4@MWCNT nano-adsorbent is crystalline and pure. y TEM and HR-TEM analysis were performed to determine the structural properties of the Fe3O4@MWCNT catalyst. As can be seen from Fig. 2, the mean particle size of the monodisperse Fe3O4@MWCNT nanoparticle was 3.24 ± 0.61 nm which is in good agreement with XRD results. In addition, Fig. 2 shows a uniform distribution of Fe3O4 over the MWCNT without any agglomeration. HR-TEM image also shows that the atomic lattice fringe of Fe3O4@MWCNT nanoparticle is consistent with the literature data (0.21 nm)4. TEM image of Fe3O4 is also given in Fig. S1. Scientific Reports \| (2019) 9:10850 \| https://doi.org/10.1038/s41598-019-47393-0 3 www.nature.com/scientificreports/ www.nature.com/scientificreports ntificreports/ Figure 3. Micro-Raman patterns of Fe3O4@MWCNTnanocomposites (D band of MWCNT near 1340.5 cm−1). igure 3. Micro-Raman patterns of Fe3O4@MWCNTnanocomposites (D band of MWCNT near 1340.5 cm−1). Figure 3. Micro-Raman patterns of Fe3O4@MWCNTnanocomposites (D band of MWCNT near 1340.5 cm−1). For further structural analysis of synthesized Fe3O4@MWCNT nano-adsorbent, Raman35 spectroscopic anal- yses were carried out. Raman spectroscopic analysis (given in Fig. 3) of Fe3O4@MWCNT nano-adsorbent, further details of the structure of Fe3O4@MWCNT nano-adsorbent were revealed in Fig. 3. The modification and struc- tural disorders were controlled by comparing the density ratios of G and D bands. ID/IG ratios for MWCNT and Fe3O4@MWCNT were found to be 0.77 and 0.98, respectively. The findings of Raman spectroscopy showed that MWCNT were functionalized with Fe3O4 36–38. D band in 1340 cm−1 region and G band in 1600cm−1 region were observed in Raman spectra of the prepared materials. Results and Discussion The highest removing amount of the Maxilon Blue 5G by the using of Fe3O4@MWCNT was also detected, as seen in Fig. 4(a). The effects of Maxilon Blue 5G concentrations on the removal efficiency. To investigate the effect of Maxilon Blue 5G dye concentration, some experiments were conducted at different initial concentrations of Maxilon Blue 5G at constant parameters such as 2 mM of H2O2 concentration, 303 K temperature, and pH of 9. By increasing the dye concentration of Maxilon Blue 5G from 0.0012 to 0.0024 g. L−1 in the sono adsorption process, the efficiency of the Maxilon Blue 5G removals increased from 50.2% to 82.1% within 120 min (Fig. 4(b)). The amount of adsorbed dye on the surface of the magnetic nano-adsorbent material is increased when the amount of dye in the solution medium increased, and this prevents absorption of energy produced due to acoustic cavitation by nano-adsorbent particles40. Hence, the percentage of OH− radicals and removing dye capacity will result in a decrease. It can be explained that the removal efficiency of intermediates, which is particularly evident as a result of the interaction of OH− molecules with dye molecules, can be reduced39. Also, it blocks active areas on the surface of Fe3O4@MWCNT as a result of high dye concentration in the solution medium. In this case, it causes the minimal growth of the OH radicals and thus, results in lower Maxilon Blue 5G removal efficiency. Besides, the nitrogen adsorption and desorption isotherms of Fe3O4@MWCNT nano-adsorbents are also given in Fig. S3 Scientific Reports \| (2019) 9:10850 \| https://doi.org/10.1038/s41598-019-47393-0 4 www.nature.com/scientificreports/ in order to explain the higher efficiency of prepared nano-adsorbents. The analysis was performed by evaluating th h t i f it d ti d d ti t i th l diti A di t th l i Figure 4. The removal efficiency of Maxilon Blue 5G using Fe3O4@MWCNT nano-adsorbents at different reaction mediums. (a) Fe3O4@MWCNT Maxilon nano-adsorbent concentrations, (b) Maxilon Blue 5G concentrations, (c) Ultrasonic wavelength, (d) H2O2 concentrations, (e) Temperatures, (f) Solution pH. Maxilon Blue 5G = 20 g L−1, [Fe3O4@MWCNT] = 0.020 g L−1, [H2O2] = 2 mM, pH = 9, time = 120 min). Figure 4. The removal efficiency of Maxilon Blue 5G using Fe3O4@MWCNT nano-adsorbents at different Figure 4. The removal efficiency of Maxilon Blue 5G using Fe3O4@MWCNT nano-adsorbents at different reaction mediums. The zero-load point of the Fe3O4@ MWCNT magnetic nano-adsorbent was determined to be 6.8 by the method specified in the literature46. When the pH of the solution lower than the zero-load point of the Fe3O4@MWCNT magnetic nano-adsorbent, the sur- face of the nano-adsorbent material is protonated. Similarly, the surface of the nano-adsorbent is deprotonated when a higher pH value applied47. For this reason, the cationic dye can be adsorbed onto the Fe3O4@MWCNT nano-adsorbent, and the surface binding domains of the nano-adsorbent material are affected. Therefore, the ionic state of the Maxilon Blue 5G molecule has great importance. At low pH, the nano-adsorbent surface charge is positively charged and H+ the ions encounter an impulsive force effectively with the Maxilon Blue 5G cations, thus causing a reduction in the amount of adsorbed dye. At higher pH values, the magnetic nano-adsorbent par- ticle increases the negatively charged density. By this way, the electrostatic attraction forces between the support material and the cationic dye can be increased48,49. Besides, as shown in Table S1, the iron ion concentration in the solution medium increased at high pH. This relates to both the absolute concentration of dissolved iron and the increased dissociation of OH− radicals of H2O2 molecules in the heterogeneous sono-Fenton process50. As a result, the presence of % OH− radicals also significantly affected the electrostatic attraction between the nano-adsorbent and the dye. The most efficient removal was achieved at an optimum pH value of 11 (Fig. 4(f)). The comparisons of some parameters studied for Maxilon Blue 5G removal using Fe3O4@MWCNT nano-adsorbent and their reusability efficiency. The experiments of removal of Maxilon Blue 5G conducted at different param- eters were carried out at pH of 9 with 10 mgL−1 of Fe3O4@MWCNT nano-adsorbent; the results of these experi- ments are given in Fig. 5a. As indicated in Fig. 5a, the efficiency of Maxilon Blue 5G removal was determined to be approximately 3.75% and 5.72% after operating the system for 120 minutes using ultrasonic wave and H2O2, respectively. The data obtained under these experimental conditions show how stable Maxilon Blue 5G is. The efficiency of Fe3O4@MWCNT magnetic nano-adsorbent in removing the Maxilon Blue 5G dye was not nearly at the desired level. Among the different compositions of the prepared nano-adsorbents as shown in Fig. 5a, Fe3O4@ MWCNT/H2O2 nano-adsorbent exhibited a best efficiency for the removal of Maxilon Blue 5G compared to the others. As seen in Fig. Results and Discussion (a) Fe3O4@MWCNT Maxilon nano-adsorbent concentrations, (b) Maxilon Blue 5G concentrations, (c) Ultrasonic wavelength, (d) H2O2 concentrations, (e) Temperatures, (f) Solution pH. Maxilon Blue 5G = 20 g L−1, [Fe3O4@MWCNT] = 0.020 g L−1, [H2O2] = 2 mM, pH = 9, time = 120 min). in order to explain the higher efficiency of prepared nano-adsorbents. The analysis was performed by evaluating the hysteresis curve of nitrogen adsorption and desorption at isothermal conditions. According to the analysis, it is known that the Fe3O4/MWCNT nanocomposites have a large surface area, namely 335 m2/g. Such large area would improve the performance of the adsorption property of the nanocomposites and very appropriate for removal of maxilon 5G. in order to explain the higher efficiency of prepared nano-adsorbents. The analysis was performed by evaluating the hysteresis curve of nitrogen adsorption and desorption at isothermal conditions. According to the analysis, it is known that the Fe3O4/MWCNT nanocomposites have a large surface area, namely 335 m2/g. Such large area would improve the performance of the adsorption property of the nanocomposites and very appropriate for removal of maxilon 5G. The effects of H2O2 concentrations, ultrasonic wavelength, temperatures, and solution pH on the removal of Maxilon Blue 5G from aqueous medium. Figure 4 also shows the effects of H2O2 concentrations (Fig. 4c), ultrasonic wavelength (Fig. 4d), temperatures (Fig. 4e), and solution pH (Fig. 4f) on the removal Maxilon Blue 5G from aqueous medium. In this study, the effect of H2O2 concentration on the elimination of the dye in the solution Scientific Reports \| (2019) 9:10850 \| https://doi.org/10.1038/s41598-019-47393-0 5 www.nature.com/scientificreports/ medium was checked. In heterogeneous Fenton-like systems, the concentration of H2O2 has a positive effect on the increase of active radicals41. To investigate the effect of H2O2 at different concentrations, experiments were conducted at the constant parameters such as 0.02 g L−1 Fe3O4@MWCNT, pH of 9 in aqueous solution and 120 minutes of reaction time. The experimental results showed that the removal efficiency of Maxilon Blue 5G was highest at 2 M concentration of H2O2 as shown in Fig. 4c. This situation is considered due to the increase in OH− released to the solution environment. The efficiency of dye elimination decreased at higher hydrogen peroxide (H2O2) concentrations. (6) One of the essential parameters for the removal of Maxilon Blue 5G dye is the amount of ultrasonic power. To determine the effect of the ultrasonic power, the same constant parameter conditions were prepared with 20 mg L−1 of Fe3O4@MWCNT, pH of 9 and 2 mM H2O2 concentration. As indicated in Fig. 4(d), the ultrasonic power effect was seen to be more effective in removal efficiency from 350 W to 450 W. It can be explained this situation on two different mechanisms. Firstly, the increase in ultrasound power increased dissolution turbu- lence. This leading to the higher release of reactive radicals and an increase in the mass transfer rate of Maxilon Blue 5G, which positively contributed to the reduction in the number of by-products present throughout the nano-adsorbent surface13. Secondly, the cleaning of the ultrasonic beam responded positively to the increase in power. It is thought that the increase of ultrasonic irradiation causes further expands of active fields on the surface of the magnetic nano-adsorbent13,45. In can be concluded that the increase in ultrasonic power results in a further increase of reactive radicals. In order to determine the optimum temperature for the adsorption of Maxilon Blue 5G on Fe3O4@MWCNT, a set of experiments at different temperatures ranging 296–323 K was performed. The results of the experiments conducted at different temperatures are given in Fig. 4e. The optimum temperature on the adsorption process was found to be 323 K. Increasing temperature effects the interaction of particle, and that increasing interactions increased the adsorption of Maxilon Blue 5G. additionally, the volumes of pores on the adsorbent is increased with increasing temperature12. These results affected positively the adsorption amount of Maxilon Blue 5G. The effect of pH solution is also a very crucial parameter in the adsorption process to gain the properties of materials investigated under ultrasonic wave iridations41,42. The results for pH effects of the solution containing 0.002 g.L−1 Fe3O4@MWCNT magnetic nanomaterials and 20 mg L−1 Maxilon Blue 5G in 120 minutes to remove the Maxilon Blue 5G are given in Fig. 4(f). The highest yield was obtained at a pH of 11. These might be explained by two reasons. The situation of the surface of nano-adsorbent affects the values of pH, and it can be explained according to the zero-load point of Fe3O4@MWCNT. Results and Discussion This is because the hydrogen peroxide (H2O2) added after a certain point in the sonocatalytic heterogeneous processes was found to interfere with the interaction between the surface of the nano-adsorbent material and the dye material. As stated in eqs (5) and (6), excessive concentration of hydrogen peroxide in the solution medium can induce OH− radical scavenging effect42 and cause reduction of radicals required for oxidation43,44. (5) (6) (5) As shown in Fig. 5b, six successive dye-extraction yields were obtained in % 88.51, 80.72, 75.56, 73.09, 70.16, and %67.85 respectively. The obtained data in Fig. 5b demonstrate the reusability of Fe3O4@MWCNT magnetic nano-adsorbents for treatment of wastewater. TEM image of used nano-adsorbents was obtained as shown in Fig. S4 and it was seen that some of the particles was agglomerated which results in the decrease of the reusability efficiency. We have also checked the content of the catalyst with the help of ICP (Inductively Coupled Plasma spectroscopy) in order to see whether there is any leaching in nanocomposite or not and we have seen that there was no leaching in nanocomposite.i ollapse on the surface of the magnetic nano-adsorbent51,52. The amount of iron and OH− radicals present on he surface of the nanoparticles increased the efficiency of the Fenton-like process depend on eqs ((1) and (2))9. fi Another most critical parameters of nano-adsorbents in the removal of dye studies is the reusability tests conducted to investigate the stability of the synthesized materials10. The stability of Fe3O4@MWCNT magnetic nano-adsorbents was investigated reusability in 6 sequential reuse at fixed parameters with 0.020 g L−1 nano-adsorbent, 20 mg L−1 Maxilon Blue 5G dye, 2 mM H2O2, pH of 9, 120 minutes. The magnetic nano-adsorbents were magnetically separated from the treated solution after each treatment and washed using ultrapure water, dried and reused for subsequent work (Fig. 5b). As shown in Fig. 5b, six successive dye-extraction yields were obtained in % 88.51, 80.72, 75.56, 73.09, 70.16, and %67.85 respectively. The obtained data in Fig. 5b demonstrate the reusability of Fe3O4@MWCNT magnetic nano-adsorbents for treatment of wastewater. TEM image of used nano-adsorbents was obtained as shown in Fig. S4 and it was seen that some of the particles was agglomerated which results in the decrease of the reusability efficiency. We have also checked the content of the catalyst with the help of ICP (Inductively Coupled Plasma spectroscopy) in order to see whether there is any leaching in nanocomposite or not and we have seen that there was no leaching in nanocomposite.i g p g p Also, the recycling of the Fe3O4@MWCNT nano-adsorbent film from water is crucial to prevent further contamination during wastewater treatment in industrial applications because the nanoadsorbent can be easily removed from the water using magnetic force. 5a, the conversion of H2O2 to free OH radicals in the Fe3O4@MWCNT/H2O2 system positively increased the oxidation process in experimental studies (6). As stated in the eq. (1) the interaction between active sites of the Fe3O4@MWCNT surface and H2O2 supplied a positive increase in the percentage of OH− radicals. In the US/Fe3O4@MWCNT/H2O2 working process, the magnetic nano-adsorbents interacted with ultrasonic waves and produced a higher contact area of magnetic nano-adsorbent as a support material50. The het- erogeneous catalytic efficiency has been enhanced because of the formation of cavitation microbubbles and their Scientific Reports \| (2019) 9:10850 \| https://doi.org/10.1038/s41598-019-47393-0 6 www.nature.com/scientificreports/ www.nature.com/scientificreports ntificreports/ Figure 5. (a) The effects of different experimental conditions on the removal of Fe3O4@MWCNT Ultrasonic waves (A), H2O2 concentrations (B), Fe3O4@MWCNT concentrations (C), Fe3O4@MWCNT/H2O2 (D), Fe3O4@ MWCNT/Ultrasonic wavelength (E), Fe3O4@MWCNT/Ultrasonic wavelength/H2O2 (F). (b) Reusability tests of Fe3O4@MWCNTnano-adsorbent in the Maxilon Blue 5G aqueous solution. (c) Absorbance change by time: Maxilon Blue 5G aqueous solution containing Fe3O4@MWCNT adsorbent at 300–500 nm. [Maxilon Blue 5G] = 20 mg L−1, [Fe3O4@MWCNT] = 0.020 g L−1, [H2O2] = 2 mM, UP = 350 W, pH = 9 and time = 120 min). Figure 5. (a) The effects of different experimental conditions on the removal of Fe3O4@MWCNT Ultrasonic waves (A), H2O2 concentrations (B), Fe3O4@MWCNT concentrations (C), Fe3O4@MWCNT/H2O2 (D), Fe3O4@ MWCNT/Ultrasonic wavelength (E), Fe3O4@MWCNT/Ultrasonic wavelength/H2O2 (F). (b) Reusability tests of Fe3O4@MWCNTnano-adsorbent in the Maxilon Blue 5G aqueous solution. (c) Absorbance change by time: Maxilon Blue 5G aqueous solution containing Fe3O4@MWCNT adsorbent at 300–500 nm. [Maxilon Blue 5G] = 20 mg L−1, [Fe3O4@MWCNT] = 0.020 g L−1, [H2O2] = 2 mM, UP = 350 W, pH = 9 and time = 120 min). collapse on the surface of the magnetic nano-adsorbent51,52. The amount of iron and OH− radicals present on the surface of the nanoparticles increased the efficiency of the Fenton-like process depend on eqs ((1) and (2))9. Another most critical parameters of nano-adsorbents in the removal of dye studies is the reusability tests conducted to investigate the stability of the synthesized materials10. The stability of Fe3O4@MWCNT magnetic nano-adsorbents was investigated reusability in 6 sequential reuse at fixed parameters with 0.020 g L−1 nano-adsorbent, 20 mg L−1 Maxilon Blue 5G dye, 2 mM H2O2, pH of 9, 120 minutes. The magnetic nano-adsorbents were magnetically separated from the treated solution after each treatment and washed using ultrapure water, dried and reused for subsequent work (Fig. 5b). The absorbance changes of Maxilon Blue 5G containing Fe3O4@ MWCNT adsorbent at 300–500 nm (Maxilon Blue 5G of 20 mg L−1, Fe3O4@MWCNT of 0.020 g L−1, H2O2 of 2 mM, UP of 350 W, pH of 9) for 120 min are shown in Fig. 5c. The maximum absorbance value was obtained as >90%. Figure 5c shows the initial and latest solution color and absorbance values for Maxilon Blue 5G aqueous solution containing Fe3O4@MWCNT adsorbent. As shown in this figure, Maxilon Blue 5G lost its color during the adsorption process after interaction with Fe3O4@MWCNT adsorbent.Fig. 6a,b shows adsorption capacity of Fe3O4 nano-adsorbent and its adsorption amount from aqueous mediums. As seen in Fig. 6a the initial con- centration of Maxilon Blue 5G was investigated in concentrations ranging 5–40 mg/L for 120 min. The highest adsorption value of Maxilon Blue 5G on Fe3O4@MWCNT nano-adsorbents was obtained as 25 mg/L. Figure 6b shows that the maximum adsorption capacity of Maxilon Blue 5G on Fe3O4@MWCNT nano-adsorbent was reached in almost 60 min. and after this time the adsorption process has reached the equilibrium. According to Scientific Reports \| (2019) 9:10850 \| https://doi.org/10.1038/s41598-019-47393-0 7 www.nature.com/scientificreports/ Figure 6. The adsorption capacities for Maxilon Blue 5G at different concentrations by the Fe3O4@MWCNT nanoparticles, the initial concentration of Maxilon Blue 5G is 5–40 mg/L and the adsorption time is 120 min; (b) Percentage removal for Maxilon Blue 5G by the Fe3O4@MWCNT nanoparticles. Figure 6. The adsorption capacities for Maxilon Blue 5G at different concentrations by the Fe3O4@MWCNT nanoparticles, the initial concentration of Maxilon Blue 5G is 5–40 mg/L and the adsorption time is 120 min; (b) Percentage removal for Maxilon Blue 5G by the Fe3O4@MWCNT nanoparticles. btained data, the Fe3O4@MWCNT magnetic nano-adsorbents proved to be a very effective nano-adsorbent to emove Maxilon Blue 5G under different parameters. Kinetic parameters and their calculation for sono -Fenton-like method. Three models were used to find the sufficient kinetic model for the adsorption of Maxilon Blue 5G using Fe3O4@MWCNT magnetic nano-adsorbent through heterogeneous under the ultrasonic irradiations. The equations used in the calculation to determine the sufficient model are given formulas53, where the t is time (min.), ki is adsorption rate constant, qe, and qt are the initial and final concentration (mol. g−1) of Maxilon Blue 5G dye, respectively. The calculation results obtained from the models are seen in Table S1. Equations of 7, 8 are the first order and second-order models, respectively54. In eq. (8); time is t, k2 is a constant rate at the adsorption equilibrium, the Maxilon Blue 5G amount is qe (mol. min−1). Equation (9) was used to calculate halftime of adsorption process for Maxilon Blue 5G with Fe3O4@MWCNT nano-adsorbent under ultrasonic wave irradiations. The eq. (10) is used to calculate the initial adsorption rate, h (mol/(g min) and in the values of t1/2, k2 and qe were calculated and given in Table S1. The initial rate of the intraparticle diffusion is calculated using eq. (11)55. − = − ln(q q ) lnq k t (7) e t e i = + t qe k q q t 1 1 (8) e e 2 2 = t k q 1 (9) e 1/2 2 = h k q (10) e 2 = + q k t C (11) t int 1/2 (7) (8) (9) = h k qe 2 (10) = + q k t C t int 1/2 = + q k t C (11) t int 1/2 (11) Table S2 shows kint values (mg (g min−1/2)−1 calculated from the intra-particle diffusion model. The studies in the literature revealed that the slopes between qt and t1/2 are multilinear; the graph of qt with t1/2 is multi- linear52. In the adsorption process of Maxilon Blue 5G containing Fe3O4@MWCNT nano-adsorbent, the first stage of the adsorption process is compatible with the intraparticle. In Fig. 6 the first portion curve exhibited the boundary layer effect in the adsorption process and the second curve shows the effect of the intraparticle and diffusion in pores. Table S2 shows the kint1,2 values. The first plot values are so high, and these values are not sufficient for the first stage. kint2 is used in the intraparticle diffusion and is compatible with the second linear plot (mol.g.mol−1/2)56. The ln [(Ct.Co−1)−1(1 + mK)] is used for obtaining R12 and R2 2 calculated values57 and its values are seen in Table S2. The model of mass transfer equations values with particle distribution equations is not appropriates for the adsorption of Maxilon Blue 5G on Fe3O4@MWCNT nano-adsorbent. The calculation of thermodynamic parameters. To calculate the activation parameters for the adsorp- tion of Maxilon Blue 5G using Fe3O4@MWCNT nano-adsorbent from the aqueous medium, Arrhenius Equation (eq. 12) and k2 values were used as shown in Fig. S5. References 1. Brookstein, D. S. Factors Associated with Textile Pattern Dermatitis Caused by Contact Allergy to Dyes, Finishes, Foams, and Preservatives. Dermatol. Clin. 27, 309–322 (2009). 2. Carneiro, P. A., Umbuzeiro, G. A., Oliveira, D. P. & Zanoni, M. V. B. Assessment of water contamination caused by a mutagenic textile effluent/dyehouse effluent bearing disperse dyes. J. Hazard. Mater. 174, 694–699 (2010).h fl yfl g p y 3. Olivares-Marín, M. et al. The development of an activated carbon from cherry stones and its use in the removal of ochratoxin A from red wine. Food Control 20, 298–303 (2009). 4. Farhadi, S., Siadatnasab, F. & Khataee, A. Ultrasound-assisted degradation of organic dyes over magnetic CoFe2O4@ZnS core-shel nanocomposite. Ultrason. Sonochem. 37, 298–309 (2017).f p 5. Modirshahla, N., Behnajady, M. A., Rahbarfam, R. & Hassani, A. Effects of Operational Parameters on Decolorization of C. I. Acid Red 88 by UV/H 2O 2 Process: Evaluation of Electrical Energy Consumption. Clean - Soil, Air, Water 40, 298–302 (2012).i 5. Modirshahla, N., Behnajady, M. A., Rahbarfam, R. & Hassani, A. Effects of Operational Parameters on Decolorization of C. I. Acid Red 88 by UV/H 2O 2 Process: Evaluation of Electrical Energy Consumption. Clean - Soil, Air, Water 40, 298–302 (2012). 6. Hassani, A. et al. Ultrasound-assisted adsorption of textile dyes using modified nanoclay: Central composite design optimization. Korean J. Chem. Eng. 33, 178–188 (2016). 6. Hassani, A. et al. Ultrasound-assisted adsorption of textile dyes using modified nanoclay: Central composite design optimization Korean J. Chem. Eng. 33, 178–188 (2016). g 7. Khataee, A., Sheydaei, M., Hassani, A., Taseidifar, M. & Karaca, S. Sonocatalytic removal of an organic dye using TiO2 Montmorillonite nanocomposite. Ultrason. Sonochem. 22, 404–411 (2015). p 8. Gürses, A., Hassani, A., Kıranşan, M., Açışlı, Ö. & Karaca, S. Removal of methylene blue from aqueous solution using by untreated lignite as potential low-cost adsorbent: Kinetic, thermodynamic and equilibrium approach. J. Water Process Eng. 2, 10–21 (2014). 9 Kh t A Gh l i P & V hid B H t F t lik i t t d it d b A l 8. Gürses, A., Hassani, A., Kıranşan, M., Açışlı, Ö. & Karaca, S. Removal of methylene blue from aqueous solution using by untreated lignite as potential low-cost adsorbent: Kinetic, thermodynamic and equilibrium approach. J. Water Process Eng. 2, 10–21 (2014). 9. Khataee, A., Gholami, P. & Vahid, B. www.nature.com/scientificreports/ www.nature.com/scientificreports/ Vice versa the adsorption processes which having enthalpies higher than 40 k J.mol−1 was considered as chemical processes58. The following eqs (12 and 13) are used to calculate the other activation parameters57. Vice versa the adsorption processes which having enthalpies higher than 40 k J.mol−1 was considered as chemical processes58. The following eqs (12 and 13) are used to calculate the other activation parameters57. = − . lnk lnA Ea R T (12) 2 = + Δ −Δ ln k T ln h S R H RT ( / ) (kb/ ) (13) 2 = − . lnk lnA Ea R T (12) 2 (12) = + Δ −Δ ln k T ln h S R H RT ( / ) (kb/ ) (13) 2 (13) Where; enthalpy, entropy, adsorption rate, Boltzmann constant, gas constant and Planck constant (6.6261.10−34 Js) are ∆H, ∆S, k2, kb, R (1.3807.10−23 JK−1) and h, respectively.h The results for these activation parameters and kinetic data are given in Table S3. Fig. S5a,b shows Arrhenius plots for calculations the adsorption parameters for removal Maxilon Blue 5B dye. The value of ∆S (entropy change) was founded to be −94 J.K.mol−1. This value indicates that the Maxilon Blue 5G dye was distributed regularly on the Fe3O4@MWCNT magnetic nano-adsorbent. The results also revealed that the adsorption mech- anism for Maxilon Blue 5G dye containing Fe3O4@MWCNT magnetic nano-adsorbent occurs spontaneously. It was determined that the sonocatalytic removal of the magnetic nano-adsorbent particle was suitable for Langmuir-Hinshelwood kinetic expression by looking at the obtained regression coefficient (R2 = 0.9930). The calculation activation parameters were performed using eq. (14) as given below; Δ = Δ − . Δ G H T S (14) (14) Δ = Δ − . Δ G H T S The calculated values of the adsorption of Maxilon Blue 5G dye on the Fe3O4@MWCNT surface were given in Table S3. Conclusion In this work, Fe3O4@MWCNT magnetic nano-adsorbent particles were synthesized by ultrasonic reduction method. The nano-adsorbent particles from the data obtained in experimental studies were found to have extreme sonocatalytic efficiency in eliminating dyes from aqueous medium under ultrasonic condition. It has been proved that Maxilon Blue 5G dye is successfully removed from the aqueous solution by using Fe3O4@MWCNT as the adsorbent material and with the help of ultrasonication, separately. The experimental process reached a disposal efficiency of %92 at pH of 9 after a 120-minute reaction period. The obtained data showed that OH. radicals play a significant role in the removal of Maxilon Blue 5G dye by the sonocatalytic method in the presence of Fe3O4@ MWCNT magnetic nano-adsorbent. Moreover, the reusability test has shown the stability of Fe3O4@MWCNT magnetic nano-adsorbents with very high sonocatalytic removal efficiency under optimum conditions. The thermodynamic parameters such as Gibbs free energy (ΔG∗), Ea, ΔH, and ΔS were calculated as −61.465, 27.01, 32.325 kJ mol−1 and 94.00 J mol−1 K−1 for removal of Maxilon Blue 5G dye, respectively. According to the calculated values of free Gibbs Energy also shows that the adsorption process occurs spontaneously. It was also determined that the most suitable kinetic model for the adsorption mechanism was intra-particle diffusion mod- els. As a result, the prepared Fe3O4@MWCNT nano-adsorbent is very effective for the removal of the dyes from industrial wastewater. Scientific Reports \| (2019) 9:10850 \| https://doi.org/10.1038/s41598-019-47393-0 In eq. (12), R is gas constant (J.K−1.mol−1), and T is temper- ature (K). The activation energy of Maxilon Blue 5G using Fe3O4 nano-adsorbent was found to be 27 k J.mol−1. Generally, the adsorption process having enthalpies less than 40 k J.mol−1 was considered as physical interactions. 8 Scientific Reports \| (2019) 9:10850 \| https://doi.org/10.1038/s41598-019-47393-0 www.nature.com/scientificreports/ g j 29. Li, T. et al. Ligand-Triggered Tunable Charge Transfer toward Multifarious Photoreduction Catalysis. J. Phys. Chem. C. https://doi. org/10.1021/acs.jpcc.8b11363 (2019). g j 0. Dai, X. C. et al. Regulating spatial charge transfer over intrinsically ultrathin-carbon-encapsulated photoanodes toward solar water splitting. J. Mater. Chem. 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Scientific Reports \| (2019) 9:10850 \| https://doi.org/10.1038/s41598-019-47393-0 10 www.nature.com/scientificreports/ Scientific Reports \| (2019) 9:10850 \| https://doi.org/10.1038/s41598-019-47393-0 Additional Information Supplementary information accompanies this paper at https://doi.org/10.1038/s41598-019-47393-0. Competing Interests: The authors declare no competing interests. Competing Interests: The authors declare no competing interests. Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Cre- ative Commons license, and indicate if changes were made. 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https://openalex.org/W2106750780	https://zenodo.org/records/1260999/files/article.pdf	English	null	Axially polarized wiggler radiation from a toroidal electron beam source	IEEE transactions on magnetics	1,998	public-domain	1,721	IEEE TRANSACTIONS ON MAGNETICS, VOL. 34, NO. 4, JULY 1998 IEEE TRANSACTIONS ON MAGNETICS, VOL. 34, NO. 4, JULY 1998 1405 M. M. Visosky Physics Department, U.S. Military Academy, West Point, NY M. M. Visosky Physics Department, U.S. Military Academy, West Point, NY Abstract - Recent work has described wigglers in which the radiating electron beam circulates azimuthally within a toroidal tube where a surrounding permanent magnet structure keeps the beam on a mean circular path and simultaneously provides the field periodicity for wiggler action.’,’ Both the mean field and the periodic one are in the axial direction so that the resulting synchrotron and wiggler radiations are both polarized in the principal toroidal plane. The present work analyzes a structure in which the synchrotron and wiggler polarizations are mutually orthogonal so that one or the other can be easily eliminated to provide essentially monochromatic radiation. This is accomplished by an azimuthally periodic arrangement of magic ring sections that form a toroidal tube. These are oriented with their axial field components constant and equal and their radial components equal but alternating radially outward and inward in direction. The constant axial field holds the electron beam in a circular path while the alternating radial field provides the wiggler action. The wiggler radiation has n times the frequency of the synchrotron radiation where n is the number of azimuthal periods per circuit. of electron oscillation. Because of the mean circular motion of the beam the wiggler radiation is accompanied by tangentially polarized synchrotron radiation with a frequency of w, = y2w, where w, is the classical frequency of an electron circulating in a magnetic field and y is the relativistic parameter [l-(vlc2]’”. w, = nw, where w, is the wiggler frequency and n the number of wiggler periods in a complete circuit. Both wiggler and synchrotron radiation are propagated tangentially through a circumferential slot cut through the toroidal shell in the equatorial plane. Fig 1. Circular wiggler configuration suggested in reference [l]. Magnetization 1\; Field f Electron Beam - - - -> Index Terms - Wigglers, Sychrotrons, Axial and Radial Polarization, Toroidal Magnets Axially Polarized Wiggler Radiation From A Toroidal Electron Beam Source H. A. Leupold and A. S. Tilak U.S. Army Research Laboratory, U.S. Military Academy, West Point, NY I. INTRODUCTIO Polarization 00, synchrotron polarization /1\ Radiation Direction .+ 1406 Fig 2. An expanded view of a single field-source for both synchrotron and uiggler fields. In this structure iron “teeth” are azimuthally spaced to modulate the field. Fig 4. Orthogonal Polarizations. Wiggler. Polarization 00, synchrotron polarization /1\ Radiation Direction .+ Fig 5. Construction of toroid with constant axial and periodic radial field. A. Orientations of successive magic cyclinder slices. B. Arrangement of slices in a toroid. Fig 4. Orthogonal Polarizations. Wiggler. Polarization 00, synchrotron polarization /1\ Radiation Direction .+ Fig 2. An expanded view of a single field-source for both synchrotron and uiggler fields. In this structure iron “teeth” are azimuthally spaced to modulate the field. In such structures the synchrotron and wiggler radiations have the same equatorial polarizations as shown in Fig 3. It may be undesirable in some cases for the polarizations to be coplanar. I. INTRODUCTIO Fig 1. Circular wiggler configuration suggested in reference [l]. Magnetization 1\; Field f Electron Beam - - - -> Free electron lasers or wigglers are periodic magnetic arrays which provide an accelerating field to an electron beam, forcing it to oscillate transversely about its translational path, thereby causing it to radiate. [ 11 Various rectilinear arrangements of both the electromagnet and permanent magnet varieties are used for this purpose. In such devices, the emitted radiant energy is derived from the translational kinetic energy of the electron beam of which only a small portion is converted to radiation in a single pass of the beam through the array. It has been suggested [1] that in a circular array the beam might make multiple passes to afford longer exposure of the electron beam to the circumferentially alternating magnetic elements as in Figure 1. Such a structure requires an additional axial field source to keep the electron beam on the desired circular path. This source may consist of either electro-magnets or massive permanent magnets. Both options entail undesirable weight and bulk and the former also requires an electric power supply together with its cooling impedimenta. In a previous paper [2] the authors described a toroidal wiggler in which the same permanent magnet source effectively provides both the synchrotron and wiggler functions, through the modulation of a single axial field. In figure 2 is pictured such an array. The cross section of its permanent magnet segments is that of a magic cylinder or ring which was chosen because the fields of such configurations are very high compared to other arrays of similar mass and bulk. The wiggler magnets produce an azimuthally periodic, radial magnetic field which causes the beam to vibrate parallel to the principal toroidal axis. The vibration gives rise to radiation with its electric vector polarized in the direction U.S. Government Work Not Protected by U.S. Copyright Fig 4. Orthogonal Polarizations. Wiggler. Polarization 00, synchrotron polarization /1\ Radiation Direction .+ Fig 5. Construction of toroid with constant axial and periodic radial field. A. Orientations of successive magic cyclinder slices. B. Arrangement of slices in a toroid. It might be necessary to provide an electric solenoid wound about the toroidal tube to furnish an azimuthal columnating field for the electron beams, although this might also be accomplished by judicious perturbations in permanent magnet shape. Fig 4. Orthogonal Polarizations. Wiggler. 111. ANALYSIS AND RESULTS For analysis a structure of moderate size and plausible applicability was chosen. Figure 6 shows the given parameters and Table 1 shows required fields and resulting frequencies as functions of electron energy in the configuration of Figure 5. A Fig 7. A. Approximation of wedged toroidal segments with disk like ones. B. Cross section of a disk set for a field of 0.867 of the maxium. A g g Note the high wiggler accelerating fields of over 7kG in the militarily interesting giga and terahertz regions. y gg g g For lower electron beam energies the axial field can be made smaller to enhance the wiggler field amplitude at its expense through the change in slice orientation. Bore Radius (ri) 1 cm Principal Toroidal Radius ( r ~ ) Outer Radius of Toroidal Tube (ro) magnetic Remanence 12 cm 2 cm 12 kG (1.2 T) &,is Fig 6. Dimensions and Parameters used in analytic calculations Fig 7. A. Approximation of wedged toroidal segments with disk like ones. B. Cross section of a disk set for a field of 0.867 of the maxium. 11. AXIALLY POLARIZED WIGGLER RADIATION SOURCES To obtain mutually orthogonal polarization for the two radiations with a single field source as in Fig 3, appropriately beveled magic ring slices are fitted together to form a toroid, the successive slices of which are oriented as shown in figure 4. The axial field component remains constant from slice to slice, thereby providing the constant centripetal force needed to keep the electrons in orbit while they emit radially polarized synchrotron radiation. The axial field component reverses direction from slice to slice so that it provides the periodic acceleration along the principal toroidal axis that gives rise to the axially polarized wiggler radiation. Hence the two radiations are polarized orthogonally and easily separable by appropriate devices such as prisms. The ratio of the field components BAD, can be adjusted by a change in the angle between the disks’ polar axes and the principal toroidal axis. Fig 3. Coplanar polarizations of wiggler and synchroton radiations. Wiggler polarization ? synchotron polarizatioq Radiation directions + Fig 5. Construction of toroid with constant axial and periodic radial field. A. Orientations of successive magic cyclinder slices. B. Arrangement of slices in a toroid. It might be necessary to provide an electric solenoid wound about the toroidal tube to furnish an azimuthal columnating field for the electron beams, although this might also be accomplished by judicious perturbations in permanent magnet shape. Fig 3. Coplanar polarizations of wiggler and synchroton radiations. Wiggler polarization ? synchotron polarizatioq Radiation directions + Fig 3. Coplanar polarizations of wiggler and synchroton radiations. Wiggler polarization ? synchotron polarizatioq Radiation directions + Fig 3. Coplanar polarizations of wiggler and synchroton radiations. Wiggler polarization ? synchotron polarizatioq Radiation directions + 1407 field as the outer. Mutual rotations in opposite senses can adjust the field to any value below maximum. Also the total field can be rotated thereby giving considerable flexibility in operational mode; ring twister, wiggler, and variable period and field strength. IV. CONCLUSIONS The wiggler and synchrotron fields of a circular free electron laser can be provided and made orthogonal by a single permanent magnet source. The field strengths provided by such a source compare favorably with those of standard sources and offer multiple circuits of the electron beam. For some structures a field and mode adjustable toroid might be practicable. Fig 6. Dimensions and Parameters used in analytic calculations Fig 6. Dimensions and Parameters used in analytic calculations TABLE 1 TABLE 1 Fields and frequencies of axially-polarizing wigglers as functions of y. Fields and frequencies of axially-polarizing wigglers as functions of [I] T.C. Marshal, Free Electron Lasers, Macmillian Inc.: New York, NY. [2] H.A. Leupold, A.S. Tilak and E. Potenziani 11. IEEE Trans. Magn., 22, No. 5, p. 3418, 1997. [3] H.A. Leupold, M. Abele and E. Potenziani 11. Journal of Applied Physics, 67, p. 4650, 1990. [4] A.B.C. Morcos, H.A. Leupold and E. Potenziani 11. IEEE Trans. Magn., vol22, No. 5, p. 1066, 1986. [5] K. Halbach, Proceedings of the Fifth International Workshop on Rare Earth-Cobalt Permanent Magnets and Their Applications, Roanoke, VA, 7-10 June 1981. University of Dayton, Magnetics KL 365. Dayton, OH 45469, p. 73, 1981. For structures in which the principal toroid radius is enough larger than the outer tube radius so that the wedge angle of the magic ring slices is small it might be practicable to approximate the wedged segments with pierced disks as in Fig. 7. If the inner segment is made to produce the same [6] H.A. Leupold and E. Potenziani 11. Journal of Applied Physics, 67 (a), p. 4653, 1990. [7] H.A. Leupold, E. Potenziani I1 and A.S. Tilak Journal of Applied Physics, 2 (1 0), p. 6856, 1994.
https://openalex.org/W3025253132	https://dr.ntu.edu.sg/bitstream/10356/146116/2/s41598-020-64800-z.pdf	English	null	Information Cascades and the Collapse of Cooperation	Scientific reports	2,020	cc-by	10,519	2020 Yang, G., Csikász‑Nagy, A., Waites, W., Xiao, G., & Cavaliere, M. (2020). Information cascades and the collapse of cooperation. Scientific Reports, 10(1), 8004‑. doi:10.1038/s41598‑020‑64800‑z Yang, G., Csikász‑Nagy, A., Waites, W., Xiao, G., & Cavaliere, M. (2020). Information cascades and the collapse of cooperation. Scientific Reports, 10(1), 8004‑. doi:10.1038/s41598‑020‑64800‑z Guoli Yang1, Attila Csikász-Nagy2,3, William Waites4, Gaoxi Xiao5 & Matteo Cavaliere6 ✉ In various types of structured communities newcomers choose their interaction partners by selecting a role-model and copying their social networks. Participants in these networks may be cooperators who contribute to the prosperity of the community, or cheaters who do not and simply exploit the cooperators. For newcomers it is beneficial to interact with cooperators but detrimental to interact with cheaters. However, cheaters and cooperators usually cannot be identified unambiguously and newcomers’ decisions are often based on a combination of private and public information. We use evolutionary game theory and dynamical networks to demonstrate how the specificity and sensitivity of those decisions can dramatically affect the resilience of cooperation in the community. We show that promiscuous decisions (high sensitivity, low specificity) are advantageous for cooperation when the strength of competition is weak; however, if competition is strong then the best decisions for cooperation are risk-adverse (low sensitivity, high specificity). Opportune decisions based on private and public information can still support cooperation but suffer of the presence of information cascades that damage cooperation, especially in the case of strong competition. Our research sheds light on the way the interplay of specificity and sensitivity in individual decision-making affects the resilience of cooperation in dynamical structured communities. Cooperation is widespread in the real world and can be observed at different scales of biological organization, ranging from genes to multi-cellular organisms and socio-technological systems1–4. However, cooperators pay a cost to benefit others. The extent to which cooperators can thrive within the system apparently contradicts the idea that only selfish behaviors are rewarded during competition between individuals. The resilience of coop- eration has been approached in different domains5 and evolutionary game theory6–8 provides a framework for studying the evolution of cooperation among unrelated individuals.h y g p g The Prisoner’s Dilemma (PD) in particular has been widely employed for investigating the sustainability of cooperation. The prisoner’s dilemma stresses the key point of the conflict of interest between what is best for the individual and what is best for the group, and thus creates a social dilemma. To solve the dilemma, several mech- anisms have been suggested to facilitate the spreading of cooperation such as direct reciprocity, indirect reciproc- ity, kin selection, group selection, and graph selection or spatial reciprocity, etc.2. https://hdl.handle.net/10356/146116 https://doi.org/10.1038/s41598‑020‑64800‑z © 2020 The Author(s). 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/. Downloaded on 24 Oct 2024 11:55:52 SGT www.nature.com/scientificreports www.nature.com/scientificreports Information Cascades and the Collapse of Cooperation N Guoli Yang1, Attila Csikász-Nagy2,3, William Waites4, Gaoxi Xiao5 & Matteo Cavaliere6 ✉ Computational Model p We consider a network of N nodes linked by a number of edges which varies over the course of the evolution of the system and where the individual success is linked to actual number and types of the neighbours15,18,19. y y g Each node is either a cooperator or defector (also referred as cheater), adopting one of the strategies of the Prisoner’s Dilemma2,8,20. A cooperator pays a cost c to distribute a benefit b to all of its neighbors. A defector pays no cost and distributes no benefit. We assume b > c > 0.hf i This can be represented by the following payoff matrix: − − ( ) C D C D b c c b 0 (1) (1) The payoff Pi received by node ni in the network is calculated as the sum of pair-wise interactions with its neighbours. The payoff Pi received by node ni in the network is calculated as the sum of pair-wise interactions with its neighbours. g Specifically, if a cooperator has m cooperative neighbours and n defective neighbours, its payoff is − − m b c nc ( ) . However, a cheater in the same neighbourhood has payoff mb.hif g yf The prosperity of a population is defined by the average payoff of all nodes present in the network. We use an exponential function with a parameter δ to define the fitness of node ni, δ = + f (1 ) i Pi where Pi is the payoff of node ni. i Each update step of the network proceeds as follows (Fig. 1). A role-model node is selected at random, with a probability proportional to its fitness (i.e., higher the fitness of a node, higher its chances to be selected as role-model). The parameter δ (selection strength) serves to determine the “strength” of the game between the nodes21,22. This means that when δ = 0, the selection probability is the same for all nodes, independently of their obtained payoff, while increasing δ makes it more likely that a node with a higher payoff is selected as role-model. g p yf A newcomer node is added into the network and with a probability 1 − μ will behave exactly as the role-model (i.e., using the same strategy) while with a probability μ will behave differently adopting the alternative strategy. www.nature.com/scientificreports/ www.nature.com/scientificreports/ We use the degree of a node for this public information, reasoning that if it is very popular, if more previous newcomers have connected to such node, then following the crowd might be a good idea. Note that the newcomer has no knowledge of why previous newcomers may have made this decision.h g y p y This is an important point: because newcomer nodes cannot know for certain whether nodes are cooperators or cheaters, and cannot know the reasons for previous newcomers connecting to a given node, they may errone- ously select to connect to a cheater node.h y This error increases the degree of that node and makes it more likely to be selected in the future. As this pro- cess continues, an information cascade can start - this same erroneous decision is copied on the basis of the public information again and again. g g To temper this tendency to simply follow the crowd, with the potential for information cascades, newcomers also have access to private information. Private information gives a newcomer node a chance to act differently, to not follow the crowd. The private information is modelled simply as two Gaussian probability distributions, φc for cooperators and φd for cheaters17. The two distributions are then used by the newcomers to distinguish (using a decision-threshold) cooperators from cheaters. Different combinations of the distributions and of the decision-thresholds lead to decisions with different specificity and sensitivity. In this paper, we fix the two dis- tributions, and change only the decision-thresholds. This allows us to study how different types of individual decisions (i.e., with different specificities and sensitivities) can affect the presence of cooperation. We investigate what happens when decisions are made based on private information alone as well as both private and public information17.fff We demonstrate that different decisions based on private information, with different trade-offs of sensitivity and specificity, can have positive or negative consequences on long term cooperation and crucially depend on the strength of the competition between cooperators and cheaters (selection strength). Decisions beneficial for cooperation can become detrimental if the selection strength changes. Moreover we show that decisions based on a combination of private and public information create information cascades17 which generally damage coopera- tion, in particular when selection strength is strong. Guoli Yang1, Attila Csikász-Nagy2,3, William Waites4, Gaoxi Xiao5 & Matteo Cavaliere6 ✉ Much work has been dedicated to the spreading of cooperation in structured populations and networks, where the promotion of cooperation is associated with the formation of cooperative clusters9–12. p In this paper we use evolutionary game theory to study a model of dynamical networks to understand how attachment choices cascade down the generations and lead, or not, to a collapse of cooperation. In this model, when a new node (newcomer) joins the network, it selects a role-model and imitates its strategy13–15. The new- comer also needs to decide its connections and, in particular, whether to establish connections with the social network of the chosen role-model, a mechanism often refereed as social inheritance14–16. A newcomer would ideally connect only to cooperative nodes and avoid detrimental connections to cheaters. The challenge for the newcomer, therefore, is to distinguish between cooperators and cheaters. g p We model this challenge as a combination two kinds of information that we call public and private17. Public information is that which can be computed by observing the network and choices that previous newcomers have made. 1Unit 66136 and College of System Engineering, National University of Defense Technology, Changsha, China. 2Faculty of Information Technology and Bionics, Pázmány Péter Catholic University, Budapest, Hungary. 3Randall Centre for Cell and Molecular Biophysics, King’s College, London, London, UK. 4School of Informatics, University of Edinburgh, Edinburgh, Scotland, UK. 5School of Electrical and Electronic Engineering, Nanyang Technological University, Singapore, Singapore. 6Department of Computing and Mathematics, Manchester Metropolitan University, Manchester, UK. ✉e-mail: m.cavaliere@mmu.ac.uk Scientific Reports \| (2020) 10:8004 \| https://doi.org/10.1038/s41598-020-64800-z Scientific Reports \| (2020) 10:8004 \| https://doi.org/10.1038/s41598-020-64800-z Computational Model Here the parameter μ means the mutation rate, which usually is a small value (we use μ = 0.0001).f p y Once the newcomer is added, edges are established in two different ways, depending on decisions which can make use of either only (i) private information or (ii) private and public information (see Fig. 1) in order to identify the cooperators from cheaters. Decisions based on private information. Private information is modelled using fixed Gaussian distribu- tions, ( , ) c c c 2 N φ µ σ = and ( , ) d d d 2 N φ µ σ = , and use the variance of the standard Gaussian distribution, σ σ = = .0 5 c d 2 2 ; we assume the distributions peaks are a distance of 1 apart, setting μ = −.0 5 c and μ = .0 5 d . Individual decisions are modelled using a fixed real number τ (called the decision-threshold) chosen in the sup- port of the private information distributions. Figure 1 shows the overlapping distributions φc (blue), φd (orange) and the vertical line denotes a possible decision-threshold between the two distributions. Connections between the added newcomer and the rest of the network are established in the following way. For each node x (either the chosen role-model or any of its neighbors) with whom a connection may be estab- lished, a sample, sx is drawn from the distribution φc or the other distribution φd accordingly as the node x is a cooperator or a defector. If τ < sx for some threshold τ, then a connection is established between the newcomer Scientific Reports \| (2020) 10:8004 \| https://doi.org/10.1038/s41598-020-64800-z www.nature.com/scientificreports/ Figure 1. Network update. (a) A role-model is selected with probability proportional to its fitness. A newcomer (white node) is added to the network (b) The newcomer imitates the behaviour of the role-model with probability 1 − μ. (c) The newcomer establishes links (dashed) with the role-model and with each node of its social network with the aim to establish links only with the cooperators (blue nodes). The decision to establish a link with a node is based on the exclusive use of private (blue and orange distributions) or on a combination of private and public information which can be used by the newcomer to distinguish cooperative nodes. Figure 1. Network update. Decisions based on private & public information. We also consider the case in which connections are dd d b d b f b h bl d f h h d d Decisions based on private & public information. We also consider the case in which connections are added based on a combination of both public and private information, which may give contradictory indications. We use a common and intuitive type of public information which is the degree of a node. In this way the public information provides information on the choices of previous newcomers: a node with many connections (high degree) has already attracted many connections from earlier newcomers and the chosen public information will indicate (perhaps erroneously) that the node is a cooperator and a connection to that node should be made.i y Specifically, let P and Q be Boolean variables of whether or not the public and private signals indicate that a connection should be made, respectively. The private signal is just as specified above, and the public signal is “does the target node x have a degree greater than the average degree of network” (i.e., kx > k).h g g g g g f ( x ) The private and public signals are then combined as follows: g g g g g f The private and public signals are then combined as follows: 1. If ∧ P Q, then connect 2. If ∧¬ P Q, then connect with probability p 3. If ¬ ∧ P Q, then connect with probability q 4. If ¬ ∧¬ P Q, then do not connect 1. If ∧ P Q, then connect 2. If ∧¬ P Q, then connect with probability p 3. If ¬ ∧ P Q, then connect with probability q 4. If ¬ ∧¬ P Q, then do not connect 1. If ∧ P Q, then connect 2. If ∧¬ P Q, then connect with probability p 3. If ¬ ∧ P Q, then connect with probability q 4. If ¬ ∧¬ P Q, then do not connect 1. If ∧ P Q, then connect 2. If ∧¬ P Q, then connect with probability p 3. If ¬ ∧ P Q, then connect with probability q 4. If ¬ ∧¬ P Q, then do not connect 4. If ¬ ∧¬ P Q, then do not connect where the parameters p and q control the weights of private information and public information when they con- flict. There is a subtlety about the public signal. Computational Model (a) A role-model is selected with probability proportional to its fitness. A newcomer (white node) is added to the network (b) The newcomer imitates the behaviour of the role-model with probability 1 − μ. (c) The newcomer establishes links (dashed) with the role-model and with each node of its social network with the aim to establish links only with the cooperators (blue nodes). The decision to establish a link with a node is based on the exclusive use of private (blue and orange distributions) or on a combination of private and public information which can be used by the newcomer to distinguish cooperative nodes. and the node x (the private information signals that x is cooperator); otherwise these two nodes would not be connected (the private information signals that x is a cheater). I hi fi d i i f i di ib i d l d diff d i i b h i In this paper we assume fixed private information distributions and analyzed different decisions by changing he decision-threshold τ.h The use of the two overlapping distributions φc, φd models the imperfect ability of the newcomer to observe and distinguish cooperative nodes from cheating ones. Decisions based on private & public information. We also consider the case in which connections are dd d b d b f b h bl d f h h d d If the choice is made on the basis of and, the threshold comparison must be done as ≥. This is because otherwise it is never possible to recover from the scenario where there are no edges or connections. Similarly, if the choice is made as or, the comparison must be strictly greater than, other- wise if the network becomes fully connected it will always remain so. Scientific Reports \| (2020) 10:8004 \| https://doi.org/10.1038/s41598-020-64800-z www.nature.com/scientificreports/ Figure 2. Decision-making based on private information and long-term cooperation. (a) The plot shows the sensitivity (red curve) and specificity (blue curve) of the decision associated to the chosen decision-threshold and the private information described by the two overlapping distributions. (b) The plot shows the amount of long-term cooperation (blue curve) and prosperity (red curve) obtained by varying the decision-threshold. Each chosen decision-threshold leads to a different decision which is associated a different specificity and sensitivity, leading to a different amount of long-term cooperation and prosperity. Results are obtained with a simulation of 108 time steps using δ = 0.001 and b/c = 10/8. Figure 2. Decision-making based on private information and long-term cooperation. (a) The plot shows the sensitivity (red curve) and specificity (blue curve) of the decision associated to the chosen decision-threshold and the private information described by the two overlapping distributions. (b) The plot shows the amount of long-term cooperation (blue curve) and prosperity (red curve) obtained by varying the decision-threshold. Each chosen decision-threshold leads to a different decision which is associated a different specificity and sensitivity, leading to a different amount of long-term cooperation and prosperity. Results are obtained with a simulation of 108 time steps using δ = 0.001 and b/c = 10/8. Node removal. In either cases (A) or (B), after the addition of a newcomer, following a Moran process20, a randomly selected node is removed from the network together with all of its connections so that number of nodes in the population is kept constant. Node removal. In either cases (A) or (B), after the addition of a newcomer, following a Moran process20, a randomly selected node is removed from the network together with all of its connections so that number of nodes in the population is kept constant. Scientific Reports \| (2020) 10:8004 \| https://doi.org/10.1038/s41598-020-64800-z Results We study the proposed computational model using numerical simulations and computer visualizations, per- formed using custom created software tool. t Simulations start from a randomly connected network of N nodes having an average connectivity = k 4. For each simulation run, we fix the decision-threshold τ to a value chosen uniformly from the range, [−2, 2]. All nodes initially adopt the same strategy and statistics are calculated by taking a single run of 108 update steps as described in Computational Model Section. We score long term cooperation (when there is no confusion, we simply say cooperation), prosperity and connectivity calculated as the sum of the fraction of cooperators, average payoff and average degree at each step, respectively, divided by the total number of steps considered in the simulation. We consider the normalized values (ranging between 0 and 1) of long term cooperation, prosperity and connectivity which are computed by dividing the respective values by the maximum possible. g p y p We also record the numbers of transitions, i.e., switches between the two states of all cooperators and all cheat- ers. A trajectory of the system obtained in a simulation, with the typical switches between all cooperators and all cheaters is shown in Fig. 1 in Supplement. g pp Moreover, keeping in mind that newcomers would like to establish connections only with cooperative nodes, we record during the simulations the number of false positive (FP), false negative (FN), true positive (TP) and true negative (TN). g If a newcomer decides to establish a connection with a cooperative node x then this constitutes a TP; if a newcomer decides to not establish a connection with a cooperative node, then it is a FN. If a newcomer decides to establish a connection with a cheater then it is a FP, while if a newcomer decides to not establish a connection with a cheater, then it is a TN. The corresponding specificity and sensitivity are defined as = specificity TN/ + TN FP ( ) and = + sensitivity TP TP FN /( ). We also refer to 1-specificity as promiscuity. Distinct values of τ (decision-threshold) imply decisions with distinct sensitivity and specificity. Also note, that specificity measures how well cheaters are recog- nised by newcomers and sensitivity shows how properly cooperators are recognised. Which kinds of decisions help cooperation?. We first analyze the model where connections are estab- lished with decisions that depend exclusively on the available private information. Which kinds of decisions help cooperation?. We first analyze the model where connections are estab- shed with decisions that depend exclusively on the available private information. We assume that all newcomers use an identical decision-threshold τ. Varying the decision threshold τ, one obtains decisions which differ for sensitivity and specificity (Fig. 2a); the sensitivity increases and the specificity decreases as the threshold increases. As one can see in Fig. 2b these differences affect the long term cooperation and prosperity present in the system, which clearly suggests that individual decisions based on an appropriate balance of specificity and sensitivity are important for the resilience of long term cooperation.f i To provide a better intuition on the effects of individual decisions (τ) on long term cooperation we plot in Fig. 3 the network snapshots for different decision-thresholds and corresponding to the four key stages which we can find in a trajectory: a network of only cooperators, of only cheaters, the typical network in an invasion of Scientific Reports \| (2020) 10:8004 \| https://doi.org/10.1038/s41598-020-64800-z www.nature.com/ ntificreports/ cheaters (in a network of cooperators) and the typical network of an invasion of coope Figure 3. Typical networks for different decision-thresholds. We present the typical netw transition from of all cooperators to a network of all cheaters, and from all cheaters to all different decision-thresholds τ. The typical stages of the invasions are: all-cooperators (A defectors (C → D), all-defectors (All-D) and defectors-to-cooperators (D → C), presented τ = 0, τ = .0 5 and τ = 1. The simulations have been obtained at weak selection with δ = and strong selection with δ = .0 1 (lower panel), and the benefit-to-cost ratio is high with www.nature.com/scientificreports/ Figure 3. Typical networks for different decision-thresholds. We present the typical networks observed during a transition from of all cooperators to a network of all cheaters, and from all cheaters to all cooperators, for different decision-thresholds τ. The typical stages of the invasions are: all-cooperators (All-C), cooperators-to- defectors (C → D), all-defectors (All-D) and defectors-to-cooperators (D → C), presented at τ = −1, τ = −.0 5, τ = 0, τ = .0 5 and τ = 1. The simulations have been obtained at weak selection with δ = .0 001 (upper panel) and strong selection with δ = .0 1 (lower panel), and the benefit-to-cost ratio is high with b/c = 10/8. Figure 3. cheaters (in a network of cooperators) and the typical network of an invasion of cooperators (in a network of cheaters). Layout of the networks is based on a physical model of springs23. Which kinds of decisions help cooperation?. We first analyze the model where connections are estab- lished with decisions that depend exclusively on the available private information. If a cheater “attaches” to a connected network of cooperators, such cheater would get a large fitness, attracting more cheating newcomers and ultimately leading to a full cheater invasion; we can see in Fig. 3 that this scenario is generally facilitated by increasing values of τ (increasing the promiscuity of the decisions). In that case, most connections to cooperators are established (lead- ing to well connected networks) but those to cheaters are also rarely avoided (leading to frequent invasions of cheaters). On the contrary, the recovery of cooperation from a network of a cheaters (All-D, D → C, All-C, Fig. 3) is fostered by a connected component of cooperators isolated in a sparse population of cheaters (with rare connec- tions between the community of cooperators and that of cheaters). This scenario is facilitated by the lower values of τ (lower sensitivity of the decision). In that case, most detrimental connections to cheaters are correctly avoided and some connections to cooperators are also established. However, as we can see in Fig. 3, the exact regime of decision-thresholds which avoid the formation of highly connected (and fragile) networks of cooperators, but still provide some sufficient connectivity between the coop- erators, depend on the selection strength δ.f We systematically explore the effects of the decision-threshold on cooperation in Fig. 4. In Fig. 4 we show how the amount of long term cooperation, the prosperity and the number of transitions change depending on the decision-threshold and the selection strength δ. In general, cooperators can be successful if they avoid connections to cheaters and connect to other cooper- ators, so the best decisions for cooperation are at some intermediate level of specificity and sensitivity. However, analyzing Fig. 4 we can observe that the opportune balance of specificity and sensitivity depends crucially on the selection strength δ. Generally, decisions which are highly promiscuous (i.e., little specificity) tend to be negative for long term cooperation - however the opportune degree of promiscuity that maximizes cooperation depends on the strength of competition between cooperators and cheaters (selection strength).hi The opportune balance of specificity and sensitivity is non trivial: detrimental decisions for cooperation can become beneficial if the selection strength changes. As we can observe in Fig. 4 when selection strength is weak, the maximum amount of long term cooperation is obtained when decisions are highly sensitive and promiscuous (Fig. 4, left panel). Which kinds of decisions help cooperation?. We first analyze the model where connections are estab- lished with decisions that depend exclusively on the available private information. Typical networks for different decision-thresholds. We present the typical networks observed during a transition from of all cooperators to a network of all cheaters, and from all cheaters to all cooperators, for different decision-thresholds τ. The typical stages of the invasions are: all-cooperators (All-C), cooperators-to- defectors (C → D), all-defectors (All-D) and defectors-to-cooperators (D → C), presented at τ = −1, τ = −.0 5, τ = 0, τ = .0 5 and τ = 1. The simulations have been obtained at weak selection with δ = .0 001 (upper panel) and strong selection with δ = .0 1 (lower panel), and the benefit-to-cost ratio is high with b/c = 10/8. cheaters (in a network of cooperators) and the typical network of an invasion of cooperators (in a network of cheaters). Layout of the networks is based on a physical model of springs23. cheaters (in a network of cooperators) and the typical network of an invasion of cooperators (in a network of cheaters). Layout of the networks is based on a physical model of springs23. Scientific Reports \| (2020) 10:8004 \| https://doi.org/10.1038/s41598-020-64800-z www.nature.com/scientificreports/ Figure 4. Cooperation, connectivity, prosperity and transitions as function of the decision threshold τ. The beneficial or detrimental effects of decisions strongly depend on the selection strength. We use weak selection with δ = .0 001 (left panel) and strong selection with δ = .0 1 (right panel), respectively. The benefit-to-cost ratio is b/c = 10/8. Results are obtained using a long run of 108 time steps. Figure 4. Cooperation, connectivity, prosperity and transitions as function of the decision threshold τ. The beneficial or detrimental effects of decisions strongly depend on the selection strength. We use weak selection with δ = .0 001 (left panel) and strong selection with δ = .0 1 (right panel), respectively. The benefit-to-cost ratio is b/c = 10/8. Results are obtained using a long run of 108 time steps. Figure 3 considers the case of large benefit-to-cost ratio (for the case of low benefit-to-cost ratio, see Fig. 2 in Supplement).h The collapse of a network of cooperators (All-C, C → D, All-D, Fig. 3) is generally facilitated by the introduc- tion of a cheater into a well connected networks of cooperators. Scientific Reports \| (2020) 10:8004 \| https://doi.org/10.1038/s41598-020-64800-z Which kinds of decisions help cooperation?. We first analyze the model where connections are estab- lished with decisions that depend exclusively on the available private information. On the other hand, these types of decisions are detrimental for cooperation when the selection strength is strong (Fig. 4, right panel). In this case, the largest amount of cooperation is obtained when the indi- viduals use decisions which are highly specific and with low sensitivity.i i When benefit-to-cost ratio is generally smaller, the amount of long term cooperation is generally lower but also in that case the regimes of decisions which facilitate cooperation depends on the selection strength, and the decision-threshold τ after which cooperation collapses tend to smaller as selection strength increases (Fig. 3 in Supplement).hi The intuition behind these results is the following one. For a given benefit-to-cost ratio b/c and a given selec- tion strength δ, the connectivity and structure of the formed network is highly dependent on the threshold τ (as seen in Fig. 3). As we can see in Fig. 4 for both cases of weak and strong selections, there are intermediate τ s where long term cooperation is maximized. These are the τ s that facilitate the formation of networks where there is a good degree of connectivity (due to the sufficiently high promiscuity), a limited number of mixed connections between cooperators and cheaters (due to sufficiently high specificity), which are detrimental for cooperation. At Scientific Reports \| (2020) 10:8004 \| https://doi.org/10.1038/s41598-020-64800-z www.nature.com/scientificreports/ the same time, these are the τ s that allow enough connections exclusively between cooperators (due to suffi- ciently high sensitivity) which are advantageous for cooperators. The optimal τ s for cooperation depend on the selection strength. Increasing τ leads to a decrease in the specificity and an increase in the sensitivity of the deci- sions. This means that, with the increase of τ, the network tends generally to get more connected due an increase of the decision promiscuity (as we can observe in Fig. 3); we can observe a general increase in network connectiv- ity and, in particular, due to the high promiscuity, an increase of the number of mixed connections between cooperative and cheating nodes (which are detrimental for cooperators and advantageous for cheaters). The det- riment caused to cooperation by these types of mixed connections can be outbalanced by the connections which are exclusively between cooperative nodes (which are facilitate by high sensitivity) when the selection strength is weak (e.g., δ = .0 001, Fig. 4 left panel). Information cascades and the collapse of cooperation. As discussed in the Introduction, in many scenarios decisions are taken by considering a combination of public and private information; in this case a new- comer may make an erroneous decision to connect to a cheater when the private and public information conflict. We will investigate the influence of private & public information on the spreading of erroneous choices, and the consequences on long term cooperation. Information cascades and the collapse of cooperation. As discussed in the Introduction, in many scenarios decisions are taken by considering a combination of public and private information; in this case a new- comer may make an erroneous decision to connect to a cheater when the private and public information conflict. We will investigate the influence of private & public information on the spreading of erroneous choices, and the consequences on long term cooperation. q g p As described above (computational model) we combine public and private information using the parameters p and q which control the decision-making of the newcomer when private and public information conflict. When p is larger than q, then the public information has a stronger weight in the decision to establish a connection, while when q is larger than p, the private information has a stronger weight in the decision.h The addition of public information can change the dynamics observed leading to the formation of information cascades17, that happen when an erroneous choice to connect (or not) to another node is made by the newcomer and such error can propagate to successive newcomers (a choice to connect is correct when the connection is established with a cooperator, while it is erroneous when established with a cheater). p ) We define two kinds of information cascades, P-cascades and N-cascades, depending on the different possibil- ities for the newcomer to establish (or not) its connections. If the choice to connect or not is erroneous, the newcomer can be part of a cascade only if the private and public information conflict: if the private information correctly indicates that a connection should be made and a connection is nevertheless not made due to the public information, then the newcomer forms part of what we call a P-cascade. Similarly, if the private information correctly indicates that a connection should not be made and a connection is nevertheless made due to the public information, then the newcomer forms part of what we call an N-cascade. The above situation is summarized in Table 1. h When a newcomer is determined as part of a cascade, its chosen role-model is then considered. If the type of the cascade, P-cascade or N-cascade is the same as that of the role-model, then the newcomer becomes part of the same cascade. Scientific Reports \| (2020) 10:8004 \| https://doi.org/10.1038/s41598-020-64800-z Which kinds of decisions help cooperation?. We first analyze the model where connections are estab- lished with decisions that depend exclusively on the available private information. However, for stronger selection (e.g., δ = .0 1, Fig. 4 right panel) the det- rimental effects for cooperation of mixed connections (facilitated when τ is large) are larger: in fact, the chances that a cheater connected to cooperators is selected as role-model (and imitated by future newcomers) are larger as δ increases. Therefore, the decision-threshold τ corresponding to the best levels of cooperation decreases (higher specificity) with the increase of δ (this is true for large, Fig. 4, or small b/c Fig. 3 in Supplement).f i Another interesting factor which is affected by the decision-threshold τ is the number of transitions between the two states of all cooperators and all cheaters. More transitions mean that the system is more unstable, with more frequent switches between the two states - cooperators invade networks of cheaters and cheaters invade networks of cooperators. In Fig. 4 we can observe that the collapse of long term cooperation is correlated to an increase in the number of transitions - in fact, as τ increases, and the specificity decreases (promiscuity increases), there are more often connections between cooperators and cheaters which makes the network more unstable, with frequent invasions of cheaters which leads to an overall increase in the number of recorded transitions. Once the network is highly connected, however, at very high τ s, the cooperators cannot anymore recover (since it becomes harder to form up isolated cooperative communities), so the networks become more stable with rare invasions of cooperators, which corresponds to the observed low level of long term cooperation (Fig. 4). We can also observe that the pros- perity (which is the long term overall payoff of the network) is maximized when there are sufficient cooperators and the network has some connectivity (only interactions produce some payoff) obtained when the specificity of the decisions is sufficiently low. Information cascades and the collapse of cooperation. As discussed in the Introduction, in many scenarios decisions are taken by considering a combination of public and private information; in this case a new- comer may make an erroneous decision to connect to a cheater when the private and public information conflict. We will investigate the influence of private & public information on the spreading of erroneous choices, and the consequences on long term cooperation. correct pub priv choice cascade C C C C 0 C NC NC NC 0 C C NC NC 0 C NC C C 0 C C NC C 0 C NC C NC P NC C C C 0 NC NC NC NC 0 NC C NC NC 0 NC NC C C 0 NC NC C NC 0 NC C NC C N Table 1. Truth table for determining when a node forms part of a P-cascade or N-cascade. The first column indicates whether the choice to connect (C) or not (NC) is correct. The second and third columns show the indication of the public and private information. The fourth column is the actual choice that is made. The final column identifies what sort of cascade this combination of inputs represents, either 0 for not a cascade, or P- cascade or N-cascade. Figure 5. Growth of Information Cascades in the presence of Public Information. P-cascade: (a) A newcomer (next to the box) is added to the network and establishes a connection with the chosen role-model (without loss of generality we assume the role-model is part of a P-cascade - shaded light red area). (b) The newcomer decides on the other connections. (c) If the connection with a cooperator is missed as consequence of public information, then the newcomer becomes part of a P-cascade. N-cascade: (d) A newcomer is added to the network and establishes a connection with the chosen role-model (we assume the role-model is part of an N-cascade - light green shaded area). (e) The newcomer decides on the other connections. (f) If the connection with a cheater is established as consequence of the public information, then the newcomer becomes part of an N-cascade. In both types of cascades, the newcomers becomes part of the same cascade of its role-model if the type of their cascades is equal. Figure 5. Growth of Information Cascades in the presence of Public Information. P-cascade: (a) A newcomer (next to the box) is added to the network and establishes a connection with the chosen role-model (without loss of generality we assume the role-model is part of a P-cascade - shaded light red area). (b) The newcomer decides on the other connections. (c) If the connection with a cooperator is missed as consequence of public information, then the newcomer becomes part of a P-cascade. Information cascades and the collapse of cooperation. As discussed in the Introduction, in many scenarios decisions are taken by considering a combination of public and private information; in this case a new- comer may make an erroneous decision to connect to a cheater when the private and public information conflict. We will investigate the influence of private & public information on the spreading of erroneous choices, and the consequences on long term cooperation. If the type differs (or the role-model is not part of any cascade) then the newcomer becomes part of a new cascade (Fig. 5).hf g The structure of the network is affected by the type of information (private or public) which is predominant in the decisions and which can determine the amount of long term cooperation (see the typical trajectories of the system, with more or less frequent switches, as function of the predominant type of information, Fig. 4 in the Supplement). pp In Fig. 6 we plot the typical networks that can be observed in a trajectory of the system, corresponding to only cooperators, only cheaters and the transients between these two stages. We fix the decision-threshold and analyze the network changes for the cases in which private information is predominant in the decisions and the cases in which public information is predominant, in the scenarios of weak and strong selection. p p g Generally, we can see from Fig. 6 that connections are more likely to be established when more public infor- mation is considered in the decisions. This high connectivity caused by the presence of public information is particularly evident in the case of strong selection (rightmost panels in Fig. 6). For a smaller b/c ratio, we observe a similar scenario where strong selection and an increasing weight of public information foster the connectivity of networks (Fig. 5 in Supplement). Scientific Reports \| (2020) 10:8004 \| https://doi.org/10.1038/s41598-020-64800-z www.nature.com/scientificreports/ correct pub priv choice cascade C C C C 0 C NC NC NC 0 C C NC NC 0 C NC C C 0 C C NC C 0 C NC C NC P NC C C C 0 NC NC NC NC 0 NC C NC NC 0 NC NC C C 0 NC NC C NC 0 NC C NC C N Table 1. Truth table for determining when a node forms part of a P-cascade or N-cascade. The first column indicates whether the choice to connect (C) or not (NC) is correct. The second and third columns show the indication of the public and private information. The fourth column is the actual choice that is made. The final column identifies what sort of cascade this combination of inputs represents, either 0 for not a cascade, or P- cascade or N-cascade. Information cascades and the collapse of cooperation. As discussed in the Introduction, in many scenarios decisions are taken by considering a combination of public and private information; in this case a new- comer may make an erroneous decision to connect to a cheater when the private and public information conflict. We will investigate the influence of private & public information on the spreading of erroneous choices, and the consequences on long term cooperation. The snapshots represent the typical networks composed by all cooperators, all cheaters and the transients between these two states. We consider strong selection (δ = .0 1) and weak selection (δ − .0 001). The benefit-to-cost ratio is b/c = 10/8. More public information is obtained using = . = . p q 0 75, 0 25. More private information is obtained with ( = . p 0 25 and = . q 0 75). Decision-threshold τ is fixed to −1. Figure 7. Information cascades with prevalent private information. We consider the scenario in which decisions are taken mostly (but not completely) based on private information ( = . = . p q 0 25, 0 75). We plot the long-term cooperation, total size of P-cascades and N-cascades. The benefit-to-cost ratio is b/c = 10/8, weak selection is δ = .0 001 (left panel) and strong selection is δ = .0 1 (right panel) are considered respectively. Figure 7. Information cascades with prevalent private information. We consider the scenario in which decisions are taken mostly (but not completely) based on private information ( = . = . p q 0 25, 0 75). We plot the long-term cooperation, total size of P-cascades and N-cascades. The benefit-to-cost ratio is b/c = 10/8, weak selection is δ = .0 001 (left panel) and strong selection is δ = .0 1 (right panel) are considered respectively. We can also observe in Fig. 7 that the collapse of cooperation is correlated with an increase of the size of − N cascades, and the increase of the size of N-cascades start at lower τ for stronger selection strength δ (Fig. 7, right panel). This is essentially caused by the fact that once an N-cascade starts (an erroneous connection is estab- lished with a cheating role-model), then the cascade will grow as long as the same role-model is chosen by more We can also observe in Fig. 7 that the collapse of cooperation is correlated with an increase of the size of − N cascades, and the increase of the size of N-cascades start at lower τ for stronger selection strength δ (Fig. 7, right panel). Information cascades and the collapse of cooperation. As discussed in the Introduction, in many scenarios decisions are taken by considering a combination of public and private information; in this case a new- comer may make an erroneous decision to connect to a cheater when the private and public information conflict. We will investigate the influence of private & public information on the spreading of erroneous choices, and the consequences on long term cooperation. N-cascade: (d) A newcomer is added to the network and establishes a connection with the chosen role-model (we assume the role-model is part of an N-cascade - light green shaded area). (e) The newcomer decides on the other connections. (f) If the connection with a cheater is established as consequence of the public information, then the newcomer becomes part of an N-cascade. In both types of cascades, the newcomers becomes part of the same cascade of its role-model if the type of their cascades is equal. We analyze in more details the consequences of public information on long term cooperation in Fig. 7. As one might intuitively expect, when private information is prevalent in the decisions, the curves are similar to the case where exclusively private information is present (Fig. 4). We analyze in more details the consequences of public information on long term cooperation in Fig. 7. As one might intuitively expect, when private information is prevalent in the decisions, the curves are similar to the case where exclusively private information is present (Fig. 4). However even a limited presence of public information already highlights a problem for long term coopera- tion: some mixed connections between cooperative and cheating nodes (which are detrimental for cooperators and advantageous for cheaters) are established due to the presence of public information. Therefore the way to balance these connections caused by public information is to increase the specificity (and consequently decreases the sensitivity) of the decisions: in fact, by comparing Figs. 4 and 7 we can observe that the maximal amount of long term cooperation is obtained at a lower τ when public information is also considered for the decisions. Scientific Reports \| (2020) 10:8004 \| https://doi.org/10.1038/s41598-020-64800-z www.nature.com/scientificreports/ Figure 6. Networks structures when private or public information is predominant in the decision snapshots represent the typical networks composed by all cooperators, all cheaters and the transie these two states. We consider strong selection (δ = .0 1) and weak selection (δ − .0 001). The bene ratio is b/c = 10/8. More public information is obtained using = . = . p q 0 75, 0 25. More private in obtained with ( = . p 0 25 and = . q 0 75). Decision-threshold τ is fixed to −1. Figure 6. Networks structures when private or public information is predominant in the decisions. Information cascades and the collapse of cooperation. As discussed in the Introduction, in many scenarios decisions are taken by considering a combination of public and private information; in this case a new- comer may make an erroneous decision to connect to a cheater when the private and public information conflict. We will investigate the influence of private & public information on the spreading of erroneous choices, and the consequences on long term cooperation. This is essentially caused by the fact that once an N-cascade starts (an erroneous connection is estab- lished with a cheating role-model), then the cascade will grow as long as the same role-model is chosen by more 9 Scientific Reports \| (2020) 10:8004 \| https://doi.org/10.1038/s41598-020-64800-z www.nature.com/scientificreports/ Figure 8. Information cascades with prevalent public information. We consider the scenario in which decisions are taken mostly (but not completely) based on public information ( = . = . p q 0 75, 0 25). We plot the long-term cooperation, total size of P-cascades and N-cascades. The benefit-to-cost ratio is b/c = 10/8, weak selection is δ = .0 001 (left panel) and strong selection is δ = .0 1 (right panel) are considered respectively. Figure 8. Information cascades with prevalent public information. We consider the scenario in which decisions are taken mostly (but not completely) based on public information ( = . = . p q 0 75, 0 25). We plot the long-term cooperation, total size of P-cascades and N-cascades. The benefit-to-cost ratio is b/c = 10/8, weak selection is δ = .0 001 (left panel) and strong selection is δ = .0 1 (right panel) are considered respectively. newcomers, and this will happen with higher chances for stronger selection strength. The case for lower b/c ratio is presented in the Supplement (Fig. 6 in Supplement). f h h h d k b d bl d f h In Fig. 8 we focus on the scenario in which decisions are taken based on public and private information with the weight of public information prevalent over the one of private information (the case of a lower benefit-to-cost ratio can be found in the Supplement Fig. 6). In general, comparing with Fig. 7, where little public information is used, we can see more dramatic effects. In the case of decisions where public information has a strong weight, long term cooperation decreases, particularly in the case of strong selection (Fig. 8, right panel). g p p y g g g p A stronger role of public information coupled to strong selection strength lead to an increase in the number of connections to cooperators at lower τs; this increases the sensitivity of the decisions for low τs (Fig. 8) because, in that regime, well connected nodes are mostly cooperators (due to the low number of false positives). Information cascades and the collapse of cooperation. As discussed in the Introduction, in many scenarios decisions are taken by considering a combination of public and private information; in this case a new- comer may make an erroneous decision to connect to a cheater when the private and public information conflict. We will investigate the influence of private & public information on the spreading of erroneous choices, and the consequences on long term cooperation. This ten- dency of cooperators to be well connected is reinforced by the presence of public information: more connected nodes tend to attract new connections. The increase in sensitivity for lower τs when public information has a strong weight makes difficult to find a balance of low sensitivity and high specificity which advantages coopera- tors (possible when mostly private information is used in the decisions, Fig. 7). In fact, as τs increases, we observe a further increase of sensitivity (as the number of false negative decreases) and a sharp decrease in specificity (as the number of false positive increases - cheaters become highly connected and attract connections due to the presence public information). This is facilitated even more by the presence of information cascades whose size rapidly increases as τ increases and that lead to frequent formation of mixed connections between cooperators and cheaters (detrimental to cooperators). This is particularly evident when selection strength is strong (Fig. 8, right panel); when selection strength is weak, the detrimental effects of public information are less dramatic, as the chances of role-model selection are less connected to actual node fitness and this minimizes the growing of information cascades when the sensitivity of the decision is sufficiently large (Fig. 9, left panel).ff fit Figure 9 summarizes the effects of public information - for different b/c and selection strengths: increasing the weight of public information in the decision is detrimental for long-term cooperation, with a more dramatic effect when the selection strength is large. Scientific Reports \| (2020) 10:8004 \| https://doi.org/10.1038/s41598-020-64800-z Discussion l d We analyzed a system where role-models are chosen by newcomers which imitate their strategy (cooperator or cheater) and their social connections inspired by the idea of social inheritance15,16. In general, for any newcomer (independently of its strategy) it is beneficial to connect to cooperators but detrimental to connect to cheaters. On the other hand, it is not possible to unambiguously identify cooperators from cheaters and the decisions to establish a connection become crucial for the resilience of cooperation. In this paper we evaluate the effects of different decisions expressed in terms of specificity and sensitivity. We show that the kinds of decisions (i.e., opportune balance of specificity and sensitivity) which maximize long term cooperation crucially depend on the degree of competition between cooperators and cheaters (selection strength). There is not really an unique kind of decisions that maximize cooperation: decisions which are beneficial for cooperation become detrimental if the selection strength changes. g g When decisions are based exclusively on private information, we identify two major classes of decisions advantageous for cooperation: promiscuous decisions (high sensitivity, low specificity) where the priority is to properly connect to other cooperators rather than to avoid cheaters, and more risk-adverse decisions (high spec- ificity, low sensitivity), where the priority is to avoid cheaters rather than connect to cooperators. Promiscuous decisions help cooperation when the selection strength is weak, while they are detrimental in the case of strong Scientific Reports \| (2020) 10:8004 \| https://doi.org/10.1038/s41598-020-64800-z 10 www.nature.com/scientificreports/ Figure 9. Public information facilitates the collapse of cooperation. We consider the scenario where mostly private information is used in the decision (left panels, with = . = . p q 0 25, 0 75), and the scenario where mostly public information is used in the decisions ( = . = . p q 0 75, 0 25 (right panels)). We plot the the average amount of cooperators obtained in a long run for different τs. We use = . b c/ 10/9 99 (first row), b/c = 10/9.9 (second row), = b c/ 10/9 (third row) and = b c/ 10/8 (bottom row). The selection strength is δ = .0 001, δ = .0 01 and δ = .0 1. Figure 9. Public information facilitates the collapse of cooperation. We consider the scenario where mostly private information is used in the decision (left panels, with = . = . Scientific Reports \| (2020) 10:8004 \| https://doi.org/10.1038/s41598-020-64800-z www.nature.com/scientificreports/ dynamical network model, our approach is general and serves as a first step to shed light on the dynamics of structured systems where the time-scale of decision-making and strategies dynamics may overlap such as bio- logical24–26 and social systems27. As the acquisition of information (public or private) is generally costly, it would be interesting to extend the considered model with an additional cost for the individuals related to their ability to acquire information (and to process them28). Furthermore the observed strategy separation (observed in Figs. 6 and 3) underlying our results has been observed in other models, e.g.3,29, which may suggest the possible general- ity of our results. y More generally, we believe that the proposed idea of coupling specificity/sensitivity trade-off of decision-making with evolutionary game theory in dynamical networks can be applied to other types of mod- els, in a variety of scenarios where has already been highlighted the role of individual decisions in preserving cooperation25,26,30–32. Received: 6 March 2020; Accepted: 20 April 2020; Published: xx xx xxxx Received: 6 March 2020; Accepted: 20 April 2020; Published: xx xx xxxx References Networks, crowds, and markets: Reasoning about a highly connected world (Cambridge University Press, 2010). ) 18. Cavaliere, M., Guoli, Y., Vincent, D. & Vasilis, D. Detecting the collapse of cooperation in evolving networks. Sci. Reports 6, 3 (2016). 19. Nax, H. H., Perc, M., Szolnoki, A. & Helbing, D. Stability of cooperation under image scoring in group interactions. Sci. reports 5, 12145 (2015). 20. Nowak, M. A. & Sigmund, K. Evolutionary dynamics of biological games. Science 303, 793–799 (2004). 21. Avilés, L. Cooperation and non-linear dynamics: an ecological perspective on the evolution of sociality. Evol. Ecol. Res. 1, 459–477 (1999). 22. Traulsen, A., Shoresh, N. & Nowak, M. A. Analytical results for individual and group selection of any intensity. Bull. mathematical biology 70, 1410 (2008). gy ( ) 23. Csárdi, G. & Nepusz, T. Igraph reference manual, https://igraph.org/c/doc/igraph-docs.pdf.html (2020).h p g p p g p g g p p 24. Cavaliere, M. & Poyatos, J. F. Plasticity facilitates sustainable growth in the commons. J. The Royal Soc. Interface 10, 20121006 (2 24. Cavaliere, M. & Poyatos, J. F. Plasticity facilitates sustainable growth in the commons. J. The Royal Soc. Interface 10, 20121006 (2013). 25. Harrington, K. I. & Sanchez, A. Eco-evolutionary dynamics of complex social strategies in microbial communities. Commun. & integrative biology 7, e28230 (2014).h h 25. Harrington, K. I. & Sanchez, A. Eco-evolutionary dynamics of complex social strategies in microbial communities. Commun. & integrative biology 7, e28230 (2014).h 26. Allen, R. C., McNally, L., Popat, R. & Brown, S. P. Quorum sensing protects bacterial co-operation from exploitation by cheats. T ISME journal 10, 1706–1716 (2016). j 27. Capraro, V. & Perc, M. Grand challenges in social physics: In pursuit of moral behavior. Front. Phys. 6, 107 (2018).h 28. Halpern, J. Y. & Pass, R. Algorithmic rationality: Game theory with costly computation. J. Econ. Theory 156, 246–268 h 29. Szolnoki, A. & Perc, M. Resolving social dilemmas on evolving random networks. EPL (Europhysics Lett. 86, 30007 (20 g g p y 30. Rand, D. G., Greene, J. D. & Nowak, M. A. Spontaneous giving and calculated greed. Nature 489, 427 (2012).f f 32. Rauch, J., Kondev, J. & Sanchez, A. Cooperators trade off ecological resilience and evolutionary stability in public goods games. J. The Royal Soc. Interface 14, 20160967 (2017). Acknowledgements g imulations were done on Azure using a Microsoft AI for Earth grant. This work is partially supported by NSSFC 019-SKJJ-C-005. References Nowak, M. A. Evolutionary dynamics (Harvard University Press, 20 y y y 2. Nowak, M. A. Five rules for the evolution of cooperation. Science 314, 1560–1563 (2006). 3. Szolnoki, A. & Perc, M. Coevolution of teaching activity promotes cooperation. New J. Phys. 10, 043036 (2008). l d l l d d 3. Szolnoki, A. & Perc, M. Coevolution of teaching activity promotes cooperation. New J. Phys. 10, 043036 (2008). l ó d T l l d d 4. Poncela, J., Gómez-Gardeñes, J., Traulsen, A. & Moreno, Y. Evolutionary game dynamics in a growing structured population. New J Phys. 11, 083031 (2009). y evin, S. Crossing scales, crossing disciplines: collective motion and collective action in the global commons. Philos. Transactions oyal Soc. B: Biol. Sci. 365, 13–18 (2010).hl y 5. Levin, S. Crossing scales, crossing disciplines: collective motion and collective action in the global commons. Philos. Transactions Royal Soc. B: Biol. Sci. 365, 13–18 (2010).hl y 6. Smith, J. M. & Price, G. The logic of animal conflict. Nature 246, 15 (1973). y 6. Smith, J. M. & Price, G. The logic of animal conflict. Nature 246, 15 (1973). h gl 7. Weibull, J. W. Evolutionary game theory (MIT press, 1997).b b g y g p p y g y 9. Perc, M. & Szolnoki, A. Coevolutionary games – a mini review. BioSystems 99, 109–125 (2010). 10. Pacheco, J. M., Vasconcelos, V. V. & Santos, F. C. Climate change governance, cooperation and self-organization. Phys. Life Re 573–586 (2014).h 11. Wang, Z., Wang, L., Szolnoki, A. & Perc, M. Evolutionary games on multilayer networks: a colloquium. The Eur. Phys. J. B 88 (2015). Perc, M. et al. Statistical physics of human cooperation. Phys. Repor 13. Jackson, M. O. Social and economic networks (Princeton university press, 2010 14. Jackson, M. O. & Rogers, B. W. Meeting strangers and friends of friends: How random are social networks? Am. Econ. Rev. 97, 890–915 (2007). ( ) 15. Cavaliere, M., Sedwards, S., Tarnita, C. E., Nowak, M. A. & Csikász-Nagy, A. Prosperity is associated with instability in dynamical networks. J. theoretical biology 299, 126–138 (2012). gy ( ) 6. Ilany, A. & Akcay, E. Social inheritance can explain the structure of animal social networks. Nat. communications 7, 12084 (2016). 16. Ilany, A. & Akcay, E. Social inheritance can explain the structure of animal social networks. Nat. communications 7, 12084 (2016). 17. Easley, D. & Kleinberg, J. Discussion l d p q 0 25, 0 75), and the scenario where mostly public information is used in the decisions ( = . = . p q 0 75, 0 25 (right panels)). We plot the the average amount of cooperators obtained in a long run for different τs. We use = . b c/ 10/9 99 (first row), b/c = 10/9.9 (second row), = b c/ 10/9 (third row) and = b c/ 10/8 (bottom row). The selection strength is δ = .0 001, δ = .0 01 and δ = .0 1. selection. In the latter case, the best decisions for cooperation are the risk-adverse ones (Fig. 4). Intuitively, when competition is strong (large selection strength) an erroneous mixed connection between a cooperator and a cheater can have large negative consequences on cooperation - in that case more risk adverse decisions, which carefully avoid cheaters, can benefit cooperation.hf yi p The addition of public information makes this effect more dramatic (Fig. 8): when the public information and private information conflict, and the decision to connect is based on a combination of the two, then an initial erroneous connection between a cooperator and a cheater can lead to the formation of information cascades17, e.g., a series of erroneous connections. Such information cascade generally facilitate connections of (cooperative and cheating) newcomers to cooperative nodes. This is advantageous for cooperative newcomers but even more for cheaters: it increases the sensitivity of the decisions, making difficult to find the opportune balance of sensi- tivity and specificity advantageous for cooperation. Though the negative effects on cooperation of public infor- mation are more evident when selection strength is strong, they can be observed independently of the selection strength and of the benefits and costs of cooperation (Fig. 9). gi p g Overall, our results demonstrate the importance of decision-making in the resilience of cooperation in structured populations: whether it is better for cooperators to focus on avoiding cheaters, or to search for other cooperators, really depends on how strong is the competition. Though our work has been focusing on a specific Scientific Reports \| (2020) 10:8004 \| https://doi.org/10.1038/s41598-020-64800-z www.nature.com/scientificreports/ Author contributions All authors contributed to the design of the research. G.Y., W.W. performed the experiments. All authors contributed to the analysis of the results. Correspondence and requests for materials should be addressed to M.C. Correspondence and requests for materials should be addressed to M.C. Reprints and permissions information is available at www.nature.com/reprints. Additional information Scientific Reports \| (2020) 10:8004 \| https://doi.org/10.1038/s41598-020-64800-z www.nature.com/scientificreports/ www.nature.com/scientificreports/ Reprints and permissions information is available at www.nature.com/reprints. Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Cre- ative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not per- mitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. © The Author(s) 2020 © The Author(s) 2020 Scientific Reports \| (2020) 10:8004 \| https://doi.org/10.1038/s41598-020-64800-z 13
https://openalex.org/W4297004970	https://www.acquaintpublications.com/get/1-4-JMCRCS2022070101 Galley_Proof-1663743550.pdf	English	null	Eczematous reaction with Meyerson’s phenomenon during treatment with imiquimod 5 % cream	Journal of medical case reports and case series	2,022	cc-by	1,468	Dear Editor, Imiquimod is an imidazoquinoline derivative with a direct agonistic activity towards toll-like receptors (TLR)-7 and TLR-8, resulting in the production of proinflammatory cytokines and stimulation of innate and acquired Th1- and Th17-weighted immune responses. [1] Additional effects of imiquimod include changes in the epidermal barrier functions, induction of migration of epidermal Langerhans cells to regional lymph nodes, and stimulation of plasmacytoid dendritic cells. [2,3] Received date: 02 July 2022; Accepted date: 26 July 2022; Published date: 21 September 2022 Received date: 02 July 2022; Accepted date: 26 July 2022; Published date: 21 September 2022 Citation: Ambrogio F, Cassano N, Vena GA, Bonamonte D, Grandolfo M, et al. (2022) Eczematous reaction with Meyerson’s phenomenon during treatment with imiquimod 5 % cream. J Med Case Rep Case Series 3(13): https://doi.org/10.38207/JMCRCS/2022/DECA03130496 Copyright: © 2022 Francesca Ambrogio. 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. Case Report Case Report M, et al. (2022) Eczematous reaction with Meyerson’s phenomenon during treatment with imiquimod 5 % cream. J Med Case Rep Case Series 3(13): https://doi.org/10.38207/JMCRCS/2022/DECA03130496 Citation: Ambrogio F, Cassano N, Vena GA, Bonamonte D, Grandolfo M, et al. (2022) Eczematous reaction with Meyerson’s phenomenon during treatment with imiquimod 5 % cream. J Med Case Rep Case Series 3(13): https://doi.org/10.38207/JMCRCS/2022/DECA03130496 Case Report Eczematous reaction with Meyerson’s phenomenon during treatment with imiquimod 5 % cream Francesca Ambrogio1, Nicoletta Cassano2, Gino Antonio Vena2, Domenico Bonamonte1, Mauro Grandolfo1, Paolo Romita1, Anna Bosco2, Silvia Mazzotta1, Caterina Foti1 1Dermatological Clinic, Department of Biomedical Science and Human Oncology, University of Bari “Aldo Moro”, Bari, Italy. 2Dermatology and Venereology Private Practice, Bari, Italy. Corresponding Author: Francesca Ambrogio, Dermatological Clinic, Department of Biomedical Science and Human Oncology, University of Bari “Aldo Moro”, Bari, Italy. Case Report A 36-year-old non-atopic male presented with an itchy eczematous dermatitis involving initially the perianal region and then other areas. He was suffering from perianal warts that were treated with imiquimod 5 % cream. The patient had initially applied the cream three times a week for 15 days and then decided to discontinue treatment that was resumed after one month. Perianal dermatitis started after about 15 days from the beginning of the second treatment course. The patient was reported to be sensitized to nickel sulfate. He applied the cream only on perianal warts and denied any accidental transfer of the cream to the other affected sites (e.g., by scratching), as well as the use of compounds containing nickel in the last weeks. Physical examination revealed eczematous dermatitis on the left forearm (Figure 1A) and perianal region, and small patches with mild erythema and fine scaling around (Figure 1B) or near some melanocytic nevi of the trunk and upper limbs. Dermoscopy did not show atypical features of such nevi. In clinical practice, local reactions are frequently seen at the site of application of topical imiquimod as an expected consequence of the drug's pro-inflammatory and immunostimulating mode of action that can also trigger immune-mediated cutaneous adverse events in a few subjects. For instance, exacerbation of psoriasis and de novo psoriasis In clinical practice, local reactions are frequently seen at the site of application of topical imiquimod as an expected consequence of the drug's pro-inflammatory and immunostimulating mode of action that can also trigger immune-mediated cutaneous adverse events in a few subjects. For instance, exacerbation of psoriasis and de novo psoriasis have been reported in patients treated with topical imiquimod. [1,4] A minority of patients can experience adverse events at non- application sites, including systemic symptoms (e.g., headache, fever, fatigue, myalgias, or nausea). [4,5]. Widespread cutaneous reactions, such as erythema multiforme, seem to be rare, [6] as well as distant inflammatory mucosal reactions. Citation: Ambrogio F, Cassano N, Vena GA, Bonamonte D, Grandolfo M, et al. (2022) Eczematous reaction with Meyerson’s phenomenon du 3(13): https://doi.org/10.38207/JMCRCS/2022/DECA03130496 Journal of Medical Case Reports and Case Series ISSN: 2692-9880 dermatitis rather than psoriasis whose peculiar hallmark is the primary involvement of plasmacytoid dendritic cells leading to a Th17-mediated response. [3] Therefore, our patient developed perianal dermatitis at the site of application of imiquimod and subsequent distant eczematous lesions with halo dermatitis around some melanocytic nevi, resembling Meyerson’s phenomenon (MP). The literature contains only anecdotal reports of eczema in patients treated with imiquimod. Taylor et al. described a severe recurring eczematous reaction at sites previously affected by eczema during imiquimod treatment, in the absence of any reaction at the site of application, suggesting a possible systemic absorption of imiquimod with enhanced T cell response and flare of the past eczematous dermatitis. [9] MP is an uncommon condition characterized by an eczematous halo surrounding a preexisting melanocytic nevus (Meyerson nevus) or numerous nonmelanocytic lesions, and even melanoma.[7,8] MP can involve multiple nevi, either separately over time or simultaneously, and generally resolves spontaneously or with the use of topical steroids or excision. Even though the pathogenesis is unknown, substantial evidence suggests that MP is immune-mediated, with the recruitment of a CD4+ T cell response. Interestingly, MP was observed in patients undergoing interferon-alpha-2b treatment. [7] There are limited data concerning imiquimod-related regional adverse effects with remote inflammatory skin reactions. Epicutaneous application of imiquimod has been used to create a murine model of psoriasis-like skin inflammation. [1] In human skin, imiquimod was shown to induce an inflammatory reaction similar to acute contact It is conceivable that the Th1/Th17 microenvironment created by imiquimod might stimulate distant eczematous reactions in predisposed patients. Keywords: imiquimod, eczema, Meyerson phenomenon Financial Disclosure: None reported. Keywords: imiquimod, eczema, Meyerson phenomenon Financial Disclosure: None reported. Conflict of Interest Disclosure: None Declared. Citation: Ambrogio F, Cassano N, Vena GA, Bonamonte D, Grandolfo M, et al. (2022) Eczematous reaction with Meyerson’s phenomenon d 3(13): https://doi.org/10.38207/JMCRCS/2022/DECA03130496 Citation: Ambrogio F, Cassano N, Vena GA, Bonamonte D, Grandolfo M, et al. (2022) Eczematous reaction with Meyerson’s phenomenon during treatment with imiquimod 5 % cream. J Med Case Rep Case Series 3(13): https://doi.org/10.38207/JMCRCS/2022/DECA03130496 Case Report [5] Figure 1: 1A) Eczematous dermatitis on the left forearm; 1B) Mild erythema and fine scaling around a melanocytic nevus of the trunk resembling Meyerson’s phenomenon Figure 1: 1A) Eczematous dermatitis on the left forearm; 1B) Mild erythema and fine scaling around a melanocytic nevus of the trunk resembling Meyerson’s phenomenon Figure 1: 1A) Eczematous dermatitis on the left forearm; 1B) Mild erythema and fine scaling around a melanocytic nevus of the trunk resembling Meyerson’s phenomenon Figure 1: 1A) Eczematous dermatitis on the left forearm; 1B) Mild erythema and fine scaling around a melanocytic nevus of the trunk resembling Meyerson’s phenomenon Meyerson’s phenomenon References imiquimod-A scoping review of the literature. Dermatol Ther. 34(1): e14355. imiquimod-A scoping review of the literature. Dermatol Ther. 34(1): e14355. 1. van der Kolk T, Assil S, Rijneveld R, Klaassen ES, Feiss G, et al. (2018) Comprehensive, Multimodal Characterization of an Imiquimod-Induced Human Skin Inflammation Model for Drug Development. Clin Transl Sci. 11(6): 607-615. 6. Chan MYL, Kennedy J, Oakley A (2017) Erythema multiforme triggered by imiquimod 5% cream. Australas J Dermatol. 58(4): e257-e258. 2. Schön MP, Schön M (2007) Imiquimod: mode of action. Br J Dermatol. 157(Suppl 2): 8-13. 2. Schön MP, Schön M (2007) Imiquimod: mode of action. Br J Dermatol. 157(Suppl 2): 8-13. 7. Loh J, Kenny P (2010) Meyerson phenomenon. J Cutan Med Surg. 14(1): 30-2. 3. Garzorz-Stark N, Lauffer F, Krause L, Thomas J, Atenhan A, et al. (2018) Toll-like receptor 7/8 agonists stimulate plasmacytoid dendritic cells to initiate TH17-deviated acute contact dermatitis in human subjects. J Allergy Clin Immunol. 141(4): 1320- 1333.e11. 3. Garzorz-Stark N, Lauffer F, Krause L, Thomas J, Atenhan A, et al. (2018) Toll-like receptor 7/8 agonists stimulate plasmacytoid dendritic cells to initiate TH17-deviated acute contact dermatitis in human subjects. J Allergy Clin Immunol. 141(4): 1320- 1333.e11. 3. Garzorz-Stark N, Lauffer F, Krause L, Thomas J, Atenhan A, et al. (2018) Toll-like receptor 7/8 agonists stimulate plasmacytoid dendritic cells to initiate TH17-deviated acute contact dermatitis in human subjects. J Allergy Clin Immunol. 141(4): 1320- 1333.e11. 8. Pižem J, Stojanovič L, Luzar B (2012) Melanocytic lesions with eczematous reaction (Meyerson's phenomenon) - a histopathologic analysis of 64 cases. J Cutan Pathol. 39(10): 901- 910. 9. Taylor CL, Maslen M, Kapembwa M (2006) A case of severe eczema following use of imiquimod 5 % cream. Sex Transm Infect. 82(3): 227-8. 4. Hanna E, Abadi R, Abbas O (2016) Imiquimod in dermatology: an overview. Int J Dermatol. 55(8): 831-844. 4. Hanna E, Abadi R, Abbas O (2016) Imiquimod in dermatology: an overview. Int J Dermatol. 55(8): 831-844. 5. Hammerl V, Parlar B, Navarini A, Gantenbein L, Väth H, et al. (2021) Mucosal side effects in patients treated with topical 5. Hammerl V, Parlar B, Navarini A, Gantenbein L, Väth H, et al. (2021) Mucosal side effects in patients treated with topical
https://openalex.org/W3030129290	https://journals.plos.org/plosgenetics/article/file?id=10.1371/journal.pgen.1008854&type=printable	English	null	Correction: Rapidly evolving protointrons in Saccharomyces genomes revealed by a hungry spliceosome	PLOS genetics	2,020	cc-by	248	PLOS GENETICS PLOS GENETICS OPEN ACCESS Citation: Talkish J, Igel H, Perriman RJ, Shiue L, Katzman S, Munding EM, et al. (2020) Correction: Rapidly evolving protointrons in Saccharomyces genomes revealed by a hungry spliceosome. PLoS Genet 16(5): e1008854. https://doi.org/10.1371/ journal.pgen.1008854 CORRECTION Correction: Rapidly evolving protointrons in Saccharomyces genomes revealed by a hungry spliceosome Jason Talkish, Haller Igel, Rhonda J. Perriman, Lily Shiue, Sol Katzman, Elizabeth M. Munding, Robert Shelansky, John Paul Donohue, Manuel Ares Jr. In the Data Availability statement and the Mapping and analysis of RNAseq data subsection of Materials and Methods, the accession number for RNAseq data deposited in GEO is listed incorrectly. The correct accession number is: GSE90105. Reference 1. Talkish J, Igel H, Perriman RJ, Shiue L, Katzman S, Munding EM, et al. (2019) Rapidly evolving protoin- trons in Saccharomyces genomes revealed by a hungry spliceosome. PLoS Genet 15(8): e1008249. https://doi.org/10.1371/journal.pgen.1008249 PMID: 31437148 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 1. Talkish J, Igel H, Perriman RJ, Shiue L, Katzman S, Munding EM, et al. (2019) Rapidly evolving protoin- trons in Saccharomyces genomes revealed by a hungry spliceosome. PLoS Genet 15(8): e1008249. https://doi.org/10.1371/journal.pgen.1008249 PMID: 31437148 PLOS Genetics \| https://doi.org/10.1371/journal.pgen.1008854 May 27, 2020 Published: May 27, 2020 Copyright: © 2020 Talkish et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. 1 / 1 PLOS Genetics \| https://doi.org/10.1371/journal.pgen.1008854 May 27, 2020
https://openalex.org/W2343291705	https://stemcellres.biomedcentral.com/track/pdf/10.1186/s13287-016-0322-3	English	null	The trans-well coculture of human synovial mesenchymal stem cells with chondrocytes leads to self-organization, chondrogenic differentiation, and secretion of TGFβ	Stem cell research & therapy	2,016	cc-by	7,656	RESEARCH Open Access The trans-well coculture of human synovial mesenchymal stem cells with chondrocytes leads to self-organization, chondrogenic differentiation, and secretion of TGFβ a Johanna Kubosch1, Emanuel Heidt1, Anke Bernstein1, Katharina Böttiger1 and Hagen Schmal2 * Correspondence: hagen.schmal@freenet.de 2Department of Orthopaedics and Traumatology, Odense University Hospital, Sdr. Boulevard 29, 5000 Odense C, Denmark 3Department of Clinical Research, University of Southern Denmark, Odense, Denmark Full list of author information is available at the end of the article Abstract Background: Synovial mesenchymal stem cells (SMSC) possess a high chondrogenic differentiation potential, which possibly supports natural and surgically induced healing of cartilage lesions. We hypothesized enhanced chondrogenesis of SMSC caused by the vicinity of chondrocytes (CHDR). Methods: Human SMSC and CHDR interactions were investigated in an in-vitro trans-well monolayer coculture over a time period of up to 21 days. Protein expression was analyzed using histology, immunostaining, or enzyme-linked immunosorbent assay. Additionally, mRNA expression was assessed by quantitative PCR. Results: After 7 days, phase-contrast microscopy revealed cell aggregation of SMSC in coculture with CHDR. Afterwards, cells formed spheres and lost adherence. However, this phenomenon was not observed when culturing SMSC alone. Fluorescence labeling showed concurrent collagen type II expression. Addition of transforming growth factor beta (TGFβ) to the cocultures induced SMSC aggregation in less time and with higher intensity. Additionally, alcian blue staining demonstrated enhanced glycosaminoglycan expression around SMSC aggregates after 1 and 2 weeks. Although TGFβ mRNA was expressed in all SMSC, the protein was measured with constantly increasing levels over 21 days only in supernatants of the cocultures. Considering the enhanced mRNA levels following supplementation with TGFβ, a positive feedback mechanism can be supposed. In line with the development of a chondrogenic phenotype, aggrecan mRNA expression increased after 7 and 14 days in the cocultures with and without TGFβ. Coculture conditions also amplified collagen type II mRNA expression after 2 weeks without and already after 1 week with TGFβ. There was no difference in collagen type I and type X expression between SMSC alone and the coculture with CHDR. Expression of both collagens increased following addition of TGFβ. mRNA data correlated with the intensity of immunofluorescence staining. Conclusions: Paracrine effects of CHDR induce a chondrogenic phenotype in SMSC possibly mimicking joint homeostasis. Coculture approaches may lead to a better understanding of cellular interactions with potential implications for cartilage repair procedures. Keywords: Synovial mesenchymal stem cell, Coculture, Differentiation, Chondrocytes, Synovium, Chondrogenesis Full list of author information is available at the end of the article © 2016 Kubosch et al. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. © 2016 Kubosch et al. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Kubosch et al. Stem Cell Research & Therapy (2016) 7:64 DOI 10.1186/s13287-016-0322-3 Kubosch et al. Stem Cell Research & Therapy (2016) 7:64 DOI 10.1186/s13287-016-0322-3 Background study was the histological observation of cell organization, the time frame, and the influencing cytokines. g Cartilage lesions have a limited capacity for repair and cause osteoarthritis (OA), so the search for treatment al- ternatives is ongoing. Many current approaches focus on the use of mesenchymal stem cells, which may play a significant role in both natural and surgically supported cartilage repair. Previously it has been described that natural cartilage repair can occur, especially in osteo- chondral defects; however, “the repair was mediated wholly by the proliferation and differentiation of mesen- chymal cells of the marrow” [1]. More recently, a pos- sible role for synovial mesenchymal stem cells (SMSC) was highlighted [2]. Injection of bone marrow-derived mesenchymal stem cells (BMSC) in osteoarthritic knees resulted in a long-term improvement of clinical outcome parameters [3], and SMSC were successfully used for arthroscopically assisted cartilage repair resulting in im- proved MRI features, histology, and clinical outcome [4]. Different studies suggested that SMSC have the best chondrogenic potential compared with mesenchymal stem cells derived from other tissue sources [5]. Adher- ence of SMSC to cartilage was mediated by hyaluronan [6], a possible mechanism for how these cells may be enriched in cartilage lesions. When SMSC migrate or are surgically placed at the site of a cartilage defect, they are in the direct vicinity of chondrocytes (CHDR) in their natural habitat. For this specific situation, coculture models are a powerful instrument to define and clarify cell–cell interactions. Until now the emphasis of SMSC/ CHDR cocultures was to show effects in acute [7] or chronic [8] inflammation. Hereby, it could be demon- strated that SMSC were able to secret typical cartilage markers such as aggrecan and decisively influence the course of inflammation. Furthermore, pellet cocultures of mesenchymal stem cells, usually bone marrow de- rived, and CHDR resulted in formation of hyaline struc- tured cartilage showing partially an even higher quality than the CHDR control group [9]. This phenomenon was independent of certain culture conditions and cell sources [10]. The disadvantage of this experimental ap- proach is the missing possibility to differentiate between paracrine effects and cell–cell interactions. The hypoth- esis of the study was that CHDR are able to induce a chondrogenic differentiation of synovial stem cells. Since we presumed paracrine signals originating from CHDR causing this phenomenon, a coculture model was chosen where the cells were separated by a filter. Background This model does not allow direct cell contact and mimics the bio- logical situation of cells collecting in a lesion with only marginal contact to the original cartilage layer. For evaluation, the markers of chondrogenesis aggrecan and collagen type II, the marker of dedifferentiation collagen type I, and the hypertrophy marker collagen type X were l d RNA d t i l l E h i f th Abstract 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. Kubosch et al. Stem Cell Research & Therapy (2016) 7:64 Page 2 of 11 Page 2 of 11 Kubosch et al. Stem Cell Research & Therapy (2016) 7:64 Methods Isolation, culture, and importance of SMSC in inflamma- tion have been described previously [7]. The cells’ prepar- ation protocols were approved by the Ethics Committee of the University of Freiburg as part of the “Tissue Bank for Research in the Field of Tissue Engineering” project (GTE-2002) and the biobank “Osteo” (AN-EK-FRBRG- 135/14). Cells from the same donors were used when comparing different culture conditions. Isolation of SMSC The cell preparation was described before [7]. Briefly, synovial tissue was gathered during knee operations with arthrotomy and arthroscopies (n = 4, male/female 2/2, average age 42.7 ± 15.0 years). The degree of OA was evaluated on X-ray images using Croft’s modification of the Kellgren and Lawrence score (KLS). Cells were used only from patients with healthy joints (KLS ≤2). The harvested tissue was kept in DMEM F-12 medium (Lonza BioWhittaker, Basel, Switzerland) at 4 °C. Within 2 h the tissue was cut into small pieces, washed, and transferred into DMEM F-12 medium with 10 % FCS (Biochrom, Berlin, Germany), 1 % penicillin/strepto- mycin (P/S) (Invitrogen, Karlsruhe, Germany), 0.5 % gentamycin (Biochrom), and 3 % collagenase P (Roche, Mannheim, Germany). The suspension was digested during the next 4 h on a shaking incubator (200 rpm) at 37 °C. Subsequently the released cells were centrifuged, washed, and seeded in expansion medium DMEM F-12 (10 % FCS, 1 % P/S, 0.5 % gentamycin). SMSC were seeded on coated T-flasks with a density of 2500–5000 cells/cm2 for expansion. The cells were frozen after reaching confluence. Thawed cells were grown and used when reaching a log phase of growth (passage 1). These cells were not further enriched and are also known as synovial fibroblasts or type B synoviocytes [11, 12]. Characterization of these cells was done by FACS showing that combined expression of the stem cell markers CD44, CD73, CD90, and CD105 was present in 76 %, but the combined expression of the negative markers CD11b, CD19, CD34, CD45, and HLA-DR reached only 6.9 ± 1.7 %. Osteogenic, adipogenic, and chondrogenic differentiation was possible using stand- ard protocols [13]. Real-time PCR Real-time PCR was carried out for SMSC only. RNA sam- ples from days 7 and 14 were transcribed into cDNA; RNA analysis was carried out for gene expression of aggrecan, TGFβ, and collagen type I, II, and X. Total mRNA was prepared using the Qiagen RNeasy kit accord- ing to the manufacturer’s instructions (Qiagen, Hilden, Germany). Total RNA (1 μg) was treated with 1 U DNAse I (Invitrogen, Karlsruhe, Germany) to remove genomic DNA. Poly-T primed cDNA synthesis was performed using 1 U reverse transcriptase III (RTIII; Invitrogen) per 1 μg RNA according to the manufacturer’s instruc- tions. TaqMan™PCR assays were performed in 384- well plates in a Roche LightCycler480 (Roche) using the Roche LightCycler Mastermix. For gene expres- sion analyses, Roche’s universal ProbeLibrary Probes Immunohistology ll f d Immunohistology Cells were fixed at –20 °C with methanol (Sigma-Aldrich, St. Louis, MO, USA) for 10 min; afterwards, they were washed with Dulbecco’s phosphate-buffered saline (DPBS; Gibco Invitrogen, Carlsbad, CA, USA). For blocking of unspecific binding sites, cells were incubated at room temperature with 5 % BSA (AppliChem GmbH, Darmstadt, Germany) in DPBS. Primary antibodies were diluted as follows: collagen type I (mouse monoclonalAb COL-1; abcam, Cambridge, UK) 1:500, collagen type II (rabbit polyclonalAb COL-2; abcam) 1:250, and collagen type X (mouse monoclonalAb COL-10; abcam) 1:750. After washing, the antibody working solutions were applied to the cells and incubated at 4 °C overnight in a humid chamber. After washing three times, the cells were incu- bated with the working solution of the secondary antibody: Alexa Fluor 488 goat anti-mouse IgG and Alexa Fluor 568 goat anti-rabbit IgG (Life Technologies, Grand Island, NY, USA) in 1 % BSA, dilution 1:250. After washing three times again, color reagent (ProLong® Gold antifade reagent DAPI; Life Technologies) was applied. An Olympus BX51 micro- scope (Olympus Deutschland GmbH) with special fluores- cence filters was used for image acquisition. The program ImageJ (Wayne Rasband, NIH; imagej.nih.gov/ij/download) facilitated overlaying of images. Isolation of CHDR Isolation of CHDR Cell preparation was described before [14]. Briefly, CHDR were gained from femoral heads during hip arthroplasty operations (n = 6, male/female 5/1, average age 79.2 ± 8.2 years). The degree of OA was evaluated on X-ray images using Croft’s modification of the KLS Page 3 of 11 Page 3 of 11 Kubosch et al. Stem Cell Research & Therapy (2016) 7:64 score. Cells from patients with advanced OA (≥grade 3) were not used for experiments. Within 8 h after surgery, the cartilage was separated from the bone and cut into small pieces, washed, and transferred into DMEM F-12 10 % FCS, 1 % P/S, 0.5 % gentamycin, and 3 % collage- nase CLS type II (Biochrom). Minced cartilaginous tissue was then enzymatically digested during the next 16 h on a shaking incubator at 37 °C with 200 rpm. Subsequently the released CHDR were centrifuged, washed, and seeded in expansion medium DMEM F-12 (Lonza BioWhittaker) (10 % FCS, 1 % P/S, 0.5 % gentamycin). Expansion of CHDR was performed by seeding them on coated T-flasks with a density of 2500–5000 cells/cm2. The cells were frozen after reaching confluence. Thawed cells were grown and used when reaching a log phase of growth (passage 1). in 24-well-plates (Corning Incorporated, Corning, NY, USA). For alcian blue staining, the cells were fixed and stained using the PAS-staining Kit (Merck Millipore, Billerica, Massachusetts, USA). Briefly, staining with alcian blue was followed by incubation with periodic acid, Schiff reagent, and hematoxylin, whereupon each step was followed by washing. After mounting, histo- logical pictures were analyzed using an Olympus BX51 microscope (Olympus Deutschland GmbH, Hamburg, Germany) with the software module Stream Motion adjusting only brightness and contrast. TGFβ enzyme-linked immunosorbent assay TGFβ levels in supernatants were analyzed by enzyme- linked immunosorbent assay (ELISA) (R&D; and Bio- Source Deutschland GmbH, Solingen, Germany) accord- ing to the manufacturers’ instructions. Briefly, this assay employs the quantitative sandwich enzyme immunoassay technique. The microplate was precoated with a specific monoclonal antibody. Supernatants were applied to the wells and, after washing, HRP-conjugated specific anti- body was added to the wells. Following the next wash, color development was proportional to the protein con- centration and calculated by comparison with a stand- ard. A colorimetric method was applied to quantify the total protein amount in the lavage fluids. Coculture conditions SMSC in the bottom and CHDR on top were separated in a trans-well culture with 0.4 μm inserts. As a basal culture medium, DMEM F-12 medium supplemented with 10 % FCS, 1 % P/S, and 0.5 % gentamycin was used. Cell viability was >95 % before starting the experiment. Half-media changes were performed three times per week. The initial seeding density was 20,000 cells/cm2. Experiments were repeated at least three times with cells from different donors (one patient for one experimental trial). The cells were not pooled. The total time of cocul- ture was 21 days maximum. There were three different groups: SMSC alone, SMSC with CHDR, and SMSC with CHDR supplemented with transforming growth factor beta (TGFβ)-3. A concentration of 10 ng/ml TGF- β3 (R&D Systems, Minneapolis, MN, USA) was added to the positive controls. Histology Cover slides were coated with poly-D-lysine (0.1 mg/ml) (Merck Millipore, Billerica, Massachusetts, USA) at 37 °C (5 % CO2) for 60 min, then washed and dried overnight. Afterwards, SMSC were grown on the slides in coculture Page 4 of 11 Kubosch et al. Stem Cell Research & Therapy (2016) 7:64 Page 4 of 11 cell density was observed in the TGFβ-treated positive control group. Figure 1c demonstrates representative slides of the single staining for collagen type X and the DAPI staining for the different groups. The percentage- wise estimation of the aggregation extent was 0 % for SMSC alone, ≥40 % for the coculture of SMSC with CHDR, and ≥90 % for the coculture supplemented with TGFβ (overview in phase-contrast microscopy). Addition- ally, cell spheres per field of view were counted resulting in 0 ± 0 aggregates/field for SMSC alone, 1 ± 0.4 aggre- gates/field for the coculture of SMSC with CHDR, and 2.25 ± 0.5 aggregates/field for the coculture supplemented with TGFβ (n = 5, 20-fold magnification). There was no difference comparing the aggregation after 7 or 14 days. and recommended Universal ProbeLibrary Reference Gene Assays were used. The cycling conditions were de- naturation (one cycle: 50 °C for 120 sec, 95 °C for 600 sec), followed by 40 amplification cycles (95 °C for 15 sec, 60 °C for 60 sec, 72 °C for 10 sec), followed by melting (one cycle: 95 °C for 10 sec, 50 °C for 30 sec, 70 °C for 1 sec), and cooling (one cycle: 40 °C for 30 sec). Data were quantified via ΔΔCT comparisons. Data were normalized by comparing genes of interest versus reference genes (GAPDH). Reaction efficiency is controlled by a relative standard curve and/or a calibrator per reaction. Real-time PCR was carried out in quadruplicate, each value repre- senting an average of four experiments. Role for TGFβ Concentrations of cytokines determined by the specific ELISA were calculated according to the manufacturers’ instructions (R&D; and Thermo Scientific, Rockford, IL, USA), creating a standard curve and reducing data with a four-parameter logistic (4-PL) curve fit using Graph- Pad Prism 5 software (GraphPad Software, Inc., La Jolla, CA, USA). All values were expressed as mean ± standard error of the mean. Statistical significance was tested nonparametrically using the Mann–Whitney U test. The values of different time points were compared in each group, and the values of one time point were compared between the groups. Statistical significance was defined as p < 0.05. TGFβ concentrations in the supernatants were measured comparing SMSC in monolayer with the coculture of SMSC and CHDR without or with TGFβ supplementa- tion (Fig. 2). As expected, the highest concentrations were observed in the positive control with TGFβ (777 ± 28 pg/ml). This is lower than the added concentrations indicating receptor immobilization of the cytokine or degradation, because supernatants were collected to- gether with medium change. Concentration levels are followed by the coculture without TGFβ supplementa- tion starting at week 1 with 68 ± 5 pg/ml and steadily in- creasing up to 183 ± 15 pg/ml at week 3. Although TGFβ was also found in the SMSC monolayer, levels were short over the detection limit. There was a statisti- cally significant difference between the levels of each time point of all groups and each time point within the cocultures (p < 0.05). Because the statistical significance reached was marginal, additional comparisons were cal- culated using a Student’s t test, resulting in p < 0.02. Fur- thermore, the values of all groups were merged independent of the time point. The comparison resulted in highly significant differences (p < 0.001) using the dir- ect comparison of groups with the Mann–Whitney U test and using the Kruskall–Wallis H test (multiple com- parisons). Considering a significance level of the direct group comparisons very close to the defined α and a possibly not complete random sample, the tests may overstate the accuracy of the results. TGFβ mRNA ex- pression was also compared over a 2-week interval (Fig. 3). A statistically significant difference was found between the TGFβ-supplemented group and both other groups (p = 0.021), but not between the different time points within each group. Results Histology SMSC were kept in monolayer cultures alone or in co- culture with CHDR. As a positive control, these cocul- tures were supplemented with TGFβ. While SMSC alone stayed separated after 1 week, an aggregation of SMSC was visible in the coculture group. The addition of TGFβ even resulted in sphere formation (Fig. 1a–c). No further time points are shown, because the spheres lost adher- ence and could no longer be comparably stained. Since spin-downs resulted in cell debris, SMSC were grown on coated cover slides allowing only alcian blue staining and phase-contrast microscopy as shown in Fig. 1d. Again, the phenomenon of cell aggregation, sphere for- mation, and loss of adherence could be observed after 1 or 2 weeks, first in the TGFβ-treated positive control and then in the coculture. The alcian blue staining, which was more intense in cell aggregates, documents the presence of glycosaminoglycans/mucopolysaccha- rides. Cell aggregation could be observed with the cells of all different donors. Figure 1a compares the different groups by overlaying immunofluorescence staining and phase-contrast microscopy images. Figure 1b shows the single staining for collagen type I and type II of the dif- ferent groups. The highest intensity but also the highest mRNA regulation of aggrecan and collagen type I, II, and X Aggrecan mRNA expression was compared in SMSC monolayer with the coculture of SMSC and CHDR with- out or with TGFβ supplementation (Fig. 4). The highest Page 5 of 11 Kubosch et al. Stem Cell Research & Therapy (2016) 7:64 Fig. 1 (See legend on next page.) Fig. 1 (See legend on next page.) Page 6 of 11 Kubosch et al. Stem Cell Research & Therapy (2016) 7:64 (See figure on previous page.) Fig. 1 a Self-organization of SMSC after 7 days in monolayer culture. SMSC alone (left) stay separated, but in coculture with CHDR (middle) an aggregation of cells is visible, and addition of TGFβ (right) results in sphere formation. Upper row shows overlaying immunofluorescence staining (green: collagen type I, red: collagen type II, blue: DAPI; scale 20 μm), and lower row the phase-contrast microscopy. b Single staining for collagen type I (upper row) and type II (lower row) of the different groups. c Single staining for collagen type X and DAPI of the different groups. d Alcian blue (AB) staining of the different groups and time points. Results Histology Here the levels were found in both cocultures at both investigated time points (without TGFβ: 4.7 ± 1.2-fold at week 1 and 6.7 ± 1.1-fold after 2 weeks, with TGFβ: 6.5 ± 0.9-fold at week 1 and 4.9 ± 1.1-fold at week 2). There is a statisti- cally significant difference between both cocultures and the monolayer, but not between the different time points within each group. Collagen type I (col1) mRNA expres- sion was also compared between SMSC monolayer with the coculture of SMSC and CHDR without or with sup- plemented TGFβ (Fig. 5). The highest values were mea- sured in the TGFβ-supplemented coculture (up to 1625 ± 219-fold). There is a statistically significant difference between the TGFβ-supplemented group and both other groups (p = 0.021), but not between the different time points within each group or SMSC alone and the non- supplemented coculture. Collagen type II (col2) mRNA expression was also examined (Fig. 6), showing the high- est values in both cocultures (up to 8.9 ± 3.2-fold). There was no statistically significant difference between the TGFβ-supplemented group and day 14 of the coculture, but these values were higher compared with SMSC alone and day 7 of the coculture (p = 0.021). There is no difference between the two time points within each group except for the coculture without TGFβ. Here the Results Histology Upper row: AB 7 days (marker 50 μm), middle row: AB 14 days (marker 100 μm), lower row: phase-contrast (PC) microscopy 14 days (marker 100 μm). CHDR chondrocytes, SMSC synovial mesenchymal stem cells, TGFβ transforming growth factor beta (Color figure online) col2 mRNA increased between the first week and the second week significantly (p = 0.043). Since collagen type II is considered the main marker for differentiated cartil- age, the results indicate increasing chondrogenic differ- entiation induced by the presence of CHDR in coculture and the addition of TGFβ. Furthermore, the mRNA of the chondrogenic hypertrophy marker collagen type X (col10) was analyzed (Fig. 7). Although we found signifi- cant values at day 7 in the plain coculture, the highest values were measured when TGFβ was supplemented (up to 3.1 ± 0.7-fold). The value in the TGFβ- supplemented group at day 7 is statistically significantly higher than at all other time points (p = 0.014). There is no difference between the different time points within the SMSC (0 caused by rounding) and the coculture groups. levels were found in both cocultures at both investigated time points (without TGFβ: 4.7 ± 1.2-fold at week 1 and 6.7 ± 1.1-fold after 2 weeks, with TGFβ: 6.5 ± 0.9-fold at week 1 and 4.9 ± 1.1-fold at week 2). There is a statisti- cally significant difference between both cocultures and the monolayer, but not between the different time points within each group. Collagen type I (col1) mRNA expres- sion was also compared between SMSC monolayer with the coculture of SMSC and CHDR without or with sup- plemented TGFβ (Fig. 5). The highest values were mea- sured in the TGFβ-supplemented coculture (up to 1625 ± 219-fold). There is a statistically significant difference between the TGFβ-supplemented group and both other groups (p = 0.021), but not between the different time points within each group or SMSC alone and the non- supplemented coculture. Collagen type II (col2) mRNA expression was also examined (Fig. 6), showing the high- est values in both cocultures (up to 8.9 ± 3.2-fold). There was no statistically significant difference between the TGFβ-supplemented group and day 14 of the coculture, but these values were higher compared with SMSC alone and day 7 of the coculture (p = 0.021). There is no difference between the two time points within each group except for the coculture without TGFβ. Discussion The main finding of the study is that human CHDR are able to induce a chondrogenic phenotype in human SMSC in a trans-well coculture. Although SMSC were kept in a monolayer, chondrogenesis leads to loss of ad- herence and formation of spheres. This was sufficiently regulated by paracrine factors; no direct cell–cell Fig. 2 TGFβ concentrations in supernatants comparing SMSC in monolayer with the coculture of SMSC and CHDR without or with TGFβ supplementation. There is a statistically significant difference between the groups and each time point within the cocultures. CHDR chondrocytes, SMSC synovial mesenchymal stem cells or SFB synovial fibroblasts, TGFβ transforming growth factor beta Fig. 2 TGFβ concentrations in supernatants comparing SMSC in monolayer with the coculture of SMSC and CHDR without or with TGFβ supplementation. There is a statistically significant difference between the groups and each time point within the cocultures. CHDR chondrocytes, SMSC synovial mesenchymal stem cells or SFB synovial fibroblasts, TGFβ transforming growth factor beta Kubosch et al. Stem Cell Research & Therapy (2016) 7:64 Page 7 of 11 Fig. 3 TGFβ mRNA expression comparing SMSC in monolayer with the coculture of SMSC and CHDR without or with TGFβ supplementation. There is a statistically significant difference between the TGFβ-supplemented group and both other groups, but not between the different time points within each group. CHDR chondrocytes, SMSC synovial mesenchymal stem cells or SFB synovial fibroblasts, TGFβ transforming growth factor beta Fig. 3 TGFβ mRNA expression comparing SMSC in monolayer with the coculture of SMSC and CHDR without or with TGFβ supplementation. There is a statistically significant difference between the TGFβ-supplemented group and both other groups, but not between the different time points within each group. CHDR chondrocytes, SMSC synovial mesenchymal stem cells or SFB synovial fibroblasts, TGFβ transforming growth factor beta interactions were necessary. The degree of cell self- assembly and sphere formation and the collagen type II and aggrecan expression were associated with the levels of TGFβ found in the supernatants. Collagen type X, a marker of chondrocyte hypertrophy, was expressed in the coculture of SMSC and CHDR when TGFβ was additionally supplemented. inflammatory regulations attributed to mesenchymal stem cells [15]. The possible high impact of coculture sys- tems on cartilage tissue engineering is documented by the increasing number of described technical solutions [16] and experimental designs [17]. Discussion In contrast to previous studies, this investigation focused on an experimental trans-well design using human synovial stem cells. This set-up was inspired by the idea that—unlike the previously suggested predominant role of bone marrow-derived pro- genitor cells for cartilage regeneration [1]—synovial stem Cocultures of mesenchymal stem cells and CHDR can result in improved chondrogenic differentiation in pel- lets [9] and immunological interactions including anti- Fig. 4 Aggrecan mRNA expression comparing SMSC in monolayer with the coculture of SMSC and CHDR without or with TGFβ supplementation. There is a statistically significant difference between both cocultures and the monolayer, but not between the different time points within each group. CHDR chondrocytes, SMSC synovial mesenchymal stem cells or SFB synovial fibroblasts, TGFβ transforming growth factor beta Fig. 4 Aggrecan mRNA expression comparing SMSC in monolayer with the coculture of SMSC and CHDR without or with TGFβ supplementation. There is a statistically significant difference between both cocultures and the monolayer, but not between the different time points within each group. CHDR chondrocytes, SMSC synovial mesenchymal stem cells or SFB synovial fibroblasts, TGFβ transforming growth factor beta Kubosch et al. Stem Cell Research & Therapy (2016) 7:64 Page 8 of 11 Fig. 5 Collagen type I (col1) mRNA expression comparing SMSC in monolayer with the coculture of SMSC and CHDR without or with TGFβ supplementation. There is a statistically significant difference between the TGFβ-supplemented group and both other groups, but not between the different time points within each group. CHDR chondrocytes, SMSC synovial mesenchymal stem cells or SFB synovial fibroblasts, TGFβ transforming growth factor beta Fig. 5 Collagen type I (col1) mRNA expression comparing SMSC in monolayer with the coculture of SMSC and CHDR without or with TGFβ supplementation. There is a statistically significant difference between the TGFβ-supplemented group and both other groups, but not between the different time points within each group. CHDR chondrocytes, SMSC synovial mesenchymal stem cells or SFB synovial fibroblasts, TGFβ transforming growth factor beta cells might be able to contribute to natural cartilage re- generation. This phenomenon has been described, but the cellular sources for repair are controversially discussed. The potential of CHDR themselves appears to be very lim- ited, as previously published outgrowth experiments have shown [18]. Discussion The value of the TGFβ-supplemented group at day 7 is statistically significantly higher than at all other time points. There is no difference between the different time points within the SMSC and the coculture groups. CHDR chondrocytes, SMSC synovial mesenchymal stem cells or SFB synovial fibroblasts, TGFβ transforming growth factor beta sphere formation), causing minimal fibrochondrogenesis (col1) or hypertrophy (col10). was accompanied by expression of cartilage markers as aggrecan and collagen type II on the protein and mRNA levels. The key role for TGFβ in the described and ob- served process of chondrogenic differentiation was not only demonstrated for BMSC [21], but also for SMSC [22]. TGFβ has been described to upregulate collagen type I mRNA in osteoblasts [23] or CHDR [24]. Furthermore, TGFβ seems to induce the cartilage hypertrophy marker collagen type X. This was confirmed by our results, but high levels of either collagen type I or type X were mainly found in the control group that was supplemented with additional TGFβ. In accordance with these data, the hypertrophic status of chondrogenically differentiated mesenchymal stem cells has previously been described to be associated with overexpression of osteogenic markers as collagen type I and alkaline phosphatase [25], explaining our positive immunostaining and the higher collagen type I mRNA expression in the TGFβ-supplemented group. The observed missing accordance of TGFβ mRNA and protein regulation in the supernatants can have different causes. First, TGFβ protein might also significantly be secreted by CHDR, which in the current experimental set-up cannot be measured separately. Secondly, protein formation in SMSC might undergo further regulatory processing and not only depend on RNA regulation. The higher levels of TGFβ mRNA in the supplemented co- culture suggest a positive feedback regulation. Chondrogenesis of SMSC was also demonstrated in a pellet coculture model using rabbit CHDR that overex- pressed TGFβ after adenoviral transfection [26]. Based on the results of our study it may be concluded that gene transfer might not be necessary, because the cocul- ture itself provides sufficient paracrine stimuli for chon- drogenic differentiation of SMSC. Similarly, TGFβ induced the chondrogenesis of SMSC with high levels of collagen type II, aggrecan, and Sox 9, and low levels of dedifferentiation or hypertrophy markers in a coculture pellet model using nucleus pulposus cells in serum-free medium [27]. Discussion Since synovial fibroblasts are located in the direct vicinity of cartilage and cartilage lesions and also exhibit stem cell characteristics [19] with the best chon- drogenic potential of different mesenchymal stem sources [5], these cells are an interesting candidate for the cellular origin of natural and spontaneous cartilage regeneration. The natural environment of cells, especially stem cells, de- termines their histological and biochemical phenotype [20]. Therefore, we hypothesized that CHDR are able to secret paracrine signals inducing a chondrogenic differen- tiation of SMSC. This was confirmed by showing spontan- eous formation of first cell aggregation and then cell sphere formation by SMSC in coculture with CHDR. This Fig. 6 Collagen type II (col2) mRNA expression comparing SMSC in monolayer with the coculture of SMSC and CHDR without or with TGFβ supplementation. There is no statistically significant difference between the TGFβ-supplemented group and day 14 of the coculture, but these values are higher compared with SMSC alone and day 7 of the coculture. There is no difference between the different time points within each group except for the coculture. CHDR chondrocytes, SMSC synovial mesenchymal stem cells or SFB synovial fibroblasts, TGFβ transforming growth factor beta Fig. 6 Collagen type II (col2) mRNA expression comparing SMSC in monolayer with the coculture of SMSC and CHDR without or with TGFβ supplementation. There is no statistically significant difference between the TGFβ-supplemented group and day 14 of the coculture, but these values are higher compared with SMSC alone and day 7 of the coculture. There is no difference between the different time points within each group except for the coculture. CHDR chondrocytes, SMSC synovial mesenchymal stem cells or SFB synovial fibroblasts, TGFβ transforming growth factor beta Kubosch et al. Stem Cell Research & Therapy (2016) 7:64 Page 9 of 11 Fig. 7 Collagen type X (col10) mRNA expression comparing SMSC in monolayer with the coculture of SMSC and CHDR without or with TGFβ supplementation. The value of the TGFβ-supplemented group at day 7 is statistically significantly higher than at all other time points. There is no difference between the different time points within the SMSC and the coculture groups. CHDR chondrocytes, SMSC synovial mesenchymal stem cells or SFB synovial fibroblasts, TGFβ transforming growth factor beta Fig. 7 Collagen type X (col10) mRNA expression comparing SMSC in monolayer with the coculture of SMSC and CHDR without or with TGFβ supplementation. Authors’ contributions 11. Jones BA, Pei M. Synovium-derived stem cells: a tissue-specific stem cell for cartilage engineering and regeneration. Tissue Eng Part B Rev. 2012;18:301–11. 11. Jones BA, Pei M. Synovium-derived stem cells: a tissue-specific stem cell for cartilage engineering and regeneration. Tissue Eng Part B Rev. 2012;18:301–11. EJK was involved in the conception and the design of the study, and the analysis and interpretation of the data, contributed to the article draft, and contributed to the final approval of the submitted article. EH was responsible for the collection, assembly, and management of data, performed the experiments, contributed to the article draft, calculated the ELISA values and some statistics, and approved the final version of the article. AB supported the laboratory work, supervised the sample analysis, was responsible for sample storage, revised the article draft critically, and approved the final version of the article. KB supported the laboratory work, revised the article draft critically, and approved the final version of the article. HS was responsible for the conception and the design of the study, obtaining funding, the analysis and interpretation of the data, tutorial of EH and EJK, writing the article, and the final approval of the submitted article. All authors take responsibility for the integrity of the work as a whole, from inception to finished article. 12. Kurth TB, Dell’accio F, Crouch V, Augello A, Sharpe PT, De Bari C. Functional mesenchymal stem cell niches in adult mouse knee joint synovium in vivo. Arthritis Rheum. 2011;63:1289–300. 12. Kurth TB, Dell’accio F, Crouch V, Augello A, Sharpe PT, De Bari C. Functional mesenchymal stem cell niches in adult mouse knee joint synovium in vivo. Arthritis Rheum. 2011;63:1289–300. 13. Pittenger MF, Mackay AM, Beck SC, Jaiswal RK, Douglas R, Mosca JD, Moorman MA, Simonetti DW, Craig S, Marshak DR. Multilineage potential of adult human mesenchymal stem cells. Science. 1999;284:143–7. 14. Schmal H, Mehlhorn AT, Fehrenbach M, Müller CA, Finkenzeller G, Südkamp NP. Regulative mechanisms of chondrocyte adhesion. Tissue Eng. 2006;12:741–50. 15. van Buul GM, Villafuertes E, Bos PK, Waarsing JH, Kops N, Narcisi R, Weinans H, Verhaar J a. N, Bernsen MR, van Osch GJVM. Mesenchymal stem cells secrete factors that inhibit inflammatory processes in short-term osteoarthritic synovium and cartilage explant culture. 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Mesenchymal stem cell therapy for knee osteoarthritis: 5 years follow-up of three patients. Int J Rheum Dis. 2016;19:219. 4. Sekiya I, Muneta T, Horie M, Koga H. Arthroscopic transplantation of synovial stem cells improves clinical outcomes in knees with cartilage defects. Clin Orthop Relat Res. 2015;473:2316. Author details 1 1Department of Orthopedics and Trauma Surgery, Albert-Ludwigs University Medical Center Freiburg, Freiburg, Germany. 2Department of Orthopaedics and Traumatology, Odense University Hospital, Sdr. Boulevard 29, 5000 Odense C, Denmark. 3Department of Clinical Research, University of Southern Denmark, Odense, Denmark. Received: 13 January 2016 Revised: 7 April 2016 Accepted: 11 April 2016 Competing interests All authors declare that they have no financial and personal relationships with other people, or organizations that could inappropriately influence (bias) their work. 10. Wu L, Prins H-J, Helder MN, van Blitterswijk CA, Karperien M. Trophic effects of mesenchymal stem cells in chondrocyte co-cultures are independent of culture conditions and cell sources. Tissue Eng Part A. 2012;18:1542–51. Abbreviations BMSC: bone marrow-derived mesenchymal stem cells; BSA: bovine serum albumin; CHDR: chondrocytes; col1: collagen type I; col10: collagen type X; col2: collagen type II; DMEM: Dulbecco’s modified Eagle’s medium; DPBS: Dulbecco’s phosphate-buffered saline; ELISA: enzyme-linked immunosorbent assay; FCS: fetal calf serum; KLS: Kellgren and Lawrence score; OA: osteoarthritis; P/S: penicillin/streptomycin; PAS-staining: Periodic acid–Schiff stain; SMSC: synovial mesenchymal stem cells; TGFβ: transforming growth factor beta. 8. Ryu J-S, Jung Y-H, Cho M-Y, Yeo JE, Choi Y-J, Kim YI, Koh Y-G. Co-culture with human synovium-derived mesenchymal stem cells inhibits inflammatory activity and increases cell proliferation of sodium nitroprusside-stimulated chondrocytes. Biochem Biophys Res Commun. 2014;447:715–20. 8. Ryu J-S, Jung Y-H, Cho M-Y, Yeo JE, Choi Y-J, Kim YI, Koh Y-G. Co-culture with human synovium-derived mesenchymal stem cells inhibits inflammatory activity and increases cell proliferation of sodium nitroprusside-stimulated chondrocytes. Biochem Biophys Res Commun. 2014;447:715–20. 9. Lettry V, Hosoya K, Takagi S, Okumura M. Coculture of equine mesenchymal stem cells and mature equine articular chondrocytes results in improved chondrogenic differentiation of the stem cells. Jpn J Vet Res. 2010;58:5–15. 9. Lettry V, Hosoya K, Takagi S, Okumura M. Coculture of equine mesenchymal stem cells and mature equine articular chondrocytes results in improved chondrogenic differentiation of the stem cells. Jpn J Vet Res. 2010;58:5–15. Discussion However, the stimulatory suc- cess of the coculture with CHDR on the chondrogenic differentiation of SMSC underlines the potential role of these cells in natural and artificially supported cartilage repair. Conclusions The vicinity of CHDR in a trans-well coculture induces a chondrogenic phenotype in SMSC. This process is associated with increased TGFβ secretion and offers possible implications for cartilage repair. This effect may play a significant role in natural and surgically in- duced cartilage repair, especially in cell therapeutic approaches. 5. Sakaguchi Y, Sekiya I, Yagishita K, Muneta T. Comparison of human stem cells derived from various mesenchymal tissues: superiority of synovium as a cell source. Arthritis Rheum. 2005;52:2521–9. 6. Baboolal TG, Mastbergen SC, Jones E, Calder SJ, Lafeber FPJG, McGonagle D. Synovial fluid hyaluronan mediates MSC attachment to cartilage, a potential novel mechanism contributing to cartilage repair in osteoarthritis using knee joint distraction. Ann Rheum Dis. 2015;75:908. 7. Pilz IH, Mehlhorn A, Dovi-Akue D, Langenmair ER, Südkamp NP, Schmal H. Development and retranslational validation of an in vitro model to characterize acute infections in large human joints. BioMed Res Int. 2014;2014:848604. Discussion All of the data emphasize the key role for TGFβ in chondrogenic differentiation for both in-vitro cultures of mesenchymal stem cells of different origin [13] and in the natural articular environment. Although the role of TGFβ seems striking, there are a few limita- tions attributed to the presented experimental set-up. First, the measured TGFβ levels in the supernatants are a summary response of all cells, which makes it The fact that exogenous TGFβ supplementation up- regulated collagen type I and type X in the SMSC with- out providing further enhancement of collagen type II or aggrecan expression is probably a dosage effect, assum- ing that the CHDR are promoting chondrogenesis in SMSC by releasing TGFβ; or it could suggest that CHDR are releasing other paracrine factors modulating the ef- fect. This has to be considered also in the light of the only marginal statistical significance regarding the in- creased TGFβ concentrations in the supernatants of the coculture. However, the CHDR alone had a chondro- genic effect (at least in terms of gene expression and Page 10 of 11 Page 10 of 11 Kubosch et al. Stem Cell Research & Therapy (2016) 7:64 impossible to differentiate the true origin. This is the na- ture of a coculture, and therefore mRNA levels were de- termined in SMSC. Unfortunately, the data were not completely conclusive, because TGFβ mRNA could also be found in SMSC alone, indicating separate regulation pathways for protein and mRNA. Secondly, the simple presence of TGFβ does not allow drawing functional conclusions. This means we observed an association of TGFβ in the supernatant and chondrogenic differenti- ation of SMSC, but the biological or mechanistic relation is not necessarily evident. However, the stimulatory suc- cess of the coculture with CHDR on the chondrogenic differentiation of SMSC underlines the potential role of these cells in natural and artificially supported cartilage repair. impossible to differentiate the true origin. This is the na- ture of a coculture, and therefore mRNA levels were de- termined in SMSC. Unfortunately, the data were not completely conclusive, because TGFβ mRNA could also be found in SMSC alone, indicating separate regulation pathways for protein and mRNA. Secondly, the simple presence of TGFβ does not allow drawing functional conclusions. This means we observed an association of TGFβ in the supernatant and chondrogenic differenti- ation of SMSC, but the biological or mechanistic relation is not necessarily evident. Acknowledgements and funding source The Research Commission of the Albert-Ludwigs University Medical Center Freiburg (SCHM1014/14) and the Federal Ministry of Education and Research Germany (01EC1001D) funded the study. The article processing charge was funded by the open access publication fund of the Albert Ludwigs University Freiburg. The Research Commission of the Albert-Ludwigs University Medical Center Freiburg (SCHM1014/14) and the Federal Ministry of Education and Research Germany (01EC1001D) funded the study. The article processing charge was funded by the open access publication fund of the Albert Ludwigs University Freiburg. 17. Wu L, Post JN, Karperien M. Engineering cartilage tissue by pellet coculture of chondrocytes and mesenchymal stromal cells. Methods Mol Biol Clifton NJ. 2015;1226:31–41. 17. Wu L, Post JN, Karperien M. Engineering cartilage tissue by pellet coculture of chondrocytes and mesenchymal stromal cells. Methods Mol Biol Clifton NJ. 2015;1226:31–41. Page 11 of 11 Kubosch et al. Stem Cell Research & Therapy (2016) 7:64 Kubosch et al. Stem Cell Research & Therapy (2016) 7:64 18. Zingler C, Carl H-D, Swoboda B, Krinner S, Hennig F, Gelse K. Limited evidence of chondrocyte outgrowth from adult human articular cartilage. Osteoarthr Cartil OARS Osteoarthr Res Soc. 2016;24:124–8. 19. Pei M, He F, Boyce BM, Kish VL. Repair of full-thickness femoral condyle cartilage defects using allogeneic synovial cell-engineered tissue constructs Osteoarthr Cartil OARS Osteoarthr Res Soc. 2009;17:714–22. 20. Leyh M, Seitz A, Dürselen L, Schaumburger J, Ignatius A, Grifka J, Grässel S. Subchondral bone influences chondrogenic differentiation and collagen production of human bone marrow-derived mesenchymal stem cells and articular chondrocytes. Arthritis Res Ther. 2014;16:453. 21. Mehlhorn AT, Schmal H, Kaiser S, Lepski G, Finkenzeller G, Stark GB, Südkamp NP. Mesenchymal stem cells maintain TGF-beta-mediated chondrogenic phenotype in alginate bead culture. Tissue Eng. 2006;12:1393–403. 22. Kim YI, Ryu J-S, Yeo JE, Choi YJ, Kim YS, Ko K, Koh Y-G. Overexpression of TGF-β1 enhances chondrogenic differentiation and proliferation of human synovium-derived stem cells. Biochem Biophys Res Commun. 2014;450:1593–9. 23. Glueck M, Gardner O, Czekanska E, Alini M, Stoddart MJ, Salzmann GM, Schmal H. Induction of osteogenic differentiation in human mesenchymal stem cells by crosstalk with osteoblasts. BioResearch Open Access. 2015;4:121–30. 24. Perrier-Groult E, Pasdeloup M, Malbouyres M, Galéra P, Mallein-Gerin F. Control of collagen production in mouse chondrocytes by using a combination of bone morphogenetic protein-2 and small interfering RNA targeting Col1a1 for hydrogel-based tissue-engineered cartilage. Tissue Eng Part C Methods. 2013;19:652–64. 25. Kubosch et al. Stem Cell Research & Therapy (2016) 7:64 Acknowledgements and funding source Mueller MB, Fischer M, Zellner J, Berner A, Dienstknecht T, Prantl L, Kujat R, Nerlich M, Tuan RS, Angele P. Hypertrophy in mesenchymal stem cell chondrogenesis: effect of TGF-beta isoforms and chondrogenic conditioning. Cells Tissues Organs. 2010;192:158–66. 26. Varshney RR, Zhou R, Hao J, Yeo SS, Chooi WH, Fan J, et al. Chondrogenesis of synovium-derived mesenchymal stem cells in gene-transferred co-culture system. Biomaterials. 2010;31:6876–91. 27. Chen S, Emery SE, Pei M. Coculture of synovium-derived stem cells and nucleus pulposus cells in serum-free defined medium with supplementation of transforming growth factor-beta1: a potential application of tissue-specific stem cells in disc regeneration. Spine. 2009;34:1272–80. 27. Chen S, Emery SE, Pei M. Coculture of synovium-derived stem cells and nucleus pulposus cells in serum-free defined medium with supplementation of transforming growth factor-beta1: a potential application of tissue-specific stem cells in disc regeneration. Spine. 2009;34:1272–80. • We accept pre-submission inquiries • Our selector tool helps you to ﬁnd the most relevant journal • We provide round the clock customer support • Convenient online submission • Thorough peer review • Inclusion in PubMed and all major indexing services • Maximum visibility for your research Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and we will help you at every step: Submit your next manuscript to BioMed Central and we will help you at every step: • We accept pre-submission inquiries • Our selector tool helps you to ﬁnd the most relevant journal • We provide round the clock customer support • Convenient online submission • Thorough peer review • Inclusion in PubMed and all major indexing services • Maximum visibility for your research Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and we will help you at every step:
https://openalex.org/W4362394701	https://figshare.com/articles/journal_contribution/Supplementary_Table_S4_from_Effect_of_Aromatase_Inhibition_on_Functional_Gene_Modules_in_Estrogen_Receptor_Positive_Breast_Cancer_and_Their_Relationship_with_Antiproliferative_Response/22448531/1/files/39899582.pdf	English	null	Supplementary Table S2 from Effect of Aromatase Inhibition on Functional Gene Modules in Estrogen Receptor–Positive Breast Cancer and Their Relationship with Antiproliferative Response	null	2,023	cc-by	21,464	within each module ent Entrez_Gene_ID Symbol Probe_Id -1 991CDC20 ILMN_1663390 -1 1476 CSTB ILMN_1761797 -1 1503 CTPS ILMN_1783285 1 3708 ITPR1 ILMN_1789505 -1 4281 MID1 ILMN_1761858 -1 4781 NFIB ILMN_1778991 -1 5433 POLR2D ILMN_1792672 1 5684 PSMA3 ILMN_1694931 -1 6317 SERPINB3 ILMN_1703855 -1 6446 SGK ILMN_1702487 -1 7159 TP53BP2 ILMN_1779706 1 7251 TSG101 ILMN_1747146 -1 8208 CHAF1B ILMN_1674231 -1 8543 LMO4 ILMN_1703487 -1 9353 SLIT2 ILMN_1676449 -1 10200 MPHOSPH6 ILMN_1746682 -1 10982 MAPRE2 ILMN_1695276 -1 25925 ZNF521 ILMN_1679747 -1 27436 EML4 ILMN_1718297 1 55970 GNG12 ILMN_1673380 1 56204 KIAA1370 ILMN_1700733 1 56890 MDM1 ILMN_1750703 -1 56942 C16orf61 ILMN_1783333 -1 58528 RRAGD ILMN_1699772 1 5825 ABCD3 ILMN_1674032 1 36ACADSB ILMN_1740920 -1 92ACVR2A ILMN_1670912 -1 8038 ADAM12 ILMN_1705689 -1 60312 AFAP ILMN_1701998 1 3899 AFF3 ILMN_1775235 1 10551 AGR2 ILMN_1814151 -1 55966 AJAP1 ILMN_1723521 -1 122481 AK7 ILMN_1684305 1 11215 AKAP11 ILMN_1693220 1 9068 ANGPTL1 ILMN_1669773 -1 26057 ANKRD17 ILMN_1712019 -1 163782 ANKRD38 ILMN_1776936 1 10053 AP1M2 ILMN_1705861 1 63941 APBA2BP ILMN_1749738 -1 147495 APCDD1 ILMN_1656951 1 51074 APIP ILMN_1793598 1 367AR ILMN_1767351 1 9828 ARHGEF17 ILMN_1754562 within each module ent Entrez_Gene_ID Symbol Probe_Id -1 991CDC20 ILMN_1663390 -1 1476 CSTB ILMN_1761797 -1 1503 CTPS ILMN_1783285 1 3708 ITPR1 ILMN_1789505 -1 4281 MID1 ILMN_1761858 -1 4781 NFIB ILMN_1778991 -1 5433 POLR2D ILMN_1792672 1 5684 PSMA3 ILMN_1694931 -1 6317 SERPINB3 ILMN_1703855 -1 6446 SGK ILMN_1702487 -1 7159 TP53BP2 ILMN_1779706 1 7251 TSG101 ILMN_1747146 -1 8208 CHAF1B ILMN_1674231 -1 8543 LMO4 ILMN_1703487 -1 9353 SLIT2 ILMN_1676449 -1 10200 MPHOSPH6 ILMN_1746682 -1 10982 MAPRE2 ILMN_1695276 -1 25925 ZNF521 ILMN_1679747 -1 27436 EML4 ILMN_1718297 1 55970 GNG12 ILMN_1673380 1 56204 KIAA1370 ILMN_1700733 1 56890 MDM1 ILMN_1750703 -1 56942 C16orf61 ILMN_1783333 -1 58528 RRAGD ILMN_1699772 1 5825 ABCD3 ILMN_1674032 1 36ACADSB ILMN_1740920 -1 92ACVR2A ILMN_1670912 -1 8038 ADAM12 ILMN_1705689 -1 60312 AFAP ILMN_1701998 1 3899 AFF3 ILMN_1775235 1 10551 AGR2 ILMN_1814151 -1 55966 AJAP1 ILMN_1723521 -1 122481 AK7 ILMN_1684305 1 11215 AKAP11 ILMN_1693220 1 9068 ANGPTL1 ILMN_1669773 -1 26057 ANKRD17 ILMN_1712019 -1 163782 ANKRD38 ILMN_1776936 1 10053 AP1M2 ILMN_1705861 1 63941 APBA2BP ILMN_1749738 -1 147495 APCDD1 ILMN_1656951 1 51074 APIP ILMN_1793598 1 367AR ILMN_1767351 1 9828 ARHGEF17 ILMN_1754562 9915 ARNT2 ILMN_1718046 22901 ARSG ILMN_1720604 419ART3 ILMN_1693218 427ASAH1 ILMN_1684054 84002 B3GNT5 ILMN_1702609 25825 BACE2 ILMN_1669323 11176 BAZ2A ILMN_1676318 8424 BBOX1 ILMN_1734929 637BID ILMN_1763386 8548 BLZF1 ILMN_1669140 25798 BRI3 ILMN_1781580 8945 BTRC ILMN_1815718 221061 FAM171A1 ILMN_1749868 84229 CCDC135 ILMN_1712067 80169 C17orf68 ILMN_1755711 29919 C18orf8 ILMN_1719097 81563 C1orf21 ILMN_1797735 114897 C1QTNF1 ILMN_1694608 55614 KIF16B ILMN_1772786 140862 C20orf82 ILMN_1772869 79669 C3orf52 ILMN_1672969 79624 C6orf211 ILMN_1798108 80129 C6orf97 ILMN_1772588 761CA3 ILMN_1740996 794CALB2 ILMN_1748840 842CASP9 ILMN_1718070 51019 CCDC53 ILMN_1715569 6362 CCL18 ILMN_1654411 1026 CDKN1A ILMN_1784602 84181 CHD6 ILMN_1781816 1124 CHN2 ILMN_1774110 9435 CHST2 ILMN_1794011 64084 CLSTN2 ILMN_1731237 1191 CLU ILMN_1724895 112616 CMTM7 ILMN_1698934 169044 COL22A1 ILMN_1784532 51692 CPSF3 ILMN_1808458 64764 CREB3L2 ILMN_1751097 1387 CREBBP ILMN_1809583 1396 CRIP1 ILMN_1742350 8531 CSDA ILMN_1782788 1439 CSF2RB ILMN_1798475 1466 CSRP2 ILMN_1660806 8722 CTSF ILMN_1750122 1521 CTSW ILMN_1794364 55917 CTTNBP2NL ILMN 1719517 -1 10659 CUGBP2 ILMN_1668652 -1 6387 CXCL12 ILMN_1791447 1 90736 FAM104B ILMN_1705848 -1 10046 CXorf6 ILMN_1680856 1 124637 CYB5D1 ILMN_1670925 1 51706 CYB5R1 ILMN_1729237 -1 1612 DAPK1 ILMN_1708340 1 51164 DCTN4 ILMN_1741564 -1 23586 DDX58 ILMN_1797001 1 26063 DECR2 ILMN_1783337 -1 163259 DENND2C ILMN_1815035 1 51635 DHRS7 ILMN_1807455 1 1741 DLG3 ILMN_1811515 1 56521 DNAJC12 ILMN_1803073 1 7802 DNALI1 ILMN_1745501 -1 26010 DNAPTP6 ILMN_1683678 -1 1788 DNMT3A ILMN_1676128 -1 1807 DPYS ILMN_1749324 -1 1824 DSC2 ILMN_1782125 -1 1827 RCAN1 ILMN_1814135 -1 1830 DSG3 ILMN_1655468 -1 56940 DUSP22 ILMN_1801331 -1 1870 E2F2 ILMN_1777233 -1 1871 E2F3 ILMN_1652143 -1 1906 EDN1 ILMN_1682775 -1 1956 EGFR ILMN_1798975 1 1964 EIF1AX ILMN_1808344 -1 9470 EIF4E2 ILMN_1738326 -1 2001 ELF5 ILMN_1813270 1 60481 ELOVL5 ILMN_1712533 -1 2019 EN1 ILMN_1709593 1 955ENTPD6 ILMN_1677955 1 2046 EPHA8 ILMN_1756989 -1 2064 ERBB2 ILMN_1694303 1 2099 ESR1 ILMN_1678535 -1 10205 MPZL2 ILMN_1752932 -1 2131 EXT1 ILMN_1794343 -1 2173 FABP7 ILMN_1804948 -1 57488 FAM62B ILMN_1807761 -1 2195 FAT ILMN_1754795 -1 2199 FBLN2 ILMN_1774602 1 2203 FBP1 ILMN_1728799 -1 114907 FBXO32 ILMN_1703955 1 2263 FGFR2 ILMN_1682270 1 163486 DENND1B ILMN_1763654 1 55638 FLJ20366 ILMN 1738989 -1 79919 FLJ22671 ILMN_1665471 -1 153830 RNF145 ILMN_1719951 1 132720 C4orf32 ILMN_1700257 -1 151195 CCNYL1 ILMN_1810069 -1 346689 FLJ44186 ILMN_1807691 1 2321 FLT1 ILMN_1705002 1 2330 FMO5 ILMN_1811632 -1 84624 FNDC1 ILMN_1734653 1 3169 FOXA1 ILMN_1766650 -1 2295 FOXF2 ILMN_1683960 -1 53826 FXYD6 ILMN_1768812 -1 2533 FYB ILMN_1796537 -1 2564 GABRE ILMN_1784706 1 2593 GAMT ILMN_1794595 1 2625 GATA3 ILMN_1727652 -1 115361 GBP4 ILMN_1771385 -1 2707 GJB3 ILMN_1652390 -1 2709 GJB5 ILMN_1735365 1 26035 GLCE ILMN_1714349 -1 2719 GPC3 ILMN_1799759 1 23171 GPD1L ILMN_1694106 1 2850 GPR27 ILMN_1744034 1 27198 GPR81 ILMN_1811272 -1 51704 GPRC5B ILMN_1786034 -1 63940 GPSM3 ILMN_1662799 -1 2886 GRB7 ILMN_1740762 1 79774 GRTP1 ILMN_1745068 1 51440 HPCAL4 ILMN_1736976 -1 3294 HSD17B2 ILMN_1808713 -1 3455 IFNAR2 ILMN_1791057 1 3480 IGF1R ILMN_1721653 -1 3600 IL15 ILMN_1724181 -1 3559 IL2RA ILMN_1683774 -1 9235 IL32 ILMN_1778010 -1 10527 IPO7 ILMN_1682647 -1 79192 IRX1 ILMN_1735353 1 10265 IRX5 ILMN_1757736 -1 3680 ITGA9 ILMN_1813117 -1 3689 ITGB2 ILMN_1654396 1 3712 IVD ILMN_1724207 1 126823 KLHDC9 ILMN_1701918 -1 3783 KCNN4 ILMN_1726320 1 200845 KCTD6 ILMN_1800942 -1 11015 KDELR3 ILMN_1722820 1 9812 KIAA0141 ILMN_1762990 -1 9914 KIAA0703 ILMN 1785413 1 57148 KIAA1219 ILMN_1733115 1 26128 KIAA1279 ILMN_1745813 1 57535 KIAA1324 ILMN_1771482 1 222223 KIAA1324L ILMN_1652371 -1 55582 KIF27 ILMN_1692572 1 11127 KIF3A ILMN_1653385 -1 3801 KIFC3 ILMN_1715900 -1 51621 KLF13 ILMN_1679929 -1 688KLF5 ILMN_1770293 -1 5655 KLK10 ILMN_1688205 -1 25818 KLK5 ILMN_1718731 -1 5653 KLK6 ILMN_1780255 -1 5650 KLK7 ILMN_1745570 -1 3872 KRT17 ILMN_1666845 -1 25984 KRT23 ILMN_1791545 -1 3852 KRT5 ILMN_1801632 -1 3854 KRT6B ILMN_1721354 -1 8942 KYNU ILMN_1746517 -1 27074 LAMP3 ILMN_1719734 -1 93273 LEMD1 ILMN_1785444 1 80774 LIMD2 ILMN_1673867 -1 3985 LIMK2 ILMN_1660624 1 89782 LMLN ILMN_1815716 -1 197135 LOC197135 ILMN_1803596 -1 57228 LOC57228 ILMN_1804415 1 90355 C5orf30 ILMN_1677292 -1 84171 LOXL4 ILMN_1754174 1 987LRBA ILMN_1652160 1 26018 LRIG1 ILMN_1707342 1 90506 LRRC46 ILMN_1768818 -1 56262 LRRC8A ILMN_1739840 -1 84230 LRRC8C ILMN_1765855 1 4049 LTA ILMN_1666732 1 10916 MAGED2 ILMN_1683576 -1 84441 MAML2 ILMN_1765729 1 9053 MAP7 ILMN_1712719 1 4137 MAPT ILMN_1710903 1 375449 LOC375449 ILMN_1738438 1 92014 MCART1 ILMN_1772492 -1 51102 MECR ILMN_1683160 -1 4240 MFGE8 ILMN_1756071 -1 6157 RPL27A ILMN_1716740 10884 MRPS30 ILMN_1726743 4604 MYBPC1 ILMN_1752075 4643 MYO1E ILMN_1773342 9NAT1 ILMN_1688071 115677 NOSTRIN ILMN_1783665 4860 NP ILMN_1777534 8828 NRP2 ILMN_1787190 9369 NRXN3 ILMN_1720935 79902 NUP85 ILMN_1669635 78990 OTUB2 ILMN_1799198 286530 P2RY8 ILMN_1768284 11240 PADI2 ILMN_1771223 56924 PAK6 ILMN_1708223 56288 PARD3 ILMN_1710524 5092 PCBD1 ILMN_1795906 54039 PCBP3 ILMN_1687216 648BMI1 ILMN_1700915 5046 PCSK6 ILMN_1802550 5156 PDGFRA ILMN_1681949 27152 INTU ILMN_1716564 5570 PKIB ILMN_1768268 5327 PLAT ILMN_1738742 5337 PLD1 ILMN_1719696 5341 PLEK ILMN_1795762 57480 PLEKHG1 ILMN_1805606 5351 PLOD1 ILMN_1684391 5357 PLS1 ILMN_1759206 25885 POLR1A ILMN_1780758 10891 PPARGC1A ILMN_1750062 5475 PPEF1 ILMN_1652017 5509 PPP1R3D ILMN_1781198 5521 PPP2R2B ILMN_1660732 639PRDM1 ILMN_1655077 5549 PRELP ILMN_1707380 57580 PREX1 ILMN_1777342 8842 PROM1 ILMN_1786720 23362 PSD3 ILMN_1717477 140885 PTPNS1 ILMN_1758146 5860 QDPR ILMN_1672443 9135 RABEP1 ILMN_1719622 5918 RARRES1 ILMN_1800091 83937 RASSF4 ILMN_1690566 1 9252 RPS6KA5 ILMN_1657515 -1 6251 RSU1 ILMN_1721657 -1 6273 S100A2 ILMN_1725852 -1 6279 S100A8 ILMN_1729801 -1 6304 SATB1 ILMN_1690646 1 51097 SCCPDH ILMN_1795839 1 57758 SCUBE2 ILMN_1684085 1 7869 SEMA3B ILMN_1653501 1 6405 SEMA3F ILMN_1761540 -1 123228 SENP8 ILMN_1767690 -1 23157 Sep-06ILMN_1661342 -1 10801 Sep-09ILMN_1769118 -1 5271 SERPINB8 ILMN_1718960 -1 5270 SERPINE2 ILMN_1655595 1 29946 SERTAD3 ILMN_1801934 1 389376 SFTPG ILMN_1703864 -1 399694 SHC4 ILMN_1807050 -1 55423 SIRPB2 ILMN_1771801 -1 56833 SLAMF8 ILMN_1667224 -1 6565 SLC15A2 ILMN_1706040 1 6512 SLC1A7 ILMN_1704629 1 6584 SLC22A5 ILMN_1699357 -1 60386 SLC25A19 ILMN_1666553 -1 7782 SLC30A4 ILMN_1653098 -1 10237 SLC35B1 ILMN_1727840 1 25800 SLC39A6 ILMN_1750394 -1 8884 SLC5A6 ILMN_1741054 1 6542 SLC7A2 ILMN_1781400 -1 6608 SMO ILMN_1782098 -1 64750 SMURF2 ILMN_1675429 -1 6627 SNRPA1 ILMN_1715179 1 28966 SNX24 ILMN_1795666 -1 6648 SOD2 ILMN_1775672 -1 6667 SP1 ILMN_1676010 -1 23648 SSBP3 ILMN_1814165 -1 8082 SSPN ILMN_1775486 -1 6489 ST8SIA1 ILMN_1664859 1 8614 STC2 ILMN_1691884 1 27148 STK36 ILMN_1693538 -1 23208 SYT11 ILMN_1789954 1 23158 TBC1D9 ILMN_1703891 -1 6934 TCF7L2 ILMN_1672486 -1 6999 TDO2 ILMN_1716859 1 7031 TFF1 ILMN_1722489 1 79875 THSD4 ILMN_1746737 -1 7077 TIMP2 ILMN_1670054 1 283578 TMED8 ILMN_1676670 1 161291 TMEM30B ILMN_1752935 1 283131 TncRNA ILMN_1657920 -1 10673 TNFSF13B ILMN_1758418 -1 23321 TRIM2 ILMN_1745079 -1 23650 TRIM29 ILMN_1657766 1 142940 TRUB1 ILMN_1744574 1 27075 TSPAN13 ILMN_1669881 1 151613 TTC14 ILMN_1696757 1 54902 TTC19 ILMN_1781260 -1 57217 TTC7A ILMN_1732535 -1 10537 UBD ILMN_1678841 1 11274 USP18 ILMN_1740200 -1 51442 VGLL1 ILMN_1703146 -1 7472 WNT2 ILMN_1694011 1 7473 WNT3 ILMN_1784840 1 7490 WT1 ILMN_1802174 -1 57510 XPO5 ILMN_1759495 1 22882 ZHX2 ILMN_1792951 1 57336 ZNF287 ILMN_1693597 -1 10320 ZNFN1A1 ILMN_1676575 Symmans et al. within each module ent Entrez_Gene_ID Symbol Probe_Id -1 991CDC20 ILMN_1663390 -1 1476 CSTB ILMN_1761797 -1 1503 CTPS ILMN_1783285 1 3708 ITPR1 ILMN_1789505 -1 4281 MID1 ILMN_1761858 -1 4781 NFIB ILMN_1778991 -1 5433 POLR2D ILMN_1792672 1 5684 PSMA3 ILMN_1694931 -1 6317 SERPINB3 ILMN_1703855 -1 6446 SGK ILMN_1702487 -1 7159 TP53BP2 ILMN_1779706 1 7251 TSG101 ILMN_1747146 -1 8208 CHAF1B ILMN_1674231 -1 8543 LMO4 ILMN_1703487 -1 9353 SLIT2 ILMN_1676449 -1 10200 MPHOSPH6 ILMN_1746682 -1 10982 MAPRE2 ILMN_1695276 -1 25925 ZNF521 ILMN_1679747 -1 27436 EML4 ILMN_1718297 1 55970 GNG12 ILMN_1673380 1 56204 KIAA1370 ILMN_1700733 1 56890 MDM1 ILMN_1750703 -1 56942 C16orf61 ILMN_1783333 -1 58528 RRAGD ILMN_1699772 1 5825 ABCD3 ILMN_1674032 1 36ACADSB ILMN_1740920 -1 92ACVR2A ILMN_1670912 -1 8038 ADAM12 ILMN_1705689 -1 60312 AFAP ILMN_1701998 1 3899 AFF3 ILMN_1775235 1 10551 AGR2 ILMN_1814151 -1 55966 AJAP1 ILMN_1723521 -1 122481 AK7 ILMN_1684305 1 11215 AKAP11 ILMN_1693220 1 9068 ANGPTL1 ILMN_1669773 -1 26057 ANKRD17 ILMN_1712019 -1 163782 ANKRD38 ILMN_1776936 1 10053 AP1M2 ILMN_1705861 1 63941 APBA2BP ILMN_1749738 -1 147495 APCDD1 ILMN_1656951 1 51074 APIP ILMN_1793598 1 367AR ILMN_1767351 1 9828 ARHGEF17 ILMN_1754562 coefficient Entrez_Gene_ID Symbol Probe_Id SET 1 27075 TSPAN13 ILMN_1669881 -1 5613 PRKX ILMN_1786834 1 51133 KCTD3 ILMN_1800220 -1 8690 JRKL ILMN_1776140 -1 2744 GLS ILMN_1798791 1 2593 GAMT ILMN_1794595 -1 2950 GSTP1 ILMN_1679809 -1 4783 NFIL3 ILMN_1707312 -1 8543 LMO4 ILMN_1703487 1 323APBB2 ILMN_1716790 1 7905 REEP5 ILMN_1758941 1 9NAT1 ILMN_1688071 1 23030 JMJD2B ILMN_1720531 1 1396 CRIP1 ILMN_1742350 1 1602 DACH1 ILMN_1755741 1 4059 BCAM ILMN_1770421 1 55686 DSU ILMN_1713679 1 9687 GREB1 ILMN_1721170 1 23116 KIAA0423 ILMN_1778876 -1 3475 IFRD1 ILMN_1667561 1 18ABAT ILMN_1805104 -1 9444 QKI ILMN 1690476 Symmans et al. within each module ent Entrez_Gene_ID Symbol Probe_Id -1 991CDC20 ILMN_1663390 -1 1476 CSTB ILMN_1761797 -1 1503 CTPS ILMN_1783285 1 3708 ITPR1 ILMN_1789505 -1 4281 MID1 ILMN_1761858 -1 4781 NFIB ILMN_1778991 -1 5433 POLR2D ILMN_1792672 1 5684 PSMA3 ILMN_1694931 -1 6317 SERPINB3 ILMN_1703855 -1 6446 SGK ILMN_1702487 -1 7159 TP53BP2 ILMN_1779706 1 7251 TSG101 ILMN_1747146 -1 8208 CHAF1B ILMN_1674231 -1 8543 LMO4 ILMN_1703487 -1 9353 SLIT2 ILMN_1676449 -1 10200 MPHOSPH6 ILMN_1746682 -1 10982 MAPRE2 ILMN_1695276 -1 25925 ZNF521 ILMN_1679747 -1 27436 EML4 ILMN_1718297 1 55970 GNG12 ILMN_1673380 1 56204 KIAA1370 ILMN_1700733 1 56890 MDM1 ILMN_1750703 -1 56942 C16orf61 ILMN_1783333 -1 58528 RRAGD ILMN_1699772 1 5825 ABCD3 ILMN_1674032 1 36ACADSB ILMN_1740920 -1 92ACVR2A ILMN_1670912 -1 8038 ADAM12 ILMN_1705689 -1 60312 AFAP ILMN_1701998 1 3899 AFF3 ILMN_1775235 1 10551 AGR2 ILMN_1814151 -1 55966 AJAP1 ILMN_1723521 -1 122481 AK7 ILMN_1684305 1 11215 AKAP11 ILMN_1693220 1 9068 ANGPTL1 ILMN_1669773 -1 26057 ANKRD17 ILMN_1712019 -1 163782 ANKRD38 ILMN_1776936 1 10053 AP1M2 ILMN_1705861 1 63941 APBA2BP ILMN_1749738 -1 147495 APCDD1 ILMN_1656951 1 51074 APIP ILMN_1793598 1 367AR ILMN_1767351 1 9828 ARHGEF17 ILMN_1754562 SET 1 5087 PBX1 ILMN_1784678 -1 3783 KCNN4 ILMN_1726320 -1 5918 RARRES1 ILMN_1800091 -1 23650 TRIM29 ILMN_1657766 1 36ACADSB ILMN_1740920 1 2625 GATA3 ILMN_1727652 -1 1612 DAPK1 ILMN_1708340 -1 1001 CDH3 ILMN_1704294 -1 3930 LBR ILMN_1724240 1 253782 LASS6 ILMN_1767662 -1 2882 GPX7 ILMN_1726030 -1 10123 ARL4C ILMN_1768391 -1 1824 DSC2 ILMN_1782125 1 6584 SLC22A5 ILMN_1699357 -1 2023 ENO1 ILMN_1710756 1 11059 WWP1 ILMN_1692092 1 388RHOB ILMN_1802205 -1 10950 BTG3 ILMN_1707339 1 3572 IL6ST ILMN_1797861 -1 50649 ARHGEF4 ILMN_1666076 -1 26227 PHGDH ILMN_1704537 1 6500 SKP1A ILMN_1711766 1 79641 ROGDI ILMN_1722738 1 7009 TEGT ILMN_1693311 -1 114908 TMEM123 ILMN_1724139 -1 23175 LPIN1 ILMN_1671554 1 115ADCY9 ILMN_1654287 1 10451 VAV3 ILMN_1657679 1 57613 KIAA1467 ILMN_1668619 -1 23321 TRIM2 ILMN_1745079 1 1153 CIRBP ILMN_1674661 1 9778 KIAA0232 ILMN_1795704 -1 55144 LRRC8D ILMN_1782878 1 55638 FLJ20366 ILMN_1738989 1 7869 SEMA3B ILMN_1653501 -1 3945 LDHB ILMN_1728132 1 7494 XBP1 ILMN_1710675 1 79875 THSD4 ILMN_1746737 1 92579 G6PC3 ILMN_1806962 -1 1871 E2F3 ILMN_1652143 1 51097 SCCPDH ILMN_1795839 1 4832 NME3 ILMN_1669456 1 3667 IRS1 ILMN_1759232 -1 6868 ADAM17 ILMN_1765779 -1 8440 NCK2 ILMN_1724718 -1 9448 MAP4K4 ILMN_1797745 1 80129 C6orf97 ILMN_1772588 1 8204 NRIP1 ILMN_1718629 1 5914 RARA ILMN_1659206 1 51466 EVL ILMN_1730622 1 26018 LRIG1 ILMN_1707342 1 4137 MAPT ILMN_1710903 1 57535 KIAA1324 ILMN_1771482 1 7357 UGCG ILMN_1736939 1 90196 SYS1 ILMN_1756590 -1 29015 SLC43A3 ILMN_1658407 1 10551 AGR2 ILMN_1814151 1 689BTF3 ILMN_1676221 -1 23603 CORO1C ILMN_1745954 -1 55689 YEATS2 ILMN_1676899 1 7162 TPBG ILMN_1756230 -1 23683 PRKD3 ILMN_1703196 -1 5610 EIF2AK2 ILMN_1706502 -1 56935 C11orf75 ILMN_1798270 -1 5912 RAP2B ILMN_1677590 1 54681 PH-4 ILMN_1672122 1 80736 SLC44A4 ILMN_1730977 1 25800 SLC39A6 ILMN_1750394 -1 83439 TCF7L1 ILMN_1733841 -1 3159 HMGA1 ILMN_1693560 1 90355 C5orf30 ILMN_1677292 1 9254 CACNA2D2 ILMN_1711049 1 5167 ENPP1 ILMN_1700888 1 987LRBA ILMN_1652160 -1 123ADFP ILMN_1660332 1 3295 HSD17B4 ILMN_1673795 1 10560 SLC19A2 ILMN_1729594 -1 51029 C1orf121 ILMN_1660840 1 2066 ERBB4 ILMN_1661857 1 51706 CYB5R1 ILMN_1729237 1 79083 MLPH ILMN_1795342 -1 55975 KLHL7 ILMN_1674458 1 1345 COX6C ILMN_1654151 -1 56888 KCMF1 ILMN_1669669 -1 4651 MYO10 ILMN_1703576 -1 246243 RNASEH1 ILMN_1726783 1 7031 TFF1 ILMN 1722489 8382 NME5 ILMN_1784783 2296 FOXC1 ILMN_1743864 23158 TBC1D9 ILMN_1703891 9135 RABEP1 ILMN_1719622 5214 PFKP ILMN_1805737 64084 CLSTN2 ILMN_1731237 7090 TLE3 ILMN_1716928 5690 PSMB2 ILMN_1764794 6715 SRD5A1 ILMN_1793241 51604 PIGT ILMN_1738759 54463 FLJ20152 ILMN_1811330 79818 ZNF552 ILMN_1700563 771CA12 ILMN_1720998 8416 ANXA9 ILMN_1755721 3480 IGF1R ILMN_1721653 8842 PROM1 ILMN_1786720 4281 MID1 ILMN_1761858 10916 MAGED2 ILMN_1683576 11240 PADI2 ILMN_1771223 10974 C10orf116 ILMN_1680110 79624 C6orf211 ILMN_1798108 2674 GFRA1 ILMN_1725678 7033 TFF3 ILMN_1811387 2203 FBP1 ILMN_1728799 27074 LAMP3 ILMN_1719734 107ADCY1 ILMN_1742073 56521 DNAJC12 ILMN_1803073 6405 SEMA3F ILMN_1761540 403ARL3 ILMN_1780444 9674 KIAA0040 ILMN_1797191 53335 BCL11A ILMN_1698224 3708 ITPR1 ILMN_1789505 64087 MCCC2 ILMN_1769264 8073 PTP4A2 ILMN_1688490 8553 BHLHB2 ILMN_1768534 221061 FAM171A1 ILMN_1749868 6732 SRPK1 ILMN_1798804 3169 FOXA1 ILMN_1766650 64943 NT5DC2 ILMN_1708743 4904 YBX1 ILMN_1669424 5104 SERPINA5 ILMN_1759910 57496 MKL2 ILMN_1690807 Desmedt et al. within each module ent Entrez_Gene_ID Symbol Probe_Id -1 991CDC20 ILMN_1663390 -1 1476 CSTB ILMN_1761797 -1 1503 CTPS ILMN_1783285 1 3708 ITPR1 ILMN_1789505 -1 4281 MID1 ILMN_1761858 -1 4781 NFIB ILMN_1778991 -1 5433 POLR2D ILMN_1792672 1 5684 PSMA3 ILMN_1694931 -1 6317 SERPINB3 ILMN_1703855 -1 6446 SGK ILMN_1702487 -1 7159 TP53BP2 ILMN_1779706 1 7251 TSG101 ILMN_1747146 -1 8208 CHAF1B ILMN_1674231 -1 8543 LMO4 ILMN_1703487 -1 9353 SLIT2 ILMN_1676449 -1 10200 MPHOSPH6 ILMN_1746682 -1 10982 MAPRE2 ILMN_1695276 -1 25925 ZNF521 ILMN_1679747 -1 27436 EML4 ILMN_1718297 1 55970 GNG12 ILMN_1673380 1 56204 KIAA1370 ILMN_1700733 1 56890 MDM1 ILMN_1750703 -1 56942 C16orf61 ILMN_1783333 -1 58528 RRAGD ILMN_1699772 1 5825 ABCD3 ILMN_1674032 1 36ACADSB ILMN_1740920 -1 92ACVR2A ILMN_1670912 -1 8038 ADAM12 ILMN_1705689 -1 60312 AFAP ILMN_1701998 1 3899 AFF3 ILMN_1775235 1 10551 AGR2 ILMN_1814151 -1 55966 AJAP1 ILMN_1723521 -1 122481 AK7 ILMN_1684305 1 11215 AKAP11 ILMN_1693220 1 9068 ANGPTL1 ILMN_1669773 -1 26057 ANKRD17 ILMN_1712019 -1 163782 ANKRD38 ILMN_1776936 1 10053 AP1M2 ILMN_1705861 1 63941 APBA2BP ILMN_1749738 -1 147495 APCDD1 ILMN_1656951 1 51074 APIP ILMN_1793598 1 367AR ILMN_1767351 1 9828 ARHGEF17 ILMN_1754562 2099 ESR1 ILMN_1678535 23158 TBC1D9 ILMN_1703891 2625 GATA3 ILMN_1727652 771CA12 ILMN_1720998 3169 FOXA1 ILMN_1766650 4602 MYB ILMN_1711894 7802 DNALI1 ILMN_1745501 18ABAT ILMN_1805104 7494 XBP1 ILMN_1710675 57758 SCUBE2 ILMN_1684085 2066 ERBB4 ILMN_1661857 9NAT1 ILMN_1688071 10551 AGR2 ILMN_1814151 987LRBA ILMN_1652160 56521 DNAJC12 ILMN_1803073 2203 FBP1 ILMN_1728799 51466 EVL ILMN_1730622 51442 VGLL1 ILMN_1703146 57496 MKL2 ILMN_1690807 7031 TFF1 ILMN_1722489 1153 CIRBP ILMN_1674661 26227 PHGDH ILMN_1704537 6648 SOD2 ILMN_1775672 55638 FLJ20366 ILMN_1738989 221061 FAM171A1 ILMN_1749868 7033 TFF3 ILMN_1811387 53335 BCL11A ILMN_1698224 79818 ZNF552 ILMN_1700563 57613 KIAA1467 ILMN_1668619 8416 ANXA9 ILMN_1755721 582BBS1 ILMN_1800590 54463 FLJ20152 ILMN_1811330 55733 HHAT ILMN_1693853 2674 GFRA1 ILMN_1725678 4478 MSN ILMN_1659895 51097 SCCPDH ILMN_1795839 54502 FLJ20273 ILMN_1689046 26018 LRIG1 ILMN_1707342 55793 FAM63A ILMN_1689552 3868 KRT16 ILMN_1736760 54961 SSH3 ILMN_1755234 60481 ELOVL5 ILMN_1712533 S ESR1.2 -1 4783 NFIL3 ILMN_1707312 1 51161 C3orf18 ILMN_1728581 -1 2296 FOXC1 ILMN_1743864 -1 6664 SOX11 ILMN_1773459 -1 5613 PRKX ILMN_1786834 -1 8543 LMO4 ILMN_1703487 1 55686 DSU ILMN_1713679 1 8100 TTC10 ILMN_1669366 -1 2617 GARS ILMN_1771026 -1 3945 LDHB ILMN_1728132 1 8382 NME5 ILMN_1784783 1 10614 HEXIM1 ILMN_1685115 1 9633 MTL5 ILMN_1661778 -1 2568 GABRP ILMN_1689146 1 23324 MAN2B2 ILMN_1768510 -1 55765 C1orf106 ILMN_1713952 1 5104 SERPINA5 ILMN_1759910 1 5174 PDZK1 ILMN_1708341 1 56674 TMEM9B ILMN_1689979 -1 1054 CEBPG ILMN_1716766 1 9120 SLC16A6 ILMN_1729691 1 79641 ROGDI ILMN_1722738 1 23303 KIF13B ILMN_1686562 -1 2173 FABP7 ILMN_1804948 1 23171 GPD1L ILMN_1694106 1 9674 KIAA0040 ILMN_1797191 1 27134 TJP3 ILMN_1659610 1 79921 TCEAL4 ILMN_1748625 1 54898 ELOVL2 ILMN_1716843 1 1345 COX6C ILMN_1654151 -1 5937 RBMS1 ILMN_1762568 1 400451 LOC400451 ILMN_1652797 -1 3898 LAD1 ILMN_1782389 1 2530 FUT8 ILMN_1741422 1 51306 C5orf5 ILMN_1673478 1 25837 RAB26 ILMN_1790317 -1 10982 MAPRE2 ILMN_1695276 1 1632 DCI ILMN_1803485 1 7905 REEP5 ILMN_1758941 1 1101 CHAD ILMN_1700652 1 323APBB2 ILMN_1716790 1 28958 CCDC56 ILMN_1789786 1 1476 CSTB ILMN 1761797 -1 8685 MARCO ILMN_1731503 1 3295 HSD17B4 ILMN_1673795 -1 11013 TMSL8 ILMN_1681737 1 51604 PIGT ILMN_1738759 -1 6663 SOX10 ILMN_1653750 -1 85377 MICALL1 ILMN_1671242 1 58495 OVOL2 ILMN_1724832 1 11001 SLC27A2 ILMN_1700831 -1 25841 ABTB2 ILMN_1753370 1 64080 RBKS ILMN_1732127 -1 375035 SFT2D2 ILMN_1668378 -1 10479 SLC9A6 ILMN_1677829 1 5002 SLC22A18 ILMN_1815581 -1 8645 KCNK5 ILMN_1766918 1 79885 HDAC11 ILMN_1684690 -1 11254 SLC6A14 ILMN_1745770 1 122616 C14orf79 ILMN_1686082 -1 79650 C16orf57 ILMN_1765880 -1 23321 TRIM2 ILMN_1745079 1 23327 NEDD4L ILMN_1733627 1 22977 AKR7A3 ILMN_1676592 -1 8581 LY6D ILMN_1814270 -1 8842 PROM1 ILMN_1786720 -1 4953 ODC1 ILMN_1748591 -1 55544 RBM38 ILMN_1727353 1 55663 ZNF446 ILMN_1743767 1 27124 PIB5PA ILMN_1777644 -1 6715 SRD5A1 ILMN_1793241 1 51809 GALNT7 ILMN_1670748 1 89927 C16orf45 ILMN_1687821 -1 1827 RCAN1 ILMN_1814135 1 51706 CYB5R1 ILMN_1729237 -1 3383 ICAM1 ILMN_1812226 -1 5806 PTX3 ILMN_1692719 1 9501 RPH3AL ILMN_1693717 -1 3613 IMPA2 ILMN_1727639 1 7568 ZNF20 ILMN_1670377 -1 6280 S100A9 ILMN_1714991 -1 22929 SEPHS1 ILMN_1673369 1 81563 C1orf21 ILMN_1797735 1 1389 CREBL2 ILMN_1660223 -1 1410 CRYAB ILMN_1729216 1 10884 MRPS30 ILMN 1726743 1 2743 GLRB ILMN_1669631 1 427ASAH1 ILMN_1684054 1 5241 PGR ILMN_1811014 1 51364 ZMYND10 ILMN_1790574 1 6926 TBX3 ILMN_1713449 -1 8531 CSDA ILMN_1782788 -1 23ABCF1 ILMN_1763875 1 819CAMLG ILMN_1714599 1 2947 GSTM3 ILMN_1736184 1 5825 ABCD3 ILMN_1674032 1 5860 QDPR ILMN_1672443 -1 59342 SCPEP1 ILMN_1767470 -1 51806 CALML5 ILMN_1749118 1 79603 LASS4 ILMN_1748057 1 21ABCA3 ILMN_1742051 1 54847 SIDT1 ILMN_1795118 1 8537 BCAS1 ILMN_1776647 -1 10874 NMU ILMN_1740250 -1 54149 C21orf91 ILMN_1698166 -1 9929 JOSD1 ILMN_1798536 -1 5317 PKP1 ILMN_1663454 -1 7388 UQCRH ILMN_1792138 -1 64764 CREB3L2 ILMN_1751097 1 10127 ZNF263 ILMN_1692620 1 80347 COASY ILMN_1753498 1 126353 C19orf21 ILMN_1683905 1 50865 HEBP1 ILMN_1802557 1 54812 AFTPH ILMN_1795996 1 64087 MCCC2 ILMN_1769264 -1 8884 SLC5A6 ILMN_1741054 1 5269 SERPINB6 ILMN_1712400 -1 4321 MMP12 ILMN_1680512 -1 8190 MIA ILMN_1745077 -1 6769 STAC ILMN_1774127 1 51368 TEX264 ILMN_1781623 1 23541 SEC14L2 ILMN_1684211 1 9185 REPS2 ILMN_1656934 1 185AGTR1 ILMN_1814327 -1 7368 UGT8 ILMN_1758816 1 399665 C9orf132 ILMN_1745112 1 12SERPINA3 ILMN_1788874 -1 55975 KLHL7 ILMN_1674458 1 25864 ABHD14A ILMN_1794213 -1 4851 NOTCH1 ILMN_1729161 1 2800 GOLGA1 ILMN_1806301 -1 8326 FZD9 ILMN_1704398 -1 6376 CX3CL1 ILMN_1654072 1 8399 PLA2G10 ILMN_1762561 1 5327 PLAT ILMN_1738742 1 22885 ABLIM3 ILMN_1656940 1 11094 C9orf7 ILMN_1814856 -1 5321 PLA2G4A ILMN_1764157 -1 57348 TTYH1 ILMN_1685569 1 123872 LRRC50 ILMN_1776967 1 10421 CD2BP2 ILMN_1807264 -1 5971 RELB ILMN_1811258 1 6833 ABCC8 ILMN_1767129 1 11122 PTPRT ILMN_1698885 -1 23650 TRIM29 ILMN_1657766 1 79629 OCEL1 ILMN_1757388 1 8722 CTSF ILMN_1750122 -1 57110 HRASLS ILMN_1655867 1 6697 SPR ILMN_1705849 -1 2919 CXCL1 ILMN_1787897 1 27250 PDCD4 ILMN_1768004 1 23245 ASTN2 ILMN_1744118 1 10265 IRX5 ILMN_1757736 -1 2824 GPM6B ILMN_1704665 -1 10644 IGF2BP2 ILMN_1702447 -1 7436 VLDLR ILMN_1675092 -1 25825 BACE2 ILMN_1669323 1 10827 C5orf3 ILMN_1675701 -1 4828 NMB ILMN_1683940 1 6720 SREBF1 ILMN_1663035 -1 10477 UBE2E3 ILMN_1669553 -1 3066 HDAC2 ILMN_1767747 1 55224 ETNK2 ILMN_1800130 -1 875CBS ILMN_1804735 -1 3872 KRT17 ILMN_1666845 1 753C18orf1 ILMN_1777436 -1 136ADORA2B ILMN_1703946 1 2013 EMP2 ILMN_1671270 1 1917 EEF1A2 ILMN_1811315 -1 3576 IL8 ILMN_1666733 -1 419ART3 ILMN_1693218 1 55650 PIGV ILMN_1762993 1 23107 MRPS27 ILMN 1711414 1 10002 NR2E3 ILMN_1699266 -1 60487 C6orf75 ILMN_1805481 -1 10656 KHDRBS3 ILMN_1691747 -1 55240 STEAP3 ILMN_1746926 1 3315 HSPB1 ILMN_1674236 1 10273 STUB1 ILMN_1756126 -1 2171 FABP5 ILMN_1696302 1 55184 C20orf12 ILMN_1664242 1 5783 PTPN13 ILMN_1652805 1 1877 E4F1 ILMN_1720287 1 11098 PRSS23 ILMN_1797776 1 10202 DHRS2 ILMN_1725726 1 80223 RAB11FIP1 ILMN_1779426 -1 79627 OGFRL1 ILMN_1715809 -1 6948 TCN2 ILMN_1740572 -1 3097 HIVEP2 ILMN_1745447 -1 8985 PLOD3 ILMN_1714350 -1 3892 KRTHB6 ILMN_1811984 -1 10575 CCT4 ILMN_1776073 1 51004 COQ6 ILMN_1783985 -1 4071 TM4SF1 ILMN_1770338 1 1718 DHCR24 ILMN_1725510 -1 1381 CRABP1 ILMN_1658040 1 9368 SLC9A3R1 ILMN_1680925 1 92104 FLJ13946 ILMN_1735822 1 9518 GDF15 ILMN_1763658 -1 6364 CCL20 ILMN_1657234 1 3306 HSPA2 ILMN_1766499 -1 79605 PGBD5 ILMN_1733831 -1 23336 DMN ILMN_1712075 -1 1356 CP ILMN_1739608 -1 54619 CCNJ ILMN_1813431 -1 9200 PTPLA ILMN_1725791 -1 51302 CYP39A1 ILMN_1680417 1 5191 PEX7 ILMN_1729650 -1 706TSPO ILMN_1754663 -1 7159 TP53BP2 ILMN_1779706 1 55218 EXDL2 ILMN_1771689 1 79669 C3orf52 ILMN_1672969 1 10140 TOB1 ILMN_1672004 1 11226 GALNT6 ILMN_1677574 1 6652 SORD ILMN_1692517 -1 3418 IDH2 ILMN_1751753 -1 10200 MPHOSPH6 ILMN_1746682 -1 7345 UCHL1 ILMN_1757387 -1 6564 SLC15A1 ILMN 1652857 1 54458 PRR13 ILMN_1795944 1 51103 NDUFAF1 ILMN_1754421 1 11042 SMA5 ILMN_1770444 1 10040 TOM1L1 ILMN_1802642 -1 1117 CHI3L2 ILMN_1705078 1 112398 EGLN2 ILMN_1768773 -1 9258 MFHAS1 ILMN_1745686 -1 2982 GUCY1A3 ILMN_1808590 -1 688KLF5 ILMN_1770293 1 1960 EGR3 ILMN_1807638 1 7993 UBXD6 ILMN_1731891 1 25823 TPSG1 ILMN_1769219 1 4485 MST1 ILMN_1707464 1 23528 ZNF281 ILMN_1802758 -1 1672 DEFB1 ILMN_1686573 -1 28960 DCPS ILMN_1740737 -1 5268 SERPINB5 ILMN_1793888 1 55450 CAMK2N1 ILMN_1794863 -1 6261 RYR1 ILMN_1682062 -1 2627 GATA6 ILMN_1695041 -1 57180 ACTR3B ILMN_1787513 1 4036 LRP2 ILMN_1723443 1 29116 MYLIP ILMN_1656111 -1 57211 GPR126 ILMN_1658815 1 4435 CITED1 ILMN_1691641 -1 54913 RPP25 ILMN_1695271 -1 9982 FGFBP1 ILMN_1785404 -1 11170 FAM107A ILMN_1743445 -1 3294 HSD17B2 ILMN_1808713 1 6583 SLC22A4 ILMN_1685057 1 79170 ATAD4 ILMN_1748970 -1 79745 CLIP4 ILMN_1759792 -1 6723 SRM ILMN_1661337 1 1360 CPB1 ILMN_1685174 1 5016 OVGP1 ILMN_1734542 -1 5271 SERPINB8 ILMN_1718960 1 347902 AMIGO2 ILMN_1725896 1 79719 FLJ11506 ILMN_1654194 1 55258 FLJ10916 ILMN_1670476 -1 8563 THOC5 ILMN_1773496 1 83464 APH1B ILMN_1767816 -1 23532 PRAME ILMN_1700031 1 6834 SURF1 ILMN 166340 -1 10525 HYOU1 ILMN_1673649 1 2232 FDXR ILMN_1799319 -1 274BIN1 ILMN_1674160 1 10307 APBB3 ILMN_1740772 -1 8986 RPS6KA4 ILMN_1756204 -1 56938 ARNTL2 ILMN_1798064 -1 9510 ADAMTS1 ILMN_1673566 -1 2770 GNAI1 ILMN_1742044 1 4350 MPG ILMN_1732821 1 863CBFA2T3 ILMN_1775637 1 2891 GRIA2 ILMN_1683504 1 10309 CCNU ILMN_1676631 -1 7037 TFRC ILMN_1674243 -1 3574 IL7 ILMN_1705769 1 55293 UEV3 ILMN_1711452 1 27165 GLS2 ILMN_1709771 1 55188 RIC8B ILMN_1663532 -1 11202 KLK8 ILMN_1735700 1 51181 DCXR ILMN_1681437 -1 827CAPN6 ILMN_1782654 -1 390RND3 ILMN_1759513 -1 54438 GFOD1 ILMN_1778240 1 10079 ATP9A ILMN_1799232 1 4285 MIPEP ILMN_1669070 -1 8324 FZD7 ILMN_1746341 1 9052 GPRC5A ILMN_1682599 -1 9508 ADAMTS3 ILMN_1703803 1 10519 CIB1 ILMN_1656899 1 7138 TNNT1 ILMN_1717297 1 51735 RAPGEF6 ILMN_1725992 -1 2591 GALNT3 ILMN_1671039 -1 2348 FOLR1 ILMN_1661733 1 2954 GSTZ1 ILMN_1684168 -1 23318 ZCCHC11 ILMN_1655137 1 10267 RAMP1 ILMN_1764754 -1 25984 KRT23 ILMN_1791545 -1 6496 SIX3 ILMN_1727537 1 786CACNG1 ILMN_1724306 1 22976 PAXIP1 ILMN_1680193 1 283232 TMEM80 ILMN_1689868 1 629CFB ILMN_1774287 1 7286 TUFT1 ILMN_1781374 -1 6271 S100A1 ILMN_1653494 1 55859 BEX1 ILMN_1702637 -1 3595 IL12RB2 ILMN_1743455 -1 2861 GPR37 ILMN_1668271 -1 55506 H2AFY2 ILMN_1705570 1 64215 DNAJC1 ILMN_1683234 -1 3096 HIVEP1 ILMN_1735548 1 23059 CLUAP1 ILMN_1800993 1 79602 ADIPOR2 ILMN_1750651 1 56683 C21orf59 ILMN_1776147 -1 22943 DKK1 ILMN_1773337 -1 6277 S100A6 ILMN_1713636 -1 65983 GRAMD3 ILMN_1734007 1 4255 MGMT ILMN_1795639 1 10406 WFDC2 ILMN_1706612 1 23552 CCRK ILMN_1806176 1 9722 NOS1AP ILMN_1710315 1 23613 ZMYND8 ILMN_1652407 -1 202AIM1 ILMN_1688625 1 51207 DUSP13 ILMN_1785592 -1 83988 NCALD ILMN_1747979 -1 2920 CXCL2 ILMN_1682636 1 8870 IER3 ILMN_1682717 1 55245 C20orf44 ILMN_1745152 1 6666 SOX12 ILMN_1736974 1 80279 CDK5RAP3 ILMN_1658579 -1 1644 DDC ILMN_1811601 1 5441 POLR2L ILMN_1670037 -1 9022 CLIC3 ILMN_1796423 1 7769 ZNF226 ILMN_1715418 1 27239 GPR162 ILMN_1730816 1 26504 CNNM4 ILMN_1655720 -1 3400 ID4 ILMN_1721758 1 1733 DIO1 ILMN_1769547 1 25915 C3orf60 ILMN_1691557 -1 1525 CXADR ILMN_1796925 -1 1475 CSTA ILMN_1669888 1 2155 F7 ILMN_1740559 1 3622 ING2 ILMN_1671265 1 25980 C20orf4 ILMN_1721225 1 8310 ACOX3 ILMN_1743396 1 54820 NDE1 ILMN_1739805 1 816 PVALB ILMN 1 23431 6271 S100A1 ILMN_1653494 55859 BEX1 ILMN_1702637 3595 IL12RB2 ILMN_1743455 2861 GPR37 ILMN_1668271 55506 H2AFY2 ILMN_1705570 64215 DNAJC1 ILMN_1683234 3096 HIVEP1 ILMN_1735548 23059 CLUAP1 ILMN_1800993 79602 ADIPOR2 ILMN_1750651 56683 C21orf59 ILMN_1776147 22943 DKK1 ILMN_1773337 6277 S100A6 ILMN_1713636 65983 GRAMD3 ILMN_1734007 4255 MGMT ILMN_1795639 10406 WFDC2 ILMN_1706612 23552 CCRK ILMN_1806176 9722 NOS1AP ILMN_1710315 23613 ZMYND8 ILMN_1652407 202AIM1 ILMN_1688625 51207 DUSP13 ILMN_1785592 83988 NCALD ILMN_1747979 2920 CXCL2 ILMN_1682636 8870 IER3 ILMN_1682717 55245 C20orf44 ILMN_1745152 6666 SOX12 ILMN_1736974 80279 CDK5RAP3 ILMN_1658579 1644 DDC ILMN_1811601 5441 POLR2L ILMN_1670037 9022 CLIC3 ILMN_1796423 7769 ZNF226 ILMN_1715418 27239 GPR162 ILMN_1730816 26504 CNNM4 ILMN_1655720 3400 ID4 ILMN_1721758 1733 DIO1 ILMN_1769547 25915 C3orf60 ILMN_1691557 1525 CXADR ILMN_1796925 1475 CSTA ILMN_1669888 2155 F7 ILMN_1740559 3622 ING2 ILMN_1671265 25980 C20orf4 ILMN_1721225 8310 ACOX3 ILMN_1743396 54820 NDE1 ILMN_1739805 5816 PVALB ILMN_1723431 60686 C14orf93 ILMN_1724897 8792 TNFRSF11A ILMN_1698952 54894 RNF43 ILMN_1700606 1 5737 PTGFR ILMN_1813341 1 1501 CTNND2 ILMN_1716875 1 7764 ZNF217 ILMN_1789841 1 8405 SPOP ILMN_1795404 1 1847 DUSP5 ILMN_1656501 1 4488 MSX2 ILMN_1766951 1 7163 TPD52 ILMN_1693367 1 25790 CCDC19 ILMN_1807976 1 5803 PTPRZ1 ILMN_1676616 1 23635 SSBP2 ILMN_1711608 1 6548 SLC9A1 ILMN_1800425 1 8187 ZNF239 ILMN_1748427 1 2588 GALNS ILMN_1737949 1 54903 MKS1 ILMN_1738949 1 55163 PNPO ILMN_1684289 1 55101 ATP5SL ILMN_1809027 1 4682 NUBP1 ILMN_1689342 1 3779 KCNMB1 ILMN_1652065 1 64849 SLC13A3 ILMN_1658519 1 4691 NCL ILMN_1695422 1 64428 NARFL ILMN_1813833 1 23266 LPHN2 ILMN_1697548 1 29104 C21orf127 ILMN_1701077 1 1783 DYNC1LI2 ILMN_1783448 1 8987 GENX-3414 ILMN_1673950 1 79852 ABHD9 ILMN_1699750 1 57586 SYT13 ILMN_1658499 1 8785 MATN4 ILMN_1662126 1 10331 B3GNT3 ILMN_1800082 1 5357 PLS1 ILMN_1759206 1 54880 BCOR ILMN_1773117 1 4139 MARK1 ILMN_1779800 1 81539 SLC38A1 ILMN_1769911 1 10810 WASF3 ILMN_1810797 1 926CD8B1 ILMN_1748601 1 50805 IRX4 ILMN_1684108 1 58513 EPS15L1 ILMN_1708369 1 6304 SATB1 ILMN_1690646 1 79446 WDR25 ILMN_1704351 1 23366 KIAA0895 ILMN_1690484 1 55699 IARS2 ILMN_1671207 t Entrez_Gene_ID Symbol Probe_Id Dunbier et al. within each module ent Entrez_Gene_ID Symbol Probe_Id -1 991CDC20 ILMN_1663390 -1 1476 CSTB ILMN_1761797 -1 1503 CTPS ILMN_1783285 1 3708 ITPR1 ILMN_1789505 -1 4281 MID1 ILMN_1761858 -1 4781 NFIB ILMN_1778991 -1 5433 POLR2D ILMN_1792672 1 5684 PSMA3 ILMN_1694931 -1 6317 SERPINB3 ILMN_1703855 -1 6446 SGK ILMN_1702487 -1 7159 TP53BP2 ILMN_1779706 1 7251 TSG101 ILMN_1747146 -1 8208 CHAF1B ILMN_1674231 -1 8543 LMO4 ILMN_1703487 -1 9353 SLIT2 ILMN_1676449 -1 10200 MPHOSPH6 ILMN_1746682 -1 10982 MAPRE2 ILMN_1695276 -1 25925 ZNF521 ILMN_1679747 -1 27436 EML4 ILMN_1718297 1 55970 GNG12 ILMN_1673380 1 56204 KIAA1370 ILMN_1700733 1 56890 MDM1 ILMN_1750703 -1 56942 C16orf61 ILMN_1783333 -1 58528 RRAGD ILMN_1699772 1 5825 ABCD3 ILMN_1674032 1 36ACADSB ILMN_1740920 -1 92ACVR2A ILMN_1670912 -1 8038 ADAM12 ILMN_1705689 -1 60312 AFAP ILMN_1701998 1 3899 AFF3 ILMN_1775235 1 10551 AGR2 ILMN_1814151 -1 55966 AJAP1 ILMN_1723521 -1 122481 AK7 ILMN_1684305 1 11215 AKAP11 ILMN_1693220 1 9068 ANGPTL1 ILMN_1669773 -1 26057 ANKRD17 ILMN_1712019 -1 163782 ANKRD38 ILMN_1776936 1 10053 AP1M2 ILMN_1705861 1 63941 APBA2BP ILMN_1749738 -1 147495 APCDD1 ILMN_1656951 1 51074 APIP ILMN_1793598 1 367AR ILMN_1767351 1 9828 ARHGEF17 ILMN_1754562 ERG 5 _ 083 1 5209 PFKFB3 ILMN_1660847 1 5366 PMAIP1 ILMN_1689525 1 5570 PKIB ILMN_1768268 1 6252 RTN1 ILMN_1687326 1 6781 STC1 ILMN_1758164 1 7031 TFF1 ILMN_1722489 1 7033 TFF3 ILMN_1811387 1 7278 TUBA3C ILMN_1679482 1 7869 SEMA3B ILMN_1653501 1 8309 ACOX2 ILMN_1685703 1 9536 PTGES ILMN_1713829 1 10742 RAI2 ILMN_1760574 1 11001 SLC27A2 ILMN_1700831 1 23541 SEC14L2 ILMN_1684211 1 23678 SGK3 ILMN_1747020 1 23682 RAB38 ILMN_1687293 1 26353 HSPB8 ILMN_1791280 1 27290 SPINK4 ILMN_1681263 1 27314 RAB30 ILMN_1790994 1 55540 IL17RB ILMN_1767523 1 56155 TEX14 ILMN_1685490 1 57580 PREX1 ILMN_1777342 1 64699 TMPRSS3 ILMN_1741768 1 80097 FAM128B ILMN_1717184 1 112714 TUBA3E ILMN_1652464 1 116844 LRG1 ILMN_1805228 1 130013 ACMSD ILMN_1727091 1 203328 SUSD3 ILMN_1785570 1 221662 RBM24 ILMN_1673941 coefficient Entrez_Gene_ID Symbol Probe_Id -1 8714 ABCC3 ILMN_1677814 -1 55902 ACSS2 ILMN_1714197 -1 101ADAM8 ILMN_1708348 1 219ALDH1B1 ILMN_1675398 1 10195 ALG3 ILMN_1711886 1 54865 GPATCH4 ILMN_1694837 1 55615 PRR5 ILMN_1692664 -1 406ARNTL ILMN_1731540 -1 56938 ARNTL2 ILMN_1798064 1 471ATIC ILMN_1673991 -1 493ATP2B4 ILMN_1680579 1 617BCS1L ILMN_1738529 1 51154 C1orf33 ILMN_1689800 1 54976 C20orf27 ILMN_1697363 -1 901CCNG2 ILMN_1747244 Bild et al. within each module ent Entrez_Gene_ID Symbol Probe_Id -1 991CDC20 ILMN_1663390 -1 1476 CSTB ILMN_1761797 -1 1503 CTPS ILMN_1783285 1 3708 ITPR1 ILMN_1789505 -1 4281 MID1 ILMN_1761858 -1 4781 NFIB ILMN_1778991 -1 5433 POLR2D ILMN_1792672 1 5684 PSMA3 ILMN_1694931 -1 6317 SERPINB3 ILMN_1703855 -1 6446 SGK ILMN_1702487 -1 7159 TP53BP2 ILMN_1779706 1 7251 TSG101 ILMN_1747146 -1 8208 CHAF1B ILMN_1674231 -1 8543 LMO4 ILMN_1703487 -1 9353 SLIT2 ILMN_1676449 -1 10200 MPHOSPH6 ILMN_1746682 -1 10982 MAPRE2 ILMN_1695276 -1 25925 ZNF521 ILMN_1679747 -1 27436 EML4 ILMN_1718297 1 55970 GNG12 ILMN_1673380 1 56204 KIAA1370 ILMN_1700733 1 56890 MDM1 ILMN_1750703 -1 56942 C16orf61 ILMN_1783333 -1 58528 RRAGD ILMN_1699772 1 5825 ABCD3 ILMN_1674032 1 36ACADSB ILMN_1740920 -1 92ACVR2A ILMN_1670912 -1 8038 ADAM12 ILMN_1705689 -1 60312 AFAP ILMN_1701998 1 3899 AFF3 ILMN_1775235 1 10551 AGR2 ILMN_1814151 -1 55966 AJAP1 ILMN_1723521 -1 122481 AK7 ILMN_1684305 1 11215 AKAP11 ILMN_1693220 1 9068 ANGPTL1 ILMN_1669773 -1 26057 ANKRD17 ILMN_1712019 -1 163782 ANKRD38 ILMN_1776936 1 10053 AP1M2 ILMN_1705861 1 63941 APBA2BP ILMN_1749738 -1 147495 APCDD1 ILMN_1656951 1 51074 APIP ILMN_1793598 1 367AR ILMN_1767351 1 9828 ARHGEF17 ILMN_1754562 MYC -1 55143 CDCA8 ILMN_1709294 -1 1017 CDK2 ILMN_1665559 1 1019 CDK4 ILMN_1689001 -1 8837 CFLAR ILMN_1789830 1 51651 PTRH2 ILMN_1811594 -1 57530 CGN ILMN_1746801 1 84916 CIRH1A ILMN_1796235 -1 64857 PLEKHG2 ILMN_1657836 -1 1277 COL1A1 ILMN_1701308 -1 1289 COL5A1 ILMN_1706505 -1 1296 COL8A2 ILMN_1674050 -1 10903 MTMR11 ILMN_1769299 -1 23564 DDAH2 ILMN_1770787 1 1662 DDX10 ILMN_1753249 1 8886 DDX18 ILMN_1756220 -1 1606 DGKA ILMN_1806863 1 1723 DHODH ILMN_1782993 -1 115817 DHRS1 ILMN_1807206 1 1736 DKC1 ILMN_1671257 -1 91404 SESTD1 ILMN_1782341 1 1844 DUSP2 ILMN_1712959 -1 2012 EMP1 ILMN_1801616 -1 54869 EPS8L1 ILMN_1685000 -1 2065 ERBB3 ILMN_1751346 1 2193 FARSLA ILMN_1778255 -1 2256 FGF11 ILMN_1719938 -1 89848 FCHSD1 ILMN_1783847 1 54663 WDR74 ILMN_1809866 1 54475 NLE1 ILMN_1724304 1 55341 LSG1 ILMN_1705746 1 55621 TRMT1 ILMN_1812940 1 55794 DDX28 ILMN_1741736 1 144577 FLJ32549 ILMN_1736995 1 124093 CCDC78 ILMN_1811397 -1 259173 ALS2CL ILMN_1702322 1 10056 FARSLB ILMN_1697304 -1 2801 GOLGA2 ILMN_1738821 -1 266977 GPR110 ILMN_1744299 -1 9982 FGFBP1 ILMN_1785404 1 7965 JTV1 ILMN_1673252 1 90161 HS6ST2 ILMN_1739429 1 3310 HSPA6 ILMN_1806165 1 51491 HSPC111 ILMN 1704055 -1 10379 ISGF3G ILMN_1745471 1 23171 GPD1L ILMN_1694106 1 23170 TTLL12 ILMN_1663113 -1 23351 KIAA0323 ILMN_1654392 1 23357 ANGEL1 ILMN_1665871 -1 57674 RNF213 ILMN_1749722 -1 57707 KIAA1609 ILMN_1668465 -1 3918 LAMC2 ILMN_1701424 1 124402 FAM100A ILMN_1733863 1 148523 C1orf51 ILMN_1793543 1 201164 LOC201164 ILMN_1731518 1 55326 AGPAT5 ILMN_1768029 -1 4053 LTBP2 ILMN_1703486 1 27346 TMEM97 ILMN_1710962 -1 9064 MAP3K6 ILMN_1694539 1 255231 MCOLN2 ILMN_1660462 1 4234 METTL1 ILMN_1815190 1 84798 C19orf48 ILMN_1759184 1 84315 MON1A ILMN_1696151 1 84331 C16orf14 ILMN_1730523 1 84291 MGC2408 ILMN_1762883 1 79081 C11orf48 ILMN_1739345 1 79080 CCDC86 ILMN_1689467 1 27042 C1orf107 ILMN_1746677 1 78991 PCYOX1L ILMN_1815951 -1 79026 AHNAK ILMN_1787375 -1 8569 MKNK1 ILMN_1750429 -1 4486 MST1R ILMN_1688154 1 10514 MYBBP1A ILMN_1806757 1 4609 MYC ILMN_1753923 -1 22861 NALP1 ILMN_1680774 1 4691 NCL ILMN_1695422 -1 4791 NFKB2 ILMN_1799062 -1 4804 NGFR ILMN_1752658 -1 64780 MICAL1 ILMN_1807972 -1 4817 NIT1 ILMN_1698330 1 4839 NOL1 ILMN_1723158 1 10528 NOL5A ILMN_1787628 1 65083 NOL6 ILMN_1754061 1 9221 NOLC1 ILMN_1800224 -1 8829 NRP1 ILMN_1699574 1 26354 GNL3 ILMN_1806106 1 4909 NTF5 ILMN 1737208 1 10606 PAICS ILMN_1773760 -1 5091 PC ILMN_1740240 1 26577 PCOLCE2 ILMN_1746888 1 56652 C10orf2 ILMN_1701243 1 23481 PES1 ILMN_1656372 1 5213 PFKM ILMN_1708180 1 5245 PHB ILMN_1692651 -1 5252 PHF1 ILMN_1746968 -1 5328 PLAU ILMN_1656057 -1 5339 PLEC1 ILMN_1744268 1 5373 PMM2 ILMN_1785336 1 87178 PNPT1 ILMN_1810608 1 80308 FLAD1 ILMN_1663667 -1 54704 PPM2C ILMN_1720631 -1 9266 PSCD2 ILMN_1721241 1 10244 RABEPK ILMN_1741957 -1 9052 GPRC5A ILMN_1682599 1 10171 RCL1 ILMN_1813766 -1 64285 RHBDF1 ILMN_1808404 1 10799 RPP40 ILMN_1810423 1 9136 RRP9 ILMN_1795758 1 56915 EXOSC5 ILMN_1659725 1 10813 UTP14A ILMN_1688725 -1 1992 SERPINB1 ILMN_1679133 1 119559 SFXN4 ILMN_1658437 1 84561 SLC12A8 ILMN_1797600 1 6573 SLC19A1 ILMN_1698996 1 3177 SLC29A2 ILMN_1730809 1 6535 SLC6A8 ILMN_1806349 -1 84189 SLITRK6 ILMN_1813197 -1 6525 SMTN ILMN_1785618 -1 57154 SMURF1 ILMN_1737672 1 6652 SORD ILMN_1692517 -1 6653 SORL1 ILMN_1759818 -1 6709 SPTAN1 ILMN_1663526 1 6723 SRM ILMN_1661337 -1 54961 SSH3 ILMN_1755234 -1 50861 STMN3 ILMN_1693425 1 152579 SCFD2 ILMN_1708891 1 6832 SUPV3L1 ILMN_1765857 1 6949 TCOF1 ILMN_1727551 1 29914 UBIAD1 ILMN_1651872 1 64216 TFB2M ILMN 1 9998 -1 7846 TUBA1A ILMN_1742981 -1 6843 VAMP1 ILMN_1737611 1 55759 WDR12 ILMN_1770692 1 26094 WDR21A ILMN_1715563 1 57510 XPO5 ILMN_1759495 -1 79413 ZBED2 ILMN_1688392 1 23246 BOP1 ILMN_1715583 1 637BID ILMN_1763386 -1 51619 UBE2D4 ILMN_1707084 -1 80761 UPK3B ILMN_1803024 -1 7052 TGM2 ILMN_1705750 -1 400077 FLJ42957 ILMN_1732143 Desmedt et al. within each module ent Entrez_Gene_ID Symbol Probe_Id -1 991CDC20 ILMN_1663390 -1 1476 CSTB ILMN_1761797 -1 1503 CTPS ILMN_1783285 1 3708 ITPR1 ILMN_1789505 -1 4281 MID1 ILMN_1761858 -1 4781 NFIB ILMN_1778991 -1 5433 POLR2D ILMN_1792672 1 5684 PSMA3 ILMN_1694931 -1 6317 SERPINB3 ILMN_1703855 -1 6446 SGK ILMN_1702487 -1 7159 TP53BP2 ILMN_1779706 1 7251 TSG101 ILMN_1747146 -1 8208 CHAF1B ILMN_1674231 -1 8543 LMO4 ILMN_1703487 -1 9353 SLIT2 ILMN_1676449 -1 10200 MPHOSPH6 ILMN_1746682 -1 10982 MAPRE2 ILMN_1695276 -1 25925 ZNF521 ILMN_1679747 -1 27436 EML4 ILMN_1718297 1 55970 GNG12 ILMN_1673380 1 56204 KIAA1370 ILMN_1700733 1 56890 MDM1 ILMN_1750703 -1 56942 C16orf61 ILMN_1783333 -1 58528 RRAGD ILMN_1699772 1 5825 ABCD3 ILMN_1674032 1 36ACADSB ILMN_1740920 -1 92ACVR2A ILMN_1670912 -1 8038 ADAM12 ILMN_1705689 -1 60312 AFAP ILMN_1701998 1 3899 AFF3 ILMN_1775235 1 10551 AGR2 ILMN_1814151 -1 55966 AJAP1 ILMN_1723521 -1 122481 AK7 ILMN_1684305 1 11215 AKAP11 ILMN_1693220 1 9068 ANGPTL1 ILMN_1669773 -1 26057 ANKRD17 ILMN_1712019 -1 163782 ANKRD38 ILMN_1776936 1 10053 AP1M2 ILMN_1705861 1 63941 APBA2BP ILMN_1749738 -1 147495 APCDD1 ILMN_1656951 1 51074 APIP ILMN_1793598 1 367AR ILMN_1767351 1 9828 ARHGEF17 ILMN_1754562 coefficient Entrez_Gene_ID Symbol Probe_Id VEGF 1 7422 VEGFA ILMN_1803882 -1 911CD1C ILMN_1654210 -1 4005 LMO2 ILMN_1800078 -1 4222 MEOX1 ILMN_1657145 1 29927 SEC61A1 ILMN_1696952 -1 6166 RPL36AL ILMN_1691599 -1 9450 LY86 ILMN_1807825 -1 22900 CARD8 ILMN_1654376 -1 1776 DNASE1L3 ILMN_1762084 1 1119 CHKA ILMN_1658504 1 22809 ATF5 ILMN_1669113 -1 23417 MLYCD ILMN_1651336 -1 23592 LEMD3 ILMN_1727361 1 51621 KLF13 ILMN_1679929 Bild et al. coefficient Entrez_Gene_ID Symbol Probe_Id E2F3 1 23456 ABCB10 ILMN_1665730 -1 89845 ABCC10 ILMN_1776119 1 9429 ABCG2 ILMN_1789641 1 253152 ABHD7 ILMN_1719498 1 109ADCY3 ILMN_1676893 1 51517 NCKIPSD ILMN_1736623 1 231AKR1B1 ILMN_1701731 1 207AKT1 ILMN_1784157 1 7915 ALDH5A1 ILMN_1715859 1 56172 ANKH ILMN_1660778 1 134549 SHROOM1 ILMN_1738322 1 576BAI2 ILMN_1773109 1 10018 BCL2L11 ILMN_1766541 1 54880 BCOR ILMN_1773117 -1 55612 C20orf42 ILMN_1696585 1 221545 C6orf136 ILMN_1813236 Desmedt et al. VEGF Bild et al. within each module ent Entrez_Gene_ID Symbol Probe_Id -1 991CDC20 ILMN_1663390 -1 1476 CSTB ILMN_1761797 -1 1503 CTPS ILMN_1783285 1 3708 ITPR1 ILMN_1789505 -1 4281 MID1 ILMN_1761858 -1 4781 NFIB ILMN_1778991 -1 5433 POLR2D ILMN_1792672 1 5684 PSMA3 ILMN_1694931 -1 6317 SERPINB3 ILMN_1703855 -1 6446 SGK ILMN_1702487 -1 7159 TP53BP2 ILMN_1779706 1 7251 TSG101 ILMN_1747146 -1 8208 CHAF1B ILMN_1674231 -1 8543 LMO4 ILMN_1703487 -1 9353 SLIT2 ILMN_1676449 -1 10200 MPHOSPH6 ILMN_1746682 -1 10982 MAPRE2 ILMN_1695276 -1 25925 ZNF521 ILMN_1679747 -1 27436 EML4 ILMN_1718297 1 55970 GNG12 ILMN_1673380 1 56204 KIAA1370 ILMN_1700733 1 56890 MDM1 ILMN_1750703 -1 56942 C16orf61 ILMN_1783333 -1 58528 RRAGD ILMN_1699772 1 5825 ABCD3 ILMN_1674032 1 36ACADSB ILMN_1740920 -1 92ACVR2A ILMN_1670912 -1 8038 ADAM12 ILMN_1705689 -1 60312 AFAP ILMN_1701998 1 3899 AFF3 ILMN_1775235 1 10551 AGR2 ILMN_1814151 -1 55966 AJAP1 ILMN_1723521 -1 122481 AK7 ILMN_1684305 1 11215 AKAP11 ILMN_1693220 1 9068 ANGPTL1 ILMN_1669773 -1 26057 ANKRD17 ILMN_1712019 -1 163782 ANKRD38 ILMN_1776936 1 10053 AP1M2 ILMN_1705861 1 63941 APBA2BP ILMN_1749738 -1 147495 APCDD1 ILMN_1656951 1 51074 APIP ILMN_1793598 1 367AR ILMN_1767351 1 9828 ARHGEF17 ILMN_1754562 E2F3 55450 CAMK2N1 ILMN_1794863 10645 CAMKK2 ILMN_1743021 896CCND3 ILMN_1668721 898CCNE1 ILMN_1795852 9134 CCNE2 ILMN_1740452 9308 CD83 ILMN_1780582 23097 CDC2L6 ILMN_1676891 1028 CDKN1C ILMN_1718565 1029 CDKN2A ILMN_1717714 1031 CDKN2C ILMN_1656415 1050 CEBPA ILMN_1715715 8534 CHST1 ILMN_1677719 9435 CHST2 ILMN_1794011 56548 CHST7 ILMN_1732831 84678 FBXL10 ILMN_1794260 25792 CIZ1 ILMN_1724659 9738 CP110 ILMN_1773200 1407 CRY1 ILMN_1760593 10675 CSPG5 ILMN_1688184 79850 FAM57A ILMN_1673899 10208 USPL1 ILMN_1662686 27065 D4S234E ILMN_1772627 1633 DCK ILMN_1651433 64858 DCLRE1B ILMN_1655382 23576 DDAH1 ILMN_1668507 26151 NAT9 ILMN_1776088 23336 DMN ILMN_1712075 23234 DNAJC9 ILMN_1799516 29980 DONSON ILMN_1758629 53820 DSCR6 ILMN_1709257 1857 DVL3 ILMN_1686411 144455 E2F7 ILMN_1798210 1917 EEF1A2 ILMN_1811315 2026 ENO2 ILMN_1765796 399474 TTMB ILMN_1682231 54566 EPB41L4B ILMN_1784967 1969 EPHA2 ILMN_1700527 2146 EZH2 ILMN_1708105 2175 FANCA ILMN_1799224 23014 FBXO21 ILMN_1745887 2237 FEN1 ILMN_1755834 2254 FGF9 ILMN_1762531 84915 C12orf34 ILMN_1772446 54808 DYM ILMN_1680130 54463 FLJ20152 ILMN_1811330 54867 FLJ20254 ILMN_1716907 92370 ACPL2 ILMN_1706598 132332 FLJ30834 ILMN_1809947 149175 MANEAL ILMN_1662147 158158 RASEF ILMN_1779517 94234 FOXQ1 ILMN_1669046 10023 FRAT1 ILMN_1781416 8321 FZD1 ILMN_1784250 2643 GCH1 ILMN_1812759 80318 GKAP1 ILMN_1736972 2743 GLRB ILMN_1669631 2774 GNAL ILMN_1746027 84634 KISS1R ILMN_1673521 57531 HACE1 ILMN_1740217 51696 HECA ILMN_1770667 23462 HEY1 ILMN_1788203 51523 CXXC5 ILMN_1745256 29901 SAC3D1 ILMN_1776674 55540 IL17RB ILMN_1767523 3553 IL1B ILMN_1775501 3625 INHBB ILMN_1685714 3643 INSR ILMN_1670918 56704 JPH1 ILMN_1693985 200845 KCTD6 ILMN_1800942 10656 KHDRBS3 ILMN_1691747 23199 KIAA0182 ILMN_1807767 26033 ATRNL1 ILMN_1786966 23030 JMJD2B ILMN_1720531 23158 TBC1D9 ILMN_1703891 55636 CHD7 ILMN_1778930 25959 KANK2 ILMN_1733226 55225 RAVER2 ILMN_1804737 116236 LOC116236 ILMN_1769390 116238 TLCD1 ILMN_1784655 125476 C18orf37 ILMN_1730294 129607 LOC129607 ILMN_1783621 151194 FAM119A ILMN_1762888 221061 FAM171A1 ILMN_1749868 90355 C5orf30 ILMN_1677292 4091 SMAD6 ILMN_1767068 79174 CRELD2 ILMN_1748707 254887 ZDHHC23 ILMN_1736901 115752 DIS3L ILMN_1795822 1609 DGKQ ILMN_1793017 130574 LYPD6 ILMN_1776724 79101 JOSD3 ILMN_1682038 79022 TMEM106C ILMN_1692511 4324 MMP15 ILMN_1718646 22808 MRAS ILMN_1748881 4602 MYB ILMN_1711894 4678 NASP ILMN_1725840 4747 NEFL ILMN_1659086 81831 NETO2 ILMN_1760849 51199 NIN ILMN_1724753 9631 NUP155 ILMN_1768293 1122 CHML ILMN_1747697 5111 PCNA ILMN_1694177 26577 PCOLCE2 ILMN_1746888 5151 PDE8A ILMN_1699767 23089 PEG10 ILMN_1763359 5209 PFKFB3 ILMN_1660847 55274 PHF10 ILMN_1752823 5253 PHF2 ILMN_1720476 57157 PHTF2 ILMN_1698220 5300 PIN1 ILMN_1776375 29941 PKN3 ILMN_1797184 23228 PLCL2 ILMN_1737025 23129 PLXND1 ILMN_1675222 5426 POLE ILMN_1740245 5427 POLE2 ILMN_1774336 8613 PPAP2B ILMN_1688868 29028 ATAD2 ILMN_1763064 11168 PSIP1 ILMN_1788701 5742 PTGS1 ILMN_1708275 11137 PWP1 ILMN_1743049 5827 PXMP2 ILMN_1799015 25837 RAB26 ILMN_1790317 51514 DTL ILMN_1745251 9401 RECQL4 ILMN_1703092 25907 RIS1 ILMN_1792455 58517 RBM25 ILMN_1768117 6337 SCNN1A ILMN_1713995 9147 SDCCAG1 ILMN_1772489 10194 SDCCAG33 ILMN_1718907 51460 SFMBT1 ILMN_1741585 6431 SFRS6 ILMN_1697469 6450 SH3BGR ILMN_1761875 23309 SIN3B ILMN_1788315 147912 SIX5 ILMN_1793672 8604 SLC25A12 ILMN_1788053 11000 SLC27A3 ILMN_1719627 9368 SLC9A3R1 ILMN_1680925 6666 SOX12 ILMN_1736974 123606 NIPA1 ILMN_1689274 6720 SREBF1 ILMN_1663035 117178 SSX2IP ILMN_1720844 201254 STRA13 ILMN_1769634 6812 STXBP1 ILMN_1728747 6874 TAF4 ILMN_1737535 7004 TEAD4 ILMN_1705301 7027 TFDP1 ILMN_1661717 7074 TIAM1 ILMN_1655577 64097 EPB41L4A ILMN_1775376 53346 TM6SF1 ILMN_1750961 7107 GPR137B ILMN_1794715 79056 PRRG4 ILMN_1661809 7128 TNFAIP3 ILMN_1702691 10612 TRIM3 ILMN_1810441 81786 TRIM7 ILMN_1756657 7368 UGT8 ILMN_1758816 10309 CCNU ILMN_1676631 7474 WNT5A ILMN_1800317 9933 KIAA0020 ILMN_1665483 7748 ZNF195 ILMN_1688515 11130 ZWINT ILMN_1778034 376267 RAB15 ILMN_1731699 388228 SBK1 ILMN_1728298 112724 RDH13 ILMN_1660723 ez_Gene_ID Symbol Probe_Id 10152 ABI2 ILMN_1724497 26225 ARL5 ILMN_1688526 55870 ASH1L ILMN_1782032 546ATRX ILMN_1666885 BetaCatenin -1 23196 FAM120A ILMN_1721089 -1 1305 COL13A1 ILMN_1761380 -1 1296 COL8A2 ILMN_1674050 -1 1462 VCAN ILMN_1687301 -1 9126 SMC3 ILMN_1718807 1 1591 CYP24A1 ILMN_1685663 -1 170506 DHX36 ILMN_1738272 -1 1778 DYNC1H1 ILMN_1808448 -1 1847 DUSP5 ILMN_1656501 -1 1997 ELF1 ILMN_1664010 -1 2058 EPRS ILMN_1783695 -1 55205 ZNF532 ILMN_1731358 -1 80205 CHD9 ILMN_1762972 1 94234 FOXQ1 ILMN_1669046 -1 10772 FUSIP1 ILMN_1658460 -1 55970 GNG12 ILMN_1673380 -1 23185 LARP5 ILMN_1766222 -1 23351 KIAA0323 ILMN_1654392 -1 23023 TMCC1 ILMN_1750539 -1 23030 JMJD2B ILMN_1720531 -1 23254 KIAA1026 ILMN_1798458 -1 23325 KIAA1033 ILMN_1782222 -1 259282 FAM44A ILMN_1775002 -1 26524 LATS2 ILMN_1703412 -1 987LRBA ILMN_1652160 -1 7798 LUZP1 ILMN_1664366 -1 79109 MAPKAP1 ILMN_1723142 -1 79101 JOSD3 ILMN_1682038 -1 8202 NCOA3 ILMN_1708805 -1 4750 NEK1 ILMN_1699459 1 25805 BAMBI ILMN_1691410 -1 2908 NR3C1 ILMN_1762085 -1 5325 PLAGL1 ILMN_1815121 -1 59338 PLEKHA1 ILMN_1662839 -1 5744 PTHLH ILMN_1785699 -1 9444 QKI ILMN_1690476 1 157869 RPESP ILMN_1808245 -1 79048 SECISBP2 ILMN_1736481 -1 6322 SCML1 ILMN_1776653 -1 9147 SDCCAG1 ILMN_1772489 -1 140890 SFRS12 ILMN_1720088 -1 6431 SFRS6 ILMN_1697469 -1 23049 SMG1 ILMN_1801383 -1 6653 SORL1 ILMN_1759818 -1 57187 THOC2 ILMN_1696883 -1 7128 TNFAIP3 ILMN 1702691 -1 7150 TOP1 ILMN_1735572 1 9830 TRIM14 ILMN_1713542 -1 619435 C8orf61 ILMN_1684576 1 860RUNX2 ILMN_1716651 Majumder et al. within each module ent Entrez_Gene_ID Symbol Probe_Id -1 991CDC20 ILMN_1663390 -1 1476 CSTB ILMN_1761797 -1 1503 CTPS ILMN_1783285 1 3708 ITPR1 ILMN_1789505 -1 4281 MID1 ILMN_1761858 -1 4781 NFIB ILMN_1778991 -1 5433 POLR2D ILMN_1792672 1 5684 PSMA3 ILMN_1694931 -1 6317 SERPINB3 ILMN_1703855 -1 6446 SGK ILMN_1702487 -1 7159 TP53BP2 ILMN_1779706 1 7251 TSG101 ILMN_1747146 -1 8208 CHAF1B ILMN_1674231 -1 8543 LMO4 ILMN_1703487 -1 9353 SLIT2 ILMN_1676449 -1 10200 MPHOSPH6 ILMN_1746682 -1 10982 MAPRE2 ILMN_1695276 -1 25925 ZNF521 ILMN_1679747 -1 27436 EML4 ILMN_1718297 1 55970 GNG12 ILMN_1673380 1 56204 KIAA1370 ILMN_1700733 1 56890 MDM1 ILMN_1750703 -1 56942 C16orf61 ILMN_1783333 -1 58528 RRAGD ILMN_1699772 1 5825 ABCD3 ILMN_1674032 1 36ACADSB ILMN_1740920 -1 92ACVR2A ILMN_1670912 -1 8038 ADAM12 ILMN_1705689 -1 60312 AFAP ILMN_1701998 1 3899 AFF3 ILMN_1775235 1 10551 AGR2 ILMN_1814151 -1 55966 AJAP1 ILMN_1723521 -1 122481 AK7 ILMN_1684305 1 11215 AKAP11 ILMN_1693220 1 9068 ANGPTL1 ILMN_1669773 -1 26057 ANKRD17 ILMN_1712019 -1 163782 ANKRD38 ILMN_1776936 1 10053 AP1M2 ILMN_1705861 1 63941 APBA2BP ILMN_1749738 -1 147495 APCDD1 ILMN_1656951 1 51074 APIP ILMN_1793598 1 367AR ILMN_1767351 1 9828 ARHGEF17 ILMN_1754562 coefficient Entrez_Gene_ID Symbol Probe_Id AKT/mTOR 1 535ATP6V0A1 ILMN_1752579 1 9474 ATG5 ILMN_1717786 1 998CDC42 ILMN_1738424 1 475ATOX1 ILMN_1670609 1 5327 PLAT ILMN_1738742 1 6427 SFRS2 ILMN_1696407 1 5987 RFP ILMN_1730005 1 5518 PPP2R1A ILMN_1810467 1 3948 LDHC ILMN_1745043 1 3838 KPNA2 ILMN_1708160 1 2990 GUSB ILMN_1669878 1 5476 PPGB ILMN_1719286 1 27069 GHITM ILMN_1728467 1 2222 FDFT1 ILMN_1741096 1 896CCND3 ILMN_1668721 1 7167 TPI1 ILMN_1707627 1 10938 EHD1 ILMN_1651832 1 3190 HNRPK ILMN_1712357 1 2171 FABP5 ILMN_1696302 1 51155 HN1 ILMN_1651461 1 7351 UCP2 ILMN_1685625 1 928CD9 ILMN_1679858 1 27166 PRELID1 ILMN_1680022 1 5214 PFKP ILMN_1805737 1 7534 YWHAZ ILMN_1801928 1 6241 RRM2 ILMN_1678669 1 50814 NSDHL ILMN_1692163 1 5695 PSMB7 ILMN_1814156 1 11258 DCTN3 ILMN_1762281 1 2937 GSS ILMN_1683462 1 9551 ATP5J2 ILMN_1750143 1 529ATP6V1E1 ILMN_1798485 1 5356 PLRG1 ILMN_1749634 1 4717 NDUFC1 ILMN_1733603 1 9516 LITAF ILMN_1713934 1 4259 MGST3 ILMN 1751956 7150 TOP1 ILMN_1735572 9830 TRIM14 ILMN_1713542 619435 C8orf61 ILMN_1684576 860RUNX2 ILMN_1716651 Gene_ID Symbol Probe_Id 535ATP6V0A1 ILMN_1752579 9474 ATG5 ILMN_1717786 998CDC42 ILMN_1738424 475ATOX1 ILMN_1670609 5327 PLAT ILMN_1738742 6427 SFRS2 ILMN_1696407 5987 RFP ILMN_1730005 5518 PPP2R1A ILMN_1810467 3948 LDHC ILMN_1745043 3838 KPNA2 ILMN_1708160 2990 GUSB ILMN_1669878 5476 PPGB ILMN_1719286 27069 GHITM ILMN_1728467 2222 FDFT1 ILMN_1741096 896CCND3 ILMN_1668721 7167 TPI1 ILMN_1707627 10938 EHD1 ILMN_1651832 3190 HNRPK ILMN_1712357 2171 FABP5 ILMN_1696302 51155 HN1 ILMN_1651461 7351 UCP2 ILMN_1685625 928CD9 ILMN_1679858 27166 PRELID1 ILMN_1680022 5214 PFKP ILMN_1805737 7534 YWHAZ ILMN_1801928 6241 RRM2 ILMN_1678669 50814 NSDHL ILMN_1692163 5695 PSMB7 ILMN_1814156 11258 DCTN3 ILMN_1762281 2937 GSS ILMN_1683462 9551 ATP5J2 ILMN_1750143 529ATP6V1E1 ILMN_1798485 5356 PLRG1 ILMN_1749634 4717 NDUFC1 ILMN_1733603 9516 LITAF ILMN_1713934 4259 MGST3 ILMN_1751956 81614 NIPA2 ILMN_1720344 4282 MIF ILMN_1716169 5604 MAP2K1 ILMN_1694240 9524 GPSN2 ILMN_1720799 3326 HSP90AB1 ILMN 1673711 4074 M6PR ILMN_1682589 527ATP6V0C ILMN_1773849 2987 GUK1 ILMN_1758398 5050 PAFAH1B3 ILMN_1762654 10055 SAE1 ILMN_1657204 6813 STXBP2 ILMN_1795426 1072 CFL1 ILMN_1705617 56478 EIF4ENIF1 ILMN_1794967 10390 CEPT1 ILMN_1676588 2539 G6PD ILMN_1697559 25994 HIGD1A ILMN_1674522 3959 LGALS3BP ILMN_1654314 4072 TACSTD1 ILMN_1673871 1366 CLDN7 ILMN_1723564 80736 SLC44A4 ILMN_1730977 57192 MCOLN1 ILMN_1764383 25825 BACE2 ILMN_1669323 54676 GTPBP2 ILMN_1694475 1803 DPP4 ILMN_1692535 6120 RPE ILMN_1699476 11035 RIPK3 ILMN_1763763 204AK2 ILMN_1716053 4289 MKLN1 ILMN_1742578 10280 OPRS1 ILMN_1717925 533ATP6V0B ILMN_1721391 64225 ATL2 ILMN_1716384 29103 DNAJC15 ILMN_1812666 79647 C1orf108 ILMN_1658337 226ALDOA ILMN_1736700 6993 DYNLT1 ILMN_1678766 637BID ILMN_1763386 10519 CIB1 ILMN_1656899 2764 GMFB ILMN_1752915 6733 SRPK2 ILMN_1657451 84245 MGC3207 ILMN_1732089 3880 KRT19 ILMN_1753924 83786 FKSG44 ILMN_1781047 7389 UROD ILMN_1740742 1717 DHCR7 ILMN_1654028 55872 PBK ILMN_1673673 6391 SDHC ILMN_1746241 5315 PKM2 ILMN_1672650 9843 HEPH ILMN_1714523 116541 MRPL54 ILMN_1658486 2820 GPD2 ILMN_1723139 6884 TAF13 ILMN_1712561 8504 PEX3 ILMN_1702034 10487 CAP1 ILMN_1797604 1508 CTSB ILMN_1696360 93974 ATPIF1 ILMN_1727332 6646 SOAT1 ILMN_1699100 55611 OTUB1 ILMN_1778017 4669 NAGLU ILMN_1694980 5836 PYGL ILMN_1696187 23603 CORO1C ILMN_1745954 80777 CYB5-M ILMN_1684321 59084 ENPP5 ILMN_1660727 5230 PGK1 ILMN_1755749 51478 HSD17B7 ILMN_1671661 890CCNA2 ILMN_1786125 5641 LGMN ILMN_1698019 10635 RAD51AP1 ILMN_1670353 10103 TSPAN1 ILMN_1747546 7064 THOP1 ILMN_1726659 100ADA ILMN_1803686 686BTD ILMN_1699728 10728 PTGES3 ILMN_1752566 192286 HIGD2A ILMN_1774334 2150 F2RL1 ILMN_1673113 8342 HIST1H2BM ILMN_1736820 23597 ACOT9 ILMN_1658995 56675 NRIP3 ILMN_1759563 7099 TLR4 ILMN_1706217 90843 TCEAL8 ILMN_1670561 10204 NUTF2 ILMN_1655046 254863 C17orf61 ILMN_1737358 3073 HEXA ILMN_1813911 3433 IFIT2 ILMN_1739428 2872 MKNK2 ILMN_1654060 5019 OXCT1 ILMN_1813544 3799 KIF5B ILMN_1788160 6945 MLX ILMN_1792207 7514 XPO1 ILMN_1725121 3898 LAD1 ILMN_1782389 6698 SPRR1A ILMN_1716591 51330 TNFRSF12A ILMN_1689004 2597 GAPDH ILMN_1802252 27075 TSPAN13 ILMN_1669881 112399 EGLN3 ILMN_1667626 726CAPN5 ILMN_1737089 57402 S100A14 ILMN_1783287 54997 TESC ILMN_1750181 10380 BPNT1 ILMN_1712718 11187 PKP3 ILMN_1753457 80324 PUS1 ILMN_1662658 8766 RAB11A ILMN_1712312 26517 TIMM13 ILMN_1686557 2673 GFPT1 ILMN_1696000 2877 GPX2 ILMN_1662776 2023 ENO1 ILMN_1710756 30001 ERO1L ILMN_1744963 4356 MPP3 ILMN_1724754 9982 FGFBP1 ILMN_1785404 119504 C10orf104 ILMN_1738955 7113 TMPRSS2 ILMN_1791123 51268 PIPOX ILMN_1743475 3055 HCK ILMN_1791771 528ATP6V1C1 ILMN_1659801 64078 SLC28A3 ILMN_1741014 4641 MYO1C ILMN_1812616 79570 NKAIN1 ILMN_1739374 26471 P8 ILMN_1790234 4488 MSX2 ILMN_1766951 3939 LDHA ILMN_1807106 10087 COL4A3BP ILMN_1680109 30844 EHD4 ILMN_1720083 3565 IL4 ILMN_1669174 55760 DHX32 ILMN_1713688 23643 LY96 ILMN_1724533 136319 MTPN ILMN_1791478 2643 GCH1 ILMN_1812759 6303 SAT1 ILMN_1753342 8521 GCM1 ILMN_1695135 58472 SQRDL ILMN_1667199 3856 KRT8 ILMN_1753584 84709 OSAP ILMN_1793025 7529 YWHAB ILMN_1694385 3098 HK1 ILMN_1727672 7422 VEGFA ILMN_1803882 5284 PIGR ILMN_1685387 112939 BTBD14B ILMN_1658231 5531 PPP4C ILMN_1750364 2821 GPI ILMN_1654465 171425 CLYBL ILMN_1663538 54107 POLE3 ILMN_1785198 9663 LPIN2 ILMN_1670028 6611 SMS ILMN_1694305 54836 BSPRY ILMN_1667077 26292 MYCBP ILMN_1659620 429ASCL1 ILMN_1701653 10452 TOMM40 ILMN_1683475 4793 NFKBIB ILMN_1690473 2017 CTTN ILMN_1744912 136ADORA2B ILMN_1703946 4363 ABCC1 ILMN_1802404 10611 PDLIM5 ILMN_1770224 1119 CHKA ILMN_1658504 117156 SCGB3A2 ILMN_1670306 5784 PTPN14 ILMN_1754830 5211 PFKL ILMN_1713037 8740 TNFSF14 ILMN_1655414 55740 ENAH ILMN_1716552 10401 PIAS3 ILMN_1675497 4000 LMNA ILMN_1696749 81490 PTDSS2 ILMN_1763723 822CAPG ILMN_1655821 3949 LDLR ILMN_1651611 3839 KPNA3 ILMN_1708427 205AK3L1 ILMN_1764090 5601 MAPK9 ILMN_1795411 5469 PPARBP ILMN_1721729 5905 RANGAP1 ILMN_1662198 5610 EIF2AK2 ILMN_1706502 54469 ZFAND6 ILMN_1778803 5583 PRKCH ILMN_1780898 54463 FLJ20152 ILMN_1811330 79774 GRTP1 ILMN_1745068 6700 SPRR2A ILMN_1795359 54205 CYCS ILMN_1730416 51474 LIMA1 ILMN_1704369 1476 CSTB ILMN_1761797 523ATP6V1A ILMN_1711516 1431 CS ILMN_1791182 5863 RGL2 ILMN_1759159 3099 HK2 ILMN_1723486 23624 CBLC ILMN_1811729 9646 CTR9 ILMN_1665164 5550 PREP ILMN_1751887 10146 G3BP1 ILMN_1690065 3434 IFIT1 ILMN_1707695 8357 HIST1H3H ILMN_1749368 51142 CHCHD2 ILMN_1815124 9689 BZW1 ILMN_1793846 9948 WDR1 ILMN_1675844 51727 CMPK1 ILMN_1738642 6307 SC4MOL ILMN_1720889 10140 TOB1 ILMN_1672004 9777 TM9SF4 ILMN_1674421 740MRPL49 ILMN_1790834 3985 LIMK2 ILMN_1660624 92259 MRPS36 ILMN_1807095 54682 MANSC1 ILMN_1652490 5578 PRKCA ILMN_1771800 8560 DEGS1 ILMN_1667430 2805 GOT1 ILMN_1656145 2224 FDPS ILMN_1804248 9262 STK17B ILMN_1798543 7086 TKT ILMN_1736597 26140 TTLL3 ILMN_1682818 10783 NEK6 ILMN_1660871 10383 TUBB2C ILMN_1780769 55041 PLEKHB2 ILMN_1698323 10159 ATP6AP2 ILMN_1677440 5226 PGD ILMN_1794165 513ATP5D ILMN_1653599 5163 PDK1 ILMN_1670256 54583 EGLN1 ILMN_1749892 3422 IDI1 ILMN_1755075 28958 CCDC56 ILMN_1789786 29887 SNX10 ILMN_1786257 6050 RNH1 ILMN_1660880 81876 RAB1B ILMN_1664030 8453 CUL2 ILMN_1669252 2739 GLO1 ILMN_1702177 150684 COMMD1 ILMN_1761242 55065 GPR172B ILMN_1683492 55620 STAP2 ILMN_1657631 30851 TAX1BP3 ILMN_1803392 92799 SHKBP1 ILMN_1765493 307ANXA4 ILMN_1711408 150290 DUSP18 ILMN_1734288 4967 OGDH ILMN_1733869 26136 TES ILMN_1765246 134147 LOC134147 ILMN_1709634 10999 SLC27A4 ILMN_1655039 5130 PCYT1A ILMN_1658310 55813 UTP6 ILMN_1778238 26355 FAM162A ILMN_1803647 23443 SLC35A3 ILMN_1653429 79815 NPAL2 ILMN_1774373 4731 NDUFV3 ILMN_1765500 6309 SC5DL ILMN_1677607 230ALDOC ILMN_1755974 58513 EPS15L1 ILMN_1708369 79370 BCL2L14 ILMN_1664235 55930 MYO5C ILMN_1808789 767CA8 ILMN_1696615 55287 TMEM40 ILMN_1705054 171177 RHOV ILMN_1698484 129642 OACT2 ILMN_1777853 54097 FAM3B ILMN_1685767 6888 TALDO1 ILMN_1746588 29088 MRPL15 ILMN_1800598 11269 DDX19B ILMN_1707123 8786 RGS11 ILMN_1763704 2013 EMP2 ILMN_1671270 47ACLY ILMN_1749014 1355 COX15 ILMN_1718309 151354 FAM84A ILMN_1784422 3135 HLA-G ILMN_1656670 5795 PTPRJ ILMN_1699851 57458 TMCC3 ILMN_1685493 1636 ACE ILMN_1779984 327APEH ILMN 1718023 5165 PDK3 ILMN_1776582 7706 TRIM25 ILMN_1754859 94240 EPSTI1 ILMN_1688566 79747 C6orf103 ILMN_1666316 6513 SLC2A1 ILMN_1809256 51075 TXNDC14 ILMN_1799367 25797 QPCT ILMN_1741727 3320 HSP90AA1 ILMN_1691097 55907 CMAS ILMN_1714082 85414 SLC45A3 ILMN_1726114 9563 H6PD ILMN_1721136 1266 CNN3 ILMN_1782439 515ATP5F1 ILMN_1672191 10970 CKAP4 ILMN_1790891 23028 AOF2 ILMN_1813840 64761 PARP12 ILMN_1718558 64859 OBFC2A ILMN_1785852 3958 LGALS3 ILMN_1747118 286262 C9orf75 ILMN_1664283 253943 YTHDF3 ILMN_1657470 283130 LOC283130 ILMN_1810727 51809 GALNT7 ILMN_1670748 4047 LSS ILMN_1715024 5336 PLCG2 ILMN_1815719 6836 SURF4 ILMN_1799055 3892 KRTHB6 ILMN_1811984 81844 TRIM56 ILMN_1666376 51495 PTPLAD1 ILMN_1729762 23089 PEG10 ILMN_1763359 162494 RHBDL3 ILMN_1716019 6049 RNF6 ILMN_1687267 79622 C16orf33 ILMN_1801118 57142 RTN4 ILMN_1748983 7037 TFRC ILMN_1674243 84899 TMTC4 ILMN_1762095 85461 TANC1 ILMN_1772278 54541 DDIT4 ILMN_1661599 84267 C9orf64 ILMN_1777318 51081 MRPS7 ILMN_1813389 4696 NDUFA3 ILMN_1784641 644BLVRA ILMN_1691436 118881 COMTD1 ILMN_1736752 5520 PPP2R2A ILMN_1788961 55276 PGM2 ILMN_1673543 57494 FAM80B ILMN_1681757 158219 TTC39B ILMN_1769290 9603 NFE2L3 ILMN_1724898 178AGL ILMN_1664006 55161 TMEM33 ILMN_1750795 1545 CYP1B1 ILMN_1693338 3157 HMGCS1 ILMN_1797728 10992 SF3B2 ILMN_1737807 55361 PI4K2A ILMN_1750549 9716 AQR ILMN_1717154 6659 SOX4 ILMN_1815745 400916 CHCHD10 ILMN_1740170 83699 SH3BGRL2 ILMN_1762764 92359 CRB3 ILMN_1754635 10043 TOM1 ILMN_1813148 9993 DGCR2 ILMN_1713301 1317 SLC31A1 ILMN_1804562 55293 UEV3 ILMN_1711452 340348 TSPAN33 ILMN_1749403 23218 NBEAL2 ILMN_1660629 10900 RUNDC3A ILMN_1756715 2585 GALK2 ILMN_1723124 10971 YWHAQ ILMN_1674385 91373 UAP1L1 ILMN_1653712 50ACO2 ILMN_1654861 6645 SNTB2 ILMN_1786766 23788 MTCH2 ILMN_1785218 6218 RPS17 ILMN_1685653 292SLC25A5 ILMN_1774062 6651 SON ILMN_1703427 1974 EIF4A2 ILMN_1685722 5209 PFKFB3 ILMN_1660847 2314 FLII ILMN_1737170 51629 SLC25A39 ILMN_1721723 _Gene_ID Symbol Probe_Id 2064 ERBB2 ILMN_1694303 93210 PERLD1 ILMN_1805636 5709 PSMD3 ILMN_1796430 5409 PNMT ILMN_1798007 55876 GSDML ILMN_1666206 1 55276 PGM2 ILMN_1673543 1 57494 FAM80B ILMN_1681757 1 158219 TTC39B ILMN_1769290 1 9603 NFE2L3 ILMN_1724898 1 178AGL ILMN_1664006 1 55161 TMEM33 ILMN_1750795 1 1545 CYP1B1 ILMN_1693338 1 3157 HMGCS1 ILMN_1797728 1 10992 SF3B2 ILMN_1737807 1 55361 PI4K2A ILMN_1750549 1 9716 AQR ILMN_1717154 1 6659 SOX4 ILMN_1815745 1 400916 CHCHD10 ILMN_1740170 1 83699 SH3BGRL2 ILMN_1762764 1 92359 CRB3 ILMN_1754635 1 10043 TOM1 ILMN_1813148 1 9993 DGCR2 ILMN_1713301 1 1317 SLC31A1 ILMN_1804562 1 55293 UEV3 ILMN_1711452 1 340348 TSPAN33 ILMN_1749403 1 23218 NBEAL2 ILMN_1660629 1 10900 RUNDC3A ILMN_1756715 1 2585 GALK2 ILMN_1723124 1 10971 YWHAQ ILMN_1674385 1 91373 UAP1L1 ILMN_1653712 1 50ACO2 ILMN_1654861 1 6645 SNTB2 ILMN_1786766 1 23788 MTCH2 ILMN_1785218 1 6218 RPS17 ILMN_1685653 1 292SLC25A5 ILMN_1774062 1 6651 SON ILMN_1703427 1 1974 EIF4A2 ILMN_1685722 1 5209 PFKFB3 ILMN_1660847 1 2314 FLII ILMN_1737170 1 51629 SLC25A39 ILMN_1721723 Desmedt et al. within each module ent Entrez_Gene_ID Symbol Probe_Id -1 991CDC20 ILMN_1663390 -1 1476 CSTB ILMN_1761797 -1 1503 CTPS ILMN_1783285 1 3708 ITPR1 ILMN_1789505 -1 4281 MID1 ILMN_1761858 -1 4781 NFIB ILMN_1778991 -1 5433 POLR2D ILMN_1792672 1 5684 PSMA3 ILMN_1694931 -1 6317 SERPINB3 ILMN_1703855 -1 6446 SGK ILMN_1702487 -1 7159 TP53BP2 ILMN_1779706 1 7251 TSG101 ILMN_1747146 -1 8208 CHAF1B ILMN_1674231 -1 8543 LMO4 ILMN_1703487 -1 9353 SLIT2 ILMN_1676449 -1 10200 MPHOSPH6 ILMN_1746682 -1 10982 MAPRE2 ILMN_1695276 -1 25925 ZNF521 ILMN_1679747 -1 27436 EML4 ILMN_1718297 1 55970 GNG12 ILMN_1673380 1 56204 KIAA1370 ILMN_1700733 1 56890 MDM1 ILMN_1750703 -1 56942 C16orf61 ILMN_1783333 -1 58528 RRAGD ILMN_1699772 1 5825 ABCD3 ILMN_1674032 1 36ACADSB ILMN_1740920 -1 92ACVR2A ILMN_1670912 -1 8038 ADAM12 ILMN_1705689 -1 60312 AFAP ILMN_1701998 1 3899 AFF3 ILMN_1775235 1 10551 AGR2 ILMN_1814151 -1 55966 AJAP1 ILMN_1723521 -1 122481 AK7 ILMN_1684305 1 11215 AKAP11 ILMN_1693220 1 9068 ANGPTL1 ILMN_1669773 -1 26057 ANKRD17 ILMN_1712019 -1 163782 ANKRD38 ILMN_1776936 1 10053 AP1M2 ILMN_1705861 1 63941 APBA2BP ILMN_1749738 -1 147495 APCDD1 ILMN_1656951 1 51074 APIP ILMN_1793598 1 367AR ILMN_1767351 1 9828 ARHGEF17 ILMN_1754562 coefficient Entrez_Gene_ID Symbol Probe_Id ERBB2 1 2064 ERBB2 ILMN_1694303 1 93210 PERLD1 ILMN_1805636 1 5709 PSMD3 ILMN_1796430 1 5409 PNMT ILMN_1798007 1 55876 GSDML ILMN_1666206 1 22794 CASC3 ILMN_1683600 1 3927 LASP1 ILMN_1665909 1 147179 WIPF2 ILMN_1786229 1 55040 EPN3 ILMN_1726893 1 5245 PHB ILMN_1692651 1 9635 CLCA2 ILMN_1655523 1 3227 HOXC11 ILMN_1697342 1 29095 ORMDL2 ILMN_1774708 1 5909 RAP1GAP ILMN_1776519 1 1573 CYP2J2 ILMN_1758731 1 26154 ABCA12 ILMN_1701642 1 3081 HGD ILMN_1737541 -1 8804 CREG1 ILMN_1680624 1 9914 KIAA0703 ILMN_1785413 -1 5129 PCTK3 ILMN_1784110 -1 54793 KCTD9 ILMN_1653612 1 404093 CUEDC1 ILMN_1676665 1 3675 ITGA3 ILMN_1685397 1 55129 TMEM16K ILMN_1767111 -1 24147 FJX1 ILMN_1746465 1 1048 CEACAM5 ILMN_1670959 -1 51375 SNX7 ILMN_1685077 coefficient Entrez_Gene_ID Symbol Probe_Id -1 89845 ABCC10 ILMN_1776119 -1 47ACLY ILMN_1749014 -1 771CA12 ILMN_1720998 -1 55870 ASH1L ILMN_1782032 -1 55612 C20orf42 ILMN_1696585 -1 157680 VPS13B ILMN_1765165 -1 1296 COL8A2 ILMN_1674050 -1 1479 CSTF3 ILMN_1736161 -1 8853 DDEF2 ILMN_1757237 -1 115817 DHRS1 ILMN_1807206 -1 9988 DMTF1 ILMN_1750075 -1 131566 DCBLD2 ILMN_1735499 -1 79813 EHMT1 ILMN_1712453 -1 2135 EXTL2 ILMN_1797950 -1 55120 FANCL ILMN_1754045 -1 64848 YTHDC2 ILMN_1725760 -1 10634 GAS2L1 ILMN_1724059 -1 10808 HSPH1 ILMN_1712888 -1 23023 TMCC1 ILMN_1750539 -1 23030 JMJD2B ILMN_1720531 -1 9388 LIPG ILMN_1689037 -1 125476 C18orf37 ILMN_1730294 -1 4047 LSS ILMN_1715024 1 4140 MARK3 ILMN 1704795 1 5245 PHB ILMN_1692651 1 9635 CLCA2 ILMN_1655523 1 3227 HOXC11 ILMN_1697342 1 29095 ORMDL2 ILMN_1774708 1 5909 RAP1GAP ILMN_1776519 1 1573 CYP2J2 ILMN_1758731 1 26154 ABCA12 ILMN_1701642 1 3081 HGD ILMN_1737541 -1 8804 CREG1 ILMN_1680624 1 9914 KIAA0703 ILMN_1785413 -1 5129 PCTK3 ILMN_1784110 -1 54793 KCTD9 ILMN_1653612 1 404093 CUEDC1 ILMN_1676665 1 3675 ITGA3 ILMN_1685397 1 55129 TMEM16K ILMN_1767111 -1 24147 FJX1 ILMN_1746465 1 1048 CEACAM5 ILMN_1670959 -1 51375 SNX7 ILMN_1685077 oefficient Entrez_Gene_ID Symbol Probe_Id -1 89845 ABCC10 ILMN_1776119 -1 47ACLY ILMN_1749014 -1 771CA12 ILMN_1720998 -1 55870 ASH1L ILMN_1782032 -1 55612 C20orf42 ILMN_1696585 -1 157680 VPS13B ILMN_1765165 -1 1296 COL8A2 ILMN_1674050 -1 1479 CSTF3 ILMN_1736161 -1 8853 DDEF2 ILMN_1757237 -1 115817 DHRS1 ILMN_1807206 -1 9988 DMTF1 ILMN_1750075 -1 131566 DCBLD2 ILMN_1735499 -1 79813 EHMT1 ILMN_1712453 -1 2135 EXTL2 ILMN_1797950 -1 55120 FANCL ILMN_1754045 -1 64848 YTHDC2 ILMN_1725760 -1 10634 GAS2L1 ILMN_1724059 -1 10808 HSPH1 ILMN_1712888 -1 23023 TMCC1 ILMN_1750539 -1 23030 JMJD2B ILMN_1720531 -1 9388 LIPG ILMN_1689037 -1 125476 C18orf37 ILMN_1730294 -1 4047 LSS ILMN_1715024 -1 4140 MARK3 ILMN_1704795 1 326625 MMAB ILMN_1713055 -1 6526 SLC5A3 ILMN_1787813 1 23645 PPP1R15A ILMN 1659936 Bild et al. within each module ent Entrez_Gene_ID Symbol Probe_Id -1 991CDC20 ILMN_1663390 -1 1476 CSTB ILMN_1761797 -1 1503 CTPS ILMN_1783285 1 3708 ITPR1 ILMN_1789505 -1 4281 MID1 ILMN_1761858 -1 4781 NFIB ILMN_1778991 -1 5433 POLR2D ILMN_1792672 1 5684 PSMA3 ILMN_1694931 -1 6317 SERPINB3 ILMN_1703855 -1 6446 SGK ILMN_1702487 -1 7159 TP53BP2 ILMN_1779706 1 7251 TSG101 ILMN_1747146 -1 8208 CHAF1B ILMN_1674231 -1 8543 LMO4 ILMN_1703487 -1 9353 SLIT2 ILMN_1676449 -1 10200 MPHOSPH6 ILMN_1746682 -1 10982 MAPRE2 ILMN_1695276 -1 25925 ZNF521 ILMN_1679747 -1 27436 EML4 ILMN_1718297 1 55970 GNG12 ILMN_1673380 1 56204 KIAA1370 ILMN_1700733 1 56890 MDM1 ILMN_1750703 -1 56942 C16orf61 ILMN_1783333 -1 58528 RRAGD ILMN_1699772 1 5825 ABCD3 ILMN_1674032 1 36ACADSB ILMN_1740920 -1 92ACVR2A ILMN_1670912 -1 8038 ADAM12 ILMN_1705689 -1 60312 AFAP ILMN_1701998 1 3899 AFF3 ILMN_1775235 1 10551 AGR2 ILMN_1814151 -1 55966 AJAP1 ILMN_1723521 -1 122481 AK7 ILMN_1684305 1 11215 AKAP11 ILMN_1693220 1 9068 ANGPTL1 ILMN_1669773 -1 26057 ANKRD17 ILMN_1712019 -1 163782 ANKRD38 ILMN_1776936 1 10053 AP1M2 ILMN_1705861 1 63941 APBA2BP ILMN_1749738 -1 147495 APCDD1 ILMN_1656951 1 51074 APIP ILMN_1793598 1 367AR ILMN_1767351 1 9828 ARHGEF17 ILMN_1754562 SRC -1 144165 PRICKLE1 ILMN_1741356 -1 150696 PROM2 ILMN_1761946 -1 5744 PTHLH ILMN_1785699 -1 6241 RRM2 ILMN_1678669 -1 79048 SECISBP2 ILMN_1736481 -1 5055 SERPINB2 ILMN_1752993 -1 6558 SLC12A2 ILMN_1720996 1 6714 SRC ILMN_1729987 -1 6744 SSFA2 ILMN_1742260 -1 10299 Mar-06ILMN_1757106 -1 7128 TNFAIP3 ILMN_1702691 -1 23270 TSPYL4 ILMN_1715821 -1 7559 ZNF12 ILMN_1784577 -1 27107 ZBTB11 ILMN_1691395 -1 51592 TRIM33 ILMN_1682316 -1 1277 COL1A1 ILMN_1701308 coefficient Entrez_Gene_ID Symbol Probe_Id 1 23157 Sep-06ILMN_1661342 -1 23461 ABCA5 ILMN_1784176 1 51703 ACSL5 ILMN_1705247 -1 135ADORA2A ILMN_1743519 1 202AIM1 ILMN_1688625 -1 205AK3L1 ILMN_1764090 -1 7915 ALDH5A1 ILMN_1715859 1 240ALOX5 ILMN_1680996 -1 10513 APPBP2 ILMN_1694875 1 367AR ILMN_1767351 1 397ARHGDIB ILMN_1678143 1 9915 ARNT2 ILMN_1718046 -1 10776 ARPP-19 ILMN_1772798 -1 440ASNS ILMN_1796417 1 481ATP1B1 ILMN_1736862 -1 8992 ATP6V0E1 ILMN_1715635 1 23215 BAT2D1 ILMN_1743137 -1 596BCL2 ILMN_1801119 1 629CFB ILMN_1774287 1 330BIRC3 ILMN_1696549 -1 51651 PTRH2 ILMN_1811594 1 684BST2 ILMN_1723480 1 10384 BTN3A3 ILMN_1733338 -1 55847 CISD1 ILMN_1761781 1 116496 C1orf24 ILMN_1667966 1 25966 C21orf25 ILMN_1652512 1 9980 DOPEY2 ILMN_1741711 Loi et al. within each module ent Entrez_Gene_ID Symbol Probe_Id -1 991CDC20 ILMN_1663390 -1 1476 CSTB ILMN_1761797 -1 1503 CTPS ILMN_1783285 1 3708 ITPR1 ILMN_1789505 -1 4281 MID1 ILMN_1761858 -1 4781 NFIB ILMN_1778991 -1 5433 POLR2D ILMN_1792672 1 5684 PSMA3 ILMN_1694931 -1 6317 SERPINB3 ILMN_1703855 -1 6446 SGK ILMN_1702487 -1 7159 TP53BP2 ILMN_1779706 1 7251 TSG101 ILMN_1747146 -1 8208 CHAF1B ILMN_1674231 -1 8543 LMO4 ILMN_1703487 -1 9353 SLIT2 ILMN_1676449 -1 10200 MPHOSPH6 ILMN_1746682 -1 10982 MAPRE2 ILMN_1695276 -1 25925 ZNF521 ILMN_1679747 -1 27436 EML4 ILMN_1718297 1 55970 GNG12 ILMN_1673380 1 56204 KIAA1370 ILMN_1700733 1 56890 MDM1 ILMN_1750703 -1 56942 C16orf61 ILMN_1783333 -1 58528 RRAGD ILMN_1699772 1 5825 ABCD3 ILMN_1674032 1 36ACADSB ILMN_1740920 -1 92ACVR2A ILMN_1670912 -1 8038 ADAM12 ILMN_1705689 -1 60312 AFAP ILMN_1701998 1 3899 AFF3 ILMN_1775235 1 10551 AGR2 ILMN_1814151 -1 55966 AJAP1 ILMN_1723521 -1 122481 AK7 ILMN_1684305 1 11215 AKAP11 ILMN_1693220 1 9068 ANGPTL1 ILMN_1669773 -1 26057 ANKRD17 ILMN_1712019 -1 163782 ANKRD38 ILMN_1776936 1 10053 AP1M2 ILMN_1705861 1 63941 APBA2BP ILMN_1749738 -1 147495 APCDD1 ILMN_1656951 1 51074 APIP ILMN_1793598 1 367AR ILMN_1767351 1 9828 ARHGEF17 ILMN_1754562 PIK3CA 56892 C8orf4 ILMN_1656369 51101 FAM164A ILMN_1789558 51806 CALML5 ILMN_1749118 10486 CAP2 ILMN_1691237 824CAPN2 ILMN_1716057 64127 NOD2 ILMN_1762594 834CASP1 ILMN_1742270 874CBR3 ILMN_1652237 875CBS ILMN_1804735 6352 CCL5 ILMN_1773352 10576 CCT2 ILMN_1701298 972CD74 ILMN_1761464 64866 CDCP1 ILMN_1724941 1048 CEACAM5 ILMN_1670959 4680 CEACAM6 ILMN_1712522 1081 CGA ILMN_1734176 79789 CLMN ILMN_1714522 1363 CPE ILMN_1731374 1384 CRAT ILMN_1728671 8531 CSDA ILMN_1782788 1472 CST4 ILMN_1752452 1488 CTBP2 ILMN_1691294 1501 CTNND2 ILMN_1716875 1602 DACH1 ILMN_1755741 23259 DDHD2 ILMN_1705871 55526 DHTKD1 ILMN_1664369 79665 DHX40 ILMN_1653047 1832 DSP ILMN_1655614 1837 DTNA ILMN_1730201 1875 E2F5 ILMN_1782551 55862 ECHDC1 ILMN_1762134 26298 EHF ILMN_1708130 1968 EIF2S3 ILMN_1665717 8507 ENC1 ILMN_1779147 22875 ENPP4 ILMN_1693675 956ENTPD3 ILMN_1775114 957ENTPD5 ILMN_1745849 2042 EPHA3 ILMN_1775931 2052 EPHX1 ILMN_1701025 2099 ESR1 ILMN_1678535 51466 EVL ILMN_1730622 58516 FAM60A ILMN_1683664 2263 FGFR2 ILMN 16822 0 1 2299 FOXI1 ILMN_1736936 1 53827 FXYD5 ILMN_1704286 -1 8323 FZD6 ILMN_1659297 1 2571 GAD1 ILMN_1660973 1 55568 GALNT10 ILMN_1681984 1 2590 GALNT2 ILMN_1814606 1 51809 GALNT7 ILMN_1670748 -1 2743 GLRB ILMN_1669631 1 2745 GLRX ILMN_1737308 1 2762 GMDS ILMN_1711227 1 9630 GNA14 ILMN_1734857 1 51280 GOLM1 ILMN_1766405 1 2239 GPC4 ILMN_1789502 1 2888 GRB14 ILMN_1793832 -1 2886 GRB7 ILMN_1740762 1 2950 GSTP1 ILMN_1679809 -1 51454 GULP1 ILMN_1802690 -1 55573 CDV3 ILMN_1810977 -1 50810 HDGFRP3 ILMN_1784256 -1 3012 HIST1H2AE ILMN_1756849 -1 8969 HIST1H2AG ILMN_1686478 -1 3109 HLA-DMB ILMN_1761733 -1 8337 HIST2H2AA3 ILMN_1659047 -1 8349 HIST2H2BE ILMN_1732071 -1 92815 HIST3H2A ILMN_1805655 1 3106 HLA-B ILMN_1703403 1 3108 HLA-DMA ILMN_1695311 1 3120 HLA-DQB2 ILMN_1741648 1 3158 HMGCS2 ILMN_1815203 -1 9456 HOMER1 ILMN_1804568 -1 22824 HSPA4L ILMN_1732468 1 3310 HSPA6 ILMN_1806165 1 3383 ICAM1 ILMN_1812226 1 3398 ID2 ILMN_1793990 -1 3400 ID4 ILMN_1721758 -1 51124 IER3IP1 ILMN_1763539 1 3321 IGSF3 ILMN_1713014 1 10788 IQGAP2 ILMN_1769433 -1 8660 IRS2 ILMN_1763809 1 11015 KDELR3 ILMN_1722820 1 23254 KIAA1026 ILMN_1798458 1 22998 LIMCH1 ILMN_1664138 1 51176 LEF1 ILMN_1679185 1 3958 LGALS3 ILMN_1747118 1 3964 LGALS8 ILMN_1664402 1 3965 LGALS9 ILMN_1715760 1 51136 LOC51136 ILMN_1688606 1 93349 LOC93349 ILMN_1658858 1 9404 LPXN ILMN_1742789 1 64167 LRAP ILMN_1743145 1 4057 LTF ILMN_1677920 1 4065 LY75 ILMN_1757011 1 23764 MAFF ILMN_1680139 1 57134 MAN1C1 ILMN_1713807 1 4233 MET ILMN_1715175 1 286527 MGC39900 ILMN_1740512 1 23531 MMD ILMN_1733937 1 4331 MNAT1 ILMN_1672361 1 4488 MSX2 ILMN_1766951 1 4493 MT1E ILMN_1718968 1 4582 MUC1 ILMN_1756992 1 4609 MYC ILMN_1753923 1 4651 MYO10 ILMN_1703576 1 9NAT1 ILMN_1688071 1 4739 NEDD9 ILMN_1758719 1 27018 NGFRAP1 ILMN_1729208 1 4824 NKX3-1 ILMN_1667162 1 4853 NOTCH2 ILMN_1656254 1 4862 NPAS2 ILMN_1765558 1 4939 OAS2 ILMN_1674063 1 51686 OAZ3 ILMN_1681892 1 4969 OGN ILMN_1790098 1 5001 ORC5L ILMN_1705093 1 5021 OXTR ILMN_1804929 1 5066 PAM ILMN_1788631 1 9659 PDE4DIP ILMN_1743773 1 10158 PDZK1IP1 ILMN_1708580 1 5178 PEG3 ILMN_1675331 1 64065 PERP ILMN_1726161 1 5203 PFDN4 ILMN_1801762 1 5213 PFKM ILMN_1708180 1 5217 PFN2 ILMN_1728258 1 51659 GINS2 ILMN_1809590 1 5281 PIGF ILMN_1808938 1 5295 PIK3R1 ILMN_1760303 1 8503 PIK3R3 ILMN_1692895 1 5327 PLAT ILMN_1738742 -1 5332 PLCB4 ILMN_1736426 -1 5350 PLN ILMN_1785523 -1 5352 PLOD2 ILMN_1771599 1 5376 PMP22 ILMN_1785646 1 8495 PPFIBP2 ILMN_1675656 -1 8493 PPM1D ILMN_1670875 1 54704 PPM2C ILMN_1720631 1 79056 PRRG4 ILMN_1661809 1 23362 PSD3 ILMN_1717477 -1 11168 PSIP1 ILMN_1788701 -1 5718 PSMD12 ILMN_1691428 1 29108 PYCARD ILMN_1698766 1 5768 QSOX1 ILMN_1758229 -1 5885 RAD21 ILMN_1748578 -1 5889 RAD51C ILMN_1695386 -1 10267 RAMP1 ILMN_1764754 1 5920 RARRES3 ILMN_1701613 1 11030 RBPMS ILMN_1708025 1 9185 REPS2 ILMN_1656934 1 5979 RET ILMN_1655610 -1 6019 RLN2 ILMN_1748529 -1 9045 RPL14 ILMN_1726460 -1 6169 RPL38 ILMN_1670633 -1 6198 RPS6KB1 ILMN_1704557 -1 58528 RRAGD ILMN_1699772 -1 22800 RRAS2 ILMN_1667519 1 23328 SASH1 ILMN_1712673 1 10647 SCGB1D2 ILMN_1714536 1 4250 SCGB2A2 ILMN_1723333 -1 6383 SDC2 ILMN_1784553 1 12SERPINA3 ILMN_1788874 -1 8910 SGCE ILMN_1674620 1 23677 SH3BP4 ILMN_1785330 1 22941 SHANK2 ILMN_1814790 1 26503 SLC17A5 ILMN_1771317 1 64754 SMYD3 ILMN_1741954 -1 150094 SNF1LK ILMN_1717639 -1 51375 SNX7 ILMN_1685077 1 6653 SORL1 ILMN_1759818 -1 79582 SPAG16 ILMN_1739192 1 9517 SPTLC2 ILMN_1704290 1 6713 SQLE ILMN 1772241 1 7056 THBD ILMN_1759787 1 7088 TLE1 ILMN_1751572 1 7107 GPR137B ILMN_1794715 1 64699 TMPRSS3 ILMN_1741768 1 8743 TNFSF10 ILMN_1801307 1 23043 TNIK ILMN_1687442 1 27324 TNRC9 ILMN_1704283 1 7164 TPD52L1 ILMN_1714383 1 7188 TRAF5 ILMN_1740493 -1 23321 TRIM2 ILMN_1745079 -1 51174 TUBD1 ILMN_1749253 -1 7295 TXN ILMN_1680314 1 7351 UCP2 ILMN_1685625 -1 10810 WASF3 ILMN_1810797 -1 51186 WBP5 ILMN_1679838 1 7474 WNT5A ILMN_1800317 -1 65986 ZBTB10 ILMN_1673222 1 677ZFP36L1 ILMN_1675448 Saale at al. within each module ent Entrez_Gene_ID Symbol Probe_Id -1 991CDC20 ILMN_1663390 -1 1476 CSTB ILMN_1761797 -1 1503 CTPS ILMN_1783285 1 3708 ITPR1 ILMN_1789505 -1 4281 MID1 ILMN_1761858 -1 4781 NFIB ILMN_1778991 -1 5433 POLR2D ILMN_1792672 1 5684 PSMA3 ILMN_1694931 -1 6317 SERPINB3 ILMN_1703855 -1 6446 SGK ILMN_1702487 -1 7159 TP53BP2 ILMN_1779706 1 7251 TSG101 ILMN_1747146 -1 8208 CHAF1B ILMN_1674231 -1 8543 LMO4 ILMN_1703487 -1 9353 SLIT2 ILMN_1676449 -1 10200 MPHOSPH6 ILMN_1746682 -1 10982 MAPRE2 ILMN_1695276 -1 25925 ZNF521 ILMN_1679747 -1 27436 EML4 ILMN_1718297 1 55970 GNG12 ILMN_1673380 1 56204 KIAA1370 ILMN_1700733 1 56890 MDM1 ILMN_1750703 -1 56942 C16orf61 ILMN_1783333 -1 58528 RRAGD ILMN_1699772 1 5825 ABCD3 ILMN_1674032 1 36ACADSB ILMN_1740920 -1 92ACVR2A ILMN_1670912 -1 8038 ADAM12 ILMN_1705689 -1 60312 AFAP ILMN_1701998 1 3899 AFF3 ILMN_1775235 1 10551 AGR2 ILMN_1814151 -1 55966 AJAP1 ILMN_1723521 -1 122481 AK7 ILMN_1684305 1 11215 AKAP11 ILMN_1693220 1 9068 ANGPTL1 ILMN_1669773 -1 26057 ANKRD17 ILMN_1712019 -1 163782 ANKRD38 ILMN_1776936 1 10053 AP1M2 ILMN_1705861 1 63941 APBA2BP ILMN_1749738 -1 147495 APCDD1 ILMN_1656951 1 51074 APIP ILMN_1793598 1 367AR ILMN_1767351 1 9828 ARHGEF17 ILMN_1754562 coefficient Entrez_Gene_ID Symbol Probe_Id PTEN -1 5728 PTEN ILMN_1701134 1 4291 MLF1 ILMN_1684439 1 1010 CDH12 ILMN_1709269 -1 145781 Gcom1 ILMN_1674151 1 4751 NEK2 ILMN_1653822 1 6612 SUMO3 ILMN_1725642 -1 2331 FMOD ILMN_1789639 1 643224 LOC643224 ILMN_1802181 -1 10399 GNB2L1 ILMN_1736500 1 7372 UMPS ILMN_1757437 1 11073 TOPBP1 ILMN_1684929 1 126731 C1orf96 ILMN_1699217 1 5052 PRDX1 ILMN_1710159 -1 5094 PCBP2 ILMN_1724825 1 330BIRC3 ILMN_1696549 1 5290 PIK3CA ILMN_1705468 -1 1265 CNN2 ILMN_1770290 -1 26123 C10orf61 ILMN_1678652 1 79132 LGP2 ILMN_1678422 1 3838 KPNA2 ILMN_1708160 1 9711 KIAA0226 ILMN_1763637 -1 8510 MMP23B ILMN_1808107 -1 80306 MED28 ILMN_1749821 -1 2043 EPHA4 ILMN_1672022 1 26973 CHORDC1 ILMN_1776337 -1 84896 ATAD1 ILMN_1654497 1 1164 CKS2 ILMN 1756326 Saale at al. within each module ent Entrez_Gene_ID Symbol Probe_Id -1 991CDC20 ILMN_1663390 -1 1476 CSTB ILMN_1761797 -1 1503 CTPS ILMN_1783285 1 3708 ITPR1 ILMN_1789505 -1 4281 MID1 ILMN_1761858 -1 4781 NFIB ILMN_1778991 -1 5433 POLR2D ILMN_1792672 1 5684 PSMA3 ILMN_1694931 -1 6317 SERPINB3 ILMN_1703855 -1 6446 SGK ILMN_1702487 -1 7159 TP53BP2 ILMN_1779706 1 7251 TSG101 ILMN_1747146 -1 8208 CHAF1B ILMN_1674231 -1 8543 LMO4 ILMN_1703487 -1 9353 SLIT2 ILMN_1676449 -1 10200 MPHOSPH6 ILMN_1746682 -1 10982 MAPRE2 ILMN_1695276 -1 25925 ZNF521 ILMN_1679747 -1 27436 EML4 ILMN_1718297 1 55970 GNG12 ILMN_1673380 1 56204 KIAA1370 ILMN_1700733 1 56890 MDM1 ILMN_1750703 -1 56942 C16orf61 ILMN_1783333 -1 58528 RRAGD ILMN_1699772 1 5825 ABCD3 ILMN_1674032 1 36ACADSB ILMN_1740920 -1 92ACVR2A ILMN_1670912 -1 8038 ADAM12 ILMN_1705689 -1 60312 AFAP ILMN_1701998 1 3899 AFF3 ILMN_1775235 1 10551 AGR2 ILMN_1814151 -1 55966 AJAP1 ILMN_1723521 -1 122481 AK7 ILMN_1684305 1 11215 AKAP11 ILMN_1693220 1 9068 ANGPTL1 ILMN_1669773 -1 26057 ANKRD17 ILMN_1712019 -1 163782 ANKRD38 ILMN_1776936 1 10053 AP1M2 ILMN_1705861 1 63941 APBA2BP ILMN_1749738 -1 147495 APCDD1 ILMN_1656951 1 51074 APIP ILMN_1793598 1 367AR ILMN_1767351 1 9828 ARHGEF17 ILMN_1754562 PTEN 1 8208 CHAF1B ILMN_1674231 1 24137 KIF4A ILMN_1799667 -1 23613 ZMYND8 ILMN_1652407 1 87178 PNPT1 ILMN_1810608 1 11004 KIF2C ILMN_1685916 -1 27122 DKK3 ILMN_1815673 1 205564 SENP5 ILMN_1675501 -1 8724 SNX3 ILMN_1740180 -1 11171 STRAP ILMN_1731194 -1 6228 RPS23 ILMN_1772459 1 54503 ZDHHC13 ILMN_1684663 1 1207 CLNS1A ILMN_1736814 1 1062 CENPE ILMN_1716279 -1 3416 IDE ILMN_1769883 1 9833 MELK ILMN_1731184 -1 5169 ENPP3 ILMN_1749131 -1 140459 ASB6 ILMN_1806705 1 51642 MRPL48 ILMN_1774580 -1 56977 STOX2 ILMN_1803256 1 5612 PRKRIR ILMN_1655622 1 10440 TIMM17A ILMN_1656798 -1 84942 WDR73 ILMN_1702592 1 3925 STMN1 ILMN_1657796 1 7307 U2AF1 ILMN_1772113 1 27316 RBMX ILMN_1723580 1 27238 GPKOW ILMN_1684197 -1 50861 STMN3 ILMN_1693425 1 1503 CTPS ILMN_1783285 -1 54556 ING3 ILMN_1806226 1 11169 WDHD1 ILMN_1756043 1 4172 MCM3 ILMN_1806818 -1 9037 SEMA5A ILMN_1656927 1 10383 TUBB2C ILMN_1780769 -1 1889 ECE1 ILMN_1672174 -1 10121 ACTR1A ILMN_1792314 1 9392 TGFBRAP1 ILMN_1696870 1 9787 DLG7 ILMN_1749829 1 151246 SGOL2 ILMN_1746699 -1 3705 ITPK1 ILMN_1715674 1 891CCNB1 ILMN_1712803 -1 83700 JAM3 ILMN_1769575 1 5984 RFC4 ILMN_1724489 S 6732 SRPK1 ILMN_1798804 5870 RAB6A ILMN_1800871 81610 C20orf129 ILMN_1781943 57380 MRS2L ILMN_1778677 3066 HDAC2 ILMN_1767747 2618 GART ILMN_1679476 25852 ARMC8 ILMN_1781151 64793 CCDC21 ILMN_1812489 23279 NUP160 ILMN_1652989 6632 SNRPD1 ILMN_1768393 10972 TMED10 ILMN_1736585 8450 CUL4B ILMN_1691535 9133 CCNB2 ILMN_1801939 7159 TP53BP2 ILMN_1779706 6434 SFRS10 ILMN_1742798 151636 DTX3L ILMN_1784380 4094 MAF ILMN_1719543 151188 ARL6IP6 ILMN_1797964 4629 MYH11 ILMN_1660086 5873 RAB27A ILMN_1665859 2999 GZMH ILMN_1731233 8681 PLA2G4B ILMN_1697629 5111 PCNA ILMN_1694177 5359 PLSCR1 ILMN_1752889 79694 MANEA ILMN_1757026 84955 NUDCD1 ILMN_1678745 10606 PAICS ILMN_1773760 4259 MGST3 ILMN_1751956 51028 VPS36 ILMN_1802519 4038 LRP4 ILMN_1675268 64844 Mar-07ILMN_1717337 200734 SPRED2 ILMN_1791232 10541 ANP32B ILMN_1684293 92342 C1orf156 ILMN_1734915 64789 C1orf176 ILMN_1783996 6397 SEC14L1 ILMN_1732575 11257 TP53AP1 ILMN_1658469 9044 BTAF1 ILMN_1791536 9232 PTTG1 ILMN_1753196 7284 TUFM ILMN_1738369 10951 CBX1 ILMN_1770244 54845 RBM35A ILMN_1749180 1 6772 STAT1 ILMN_1690105 1 387103 C6orf173 ILMN_1763907 1 6426 SFRS1 ILMN_1795341 -1 2767 GNA11 ILMN_1739781 -1 9897 KIAA0196 ILMN_1728676 1 3608 ILF2 ILMN_1745172 -1 25794 FSCN2 ILMN_1795472 1 9928 KIF14 ILMN_1808071 1 6941 TCF19 ILMN_1682008 1 29028 ATAD2 ILMN_1763064 1 3833 KIFC1 ILMN_1764840 -1 89122 TRIM4 ILMN_1792265 1 219285 SAMD9L ILMN_1748256 1 699BUB1 ILMN_1736090 -1 8576 STK16 ILMN_1803774 1 8532 CPZ ILMN_1766780 1 7936 RDBP ILMN_1765532 1 10589 DRAP1 ILMN_1733048 1 219988 PATL1 ILMN_1680782 -1 374868 ATP9B ILMN_1658684 -1 8848 TSC22D1 ILMN_1692177 -1 10540 DCTN2 ILMN_1662232 -1 3983 ABLIM1 ILMN_1785424 1 5889 RAD51C ILMN_1695386 1 6790 AURKA ILMN_1680955 -1 10975 UQCR ILMN_1745049 1 55827 IQWD1 ILMN_1670000 1 60313 GPBP1L1 ILMN_1662719 -1 55662 HIF1AN ILMN_1681812 -1 54531 MIER2 ILMN_1766159 -1 11329 STK38 ILMN_1799153 1 6619 SNAPC3 ILMN_1803611 1 157313 CDCA2 ILMN_1660654 -1 11337 GABARAP ILMN_1692338 -1 5159 PDGFRB ILMN_1815057 -1 5797 PTPRM ILMN_1744937 1 3796 KIF2A ILMN_1734476 1 9493 KIF23 ILMN_1811472 1 7444 VRK2 ILMN_1750088 1 29979 UBQLN1 ILMN_1688622 1 29127 RACGAP1 ILMN_1702140 -1 80031 SEMA6D ILMN_1814494 1 4175 MCM6 ILMN_1766634 -1 9252 RPS6KA5 ILMN_1657515 1 51773 RSF1 ILMN_1668834 1 54534 MRPL50 ILMN_1664833 1 79572 ATP13A3 ILMN_1663684 1 54625 PARP14 ILMN_1691731 -1 55101 ATP5SL ILMN_1809027 -1 1311 COMP ILMN_1677636 1 8317 CDC7 ILMN_1741801 -1 10206 TRIM13 ILMN_1674260 1 6596 SMARCA3 ILMN_1673820 Farmer et al. within each module ent Entrez_Gene_ID Symbol Probe_Id -1 991CDC20 ILMN_1663390 -1 1476 CSTB ILMN_1761797 -1 1503 CTPS ILMN_1783285 1 3708 ITPR1 ILMN_1789505 -1 4281 MID1 ILMN_1761858 -1 4781 NFIB ILMN_1778991 -1 5433 POLR2D ILMN_1792672 1 5684 PSMA3 ILMN_1694931 -1 6317 SERPINB3 ILMN_1703855 -1 6446 SGK ILMN_1702487 -1 7159 TP53BP2 ILMN_1779706 1 7251 TSG101 ILMN_1747146 -1 8208 CHAF1B ILMN_1674231 -1 8543 LMO4 ILMN_1703487 -1 9353 SLIT2 ILMN_1676449 -1 10200 MPHOSPH6 ILMN_1746682 -1 10982 MAPRE2 ILMN_1695276 -1 25925 ZNF521 ILMN_1679747 -1 27436 EML4 ILMN_1718297 1 55970 GNG12 ILMN_1673380 1 56204 KIAA1370 ILMN_1700733 1 56890 MDM1 ILMN_1750703 -1 56942 C16orf61 ILMN_1783333 -1 58528 RRAGD ILMN_1699772 1 5825 ABCD3 ILMN_1674032 1 36ACADSB ILMN_1740920 -1 92ACVR2A ILMN_1670912 -1 8038 ADAM12 ILMN_1705689 -1 60312 AFAP ILMN_1701998 1 3899 AFF3 ILMN_1775235 1 10551 AGR2 ILMN_1814151 -1 55966 AJAP1 ILMN_1723521 -1 122481 AK7 ILMN_1684305 1 11215 AKAP11 ILMN_1693220 1 9068 ANGPTL1 ILMN_1669773 -1 26057 ANKRD17 ILMN_1712019 -1 163782 ANKRD38 ILMN_1776936 1 10053 AP1M2 ILMN_1705861 1 63941 APBA2BP ILMN_1749738 -1 147495 APCDD1 ILMN_1656951 1 51074 APIP ILMN_1793598 1 367AR ILMN_1767351 1 9828 ARHGEF17 ILMN_1754562 coefficient Entrez_Gene_ID Symbol Probe_Id Stroma.1 1 1634 DCN ILMN_1768227 1 1462 VCAN ILMN_1687301 1 1009 CDH11 ILMN_1672611 1 1281 COL3A1 ILMN_1773079 1 2191 FAP ILMN_1741468 1 5176 SERPINF1 ILMN_1685078 1 2200 FBN1 ILMN_1783182 1 5157 PDGFRL ILMN_1680339 1 1513 CTSK ILMN_1758895 1 5654 HTRA1 ILMN_1676563 1 54829 ASPN ILMN_1801583 1 6678 SPARC ILMN_1796734 1 1290 COL5A2 ILMN_1729117 1 4016 LOXL1 ILMN_1734950 1 4313 MMP2 ILMN_1762106 1 10418 SPON1 ILMN_1791890 1 6424 SFRP4 ILMN_1810172 1 9358 ITGBL1 ILMN_1653719 1 800CALD1 ILMN_1717990 1 51226 COPZ2 ILMN_1667361 1 4237 MFAP2 ILMN_1787981 1 23452 ANGPTL2 ILMN_1772612 1 5328 PLAU ILMN_1656057 1 1278 COL1A2 ILMN_1785272 1 10234 LRRC17 ILMN_1652826 1 114899 C1QTNF3 ILMN_1768925 1 6591 SNAI2 ILMN_1655740 1 5118 PCOLCE ILMN_1707070 1 10631 POSTN ILMN_1790761 1 1842 ECM2 ILMN_1746763 1 2192 FBLN1 ILMN 1700541 Farmer et al. Stroma.1 1 5159 PDGFRB ILMN_1815057 1 2619 GAS1 ILMN_1772910 1 1293 COL6A3 ILMN_1706643 1 5919 RARRES2 ILMN_1811873 1 1291 COL6A1 ILMN_1732151 1 715C1R ILMN_1677198 1 4692 NDN ILMN_1730845 1 7043 TGFB3 ILMN_1687652 1 4035 LRP1 ILMN_1669772 1 1300 COL10A1 ILMN_1672776 1 1809 DPYSL3 ILMN_1679262 1 25903 OLFML2B ILMN_1765557 1 4323 MMP14 ILMN_1774739 1 51339 DACT1 ILMN_1798984 1 54587 MXRA8 ILMN_1809868 1 7058 THBS2 ILMN_1678842 Desmedt et al. coefficient Entrez_Gene_ID Symbol Probe_Id CASP3 1 836CASP3 ILMN_1756787 1 10393 ANAPC10 ILMN_1770378 1 7738 ZNF184 ILMN_1678431 -1 3728 JUP ILMN_1733811 -1 8237 USP11 ILMN_1664537 -1 402ARL2 ILMN_1787879 1 25978 CHMP2B ILMN_1683698 -1 6301 SARS ILMN_1706949 -1 55361 PI4K2A ILMN_1750549 -1 5977 DPF2 ILMN_1734317 Bild et al. coefficient Entrez_Gene_ID Symbol Probe_Id RAS -1 19ABCA1 ILMN_1766054 1 101ADAM8 ILMN_1708348 -1 11096 ADAMTS5 ILMN_1671747 1 154ADRB2 ILMN_1695590 1 51129 ANGPTL4 ILMN_1707727 1 384ARG2 ILMN_1800898 -1 23229 ARHGEF9 ILMN_1690634 1 56938 ARNTL2 ILMN_1798064 1 490ATP2B1 ILMN_1813399 1 84002 B3GNT5 ILMN_1702609 1 11332 ACOT7 ILMN_1740265 -1 604BCL6 ILMN_1737314 1 7851 MALL ILMN_1754766 1 650BMP2 ILMN_1722718 1 55612 C20orf42 ILMN_1696585 1 135398 C6orf141 ILMN_1761762 1 10221 TRIB1 ILMN_1767754 1 805CALM2 ILMN 1687858 Bild et al. Farmer et al. Stroma.1 RAS 813CALU ILMN_1727194 835CASP2 ILMN_1736568 6364 CCL20 ILMN_1657234 8900 CCNA1 ILMN_1726415 64866 CDCP1 ILMN_1724941 23529 CLCF1 ILMN_1661197 85301 COL27A1 ILMN_1788377 1316 KLF6 ILMN_1702995 1453 CSNK1D ILMN_1737198 1454 CSNK1E ILMN_1724363 84951 TNS4 ILMN_1716370 2919 CXCL1 ILMN_1787897 2920 CXCL2 ILMN_1682636 2921 CXCL3 ILMN_1709350 1545 CYP1B1 ILMN_1693338 1594 CYP27B1 ILMN_1740418 120227 CYP2R1 ILMN_1762498 1604 CD55 ILMN_1800540 10521 DDX17 ILMN_1724114 8061 FOSL1 ILMN_1771841 54441 STAG3L1 ILMN_1667315 28514 DLL1 ILMN_1743373 1743 DLST ILMN_1773228 1839 HBEGF ILMN_1756777 1843 DUSP1 ILMN_1781285 1846 DUSP4 ILMN_1734387 1847 DUSP5 ILMN_1656501 1848 DUSP6 ILMN_1677466 9170 EDG4 ILMN_1662741 1947 EFNB1 ILMN_1654563 1958 EGR1 ILMN_1762899 10938 EHD1 ILMN_1651832 1969 EPHA2 ILMN_1700527 9592 IER2 ILMN_1700584 26268 FBXO9 ILMN_1702928 2263 FGFR2 ILMN_1682270 55700 RPRC1 ILMN_1733348 65979 PHACTR4 ILMN_1736548 79977 GRHL2 ILMN_1748424 79993 ELOVL7 ILMN_1740213 201799 TMEM154 ILMN_1683494 121227 LRIG3 ILMN_1699808 1 2353 FOS ILMN_1669523 1 94234 FOXQ1 ILMN_1669046 1 50486 G0S2 ILMN_1691846 -1 2619 GAS1 ILMN_1772910 1 2707 GJB3 ILMN_1652390 1 2709 GJB5 ILMN_1735365 1 2710 GK ILMN_1725471 -1 113263 GLCCI1 ILMN_1781431 1 51228 GLTP ILMN_1768094 1 54676 GTPBP2 ILMN_1694475 1 9982 FGFBP1 ILMN_1785404 -1 3008 HIST1H1E ILMN_1746435 1 3099 HK2 ILMN_1723486 -1 9324 HMGN3 ILMN_1731984 -1 3223 HOXC6 ILMN_1794492 1 10855 HPSE ILMN_1779547 1 3265 HRAS ILMN_1773751 1 8870 IER3 ILMN_1682717 1 3589 IL11 ILMN_1788107 1 3598 IL13RA2 ILMN_1688722 1 3553 IL1B ILMN_1775501 1 3576 IL8 ILMN_1666733 1 3628 INPP1 ILMN_1667239 1 50640 PNPLA8 ILMN_1680223 -1 153572 IRX2 ILMN_1782412 1 3673 ITGA2 ILMN_1665792 1 3710 ITPR3 ILMN_1815500 1 3775 KCNK1 ILMN_1718863 1 3783 KCNN4 ILMN_1726320 1 9938 ARHGAP25 ILMN_1777998 -1 9847 KIAA0528 ILMN_1682572 -1 57507 ZNF608 ILMN_1712798 1 85450 ITPRIP ILMN_1805192 1 688KLF5 ILMN_1770293 1 26092 TOR1AIP1 ILMN_1729318 1 3949 LDLR ILMN_1651611 -1 3964 LGALS8 ILMN_1664402 1 10184 LHFPL2 ILMN_1811077 1 3976 LIF ILMN_1780599 1 119548 PNLIPRP3 ILMN_1678655 1 120224 TMEM45B ILMN_1771120 1 129642 OACT2 ILMN_1777853 4084 MXD1 ILMN_1701957 64332 NFKBIZ ILMN_1719695 81631 MAP1LC3B ILMN_1703244 4170 MCL1 ILMN_1710028 4237 MFAP2 ILMN_1787981 84803 AGPAT9 ILMN_1794875 10793 ZNF273 ILMN_1775073 84985 FAM83A ILMN_1796479 115572 FAM46B ILMN_1808011 78996 C7orf49 ILMN_1740903 58508 MLL3 ILMN_1736165 4323 MMP14 ILMN_1774739 57509 MTUS1 ILMN_1663648 4615 MYD88 ILMN_1738523 89795 NAV3 ILMN_1714600 10397 NDRG1 ILMN_1809931 4781 NFIB ILMN_1778991 9221 NOLC1 ILMN_1800224 4907 NT5E ILMN_1697220 4953 ODC1 ILMN_1748591 9943 OXSR1 ILMN_1811489 10135 PBEF1 ILMN_1653871 22822 PHLDA1 ILMN_1687978 7262 PHLDA2 ILMN_1671557 5266 PI3 ILMN_1693192 8554 PIAS1 ILMN_1780598 5293 PIK3CD ILMN_1766275 5292 PIM1 ILMN_1809933 9518 GDF15 ILMN_1763658 5329 PLAUR ILMN_1691508 5362 PLXNA2 ILMN_1680617 5383 PMS2L5 ILMN_1759901 10687 PNMA2 ILMN_1790778 5473 PPBP ILMN_1767281 10105 PPIF ILMN_1809607 23645 PPP1R15A ILMN_1659936 5621 PRNP ILMN_1737988 5743 PTGS2 ILMN_1677511 5744 PTHLH ILMN_1785699 5791 PTPRE ILMN_1734543 5806 PTX3 ILMN_1692719 5817 PVR ILMN_1677305 6385 SDC4 ILMN_1666181 10509 SEMA4B ILMN_1672589 54910 SEMA4C ILMN_1728432 1992 SERPINB1 ILMN_1679133 5055 SERPINB2 ILMN_1752993 5268 SERPINB5 ILMN_1793888 83667 SESN2 ILMN_1751598 2810 SFN ILMN_1806607 6502 SKP2 ILMN_1665538 9123 SLC16A3 ILMN_1808982 6574 SLC20A1 ILMN_1672662 6515 SLC2A3 ILMN_1775708 6548 SLC9A1 ILMN_1800425 6525 SMTN ILMN_1785618 8651 SOCS1 ILMN_1774733 81848 SPRY4 ILMN_1741168 23524 SRRM2 ILMN_1764460 6764 ST5 ILMN_1773470 6804 STX1A ILMN_1791371 6926 TBX3 ILMN_1713449 7980 TFPI2 ILMN_1667630 7039 TGFA ILMN_1805175 7042 TGFB2 ILMN_1812526 7076 TIMP1 ILMN_1711566 8797 TNFRSF10A ILMN_1721316 8795 TNFRSF10B ILMN_1699265 51330 TNFRSF12A ILMN_1689004 27327 TNRC6A ILMN_1714622 7150 TOP1 ILMN_1735572 10346 TRIM22 ILMN_1779252 8848 TSC22D1 ILMN_1692177 123016 TTC8 ILMN_1652309 7277 TUBA4A ILMN_1784300 6675 UAP1 ILMN_1742461 7378 UPP1 ILMN_1798256 57216 VANGL2 ILMN_1715647 7422 VEGFA ILMN_1803882 81857 MED25 ILMN_1811823 79413 ZBED2 ILMN_1688392 7538 ZFP36 ILMN_1720829 7745 ZNF192 ILMN_1811755 84879 MFSD2 ILMN_1798284 G 1303 COL12A1 ILMN_1733756 2043 EPHA4 ILMN_1672022 _Gene_ID Symbol Probe_Id 22974 TPX2 ILMN_1792494 9055 PRC1 ILMN_1728934 2305 FOXM1 ILMN_1716400 983CDC2 ILMN_1747911 60436 TGIF2 ILMN_1709044 55969 C20orf24 ILMN_1679195 4171 MCM2 ILMN_1681503 3015 H2AFZ ILMN_1707858 7153 TOP2A ILMN_1686097 5111 PCNA ILMN_1694177 11065 UBE2C ILMN_1714730 9833 MELK ILMN_1731184 9319 TRIP13 ILMN_1796589 9918 NCAPD2 ILMN_1775008 4176 MCM7 ILMN_1663195 10535 RNASEH2A ILMN_1810901 10635 RAD51AP1 ILMN_1670353 10112 KIF20A ILMN_1695658 8318 CDC45L ILMN_1808347 4085 MAD2L1 ILMN_1777564 9700 ESPL1 ILMN_1742145 9133 CCNB2 ILMN_1801939 2237 FEN1 ILMN_1755834 7272 TTK ILMN_1788166 22948 CCT5 ILMN_1706246 5984 RFC4 ILMN_1724489 29028 ATAD2 ILMN_1763064 9793 CKAP5 ILMN_1748770 23165 NUP205 ILMN_1673962 991CDC20 ILMN_1663390 1164 CKS2 ILMN_1756326 6241 RRM2 ILMN_1678669 1994 ELAVL1 ILMN_1685816 891CCNB1 ILMN_1712803 6240 RRM1 ILMN_1771593 9212 AURKB ILMN_1684217 2956 MSH6 ILMN_1729051 2146 EZH2 ILMN_1708105 1503 CTPS ILMN_1783285 1736 DKC1 ILMN_1671257 11339 OIP5 ILMN_1759277 55143 CDCA8 ILMN_1709294 9232 PTTG1 ILMN 1753196 Carter et al. Farmer et al. Stroma.1 CIN70 Carter et al. CIN70 1 55165 CEP55 ILMN_1747016 1 3014 H2AFX ILMN_1785756 1 55907 CMAS ILMN_1714082 1 23397 BRRN1 ILMN_1657600 1 55388 MCM10 ILMN_1706194 1 25804 LSM4 ILMN_1788099 1 55723 ASF1B ILMN_1695414 1 11130 ZWINT ILMN_1778034 1 55872 PBK ILMN_1673673 1 55055 ZWILCH ILMN_1700378 1 83461 CDCA3 ILMN_1737728 1 1894 ECT2 ILMN_1719307 1 990CDC6 ILMN_1785914 1 7374 UNG ILMN_1683120 1 23788 MTCH2 ILMN_1785218 1 5885 RAD21 ILMN_1748578 1 86ACTL6A ILMN_1749297 1 2821 GPI ILMN_1654465 1 84306 PDCD2L ILMN_1706149 1 6427 SFRS2 ILMN_1696407 1 3068 HDGF ILMN_1765621 1 29107 NXT1 ILMN_1760280 1 4751 NEK2 ILMN_1653822 1 1717 DHCR7 ILMN_1654028 1 6790 AURKA ILMN_1680955 1 4706 NDUFAB1 ILMN_1755521 1 23306 TMEM194 ILMN_1725880 1 24137 KIF4A ILMN_1799667 Teschendorff et al. coefficient Entrez_Gene_ID Symbol Probe_Id Immune.1 1 608TNFRSF17 ILMN_1768016 1 712C1QA ILMN_1657181 1 3134 HLA-F ILMN_1762861 1 4063 LY9 ILMN_1725682 -1 6696 SPP1 ILMN_1651354 1 6846 XCL2 ILMN_1666257 Desmedt et al. Farmer et al. Stroma.1 coefficient Entrez_Gene_ID Symbol Probe_Id AURKA 1 6790 AURKA ILMN_1680955 1 11065 UBE2C ILMN_1714730 1 9133 CCNB2 ILMN_1801939 1 1058 CENPA ILMN_1801257 1 332BIRC5 ILMN_1710082 1 11004 KIF2C ILMN_1685916 1 10112 KIF20A ILMN_1695658 1 991CDC20 ILMN_1663390 1 2305 FOXM1 ILMN_1716400 1 891CCNB1 ILMN_1712803 22974 TPX2 ILMN_1792494 9088 PKMYT1 ILMN_1766658 54478 FAM64A ILMN_1728972 4751 NEK2 ILMN_1653822 24137 KIF4A ILMN_1799667 23397 BRRN1 ILMN_1657600 9319 TRIP13 ILMN_1796589 4085 MAD2L1 ILMN_1777564 9156 EXO1 ILMN_1673721 10615 SPAG5 ILMN_1768291 7083 TK1 ILMN_1806037 6491 STIL ILMN_1807232 6241 RRM2 ILMN_1678669 55839 CENPN ILMN_1720526 7298 TYMS ILMN_1806040 641BLM ILMN_1709484 4171 MCM2 ILMN_1681503 1164 CKS2 ILMN_1756326 79682 MLF1IP ILMN_1679438 10129 FRY ILMN_1678437 51659 GINS2 ILMN_1809590 10212 DDX39 ILMN_1747303 3925 STMN1 ILMN_1657796 79801 SHCBP1 ILMN_1709162 3014 H2AFX ILMN_1785756 10535 RNASEH2A ILMN_1810901 5984 RFC4 ILMN_1724489 55970 GNG12 ILMN_1673380 1033 CDKN3 ILMN_1666305 55388 MCM10 ILMN_1706194 55257 C20orf20 ILMN_1790136 1163 CKS1B ILMN_1719256 8914 TIMELESS ILMN_1735093 54821 ERCC6L ILMN_1790992 23371 TENC1 ILMN_1685042 8544 PIR ILMN_1761247 8317 CDC7 ILMN_1741801 2331 FMOD ILMN_1789639 51512 GTSE1 ILMN_1771039 6424 SFRP4 ILMN_1810172 55353 LAPTM4B ILMN_1680196 8404 SPARCL1 ILMN_1795251 990CDC6 ILMN_1785914 7043 TGFB3 ILMN 1687652 -1 79838 TMC5 ILMN_1730117 1 84823 LMNB2 ILMN_1708101 -1 83989 C5orf21 ILMN_1654542 -1 1793 DOCK1 ILMN_1715789 -1 9358 ITGBL1 ILMN_1653719 1 8836 GGH ILMN_1681754 -1 57088 PLSCR4 ILMN_1757338 -1 6642 SNX1 ILMN_1693998 -1 4969 OGN ILMN_1790098 -1 90627 STARD13 ILMN_1660451 1 11260 XPOT ILMN_1743711 1 22827 PUF60 ILMN_1779404 1 9793 CKAP5 ILMN_1748770 -1 2791 GNG11 ILMN_1782419 1 55247 NEIL3 ILMN_1757697 -1 10234 LRRC17 ILMN_1652826 -1 9353 SLIT2 ILMN_1676449 1 1841 DTYMK ILMN_1716445 1 9631 NUP155 ILMN_1768293 1 5424 POLD1 ILMN_1652580 1 6631 SNRPC ILMN_1741997 -1 10186 LHFP ILMN_1696627 1 4521 NUDT1 ILMN_1735692 -1 3479 IGF1 ILMN_1709613 1 4172 MCM3 ILMN_1806818 -1 2205 FCER1A ILMN_1710386 1 55732 C1orf112 ILMN_1727540 -1 9077 DIRAS3 ILMN_1688877 1 5557 PRIM1 ILMN_1694502 1 54963 UCKL1 ILMN_1800463 1 54512 EXOSC4 ILMN_1745271 -1 79901 CYBRD1 ILMN_1712305 -1 10161 P2RY5 ILMN_1786429 1 29097 CNIH4 ILMN_1714759 1 6513 SLC2A1 ILMN_1809256 1 51123 ZNF706 ILMN_1702384 -1 857CAV1 ILMN_1687583 1 51110 LACTB2 ILMN_1660635 1 51204 CCDC44 ILMN_1685112 1 54845 RBM35A ILMN_1749180 -1 283ANG ILMN_1760727 -1 79652 C16orf30 ILMN_1751559 OLFML ILMN 79838 TMC5 ILMN_1730117 84823 LMNB2 ILMN_1708101 83989 C5orf21 ILMN_1654542 1793 DOCK1 ILMN_1715789 9358 ITGBL1 ILMN_1653719 8836 GGH ILMN_1681754 57088 PLSCR4 ILMN_1757338 6642 SNX1 ILMN_1693998 4969 OGN ILMN_1790098 90627 STARD13 ILMN_1660451 11260 XPOT ILMN_1743711 22827 PUF60 ILMN_1779404 9793 CKAP5 ILMN_1748770 2791 GNG11 ILMN_1782419 55247 NEIL3 ILMN_1757697 10234 LRRC17 ILMN_1652826 9353 SLIT2 ILMN_1676449 1841 DTYMK ILMN_1716445 9631 NUP155 ILMN_1768293 5424 POLD1 ILMN_1652580 6631 SNRPC ILMN_1741997 10186 LHFP ILMN_1696627 4521 NUDT1 ILMN_1735692 3479 IGF1 ILMN_1709613 4172 MCM3 ILMN_1806818 2205 FCER1A ILMN_1710386 55732 C1orf112 ILMN_1727540 9077 DIRAS3 ILMN_1688877 5557 PRIM1 ILMN_1694502 54963 UCKL1 ILMN_1800463 54512 EXOSC4 ILMN_1745271 79901 CYBRD1 ILMN_1712305 10161 P2RY5 ILMN_1786429 29097 CNIH4 ILMN_1714759 6513 SLC2A1 ILMN_1809256 51123 ZNF706 ILMN_1702384 857CAV1 ILMN_1687583 51110 LACTB2 ILMN_1660635 51204 CCDC44 ILMN_1685112 54845 RBM35A ILMN_1749180 283ANG ILMN_1760727 79652 C16orf30 ILMN_1751559 56944 OLFML3 ILMN_1727532 3297 HSF1 ILMN_1665621 27235 COQ2 ILMN_1756572 2487 FRZB ILMN_1716246 1 3251 HPRT1 ILMN_1736940 1 5119 PCOLN3 ILMN_1709439 1 6839 SUV39H1 ILMN_1781479 -1 27303 RBMS3 ILMN_1665040 -1 10468 FST ILMN_1700081 -1 26289 AK5 ILMN_1690676 1 55038 CDCA4 ILMN_1684045 1 7283 TUBG1 ILMN_1695731 1 23212 RRS1 ILMN_1767658 1 65094 JMJD4 ILMN_1692896 1 55379 LRRC59 ILMN_1786259 -1 10956 OS9 ILMN_1674254 1 51022 GLRX2 ILMN_1680727 1 54915 YTHDF1 ILMN_1753885 -1 54861 SNRK ILMN_1794399 1 79000 C1orf135 ILMN_1787280 -1 79776 ZFHX4 ILMN_1657606 -1 79971 GPR177 ILMN_1671260 1 7718 ZNF165 ILMN_1806502 1 201254 STRA13 ILMN_1769634 -1 1848 DUSP6 ILMN_1677466 -1 9037 SEMA5A ILMN_1656927 1 5433 POLR2D ILMN_1792672 -1 29087 THYN1 ILMN_1781408 -1 79864 C11orf63 ILMN_1680659 -1 358AQP1 ILMN_1815081 1 6634 SNRPD3 ILMN_1794599 -1 2621 GAS6 ILMN_1779558 1 56270 WDR45L ILMN_1719140 -1 5187 PER1 ILMN_1653125 -1 2098 ESD ILMN_1720285 1 81887 LAS1L ILMN_1774890 -1 1811 SLC26A3 ILMN_1760087 1 54535 CCHCR1 ILMN_1674051 1 55526 DHTKD1 ILMN_1664369 -1 57161 PELI2 ILMN_1780132 -1 2353 FOS ILMN_1669523 -1 51279 C1RL ILMN_1733288 1 60436 TGIF2 ILMN_1709044 1 3028 HADH2 ILMN_1758275 1 26519 TIMM10 ILMN_1765332 -1 25960 GPR124 ILMN_1773059 -1 10252 SPRY1 ILMN_1651610 1 6199 RPS6KB2 ILMN_1761175 1 55630 SLC39A4 ILMN_1706386 -1 7049 TGFBR3 ILMN 1784287 1 8607 RUVBL1 ILMN_1693108 -1 2581 GALC ILMN_1799744 -1 862RUNX1T1 ILMN_1812795 1 8458 TTF2 ILMN_1810228 1 9775 EIF4A3 ILMN_1667043 1 3181 HNRPA2B1 ILMN_1796491 1 26039 SS18L1 ILMN_1676625 -1 10580 SORBS1 ILMN_1749792 -1 7056 THBD ILMN_1759787 -1 8322 FZD4 ILMN_1743367 -1 1003 CDH5 ILMN_1719236 -1 2152 F3 ILMN_1797009 -1 55068 ENOX1 ILMN_1713471 1 64785 GINS3 ILMN_1754272 1 79042 TSEN34 ILMN_1673111 1 8805 TRIM24 ILMN_1798032 1 1478 CSTF2 ILMN_1653129 -1 57125 PLXDC1 ILMN_1652878 -1 22998 LIMCH1 ILMN_1664138 1 79915 C17orf41 ILMN_1686835 -1 7026 NR2F2 ILMN_1745785 -1 7474 WNT5A ILMN_1800317 -1 55857 C20orf19 ILMN_1779536 -1 114625 ERMAP ILMN_1715013 -1 8857 FCGBP ILMN_1718984 -1 26872 STEAP1 ILMN_1663575 1 29844 TFPT ILMN_1709451 1 4719 NDUFS1 ILMN_1728810 -1 4013 VWA5A ILMN_1764769 1 3396 ICT1 ILMN_1734508 -1 397ARHGDIB ILMN_1678143 1 10436 EMG1 ILMN_1797074 1 51582 AZIN1 ILMN_1656682 1 10598 AHSA1 ILMN_1703617 1 333APLP1 ILMN_1763834 1 51142 CHCHD2 ILMN_1815124 -1 27123 DKK2 ILMN_1695506 -1 55020 FLJ20699 ILMN_1692464 -1 23460 ABCA6 ILMN_1701011 -1 64321 SOX17 ILMN_1671092 -1 7098 TLR3 ILMN_1689578 1 6338 SCNN1B ILMN_1740917 1 3692 EIF6 ILMN 1787410 -1 79618 HMBOX1 ILMN_1720059 1 8772 FADD ILMN_1758658 1 9986 RCE1 ILMN_1685002 1 58500 ZNF250 ILMN_1757230 -1 11081 KERA ILMN_1732678 1 7064 THOP1 ILMN_1726659 -1 55799 CACNA2D3 ILMN_1754076 -1 49855 SCAPER ILMN_1811178 1 54606 DDX56 ILMN_1679405 1 7164 TPD52L1 ILMN_1714383 1 80775 TMEM177 ILMN_1756402 -1 667DST ILMN_1703913 1 2781 GNAZ ILMN_1766182 1 23464 GCAT ILMN_1724437 1 79763 ISOC2 ILMN_1769158 -1 4649 MYO9A ILMN_1690614 1 53820 DSCR6 ILMN_1709257 1 3638 INSIG1 ILMN_1793474 1 11171 STRAP ILMN_1731194 1 10992 SF3B2 ILMN_1737807 1 6832 SUPV3L1 ILMN_1765857 1 55922 NKRF ILMN_1707534 1 10557 RPP38 ILMN_1680386 -1 3216 HOXB6 ILMN_1768101 -1 54785 C17orf59 ILMN_1789643 -1 1933 EEF1B2 ILMN_1705714 1 8161 COIL ILMN_1688034 -1 594BCKDHB ILMN_1764166 1 6286 S100P ILMN_1801216 1 3954 LETM1 ILMN_1710668 1 51087 YBX2 ILMN_1755354 1 10953 TOMM34 ILMN_1721128 Creighton et al. Farmer et al. Stroma.1 coefficient Entrez_Gene_ID Symbol Probe_Id obesity 1 51ACOX1 ILMN_1750158 1 345APOC3 ILMN_1722070 1 1016 CDH18 ILMN_1733669 1 1415 CRYBB2 ILMN_1760708 1 1577 CYP3A5 ILMN_1810942 1 1815 DRD4 ILMN_1773348 1 2147 F2 ILMN_1671753 1 2192 FBLN1 ILMN_1700541 1 2918 GRM8 ILMN_1729212 1 3268 HRBL ILMN_1703228 1 3483 IGFALS ILMN_1673488 1 3769 KCNJ13 ILMN_1709847 1 4352 MPL ILMN 1776640 4916 NTRK3 ILMN_1687967 5224 PGAM2 ILMN_1666889 5337 PLD1 ILMN_1719696 5723 PSPH ILMN_1776105 6362 CCL18 ILMN_1654411 6545 SLC7A4 ILMN_1774229 6645 SNTB2 ILMN_1786766 6654 SOS1 ILMN_1767135 6693 SPN ILMN_1801040 6716 SRD5A2 ILMN_1788895 6902 TBCA ILMN_1726239 7104 TM4SF4 ILMN_1792404 7184 HSP90B1 ILMN_1686328 8876 VNN1 ILMN_1712365 8968 HIST1H3F ILMN_1788489 9509 ADAMTS2 ILMN_1796628 9776 KIAA0652 ILMN_1692306 9948 WDR1 ILMN_1675844 10435 CDC42EP2 ILMN_1652777 10538 BATF ILMN_1668822 10741 RBBP9 ILMN_1786050 11009 IL24 ILMN_1774685 11255 HRH3 ILMN_1694273 11338 U2AF2 ILMN_1768930 25788 RAD54B ILMN_1722127 26150 RIBC2 ILMN_1680349 27240 SIT1 ILMN_1788531 27309 ZNF330 ILMN_1710873 29122 TSP50 ILMN_1775615 51512 GTSE1 ILMN_1771039 55658 RNF126 ILMN_1669572 55764 IFT122 ILMN_1742379 63925 ZNF335 ILMN_1758984 64097 EPB41L4A ILMN_1775376 65080 MRPL44 ILMN_1671452 84168 ANTXR1 ILMN_1670379 90634 CG018 ILMN_1799487 90861 HN1L ILMN_1706118 94081 SFXN1 ILMN_1713680 201501 APM-1 ILMN_1796737 25ABL1 ILMN_1713732 148ADRA1A ILMN_1799019 -1 398ARHGDIG ILMN_1711278 -1 488ATP2A2 ILMN_1655884 -1 582BBS1 ILMN_1800590 -1 659BMPR2 ILMN_1789095 -1 687KLF9 ILMN_1778523 -1 901CCNG2 ILMN_1747244 -1 905CCNT2 ILMN_1722522 -1 1102 RCBTB2 ILMN_1771695 -1 1107 CHD3 ILMN_1740764 -1 1195 CLK1 ILMN_1679727 -1 1203 CLN5 ILMN_1778203 -1 1385 CREB1 ILMN_1700138 -1 1386 ATF2 ILMN_1748271 -1 1387 CREBBP ILMN_1809583 -1 1407 CRY1 ILMN_1760593 -1 1488 CTBP2 ILMN_1691294 -1 1495 CTNNA1 ILMN_1804854 -1 1523 CUTL1 ILMN_1687567 -1 1655 DDX5 ILMN_1805344 -1 1678 TIMM8A ILMN_1722239 -1 1741 DLG3 ILMN_1811515 -1 1778 DYNC1H1 ILMN_1808448 -1 1781 DYNC1I2 ILMN_1773847 -1 1831 TSC22D3 ILMN_1748124 -1 1846 DUSP4 ILMN_1734387 -1 1848 DUSP6 ILMN_1677466 -1 1859 DYRK1A ILMN_1807735 -1 1948 EFNB2 ILMN_1703852 -1 1964 EIF1AX ILMN_1808344 -1 1998 ELF2 ILMN_1691559 -1 2009 EML1 ILMN_1787972 -1 2071 ERCC3 ILMN_1740216 -1 2145 EZH1 ILMN_1791436 -1 2181 ACSL3 ILMN_1666096 -1 2186 FALZ ILMN_1692021 -1 2188 FANCF ILMN_1682724 -1 2264 FGFR4 ILMN_1730355 -1 2309 FOXO3A ILMN_1681703 -1 2487 FRZB ILMN_1716246 -1 2581 GALC ILMN_1799744 -1 2737 GLI3 ILMN_1685871 -1 2776 GNAQ ILMN_1741482 -1 2800 GOLGA1 ILMN_1806301 -1 2820 GPD2 ILMN_1723139 -1 2887 GRB10 ILMN_1669617 -1 3021 H3F3B ILMN 1695706 -1 3030 HADHA ILMN_1712751 -1 3099 HK2 ILMN_1723486 -1 3184 HNRNPD ILMN_1751368 -1 3382 ICA1 ILMN_1694399 -1 3398 ID2 ILMN_1793990 -1 3431 SP110 ILMN_1731418 -1 3551 IKBKB ILMN_1727142 -1 3572 IL6ST ILMN_1797861 -1 3621 ING1 ILMN_1808863 -1 3632 INPP5A ILMN_1735224 -1 3708 ITPR1 ILMN_1789505 -1 3720 JARID2 ILMN_1764177 -1 4026 LPP ILMN_1651254 -1 4087 SMAD2 ILMN_1771203 -1 4130 MAP1A ILMN_1772366 -1 4149 MAX ILMN_1706546 -1 4179 CD46 ILMN_1815689 -1 4205 MEF2A ILMN_1661888 -1 4211 MEIS1 ILMN_1705685 -1 4351 MPI ILMN_1761262 -1 4608 MYBPH ILMN_1705297 -1 4735 Sep-02ILMN_1740252 -1 4739 NEDD9 ILMN_1758719 -1 4750 NEK1 ILMN_1699459 -1 4752 NEK3 ILMN_1695020 -1 4780 NFE2L2 ILMN_1790909 -1 4781 NFIB ILMN_1778991 -1 4801 NFYB ILMN_1750005 -1 4820 NKTR ILMN_1721621 -1 4850 CNOT4 ILMN_1772677 -1 4854 NOTCH3 ILMN_1755832 -1 4928 NUP98 ILMN_1732776 -1 4929 NR4A2 ILMN_1782305 -1 4999 ORC2L ILMN_1701440 -1 5048 PAFAH1B1 ILMN_1722276 -1 5062 PAK2 ILMN_1676385 -1 5094 PCBP2 ILMN_1724825 -1 5108 PCM1 ILMN_1690487 -1 5204 PFDN5 ILMN_1755536 -1 5213 PFKM ILMN_1708180 -1 5253 PHF2 ILMN_1720476 -1 5289 PIK3C3 ILMN_1687896 -1 5350 PLN ILMN_1785523 -1 5411 PNN ILMN 1721703 _ 3099 HK2 ILMN_1723486 3184 HNRNPD ILMN_1751368 3382 ICA1 ILMN_1694399 3398 ID2 ILMN_1793990 3431 SP110 ILMN_1731418 3551 IKBKB ILMN_1727142 3572 IL6ST ILMN_1797861 3621 ING1 ILMN_1808863 3632 INPP5A ILMN_1735224 3708 ITPR1 ILMN_1789505 3720 JARID2 ILMN_1764177 4026 LPP ILMN_1651254 4087 SMAD2 ILMN_1771203 4130 MAP1A ILMN_1772366 4149 MAX ILMN_1706546 4179 CD46 ILMN_1815689 4205 MEF2A ILMN_1661888 4211 MEIS1 ILMN_1705685 4351 MPI ILMN_1761262 4608 MYBPH ILMN_1705297 4735 Sep-02ILMN_1740252 4739 NEDD9 ILMN_1758719 4750 NEK1 ILMN_1699459 4752 NEK3 ILMN_1695020 4780 NFE2L2 ILMN_1790909 4781 NFIB ILMN_1778991 4801 NFYB ILMN_1750005 4820 NKTR ILMN_1721621 4850 CNOT4 ILMN_1772677 4854 NOTCH3 ILMN_1755832 4928 NUP98 ILMN_1732776 4929 NR4A2 ILMN_1782305 4999 ORC2L ILMN_1701440 5048 PAFAH1B1 ILMN_1722276 5062 PAK2 ILMN_1676385 5094 PCBP2 ILMN_1724825 5108 PCM1 ILMN_1690487 5204 PFDN5 ILMN_1755536 5213 PFKM ILMN_1708180 5253 PHF2 ILMN_1720476 5289 PIK3C3 ILMN_1687896 5350 PLN ILMN_1785523 5411 PNN ILMN_1721703 5420 PODXL ILMN_1711311 5495 PPM1B ILMN 1734991 -1 5500 PPP1CB ILMN_1736942 -1 5520 PPP2R2A ILMN_1788961 -1 5525 PPP2R5A ILMN_1738784 -1 5527 PPP2R5C ILMN_1789283 -1 5529 PPP2R5E ILMN_1666761 -1 5573 PRKAR1A ILMN_1738632 -1 5635 PRPSAP1 ILMN_1768449 -1 5664 PSEN2 ILMN_1714417 -1 5683 PSMA2 ILMN_1682553 -1 5863 RGL2 ILMN_1759159 -1 5903 RANBP2 ILMN_1654866 -1 5921 RASA1 ILMN_1725312 -1 5925 RB1 ILMN_1696591 -1 5930 RBBP6 ILMN_1665135 -1 5935 RBM3 ILMN_1698213 -1 5936 RBM4 ILMN_1709042 -1 6171 RPL41 ILMN_1710001 -1 6314 ATXN7 ILMN_1688602 -1 6397 SEC14L1 ILMN_1732575 -1 6416 MAP2K4 ILMN_1739534 -1 6418 SET ILMN_1742238 -1 6421 SFPQ ILMN_1769931 -1 6430 SFRS5 ILMN_1761996 -1 6431 SFRS6 ILMN_1697469 -1 6432 SFRS7 ILMN_1778836 -1 6595 SMARCA2 ILMN_1791702 -1 6604 SMARCD3 ILMN_1792521 -1 6642 SNX1 ILMN_1693998 -1 6651 SON ILMN_1703427 -1 6668 SP2 ILMN_1796208 -1 6738 TROVE2 ILMN_1738909 -1 6777 STAT5B ILMN_1777783 -1 6812 STXBP1 ILMN_1728747 -1 6907 TBL1X ILMN_1744795 -1 6938 TCF12 ILMN_1669832 -1 7025 NR2F1 ILMN_1786197 -1 7072 TIA1 ILMN_1712634 -1 7109 TMEM1 ILMN_1778464 -1 7158 TP53BP1 ILMN_1739234 -1 7187 TRAF3 ILMN_1793154 -1 7204 TRIO ILMN_1657521 -1 7216 TRO ILMN_1691648 1 26 TTC3 ILMN 1 2860 1 7433 VIPR1 ILMN_1707959 -1 7503 XIST ILMN_1764573 -1 7507 XPA ILMN_1787591 -1 7559 ZNF12 ILMN_1784577 -1 7561 ZNF14 ILMN_1692145 -1 7639 ZNF85 ILMN_1747824 -1 7644 ZNF91 ILMN_1802053 -1 7694 ZNF135 ILMN_1726368 -1 7702 ZNF143 ILMN_1674399 -1 7716 VEZF1 ILMN_1705310 -1 7727 ZNF174 ILMN_1719202 -1 7728 ZNF175 ILMN_1675788 -1 7733 ZNF180 ILMN_1696319 -1 7743 ZNF189 ILMN_1806809 -1 7750 ZMYM2 ILMN_1661636 -1 7763 ZFAND5 ILMN_1795228 -1 7767 ZNF224 ILMN_1661293 -1 7771 ZNF228 ILMN_1815885 -1 7779 SLC30A1 ILMN_1745021 -1 7840 ALMS1 ILMN_1709155 -1 7874 USP7 ILMN_1729319 -1 8125 ANP32A ILMN_1803500 -1 8289 ARID1A ILMN_1797341 -1 8451 CUL4A ILMN_1695792 -1 8452 CUL3 ILMN_1653670 -1 8467 SMARCA5 ILMN_1713163 -1 8473 OGT ILMN_1697639 -1 8491 MAP4K3 ILMN_1813120 -1 8500 PPFIA1 ILMN_1800164 -1 8502 PKP4 ILMN_1749410 -1 8527 DGKD ILMN_1765326 -1 8554 PIAS1 ILMN_1780598 -1 8604 SLC25A12 ILMN_1788053 -1 8621 CDC2L5 ILMN_1778557 -1 8648 NCOA1 ILMN_1669033 -1 8658 TNKS ILMN_1657891 -1 8725 C19orf2 ILMN_1798728 -1 8777 MPDZ ILMN_1707649 -1 8805 TRIM24 ILMN_1798032 -1 8863 PER3 ILMN_1732519 -1 8864 PER2 ILMN_1738095 -1 8874 ARHGEF7 ILMN_1796912 -1 8880 FUBP1 ILMN_1776552 -1 8888 MCM3AP ILMN_1784766 -1 8897 MTMR3 ILMN_1739641 -1 8899 PRPF4B ILMN 1760281 -1 8925 HERC1 ILMN_1786211 -1 9140 ATG12 ILMN_1673898 -1 9202 ZMYM4 ILMN_1688404 -1 9276 COPB2 ILMN_1684385 -1 9282 CRSP2 ILMN_1739956 -1 9295 SFRS11 ILMN_1657790 -1 9320 TRIP12 ILMN_1720241 -1 9400 RECQL5 ILMN_1697682 -1 9414 TJP2 ILMN_1664978 -1 9439 MED23 ILMN_1690999 -1 9445 ITM2B ILMN_1713733 -1 9451 EIF2AK3 ILMN_1724984 -1 9491 PSMF1 ILMN_1671696 -1 9583 ENTPD4 ILMN_1806304 -1 9611 NCOR1 ILMN_1700473 -1 9617 MTRF1 ILMN_1654918 -1 9637 FEZ2 ILMN_1739586 -1 9649 RALGPS1 ILMN_1674135 -1 9655 SOCS5 ILMN_1785286 -1 9665 LKAP ILMN_1793371 -1 9666 DZIP3 ILMN_1763200 -1 9716 AQR ILMN_1717154 -1 9724 UTP14C ILMN_1686645 -1 9736 USP34 ILMN_1739454 -1 9759 HDAC4 ILMN_1764396 -1 9816 KIAA0133 ILMN_1696556 -1 9830 TRIM14 ILMN_1713542 -1 9849 ZNF518 ILMN_1742541 -1 9851 KIAA0753 ILMN_1680010 -1 9852 EPM2AIP1 ILMN_1682658 -1 9857 CEP350 ILMN_1742400 -1 9874 TLK1 ILMN_1811029 -1 9878 C14orf92 ILMN_1743131 -1 9879 DDX46 ILMN_1727001 -1 9896 FIG4 ILMN_1800889 -1 9906 SLC35E2 ILMN_1651209 -1 9913 SUPT7L ILMN_1801488 -1 9962 SLC23A2 ILMN_1746578 -1 9969 THRAP1 ILMN_1806184 -1 9984 THOC1 ILMN_1802157 -1 9988 DMTF1 ILMN_1750075 -1 10001 MED6 ILMN_1654543 1 100 9 ATP9A ILMN 1 99232 -1 10137 RBM12 ILMN_1797698 -1 10142 AKAP9 ILMN_1779315 -1 10157 AASS ILMN_1678323 -1 10181 RBM5 ILMN_1786893 -1 10254 STAM2 ILMN_1781135 -1 10280 OPRS1 ILMN_1717925 -1 10284 SAP18 ILMN_1752793 -1 10285 SMNDC1 ILMN_1691789 -1 10299 Mar-06ILMN_1757106 -1 10314 LANCL1 ILMN_1703697 -1 10324 KBTBD10 ILMN_1690866 -1 10336 PCGF3 ILMN_1803454 -1 10363 HMG20A ILMN_1719006 -1 10384 BTN3A3 ILMN_1733338 -1 10401 PIAS3 ILMN_1675497 -1 10427 SEC24B ILMN_1745469 -1 10443 PFAAP5 ILMN_1797893 -1 10447 FAM3C ILMN_1777261 -1 10512 SEMA3C ILMN_1695475 -1 10513 APPBP2 ILMN_1694875 -1 10520 ZNF211 ILMN_1811195 -1 10602 CDC42EP3 ILMN_1736327 -1 10605 PAIP1 ILMN_1731775 -1 10614 HEXIM1 ILMN_1685115 -1 10628 TXNIP ILMN_1697448 -1 10651 MTX2 ILMN_1794046 -1 10664 CTCF ILMN_1688638 -1 10677 AVIL ILMN_1674265 -1 10724 MGEA5 ILMN_1686750 -1 10765 JARID1B ILMN_1755727 -1 10768 AHCYL1 ILMN_1770412 -1 10781 ZNF266 ILMN_1753782 -1 10892 MALT1 ILMN_1730986 -1 10912 GADD45G ILMN_1651498 -1 10915 TCERG1 ILMN_1706839 -1 10920 COPS8 ILMN_1756065 -1 10949 HNRNPA0 ILMN_1753279 -1 10956 OS9 ILMN_1674254 -1 10983 CCNI ILMN_1691942 -1 10992 SF3B2 ILMN_1737807 -1 11120 BTN2A1 ILMN_1792070 -1 11177 BAZ1A ILMN_1742230 -1 11189 TNRC4 ILMN_1681234 -1 11212 PROSC ILMN_1748908 -1 11215 AKAP11 ILMN_1693220 -1 11325 DDX42 ILMN 1695983 -1 22826 DNAJC8 ILMN_1698258 -1 22828 RBM16 ILMN_1681675 -1 22856 CHSY1 ILMN_1791576 -1 22863 KIAA0831 ILMN_1678808 -1 22868 FASTKD2 ILMN_1728094 -1 22880 MORC2 ILMN_1661111 -1 22884 WDR37 ILMN_1796464 -1 22887 FOXJ3 ILMN_1657188 -1 22902 RUFY3 ILMN_1730931 -1 22906 TRAK1 ILMN_1754629 -1 22908 SACM1L ILMN_1765019 -1 22909 MTMR15 ILMN_1778734 -1 22930 RAB3GAP1 ILMN_1739876 -1 22934 RPIA ILMN_1714809 -1 22936 ELL2 ILMN_1655930 -1 22941 SHANK2 ILMN_1814790 -1 22950 SLC4A1AP ILMN_1750876 -1 22981 NLP ILMN_1789094 -1 22982 DIP2C ILMN_1676062 -1 22990 PCNX ILMN_1740010 -1 22992 FBXL11 ILMN_1668414 -1 23001 WDFY3 ILMN_1697493 -1 23014 FBXO21 ILMN_1745887 -1 23036 ZNF292 ILMN_1714412 -1 23063 KIAA0261 ILMN_1778792 -1 23071 TXNDC4 ILMN_1772156 -1 23080 KIAA0241 ILMN_1722292 -1 23091 KIAA0853 ILMN_1761384 -1 23099 ZBTB43 ILMN_1731113 -1 23113 PARC ILMN_1806010 -1 23116 KIAA0423 ILMN_1778876 -1 23122 CLASP2 ILMN_1770548 -1 23126 POGZ ILMN_1652929 -1 23171 GPD1L ILMN_1694106 -1 23174 ZCCHC14 ILMN_1743456 -1 23185 LARP5 ILMN_1766222 -1 23190 UBXD2 ILMN_1781097 -1 23196 FAM120A ILMN_1721089 -1 23198 PSME4 ILMN_1763540 -1 23219 FBXO28 ILMN_1812776 -1 23230 VPS13A ILMN_1800721 -1 23232 TBC1D12 ILMN_1743324 -1 23244 PDS5A ILMN_1701069 -1 23253 ANKRD12 ILMN_1661833 -1 23262 HISPPD1 ILMN_1785520 -1 23270 TSPYL4 ILMN 1715821 _ -1 23294 ANKS1A ILMN_1813669 -1 23300 ATMIN ILMN_1772540 -1 23303 KIF13B ILMN_1686562 -1 23304 UBR2 ILMN_1663489 -1 23326 USP22 ILMN_1666609 -1 23327 NEDD4L ILMN_1733627 -1 23332 CLASP1 ILMN_1700507 -1 23341 DNAJC16 ILMN_1717078 -1 23347 SMCHD1 ILMN_1808148 -1 23348 DOCK9 ILMN_1780732 -1 23358 USP24 ILMN_1684594 -1 23360 FNBP4 ILMN_1750911 -1 23365 ARHGEF12 ILMN_1810712 -1 23369 PUM2 ILMN_1722345 -1 23398 PPWD1 ILMN_1776726 -1 23405 DICER1 ILMN_1772692 -1 23435 TARDBP ILMN_1677532 -1 23451 SF3B1 ILMN_1813603 -1 23462 HEY1 ILMN_1788203 -1 23484 LEPROTL1 ILMN_1752591 -1 23499 MACF1 ILMN_1807808 -1 23509 POFUT1 ILMN_1776076 -1 23522 MYST4 ILMN_1698441 -1 23543 RBM9 ILMN_1745130 -1 23585 TMEM50A ILMN_1745368 -1 23592 LEMD3 ILMN_1727361 -1 23598 PATZ1 ILMN_1759297 -1 23637 RABGAP1 ILMN_1724438 -1 23741 EID1 ILMN_1775269 -1 23774 BRD1 ILMN_1730019 -1 24149 ZNF318 ILMN_1792305 -1 25836 NIPBL ILMN_1759062 -1 25896 INTS7 ILMN_1718354 -1 25924 MYRIP ILMN_1668125 -1 25949 SYF2 ILMN_1660186 -1 25957 C6orf111 ILMN_1742099 -1 26018 LRIG1 ILMN_1707342 -1 26035 GLCE ILMN_1714349 -1 26040 SETBP1 ILMN_1720513 -1 26054 SENP6 ILMN_1761731 -1 26058 TNRC15 ILMN_1750718 -1 26190 FBXW2 ILMN_1661479 -1 26273 FBXO3 ILMN_1749641 -1 26504 CNNM4 ILMN_1655720 -1 26747 NUFIP1 ILMN_1813800 _ -1 26993 AKAP8L ILMN_1768962 -1 27072 VPS41 ILMN_1768486 -1 27241 BBS9 ILMN_1794956 -1 27244 SESN1 ILMN_1800626 -1 27332 ZNF638 ILMN_1791379 -1 27342 RABGEF1 ILMN_1760884 -1 28981 IFT81 ILMN_1655504 -1 29066 ZC3H7A ILMN_1693227 -1 29072 HYPB ILMN_1769473 -1 29079 MED4 ILMN_1664641 -1 29994 BAZ2B ILMN_1720850 -1 50717 WDR42A ILMN_1717905 -1 50853 VILL ILMN_1778650 -1 51111 SUV420H1 ILMN_1692026 -1 51112 TTC15 ILMN_1693317 -1 51143 DYNC1LI1 ILMN_1703844 -1 51276 ZNF571 ILMN_1706709 -1 51306 C5orf5 ILMN_1673478 -1 51315 KRCC1 ILMN_1745620 -1 51447 IHPK2 ILMN_1683328 -1 51542 VPS54 ILMN_1761086 -1 51552 RAB14 ILMN_1735765 -1 51592 TRIM33 ILMN_1682316 -1 51594 NAG ILMN_1686392 -1 51603 KIAA0859 ILMN_1790575 -1 51616 TAF9L ILMN_1784880 -1 51626 DYNC2LI1 ILMN_1811836 -1 51631 LUC7L2 ILMN_1747099 -1 51646 YPEL5 ILMN_1711069 -1 51652 VPS24 ILMN_1683827 -1 51742 ARID4B ILMN_1761334 -1 51780 JMJD1B ILMN_1706539 -1 51809 GALNT7 ILMN_1670748 -1 53340 SPA17 ILMN_1678423 -1 54103 LOC54103 ILMN_1772064 -1 54468 FLJ20323 ILMN_1680644 -1 54482 CCDC76 ILMN_1659786 -1 54520 FLJ10996 ILMN_1800392 -1 54529 ASNSD1 ILMN_1722873 -1 54530 C1orf218 ILMN_1789436 -1 54556 ING3 ILMN_1806226 -1 54585 LZTFL1 ILMN_1815705 -1 54665 RSBN1 ILMN_1791097 -1 54741 LEPROT ILMN_1661537 -1 54778 RNF111 ILMN_1767454 1 54780 NSMCE4A ILMN_1679134 -1 54816 ZNF280D ILMN_1767142 -1 54819 ZCCHC10 ILMN_1769104 -1 54832 VPS13C ILMN_1809582 -1 54861 SNRK ILMN_1794399 -1 54878 DPP8 ILMN_1759801 -1 54880 BCOR ILMN_1773117 -1 54885 TBC1D8B ILMN_1701604 -1 54902 TTC19 ILMN_1781260 -1 54934 C12orf41 ILMN_1713189 -1 55000 TUG1 ILMN_1682783 -1 55006 FLJ20628 ILMN_1706558 -1 55030 FBXO34 ILMN_1765060 -1 55035 NOL8 ILMN_1689747 -1 55037 PTCD3 ILMN_1714424 -1 55082 FLJ10154 ILMN_1788468 -1 55112 WDR60 ILMN_1793290 -1 55119 PRPF38B ILMN_1799299 -1 55125 CEP192 ILMN_1703754 -1 55187 VPS13D ILMN_1656840 -1 55198 DIP13B ILMN_1765076 -1 55251 PCMTD2 ILMN_1767848 -1 55256 ADI1 ILMN_1795671 -1 55295 KLHL26 ILMN_1805330 -1 55333 SYNJ2BP ILMN_1697793 -1 55425 KIAA1704 ILMN_1749676 -1 55435 C4orf16 ILMN_1758427 -1 55556 ENOSF1 ILMN_1803676 -1 55578 FAM48A ILMN_1669555 -1 55596 ZCCHC8 ILMN_1775793 -1 55614 KIF16B ILMN_1772786 -1 55634 ZNF673 ILMN_1710768 -1 55665 URG4 ILMN_1777811 -1 55683 FLJ10081 ILMN_1663571 -1 55758 RCOR3 ILMN_1682095 -1 55763 EXOC1 ILMN_1793766 -1 55769 ZNF83 ILMN_1716921 -1 55786 ZNF415 ILMN_1664292 -1 55818 JMJD1A ILMN_1722532 -1 55893 ZNF395 ILMN_1772876 -1 55930 MYO5C ILMN_1808789 -1 55937 APOM ILMN_1713438 -1 55958 KLHL9 ILMN_1664466 -1 56255 TXNDC13 ILMN_1702759 -1 56339 METTL3 ILMN_1655635 -1 56890 MDM1 ILMN 1750703 1 56967 C14orf132 ILMN_1713744 -1 57020 MGC16824 ILMN_1729450 -1 57035 C1orf63 ILMN_1749915 -1 57050 UTP3 ILMN_1745423 -1 57209 ZNF248 ILMN_1662192 -1 57214 KIAA1199 ILMN_1813704 -1 57337 SENP7 ILMN_1778294 -1 57396 CLK4 ILMN_1695853 -1 57496 MKL2 ILMN_1690807 -1 57498 KIDINS220 ILMN_1679232 -1 57509 MTUS1 ILMN_1663648 -1 57556 SEMA6A ILMN_1713529 -1 57599 WDR48 ILMN_1762103 -1 57711 ZNF529 ILMN_1660193 -1 57728 WDR19 ILMN_1655117 -1 57798 GATAD1 ILMN_1710863 -1 58517 RBM25 ILMN_1768117 -1 60625 DHX35 ILMN_1733667 -1 63917 GALNT11 ILMN_1651819 -1 63920 LOC63920 ILMN_1727553 -1 63931 MRPS14 ILMN_1779423 -1 64112 MOAP1 ILMN_1804988 -1 64766 S100PBP ILMN_1663577 -1 64795 RMND5A ILMN_1714093 -1 64844 Mar-07ILMN_1717337 -1 64848 YTHDC2 ILMN_1725760 -1 64860 ARMCX5 ILMN_1741156 -1 65110 UPF3A ILMN_1671742 -1 65117 RSRC2 ILMN_1724431 -1 65982 ZSCAN18 ILMN_1654946 -1 66008 TRAK2 ILMN_1781691 -1 79048 SECISBP2 ILMN_1736481 -1 79072 FASTKD3 ILMN_1750160 -1 79088 ZNF426 ILMN_1673804 -1 79443 FYCO1 ILMN_1709032 -1 79612 NARG1L ILMN_1801109 -1 79635 CCDC121 ILMN_1739054 -1 79664 NARG2 ILMN_1718830 -1 79673 ZNF329 ILMN_1689059 -1 79684 C11orf61 ILMN_1773148 -1 79726 WDR59 ILMN_1795428 -1 79738 C12orf58 ILMN_1741985 -1 79868 CXorf45 ILMN_1724341 -1 79882 ZC3H14 ILMN_1785292 -1 79960 PHF17 ILMN_1736015 -1 79986 ZNF702 ILMN 1663281 _ -1 57020 MGC16824 ILMN_1729450 -1 57035 C1orf63 ILMN_1749915 -1 57050 UTP3 ILMN_1745423 -1 57209 ZNF248 ILMN_1662192 -1 57214 KIAA1199 ILMN_1813704 -1 57337 SENP7 ILMN_1778294 -1 57396 CLK4 ILMN_1695853 -1 57496 MKL2 ILMN_1690807 -1 57498 KIDINS220 ILMN_1679232 -1 57509 MTUS1 ILMN_1663648 -1 57556 SEMA6A ILMN_1713529 -1 57599 WDR48 ILMN_1762103 -1 57711 ZNF529 ILMN_1660193 -1 57728 WDR19 ILMN_1655117 -1 57798 GATAD1 ILMN_1710863 -1 58517 RBM25 ILMN_1768117 -1 60625 DHX35 ILMN_1733667 -1 63917 GALNT11 ILMN_1651819 -1 63920 LOC63920 ILMN_1727553 -1 63931 MRPS14 ILMN_1779423 -1 64112 MOAP1 ILMN_1804988 -1 64766 S100PBP ILMN_1663577 -1 64795 RMND5A ILMN_1714093 -1 64844 Mar-07ILMN_1717337 -1 64848 YTHDC2 ILMN_1725760 -1 64860 ARMCX5 ILMN_1741156 -1 65110 UPF3A ILMN_1671742 -1 65117 RSRC2 ILMN_1724431 -1 65982 ZSCAN18 ILMN_1654946 -1 66008 TRAK2 ILMN_1781691 -1 79048 SECISBP2 ILMN_1736481 -1 79072 FASTKD3 ILMN_1750160 -1 79088 ZNF426 ILMN_1673804 -1 79443 FYCO1 ILMN_1709032 -1 79612 NARG1L ILMN_1801109 -1 79635 CCDC121 ILMN_1739054 -1 79664 NARG2 ILMN_1718830 -1 79673 ZNF329 ILMN_1689059 -1 79684 C11orf61 ILMN_1773148 -1 79726 WDR59 ILMN_1795428 -1 79738 C12orf58 ILMN_1741985 -1 79868 CXorf45 ILMN_1724341 -1 79882 ZC3H14 ILMN_1785292 -1 79960 PHF17 ILMN_1736015 -1 79986 ZNF702 ILMN_1663281 -1 80095 ZNF606 ILMN_1681829 -1 80204 FBXO11 ILMN_1713682 -1 80205 CHD9 ILMN_1762972 -1 80207 OPA3 ILMN_1652819 -1 80208 KIAA1840 ILMN_1665049 -1 80213 TM2D3 ILMN_1761120 -1 80232 WDR26 ILMN_1719343 -1 80345 ZSCAN16 ILMN_1809566 -1 81575 APOLD1 ILMN_1723522 -1 81578 COL21A1 ILMN_1732850 -1 81608 FIP1L1 ILMN_1768743 -1 83759 RBM4B ILMN_1743104 -1 84099 ID2B ILMN_1658904 -1 84162 KIAA1109 ILMN_1694357 -1 84901 NFATC2IP ILMN_1725441 -1 84939 MUM1 ILMN_1764764 -1 91647 ATPAF2 ILMN_1727390 -1 91746 YTHDC1 ILMN_1707506 -1 93643 TJAP1 ILMN_1743763 -1 94104 C21orf66 ILMN_1682896 -1 94134 ARHGAP12 ILMN_1753500 -1 94239 H2AFV ILMN_1706784 -1 114625 ERMAP ILMN_1715013 -1 114882 OSBPL8 ILMN_1782459 -1 116987 CENTG2 ILMN_1807407 -1 120526 ZCSL3 ILMN_1685930 -1 126231 ZNF573 ILMN_1658080 -1 130074 LOC130074 ILMN_1677122 -1 146057 TTBK2 ILMN_1705834 -1 150759 LOC150759 ILMN_1755455 -1 158427 C9orf97 ILMN_1684520 -1 171023 ASXL1 ILMN_1726025 -1 200576 PIP5K3 ILMN_1671108 -1 200734 SPRED2 ILMN_1791232 -1 203197 C9orf91 ILMN_1705313 -1 220988 HNRPA3 ILMN_1761083 -1 221078 NSUN6 ILMN_1664646 -1 221395 GPR116 ILMN_1728785 -1 221443 C6orf130 ILMN_1804834 -1 222161 DKFZP586I1420 ILMN_1803856 -1 246243 RNASEH1 ILMN_1726783 -1 253959 GARNL1 ILMN_1741391 -1 285189 PLGLA1 ILMN_1751811 -1 286451 YIPF6 ILMN_1803939 -1 375056 UNQ6077 ILMN_1672043 -1 388397 LOC388397 ILMN 1678312 -1 388969 LOC388969 ILMN_1679771 -1 440026 TMEM41B ILMN_1678004 -1 644450 LOC644450 ILMN_1715439 -1 651633 LOC651633 ILMN_1739583 Sotiriou et al. Farmer et al. Stroma.1 coefficient Entrez_Gene_ID Symbol Probe_Id GGI 1 3838 KPNA2 ILMN_1708160 1 8140 SLC7A5 ILMN_1720373 1 4141 MARS ILMN_1799819 1 7334 UBE2N ILMN_1793651 1 10212 DDX39 ILMN_1747303 1 4605 MYBL2 ILMN_1709020 1 332BIRC5 ILMN_1710082 1 4171 MCM2 ILMN_1681503 1 5347 PLK1 ILMN_1736176 1 2305 FOXM1 ILMN_1716400 1 9133 CCNB2 ILMN_1801939 1 27338 UBE2S ILMN_1808591 1 991CDC20 ILMN_1663390 1 11065 UBE2C ILMN_1714730 1 10535 RNASEH2A ILMN_1810901 1 8914 TIMELESS ILMN_1735093 1 10615 SPAG5 ILMN_1768291 1 983CDC2 ILMN_1747911 1 4001 LMNB1 ILMN_1672988 1 4085 MAD2L1 ILMN_1777564 1 890CCNA2 ILMN_1786125 1 9232 PTTG1 ILMN_1753196 1 3149 HMGB3 ILMN_1733519 1 701BUB1B ILMN_1733950 1 9787 DLG7 ILMN_1749829 1 11130 ZWINT ILMN_1778034 1 9319 TRIP13 ILMN_1796589 1 6790 AURKA ILMN_1680955 1 10403 NDC80 ILMN_1664511 1 1017 CDK2 ILMN_1665559 1 51512 GTSE1 ILMN_1771039 1 3832 KIF11 ILMN_1794539 1 9156 EXO1 ILMN_1673721 1 10024 TROAP ILMN_1700337 1 993CDC25A ILMN_1711005 1 4521 NUDT1 ILMN 1735692 388969 LOC388969 ILMN_1679771 440026 TMEM41B ILMN_1678004 644450 LOC644450 ILMN_1715439 651633 LOC651633 ILMN_1739583 Gene_ID Symbol Probe_Id 3838 KPNA2 ILMN_1708160 8140 SLC7A5 ILMN_1720373 4141 MARS ILMN_1799819 7334 UBE2N ILMN_1793651 10212 DDX39 ILMN_1747303 4605 MYBL2 ILMN_1709020 332BIRC5 ILMN_1710082 4171 MCM2 ILMN_1681503 5347 PLK1 ILMN_1736176 2305 FOXM1 ILMN_1716400 9133 CCNB2 ILMN_1801939 27338 UBE2S ILMN_1808591 991CDC20 ILMN_1663390 11065 UBE2C ILMN_1714730 10535 RNASEH2A ILMN_1810901 8914 TIMELESS ILMN_1735093 10615 SPAG5 ILMN_1768291 983CDC2 ILMN_1747911 4001 LMNB1 ILMN_1672988 4085 MAD2L1 ILMN_1777564 890CCNA2 ILMN_1786125 9232 PTTG1 ILMN_1753196 3149 HMGB3 ILMN_1733519 701BUB1B ILMN_1733950 9787 DLG7 ILMN_1749829 11130 ZWINT ILMN_1778034 9319 TRIP13 ILMN_1796589 6790 AURKA ILMN_1680955 10403 NDC80 ILMN_1664511 1017 CDK2 ILMN_1665559 51512 GTSE1 ILMN_1771039 3832 KIF11 ILMN_1794539 9156 EXO1 ILMN_1673721 10024 TROAP ILMN_1700337 993CDC25A ILMN_1711005 4521 NUDT1 ILMN_1735692 2237 FEN1 ILMN_1755834 9700 ESPL1 ILMN_1742145 7272 TTK ILMN_1788166 9833 MELK ILMN_1731184 1058 CENPA ILMN_1801257 Sotiriou et al. Farmer et al. Stroma.1 GGI 9134 CCNE2 ILMN_1740452 1062 CENPE ILMN_1716279 641BLM ILMN_1709484 9928 KIF14 ILMN_1808071 3161 HMMR ILMN_1781942 1063 CENPF ILMN_1664516 22948 CCT5 ILMN_1706246 84790 TUBA1C ILMN_1663388 11004 KIF2C ILMN_1685916 9212 AURKB ILMN_1684217 699BUB1 ILMN_1736090 3833 KIFC1 ILMN_1764840 1033 CDKN3 ILMN_1666305 6241 RRM2 ILMN_1678669 22974 TPX2 ILMN_1792494 10376 TUBA1B ILMN_1800261 4288 MKI67 ILMN_1734827 4173 MCM4 ILMN_1771558 23397 BRRN1 ILMN_1657600 11339 OIP5 ILMN_1759277 3015 H2AFZ ILMN_1707858 6472 SHMT2 ILMN_1661264 8833 GMPS ILMN_1771734 891CCNB1 ILMN_1712803 55969 C20orf24 ILMN_1679195 9055 PRC1 ILMN_1728934 51203 NUSAP1 ILMN_1726720 24137 KIF4A ILMN_1799667 56942 C16orf61 ILMN_1783333 55165 CEP55 ILMN_1747016 29095 ORMDL2 ILMN_1774708 64151 NCAPG ILMN_1751444 55355 DKFZp762E1312 ILMN_1703906 10112 KIF20A ILMN_1695658 79682 MLF1IP ILMN_1679438 79075 DSCC1 ILMN_1776577 56992 KIF15 ILMN_1809440 10721 POLQ ILMN_1740291 55839 CENPN ILMN_1720526 259266 ASPM ILMN_1815184 55010 C12orf48 ILMN_1727055 55388 MCM10 ILMN_1706194 83461 CDCA3 ILMN_1737728 55143 CDCA8 ILMN 1709294 1 29127 RACGAP1 ILMN_1702140 -1 10129 FRY ILMN_1678437 -1 8100 TTC10 ILMN_1669366 -1 1524 CX3CR1 ILMN_1745788 -1 90627 STARD13 ILMN_1660451 -1 3913 LAMB2 ILMN_1752968 -1 7178 TPT1 ILMN_1789614 -1 79901 CYBRD1 ILMN_1712305 -1 27244 SESN1 ILMN_1800626 -1 582BBS1 ILMN_1800590 -1 56912 C11orf60 ILMN_1677080 -1 55650 PIGV ILMN_1762993 -1 79846 FLJ21062 ILMN_1790315 -1 79864 C11orf63 ILMN_1680659 -1 57728 WDR19 ILMN_1655117 -1 54737 MPHOSPH8 ILMN_1759942 Desmedt et al. coefficient Entrez_Gene_ID Symbol Probe_Id Stroma.2.PLAU 1 526ATP6V1B2 ILMN_1787705 1 602BCL3 ILMN_1662437 1 649BMP1 ILMN_1800412 1 871SERPINH1 ILMN_1751028 1 1281 COL3A1 ILMN_1773079 1 1290 COL5A2 ILMN_1729117 1 1291 COL6A1 ILMN_1732151 1 1627 DBN1 ILMN_1769926 1 1774 DNASE1L1 ILMN_1787709 1 1833 EPYC ILMN_1677567 1 2037 EPB41L2 ILMN_1805448 1 2050 EPHB4 ILMN_1734624 1 2191 FAP ILMN_1741468 1 2720 GLB1 ILMN_1790862 1 3671 ISLR ILMN_1747593 1 4205 MEF2A ILMN_1661888 1 4237 MFAP2 ILMN_1787981 1 4314 MMP3 ILMN_1784459 1 4323 MMP14 ILMN_1774739 1 4430 MYO1B ILMN_1797585 1 4681 NBL1 ILMN_1770986 1 4811 NID1 ILMN_1674719 1 4921 DDR2 ILMN_1730553 1 5155 PDGFB ILMN_1775822 1 5159 PDGFRB ILMN_1815057 1 5328 PLAU ILMN_1656057 1 5476 PPGB ILMN_1719286 -1 6141 RPL18 ILMN_1695598 -1 6233 RPS27A ILMN 1755883 Desmedt et al. Farmer et al. Stroma.1 Stroma.2.PLAU 6591 SNAI2 ILMN_1655740 6876 TAGLN ILMN_1778668 7042 TGFB2 ILMN_1812526 7070 THY1 ILMN_1779875 7421 VDR ILMN_1794337 8038 ADAM12 ILMN_1705689 8408 ULK1 ILMN_1705144 8459 TPST2 ILMN_1695432 8481 OFD1 ILMN_1785266 8507 ENC1 ILMN_1779147 8904 CPNE1 ILMN_1670841 9180 OSMR ILMN_1724593 9260 PDLIM7 ILMN_1690125 9697 TRAM2 ILMN_1788783 9849 ZNF518 ILMN_1742541 9945 GFPT2 ILMN_1655236 10409 BASP1 ILMN_1651826 10487 CAP1 ILMN_1797604 10993 SDS ILMN_1811114 23166 STAB1 ILMN_1682530 23299 BICD2 ILMN_1651375 23452 ANGPTL2 ILMN_1772612 23645 PPP1R15A ILMN_1659936 23705 CADM1 ILMN_1680132 25903 OLFML2B ILMN_1765557 26585 GREM1 ILMN_1752965 27295 PDLIM3 ILMN_1695299 51330 TNFRSF12A ILMN_1689004 51463 GPR89A ILMN_1804539 51592 TRIM33 ILMN_1682316 54587 MXRA8 ILMN_1809868 55033 FKBP14 ILMN_1665243 55035 NOL8 ILMN_1689747 55742 PARVA ILMN_1756408 57158 JPH2 ILMN_1718941 59286 UBL5 ILMN_1691379 93974 ATPIF1 ILMN_1727332 157567 ANKRD46 ILMN_1712298 Entrez_Gene_ID Symbol Probe_Id 32ACACB ILMN_1763852 107ADCY1 ILMN_1742073 191AHCY ILMN_1657862 Creighton et al. Farmer et al. Stroma.1 MAPK -1 521ATP5I ILMN_1803509 -1 587BCAT2 ILMN_1695110 -1 596BCL2 ILMN_1801119 -1 617BCS1L ILMN_1738529 -1 694BTG1 ILMN_1775743 -1 744MPPED2 ILMN_1676526 -1 776CACNA1D ILMN_1681223 -1 839CASP6 ILMN_1694877 -1 1164 CKS2 ILMN_1756326 -1 1326 MAP3K8 ILMN_1741159 -1 1345 COX6C ILMN_1654151 -1 1373 CPS1 ILMN_1792748 -1 1434 CSE1L ILMN_1706238 -1 1468 SLC25A10 ILMN_1804090 -1 1479 CSTF3 ILMN_1736161 -1 1573 CYP2J2 ILMN_1758731 -1 1789 DNMT3B ILMN_1794692 -1 1952 CELSR2 ILMN_1711208 -1 1960 EGR3 ILMN_1807638 -1 2001 ELF5 ILMN_1813270 -1 2099 ESR1 ILMN_1678535 -1 2122 EVI1 ILMN_1803367 -1 2262 GPC5 ILMN_1688886 -1 2264 FGFR4 ILMN_1730355 -1 2317 FLNB ILMN_1752635 -1 2494 NR5A2 ILMN_1811303 -1 2576 GAGE4 ILMN_1715638 -1 2581 GALC ILMN_1799744 -1 2960 GTF2E1 ILMN_1655921 -1 3321 IGSF3 ILMN_1713014 -1 3358 HTR2C ILMN_1767145 -1 3398 ID2 ILMN_1793990 -1 3643 INSR ILMN_1670918 -1 3646 EIF3S6 ILMN_1739257 -1 3682 ITGAE ILMN_1683927 -1 3705 ITPK1 ILMN_1715674 -1 3800 KIF5C ILMN_1773837 -1 3860 KRT13 ILMN_1721218 -1 4048 LTA4H ILMN_1690342 -1 4092 SMAD7 ILMN_1815941 -1 4171 MCM2 ILMN_1681503 -1 4477 MSMB ILMN_1699243 1 4528 MTIF2 ILMN 1765520 -1 4685 NCAM2 ILMN_1749962 -1 4702 NDUFA8 ILMN_1759729 -1 4729 NDUFV2 ILMN_1766496 -1 4883 NPR3 ILMN_1665033 -1 4929 NR4A2 ILMN_1782305 -1 5025 P2RX4 ILMN_1813846 -1 5087 PBX1 ILMN_1784678 -1 5096 PCCB ILMN_1761010 -1 5110 PCMT1 ILMN_1671621 -1 5121 PCP4 ILMN_1682326 -1 5174 PDZK1 ILMN_1708341 -1 5191 PEX7 ILMN_1729650 -1 5241 PGR ILMN_1811014 -1 5289 PIK3C3 ILMN_1687896 -1 5298 PI4KB ILMN_1666597 -1 5335 PLCG1 ILMN_1740160 -1 5425 POLD2 ILMN_1746252 -1 5627 PROS1 ILMN_1671928 -1 5710 PSMD4 ILMN_1728355 -1 5885 RAD21 ILMN_1748578 -1 5966 REL ILMN_1766085 -1 5979 RET ILMN_1655610 -1 5981 RFC1 ILMN_1792986 -1 6019 RLN2 ILMN_1748529 -1 6138 RPL15 ILMN_1762747 -1 6141 RPL18 ILMN_1695598 -1 6175 RPLP0 ILMN_1709880 -1 6185 RPN2 ILMN_1693421 -1 6205 RPS11 ILMN_1740587 -1 6227 RPS21 ILMN_1800573 -1 6383 SDC2 ILMN_1784553 -1 6387 CXCL12 ILMN_1791447 -1 6445 SGCG ILMN_1659649 -1 6565 SLC15A2 ILMN_1706040 -1 6605 SMARCE1 ILMN_1747857 -1 6744 SSFA2 ILMN_1742260 -1 6785 ELOVL4 ILMN_1737965 -1 6947 TCN1 ILMN_1768469 -1 7031 TFF1 ILMN_1722489 -1 7074 TIAM1 ILMN_1655577 -1 7155 TOP2B ILMN_1777663 -1 7164 TPD52L1 ILMN_1714383 -1 8187 ZNF239 ILMN_1748427 -1 8270 DXS9879E ILMN_1708151 -1 8604 SLC25A12 ILMN_1788053 -1 8645 KCNK5 ILMN_1766918 -1 8663 EIF3C ILMN_1725984 -1 8723 SNX4 ILMN_1738736 -1 8776 MTMR1 ILMN_1741556 -1 8777 MPDZ ILMN_1707649 -1 8890 EIF2B4 ILMN_1671921 -1 8934 RAB7L1 ILMN_1750202 -1 9045 RPL14 ILMN_1726460 -1 9050 PSTPIP2 ILMN_1713058 -1 9053 MAP7 ILMN_1712719 -1 9063 PIAS2 ILMN_1658302 -1 9125 RQCD1 ILMN_1679415 -1 9289 GPR56 ILMN_1756931 -1 9368 SLC9A3R1 ILMN_1680925 -1 9467 SH3BP5 ILMN_1814773 -1 9656 MDC1 ILMN_1814122 -1 9668 ZNF432 ILMN_1693788 -1 9687 GREB1 ILMN_1721170 -1 9779 TBC1D5 ILMN_1767433 -1 9797 TATDN2 ILMN_1734138 -1 9847 KIAA0528 ILMN_1682572 -1 9931 HELZ ILMN_1762835 -1 10090 UST ILMN_1700024 -1 10125 RASGRP1 ILMN_1768958 -1 10140 TOB1 ILMN_1672004 -1 10247 HRSP12 ILMN_1807633 -1 10273 STUB1 ILMN_1756126 -1 10276 NET1 ILMN_1758311 -1 10295 BCKDK ILMN_1693394 -1 10370 CITED2 ILMN_1663092 -1 10395 DLC1 ILMN_1698020 -1 10497 UNC13B ILMN_1669747 -1 10634 GAS2L1 ILMN_1724059 -1 10675 CSPG5 ILMN_1688184 -1 10732 TCFL5 ILMN_1814247 -1 10758 TRAF3IP2 ILMN_1701514 -1 10856 RUVBL2 ILMN_1708808 -1 10884 MRPS30 ILMN_1726743 -1 10907 TXNL4A ILMN_1790218 PAPOLA ILMN 1 11108 PRDM4 ILMN_1691432 -1 11232 POLG2 ILMN_1671004 -1 11259 DOC1 ILMN_1738578 -1 22828 RBM16 ILMN_1681675 -1 22909 MTMR15 ILMN_1778734 -1 23007 PLCH1 ILMN_1668409 -1 23067 KIAA1076 ILMN_1813573 -1 23080 KIAA0241 ILMN_1722292 -1 23118 MAP3K7IP2 ILMN_1672331 -1 23171 GPD1L ILMN_1694106 -1 23219 FBXO28 ILMN_1812776 -1 23229 ARHGEF9 ILMN_1690634 -1 23244 PDS5A ILMN_1701069 -1 23357 ANGEL1 ILMN_1665871 -1 23394 ADNP ILMN_1657993 -1 23443 SLC35A3 ILMN_1653429 -1 23521 RPL13A ILMN_1713369 -1 23549 DNPEP ILMN_1691393 -1 23563 CHST5 ILMN_1759614 -1 23576 DDAH1 ILMN_1668507 -1 23779 ARHGAP8 ILMN_1656276 -1 25840 METTL7A ILMN_1656285 -1 25926 NOL11 ILMN_1688971 -1 25980 C20orf4 ILMN_1721225 -1 26097 C1orf77 ILMN_1736234 -1 26121 PRPF31 ILMN_1719204 -1 26190 FBXW2 ILMN_1661479 -1 26353 HSPB8 ILMN_1791280 -1 26578 OSTF1 ILMN_1742456 -1 27250 PDCD4 ILMN_1768004 -1 27258 LSM3 ILMN_1719032 -1 28956 MAPBPIP ILMN_1756352 -1 28978 TMEM14A ILMN_1785191 -1 29104 C21orf127 ILMN_1701077 -1 29796 UCRC ILMN_1781986 -1 29902 C12orf24 ILMN_1753781 -1 29964 C6orf49 ILMN_1695868 -1 50865 HEBP1 ILMN_1802557 -1 51074 APIP ILMN_1793598 -1 51098 IFT52 ILMN_1673172 -1 51141 INSIG2 ILMN_1797946 -1 51185 CRBN ILMN_1668582 -1 51433 ANAPC5 ILMN_1723177 -1 51491 HSPC111 ILMN_1704055 -1 51499 TRIAP1 ILMN_1774083 -1 51526 C20orf111 ILMN 1676827 1 51550 CINP ILMN_1765257 -1 51669 TMEM66 ILMN_1780141 -1 54332 GDAP1 ILMN_1730897 -1 54470 ARMCX6 ILMN_1716026 -1 54665 RSBN1 ILMN_1791097 -1 54788 DNAJB12 ILMN_1758545 -1 54840 APTX ILMN_1726752 -1 55005 RMND1 ILMN_1694533 -1 55010 C12orf48 ILMN_1727055 -1 55061 SUSD4 ILMN_1760533 -1 55081 IFT57 ILMN_1811636 -1 55135 WDR79 ILMN_1693669 -1 55245 C20orf44 ILMN_1745152 -1 55256 ADI1 ILMN_1795671 -1 55347 ABHD10 ILMN_1770031 -1 55508 SLC35E3 ILMN_1749521 -1 55573 CDV3 ILMN_1810977 -1 55661 DDX27 ILMN_1760218 -1 55709 KBTBD4 ILMN_1687092 -1 55726 C12orf11 ILMN_1723222 -1 55734 ZFP64 ILMN_1797801 -1 55764 IFT122 ILMN_1742379 -1 55769 ZNF83 ILMN_1716921 -1 55812 SPATA7 ILMN_1652409 -1 55915 LANCL2 ILMN_1708009 -1 56034 PDGFC ILMN_1683023 -1 56943 ENY2 ILMN_1655536 -1 56994 CHPT1 ILMN_1729112 -1 57007 CMKOR1 ILMN_1798360 -1 57020 MGC16824 ILMN_1729450 -1 57380 MRS2L ILMN_1778677 -1 57473 ZNF512B ILMN_1766346 -1 57613 KIAA1467 ILMN_1668619 -1 60436 TGIF2 ILMN_1709044 -1 60598 KCNK15 ILMN_1788421 -1 64172 OSGEPL1 ILMN_1658909 -1 64400 AKTIP ILMN_1665982 -1 64699 TMPRSS3 ILMN_1741768 -1 64769 C1orf149 ILMN_1714854 -1 64919 BCL11B ILMN_1665761 -1 65055 REEP1 ILMN_1733746 -1 65243 ZNF643 ILMN_1799086 -1 79158 GNPTAB ILMN_1736757 -1 79602 ADIPOR2 ILMN_1750651 -1 79624 C6orf211 ILMN_1798108 -1 79665 DHX40 ILMN 1653047 79695 GALNT12 ILMN_1735157 79833 GEMIN6 ILMN_1707484 79882 ZC3H14 ILMN_1785292 79989 TTC26 ILMN_1676555 80129 C6orf97 ILMN_1772588 80173 IFT74 ILMN_1777449 80303 EFHD1 ILMN_1779448 81550 TDRD3 ILMN_1745811 83752 LONP2 ILMN_1691480 84909 C9orf3 ILMN_1674629 87ACTN1 ILMN_1798892 133ADM ILMN_1708934 391RHOG ILMN_1739792 411ARSB ILMN_1712587 444ASPH ILMN_1693771 677ZFP36L1 ILMN_1675448 727C5 ILMN_1746819 824CAPN2 ILMN_1716057 831CAST ILMN_1672947 967CD63 ILMN_1753468 1647 GADD45A ILMN_1694075 1728 NQO1 ILMN_1720282 1729 DIAPH1 ILMN_1757358 1874 E2F4 ILMN_1761828 1969 EPHA2 ILMN_1700527 1999 ELF3 ILMN_1769201 2000 ELF4 ILMN_1652082 2034 EPAS1 ILMN_1760034 2101 ESRRA ILMN_1774272 2118 ETV4 ILMN_1720822 2119 ETV5 ILMN_1739222 2185 PTK2B ILMN_1815522 2280 FKBP1A ILMN_1683969 2286 FKBP2 ILMN_1807563 2355 FOSL2 ILMN_1725175 2495 FTH1 ILMN_1683146 2771 GNAI2 ILMN_1775762 2810 SFN ILMN_1806607 3106 HLA-B ILMN_1703403 3107 HLA-C ILMN_1721113 3133 HLA-E ILMN_1765258 3134 HLA-F ILMN_1762861 3956 LGALS1 ILMN_1723978 3959 LGALS3BP ILMN_1654314 3988 LIPA ILMN_1718063 3992 FADS1 ILMN_1670134 3995 FADS3 ILMN_1716847 4000 LMNA ILMN_1696749 4862 NPAS2 ILMN_1765558 5048 PAFAH1B1 ILMN_1722276 5152 PDE9A ILMN_1683063 5337 PLD1 ILMN_1719696 5359 PLSCR1 ILMN_1752889 5526 PPP2R5B ILMN_1693975 5578 PRKCA ILMN_1771800 5583 PRKCH ILMN_1780898 5827 PXMP2 ILMN_1799015 5836 PYGL ILMN_1696187 5937 RBMS1 ILMN_1762568 5971 RELB ILMN_1811258 6286 S100P ILMN_1801216 6303 SAT1 ILMN_1753342 6305 SBF1 ILMN_1703246 6583 SLC22A4 ILMN_1685057 6672 SP100 ILMN_1690920 6892 TAPBP ILMN_1742450 7126 TNFAIP1 ILMN_1655429 7422 VEGFA ILMN_1803882 7461 CYLN2 ILMN_1798846 7538 ZFP36 ILMN_1720829 8507 ENC1 ILMN_1779147 8714 ABCC3 ILMN_1677814 9138 ARHGEF1 ILMN_1772370 9568 GABBR2 ILMN_1660718 9582 APOBEC3B ILMN_1691457 9961 MVP ILMN_1803277 10133 OPTN ILMN_1717586 10205 MPZL2 ILMN_1752932 10263 CDK2AP2 ILMN_1690653 10331 B3GNT3 ILMN_1800082 10410 IFITM3 ILMN_1805750 10847 SRCAP ILMN_1750641 11097 NUPL2 ILMN_1789616 11138 TBC1D8 ILMN_1735495 11182 SLC2A6 ILMN_1778321 22927 HABP4 ILMN 1792384 1 23764 MAFF ILMN_1680139 1 26010 DNAPTP6 ILMN_1683678 1 27242 TNFRSF21 ILMN_1738006 1 29109 FHOD1 ILMN_1651776 1 51097 SCCPDH ILMN_1795839 1 51129 ANGPTL4 ILMN_1707727 1 51400 PPME1 ILMN_1732772 1 51657 STYXL1 ILMN_1718822 1 53838 C11orf24 ILMN_1808163 1 55080 TAPBPL ILMN_1805449 1 55652 FLJ20489 ILMN_1758279 1 55861 C20orf35 ILMN_1730612 1 56288 PARD3 ILMN_1710524 1 56829 ZC3HAV1 ILMN_1729973 1 56935 C11orf75 ILMN_1798270 1 57154 SMURF1 ILMN_1737672 1 60673 C12orf44 ILMN_1772527 1 64063 PRSS22 ILMN_1776925 1 64761 PARP12 ILMN_1718558 1 64787 EPS8L2 ILMN_1691047 1 65983 GRAMD3 ILMN_1734007 1 79094 CHAC1 ILMN_1739241 1 79174 CRELD2 ILMN_1748707 1 79567 FAM65A ILMN_1680037 1 79837 PIP4K2C ILMN_1787308 1 80761 UPK3B ILMN_1803024 1 81848 SPRY4 ILMN_1741168 1 85363 TRIM5 ILMN_1704972 1 93664 CADPS2 ILMN_1684461 1 206358 SLC36A1 ILMN_1787826 Creighton et al. Farmer et al. Stroma.1 coefficient Entrez_Gene_ID Symbol Probe_Id IGF1 1 5104 SERPINA5 ILMN_1759910 1 16AARS ILMN_1662364 1 6950 TCP1 ILMN_1776347 1 47ACLY ILMN_1749014 1 81ACTN4 ILMN_1725534 1 87ACTN1 ILMN_1798892 1 226ALDOA ILMN_1736700 1 22906 TRAK1 ILMN_1754629 1 440ASNS ILMN_1796417 1 444ASPH ILMN_1693771 1 578BAK1 ILMN_1805990 1 617BCS1L ILMN_1738529 1 645BLVRB ILMN_1797793 1 655BMP7 ILMN_1741566 Creighton et al. Farmer et al. Stroma.1 IGF1 762CA4 ILMN_1695157 816CAMK2B ILMN_1694997 833CARS ILMN_1696066 896CCND3 ILMN_1668721 955ENTPD6 ILMN_1677955 1026 CDKN1A ILMN_1784602 1051 CEBPB ILMN_1693014 1054 CEBPG ILMN_1716766 1075 CTSC ILMN_1696347 1147 CHUK ILMN_1677041 1152 CKB ILMN_1671478 1175 AP2S1 ILMN_1662426 1317 SLC31A1 ILMN_1804562 1465 CSRP1 ILMN_1811921 1468 SLC25A10 ILMN_1804090 1503 CTPS ILMN_1783285 1515 CTSL2 ILMN_1748352 1534 CYB561 ILMN_1771179 1537 CYC1 ILMN_1815115 1594 CYP27B1 ILMN_1740418 1622 DBI ILMN_1715120 1717 DHCR7 ILMN_1654028 1743 DLST ILMN_1773228 1789 DNMT3B ILMN_1794692 1837 DTNA ILMN_1730201 1847 DUSP5 ILMN_1656501 1848 DUSP6 ILMN_1677466 1850 DUSP8 ILMN_1743204 1857 DVL3 ILMN_1686411 1960 EGR3 ILMN_1807638 1965 EIF2S1 ILMN_1739821 1978 EIF4EBP1 ILMN_1767324 2021 ENDOG ILMN_1722309 2030 SLC29A1 ILMN_1723971 2055 CLN8 ILMN_1701094 2119 ETV5 ILMN_1739222 2146 EZH2 ILMN_1708105 2161 F12 ILMN_1671766 2184 FAH ILMN_1781536 2222 FDFT1 ILMN_1741096 2224 FDPS ILMN_1804248 2274 FHL2 ILMN_1668411 2582 GALE ILMN_1792168 2617 GARS ILMN_1771026 2618 GART ILMN_1679476 2644 GCHFR ILMN_1694780 2810 SFN ILMN_1806607 3015 H2AFZ ILMN_1707858 3035 HARS ILMN_1763523 3145 HMBS ILMN_1726306 3157 HMGCS1 ILMN_1797728 3163 HMOX2 ILMN_1658807 3241 HPCAL1 ILMN_1764850 3251 HPRT1 ILMN_1736940 3376 IARS ILMN_1733956 3396 ICT1 ILMN_1734508 3422 IDI1 ILMN_1755075 3475 IFRD1 ILMN_1667561 3556 IL1RAP ILMN_1686884 3638 INSIG1 ILMN_1793474 3654 IRAK1 ILMN_1738397 3661 IRF3 ILMN_1765649 3682 ITGAE ILMN_1683927 3730 KAL1 ILMN_1750373 3831 KNS2 ILMN_1654653 3836 KPNA1 ILMN_1751859 3898 LAD1 ILMN_1782389 3949 LDLR ILMN_1651611 3992 FADS1 ILMN_1670134 3995 FADS3 ILMN_1716847 4016 LOXL1 ILMN_1734950 4256 MGP ILMN_1651958 4319 MMP10 ILMN_1741847 4552 MTRR ILMN_1718932 4597 MVD ILMN_1657550 4605 MYBL2 ILMN_1709020 4728 NDUFS8 ILMN_1794132 4860 NP ILMN_1777534 4900 NRGN ILMN_1705686 5029 P2RY2 ILMN_1723535 5106 PCK2 ILMN_1671791 5214 PFKP ILMN_1805737 5277 PIGA ILMN_1705985 5373 PMM2 ILMN_1785336 5723 PSPH ILMN_1776105 5831 PYCR1 ILMN_1796013 5833 PCYT2 ILMN_1652846 5983 RFC3 ILMN_1784860 5985 RFC5 ILMN_1659364 6182 MRPL12 ILMN_1699603 6197 RPS6KA3 ILMN_1652736 6199 RPS6KB2 ILMN_1761175 6301 SARS ILMN_1706949 6319 SCD ILMN_1689329 6472 SHMT2 ILMN_1661264 6510 SLC1A5 ILMN_1707720 6520 SLC3A2 ILMN_1679041 6535 SLC6A8 ILMN_1806349 6541 SLC7A1 ILMN_1683859 6576 SLC25A1 ILMN_1813671 6624 FSCN1 ILMN_1808707 6627 SNRPA1 ILMN_1715179 6674 SPAG1 ILMN_1658731 6721 SREBF2 ILMN_1669615 6723 SRM ILMN_1661337 6897 TARS ILMN_1666498 6949 TCOF1 ILMN_1727551 7083 TK1 ILMN_1806037 7262 PHLDA2 ILMN_1671557 7264 TSTA3 ILMN_1697777 7277 TUBA4A ILMN_1784300 7283 TUBG1 ILMN_1695731 7347 UCHL3 ILMN_1660111 7407 VARS ILMN_1696601 7422 VEGFA ILMN_1803882 7443 VRK1 ILMN_1805828 7453 WARS ILMN_1727271 7791 ZYX ILMN_1701875 7804 LRP8 ILMN_1677765 7866 IFRD2 ILMN_1666194 8078 USP5 ILMN_1671494 8140 SLC7A5 ILMN_1720373 8260 ARD1A ILMN_1721977 8273 SLC10A3 ILMN_1712887 8402 SLC25A11 ILMN_1664168 8407 TAGLN2 ILMN_1700426 8669 EIF3S1 ILMN_1815345 8682 PEA15 ILMN_1771376 8726 EED ILMN_1710150 8818 DPM2 ILMN_1732049 8829 NRP1 ILMN_1699574 8877 SPHK1 ILMN_1702032 8891 EIF2B3 ILMN_1689233 8900 CCNA1 ILMN_1726415 8974 P4HA2 ILMN_1795778 8985 PLOD3 ILMN_1714350 8986 RPS6KA4 ILMN_1756204 9040 UBE2M ILMN_1701331 9129 PRPF3 ILMN_1715392 9141 PDCD5 ILMN_1668425 9145 SYNGR1 ILMN_1727805 9238 TBRG4 ILMN_1783728 9260 PDLIM7 ILMN_1690125 9270 ITGB1BP1 ILMN_1665107 9361 LONP1 ILMN_1766125 9455 HOMER2 ILMN_1671486 9466 IL27RA ILMN_1688152 9631 NUP155 ILMN_1768293 9636 ISG15 ILMN_1813289 9738 CP110 ILMN_1773200 9830 TRIM14 ILMN_1713542 9933 KIAA0020 ILMN_1665483 9948 WDR1 ILMN_1675844 10038 PARP2 ILMN_1775885 10061 ABCF2 ILMN_1781999 10105 PPIF ILMN_1809607 10184 LHFPL2 ILMN_1811077 10273 STUB1 ILMN_1756126 10425 ARIH2 ILMN_1792825 10452 TOMM40 ILMN_1683475 10469 TIMM44 ILMN_1784031 10528 NOL5A ILMN_1787628 10573 MRPL28 ILMN_1694950 10644 IGF2BP2 ILMN_1702447 10807 SDCCAG3 ILMN_1803997 10808 HSPH1 ILMN_1712888 10897 YIF1A ILMN_1712975 10950 BTG3 ILMN_1707339 11151 CORO1A ILMN_1713749 11187 PKP3 ILMN_1753457 11247 NXPH4 ILMN_1741214 11260 XPOT ILMN_1743711 11266 DUSP12 ILMN_1767892 11322 TMC6 ILMN_1794677 11332 ACOT7 ILMN_1740265 11340 EXOSC8 ILMN_1756162 22843 PPM1E ILMN_1708508 22913 RALY ILMN_1690610 23043 TNIK ILMN_1687442 23165 NUP205 ILMN_1673962 23234 DNAJC9 ILMN_1799516 23381 SMG5 ILMN_1721735 23516 SLC39A14 ILMN_1764629 23555 TSPAN15 ILMN_1803850 23590 PDSS1 ILMN_1703324 23594 ORC6L ILMN_1731070 23649 POLA2 ILMN_1696713 23657 SLC7A11 ILMN_1655229 25888 ZNF473 ILMN_1782730 26155 NOC2L ILMN_1716428 26168 SENP3 ILMN_1751147 26227 PHGDH ILMN_1704537 26271 FBXO5 ILMN_1710676 26471 P8 ILMN_1790234 26472 PPP1R14B ILMN_1651406 26521 TIMM8B ILMN_1738938 26973 CHORDC1 ILMN_1776337 27341 RRP7A ILMN_1688178 28231 SLCO4A1 ILMN_1743701 28960 DCPS ILMN_1740737 29107 NXT1 ILMN_1760280 29763 PACSIN3 ILMN_1682957 29893 PSMC3IP ILMN_1675321 29901 SAC3D1 ILMN_1776674 29915 HCFC2 ILMN_1674297 9807 IHPK1 ILMN_1788593 29960 FTSJ2 ILMN_1815933 51016 FAM158A ILMN_1755677 51018 CGI-115 ILMN_1685661 51022 GLRX2 ILMN_1680727 51087 YBX2 ILMN_1755354 51109 RDH11 ILMN_1768719 51236 C8orf30A ILMN_1658577 51330 TNFRSF12A ILMN_1689004 51400 PPME1 ILMN_1732772 192286 HIGD2A ILMN_1774334 51728 POLR3K ILMN_1801664 53838 C11orf24 ILMN_1808163 54205 CYCS ILMN_1730416 54442 KCTD5 ILMN_1672728 54476 TRIAD3 ILMN_1729980 54541 DDIT4 ILMN_1661599 54663 WDR74 ILMN_1809866 54849 DEF8 ILMN_1767509 91860 CALML4 ILMN_1652389 55038 CDCA4 ILMN_1684045 55061 SUSD4 ILMN_1760533 55071 C9orf40 ILMN_1711799 55110 MAGOHB ILMN_1759985 55111 PLEKHJ1 ILMN_1697701 55168 MRPS18A ILMN_1730391 55176 SEC61A2 ILMN_1779381 55224 ETNK2 ILMN_1800130 55312 RFK ILMN_1690780 55341 LSG1 ILMN_1705746 55379 LRRC59 ILMN_1786259 55388 MCM10 ILMN_1706194 55630 SLC39A4 ILMN_1706386 55700 RPRC1 ILMN_1733348 55720 TSR1 ILMN_1775761 55969 C20orf24 ILMN_1679195 55975 KLHL7 ILMN_1674458 56894 AGPAT3 ILMN_1678548 56935 C11orf75 ILMN_1798270 56942 C16orf61 ILMN_1783333 57060 PCBP4 ILMN_1728498 57102 C12orf4 ILMN_1800167 57418 WDR18 ILMN_1694479 57596 BEGAIN ILMN_1744593 57706 DENND1A ILMN_1727315 57761 TRIB3 ILMN_1787815 58526 MID1IP1 ILMN_1668960 64782 ISG20L1 ILMN_1814282 65990 FAM173A ILMN_1773780 79000 C1orf135 ILMN_1787280 79006 METRN ILMN_1712583 79050 NOC4L ILMN_1733107 79071 ELOVL6 ILMN_1700546 79080 CCDC86 ILMN_1689467 79094 CHAC1 ILMN_1739241 79154 MGC4172 ILMN_1756701 79174 CRELD2 ILMN_1748707 79180 EFHD2 ILMN_1761463 79412 KREMEN2 ILMN_1795710 79573 TTC13 ILMN_1745573 79581 GPR172A ILMN_1664723 79609 C14orf138 ILMN_1781102 79693 YRDC ILMN_1736008 79710 MORC4 ILMN_1721526 79711 IPO4 ILMN_1772988 80178 FLJ13909 ILMN_1656452 80179 MYO19 ILMN_1750711 80742 PRR3 ILMN_1750167 81034 SLC25A32 ILMN_1683212 81873 ARPC5L ILMN_1800844 81894 SLC25A28 ILMN_1790472 83743 GRWD1 ILMN_1742166 84065 C1orf160 ILMN_1793632 84549 MAK16 ILMN_1777139 84617 TUBB6 ILMN_1697132 84790 TUBA1C ILMN_1663388 89797 NAV2 ILMN_1675151 92170 MTG1 ILMN_1775328 112398 EGLN2 ILMN_1768773 115201 ATG4A ILMN_1812161 116496 C1orf24 ILMN_1667966 116832 RPL39L ILMN_1712413 203068 TUBB ILMN_1665583 246243 RNASEH1 ILMN_1726783 353189 SLCO4C1 ILMN_1686464 9NAT1 ILMN_1688071 36ACADSB ILMN_1740920 38ACAT1 ILMN_1800008 104ADARB1 ILMN_1679797 107ADCY1 ILMN_1742073 120ADD3 ILMN_1814526 123ADFP ILMN_1660332 143PARP4 ILMN 1776464 -1 224ALDH3A2 ILMN_1794825 -1 288ANK3 ILMN_1657016 -1 317APAF1 ILMN_1659463 -1 318NUDT2 ILMN_1778347 -1 328APEX1 ILMN_1661886 -1 360AQP3 ILMN_1651574 -1 549AUH ILMN_1740349 -1 596BCL2 ILMN_1801119 -1 604BCL6 ILMN_1737314 -1 638BIK ILMN_1770505 -1 665BNIP3L ILMN_1718961 -1 694BTG1 ILMN_1775743 -1 753C18orf1 ILMN_1777436 -1 831CAST ILMN_1672947 -1 857CAV1 ILMN_1687583 -1 900CCNG1 ILMN_1694491 -1 901CCNG2 ILMN_1747244 -1 1102 RCBTB2 ILMN_1771695 -1 1200 TPP1 ILMN_1729234 -1 1299 COL9A3 ILMN_1740155 -1 1315 COPB1 ILMN_1699112 -1 1326 MAP3K8 ILMN_1741159 -1 1362 CPD ILMN_1703074 -1 1604 CD55 ILMN_1800540 -1 29935 RPA4 ILMN_1798880 -1 1733 DIO1 ILMN_1769547 -1 1942 EFNA1 ILMN_1775903 -1 1962 EHHADH ILMN_1751877 -1 1974 EIF4A2 ILMN_1685722 -1 1992 SERPINB1 ILMN_1679133 -1 2053 EPHX2 ILMN_1709237 -1 2065 ERBB3 ILMN_1751346 -1 2066 ERBB4 ILMN_1661857 -1 2073 ERCC5 ILMN_1795495 -1 2186 FALZ ILMN_1692021 -1 2188 FANCF ILMN_1682724 -1 2258 FGF13 ILMN_1671777 -1 2263 FGFR2 ILMN_1682270 -1 2273 FHL1 ILMN_1805842 -1 2308 FOXO1A ILMN_1738816 -1 2330 FMO5 ILMN_1811632 -1 2353 FOS ILMN_1669523 -1 2515 ADAM2 ILMN_1662675 -1 2517 FUCA1 ILMN_1752728 -1 2745 GLRX ILMN_1737308 -1 2752 GLUL ILMN_1653496 -1 2799 GNS ILMN_1744517 -1 2804 GOLGB1 ILMN_1747935 -1 2888 GRB14 ILMN_1793832 -1 2896 GRN ILMN_1724250 -1 2990 GUSB ILMN_1669878 -1 3006 HIST1H1C ILMN_1757406 -1 3012 HIST1H2AE ILMN_1756849 -1 3017 HIST1H2BD ILMN_1758623 -1 3155 HMGCL ILMN_1688095 -1 3176 HNMT ILMN_1751789 -1 3321 IGSF3 ILMN_1713014 -1 3358 HTR2C ILMN_1767145 -1 3431 SP110 ILMN_1731418 -1 3476 IGBP1 ILMN_1717165 -1 3554 IL1R1 ILMN_1810584 -1 3667 IRS1 ILMN_1759232 -1 1506 CTRL ILMN_1664863 -1 3764 KCNJ8 ILMN_1735779 -1 3778 KCNMA1 ILMN_1688785 -1 3781 KCNN2 ILMN_1685636 -1 3797 KIF3C ILMN_1797277 -1 3912 LAMB1 ILMN_1658709 -1 3964 LGALS8 ILMN_1664402 -1 4005 LMO2 ILMN_1800078 -1 4048 LTA4H ILMN_1690342 -1 4126 MANBA ILMN_1800733 -1 4128 MAOA ILMN_1663640 -1 4311 MME ILMN_1678170 -1 4329 ALDH6A1 ILMN_1785284 -1 4477 MSMB ILMN_1699243 -1 4501 MT1X ILMN_1775170 -1 4601 MXI1 ILMN_1794074 -1 4602 MYB ILMN_1711894 -1 4610 MYCL1 ILMN_1764443 -1 4753 NELL2 ILMN_1725417 -1 4781 NFIB ILMN_1778991 -1 4886 NPY1R ILMN_1799878 -1 4889 NPY5R ILMN_1718198 -1 4982 TNFRSF11B ILMN_1676663 -1 5046 PCSK6 ILMN_1802550 1 5087 PBX1 ILMN 1784678 -1 5334 PLCL1 ILMN_1718852 -1 5349 FXYD3 ILMN_1791580 -1 5376 PMP22 ILMN_1785646 -1 5498 PPOX ILMN_1673798 -1 5538 PPT1 ILMN_1669273 -1 5547 PRCP ILMN_1769091 -1 5627 PROS1 ILMN_1671928 -1 5733 PTGER3 ILMN_1759840 -1 5734 PTGER4 ILMN_1795930 -1 5771 PTPN2 ILMN_1686731 -1 5783 PTPN13 ILMN_1652805 -1 5826 ABCD4 ILMN_1746032 -1 5926 ARID4A ILMN_1810229 -1 5993 RFX5 ILMN_1741200 -1 6019 RLN2 ILMN_1748529 -1 6038 RNASE4 ILMN_1776602 -1 6253 RTN2 ILMN_1749115 -1 6414 SEPP1 ILMN_1785071 -1 6642 SNX1 ILMN_1693998 -1 6658 SOX3 ILMN_1653055 -1 6672 SP100 ILMN_1690920 -1 6814 STXBP3 ILMN_1764168 -1 7026 NR2F2 ILMN_1745785 -1 7035 TFPI ILMN_1707124 -1 7045 TGFBI ILMN_1663866 -1 7078 TIMP3 ILMN_1773664 -1 7107 GPR137B ILMN_1794715 -1 7181 NR2C1 ILMN_1728983 -1 7306 TYRP1 ILMN_1699692 -1 7456 WASPIP ILMN_1689645 -1 7508 XPC ILMN_1790807 -1 7580 ZNF32 ILMN_1695362 -1 7770 ZNF227 ILMN_1721034 -1 7844 RNF103 ILMN_1752677 -1 7852 CXCR4 ILMN_1801584 -1 8031 NCOA4 ILMN_1773906 -1 8204 NRIP1 ILMN_1718629 -1 8269 CXorf12 ILMN_1655022 -1 8339 HIST1H2BG ILMN_1716195 -1 8481 OFD1 ILMN_1785266 -1 8502 PKP4 ILMN_1749410 -1 8519 IFITM1 ILMN_1801246 1 8720 MBTPS1 ILMN_1767256 -1 8722 CTSF ILMN_1750122 -1 8800 PEX11A ILMN_1690695 -1 8835 SOCS2 ILMN_1798926 -1 8934 RAB7L1 ILMN_1750202 -1 8991 SELENBP1 ILMN_1680652 -1 9111 NMI ILMN_1739541 -1 9185 REPS2 ILMN_1656934 -1 9187 SLC24A1 ILMN_1747395 -1 9204 ZMYM6 ILMN_1657632 -1 9236 CCPG1 ILMN_1794190 -1 9337 CNOT8 ILMN_1736068 -1 9338 TCEAL1 ILMN_1742341 -1 9367 RAB9A ILMN_1750409 -1 9419 CRIPT ILMN_1813256 -1 9439 MED23 ILMN_1690999 -1 9444 QKI ILMN_1690476 -1 9445 ITM2B ILMN_1713733 -1 9467 SH3BP5 ILMN_1814773 -1 9512 PMPCB ILMN_1728660 -1 9517 SPTLC2 ILMN_1704290 -1 9525 VPS4B ILMN_1792587 -1 9528 TMEM59 ILMN_1792508 -1 9630 GNA14 ILMN_1734857 -1 9638 FEZ1 ILMN_1779071 -1 9649 RALGPS1 ILMN_1674135 -1 9665 LKAP ILMN_1793371 -1 9743 RICS ILMN_1722726 -1 9764 KIAA0513 ILMN_1693233 -1 9766 KIAA0247 ILMN_1786779 -1 9767 PHF16 ILMN_1790518 -1 9772 KIAA0195 ILMN_1669196 -1 9779 TBC1D5 ILMN_1767433 -1 9821 RB1CC1 ILMN_1736796 -1 9847 KIAA0528 ILMN_1682572 -1 9886 RHOBTB1 ILMN_1731831 -1 9906 SLC35E2 ILMN_1651209 -1 9936 CD302 ILMN_1716797 -1 9946 CRYZL1 ILMN_1719290 -1 10023 FRAT1 ILMN_1781416 -1 10040 TOM1L1 ILMN_1802642 -1 10106 CTDSP2 ILMN_1692962 -1 10129 FRY ILMN_1678437 -1 10133 OPTN ILMN_1717586 -1 10142 AKAP9 ILMN_1779315 -1 10179 RBM7 ILMN 1687057 -1 10267 RAMP1 ILMN_1764754 -1 10282 BET1 ILMN_1684042 -1 10284 SAP18 ILMN_1752793 -1 10314 LANCL1 ILMN_1703697 -1 10325 RRAGB ILMN_1741219 -1 10370 CITED2 ILMN_1663092 -1 10397 NDRG1 ILMN_1809931 -1 10451 VAV3 ILMN_1657679 -1 10610 ST6GALNAC2 ILMN_1658706 -1 10742 RAI2 ILMN_1760574 -1 10753 CAPN9 ILMN_1731073 -1 10826 C5orf4 ILMN_1728742 -1 10916 MAGED2 ILMN_1683576 -1 10924 SMPDL3A ILMN_1796349 -1 10928 RALBP1 ILMN_1791840 -1 10957 PNRC1 ILMN_1789955 -1 11045 UPK1A ILMN_1655637 -1 11046 SLC35D2 ILMN_1706571 -1 11069 RAPGEF4 ILMN_1661802 -1 11152 WDR45 ILMN_1756146 -1 11345 GABARAPL2 ILMN_1796458 -1 22882 ZHX2 ILMN_1792951 -1 22920 KIFAP3 ILMN_1697884 -1 23097 CDC2L6 ILMN_1676891 -1 23158 TBC1D9 ILMN_1703891 -1 23240 KIAA0922 ILMN_1712254 -1 23321 TRIM2 ILMN_1745079 -1 23327 NEDD4L ILMN_1733627 -1 23351 KIAA0323 ILMN_1654392 -1 23401 FRAT2 ILMN_1788213 -1 23411 SIRT1 ILMN_1739083 -1 23424 TDRD7 ILMN_1689977 -1 23503 ZFYVE26 ILMN_1798061 -1 23530 NNT ILMN_1806634 -1 23548 OSRF ILMN_1807088 -1 23550 PSD4 ILMN_1699035 -1 23554 TSPAN12 ILMN_1665572 -1 23588 KLHDC2 ILMN_1741204 -1 1122 CHML ILMN_1747697 -1 23678 SGK3 ILMN_1747020 -1 24149 ZNF318 ILMN_1792305 -1 25805 BAMBI ILMN_1691410 METTL A ILMN 1 25913 POT1 ILMN_1692041 -1 25949 SYF2 ILMN_1660186 -1 25996 REXO2 ILMN_1749009 -1 26018 LRIG1 ILMN_1707342 -1 26273 FBXO3 ILMN_1749641 -1 26509 FER1L3 ILMN_1810289 -1 26751 SH3YL1 ILMN_1712231 -1 27010 TPK1 ILMN_1804629 -1 27072 VPS41 ILMN_1768486 -1 27113 BBC3 ILMN_1729645 -1 27241 BBS9 ILMN_1794956 -1 27250 PDCD4 ILMN_1768004 -1 27316 RBMX ILMN_1723580 -1 28965 SLC27A6 ILMN_1797666 -1 29071 C1GALT1C1 ILMN_1751234 -1 29099 COMMD9 ILMN_1808821 -1 29760 BLNK ILMN_1724527 -1 29799 YPEL1 ILMN_1795338 -1 29887 SNX10 ILMN_1786257 -1 29995 LMCD1 ILMN_1754969 -1 51019 CCDC53 ILMN_1715569 -1 51084 CRYL1 ILMN_1714397 -1 51172 NAGPA ILMN_1675266 -1 51227 DSCR5 ILMN_1764871 -1 51231 VRK3 ILMN_1771697 -1 51292 GMPR2 ILMN_1677919 -1 51309 ARMCX1 ILMN_1788192 -1 51315 KRCC1 ILMN_1745620 -1 51363 GALNAC4S-6ST ILMN_1670926 -1 51447 IHPK2 ILMN_1683328 -1 51474 LIMA1 ILMN_1704369 -1 51594 NAG ILMN_1686392 -1 51626 DYNC2LI1 ILMN_1811836 -1 51646 YPEL5 ILMN_1711069 -1 51696 HECA ILMN_1770667 -1 54014 BRWD1 ILMN_1801866 -1 54453 RIN2 ILMN_1769546 -1 54621 FLJ20674 ILMN_1778318 -1 54664 TMEM106B ILMN_1668943 -1 54665 RSBN1 ILMN_1791097 -1 54769 DIRAS2 ILMN_1742846 -1 54800 KLHL24 ILMN_1685297 -1 54816 ZNF280D ILMN_1767142 -1 54974 ICF45 ILMN_1792620 -1 55008 HERC6 ILMN_1654639 -1 55062 WIPI1 ILMN 1781386 _ -1 55196 C12orf35 ILMN_1726289 -1 55204 GOLPH3L ILMN_1655570 -1 55213 RCBTB1 ILMN_1695317 -1 55218 EXDL2 ILMN_1771689 -1 55286 C4orf19 ILMN_1795838 -1 55296 TBC1D19 ILMN_1692844 -1 55315 SLC29A3 ILMN_1717326 -1 55332 FLJ11259 ILMN_1669376 -1 55501 CHST12 ILMN_1735058 -1 55510 DDX43 ILMN_1813893 -1 55625 ZDHHC7 ILMN_1730568 -1 55742 PARVA ILMN_1756408 -1 55763 EXOC1 ILMN_1793766 -1 55788 LMBRD1 ILMN_1652128 -1 55793 FAM63A ILMN_1689552 -1 55812 SPATA7 ILMN_1652409 -1 55818 JMJD1A ILMN_1722532 -1 55827 IQWD1 ILMN_1670000 -1 55830 GLT8D1 ILMN_1713290 -1 55837 C14orf11 ILMN_1741513 -1 55857 C20orf19 ILMN_1779536 -1 55900 ZNF302 ILMN_1652120 -1 56603 CYP26B1 ILMN_1812297 -1 56898 BDH2 ILMN_1673360 -1 56922 MCCC1 ILMN_1760174 -1 56997 CABC1 ILMN_1731064 -1 57007 CMKOR1 ILMN_1798360 -1 57088 PLSCR4 ILMN_1757338 -1 57326 PBXIP1 ILMN_1814315 -1 57419 SLC24A3 ILMN_1663519 -1 57509 MTUS1 ILMN_1663648 -1 57515 SERINC1 ILMN_1741613 -1 57599 WDR48 ILMN_1762103 -1 57728 WDR19 ILMN_1655117 -1 57758 SCUBE2 ILMN_1684085 -1 57763 ANKRA2 ILMN_1710655 -1 58488 PCTP ILMN_1802257 -1 58497 PRUNE ILMN_1728914 -1 59348 ZNF350 ILMN_1744986 -1 60686 C14orf93 ILMN_1724897 -1 63892 THADA ILMN_1811624 -1 63897 ABC1 ILMN_1791106 -1 63901 FAM111A ILMN_1778845 -1 63920 LOC63920 ILMN_1727553 -1 64430 C14orf135 ILMN 1680781 1 64786 TBC1D15 ILMN_1803941 -1 64798 DEPDC6 ILMN_1756685 -1 64927 TTC23 ILMN_1690320 -1 65018 PINK1 ILMN_1666924 -1 65084 TMEM135 ILMN_1700202 -1 79083 MLPH ILMN_1795342 -1 79090 TRAPPC6A ILMN_1775703 -1 79443 FYCO1 ILMN_1709032 -1 79611 ACSS3 ILMN_1659885 -1 79664 NARG2 ILMN_1718830 -1 79665 DHX40 ILMN_1653047 -1 79738 C12orf58 ILMN_1741985 -1 79762 C1orf115 ILMN_1674817 -1 79789 CLMN ILMN_1714522 -1 79864 C11orf63 ILMN_1680659 -1 79901 CYBRD1 ILMN_1712305 -1 79983 POF1B ILMN_1760705 -1 80195 C10orf57 ILMN_1672717 -1 80230 RUFY1 ILMN_1659777 -1 80254 CEP63 ILMN_1787808 -1 80303 EFHD1 ILMN_1779448 -1 80344 WDR23 ILMN_1765825 -1 81031 SLC2A10 ILMN_1663351 -1 81578 COL21A1 ILMN_1732850 -1 81603 TRIM8 ILMN_1746704 -1 81627 C1orf25 ILMN_1665300 -1 83439 TCF7L1 ILMN_1733841 -1 84124 ZNF394 ILMN_1807514 -1 84159 ARID5B ILMN_1721626 -1 89910 UBE3B ILMN_1752027 -1 90362 C8orf72 ILMN_1679217 -1 90864 SPSB3 ILMN_1682864 -1 93129 ORAI3 ILMN_1736628 -1 112611 RWDD2A ILMN_1722156 -1 113791 PIK3IP1 ILMN_1719986 -1 114625 ERMAP ILMN_1715013 -1 114876 OSBPL1A ILMN_1773063 -1 114883 OSBPL9 ILMN_1785167 -1 123872 LRRC50 ILMN_1776967 -1 259230 SGMS1 ILMN_1740505 -1 286148 DPY19L4 ILMN_1775573 ent Entrez_Gene_ID Symbol Probe_Id -1 163AP2B1 ILMN_1681998 1 1058 CENPA ILMN_1801257 1 1284 COL4A2 ILMN_1724994 1 1633 DCK ILMN 1651433 1894 ECT2 ILMN_1719307 2131 EXT1 ILMN_1794343 2781 GNAZ ILMN_1766182 2947 GSTM3 ILMN_1736184 3488 IGFBP5 ILMN_1750324 4175 MCM6 ILMN_1766634 4318 MMP9 ILMN_1796316 5019 OXCT1 ILMN_1813544 5984 RFC4 ILMN_1724489 6515 SLC2A3 ILMN_1775708 7043 TGFB3 ILMN_1687652 8476 CDC42BPA ILMN_1781472 8659 ALDH4A1 ILMN_1656368 8817 FGF18 ILMN_1797030 8833 GMPS ILMN_1771734 8840 WISP1 ILMN_1713499 9055 PRC1 ILMN_1728934 9134 CCNE2 ILMN_1740452 9833 MELK ILMN_1731184 10403 NDC80 ILMN_1664511 10455 PECI ILMN_1761131 10531 PITRM1 ILMN_1698072 10874 NMU ILMN_1740250 23594 ORC6L ILMN_1731070 27113 BBC3 ILMN_1729645 51203 NUSAP1 ILMN_1726720 51377 UCHL5 ILMN_1731612 51514 DTL ILMN_1745251 51560 RAB6B ILMN_1752299 55321 C20orf46 ILMN_1751228 55351 STK32B ILMN_1736356 56942 C16orf61 ILMN_1783333 57110 HRASLS ILMN_1655867 57211 GPR126 ILMN_1658815 57758 SCUBE2 ILMN_1684085 58475 MS4A7 ILMN_1743932 79132 LGP2 ILMN_1678422 79888 LPCAT1 ILMN_1702171 81624 DIAPH3 ILMN_1695226 83879 CDCA7 ILMN_1737184 85453 TSPYL5 ILMN_1737972 91283 C9orf30 ILMN_1714278 1 160897 ITR ILMN_1738712 1 169714 QSCN6L1 ILMN_1789384 Desmedt et al. Farmer et al. Stroma.1 coefficient Entrez_Gene_ID Symbol Probe_Id Immune.2.STAT1 1 6772 STAT1 ILMN_1690105 1 3627 CXCL10 ILMN_1791759 1 6890 TAP1 ILMN_1751079 1 6373 CXCL11 ILMN_1719597 1 3620 INDO ILMN_1656310 1 4283 CXCL9 ILMN_1745356 1 4599 MX1 ILMN_1662358 1 27074 LAMP3 ILMN_1719734 1 9636 ISG15 ILMN_1813289 1 64108 RTP4 ILMN_1788013 1 55008 HERC6 ILMN_1654639 1 10964 IFI44L ILMN_1723912 1 4600 MX2 ILMN_1754619 1 3437 IFIT3 ILMN_1701789 1 51191 HERC5 ILMN_1729749 1 91543 RSAD2 ILMN_1657871 1 23586 DDX58 ILMN_1797001 1 6352 CCL5 ILMN_1773352 1 27299 ADAMDEC1 ILMN_1716909 1 914CD2 ILMN_1695025 1 55601 FLJ20035 ILMN_1795181 1 10866 HCP5 ILMN_1803945 1 9111 NMI ILMN_1739541 1 9806 SPOCK2 ILMN_1656287 1 6355 CCL8 ILMN_1772964 1 10346 TRIM22 ILMN_1779252 1 4069 LYZ ILMN_1815205 1 3659 IRF1 ILMN_1708375 1 3902 LAG3 ILMN_1813338 1 9595 PSCDBP ILMN_1746864 1 22797 TFEC ILMN_1711332 1 10537 UBD ILMN_1678841 1 11262 SP140 ILMN_1778599 1 1075 CTSC ILMN_1696347 1 2537 G1P3 ILMN_1687384 1 7941 PLA2G7 ILMN_1701195 1 917CD3G ILMN_1717197 1 1890 ECGF1 ILMN 1690939 160897 ITR ILMN_1738712 169714 QSCN6L1 ILMN_1789384 Gene_ID Symbol Probe_Id 6772 STAT1 ILMN_1690105 3627 CXCL10 ILMN_1791759 6890 TAP1 ILMN_1751079 6373 CXCL11 ILMN_1719597 3620 INDO ILMN_1656310 4283 CXCL9 ILMN_1745356 4599 MX1 ILMN_1662358 27074 LAMP3 ILMN_1719734 9636 ISG15 ILMN_1813289 64108 RTP4 ILMN_1788013 55008 HERC6 ILMN_1654639 10964 IFI44L ILMN_1723912 4600 MX2 ILMN_1754619 3437 IFIT3 ILMN_1701789 51191 HERC5 ILMN_1729749 91543 RSAD2 ILMN_1657871 23586 DDX58 ILMN_1797001 6352 CCL5 ILMN_1773352 27299 ADAMDEC1 ILMN_1716909 914CD2 ILMN_1695025 55601 FLJ20035 ILMN_1795181 10866 HCP5 ILMN_1803945 9111 NMI ILMN_1739541 9806 SPOCK2 ILMN_1656287 6355 CCL8 ILMN_1772964 10346 TRIM22 ILMN_1779252 4069 LYZ ILMN_1815205 3659 IRF1 ILMN_1708375 3902 LAG3 ILMN_1813338 9595 PSCDBP ILMN_1746864 22797 TFEC ILMN_1711332 10537 UBD ILMN_1678841 11262 SP140 ILMN_1778599 1075 CTSC ILMN_1696347 2537 G1P3 ILMN_1687384 7941 PLA2G7 ILMN_1701195 917CD3G ILMN_1717197 1890 ECGF1 ILMN_1690939 51316 PLAC8 ILMN_1653026 10875 FGL2 ILMN_1746471 3003 GZMK ILMN_1710734 962CD48 ILMN_1753326 6775 STAT4 ILMN 1785202 2841 GPR18 ILMN_1780368 5026 P2RX5 ILMN_1677793 10437 IFI30 ILMN_1682846 4068 SH2D1A ILMN_1705892 7805 LAPTM5 ILMN_1772359 969CD69 ILMN_1651316 5778 PTPN7 ILMN_1715214 3394 IRF8 ILMN_1666594 11040 PIM2 ILMN_1727624 51513 ETV7 ILMN_1759014 29909 GPR171 ILMN_1761166 5720 PSME1 ILMN_1806017 330BIRC3 ILMN_1696549 356FASLG ILMN_1781824 8519 IFITM1 ILMN_1801246 24138 IFIT5 ILMN_1696654 3689 ITGB2 ILMN_1654396 11118 BTN3A2 ILMN_1676528 3059 HCLS1 ILMN_1727402 6398 SECTM1 ILMN_1652277 55843 ARHGAP15 ILMN_1777882 22914 KLRK1 ILMN_1722991 10261 IGSF6 ILMN_1689415 1880 EBI2 ILMN_1798706 26034 PIP3-E ILMN_1796497 29887 SNX10 ILMN_1786257 79132 LGP2 ILMN_1678422 684BST2 ILMN_1723480 55337 FLJ11286 ILMN_1750400 341APOC1 ILMN_1789007 51237 PACAP ILMN_1662878 445347 TARP ILMN_1723944 56829 ZC3HAV1 ILMN_1729973 23564 DDAH2 ILMN_1770787 23547 LILRA4 ILMN_1702385 10148 EBI3 ILMN_1802653 3823 KLRC3 ILMN_1658387 50856 CLEC4A ILMN_1709204 959CD40LG ILMN_1659077 7409 VAV1 ILMN_1717334 2745 GLRX ILMN_1737308 54ACP5 ILMN_1761728 1 3606 IL18 ILMN_1778457 -1 1942 EFNA1 ILMN_1775903 1 10125 RASGRP1 ILMN_1768958 1 9985 REC8L1 ILMN_1751886 -1 10126 DNAL4 ILMN_1801845 t Entrez_Gene_ID Symbol Probe_Id -1 5728 PTEN ILMN_1701134 1 4291 MLF1 ILMN_1684439 1 1010 CDH12 ILMN_1709269 -1 145781 Gcom1 ILMN_1674151 1 4751 NEK2 ILMN_1653822 1 6612 SUMO3 ILMN_1725642 -1 2331 FMOD ILMN_1789639 1 643224 LOC643224 ILMN_1802181 -1 10399 GNB2L1 ILMN_1736500 1 7372 UMPS ILMN_1757437 1 11073 TOPBP1 ILMN_1684929 1 126731 C1orf96 ILMN_1699217 1 5052 PRDX1 ILMN_1710159 -1 5094 PCBP2 ILMN_1724825 1 330BIRC3 ILMN_1696549 1 5290 PIK3CA ILMN_1705468 -1 1265 CNN2 ILMN_1770290 -1 26123 C10orf61 ILMN_1678652 1 79132 LGP2 ILMN_1678422 1 3838 KPNA2 ILMN_1708160 1 9711 KIAA0226 ILMN_1763637 -1 8510 MMP23B ILMN_1808107 -1 80306 MED28 ILMN_1749821 -1 2043 EPHA4 ILMN_1672022 1 26973 CHORDC1 ILMN_1776337 -1 84896 ATAD1 ILMN_1654497 1 1164 CKS2 ILMN_1756326 1 8208 CHAF1B ILMN_1674231 1 24137 KIF4A ILMN_1799667 -1 23613 ZMYND8 ILMN_1652407 1 87178 PNPT1 ILMN_1810608 1 11004 KIF2C ILMN_1685916 -1 27122 DKK3 ILMN_1815673 1 205564 SENP5 ILMN_1675501 -1 8724 SNX3 ILMN_1740180 -1 11171 STRAP ILMN_1731194 -1 6228 RPS23 ILMN_1772459 1 54503 ZDHHC13 ILMN_1684663 1 1207 CLNS1A ILMN_1736814 1 1062 CENPE ILMN 1716279 Saal et al. Farmer et al. Stroma.1 PTEN 3416 IDE ILMN_1769883 9833 MELK ILMN_1731184 5169 ENPP3 ILMN_1749131 140459 ASB6 ILMN_1806705 51642 MRPL48 ILMN_1774580 56977 STOX2 ILMN_1803256 5612 PRKRIR ILMN_1655622 10440 TIMM17A ILMN_1656798 84942 WDR73 ILMN_1702592 3925 STMN1 ILMN_1657796 7307 U2AF1 ILMN_1772113 27316 RBMX ILMN_1723580 27238 GPKOW ILMN_1684197 50861 STMN3 ILMN_1693425 1503 CTPS ILMN_1783285 54556 ING3 ILMN_1806226 11169 WDHD1 ILMN_1756043 4172 MCM3 ILMN_1806818 9037 SEMA5A ILMN_1656927 10383 TUBB2C ILMN_1780769 1889 ECE1 ILMN_1672174 10121 ACTR1A ILMN_1792314 9392 TGFBRAP1 ILMN_1696870 9787 DLG7 ILMN_1749829 151246 SGOL2 ILMN_1746699 3705 ITPK1 ILMN_1715674 891CCNB1 ILMN_1712803 83700 JAM3 ILMN_1769575 5984 RFC4 ILMN_1724489 5718 PSMD12 ILMN_1691428 8897 MTMR3 ILMN_1739641 23111 SPG20 ILMN_1801236 29105 C16orf80 ILMN_1707100 6732 SRPK1 ILMN_1798804 5870 RAB6A ILMN_1800871 81610 C20orf129 ILMN_1781943 57380 MRS2L ILMN_1778677 3066 HDAC2 ILMN_1767747 2618 GART ILMN_1679476 25852 ARMC8 ILMN_1781151 64793 CCDC21 ILMN_1812489 23279 NUP160 ILMN_1652989 6632 SNRPD1 ILMN_1768393 10972 TMED10 ILMN_1736585 8450 CUL4B ILMN_1691535 9133 CCNB2 ILMN 1801939 7159 TP53BP2 ILMN_1779706 6434 SFRS10 ILMN_1742798 151636 DTX3L ILMN_1784380 4094 MAF ILMN_1719543 151188 ARL6IP6 ILMN_1797964 4629 MYH11 ILMN_1660086 5873 RAB27A ILMN_1665859 2999 GZMH ILMN_1731233 8681 PLA2G4B ILMN_1697629 5111 PCNA ILMN_1694177 5359 PLSCR1 ILMN_1752889 79694 MANEA ILMN_1757026 84955 NUDCD1 ILMN_1678745 10606 PAICS ILMN_1773760 4259 MGST3 ILMN_1751956 51028 VPS36 ILMN_1802519 4038 LRP4 ILMN_1675268 64844 Mar-07ILMN_1717337 200734 SPRED2 ILMN_1791232 10541 ANP32B ILMN_1684293 92342 C1orf156 ILMN_1734915 64789 C1orf176 ILMN_1783996 6397 SEC14L1 ILMN_1732575 11257 TP53AP1 ILMN_1658469 9044 BTAF1 ILMN_1791536 9232 PTTG1 ILMN_1753196 7284 TUFM ILMN_1738369 10951 CBX1 ILMN_1770244 54845 RBM35A ILMN_1749180 3192 HNRNPU ILMN_1718571 54838 C10orf26 ILMN_1658830 1848 DUSP6 ILMN_1677466 10963 STIP1 ILMN_1745906 6772 STAT1 ILMN_1690105 387103 C6orf173 ILMN_1763907 6426 SFRS1 ILMN_1795341 2767 GNA11 ILMN_1739781 9897 KIAA0196 ILMN_1728676 3608 ILF2 ILMN_1745172 25794 FSCN2 ILMN_1795472 9928 KIF14 ILMN_1808071 6941 TCF19 ILMN_1682008 29028 ATAD2 ILMN_1763064 3833 KIFC1 ILMN 1764840 1 699BUB1 ILMN_1736090 -1 8576 STK16 ILMN_1803774 1 8532 CPZ ILMN_1766780 1 7936 RDBP ILMN_1765532 1 10589 DRAP1 ILMN_1733048 1 219988 PATL1 ILMN_1680782 -1 374868 ATP9B ILMN_1658684 -1 8848 TSC22D1 ILMN_1692177 -1 10540 DCTN2 ILMN_1662232 -1 3983 ABLIM1 ILMN_1785424 1 5889 RAD51C ILMN_1695386 1 6790 AURKA ILMN_1680955 -1 10975 UQCR ILMN_1745049 1 55827 IQWD1 ILMN_1670000 1 60313 GPBP1L1 ILMN_1662719 -1 55662 HIF1AN ILMN_1681812 -1 54531 MIER2 ILMN_1766159 -1 11329 STK38 ILMN_1799153 1 6619 SNAPC3 ILMN_1803611 1 157313 CDCA2 ILMN_1660654 -1 11337 GABARAP ILMN_1692338 -1 5159 PDGFRB ILMN_1815057 -1 5797 PTPRM ILMN_1744937 1 3796 KIF2A ILMN_1734476 1 9493 KIF23 ILMN_1811472 1 7444 VRK2 ILMN_1750088 1 29979 UBQLN1 ILMN_1688622 1 29127 RACGAP1 ILMN_1702140 -1 80031 SEMA6D ILMN_1814494 1 54014 BRWD1 ILMN_1801866 1 79894 ZNF672 ILMN_1669094 1 9582 APOBEC3B ILMN_1691457 -1 221981 LOC221981 ILMN_1653057 1 4175 MCM6 ILMN_1766634 -1 9252 RPS6KA5 ILMN_1657515 1 51773 RSF1 ILMN_1668834 1 54534 MRPL50 ILMN_1664833 1 79572 ATP13A3 ILMN_1663684 1 54625 PARP14 ILMN_1691731 -1 55101 ATP5SL ILMN_1809027 -1 1311 COMP ILMN_1677636 1 8317 CDC7 ILMN_1741801 1 10206 TRIM13 ILMN 16 4260
https://openalex.org/W1968540097	https://europepmc.org/articles/pmc4125812?pdf=render	English	null	Weight Loss Programs May Have Beneficial or Adverse Effects on Fat Mass and Insulin Sensitivity in Overweight and Obese Black Women	Journal of racial and ethnic health disparities	2,014	cc-by	5,516	Abstract Objective Weight loss interventions have produced little change in insulin sensitivity in black women, but mean data may obscure metabolic benefit to some and adverse effects for others. Accordingly, we analyzed insulin sensitivity relative to fat mass change following a weight loss program. Design and Methods Fifty-four black women (BMI range 25.9 to 54.7 kg/m2) completed the 6-month program that included nutrition information and worksite exercise facilities. Fat mass was measured by dual-energy X-ray absorptiometry, and insulin sensitivity index (SI) was calculated from an insulin-modified intravenous glucose tolerance test using the minimal model. Conclusions Black women in a weight loss program who lose fat mass may have improved insulin sensitivity, but fat mass gain with diminished sensitivity is common. Additional sup- port for participants who fail to achieve fat mass loss early in an intervention may be required for success. Results Baseline SI (range 0.74 to 7.58 l/mU−1•min−1) was inversely associated with fat mass (r= −0.516, p<0.001), inde- pendent of age. On average, subjects lost fat mass (baseline Keywords Obesity . insulin sensitivity . women . race . intervention B. Leon: G. Zalos: T. M. Powell-Wiley: R. O. Cannon III (*) Cardiovascular and Pulmonary Branch, National Heart, Lung, and Blood Institute, Building 10-CRC Room 5-3330, 10 Center Drive, Bethesda, MD 20892, USA e-mail: cannonr@nih.gov J. Racial and Ethnic Health Disparities (2014) 1:140–147 DOI 10.1007/s40615-014-0006-6 J. Racial and Ethnic Health Disparities (2014) 1:140–147 DOI 10.1007/s40615-014-0006-6 Weight Loss Programs May Have Beneficial or Adverse Effects on Fat Mass and Insulin Sensitivity in Overweight and Obese Black Women Benjamin Leon & Bernard V. Miller III & Gloria Zalos & Amber B. Courville & Anne E. Sumner & Tiffany M. Powell-Wiley & Mary F. Walter & Myron A. Waclawiw & Richard O. Cannon III Received: 16 October 2013 /Revised: 17 December 2013 /Accepted: 2 January 2014 /Published online: 5 March 2014 # The Author(s) 2014. This article is published with open access at Springerlink.com 40.8±12.4 to 39.4±12.6 kg [mean±SD], P<0.01), but 17 women (32 %) actually gained fat mass. SI for the group was unchanged (baseline 3.3±1.7 to 3.2±1.6, P=0.67). However, the tertile with greatest fat mass loss (−3.6 kg, range −10.7 to −1.7 kg) improved insulin sensitivity (SI +0.3±1.2), whereas the tertile with net fat mass gain (+0.9 kg, range −0.1 to + 3.8 kg) had reduced insulin sensitivity (SI −0.7±1.3) from baseline values (P<0.05 by ANOVA). Introduction The obesity epidemic has steadily worsened as nearly 70 % of adults in the USA are at least overweight and more than one- third are obese [1, 2]. The prevalence of obesity is the highest among underrepresented minority populations, particularly blacks who have the highest age-adjusted rate of obesity na- tionwide [1, 2]. Additionally, blacks also have a disproportion- ately high prevalence of type 2 diabetes and are at greater risk of developing associated cardiovascular complications including coronary artery disease, myocardial infarction, stroke, conges- tive heart failure, and peripheral vascular disease [3–6]. M. A. Waclawiw Office of Biostatistics Research, National Heart, Lung, and Blood Institute, Bethesda, MD, USA M. A. Waclawiw Office of Biostatistics Research, National Heart, Lung, and Blood Institute, Bethesda, MD, USA M. A. Waclawiw Office of Biostatistics Research, National Heart, Lung, and Blood Institute, Bethesda, MD, USA B. V. Miller III: A. E. Sumner Diabetes, Endocrinology and Obesity Branch, Clinical Center; National Institutes of Health, Bethesda, MD, USA M. F. Walter Core for Clinical Laboratory Services, Clinical Center; National Institutes of Health, Bethesda, MD, USA M. F. Walter Core for Clinical Laboratory Services, Clinical Center; National Institutes of Health, Bethesda, MD, USA Major contributors to the growing obesity epidemic are inactivity and excess energy intake [7]. Evidence suggests that the work place contributes to the prevalence of obesity as employees in the USA are sedentary for large portions of A. B. Courville Nutrition Department, Clinical Center; National Institutes of Health, Bethesda, MD, USA A. B. Courville Nutrition Department, Clinical Center; National Institutes of Health, Bethesda, MD, USA A. B. Courville Nutrition Department, Clinical Center; National Institutes of Health, Bethesda, MD, USA J. Racial and Ethnic Health Disparities (2014) 1:140–147 141 the workday [7, 8]. To counter the mounting obesity rates, many organizations have initiated wellness programs at the work site to achieve weight loss by encouraging exercise and reduced caloric intake [9–14]. While many of these programs have been effective in promoting weight loss and improving health measures among whites, black women have been less successful in achieving these ends [15]. Therefore, we hy- pothesized that an intervention at the worksite that provides women with healthful information, either through interactive group sessions or internet-based tools, as well as exercise resources in the work place would enable fat mass loss and improve insulin-mediated glucose metabolism in overweight and obese black women. Methods and Procedures Study population Overweight (body mass index [BMI] 25 to <30 kg/m2) and obese (BMI≥30 kg/m2) nondiabetic (fasting glucose <126 mg/dL) black (by self-report) female employees of the National Institutes of Health (NIH), Bethesda, Mary- land were enrolled. Participants were recruited by flyers dis- tributed across the Bethesda campus of NIH and self- identified as healthy without participation in structured exer- cise or weight loss programs and weight stable (fluctuation in weight < 5 %) over the previous 3 months. Women were excluded from participation if screening blood work revealed anemia (hemoglobin <11 g/dL), liver, kidney, or thyroid dis- ease. Prescription medications at stable doses for at least 2 months – including hormonal preparations for thyroid dys- function or estrogen preparations (i.e., birth control or post- menopausal hormone therapy) – were permitted, but a change in medications during the study was prespecified as an exclu- sion criterion from further participation due to the potential of confounding main outcome measures. The protocol was ap- proved by the Institutional Review Board of the National Heart Lung and Blood (NHLBI) and registered in www. ClinicalTrials.gov (NCT00666172) prior to study initiation. All subjects provided informed consent. Body weight (to the nearest 0.1 kg, Scale-Tronix 5702, Carol Stream, IL) and height (by stadiometry to the nearest 0.1 cm, Seca 242, Hanover, MD) were measured in light- weight clothing. Body fat (%) and truncal fat (%) were mea- sured by dual-energy X-ray absorptiometry (DXA; iDXA Software Encore 11.10, GE Lunar Medical Systems, Madi- son, WI). Fat mass (kg) and % truncal fat mass were calculat- ed using the bodyweight and DXA measurements. Exercise performance was measured during a graded treadmill exercise test [18] with a SensorMedics Vmax Spectra 229c metabolic cart (CareFusion, San Diego, CA) for the analysis of oxygen consumption at peak exercise (VO2 peak). In addition, women were asked to record daily pedometer counts and track the amount of time walking and using aerobic exercise machines each day throughout the 6-month study. Activity diaries were submitted on a weekly basis. A reduced sample insulin-modified intravenous glucose tolerance test (FSIGT) was used in conjunction with the minimal model of glucose kinetics to determine an insulin sensitivity index (SI) (MinMOD Millenium v6.02, Los Angeles, CA) [19, 20]. Testing was performed at 8 AM fol- lowing a 12-h fast after placement of intravenous lines in both antecubital veins. Introduction Specifically, we proposed that this decrease in fat mass would improve insulin sensitivity for those women who completed the diet and exercise program. card-key access to private fitness rooms located in three buildings on campus, each equipped with aerobic exercise equipment (e.g., treadmill, elliptical machine, and supine bi- cycle). Participants were also encouraged to continue physical activity – especially walking – on nonwork days. Enrollees were randomized into either intervention or control groups: Intervention participants attended nutrition education sessions (weekly for the first 3 months, monthly for the last 3 months) conducted by a registered dietitian, whereas control subjects were not provided this instruction. Subjects were compensat- ed for their time and inconvenience of testing. Testing All tests were performed at the NIH Clinical Center, Bethesda, Maryland at baseline and at 6 months following an overnight fast. Premenopausal women were scheduled for testing within the first 10 days of their menstrual cycle. Base- line and 6 month visits included collection and review of 3- day food records by the registered dietitians. The food records, which included two work days and one nonwork day, were reviewed for accuracy by dietitians and then analyzed using the Nutrition Data System for Research (NDS-R versions 2009–2011, Minneapolis, MN) [17]. Methods and Procedures After obtaining baseline glucose and insulin samples, dextrose (0.3 g/kg) was injected at time 0 over approximately1 min. A bolus of insulin (0.03 units/kg) was injected rapidly at 20 min. Blood samples were taken for determination of glucose and insulin concentrations at 0, 2, 4, 8, 19, 22, 30, 40, 50, 70, 100, and 180 min. The acute insulin response to glucose (AIRg) was determined by the area under the insulin concentration curve above basal insulin concentration between 0 and 10 min. Study Design All participants were provided internet-based nutrition and exercise information created by NHLBI for employees (recent version can be found at http://apps.nhlbi. nih.gov/keepthebeat) that included recommendations from the Department of Health and Human Services and the US Department of Agriculture [16]. The web site included walking paths around the NIH Bethesda campus, sample menus, healthful lifestyle information, and tools for counting calories. Each participant was given a pedometer (Walk4Life, Plainfield, IL) with instructions to increase average daily step count by 5,000 steps over their baseline average and given 142 J. Racial and Ethnic Health Disparities (2014) 1:140–147 determined by multivariable regression analysis. All adjusted model analyses were performed using the SAS statistical anal- ysis package (SAS User’s Guide: Statistics, Version 9 Edition: SAS Institute Inc, Cary, NC). Skewed data were log trans- formed. A P-value ≤0.05 was considered statistically signifi- cant. Nonskewed data are reported as mean±standard devia- tion. Tertiles of fat mass change are reported as mean and range of values within each tertile. Leptin was measured in serum by ELISA (Millipore, Bil- lerica, MA). The minimum detectable concentration was 0.5 ng/ml, with intraassay CV 3.7 % and interassay CV 4.0 %. Adiponectin was measured in serum by ELISA (R&D Systems, Minneapolis, MN). The minimum detectable concentration was 0.25 ng/ml, with intraassay CV 3.5 % and interassay CV 6.5 %. y One hundred forty-one black women were assessed for eligibility in this protocol, and 114 were enrolled for partici- pation (Fig. 1). Of the 114 participants who underwent base- line testing, 28 women were excluded from analysis due to failed baseline or follow-up FSIGT. Reasons for failed FSIGT’s included hemolyzed samples (n=21), inability to place intravenous lines in antecubital veins of both arms (n= 4), improper testing technique (n=2), and poor data fit for predicted glucose disposal (n=1). In addition, 32 women withdrew from the study prior to completing 6 months partic- ipation. Results For the 54 subjects included in the analysis for this report, the average age at baseline was 45±10 years with an average BMI of 34.2±6.0 kg/m2 (range 25.9 to 54.7 kg/m2); 70 % had a BMI≥30 kg/m2. Menopausal status of the subjects was 37 premenopausal [five on hormonal contraception therapy (14 %)], 17 perimenopausal, or postmenopausal [two on hormonal therapy (12 %)]. Energy intake for the group at baseline was 2,024±614 kcal/day. Baseline measures of fat mass, exercise performance, insulin sensitivity, and adipokines leptin and adiponectin are shown in Table 1. At baseline, SI for the 54 subjects ranged from 0.74 to 7.58 L•mU−1•min−1 and was inversely associated with total and % truncal fat masses when adjusted for age (Fig. 2). Additionally, SI was positively associated with exercise per- formance by VO2 peak at baseline (r=0.448, P<0.001). Lep- tin was positively associated with total fat mass (r=0.652, P<0.001) and % truncal fat mass (r=0.643, P<0.001), and inversely associated with SI (r= −0.492, P<0.001). Adiponectin levels, however, were not related to baseline fat mass (either total or % truncal) or SI (all P>0.208). Endoge- nous insulin response, AIRg, was positively associated with total fat mass (r=0.310, P=0.024) but not with % truncal fat mass (P=0.200). Statistical Analysis As it was anticipated that not all subjects would lose weight and fat mass, it was planned that changes in insulin sensitivity would be analyzed for those who lost the most fat mass compared with those who did not lose fat mass. Accordingly, the primary analysis for this report was change in insulin sensitivity by tertile of change in fat mass for women who had measures both at baseline and at 6-month completion of the protocol by analysis of variance (ANOVA) with posthoc testing among tertiles, if statistically significant. Since no sig- nificant difference was determined in weight loss (−2.1±3.5 kg vs. −1.1±3.9 kg, P=0.167) following program completion between those subjects randomized to attend group nutrition instructional sessions (n=26) and those who were provided internet-based nutrition and exercise information only (n=28), the groups were combined for this analysis. The contribution of exercise performance to insulin sensitivity at baseline and fol- lowing program completion was also investigated. Paired or unpaired t-tests for continuous data and chi-square proportion- ality tests were performed using InStat biostatistics software (Version 3.06, 2003). Methods and Procedures Thus, 54 subjects underwent testing for fat mass and insulin sensitivity at baseline and at 6 months. To evaluate whether the characteristics of these 54 subjects differed from the remaining subjects who either had incomplete data or dropped out of the study (Fig. 1), we compared all baseline variables. The two groups did not differ in age or baseline BMI, total fat mass, % truncal fat mass, insulin, glucose, or SI (all P>0.1). Those that dropped out or with incomplete data, however, had significantly lower baseline VO2 peak than those who completed the study (20.7±4.4 vs. 22.8±4.9 mL O2/kg/min, P=0.019). Results Univariate associations among fat mass measurements, adipokines, exercise performance, and insulin sensitivity were performed using either Pearson’s correlation or Spearman’s rank correlation, as appropriate. Independent pre- dictors of change in SI following program completion were At program completion, reduction in energy intake (−339± 653 kcal/day P<0.001) was determined for the group, based on analyses of baseline and 6-month 3-day food records. On average, subjects reported the following activity measures: time devoted to daily walking 20±24 min/day and to aerobic exercise 8±8 min/day, with pedometer counts 6,131±3,596 steps/day. Six-month measures of weight, fat mass, exercise performance, insulin sensitivity, and adipokine levels are shown in Table 1. Significant reductions in weight (−1.6± 3.7 kg, range −11.6 to +5.5 kg; P<0.01), total fat mass (−1.4± 2.8 kg, range −10.7 to +3.8 kg; P<0.01) and % truncal mass (−1.1±2.7 kg, range −10.9 to +3.4 kg P<0.01), improvement in VO2 peak (+1.0±3.1 mL O2/kg/min, range +9.6 to −4.1 mL O2/kg/min; P<0.001) and reduction in leptin levels (−9.1± 27.2 ng/mL, range −116.6 to +50.2 ng/mL; P<0.001) were determined for subjects at 6 months. Improvement in SI from baseline to 6-month follow-up was significantly associated 143 J. Racial and Ethnic Health Disparities (2014) 1:140–147 Fig. 1 Flow chart of participant participation Fig. 1 Flow chart of participant participation with reductions in both total FM (r= −0.348, P=0.010) and % truncal FM (r= −0.423, P=0.001). There was no association between the change in VO2 peak and change in SI. Reduction in % truncal fat mass was associated with reduction in leptin (r=0.381, P=0.008). Reduction in leptin was associated with improvement in SI (r= −0.337, P=0.021). Change in AIRg showed a trend toward significance with the change in total fat mass (r=0.238, P=0.087) but not with the change in % truncal fat mass (P=0.135) following completion of the program. pedometer counts (5,574±2,891 vs. 6,394±3,895 steps/day) similar to the 37 who lost fat mass (all P>0.2). Weight gain was observed with similar frequency among women who were overweight (4 of 16) or obese (11 of 38, P=0.729) at the beginning of the program. There was a trend toward fewer women experiencing weight gain in the group randomized to attend nutrition instructional sessions (4 of 26) vs. those who were provided internet-based nu- trition and exercise information only (11 of 28, P= 0.098). Results When participants were analyzed by tertiles of fat mass change and % truncal fat mass change, the tertile with the greatest fat mass loss improved insulin sensitivity in contrast to the tertile with net fat mass gain in which SI worsened (Fig. 3). By multivariable Although on average subjects lost total fat mass and % truncal fat mass, 17 women (32 %) actually gained fat mass despite reporting energy intake reduction (−483±697 vs. −287±623 kcal/day), time devoted to daily walking (27±25 vs. 17±24 min) and aerobic exercise (7±5 vs. 9±10 min), and J. Racial and Ethnic Health Disparities (2014) 1:140–147 144 regression analysis, changes in either total fat mass (β= −0.038, P=0.039) or % truncal fat mass (β= −0.047, P= 0.019) were independent predictors of change in SI, after adjustment for age, change in leptin, and change in VO2 peak. To help identify possible indicators of change in fat mass following program participation, subject characteristics of the tertile with the greatest fat mass loss were compared to the tertile with net fat mass gain. There were no significant differences between the two groups in age or baseline weight, BMI, hormonal status (premenopaus- al, perimenopausal, or postmenopausal), total fat mass, % truncal fat mass, VO2 peak, adipokine levels, glucose and insulin levels, SI, or AIRg (all P>0.2). Although the women in the tertile with the greatest fat mass loss had a slightly greater proportion of participants in the intervention group with dietitian-led classes vs. the tertile with net fat mass gain (61 vs. 44 %), this did not achieve statistical significance (P=0.317). Table 1 Data for 54 overweight and obese black women who completed the 6-month weight loss program Baseline 6 months P-value Adipositya Weight (kg) 92.6±18.1 91.1±18.9 0.003 Total fat mass (kg) 40.8±12.4 39.4±12.6 <0.001 % Truncal fat mass (%) 46.4±6.6 45.2±6.7 0.003 Exercise performancea Peak VO2 (mL/kg/min) 22.8±4.9 23.8±5.3 0.028 Adipokinesa Leptin (ng/ml) 61.1±37.1 52.1±24.2 0.027 Adiponectin (μg/ml) 5.6±3.6 5.7±2.9 0.389 Insulin sensitivitya Insulin (μU/ml) 7.2±5.4 6.9±6.4 0.077 Glucose (μU/ml) 91±9 88±10 0.060 Insulin sensitivity index (SI) 3.30±1.71 3.21±1.56 0.665 AIRg 529±461 548±455 0.335 a Values represented as mean±SD Fig. 2 Baseline correlations between insulin sensitivity index and total fat mass (top) and % truncal fat mass (bottom), adjusted for age 144 J. Results Racial and Ethnic Health Disparities (2014) 1:140–147 Table 1 Data for 54 overweight and obese black women who completed the 6-month weight loss program Baseline 6 months P-value Adipositya Weight (kg) 92.6±18.1 91.1±18.9 0.003 Total fat mass (kg) 40.8±12.4 39.4±12.6 <0.001 % Truncal fat mass (%) 46.4±6.6 45.2±6.7 0.003 Exercise performancea Peak VO2 (mL/kg/min) 22.8±4.9 23.8±5.3 0.028 Adipokinesa Leptin (ng/ml) 61.1±37.1 52.1±24.2 0.027 Adiponectin (μg/ml) 5.6±3.6 5.7±2.9 0.389 Insulin sensitivitya Insulin (μU/ml) 7.2±5.4 6.9±6.4 0.077 Glucose (μU/ml) 91±9 88±10 0.060 Insulin sensitivity index (SI) 3.30±1.71 3.21±1.56 0.665 AIRg 529±461 548±455 0.335 a Values represented as mean±SD Table 1 Data for 54 overweight and obese black women who completed the 6-month weight loss program regression analysis, changes in either total fat mass (β= −0.038, P=0.039) or % truncal fat mass (β= −0.047, P= 0.019) were independent predictors of change in SI, after adjustment for age, change in leptin, and change in VO2 peak. To help identify possible indicators of change in fat mass following program participation, subject characteristics of the tertile with the greatest fat mass loss were compared to the tertile with net fat mass gain. There were no significant differences between the two groups in age or baseline weight, BMI, hormonal status (premenopaus- al, perimenopausal, or postmenopausal), total fat mass, % truncal fat mass, VO2 peak, adipokine levels, glucose and insulin levels, SI, or AIRg (all P>0.2). Although the women in the tertile with the greatest fat mass loss had a slightly greater proportion of participants in the intervention group with dietitian-led classes vs. the tertile with net fat mass gain (61 vs. 44 %), this did not achieve statistical significance (P=0.317). had a slightly greater proportion of participants in the intervention group with dietitian-led classes vs. the tertile with net fat mass gain (61 vs. 44 %), this did not achieve statistical significance (P=0.317). Glucose (μU/ml) 91±9 88±10 0.060 Insulin sensitivity index (SI) 3.30±1.71 3.21±1.56 0.665 AIRg 529±461 548±455 0.335 a Values represented as mean±SD Fig. 2 Baseline correlations between insulin sensitivity index and total fat mass (top) and % truncal fat mass (bottom), adjusted for age J. Racial and Ethnic Health Disparities (2014) 1:140–147 J. Racial and Ethnic Health Disparities (2014) 1:140–147 145 Fig. 3 Tertiles of change in total (upper panel) and percent truncal fat mass (lower panel) and change in insulin sensitivity after completion of the 6-month weight loss program Discussion We conclude that fat mass loss can be achieved in over- weight and obese nondiabetic black women through partici- pation in a worksite weight loss program, which can signifi- cantly improve insulin sensitivity. However, fat mass gain and worsened insulin sensitivity were also frequent among our participants despite self-reports of compliance. As a result, participation by black women in weight loss programs could paradoxically contribute to diabetes risk in some subjects, who may require special attention early in these programs to achieve success. Regardless of the reason for fat mass gain by some of our subjects, the metabolic consequence was apparent in the form of reduction in insulin sensitivity. Baseline characteristics of our participants did not identify who would lose fat mass and improve insulin sensitivity and who would gain fat mass with diminished insulin sensitivity at completion of the program. More detailed phenotyping of novel adipokines and other biomarkers involved in regulation of body weight in future work may identify mechanisms by which study participants may have been resistant to fat mass loss, as has been seen in studies of weight regain [29]. Disclosure This research was supported by the intramural research programs of the National Heart, Lung, and Blood Institute, the National Institute of Diabetes, and Digestive and Kidney Diseases, and the Clinical Center, National Institutes of Health. No author has any conflict of interest. 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. Our findings of fat mass gain with reduced insulin sensi- tivity in almost a third of participants indicate that some may require additional attention to achieve weight and fat mass loss. One approach that has been proposed to identify and focus on those participants that are motivated to lose weight but are not successfully doing so is a stepped-care design [30, 31]. In a stepped-care intervention, participants that are not attaining short-term weight loss goals are prescribed addition- al resources and nutrition counseling to help them achieve their long-term weight loss goals. Discussion Although all participants were provided with the same fitness resources including access to exercise rooms at the worksite and pedometers to incentivize exercise, those who lost fat mass had significantly greater improvement in exercise performance despite reporting simi- lar time spent in daily aerobic activity and pedometer counts to those who gained fat mass. It is possible that those who gained fat mass either engaged in physical activity with lower energy expenditure compared with those who lost fat mass or in- creased caloric intake as a result of over-overestimating calo- ries consumed with relatively low-intensity exercise. Addi- tionally, errors in assessing – and reporting – nutritional com- position of diet and daily caloric intake may have also played a role in the increased fat mass experienced by several of our participants. It is possible that the 3-day food records used at the beginning and end of each participant’s participation might not have been indicative of energy intake throughout the 6- month period. Use of more reliable food assessment tools such as the picture recording technique reported by Six et al. [27] may limit the discrepancy between the foods consumed and those reported in weight loss studies similar to ours [28]. It is possible that fat mass loss may be achieved by the use of portable devices to self-monitor diet and physical activity as reported in the Move! Trial conducted in a Veterans Admin- istration cohort [32]. In addition, individualized counseling or motivational interviewing might have increased compliance and achieved greater success in our participants [33]. An important message of this study is the demonstration that relatively modest fat mass loss achieved by most black women improved insulin sensitivity in a population that is at high risk of developing diabetes and associated complications. A limitation of the study is that the duration was only 6 months: Continuation for a longer period might have result- ed in greater fat mass loss and further improvement in insulin sensitivity but might have been associated with even greater weight and fat mass gain with worsened metabolic conse- quences in some participants. In addition, as this report eval- uates the change in fat mass and insulin sensitivity only in overweight and obese black women, these findings cannot be extended to men or to other racial groups. Discussion improved insulin sensitivity but with greater weight loss than achieved by most of our participants [24–26]. Our principal finding was that fat mass loss through partici- pation in a worksite weight loss program can significantly improve insulin sensitivity (increase in SI) in overweight and obese nondiabetic black women. By multivariable regression analysis, fat mass loss – both total and truncal – was a significant predictor of increase in SI, independent of changes in leptin or improvement in exercise performance, both of which have been shown to improve insulin sensitivity [21–23]. We found no evidence of improved insulin secretion with fat mass loss as no difference was ob- served in the AIRg in the initial minutes of the FSIGT between the tertile of subjects with the greatest fat mass loss compared to the tertile with net fat mass gain. Our findings of improved insulin sensitivity with relatively modest fat mass reduction extend observations in other studies which have enrolled blacks that reported A disconcerting finding in our study was that nearly one- third of the overweight and obese black subjects who underwent testing for fat mass and insulin sensitivity at base- line and at 6 months actually gained fat mass with associated adverse metabolic consequences. Specifically, a decrease in insulin sensitivity was observed in the tertile of women with net fat mass gain despite self-reports of reduced caloric intake and increased physical activity similar to that of the tertile who lost the most fat mass. One reason why some of the partici- pants gained fat mass over the course of this study despite provision of resources to enable weight loss could be loss of interest in the program. As reviewed by Fitzgibbons et al. [15], weight loss trials – including those with black women – often have high attrition similar to the nearly 30 % dropout rate observed in our study. However, the participants we report in this paper – including those who gained fat mass – all J. Racial and Ethnic Health Disparities (2014) 1:140–147 146 completed the 6-month program, suggesting commitment to the goals of the study. An alternative explanation for fat mass gain by many subjects in our study may be misinterpretation of the energy expenditure associated with physical activity performed by participants. Discussion Thus, Jakicic and colleagues [31] tested a stepped-care model in a randomized trial of 363 overweight and obese adults (83 % female, 33 % nonwhite) that included increased contact frequency (group sessions, mail, telephone, and individual sessions) and even meal re- placements for participants missing weight loss goals at 3- month intervals. 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https://openalex.org/W3199727879	https://link.springer.com/content/pdf/10.1007/s12027-021-00687-1.pdf	English	null	Digital investigations: relevance and confidence in disclosure	ERA-Forum	2,021	cc-by	6,747	P. Anderson philip.anderson@northumbria.ac.uk ERA Forum (2021) 22:587–599 https://doi.org/10.1007/s12027-021-00687-1 ERA Forum (2021) 22:587–599 https://doi.org/10.1007/s12027-021-00687-1 ARTICLE ARTICLE Abstract The ﬁeld of digital forensics has grown exponentially to include a variety of dig- ital devices on which digitally stored information can be processed and used for different types of crimes. As a result, as this growth continues, new challenges for those conducting digital forensic examinations emerge. Digital forensics has become mainstream and grown in importance in situations where digital devices used in the commission of a crime need examining. This article reviews existing literature and highlights the challenges while exploring the lifecycle of a mobile phone examination and how the disclosure and admissibility of digital evidence develops. Keywords Digital investigations · Disclosure · Relevance · Transparency · Fair trial Keywords Digital investigations · Disclosure · Relevance · Transparency · Fair trial Digital investigations: relevance and conﬁdence in disclosure Philip Anderson1 ·Dave Sampson1 ·Seanpaul Gilroy2 Accepted: 7 September 2021 / Published online: 21 September 2021 © The Author(s) 2021 1 Dept. of Computer and Information Sciences, Northumbria University, Newcastle upon Tyne, UK 2 Newcastle upon Tyne, UK 1.2 Digital investigation guidelines and standards The Association of Chief Police Ofﬁcers (ACPO) guide to good practice during dig- ital investigations fundamentally consists of four principles. These guiding ‘princi- ples’ are relied upon by digital investigators, police ofﬁcers and staff, and those work- ing within the private sector. Lallie and Pimlott [3] identify the use of the four key principles within Digital Forensic Investigations, highlighting their adoption as part of any standard investigation not only within the UK but within most of Europe. The four ACPO principles are as follows: • Principle 1: No action taken by law enforcement agencies, persons employed within those agencies or their agents should change data which may subsequently be relied upon in court. • Principle 1: No action taken by law enforcement agencies, persons employed within those agencies or their agents should change data which may subsequently be relied upon in court. • Principle 2: In circumstances where a person ﬁnds it necessary to access original data, that person must be competent to do so and be able to give evidence explain- ing the relevance and the implications of their actions. • Principle 3: An audit trail or other record of all processes applied to digital evi- dence should be created and preserved. An independent third party should be able to examine those processes and achieve the same result. • Principle 4: The person in charge of the investigation has overall responsibility for ensuring that the law and these principles are adhered to [4]. • Principle 4: The person in charge of the investigation has overall responsibility for ensuring that the law and these principles are adhered to [4]. The ﬁrst two principles might seem contradictory, but they have been developed in such a way that adherence will not impede a digital investigation. Many cases, es- pecially involving smartphones, dictate that evidence cannot be secured or analysed without any changes being made. Hence, the second principle allows for changes to be made by a competent individual. If this principle was not present in the guide- lines, many investigations could not be completed while maintaining compliance. Consequently, the admissibility of any presented evidence could quickly be called into question by the opposition in court [4]. At the same time that these guidelines were being published by the ACPO, the National Institute for Standards and Technology released their ofﬁcial recommenda- tions for best practice in the ﬁeld of digital forensics. 1.1 Digital evidence Owen and Thomas deﬁne forensics as the use of science to provide facts in the pro- cess of identifying, recovering and reconstructing evidence [1]. Therefore, the aim of computer or digital forensics can be described as the preservation, identiﬁcation, extraction, interpretation, and presentation of computer data which can be used by a court of law [2]. Digital forensic evidence consists of exhibits, each consisting of a sequence of bits, presented by witnesses in a legal matter, to help jurors establish the P. Anderson et al. 588 facts of the case and to support or refute legal theories of the case. Traditional ac- quisition tools produce an image that is a duplicate of the original media. The image will include all regions of the original media, even those that are blank, unused, or irrelevant to the investigation. It will also include large portions devoted to operating systems of third-party applications and programs supplied. 1.3 Digital crimes: smartphones The landscape of digital crime has been drastically changed by the widespread popu- larity of smartphones, with an increasing number of mobile devices being involved in criminal investigations. The underlying technology is rapidly evolving and has pre- sented a new set of challenges for digital forensics investigators [8]. Over the last 20 years, various eras of mobile forensics have been identiﬁed which reﬂect the state of the discipline at that time. These stages consist of the early era pre-2007, at the ﬁrst turning point for smartphone popularity. 2011-2013 saw the rapidly increasing pop- ularity of smartphones across the globe, with further inclusions within the criminal investigations setting. Finally, 2014 onwards saw the exponential increase in smart- phone usage in computer crime [9]. Smartphones are potentially very valuable within a forensic investigation, due to the wealth of data they can hold regarding user activity and events [10]. 1.2 Digital investigation guidelines and standards The NIST guidelines provided an extensive set of recommendations for forensic examiners carrying out forensic investigations at the time. The publication includes both investigative models and principles, as well as practical knowledge that can be directly applied to various chal- lenges during an investigation [5]. In addition to the ACPO Principles and NIST guidelines, Digital Forensic Investi- gators in the UK faced a new set of regulatory guidelines around investigative meth- ods and tools. The UK Forensic Science Regulator introduced ISO17025, a nation- ally recognised laboratory standard for the testing and validation of methods and Digital investigations: relevance and conﬁdence in disclosure 589 tools which became a mandatory accreditation for Digital Forensic labs in 2017 [6]. ISO17025 details the technical and quality requirements that forensic labs must abide by, such as when a forensic practitioner conducts a method that does not conform to the standard [5]. If a digital forensic unit is not accredited under ISO17025, or they use a method that has not been assessed by the United Kingdom Accreditation Service (UKAS) for its conformance with ISO17025, they must detail their non-conformance when presenting their evidence. In the same way that other forms of evidence need to be handled appropriately, there are several standards and industry guidelines to be considered while interacting with digital evidence. Since the relevant publications have now been implemented for many years, a failure to adhere will likely cause a dispute of admissibility during its presentation in a courtroom. More recently the College of Policing [7] developed its own set of guidelines around the examination of digital devices, the ‘Associate Professional Practice’ guid- ance in the form of 10 principles. When writing the principles, the College of Policing acknowledges the consideration of a report by the Information Commissioners Ofﬁce, relevant legislation, and a recent Court of Appeal judgments to ensure police ofﬁcers and staff obtain evidence from digital devices fairly and lawfully while maintaining an individual’s right to privacy and a fair trial. 2.1 Digital forensic lifecycle A model will typically be structured into several stages, each of which contains the appropriate methodologies to be used across the investigation’s timeline. Different methodologies store the relevant actions to be taken during a stage of an investiga- tion. These models are generic so that they can be adapted to guide a broad range of scenarios. Since our digital landscape has drastically changed, older models need to be adapted along with the development of new frameworks. Recent developments in digital forensics models have focused on speciﬁc areas of the discipline, such as creating models for smartphone forensics. Newer research aims to modernise process models to mimic the advancements in digital forensics [11]. To understand the complexities, those involved in the disclosure of digital data must understand the lifecycle of a Digital Forensic Investigation. Whilst the lifecycle may vary between organisations across the globe, the fundamental lifecycle often used remains very similar and comprises of several key stages [12]. 1. Identiﬁcation – Stage one involves investigators identifying sources of informa- tion; this could be from an array of digital devices including computers, mobile phones, tablets, and SIM cards. 1. Identiﬁcation – Stage one involves investigators identifying sources of informa- tion; this could be from an array of digital devices including computers, mobile phones, tablets, and SIM cards. 2. Preservation – Stage two involves investigators safeguarding electronic informa- tion and preserving the crime scene. 3. Collection – Stage three involves investigators collecting the devices from the crime scene. Once collected they are then responsible for extracting the data from the device and creating a forensic copy, where possible in a forensically sound manner maintaining evidential integrity. 4. Analysis – Once the data has been successfully extracted, the investigator is then responsible for conducting an analysis of the recovered data; this could include an analysis of both system- and user-generated information which may be useful and assist in drawing conclusions. 5. Reporting – The ﬁnal stage involves the investigator generating a report document- ing the result of their analysis of the digital data. This also ensures that a third party is able to repeat their actions and obtain the same results where required. 5. Reporting – The ﬁnal stage involves the investigator generating a report document- ing the result of their analysis of the digital data. 2 Examination of digital devices As the ﬁeld of Digital Forensics grows and forensic software providers enhance their capability for recovering data from digital devices, the complexity of these exami- nations increases. Whilst this is prevalent across the entire ﬁeld of digital forensics, it is arguably most prevalent in the forensic examination of mobile devices such as mobile phones, tablets, and satellite navigation systems due to the complexities and challenges posed during such investigations. P. Anderson et al. 590 2.1 Digital forensic lifecycle This also ensures that a third party is able to repeat their actions and obtain the same results where required. 2.2 Mobile phone forensics The mobile phone market has a number of different vendors, such as Apple, Sam- sung, and Huawei, all of which ﬁght to dominate the market. These different vendors’ devices often store data in different ways and have a variety of operating systems alongside proprietary ﬁle systems. These variables themselves present an ongoing challenge the ﬁeld of Digital Forensics and as a result, the ﬁeld must constantly adapt to ensure that new emerging technologies can be collected, analysed, and reported upon [13]. During the collection stage, the Digital Forensic Investigator can employ several techniques to extract data from mobile devices, as outlined in Fig. 1 [14]: Whilst all methods of extraction may not be possible for every model of device, it is common practice that a single mobile phone device may be subjected to several different extraction types to maximise forensic opportunities by extracting all of the Digital investigations: relevance and conﬁdence in disclosure 591 Fig. 1 Mobile Phone Extraction Pyramid required data. It is the responsibility of the allocated Digital Forensic Investigator to understand these techniques and identify the most suitable on a case-by-case basis. 2 3 C t d Fig. 1 Mobile Phone Extraction Pyramid required data. It is the responsibility of the allocated Digital Forensic Investigator to understand these techniques and identify the most suitable on a case-by-case basis. required data. It is the responsibility of the allocated Digital Forensic Investigator to understand these techniques and identify the most suitable on a case-by-case basis. 2.3 Case study To fully understand the problems posed when dealing with disclosure in Digital Forensics, the case study documented below follows a basic submission to a Digi- tal Forensic Unit as part of an ongoing homicide investigation. As part of this case study, the investigation team have submitted a single mobile device to their local Digital Forensic Unit for examination. Digital Forensic Investigations present several unique challenges to investigators. In an attempt to overcome these challenges, inves- tigators often employ the use of Digital Evidence Strategies, also commonly known as Digital Investigative Strategies [4]. The Association of Chief Police Ofﬁcers high- lights several key stages when developing a Digital Investigative Strategy [4]: • Data Capture and search and seizure at crime scenes; • Data Capture and search and seizure at crime scenes; • Data Examination; • Data Interpretation; • Data Reporting; • Interview of Witness and Suspects. Digital Evidence strategies allow investigators to set parameters such as time frames that are proportionate to the facts and assist in overcoming the challenges presented by the large volume of data stored on digital devices and associated storage services [15]. The use of Digital Evidence Strategies allows the examination of digital devices to be both targeted and proportionate and streamline the forensic process. The submission contains a Digital Evidence Strategy which requests the following key data from the device: P. Anderson et al. 592 • SMS/MMS Messages • Incoming & Outgoing Call Data • Pictures & Videos It is widely recognised within the ﬁeld of Digital Forensic Investigations that inves- tigators have a variety of forensic tools at their disposal that can be used throughout the extraction and analysis stage of the investigation [15]. y g g The Digital Forensic Investigator allocated to the case may have utilised forensic “Tool A” to conduct the following forensic extractions of the device: • Logical • File system During a review of the two forensic extractions undertaken using forensic “Tool A”, it may become apparent to the Digital Forensic Investigator that the SMS messages required by the investigation team were not recovered, and again this is not uncom- mon when conducting an examination of mobile devices as highlighted by the Crown Prosecution Service [16]. As a result, they may opt to use forensic “Tool B” to con- duct a further two extractions in an attempt to recover the required SMS messages: • Logical Extraction • File System 3 Principles of disclosure The disclosure regime in England and Wales is governed by the Criminal Procedure and Investigations Act (CPIA) 1996 [18]. According to guidelines published by the Attorney General, the CPIA 1996 “aims to ensure that criminal investigations are conducted in a fair, objective and thorough manner” [19]. Further, the guidelines acknowledge that “[a] fair trial is the proper object and expectation of all participants in the trial process. Fair disclosure to the accused is an inseparable part of a fair trial” [20]. This explicit link between effective disclosure and the fair trial rights of the accused mean that failures of disclosure by the prosecution can have serious implications under Article 6 European Convention on Human Rights (ECHR [21]). “Fairness ordinarily requires that any material held by the prosecution which weakens its case or strengthens that of the defendant, if not relied on as part of its formal case against the defendant, should be disclosed to the defence. Bitter experience has shown that miscarriages of justice may occur where such material is withheld from disclosure. The golden rule is that full disclosure of such material should be made.” - House of Lords, R v H and C [22]. “Fairness ordinarily requires that any material held by the prosecution which weakens its case or strengthens that of the defendant, if not relied on as part of its formal case against the defendant, should be disclosed to the defence. Bitter experience has shown that miscarriages of justice may occur where such material is withheld from disclosure. The golden rule is that full disclosure of such material should be made.” - House of Lords, R v H and C [22]. CPIA 1996 endeavours to set standards and procedures to regulate the investigation process and manage the recording and retention of all material found or generated in the course of an investigation. Core roles and responsibilities are deﬁned with regulatory duties to achieve the desired outcomes. CPIA 1996 endeavours to set standards and procedures to regulate the investigation process and manage the recording and retention of all material found or generated in the course of an investigation. Core roles and responsibilities are deﬁned with regulatory duties to achieve the desired outcomes. • Logical Extraction • File System Both “Tool A” and “Tool B” may have failed to recover the SMS messages required by the investigation team; this is not a fault with the software but instead a limitation of the forensic software for that given device. To further complicate matters, device support across forensic tools differs heavily and as a result “Tool C” may offer a more complex type of extraction such as a Physical, as outlined within the Mobile Phone Extraction Pyramid, which both “Tool A” and “Tool B” did not offer. As part of this case study, consider that the physical extraction conducted by “Tool C” successfully recovered the required SMS messages. As a result of the above extractions the Digital Forensic Investigator now has the below extractions ready to report upon: • Logical (Obtained using forensic tool A) • File System (Obtained using forensic tool A) • Logical (Obtained using forensic tool B) • File System (Obtained using forensic tool B) • Physical (Obtained using forensic tool C) • Logical (Obtained using forensic tool A) • File System (Obtained using forensic tool A) • Logical (Obtained using forensic tool B) • File System (Obtained using forensic tool B) • Physical (Obtained using forensic tool C) Digital Forensic Investigators often merge multiple mobile phone extractions; this provides the Digital Forensic Investigator with the ability to produce a uniﬁed report (such as PDF or Excel) for the investigation team to review as part of “Stage 5: Reporting” [17]. Merging forensic extractions has several different beneﬁts, including reducing the amount of time spent reviewing numerous extraction types as well as removing du- plicate records. A logical, ﬁle system, and physical extraction may recover the same SMS 3 times; merging the records would ensure that the investigation team are only required to review this a single time, ultimately leading to more streamlined investi- gations - beneﬁcial in today’s world of “big data” [16]. 593 Digital investigations: relevance and conﬁdence in disclosure As Digital Forensic Investigators and legal practitioners, robust procedures must be developed and maintained into department Quality Management Systems to en- sure that data held as part of the Digital Forensic Investigation is disclosed correctly. Forensic tools store data in proprietary formats which can often only be accessed by the use of forensic tools which require specialist training; disclosing this data presents us with the challenge of interpreting it away from Digital Forensic Laboratories. 3 Principles of disclosure In R v Malook [23] the England and Wales Court of Appeal held that the duty to record applies not only to material evidence seized by the police during an in- vestigation but equally to “documentation produced by the police in the course of investigations in contradistinction to pre-existing material seized by a police force.” Further, the court held that “Proper record-keeping in an investigation is essential to the integrity of an investigation, to public conﬁdence in police investigations and the proper administration of justice [24]. All investigators have a responsibility to carry out the duties imposed under CPIA 1996, including recording relevant information and retaining records of the informa- tion and other material from the outset of the investigation. The most important of these responsibilities is for an investigator to pursue all reasonable lines of enquiry, whether these point towards or away from a suspect. What is reasonable in each case will depend on the particular circumstances. For example, where the material is held on a computer, it is for the investigator to decide which material on the computer it is reasonable to enquire into, and in what manner. Material may be relevant to an inves- tigation if it appears that it has some bearing on any offence under investigation or 594 P. Anderson et al. any person being investigated, or on the surrounding circumstances of the case unless it is incapable of having any impact on the case [25]. any person being investigated, or on the surrounding circumstances of the case unless it is incapable of having any impact on the case [25]. The ofﬁcer in charge of an investigation is responsible for directing and focusing the investigation, setting the parameters for the lines of enquiry. They are also re- sponsible for ensuring that there are proper processes and procedures in place in the investigation for the recording of information and retaining records of other mate- rial. Some information will be used in the prosecution and will form the evidence of the case. The remaining information is referred to as unused material. This material is relevant to the case but is not being used as part of the prosecution evidence presented to the Court. The disclosure ofﬁcer [26] will have responsibility for ensuring that material gen- erated during an investigation is properly recorded on a Schedule of Unused Material [27]. 3 Principles of disclosure The revised Code of Practice to the CPIA 1996 requires that the disclosure of- ﬁcer should ensure that each item of material is listed separately on the schedule and is numbered consecutively. The description of each item should make clear the na- ture of the item and should contain sufﬁcient detail to enable the prosecutor to decide whether they need to inspect the material before deciding whether or not it should be disclosed [28]. CPIA 1996 establishes a disclosure test that requires the prosecution to disclose any unused material that might reasonably be considered capable of undermining the prosecution case or of assisting the case for the accused [29]. The police and CPS in England and Wales are independent of each other, but each depend on the other performing their respective disclosure obligations. When a charge is brought against a person, the prosecution must serve the evidence that it will rely on in court to prove its case. The prosecution also has an initial disclosure duty which obliges the pros- ecutor to disclose to the defendant any unused material that satisﬁes the disclosure test. The prosecution’s duty to disclose material is determined by the application of s.3 CPIA 1996. The disclosure of sensitive material [30] may be withheld on public interest grounds under a Public Interest Immunity (PII) application. In R v H [31] Lord Bingham of Cornhill eruditely summarised the operation of PII in the follow- ing terms: “Circumstances may arise in which material held by the prosecution and tending to undermine the prosecution or assist the defence cannot be disclosed to the defence, fully or even at all, without the risk of serious prejudice to an important public interest.” All parties, the investigator, the disclosure ofﬁcer, and the prosecutor have a con- tinuing duty to keep disclosure under review throughout the life of a case. As a part of the defence case, they serve a defence statement setting out the nature of the de- fence and request any material which could reasonably assist their case. This request is incorporated within the ongoing evaluation of the material held. Disclosure of digital evidence ﬁts within the framework of CPIA 1996; in principle the digital data is material. 4 The disclosure challenges Identiﬁcation – To support the compiling of a Disclosure Management Document to facilitate a robust digital disclosure procedure the early creation of a digital strategy is required within every investigation. This can then be used as a guide and a rationale for the seizure and more importantly the non-seizure of digital items. Some devices’ non-seizure can be dictated by the sheer volume of the device or the volatile nature of the data. Other devices’ non-seizure may be mandated in respect of a victim’s device, or subjectively in each case, for example following the onsite triage of a child’s laptop to eliminate it from the enquiries due to its necessity for educational support or other reasons. Historically, all digital devices would be seized for evaluation and potential exami- nation as a matter of course. This process is still valid if it is justiﬁed and documented. But the modern digital world requires a more efﬁcient digital data seizure approach. What are the relevant lines of the enquiry of the investigation and, within that, what are the date parameters? The validity of the seizure of a computer base unit stored and undisturbed for a signiﬁcant period at a scene where the alleged offending has occurred in a recent timeframe would appear disproportionate to the investigation. For this issue, there is not a default answer, it is in certain circumstances acceptable to seize all digital media. But with this they should also expect to have to justify their course of actions, explain the relevance of the items to the investigation, and state what they intend to do with the items. If the intention is to not submit them for examination, then surely the question should be asked – why seize them in the ﬁrst place? Similarly, the seize-all policy cannot be applied to complainants’ and wit- nesses’ digital devices. The right of privacy of an individual must be balanced against the suspect’s right to a fair trial. The examination of complainants’ and witnesses’ de- vices must be done with their informed consent. They need to be fully aware of how the device is going to be examined, the data desired to be extracted, and to what ends that information is to be interrogated and shared. 3 Principles of disclosure The challenge lies in the volume of the digital data and the additional challenges this raises in the identiﬁcation of relevant data, as well as the ethical challenge posed by the processes to accessing and interpreting the data. As stated by Alison Saunders DPP – “while the principles remain unchanged, our working practices have had to respond to several signiﬁcant developments... Criminal justice system-wide initiatives and, other changes, such as the unprecedented rise in Digital investigations: relevance and conﬁdence in disclosure 595 the volume of digital material created in criminal investigations, could not easily have been foreseen, and a fundamental review of the manual (CPIA 1996) has been undertaken” [32]. 4 The disclosure challenges This requirement needs to follow the relevant lines of enquiry of the investigation, with the owner informed that it is not always possible to extract the exact data as required and that data outside of the requirement may need to be extracted to allow for further investigation and recovery of the relevant material [33]. Preservation and collection – Data is volatile and the historic storage of data on a single hard drive connected to a single device is of a rapidly disappearing era. In the modern home, there is now a myriad of digital devices utilising wireless and mobile network connectivity to provide the always-on requirement of contemporary society. This presents a new world of challenges to the safe and secure presentation of data. Volatile data is any kind of data that is available while a digital device is powered on and could be lost once the machine is turned off. The capturing of live data may con- tain information that through correct collection and analysis may become evidence 596 P. Anderson et al. within an investigation that may be critical. Similarly, cloud data storage can often be split across multiple different devices and infrastructures, resulting in evidence be- ing difﬁcult to ﬁnd and, more importantly, to preserve in an efﬁcient timeframe. Once identiﬁed, the evidence must be recovered and preserved in a forensically sound man- ner. Data storage has the potential to be so vast that acquisition for analysis becomes unreasonable; this requires a different approach to examining the preserved data in situ and presents a greater need for all investigating bodies on both sides of a case to streamline the analysis process by agreeing on common requirements to feed the data interrogation. g The examination and analysis of digital media does not change data but identi- ﬁes from within the data what is deemed relevant. This can be a fraught process with some digital material initially assessed as irrelevant data on its own merit, but this data may be metadata [34] with the ability to add value and substance to other identiﬁed relevant data. This can be very relevant and making the initially disregarded data sig- niﬁcant to an investigation. As the disclosure process itself is a live ongoing process throughout an investigation, the review and identiﬁcation of relevance within digital material must be ﬂuid and adaptable. 4 The disclosure challenges Collaboration is key to allow for all parties to have a thorough understanding of what digital material is held and, when lawfully allowed an understanding of the content of the digital material so that all parties are fully informed of the material’s relevance and value to their investigation. The agree- ment of search parameters and search deﬁnitions supported by how the searching is to be completed is seldom reached in a timeframe which would allow the process to be completed, reviewed, and where necessary challenged. Search parameters cannot be set so wide as to fail to reduce the data sets being examined or so narrow as to yield minimal content. Within digital data, there is the potential for a person’s whole life to be laid bare in a raw format. Uncontrolled access to this data cannot be granted due to the necessity of protecting the individual’s right to a private life. The data should be accessed in a manner that allows for the examination of relevant information only, not - as is currently the case when a signiﬁcant date in the criminal justice process ap- proaches and acts as a trigger – in a manner giving unfettered access in contravention of CPIA 1996. 5 Conclusions If during the course of an investigation new lines of enquiry are identiﬁed, the Digital Evidence Strategy could be altered to include them, for example through further keyword searches across the raw data. Due to the complex nature of Digital Forensic Investigations, it is vital that all parties collaborate in order to ensure that Digital Evidence is disclosed in the correct manner. Investigation teams may only receive tailored reports such as PDF or Excel documents that have been produced as part of the supplied Digital Evidence Strategy. The Digital Forensic team will retain the raw case ﬁles generated during the “collec- tion” and “analysis” stage of the lifecycle which is likely to contain a wealth of data in comparison to the tailored reports. These raw case ﬁles often require commercial forensic tools and specialist training to analyse and interpret so that reports can be produced; ensuring that these raw case ﬁles are disclosed during an investigation is therefore vital “to ensure that criminal investigations are conducted in a fair, objective and thorough manner” [35]. A robust system should be employed to ensure that all material during the forensic examination is disclosed correctly. The uncontrolled access to and examination of digital devices is correctly being challenged by society as a breach of privacy. The modern mobile phone contains a link or direct access route to every part of an individual’s life from banking to social media proﬁles and beyond. New regimes and requirements are emerging and proving additional challenges to the seizure and examination of such devices. This requirement will grow and, as it is initially tested within the criminal justice system, will raise further exacting considerations for both disclosure and beyond. The success of the disclosure process is fundamental to underpinning conﬁdence in the criminal justice system. The deﬁned structures and processes of the disclosure regime are in principle robust and correct but they need to be constantly reviewed in order to keep pace with the continually changing digital landscape and must be correctly applied by all parties. Collaboration is key across all interested parties in order to enable a fair trial to be held with appropriate interrogation of the disputed facts within an investigation, supported by the knowledge that all parties have a fun- damentally clear awareness of all other data held. A disclosure process has to be adopted early in order for it to be successful. 5 Conclusions Digital evidence poses signiﬁcant challenges and continues to grow in size, com- plexity and importance. In recent years, investigations involving digital devices have exponentially increased to include many and varied types of digital devices on which digitally stored information can be stored and processed. As a result of this, digital ev- idence has been presented in a growing number of criminal and civil court cases over the last decade. Because of how digital evidence is obtained, examined, analysed, and presented in court, it is often challenged, with parties contesting how the digital device was acquired or arguing that it was examined and analysed ineffectively. Digital evidence must meet the same standards as other scientiﬁc and technical evidence in order to be admissible in court. Standards and guidelines are a necessary requirement to ensure that the courts and legal system are safeguarded from poor Digital investigations: relevance and conﬁdence in disclosure 597 digital evidence. The recent ISO17025 standard, however, does not directly assess an individual’s expertise and competence, instead, it addresses the competency of the digital forensic lab and the tools used to conduct digital device examination. In some instances, digital evidence is omitted on the basis that it was obtained incorrectly or examined ineffectively. digital evidence. The recent ISO17025 standard, however, does not directly assess an individual’s expertise and competence, instead, it addresses the competency of the digital forensic lab and the tools used to conduct digital device examination. In some instances, digital evidence is omitted on the basis that it was obtained incorrectly or examined ineffectively. y In the United Kingdom, various laws and rules govern the admissibility of digi- tal evidence in courts. Understanding how digital evidence is obtained affects how judges and juries weigh the importance of this evidence when it is presented in court. It could be said that to fairly and impartially assess the value of digital evidence, judges and juries should understand the basic functions of digital devices, such as computers and the software from which any evidence is extracted. p y The use of Digital Evidence Strategies within a Digital Forensic Examination is becoming increasingly more important and offers a useful tool to investigators to assist with the disclosure of Digital Evidence. Whilst these strategies are dynamic and may change as the investigation progresses, they ensure that the examination is targeted and proportionate. 5 Conclusions Any delay results in an ongoing process 598 P. Anderson et al. of catch-up, which has consequences at each stage of the criminal justice process which can and do culminate in failings by one or all parties at a critical evidential stage, to the detriment of all involved. 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. 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Attorney General’s Ofﬁce, Attorney General’s Guidelines on Disclosure, December 2013, page 4, para 7. https://www.gov.uk/government/publications/attorney-generals-guidelines-on-disclosure- 2013. Accessed 9 July 2021 y 21. Article 6 European Convention on Human Rights. https://www.echr.coe.int/Documents/Convention_ ENG.pdf. Accessed 9 July 2021 22. House of Lords, R v H and C [2004] UKHL 3. https://publications.parliament.uk/pa/ld200304/ ldjudgmt/jd040205/hc-1.htm. Accessed 9 July 2021 23. R v Malook [2011] EWCA Crim 254. https://www.bailii.org/ew/cases/EWCA/Crim/2011/254.html. Accessed 9 July 2021 24. R v Malook [2011] EWCA Crim 254, [35]. https://www.bailii.org/ew/cases/EWCA/Crim/2011/25 html. Accessed 9 July 2021 25. Section 3(1)(a) of the CPIA,1. https://www.cps.gov.uk/legal-guidance/disclosure-guide-reasonable- lines-enquiry-and-communications-evidence. Accessed 9 July 2021 26. Section 3(1)(a) of the CPIA,2. https://www.cps.gov.uk/legal-guidance/disclosure-guide-reasonabl lines-enquiry-and-communications-evidence. Accessed 9 July 2021 7. Form MG6C, see CPS Disclosure Manual (revised 14th Dec. 2018), Chap. 6 and NPCC, CPS, Na- tional Disclosure Standards, March 2019. https://www.cps.gov.uk/sites/default/ﬁles/documents/legal_ 27. Form MG6C, see CPS Disclosure Manual (revised 14th Dec. 2018), Chap. 6 and NPCC, CPS, Na- tional Disclosure Standards, March 2019. https://www.cps.gov.uk/sites/default/ﬁles/documents/legal_ guidance/National-Disclosure-Standards-2018.pdf. Accessed 9 July 2021 p p g guidance/National-Disclosure-Standards-2018.pdf. Accessed 9 July 2021 28. Revised Code of Practice to the CPIA 1996 (s.23(1)), para 6.11. https://www.gov.uk/government/ publications/criminal-procedure-and-investigations-act-code-of-practice. Accessed 9 July 2021 29. Criminal Procedure and Investigations Act 1996, s.3 (as amended by Criminal Justice Act 2003 (c.44)). https://www.cps.gov.uk/legal-guidance/disclosure-guide-reasonable-lines-enquiry-and- communications-evidence. Accessed 9 July 2021 y 30. Revised Code of Practice to the CPIA 1996 (s.23(1)), para 6.14. https://www.gov.uk/governmen publications/criminal-procedure-and-investigations-act-code-of-practice. Accessed 9 July 2021 31. R v H [2004] UKHL 4. https://publications.parliament.uk/pa/ld200607/ldjudgmt/jd070228/rvh-4.htm. Accessed 9 July 2021 y 32. Crown Prosecution Service and National Police Chiefs’ Council - Action on Disclosure. https://www. cps.gov.uk/sites/default/ﬁles/documents/legal_guidance/Disclosure%20Manual_0.pdf. Accessed 9 July 2021 y 33. NPCC – Data Processing Notice DPNb - Witness information sheet. https://news.npcc.police.u resources/dpnb-witness-information-sheet. Accessed 9 July 2021 34. Metadata – Practice direction 31b Disclosure of electronic documents. Para 28. https://www.justice. gov.uk/courts/procedure-rules/civil/rules/part31/pd_part31b. Accessed 28 July 2021 35. Attorney General’s Guidelines on Disclosure. https://assets.publishing.service.gov.uk/government/ uploads/system/uploads/attachment_data/ﬁle/262994/AG_Disclosure_Guidelines_-_December_ 2013.pdf. Accessed 28 July 2021 Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional afﬁliations.
https://openalex.org/W4255017516	https://www.qeios.com/read/15F3WZ/pdf	English	null	Can Dosing Unit	Definitions	2,020	cc-by	61	Qeios · Definition, February 7, 2020 Open Peer Review on Qeios Open Peer Review on Qeios Can Dosing Unit National Cancer Institute National Cancer Institute Qeios ID: 15F3WZ · https://doi.org/10.32388/15F3WZ Source National Cancer Institute. Can Dosing Unit. NCI Thesaurus. Code C48479. A dosing unit equal to the amount of active ingredient(s) contained in a can. Qeios ID: 15F3WZ · https://doi.org/10.32388/15F3WZ 1/1
https://openalex.org/W4393980945	https://www.bmjour.ru/jour/article/download/197/134	Russian	null	CLINICAL CASE OF KUGELBERG – WELANDER DISEASE (SPINAL MUSCULAR ATROPHY TYPE III)	Bajkalʹskij medicinskij žurnal	2,024	cc-by	5,269	Baikal Medical Journal, 2024, Vol. 3, No1 Baikal Medical Journal, 2024, Vol. 3, No1 Байкальский медицинский журнал, 2024, Том 3, № 1 Baikal Medical Journal, 2024, Vol. 3, No1 кальский медицинский журнал, 2024, Том 3, № 1 Быков Ю.Н., Васильев Ю.Н., Загвозкина Т.Н., Аникина И.В., Тарасова Т.А., Василькова С.В., Плеханова Ю.С. Быков Ю.Н., Васильев Ю.Н., Загвозкина Т.Н., Аникина И.В., Тарасова Т.А., Василькова С.В., Плеханова Ю.С. ФГБОУ ВО «Иркутский государственный медицинский университет» Минздрава России (664003, г. Иркутск, ул. Красного Восстания, 1, Россия) Клинические случаи Case reports Ключевые слова: спинальная мышечная атрофия Кугельберга – Веландера, молекулярно-генетическое ис следование, миопатия Эрба – Рота РЕЗЮМЕ Актуальность. Дифференциальная диагностика нервно-мышечных заболеваний является одним из самых сложных разделов в неврологии. Особое значение имеет проведение молекулярно-генетического исследо вания у этих больных. Представлен клинический случай спинальной мышечной атрофии 5q III типа (болезнь Кугельберга – Веландера), подтверждённый молекулярно-генетическим обследованием. Актуальность. Дифференциальная диагностика нервно-мышечных заболеваний является одним из самых сложных разделов в неврологии. Особое значение имеет проведение молекулярно-генетического исследо вания у этих больных. Представлен клинический случай спинальной мышечной атрофии 5q III типа (болезнь Кугельберга – Веландера), подтверждённый молекулярно-генетическим обследованием. Описание клинического случая. В статье описано клиническое наблюдение пациентки с первоначальным диагнозом миопатии Эрба – Рота. Проведено генетическое исследование. В результате анализа наличия экзона 7 генов SMN1/SMN2 зарегистрировано отсутствие сигнала, соответствующего экзону 7 гена SMN1. Поиск делеций в гене SMN1 показал, что у пробанда зарегистрирована делеция экзонов 7–8 гена SMN1 в гомозиготном состоянии. Делеция экзонов 7–8 гена SMN1 в гомозиготном состоянии является причиной проксимальной спинальной мышечной атрофии 5q. В результате исследования по определению числа копий генов SMN1, SMN2 зарегистрировано 0 копий экзонов 7–8 гена SMN1, 4 копии экзонов 7–8 гена SMN2, что подтверждает диагноз спинальной мышечной атрофии 5q III типа. Описание клинического случая. В статье описано клиническое наблюдение пациентки с первоначальным диагнозом миопатии Эрба – Рота. Проведено генетическое исследование. В результате анализа наличия экзона 7 генов SMN1/SMN2 зарегистрировано отсутствие сигнала, соответствующего экзону 7 гена SMN1. Поиск делеций в гене SMN1 показал, что у пробанда зарегистрирована делеция экзонов 7–8 гена SMN1 в гомозиготном состоянии. Делеция экзонов 7–8 гена SMN1 в гомозиготном состоянии является причиной проксимальной спинальной мышечной атрофии 5q. В результате исследования по определению числа копий генов SMN1, SMN2 зарегистрировано 0 копий экзонов 7–8 гена SMN1, 4 копии экзонов 7–8 гена SMN2, что подтверждает диагноз спинальной мышечной атрофии 5q III типа. Заключение. Взрослым пациентам, страдающим нервно-мышечными заболеваниями, рекомендовано про ведение подтверждающей ДНК-диагностики для назначения патогенетического лечения, что увеличивает их шансы на выживание. У лиц молодого возраста с клинически выставленным диагнозом нервно-мышечного заболевания обязательно проведение генетического исследования для уточнения диагноза и дальнейшей тактики лечения. Ключевые слова: спинальная мышечная атрофия Кугельберга – Веландера, молекулярно-генетическое ис следование, миопатия Эрба – Рота Для цитирования: Быков Ю.Н., Васильев Ю.Н., Загвозкина Т.Н., Аникина И.В., Тарасова Т.А., Василько ва С.В., Плеханова Ю.С. Клинический случай болезни Кугельберга – Веландера (спинальной мышечной атрофии III типа). Байкальский медицинский журнал. 2024; 3(1): 45-52. doi: 10.57256/2949-0715-2024-3-1- 45-52 Для цитирования: Быков Ю.Н., Васильев Ю.Н., Загвозкина Т.Н., Аникина И.В., Тарасова Т.А., Василько ва С.В., Плеханова Ю.С. Клинический случай болезни Кугельберга – Веландера (спинальной мышечной атрофии III типа). Для цитирования: Быков Ю.Н., Васильев Ю.Н., Загвозкина Т.Н., Аникина И.В., Тарасова Т.А., Василько ва С.В., Плеханова Ю.С. Клинический случай болезни Кугельберга – Веландера (спинальной мышечной атрофии III типа). Байкальский медицинский журнал. 2024; 3(1): 45-52. doi: 10.57256/2949-0715-2024-3-1- 45-52 РЕЗЮМЕ Байкальский медицинский журнал. 2024; 3(1): 45-52. doi: 10.57256/2949-0715-2024-3-1- 45-52 Клинические случаи Case reports 45 дицинский журнал, 2024, Том 3, № 1 Baikal Medical Journal, 2024, Vol. 3, No 1 Байкальский медицинский журнал, 2024, Том 3, № 1 Baikal Medical Journal, 2024, Vol. 3, No 1 Baikal Medical Journal, 2024, Vol. 3, No 1 йкальский медицинский журнал, 2024, Том 3, № 1 Bykov Yu.N., Vasyliev Yu.N., Zagvozkina T.N., Anikina I.V., Tarasova T.A., Vasilkova S.V., Plekhanova Yu.S. Irkutsk State Medical University (664003, Irkutsk, Krasnogo Vosstaniya str., 1, Russian Federation) ABSTRACT Background. Differential diagnosis of neuromuscular diseases is one of the most difficult areas in neurology. Molecular genetic research in these patients is of particular importance. The article presents a clinical case of 5q spinal muscular atrophy type III (Kugelberg – Welander disease), confirmed by molecular genetic testing. Background. Differential diagnosis of neuromuscular diseases is one of the most difficult areas in neurology. Molecular genetic research in these patients is of particular importance. The article presents a clinical case of 5q spinal muscular atrophy type III (Kugelberg – Welander disease), confirmed by molecular genetic testing. Description of the clinical case. The article presents the clinical observation of a patient with an initial diagnosis of Erb – Roth myopathy. A genetic study was carried out. The analysis of the presence of exon 7 of the SMN1/SMN2 genes showed the absence of a signal corresponding to exon 7 of the SMN1 gene. A search for deletions in the SMN1 gene showed the homozygous deletion of exons 7–8 of the SMN1 gene in the proband. Homozygous deletion of exons 7–8 of the SMN1 gene causes 5q proximal spinal muscular atrophy. As a result of determining the num ber of copies of the SMN1 and SMN2 genes, 0 copies of exons 7–8 of the SMN1 gene and 4 copies of exons 7–8 of the SMN2 gene were registered, which confirms the diagnosis of 5q spinal muscular atrophy type III. Conclusion. Adult patients suffering from neuromuscular diseases are recommended to undergo confirmatory DNA diagnostics to prescribe pathogenetic treatment, which increases their chances of survival. In young people with a clinical p y yp ( g g ), y g g Description of the clinical case. The article presents the clinical observation of a patient with an initial diagnosis of Erb – Roth myopathy. A genetic study was carried out. The analysis of the presence of exon 7 of the SMN1/SMN2 genes showed the absence of a signal corresponding to exon 7 of the SMN1 gene. A search for deletions in the SMN1 gene showed the homozygous deletion of exons 7–8 of the SMN1 gene in the proband. Homozygous deletion of exons 7–8 of the SMN1 gene causes 5q proximal spinal muscular atrophy. words: Kugelberg – Welander spinal muscular atrophy, molecular genetic study, Erb – Roth myopathy CLINICAL CASE OF KUGELBERG – WELANDER DISEASE (SPINAL MUSCULAR ATROPHY TYPE III) Bykov Yu.N., Vasyliev Yu.N., Zagvozkina T.N., Anikina I.V., Tarasova T.A., Vasilkova S.V., Plekhanova Yu.S. ВВЕДЕНИЕ ющая боль в теле, которую облегчает горячая ван на. При ходьбе возникают падения из-за слабости в ногах. Дрожание в руках, ногах при физической нагрузке. Подёргивания в мышцах всего тела. Чув ство нехватки воздуха после физической нагруз ки, при подъёме на второй этаж. Проще спускать ся по лестнице, чем подниматься. ющая боль в теле, которую облегчает горячая ван на. При ходьбе возникают падения из-за слабости в ногах. Дрожание в руках, ногах при физической нагрузке. Подёргивания в мышцах всего тела. Чув ство нехватки воздуха после физической нагруз ки, при подъёме на второй этаж. Проще спускать ся по лестнице, чем подниматься. С п и н а л ь н а я м ы ш е ч н а я а т р о ф и я (СМА) – это тяжёлое аутосомно-рецессивное нервно-мышечное заболевание, характеризующе еся прогрессирующими симптомами вялого па ралича и мышечной атрофии вследствие дегене рации α-мотонейронов передних рогов спинного мозга [1]. Выделяют 0, I, II, III и IV типы СМА [2]. Ювенильная форма СМА относится к III типу; к её возникновению приводят мутации в теломер ной копии 5-й хромосомы (5q) [3]. Анамнез. Установлено, что с 7 лет стала уста вать на уроках физкультуры, но к врачам не об ращались. В 5-м классе после летних каникул не смогла сделать приседания, из положения «сидя» поднималась с помощью рук. Обратились к неврологу. Консультировалась у невролога, был заподозрен диагноз «миопатия». Ежегодно прохо дила лечение в неврологическом отделении Ива но-Матрёнинской детской клинической боль ницы (Иркутск). Выставлен диагноз: Миопатия Эрба – Рота. Генетическое обследование не про водилось. Биопсия мышц не проводилась. На блюдалась у детского невролога по месту житель ства, периодически проходила амбулаторное ле чение: эссенциале, витамины группы В, Е, акто вегин, милдронат. Наблюдался незначительный эффект от лечения. Со слов пациентки, две род ные сестры страдают подобными проявлениями, «слабой физической подготовкой». Обследована одна родная сестра пациентки: выявлены клини ческие признаки подобного нервно-мышечного заболевания. Рекомендовано молекулярно-гене тическое исследование сестры. В феврале 2021 г. перенесла COVID-19, после чего появилась одыш ка, стала быстрее уставать, но с данными жало бами к врачу не обращалась. Также стала отме чать усиление слабости в ногах, стала часто па дать из-за слабости при обычной физической на грузке. Обратилась к неврологу в поликлинику, после осмотра рекомендовано стационарное ле чение. В сентябре 2021 г. хотела сдать экзамен на водительские права; при прохождении меди цинского осмотра было отказано в выдаче поло жительного медицинского заключения из-за диа гноза «миопатия». Тогда больная по собственной инициативе 15.11.2022 прошла электронейроми ографию (ЭНМГ). Клинические случаи Case reports ВВЕДЕНИЕ При игольчатой электромио грамме (ЭМГ) (nn. tibialis dex. et sin.) зарегистри рована спонтанная активность в виде потенциа лов фибрилляций; амплитуда и длительность по тенциалов двигательных единиц (ПДЕ) повышена; амплитуда интерференционного паттерна с разре жением кривой, что характерно для нейрогенно го процесса. В ф 2023 Распространённость проксимальной спиналь ной мышечной атрофии составляет 1 на 6000– 10000 новорождённых [4]. Данные по распро странённости заболевания в Российской Феде рации отсутствуют. Частота носительства забо левания – 1/40–1/50 в популяции в целом [3]. По данным ФГБНУ «Медико-генетический науч ный центр имени академика Н.П. Бочкова», ча стота носительства мутации в гене SMN1 в Рос сии – 1/36 человек, расчётная частота рождения ребёнка со СМА – 1 на 5184 новорождённых [5]. Клинически СМА проявляются слабостью, атрофией и фасцикуляциями в мышцах. В боль шинстве случаев наблюдается симметричная слабость проксимальной мускулатуры, и лишь для редких вариантов характерно поражение дис тальных мышц, асимметричное поражение или во влечение бульбарной мускулатуры. Нарушений чувствительности, пирамидных знаков нет [6]. СМА III типа (болезнь Кугельберга – Велан дера) начинается в возрасте 2–17 лет. Тип насле дования – аутосомно-рецессивный. Заболевание в большинстве случаев прогрессирует медлен но. Многие больные доживают до взрослого воз раста, сохраняя способность к самостоятельному передвижению, и имеют нормальную продолжи тельность жизни, но некоторые утрачивают спо собность к передвижению в подростковом возрас те. Тяжёлые осложнения в виде дисфагии и сла бости дыхательных мышц наблюдаются редко [7]. Байкальский медицинский журнал, 2024, Том 3, № 1 Байкальский медицинский журнал, 2024, Том 3, № 1 Baikal Medical Journal, 2024, Vol. 3, No 1 Байкальский медицинский журнал, 2024, Том 3, № 1 ABSTRACT As a result of determining the num ber of copies of the SMN1 and SMN2 genes, 0 copies of exons 7–8 of the SMN1 gene and 4 copies of exons 7–8 of the SMN2 gene were registered, which confirms the diagnosis of 5q spinal muscular atrophy type III. C l i Ad lt ti t ff i f l di d d t d fi t DNA Conclusion. Adult patients suffering from neuromuscular diseases are recommended to undergo confirmatory DNA diagnostics to prescribe pathogenetic treatment, which increases their chances of survival. In young people with a clinical diagnosis of neuromuscular disease, genetic testing is required to clarify the diagnosis and further treatment tactics. Key words: Kugelberg – Welander spinal muscular atrophy, molecular genetic study, Erb – Roth myopathy For citation: Bykov Yu.N., Vasyliev Yu.N., Zagvozkina T.N., Anikina I.V., Tarasova T.A., Vasilkova S.V., Plekhano va Yu.S. Clinical case of Kugelberg – Welander disease (spinal muscular atrophy type III). Baikal Medical Journal. 2024; 3(1): 45-52. doi: 10.57256/2949-0715-2024-3-1-45-52 Клинические случаи Case reports Клинические случаи Case reports КЛИНИЧЕСКИЙ ПРИМЕР Сила в ногах в дистальных отделах: 4 балла с двух сторон. Тонус мышц в конечностях: в руках гипото нус, в ногах гипотонус. Псевдогипертрофия икро ножных мышц. Движения в шейном отделе позво ночника не ограничены. Карпорадиальные рефлек сы не вызываются. Рефлексы с бицепса резко сни жены, равные. Рефлексы с трицепса не вызывают ся. Коленные рефлексы не вызываются. Ахилловы рефлексы не вызываются. Подошвенные рефлек сы не вызываются. Брюшные рефлексы вызывают ся, равные, умеренные. Стопные патологические рефлексы: нет. Кистевые патологические рефлек сы: нет. Клонусы стоп: нет. Менингеальные сим птомы: нет. Болевая чувствительность не измене на. Температурная чувствительность не измене на. Глубокие виды чувствительности не измене ны. Вибрационная чувствительность не изменена. Мышечно-суставное чувство не изменено. Пальце- носовая координационная проба: выполняет удов летворительно, с двух сторон, тремор пальцев вы тянутых рук. Пяточно-коленная координационная проба: выполняет затруднённо из-за пареза, с обе их сторон. Атаксия: нет. Поза Ромберга: покачива ется, из позы не выходит. Походка утиная. Адиа дохокинеза нет. Незначительно выраженный ско лиоз поясничного отдела позвоночника. Пальпа ция позвоночника по остистым отросткам безбо лезненна. Дефанс мышц спины: нет. Пальпация окципитальных точек безболезненна. Симптомы натяжения отрицательные. Вегетативный статус: влажность кожи в пределах возрастной нормы, ор тостатическая проба – пульс относительно замед лен, дермографизм розовый, тип реакции сердеч но-сосудистой системы на физическую нагрузку – гипертонический, влажность кожи в пределах возрастной нормы, гипергидроз дистальный, саль ность кожи нормальная. Расширенная шкала мо торных функций HFMSE (Hammersmith Functional Motor Scale – Expanded) – 57 баллов [8]. Симптом Говерса положительный. Д б Неврологический статус. I пара – обоняние: норма. II пара – зрение: норма. II, IV, VI пары – зрачки круглые, средней величины, D = S, движе ние глазных яблок в полном объёме. Зрачковые ре акции на свет живые. Зрачковые реакции на кон вергенцию живые. Зрачковые реакции на аккомо дацию живые. Птоз: нет. Косоглазие: нет. Дипло пия: нет. V пара – точки выхода тройничного нерва безболезненные. Чувствительность на лице сохра нена. Жевательную мускулатуру напрягает доста точно. Вкус на языке не изменён. VII пара – лицо симметричное. Функция мимической мускулатуры достаточная. VIII пара – слух сохранён. Нистагма нет. Головокружения нет. IX, X пары – голос не из менён. Глотание не нарушено. Дужки мягкого нёба стоят симметрично, при фонации подвижны. Гло точный рефлекс сохранен. XI пара – движения го ловы не ограничены. Плечи поднимает одинаково с обеих сторон. XII пара – язык по средней линии. КЛИНИЧЕСКИЙ ПРИМЕР Клинический случай пациентки И., 36 лет, которая в феврале 2023 г. была госпитализирова на в неврологическое отделение клиники нерв ных болезней ФГБОУ ВО «Иркутский государ ственный медицинский университет» Минздрава России (ФГБОУ ВО ИГМУ Минздрава России) для уточнения диагноза и симптоматического ле чения (было получено информированное согла сие пациентки). В феврале 2023 г. впервые госпитализирована в неврологическое отделение клиник ФГБОУ ВО ИГМУ Минздрава России. Физикальное исследование. Температура 36,5 °C. Рост 154 см, вес 50 кг. Индекс массы тела 21,08. Состояние пациентки удовлетворительное. Со знание ясное. Положение больной активное. Тип конституции – нормостеник. Осанка: незначи Жалобы. Слабость в ногах и руках в прокси мальных отделах, больше в ногах; слабость в спи не. При физической нагрузке возникают судоро ги в ногах, руках, теле, после чего возникает но 47 Байкальский медицинский журнал, 2024, Том 3, № 1 Baikal Medical Journal, 2024, Vol. 3, Baikal Medical Journal, 2024, Vol. 3, No 1 Байкальский медицинский журнал, 2024, Том 3, № 1 Атрофии мышц языка нет. Фасцикуляция мышц языка: есть. Рефлексы орального автоматизма: нет. Объём активных движений в руках не ограничен. Объём активных движений в ногах не ограничен. Верхняя проба Барре: отрицательная с обеих сто рон. Нижняя проба Барре: положительная с обе их сторон. Сила в руках в проксимальных отде лах: 4 балла с двух сторон. Сила в руках в дисталь ных отделах: 4,5 балла с двух сторон. Сила в ногах в проксимальных отделах: 3 балла с двух сторон. Сила в ногах в дистальных отделах: 4 балла с двух сторон. Тонус мышц в конечностях: в руках гипото нус, в ногах гипотонус. Псевдогипертрофия икро ножных мышц. Движения в шейном отделе позво ночника не ограничены. Карпорадиальные рефлек сы не вызываются. Рефлексы с бицепса резко сни жены, равные. Рефлексы с трицепса не вызывают ся. Коленные рефлексы не вызываются. Ахилловы рефлексы не вызываются. Подошвенные рефлек сы не вызываются. Брюшные рефлексы вызывают ся, равные, умеренные. Стопные патологические рефлексы: нет. Кистевые патологические рефлек сы: нет. Клонусы стоп: нет. Менингеальные сим птомы: нет. Болевая чувствительность не измене на. Температурная чувствительность не измене на. Глубокие виды чувствительности не измене ны. Вибрационная чувствительность не изменена. Мышечно-суставное чувство не изменено. Пальце- носовая координационная проба: выполняет удов летворительно, с двух сторон, тремор пальцев вы тянутых рук. Пяточно-коленная координационная проба: выполняет затруднённо из-за пареза, с обе их сторон. Атаксия: нет. Поза Ромберга: покачива ется, из позы не выходит. Походка утиная. Адиа дохокинеза нет. Незначительно выраженный ско лиоз поясничного отдела позвоночника. Пальпа ция позвоночника по остистым отросткам безбо лезненна. КЛИНИЧЕСКИЙ ПРИМЕР Дефанс мышц спины: нет. Пальпация окципитальных точек безболезненна. Симптомы натяжения отрицательные. Вегетативный статус: влажность кожи в пределах возрастной нормы, ор тостатическая проба – пульс относительно замед лен, дермографизм розовый, тип реакции сердеч но-сосудистой системы на физическую нагрузку – гипертонический, влажность кожи в пределах возрастной нормы, гипергидроз дистальный, саль ность кожи нормальная. Расширенная шкала мо торных функций HFMSE (Hammersmith Functional Motor Scale – Expanded) – 57 баллов [8]. Симптом Говерса положительный. Дополнительное обследование К ф ф 386 00 МЕ/ тельно выраженный сколиоз поясничного отдела позвоночника. Оценка состояния кожных покро вов и видимых слизистых оболочек: телесного цве та, обычной влажности, без патологических вы сыпаний. Состояние подкожно-жировой клетчат ки: нормальное, псевдогипертрофия мышц голе ней. Наличие пастозности; отёков нет. Результаты пальпации лимфатических узлов: не пальпируют ся. Варикозное расширение вен: нет. Щитовидная железа в пределах нормы. Форма грудной клетки: нормальная. Деформация грудной клетки: нет. Тип дыхания – грудной. Частота дыхательных движе ний – 17. Насыщение крови кислородом (сатура ция) – 99 %. Незначительная одышка при ходь бе. Результаты аускультации лёгких: дыхание про водится по всем отделам. Хрипов нет. Результаты перкуссии и аускультации сердца: перкуторно гра ницы сердца не изменены. Аускультативно: тоны сердца ритмичные, ясные, патологических шумов не определяется. Частота сердечных сокращений и пульса – 63 ударов в минуту. Пульс ритмичный. Артериальное давление (АД) – 110/70 мм рт. ст. Язык чистый, влажный. Живот обычной формы. Результаты пальпации органов брюшной полости: пальпация безболезненна. Печень по краю рёбер ной дуги. Селезёнка не пальпируется. Перкутор но границы селезёнки в пределах нормы. Пальпа ция кишечника безболезненная. Осмотр пояснич ной области: патологических изменений не опре деляется. Пальпация почек: область проекции по чек внешне не изменена. Симптом поколачивания отрицательный с обеих сторон. Диурез в пределах нормы. Оценка характера системы мочеиспуска ния: со слов пациентки, свободное, не затруднено, произвольное, безболезненное. Оценка характера стула и кратности дефекации: со слов пациентки, нормальный, оформленный, без патологических включений, 1–2 раза в день. Наличие симптомов раздражения брюшины: не определяются. Нали чие менингеальных симптомов: не определяются. Атрофии мышц языка нет. Фасцикуляция мышц языка: есть. Рефлексы орального автоматизма: нет. Объём активных движений в руках не ограничен. Объём активных движений в ногах не ограничен. Верхняя проба Барре: отрицательная с обеих сто рон. Нижняя проба Барре: положительная с обе их сторон. Сила в руках в проксимальных отде лах: 4 балла с двух сторон. Сила в руках в дисталь ных отделах: 4,5 балла с двух сторон. Сила в ногах в проксимальных отделах: 3 балла с двух сторон. Дополнительное обследование Креатинфосфокиназа – 386,00 МЕ/л (26.01.2023), референсное значение для женщин – 26,00–140,00 МЕ/л. При проведении игольчатой ЭМГ 28.03.2023 (m. deltoideus dextra) зарегистрирована спонтанная активность в виде потенциалов фибрилляций; ам плитуда и длительность ПДЕ повышены; полифазия Клинические случаи Case reports 48 Байкальский медицинский журнал, 2024, Том 3, № 1 Baikal Medical Journal, 2024, Vol. 3, No 1 Байкальский медицинский журнал, 2024, Том 3, № 1 Baikal Medical Journal, 2024, Vol. 3, No 1 м 3, № 1 Baikal Medical Journal, 2024, Vol. 3, No 1 Байкальский медицинский журнал, 2024, Том 3, № 1 Baik Baikal Medical Journal, 2024, Vol. 3, No 1 Байкальский медицинский журнал, 2024, Том 3, № 1 Baikal Medical Journal, 2024, Vol. 3, No 1 Baikal Medical Journal, 2024, Vol. 3, No 1 ции диагноза СМА. Особенностью случая явля ется относительно благоприятный тип течения заболевания с дебютом после 18 месяцев (СМА III типа). Первые проблемы в двигательной сфе ре появились в школьном возрасте. Больная кли нически неоднократно обследовалась и была пра вильно определена группа патологии – нервно- мышечные заболевания. Клиническая картина в значительной степени мимикрировала миопа тию Эрба – Рота. Относительно мягкий харак тер заболевания, отсутствие внедрения в рутин ную клиническую практику молекулярно-гене тического исследования не способствовали чёт кой верификации нозологии. Проведённая впер вые в 2022 г. ЭМГ выявила наличие спонтанной активности в виде потенциалов фибрилляций. С этого момента в диагностический алгоритм было включено молекулярно-генетическое ис следование [2, 9, 10]. Подтверждённый диагноз СМА III типа, согласно клиническим рекомен дациям, предусматривает проведение патогене тической терапии с назначением препарата ну синерсена [3, 9, 10]. Выполнена компьютерная томография позвоноч ника (один отдел) (29.03.2023). Заключение: Деге неративно-дистрофические изменения пояснично- крестцового отдела позвоночника. Умеренная ско лиотическая деформация поясничного отдела по звоночника. Ретропсевдоспондилолистез L3, 1 ст. МСКТ-картина дорзальной равномерной протру зии межпозвонкового диска L5–S1. Камень пра вой почки. Проведён генетический анализ на выявление спи нальной мышечной атрофии. Анализ наличия экзона 7 генов SMN1/SMN2 (03.02.2023): зарегистрировано отсутствие сигнала, соответствующего экзону 7 гена SMN1. Поиск делеций в гене SMN1 (07.02.2023): в ре зультате исследования у пробанда зарегистрирова на делеция экзонов 7–8 гена SMN1 в гомозиготном состоянии. Делеция экзонов 7–8 гена SMN1 в го мозиготном состоянии является причиной прок симальной спинальной мышечной атрофии 5q. Определение числа копий генов SMN1, SMN2 (21.02.2023): зарегистрировано 0 копий экзонов 7–8 гена SMN1, 4 копии экзонов 7–8 гена SMN2, что подтверждает диагноз спиналь ной мышечной атрофии 5q III типа. Учитывая наличие генетически подтверждён ной спинальной мышечной атрофии, которая носит неуклонно прогрессирующий жизнеугро жающий характер, незначительную сколиотиче скую деформацию, фертильный возраст, с целью предотвращения прогрессирования заболевания, стабилизации двигательных функций, сохранения самообслуживания показано постоянное длитель ное патогенетическое лечение по жизненным по казаниям. Предпочтителен препарат нусинерсен, 12 мг интратекально, по схеме. Больная направ лена в специализированный центр для проведе ния патогенетического лечения. Пациентка про должает находиться под динамическим наблюде нием в клинике нервных болезней ФГБОУ ВО ИГМУ Минздрава России. Лечение: цитипигам 1 драже 3 раза в сутки; мексидол 4,0 мл на 200,0 мл физиологического раствора внутривенно капельно № 8; кортексин 10 мг на 2,0 физиологического раствора внутри мышечно № 8; ЛФК и физиолечение. Субъектив но: сохраняется слабость в ногах и руках в прокси мальных отделах, больше в ногах; слабость в спи не. Байкальский медицинский журнал, 2024, Том 3, № 1 Baikal Medical Journal, 2024, Vol. 3, No 1 На фоне лечения отмечает некоторую стабили зацию состояния, в настоящее время при привыч ной физической нагрузке не возникают судороги в ногах, руках, теле; нет дрожания в руках и ногах; не появляется чувство нехватки воздуха после фи зической нагрузки; боль в теле не беспокоит; го ловная боль на момент осмотра не беспокоит. Па циентка выписана под диспансерное наблюдение у специалистов по месту жительства с контролем уровня креатинфосфокиназы (КФК). ЗАКЛЮЧЕНИЕ Рекомендовано: продолжить амбулаторно приём капсул тиоктовой кислоты 600 мг утром до 3 месяцев; курсы тиоктовой кислоты 1–2 раза в год; курс ипидакрина 20 мг 3 раза в день до 2 ме сяцев; курсы ипидакрина несколько раз в год с пе рерывом между курсами в 1–2 месяца. ЛФК регу лярно. Курсы физиотерапии, массажа при отсут ствии противопоказаний 1–2 раза в год. Гиполи пидемическая диета. Контроль КФК амбулатор но через 6 месяцев, исследование уровня 25-ОН витамина D. Консультация генетика. У лиц молодого возраста с клинически вы ставленным диагнозом нервно-мышечного забо левания или подозрением на таковое проведение генетического обследования является обязатель ным. В частности, при проведении дифферен циального диагноза со спинальной мышечной атрофией должно проводиться молекулярно-ге нетическое исследование мутаций в гене SMN1. Верификация диагноза позволяет выбрать пра вильную тактику лечения и проводить патоге нетическое лечение, что способствует стабили зации состояния и улучшению прогноза у таких пациентов. Также необходимо генетическое те стирование сиблингов пациента с нервно-мы шечным заболеванием для выявления у них по добных мутаций. Байкальский медицинский журнал, 2024, Том 3, № 1 Байкальский медицинский журнал, 2024, Том 3, № 1 в сторону высоких амплитуд и низких частот – ха рактерно для нейрогенного процесса (рис. 1). Проведено исследование неспровоцированных ды хательных объёмов и потоков (29.03.2023). Показа тель жизненной ёмкости лёгких (ЖЕЛ) – 81 %. По казатель объёма форсированного выдоха (ОФВ1) – 99 %. Соотношение ОФВ1/ФЖЕЛ – 92,4. Заклю чение: нормальная спирометрия. в сторону высоких амплитуд и низких частот – ха рактерно для нейрогенного процесса (рис. 1). Проведено исследование неспровоцированных ды хательных объёмов и потоков (29.03.2023). Показа тель жизненной ёмкости лёгких (ЖЕЛ) – 81 %. По казатель объёма форсированного выдоха (ОФВ1) – 99 %. Соотношение ОФВ1/ФЖЕЛ – 92,4. Заклю чение: нормальная спирометрия. ПДЕ незначительная, амплитуда интерференцион ного паттерна с разрежением кривой – характерная для нейрогенного процесса. При игольчатой ЭМГ (m. tibialis anterior sinistra) зарегистрирована спонтан ная активность в виде потенциалов фибрилляций, положительных острых волн; амплитуда и длитель ность ПДЕ повышены; полифазия ПДЕ умеренная, турно-амплитудный анализ со смещением «облака» РИС. 1. Электромиография пациентки И., 36 лет РИС. 1. Электромиография пациентки И., 36 лет Клинические случаи Case reports Клинические случаи C 49 49 Байкальский медицинский журнал, 2024, Том 3, № 1 Baikal Medical Journal, 2024, Vol. 3, No 1 ОБСУЖДЕНИЕ Представленный клинический случай демон стрирует достаточно поздний этап верифика Клинические случаи Case reports 50 Байкальский медицинский журнал, 2024, Том 3, № 1 Baikal Medical Journal, 2024, Vol. 3, No 1 Конфликт интересов Авторы декларируют отсутствие явных и потенциальных конфлик тов интересов, связанных с публикацией настоящей статьи. The authors declare no apparent or potential conflict of interest related to the publication of this article. The authors declare no apparent or potential conflict of interest related to the publication of this article. Informed consent for publication Авторы получили письменное согласие пациента на анализ и пу бликацию медицинских данных. Авторы получили письменное согласие пациента на анализ и пу бликацию медицинских данных. Written consent was obtained from the patient for publication of rele vant medical information within the manuscript. Written consent was obtained from the patient for publication of rele vant medical information within the manuscript. Соответствие принципам этики The study was approved by the local ethics committee. The ap proval and procedure for the protocol were obtained in accordance with the principles of the Helsinki Convention. Протокол исследования был одобрен локальным этическим коми тетом. Одобрение и процедуру проведения протокола получали по принципам Хельсинкской конвенции. 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Забненкова В.В., Дадали Е.Л., Руденская Г.Е., Галкина В.А., Федотов В.П., Поляков А.В. Анализ фено- ЛИТЕРАТУРА / REFERENCES генотипической корреляции у российских больных спи нальной мышечной атрофией I–IV типа. Медицинская генетика. 2012; 1(115): 15-21. [Zabnenkova VV, Dadali EL, Rudenskaya GE, Galkina VA, Fedotov VP, Polyakov AV. Analysis of pheno-genotypic correlations in Russian patients with spinal muscular atrophy I–IV. Medical Genetics. 2012; 1(115): 15-21. (In Russ.)]. 1. Левин О.С., Штульман Д.Р. Неврология: справоч ник практического врача; 14-е изд. М.: МЕДпресс-ин форм; 2022. [Levin OS, Stulman DR. Neurology: handbook for practicing physician; 14th ed. Moscow: MEDpress-inform; 2022. (In Russ.)]. 2. Влодавец Д.В., Харламов Д.А., Артемьева С.Б., Белоусова Е.Д. Федеральные клинические рекомендации (протоколы) по диагностике и лечению спинальных мы шечных атрофий у детей. М.; 2013. [Vlodavets DV, Khar lamov DA, Artemieva SB, Belousova ED. Federal clinical guidelines (protocols) for the diagnosis and treatment of spinal muscular atrophy in children. Moscow; 2013. (In Russ.)]. 6. Фельдман Е. Атлас нервно-мышечных болезней: Практическое руководство. М.: Практическая медицина; 2017. [Feldman E. Atlas of neuromuscular diseases: A practical guide. Moscow: Prakticheskaya meditsina; 2017. (In Russ.)]. 7. Гехт А.Б., Касаткина Л.Ф., Санадзе А.Г. Атлас нервно-мышечных болезней. Клинические примеры. М.: ООО «Буки-Веди»; 2022 [Gekht AB, Kasatkina LF, San adze AG. Atlas of neuromuscular diseases. Clinical cases. Moscow: Buki-Vedi; 2022. (In Russ.)]. 3. Артемьева С.Б., Белоусова Е.Д., Влодавец Д.В., Вольский Г.Б., Германенко О.Ю., Грознова О.С., и др. Клинические рекомендации. Проксимальная спинальная мышечная атрофия 5q. М., 2020. [Artemieva SB, Belou sova ED, Vlodavets DV, Volskiy GB, Germanenko OYu, Groznova OS, et al. 5q proximal spinal muscular atrophy. Moscow; 2020. (In Russ.)]. 3. Артемьева С.Б., Белоусова Е.Д., Влодавец Д.В., Вольский Г.Б., Германенко О.Ю., Грознова О.С., и др. Клинические рекомендации. Проксимальная спинальная мышечная атрофия 5q. М., 2020. [Artemieva SB, Belou sova ED, Vlodavets DV, Volskiy GB, Germanenko OYu, Groznova OS, et al. 5q proximal spinal muscular atrophy. Moscow; 2020. (In Russ.)]. 8. Расширенная шкала оценки моторных функций боль ницы Хаммерсмит (HFMSE). Методическое руководство и оценочные листы [The Extended Hammersmith Hospital Mo tor Function Assessment Scale (HFMSE). Methodological guide line and evaluation sheets. (In Russ.)]. URL: https://ormiz.ru/ adm/files/janssen/HFMSE.pdf [дата доступа: 04.02.2024]. 8. Расширенная шкала оценки моторных функций боль ницы Хаммерсмит (HFMSE). Методическое руководство и оценочные листы [The Extended Hammersmith Hospital Mo tor Function Assessment Scale (HFMSE). Methodological guide line and evaluation sheets. (In Russ.)]. URL: https://ormiz.ru/ adm/files/janssen/HFMSE.pdf [дата доступа: 04.02.2024]. 4. Селивёрстов Ю.А., Клюшников С.А., Иллари ошкин С.Н. Спинальные мышечные атрофии: понятие, дифференциальная диагностика, перспективы лечения. Нервные болезни. 2015; (3): 9-17 [Seliverstov YuA, Klyush nikov SA, Illarioshkin SN. Источник финансирования Авторы декларируют отсутствие внешнего финансирования для проведения исследования и публикации статьи. The authors declare no external funding for the study and publication of the article. The authors declare no external funding for the study and publication of the article. Baikal Medical Journal, 2024, Vol. 3, No 1 Baikal Medical Journal, 2024, Vol. 3, No 1 Tatiana A. Tarasova – Neurologist at the Neurological Department of Clinics, Irkutsk State Medical University. ORCID: 0009-0001-4576- 785X Тарасова Татьяна Александровна – врач-невролог неврологи ческого отделения клиник, ФГБОУ ВО «Иркутский государствен ный медицинский университет» Минздрава России. ORCID: 0009- 0001-4576-785X Svetlana V. Vasilkova – Neurologist at the Neurological Department of Clinics, Irkutsk State Medical University. ORCID: 0000-0002-9208- 9983 Svetlana V. Vasilkova – Neurologist at the Neurological Department of Clinics, Irkutsk State Medical University. ORCID: 0000-0002-9208- 9983 Василькова Светлана Владимировна – врач-невролог не врологического отделения клиник, ФГБОУ ВО «Иркутский го сударственный медицинский университет» Минздрава России. ORCID: 0000-0002-9208-9983 Yulia S. Plekhanova – Neurologist at the Neurological Department of Clinics, Irkutsk State Medical University. ORCID: 0009-0005-4023- 376X Плеханова Юлия Станиславовна – врач-невролог неврологи ческого отделения клиник, ФГБОУ ВО «Иркутский государствен ный медицинский университет» Минздрава России. ORCID: 0009- 0005-4023-376X Клинические случаи Case reports Corresponding author Corresponding author Yury N. Bykov, bykov1971@mail.ru Received 04.02.2024 Accepted 12.02.2024 Published 10.03.2024 Information about the authors Информация об авторах Быков Юрий Николаевич – д.м.н., профессор, заведующий ка федрой нервных болезней, ФГБОУ ВО «Иркутский государствен ный медицинский университет» Минздрава России. ORCID: 0000- 0002-7836-5179 Yury N. Bykov – Dr. Sci. (Med.), Professor, Head of the Department of Nervous Diseases, Irkutsk State Medical University. ORCID: 0000- 0002-7836-5179 Yury N. Vasyliev – Cand. Sci. (Med.), Associate Professor at the De partment of Nervous Diseases, Irkutsk State Medical University. ORCID: 0000-0003-0181-3292 Васильев Юрий Николаевич – к.м.н., доцент кафедры нервных болезней, ФГБОУ ВО «Иркутский государственный медицинский университет» Минздрава России. ORCID: 0000-0003-0181-3292 Tatiana N. Zagvozkina – Head of the Neurological Department of Clinics, Irkutsk State Medical University. ORCID: 0009-0007-2213- 9133 Загвозкина Татьяна Николаевна – заведующая неврологиче ским отделением клиник, ФГБОУ ВО «Иркутский государствен ный медицинский университет» Минздрава России. ORCID: 0009- 0007-2213-9133 Irina V. Anikina – Neurologist at the Neurological Department of Clin ics, Irkutsk State Medical University. ORCID: 0009-0008-5109-5886 Irina V. Anikina – Neurologist at the Neurological Department of Clin ics, Irkutsk State Medical University. ORCID: 0009-0008-5109-5886 Аникина Ирина Викторовна – врач-невролог неврологическо го отделения клиник, ФГБОУ ВО «Иркутский государственный ме дицинский университет» Минздрава России. ORCID: 0009-0008- 5109-5886 Клинические случаи Case reports 51 Вклад авторов The authors participated equally in the preparation of the publica tion: concept development, obtaining and analyzing factual data, writ ing and editing the text of the article, checking and approving the text of the article. The authors declare their authorship to be in compliance with the international ICMJE criteria. Авторы в равной степени участвовали в подготовке публикации: разработка концепции статьи, получение и анализ фактических данных, написание и редактирование текста статьи, проверка и ут верждение текста статьи. Авторы декларируют соответствие сво его авторства международным критериям ICMJE. Для переписки Для переписки Быков Юрий Николаевич, bykov1971@mail.ru Быков Юрий Николаевич, bykov1971@mail.ru Received 04.02.2024 Accepted 12.02.2024 Published 10.03.2024 Получена 04.02.2024 Принята 12.02.2024 Опубликована 10.03.2024 Получена 04.02.2024 Принята 12.02.2024 Опубликована 10.03.2024 Клинические случаи Case reports
https://openalex.org/W4291497262	https://zenodo.org/records/898027/files/599039720.pdf	English	null	Study on length–Weight Relationship and Condition Factor of Capoeta trutta in Gamasiab River (Iran)	Zenodo (CERN European Organization for Nuclear Research)	2,015	cc-by	1,758	g ( ), RESULTS AND DISCUSSION In the present study, Total length and weight ranged from 10.5 to 19.8mm and 11.86 to 76.82g, respectively. Average total length and weight was 14.41±0.91 (±SD) mm and 34.7±0.86 (±SD) g, respectively. Length-weight relationship was W= 0.009L3.054 (Figure 1). The obtained b value for LWR was 3.054. The fish exhibited positive allometric growth pattern. The LWR was highly significant (P<0.01), with coefficient of determination values = 0.97. There was a higher correlation coefficient value in the length-weight for C. trutta. 3/ Issue 4 ISSN: 2348 – 7313 1 Zoology Volume 3/ Issue 4 ISSN: 2348 – 7313 1 er of the well-being, state of the fish tions [8]. The results of condition compares the wellbeing of fish been under taken to establish a and condition factor of C. trutta in will be helpful in the future studies HODS f C. trutta were randomly sampled Electrofishing on September 2014. The present study has been under taken to establish a length-weight relationship and condition factor of C. trutta in Gamasiab River. This study will be helpful in the future studies on C. trutta in this region. INTRODUCTION The total lengths were measured with a digital slide caliper. Also, body weights were measured to the nearest 0.01 g using a digital weighing scale. The length-weight relationship was estimated by using the equation provided by Ricker (1973): W=aLb [10], where W=Weight of fish (g), L= Total length of fish (cm), b = Regression coefficient or slope and a = Regression constant or intercept. The condition factor was calculated using the means of the total length and weight of fish as provided by Gayanilo and Pauly (1997) with the equation: K =100w/L3 [11], where W= Mean body weight of fish (g), L= Mean total length of fish (cm), K= condition factor. The genus Capoeta is found in Eastern Europe and southwestern Asia [1] and contains about 10 species, of which 7 occur in Iran [2]. Capoeta trutta belongs to the genus Capoeta and Cyprinidae family [3]. In the specie, the mouth is slightly arched or even straight in ventral view. In addition to, the horny edge to the lower jaw is usually well-developed but may be lost in preserved specimens [2]. This species is found in the Tigris-Euphrates basins from Iran. C. trutta is present in Dez River near Dezful and Gheshlagh Dam Lake [2-3]. Length-weight relationship (LWR) of fishes are important in fisheries and fish biology because they allow the estimation of the average weight of the fish of a given length group by establishing a mathematical relation between them [4]. LWR helps to figure out the reproduction history, condition, life history, health of fish and general conditions of fish species [5]. The length-weight relationship is providing information on the condition of fish and to determine whether growth pattern was isometric or allometric [6]. Therefore, Length–weight Relationship (LWR) is useful tool in fish growth pattern or age determination and fishery assessment [7]. The condition factor (k) is a quantitative parameter of the well-being, state of the fish and reflects feeding conditions [8]. The results of condition factor can be used to compares the wellbeing of fish populations [9]. Study on length–Weight Relationship and Condition Factor of Capoeta trutta in Gamasiab River (Iran) Alireza Radkhah1, hashem nowferesti2 and shaghayegh asgardun2 1University of Tehran, Faculty of Natural Resources, Department of Fisheries, Karaj, Iran 2University of Gorgan, Faculty of Natural Resources, Department of Fisheries, Gorgan, Iran Corresponding author: Alireza Radkhah, E-mail: radkhahalireza@yahoo.com Received: October 12, 2015, Accepted: November 23, 2015, Published: November 23, 2015. ABSTRACT The present study aims to study the length-weight relationship and condition factor of Capoeta trutta in Gamasiab River from Iran. A total of 40 individuals of C. trutta were sampled from Gamasiab River by Electro fishing on September 2014. Total Length (cm) and body weight (g) for each specimen was taken by a digital slide caliper and balance, respectively. In the present study, Total length and weight ranged from 10.5 to 19.8 mm and 11.86 to 76.82 g, respectively. Average total length and weight was 14.41±0.91 (SD) mm and 34.7±0.86 (SD) g, respectively. The b value was 3.054 that is significantly larger than 3, indicating a positive allometric growth for the considered group. In this study, condition factor (K) of fish was 0.93. This study provides basic information on C. trutta for fishery biologists in Iran. g ds: Length, Weight, Relationship, Condition factor, C. trutta. y g Key words: Length, Weight, Relationship, Condition factor, C. trutta. Study on length–Weight Relationship and Condition Factor of Capoeta trutta in Gamasiab River (Iran) Alireza Radkhah1, hashem nowferesti2 and shaghayegh asgardun2 1University of Tehran, Faculty of Natural Resources, Department of Fisheries, Karaj, Iran 2University of Gorgan, Faculty of Natural Resources, Department of Fisheries, Gorgan, Iran Corresponding author: Alireza Radkhah, E-mail: radkhahalireza@yahoo.com Received: October 12, 2015, Accepted: November 23, 2015, Published: November 23, 2015. JOURNAL OF ADVANCED BOTANY AND ZOOLOGY Journal homepage: http://scienceq.org/Journals/JABZ.php Research Article Open Access JOURNAL OF ADVANCED BOTANY AND ZOOLOGY Journal homepage: http://scienceq.org/Journals/JABZ.php MATERIALS AND METHODS A total of 40 individuals of C. trutta were randomly sampled from Gamasiab River by Electrofishing on September 2014. ISSN: 2348 – 7313 J. of Advanced Botany and Zoology Volume 3/ Issue 4 Fig. 1: The Length-weight relationship curve for C. trutta in Gamasiab River. Fig. 1: The Length-weight relationship curve for C. trutta in Gamasiab River. 4. 4. J.E. Beyer, On length-weight relationships: Part II. Computing mean weights from length statistics, Fishbyte. 9 (2) (1991) 50-54. 5. Length-weight parameters are affected by a series of parameters such as season, habitat, health, habitat, diet, gonad maturity, degree of stomach fullness, sex, health and preservation technique [12]. Moradinasab et al. (2012) [13] study length-length relationship of H. leucisculus in Anzali wetland. In this study, the values of a, b and r2 were 0.409, 0.927 and 0.93 respectively for length-length relationship of H. leucisculus from the Anzali wetland. Hashemzadeh Segherloo et al. (2015) [14] study length-weight relationships of Garra rufa, in the Tigris and Persian Gulf basins of Iran. In their study, the value of exponent b ranged from 2.74 to 3.19 with average of 2.99 in the Tigris basin and 2.96 in the Persian Gulf basin which was in normal range (2.5-3.5). Therefore, in our Study, According to Hashemzadeh Segherloo et al. (2015) [14] the b value was in normal range. Haque and Biswas (2014) [15] study length-weight relationship of Botia dario from wetland of Sivasagar district. In this study, the ‘b’ value ranged from 3.06 in male and 3.128 in female lengthwise. Seasonally, ‘b’ value for male is 2.02 to 3.45 and for female is 2.4 to 3.17. 5. 5. G.W. Nikolsky, The ecology fishes. Academic Press, London and New York, 1963, pp. 1-352. 6. 6. W. E. Ricker, Computations and interpretation of biological statistics of fish populations, Bulletin of the Fisheries Research Board of Canada. 191 (1975) 382. 7. 7. P.C.G. Pepple, C.O. Ofor, Length-Weight relationship of Heleterobranchus longifilis reared in earthen Ponds, Nigerian Journal of Fisheries. 8(2) (2011) 315-321. 8. 8. E.D. LeCren, The length-weight relationship and seasonal cycle in gonad weight and condition in the perch (Percafluviatilis), Journal of Animal Ecology. 20 (1951) 201-219. 9. 9. T. Bagenal, F.W. Tesch, Age and Growth in Method of Assessment of Fish Production in Fresh water. IBP Handbook, Blackwell Scientific Press, Oxford, 1978. 10. 10. W. E. Ricker, Linear regressions in fishery research, Journal of Fisheries Research Board of Canada. MATERIALS AND METHODS 30 (1973) 409-434. 11. 11. F.C. Gayanilo, D. Pauly, Fao-Iclarm Stock Assessment Tools (FiSAT). FAO Computerized Information Series, (Fisheries). 8 (1997): 262. In the present study, condition factor (K) of fish was 0.93. According to LeCren (1951) [8] the relative condition factor (K) is an indicator of general well-being of the fish. Tabassum et al. (2015) [16] study condition factor of Hemiramphus archipelagicus (Family: Hemiramphidae) from Karachi Coast in Pakistan. In this study, the highest mean condition factor (0.257 ± 0.031) was recorded in samples of length of 11-20 cm. Also, the lowest mean condition factor (0.197 ± 0.016) was recorded in 21-30 cm. The results showed that K varied mainly with size classes. The condition factor of fishes influenced by a number of factors such as the spawning and maturity, sex, availability of feeds and season [17]. 12. 12. R.J. Wooten, Ecology of teleost fishes. Kluwer Academic Publishers, Dordrecht, the Netherlands, 1990, pp. 1-404. 13. 13. G. Moradinasab, M. Daliri, R. Ghorbani, S.Y. Paighambari, R. Davoodi, Length-weight and length-length relationships, Relative condition factor and Fulton’s condition factor of Five Cyprinid species in Anzali wetland, southwest of the Caspian Sea, Caspian J. Env. Sci. 10(1) (2012) 25- 31. 14. 14. I. HashemzadehSegherloo, N. Tabatabaei, A. Mansouri, A. Abdoli, M. Ghalenoei, K. Golzarianpour, Length-weight relationships of Garrarufa, in the Tigris and Persian Gulf basins of Iran, International Journal of Aquatic Biology. 3(1) (2015) 25-27. CONCLUSION The present study provides useful information for biologists and the direction for the future management of C. trutta in the Gamasiab River (Iran). 15. 15. S. Haque, S.P. Biswas, Length-Weight relationship and condition factor of Botiadario (Hamilton-Buchanan) from Sivasagar District, International Journal of Fisheries and Aquatic Studies. 2(1) (2014) 244-247. REFERENCES 1. 1. P. Banarescu, B.W. Coad, Cyprinids of Eurasia. In: Winfield I.J. and Nelson J.S (Eds.), Systematics, Biology and Exploitation. Chapman and Hall, London, 1991, pp. 1-4. 16. 16. S. Tabassum, F. Yousuf, N. Elahi, A. Raza, S. Arif, N. Fatima, Length weight relationship and condition factor of Hemiramphus archipelagicus Collette and Parin, 1978. (Family: Hemiramphidae) from Karachi Coast, Pakistan, International Journal of Fauna and Biological Studies. 2(2) (2015) 53-56. 2. 2. B.W. Coad, Freshwater fishes of Iran, Available at: www.briancoad.com, 2015. 3. 3. A. Abdoli, The Inland Water Fishes of Iran. Iranian Museum of Nature and Wildlife. Tehran, Iran, 2000, pp. 1-378. 17. 17. E.G. Khallaf, M. Athuman, The biology of Oreochromis niloticus in Polluted Canal, Ecotoxicology. 12 (2003) 405-416. Citation: Alireza Radkhah et al (2015). Study on length–Weight Relationship and Condition Factor of Capoeta trutta in Gamasiab River (Iran). J. of Advanced Botany and Zoology, V3I4. DOI: 10.15297/JABZ.V3I4.07. Citation: Alireza Radkhah et al (2015). Study on length–Weight Relationship and Condition Factor of Capoeta trutta in Gamasiab River (Iran). J. of Advanced Botany and Zoology, V3I4. DOI: 10.15297/JABZ.V3I4.07. Copyright: © 2015 Alireza Radkhah. 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. ISSN: 2348 – 7313 J. of Advanced Botany and Zoology Volume 3/ Issue 4 2
https://openalex.org/W2608001712	https://europepmc.org/articles/pmc5481554?pdf=render	English	null	Heat-shock protein 90 (Hsp90) promotes opioid-induced anti-nociception by an ERK mitogen-activated protein kinase (MAPK) mechanism in mouse brain	Journal of biological chemistry/The Journal of biological chemistry	2,017	cc-by	15,798	Heat-shock protein 90 (Hsp90) promotes opioid-induced anti-nociception by an ERK mitogen-activated protein kinase (MAPK) mechanism in mouse brain Received for publication,November 23, 2016, and in revised form, April 13, 2017 Published, Papers in Press,April 27, 2017, DOI 10 Wei Lei‡, Nathan Mullen§, Sarah McCarthy§, Courtney Brann§, Philomena Richard§, James Cormier§, Katie Edwards§, Edward J. Bilsky§¶, and X John M. Streicher‡1 From the ‡Department of Pharmacology, College of Medicine, University of Arizona, Tucson, Arizona 85724, the §Department of Biomedical Sciences, College of Osteopathic Medicine, University of New England, Biddeford, Maine 04005, and the ¶Department of Biomedical Sciences, College of Osteopathic Medicine, Pacific Northwest University, Yakima, Washington 98901 Edited by F. Anne Stephenson Recent advances in developing opioid treatments for pain with reduced side effects have focused on the signaling cascades of the -opioid receptor (MOR). However, few such signaling targets have been identified for exploitation. To address this need, we explored the role of heat-shock protein 90 (Hsp90) in opioid-induced MOR signaling and pain, which has only been studied in four previous articles. First, in four cell models of MOR signaling, we found that Hsp90 inhibition for 24 h with the inhibitor 17-N-allylamino-17-demethoxygeldanamycin (17-AAG) had different effects on protein expression and opioid signaling in each line, suggesting that cell models may not be reliable for predicting pharmacology with this protein. We thus developed an in vivo model using CD-1 mice with an intracere- broventricular injection of 17-AAG for 24 h. We found that Hsp90 inhibition strongly blocked morphine-induced anti-no- ciception in models of post-surgical and HIV neuropathic pain but only slightly blocked anti-nociception in a naive tail-flick model, while enhancing morphine-induced precipitated with- drawal. Seeking a mechanism for these changes, we found that Hsp90 inhibition blocks ERK MAPK activation in the periaque- ductal gray and caudal brain stem. We tested these signaling changes by inhibiting ERK in the above-mentioned pain models and found that ERK inhibition could account for all of the changes in anti-nociception induced by Hsp90 inhibition. Taken together, these findings suggest that Hsp90 promotes opioid-induced anti-nociception by an ERK mechanism in mouse brain and that Hsp90 could be a future target for improv- ing the therapeutic index of opioid drugs. annual economic cost of $600 billion (1). Moderate to severe cancer and non-cancer chronic pain seriously degrades patient quality of life and interferes with daily activities (2). 2 The abbreviations used are: MOR, -opioid receptor; arr2, -arrestin2; Hsp, heat-shock protein; CCI, chronic constriction injury; 17-AAG, 17-N-allyl- amino-17-demethoxygeldanamycin; pERK, phosphorylated ERK; tERK, total ERK; LPA, lysophosphatidic acid; i.c.v., intracerebroventricular; PAG, periaqueductal gray; s.c., subcutaneous; GPCR, G-protein-coupled recep- tor; ANOVA, analysis of variance; AUC, area under the curve; DAMGO, [D-Ala2, N-MePhe4, Gly2-ol]-enkephalin. This work was supported by the University of New England and the University of Arizona and by National Institutes of Health Pilot Project Grant P20GM103643 (Ian Meng PI) under an institutional COBRE award. The authors declare that they have no conflicts of interest with the contents of this article. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. cros cros ARTICLE Heat-shock protein 90 (Hsp90) promotes opioid-induced anti-nociception by an ERK mitogen-activated protein kinase (MAPK) mechanism in mouse brain There are multiple drug classes for the treatment of chronic pain, includ- ing tricyclic antidepressants and gabapentinoids, but often the only efficacious option for serious pain is opioid drugs such as morphine or fentanyl. Although efficacious, opioid drugs induce a constellation of side effects, including abuse liability, tolerance, constipation, and respiratory depression (3, 4). These limitations have spurred a search for new analgesic drugs with a reduction or elimination of side effects, which has persisted for more than 100 years with little success (5). Recent advances in the understanding of signal transduction downstream of the -opioid receptor (MOR)2 have suggested new approaches for drug discovery. Notably, the ubiquitous signaling regulator -arrestin2 (arr2) has been found to reduce anti-nociception (6) and promote opioid side effects (4, 7), leading to the development of biased ligands that activate MOR without recruiting arr2, one of which is currently in clinical trials (8–11). Although promising, this signal transduc- tion-focused approach is limited by the availability of signaling targets to exploit; some targets such as Raf-1 (12) and RGS proteins (13) have been shown to regulate opioid side effects, but they have not been exploited for drug discovery to our knowledge, and few other such targets are known. To address this need and improve our chances of developing new opioid therapies, we investigated the ubiquitous and cru- cial signaling regulator Hsp90 as a potential regulator of opioid signaling and behavioral responses. Hsp90 is a chaperone pro- tein that prevents protein aggregation and degradation. How- ever, it can also facilitate the activation of signal transduction molecules, including kinases, by promoting a conformation that is ideal for activation (14, 15). Hsp90, present in all cell types, makes up 2–3% of total cell protein content and up to 4–5% during heat stress. The activity of Hsp90 is regulated and Chronic pain is a serious public health problem in the United States, with an estimated 100 million affected people and an 1 To whom correspondence should be addressed: Dept. of Pharmacology, College of Medicine, University of Arizona, Box 245050, Life Sciences North 563, 1501 N. Campbell Ave., Tucson, AZ 85724. Tel.: 520-626-7495; Fax: 520-626-4182; E-mail: jstreicher@email.arizona.edu. 1 To whom correspondence should be addressed: Dept. of Pharmacology, College of Medicine, University of Arizona, Box 245050, Life Sciences North 563, 1501 N. Campbell Ave., Tucson, AZ 85724. Tel.: 520-626-7495; Fax: 520-626-4182; E-mail: jstreicher@email.arizona.edu. Heat-shock protein 90 in opioid signaling and pain MOR itself and of the signaling regulator arr2 and the kinase Akt. Unlike Hsp70, however, the regulation of these protein levels differed considerably by cell line; the MOR was decreased in CHO cells but unaltered in HEK, arr2 levels were decreased in U2OS but not CHO cells, and Akt levels were decreased in CHO cells but not U2OS (Fig. 1B). directed by co-chaperone proteins, including Hop/STIP1 and Cdc37, which are more selective by tissue and signaling type than Hsp90 itself (14, 16). Because of this selectivity, co-chap- erone proteins may represent future targets for modulation that could decrease the side effects of modulating Hsp90 itself, as has been done for Hsp70 (17). directed by co-chaperone proteins, including Hop/STIP1 and Cdc37, which are more selective by tissue and signaling type than Hsp90 itself (14, 16). Because of this selectivity, co-chap- erone proteins may represent future targets for modulation that could decrease the side effects of modulating Hsp90 itself, as has been done for Hsp70 (17). p ( ) Hsp90 has only been studied in two pain models to date. First, it has been found that Hsp90 inhibition reverses diabetes- induced mechanical hypoalgesia (18). Second, it has been shown that Hsp90 inhibition reverses chronic constriction injury (CCI)-induced mechanical allodynia and potentiates morphine anti-nociception in this model, whereas Hsp90 acti- vation has the opposite effect when combined with lipopolysac- charide treatment (19). Only two studies have examined the role of Hsp90 specifically in MOR signal transduction and opi- oid response. One study found in an in vitro model that Hsp90 inhibition reduces adenylyl cyclase superactivation, a cellular marker of dependence (20). An article by the Devi group found that Hsp90 is up-regulated by chronic morphine treatment in the presynaptic terminal and that administration of the Hsp90 inhibitor geldanamycin in the periphery immediately prior to naloxone precipitation reduces the somatic signs of withdrawal (21). This limited evidence from the literature suggests that Hsp90 has a role in regulating MOR signal transduction and opioid behavioral responses. However, in the CCI and depen- dence in vivo studies (19, 21) the Hsp90 inhibitor was adminis- tered 3 h or less prior to measuring pain or withdrawal, which may not be a good model for chronic pain treatment. Hsp90 inhibition in mouse models of opioid-induced behavior For the in vivo studies, mice were treated with 17-AAG by the i.c.v. route to isolate the role of Hsp90 specifically in the brain, with a 24-h recovery time as described above, to more closely model chronic pain treatment. The i.c.v. route results in higher concentrations of injected drug molecules around the wall of the ventricles and associated areas, which have high levels of opioid receptors and are known to be important in producing various effects of opioid drugs such as morphine (e.g. anti-no- ciception, physical dependence, etc.). However, we could not find in the literature that 17-AAG had been administered by the i.c.v. route, although Hsp70 is up-regulated in the brain in response to systemic 17-AAG (25). We thus performed a dose- response study of i.c.v. 17-AAG in male CD-1 mice, with a read- out of Hsp70 up-regulation by Western blotting (Fig. 2). We found robust dose-dependent Hsp70 up-regulation in the peri- aqueductal gray (PAG), a region high in MOR expression and critical for pain regulation (26), with an approximate A50 of 0.084 nmol. Based on these results, we chose the approximate A90 dose of 0.5 nmol for further studies, which reduced the risk of off-target effects from using the highest dose tested (5 nmol). We then tested the effects of Hsp90 inhibition on opioid response in several pain and dependence/withdrawal models. We first established a clinically relevant post-surgical pain model by paw incision (27) with concurrent 17-AAG injection and a 24-h recovery. Pain response and allodynia were mea- sured by mechanical stimulation with Von Frey filaments (28). The animals showed a robust allodynia post-surgery that was not altered by 17-AAG treatment (Fig. 3A). The vehicle-treated animals showed a robust time-dependent anti-nociception in response to systemic morphine (1, 3.2, and 10 mg/kg s.c.), which was strongly blocked by 17-AAG treatment (area under the curve (AUC) reduction of 70.2, 81.5, and 67.0% at 1, 3.2, and For the in vivo studies, mice were treated with 17-AAG by the i.c.v. route to isolate the role of Hsp90 specifically in the brain, with a 24-h recovery time as described above, to more closely model chronic pain treatment. The i.c.v. route results in higher concentrations of injected drug molecules around the wall of the ventricles and associated areas, which have high levels of opioid receptors and are known to be important in producing various effects of opioid drugs such as morphine (e.g. 10414 J. Biol. Chem. (2017) 292(25) 10414–10428 © 2017 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A. Heat-shock protein 90 in opioid signaling and pain This heterogeneity continued when we analyzed the phos- phorylation and activation of ERK (pERK) in these cells in response to DAMGO after Hsp90 inhibition (total ERK levels (tERK) were not altered by Hsp90 inhibition (Fig. 1A)). In CHO cells, the per-unit ERK activation was strongly increased by Hsp90 inhibition in response to DAMGO and the endogenous GPCR agonist lysophosphatidic acid (LPA) (Fig. 1C). However, in HEK cells the pattern of ERK activation was not changed by Hsp90 inhibition, whereas it was decreased in U2OS cells. Lastly, the SH-SY5Y cells displayed an unusual ERK activation pattern whereby the unstimulated baseline was strongly increased but ERK was not stimulated above that elevated base- line by either DAMGO or LPA. As summarized in Table 1, Hsp90 inhibition showed heterogeneous results in different cell lines, which suggested that in vitro models would not be reliable in studying the role of Hsp90 in the opioid system and would necessitate the use of relevant in vivo models for the study of this protein. Hsp90 inhibition in mouse models of opioid-induced behavior anti-no- ciception, physical dependence, etc.). However, we could not find in the literature that 17-AAG had been administered by the i.c.v. route, although Hsp70 is up-regulated in the brain in response to systemic 17-AAG (25). We thus performed a dose- response study of i.c.v. 17-AAG in male CD-1 mice, with a read- out of Hsp70 up-regulation by Western blotting (Fig. 2). We found robust dose-dependent Hsp70 up-regulation in the peri- aqueductal gray (PAG), a region high in MOR expression and critical for pain regulation (26), with an approximate A50 of 0.084 nmol. Based on these results, we chose the approximate A90 dose of 0.5 nmol for further studies, which reduced the risk of off-target effects from using the highest dose tested (5 nmol). h d h ff f h b d In our current studies, we tested the role of Hsp90 in MOR signaling and pain using the selective inhibitor 17-AAG (22) on a 24-h treatment regimen to more closely model chronic pain treatment. The results from both in vitro and in vivo experi- ments indicated strong impacts of Hsp90 inhibition on opioid- induced signaling and pain behaviors, showing that Hsp90 regulates pain behavior through an ERK mechanism. These findings suggest that Hsp90 could be a target for promoting analgesia and reducing the side effects of opioid therapy. Hsp90 inhibition in cell models of MOR signaling We first sought to explore the role of Hsp90 in regulating MOR signaling in four in vitro models. These include Chinese hamster ovary (CHO), human embryonic kidney (HEK), and human bone osteosarcoma (U2OS) cell lines stably expressing the human MOR (see “Experimental procedures” for cell line details) and the human neuroblastoma cell line SH-SY5Y, which endogenously expresses the MOR and -opioid receptor (DOR) (23). All cells were treated with 1 M 17-AAG for 24 h and then stimulated with the MOR full agonist DAMGO ([D- Ala2, N-MePhe4, Gly2-ol]-enkephalin) for 10 min; signaling protein expression and signal transduction were analyzed by Western blotting. We then tested the effects of Hsp90 inhibition on opioid response in several pain and dependence/withdrawal models. We first established a clinically relevant post-surgical pain model by paw incision (27) with concurrent 17-AAG injection and a 24-h recovery. Pain response and allodynia were mea- sured by mechanical stimulation with Von Frey filaments (28). The animals showed a robust allodynia post-surgery that was not altered by 17-AAG treatment (Fig. 3A). The vehicle-treated animals showed a robust time-dependent anti-nociception in response to systemic morphine (1, 3.2, and 10 mg/kg s.c.), which was strongly blocked by 17-AAG treatment (area under the curve (AUC) reduction of 70.2, 81.5, and 67.0% at 1, 3.2, and For signaling protein expression changes, we found that Hsp70 was induced in all four cell lines, which is a canonical effect of this class of Hsp90 inhibitors and serves as a marker of Hsp90 inhibition (Fig. 1, A and B) (24, 25). We also found that Hsp90 inhibition altered the protein expression levels of the 10415 J. Biol. Chem. (2017) 292(25) 10414–10428 10415 Table 1 nociception in 17-AAG-treated mice at 10 mg/kg morphine but not at 1 and 3.2 mg/kg, suggesting that this shift represents a change in potency (not efficacy) of the opioid system in this pain model with 17-AAG treatment. Summary of the effects of Hsp90 inhibition in vitro The results of Fig. 1 are summarized here. Stim, stimulation; triangle, in inverted triangle, decreased; —, no change; blank, not investigated. Seeking to determine whether this effect of 17-AAG was con- sistent across pain models, we next tested the effects of the inhibitor in a model of thermal tail-flick anti-nociception in naive mice treated with i.c.v. DAMGO. Tail-flick baselines were not altered by 17-AAG treatment, whereas both vehicle- and 17-AAG-treated mice displayed robust anti-nociception in response to 0.01, 0.032, and 0.1 nmol of DAMGO i.c.v. (Fig. 3C). 17-AAG treatment reduced the extent of the anti-nociception but by far less than in the paw-incision model, with an AUC increase of 5.3% at 0.01 nmol and a reduction of 19.6 and 18.2% at 0.032 and 0.1 nmol (Fig. 3C and Table 2 compared with a 67.0–81.5% reduction with paw incision). As described above, the AUC values of the individual mice were used to construct dose-response curves (Fig. 3D). Analysis of these curves revealed an A50 of 0.095 nmol for vehicle-treated mice and an A50 of 0.251 nmol for 17-AAG-treated mice; this was a 2.64-fold shift, far less than the 131.2-fold shift for paw incision (Table 2). Because the magnitude of the anti-nociception was equal for both vehicle- and 17-AAG-treated mice at the low dose of 0.01 nmol, we concluded that 17-AAG treatment reduces the effi- cacy (not potency) of the opioid system in this pain model. y y y Both the paw-incision and tail-flick models represent acute pain, with durations of 24 h or less. To investigate the role of Hsp90 in chronic pain anti-nociception, we established a mouse model of HIV peripheral neuropathy (29). The pain state was induced by intrathecal injection of HIV gp120 protein at days 1, 3, and 5, which induced a progressive and strong mechanical allodynia over a 3-week treatment period (Fig. 3E). We injected 17-AAG on day 20 as described above and mea- sured mechanical allodynia as for the paw-incision experiments on day 21 in response to 10 mg/kg morphine. As for the other two pain models, 17-AAG treatment did not alter the pain base- line. Figure 1. Hsp90 inhibition has multivariate effects on protein expression and signal transduction in vitro. Four different cell models of MOR signal transduction were treated with 1 M 17-AAG or vehicle for 24 h, serum-starved (see “Experimental procedures”), and treated with vehicle, 10 M DAMGO, or 10 M LPA (a non-opioid GPCR endogenous agonist) for 10 min. Results were measured by Western blot, and analyzed as reported under “Experimental procedures.” The sample size reported in each experiment graph represents an independent technical replicate consisting of two or more averaged replicate wells counted as n 1. All data are reported as the mean S.D. A, representative Western blots for each target are shown, with vehicle (Veh) or 17-AAG (AAG) treatment. The cell line from which each representative blot was taken is indicated below the target name. The numbers with arrows on the left side of each image represents the position of the nearest molecular weight markers. These images also indicate that GAPDH and tERK were not altered by 17-AAG treatment(datanotshown).B,theexpressionlevelsoffourseparatesignalingproteinsnormalizedtoGAPDHandvehiclecontrolwereanalyzedineachcellline as labeled. , , and *, p 0.05, 0.001, and 0.0001, respectively, versus same cell line vehicle control by unpaired 2-tailed t test. Only Hsp70 shows consistent regulation across cell lines with Hsp90 inhibition. C, signal transduction via ERK phosphorylation is reported for each cell line in a separate graph. The pERK signal was normalized to tERK and, in the case of CHO, was normalized further to MOR levels as they were decreased. The pERK/tERK signal was further normalized to the vehicle:vehicle group. and *, p 0.05 and 0.01 versus same pretreatment group (vehicle, 17-AAG) vehicle control; #, ##, ###, and ####, p 0.05, 0.01, 0.001, and 0.0001, respectively, versus vehicle:vehicle group; both sets by two-way ANOVA with Fisher’s least significant difference post-hoc test. Each line showed a different alteration in the ERK signaling pattern after Hsp90 inhibition, and both DAMGO and LPA showed similar behavior. Heat-shock protein 90 in opioid signaling and pain 10416 J. Biol. Chem. (2017) 292(25) 10414–10428 Table 1 with a dose response of 17-AAG or an equalized vehicle d ll d t f 24 h Th i ifi d d th PAG di Figure 2. Hsp90 inhibitor (17-AAG) dose response in mouse brain. Mice 10 mg/kg with 17-AAG) (Fig. 3A and Table 2). The AUC values from individual mice at the three dosages were used to con- struct dose-response curves (Fig. 3B). Analysis of these curves showed an A50 of 3.98 mg/kg for vehicle-treated mice and an A50 of 522.2 mg/kg for 17-AAG-treated mice, a 131.2-fold shift (Table 2). We observed strongly reduced but still present anti- Heat-shock protein 90 in opioid signaling and pain Table 1 Summary of the effects of Hsp90 inhibition in vitro The results of Fig. 1 are summarized here. Stim, stimulation; triangle, increased; inverted triangle, decreased; —, no change; blank, not investigated. Figure 2. Hsp90 inhibitor (17-AAG) dose response in mouse brain. Mice were injected i.c.v. with a dose response of 17-AAG or an equalized vehicle and allowed to recover for 24 h. The mice were sacrificed, and the PAG dis- sected, removed, and analyzed by Western blotting. Representative blots of Hsp70 and GAPDH are shown with treatment and molecular weight markers indicated. The Hsp70 signal is normalized to GAPDH and further to vehicle control. GAPDH was not altered by 17-AAG treatment. The data were reported as the mean S.D. Linear regression was performed on the data points, and the A50 (0.084 nmol) was calculated as described under “Experi- mental procedures.” The reported sample size is for experiments performed on individual mice (biological replicates) over two technical replicates on different days with different mouse cohorts by the same experimenter. Based on this experiment, 0.5 nmol of 17-AAG was chosen for further experiments. Table 1 However, 17-AAG treatment completely abolished anti- nociception in response to 10 mg/kg morphine s.c. (AUC reduction of 97.6%) (Fig. 3F and Table 2). This result demon- strates that Hsp90 has a strong role in chronic pain anti-noci- ception, likely stronger than in acute pain. Dose-response experiments could not be completed in this model, as the motor-activating effects of the next half-log dose of morphine (32 mg/kg) were too strong for us to reliably measure mechan- ical thresholds. Figure 2. Hsp90 inhibitor (17-AAG) dose response in mouse brain. Mice were injected i.c.v. with a dose response of 17-AAG or an equalized vehicle and allowed to recover for 24 h. The mice were sacrificed, and the PAG dis- sected, removed, and analyzed by Western blotting. Representative blots of Hsp70 and GAPDH are shown with treatment and molecular weight markers indicated. The Hsp70 signal is normalized to GAPDH and further to vehicle control. GAPDH was not altered by 17-AAG treatment. The data were reported as the mean S.D. Linear regression was performed on the data points, and the A50 (0.084 nmol) was calculated as described under “Experi- mental procedures.” The reported sample size is for experiments performed on individual mice (biological replicates) over two technical replicates on different days with different mouse cohorts by the same experimenter. Based on this experiment, 0.5 nmol of 17-AAG was chosen for further experiments. Figure 2. Hsp90 inhibitor (17-AAG) dose response in mouse brain. Mice were injected i.c.v. with a dose response of 17-AAG or an equalized vehicle and allowed to recover for 24 h. The mice were sacrificed, and the PAG dis- sected, removed, and analyzed by Western blotting. Representative blots of Hsp70 and GAPDH are shown with treatment and molecular weight markers indicated. The Hsp70 signal is normalized to GAPDH and further to vehicle control. GAPDH was not altered by 17-AAG treatment. The data were reported as the mean S.D. Linear regression was performed on the data points, and the A50 (0.084 nmol) was calculated as described under “Experi- mental procedures.” The reported sample size is for experiments performed on individual mice (biological replicates) over two technical replicates on different days with different mouse cohorts by the same experimenter. Based on this experiment, 0.5 nmol of 17-AAG was chosen for further experiments. Figure 2. Hsp90 inhibitor (17-AAG) dose response in mouse brain. Mice were injected i.c.v. Heat-shock protein 90 in opioid signaling and pain The reported sample size of individual mice includes every HIV neuropathy mouse used in this study combined with six technical replicates by the same experimenter on different days. F, HIV neuropathy was established as described in E, with pre-gp120, day 19, and day 21 post-i.c.v. injection baselines noted. On day 20, vehicle or 17-AAG was injected i.c.v. as above followed by 10 mg/kg morphine s.c. on day 21. 17-AAG had no effect on pain baselines but abolished (AUC reduction of 97.6% (Table 2)) morphine anti-nociception. and ***, p 0.05 and 0.0001 versus same time point by two-way ANOVA with Fisher’s least significant difference post-hoc test. The reported sample size of individual mice includes two technical replicates performed on different days by the same experimenter. Both hind paws showed extremely similar results, and thus only the data from the left paw is shown (data not shown) Figure 3. Hsp90 inhibition decreases opioid-induced anti-nociception. All data are reported as the mean S.D. A, post-surgical pain was induced by paw-incision surgery, and mechanical thresholds were measured with Von Frey filaments as described under “Experimental procedures.” Vehicle or 17-AAG (0.5 nmol) was injected i.c.v. concurrent with surgery. After 24 h, morphine was injected (1, 3.2, or 10 mg/kg s.c.), and mechanical thresholds were measured over a time course. Each dose is reported in a separate graph. , , , and *** (same with # and $), p 0.05, 0.01, 0.001, and 0.0001, respectively, versus same time point by two-way ANOVA with Fisher’s least significant difference post-hoc test. AUC analysis results are summarized in Table 2. The reported sample size of individual mice includes two to three technical replicates for each dose on different days by the same experimenter. B, the AUC values from each individual mouse in A were used to construct dose-response curves, and linear regression analysis was performed to calculate the A50 values (see “Experimental procedures” and results in Table 2). Vehicle 3.98 mg/kg and 17-AAG 522.2 mg/kg, a 131.2-fold shift. The results suggest a strong decrease in potency for the system. C, 52 °C tail-flick baselines were determined as reported under “Experimental procedures” prior to and 24 h post-i.c.v. injection of vehicle or 0.5 nmol of 17-AAG. DAMGO (0.01, 0.032, or 0.1 nmol) was then injected i.c.v., and tail-flick latencies were determined over a time course. J. Biol. Chem. (2017) 292(25) 10414–10428 10417 Figure 3. Hsp90 inhibition decreases opioid-induced anti-nociception. All data are reported as the mean S.D. A, post-surgical pain was induced by paw-incision surgery, and mechanical thresholds were measured with Von Frey filaments as described under “Experimental procedures.” Vehicle or 17-AAG (0.5 nmol) was injected i.c.v. concurrent with surgery. After 24 h, morphine was injected (1, 3.2, or 10 mg/kg s.c.), and mechanical thresholds were measured over a time course. Each dose is reported in a separate graph. , , , and ** (same with # and $), p 0.05, 0.01, 0.001, and 0.0001, respectively, versus same time point by two-way ANOVA with Fisher’s least significant difference post-hoc test. AUC analysis results are summarized in Table 2. The reported sample size of individual mice includes two to three technical replicates for each dose on different days by the same experimenter. B, the AUC values from each individual mouse in A were used to construct dose-response curves, and linear regression analysis was performed to calculate the A50 values (see “Experimental procedures” and results in Table 2). Vehicle 3.98 mg/kg and 17-AAG 522.2 mg/kg, a 131.2-fold shift. The results suggest a strong decrease in potency for the system. C, 52 °C tail-flick baselines were determined as reported under “Experimental procedures” prior to and 24 h post-i.c.v. injection of vehicle or 0.5 nmol of 17-AAG DAMGO (0 01, 0 032, or 0 1 nmol) was then injected i c v , and tail-flick latencies were determined over a time course * and ** p 0 05 and Heat-shock protein 90 in opioid signaling and pain 10418 J. Biol. Chem. (2017) 292(25) 10414–10428 Heat-shock protein 90 in opioid signaling and pain * and ** p 0.05 and 0.01, respectively, versus 0.1 nmol time point; #, p 0.05 versus 0.032 nmol time point; both by two-way ANOVA with Fisher’s least significant difference post-hoctest.AUCanalysisresultsaresummarizedinTable2.Thereportedsamplesizeofindividualmiceincludesthreetechnicalreplicatesforeachdosefrom two different experimenters at different institutions; each replicate showed very similar results. D, dose-response curves were created and analyzed from C using the same method as described in B. The results are summarized in Table 2. Vehicle 0.095 nmol and 17-AAG 0.251 nmol, a 2.64-fold shift. The results suggest a modest decrease in efficacy for the system. E, HIV peripheral neuropathy was established using the method outlined under “Experimental proce- dures.” This figure demonstrates progressive, severe, mechanical allodynia, measured as described in A, over a 21-day treatment period. Both hind paws were measured, and the injection days were noted (Inj). The reported sample size of individual mice includes every HIV neuropathy mouse used in this study combined with six technical replicates by the same experimenter on different days. F, HIV neuropathy was established as described in E, with pre-gp120, day 19, and day 21 post-i.c.v. injection baselines noted. On day 20, vehicle or 17-AAG was injected i.c.v. as above followed by 10 mg/kg morphine s.c. on day 21. 17-AAG had no effect on pain baselines but abolished (AUC reduction of 97.6% (Table 2)) morphine anti-nociception. * and ***, p 0.05 and 0.0001 versus same time point by two-way ANOVA with Fisher’s least significant difference post-hoc test. The reported sample size of individual mice includes two technical replicates performed on different days by the same experimenter. Both hind paws showed extremely similar results, and thus only the data from the left paw is shown (data not shown). Heat-shock protein 90 in opioid signaling and pain Heat-shock protein 90 in opioid signaling and pain Figure 3. Hsp90 inhibition decreases opioid-induced anti-nociception. All data are reported as the mean S.D. A, post-surgical pain was induced by paw-incision surgery, and mechanical thresholds were measured with Von Frey filaments as described under “Experimental procedures.” Vehicle or 17-AAG (0.5 nmol) was injected i.c.v. concurrent with surgery. After 24 h, morphine was injected (1, 3.2, or 10 mg/kg s.c.), and mechanical thresholds were measured over a time course. Each dose is reported in a separate graph. , , , and *** (same with # and $), p 0.05, 0.01, 0.001, and 0.0001, respectively, versus same time point by two-way ANOVA with Fisher’s least significant difference post-hoc test. AUC analysis results are summarized in Table 2. The reported sample size of individual mice includes two to three technical replicates for each dose on different days by the same experimenter. B, the AUC values from each individual mouse in A were used to construct dose-response curves, and linear regression analysis was performed to calculate the A50 values (see “Experimental procedures” and results in Table 2). Vehicle 3.98 mg/kg and 17-AAG 522.2 mg/kg, a 131.2-fold shift. The results suggest a strong decrease in potency for the system. C, 52 °C tail-flick baselines were determined as reported under “Experimental procedures” prior to and 24 h post-i.c.v. injection of vehicle or 0.5 nmol of 17-AAG. DAMGO (0.01, 0.032, or 0.1 nmol) was then injected i.c.v., and tail-flick latencies were determined over a time course. * and ** p 0.05 and 0.01, respectively, versus 0.1 nmol time point; #, p 0.05 versus 0.032 nmol time point; both by two-way ANOVA with Fisher’s least significant difference post-hoctest.AUCanalysisresultsaresummarizedinTable2.Thereportedsamplesizeofindividualmiceincludesthreetechnicalreplicatesforeachdosefrom two different experimenters at different institutions; each replicate showed very similar results. D, dose-response curves were created and analyzed from C using the same method as described in B. The results are summarized in Table 2. Vehicle 0.095 nmol and 17-AAG 0.251 nmol, a 2.64-fold shift. The results suggest a modest decrease in efficacy for the system. E, HIV peripheral neuropathy was established using the method outlined under “Experimental proce- dures.” This figure demonstrates progressive, severe, mechanical allodynia, measured as described in A, over a 21-day treatment period. Both hind paws were measured, and the injection days were noted (Inj). Table 2 The sample size reported for individual mice consisted of one technical replicate. 17-AAG had no effect on Rotarod performance (p 0.05). Data are reported as the mean S.D. We next tested the effects of Hsp90 inhibition on the opioid side effects of dependence and withdrawal. This was performed first in an acute model of dependence and withdrawal, with 17-AAG treatment for 20 h as described above, a single 100 mg/kg s.c. injection of morphine with a 4-h recovery, and the precipitation of withdrawal with 10 mg/kg naloxone i.p. (Fig. 4A). We also tested 17-AAG in a model of chronic dependence and withdrawal, with an escalating dosage protocol over 4 days (see “Experimental procedures”) with daily re-injection of 17-AAG, and precipitation of withdrawal on day 4 with 30 mg/kg naloxone i.p. (Fig. 4B). In both models of dependence and withdrawal, 17-AAG treatment significantly increased the production of withdrawal-associated feces and urine without altering jumping behavior. The above results suggest that Hsp90 inhibition with 17-AAG worsens the opioid thera- peutic index by decreasing anti-nociception and increasing side effects. Lastly, to determine whether the above results were due to off-target motor, sedative, or similar effects, we measured the effect of 17-AAG treatment on performance in the rotarod test (30). Both 17-AAG and vehicle-treated mice had unaltered per- formance in this test, suggesting that the above results are spe- cific to the opioid system and are not off-target (Fig. 4C). Table 2 The sample size reported for individual mice consisted of one technical replicate. 17-AAG had no effect on Rotarod performance (p 0.05). Data are reported as the mean S.D. Figure 4. Hsp90 inhibition increases opioid-induced withdrawal symptoms. Acute (A) and chronic (B) morphine dependence and withdrawal paradigms were established with concurrent vehicle and 17-AAG (0.5 nmol) i.c.v. injections as described under “Experimental procedures.” Withdrawal was precipitated with 10 mg/kg (acute) or 30 mg/kg (chronic) naloxone i.p. with behaviors recorded for 20 min. Jumps and urine/feces weight are reported for each paradigm as the mean S.D. , p 0.05 versus the same measure vehicle control by unpaired two-tailed t test; n 24–29 mice/group for each paradigm; three technical replicates were used for each paradigm by the same experimenter using different mouse cohorts on different days. 17-AAG treatment increased urine and feces weight, but not jumps, in both paradigms. C, mice were exposed to three 3-min training sessions on a Rotarod device followed by vehicle or 0.5 nmol of 17-AAG injected i.c.v. and a 24-h recovery. Another 3-min trial was then performed with acceleration from 4 to 16 rpm and no further treatment. The sample size reported for individual mice consisted of one technical replicate. 17-AAG had no effect on Rotarod performance (p 0.05). Data are reported as the mean S.D. Figure 4. Hsp90 inhibition increases opioid-induced withdrawal symptoms. Acute (A) and chronic (B) morphine dependence and withdrawal paradigms were established with concurrent vehicle and 17-AAG (0.5 nmol) i.c.v. injections as described under “Experimental procedures.” Withdrawal was precipitated with 10 mg/kg (acute) or 30 mg/kg (chronic) naloxone i.p. with behaviors recorded for 20 min. Jumps and urine/feces weight are reported for each paradigm as the mean S.D. , p 0.05 versus the same measure vehicle control by unpaired two-tailed t test; n 24–29 mice/group for each paradigm; three technical replicates were used for each paradigm by the same experimenter using different mouse cohorts on different days. 17-AAG treatment increased urine and feces weight, but not jumps, in both paradigms. C, mice were exposed to three 3-min training sessions on a Rotarod device followed by vehicle or 0.5 nmol of 17-AAG injected i.c.v. and a 24-h recovery. Another 3-min trial was then performed with acceleration from 4 to 16 rpm and no further treatment. Heat-shock protein 90 in opioid signaling and pain We next tested the effects of Hsp90 inhibition on the opioid id ff t f d d d ithd l Thi f d Hsp90 inhibition with 17-AAG worsens the opioid thera- ti i d b d i ti i ti d i i Figure 4. Hsp90 inhibition increases opioid-induced withdrawal symptoms. Acute (A) and chronic (B) morphine dependence and withdrawal paradigms were established with concurrent vehicle and 17-AAG (0.5 nmol) i.c.v. injections as described under “Experimental procedures.” Withdrawal was precipitated with 10 mg/kg (acute) or 30 mg/kg (chronic) naloxone i.p. with behaviors recorded for 20 min. Jumps and urine/feces weight are reported for each paradigm as the mean S.D. , p 0.05 versus the same measure vehicle control by unpaired two-tailed t test; n 24–29 mice/group for each paradigm; three technical replicates were used for each paradigm by the same experimenter using different mouse cohorts on different days. 17-AAG treatment increased urine and feces weight, but not jumps, in both paradigms. C, mice were exposed to three 3-min training sessions on a Rotarod device followed by vehicle or 0.5 nmol of 17-AAG injected i.c.v. and a 24-h recovery. Another 3-min trial was then performed with acceleration from 4 to 16 rpm and no further treatment. The sample size reported for individual mice consisted of one technical replicate. 17-AAG had no effect on Rotarod performance (p 0.05). Data are reported as the mean S.D. Table 2 Summary of in vivo anti-nociception assays The relevant results of Figs. 3 and 6 are summarized here. AUC, change in AUC percentage, either increased () or decreased ( ) with treatment. The A50 values are derived from the dose-response curves shown in Fig. 3. Veh, vehicle. Heat shock protein 90 in opioid signaling and pain Hsp90 inhibition alters protein expression and signal transduction in the brain In an effort to explore the mechanism of Hsp90 regulation of the above-described opioid-induced behaviors, we measured Table 2 Summary of in vivo anti-nociception assays Summary of in vivo anti-nociception assays The relevant results of Figs. 3 and 6 are summarized here. AUC, change in AUC percentage, either increased () or decreased ( ) with treatment. The A50 values are derived from the dose-response curves shown in Fig. 3. Veh, vehicle. assays mmarized here. AUC, change in AUC percentage, either increased () or decreased ( ) with treatment. The A50 values are wn in Fig. 3. Veh, vehicle. Summary of in vivo anti nociception assays The relevant results of Figs. 3 and 6 are summarized here. AUC, change in AUC percentage, either increased () or decreased ( ) with treatment. The A50 values are derived from the dose-response curves shown in Fig. 3. Veh, vehicle. Summary of in vivo anti nociception assays The relevant results of Figs. 3 and 6 are summarized here. AUC, change in AUC percentage, either increased () or decreased ( ) with treatment. The A50 values are derived from the dose-response curves shown in Fig. 3. Veh, vehicle. The relevant results of Figs. 3 and 6 are summarized here. AUC, change in AUC percentage, either increased () or decreased ( ) with treatment. The A50 values are derived from the dose-response curves shown in Fig. 3. Veh, vehicle. derived from the dose-response curves shown in Fig. 3. Veh, vehicle. Figure 4. Hsp90 inhibition increases opioid-induced withdrawal symptoms. Acute (A) and chronic (B) morphine dependence and withdrawal paradigms were established with concurrent vehicle and 17-AAG (0.5 nmol) i.c.v. injections as described under “Experimental procedures.” Withdrawal was precipitated with 10 mg/kg (acute) or 30 mg/kg (chronic) naloxone i.p. with behaviors recorded for 20 min. Jumps and urine/feces weight are reported for each paradigm as the mean S.D. , p 0.05 versus the same measure vehicle control by unpaired two-tailed t test; n 24–29 mice/group for each paradigm; three technical replicates were used for each paradigm by the same experimenter using different mouse cohorts on different days. 17-AAG treatment increased urine and feces weight, but not jumps, in both paradigms. C, mice were exposed to three 3-min training sessions on a Rotarod device followed by vehicle or 0.5 nmol of 17-AAG injected i.c.v. and a 24-h recovery. Another 3-min trial was then performed with acceleration from 4 to 16 rpm and no further treatment. 10418 J. Biol. Chem. (2017) 292(25) 10414–10428 10418 J. Biol. Chem. (2017) 292(25) 10414–10428 Heat-shock protein 90 in opioid signaling and pain Heat-shock protein 90 in opioid signaling and pain brain protein expression and ERK and Akt signal transduction in the PAG and caudal brain stem (pons and medulla) by West- ern blotting after 24 h of 17-AAG treatment and 0.1 nmol of DAMGO administered i.c.v. for 10 min. As expected, we found up-regulation of Hsp70 in both brain regions in response to 17-AAG (Fig. 5, A–C). Contrary to some of our in vitro findings shown in Fig. 1B, we did not observe changes in MOR, arr2, or STAT3. Importantly for our analysis, 17-AAG treatment did not change the protein expression levels of GAPDH, tERK, or tAkt (Fig. 5, A–C, and data not shown). stimulation with 17-AAG treatment is the mechanism for the reduction of tail-flick anti-nociception seen with 17-AAG treatment. We then extended these results to the paw-incision model to determine whether ERK signaling would explain the results there as well. We performed paw-incision surgery as described above, except without the 17-AAG i.c.v. injection. After the 24-h recovery time, the animals displayed significant mechan- ical allodynia as measured by Von Frey filaments. The mice were then i.c.v. injected with 5 g of U0126 (or vehicle control) with a 15-min treatment time followed by 1 mg/kg morphine s.c. We observed that the vehicle-treated mice displayed ro- bust time-dependent anti-nociception, as expected. We also observed that U0126 blocked nearly all morphine anti-nocice- ption in this model, with an AUC reduction of 89.2% (Fig. 6C and Table 2). Again, the U0126 results showed a similar pattern to that of 17-AAG (results from Fig. 3A), with similar AUC reductions (89.2% for U0126 versus 70.2% for 17-AAG). Both brain regions also displayed an unusual pattern of ERK signaling in response to 17-AAG whereby the pERK baseline was increased to the same level as DAMGO stimulation in the vehicle-treated brains, but stimulation above that baseline in response to DAMGO was completely lost (Fig. 5, D and E). This was a similar pattern of signaling as observed in SH-SY5Y cells treated with 17-AAG (Fig. 1C). We also examined pAkt levels in both regions and found a pattern similar to but less robust than the ERK signaling pattern (Fig. 5, F and G). We also tested the role of ERK in our HIV peripheral neurop- athy model. Peripheral neuropathy was established as described above with a 21-day treatment period. On day 21, the mice received vehicle or U0126 i.c.v. Heat-shock protein 90 in opioid signaling and pain as described above, with a 15-min treatment time followed by 10 mg/kg morphine s.c. Again, we found that ERK inhibition strongly blocked mor- phine anti-nociception in this model, with an AUC reduction of 99.7% (Fig. 6D and Table 2). This was again very similar to the 17-AAG treatment AUC reduction value of 97.6% in this model (Table 2). Taken together, our data suggest that the loss of ERK stimulation caused by 17-AAG treatment is the mechanism by which 17-AAG reduces opioid anti-nociception in the paw- incision, tail-flick, and HIV peripheral neuropathy models. As a control, we also examined ERK activation in mice treated with 5 nmol of 17-AAG, the highest dose tested in our dose-response experiment shown in Fig. 2. The ERK pattern observed in this experiment was identical to that observed with 0.5 nmol of 17-AAG, with no increase in the magnitude of sig- naling, further justifying the choice of 0.5 nmol of 17-AAG for our studies (Fig. 5H). Determining the mechanism of Hsp90 regulation of anti-nociception In our signaling studies of mouse brain, we determined that Hsp70 expression and ERK signaling were altered by 17-AAG treatment. We thus tested these two proteins as mechanisms for Hsp90 regulation of anti-nociception. First, we tested the role of Hsp70 by combining Hsp90 inhibition by 17-AAG with Hsp70 inhibition by pifithrin-, a selective Hsp70 inhibitor. 17-AAG and 50 g of pifithrin-, a previously established i.c.v. dose of this inhibitor (31), were co-injected i.c.v. with a 24-h recovery (along with single drug and vehicle controls). The mice were then i.c.v. injected with 0.1 nmol of DAMGO i.c.v., and a tail-flick assay performed as described above. We found that pifithrin- had no effect on the tail-flick response, with indistinguishable vehicle and pifithrin groups (AUC of 100 and 100.5%), and indistinguishable 17-AAG and 17-AAG/pifithrin groups (AUC of 76.3 and 78.7%) (Fig. 6A). This finding suggests that up-regulated Hsp70 is not responsible for the anti-nocice- ption changes we observed. 10419 J. Biol. Chem. (2017) 292(25) 10414–10428 10419 Heat-shock protein 90 in opioid signaling and pain J. Biol. Chem. (2017) 292(25) 10414–10428 10421 Discussion During the course of this study, we first sought to establish an in vitro model for Hsp90 regulation of MOR signaling, as Hsp90 is expressed ubiquitously. We found that changes in both pro- tein expression and ERK signal transduction varied consider- ably between the four cell models used (Fig. 1). We took this to mean that the context of Hsp90 activity is important in deter- mining which proteins Hsp90 regulates and thus presumably the resultant signal transduction and opioid-induced behav- iors. This may be due to differential expression of Hsp90 co- chaperones (Hsp70, Hsp40, Hop/STIP1, Cdc37, etc.) in differ- ent cell and tissue types (14, 16, 33). These co-chaperones, particularly Hop/STIP1 and Cdc37, are very important in directing the activity and targeting of Hsp90 and should be investigated in the context of the brain, both to determine the mechanism of Hsp90 regulation of MOR signaling and opioid- induced behaviors and as potential targets for therapeutic intervention. Targeting the co-chaperone complex may be more selective by tissue type, thus reducing the potential side effects of targeting the ubiquitously expressed Hsp90. An anal- ogous approach has been used to design Hsp70-targeted ther- apeutics and could be adapted for Hsp90 and pain (17). We note, however, that the neuroblastoma cell line SH-SY5Y dis- played a similar ERK signaling profile after 17-AAG treatment to that we observed in the brain (Figs. 1C and 5, D and E). SH-SY5Y cells may express a similar profile of co-chaperones as For ERK signaling, we reasoned that the loss of DAMGO stimulation seen with 17-AAG treatment (Fig. 5, D and E) was more critical for acute anti-nociception than the elevated base- line. We thus modeled the change in ERK signaling seen with 17-AAG by i.c.v. injection of 5 g of U0126, a selective ERK inhibitor used at this i.c.v. dose previously (32). Vehicle or U0126 was i.c.v. injected with a 15-min treatment time followed by 0.1 nmol of DAMGO i.c.v. and a tail-flick assay as described above. We observed that U0126 reduced tail-flick anti-nocice- ption with a pattern very similar to 17-AAG (Fig. 6B and Table 2) and a very similar AUC reduction of 23.5% (versus 18.2% for 17-AAG in Fig. 3C). These results suggest that the loss of ERK 10420 10420 J. Biol. Chem. (2017) 292(25) 10414–10428 Heat-shock protein 90 in opioid signaling and pain Heat-shock protein 90 in opioid signaling and pain injected as above with a 15 min treatmenttime.Thiswasfollowedby10mg/kgmorphines.c.,andmechanicalthresholdsweremeasuredinatimecourse.,**p0.05,0.0001versussame time point by 2 Way ANOVA with Fisher’s least significant difference post-hoc test. AUC analysis revealed that U0126 reduced morphine anti-nociception by 99.7%, very similar to the value for 17-AAG (Table 2). The sample size of individual mice reported consisted of three technical replicates performed by the same experimenter on different days. Both hind paws showed very similar results; only the left paw is reported here (data not shown). Figure 5. Hsp90 inhibition induces protein expression and signaling changes in mouse brain. Mice were injected i.c.v. with vehicle or 0.5 nmol of 17-AAG, allowed to recover for 24 h, and then injected i.c.v. with vehicle (Veh) or 0.1 nmol of DAMGO for 10 min. Brains were extracted and analyzed by Western blot analysisasreportedunder“Experimentalprocedures.”AlldataarereportedasthemeanS.D.A,representativeblotsfromeachproteintargetareshown,with the treatment and molecular weight markers indicated as described in the legend for Fig. 1. PAG (B) and caudal brain stem (pons and medulla) (C) protein expression changes are indicated. The signal for each target is normalized to GAPDH and further normalized to the vehicle control for each. MOR and arr2 in the PAG were measured by immunoprecipitation (see “Experimental procedures”). , p 0.05 versus same target vehicle control by unpaired 2-tailed t test. The reported sample size of the individual mice consists of two technical replicates performed by two different experimenters at different institutions. Only Hsp70 expression was altered by 17-AAG treatment in both brain regions, contrary to some of the results reported in the legend for Fig. 1. Importantly, GAPDH, tERK, and tAkt protein levels were not changed (see A; GAPDH and tAkt quantified data not shown). PAG (D) and caudal brain stem (E) ERK signaling changes are indicated.pERKwasnormalizedtothetERKsignalandfurthernormalizedtothevehicle:vehiclegroup.,,and**,p0.01,0.001,and0.0001,respectively, versus vehicle:vehicle group by Fisher’s least significant difference post-hoc test. The reported sample size of individual mice for both D and E consisted of two technical replicates each performed by different experimenters at different institutions. Hsp90 inhibition increased the ERK signaling baseline, and stimulation above that baseline by DAMGO was completely lost in both brain regions. PAG (F) and caudal brain stem (G) Akt signaling changes are indicated. Akt was analyzed as described for ERK from the same samples and with the same technical replicates. and , p 0.05 and 0.01 versus vehicle:vehicle group by Fisher’s least significant difference post-hoc test. Heat-shock protein 90 in opioid signaling and pain Heat-shock protein 90 in opioid signaling and pa J. Biol. Chem. (2017) 292(25) 10414–10428 10421 Figure 6. ERK inhibition recapitulates the effects of Hsp90 inhibition on anti-nociception. Tail-flick, HIV neuropathy/Von Frey, and paw-incision/Von Frey tests were performed as described in the legend for Fig. 3 and under “Experimental procedures.” Data are reported as the mean S.D. A, vehicle or 17-AAG (0.5 nmol) combined with pifithrin- (50 g), as well as single injection controls, were injected i.c.v. with a 24-h recovery period. 0.1 nmol of DAMGO was then injected i.c.v., and tail-flick latencies were determined in a time course. and **, p 0.01 and 0.001 for vehicle versus pifithrin/17-AAG; #, ##, and ###, p 0.05, 0.01, and 0.001, respectively, for pifithrin versus pifithrin/17-AAG group; both at the same time point using a two-way ANOVA with Fisher’s least significant differencepost-hoctest.AUCanalysisrevealed:17-AAG,23.7%decrease;pifithrin,0.5%increase;pifithrin/17-AAG,21.3%decrease.Pifithrinhadnoeffectalone or in combination with 17-AAG on tail-flick anti-nociception. The sample size of individual mice reported consisted of two technical replicates by the same experimenter on different days. B, vehicle or 5 g of U0126 was injected i.c.v. with a 15-min treatment period followed by 0.1 nmol of i.c.v. DAMGO and a tail-flick time course. , p 0.05 versus same time point via two-way ANOVA with a Fisher’s least significant difference post-hoc test. AUC analysis revealed a decrease of 23.5% with U0126, very similar to the AUC values for 17-AAG treatment (Table 2). The sample size of individual mice reported consisted of two technical replicates performed on different days by the same experimenter. C, paw-incision surgery and pre- and post-surgery mechanical withdrawal thresholdswereperformedinmice.5gofU0126orvehiclecontrolwasinjectedi.c.v.witha15-mintreatmenttime.Morphine(1mg/kgs.c.)wastheninjected, and withdrawal thresholds were determined over a time course. and *, p 0.01 and 0.0001 versus same time point via two-way ANOVA with a Fisher’s Least Significant Difference post-hoc test. AUC analysis revealed that U0126 reduced anti-nociception by 89.2%, very similarly to the reduction caused by 17-AAG above (Table 2). The sample size of individual mice reported consisted of 2 technical replicates performed by the same experimenter on different days. D, HIV neuropathy was induced as in Fig. 3 and under “Experimental procedures.” On day 21, U0126 or vehicle control was i.c.v. injected as above with a 15 min treatmenttime.Thiswasfollowedby10mg/kgmorphines.c.,andmechanicalthresholdsweremeasuredinatimecourse.,**p0.05,0.0001versussame time point by 2 Way ANOVA with Fisher’s least significant difference post-hoc test. AUC analysis revealed that U0126 reduced morphine anti-nociception by 99.7%, very similar to the value for 17-AAG (Table 2). Heat-shock protein 90 in opioid signaling and pain Heat-shock protein 90 in opioid signaling and pain . ERK inhibition recapitulates the effects of Hsp90 inhibition on anti-nociception. Tail-flick, HIV neuropathy/Von Fre f d d ib d i h l d f i d d “ i l d ” d h Figure 6. ERK inhibition recapitulates the effects of Hsp90 inhibition on anti-nociception. Tail-flick, HIV neuropathy/Von Frey, and paw-incision/Von Frey tests were performed as described in the legend for Fig. 3 and under “Experimental procedures.” Data are reported as the mean S.D. A, vehicle or 17-AAG (0.5 nmol) combined with pifithrin- (50 g), as well as single injection controls, were injected i.c.v. with a 24-h recovery period. 0.1 nmol of DAMGO was then injected i.c.v., and tail-flick latencies were determined in a time course. and **, p 0.01 and 0.001 for vehicle versus pifithrin/17-AAG; #, ##, and ###, p 0.05, 0.01, and 0.001, respectively, for pifithrin versus pifithrin/17-AAG group; both at the same time point using a two-way ANOVA with Fisher’s least significant differencepost-hoctest.AUCanalysisrevealed:17-AAG,23.7%decrease;pifithrin,0.5%increase;pifithrin/17-AAG,21.3%decrease.Pifithrinhadnoeffectalone or in combination with 17-AAG on tail-flick anti-nociception. The sample size of individual mice reported consisted of two technical replicates by the same experimenter on different days. B, vehicle or 5 g of U0126 was injected i.c.v. with a 15-min treatment period followed by 0.1 nmol of i.c.v. DAMGO and a tail-flick time course. , p 0.05 versus same time point via two-way ANOVA with a Fisher’s least significant difference post-hoc test. AUC analysis revealed a decrease of 23.5% with U0126, very similar to the AUC values for 17-AAG treatment (Table 2). The sample size of individual mice reported consisted of two technical replicates performed on different days by the same experimenter. C, paw-incision surgery and pre- and post-surgery mechanical withdrawal thresholdswereperformedinmice.5gofU0126orvehiclecontrolwasinjectedi.c.v.witha15-mintreatmenttime.Morphine(1mg/kgs.c.)wastheninjected, and withdrawal thresholds were determined over a time course. and *, p 0.01 and 0.0001 versus same time point via two-way ANOVA with a Fisher’s Least Significant Difference post-hoc test. AUC analysis revealed that U0126 reduced anti-nociception by 89.2%, very similarly to the reduction caused by 17-AAG above (Table 2). The sample size of individual mice reported consisted of 2 technical replicates performed by the same experimenter on different days. D, HIV neuropathy was induced as in Fig. 3 and under “Experimental procedures.” On day 21, U0126 or vehicle control was i.c.v. 10422 J. Biol. Chem. (2017) 292(25) 10414–10428 Heat-shock protein 90 in opioid signaling and pain The sample size of individual mice reported consisted of three technical replicates performed by the same experimenter on different days. Both hind paws showed very similar results; only the left paw is reported here (data not shown). Heat-shock protein 90 in opioid signaling and pain Heat-shock protein 90 in opioid signaling and pain This loss of stimulation may account for the loss of opioid anti-nociception seen in three pain models. The molecular mechanisms link- ingHsp90toERKandERKtoanti-nociceptionandpotentiallytodependence/ withdrawal are currently unknown. We also note in this figure that Hsp90 effects on signaling kinases are likely to impact other receptor systems, including GPCRs and receptor tyrosine kinases. Stimulating these systems while Hsp90 is inhibited could cause a variety of other physiological effects. Figure 7. Summary model of the role of Hsp90 in opioid signaling and behavior. This model summarizes our findings that Hsp90 inhibition causes ERK MAPK to lose the ability to be stimulated through opioid-receptor acti- vation in the brain. This loss of stimulation may account for the loss of opioid anti-nociception seen in three pain models. The molecular mechanisms link- ingHsp90toERKandERKtoanti-nociceptionandpotentiallytodependence/ withdrawal are currently unknown. We also note in this figure that Hsp90 effects on signaling kinases are likely to impact other receptor systems, including GPCRs and receptor tyrosine kinases. Stimulating these systems while Hsp90 is inhibited could cause a variety of other physiological effects. 17-AAG treatment caused an elevation in the ERK signaling baseline in both the PAG and the caudal brain stem, which was unable to be stimulated above that baseline with DAMGO (Fig. 5). We also saw similar results with the kinase Akt. By using the ERK-selective inhibitor U0126 to model this effect, we were able to completely account for the anti-nociceptive changes caused by 17-AAG in tail-flick, post-surgical, and HIV neurop- athy pain, with nearly identical curve shapes and AUC reduc- tions (Fig. 6 and Table 2). Hsp70 was also up-regulated by 17-AAG treatment, but an Hsp70 inhibitor had no effect on tail-flick anti-nociception (Fig. 6A). Taken together, our results suggest that Hsp90 promotes anti-nociception in these pain models by maintaining the ability of ERK to be stimulated by opioid drugs. Our findings are summarized in Fig. 7. The mag- nitude of the ERK effect is different in the different pain models, with a small contribution to tail-flick anti-nociception and a very large or even total contribution to post-surgical and HIV neuropathy anti-nociception. These findings should be ex- plored in greater detail. From the literature, it is clear that ERK has a substantial role in the pathophysiology of pain states and opioid-receptor function and side effects. Heat-shock protein 90 in opioid signaling and pain However, that role appears to be very context-dependent, as ERK located in differ- ent circuits or regions or manipulated in different models can have different or opposing effects (36–41). We found 5 stud- ies each that tested ERK specifically in paw-incision and tail- flick pain, and none of these tested these models as we did, specifically by testing ERK in the opioid anti-nociceptive response. We could find no studies that tested the role of ERK in HIV neuropathic pain. Thus our findings contribute to the growing understanding of the role of ERK in pain and opioid response, which should be further investigated for possible therapeutic use. Hsp90 inhibition was even more effective in blocking opioid anti-nociception in the HIV neuropathy model than in either the paw-incision or tail-flick models (Fig. 3 and Table 2). Pain is induced in this model from the pro-inflammatory activity of the gp120 protein in the spinal cord (29). This may further indicate that Hsp90 is more effective in descending modulatory circuits to the spinal cord than in central circuits. These results also demonstrate that Hsp90 is involved in anti-nociception against multiple pain types (mechanical, thermal, inflammatory, and neuropathic) and in both acute and chronic pain states. Fur- ther investigation should focus on the molecular and circuit mechanisms by which Hsp90 regulates this broad array of anti-nociception. We also found that Hsp90 inhibition worsened gastrointes- tinal effects, but not jumping behavior, in both acute and chronic morphine dependence and withdrawal (Fig. 4, A and B). Hsp90 thus has effects beyond anti-nociception, and Hsp90 inhibition worsens the opioid therapeutic index (decreasing anti-nociception and increasing side effects). These effects are likely specific to the opioid system and not off-target, as Hsp90 inhibition did not alter anti-nociceptive naive, paw-incision, or HIV neuropathy baselines or performance in the rotarod test (Fig. 4C). These results also imply that an Hsp90 activator could improve the opioid therapeutic index, which could be a basis for improving opioid therapy. Tamoxifen (35) and DMSO (19) are both Hsp90 activators, which we will use in future studies to test this hypothesis. Long-term, new and more selective Hsp90 activators need to be developed through drug discov- ery and development. This study suggests that the mechanism for Hsp90 regulation of anti-nociception is through the signaling kinase ERK. Heat-shock protein 90 in opioid signaling and pain Heat-shock protein 90 in opioid signaling and pain Figure 7. Summary model of the role of Hsp90 in opioid signaling and behavior. This model summarizes our findings that Hsp90 inhibition causes ERK MAPK to lose the ability to be stimulated through opioid-receptor acti- vation in the brain. This loss of stimulation may account for the loss of opioid anti-nociception seen in three pain models. The molecular mechanisms link- ingHsp90toERKandERKtoanti-nociceptionandpotentiallytodependence/ withdrawal are currently unknown. We also note in this figure that Hsp90 effects on signaling kinases are likely to impact other receptor systems p p g g p found in the brain and should be investigated as a potential cell model for Hsp90 activity in the brain. We also found that Hsp90 inhibition strongly or completely blocked morphine anti-nociception in the clinically relevant paw-incision post-surgical pain and HIV peripheral neuropa- thy pain models and slightly blocked DAMGO anti-nocicep- tion in the naive tail-flick model (Fig. 3). These results are strik- ing and strongly implicate Hsp90 for a role in regulating opioid anti-nociception in both acute and chronic pain states. It’s also notable that Hsp90 inhibition in the brain itself was able to block (79.3% reduction) systemic morphine anti-nociception from a peripherally induced pain state in the paw-incision model. This may indicate that Hsp90 in brain descending mod- ulatory circuits is more critical than in the spinal circuits or primary afferents, at least in this particular model. Along these lines, Hsp90 inhibition only slightly reduced centrally adminis- tered DAMGO anti-nociception in the tail-flick model. This may also indicate a more important role for Hsp90 in descend- ing modulatory circuits rather than centrally mediated affective anti-nociception (34), but this remains to be demonstrated. Because the two pain models differed by drug, opioid route, and pain model/type, these other variables must also be explored and may explain the differences seen. Morphine was chosen for the paw-incision and HIV neuropathy models due to clinical relevance of the drug and route, and DAMGO was chosen for the tail-flick model as a highly MOR-selective drug that would remain in the brain, helping to isolate mechanisms of action. Figure 7. Summary model of the role of Hsp90 in opioid signaling and behavior. This model summarizes our findings that Hsp90 inhibition causes ERK MAPK to lose the ability to be stimulated through opioid-receptor acti- vation in the brain. J. Biol. Chem. (2017) 292(25) 10414–10428 10423 Heat-shock protein 90 in opioid signaling and pain Hsp90 inhibition induced a very similar, albeit less robust pattern in Akt as in ERK. H, in a control experiment, ERK signaling was assessed in mice injected i.c.v. with the highest dose of 17-AAG tested in the dose response (5 nmol). PAG ERK signaling in these mice was assessed as described in D and E. , p 0.05 versus vehicle:vehicle group by Fisher’s least significant difference post-hoc test. 5 nmol of 17-AAG induces the same ERK pattern as seen in D and E with no increased magnitude of the effect, suggesting that 0.5 nmol has a maximal effect on brain signaling, justifying the use of the lower dose. The reported sample size of individual mice consisted of one technical replicate. 10422 J. Biol. Chem. (2017) 292(25) 10414–10428 Heat-shock protein 90 in opioid signaling and pain From known roles in the literature and our data showing changes in ERK and Akt generally in the brain, it is very likely 10423 J. Biol. Chem. (2017) 292(25) 10414–10428 10423 Materials y p g As discussed above, Hsp90 has been investigated in 4 studies for its role in pain and opioid response. The Devi group (21) found that Hsp90 inhibition immediately prior to precipitation of morphine withdrawal reduced jumping behavior; and a sep- arate group found that Hsp90 inhibition blocked adenylyl cyclase superactivation, a marker of dependence, in a cell model (20). In contrast, we found that Hsp90 inhibition promoted the gastrointestinal effects of dependence and withdrawal (Fig. 4, A and B). For the Devi study (21), the differences observed may be because they applied the inhibitor immediately prior to with- drawal via the i.p route, whereas we applied inhibitor chroni- cally using the i.c.v. route. We suggest that chronic treatment may more closely model chronic pain treatment, but these dif- ferences are still notable. We will investigate the chronic appli- cation of 17-AAG systemically in future studies to explore these differences. For the adenylyl cyclase superactivation study, Koshimizu et al. (20) applied the inhibitor chronically as did we. However, as we discovered in our in vitro studies (Fig. 1), the effects of Hsp90 inhibition can be highly heterogeneous between lines. The SK-N-SH cells used in that study may or may not accurately model Hsp90 in the brain. Further investi- gation into the role of Hsp90 in dependence and withdrawal is needed. 17-AAG, DAMGO, LPA, naloxone, and U0126 were all pur- chased from Tocris (subsidiary of R&D Systems, Minneapolis, MN). Pifithrin- was purchased from Sigma-Aldrich. Mor- phine sulfate pentahydrate was obtained through the National Institute on Drug Abuse Drug Supply Program and distributed through the Research Triangle Institute. Gp120 IIIb protein was obtained from SPEED BioSystems, LLC (Gaithersburg, MD, catalog no. YCP1549). 17-AAG, U0126, and pifithrin- were prepared as stock solutions in DMSO, and DAMGO and LPA were prepared as stock solutions in water. Naloxone and morphine were prepared fresh for each experiment in USP saline. Appropriate vehicle controls were used for each experi- ment: 0.1% DMSO in serum-free culture media for 17-AAG and DAMGO in the culture experiments; 10% DMSO in water for 17-AAG i.c.v. injections; water for DAMGO i.c.v. injections; USP saline for systemic morphine and naloxone injections; and 10% DMSO, 10% Tween-80, and 80% USP saline for the 17-AAG/pifithrin- and U0126 i.c.v. injections. Heat-shock protein 90 in opioid signaling and pain In summary, we have demonstrated that Hsp90 promotes opioid anti-nociception in three different pain models by an ERK-dependent mechanism. Based on our signaling findings in the brain, this is likely accomplished by Hsp90 maintaining ERK in a competent state for opioid signal transduction activation. Despite the role of Hsp90 as a ubiquitous and crucial signaling regulator, very little work has been done on the role of this protein in opioid signal transduction and pain. Our results thus make a significant contribution to this field and provide the rationale for testing Hsp90 activators as therapies to enhance the opioid therapeutic index. Future studies will expand on the molecular mechanisms downstream of Hsp90 and ERK respon- sible for these effects and the specific cell types, regions, and circuits in the brain responsible for the effects of Hsp90 on opioid signal transduction and anti-nociception. that numerous other receptor systems are affected by Hsp90 inhibition. These could include receptor tyrosine kinases, other GPCRs, and other receptor systems generally. Specificity to the opioid system was achieved in this study by using selective opi- oid drugs and assessing opioid-mediated behaviors. This repre- sents an opportunity, in that our model will be useful for explor- ing the role of Hsp90 in many brain systems and responses. This also represents a challenge to developing Hsp90 as a therapeu- tic target, as there are likely to be off-target effects. As discussed above, targeting selective co-chaperones could be a means of decreasing off-target interactions (17). In addition, biased ligands or similar approaches could be developed to manipulate the Hsp90-regulated signaling cascade downstream of a spe- cific receptor. This would be analogous to the method used to target another universal regulator, arr2 (8). The likelihood that Hsp90 affects other receptor systems is also included in the summary presented in Fig. 7. Heat-shock protein 90 in opioid signaling and pain day, the signaling experiment mice had the incision reopened, and an i.c.v. injection of 0.1 nmol of DAMGO was done as above using the same site. After 10 min, the mice were sacrificed by rapid cervical dislocation. The dose-response mice proceeded immediately to sacrifice after 24 h. For both groups of mice, the brains were removed and dissected on an ice-cooled block. PAG and caudal brainstems (pons and medulla) were removed and rapidly frozen on liquid nitrogen. Protein was extracted using the same lysis buffer and general protocol as described above, except that a Polytron homogenizer was used to disrupt the tissue (maximum speed, 3 10 s, on ice) for PAG, and a 1-ml glass/glass Dounce homogenizer was used for the caudal brain stem. For a subset of the brain signaling experi- ments, MOR and arr2 were immunoprecipitated from PAG using the protocol reported by Schmid and Bohn (45). The cells were all maintained in growth medium (DMEM/ F12 for CHO and SH-SY5Y cells and MEM for HEK and U2OS cells) with 10% heat-inactivated FBS (Gibco brand, Thermo- Fisher Scientific) and 1 penicillin/streptomycin. The CHO and HEK cells were further maintained with 500 g/ml G418, and the U20S cells with 200 g/ml G418 and 100 g/ml Zeocin. All lines were incubated at 37 °C in a 5% CO2 humidified incu- bator. All lines were monitored for mycoplasma contamination by DAPI staining and microscopy, and all cells were mycoplas- ma-negative for these experiments. g p For experiments, the cells were plated in 6-well plates in a minimum of two replicate wells/condition (averaged for a tech- nical replicate of n 1), recovered overnight, and then treated with 1 M 17-AAG or vehicle for a further 24 h. Then the cells were serum-starved with the appropriate serum-free culture medium: 30 min for HEK, 60 min for CHO, and 24 h for U2OS and SH-SY5Y cells (serum starvation concurrent with 17-AAG treatment for U2OS and SH-SY5Y cells). The cells were then treated with vehicle, 10 M DAMGO, or 10 M LPA for 10 min. Dose-response and brain-signaling experiments For the dose-response experiments, the mice were injected i.c.v. with 0.05, 0.5, or 5 nmol of 17-AAG or vehicle. For the brain signaling experiments, the mice were injected with 0.5 nmol of 17-AAG i.c.v. This was performed by anesthetizing the mice with 2% isoflurane in standard air and incising the skin by 5 mm with a no. 10 scalpel blade. The i.c.v. injection was made using a jacketed 10-l Hamilton syringe in a 5-l maxi- mum volume, 1 mm posterior and 1 mm lateral to bregma and 2.5 mm deep. The skin was closed with tissue glue, and the mice were allowed to recover for 24 h in their home cage. The next Heat-shock protein 90 in opioid signaling and pain The cells were washed in ice-cold PBS and then collected with a plastic cell scraper in the presence of lysis buffer (20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 2 mM EDTA, 0.1% SDS, 1% Nonidet P-40 or equivalent, 0.25% deoxycholate, 1 mM sodium orthovanadate, 1 mM PMSF, 1 mM sodium fluoride, and a pro- tease inhibitor mixture from EMD Millipore, Billerica, MA). The lysates were sheared twice with a 27-gauge needle and cen- trifuged at 13,000 rpm at 4 °C for 10 min, and the supernatants were stored at 80 °C. Western blotting and blot analysis Cell and brain protein lysates were quantified with a BCA protein quantitation assay using the manufacturer’s protocol (Bio-Rad). The protein was run on precast Tris-glycine gels using a mini-PROTEAN Tetra Western blotting apparatus (Bio-Rad) following the manufacturer’s instructions. The gels were transferred to nitrocellulose membrane (Bio-Rad) using a wet transfer system (30 V, minimum of 1 h on ice). The blots were blocked with 5% nonfat dry milk in TBS and incubated with primary antibody in 5% BSA in TBS 0.1% Tween-20 (TBST) overnight rocking at 4 °C. The blots were then washed three times for 5 min in TBST, incubated with secondary anti- body (see below) in 5% milk in TBST for 1 h of rocking at room temperature, washed again, and imaged with a LiCor Odyssey infrared imaging system (LiCor, Lincoln, NE). Some blots were then stripped with 25 mM glycine-HCl and 1% SDS, pH 2.0, for 30–60 min of rocking at room temperature prior to being washed and re-exposed to primary antibody. Cell lines, culture, and in vitro experiments Parental CHO-K1, HEK, and SH-SY5Y cells were obtained from American Type Culture Collection (ATCC, Manassas, VA). Human MOR--arrestin2EGFP-U2OS cells were a kind gift from Dr. Larry Barak of the Duke University Addiction Research GPCR Assay Bank (42). The SH-SY5Y and U2OS cells were used without further modification. The CHO and HEK cells were developed into stable cell lines by electroporation of a 3X-HA N-terminally tagged human MOR expression con- struct (Genecopoeia, Rockville, MD) and by selection with 500 g/ml G418. The populations were enriched for MOR expres- sion by two cycles of flow cytometry, selecting the top 2–3% of the population in each round. The resulting stable, high-ex- pressing cell lines were characterized by immunocytochemistry and Western blot analysis of ERK activation in response to DAMGO, and the CHO line was further characterized by satu- ration radioligand binding with [3H]diprenorphine (KD 2.81 0.72 nM; Bmax 435 76 fmol/mg; n 3 independent experiments). Both lines were also characterized in multiple signal transduction assays for MOR function for other projects (data not shown). For the pain studies, it was found that Hsp90 inhibition reversed diabetes-induced sensory hypoalgesia in mice (18) and that Hsp90 inhibition reversed CCI-induced mechanical allo- dynia and potentiated morphine anti-nociception (19). The hypoalgesia results are too orthogonal to our model for a direct comparison, but the reduction in pain and enhancement of morphine anti-nociception in the CCI model is opposed to our results, particularly our findings with HIV neuropathy, as CCI is also a chronic neuropathic pain model. Important differences between our studies include that the Hsp90 inhibitor was given systemically over a 3-h or less time scale, whereas we adminis- tered inhibitor i.c.v. for 24 h. CCI also has a different mecha- nism of pain induction than HIV neuropathy. Either of these differences could explain the discrepancy, and future studies will explore CCI neuropathic pain and different routes of administration in our pain models. Given the importance of context to Hsp90 function, future therapy may need to be closely tailored to the specific pain state of the patient. 10424 J. Biol. Chem. (2017) 292(25) 10414–10428 10424 Animals Male CD-1 mice in age-matched controlled cohorts from 4–8 weeks of age were used for all experiments and were obtained from Charles River Laboratories (Wilmington, MA). CD-1 (a.k.a. ICR) mice are commonly used in opioid research as a line with a strong response to opioid drugs (e.g. see Refs. 43 and 44). Mice were allowed to recover for a minimum of 5 days after shipment before being used in experiments. The mice were kept in AAALAC-accredited vivaria at the University of New England and the University of Arizona under temperature control and 12-h light/dark cycles with food (standard lab chow) and water available ad libitum. No more than five mice were kept in a cage. The animals were monitored daily, includ- ing after surgical procedures, by trained veterinary staff. All experiments performed were in accordance with IACUC-ap- proved protocols at both the University of New England and the University of Arizona. The resulting image bands were quantified using Scion Image (based on NIH Image). All images were quantified in the linear signal range, which is easier to ensure because the Odys- sey imager is a dynamic imager that allows for fine control of exposure. Scion Image also indicated any saturated pixels. The intensity levels of all proteins except ERK and Akt were normal- ized to the housekeeping protein GAPDH, the pERK signal was normalized to the tERK signal, and pAkt was normalized to tAkt, with both measured from the same blot as the primary target. Separate quantitation of GAPDH, tERK, and tAkt indi- cated that the drug treatments used did not change the expres- sion levels of these proteins (Fig. 5, A–C, data not shown). The normalized intensities were further normalized to a vehicle control present on the same blot. Antibodies All antibodies were validated prior to use by in vitro experi- ments using a cell line expressing the target in question with a negative control cell line that did not express the target in ques- tion or, in the case of Hsp70 and ERK, by treatment with a drug as a positive control. Hsp70 and ERK were validated by proper response to the drug (17-AAG and DAMGO/10% FBS/LPA); the other targets were validated by comparison with the nega- tive control. Tail-flick assay Pre-injection tail-flick baselines were determined in a 52 °C tail-flick assay with a 10-s cutoff time. The mice were then injected i.c.v. with 17-AAG/pifithrin- or U0126 with a 24-h (17-AAG/pifithrin-) or 15-min (U0126) treatment time. 24 h post-injection baselines were determined for the 17-AAG experiments. The mice were then injected i.c.v. with 0.01, 0.032, or 0.1 nmol of DAMGO, and tail-flick latencies were deter- Paw incision and mechanical allodynia Mechanical thresholds were determined prior to surgery using calibrated Von Frey filaments (Ugo Basile, Varese, Italy) with the up-down method and four measurements after the first response per mouse (28). The mice were housed in a home- made apparatus with Plexiglas walls and ceiling and a wire- mesh floor (3-inch wide 4-inch long 3-inch high with 0.25- inch wire mesh). The surgery was then performed by anesthesia with 2% isoflurane in standard air, preparation of the left plantar hind paw with iodine and 70% ethanol, and a 5-mm incision made through the skin and fascia with a no. 11 scalpel. The muscle was elevated with curved forceps leaving the origin and insertion intact, and the muscle was split lengthwise using the scalpel. The wound was then closed with 5-0 polyglycolic acid sutures. For the 17-AAG experiments, the mice were then injected i.c.v. and left to recover for 24 h. For the U0126 exper- iments, the mice went directly to recovery. The next day, the mechanical threshold was again determined as described above, and i.c.v. injections took place for the U0126 experi- ments with a 15-min treatment time. Both the 17-AAG and the U0126 mice were then injected with 1, 3.2, or 10 mg/kg mor- phine s.c., and mechanical thresholds were determined over a time course. No animals were excluded from these studies. Heat-shock protein 90 in opioid signaling and pain The antibodies used were: Hsp70 (Cell Signaling 4872S, lot 4, rabbit, 1:1000), GAPDH (ThermoFisher MA5-15738, lot PI209504, mouse, 1:1000), STAT3 (Cell Signaling 9139S, lot 7, mouse, 1:1000), pERK (Cell Signaling 4370S, lot 12, rabbit, 1:1000), tERK (Cell Signaling 4696S, lot 16, mouse, 1:1000), pAkt (Cell Signaling 9018P, lot 3, rabbit, 1:1000), tAkt (Cell Signaling 2920S, lot 3, mouse, 1:1000), MOR (Abcam ab10275, lot GR81301-2, 1:500 for immunoprecipitation), -arrestin2 (Abcam ab54790, rabbit, 1:500 for immunoprecipitation), -ar- restin2 (Cell Signaling 3857S, 2, rabbit, 1:1000 for Western blot- ting), HA tag for in vitro MOR (Cell Signaling 3724S, lot 3, rabbit, 1:1000), secondary GM680 (LiCor 926-68020, lot C50721-02, goat, 1:10,000–1:20,000), and secondary GR800 (LiCor 926-32211, lot C50602–05, goat, 1:10,000–1:20,000). mined over a time course. After completing the study, the mice that did not respond to DAMGO were excluded from the anal- ysis, suggesting a missed i.c.v. injection, as it was clear that the 17-AAG and other treatments did not cause a large decrease in anti-nociception. Dependence and withdrawal For the acute dependence and withdrawal paradigm, mice were injected i.c.v. with 17-AAG or vehicle and left to recover for 20 h. They were then injected with 100 mg/kg morphine s.c. and left to recover for 4 h, and then withdrawal was precipitated with 10 mg/kg naloxone i.p. For the chronic dependence and withdrawal paradigm, a 4-day escalating-dosage protocol was used. Morphine was injected twice daily, with 1 A90 on day 1, 2 A90 on day 2, 3.5 A90 on day 3, and finally one injection of 3.5 A90 on day 4 followed 4 h later by 30 mg/kg naloxone i.p. 17-AAG or vehicle was injected i.c.v. daily from days 0–3. For both paradigms, behavior was observed in 16-inch-high 6-inch-deep Plexiglas cylinders for 20 min. The number of jumps was recorded, and the weight of the urine and feces was recorded at the end of the observation period. One whole tech- nical replicate of the chronic dependence and withdrawal was excluded (both treatment groups) because the majority of the vehicle-treated animals did not display any withdrawal behav- ior. No animals were excluded from the acute dependence and withdrawal studies. Rotarod test Mice were subjected to three training trials of 3 min each on a Rotarod device, with the machine off for trial 1, the machine on but not rotating for trial 2, and the machine rotating at 4 rpm for trial 3 (30). An automatic timer in the unit was used to record fall latencies with a 3-min maximum time. The mice were then injected i.c.v. with 17-AAG or vehicle and allowed to recover for 24 h, and another 3-min Rotarod trial was per- formed without additional treatments or interventions. This trial was done with an accelerating 4–16 rpm over the 3-min trial time. No mice were excluded from these studies. HIV peripheral neuropathy HIV peripheral neuropathy was induced by intrathecal injec- tion of gp120 protein (15 ng/l in 0.1 M PBS and 0.1% BSA, 7-l volume) using an established protocol (29) on days 1, 3, and 5. Mechanical thresholds were monitored every 2 days on both hind paws using Von Frey filaments as described above. For the 17-AAG experiments, 17-AAG was injected i.c.v. as described above on day 20 with a 24-h treatment time. For the U0126 experiments, U0126 was injected i.c.v. on day 21 with a 15-min treatment time. For both experiments, morphine (10 mg/kg s.c.) was then injected, and mechanical thresholds were mea- sured over a time course on both hind paws. Both hind paws gave highly similar results, so only the morphine results from the left paw are shown (data not shown). No animals were excluded from these studies. Behavioral experiments Prior to any behavioral experiment or testing, the animals were brought to the testing room in their home cages for at least 1 h for acclimation. Testing always occurred within the same approximate time of day between experiments, and environ- mental factors (noise, personnel, and scents) were minimized. All testing apparatus (cylinders, grid boxes, etc.) were cleaned between uses. The experimenter was blinded to treatment group by another laboratory member delivering coded drug vials, which were then decoded after collection of all data. J. Biol. Chem. (2017) 292(25) 10414–10428 10425 Heat-shock protein 90 in opioid signaling and pain Heat-shock protein 90 in opioid signaling and pain were reported raw without maximum possible effect (MPE) or other normalization. Biological and technical replicates are described in the figure legends. Comparisons between two groups (protein expression and withdrawal) were performed by unpaired 2-tailed t tests. Comparisons of more than two groups (ERK and Akt signaling, paw incision, tail flick, HIV neuropa- thy, and rotarod) were performed by two-way ANOVA with Fisher’s least significant difference post-hoc test. In all cases, significance was defined as p 0.05. For the dose-response experiments, the A50 was calculated by performing linear regression analysis of the data points and solving for A50 by the equation y (logA50), where y is the effect at the desired percentage (i.e. 1.6 at the A50 in the 17-AAG dose-response experiment), is the y intercept of the line, and is the slope. The stimulation caused by 5 nmol of 17-AAG was assumed to be the maximum stimulation for the 17-AAG dose-response analysis. AUC analysis was performed in the tail-flick, paw- incision, and HIV neuropathy experiments by setting the low- est point from both summary curves as the baseline in each analysis. 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https://openalex.org/W2773893960	https://aacr.figshare.com/articles/journal_contribution/Supplementary_information_from_Inhibition_of_ID1_BMPR2_Intrinsic_Signaling_Sensitizes_Glioma_Stem_Cells_to_Differentiation_Therapy/22467240/1/files/39918651.pdf	English	null	Inhibition of ID1–BMPR2 Intrinsic Signaling Sensitizes Glioma Stem Cells to Differentiation Therapy	Clinical cancer research	2,018	cc-by	2,783	Supplementary Information 1 Inhibition of ID1-BMPR2 intrinsic signaling sensitizes glioma stem cells to 2 differentiation therapy 3 Xiong Jin1,2, Xun Jin3,4,5, Leo J Y Kim3, Deobrat Dixit3, Hee-Young Jeon1,2, Eun-Jung Kim1,2, Jun- 4 Kyum Kim1,2, Seon Yong Lee1, Jinlong Yin6, Jeremy N. Rich3, and Hyunggee Kim1,2 5 1Department of Biotechnology, College of Life Sciences and Biotechnology, Korea University, 6 Seoul 02841, Republic of Korea. 7 2Institute of Animal Molecular Biotechnology, Korea University, Seoul 02841, Republic of 8 Korea. 9 3Department of Stem Cell Biology and Regenerative Medicine, Lerner Research Institute, 10 Cleveland Clinic, Cleveland, OH 44195, USA. 11 4Tianjin Medical University Cancer Institute and Hospital, Tianjin 300060, China. 12 5Institute of Translational Medicine, First Affiliated Hospital of Wenzhou Medical University, 13 Wenzhou 325015, Zhejiang, China. 14 6Specific Organ Cancer Devision, Research Institute and hospital, National Cancer Center, 15 Goyang 10408, Republic of Korea. 16 17 Corresponding authors: H Kim, Department of Biotechnology, College of Life Sciences and 18 Biotechnology, Korea University, 145 Anam-Ro, Seongbuk-Gu, Seoul 02841, Republic of Korea. 19 Phone: +82 2 3290 3059; Fax: +82 2 953 0737; E-mail: hg-kim@korea.ac.kr; 20 X Jin, Department of Stem Cell Biology and Regenerative Medicine, Lerner Research Institute, 21 Cleveland Clinic, Cleveland, OH 44195, USA. Tel: +1 216 636 1008; Fax: +1 216 636 5454; E- 22 mail: jinx2354@gmail.com 23 24 Xiong Jin and Xun Jin contributed equally to this work. 25 26 Supplementary Information 1 Inhibition of ID1-BMPR2 intrinsic signaling sensitizes glioma stem cells to 2 differentiation therapy 3 Xiong Jin1,2, Xun Jin3,4,5, Leo J Y Kim3, Deobrat Dixit3, Hee-Young Jeon1,2, Eun-Jung Kim1,2, Jun- 4 Kyum Kim1,2, Seon Yong Lee1, Jinlong Yin6, Jeremy N. Rich3, and Hyunggee Kim1,2 5 1Department of Biotechnology, College of Life Sciences and Biotechnology, Korea University, 6 Seoul 02841, Republic of Korea. 7 2Institute of Animal Molecular Biotechnology, Korea University, Seoul 02841, Republic of 8 Korea. 9 3Department of Stem Cell Biology and Regenerative Medicine, Lerner Research Institute, 10 Cleveland Clinic, Cleveland, OH 44195, USA. 11 4Tianjin Medical University Cancer Institute and Hospital, Tianjin 300060, China. 12 5Institute of Translational Medicine, First Affiliated Hospital of Wenzhou Medical University, 13 Wenzhou 325015, Zhejiang, China. 14 6Specific Organ Cancer Devision, Research Institute and hospital, National Cancer Center, 15 Goyang 10408, Republic of Korea. 16 17 Corresponding authors: H Kim, Department of Biotechnology, College of Life Sciences and 18 Biotechnology, Korea University, 145 Anam-Ro, Seongbuk-Gu, Seoul 02841, Republic of Korea. 19 Phone: +82 2 3290 3059; Fax: +82 2 953 0737; E-mail: hg-kim@korea.ac.kr; 20 X Jin, Department of Stem Cell Biology and Regenerative Medicine, Lerner Research Institute, 21 Cleveland Clinic, Cleveland, OH 44195, USA. Tel: +1 216 636 1008; Fax: +1 216 636 5454; E- 22 mail: jinx2354@gmail.com 23 24 Xiong Jin and Xun Jin contributed equally to this work. 25 26 differentiation therapy 2Institute of Animal Molecular Biotechnology, Korea University, Seoul 02841, Republic of 8 Korea. 9 3Department of Stem Cell Biology and Regenerative Medicine, Lerner Research Institute, 10 Cleveland Clinic, Cleveland, OH 44195, USA. 11 4Tianjin Medical University Cancer Institute and Hospital, Tianjin 300060, China. 12 5Institute of Translational Medicine, First Affiliated Hospital of Wenzhou Medical University, 13 Wenzhou 325015, Zhejiang, China. 14 6Specific Organ Cancer Devision, Research Institute and hospital, National Cancer Center, 15 Goyang 10408, Republic of Korea. 16 6Specific Organ Cancer Devision, Research Institute and hospital, National Cancer Center, 15 Goyang 10408, Republic of Korea. 16 Corresponding authors: H Kim, Department of Biotechnology, College of Life Sciences and 18 Biotechnology, Korea University, 145 Anam-Ro, Seongbuk-Gu, Seoul 02841, Republic of Korea. 19 Phone: +82 2 3290 3059; Fax: +82 2 953 0737; E-mail: hg-kim@korea.ac.kr; 20 X Jin, Department of Stem Cell Biology and Regenerative Medicine, Lerner Research Institute, 21 Cleveland Clinic, Cleveland, OH 44195, USA. Tel: +1 216 636 1008; Fax: +1 216 636 5454; E- 22 mail: jinx2354@gmail.com 23 26 1 Figure S1-S7 1 Table S1-S4 2 Figure S1-S7 1 Table S1-S4 2 2 2 3 Figure S1. A potential function of ID1 in lineage differentiation as analyzed by means of a 1 public dataset of GBM (Related to Figure 1) 2 Figure S1. A potential function of ID1 in lineage differentiation as analyzed by means of a 1 public dataset of GBM (Related to Figure 1) 2 (A-C) Correlation between the ID family and three lineage differentiation signatures: astrocytic, 3 mature oligodendrocytic, and neuronal in (A) Gravendeel (n = 284), (B) Pola.network (n = 71), 4 and (C) TCGA-GBMLGG dataset (n = 669) GBM datasets (P < 0.05, P < 0.01, t-test). (D) 5 mRNA expression levels of ID1 in control GSC8 and GSC2 and ID1-knocked down GSC8 and 6 GSC2 cells shown in microarray (GSE40614) (P < 0.01, t-test). (E) Immunofluorescence 7 image showing BMPR2 (red)- and Nestin (green)-positive cells in primary GBM specimen. DAPI 8 (blue) stained the nuclei. 9 10 4 1 2 5 Figure S2. ID1 represses BMP4-mediated lineage differentiation. (Related to Figure 2) 1 Figure S2. ID1 represses BMP4-mediated lineage differentiation. (Related to Figure 2) 1 (A) The inhibition rate in terms of the neurosphere-forming ability of GSC8-CON and GSC8-ID1 2 cells treated with BMP4. Error bar: +/- SEM, n = 6, P < 0.01 for CON vs. ID1, t-test. (B, C) 3 BMPR2 and phospho-SMAD1, -5, and -8 protein levels in Ink4a/Arf-/- astrocytes (B) and GSC8 4 cells (C) expressing Control, ID1, or ID1-BMPR2. (D, E) A pairwise chi-squared test was 5 performed on the results of the assay for the single-cell in vitro limiting dilution neurosphere- 6 forming ability of Ink4a/Arf-/- astrocytes (D) and GSC8 cells (E) that are presented in Figures 3E 7 and 3F. (F) ID1 protein levels in 528 GSC cells expressing Control or ID1. (G) Dose-dependent 8 neurosphere-forming ability of 528-CON and 528-ID1 cells treated with BMP4. Error bar: +/- 9 SEM, n = 6, P < 0.01 for HCl treatment vs. BMP4 treatment [50 or 100 ng/ml], t-test. (H) The 10 proportions of Nestin+, Sox2+, S100β+, and Tuj1+ cells among 528-CON and 528-ID1 cells 11 treated with HCl and BMP4 (50 ng/ml), as analyzed by FACS. Error bar: +/- SEM, n = 3, P < 12 0.01, t-test. (I) BMPR2 and phospho-SMAD1, -5, and -8 protein levels in 528 cells expressing 13 Control, ID1, or ID1-BMPR2. (J) Single-cell in vitro limiting dilution neurosphere-forming ability 14 of 528 cells expressing Control, ID1, or ID1-BMPR2. W/o = without, n = 24, P < 0.01, t-test. 15 (K) Expression levels of Nestin (stem cell), Sox2 (stem cell), S100β (astrocyte), and Tuj1 (neuron) 16 mRNAs in 528 cells expressing Control, ID1, or ID1-BMPR2 were determined by q-RT-PCR. 17 Error bar: +/- SEM n = 3 P < 0 01 t-test 18 19 6 1 Figure S3. Blockade of miR-17 and miR-20a suppresses ID1-mediated BMPR2 repression 2 (Related to Figure 3) 3 1 Figure S3. Blockade of miR-17 and miR-20a suppresses ID1-mediated BMPR2 repression 2 (Related to Figure 3) 3 (A, B) mRNA expression levels of BMPR2 in Ink4a/Arf-/- astrocyte-ID1 cells (A) and GSC8 cells 4 (B) transduced with antisense miR-17 and miR-20a (50 nM) were determined by qRT-PCR 5 (Error bar: +/- SEM, n = 3, P < 0.01, t-test). Figure S2. ID1 represses BMP4-mediated lineage differentiation. (Related to Figure 2) 1 6 (A, B) mRNA expression levels of BMPR2 in Ink4a/Arf-/- astrocyte-ID1 cells (A) and GSC8 cells 4 (B) transduced with antisense miR-17 and miR-20a (50 nM) were determined by qRT-PCR 5 (Error bar: +/- SEM, n = 3, P < 0.01, t-test). 6 7 7 7 2 8 Figure S4. Effects of c-Myc and WNT/SHH signaling activated by ID1 on the neurosphere- 1 forming ability and their downstream target gene expression (Related to Figure 4) 2 (A, B) The single-cell in vitro limiting dilution neurosphere-forming ability of Ink4a/Arf-/- 3 astrocytes (A) and GSC8 cells (B) expressing Control, MYC, ID1, or ID1-shMYC (W/o = without, 4 n = 24, P < 0.01, t-test). (C, D) A pairwise chi-squared test was performed on the results of 5 Fig. S4A and S4B. (E, F) Expression of WNT’s and SHH’s downstream target genes in Ink4a/Arf- 6 /- astrocytes (E) and GSC8 cells (F) expressing Control or ID1 were detected using qRT-PCR 7 (Error bar: +/- SEM. n = 3. P < 0.05, P < 0.01, t-test). 8 Figure S4. Effects of c-Myc and WNT/SHH signaling activated by ID1 on the neurosphere- 1 forming ability and their downstream target gene expression (Related to Figure 4) 2 (A, B) The single-cell in vitro limiting dilution neurosphere-forming ability of Ink4a/Arf-/- 3 astrocytes (A) and GSC8 cells (B) expressing Control, MYC, ID1, or ID1-shMYC (W/o = without, 4 n = 24, P < 0.01, t-test). (C, D) A pairwise chi-squared test was performed on the results of 5 Fig. S4A and S4B. (E, F) Expression of WNT’s and SHH’s downstream target genes in Ink4a/Arf- 6 /- astrocytes (E) and GSC8 cells (F) expressing Control or ID1 were detected using qRT-PCR 7 (Error bar: +/- SEM. n = 3. P < 0.05, P < 0.01, t-test). 8 9 9 9 9 1 2 10 Figure S5. Effects of combined treatments with WNT/SHH inhibitors and BMP4 on 1 expression of stem cell markers and differentiated-cell markers (Related to Figure 5) 2 (A) Luciferase reporter assay in Ink4a/Arf-/- astrocyte-ID1 and GSC8 cells revealed that JW67 3 suppressed WNT signaling (pTOP-luciferase activity) in a dose-dependent manner. Error bar: 4 +/- SEM, n = 3, P < 0.01, t-test. (B) The luciferase reporter assay in Ink4a/Arf-/- astrocyte-ID1 5 and GSC8 cells revealed that GANT61 suppressed SHH signaling (Gli-luciferase activity) in a 6 dose-dependent manner. Error bar: +/- SEM, n = 3, P < 0.01, t-test. (C) Dot plot images 7 showing the proportions of Nestin+, Sox2+, S100β+, Tuj1+, and O4+ cells presented in Fig. 5J. 8 Dots in each colored box represent a shifted proportion. (D) BMPR2, phospho-SMAD1, -5, and 9 -8, and c-Myc protein levels in Ink4a/Arf-/- astrocyte-ID1 cells treated with JQ1 (2.5 μM) or 10 10058-F4 (10 μM). (E) Effects of BMP4 (50 ng/ml) and JQ1 (2.5 µM) alone or in combination 11 on the neurosphere-forming ability of Ink4a/Arf-/- astrocyte-ID1 cells. Error bar: +/- SEM, n = 12 24, P < 0.05, P < 0.01, t-test. (F) Proportions of Nestin+, Sox2+, S100β+, and Tuj1+ cells 13 among Ink4a/Arf-/- astrocyte-ID1 cells treated with BMP4 (50 ng/ml) and JQ1 (2.5 µM) alone 14 or in combination were analyzed by FACS. Error bar: +/- SEM, n = 24, P < 0.01, t-test. 15 16 11 1 12 12 Figure S6. Effects of combined treatments with WNT/SHH inhibitors and BMP4 on 1 expression of stem cell markers and differentiated-cell markers in xenograft tumors (Related 2 to Figure 6) 3 Figure S6. Effects of combined treatments with WNT/SHH inhibitors and BMP4 on 1 expression of stem cell markers and differentiated-cell markers in xenograft tumors (Related 2 to Figure 6) 3 (A) In case of Ink4a/Arf-/- astrocytes-ID1 cells, control and BMP4-treated mice was sacrificed 4 at days 25 after implantation and in this time point, JW67+GANT61- or all three reagents- 5 treated mice did not give rise to tumors. Thus, later two groups were sacrificed at days 80 6 after implantation when they displayed neurological symptom. Representative images of H&E 7 staining and immunohistochemistry (IHC) showing Nestin-, Sox2-, GFAP-, Tuj1-, O4-positive 8 cell populations in the orthotopical brain tumor samples. Quantification of cell populations 9 expressing stem cell and lineage differentiation markers was shown in Figure 7A. Scale bar for 10 H&E = 1 mm; Scale bar for IHC = 50 μm. (B) In case of GSC8 cells, all groups of mice were 11 sacrificed at days 50 after implantation when control mice showed neurological symptom. 12 Representative images of H&E staining and immunohistochemistry showing Nestin-, Sox2-, 13 GFAP-, Tuj1-, O4-positive cell populations in the orthotopical brain tumor samples. 14 Quantification of cell populations expressing stem cell and lineage differentiation markers was 15 shown in Figure 6B. Scale bar for H&E = 1 mm; Scale bar for IHC = 50 μm. 16 17 13 1 2 14 Figure S7. Therapeutic effects of combined treatments with Myc inhibitors and BMP4 on 1 tumorigenesis and expression of stem cell markers and differentiated-cell markers (Related 2 to Figure 6) 3 Figure S7. Therapeutic effects of combined treatments with Myc inhibitors and BMP4 on 1 tumorigenesis and expression of stem cell markers and differentiated-cell markers (Related 2 to Figure 6) 3 (A) Survival of brain tumor-bearing mice orthotopically implanted with 103 Ink4a/Arf-/- 4 astrocyte-ID1 cells administrated intraperitoneally with BMP4 (50 mg/kg/3 days) and JQ1 (50 5 mg/kg/3 days) alone or in combination along with elacridar (100 mg/kg/3 days) at 24 days 6 after tumor cell implantation. The elacridar, a potent inhibitor of P-glycoprotein (ABCB1), was 7 used to penetrate the blood-brain barrier and allow drugs to accumulate in the brain. n = 6, 8 P < 0.01, t-test. (B) Representative immunohistochemistry images showing Nestin-, Sox2-, 9 GFAP-, and Tuj1-positive cells in brain tumor samples obtained at 1 day after BMP4 or JQ1 10 administration. Scale bar = 50 μm. (C) Quantification of cell populations expressing stem cell 11 and lineage differentiation markers was measured by MetaMorph. Error bar: +/- SEM, n = 5, 12 P < 0.01, t-test. 13 (A) Survival of brain tumor-bearing mice orthotopically implanted with 103 Ink4a/Arf-/- 4 astrocyte-ID1 cells administrated intraperitoneally with BMP4 (50 mg/kg/3 days) and JQ1 (50 5 mg/kg/3 days) alone or in combination along with elacridar (100 mg/kg/3 days) at 24 days 6 after tumor cell implantation. The elacridar, a potent inhibitor of P-glycoprotein (ABCB1), was 7 used to penetrate the blood-brain barrier and allow drugs to accumulate in the brain. n = 6, 8 P < 0.01, t-test. (B) Representative immunohistochemistry images showing Nestin-, Sox2-, 9 GFAP-, and Tuj1-positive cells in brain tumor samples obtained at 1 day after BMP4 or JQ1 10 administration. Scale bar = 50 μm. (C) Quantification of cell populations expressing stem cell 11 and lineage differentiation markers was measured by MetaMorph. Error bar: +/- SEM, n = 5, 12 P < 0.01, t-test. 13 14 15 Table S1. Antisense-microRNAs and shRNA used in this study 1 2 3 Primer Sequence (5¢ to 3¢) Antisense negative control CTTTTTGCGGTCTGGGCTTGC Antisense miR-17 CTACCTGCACTGTAAGCACTTTG Antisense miR-20a CTACCTGCACTATAAGCACTTTA shMYC CAGTTGAAACACAAACTTGAA Table S1. Antisense-microRNAs and shRNA used in this study 1 16 Table S2. Antibodies used in this study 1 2 WB, Western blot; FACS, Fluorescence-activated cell sorting analysis; IHC, 3 Immunohistochemistry; ChIP, Chromatin immunoprecipitation. ohistochemistry; ChIP, Chromatin immunoprecipitation. Figure S7. Therapeutic effects of combined treatments with Myc inhibitors and BMP4 on 1 tumorigenesis and expression of stem cell markers and differentiated-cell markers (Related 2 to Figure 6) 3 4 Antibody Origin Designation Experiment β-actin Santa Cruz Biotechnology C-4 WB ID1 Biocheck 37-2 WB BMPR2 Santa Cruz Biotechnology H-300 WB, IF Smad 1,5,8 Santa Cruz Biotechnology N-18 WB P-Smad 1,5,8 Cell signaling Ser463/465 WB c-Myc Santa Cruz Biotechnology C-33 WB, ChIP, IHC Gli2 Santa Cruz Biotechnology H-300 WB β-catenin Santa Cruz Biotechnology C-18 WB Nestin Sigma N5413 FACS, IF, IHC Sox2 R&D systems AF2018 FACS, IHC S100β Sigma S2644 FACS GFAP NOVUS NBP2-29415 IHC Tuj1 Neuromics MO15013 FACS, IHC O4 Merck Millipore 81 FACS, IHC H3K9ac Emd Millipore 07-352 ChIP 2 WB, Western blot; FACS, Fluorescence-activated cell sorting analysis; IHC, 3 Immunohistochemistry; ChIP, Chromatin immunoprecipitation. 4 17 Table S3. PCR primer sequences (5¢ to 3¢) 1 Primer Forward Reverse 18S CAGCCACCCGAGATTGAGCA TAGTAGCGACGGGCGGTGTG BMPR2 AGACTGTTGGGACCAGGATG TTGCGTTCATTCTGCATAGC MYC TCGGATTCTCTGCTCTCCTC CGCCTCTTGACATTCTCCTC NES AGAGGGAGGACAAAGTCCCT CACTTCCTCAGACTGCTCCA SOX2 CAAGATGCACAACTCGGAGA CGGGGCCCGTATTTATAATC S100B TCAAAGAGCAGGAGGTTGTG TCGTGGCAGGCAGTAGTAAC TUJ1 ACGACGCTGAAGGTGTTCAT AGTGTGAAAACTGCGACTGC COX2 CCCTTCTGCCTGACACCTTT TTCTGTACTGCGGGTGGAAC AXIN2 TGTGGGCAGTAAGAAACAGC GGTTCTCGGGAAATGAGGTA MYCN ATGCACCCCCACAGAAGAAG CTCCGAGTCAGAGTTTCGGG LGR5 GACTTTAACTGGAGCACAGA AGCTTTATTAGGGATGGCAA CCND1 AAGCTGTGCATCTACACCGA CTTGAGCTTGTTCACAGGA BMI1 TATGCAGCTCATCCTTCTGC TTCCGATCCAATCTGTTCTG HES1 GGCCAGTTTGCTTTCCTCAT TTTAGAGTCCGGAGGGAAGA GLI1 AGAGTAGGGAATCTCATCCATCA TGATGCAGTTCCTTTATTATCAGG PTCH1 CGCATAGCGTGTTTGAAAGT GTATTTGTGCATTGGGCTCC Gapdh AAACCCATAAGTGAAATGGG GCTTCCACAGGGATGTTTAT Bmpr2 GACGCTTTTCTCGTTTGTGG CAGGAAGCTTTGGGAAACAG Myc AAAACGACAAGAGGCGGACA ATCACTACCTTGGGGGCCTT Nes GGCATCCCTTTACAGGAACA TAATCTTCCCCTGAGGACCA Sox2 AGCTCGCAGACCTACATGAA AGTGGGAGGAAGAGGTAACC S100b CAGAGACCCTTAATTCCCCA GTTCCTGGAGGACAGAGGAG Tuj1 CCCAAGTGAAGTTGCTCGCA ACAGAGCCAAGTGGACTCAC Cox2 AATACTGGAAGCCGAGCACC TGGCTGTTTTGGTAGGCTGT Axin2 GCAAGTCCAAGCCCCATAGT GGGTCCTGGGTAAATGGGTG Mycn TGGGTGGCCTCACTCCTAAT GCCGTGCTGTAGTTTTTCGT 18 Lgr5 ATCTCCTGTCGTCCTTCCCT CTGTAAGGCTCGGTTCCCTG Ccnd1 GCCTACAGCCCTGTTACCTG ATTTCATCCCTACCGCTGTG Bmi1 ATGCTGGAGAGCTGGAAAGT AATGTGAGGGAACTGTGGGT Hes1 ACACCGGACAAACCAAAGAC TGATCTGGGTCATGCAGTTG Gli1 AAGAGGCTGGCAAAGTTGTT TGTTTGGACCGACACTGTTT Ptch1 CAGACATCAGCCTCCCTTGA AGAAGCCGTCACAGTGGTGA MIR17HG TACGCGGAGAATCGCAGG GACTGCAAAGTGCCCGC 2 19 Table S4. Primers used for BMPR2 promoter and 3¢-UTR constructs (5ʹ to 3ʹ) 1 A promoter position was defined relative to the transcription start site, and the 3¢-UTR 2 position was defined relative to the translation stop codon. 3 4 Position Forward Reverse promoter -1943 to +10 TCCCCCGGGGGAGACCTTTT GGGCAAAACAAA GAAGATCTTCCGAGCAGACCAG ACACAAAG 3¢-UTR +111 to +662 ACCCCTGCAACAAAGACTTG TAGCTGCTTCTCCTGTGCAA Table S4. Primers used for BMPR2 promoter and 3¢-UTR constructs (5ʹ to 3ʹ) 1 A promoter position was defined relative to the transcription start site, and the 3¢-UTR 2 position was defined relative to the translation stop codon. 3 4 Position Forward Reverse promoter -1943 to +10 TCCCCCGGGGGAGACCTTTT GGGCAAAACAAA GAAGATCTTCCGAGCAGACCAG ACACAAAG 3¢-UTR +111 to +662 ACCCCTGCAACAAAGACTTG TAGCTGCTTCTCCTGTGCAA Table S4. Primers used for BMPR2 promoter and 3¢-UTR constructs (5ʹ to 3ʹ) 1 4 20
https://openalex.org/W2799366056	https://bmcgenomics.biomedcentral.com/track/pdf/10.1186/s12864-018-4605-1	English	null	Genome-wide transcriptional analyses in Anopheles mosquitoes reveal an unexpected association between salivary gland gene expression and insecticide resistance	BMC genomics	2,018	cc-by	10,483	Isaacs et al. BMC Genomics (2018) 19:225 https://doi.org/10.1186/s12864-018-4605-1 Isaacs et al. BMC Genomics (2018) 19:225 https://doi.org/10.1186/s12864-018-4605-1 Genome-wide transcriptional analyses in Anopheles mosquitoes reveal an unexpected association between salivary gland gene expression and insecticide resistance Alison T. Isaacs1* , Henry D. Mawejje2, Sean Tomlinson1, Daniel J. Rigden3 and Martin J. Donn * Correspondence: alisonisaacs@outlook.com 1Department of Vector Biology, Liverpool School of Tropical Medicine, Liverpool, UK Full list of author information is available at the end of the article © 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. Abstract Background: To combat malaria transmission, the Ugandan government has embarked upon an ambitious programme of indoor residual spraying (IRS) with a carbamate class insecticide, bendiocarb. In preparation for this campaign, we characterized bendiocarb resistance and associated transcriptional variation among Anopheles gambiae s.s. mosquitoes from two sites in Uganda. Results: Gene expression in two mosquito populations displaying some resistance to bendiocarb (95% and 79% An. gambiae s.l. WHO tube bioassay mortality in Nagongera and Kihihi, respectively) was investigated using whole- genome microarrays. Significant overexpression of several genes encoding salivary gland proteins, including D7r2 and D7r4, was detected in mosquitoes from Nagongera. In Kihihi, D7r4, two detoxification-associated genes (Cyp6m2 and Gstd3) and an epithelial serine protease were among the genes most highly overexpressed in resistant mosquitoes. Following the first round of IRS in Nagongera, bendiocarb-resistant mosquitoes were collected, and real-time quantitative PCR analyses detected significant overexpression of D7r2 and D7r4 in resistant mosquitoes. A single nucleotide polymorphism located in a non-coding transcript downstream of the D7 genes was found at a significantly higher frequency in resistant individuals. In silico modelling of the interaction between D7r4 and bendiocarb demonstrated similarity between the insecticide and serotonin, a known ligand of D7 proteins. A meta-analysis of published microarray studies revealed a recurring association between D7 expression and insecticide resistance across Anopheles species and locations. Conclusions: A whole-genome microarray approach identified an association between novel insecticide resistance candidates and bendiocarb resistance in Uganda. In addition, a single nucleotide polymorphism associated with this resistance mechanism was discovered. The use of such impartial screening methods allows for discovery of resistance candidates that have no previously-ascribed function in insecticide binding or detoxification. Characterizing these novel candidates will broaden our understanding of resistance mechanisms and yield new strategies for combatting widespread insecticide resistance among malaria vectors. Keywords: Anopheles gambiae, Whole-genome microarray, Salivary gland proteins, Indoor residual spraying, Bendiocarb, Pyrethroids, Long-lasting insecticidal nets, Malaria control Background were observed following the universal LLIN distribu- tion campaign. Malaria interventions deployed across sub-Saharan Africa over the past 15 years have had an extraordinary impact upon disease transmission, halving the prevalence of Plasmodium falciparum infection in endemic regions [1]. Insecticide-based control approaches, such as long-lasting insecticidal nets (LLINs) and indoor residual spraying (IRS) were by far the largest contributors to this outcome. As the use of these vector control tools has increased, so too has the emergence of insecticide-resistant mosquito populations. Mosquito populations resistant to pyre- throids, the only class of insecticide approved for use in LLINs, are starting to outnumber susceptible populations [2]. Furthermore, resistance to other insecticide classes, such as carbamates and organophosphates, is increasingly reported [3, 4]. To combat high levels of malaria transmission in Nagongera, an IRS campaign using the carbamate class insecticide bendiocarb was initiated in December 2014. We collected mosquito specimens prior to IRS deploy- ment to measure pre-existing bendiocarb resistance (95 and 79% An. gambiae s.l. WHO tube bioassay mor- tality in Nagongera and Kihihi, respectively) and iden- tify associated mechanisms present in An. gambiae s.s. We then collected samples in the months following the first round of IRS to detect whether these insecticide resist- ance expression phenotypes persisted. Bendiocarb-resistant An. gambiae s.s. in Nagongera did not display any of the commonly observed resistance mechanisms, such as muta- tions in the insecticide target site (Ace1-119S) or overex- pression of detoxification enzymes. Instead, a significant association between bendiocarb resistance and overexpres- sion of two salivary gland genes, D7r2 and D7r4, was ob- served both before and after IRS deployment. Further research is needed to elucidate the role of D7 proteins in bendiocarb resistance, however in silico models support the hypothesis that these proteins are capable of binding insecticide, suggesting that they may play a role in trans- port or sequestration. Malaria is a critical public health challenge in Uganda, with the estimated number of annual cases ranking fourth highest in the world [5]. The investigation de- scribed herein is part of a comprehensive malaria sur- veillance program conducted since 2011 in three areas of Uganda: Walukuba, Kihihi, and Nagongera [6]. Dur- ing this time, an LLIN distribution campaign has been successful in increasing bed net ownership. A prospect- ive observational study was performed to measure the impact of this campaign upon malaria transmission [7]. * Correspondence: alisonisaacs@outlook.com 1Department of Vector Biology, Liverpool School of Tropical Medicine, Liverpool, UK Full list of author information is available at the end of the article © 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. Isaacs et al. BMC Genomics (2018) 19:225 Page 2 of 12 Background In 2013, the proportion of households in Nagongera with at least one LLIN increased from 71 to 95.5%. In 2014, the proportion of households in Kihihi with at least one LLIN increased from 37.5 to 86.5%. Through- out the study period, no evidence of a shift in vector species composition was observed, with An. gambiae s.s. remaining the primary vector in these communities. In- secticide resistance testing in both Kihihi and Nagongera revealed high levels of pyrethroid resistance in this spe- cies (24–40% mortality), and lower levels of bendiocarb resistance (70 and 83% mortality, respectively) in 2014. This high level of pyrethroid resistance may partially explain why only modest declines in malaria metrics Insecticide resistance detection Insecticide resistance detection WHO tube bioassays were performed between January and May 2014 using An. gambiae s.l. mosquitoes collected as larvae in Nagongera and Kihihi (Fig. 1). An. gambiae s.s. mortality levels for these DDT, delta- methrin, permethrin, bendiocarb, fenitrothion, and malathion WHO tube bioassays have been previously reported [7]. Species identification PCR revealed dif- fering species compositions in the two sites: 68% of mosquitoes in Nagongera (n = 925) were identified as An. gambiae s.s. and 32% were identified as Anopheles arabiensis, while those from Kihihi were exclusively Fig. 1 Map of Uganda showing study sites. Larval collections were completed in both Kihihi and Nagongera in January–May 2014. Larval collections were completed in Nagongera in January–May 2015. The geographical origin of the Kisumu laboratory strain of susceptible mosquito is also indicated. The figure was made by the authors using Google Maps (maps.google.com) Fig. 1 Map of Uganda showing study sites. Larval collections were completed in both Kihihi and Nagongera in January–May 2014. Larval collections were completed in Nagongera in January–May 2015. The geographical origin of the Kisumu laboratory strain of susceptible mosquito is also indicated. The figure was made by the authors using Google Maps (maps.google.com) Page 3 of 12 Isaacs et al. BMC Genomics (2018) 19:225 Page 3 of 12 An. gambiae s.s (n = 556). All survivors of bendiocarb exposure whose species could be identified were An. gambiae s.s. as well as the Kisumu laboratory strain of insecticide- susceptible mosquito originating from western Kenya (Fig. 1). Comparison between sympatric resistant and unex- posed mosquitoes controls for geographic origin and life history, while incorporation of the Kisumu strain permits comparison to a fully susceptible strain. Four biological rep- licates of each treatment were hybridized to whole-genome microarrays using an interwoven loop design. This strategy allowed for identification of genes whose expression was highest in resistant mosquitoes, intermediate in unexposed mosquitoes (of which an estimated 79–95% are suscep- tible), and lowest in the fully susceptible Kisumu strain. To investigate the observed bendiocarb resistance phenotype, resistant and unexposed individuals were tested for a mutation in the target of the insecticide, acetylcholinesterase-1. This G119S mutation is wide- spread in West African An. gambiae s.s [8, 9]. A TaqMan assay detected no instance of this mutation in resistant females from either location (n = 40), however one het- erozygous 119G:119S female from Nagongera was de- tected among unexposed samples (n = 40). Insecticide resistance detection Its genotype was confirmed by Sanger sequencing. Several genes met these criteria in addition to display- ing greater than 1.2-fold overexpression in resistant vs. unexposed control mosquitoes and an ANOVA F-test P value < 0.05 (Table 1). This filtering strategy relied pri- marily upon directionality, as the ANOVA F-test P value yielded a high number of significant hits. In samples from Kihihi, the transcripts most overexpressed in Microarray results were filtered to select genes whose expression was highest in resistant mosquitoes, intermediate in unexposed mosquitoes (of which an estimated 79–95% are susceptible), and lowest in the fully susceptible strain. Furthermore, genes with less than 1.2-fold overexpression in resistant vs. unexposed control mosquitoes or those with an ANOVA F-test P value > 0.05 were excluded. The 10 most highly overexpressed genes (resistant vs. unexposed) from each location are displayed above. Differential gene expression associated with bendiocarb resistance In contrast, resistant mosquitoes from Nagongera displayed overexpression of genes encoding a cuticular protein and several salivary gland proteins. Four genes were overexpressed in mos- quitoes from both locations: two of unknown function (AGAP009918 and AGAP004528), apyrase, and D7r4. Resistance-associated overexpression of D7r2 was also detected in both sites, however it was excluded from the Kihihi candidate gene list by our filtering strategy (2.1 fold change resistant vs. unexposed, ANOVA F- test P value = 0.08). p Additional bendiocarb WHO tube bioassays were per- formed following the bendiocarb IRS campaign imple- mented between December 2014 and February 2015 in Nagongera. Species identification PCR revealed a de- crease in the proportion of An. gambiae s.s. mosquitoes, with only 27% identified as An. gambiae s.s. (n = 89) (Fisher’s exact test, two-sided p < 0.0001). Surprisingly, bioassay mortality observed in May 2015 (98%) was significantly higher than that observed in May 2014 (95%) (Fisher’s exact test, two-sided p = 0.004, May 2014 n = 411, May 2015 n = 520). A TaqMan assay de- tected no instance of the G119S target-site mutation in either resistant or unexposed control mosquitoes (n = 27). Real-time quantitative PCR analyses performed on indi- vidual mosquitoes detected overexpression of both D7r2 and D7r4 in resistant individuals (Two-tailed Mann- Whitney, D7r2 p = 0.002, D7r4 p = 0.005) (Fig. 3). Comparison of D7r2 and D7r4 expression in these in- dividuals revealed a highly significant correlation (Spearman r = 0.9075, p < 0.0001), which supports the hypothesis that these genes are co-regulated. Real-time quantitative PCR analyses did not detect overexpression of gSG1a, apyrase, GSG7, or CPLCX2 in resistant mosqui- toes (Two-tailed Mann-Whitney, p > 0.05), To validate these findings, real-time quantitative PCR (qPCR) was used to measure expression of nine resist- ance candidates in the same pooled RNA samples that were assayed by whole-genome microarray (Fig. 2). For each collection site, the five genes mostly highly overex- pressed in bendiocarb-resistant mosquitoes were evalu- ated. When functional primers could not be identified or gene function was unknown, the analyses were expanded to include additional candidates (gSG1a, apyrase, GSG7). Additional resistant mosquitoes from the Kihihi collec- tions were also included: one additional unexposed con- trol pool and either 3 additional resistant pools (GSTD3, D7r4) or 4 additional resistant pools (ESP, CYP6M2). Differential gene expression associated with bendiocarb resistance Whole-genome microarray analyses were performed to compare gene expression among resistant and unexposed An. gambiae s.s. mosquitoes from Nagongera and Kihihi Table 1 Genes highly overexpressed in bendiocarb-resistant An. gambiae s.s. mosquitoes, as measured by whole-genome microarrays Location Transcript ID Gene name Fold change resistant vs. unexposed p-value Fold change resistant vs. Kisumu p-value Nagongera AGAP003334-RA CPLCX2 2.4 2.97E-03 2.4 2.92E-03 AGAP008282-RA D7r2 2.4 4.95E-02 7.2 9.22E-05 AGAP002198-RA glycine N-methyltransferase 2.1 6.79E-03 2.5 1.34E-03 AGAP000603-RA Unknown 1.9 3.32E-02 2.5 3.84E-03 AGAP008281-RA D7r4 1.9 1.97E-02 2.8 3.98E-04 AGAP000611-RA gSG1a 1.7 2.49E-04 1.8 8.17E-05 AGAP011971-RA apyrase 1.7 2.01E-02 3.1 3.28E-05 AGAP009623-RA glyceraldehyde 3-phosphate dehydrogenase 1.7 4.63E-03 2.0 8.13E-04 AGAP008216-RA GSG7 1.7 4.99E-04 1.8 2.34E-04 AGAP011368-RA OBP57 1.5 4.18E-02 1.9 4.97E-03 Kihihi AGAP010240-RA ESP 2.5 1.06E-04 4.6 6.77E-08 AGAP008212-RA Cyp6m2 2.3 1.75E-03 3.7 1.01E-05 AGAP004382-RA Gstd3 2.2 1.25E-03 3.0 4.82E-05 AGAP008281-RA D7r4 2.1 6.84E-03 2.8 3.24E-04 AGAP008358-RA Cyp4h17 2.0 2.01E-03 2.9 1.86E-05 AGAP001956-RA Niemann-Pick Type C-2 2.0 7.01E-03 5.1 2.93E-07 AGAP005501-RA dehydrogenase/reductase SDR family member 11 precursor 2.0 1.08E-03 3.4 6.42E-07 AGAP005498-RB phospholipid scramblase 2 1.9 2.78E-06 3.0 2.87E-10 AGAP005645-RA dehydrogenase/reductase SDR family member 11 precursor 1.9 7.48E-04 2.8 2.03E-06 AGAP010066-RA Unknown 1.9 1.38E-05 2.4 6.83E-08 Microarray results were filtered to select genes whose expression was highest in resistant mosquitoes, intermediate in unexposed mosquitoes (of which an estimated 79–95% are susceptible), and lowest in the fully susceptible strain. Furthermore, genes with less than 1.2-fold overexpression in resistant vs. unexposed control mosquitoes or those with an ANOVA F-test P value > 0.05 were excluded. The 10 most highly overexpressed genes (resistant vs. unexposed) from each location are displayed above. ghly overexpressed in bendiocarb-resistant An. gambiae s.s. mosquitoes, as measured by whole-genome Microarray results were filtered to select genes whose expression was highest in resistant mosquitoes, intermediate in unexposed mosquitoes (of which an estimated 79–95% are susceptible), and lowest in the fully susceptible strain. Furthermore, genes with less than 1.2-fold overexpression in resistant vs. unexposed control mosquitoes or those with an ANOVA F-test P value > 0.05 were excluded. The 10 most highly overexpressed genes (resistant vs. unexposed) from each location are displayed above. Isaacs et al. BMC Genomics (2018) 19:225 Page 4 of 12 Bendiocarb resistance post-IRS resistant mosquitoes were those encoding an epithelial serine protease (ESP) (AGAP010240), the cytochrome P450 Cyp6m2, the glutathione S transferase Gstd3, and the salivary gland protein D7r4. Differential gene expression associated with bendiocarb resistance Among the Kihihi candidates, overexpression of all four genes was confirmed (Two-tailed Mann-Whitney, ESP, Cyp6m2, Gstd3 p < 0.05, D7r4 p < 0.01). Resistant mos- quitoes from Nagongera displayed overexpression of D7r2, D7r4, gSG1a, and CPLCX2 relative to unexposed controls (Two-tailed Mann-Whitney p < 0.05). Overex- pression of apyrase and GSG7 was not validated by qPCR (Two-tailed Mann-Whitney, p > 0.05). Silencing of D7r2 and D7r4 Members of the D7 family are among the most highly- expressed salivary proteins in An. gambiae, and aid in blood feeding by scavenging host amines that induce vasoconstriction, platelet aggregation, and pain [10, 11]. In An. gambiae, three long (D7L1–3) and five short D7 (D7r1–5) have been identified, and their expression is limited to female mosquitoes [10, 12]. RNAi- induced knockdown of D7r2 and D7r4 expression via Fig. 2 Expression of insecticide resistance candidates in An. gambiae s.s. mosquitoes collected in 2014, measured by real-time quantitative PCR. Gene expression in mosquitoes collected from Kihihi (a) and Nagongera (b). Bendiocarb-resistant mosquitoes were selected using a standard WHO tube bioassay. Unexposed mosquitoes were placed in a tube with a control paper. Each RNA sample was extracted from pools of five mosquitoes. The y-axis depicts the level of transcript in each sample relative to the unexposed control group. The median value for each treatment is indicated by a line. Two-tailed Mann-Whitney p < 0.05 () or p < 0.01 () Fig. 2 Expression of insecticide resistance candidates in An. gambiae s.s. mosquitoes collected in 2014, measured by real-time quantitative PCR. Gene expression in mosquitoes collected from Kihihi (a) and Nagongera (b). Bendiocarb-resistant mosquitoes were selected using a standard WHO tube bioassay. Unexposed mosquitoes were placed in a tube with a control paper. Each RNA sample was extracted from pools of five mosquitoes. The y-axis depicts the level of transcript in each sample relative to the unexposed control group. The median value for each treatment is indicated by a line. Two-tailed Mann-Whitney p < 0.05 () or p < 0.01 () Isaacs et al. BMC Genomics (2018) 19:225 Page 5 of 12 Fig. 3 Expression of insecticide resistance candidates in An. gambiae s.s. mosquitoes collected in Nagongera in January–May 2015, measured by real-time quantitative PCR. Resistant and unexposed mosquitoes were selected as described in Fig. 2. Each RNA sample was extracted from an individual mosquito. The y-axis depicts the level of transcript in each sample relative to the unexposed control group. The median value for each treatment is indicated by a line. Two-tailed Mann-Whitney p < 0.01 () Fig. 3 Expression of insecticide resistance candidates in An. gambiae s.s. mosquitoes collected in Nagongera in January–May 2015, measured by real-time quantitative PCR. Resistant and unexposed mosquitoes were selected as described in Fig. 2. Each RNA sample was extracted from an individual mosquito. Silencing of D7r2 and D7r4 The y-axis depicts the level of transcript in each sample relative to the unexposed control group. The median value for each treatment is indicated by a line. Two-tailed Mann-Whitney p < 0.01 (**) promoter activity in transgenic fruit flies, where it repro- duced the tissue-specificity of D7r4 expression [13]. Seven sequences derived from five individuals displaying low levels of D7r4 expression (fold change 1–2 relative to unexposed controls) were compared to twenty-three sequences derived from twelve individuals displaying high levels of D7r4 expression (fold change 4–216 rela- tive to unexposed controls). The amplified sequences varied widely in size, ranging from 1.2 to 1.8 kilobase pairs (Additional file 2). Variant base pairs identified by alignment were counted for low and high-expressing in- dividuals. To identify a variant base pair associated with high expression, these results were filtered for variants appearing in at least five high expression individuals, and absent from low expression individuals. This thresh- old was set based on the hypothesis that A) high expres- sion is a dominant trait, and therefore could be present at a minimum in only half of high expression alleles; B) the variant bases conferring high expression levels may differ among the population, and therefore may be iden- tical in only a fraction of high expression individuals. This filtering strategy did not identify any variants asso- ciated with expression of D7r4. These low and high ex- pression sequence sets were then analysed for relative enrichment of known transcription factor binding motifs using the Analysis of Motif Enrichment (AME) tool [14]. A search of these sequences for motifs from the JASPAR CORE Insects database did not detect any significant enrichment of motifs in high expression sequences (Wilcoxon rank-sum test, p > 0.05). intrathoracic injection was performed to test whether these transcripts could be silenced. Kisumu strain mosquitoes were injected with either double- stranded RNA (dsRNA) targeting the D7r2 transcript (dsD7r2), the D7r4 transcript (dsD7r4) or control dsRNA containing green fluorescent protein se- quence (dsGFP). Attempts to silence these genes yielded highly variable results (32–89% reduction in transcript abundance) as well as off-target silencing of other D7 genes, and thus were not pursued further. D7 family genes are overexpressed in multiple species of insecticide-resistant mosquitoes across Africa D7 family genes are overexpressed in multiple species of insecticide-resistant mosquitoes across Africa q A meta-analysis of published microarray data was per- formed to evaluate whether D7 expression is associated with insecticide resistance in other species of Anopheles mosquitoes. A PubMed and Google Scholar search for publications containing microarray data from insecticide-resistant Anopheles mosquitoes yielded 35 re- sults. Fourteen of these publications described genome- wide microarrays performed on whole female mosqui- toes selected with insecticides. Eight of the fourteen studies analysed documented a significant association between expression of D7 genes and insecticide resist- ance. Overexpression of both the short and long form D7 genes has been previously associated with resistance to two insecticide classes: carbamates (bendiocarb), and pyrethroids (etofenprox, permethrin, deltamethrin, lambda-cyhalothrin) in four mosquito species (Anopheles funestus, Anopheles gambiae s.s, Anopheles coluzzi, Anopheles arabiensis) (Table 2). All five known short form D7 proteins and two long form D7 proteins (D7L1 and 2) were associated with insecticide resistance. In most instances, overexpression of D7 was observed, how- ever underexpression of D7r2 and a long form D7 were seen in etofenprox- and lambda-cyhalothrin-resistant An. funestus from Zambia [18]. The hypothesis that D7 expres- sion is associated with insecticide resistance is also sup- ported by longitudinal data. A deltamethrin-resistant population of An. coluzzi collected from Burkina Faso dis- played overexpression of D7L2 in 2011, and even higher ex- pression in 2012, concomitant with an increase in Identification of resistance- and overexpression- associated single nucleotide polymorphisms A subset of bendiocarb-resistant and unexposed females were examined to determine whether resistance was as- sociated with non-synonymous mutations in the coding sequence of either D7r2 or D7r4. Six variant amino acids were detected in the D7r4 coding region of four resistant individuals, however no association with resistance was detected (Additional file 1). Within three resistant indi- viduals whose D7r2 coding region was sequenced, no amino acids varying from the An. gambiae reference genome were detected (Additional file 1). To identify nucleic acid variants responsible for the el- evated D7r2 and D7r4 expression levels observed in bendiocarb-resistant mosquitoes, two nearby genomic regions were examined for the presence of associated single nucleotide polymorphisms (SNPs). First, the D7r2/D7r4 intergenic regions from resistant and unex- posed mosquitoes displaying a wide range of expression levels were amplified and sequenced. This D7r2/D7r4 intergenic region has previously been tested for its Next, a region downstream of the short D7 cassette that encodes an apparently non-coding transcript was examined for expression-associated SNPs. It has been Isaacs et al. BMC Genomics (2018) 19:225 Page 6 of 12 previously speculated that this transcript may have a role in regulation of D7 expression [12]. For this analysis, the sample set was expanded to include all An. gambiae s.s. (n = 28) from the 2015 Nagongera collections whose ex- pression had been measured. Thirteen individuals dis- playing low levels of D7r4 expression (fold change 0–2 relative to unexposed controls) were compared to fifteen individuals displaying high levels of D7r4 expression (fold change 3–216 relative to unexposed controls). Fol- lowing molecular cloning, one sequence per individual was included in the comparison. A cytosine/thymine SNP at genomic location 3R:8564156 displayed an asso- ciation with both D7 expression level and bendiocarb re- sistance (Additional file 3). A real-time quantitative PCR using a locked nucleic acid probe was used to genotype the 28 individuals in the expanded sample set, 14 additional individuals from the 2015 Nagongera col- lections, and 40 individuals from the 2014 Nagongera collections. A statistically significant association be- tween the 3R:8564156 SNP and bendiocarb resistance was detected, with 15/38 resistant and 7/44 unex- posed individuals displaying the SNP (Fisher’s exact test, p = 0.03, Odds ratio = 3, n = 82). In contrast, this SNP was less prevalent in mosquitoes from the 2014 Kihihi collections, appearing in only 2/24 resistant and 2/ 25 unexposed individuals (n = 49). Identification of resistance- and overexpression- associated single nucleotide polymorphisms No mosquito homozy- gous for the non-reference nucleotide was identified. previously speculated that this transcript may have a role in regulation of D7 expression [12]. For this analysis, the sample set was expanded to include all An. gambiae s.s. (n = 28) from the 2015 Nagongera collections whose ex- pression had been measured. Thirteen individuals dis- playing low levels of D7r4 expression (fold change 0–2 relative to unexposed controls) were compared to fifteen individuals displaying high levels of D7r4 expression (fold change 3–216 relative to unexposed controls). Fol- lowing molecular cloning, one sequence per individual was included in the comparison. A cytosine/thymine SNP at genomic location 3R:8564156 displayed an asso- ciation with both D7 expression level and bendiocarb re- sistance (Additional file 3). A real-time quantitative PCR using a locked nucleic acid probe was used to genotype the 28 individuals in the expanded sample set, 14 additional individuals from the 2015 Nagongera col- lections, and 40 individuals from the 2014 Nagongera collections. A statistically significant association be- tween the 3R:8564156 SNP and bendiocarb resistance was detected, with 15/38 resistant and 7/44 unex- posed individuals displaying the SNP (Fisher’s exact test, p = 0.03, Odds ratio = 3, n = 82). In contrast, this SNP was less prevalent in mosquitoes from the 2014 Kihihi collections, appearing in only 2/24 resistant and 2/ 25 unexposed individuals (n = 49). No mosquito homozy- gous for the non-reference nucleotide was identified. Fig. 4 Comparison between serotonin binding to D7r4 protein and the predicted mode of binding of bendiocarb. Serotonin, as visualised in the crystal structure (PDB code 2qeh; [15]), is displayed on the left, and the ROSIE server [16, 17] predicted pose of bendiocarb for the same protein on the right, each in a stick representation. Ligand atoms are coloured white (carbon), red (oxygen) or blue (nitrogen). Solvent- accessible protein surfaces were calculated within PyMOL using the default solvent molecule radius of 1.4 Å. Surface contributed by carbon atoms is coloured green in the serotonin complex and yellow in the bendiocarb complex. In both, red and blue are used for surface contributed by oxygen or nitrogen atoms, respectively. The figure was made with PyMOL (pymol.org) In silico modelling Th l susceptible colony cD7r2 and long form D7 underexpressed in resistant vs. susceptible colony p p , p py p , control vs. susceptible colony bD7r3 overexpressed in bendiocarb-resistant vs. unexposed control, underexpressed in unexposed control vs. susceptible colony cD7r2 and long form D7 underexpressed in resistant vs. susceptible colony have been false positives. Our results demonstrate an asso- ciation between D7 overexpression and bendiocarb resist- ance in An. gambiae s.s. mosquitoes, however further studies are needed to confirm whether D7 overexpression is a causative factor in resistance, or instead closely associ- ated with a resistance-conferring variant. insecticide resistance [19]. In this same study, a comparison of the highly-resistant VK7 strain and the moderately resistant Tengrela strain identified D7L2 as the second mostly highly overexpressed gene in the more resistant strain. Furthermore, when permethrin-resistant An. arabiensis from Uganda were compared to the Dongola susceptible laboratory strain, D7r2 was the most highly- overexpressed gene [20]. The hypothesis that D7 family proteins are directly involved in bendiocarb resistance is supported by four key observations: (1) Overexpression of D7r4 in resist- ant mosquitoes from both Nagongera and Kihihi (2) Overexpression of D7 genes in mosquitoes collected from Nagongera both pre- and post-IRS intervention (3) Association between a D7-adjacent SNP and both D7 overexpression and bendiocarb resistance (4) The ability of the D7r4 protein structure (and probably D7r2 too) to accommodate bendiocarb in the central binding pocket, in a fashion that resembles that observed experimentally for the chemically similar serotonin. In silico modelling Th l The crystal structure of D7r4 protein and a modelled structure of D7r2 were used to assess the possibility of direct binding of bendiocarb to these proteins. Having noted a strong chemical structural similarity between bendiocarb and serotonin, as visualised in complex with D7r4 [15], we confirmed that bendiocarb could be manually positioned in the D7r4 pocket in a similar binding conformation as serotonin, where it was well ac- commodated with minimal steric clashes. In order to strengthen this hypothesis more objectively, computa- tional small molecule docking was done at the ROSIE server [16, 17], which indeed predicted a conformation similar to that derived manually (Fig. 4). Although D7r2 and D7r4 are relatively distantly related, sharing only 32% sequence identity, they align with only a single one residue insertion in the former vs the latter. This, along with the conservation of most cavity-lining residues, ensures a comparatively reliable prediction of the cavity shape and size in D7r2, although not of the quality desirable for automated docking methods. Interestingly, D7r2 is predicted to have a distinctly larger cavity, the dif- ference arising principally from the replacement of two residues, Phe 110 and Leu 43 in D7r4, with the smaller Val in both cases (Additional file 4). Isaacs et al. BMC Genomics (2018) 19:225 Page 7 of 12 Table 2 Whole-genome microarray studies in which D7 expression was associated with insecticide resistance Mosquito Species Country of collection Insecticide D7 gene Reference An. funestus Malawi Bendiocarb, Permethrin D7r1, D7r2a, D7r3b [48] An. funestus Mozambique, Malawi Permethrin D7r4 [49] An. funestus Zambia Deltamethrin D7r1 [18] An. funestus Zambia Etofenprox, Lambda-cyhalothrin D7r2, long form D7c [18] An. funestus Senegal Lambda-cyhalothrin D7r1 [49] An. coluzzi Burkina Faso, Cote d’Ivoire Deltamethrin D7L2 [19] An. gambiae s.s. Zambia Deltamethrin D7r1, D7r2, D7r3, D7r5, D7L2 [18] An. arabiensis Sudan Permethrin D7r2, D7r4 [50] An. arabiensis Uganda Permethrin D7r2, D7r4, D7L1 [20] An. arabiensis Zanzibar Lambda-cyhalothrin D7r4 [51] aD7r2 overexpressed in bendiocarb-resistant vs. unexposed control, overexpressed in pyrethroid-resistant vs. unexposed control, and underexpressed in unexposed control vs. susceptible colony bD7r3 overexpressed in bendiocarb-resistant vs. unexposed control, underexpressed in unexposed control vs. susceptible colony cD7r2 and long form D7 underexpressed in resistant vs. susceptible colony D7r2 overexpressed in bendiocarb-resistant vs. unexposed control, overexpressed in pyrethroid-resistant vs. unexposed control, an control vs. susceptible colony bD7r3 overexpressed in bendiocarb-resistant vs. unexposed control, underexpressed in unexposed control vs. Discussion BMC Genomics (2018) 19:225 be needed to determine whether the apparently non- coding transcript downstream of the short D7 cassette regulates expression of these genes. Our ability to test for an association between this SNP and bendiocarb re- sistance was limited by the fact that unexposed controls contain a mixture of resistant and susceptible individuals. However, the increase in SNP frequency among resistant mosquitoes following implementation of bendiocarb IRS supports our hypothesis that this SNP is associated with both D7 overexpression and bendiocarb resistance. The 3R:8564156 SNP was found in approximately half of the D7-overexpressing individuals tested, indicating that other SNPs conferring this phenotype remain to be discovered. Additional putative candidate resistance polymor- phisms within this locus are being investigated using the Anopheles gambiae 1000 genomes dataset available for this region [23]. Preliminary analyses have detected no selective sweeps in the region of the D7 genes, con- sistent with the hypothesis that multiple SNPs could con- fer D7-mediated insecticide resistance. Such resistance- associated SNPs could enable surveillance of mosquito populations and testing of archival DNA samples to meas- ure the spread of this resistance mechanism. hydrophobic substances within its large hydrophobic cavity [27]. The crystal structure of D7r4 revealed that its hydrophobic binding pocket has polar or charged side chains that can form hydrogen bonds with ligand functional groups in varied arrangements, resulting in a broad ligand specificity [15]. Isothermal titration calori- metry, which has been used to measure binding of D7 protein binding to biogenic amines, may be a suitable method for testing the ability of these proteins to bind bendiocarb and pyrethroids [10]. Silencing the expression of D7 proteins yielded only moderate decreases in D7 transcript, which could be due to both transcript abundance and the previously- documented inefficiency of RNAi in mosquito salivary glands [28, 29]. Overexpression of D7r2 and D7r4 from a transgene will provide a more robust tool for investi- gating the role of these proteins in insecticide resistance. In particular, this method would allow us to test the pro- moter activity of putative D7 regulatory sequences, and test whether the tissue-specificity of D7 expression im- pacts insecticide resistance. Whether D7 proteins are directly responsible for in- secticide resistance or simply closely-associated markers, their role in mosquito blood-feeding makes this overex- pression phenotype relevant to malaria transmission. Discussion Real-time quantitative PCR analyses of mosquitoes col- lected post-IRS detected instances of extremely high levels of D7r2 and D7r4 expression. D7 proteins are esti- mated to compose at least 5–20% of salivary protein [10], thus resistant mosquitoes with even modest levels of overexpression would be expected to have large quan- tities of these proteins in their saliva. Increased D7 ex- pression may alter the efficiency of blood-feeding, impacting the likelihood of parasite transmission. Previ- ously, knockdown of another D7 gene, D7L2, was shown to decrease blood feeding capacity and increase probing time in An. gambiae mosquitoes [29]. Furthermore, it was recently reported that a genetically modified D7 knock out Aedes aegypti mosquito was significantly less susceptible to an avian malaria parasite, Plasmodium gallinaceum [30]. Exploration of blood feeding in D7- overexpressing transgenic mosquitoes will allow for a more precise measurement of feeding success, blood meal size, and probing time. Blood-feeding behaviour in resistant mosquitoes is an important characteristic to measure, as it impacts both their reproductive success and vector competence. p Despite repeated appearances among the top micro- array hits for genes overexpressed in resistant mosqui- toes, the D7 protein family has never been functionally validated as an insecticide resistance candidate. The crystal structure of D7r4 revealed that the short form D7 proteins are structurally related to arthropod odorant- binding proteins, and exhibit a single binding site [15]. In a recent study, a D7 protein from the Aedes aegypti mos- quito was observed to bind dengue virions and envelope protein, suggesting that this family of proteins may have a broader range of associated phenotypes than previously thought [24]. We hypothesize that if D7 proteins at least partially confer insecticide resistance, they are more likely to do so by binding and sequestering insecticide or insecticide metabolites rather than by any direct de- toxification. This hypothesis is strongly supported by the structural compatibility of the D7r4 protein’s cen- tral binding pocket and bendiocarb. The D7r2 protein is likely to have a larger and differently shaped pocket, potentially suggesting that it has an overlapping but distinct specificity for bound ligands. Preliminary dock- ing experiments suggest that the larger pocket found in D7r2 may permit binding of pyrethroids such as permeth- rin, however there is insufficient structural information for the D7r2 protein to make strong conclusions. Discussion Recent campaigns to control malaria transmission in Uganda through a combination of LLIN distribution and IRS have been highly effective in decreasing malaria metrics, however our sampling of mosquito larval popu- lations indicates that a reservoir of bendiocarb-resistant An. gambiae s.s. persists [7]. Measures of human biting rate decreased significantly after initiation of the IRS programme in Nagongera, indicating that mosquito pop- ulations were suppressed [7]. Furthermore, surveillance using CDC light traps did not detect a change in the relative abundance of mosquito species. In contrast, our sampling of larval pools revealed a decrease in the fre- quency of An. gambiae s.s. mosquitoes following IRS, suggesting that this species was disproportionately af- fected, perhaps due to it being more anthrophophilic than An. arabiensis [21]. p y y We hypothesize that D7 overexpression is symptomatic of a disruption in the tissue-specificity of D7 expression which allows these proteins to interact with insecticides in tissues other than the salivary glands. While expression of D7 genes has primarily been associated with the salivary glands, lower expression levels have been detected in tissues such as the malpighian tubules [22]. Our sequencing of the D7r2/D7r4 intergenic region, which has previously been shown to direct salivary gland-specific expression [13], revealed high sequence diversity. In future, we will meas- ure D7 expression in the dissected tissues of bendiocarb- resistant mosquitoes to establish whether mutations that confer overexpression also alter tissue-specificity. It is likely that several different mutations circulate within this population of An. gambiae s.s. and collectively confer bendiocarb resistance, however one resistance- associated phenotype that was observed both before and after IRS deployment is overexpression of genes encoding the D7r2 and D7r4 salivary gland proteins. The overex- pression of apyrase, GSG7, gSG1a, and CPLCX2, detected in Nagongera in 2014 but not in 2015, may have associ- ated fitness costs that caused these phenotypes to be eliminated from the local mosquito population, or may Importantly, the identification of a D7 overexpression- associated SNP provides evidence that the high level of D7 expression observed in resistant mosquitoes is not simply a reaction to insecticide exposure, but is associ- ated with an identifiable genotype. Further studies will Isaacs et al. BMC Genomics (2018) 19:225 Page 8 of 12 Page 8 of 12 Isaacs et al. BMC Genomics (2018) 19:225 Isaacs et al. Discussion Such promiscuous binding of insecticides has precedent in cytochrome P450 enzymes such as CYP6M2, which me- tabolizes type I and II pyrethroids as well at DDT [25, 26]. Furthermore, the ability to bind structurally diverse molecules has been demonstrated in proteins such as the bacterial chaperonin GroEL, which binds various Conclusions The replicated association between expression of D7 genes and bendiocarb resistance across multiple geo- graphic locations and multiple collection years supports the hypothesis that D7 proteins confer insecticide resist- ance. Further study of the function of these proteins in Isaacs et al. BMC Genomics (2018) 19:225 Page 9 of 12 Page 9 of 12 [25, 35]. Normalization of microarray data was performed using the Limma package within R software [36]. Analysis of signal intensities was then completed using the MAA- NOVA package [37]. Four of the forty hybridizations (Kihihi resistant #3 cy5/Kihihi unexposed #3 cy3, Kisumu #3 cy5/Nagongera resistant #4 cy3, Nagongera control #1 cy5/Kihihi resistant #2 cy3, Nagongera control #3 cy5/ Kihihi resistant #4 cy3) were excluded from the analysis due to the poor quality of the hybridizations, which yielded aberrant Cy3/Cy5 spectra. Full microarray results are presented in Additional file 6. The results of these ana- lyses were then filtered to identify probes that: 1. Dis- played statistical significance (P value < 0.05) in an ANOVA F-test of the resistant vs. unexposed individuals comparison; 2. Displayed a log2 fold change greater than 0.3 in the resistant vs. unexposed individuals comparison; 3. Displayed the greatest fold change in the resistant vs. Kisumu individuals comparison (relative to resistant vs. unexposed and unexposed vs. Kisumu comparisons). All microarray data have been deposited in ArrayExpress (accession no. E-MTAB-6280). insecticide resistance, and the impact of their overexpres- sion on mosquito blood-feeding behaviour is needed. This study demonstrates the value of a whole-genome ap- proach to detecting insecticide resistance, as it identified genes that have no known detoxification function and have never before been studied for their role in insecticide resistance. Screening for novel insecticide resistance can- didates is necessary, as even the most well-characterized resistance mutations explain only a small fraction of a re- sistance phenotype [31]. Continued surveillance of insecticide resistance and investigation of the underlying mechanisms are a key component of vector control in malaria-endemic coun- tries. The recent success of the IRS campaign in Nagongera underscores the value of insecticides in mal- aria control and the importance of preserving their efficacy. Ace1 TaqMan DNA extracted from individual legs was used as tem- plate for a TaqMan assay to detect the G119S mutation in the Ace1 gene [38]. Each 10 μl reaction contained 1× Sensimix (Bioline), 1× primer/probe mix and 1 μl tem- plate DNA. An Agilent MX3005p real-time PCR ma- chine was used to run the following program: 95 °C for 10 min followed by 40 cycles of 95 °C for 10 s and 60 °C for 45 s. Genotypes were called from endpoint HEX and FAM fluorescence using MxPro software. Mosquito collection and insecticide resistance phenotyping WHO tube bioassays were performed in January, March, and May 2015, as described previously [32]. Briefly, mos- quito larvae were collected from various breeding sites using the dipping method, then transferred to an insect- ary in Nagongera for rearing. Adult mosquitoes were fed on a 10% sugar solution and identified as belonging to the An. gambiae species complex using morphological keys. Female mosquitoes were exposed to 0.1% bendio- carb or control papers according to standard WHO tube bioassay protocols [33]. Real-time quantitative PCR For all bendiocarb-resistant and unexposed mosquito samples, chelex-extracted DNA from individual legs was used as template for a species identification PCR [34]. Mosquitoes from the Kisumu laboratory strain were in- cluded as fully susceptible control samples. RNA was extracted from pools of five An. gambiae s.s. females using an RNAqueous®-4PCR Total RNA Isolation Kit (Ambion) according to the manufacturer’s protocol. For each sample type (Nagongera resistant and unexposed, Kihihi resistant and unexposed, Kisumu), four RNA pools were extracted. A Nanodrop spectrophotometer (Nanodrop Technologies) and an Agilent 2100 Bioanaly- ser (Agilent Technologies) were used to measure the quality and quantity of RNA samples. RNA labelling, as well as array hybridization, washing, scanning, and fea- ture extraction were performed as previously described [25]. Five 8 × 15 K Agilent whole-transcriptome An. gambiae microarray chips (A-MEXP-2196) were used in an interwoven loop design to compare four biological rep- licates each of resistant, unexposed, and laboratory mos- quitoes with maximal statistical power (Additional file 5) The expression of nine genes was evaluated using qPCR. Following species identification, RNA was extracted from individual females collected in January, March, and May 2015 using a Zymo Quick-RNA MiniPrep kit (Zymo Research) according to the manufacturer’s protocol. Invitrogen SuperScript III Reverse Transcriptase (Invi- trogen) and 200 nanograms of template RNA were used to perform cDNA synthesis reactions. Following cDNA purification with a QIAquick PCR Purification Kit (QIA- GEN), triplicate qPCR reactions were prepared using SYBR Brilliant III (Agilent Technologies) and 300 nM each primer (Additional file 7). Primers were designed using the Primer-BLAST tool (http://www.ncbi.nlm.nih. gov/tools/primer-blast/) and evaluated for their effi- ciency over a ten-fold serial dilution. Primer efficiencies ranged between 89 and 110%, and were included in cal- culations of fold change. Amplification was performed and analysed using an Agilent Mx3005P QPCR System. The thermal profile was as follows: 1 cycle 95C for 3 min, 40 cycles of 95C for 10 s followed by 60C for Isaacs et al. BMC Genomics (2018) 19:225 Page 10 of 12 Page 10 of 12 amplified and cloned as described as above. Assem- bled sequences of PCR amplification products are presented in Additional file 3. 10 s. Gene expression relative to two housekeeping genes (rpS7 and GDPH) was calculated using the REST 2009 software tool [39]. Using this tool, the transcript abundance in each sample was calculated relative to the group of unexposed control samples. Real-time quantitative PCR p Leg DNA from individual females collected from Nagongera in 2014 was used as template for amplifica- tion of the D7r2 and D7r4 coding sequences. The D7r4 coding region of four resistant and four unexposed fe- males was amplified using the following primers: D7r4seqF (5’ GTAATTCTGAAGATCAAGGTGTG 3′) and D7r4seqR (5’ CGTGCCTTTGAACGCTACAT 3′) or D7r4seqF2 (5’ AGGTAATGGATCATGAAGTAAG TCT 3′) and D7r4seqR2 (5’ TGAACGCTACATCT GTTTTCA 3′). Additional sequences the D7r2/D7r4 intergenic region (described above) was used to assem- ble partial or full coding sequences. The D7r2 coding region of three resistant females was amplified using the following primers: D7r2seqF (5’ TCGCAGTATAAAAGG CAGTATCT 3′) and D7r2seqR (5’ TGCATCATTGT TCCTTTTGCT 3′). PCR products were amplified and cloned as described as above. Assembled sequences of PCR amplification products are presented in Additional file 1. RNAi-induced gene silencing g g Double-stranded RNAs targeting the D7r2 and D7r4 genes was synthesized using an Applied Biosystems MEGAscript RNAi kit according to the manufacturer’s protocol. The template for dsD7r2 and dsD7r4 synthesis was PCR-amplified from a mixed sample of Nagongera mosquito cDNA, then cloned using a Thermo scientific CloneJET PCR cloning kit. Kisumu strain mosquitoes age 2–3 days were drawn haphazardly from a colony cage. A Drummond nano-injector was used to inject 101 nL of solution containing 500 nanograms dsD7r2, 1 microgram dsD7r4, or an equivalent amount of dsGFP into the thorax of cold-anaesthetized mosquitoes. The first day post-injection, five males from the colony cage were added to each cage to provide an additional oppor- tunity for mating. Five females were removed from each cage at 24, 48, 72, and 96 h post-injection for RNA ex- traction. The sequences of primers used in dsRNA syn- thesis and qPCR analyses of knockdown individuals are listed in Additional file 7. Expression of D7r1–4 was evaluated using RNA extracted from pools of 5 carcasses. D7 SNP genotyping A real-time quantitative PCR using a locked nucleic acid probe was designed for genotyping individuals for the 3R:8564156 C/T SNP located in Contig 709. The PCR primers TM709F (5’ GCCCGCGTAATAGGGATTATGT 3′) and TM709R (5’ TTTTTCGCTGGGAAGTTGGTC 3′) were used in combination with the following probes: 709Alt (5’ CGCC+A + T + GGAGA 3′) labelled with FAM, 709Ref (5’ CGCC+A + C + GGAGA) labelled with HEX. Each 10 μl reaction contained 1× Sensimix (Bioline), 0.5 μM each primer, 0.5 μM each probe, and 1 μl template DNA. An Agilent MX3005p real-time PCR machine was used to run the following program: 95 °C for 10 min followed by 40 cycles of 92 °C for 15 s and 57 °C for 1 min. Genotypes were called from endpoint HEX and FAM fluorescence using MxPro software. A real-time quantitative PCR using a locked nucleic acid probe was designed for genotyping individuals for the 3R:8564156 C/T SNP located in Contig 709. The PCR primers TM709F (5’ GCCCGCGTAATAGGGATTATGT 3′) and TM709R (5’ TTTTTCGCTGGGAAGTTGGTC 3′) were used in combination with the following probes: 709Alt (5’ CGCC+A + T + GGAGA 3′) labelled with FAM, 709Ref (5’ CGCC+A + C + GGAGA) labelled with HEX. Each 10 μl reaction contained 1× Sensimix (Bioline), 0.5 μM each primer, 0.5 μM each probe, and 1 μl template DNA. An Agilent MX3005p real-time PCR machine was used to run the following program: 95 °C for 10 min followed by 40 cycles of 92 °C for 15 s and 57 °C for 1 min. Genotypes were called from endpoint HEX and FAM fluorescence using MxPro software. Sequencing of D7-coding and adjacent genomic regions Leg DNA from individual females collected in March and May 2015 was used as template for amplification of the D7r2/D7r4 intergenic region. Forward (5’ GCTACT GAAGGCTGGCAAGA 3′) and reverse (5’ CGGATC TCGCACAGTCTACT 3′) primers were designed to bind within the D7r2 and D7r4 coding sequences, respect- ively. A high-fidelity Taq polymerase, either Thermo Scientific Phusion Hot Start II High-Fidelity DNA poly- merase or NEB Q5 High-Fidelity DNA polymerase, was used according to the manufacturer’s protocol. A Promega Wizard SV gel and PCR clean-up system was used to pur- ify individual PCR amplification products. A Thermo Scientific CloneJET PCR cloning kit was used to clone se- lect PCR amplification products prior to sequencing. As- sembled sequences of PCR amplification products are presented in Additional file 2. Statistical analyses GraphPad Prism version 5.04 for Windows statistical software (GraphPad Software) was used to perform Fisher’s exact, Mann-Whitney and Spearman tests for statistical significance. The lower and limits of the 95% confidence interval for a proportion were calculated using VassarStats (www.vassarstats.net). In silico methods Structure modelling of the D7r2 protein was carried out using the crystal structure of D7r4 protein bound to serotonin (PDB code 2qeh; [15]) as a template. HHpred [40, 41] was used to align the target and template, which shared 32% sequence identity, producing an alignment covering residues 3–146 of the D7r2 protein. MODEL- LER [42] was used to generate 40 models from this alignment and DOPE scores [43] used to select the best model. Leg DNA from individual females collected from Nagongera in 2015 was used as template for amplifica- tion of a region downstream of the short D7 cassette. The genomic region encoding a non-coding transcript, identified by Arca et al. as “Contig 709”, was amplified using the following primers: 709F (5’ ACCTATCCAT CAGTTCCACCAC 3′) and 709R (5’ CAGGCCTAAT CTGTGGCAGT 3′) [12]. PCR products were PyMOL (pymol.org) was used for manual positioning of bendiocarb into D7 protein structures. Automated small molecule docking was then done at the ROSIE/ Rosetta [16, 17] server. PDBSUM [44] was used to ob- tain a precalculated definition of the cavity shape and Page 11 of 12 Page 11 of 12 Isaacs et al. BMC Genomics (2018) 19:225 volume of the D7r4 crystal structure, and ProFunc [45] used for similar calculations on model structures. volume of the D7r4 crystal structure, and ProFunc [45] used for similar calculations on model structures. Funding Meta-analysis of Anopheles genome-wide microarray data To identify publications containing microarray data col- lected from insecticide-resistant Anopheles mosquitoes, a search was performed on the PubMed website (date of search: December 4, 2016) with the following search terms: Anopheles AND insecticide resistance AND microarray. These 32 results were filtered to select publications in which genome-wide microarrays were performed on whole female mosquitoes that had been selected with insecticides. Three microarrays were used in these publications: the 4 × 44 k (ArrayExpress accession number A-MEXP-2245) and the 8 × 60 k (A-MEXP-2374) microarrays were used to assay An. funestus mosquitoes, and the 8 × 15 k (A-MEXP- 2196) microarray was used to assay An. gambiae s.s. and An. arabiensis mosquitoes [25, 46, 47]. Additional searches on Google Scholar using these microarray identification numbers yielded three publications not listed in the PubMed query. A list of the 35 PubMed and Google Scholar search results is provided in Additional file 8. This work was supported by the National Institute of Allergy and Infectious Diseases at the National Institutes of Health (U19AI089674). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Acknowledgements We thank Dr. Simon Jochems for guidance in writing sequence analysis R scripts, and for critical revision of the manuscript. Author details 1 f 1Department of Vector Biology, Liverpool School of Tropical Medicine, Liverpool, UK. 2Infectious Diseases Research Collaboration, Kampala, Uganda. 3Institute of Integrative Biology, University of Liverpool, Liverpool, UK. 4Malaria Programme, Wellcome Trust Sanger Institute, Cambridge, UK. 4Malaria Programme, Wellcome Trust Sanger Institute, Cambridge, UK. Additional files Received: 6 November 2017 Accepted: 14 March 2018 Received: 6 November 2017 Accepted: 14 March 2018 Additional file 1: Partial and complete D7r2 and D7r4 coding sequences, from Nagongera mosquitoes. (DOCX 12 kb) Additional file 2: FASTA sequences of amplified D7r2/D7r4 intergenic regions, from Nagongera mosquitoes. (DOCX 22 kb) Additional file 3: FASTA sequences of region encoding Contig 709 transcript, from Nagongera mosquitoes. (DOCX 20 kb) Additional file 4: Comparison of the cavity binding serotonin in the D7r4 crystal structure (PDB code 2qeh; [15]) and the larger cavity predicted for modelled D7r2, largely due to the replacement of Phe110 and Leu43 with Val residues. The figure was made with PyMOL (pymol.org). (PNG 1766 kb) Additional file 5: Diagram of hybridizations performed in the microarray analysis. (PDF 117 kb) Additional file 6: Complete microarray results. (XLSX 7082 kb) Additional file 7: Sequences of primers used in qPCR analyses and dsRNA synthesis. (XLSX 11 kb) Additional file 8: Articles considered in the meta-analysis of Anopheles insecticide resistance microarrays. (DOCX 20 kb) Additional file 1: Partial and complete D7r2 and D7r4 coding sequences, from Nagongera mosquitoes. (DOCX 12 kb) Additional file 1: Partial and complete D7r2 and D7r4 coding sequences, from Nagongera mosquitoes. (DOCX 12 kb) 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. Availability of data and materials All data generated or analysed during this study are included in this published article and its supplementary information files. Competing interests Th h d l h Competing interests The authors declare that they have no competing interests. Consent for publication Not applicable Consent for publication Not applicable Competing interests The authors declare that they have no competing interests. References Additional file 2: FASTA sequences of amplified D7r2/D7r4 intergenic regions, from Nagongera mosquitoes. (DOCX 22 kb) 1. Bhatt S, Weiss DJ, Cameron E, Bisanzio D, Mappin B, Dalrymple U, Battle KE, Moyes CL, Henry A, Eckhoff PA, et al. The effect of malaria control on Plasmodium falciparum in Africa between 2000 and 2015. Nature. 2015; 526(7572):207–11. 1. Bhatt S, Weiss DJ, Cameron E, Bisanzio D, Mappin B, Dalrymple U, Battle KE, Moyes CL, Henry A, Eckhoff PA, et al. The effect of malaria control on Plasmodium falciparum in Africa between 2000 and 2015. Nature. 2015; 526(7572):207–11. Additional file 3: FASTA sequences of region encoding Contig 709 transcript, from Nagongera mosquitoes. (DOCX 20 kb) 2. Ranson H, Lissenden N. Insecticide resistance in African Anopheles mosquitoes: a worsening situation that needs urgent action to maintain malaria control. Trends Parasitol. 2016;32(3):187–96. 3. Matowo J, Kitau J, Kaaya R, Kavishe R, Wright A, Kisinza W, Kleinschmidt I, Mosha F, Rowland M, Protopopoff N. Trends in the selection of insecticide resistance in Anopheles gambiae s.L. mosquitoes in Northwest Tanzania during a community randomized trial of longlasting insecticidal nets and indoor residual spraying. 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ST contributed to RNAi-induced knockdown experiments. DR designed and carried out in silico modelling experiments. MD conceived of the study, participated in its design and coordination, and helped to draft the manuscript. All authors read and approved the final manuscript. Ethics approval and consent to participate Not applicable Ethics approval and consent to participate Not applicable 7. Katureebe A, Zinszer K, Arinaitwe E, Rek J, Kakande E, Charland K, Kigozi R, Kilama M, Nankabirwa J, Yeka A, et al. Measures of malaria burden after long- lasting insecticidal net distribution and indoor residual spraying at three sites in Uganda: a prospective observational study. PLoS Med. 2016;13(11):e1002167. Abbreviations f CDC: Center for disease control; dsRNA: Double-stranded RNA; ESP: Epithelial serine protease; IRS: Indoor residual spraying; LLINs: Long-lasting insecticidal nets; qPCR: Real-time quantitative PCR; SNP: Single nucleotide polymorphism; WHO: World Health Organization 7. 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https://openalex.org/W3173819558	https://westminsterresearch.westminster.ac.uk/download/970c591df87d4cfbdb0b9ac77cfec1210795c03af87b31468d0f4c4dd1f7c032/14974030/s41588-021-00891-2.pdf	English	null	Reconstructing single-cell karyotype alterations in colorectal cancer identifies punctuated and gradual diversification patterns	Nature genetics	2,021	cc-by	20,550	1Molecular Cancer Research, Center for Molecular Medicine, University Medical Center Utrecht, Utrecht University, Utrecht, the Netherlands. 2Oncode Institute, Utrecht, the Netherlands. 3Medical Cell Biophysics, TechMed Centre, University of Twente, Enschede, the Netherlands. 4Department of Genetics, Center for Molecular Medicine, University Medical Center Utrecht, Utrecht University, Utrecht, the Netherlands. 5Department of Cell and Developmental Biology, University College London, London, UK. 6UCL Genetics Institute, University College London, London, UK. 7Hubrecht Institute, KNAW, Utrecht, the Netherlands. 8University Medical Center Utrecht, Utrecht, the Netherlands. 9Centre for Genomics and Computational Biology, Barts Cancer Institute, Barts and the London School of Medicine and Dentistry, Queen Mary University of London, London, UK. 10UCL Cancer Institute, UCL, London, UK. 11Hartwig Medical Foundation, Amsterdam, the Netherlands. 12Evolutionary Genomics and Modelling Lab, Centre for Evolution and Cancer, The Institute of Cancer Research, London, UK. 13These authors contributed equally: Yannik Bollen, Ellen Stelloo. ✉e-mail: h.j.g.snippert@umcutrecht.nl Reconstructing single-cell karyotype alterations in colorectal cancer identifies punctuated and gradual diversification patterns Crucially, PDTO models are the closest representatives of human tumors that are compatible with high-spatiotemporal-resolution imaging of cells, enabling live microscopy of clonal tumor outgrowth. Patient-derived tumor organoids (PDTOs) offer a unique opportunity to address these fundamental questions. PDTOs are three-dimensional (3D) mini-organs derived from primary tumor tissue. PDTOs maintain the histopathological features of the native tumor, including high concordance in somatic mutations and tran- scriptome and drug response profiles15–17. Crucially, PDTO models are the closest representatives of human tumors that are compatible with high-spatiotemporal-resolution imaging of cells, enabling live microscopy of clonal tumor outgrowth. How an aneuploid tumor genome evolves over time remains incompletely resolved. Although single-cell sequencing from patient samples captures genetic diversity in human tumors4,5, the evolutionary history of an aneuploid genome is difficult to deter- mine, as the two critical parameters that fuel its progression—ongo- ing chromosomal instability (CIN) and selection pressures—are dynamically intertwined3,6–8. Consequently, general models of tumor evolution are still under debate, in part because the karyotype alter- ation rate and the patterns by which de novo karyotypes emerge and propagate within human tumors are not well understood6,8,9. These parameters in particular are difficult to extract from patient mate- rial since it provides limited to no information on genetic interme- diates and the timescales that separate individual tumor subclones (the number of cell generations). For instance, punctuated bursts of Reconstructing single-cell karyotype alterations in colorectal cancer identifies punctuated and gradual diversification patterns Yannik Bollen 1,2,3,13, Ellen Stelloo2,4,13, Petra van Leenen 1,2, Myrna van den Bos 1,2, Bas Ponsioen1,2, Bingxin Lu 5,6, Markus J. van Roosmalen4, Ana C. F. Bolhaqueiro2,7,8, Christopher Kimberley9, Maximilian Mossner 9, William C. H. Cross9,10, Nicolle J. M. Besselink 2,4, Bastiaan van der Roest 2,4, Sander Boymans 2,4, Koen C. Oost1,2, Sippe G. de Vries1,2, Holger Rehmann1, Edwin Cuppen2,4,11, Susanne M. A. Lens1,2, Geert J. P. L. Kops 2,7,8, Wigard P. Kloosterman4, Leon W. M. M. Terstappen 3, Chris P. Barnes 5,6, Andrea Sottoriva 12, Trevor A. Graham 9 and Hugo J. G. Snippert 1,2 ✉ Central to tumor evolution is the generation of genetic diversity. However, the extent and patterns by which de novo karyotype alterations emerge and propagate within human tumors are not well understood, especially at single-cell resolution. Here, we present 3D Live-Seq—a protocol that integrates live-cell imaging of tumor organoid outgrowth and whole-genome sequencing of each imaged cell to reconstruct evolving tumor cell karyotypes across consecutive cell generations. Using patient-derived colorectal cancer organoids and fresh tumor biopsies, we demonstrate that karyotype alterations of varying complexity are prevalent and can arise within a few cell generations. Sub-chromosomal acentric fragments were prone to replication and col- lective missegregation across consecutive cell divisions. In contrast, gross genome-wide karyotype alterations were gener- ated in a single erroneous cell division, providing support that aneuploid tumor genomes can evolve via punctuated evolution. Mapping the temporal dynamics and patterns of karyotype diversification in cancer enables reconstructions of evolutionary paths to malignant fitness. A Th A neuploidy—defined as a cell having an abnormal num- ber of chromosomes or sub-chromosomal fragments—is among the most common features of human cancers1. The prevalence of aneuploidy and the detection of recurrent copy-number alterations (CNAs) across cancer types indicate that genome-wide aneuploidy plays an active role in promoting malig- nant phenotypes2,3. karyotype alterations in human cancers have been described10–13, but the exact timescale and prevalence of such events remain elusive14. karyotype alterations in human cancers have been described10–13, but the exact timescale and prevalence of such events remain elusive14. Patient-derived tumor organoids (PDTOs) offer a unique opportunity to address these fundamental questions. PDTOs are three-dimensional (3D) mini-organs derived from primary tumor tissue. PDTOs maintain the histopathological features of the native tumor, including high concordance in somatic mutations and tran- scriptome and drug response profiles15–17. R Reconstructing single-cell karyotype alterations in colorectal cancer identifies punctuated and gradual diversification patterns Nature Genetics \| VOL 53 \| August 2021 \| 1187–1195 \| www.nature.com/naturegenetics Results Sub-chromosomal CNAs were counted as events when represented in more than one cell. Parallel emergence of de novo CNAs involving chromosome 1q (lineages I and II) was determined by co-occurrence of a sub-chromosomal CNA in chromosome 18. Scale bar, 10 μm. c, As in b. The sequencing data of 58 PDTO-19b cells are shown (87% recovery). A total of 16 cells showed de novo CNAs. The bottom panel shows a population of polyploid cells with large deviations from the core karyotype. Two cells show reciprocal gains and losses across their genome (lineage III). Scale bar, 10 μm. d, Graph indicating the number of CNA events per dataset (eight datasets). The dataset size is indicated within each circle. Events include reciprocal CNAs, non-reciprocal whole-chromosome gains or losses and non-reciprocal sub-chromosomal CNAs represented in more than one cell. Hopeful monster karyotypes were excluded from this analysis. CNAs that occurred in the same cell division were considered as one event. Fig. 1 \| Chromosomal CNAs emerge and propagate during clonal PDTO outgrowth. a, Schematic of the procedure to pe stable) and PDTO-19b (microsatellite unstable)15. Sequencing data from both PDTO lines displayed de novo whole-chromosome as well as sub-chromosomal CNAs among cells (Fig. 1b,c and Extended Data Figs. 1 and 2), in agreement with our previous study showing that both PDTOs are in CIN7. Capturing the entire popula- tion of cells from individual clonal PDTOs has the critical advan- tage of detecting reciprocal copy-number gains and losses between cells, indicating that the two lineages are progeny of the same ances- tral CNA event (Fig. 1b,c, red boxes; >1 cell with the same CNA indicates propagation). Across datasets, CNA events occurred at a rate of approximately one in ten cell divisions for both PDTO-9 and PDTO-19b (Fig. 1d). Notably, we identified instances where identical CNAs emerged from independent events within a single PDTO-9 organoid, which were discriminated by the co-occurrence of additional CNAs that prohibited the assembly of a coherent single phylogeny (Fig. 1b (lineages I and II) and Extended Data Fig. 1b (lineages I and II)). Indeed, we frequently detected cell divisions where multiple CNAs arose simultaneously. Extreme cases were represented by a subset of PDTO-19b cells with highly aberrant karyotypes that could be classified as hopeful monsters18,19, defined as a genome-wide set of changes in ploidy that are likely to substan- tially alter fitness. Results The sequencing data of 58 PDTO-19b cells are shown (87% recovery). A total of 16 cells showed de novo CNAs. The bottom panel shows a population of polyploid cells with large deviations from the core karyotype. Two cells show reciprocal gains and losses across their genome (lineage III). Scale bar, 10 μm. d, Graph indicating the number of CNA events per dataset (eight datasets). The dataset size is indicated within each circle. Events include reciprocal CNAs, non-reciprocal whole-chromosome gains or losses and non-reciprocal sub-chromosomal CNAs represented in more than one cell. Hopeful monster karyotypes were excluded from this analysis. CNAs that occurred in the same cell division were considered as one event. a b 1 2 3 4 5 6 7 8 9 10 11 12 14 13 15 17 16 18 19 20 2122 X I II Core (44 cells) 5N 6N 7N+ 2N 1N 3N 4N PDTO-19b 58/67 cells (87%) Clonal outgrowth Tumor organoid dissociation Single-cell picking 1 2 3 4 5 6 7 8 9 10 11 12 14 13 15 17 16 18 19 20 2122 X Number of CNA events 62/67 cells (93%) PDTO-9 c 2 4 PDTO-9 PDTO-19b 2N 1N 3N 4N 5N CNAs (18 cells) Core (32 cells) CNAs (16 cells) Polyploid (10 cells) 2N 1N 3N 4N 6N 0 6 8 10 12 14 16 82 62 64 33 58 40 52 21 III d Chromosome Chromosome b b 4 5 6 7 8 9 10 11 12 14 13 15 17 16 18 19 20 2122 X 62/67 cells (93%) PDTO-9 Chromosome PDTO-19b 58/67 cells (87%) 5 6 7 8 9 10 11 12 14 13 15 17 16 18 19 20 2122 X III Chromosome Polyploid (10 cells) Fig. 1 \| Chromosomal CNAs emerge and propagate during clonal PDTO outgrowth. a, Schematic of the procedure to perform single-cell whole-genome sequencing of the entire cell population from individual organoids. Clonal PDTO structures are mechanically dissociated into single cells before manual picking of individual cells for prospective genome-wide CNA analysis. b, Karyotype heatmap showing 62 cells derived from a clonal PDTO-9 structure expressing transgenic H2B-Dendra2 and consisting of 67 cells (93% recovery). A total of 18 cells showed de novo CNAs. Reciprocal gains and losses are indicated with solid red boxes. Dashed boxes indicate CNA events where a reciprocal loss or gain is missing. Results De novo CNAs emerge and propagate during PDTO outgrowth. To investigate the extent to which de novo chromosomal CNAs emerge and propagate during clonal outgrowth of a single tumor cell, we developed a protocol that allows single-cell whole-genome sequencing of the entire cell population derived from a single organoid (Fig. 1a). Using this protocol, we repeatedly sequenced a high fraction (~90%) of cells from clonal tumor organoids of two patients with colorectal cancer (CRC): PDTO-9 (microsatellite Nature Genetics \| VOL 53 \| August 2021 \| 1187–1195 \| www.nature.com/naturegenetics 1187 Nature Genetics \| VOL 53 \| August 2021 \| 1187–1195 ature Genetics \| VOL 53 \| August 2021 \| 1187–1195 \| www.nature.co Articles NatUrE GEnEtICS a b 1 2 3 4 5 6 7 8 9 10 11 12 14 13 15 17 16 18 19 20 2122 X I II Core (44 cells) 5N 6N 7N+ 2N 1N 3N 4N PDTO-19b 58/67 cells (87%) Clonal outgrowth Tumor organoid dissociation Single-cell picking 1 2 3 4 5 6 7 8 9 10 11 12 14 13 15 17 16 18 19 20 2122 X Number of CNA events 62/67 cells (93%) PDTO-9 c 2 4 PDTO-9 PDTO-19b 2N 1N 3N 4N 5N CNAs (18 cells) Core (32 cells) CNAs (16 cells) Polyploid (10 cells) 2N 1N 3N 4N 6N 0 6 8 10 12 14 16 82 62 64 33 58 40 52 21 III d Chromosome Chromosome Fig. 1 \| Chromosomal CNAs emerge and propagate during clonal PDTO outgrowth. a, Schematic of the procedure to perform single-cell whole-genome sequencing of the entire cell population from individual organoids. Clonal PDTO structures are mechanically dissociated into single cells before manual picking of individual cells for prospective genome-wide CNA analysis. b, Karyotype heatmap showing 62 cells derived from a clonal PDTO-9 structure expressing transgenic H2B-Dendra2 and consisting of 67 cells (93% recovery). A total of 18 cells showed de novo CNAs. Reciprocal gains and losses are indicated with solid red boxes. Dashed boxes indicate CNA events where a reciprocal loss or gain is missing. Sub-chromosomal CNAs were counted as events when represented in more than one cell. Parallel emergence of de novo CNAs involving chromosome 1q (lineages I and II) was determined by co-occurrence of a sub-chromosomal CNA in chromosome 18. Scale bar, 10 μm. c, As in b. The sequencing data of 58 PDTO-19b cells are shown (87% recovery). Nature Genetics \| VOL 53 \| August 2021 \| 1187–1195 \| www.nature.com/naturegenetics Results A total of 16 cells showed de novo CNAs. The bottom panel shows a population of polyploid cells with large deviations from the core karyotype. Two cells show reciprocal gains and losses across their genome (lineage III). Scale bar, 10 μm. d, Graph indicating the number of CNA events per dataset (eight datasets). The dataset size is indicated within each circle. Events include reciprocal CNAs, non-reciprocal whole-chromosome gains or losses and non-reciprocal sub-chromosomal CNAs represented in more than one cell. Hopeful monster karyotypes were excluded from this analysis. CNAs that occurred in the same cell division were considered as one event. Number of CNA events 2 4 PDTO-9 PDTO-19b 0 6 8 10 12 14 16 82 62 64 33 58 40 52 21 d a b 1 2 3 4 5 6 7 8 9 10 11 12 14 13 15 17 16 18 19 20 2122 X I II Core (44 cells) 5N 6N 7N+ 2N 1N 3N 4N PDTO-19b 58/67 cells (87%) Clonal outgrowth Tumor organoid dissociation Single-cell picking 1 2 3 4 5 6 7 8 9 10 11 12 14 13 15 17 16 18 19 20 2122 X Number of CNA events 62/67 cells (93%) PDTO-9 c 2 4 PDTO-9 PDTO-19b 2N 1N 3N 4N 5N CNAs (18 cells) Core (32 cells) CNAs (16 cells) Polyploid (10 cells) 2N 1N 3N 4N 6N 0 6 8 10 12 14 16 82 62 64 33 58 40 52 21 III d Chromosome Chromosome Fig. 1 \| Chromosomal CNAs emerge and propagate during clonal PDTO outgrowth. a, Schematic of the procedure to perform single-cell whole-genome sequencing of the entire cell population from individual organoids. Clonal PDTO structures are mechanically dissociated into single cells before manual picking of individual cells for prospective genome-wide CNA analysis. b, Karyotype heatmap showing 62 cells derived from a clonal PDTO-9 structure expressing transgenic H2B-Dendra2 and consisting of 67 cells (93% recovery). A total of 18 cells showed de novo CNAs. Reciprocal gains and losses are indicated with solid red boxes. Dashed boxes indicate CNA events where a reciprocal loss or gain is missing. Sub-chromosomal CNAs were counted as events when represented in more than one cell. Parallel emergence of de novo CNAs involving chromosome 1q (lineages I and II) was determined by co-occurrence of a sub-chromosomal CNA in chromosome 18. Scale bar, 10 μm. c, As in b. Results Two of these cells displayed extensive reciprocal CNAs across their genomes (Fig. 1c, lineage III), indicating that they were the progeny of a single catastrophic cell division. The sum of both karyotypes pointed to a genome-duplicated ancestor, in line with models suggesting that extreme aneuploid states often evolve from unstable tetraploid intermediates20 (Extended Data Fig. 3a). Collectively, these data indicate that de novo CNAs readily arise and propagate during PDTO outgrowth. Furthermore, we detected several instances of punctuated karyotype alterations that involved multiple chromosomes in a single-cell division, with hopeful mon- sters representing the most extreme cases. Integrating live-cell imaging and single-cell sequencing data using 3D Live-Seq. To further investigate the dynamics of de novo CNAs as they arise and propagate across cell generations, we devel- oped 3D Live-Seq—a protocol inspired by the LookSeq strategy21. 3D Live-Seq incorporates confocal live-cell imaging data of a grow- ing PDTO structure and single-cell sequencing data of the entire imaged cell population, to allow a precise reconstruction of evolving tumor karyotypes across consecutive cell generations. The expres- sion of transgenic H2B-Dendra2 (ref. 22) is sufficiently bright to support long-term confocal imaging with remarkable spatiotempo- ral resolution. Importantly, cells of interest can be photoconverted Nature Genetics \| VOL 53 \| August 2021 \| 1187–1195 \| www.nature.com/naturegenetics 1188 Articles NatUrE GEnEtICS from green to red Dendra2 fluorescence to serve as a reference land- mark for the direct integration of imaging and sequencing datasets. carrying five copies of chromosome 6q while its sister cell, identi- fied by the reciprocal CNA of chromosome 1q, displayed only one copy of chromosome 6q. Given the structural constraints set forth by the mitotic tree, any conventional phylogenetic interpretation that could explain the origin of additional chromosome 6q copies appeared unlikely. Chromatin errors during mitosis are phenotypes of de novo CNAs. We first implemented 3D Live-Seq to investigate whether de novo CNAs correlate with specific chromatin errors during mito- sis. Collectively, we generated high-resolution imaging data of 64 cell divisions, each from individual tumor organoids (PDTO-9), to score their chromatin phenotype. We then sequenced both daughter cells along with a bulk sample consisting of the remain- ing cells of the dissociated PDTO structure (Fig. 2a). As expected, de novo CNAs were almost exclusively generated among daughter cells of cell divisions that involved either a chromatin bridge or lag- ging chromatin (Fig. 2b and Supplementary Note 1). Results Notably, no indications of chro- mothripsis21,29 were found within the amplified chromosome 9q frag- ment (Extended Data Fig. 5), suggesting that micronuclear envelope integrity was maintained30. Similar sub-chromosomal amplifications were observed in additional datasets (Extended Data Fig. 1 (lineages III and IV) and Extended Data Fig. 6, (lineage I). In each case, the amplified sub-chromosomal fragments were acentric, suggesting a model where a lack of spindle microtubule attachment results in non-disjunction of the replicated acentric fragments. During ana- phase, the unattached replicated fragments are collectively displaced to one daughter cell, probably resulting in a stochastic nuclear or micronuclear containment. While the impact of amplified acentric fragments on tumorigenesis and tumor evolution is speculative, it is conceivable that amplified acentric chromosomal fragments act as substrates for chromothriptic events, contribute to the formation of extrachromosomal DNA or eventually fuse to chromosomes, result- ing in a stable inheritance of the amplified segment. While these data confirm that chromatin errors are distinct phe- notypes of karyotype diversification, chromatin bridges in particu- lar often failed to generate detectable CNAs (Fig. 2b). In contrast with artificially induced chromatin bridges25, naturally occurring chromatin bridges in PDTOs are likely to reflect a more com- plete spectrum of underlying causalities, of which a subset may be resolved at high fidelity, while other cases may give rise to mutations that are below our detection threshold (Supplementary Note 2). 3D Live-Seq captures karyotype alterations across cell gen- erations. Next, we applied 3D Live-Seq to study evolving tumor genomes across multiple consecutive cell divisions. To showcase the feasibility of our protocol, we recorded the unperturbed outgrowth of an individual PDTO-9 organoid across three cell generations (from two to 13 cells) and reconstructed the true mitotic tree with a detailed characterization of mitotic fidelity for each cell division (Fig. 2c). Before single-cell isolation, we selected one cell of inter- est for photoconversion based on a lagging chromatin phenotype during its preceding cell division (Fig. 2c, cell number 1.1.1.1). Single-cell sequencing data from 12 out of 13 cells displayed several de novo CNAs across three lineages (Fig. 2c, lineages I–III). Using the sequencing result of the photoconverted cell as a landmark and the mitotic tree as a structural constraint on potential phylogenetic solutions of lineage I, we mapped a consecutive missegregation of chromosome 7 to the highlighted branch within the mitotic tree (Fig. 2c, branch 1.1). Results Furthermore, chromatin bridges generally resulted in sub-chromosomal CNAs, whereas whole-chromosome missegregations were mainly asso- ciated with lagging chromatin. This bias was consistent with reports showing that chromatin bridges primarily represent the stretching of fused or dicentric chromosomes23, likely to result in sub-chromosomal fragments when broken. To exclude that the expression of transgenic H2B substantially exacerbates CIN pheno- types, we generated a CRISPR knock-in24 of Dendra2 at the carboxy terminus of the HIST1H2BC gene (Extended Data Fig. 4a,b). In a direct comparison, both lines showed clear CIN phenotypes with similar error-type frequency distributions (Extended Data Fig. 4c). The lower overall CIN rate in the knock-in line could be attributed to the substantially reduced brightness of the knock-in, which ham- pers effective scoring of subtle chromatin errors, as well as to clonal differences between lines7 (Extended Data Fig. 4d). Furthermore, single cells isolated from genetically unmodified PDTOs (using the DNA dye Syto 11) that had not been exposed to live-cell imaging procedures, displayed a similar number of de novo CNAs compared with other PDTO datasets (Extended Data Figs. 1d and 2b,c). Replication and collective missegregation of acentric chromo- somal fragments. We encountered a similar amplification of chro- mosome 9q (chromosome 9q21.33-ter) within the photoconverted lineage of another PDTO-9 3D Live-Seq dataset (25 cells; 100% recovery), which allowed accurate mapping of its phylogenetic ori- gin across four cell generations (Fig. 3a, lineage I). The amplifica- tion of chromosome 9q in one of the photoconverted cells and the reciprocal loss across all other cells within the lineage could only be reconciled by two cycles of replication and collective missegregation of an acentric chromosome 9q fragment (Supplementary Note 3). Chromatin phenotypes along the photoconverted branch suggested that replicated chromosome 9q fragments were missegregated dur- ing consecutive divisions 1.1 and 1.1.1, but were probably shielded from a third round of replication before division 1.1.1.1 by micro- nuclear containment (asterisk), which are known to be associated with replication defects26–28. Next, we performed additional deep sequencing of cell 1 (five copies), cells 2 and 3 (one copy) and a cell with the core karyotype (diploid), to extract single-nucleotide vari- ants (SNVs) unique for the chromosome 9q21.33-ter region of each parental chromatid. As expected, the cell containing an amplifica- tion of chromosome 9q displayed a biased variant allele frequency of SNVs in favor of one parental allele and at a degree consistent with two rounds of replication (Fig. 3b). Results Divisions (%) c 10 20 30 50 60 70 40 80 117 90 100 110 2–6 1 1.1.1 1.1 1.2 1 2 1.1.2 1.1.1.1 1.1.1.2 Chromosome 7 Chromosome 7 7 Chromosome 7 Chromosome 7 Chromosome 7 Chromosome 7 2.1 2.2 Chromosome 7 1.2.1 1.2.1 1.2.1.1 1.2.1.2 2.1.1 2.1.1.1 2.1.1.2 2.1.2 2.1.2.1 2.1.2.2 2.2.1 2.2.1.1 2.2.1.2 2.2.2 Chromosome 7 Sequence number Time (h) 4 5 6 7 8 9 10 11 12 14 13 15 17 16 18 19 20 2122 X I II III 2N 1N 3N 4N 5N Chromosome Fig. 2 \| Capturing karyotype diversification across consecutive cell generations using 3D Live-Seq. a, Top: schematic representing confocal imaging of individual PDTO-9 organoids expressing H2B-Dendra (green) to capture high-spatiotemporal-resolution recordings of individual cell divisions, followed by photoconversion of cells of interest to red fluorescence before single-cell isolation. Bottom: representative imaging stills of the procedure. Right: CNA plots from chromosome 7 to chromosome 16 are shown for photoconverted daughter cells 1 and 2 (top two lanes), as well as the remaining single cells of the organoid combined in lane 3 as reference material. The red box indicates the reciprocal gain and loss of chromosome 15, matching the lagging chromatin phenotype. The dots represent measured CNAs per bin (1 Mb). The average deviation from the diploid genome is indicated by color coding (blue = loss; orange = gain). Scale bars, 10 μm. b, Bar graph representing the fraction of PDTO-9 cell divisions that resulted in reciprocal whole-chromosome or sub-chromosomal CNAs among daughter cells per chromatin error class. In total, 37 normal (N) divisions, 18 divisions displaying chromatin bridges (CB) and nine divisions with lagging chromatin (LC) were captured. c, 3D Live-Seq dataset of a PDTO-9 organoid consisting of 13 cells. Top: representative stills of the growing PDTO-9 structure, with nuclei coded in false color by depth. Middle: reconstruction of the true mitotic tree containing representative stills of 3D-rendered anaphases. The onset of anaphase is indicated by arrowheads in relation to the time axis. Bottom: karyotype heatmap of 12 cells (93% recovery) isolated from the imaged PDTO-9 organoid. A consecutive missegregation of chromosome 7 (lineage I) was mapped to the highlighted branch of the mitotic tree using the photoconverted cell (white arrow in top panel) as a reference landmark. Anaphase stills showing lagging chromatin are indicated with red arrows. Chromosome cartoons along the mitotic tree indicate copy-number changes across cell generations. Results Nature Genetics \| VOL 53 \| August 2021 \| 1187–1195 \| www.nature.com/naturegenetics 1189 Articles NatUrE GEnEtICS 10 20 30 50 60 70 40 80 117 90 100 110 2–6 1 1.1.1 1.1 1.2 1 2 1.1.2 1.1.1.1 1.1.1.2 Chromosome 7 Chromosome 7 7 Chromosome 7 Chromosome 7 Chromosome 7 Chromosome 7 2.1 2.2 34 0 c Chromosome 7 1.2.1 1.2.1 1.2.1.1 1.2.1.2 2.1.1 2.1.1.1 2.1.1.2 2.1.2 2.1.2.1 2.1.2.2 2.2.1 2.2.1.1 2.2.1.2 2.2.2 Chromosome 7 Sequence number 20 µm b 405 nm Dendra2 (red) Dissociated PDTO Dendra2 (merged) Copy number 15 16 14 13 12 11 10 9 8 7 15 16 14 13 12 11 10 9 8 7 6 4 2 0 6 4 2 0 6 4 2 0 15 16 14 13 12 11 10 9 8 7 a N (37) CB (18) LC (9) Error type (number of divisions) Divisions (%) 25 50 75 100 No CNA Sub-chro Whole ch Sub- + w chromoso Chromosome Time (h) Depth (µm) b 405 nm Dendra2 (red) Dissociated PDTO Dendra2 (merged) Copy number 15 16 14 13 12 11 10 9 8 7 15 16 14 13 12 11 10 9 8 7 6 4 2 0 6 4 2 0 6 4 2 0 15 16 14 13 12 11 10 9 8 7 a N (37) CB (18) LC (9) Error type (number f di i i ) Divisions (%) 25 50 75 100 No CNA Sub-chrom Whole chro Sub- + who chromosom Chromosome b N (37) CB (18) LC (9) Error type (number of divisions) Divisions (%) 25 50 75 100 No CNA Sub-chromosome Whole chromosome Sub- + whole chromosome 405 nm Dendra2 (red) Dissociated PDTO Dendra2 (merged) a Copy number 15 16 14 13 12 11 10 9 8 7 15 16 14 13 12 11 10 9 8 7 6 4 2 0 6 4 2 0 6 4 2 0 15 16 14 13 12 11 10 9 8 7 Chromosome 1 2 3 4 5 6 7 8 9 10 11 12 14 13 15 17 16 18 19 20 2122 X I II III 1 12 10 20 30 50 60 70 40 80 117 90 100 110 2–6 1 1.1.1 1.1 1.2 1 2 1.1.2 1.1.1.1 1.1.1.2 Chromosome 7 Chromosome 7 7 Chromosome 7 Chromosome 7 Chromosome 7 Chromosome 7 2.1 2.2 2N 1N 3N 4N 5N 34 0 c Chromosome 7 1.2.1 1.2.1 1.2.1.1 1.2.1.2 2.1.1 2.1.1.1 2.1.1.2 2.1.2 2.1.2.1 2.1.2.2 2.2.1 2.2.1.1 2.2.1.2 2.2.2 Chromosome 7 Sequence number 20 µm Dendra2 (red) Dissociated PDTO Dendra2 (merged) Copy number 15 16 14 13 12 11 10 9 8 7 15 16 14 13 12 11 10 9 8 7 4 2 0 6 4 2 0 6 4 2 0 15 16 14 13 12 11 10 9 8 7 N (37) CB (18) LC (9) Error type (number of divisions) Divisions (%) 25 50 75 Sub-chromo Whole chrom Sub- + whol chromosom 12/13 cells (92%) Chromosome Time (h) Chromosome Depth (µm) Fig. Results In agreement with our previous data, divisions at which chromosome 7 missegregated displayed a lagging chro- matin phenotype migrating toward the daughter cell that gained a copy of chromosome 7 (Fig. 2c, red arrows). The remaining de novo CNAs could not be mapped with high accuracy as their potential phylogenetic solutions matched multiple branches of the mitotic tree (Fig. 2c, lineages II and III). Lineage III displayed an unusual CNA of chromosome 6q (chromosome 6q24.2-ter) with one cell Gross karyotype alterations result from multipolar spindle defects. While CNA events generally involve one or a few chromo- somes, we identified two cells that displayed extensive reciprocal CNAs across their genomes, and their karyotypes can be classified as hopeful monsters (Fig. 3a, lineage II). In contrast with the popu- lation of hopeful monsters that we previously detected in PDTO- 19b, these cells displayed an unbalanced distribution of the core karyotype of lineage I without signs of previous genome duplica- tion, demonstrating that near-haploid and near-triploid genomes can be generated in a single division without tetraploid intermedi- ates20. The uneven distribution of chromosomes matched the sub- stantial difference in chromatin mass seen between daughter cells of division 1.1.2.2. Furthermore, the irregular shape of the meta- phase plate indicated a multipolar spindle defect as a cause of the unbalanced chromosome distribution. Multipolar spindle defects are associated with a loss of spindle pole integrity31 or supernumer- ary centrosomes. Indeed, the distribution of a replicated genome among excess spindle poles is likely to instigate a genome-wide mis- allocation of chromosomes. Nature Genetics \| VOL 53 \| August 2021 \| 1187–1195 \| www.nature.com/naturegenetics Results 2 \| Capturing karyotype diversification across consecutive cell generations using 3D Live-Seq. a, Top: schematic representing confocal imaging of individual PDTO-9 organoids expressing H2B-Dendra (green) to capture high-spatiotemporal-resolution recordings of individual cell divisions, followed by photoconversion of cells of interest to red fluorescence before single-cell isolation. Bottom: representative imaging stills of the procedure. Right: CNA plots chromosome 7 to chromosome 16 are shown for photoconverted daughter cells 1 and 2 (top two lanes), as well as the remaining single cells of the organoid combined in lane 3 as reference material. The red box indicates the reciprocal gain and loss of chromosome 15, matching the lagging chromatin phenotype. The dots represent measured CNAs per bin (1 Mb). The average deviation from the diploid genome is indicated by color coding (blue = loss; orange = gain) Scale bars, 10 μm. b, Bar graph representing the fraction of PDTO-9 cell divisions that resulted in reciprocal whole-chromosome or sub-chromosomal CNAs among daughter cells per chromatin error class. In total, 37 normal (N) divisions, 18 divisions displaying chromatin bridges (CB) and nine divisions with lag chromatin (LC) were captured. c, 3D Live-Seq dataset of a PDTO-9 organoid consisting of 13 cells. Top: representative stills of the growing PDTO-9 structur with nuclei coded in false color by depth. Middle: reconstruction of the true mitotic tree containing representative stills of 3D-rendered anaphases. The ons of anaphase is indicated by arrowheads in relation to the time axis. Bottom: karyotype heatmap of 12 cells (93% recovery) isolated from the imaged PDTO- organoid. A consecutive missegregation of chromosome 7 (lineage I) was mapped to the highlighted branch of the mitotic tree using the photoconverted c (white arrow in top panel) as a reference landmark. Anaphase stills showing lagging chromatin are indicated with red arrows. Chromosome cartoons along mitotic tree indicate copy-number changes across cell generations. Lineages II and III cannot be accurately mapped onto the mitotic tree. Results Top: representative stills of the growing PDTO-9 structure with nuclei coded in false color by depth. The final still shows two photoconverted cells (white arrows) that were the progeny of branch 1.1 of the mitotic tree. Branch 1.1 is highlighted (dashed red box) in the full tree structure in the top left of the middle panel and enlarged as the main image in the middle panel, with 3D-rendered stills of each anaphase. The onset of anaphase is indicated by arrowheads in relation to the time axis. Bottom: karyotype heatmap of all cells mapped to branch 1.1 (cells 1–7) and a reference core karyotype of the imaged PDTO. The sequence numbers of the mapped sequencing results in relation to the mitotic tree are indicated, including the photoconversion state of each cell. Chromosome cartoons along enlarged branch 1.1 indicate reconstructed copy-number changes across cell generations. The inset of cell division 1 shows a lagging chromatin structure, as indicated by the red arrow. The asterisk highlights the presence of a micronucleus in cell 1.1.1.1 that persists until nuclear envelope breakdown. b, The variant allele frequency (VAF) distribution of variants located on the acentric chromosome 9q fragment (Chr. 9q21.33-ter) is consistent with two rounds of replication and collective missegregation for cell number 1. The VAF of SNVs located on the missegregated chromosome 9q21.33-ter region is 0% for cell numbers 2 and 3 (only one copy of chromosome 9q) and 50% for a cell with the core karyotype. Cell number 1 shows nearly 80% VAF for these SNVs, indicative that four of the five chromosome 9q21.33-ter copies originated from the same parental allele. The dashed red line indicates the median. 1–4 displayed the same loss of chromosome 1p and were mapped to branch 1.1.1. The difference in ploidy between branches 1.1.1 and 1.1.2 and the absence of nuclear envelope breakdown and a prolonged cell cycle time of cell 1.1.2 clearly support re-replication. Mitotic slippage similarly results in a genome-duplicated cell state and, in contrast with re-replication events, can readily be recognized by sequential chroma- tin condensation and de-condensation without entering anaphase. and trace mitotic slippage events. Although apoptosis was a more frequent outcome, we scored a high number of cells entering mitosis following a previous mitotic slippage event. Multipolar spindle defects were common among these downstream cell divisions, with 3 out of 12 cells displaying three or more spindle poles (Extended Data Fig. 7a,b). Results Lineages II and III cannot be accurately mapped onto the mitotic tree. diversification across consecutive cell generations using 3D Live-Seq. a, Top: schematic representing confocal imaging of Since centrosome amplification is frequently associated with whole-genome doubling32, we hypothesized that genome-duplicated cells, resulting from mitotic entry and exit defects, could serve as effi- cient substrates for the generation of hopeful monster karyotypes by frequently instigating multipolar spindle defects. Indeed, we captured a tripolar division following a re-replication event, which generated three daughter cells with reciprocal hopeful monster karyotypes (Fig. 4a (lineage I) and Supplementary Note 3). Collectively, their genomes aggregate to a twice-replicated core karyotype with a de novo loss of chromosome 1p (1pter–p34.2) (Extended Data Fig. 3b). Cells 1190 Articles NatUrE GEnEtICS Articles NatUrE GEnEtICS 10 20 30 50 60 70 40 80 117 90 100 110 3 6 7 Chromosome 9 Chromosome 9 Chromosome 9 Chromosome 9 Chromosome 21 1/2 1/2 Chromosome 9 Chromosome 21 4/5 4/5 1.1.2.1 1.1.2.2 1.1 1.1.1 1.1.2 1.1.1.1 Chromosome 9 Chromosome 9 Multipolar Sequence number 1 1.1.1.1.1 1.1.1.1.2 1.1.1.2 1.1.2.1.1 1.1.2.1.2 1.1.2.2.1 1.1.2.2.2 1 2 3 4 5 6 7 8 9 10 11 12 14 13 15 17 16 18 19 20 2122 X I II b Chr. 9q21.33-ter Chr. 9q21.33-ter Chr. 9q21.33-ter Sequence no. 1 Core Sequence no. 2/3 a 0N 2N 1N 5N 3N 4N 6N Micronucleus Core 7 1 64 Depth (µm) 0 20 µm 2.5 2.0 1.5 1.0 Density 0.5 0 0 0.25 0.50 0.75 1.00 0 0.25 0.50 0.75 1.00 0 VAF 0.25 0.50 0.75 1.00 Time (h) Chromosome Articles 10 20 30 50 60 70 40 80 117 90 100 110 3 6 7 Chromosome 9 Chromosome 9 Chromosome 9 Chromosome 9 Chromosome 21 1/2 1/2 Chromosome 9 Chromosome 21 4/5 4/5 1.1.2.1 1.1.2.2 1.1 1.1.1 1.1.2 1.1.1.1 Chromosome 9 Chromosome 9 Multipolar Sequence number 1 1.1.1.1.1 1.1.1.1.2 1.1.1.2 1.1.2.1.1 1.1.2.1.2 1.1.2.2.1 1.1.2.2.2 1 2 3 4 5 6 7 8 9 10 11 12 14 13 15 17 16 18 19 20 2122 X I II b Chr. 9q21.33-ter Chr. 9q21.33-ter Chr. 9q21.33-ter Sequence no. 1 Core Sequence no. 2/3 a 0N 2N 1N 5N 3N 4N 6N Micronucleus Core 7 1 64 Depth (µm) 0 20 µm 2.5 2.0 1.5 1.0 Density 0.5 0 0 0.25 0.50 0.75 1.00 0 0.25 0.50 0.75 1.00 0 VAF 0.25 0.50 0.75 1.00 Time (h) Chromosome Fig. Results 3 \| Acentric chromosomal fragments are prone to cycles of replication and collective missegregation. a, 3D Live-Seq dataset of a PDTO-9 organoid consisting of 25 cells (100% recovery). Top: representative stills of the growing PDTO-9 structure with nuclei coded in false color by depth. The final still shows two photoconverted cells (white arrows) that were the progeny of branch 1.1 of the mitotic tree. Branch 1.1 is highlighted (dashed red box) in the full tree structure in the top left of the middle panel and enlarged as the main image in the middle panel, with 3D-rendered stills of each anaphase. The onset of anaphase is indicated by arrowheads in relation to the time axis. Bottom: karyotype heatmap of all cells mapped to branch 1.1 (cells 1–7) and a reference core karyotype of the imaged PDTO. The sequence numbers of the mapped sequencing results in relation to the mitotic tree are indicated, including the photoconversion state of each cell. Chromosome cartoons along enlarged branch 1.1 indicate reconstructed copy-number changes across cell generations. The inset of cell division 1 shows a lagging chromatin structure, as indicated by the red arrow. The asterisk highlights the presence of a micronucleus in cell 1.1.1.1 that persists until nuclear envelope breakdown. b, The variant allele frequency (VAF) distribution of variants located on the acentric chromosome 9q fragment (Chr. 9q21.33-ter) is consistent with two rounds of replication and collective missegregation for cell number 1. The VAF of SNVs located on the missegregated chromosome 9q21.33-ter region is 0% for cell numbers 2 and 3 (only one copy of chromosome 9q) and 50% for a cell with the core karyotype. Cell number 1 shows nearly 80% VAF for these SNVs, indicative that four of the five chromosome 9q21.33-ter copies originated from the same parental allele. The dashed red line indicates the median. 64 Depth (µm) 0 20 µm 10 20 30 50 60 70 40 80 117 90 100 110 3 6 7 Chromosome 9 Chromosome 9 Chromosome 9 Chromosome 9 Chromosome 21 1/2 1/2 Chromosome 9 Chromosome 21 4/5 4/5 1.1.2.1 1.1.2.2 1.1 1.1.1 1.1.2 1.1.1.1 Chromosome 9 Chromosome 9 Multipolar Sequence number 1 1.1.1.1.1 1.1.1.1.2 1.1.1.2 1.1.2.1.1 1.1.2.1.2 1.1.2.2.1 1.1.2.2.2 Micronucleus Time (h) b Fig. 3 \| Acentric chromosomal fragments are prone to cycles of replication and collective missegregation. a, 3D Live-Seq dataset of a PDTO-9 organoid consisting of 25 cells (100% recovery). Nature Genetics \| VOL 53 \| August 2021 \| 1187–1195 \| www.nature.com/naturegenetics Results A PDTO with a photoconverted slippage cell was cultured overnight. The final still shows two photoconverted daughter cells (white arrows) with a similar chromatin mass. Bottom: karyotype heatmap of both daughter cells and the remaining PDTO cells (core). Daughter cells showed polyploidy (8 N) but roughly maintained the core karyotype. Heatmap colour coding indicates deviations with respect to the ploidy of the core karyotype (2N) or daughter cells (8N). The red boxes indicate reciprocal gains and losses. Lineage II: as described for lineage I, but showing a multipolar spindle defect after mitotic slippage resulting in hopeful monster karyotypes among three d ht ll ( hit ) S i d t bt i d f t t f th d ht ll d di l id k t lt ti 42 Depth (µm) 0 20 µm b Fig. 4 \| Genome duplication events are substrates for hopeful monster karyotypes. a, 3D Live-Seq dataset of a PDTO-9 organoid consisting of 19 cells (95% recovery). Top: representative stills of the growing PDTO-9 structure with nuclei coded in a false color by depth. Photoconverted cells are indicated in the final still (white arrows). One photoconverted cell was mapped to branch 1.1 of the mitotic tree, as highlighted (dashed red box) in the full tree structure at the top left of the middle panel and enlarged in the main image of the middle panel, with 3D-rendered stills of each anaphase. The onset of anaphase is indicated by arrowheads in relation to the time axis. Bottom: karyotype heatmap of all cells mapped to branch 1.1 (all cells share a loss of chromosome 1p; chromosome cartoon) and a reference core karyotype of the imaged PDTO. Three cells, including the photoconverted cell, showed reciprocal hopeful monster karyotypes (I) and were mapped to tripolar division 1.1.2 using the photoconversion reference landmark. Cell 1.1.2 underwent re-replication before anaphase, as indicated by the difference in ploidy between branches 1.1.1 and 1.1.2. b, Daughter cells post-mitotic slippage maintain polyploidy of the genome-duplicated ancestor. Lineage I: representative imaging stills (top) of a binucleated cell undergoing mitotic slippage. A PDTO with a photoconverted slippage cell was cultured overnight. The final still shows two photoconverted daughter cells (white arrows) with a similar chromatin mass. Bottom: karyotype heatmap of both daughter cells and the remaining PDTO cells (core). Daughter cells showed polyploidy (8 N) but roughly maintained the core karyotype. Results To confirm that multipolar spindle defects resulting from upstream mitotic slippage generate progeny with hopeful monster karyotypes, we photoconverted cells after mitotic slippage and isolated the progeny after their next 1–4 displayed the same loss of chromosome 1p and were mapped to branch 1.1.1. The difference in ploidy between branches 1.1.1 and 1.1.2 and the absence of nuclear envelope breakdown and a prolonged cell cycle time of cell 1.1.2 clearly support re-replication. Mitotic slippage similarly results in a genome-duplicated cell state and, in contrast with re-replication events, can readily be recognized by sequential chroma- tin condensation and de-condensation without entering anaphase. To investigate cell fate after mitotic slippage, we recorded confocal imaging data of PDTO-9 organoids for 72 h to identify Nature Genetics \| VOL 53 \| August 2021 \| 1187–1195 \| www.nature.com/naturegenetics Nature Genetics \| VOL 53 \| August 2021 \| 1187–1195 \| www.nature.com/naturegenetics 1191 Articles NatUrE GEnEtICS 0N 2N 1N 5N+ 3N 4N 1 2 3 4 5 6 7 8 9 10 11 12 14 13 15 17 16 18 19 20 21 22 X a 1 1.1.1.1.1 1.1.1.1.2 1.1.1.2.1 1.1.1.2.2 1.1.2.1 1.1.2.3 1.1.2.2 1.1 1.1.1 1.1.2 1.1.1.1 1.1.1.2 10 20 30 50 60 70 40 80 136 90 100 110 120 130 Replication Re-replication Core Multipolar 42 Depth (µm) 0 20 µm Core 1 2 3 4 5 10 11 12 14 13 15 17 16 18 19 20 21 22 X +1 +2 +3> –2 –1 Core 1 2 3 4 5 6 7 8 9 10 11 12 14 13 15 17 16 18 19 20 21 22 X 0N 2N 1N 5N 3N 4N 6N I 1 7 <–3 10 µM 39:07 h 11:00 h 18:19 h 01:30h b 01:00 h 02:30 h 00:00 h On culture Mitotic slippage 00:00 h 00:20 h 02:50 h 04:10 h 15:50 h 17:09 h 39:35 h On culture Mitotic slippage 6 7 8 9 I II I II (8N) Polyploidy Hopeful monster karotypes 10 µM Chromosome 1 Time (h) Chromosome Chromosome Chromosome Fig. 4 \| Genome duplication events are substrates for hopeful monster karyotypes. a, 3D Live-Seq dataset of a PDTO-9 organoid consisting of 19 cells (95% recovery). Top: representative stills of the growing PDTO-9 structure with nuclei coded in a false color by depth. Photoconverted cells are indicated in the final still (white arrows). Results One photoconverted cell was mapped to branch 1.1 of the mitotic tree, as highlighted (dashed red box) in the full tree structure at the top left of the middle panel and enlarged in the main image of the middle panel, with 3D-rendered stills of each anaphase. The onset of anaphase is indicated by arrowheads in relation to the time axis. Bottom: karyotype heatmap of all cells mapped to branch 1.1 (all cells share a loss of chromosome 1p; chromosome cartoon) and a reference core karyotype of the imaged PDTO. Three cells, including the photoconverted cell, showed reciprocal hopeful monster karyotypes (I) and were mapped to tripolar division 1.1.2 using the photoconversion reference landmark. Cell 1.1.2 underwent re-replication before anaphase, as indicated by the difference in ploidy between branches 1.1.1 and 1.1.2. b, Daughter cells post-mitotic slippage maintain polyploidy of the genome-duplicated ancestor. Lineage I: representative imaging stills (top) of a binucleated cell undergoing mitotic slippage. A PDTO with a photoconverted slippage cell was cultured overnight. The final still shows two photoconverted daughter cells (white arrows) with a similar chromatin mass. Bottom: karyotype heatmap of both daughter cells and the remaining PDTO cells (core). Daughter cells showed polyploidy (8 N) but roughly maintained the core karyotype. Heatmap colour coding indicates deviations with respect to the ploidy of the core karyotype (2N) or daughter cells (8N). The red boxes indicate reciprocal gains and losses. Lineage II: as described for lineage I, but showing a multipolar spindle defect after mitotic slippage resulting in hopeful monster karyotypes among three daughter cells (white arrows). Sequencing data were obtained from two out of three daughter cells and display gross genome-wide karyotype alterations relative to the core karyotype. Results Articles NatUrE GEnEtICS 0N 2N 1N 5N+ 3N 4N 1 2 3 4 5 6 7 8 9 10 11 12 14 13 15 17 16 18 19 20 21 22 X a 1 1.1.1.1.1 1.1.1.1.2 1.1.1.2.1 1.1.1.2.2 1.1.2.1 1.1.2.3 1.1.2.2 1.1 1.1.1 1.1.2 1.1.1.1 1.1.1.2 10 20 30 50 60 70 40 80 136 90 100 110 120 130 Replication Re-replication Core Multipolar 42 Depth (µm) 0 20 µm Core 1 2 3 4 5 10 11 12 14 13 15 17 16 18 19 20 21 22 X +1 +2 +3> –2 –1 Core 1 2 3 4 5 6 7 8 9 10 11 12 14 13 15 17 16 18 19 20 21 22 X 0N 2N 1N 5N 3N 4N 6N I 1 7 <–3 10 µM 39:07 h 11:00 h 18:19 h 01:30h b 01:00 h 02:30 h 00:00 h On culture Mitotic slippage 00:00 h 00:20 h 02:50 h 04:10 h 15:50 h 17:09 h 39:35 h On culture Mitotic slippage 6 7 8 9 I II I II (8N) Polyploidy Hopeful monster karotypes 10 µM Chromosome 1 Time (h) Chromosome Chromosome Chromosome Fig. 4 \| Genome duplication events are substrates for hopeful monster karyotypes. a, 3D Live-Seq dataset of a PDTO-9 organoid consisting of 19 cells (95% recovery). Top: representative stills of the growing PDTO-9 structure with nuclei coded in a false color by depth. Photoconverted cells are indicated in the final still (white arrows). One photoconverted cell was mapped to branch 1.1 of the mitotic tree, as highlighted (dashed red box) in the full tree structure at the top left of the middle panel and enlarged in the main image of the middle panel, with 3D-rendered stills of each anaphase. The onset of anaphase is indicated by arrowheads in relation to the time axis. Bottom: karyotype heatmap of all cells mapped to branch 1.1 (all cells share a loss of chromosome 1p; chromosome cartoon) and a reference core karyotype of the imaged PDTO. Three cells, including the photoconverted cell, showed reciprocal hopeful monster karyotypes (I) and were mapped to tripolar division 1.1.2 using the photoconversion reference landmark. Cell 1.1.2 underwent re-replication before anaphase, as indicated by the difference in ploidy between branches 1.1.1 and 1.1.2. b, Daughter cells post-mitotic slippage maintain polyploidy of the genome-duplicated ancestor. Lineage I: representative imaging stills (top) of a binucleated cell undergoing mitotic slippage. Nature Genetics \| VOL 53 \| August 2021 \| 1187–1195 \| www.nature.com/naturegenetics Results The total CIN rates were analyzed by chi-squared test (P = 0.56; not significant (NS)). b, Fate analysis of PDTO-9 progeny resulting from normal (N) or erroneous (E) cell divisions per growth stage. Alive progeny were non-proliferative until the end of the imaging window and this inactive period exceeded the average cell cycle length. The numbers of scored events are indicated in brackets. The difference in apoptotic rates was statistically significant, as determined by chi-squared test (P = 0.014 (young) and P = 0.0026 (mature)). c, As in b, but the fate analysis is shown for normal (N), chromatin bridge (CB), lagging chromatin (LC) and multipolar cell divisions (MP). d, Ratio between the cell cycle duration before a division and the progeny’s cell cycle duration (averaged) after that division. Data from young and mature PDTOs were pooled and subdivided by mitotic fidelity. The difference in cell cycle duration ratios between normal (94 branches) and erroneous divisions (76 branches) was not significant (data not normally distributed; P = 6.104 × 10−13 (Saphiro); P = 0.64 (two-sided Wilcoxon test)). In the box and whisker plots, the boxes represent quartiles 2 and 3, the horizontal lines represent median values and the whiskers represent minimum and maximum values within 1.5× the interquartile range. The data points indicate outlier values that deviate by more than 1.5× the interquartile range. e, Top: schematic of karyotype analysis of successfully formed organoids (bulk) grown from single (fit) cells derived from a parental (clonal) PDTO. Bottom: karyotype heatmaps of sequenced PDTOs (bulk; similar sized) as a proxy for the karyotype of the seeding cell. In total, 67 organoids shared the reference PDTO-9 core karyotype, seven were polyploid and five had de novo localized CNAs. are constantly being generated, upon which selection pressures act to shape the genomic evolution of advanced human cancers). Gross deviations of the core karyotype are generally less fit. To investigate cell intrinsic selection pressures against de novo karyotypes in closer detail, we captured a high number of indi- vidual cell lineages in PDTOs during early stages of outgrowth (days 4–6; up to ~30 cells in size) and mature stages (days 9–11; hundreds of cells). Overall, the level of CIN remained constant from young to mature organoids (Fig. 5a). Results Articles NatUrE GEnEtICS b c d 100 Percentage of progeny 75 50 25 0 100 75 50 25 0 Percentage of progeny Young Mature Young Mature N E N E Type of division N CB LC MP N CB LC MP Type of division (249)(218) (181) (148) (249)(158) (32) (28) (181) (96) (44) (8) Apoptosis Alive Mitosis Fate Clonal outgrowth Dissociation Clonal outgrowth Bulk PDTO sequencing e 1 2 3 4 5 6 7 8 9 10 11 12 14 13 15 17 16 18 19 20 21 22 X 0N 2N 1N 5N 3N 4N 6N+ Core (67 organoids) CNAs (5 organoids) Polyploid (7 organoids) N E 0 1 2 3 Cell cycle duration ratio (before/after) Apoptosis Alive Mitosis Fate Type of division NS a Young Mature 40 30 20 10 0 Mitotic slippage Multipolar spindle Lagging chromatin Chromatin brige 50 (361) (288) NS CIN rate (%) Chromosome c 100 75 50 25 0 Percentage of progeny Young Mature N CB LC MP N CB LC MP Type of division (249)(158) (32) (28) (181) (96) (44) (8) Apo Aliv Mito Fate c d 100 75 50 25 0 Percentage of progeny Young Mature N CB LC MP N CB LC MP Type of division (249)(158) (32) (28) (181) (96) (44) (8) Apoptosis Alive Mitosis Fate N E 0 1 2 3 Cell cycle duration ratio (before/after) Type of division NS a Young Mature 40 30 20 10 0 Mitotic slippage Multipolar spindle Lagging chromatin Chromatin brige 50 (361) (288) NS CIN rate (%) d N E 0 1 2 3 Cell cycle duration ratio (before/after) Type of division NS b 100 Percentage of progeny 75 50 25 0 Young Mature N E N E Type of division (249)(218) (181) (148) Apoptosis Alive Mitosis Fate Percentage of progeny Clonal outgrowth Dissociation Clonal outgrowth Bulk PDTO sequencing e 1 2 3 4 5 6 7 8 9 10 11 12 14 13 15 17 16 18 19 20 21 22 X 0N 2N 1N 5N 3N 4N 6N+ Core (67 organoids) CNAs (5 organoids) Polyploid (7 organoids) Chromosome e Bulk PDTO sequencing Fig. 5 \| Novel karyotypes frequently remain proliferative and can seed new PDTOs. a, CIN phenotype of PDTO-9 expressing transgenic H2B-Dendra2 during early and mature stages of outgrowth. The numbers of scored divisions are indicated in brackets. Results In the box and whisker plots, the boxes represent quartiles 2 and 3, the horizontal lines represent median values and the whiskers represent minimum and maximum values within 1.5× the interquartile range. The data points indicate outlier values that deviate by more than 1.5× the interquartile range. e, Top: schematic of karyotype analysis of successfully formed organoids (bulk) grown from single (fit) cells derived from a parental (clonal) PDTO. Bottom: karyotype heatmaps of sequenced PDTOs (bulk; similar sized) as a proxy for the karyotype of the seeding cell. In total, 67 organoids shared the reference PDTO-9 core karyotype, seven were polyploid and five had de novo localized CNAs. Results Heatmap colour coding indicates deviations with respect to the ploidy of the core karyotype (2N) or daughter cells (8N). The red boxes indicate reciprocal gains and losses. Lineage II: as described for lineage I, but showing a multipolar spindle defect after mitotic slippage resulting in hopeful monster karyotypes among three daughter cells (white arrows). Sequencing data were obtained from two out of three daughter cells and display gross genome-wide karyotype alterations relative to the core karyotype. that follow mitotic slippage appeared fairly normal (bipolar) (Fig. 4b (lineage I) and Extended Data Fig. 7a,b), mitotic slippage may represent an important pathway for whole-genome doubling during carcinogenesis and tumor progression, which is a feature of many human cancers2,3,33. cell division. As expected, when two daughter cells were gener- ated with equal chromatin mass, we detected polyploidy (including a few reciprocal CNAs) but no genome-wide misallocation of chromosomes (Fig. 4b, lineage I). In contrast, when downstream division resulted in three daughter cells, indicating a multipolar spindle defect, the isolated cells displayed hopeful monster karyo- types (Fig. 4b, lineage II). Cells with novel karyotypes frequently remain proliferative. As presented here, 3D Live-Seq of PDTOs enables the reconstruction of karyotype alterations across multiple consecutive cell genera- tions. It reveals the immediate genomic consequences of CIN (that is, the temporal dynamics and patterns by which new karyotypes Collectively, these data demonstrate that genome duplication events, irrespective of their origin, can act as unstable intermedi- ates in the generation of progeny with gross genome-wide karyo- type alterations. Results In addition, since the majority of cell divisions Nature Genetics \| VOL 53 \| August 2021 \| 1187–1195 \| www.nature.com/naturegenetics Nature Genetics \| VOL 53 \| August 2021 \| 1187–1195 \| www.nature.com/naturegenetics 1192 Articles NatUrE GEnEtICS b c d 100 Percentage of progeny 75 50 25 0 100 75 50 25 0 Percentage of progeny Young Mature Young Mature N E N E Type of division N CB LC MP N CB LC MP Type of division (249)(218) (181) (148) (249)(158) (32) (28) (181) (96) (44) (8) Apoptosis Alive Mitosis Fate Clonal outgrowth Dissociation Clonal outgrowth Bulk PDTO sequencing e 1 2 3 4 5 6 7 8 9 10 11 12 14 13 15 17 16 18 19 20 21 22 X 0N 2N 1N 5N 3N 4N 6N+ Core (67 organoids) CNAs (5 organoids) Polyploid (7 organoids) N E 0 1 2 3 Cell cycle duration ratio (before/after) Apoptosis Alive Mitosis Fate Type of division NS a Young Mature 40 30 20 10 0 Mitotic slippage Multipolar spindle Lagging chromatin Chromatin brige 50 (361) (288) NS CIN rate (%) Chromosome Fig. 5 \| Novel karyotypes frequently remain proliferative and can seed new PDTOs. a, CIN phenotype of PDTO-9 expressing transgenic H2B-Dendra2 during early and mature stages of outgrowth. The numbers of scored divisions are indicated in brackets. The total CIN rates were analyzed by chi-squared test (P = 0.56; not significant (NS)). b, Fate analysis of PDTO-9 progeny resulting from normal (N) or erroneous (E) cell divisions per growth stage. Alive progeny were non-proliferative until the end of the imaging window and this inactive period exceeded the average cell cycle length. The numbers of scored events are indicated in brackets. The difference in apoptotic rates was statistically significant, as determined by chi-squared test (P = 0.014 (young) and P = 0.0026 (mature)). c, As in b, but the fate analysis is shown for normal (N), chromatin bridge (CB), lagging chromatin (LC) and multipolar cell divisions (MP). d, Ratio between the cell cycle duration before a division and the progeny’s cell cycle duration (averaged) after that division. Data from young and mature PDTOs were pooled and subdivided by mitotic fidelity. The difference in cell cycle duration ratios between normal (94 branches) and erroneous divisions (76 branches) was not significant (data not normally distributed; P = 6.104 × 10−13 (Saphiro); P = 0.64 (two-sided Wilcoxon test)). Results a, Karyotype heatmap showing 112 cells (45% recovery) derived from an excised fragment of a single CRC gland isolated from a primary tumor biopsy of patient C274. The remaining gland structure was isolated as bulk (core). A total of 33 cells showed CNAs that deviated from the core karyotype. Glands were stained with Syto 11 to support imaging (right) and single-cell i l ti R i l CNA f h 2 d ti f th l i i di t d (li I) S l ll h id k t lt ti 77/~400 cells (19%) 100 µm Patient C575 (wild type) 3N 2N 4N 1 2 3 4 5 6 7 8 9 10 11 12 14 13 15 17 16 18 19 20 2122 X Core CNAs (4 cells) Wild type (73 cells) b Chromosome Fig. 6 \| Punctuated and localized karyotype alterations are ongoing in mature CRC. a, Karyotype heatmap showing 112 cells (45% recovery) derived from an excised fragment of a single CRC gland isolated from a primary tumor biopsy of patient C274. The remaining gland structure was isolated as bulk (core). A total of 33 cells showed CNAs that deviated from the core karyotype. Glands were stained with Syto 11 to support imaging (right) and single-cell isolation. Reciprocal CNA of chromosome 2 and propagation of the loss is indicated (lineage I). Several cells show gross genome-wide karyotype alterations (lineage II). b, As in a. A total of 77 single cells were isolated from a wild-type intestinal crypt of patient C575. Four cells showed de novo CNAs of the healthy diploid karyotype. The insets to the right show the excised fragment, obtained by cutting along the solid white lines shown in the main figure. fit. In contrast, the majority of cells with localized karyotype altera- tions that result from chromatin bridges or lagging chromatin phenotypes clearly remain proliferative, although these cells seem to experience a subtle fitness cost in the aggregate. sensitivity of our assay, in agreement with an earlier report on basal CIN levels in healthy colon tissue35 (Fig. 6b). Notably, by capturing a large fraction of clonal tumor cells (44%), we detected the emer- gence and propagation of a de novo CNA in vivo (Fig. 6a, lineage I). Moreover, we readily identified cells with hopeful monster karyo- types (Fig. Results Apoptosis was read- ily detected during both stages of outgrowth and, in agreement with previous reports7, we detected elevated levels of apoptosis among the progeny of erroneous cell divisions, in particular after multipolar spindle defects (Fig. 5b,c and Extended Data Fig. 7c). For cells that remained proliferative, the apparent slight shift towards longer cell cycle durations immediately after an error was not significant (Fig. 5d; Wilcoxon test, P = 0.64). However, a comprehensive analysis of mitotic tree structures using our derived likelihood ratio test was, again, able to indicate evidence for a decrease in birth rate following mitotic errors (P = 5 × 10−6; Supplementary Note 4). Our 3D Live-Seq datasets suggest that intrinsic negative selec- tion against de novo karyotypes, including grossly altered hopeful monster karyotypes, is often not absolute or instantaneous. To derive quantitative evidence for the strength of selection experienced by novel karyotypes, we used a stochastic branching process model to simulate CNA evolution and clonal selection within organoids using cell cycle parameters and measures of karyotype diversity extracted from our datasets. The results were best explained by a neutral drift model, although the power to detect subtle selection pressures was restricted due to the limited dataset size (Extended Data Fig. 8a–d). To further explore selective forces in 3D Live-Seq data, we devel- oped a likelihood ratio test to assess whether there was evidence for a change in birth rate following mitotic events. Using this approach, we were able to aggregate all four mitotic trees of PDTO-9 and found evidence for a decrease in birth rate after a mitotic error (P = 0.029; Supplementary Note 4). Taken together, this implied that many de novo karyotypes remain proliferative over a timescale of a few cell generations and experience neutral or subtle negative fitness effects. Results 6a, lineage II), further demonstrating that the genera- tion of cells with genome-wide karyotype alterations is ongoing at advanced cancer stages. Cells with novel karyotypes can seed new PDTOs. To func- tionally probe the fitness of novel karyotypes, we assessed the organoid-forming capacity of single tumor cells derived from one parental clonal PDTO. Subsequent karyotype analysis of the newly formed individual organoids (bulk) acts as a proxy for the karyo- type of the seeding single cell and can confirm sustained fitness of de novo CNAs. Indeed, cells with (sub-)chromosomal karyo- type alterations and even whole-genome duplications were able to develop into new PDTOs (Fig. 5e and Extended Data Fig. 8e), in line with our general observations, demonstrating that from a cell intrinsic and short-term perspective, some cells with novel karyo- types maintain a high level of fitness. Results Thus, a subset of de novo karyotypes are subject to stringent intrinsic negative selection, with large deviations from the core karyotype originating from multipolar spindle defects being least Nature Genetics \| VOL 53 \| August 2021 \| 1187–1195 \| www.nature.com/naturegenetics 1193 Articles NatUrE GEnEtICS 1 2 3 4 5 6 7 8 9 10 11 12 14 13 15 17 16 18 19 20 2122 X Core (79 cells) 112/251 cells (45%) Patient C274 (carcinoma) I CNAs (33 cells) II 50 µm 4N 3N 5N 6N+ 2N 77/~400 cells (19%) 100 µm Patient C575 (wild type) 3N 2N 4N 1 2 3 4 5 6 7 8 9 10 11 12 14 13 15 17 16 18 19 20 2122 X Core CNAs (4 cells) Wild type (73 cells) b a Chromosome Chromosome Fig. 6 \| Punctuated and localized karyotype alterations are ongoing in mature CRC. a, Karyotype heatmap showing 112 cells (45% recovery) derived from an excised fragment of a single CRC gland isolated from a primary tumor biopsy of patient C274. The remaining gland structure was isolated as bulk (core). A total of 33 cells showed CNAs that deviated from the core karyotype. Glands were stained with Syto 11 to support imaging (right) and single-cell isolation. Reciprocal CNA of chromosome 2 and propagation of the loss is indicated (lineage I). Several cells show gross genome-wide karyotype alterations (lineage II). b, As in a. A total of 77 single cells were isolated from a wild-type intestinal crypt of patient C575. Four cells showed de novo CNAs of the healthy diploid karyotype. The insets to the right show the excised fragment, obtained by cutting along the solid white lines shown in the main figure. 1 2 3 4 5 6 7 8 9 10 11 12 14 13 15 17 16 18 19 20 2122 X Core (79 cells) 112/251 cells (45%) Patient C274 (carcinoma) I CNAs (33 cells) II 50 µm 4N 3N 5N 6N+ 2N a Chromosome a 112/251 cells (45%) 50 µm 77/~400 cells (19%) 100 µm Patient C575 (wild type) 3N 2N 4N 1 2 3 4 5 6 7 8 9 10 11 12 14 13 15 17 16 18 19 20 2122 X Core CNAs (4 cells) Wild type (73 cells) b Chromosome Fig. 6 \| Punctuated and localized karyotype alterations are ongoing in mature CRC. Nature Genetics \| VOL 53 \| August 2021 \| 1187–1195 \| www.nature.com/naturegenetics Articles Articles NatUrE GEnEtICS division, providing support for punctuated evolution of malignant karyotypes during the earliest stages of carcinogenesis13,14,36. 19. Markowetz, F. A saltationist theory of cancer evolution. Nat. Genet. 48, 1102–1103 (2016). 20. Laughney, A. M., Elizalde, S., Genovese, G. & Bakhoum, S. F. 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Sottoriva, A. et al. A Big Bang model of human colorectal tumor growth. Nat. Genet. 47, 209–216 (2015). 11. Field, M. G. et al. Punctuated evolution of canonical genomic aberrations in uveal melanoma. Nat. Commun. 9, 116 (2018). 12. Baca, S. C. et al. Punctuated evolution of prostate cancer genomes. Cell 153, 666–677 (2013). Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. 13. Gao, R. et al. Punctuated copy number evolution and clonal stasis in triple-negative breast cancer. Nat. Genet. 48, 1119–1130 (2016). Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adap- tation, 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 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 license, visit http://creativecommons. org/licenses/by/4.0/. Discussion Our work reveals the genomic consequences of CIN across consecu- tive cell generations. By reconstructing intermediate genomic states, we demonstrate that both genome-wide (punctuated) and localized (singular, Extended Data Fig. 10) karyotype alterations are ongoing and prevalent in advanced CRC. The substantially reduced fitness of many hopeful monster karyotypes is not surprising given the extent of their karyotype alterations. Nevertheless, low probabili- ties of increased fitness can still be of potential impact considering that tumors can consist of hundreds of billions of cells9. Thus, while most diversity provides incremental variation around a proven-fit aneuploid genome, millions of hopeful monster karyotypes repre- sent extreme, potentially adaptive phenotypes within a fitness land- scape. The ongoing generation of karyotype diversity in advanced cancers is in agreement with recent analyses of tumor genomes from large patient cohorts2,3. However, as most of these studies measure the net outcome of evolution, it remains difficult to disentangle the rate at which karyotype diversity is generated and the selection pres- sures that act on them to shape an aneuploid landscape over time. Therefore, our insights into the alteration rate and diversification pat- terns of tumor karyotypes provide valuable parameters to understand the historic trajectories and temporal dynamics of an evolving aneu- ploid genome. In addition, our work suggests that aneuploid tumor karyotypes can, in principle, be generated in a single erroneous cell Punctuated and localized karyotype alterations are ongoing in mature tumors. To investigate whether advanced CRCs generate a similar degree of karyotype diversity in vivo before the reshaping of the karyotypic landscape as a consequence of selection, we adapted our sequencing methodology to measure karyotype diversity within clonal lineages of freshly frozen human CRC samples. To maximize recent ancestry within the sampled cell population, we excised small fragments from individual CRC glands, which are analogous to clonal intestinal crypts of the normal epithelium34. Subsequent single-cell isolation and prospective genome analyses displayed substantial karyotype diversity between cells, with almost 30% of cells carrying CNAs that deviated from the core gland karyotype—a number that was in agreement with our PDTO data (Fig. 6a and Extended Data Fig. 9). In contrast, only a few single cells from a wild-type intestinal crypt displayed de novo CNAs, supporting the Nature Genetics \| VOL 53 \| August 2021 \| 1187–1195 \| www.nature.com/naturegenetics 1194 References 26. Soto, M., Garcia-Santisteban, I., Krenning, L., Medema, R. H. & Raaijmakers, J. A. Chromosomes trapped in micronuclei are liable to segregation errors. J. Cell Sci. https://doi.org/10.1242/jcs.214742 (2018). 1. Taylor, A. M. et al. Genomic and functional approaches to understanding cancer aneuploidy. Cancer Cell 33, 676–689.e3 (2018). 1. Taylor, A. M. et al. Genomic and functional approaches to understanding cancer aneuploidy. Cancer Cell 33, 676–689.e3 (2018). 2. Priestley, P. et al. Pan-cancer whole-genome analyses of metastatic solid tumours. Nature 575, 210–216 (2019). 2. Priestley, P. et al. Pan-cancer whole-genome analyses of metastatic solid tumours. Nature 575, 210–216 (2019). 27. Terradas, M., Martin, M. & Genesca, A. Impaired nuclear functions in micronuclei results in genome instability and chromothripsis. Arch. Toxicol. 90, 2657–2667 (2016). 3. Watkins, T. B. K. et al. Pervasive chromosomal instability and karyotype order in tumour evolution. Nature https://doi.org/10.1038/s41586-020-2698-6 (2020).i 28. Crasta, K. et al. DNA breaks and chromosome pulverization from errors in mitosis. Nature 482, 53–58 (2012). ( ) 29. Liu, S. et al. Nuclear envelope assembly defects link mitotic errors to chromothripsis. Nature 561, 551–555 (2018). 4. Laks, E. et al. Clonal decomposition and DNA replication states defined by scaled single-cell genome sequencing. Cell 179, 1207–1221.e22 (2019). 29. Liu, S. et al. Nuclear envelope assembly defect chromothripsis. Nature 561, 551–555 (2018). chromothripsis. Nature 561, 551–555 (2018). 5. Navin, N. et al. Tumour evolution inferred by single-cell sequencing. Nature 472, 90–94 (2011). 30. Hatch, E. M., Fischer, A. H., Deerinck, T. J. & Hetzer, M. W. Catastrophic nuclear envelope collapse in cancer cell micronuclei. Cell 154, 47–60 (2013) 6. Zahir, N., Sun, R., Gallahan, D., Gatenby, R. A. & Curtis, C. Characterizing the ecological and evolutionary dynamics of cancer. Nat. Genet. 52, 759–767 (2020). 31. Maiato, H. & Logarinho, E. Mitotic spindle multipolarity without centrosome amplification. Nat. Cell Biol. 16, 386–394 (2014). i 32. Godinho, S. A., Kwon, M. & Pellman, D. Centrosomes and cancer: how cancer cells divide with too many centrosomes. Cancer Metastasis Rev. 28, 85–98 (2009). 7. Bolhaqueiro, A. C. F. et al. Ongoing chromosomal instability and karyotype evolution in human colorectal cancer organoids. Nat. Genet. 51, 824–834 (2019). 33. Bielski, C. M. et al. Genome doubling shapes the evolution and prognosis of advanced cancers. Nat. Genet. 50, 1189–1195 (2018). 8. Davis, A., Gao, R. & Navin, N. Tumor evolution: linear, branching, neutral or punctuated. Biochim. Biophys. Acta Rev. Cancer 1867, 151–161 (2017). 8. Methods Cells expressing H2B-Dendra2-IRES-Puromycin were selected by supplementing the culture media with puromycin dihydrochloride (Santa Cruz; 2 μg ml−1). Organoid-forming capacity of PDTO-9 cells. PDTO-9 organoids were cultured and dissociated as previously described. Single cells obtained from a single parental clonal organoid were reseeded and cultured for 9 d as previously described. Organoids of similar size were isolated and individually bulk sequenced as a proxy for the karyotype of the seeding cell. CRISPR gene tagging. TV-hHIST1H2BC-Dendra2 was generated by Golden Gate assembly37 of hH2BC_UHA and hH2BC_DHA (gBlocks; IDT) into TVBB-Dendra2 (Supplementary Table 1). To knock-in Dendra2 at the carboxy terminus of the human HIST1H2BC locus, 1 × 106 PDTO-9 cells were co-electroporated with 7.5 μg Cas9 D10A nickase (Addgene; 48141) and 7.5 μg TV-hHIST1H2BC-Dendra2 using the NEPA21 Super Electroporator (Nepagene) following described conditions38. A bulk knock-in culture was established by fluorescence-activated cell sorting of a Dendra2-positive cohort 18 d post-electroporation. Site-specific integration was confirmed by a genotyping PCR on bulk genomic DNA extract using locus-specific primer sets (Supplementary Table 1). Fresh-frozen CRC tissue specimens. Fresh-frozen tissue samples were collected from University College London Hospitals under ethical approval 11/LO/1613 and via the University College London Hospital Biobank (15/YH/0311) and processed as previously described41. All surgically resected samples were collected from patients who had given informed consent. Single-cell isolation of CRC glands. Individual tumor glands were mechanically separated from thawed CRC tissue pieces using a fluorescence stereo microscope (SMZ18; Nikon) and transferred to a WillCo dish containing PBS and 1:1,000 Syto 11 (S7573; Thermo Fisher Scientific) for image acquisition (LSM510; Zeiss). A small fragment of individual CRC glands was resected for single-cell isolation. Raw Z-stack data were analyzed with Fiji (ImageJ) to obtain the exact cell number of the resected fragment, and a 3D render of the imaged gland was generated using Imaris version 9.3 image analysis software. Single cells of the resected fragment were subsequently isolated as described for PDTOs. The remaining part of the gland was transferred to a single PCR tube as a bulk sample. Karyotyping. PDTOs were treated with 0.1 µg ml−1 colcemid (Thermo Fisher Scientific) in culture medium for 12 h. Then, PDTOs were dissociated into single cells using TrypLE Express, incubated in a hypotonic 27 mM trisodium citrate solution at 37 °C for 10 min and fixed in methanol:acetic acid solution (3:1). Single-cell sequencing. Single-cell sequencing was performed on clonal PDTOs with and without photoconverted daughter cells and on CRC glands. Single-cell sequencing of clonal PDTOs and photoconverted daughter cells. For cells expressing H2B-Dendra2-IRES-Puromycin, single-cell lysis and whole-genome amplification (WGA) were performed using the REPLI-G single-cell kit (Qiagen) according to the manufacturer’s instructions. A positive control (multiple organoids or HEK293T cells) and negative control (3 µl H2O) were included during each WGA reaction. Amplified DNA was purified by phenol:chloroform:isoamyl alcohol (25:24:1) treatment, followed by precipitation with ethanol. To assess the quality of amplified DNA, a multiplex PCR was used to simultaneously amplify nine loci across the human genome in each single cell: SCYL1, PPP5C, JMJD6, ACTR10, ROCK2, SCAP, SCAMP3, SCARB2 and XPOT42. DNA libraries of each single cell, with two or more amplified products in the multiplex PCR, were constructed with the TruSeq Nano DNA library preparation kit (Illumina). The concentration of all libraries was quantified using a Qubit dsDNA HS Assay kit. Library fragment size profiling was performed using the Agilent TapeStation system using Agilent High Sensitivity D1000 ScreenTapes (Agilent Technologies), and subsequently all libraries with unique indices were pooled equimolarly. To support long-term imaging of organoid outgrowth, a dissociated PDTO suspension was filtered twice through a CellTrics 10-μm sieve (Sysmex) to obtain a pure single-cell suspension. Single cells were seeded in BME-coated glass-bottom WillCo dishes as described previously. At 30 h after seeding, the outgrowth of cell doublets was imaged (3-min frame rate; z step = 1 µm). The culture media was refreshed each day to prevent excessive evaporation. Raw data were converted to videos using an ImageJ macro as described39,40. Lineage traces were generated manually. Single-cell sequencing of clonal PDTOs and CRC glands. For cells stained with Syto 11, cell lysis was performed with 0.63 mUA protease (Qiagen) and 1× NEBuffer 4 at 50 °C for 2 h, followed by a protease inactivation incubation for 20 min at 80 °C. DNA libraries were prepared from individual cells with a modified KAPA HyperPlus kit protocol (one-seventh of all volumes; Roche). Cleaned-up pooled single-cell libraries were quantified using a Qubit dsDNA HS Assay kit and analyzed using the Agilent 2100 Bioanalyzer HS kit. Spinning disk live-cell imaging. To support long-term imaging of organoid outgrowth using a spinning disk confocal system (Nikon), 3-d-old organoids carrying a HIST1H2BC-Dendra2 knock-in or expressing transgenic H2B-Dendra were cultured and seeded in BME-coated glass-bottom WillCo dishes as previously described. Methods grown into structures of 60–100 cells before harvesting. Before single-cell isolation, a single PDTO was transferred to a WillCo dish containing phosphate-buffered saline (PBS) (and 1:1,000 Syto 11 (S7573; Thermo Fisher Scientific) for PDTOs lacking H2B-Dendra2-IRES-Puromycin expression) for image acquisition (LSM510; Zeiss). Raw Z-stack data were analyzed with Fiji (ImageJ) to obtain the exact cell number, and a 3D render of the imaged organoid was generated using Imaris version 9.3 image analysis software. To isolate single cells, the imaged organoid was mouth-pipetted to a drop of PBS on a siliconized object slide (Sigmacote; Sigma–Aldrich) and mounted on a fluorescence stereo microscope (SMZ18; Nikon) equipped with a Sola LED (Lumencor) for fluorescence-assisted single-cell picking. The organoid was transferred to a 20-μl drop of trypsin-EDTA (Lonza) for dissociation (30 s). After removing trypsin-EDTA, PBS containing 0.5% bovine serum albumin (Sigma–Aldrich) was added immediately. The organoid structure was further dissociated by repeated mouth-pipetting using a 30-μm glass capillary (custom made). Single cells were transferred to PCR tubes containing either 30 μl mineral oil (Affymetrix) or 4 μl H2O and each tube was immediately spun down and stored ice-cold. Photoconverted daughter cells were isolated as previously described and visualized with a custom filter set on SMZ18 (FF01- 433/24, FF458-Di02 and FF01-482/35). The remaining cells were pooled and transferred to a single PCR tube as a bulk sample. An overview of the single-cell datasets is presented in Supplementary Table 2. Patient-derived organoid culture. CRC patient-derived organoids with the identifiers p9T and p19bT were obtained from a previously established biobank and published study15. PDTO cultures were maintained at 37 °C with 5% CO2 atmosphere, as previously described15. The culture medium contained advanced DMEM/F-12 (Gibco) supplemented with penicillin/streptomycin (Lonza; 10 U ml−1), GlutaMAX (Gibco; 1×), HEPES buffer (Gibco; 10 mM), Noggin-conditioned medium (10%), R-spondin1-conditioned medium (10%), B-27 (Gibco; 1×), nicotinamide (Sigma–Aldrich; 10 mM), N-acetylcysteine (Sigma–Aldrich; 1.25 mM), SB202190 (Gentaur; 10 μM), A83-01 (Tocris; 500 nM) and recombinant human epidermal growth factor (PeproTech; 50 ng ml−1). PDTOs were passaged weekly and maintained below passage 10. Briefly, PDTOs were dissociated using trypsin-EDTA (Sigma–Aldrich) and seeded in Cultrex Reduced Growth Factor Basement Membrane Extract (BME), Type 2 in a pre-warmed 24-well plate. ROCK inhibitor Y-27632 (Gentaur; 10 μM) was added to the culture medium upon plating for 2 d. Generation of H2B-Dendra2 PDTO lineages. PDTO cultures were transduced with lentivirus carrying H2B-Dendra2-IRES-Puromycin (pLV-H2B-Den2-IRES-Puro was a gift from J. van Rheenen). Articles NatUrE GEnEtICS Methods Fixed cells were dropped on a microscope slide, mounted with DAPI-containing Vectashield and imaged on a Zeiss Axio Imager Z1 microscope with a 63× objective. In total, 36 metaphase spreads were imaged for each PDTO line and quantified by manual chromosome counting (Extended Data Fig. 10a). Confocal live-cell microscopy and image analysis. To support live-cell microscopy of cell divisions and subsequent photoconversion of daughter cells, PDTO cultures expressing transgenic H2B-Dendra2-IRES-Puromycin were passaged 5–7 d before imaging. PDTOs were harvested 24 h before imaging and resuspended in an ice-cold mix of culture media containing 50% vol/vol BME. The organoid suspension was then seeded in an ice-cold glass-bottom WillCo dish (WillCo Wells) coated with a thin film of BME. PDTOs were allowed to settle on ice before BME polymerization at 37 °C and the addition of culture media. Cells about to go into mitosis were identified by chromosome condensation and imaged until completion of mitosis with a confocal laser-scanning microscope (Leica SP8X, z step 1 µm, 30-s frame rate) equipped with atmospheric and temperature control. Daughter cells of imaged cell divisions were marked via photoconversion of Dendra2 (405 nm laser; 1 min; 10% laser power; per region of interest). Imaging data were analyzed and 3D rendered with Imaris version 9.3 image analysis software (Oxford Instruments). Single-cell sequencing. Single-cell sequencing was performed on clonal PDTOs with and without photoconverted daughter cells and on CRC glands. Nature Genetics \| VOL 53 \| August 2021 \| 1187–1195 \| www.nature.com/naturegenetics Nature Genetics \| VOL 53 \| August 2021 \| 1187–1195 \| www.nature.com/naturegenetics Nature Genetics \| VOL 53 \| August 2021 \| 1187–1195 \| www.nature.com/naturegenetics 1195 Articles References 37. Koo, B. K. Generation of FLIP and FLIP-FlpE targeting vectors for biallelic conditional and reversible gene knockouts in mouse and human cells. Methods Mol. Biol. 1842, 255–264 (2018).fi 38. Fujii, M., Matano, M., Nanki, K. & Sato, T. Efficient genetic engineering of human intestinal organoids using electroporation. Nat. Protoc. 10, 1474–1485 (2015). 39. Drost, J. et al. Sequential cancer mutations in cultured human intestinal stem cells. Nature 521, 43–47 (2015). For cell numbers 1–3 and 15 of the clonal PDTO dataset presented in Fig. 3 with 15× or 30× coverage, SNV and indel calling was performed using GATK HaplotypeCaller. The allele frequency of variants located on chromosome 9 (genomic location = 89502430–141213431) in sequences 1 (SC11-20190409_1) and 15 (SC15-20190409_1) (core) were calculated by subtracting all of the variants present on the allele of sequences 2 (SC10-20190409_1) and 3 (SC17-20190409_1) (Fig. 3b). Density plots of the allele frequencies of variants were generated using the R package ggplot2. Manta (version 0.29.5) was used with standard settings to detect structural variants. To exclude structural variants introduced by the WGA (short-range chimeras of the inverted orientation), we established a threshold of structural variant length for which a similar count in inverted and non-inverted structural variants was observed. Next, we estimated the frequency of structural variants (subdivided based on threshold structural variant length) on all control chromosomes and on the missegregated chromosome to determine enrichment of structural variants in the missegregated chromosome. 40. Verissimo, C. S. et al. Targeting mutant RAS in patient-derived colorectal cancer organoids by combinatorial drug screening. eLife https://doi. org/10.7554/eLife.18489 (2016). 41. Cross, W. et al. Stabilising selection causes grossly altered but stable karyotypes in metastatic colorectal cancer. Preprint at bioRxiv https://doi.org/10.1101/2020.03.26.007138 (2020). 42. Swennenhuis, J. F. Unmasking Circulating Tumor Cells. PhD thesis, Univ. Twente (2017). 43. Garvin, T. et al. Interactive analysis and assessment of single-cell copy-number variations. Nat. Methods 12, 1058–1060 (2015).i 44. Williams, M. J. et al. Quantification of subclonal selection in cancer from bulk sequencing data. Nat. Genet. 50, 895–903 (2018). 45. Toni, T., Welch, D., Strelkowa, N., Ipsen, A. & Stumpf, M. P. Approximate Bayesian computation scheme for parameter inference and model selection in dynamical systems. J. R. Soc. Interface 6, 187–202 (2009). Parameter estimation and model selection. We used a stochastic birth–death branching process to model the outgrowth of PDTOs (Supplementary Note 4). References Since apoptotic events were rare in the 3D Live-Seq datasets, we modeled only cell birth with rate b (per day). During each cell division, n1 reciprocal chromosome-level CNAs and n2 reciprocal arm-level CNAs were introduced to daughter cells, where n1 = ~Pois(μ1) and n2 = ~Pois(μ2). We assumed two models of evolution: a neutral model where all cells have the same fitness; and a selection model in which cells with novel mutation(s) have a selective (dis) advantage, s, given by bd bp = 1 + s, where bp and bd are the birth rate of the Parameter estimation and model selection. We used a stochastic birth–death branching process to model the outgrowth of PDTOs (Supplementary Note 4). Since apoptotic events were rare in the 3D Live-Seq datasets, we modeled only cell birth with rate b (per day). During each cell division, n1 reciprocal chromosome-level CNAs and n2 reciprocal arm-level CNAs were introduced to daughter cells, where n1 = ~Pois(μ1) and n2 = ~Pois(μ2). We assumed two models of evolution: a neutral model where all cells have the same fitness; and a selection model in which cells with novel mutation(s) have a selective (dis) advantage, s, given by bd bp = 1 + s, where bp and bd are the birth rate of the parent and daughter cells respectively. The model parameters, θ = (μ1,μ2,b,s), were inferred using approximate Bayesian computation with sequential Monte Carlo sampling44,45. Using simulated data, we validated that the true parameters were inferred accurately. For datasets without recorded mitotic trees, we used single-cell copy-number profiles to estimate mutation rates under the neutral model (assuming b = 0.5). For datasets with mitotic trees, we estimated all parameters under both models and selected the model that best fit the data using deviance information criteria46. We also examined the power of our approach to detect negative selection at varying selection strengths and cell population sizes. This was done by simulating 100 datasets for each parameter setting, with the parameters estimated from real data (μ1 = 0.1, μ2 = 0.1 and b = 0.4) using the approximate Bayesian computation rejection algorithm, and computing deviance information criteria under the neutral and selection models for each dataset. y y f 46. François, O. & Laval, G. Deviance information criteria for model selection in approximate Bayesian computation. Stat. Appl. Genet. Mol. Biol. https://doi.org/10.2202/1544-6115.1678 (2011). Code availability Full pipeline descriptions and settings used for mapping are available at https:// github.com/UMCUGenetics/IAP. Ginkgo, for the analysis and assessment of single-cell CNAs, is publicly available at https://github.com/robertaboukhalil/ ginkgo. The code for modeling real data (the stochastic branching process and likelihood ratio test) is freely available at https://github.com/ucl-cssb/CIN_PDO or https://doi.org/10.5281/zenodo.4762533. To identify high-confidence copy-number variations, regions of at least 25 Mb (excluding bins greater than 5 Mb) with copy-number values deviating >0.6 from the average ploidy were considered to indicate losses or gains. Copy-number variations smaller than 25 Mb were only included if both the loss and gain of that particular region were identified in the clonally expanded organoid, photoconverted daughter cells or CRC gland. To avoid missing copy-number variations, less confident variations (>0.3 and ≤0.6 below or above the average ploidy) with >70% reciprocal overlap between high-confidence variations were reviewed. Heatmaps were generated using the R (version 3.4.3) package ggplot2. All de novo CNA events observed in PDTO datasets are shown in Extended Data Fig. 10b using the R package karyoploteR. NatUrE GEnEtICS Genomic analysis. Sequence reads were aligned to the human genome reference (hg19/GRCh37) using the Burrows–Wheeler Aligner mapping tool (BWA-MEM; version 0.7.15). Duplicated sequence reads were marked by Sambamba (version 0.6.5) and realigned using the Genome Analysis Toolkit IndelRealigner (GATK; version 3.8), and sequence read quality scores were recalibrated with GATK BaseRecalibrator. European Bioinformatics Institute and Centre for Genomic Regulation (https:// ega-archive.org) under accession number EGAS00001003812. Data access requests will be evaluated by the University Medical Center Utrecht Department of Genetics Data Access Board (EGAC00001000432) and transferred on completion of an agreement and authorization by the medical ethical committee at the University Medical Center Utrecht at the request of Hubrecht Organoid Technology to ensure compliance with the Dutch Medical Research Involving Human Subjects Act. The copy-number segment calls of all sequenced single cells are publicly available on Zenodo (https://doi.org/10.5281/zenodo.4732372). The copy-number status of each single cell was analyzed using Ginkgo43. Briefly, the created BAM files were converted to BED format files using BEDtools (version 2.25.0). Then, the aligned reads were binned into 1-Mb variable-length intervals across the genome, normalized and corrected for GC biases, and segmented in regions with an equal copy-number status. Thereafter, a final integer copy-number profile was assigned to each single cell. Single cells that exhibited low-quality sequencing data, defined as high variation between copy-number ratios of segmented regions, were excluded. One exception was made for cell number 9 of dataset 04032020, as this cell clearly belonged to the lineage with segmental loss of chromosome 1p. Acknowledgements Representative snapshots of organoid outgrowth were extracted from imaging data at evenly distributed time points across the imaging span. The numbers of samples analyzed are indicated when applicable. Statistics and reproducibility. Imaging stills in the manuscript represent (time-compressed) representations of the entire captured imaging data. Representative snapshots of organoid outgrowth were extracted from imaging data at evenly distributed time points across the imaging span. The numbers of samples analyzed are indicated when applicable. Author contributions H.J.G.S., W.P.K., Y.B. and K.C.O. conceived of and initiated the project. Y.B., E.S. and P.v.L. designed, performed and quantified most of the experiments. Y.B., P.v.L. and M.v.d.B. maintained the organoid culture and performed the genome editing, confocal imaging and single-cell isolation. E.S. and N.J.M.B. performed the WGA and library preparations. E.S. performed sequencing data analysis and chromosome spreads. M.J.v.R., B.v.d.R. and S.B. helped with the computational analysis. A.C.F.B. performed imaging and quantification of the Dendra2 knock-in organoids. B.P. provided imaging support and ImageJ macros. S.G.d.V. assisted with cloning. B.L., Reporting Summary. Further information on research design is available in the Nature Research Reporting Summary linked to this article. Acknowledgements We thank members of the Snippert, Terstappen, Kloosterman, Cuppen, Sottoriva and Graham laboratories for reagents, suggestions and discussions, and A. van Oudenaarden and J. Korving for providing custom glass capillaries. We thank the Utrecht Sequencing Facility and Hartwig Medical Foundation for providing sequencing services and data. The Utrecht Sequencing Facility is subsidized by the University Medical Center Utrecht, Hubrecht Institute, Utrecht University and The Netherlands X-omics Initiative (NWO). We thank the University College London Hospital (UCLH) Biobank, led by M. Rodriguez-Justo, for providing patient materials. This work was supported by the Wellcome Trust (202778/B/16/Z to A.S.; 202778/Z/16/Z to T.A.G., supporting C.K. and W.C.H.C.; 105104/Z/14/Z to the Centre for Evolution and Cancer, The Institute of Cancer Research; and 097319/Z/11/Z to C.P.B., supporting B.L.) and Cancer Research UK (A22909 to A.S.; and A19771 to T.A.G.). A.S. and T.A.G. received support from the US National Institutes of Health National Cancer Institute (U54 CA217376), supporting M.M. This work was facilitated by the Oncode Institute, which is partly financed by the Dutch Cancer Society and was funded by the Gieskes Strijbis Foundation (1816199), a grant from the Dutch Cancer Society (UU 20130-6070), an ERC starting grant (IntratumoralNiche) and Health Holland to H.J.G.S. Y.B. was supported by a strategic alliance between the University of Twente and UMC Utrecht on Advanced Biomanufacturing (to L.W.M.M.T. and H.J.G.S.). p parent and daughter cells respectively. The model parameters, θ = (μ1,μ2,b,s), were inferred using approximate Bayesian computation with sequential Monte Carlo sampling44,45. Using simulated data, we validated that the true parameters were inferred accurately. For datasets without recorded mitotic trees, we used single-cell copy-number profiles to estimate mutation rates under the neutral model (assuming b = 0.5). For datasets with mitotic trees, we estimated all parameters under both models and selected the model that best fit the data using deviance information criteria46. We also examined the power of our approach to detect negative selection at varying selection strengths and cell population sizes. This was done by simulating 100 datasets for each parameter setting, with the parameters estimated from real data (μ1 = 0.1, μ2 = 0.1 and b = 0.4) using the approximate Bayesian computation rejection algorithm, and computing deviance information criteria under the neutral and selection models for each dataset. Statistics and reproducibility. Imaging stills in the manuscript represent (time-compressed) representations of the entire captured imaging data. Nature Genetics \| www.nature.com/naturegenetics Single-cell sequencing. Single-cell sequencing was performed on clonal PDTOs with and without photoconverted daughter cells and on CRC glands. The outgrowth was captured for 72 h on days 4–6 and days 9–11 (4.5-min frame rate; z step = 1.4 µm). The imaging data were analyzed with Fiji (ImageJ). Low-coverage whole-genome single-end 75-base pair sequencing was performed on an Illumina NextSeq 500 aimed at generating ~5 million reads per single cell for the WGA TruSeq libraries and more than 100,000 reads for the KAPA libraries. For a subset of WGA TruSeq libraries (cell numbers 1–3 and 15 of the clonal PDTO dataset presented in Fig. 3), whole-genome paired-end sequencing (2 × 150 base pairs) was performed on an Illumina NovaSeq 6000 to a coverage of 15× (cell numbers 2 and 3) or 30× (cell numbers 1 and 15). Single-cell isolation of PDTOs. To generate clonal PDTOs, a dissociated PDTO suspension was filtered twice through a CellTrics 10-μm sieve to obtain a pure single-cell suspension and seeded at medium density in BME. Clonal PDTOs were Nature Genetics \| www.nature.com/naturegenetics Articles NatUrE GEnEtICS Additional information C.P.B. and T.A.G. designed and performed the mathematical modeling. S.M.A.L., A.S., T.A.G., E.C., H.R., C.P.B., W.C.H.C., G.J.P.L.K. and W.P.K. assisted with data interpretation. T.A.G., M.M., C.K. and W.C.H.C. provided and processed the human cancer glands. L.W.M.M.T. and H.J.G.S. obtained funding. Y.B., E.S. and H.J.G.S. wrote the manuscript. All authors edited and approved the manuscript. Additional information Extended data is available for this paper at https://doi.org/10.1038/s41588-021-00891-2. Supplementary information The online version contains supplementary material available at https://doi.org/10.1038/s41588-021-00891-2. Correspondence and requests for materials should be addressed to H.J.G.S. Peer review information Nature Genetics thanks Karuna Ganesh and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Reprints and permissions information is available at www.nature.com/reprints. Additional information Extended data is available for this paper at https://doi.org/10.1038/s41588-021-00891-2. Supplementary information The online version contains supplementary material available at https://doi.org/10.1038/s41588-021-00891-2. Correspondence and requests for materials should be addressed to H.J.G.S. Peer review information Nature Genetics thanks Karuna Ganesh and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Reprints and permissions information is available at www.nature.com/reprints. Articles C.P.B. and T.A.G. designed and performed the mathematical modeling. S.M.A.L., A.S., T.A.G., E.C., H.R., C.P.B., W.C.H.C., G.J.P.L.K. and W.P.K. assisted with data interpretation. T.A.G., M.M., C.K. and W.C.H.C. provided and processed the human cancer glands. L.W.M.M.T. and H.J.G.S. obtained funding. Y.B., E.S. and H.J.G.S. wrote the manuscript. All authors edited and approved the manuscript. Competing interests The authors declare no competing interests. Additional information Extended data is available for this paper at https://doi.org/10.1038/s41588-021-00891-2. Supplementary information The online version contains supplementary material available at https://doi.org/10.1038/s41588-021-00891-2. Correspondence and requests for materials should be addressed to H.J.G.S. Peer review information Nature Genetics thanks Karuna Ganesh and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Reprints and permissions information is available at www.nature.com/reprints. Nature Genetics \| www.nature.com/naturegenetics Data availability BAM files of the single-cell sequencing data are available through controlled access at the European Genome-phenome Archive (EGA), which is hosted by the Nature Genetics \| www.nature.com/naturegenetics Competing interests The authors declare no competing interests. Articles NatUrE GEnEtICS Extended Data Fig. 1 \| Full and additional whole population PDTO sequencing datasets of PDTO-9. a, Full PDTO-9 dataset of experiment shown in Fig. 1b. Karyotype heatmap showing 62 cells derived from a clonal PDTO-9 structure expressing transgenic H2B-Dendra2 and consisting of 67 cells (93% recovery). Reciprocal gains and losses are indicated with red boxes. Dashed boxes indicate CNA events where a reciprocal loss or gain is missing. Sub-chromosomal CNAs were counted as events when represented in more than one cell. Scalebar is 10μm. b, As in a. 64 out of 72 cells provided quality sequence (89% recovery). Parallel emergence of de novo whole-chromosome missegregations of Chr.15 (I, II) was determined by co-occurrence of a sub-chromosomal CNA in Chr.3. c, As in a. 33 out of 37 cells provided quality sequence (89% recovery). Two lineages show amplified sub-chromosomal CNAs in one cell, while all other cells in the lineage show a reciprocal loss of the same region (III. IV). Consistent with replication and collective missegregation of acentric chromosomal fragments. See Fig. 3. d, As in a. Genetically unmodified PDTO-9 organoid. Cells were stained with Syto 11 to allow single-cell picking. 85 out of 91 cells provided quality sequence (93% recovery). Extended Data Fig. 1 \| Full and additional whole population PDTO sequencing datasets of PDTO-9. a, F Extended Data Fig. 1 \| Full and additional whole population PDTO sequencing datasets of PDTO-9. a, Full PDTO-9 dataset of experiment shown in Fig. 1b. Karyotype heatmap showing 62 cells derived from a clonal PDTO-9 structure expressing transgenic H2B-Dendra2 and consisting of 67 cells (93% recovery). Reciprocal gains and losses are indicated with red boxes. Dashed boxes indicate CNA events where a reciprocal loss or gain is missing. Sub-chromosomal CNAs were counted as events when represented in more than one cell. Scalebar is 10μm. b, As in a. 64 out of 72 cells provided quality sequence (89% recovery). Parallel emergence of de novo whole-chromosome missegregations of Chr.15 (I, II) was determined by co-occurrence of a sub-chromosomal CNA in Chr.3. c, As in a. 33 out of 37 cells provided quality sequence (89% recovery). Two lineages show amplified sub-chromosomal CNAs in one cell, while all other cells in the lineage show a reciprocal loss of the same region (III. IV). Consistent with replication and collective missegregation of acentric chromosomal fragments. See Fig. 3. Nature Genetics \| www.nature.com/naturegenetics Nature Genetics \| www.nature.com/naturegenetics Competing interests d, As in a. Genetically unmodified PDTO-9 organoid. Cells were stained with Syto 11 to allow single-cell picking. 85 out of 91 cells provided quality sequence (93% recovery). Nature Genetics \| www.nature.com/naturegenetics Articles NatUrE GEnEtICS Extended Data Fig. 2 \| Full and additional whole population PDTO sequencing datasets of PDTO-19b. a, Full PDTO-19b dataset of experiment shown in Fig. 1c. Karyotype heatmap showing 58 cells derived from a clonal PDTO-19b structure expressing transgenic H2B-Dendra2 and consisting of 67 cells (87% recovery). Reciprocal gains and losses are indicated with red boxes. Dashed boxes indicate CNA events where a reciprocal loss or gain is missing. Sub-chromosomal CNAs were counted as events when represented in more than one cell. The bottom panel shows a population of polyploid cells with large deviations from the core karyotype (hopeful monsters). Two cells show reciprocal gains and losses across their genome (red brackets). Scalebar is 10μm. b, As in a. Genetically unmodified PDTO-19b organoid. Cells were stained with Syto 11 to allow single cell picking. 40 out of 45 cells provided quality sequence (89% recovery). c, As in a. Genetically unmodified PDTO-19b organoid. Cells were stained with Syto 11 to allow single cell picking. 52 out of 70 cells provided quality sequence (74% recovery). d, As in a. 21 out of 29 cells were sequenced (72% recovery). The previously reported TP53 mutation (chr 17: 7577121 G > A) for PDTO-19b [van de Wetering, 2016] was confirmed in all sequenced clonal organoids. Articles NatUrE GEnEtICS Extended Data Fig. 2 \| Full and additional whole population PDTO sequencing datasets of PDTO-19b. a, Full PDTO-19b dataset of experiment shown in Fig. 1c. Karyotype heatmap showing 58 cells derived from a clonal PDTO-19b structure expressing transgenic H2B-Dendra2 and consisting of 67 cells (87% recovery). Reciprocal gains and losses are indicated with red boxes. Dashed boxes indicate CNA events where a reciprocal loss or gain is missing. Sub-chromosomal CNAs were counted as events when represented in more than one cell. The bottom panel shows a population of polyploid cells with large deviations from the core karyotype (hopeful monsters). Two cells show reciprocal gains and losses across their genome (red brackets). Scalebar is 10μm. b, As in a. Genetically unmodified PDTO-19b organoid. Cells were stained with Syto 11 to allow single cell picking. 40 out of 45 cells provided quality sequence (89% recovery). c, As in a. Genetically unmodified PDTO-19b organoid. Competing interests Cells were stained with Syto 11 to allow single cell picking. 52 out of 70 cells provided quality sequence (74% recovery). d, As in a. 21 out of 29 cells were sequenced (72% recovery). The previously reported TP53 mutation (chr 17: 7577121 G > A) for PDTO-19b [van de Wetering, 2016] was confirmed in all sequenced clonal organoids. Extended Data Fig. 2 \| Full and additional whole population PDTO sequencing datasets of PDTO-19b. a, Full PDTO-19b dataset of experiment shown in Fig. 1c. Karyotype heatmap showing 58 cells derived from a clonal PDTO-19b structure expressing transgenic H2B-Dendra2 and consisting of 67 cells (87% recovery). Reciprocal gains and losses are indicated with red boxes. Dashed boxes indicate CNA events where a reciprocal loss or gain is missing. Sub-chromosomal CNAs were counted as events when represented in more than one cell. The bottom panel shows a population of polyploid cells with large deviations from the core karyotype (hopeful monsters). Two cells show reciprocal gains and losses across their genome (red brackets). Scalebar is 10μm. b, As in a. Genetically unmodified PDTO-19b organoid. Cells were stained with Syto 11 to allow single cell picking. 40 out of 45 cells provided quality sequence (89% recovery). c, As in a. Genetically unmodified PDTO-19b organoid. Cells were stained with Syto 11 to allow single cell picking. 52 out of 70 cells provided quality sequence (74% recovery). d, As in a. 21 out of 29 cells were sequenced (72% recovery). The previously reported TP53 mutation (chr 17: 7577121 G > A) for PDTO-19b [van de Wetering, 2016] was confirmed in all sequenced clonal organoids. Nature Genetics \| www.nature.com/naturegenetics Articles NatUrE GEnEtICS Articles NatUrE GEnEtICS Articles NatUrE GEnEtICS Extended Data Fig. 3 \| Copy-number profiles of hopeful monster karyotypes. a, Reciprocal copy-number profiles of two PDTO-19b cells with hopeful monster karyotypes are shown (Fig. 1c, III). Since the ancestor karyotype of these two cells is unclear, reciprocity between hopeful monster karyotypes refers to the observation that all chromosomal segments add up to an even number. Grey dots indicate bin ratios (Bin size: 1 Mbp). Integer copy-number states are indicated with black (no deviation of a tetraploid state), blue (loss) and orange (gain) lines. Below: The copy-number profile of the merged sequencing data of the two hopeful monsters mimics a duplicated core karyotype of the sequenced PDTO-19b organoid. Nature Genetics \| www.nature.com/naturegenetics Competing interests Orange boxes indicate CNAs in the merged profile that deviate from a duplication of the core karyotype, indicating that these CNAs were present in the ancestor cell. b, Reciprocal copy-number profiles of three PDTO-9 cells with hopeful monster karyotypes are shown (Fig. 4a). Grey dots indicate bin ratios (Bin size: 1 Mbp). Integer copy-number states are indicated with black (no deviation of a tetraploid state), blue (loss) and orange (gain) lines. Adding up the copy-number state of chromosome segments across all three hopeful monster karyotypes results in a twice replicated genome (‘ploidy 8’, not shown). Below: The copy-number profile of the merged sequencing data mimics the core karyotype carrying a de novo loss of 1pter-p34.2 (orange box). Extended Data Fig. 3 \| Copy-number profiles of hopeful monster karyotypes. a, Reciprocal copy-number profiles of two PDTO-19b cells with hopeful monster karyotypes are shown (Fig. 1c, III). Since the ancestor karyotype of these two cells is unclear, reciprocity between hopeful monster karyotypes refers to the observation that all chromosomal segments add up to an even number. Grey dots indicate bin ratios (Bin size: 1 Mbp). Integer copy-number states are indicated with black (no deviation of a tetraploid state), blue (loss) and orange (gain) lines. Below: The copy-number profile of the merged sequencing data of the two hopeful monsters mimics a duplicated core karyotype of the sequenced PDTO-19b organoid. Orange boxes indicate CNAs in the merged profile that deviate from a duplication of the core karyotype, indicating that these CNAs were present in the ancestor cell. b, Reciprocal copy-number profiles of three PDTO-9 cells with hopeful monster karyotypes are shown (Fig. 4a). Grey dots indicate bin ratios (Bin size: 1 Mbp). Integer copy-number states are indicated with black (no deviation of a tetraploid state), blue (loss) and orange (gain) lines. Adding up the copy-number state of chromosome segments across all three hopeful monster karyotypes results in a twice replicated genome (‘ploidy 8’, not shown). Below: The copy-number profile of the merged sequencing data mimics the core karyotype carrying a de novo loss of 1pter-p34.2 (orange box). NatUrE GEnEtICS Extended Data Fig. 4 \| Transgenic H2B expression does not exacerbate CIN. a, The HIST1H2BC locus, coding for most abundant Histone H2B, was targeted to fluorescently tag H2B1C with Dendra2. Cas9 nickase (D10A) was targeted to the endogenous STOP codon, as well as both sites of the homology arms flanking the donor template. Competing interests b, 18 days post electroporation, H2B-Dendra2 positive cells were isolated using FACS. Overall knock-in efficiency of 1.7% (not corrected for electroporation efficiency of ~10%). c, Similar CIN phenotypes of PDTO-9 expressing transgenic H2B-Dendra2 or carrying a HIST1H2BC-Dendra2 knock-in. 649 divisions across 34 organoids and 183 divisions across 27 organoids were scored from the transgenic and knock-in line respectively. Difference in scored chromatin error rates is statistically significant (Chi square test, p = 0.031), although the error rate in the knock-in line is likely underreported due to the lower brightness of endogenous H2B-Dendra2 expression. d, Fluorescent signal of H2B-Dendra was compared between PDTO-9 lines expressing transgenic H2B-Dendra2 (n = 95 cells, 19 organoids) or carrying a HIST1H2BC-Dendra2 knock-in (n = 100 cells, 20 organoids). Data are represented as box-and-whisker plots; boxes represent quartiles 2 and 3, the horizontal line represents the mean and whiskers represent minima and maxima within the 1.5× interquartile range. Extended Data Fig. 4 \| Transgenic H2B expression does not exacerbate CIN. a, The HIST1H2BC locus, coding for most abundant Histone H2B, was targeted to fluorescently tag H2B1C with Dendra2. Cas9 nickase (D10A) was targeted to the endogenous STOP codon, as well as both sites of the homology arms flanking the donor template. b, 18 days post electroporation, H2B-Dendra2 positive cells were isolated using FACS. Overall knock-in efficiency of 1.7% (not corrected for electroporation efficiency of ~10%). c, Similar CIN phenotypes of PDTO-9 expressing transgenic H2B-Dendra2 or carrying a HIST1H2BC-Dendra2 knock-in. 649 divisions across 34 organoids and 183 divisions across 27 organoids were scored from the transgenic and knock-in line respectively. Difference in scored chromatin error rates is statistically significant (Chi square test, p = 0.031), although the error rate in the knock-in line is likely underreported due to the lower brightness of endogenous H2B-Dendra2 expression. d, Fluorescent signal of H2B-Dendra was compared between PDTO-9 lines expressing transgenic H2B-Dendra2 (n = 95 cells, 19 organoids) or carrying a HIST1H2BC-Dendra2 knock-in (n = 100 cells, 20 organoids). Data are represented as box-and-whisker plots; boxes represent quartiles 2 and 3, the horizontal line represents the mean and whiskers represent minima and maxima within the 1.5× interquartile range. Extended Data Fig. 4 \| Transgenic H2B expression does not exacerbate CIN. a, The HIST1H2BC locus, codin Extended Data Fig. 4 \| Transgenic H2B expression does not exacerbate CIN. Nature Genetics \| www.nature.com/naturegenetics Nature Genetics \| www.nature.com/naturegenetics Competing interests a, The HIST1H2BC locus, coding for most abundant Histone H2B, was targeted to fluorescently tag H2B1C with Dendra2. Cas9 nickase (D10A) was targeted to the endogenous STOP codon, as well as both sites of the homology arms flanking the donor template. b, 18 days post electroporation, H2B-Dendra2 positive cells were isolated using FACS. Overall knock-in efficiency of 1.7% (not corrected for electroporation efficiency of ~10%). c, Similar CIN phenotypes of PDTO-9 expressing transgenic H2B-Dendra2 or carrying a HIST1H2BC-Dendra2 knock-in. 649 divisions across 34 organoids and 183 divisions across 27 organoids were scored from the transgenic and knock-in line respectively. Difference in scored chromatin error rates is statistically significant (Chi square test, p = 0.031), although the error rate in the knock-in line is likely underreported due to the lower brightness of endogenous H2B-Dendra2 expression. d, Fluorescent signal of H2B-Dendra was compared between PDTO-9 lines expressing transgenic H2B-Dendra2 (n = 95 cells, 19 organoids) or carrying a HIST1H2BC-Dendra2 knock-in (n = 100 cells, 20 organoids). Data are represented as box-and-whisker plots; boxes represent quartiles 2 and 3, the horizontal line represents the mean and whiskers represent minima and maxima within the 1.5× interquartile range. Nature Genetics \| www.nature.com/naturegenetics Articles NatUrE GEnEtICS Articles NatUrE GEnEtICS Extended Data Fig. 5 \| Replication and collective missegregation of Chr.9q21.33-ter did not result in chromothripsis. a, Length distribution of inverted and non-inverted structural variants (SVs) in four selected single cells of the dataset presented in Fig. 3 (cell 1 (5 copies of a chr9.q fragment)), cell 2 and 3 (1 copy) and a cell with the core karyotype (diploid)). The enrichment of short-range inverted SVs can be explained by the WGA reaction. A similar count in inverted and non-inverted SVs was observed for SVs with a length ≥10 kb. b, The frequency of SVs (length ≥ 10 kb) on all control chromosomes and on the missegregated chr.9q21.33-ter fragment. No enrichment of SVs was observed in the chr.9q21.33-ter region for Cell No. 1 vs. Cell No. 2/3 which suggests that chromothripsis did not occur (p = 1, two-sided Fisher’s exact test). Extended Data Fig. 5 \| Replication and collective missegregation of Chr.9q21.33-ter did not result in chromothripsis. a, Length distribution of inverted and non-inverted structural variants (SVs) in four selected single cells of the dataset presented in Fig. 3 (cell 1 (5 copies of a chr9.q fragment)), cell 2 and 3 (1 copy) and a cell with the core karyotype (diploid)). The enrichment of short-range inverted SVs can be explained by the WGA reaction. A similar count in inverted and non-inverted SVs was observed for SVs with a length ≥10 kb. b, The frequency of SVs (length ≥ 10 kb) on all control chromosomes and on the missegregated chr.9q21.33-ter fragment. No enrichment of SVs was observed in the chr.9q21.33-ter region for Cell No. 1 vs. Cell No. 2/3 which suggests that chromothripsis did not occur (p = 1, two-sided Fisher’s exact test). Articles NatUrE GEnEtICS Extended Data Fig. 6 \| 3D Live-Seq dataset of a PDTO-9 organoid consisting of 23 cells. a, 3D Live-Seq dataset of a PDTO-9 organoid consisting of 23 cells of which 21 cells were whole-genome sequenced (91% recovery). Top: representative stills of the growing PDTO-9 structure with nuclei in false color depth code. Below: full mitotic tree with 3D-rendered stills of each anaphase. Onset of anaphase is indicated by arrowheads in relation to the time axis (in hours). Bottom panel shows a karyotype heatmap of all cells of the imaged PDTO. Lineage I: one cell carries four copies of Chr1q24.2-ter and three cells carry the reciprocal loss. Nature Genetics \| www.nature.com/naturegenetics Articles NatUrE GEnEtICS b, Minimal phylogenic structure of lineage I, which only fits branch 1.2 (highlighted in orange) of the mitotic tree. Obtaining four copies of Chr1q24.2-ter involves one round of replication and collective missegregation of the Chr1q fragments (Fig. 3). Extended Data Fig. 6 \| 3D Live-Seq dataset of a PDTO-9 organoid consisting of 23 cells. a, 3D Live-Seq dataset of a PDTO-9 organoid consisting of 23 cells of which 21 cells were whole-genome sequenced (91% recovery). Top: representative stills of the growing PDTO-9 structure with nuclei in false color depth code. Below: full mitotic tree with 3D-rendered stills of each anaphase. Onset of anaphase is indicated by arrowheads in relation to the time axis (in hours). Bottom panel shows a karyotype heatmap of all cells of the imaged PDTO. Lineage I: one cell carries four copies of Chr1q24.2-ter and three cells carry the reciprocal loss. b, Minimal phylogenic structure of lineage I, which only fits branch 1.2 (highlighted in orange) of the mitotic tree. Obtaining four copies of Chr1q24.2-ter involves one round of replication and collective missegregation of the Chr1q fragments (Fig. 3). Extended Data Fig. 6 \| 3D Live-Seq dataset of a PDTO-9 organoid consisting of 23 cells. a, 3D Live-Seq dataset of a PDTO-9 organoid consisting of 23 cells of which 21 cells were whole-genome sequenced (91% recovery). Top: representative stills of the growing PDTO-9 structure with nuclei in false color depth code. Below: full mitotic tree with 3D-rendered stills of each anaphase. Onset of anaphase is indicated by arrowheads in relation to the time axis (in hours). Bottom panel shows a karyotype heatmap of all cells of the imaged PDTO. Lineage I: one cell carries four copies of Chr1q24.2-ter and three cells carry the reciprocal loss. b, Minimal phylogenic structure of lineage I, which only fits branch 1.2 (highlighted in orange) of the mitotic tree. Obtaining four copies of Chr1q24.2-ter involves one round of replication and collective missegregation of the Chr1q fragments (Fig. 3). Extended Data Fig. 6 \| 3D Live-Seq dataset of a PDTO-9 organoid consisting of 23 cells. a, 3D Live-Seq dataset of a PD Artic rE GEnEtICS d Data Fig. 7 \| Fate of mitotic slippage events. a, Bar graph showing the fate of traced mitotic slippage events in PDTO-9 expressing transg dra2. 99 mitotic slippage events were traced, each from independent organoids. Nature Genetics \| www.nature.com/naturegenetics Articles NatUrE GEnEtICS 28 slippage events resulted in downstream apoptosis or m included in the statistics. Mitotic events were segregated into bipolar or multipolar spindles based on their chromatin phenotype. b, Imag ing the fate of all mitotic cells (n = 12). Spindle polarity is indicated per division. Scalebar is 10μm. White arrows indicate daughter nuclei. c of progeny from 19 multipolar spindle divisions of any origin. Articles NatUrE GEnEtICS Artic UrE GEnEtICS ed Data Fig. 7 \| Fate of mitotic slippage events. a, Bar graph showing the fate of traced mitotic slippage events in PDTO-9 expressing transg endra2. 99 mitotic slippage events were traced, each from independent organoids. 28 slippage events resulted in downstream apoptosis or Extended Data Fig. 7 \| Fate of mitotic slippage events. a, Bar graph showing the fate of traced mitotic slippage events in PDTO-9 expressing transgenic H2B-Dendra2. 99 mitotic slippage events were traced, each from independent organoids. 28 slippage events resulted in downstream apoptosis or mitosis and were included in the statistics. Mitotic events were segregated into bipolar or multipolar spindles based on their chromatin phenotype. b, Imaging stills tracing the fate of all mitotic cells (n = 12). Spindle polarity is indicated per division. Scalebar is 10μm. White arrows indicate daughter nuclei. c, Fate analysis of progeny from 19 multipolar spindle divisions of any origin. Nature Genetics \| www.nature.com/naturegenetics Articles NatUrE GEnEtICS Articles NatUrE GEnEtICS Extended Data Fig. 8 \| Model selection and cell fate. a, Stochastic branching process model of mitosis in a PDTO. Cell division rate = b (per day). Per division, de novo reciprocal chromosomal (chr-level) or sub-chromosomal (arm-level) CNAs accumulate at rates μ1 and μ2, respectively. Division branches: normal (black), chr-level CNAs (blue), arm-level CNAs (green). Under a selection model de novo CNAs may confer selective (dis)advantages (cells in green and blue). The heatmap on the right shows copy number states of cells in the final population (Grey: 2 N; Light red: 3 N; Dark red: 4 N; Light blue: 1 N). Chr. N copy numbers (orange rectangular box) correspond to chromosomes depicted on the left. Summary statistics were derived from the mitotic tree and copy number states, and used for inference with approximate Bayesian computation (ABC). b, CNA rates and cell division rates estimated from seven individual PDTOs (four PDTOs with mitotic trees) using ABC under the neutral model. Articles NatUrE GEnEtICS Violin plots (posterior distributions) and box plots were generated from n = 500 independent samples. Box plots: boxes represent quartiles 2 and 3, horizontal lines represents median; whiskers extend to 1.5× of the interquartile range; data beyond interquartile range are plotted individually. c, Model selection using deviance information criteria (DIC, smaller values indicate better support) suggests the neutral model is better supported (100 replicates). Box plot criteria as in b. d, Power analysis with DIC on the detectability of negative selection in simulated data (n = 100 per parameter setting). Top: number of times each model is supported with different selection strengths and cell numbers. Bottom: differences of DIC values computed under the two models. With increasing strength of negative selection or more cells, the power to detect negative selection increases. Box plots as in c. e, As in Fig. 5e but for an independent parental organoid. 28 organoids share the reference PDTO-9 core karyotype, 4 were polyploid and 2 had de novo localized CNAs. Extended Data Fig. 8 \| Model selection and cell fate. a, Stochastic branching process model of mitosis in a PDTO. Cell division rate = b (per day). Per division, de novo reciprocal chromosomal (chr-level) or sub-chromosomal (arm-level) CNAs accumulate at rates μ1 and μ2, respectively. Division branches: normal (black), chr-level CNAs (blue), arm-level CNAs (green). Under a selection model de novo CNAs may confer selective (dis)advantages (cells in green and blue). The heatmap on the right shows copy number states of cells in the final population (Grey: 2 N; Light red: 3 N; Dark red: 4 N; Light blue: 1 N). Chr. N copy numbers (orange rectangular box) correspond to chromosomes depicted on the left. Summary statistics were derived from the mitotic tree and copy number states, and used for inference with approximate Bayesian computation (ABC). b, CNA rates and cell division rates estimated from seven individual PDTOs (four PDTOs with mitotic trees) using ABC under the neutral model. Violin plots (posterior distributions) and box plots were generated from n = 500 independent samples. Box plots: boxes represent quartiles 2 and 3, horizontal lines represents median; whiskers extend to 1.5× of the interquartile range; data beyond interquartile range are plotted individually. c, Model selection using deviance information criteria (DIC, smaller values indicate better support) suggests the neutral model is better supported (100 replicates). Box plot criteria as in b. Nature Genetics \| www.nature.com/naturegenetics Articles NatUrE GEnEtICS d, Power analysis with DIC on the detectability of negative selection in simulated data (n = 100 per parameter setting). Top: number of times each model is supported with different selection strengths and cell numbers. Bottom: differences of DIC values computed under the two models. With increasing strength of negative selection or more cells, the power to detect negative selection increases. Box plots as in c. e, As in Fig. 5e but for an independent parental organoid. 28 organoids share the reference PDTO-9 core karyotype, 4 were polyploid and 2 had de novo localized CNAs. Nature Genetics \| www.nature.com/naturegenetics Articles NatUrE GEnEtICS Articles NatUrE GEnEtICS Extended Data Fig. 9 \| See next page for caption. Nature Genetics \| www.nature.com/naturegenetics Extended Data Fig. 9 \| See next page for caption. Nature Genetics \| www.nature.com/naturegenetics Extended Data Fig. 9 \| See next page for caption. Nature Genetics \| www.nature.com/naturegenetics Nature Genetics \| www.nature.com/naturegenetics Extended Data Fig. 9 \| Single-cell sequencing datasets of CRC glands. a, Karyotype heatmap showing 112 cells (45% recovery) derived from an excised fragment of a single CRC gland isolated from a primary tumor biopsy of patient C274 (inset). Core: bulk karyotype analysis of the remaining gland structure. (I) Reciprocal loss, gain and propagation of Chr.2. (II) Cells with gross genome wide karyotype alterations. b, As in a. 47 single cells were isolated from an excised fragment of a single CRC gland, containing approximately ~500 cells based on fragment size. Two cells in the sequenced population are polyploid. c) Box-and-whisker plots of PDTO-9, PDTO-19b and C274 representing the fraction of cells per dataset with de novo CNAs. Boxes represent quartiles 2 and 3, the horizontal line represents the mean and whiskers extend to the minimum and maximum within the 1.5× interquartile range. All data points are shown. Non-significant, p > 0.05 (p = 0.902, one-way ANOVA). NatUrE GEnEtICS Extended Data Fig. 9 \| Single-cell sequencing datasets of CRC glands. a, Karyotype heatmap showing 112 cells (45% recovery) derived from an excised fragment of a single CRC gland isolated from a primary tumor biopsy of patient C274 (inset). Core: bulk karyotype analysis of the remaining gland structure. (I) Reciprocal loss, gain and propagation of Chr.2. (II) Cells with gross genome wide karyotype alterations. b, As in a. 47 single cells were isolated from an excised fragment of a single CRC gland, containing approximately ~500 cells based on fragment size. Two cells in the sequenced population are polyploid. c) Box-and-whisker plots of PDTO-9, PDTO-19b and C274 representing the fraction of cells per dataset with de novo CNAs. Boxes represent quartiles 2 and 3, the horizontal line represents the mean and whiskers extend to the minimum and maximum within the 1.5× interquartile range. All data points are shown. Non-significant, p > 0.05 (p = 0.902, one-way ANOVA). Articles NatUrE GEnEtICS Articles NatUrE GEnEtICS Extended Data Fig. 10 \| Overview of CNA events per chromosome for PDTO-9 and PDTO-19. a, Genome-wide map of all de novo CNA events that were observed in eight PDTO-9 and four PDTO-19b datasets (respectively, 310 and 158 analysed single cells), and 64 PDTO-9 cell divisions (128 analysed single cells). Events include reciprocal CNAs, non-reciprocal whole-chromosome gains or losses and non-reciprocal sub-chromosomal CNAs represented in more than one cell. Hopeful monster karyotypes were excluded for this representation. Centromeric and other complex regions of chromosomes are depicted in red. b, Metaphase spread-based karyotyping of 36 tumor cells from PDTO-9 and PDTO-19b stained with DAPI to confirm their near-diploid genome. Each dot represents a tumor cell, center bar and error bars represent the mean and standard deviation. Scalebar is 5 μm. Extended Data Fig. 10 \| Overview of CNA events per chromosome for PDTO-9 and PDTO-19. a, Genome-wide map of all de novo CNA events that were observed in eight PDTO-9 and four PDTO-19b datasets (respectively, 310 and 158 analysed single cells), and 64 PDTO-9 cell divisions (128 analysed single cells). Events include reciprocal CNAs, non-reciprocal whole-chromosome gains or losses and non-reciprocal sub-chromosomal CNAs represented in more than one cell. Hopeful monster karyotypes were excluded for this representation. Centromeric and other complex regions of chromosomes are depicted in red. b, Metaphase spread-based karyotyping of 36 tumor cells from PDTO-9 and PDTO-19b stained with DAPI to confirm their near-diploid genome. Nature Genetics \| www.nature.com/naturegenetics NatUrE GEnEtICS Each dot represents a tumor cell, center bar and error bars represent the mean and standard deviation. Scalebar is 5 μm. Extended Data Fig. 10 \| Overview of CNA events per chromosome for PDTO-9 and PDTO-19. a, Genome-wide map of all de novo CNA events that were observed in eight PDTO-9 and four PDTO-19b datasets (respectively, 310 and 158 analysed single cells), and 64 PDTO-9 cell divisions (128 analysed single cells). Events include reciprocal CNAs, non-reciprocal whole-chromosome gains or losses and non-reciprocal sub-chromosomal CNAs represented in more than one cell. Hopeful monster karyotypes were excluded for this representation. Centromeric and other complex regions of chromosomes are depicted in red. b, Metaphase spread-based karyotyping of 36 tumor cells from PDTO-9 and PDTO-19b stained with DAPI to confirm their near-diploid genome. Each dot represents a tumor cell, center bar and error bars represent the mean and standard deviation. Scalebar is 5 μm. Nature Genetics \| www.nature.com/naturegenetics
https://openalex.org/W3015452061	https://europepmc.org/articles/pmc7232517?pdf=render	English	null	Talkin’ Toxins: From Coley’s to Modern Cancer Immunotherapy	Toxins	2,020	cc-by	14,507	Received: 11 March 2020; Accepted: 7 April 2020; Published: 9 April 2020 Abstract: The ability of the immune system to precisely target and eliminate aberrant or infected cells has long been studied in the ﬁeld of infectious diseases. Attempts to deﬁne and exploit these potent immunological processes in the ﬁght against cancer has been a longstanding eﬀort dating back over 100 years to when Dr. William Coley purposefully infected cancer patients with a cocktail of heat-killed bacteria to stimulate anti-cancer immune processes. Although the ﬁeld of cancer immunotherapy has been dotted with skepticism at times, the success of immune checkpoint inhibitors and recent FDA approvals of autologous cell therapies have pivoted immunotherapy to center stage as one of the most promising strategies to treat cancer. This review aims to summarize historic milestones throughout the ﬁeld of cancer immunotherapy as well as highlight current and promising immunotherapies in development. Keywords: cancer; immunotherapy; vaccine; immune checkpoint inhibitors; adoptive cell therapy; cytokine therapy; Coley’s Toxins Key Contribution: This review summarizes the pivotal milestones in cancer immunotherapy development from Coley’s Toxins to modern day. Review Talkin’ Toxins: From Coley’s to Modern Cancer Immunotherapy Robert D. Carlson †, John C. Flickinger, Jr. † and Adam E. Snook * Department of Pharmacology and Experimental Therapeutics, Thomas Jeﬀerson University, 1020 Locust Street, Philadelphia, PA 19107, USA; Robert.Carlson@jeﬀerson.edu (R.D.C.); John.Flickinger@jeﬀerson.edu (J.C.F.J.) * Correspondence: adam.snook@jeﬀerson.edu; Tel.: +1-215-503-7445 † These authors contributed equally to this work. Department of Pharmacology and Experimental Therapeutics, Thomas Jeﬀerson University, 1020 Locust Street, Philadelphia, PA 19107, USA; Robert.Carlson@jeﬀerson.edu (R.D.C.); John.Flickinger@jeﬀerson.edu (J.C.F.J.) * Correspondence: adam.snook@jeﬀerson.edu; Tel.: +1-215-503-7445 † These authors contributed equally to this work. Received: 11 March 2020; Accepted: 7 April 2020; Published: 9 April 2020 1. Introduction The understanding of immune system governance in neoplastic growth and development has made signiﬁcant leaps in recent years [1], but its origins can be traced back well over a century ago. Incidence of tumors spontaneously regressing following infectious or pyretic periods have been described throughout history [2–4]. However, advancements made in histological diagnosis and assessment of tumor malignancies over the past 100 years have given credence to these claims of immune system modulation in cancer. toxins toxins toxins 2.1. The Story of Coley’s Toxins William Coley, often regarded as the “Father of Immunotherapy”, was a bone surgeon in New York from 1890–1936 who famously developed a cocktail of heat-killed bacteria, called “Coley’s Toxins”, to treat patients with osteosarcoma. Inspiration for developing this treatment apparently started with one of his ﬁrst patients, a young woman with osteosarcoma of the hand. Despite his surgical intervention (amputation of the forearm), she succumbed to metastatic disease within months of the operation. This episode had a profound impact on Coley and motivated him to learn more about her disease. He began by reviewing hospital medical records from ninety sarcoma patients, an analysis he later published [6]. While conducting his review, one patient’s course of disease was of particular intrigue. Coley came across the description of a patient with an inoperable sarcoma whose tumor completely regressed after developing erysipelas [7], a type of skin infection [8]. Upon reading this account, Coley wondered if it was possible to induce erysipelas in patients as a means to treat cancer. Fortunately for Coley, a German surgeon named Friedrich Fehleisen had only a few years earlier, in 1883, identiﬁed Streptococcus pyogenes as the bacterium responsible for erysipelas [9]. Thus, Coley was able to test his hypothesis and began injecting sarcoma patients with Streptococcus pyogenes, a primitive version of what would later be named Coley’s Toxins. Over the course of Coley’s career, from 1888–1933, he tested over a dozen diﬀerent preparations of his toxin. Developing his infamous toxin required striking a balance between safety and eﬃcacy. Indeed, early preparations were highly variable. Some preparations were impotent and failed to produce any signs of infection while other preparations were highly infectious and led to mortality [10]. Eventually, Coley settled on a combination of heat-killed Streptococcus pyogenes and Serratia marcescens [11]. Although Coley was not the ﬁrst person to make a connection between infection and cancer regression, nor the ﬁrst to inject bacteria into a patient as a means to mediate tumor rejection, Coley’s eﬀorts were the most comprehensive and inﬂuential. In total, it is estimated that Coley himself injected more than 1000 cancer patients and published over 150 papers related to the topic [11]. Coley reported remarkable success with his toxins and published many reports of his toxins inducing tumor regression [12,13]. However, at the time, his ﬁndings were highly controversial and were met with harsh criticism by many of his colleagues. 2. Pivotal Observations in Cancer Immunotherapy It is possible that cancer has existed ever since the evolution from unicellular organisms into multicellular entities. However, the oldest record of cancer to date is from a 240 million-year-old fossil containing a shell-less stem turtle, Pappochelys rosinae, with evidence of osteosarcoma [5]. Until recently, the treatment of cancer has historically focused on tumor excision, cytotoxic chemotherapeutic agents, and radiation therapy. Only after the turn of the 21st century did immunotherapy to treat cancer take stage [Figure 1]. Toxins 2020, 12, 241; doi:10.3390/toxins12040241 www.mdpi.com/journal/toxins www.mdpi.com/journal/toxins Toxins 2020, 12, 241 2 of 23 2 of 23 Figure 1. Milestones in the History of Cancer Immunotherapy. Figure 1. Milestones in the History of Cancer Immunotherapy. Figure 1. Milestones in the History of Cancer Immunotherapy. Figure 1. Milestones in the History of Cancer Immunotherapy. Toxins 2020, 12, 241 3 of 23 2.1. The Story of Coley’s Toxins Notable critiques include those in the Journal of the American Medical Association in 1894 issuing a statement criticizing the use of his toxins as well as the FDA re-categorizing of “Coley’s Toxins” in 1963 as an investigational drug that lacked safety and eﬃcacy data, despite over 70 years of use and numerous publications [11]. This recategorization made it illegal to prescribe Coley’s Toxins outside of clinical trial testing. In the end, history would be on the side of William Coley. Years after his death, his toxins were re-evaluated in a controlled trial and were demonstrated to mediate antitumor eﬀects [14]. Moreover, advancements in fundamental understanding of cancer and the immune system have allowed his ﬁndings to become more widely accepted and to lay a foundation for future studies of cancer immunotherapy. 2.2. Evidence the Immune System Targets Cancer Although Coley never fully understood the mechanism by which his toxins functioned, he gathered substantial evidence linking the immune system and cancer. Further clarity and development of this connection would come years later in the form of the immunosurveillance hypothesis. The idea that the immune system possesses a capacity to recognize and eliminate cancer cells was ﬁrst postulated by Paul Ehrlich in 1909 [15]. While direct experimental evidence during this time period was lacking, Ehrlich reasoned that the incidence of cancer is relatively low but that the formation of aberrant cells is a common phenomenon, suggesting the existence of a host defense system against cancer. Over 50 years later, these ideas were further developed by Burnet and Thomas and formally coined the “immune surveillance” hypothesis [16,17]. Early experimental evidence for the existence of tumor-specific immunity derives from transplantation studies. In 1943, Luwik Gross utilized methylcholanthrene (MCA) to chemically induce sarcoma in a C3H mouse and then transplanted this sarcoma into syngeneic mice. While inoculation with high doses of 4 of 23 Toxins 2020, 12, 241 tumor cells often killed mice, Gross found that inoculation with low doses of tumor cells led to a period of growth followed by gradual tumor regression. In these surviving mice, tumor challenge using high doses of tumor cells invariably led to rejection, suggesting these animals developed immunity to the tumor [18]. Further support for immunosurveillance comes from a seminal study by Prehn and Main in 1953. In these studies, an array of sarcomas from multiple syngeneic mice were generated using MCA. Prehn and Main found that inoculation of a mouse with sarcoma from one source protected that mouse from future challenge using the same sarcoma source but did not protect against challenge using sarcoma derived from a different mouse [19]. Moreover, Prehn and Main demonstrated that transplantation of skin tissue from a donor mouse did not sensitize the recipient mouse to the donor’s sarcoma, directly addressing a common critique at the time that rejection was mediated by subtle differences in genetic backgrounds. Collectively, these studies further supported the existence of tumor-specific immunity, adding the nuance that tumor antigens are highly unique to a tumor even in tumors of the same histological type, induced by the same chemical means, and from mice of the same genetic background [19]. 2.2. Evidence the Immune System Targets Cancer y g g While studies in partially immunocompromised mouse models over the following decades failed to support the immunosurveillance hypothesis, deﬁnitive demonstration of immunosurveillance came in the early 2000s following a series of studies conducted in novel, speciﬁcally immunocompromised, mouse strains. In 2001, Robert Schreiber’s group compared the incidence of spontaneous neoplasms between wild-type and Rag2−/−mice (Rag2 encodes a protein necessary for somatic recombination and thus Rag2−/−mice lack mature T and B lymphocytes) [20]. In mice over 15 months old, fewer than 20% of wild-type mice contained neoplastic disease while 100% of surveyed Rag2−/−mice developed spontaneous neoplastic lesions in various tissues, suggesting functional T and B lymphocytes suppress the development of cancer. Moreover, the same study observed that Rag2−/−mice, as well as Ifngr1−/− and Stat1−/−mice, which are deﬁcient in vital immune signaling pathways, develop higher incidences of sarcoma compared to wild-type mice in MCA-induced tumor models [21]. Similarly, higher incidences of MCA-induced tumors were reported by additional investigators using mice deﬁcient in other vital immune-signaling molecules such as perforin or TNF-related apoptosis-inducing ligand (TRAIL) [22,23]. These experimental studies in mice are mirrored by clinical evidence that humans with compromised immune systems develop higher incidences of cancer. Indeed, individuals born with genetic defects in immune-related genes develop higher incidences of lymphoma [24]. Moreover, people with otherwise normal immune function who acquire AIDS infection or transplant patients who receive immunosuppressive drugs are both at higher risk for developing Non-Hodgkin’s lymphoma and virus-induced Kaposi Sarcoma [25,26]. Since the emergence of the immunosurveillance hypothesis, the interplay between the immune system and cancer has been further reﬁned and renamed as the process of “immunoediting” [27]. Immunoediting posits that the immune system and cancer intersect at three stages: elimination, equilibrium, and escape. During the elimination stage, the immune system recognizes and destroys many, but not all aberrant cells. During equilibrium, the immune system and the tumor exert opposing forces, eﬀectively resulting in containment of the tumor. Over time, as the cancer acquires additional mutations and as the immune system exerts a selective pressure eliminating immunogenic cells and leaving behind non-immunogenic cells, the cancer eventually fully escapes immune surveillance. In this ﬁnal escape stage, the cancer has fully circumvented detection by the immune system and undergoes rapid and uncontrolled growth. Evidence for equilibrium and escape stages is supported by experiments in mice with stagnant tumor sizes who then undergo rapid growth after immune cell depletion [28]. 3.1. Cytokine Therapy Prior to understanding their therapeutic immunomodulatory potential in cancer, cytokines were ﬁrst recognized as systemic soluble factors that could regulate lymphocyte function and inﬂammatory responses. In 1972, a group from Yale University School of Medicine ﬁrst characterized a “lymphocyte activating factor” that spurred lymphocyte proliferation in response to soluble agents released by other syngeneic immune cells [37]. These agents were partially puriﬁed from antigen-stimulated, lymphocyte-conditioned media and characterized further as “T-cell growth factor” that could support cytotoxic T cells capable of killing autologous leukemic myeloblasts [38]. Shortly thereafter, this key growth factor was deﬁnitively puriﬁed and described as interleukin-2 (IL-2) [39], which not only allowed T lymphocytes to be cultured in large quantities ex vivo, but also allowed recombinant IL-2 to be administered as a high-dose single-agent [40], or used in tandem with preconditioned and transplanted cancer-speciﬁc lymphoid cells [41,42]. A comprehensive report published in 1987 by the NCI’s Surgery Branch documented objective responses to high-dose IL-2 and regression of tumors in patients with metastatic melanoma and renal cell cancer [43]. Adjustments made to IL-2 dose scheduling would largely combat acute toxicities, the most prominent being capillary leak syndrome and hypovolemia [44]. These results and safety measures would spur numerous and larger cohort studies utilizing IL-2 in a metastatic setting [45,46], culminating with FDA approval of high-dose intravenous IL-2 for patients with metastatic renal cancer in 1992, and metastatic melanoma several years later. This would be noted as one of the ﬁrst FDA-approved cancer immunotherapies [47]. er. This would be noted as one of the ﬁrst FDA-approved cancer immunotherapies [47]. Other cytokines that have demonstrated translational applications include interleukin-15 (IL-15), interferon-alpha (IFNα), and granulocyte macrophage colony-stimulating factor (GM-CSF). While both structurally similar and capable of stimulating early T-cell proliferation and NK cell engagement much like IL-2, IL-15 additionally supports memory CD8+ T-cell persistence, a known mediator of long-term antitumor immunity [48]. IL-15 has also proven to be an eﬀective mediator of antitumor protection in murine models of cancer [49–52]. In support of these claims, phase 1 clinical trials utilizing recombinant IL-15 alone, and in conjunction with B-cell-depleting antibodies, are currently underway for treating both solid and liquid tumors, respectively [NCT01021059, NCT03759184]. 3. Immunomodulatory Agents Immunomodulators comprise a variety of therapeutic agents and treatment strategies that aid in normalizing, or in the context of cancer, re-engaging or boosting immune cell function to counter uncontrolled cell proliferation. By deﬁnition, a tumor that presents in the clinic can be said to have escaped normal immune control, even if tumor-reactive T cells are detected in the blood or have inﬁltrated the tumor tissue [30]. Although tumor cells themselves possess intrinsic immunosuppressive behaviors, such as cultivating a hypoxic microenvironment [31,32] or generating lactic acid [33], the majority of suppressive inﬂuence comes from the normal functions of suppressive immune cells, cytokines, and inhibitory surface molecules [34,35]. Under normal physiological circumstances, these mechanisms suppress T-cell priming and cytotoxic T-cell function to stave oﬀunwarranted autoinﬂammatory responses [36]. Therefore, many of the immunomodulatory agents described herein aim to directly oppose these immunosuppressive mechanisms and to re-engage the immune system. 2.2. Evidence the Immune System Targets Cancer Additionally, tumors arising from immunocompromised mice are more frequently rejected when transplanted into a wild-type host compared to tumors arising from immunocompetent mice [21], partly reﬂecting immune-induced antigen loss in the presence of an intact immune system [29] (immunoediting). Thus, the fundamental goal of cancer immunotherapy is to overcome the years to decades of immunoediting to generate antitumor immunity that is suﬃcient to completely eliminate the patient’s cancer and cure their disease. 5 of 23 Toxins 2020, 12, 241 3.2. Immune Checkpoint Inhibitors Suppression of immune cell activation, inﬁltration, and eﬀector functions required for tumor cell clearance can be largely attributed to the immunosuppressive conditions within the tumor microenvironment [69]. In several studies, “dysfunctional” CD8+ T cells were retrieved from patient tumors and nearby draining lymph nodes and said to be lacking the expected diﬀerentiation proﬁles [70], or impaired by the accumulation of repressive Foxp3+ regulatory T cells (Tregs) [71]. A similar phenomenon was originally described in mice infected with a lymphocytic choriomeningitis virus, whereby chronic antigen stimulation induced an “exhausted” T-cell state proceeded by T-cell receptor (TCR) downregulation [72]. The discovery of other requisite activating co-stimulatory signals in addition to canonical antigen stimulation with the TCR [73], which was now known to be insuﬃcient for fully functional T-cell activation [74], gave clues to the complex nature of balancing activation with self-antigen tolerance [75]. Immune homeostasis is dependent upon these co-stimulatory/co-inhibitory receptor-ligand interactions, which in the correct context, safeguard against chronic immune activation and excessive inﬂammatory responses [76]. Addressing these phenomena directly, immune checkpoint blockade selectively restricts these co-inhibitory signaling mechanisms that have been co-opted by cancer cells, thereby enhancing antitumor T-cell activity. Although initially identiﬁed in 1987 [77], the 1994 discovery of cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) co-inhibitory receptor pairing with the B7 co-stimulatory ligand is perhaps the most substantial [78]. Upregulation of CTLA-4 on both CD4+ and CD8+ T lymphocytes was identiﬁed as a negative regulator of T-cell activation and eﬀector functions [79,80], while murine models deﬁcient in CTLA-4 experienced massive lymphoproliferation and tissue inﬁltration due to over-activation of resident T cells [81,82]. In the late 1990s, Dr. James Allison’s group at University of California, administered an inhibitory antibody to block the CTLA-4 co-inhibitory synapse in mice burdened with tumors. Both orthotopic and pre-established tumor cells were rejected following administration of the anti-CTLA-4 antibody, indicating that blockade of inhibitory signals associated with the co-stimulatory pathway can enhance antitumor immunity [83]. These indications prompted the application of CTLA-4 blockade in patients with stage III/IV unresectable melanoma with remarkable success [84], culminating in the 2011 FDA approval of the anti-CTLA-4 monoclonal antibody (mAb), ipilimumab, as an adjuvant therapy for patients with cutaneous melanoma. Retrospective studies have revealed marked increases in survival beneﬁt compared to traditional chemotherapy regimens [85,86], with modest gains observed in other solid tumor types currently in various phases of clinical trials [87]. 3.1. Cytokine Therapy IFNα, another cytokine originally described in the context of mediating antiviral immune response [53,54], was also identiﬁed to inhibit tumor neovasculature, upregulate MHC class I expression, mediate dendritic cell maturation, activate B and T cells, and induce apoptosis—all favorable antitumor attributes [55]. Thanks to the eﬀorts of blood banking that began in the late 1970s [56], adequate quantities of puriﬁed IFNα spurred a burst of clinical evaluations in patients with hematological malignancies [57,58] and solid tumors, such as renal cell cancers and malignant melanoma [59,60]. These trials culminated in FDA approval of IFNα as an adjuvant therapy ﬁrst in rare forms of leukemia, and later in patients with high-risk stage II and stage III melanoma [61]. Asthenamesuggests, GM-CSFwasoriginallyidentifiedasaregulatorofgranulocyteandmacrophage differentiation, as well as general hematopoiesis of multi-lineage progenitors [62]. However, in 1993, 6 of 23 Toxins 2020, 12, 241 Dranoff and colleagues transduced B16 melanoma cells with ten known pro-inflammatory mediators, vaccinated mice with these constructs, and then challenged them with live B16 cells. Of the ten, GM-CSF conferred the largest magnitude of antitumor immunity [63]. These findings prompted strategies to deliver GM-CSF to patients, either by vaccinating patients with irradiated tumor cells engineered to secrete the cytokine [64], or by single-agent dosing [65]. Although the exploration of GM-CSF-expressing tumor vaccines has waned in recent years due to limited clinical efficacy [66], combination strategies employing recombinant GM-CSF with other immunomodulatory agents, such as checkpoint inhibitors [67] and additional cytokines [68] have enhanced overall survival in melanoma patient trials. 3.2. Immune Checkpoint Inhibitors In an eﬀort to identify genes associated with apoptosis, Dr. Tasuku Honjo’s group at Kyoto University discovered programmed cell death protein 1 (PD-1), a lymphoid cell surface protein that the group hypothesized to be a cell-death inducer [88]. Several years later in 1999, the same group generated a PD-1 deﬁcient mouse model that spontaneously developed several autoimmune-like symptoms and systemic graft-versus-host-like disease [89]. Like CTLA-4, PD-1 was identiﬁed as a negative regulator of adaptive immune responses. PD-1 ligand 1 (PD-L1) was discovered that same year at the Mayo Clinic and characterized as functionally homologous to the CTLA-4 ligand, B7, but co-stimulated T cells through some additional unknown receptor [90], later identiﬁed to be PD-1 [91]. Engagement of PD-1 with its ligand prevented T-cell proliferation and cytokine production when synthetically stimulated, identifying it as an intrinsic inhibitory mechanism of autoreactive lymphocyte 7 of 23 Toxins 2020, 12, 241 activation [91]. PD-L1 surface expression on tumor cells was also discovered to suppress the cytolytic eﬀector functions of CD8+ T cells, with additional speculation that PD-1/PD-L1 blockade could serve as an eﬀective strategy to combat tumor cell escape [92]. Speculation became reality when several groups tested PD-L1 blockade in murine tumor models and concluded that antibodies directed at this co-stimulatory interaction could enhance cancer immunotherapy [93–95]. In one step closer to the clinic, PD-L1 was determined to be a prognostic marker of patient outcome, with higher levels of ligand in resected specimens correlated with poorer patient survival [96]. Within the past 10 years, several high-proﬁle trials employing anti-PD-1/PD-L1 mAbs under various conditions, dosing strategies, and cancer types, have indicated that blockade of this co-inhibitory pathway is both well-tolerated and associated with durable objective responses in patients [97–99]. Consequently, FDA approval was granted ﬁrst to nivolumab, a humanized PD-1 mAb for metastatic melanoma in 2014, and subsequently for pembrolizumab, a PD-1 mAb alternative. Both therapies elicited greater overall patient survival compared to their anti-CTLA-4 counterpart [100,101]. In 2016, a third antibody was developed, this time directed at the PD-L1 ligand to treat patients with urothelial carcinoma and non-small cell lung cancer with much success [102,103]. This fully humanized anti-PD-L1 mAb, atezolizumab, was granted FDA approval for bladder cancer patients ineligible for traditional cisplatin-based chemotherapies [104]. Recent studies have expanded the number of indications for anti-PD-1/PD-L1 blockade alone [105], and in combination with anti-CTLA-4 [106], both proving to be summarily eﬃcacious. 3.2. Immune Checkpoint Inhibitors Although CTLA-4 and PD-1 blockade strategies have demonstrated unprecedented clinical success and accelerated FDA approval, there remains a population of non-responders. These individuals either fail to respond to checkpoint blockade from treatment onset due to innate resistance mechanisms, or acquire secondary resistance resulting in relapse. Retrospective studies of large-cohort clinical trials may expose novel biomarkers capable of predicting resistance to checkpoint therapies [107]. Additional co-inhibitory receptors, each with unique functions, have since been identiﬁed to inﬂuence negative immune regulation by various mechanisms [108]. Likewise, recent ﬁndings have demonstrated that the resident gut microbiome has the ability to inﬂuence patient responses to checkpoint blockade, with individuals who had consumed oral antibiotics prior to therapy experiencing poorer anti-PD-1 responses [109,110]. Nonetheless, immune checkpoint inhibitors continue to represent the vast majority of new immunotherapies for the treatment of cancer. These therapies would not be possible without seminal discoveries made in the blockade of negative immune regulatory elements by Drs. James Allison and Tasuku Honjo, for which they were awarded the 2018 Nobel Prize in Physiology or Medicine. 4. Vaccines While immunomodulatory agents broadly stimulate the immune system, cancer vaccines aim to more precisely steer an immune response towards cancer. At its purest form, a cancer vaccine consists of one or more tumor antigens combined with an adjuvant to enhance the immune response. As will later be described, the type of tumor antigen, delivery method of the antigen, and adjuvant varies greatly. Cancer vaccines can be administered as a therapeutic vaccine in patients with existing malignancies or as a preventive vaccine in healthy or high-risk individuals (primary prevention) or patients in remission (secondary prevention) to protect against future tumor development or recurrence, respectively. 4.2. Therapeutic Cancer Vaccines In 1995 and 1996, the ﬁrst clinical trials testing cancer vaccines against tumor-associated antigens were published. These trials utilized either peptide vaccines [125], or peptide-pulsed dendritic-cell vaccines composed of patient-derived dendritic cells that have been incubated with a peptide prior to re-infusion [126,127]. Other popular methods of cancer vaccination include the use of recombinant viral and bacterial vectors. As microorganisms potently stimulate the immune system, the use of these vectors to deliver tumor antigen in the context of an infection is hypothesized to enhance antitumor immune responses. Common vectors used to deliver tumor antigens include poxvirus [128], adenovirus [129], Salmonella typhimurium [130], and Listeria monocytogenes [131]. As methods of gene therapy have advanced over the years, the use of DNA and RNA vaccines has become increasingly common [132]. Despite thousands of cancer vaccine clinical trials, only one therapeutic cancer vaccine, Sipuleucel-T, is FDA-approved [133]. Sipuleucel-T is an autologous cell vaccine composed of patient peripheral blood mononuclear cells (PBMCs) pulsed with a chimeric protein consisting of the tumor-associated antigen prostate alkaline phosphatase (PAP) fused to the immunomodulating cytokine GM-CSF. A phase III clinical trial in men with metastatic castration-resistant prostate cancer found that three infusions of Sipuleucel-T led to the induction of PAP-speciﬁc immune responses and a 4.1-month improvement in median survival [134]. Limited success in therapeutic settings may be, in part, attributed to poor immunogenicity of the vaccine target and immunosuppressive tumor microenvironments. One approach to overcome poorly immunogenic tumor-associated antigens is the recent trend towards targeting neoantigens. Compared to tumor-associated antigens, neoantigens may be more immunogenic due to a lack of immunological tolerance mechanisms [135]. Until recently, the identiﬁcation of neoantigens was impractical as the cost and time to sequence patient genomes for unique mutations presented a formidable barrier. However, with advancements in next-generation sequencing, it has become feasible to sequence a patient’s normal and tumor genome to identify unique tumor-speciﬁc antigens. Personalized therapeutic neoantigen vaccines have shown promise in phase I trials for melanoma [136] and glioblastoma [137]. However, these neoantigen vaccines are in early clinical testing, and thus the eﬃcacy and feasibility of this approach is yet to be determined. 4.1. Tumor Antigens Initial attempts at vaccination occurred before the identiﬁcation of speciﬁc tumor antigens. These trials utilized cellular-based vaccines consisting of modiﬁed or irradiated tumor cells derived from a patient (autologous) or from a cancer cell line (allogeneic) injected with adjuvant [111–113]. With the identiﬁcation of the ﬁrst human tumor antigen, MAGE-1, by Thierry Boon’s group in 1991, a more reﬁned approach of vaccinating against speciﬁc targets was born [114]. Since then, over 75 tumor-associated antigens have been identiﬁed [115]. There are two categories of tumor antigens: tumor-associated antigens and tumor-speciﬁc antigens. By deﬁnition, tumor-associated antigens share expression with some normal tissues while tumor-speciﬁc antigens are unique to cancer cells. Notable 8 of 23 Toxins 2020, 12, 241 examples of tumor-associated antigens that have been a focus of multiple immunotherapies include the cancer-testis antigens NY-ESO-1 [116] and MAGE-1 [117], which are expressed by germ cells and ectopically re-expressed in cancers; the oncofetal antigens CEA [118] and alpha-1-fetoprotein [119], which are present during fetal development and re-expressed by cancers; diﬀerentiation antigens, such as prostate-speciﬁc antigen (PSA) [120] and CD19 [121], which are expressed by cells derived from a speciﬁc tissue-type and retained in cancers; and antigens that are over-expressed by cancers relative to normal tissue, including HER2 [122] and telomerase [123]. In contrast to tumor-associated antigens, which share expression with healthy tissue, tumor-speciﬁc antigens are exclusively expressed by tumors. Tumor-speciﬁc antigens, also known as neoantigens, are mutated peptides created by unique genetic aberrations or may be viral antigens in the case of virus-associated cancers [124]. 4.4. Oncolytic Virotherapy An emerging immunotherapeutic strategy that is often categorized as a cancer vaccine is the use of oncolytic viruses. Oncolytic viruses preferentially infect and kill tumor cells compared to normal tissue. Selective infection of tumor cells is achieved through a combination of factors including the overexpression of viral receptors on tumor cells which can facilitate viral entry, a proliferative cell cycle that promotes viral replication, and a tumor cell deﬁciency in type I interferon production leading to limited viral clearance [143]. In addition to mediating tumor regression by direct cell lysis, viral infection activates components of the innate and adaptive immune system, thereby contributing further to tumor cell death. For example, oncolytic viral infection activates NK cells to clear virally-infected tumor cells [144]. Moreover, immunogenic cell death of virally infected tumor cells releases both tumor-associated antigens and neoantigens that can be acquired and presented by antigen-presenting cells, leading to the induction of antitumor CD8+ T cell responses (an approach often described as “in situ vaccination”) [145,146]. The potential of oncolytic virotherapy was ﬁrst noted by George Dock in 1904. Similar to Coley, Dock noticed that a patient with acute leukemia underwent remission after acquiring an inﬂuenza infection [147]. Many other case reports followed over the years, eventually leading to hundreds of clinical trials testing oncolytic viruses [148]. In 2015, the ﬁrst oncolytic viral therapy, talimogene laherparepvec (“T-VEC”), was approved by the FDA for use in metastatic melanoma [143,149]. T-VEC is an attenuated herpes simplex virus harboring various genetic deletions and insertions designed to enhance the antitumor immune response, such as the deletion of an immune-evasive viral gene ICP47 and the insertion of a human GM-CSF gene [145]. Compared to GM-CSF administration alone, T-VEC led to a 4.4 month increase in median survival in a phase III trial in patients with advanced and metastatic melanoma [143,149]. 4.3. Preventive Cancer Vaccines Recently, there has been a trend towards testing cancer vaccines as preventive therapies. Vaccination in preventive settings may be preferable to therapeutic ones as it may allow for the induction of antitumor immunity before the development of immunosuppressive microenvironments [138]. This strategy has shown promise against multiple viral-based cancers. Indeed, vaccination against oncogenic viruses including hepatitis B and human papillomavirus have led to reductions in hepatic [139] and cervical [140] cancers, respectively. However, for non-virally associated cancers, a target antigen and clinical setting to administer preventive vaccines is often less clear. For example, vaccinating a healthy patient against a tumor-associated antigen may carry an unnecessary risk of autoimmunity. Additionally, preventive 9 of 23 Toxins 2020, 12, 241 vaccination against neoantigens, while reducing the risk of autoimmunity, may be impractical as neoantigens are often widely variable between patients. However, preventive vaccination in some settings may be possible. One such example is vaccination against the mucin 1 (MUC1) antigen in patients at high-risk of colorectal cancer. In tumors, MUC1 is hypoglycosylated relative to normal tissues, allowing for the induction of selective antitumor responses [141]. A phase I/II study in patients with a history of colorectal adenoma demonstrated MUC1 immunogenicity and a phase II trial investigating the ability of MUC1 vaccine to prevent adenoma recurrence is currently ongoing [142]. 5.1. Tumor-Inﬁltrating Lymphocytes and Engineered T-Cell Receptors 5.1. Tumor-Inﬁltrating Lymphocytes and Engineered T-Cell Receptors The antitumor activity of T lymphocytes was ﬁrst elucidated in the 1950s and 1960s with seminal discoveries made in allograft rejection of sarcomas in experimental rodent models [150,151]. In 1953, Mitchison investigated the passive transfer of tumor immunity via transplantation of lymph nodes from mice inoculated with lymphosarcomas to equivalently challenged, but non-inoculated, mice [152]. A decade later, two groups made similar observations of transferrable tumor immunity by isolating and transplanting the cells of lymphatic tissues, rather than the organs themselves. Cells were collected from the lymphatic ducts and spleens of donor animals previously immunized with sarcoma cells that developed palpable tumors. Administration of those lymphoid cells back into syngeneic and non-syngeneic recipients inoculated with tumors, saw sustained regression indicating that these lymphocytes were “educated” by prior exposure to tumor antigens [153,154]. The means to exploit these T lymphocytes for their antitumor potential was limited by the inherent diﬃculty of expanding cells ex vivo. In 1976, a group at the NIH ﬁrst described the co-culture of isolated human bone marrow with conditioned media containing IL-2 that could induce growth and diﬀerentiation of bone marrow cells to T lymphocyte precursors [155]. With the advent of commercially synthesized IL-2, T lymphocytes could now be cultured in large quantities, or in the 10 of 23 Toxins 2020, 12, 241 context of an adjuvant, to boost the therapeutic eﬀects of tumor-sensitized and adoptively transferred T lymphocytes [156]. This subset of cytokine-activated lymphocytes was identiﬁed to be among those inﬁltrating the stroma of transplanted tumors. Dr. Steven Rosenberg’s group conﬁrmed that tumor-inﬁltrating lymphocytes (TILs) isolated from resected tumor could recognize syngeneic tumor cells in vitro [157], as well as mediate durable antitumor responses when re-administered back into autologous animal models [158] and cancer patients with metastatic disease [159]. The relatively pure populations of CD8+ and CD4+ T cells cultured from resected tumors appeared to dissipate quickly when returned to patients, meaning that therapeutic responses were often transient. However, in 2002, prior application of a lymphodepleting, nonmyeloablative chemotherapy regimen, originally designed for allogeneic bone marrow transplantation [160], greatly enhanced TIL engraftment and clonal persistence in patients, with some individuals harboring up to 80% CD8+ T cells many months post-infusion [161,162]. Shortly thereafter, another milestone was achieved when the Rosenberg group retrovirally transduced patient-derived peripheral blood lymphocytes with a TCR recognizing the common melanoma antigen, MART-1. 5.1. Tumor-Inﬁltrating Lymphocytes and Engineered T-Cell Receptors Objective cancer regression was observed in 2 out of 15 patients (13%) when engineered T lymphocytes were adoptively transferred back into patients [163], with a subsequent report demonstrating an improved 30% objective response rate [164]. Additional trials employing engineered TCRs targeting NY-ESO-1 in synovial cell sarcoma [165], the GD2 disialoganglioside in neuroblastoma [166], and carcinoembryonic antigen (CEA) in colorectal cancer [167] demonstrated objective clinical responses, thereby broadening the application to additional tumor types. More recently, personalized strategies using whole-exome sequencing of patient tumors has given researchers the ability to target neoantigens with high speciﬁcity [168,169]. Classically unmanipulated TIL therapy will continue to serve a patient population with shared and broadly targetable antigens [170], while more nuanced TILs recognizing neoantigens will continue to pace evolving therapies in the age of personalized medicine [171]. 5.2. Chimeric Antigen Receptor T Cell (CAR-T Cell) Therapy Although adoptive transfer of tumor-sensitized and antigen-reactive TILs with prefatory lymphodepletion and IL-2 dosing regimens had proven eﬀective in the clinic, patient responses were often transient: shrinkage in metastatic lesions could occur, without objective response to treatment [172]. Native TCRs are often limited by their ability to recognize post-translationally or aberrantly modiﬁed proteins, such as those observed in tumor-associated antigens of malignant cells [173,174]. Likewise, T cells dependent on antigen presentation by MHC molecules are routinely hindered by MHC class I downregulation, a selective mechanism of tumor immune escape [175]. In 1989, an Israeli group at the Weizmann Institute of Science devised the ﬁrst proof-of-concept strategy using an engineered chimeric antigen receptor (CAR) to circumvent the need for MHC-mediated antigen presentation for T-cell activation. By combining the intracellular T-cell receptor circuitry with the antigen-recognizing variable domains of an antibody raised against 2,4,6-trinitrophenyl (TNP), the researchers were able to elicit a non-MHC-restricted response in transfected T lymphocytes to target cells bearing TNP on their surface [176]. The unprecedented antibody-type speciﬁcity, now liberated from MHC presentation and paired to eﬀector T-cell function, could conceivably target post-translationally modiﬁed proteins characteristic of tumor cells undergoing selection or escape. Several years later, the same group successfully generated CARs directed towards HER2, a cell surface antigen commonly overexpressed in adenocarcinomas. These CAR-T cells selectively lysed HER2+ cancer cells in vitro, providing evidence that mAbs directed towards common tumor cell antigens, could be reassembled into single chain variable fragments (scFvs) to facilitate immune eﬀector function directly [177]. That same year, a joint venture between Weizmann Institute and the NIH expanded the spectrum of available targets by targeting folate receptors commonly overexpressed in ovarian carcinoma cells and further demonstrating the potential of adoptively transferring these CAR-T cells into cancer patients [178]. Toxins 2020, 12, 241 11 of 23 In an eﬀort to improve CAR-T cell activation, CD28 costimulatory molecules were added in a single tandem gene product with the intracellular CD3ζ-chain. Tumor cells often lack costimulatory molecules entirely, a barrier to persistent activation in ﬁrst-generation CAR-T cells. Much like conventional T cells, an “exhausted” phenotype was observed in T cells expressing ﬁrst-generation constructs encountering tumor cells in excess. In contrast, second-generation CAR-T cells containing additional built-in CD28 costimulatory moieties demonstrated superior signaling functionality, persistence, and cytokine production [179,180], as well as antitumor activity [181]. 5.2. Chimeric Antigen Receptor T Cell (CAR-T Cell) Therapy Over the next decade, second-generation CARs would be the basis for many ﬁrst-in-human studies: ﬁrst targeting carbonic anhydrase IX (CAIX), an antigen commonly overexpressed in renal cell carcinoma (RCC), and shortly thereafter, the ovarian cancer–associated antigen α-folate receptor (FR). CAIX-directed CAR-T cells produced grade 2–4 liver toxicity in patients due to CAIX self-antigen present in normal bile duct epithelium, with no overall response in RCC tumors [182]. Likewise, FR-directed CAR-T cells in a parallel phase 1 study, produced no reduction in ovarian tumor burden, albeit with lower grade 1–2 toxicity and no detectable oﬀ-tumor or oﬀ-target responses [183]. Major clinical breakthroughs were not seen until several years later when CAR-T cell therapy strategy switched from targeting primarily solid tumors, to liquid tumors, such as B-cell lymphomas and leukemias. In 2008, a group at University of Washington pioneered a proof-of-concept clinical trial in which refractory B-cell lymphoma patients received 20 infusions of autologous CD20-directed CAR-T cells. Treatment was well-tolerated in patients, with minimal toxicities and modiﬁed T cells persisting up to 9 weeks. Of the seven patients, two were noted as having complete response to treatment [184]. With phase 1 clinical trials rapidly taking shape around B-cell targets, methods for manufacturing and validating clinical-grade autologous CAR-T cells were developed to support increasing demand [185]. Shortly thereafter, the Rosenberg group within the NCI’s Surgery Branch, demonstrated in vivo antigen-speciﬁc activity of CAR-T cells directed towards the B-cell-speciﬁc antigen, CD19, in advanced-stage follicular lymphoma [186]. Paralleling this seminal study, Dr. Carl June’s group at the University of Pennsylvania demonstrated speciﬁc and eﬀective on-target killing of CD19+ malignant B cells in patients with advanced chronic lymphocytic leukemia (CLL) using CD19-directed CAR-T cells. In that study, two out of three patients experienced complete remission of disease, with a portion of CAR-T cells retaining potent eﬀector function six months after initial infusion, indicating a possible memory CAR-T cell phenotype [187,188]. The CAR employed by the University of Pennsylvania possessed a 4-1BB (CD137) costimulatory domain, rather than CD28, that promoted in vivo persistence and antileukemic function that outperformed conventional CARs with either CD3ζ and CD28 costimulatory molecules or CD3ζ alone [187,189]. Over the next few years, both groups continued to advance the ﬁeld by targeting various CD19+ hematological malignancies with great success. However, unanticipated and oftentimes severe neurological toxicities in the form of cytokine release syndrome were observed in patients. 5.2. Chimeric Antigen Receptor T Cell (CAR-T Cell) Therapy This toxicity can manifest as fevers, headaches, aphasia, and in some cases, hallucinations, delirium, and seizures [190]. Cytokine blockade strategies to control the abundance of systemically released cytokines, namely administering the IL-6-blocking antibody tocilizumab with and without corticosteroids, were developed to combat acute neural toxicity [191–195]. Although major clinical gains have been achieved with CD19+ hematologic malignancies, the same successes have yet to be fully realized in solid tumors. Despite abundant antigenic heterogeneity, diﬃculties in traﬃcking to tumor sites, and an intrinsic immunosuppressive tumor microenvironment [196,197], CAR-T cell therapies against solid tumor malignancies have entered early-phase clinical trials with varying degrees of success [Table 1]. 12 of 23 Toxins 2020, 12, 241 Table 1. Summary of active clinical trials for CAR-T cell therapy in solid tumors. Table 1. Summary of active clinical trials for CAR-T cell therapy in solid tumors. Antigen Target Cancer Type Phase ClinicalTrials.gov Designation CD117 Osteoid Sarcoma, Ewing Sarcoma I/II NCT03356782 CD133 Liver, Pancreatic, Brain, Breas, Ovarian, Colorectal, Acute Myeloid and Lymphoid Leukemias I/II NCT02541370 Osteoid Sarcoma, Ewing Sarcoma I/II NCT03356782 CD171 Neuroblastoma, Ganglioneuroblastoma, I NCT02311621 CEA Colorectal I/II NCT02959151 Lung, Colorectal, Gastric, Breast, Pancreatic I NCT02349724 EGFR Colorectal I/II NCT03152435 EGFRvIII Glioma, Glioblastoma, Gliosarcoma I/II NCT01454596 EpCAM Colon, Esophageal, Pancreatic, Prostate, Gastric, Hepatic I/II NCT03013712 EphA2 Glioma I/II NCT02575261 ErbB Head and Neck I/II NCT01818323 FRα Urothelial Bladder I/II NCT03185468 GD2 Glioma I/II NCT03252171 Neuroblastoma I/II NCT03373097 I NCT01822652 II NCT02765243 Cervical I/II NCT03356795 Osteoid Sarcoma, Ewing Sarcoma I/II NCT03356782 Sarcoma, Osteosarcoma, Neuroblastoma, Melanoma I NCT02107963 GPC3 Hepatocellular Carcinoma I/II NCT02723942 NCT02959151 NCT03084380 HER2 Breast I/II NCT02547961 Sarcoma I NCT00902044 IL-13Rα2 Glioma, Glioblastoma I NCT02208362 Mesothelin Pancreatic I/II NCT02959151 Cervical I/II NCT03356795 Advanced Solid Tumors I/II NCT03615313 Pancreatic, Ovarian, Mesothelioma I NCT02159716 Malignant Pleural Disease, Mesothelioma, Lung, Breast I NCT02414269 MUC1 Cervical I/II NCT03356795 Esophageal I/II NCT03706326 Non-Small Cell Lung I/II NCT03525782 Osteoid Sarcoma, Ewing Sarcoma I/II NCT03356782 Intrahepatic Cholangiocarcinoma I/II NCT03633773 MUC-16 Ovarian I NCT02498912 PSMA Urothelial Bladder I/II NCT03185468 Cervical I/II NCT03356795 Prostate I NCT01140373 Toxins 2020, 12, 241 13 of 23 13 of 23 6. Conclusions Conﬂicts of Interest: The authors declare no conﬂict of interest. Conﬂicts of Interest: The authors declare no conﬂict of interest. 6. Conclusions The late 19th century observation that tumors could be treated with cocktails of heat-killed bacteria has proven highly inﬂuential. Unbeknownst to William Coley and his contemporaries, this would prove to be one of the ﬁrst documented cases of tumor regression by induced activation of the immune system. Coley’s legacy would help spur subsequent hypotheses of immunosurveillance mechanisms capable of mediating steady-state tumor recognition and elimination. Over the next century, exploitation of these mechanisms was to become a major priority as immunotherapies continued to evolve. Treatment regimens using recombinant cytokines that activate immune cell proliferation and eﬀector functions are eﬃcacious in treating selected patient populations. Likewise, strategies employing immune checkpoint blockade against tumor cells that express co-inhibitory molecules have reached clinical milestones once thought to be unachievable. Vaccines against tumor-associated antigens have demonstrated clinical beneﬁt, with applications turning towards neoantigens as patient-speciﬁc tumor sequencing becomes feasible. FDA approval of the oncolytic virotherapy, T-VEC, may also oﬀer another option for locally-acting immune stimulation and antitumor activity when resection, chemotherapy, or radiation are not amenable. Moreover, the success of CAR-T cell therapy in patients with hematological malignancies has established adoptive cell therapy as a viable treatment modality. As the costs associated with patient and tumor genome sequencing continue to decrease, the rapidly evolving “omics”-level of data acquisition and processing may enable precise treatment strategies for these patients. Deconvoluting patient-speciﬁc tumor heterogeneity with the assistance of “big data” may enable clinicians and researchers to select the best candidate immunotherapy from the start, while taking proactive approaches to overcome resistance mechanisms in an adaptive tumor microenvironment [198]. The previous ~150 years of immuno-oncology research without signiﬁcant clinical success has now enabled “hockey stick” growth in exploration of the safety and eﬃcacy of immune-centric therapies in clinical trials. Just as William Coley’s fundamental discoveries have shaped modern cancer immunotherapy, so too shall current eﬀorts inﬂuence the future of cancer treatment. Author Contributions: R.D.C., J.C.F.J., and A.E.S. conceived the review, R.D.C. and J.C.F.J. wrote the manuscript, and A.E.S. revised. All authors have read and agreed to the published version of the manuscript. 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https://openalex.org/W2472849262	https://europepmc.org/articles/pmc4947047?pdf=render	English	null	Calculation of x-ray scattering patterns from nanocrystals at high x-ray intensity	Structural dynamics	2,016	cc-by	6,570	STRUCTURAL DYNAMICS 3, 054101 (2016) (Received 29 March 2016; accepted 1 July 2016; published online 13 July 2016) (Received 29 March 2016; accepted 1 July 2016; published online 13 July 2016) We present a generalized method to describe the x-ray scattering intensity of the Bragg spots in a diffraction pattern from nanocrystals exposed to intense x-ray pulses. Our method involves the subdivision of a crystal into smaller units. In order to calculate the dynamics within every unit, we employ a Monte-Carlo-molecular dynamics-ab-initio hybrid framework using real space periodic boundary conditions. By combining all the units, we simulate the diffraction pattern of a crystal larger than the transverse x-ray beam proﬁle, a situation commonly encountered in femtosecond nanocrystallography experiments with focused x-ray free-electron laser radiation. Radiation damage is not spatially uniform and depends on the ﬂuence associated with each speciﬁc region inside the crystal. To investigate the effects of uniform and non-uniform ﬂuence distribution, we have used two different spatial beam proﬁles, Gaussian and ﬂattop. V C 2016 Author(s). All article content, except where otherwise noted, is licensed under a Creative Commons Attribution (CC BY) license (http:// creativecommons.org/licenses/by/4.0/). [http://dx.doi.org/10.1063/1.4958887] Calculation of x-ray scattering patterns from nanocrystals at high x-ray intensity Malik Muhammad Abdullah,1,2,3 Zoltan Jurek,1,2 Sang-Kil Son,1,2 and Robin Santra1,2,3 Malik Muhammad Abdullah,1,2,3 Zoltan Jurek,1,2 Sang-Kil Son,1,2 and Robin Santra1,2,3 1Center for Free-Electron Laser Science, DESY, Notkestrasse 85, 22607 Hamburg, Germany 2The Hamburg Centre for Ultrafast Imaging, Luruper Chaussee 149, 22761 Hamburg, Germany 3Department of Physics, University of Hamburg, Jungiusstrasse 9, 20355 Hamburg, Germany Malik Muhammad Abdullah,1,2,3 Zoltan Jurek,1,2 Sang-Kil Son,1,2 and Robin Santra1,2,3 1Center for Free-Electron Laser Science, DESY, Notkestrasse 85, 22607 Hamburg, Germany 2The Hamburg Centre for Ultrafast Imaging, Luruper Chaussee 149, 22761 Hamburg, Germany 3Department of Physics, University of Hamburg, Jungiusstrasse 9, 20355 Hamburg, Germany 1Center for Free-Electron Laser Science, DESY, Notkestrasse 85, 22607 Hamburg, Germany 2The Hamburg Centre for Ultrafast Imaging, Luruper Chaussee 149, 22761 Hamburg, Germany 3Department of Physics, University of Hamburg, Jungiusstrasse 9, 20355 Hamburg, Germany I. INTRODUCTION With the advent of x-ray free electron laser (XFEL) sources,1 studies of structural determi- nation of biomolecules2–5 have gained a new boost. XFELs provide intense radiation of a wave- length comparable to atomic scales. The characteristics of XFEL radiation and associated sam- ple environments have triggered the development of new data collection methods such as serial femtosecond crystallography6 (SFX). The ultimate goal and dream is to perform atomic resolu- tion single particle imaging.7–11 Sample damage by x-rays and low signal to noise ratio at high photon momentum transfer limit the resolution of structural studies on non-repetitive structures such as individual biomolecules or cells.7,12 Therefore, at high resolution, SFX is currently still a better option to use. XFELs deliver intense femtosecond pulses that promise to yield high- resolution diffraction data of nanocrystals (200 nm to 2 lm in size) before the destruction of the sample by radiation damage.13,14 In SFX, a complete data-set can be obtained by exposing thousands of randomly oriented, individual crystals of proteins to the x-ray beam. For imaging proteins and viruses at atomic resolution, one calls for high intensity and short x-ray pulses.7,15–19 The shortcoming of high intensities is the rapid ionization of the atoms on the few femtosecond timescale, which affects the structure of the system. This radiation induced damage changes the atomic form factors20,21 and may induce signiﬁcant atomic displacement on longer times. Finally, radiation damage changes the scattering pattern. For a comprehensive theoretical study of signal formation in an SFX experiment, one needs to simulate (i) the radia- tion induced dynamics of the sample and (ii) pattern formation based on the dynamics. During the past decade, several models have been developed for studying the time evolution of small and large samples irradiated by XFEL pulses.22–30 We use XMDYN,31,32 a Monte-Carlo molec- ular-dynamics based code developed by the authors. In the theoretical study presented here, we 2329-7778/2016/3(5)/054101/10 054101-2 Abdullah et al. Struct. Dyn. 3, 054101 (2016) Struct. Dyn. 3, 054101 (2016) consider a micron-size crystal in a 100 nm focus beam, a scenario where a nanocrystalline sample experiences ﬂuences as high as to be used in single particle imaging experiments. As a consequence, the x-ray ﬂuence is non-uniform throughout the sample. This may also have its imprint in the scattering pattern. A. Radiation damage simulation XMDYN31–33 has been originally developed for modeling ﬁnite-size systems irradiated by an XFEL pulse. It unites a Monte-Carlo description of ionizations with a classical molecular- dynamical treatment of particle dynamics. XMDYN keeps track of the conﬁguration of the bound electrons in neutral atoms and atomic ions. These conﬁgurations change dynamically be- cause of different atomic processes like inner and outer-shell photoionization, Auger and ﬂuo- rescence decay and collisional (secondary) ionization. In order to treat x-ray-atom interactions, XMDYN uses the XATOM21,34 toolkit, which is an ab-initio framework based on non-relativistic quantum electrodynamics and perturbation the- ory. XATOM provides rates and cross-sections of x-ray-induced processes such as photoioniza- tion, Auger decay, and x-ray ﬂuorescence. XMDYN employs XATOM data, keeps track of all the ionization events along with the electron conﬁguration of each atom, calculates impact ioni- zation and recombination, and follows the trajectories of all the ionized electrons and atoms solving the classical equations of motion numerically. The framework based on these micro- scopic processes can describe complex many-body phenomena in ionized systems such as nano- plasma formation, charge screening, thermalization of electrons through collisions and thermal emission.32 In the current study, chemical bonds between carbon atoms are not considered. This is a good approximation when the ﬂuence is high enough to cause severe ionization in the sys- tem early in the pulse. The immediate ionization of the atoms leads to fast bond breaking that allows their exclusion in simulations.31 I. INTRODUCTION The bottleneck one faces is that it is computationally not feasi- ble to simulate a system with realistic size using tools which are capable to follow the dynam- ics of each atom, required for imaging studies. Therefore, we present an approach that involves the division of a crystal into smaller units (super-cells) and the calculation of their dynamics in- dividually using periodic boundary conditions (PBC). In order to investigate the effect of inho- mogeneous spatial ﬂuence distribution, the super-cells are subjected to different ﬂuences. Then we combine all the super-cells to form a nano-crystal and construct the scattering pattern under the inﬂuence of uniform (within the irradiated part of the sample) and non-uniform spatial beam proﬁles. We study and compare these two scenarios. B. Super-cell approach The symbol 0 represents the exclusion of the term j ¼ i if and only if n ¼ 0. The summation in Eq. (1) is not only computationally very expensive because of the formally inﬁnite sum but is also conditionally convergent which states that the result depends upon the order of summation. To overcome this problem, we follow a route used often in the literature for spatially periodic systems, the method of minimum image convention.37 According to the convention: (i) when evaluating Eq. (1), we do not use the same super-cell division of the PBC crystal for all par- ticles, but we always shift the boundaries so that the selected particle appears in the center; (ii) we consider only n ¼ 0 terms. The former choice ensures that no jump happens in the potential energy when a particle crosses a super-cell boundary and therefore “jumps” in the evaluation from one border of the cell to the opposite. The latter is a minimum choice considering interac- tions between a selected particle with the closest copy of the others only. Finally, one can as- semble the entire real crystal from the individually simulated super-cells to model the whole dynamics. While in this way modeling becomes feasible even without the need of super- computers, we should also note a shortcoming of the approach: we do not allow particle trans- port, in particular, electron transport between the super-cells. For biologically relevant light elements, Auger and secondary electrons have energies Ekin 300 eV, which yields a short mean free path in a dense environment. Therefore, such electrons may travel only to neighbor- ing super-cells experiencing similar ﬂuences during the irradiation, so that the effect of net transport may be negligible. On the other hand, photoelectrons have an energy almost as high as the photon energy. Hence, they are fast and have a long mean free path: they can leave super-cells located at high ﬂuences regions and can affect super-cells at larger distances experi- encing lower ﬂuences. We will overcome this shortcoming of the model in the future. B. Super-cell approach The dimensions of the interaction volume are deﬁned by the intersection of the x-ray beam and the crystal, therefore, its dimensions are determined by the focal area (100 100 nm2) and the thickness of the crystal along the beam propagation direction (lm). The number of atoms within this volume is of the order of 109. This number is formidably large: it is not feasi- ble to simulate the whole system by a single XMDYN run. In order to overcome this barrier, we propose the procedure of dividing the whole crystal into smaller units. These super-cells may contain several crystallographic unit cells. We follow the dynamics within each super-cell driven by the local ﬂuence (assumed to be uniform throughout the super-cell) individually. For this purpose, we have developed an extension to XMDYN that applies PBC35,36 to a super-cell, accounting also for the effect of the environment surrounding it. Within the concept of PBC, a hypothetic crystal is constructed as a periodic extension of a selected super-cell. The total Coulomb interaction energy for a super-cell includes all the inter- actions within the given cell as well as pair interactions when one particle is in the cell while the other is in a periodic image within the super-cell based hypothetic lattice (PBC-crystal). Formally, 54101-3 Abdullah et al. Struct. Dyn. 3, 054101 (2016) 054101-3 Abdullah et al. Struct. Dyn. 3, 054101 (2016) E ¼ 1 4pe0 1 2 X n X N i¼1 X N j¼1 0 qiqj jrij þ nLj ; (1) (1) where N represents the total number of particles in the super-cell, qi is the charge of the ith par- ticle, e0 is the dielectric constant, L represents the dimension of the cell (here assumed to be a cube), nL ¼ n1c1 þ n2c2 þ n3c3, where c1; c2; c3 represent basis vectors of the PBC-crystal, and n1, n2, n3 are integers indexing the periodic images. Hence, jrij þ nLj is the distance between the ith particle in the central super-cell (n ¼ 0) and jth particle in the super-cell indexed by n. The symbol 0 represents the exclusion of the term j ¼ i if and only if n ¼ 0. The summation in Eq. (1) is not only computationally very expensive because of the formally inﬁnite sum but is also conditionally convergent which states that the result depends upon the order of summation. B. Super-cell approach To overcome this problem, we follow a route used often in the literature for spatially periodic systems, the method of minimum image convention.37 According to the convention: (i) when evaluating Eq. (1), we do not use the same super-cell division of the PBC crystal for all par- ticles, but we always shift the boundaries so that the selected particle appears in the center; (ii) we consider only n ¼ 0 terms. The former choice ensures that no jump happens in the potential energy when a particle crosses a super-cell boundary and therefore “jumps” in the evaluation from one border of the cell to the opposite. The latter is a minimum choice considering interac- tions between a selected particle with the closest copy of the others only. Finally, one can as- semble the entire real crystal from the individually simulated super-cells to model the whole dynamics. While in this way modeling becomes feasible even without the need of super- computers, we should also note a shortcoming of the approach: we do not allow particle trans- port, in particular, electron transport between the super-cells. For biologically relevant light elements, Auger and secondary electrons have energies Ekin 300 eV, which yields a short mean free path in a dense environment. Therefore, such electrons may travel only to neighbor- ing super-cells experiencing similar ﬂuences during the irradiation, so that the effect of net transport may be negligible. On the other hand, photoelectrons have an energy almost as high as the photon energy. Hence, they are fast and have a long mean free path: they can leave super-cells located at high ﬂuences regions and can affect super-cells at larger distances experi- encing lower ﬂuences. We will overcome this shortcoming of the model in the future. where N represents the total number of particles in the super-cell, qi is the charge of the ith par- ticle, e0 is the dielectric constant, L represents the dimension of the cell (here assumed to be a cube), nL ¼ n1c1 þ n2c2 þ n3c3, where c1; c2; c3 represent basis vectors of the PBC-crystal, and n1, n2, n3 are integers indexing the periodic images. Hence, jrij þ nLj is the distance between the ith particle in the central super-cell (n ¼ 0) and jth particle in the super-cell indexed by n. D. XSINC: Scattering pattern simulation In order to construct the scattering pattern, Eq. (2) cannot be used directly as the PI and r conﬁguration space is too large. However, by calculating realizations of super-cell dynamics with XMDYN, a Monte-Carlo sampling of the distribution PI;rðF; x; tÞ represented in Eq. (2) becomes feasible. To construct the time evolution of the crystal through global conﬁgurations and to calculate patterns, we used the following strategy, implemented in the code XSINC (x- ray scattering in nano-crystals). y g y We discretize the ﬂuence space and calculate many super-cell trajectories for each ﬂuence value with XMDYN. XSINC selects randomly a trajectory for each super-cell within the crystal (a local realization), so that the corresponding ﬂuence values are matching the best. These tra- jectories describe the local time evolution of the super-cells and together they form a global re- alization of the crystal. Then, taking into account the spatial and temporal pulse proﬁles, XSINC calculates the scattering amplitudes and intensities for the global conﬁguration at differ- ent times based on the corresponding snapshots. Finally, the incoherent sum of these patterns corresponds to a time integrated pattern measured at in a single-shot experiment. In our calcula- tion, we perform a dense sampling of the ﬂuence space. As a consequence, two neighboring super-cells experience very similar ﬂuence. Therefore, it is a good approximation to take into account the direct effect of the neighboring cells by applying periodic boundary conditions and this construction leads to a realistic global trajectory. In the scheme above, several parameters are convergence parameters of the method (Table I). Results are considered converged when characteristic properties of the Bragg peaks, such as the width and height of the intensity distri- bution in reciprocal space, converge during monotonic increase (or decrease) of the parameter. As an example, Figures 1(a) and 1(b) illustrate the convergence of the time integrated peak intensity as a function of the number of local (super-cell) realizations per ﬂuence point for the reﬂection (1 1 1) for the Gaussian and ﬂattop spatial proﬁle cases. We note that convergence implicitly depends on the total number of different realizations used to build a global realiza- tion. Therefore, in the Gaussian case, where 350 different ﬂuence points are used, convergence starts at a much smaller value. TABLE I. Convergence parameters for calculating scattering intensity with XSINC and their values in the current study. C. Scattering intensity Although during a single shot experiment the sample may undergo signiﬁcant changes, the scattering patterns are static: they accumulate diffracted signal over the whole pulse. Further, the signal may contain an imprint of a spatially non-uniform intensity proﬁle. Formally, the scattering intensity at a speciﬁc reﬂection described by reciprocal vector Q, including the inte- gration over time and the subdivision of the crystal volume into super-cells according to the approach introduced in Section II B, reads dI Q; F; x ð Þ dX ¼ C X ð Þ ð1 1 dt g tð Þ X I;r PI;r F; x; t ð Þ X l ﬃﬃﬃﬃﬃﬃﬃ F l p eiQRl X X X NX j¼1 fX;Il X;j Q; x ð Þ eiQrl X;j 2 : dI Q; F; x ð Þ dX ¼ C X ð Þ ð1 1 dt g tð Þ X I;r PI;r F; x; t ð Þ X l ﬃﬃﬃﬃﬃﬃﬃ F l p eiQRl X X X NX j¼1 fX;Il X;j Q; x ð Þ eiQrl X;j 2 : (2) (2) In this equation, Q is the momentum transfer, F ¼ fF lg is the x-ray ﬂuence distribution through- out the crystal, the index l runs over all super-cells, and x is the photon energy. C(X) is a factor depending the polarization of the x-ray pulse, and g(t) represents the normalized temporal envelope. fX;Il X;j is the atomic form factor of the jth atom of species X in the lth super-cell, Il X;j is the associat- ed electronic conﬁguration, I ¼ fIl X;jg denotes a global electronic conﬁguration, rl X;j represents the position vector of the jth atom of species X in the lth super-cell, and r ¼ frl X;jg indicates the set of all atomic positions. NX represents the total number of atoms for species X within a super-cell. PI,r represents the probability distribution of electronic conﬁguration I and atomic positions r, and Rl represents the position of the lth super-cell. The atomic form factor 054101-4 Abdullah et al. Struct. Dyn. 3, 054101 (2016) 054101-4 Abdullah et al. Struct. Dyn. 3, 054101 (2016) fX;IX j ðQ; xÞ ¼ f 0 X;Il X;jðQÞ þ f 0 X;Il X;jðxÞ þ i f 00 X;Il X;jðxÞ (3) (3) includes the dispersion corrections f 0 X;Il X;jðxÞ and i f 00 X;Il X;jðxÞ. C. Scattering intensity This dispersion correction can be includes the dispersion corrections f 0 X;Il X;jðxÞ and i f 00 X;Il X;jðxÞ. This dispersion correction can be neglected when the applied photon energy is high above the ionization edges, which is fulﬁlled in our study. Note that the summation over ﬃﬃﬃﬃﬃﬃﬃ F l p appears inside the modulus square in Eq. (2). The scattering amplitude from the lth super-cell is proportional to the x-ray ﬁeld amplitude (/ ﬃﬃﬃﬃﬃﬃﬃ F l p ) in that super-cell. A key assumption when performing the coherent sum in Eq. (2) is that the entire crystal is illuminated coherently, a condition that is fulﬁlled considering realistic XFEL beam parameters and crystal sizes. D. XSINC: Scattering pattern simulation Convergence parameters Gaussian case Flattop case Number of crystallographic unit cells in a super-cell 5 5 5 5 5 5 Number of ﬂuence points 350 1 Number of local realizations (XMDYN trajectories) per ﬂuence point 5 150 Number of assembled global realizations 10 10 Depth of the crystal in beam propagation direction 1 Thickness of the super-cell lattice constant 1 Thickness of the super-cell lattice constant Number of snapshots 28 28 onvergence parameters for calculating scattering intensity with XSINC and their values in the current study. 054101-5 Abdullah et al. Struct. Dyn. 3, 054101 (2016) FIG. 1. Convergence of time integrated peak intensity for the reﬂection (1 1 1) as a function of the number of realizations per ﬂuence point: (a) for the Gaussian case and (b) the ﬂattop case. For the Gaussian case, 350 different ﬂuences points are used to calculate the time integrated intensity. FIG. 1. Convergence of time integrated peak intensity for the reﬂection (1 1 1) as a function of the number of realizations per ﬂuence point: (a) for the Gaussian case and (b) the ﬂattop case. For the Gaussian case, 350 different ﬂuences points are used to calculate the time integrated intensity. A. Simulation setup In our investigations, we consider a diamond cube of a size of 1 lm. We investigate the cases of ﬂattop and Gaussian beam proﬁles (Fig. 2). Other parameters of the pulses are the same in both cases: photon energy is 10 keV, total number of photons per x-ray pulse is 1 1012, the temporal pulse envelope is Gaussian with a duration of 10 fs FWHM, and focus size is 100 100 nm2 FWHM. The size of the diamond unit cell is a ¼ b ¼ c ¼ 3.57 A˚ contain- ing 8 carbon atoms. The parameter choices listed in Table I yield converged results. B. Radiation damage The coherent scattering patterns depend on the presence of the atomic bound electrons as well as on the atomic positions. The XMDYN and XATOM simulations allow to analyze their change due to radiation damage for both diamond and for the isolated carbon atom cases. FIG. 2. Radial ﬂuence distributions in the current study: Gaussian proﬁle (spatially non-uniform case) and ﬂattop proﬁle (uniform within the irradiated part of the crystal). The focal size is 100 nm in both cases, and the pulse energy is also con- sidered to be same. FIG. 2. Radial ﬂuence distributions in the current study: Gaussian proﬁle (spatially non-uniform case) and ﬂattop proﬁle (uniform within the irradiated part of the crystal). The focal size is 100 nm in both cases, and the pulse energy is also con- sidered to be same. 054101-6 Abdullah et al. Struct. Dyn. 3, 054101 (2016) Struct. Dyn. 3, 054101 (2016) Struct. Dyn. 3, 054101 (2016) Radiation damage is initiated by atomic photoionization events. In case of isolated carbon atoms, Auger decays contribute approximately to the same extent to the overall ionization. At the maximum ﬂuence in our study, 35% of the atoms are photoionized (Fig. 3(a)). Although the absorbed energy is 10 keV per photon, almost all of this energy is taken away from the atom by the high energy photoelectron. The picture is different when the atom is embedded in a crystal environment (Fig. 3(c)). The high energy photoelectrons stay within the medium and distribute their energy by causing further ionization via secondary ionization events. As a con- sequence, neutral atoms disappear early in the pulse and by the end even fully stripped carbon ions (C6þ) appear. Many electrons are promoted to (quasi-)free states within the sample. This also illustrates the importance of secondary ionizations in the progress of radiation damage in a dense environment.38–40 In the center of focus, the sample absorbs 3.5 keV energy per atom that heats up the plasma electrons beside the ionizations. Despite the high charge states, recom- bination remains negligible during the pulse (number of events less than 1% per atom in the simulation) due to the extreme conditions. Figure 4 represents the mean displacement of the carbon atoms during the pulse. The aver- age atomic displacement is much below the maximum achievable resolution, 1.2 A˚ at 10 keV, even at the highest ﬂuence. B. Radiation damage This suggests that the patterns are affected predominantly due to the bound-electron loss through the modiﬁcation of atomic scattering form factors. Despite the heavy ionization, atomic displacements remain negligible during the ultrashort pulse duration due to the highly symmetrical sample environment. We note here again that in our calculations we neglected the chemical bonds. In low ﬂuence regions bonds may survive and stabilize the structure against the emerging Coulomb forces. As the observed displacements are far below the resolution even without any stabilization due to bonds, bondless modeling of the current scenario is applicable. FIG. 3. Ionization dynamics of carbon atoms at different ﬂuences: time dependent charge state populations of isolated car- bon atoms calculated with XATOM for (a) F high ¼ 1 1014 lm2 and (b) F mid ¼ 4:5 1013 lm2. Similarly, time depen- dent charge state populations of carbon atoms in diamond calculated with XMDYN for (c) F high and (d) F mid. Secondary ionization events enhance the overall ionization in a dense environment. The x-ray pulse with 10 fs FWHM temporal pro- ﬁle is centered at t ¼ 0 fs. FIG. 3. Ionization dynamics of carbon atoms at different ﬂuences: time dependent charge state populations of isolated car- bon atoms calculated with XATOM for (a) F high ¼ 1 1014 lm2 and (b) F mid ¼ 4:5 1013 lm2. Similarly, time depen- dent charge state populations of carbon atoms in diamond calculated with XMDYN for (c) F high and (d) F mid. Secondary ionization events enhance the overall ionization in a dense environment. The x-ray pulse with 10 fs FWHM temporal pro- ﬁle is centered at t ¼ 0 fs. Struct. Dyn. 3, 054101 (2016) 054101-7 Abdullah et al. FIG. 4. Mean displacement of the atoms for ﬂuences F high ¼ 1 1014 lm2 (red dots), F mid ¼ 4:5 1013 lm2 (blue dots), and F low ¼ 6:0 1012 lm2 (green dots). The Gaussian temporal pulse envelope is also depicted with the dashed black line. F high is the ﬂuence for the ﬂattop proﬁle, which is also the maximum ﬂuence in the present study. F mid and F low are two values representing intermediate and low ﬂuences taken from the Gaussian proﬁle case. The mean atomic dis- placement remains below the achievable resolution (1.2 A˚ ) at 10 keV for all the cases. FIG. 4. B. Radiation damage Mean displacement of the atoms for ﬂuences F high ¼ 1 1014 lm2 (red dots), F mid ¼ 4:5 1013 lm2 (blue dots), and F low ¼ 6:0 1012 lm2 (green dots). The Gaussian temporal pulse envelope is also depicted with the dashed black line. F high is the ﬂuence for the ﬂattop proﬁle, which is also the maximum ﬂuence in the present study. F mid and F low are two values representing intermediate and low ﬂuences taken from the Gaussian proﬁle case. The mean atomic dis- placement remains below the achievable resolution (1.2 A˚ ) at 10 keV for all the cases. Effect of the PBC approach on the dynamics. While ionic motion is negligible during the pulse, fast photoelectrons can travel long distances. However, PBC conﬁnes all plasma elec- trons artiﬁcially within the supercell they have been created in. Neglecting particle transport may lead to error in (i) local plasma electron density and (ii) local energy density. Whenever a photoelectron is ejected it leaves behind a positive charge located on an ion. If we consider Coulomb interaction only, a positive space charge would build up in a central cylinder because of photoelectron escape. Photoelectron trapping within the interaction volume would start early in the pulse, at an average ion charge as low as þ0.005. An analogous phenomenon was dis- cussed for ﬁnite samples in the literature.29 However, photoelectrons cause secondary ionization as well, so an atomic bound electron is promoted to a low energy continuum state. If this slow electron was created in an outer region, it can efﬁciently contribute to the screening of the space charge the photoelectron left behind. Based on these arguments we can conclude that (i) considering the interaction region to be neutral is a good approximation and (ii) in all regions we overestimate the energy density by conﬁning fast photoelectrons within a supercell. Similarly, as the Coulomb forces are the driving forces of the ionic motions, we may also over- estimate the atomic/ionic displacements. In our study eventually the effect on the scattering sig- nal is relevant, as will be discussed in Sec. III C. C. Scattering with damage In this section, we analyze the changes of the Bragg peak intensity proﬁles in reciprocal space due to the severe radiation damage. In Figs. 5(a) and 5(b), snapshots of the 1D Bragg peak proﬁles in reciprocal space are depicted for the reﬂection Q ¼ (1 1 1) for Gaussian and ﬂattop spatial beam proﬁles, respectively. Two apparent features can be seen, valid for other reﬂections as well. (i) The width of the Bragg peak does not change during the pulse. This is consistent with the expectation based on the negligible ion displacements: no visible Debye-Waller-like broad- ening occurs. However, the widths are different for the Gaussian and ﬂattop cases. The rea- son is the difference between the size of the illuminated parts of the crystal. In the ﬂattop 054101-8 Abdullah et al. Struct. Dyn. 3, 054101 (2016) FIG. 5. Snapshots of the scattering intensity for reﬂection (1 1 1) along the Qy ¼ Qz ¼ 1 ˚A 1 line in reciprocal space: (a) Gaussian spatial beam proﬁle, (b) ﬂattop spatial beam proﬁle. Solid and dashed lines with the same color correspond to the same instantaneous irradiating x-ray intensities. Note that the negative and the corresponding positive times are of equal in- tensity during the rise and fall of the pulse envelope. (c) and (d) Total time integrated scattering signal for Gaussian and ﬂattop spatial beam proﬁles, respectively. Note the different vertical axis scales. FIG. 5. Snapshots of the scattering intensity for reﬂection (1 1 1) along the Qy ¼ Qz ¼ 1 ˚A 1 line in reciprocal space: (a) Gaussian spatial beam proﬁle, (b) ﬂattop spatial beam proﬁle. Solid and dashed lines with the same color correspond to the same instantaneous irradiating x-ray intensities. Note that the negative and the corresponding positive times are of equal in- tensity during the rise and fall of the pulse envelope. (c) and (d) Total time integrated scattering signal for Gaussian and ﬂattop spatial beam proﬁles, respectively. Note the different vertical axis scales. proﬁle case, the focus size deﬁnes strictly the region exposed. On the other hand, a Gaussian proﬁle has no sharp edge and therefore illuminate a larger region, yielding a nar- rower Bragg peak and a larger effective crystal size. (ii) Snapshots of the Bragg peak intensities behave differently for ﬂattop and Gaussian beams. IV. CONCLUSIONS We presented a methodology for the simulation of x-ray scattering patterns from serial femtosecond crystallography experiments with a high intensity x-ray beam. Our approach includes the simulation of radiation damage within the sample with the codes XMDYN and XATOM as well as the calculation of the patterns using the code XSINC. In the approach the crystal is divided into smaller units. The time evolution (the radiation damage process) of these units is calculated using periodic boundary conditions. Finally, a nanocrystal is assembled from the small units for the calculation of the patterns integrated over the pulse. As a demonstration, we investigated spatial pulse proﬁle effects on the Bragg peaks for a diamond nanocrystal. We found that if a Gaussian proﬁle is used (assuming realistic XFEL parameters such as tight focus and ultrashort pulse duration), the time integrated signal intensity is reduced only by a small amount compared to the damage-free case. For a ﬂattop proﬁle, the decrease is much more signiﬁcant. The intensity reduction is due to the change of the form fac- tors caused by ionizations. In both cases, the width of the Bragg peak was connected to the size of the illuminated region in the crystal, but was not affected by damage. We analyzed the shortcoming of the periodic boundary condition approach. The method overestimates radiation damage in the interaction region, so it gives an upper bound to the effect of radiation damage on the patterns. In the future, the simulation method developed here is to be applied to more complex scenarios. C. Scattering with damage The snapshots of the Bragg intensities depend not only on the scattering power of the sam- ple but also on the instantaneous x-ray intensity. However, as the instantaneous x-ray inten- sities are equal at the same time before and after the maximum of the pulse, a direct comparison of the corresponding snapshots of the Bragg proﬁles reﬂects exclusively the ef- fect of different damage extents. In the Gaussian proﬁle case, these corresponding curves show small difference only, indicating that a signiﬁcant contribution is coming from regions in the crystal suffering little damage (Fig. 5(a)). In contrast, applying a ﬂattop pulse proﬁle, the scattering pattern is formed only from extensively ionized parts of the crystal. A consequence of the loss of atomic bound-electrons is the decrease of the atomic form fac- tors yielding signiﬁcant signal drop for longer times (Fig. 5(b)). The above ﬁndings are reﬂected by the time integrated signals that correspond to the situa- tion one would encounter in an experiment (1D cut: Figs. 5(c) and 5(d); 2D cut: Fig. 6). Note that for the Gaussian spatial proﬁle, there is only a small decrease of the signal compared to the ideal (no damage) case. Effect of the PBC approach on the x-ray scattering patterns. In Section III B, we discussed that the PBC approximation overestimates ionization and atomic displacements, and therefore radiation damage throughout the sample. It means that the method gives an upper bound to the effect of radiation damage on the scattering patterns. A trivial lower bound is the case without any radiation damage. 054101-9 Abdullah et al. Struct. Dyn. 3, 054101 (2016) FIG. 6. Contour plot for the Bragg spot of reﬂection (1 1 1) in the Qz ¼ 1 ˚A 1 plane in reciprocal space: (a) Gaussian beam proﬁle; (b) ﬂattop beam proﬁle. FIG. 6. Contour plot for the Bragg spot of reﬂection (1 1 1) in the Qz ¼ 1 ˚A 1 plane in reciprocal space: (a) Gaussian beam proﬁle; (b) ﬂattop beam proﬁle. 1N. Berrah and P. H. Bucksbaum, Sci. Am. 310, 64–71 (2014). 2 2H. N. Chapman et al., Nature 470, 73–77 (2011). 3 1N. Berrah and P. H. Bucksbaum, Sci. Am. 310, 64–71 (2014). 2H. N. Chapman et al., Nature 470, 73–77 (2011). 3A. Barty et al., Nature Photon. 6, 35–40 (2012). 4S. 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https://openalex.org/W2898943629	https://www.scielo.br/j/clin/a/sHFv57qzTsKzNtfS5ghTzpx/?lang=en&format=pdf	English	null	Histomorphometric analysis of the skin of women during the reproductive period	Clinics	2,018	cc-by	3,270	Heraldo Carlos Borges Inforzato,I Adriana Aparecida Ferraz Carbonel,II,* Ricardo Santos Simo˜ es,III Gisela Rodrigues da Silva Sasso,II Patricia Daniele Azevedo Lima,IV Jose´ Maria Soares-Ju´ nior,III Lydia Masako Ferreira,I Manuel de Jesus Simo˜ esII I Divisao de Cirurgia Plastica, Universidade Federal de Sao Paulo (EPM/UNIFESP), Sao Paulo, SP, BR. II Departamento de Morfologia e Genetica, Universidade Federal de Sao Paulo (EPM/UNIFESP), Sao Paulo, SP, BR. III Departamento de Ginecologia e Obstetricia, Faculdade de Medicina FMUSP, Universidade de Sao Paulo, Sao Paulo, SP, BR. IV Department of Cellular and Molecular Medicine, University of Ottawa, Ottawa, Canada. Inforzato HC, Carbonel AA, Simo˜ es RS, Sasso GR, Azevedo Lima PD, Soares-Ju´ nior JM, et al. Histomorphometric analysis of the skin of women during the reproductive period. Clinics. 2018;73:e387 Inforzato HC, Carbonel AA, Simo˜ es RS, Sasso GR, Azevedo Lima PD, Soares-Ju´ nior JM, et al. Histomorphometric analysis o reproductive period. Clinics. 2018;73:e387 Corresponding author. E-mail: adricarbonellﬁsio@hotmail.com Corresponding author. E-mail: adricarbonellﬁsio@hotmail.com OBJECTIVES: The aim of this study was to evaluate the histomorphometry of the skin of women during the reproductive period according to the Fitzpatrick classiﬁcation. OBJECTIVES: The aim of this study was to evaluate the histomorphometry of the skin of women during the reproductive period according to the Fitzpatrick classiﬁcation. METHODS: Thirty women aged 30 to 45 years were included in this study. We studied the surgical sites of extracted nevi. The material was processed for routine histology and then stained with haematoxylin and eosin as well as Picrosirius red. Four-micrometre histological sections were analysed according the Fitzpatrick criteria (skin pigmentation). The skin thickness and collagen concentration were determined for the reticular dermal skin. The data were statistically analysed with ANOVA. RESULTS: Fitzpatrick skin types I and II were thicker than the other skin types. RESULTS: Fitzpatrick skin types I and II were thicker than the other skin types. CONCLUSIONS: Our data suggest that white skin may be less thick than dark sk CONCLUSIONS: Our data suggest that white skin may be less thick than dark skin. KEYWORDS: Skin; Epidermis; Dermis; Collagen. ’ INTRODUCTION suggest that there is no difference in skin thickness between black and white people (4,11-13). Since the appearance of the first human ancestor, our skin has played a significant role as an organ responsible for protec- tion, vitamin D3 synthesis, thermoregulation and protec- tion against folic acid lyses (1-4). Shuster et al. (14) suggest that women’s skin thickness is continuous until they reach 50 years of age. This character- istic is not observed in male skin, even though women’s skin contains less collagen. Oriá et al. (15) also suggested that skin thickness decreases with human age because of hormone changes. Edwards and Duntley (5) were the first to quantify human skin pigmentation. They used a spectrophotometer to mea- sure the visible spectrum of their own pigmentation. How- ever, Schulze (6), in 1956, was the first to consider human skin according its reaction to UV rays. g Montagna and Carlisle (16) did not observe a difference in facial skin thickness between black and white women. Based on these data, this study conducted a morphological and morphometric study (thickness and collagen concentra- tion) of female skin during the reproductive period based on the Fitzpatrick classification method. g y Fitzpatrick (7) established the classification criteria related to reactions to sun rays based on a specific need to classify white-skinned people undergoing phototherapy. Currently, the most frequently applied skin classification method is the one established by Fitzpatrick (8). He presen- ted a classification based on six different skin types (9,10). ORIGINAL ARTICLE ORIGINAL ARTICLE ’ DISCUSSION The human species has undergone considerable misce- genation that has resulted in the appearance of different skin shades, from white to black. Considering that skin shade can be associated with various pathological statuses, it is necessary to understand the architecture of the skin and establish a relationship between skin architecture and skin colour. Various studies have attempted to classify the skin, but the most frequently referenced is that of Fitzpatrick (7), who presented his first classification in 1975. He observed adverse effects when chemo- and phototherapy were used to treat psoriasis in people with different skin shades. In 1988, Fitzpatrick (8) presented a new classification that was adop- ted in this and other studies (18,19). The same procedure was applied to determine dermal thickness. The upper limit was the under-portion of the under-face of basal layer, and the under limit was the subcutaneous adipose tissue. The section chosen for these measures was the under-section of the epidermal papilla. Women’s skin was analysed in this study as women represent the patient population most likely to seek skin treatments and plastic surgery. Some limits were established to avoid deep hormonal interferences: the study participants were limited to women aged between 20 and 45 years with regular menstrual cycles. p p p To determine the collagen concentration, the Picrosirius red-stained fragments were used. Ten images from the reticular dermis were taken from the middle dermis portion of each woman. The image capture system was the same as that described above. These images were analysed using Image-Pro-Pluss software version 4.5. g y Changes in skin characteristics during ageing are fre- quently determined by environmental and extrinsic factors, such as UV radiation. Therefore, the skin analyses performed in this study used samples collected from the dorsal zone of eumenorrheic women. This zone was chosen because it is not exposed to the environment full-time and because it presents a well-defined lucidum stratum that can make the epidermis measurements easier. Morphological results Among fragments of the same skin type, there were no significant morphological differences. However, some differ- ences between the epidermis and dermis thickness were observed. Thus, well-defined epidermis and dermis limits were observed in all skin types. Skin types I and II presented a thin epidermis and well- defined epidermal papilla compared with the other types. These types had a lower quantity of granulose and spiny keratinocytes compared to the other types (Figure 1). In the type III and IV epidermis, melanin was clearly present in the basal and spiny keratinocyte cytoplasm layer (Figure 1). Women classified with skin types V and VI and presenting with a dysplastic nevus were not identified in the sample and were not studied. This pathology is very rare in people with darker skin because they have greater natural protec- tion from melanin (17). The presence of loose connective tissue between the dermal papilla of all studied types was noted. Numerous collagen fibres were regularly distributed under the dermal exten- sion (reticular region). After the local application of lidocaine (2%) and adrenalin (1:200.000), skin fragments were extracted from the free surgical margins of a dysplasia nevus in the dorsal region. The fragments were immersed in 10% formaldehyde and phosphate buffer for 12 hours and then processed for paraffin inclusion. The fractions (3 mm) were submitted to staining with haematoxylin and eosin (H.E.) and Picrosirius red (Sirius Red F3BA - Sigma-Aldrich Corp., St. Louis, MO). In skin types I and II, numerous light gaps were identified between the collagen fibre bundles. In skin types III and IV, these bundles were thicker. Morphometric analysis The thickness of the H.E.-stained fractions was measured on images taken with image-capture equipment. The images were obtained with a high-resolution camera (AxioCam – Carl Zeisss) attached to a Zeiss microscope (Carl Zeisss) that transmitted these images to a Pentium 4 computer with 502 megabytes of RAM memory working on the Windows XP Professionals platform.AxioVision Rel 4.2 (Carl Zeisss) software was employed for the skin measures. It was stan- dardized with a millimetric scale plate (Carl Zeisss) for the several objectives used (4, 10, 40X). This procedure was perfor- med for 20 epidermal measures from each woman limited by the under-face of the basal layer (the portion in contact with the basal membrane) and the outermost face of the stratum granulosum (the layer in which the nucleus is under the lucidum stratum) in different sections of the same plate. The section chosen was the outermost part of the dermal papilla. Morphometric results Analyses of epidermis (p=0.18) and dermis (p=0.32) thick- ness and collagen concentration (p=0.458) showed no discre- pancies among the skin types studied. The Bartlett test was applied to elucidate the homoscedasticity principle (Table 2). ’ MATERIALS AND METHODS yp Regarding morphological aspects, there are rare studies in the literature that mention the different types of skin and their histomorphometric characteristics. A majority of studies The procedure applied in this study was first submitted to and approved by the UNIFESP/EPM Ethics Committee, and it was carried out after the women’s assent was obtained. Skin samples were collected from women at the Santa Rita Clinical Centre in Guarujá/São Paulo and analysed. Copyright & 2018 CLINICS – This is an Open Access article distributed under the terms of the Creative Commons License (http://creativecommons.org/licenses/by/ 4.0/) which permits unrestricted use, distribution, and reproduction in any medium or format, provided the original work is properly cited. Skin samples were taken from 30 eumenorrheic women aged 20 to 45 years. Their skin had previously been classified by two observers according to the parameters established by Fitzpatrick (10) (Table 1). Some exclusion factors were defined, including skin and collagen diseases, the use of hormone-based medications, the absence of menstruation and a lack of exposure to the sun exposure in the last 30 days or more. No potential conﬂict of interest was reported. Received for publication on October 25, 2017. Accepted for publi- cation on June 5, 2018 1 Table 1 - Skin types according to Fitzpatrick (8) skin type classiﬁcation in relation to the ﬁrst solar exposure of the summer. Skin Pigmentation Skin reaction to the ﬁrst summer exposure I White Burns and never tans II White In general, burns but can tan with difﬁculty III White Sometimes burns, can get moderately tanned IV Light brown Sporadically burns but tans easily V Light brown Tans very easily and never gets burned VI Black Tans very easily Skin of women during reproductive period Inforzato HCB et al. CLINICS 2018;73:e387 Table 1 - Skin types according to Fitzpatrick (8) skin type classiﬁcation in relation to the ﬁrst solar exposure of the summer. Statistical analyses One-way ANOVA was used to analyse data analysis of skin thickness and collagen concentration data. The dermis and epidermis thickness in the same group was statistically compared using the Bartlett test, while the Bonferroni test was used for comparisons between the different types of skin. The same scheme was followed for the collagen con- centration analyses. The 30 patients were well distributed among skin types I to IV. There were no patients with skin types V and VI because the skin fragments were taken from the free surgical margins of excised dysplastic nevi, and this type of patho- logy is rare among people with those skin types. 2 Figure 1 - Photomicrographs of Fitzpatrick skin types I, II, III and IV in women. CLINICS 2018;73:e387 Skin of women during reproductive perio Inforzato HCB et a CLINICS 2018;73:e387 Skin of women during reproductive perio Inforzato HCB et a Skin of women during reproductive period Inforzato HCB et al. Skin of women during reproductive period Inforzato HCB et al. CLINICS 2018;73:e387 Figure 1 - Photomicrographs of Fitzpatrick skin types I, II, III and IV in women. Figure 1 - Photomicrographs of Fitzpatrick skin types I, II, III and IV in women. Table 2 - Morphometric analysis of Fitzpatrick skin types I, II, III and IV (8). Table 2 - Morphometric analysis of Fitzpatrick skin types I, II, III and IV (8). Skin types according to the Fitzpatrick classiﬁcation Type I (n=6) Type II (n=8) Type III (n=10) Type IV (n=6) Skin thickness (mm) 2.99±0.22 3.49±0.37 3.91±0.21 4.30±0.82 Epidermis thickness (mm) 40.39±1.05 44.10±1.07 50.49±2.11 58.72±1.54 Dermis thickness (mm) 2.97±0.21 3.45±0.35 3.86±0.19 4.24±0.71 Collagen in the reticular dermis (%) 61.83±5.58 67.66±2.89 70.38±2.45 64.44±2.74 Histological differences between the black and white skin have been reported in other articles. A few of these make some reference to the intermediary pigmentation. Lu et al. (20) were the first authors to make an analogy between the Fitzpatrick classification method and the melanin granule distribution in keratinocytes of different skin types. This analogy seems to be logical and suitable for clinical practice. Significant differences in the thickness of the skin and its layers (dermis and epidermis) were found between the different skin types classified according to Fitzpatrick. Skin type I was the thinnest, and skin type IV was the thickest. ’ REFERENCES between types I and II and types III and IV. It is important to reiterate that this method evaluates the collagen concentra- tion of a predetermined fraction of the study samples. 1. Quevedo WC, Fitzpatrick TB, Pathak MA, Jimbow K. Role of light in human skin color viariation. Am J Phys Anthropol. 1975;43(3):393-408, http://dx.doi.org/10.1002/ajpa.1330430321. p y p The data obtained could be useful to surgical and clinical practices because it indicates the patient groups who have more or less protection against the environment because their epidermis is thinner or thicker. This could be one more factor that explains why white skin is more inclined toward skin cancer, which confirms the information found in the medical literature. Considering skin healing, darker skin tends to respond faster to healing after surgical incisions, which reflects the higher keloid incidence in this group. However, the dermis of people with skin types I and II may have less tensile strength than the dermis of people with skin types III and IV. p // g/ / jp 2. Jablonski NG, Chaplin G. The evolution of human skin coloration. J Hum Evol. 2000;39(1):57-106, http://dx.doi.org/10.1006/jhev.2000.0403. a p g jp 2. Jablonski NG, Chaplin G. The evolution of human skin coloration. J Hum Evol. 2000;39(1):57-106, http://dx.doi.org/10.1006/jhev.2000.0403. p g jp . Jablonski NG, Chaplin G. The evolution of human skin colorat Evol. 2000;39(1):57-106, http://dx.doi.org/10.1006/jhev.2000.040 h d l l a d l 3. Linton R. O homem. Traduc¸ão Lavínia Vilela. 12a Ed. São Paulo. Martins Fontes, 2000. 4. Langaney A, Cloítes J, Guilaine J, Simonnet D. A mais bela história do homem. Traduc¸ão Maria Helena Kühner. 1a Ed. Rio de Janeiro; DIFEL. 2002. 5. Edwards EA, Duntley SQ. The pigment and color of human skin. Am Anat. 1939;65:1-33, http://dx.doi.org/10.1002/aja.1000650102. p g j 6. Einige SR. Versuche und Bemerkungen zum Problem der Handelsue- blichen Lichtschutzmittel. Parf u Kosm. 1956;37:310-5. blichen Lichtschutzmittel. Parf u Kosm. 1956;37:310-5. 7. Fitzpatrick TB. Soleil et peau. J Med Esthet. 1975;2:33-4. 8. Fitzpatrick TB. The validity and practicality of sun-reactive skin types I through VI. Arch Dermatol. 1988;124(6):869-71, http://dx.doi.org/10.1001/ archderm.1988.01670060015008. These data can be applied in surgical and clinical areas in relation to protection factors, healing and laser and cosmetic treatments for the different skin types. 9. Jimbow K, Quevedo WCJr, Fitzpatrick TB, Szabo G. Some aspects of melanin biology: 1950-1975. J Invest Dermatol. 1976;67(1):72-89, http:// dx.doi.org/10.1111/1523-1747.ep12512500. g p 10. Fitzpatrick TB. Ultraviolet-induced pigmentary changes: benefits and hazards. ’ ACKNOWLEDGMENTS p 18. Sadick NS, Smoller B. A study examining the safety and efficacy of a fractional laser in the treatment of photodamage on the hands. J Cosmet Laser Ther. 2009;11(1):29-33, http://dx.doi.org/10.1080/147641708026 12992. We wish to thank Elizabeth Micai and Paulo Celso Franco for technical support. 19. Sachdeva S. Fitzpatrick skin typing: applications in dermatology. Indian J Dermatol Venereol Leprol. 2009;75(1):93-6, http://dx.doi.org/10.4103/ 0378-6323.45238. ’ REFERENCES Curr Probl Dermatol. 1986;15:25-38, http://dx.doi.org/10.1159/ issn.1421-5721. Regarding the clinical application of our findings, we believe that there are biological differences in skin. Thicker skin is more resistant to stress and tends to wrinkle less because it has a higher collagen fibre content. Furthermore, there may be differences in the absorption of topical pharma- cological substances and a great tendency toward collagen and fibroblast proliferation, which may help during the cicatricial process or explain the high number of keloids among patients with these skin types. Such characteristics are due to a high quantity of stem cells, fibroblasts, collagen fibres, and other elements of the extracellular matrix, such as structural proteins and glycosaminoglycans which contribute to different inflammatory processes (21). 11. Olson RL, Gaylor J, Everett MA. Skin color, melanin, and erythema. Arch Dermatol. 1973;108(4):541-4, http://dx.doi.org/10.1001/archderm.1973. 01620250029008. 12. Weigand DA, Haygood C, Gaylor JR. Cell layers and density of Negro and Caucasian stratum corneum. J Invest Dermatol. 1974;62(6):563-8, http://dx.doi.org/10.1111/1523-1747.ep12679412. p g p 13. Rubegni P, Cevenini G, Barbini P, Flori ML, Fimiani M, Andreassi L. Quantitative characterization and study of the relationship between constitutive-facultative skin color and phototype in Caucasians. Photo- chem Photobiol. 1999;70(3):303-7. 14. Shuster S, Black MM, McVitie E. The influence of age and sex on skin thickness, skin collagen and density. Br J Dermatol. 1975;93(6):639-43, http://dx.doi.org/10.1111/j.1365-2133.1975.tb05113.x. 15. Oriá RB, Ferreira FV, Santana EN, Fernandes MR, Brito GA. Estudo das alterac¸ões relacionadas com a idade na pele humana, utilizando métodos de histo-morfometria e autofluorescência. An Bras Dermatol. 2003;78(4): 425-34, http://dx.doi.org/10.1590/S0365-05962003000400004. y p Our data analysing the skin of Brazilian women consid- ered Brazil as a mixed country with great variation in ethnic groups. According to the Fitzpatrick classification method, lighter skin is thinner. However, there was no difference in the collagen concentration in the dermis according to skin type. 425-34, http://dx.doi.org/10.1590/S0365-05962003000400004 p g 16. Montagna W, Carlisle K. The architecture of black and white facial skin. J Am Acad Dermatol. 1991;24(6 Pt 1):929-37, http://dx.doi.org/10.1016/ 0190-9622(91)70148-U. 17. Joel L. Spitz, editor. Genodermatoses. A Clinical Guide to Genetic Skin Disorders. Philadelphia, Baltimore, New York: Lippincott Williams and Wilkins, 2005. Format: hard cover, one volume. Second edition. Pages 400, chapters 13. ISBN 0-7817-4088-6. CLINICS 2018;73:e387 CLINICS 2018;73:e387 Statistical analyses The majority of authors of previous studies had studied the skin from corpses, and they focused on neither age nor gender. In addition, there was no standardization of the region from which the skin samples were collected. It is important to clarify that the thickness and viscoelastic properties of the skin depend on the quantity of the material quantity in the dermis and its structural organization (11,12). p Several studies of melanocytes have suggested that mela- nosomes differ between black and white people. In 1991, Montagna and Carlisle (16) demonstrated that the epidermis of black skin has thicker collagen fibres, larger fibroblasts and larger melanosomes. It should be noted that this study included samples from white, light brown and black skin. The use of three skin types reinforces the need for parameters for histological studies of the different skin types. Instead limited studies to three skin types, this new parameter must reflect a larger diversity that is complementary to anthro- pological studies. To determine the collagen concentration in the dermis, Picrosirius staining based, which is based on Sirius Red staining, was applied. As this stain is strongly acidic, it reacts with the amine groups of the lysine molecules in the collagen structure. Microscopic analysis revealed that the collagen from skin types I and II is similar in morphology and concentration. The same is true for skin types III and IV; however, no significant differences (p=0.458) were observed 3 Skin of women during reproductive period Inforzato HCB et al. Skin of women during reproductive period Inforzato HCB et al. ’ AUTHOR CONTRIBUTIONS Inforzato HC and Carbonel AA were responsible for general supervision, data acquisition and writing and revising the manuscript. Simões RS, Azevedo Lima PD and Sasso GR were responsible for discussion, writing and ﬁnal preparation of the manuscript. Soares-Júnior JM, Ferreira LM and Simões MJ were responsible for the ﬁnal preparation of the manuscript. 20. Lu H, Edwards C, Gaskell S, Pearse A, Marks R. Melanin content and distribution in the surface corneocyte with skin phototypes. Br J Dermatol. 1996;135(2):263-7, http://dx.doi.org/10.1111/j.1365-2133.1996. tb01157.x. 21. Weigand DA, Haygood C, Gaylor JR. Cell layers and density of Negro and Caucasian stratum corneum. J Invest Dermatol. 1974;62(6):563-8, http://dx.doi.org/10.1111/1523-1747.ep12679412. 4
https://openalex.org/W4362479428	https://figshare.com/articles/journal_contribution/Supplementary_Figure_1_from_Epithelial_Mesenchymal_Transition_and_Stem_Cell_Markers_in_Patients_with_HER2-Positive_Metastatic_Breast_Cancer/22496455/1/files/39954475.pdf	English	null	Supplementary Figure 1 from Epithelial–Mesenchymal Transition and Stem Cell Markers in Patients with HER2-Positive Metastatic Breast Cancer	null	2,023	cc-by	3	Supplementary Figure 1
https://openalex.org/W4281610121	https://link.springer.com/content/pdf/10.1007/s00586-022-07235-6.pdf	English	null	Muscle spindles of the multifidus muscle undergo structural change after intervertebral disc degeneration	European spine journal	2,022	cc-by	7,407	Abstract Purpose Proprioceptive deficits are common in low back pain. The multifidus muscle undergoes substantial structural change after back injury, but whether muscle spindles are affected is unclear. This study investigated whether muscle spindles of the multifidus muscle are changed by intervertebral disc (IVD) degeneration in a large animal model.i Purpose Proprioceptive deficits are common in low back pain. The multifidus muscle undergoes substantial structural change after back injury, but whether muscle spindles are affected is unclear. This study investigated whether muscle spindles of the multifidus muscle are changed by intervertebral disc (IVD) degeneration in a large animal model. Methods IVD degeneration was induced by partial thickness annulus fibrosus lesion to the L3-4 IVD in nine sheep. Mul- tifidus muscle tissue at L4 was harvested at six months after lesion and from six age /sex matched naïve control animals p p pi pi g g after back injury, but whether muscle spindles are affected is unclear. This study investigated whether muscle spindles of the multifidus muscle are changed by intervertebral disc (IVD) degeneration in a large animal model. Methods IVD degeneration was induced by partial thickness annulus fibrosus lesion to the L3-4 IVD in nine sheep. Mul- tifidus muscle tissue at L4 was harvested at six months after lesion, and from six age-/sex-matched naïve control animals. Muscle spindles were identified in Van Gieson’s-stained sections by morphology. The number, location and cross-sectional area (CSA) of spindles, the number, type and CSA of intrafusal fibers, and thickness of the spindle capsule were measured. Immunofluorescence assays examined Collagen I and III expression.i i Methods IVD degeneration was induced by partial thickness annulus fibrosus lesion to the L3-4 IVD in nine sheep. Mul- tifidus muscle tissue at L4 was harvested at six months after lesion, and from six age-/sex-matched naïve control animals. Muscle spindles were identified in Van Gieson’s-stained sections by morphology. The number, location and cross-sectional area (CSA) of spindles, the number, type and CSA of intrafusal fibers, and thickness of the spindle capsule were measured. Immunofluorescence assays examined Collagen I and III expression.i l Results Multifidus muscle spindles were located centrally in the muscle and generally near connective tissue. There were no differences in the number or location of muscle spindles after IVD degeneration and only changes in the CSA of nuclear chain fibers. Abstract The thickness of connective tissue surrounding the muscle spindle was increased as was the expression of Col- lagen I and III. Conclusion Changes to the connective tissue and collagen expression of the muscle spindle capsule are likely to impact their mechanical properties. Changes in capsule stiffness may impact the transmission of length change to muscle spindles and thus transduction of sensory information. This change in muscle spindle structure may explain some of the proprioceptive deficits identified with low back pain. Keywords Multifidus · Muscle spindle · Connective tissue · Intervertebral disc degeneration · Proprioception * Paul W. Hodges p.hodges@uq.edu.au Muscle spindles of the multifidus muscle undergo structural change after intervertebral disc degeneration Gregory James1 · Carla Stecco2 · Linda Blomster1 · Leanne Hall1 · Annina B. Schmid1,3 · Cindy Christopher B. Little4 · James Melrose4,5 · Paul W. Hodges1,6 Received: 19 October 2021 / Revised: 14 March 2022 / Accepted: 17 April 2022 / Published online: 27 May 2022 © The Author(s) 2022 European Spine Journal (2022) 31:1879–1888 https://doi.org/10.1007/s00586-022-07235-6 European Spine Journal (2022) 31:1879–1888 https://doi.org/10.1007/s00586-022-07235-6 ORIGINAL ARTICLE Introduction 1 Centre of Clinical Research Excellence in Spinal Pain, Injury and Health, School of Health & Rehabilitation Sciences, University of Queensland, Brisbane, QLD 4072, Australia Low back pain (LBP) is the foremost cause of disability [1]. LBP and/or injury induces structural [2–7] and functional [8] changes to the paraspinal muscles, that depend on the stage of chronicity. Changes in the structure of the multifidus are likely to contribute to altered muscle function and vice versa [9] and provide a plausible link to chronic LBP. Mus- cle spindles provide a vital contribution to proprioception, the sense of body position and movement, and are necessary for control of movement and for health of the musculoskel- etal system [10] including the spine [11]. The high density of muscle spindles in the multifidus has been interpreted to suggest a critical sensory role for this muscle [12]. Proprio- ception is impaired in LBP (e.g., reduced positional sense and difficulty in repositioning [13]), and likely underpins sub-optimal control of spinal motion [14] with potential for 2 Human Anatomy and Movement Science, University of Padua, Padua, Italy 3 Nuffield Department of Clinical Neurosciences, Oxford University, Oxford, UK 4 Raymond Purves Bone and Joint Research Laboratories, Kolling Institute of Medical Research, Institute of Bone and Joint Research, The Royal North Shore Hospital, University of Sydney, St Leonards, NSW, Australia 5 Graduate School of Biomedical Engineering, University of New South Wales, Sydney, Australia 6 Centre of Clinical Research Excellence in Spinal Pain, Injury and Health, School of Health and Rehabilitation Sciences, The University of Queensland, Brisbane, QLD 4072, Australia (0123 1 3456789) 3 1880 European Spine Journal (2022) 31:1879–1888 tissue surrounding the muscle spindles capsule and, 2) alter the collagen composition of the capsule. Similar to observa- tions for muscular dystrophy, we predicted that IVD injury would not alter the number or location of the spindles in the multifidus muscle or induce changes to the number or cross-sectional area (CSA) of the intrafusal muscle fibers. a causal relationship to injury [10, 15]. Although spindles have structural similarities to extrafusal muscle (i.e., muscle excluding muscle spindles), whether they undergo similar structural change in response to LBP and injury has not been investigated. Muscle spindles modify their discharge in response to changes in length or speed of length change in skeletal muscle [10]. Animals The animals in this study were utilized in a parallel study examining IVD degeneration [18]. For the present study, tissue was harvested from fifteen merino sheep (aged 3–4 years) that had or had not been exposed to an IVD lesion, but no other treatment. Tissue from these animals has been used for previous experiments [2, 19–21]. Institu- tional ethical approval was obtained for all procedures and experiments performed in this study. Muscle spindle structure changes with ageing and some musculoskeletal disorders such as muscular dystrophy. In both cases, the connective tissue equatorial capsule is thick- ened [10], but evidence of changes to intrafusal fibers is con- flicting. In animal models of muscular dystrophy, intrafusal muscle fibers are unaffected, despite substantial extrafusal muscle fiber atrophy. In humans studies of ageing, spindles include fewer intrafusal muscle fibers and show signs of den- ervation [10]. Changes to connective tissue, even without changes to intrafusal fibers, might affect spindle function secondary to changes in mechanics, with potential contri- bution to the compromised proprioception reported in both conditions. Surgical procedure and tissue harvesting Nine sheep (injured group) were assigned to the ‘injury group’. These animals received a single 6 × 20 mm lesion on the L1-2, L3-4 and L5-6 IVDs, as previously described [19]. This lesion induces progressive degeneration of the injured IVDs in a manner that replicates the molecular components of human IVD degeneration [22]. Post-surgery, the animals were housed individually and closely monitored to ensure their health and well-being. The animals were then housed in open paddocks with 6 animals assigned to the ‘control group,’ that did not undergo any surgical procedures. Six months post-surgery, the animals were euthanized and the multifidus muscle adjacent to the L4 spinous process was harvested. The tissue underwent fixation in 4% paraformal- dehyde and dehydration before being embedded in paraffin. Transverse tissue Sects. (7 μm) were obtained and used for all experimental procedures. Altered mechanics of the spindle could plausibly impact the potential for spindles to transduce information regarding muscle length change and might explain the proprioceptive deficits frequently reported in individuals with LBP [13, 15, 16]. Extrafusal multifidus muscle undergoes substantial structural change (fibrosis, change in collagen subtypes, fatty infiltration, atrophy and fiber-type change of the extrafusal fibers) as a result of neural and immune mechanisms [9]. These changes are induced by spine injury, even when the muscle is uninjured. Although direct injury to spindles and other mechanoreceptors in conjunction with spine injury has been suggested to cause proprioceptive deficits [17], the potential for uninjured muscle spindles to undergo simi- lar structural adaption to the extrafusal fibers has not been investigated in LBP. This proposal is strengthened by the presence of changes in structure of muscle spindles (and associated proprioceptive deficits) in aging muscular and dystrophy [10]. Introduction They consist of a connective tissue-covered central fluid capsule in the equatorial region of the spindle and two polar regions that extend parallel to the extrafusal muscle fibers [10]. The capsule includes specialized mus- cle fibers (intrafusal fibers) that have non-contractile mid- regions where sensory nerve endings of primary and sec- ondary afferents terminate. There are two distinct subtypes of intrafusal fibers, nuclear chain and nuclear bag fibers. Nuclear chain fibers have a smaller diameter and are short (restricted to the equatorial region). Nuclear bag fibers have larger diameter, include an expanded mid region, and extend beyond the capsule to the poles of the spindle. 1 3 Van Gieson’s stain and analysis The multifidus muscle was divided into 3 regions in the dorsolateral and 3 regions in the medi- olateral dimension. a and b show the proportion of muscle spindles in each of the regions for the control and IVD degeneration groups in sections with Van Gieson’s staining. c and d show the proportions in each third in the dorsolateral and mediolateral axes. There was no dif- ference between groups in any location measure. D—dorsal; V—ven- tral; M—medial; L—lateral; IVD—intervertebral disc B IVD degeneration grou Nuclear bag fibre Extrafusal muscle fibre Capsule thickness measure Boundary for CSA measure Nuclear chain fibre Connective tissue Nuclear bag fibre A Equatorial muscle spindle B Polar muscle spindle Boundary for extrafusal muscle & connective tissue measure Fig. 1 Methods for quantification of muscle spindle parameters. a shows features of an equatorial spindle and associated measures of spindle cross-sectional area (CSA) and capsule thickness at eight equidistant locations. The boundary used for quantification of sur- rounding muscle and connective tissue (two times CSA of muscle spindle is shown. b shows features of a polar spindle with no nuclear chain fibers and narrowed ends of nuclear bag fibers A Control Group B IVD degeneration group Nuclear bag fibre Extrafusal muscle fibre Capsule thickness measure Boundary for CSA measure Nuclear chain fibre Connective tissue A Equatorial muscle spindle Boundary for extrafusal muscle & connective tissue measure B IVD degeneration group B A Control Group Equatorial muscle spindle A Equatorial muscle spindle A D Mediolateral axis Spindle location along axis (%) Dorsal third Central third Ventral third Medial third Central third Lateral third Control IVD degeneration C Dorsolateral axis D Mediolateral axis D C Nuclear bag fibre B Polar muscle spindle B Polar muscle spindle Polar muscle spindle Fig. 2 Location of muscle spindles. The multifidus muscle was divided into 3 regions in the dorsolateral and 3 regions in the medi- olateral dimension. a and b show the proportion of muscle spindles in each of the regions for the control and IVD degeneration groups in sections with Van Gieson’s staining. c and d show the proportions in each third in the dorsolateral and mediolateral axes. There was no dif- ference between groups in any location measure. D—dorsal; V—ven- tral; M—medial; L—lateral; IVD—intervertebral disc Fig. 1 Methods for quantification of muscle spindle parameters. Van Gieson’s stain and analysis Slides underwent three 2-min xylene and ethanol washes to remove wax and rehydrate the sections. The slides were then bathed in running water (2 min), Weigert’s haema- toxylin (10 min) and Van Gieson’s solution (1 min). The stained sections were dehydrated, mounted and imaged (Imagescope, Leica). All muscle sections were closely examined to identify the total number and location of all muscle spindles. Spindles were identified by the presence of intrafusal muscle fibers surrounded by a connective tis- sue capsule (Fig. 1). Spindles in the multifidus muscle were examined in six ways illustrated in Fig. 1. First, the Using an established intervertebral disc (IVD) degenera- tion model, this study aimed to test the hypotheses that IVD degeneration would: 1) increase the thickness of connective 1 3 European Spine Journal (2022) 31:1879–1888 1881 Spindle location along axis (%) Dorsal third Central third Ventral third Medial third Central third Lateral third Control IVD degeneration A Control Group B IVD degeneration group C Dorsolateral axis D Mediolateral axis Fig. 2 Location of muscle spindles. The multifidus muscle was divided into 3 regions in the dorsolateral and 3 regions in the medi- olateral dimension. a and b show the proportion of muscle spindles in each of the regions for the control and IVD degeneration groups in sections with Van Gieson’s staining. c and d show the proportions in each third in the dorsolateral and mediolateral axes. There was no dif- ference between groups in any location measure. D—dorsal; V—ven- tral; M—medial; L—lateral; IVD—intervertebral disc Nuclear bag fibre Extrafusal muscle fibre Capsule thickness measure Boundary for CSA measure Nuclear chain fibre Connective tissue Nuclear bag fibre A Equatorial muscle spindle B Polar muscle spindle Boundary for extrafusal muscle & connective tissue measure Fig. 1 Methods for quantification of muscle spindle parameters. a shows features of an equatorial spindle and associated measures of spindle cross-sectional area (CSA) and capsule thickness at eight equidistant locations. The boundary used for quantification of sur- rounding muscle and connective tissue (two times CSA of muscle spindle is shown. b shows features of a polar spindle with no nuclear chain fibers and narrowed ends of nuclear bag fibers Spindle location along axis (%) Dorsal third Central third Ventral third Medial third Central third Lateral third Control IVD degeneration A Control Group B IVD degeneration group C Dorsolateral axis D Mediolateral axis Fig. 2 Location of muscle spindles. Immunofluorescence assay and analysis Slides were dewaxed and rehydrated using a series of three 2-min xylene and ethanol washes and blocked with 2% Bovine Serum Albumin in Tris-buffered saline (TBS). The sections were incubated in Ethylenediami- netetraacetic acid at pH 8.0 for 45 min at 90° Celsius. The sections were washed 3 × TBS before incubation overnight in Col I (1:200, AB6308, Abcam) and Col III (1:200, ab7778, Abcam) antibodies. Finally, sections were mounted with mounting media containing DAPI. No pri- mary antibody controls were used. All slides were imaged using a AxioScan Z1 Scanner (Zeiss) and Collagen I and III staining intensity analysis was performed using ImageJ (NIH, USA). First, the mean grey value of 5 muscle fib- ers surrounding each spindle were averaged to determine the level of background staining. Next, the area and grey value of the pixels within the connective tissue capsule were determined. The pixels with grey values equal to or below the level of background staining were removed from the analysis. Mean grey value of the pixels within the capsule was determined and the staining intensity of Collagen I and III staining was calculated as the product of capsule area and mean grey value. Analysis was per- formed with the investigator blinded to group. Spindle location data were compared between groups and regions using a 2-way ANOVA analysis with a Sidak post hoc analysis. All remaining data were compared between IVD degeneration and control groups using t-tests. Sig- nificance was set at P < 0.05. All data are presented as mean ± SEM in text, tables and figures. Results In total, 218 muscle spindles were identified. Of these, 117 were sectioned through the polar region of the spindle and were excluded from further measurement. The equatorial region was identified for 111 spindles. Spindles were either solitary (34.9%) or in groups of 2–6 spindles (65.1%). Van Gieson’s stain and analysis a shows features of an equatorial spindle and associated measures of spindle cross-sectional area (CSA) and capsule thickness at eight equidistant locations. The boundary used for quantification of sur- rounding muscle and connective tissue (two times CSA of muscle spindle is shown. b shows features of a polar spindle with no nuclear chain fibers and narrowed ends of nuclear bag fibers multifidus muscle was divided into three segments along the mediolateral and dorsoventral axes (Fig. 2). The loca- tion of each spindle was recorded with respect to these segments. Second, the proximity of each spindle to a major fascial element was also examined. Each spindle within 500 μm of a connective tissue segment thicker than 100 μm was identified as being in “close proximity.” Third, the type, number and CSA of each intrafusal muscle fiber were determined for all spindles. Muscle spindles were classified as nuclear bag or chain on the basis of the rela- tive diameter of intrafusal fibers. Based on the anatomy of spindles [10], those that contained both nuclear bag and chain intrafusal muscle fibers were classified as equato- rial spindles, whereas those that contained only nuclear bag fibers were classified as polar spindles. Polar spindles were only analyzed for number and location as the area is highly variable depending where the section was made. Fourth, the CSA of the equatorial spindles was determined by drawing a line along the internal edge of the connec- tive tissue capsule and calculating the contained CSA. Fifth, the thickness of the connective tissue capsule was quantified as the distance between the external edge of the capsule to the internal capsule at 8 equidistant posi- tions around the spindle. Sixth, the proportion of muscle and connective tissue surrounding the spindles was deter- mined. A region of interest (ROI) was set at twice the size of the average equatorial spindle CSA (calculated from all spindles in the present study; 10,500 μm2) and cen- tered over the spindle. Muscle and connective tissue were individually selected and quantified using the threshold function in ImageJ (NIH, USA). Data are presented as a percentage of area positive for muscle or connective tissue in the ROI. 1 3 3 1882 European Spine Journal (2022) 31:1879–1888 Fig. 3 Location of muscle spindles in each muscle sample. Data are shown for all animals in the (a) control and (b) intervertebral disc (IVD) degeneration groups. Using Van Gieson’s stain connective tissue elements are pink and muscle is orange. D—dorsal; V—ven- tral; M—medial; L—lateral; Black star—single spindle; Blue star—compound spindle Muscle spindle number and location Figures 2 and 3 show the location of muscle spindles by region and for each muscle sample, respectively. Spindles in the multifidus muscle are primarily located in the cen- tral third of the muscle along both the dorsoventral and mediolateral axes (Table 2). Of muscle spindles, 59.2% 1 3 Fig. 3 Location of muscle spindles in each muscle sample. Data are shown for all animals in the (a) control and (b) intervertebral disc (IVD) degeneration groups. Using Van Gieson’s stain connective tissue elements are pink and muscle is orange. D—dorsal; V—ven- tral; M—medial; L—lateral; Black star—single spindle; Blue star—compound spindle A Control Group B IVD degeneration group B B A 1 3 European Spine Journal (2022) 31:1879–1888 1883 Intrafusal fiber analysis Neither the number of nuclear bag fibers per spindle nor their CSAs were different between control and IVD degen- eration groups in equatorial spindles in the multifidus muscle (Table 1; Fig. 5a and c). Although the number of nuclear chain fibers did not differ, their CSA was greater in the IVD degeneration group (Table 2; Fig. 5b and d). A Control Group B IVD degeneration group Fig. 4 Location of muscles spindles relative to connective tissue for representative animals in the (a) control and (b) intervertebral disc (IVD) degeneration groups. Using Van Gieson’s stain connective tis- sue elements are pink and muscle is orange. The left panels show the location of each numbered spindle or cluster of spindles and the right panel shows each at higher magnification. Note the close proximity of most spindles to dense connective tissue in the muscle. D—dorsal; V—ventral; M—medial; L—lateral; IVD—intervertebral disc. Cali- bration—150 μm Table 1 Group means and statistical analysis of spindle location data between Control and IVD degeneration groups Values in bold are statistically significant G, group; L, location Control IVD degeneration Main effects p-value Dorsoventral axis G: 0.40 L: 0.005 Dorsal third (%) 31.1 ± 7.3 26.3 ± 7.6 Central third (%) 50.6 ± 9.4 52.5 ± 5.7 Ventral third (%) 18.3 ± 9.5 21.2 ± 5.3 Mediolateral axis G: 0.55 L: < 0.001 Medial third (%) 5.0 ± 2.6 3.7 ± 1.5 Central third (%) 65.9 ± 9.6 68.3 ± 5.0 Lateral third (%) 29.0 ± 8.8 28.1 ± 5.5 No. bag fibres No. Muscle spindle number and location chain fibres Bag fibre CSA (µm2) Chain fibre CSA (µm2) A C D B A Control Group Table 1 Group means and statistical analysis of spindle location data between Control and IVD degeneration groups Values in bold are statistically significant G, group; L, location Control IVD degeneration Main effects p-value Dorsoventral axis G: 0.40 L: 0.005 Dorsal third (%) 31.1 ± 7.3 26.3 ± 7.6 Central third (%) 50.6 ± 9.4 52.5 ± 5.7 Ventral third (%) 18.3 ± 9.5 21.2 ± 5.3 Mediolateral axis G: 0.55 L: < 0.001 Medial third (%) 5.0 ± 2.6 3.7 ± 1.5 Central third (%) 65.9 ± 9.6 68.3 ± 5.0 Lateral third (%) 29.0 ± 8.8 28.1 ± 5.5 Table 1 Group means and statistical analysis of spindle location data between Control and IVD degeneration groups A A Intrafusal fiber analysis B IVD degeneration group IVD degeneration group B Neither the number of nuclear bag fibers per spindle nor their CSAs were different between control and IVD degen- eration groups in equatorial spindles in the multifidus muscle (Table 1; Fig. 5a and c). Although the number of nuclear chain fibers did not differ, their CSA was greater in the IVD degeneration group (Table 2; Fig. 5b and d). No. bag fibres Control IVD degen. Control IVD degen. No. chain fibres Bag fibre CSA (µm2) Chain fibre CSA (µm2) A C D B Fig. 5 Number and cross-sectional area of intrafusal fibers for mus- cle spindles in control and (b) intervertebral disc degeneration (IVD degen.) groups. Data are shown for (a) and (c) nuclear bag and (b) and (d) nuclear chain fibers No. bag fibres No. chain fibres A B B Control IVD degen. Control IVD degen. Bag fibre CSA (µm2) Chain fibre CSA (µm2) C D Fig. 4 Location of muscles spindles relative to connective tissue for representative animals in the (a) control and (b) intervertebral disc (IVD) degeneration groups. Using Van Gieson’s stain connective tis- sue elements are pink and muscle is orange. The left panels show the location of each numbered spindle or cluster of spindles and the right panel shows each at higher magnification. Note the close proximity of most spindles to dense connective tissue in the muscle. D—dorsal; V—ventral; M—medial; L—lateral; IVD—intervertebral disc. Cali- bration—150 μm were in close proximity to a major fascial element in the muscle (see Fig. 4 for a representative sample from each group). No differences were detected in the number of spindles in the multifidus muscle or any feature of their location between the control and IVD degeneration groups (Table 2). Fig. 5 Number and cross-sectional area of intrafusal fibers for mus- cle spindles in control and (b) intervertebral disc degeneration (IVD degen.) groups. Discussion This is the first study to examine the potential impact of low back injury, specifically IVD injury, on morphology of muscle spindles in the multifidus muscle. The absence of differences in spindle number, location, CSA and intrafusal fiber number confirm that spindles do not undergo atrophy within six months after injury. Increased thickness of the connective tissue capsule and Collagen I and III expression provides evidence of fibrotic muscle spindle changes with IVD degeneration. Structural change to the muscle spindles would modify their mechanical properties, with potential to interfere with spindle sensory function. This novel obser- vation provides a potential explanation for proprioceptive deficits and changes to motor control in IVD degeneration. Structural changes to muscle spindles with experimental IVD degeneration The absence of signs of atrophy/loss of muscle spindles or intrafusal fibers in the multifidus in this model of IVD degeneration parallels animal models of muscular dystro- phy. Those models induce substantial extrafusal fiber atro- phy except in regions directly surrounding the spindles [25]. Preservation of spindles and surrounding extrafusal muscle has been interpreted to indicate a process of active sparing from degeneration [26]. We also report no obvious altera- tions to muscle and connective tissue in the region surround- ing the spindles, despite the increase in connective tissue and muscle fibers changes in multifidus reported in this model [2]. In contrast to muscular dystrophy, aging induces intrafusal fiber atrophy, increased capsule CSA and spindle loss. Taken together this implies that muscle spindle changes Muscle and connective tissue analysis The thickness of the connective tissue layer surrounding the equatorial spindle capsules was greater in the multifi- dus muscle of animals with IVD degeneration than control animals (Table 2; Fig. 6a). IVD injury did not significantly change the proportion of muscle or connective tissue sur- rounding the spindles (Table 2; Fig. 6c and d). No differ- ences were identified in the CSA of equatorial spindles (Table 2; Fig. 6b). The intensity of Collagen I and III stain- ing was significantly greater in the connective tissue capsule of equatorial spindles in the IVD injury group than controls (Table 2; Fig. 6e and f, 7). Most muscle spindles were close to major areas of con- nective tissue. Although attachment of spindles to the per- imysial fascia that surrounds bundles of muscle fibers is well known [24], their relationship to other fascial structures has not been described. This may have functional significance for sensation and control of distribution of tension [24]. Examination of the relationship between spindle location and multifidus proprioception requires investigation. Intrafusal fiber analysis Data are shown for (a) and (c) nuclear bag and (b) and (d) nuclear chain fibers 1 3 European Spine Journal (2022) 31:1879–1888 1884 means and s of spindle ntrol and IVD ups Values in bold are statistically significant CSA, cross-sectional area; IVD, intervertebral disk Control IVD degeneration p-values Total spindle number (n) 14.7 ± 3.1 14.4 ± 1.1 0.94 Spindles close to connective tissue (%) 58.8 ± 6.9 59.4 ± 6.0 0.95 Number of bag fibers (n) 2.7 ± 0.1 2.9 ± 0.1 0.25 Number of chain fibers (n) 3.6 ± 0.2 3.3 ± 0.2 0.31 Bag fiber CSA (μm2) 316.7 ± 29.0 343.6 ± 21.6 0.45 Chain fiber CSA (μm2) 70.8 ± 6.4 92.8 ± 6.5 0.020 Spindle capsule CSA (μm2) 8461 ± 1054 12,151 ± 1521 0.061 Capsule connective tissue thickness (μm) 7.5 ± 0.4 12.2 ± 0.6 < 0.001 Muscle tissue surrounding spindles (%) 47 ± 3.1 52.2 ± 2.3 0.48 Connective tissue surrounding spindles (%) 13.5 ± 1.6 16.2 ± 1.7 0.28 Collagen I staining intensity (fold difference) 1 ± 0.3 2.3 ± 0.3 0.005 Collagen III staining intensity (fold difference) 1 ± 0.2 2.1 ± 0.4 0.006 the cervical region of humans, which showed spindles pri- marily located in the anterolateral region of the multifidus muscle [23]. This differs from the location centrally along the dorsoventral and mediolateral axis of the lumbar mus- cle in sheep. It is not clear whether this is a region or spe- cies difference. Few muscle spindles are present in the most medial/ventral region of the multifidus which is the region that undergoes the most profound structural changes in mod- els of IVD degeneration [2, 19]. Table 2 Group means and statistical analysis of spindle data between Control and IVD degeneration groups Limitations As muscle spindles were examined in a single section multifi- dus for each animal it was not possible to quantify the location of all spindles in the muscle. Polar spindles were identified but were not included in detailed analysis of because of the large variation in CSA depending on the location of the section. It is important to consider that there are anatomical differences of the multifidus muscle between bipedal humans and quadru- pedal sheep that suggest caution for translation of the present findings to humans. For instance, unlike human multifidus which has a large proportion of slow twitch muscle fibers [33], sheep multifidus muscle has predominately fast type muscle fibers [2, 19]. As some functional and structural properties of spindles differ between slow and fast muscles [34], this may impact the translation of these findings to humans. Functional changes in multifidus and proprioception were not evaluated in the sheep in this study, and so direct correlation with muscle spindle changes was not possible. Fig. 6 Connective tissue, capsule collagen and surrounding tissue measures. Data are shown for control and intervertebral disc (IVD) degeneration groups. a Connective tissue thickness averaged over measures and eight equidistant sites. b Muscle spindle capsule thick- ness. c Muscle tissue and d connective tissue proportion surrounding the muscle spindles. e Collagen I expression. f Collagen II expres- sion. IVD—intervertebral disc observed in our injury model are not attributable to acceler- ated aging and imply an active process. Increased CSA of intrafusal fibers has been reported previously after issues such as denervation [27], which should not be present in out model, and requires further investigation. Whether atrophy of spindles and intrafusal fibers occurs in the chronic stage has not been studied. Anatomy of multifidus muscle spindles Muscle tissue surrounding spindle (%) Connective tissue surrounding spindle (%) C D C Collagen I Fold difference to control Collagen III Fold difference to control E Control IVD degen. Control IVD degen. F Anatomy of multifidus muscle spindles The anatomical location of muscle spindles has not been reported for the lumbar multifidus. Data are available for 3 European Spine Journal (2022) 31:1879–1888 1885 Muscle tissue surrounding spindle (%) Connective tissue surrounding spindle (%) Spindle capsule (um2) Connective tissue thickness (um) Collagen I Fold difference to control Collagen III Fold difference to control C B E Control IVD degen. Control IVD degen. D A F Fig. 6 Connective tissue, capsule collagen and surrounding tissue measures. Data are shown for control and intervertebral disc (IVD) degeneration groups. a Connective tissue thickness averaged over measures and eight equidistant sites. b Muscle spindle capsule thick- ness. c Muscle tissue and d connective tissue proportion surrounding the muscle spindles. e Collagen I expression. f Collagen II expres- sion. IVD—intervertebral disc Spindle capsule (um2) Connective tissue thickness (um) B A expression is similar to the fibrosis observed throughout the multifidus muscle (increased total connective tissue CSA [2] and epimysium (fascia that surrounds muscles) thickening [3]) in models of IVD degeneration. Fibrotic changes of the extrafusal multifidus muscle are thought to be promoted by inflammation [19, 21, 28]. Increased levels of pro-inflamma- tory cytokines are present in muscular dystrophy and aging [29], which also display spindle capsule thickening. A Thickening of the capsule has been considered as an adap- tive, rather than maladaptive change, to protect the intrafusal muscle fibers [10]. Although plausible, fibrosis would also impact tissue mechanics. Muscle spindles are responsive to length change and rate of length change [10]. Although lit- tle data exists quantifying the relationship between capsule thickness and spindle function, modified stiffness of capsule would change deformation properties with length change. Pre- vious work has highlighted the sensitivity of muscle spindles to even minor changes in stiffness of related structures, such as the thixotropic properties of muscle after contraction [30]. As Collagen I is a less elastic collagen subtype, its increased expression could impact the exposure of the sensory endings in the muscle spindle length changes, and thus their signalling of such events. Such modification of responsiveness of muscle spindles could underlie the proprioceptive deficits LBP. Mod- ified afferent signals from the multifidus muscle have been identified as a primary mechanism for these proprioceptive defects [31, 32]. Direct evaluation of the impact of capsule collagen and thickness on spindle sensitivity is required. Impact of connective tissue changes on function Author contributions Conceptualization: Paul Hodges, Greg James, Carla Stecco; Methodology: Paul Hodges, Greg James, Linda Blomster, Leanne Hall, Annina Schmid, Cindy Shu, Christopher Little, James Melrose; Formal analysis and investigation: Paul Hodges, Greg James, Carla Stecco, Linda Blomster, Leanne Hall, Annina Schmid, Cindy Shu; Writing—original draft preparation: Paul Hodges, Greg James; Writing—review and editing: All authors; Funding acquisition: Paul Hodges. Funding Open Access funding enabled and organized by CAUL and its Member Institutions. inner and outer border of the connective tissue capsule. The boxed regions in (a to d) are magnified in (e to h) Calibra- tion—50 μm C D E F H G C Control IVD degen. IVD degen. C D C F E Control H H Impact of connective tissue changes on function Alterations to the connective tissue capsule of multifidus muscle spindles are likely to impact their function. Increased spindle capsule thickness and increased Collagen I and III 1 3 European Spine Journal (2022) 31:1879–1888 Author contributions Conceptualization: Paul Hodges, Greg James, Carla Stecco; Methodology: Paul Hodges, Greg James, Linda Blomster, ll A i S h id Ci d Sh Ch i h i l B A C D E F H G Control Control IVD degen. IVD degen. Collagen l Collagen IIl 1886 European Spine Journal (2022) 31:1879–1888 European Spine Journal (2022) 31:1879–1888 ealed that the muscle spindles ns in a model of IVD degenera- terations and their impact on the and spine control in individuals nvestigation. as provided by the National Health and ustralia (APP1091302; APP1194937). Author contributions Conceptualization: Paul Hodges, Greg James, Carla Stecco; Methodology: Paul Hodges, Greg James, Linda Blomster, Leanne Hall, Annina Schmid, Cindy Shu, Christopher Little, James Melrose; Formal analysis and investigation: Paul Hodges, Greg James, Carla Stecco, Linda Blomster, Leanne Hall, Annina Schmid, Cindy Shu; Writing—original draft preparation: Paul Hodges, Greg James; Writing—review and editing: All authors; Funding acquisition: Paul Hodges. Funding Open Access funding enabled and organized by CAUL and its Member Institutions. B A C D E F H G Control Control IVD degen. IVD degen. Collagen l Collagen IIl B Collagen IIl Fig. 7 Collagen I (blue) and collagen III (red) staining of muscle spindle capsules. Images are shown for representative animals from the control (a and b) and intervertebral disc (IVD) degeneration (c and d) groups. The dashed lines delineate the inner and outer border of the connective tissue capsule. The boxed regions in (a to d) are magnified in (e to h) Calibra- tion—50 μm B Conclusion This investigation has revealed that the muscle spindles undergo structural alterations in a model of IVD degenera- tion. The causes of these alterations and their impact on the multifidus muscle function and spine control in individuals with LBP requires further investigation. Acknowledgements Funding was provided by the National Health and Medical Research Council of Australia (APP1091302; APP1194937). Funding Open Access funding enabled and organized by CAUL and its Member Institutions. References 15. Pinto SM, Cheung JPY, Samartzis D, Karppinen J, Zheng YP, Pang MYC, Wong AYL (2020) Differences in propriocep- tion between young and middle-aged adults with and without chronic low back pain. Front Neurol 11:605787. https://doi.org/ 10.3389/fneur.2020.605787 1. 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BMC Musculoskelet Disord 18:12. https://doi.org/10.1186/ s12891-016-1376-1 Conclusion Author contributions Conceptualization: Paul Hodges, Greg James, Carla Stecco; Methodology: Paul Hodges, Greg James, Linda Blomster, Leanne Hall, Annina Schmid, Cindy Shu, Christopher Little, James Melrose; Formal analysis and investigation: Paul Hodges, Greg James, Carla Stecco, Linda Blomster, Leanne Hall, Annina Schmid, Cindy Shu; Writing—original draft preparation: Paul Hodges, Greg James; Writing—review and editing: All authors; Funding acquisition: Paul Hodges. This investigation has revealed that the muscle spindles undergo structural alterations in a model of IVD degenera- tion. The causes of these alterations and their impact on the multifidus muscle function and spine control in individuals with LBP requires further investigation. Funding Open Access funding enabled and organized by CAUL and its Member Institutions. 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https://openalex.org/W2998477174	https://europepmc.org/articles/pmc7093813?pdf=render	English	null	Data from brain activity during visual working memory replicates the correlation between contralateral delay activity and memory capacity	Data in brief	2,020	cc-by	2,605	Data from brain activity during visual working memory replicates the correlation between contralateral delay activity and memory capacity Data from brain activity during visual working memory replicates the correlation between contralateral delay activity and memory capacity Mario Villena-Gonzalez a, b, , Ivan Rubio-Venegas a, Vladimir Lopez a a Escuela de Psicología, Pontiﬁcia Universidad Catolica de Chile, Santiago, CP: 7820436, Chile b Laboratorio de Neurociencia Cognitiva y Social, Facultad de Psicología, Universidad Diego Portales, Santiago, Chile Contents lists available at ScienceDirect Contents lists available at ScienceDirect https://doi.org/10.1016/j.dib.2019.105042 2352-3409/© 2019 The Author(s). Published by Elsevier Inc. This is an open access article under the CC BY license (http:// creativecommons.org/licenses/by/4.0/). Data in brief 28 (2020) 105042 Data in brief 28 (2020) 105042 Corresponding author. Escuela de Psicología, Pontiﬁcia Universidad Catolica de Chile, Santiago, CP: 7820436, Chile. E-mail address: mvillena@ug.uchile.cl (M. Villena-Gonzalez). Value of the Data These data are useful because they show that even by changing some experimental parameters, the results from an important research in the ﬁeld of working memory remain consistent and replicable Researchers in the ﬁeld of neurophysiology of memory can beneﬁt from these data because provides further conﬁrmation on the brain potentials related to memory load and ensure reliability of the results regardless of physical parameters of stimuli. Researchers in the ﬁeld of neurophysiology of memory can beneﬁt from these data because provides further conﬁrmation on the brain potentials related to memory load and ensure reliability of the results regardless of physical parameters of stimuli. These data could be useful to complement the discussion about the relationship between ERP amplitude measurements and behavior, but also to encourage design modiﬁcations in order to explore other features of the phenomenon of working memory These data could be useful to complement the discussion about the relationship between ERP amplitude measurements and behavior, but also to encourage design modiﬁcations in order to explore other features of the phenomenon of working memory y Since replication is an essential issue in science, we believe these data are valuable because provide support and infor- mation about reliability of results in the ﬁeld of cognitive neuroscience y Since replication is an essential issue in science, we believe these data are valuable because provide support and infor- mation about reliability of results in the ﬁeld of cognitive neuroscience a r t i c l e i n f o Article history: Received 15 November 2019 Received in revised form 11 December 2019 Accepted 16 December 2019 Available online 23 December 2019 This article provides data from statistical analysis of event-related brain potentials (ERPs) and behavioural performance from 23 participants during a working memory task. Speciﬁcally, we used the change detection task from Vogel 2004, using the same timing but a modiﬁed size and distance between stimuli. Contralateral delay activity (CDA) was calculated from posterior parieto-occipital electrodes and then it was compared between conditions with different memory load (one, two and four items). Working mem- ory capacity (WMC) was calculated from behavioural data using the formula developed by Pashler (1988). Correlation was per- formed between WMC and the CDA amplitude difference (from two to four items). The correlation replicates the results from the original paper of Vogel 2004 [1], even though some parameters are different from the original design. Keywords: Working memory Memory capacity Event related potential Contralateral delay activity Memory load Change detection task © 2019 The Author(s). Published by Elsevier Inc. This is an open access article under the CC BY license (http://creativecommons. org/licenses/by/4.0/). https://doi.org/10.1016/j.dib.2019.105042 2352-3409/© 2019 The Author(s). Published by Elsevier Inc. This is an open access article under the CC BY license (http:// creativecommons.org/licenses/by/4.0/). M. Villena-Gonzalez et al. / Data in brief 28 (2020) 105042 2 Speciﬁcations Table Subject Cognitive Neuroscience Speciﬁc subject area Neurophysiology of the Visuo-Spatial Working Memory Type of data Raw dataset Text ﬁle Graph Figure Table How data were acquired Electroencephalography (EEG) Biosemi active two 64 electrodes Data format Raw (BDF ﬁle) Analysed Filtered Parameters for data collection ERP was calculated time-locked to the memory array appearance, for three different memory load conditions; one item, two items and four items. Description of data collection Continuous EEG data was recorded during the experiment and data were off-line resampled to 1024 Hz, re-referenced to mastoids, ﬁltered between 0.01 and 20 Hz and epoched for each experimental condition. Event-related potential (ERP) was calculated from contralateral and ipsilateral hemisphere at the posterior parieto-occipital electrodes. Subtraction between contralateral and ipsilateral signal was performed in order to obtain contralateral delay activity (CDA) Data source location Institution: Pontiﬁcal Catholic University of Chile City/Town/Region: Santiago Country: Chile Data accessibility Repository name: Mendeley Data Data identiﬁcation number: 10.17632/j2v7btchdy.1 Direct URL to data: https://doi.org/10.17632/j2v7btchdy.1 1. Data description Dataset from Mendeley repository contains EEG raw data ﬁles from 23 participants while they performed a change detection task used in Vogel 2004 [1]. The task involved three different memory loads (one, two and four items). This data ﬁles can be found in BDF format, which can be visualized using MATLAB software (The Mathworks, Inc.), EEGLAB toolbox [2] and the data import extension BIOSIG. Fig. 1 shows an example of the visualization for one trial of the raw ﬁle. Details of the speciﬁc stimuli coding can be found in Table 1. Data shown in the present article is from the above-mentioned dataset, but after all the processing described in the following methods section (EEG Data pre-processing, ERP calculation and Statistics). In the section “Behavioral and electrophysiological data”, the total accuracy for the task and working memory capacity (WMC) can be found in the text, as the average across participants with the corre- sponding standard deviation. The EEG activity associated with memory retention known as Contralateral-delay activity (CDA) can be seen in Fig. 2 (left). This waveform is shown for three different number of items. M. Villena-Gonzalez et al. / Data in brief 28 (2020) 105042 3 Fig. 1. Visualization of a sample trial from the raw ﬁle. The ﬁrst stimuli code is the arrow cue (irrelevant for further EEG analysis) the second one is for the memory array and the last one is the response code indicating correct or incorrect answers. Fig. 1. Visualization of a sample trial from the raw ﬁle. The ﬁrst stimuli code is the arrow cue (irrelevant for further EEG analy the second one is for the memory array and the last one is the response code indicating correct or incorrect answers. Fig. 1. Visualization of a sample trial from the raw ﬁle. The ﬁrst stimuli code is the arrow cue (irrelevant for further EEG analysis), the second one is for the memory array and the last one is the response code indicating correct or incorrect answers. Statistics from Pearson correlation test can also be found in the text. This Correlation was calculated for the increase in amplitude of CDA between two and four items and WMC. This correlation plot can be found in the Fig. 2 (right). We also provide the statistics for correlation between the magnitude of CDA increase between one and four items and WMC. 2. Experimental design, materials, and methods 2.1. Participants 2.1. Participants Twenty-three volunteers (15 women) were recruited for the study (mean age ¼ 26.17, SD ¼ 6.69). All participants had normal or corrected-to-normal vision and reported no color-vision deﬁciency. 2.2. Stimuli and procedure 2.2. Stimuli and procedure All stimuli were presented on a computer screen with gray background situated 120 cm away from the participant. Psychopy software [3] was used to design the experiment and display the stimuli. The design of the experiment is the same change detection task used in Vogel (2004) with varia- tions in size and distances of the stimuli. At the beginning of each trial, a central arrow cue instructed the participants to remember the items in either the left or the right hemiﬁeld. Afterwards, a memory array appeared with the items (100 ms), which can be composed by one item (low memory load), two items (medium memory load) or four items (high memory load). After a retention period of 900 ms, a test array appeared in which the color of one square was different from the corresponding item in the memory array in 50% of trials; the colours of the two arrays were identical on the remaining trials. Participants had to report if color squares were identical or different between memory array and test array. The task was composed by 96 trials, with 32 trials for each condition of memory load (one, two and four items). All stimulus arrays were presented within two 7.2 13.15 rectangular regions that were centered 5.4 to the left and right of a central ﬁxation cross on a gray background. Each memory array consisted of 1, 2 or 4 colored squares (1.17 1.17) in each hemiﬁeld. Each square was selected at random from a set of seven highly discriminable colours (red, blue, violet, green, yellow, black and white), and a given colour could appear no more than twice within an array. Stimulus positions were randomized on each trial, with the constraint that the distance between squares within a hemiﬁeld was at least 3.5 (centre to centre). Table 1 Table 1 Details of the speciﬁc stimuli coding for memory array and participants responses. Memory array codes Participant responses Left cued stimuli Right cued stimuli Correct answer Incorrect answer One item 11 or 12 10 or 13 60 61 Two items 21 or 22 20 or 23 Three items 41 or 42 40 or 43 M. Villena-Gonzalez et al. / Data in brief 28 (2020) 105042 4 Fig. 2. Contralateral delay activity (CDA) correlates with Working Memory Capacity (WMC). Waveform of CDA for different number of items during memory retention (left). Correlation between the amplitude increase between two and four items and the WMC across participants (right). Fig. 2. Contralateral delay activity (CDA) correlates with Working Memory Capacity (WMC). Waveform of CDA for different number of items during memory retention (left). Correlation between the amplitude increase between two and four items and the WMC across participants (right). Participants had no history of drug abuse, neurological or psychiatric conditions. The protocol was approved by the Ethics Committee of Pontiﬁcia Universidad Catolica de Chile. All subjects gave written informed consent in accordance with the Declaration of Helsinki. All experiments were performed at the Laboratory of experimental psychology of this University. 2.6. ERP calculation For ERP analysis, a 2nd order inﬁnite impulse response (IIR) Butterworth ﬁlter was used for band- pass ﬁltering continuous EEG data, with a half amplitude cut-off frequency of 0.01 Hz and 20 Hz. We calculated contralateral delay activity by averaging the activity recorded at right posterior electrode sites (TP8, CP6, CP4, CP2, P2, P4, P6, P8, P10, PO8, PO4, O2) when participants were cued to remember the left side of the memory array with the activity recorded from the left posterior electrode sites (TP7, CP5, CP3, CP1, P1, P3, P5, P7, P9, PO7, PO3, O1) when they were cued to remember the right side. Contralateral delay activity (CDA) was measured as the difference in mean amplitude between the ipsilateral and contralateral waveforms, with a measurement window of 300e900 ms after the onset of the memory array. CDA was calculated separately for memory array of one, two and four items. 2.5. EEG data pre-processing 2.5. EEG data pre-processing Data pre-processing was performed using Matlab R2018b (The Mathworks, Inc.) with EEGLAB 14_1_2b toolbox [2]. The signal was down-sampled off-line at 1024 Hz. Because of hardware setup constraints, all electrodes were referenced to CMS and DRL during acquisition, but off-line re-refer- enced to averaged mastoids. The EEG signal was segmented in epochs of 1400 ms length, selecting the 200 ms preceding the memory array appearance up to 1200 ms after that (based on codes from Table 1). Baseline correction was carried out using the 200 ms time window preceding stimulus appearance. Only correct trials were analysed. Muscular and movement artefacts detection was performed on epoched data by manual inspection, blind to condition. All epochs with artefacts were rejected. Independent component analysis (ICA) was performed using EEGLAB toolbox. Afterwards, the independent component associated with blinks was rejected for each participant. 2.7. Statistics We calculated the difference in amplitude of CDA between two and four items, for each participant. Pearson correlation test was performed to assess correlation between the difference in CDA and WMC for each participant. This analysis was performed using SPSS software. 2.8. Behavioural and electrophysiological data 2.4. EEG recording EEG data was obtained using 64 electrodes (Biosemi ® ActiveTwo) arranged according to the in- ternational 10/20 extended system. Horizontal and vertical eye movements were monitored using four external electrodes. Horizontal EOG was recorded bipolarly from the outer canthi of both eyes and vertical EOG was recorded from above and below of the participant's right eye. Two additional external electrodes were placed on the right and left mastoid to be used for later re-referencing. M. Villena-Gonzalez et al. / Data in brief 28 (2020) 105042 5 5 2.3. Behavioural data processing Working memory capacity (WMC) was calculated using the formula developed by Pashler (1988). The formula is WMC ¼ S ðHF 1FÞ, where H is the observed hit rate, F the false alarm rate and S is the higher set size (maximum number of to-be-remembered items). The total accuracy was calculated as the hit rate for the whole experiment including all set sizes conditions. M. Villena-Gonzalez et al. / Data in brief 28 (2020) 105042 Acknowledgments This research was supported by FONDECYT POSTDOCTORADO No. 3180295 to MVG. 2.8. Behavioural and electrophysiological data Participants performed the experiment with a total accuracy of 85.6 þ 7% (mean þ SD). The working memory capacity (WMC) was 2.3 þ 0.8 (mean þ SD). We replicated the results from Vogel 2004, showing that the amplitude of the contralateral delay activity (CDA) is sensitive to the number of items in the memory array (Fig. 2, left). Speciﬁcally, the amplitude of CDA increase with the number of items that participants had to maintain in visual working memory. Furthermore, the increase in amplitude of CDA between two and four items was correlated with WMC calculated for each participant (r ¼ 0.4485; p ¼ 0.0159). We can observe in Fig. 2 (right) that participants with high WMC showed larger amplitude increase in CDA between two and four items, compared with participants with low WMC. We also observed this correlation between the magnitude of CDA increase between one and four items and WMC (r ¼ 0.4606; p ¼ 0.0135). M. Villena-Gonzalez et al. / Data in brief 28 (2020) 105042 6 Conﬂict of Interest The authors declare that they have no known competing ﬁnancial interests or personal relation- ships that could have appeared to inﬂuence the work reported in this paper. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.dib.2019.105042. Supplementary data to this article can be found online at https://doi.org/10.1016/j.dib.2 [3] J. Peirce, J.R. Gray, S. Simpson, M. MacAskill, R. H€ochenberger, H. Sogo, E. Kastman, J.K. Lindeløv, PsychoPy2: experiments in behavior made easy, Behav. Res. Methods 51 (2019) 195e203, https://doi.org/10.3758/s13428-018-01193-y. (2004) 748 751, https://doi.org/10.1038/nature02447. [2] A. Delorme, S. Makeig, EEGLAB: an open source toolbox for analysis of single-trial EEG dynamics including independent component analysis, J. Neurosci. Methods 134 (2004) 9e21, https://doi.org/10.1016/j.jneumeth.2003.10.009. References [1] E.K. Vogel, M.G. Machizawa, Neural activity predicts individual differences in visual working memory capacity, Nature 428 (2004) 748e751, https://doi.org/10.1038/nature02447. p y , J ( ) , p // g/ /j j [3] J. Peirce, J.R. Gray, S. Simpson, M. MacAskill, R. H€ochenberger, H. Sogo, E. Kastman, J.K. Lindeløv, PsychoPy2: experiments in behavior made easy, Behav. Res. Methods 51 (2019) 195e203, https://doi.org/10.3758/s13428-018-01193-y.
https://openalex.org/W4385231496	https://www.researchsquare.com/article/rs-3189341/latest.pdf	English	null	Associations of dichlorophenol with metabolic syndrome based on multivariate-adjusted logistic regression: A U.S. nationwide population-based study 2003-2016	Research Square (Research Square)	2,023	cc-by	7,735	Associations of dichlorophenol with metabolic syndrome based on multivariate-adjusted logistic regression: A U.S. nationwide population-based study 2003-2016 Cai Jing Zhejiang Police College Zhichao Yang Zhejiang Police College Sen Zhao Zhejiang Police College Xing Ke (  kexing@zjjcxy.cn ) Zhejiang Police College Associations of dichlorophenol with metabolic syndrome based on multivariate-adjusted logistic regression: A U.S. nationwide population-based study 2003-2016 Cai Jing Associations of dichlorophenol with metabolic syndrome based on multivariate-adjusted logistic regression: A U.S. nationwide population-based study 2003-2016 Cai Jing Methods We included 10,428 participants (5,084 men and 5,344 women) adults (aged ≥ 20 years) from the National Health and Nutrition Examination Survey (2003–2016). The cases of MetS were diagnosed by NCEP/ATPIII. Logistic regression models were conducted to calculate the odds ratios (ORs) and 95% confidence intervals (CIs) of MetS prevalence. Background Para-dichlorobenzene (p-DCB) exposure associated with oxidative stress has indeed raised public concerns. However, whether p-DCB is linked with metabolic syndrome (MetS) remains unclear. This study aimed to examine the associations of exposure to p-DCB with MetS prevalence. Results We documented 2,861 (27.1%) MetS cases. After adjustment for the potential risk factors, the ORs (95% CI) of MetS prevalence across the quartile of urinary 2,5-dichlorophenol (2,5-DCP) were 1.09 (0.93–1.28), 1.22 (1.00-1.49), and 1.34 (1.04–1.73). Moreover, 2,5 DCP is significantly associated with a higher prevalence of abdominal obesity. We further examined that 2,5 DCP was correlated with higher systolic blood pressure, waist circumference, and glycohemoglobin and a lower high density cholesterol. In addition, the significant positive associations between 2,5 DCP and MetS were robust in the subgroup and sensitivity analyses. Conclusion These findings indicated that increased urinary p-DCB concentration, especially 2,5 DCP, had a higher MetS prevalence. B k d These findings indicated that increased urinary p-DCB concentration, especially 2,5 D Research Article Keywords: Para-dichlorobenzene, 2,5-dichlorophenol, Urine, Metabolic syndrome, Population-based study Posted Date: July 25th, 2023 DOI: https://doi.org/10.21203/rs.3.rs-3189341/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Additional Declarations: No competing interests reported. Additional Declarations: No competing interests reported. Version of Record: A version of this preprint was published at Environmental Health on December 15th, 2023. See the published version at https://doi.org/10.1186/s12940-023-01037-z. Page 1/16 Page 1/16 Study participants NHANES is a national representation, multi-year cycle, multi-stage sample design, and cross-sectional study among the US noninstitutionalized civilian [17]. In this study, we aggregated data from seven survey periods on p-DCB from seven circles, including 2003–2004, 2005–2006, 2007–2008, 2009–2010, 2011–2012, 2013–2014, and 2015–2016 circles. Out of the initial NHANES dataset, consisting of 79,648 participants, we excluded 39,749 subjects to focus our analysis exclusively on the adult population aged 20 years or older. Additionally, we further excluded participants without data on 2,4-DCP or 2,5-DCP (n = 29,341). Finally, 10,428 participants (5084 men and 5344 women) were included in the current study. f Assessment of p-DCB Urinary 2,5-DCP and 2,4 DCP were measured to evaluate the level of p-DCB exposure. Urine samples of each individual were collected and stored at − 20°C for further study. The preparation, extraction, and measurement of urine samples were documented in the NHANES website [18]. In detail, the urine sample was treated using the on-line solid phase extraction (SPE) and the concentration of 2,5-DCP and 2,4 DCP were measured by HPLC linked tandem mass spectrometry [19]. The lower limit of detection (LLOD) for 2,5-DCP and 2,4 DCP were 0.2 ng/ml. The urinary creatinine was measured using the Roche/Hitachi Modular P Chemistry Analyzer and used to adjust the concentration of p-DCB exposure. Background Metabolic syndrome (MetS) is a group of risk factors that contain high blood pressure, hyperlipidemia, and disturbance of glucose homeostasis [1]. The prevalence of MetS has become an increasing public health burden, which affects about a quarter of adults worldwide [2]. Epidemiological evidence from the NHANES study, the prevalence of MetS has continued to rise and reached 34.7% in 2011–2012 due to the increased prevalence of overweight and obesity rates in adults [3, 4]. The development of MetS influences individual life and leads to further cardiometabolic disease, including cardiovascular disease (CVD) [5] and type 2 diabetes (T2D) [6]. Thus, it is warranted to prevent the deteriorating development of MetS. Although, MetS was mainly impacted by dietary and lifestyle factors, such as excessive energy intake and lack of exercise[1], increasing evidence suggested that Environmental pesticides, such as Para- dichlorobenzene (p-DCB), also have the potential to increase MetS prevalence [7, 8]. p-DCB, an organic compound, is poorly soluble in water and has been widely used as a disinfectant, pesticide, and deodorant [9, 10]. Similar to some hydrocarbons, p-DCB exists in new buildings, restrooms, and the air of households [11]. The International Agency for Research on Cancer (IARC) suggested that p-DCB may reasonably be a carcinogen base on animal evidence [10]. People may be exposed to p-DCB in mothballs, toilet deodorizer blocks, and air fresheners [12]. 2,5-dichlorophenol (2,5-DCP) and 2,4-Dichlorophenol (2,4-DCP) are the major metabolites of p-DCB [13, 14]. Given that 2,5-DCP is readily detectable at low concentrations, it is well suited for monitoring daily exposure to p-DCB [13]. NHANES study reported that 2,4-DCP and 2,5-DCP were found in 64% and 98% of U.S. adult urinary samples [15]. Page 2/16 Page 2/16 Page 2/16 The global community has expressed significant concern regarding the potential health hazards posed by p-DCB pesticides. Epidemiological studies reported that higher 2,5-DCP levels were related to a higher obesity prevalence [16]. Another cross-sectional study from U.S adults also determined the positive association between 2,5-dichlorophenol and diabetes [11]. Emerging evidence found 2,5 DCP and 2,4 DCP may increase the prevalence of hypertriglyceridemia in Mexican women [8]. These studies suggested that p-DCB and its metabolites are linked with the risk factors of MetS. Background Although, the previous study showed a positive association of 2,5 DCP with MetS prevalence among non-diabetic adults [7], the small size sample (n = 1,706) and insufficient assessment (lacking sensitivity analysis) make it difficult to account for the robustness of the results. Moreover, the association between p-DCB exposure and MetS among the general population remains unclear. To address the above-mentioned gaps in knowledge, we analyzed data from 10,428 participants from the NHANES (2003–2016) to evaluate the associations of internal exposures to p-DCB with MetS prevalence. We also conducted subgroup analyses and sensitivity analyses to verify the robustness of the results. Covariates Baseline information on age, sex, race, education, family poverty-income ratio (PIR), physical activity, smoking, and alcohol drinking status, and medical history were collected by a household interview questionnaire. The smoking status was assessed using the concentration of serum cotinine (ng/mL) [21]. Participants self-reported cases of CVD or cancer at the baseline for diagnosis. The total energy and fat intake were assessed using the average of two consecutive 24-h diet questionnaires. (5) HDL-C < 40 mg/dL for males or < 50 mg/dL for females (5) HDL-C < 40 mg/dL for males or < 50 mg/dL for females. (5) HDL-C < 40 mg/dL for males or < 50 mg/dL for females. (5) HDL-C < 40 mg/dL for males or < 50 mg/dL for females. Statistical analysis Due to the skewness of the data, the urinary concentrations of 2,5-DCP and 2,4-DCP were transformed using the natural logarithm (ln). This transformation helps to normalize the distribution of the data and improve its statistical analysis. Total dichlorophenol was calculated using the sum of 2,5-DCP and 2,4 DCP. We conducted continuous (each 1-unit increase) and categorical (across quartiles) analyses to assess the odds ratios (ORs) and confidence intervals (CIs) of MetS prevalence related to p-DCB exposure using multivariate-adjusted logistic regression models. The first model was adjusted urinary creatinine concentration, model 2 was further adjusted sex, age, and race, model 3 was adjusted model 2 plus education, PIR, physical activity, smoking, and drinking status, and model 4 (full model) was adjusted for total energy intake and total fat intake based on model 3. We utilized restricted cubic spline models to investigate the dose- response of p-DCB exposure with MetS. We further tested associations between p-DCB exposure and MetS components. Moreover, Additionally, we examined the correlation between p-DCB exposure and MetS indicators, such as systolic blood pressure (SBP), FPG, triglycerides, waist circumference, glycohemoglobin, and HDL-C. In addition, subgroup and sensitivity analyses were conducted to test the robustness of the current result. Firstly, we investigated whether the associations changed stratified by age, sex, education, PIR, physical activity, smoking status, and drinking status. Subgroup analysis was used to assess whether the associations observed were consistent across different subgroups or if there were any subgroup-specific effects. The interaction was evaluated using the likelihood- ratio test. Additionally, sensitivity analyses were performed by accounting for CVD history, cancer history, and hypoglycemic agents. In addition, pregnant individuals, those with extreme total energy intake, and individuals with extreme BMI were excluded to enhance the validity of our findings. In addition, subgroup and sensitivity analyses were conducted to test the robustness of the current result. Firstly, we investigated whether the associations changed stratified by age, sex, education, PIR, physical activity, smoking status, and drinking status. Subgroup analysis was used to assess whether the associations observed were consistent across different subgroups or if there were any subgroup-specific effects. The interaction was evaluated using the likelihood- ratio test. Additionally, sensitivity analyses were performed by accounting for CVD history, cancer history, and hypoglycemic agents. In addition, pregnant individuals, those with extreme total energy intake, and individuals with extreme BMI were excluded to enhance the validity of our findings. Statistical analysis All statistical analyses were performed using SAS version 9.4 (SAS Institute Inc., Cary, NC). A two-sided p-value less than 0.05 was considered statistically significant. Ascertainment of outcome Data on waist circumference (cm), fasting plasma glucose (FPG) (mg/dL), triglycerides (mg/dL), and high-density lipoprotein cholesterol (HDL-C; mg/dL) have been described on the NHANES website. The case of MetS was identified using the criteria of National Cholesterol Education Program Adult Treatment Panel III guidelines (NCEP/ATP III) [20]. Participants who matched three or more of the following five criteria were diagnosed as having MetS: (1) systolic blood pressure (SBP) ≥ 130 mmHg or diastolic blood pressure (DBP) ≥ 85 mmHg, or use of antihypertensive agents; 2) FPG ≥ 110 mg/dL, use of insulin or hypoglycemic drugs, or diagnosis of diabetes; Page 3/16 (3) waist circumference ≥ 102 cm for males or 88 cm for females; Page 3/16 (3) waist circumference ≥ 102 cm for males or 88 cm for females; (4) triglycerides ≥ 150 mg/dL; (4) Population characteristics In the NHANES study, among the 10,428 participants, an average of age 49.0 ± 17.8 years, 2,861 (27.1%) participants developed the MetS. Table 1 concluded the baseline characteristics of the current population classified by MetS status. Individuals with MetS were more often older and had higher levels of SBP, fasting glucose, waist circumference, and triglycerides, compared with not MetS patients. They were less likely to be male, educated, physically active, and alcohol drinkers and had a lower income and total energy intake. Moreover, MetS participants had higher concentrations of 2,5- dichlorophenol, 2,4-dichlorophenol, and total dichlorophenol compared with the healthy population (mean: 247.1 µg/L versus 151.7 µg/L, 7.3 versus 4.5, and 254.5 versus 156.2, respectively) Page 4/16 Page 4/16 Table 1 Population characteristics by metabolic syndrome status in NHANES 2003–2016 (n = 10,428) Data are expressed as mean ± SD or numbers with percentages. PIR, poverty-income ratio. Characteristics Overall (n = 10,428) Non-MetS (n = 7,607) MetS (n = 2,821) Age (years) 49.0 ± 17.8 46.0 ± 17.8 57.0 ± 15.4 Male (%) 48.8 49.4 47.0 Education levels (%) Under high school 10.9 9.5 14.6 High school 23.1 22.6 24.3 Above high school 51.1 53.9 43.5 Race/ethnicity (%) Mexican American 15.7 15.2 16.9 Non-Hispanic white 42.6 42.3 43.2 Non-Hispanic black 21.9 21.4 23.3 Others 19.8 21.0 16.6 PIR < 1.52 43.7 42.3 47.4 1.52 to 3.48 27.6 27.4 28.2 > 3.48 28.7 30.3 24.4 Smoking Status (%) Never 29.22 28.38 31.48 Former 46.74 47.4 44.95 Active 24.04 24.21 23.57 Alcohol Drinking Status (%) Never 16.96 14.32 24.1 Active 60.94 63.95 52.82 Physical activity (%) Never 34.42 31.19 43.11 Moderate 30.99 30.09 33.43 Vigorous 34.15 38.33 22.86 Cancer Yes 90.97 92.4 87.1 No 9 03 7 6 12 9 Table 1 teristics by metabolic syndrome status in NHANES 2003–2016 (n = 10,428) Data are sed as mean ± SD or numbers with percentages. PIR, poverty-income ratio. Population characteristics Page 5/16 Page 5/16 Characteristics Overall (n = 10,428) Non-MetS (n = 7,607) MetS (n = 2,821) Yes 10.79 7.11 20.73 No 89.21 92.89 79.27 Indicator of Metabolic Syndrome Systolic blood pressure (mmHg) 123.9 ± 18.5 120.9 ± 17.2 132.0 ± 19.2 Diastolic blood pressure (mmHg) 69.7 ± 13.1 69.2 ± 12.2 70.7 ± 15.1 Fasting glucose (mg/dL) 109.2 ± 36.2 99.8 ± 22.0 129.0 ± 49.8 Waist circumference (cm) 99.2 ± 16.4 94.7 ± 14.7 111.2 ± 14.5 Triglycerides (mg/dL) 127.8 ± 104.3 100.5 ± 69.5 185.5 ± 137.0 High density cholesterol (mg/dL) 53.1 ± 16.2 56.5 ± 16.2 43.7 ± 12.0 Diet Total energy intake, kcal/day 2113.8 ± 1017.0 2164.8 ± 1036.0 1978.2 ± 951.7 Total fat intake, g/day 79.8 ± 47.3 81.1 ± 48.1 76.3 ± 44.8 Dichlorophenol biomarkers 2,5-dichlorophenol (µg/L) 177.5 ± 1215.7 151.7 ± 1125.4 247.1 ± 1429.1 2,4-dichlorophenol (µg/L) 5.3 ± 32.2 4.5 ± 27.8 7.3 ± 41.8 Total Dichlorophenol (µg/L) 182.8 ± 1246.8 156.2 ± 1152.1 254.5 ± 1469.9 Associations of p-DCB biomarkers with MetS and its components Associations of p-DCB biomarkers with MetS and its components After adjusting the urinary creatinine, a 1-unit increase in 2,5-DCP and total dichlorophenol was related to a 5% higher MetS prevalence (Table 2). After adjustment for the lifestyle and dietary factors (model 4), the association between the total dichlorophenol and MetS prevalence is not significant, while the higher 2,5-DCP concentrations still had a higher prevalence of MetS. In the category analyses, we also observed significant and positive associations between the 2,5- DCP exposure and MetS prevalence. The multivariate-adjusted ORs (95% CIs) of MetS across increasing quartiles were 1.09 (0.93–1.28), 1.22 (1.00-1.49), and 1.34 (1.04–1.73) for 2,5-DCP in the full model (model 4, P = 0.018 for trend). Moreover, the restricted cubic spline model including p-DCB biomarkers as continuous variables to assess the dose- response relation between p-DCB exposure and MetS prevalence showed similar trends (Fig. 1). To investigate the potential mechanism of p-DCB inducing the MetS prevalence, we also conducted the analyses on the association between p-DCB biomarkers and MetS components (Table 3). Individuals with cases of elevated blood pressure (EBP), high fasting glucose (HFG), abdominal obesity, hypertriglyceridemia, and low HDL-C were 4,831 (46.3%), 1,663 (15.9%), 5,428 (15.9%), 1,194 (11.4%), and 3,569 (34.2%), respectively. Population characteristics The higher exposure of 2,5-DCP was positively associated with a higher prevalence of abdominal obesity (ORQ4vsQ1=1.23, 95% CI: 1.03–1.48, P = 0.017 for trend), whereas the levels of total dichlorophenol had a higher prevalence of HFG (ORQ4vsQ1=1.25, 95% CI: 0.96–1.62, P = 0.040 for trend). Page 6/16 Table 2 Multivariate-adjusted Odds ratios (95% CI) of associations between dichlorophenol biomarkers and the prevalence of MetS in NHANES 2003–2016 (n = 10,428) Biomarkers Effect estimates by continuous metabolites Effect estimates (95% CI) by quantiles of metabolites Q1 Q2 Q3 Q4 P- trend 2,5-dichlorophenol (µg/L) < 1.5 1.6–5.4 5.5–25.2 > 25.3 Case/N 2,821/10,428 632/2,644 696/2,569 710/2610 783/2,605 Model 1 1.05 (1.02–1.08) 1.00 1.10 (0.94– 1.29) 1.21 (1.00- 1.46) 1.26 (1.06– 1.51) 0.009 Model 2 1.03 (1.00-1.06) 1.00 1.11 (0.95– 1.30) 1.22 (1.01– 1.47) 1.30 (1.09– 1.56) 0.005 Model 3 1.02 (0.99–1.06) 1.00 1.08 (0.92– 1.26) 1.16 (0.97– 1.39) 1.21 (1.00- 1.45) 0.039 Model 4 1.05 (1.00-1.10) 1.00 1.09 (0.93– 1.28) 1.22 (1.00- 1.49) 1.34 (1.04– 1.73) 0.018 2,4-dichlorophenol (µg/L) < 0.3 0.4–0.7 0.8–1.8 > 1.9 Case/N 2,821/10,428 697/2,704 683/2,536 711/2,644 730/2,544 Model 1 1.04 (0.99–1.08) 1.00 1.04 (0.88– 1.23) 1.04 (0.86– 1.25) 1.09 (0.90– 1.31) 0.440 Model 2 0.99 (0.95–1.04) 1.00 1.04 (0.88– 1.23) 1.04 (0.87– 1.26) 1.09 (0.91– 1.32) 0.387 Model 3 0.98 (0.94–1.03) 1.00 1.01 (0.85– 1.21) 1.01 (0.84– 1.21) 1.01 (0.83– 1.21) 0.979 Model 4 0.98 (0.94–1.03) 1.00 0.96 (0.80– 1.14) 0.91 (0.74– 1.13) 0.83 (0.65– 1.06) 0.997 Total Dichlorophenol (µg/L) < 2.1 2.1–6.4 6.4–26.9 > 27 Case/N 2,821/10,428 639/2,637 686/2,560 713/2,623 783/2,608 Model 1 1.05 (1.02–1.08) 1.00 1.06 (0.90– 1.25) 1.19 (1.00- 1.41) 1.25 (1.05– 1.49) 0.008 Model 2 1.03 (0.99–1.06) 1.00 1.07 (0.91– 1.26) 1.20 (1.01– 1.42) 1.28 (1.07– 1.54) 0.005 CI, confidence interval; OR, Odd ratio; Q, quintile; PIR, poverty-income ratio; Model 1 was adjusted urinary creatinine concentration Model 2 was adjusted for covariates in Model l plus age (years), gender (male or female) and race (non-Hispanic Black, non-Hispanic White, Mexican American, or others). Population characteristics Model 3 was adjusted for covariates in Model 2 plus education (under high school, high school, or above high school), PIR (< 1.52, 1.52 to 3.48, or > 3.48), physical activity (never, moderate, or vigorous), smoking (non-smoker, former smoker, or active smoker), and drinking (abstainer or active drinker) status Model 4 was adjusted for covariates in Model 3 plus total energy intake and total fat intake Page 7/16 Page 7/16 Page 7/16 Biomarkers Effect estimates by continuous metabolites Effect estimates (95% CI) by quantiles of metabolites Q1 Q2 Q3 Q4 P- trend Model 3 1.02 (0.98–1.05) 1.00 1.03 (0.88– 1.22) 1.14 (0.96– 1.34) 1.18 (0.99– 1.42) 0.046 Model 4 1.02 (0.98–1.05) 1.00 1.02 (0.86– 1.21) 1.13 (0.95– 1.33) 1.17 (0.98– 1.41) 0.053 CI, confidence interval; OR, Odd ratio; Q, quintile; PIR, poverty-income ratio; Model 1 was adjusted urinary creatinine concentration Model 2 was adjusted for covariates in Model l plus age (years), gender (male or female) and race (non-Hispanic Black, non-Hispanic White, Mexican American, or others). Population characteristics Model 3 was adjusted for covariates in Model 2 plus education (under high school, high school, or above high school), PIR (< 1.52, 1.52 to 3.48, or > 3.48), physical activity (never, moderate, or vigorous), smoking (non-smoker, former smoker, or active smoker), and drinking (abstainer or active drinker) status Model 4 was adjusted for covariates in Model 3 plus total energy intake and total fat intake CI, confidence interval; OR, Odd ratio; Q, quintile; PIR, poverty-income ratio; Page 8/16 Table 3 Multivariate-adjusted odds ratios (95% CI) for associations between dichlorophenol biomarkers and individual components of MetS prevalence in NHANES 2003–2016 Biomarkers Elevated blood pressure (n = 4,831) High fasting glucose (n = 1,663) Abdominal obesity (n = 5,428) Hypertriglyceridemia (n = 1,194) Low HDL-C (n = 3,569) OR (95%CI) ptrend OR (95%CI) ptrend OR (95%CI) ptrend OR (95%CI) ptrend OR (95%CI) ptrend 2,5-dichlorophenol (µg/L) 0.862 0.202 0.017 0.378 0.228 Q1 1.00 1.00 1.00 1.00 1.00 Q2 0.97 (0.82– 1.14) 0.97 (0.81– 1.17) 1.13 (0.97–1.31) 1.16 (0.94–1.44) 0.98 (0.83– 1.15) Q3 1.08 (0.91– 1.28) 1.12 (0.89– 1.40) 1.20 (1.02–1.40) 1.23 (0.99–1.52) 1.11 (0.95– 1.30) Q4 0.98 (0.80– 1.19) 1.13 (0.89– 1.44) 1.23 (1.03–1.48) 1.06 (0.86–1.29) 1.07 (0.89– 1.29) 2,4-dichlorophenol (µg/L) 0.479 0.159 0.318 0.235 0.056 Q1 1.00 1.00 1.00 1.00 1.00 Q2 1.02 (0.85– 1.22) 1.09 (0.85– 1.40) 0.97 (0.83–1.14) 1.05 (0.83–1.33) 1.00 (0.86– 1.16) Q3 1.05 (0.87– 1.26) 1.15 (0.85– 1.55) 0.99 (0.84–1.17) 0.93 (0.72–1.19) 0.94 (0.81– 1.10) Q4 0.91 (0.74– 1.11) 1.26 (0.92– 1.72) 0.90 (0.75–1.07) 0.88 (0.68–1.15) 0.86 (0.72– 1.01) Total dichlorophenol (µg/L) 0.143 0.040 0.062 0.580 0.419 Q1 1.00 1.00 1.00 1.00 1.00 Q2 1.03 (0.89– 1.20) 1.02 (0.83– 1.27) 0.95 (0.83–1.09) 1.25 (0.98–1.59) 0.95 (0.81– 1.11) CI, confidence interval; OR, Odd ratio; Q, quintile; PIR, poverty-income ratio; Model 1 was adjusted urinary creatinine concentration Model 2 was adjusted for covariates in Model l plus age (years), gender (male or female) and race (non-Hispanic Black, non-Hispanic White, Mexican American, or others). Subgroup analyses and sensitivity analyses Subgroup analyses and sensitivity analyses In the subgroup analyses, we observed the positive associations between 2,5-DCP concentration were similarly stratified by age, sex, education, income, exercise, smoking or drinking status (all P for interaction > 0.05) (Fig. 2). In the sensitivity analyses, the observed associations were not substantially influenced by the additional adjustment for CVD and cancer or the use of lower sugar drug (Table S2). The consistent results after excluding individuals with extreme energy intake, BMI, or pregnancy supported the validity of the positive associations between 2,5-DCP exposure and MetS prevalence. (Table S3). Association between p-DCB biomarkers and MetS indicators We further conducted the analyses to test the relation between p-DCB Biomarkers and MetS factors (Table S1). The 2,5- DCP concentration was associated with higher SBP (r = 0.022, P = 0.027), waist circumference (r = 0.099, P < 0.001), glycohemoglobin (β = 0.190, P < 0.001), and lower HDL-C (r=-0.059, P < 0.001) after adjusting for the full covariates. The associations of 2,4 DCP with MetS indicators including waist circumference (r = 0.080, P < 0.001) and lower HDL-C (r=-0.039, P < 0.001) showed a similar trend. Besides, positive correlations were found between total dichlorophenol and waist circumference (r = 0.094, P < 0.001) and glycohemoglobin (r = 0.023, P = 0.021) while a negative association for HDL-C (r=-0.053, P < 0.001). Population characteristics Model 3 was adjusted for covariates in Model 2 plus education (under high school, high school, or above high school), PIR (< 1.52, 1.52 to 3.48, or > 3.48), physical activity (never, moderate, or vigorous), smoking (non-smoker, former smoker or active smoker) and drinking (abstainer or active drinker) status Table 3 Page 9/16 Page 9/16 Biomarkers Elevated blood pressure (n = 4,831) High fasting glucose (n = 1,663) Abdominal obesity (n = 5,428) Hypertriglyceridemia (n = 1,194) Low HDL-C (n = 3,569) OR (95%CI) ptrend OR (95%CI) ptrend OR (95%CI) ptrend OR (95%CI) ptrend OR (95%CI) ptrend Q3 1.12 (0.96– 1.30) 1.25 (0.99– 1.59) 1.10 (0.93–1.29) 1.15 (0.92–1.43) 1.09 (0.93– 1.28) Q4 1.13 (0.94– 1.35) 1.25 (0.96– 1.62) 1.17 (0.97–1.40) 1.07 (0.88–1.31) 1.03 (0.85– 1.25) CI, confidence interval; OR, Odd ratio; Q, quintile; PIR, poverty-income ratio; Model 1 was adjusted urinary creatinine concentration Model 2 was adjusted for covariates in Model l plus age (years), gender (male or female) and race (non-Hispanic Black, non-Hispanic White, Mexican American, or others). Model 3 was adjusted for covariates in Model 2 plus education (under high school, high school, or above high school), PIR (< 1.52, 1.52 to 3.48, or > 3.48), physical activity (never, moderate, or vigorous), smoking (non-smoker, former smoker, or active smoker), and drinking (abstainer or active drinker) status Model 4 was adjusted for covariates in Model 3 plus total energy intake and total fat intake Discussion A previous study conducted by Wei et al (2016) reported that higher 2,5-DCP concentration showed a significant association with diabetes development (OR: 1.59, 95% CI: 1.06–2.40) [11]. Our findings also found that total dichlorophenol was related to a 25% higher prevalence of diabetes (Table 3). The positive correlation between the total dichlorophenol, especially 2,5-DCP and glycohemoglobin may explain the adverse effects of dichlorophenol for the risk of developing diabetes [22]. Another work based on the NHANES study (2007–2010) found that 2,5-DCP had an 84% (95% CI: 26%-170%) higher prevalence of CVD after adjusting potential confounders [23]. Consistently, we found that higher urinary 2,5-DCP levels are positively associated with systolic blood pressure. Additionally, epidemiological evidence revealed a significant and positive association between p-DCB exposure and obesity risk. A previous cross- sectional study collected the data from NHANES (2005–2008) to investigate the association between dichlorophenol pesticides and the prevalence of obesity [16]. Consistent with our results, Wei et al. demonstrated that p-DCB metabolite 2,5-DCP not 2,4-DCP was positively associated with obesity prevalence [16]. A recent study based on Korean girls also reported that chlorophenol exposure had a higher risk of obesity by affecting waist circumference [24]. In the current study, we also revealed the significant and positive relation between the 2,5-DCP and 2,4 DCP and waist circumference. Overall, these epidemiological studies suggested that p-DCB exposure may pose a potential risk for the development of metabolic disorders. Additional research is warranted to enhance our understanding of the underlying mechanisms by which p-DCB exerts metabolic toxicity and to develop strategies for reducing exposure to this chemical in household products. The metabolism of para-dichlorobenzene (p-DCB) in humans and animals involves oxidation, reduction, and conjugation reactions that result in the formation of several metabolites [25]. The primary metabolites of p-DCB include 2,5- dichlorophenol (2,5-DCP), 2,4-dichlorophenol (2,4-DCP), and 4-chlorophenol (4-CP) [26]. These metabolites are formed through the oxidative dechlorination of p-DCB by cytochrome P450 enzymes in the liver, followed by conjugation with glucuronic acid or sulfate in the liver and kidneys [25]. 2,5-DCP is the major metabolite of p-DCB and is excreted in the urine, accounting for approximately 90% of the dose in humans [15]. Other minor metabolites of p-DCB include 2,6- dichlorophenol, 3,5-dichlorocatechol, and 3-chlorocatechol, which are formed through further oxidation and cleavage reactions [26]. Discussion This study examined the relations between p-DCB Biomarkers and the prevalence of MetS and its indicators. Using the data of 10,428 participants with 2,861 cases of MetS, we observed that higher 2,5-DCP levels were positively associated with MetS prevalence. After adjusting demographic, lifestyle, and dietary confounders, individuals in the highest versus lowest quartiles of 2,5-DCP concentrations had a 34% higher prevalence of MetS. Moreover, the 2,5-DCP exposure was associated with higher abdominal obesity prevalence and the increase of MetS factors including systolic blood pressure, waist circumference, and glycohemoglobin. Page 10/16 Recently, numerous epidemiological studies linking p-DCB exposure to chronic health burdens have attracted global concern [8, 11, 16]. A previous study conducted by Wei et al (2016) reported that higher 2,5-DCP concentration showed a significant association with diabetes development (OR: 1.59, 95% CI: 1.06–2.40) [11]. Our findings also found that total dichlorophenol was related to a 25% higher prevalence of diabetes (Table 3). The positive correlation between the total dichlorophenol, especially 2,5-DCP and glycohemoglobin may explain the adverse effects of dichlorophenol for the risk of developing diabetes [22]. Another work based on the NHANES study (2007–2010) found that 2,5-DCP had an 84% (95% CI: 26%-170%) higher prevalence of CVD after adjusting potential confounders [23]. Consistently, we found that higher urinary 2,5-DCP levels are positively associated with systolic blood pressure. Additionally, epidemiological evidence revealed a significant and positive association between p-DCB exposure and obesity risk. A previous cross- sectional study collected the data from NHANES (2005–2008) to investigate the association between dichlorophenol pesticides and the prevalence of obesity [16]. Consistent with our results, Wei et al. demonstrated that p-DCB metabolite 2,5-DCP not 2,4-DCP was positively associated with obesity prevalence [16]. A recent study based on Korean girls also reported that chlorophenol exposure had a higher risk of obesity by affecting waist circumference [24]. In the current study, we also revealed the significant and positive relation between the 2,5-DCP and 2,4 DCP and waist circumference. Overall, these epidemiological studies suggested that p-DCB exposure may pose a potential risk for the development of metabolic disorders. Additional research is warranted to enhance our understanding of the underlying mechanisms by which p-DCB exerts metabolic toxicity and to develop strategies for reducing exposure to this chemical in household products. Recently, numerous epidemiological studies linking p-DCB exposure to chronic health burdens have attracted global concern [8, 11, 16]. Discussion Previous studies showed that skin contact with 2,5-dichlorophenol can cause irritation and inflammation, and prolonged exposure can result in skin sensitization [27]. Moreover, 2,5-dichlorophenol may be associated with adverse effects on the endocrine system, which regulates hormone production, leading to reproductive and developmental problems, thyroid dysfunction, and other health issues [8, 23, 28]. In the current study, we also found that 2,5-DCP exposure was associated with dysglycolipidosis, thus leading to the MetS prevalence. On the other hand, 2,4- DCP has been shown to induce oxidative stress and inflammatory responses [29, 30]. Exposure to 2,4-DCP has been found to increase intracellular oxidative stress substances such as superoxide dismutase, catalase, and glutathione peroxidase [31]. These increased substances can lead to oxidative stress reactions and damage to cell components such as membranes, proteins, and DNA [32]. Our results of non-significant association of 2,4-DCP with MetS prevalence may be attributed to too low concentration of 2,4-DCP (5.3 ug/L). Overall, 2,5-DCP and 2,4-DCP can serve as biomarkers of p-DCB, used to assess the level of exposure, and the documented positive relationship of p-DCB and MetS prevalence could be mainly explained by the toxic effects of 2,5-DCP. The positive association between 2,5-DCP and MetS prevalence in this study can be linked to the pathophysiology of dysglycolipidosis. Evidence has revealed that p-DCB affected thyroid gland functions and was negatively associated with free thyroxine [33], leading to a higher risk of MetS [34]. Previous studies also found that 2,5-DCP may lead to metabolic risk by the disturbance of glycolipid homeostasis consistent with the current study that 2,5-DCP concentrates were associated with the higher glycohemoglobin level [11] and lower HDL-C [7]. Dyslipidemia, such as TG/HDL-C, has become an important marker for the development of MetS risk [35]. The latest meta-analyses also concluded that pesticide exposure increased the risk by altering the HDL-C levels [36, 37]. Insulin resistance can disrupt the balance of Page 11/16 Page 11/16 Page 11/16 glucose metabolism and result in chronic hyperglycemia, which leads to oxidative stress[38] and causes an inflammatory response [39] that contributes to cellular damage [40]. Moreover, insulin resistance can also alter systemic lipid metabolism and thus causing the MetS [41]. Further cohort or case-control research is warranted to investigate the potential mechanisms of p-DCB exposure associated with higher MetS prevalence. Discussion glucose metabolism and result in chronic hyperglycemia, which leads to oxidative stress[38] and causes an inflammatory response [39] that contributes to cellular damage [40]. Moreover, insulin resistance can also alter systemic lipid metabolism and thus causing the MetS [41]. Further cohort or case-control research is warranted to investigate the potential mechanisms of p-DCB exposure associated with higher MetS prevalence. Our study has several strengths. We provided the largest and most extensive evaluation (n = 10,428) on the associations of the 2,5-DCP ratio, a urinary biomarker of exposure to p-DCB, with MetS and its components. Meanwhile, the high correlation between p-DCB biomarker and glycolipid indicators, such as waist circumference, glycohemoglobin, and HDL-C suggests the potential causality of the relation between p-DCB exposure and MetS prevalence. Moreover, excluding the missing value of p-DCB biomarkers (including 2,5-DCP and 2,4-DCP) and MetS indexes (such as TC, TG, HDL-C, and FPG) could effectively assess relations between p-DCB and MetS prevalence. Finally, comprehensive information of covariates, including demographic, lifestyle, and dietary factors, used in the current study can allow us to investigate the realistic associations between p-DCB and MetS. In addition, some limitations are worth discussing. First, in NHANES, one time point urine sample was used to determine the concentration of 2,5-DCP and 2,4-DCP. Although we have adjusted the urinary creatinine in the model for better evaluation of p-DCB exposure [42], it might not represent long-term exposure to p-DCB. Thus, the repeat measurement of p-DCB exposure biomarkers or measurement of biomarkers in the blood is warranted to confirm the current results. Second, although we have strictly controlled for lifestyle and dietary factors in the multivariate-adjusted model, residual confoundings such as measurement and self- report errors were inevitable. Third, the generalizability of our findings was restricted to American descent. Finally, due to observational nature, the causality of the association between p-DCB exposure and MetS remains unclear. Thus, prospective studies and animal experiments need to elucidate the potential mechanism in the future. Conclusions In this study, p-DCB exposure biomarkers, 2,5-DCP, were significantly positively associated with a higher prevalence of MetS among U.S. adults. Notably, highly positive correlation between 2,5-DCP and lower HDL-C and higher glycohemoglobin suggested the potential mechanism of p-DCB exposure induced glycolipid metabolism and cause the developing MetS. Further long-time follow up studies are warranted to verify our results and investigate potential mechanisms. Competing interests No conflict of interest with respect to this manuscript to disclose. Consent for publication Not applicable. Declarations Author contributions: XK conceived and designed the study. JC, ZCY, and SZ did the data cleaning, analysis and interpretation. JC wrote the manuscript. ZCY provided statistical expertise and assistance. JC, ZCY, and SZ helped with interpretation of the results and provided revision and critical comments on the manuscript. All authors contributed to the interpretation of the data and critical revision of the manuscript for important intellectual content and approved the final draft. XK were involved in data acquisition. XK is the guarantor. Funding: The Science and Technology Department of Zhejiang Province (no. LGC21B050001), and Leading Talents Training Program of universities of Zhejiang Province. The funders had no role in design and conduct of the study; collection, management, analysis, and interpretation of the data; preparation, review, and approval of the manuscript; or the decision to submit the manuscript for publication. Data Availability Statement: All data are open access and available for download at url: https://www.cdc.gov/nchs/nhanes/index.htm (accessed on 18 June 2023). Ethics approval and consent to participate Page 12/16 Page 12/16 Not applicable. Not applicable. Not applicable. References 1. Eckel RH, Grundy SM, Zimmet PZ: The metabolic syndrome. Lancet 2005, 365(9468):1415-1428. 2. International Diabetes Federation (IDF): The IDF consensus worldwide definition of the metabolic syndrome. E- Publishing 2006. 2. 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Barr DB, Wilder LC, Caudill SP, Gonzalez AJ, Needham LL, Pirkle JL: Urinary creatinine concentrations in the U.S. population: implications for urinary biologic monitoring measurements. Environmental health perspectives 2005, 113(2):192-200. 42. Barr DB, Wilder LC, Caudill SP, Gonzalez AJ, Needham LL, Pirkle JL: Urinary creatinine concentrations in the U.S. population: implications for urinary biologic monitoring measurements. Environmental health perspectives 2005, 113(2):192-200. Figure 2 Subgroup analyses for the associations between the Para-dichlorobenzene and the prevalence of MetS in NHANES 2003-2016. Adjusted covariates: creatinine concentration, age (years), gender (male or female), race (non-Hispanic Black, non-Hispanic White, Mexican American, or others), education (under high school, high school, or above high h l) PIR ( 1 52 1 52 3 48 3 48) h i l i i ( d i ) ki ( k f Subgroup analyses for the associations between the Para-dichlorobenzene and the prevalence of MetS in NHANES 2003 2016 Adjusted covariates: creatinine concentration age (years) gender (male or female) race (non Hispanic Subgroup analyses for the associations between the Para-dichlorobenzene and the prevalence of MetS in NHANES 2003-2016. Adjusted covariates: creatinine concentration, age (years), gender (male or female), race (non-Hispanic Black, non-Hispanic White, Mexican American, or others), education (under high school, high school, or above high school), PIR (< 1.52, 1.52 to 3.48, or > 3.48), physical activity (never, moderate, or vigorous), smoking (non-smoker, forme smoker, or active smoker), drinking (abstainer or active drinker) status, total energy intake and total fat intake. Shaded areas represent 95% confidence intervals. CI, confidence interval; PIR, poverty-income ratio, OR, odds ratio; MetS, metabolic syndrome. Figures Page 15/16 Figures Figure 1 Associations between log-transformed Para-dichlorobenzene biomarkers and the prevalence of MetS. Odds ratios were estimated by restricted-cubic-spline regression after adjustment for creatinine concentration, age (years), gender (male or female), race (non-Hispanic Black, non-Hispanic White, Mexican American, or others), education (under high school, high g g Figure 1 Page 15/16 Associations between log-transformed Para-dichlorobenzene biomarkers and the prevalence of MetS. Odds ratios were estimated by restricted-cubic-spline regression after adjustment for creatinine concentration, age (years), gender (male or female), race (non-Hispanic Black, non-Hispanic White, Mexican American, or others), education (under high school, high Page 15/16 Associations between log-transformed Para-dichlorobenzene biomarkers and the prevalence of MetS. Odds ratios were estimated by restricted-cubic-spline regression after adjustment for creatinine concentration, age (years), gender (male or female), race (non-Hispanic Black, non-Hispanic White, Mexican American, or others), education (under high school, high Page 15/16 estimated by restricted-cubic-spline regression after adjustment for creatinine conce female), race (non-Hispanic Black, non-Hispanic White, Mexican American, or others school, or above high school), PIR (< 1.52, 1.52 to 3.48, or > 3.48), physical activity (never, moderate, or vigorous), smoking (non-smoker, former smoker, or active smoker), drinking (abstainer or active drinker) status, total energy intake and total fat intake. Shaded areas represent 95% confidence intervals. CI, confidence interval; PIR, poverty-income ratio, OR, odds ratio; MetS, metabolic syndrome. smoking (non-smoker, former smoker, or active smoker), drinking (abstainer or active drinker) status, total energy intake and total fat intake. Shaded areas represent 95% confidence intervals. CI, confidence interval; PIR, poverty-income ratio, OR, odds ratio; MetS, metabolic syndrome. SupplMaterWanetaljfEnvironmentalHealth.docx Supplementary Files This is a list of supplementary files associated with this preprint. Click to download. SupplMaterWanetaljfEnvironmentalHealth.docx Page 16/16
https://openalex.org/W2626347287	https://birmingham.elsevierpure.com/files/41998271/1_s2.0_S1388248117301704_main.pdf	English	null	Electrochemical sulfidation of WS 2 nanoarrays: Strong dependence of hydrogen evolution activity on transition metal sulfide surface composition	Electrochemistry communications	2,017	cc-by	5,042	Electrochemical sulfidation of WS2 nanoarrays Escalera-López, Daniel; Griffin, Ross; Isaacs, Mark; Wilson, Karen; Palmer, Richard E.; Rees, Neil V. DOI: 10.1016/j.elecom.2017.06.016 License: Creative Commons: Attribution (CC BY) Document Version Publisher's PDF, also known as Version of record Citation for published version (Harvard): Escalera-López, D, Griffin, R, Isaacs, M, Wilson, K, Palmer, RE & Rees, NV 2017, 'Electrochemical sulfidation of WS2 nanoarrays: strong dependence of hydrogen evolution activity on transition metal sulfide surface composition', Electrochemistry Communications, vol. 81, pp. 106-111. https://doi.org/10.1016/j.elecom.2017.06.016 Document Version Publisher's PDF, also known as Version of record Citation for published version (Harvard): Escalera-López, D, Griffin, R, Isaacs, M, Wilson, K, Palmer, RE & Rees, NV 2017, 'Electrochemical sulfidation of WS2 nanoarrays: strong dependence of hydrogen evolution activity on transition metal sulfide surface composition', Electrochemistry Communications, vol. 81, pp. 106-111. https://doi.org/10.1016/j.elecom.2017.06.016 Citation for published version (Harvard): Escalera-López, D, Griffin, R, Isaacs, M, Wilson, K, Palmer, RE & Rees, NV 2017, 'Electrochemical sulfidation of WS2 nanoarrays: strong dependence of hydrogen evolution activity on transition metal sulfide surface composition', Electrochemistry Communications, vol. 81, pp. 106-111. https://doi.org/10.1016/j.elecom.2017.06.016 Link to publication on Research at Birmingham portal General rights U l li General rights Unless a licence is specified above, all rights (including copyright and moral rights) in this document are retained by the authors and/or the copyright holders. 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A R T I C L E I N F O Keywords: Transition metal dichalcogenides Hydrogen evolution Sulﬁdation Tungsten disulﬁde Nanoarrays The activity of transition metal sulﬁdes for the hydrogen evolution reaction (HER) can be increased by sulfur- enrichment of active metal-sulﬁde sites. In this report, we investigate the electrochemical sulﬁdation of atmo- spherically aged WS2 nanoarrays with respect to enhancing HER activity. In contrast to MoS2, it is found that sulﬁdation diminishes HER activity. Electrochemical and XPS experiments suggest the involvement of insoluble tungsten oxides in the altered HER and electron transfer properties. This demonstrates the strong dependence of the transition metal dichalcogenide (TMD) composition with the successful sulfur incorporation and subsequent HER activity. monitoring surface composition, morphology, and electron transfer properties over a one month period by XPS, SEM, and voltammetric experiments. 1. Introduction The demand for sustainable sources of electrochemical hydrogen production [1] has triggered the development of the abundant and low- cost TMDs as substitutes to the best performing platinum group metal catalysts for the hydrogen evolution reaction (HER) [2–5]. Electrochemical sulﬁdation of WS2 nanoarrays: Strong dependence of hydrogen evolution activity on transition metal sulﬁde surface composition Daniel Escalera-Lópeza,b, Ross Griﬃnb, Mark Isaacsc, Karen Wilsonc, Richard E. P Neil V. Reesa,⁎ a Centre for Hydrogen and Fuel Cell Research, School of Chemical Engineering, University of Birmingham, Birmingham B15 2TT, UK b School of Physics and Astronomy, University of Birmingham, Birmingham B15 2TT, UK c European Bioenergy Research Institute, Aston University, Birmingham B4 7ET, UK d College of Engineering, Swansea University, Fabian Way, Swansea SA1 8EN, UK ⁎ Corresponding author. E-mail address: n.rees@bham.ac.uk (N.V. Rees). Contents lists available at ScienceDirect Contents lists available at ScienceDirect http://dx.doi.org/10.1016/j.elecom.2017.06.016 Received 30 May 2017; Received in revised form 15 June 2017; Accepted 15 June 2017 ⁎ Corresponding author. E-mail address: n.rees@bham.ac.uk (N.V. Rees). Available online 16 June 2017 1388-2481/ © 2017 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license (http:// Take down policy Take down policy While the University of Birmingham exercises care and attention in making items available there are rare occasions when an item has been uploaded in error or has been deemed to be commercially or otherwise sensitive. If you believe that this is the case for this document, please contact UBIRA@lists.bham.ac.uk providing details and we will remove access to the work immediately and investigate. Download date: 24. Oct. 2024 Electrochem istry Com m unications 81 (2017) 106–111 http://dx.doi.org/10.1016/j.elecom.2017.06.016 Received 30 May 2017; Received in revised form 15 June 2017; Accepted 15 June 2017 ⁎ Corresponding author. E-mail address: n.rees@bham.ac.uk (N.V. Rees). Available online 16 June 2017 1388-2481/ © 2017 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecom m ons.org/licenses/BY/4.0/). e Authors. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecom m ons.org/licenses/BY/4.0/). 2.2. Electrochemical characterization The application of this method to WS2 was evaluated by monitoring HER performance, oxidation state, and electron transfer properties over a one month period following this sulﬁdation treatment on previously tested, atmospherically aged WS2 samples. This provided the following observations: (i) freshly sulﬁdated samples did not necessarily present enhanced HER performances compared with pre-sulﬁdated samples, and (ii) the samples' HER peak current, after correction for roughness factor, was inferior after a 3-week environmental exposure compared to the pre-sulﬁdated, atmospherically-exposed state. Investigation of heterogeneous electron transfer (HET) rates was performed under the same conditions as of Pumera et al. for ease of reference [5,13], by acquiring ﬁve CVs in 10 mM K4Fe(CN)6/K3Fe(CN) 6 (BioUltra ≥99.5%, Sigma-Aldrich) electrolyte supported by a pH 7.2 phosphate buﬀer (50 mM potassium phosphate monobasic/potassium phosphate dibasic trihydrate, ≥99%, Sigma-Aldrich) at scan rates from 10 to 300 mV s−1. Experimental HET rate constants (kapp O) were cal- culated by correlating the peak-to-peak separation of the Fe(CN)6 4−/Fe (CN)6 3−redox couple with the dimensionless parameter ψ, using the literature methods of Nicholson and Shain (ψ≈0.1 and ΔEP < 210 mV) [27], and Klinger and Kochi (ψ < 0.1 and ΔEP > 210 mV) [28]. A diﬀusion coeﬃcient of 7.26 × 10−6 cm2 s−1 for the [Fe(CN)6]4−/3−redox pair was used in the calculation [29], assuming α = 0.5 For the nanoarrays under study, Case 4 diﬀusion behaviour (1D) was observed [30]. All electrolytes were freshly pre- pared with ultrapure water (resistivity not < 18.2 MΩ∙cm, Millipore Milli-Q Direct 8), and thoroughly purged with N2 (Oxygen-free grade, BOC Gases plc), and experiments run under a N2 atmosphere. All glassware was cleaned with a dilute solution of KMnO4 in concentrated H2SO4 followed by rinsing with ultrapure water. Both phenomena can be understood by changes in oxidation state revealed by XPS. For the 31 ± 1 s (R = 2, Z = 6.4) plasma-etched WS2 sample, the peak current decays to half of its initial value following sulﬁdation (Fig. 1a). This is correlated to a decrease in the total S:W ratio (from ca. 2:1 to 1.5:1, see Fig. 2e), and the appearance of WO2 at the crystal surface up to ca. 24% (W 4f7/2/W 4f5/2 doublet lies at binding energies of ca. 33 and 35.2 eV, respectively; Figs. 1c, 2a) [31]. Previous reports on bulk and chemically-exfoliated WS2 crystals suggest that incorporation of WO2 is detrimental for the HER [18,35]. 2.2. Electrochemical characterization For reinstating, or improving, the initial HER activity of atmo- spherically-exposed WS2 samples, a solution-phase method previously demonstrated for MoS2 nanoarrays [20] was used. This requires voltage cycling of the TMD samples in a pH 3 solution containing 10 mM Na2S2O3 and 0.1 M Na2SO4 whereby S2O3 2−spontaneously decom- poses to form colloidal sulfur [33]. Electrochemical measurements were carried out with a PC-con- trolled PGSTAT128N potentiostat (Metrohm Autolab B·V, Netherlands) in a thermostatted three-electrode electrochemical cell (23 ± 2 °C). The electrodes used were: a bright Pt mesh counter (Alfa Aesar Ltd., UK), a double junction saturated Ag/AgCl reference (electrochemical sulﬁdation experiments, Sigma-Aldrich), a saturated calomel (SCE) re- ference electrode (HER experiments, BAS Inc., Japan) and TMD-mod- iﬁed GC stubs connected to a rotating disk working electrode (OrigaLys ElectroChem SAS, France). + ⇌ + − + − S O (aq) H (aq) S (s) HSO 2 3 2 3 An anodic sweep to fully oxidize the TMD surface, was followed by a cathodic scan to maximize sulfur incorporation onto the TMD surface by reduction HER experiments were carried out in a 2 mM HClO4 (ACS ≥70%, Sigma-Aldrich), 0.1 M NaClO4 (ACS ≥98%, Sigma-Aldrich) solution using a range of voltage scan rates (2–1200 mV s−1). Preconditioning of TMD electrodes prior to HER experiments was via 10 cycles from −0.045 to −1.645 V (vs SCE) at a voltage scan rate of 50 mV s−1. Additional capacitance (voltage range −0.2 to 0.2 V vs. NHE, scan rates 10–500 mV s−1) and impedance measurements (voltage range 0 to −1.645 V vs. SCE, frequency range 10−1 to 105 Hz, voltage ampli- tude 10 mV) were performed alongside all HER measurements to apply roughness factor and iR compensation corrections. HER potentials are referenced versus the normal hydrogen electrode (NHE) by means of Nernstian shift correction (ENHE = 0.242 V + 0.059 pH). + ⇌+ − − S (s) 2e S (aq) 2 For MoS2, electrochemically-induced surface oxidation (at E > +1 V vs Ag/AgCl) yields the acid-soluble MoO4 2−species [34]. Sulfur incorporation after surface oxidation suggests that MoO4 2− species assist in the overall sulﬁdation mechanism. For WS2, the cyclic voltammogram obtained during the sulﬁdation treatment is similar to that of MoS2 [20]. 2.1. Fabrication To improve their HER activity, research has focussed on the pre- paration of S-rich TMD structures which surpass the 1:2 M:X stoichio- metry found in bulk materials [6–8]. Such sulfur enrichment aims to incorporate more bridging S2 2−and terminal S2−moieties into the TMD structure; both consistently reported as being the active sites in- volved in proton adsorption and desorption [9,10]. Enhanced HER performance has been reported for S-rich structures such as amorphous MoS2+x [11–15] and WS2.64 electrodeposited thin ﬁlms [9], as well as on wet chemical synthesis-prepared MoS2+x [16,17] or MX3/MX2 physical mixtures [18]. However, some of the proposed structures ex- hibit diminished HER performances after atmospheric or electro- chemically-induced sulfur depletion [14,18] or impurities presence [19]. The plasma-etch fabrication method used is based on a literature method [22–24], recently reported in TMDs for electrocatalytic appli- cations [20,21,25]. In short, WS2 (defect-free, 99.9995% purity, 2D Semiconductors USA) crystals cut into rectangles of approximately 1.5 × 5 mm were aﬃxed to glassy carbon (GC) type 2 stubs (7 mm diameter, 2 mm thick, Alfa Aesar, UK) with carbon tape. A 20 μL mixture of a 216 ± 4 nm diameter polystyrene-latex nanosphere (NS) suspension (3000 Series Nanosphere, 1 wt% in water, Thermo Scientiﬁc, UK) with absolute ethanol in a 1:1 vol. ratio was transferred to a silicon wafer (previously cleaned with piranha solution and oxygen plasma) to form a self-as- sembled, hexagonal close-packed, NS monolayer. The NS monolayer was transferred onto the liquid interface of a water-ﬁlled Petri dish containing the TMD-modiﬁed GC stubs, and the supernatant extracted with a syringe to promote NS deposition onto the TMD surface. We report the use of a one step, room temperature electrochemical sulﬁdation method initially developed for MoS2 [20], for sulfur-en- riching WS2. In particular, atmospherically-aged WS2 nanocone arrays which are of interest due to their enhanced electrocatalytic properties [21]. Changes in the electrocatalytic behaviour are understood via NS-modiﬁed TMDs etching was carried out in an Oxford Electrochem istry Com m unications 81 (2017) 106–111 D. Escalera-López et al. CASA XPS version 2.3.18PR1.0, with spectral energy corrected to the adventitious C 1 s peak at 284.6 eV. Shirley backgrounds were applied to high resolution peaks before being ﬁtted with individual compo- nents. 2.1. Fabrication W 4f spectra were ﬁt using a FWHM of 0.98 eV, peak area ratios of 4:3, doublet separations of 2.17 eV and Gaussian-Lorentz (30) line- shape, with W 4f7/2 WS2 2H (32.7 eV), W 4f7/2 WS2 1 T (31.7 eV) and W 4f7/2 WO2 (33.1 eV) components, whilst the overlapping W 5p3/2 feature was ﬁtted with a FWHM of 2 eV, a Gaussian-Lorentz (30) lineshape and a binding energy of 38.1 eV. Sulfur 2p peaks were ﬁtted with a FWHM of 1.03 eV, peak area ratios of 2:1 and a binding energy for S 2p3/2 WS2 of 162.3 eV [31,32]. Instruments Plasmalab NGP 80 ICP/RF etcher. Isotropic oxygen plasma etching (40 s, 30 sccm O2 ﬂow rate, 100 W RF power) was performed as an NS shrinking step. Afterwards, exposure to an anisotropic plasma etching mixture of SF6/C4F8 (20 sccm/30 sccm, 200 W ICP, 20 W RF power) for variable times (15–60 s) was carried out to obtain nanocone arrays with variable aspect ratios. Pressures of 15 mTorr and tem- peratures of 20 °C were used in both etching steps. SEM micrographs of the electrochemically tested samples before/after sulﬁdation were ac- quired to determine the aspect ratio (nanocone height/base diameter), interspacing, and dimensionless radial (R, domain radius/nanocone radius) and normal (Z, nanocone height/nanocone radius) coordinates [26] of the individual nanostructures in the array (XL 30 SFEG and JEOL 7100 FEG-SEM, 5 kV, tilt angles from 45° to 85°). 2.2. Electrochemical characterization In the case of the 31 ± 1 s sample, this is supported by the changed HER kinetics (Tafel slope increase from 100 to 185 mV dec−1, Fig. 3c) and higher onset potentials (\|ηonset\| from 173 to 207 mV). Conversely, the 61 ± 1 s plasma-etched WS2 sample presented higher peak currents (Fig. 1b) and kinetics (Tafel slope 130 vs. initial 210 mV dec−1, Fig. 3d) following sulﬁdation, despite the decay in the total S:W ratio (from ca. 2:1 to 1.88:1, see Fig. 2f) and the 14% increase in surface WO2 content (Figs. 1d and 2c). This initially non-linear trend is found to be linked to the S:W ratio, if calculated solely using the W4+ XPS components characteristic of WS2. Sulfur-rich S:W ratios promote enhanced HER performance and vice versa. Maximum peak currents coincide with the highest sulfur-to-metal ratios for both 31 ± 1 s (S:W = 2.08:1, jp ≈9 mA cm−2, day 8) and 61 ± 1 s (S:W = 2.18:1, j ≈1.6 mA cm−2, freshly sulﬁdated) samples. After these peak values, both post-sulﬁdated 31 ± 1 s and 61 ± 1 s etched samples exhibited 4. Conclusions The cathodic feature appearing at E ca. −0.4 V vs NHE in the HER experiments (Fig. 3a and b) is ascribed to the diﬀusion decay peak proﬁle of proton reduction catalysed by the WS2 active sites, char- acteristic of the fully-supported, low proton concentration electrolyte used [36,37]. Indeed, the resolution of this peak also seems correlated with the S2−:W4+ ratio, and consequently to the active sites present. In contrast to MoS2, the application of the solution-phase, room- temperature electrochemical sulﬁdation method to obtain S-rich structures did not lead to S-rich WSx but to S-deﬁcient WSx structures with high WO2 surface content. The inferior HER performances but improved electron transfer properties are in agreement with the detri- mental eﬀect reported after WO2 incorporation into WS2 for the HER catalysis. The unsuccessful incorporation of electroreduced sulﬁde in the WSx structure is suspected to arise from the nature of the sulﬁdation mechanism: redeposition of acid-soluble MoO4 2−species for MoX2 improves S2−incorporation onto the surface, which is not possible in the case of WX2 as the WOx compounds formed are acid insoluble. This demonstrates the key role of the nature of the TMDs in the successful electrochemical incorporation of sulfur in their structure, and reveals that an electrochemistry-based sulﬁdation method universally applic- able for any TMDs remains to be developed. With regard to the electron transfer kinetics, both samples exhibit higher kapp O values (≈4 × 10−5 cms−1) after undergoing the sulﬁda- tion treatment (Fig. 1e–f). This agrees with literature reports which found enhanced electrical conductivities of WOx species vs. WS2 [38], beneﬁcial for mediating in the redox chemistry of surface sensitive species such as Fe(CN)6 4−/Fe(CN)6 3−. A gradual decrease in kapp O values after two weeks of testing correlates with reduced WO2 content at the crystal surface, whereas restored kapp O values after three weeks can be attributed to the higher WS2 metallic 1T polymorph contents (ca. 10–15%). These results suggest that this sulﬁdation method does not in- corporate sulfur into the atmospherically aged WS2 samples. Instead, it promotes the appearance of WOx moieties at the WSx surface which are reduced in the cathodic sweep. We hypothesize that in general, the sulfur incorporation is only eﬀective when the electro-oxidative step of TMDs forms acid-soluble species, as sulfur incorporation into atmo- spherically-aged MoS2 crystals was optimal when the cathodic voltage vertex surpassed the reduction potential of the TMD oxidised species (MoO4 2−) [20]. 2.3. XPS measurements A Kratos Axis HSi X-ray photoelectron spectrophotometer (Aston University) ﬁtted with a charge neutraliser and operated using a Mg Kα (1253.6 eV) achromatic radiation, was used to record spectra at a pressure of < 1 × 10−9 Torr using a spot size of 100 μm. Pass energies used were: 160 eV for survey spectra, and 20 eV for high resolution scans of speciﬁc energy regions. Data processing was performed using 107 Electrochem istry Com m unications 81 (2017) 106–111 D. Escalera-López et al. Fig. 1. Comparison of: a)–b) roughness-factor corrected HER peak current densities at υ = 25 mV s−1, c)–d) WO2 surface content (left axis) and S2−:W4+ XPS atomic photoemission ratios (right axis), and e)–f) kapp O values for the [Fe(CN)6]4−/[Fe(CN)6]3−redox probe, for atmospherically aged, sulﬁdation treated plasma-etched WS2 samples 31 ± 1 s (R = 2, Z = 6.4, ﬁrst row) and 61 ± 1 s (second row), after weekly electrochemical testing over a three-week ambient exposure period. p y ( ) Fig. 1. Comparison of: a)–b) roughness-factor corrected HER peak current densities at υ = 25 mV s−1, c)–d) WO2 surface content (left axis) and S2−:W4+ XPS atomic photoemission ratios (right axis), and e)–f) kapp O values for the [Fe(CN)6]4−/[Fe(CN)6]3−redox probe, for atmospherically aged, sulﬁdation treated plasma-etched WS2 samples 31 ± 1 s (R = 2, Z = 6.4, ﬁrst row) and 61 ± 1 s (second row), after weekly electrochemical testing over a three-week ambient exposure period. during the electro-oxidative step are insoluble at pH ≤3 [39], coin- ciding with the optimized pH value for the sulﬁdation electrolyte (pH 3). Consequently, the electroreduced sulfur cannot be incorporated into the WOx structure, and would dissolve under acidic conditions [40]. Hence, we predict that the electrochemical solvent-phase sulﬁ- dation method is only suited for MoX2 (X = S, Se) rather than for WX2 (X = S, Se). an HER current decrease in subsequent electrochemical testing to va- lues lower or comparable with the freshly sulﬁdated state, due to lower S:W ratios. This accords with previous investigations which correlated higher sulfur content in TMDs with improved hydrogen turnover fre- quencies [12,14], and sulfur-depleted W-edge sites of electro-oxidised WS2 with poor catalytic activity [35]. We hypothesize that the elec- trochemically-induced restructuring gradually depletes the WO2 phase, initially exposing underlying WS2 with high active site densities which are later reconstructed during atmospheric and experimental conditions to a more homogeneous nanostructure (Fig. 2.3. XPS measurements 2g–h). 4. Conclusions In the case of WS2, the oxidised WOx species generated Acknowledgments We thank the EPSRC for ﬁnancial support via fellowship (REP, EP/ L015749/1) and the Centre for Doctoral Training in Fuel Cells and their Fuels (DE-L, NVR, EP/G037116/1). The authors also thank Martin Roe and Nigel Neate from the Nanoscale and Microscale Research Centre (NMRC, EP/L022494/1) for the SEM micrograph acquisition (JEOL 7100F FEG-SEM.) 108 Electrochem istry Com m unications 81 (2017) 106–111 D. Escalera-López et al. (caption on 109 (caption on next page) 109 Electrochem istry Com m unications 81 (2017) 106–111 D. Escalera-López et al. Fig. 2. Stacked high-resolution XPS spectra of W 4f and S 2p for a)–b) 31 ± 1 s (R = 2, Z = 6.4) and c)–d) 61 ± 1 s atmospherically aged, sulﬁdation treated, plasma-etched WS2 samples over a three-week ambient exposure period. e)–f) Comparison of total S:W XPS atomic photoemission ratios. Representative SEM micrographs g) before and h) after solution phase-sulﬁdation. Fig. 2. Stacked high-resolution XPS spectra of W 4f and S 2p for a)–b) 31 ± 1 s (R = 2, Z = 6.4) and c)–d) 61 ± 1 s atmospherically aged, sulﬁdation treated, plasma-etched WS2 samples over a three-week ambient exposure period. e)–f) Comparison of total S:W XPS atomic photoemission ratios. Representative SEM micrographs g) before and h) after solution phase sulﬁdation Fig. 2. Stacked high-resolution XPS spectra of W 4f and S 2p for a)–b) 31 ± 1 s (R = 2, Z = 6.4) and c)–d) 61 ± 1 s atmospherically aged, sulﬁdation treated, plasma-etched WS2 samples over a three-week ambient exposure period. e)–f) Comparison of total S:W XPS atomic photoemission ratios. Representative SEM micrographs g) before and h) after solution phase sulﬁdation Fig. 3. Left column: Linear sweep voltammograms in the 0 to −1.2 V voltage range of a) 31 ± 1 s (R = 2, Z = 6.4) and b) 61 ± 1 s atmospherically aged, sulﬁdation treated plasma- etched WS2 samples over a three week ambient exposure period. Right column: Tafel plots (η vs. log∣jgeom∣) of c) 31 ± 1 s (R = 2, Z = 6.4) and d) 61 ± 1 s atmospherically aged, sulﬁdation treated plasma-etched WS2 samples over a three week ambient exposure period. Labels: pre-sulﬁdated (black), post-sulﬁdated (red), 8-day atmosphere exposed (green), 15-day atmosphere exposed (blue) and 22-day atmosphere exposed (magenta). References [1] J.A. Turner, Sustainable hydrogen production, Science 305 (2004) 972–974, http:// dx.doi.org/10.1126/science.1103197. [10] B. Hinnemann, P. Moses, J. Bonde, K.P. Jørgensen, J.H. Nielsen, S. Horch, et al., Biomimetic hydrogen evolution: MoS2 nanoparticles as catalyst for hydrogen evo- lution, J. Am. Chem. 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https://openalex.org/W4252167074	https://journals.umcs.pl/rh/article/download/3218/2414	Polish	null	Zakład Archiwistyki UMCS współorganizatorem konferencji w Moskwie	Res Historica	2,016	cc-by	1,435	Zakład Archiwistyki UMCS współorganizatorem konfe- rencji w Moskwie Zakład Archiwistyki od kilku lat stara się współpracować z czoło- wymi uczelniami rosyjskimi zajmującymi się kształceniem archiwistów. W roku 2011 dzięki inicjatywie jego pracowników UMCS podpisał umo- wę o współpracy z Federalnym Uniwersytetem imienia Pierwszego Prezydenta Rosji B. M. Jelcyna w Jekaterynburgu, drugą uczelnią pod względem hierarchii i tradycji, zajmującą się kształceniem archiwistów. Wspólnie z jej pracownikami udało się zrealizować kilka ważnych przed- sięwzięć, o których systematycznie informowałem na łamach „Wiadomo- ści Uniwersyteckich”. W roku ubiegłym dzięki inicjatywie pracownika Zakładu dr. A. Góraka, po długo trwających zabiegach, sfinalizowano po- dobną umową o współpracy z najważniejszym ośrodkiem przygotowują- cym przyszłe kadry archiwalne w Federacji Rosyjskiej, jakim jest Rosyjski Państwowy Uniwersytet Humanistyczny (Rossyjskij Gosudarstwiennyj Gumanitarnyj Uniwiersytiet – RGGU) w Moskwie. yj y ) Uczelnia ta wywodzi się ze słynnego Instytutu Historyczno-Archiwal- nego powstałego w roku 1931 i stanowiącego jedną z pierwszych uczelni archiwistycznych w Europie, a pierwszą w Europie Wschodniej. Obecnie ma ona charakter elitarny i kształci studentów na wszystkich kierunkach humanistycznych i społecznych (historia, socjologia, politologia), a tak- że na kierunkach prawa i ekonomii. W Federacji Rosyjskiej panują inne zasady rekrutacji na studia. Państwowe uczelnie nie mogą przyjmować na studia wszystkich zainteresowanych absolwentów szkół wyższych, ale jedynie ich małą liczbę. Każdego roku określa je dla poszczególnej uczel- ni Ministerstwo Oświaty i Nauki i to zarówno na studiach bezpłatnych (tzw. budżetowych), jak i komercyjnych (opłacanych przez studiujących). W ten sposób faktycznie ogranicza się wydatki na szkolnictwo wyższe, ale jednocześnie steruje się dopływem absolwentów wyższych uczelni na rynek pracy, ograniczając wśród nich bezrobocie. Uboczny skutek tych rozwiązań stanowiło wzmocnienie prestiżu starych uczelni, takich jak Uniwersytet Moskiewski im. M.W. Łomonosowa czy RGGU, które kształ- cąc niewielką liczbę studentów w stosunku do możliwości, mogły dzięki temu podnieść poziom dydaktyki. Polskich i rosyjskich archiwistów łączy nie tylko sąsiedztwo, ale i wspólna historia wynikająca z dziedzictwa komunizmu. W PRL tak jak w Związku Sowieckim archiwa były państwowe i scentralizowane. Od- 402 Sprawozdania działywały one na system biurowości urzędów oraz przedsiębiorstw, opracowując instrukcje kancelaryjne oraz wykazy akt, dlatego musiały podejmować badania nad organizacją pracy kancelaryjnej współczesnych instytucji i zakładów pracy, a także zajmować się zagadnieniami klasyfi- kacji akt oraz ich wartościowania. Drugi wspólny obszar badawczy, jed- noczący polskich i rosyjskich archiwistów oraz historyków, to dzieje ad- ministracji rosyjskiej, która w XIX i na początku XX wieku stanowiła pod- stawę władzy carskiej w Królestwie Polskim. Zakład Archiwistyki UMCS współorganizatorem konfe- rencji w Moskwie Współpraca z moskiewskimi uczelniami jest również korzystna dla polskich badaczy, ponieważ uła- twia im prowadzenie poszukiwań źródłowych w tamtejszych archiwach i bibliotekach posiadających różnorodne i cenne materiały archiwalne. W roku 2015 już uczyniono pierwszy krok w realizacji zawartej umo- wy, przygotowując konferencję, której współorganizatorem był Zakład Archiwistyki UMCS. Stało się to jeszcze przed połączeniem z Zakładem Nauk Pomocniczych Historii. Szczęśliwie organizacji tego przedsięwzię- cia nie przeszkodziło napięcie w stosunkach polsko-rosyjskich, spowodo- wane aneksją Krymu przez Rosję i wojną na wschodzie Ukrainy. Współ- praca między UMCS a RGGU ma charakter naukowy, a obie strony nie nadają jej żadnych treści politycznych. Uważamy, że nawet w tak trudnej sytuacji, jaka panuje obecnie w naszej części Europy, należy podtrzymy- wać kontakty naukowe i kulturalne z uczelniami rosyjskimi, jeśli nie słu- żą celom polityczno-propagandowym. Mogą one stanowić kapitał, który będzie procentować w przyszłości, gdy relacje miedzy naszymi krajami poprawią się. Zdajemy sobie sprawę, że zaostrzenie stosunków między obu krajami w przypadku dalszej eskalacji konfliktu na Ukrainie może w praktyce uniemożliwić współpracę naszych uczelni, ale dopóki to nie nastąpi, będzie ona realizowana dla wspólnych korzyści naukowych. p p y y y Przedmiotem konferencji, która odbyła się w dniach 19–20 marca 2015 r. w Moskwie, stał się szeroko rozumiany problem zarządzania do- kumentacją w aspekcie współczesnym i historycznym. Było to już dru- gie tego rodzaju sympozjum (pierwsze odbyło się w roku 2013). Tak jak poprzednie, miało też swojego patrona, którym była profesor Tatiana Wiaczesławowna Kuzniecowa, zmarła w roku 2012 twórczyni Katedry Dokumentoznawstwa RGGU. Katedra ta kierowana jest obecnie przez dynamiczną doc. Tatianę Aleksandrownę Bykową, która stała się głów- nym organizatorem tego przedsięwzięcia. Jej partnerem ze strony Za- kładu Archiwistyki UMCS był dr Artur Górak. Udział naszego zakładu polegał na współtworzeniu programu spotkania, wygłoszeniu referatów w części plenarnej jak i w sekcjach, aktywnym uczestnictwie w dyskusjach odbywających się w obradach sekcyjnych, a także złożeniu tekstów refe- 403 Sprawozdania ratów do przygotowywanego tomu pokonferencyjnego, również przez pracowników, którzy nie mogli przyjechać do Moskwy. W konferencji uczestniczyło kilkudziesięciu specjalistów z terenu Fe- deracji Rosyjskiej, chociaż najwięcej referentów reprezentowało uczelnie moskiewskie oraz Wszechrosyjski Naukowo-Badawczy Instytut Doku- mentoznawstwa i Archiwistyki, będący drugim współorganizatorem tego sympozjum, a zarazem prowadzący badania nad dokumentacją archiwal- ną oraz metodami opracowania materiałów archiwalnych, w tym także standardami w zakresie informatyzacji zasobu archiwalnego. Instytut ści- śle współpracuje z RGGU, a niektórzy jego pracownicy prowadzą zajęcia dydaktyczne ze studentami tej uczelni. Zakład Archiwistyki UMCS współorganizatorem konfe- rencji w Moskwie Pierwszego dnia konferencji (19 marca) w obradach plenarnych wy- stąpiło dwóch pracowników Zakładu. Jego kierownik dr hab. prof. UMCS Krzysztof Skupieński wygłosił referat o dokumencie jako obiekcie badań kultury. Dokonał w nim charakterystyki nowego podejścia w badaniach dokumentów, polegającego na wykorzystywaniu w nich treści użytecz- nych dla badaczy kultury. Wcześniej dokumenty były traktowane jako wytwory o funkcjach prawnych, głównie służące do wprowadzania no- wego stanu prawnego lub jego poświadczania. Referent ten zwięźle scha- rakteryzował też najważniejsze prace historyków zachodnich oraz pol- skich, reprezentujące ten kierunek. W trakcie obrad plenarnych wystąpił też inny pracownik Zakładu dr Piotr Dymmel, kierujący na co dzień lubelskim archiwum państwo- wym. Przedstawił on rosyjskim słuchaczom najważniejsze elementy wprowadzanego do polskich archiwów systemu elektronicznego zarzą- dzania dokumentacją (EZD). Stanowi on swego rodzaju rewolucję kan- celaryjną, ponieważ umożliwia zastąpienie dokumentacji papierowej przez elektroniczną, co oczywiście przynosi znaczące korzyści w postaci szybkiego wyszukiwania potrzebnych pism, łatwiejszego do nich dostępu oraz możliwości kontrolowania toku załatwiania sprawy przez referen- tów. Zastosowanie tego systemu pociąga za sobą jednak coraz większe koszty związane z potrzebą regularnej modernizacji sprzętu i oprogramo- wania, a także instalowania coraz droższych serwerów. Następnego dnia (20 marca) odbywały się już wyłącznie obrady w sek- cjach. W pierwszej, poświęconej dziejom dokumentu oraz kancelarii, prof. dr hab. Janusz Łosowski dokonał charakterystyki dokumentacji polskich komisji porządkowych cywilno-wojskowych działających w schyłkowym okresie Rzeczypospolitej w latach 1790–1792. Posługiwały się one tak zwa- nymi aktami czynności, które referent uznał za system kancelaryjny róż- niący się od panującej wcześniej księgi wpisów jak i systemów akt spraw, stosowanych przez organy władzy administracyjnej państw zaborczych. 404 Sprawozdania We wcześniejszych pracach naukowych nie zajmowano się zarówno orga- nizacją pracy kancelaryjnej tych komisji, jak i nie traktowano akt czynno- ści jako odmiennego systemu kancelaryjnego. j g y yj g Akcenty lubelskie były najbardziej widoczne w sekcji czwartej, sku- piającej referaty poświęcone antropologii biurokracji i cieszącej się też naj- większym zainteresowaniem uczestników. Jej obrady prowadził dr Artur Górak wraz z dr Tatianą Kandaurową z RGGU. Przedstawił on również referat dotyczący kadry kierowniczej urzędów rosyjskiej administracji specjalnej w Królestwie Polskim, przygotowany wspólnie dr. Krzyszto- fem Latawcem z Zakładu Historii Krajów Europy Wschodniej Instytutu Historii UMCS. Zakład Archiwistyki UMCS współorganizatorem konfe- rencji w Moskwie W swoim wystąpieniu wykazał, że kierownicy tego dzia- łu administracji, czyli urzędów skarbowych, włościańskich, zajmujących się przeprowadzeniem uwłaszczenia chłopów, pocztowych, inspekcji fabrycznej, administracji celnej, kolejowej i leśnej w przeciwieństwie do organów administracji ogólnej (rządy gubernialne i zarządy powiatowe) byli lepiej wykształceni i posiadali wyższe kwalifikacje fachowe oraz re- prezentowali wyższy poziom moralny. Nie był to przypadek, lecz swo- ista prawidłowość, wynikająca z polityki personalnej prowadzonej przez władze rosyjskie. Zaborcom zależało na sprawnym funkcjonowaniu tego rodzaju administracji, dlatego musieli powierzać funkcje kierownicze kompetentnym urzędnikom. Inaczej było w urzędach administracji ogól- nej, w których o nominacji na stanowisko kierownicze decydowały przede wszystkim kwalifikacje polityczne, a nie fachowość. i Obradom konferencji przysłuchiwali się także studenci różnych kie- runków naszej uczelni z wydziałów Politologii oraz Humanistycznego. Referenci z UMCS mieli więc jedyną w swoim rodzaju okazję wystąpienia za granicą nie tylko przed koleżankami i kolegami z zagranicznych uczel- ni, ale także przed swoimi studentami. Pobyt studentów, zorganizowa- ny przez dr. A. Góraka, został sfinansowany ze środków Europejskiego Funduszu Społecznego i stanowił istotny składnik programu specjalno- ści Zarządzanie danymi w instytucjach i firmach. Część studentów (huma- niści) wyjazdem do Moskwy wieńczyła trwającą cztery semestry naukę w ramach tej specjalności. W trakcie pobytu w stolicy Rosji nasi studenci nie tylko poznawali miasto i jego zabytki, ale także zwiedzili Rosyjskie Państwowe Archiwum Akt Dawnych, posiadające najstarszy zasób akto- wy w Rosji. Złożyli też wizytę we wspomnianym już Wszechrosyjskim Instytucie Naukowo-Badawczym Dokumentoznawstwa i Archiwistyki, zapoznając się z jego działalnością. Ta udana pod każdym względem kon- ferencja stanowi zapowiedź owocnej współpracy UMCS i RGGU. J Ł ki Janusz Łosowski (Instytut Historii UMCS w Lublinie) Janusz Łosowski (Instytut Historii UMCS w Lublinie)
https://openalex.org/W3201339288	https://www.nature.com/articles/s41598-021-97947-4.pdf	English	null	Local photoreceptor cell death differences in the murine model of retinal detachment	Scientific reports	2,021	cc-by	10,149	Daniel E. Maidana1,2, Lucia Gonzalez‑Buendia1, Joan W. Miller1 & Demetrios G. Vavvas1 Daniel E. Maidana1,2, Lucia Gonzalez‑Buendia1, Joan W. Miller1 & Demetrios G. Vavvas1* To investigate local cell death differences in the attached and detached retina at different regions in a murine experimental retinal detachment model. Subretinal injection of sodium hyaluronate was performed in eight-week-old C57BL/6J mice. Retinal regions of interest were defined in relation to their distance from the peak of the retinal detachment, as follows: (1) attached central; (2) attached paracentral; (3) detached apex; and (4) detached base. At day 0, the outer nuclear layer cell count for the attached central, attached paracentral, detached apex, and detached base was 1247.60 ± 64.62, 1157.80 ± 163.33, 1264.00 ± 150.7, and 1013.80 ± 67.16 cells, respectively. There were significant differences between the detached base vs. attached central, and between detached base vs. detached apex at day 0. The detached apex region displayed a significant and progressive cell count reduction from day 0 to 14. In contrast, the detached base region did not show progressive retinal degeneration in this model. Moreover, only the detached apex region had a significant and progressive cell death rate compared to baseline. Immediate confounding changes with dramatic differences in cell death rates are present across regions of the detached retina. We speculate that mechanical and regional differences in the bullous detached retina can modify the rate of cell death in this model. The separation of the neurosensory retina from the retinal pigment epithelium (RPE), or retinal detachment (RD), decreases the survival and alters the function of photoreceptors1–3. As the subretinal space is filled with fluid, debris, and infiltrating cells, and the gap between the outer retina and the choriocapillaris is increased, there is a decline in the availability of nutrients needed by the photoreceptors. This shortage, in context with the metabolic demands and fragility of photoreceptors, culminates in rod and cone cell death, which can occur as early as 12 hours post-detachment4. It has been previously demonstrated in a feline retinal detachment model, that the oxygen profile in the detached retina shares some features with the attached retina. However, the presence of a subretinal space increases the latency of oxygen delivery to the photoreceptors from the choriocapillaris5. Despite ongoing cell death, photoreceptors subsequently adapt by modifying their oxygen consumption and metabolism, reducing the rate of cell death3,6. g To further understand photoreceptor cell death, and consequently develop new therapies, experimental animal models have been developed. www.nature.com/scientificreports www.nature.com/scientificreports 1Retina Service, Angiogenesis Lab, Massachusetts Eye and Ear Infirmary, Harvard Medical School, 243 Charles Street, Boston, MA 02114, USA. 2Department of Ophthalmology and Visual Sciences, University of Illinois at Chicago, Chicago, IL, USA. email: demetrios_vavvas@meei.harvard.edu Scientific Reports \| (2021) 11:18798 Materials and methods Animals. All animals used in experiments and breeding adhered to the statement of the Association for Research in Vision and Ophthalmology (ARVO). Animal protocols were reviewed and approved by the Animal Care Committee of the Massachusetts Eye and Ear Infirmary. All experiments were performed in accordance with institutional regulations. This study was carried out in compliance with the ARRIVE guidelines. C57BL/6J mice were purchased from The Jackson Laboratories (Bar Harbor, ME). Mice were maintained in a standard 12-hour light/dark cycle. Retinal detachment model. Eight-week-old male C57BL/6J mice were anesthetized with an intraperito- neal injection of a mixture of 2,2,2-tribromoethanol and 2-methyl-2-butanol at a dose of 125 mg/kg, as previ- ously described7. Briefly, a Hamilton injector (Hamilton Company, Reno, NV) with a 34-gauge needle was intro- duced through a nasal sclerotomy into the subretinal space. Sodium hyaluronate (3.5 µL) was gently injected to detach the neurosensory retina from the underlying RPE. The nasal sclerotomy was sealed with surgical glue. Eyes with haemorrhage, leakage, or cataract were excluded from further analysis. A topical ointment containing bacitracin was applied after the procedure7. Tissue processing and immunofluorescence. Mice were euthanized at the following timepoints: (1) immediately after detachment (baseline, day 0); (2) day 1; (3) day 7; (4) day 14. Eyes were enucleated immedi- ately after euthanasia and cryopreserved using O.C.T. (Optimal Cutting Temperature, Sakura Finetek, Torrance, CA) at − 20 °C. Cryosections of 10 µm thickness at 1000 µm distance from the first section of the specimen of each sample were cut, mounted, and dried at room temperature (Fig. 1). Slides were frozen at − 80 °C for further processing. Samples were fixed with paraformaldehyde 4% for 30 min. Following two subsequent washes with phosphate-buffered saline, slides were counterstained with 4′,6-diamidine-2′-phenylindole dihydrochloride (Catalogue # D1306, ThermoFisher Scientific, Waltham, MA) for 5 min. Slides were washed with phosphate- buffered saline and mounted with Fluoromount-G Mounting Medium (Catalogue # 00-4958-02, ThermoFisher Scientific, Waltham, MA). Slides were imaged as whole-eye tiles with Zeiss AXIO Imager M2 (Carl Zeiss Inc., Thornwood, NY) fluorescence microscope, and exported in TIFF format for further analysis. Local cell death quantitation. The ONL cell count was assessed in an automated manner as previously described by Byun et al.9 (Fig. 1B). The ONL and INL thickness was quantitated with the ThicknessTool auto- mated ImageJ plugin using a 1-pixel calliper interval (Fig. 1C)12. Daniel E. Maidana1,2, Lucia Gonzalez‑Buendia1, Joan W. Miller1 & Demetrios G. Vavvas1 Amongst many experimental animal models of retinal degeneration, the murine retinal detachment model has been widely used given its reproducibility and feasibility to evaluate dif- ferent cell death pathways, genetic manipulation, or neuroprotective agents4. Subretinal injection of sodium hyaluronate 1% is commonly used to detach the neurosensory retina from the RPE7. This viscoelastic material induces a retinal detachment of consistent height and duration, in comparison to subretinal saline injection which is prone to reabsorption by the RPE and subsequent RD shallowing or complete resolution7. The outcome of the viscoelastic mediated RD is almost exclusive cell death in the photoreceptor layer6. As photoreceptor cell death progresses, the ONL cell count and outer nuclear layer (ONL)/inner nuclear layer (INL) thickness ratio are reduced8,9. Nonetheless, even with this predictable experimental model, the pattern and rate of photorecep- tor cell death is neither uniform nor constant in the detached murine retina4,10,11. Despite this being a common knowledge among investigators invested in this field, to date, the regional cell death differences in the detached murine retina have not yet been addressed. y In this work, we investigated the local cell death in a murine experimental retinal detachment model. We analysed the attached and detached retina in different regions and compared the rate of photoreceptor cell death by ONL cell count, ONL/INL ratio, and cell death rate. We found significant regional differences in the detached retina, measured by these three outcome measures. Given this data, we developed three case studies to illustrate 1Retina Service, Angiogenesis Lab, Massachusetts Eye and Ear Infirmary, Harvard Medical School, 243 Charles Street, Boston, MA 02114, USA. 2Department of Ophthalmology and Visual Sciences, University of Illinois at Chicago, Chicago, IL, USA. email: demetrios_vavvas@meei.harvard.edu Scientific Reports \| (2021) 11:18798 \| https://doi.org/10.1038/s41598-021-97947-4 www.nature.com/scientificreports/ common technical pitfalls in cell death analysis in this model. We expect this work will unveil these potential drawbacks and be useful for researchers in this field to detect significant photoreceptor cell death. common technical pitfalls in cell death analysis in this model. We expect this work will unveil these potential drawbacks and be useful for researchers in this field to detect significant photoreceptor cell death. Materials and methods A retina thickness heatmap was developed as a custom script for the ImageJ platform to display qualitative retinal thickness changes across the detached and attached retina in whole-eye tiles. The relative heatmap color-coded and identified two regions in the detached retina with varying ONL thickness. Two central and paracentral regions in the attached retina were selected for comparative analysis. These four regions of interest with corresponding images from each tile with an ONL length of 400 µm were extracted by a masked observer (Fig. 1D). Retinal regions of interest were defined relative to their distance from the peak of the retinal detachment, as follows: (1) attached central; (2) attached para- central; (3) detached apex, thus contiguous to the highest point or peak of the RD; and (4) detached base, at a distance of 500 µm from the RD peak. The rate of photoreceptor cell death was estimated by a regression model and expressed as ONL cell count (cells), or ONL/INL ratio (units) change per day. The height of the detachment was measured with a straight calliper as the distance from the RPE to the ONL, perpendicular to each of these cell layers. Retinal detachment degeneration criteria. The retinal detachment model causes photoreceptor cell death, which results in ONL cell count reduction and subsequent ONL thinning. Given that the INL is not sub- ject to significant degeneration, the ONL/INL ratio is commonly used to normalize the ONL thickness, given the variations which may occur due to obliquity in tissue sectioning. Taking into account the retinal degeneration that occurs in RD, we proposed a priori the following criteria to determine cell death and degeneration in this model. . Absence of significant ONL cell count reduction in the detached retina regions compared to baseline. Ab f i ifi ONL/INL i d i i h d h d i i d b li Absence of significant ONL cell count reduction in the detached retina regions compared to baseline. Absence of significant ONL/INL ratio reduction in the detached retina regions compared to baseline. i 2. Absence of significant ONL/INL ratio reduction in the detached retina regions compared to baseline. ll d h d h d h h d b l gi g . Progressive ONL cell count reduction in the detached retina through timepoints compared to baseline. Progressive ONL/INL ratio reduction in the detached retina through timepoints compared to baseline i 3. Materials and methods Progressive ONL cell count reduction in the detached retina through timepoints compared to baseline. O / d h d h d h h d b l i 3. Progressive ONL cell count reduction in the detached retina through timepoints compared to baseline. g g p p . Progressive ONL/INL ratio reduction in the detached retina through timepoints compared to baseline. The ONL cell count and ONL/INL ratio will be assessed in absolute values (a) and normalized to the attached etina (b). Statistical analyses. Statistical analysis was performed with SAS software version 11.2.0 (SAS, Cary, NC). Normality was assessed with Shapiro–Wilk test. Statistical significance for differences between groups was deter- mined with one-way ANOVA with Tukey post-hoc correction for multiple comparisons, and T-test for two- group comparisons. Error variance in cell death rate modelling was determined by F-Test. Results are expressed as mean ± standard deviation (SD). A p value of < 0.050 was considered statistically significant. Scientific Reports \| (2021) 11:18798 \| https://doi.org/10.1038/s41598-021-97947-4 www.nature.com/scientificreports/ Results Retinal architecture changes immediately after retinal detachment induction. To evaluate the changes in retinal architecture in this model, we first analysed the baseline changes immediately after the induc- ti f ti l d t h t (b li d 0) F thi i d th ti ti i it ONL Heatmap RD Day 0 Detached (Apex) Detached (Base) Attached (Paracentral) Attached (Central) RD Peak Retinal Detachment Attached Detached ONL & INL Thickness ONL Cell Count A ONL Count Day 0 Cell Count 0 375 750 1,125 1,500 * Thickness Ratio Day 0 ONL/INL Ratio 1.0 1.3 1.6 1.8 2.1 Attached (Central) Attached (Paracentral) Detached (Apex) Detached (Base) B C D E F Figure 1. Retinal detachment changes immediately after retinal detachment (baseline, day 0). (A) Representative fundus photo of a murine retinal detachment (RD) and sectioning planes (dashed arrows). (B) Cryosection image of a 400 µm ONL segment for ONL Cell Count at baseline analysis. (C) Cryosection image of a 400 µm ONL segment for outer (ONL) and inner nuclear layer (INL) thickness measurement. (D) Representative cryosection whole-retina tile image of a retinal detachment relative ONL thickness heatmap. Regions of interest used for analysis (dotted boxes). (E) ONL cell count analysis. (F) ONL/INL thickness ratio analysis. Scale bar = 400 µm (D). p < 0.05 vs. attached central region (n = 4–7). Materials and methods Retinal detachment changes immediately after retinal detachment (baseline, day 0). (A) Representative fundus photo of a murine retinal detachment (RD) and sectioning planes (dashed arrows). (B) Cryosection image of a 400 µm ONL segment for ONL Cell Count at baseline analysis. (C) Cryosection image of a 400 µm ONL segment for outer (ONL) and inner nuclear layer (INL) thickness measurement. (D) Representative cryosection whole-retina tile image of a retinal detachment relative ONL thickness heatmap. Regions of interest used for analysis (dotted boxes). (E) ONL cell count analysis. (F) ONL/INL thickness ratio analysis. Scale bar = 400 µm (D). p < 0.05 vs. attached central region (n = 4–7). Materials and methods ONL Heatmap RD Day 0 Detached (Apex) Detached (Base) Attached (Paracentral) Attached (Central) RD Peak Retinal Detachment Attached Detached ONL & INL Thickness ONL Cell Count A B C D Retinal Detachment Attached Detached ONL & INL Thickness ONL Cell Count A B C ONL Heatmap RD Day 0 Detached (Apex) Detached (Base) Attached (Paracentral) Attached (Central) RD Peak D ONL Heatmap RD Day 0 Retinal Detachment A B ONL Count Day 0 Cell Count 0 375 750 1,125 1,500 * Thickness Ratio Day 0 ONL/INL Ratio 1.0 1.3 1.6 1.8 2.1 Attached (Central) Attached (Paracentral) Detached (Apex) Detached (Base) E F Figure 1. Retinal detachment changes immediately after retinal detachment (baseline, day 0). (A) Representative fundus photo of a murine retinal detachment (RD) and sectioning planes (dashed arrows). (B) Cryosection image of a 400 µm ONL segment for ONL Cell Count at baseline analysis. (C) Cryosection image of a 400 µm ONL segment for outer (ONL) and inner nuclear layer (INL) thickness measurement. (D) Representative cryosection whole-retina tile image of a retinal detachment relative ONL thickness heatmap. Regions of interest used for analysis (dotted boxes). (E) ONL cell count analysis. (F) ONL/INL thickness ratio analysis Scale bar=400 µm (D) p<0 05 vs attached central region (n=4–7) ONL Count Day 0 Cell Count 0 375 750 1,125 1,500 Thickness Ratio Day 0 ONL/INL Ratio 1.0 1.3 1.6 1.8 2.1 Attached (Central) Attached (Paracentral) Detached (Apex) Detached (Base) E F Thickness Ratio Day 0 ONL/INL Ratio 1.0 1.3 1.6 1.8 2.1 Attached (Central) Attached (Paracentral) Detached (Apex) Detached (Base) F ONL Count Day 0 Cell Count 0 375 750 1,125 1,500 * E E Figure 1. Retinal detachment changes immediately after retinal detachment (baseline, day 0). (A) Figure 1. Retinal detachment changes immediately after retinal detachment (baseline, day 0). (A) Representative fundus photo of a murine retinal detachment (RD) and sectioning planes (dashed arrows). (B) Cryosection image of a 400 µm ONL segment for ONL Cell Count at baseline analysis. (C) Cryosection image of a 400 µm ONL segment for outer (ONL) and inner nuclear layer (INL) thickness measurement. (D) Representative cryosection whole-retina tile image of a retinal detachment relative ONL thickness heatmap. Regions of interest used for analysis (dotted boxes). (E) ONL cell count analysis. (F) ONL/INL thickness ratio analysis. Scale bar = 400 µm (D). p < 0.05 vs. attached central region (n = 4–7). Figure 1. www.nature.com/scientificreports/ Moreover, given the significant reduction in the ONL cell count and ONL thickness in the detached base region at baseline, we hypothesize that this detached region may be subject to more mechanical stretching during detachment than the detached apex area, most likely given that the retinal detachment is induced in the peripheral retina close to the ora serrata. We speculate that the ONL/INL ratio failed to detect significant differences most likely due to a concurrent ONL and INL stretching. Progressive photoreceptor cell death in retinal detachment. Considering these differences in the detached area, we constructed a relative ONL thickness heatmap 14 days following retinal detachment to bet- ter evaluate the degree of retinal degeneration induced by this model. Interestingly, the relative heatmap sug- gested lower ONL thickness values in the detached apex in comparison to the detached base area (Fig. 2A). Moreover, we observed slight changes between the detached base area compared to both attached areas, even at this timepoint. To further confirm this observation, we analysed the longitudinal changes in both detached apex and detached base areas from day 0 to day 14 (Fig. 2). Of note, no significant changes were observed in the attached retina (central and paracentral) throughout these timepoints compared to baseline. For the detached apex region, the absolute ONL cell count at day 0, 1, 7, and 14 was 1264.00 ± 150.71, 984.40 ± 158.07, 785.80 ± 89.18, 709.20 ± 108.30 cells, respectively (Fig. 2B). There were significant differences between day 0 vs. day 1 (p = 0.0171), day 0 vs. day 7 (p = 0.0001), and day 1 vs. day 14 (p < 0.0001). The ONL/INL ratio in the detached apex region at day 0, 1, 7, and 14 was 1.98 ± 0.16, 1.36 ± 0.31, 1.05 ± 0.20, and 1.08 ± 0.23, respectively (Fig. 2C). There were significant differences between day 0 vs. day 1 (p = 0.0006), day 0 vs. day 7 (p < 0.0001), and day 0 vs. day 14 (p < 0.0001). These results indicate that the detached apex area displays progressive cell count reduction from day 0 to 14. Thus, this area fulfilled criteria 3 and 4, and therefore it is appropriate to determine progressive retinal degeneration in this model. www.nature.com/scientificreports/ p g g For the detached base area, the absolute ONL cell count at day 0, 1, 7, and 14 was 1013.80 ± 67.16, 1017.60 ± 163.62, 1001.00 ± 27.93, and 954.00 ± 122.38, respectively (Fig. 2B). There were no significant differ- ences between day 0 and any later timepoint in this detached region. The ONL/INL ratio in the detached base region at day 0, 1, 7, and 14 was 1.85 ± 0.15, 1.79 ± 0.23, 1.66 ± 0.15, 1.81 ± 0.24, respectively (Fig. 2C). There were no significant differences between day 0 and any later timepoint in this detached region. These results suggest that the detached base area did not pass criteria 3 and 4, therefore was unfit to determine progressive retinal degeneration in this retinal detachment model.f g Finally, given the aspect of the detached bullous retina, we hypothesized that the observed differences in ONL count and ONL/INL ratio between the detached apex and detached base were not entirely dependent on the height of the retinal detachment. At baseline, the distance from the RPE to the ONL, or height of retinal detach- ment, was 1126.75 ± 62.02 µm for the detached apex and 1171.03 ± 48.14 µm for the detached base. On day 14, the height of retinal detachment was 1120.77 ± 223.85 µm for the detached apex and 1121.77 ± 144.17 µm for the detached base. There were no statistically significant differences in the height of retinal detachment between the detached apex and detached based at baseline (p = 0.244) or day 14 (p = 0.993). Collectively, these results suggest that the observed local differences in photoreceptor cell death are not entirely due to the height of the retinal detachment, which may suggest different adaptive mechanisms in these regions. Moreover, there were no statistically significant differences in the height of retinal detachment from baseline compared to day 14 in the detached apex (p = 0.956) or detached base (p = 0.552). These results further demonstrate that the retinal detachment achieved with this model is stable and reproducible. Photoreceptor cell death rate in retinal detachment. Next, we calculated the rate of photoreceptor cell death per day in all regions by ONL cell count and ONL/INL ratio (Fig. 3A,B). www.nature.com/scientificreports/ of the detached retina (RD) to the attached central retina (AR). The normalized RD/AR ONL cell count at baseline was 1.01 ± 0.10 for the detached apex, and 0.81 ± 0.07 for the detached base area. There were significant differences between the detached base vs. attached central (p = 0.0020), detached base and attached paracentral (p = 0.0.0020); and detached base vs. detached apex (p = 0.0011). These results indicate that a significant absolute and normalized ONL cell count reduction can be observed in the detached base region immediately after detach- ment, before substantial cell death can take place. In addition, only the detached apex region matched criteria 1a and 1b, whereas detached base failed to do so. This reduction in cell count could be consequence of the retina being reshaped, in part due to the viscoelastic’s mechanical properties and its delivery to the subretinal space.h The absolute ONL/INL ratio for the central attached retina, paracentral attached retina, detached apex, and detached base at day 0, was 2.00 ± 0.17, 1.92 ± 0.23, 1.98 ± 0.15, and 1.84 ± 0.14, respectively (Fig. 1F). There were no significant differences between any of these regions. The normalized ONL/INL ratio was 0.99 ± 0.13 for the detached apex, and 0.93 ± 0.14 for the detached base area, with no significant differences between any of these regions and the attached retina. These results indicate the ONL/INL was not capable of detecting differences between these detached regions. Thus, both the detached apex and base areas matched criteria 2a and 2b.h gh Nonetheless, we performed a subgroup analysis of the ONL thickness at baseline. The absolute ONL thickness for the central attached retina, paracentral attached retina, detached apex, and detached base was 94.20 ± 7.08, 85.43 ± 11.78, 88.99 ± 7.42, and 79.96 ± 7.93 µm, respectively. There was a significant difference only between the detached base vs. attached central area (p = 0.0268). We calculated the normalized ONL thickness per tile using the attached central retina, as described above. The normalized ONL thickness was 0.94 ± 0.09 for the detached apex, and 0.85 ± 0.08 for the detached base region. Similarly, there was a significant difference only between the detached base vs. attached central area (p = 0.0056). Taken together, these results indicate that only detached apex region passed criteria 1 and 2. Results R i l Retinal architecture changes immediately after retinal detachment induction. To evaluate the changes in retinal architecture in this model, we first analysed the baseline changes immediately after the induc- tion of a retinal detachment (baseline or day 0). For this purpose, we imaged the entire retina using composite tiles. We then quantitated the ONL thickness in the entire attached and detached retina using an automated reti- nal thickness measurement plugin for ImageJ12. Subsequently, we constructed a retina thickness heatmap using the thickness calliper values. The relative heatmap clearly showed lower ONL thickness values in the detached retina in comparison to the attached retina immediately after detachment (Fig. 1D). Moreover, we observed sub- tle changes in the heatmap values between two regions in the detached retina, namely the apex and base region of the detached retina. These results suggest that the induction of retinal detachment can subtly modify the outer nuclear layer thickness measured at baseline, before the induction of substantial cell death. y We further assessed this observation by automated ONL cell counting and ONL/INL ratio quantitation. At day 0, the absolute ONL cell count for the central attached retina, paracentral attached retina, detached apex, and detached base was 1247.60 ± 64.62, 1157.80 ± 163.33, 1264.00 ± 150.7, and 1013.80 ± 67.16 cells, respectively (Fig. 1E). There were significant differences between the detached base vs. attached central area (p = 0.0335), and between the detached base vs. detached apex area (p = 0.0219). Moreover, we normalized the ONL cell counts https://doi.org/10.1038/s41598-021-97947-4 Scientific Reports \| (2021) 11:18798 \| www.nature.com/scientificreports/ cientificreports/ ONL/INL Thickness Ratio ONL/INL Ratio 0 0.6 1.2 1.8 2.4 Day 0 Day 1 Day 7 Day 14 ONL Cell Count Cell Count 0 375 750 1,125 1,500 Day 0 Day 1 Day 7 Day 14 Attached (Central) Attached (Paracentral) Detached (Apex) Detached (Base) D E * * * * * ONL Heatmap RD Day 14 A B Distance RPE ONL Distance (µm) 0 325 650 975 1,300 Detached Apex Detached Base NS Day 0 Distance (µm) 0 325 650 975 1,300 Detached Apex Detached Base NS Day 14 RPE C Figure 2. Progressive outer nuclear layer (ONL) degeneration following retinal detachment. (A) Representative cryosection whole-retina tile image of a retinal detachment relative ONL thickness heatmap. Regions of interest used for analysis (dotted boxes), and retinal detachment height callipers (dashed arrows). (B) Height of retinal detachment at baseline. (C) Height of retinal detachment at day 14. (D) ONL cell count analysis. (E) ONL/INL thickness ratio analysis. Scale bar = 400 µm (A). p < 0.05 vs. baseline day 0 between groups (n = 4–7). The cell death rate by ONL/INL ratio for the central attached retina paracentral attached retina detached apex ONL/INL Thickness Ratio ONL/INL Ratio 0 0.6 1.2 1.8 2.4 Day 0 Day 1 Day 7 Day 14 ONL Cell Count Cell Count 0 375 750 1,125 1,500 Day 0 Day 1 Day 7 Day 14 Attached (Central) Attached (Paracentral) Detached (Apex) Detached (Base) D E * * * * * ONL Heatmap RD Day 14 A B Distance RPE ONL Distance (µm) 0 325 650 975 1,300 Detached Apex Detached Base NS Day 0 Distance (µm) 0 325 650 975 1,300 Detached Apex Detached Base NS Day 14 RPE C Figure 2. Progressive outer nuclear layer (ONL) degeneration following retinal detachment. (A) Representativ cryosection whole-retina tile image of a retinal detachment relative ONL thickness heatmap. Regions of interest used for analysis (dotted boxes), and retinal detachment height callipers (dashed arrows). (B) Height of retinal detachment at baseline. (C) Height of retinal detachment at day 14. (D) ONL cell count analysis. (E) ONL/INL thickness ratio analysis. Scale bar = 400 µm (A). p < 0.05 vs. baseline day 0 between groups (n = 4–7). ONL Heatmap RD Day 14 A B Distance RPE ONL Distance (µm) 0 325 650 975 1,300 Detached Apex Detached Base NS Day 0 Distance (µm) 0 325 650 975 1,300 Detached Apex Detached Base NS Day 14 RPE C ONL Heatmap RD Day 14 A RPE B Distance RPE ONL Distance (µm) 0 325 650 975 1,300 Detached Apex Detached Base NS Day 0 ONL Heatmap RD Day 14 B C Day 0 Distance (µm) 0 325 650 975 1,300 Detached Apex Detached Base NS Day 14 C ONL Cell Count Cell Count 0 375 750 1,125 1,500 Day 0 Day 1 Day 7 Day 14 Attached (Central) Attached (Paracentral) Detached (Apex) Detached (Base) D * * D ONL/INL Thickness Ratio ONL/INL Ratio 0 0.6 1.2 1.8 2.4 Day 0 Day 1 Day 7 Day 14 E * * * E Figure 2. Progressive outer nuclear layer (ONL) degeneration following retinal detachment. (A) Representative cryosection whole-retina tile image of a retinal detachment relative ONL thickness heatmap. Regions of interest used for analysis (dotted boxes), and retinal detachment height callipers (dashed arrows). (B) Height of retinal detachment at baseline. (C) Height of retinal detachment at day 14. (D) ONL cell count analysis. (E) ONL/INL thickness ratio analysis. www.nature.com/scientificreports/ The cell death rate by ONL cell count for the central attached retina, paracentral attached retina, detached apex, and detached base region was − 2.34 (p = 0.4353), − 1.90 (p = 0.7482), − 33.40 (p < 0.0001), and − 4.35 (p = 0.3020), respectively (Fig. 3A). https://doi.org/10.1038/s41598-021-97947-4 Scientific Reports \| (2021) 11:18798 \| www.nature.com/scientificreports/ cientificreports/ ONL/INL Thickness Ratio ONL/INL Ratio 0 0.6 1.2 1.8 2.4 Day 0 Day 1 Day 7 Day 14 ONL Cell Count Cell Count 0 375 750 1,125 1,500 Day 0 Day 1 Day 7 Day 14 Attached (Central) Attached (Paracentral) Detached (Apex) Detached (Base) D E * * * * * * ONL Heatmap RD Day 14 A B Distance RPE ONL Distance (µm) 0 325 650 975 1,300 Detached Apex Detached Base NS Day 0 Distance (µm) 0 325 650 975 1,300 Detached Apex Detached Base NS Day 14 RPE C Figure 2. Progressive outer nuclear layer (ONL) degeneration following retinal detachment. (A) Representative cryosection whole-retina tile image of a retinal detachment relative ONL thickness heatmap. Regions of interest used for analysis (dotted boxes), and retinal detachment height callipers (dashed arrows). (B) Height of retinal detachment at baseline. (C) Height of retinal detachment at day 14. (D) ONL cell count analysis. (E) ONL/INL thickness ratio analysis. Scale bar = 400 µm (A). p < 0.05 vs. baseline day 0 between groups (n = 4–7). For this purpose, the investigator will compare the ONL cell count and ONL/INL ratio in the detached apex or base as a ratio of the attached retina per timepoint. We will use the attached central retina area for normalization purposes (Fig. 3C,D). p p g If the investigator uses the detached apex area, the normalized ONL cell count at day 0, 1, 7, and 14 will be 1.01 ± 0.10, 0.80 ± 0.17, 0.61 ± 0.07, and 0.61 ± 0.06, respectively (Fig. 3C). There will be significant differences between day 0 vs. day 7 (p = 0.0003), day 0 vs. day 14 (p = 0.0005). If the ONL/INL ratio is expressed as a func- tion of the attached retina, this normalized ONL/INL ratio at day 0, 1, 7, and 14 will be 0.99 ± 0.13, 0.65 ± 0.13, 0.50 ± 0.13, and 0.51 ± 0.13, respectively (Fig. 3D). There will be significant differences between day 0 vs. day 1 (p = 0.0008), day 0 vs. day 7 (p < 0.0001), day 0 vs. day 14 (p < 0.0001). p y y p y y p However, if the investigator uses the detached base area, the normalized ONL cell count at day 0, 1, 7, and 14 will be 0.81 ± 0.07, 0.79 ± 0.16, 0.78 ± 0.03, and 0.79 ± 0.10 respectively (Fig. 3C). There will be no significant differences between day 0 and any later timepoint in this detached region. The normalized ONL/INL ratio at day 0, 1, 7, and 14 will be 0.93 ± 0.14, 0.87 ± 0.15, 0.80 ± 0.14, and 0.86 ± 0.11, respectively (Fig. 3D). There will be no significant differences between day 0 and any later timepoint in this detached region.i gif y y p g In summary, if the investigator chooses the detached base region, it will fail to demonstrate significant true differences between the detached and attached retina. In essence, it will fail to reject of a false null hypothesis, thus falling into a type II error. Case 2: two‑group comparison. Another investigator in this case, will try to shortcut his experiment by com- paring only two timepoints. It will compare the detached retina at day 0 vs day 7 or 14, using a two-group mean statistical comparison test (Fig. 4A,B).i If the investigator uses the detached apex area (Fig. cientificreports/ ONL/INL Thickness Ratio ONL/INL Ratio 0 0.6 1.2 1.8 2.4 Day 0 Day 1 Day 7 Day 14 ONL Cell Count Cell Count 0 375 750 1,125 1,500 Day 0 Day 1 Day 7 Day 14 Attached (Central) Attached (Paracentral) Detached (Apex) Detached (Base) D E * * * * * ONL Heatmap RD Day 14 A B Distance RPE ONL Distance (µm) 0 325 650 975 1,300 Detached Apex Detached Base NS Day 0 Distance (µm) 0 325 650 975 1,300 Detached Apex Detached Base NS Day 14 RPE C Figure 2. Progressive outer nuclear layer (ONL) degeneration following retinal detachment. (A) Representative cryosection whole-retina tile image of a retinal detachment relative ONL thickness heatmap. Regions of interest used for analysis (dotted boxes), and retinal detachment height callipers (dashed arrows). (B) Height of retinal detachment at baseline. (C) Height of retinal detachment at day 14. (D) ONL cell count analysis. (E) ONL/INL thickness ratio analysis. Scale bar = 400 µm (A). p < 0.05 vs. baseline day 0 between groups (n = 4–7). Scale bar = 400 µm (A). p < 0.05 vs. baseline day 0 between groups (n = 4–7). The cell death rate by ONL/INL ratio for the central attached retina, paracentral attached retina, detached apex, and detached base region was 0.01 (p = 0.4701), 0.01 (p = 0.1491), − 0.05 (p = 0.0002), and − 0.01 (p = 0.6808), respectively (Fig. 3B). Collectively, these results suggest that only the detached apex region had a significant and progressive cell death rate compared to baseline, with an estimate of 33.40 photoreceptors dying per day and a 0.05 reduction of ONL/INL ratio per day. Case studies. In summary, the detached apex outperformed the detached base area in detecting expected and significant differences between the attached and detached retina by means of common outcome variables in Scientific Reports \| (2021) 11:18798 \| https://doi.org/10.1038/s41598-021-97947-4 www.nature.com/scientificreports/ Days 0 2 4 6 8 10 12 14 ONL/INL Ratio 0.5 1.0 1.5 2.0 2.5 3.0 AR-C AR-P RD-A RD-B Days 0 2 4 6 8 10 12 14 ONL Cell Count 400 600 800 1000 1200 1400 ONL Cell Death Rate Days 0 2 4 6 8 10 12 14 Normalized ONL/INL Thickness 0.4 0.5 0.6 0.7 0.8 0.9 1.0 AR-C AR-P DR-C DR-P Days 0 2 4 6 8 10 12 14 Normalized Cell Count 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1 ONL/INL Ratio Death Rate * * * * * * * Attached (Central) Attached (Paracentral) Detached (Apex) Detached (Base) A B C D Figure 3. Cell death rate and normalized cell death following retinal detachment. (A) ONL cell death rate. (B) ONL/INL thickness ratio cell death rate. (C) Case 1: normalized ONL cell count. (D) Case 1: normalized ONL/ INL thickness ratio. p < 0.05 (n = 4–7). cientificreports/ ONL/INL Thickness Ratio ONL/INL Ratio 0 0.6 1.2 1.8 2.4 Day 0 Day 1 Day 7 Day 14 ONL Cell Count Cell Count 0 375 750 1,125 1,500 Day 0 Day 1 Day 7 Day 14 Attached (Central) Attached (Paracentral) Detached (Apex) Detached (Base) D E * * * * * ONL Heatmap RD Day 14 A B Distance RPE ONL Distance (µm) 0 325 650 975 1,300 Detached Apex Detached Base NS Day 0 Distance (µm) 0 325 650 975 1,300 Detached Apex Detached Base NS Day 14 RPE C Figure 2. Progressive outer nuclear layer (ONL) degeneration following retinal detachment. (A) Representative cryosection whole-retina tile image of a retinal detachment relative ONL thickness heatmap. Regions of interest used for analysis (dotted boxes), and retinal detachment height callipers (dashed arrows). (B) Height of retinal detachment at baseline. (C) Height of retinal detachment at day 14. (D) ONL cell count analysis. (E) ONL/INL thickness ratio analysis. Scale bar = 400 µm (A). p < 0.05 vs. baseline day 0 between groups (n = 4–7). 0 2 4 6 8 10 12 14 ONL Cell Count 400 600 800 1000 1200 1400 ONL Cell Death Rate A 0 2 4 6 8 10 12 14 ONL/INL Ratio 0.5 1.0 1.5 2.0 2.5 3.0 AR-C AR-P RD-A RD-B ONL/INL Ratio Death Rate * Attached (Central) Attached (Paracentral) Detached (Apex) Detached (Base) B 3 B A B D Days 0 2 4 6 8 10 12 14 Days 0 2 4 6 8 10 12 14 Normalized ONL/INL Thickness 0.4 0.5 0.6 0.7 0.8 0.9 1.0 AR-C AR-P DR-C DR-P * * * D Days 0 2 4 6 8 10 12 14 Days 0 2 4 6 8 10 12 14 Normalized Cell Count 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1 * * C C Figure 3. Cell death rate and normalized cell death following retinal detachment. (A) ONL cell death rate. (B) ONL/INL thickness ratio cell death rate. (C) Case 1: normalized ONL cell count. (D) Case 1: normalized ONL/ INL thickness ratio. p < 0.05 (n = 4–7). retinal degeneration models such as ONL cell count and ONL/INL ratio. In addition, the detached base region appears to be subject to a more pronounced mechanical stretching during the induction of the retinal detach- ment. Moreover, this is further compromised by a lower rate in ONL cell count and ONL/INL ratio reduction. Given these significant findings, we propose three case studies to explore different methods and emulate several researchers’ approach in this model. We will assess the capacity of the chosen variables and group comparisons to detect true and significant differences in retinal detachment. retinal degeneration models such as ONL cell count and ONL/INL ratio. In addition, the detached base region appears to be subject to a more pronounced mechanical stretching during the induction of the retinal detach- ment. Moreover, this is further compromised by a lower rate in ONL cell count and ONL/INL ratio reduction. Given these significant findings, we propose three case studies to explore different methods and emulate several researchers’ approach in this model. We will assess the capacity of the chosen variables and group comparisons to detect true and significant differences in retinal detachment. Case 1: normalized ONL count. We will assume that the investigator, in this case will evaluate the detached retina at different timepoints compared to day 0. cientificreports/ ONL/INL Thickness Ratio ONL/INL Ratio 0 0.6 1.2 1.8 2.4 Day 0 Day 1 Day 7 Day 14 ONL Cell Count Cell Count 0 375 750 1,125 1,500 Day 0 Day 1 Day 7 Day 14 Attached (Central) Attached (Paracentral) Detached (Apex) Detached (Base) D E * * * * * ONL Heatmap RD Day 14 A B Distance RPE ONL Distance (µm) 0 325 650 975 1,300 Detached Apex Detached Base NS Day 0 Distance (µm) 0 325 650 975 1,300 Detached Apex Detached Base NS Day 14 RPE C Figure 2. Progressive outer nuclear layer (ONL) degeneration following retinal detachment. (A) Representative cryosection whole-retina tile image of a retinal detachment relative ONL thickness heatmap. Regions of interest used for analysis (dotted boxes), and retinal detachment height callipers (dashed arrows). (B) Height of retinal detachment at baseline. (C) Height of retinal detachment at day 14. (D) ONL cell count analysis. (E) ONL/INL thickness ratio analysis. Scale bar = 400 µm (A). p < 0.05 vs. baseline day 0 between groups (n = 4–7). A B A Retinal Detachment Apex Retinal Detachment Base 0 375 750 1,125 1,500 0 7 Days ONL Cell Count 0 375 750 1,125 1,500 0 14 Days * ONL Cell Count 0 375 750 1,125 1,500 0 375 750 1,125 1,500 0 7 Days 0 14 Days NS NS ONL/INL Thickness 0.5 0.8 1.2 1.5 1.8 I II I II ONL Cell Count 0 300 600 900 1,200 Retinal Detachment Day 7 C NS NS ONl Cell Count 0 300 600 900 1,200 ONL/INL Thickness 0.5 0.8 1.2 1.5 1.8 Retinal Detachment Day 14 I II I II D NS NS C D C Figure 4. Cases 2 and 3. Case 2: Two-group outer nuclear layer (ONL) cell count comparison between baseline and day 7 or 14 following retinal detachment. (A) Retinal detachment apex area. (B) Retinal detachment base area. Case 3: Alternate use of apex and base region in retinal detachment. Images from detached apex and base areas were pooled and randomly shuffled to two groups, I and II. (C) Comparison between groups I and II at baseline and day 7. (D) Comparison between groups I and II at baseline and day 14. p < 0.05 vs. baseline, NS not significant vs. baseline (n = 5–7). also fail to demonstrate significant differences between day 0 vs. day 7 (p < 0.2820), day 0 vs. day 14 (p < 0.9902). In summary, a two-tailed two-group comparison will also fail to demonstrate any significant differences in the detached base region. Case 3: alternate use of detached apex or base areas. Finally, the investigator in this case, will compare the effect of treatment compared to a control, on day 7 or 14. However, the researcher is not aware of the regional cell death differences in the detached retina between the detached apex and base areas; therefore, it will acquire images from the detached retina at these regions indistinctively. To simulate this selection, we pooled detached apex and detached base images from day 7 or 14 and randomly allocated them to two groups labelled I for the control group, and II for the treatment group (Fig. 4C,D). g g g On day 7, the ONL cell count for groups I and II was 867.83 ± 146.98 and 859.50 ± 151.73 cells, respectively (Fig. 4C). No significant differences were observed between group I and II (p = 0.9249, T-test). cientificreports/ ONL/INL Thickness Ratio ONL/INL Ratio 0 0.6 1.2 1.8 2.4 Day 0 Day 1 Day 7 Day 14 ONL Cell Count Cell Count 0 375 750 1,125 1,500 Day 0 Day 1 Day 7 Day 14 Attached (Central) Attached (Paracentral) Detached (Apex) Detached (Base) D E * * * * * ONL Heatmap RD Day 14 A B Distance RPE ONL Distance (µm) 0 325 650 975 1,300 Detached Apex Detached Base NS Day 0 Distance (µm) 0 325 650 975 1,300 Detached Apex Detached Base NS Day 14 RPE C Figure 2. Progressive outer nuclear layer (ONL) degeneration following retinal detachment. (A) Representative cryosection whole-retina tile image of a retinal detachment relative ONL thickness heatmap. Regions of interest used for analysis (dotted boxes), and retinal detachment height callipers (dashed arrows). (B) Height of retinal detachment at baseline. (C) Height of retinal detachment at day 14. (D) ONL cell count analysis. (E) ONL/INL thickness ratio analysis. Scale bar = 400 µm (A). p < 0.05 vs. baseline day 0 between groups (n = 4–7). 4A), the ONL cell count will demonstrate significant differences between day 0 vs. day 7 (p < 0.0001), day 0 vs. day 14 (p < 0.0001). The ONL/INL ratio will demon- strate similar significant differences between day 0 vs. day 7 (p < 0.0001), day 0 vs. day 14 (p < 0.0001). If the investigator uses the detached base area (Fig. 4B), the ONL cell count will fail to demonstrate significant dif- ferences between day 0 vs. day 7 (p < 0.8543), day 0 vs. day 14 (p < 0.3962). Moreover, the ONL/INL ratio will https://doi.org/10.1038/s41598-021-97947-4 Scientific Reports \| (2021) 11:18798 \| www.nature.com/scientificreports/ ONL/INL Thickness 0.5 0.8 1.2 1.5 1.8 ONl Cell Count 0 300 600 900 1,200 ONL/INL Thickness 0.5 0.8 1.2 1.5 1.8 0 375 750 1,125 1,500 0 7 Days ONL Cell Count 0 375 750 1,125 1,500 0 14 Days Retinal Detachment Apex ONL Cell Count 0 375 750 1,125 1,500 0 375 750 1,125 1,500 0 7 Days 0 14 Days I II I II ONL Cell Count 0 300 600 900 1,200 Retinal Detachment Base Retinal Detachment Day 7 Retinal Detachment Day 14 I II I II A B C D * NS NS NS NS NS NS Figure 4. Cases 2 and 3. Case 2: Two-group outer nuclear layer (ONL) cell count comparison between baseline and day 7 or 14 following retinal detachment. (A) Retinal detachment apex area. (B) Retinal detachment base area. Case 3: Alternate use of apex and base region in retinal detachment. Images from detached apex and base areas were pooled and randomly shuffled to two groups, I and II. (C) Comparison between groups I and II at baseline and day 7. (D) Comparison between groups I and II at baseline and day 14. p < 0.05 vs. baseline, NS not significant vs. baseline (n = 5–7). cientificreports/ ONL/INL Thickness Ratio ONL/INL Ratio 0 0.6 1.2 1.8 2.4 Day 0 Day 1 Day 7 Day 14 ONL Cell Count Cell Count 0 375 750 1,125 1,500 Day 0 Day 1 Day 7 Day 14 Attached (Central) Attached (Paracentral) Detached (Apex) Detached (Base) D E * * * * * ONL Heatmap RD Day 14 A B Distance RPE ONL Distance (µm) 0 325 650 975 1,300 Detached Apex Detached Base NS Day 0 Distance (µm) 0 325 650 975 1,300 Detached Apex Detached Base NS Day 14 RPE C Figure 2. Progressive outer nuclear layer (ONL) degeneration following retinal detachment. (A) Representative cryosection whole-retina tile image of a retinal detachment relative ONL thickness heatmap. Regions of interest used for analysis (dotted boxes), and retinal detachment height callipers (dashed arrows). (B) Height of retinal detachment at baseline. (C) Height of retinal detachment at day 14. (D) ONL cell count analysis. (E) ONL/INL thickness ratio analysis. Scale bar = 400 µm (A). *p < 0.05 vs. baseline day 0 between groups (n = 4–7). The ONL/INL ratio for the group I was 1.32 ± 0.43 and 1.38 ± 0.30 for group II. Similarly, no significant differences were observed between groups I and II (p = 0.7906, T-test). On day 14, the ONL cell count for groups I and II was 864.60 ± 175.50 and 798.60 ± 175.03 cells, respectively (Fig. 4D). No significant differences were observed between groups I and II (p = 0.5680, T-test). The ONL/INL ratio for the group I was 1.32 ± 0.49 and 1.57 ± 0.38 for group IΙ. Similarly, no significant differences were observed between groups I and II (p = 0.3593).h gif g p p These results suggest that lack of consistency in RD region selection for further analysis can cancel previ- ously observed significant differences between these two regions on day 7 in the ONL cell count (p = 0.0033) and ONL/INL ratio (p < 0.0001). Similarly, differences at day 14 in the ONL cell count (p = 0.0103) and ONL/INL ratio (p = 0.0003) would also be masqueraded. In summary, if the investigator neglects the regional cell death differences in this model, it will fail to reject a false null hypothesis. www.nature.com/scientificreports/ have previously demonstrated, in a cone-dominant ground squirrel experimental retinal detachment, a similar photoreceptor cell death percentage across three different detached regions15. In the ground squirrel, the overall photoreceptor population is composed of a majority of cones (86%) and a few remaining rods (14%)15,16. In our work, we used the C57BL/6J mice, which has a ratio of 34 rods to 1 cone at an eccentricity of 20° for the optic nerve, similar to 30 rods to 1 cone in humans17. We believe these similarities in the rod/cone ratio can further support the applicability of this experimental model.h The height of retinal detachment has been posed as a critical variable in cell death induction in this injury. Increased distance of photoreceptors to the RPE and choriocapillaris, and thus less availability of nutrients and potentially oxygen, is the most plausible mechanism to trigger cell death pathways. As the retina is detached, the presence of a subretinal space increases the latency of oxygen delivery to the photoreceptors from the choriocapillaris5. However, as the choriocapillaris flows continue to be uninterrupted, we speculate that diffusion into the subretinal space should be maintained. Therefore, the initial oxygen partial pressure should eventually reach an equilibrium with the plasma oxygen partial pressure. However, the time to equilibrium is critical and probably not prompt enough to provide adequate metabolic support to photoreceptors initially. Furthermore, oxygen consumption by photoreceptors maybe altered and infiltrating subretinal immune cells may also consume part of the oxygen originally destined for photoreceptors. Still, given the fact that cell death kinetics slow down over time10 and photoreceptor cell loss is observed to reach a plateau, we hypothesize that a combination of moderate O2 and nutrient supply and other local factors can contribute to rescue the retina in this injury model. Such a premise is insufficient to address the differences between different regions in the retina. It remains elusive why photoreceptors in the detached base display a different cell death as compared to the detached apex area. We can speculate about the role of mechanical factors for these disparities. However, there may be other regional factors related to oxygen consumption and metabolic adaptation on the retina, which may be further affected by the local densities of rods/cones or glial cells, or even unknown factors.h g There are several limitations to this study. First, we used axial cryosections to evaluate the morphology of the attached retina. www.nature.com/scientificreports/ towards the central retina, it is expected that a certain degree of dragging and stretching of this initial peripheral detached region will occur to accommodate this added subretinal volume (~ 3 to 4 µL). In addition, once the detached retina is in apposition with the large murine lens, this latter structure may act as a partially opposing vector to the bullous detachment vector. Hence, part of the detachment vector can be neutralized by this lens vector, at the expense of compacting the retinal tissues. However, the remainder unopposed detachment vector contributes to stretching, which is more evident in the detached base than detached apex, probably given the more central location of the latter region. Finally, we speculate that the lesser amount of stretching at the detached apex area and apex itself could be due to an already attenuated RD force vector in the central detached retina given the injected volume, and probably a different magnitude and direction of the lens vector given the smaller radius of contact between these two structures at the apex.f towards the central retina, it is expected that a certain degree of dragging and stretching of this initial peripheral detached region will occur to accommodate this added subretinal volume (~ 3 to 4 µL). In addition, once the detached retina is in apposition with the large murine lens, this latter structure may act as a partially opposing vector to the bullous detachment vector. Hence, part of the detachment vector can be neutralized by this lens vector, at the expense of compacting the retinal tissues. However, the remainder unopposed detachment vector contributes to stretching, which is more evident in the detached base than detached apex, probably given the more central location of the latter region. Finally, we speculate that the lesser amount of stretching at the detached apex area and apex itself could be due to an already attenuated RD force vector in the central detached retina given the injected volume, and probably a different magnitude and direction of the lens vector given the smaller radius of contact between these two structures at the apex.f p Given the different cell death kinetics of cones and rods in retinal degeneration14, their response to retinal detachment can vary, in context with the various rod/cone ratio across species. Interestingly, Linberg et al. www.nature.com/scientificreports/ We believe it is necessary to have a detached and attached region in the same slide, to obtain a normalized ratio if needed, as described above. However, investigators have various ways of processing the enucleated eyes. While some perform sagittal or coronal cryosections, axial cryosections allow us to evaluate the true height of the detachment, otherwise not feasible by other methods, as these former two sectioning strategies do not necessarily have the attached retina in the same section. Second, we use a cropped section of 400 µm to assess cell death. Though a larger area may seem more appropriate, regional differences between detached apex and detached base areas may mask subtle photoreceptor cell count reduction by merging these two regions, which present different cell death rate. Finally, regarding the area between the retinal detachment apex and the attached retina, we refrained from using this region due to the following concerns: (1) the area or region of interest in this initial portion of the detached retina was often irregular and did not always measure at least 400 µm, and thus did have smaller area values which could induce another source of bias; (2) investigators are prone to analyse photoreceptor survival and outcomes in the detached retina, usually in contact with the lens; (3) a critical factor in the differences between the detached apex and detached base is that both areas are in contact with the lens, and most importantly equidistant and parallel to the RPE, which argues towards comparable oxygen and nutri- ent delivery to these areas from the choriocapillaris. The area between the attached retina and detached apex is perpendicular to the RPE, and may receive different oxygen and nutrient delivery across its area.t p p yf yg y In summary, baseline changes can be observed in the retina immediately after detachment. Moreover, cell death rates differ across regions in the detached retina, most likely given a combination of mechanical and meta- bolic adaptive changes in the photoreceptor layer. We encourage the use of axial cryosections with a detached and attached retina in the same section to normalize their respective ONL cell count and ONL/INL ratio. In addition, cell death analysis performed in the detached apex region can detect true retinal degeneration. We believe this systematic approach to evaluating photoreceptor cell death can help reduce biases and advance knowledge in photoreceptor cell death in retinal disease models. Discussion In this study, we analysed the morphological changes in the retina after detachment. We evaluated the ONL cell count and ONL/INL ratio in the entire retina at baseline and observed significant morphological changes between RD regions immediately after detachment. Moreover, we identified two regions in the detached retina subject to different cell death rates. We speculate that mechanical and regional differences in the bullous detached retina can modify the rate of cell death in this model.h y The murine retinal detachment model has been widely established to study cell death mechanisms in the retina6,7,13. As sodium hyaluronate is injected into the subretinal space close to the ora serrata, it detaches the ret- ina from the periphery towards the equator and posterior pole. As the bullous RD force vector moves posteriorly Scientific Reports \| (2021) 11:18798 \| https://doi.org/10.1038/s41598-021-97947-4 www.nature.com/scientificreports/ References The topography of rod and cone photoreceptors in the retina of the ground squirrel. Vis. Neurosci. 15, 685–691 (1998). q , ( ) 17. Volland, S., Esteve-Rudd, J., Hoo, J., Yee, C. & Williams, D. S. A comparison of some organizational characteristics of the mouse central retina and the human macula. PLoS ONE 10, 1–13 (2015). q 7. Volland, S., Esteve-Rudd, J., Hoo, J., Yee, C. & Williams, D. S. A comparison of some organizational characteristics of the mouse central retina and the human macula. PLoS ONE 10, 1–13 (2015). Competing interests h p g The authors declare no competing interests. Literature searchh The biomedical literature from MEDLINE was searched through PubMed on March 20, 2020. The following terms were used: retinal detachment, photoreceptor, cell death, mouse, and regional difference. Only results in the English language were selected for further analysis. Received: 2 April 2021; Accepted: 5 August 2021 https://doi.org/10.1038/s41598-021-97947-4 Scientific Reports \| (2021) 11:18798 \| www.nature.com/scientificreports/ Funding Th k This work was supported by Bayer Healthcare Global Ophthalmology Awards Program (DEM); the Yeatts Family Foundation (DGV, JWM); Loefflers family foundation (JWM, DGV); a Macula Society Research Grant award (DGV); a Physician Scientist Award (DGV) and an Unrestricted Grant (JWM) from the Research to Prevent Blindness Foundation; NEI Grant R21EY023079-01A1 (DGV); and NEI Grant EY014104 (MEEI Core Grant). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Author contributions D.E.M. and D.G.V. designed research. D.E.M. performed the retinal detachment model, developed the Thickness Heatmap script, automated measurements, statistical analysis, and drafted the manuscript. L.G.B. performed the retinal detachment model, processed eyes, and imaged sections. J.W.M. and D.G.V. made substantial intellectual contributions to the research design and manuscript. References Heat shock protein 70 (HSP70) is critical for the photoreceptor stress response after retinal detachment via modulating anti-apoptotic Akt kinase. Am. J. Pathol. 178, 1080–1091 (2011).hi p g 1. Kayama, M. et al. Heat shock protein 70 (HSP70) is critical for the photoreceptor stress response after retinal detachment via modulating anti-apoptotic Akt kinase. Am. J. Pathol. 178, 1080–1091 (2011).hi g p p ( ) 2. Maidana, D. E. et al. ThicknessTool: automated ImageJ retinal layer thickness and profile in digital images. Sci. Rep. 10, (2020). 12. Maidana, D. E. et al. ThicknessTool: automated ImageJ retinal layer thickness and profile in digital images. Sci. Rep. 10, (2020). 13. Anderson, D. H., Guerin, C. J., Erickson, P. A., Stern, W. H. & Fisher, S. K. Morphological recovery in the reattached retina. Investig. Ophthalmol. Vis. Sci. 27, 168–183 (1986). 12. Maidana, D. E. et al. ThicknessTool: automated ImageJ retinal layer thickness and profile in digital images. Sci. Rep. 10, (2020). 13. Anderson, D. H., Guerin, C. J., Erickson, P. A., Stern, W. H. & Fisher, S. K. Morphological recovery in the reattached retina. Investig. Ophthalmol. Vis. Sci. 27, 168–183 (1986). h g y pi g g p 3. Anderson, D. H., Guerin, C. J., Erickson, P. A., Stern, W. H. & Fisher, S. K. Morphological recovery in the reattached retina. Investig Ophthalmol. Vis. Sci. 27, 168–183 (1986). p ( ) 14. Murakami, Y. et al. Receptor interacting protein kinase mediates necrotic cone but not rod cell death in a mouse model of inherited degeneration. Proc. Natl. Acad. Sci. USA 109, 14598–14603 (2012). p 4. Murakami, Y. et al. Receptor interacting protein kinase mediates necrotic cone but not rod cell death in a mouse model of inherited degeneration. Proc. Natl. Acad. Sci. USA 109, 14598–14603 (2012). g 5. Linberg, K. A., Sakai, T., Lewis, G. P. & Fisher, S. K. Experimental retinal detachment in the cone-dominant ground squirrel retina Morphology and basic immunocytochemistry. Vis. Neurosci. 19, 603–619 (2002).h g 15. Linberg, K. A., Sakai, T., Lewis, G. P. & Fisher, S. K. Experimental retinal detachment in the co Morphology and basic immunocytochemistry. Vis. Neurosci. 19, 603–619 (2002).h p gy y y 6. Kryger, Z., Galli-Resta, L., Jacobs, G. H. & Reese, B. E. The topography of rod and cone photoreceptors in the retina of the ground squirrel. Vis. Neurosci. 15, 685–691 (1998). 16. Kryger, Z., Galli-Resta, L., Jacobs, G. H. & Reese, B. E. References References 1. Fisher, S. K., Lewis, G. P., Linberg, K. A. & Verardo, M. R. Cellular remodeling in mammalian retina: Results from studies of experimental retinal detachment. Prog. Retin. Eye Res. 24, 395–431 (2005). 1. Fisher, S. K., Lewis, G. P., Linberg, K. A. & Verardo, M. R. Cellular remodeling in mammalian retina: Results from studies of experimental retinal detachment. Prog. Retin. Eye Res. 24, 395–431 (2005). 2. Murakami, Y. et al. Photoreceptor cell death and rescue in retinal detachment and degenerations. Prog. Retin. Eye Res. 37, 114–140 (2013). ( ) 3. Stone, J. et al. Mechanisms of photoreceptor death and survival in mammalian retina. Prog. Retin. Eye Res. 18, 689–735 (1999). p p g y 4. Trichonas, G. et al. Receptor interacting protein kinases mediate retinal detachment-induced photoreceptor necrosis and com- pensate for inhibition of apoptosis. Proc. Natl. Acad. Sci. USA 107, 21695–21700 (2010). p p p ( ) 5. Wang, S. & Linsenmeier, R. A. Hyperoxia improves oxygen consumption in the detached feline retina. Investig. Ophthalmol. Vis Sci. 48, 1335–1341 (2007). 6. Cook, B., Lewis, G. P., Fisher, S. K. & Adler, R. Apoptotic photoreceptor degeneration in experimental retinal detachment. Invest Ophthalmol. Vis. Sci. 36, 990–996 (1995). p ( ) 7. Matsumoto, H., Miller, J. W. & Vavvas, D. G. Retinal detachment model in rodents by subretinal injection of sodium hyaluronate. J. Vis. Exp. https://doi.org/10.3791/50660 (2013).t p 7. Matsumoto, H., Miller, J. W. & Vavvas, D. G. Retinal detachment model in rodents by subretinal injection of sodium hyaluronate J. Vis. Exp. https://doi.org/10.3791/50660 (2013).t p p g 8. Byun, J. et al. Quantifying structural distortions in retinal tissue before and after injury. In Workshop on Multiscale Biologica Imaging, Data Mining & Informatics, Santa Barbara, CA, USA 10–11 (2006). g g g f 9. Byun, J. et al. Automated tool for the detection of cell nuclei in digital microscopic images: Application to retinal images. Mol. Vis. 12, 949–960 (2006). l d ff h ll d h ft l d h h h l l g g g f 9. Byun, J. et al. Automated tool for the detection of cell nuclei in digital microscopic images: Application to retinal images. Mol. Vis. 12, 949–960 (2006). 0. Matsumoto, H. et al. Strain difference in photoreceptor cell death after retinal detachment in mice. Invest. Ophthalmol. Vis. Sci https://doi.org/10.1167/iovs.14-14238 (2014).t https://doi.org/10.1167/iovs.14-14238 (2014). 11. Kayama, M. et al. Additional information d d f Correspondence and requests for materials should be addressed to D.G.V. Reprints and permissions information is available at www.nature.com/reprints. Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. 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https://openalex.org/W4297493090	https://www.journals.vu.lt/teise/article/download/29446/28483	English	null	Criminological Activity of Forensic Science Institutions: the Concept of Implementation in Ukraine	Teisė	2,022	cc-by	9,159	Hanna Spitsyna https://orcid.org/0000-0002-6677-6915 Doctor of Law, Professor manager at the Law Department of National Aerospace University – «Kharkiv Aviation Institute» NAU «KhAI», Chkalova Street, 17, Kharkiv 61070, Ukraine E-mail: spitsyna_hanna@ukr.net https://orcid.org/0000-0002-6677-6915 Doctor of Law, Professor manager at the Law Department of National Aerospace University – «Kharkiv Aviation Institute» NAU «KhAI», Chkalova Street, 17, Kharkiv 61070, Ukraine E-mail: spitsyna_hanna@ukr.net https://orcid.org/0000-0002-6677-6915 Doctor of Law, Professor manager at the Law Department of National Aerospace University – «Kharkiv Aviation Institute» NAU «KhAI», Chkalova Street, 17, Kharkiv 61070, Ukraine E-mail: spitsyna_hanna@ukr.net The article analyzes the conceptual principles, opinions and ideas for understanding the essence of forensic science private theory, namely: forensic prevention concept is considered. The empirical prerequisites for the emergence and develop- ment of forensic research have been investigated: accumulation of empirical facts in the theory of forensic science and other sciences, manifestation of integration processes. Theoretical and applied foundations of expert prevention private theory are formulated. Keywords: Prevention, criminalistics, forensic science, forensic prevention, forensic science general theory, forensic prevention private theory, specific expertise, forensic institutions, expert. Teisė ISSN 1392-1274 eISSN 2424-605 2022, Vol. 124, pp. 67–80 DOI: https://doi.org/10.15388/Teise.2022.124. Contents lists available at Vilnius University Press Teisė ISSN 1392-1274 eISSN 2424-605 2022, Vol. 124, pp. 67–80 DOI: https://doi.org/10.15388/Teise.2022.124. Contents lists available at Vilnius University Press Contents lists available at Vilnius University Press ISSN 1392-1274 eISSN 2424-6050 DOI: https://doi.org/10.15388/Teise.2022.124.5 Criminological Activity of Forensic Science Institutions: the Concept of Implementation in Ukraine Nataliia Filipenko https://orcid.org/0000-0001-9469-3650 Doctor of Law, Associate Professor Senior researcher at Vilnius University, Faculty of Law 306 room (Dean’s Office) Saulėtekis av. 9 – I block, LT-10222 Vilnius, Lithuania Phone: (+370 5) 236 61 85 E-mail: tf@tf.vu.lt Professor at the Law Department of National Aerospace University – «Kharkiv Aviation Institute» NAU «KhAI», Chkalova Street, 17, Kharkiv 61070, Ukraine E-mail: filipenko_natalia@ukr.net Nataliia Filipenko https://orcid.org/0000-0001-9469-3650 Doctor of Law, Associate Professor Senior researcher at Vilnius University, Faculty of Law 306 room (Dean’s Office) Saulėtekis av. 9 – I block, LT-10222 Vilnius, Lithuania Phone: (+370 5) 236 61 85 E-mail: tf@tf.vu.lt Professor at the Law Department of National Aerospace University – «Kharkiv Aviation Institute» NAU «KhAI», Chkalova Street, 17, Kharkiv 61070, Ukraine E-mail: filipenko_natalia@ukr.net Nataliia Filipenko Received: 24/03/2022. Accepted: 22/06/2022 Copyright © 2022 Nataliia Filipenko, Hanna Spitsyna. Published by Vilnius University Press 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: 24/03/2022. Accepted: 22/06/2022 Introduction The conceptual and methodological model of research of criminological activity of forensic science institutions should be based on use of modern methods of scientific analysis based on the integrated use of a set of general and special approaches to scientific cognition management and implementation. At the same time, to understand the essence of such a complex phenomenon as forensic prevention, current state clarification, analysis and forecasting of trends and prospects for its development, it is important to study the history of the emergence and development of the scientific category. Effect- iveness of the crime investigation process largely depends on the results of forensic researches. The current period of development of legal science in Ukraine is associated with the need to solve a number of problems, among which a significant place is occupied by issues of study and creative analysis of scientific developments, ways to use scientific and technical achievements, identifying promising areas of research and more. Consideration of these issues is impossible without a deep, unbiased study of the history of science in general and its separate fields. This is especially true of applied sciences, the results of scientific researches that can be relatively quickly implemented in specific practical activities. The general theoretical and applied sciences include criminalistics and forensic science that are designed to provide their recommendations for the practice of combating crime.i Scientific achievements not only enrich the theoretical achievements of science, but also determine the further path of development of practice, provide scientific principles for optimizing such activities. In law enforcement and law enforcement fields, theoretical understanding of ways to improve practice is particularly important because the possibilities for experimentation, as a form of theoretical forecasting verification, are very limited. This is especially true of preventive activity that is a general term and covers all types of impact on crime. By general prevention we mean one of the areas of social management which is to prevent and stop specific crimes and crime itself as a social phenomenon. In other words, crime as an integral part of development and functioning of society, develops, professes and implements its own interests, generates the latest properties that come into conflict with the values protected by law. There can be no compromises in this confrontation, because in the event of a loss, the state and society self-destruct. Kriminalistikos mokslo institucijų kriminologinė veikla: įgyvendinimo Ukrainoje koncepcija Šiame straipsnyje analizuojami konceptualūs principai, nuomonės ir idėjos, padedančios suprasti kriminalistikos mokslo privačiosios teorijos esmę, būtent: nagrinėjama kriminalistinės prevencijos idėja. Ištirtos kriminalistinių tyrimų atsiradimo ir plėtros empirinės prielaidos: empirinių faktų kriminalistikos ir kitų mokslų teorijoje kaupimas, integracinių procesų raiška. Suformuluoti teoriniai ir taikomieji ekspertinės prevencijos privačiosios teorijos pagrindai. Pagrindiniai žodžiai: prevencija, kriminalistika, kriminalistikos mokslas, kriminalistinė prevencija, bendroji kriminalistikos mokslo teorija, kriminalistinės prevencijos privačioji teorija, specialioji ekspertizė, kriminalistikos institucijos, ekspertai. 67 ISSN 1392-1274 eISSN 2424-6050 Teisė. 2022, t. 124 Main Content Presentation The nature of the knowledge of each science is determined solely by its subject matter as part of the objective reality that a particular science studies. The use of knowledge of other sciences does not change their topics, this knowledge is only adapted to solve the tasks assigned to this science. While developing theoretical foundations and corresponding recommendations addressed to expert practice, the theory of forensic science uses the so-called “specific expertise” and transforms them into legal knowledge, which optimizes the implementation of special knowledge in legal proceedings (Segai, 2001, р. 10). Criminology and forensic science indirectly including sectoral forensic knowledge and practical types of forensic science, serve expert practice indirectly, by developing general theoretical principles and origins of modern forensic methodology including standard methods based on basic tools and modern computer technology, should play the role of methodological guidelines in the construction of industry expert disciplines and their inherent expert methods, techniques and technologies.i Ranking and purpose of forensic examination, as a theoretical category is as follows: first, having a dual nature, it should take over the scientific service of all aspects of practical forensic activity, in the context of developing practical procedural recommendations for forensic experts in all types of proceedings (criminal, administrative, civil, etc.); secondly, in close cooperation with procedural sci- ence, forensic examination should actively improve the legal framework of state policy in the field of forensic justice; thirdly, it cannot be outside the development of scientific principles of organizational and managerial aspect of experts, which would ensure the most effective functioning of state forensic institutions as an important component of the judiciary and the judiciary ; fourth, forensic examina- tion, constantly evolving and changing, prepares a large array of modern specialists, both scientists and professionals. On the basis of criminalistics theories of identification and recognition scientific foundations that were laid by S. M. Potapov (Potapov, 1946); theories, methods and techniques of forensic identi- fication, diagnosis and situation were formed that differ significantly from investigative methods of identification, recognition, situational analysis of crime. The tactics of appointing an examination in terms of content do not coincide with the concept of tactics of conducting an examination. Introduction Professional legal sources have repeatedly expressed the opinion that the fight against crime is a special kind of interaction between two opposing parties of social life (Filipenko N. (2020) Criminological activity of forensic science institutions of Ukraine). As experts rightly noted in this regard, preventive activities cover three areas: As experts rightly noted in this regard, preventive activities cover three areas: 1) general organization of such activities: set of organizational (accounting, registration), man- agement (forecasting, planning, coordination, definition of strategy and tactics), preventive (implementation of programs and plans, implementation of preventive measures), control (study of practice, crime trends) actions of various bodies and institutions that interact with each other to achieve common results; 2) law enforcement activity consisting in the implementation by specially authorized state bodies of measures provided by law to prevent the development of criminal intent in the early stages of the crime, identify signs of crimes, identify those who committed them, bring these people to justice, restore violated rights, freedoms and the legitimate interests of the people and the compensation of damages from criminal acts; 3) crime prevention, which means the implementation of economic, political, ideological, educational, legal and other measures to combat crime, is the activity to identify and eliminate the causes of crime, certain types and groups of crimes, specific crimes to prevent the completion of crimes at different stages development of criminal behavior (Filipenko, Spitsyna, 2020, р. 410). 68 Nataliia Filipenko, Hanna Spitsyna. Criminological Activity of Forensic Science Institutions: the Concept of Implementation in Ukraine Despite a declining trend in crime in recent years, the level of latent crime remains quite high, new types of crime are constantly appearing, primarily related to the development of the Internet, the improvement of IT technologies, the globalization of crime, and so on. l Despite a declining trend in crime in recent years, the level of latent crime remains quite high, new types of crime are constantly appearing, primarily related to the development of the Internet, the improvement of IT technologies, the globalization of crime, and so on. Another major factor influencing the course and effectiveness of preventive activities is the problem of timely recording and initiation of criminal cases or misdemeanors based on available information. Introduction The vagueness and complexity of disguised crimes, impossibility of identifying the perpetrators, the shortcomings of the regulatory framework of law enforcement agencies, the imperfection of the in- teraction of forensic officers with investigators at the stage of criminal proceedings greatly affect the preventive activities, its efficiency and effectiveness in modern conditions. Main Content Presentation Quite a different meaning includes the use of specific expertise by investigators in the criminalistics methods of investigation of certain categories of crimes and methods of using specific expertise by an expert who professionally “serves” specific types of crimes (forensic doctor, auto expert, motor mechanic, and others). The professional knowledge of investigator and the expert specific expertise on the crim- inalistic characteristics of the crime and the methods of including this knowledge by everyone in their professional activities have both common and distinctive features. 69 ISSN 1392-1274 eISSN 2424-6050 Teisė. 2022, t. 124 Currently we see a tendency to gradually update forensic science as a theoretical category, its transformation into a more capacious than before, a kind of theoretical knowledge that serves not only the law but also the needs of management. It is about the active extension of forensic activity not only in the field of civil commercial or administrative proceedings but also in other areas of public activ transformation into a more capacious than before, a kind of theoretical knowledge that serves not only the law but also the needs of management. It is about the active extension of forensic activity not only in the field of civil, commercial or administrative proceedings but also in other areas of public activ- ity that require professional expertise to make important decisions in the field of law, management, economics (Segai, 2001, р. 12). An important condition for improving the effectiveness of preventive activities is to intensify the use of scientific and technological progress in the detection and investigation of crimes. Forensic sci- ence as a synthetic branch of knowledge very productively contributes to introduction into investigat- ive practice of modern advances in science and technology. However, the problems of interconnection and interaction of forensic science and investigative practice are among the most complex in forensic theory and practice. The solution of these problems largely depends on an adequate understanding of the dynamic processes within science itself associated with its development, changing priorities, updating the knowledge itself (Filipenko, Shynkarenko, Tsymbalistyi, 2021). The growing interest in science as a higher form of knowledge of objective reality by philosophers, sociologists, lawyers, social scientists is a characteristic feature of the current stage of development of scientific and technological progress. Among the issues consideration of which largely provides an adequate understanding of science, a significant place is occupied by the problem of its topic and object. Main Content Presentation The issue of the science topic includes a large number of nuances, a wide range of phenom- ena and circumstances that are constantly changing making it an “eternal” problem for each field of knowledge. Science goes through several stages in its development. The process of science evolution can be divided into several periods: a) genesis of a certain system of knowledge; b) knowledge differ- entiation; c) knowledge integration. Formation of a scientific discipline is impossible without defining the range of its interests. Further development of science objectively leads to its differentiation, the formation of new branches of knowledge that requires the specification of the subject of so-called “maternal” science and separation from its independent areas, clarification between them the topic of scientific cognition (Filipenko, Bublikov, Obolientseva-Krasyvska, 2021). Further development involves the integration of sciences. Given that the outlined stages are in- terdependent, do not have clear time limits, include both differential and integration processes, the relevance of the study of the subject of science with its development does not decrease but increases. Manifestations of these provisions have their own specifics in relation to specific areas of knowledge and especially in relation to young applied sciences including forensic science. The origins of this rather complex problem lie in the natural difference between the object of science and its topic. This is an interesting theoretical collision because many legal sciences study the same events or phenomena that may coincide or be similar in content. It is for the purpose of a clear delineation of sciences in the context of the coincidence of the studied phenomena that the theoretical concepts topic of science and object of science were introduced into scientific circulation. The topic is a part, a side of this or that concrete aspect of object investigated by the given science; this is the range of the most important studying issues. If the object is usually common to a number of sciences, the subject of one science cannot coincide with the topic of another. The object of science is understood as a holistic system with a certain structural organization with different levels of internal and external relationships and interdependencies having not only a certain stability but also its own dynamics of development. Main Content Presentation The existence of a common object of research for different sciences is emphasized by the ma- jority of criminologists who are specially engaged in the study of the problem of expert institutions 70 Nataliia Filipenko, Hanna Spitsyna. Criminological Activity of Forensic Science Institutions: the Concept of Implementation in Ukraine (Criminalistics and forensic examination, 2018). In our opinion, the long discussions of legal lawyers that continues in our time, about the fields of interests of criminalistics, criminal procedure, forensic science are largely caused by the fact that the object of research is included in the system of a science. It should be noted that the object of science is a certain part (fragment) of objective reality having a qualitative certainty and homogeneity that opposes the subject of activity, cognition. Based on this, the system of science includes not the object of study but knowledge about it in the form of concepts, positions, ideas, views, theories, hypotheses reflecting the real object, its qualities, features, relation- ships, connections, the laws of its formation, development and functioning. The object of science cannot remain unchanged. Its evolution highlights new, previously unknown aspects and circumstances. On the other hand, the development of science itself not only expands the range of knowledge about a particular object, but also opens previously unknown aspects, con- nections, relationships and, as a consequence, may develop so that the capabilities of the existing complex of sciences that study a particular object (the variety of qualities of the object, its multifa- ceted nature, objectively cannot be studied by a single science) is not enough to study . Therefore, the most effective and most promising research are cross-sectoral, butt, which include a system, a set of certain knowledge, approaches, methods of different fields of knowledge. Such sciences include forensic science. In addition, the knowledge about object and topic of science is evolving. It is sup- plemented by new provisions, clarifications, in some cases, the system of ideas about the object, its elements or characteristics changes significantly. The dynamism of the object of cognition, develop- ment of science objectively leads to a significant increase in knowledge about the field under study. In this regard, the problem of clarifying the subject of science and its object is constantly updated, as the subject of science, just formed in the process of cognition, being an idealized object of science, its theoretical model. Main Content Presentation General theoretical and methodological problems of forensic examinations and their previous articles, as well as further clarifications give a complete picture of their concept of a new field of knowledge that they called forensic expertology. These scientists define it as a branch of legal science that studies the laws, methodology, process of formation and development of scientific bases of forensic science, as well as examines their objects allowing to have an idea of the subject of science and its place in the system of scientific knowledge. Forensic expertology should explore the general patterns and methodological problems of the theory of forensic science and not fall to the level of certain types of expertise as a practical specific activity (Vinberg, Malakhovskaya, 1979). Forensic expertology is a science that studies the patterns of functioning of forensic activity, patterns and methodology of formation and development of examinations, patterns of research of objects of examination, carried out on the basis of specific expertise transformed on the basis of branch sciences into the system of scientific methods, techniques and means of solving expert problems. Within the framework of legal regulation, it should be defined as a synthetic, applied legal science that by its genesis and general research object (criminal activity) belongs to the cycle of criminal law sciences.i As noted in the scientific works devoted to the development of forensic science genesis from the part of criminalistics to an independent scientific category, the general idea of its creation was the main (fundamental) pattern: for all the individual differences of examinations of different genera and species, they all have many common positions expressed in their purpose, theoretical justification, sources, stages of development, functioning, regulation, organization. Main Content Presentation Thus, the problem of the subject of science cannot, in our opinion, belong to the circle of unconditionally solved . Each stage of development makes its own adjustments, opens up new aspects and aspects of this fundamental concept. Adequate understanding of the subject of forensic science, the essence of its subsystems and separate theories is of fundamental importance, because their definition specifies the area of objective reality that is the substantive basis of this area of knowledge, namely the activity the object of which is expert research. The private theory of expert prevention did not arise out of nowhere. Its development was pre- ceded by a long and painstaking work on the development of the general theory of forensic science. The fact that the private theory of forensic prevention studies almost the same object as the general theory of forensic examination, it seems appropriate to pre-analyze the content of general theory topic. In this case, it is taken into account that this theory itself is undergoing significant qualitative changes: in the legal regulation of forensic activities; expanding the scope of specific expertise; accu- mulation and systematization of new factual material, etc.i The process of forming scientific ideas about the subject of the general theory of forensic science has a long history and is not completed so far. A large number of criminologists (M. Segai, P. Bilenchuk, V. Prokhorov-Lukin, A. Ishchenko, N. Klimenko and others) recognized forensic science as an inde- pendent branch of legal science, but differed in their interpretation of its ranking in the system of other fields of knowledge. They believe that its further formation requires a clearer definition of the object and topic of new science, its conceptual foundations, its methodology that should be based on the model of expert cognition and intersectoral teachings for their ontological and epistemological guidelines.ii For example, defining the place of forensic expertise in the system of scientific knowledge, А. Eisman noted that it is rather separate part of criminalistics having a specific issue, topic and task. It should be considered a doctrine of forensic science in the criminalistics system (Eisman, 1980). The book by 71 ISSN 1392-1274 eISSN 2424-6050 Teisė. 2022, t. 124 A. Winberg and N. Malakhovskaya: Forensic expertology. Main Content Presentation Bychkov, is a system of theoretical knowledge that reflects at the meta-subject level the general patterns of development of the scientific basis of forensic science and expert activity (Bychkova, 1992, р. 124). The subject of the general theory of forensic examination, according to S. F. Bychkov, is a system of theoretical knowledge that reflects at the meta-subject level the general patterns of development of the scientific basis of forensic science and expert activity (Bychkova, 1992, р. 124). One of the main factors that significantly affect the study of expert prevention is to take it as a whole system to reveal its essence, qualitative specificity, its inherent systemic integration qualities (composition, structure, internal and external organization, relationship of components). The emphasis is on the interaction and interdependence of individual components of the system, the specifics of the functioning of the whole system as a whole . It is necessary to distinguish between the overall qualities of the system and the individual qualities of consisting elements because they can differ significantly. The solution of this problem required, in turn, the development of a private theory of forensic prevention. All attempts of theoretical generalizations concerning separate elements of preventive activity without intention to consider them as components of one system, without application of the scientific analysis of all the system as a whole (for example, interconnected processes occurring in this system, their hierarchical communications), failed to build a private theory of forensic prevention.i The creation of theory always marks a qualitatively new stage in the development of scientific knowledge and its positive impact on practice. The private theory of forensic prevention is designed to systematize empirical knowledge and available theoretical generalizations, to describe and explain it, to reveal the patterns of its functioning and development. However, private theory development of forensic prevention required a solution to the issue in which field of knowledge, in the light of which general theory, it can be created and developed. The theory includes several provisions expressing regular connections. Moreover, these provisions are united by one common principle reflecting the fundamental laws of the subject (or set of phenomena). If there is no unifying general principle, then no set of scientific propositions that reflect the natural connections will not be a scientific theory (Bychkova, 1992). A similar opinion is held by other scientists. Main Content Presentation The conditions that contributed to the solution of the problem of creating a forensic expertology are: (1) availability of a large body of empirical material in certain genera (types) of examinations, the creation on this basis of separate theories of these examinations, which reflected their scientific basis and patterns; (2) development of principles, methodological, legal and organizational bases of various kinds of forensic examinations, allocation from this volume of that general that should be inherent in any kind of examination, including that created ; (3) the presence of intermediate theoretical developments on certain problems of forensic examination, which are reflected in monographs, articles, educational and methodological literature; (4) the system of methods and techniques of expert research, which is constantly improved and is a reflection of general scientific and technological progress; (5) the presence of a developed system of state forensic institutions in various departments of the country, coordinating their practical and scientific activities. Thus, the purpose of forensic expertology is primarily to systematize the accumulated so far categories of all kinds (types) of forensic examinations, namely: concept of the forensic examination topic, objects of research, expert tasks, expert specific expertise, concept of expert competence and competence, technology of expert research, etc. (Synopsis of lectures on the subject “Expertology” for law students, 2016). As I. A. Aliiev rightly noted, the general theory of forensic examination should reflect the patterns of origin and development of examinations of different classes, genera, species with aim to synthesize and systematize the knowledge about forensic examination as a single system about what processes take place in . It should reflect the concept of the subject, general methodology, training on objects, subjects, expert tasks and methods of expert research, theoretical aspects of infrastructure and system-functional analysis of expert activities, as well as prospects for the creation and development of private theories of forensic science. In this case, it should be a legal interdisciplinary theory, substantively related to the criminal process, criminology, with other sciences, including specialized, which form the basis of examinations of different classes, kinds (Aliev, 1990). 72 Nataliia Filipenko, Hanna Spitsyna. Criminological Activity of Forensic Science Institutions: the Concept of Implementation in Ukraine The subject of the general theory of forensic examination, according to S. F. Main Content Presentation For example, considering the relationship between general theory and private theories of individual genera and types of examinations, T. V Averyanova and I. A. Aliiev noted that private theories are based on the provisions of general theory and contain as source premises those data that correspond to specifics of certain types or kinds of forensic examinations. In addition to these data of general importance, the content of private theory consists of specific scientific bases of this kind, type of ex- pertise, characteristics of typical research methods and typical or typical methods used to solve again typical for this kind or type of expert tasks. These authors, characterizing private theories, note that they may differ in the level of scientific generalization. In their hierarchy, one can imagine theories of class, kinds, types and even a subtypes of forensic examinations and each subsequent one in this series differs in greater concretization from the previous one, retaining in its content some provisions common to all these theories (Aliev, Averyanova, 1992). The basis for the formation of the theory of forensic prevention was a discussion of crime hindering unfolded in the early 60s of last century. Until then, the question of forensic (including expert) prevention in the literature has not been raised. Only some forensic specialists (V. P. Kolmakov, I. Ya. Friedman, V. F. Zudin, I. A. Aliiev) began to consider the basics of forensic prevention in their research papers. In the research papers of R. S. Belkin devoted to the consideration of private forensic theories, it was argued that in their content they can be more general and less general, reflecting, respectively, a larger or smaller subject area, more or less significant group of phenomena and processes (Belkin, 1997, р. 172).i One of the first to develop the provisions of the private theory of forensic prevention was a prom- inent Ukrainian scientist V. P. Kolmakov (Коlmakov, 1961). However, his work was met with great criticism by some scientists, especially criminologists. One of the ardent opponents of V. P. Kolmakov 73 ISSN 1392-1274 eISSN 2424-6050 Teisė. 2022, t. 124 was I. Ya. Friedman. However, devoting much time to the study of this question and accumulating a certain theoretical array of information, he was forced to agree with V. P. Main Content Presentation Kolmakov concluding that this particular theory has a right to exist because it is a part of the criminalistics topic, is a part of its general scientific theory, but he proposed the structure and content of this doctrine (Fridman, 1976, р. 63). Therefore, other theorists in the field of criminalistics and forensic science considered in their work issues of legal, organizational and methodological nature of expert prevention, paid considerable attention to the problems of monitoring the implementation of recommendations given in the conclusions of experts and sent to law enforcement agencies and other officials whose functional responsibilities include the implementation of such recommendations. Later, analyzing the theoretical achievements of predecessors and a large array of empirical material, I. A. Aliiev substantiated the need to construct a private theory of expert prevention, defined its principles and functions, content and structure, as well as patterns of its construction and outlined the interaction of its components with other preventive activity (Aliev, 1987, р. 21). In this regard, R. S. Belkin pointed out that in the context of the general theory of forensic science, the object of which is practical expert activity, private theory has a right to exist because it reflects an independent and also very important direction of expert practice, which can rightly be called crime hindering (Belkin, 1997, р. 161). Like most private theories, the theory of expert prevention develops and implements its own system of concepts that are a reflection of reality. After analyzing the opinions of scientists who have previously considered these issues, we can say that the system of concepts of expert prevention can be divided into three major blocks: basic, intermediate and integral. Basic concepts of the of forensic prevention theory in most cases coincide with those used in the general theory of forensic science: specific expertise, forensic expert, forensic science, expert conclu- sion, forensic research. The second, intermediate, block of the system of concepts of forensic prevention theory includes already more specific concepts inherent in this particular theory: tasks of judicial prevention, expert preventive activity, recommendations of the expert on elimination (minimization) of the reasons and conditions which have promoted crime commission, revealing of determinants of crime, etc (Filipenko, Bublikov, Obolientseva-Krasyvska, 2021). Main Content Presentation The integral level of concepts corresponds to: set of separate preventive measures used by the expert in the course of his professional activity, structural and functional basis of preventive expert activity, subjects of expert preventive activity, models of preventive expert activity, etc. Modern domestic scholars, studying the place and role of expert prevention in the system of legal sciences, and in particular, forensic expertise, considered it mostly in the context of conducting specific forensic examinations. After their analyzing we see that most scholars understood expert prevention as the establishment on the basis of special knowledge of facts containing data on the circumstances of the event that contributed to the crime (offense) and the transformation of these data through willful acts of legal persons with the ultimate goal of eliminating these circumstances. present and future or minimizing (Bordyugov, 2008). For example, developing the scientific postulates of forensic expertise, I. V. Pyrih believes that expert prevention is part of its conceptual framework (Pirig, 2015). Theoretical foundations of expert security of pre-trial investigation) and stressed that prevention is the use of these examinations to purposefully influence specific individuals, causes and conditions of crime. Unfortunately, in most of the analyzed works the problematic issues of preventive activity of a forensic expert are considered mainly in the issues of using the conclusions and results of forensic examinations. We believe that this approach is disproportionate. In this regard, we agree with the views 74 Nataliia Filipenko, Hanna Spitsyna. Criminological Activity of Forensic Science Institutions: the Concept of Implementation in Ukraine of A. A. Rusetskyi and O. P. Uhrovetskyi who emphasized that the logic of building the latest model of combating crime involves the introduction of the use of expert results at all stages of combating crime, namely to form optimal organizational tactical models of actions of operatives and investigators at the stages of search and detection of signs of criminal activity, preventive measures and during the pre-trial investigation of criminal proceedings. In their opinion, forensic prevention of crimes is a component of counteraction to crimes, which consists in the use of special knowledge aimed at improving the theoretical, legal, organizational and tactical bases of measures, investigative and covert criminal invest- igation measures in order to identify and eliminate the circumstances of criminal offenses (Rusetsky, Ugrovetsky, 2018, р. 85). Main Content Presentation We do not completely agree with this definition, as we believe that expert prevention, first of all, is an independent activity of a forensic expert, and, secondly, it cannot serve covert investigative actions. In our opinion, this is a professional activity of a forensic expert aimed at eliminating or minimizing the determinants of crime. Based on the fact that by the determinants of crime we mean a set of internal predispositions of a person (his criminal motivation) and external factors that create a certain basis for a person to commit an illegal act. Therefore, the determinants of crime, to a greater extent are legal concepts, because they are based on violations of legal, social or technical norms. It is these shortcomings that the court reveals when considering cases and takes measures to their minimizing or eliminating (Filipenko, 2019) Identification of crime determinants while performing forensic expert activities), we want to offer our own definition of forensic prevention. In our opinion, this is based on laws and regulations of the forensic expert aimed at identifying the determinants that contributed to the commission of a particular crime and development of measures to their eliminating (minimizing) using specific expertise (Filipenko, Bublikov, 2020). Preventive activity should be carried out: while carrying out forensic examinations regarding specific criminal, administrative or civil cases, by means of generalizing expert and forensic practice; while scientific research on problems of expert prevention; by providing, on the basis of specialized knowledge, scientific and practical assistance to bodies and organizations in identifying circumstances contributing to crime commission, etc. (Filipenko, Ugrovetsky, Sharapova, 2019). Having understood the essence of forensic prevention, we will consider in more detail its main components. These are the topic, object and principles of forensic prevention. Having analyzed the results of discussion and statements of scientists on the concept of expert prevention topic and based on our own researches (Filipenko, 2019), we give our understanding of this theoretical definition. Main Content Presentation In our opinion, the subject of private theory of forensic prevention consists of theoretical, normative, legal and organizational laws of preventive activity; regularities of origin and formation of the bases of preventive activity on the basis of the uniform methodology, the unified conceptual device; restructuring and adjustment of theoretical categories taking into account the constant updating, modernization and modification of special expertise; normative, legal and organizational support of preventive expert activity; common language of scientific terminology and unification of preventive standards, etc. Disclosing the concept of private theory object of expert prevention, we want to note that they can be classified into: al objects (material media of information about the criminal event, for example, documents • general objects (material media of information about the criminal event, for example, documents provided for examination by an expert; objects of the surrounding world, referrals for examination for comparison, etc.); ded for examination by an expert; objects of the surrounding world, referrals for examination mparison, etc.); • characteristic objects (special objects of kinds and types of forensic examinations: for example, accounting documents, audit reports, etc.);ii • characteristic objects (special objects of kinds and types of forensic examinations: for example, accounting documents, audit reports, etc.);ii • specific objects (objects examined by an expert during a specific forensic examination) 75 ISSN 1392-1274 eISSN 2424-6050 Teisė. 2022, t. 124 Based on the content and scope of the studied objects of expert prevention, it can be argued that the problems solved within the framework of this particular theory are divided into two groups: general and special. The general task of the private theory of expert prevention should be considered the practice of special subjects aimed at combating crime by developing scientific provisions for the use of forensic science in crime prevention. After analyzing the views of scientists (Klimenko, 2014), we believe that the special tasks include: establishing the causes and conditions that contribute to the commission of crimes, when conducting expert research; development of methods and means of obtaining reliable knowledge, which provided forensic experts with new opportunities to establish the determinant of crime; development and improvement of measures to increase the effectiveness of general interaction and coordination of activities between employees of forensic institutions and other subjects of pre- ventive activities; introduction of elements of preventive activity in civil, economic, arbitration and administrative proceedings, etc. Main Content Presentation The main principles of the expert prevention private theory are: the principle of causality (research on causal relationships, causal actions of the expert to establish criminogenic factors and develop pro- posals for their elimination); the principle of heredity (that is, the private theory of expert prevention implements the laws of criminalistics prevention theory), etc. Also, the allocation of expert prevention in an independent system is based on the principle of a systematic approach as one of the most promising areas in the development of social research meth- odology. While such development, a variant of solving a specific system problem should be chosen, namely: isolation of the object under research. This isolation has a dualistic nature: on the one hand, we can identify the levels of the social system, and on the other, determine the sphere of public life, where this system will be the most viable and dynamic. If we are talking about the system of forensic prevention, in our opinion, the most promising is its distribution (isolation of its elements) at different levels. This will allow the most effective consider- ation of the forensic prevention system as a single conglomerate of data and will analyze the existing value regulations in the system and the specifics of their implementation. Such regulations (markers or value invariants) for the expert prevention system are the social, legal and ethical obligation to take preventive measures by employees of forensic science institutions of Ukraine. After all, it is forensic experts who have the opportunity to establish the causes and conditions of crimes, applying specific expertise in practice, have the opportunities to develop and implement a set of special preventive measures. The effect of this special complex invariant of this system extends to the entire forensic science institution. Conclusions Based on the results of the analysis of professional sources on this issue and taking into account our own works, we note that: Based on the results of the analysis of professional sources on this issue and taking into account our own works, we note that: 1. The genesis of forensic science as a scientific and theoretical category convincingly shows that it is in the process of continuous development and a constant factor in its evolution are the processes of differentiation and integration of knowledge in this subject area. In practice, this means the constant emergence of new areas of scientific knowledge in-depth development of which often leads to the emergence of new theories within the basic science. Such theories, having received a certain degree of autonomy, deepen (clarify) knowledge within their subject, and at a new theoretical level are embedded in the fabric of basic science, enriching it. In our opinion, such a process occurs with the system of knowledge about forensic prevention private theory. 2. Forensic prevention is a mandatory element of expert technology considered by forensic scientists and proceduralists and leading forensic experts as a scientific category. This category is based on expert initiative provided by procedural law for the implementation of the expert’s right to emphasize in his opinion the facts revealed during the examination, relevant to the case and prevention as an expert’s activity to identify circumstances that contributed (could contribute) to the commission study of an offense and about which he was not asked questions . 3. The development and deepening of the forensic prevention private theory allows us to formulate the basic patterns inherent in the preventive processes of forensic science. Main Content Presentation Thus, the system forming factors of forensic prevention are: implementation of preventive tasks using specific expertise; coordination, information and methodological support of preventive meas- ures; unification of the structure of interconnected subjects of preventive activity; normatively fixed subjects of preventive activity; uniformity of functional support of preventive measures of forensic science institutions of the system of the Ministry of Justice of Ukraine, the Ministry of Internal Affairs.i The possibility and regularity of the use of specific expertise by forensic experts in the implement- ation of preventive activities is not disputed, because the possibility of forensic science in identifying circumstances that contribute to the commission of crimes, as well as in developing measures to eliminate them are of paramount importance. 76 Nataliia Filipenko, Hanna Spitsyna. Criminological Activity of Forensic Science Institutions: the Concept of Implementation in Ukraine Conclusions These include: organizational and methodological bases of forensic prevention; regularity of obtaining, as a result of preventive activity, information about the facts or circumstances that contribute to the commission of crimes and the elimination or minimization of their impact on public life ; regularity of growth of author- ity of forensic institution in the conditions of acceleration of scientific and technical progress and perception of results of their activity by the persons who do not possess the corresponding specific expertise; unification of methods and means of preventive activity and support of its invariance in the legal field of expert activity; formation of a specific conceptual apparatus, the language of theory adapting concepts borrowed from other related fields of knowledge to the preventive activities of forensic science institutions. 4. The scope of knowledge systematized by a theory, as well as its place in the general system of science, is determined by its topic. In science, quite often the subject of theory is expressed through a system of studied patterns, generalized and formulated in the form of a definition. In our opinion, the subject of the of forensic prevention private theory are theoretical, normative-legal and organ- izational laws of preventive activity; regularities of origin and formation of the bases of preventive activity on the basis of the uniform methodology, the unified conceptual device; restructuring and adjustment of theoretical categories taking into account the constant updating, modernization and modification of specific expertise; normative-legal and organizational support of preventive expert activity; common language of scientific terminology and unification of preventive standards, etc. 5. By forensic prevention we mean the activities of a forensic expert based on laws and bylaws, aimed at identifying determinants that contributed to the commission of a particular crime, and the development of measures to eliminate (minimize) them using specific expertise. 5. By forensic prevention we mean the activities of a forensic expert based on laws and bylaws, aimed at identifying determinants that contributed to the commission of a particular crime, and the development of measures to eliminate (minimize) them using specific expertise. 6. Conclusions Despite the spread of certain elements of forensic prevention in the practice of forensic science in- stitutions, to date, the development of theoretical foundations, conceptual approaches to its structure and methods of construction are still at an insufficient level of development. Therefore, we consider it necessary to propose a definition of the central concept in the concept of forensic prevention private theory: it is a category with which the set of its characteristics fulfills the requirements of the legal system of the state in the implementation of preventive activities. 9. Despite the spread of certain elements of forensic prevention in the practice of forensic science in- stitutions, to date, the development of theoretical foundations, conceptual approaches to its structure and methods of construction are still at an insufficient level of development. Therefore, we consider it necessary to propose a definition of the central concept in the concept of forensic prevention private theory: it is a category with which the set of its characteristics fulfills the requirements of the legal system of the state in the implementation of preventive activities. Conclusions The topic of forensic prevention private theory are theoretical, normative, legal and organiza- tional laws of preventive activity; regularities of origin and formation of the bases of preventive activity on the basis of the uniform methodology, the unified conceptual device; restructuring and adjustment of theoretical categories taking into account the constant updating, modernization and 77 ISSN 1392-1274 eISSN 2424-6050 Teisė. 2022, t. 124 modification of special expertise; normative, legal and organizational support of preventive expert activity; common language of scientific terminology and unification of preventive standards, etc. modification of special expertise; normative, legal and organizational support of preventive expert activity; common language of scientific terminology and unification of preventive standards, etc. 7. Disclosing the concept of private theory object of expert prevention, we want to note that they can be classified into: general objects (material media of information about the criminal event, for example, documents provided for examination by an expert; objects of the surrounding world, referrals for examination for comparison, etc.); characteristic objects (special objects of kinds and types of forensic examinations: for example, accounting documents, audit reports, etc.); specific objects (objects examined by an expert during a specific forensic examination).i 8. The significance of the forensic prevention private theory lies in the generalization of the direct experience of the organization of such activities that can be expressed in the form of a system of principles. In our opinion, they are formed by: principle of legality and observance of human and civil rights and freedoms; principle of professional independence and professional ethics of experts; principle of confidentiality; principle of leadership of the head of the forensic science institution; principle of qualification and involvement of all staff; principle of continuous improvement of preventive activities; factual principle of decision making, etc. 9. Despite the spread of certain elements of forensic prevention in the practice of forensic science in- stitutions, to date, the development of theoretical foundations, conceptual approaches to its structure and methods of construction are still at an insufficient level of development. 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Available at: https://library.pp-ss.pro/index. php/ndippsn_20201120/article/view/filipenko. Filipenko, N. (2020). Nataliia Filipenko (Vilnius University) (Vilnius University) (National Aerospace University – «Kharkiv Aviation Institute») Hanna Spitsyna (National Aerospace University – «Kharkiv Aviation Institute») The article analyzes the conceptual principles, opinions and ideas for understanding the essence of forensic science private theory, namely: forensic prevention concept is considered. The empirical prerequisites for the emergence and development 79 ISSN 1392-1274 eISSN 2424-6050 Teisė. 2022, t. 124 of forensic research have been investigated: accumulation of empirical facts in the theory of forensic science and other sciences, and manifestation of integration processes. Theoretical and applied foundations of expert prevention private theory are formulated. Its concept, structure, subject, object and tasks are considered and analyzed. Its place and role in the theory of criminalistics and forensic science are clarified. It is noted that the study of the current state of the theory and practice of private expert theory has allowed to establish that it has a common beginning and justification. Private theories, as systems of interconnected components, include: idea, principles, large empirical material that is the theory cornerstone, general and specific tasks, functions and purpose of the theory. Nataliia Filipenko (Vilniaus universitetas) ( ) (Nacionalinis kosminės erdvės universitetas – Charkovo aviacijos institutas) Hanna Spitsyna (Nacionalinis kosminės erdvės universitetas – Charkovo aviacijos institutas) S a n t r a u k a Šiame straipsnyje analizuojami konceptualūs principai, nuomonės ir idėjos, padedančios suprasti kriminalistikos mokslo privačiosios teorijos esmę, būtent: nagrinėjama kriminalistinės prevencijos idėja. Ištirtos kriminalistinių tyrimų atsiradimo ir plėtros empirinės prielaidos: empirinių faktų kriminalistikos ir kitų mokslų teorijoje kaupimas, integracinių procesų raiška. Suformuluoti teoriniai ir taikomieji ekspertinės prevencijos privačiosios teorijos pagrindai. Nagrinėjama ir analizuojama jos samprata, struktūra, subjektas, objektas ir uždaviniai. Aiškinama jos vieta ir vaidmuo kriminalistikos mokslo teorijoje. Pažymima, kad ekspertinės prevencijos privačiosios teorijos ir praktikos dabartinės būklės tyrimas leido nustatyti, kad jos turi bendrą pradžią ir pagrindimą. Privačiosios teorijos, kaip susijusių komponentų sistemos, apima: idėją, principus, didelę empirinę medžiagą, kuri yra teorijos pagrindas, bendruosius ir specialiuosius uždavinius, teorijos funkcijas ir tikslą. Nataliia Filipenko, Doctor of Law, Associate Professor, рrofessor at the Law Department of National Aerospace University – «Kharkiv Aviation Institute»; scientific interests: criminalistics, criminology, forensic science Nataliia Filipenko, teisės mokslų daktarė, docentė, Nacionalinio kosminės erdvės universiteto – Charkovo aviacijos institutas Teisės departamento dėstytoja. Moksliniai interesai: kriminalistika, kriminologija, kriminalistikos mokslas. Hanna Spitsyna, Doctor of Law, Professor, manager at the Law Department of National Aerospace Uni- versity – «Kharkiv Aviation Institute»; scientific interests: criminalistics, criminology, forensic science Hanna Spitsyna, teisės mokslų daktarė, profesorė, Nacionalinio kosminės erdvės universiteto – Charko- vo aviacijos institutas Teisės departamento vedėja. Moksliniai interesai: kriminalistika, kriminologija, kriminalistikos mokslas. Hanna Spitsyna, Doctor of Law, Professor, manager at the Law Department of National Aerospace Un ersity – «Kharkiv Aviation Institute»; scientific interests: criminalistics, criminology, forensic science 80
https://openalex.org/W4220709977	https://digibug.ugr.es/bitstream/10481/74122/1/A-Mixed-Heterobimetallic.pdf	English	null	A Mixed Heterobimetallic Y/Eu-MOF for the Cyanosilylation and Hydroboration of Carbonyls	Catalysts	2,022	cc-by	7,255	Citation: Echenique-Errandonea, E.; López-Vargas, M.E.; Pérez, J.M.; Rojas, S.; Choquesillo-Lazarte, D.; Seco, J.M.; Fernández, I.; Rodríguez-Diéguez, A. A Mixed Heterobimetallic Y/Eu-MOF for the Cyanosilylation and Hydroboration of Carbonyls. Catalysts 2022, 12, 299. https://doi.org/10.3390/ catal12030299 Keywords: heterobimetallic; metal-organic framework; cyanosilylation; hydroboration; yttrium; europium Article A Mixed Heterobimetallic Y/Eu-MOF for the Cyanosilylation and Hydroboration of Carbonyls Estitxu Echenique-Errandonea 1,† , Mireya E. López-Vargas 2,†, Juana M. Pérez 2 , Sara Rojas 3, Duane Choquesillo-Lazarte 4 , José M. Seco 1, Ignacio Fernández 2,* and Antonio Rodríguez-Diéguez 3,* 1 Departamento de Química Aplicada, Universidad del País Vasco UPV/EHU, Paseo Manuel Lardizabal, N◦3, 1 Departamento de Química Aplicada, Universidad del País Vasco UPV/EHU, Paseo Manuel Lardizabal, N◦3, 20018 Donostia-San Sebastián, Spain; estitxu.echenique@ehu.eus (E.E.-E.); josemanuel.seco@ehu.es (J.M.S.) 2 Department of Chemistry and Physics Research Centre CIAIMBITAL University of Almería Departamento de Química Aplicada, Universidad del País Vasco UPV/EHU, Paseo Manuel Lardizabal, N 3, 20018 Donostia-San Sebastián, Spain; estitxu.echenique@ehu.eus (E.E.-E.); josemanuel.seco@ehu.es (J.M.S.) 2 Department of Chemistry and Physics, Research Centre CIAIMBITAL, University of Almería, Carretera Sacramento s/n, 04120 Almería, Spain; mireyaestelalv@gmail.com (M.E.L.-V.); jperez.galera88@gmail.com (J.M.P.) 3 Departamento de Química Inorgánica, Facultad de Ciencias, Universidad de Granada, Av. Fuentenueva s/n 18071 Granada, Spain; srojas@ugr.es p j g 4 Laboratorio de Estudios Cristalográﬁcos, IACT, CSIC-UGR, Av. Las Palmeras N◦4, Armilla, p j g 4 Laboratorio de Estudios Cristalográﬁcos, IACT, CSIC-UGR, Av. La 18100 Granada, Spain; duane.choquesillo@csic.es 4 Laboratorio de Estudios Cristalográﬁcos, IACT, CSIC-UGR, Av. Las Palmeras N◦4, Armilla, 18100 Granada, Spain; duane.choquesillo@csic.es 4 Laboratorio de Estudios Cristalográﬁcos, IACT, CSIC-UGR, Av. Las Palmeras N◦4, Armilla, 18100 G d S i d h ill @ i p q * Correspondence: ifernan@ual.es (I.F.); antonio5@ugr.es (A.R.-D.) * Correspondence: ifernan@ual.es (I.F.); antonio5@ugr † These authors contributed equally to this work. Abstract: Herein, to the best of our knowledge, the ﬁrst heterobimetallic Y/Eu porous metal–organic framework (MOF), based on 3-amino-4-hydroxybenzoic acid (H2L) ligand, with the following for- mulae {[Y3.5Eu1.5L6(OH)3(H2O)3]·12DMF}n (in advance, namely Y/Eu-MOF), is described. The three-dimensional structure has been synthesized by solvothermal routes and thoroughly character- ized, by means of single crystal X-ray diffraction, powder X-ray diffraction, electronic microscopy, ICP-AES, electrophoretic mobility, and FTIR spectra. Intriguingly, the porous nature allows for coor- dinated solvent molecules displacement, yielding unsaturated metal centers, which can act as a Lewis acid catalyst. This novel supramolecular entity has been tested in cyanosilylation and hydroboration reactions on carbonyl substrates of a diverse nature, exhibiting an extraordinary activity. catalysts catalysts catalysts catalysts 1. Introduction Metal-organic frameworks are multifunctional, inorganic–organic systems composed of organic ligands and metallic nodes and, since the 1990s, have emerged as promising materials for inﬁnite applications, among them heterogeneous catalysis [1,2]. Their intrinsic porosity, along with easy tunability and functionalization, are some of the appealing characteristics to be employed in heterogeneous catalysis [3]. The structural porosity provides a conﬁned space to which substrates have access and catalysis can take place. Furthermore, the MOF’s robustness provides stable skeletons, where unsaturated metallic centers can act as a Lewis acid catalyst, subsequent displacement of coordinated solvent molecules could occur, and the further activation of the substrates already accessed through the channels might help the catalysis evolve [4]. Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional afﬁl- iations. In this context, cyanosilylation and hydroboration reactions catalyzed by heteroge- neous catalysts, such as MOFs, are presented as interesting approaches for the formation of cyanohydrins and alcohols [5,6]. The cyanosilylation reaction is an important synthetic tool for C–C bond formation in organic systems, since it gives access to key intermediates of great interest in the ﬁeld of pesticides and medical application, among which α-hydroxy acids, α-hydroxy ketones, α-amino acids, and β-amino alcohols can be mentioned [7,8]. 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/). Nonetheless, within this frame of reference, the number of reported Ln-MOF acting as a heterogeneous catalyst in the cyanosilylation reaction results were relatively scarce https://www.mdpi.com/journal/catalysts Catalysts 2022, 12, 299. https://doi.org/10.3390/catal12030299 Catalysts 2022, 12, 299 2 of 10 (Table S10 summarizes the reported examples exhibiting activity, so far) [5,9–24]. Moreover, to the best of our knowledge, lanthanide-based heterobimetallic MOFs have never been applied. In the ﬁeld of hydroboration, the context is even more unexplored. The synthesis of alcohols through this route has been explored via MOF systems based on Ti, Fe, Co, and Mg [25–30]; however, there are no precedents with these supramolecular entities built on lanthanides precursors. Therefore, in this work, the ﬁrst example of yttrium and europium- based mixed MOF, with the formula {[Y3.5Eu1.5L6(OH)3(H2O)3]·12DMF}n, is presented, taking the strong background of our group in the examination of these supramolecular systems in catalysis [15,16]. 1. Introduction The heterobimetallic Y/Eu-MOF presented herein was designed when looking for a synergy and enhanced catalytic activity, with respect to their isostructural pristine counter- parts, which were able to exhibit activity for seven cycles, with very low catalytic loading of 0.5 mol%, no catalyst leaking, TOF values of 106 h−1 and 1031 h−1, and preferred suitability towards aldehydic and ketonic substrates for Y-MOF [15] and Eu-MOF, respectively. Taking all into consideration, our interest lies in exploring the catalytic activity of this novel heterobimetallic MOF, based on 3-amino-4-hydroxybenzoic acid polytopic ligand, in the cyanosilylation and hydroboration reactions of aldehydes and ketones of diverse nature under solvent-free and environmentally friendly conditions, in order to eventually conclude whether catalytic activity enhances, with respect to initial counterparts. 2.1. Preparation of the Catalyst The solvothermal reaction between 3-amino-4-hydroxybenzoic acid (H2L) ligand and Y-Eu metal mixture in a basic media yields a three-dimensional, porous, metal–organic framework that crystallizes in hexagonal P63/m space group. The asymmetric unit of this supramolecular entity is composed of a deprotonated ligand molecule, two metal atoms with special positions (in addition to a coordinated water molecule), and a hydroxyl bridge, which acts as connector among neighboring metallic centers. Note that, taking into consideration that metals are randomly distributed within the crystal structure, a tentative formula is presented, which is corroborated by bulk properties of the material (see ICP-AES results in Table S2 or SEM-EDX results in Figure S3). g Therefore, regarding the metal environment, two surroundings are described: nine coordinated MN3O6 and eight coordinated MO8 environments, which, according to con- tinuous shape measurements, conﬁrm the spherical capped square antiprism (TCTPR-9) and triangular dodecahedron (TDD-8) polyhedra, respectively. The former coordination environment is composed of three hydroxyl oxygens and amino atoms from L in addition to three additional oxygen atoms belonging to ligand hydroxyl group. The latter metallic nucleus is composed of the coordination of a water solvent molecule, an oxygen belonging to a hydroxyl bridge, along with six oxygen atoms, corresponding to two ligand hydroxyl and two carboxylate moieties. y As aforementioned, hydroxyl bridges µ3-OH−join metallic centers constructing M5(OH)3 secondary building units in the structure, being the ligand connector, among different sbu. Regarding the connectivity of the supramolecular entity, from the topological point of view, six-connected nodes are described, which, according to TOPOS software [31] examination, the framework disposed of the acs network with the (49·66) point symbol. Intrinsic porosity, characterized by three dimensional micropores of 10.1 × 13.3 Å of Y/Eu- MOF (according to PLATON-v1.18 [32] cavities, corresponds to 19% of the volume of the structure) and enables solvent crystallization molecules to be trapped, whiting the pores; concretely, DMF molecules have been demonstrated to be located in the microchannels (Figure 1). Catalysts 2022, 12, 299 3 of 10 g micro- Figure 1. Perspective view of the (a) metal coordination environments found in heterobimetallic compound Y/Eu-MOF. (b) View of the pentametallic nodous, where M5(OH)3 sbu are appreciable. (c) View for the formation of the 1 D channels; (d) the cavities present in the structure along the a axis. Color code: Y (green), Eu (purple), C (grey), O (red), N (blue), and H (white). 2.1. Preparation of the Catalyst Note that, due to the altering and randomly oriented disposition metals, an approximated representation has been performed, taking into consideration proposed formula of {[Y3.5Eu1.5L6(OH)3(H2O)3]·12DMF}n. Figure 1. Perspective view of the (a) metal coordination environments found in heterobimetallic compound Y/Eu-MOF. (b) View of the pentametallic nodous, where M5(OH)3 sbu are appreciable. (c) View for the formation of the 1 D channels; (d) the cavities present in the structure along the a axis. Color code: Y (green), Eu (purple), C (grey), O (red), N (blue), and H (white). Note that, due to the altering and randomly oriented disposition metals, an approximated representation has been performed, taking into consideration proposed formula of {[Y3.5Eu1.5L6(OH)3(H2O)3]·12DMF}n. Figure 1. Perspective view of the (a) metal coordination environments found in heterobimetallic compound Y/Eu-MOF. (b) View of the pentametallic nodous, where M5(OH)3 sbu are appreciable. (c) View for the formation of the 1 D channels; (d) the cavities present in the structure along the a axis. Color code: Y (green), Eu (purple), C (grey), O (red), N (blue), and H (white). Note that, due to the altering and randomly oriented disposition metals, an approximated representation has been performed, taking into consideration proposed formula of {[Y3.5Eu1.5L6(OH)3(H2O)3]·12DMF}n. Figure 1. Perspective view of the (a) metal coordination environments found in heterobimetallic compound Y/Eu-MOF. (b) View of the pentametallic nodous, where M5(OH)3 sbu are appreciable. (c) View for the formation of the 1 D channels; (d) the cavities present in the structure along the a axis. Color code: Y (green), Eu (purple), C (grey), O (red), N (blue), and H (white). Note that, due to the altering and randomly oriented disposition metals, an approximated representation has been performed, taking into consideration proposed formula of {[Y3.5Eu1.5L6(OH)3(H2O)3]·12DMF}n. 2.2. Chemical Composition 2.2. Chemical Composition Single-crystal X-ray diffraction allowed for proposing an approximated formula of the heterobimetallic catalyst, which was further confirmed by FT-IR, ICP-AES, PXRD, and SEM-EDX. For a detailed explanation of PXRD and FT-IR, please check supporting infor- mation, Figures S1 and S2, respectively. Single-crystal X-ray diffraction allowed for proposing an approximated formula of the heterobimetallic catalyst, which was further conﬁrmed by FT-IR, ICP-AES, PXRD, and SEM-EDX. For a detailed explanation of PXRD and FT-IR, please check supporting information, Figures S1 and S2, respectively. mation, Figures S1 and S2, respectively. ICP-AES analysis, conducted in the heterobimetallic compound Y/Eu-MOF, exhib- ited an yttrium content of 1672 mg/L (corresponding to 18.8 mM of Y3+) and europium content of 1268 mg/L (8.34 mM Eu3+). The calculated Y to Eu relationship gives 2.25, which is very much in line with the proposed formula, where a proportion of 2.33 is expected. Additionally, SEM-EDX mapping confirms that both metals are present in single crystal. However, the mapping spectrum, performed in one single crystal, showed relatively higher Y3+ to Eu3+ proportions; for the crystal in which spectrum was collected, a relation- ship of 3.25, regarding to Y to Eu, was calculated. This is due to the uneven distribution of metals throughout the catalyst structure. Nonetheless, the proposed formula, which is derived by single crystal X-ray diffraction, powder X-ray diffraction, SEM–EDX, and ICP- g p y ICP-AES analysis, conducted in the heterobimetallic compound Y/Eu-MOF, exhibited an yttrium content of 1672 mg/L (corresponding to 18.8 mM of Y3+) and europium content of 1268 mg/L (8.34 mM Eu3+). The calculated Y to Eu relationship gives 2.25, which is very much in line with the proposed formula, where a proportion of 2.33 is expected. Additionally, SEM-EDX mapping conﬁrms that both metals are present in single crystal. However, the mapping spectrum, performed in one single crystal, showed relatively higher Y3+ to Eu3+ proportions; for the crystal in which spectrum was collected, a relationship of 3.25, regarding to Y to Eu, was calculated. This is due to the uneven distribution of metals throughout the catalyst structure. Nonetheless, the proposed formula, which is derived by single crystal X-ray diffraction, powder X-ray diffraction, SEM–EDX, and ICP-AES, is consistent and summarizes the best approximation for data obtained in the aforementioned techniques. 2.3. Chemical Stability and Electrophoretic Behavior The highest negative value of the ζ-potential, obtained with Y/Eu-MOF, was ached at pH 10 (−35.8 ± 0.5 mV, electrophoretic mobility of −2.252 ± 0.035 µmcm/Vs), ggesting that: (i) the dissociation of external acidic groups have occurred, conferring the verall negative charge (Figures S8 and S9, Table S7); (ii) the MOF particles will tend to pel each other, and there will be no tendency for the particles to come together at this sic pH, due to this large value of ζ-potential. However, when reducing the pH of the lutions, the ζ-potentials values started to increase, evidencing an isoelectric point be- ween pH 4 and 5. Similar results were previously reported by our group. The Eu-MOF talyst, with the highest negative value of ζ-potential, was also obtained at pH 10 (−25.0 0.6 mV); however, in this case, the isoelectric point was obtained between pH 8 and 9 igure 2). From an electrophoretic behavior point of view, the mixed nature of MOF con- rs both a higher stability, as a function of pH, and less tendency to aggregate. igure 2. Comparison of ζ-potential behavior, as a function of pH, obtained with Y/Eu-MOF and 5.9 -1.0 -5.1 -6.7 -8.5 -18.1 -35.8 2.4 1.1 1.3 6.3 4.0 -6.9 -25.0 -45 -35 -25 -15 -5 5 15 4 5 6 7 8 9 10 ζ-Potential (mV) pH Y/Eu-MOF Eu-MOF Figure 2. Comparison of ζ-potential behavior, as a function of pH, obtained with Y/Eu-MOF and Eu-MOF. All the measurements were performed with a constant conductivity of 330 µS/cm. gure 2. Comparison of ζ-potential behavior, as a function of pH, obtained with Y/Eu-MOF and Figure 2. Comparison of ζ-potential behavior, as a function of pH, obtained with Y/Eu-MOF and Eu-MOF. All the measurements were performed with a constant conductivity of 330 µS/cm. -MOF. All the measurements were performed with a constant conductivity of 330 µS/cm. In order to determine the particle size distribution of the catalyst in the suspended d deposited fraction, optical microscope images were assayed (Figures S4 and S5). The talyst was suspended in distilled water; the suspension was shaken vigorously and sub- quently left for sedimentation for 5 min, and the suspended fraction separated. The de- osited fraction was composed of 68% of the total amount of catalyst initially weighted, ving an average particle size distribution of 12 ± 6 µm (Figure S4). 2.3. Chemical Stability and Electrophoretic Behavior ζ-potential is one of the fundamental parameters normally used to measure the mag- nitude of the electrostatic (or charge) repulsion or attraction between particles, being an Catalysts 2022, 12, 299 4 of 10 ag- 4 of 10 ag- indirect estimate of the surface charge density of the systems and helping to determine the repulsive interactions between colloidal particles, as well as the tendency of aggregation. Systems with high ζ-potential (−/+) are considered electrically stable, whereas colloids with low ζ-potential (−/+) are usually referred to as systems that tend to aggregate and further precipitate. In this study, measurements were always conducted at a ﬁxed conductivity of 330 µS/cm, which is equivalent to a concentration of about 3.7 mM NaCl (Figure S7). The highest negative value of the ζ-potential, obtained with Y/Eu-MOF, was reached at pH 10 (−35.8 ± 0.5 mV, electrophoretic mobility of −2.252 ± 0.035 µmcm/Vs), suggesting that: (i) the dissociation of external acidic groups have occurred, conferring the overall negative charge (Figures S8 and S9, Table S7); (ii) the MOF particles will tend to repel each other, and there will be no tendency for the particles to come together at this basic pH, due to this large value of ζ-potential. However, when reducing the pH of the solutions, the ζ-potentials values started to increase, evidencing an isoelectric point between pH 4 and 5. Similar results were previously reported by our group. The Eu-MOF catalyst, with the highest negative value of ζ-potential, was also obtained at pH 10 (−25.0 ± 0.6 mV); however, in this case, the isoelectric point was obtained between pH 8 and 9 (Figure 2). From an electrophoretic behavior point of view, the mixed nature of MOF confers both a higher stability, as a function of pH, and less tendency to aggregate. direct estimate of the surface charge density of the systems and helping to determine e repulsive interactions between colloidal particles, as well as the tendency of aggrega- on. Systems with high ζ-potential (−/+) are considered electrically stable, whereas colloids ith low ζ-potential (−/+) are usually referred to as systems that tend to aggregate and rther precipitate. In this study, measurements were always conducted at a fixed con- uctivity of 330 µS/cm, which is equivalent to a concentration of about 3.7 mM NaCl (Fig- e S7). 2.3. Chemical Stability and Electrophoretic Behavior The suspended frac- on showed an average particle size of 8 ± 4 µm (Figure S5). Obtained values are relatively milar to SEM images, in which single crystals and larger aggregates are appreciated. In order to determine the particle size distribution of the catalyst in the suspended and deposited fraction, optical microscope images were assayed (Figures S4 and S5). The catalyst was suspended in distilled water; the suspension was shaken vigorously and subsequently left for sedimentation for 5 min, and the suspended fraction separated. The deposited fraction was composed of 68% of the total amount of catalyst initially weighted, having an average particle size distribution of 12 ± 6 µm (Figure S4). The suspended fraction showed an average particle size of 8 ± 4 µm (Figure S5). Obtained values are relatively similar to SEM images, in which single crystals and larger aggregates are appreciated. Taking into consideration the average particle size distribution in the suspension, it is suggested that single-crystals remain relatively well-suspended, and aggregates of superior size tend to deposit. 2.4. Study of the Catalytic Activity Scope of the cyanosilylation reaction using aldehydes and ketones. Scheme 1. Scope of the cyanosilylation reaction using aldehydes and ketones. As a continuation of the study of the catalytic activity of Y/Eu-MOF, we decided to go one step forward and test, for the first time, the hydroboration reaction of carbonyl compounds with a lanthanide based-MOF catalyst. For that, the optimization of the reac- tion conditions was carried out (Table S8). First of all, the reaction was tested with solvents of different natures, as well as without solvent (entries 1–5), obtaining the best result in the absence of solvent (entry 1, Table S8). Finally, the amount of catalyst was also tested (entries 6–8), observing that, with a slight increase from 0.35 to 0.5 mol%, the reaction took place with full conversion (entry 6). As a continuation of the study of the catalytic activity of Y/Eu-MOF, we decided to go one step forward and test, for the ﬁrst time, the hydroboration reaction of carbonyl compounds with a lanthanide based-MOF catalyst. For that, the optimization of the reaction conditions was carried out (Table S8). First of all, the reaction was tested with solvents of different natures, as well as without solvent (entries 1–5), obtaining the best result in the absence of solvent (entry 1, Table S8). Finally, the amount of catalyst was also tested (entries 6–8), observing that, with a slight increase from 0.35 to 0.5 mol%, the reaction took place with full conversion (entry 6). p ( y ) With the optimal reaction conditions in hand, the scope of the reaction was evaluated again, using a broad range of different ketones (Scheme 2). The use of electron-donating p y With the optimal reaction conditions in hand, the scope of the reaction was evaluated again, using a broad range of different ketones (Scheme 2). The use of electron-donating groups in the para-position of the aromatic ring negatively inﬂuenced the catalytic reaction, causing the reaction time to increase from 24 to 48 or even 120 h, in order to obtain moderate conversions (45–67%). This inﬂuence on the increase in reaction time did not take place when electron-withdrawing substituents were used, observing that, regardless of the position of the aromatic ring in which the substituents were found, yields were good (71–90%). Furthermore, heteroaromatic ketones reached good results in only 24 h of reaction (88%). 2.4. Study of the Catalytic Activity The catalytic activity of Y/Eu-MOF (0.5 mol%) was initially evaluated in the cyanosi- lylation reaction of carbonyl compounds (Y/Eu-MOF) using TMSCN as the nucleophile, solvent free reaction conditions, room temperature, and N2 atmosphere. These conditions Catalysts 2022, 12, 299 5 of 10 yanosi- hil 5 of 10 yanosi- hil have been already reported in our previous works, with monometallic Y or Eu MOF syn- thetized with the same ligand H2L (3-amino-4-hydroxybenzoic acid) [15], obtaining only 8% conversion after 14 h when the blank reaction was tested. have been already reported in our previous works, with monometallic Y or Eu MOF syn- thetized with the same ligand H2L (3-amino-4-hydroxybenzoic acid) [15], obtaining only 8% conversion after 14 h when the blank reaction was tested. The ope of the ea tio a e aluated ith fi e hi hly ea ti e aldehyde of dif The scope of the reaction was evaluated with ﬁve highly reactive aldehydes of different natures, such as aromatic, heteroaromatic, and aliphatic, demonstrating that independently of the nature of the substituent, the reaction takes place with very good to excellent conversions (89–99%), after only 24 h (Scheme 1). Later on, less reactive and sterically demanding ketones were tested, observing a signiﬁcant decrease in conversion (74–77%), when electron-donating or -withdrawing substituents were located at the para-position, whereas, again, excellent conversion were achieved with aromatic, heteroaromatic, and aliphatic substrates (91–99%) (Scheme 1). These results overpass the ones published, until the moment for other groups [21–23], and have similar catalytic behaviour with our pristine Eu-MOF. The scope of the reaction was evaluated with five highly reactive aldehydes of dif- ferent natures, such as aromatic, heteroaromatic, and aliphatic, demonstrating that inde- pendently of the nature of the substituent, the reaction takes place with very good to ex- cellent conversions (89–99%), after only 24 h (Scheme 1). Later on, less reactive and steri- cally demanding ketones were tested, observing a significant decrease in conversion (74– 77%), when electron-donating or -withdrawing substituents were located at the para-po- sition, whereas, again, excellent conversion were achieved with aromatic, heteroaromatic, and aliphatic substrates (91–99%) (Scheme 1). These results overpass the ones published, until the moment for other groups [21–23], and have similar catalytic behaviour with our pristine Eu-MOF. Scheme 1. Scope of the cyanosilylation reaction using aldehydes and ketones. Scheme 1. Scope of the cyanosilylation reaction using aldehydes and ketones. Scheme 1. 2.4. Study of the Catalytic Activity The steric hindrance of ketones, such as 1j and 1p, needed superior reaction times of 48 and 120 h, respectively, in order to achieve full conversion. Aliphatic ketones, with different natures, were tested and provided good results (95–99%). Then, we decided to test cyclic ketones, which are usually less employed, obtaining moderate results Catalysts 2022, 12, 299 6 of 10 6 of 10 (61–63%). Finally, the chemoselectivity of the reaction was pursued by employing ketones bearing double bonds and ester groups, achieving, in both cases, the speciﬁc reduction of the carbonyls; although, in the latter, the conversion was lower than 5%, due to the poor solubility of the substrate in HBpin. (61–63%). Finally, the chemoselectivity of the reaction was pursued by employing ketones bearing double bonds and ester groups, achieving, in both cases, the speciﬁc reduction of the carbonyls; although, in the latter, the conversion was lower than 5%, due to the poor solubility of the substrate in HBpin. Scheme 2. Scope of the hydroboration reaction of ketones. Reaction carried out for 24 h. a Reaction carried out during 24 h; b Reaction carried out during 48 h; c Reaction carried out during 120 h. Scheme 2. Scope of the hydroboration reaction of ketones. Reaction carried out for 24 h. a Reaction carried out during 24 h; b Reaction carried out during 48 h; c Reaction carried out during 120 h. Another important issue that needed to be addressed was if our catalysis was the object of hidden boron catalysis [33]. To distinguish between “true” catalysis and BH3 catalysis, for reactions using HBpin, we performed two complementary experiments. With the help of 1H and 11B NMR, we pursued the possible MOF-promoted BH3 formation by monitoring the mixture of our MOF catalyst and HBpin under catalytic conditions. The lack of BH3 signals was evidenced during the whole experiment, proving that our MOF catalyst does not promote formation of BH3. In addition, we performed the hydroboration of acetophenone with BH3·THF, instead of with HBpin, showing no substrate conversion and, therefore, further verifying that, under our catalytic conditions, hidden boron catalysis does not exist. 2.5. Recyclability and Leaching Studies of the Catalyst 1 Recyclability tests were also investigated, in order to study the heterogeneous na- ture of the catalyst. After standard reaction conditions, using acetophenone as substrate (Schemes S1 and S2), the catalyst was isolated from the reaction mixture via centrifugation and washed several times with dichloromethane. The catalyst was dried under vacuum Catalysts 2022, 12, 299 7 of 10 7 of 10 and then charged with a new set of reagents, in order to evaluate its recyclability. The corre- sponding products were obtained in a 28% and 61% for cyanosilylation and hydroboration, respectively, and, in another consecutive cycle, 0% and 15% of conversion, respectively, was achieved (Figure S10). A leaching test was also carried out, in the case of the hydroboration reaction (Scheme S3). For that, after the ﬁrst and second reaction of recyclability test, the catalyst was removed by centrifugation and supernatant was ﬁltered through a plug of celite and dried under vacuum. Then, 1-(pyridin-2-yl)ethan-1-one (1i) and HBpin were added to the crude of the corresponding reaction cycle, and the reaction was stirred under inert N2 atmosphere at room temperature for 24 h. After that time, an aliquot was analysed by 1H NMR, obtaining, in the ﬁrst cycle, a 38% of conversion and, in the second cycle, a 13% of product, corroborating the leaching of Y or Eu. 2.6. Green Chemistry Metrics Green chemistry metrics such as atomic economy (AE), mass intensity (MI), reaction mass efﬁciency (RME), and carbon efﬁciency (CE) were calculated for both reactions studied (Table S9), in order to evaluate if the reactions are eco-friendly and can overcome environmental and health problems. The obtained values for cyanosilylation reaction were 100% of AE, 1.127 for MI, 95.7% for RME, and 96.8% for CE. These results are comparable to those described previously for related lanthanide-based MOFs [15,16,19,20]. In the case of the hydroboration reaction, these values decreased considerably, down to 49.1%, 2.406, 46.8%, and 52.1% for AE, MI, RME, and CE, respectively, due to the ﬁnal hydrolysis and consequent loss of atoms in the ﬁnal product (Table S9). 3.1. Materials All experiments involving moisture-sensitive compounds were performed under an inert atmosphere of N2 using standard techniques. Unless otherwise indicated, reagents and substrates were purchased from commercial sources and used as received. Metallic precursors, such as europium (III) nitrate pentahydrate (99.9%, Alfa Aesar), and yttrium (III) nitrate hexahydrate (99.9% of purity, Fluorochem), were used as received, as well as 3- amino-4-hydroxybenzoic acid ligand (H2L, C7H7NO3, 97% of purity), which was purchased from Fluorochem. Solvents not required to be dry were purchased as technical grade and used as received. Conversion values relative to the limiting reagent were calculated from the 1H NMR spectra of the reaction crudes. Isolated products of cyanosilylation reaction were obtained after centrifugation (12,300 rpm, 5 min, 20 ◦C) and washed with dichloromethane (2 × 0.5 mL), in order to remove the catalyst or column chromatography in silica gel using hexane as eluent. For the hydroboration reaction, ﬁrst of all the hydrolysis of the product was carried out adding NaOH (0.1 M, 0.5 mL) and Et2O (0.5 mL) and stirring the mixture overnight; after that, the catalyst was removed via centrifugation (12,300 rpm, 5 min, 20 ◦C) and washed with dichloromethane (2 × 0.5 mL). 3.2. Synthesis of Catalyst and General Procedures of Catalysis 4. Conclusions All in all, to the best of our knowledge, we report the ﬁrst mixed heterobimetallic Y/Eu-MOF, based on a 3-amino-4-hydroxylbenzoic acid ligand, with impressive catalytic efﬁciency in the cyanosilylation and hydroboration reaction of carbonyl compounds, but reduced recyclability, compared to the related MOFs. The catalyst has been thoroughly char- acterized by means of X-ray crystallography, FTIR, ICP-AES, and PXRD; its electrophoretic behavior, as a function of pH, has been also assayed, showing a negative surface charge with a very low tendency to aggregate. Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/catal12030299/s1. Table S1: Elemental analysis of compounds Y/Eu-MOF. Table S2: ICP-AES results of compound Y/Eu-MOF. Table S3: Crystallographic data and structure reﬁnement details of compound Y/Eu-MOF. Table S4: Selected bond lengths (Å) and angles (◦) for compound Y/Eu-MOF. Table S5: Table of the continuous Shape Measurements for the MN3O6 coordination environment. Table S6: Table of the continuous Shape Measurements for the MO8 coordination environment. Table S7: Electrophoretic mobility and ζ-potential dependence, with the pH of the Y/Eu-MOFs particles dispersed in water. Conductivity ﬁxed at 330 µS/cm. Table S8: Optimization of the reaction conditions in the hydroboration reaction. Table S9: Green metrics calculated for Y/Eu-MOF catalyst. Table S10: Catalytic cyanosilylation of benzaldehyde performances of Ln-MOFs, as reported in the literature. Figure S1: Figure of the pattern matching analysis and experimental PXRD for Y/Eu-MOF. Figure S2: Figure of the infrared spectra of the ligand and Y/Eu-MOF. Figure S3: SEM and EDS mapping of bulk material of Y/Eu-MOF. Figure S4: Images and particle size distribution (an overall of 250 particles) in the deposited fraction of Y/Eu-MOF catalyst non-suspended in water (about a 68% of the total amount), determined from optical microscope images. Figure S5: Images and particle size distribution (an overall of 250 particles) of Y/Eu-MOF crystals in the fraction steadily suspended in water (about a 32% of the total amount), determined from optical microscope images. Figure S6: Comparation of the particle size distribution of Y/Eu-MOF in the fraction steadily suspended in water and the non-suspended, determined from optical microscope images. Figure S7: Calibration line of conductivity (µS/cm) vs [NaCl] (mol/L). Figure S8: ζ-potential (mV) dependence with the pH of the Y/Eu-MOF. All the measurements were performed with constant conductivity of 330 µS/cm. Figure S9: Electrophoretic mobility (µm·cm/V·s) dependence with the pH of the Y/Eu-MOF. All the measurements were performed with constant conductivity of 330 µS/cm. 3.2. Synthesis of Catalyst and General Procedures of Catalysis Synthesis of {[Y3.5Eu1.5L6(OH)3(H2O)3]·12DMF}n, namely Y/Eu-MOF: The 3-amino- 4-hydroxybenzoic acid (0.010 g, 0.0625 mmol) was dissolved in 0.2 mL of DMF containing 10 µL of Et3N (0.072 mmol). In a separate vial, Eu(NO3)3·5H2O (0.006 g, 0.0145 mmol) and Y(NO3)3·6H2O (0.011 g, 0.0289 mmol) salts were dissolved into a 0.8 mL of distilled water. After dissolution, 0.2 mL of DMF and 0.8 mL of H2O were added to the metal and ligand solutions, respectively. Finally, the metal solution was added dropwise and under continuous agitation to the ligand solution. The obtained brown solution was introduced to the oven at 100 ◦C for 2 h in a screw-capped vial (6 mL) to yield hexagonal single crystals. For the reaction scaling up synthetic procedure, consult the supporting material. General procedure for the cyanosilylation reaction: In a 1 mL vial, with a septum screw capped, equipped with a stirring bar, Y/Eu-MOF catalyst (0.5 mol%) was weighed. Subsequently, the corresponding amount of carbonylic compound 1 (0.25 mmol) was Catalysts 2022, 12, 299 8 of 10 added, followed by trimethylsilyl cyanide (TMSCN) (34 µL, 0.275 mmol, 1.1 equiv.), and the reaction was stirred under inert N2 atmosphere at room temperature for 24 h. Once the reaction was ﬁnished, the catalyst was removed by centrifugation (12,300 rpm, 5 min) and washed with DCM (2 × 0.5 mL), obtaining the corresponding pure products 2 and 3, after removal of the solvent with rotary evaporator. When not full conversion was reached, the product was puriﬁed by column chromatography using hexane as eluent. General procedure for the hydroboration reaction: In a 1 mL vial, with a septum screw capped, equipped with a stirring bar, Y/Eu-MOF catalyst (0.5 mol%) was weighed. Subsequently, the corresponding amount of carbonylic compound 1 (0.25 mmol) was added, followed by pinacolborane (HBpin) (40 µL, 0.275 mmol, 1.1 equiv.), and the reaction was stirred under inert N2 atmosphere at room temperature for the corresponding time, as indicated on the table. After that time, the hydrolysis of the ﬁnal product was carried out adding NaOH (0.1 M, 0.5 mL) and Et2O (0.5 mL), and the mixture was stirred overnight. Once the reaction was ﬁnished, the catalyst was removed by centrifugation (12,300 rpm, 5 min) and washed with DCM (2 × 0.5 mL), obtaining the corresponding product 4 after removal of the solvent with rotary evaporator. When not full conversion was reached the product was puriﬁed by column chromatography. 4. Conclusions Figure S10: Study of the recyclability of Y/Eu-MOF (0.5 mol%) catalyst on the cyanosilylation and hydroboration reaction of acetophenone as carbonyl substrate. Figure S11: Analysis of the TOF (h−1) obtained in the cyanosilylation reaction of acetophenone at different times of reaction with Y/Eu-MOF (0.5 mol%), with the optimized reaction conditions. Figure S12: Analysis of the TOF (h−1) obtained in the hydroboration reaction acetophenone at different times of reaction with Y/Eu-MOF (0.5 mol%), with the optimized reaction conditions. Scheme S1: Reaction conditions Catalysts 2022, 12, 299 9 of 10 9 of 10 used for the study of recyclability of Y/Eu-MOF catalysts in the cyanosilylation reaction. Scheme S2: Reaction conditions used for the study of recyclability of Y/Eu-MOF catalysts in the hydroboration reaction. Scheme S3: Leaching test, carried out after the ﬁrst and second cycles. Author Contributions: Conceptualization, I.F. and A.R.-D.; methodology, M.E.L.-V., J.M.P. and E.E.-E.; software, S.R., D.C.-L. and J.M.S.; validation, J.M.P., S.R. and J.M.S.; formal analysis, A.R.-D.; investigation, M.E.L.-V., J.M.P. and E.E.-E.; resources, I.F., J.M.S. and A.R.-D.; data curation, S.R. and D.C.-L.; writing-original draft preparation, J.M.P. and E.E.-E.; writing-review and editing, J.M.P. and S.R.; visualization, I.F.; supervision, A.R.-D.; project administration, I.F.; funding acquisition, A.R.-D., I.F. and J.M.S. All authors have read and agreed to the published version of the manuscript. Funding: This research has been funded by the State Research Agency (grants CTQ2017-84334-R and PGC2018-102052-B-C21) of the Spanish Ministry of Science, Innovation and Universities, the European Union (European Regional Development Fund—ERDF), Junta de Andalucía (P20_01041, UAL2020-AGR-B1781, B-FQM-734-UGR20 and FQM-394). 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https://openalex.org/W2806213906	https://dash.harvard.edu/bitstream/1/37298329/1/6007290.pdf	English	null	Updates on Prevention of Cardioembolic Strokes	Journal of stroke	2,018	cc-by	15,171	Terms of Use This article was downloaded from Harvard University’s DASH repository, and is made available under the terms and conditions applicable to Other Posted Material, as set forth at http:// nrs.harvard.edu/urn-3:HUL.InstRepos:dash.current.terms-of-use#LAA Permanent link http://nrs.harvard.edu/urn-3:HUL.InstRepos:37298329 Citation Topcuoglu, Mehmet Akif, Liping Liu, Dong-Eog Kim, and M. Edip Gurol. 2018. “Updates on Prevention of Cardioembolic Strokes.” Journal of Stroke 20 (2): 180-196. doi:10.5853/ jos.2018.00780. http://dx.doi.org/10.5853/jos.2018.00780. Published Version doi:10.5853/jos.2018.00780 Updates on Prevention of Cardioembolic Strokes Mehmet Akif Topcuoglu,a Liping Liu,b Dong-Eog Kim,c M. Edip Gurold aDepartment of Neurology, Hacettepe University, Ankara, Turkey bDepartment of Neurology, Beijing Tiantan Hospital, Capital Medical University, Beijing, China cDepartment of Neurology, Dongguk University Ilsan Hospital, Dongguk University College of Medicine, Goyang, Korea dDepartment of Neurology, Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA Mehmet Akif Topcuoglu,a Liping Liu,b Dong-Eog Kim,c M. Edip Gurold aDepartment of Neurology, Hacettepe University, Ankara, Turkey bDepartment of Neurology, Beijing Tiantan Hospital, Capital Medical University, Beijing, China cDepartment of Neurology, Dongguk University Ilsan Hospital, Dongguk University College of Medicine, Goyang, Korea dDepartment of Neurology, Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA Correspondence: M. Edip Gurol Department of Neurology, Massachusetts General Hospital, Hemorrhagic Stroke Research Program, 175 Cambridge Street, Suite 300, Boston, MA 02114, USA Tel: +1-617-726-8459 Fax: +1-506-700-2420 E-mail: edip@mail.harvard.edu Correspondence: M. Edip Gurol Department of Neurology, Massachusetts General Hospital, Hemorrhagic Stroke Research Program, 175 Cambridge Street, Suite 300, Boston, MA 02114, USA Tel: +1-617-726-8459 Fax: +1-506-700-2420 E-mail: edip@mail.harvard.edu Received: March 13, 2018 Revised: May 16, 2018 Accepted: May 23, 2018 Cardiac embolism continues to be a leading etiology of ischemic strokes worldwide. Although pathologies that result in cardioembolism have not changed over the past decade, there have been significant advances in the treatment and stroke prevention methods for these conditions. Atrial fibrillation remains the prototypical cause of cardioembolic strokes. The availability of new long-term monitoring devices for atrial fibrillation detection such as insertable cardiac monitors has allowed accurate detection of this leading cause of cardioembolism. The non-vitamin K an- tagonist oral anticoagulants have improved our ability to prevent strokes for many patients with non-valvular atrial fibrillation (NVAF). Advances in left atrial appendage closure and the U.S. Food and Drug Administration approval of the WATCHMAN (Boston Scientific) device for stroke prevention in NVAF patients who have an appropriate rationale for a nonpharmacological alter- native, have revolutionized the field and provided a viable option for patients at higher hemor- rhagic risk. The role of patent foramen ovale closure for secondary prevention in selected pa- tients experiencing cryptogenic ischemic strokes at a relatively young age has become clearer thanks to the very recent publication of long-term outcomes from three major studies. Updates on Prevention of Cardioembolic Strokes Advances in the management of infective endocarditis, heart failure, valvular diseases, and coronary artery disease have significantly changed the management of such patients, but have also revealed new concerns related to assessment of ischemic versus hemorrhagic risk in the setting of anti- thrombotic use. The current review article aims to discuss these advances especially as they per- tain to the stroke neurology practice. Received: March 13, 2018 Revised: May 16, 2018 Accepted: May 23, 2018 Keywords Ischemic stroke; Cardioembolism; Atrial fibrillation; Anticoagulants; Left atrial appendage closure; Patent foramen ovale closure Copyright © 2018 Korean Stroke Society This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. Share Your Story The Harvard community has made this article openly available. Please share how this access benefits you. Submit a story . Accessibility Journal of Stroke 2018;20(2):180-196 https://doi.org/10.5853/jos.2018.00780 Special Review Copyright © 2018 Korean Stroke Society This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. Copyright © 2018 Korean Stroke Society Correspondence: M. Edip Gurol Department of Neurology, Massachusetts General Hospital, Hemorrhagic Stroke Research Program, 175 Cambridge Street, Suite 300, Boston, MA 02114, USA Tel: +1-617-726-8459 Fax: +1-506-700-2420 E-mail: edip@mail.harvard.edu Correspondence: M. Edip Gurol Department of Neurology, Massachusetts General Hospital, Hemorrhagic Stroke Research Program, 175 Cambridge Street, Suite 300, Boston, MA 02114, USA Tel: +1-617-726-8459 Fax: +1-506-700-2420 E-mail: edip@mail.harvard.edu Received: March 13, 2018 Revised: May 16, 2018 Accepted: May 23, 2018 Introduction The United States guidelines also recommend a shared decision-making approach where the responsible clinician dis- cusses the advantages (prevention of embolism and associated strokes) as well as potential risks (major hemorrhage) of OAC medications with the patient. The same guidelines also suggest periodic review and discussion of the changing risk/benefit profile of the preventive treatment over time. Examples to such changes might include: starting a patient with initially low CHA2DS2-VASc on anticoagulation when the risk increases with age or as a result of the appearance of a new risk factor; con- sidering a patient on OAC for LAAC after sustaining ICH or manifesting any marker of high ICH risk such as brain micro- bleeds. A multidisciplinary approach that includes the cardiolo- gist, neurologist and other specialists when needed, is helpful for optimal management of AF patients with complications such as hemorrhagic risk. One revolutionary advance has been a better understanding of the causes and risk of another and much more severe type of stroke—intracerebral hemorrhage (ICH). ICHs constitute one- third of all strokes worldwide and carry a high risk of death and severe disability.3,7 Oral anticoagulants (OAC) including warfarin and NOACs are common causes of severe ICHs. Neuroimaging markers that can predict first-time or recurrent ICH risk have been validated.3 Neurologists who are familiar with both isch- emic and hemorrhagic stroke management need to balance the risks and benefits of different medical and nonpharmacological CE prevention approaches in conjunction with the cardiologists and other medical specialists. The most relevant markers of ICH risk are discussed in a separate paper on prevention of lacunar and hemorrhagic strokes published in this same issue of the Journal of Stroke.8 The current article will review advances in CE stroke prevention emphasizing the aspects most relevant to the practicing neurologists and stroke specialists. AF is also classified based on its temporal pattern as perma- nent, persistent, or paroxysmal. The paroxysmal category can be missed among inpatients even after an AF-related embolic stroke, and longer duration outpatient monitoring (≥72 hours) is therefore recommended. It is important to correctly diagnose or rule out AF as both failure to take appropriate prevention measures and the unnecessary use of OAC have serious conse- quences—embolic stroke and ICH respectively. Introduction Atrial fibrillation (AF) remains the prototypical cause of CE. Ex- ternal prolonged monitoring methods have allowed better de- tection of paroxysmal AF, and more recently, insertable cardiac monitors (ICMs) have improved accurate detection of even brief AF episodes for up to 3 years.1 Although not so novel, the non- vitamin K antagonist oral anticoagulants (NOAC) have im- proved stroke prevention in patients with non-valvular atrial fi- brillation (NVAF).1,2 The role of the left atrial appendage (LAA) in NVAF-related CE has become clearer and there have been Cardiac embolism (CE) is a leading etiology of ischemic strokes, the cause of 25% to 40% of cerebral infarctions worldwide. Cardioembolic strokes are associated with poor outcomes and relatively high recurrence rates compared to other ischemic stroke causes. The major causes of CE have not changed over the past decade, but significant advances in their primary treatment and stroke prevention methods have been achieved. pISSN: 2287-6391 • eISSN: 2287-6405 180 http://j-stroke.org Vol. 20 / No. 2 / May 2018 Vol. 20 / No. 2 / May 2018 major advances in left atrial appendage closure (LAAC) result- ing in the recent U.S. Food and Drug Administration (FDA) ap- proval of the WATCHMAN (Boston Scientific, Marlborough, MA, USA) device for stroke prevention in patients with appropriate rationale for nonpharmacological alternatives.3 Recent publica- tion of long-term follow up of patent foramen ovale (PFO) clo- sure trials have changed the landscape in that field resulting in FDA approval of this approach for secondary stroke preven- tion.4-6 Advances in the procedural management of infective endocarditis (IE), heart failure (HF), valvular disease, and coro- nary artery disease have resulted in improvements but have also revealed new concerns related to assessment of ischemic versus hemorrhagic risk in the setting of antithrombotic use. 2 to 3 is used for most other patients with AF. Embolic risk scores such as CHADS2, CHA2DS2-VASc, and ATRIA provide esti- mates of the risk of ischemic strokes in NVAF with some limi- tations.1,13-15 Despite the tendency to overestimate embolic risk, CHA2DS2-VASc has become the most commonly used embolic risk score recommended by both European and American AF management guidelines.9,10,15,16 The cutoff score for using OACs in NVAF is ≥1 per European guidelines and ≥2 per the US guidelines; patients with a score of 1 can be managed with either OAC or antiplatelet per the latter. Introduction A recent major advance in the field was the development of ICMs that were shown to be superior to conventional strategies for detecting AF.17 ICMs can be implanted with a very low complication risk (<1%) even as an outpatient and provide highly sensitive AF monitoring for 2 to 3 years (Figure 1).18 Other available outpa- tient monitoring strategies include Holter (24 to 72 hours), mobile cardiac outpatient telemetry devices (up to 4 weeks) and electrocardiographic patch devices (up to 4 weeks).1 These external devices have a relative advantage of ease of use and lower cost, but require patient compliance, have reduced sen- sitivity, and cannot be used for rhythm monitoring for longer than 2 to 4 weeks. The median time to detection of AF using ICM in a large randomized controlled trial (RCT) and the largest available observational cohort was 84 days (interquartile range https://doi.org/10.5853/jos.2017.00780 Topcuoglu et al. Prevention of Cardioembolic Strokes Topcuoglu et al. Prevention of Cardioembolic Strokes Figure 1. Placement of an insertable cardiac monitor. The insertable cardi- ac monitor is placed under the skin using an injection system through a simple incision. Reproduced with permission of Medtronic, Inc. (http:// www.medtronic.com). es the risk of ICH by 5 folds compared to placebo and 2 folds compared to aspirin.22 In NVAF patients without past history or high risk for ICH, warfarin is superior to placebo and aspirin for preventing all strokes but not vascular death or all cause deaths. Warfarin therefore has been the gold standard for stroke prevention in NVAF patients who do not have contrain- dications for long-term anticoagulation. A direct thrombin inhibitor (dabigatran) and three factor Xa inhibitors (rivaroxaban, apixaban, edoxaban) collectively called NOACs have been approved for the same indication (Table 1).23- 27 These drugs are noninferior to warfarin for overall stroke prevention in AF, and pose lower ICH risk.23 Dabigatran and ri- varoxaban however, have shown higher gastrointestinal hem- orrhage risk. These NOACs have shown a trend for mortality benefit compared to warfarin. They have multiple other advan- tages including ease of use. Their effect starts within 1 to 2 hours after the first dose and they have less food/drug interac- tions than warfarin. They do not require blood draws as their effect is fairly predictable in patients without renal failure. Their effect wears off after 24 to 48 hours of the last dose, which could be an advantage in case of bleeding or if surgery is needed, but also potentially a disadvantage for risk of embo- lism in patients with poor compliance (even when 1 to 2 doses are missed). Another advantage is that they have been proven to have similar efficacy to warfarin in the presence of comor- bidities not uncommonly seen with AF such as mild HF and history of cardioversion. Figure 1. Placement of an insertable cardiac monitor. The insertable cardi- ac monitor is placed under the skin using an injection system through a simple incision. Reproduced with permission of Medtronic, Inc. (http:// www.medtronic.com). Figure 1. Placement of an insertable cardiac monitor. The insertable cardi- ac monitor is placed under the skin using an injection system through a simple incision. Reproduced with permission of Medtronic, Inc. (http:// www.medtronic.com). Topcuoglu et al. Prevention of Cardioembolic Strokes Idarucizumab is a humanized monoclonal antibody fragment that binds dabigatran, and is currently FDA-approved as a re- versal agent for this direct thrombin inhibitor.28 Andexanet alfa, a modified recombinant derivative of factor Xa, was recently approved by FDA as a candidate reversal agent for rivaroxaban and apixaban.29 The clinical studies of both Idarucizumab and andexanet alfa provided evidence for reversal of the laboratory findings associated with the respective NOACs. However, none of these studies were randomized and we do not have evidence for the clinical efficacy of these reversal agents. It is important to use available reversal agents for NOAC-related hemorrhages, but it is unlikely that these drugs will improve outcomes espe- cially in ICHs that are already sizeable or at a critical brain lo- cation upon presentation. [IQR], 18 to 265) and 112 days (IQR, 35 to 293), respective- ly.17,19 Long-term (2 to 3 years) monitoring with ICM is thus an attractive approach with high accuracy for AF detection, with minimal patient compliance. All ischemic stroke patients should receive at least one electrocardiogram (EKG) and should ideally be kept on telemetry during hospital stay. For patients with non-lacunar infarcts, long-term monitoring should be considered if no other clear stroke etiology is found. Starting all such patients on a NOAC without confirming the presence of AF does not prevent ischemic strokes better than aspirin alone and poses increased risk of brain hemorrhages by 4 to 6.5 folds.20 There is a growing body of literature on the mini- mum duration of AF before occurrence of embolic events but until we obtain data from properly designed RCTs, even a short duration (1 to 2 minutes) of unprovoked AF should trigger con- sideration of preventive measures especially in high risk pa- tients such as ischemic stroke survivors. Disadvantages of NOACs include higher risk of gastrointesti- nal side effects especially hemorrhage, safety concerns in older adults with renal failure, higher cost, and poor compliance as a result of these problems. The FDA has issued warnings against NOAC use in patients with mechanical valves as well as in- creased risk of thromboembolic events even in case of 1 to 2 missed doses. Recent real-world data also showed that more Life-long OAC use is currently the norm for stroke prevention in patients with AF who are not at high risk of hemorrhage. 182 http://j-stroke.org Updates on stroke prevention in atrial fibrillation AF, whether related to rheumatic valve disease or not, remains the most important cause of CE worldwide.9 The prevalence of AF increases with age and it is closely linked to vascular risk factors such as hypertension, diabetes mellitus, coronary artery disease, HF, thyroid dysfunction, sleep apnea, obesity, smoking, and alcohol consumption.1,10 About 2% of the population younger than 65 years, and 9% of people aged 65 years or old- er have AF in the United States.11 The presence of AF increases the risk of stroke by about 5-fold in all age groups.12 Warfarin remains the only approved medication for valvular AF; used with dose-adjustment based on target international normal- ized ratio (INR). Concurrent aspirin and warfarin use with INR targets of 2.5 to 3.5 is recommended for patients with me- chanical heart valves, while warfarin alone with INR target of http://j-stroke.org 181 https://doi.org/10.5853/jos.2017.00780 https://doi.org/10.5853/jos.2017.00780 Topcuoglu et al. Prevention of Cardioembolic Strokes Warfarin decreases stroke risk by 64% compared to placebo, and 47% compared to aspirin in NVAF.21 Warfarin also increas- 182 http://j-stroke.org https://doi.org/10.5853/jos.2017.00780 https://doi.org/10.5853/jos.2017.00780 Vol. 20 / No. 2 / May 2018 Table 1. Summary of non-vitamin K antagonist oral anticoagulant studies Table 1. Summary of non-vitamin K antagonist oral anticoagulant studies Study Patients NOAC Comparison Outcome Conclusions for NOAC vs. warfarin Connolly et al. (2009)24 RE-LY trial NVAF, mean CHADS2=2.1, no prior ICH, CrCl ≥30 mL/min Dabigatran 150 mg twice daily (n=6,076) Warfarin, target INR=2–3, mean TTR=64% (n=6,022) Stroke, systemic embolism, death, major bleeds (ICH, GI) during 2 years of follow- up Noninferior for stroke/embolism & ma- jor bleeds, lower ICH risk but higher for GI bleeds, MI, GI upset with dabig- atran, permanent discontinuation (21.2% vs. 16.6%) Patel et al. (2011)25 ROCKET AF trial NVAF, mean CHADS2=3.5, no prior ICH, CrCl ≥30 mL/min Rivaroxaban 20 mg once daily (n=7,131) Warfarin, target INR=2–3, mean TTR=55% (n=7,133) Stroke, systemic embolism, death, major bleeds (ICH, GI) during 1.94 years of fol- low-up Noninferior for stroke/embolism & ma- jor bleeds, lower ICH risk but higher for GI bleed with rivaroxaban, perma- nent discontinuation (23.7% vs. 22.2%) Granger et al. (2011)26 ARISTOTLE trial NVAF, mean CHADS2=2.1, no prior ICH, CrCl ≥25 mL/min Apixaban 5 mg twice daily (n=9,120) Warfarin, target INR=2–3, mean TTR=62% (n=9,081) Stroke, systemic embolism, death, major bleeds (ICH, GI) during 1.8 years of fol- low-up Noninferior for stroke/embolism & ma- jor bleeds, lower ICH risk with apixa- ban, permanent discontinuation (25.3% vs. 27.5%) Giugliano et al. (2013)27 ENGAGE AF-TIMI 48 trial NVAF, mean CHADS2=2.8, no prior ICH, CrCl ≥30 mL/min Edoxaban 60 mg once daily (n=7,035) Warfarin, target INR=2–3, mean TTR=66% (n=7,036) Stroke, systemic embolism, death, major bleeds (ICH, GI) during 2.8 years of fol- low-up Noninferior for stroke/embolism & ma- jor bleeds, lower ICH risk with edoxa- ban, permanent discontinuation (34.4% vs. Topcuoglu et al. Prevention of Cardioembolic Strokes 34.5%) NOAC, non-vitamin K antagonist oral anticoagulant; RE-LY, The Randomized Evaluation of Long-Term Anticoagulation Therapy; NVAF, nonvalvular atrial fibril- lation; CHADS2, congestive heart failure, hypertension, age ≥75 years, diabetes mellitus, stroke (double weight); CrCl, creatinine clearance; INR, international normalized ratio; TTR, time in therapeutic range; ICH, intracranial hemorrhage; GI, gastrointestinal; MI, myocardial infarction; ROCKET AF, The Rivaroxaban Once Daily Oral Direct Factor Xa Inhibition Compared with Vitamin K Antagonism for Prevention of Stroke and Embolism Trial in Atrial Fibrillation; ARISTOTLE, Apixaban for Reduction in Stroke and Other Thromboembolic Events in Atrial Fibrillation; ENGAGE AF-TIMI 48, The Effective Anticoagulation with Factor Xa Next Generation in Atrial Fibrillation—Thrombolysis in Myocardial Infarction 48 trial. Noninferior for stroke/embolism & ma- jor bleeds, lower ICH risk but higher for GI bleeds, MI, GI upset with dabig- atran, permanent discontinuation (21.2% vs. 16.6%) Noninferior for stroke/embolism & ma- jor bleeds, lower ICH risk with edoxa- ban, permanent discontinuation (34.4% vs. 34.5%) NOAC, non-vitamin K antagonist oral anticoagulant; RE-LY, The Randomized Evaluation of Long-Term Anticoagulation Therapy; NVAF, nonvalvular atrial fibril- lation; CHADS2, congestive heart failure, hypertension, age ≥75 years, diabetes mellitus, stroke (double weight); CrCl, creatinine clearance; INR, international normalized ratio; TTR, time in therapeutic range; ICH, intracranial hemorrhage; GI, gastrointestinal; MI, myocardial infarction; ROCKET AF, The Rivaroxaban Once Daily Oral Direct Factor Xa Inhibition Compared with Vitamin K Antagonism for Prevention of Stroke and Embolism Trial in Atrial Fibrillation; ARISTOTLE, Apixaban for Reduction in Stroke and Other Thromboembolic Events in Atrial Fibrillation; ENGAGE AF-TIMI 48, The Effective Anticoagulation with Factor Xa Next Generation in Atrial Fibrillation—Thrombolysis in Myocardial Infarction 48 trial. NOAC, non-vitamin K antagonist oral anticoagulant; RE-LY, The Randomized Evaluation of Long-Term Anticoagulation Therapy; NVAF, nonvalvular atrial fibril- lation; CHADS2, congestive heart failure, hypertension, age ≥75 years, diabetes mellitus, stroke (double weight); CrCl, creatinine clearance; INR, international normalized ratio; TTR, time in therapeutic range; ICH, intracranial hemorrhage; GI, gastrointestinal; MI, myocardial infarction; ROCKET AF, The Rivaroxaban Once Daily Oral Direct Factor Xa Inhibition Compared with Vitamin K Antagonism for Prevention of Stroke and Embolism Trial in Atrial Fibrillation; ARISTOTLE, Apixaban for Reduction in Stroke and Other Thromboembolic Events in Atrial Fibrillation; ENGAGE AF-TIMI 48, The Effective Anticoagulation with Factor Xa Next Generation in Atrial Fibrillation—Thrombolysis in Myocardial Infarction 48 trial. Topcuoglu et al. Prevention of Cardioembolic Strokes LAA is known to be the site of thrombus in over 90% of NVAF patients with an embolic event.32 The complex morpho- logical features of the LAA are a possible explanation for this.33 Several different methods have been developed to exclude LAA from left atrium, and therefore systemic circulation. These methods include pure endocardial closure (least invasive), hy- brid endocardial and epicardial exclusion, and surgical oblitera- tion (Table 2 and Figure 2). Only one endocardial approach, WATCHMAN device, has been studied in phase 3 RCTs against warfarin for clinical stroke prevention (Figure 2A). The first study, Watchman Left Atrial Appendage System for Embolic Protection in Patients with Atrial Fibrillation (PROTECT AF), showed both non-inferiority and superiority of this approach for overall stroke prevention as well as significant reductions in ICH and mortality when compared to the warfarin arm.34 The second RCT was not powered to demonstrate clinical benefit but its analysis failed to show noninferiority to warfarin de- spite similar numerical event numbers, mainly due to an unex- pectedly low stroke rate in the warfarin arm.35 LAAC using WATCHMAN was approved by FDA for NVAF patients who need anticoagulation for stroke prevention and have an appropriate rationale to seek a nonpharmacological alternative to warfa- rin.3 These patients typically need OAC for 6 weeks after the procedure to prevent device thrombosis, followed by dual anti- platelets for another 4.5 months after which they are main- than half of patients receiving lower doses of NOACs, mostly for fear of side effects, did not have any indication for the low- er dose.30 It is important to use the appropriate doses of these medications as the use of lower and higher doses are associat- ed with elevated ischemic and hemorrhagic risks, respectively. All OACs, including NOACs, significantly increase the risk of ICH and other major and minor bleeding risks.31 None of the phase 3 NOAC studies included patients with past history of ICH or known high bleeding risk, so the effects of NOACs on such patients are unknown. https://doi.org/10.5853/jos.2017.00780 Noninferior for all stroke/embolism; su- perior for mortality and ICH preven- tion against warfarin Successful implantation (97.3%), cardi- ac tamponade (1.24%), procedure-re- lated stroke (0.86%), device emboliza- tion (0.77%), procedure-related deaths (0.76%), annual stroke rate (2.3%) Procedural safety and feasi- bility, 13-month follow-up Successful implantation (95.6%), cardi- ac tamponade (1.02%), procedure-re- lated stroke (0.078%), device emboli- zation (0.24%), procedure-related deaths (0.078%) Procedural performance and complication rates within 7 days Procedural performance and complication rates within 7 days Successful implantation (95.6%), cardi- ac tamponade (1.02%), procedure-re- lated stroke (0.078%), device emboli- zation (0.24%), procedure-related deaths (0.078%) Procedural performance and complication rates within 7 days c) antithrombotic protocol includes warfarin for 6 weeks followed by clopidogrel for 4.5 months and indefinite aspirin use. ) antithrombotic protocol includes aspirin and clopidogrel for 3 months followed by indefinite aspirin use. Post AMPLATZER (St. Jude Medical) antithrombotic protocol includes aspirin and clopidogrel for 3 months followed by indefinite aspirin use. LAAC, left atrial appendage closure; PROTECT AF, Watchman Left Atrial Appendage System for Embolic Protection in Patients with Atrial Fibrillation; NVAF, nonvalvular atrial fibrillation; CHADS2, congestive heart failure, hypertension, age ≥75 years, diabetes mellitus, stroke (double weight); LAA, left atrial append- age; INR, international normalized ratio; TTR, time in therapeutic range; ICH, intracranial hemorrhage; GI, gastrointestinal; PREVAIL, Watchman LAA Closure Device in Patients with Atrial Fibrillation Versus Long Term Warfarin Therapy; FDA, U.S. Food and Drug Administration. Figure 2. Left atrial appendage (LAA) closure devices and schematics of their deployment. Different types of LAA closure devices are seen. Endocardial devices in- clude (A) WATCHMAN™ (image provided courtesy of Boston Scientific, c2018 Boston Scientific Corporation or its affiliates, http://www.bostonscientific.com) and (B) AMPLATZER™ AMULET™ (reproduced with permission of St. Jude Medical, c2018, https://www.sjmglobal.com). (C) The hybrid (endocardial and epicardial) LAR- IAT™ suture delivery system for LAA exclusion (reproduced with permission of SENTREHEART, c2018, http://www.sentreheart.com) and (D) AtriClip™ for surgical clipping (reproduced with permission of AtriCure, c2018, https://www.atricure.com). Devices are trademarks of their respective companies, all rights reserved. A C B D B B A C D Figure 2. Left atrial appendage (LAA) closure devices and schematics of their deployment. Different types of LAA closure devices are seen. Endocardial devices in- clude (A) WATCHMAN™ (image provided courtesy of Boston Scientific, c2018 Boston Scientific Corporation or its affiliates, http://www.bostonscientific.com) and (B) AMPLATZER™ AMULET™ (reproduced with permission of St. Jude Medical, c2018, https://www.sjmglobal.com). (C) The hybrid (endocardial and epicardial) LAR- IAT™ suture delivery system for LAA exclusion (reproduced with permission of SENTREHEART, c2018, http://www.sentreheart.com) and (D) AtriClip™ for surgical clipping (reproduced with permission of AtriCure, c2018, https://www.atricure.com). Devices are trademarks of their respective companies, all rights reserved. 184 http://j-stroke.org Topcuoglu et al. Prevention of Cardioembolic Strokes Table 2. Summary of left atrial appendage closure studies Study Patients Intervention Comparison Outcome Conclusions for LAAC Reddy et al. (2014)34 PROTECT AF trial NVAF with mean CHADS2=2.2 LAA closure with the WATCHMAN® device, antithrom- botic protocol (n=463) Warfarin, target INR=2–3, mean TTR=66% (n=244) Stroke, systemic embolism, death, major bleeds (ICH, GI) during 3.8 years of fol- low-up Noninferior for all stroke/embolism; su- perior for mortality and ICH preven- tion against warfarin Holmes et al. (2014)35 PREVAIL trial NVAF with mean CHADS2=2.6 LAA closure with the WATCHMAN® device, antithrom- botic protocol (n=269) Warfarin, target INR=2–3, mean TTR=68% (n=138) Early/late (18 months) safety and efficacy Improved procedural safety, noninferior for prevention of ischemic stroke and systemic embolism >7 days post-pro- cedure against warfarin Tzikas et al. (2016)37 A retrospective obser- vational study NVAF with mean CHADS2=2.8 LAA closure with the AMPLATZER® Cardiac Plug, an- tithrombotic pro- tocol (n=1,047) Not available Procedural safety and feasi- bility, 13-month follow-up Successful implantation (97.3%), cardi- ac tamponade (1.24%), procedure-re- lated stroke (0.86%), device emboliza- tion (0.77%), procedure-related deaths (0.76%), annual stroke rate (2.3%) Reddy et al. (2017)36 A national clinical registry study in the U.S. NVAF, consecu- tive WATCH- MAN® cases af- ter FDA ap- proval LAA closure with the WATCHMAN® device, antithrom- botic protocol (n=3,822) Not available Procedural performance and complication rates within 7 days Successful implantation (95.6%), cardi- ac tamponade (1.02%), procedure-re- lated stroke (0.078%), device emboli- zation (0.24%), procedure-related deaths (0.078%) Post WATCHMAN (Boston Scientific) antithrombotic protocol includes warfarin for 6 weeks followed by clopidogrel for 4.5 months and indefinite aspirin use. Post AMPLATZER (St. Jude Medical) antithrombotic protocol includes aspirin and clopidogrel for 3 months followed by indefinite aspirin use. LAAC, left atrial appendage closure; PROTECT AF, Watchman Left Atrial Appendage System for Embolic Protection in Patients with Atrial Fibrillation; NVAF, nonvalvular atrial fibrillation; CHADS2, congestive heart failure, hypertension, age ≥75 years, diabetes mellitus, stroke (double weight); LAA, left atrial append- age; INR, international normalized ratio; TTR, time in therapeutic range; ICH, intracranial hemorrhage; GI, gastrointestinal; PREVAIL, Watchman LAA Closure Device in Patients with Atrial Fibrillation Versus Long Term Warfarin Therapy; FDA, U.S. Food and Drug Administration. Topcuoglu et al. Prevention of Cardioembolic Strokes Even in survivors of embolic ap- pearing cerebral infarct at low baseline ICH risk, NOAC use was associated with a 4- to 6.5-fold higher risk of brain hemor- rhages compared to aspirin in a recently published RCT (NAVI- GATE ESUS).20 It is also well-known that NOAC-related ICH have very high mortality and disability rates, similar to warfa- rin-related ICH.3,16 It is therefore important to consider stroke prevention measures that do not rely on long-term anticoagu- lation in patients who have a higher hemorrhagic risk. Such patients include survivors of any type of ICH, people having imaging markers of higher ICH risk such as microbleeds, corti- cal superficial siderosis, moderate-to-severe white matter dis- ease, older adults with dementia or high risk of falling, and pa- tients with past history of bleeding in a different organ system. The neurologic conditions that increase ICH risk are further de- tailed in another article from this issue and a recent review.3,8 http://j-stroke.org 183 /j-stroke.org 183 https://doi.org/10.5853/jos.2017.00780 https://doi.org/10.5853/jos.2017.00780 https://doi.org/10.5853/jos.2017.00780 https://doi.org/10.5853/jos.2017.00780 Vol. 20 / No. 2 / May 2018 tained on aspirin. WATCHMAN is commonly used in NVAF pa- tients at higher hemorrhagic risk and the post-marketing expe- rience has shown a favorable safety profile (Table 1).36 monitoring with ICM in addition to routinely performed brain magnetic resonance imaging (MRI) and cardiovascular testing such as computed tomography angiography (CTA)/magnetic resonance angiography (MRA) and echocardiography. If one of these more common etiologies is confirmed, preventive mea- sures should be selected accordingly. Once the known stroke etiologies are ruled out, the potential cause-and-effect rela- tionship between PFO and stroke should be reviewed. This sec- tion thus starts with a concise but adequately detailed review of the connection between PFO and stroke. AMPLATZER AMULET (St. Jude Medical, St. Paul, MN, USA) device (Figure 2B), a different type of pure endocardial LAA Oc- cluder (St. Jude Medical) showed good safety profile in one ob- servational study; an RCT comparing its safety and efficacy against WATCHMAN is currently underway.37 Another interest- ing feature of AMPLATZER AMULET device is that the observa- tional studies mostly used 3 months of dual antiplatelets with acceptable safety profile. LARIAT suture delivery system (Sen- treHeart, Redwood City, CA, USA) provides a hybrid approach that involves both endocardial and epicardial access, that does not leave any foreign object within the heart (Figure 2C).38 As a result, the observational studies used only aspirin after suc- cessful LAA exclusion with LARIAT. Currently the AMAZE trial aimed at comparing the safety and efficacy of LARIAT based LAA ligation together with pulmonary vein isolation (PVI) to PVI without LAA exclusion is ongoing. This study will provide information on LARIAT procedure use in conjunction with PVI compared to PVI alone. Another device, the AtriClip (AtriCure, Mason, OH, USA) is used during cardiac surgeries to clip the LAA (Figure 2D). The device has shown higher success rates (>95%) than surgical ligation or stapling.39 Overall, the proce- dural risks such as pericardial effusion, device embolization, stroke, and death should be discussed with every patient plan- ning to undergo LAAC. LAAC methods have become an impor- tant aspect of stroke prevention in NVAF patients at high hem- orrhagic risk. PFO is an interatrial foramen that provides a conduit for left atrial blood flow from the right atrium in fetal life, expected to close soon after initiation of breathing at birth. https://doi.org/10.5853/jos.2017.00780 However, com- plete closure does not happen in about one-fourth of the gen- eral population.42 When PFO remains open, it is not a real hole- like aperture, but rather a door-shaped channel. The blood pas- sage through this door, called as right-to-left shunt (RLS), oc- curs after increase in the right atrial pressures if not present in the resting state. The major mechanism of ischemic stroke in the setting of PFO is “paradoxical embolism,” in which the thromboembolic material originating from veins in the leg reaches the cerebral circulation after passing through the PFO because of RLS. Ischemic strokes may also be caused by embo- lism due to in situ thrombus formation in the PFO channel and/ or PFO-related atrial cardiomyopathy and/or arrhythmia asso- ciated with the latter. Definitive prevention of recurrent paradoxical embolism is achieved by closure of the PFO (Figure 3). Therefore, clarifica- tion of the role of PFO in the occurrence of otherwise idiopath- ic cerebral embolism is of critical importance. Broadly, ischemic stroke related to PFO can occur as a result of either: (1) throm- bus formation in or around the PFO or (2) more commonly from paradoxical embolism of thrombus formed in the venous system traveling through the PFO because of RLS. The final stroke prevention approach in NVAF should always be determined based on a shared decision-making meeting with the patient and family, after a thorough discussion of concurrent risks and benefits of different approaches. https://doi.org/10.5853/jos.2017.00780 Jude Medical) with Medical Treatment in Patients with Cryptogenic Embolism; CLOSE, Patent Foramen Ovale Closure or Anticoagulants Versus Antiplatelet Therapy to Prevent Stroke Recurrence; DAPT, dual antiplatelet therapy (aspirin and clopidogrel); RESPECT, Randomized Evaluation of Recurrent Stroke Comparing PFO Closure to Established Current Standard of Care Treatment; Gore REDUCE Clinical Study, GORE® HELEX® Septal Occluder and GORE® CARDIOFORM Septal Occluder for Patent Foramen Ovale (PFO) Closure in Stroke Patients. CLOSURE I, Evaluation of the STARFlex Septal Closure System (NMT Medical Inc.) in Patients with a Stroke and/or Transient Ischemic Attack due to Presumed Paradoxical Embolism through a Patent Foramen Ovale; PFO, patent foramen ovale; TIA, transient ischemic attack; PC trial, Clinical Trial Comparing Percutaneous Closure of Patent Foramen Ovale Using the AMPLATZER (St. Jude Medical) PFO Occluder (St. Jude Medical) with Medical Treatment in Patients with Cryptogenic Embolism; CLOSE, Patent Foramen Ovale Closure or Anticoagulants Versus Antiplatelet Therapy to Prevent Stroke Recurrence; DAPT, dual antiplatelet therapy (aspirin and clopidogrel); RESPECT, Randomized Evaluation of Recurrent Stroke Comparing PFO Closure to Established Current Standard of Care Treatment; Gore REDUCE Clinical Study, GORE® HELEX® Septal Occluder and GORE® CARDIOFORM Septal Occluder for Patent Foramen Ovale (PFO) Closure in Stroke Patients. CLOSURE I, Evaluation of the STARFlex Septal Closure System (NMT Medical Inc.) in Patients with a Stroke and/or Transient Ischemic Attack due to Presumed Paradoxical Embolism through a Patent Foramen Ovale; PFO, patent foramen ovale; TIA, transient ischemic attack; PC trial, Clinical Trial Comparing Percutaneous Closure of Patent Foramen Ovale Using the AMPLATZER (St. Jude Medical) PFO Occluder (St. Jude Medical) with Medical Treatment in Patients with Cryptogenic Embolism; CLOSE, Patent Foramen Ovale Closure or Anticoagulants Versus Antiplatelet Therapy to Prevent Stroke Recurrence; DAPT, dual antiplatelet therapy (aspirin and clopidogrel); RESPECT, Randomized Evaluation of Recurrent Stroke Comparing PFO Closure to Established Current Standard of Care Treatment; Gore REDUCE Clinical Study, GORE® HELEX® Septal Occluder and GORE® CARDIOFORM Septal Occluder for Patent Foramen Ovale (PFO) Closure in Stroke Patients. taking instruments such as the trumpet.45 The presence of these factors should be assessed whenever PFO is considered as the cause for an ischemic stroke. Demonstration of RLS at rest or with Valsalva on cardiac ultrasonography confirms the propensity for paradoxical embolism. right atrial and pulmonary artery pressures. This may be per- manent, or it may only be elevated temporarily during the on- set of stroke. 5 complicati tion (6.6% (3.2-year m CLOSURE I, Evaluation of the STARFlex Septal Closure System (NMT Medical Inc.) in Patients with a Stroke and/or Transient Is Paradoxical Embolism through a Patent Foramen Ovale; PFO, patent foramen ovale; TIA, transient ischemic attack; PC trial, Clinic Closure of Patent Foramen Ovale Using the AMPLATZER (St. Jude Medical) PFO Occluder (St. Jude Medical) with Medical Treatm Embolism; CLOSE, Patent Foramen Ovale Closure or Anticoagulants Versus Antiplatelet Therapy to Prevent Stroke Recurrence; (aspirin and clopidogrel); RESPECT, Randomized Evaluation of Recurrent Stroke Comparing PFO Closure to Established Current St REDUCE Clinical Study, GORE® HELEX® Septal Occluder and GORE® CARDIOFORM Septal Occluder for Patent Foramen Ovale (PFO Table 3. Summary of multicenter randomized clinical trials on patent foramen ovale closure Study Patients Intervention Comparison Outcome Conclusions Furlan et al. (2012)40 CLOSURE I trial PFO with recent (<6 months) crypto- genic stroke or TIA (18–60 years old) PFO closure with the STARFlex Septal Closure System®, clopidogrel for 6 months & aspirin in- definitely (n=447) Warfarin or as- pirin or both (n=462) A composite of stroke/ TIA, death Lower rate of composite end point in clo- sure group (5.5% vs. 6.8%) but statisti- cally not significant (2-year mean follow- up) Meier et al. (2013)41 PC trial PFO with cryptogenic stroke, TIA, or a pe- ripheral thrombo- embolic event (<60 years old) PFO closure with AM- PLATZER PFO Occluder®, ticlopidine/clopidogrel for 1–6 months & aspi- rin for ≥5 months (n=204) Antiplatelet therapy or oral anticoagula- tion (n=210) A composite of death, nonfatal stroke, TIA, or peripheral embo- lism Lower rate of composite end point in clo- sure group (3.4% vs. 5.2%) but statisti- cally not significant (4-year mean follow- up) Mas et al. (2017)4 CLOSE trial PFO with recent (<6 months) stroke at- tributed to PFO, and atrial septal aneu- rysm or large inter- atrial shunt (16–60 years old) PFO closure, DAPT for 3 months followed by antiplatelet therapy in- definitely (n=238) Antiplatelet therapy only arm (n=235) & oral anticoag- ulation arm (n=187) Occurrence of fatal or nonfatal stroke Significantly lower stroke risk in closure group compared to antiplatelet arm (0% vs. 6%) but an increased risk of atrial fi- brillation after closure (4.6% vs. 0.9%). Stroke rate 1.5% in anticoagulation group vs. 3.8% in the matched antiplatelet-only subcohort (5-year mean follow-up) Saver et al. Table 3. Summary of multicenter randomized clinical trials on patent foramen ovale closure Study Patients Intervention Comparison Outcome Furlan et al. (2012)40 CLOSURE I trial PFO with recent (<6 months) crypto- genic stroke or TIA (18–60 years old) PFO closure with the STARFlex Septal Closure System®, clopidogrel for 6 months & aspirin in- definitely (n=447) Warfarin or as- pirin or both (n=462) A composite of stroke/ TIA, death Lower rate o sure group cally not s up) Meier et al. (2013)41 PC trial PFO with cryptogenic stroke, TIA, or a pe- ripheral thrombo- embolic event (<60 years old) PFO closure with AM- PLATZER PFO Occluder®, ticlopidine/clopidogrel for 1–6 months & aspi- rin for ≥5 months (n=204) Antiplatelet therapy or oral anticoagula- tion (n=210) A composite of death, nonfatal stroke, TIA, or peripheral embo- lism Lower rate o sure group cally not s up) Mas et al. (2017)4 CLOSE trial PFO with recent (<6 months) stroke at- tributed to PFO, and atrial septal aneu- rysm or large inter- atrial shunt (16–60 years old) PFO closure, DAPT for 3 months followed by antiplatelet therapy in- definitely (n=238) Antiplatelet therapy only arm (n=235) & oral anticoag- ulation arm (n=187) Occurrence of fatal or nonfatal stroke Significantly group com vs. 6%) bu brillation a Stroke rate 1 vs. 3.8% in subcohort Saver et al. (2017)5 RESPECT trial PFO with cryptogenic ischemic stroke (<270 days) (18–60 years old) PFO closure with the AMPLATZER PFO Oc- cluder®, DAPT for 1 month followed by as- pirin only for 5 months, then antithrombotic use per treating physi- cian (n=499) Any antiplatelet therapy or oral anticoagula- tion (n=481) A composite of recur- rent nonfatal or fatal ischemic stroke, or early death after ran- domization Significantly emic strok venous thr arm (3.4% follow-up) Søndergaard et al. (2017)6 Gore RE- DUCE Clinical Study PFO with cryptogenic stroke (<180 days), 81% with moder- ate/large interatrial shunts (18–59 years old) PFO closure with the He- lex Septal Occluder® or the Cardioform Septal Occluder®, 300 mg clopidogrel load then antiplatelet monother- apy (n=441) Any antiplatelet monotherapy (n=223) Co-primary end points: (1) Clinical ischemic stroke, (2) composite of clinical ischemic stroke or silent brain infarction detected on imaging Significantly (1.4% vs. Søndergaard et al. (2017)6 Gore RE- DUCE Clinical Study A composite of death, nonfatal stroke, TIA, or peripheral embo- lism Occurrence of fatal or nonfatal stroke (2017)5 RESPECT trial PFO with cryptogenic ischemic stroke (<270 days) (18–60 years old) PFO closure with the AMPLATZER PFO Oc- cluder®, DAPT for 1 month followed by as- pirin only for 5 months, then antithrombotic use per treating physi- cian (n=499) Any antiplatelet therapy or oral anticoagula- tion (n=481) A composite of recur- rent nonfatal or fatal ischemic stroke, or early death after ran- domization Significantly lower rate of recurrent isch- emic strokes (3.6% vs. 5.8%) but higher venous thromboembolism in the closure arm (3.4% vs. 0.8%) (5.9-year median follow-up) Søndergaard et al. PFO with cryptogenic PFO closure with the He- Any antiplatelet Co-primary end points: Significantly lower clinical ischemic stroke Table 3. Summary of multicenter randomized clinical trials on patent foramen ovale closure A composite of death, nonfatal stroke, TIA, or peripheral embo- lism Lower rate of composite end point in clo- sure group (3.4% vs. 5.2%) but statisti- cally not significant (4-year mean follow- up) Significantly lower stroke risk in closure group compared to antiplatelet arm (0% vs. 6%) but an increased risk of atrial fi- brillation after closure (4.6% vs. 0.9%). Stroke rate 1.5% in anticoagulation group vs. 3.8% in the matched antiplatelet-only subcohort (5-year mean follow-up) Significantly lower rate of recurrent isch- emic strokes (3.6% vs. 5.8%) but higher venous thromboembolism in the closure arm (3.4% vs. 0.8%) (5.9-year median follow-up) Significantly lower stroke risk in closure group compared to antiplatelet arm (0% vs. 6%) but an increased risk of atrial fi- brillation after closure (4.6% vs. 0.9%). Stroke rate 1.5% in anticoagulation group vs. 3.8% in the matched antiplatelet-only subcohort (5-year mean follow-up) Significantly lower rate of recurrent isch- emic strokes (3.6% vs. 5.8%) but higher venous thromboembolism in the closure arm (3.4% vs. 0.8%) (5.9-year median follow-up) Any antiplatelet therapy or oral anticoagula- tion (n=481) A composite of recur- rent nonfatal or fatal ischemic stroke, or early death after ran- domization Significantly lower rate of recurrent isch- emic strokes (3.6% vs. 5.8%) but higher venous thromboembolism in the closure arm (3.4% vs. 0.8%) (5.9-year median follow-up) Søndergaard et al. Secondary stroke prevention in patients with patent foramen ovale The presence of high risk PFO features might be associated with thrombus formation in situ. These features include several morphological and physiological characteristics such as large flow of RLS, presence of long, wide or irregular PFO channel, massive shunting at rest, atrial septal hypermobility, atrial sep- tal aneurysm (ASA), complex right atrial structures such as Eu- stachian valve and Chiari’s network.43 Probably, flow grade of RLS is the most important one in terms of paradoxical embo- lism, but presence of any of the other factors can suggest PFO as the site of thrombus formation.44 PFO is frequently detected in patients with ischemic stroke. While PFO is convincingly responsible for the etiology in some stroke patients, it may also be considered an incidental finding in others. PFO has regained attention in stroke practice after announcement of the positive percutaneous PFO closure stud- ies in 2017 (Table 3 and Figure 3).4-6,40,41 However, incorporating this preventive approach into daily clinical practice is challeng- ing. Well-known causes of ischemic stroke such as large vessel disease, AF and cerebral small vessel disease should be appro- priately ruled out with detailed testing before attributing a stroke to PFO. Such testing might involve long-term cardiac Paradoxical embolism of clot formed somewhere in the ve- nous system would require demonstration of RLS and evidence of venous clot formation or at least a propensity to develop clot formation. The first criterion thus would be elevation of http://j-stroke.org 185 https://doi.org/10.5853/jos.2017.00780 (2017)6 Gore RE- DUCE Clinical Study PFO with cryptogenic stroke (<180 days), 81% with moder- ate/large interatrial shunts (18–59 years old) PFO closure with the He- lex Septal Occluder® or the Cardioform Septal Occluder®, 300 mg clopidogrel load then antiplatelet monother- apy (n=441) Any antiplatelet monotherapy (n=223) Co-primary end points: (1) Clinical ischemic stroke, (2) composite of clinical ischemic stroke or silent brain infarction detected on imaging Significantly lower clinical ischemic stroke (1.4% vs. 5.4%) but higher rates of device complications (1.4%) and atrial fibrilla- tion (6.6% vs. 0.4%) in the closure arm (3.2-year median follow-up) CLOSURE I, Evaluation of the STARFlex Septal Closure System (NMT Medical Inc.) in Patients with a Stroke and/or Transient Ischemic Attack due to Presumed Paradoxical Embolism through a Patent Foramen Ovale; PFO, patent foramen ovale; TIA, transient ischemic attack; PC trial, Clinical Trial Comparing Percutaneous Closure of Patent Foramen Ovale Using the AMPLATZER (St. Jude Medical) PFO Occluder (St. Jude Medical) with Medical Treatment in Patients with Cryptogenic Embolism; CLOSE, Patent Foramen Ovale Closure or Anticoagulants Versus Antiplatelet Therapy to Prevent Stroke Recurrence; DAPT, dual antiplatelet therapy (aspirin and clopidogrel); RESPECT, Randomized Evaluation of Recurrent Stroke Comparing PFO Closure to Established Current Standard of Care Treatment; Gore REDUCE Clinical Study, GORE® HELEX® Septal Occluder and GORE® CARDIOFORM Septal Occluder for Patent Foramen Ovale (PFO) Closure in Stroke Patients. Significantly lower clinical ischemic stroke (1.4% vs. 5.4%) but higher rates of device complications (1.4%) and atrial fibrilla- tion (6.6% vs. 0.4%) in the closure arm (3.2-year median follow-up) Any antiplatelet monotherapy (n=223) tiplatelet otherapy 23) Co-primary end points: (1) Clinical ischemic stroke, (2) composite of clinical ischemic stroke or silent brain infarction detected on imaging Co-primary end points: (1) Clinical ischemic stroke, (2) composite of clinical ischemic stroke or silent brain infarction detected on imaging CLOSURE I, Evaluation of the STARFlex Septal Closure System (NMT Medical Inc.) in Patients with a Stroke and/or Transient Ischemic Attack due to Presumed Paradoxical Embolism through a Patent Foramen Ovale; PFO, patent foramen ovale; TIA, transient ischemic attack; PC trial, Clinical Trial Comparing Percutaneous Closure of Patent Foramen Ovale Using the AMPLATZER (St. Jude Medical) PFO Occluder (St. Any antiplatelet therapy or oral anticoagula- tion (n=481) A composite of recur- rent nonfatal or fatal ischemic stroke, or early death after ran- domization Therefore, any temporary maneuver increasing pressures in the right heart (Valsalva maneuver) such as strain, heavy lifting, forced defecation, coitus, and diving should be questioned carefully in every case with suspicion of paradoxi- cal embolism. Chronic and effortful coughing may also result in increased right atrial and pulmonary pressures. Permanent pulmonary hypertension causes include chronic obstructive pulmonary disease, obstructive sleep apnea, chronic and/or massive pulmonary thromboembolism (PTE), right-sided acute myocardial infarction (AMI), adult respiratory distress syn- drome, and the trumpet player syndrome, which is an occupa- tional condition due to chronic and zealous playing of breath- The second set of criteria for paradoxical embolism are about the presence of deep vein thrombosis (DVT) and/or PTE or dem- onstration of a hypercoagulable state. For these purposes, the presence of DVT in the lower extremity should be investigated by valid methods such as compression ultrasonography, com- puted tomography, MRI, contrast venography, or scintigraphy in all cases with cryptogenic stroke and PFO. The presence of thrombus in pelvic, iliac, and caval veins should be assessed by pelvic magnetic resonance venography (MRV) or computed to- 186 http://j-stroke.org 186 http://j-stroke.org https://doi.org/10.5853/jos.2017.00780 https://doi.org/10.5853/jos.2017.00780 Vol. 20 / No. 2 / May 2018 Figure 3. Schematic of patent foramen ovale (PFO) closure. Schematic illustrating the endovascular procedure used to close a PFO using AMPLATZER™ PFO Occluder (St. Jude Medical). (A) The catheter is inserted through the PFO, (B, C) followed by expansion of the left sided disc and (D) deployment of the device to occlude the PFO (showing the device in place). AMPLATZER and St. Jude Medical are trademarks of St. Jude Medical, LLC or its related companies. Repro- duced with permission of St. Jude Medical, c2018 (https://www.sjmglobal.com). All rights reserved. A C B D B D A B C D Figure 3. Schematic of patent foramen ovale (PFO) closure. Schematic illustrating the endovascular procedure used to close a PFO using AMPLATZER™ PFO Occluder (St. Jude Medical). (A) The catheter is inserted through the PFO, (B, C) followed by expansion of the left sided disc and (D) deployment of the device to occlude the PFO (showing the device in place). AMPLATZER and St. Jude Medical are trademarks of St. Jude Medical, LLC or its related companies. Repro- duced with permission of St. Jude Medical, c2018 (https://www.sjmglobal.com). All rights reserved. suction and any laparoscopy carry high stroke risk if RLS is present. Another important point regarding DVT testing is that DVT may develop very rapidly after stroke onset, and it is abso- lutely necessary to carry out the examination within the first few days after stroke (or immobility) onset. If the DVT is de- tected later, it cannot be established whether the DVT is either the cause or the result of the stroke. Even if the initial stroke is not due to PFO, the development of DVT confers a high-risk status for stroke recurrence in paretic stroke patients with PFO. Ttranscatheter PFO closure may sometimes be considered in such situations as well. mographic venography.46 Pelvic MRV can also show the pres- ence of May-Thurner anatomy characterized by compression of the left common iliac vein by the overlying right common iliac artery, a condition that can result in compression of the com- mon venous outflow tract of the left lower extremity increas- ing the risk of DVT. Plasma D-dimer level elevation can be used as a screening tool for DVT diagnosis.47 If DVT cannot be docu- mented, this criterion may be met by the presence of DVT risk factors, especially useful in non-acute situations. In other words, the inability to demonstrate DVT does not rule out the possibility of paradoxical embolism. In this context, immobility, occult hereditary thrombophilia, oral contraceptive use, preg- nancy and postpartum period, and May-Thurner anatomy in- crease the risk. Current options to prevent ischemic stroke recurrence attrib- uted to PFO include antiplatelet medications, anticoagulants, and transcatheter PFO closure. The choice between antiplatelet and anticoagulant agents for secondary prophylaxis is based on the status of the systemic and hematological factors. It should be noted that there is no significant difference for ischemic pre- vention between the two groups of drugs in the absence of thrombophilia or DVT/PTE.50 Long-term use of OACs would in- crease the risk of hemorrhagic complications and most impor- tantly fatal/disabling ICH.3 On the other hand, if a hypercoagula- ble state that requires life-long anticoagulation such as anticar- diolipin antibody syndrome is found, long-term anticoagulation is the only option although PFO closure might still have a role in these situations. It is important to remember that medical man- For every patient, activated protein C resistance, factor-5 Leiden mutation, antiphospholipid antibody titers, prothrombin 20210 polymorphism, homocysteine levels, and associated methylenetetrahydrofolate reductase mutations, should be tested for thrombophilia detection. https://doi.org/10.5853/jos.2017.00780 Protein S, protein C, anti- thrombin III and factor VIII concentrations and/or activities should be studied in patients without these factors.48 Long plane travel (“economy-class stroke syndrome”) is a risk for embolism in patients with PFO.49 In addition, several surgical procedures such as neurosurgical posterior fossa surgery, intra- medullary orthopedic surgery, lower limb venous surgery, lipo- http://j-stroke.org 187 http://j-stroke.org 187 Topcuoglu et al. Prevention of Cardioembolic Strokes agement should involve aggressive detection and control of all vascular risk factors in patients with PFO and stroke. obtained during the index stroke, documenting one or more previous embolic (cortical) infarcts, contraindications to use of long-term anticoagulants or antiplatelets and high-risk fea- tures of PFO would support PFO closure especially in the young patient with high-flow RLS. Waiting for recurrence, that is, a second stroke with associated disability/death risks, would not be rational in these cases. It is appropriate to reiterate that there are no data suggesting that every PFO should be closed following the first embolic event. PFO closure for primary pro- phylaxis is not recommended for any reason including migraine with aura in subjects who never experienced clinical paradoxi- cal embolism, as there are no objective data showing the ben- efit of closure in these situations. Five recently-published RCTs showed that transcatheter PFO closure plus antiplatelet treatment is superior to antiplatelet therapy alone for secondary stroke prevention (Table 3 and Fig- ure 3).4-6,40,41 These studies showed that the frequency of recur- rent embolic stroke can be reduced from 1.1 to 0.53 per 100 patient-years with the transcatheter PFO closure, corresponding to 50% relative risk reduction, 2.11% absolute risk reduction, and a number needed to treat of 46.5 over about 4 years follow- up.51-53 There was no significant difference in the risk of tran- sient ischemic attack, mortality and bleeding rates between transcatheter PFO closure and antiplatelet-only approaches. The most noticeable adverse effect during post-transcatheter PFO closure period is an increase in the frequency of AF (1.3 vs. 0.25 in 100-patient years, referring to an approximately five times relative risk increase).51 Many of the AF episodes are transient and limited to the periprocedural phase. Indeed, three quarters of these post-transcatheter PFO closure AF episodes occur within 45 days, and almost all disappear in up to 6 months. It is recommended for these patients to receive anticoagulants for 6 months, provided that persistent normalization of the rhythm can be confirmed. The studies assessing PFO closure used only a simple EKG or short-term Holter to rule out concomitant AF; so, the extent of AF in these patients is not well-known. In addi- tion, an increase in venous thromboembolism frequency was noted in cases of switching anticoagulants to antiplatelets after PFO closure in patients with thrombophilia. In patients with stroke due to PFO, the risk of embolism re- currence is very low after complete PFO closure. It is well known that shunt retention after percutaneous PFO closure is about 10%. However, these residual shunts do not lead to an increased embolism risk because their sizes are very small, and their apertures are meshed with the device.56 Therefore, if there is an ischemic stroke recurrence after percutaneous PFO clo- sure, it is more likely to be related to a different etiologic mechanism. It should be remembered that PFO closure would only prevent ischemic strokes attributable to PFO and decrease the risk of bleeding related to long-term anticoagulant use. In conclusion, the PFO stroke relationship must be carefully as- sessed for each patient, and treatment decisions should involve a multidisciplinary team as well as a formal shared decision- making process. Despite the high popularity in medical and lay social media, these recent data did not change routine clinical practice in PFO management. Some of the concerns voiced are that: (1) none of these studies was completely blinded; (2) the rates of lost to follow-up and withdrawal of consent were high; (3) the enrollment criteria were heterogenous and restrictive; and (4) different devices and especially different antithrombotic regi- mens were used.54,55 Vol. 20 / No. 2 / May 2018 rebral MRI may be considered in all patients with left-sided IE even in the absence of CNS signs and symptoms. In a seminal French study, MRI documented at least one category of lesions in 82% of 106 IE patients, including ischemic lesions in 68, mi- crohemorrhages in 74, and silent aneurysms in 10, albeit only 12% of them had acute neurologic symptoms.62 Brain MRI findings (microinfarcts, cerebral microbleeds [CMBs], ICMAs, and leptomeningeal contrast enhancement) may assist in not only tailoring subsequent medical and surgical management, but also clarification of ischemic/hemorrhagic risks. MRI fre- quently shows multi-territorial cortical and subcortical embolic infarcts of various diameter in IE. The more specific forms are numerous, multi-territorial, punctate infarcts creating a “star- ry-sky view on diffusion-weighted imaging (DWI)” and combi- nation of multiple microinfarcts and microabscesses.63 Clinical- ly, multifocal and poorly localized symptoms and signs are fre- quent. Focal findings and encephalopathy can be seen together. A non-focal embolic encephalopathy is a less common, but more typical manifestation of IE.63 sulting in a more rapidly progressive and destructive infection. The most common cause of subacute NVIE is viridans group streptococcus (VGS), which causes an erosive IE that frequently develops on previously diseased and deformed valves. The most common cause of PVIE is coagulase negative staphylococcus.58 Significant systemic symptoms of IE are related to fever, HF, valve insufficiency, cerebral and peripheral embolism. Neuro- logical involvement associated with IE is common, and diverse. Approximately half of IE patients have neurological complaints at admission including focal deficits, encephalopathy, and epi- leptic seizures. Cerebral abscesses and meningitis are not un- common. Symptomatic neurovascular events include septic (infectious) cerebral embolism, central nervous system (CNS) hemorrhages, and mycotic aneurysms. Neurological complica- tions are more prevalent in the early course, usually before ini- tiation of the first dose of antibiotics, and their occurrence generally indicates poorer prognosis. In this section, we will fo- cus on cerebral septic embolism, IE-related CNS hemorrhages, and intracranial mycotic aneurysms (ICMAs). With advances in MRI techniques, previously less well-known pathologies such as microbleeds, superficial siderosis and smaller infarcts are commonly detected nowadays in IE clinical practice necessitat- ing neurology consultations. Acute ischemic stroke management in IE differs from other diseases in many respects. Firstly, standard IV tissue plasmino- gen activator administration in the acute phase is not safe due to high bleeding rates so this approach should be avoided.64 Experience indicates that mechanical thrombectomy can be performed more safely in the setting of IE.65 In addition, anti- coagulant treatment is highly perilous before complete control of infection (confirmed by three negative blood cultures) and confirmation of the lack of mycotic aneurysms or other pathol- ogy associated with high ICH risk. The risk of CNS hemorrhagic complications due to anticoagulants may not be completely excluded even with documentation of absence of cerebral sep- tic vasculitis and/or mycotic microaneurysms with cerebral catheter angiography. While it might decrease the risk of non- infected embolism from concomitant AF, if present, anticoagu- lation does not prevent septic emboli and associated infarcts. Antiplatelets have no role. In these patients, valve surgery can be performed after stabilization of the infection and ruling out a high hemorrhagic risk cerebral pathology such as ICMA. The most important issues are always rapid diagnosis and initiation of appropriate antibiotics in addition to source control. The frequency of systemic embolism in IE is 22% to 50%. Approximately two-thirds of these embolizations involve cere- bral circulation. The middle cerebral artery (MCA) territory is affected in up to 90% of IE cases with stroke. Diffusion MRI studies have shown that cerebral microembolisms are present in almost every IE case, albeit mostly clinically occult.59 Mitral valve involvement is associated with a high risk of stroke, es- pecially if anterior leaflets are infected. SA, candida, and HACEK (Haemophilus species, Aggregatibacter species, Cardio- bacterium hominis, Eikenella corrodens, and Kingella species) group organisms are linked to higher embolism risk. Response of vegetation to the antibiotic treatment is critical. Growth of vegetation under antibiotic treatment without shrinkage after 4 to 8 weeks of treatment are significant risk factors along with several morphological vegetation features such as high mobility, irregular shape, sessile, and loose attachment. Embo- lism risk increases if the size of left-sided vegetation is greater than 1 cm, albeit it is stated that this may be a more relevant criterion for VGS IE.60 Stroke frequency is significantly reduced after 2 to 3 weeks of effective IV antibiotic therapy. Such de- crease in risk is independent of IE location and offending mi- croorganism type.58 Non-infectious CE may also occur in the setting of IE due to associated cardiac diseases. https://doi.org/10.5853/jos.2017.00780 Stroke in infective endocarditis IE is a microbial infection of the cardiac endocardial surface including valves (native and prosthetic) and pacemakers/im- plantable cardioverter-defibrillator (ICD) wires. The vast major- ity of IEs are bacterial, while only a few are fungal. Culture re- mains negative in up to one-fifth of IE cases. The incidence of IE is about 3 to 10 per 100,000. A predisposing heart disease is found in approximately 75% of cases.57 Various IE classifica- tions are available. For practical purposes, IE is divided into four groups: (1) left-sided native valve IE (NVIE); (2) left-sided prosthetic valve IE (PVIE), further classified as early, if devel- oped in the first year after valve implantation, or late thereaf- ter; (3) right-sided IE; and (4) device-related IE. Right-sided IE is usually associated with intravenous (IV) drug use and causes fewer neurovascular complications if confined to the right heart. The most frequent cause of acute IE, which is overall less common (up to 20%) compared to other type IEs is Staphylo- coccus aureus (SA). SA-IE usually occurs in natural valves, re- In the light of the findings summarized above, we believe that transcatheter PFO closure is indicated in a number of clin- ical scenarios. In case of PFO and otherwise cryptogenic embo- lism, PFO should be closed if recurrence occurs despite medical treatment. These patients should continue taking antiplatelet medication thereafter. Anticoagulant therapy should be used after first embolic event in cases with thrombophilia or docu- mented DVT/PTE. If a recurrence occurs despite well-managed anticoagulation, the PFO should then be closed. Standard thrombosis management guidelines should be applied for medical treatment selection. PFO closure may be reasonable in some instances after the first embolic episode. Cerebral MRI, 188 http://j-stroke.org https://doi.org/10.5853/jos.2017.00780 The frequency of hemorrhagic stroke is lower than ischemic events in IE but indeed outcomes of ICH are dismal. Almost ev- ery type of CNS hemorrhage such as intraparenchymal, sub- arachnoid (SAH), subdural hematomas and hemorrhagic infarc- tion can be seen in IE. Mitral valve involvement, SA positivity and large (≥30 mm) vegetations are usually linked to CNS hem- orrhage.66 Anticoagulation before infection control and before ruling out ICMA or other high-risk lesions is a well-recognized An important current discussion topic in IE is about the value of cerebral MRI in the clinical decision-making processes.61 Ce- http://j-stroke.org 189 https://doi.org/10.5853/jos.2017.00780 Topcuoglu et al. Prevention of Cardioembolic Strokes risk factor for CNS hemorrhage. Importantly, the cardiac and neurological prognosis of IE-related hematomas is poor. and fragile. Rupture rate is variable (between 2% to 80%) with an average of 50%. However, according to some authorities, rupture risk may be lower than these estimates. No predictive factors for mycotic aneurysm rupture have been identified. It is well documented that the size of the aneurysm is not directly related to the risk of rupture.74 The average diameter of ICMAs is around 4 mm. The shapes are generally fusiform and irregu- lar. Saccular geometry is less common. The most typical feature is their dynamic morphology; change in shape, growth, shrink- age and emergence of new aneurysms during follow-up strongly suggests an infectious cause.75 ICMAs mostly develop in the distal cerebral vasculature. The main cause of these dis- tal mycotic aneurysms is septic embolism. Rare occurrence of proximal mycotic aneurysms is usually associated with focal meningitis. MCA territory is involved in 70% of the cases. IC- MAs are multiple in about one-fourth of patients.72,74 Strepto- cocci species are the most frequent causative organisms, found in approximately 50% of patients with ICMA in whom a mi- croorganism could be detected. SA is isolated in about 10% of these patients.60 Two types of CNS hemorrhage merit further mentioning. The first is convexal, or cortical SAH (frequency, about 10%), which may be related to distal mycotic microaneurysm or septic arte- ritis. In cases with MRI-documented convexal SAH or cortical siderosis, catheter angiography should be performed.67 The other subtle hemorrhagic pathology is CMBs which have a documented frequency of around 60% in IE based on consecu- tive MRI studies. These CMBs have been linked to PVIE rather than prior anticoagulant use.68 IE-associated CMBs show sev- eral different features such as more superficial localization, larger size and heterogeneous appearance.69 These are not usu- ally due to mycotic aneurysms, but inflammatory distal arteritis from septic microembolism. The number and localization of CMBs do not correlate with DWI positive microinfarcts. The presence of CMBs is a strong predictor of impending CNS hem- orrhage in early phases of IE. However, after successful control of infection, this risk disappears and no further increase in CMBs is anticipated. After full infection control, the risk of long-term anticoagulation might also be reduced to a reason- able degree. Therefore, CMBs may not be considered as a con- crete contraindication to anticoagulant use when indicated in these cases, once the infection is adequately treated.70 The main component of ICMA management is parenteral antibiotics.57 With intensive antibiotic therapy, the aneurysm is expected to shrink and subsequently disappear. However, oblit- eration of an ICMA should be performed when an open-heart surgery such as emergency valve replacement is needed. Since anticoagulation is not permitted and HF usually worsens dur- ing post-craniotomy period, endovascular treatment methods are preferred to neurosurgical approaches.58 Unless ICMAs are located in eloquent areas such as motor cortex, occlusion of the parent artery proximal to the aneurysm is often the proce- dure of choice.58 This approach is not always easy whenever such artery supplies a functionally important region. Discussing these difficult situations between neurology, cardiology, inter- ventional neurology, and cardiothoracic surgery experts and involving the patient in clinical decision making is important. The incidence of ICMA in IE is between 1% to 10%.71,72 IC- MAs are more commonly seen in patients with sepsis, human immunodeficiency virus, and IV drug use and relative immuno- compromised states such as diabetes. Not only incidence, but also risk of rapid growth and rupture increases in immune-sup- pressed IE patients.71 Clinical manifestations of ICMAs are high- ly variable, similar to other neurovascular manifestations of IE. Fever is seen in only 20% of cases with ICMAs, while focal neurological deficit and meningeal irritation signs are noted in 40% along with headache in 60%. ICMAs present with SAH in 40%, infarct in 30%, and lobar hemorrhage in another 30%, usually in combination. Of note, symptomatic infected cerebral emboli frequently, but not invariably, precede the ICMA forma- tion. In cases with CNS hemorrhage in the setting of IE, the likelihood of ICMA is around 20%, and 1% in non-hemorrhagic cases. A catheter angiography should therefore be performed for every case with IE associated CNS hemorrhage. CTA cannot replace catheter angiography as the sensitivity of CTA varies between 45% to 85% if the diameter of the aneurysm is 3 mm or less, albeit it might reach 100% for aneurysms greater than 3 mm.73 ICMAs are always linked to poor prognosis in IE. Their rupture corresponds to approximately 80% mortality rate, while mortality is about 30% in patients with unruptured ICMAs.72 Valve replacement and stroke Life-long OAC therapy is mandatory in the setting of prosthetic mechanical heart valves to prevent valve thrombosis, systemic and cerebral embolism. The incidence of major bleeding is around 1.4 per 100 patient-years in patients treated with cou- marin derivatives.81 In the setting of major risk for anticoagu- lant-related bleeding, such as elderly with cerebral amyloid angiopathy, a primary valvular repair, bioprosthesis, or trans- catheter aortic valve replacement are preferred instead of me- chanical valve. It is also reasonable to insert MRI-compatible prostheses in patients with a high risk of stroke. For HF patients with AF, anticoagulation is generally recom- mended based on other embolic risk factors and the absence of concurrent hemorrhagic risk. If no AF, then the benefit of anti- coagulation is not clear. In a meta-analysis of four trials, in- cluding Warfarin versus Aspirin in Reduced Cardiac Ejection Fraction (WARCEF),78 warfarin reduced ischemic stroke fre- quency from 1.36 to 0.72 events per 100 patient-years com- pared to aspirin. Unfortunately, this potential benefit was ne- gated by a significant increase in major bleeding rates from 0.87 to 1.78 events per 100 patient-years. In addition, no im- provement has been observed in survival rates with warfarin.76 However, NOACs, with well-established lower bleeding rates, may be potentially useful for this indication and merit further research. COMMANDER HF is a currently ongoing phase III trial aiming to test rivaroxaban versus placebo for reducing the risk of death, myocardial infarction (MI), or stroke in patients with chronic HF and significant coronary artery disease after a re- cent HF-related hospitalization. In observational studies, reinstitution of anticoagulation was associated with a lower risk of thromboembolic complications compared to no anticoagulation.82 In cases who need to re- sume oral anticoagulation after a hemorrhage, the risk of re- bleeding might be reduced with more strict INR control, effec- tive blood pressure management, careful countermeasures against falls, avoidance of unnecessary addition of antiplatelet medicines and structured patient education including diet and medication interactions.83 Self-monitoring or self-management of oral anticoagulation therapy such as home-based testing can improve life quality and decrease risk of thromboembolic complications. Of note, all-cause mortality is reduced with self-management but not self-monitoring. Heart failure and stroke HF is currently grouped into two: HF with reduced ejection fraction (EF), previously called “systolic HF,” and HF with pre- served EF, previously called “diastolic HF.” Low EF is diagnosed when it is less than 40%, while normal EF is higher than 50%. Values from 40% to 50% are described as “mid-range” EF.76 Prevalence of both types of HF is high, between 10% to 25%, in stroke cohorts. Functional outcomes and survival rates are significantly worse in stroke patients with HF. Major risk factors of HF are hypertension, coronary artery disease, heart valve dis- eases, AF and diabetes, all of which are also important risk/etio- The cerebral mycotic aneurysms are typically thin-walled 190 http://j-stroke.org https://doi.org/10.5853/jos.2017.00780 https://doi.org/10.5853/jos.2017.00780 Vol. 20 / No. 2 / May 2018 risk when LVADP is considered. risk when LVADP is considered. logical factors for stroke individually. Risk factors for stroke in HF patients without AF are previous stroke history, severity of HF symptoms, insulin-dependent diabetes mellitus, body mass in- dex, and age. Elevation of B-type natriuretic peptide may also indicate an increased risk.77 In addition to frequently coexistent pathologies such as atherosclerotic arterial disease and AF, HF can cause stroke itself via intracardiac stasis related thrombo- embolism along with low-cardiac output-related cerebral hypo- perfusion and reduced cerebral autoregulation capacity.76 Valve replacement and stroke Importantly, self- monitoring or self-management have no effects on the occur- rence of major hemorrhage.84 Standard management of HF include diuretics such as min- eralocorticoid receptor antagonist, thiazide and loop diuretics along with optimization of comorbidities such as pulmonary disease, anemia, and sleep disordered breathing. For HF with reduced EF, effective hypertension control with β blocker, an- giotensin converting enzyme inhibitor, angiotensin II receptor blocker, angiotensin receptor-neprilysin inhibitor, mineralocor- ticoid receptor antagonist, and nitrates are usually suggested. Cardiac revascularization with angioplasty or bypass surgery and valvular repair, if needed, may be useful in selected pa- tients. ICDs can be used for prevention of sudden cardiac death. Cardiac resynchronization therapy with biventricular pacing may also improve survival in some patients. It is import- ant to note that use of left ventricular assist device pumps (LVADP) causes significant coagulation activation and dimin- ished pulsatility and therefore mandates anticoagulation as well as antiplatelet use.79 Newer LVADP such as the Heart- Mate®3 (St. Jude Medical) decrease pump thrombosis, but not stroke rates.80 Clinicians should be aware of the neurological risk of high-intensity warfarin and combination antithrombotic use, especially for HF patients at high baseline hemorrhagic Generally, OAC agents are restarted in 4 to 8 weeks after an ICH when there are no alternatives to reduce the risk of sys- temic/cerebral thromboembolism due to thrombosis of the prosthetic valves. In such cases, the period before restarting anticoagulation should be individualized, taking into account both ischemic and hemorrhagic risks. For cases with high risk of thromboembolism due to mechanical valves, it is imperative to wait at least for the stabilization of the size of the ICH but OACs may need to be resumed as early as 7 days after the brain hemorrhage. https://doi.org/10.5853/jos.2017.00780 Disclosure All patients with coronary artery disease should receive opti- mal treatment to correct vascular risk factors including hyper- tension, diabetes, hyperlipidemia, smoking, and sedentary life style to name a few. Concurrent morbidities such as AF, other dysrhythmias and HF should be optimally treated. The choice and duration of antithrombotic treatment should be primarily determined based on the type of coronary intervention that the patient might need such as balloon angioplasty, stenting, or coronary artery bypass grafting.87 The authors have no financial conflicts of interest. References 1. Gokcal E, Pasi M, Fisher M, Gurol ME. 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Multidisciplinary collaborations also require familiarity with these advances, as parties involved (neurologist, cardiolo- gist, cardiothoracic surgeon) should be able to understand the available approaches and challenges in order to provide the best clinical management to the individual patient. Acknowledgments This work was made possible by grants from the National Insti- tute of Health (M. Edip Gurol, NS083711). Dong-Eog Kim was supported by Ministry of Health & Welfare (HI12C1847; Korea Healthcare Technology R&D Project), funded by the Korean government, Republic of Korea. Recent studies have shed light on use of simplified short- term anticoagulation regimens in patients with AF who undergo percutaneous coronary interventions. The use of a lower dose NOAC (rivaroxaban or dabigatran) and single antiplatelet agent provided comparable results to the standard triple therapy that includes warfarin, aspirin and clopidogrel in these patients, in terms of safety and efficacy.88,89 Another recent study evaluated the effects of rivaroxaban with or without aspirin in stable car- diovascular disease, in a design that not only excluded patients with past history of ICH but also symptomatic lacunar cerebral infarcts, to decrease anticoagulant-related ICHs. This study showed that in patients with stable cardiovascular disease (>90% of them with coronary artery disease), the combination of rivaroxaban 2.5 mg twice daily and aspirin prevented a com- posite outcome of cardiovascular death, stroke, or MI more than aspirin alone (4.1% vs. 5.4%). 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https://openalex.org/W3087243951	https://www.nature.com/articles/s41419-020-02985-x.pdf	English	null	NLRP3 inflammasome in endothelial dysfunction	Cell death and disease	2,020	cc-by	13,742	Facts ● Noncoding RNA, as an emerging disease biomarker, may regulate endothelial function by mediating the NLRP3 inﬂammasome signaling pathway. ● NLRP3 inﬂammasome is involved in a wide range of pathological conditions and diseases as intracellular innate immune sensors. ● Certain drugs, such as statins, hypoglycemic agents, and other anti-inﬂammatory or antioxidant drugs, can improve vascular dysfunction by inhibiting the NLRP3 inﬂammasome signaling pathway. ● NLRP3 inﬂammasome-mediated inﬂammation and ● NLRP3 inﬂammasome-mediated inﬂammation and Correspondence: Tao Yu (yutao0112@qdu.edu.cn) or Xian-ming Chu (18661801698@163.com) 1Department of Cardiology, The Afﬁliated Hospital of Qingdao University, Qingdao 266000, China 2Department of lmmunology, School of Basic Medicine, Qingdao University, Qingdao 266071, China Full list of author information is available at the end of the article These authors contributed equally: Baochen Bai, Yanyan Yang Edited by Y. Shi Bai et al. Cell Death and Disease (2020) 11:776 https://doi.org/10.1038/s41419-020-02985-x Bai et al. Cell Death and Disease (2020) 11:776 https://doi.org/10.1038/s41419-020-02985-x Cell Death & Disease Cell Death & Disease Op e n A c c e s s NLRP3 inﬂammasome in endothelial dysfunction Baochen Bai1, Yanyan Yang2, Qi Wang1, Min Li3, Chao Tian1, Yan Liu3, Lynn Htet Htet Aung 3, Pei-feng Li3, Tao Yu3,4 and Xian-ming Chu1,5 Abstract bst act Inﬂammasomes are a class of cytosolic protein complexes. They act as cytosolic innate immune signal receptors to sense pathogens and initiate inﬂammatory responses under physiological and pathological conditions. The NLR-family pyrin domain-containing protein 3 (NLRP3) inﬂammasome is the most characteristic multimeric protein complex. Its activation triggers the cleavage of pro-interleukin (IL)-1β and pro-IL-18, which are mediated by caspase-1, and secretes mature forms of these mediators from cells to promote the further inﬂammatory process and oxidative stress. Simultaneously, cells undergo pro-inﬂammatory programmed cell death, termed pyroptosis. The danger signals for activating NLRP3 inﬂammasome are very extensive, especially reactive oxygen species (ROS), which act as an intermediate trigger to activate NLRP3 inﬂammasome, exacerbating subsequent inﬂammatory cascades and cell damage. Vascular endothelium at the site of inﬂammation is actively involved in the regulation of inﬂammation progression with important implications for cardiovascular homeostasis as a dynamically adaptable interface. Endothelial dysfunction is a hallmark and predictor for cardiovascular ailments or adverse cardiovascular events, such as coronary artery disease, diabetes mellitus, hypertension, and hypercholesterolemia. The loss of proper endothelial function may lead to tissue swelling, chronic inﬂammation, and the formation of thrombi. As such, elimination of endothelial cell inﬂammation or activation is of clinical relevance. In this review, we provided a comprehensive perspective on the pivotal role of NLRP3 inﬂammasome activation in aggravating oxidative stress and endothelial dysfunction and the possible underlying mechanisms. Furthermore, we highlighted the contribution of noncoding RNAs to NLRP3 inﬂammasome activation-associated endothelial dysfunction, and outlined potential clinical drugs targeting NLRP3 inﬂammasome involved in endothelial dysfunction. Collectively, this summary provides recent developments and perspectives on how NLRP3 inﬂammasome interferes with endothelial dysfunction and the potential research value of NLRP3 inﬂammasome as a potential mediator of endothelial dysfunction. pyroptosis play a pivotal role in endothelial dysfunction. Introduction The innate immune system is the primary mechanism by which most organisms respond immediately to infec- tions or injury. Pattern-recognition receptors (PRRs) in the host are activated, which recognize molecules released by pathogens or damaged cells. These molecular signals are known as pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs)1. The PRR family has many members, including Toll-like receptors (TLRs), NOD-like receptors (NLRs), C-type lectin receptors, retinoic acid-inducible gene (RIG)-I-like receptors (RLRs), as well as several intracel- lular DNA sensors2. Innate immune cells play critical roles in PRR-initiated innate inﬂammatory response, but the effect of nonimmune cells, such as endothelial cells (ECs), is also a force to be reckoned with3. The tran- scriptional upregulation of pro-inﬂammatory genes by PRRs triggers a cascade of inﬂammatory responses. Although inﬂammation has the beneﬁcial effect of limit- ing cellular and organ damage, disruption in its regulation may result in a sustained inﬂammatory response and ultimately, local or systemic inﬂammation4. ● What is the molecular mechanism underlying NLRP3 inﬂammasome-related endothelial dysfunction? ● What is the molecular mechanism underlying NLRP3 inﬂammasome-related endothelial dysfunction? © The Author(s) 2020 OpenAccessThisarticleislicensedunderaCreativeCommonsAttribution4.0InternationalLicense,whichpermitsuse,sharing,adaptation,distributionandreproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. Ofﬁcial journal of the Cell Death Differentiation Association Ofﬁcial journal of the Cell Death Differentiation Association Bai et al. Cell Death and Disease (2020) 11:776 Page 2 of 18 ● How does NLRP3 inﬂammation perceive information about different mediators? ● How does NLRP3 inﬂammation perceive information about different mediators? ● How does NLRP3 inﬂammation perceive information about different mediators? changes eventually lead to endothelial dysfunction8. Interleukin (IL)-1β is an important pro-inﬂammatory cytokine released during the endothelial inﬂammatory response9. Interestingly, activation of NLRP3 inﬂamma- some can produce high numbers of IL-1β. ● Whether different agonists affect endothelial function through a single cascade of events or through distinct pathways to activate NLRP3 inﬂammation? ● Do other inﬂammasomes such as NLRP1, NLRC4, NLRP6, AIM2, noncanonical inﬂammasomes, and upstream pathways affect endothelial function? Additionally, high-quality research has shown that NLRP3 inﬂammasome activation is correlated with mul- tiple chronic inﬂammatory diseases and metabolic dis- orders, including obesity, hypertension, diabetes, atherosclerosis, neuroinﬂammation, retinopathy, stroke, and cancer10–16. These diseases also have a close con- nection with the dysregulation of the endothelium17. These studies indicate that the activation of NLRP3 inﬂammasome in ECs under pathophysiological condi- tions may aggravate endothelial dysfunction, leading to various diseases. In recent years, endothelial function research has increasingly focused on the regulatory role of NLRP3 inﬂammasome. In this review, we attempt to summarize the latest progress and development trends in the NLRP3 inﬂammasome research, highlighting its role in redox regulation and endothelial dysfunction. Also, we emphasize the current knowledge of the factors that mediate and inﬂuence NLRP3-related endothelial dys- function and potential therapeutic targets, which may be helpful for the treatment of endothelial dysfunction- related diseases. Ofﬁcial journal of the Cell Death Differentiation Association Inﬂammasome family The inﬂammasome is a molecular platform that drives effector caspase-1 activation, which is assembled by NLRs, AIM2-like receptors (ALRs), or pyrin that can directly or indirectly (via the adaptor apoptosis-associated speck-like protein containing a CARD (ASC)), activate caspase-1 (Fig. 1)18. Structurally, the N-terminal domain of these sensors includes a CARD or a pyrin domain (PYD). The adaptor ASC is composed of a PYD and a CARD, while caspase-1 contains a CARD19. Recently, studies have elucidated that NLRP1, NLRP3, NLRP6, AIM2, and pyrin carry a PYD in their N-terminal region, whereas NLRP1b and NLRC4 contain a CARD. The sensor protein, con- taining a PYD, binds to the PYD of ASC, allowing the ASC to activate caspase-1 by interacting with the CARD of pro-caspase-1. In contrast, a CARD-containing sensor protein may activate caspase-1 by directly binding to the CARD of pro-caspase-1 without ASC20. However, the presence of ASC can enhance the assembly of the sensor protein containing a CARD and the activation of caspase- 1 (Fig. 2)21. The inﬂammasome, a type of PRR, constitutes an essential component of the innate immunity5. Abnormal activation of inﬂammasomes is the pathogenesis of var- ious inﬂammatory diseases6. The inﬂammasome is a high- molecular-weight protein complex that acts as a cytosolic innate immune signaling receptor that senses PAMPs or DAMPs and mediates a highly inﬂammatory state. The ﬁrst inﬂammasome was discovered in 20025 following which, various inﬂammasomes have been identiﬁed, including NLR-family pyrin domain-containing protein (NLRP) 1, NLRP3, NLRP6, NLR-family caspase recruit- ment domain (CARD)-containing protein 4 (NLRC4), absent in melanoma 2 (AIM2), and pyrin inﬂamma- somes7. Among them, the NLRP3 inﬂammasome is the most well-characterized, largest multimeric protein complex to date. Microvascular ECs at a site of inﬂammation are both active participants and regulators of inﬂammatory pro- cesses. During the transition from acute inﬂammation to chronic inﬂammation or from innate immunity to adap- tive immunity, the characteristics of ECs change through EC activation, rapid recruitment of neutrophils, and increased vascular leakage of plasma proteins. These Furthermore, different sensor proteins respond to dif- ferent activators (Fig. 2). NLRP1 inﬂammasome is acti- vated by speciﬁc pathogens, such as anthrax lethal toxin, muramyl dipeptide (MDP), Toxoplasma gondii, Shigella ﬂexneri, and Listeria monocytogenes, which cause pro- teolysis of the NLRP1 N terminal21,22. NLRC4 Ofﬁcial journal of the Cell Death Differentiation Association Bai et al. Cell Death and Disease (2020) 11:776 Page 3 of 18 Fig. Inﬂammasome family 1 Domain structure of representative inﬂammasome. Inﬂammasome is a protein complex formed by the aggregation of inﬂammasome sensor, adaptor protein ASC, and effector protein caspase-1. PYD pyrin domain, NBD nucleotide-binding domain, LRR leucine-rich-repeat domain, FIIND function-to-ﬁnd domain, CARD caspase activation and recruitment domain, C–C coiled-coil domain, B B-box domain, BIR baculovirus inhibitor of apoptosis repeat, ASC apoptosis-associated speck-like protein containing a CARD, CASP1 caspase-1. Fig. 1 Domain structure of representative inﬂammasome. Inﬂammasome is a protein complex formed by the aggregation of inﬂammasome sensor, adaptor protein ASC, and effector protein caspase-1. PYD pyrin domain, NBD nucleotide-binding domain, LRR leucine-rich-repeat domain, FIIND function-to-ﬁnd domain, CARD caspase activation and recruitment domain, C–C coiled-coil domain, B B-box domain, BIR baculovirus inhibitor of apoptosis repeat, ASC apoptosis-associated speck-like protein containing a CARD, CASP1 caspase-1. inﬂammasome can be activated by bacterial ligands, including ﬂagellin and components of the type III secre- tion system (T3SS), which are sensed and bound by NOD- like receptor family apoptosis-inhibitory protein (NAIP) to induce NLRP4 oligomerization23. NLRP6, one of the newest and less-researched members of the NLR family, is overexpressed explicitly in human and mouse intestinal epithelial cells. Microbial metabolites secreted by intest- inal commensal bacteria can modulate NLRP6 activity and IL-18 secretion24. Moreover, certain pathogens, such as Listeria monocytogenes, Salmonella typhimurium, and Staphylococcus aureus, have been shown to activate the NLRP6 inﬂammasome as part of the host defense25. AIM2 acts as a cytoplasmic sensor that can detect and directly bind to double-stranded (ds) DNA of any sequence pro- duced by pathogens or cellular perturbations, inducing the capability of self-oligomerization and then recruiting ASC for activation of inﬂammasome26. In 2004, pyrin was ﬁrst shown to self-assemble into an inﬂammasome by recognizing the inactivation of RhoA GTPase, a molecular switch that controls the dynamics of the cytoskeleton27. Recently, studies found that bacterial toxins can modify the Switch-I region of RhoA and trigger pyrin activation, such as TcdA and TcdB secreted from Clostridium difﬁ- cile27. Additionally, a most recent study demonstrated that bile acid analogs of microbial origin, such as BAA473 and BAA485, can also activate the pyrin inﬂammasome28. NLRP3 inﬂammasome is triggered by numerous stimuli, inﬂammasome can be activated by bacterial ligands, including ﬂagellin and components of the type III secre- tion system (T3SS), which are sensed and bound by NOD- like receptor family apoptosis-inhibitory protein (NAIP) to induce NLRP4 oligomerization23. Inﬂammasome family NLRP6, one of the newest and less-researched members of the NLR family, is overexpressed explicitly in human and mouse intestinal epithelial cells. Microbial metabolites secreted by intest- inal commensal bacteria can modulate NLRP6 activity and IL-18 secretion24. Moreover, certain pathogens, such as Listeria monocytogenes, Salmonella typhimurium, and Staphylococcus aureus, have been shown to activate the NLRP6 inﬂammasome as part of the host defense25. AIM2 acts as a cytoplasmic sensor that can detect and directly bind to double-stranded (ds) DNA of any sequence pro- duced by pathogens or cellular perturbations, inducing the capability of self-oligomerization and then recruiting ASC for activation of inﬂammasome26. In 2004, pyrin was ﬁrst shown to self-assemble into an inﬂammasome by recognizing the inactivation of RhoA GTPase, a molecular switch that controls the dynamics of the cytoskeleton27. Recently, studies found that bacterial toxins can modify the Switch-I region of RhoA and trigger pyrin activation, such as TcdA and TcdB secreted from Clostridium difﬁ- cile27. Additionally, a most recent study demonstrated that bile acid analogs of microbial origin, such as BAA473 and BAA485, can also activate the pyrin inﬂammasome28. NLRP3 inﬂammasome is triggered by numerous stimuli, including endogenous molecules, crystalline substances, pathogenic microbes, and ATP. NLRP3 inﬂammasome functions as a signal integrator to sense several cellular signals, including ion ﬂuxes such as potassium (K+) efﬂux and calcium (Ca2+) inﬂux, lysosomal leakage, mitochon- drial dysfunction, and ROS production29. Overall, different inﬂammasomes play unique roles in the defense against speciﬁc pathogens. Overactivation of inﬂammasomes can lead to signiﬁcant inﬂammatory responses and pathological changes that are closely rela- ted to autoimmunity and autoinﬂammation. Therefore, inﬂammasomes are involved in a wide range of patholo- gical conditions and diseases as intracellular innate immune sensors for conditions like infection or aseptic inﬂammation, metabolic syndrome, age-related chronic inﬂammatory disease, and autoimmune disease7,10. Ofﬁcial journal of the Cell Death Differentiation Association NLRP3 inﬂammasome activation NLRP3 inﬂammasome contains innate immune sensor NLRP3, adaptor molecule ASC, and effector protease pro- caspase-130. The activation of NLRP3 inﬂammasome is essentially the autocatalytic activation of caspase-1. Once activated, NLRP3 acts as a sensor molecule that occurs in self-oligomerization and recruits ASC via homotypic PYD–PYD interaction, which induces the assembly of ASC into large speck-like structures. Subsequently, aggregated ASC recruits pro-caspase-1 via CARD–CARD contact, leading to autocatalytic activation of caspase-1. Ofﬁcial journal of the Cell Death Differentiation Association Bai et al. Cell Death and Disease (2020) 11:776 Page 4 of 18 Fig. 2 The canonical inﬂammasome activation pathway occurs by sensing diverse damage-associated molecular patterns (DAMPs). a NLRP3, NLRP6, AIM2, and pyrin, c to activate caspase-1 by interacting with the CARD of pro-CASP1. Activated caspase-1 t gasdermin D (GSDMD), and then releases the N-terminal domain of GSDMD to induce NLRP1b and NLRC4, containing a CARD, activate caspase-1 by directly binding the CAR scaffold. The presence of ASC can enhance the assembly of the sensor protein contain process after caspase-1 activation is the same as part A. g diverse pathogen-associated molecular patterns (PAMPs) and d pyrin, containing a PYD, binds to the PYD of ASC, allowing the ASC spase-1 triggers the cleavage of pro-interleukin (IL)-1β, pro-IL-18, and to induce pyroptosis, followed by the release of IL-1β and IL-18. b g the CARD of pro-caspase-1 without ASC or binding the paired ASC n containing a CARD and the activation of caspase-1. The molecular Fig. 2 The canonical inﬂammasome activation pathway occurs by sensing diverse pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs). a NLRP3, NLRP6, AIM2, and pyrin, containing a PYD, binds to the PYD of ASC, allowing the ASC to activate caspase-1 by interacting with the CARD of pro-CASP1. Activated caspase-1 triggers the cleavage of pro-interleukin (IL)-1β, pro-IL-18, and gasdermin D (GSDMD), and then releases the N-terminal domain of GSDMD to induce pyroptosis, followed by the release of IL-1β and IL-18. b NLRP1b and NLRC4, containing a CARD, activate caspase-1 by directly binding the CARD of pro-caspase-1 without ASC or binding the paired ASC scaffold. The presence of ASC can enhance the assembly of the sensor protein containing a CARD and the activation of caspase-1. The molecular process after caspase-1 activation is the same as part A. Fig. 2 The canonical inﬂammasome activation pathway occurs by sensing diverse pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs). NLRP3 inﬂammasome activation a NLRP3, NLRP6, AIM2, and pyrin, containing a PYD, binds to the PYD of ASC, allowing the ASC to activate caspase-1 by interacting with the CARD of pro-CASP1. Activated caspase-1 triggers the cleavage of pro-interleukin (IL)-1β, pro-IL-18, and gasdermin D (GSDMD), and then releases the N-terminal domain of GSDMD to induce pyroptosis, followed by the release of IL-1β and IL-18. b NLRP1b and NLRC4, containing a CARD, activate caspase-1 by directly binding the CARD of pro-caspase-1 without ASC or binding the paired ASC scaffold. The presence of ASC can enhance the assembly of the sensor protein containing a CARD and the activation of caspase-1. The molecular process after caspase-1 activation is the same as part A. Ofﬁcial journal of the Cell Death Differentiation Association Page 5 of 18 Page 5 of 18 Bai et al. Cell Death and Disease (2020) 11:776 The function of activated caspase-1 heterotetramers is proteolytic activation of the pro-inﬂammatory cytokines IL-1β and IL-18, and of a soluble cytosolic protein gas- dermin D (GSDMD). Upon proteolysis, the oligomerized gasdermin N can bind membrane lipids and form mem- brane pores to mediate the nonconventional secretion of IL-1β and IL-18. In parallel, cells undergo a pro- inﬂammatory type of cell death known as pyroptosis17,31,32. signaling organelles that play a crucial role in endothelial function, including subcellular location, dynamics, bio- genesis, mitophagy, autophagy, ROS production, calcium homeostasis, regulation of EC death, and heme synth- esis40. Intriguingly, the accumulation of damaged mito- chondria has also been proposed to be essential for NLRP3 activation. In addition to increased mtROS, the exposure of mtDAMPs, such as mtDNA and cardiolipin to the cytosol, alterations in metabolite levels, and mito- chondrial antiviral signaling protein, can also contribute to NLRP3 activation38. y Multiple cellular signals are thought to account for NLRP3 inﬂammasome activation, including ion ﬂuxes such as K+ efﬂux, Cl−efﬂux, Ca2+ inﬂux, and Na+ inﬂux, lyso- somal leakage, mitochondrial dysfunction, and ROS pro- duction33. These various upstream signaling pathways may be interrelated or independent (Fig. 3). Additionally, non- canonical NLRP3 inﬂammasome activation and alternative NLRP3 inﬂammasome activation are two other forms of activation34. Noncanonical NLRP3 inﬂammasome activa- tion is induced by cytosolic LPS that is sensed by non- canonical caspases 4/5 in humans and caspase-11 in mice. Subsequently, caspases 4/5/11 are activated to cleave GSDMD, resulting in K+ efﬂux and pyroptosis35. NLRP3 inﬂammasome activation Alter- native NLRP3 inﬂammasome activation is species-speciﬁc, as it only occurs in human and porcine monocytes. This activation is induced by TLR4–TIR-domain-containing adaptor-inducing interferon-β (TRIF)–receptor-interacting serine/threonine-protein kinase 1 (RIPK1)–Fas-associated protein with death domain (FADD)–CASP8 signaling pathway. Of note, this pathway does not depend on K+ efﬂux and does not induce ASC speck formation and pyroptosis36. ROS also bridge the interaction between endoplasmic reticulum (ER) stress and NLRP3 inﬂammasome activation (Fig. 3). ER stress has been proven to activate NLRP3 inﬂammasome and promote the development of endothelial dysfunction. Speciﬁcally, ER stress-induced protein mis- folding, imbalance of sterol synthesis and distribution, and release of Ca2+ and ROS can trigger NLRP3 inﬂammasome activation41. Meanwhile, multiple molecular pathways dur- ing severe ER stress are also involved in the activation of the NLRP3 inﬂammasome, including p38 mitogen-activated protein kinase (MAPK) pathway, Jun-N-terminal kinase (JNK) signaling, X-box-binding protein-1 (XBP1), CCAAT/ enhancer-binding protein–homologous protein (CHOP), NF-κB, and TXNIP signaling pathways, which are terminal signals in the unfolded protein response (UPR). The func- tion of UPR is to detect protein misfolding to restore ER homeostasis under cellular perturbations41. Additionally, several studies have reported that ER, as an intracellular metabolic regulator, plays a pivotal role in endothelial dysfunction42–44. ER stress also acts as an active inﬂam- matory trigger, accelerating the progression of endothelial dysfunction via inﬂammation and oxidative stress45. Besides, ER Ca2+ released from stressed cells increases the mtROS production and ampliﬁes the activation of the NLRP3 inﬂammasome46. Accordingly, the mutual promo- tion between mitochondrial dysfunction and ER stress contributes to the activation of NLRP3 inﬂammasome and endothelial dysfunction, both of which are involved in oxidative stress and the production of ROS. Among the aforementioned multiple activation mechanisms, ROS bridges the interaction between NLRP3 inﬂammasome and endothelial dysfunction. Speciﬁcally, ROS are one of the ﬁrst intermediates produced by many known NLRP3 inﬂammasome activators, and are involved in the mechanisms that trigger the formation and acti- vation of the NLRP3 inﬂammasome, thereby promoting tissue inﬂammation and activating immune response37. Thioredoxin-interacting protein (TXNIP), nuclear factor- kappaB (NF-κB), and the transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) are proteins involved in the response to oxidative stress, which links ROS to NLRP3 activation (Fig. 3)38. Moreover, aberrant production of ROS may increase nitric oxide (NO) cata- bolism, leading to a decrease in NO bioavailability. NLRP3 inﬂammasome activation This imbalance in NO–ROS leads to the expression of inﬂammation-related genes and upregulation of inﬂam- matory proteins, which in turn destroys endothelial function39. Implications of NLRP3 inﬂammasome activation in endothelial dysfunction NLRP3 inﬂammasome activation in endothelial inﬂammation Generally, microbes, exogenous and endogenous crys- tals, and metabolic dysbiosis could trigger NLRP3 inﬂammasome activation, which initiates the secretion of mature forms of IL-1β and IL-18 from cells to promote further inﬂammatory processes and oxidative stress in the endothelium (Fig. 4)47. This intense inﬂammasome acti- vation increases the release of new DAMPs, forming a negative-feedback loop48. The cytokine output of inﬂammasomes, speciﬁcally IL-1β, is a key inﬂammatory ROS production in the mitochondria is the primary source of cellular ROS. Indeed, mitochondrial ROS (mtROS) are involved in NLRP3 inﬂammasome activation (Fig. 3). The endothelial mitochondria act as critical Ofﬁcial journal of the Cell Death Differentiation Association Bai et al. Cell Death and Disease (2020) 11:776 Page 6 of 18 Fig. 3 Reactive oxygen species (ROS) contribute to the NLRP3 inﬂammasome activation in endothelial cells. A wide range of pathogen- associated molecular patterns (PAMPs) or damage-associated molecular patterns (DAMPs) trigger NLRP3 inﬂammasome activation by inducing potassium (K+) efﬂux, calcium (Ca2+) inﬂux, lysosomal leakage, mitochondrial dysfunction, and ROS production. ROS mainly derived from endoplasmic reticulum (ER) stress, damaged mitochondria, and NADPH oxidase. ER stress could activate the NF-κB, TXNIP, and SREBP signaling pathways, Ca2+ release, and ROS production. Moreover, athero-prone ﬂow can also mediate SREBP signaling pathways. The release of mtROS and mtDNA in damaged mitochondria might activate NLRP3 inﬂammasome. Furthermore, mitochondrial antiviral signaling protein (MAVS) is capable of molulating the recruitment and localization of NLRP3. However, NF-E2-related factor 2 (Nrf2) activated under ROS-induced stress conditions can inhibit NLRP3 inﬂammasome activation. Taken together, ROS is an intermediate factor involved in multiple signaling pathways and can trigger the activation of NLRP3 inﬂammasome. Fig. 3 Reactive oxygen species (ROS) contribute to the NLRP3 inﬂammasome activation in endothelial cells. A wide range of pathogen- associated molecular patterns (PAMPs) or damage-associated molecular patterns (DAMPs) trigger NLRP3 inﬂammasome activation by inducing potassium (K+) efﬂux, calcium (Ca2+) inﬂux, lysosomal leakage, mitochondrial dysfunction, and ROS production. ROS mainly derived from endoplasmic reticulum (ER) stress, damaged mitochondria, and NADPH oxidase. ER stress could activate the NF-κB, TXNIP, and SREBP signaling pathways, Ca2+ release, and ROS production. Moreover, athero-prone ﬂow can also mediate SREBP signaling pathways. The release of mtROS and mtDNA in damaged mitochondria might activate NLRP3 inﬂammasome. Furthermore, mitochondrial antiviral signaling protein (MAVS) is capable of molulating the recruitment and localization of NLRP3. However, NF-E2-related factor 2 (Nrf2) activated under ROS-induced stress conditions can inhibit NLRP3 inﬂammasome activation. Ofﬁcial journal of the Cell Death Differentiation Association Implications of NLRP3 inﬂammasome activation in endothelial dysfunction NLRP3 inﬂammasome activation in endothelial inﬂammation Taken together, ROS is an intermediate factor involved in multiple signaling pathways and can trigger the activation of NLRP3 inﬂammasome. NLRP3 inﬂammasome, including the endogenous med- ium. For example, visfatin has been demonstrated to promote endothelial inﬂammation as a pro-inﬂammatory adipokine51. Xia et al. found that NLRP3 inﬂammasome activation is an underlying cause of visfatin-induced endothelial inﬂammation52. This inﬂammatory response can cause endothelial dysfunction, initiating athero- sclerosis during obesity. Moreover, visfatin has also been shown to increase neointimal formation both in vitro and in vivo, which is partly attributed to the formation and activation of NLRP3 inﬂammasome and IL-1β secretion52. NLRP3 inﬂammasome activation is also involved in mediator in response to microbial invasion and tissue damage. EC is a target cell of IL-1β, and it also produces IL-1β during inﬂammation49. The activation of IL-1β can trigger the activation of secondary inﬂammatory media- tors, such as IL-6 and C-reactive protein50. Moreover, IL- 1β can also promote the secretion of adhesion molecules and chemokines in ECs, inducing a potent pro- inﬂammatory response50. Endothelial inﬂammation may initiate the occurrence and progression of endothelial dysfunction and promote one another in subsequent processes. A growing number of agonists have been demonstrated to promote vascular inﬂammation by activating the NLRP3 inﬂammasome activation is also involved in exogenous substance-mediated endothelial inﬂammation. Ofﬁcial journal of the Cell Death Differentiation Association Bai et al. Cell Death and Disease (2020) 11:776 Page 7 of 18 Xia et al. demonstrated that NLRP3 inﬂammasome acti- vation is involved in tetrachlorobenzoquinone (TCBQ)- induced endothelial inﬂammation53. TCBQ not only promotes the post-translational activation of NLRP3 and IL-1β secretion in HUVECs, but also downregulates the ubiquitination of NLRP3, which facilitates its activation, as an active metabolite of pentachlorophenol (PCP). This study also reported that K+ efﬂux, mtROS production, Fig. 4 (See legend on next page.) Fig. 4 (See legend on next page.) IL-1β secretion in HUVECs, but also downregulates the ubiquitination of NLRP3, which facilitates its activation, as an active metabolite of pentachlorophenol (PCP). This study also reported that K+ efﬂux, mtROS production, Xia et al. demonstrated that NLRP3 inﬂammasome acti- vation is involved in tetrachlorobenzoquinone (TCBQ)- induced endothelial inﬂammation53. TCBQ not only promotes the post-translational activation of NLRP3 and Ofﬁcial journal of the Cell Death Differentiation Association Bai et al. Cell Death and Disease (2020) 11:776 Page 8 of 18 (see ﬁgure on previous page) Fig. Implications of NLRP3 inﬂammasome activation in endothelial dysfunction NLRP3 inﬂammasome activation in endothelial inﬂammation 4 The role of NLRP3 inﬂammasome activation in endothelial dysfunction. a The secretion of mature forms of IL-1β, IL-18, and HMGB1 is the result of activation of NLRP3 inﬂammasome. These mediators possess properties of pro-inﬂammatory activation. Therefore, NLRP3 inﬂammasome can induce a potent inﬂammatory response, oxidative stress, and pro-inﬂammatory cell death called pyroptosis. b Inﬂammation and oxidative stress can induce endothelium DNA damage, activate NF-κB signaling pathway, increase P53/P21/P16 transcription, and inhibit autophagy, which may promote the process of endothelial cell senescence. Senescent cells can secrete senescence-associated secretory phenotype (SASP), including pro- inﬂammatory mediators, which may promote endothelial dysfunction and eventually lead to vascular sclerosis. c IL-1β binding to its cell surface receptor IL-1 receptor 1 (IL-1R1) recruits IL-1 receptor accessory protein (IL-1RacP) to activate intracellular signaling molecules, including myeloid differentiation factor 88 (MyD88), IL-1 receptor-associated kinase 1/4 (IRAK1/4), and TNF receptor-associated factor (TRAF), which then causes NF-κB activation. IL-18 binding to its cell surface receptor IL-18 receptor α chain (IL-18Rα) recruits IL-18 receptor β chain (IL-18Rβ) to activate similar intracellular signaling molecules. HMGB1 can also activate the NF-κB signaling pathway, which is downstream of toll-like receptor (TLR)2/4 activation. The activation of NF-κB signaling pathway increases the secretion of pro-inﬂammatory mediators such as cytokines and chemokines to mediate the adhesion of leukocyte and promote leukocyte extravasation. Additionally, the binding of HMGB1 to the RAGE receptor leads to the activation of downstream p38 MAP kinase, resulting in phosphorylation of the actin-binding protein Hsp27 and caldesmon, which causes actin stress ﬁbers to form, cytoskeletal remodeling, and endothelial contraction. All of these reactions increase endothelial permeability by altering cell contractility and disrupting intercellular connections. and mtDNA damage might contribute to TCBQ-induced NLRP3 inﬂammasome activation53. Further research revealed that GSDMD and mixed-lineage kinase domain- like (MLKL) play crucial roles in TCBQ-induced atypical inﬂammatory pathway activation54. The results showed that TCBQ aggravated NLRP3 inﬂammasome activation via the disruption of intracellular and extracellular ion homeostasis, which was caused by the leakage of GSDMD and MLKL and the release of cell contents54. Many ago- nists that target the NLRP3 inﬂammasome signaling pathway and facilitate endothelial dysfunction are listed in Table 1. inﬂammasome activation plays a crucial role in this pro- cess57. Chen et al. illustrated a new molecular mechanism of EC death and inﬂammation caused by cadmium (Cd), an independent risk factor for various cardiovascular diseases. NLRP3 inﬂammasome activation in endothelial cell death NLRP3 inﬂammasome activation in endothelial cell death Typically, cell death mainly includes apoptosis and necrosis; however, an emerging type of cell-intrinsic death has recently been found, termed pyroptosis55. Pyroptosis, an inﬂammatory programmed cell death process, is another functional outcome of NLRP3 inﬂammasome activation, which is mediated by the membrane pore- forming activity of the GSDMD N-terminal domain released by caspase-1 cleavage56. Pyroptosis is manifested by cell membrane rupture, cellular lysis, and release of pro-inﬂammatory contents, including IL-1β, IL-18, as well as high-mobility group box 1 (HMGB1), signifying the inﬂammatory nature of pyroptosis, and distinguishing pyroptosis from other forms of cell death55. NLRP3 inﬂammasome-mediated pyroptosis has been identiﬁed as a potential cause of EC death (Fig. 4). Ofﬁcial journal of the Cell Death Differentiation Association Implications of NLRP3 inﬂammasome activation in endothelial dysfunction NLRP3 inﬂammasome activation in endothelial inﬂammation Cd exposure induces EC death and inﬂammatory response via mitochondrial ROS-mediated NLRP3 acti- vation and pyroptosis in HUVECs58. py p A recent study reported that genetic copy number variations (CNVs) might be linked to the activation of NLRP3 inﬂammasome and endothelial pyroptosis. Chen et al. found that the human neutrophil peptide (HNP)- encoding gene (DEFA1/DEFA3) CNVs play speciﬁc roles in sepsis59. Interestingly, they reported that transgenic mice carrying more copies of the DEFA1/DEFA3 suffer from more severe sepsis and mortality than those with low copy numbers of DEFA1/DEFA3 and wild-type mice, which is caused by broader endothelial barrier dysfunc- tion and EC pyroptosis. Also, in a mouse lung micro- vascular endothelial cell line model, it was reported that HNP-1 induces EC pyroptosis and endothelial barrier dysfunction by directly targeting the purinergic receptor P2X ligand-gated ion channel 7 (P2X7) and subsequently activating the canonical NLRP3/caspase-1 pathway. Functional blockade of HNP1–3 alleviates endothelial pyroptosis and protects the mice from sepsis59. This study provided a novel molecular mechanism for NLRP3 acti- vation to mediate endothelial pyroptosis. NLRP3 inﬂammasome activation in endothelial barrier dysfunction Our previous study reported that NLRP3-mediated pyroptosis is involved in EC death caused by certain sti- muli. Acrolein, an exogenous toxin, is produced in response to environmental pollution. Jiang et al. reported that acrolein-induced EC death is attributed to a decrease in ROS-dependent autophagy, which promotes NLRP3 inﬂammasome activation and pyroptosis in HUVECs. It is generally known that autophagy is a negative regulator of NLRP3 inﬂammasome activation. Consequently, NLRP3 The vascular ECs cover the intima of blood vessels, forming a semipermeable barrier between circulating blood and extravascular matrix. This barrier maintains the transport of solutes, ﬂuids, and cells by coordinating the opening and closing of cell junctions60. The endothelial barrier dysfunction is characterized by the loss of local contact between ECs, and extravasation of plasma pro- teins, cells, or solutes61. Sustained inﬂammatory Ofﬁcial journal of the Cell Death Differentiation Association Bai et al. Cell Death and Disease (2020) 11:776 Page 9 of 18 Table 1 Summary of agonists targeting NLRP3 inﬂammasome signaling pathway and facilitating endothelial dysfunction. Agonist Category Effects and potential mechanisms Disease Referen TMAO Choline metabolite •↑Endothelial inﬂammation •↑ROS–TXNIP–NLRP3 pathway •↓eNOS expression •↓NO roduction AS 124 Visfatin Adipokine •↑Endothelial inﬂammation •↑Neointimal formation •↑NLRP3 formation and activation Obesity 52 Acetate, propionate SCFAs •↑Endothelial inﬂammation •↑Neointimal formation •↑NLRP3 formation and activation AS 125 TCBQ Toxicant •↑Endothelial inﬂammation •↑NLRP3 activation, IL-1β secretion •↑NLRP3 deubiquitination •↑GSDMD and MLKL leakage / 53,54 LCWE L. casei cell wall extract •↑Endothelial inﬂammation •↑Lysosome membrane permeability, cathepsin B release •↑NLRP3 formation and activation Coronary arteritis 126 Heme Iron porphyrins •↑Endothelial inﬂammation •↑NLRP3 activation, IL-1β secretion Hemolytic diseases 127 Endothelial GPR124 Orphan receptor •↑Atherosclerosis progression •↑Nitrosative stress •↑NLRP3 activation AS 128 AGEs Glycation products •↑Endothelial inﬂammation •↑ROS/TXNIP/NLRP3 pathway •↓EPC numbers, NO level, and eNOS expression Chronic kidney disease 129 SREBP2 Transcription factor •↑Endothelial inﬂammation •↑The production of toxic lipids, cholesterol crystals, and necrotic cells •↑NLRP3 activation AS 130 Adora2a Adenosine receptor •↑Cerebral endothelial inﬂammation •↑NLRP3 activation Ischemic stroke 131 Prdx6-aiPLA2 activity Enzyme •↑Endothelial inﬂammation •↑Endothelial NOX2 activation •↑ROS/NF-κB/NLRP3 pathway ALI 132 PGE2 Prostaglandin •↑Endothelial inﬂammation, apoptosis •↑NLRP3 activation •↑Inﬂammatory chemokines Diabetic retinopathy 133 Acrolein Unsaturated aldehyde •↑Pyroptosis •↓Cell migration •↓ROS-dependent autophagy •↑NLRP3 activation AS 57 Cadmium Metal toxicant •↑Endothelial inﬂammation, pyroptosis •↑mtROS-mediated NLRP3 activation CVD 58 Nicotine Alkaloid •↑Endothelial inﬂammation, pyroptosis •↑ROS production, NLRP3 activation AS 134 Table 1 Summary of agonists targeting NLRP3 inﬂammasome signaling pathway and facilitating endothelial dysfunction. NLRP3 inﬂammasome activation in endothelial barrier dysfunction Table 1 Summary of agonists targeting NLRP3 inﬂammasome signaling pathway and facilitating endothelial eting NLRP3 inﬂammasome signaling pathway and facilitating endothelial SREBP2 Transcription factor Ofﬁcial journal of the Cell Death Differentiation Association Bai et al. Cell Death and Disease (2020) 11:776 Bai et al. Cell Death and Disease (2020) 11:776 Page 10 of 18 Table 1 continued Agonist Category Effects and potential mechanisms Disease References •↑Endothelial barrier dysfunction •↑Cathepsin B-dependent NLRP3 activation •↑HMGB1 release 69 •↑Endothelial barrier dysfunction •↑Cathepsin B-dependent NLRP3 activation •↑HMGB1 release of HMGB1 to the RAGE receptor, leading to downstream p38 MAPK activation as well as actin-binding protein Hsp27 phosphorylation67. Interestingly, HMGB1 was also demonstrated to increase the secretion of pro- inﬂammatory mediators such as cytokines and chemo- kines via mediating NF-κB signaling pathway, at the downstream of TLR2 and TLR4 activation. This may indirectly cause endothelial barrier dysfunction. However, this needs to be thoroughly investigated in the future67. activation of the endothelium initiates the secretion of various inﬂammatory mediators, including IL-1, tumor necrosis factor (TNF), vascular endothelial growth factor, histamine, and bradykinin, leading to the internalization of VE cadherin and disruption of inter-endothelial junc- tions62. Therefore, endothelial barrier dysfunction is considered to be an important mechanism underlying many diseases associated with inﬂammation, where inﬂammatory mediators activate diverse signaling path- ways in the endothelium, resulting in the increased endothelial permeability and inappropriate loss of solutes and cells62. Previous studies have unraveled that endothelial barrier dysfunction induced by certain risk factors is linked to NLRP3 inﬂammasome activation, which is achieved via the release of HMGB1. Chen et al. reported that the activation of NLRP3 inﬂammasome and subsequent release of HMGB1 are the underlying causes of inter- cellular junction fracture in mouse vascular ECs (MVECs) treated with high glucose (HG)68. In contrast, NLRP3 deﬁciency has a protective effect on intercellular junction interruption in the coronary arterial ECs of diabetic mice68. Zhang et al. revealed that NLRP3 inﬂammasome is involved in triggering nicotine-induced endothelial barrier dysfunction69. This study suggested that nicotine enhances NLRP3 inﬂammasome activation and causes the release of HMGB1, leading to the damage of inter- endothelial junction and hyperpermeability69. and cells . Moreover, IL-1β, IL-18, and HMGB1 released by NLRP3 activation may activate NF-κB pathway, which in turn promotes the transcriptional activation of chemo- kines and adhesion molecules, increases leukocyte adhe- sion, and changes in cell permeability, thereby playing a crucial role in endothelial barrier dysfunction. NLRP3 inﬂammasome activation in endothelial barrier dysfunction IL-1β and IL-18 are products of NLRP3 inﬂammasome activation. As inﬂammatory mediators, they can enhance the expression of adhesion molecules and chemokines in the endothelium via binding to their cell surface receptors respectively, and activating the NF-κB pathway (Fig. 4)63. Endothelial NF-κB activation initiates transcriptional activation of numerous genes, including chemokines and adhesion molecules64. These reactions can increase the adhesion and rolling of leukocyte and promote leukocyte extravasation. Additionally, activated leukocytes can secrete cytokines that mediate persistent inﬂammatory responses, such as IL-1 or TNF, which can increase vas- cular endothelial permeability by altering cell contractility and disrupting intercellular connections62. Ofﬁcial journal of the Cell Death Differentiation Association NLRP3 inﬂammasome activation in endothelial cells’ senescence Tissue aging is often accompanied by chronic inﬂam- mation, and the same goes for endothelial senescence70. By inducing vascular structural and functional changes, EC senescence actively regulates aging-associated vascular dysfunction (Fig. 4)71. Speciﬁcally, the disruption of the cell cycle increases ROS and oxidative stress, vascular inﬂam- mation, causes impaired Ca2+ signaling, high serum uric acid levels, and activates renin–angiotensin–aldosterone system, which is closely related to the premature senescence of ECs71. Recently, NLRP3 inﬂammasome has been demonstrated to correlate mild systemic inﬂammation with age-related functional decline72. NLRP3 inﬂammasome can sense the accumulation of DAMPs during aging and med- iate the pro-inﬂammatory cascade both inside and outside the brain, which is independent of the noncanonical caspase-11 pathway72. Inhibiting abnormal NLRP3 inﬂam- masome activity during aging reduces age-associated innate p g HMGB1 is closely related to NLRP3 inﬂammasome activation and endothelial barrier dysfunction (Fig. 4). NLRP3 inﬂammasome activation can also release DAMPs such as HMGB1 to the extracellular environment65. Extracellular release of HMGB1 may be caused by non- canonical mechanisms along with IL-1β and IL-18, or it may be a passive consequence of cell lysis during pyr- optosis66. Extracellular HMGB1 can bind to its receptors, including toll-like receptor 2 (TLR2), TLR4, and RAGE, thereby inducing inﬂammation and repair responses66. Moreover, HMGB1 can induce endothelial barrier dis- ruption by increasing the contractile activity and endo- thelial permeability. These effects are mediated by binding Ofﬁcial journal of the Cell Death Differentiation Association Bai et al. Cell Death and Disease (2020) 11:776 Page 11 of 18 Page 11 of 18 the NLRP3 inﬂammasome signaling pathway; these stu- dies were summarized in this section (see Table 2). immune activation, attenuates multiple age-related chronic diseases, and prolongs the health span, consistent with the previous results72. Furthermore, Youm et al. showed that NLRP3 inﬂammasome activation also causes age-related thymic involution and immunosenescence73. Some exploratory research has demonstrated the potential of ncRNAs as clinical biomarkers or drug targets in various diseases78,84–86. Additionally, the distinctive roles of ncRNAs in the regulation of endothelial function have been widely explored87–92. Nevertheless, research on ncRNAs regulating endothelial function via the NLRP3 inﬂammasome signaling pathway is still limited (Fig. 5). ncRNAs tested in clinical samples have more potential as disease biomarkers in clinical applications. A great deal of clinical validation and mechanistic research is needed to elucidate the roles of ncRNAs regulating NLRP3 inﬂam- masome signaling pathway in endothelial function. Clinical drugs and NLRP3 inﬂammasome and endothelial dysfunction-related diseases Statins Statins are widely applied in the clinics because of their lipid-level-lowering properties. The functions of statins have been extended to include anticoagulant, anti- inﬂammatory, and immunomodulatory, suggesting that statins may have other beneﬁts besides cholesterol reduction, as numerous clinical studies showed93. Wu et al. found that atorvastatin inhibited EC pyroptosis via increasing the expression of lncRNA NEXN-antisense RNA 1 (AS1) and its cognate gene NEXN, and inhibiting the expression of NLRP3 and pyroptosis-related mole- cules such as caspase-1 and IL-1β94. Simvastatin has been demonstrated to ameliorate endothelial dysfunction by inhibiting the activation of NLRP3 inﬂammasome in HG conditions. Mechanistically, simvastatin treatment remarkably enhanced the expression of zonula occluden-1 (ZO-1) and VE cadherin by inhibiting the NLRP3 inﬂammasome-dependent HMGB1 release, leading to the alleviation of vascular endothelial hyperpermeability95. Additionally, simvastatin can also exert an ather- oprotective action via modulating the endothelial kruppel-like factor 2 (Klf2)-forkhead box P transcription factor 1 (Foxp1)–NLRP3 inﬂammasome network to sup- press NLRP3 inﬂammasome activation96. These studies shed light on new mechanisms of statins to improve atherosclerosis and diabetic angiopathy by inhibiting NLRP3 inﬂammasome activation. Recently, some pieces of literature have reported that some beneﬁcial substances can reduce the senescence of ECs, at least in part, by inhibiting NLRP3 activation. Sun et al. demonstrated that purple sweet potato color (PSPC), one type of ﬂavonoid derived from purple sweet potato, ameliorates endothelium senescence by restraining ROS production and NLRP3 inﬂammasome activation, and then reduces atherosclerotic lesions in insulin-resistant mice76. Additionally, they further investigated the corre- lation between NLRP3 inﬂammasome and autophagy and the underlying mechanisms of EC senescence. It was found that PSPC inhibits endothelial senescence via enhancing autophagy and keeping inﬂammasome activa- tion in check77. With the clariﬁcation of the molecular mechanism and role of NLRP3 inﬂammasome in EC aging in the future, we believe that more drugs will target the NLRP3 inﬂammasome signaling pathway to treat endo- thelial aging-related diseases. NLRP3 inﬂammasome activation in endothelial cells’ senescence y A most recent study demonstrated that NLRP3 inﬂammasome-mediated IL-1β has a pathogenic role in multiple distinct ocular aging diseases74. Nevertheless, little is known about whether NLRP3 inﬂammasome contributes to EC senescence and the underlying mole- cular mechanisms. Yin et al. explored the roles and potential mechanisms of NLRP3 inﬂammasome in EC senescence75. It was found that NLRP3 inﬂammasome activation promotes bleomycin-induced EC senescence by increasing the IL-1β secretion. Additionally, secreted IL- 1β also signiﬁcantly upregulates the expression of the senescence-related marker p53/p21 protein. During these processes, ROS plays a key role in inducing TXNIP–NLRP3 interaction75. The molecular mechanism and role of NLRP3 inﬂammasome in endothelial senes- cence remain to be further studied. Noncoding RNAs (ncRNAs) targeting the NLRP3 inﬂammasome signaling pathway in endothelial dysfunction ncRNAs are a broad spectrum of RNA molecules that have high transcriptional activity, and regulatory and structural functions, but do not encode proteins. ncRNAs, such as microRNAs (miRNAs), long noncoding RNAs (lncRNAs), and circular RNAs (circRNAs), are novel regulators of cardiovascular risk factors and cell func- tions78–81. Additionally, ncRNAs have been demonstrated to precisely control gene expression and have gradually become indispensable regulators of inﬂammation and immunity82,83. Several studies have provided evidence that ncRNAs may regulate endothelial function by mediating Hypoglycemic agents In recent years, studies have found that antidiabetic drugs can reduce endothelial disorders via blocking NLRP3 inﬂammasome activation, providing the potential for treating diabetic vasculopathy. The classiﬁcation of these drugs based on pharmacological mechanisms is summarized as follows. Dipeptidyl peptidase-4 (DPP-4) inhibitors are a class of effective hypoglycemic agents for the treatment of type 2 Ofﬁcial journal of the Cell Death Differentiation Association Page 12 of 18 Bai et al. Cell Death and Disease (2020) 11:776 Table 2 Summary of ncRNAs targeting NLRP3 inﬂammasome signaling pathway in endothelial dysfunction. ncRNA Target gene Effect on NLRP3 activation Functions Disease/research context References miR-22 NLRP3 Down •↓EC apoptosis •↓Pro-inﬂammatory cytokines •↑Tube formation Coronary atherosclerosis 135 miR-495 NLRP3 Down •↓EC inﬂammation •↓EC apoptosis •↑EC proliferation Myocardial ischemia/ reperfusion injury 136 miR-15a FOXO1 Down •↓EC inﬂammation Diabetic retinopathy 137 miR-126 HMGB1 Down •↓EC inﬂammation Diabetic retinopathy 138 miR-590-3p NLRP1, NOX4 Down •↓EC inﬂammation, pyroptosis Diabetic retinopathy 139 miR-20a TXNIP, TLR4 Down •↓EC inﬂammation •↓ROS generation Atherosclerosis 140 miR-30c-5p FOXO3 Down •↓EC inﬂammation, pyroptosis Atherosclerosis 141 miR-383-3p IL1R2 Down •↓EC inﬂammation •↓EC apoptosis •↑Tube formation Coronary atherosclerosis 142 miR-20b TXNIP Down •↓EC senescence H2O2-induced EC senescence 143 miR-101-3p Bim Down •↓EC apoptosis Serum deprivation-induced EC apoptosis 144 miR-200a-3p Keap1, NLRP3 Up •↑EC inﬂammation Sepsis-induced brain injury 145 miR-92a SIRT1, KLF2, and KLF4 Up •↑Endothelial innate immunity •↓NO bioavailability Atherosclerosis 146 miR-125a-5p TET2 Up •↑EC inﬂammation, pyroptosis Atherosclerosis 147 LncRNA MALAT1 miR-22 Up •↑EC inﬂammation, pyroptosis Atherosclerosis 148 LncRNA MEG3 miR-223 Up •↑EC pyroptosis Atherosclerosis 149 LncRNA NEXN-AS1 / Down •↓EC pyroptosis Atherosclerosis 94 Hsa_circ_0068087 miR-197 Up •↑EC inﬂammation •↑Tube formation T2DM 150 ary of ncRNAs targeting NLRP3 inﬂammasome signaling pathway in endothelial dysfunction. ome signaling pathway in endothelial dysfunction Table 2 Summary of ncRNAs targeting NLRP3 inﬂammasome signaling pathway in endothel mitochondrial function and recovering eNOS levels, indicating its protective roles on endothelial dysfunc- tion97. Recently, anagliptin, a novel DPP-4 inhibitor licensed for treating T2DM, was found to restore HG- induced endothelial dysfunction via SIRT1-dependent inhibition of NLRP3 inﬂammasome activation and sup- pression of NOX4–ROS–TXNIP–NLRP3 signaling, which suggests that anagliptin may have broad pharma- cological effects in cardiovascular diseases and other metabolic disorders98. diabetes mellitus (T2DM). DPP-4 inhibitors have proven to be effective in improving endothelial function, redu- cing oxidative and pro-inﬂammatory states, suggesting beneﬁcial effects on cardiovascular function. Qi et al. Ofﬁcial journal of the Cell Death Differentiation Association Other anti-inﬂammatory or antioxidant drugs Fig. 5 The involvement of noncoding RNAs (ncRNAs) in regulating NLRP3 inﬂammasome-mediated endothelial dysfunction and diseases. Schematic representation of ncRN Other anti-inﬂammatory or antioxidant drugs have also been investigated in different disease contexts. Aspirin is one of the most commonly used drugs for the secondary pre- vention of cardiovascular disease104. Zhou et al. found that aspirin protects the expression of ZO-1 and ZO-2 by inhi- biting NLRP3 inﬂammasome formation and activation in LPS-induced MVECs and coronary arterial endothelium, thereby alleviating endothelial gap junction dysfunction105. This study unraveled a novel mechanism of aspirin in ame- liorating endothelial dysfunction by blocking redox signaling and NLRP3 inﬂammasome activation, providing a new viewpoint on the clinical potential of aspirin in the early prevention of cardiovascular diseases105. The mitochondrion- targeting antioxidant mitoquinone (MitoQ) was found to inhibit endothelial inﬂammation and barrier injury in cigar- ette smoke extract (CSE)-treated HUVECs via restoring mitochondrial damage106. Mechanistically, the protective effects of MitoQ depend on suppressing the internalization of VE cadherin and cytoskeleton remodeling, and inhibiting the activation of NF-κB/NLRP3 inﬂammasome pathway, as well as restraining the production of mtROS and autophagy106. dysfunction and diseases. Schematic representation of ncRNAs upregulated or downregulated in multiple endothelial dysfunction- related disease models. These ncRNAs mediate endothelial dysfunction by directly or indirectly activating the NLRP3 inﬂammasome signaling pathway. The reported diseases mainly include myocardial ischemia/reperfusion (I/R) injury, sepsis-induced brain injury, type 2 diabetes mellitus (T2MD), diabetic retinopathy, and atherosclerosis. Moreover, miR-20b can be downregulated in H2O2- treated endothelial cells (ECs) and induces EC senescence by activating NLRP3 inﬂammasome. In addition, miR-101-3p is downregulated in serum deprivation-induced EC apoptosis, which is partially induced by activating the NLRP3 inﬂammasome signaling pathway. In this ﬁgure, the red upward arrow indicates upward adjustment, and the black downward arrow indicates downward adjustment. Dulaglutide, a kind of glucagon-like peptide-1 receptor agonist (GLP-1 RA), has been approved for use in the treatment of T2DM. Luo et al. found that dulaglutide inhibits HG-induced endothelial dysfunction via SIRT1- dependent inhibition of NLRP3 inﬂammasome activation and expression of NOX4 and TXNIP99. p Acarbose, a well-known α-glucosidase inhibitor, is a postprandial acting antidiabetic drug. It has been reported that acarbose has protective effects against vascular endothelial dysfunction by inhibiting the production of NOX4 oxidase-dependent O2 •−, which contributes to blocking NLRP3 inﬂammasome activation100. Other anti-inﬂammatory or antioxidant drugs Further- more, the reduced expression of NLRP3 inﬂammasome is also involved in the amelioration of vascular hyperper- meability by acarbose, which is attributed to the enhanced expression of junction protein ZO-1 and VE cadherin100. Fenoﬁbrate is a selective agonist of peroxisome proliferator-activated receptor α (PPARα) that prevents the progression of microvascular complications in T2DM. Deng et al. found that fenoﬁbrate promotes wound healing via alleviating EPC dysfunction and stimulating angiogenesis in diabetic mice induced by streptozotocin All these drugs ameliorate endothelial dysfunction by inhibiting NLRP3 inﬂammasome activation. Accordingly, we summarized the drugs targeting NLRP3 inﬂamma- some signaling pathway and their targets in endothelial dysfunction-related diseases (Fig. 6). However, it is necessary to further study the molecular mechanism of these drugs inhibiting NLRP3 inﬂammasome pathway. Together, these studies provide new perspectives on the pharmacological mechanisms of drugs and therapeutic strategies for endothelial dysfunction-related diseases. Hypoglycemic agents found that DPP-4 inhibitor vildagliptin ameliorates endothelial dysfunction, induced by high free fatty acid (FFA) levels through inhibiting the AMPK–NLRP3–HMGB1 pathway97. Moreover, vilda- gliptin also reduces the cellular lactate dehydrogenase (LDH) release and ROS levels, as well as protecting Ofﬁcial journal of the Cell Death Differentiation Association Page 13 of 18 Bai et al. Cell Death and Disease (2020) 11:776 (STZ), the effects of which are attributed to the inhibition of NLRP3 inﬂammasome pathway101. Fig. 5 The involvement of noncoding RNAs (ncRNAs) in regulating NLRP3 inﬂammasome-mediated endothelial dysfunction and diseases. Schematic representation of ncRNAs upregulated or downregulated in multiple endothelial dysfunction- related disease models. These ncRNAs mediate endothelial dysfunction by directly or indirectly activating the NLRP3 inﬂammasome signaling pathway. The reported diseases mainly include myocardial ischemia/reperfusion (I/R) injury, sepsis-induced brain injury, type 2 diabetes mellitus (T2MD), diabetic retinopathy, and atherosclerosis. Moreover, miR-20b can be downregulated in H2O2- treated endothelial cells (ECs) and induces EC senescence by activating NLRP3 inﬂammasome. In addition, miR-101-3p is downregulated in serum deprivation-induced EC apoptosis, which is partially induced by activating the NLRP3 inﬂammasome signaling pathway. In this ﬁgure, the red upward arrow indicates upward adjustment, and the black downward arrow indicates downward adjustment. Additionally, cilostazol, a phosphodiesterase-3 inhibitor, also alleviates the adverse vascular effects of living with dia- betes. Cilostazol signiﬁcantly reduces NLRP3 inﬂammasome activation and the activity of NOX4, TXNIP, HMGB1, IL-1β, and IL-18 in HAECs induced with FFA. Cilostazol also protected the function of SIRT1, which serves to limit the activity of NLRP3 inﬂammasome102. Glibenclamide is a sulfonylurea drug widely prescribed to treat T2DM. Studies have discovered that glibenclamide attenuates blood–brain barrier (BBB) disruption in experi- mental intracerebral hemorrhage model by inhibiting NLRP3 inﬂammasome activation in microvessel ECs, thereby main- taining the integrity of the BBB103. Ofﬁcial journal of the Cell Death Differentiation Association Conclusions In recent years, a group of evidence has accumulated linking NLRP3 inﬂammasome activation to the control of endothelial dysfunction. The ﬁndings reviewed here highlight the tremendous potential of direct or indirect modulation of NLRP3 inﬂammasome activation in combating various endothelial dysfunctions. When ECs are stimulated by exo- genous substances or endogenous mediators, in addition to triggering oxidative stress, ER stress, mitochondrial dys- function, and immune activation, these effects may also trigger a common signaling pathway of NLRP3 inﬂamma- some activation, thereby exacerbating endothelial dysfunc- tion. Of note, emerging literature indicates that certain well- known drugs, such as statins, hypoglycemic agents, and other anti-inﬂammatory or antioxidant drugs, can improve vas- cular dysfunction by inhibiting NLRP3 inﬂammasome sig- naling pathway. We have also summarized ncRNAs that involved in the regulation of endothelial function by directly or indirectly regulating NLRP3 inﬂammasome activation; this helps to understand the molecular mechanisms underlying NLRP3 inﬂammasome-related endothelial dysfunction, and may provide novel targets for the development of future therapeutics. Fig. 6 Drugs targeting NLRP3 inﬂammasome signaling pathway and their targets in endothelial dysfunction-related diseases. These drugs, including statins, hypoglycemic agents, and other anti- inﬂammatory or antioxidant drugs, ameliorate endothelial dysfunction by inhibiting NLRP3 inﬂammasome signaling pathway. The drugs on the left of the ﬁgure have been proved to inhibit NLRP3 inﬂammasome activation by mediating different targets, while the targets of the drug on the right of the ﬁgure still need further study to verify. Fig. 6 Drugs targeting NLRP3 inﬂammasome signaling pathway and their targets in endothelial dysfunction-related diseases. These drugs, including statins, hypoglycemic agents, and other anti- inﬂammatory or antioxidant drugs, ameliorate endothelial dysfunction by inhibiting NLRP3 inﬂammasome signaling pathway. The drugs on the left of the ﬁgure have been proved to inhibit NLRP3 NLRP3 inﬂammasome inhibitors. A large number of inhibitors have been demonstrated to improve endothelial dysfunction by inhibiting NLRP3 inﬂammasome signaling pathway, including endogenous and exogenous inhibitors, but with low speciﬁcity107–109. Here, we show the potential of speciﬁc NLRP3 inhibitors to ameliorate endothelial dysfunction (Table 3), which are brieﬂy dis- cussed below. NLRP3 inﬂammasome inhibitors. A large number of inhibitors have been demonstrated to improve endothelial dysfunction by inhibiting NLRP3 inﬂammasome signaling pathway, including endogenous and exogenous inhibitors, but with low speciﬁcity107–109. Here, we show the potential of speciﬁc NLRP3 inhibitors to ameliorate endothelial dysfunction (Table 3), which are brieﬂy dis- cussed below. NLRP3 inﬂammasome-speciﬁc pharmacological inhibitors for the treatment of endothelial dysfunction NLRP3 inﬂammasome is involved in the progress of a variety of disorders, which promotes the discovery of Ofﬁcial journal of the Cell Death Differentiation Association Bai et al. Cell Death and Disease (2020) 11:776 Page 14 of 18 Fig. 6 Drugs targeting NLRP3 inﬂammasome signaling pathway and their targets in endothelial dysfunction-related diseases. These drugs, including statins, hypoglycemic agents, and other anti- inﬂammatory or antioxidant drugs, ameliorate endothelial dysfunction by inhibiting NLRP3 inﬂammasome signaling pathway. The drugs on the left of the ﬁgure have been proved to inhibit NLRP3 inﬂammasome activation by mediating different targets, while the targets of the drug on the right of the ﬁgure still need further study to verify. Hence, NLRP3 inﬂammasome-speciﬁc pharmacological inhibitors may be the optimal choice for the treatment of endothelial dysfunction, providing a new strategy to treat endothelial dysfunction-related disease. In the future, the effects of these inhibitors are needed to be veriﬁed at the animal level and in clinical trials. Ofﬁcial journal of the Cell Death Differentiation Association Conclusions MCC950 is one of the NLRP3 inﬂammasome-speciﬁc inhibitors by blocking ASC oligomerization and NLRP3 ATP hydrolysis110,111. A previous study reported that MCC950 treatment alleviated HG-induced human retinal endothelial cell (HREC) dysfunction probably in part by suppressing the NEK7–NLRP3 interaction112. Treatment of MCC950 also attenuated diabetes-related vascular dysfunction in diabetic mice model113. Moreover, treatment of diabetic rats after stroke with MCC950 ameliorated cognitive function, vas- cular permeability, and neurovascular remodeling114. More recently, it has been found that sepsis-related endothelial dysfunction was also improved with MCC950 intervention in in vivo and in vitro models115,116. Additionally, CY-09, OLT1177, 3,4-methylenedioxy-β-nitrostyrene (MNS), N- [3′,4′-dimethoxycinnamoyl]-anthranilic acid (Tranilast), and oridonin are also speciﬁc inhibitors of NLRP3 inﬂam- masome33,117. Remarkably, a recent study found that tranilast exhibited antivascular inﬂammation and anti- atherosclerosis properties via increasing NLRP3 ubiqui- tination and impeding NLRP3 inﬂammasome activa- tion118. Besides, oridonin was found to blunt endothelial inﬂammation through restraining the activation of MAPK and NF-κB signaling pathways119. It was also reported that oridonin diminished cell migration and angiogenesis in VEGF-treated HUVECs120. With the advancement of the research, our under- standing of potential mechanisms that affect endothelial function through the NLRP3 inﬂammasome activation pathway is expanding. Meanwhile, the interactions between endothelial dysfunction and the NLRP3 inﬂammasome-regulated pathways may open up a new avenue for the treatment of cardiovascular diseases. While our understanding of the inﬂuence of NLRP3 inﬂamma- some activation on endothelial dysfunction is con- tinuously growing, our ideas on how NLRP3 inﬂammation perceives information about different mediators are still limited. Furthermore, it is unclear whether different agonists affect endothelial function through a single cascade of events or through distinct pathways to activate NLRP3 inﬂammation. In parallel, the effects of other inﬂammasomes, such as NLRP1, NLRC4, NLRP6, and AIM2, noncanonical inﬂammasomes, and upstream pathways on endothelial function, have not been thoroughly investigated so far. Of the ncRNAs that regulate NLRP3 inﬂammasome pathway, miRNAs are currently the most studied. Accu- mulating research has indicated that miRNAs have tre- mendous potential as an indicative molecular biomarker or drug target in the diagnosis and treatment of diseases Ofﬁcial journal of the Cell Death Differentiation Association Page 15 of 18 Bai et al. Cell Death and Disease (2020) 11:776 Table 3 Potential speciﬁc NLRP3 inﬂammasome inhibitors for the treatment of endothelial dysfunction. Publisher’s note S i N 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: 7 May 2020 Revised: 31 August 2020 Accepted: 4 September 2020 Excitingly, the expanding research on the role of NLRP3 inﬂammasome in endothelial dysfunction will hopefully enrich the understanding of the vital role of NLRP3 inﬂammasome in endothelial dysfunction-related inﬂam- matory diseases. Furthermore, elucidating the molecular mechanisms of such interactions will be favorable for developing novel prevention approaches and more effec- tive therapeutic strategies for diseases related to endo- thelial dysfunction, development of which is an exciting challenge and goal for future research. Conﬂict of interest The authors declare that they have no conﬂict of interest. Acknowledgements h k 5. Martinon, F., Burns, K. & Tschopp, J. The inﬂammasome: a molecular platform triggering activation of inﬂammatory caspases and processing of proIL-beta. Mol. Cell 10, 417–426 (2002). Acknowledgements This work was supported by The National Natural Science Foundation of China (grant nos. 81870331, 31701208, and 91849209), The Natural Science Foundation of Shandong Province (grant no. ZR2017MC067), The Project of Shandong Province Higher Educational Science and Technology Program (no. J18KA285), and The Qingdao municipal science and technology bureau project (grant no. 18-2-2-65-jch). 6. Franchi, L., Eigenbrod, T., Munoz-Planillo, R. & Nunez, G. The inﬂammasome: a caspase-1-activation platform that regulates immune responses and disease pathogenesis. Nat. Immunol. 10, 241–247 (2009). 7. Prochnicki, T. & Latz, E. Inﬂammasomes on the crossroads of innate immune recognition and metabolic control. Cell Metab. 26, 71–93 (2017). 7. Prochnicki, T. & Latz, E. Inﬂammasomes on the crossroads of innate immune recognition and metabolic control. Cell Metab. 26, 71–93 (2017). 8. Pober, J. S. & Sessa, W. C. Evolving functions of endothelial cells in inﬂam- mation. Nat. Rev. Immunol. 7, 803–815 (2007). 8. Pober, J. S. & Sessa, W. C. Evolving functions of endothelial cells in inﬂam- mation. Nat. Rev. Immunol. 7, 803–815 (2007). Conclusions Agents Targets Host Functions Disease References MCC950 ASC oligomerization HRECs •↑Endothelial viability •↓Endothelial apoptosis Diabetic retinopathy 112 BMVECs •↓BBB permeability •↑Vascular integrity Diabetes mellitus 114 Diabetic mice •↓Vascular dysfunction •↑ACh vasodilation Diabetes mellitus 113 CLP rat •↑Endothelial permeability Sepsis 115 HUVECs/CLP mice •↑Aortic vasodilation •↑p-eNOS expression Sepsis 116 Oridonin Cysteine 279 of NLRP3 HUVECs •↓Vascular inﬂammation Vascular inﬂammation 119 HUVECs •↓Angiogenesis Breast cancer 120 Tranilast NLRP3 oligomerization ApoE−/−mice •↓Vascular inﬂammation •↓Atherosclerosis Atherosclerosis 118 Potential speciﬁc NLRP3 inﬂammasome inhibitors for the treatment of endothelial dysfunction. Translational Medicine, The Afﬁliated Hospital of Qingdao University, Qingdao 266021, China. 4Department of Cardiac Ultrasound, The Afﬁliated hospital of Qingdao University, Qingdao 266000, China. 5Department of Cardiology, The Afﬁliated Cardiovascular Hospital of Qingdao University, Qingdao 266032, China related to NLRP3 dysfunction. Nevertheless, plenty of thorny issues remain to be resolved to achieve this goal, including poor target speciﬁcity, poor miRNA stability, and the side effects involving liver damage121. Also, their effects may be restricted and removed by the kidney and liver due to the absence of protection around the carrier molecule122. Besides, the innate immune system may recognize and eliminate miRNA drugs123. However, it is encouraging that miRNA drug efﬁcacy, delivery, and safety issues are currently being addressed. Shortly, miRNAs as therapeutic targets will be the focus of most clinical research. 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https://openalex.org/W2094542600	https://www.cambridge.org/core/services/aop-cambridge-core/content/view/0896F00338F1AACAE83FF0D7AF9EE07A/S0955603600111158a.pdf/div-class-title-alprazolam-and-panic-disorder-div.pdf	English	null	Alprazolam and panic disorder	Psychiatric bulletin of the Royal College of Psychiatrists/Psychiatric bulletin	1,991	cc-by	2,092	682 682 Finchand Ramsay and expressed suicidal thoughts. There were no biological features of depression. The initial diagnosis was of an unspecified personality disorder. and expressed suicidal thoughts. There were no biological features of depression. The initial diagnosis was of an unspecified personality disorder. merely arriving at the correct psychiatric diagnosis and disposal. At another level, the group has assisted in facilitating practical communication between mem bers of the team allowing them to make maximum use of their different areas of expertise. The group has also functioned as a ward round, providing fuller coverage of the overdose service. As a result, our scarce resources have been shared and patients get maximum benefit from them. Breaking down inter professional barriers has helped to prevent rivalries emerging between different staff members. The group has also contributed to the working relation ship of the liaison service with medical and casualty staff who appreciate their patients being contained and not rejected. p p y We helped him find bed and breakfast accommodation. However, three days later we learnt that he had been evicted. He returned to Out-patients again; on the first occasion he cut his wrists in the waiting area, but, perhaps surprisingly, did engage in treatment and later came to accept referral to the Day Hospital. This was a young man who provoked feelings of hopelessness and futility in anyone who tried to help him; all previous interventions had been rejected or sabotaged by him. Presentation of his case to the group helped the social worker who had seen him to understand some of the reactions stemming from those feelings in herself and to recognise that this man could not deal with being alone all day even if he was given bed and breakfast accommodation. He was now offered additional psychological support that was more appropriate to his needs. j Taking these patients seriously when they come into hospital has given us an opportunity to intervene at a time of crisis which might not otherwise be poss ible, however much in need of help a person may be. The group may help us to prevent the development of more serious problems in these vulnerable patients. Comment This multidisciplinary supervision group has encour aged a wide ranging approach going beyond the medical psychiatric diagnostic model. Although the patients have been traumatised, the interview allows an opportunity to intervene at a time of acute crisis when both the acuteness and the crisis are being denied by the patient. Within the group overall emphasis is given to understanding an individual's experiences of his or her situation within a broadly psychodynamic developmental model, rather than Acknowledgement We would like to thank Consultant Psychotherapist, Dr Peter Shoenberg, and members of the group, in particular, the current leader and founder Egle Laufer, and fellow member Dr Tim Read for their comments. Psychiatric Bulletin (1991), 15,682-683 panic disorder. Few other psychotropic compounds have received such concentrated coverage. What, if anything, emerges from the studies reported? panic disorder. Few other psychotropic compounds have received such concentrated coverage. What, if anything, emerges from the studies reported? Very little it would seem. Both sets of studies use a profusion of rating scales and statistical techniques. Both analyse in detail the trends in various sub-scales https://doi.org/10.1192/pb.15.11.682 Published online by Cambridge University Press Alprazolam and panic disorder There have been several publications to this effectin recent years. The findings have been delivered at a number of conferences. Yet, no reference to them is made in this supplement. All references to the cognitive therapy of panic disorder date from 1985, which seems quite surprising. which is liable to be accompanied by a relapse of the disorder on tapering of therapy, particularly in the case of benzodiazepine therapy, cognitive therapy, it would appear, achieves success rates approaching 90%. There have been several publications to this effectin recent years. The findings have been delivered at a number of conferences. Yet, no reference to them is made in this supplement. All references to the cognitive therapy of panic disorder date from 1985, which seems quite surprising. of the scales that have been used. There are intention- to-treat analyses, complÃ©teranalyses and point analyses. Despite this and despite many "significant" findings, little of true significance appears to emerge from these studies. Alprazolam and imipramine are both superior to placebo but this hardly remarkable. Alprazolam has a quicker onset of action than imipramine. Again, something that could have been predicted before either the US or cross-national collaborative panic studies began. At the end of the day, it is not clear that anything more has been demonstrated other than alprazolam produces a benzodiazepine type anxiolysis, whereas imipramine produces an anxiolysis of the type found with drugs active on the 5HT system. We still await good phenomenological distinctions between these two forms of anxiolysis. q p g The cognitive therapy findings are of importance for a reason other than their apparent superiority to either alprazolam or imipramine in the treatment of panic disorder. A stated aim of the US and cross- national collaborative studies of the efficacyof alpra zolam in panic-disorder was to determine whether panic-disorder was a valid nosological entity distinct from agoraphobia and other anxiety states. There is little evidence from the reports of the research on alprazolam that bears on this issue and no evidence of any specificity of alprazolam to panic disorder. At present the best evidence for the validity of panic disorder as an independent nosological entity lies in the high rate of response to a specific set of cognitive interventions. Indeed, perhaps the most interesting finding of the APS reports resulted from an analysis of the characteristics of placebo responders. Alprazolam and panic disorder Individuals who responded well on placebo far from being the weak, dependent, suggestible persons who are some times seemingly conjured up by the notion of a placebo responder, had in actual fact greater strength of personality and coherence of egostructure than those who failed to show such a response. Unfortu nately this interesting, indeed "significant", finding may not get the attention it deserves owing to its being buried among a mass of largely unrelated details in a supplement sponsored by a drug company. There is, perhaps, one further significant aspect to these studies on alprazolam and panic disorder. Despite a lack of evidence that alprazolam is particu larly efficacious for panic-disorder or specific to it, recent advertisements for this compound in the American journals have made much of its being the only drug with a specific licence for use in panic- disorder. This licence was, of course, obtained on the back of the American and cross-national studies referred to above. pp p y g p y What is perhaps equally interesting about the recent APS supplement is what it fails to mention. Following the series of articles in Archivesof General Psychiatry, a cross-national collaborative letter was written to that Journal by Isaac Marks and several others (1989) criticising the interpretation put on the results. Their conclusion was that, contrary to the tenor of the papers published, given the results cited, alprazolam was of dubious benefit in panic disorder. This provoked a spirited response from Gerald Klerman and co-investigators on the US study of alprazolam. Nowhere in the APS supplement are the issues raised by Marks and others referred to. DAVIDHEALY Academic Sub-Department of Psychological Medicine North Wales Hospital Denbigh, ClwydLLlo 5SS Alprazolam and panic disorder This provoked a spirited response from Gerald Klerman and co-investigators on the US study of alprazolam. Nowhere in the APS supplement are the issues raised by Marks and others referred to. A further omission are the recent reports of an efficacy of cognitive therapy for panic disorder. In contrast to the drug treatment of panic disorder, which at best achieves 60-70% response rates and which is liable to be accompanied by a relapse of disorder on tapering of therapy, particularly in case of benzodiazepine therapy, cognitive therapy would appear, achieves success rates approachi 90%. There have been several publications to t effectin recent years. The findings have been delive at a number of conferences. Yet, no reference to th is made in this supplement. All references to cognitive therapy of panic disorder date from 19 which seems quite surprising. The cognitive therapy findings are of importan for a reason other than their apparent superiority either alprazolam or imipramine in the treatment panic disorder. A stated aim of the US and cro national collaborative studies of the efficacyof alp zolam in panic-disorder was to determine whet panic-disorder was a valid nosological entity disti from agoraphobia and other anxiety states. There little evidence from the reports of the research alprazolam that bears on this issue and no evide of any specificity of alprazolam to panic disorder. present the best evidence for the validity of pa disorder as an independent nosological entity lie the high rate of response to a specific set of cognit interventions. There is, perhaps, one further significant asp to these studies on alprazolam and panic disord Despite a lack of evidence that alprazolam is partic larly efficacious for panic-disorder or specific to recent advertisements for this compound in American journals have made much of its being only drug with a specific licence for use in pan disorder. This licence was, of course, obtained on back of the American and cross-national stud referred to above. DAVIDHEA Academic Sub-Department of Psychological Medicine North Wales Hospital Denbigh, ClwydLLlo 5SS References Acta Psychiatrica Scandinavia (\99\)ftt, Supp. No. 36 Archives of GeneralPsychiatry (1988)45,407^50. MARKS,I. M., DE ALBUQUERQUE, A., COTTEAUX,J. e (1989)Archivesof General Psychiatry. 46,668-670. which is liable to be accompanied by a relapse of the disorder on tapering of therapy, particularly in the case of benzodiazepine therapy, cognitive therapy, it would appear, achieves success rates approaching 90%. https://doi.org/10.1192/pb.15.11.682 Published online by Cambridge University Press Alprazolam and panic disorder In May 1988, the greater part of an issue of Archives of Generat Psychiatry was given over to reports of the results of a clinical trial of alprazolam for panic disorder. More recently, in March 1991,supplement no 365, of Ada Psychiatrica Scandinavica (APS) was devoted to a further trial of alprazolam for y g g p Very little it would seem. Both sets of studies use a profusion of rating scales and statistical techniques. Both analyse in detail the trends in various sub-scales https://doi.org/10.1192/pb.15.11.682 Published online by Cambridge University Press 683 Alprazolam andpanic disorder of the scales that have been used. There are intention- to-treat analyses, complÃ©teranalyses and point analyses. Despite this and despite many "significant" findings, little of true significance appears to emerge from these studies. Alprazolam and imipramine are both superior to placebo but this hardly remarkable. Alprazolam has a quicker onset of action than imipramine. Again, something that could have been predicted before either the US or cross-national collaborative panic studies began. At the end of the day, it is not clear that anything more has been demonstrated other than alprazolam produces a benzodiazepine type anxiolysis, whereas imipramine produces an anxiolysis of the type found with drugs active on the 5HT system. We still await good phenomenological distinctions between these two forms of anxiolysis. Indeed, perhaps the most interesting finding of the APS reports resulted from an analysis of the characteristics of placebo responders. Individuals who responded well on placebo far from being the weak, dependent, suggestible persons who are some times seemingly conjured up by the notion of a placebo responder, had in actual fact greater strength of personality and coherence of egostructure than those who failed to show such a response. Unfortu nately this interesting, indeed "significant", finding may not get the attention it deserves owing to its being buried among a mass of largely unrelated details in a supplement sponsored by a drug company. What is perhaps equally interesting about the recent APS supplement is what it fails to mention. Following the series of articles in Archivesof General Psychiatry, a cross-national collaborative letter was written to that Journal by Isaac Marks and several others (1989) criticising the interpretation put on the results. Their conclusion was that, contrary to the tenor of the papers published, given the results cited, alprazolam was of dubious benefit in panic disorder. References References Acta Psychiatrica Scandinavia (\99\)ftt, Supp. No. 365. Archives of GeneralPsychiatry (1988)45,407^50. MARKS,I. M., DE ALBUQUERQUE, A., COTTEAUX,J. et al (1989)Archivesof General Psychiatry. 46,668-670. A further omission are the recent reports of an efficacy of cognitive therapy for panic disorder. In contrast to the drug treatment of panic disorder, which at best achieves 60-70% response rates and https://doi.org/10.1192/pb.15.11.682 Published online by Cambridge University Press
https://openalex.org/W4230063995	https://figshare.com/articles/thesis/A_Cluster-Based_Browsing_Model_For_QoS-Aware_Web_Service_Selection/14655468/1/files/28137294.pdf	English	null	A Cluster-Based Browsing Model For QoS-Aware Web Service Selection	null	2,021	cc-by	22,449	1-1-2010 A Cluster-Based Browsing Model For QoS-Aware Web Service Selection Kian Farsandaj Ryerson University Follow this and additional works at: http://digitalcommons.ryerson.ca/dissertations Part of the Computer Sciences Commons Part of the Computer Sciences Commons Ryerson University Digital Commons @ Ryerson Theses and dissertations 1-1-2010 A Cluster-Based Browsing Model For QoS-Aware Web Service Selection Kian Farsandaj Ryerson University Ryerson University Digital Commons @ Ryerson Recommended Citation Farsandaj, Kian, "A Cluster-Based Browsing Model For QoS-Aware Web Service Selection" (2010). Theses and dissertations. Paper 1334. This Thesis is brought to you for free and open access by Digital Commons @ Ryerson. It has been accepted for inclusion in Theses and dissertations by an authorized administrator of Digital Commons @ Ryerson. For more information, please contact bcameron@ryerson.ca. Date ……………………. AUTHOR’S DECLARATION I hereby declare that I am the sole author of this thesis. This is a true copy of the thesis, including any required final revisions, as accepted by my examiners. I understand that my thesis may be made electronically available to the public. I authorize Ryerson University to lend this thesis to other institutions or individuals for the purpose of scholarly research. Kian Farsandaj Date ……………………. I further authorize Ryerson University to reproduce this thesis by photocopying or by other means, in total or in part, at the request of other institutions or individuals for the purpose of scholarly research. Kian Farsandaj Signature ………………….. Date ……………………. Kian Farsandaj ii ii Kian Farsandaj Department of Computer Science, 2010 Ryerson University CLUSTER-BASED BROWSING MODEL FOR QOS-AWARE WEB SERVICE SELECTION Kian Farsandaj ABSTRACT In the last decade, selecting suitable web services based on users’ requirements has become one of the major subjects in the web service domain. Many research works have been done – either based on functional requirements, or focusing more on Quality of Service (QoS)- based selection. We believe that searching is not the only way to implement the selection. Selection could also be done by browsing, or by a combination of searching and browsing. In this thesis, we propose a browsing method based on the Scatter/Gather model, which helps users gain a better understanding of the QoS value distribution of the web services and locate their desired services. Because the Scatter/Gather model uses cluster analysis techniques and web service QoS data is best represented as a vector of intervals, or more generically a vector of symbolic data, we apply a symbolic clustering algorithm and implement different variations of the Scatter/Gather model. Through our experiments on both synthetic and real datasets, we identify the most efficient (based on the processing time) and effective implementations. iii ACKNOWLEDGEMENTS It is my pleasure to thank those who made this thesis possible. This thesis would not have been possible without my supervisor’s supports. I would like to show my gratitude to Dr. Cherie Ding, who gave me the opportunity to work with her and do my research in a very interesting area with her supports and guidance. She constantly helped me solve various problems I encountered during the course of my graduate study, and she is always patient and shows great concern on my situation. I would like to express my deepest appreciation to Dr. Alireza Sadeghian, who has always helped me with his wise advices in different circumstances, made available his support in a number of ways, and with his kindly attitudes encouraged me in different parts of my study. It is an honor for me to thank my committee members Dr. Alireza Sadeghian, Dr. Isaac Woungang, and Dr. Alex Ferworn for their valuable time, and their every appreciated support during my education. I would like to thank Ryerson University, Department of Computer Science, and all professors and staffs, especially Dr. Santos, Dr. Abhari, Maria Landau, and others for giving me the chance of continuing my education in my very interested area, supporting me in every part of my study, and helping me throughout the whole period of my study here. I owe my deepest gratitude to my late father, who was always an encouragement and a powerful help angel for me in every part of my life, my kindest mother who provided me a safest environment with her extreme supports, and my patient faithful wife Marjan who tolerated everything with love and kindness, and was a precious motivation in my whole life. I am indebted to many of my friends – Preethy Sabamoorthy, Shilpi Verma, and Keyvan Tirdad for their supports anytime and anywhere they could. And I am grateful to Dr. Mehrdad Tirandazian as my special friend who always gave me valuable advices, and supported me in every period of my study. iv iv TABLE OF CONTENTS A CLUSTER-BASED BROWSING MODEL FOR QoS-AWARE WEB SERVICE SELECTION ........... i AUTHOR’S DECLARATION ..................................................................................................................... ii ABSTRACT ................................................................................................................................................. iii ACKNOWLEDGEMENTS ......................................................................................................................... iv LIST OF TABLES ...................................................................................................................................... vii LIST OF FIGURES ................................................................................................................................... viii LIST OF ACRONYMS ............................................................................................................................... ix CHAPTER 1 ................................................................................................................................................ 1 INTRODUCTION ........................................................................................................................................ 1 1.1 Background ..................................................................................................................................... 1 1.2 Motivation and Objectives .............................................................................................................. 3 1.3 Thesis Structure .............................................................................................................................. 7 CHAPTER 2 ................................................................................................................................................ 8 LITERATURE REVIEW ............................................................................................................................. 8 2.1 Non-Functional Properties and Quality of Services ....................................................................... 8 2.2 QoS-Based Web Service Selection ............................................................................................... 10 2.3 Cluster Analysis ............................................................................................................................ 14 2.3.1 Data Clustering Techniques ............................................................................................... 15 2.3.2 Data Clustering for Interval or Symbolic Data .................................................................. 16 2.4 Scatter/Gather Model .................................................................................................................... 19 2.5 Chapter Summary ......................................................................................................................... 20 CHAPTER 3 .............................................................................................................................................. 21 A SCATTER/GATHER MODEL FOR QoS BROWSING ....................................................................... 21 3.1 Scatter/Gather: A Cluster-Based Browsing System ..................................................................... 21 3.2 QoS Data Representation .............................................................................................................. 25 3.3 Symbolic Dynamic Clustering Algorithm (SCLUST) .................................................................. 26 v 3.4 A Browsing Model for QoS-Aware Web Service Selection......................................................... 29 3.4.1 The Original Scatter/Gather Implementation for QoS Browsing ...................................... 30 3.4.2 Iterative SCLUST .............................................................................................................. 31 3.4.3 The Improved Iterative SCLUST ...................................................................................... 32 3.4.4 The Improved LAIR2 Algorithm ....................................................................................... 33 3.5 Chapter Summary ......................................................................................................................... 38 CHAPTER 4 .............................................................................................................................................. 39 EXPERIMENTS AND RESULTS ............................................................................................................. 39 4.1 Framework and Testing Environment .......................................................................................... 39 4.2 Experiment Design ....................................................................................................................... 40 4.3 Evaluation of the Results .............................................................................................................. 40 4.3.1 Rand Index Calculation ..................................................................................................... 41 4.4 Data Generation ............................................................................................................................ 42 4.5 Experiment-1: Choosing Appropriate Distance Functions and Linkage Methods ....................... 44 4.6 Experiment-2: Different Datasets, Attributes, and Number of Clusters ....................................... 47 4.7 Experiment-3: A Real QoS Dataset .............................................................................................. 60 4.8 Implementation of the Cluster-Based Browsing Model ............................................................... 63 4.9 Chapter Summary ......................................................................................................................... 68 CHAPTER 5 .............................................................................................................................................. 70 CONCLUSION AND FUTURE WORKS ................................................................................................. 70 5.1 Conclusion .................................................................................................................................... 70 5.2 Future Works ................................................................................................................................ 71 APPENDIX A: PARAMETERS FOR DATA GENERATION ................................................................. 73 REFERENCES ........................................................................................................................................... 76 vi LIST OF TABLES Table 1 – Input Parameters for Dataset-2 Containing Distinct Clusters and Distinct Sub-Clusters ........... 42 Table 2 – Input Parameters for Dataset-5 Containing Distinct Clusters with Indistinct Sub-Clusters ....... 43 Table 3 – Input Parameters for Dataset-8 Containing Distinct Clusters with Redundancy ........................ 44 Table 4 – Results for Dataset-4 Using All Distance Metrics and Linkage Methods .................................. 46 Table 5 – Results for Dataset-1 with 300 Data ........................................................................................... 48 Table 6 – Results for Dataset-2 with 3000 Data ......................................................................................... 48 Table 7 – Results for Dataset-3 with 30000 Data ....................................................................................... 48 Table 8 – Results for Dataset-4 with 300 Data ........................................................................................... 52 Table 9 – Results for Dataset-5 with 3000 Data ......................................................................................... 52 Table 10 – Results for Dataset-6 with 30000 Data ..................................................................................... 52 Table 11 – Results for Dataset-8 with 3000 Data ....................................................................................... 54 Table 12 – Results for Dataset-9 with 3000 Data ....................................................................................... 56 Table 13 – Results for Dataset-1 with 9 Clusters ........................................................................................ 58 Table 14 – Results for Dataset-7 with 3000 Data ....................................................................................... 59 Table 15 – Results for Dataset-10 (Real World Dataset) with 281 Data .................................................... 61 vii LIST OF FIGURES Figure 1 – Web Service Architecture ........................................................................................................... 2 Figure 2 – The Example of Scatter/Gather ................................................................................................ 22 Figure 3 – Sample of tModel ..................................................................................................................... 25 Figure 4 – Selection of Initial Representatives .......................................................................................... 33 Figure 5 – How to Select Clusters along the Hierarchy ............................................................................. 36 Figure 6 – The Distribution of Data in Dataset-4 ...................................................................................... 45 Figure 7 – Comparing Clustering Time on Two Levels for Dataset 1, 2, and 3 ........................................ 51 Figure 8 – Comparing Clustering Time on Two Levels for Dataset 4, 5, and 6 ........................................ 53 Figure 9 – Results for the Experiments on Dataset-8 (Level 1 ................................................................... 55 Figure 10 – Comparing Clustering Time on Two Levels for Dataset 2, 8, and 9 ...................................... 57 Figure 11 – Comparing Clustering Time on Two Levels for Dataset 1 with 3 Clusters and 9 Clusters .... 58 Figure 12 – Comparing the Accuracy for Dataset 2 and 7 ......................................................................... 60 Figure 13 – Results for Experiments on Dataset-10 .................................................................................. 62 Figure 14 – Set Data Section ..................................................................................................................... 64 Figure 15 – Sample Dataset ....................................................................................................................... 64 Figure 16 – Algorithms, Distance Metrics, and the Linkage Methods Sections ........................................ 65 Figure 17 – Command Button Part ............................................................................................................ 65 Figure 18 – The Clustering Result of the First Level of Browsing ............................................................ 66 Figure 19 – Rand Index Calculation .......................................................................................................... 67 Figure 20 – Showing Cluster Information ................................................................................................. 68 viii LIST OF ACRONYMS SOA Service Oriented Architecture UDDI Universal Description, Definition, and Integration WSDL Web Service Description Language SOAP Simple Object Access Protocol QoS Quality of Service SLA Service Level Agreements SCLUST (ISC) Symbolic Dynamic Clustering Algorithm QML Quality of Service Modeling Language WSML Web Service Management Language WSLA Web Service Level Agreement Language WSOL Web Service Offer Language NLA New LAIR2 (Hk-Means) S/G The original Scatter/Gather technique ISC2 The improved ISC algorithm EU Euclidean CB City-block HD Hausdorff ix 1.1 Background In recent years, Service Oriented Architecture (SOA) has made an influence which changes the way the enterprise software is developed and deployed, and become an opportunity for enterprises to more easily keep up with the rapidly changing market conditions, and conduct transactions with their business partners. SOA offers some major benefits in scalability, reusability, loose coupling, and platform independence. It allows enterprises to focus more on business processes as well as the application itself instead of the pure software development. Web service architecture is one special implementation of SOA. Web service is described as a self-contained software system which could be loosely coupled with other services to assemble a complex business application [1, 2]. Web services as a new standard technology consist of three main components: service descriptions which contain interface definitions, mechanisms to access or consume services by invoking their interfaces, and the implementations of the services which are the code behind the interfaces. A set of standards and protocols are widely used for web services, which enables them to communicate across different platforms and different languages, namely: Web Service Description Language (WSDL); Universal Description, Definition, and Integration (UDDI); and Simple Object Access Protocol (SOAP) [1]. 1 1 According to the said protocols, the typical architecture of web services is constructed based on three entities: service provider, service requester, and service registry, and they interact with each other in the “publish, find, and bind” process. In this process, a service provider provides access to a web service by creating and publishing it in a registry such as a UDDI registry, which is responsible for maintaining the functional description of web services [3, 4, 5]. Later, a service requester searches through the registry to find the desired web service which meets the requirements, and then continues to bind with the selected service through the defined interface (Figure 1). Figure 1- Web Service Architecture [1, 3, 4] Publish Find Bind and Execute Registry Service Provider Service Requestor Service Description Service Client Service Description Registry Find Publish Service Client Bind and Execute Service Requestor Service Provider Figure 1- Web Service Architecture [1, 3, 4] From the above discussion, we could see that the service discovery and selection component in a service registry is a key element of the architecture which leads service requesters to find and integrate the desired services offered by providers [6]. 1.1 Background In this thesis, we study how to improve this selection process. 2 2 1.2 Motivation and Objectives Generally speaking, in order to describe or understand a service, there are two key components, referred to as its functional, and its non-functional properties. Usually, the former can be inferred from the name and documentation of the service, the input/output parameters and other behaviour information, whereas the latter mainly includes the quality properties such as performance or security related ones, as well as other properties such as the cost, reputation, etc. In this thesis, to simplify the discussion, we use the term Quality of Service (QoS) to represent all non-functional properties [7, 8]. When more and more services are published online, there could be multiple services implementing the same function, therefore, the non-functional properties will be crucial to differentiate those services. However, the current UDDI registry was not designed to support non-functional attributes of web services, and hence, it limits the service selection to only functional requirements [5]. Considering that the QoS data are collected from the Service Level Agreements (SLA) or real-time monitoring engines, there could be multiple invocation instances and multiple versions of SLAs on the same service, which result in a large amount of QoS data. There could be a big challenge about how to organize, browse, search or analyze the QoS data of web services. There have been many research works on how to expand the current architecture model to support the QoS aware service selection. However, few of them considered the problem of organizing and navigating the QoS data. Therefore, the above-mentioned described the motivation of our research. 3 3 There are situations where QoS browsing is very helpful. First, it could give service requesters some idea about the QoS value distribution of the available services. Oftentimes, in order to search a service, requesters need to formulate a QoS query and submit it to a service selection system. If the query is not accurate, the returning results may not be accurate. For instance, a service requester wants to find a service with a high reliability level, and thus the request is stated as "reliability > 99%"; however, none of the services in the registry achieves this level, and the maximum reliability is 97%. In this case, no matching result could be returned although the requester can accept a service with reliability 97% as long as it is the highest available one [7]. The problem here is an inappropriate selection of QoS values in the query. 1.2 Motivation and Objectives If the requester could browse the QoS data before submitting the query, the problem could be solved. Secondly, browsing could help service providers understand the actual demand from requesters. For instance, a service provider wants to publish a newly developed service, and in order to attract more users, the guaranteed quality level specified in the service description is very high based on the assumption that the higher quality level is always preferred. Although it does attract some users, the volume of invocation is not high enough to balance the investment on resources for hosting and delivering high quality services. The problem here is that the provider does not understand the real requirement from the users. For this particular service, maybe most of the requesters do not have a demand for a high quality level. Instead, the lower cost is always preferred. If the provider could browse the actual QoS data of services with similar functionality, the decision on service quality levels could be adjusted accordingly. 4 4 The purpose of this thesis is to investigate some effective ways of browsing the service QoS data. The knowledge gained from the browsing process could help the search and analysis process. Scatter/Gather [9] is a well-known and well-studied browsing model on large document collections. The main idea is that the system scatters the given set of documents into a small number of clusters and presents summaries to users; based on their interests, the users choose one or more clusters, then the system gathers documents from the chosen clusters and scatters them into a few clusters again, and the whole process gets iterated, starting from the complete initial collection, and gradually narrowing down to user-desired documents. The process mainly uses the clustering algorithms for its implementation. We believe that this model can also be used to browse the QoS data. It could help users (either providers or requestors) to have a clear view of the QoS distribution of the service set. The number of iterations is decided by the users' granularity requirements. There are a few main differences between the document collection and the set of services. Firstly, both the document and the service QoS data can be represented as a vector. 1.2 Motivation and Objectives The size of the document vector is usually the number of terms in the whole vocabulary, whereas the size of the QoS vector is the number of QoS attributes that the system can support. The former value is usually much bigger than the latter value. Secondly, the size of the service set under study is usually much smaller than the size of the document collection because the number of online documents is far more than that of the published services. This situation will probably remain the same even in the foreseen future, due to the higher complexity of developing services and the higher cost of hosting, delivering, or using services. Thirdly, for a QoS vector, each dimension has a different meaning because it represents different QoS attributes, a different data type [10] 5 (e.g. single numerical, interval, Boolean, categorical, etc.) and a different value range (e.g. reliability is from 0 to 100%, the authentication is either 0 or 1, the response time is from 0 to 2 seconds, etc.), whereas the dimension in a document vector has the same meaning, same data type and same value range which usually represents the term-frequency-decided weight. Fourthly, similarity-based measurements are more commonly used for document clustering and it is also believed to have a better performance [11]. However, for QoS clustering, since it is more appropriate to represent QoS data as symbolic data, the distance-based measurements are more common. Because of all these differences, it is necessary to re-study the Scatter/Gather model in this new context [7]. In this thesis, based on the features of the QoS data, we propose to use the Scatter/Gather as our browsing model, and a symbolic data clustering algorithm as its clustering component. We have four different implementations of the browsing model based on two originals, and two improved algorithms. The original algorithms consist of the original Scatter/Gather and the iterative Symbolic Dynamic Clustering Algorithm (SCLUST) [12]; furthermore, our improved algorithms include the improved SCLUST and the Hk-means (NLA) algorithm [13], which is implemented based on the improvement on the LAIR2 algorithm [14]. For the improved techniques, we intended to increase the accuracy, as well as the efficiency (based on the processing time) of our clustering results. Therefore, their performances are tested and compared using some synthetic QoS datasets. 1.2 Motivation and Objectives Based on our experimental results, the Scatter/Gather model is very effective on QoS browsing, and different implementations achieve different levels of performance. To the best of our knowledge, applying the Scatter/Gather model to QoS browsing and searching is a novel idea, and using this model to browse the symbolic data set is also a new idea. 6 LITERATURE REVIEW The scope of this chapter is to give more detailed information on: i) different QoS properties, ii) different QoS-based service discovery and selection models, iii) overview of data clustering algorithms, different data types such as interval data or symbolic data and symbolic clustering algorithms, iv) the Scatter/Gather model. 1.3 Thesis Structure The rest of the thesis is structured as follows: In Chapter 2, the background information and related works are discussed. Firstly, we describe different QoS properties of web services, including classification and taxonomy of QoS properties in different perspectives. Secondly, we review research works related to QoS-aware web service discovery and selection, with different frameworks. Thirdly, cluster analysis techniques are described by reviewing partitioning and hierarchical data clustering methods and symbolic data clustering. Finally, the Scatter/Gather framework, which is the backbone of our research, is reviewed. In Chapter 3, all features of the Scatter/Gather model, which were proposed using different algorithms, are described. Subsequently, after discussing different distance functions, which are used to calculate distances between two vectors of intervals (which represent QoS attributes of web services), we describe the concept of the cluster homogeneity, and our proposed framework consisting of four techniques (the original Scatter/Gather, the iterative dynamic symbolic clustering and its improved version, and the improved LAIR2 algorithm). In Chapter 4, we explain our experimental design, the features of the datasets we used in our experiments. We also present the results of executing our framework on 10 different synthetic datasets and a real dataset. We then continue with results analysis, comparison of different approaches, and further discussion on the implications from these results. Finally, in Chapter 5, we summarize our work and the conclusions we draw from the experiments. Furthermore, we describe the future works that can be done on QoS-aware web service selection. 7 7 2.1 Non-Functional Properties and Quality of Services As previously mentioned in the Chapter 1, web service requirements are categorized into two types: functional requirements, and non-functional requirements. Functional requirements of services describe the functionality and behaviour of the service which can be described as tasks, activities, users’ goals, and in general what the system is expected to do. On the other hand, non- functional requirements contain qualities and characteristics of services, which can affect the users’ satisfaction level with a specific web service. We use the term “QoS” to represent all non- functional properties [15, 16]. Generally, QoS attributes can be classified into different groups based on different perspectives. For example, in [17], QoS is categorized into metrics (which describe quantifiable parameters) and policies. Metrics are further divided into performance specifications, security levels, and relative importance levels. Moreover, policies are divided into management policies, and level of service. In [18], QoS is classified into technical qualities and managerial qualities. 8 There are some general and common attributes (such as price) which are independent of the domain the specific web service belongs to. The domain-independent attributes can also be categorized into different groups. For example, in [8] these attributes have been categorized into four groups as follows: - Performance: Processing Time/Execution Time, Latency, Throughput, and Response Time; - Dependability: Availability, Accessibility, Accuracy, Reliability, Capacity, Integrity, Stability/Exception Handling, Robustness/Flexibility, Regulatory/Interoperability, and Scalability; - Security: Authentication, Authorization, Non-Repudiation, Integrity, Encryption, Traceability/Auditability, and Confidentiality/Privacy; - Security: Authentication, Authorization, Non-Repudiation, Integrity, Encryption, Traceability/Auditability, and Confidentiality/Privacy; - Application-specific metrics. - Application-specific metrics. In the above taxonomy, cost is not considered as a metric or a QoS attribute. However, some of papers do include it as a QoS metric. For example, the QoS attributes of web services are classified into the following groups in [5]: - Runtime Related attributes such as: scalability, capacity, reliability, robustness/flexibility, exception handling, accuracy, and performance (which can be sub-classified into response time, latency, throughput, execution time, and transaction time); - Transaction Support Related attributes such as: integrity, isolation, durability, atomicity, and consistency; 9 - Configuration Management and Cost Related attributes such as: regulatory, cost, reputation, completeness, supported standard, stability/change, and guaranteed messaging requirements; - Security Related attributes such as: authentication, authorization, traceability/audit- ability, confidentiality, accountability, data encryption, and non-repudiation. Network Related QoS is added to the above classification in [19], which includes network delay, delay variation, and packet loss, to name a few. 2.1 Non-Functional Properties and Quality of Services The reason of applying different taxonomies and classifications on quality aspects of web services on different researches is to make QoS metrics well organized and have a simpler view for further analysis, so as to be able to use them in different scenarios such as web service selection [8]. 2.2 QoS-Based Web Service Selection Current web service technology based on the UDDI model limits the service discovery and selection to functional requirements only, which causes the problem of selecting services with same functionalities but different qualities [20, 7]. In this regard, QoS-aware methods considering non-functional attributes of web services have been proposed to resolve the weaknesses found in the UDDI keyword-based search [21]. The examples of such solutions include: (1) extending UDDI with the consideration of QoS information embedded into the tModel, (2) defining a QoS repository as a QoS broker to maintain and interact with QoS information [5, 16, 22, 23]. 10 Furthermore, in order to monitor or extract the QoS data from different sources such as SLAs or users’ feedbacks, third parties such as different agents can be involved [24, 25]. For instance, in [26], QoS negotiation and web service selection are implemented with a multi-agent computational paradigm; here, the implemented agents by service provider can negotiate with the agent implemented by the requester for the SLA configuration in the service selection process. In this regard, several languages which use abstract syntaxes or HTML language have been proposed to express QoS information of web services such as Quality of Service Modeling Language (QML) [27], Web Service Management Language (WSML) [28], Web Service Level Agreement Language (WSLA) [29], and Web Service Offer Language (WSOL) [30]. In the following, we study different proposed frameworks or publish and selection models which tried to overcome the above mentioned issues by taking into account the QoS requirements of web services. In [5] a new regulated model for web service discovery, based on current publish-find- bind model (Figure. 1) is proposed. In order to overcome the inability of supporting the QoS requirements from the UDDI registry, and to improve web service selection based on users’ preferences, the authors employed QoS attributes of web services as constraints in the search process. Therefore, the proposed model which can co-exist with the current UDDI registry includes both functional description and non-functional information of web services in the repository. During the publication process, the QoS information is checked and validated by a certifier, thus stored in the certifier’s repository after the approval is granted. The UDDI registry needs to check the registered certification with the certifier before it can be stored in the repository. 2.2 QoS-Based Web Service Selection 11 In [10], a multiple criteria decision making technique – called Analytic Hierarchy Process (AHP) – has been proposed to rank the services based on their QoS values. Furthermore, QoS ontology – WS-QoSOnto, is proposed to semantically describe the QoS information of web services. The technique is based on the following four phases: 1) formulate AHP by putting every QoS attribute and candidate web services together, in order to construct a hierarchy, 2) compute the normalized weight vector of QoS attributes in each QoS group which are used in the next phase, 3) define the relative ranking of each web service by computing the Eigenvector [31] of each attribute, 4) aggregate the web service rankings for all QoS groups by building the ranking matrix. In [32], another QoS-based web service discovery model which uses ontology to describe QoS information is presented. The approach combines constraint programming with semantic matchmaking method, in order to select web services with different QoS levels. In the proposed framework, the entire process of service discovery and selection consists of three layers: semantic matchmaking layer, constraint programming layer, and QoS selection layer. In the first layer the description logic (DL) reasoning is applied to check if the QoS attributes of the candidate services are semantically matched with the request (i.e. price & cost). The second layer which deals with QoS values converts every requested QoS condition into a set of constraints by adopting Constraint Programming (CP) method. In the third layer, an optimizing algorithm is applied to sort the candidate web services based on the total values computed by quantifying the semantic description of their QoS parameters and multiplication of each value with its related weight. Liu et al. [33] presented a new dynamic and extensible QoS-driven model for web service discovery and selection based on users’ or requesters’ feedback, depending on the characteristics 12 of QoS criteria. The model contains a QoS registry in charge of evaluating the advertized web services based on their QoS information. This evaluation is done by: (a) generating a matrix based on web services and their QoS criteria, (b) normalizing the matrix with the purposes of enabling the uniform measurement of quality attributes independently, and (c) providing uniform indices for QoS criteria, grouping them, and setting a threshold for each group. 2.2 QoS-Based Web Service Selection This proposed framework is an extensible model, and any new QoS criteria (generic or domain specific) can be easily added to the system. It is preference-based, and it has a fair and open QoS computation mechanism. Skoutas et al. [34] presented a multiple criteria matching algorithm which retrieves the k most dominant web services, and then ranked them based on their degree of matching. The model used three ranking criteria to match web service descriptions with the requests, using multiple similarity measurements. Based on these criteria, three algorithms are presented in the paper, including ranking by dominated score, ranking by dominating score, and ranking by dominance score. The concept of top-k dominant web service selection problem is formalized in the paper, and the computation of k most dominant web services is presented. In [23], a QoS-aware web service discovery approach which employs matching and ranking algorithms based on user’s preferences for both functional and non-functional information of web services is proposed. The model presents a new UDDI tModel with an external file that can be hosted by the service provider or other third party, to store the QoS information of web services. QoS requirements from service consumers are divided into optional and compulsory requirements including different features such as attribute name, attribute type, attribute value, attribute unit, constraints, direction, weight, and relationship, etc. Subsequently, a matching algorithm is applied to locate a set of web services which satisfy the consumers’ QoS 13 requirements. Finally, a ranking algorithm is employed to find the most matched services with all the desired preferences. In [35], a two-way matchmaking framework is proposed in order to overcome the problem of checking consistency between offers and requests. In order to automate the process of service selection, with consideration of two-way matchmaking context, all requests and offers should be mapped to constraint satisfaction problems. This would be carried out by mapping each parameter to a variable, and mapping every condition to the related constraints. After checking consistency of both sides, the pessimistic conformance – which is based on when all possible values satisfy the requirements – is evaluated. Finally, the best offer is selected by choosing an optimal offer which is the one with the maximum value of the calculated minimum values of all conformant offers. 2.3. Cluster Analysis Data clustering can be described as an unsupervised classification of pattern or data items into some groups. In other words, grouping similar data objects into the same clusters based on their similarities is referred to as cluster analysis. Cluster analysis is well studied in many different disciplines such as statistics, machine learning, neural networks, data visualization, high performance computing, as well as databases and data warehouses, etc. It is believed that data clustering algorithms can extract interesting patterns from a large amount of data by dividing it into different groups based on certain similarity measures. Therefore, one of the most important subjects in cluster analysis is to understand the spatial relationships between data objects in each cluster, such as dense or sparse regions in a dataset [36]. 14 2.3.1 Data Clustering Techniques Clustering methods could be organized into different categories such as: partitioning methods, hierarchical methods, density based methods, grid-based methods, model-based methods, methods for high-dimensional data (such as frequent pattern-based methods), and constraint-based clustering [37]. The focus of our work is on partitioning methods and hierarchical clustering methods. The most well-known partitioning method is the k-means algorithm, which groups data into a number of clusters, based on their similarities. It starts with k random initial prototypes, keeps assigning data objects to their closest prototypes based on their similarities or distances, and re-calculates cluster’s mean which is considered as the prototypes of clusters in any iteration, until the square error criterion function converges. This criterion is based on minimizing the total sum of dissimilarities between each data object and the correspondent cluster’s prototype [12, 37, 38]. The main issues with k-means algorithm are its sensibility to outliers, and its lack of knowledge of the number of initial clusters (k). Hierarchical methods which are classified into two different types: agglomerative and divisive methods. These methods group data objects into a tree of clusters. For instance, the agglomerative (bottom-up type) algorithm starts with placing every data object in their own cluster, and iterates by joining most similar pair of clusters based on some criteria. This process ends when every data object is placed in one single cluster or when the desired number of clusters is obtained. On the other hand, the divisive or top-down type algorithm does the reverse, it starts with all objects in one single cluster, and splits the clusters into smaller pieces until each object is placed in its own cluster [37, 39]. 15 The splitting and merging of pairs of clusters depend on the linkage methods, which can be categorized as single linkage, complete linkage, and average linkage methods. In single- linkage clustering method, the shortest distance between each cluster’s individual and any member of the other cluster, which is defined as the highest similarity between them, is considered. In contrast, in complete-linkage method, the largest distance from any data object in one cluster and any object in the other cluster is considered. Average-linkage clustering method considers the average distance between any member of one cluster and any data object in the other cluster. 2.3.1 Data Clustering Techniques Each of these methods has its own characteristics, for example complete-linkage algorithm generates the compact clusters, single-linkage method is sensitive to chaining individuals, thus suffers from this effect, but overall, single-linkage method is more adaptable than the other linkage methods [39]. 2.3.2. Data Clustering for Interval or Symbolic Data The traditional data clustering can be extended to deal with symbolic type of data such as set of intervals, lists, structured variables, categories, and so on, which are described as a unified and continuous set of values by means of relationship. Many approaches have been proposed in order to define the similarity between the symbolic data and perform the clustering tasks on the symbolic dataset [11, 40, 41]. Since the interval data is the most common symbolic data type, our review will focus on interval clustering algorithms although most of them can be generalized to more generic symbolic data. In [42], an adaptive dynamic clustering method for interval data is proposed. This method uses Euclidean metric to calculate the distance between individuals and their correspondent cluster’s representatives, and aims to minimize the adequacy criterion that measures the fitting 16 between the clusters and their representatives. There are two steps involved. In the representation step, first the partition of k clusters and the vector of weights are fixed, the clusters’ representatives which minimize the criterion function are updated, and then the partition of k clusters and their representatives are fixed, and the vectors of weights which minimize the criterion are updated. In the allocation step, both vectors of weights and cluster representatives are fixed, and the clusters which minimize the adequacy criterion are updated. In [41], two adaptive dynamic data clustering methods for symbolic data which are presented by vector of intervals, based on city-block distance are introduced. The adaptive dynamic clustering algorithm, apart from the initialization step (which is the step that the partitions are chosen by randomly selecting k distinct objects as the initial prototypes), has two main steps: the allocation step and the representation step. The allocation step, similar to the standard dynamic clustering algorithm, attempts to assign data objects to the correspondent classes based on their class prototypes. In the representation step, class prototypes are computed based on the individuals’ assignments in the previous step. The process is iterated until the converging of the criterion function is achieved. In any iteration of the above mentioned process, an adaptive distance is defined for each cluster depending on its structure; therefore, the adequacy criterion is locally optimized based on the fitting between the clusters and their prototypes. 2.3.2. Data Clustering for Interval or Symbolic Data The adaptive distance function used in this method is based on two different types of distance functions: one-component adaptive city-block distance, and two-component adaptive city-block distance. The difference between these two functions is that in the two-component adaptive function, the lower bound and the upper bound of the intervals are managed independently, whereas in the one-component function, both are considered mutually. 17 17 In [43], an iterative dynamical clustering algorithm using the Hausdorff distance measurement is presented. This method again has two steps. In the representation step which is followed by the initialization phase (choosing k distinct data objects), each cluster’s prototype is computed, which minimizes the criterion function based on the Hausdorff distance metric. Then, in allocation step each individual is assigned to their correspondent class prototype which is the closest cluster’s representative to the individual. The algorithm is iterated until the adequacy criterion is converged to the minimum value. In several other papers, similar distance measurements such as Euclidean, City-Block (Manhattan), and Hausdorff are employed to deal with the clustering of interval data or the vectors of intervals. In [12], two dynamic clustering methods are presented. In first method vectors of intervals are compared in order to minimize the adequacy criterion based on Hausdorff distance metric. The second method employs the weight function, and uses two-component dissimilarity based on Hausdorff distance to compare different vectors of intervals. In [40], a hierarchical symbolic clustering algorithm using generalized Minkowski measurement for symbolic data is presented. The algorithm which works based on single linkage method, and uses both similarity and dissimilarity values, is applicable to mixed types of symbolic data including quantitative data such as ratio, absolute, and interval values, and qualitative data consisting of nominal, ordinal, and combinational values. In [44], a fuzzy clustering algorithm which uses a non-adaptive Euclidean distance for interval data is presented. The method which is a non-hierarchical clustering method, aims at providing a fuzzy partition of clusters with different dynamic distances assigned to each cluster in order to be compared with their prototypes iteratively. 18 2.4. Scatter/Gather Model In 1992, Cutting et al. [9] presented a cluster based approach to browse large document collections. They proposed a browsing model called Scatter/Gather which uses document clustering as the main operation. In any iteration of the approach, the system scatters the dataset into some groups of data and shows their summary to the user. When the user selects a cluster or a number of clusters, system re-clusters the selected data, and again shows their summary of newly clustered data to the user. The Scatter/Gather model consists of two phases: offline and online phases. In the offline phase, which uses Fractionation technique, in any iteration of clustering process a dataset is divided into a specific number of buckets. Then, by using hierarchical clustering method for each bucket, data objects are agglomerated into a specified number of clusters. Considering each generated cluster in each bucket as new individuals, the algorithm iterates with new number of data objects, until required clusters’ centres are obtained. Later, every data object is assigned to each centre to build desired clusters. Finally, by applying the Split/Join refinement method and repeating the process several times, the offline phase will terminate. The online phase uses the Buckshot technique due to its fast processing time. In this phase, number of data objects is randomly selected to agglomerate, where k denotes the number of clusters and n is the number of data in a dataset. After achieving the required clusters’ centres, every individual is assigned to these centres, and then, the Split/Join refinement algorithm is applied to improve the quality of the clustering result [9]. 19 CHAPTER 3 In this chapter we present our framework inspired by the Scatter/Gather browsing model. The proposed framework aims to organize the QoS data and help users browse through it in order to understand the QoS value distribution of available web services, then locate and select the desired services. Its main building block is the clustering component. And the web service QoS data are mainly considered as the interval data, or more generic symbolic data. 2.5. Chapter Summary In this chapter, we reviewed a few aspects of the related works, including QoS taxonomies from different perspectives, various QoS-aware service selection algorithms and frameworks, data clustering techniques and interval clustering algorithms, and Scatter/Gather as a cluster-based browsing model. In the last section of the chapter, in order to have an introduction to the basis of our research, the Scatter/Gather model was studied. Although the original model is for document clustering, potentially, the model is extensible and can be applied to different areas such as browsing QoS data of web services as presented in this thesis. Due to the different context of our application, it is necessary to make some changes to the original model, such as using symbolic clustering instead of normal clustering algorithms in both online and offline phases. 20 3.1. Scatter/Gather: A Cluster-Based Browsing system As mentioned in Chapter 2, the Scatter/Gather approach was first presented by Cutting et al. [9] in 1992, and was aimed to browse a large number of documents. This method uses data clustering to separate documents into different groups based on their topics, and shows their summary to the user. Each time the user selects one or more clusters based on his/her interest, the system gathers the documents from the chosen clusters, scatters them by re-clustering them into the required number of clusters, and then shows the summary of the newly generated clusters to the user again. This narrowing down process is repeated until the user’s satisfaction is met, and the desired data categories are achieved. Figure 2 indicates the process of scattering and gathering documents from the collection of New York Times news stories. 21 Figure 2- The Example of Scatter/Gather [45, 46] Figure 2- The Example of Scatter/Gather [45, 46] In any iteration, the user selects one or more clusters based on summaries of clustered documents presented as their topics, and asks for new clustered information based on the latest selected data. As in the above example, in the first iteration three topics “Iraq”, “Oil”, and “Germany” are selected, and the system scatters the new dataset by clustering data into another eight clusters on the second level. Next, the user chooses two clusters “Pakistan” and “Africa”, and demands a clustering of the chosen data. In the next iteration, eight clusters are generated again and are presented to the user [9, 45, 46]. 22 The Scatter/Gather model has two phases: the offline and online phases. In both phases, the agglomerative hierarchical clustering is first employed to cluster data into the desired number of clusters as the initial centres. In the offline phase which has a more accurate but slower mechanism than the online phase, the system uses an algorithm called Fractionation for finding the initial centres. It breaks the dataset into a number of buckets, in order to find k centres. The single-linkage similarity measurement is used to merge data objects in each bucket into a certain number of clusters, which are considered as the new data objects for the next iteration of the Fractionation process. After achieving the desired number of centres, the Assign-to-Nearest algorithm is used to assign each data object to the closest cluster’s centre, and this step will be repeated three times. 3.1. Scatter/Gather: A Cluster-Based Browsing system Finally, another algorithm referred to as the Split/Join refinement algorithm is applied to improve the accuracy of the result. In the online phase, which is based on user interactions, the system uses another algorithm called Buckshot to find the initial centres, re-clusters data, and then shows their summary to the user. In this algorithm, the system randomly selects number of data objects from the dataset, and then agglomerates them into the desired number of clusters, where k denotes the required number of clusters, and n represents the total number of data objects in the dataset. After having k cluster centres, the Assign-to-Nearest algorithm is used to assign data objects to their closest cluster’s centre, which is repeated three times. Finally, the Split/Join refinement is applied. The Split/Join refinement is a process of merging and splitting clusters based on their similarities. First, each cluster is divided into two sub-clusters using the Buckshot algorithm (without the refinement part) with k = 2. In this way, the data objects with the lowest similarities are placed in different clusters. Second, each pair of clusters with the highest similarity is joined 23 (agglomerated) together by calculating the distance between two clusters using the single-linkage clustering method, to make one single cluster. This process improves the clustering result, and generates more accurate clusters. The algorithm of the Scatter/Gather could be improved by making changes in both offline and online phases to make the whole process more efficient [14, 47, 48]. In 2007, Liu, Mostafa, and Ke [48] proposed an improved Scatter/Gather model. This improved model constructs a hierarchy of documents using a hierarchical clustering technique (agglomerative or divisive) during the offline phase and the information of all levels of the hierarchy is maintained in a specific table. The previous knowledge from the first phase is used to find the required clusters in the second phase, instead of gathering and re-clustering the selected documents from scratch. When the required number of clusters is k and the number of clusters selected by the user is k’ (k < k’), the system scans the hierarchy table from bottom to top (or the hierarchy from top to bottom), until the first cluster pairs which contain all data points selected by the user are found. Then, the cluster pairs are split by removing the entry from the table and adding its two sub- clusters’ entries. 3.2. QoS Data Representation In most of the researches the QoS values are represented as single numeric values such as the following example [7]: 3.1. Scatter/Gather: A Cluster-Based Browsing system This process is iterated until k clusters are obtained [47, 48]. In order to further reduce the computational time, and increase the efficiency of the first phase clustering, another algorithm, referred to as LAIR2 [14], has been proposed which uses k- means to split each cluster. In the first phase (offline phase) of this algorithm, a hierarchy using bisecting k-means (k = 2) is constructed, instead of the agglomerative hierarchical clustering. This means that in every iteration, each cluster is split into two sub-clusters using the k-means algorithm; instead of using linkage methods (in hierarchical clustering) to divide clusters based on the similarity between pairs of data objects. With this modification, the result that was 24 obtained is several hundred times faster than the previous versions. The second phase which is the online phase works similarly to the previous version of LAIR2 algorithm as explained above. Figure 3 – Sample of a tModel [7] As it is indicated in Figure 3, the response time value for the specified web service has been defined as 0.05. However, it may not be the true representation of the actual values and we may have an information loss. Because the response time for a web service might be different in different invocations, depending on the network speed and other factors, it would be more accurate if it could be defined as a value range with an upper bound and a lower bound, which is also more reasonable for providers. Even for this value, an interval such as [0, 0.05] will be more accurate than the single numeric value. It is also similar for other QoS attributes, e.g. availability can be represented as [99.99, 100]. For attributes with single numeric values, they can also be easily converted to interval data, e.g. authentication: (1, 1). 25 We believe that this observation is true for many different QoS attributes. Therefore, the interval data would be a more appropriate type to represent the QoS values. As pointed out by [10, 32], QoS values could also be Boolean or enumeration or other types. So the symbolic data is the most appropriate type to represent the QoS attribute. In the rest of the thesis, we will mainly focus on the interval data representation of the QoS values. Any discussion on interval data can be expanded to the more generic symbolic data. 3.3. Symbolic Dynamic Clustering Algorithm (SCLUST) The main component of the Scatter/Gather model is the clustering algorithm. Since the original application of the model is on the document collection, the data object of the clustering algorithm is a document, which is usually represented as a vector of numerical values. As pointed out in the previous section it would be more appropriate to represent QoS data of a service as a vector of symbolic values. Therefore, those popular clustering algorithms which work most effectively for document collections may not work equally well for the symbolic dataset. In order to apply the Scatter/Gather model effectively to the QoS data browsing, it is necessary to choose clustering algorithms which work best for the symbolic data. Both partitioning and hierarchical algorithms have been used for the symbolic data clustering. Among these algorithms, the most commonly used and well studied one is the Symbolic Dynamic Clustering Algorithm (SCLUST) [12, 43, 49]. The main idea and the steps of the algorithm are similar to those of the k-means algorithm; however, it is catered for symbolic data. Below, we will use our QoS dataset as an example to show the steps of the algorithm. Let QS = {Q1, Q2, …, Qn} be a set of n QoS vectors and each QoS vector includes values of p attributes. Each QoS vector Qi (i = 1, 2, …, n) is represented as 26 where and (j = 1, 2, …, p) represent respectively the lower and upper bounds of interval values for the j th QoS attribute of this vector [7]. The algorithm can be divided into three steps consisting of the initialization step, representation step, and the allocation step. In the initialization step, k distinct vectors which are the initial prototypes of the partition , are randomly selected. Then, all remaining QoS vectors are assigned to their clusters according to their proximities to the cluster prototypes, based on a certain distance function which will be defined later, to build the initial partitions. In the representation step, the prototypes of the generated clusters where is the median of and is the median of , are computed. In the third step, which is the allocation step, every QoS vector is assigned to the closest prototypes to build the new clusters. 3.3. Symbolic Dynamic Clustering Algorithm (SCLUST) Finally, the last two steps (the representation and the allocation steps) are iterated until the criterion function converges, and a satisfactory result is achieved [7, 11, 12]. The adequacy criterion is defined as below, (1) (1) (1) where (CQi, Gi) is a dissimilarity or distance measure between a QoS vector CQi Ci and the cluster prototype Gi of Ci. There have been many distance functions which have been defined in the past. In this thesis, we mainly use three of them. Their definitions are given in the following paragraphs. 27 The first one is the Euclidean distance measurement [37, 42, 44] and its formula is given The first one is the Euclidean distance measurement [37, 42, 44] and its formula is given The first one is the Euclidean distance measurement [37, 42, 44] and its formula is given by: (2) (2) The second one is Manhattan or city block distance metric [37, 41], obtained as: The second one is Manhattan or city block distance metric [37, 41], obtained as: The second one is Manhattan or city block distance metric [37, 41], obtained as: (3) (3) The third one is the Hausdorff distance metric [12, 43, 49]. Basically, the Hausdorff distance between two sets A, B ℜ is computed as follows: where: (4) (5) (4) where: (5) (5) Therefore, the distance between two QoS attribute interval values can be calculated using the following function, where and : (6) (6) By considering and as vectors of p intervals and , the Hausdorff distance between these two vectors is calculated as: By considering and as vectors of p intervals and , the Hausdorff distance between these two vectors is calculated as: (7) (7) 28 3.4. A Browsing Model for QoS-Aware Web Service Selection 3.4. A Browsing Model for QoS-Aware Web Service Selection In this section, we present a framework consisting of different implementations of the Scatter/Gather model, based on the clustering of the QoS attributes of the web services which are represented by the vector of intervals. By using a browsing system, the user can interact with the system, learn about the actual value ranges of the QoS attributes of available services, narrow down to a few attributes or a few value ranges, and eventually locate the desired services, or sometimes be prepared with enough knowledge to switch to a searching process. In this model, in order to ease the process of browsing, the QoS data are clustered in the offline phase and the summary of the results is shown to the user. Therefore, the user can select one or more clusters depending on their needs, and ask the system to repeat the process based on the user’s selected clusters in the online phase of the algorithm. This process gets iterated until satisfactory results based on the user’s preferences are achieved. In the above-mentioned process, each cluster has the following representative information included in its summary: the size of the cluster (i.e. how many services), the range of QoS values (i.e. the minimum value within the cluster and the maximum value), the prototype, the service with QoS values closest to the prototype (prototype is the calculated centre of the cluster, not the real vector), and the homogeneity [12] of the cluster. The homogeneity criterion is used to measure the density and quality of the cluster. Its calculation is discussed later. The framework is implemented based on the following techniques: (1) the original Scatter/Gather model with normal clustering algorithms being replaced by the interval clustering algorithms, (2) the iterative SCLUST, (3) the improved iterative SCLUST, and (4) the improved LAIR2 algorithm (Hk-Means). 29 3.4.1. The Original Scatter/Gather Implementation for QoS Browsing The model works based on the original Scatter/Gather algorithm, with the difference that the input parameters consist of the QoS attributes of the web services. Each of these attributes is represented as an interval, which is a range of real numbers, and thus, each service is represented as a vector of intervals in the system. Similar to the original Scatter/Gather model, the algorithm consists of offline phase and online phase. In offline phase, the QoS vectors of the web services are clustered into the specified (k) number of clusters and their summaries are shown to the user. In this phase, the Fractionation algorithm is applied to break the dataset (n data objects) into b buckets of the size m > k, in each iteration. The initial value of m is defined as and b is calculated as k / m. By considering p (here we choose p = 2) as the number of desired clusters in each bucket, and the value of reduction in the dataset as k / p, the data objects are agglomerated into p clusters in each bucket separately. The process is iterated by calculating the new values for m, b, and the reduction value, and considering each generated cluster as a new individual in the object space, until the required number of clusters is generated. In this step, after defining all generated clusters’ centres, the Assign-to-Nearest algorithm is applied to assign every data object into their closest centres. The distance between each data object and the correspondent cluster’s centre is calculated using one of the previously mentioned distance functions. This process is repeated three times, and then the Split/Join refinement is applied, to improve the accuracy of the clustering result. 30 In the online phase, the Buckshot algorithm is used to group the dataset into k clusters. In this regard, data objects are randomly selected, and agglomerated into the desired number of clusters. At this time, after calculating the centre for each cluster, the Assign-to-Nearest algorithm is used three times to assign data objects to the closest centre based on their distances. Finally, the Split/Join refinement algorithm is applied to improve the accuracy of the result. In the split and join refinement part, all data clusters are split into two sub-clusters using the Buckshot algorithm with k = 2. 3.4.1. The Original Scatter/Gather Implementation for QoS Browsing In this step, objects are selected, and then agglomerated into the two separate clusters using the hierarchical interval clustering algorithm. With two cluster centres, the system then keeps assigning all individuals to their correspondent clusters based on their distances. In the join part, the clusters with the highest similarity are merged into a single cluster, using one of the mentioned linkage methods. 3.4.2. Iterative SCLUST The iterative SCLUST algorithm is a variation of the Scatter/Gather model, in which the initial centre finding and the Split/Join refinement parts have been removed, and replaced with the calculation of the adequacy criterion. Because the initial centre finding algorithm such as Fractionation or Buckshot takes extra processing time, the efficiency of the system will be affected. We would like to check whether the accuracy of the system will be largely affected when it is removed. In this model, the same clustering process is done in both phases. However, in order to increase the processing speed (due to having a large number of data at the beginning), we still separate the process into the non-interaction phase (or offline phase) and the user interaction phase (or online phase). In both phases, the QoS dataset is clustered into a certain number of 31 clusters, and the summary of each cluster is shown to the user. Then, the user can select one or more clusters and ask the system to repeat the process based on newly selected data. Each time a dataset is introduced as an input to the system, k objects (vectors) as the clusters’ representatives are randomly selected from the dataset, and all remaining objects are iteratively assigned to the closest cluster’s representative based on their distances. The new clusters’ representatives are defined by calculating the centroids of the clusters, which are the mean vectors of all data objects’ values in each cluster. Each time after all individuals are assigned to their corresponding clusters, the criterion function is computed, and the result is compared to its previous saved value in the last iteration. If they were identical, it means that the criterion function converges to a certain value and the algorithm stops. The adequacy criterion is computed based on criterion (8), which is the value of the sum of all square distances (in this formula) between every data object and its corresponding cluster’s representative [7, 11, 12]. (8) (8) 3.4.3. The Improved Iterative SCLUST 3.4.3. The Improved Iterative SCLUST One of the most important issues which may cause a decrease in the accuracy of the clustering result based on the SCLUST algorithm is that, most of the time, the adequacy criterion function may converge to a local optimum, due to the random selection of the initial clusters’ representatives. In this regard, in order to reduce the chance of convergence of the criterion function to a local optimum, we use an initial centre finding algorithm to predict the closest 32 initial prototypes to the real centres of each cluster, instead of randomly selecting the initial clusters’ representative (Figure 4). To avoid the long processing time of the Fractionation algorithm in the original Scatter/Gather model, we use the Buckshot algorithm in both phases. 1- Random selection 2- Buckshot selection Figure 4- Selection of Initial Representatives 2- Buckshot selection 1- Random selection 2- Buckshot selection Figure 4- Selection of Initial Representatives The Buckshot algorithm is employed to select number of QoS vectors from the dataset, and agglomerate them into the required number of clusters, using the agglomerative hierarchical interval clustering method, based on the single-linkage similarity measurement. Then, by having the initial centres for k clusters, the algorithm does the same as the SCLUST algorithm, and keeps assigning every object to the closest centres, based on one of the introduced distance functions. Finally, after the convergence of the criterion function, the Split/Join process (without Assign-to-Nearest part) is applied to improve the accuracy of the result. 3.4.4. The Improved LAIR2 Algorithm Because the LAIR2 algorithm has been proved to be much more efficient than the original Scatter/Gather algorithm, and the efficiency is really an important factor for an interactive application, in this thesis, we will also implement LAIR2 for our QoS browsing 33 system. The online phase of LAIR2 is mainly based on the pre-built hierarchy, and therefore the accuracy of the system may not be as good as the original model. Thus, we would like to investigate the possible ways to improve the accuracy of the LAIR2 algorithm while keeping a similar level of efficiency. One of the most important subjects in cluster analysis is to understand the spatial relationships between data objects in each cluster, such as dense or sparse regions in a dataset [50]. It becomes a problem, when some clustering algorithms do not obey these relationships and distributions. For example, when a clustering algorithm creates a cluster with uniformly distributed data, it would be advisable to stop splitting that cluster. Or if it is a cluster with some sparse regions, then it should be split into a few sub-clusters. When we calculate their homogeneity or quality values, the latter one has a lower value than the former one. So we may say that the cluster with the lowest quality or homogeneity should be split first. It would be very desirable to use this principle to control the splitting process for our clustering algorithms. In this section, we propose a new approach to using the homogeneity and the quality of partitions to control the clustering process, and consequently to improve the accuracy of the LAIR2 algorithm. The proposed algorithm is divided into offline and online phases. In the offline phase, the algorithm begins with placing every data object in one single cluster, and then divides the cluster into smaller clusters using the improved SCLUST algorithm with k = 2 to construct a hierarchy, with a specific index for each generated cluster. In any iteration, we use the Split/Join refinement to improve the accuracy of the generated clusters. The process iterates until each object forms its own cluster, or satisfies a certain termination condition. The mentioned steps of the algorithm for the offline phase are as follows: 34 1. Use buckshot algorithm to select number of prototypes; 2. Agglomerate the selected prototypes until 2 cluster centres are achieved; 3. 3.4.4. The Improved LAIR2 Algorithm Assign every data objects to the closest initial representatives ; 4. Calculate new cluster’s centre for each cluster; 5. Assign all objects to the correspondent clusters; 6. Calculate the criterion function; 7. Repeat last three steps (4, 5, and 6) until the criterion function converges; 8. Use Split/Join refinement to achieve a better result; 9. Give the generated clusters corresponding indices, specify the relationship betw parent clusters and their children, and repeat the algorithm for all existing clus (starting from step 1); 10. Stop the process when all data objects are placed in their own clusters or satis certain termination condition. 5. Assign all objects to the correspondent clusters; 7. Repeat last three steps (4, 5, and 6) until the criterion function converges; 9. Give the generated clusters corresponding indices, specify the relationship between parent clusters and their children, and repeat the algorithm for all existing clusters (starting from step 1); 10. Stop the process when all data objects are placed in their own clusters or satisfy a certain termination condition. The second phase of the algorithm, i.e. the online phase, works based on the user’s interaction. It searches the hierarchy which was structured in the offline phase to find the desired number of clusters chosen by the user. Here, the problem appears when the system searches for the desired number of clusters through the hierarchy. For instance, as shown in Figure 5 (1), the desired number of clusters is chosen as three. Hence, in level 2 of the hierarchy, once the first two clusters are found, the system is unable to select which cluster should be split first, and to which sub-cluster it should move (to return the three desired clusters), in order to have the most accurate results [13]. To solve this problem, we calculate the quality or homogeneity of the cluster in the search process, and sort the available clusters in ascending order based on their homogeneities. 35 As a result, the cluster with the lowest quality is placed on the top of our cluster list. At each iteration, in order to select the sub-clusters related to the parent cluster containing the data chosen by the user, we sort out all available clusters based on their homogeneities, and the cluster with the lowest homogeneity is selected as shown in Figure 5 (2). 3.4.4. The Improved LAIR2 Algorithm 1 2 2 3 3 4 1 2 (1) (2) Figure 5- How to Select Clusters along the Hierarchy [13] (1) (2) Figure 5- How to Select Clusters along the Hierarchy [13] The mentioned steps of the algorithm for the second phase, after choosing one or more clusters by user, are as follows: 1. Calculate the selected clusters’ homogeneities, and sort them in ascending order; 2. Search through the hierarchy for the first cluster in the list; 3. Replace the cluster with 2 leaf sub-clusters; 4. Repeat the process until the desired number of clusters is achieved. 36 Hence, in contrast to the LAIR2 algorithm, in which the search moves to the next level of hierarchy after all clusters in previous levels are split, our proposed system is able to choose any sub-cluster in any levels, and moves down in a single branch as deep as necessary to find the cluster with the lowest homogeneity [13]. Another problem with the LAIR2 algorithm is that, every time, when the user selects a cluster to be scattered, the search process restarts from the first level of the hierarchy, and this drastically slows down the processing time especially for a large dataset. However, in our proposed approach, the search always begins from the minimum index of the clusters selected by the user (which was specified for each cluster in the previous phase) in the hierarchy. Thus, the duration of the search process would be almost the same at any iteration, even for a dataset with a large amount of data [13]. In order to calculate the homogeneity of clusters, we use a generalized criterion proposed in [51], which decomposes the total inertia into between-cluster and within-cluster inertia. The adequacy between a partition P and a vector L of k prototypes is measured by (9), which is defined as the sum on k clusters and on every object of dissimilarities [12]. (9) (9) If we use the Hausdorff distance measurement as our distance metric, we have the following adequacy criteria. (10) (10) 37 (11) (11) Therefore, the homogeneity or the quality of each cluster is calculated as: Therefore, the homogeneity or the quality of each cluster is calculated as: (12) where denotes the prototype of the cluster ; is the mean of n vectors of QoS attributes. where denotes the prototype of the cluster ; is the mean of n vectors of QoS attributes. CHAPTER 4 In this Chapter, different types of datasets are used to check the flexibility of our techniques, and compare them to each other based on some of the clustering requirements which were described in [37] such as: – Scalability, – Ability to deal with different types of attributes, – Discovery of clusters with arbitrary shape, – Minimal requirements for domain to determine input parameters, – Ability to deal with noisy data, – insensitivity to the order of input records, – High dimensionality,– Constraint-based clustering, and – Interpretability and usability. Furthermore, the results of the experiments for all algorithms of our framework are illustrated and discussed. 3.5. Chapter Summary In this chapter, we described: (1) the original document-based Scatter/Gather model, (2) the LAIR2 model which is based on an improvement on the Scatter/Gather algorithm, (3) different distance functions based on interval data used in our methods and the SCLUST algorithm, and (4) our proposed framework consisting of four different implementations of the Scatter/Gather model. The first method only changes the original Scatter/Gather on its clustering component, with all clustering algorithms changed for symbolic data. In the second method, the SCLUST algorithm is adopted to cluster QoS data in the offline phase, and does the same, in order to deal with user interaction in the online phase. The third model improves the second model by adding Buckshot as the initial centre finding algorithm. And finally, in the forth model which is an improved version of the LAIR2 algorithm, a hierarchy is constructed using the bisecting SCLUST algorithm, and in the online phase the homogeneity of the cluster is used to sort the selected clusters based on their homogeneity in ascending order, so as to improve the accuracy of the clustering results. 38 4.2. Experiment Design Generally, all the experiments which are described in the next sections are based on the following test scenarios: 1) Test the algorithms to find out which distance metric, and the linkage method (for hierarchical interval clustering) is more suitable to be used for our QoS dataset; 2) Compare four algorithms based on the following testing conditions, with different datasets: 1.1) Synthetic datasets (distinct or overlapped) with similar distribution but different sizes (when the number of data is increased); 1.2) Synthetic datasets with similar distribution but different numbers of attributes (when the number of attributes is increased); 1.3) Similar datasets but different number of required clusters (when the number of clusters is increased); 2) Compare four algorithms based on the following testing conditions, with different datasets: 1.1) Synthetic datasets (distinct or overlapped) with similar distribution but different sizes (when the number of data is increased); 1.2) Synthetic datasets with similar distribution but different numbers of attributes (when the number of attributes is increased); 1.3) Similar datasets but different number of required clusters (when the number of clusters is increased); 3) Conduct the experiment on a real QoS dataset. 3) Conduct the experiment on a real QoS dataset. 4.1. Framework and Testing Environment The framework has been implemented as a windows-based application, using C# language, in Microsoft Visual Studio 2008 environment with .Net framework 3.5, to help users select their desired web services, based on the combination of the preferred QoS parameters. Furthermore, the experiments were done with a machine configured as an Intel dual Core CPU 6300 with speed of 1.86 GHz, 1 GB RAM, with Microsoft Windows XP Professional 2002 as the platform. 39 4.3. Evaluation of the Results The following describes the three measurements we used to evaluate our algorithms and their clustering results: (1) Runtime Duration shows the processing time from when the algorithm’s function is called, to when it is finished and back to the next line of the calling function. (2) Cluster’s Homogeneity or quality which was described in 3.4.4 is to measure the density of a cluster, with the value range between 0 and 1 (the higher the value, the better the quality). (3) Rand Index, which is discussed in the next section, is a concept for defining the accuracy of the clustering result, in compared to a predefined clustered dataset. 40 4.3.1. Rand Index Calculation Rand index is one of the most common ways to measure the accuracy of the clustering result, based on the calculation of different possibilities (or decisions), and the assignment errors which happened during the process, such as assigning a data object which does not belong to a specific cluster. These possibilities, which are based on testing each pair of data objects in all existing clusters, are divided into four action types: – true positive (TP) is when two similar data objects are assigned to the same cluster, – true negative (TN) is a situation in which two dissimilar data objects are assigned to different clusters, – false positive (FP) happens when two dissimilar objects are assigned to the same cluster, and finally – false negative (FN) is when two similar objects are assigned to different clusters. Based on this, Rand Index (RI) is defined as follows. Given a set of n elements, and are two partitions of S. Rand Index is calculated by the following formula, and Index is calculated by the following formula, (13) (13) where a denotes the number of pairs of objects in S, that are placed in the same cluster in U and V, b is the number of pair of objects which are placed in the same cluster in U, but in different cluster in V, c is the number of pairs of objects which are placed in different cluster in U but the same cluster in V, d is the number of pairs of objects which are placed in the different cluster in U and V [46, 52]. 41 4.4. Data Generation As previously mentioned, in order to illustrate the differences between the four algorithms, we used different datasets with different sizes and distributions, generated by MATLAB. In this regard, 9 datasets with their data points which follow the multivariate normal distribution pattern using different mean vectors μ and specified covariance values σ, were employed. As an example, we have chosen three synthetic datasets to demonstrate their overall data distributions based on specific mean vectors and covariance values. The rest of the datasets and their input parameters could be found in Appendix A. The first simulated dataset including randomly generated 3,000 data follows a multivariate normal distribution, in which the data has been considered as a vector of three intervals, consisting of Cost ($), Response time (ms), and Reliability (%) respectively (Table 1). Nine main data vectors have been chosen as the initial mean vectors for data generations to create nine distinct clusters using their own covariance values. At the end, based on what was mentioned, three distinct clusters (each consisting of 1,000 data vectors), in which each cluster contains three sub-clusters containing 330, 330, 340 data vectors respectively, were generated. Cost ($) Response Time (ms) Reliability (%) µ σ µ σ µ σ Cluster 1 (1000) (330,330,340) 55 2.0 100 2.0 10 1.0 80 2.0 140 1.8 13 1.2 125 2.0 170 3.0 17 0.9 Cluster 2 (1000) (330,330,340) 310 3.0 400 3.0 42 0.8 360 3.6 450 2.4 45 1.3 420 4.0 510 4.0 47 0.6 Cluster 3 (1000) (330,330,340) 660 2.5 760 3.6 82 1.2 700 3.9 800 3.0 85 1.0 740 2.0 830 2.0 88 0.9 Table 1 – Input Parameters for Dataset-2 Containing Distinct Clusters and Distinct Sub-Clusters Parameters for Dataset-2 Containing Distinct Clusters and Distinct Sub-Clusters Table 1 – Input Parameters for Dataset-2 Containing Distinct Clusters and Distin 42 In the second dataset, nine initial mean vectors containing 3,000 randomly generated data vectors based on multivariate normal distribution, have been chosen to create three distinct clusters, which include three indistinct or overlapped sub-clusters, all having closer mean vectors and larger covariance values (Table 2). Therefore, each predefined distinct cluster contains 1,000 data objects which are subdivided into three overlapped sub-clusters consisting of 330, 330, 340 data vectors respectively. 4.4. Data Generation Cost ($) Response Time (ms) Reliability (%) µ σ µ Cluster 1 (1000) (330,330,340) 150 10 100 4 50 2 170 9 110 5 55 3 200 12 120 6 57 3 Cluster 2 (1000) (330,330,340) 410 10 250 4 66 2 430 20 260 6 69 2.5 460 15 275 7 73 3 Cluster 3 (1000) (330,330,340) 700 12 370 8 80 3 730 11 385 10 85 2 750 8 400 10 88 3 Table 2 – Input Parameters for Dataset-5 Containing Distinct Cluste with Indistinct Sub-Clusters Cost ($) Response Time (ms) Reliability (%) µ σ µ Cluster 1 (1000) (330,330,340) 150 10 100 4 50 2 170 9 110 5 55 3 200 12 120 6 57 3 Cluster 2 (1000) (330,330,340) 410 10 250 4 66 2 430 20 260 6 69 2.5 460 15 275 7 73 3 Cluster 3 (1000) (330,330,340) 700 12 370 8 80 3 730 11 385 10 85 2 750 8 400 10 88 3 Table 2 – Input Parameters for Dataset-5 Containing Distinct Clusters with Indistinct Sub-Clusters Table 2 – Input Parameters for Dataset-5 Containing Distinct Clusters with Indistinct Sub-Clusters In the third dataset, generated with the same characteristics of the previous examples, there are 3,000 data objects, represented as the vectors of six interval data which have the six QoS attributes including: Cost ($), Response time (ms), Reliability (%), Availability (%), Accessibility (%), and Security (%) respectively (Table 3). In this regard, three initial mean vectors of six interval values were defined to generate three predefined distinct clusters, in which each cluster contains three sub-clusters and some of the elements of data vectors in one cluster are overlapped with some elements of data vectors in other two clusters. It means that, we may have different data vectors with the same similarity between their attributes, placed in different 43 clusters. The idea of using this type of dataset is to check the accuracy of the vector clustering when there exists redundant data placed in different clusters. 4.4. Data Generation Cost ($) Response Time (ms) Reliability (%) µ σ µ Cluster 1 (1000) (330,330,340) 150 2 100 3 45 2 170 2 100 3 50 1.5 200 2.5 120 2 55 1 Cluster 2 (1000) (330,330,340) 310 2 250 3 64 1.2 325 3 275 2 69 1 350 3.5 275 2.5 76 1.2 Cluster 3 (1000) (330,330,340) 420 2 100 4 83 1 435 3 160 3 89 0.5 460 =3 275 3 95 1 Availability (%) Accessibility (%) Security (%) µ σ µ Cluster 1 (1000) (330,330,340) 82 1.5 100 0 48 2 89 1.8 92 3 54 3 95 1 95 1 60 3 Cluster 2 (1000) (330,330,340) 62 1.8 58 2 72 2 77 1 65 2 77 1.8 70 1.5 50 1.8 83 2 Cluster 3 (1000) (330,330,340) 52 1.2 75 1.2 88 1.1 45 1.2 80 0.8 96 1 40 1.5 85 1.2 54 3 Table 3 – Input Parameters for Dataset-8 Containing Distinct Clusters with Redundancy 3 – Input Parameters for Dataset-8 Containing Distinct Clusters with Redundancy 4.5. Experiment-1: Choosing Appropriate Distance Functions and Linkage Methods To shorten the names of the algorithms, we use the following abbreviations for the presented techniques: S/G for the original Scatter/Gather technique, ISC for the iterative 44 SCLUST algorithm, ISC2 for the improved ISC algorithm, and NLA (New LAIR2) for the improved LAIR2 algorithm based on interval data. This notation will be used in the rest of the thesis. The application is able to execute each method based on three different distance metrics for the interval data including: Euclidean (EU), City-block (CB), and Hausdorff (HD). Furthermore, it can be run based on three different linkage methods for hierarchical interval clustering algorithm used in the original Scatter/Gather, including single linkage, average linkage, and complete linkage methods. Therefore, in order to choose the most appropriate methods, we tested our algorithms based on different distance metrics and linkage methods separately (Table 4). We have used a few datasets with different distribution patterns to conduct this experiment. Below is an example dataset (Dataset 4), containing three distinct clusters with three overlapped sub-clusters which is illustrated as below (Figure 6). Figure 6 – The Distribution of Data in Dataset 4 Figure 6 – The Distribution of Data in Dataset 4 45 S/G Single Average Complete EU CB HD EU CB HD EU CB HD Duration 0.2.752 0.2.755 0.2.738 0.3.881 0.3.856 0.3.867 0.2.797 0.2.805 0.2.803 Homogeneity 0.989 0.989 0.989 0.989 0.989 0.989 0.989 0.989 0.989 Rand Index 1 1 1 1 1 1 1 1 1 ISC2 ISC NLA EU CB HD EU CB HD EU CB HD Duration 0.0.277 0.0.280 0.0.277 0.0.228 0.0.209 0.0.184 0.7.897 0.7.886 0.7.866 Homogeneity 0.989 0.963 0.989 0.94 0.941 0.989 0.989 0.989 0.989 Rand Index 1 0.963 1 0.934 0.934 1 1 1 1 Table 4 – Results for Dataset-4 Using All Distance Functions and Linkage Methods When we compare different distance functions, the lowest duration is consistently from the Hausdorff distance measurement for these four algorithms. Accuracy-wise, there is no obvious winner. Hausdorff usually performs well regarding its rand index values. Based on our literature review, Hausdorff was advocated as the most commonly used distance function among the three. Therefore, we will use it in our experiment later. Comparing different linkage methods for Scatter/Gather algorithm, single linkage method consistently performs the best. According to these results, we are going to use the following setting in the rest of the experiments: - Distance metric: Hausdorff. 4.6. Experiment-2: Different Datasets, Attributes, and Number of Clusters Altogether there are 9 datasets. With the different datasets, we could test the performance change in different scenarios. We have defined three groups of testing scenarios: changing the size of the dataset, changing the number of QoS attributes, and changing the number of clusters (e.g. k value). In this section, the results from different testing scenarios are presented and discussed. In most cases, we did our experiments for two levels of browsing (2 iterations) – initial level and one level after user selection. In the first level or initial level, we repeated each clustering algorithm for a number of times, and calculate the average values of the clustering results. In second level, by choosing the clustering result from the previous level, we execute the application for a few times and get the average value for results. Methods - Linkage method for Hierarchical clustering: Single-linkage method, as it was briefly mentioned in section 2.3.1, about the more adaptability of the single-linkage in comparison to other linkage methods [39]. - Linkage method for Hierarchical clustering: Single-linkage method, as it was briefly mentioned in section 2.3.1, about the more adaptability of the single-linkage in comparison to other linkage methods [39]. 46 - Number of runs: 10 for the datasets with 300 data objects, and 5 for the datasets with more than 1000 data points, and 2 for the datasets with 30,000 data objects. - Number of runs: 10 for the datasets with 300 data objects, and 5 for the datasets with more than 1000 data points, and 2 for the datasets with 30,000 data objects. - Number of runs: 10 for the datasets with 300 data objects, and 5 for the datasets with more than 1000 data points, and 2 for the datasets with 30,000 data objects. Test1: Similar Distributed Datasets with Different Sizes The following datasets contain 300, 3000, and 30000 QoS vectors respectively, and each vector includes three QoS attributes. There are three distinct clusters on the first level, and each cluster consists of three distinct sub-clusters on the second level. Below Table 5-7 show the values of three measurements – duration, homogeneity and rand index in the first and second iteration, when identifying the first level and second level sub-clusters. Test1: Similar Distributed Datasets with Different Sizes 47 Table 5 – Results for Dataset-1 with 300 Data Run: 10 Dataset-1 Data 300 100 Iteration 1 2 Duration Homogeneity Rand Index Duration Homogeneity Rand Index S/G 0.2.667 0.98 1 0.0.261 0.999 1 ISC 0.0.164 0.917 0.898 0.0.061 0.997 1 ISC2 0.0.242 0.98 1 0.0.091 0.999 1 NLA 0.6.519 0.955 0.984 0.0.016 0.999 1 Table 6 – Results for on Dataset-2 with 3,000 Data Run: 5 Dataset-2 Data 3000 1000 Iteration 1 2 Duration Homogeneity Rand Index Duration Homogeneity Rand Index S/G 0.45.605 0.998 1 0.2.717 0.996 1 ISC 0.1.891 0.998 1 0.0.775 0.916 0.89 ISC2 0.2.641 0.998 1 0.0.875 0.996 1 NLA 1.34.520 0.998 1 0.0.121 0.996 1 Table 7 – Results for Dataset-3 with 30,000 Data Run: 2 Dataset-3 Data 30000 10000 Iteration 1 2 Rand Rand Run: 10 Dataset-1 Data 300 100 Iteration 1 2 Duration Homogeneity Rand Index Duration Homogeneity Rand Index S/G 0.2.667 0.98 1 0.0.261 0.999 1 ISC 0.0.164 0.917 0.898 0.0.061 0.997 1 ISC2 0.0.242 0.98 1 0.0.091 0.999 1 NLA 0.6.519 0.955 0.984 0.0.016 0.999 1 Table 6 – Results for on Dataset-2 with 3,000 Data Run: 5 Dataset-2 Data 3000 1000 Iteration 1 2 Duration Homogeneity Rand Index Duration Homogeneity Rand Index S/G 0.45.605 0.998 1 0.2.717 0.996 1 ISC 0.1.891 0.998 1 0.0.775 0.916 0.89 ISC2 0.2.641 0.998 1 0.0.875 0.996 1 NLA 1.34.520 0.998 1 0.0.121 0.996 1 Table 6 – Results for on Dataset-2 with 3,000 Data Table 7 – Results for Dataset-3 with 30,000 Data Run: 2 Dataset-3 Data 30000 10000 Iteration 1 2 Duration Homogeneity Rand Index Duration Homogeneity Rand Index S/G 84.48.501 0.811 0.891 2.0.921 0.999 1 ISC 0.22.078 0.77 0.864 0.9.159 0.975 0.898 ISC2 0.32.547 0.98 1 0.9.937 0.999 1 NLA 28.57.766 0.917 0.962 0.0.875 0.992 0.996 Table 7 – Results for Dataset-3 with 30,000 Data 48 From these tables, it can be observed that both S/G and NLA take much longer processing time on the first level than ISC and ISC2, whereas the time difference on the second level is much smaller and within an acceptable range. Test1: Similar Distributed Datasets with Different Sizes The processing time for NLA is higher than that for S/G in the smaller datasets with 300 and 3000 vectors, however by increasing the size of the dataset, the duration time for S/G drastically increases to almost two times higher than NLA for the dataset with 30000 vectors due to more iterations in the refinement part, which uses the hierarchical clustering method. The clustering time on the second level from NLA is always the smallest among all four methods. Since for the interactive browsing, the offline processing time is not very important, whereas the online clustering time is really crucial to attract users to use the browsing system. From this perspective, NLA – our improved version of LAIR2 algorithm, is the most efficient implementation of Scatter/Gather model for the QoS interval dataset. It reconfirms the conclusion from [13, 14]. When we check the homogeneity and rand index, ISC usually gets one of the worst results due to its weakness (the convergence of the criterion function to a local optimum). Since all the other three methods use certain ways to find initial centres which are closer to the real centres, whereas ISC just randomly chooses the initial centres, it indicates the effectiveness of adding the initial centre finder component in the clustering algorithm. ISC sometimes suffers from getting stuck in a local optimum. Duration of ISC2 on both levels is always longer than that of ISC, which is mainly due to the additional time used for identifying the initial centres. But they get very close to each other when the size of the dataset is increased. The main reason is that in ISC by choosing imperfect 49 49 initial prototypes (e.g. choosing k similar objects which belong to the same cluster as the initial cluster representatives), the process iterates several times more to find the optimum centroids, whereas in ISC2, by choosing the most optimum initial cluster representatives at the beginning of the process, much faster iterations can be achieved to reach the final clustering result. Both homogeneity and rand index of ISC2 are better than those of ISC, which indicates that although the processing time of ISC2 is longer, its accuracy is also higher. When the system requires a high clustering accuracy, ISC2 would be preferred than ISC. The best homogeneity and rand index results on both levels are from ISC2. Test1: Similar Distributed Datasets with Different Sizes S/G also performs very well in the first two datasets, and it is slightly worse on the first level results for the third dataset. NLA could always achieve a reasonable quality result which is usually not too much worse than the best result. Due to its bisecting splitting behaviour (based on k = 2 instead of 3), it may have some small errors in the clustering result which are usually negligible. ISC performs consistently the worst on the two accuracy metrics. In conclusion, for these three datasets, if we want to choose an implementation which is best efficiency-wise, it is NLA, and if we want to choose one with the best accuracy, it is ISC2. Overall speaking, NLA is most preferred for online interactive browsing because of its shortest online clustering time and a high level of accuracy (although not the best). Figure 7 below shows the comparison of four methods when the size of the dataset increases. It is based on dataset 1, 2 and 3. 50 Figure 7 – Comparing Clustering Time on Two Levels for Dataset 1, 2, and 3 Figure 7 – Comparing Clustering Time on Two Levels for Dataset 1, 2, and 3 From this figure, it is observed` that if we look at the clustering time for level 1, which basically measures the efficiency of the offline clustering phase, both ISC and ISC2 are better than the other two algorithms. However, if we look at the time for level 2, which measures the online phase efficiency, NLA is the best whereas S/G is the worst. When the size of the dataset increases, the time increases almost linearly for all algorithms. If we compare the homogeneity and rand index results on datasets with increasing sizes based on Table 5-7, this kind of linear relationship may not hold. The lower value of homogeneity on dataset 3 is mainly due to the existence of three sub-clusters within each cluster. And the value of rand index is usually controlled by the data distribution pattern. When there is more overlapping between clusters, the rand index value is generally lower. 51 Tables 8-10 show the experimental results of similar tests. The difference is that in these datasets, although the first level clusters are distinct, the second level sub-clusters are overlapped or indistinct. Our aim was to test our techniques for different distribution patterns. Test1: Similar Distributed Datasets with Different Sizes Table 8 – Results for Dataset-4 with 300 Data Run: 10 Dataset-4 Data 300 100 Iteration 1 2 Duration Homogeneity Rand Index Duration Homogeneity Rand Index S/G 0.2.666 0.989 1 0.0.261 0.998 0.775 ISC 0.0.188 0.94 0.934 0.0.102 0.998 0.836 ISC2 0.0.261 0.989 1 0.0.116 0.998 0.917 NLA 0.6.616 0.989 1 0.0.016 0.998 0.954 Table 8 – Results for Dataset-4 with 300 Data Table 8 – Results for Dataset-4 with 300 Data Duration Homogeneity Index Duration Homogeneity Index S/G 0.2.666 0.989 1 0.0.261 0.998 0.775 ISC 0.0.188 0.94 0.934 0.0.102 0.998 0.836 ISC2 0.0.261 0.989 1 0.0.116 0.998 0.917 NLA 0.6.616 0.989 1 0.0.016 0.998 0.954 Table 9 – Results for Dataset-5 with 3,000 Data Run: 5 Dataset-5 Data 3000 1000 Iteration 1 2 Duration Homogeneity Rand Index Duration Homogeneity Rand Index S/G 0.45.262 0.989 1 0.2.749 0.998 0.693 ISC 0.1.479 0.989 1 0.1.072 0.998 0.784 ISC2 0.2.038 0.989 1 0.1.182 0.999 0.813 NLA 1.34.776 0.989 1 0.0.141 0.999 0.81 Table 10 – Results for Dataset-6 with 30,000 Data Run: 2 Dataset-6 Data 30000 10000 Iteration 1 2 Duration Homogeneity Rand Index Duration Homogeneity Rand Index S/G 79.40.562 0.989 1 1.53.531 0.978 0.899 ISC 0.48.984 0.741 0.898 0.14.958 0.983 0.914 ISC2 0.59.343 0.989 1 0.13.781 0.998 0.968 NLA 29.26.219 0.989 1 0.0.903 0.998 0.942 Table 9 – Results for Dataset-5 with 3,000 Data Run: 5 Dataset-5 Data 3000 1000 Iteration 1 2 Duration Homogeneity Rand Index Duration Homogeneity Rand Index S/G 0.45.262 0.989 1 0.2.749 0.998 0.693 ISC 0.1.479 0.989 1 0.1.072 0.998 0.784 ISC2 0.2.038 0.989 1 0.1.182 0.999 0.813 NLA 1.34.776 0.989 1 0.0.141 0.999 0.81 Table 9 – Results for Dataset-5 with 3,000 Data Table 10 – Results for Dataset-6 with 30,000 Data Run: 2 Dataset-6 Data 30000 10000 Iteration 1 2 Duration Homogeneity Rand Index Duration Homogeneity Rand Index S/G 79.40.562 0.989 1 1.53.531 0.978 0.899 ISC 0.48.984 0.741 0.898 0.14.958 0.983 0.914 ISC2 0.59.343 0.989 1 0.13.781 0.998 0.968 NLA 29.26.219 0.989 1 0.0.903 0.998 0.942 Table 10 – Results for Dataset-6 with 30,000 Data 52 We could almost get the same conclusion as the previous set of experiments. Efficiency- wise, ISC achieves the best performance on the first level and NLA achieves the best performance on the second level. Test1: Similar Distributed Datasets with Different Sizes Accuracy-wise, ISC2 again has the highest rand index and homogeneity values on both levels, NLA is the second best, and S/G also has a pretty good performance. There is an obvious drop on the rand index value for all the datasets on the second level from all four methods, which is mainly due to the higher level of overlapping between those sub-clusters. Generally speaking, ISC2 and NLA could achieve a better rand index value on the second level than S/G and ISC. The homogeneity values are still high despite the overlapping between the sub-clusters. Figure 8 below shows the comparison of four methods when the size of the dataset increases for dataset 4, 5 and 6. Figure 8 – Comparing Clustering Time on Two Levels for Dataset 4, 5, and 6 Figure 8 – Comparing Clustering Time on Two Levels for Dataset 4, 5, and 6 Again, there is a close-to-linear relationship between the duration and the size of the dataset. On the first level, the lowest duration belongs to ISC and the duration from ISC2 is very close to it. Also, the processing time for the original Scatter/Gather starts with a value which is 53 lower than NLA, but in 30,000 dataset it increases more than NLA. As for the second level, NLA consistently performs the best and S/G the worst. lower than NLA, but in 30,000 dataset it increases more than NLA. As for the second level, NLA consistently performs the best and S/G the worst. Test2: Same Datasets with Different Number of Attributes Table 11 shows the results of the experiment for a dataset with 3000 vectors of six QoS attributes (Dataset 8). As previously mentioned, the purpose of this test is to check how the performance is changed when the number of attributes is increased. The dataset has three distinct predefined clusters and three distinct sub-clusters for each. Table 11 – Results for Dataset-8 with 3,000 Data Run: 5 Dataset-8 Data 3000 1000 Iteration 1 2 Duration Homogeneity Rand Index Duration Homogeneity Rand Index S/G 1.18.646 0.812 0.819 0.4.078 0.999 1 ISC 0.2.016 0.838 0.889 0.0.617 0.999 1 ISC2 0.3.076 0.875 0.908 0.0.971 0.999 1 NLA 2.1.052 0.893 1 0.0.161 0.999 1 Table 11 – Results for Dataset-8 with 3,000 Data In this test, the most accurate clustering results in both iterations belong to NLA with the rand index of 1. The next most accurate clustering algorithm is ISC2. The lowest processing time is from ISC in the first iteration and NLA in the second iteration. The change does not have a big effect on homogeneity and rand index values on both levels. Regarding the duration, it is higher in the first iteration for all four methods, whereas in the second iteration, S/G and NLA get a longer duration time, but ISC and ISC2 are not largely affected by the change. 54 54 In the following figure (Figure 9), the clustering results of all methods are illustrated by 3D figures. We could see that every method would generate different clustering result. When comparing the original data distribution with the result, intuitively, ISC2 and NLA give the best result, and results from both ISC and S/G are not so good. result, and results from both ISC and S/G are not so good. (4) S/G Figure 9 – Results for experiments on dataset-8 (Level 1) (1) ISC2 (3) ISC (2) NLA (2) NLA (3) ISC (4) S/G Figure 9 – Results for experiments on dataset-8 (Level 1) 55 The next experiment is based on a dataset (Dataset 9) with 3000 vectors of nine QoS attributes, consisting of three distinct clusters and three distinct sub-clusters for each cluster. The aim of this experiment is again to check how the performance is changed when the number of attributes is increased. The results are shown in Table 12. Test2: Same Datasets with Different Number of Attributes Table 12 – Results for Dataset-9 with 3,000 Data Run: 5 Dataset-9 Data 3000 1000 Iteration 1 2 Duration Homogeneity Rand Index Duration Homogeneity Rand Index S/G 1.48.615 0.959 1 0.5.083 0.998 0.878 ISC 0.2.026 0.956 1 0.0.708 0.998 0.808 ISC2 0.3.589 0.956 1 0.1.125 0.999 1 NLA 2.19.792 0.956 1 0.0.208 0.999 1 Table 12 – Results for Dataset-9 with 3,000 Data Above table clearly indicates that in first level, the efficiency of ISC is higher than the rest of the algorithms in the order of ISC2, S/G, and NLA. However, in the second level, NLA is the fastest one in comparison to other techniques, followed by ISC and ISC2 which are not very different in their processing time. The original Scatter/Gather in this level is the slowest one mainly due to its three times of repeating the Assign-to-Nearest process. Regarding to their accuracy levels, both ISC2 and NLA are equally well. Overall speaking, NLA is the best one considering its fastest online processing time and high accuracy of the clustering results. Figure 10 below shows the relationship between the number of attributes and the duration. We could see that in the first level of browsing, the most efficient algorithm is ISC followed by ISC2, S/G, and NLA and their durations increase almost linearly with the increasing of the number of attributes. However, in the second level the most efficient one is NLA, and the 56 next efficient algorithms respectively are listed as: ISC, ISC2, and S/G. In the second level, for ISC and ISC2, the changes of the duration from dataset-8 with 6 attributes to dataset-9 with 9 attributes are small compared to those from 3 attributes to 6 attributes. For S/G and NLA, the change is almost linear. Figure 10 – Comparing Clustering Time on Two Levels for Dataset 2, 8, and 9 Figure 10 – Comparing Clustering Time on Two Levels for Dataset 2, 8, and 9 Again, if we compare the homogeneity and rand index values for dataset 2, 8 and 9 (Table 6, 11, 12), the change is not linear, and it is mainly controlled by the data distribution patterns. Test3: Same Size Datasets with Different Numbers of Clusters The aim of the next experiment is to test how each one of our proposed algorithms deals with the different number of clusters (k values). We use Dataset 1 for the test. In our previous result (Table 5), three clusters are generated in the first level, and three sub-clusters are generated 57 for each cluster in the second level. Now we change the k value to 9 for both levels to compare their performances. Table 13 shows the results. Table 13 – Results for Dataset-1 with 9 Clusters Run: 5 Dataset-1 Data 300 300 Iteration 1 2 Duration Homogeneity Rand Index Duration Homogeneity Rand Index S/G 0.3.057 0.997 0.891 0.0.276 0.998 0.952 ISC 0.0.266 0.988 0.885 0.0.078 0.997 0.927 ISC2 0.0.365 0.999 1 0.0.132 0.999 0.982 NLA 0.7.703 0.999 0.912 0.0.031 0.998 0.961 Table 13 – Results for Dataset-1 with 9 Clusters Again, we could get similar conclusions as before. NLA is the most efficient one for online clustering and ISC2 is the most accurate one for both levels. Figure 11 below shows the comparison of the processing time for dataset-1 when the number of clusters is 3 and 9. Figure 11 – Comparing Clustering Time on Two Levels for Dataset 1 with 3 Clusters and 9 Clusters Figure 11 – Comparing Clustering Time on Two Levels for Dataset 1 with 3 Clusters and 9 Clusters 58 We could see that when the number of clusters is changed from 3 to 9, its processing time is also increased for both levels. The degree of increase in the first level is very obvious, and we could see a considerable jump (but not that big) in all techniques. The increase in the second level is not that obvious. Test4: Same Size Datasets with Different Distributions Our next experiment is based on a dataset (dataset 7) including three predefined indistinct clusters. The dataset contains 3000 data vectors with four QoS attributes. Only one level clustering is done on this dataset. The purpose of this test is to check how the accuracy is affected when the overlapping degree is higher, and we also want to compare the accuracy of each technique for this kind of distribution pattern. The results are shown in Table 14. Table 14 – Results for Dataset-7 with 3,000 Data Table 14 – Results for Dataset-7 with 3,000 Data Run: 5 Dataset-7 Data 3000 Iteration 1 Duration Homogeneity Rand Index S/G 0.56.854 0.383 0.547 ISC 0.3.375 0.578 0.741 ISC2 0.4.594 0.589 0.772 NLA 1.54.786 0.581 0.774 Above table (Table 14) shows how these algorithms deal with a dataset with a high overlapping degree between clusters. Compared with the results from Dataset 2 and 5 (as shown in Table 6 and 9), which also contain 3000 QoS vectors, the homogeneity and rand index values are much lower, and the duration for ISC and ISC2 is also slightly higher. We believe that the high overlapping is the main contributing factor to the lower accuracy level, and the longer 59 duration time is due to the larger number of elements of the QoS vectors (four attributes for this dataset). Regarding the comparison between four algorithms, as it is clearly indicated in the table, the techniques with the most accurate clustering results are sorted as follows: NLA, ISC2, ISC, and S/G. However, the lowest duration belongs to the ISC and the highest is for the NLA. Figure 12 depicts the comparison of the accuracy of dataset 2 and 7, based on their total clusters homogeneity and rand index for the first level. We could see that when the degree of overlapping between clusters is increased, both homogeneity and rand index values are lower. Figure 12 – Comparing the Accuracy for Dataset 2 and 7 Figure 12 – Comparing the Accuracy for Dataset 2 and 7 4.7. Experiment-3: A Real QoS Dataset Our last experiment is based on a real dataset (QWS dataset) which consists of 2,507 web services and their QoS measurements. The data was obtained in 2008 [53, 54, 55]. Since our 60 browsing system is mainly for services with similar functionalities, we manually assign keywords to all the services, and sort the services based on their functionalities. browsing system is mainly for services with similar functionalities, we manually assign keywords to all the services, and sort the services based on their functionalities. After checking the functional keywords which contain a good number of services, we chose the topic “bioinformatics” with its 281 services for our testing. We chose three QoS attributes, namely: Reliability, Successability, and Throughput. Table 15 shows the results of the experiments for four methods to be compared based on their processing time and their total clusters homogeneities. Due to the lack of any predefined clusters related to this dataset, we couldn’t calculate their Rand Index values. Table 15 – Results for Dataset-10 (Real World Dataset) with 281 Data Table 15 – Results for Dataset-10 (Real World Dataset) with 281 Data Run: 10 Dataset-10 Data 281 60 Iteration 1 2 Duration Homogeneity Duration Homogeneity S/G 0.1.512 0.599 0.0.350 0.9972 ISC 0.0.156 0.623 0.0.053 0.9984 ISC2 0.0.298 0.673 0.0.156 0.9987 NLA 0.5.811 0.673 0.0.016 0.9989 Above table (Table 15) indicates that ISC2 and NLA generated the clusters with the highest homogeneities in compare to other two algorithms, and similar to the previous experiments ISC has the lowest duration in level 1 and NLA has the lowest duration in level 2. The clustering results are displayed in the following 3D figures (Figure 13). 61 From Figure 13, it is observed that ISC2 and NLA get the similar results. However, their results are quite different from those from the other two algorithms. By looking at the actual data (1) ISC2 (2) NLA (3) ISC (4) S/G Figure 13 – Results for Experiments on Dataset-10 (1) ISC2 (2) NLA (3) ISC (4) S/G (3) ISC (4) S/G Figure 13 – Results for Experiments on Dataset-10 Figure 13 – Results for Experiments on Dataset-10 From Figure 13, it is observed that ISC2 and NLA get the similar results. However, their results are quite different from those from the other two algorithms. 4.7. Experiment-3: A Real QoS Dataset By looking at the actual data 62 distribution as shown in the 3D figure, the result from ISC2 and NLA is more accurate to reflect the spatial relationship between those data objects. distribution as shown in the 3D figure, the result from ISC2 and NLA is more accurate to reflect the spatial relationship between those data objects. 4.8. Implementation of the Cluster-Based Browsing Model In this section, we describe the implemented browsing system, and discuss about its capabilities by showing some snapshots from the user interface. As mentioned in the previous chapter, the application which is a cluster-based browsing system has the potential to help users select their preferred web services based on the combination of their QoS attributes. The system groups the available services to the desired number of clusters, based on the user selected algorithm, the distance metric, as well as the linkage method (if the related algorithm is selected as S/G). Afterwards, the summary of each cluster is shown to the user. User can select one or more clusters which are considered as the new input data to be clustered in the next iteration. This process is continued until the user satisfaction is achieved. Additionally, user can change the number of clusters in each level of browsing, or get back to the previous level by clicking on the related buttons. The user interface consists of eight sections: Set Data, Algorithms, Distance Metrics, Linkage Methods, Summaries, Parent clusters, Generated Clusters, and Cluster Details. Set Data section contains the following three parts: Browse, Number of Clusters, and Order of Input Data. When start, user can select the specific file containing the QoS vectors of the available web services, by pressing the browsing button (Figure 14). 63 Figure 14 – Set Data Section Figure 14 – Set Data Section When saving the data, the interval’s lower bound and upper bound are separated by commas “,”, intervals of different QoS attributes are separated with semicolons “;”, and each vector is placed on a new line (Figure 15). Figure 15 – Sample Dataset Figure 15 – Sample Dataset By choosing the specific dataset, the users can define their required number of clusters by typing it in the related text box. Furthermore, the order of data can be changed to random if the algorithm is sensitive to the order of input data. In the next section, the preferred algorithm is chosen from the list, as well as the specific distance metric which is needed to be used in the clustering process (Figure 16). 4.8. Implementation of the Cluster-Based Browsing Model 64 Figure 16 – Algorithms, Distance Metrics, and the Linkage Methods Sections Depending on the selected technique (whether S/G or the others), the linkage methods used in the original Scatter/Gather technique is chosen from the related panel, which are disabled when other techniques are selected. The other part is the execution button, containing Scatter/Gather command button, Back button, Run and Export, Reset, Rand Index, and Export Buttons (Figure 17). Figure 17 – Command Button Part Figure 17 – Command Button Part After defining the address of the specific dataset, the required number of clusters, distance metric, and linkage method, the input data is scattered into different clusters, and their summaries are shown to the user in the related sections, by pressing the Scatter/Gather button, as illustrated in Figure 18. Later, we can clear the results from the user interface and the application’s memory by clicking the Reset button. 65 Figure 18 – The Clustering Result of the First Level of Browsing Figure 18 – The Clustering Result of the First Level of Browsing By pressing the Rand Index button, the system could calculate the rand index value so that it could help us measure the accuracy of the clustering result. When pressing this button another window is opened to get the address of the predefined clustered dataset from user to be compared with the result of the generated clusters by the application (Figure 19). The result which is a real number between 0 and 1 is shown in the summaries section. 66 Figure 19 – Rand Index Calculation Figure 19 – Rand Index Calculation Figure 20 – Showing Cluster Information Finally, the user can click on the export button, after finding the most preferred web services which are close to the user’s need. The specific files are stored in the location which its address is defined in the Set Data section. Figure 19 – Rand Index Calculation The other two buttons (Run and Export, Export) are responsible for exporting the generated clusters to external text files, containing all clustering information such as cluster ranges, cluster representatives, three closest individuals to each centre, cluster homogeneity, processing time, size, and some other information such as name of the technique, distance metric, etc. The difference between Run and Export, and the Export button is that the former one runs the algorithm a number of times, then exports the average values for all clustering results as well as different text files containing each cluster’s data, whereas the latter one only exports those files without executing the program. By clicking each cluster’s summary in the Generated Clusters section, the detailed information is shown in the Clusters Details section. Furthermore, we can see the data of each cluster by double clicking on each of those clusters in the Generated Clusters list (Figure 20). In order to continue the browsing of the web service information, we can simply select the specific cluster by clicking on the checkboxes beside each cluster’s information in the Generated Clusters lists, and press the Scatter/Gather button. Then, the application returns new clustering result based on the latest selection. Each time the user can return to the previous level by clicking on Back button, and repeat the browsing process. 67 Figure 20 – Showing Cluster Information 4.9. Chapter Summary In this chapter, we tested our algorithms based on 9 synthetic datasets with different distribution of data, and one real dataset containing the QoS information of the real web services, in two levels of browsing. The datasets were based on randomly generated vectors of interval data which followed a multivariate normal distribution, and contained three predefined clusters 68 in which each cluster included three sub-clusters. The difference between datasets were in their cluster distances (whether distinct or indistinct), their sizes, complexities, and the number of elements of each data vectors. We tested our algorithms based on the above mentioned datasets, measured the accuracy of each technique by calculating the cluster homogeneities and their rand indices, computed the processing time and the duration for each algorithm, and compared them to each other. We showed the results on separated tables and plotted some charts trying to find the relationship between the processing time and a few different changes on the dataset. Furthermore, the application is explained by showing the flow of the user interfaces. 69 5.1 Conclusion In this thesis, we presented a cluster-based browsing framework for the QoS-aware web service selection consisting of the following algorithms: (1) Original Scatter/Gather (S/G), (2) Iterative SCLUST (ISC), (3) Improved Iterative SCLUST (ISC2), and (4) Improved LAIR2 (NLA). The proposed framework, which is based on the Scatter/Gather browsing model, uses the above mentioned techniques to cluster the vectors of interval data, in which each interval represents a QoS attribute of a web service. In order to compare the proposed methods to one another, we used various datasets which were generated based on different scenarios but all following a multivariate normal distribution. Depending on the scenario, each dataset contains three distinct or indistinct clusters, in which each cluster consists of three other distinct or indistinct sub-clusters. Furthermore, by executing the application a number of times on each dataset, and further calculating the averages of the generated values of the clustering results, such as process duration, cluster homogeneity, and rand index for each technique, certain results were achieved and shown in different tables for comparison. We compared the accuracy and the efficiency of the clustering results for each technique when dealing with different types and sizes of datasets. The results of the testing based on the original Scatter/Gather showed that the algorithm (S/G) is more suitable for datasets with some distinct clusters, rather than the overlapped datasets. Moreover, by increasing the size of datasets 70 the processing duration of the algorithm is drastically increased, especially in the first level of browsing or offline phase (which uses the Fractionation method). ISC is the fastest algorithm among our four techniques in the offline phase, but it suffers from getting stuck in a local optimum, which is caused by the convergence of the adequacy criterion; therefore, it generates poor clusters in some of the executions of the technique (or the iterations of the clustering). Hence, in order to increase the accuracy of the ISC algorithm, we applied some modifications on the technique, which include choosing more than k initial prototypes and agglomerating them to k centroids, and adding a refinement part at the end of the clustering process. The improved ISC algorithm (ISC2) is more accurate and stable than the other techniques (ISC and the original Scatter/Gather), but its processing time is approximately one and a half times more than the ISC. 5.1 Conclusion The problem with all those techniques appears in large datasets and in the online phase, when the number of data in the selected clusters is not small. By comparing the improved LAIR2 algorithm (NLA) which used the modified version of ISC to build the hierarchy, to other algorithms in the first level of browsing (offline phase), it was observed that the results are almost similar to the improved ISC, but the duration is higher than that both in the improved and the original ISC, and lower than that of the original Scatter/Gather. In contrast, this algorithm (NLA) is more efficient (e.g. with the processing time of less than a second for 10,000 data) and more accurate than other techniques in the second phase. 5.2 Future Works For future work, we intend to continue our research in the following three directions: (1) Employ Fuzzy C-Means Clustering (FCM), as an extension of the ISC algorithm, to discover soft clusters, especially in the first level of browsing (offline phase). By choosing fuzzy 71 clustering for the dataset containing vectors of intervals, we can overcome the issue with the datasets consisting of the overlapped clusters, due to their symbolic (interval) nature of data. Having similar performance to the popular SCLUST algorithm, we can obtain fuzzy output which can be more sensible for the user to deal with. (2) Predict the number of initial prototypes which presents the number of clusters in different datasets, depending on the distribution of data. Determining the number of clusters is one of the main issues in different clustering algorithms. In this regard, many techniques have been proposed to overcome this issue, such as grid-based and density-based clustering methods, or even by the calculation of the within-cluster qualities or homogeneities. Therefore, in order to make the application simpler for the user, we can let the program decide how many clusters are more suitable for the specific type of dataset. (3) Increase the ability of the system to support various value types of QoS properties of the web services, by moving beyond the interval data to more generic symbolic data. As a matter of fact, web services contain different QoS attributes with various value types such as: fuzzy value type, multiple value type (consisting of list, set, range, and vector), and single value type (enumeration, string, numeric, ordinal, nominal, and Boolean). Therefore, in order to make our framework more flexible for dealing with different value types of QoS attributes, in the future, we will provide new techniques to support different types of data. 72 GENERATION Cost ($) Response Time (ms) Reliability (%) µ σ µ σ µ σ Cluster 1 (100) (33,33,34) 55 2.0 100 2.0 10 1.0 80 2.0 140 1.8 17 1.2 125 2.0 170 3.0 23 0.9 Cluster 2 (100) (33,33,34) 310 3.0 400 3.0 42 0.8 360 3.6 450 2.4 49 1.3 420 4.0 510 4.0 56 0.6 Cluster 3 (100) (33,33,34) 660 2.5 760 3.6 77 1.2 700 3.9 800 3.0 83 1.0 740 2.0 830 2.0 89 0.9 Table 16 – Dataset-1, 300 three attribute QoS vectors, containing three distinct cl with three distinct sub-clusters 16 – Dataset-1, 300 three attribute QoS vectors, containing three distinct clusters with three distinct sub-clusters Table 17 – Dataset-3, 30,000 three attribute QoS vectors, containing three distinct clusters with three distinct sub-clusters Table 17 – Dataset-3, 30,000 three attribute QoS vectors, containing three distinct clusters with three distinct sub-clusters Cost ($) Response Time (ms) Reliability (%) µ σ µ σ µ σ Cluster 1 (10000) (3300,3300,3400) 55 2.0 100 2.0 10 1.0 80 4 140 1.8 17 1.2 125 4 170 3.0 23 0.9 Cluster 2 (10000) (3300,3300,3400) 310 3.0 400 3.0 42 0.8 360 3.6 450 2.4 49 1.3 420 4.0 510 4.0 56 0.6 Cluster 3 (10000) (3300,3300,3400) 660 2.5 760 3.6 77 1.2 700 3.8 800 3.0 83 1.0 740 2.0 830 2.0 89 0.9 Table 17 – Dataset-3, 30,000 three attribute QoS vectors, containing three distin clusters with three distinct sub-clusters 73 Cost ($) Response Time (ms) Reliability (%) µ σ µ σ µ σ Cluster 1 (100) (33,33,34) 55 2.0 100 2.0 10 1.0 80 2.0 140 1.8 17 1.2 125 2.0 170 3.0 23 0.9 Cluster 2 (100) (33,33,34) 310 3.0 400 3.0 42 0.8 360 3.6 450 2.4 49 1.3 420 4.0 510 4.0 56 0.6 Cluster 3 (100) (33,33,34) 660 2.5 760 3.6 77 1.2 700 3.9 800 3.0 83 1.0 740 2.0 830 2.0 89 0.9 Table 18 – Dataset-4, 300 three attribute QoS vectors, containing three distinct c with three indistinct sub-clusters Table 18 – Dataset-4, 300 three attribute QoS vectors, containing three distinct clusters with three indistinct sub-clusters Table 19 – Dataset-6, 30,000 three attribute QoS vectors, containing three distinct clusters with three indistinct sub-clusters Table 19 – Dataset-6, 30,000 three attribute QoS vectors, containing three distinct clusters with three indistinct sub-clusters Cost ($) Response Time (ms) Reliability (%) µ σ µ σ µ σ Cluster 1 (10000) (3300,3300,3400) 150 10 100 4 50 2 170 9 110 5 55 3 200 12 120 6 57 3 Cluster 2 (10000) (3300,3300,3400) 410 10 250 4 66 2 430 15 260 4 69 2.5 460 20 275 7 73 3 Cluster 3 (10000) (3300,3300,3400) 700 12 370 8 80 3 730 11 385 10 85 2 750 8 400 10 88 3 Table 19 – Dataset-6, 30,000 three attribute QoS vectors, containing three distin clusters with three indistinct sub-clusters Cost ($) Response Time (ms) Reliability (%) Availability (%) µ σ µ σ µ σ µ σ Cluster 1 (3000) (1000,1000, 1000) 50 20 120 30 78 10 80 5 100 35 150 20 85 7 85 5 75 28 180 20 90 3 88 3 Table 20 – Dataset-7, 3000 four attribute QoS vectors, containing three overlapped clusters Cost ($) Response Time (ms) Reliability (%) Availability (%) µ σ µ σ µ σ µ σ Cluster 1 (3000) (1000,1000, 1000) 50 20 120 30 78 10 80 5 100 35 150 20 85 7 85 5 75 28 180 20 90 3 88 3 Table 20 – Dataset-7, 3000 four attribute QoS vectors, containing three overlapped clusters Dataset-7, 3000 four attribute QoS vectors, containing three overlapped clusters Table 20 – Dataset-7, 3000 four attribute QoS vectors, containing three overlapp 74 Table 21 – Dataset-9, 3000 nine attribute QoS vectors, containing three distinct clusters with three distinct sub-clusters Cost ($) Response Time (ms) Reliability (%) µ σ µ σ µ σ Cluster 1 (10000) (3300,3300,3400) 70 1.8 15 1.8 45 2 95 1.5 30 1.5 50 1.5 115 1.5 50 2 55 1 Cluster 2 (10000) (3300,3300,3400) 250 2 250 2 64 1.2 270 1.5 275 1.8 9 1 285 1.8 288 2 76 1.2 Cluster 3 (10000) (3300,3300,3400) 340 1.8 100 2 83 1 365 2 120 1.5 89 0.5 380 2 140 2 95 1 Availability (%) Accessibility (%) Security (%) µ σ µ σ µ σ Cluster 4 (10000) (3300,3300,3400) 45 1.2 46 1.8 45 2 52 1 51 1.5 50 1.5 57 1 57 1 55 1 Cluster 5 (10000) (3300,3300,3400) 66 1.3 67 1.5 64 1.2 71 1 74 1.2 69 1 77 1.2 79 1 76 1.2 Cluster 6 (10000) (3300,3300,3400) 84 1.5 87 1.2 83 1 90 1 92 1 89 0.5 96 0.8 97 1 95 1 Compliance (%) Latency (ms) Documentation (%) µ σ µ σ µ σ Cluster 7 (10000) (3300,3300,3400) 82 1.2 4 0.5 40 1.5 89 1.2 10 1.5 46 1.5 95 1 18 2 52 1.5 Cluster 8 (10000) (3300,3300,3400) 60 1.4 40 1.5 62 1 66 1 46 1 69 1 71 1.5 52 1.2 75 1.2 Cluster 9 (10000) (3300,3300,3400) 52 1.2 75 1.2 84 1.1 45 1 80 0.8 90 1.3 40 1.3 85 1.2 97 1 Q g with three distinct sub-clusters 75 References [1] J. 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https://openalex.org/W2264135922	https://europepmc.org/articles/pmc4891376?pdf=render	English	null	Evaluating metabolites in patients with major depressive disorder who received mindfulness-based cognitive therapy and healthy controls using short echo MRSI at 7 Tesla	Magma	2,016	cc-by	8,002	Magn Reson Mater Phy (2016) 29:523–533 DOI 10.1007/s10334-016-0526-7 RESEARCH ARTICLE Abstract Objectives Our aim was to evaluate differences in metab- olite levels between unmedicated patients with major depressive disorder (MDD) and healthy controls, to assess changes in metabolites in patients after they completed an 8-week course of mindfulness-based cognitive therapy (MBCT), and to exam the correlation between metabolites and depression severity. Conclusions This study has successfully evaluated regional differences in metabolites for patients with MDD who received MBCT treatment and in controls using 7 Tesla MRSI. Materials and methods Sixteen patients with MDD and ten age- and gender-matched healthy controls were stud- ied using 3D short echo-time (20 ms) magnetic resonance spectroscopic imaging (MRSI) at 7 Tesla. Relative metabo- lite ratios were estimated in five regions of interest corre- sponding to insula, anterior cingulate cortex (ACC), cau- date, putamen, and thalamus. Keywords Major depressive disorder · MBCT · Magnetic resonance spectroscopic imaging · 7 Tesla Results In all cases, MBCT reduced severity of depres- sion. The ratio of total choline-containing compounds/ total creatine (tCr) in the right caudate was significantly increased compared to that in healthy controls, while ratios Evaluating metabolites in patients with major depressive disorder who received mindfulness‑based cognitive therapy and healthy controls using short echo MRSI at 7 Tesla Yan Li1 · Angela Jakary1 · Erin Gillung2 · Stuart Eisendrath2 · Sarah J. Nelson1,3 · Pratik Mukherjee1 · Tracy Luks1 Yan Li1 · Angela Jakary1 · Erin Gillung2 · Stuart Eisendrath2 · Sarah J. Nelson1,3 · P tik M kh j 1 T L k 1 Received: 15 October 2015 / Revised: 5 January 2016 / Accepted: 6 January 2016 / Published online: 9 February 2016 © The Author(s) 2016. This article is published with open access at Springerlink.com of N-acetyl aspartate (NAA)/tCr in the left ACC, myo- inositol/tCr in the right insula, and glutathione/tCr in the left putamen were significantly decreased. At baseline, the severity of depression was negatively correlated with my- inositol/tCr in the left insula and putamen. The improve- ment in depression severity was significantly associated with changes in NAA/tCr in the left ACC. Conclusions This study has successfully evaluated regional differences in metabolites for patients with MDD who received MBCT treatment and in controls using 7 Tesla MRSI. of N-acetyl aspartate (NAA)/tCr in the left ACC, myo- inositol/tCr in the right insula, and glutathione/tCr in the left putamen were significantly decreased. At baseline, the severity of depression was negatively correlated with my- inositol/tCr in the left insula and putamen. The improve- ment in depression severity was significantly associated with changes in NAA/tCr in the left ACC. 1 Department of Radiology and Biomedical Imaging, University of California, San Francisco, Radiology Box 2532, Byers Hall, 1700 4th Street, San Francisco, CA 94158‑2532, USA 2 Department of Psychiatry, University of California, San Francisco, San Francisco, CA, USA Presented at the Annual Meeting of International Society for Magnetic Resonance in Medicine, Salt Lake City, Utah, USA, April 2013. 3 Department of Bioengineering and Therapeutic Sciences, University of California, San Francisco, San Francisco, CA, USA * Yan Li yan.li@ucsf.edu Study population Sixteen depressed patients (11 F/5 M, 30 ± 7 years) who met the Diagnostic and Statistical Manual of Mental Dis- orders, Fourth Edition (DSM-IV) criteria for MDD and who had been medication free for at least 6 weeks, and ten age- and gender-matched healthy controls (7 F/3 M, 32 ± 9 years) who had no history of neurologic illness, traumatic brain injury, or DSM-IV Axis I or II diagnosis were recruited into this study. Individuals with alcohol or substance abuse or dependence within the last 3 months, inadequate English language comprehension, or significant medical conditions or contraindications to MR systems were excluded, as were individuals who currently medi- tated once or more a week or who practiced yoga twice or more per week. Each participant was given written informed consent in accordance with the University of California San Francisco (UCSF) IRB procedures. Baseline and post-MBCT MRSI data were successfully acquired from nine patients. Five additional patients had baseline MRSI data only. Among these five patients, two dropped out of the study during MBCT treatment, one declined to participate in the post- MBCT MR scans, and two became pregnant during MBCT treatment. Two patients only had post-MBCT MRSI scans for purely logistical reasons. Multiple studies using single-voxel and spectroscopic imaging have been performed at lower field strengths in patients with MDD and have reported regional and/or global metabolite differences. Although the overall consen- sus is that there is hypometabolism in patients compared to healthy controls, there was some variability in the results obtained [10]. Efficacy of other MDD treatments was evaluated using MRS in different brain regions. Changes in metabolites, such as Glu, NAA, and tCho, were found to be correlated with response to pharmacotherapy or anti- depressant stimulation techniques [11]. Regions of inter- est (ROI) for these studies involved locations in the basal ganglia, limbic system, frontal cortex, and occipital cortex. Multivoxel 3D magnetic resonance spectroscopic imaging (MRSI) is of interest because it allows characterization of the spatial extent and metabolic properties within multiple regions of the brain. Depressive symptoms were evaluated using the Hamil- ton Depression Severity Rating 17-item scale (HAMD-17) in all participants at baseline. Fourteen of the 16 patients completed the manual and were reassessed using the HAMD-17 within 2 weeks of completing treatment (post- MBCT). Introduction Major depressive disorder (MDD) affects about 6.7 % of the adults in the United States per year and is the leading cause of disability for people from 15 to 44 years old. The most com- mon treatments include antidepressant medications and psy- chotherapy. Most patients with MDD have recurrent episodes and often experience treatment failure under current thera- pies, which severely impact their quality of life. Mindfulness- based cognitive therapy (MBCT), a method for integrating cognitive behavioral therapy with mindfulness meditation, teaches patients how to disengage from habitual “automatic” dysfunctional cognitive routines, in particular depression- related ruminative thought patterns. MBCT was first used for preventing the relapse of depression [1] and has shown its efficacy in reducing depressive recurrence [2] and treat- ing acute [3], chronic [4], and treatment-resistant depression [5] as an augmentation. These findings make MBCT treat- ment a potential valuable stand-alone treatment for MDD. 2 Department of Psychiatry, University of California, San Francisco, San Francisco, CA, USA 3 Department of Bioengineering and Therapeutic Sciences, University of California, San Francisco, San Francisco, CA, USA 1 3 524 Magn Reson Mater Phy (2016) 29:523–533 Although the etiology and neuropathology of MDD is still not fully understood, it is thought to be associ- ated with chemical changes that involve multiple brain circuits. Proton magnetic resonance spectroscopy (MRS) is a powerful tool for noninvasively investigat- ing brain metabolism. Spectra acquired with long echo times (TEs) provide estimates of metabolite levels, such as total choline-containing compounds (tCho, reflect- ing membrane synthesis), total creatine (tCr, reflect- ing cellular bioenergetics), and N-acetyl aspartate (NAA, a neuronal marker). At short TEs, metabolites such as myo-inositol (mI), glutamate (Glu), and glu- tamine (Gln) also appear, but overlap between peaks can make it difficult to obtain accurate quantification. Spectral editing methods using J-coupling differences allow unobstructed detection of glutathione (GSH) and γ-aminobutyric acid (GABA) but require acquisition from a relatively large region and/or relatively long acquisition time. High field MR systems, such as 7 Tesla (7T) scanners, offer advantages in higher signal- to-noise ratio (SNR) and enhanced spectral quantifica- tion for all of these metabolites [6]. Other brain metab- olites, such as N-acetyl-aspartyl glutamate (NAAG), glycine (Gly), and glucose that have much lower con- centration and/or overlap with the major peaks, can also be detected at high field using ultrashort TE (≤10 ms) MRS or TE-optimized sequences [7–9]. efficacy of treatment based upon standard clinical criteria [12]. Study population Remission was identified by scores ≤7 on the post-MBCT HAMD-17, and the reduction rate was cal- culated by changes between baseline and post-MBCT HAMD-17 scores divided by the baseline scores. The purpose of this study was to compare relative levels of metabolites between unmedicated patients with MDD and healthy controls, to evaluate the relationship between metabolite levels and disease severity, and to examine the association between changes in metabolite levels and the outcome of completed MBCT treatment for these patients using 3D short echo-time MRSI at 7T. The patient popu- lation comprised a subset of individuals who participated in a previously published project designed to evaluate the Introduction ROIs within the basal ganglia, limbic system, and frontal cortex were selected for analysis because they have been identified as being relevant for studying psychiatric diseases previously. 3 MR acquisitions movement between the two acquisitions. Segmentation of the brain was performed automatically on these images using Harvard–Oxford cortical and subcortical structural atlases [20, 21]. ROIs used in the analysis of the 3D MRSI consisted of insula, medial frontal gyrus (MFG), anterior cingulate cortex (ACC), caudate, putamen, pallidum, and thalamus. The segmented ROIs were then aligned to the orientation of the 3D MRSI and down-sampled to the reso- lution of spectral data. Median metabolite ratios from vox- els overlapping by at least 40 % with the anatomical ROIs were included in the analysis. An example of ROIs overlaid on the 3D T1-weighted images is shown in Fig. 1a. The signal from the MFG was partially suppressed by the outer- volume suppression bands; the small size of the pallidum meant that overlapping voxels may contain a relatively high portion of other tissue. These two regions were therefore excluded from further analysis. The remaining five ROIs were analyzed and divided into right- and left-hemisphere sides. All MR scans were performed using a 32-channel receive-only array with a volume-transmit head coil (NOVA Medical, Wilmington, MA, USA) on a GE 7T MR950 scanner (GE Healthcare, Waukesha, WI, USA). Anatomical imaging consisted of a T1-weighted sag- ittal scout (TR/TE = 6/2 ms), 3D T1-weighted inver- sion recovery-prepared spoiled gradient echo (IR SPGR) [TR/TE/inversion time (TI) = 6/2/600 ms, matrix size = 256 × 256 × 192 FOV = 256 × 256 × 192 mm3, voxel size = 1 × 1 × 1 mm3), and 3D T2-weighted fast spin echo (FSE-Cube) (TR/TE = 6000/60 ms, matrix size = 512 × 512, FOV = 256 × 256 mm2, voxel size = 0.5 × 0.5 mm2, slice thickness/overlap = 2/1 mm, acceleration factor = 3) images. Optimization of high order shimming and transmit gains were performed prior to spectral data acquisitions. 3D H-1 MRSI was obtained using spectrally selective adiabatic inversion recovery lipid suppression, chemi- cal shift selective (CHESS) water suppression, automati- cally prescribed very selective suppression (VSS) outer volume suppression, and spin-echo slice selection with TE/TR = 20/2000 ms, spectra array = 16–20 × 22 × 8, and nominal spatial resolution = 1 cm3 [15]. Total acqui- sition time was 8.77 min for 16 × 22 × 8 or 10.96 min for 20 × 22 × 8 spectral arrays when applying an inter- leaved flyback echo-planar trajectory [16] in the anterior/ posterior direction of the FOV. MR postprocessing Postprocessing was performed using a previously pub- lished methodology [15]. Spectral arrays acquired with the interleaved flyback readout trajectory were combined and reordered to a rectilinear grid [16], processed with phase and frequency corrections individually for each coil, com- bined by weighting with coil sensitivities, and then quanti- fied using LCModel [17]. Metabolite signals for the basis set were generated using NMR-SCOPE [18] with prior knowledge of chemical shift and J-coupling information [19]. Only those voxels with relative Cramer–Rao lower bounds (CRLBs) <10 % for tCho, tCr, and NAA, and 20 % for Glu, Gln, GABA, mI, Gly, and GSH, were included in the analysis. MR acquisitions Slice direction of spectra data was placed parallel to the anterior commissure (AC)– posterior commissure (PC) line with full coverage of the thalamus (Fig. 1a). The variation in radio-frequency (RF) excitation due to B1 inhomogeneities was minimized by adjusting both the transmitter gain and relative RF power of CHESS, 90°, and 180° pulses. Mindfulness‑based cognitive therapy (MBCT) methods MBCT treatment consisted of 8 weekly classes each last- ing 2.25 h, and participants were also asked to complete 45 min of homework 6 days per week. This 8-week MBCT group intervention used a clinician and participant manual developed by Segal et al. [13] with modifications for cur- rent depression, and was described in Eisendrath et al. [14]. 1 3 525 Magn Reson Mater Phy (2016) 29:523–533 Statistics Statistical analysis was performed using R (www.r-project. org). Given the small sample size of the study popula- tion, nonparametric statistic methods were used for data analysis, and the number of participants in each group was required to be more than seven. Adjustment for mul- tiple comparisons was not performed due to the explora- tory nature of the study. A p value of <0.05 was consid- ered as significant at this stage. Wilcoxon signed-rank tests were used to test differences in HAMD-17 scores between baseline and post-MBCT evaluations. Differences in base- line metabolite ratios between patients and controls were assessed using Wilcoxon rank-sum tests, and the effects of MBCT on metabolite ratios were assessed using Wil- coxon signed-rank tests. Spearman rank-correlation coeffi- cients were calculated to determine the association between baseline metabolite ratios and baseline HAMD-17 scores, between baseline metabolite ratios and reduction rates on HAMD-17 scores, and between changes in metabolite ratios and reduction rates in HAMD-17 scores. Participant characteristics and treatment outcomes The characteristics of study participants are summarized in Fig. 2, which also shows changes in HAMD-17 in all patients. At baseline, the median HAMD-17 for patients was 18 (range 13–24) and declined to 8 (range 2–18) after the 8-week MBCT treatment. All 14 patients who completed the MBCT treatment demonstrated reduced The 3D MRSI data were referenced to the 3D T1-weighted images by assuming that there was no 1 526 Magn Reson Mater Phy (2016) 29:523–533 MD-17 scores, six achieved remission, and eight had a ti th HAMD 17 >50 % Th diff MR characteristics 4.0 3.0 2.0 Chemical Shift (ppm) NAA Glu tCr tCho tCr-CH2 GABA Glx mI GSH Glu CRLB = 8% Gln CRLB = 19% GSH CRLB = 8% GABA CRLB = 12% a b 1 a Location of 3D magnetic resonance spectroscopic imag- (MRSI) prescription and regions of interest (ROIs) that were mented using Harvard–Oxford cortical and subcortical atlases, rsely transformed from the MNI space and then resampled to the resolution of spectra overlaid on T1-weighted images. b MRS (with baseline between 1.6 and 4.2 ppm) from a patient with depressive disorder (MDD) and an example of fitted spectra LCModel in the selected voxel a b b 3.0 Chemical Shift (ppm) Fig. 1 a Location of 3D magnetic resonance spectroscopic imag- ing (MRSI) prescription and regions of interest (ROIs) that were segmented using Harvard–Oxford cortical and subcortical atlases, inversely transformed from the MNI space and then resampled to the resolution of spectra overlaid on T1-weighted images. b MRSI data (with baseline between 1.6 and 4.2 ppm) from a patient with major depressive disorder (MDD) and an example of fitted spectra from LCModel in the selected voxel MR characteristics HAMD-17 scores, six achieved remission, and eight had a reduction on the HAMD-17 score >50 %. The difference in HAMD-17 scores between baseline and post-MBCT was highly significant (p < 0.001). The presence of obvious imaging abnormalities was ruled out based on FSE-Cube images by a neuroradiologist. An 1 3 Magn Reson Mater Phy (2016) 29:523–533 527 HC MDD at baseline MDD at baseline (paired) MDD post-MBCT (paired) MDD post-MBCT tCho/tCr Glu/tCr mI/tCr GSH/tCr NAA/tCr R Caudate L Caudate R Insula L Putamen L ACC 2.0 1.8 1.6 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0 <0.05 Fig. 3 Significant differences in metabolite ratios between patients with major depressive disorders (MDD) and controls at baseline and between patients at baseline and after mindfulness-based cognitive therapy (MBCT) in the R caudate, L caudate, R insula, L putamen, and L anterior cingulate cortex (ACC). (R right; L left) HC MDD at baseline MDD at baseline (paired) MDD post-MBCT (paired) MDD post-MBCT tCho/tCr Glu/tCr mI/tCr GSH/tCr NAA/tCr R Caudate L Caudate R Insula L Putamen L ACC 2.0 1.8 1.6 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0 <0.05 25 20 15 10 5 0 Baseline Post-MBCT Baseline MRSI only Post-MBCT MRSI only Two MRSI scans HAMD-17 Fig. 3 Significant differences in metabolite ratios between patients with major depressive disorders (MDD) and controls at baseline and between patients at baseline and after mindfulness-based cognitive therapy (MBCT) in the R caudate, L caudate, R insula, L putamen, and L anterior cingulate cortex (ACC). (R right; L left) MDD Patients Health Controls Number of subjects 16 10 Age (years) 30.2±6.7 32.1±8.8 Education (years) 14.9±2.2 17.6±2.1* HAMD-17 Scores 19.3±3.5 8.4±3.7** 1.3±1.0* Reduction Rate 57.2±17.2%** * 1 subject had unknow information ** 2 patients dropped out of the study MDD Patients Health Controls MDD Patients Health Controls vs. 0.66 ± 0.06, p = 0.042], GSH/tCr in the left puta- men (0.23 ± 0.06 vs. 0.28 ± 0.05, p = 0.044), NAA/tCr in the left ACC (1.27 ± 0.14 vs. 1.41 ± 0.21, p = 0.044). The level of tCho/tCr in the right caudate (0.26 ± 0.07 vs. 0.21 ± 0.07, p = 0.041) was significantly higher at base- line in patients with MDD than in controls. Differences in metabolite ratios between groups Figure 3 summarizes ROIs that had significant differences in metabolite ratios between groups. No differences were detected in the thalamus. MR characteristics * 1 subject had unknow information ** 2 patients dropped out of the study * 1 subject had unknow information ** 2 patients dropped out of the study Table 2 demonstrates median and IQR of tCho/tCr, NAA/tCr, mI/tCr, Glu/tCr, GABA/tCr, and GSH/tCr from patients at baseline and post-treatment. After com- pleting MBCT treatment, the levels of NAA/tCr in the left ACC were significantly increased (1.26 ± 0.10 vs. 1.42 ± 0.17, signed-rank, p = 0.018). Although tCho/ tCr in the right caudate (0.25 ± 0.06 vs. 0.20 ± 0.02, signed-rank, p = 0.084) were not significantly different from that at baseline in patients who had both baseline and post-MBCT MRSI scans (nine patients), they were statistically decreased (0.26 ± 0.07 vs. 0.19 ± 0.02, rank- sum, p = 0.004) when comparing all patients between baseline and post-MBCT (14 vs. 11 patients). Glu/tCr in the left caudate (0.93 ± 0.18 vs. 1.09 ± 0.14, rank-sum, p = 0.026), mI/tCr in right caudate (0.62 ± 0.14 vs. 0.48 ± 0.07, rank-sum, p = 0.017), and tCho/tCr in the right putamen (0.22 ± 0.04 vs. 0.18 ± 0.02, rank-sum, p = 0.011) were also significantly different from baseline to post-treatment. Fig. 2 Characteristics of study participants. Age, education, and Hamilton Depression Severity Rating 17-item scale (HAMD-17) scores are summarized for patients and healthy controls. All patients demonstrated reduced HAMD-17 scores after completing mindful- ness-based cognitive therapy (MBCT). HAMD-17 scores for those who did or did not have 3D magnetic resonance spectroscopic imag- ing (MRSI) scans are shown in different shapes of markers example of 3D MRSI data from a patient and the ROIs segmented using the Harvard–Oxford cortical and subcor- tical structural atlases are illustrated in Fig. 1b. Note that the baseline was not removed from the spectra shown and that the CRLB estimates of reliability of Glu, GSH, and mI measures obtained from LCModel were relatively small. Correlations between metabolite ratios and HAMD‑17 scores Table 1 gives median and interquartile range (IQR) for tCho/tCr, NAA/tCr, mI/tCr, Glu/tCr, GABA/tCr, and GSH/tCr in each ROI from patients and controls at base- line. When compared to values in controls, metabolite ratio levels of the following were significantly lower at baseline in patients: mI/tCr in the right insula [patients vs. controls, mean ± standard deviation (SD), 0.60 ± 0.07 Figure 4 shows the regions in which there were signifi- cant associations between metabolite ratios and HAMD- 17 scores. Correlations between metabolite ratios and HAMD‑17 scores Higher HAMD-17 scores at baseline were sig- nificantly associated with lower levels of mI/tCr in the left insula (correlation coefficient, r = −0.59, p = 0.026, 1 3 528 Magn Reson Mater Phy (2016) 29:523–533 Table 1 Metabolite ratios [median (interquartile range (IQR)] within regions of interest (ROIs) in patients with MDD and healthy controls (HC) at baseline Metabolite values in bold were significant between MDD and HC MDD major depressive disorder, tCho/tCr total choline-containing compounds/total creatine, NAA N-acetyl aspartate, Gly glycine, mI myo-inositol, Glu glutamate, Gln glutamine, GSH glu- tathione, GABA γ-aminobutyric acid tCho/tCr NAA/tCr mI/tCr Glu/tCr GABA/tCr GSH/tCr MDD HC MDD HC MDD HC MDD HC MDD HC MDD HC Left insula 0.20 (0.03) 0.21 (0.03) 1.19 (0.11) 1.32 (0.24) 0.65 (0.07) 0.64 (0.07) 1.14 (0.12) 1.21 (0.21) 0.28 (0.08) 0.28 (0.04) 0.28 (0.08) 0.34 (0.04) Right insula 0.22 (0.02) 0.22 (0.03) 1.25 (0.21) 1.25 (0.20) 0.61 (0.11) 0.68 (0.09) 1.21 (0.15) 1.29 (0.15) 0.30 (0.14) 0.30 (0.08) 0.29 (0.04) 0.34 (0.06) Left ACC 0.23 (0.04) 0.24 (0.04) 1.22 (0.18) 1.32 (0.25) 0.68 (0.14) 0.69 (0.23) 1.31 (0.20) 1.44 (0.17) 0.32 (0.10) 0.40 (0.16) 0.27 (0.08) 0.23 (0.04) Right ACC 0.24 (0.05) 0.24 (0.03) 1.22 (0.25) 1.29 (0.30) 0.68 (0.12) 0.72 (0.15) 1.29 (0.11) 1.36 (0.16) 0.37 (0.10) 0.40 (0.16) 0.24 (0.03) 0.27 (0.07) Left caudate 0.23 (0.09) 0.21 (0.04) 1.18 (0.20) 1.22 (0.14) 0.56 (0.19) 0.50 (0.24) 0.96 (0.25) 1.16 (0.38) 0.34 (0.12) 0.31 (0.12) 0.30 (0.10) 0.29 (0.05) Right caudate 0.25 (0.05) 0.21 (0.05) 1.18 (0.40) 1.17 (0.46) 0.57 (0.15) 0.49 (0.16) 1.05 (0.40) 0.87 (0.25) 0.37 (0.11) 0.32 (0.12) 0.27 (0.12) 0.30 (0.12) Left putamen 0.19 (0.06) 0.22 (0.03) 1.22 (0.29) 1.34 (0.39) 0.48 (0.12) 0.50 (0.11) 1.20 (0.23) 1.02 (0.14) 0.30 (0.17) 0.32 (0.08) 0.22 (0.09) 0.30 (0.05) Right putamen 0.22 (0.07) 0.25 (0.04) 1.38 (0.35) 1.35 (0.36) 0.44 (0.13) 0.50 (0.18) 1.08 (0.30) 0.90 (0.18) 0.29 (0.11) 0.27 (0.21) 0.28 (0.07) 0.32 (0.08) Left thalamus 0.23 (0.03) 0.24 (0.04) 1.42 (0.11) 1.46 (0.23) 0.61 (0.08) 0.68 (0.22) 1.04 (0.12) 1.02 (0.07) 0.32 (0.05) 0.34 (0.10) 0.22 (0.03) 0.21 (0.03) Right thalamus 0.24 (0.04) 0.22 (0.04) 1.36 (0.24) 1.45 (0.45) 0.62 (0.15) 0.71 (0.11) 1.15 (0.21) 1.22 (0.33) 0.33 (0.04) 0.36 (0.01) 0.23 (0.04) 0.24 (0.04) 1 3 529 Magn Reson Mater Phy (2016) 29:523–533 Table 2 Median [interquartile range (IQR)] of metabolite ratios within regions of interest (ROIs) in patients with MDD at baseline and post-treatment Metabolite values in bold were significantly different between baseline and post-treatment MDD major depressive disorder, tCho/tCr total choline-containing compounds/total creatine, NAA N-acetyl aspartate, Gly glycine, mI myo-inositol, Glu glutamate, Gln glutamine, GSH glu- tathione, GABA γ-aminobutyric acid tCho/tCr NAA/tCr mI/tCr Glu/tCr GABA/tCr GSH/tCr Pre Post Pre Post Pre Post Pre Post Pre Post Pre Post Left insula 0.20 (0.03) 0.19 (0.03) 1.19 (0.11) 1.24 (0.09) 0.65 (0.07) 0.67 (0.08) 1.14 (0.12) 1.25 (0.29) 0.28 (0.08) 0.30 (0.07) 0.28 (0.08) 0.29 (0.05) Right insula 0.22 (0.02) 0.20 (0.04) 1.25 (0.21) 1.24 (0.11) 0.61 (0.11) 0.63 (0.12) 1.21 (0.15) 1.32 (0.22) 0.30 (0.14) 0.33 (0.08) 0.29 (0.04) 0.33 (0.07) Left ACC 0.23 (0.04) 0.24 (0.05) 1.22 (0.18) 1.31 (0.17) 0.68 (0.14) 0.70 (0.13) 1.31 (0.20) 1.40 (0.24) 0.32 (0.10) 0.36 (0.10) 0.27 (0.08) 0.25 (0.03) Right ACC 0.24 (0.05) 0.26 (0.07) 1.22 (0.25) 1.33 (0.09) 0.68 (0.12) 0.75 (0.12) 1.29 (0.11) 1.45 (0.20) 0.37 (0.10) 0.37 (0.10) 0.24 (0.03) 0.27 (0.05) Left caudate 0.23 (0.09) 0.19 (0.05) 1.18 (0.20) 1.24 (0.31) 0.56 (0.19) 0.49 (0.10) 0.96 (0.25) 1.10 (0.18) 0.34 (0.12) 0.36 (0.19) 0.30 (0.10) 0.29 (0.10) Right caudate 0.25 (0.05) 0.20 (0.03) 1.18 (0.40) 1.13 (0.24) 0.57 (0.15) 0.46 (0.09) 1.05 (0.40) 1.09 (0.16) 0.37 (0.11) 0.30 (0.05) 0.27 (0.12) 0.30 (0.13) Left putamen 0.19 (0.06) 0.19 (0.04) 1.22 (0.29) 1.22 (0.11) 0.48 (0.12) 0.51 (0.11) 1.20 (0.23) 1.05 (0.05) 0.30 (0.17) 0.34 (0.16) 0.22 (0.09) 0.26 (0.03) Right putamen 0.22 (0.07) 0.18 (0.02) 1.38 (0.35) 1.14 (0.08) 0.44 (0.13) 0.45 (0.11) 1.08 (0.30) 1.00 (0.24) 0.29 (0.11) 0.27 (0.09) 0.28 (0.07) 0.29 (0.06) Left thalamus 0.23 (0.03) 0.23 (0.04) 1.42 (0.11) 1.44 (0.26) 0.61 (0.08) 0.59 (0.15) 1.04 (0.12) 1.05 (0.18) 0.32 (0.05) 0.30 (0.05) 0.22 (0.03) 0.23 (0.07) Right thalamus 0.24 (0.04) 0.22 (0.04) 1.36 (0.24) 1.46 (0.27) 0.62 (0.15) 0.60 (0.15) 1.15 (0.21) 1.10 (0.21) 0.33 (0.04) 0.28 (0.09) 0.23 (0.04) 0.24 (0.06) 1 530 N = 14) and left putamen (r = −0.65, p = 0.021, N = 12) at baseline. Discussion 26 24 20 20 18 16 14 12 10 0.40 0.50 0.60 0.70 0.80 mI/tCr in left Insula at the baseline HAMD-17 at the baseline r = - 0.59 p = 0.021 This study has successfully demonstrated the application of 7T H-1 MRSI to healthy controls and to patients with MDD before and after treatment with MBCT. As reported by Eisendrath et al. [12] for the larger study that our popu- lation was drawn from, MBCT greatly reduced the severity of depression. Changes in metabolite levels after complet- ing MBCT treatment, and the correlation between metabo- lite levels and depression severity are of interest for future use in evaluating individuals who may benefit from this or other, similar therapies. The main finding in this respect is that differences in metabolite ratios observed in patients at baseline were normalized after treatment and became similar to the levels in healthy controls. To our knowledge, this is the first study showing differences in metabolites in patients with MDD who were treated with MBCT alone. 0.30 0.40 0.50 0.60 mI/tCr in left Putamen at the baseline 26 24 20 20 18 16 14 12 10 HAMD-17 at the baseline r = -0.65 p = 0.021 The improved sensitivity and spectral resolution of the 7T scanner is important for optimizing data quality using in vivo MRSI [8, 22]. The method used in this study to estimate metabolite levels was demonstrated previously and has been applied in other patient populations [15]. To avoid the time penalty of acquiring an additional 3D MRSI acquisition with unsuppressed water for scaling metabolite signals, we chose to reference levels of the metabolites of interest to the levels of tCr in the same voxel. Prior studies have shown that these acquisition and postprocessing meth- ods are able to provide uniform and reliable quantification in the entire spectral array for metabolite ratios, including tCho/tCr, NAA/tCr, Glu/tCr, Gln/tCr, GSH/tCr, mI/tCr, Gly/tCr, and GABA/tCr, within a clinically reasonable total acquisition time of ~10 min [15]. -0.50 -0.30 -0.10 0.10 Changes on NAA/tCr in left ACC Reduction on HMAD-17 0.75 0.65 0.55 0.45 0.35 0.25 r = -0.93 p = 0.002 Previous studies evaluating metabolites in MDD used a single-voxel MRS acquisition with a large voxel size and pro- duced variable results [10]. Discussion This may be due to differences in the patient population or in tissue components studied and the degree of partial volume that was present. Multi-voxel 3D MRSI has the advantage of providing an assessment of spatial distribution of metabolites. In this study, 3D MRSI was prescribed within a 4-cm excitation slab that was paral- lel to the AC-PC line and had full coverage of the thalamus. Five types of ROIs were defined within frontal and limbic lobes, insular and subcortical regions. The apriori selection of ROIs was limited by the position of the excitation slab such that the cortex was partially suppressed by the eight automati- cally prescribed outer-volume lipid suppression and the spa- tial resolution (1 cm3) used for data acquisition (see Fig. 1). In an attempt to provide robust measures, we used median metabolite levels from voxels that overlapped by at least 40 % with the anatomic ROIs being considered. Previous studies evaluating metabolites in MDD used a single-voxel MRS acquisition with a large voxel size and pro- duced variable results [10]. This may be due to differences in the patient population or in tissue components studied and the degree of partial volume that was present. Multi-voxel 3D MRSI has the advantage of providing an assessment of spatial distribution of metabolites. In this study, 3D MRSI was prescribed within a 4-cm excitation slab that was paral- lel to the AC-PC line and had full coverage of the thalamus. i Fig. 4 Significant associations between metabolite ratios and Ham- ilton Depression Severity Rating 17-item scale (HAMD-17) scores at baseline in patients (top, middle) and between percent changes on metabolite ratios and reduction on HAMD-17 scores after completing MBCT (bottom). The correlation coefficients and p values were cal- culated using Spearman rank tests N = 14) and left putamen (r = −0.65, p = 0.021, N = 12) at baseline. Increases in NAA/tCr from baseline to post- MBCT were significantly associated with a reduction in HAMD-17 scores after completing MBCT in the left ACC (r = −0.93, p = 0.002, N = 8). N = 14) and left putamen (r = −0.65, p = 0.021, N = 12) at baseline. Increases in NAA/tCr from baseline to post- MBCT were significantly associated with a reduction in HAMD-17 scores after completing MBCT in the left ACC (r = −0.93, p = 0.002, N = 8). Correlations between metabolite ratios and HAMD‑17 scores Increases in NAA/tCr from baseline to post- MBCT were significantly associated with a reduction in 26 24 20 20 18 16 14 12 10 0.40 0.50 0.60 0.70 0.80 mI/tCr in left Insula at the baseline HAMD-17 at the baseline 0.30 0.40 0.50 0.60 mI/tCr in left Putamen at the baseline 26 24 20 20 18 16 14 12 10 HAMD-17 at the baseline -0.50 -0.30 -0.10 0.10 Changes on NAA/tCr in left ACC Reduction on HMAD-17 0.75 0.65 0.55 0.45 0.35 0.25 r = - 0.59 p = 0.021 r = -0.65 p = 0.021 r = -0.93 p = 0.002 Fig. 4 Significant associations between metabolite ratios and Ham- ilton Depression Severity Rating 17-item scale (HAMD-17) scores at baseline in patients (top, middle) and between percent changes on metabolite ratios and reduction on HAMD-17 scores after completing MBCT (bottom). The correlation coefficients and p values were cal- culated using Spearman rank tests Magn Reson Mater Phy (2016) 29:523–533 530 26 24 20 20 18 16 14 12 10 0.40 0.50 0.60 0.70 0.80 mI/tCr in left Insula at the baseline HAMD-17 at the baseline 0.30 0.40 0.50 0.60 mI/tCr in left Putamen at the baseline 26 24 20 20 18 16 14 12 10 HAMD-17 at the baseline r = - 0.59 p = 0.021 r = -0.65 p = 0.021 Discussion This is consistent with our findings of sig- nificantly increased tCho/tCr in the right caudate in MDD, which decreased to levels in controls after com- pleting MBCT treatment. Although an elevation of tCho/ tCr in the basal ganglia has been reported after 8 weeks of fluoxetine treatment in patients who responded to the therapy [25], no controls were included in that study. Elevated tCho is thought to be associated with increased cell density and/or membrane turnover. The alteration in tCho observed in our study suggests abnormalities in myelination [26] and/or glial function [27], which are associated with MDD, and/or altered cellular signal- transduction pathways. p y NAA is found only in the brain and spinal cord and is synthesized and stored in neurons but hydrolyzed in oligo- dendrocytes. Since NAA is a neuronal marker, any pathol- ogy with a loss of neurons or function results in reduced NAA levels. Vythilingam et al. [24] reported significantly decreased NAA/tCr in the caudate in patients with MDD. NAA has also been studied in the ACC and the frontal lobe in patients with MDD [28–30], and prior observed that lower NAA in the ACC and decreased NAA/tCr in the MFG at baseline were normalized after antidepressant treatment [30, 31]. This is similar to our results for patients who com- pleted MBCT treatment. Normalization of NAA/tCr within an 8-week treatment period indicates that MDD is more likely to be associated with neuronal dysfunction rather than neuronal loss [32, 33]. We found that significant correlations with percent changes of HAMD-17 scores between baseline and after MBCT treatment were found only for NAA/tCr levels in the left ACC, suggesting that NAA/tCr is a good marker for evaluating response to therapy. GABA is a main inhibitory neurotransmitter within the brain. Previous studies reported lower GABA in the occipi- tal cortex in MDD patients [40] and an increase in GABA with antidepressant treatment [41]. Reduction in GABA is possibly due to impaired GABAergic function and/or decreased GABA synthesis [42]. The occipital cortex was not analyzed in our study because it was not covered by the excitation slab. Although decreased GABA was previously reported in ACC [43], there were no significant differences in GABA/tCr in ACC in our study. Discussion The tCho peak resonates at 3.22 ppm at in vivo spec- trum and includes signals from Cho, phosphocholine, glycerophosphocholine, and acetylcholine. Previous 1 3 531 Magn Reson Mater Phy (2016) 29:523–533 Glu is a main excitatory neurotransmitter in the brain and is converted to Gln by the reabsorption by neurons or reuptake by astrocytes in order to avoid excitotoxicity. It is difficult to separate Glu from Gln in the short TE spectra at 3T, and Glx is typically designated as the sum of Glu and Gln peaks, with Glu as the majority component. A previous study found that Glx was lower in the dorsolateral prefron- tal cortex (DLPFC) of MDD patients compared to healthy controls [37] and that Glu was increased in responders after treatment with 10 days of high-frequency transcranial mag- netic stimulation (TMS) [38]. Although Glu/tCr in the left caudate at baseline was not significantly different than that in controls in our study (p = 0.051), it returned to normal levels after the 8-week treatment with MBCT. Gln is also a precursor for GSH, an antioxidant that prevents damage from reactive oxygen species. The role of GSH in MDD has not been widely studied. A previous study showed no significant correlation between GSH and depression or mania for patients with bipolar disorder [39]. Our findings of decreased GSH/tCr in insula and putamen indicate that GSH may play an important role in MDD. studies showed that the level of tCho/tCr was increased in the basal ganglion in patients with MDD [23, 24] and that it decreased with antidepressant treatment (nefazo- done) [23]. This is consistent with our findings of sig- nificantly increased tCho/tCr in the right caudate in MDD, which decreased to levels in controls after com- pleting MBCT treatment. Although an elevation of tCho/ tCr in the basal ganglia has been reported after 8 weeks of fluoxetine treatment in patients who responded to the therapy [25], no controls were included in that study. Elevated tCho is thought to be associated with increased cell density and/or membrane turnover. The alteration in tCho observed in our study suggests abnormalities in myelination [26] and/or glial function [27], which are associated with MDD, and/or altered cellular signal- transduction pathways. studies showed that the level of tCho/tCr was increased in the basal ganglion in patients with MDD [23, 24] and that it decreased with antidepressant treatment (nefazo- done) [23]. Conclusion In conclusion, results of our study demonstrate differences in tCho/tCr, NAA/tCr, Glu/tCr, mI/tCr, GABA/tCr, and GSH/tCr in patients with MDD compared to healthy con- trols and demonstrated changes in these ratios associated with MBCT treatment and reduction in depression sever- ity. Regional differences in metabolite ratios also existed in patients with MDD. These results reflect the complexity of biochemical changes associated with disease processes and treatment effects. Future studies in a larger randomized controlled study population will be helpful for interpreting changes in metabolite ratios and understanding how thera- peutic interventions impact MDD. Such information could also be valuable for determining at an early stage whether patients are responding to therapy or whether a new treat- ment strategy should be considered. in primary care. BMC Psychiatry 6:14 4. Barnhofer T, Crane C, Hargus E, Amarasinghe M, Winder R, Williams JM (2009) Mindfulness-based cognitive therapy as a treatment for chronic depression: a preliminary study. Behav Res Ther 47(5):366–373 5. Eisendrath SJ, Delucchi K, Bitner R, Fenimore P, Smit M, McLane M (2008) Mindfulness-based cognitive therapy for treatment-resistant depression: a pilot study. Psychother Psycho- som 77(5):319–320 6. Tkac I, Oz G, Adriany G, Ugurbil K, Gruetter R (2009) In vivo 1H NMR spectroscopy of the human brain at high magnetic fields: metabolite quantification at 4T vs. 7T. Magn Reson Med 62(4):868–879 7. Choi C, Douglas D, Hawesa H, Jindal A, Storey C, Dimitrov I (2009) Measurement of glycine in human prefrontal brain by point-resolved spectroscopy at 7.0 tesla in vivo. Magn Reson Med 62(5):1305–1310 8. Mekle R, Mlynarik V, Gambarota G, Hergt M, Krueger G, Gruet- ter R (2009) MR spectroscopy of the human brain with enhanced signal intensity at ultrashort echo times on a clinical platform at 3T and 7T. Magn Reson Med 61(6):1279–1285 Acknowledgments This research was supported by a technology development research grant from GE Healthcare. g 9. An L, Li S, Wood ET, Reich DS, Shen J (2014) N-acetyl-aspar- tyl-glutamate detection in the human brain at 7 Tesla by echo time optimization and improved Wiener filtering. Magn Reson Med 72(4):903–912 Author contribution Gillung and Eisendrath recorded clinical data, Luks formed the MR protocol, Jakary and Li collected MR data, Nel- son setup processing scripts, and Mukherjee interpreted MR images. Li carried out data analysis and wrote the first draft of the manu- script. All coauthors contributed to interpretation of results and have approved the manuscript. 10. Discussion This study supports the effectiveness of the MBCT treat- ment as a monotherapy for acute MDD and the effectiveness of H-1 MRSI to detect metabolite changes associated with successful MBCT treatment for MDD. Although the number of patients was relatively small, a number of key results that are consistent with our understanding of brain chemistry were obtained. Another limitation is that the significant changes in metabolite ratios could have been caused by alteration in tCr level rather than in other metabolites. The tCr, which has peaks at 3.0 and 3.9 ppm, includes Cr and phosphocreatine, which are involved in adenosine triphosphate (ATP) metab- olism. Although some previous studies found no significant differences in tCr in patients with MDD [28], others reported a significant increase in tCr compared to controls [34]. Imple- menting and adding a fast MRSI acquisition for unsuppressed water may be useful to better understand the roles of tCr in MDD, as well as the other metabolites that were observed dif- ferent in this study. However, our results suggest that using 3D MRSI at 7T in conjunction with metabolite ratios may be a time-efficient strategy for differentiating patients from healthy controls and following changes due to treatment. The mI is predominately located within astrocytes and is a precursor for the phosphatidylinositol second-messenger system, which is also presumed to act as an osmoregula- tor. Significantly decreased mI/tCr levels were previously reported in the frontal lobe in patients with MDD [29, 34], but there have been limited studies of these chemicals in the basal ganglia and insular cortex. The insula plays a role in consciousness and subjective feelings [35], and studies in MDD using functional MRI (fMRI) have detected abnor- mal interoceptive activity within this region [36]. In our study, decreased mI/tCr was found in the insula in MDD relative to controls, which may indicate glial dysfunction and/or a reduced number of glial cells in patients with MDD [32, 33]. HAMD-17 scores at baseline were signif- icantly correlated with mI/tCr in the insula and putamen, suggesting that mI/tCr may be associated with the severity of depression. 1 3 3 3 532 Magn Reson Mater Phy (2016) 29:523–533 3. Finucane A, Mercer SW (2006) An exploratory mixed methods study of the acceptability and effectiveness of mindfulness-based cognitive therapy for patients with active depression and anxiety in primary care. BMC Psychiatry 6:14 Informed consent Informed consent was obtained from all partici- pants. Informed consent Informed consent was obtained from all partici- pants. 14. Eisendrath SJ, Gillung EP, Delucchi KL, Chartier M, Mathalon DH, Sullivan JC, Segal ZV, Feldman MD (2014) Mindfulness- based cognitive therapy (MBCT) versus the health-enhancement program (HEP) for adults with treatment-resistant depression: a randomized control trial study protocol. BMC Complement Altern Med 14:95 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. 15. Li Y, Larson P, Chen AP, Lupo JM, Ozhinsky E, Kelley D, Chang SM, Nelson SJ (2015) Short-echo three-dimensional H-1 MR spectroscopic imaging of patients with glioma at 7 tesla for char- acterization of differences in metabolite levels. J Magn Reson Imaging 41(5):1332–1341 16. Cunningham CH, Vigneron DB, Chen AP, Xu D, Nelson SJ, Hurd RE, Kelley DA, Pauly JM (2005) Design of flyback echo- planar readout gradients for magnetic resonance spectroscopic imaging. Magn Reson Med 54(5):1286–1289 Conclusion Yildiz-Yesiloglu A, Ankerst DP (2006) Review of 1H magnetic resonance spectroscopy findings in major depressive disorder: a meta-analysis. Psychiatry Res 147(1):1–25 11. Caverzasi E, Pichiecchio A, Poloni GU, Calligaro A, Pasin M, Palesi F, Castellazzi G, Pasquini M, Biondi M, Barale F, Basti- anello S (2012) Magnetic resonance spectroscopy in the evalua- tion of treatment efficacy in unipolar major depressive disorder: a review of the literature. Funct Neurol 27(1):13–22 Compliance with ethical standards Conflict of interest This study was funded by GE Healthcare. Conflict of interest This study was funded by GE Healthcare. 12. Eisendrath SJ, Gillung E, Delucchi K, Mathalon DH, Yang TT, Satre DD, Rosser R, Sipe WEB, Wolkowitz OM (2015) A pre- liminary study: efficacy of mindfulness-based cognitive therapy versus sertraline as first-line treatments for major depressive dis- order. Mindfulness 6(3):475–482 Ethical standards All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Declaration of Helsinki and its later amendments or comparable ethi- cal standards. 13. Segal Z, Williams JM, Teasdale JD (2002) Mindfulness-based cognitive therapy for depression: a new approach to preventing replase. The Guilford press, New York References 1. Teasdale JD, Segal ZV, Williams JM, Ridgeway VA, Soulsby JM, Lau MA (2000) Prevention of relapse/recurrence in major depression by mindfulness-based cognitive therapy. J Consult Clin Psychol 68(4):615–623 17. Provencher SW (1993) Estimation of metabolite concentrations from localized in vivo proton NMR spectra. Magn Reson Med 30(6):672–679 2. Ma SH, Teasdale JD (2004) Mindfulness-based cognitive ther- apy for depression: replication and exploration of differential relapse prevention effects. 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W4223463632.txt	https://journals.wlb-stuttgart.de/ojs/index.php/sh/article/download/1608/1694	de		Zur Sache: Initiative zur Modernisierung des Schwäbischen Heimatbundes	Schwäbische Heimat	2,022	cc-by	808	Josef Kreuzberger Zur Sache: Initiative zur Modernisierung des Schwäbischen Heimatbundes Liebe Mitglieder des Schwäbischen Heimatbundes, zur Zeit läuft in unserem Verein ein für viele Mitglieder neuartiger Diskussionsprozess. Im Heft 1/2017 habe ich mich in einem Beitrag »Zur Sache« zur Zukunft von Heimatverbänden geäußert. Nun hat ein Kreis von Mitgliedern, die sich besorgt zeigen und sich unter dem Signum «Hohenstaufenkreis» zusammengefunden haben, seine Ansichten hierzu in einem Beitrag »Zur Sache« im Heft 2/2017 ausführlich dargelegt. Dazu möchte ich, auch im Namen des Vorstandes, gerne Stellung nehmen. Der Schwäbische Heimatbund hat ein Ihnen allen wohlbekanntes strukturelles Problem: unsere Altersstruktur, verbunden mit dem stetigen Rückgang der Mitgliederzahlen und den damit zwangsläufig verbundenen enger werdenden finanziellen Spielräumen. Damit sind wir nicht allein: Fast alle Heimatverbände bundesweit und auch zahllose andere Vereine haben dasselbe Problem. Wir hatten im Jahr 2002 knapp unter 6.000 Mitglieder und Ende 2016 noch ca. 4.400. Die Mitgliederzahl sinkt also seit 15 Jahren um ca. 100 pro Jahr – und dies trotz z.B. 77 neuer Mitglieder im Jahr 2016. Wir alle sind von der Notwendigkeit von Heimatverbänden gerade auch in der heutigen Zeit überzeugt. Die Beschäftigung mit dem Thema «Heimat» halte ich nach wie vor für wichtig und richtig, es hat Konjunktur und liegt unter dem Stichwort «Heimat 4.0 – Wiederentdeckung von Heimat als Gegenpol zu Globalisierung und Entwurzelung» auch klar im Trend. Die große Frage ist nur, wie wir dieses generelle Interesse, durchaus auch bei der Jugend, für den Schwäbischen Heimatbund nutzbar machen können? Hier setzt nun auch die neue Initiative an. Vieles von dem Vorgeschlagenen kann ich nur unterstreichen: Wer hätte nicht gern mehr Öffentlichkeitsarbeit, Stellungnahmen zu politisch kontroversen Angelegenheiten, Einbindung neuer Medien, Bildung von Foren und Diskussionsrunden und Schaffung einer Mitmachkultur auf allen Ebenen. «Alte Zöpfe» zum Abschneiden haben wir zwar nicht, aber die Setzung neuer Prioritäten ist sicher ein Diskussionsthema. All dies ist nicht neu. Hierzu wurden in den letzten Jahren stapelweise Papiere erarbeitet. Dazu haben wir vor nicht allzu langer Zeit eine Vorstandsklausur durchgeführt, und es wurde darüber mit den Kollegen und Kolleginnen vom Landesverein Badische Heimat zwei Tage lang teilweise unter wissenschaftlicher Begleitung ausführlich diskutiert. Ergänzend haben wir eine Arbeitsgruppe «Mitgliederwerbung und Öffentlichkeitsarbeit» unter Leitung unseres Schatzmeisters eingesetzt, die bisher zweimal getagt hat und deren Ergebnisse, z.B. eine Werbeaktion bei Lehrern, umgesetzt werden. Sie wird ihre Arbeit fortsetzen und sich immer wieder zu punktuellen Themen in wechselnder Besetzung zusammenfinden. Das große Problem ist die Umsetzung all dieser guten und richtigen Ideen und Vorschläge. Der Vorstand arbeiSchwäbische Heimat 2017/3 tet ehrenamtlich, und die Geschäftsstelle ist im Alltagsgeschäft voll ausgelastet. Wir brauchen deshalb engagierte Mitglieder, die sich zusätzlich ehrenamtlich mit einbringen oder die gute Ideen zur Mittelgewinnung haben. Denn über allem steht immer auch die Frage: Wer soll das alles bezahlen? Dieser Frage darf ein verantwortlicher Vorstand, aber auch eine verantwortungsvoll agierende Mitgliederinitiative, nicht ausweichen. Wir müssen uns immer wieder kritisch fragen, ob wir noch das Richtige machen oder ob wir nicht das eine oder andere künftig sein lassen sollen, um Spielräume für Neues zu gewinnen. Allerdings sind unsere regelmäßigen Veranstaltungen und Projekte, wie z.B. Denkmalund Kulturlandschaftspreis, Vortragsreihe und Tagungen, Exkursionen und Studienreisen ein wichtiger Teil unseres Vereinslebens, und ich bin davon überzeugt, dass sie auch ein Stück weit die Identität des Schwäbischen Heimatbundes ausmachen und seine Außenwahrnehmung prägen, zumal sie in der Regel von Sponsoren unterstützt werden und überörtliche Beachtung finden. Ein Thema ist mir besonders wichtig: Die Verschränkung mit den Orts- und Regionalgruppen muss in Zukunft verstärkt im Fokus der Vereinsarbeit stehen. Sie verkörpern den Heimatbund vor Ort und verfügen über Detailkenntnisse. Sie können dem Vorstand und der Geschäftsstelle wichtige Informationen liefern und Handlungshinweise geben. Wir haben deshalb die Geschäftsstelle gebeten, nach Möglichkeit künftig verstärkt mit den Orts- und Regionalgruppen zusammenzuarbeiten, und für den nächsten frei werdenden Vorstandsposten wird sich hoffentlich ein Vertreter der Ortsund Regionalgruppen zur Wahl stellen. Und ich wiederhole gerne an dieser Stelle die dringende Bitte an alle Orts- und Regionalgruppen, an alle Mitglieder, jede Möglichkeit zu nützen, für den Schwäbischen Heimatbund zu werben, und durch ihre Begeisterung für die kulturelle Vielfalt unseres Landes, für seine Schönheit, für seine reiche Kulturgeschichte neue Mitglieder zu gewinnen. Denn es gibt nur einen Verein, der die landeskulturelle Einheit unseres schwäbischen Landes nach seinen bau- und kunstgeschichtlichen Aspekten, nach seinen natürlichen Gegebenheiten im Natur- und Landschaftsschutz und in seinem übergreifenden geschichtlichen Zusammenhang als ureigenstes gesamthaftes Vereinsziel hat und als Einheit sieht: unseren Schwäbischen Heimatbund. Ich begrüße es außerordentlich, dass der «Hohenstaufenkreis» die zentralen Vereinsanliegen und -sorgen aufgreift und deren Diskussion nicht – wie seither – allein dem Vorstand überlässt. Dem Vorschlag, Probleme und Lösungen auf einem gemeinsam veranstalteten «Zukunftskongress» zu diskutieren, stimmen wir gerne zu – allerdings in der Erwartung, dass sich die Mitglieder und Unterstützer der Initiative dann bei der Umsetzung der erarbeiteten Vorschläge ehrenamtlich in die Pflicht nehmen lassen. Dann können wir der Zukunft des Schwäbischen Heimatbundes getrost ins Auge sehen. 259
https://openalex.org/W2969557658	https://inria.hal.science/hal-02553860/document	English	null	Tilt Space: A Systematic Exploration of Mobile Tilt for Design Purpose	Lecture notes in computer science	2,019	cc-by	9,973	To cite this version: Chuanyi Liu, Ningning Wu, Jiali Zhang, Wei Su. Tilt Space: A Systematic Exploration of Mobile Tilt for Design Purpose. 17th IFIP Conference on Human-Computer Interaction (INTERACT), Sep 2019, Paphos, Cyprus. pp.497-517, 10.1007/978-3-030-29387-1_29. hal-02553860 Distributed under a Creative Commons Attribution 4.0 International License HAL Id: hal-02553860 https://inria.hal.science/hal-02553860v1 Submitted on 24 Apr 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. Distributed under a Creative Commons Attribution 4.0 International License Keywords: Mobile · Tilt · Design space. 1 Introduction Smartphones are essential devices for us. We use them in our work, study, and life every day. A touch screen is its basic interactive component of a smartphone. Touch screens provide us simple and intuitive interactive methods, however, cou- pled with some limitations, e.g., “fat ﬁnger” [31] and hand occlusion [36]. Besides these limitations, other issues can be caused by various application scenarios of smartphones. When walking on the road, we may risk a traﬃc accident watch- ing the screen of a smartphone. An eyes-free interaction method is more suitable under these conditions. Sometimes, not both of one’s hands are available to ma- nipulate a mobile phone, e.g., when s/he is taking a bus or holding a bag in one hand. But one-handed thumb manipulation of a touchscreen-based mobile device is typically awkward, since one-handed thumb interaction limits the accessible area on the screen [16]. It is worth resolving these issues of a smartphone, especially for some con- ditions, e.g., on a bus or the road. Built-in tilt sensors provide an independent input channel for portable devices. Tilt gestures map 3D angles of a portable device as primitive input with interaction commands. Tilt gestures have the po- tential to resolve these issues, since we can feel a mobile phone’s posture by our proprioception without watching the screen, and operate the mobile phone with an eyes-free method and unimanually. It has long been interests to many researchers exploring tilt as primitive input for interfaces. Since Rekimoto [27] used the tilt of a small screen device as input, researchers have developed a variety of tilt-based interaction techniques. Early research on tilt focused on using tilt as an additional input channel for mobile devices to implement speciﬁc interactive techniques, such as menu navigation [27], scrolling [10, 22], panning and zooming of maps [17, 35], document and photo browsing [4, 25], and text entry [23, 38, 29]. However, these studies mainly focused on what can be done with the tilt input channel. Few studies considered human ability on controlling mobile tilt input and the design space of tilt-based interaction, especially with an eyes-free method. In the study, we systematically investigated human ability in controlling tilt input of a mobile phone. 1 Introduction The major objective of our research is to determine eﬀects of diﬀerent visual feedback levels on human ability in controlling tilt input and the upper bound of the human ability, especially under the NV condition. Tilt Space: A Systematic Exploration of Mobile Tilt for Design Purpose Chuanyi Liu[0000−0001−8237−8979], Ningning Wu, Jiali Zhang, and Wei Su Chuanyi Liu[0000−0001−8237−8979], Ningning Wu, Jiali Zhang, and Wei Su School of Information Science & Engineering, Lanzhou University,Lanzhou,China liuchuanyi96@hotmail.com,{wunn16,suwei}@lzu.edu.cn,jennyfocus@163.com Abstract. Various application scenarios of a smartphone sometimes re- quire one-handed and/or eyes-free interaction. Tilt-based interfaces have the potential to meet these requirements. Taking multiple application scenarios into account, we conducted an experiment to systematically investigate human ability in controlling tilt input of a mobile phone. Three visual feedback levels, i.e., fully visual feedback (FV ), partially visual feedback (PV ), and no visual feedback (NV ), were investigated. Under the NV condition, the participants performed a task using an eyes-free method. The results revealed that trials were performed the fastest but were the most error-prone under the NV condition. The par- ticipants could easily distinguish 4 tilt orientation levels (TOLs) and 2 tilt magnitude levels (TMLs) or 8 TOLs and 2 TMLs under the NV condition with tolerance of an error rate 10% or 15%, respectively. We also found out that the participants’ abilities to control tilt input were related to tilt orientation directions. The results have some implications for non-visual interface designs using tilt as primitive input. C. Liu et al. C. Liu et al. 2 2.2 Speciﬁc Implementation Examples Some researchers studied tilt-based text entry [5, 15, 23, 29, 38, 39]. These studies typically divided letters into groups and disambiguated letter selection through device tilt. Various UI tasks, such as scrolling [2, 10, 22, 32], document browsing [6], menu navigation [27], and display orientation switch [11], using tilt control were investigated. Pietroszek et al. [24] utilized a mobile device to perform 3D interaction on large displays. Geronimo et al. [8] also focused their study on controlling separated displays using mobile devices. They designed a tilting in- teraction framework for rapid developing web-based applications. Homaeian et al. [12] explored tilt input for data browsing techniques in cross-device environ- ments, and found that tilt facilitated access to out-of-reach data. Kurosawa et al. [18] presented combination of a tilt operation and electromyography on smart watches using tilt to set the cursor’s motion direction. Sun et al. [33] proposed a wrist-to-ﬁnger input approach that enabled one-handed and touch-free target selection on smart watches. Tilt-based mobile games [3, 13, 21, 40] have been popular for years. Typically, using tilt could not improve interaction performance in these games, but tilt interaction gave users more challenges and fun. 2.1 General Human Performance of Tilt Control Teather and Mackenzie [20] proved tilt conformed to Fitts’ law [7] through ISO 9241-9 [14] 2D pointing task. Later, the authors extended their work to compare position- and velocity-control for tilt-based interaction with the similar 2D point- ing task [34]. They found out that position-control performed approximately 2 times faster than velocity-control and had higher pointing throughput. Wang et al. [37] also studied tilt control from viewpoint of Fitts’ law; they didn’t use an accelerometer for tilt input but vision-based motion tracking. Sad and Poirier [28] conﬁrmed tilt-based scrolling and pointing tasks conformed to Fitts’ law without reporting pointing throughput. Rahman et al. [26] systematically investigated human ability of tilt control according to the human wrist movement. They found out the subjects could control comfortably at least 16 levels on the supination/pronation axis. Baglioni et al. [1] explored human control ability on tilt input with a jerk gesture. Guo and Paek [9] studied human control ability of tilt on smartwatches, and found out that OjectPoint performed better. Shima et al. [30] evaluated the performance of tilting operations on wrist-worn devices. All these literature oﬀered valuable cues for tilt-based interaction design, however, none of them had taken eyes-free method into account (to our knowl- edge). In our study, we explored tilt control according to diﬀerent visual feedback levels, especially the eyes-free operation method was considered. 2 Related Work Tilt-based interaction has long been of interests to researchers of HCI. These literature can be divided roughly into two groups: studies on general human performance of tilt control and on speciﬁc implementation examples using tilt input. Tilt Space 3 3 Experiment We base our experiment on an understanding of the mobile device application contexts. Figure 1 shows the three axes of rotation of a smartphone and the tilt C. Liu et al. 4 motion space. The built-in tilt sensor can be used to detect the pitch and roll orientations and magnitude of a mobile device by analyzing the projection of the constant gravity acceleration on the three axes. But tilt sensor does not allow the determination of the static yaw orientation [11]. However, the range of motion provides by the tilt sensor may not always be conducive to tilt control due to the ergonomic limitations of human wrists and the diﬃculty in obtaining visual feedback in “extreme” angles (e.g., when a device’s screen is turned leftward or rightward from a horizontal posture to vertical). But under the eyes-free condition, users can control the tilt by pro- prioception and memory without relying on visual feedback; arms can be used besides wrists to enlarge the tilt space. In other words, the visual and ergonomic limitations are alleviated under the eyes-free condition. Fig. 1: The three axes of the device rotations and the three corresponding angles. Fig. 2: The tilt space is divided into 4, 6, 8, and 12 TOLs (from left to right) together with 2 TMLs. Fig. 2: The tilt space is divided into 4, 6, 8, and 12 TOLs (from left to right) together with 2 TMLs. Fig. 2: The tilt space is divided into 4, 6, 8, and 12 TOLs (from left to right) together with 2 TMLs. Fig. 1: The three axes of the device rotations and the three corresponding angles. Participants Twelve volunteers,10 males and 2 females, ranging in age from 22 to 32 years (Mean = 25.3, SD = 3.8), participated in the experiment. All the participants were recruited from the computer department in the local university, and they were all familiar with manipulation of smartphones and right-handed. All of them had normal or corrected to normal vision. Although none of the participants had ever used tilt to perform subtle pointing or selection tasks before, they all had prior experience with tilt-based interfaces such as gravity sensor games. 3.1 Apparatus The experiment was conducted using a Huawei honor 5c smartphone with a built-in 3-axis accelerometer running Google’s Android 6.0 operating system. The display resolution was 1920 × 1080 pixels. In addition, a Lenovo YT3-X50F 10.1-inch tablet with android 5.1.1 OS was used to inform the user a target under the no-visual-feedback condition. The experimental software was developed in Java using the Android SDK. The accelerometer was used to detect tilt magnitude and tilt orientation of the smartphone, sampling at a rate of 50 samples per second. Tilt Space 5 3.3 Task and Stimuli The human control of tilt on handheld devices was decomposed into the control of tilt orientation and the control of tilt magnitude. The tilt orientation (0° to 360°) was linearly divided into four levels: 4, 6, 8, and 12 with angular intervals of 90°, 60°, 45°, and 30°, respectively. The motion space of tilt magnitude (0° to 90°) was linearly divided into three levels: 2, 3, and 4 with angular intervals of 45°, 30°, and 22.5°, respectively. A circle, standing for the tilt space, was divided into m(the number of tilt orientation levels, TOLs, tilt orientation hereafter referred to as TO ) ×n(the number of tilt magnitude levels, TMLs, tilt magnitude hereafter referred to as TM ) districts (e.g., 4TOLs×2TMLs = 8 districts, see Fig. 2 left). One of these districts was randomly (across trials) selected to be a target. There were three visual feedback levels (visual feedback and its level hereafter referred to as VF and VFL, respectively, see Fig. 3): fully visual (FV ), partially visual (PV ), and no visual (NV ). (a) FV (b) PV (c) NV Fig. 3: Diﬀerent visual feedback conditions. FV : a target district was ﬁlled in green and a district holding the pointer in gray, the blue pointer is always visible; PV (the dashed lines were not shown in the experiment): a target district was ﬁlled in green, the blue pointer disappears once tilting began; NV : districts were shown statically during a trial in the tablet PC. (a) FV (b) PV (c) NV (a) FV (b) PV (c) NV (c) NV Fig. 3: Diﬀerent visual feedback conditions. FV : a target district was ﬁlled in green and a district holding the pointer in gray, the blue pointer is always visible; PV (the dashed lines were not shown in the experiment): a target district was ﬁlled in green, the blue pointer disappears once tilting began; NV : districts were shown statically during a trial in the tablet PC. C. Liu et al. 6 Under the FV and PV conditions, the cursor was displayed as a blue pointer, which was painted from the center of the device’s display (i.e., the center of the circle, denoted as C). Tilt magnitude and tilt orientation were computed to the pointer’s length and orientation, respectively. 3.3 Task and Stimuli The tilt magnitude of device was linearly mapped to the pointer’s length which varied between 0 and the maximum (the radius length of the largest circle) when a participant tilted the phone from horizontal to vertical. Under the NV condition, the pointer dimensions were calculated using the same method, but the pointer had no visual feedback (i.e., the pointer was hidden). To conﬁrm selection of a target, a participant manipulated tilt input to adjust the pointer’s orientation and length to make it enter the target district, and kept the pointer in the district over the time threshold. The threshold was set to be one second vs. the largest selection delay ,500ms, in [34]: the dwell time was intentionally doubled to discriminate target selection from unintentional entry and disambiguate target selection especially for NV condition. Similarly, to minimize unintentional selection and reduce selection diﬃculty, minute tilt magnitude variation (within 5°) was also eliminated from target selection. A non-target district was determined whether selected using the same method. Under FV condition (Fig. 3a), visual feedback was continuous during the whole process of a trial. All the districts were painted with gray-lined borders; a target district was ﬁlled in green, while the current district (holding the pointer) in gray. The pointer was painted in blue. When being selected, a target district was ﬁlled in red, while a non-target in yellow. Under PV condition (Fig. 3b), only a target district was continuously dis- played and the pointer was visible only at the beginning of a trial. Once tilting began, the pointer disappeared. A participant manipulated the hidden pointer to meet the target upon their proprioception and spatial perception and cognition. Once the pointer entered the target, the target color changed from green to gray. When the trial was performed correctly, the target was colored in red; otherwise, the selected non-target district became visible and was ﬁlled in yellow. There was no visual feedback under NV condition (Fig. 3c), i.e., the par- ticipants performed trials using an eyes-free method. A target was displayed on the tablet screen using a FV method except the pointer, but the surface was static during a trial (Fig. 4b). The tablet was connected to the smartphone via Blue-tooth. The trial sequence in the tablet was synchronized with that in the mobile phone. The participants selected a target completely upon their proprio- ception, kinesthetic memory, and spatial perception and cognition. 3.3 Task and Stimuli This scenario was similar to the expert mode of the Marking Menus [19]. Under any condition, a pleasure or alarm sound was played when a district was selected correctly or incorrectly, respectively. 12 participants× 3 blocks× 3.4 Procedure and Design 8 3 V FLs× (4 + 6 + 8 + 12) TOLs× (2 + 3 + 4) TMLs × 2 repetitions = 58320 target selection trials. 2 repetitions The participants were instructed to complete each trial “as quickly and ac- curately as possible”. After performing all experimental trials, the participants were investigated for their subjective comments on the three VFLs with a ques- tionnaire where they rated the usability, ease of learning, hand and eye fatigue along a Likert scale (each aspect with seven rating levels, where 1 represented the worst and 7 the best). In total, the experiment lasted approximately 3 hours for each participant. “×” means interaction eﬀects. 3.4 Procedure and Design The experiment used a within-subjects full factorial design. The experimental factors and their levels were: Tilt Space 7 (b) NV Tilt Space 7 (a) FV (b) NV Fig. 4: A participant was performing experimental trials. Tilt Space 7 (a) FV (a) FV (b) NV (b) NV Fig. 4: A participant was performing experimental trials. VF: FV, PV, NV. TO: 4, 6, 8, 12. TM : 2, 3, 4. Before the experiment, each of the participants had been told they had the right to freely quit the experiment at any time. The participants were allowed to have a rest between any two experimental conditions, and they had been told they were not allowed to do the experiment when they felt tired. A longer rest between experimental blocks was mandatory. After given a detailed description about the task, participants spent a few minutes in familiarizing themselves with tilt control and the three visual feedback conditions. The participants performed the experiment seated, and all of them held the device with their right hands (see Fig. 4). g ( g ) In the formal experiment, the participants were required to complete three blocks of trials. A Latin square was used to counterbalance the order eﬀorts of the three VFLs. For each feedback condition, the participants performed target selection beginning from TOL4 and TML2. The rationale for the sequential de- sign is that interaction performance does reduce signiﬁcantly with the increase of levels of the two factors according to the previous work [26] and our observation. So the major objective of this study is not to determine whether there exists signiﬁcantly diﬀerent eﬀects between these factor levels on interaction perfor- mance measures but to ﬁnd out the upper bound of human ability to control tilt input according to the two experimental factors. The target appeared randomly among all the districts with 2 repetitions at the same location. The TOLs and the TMLs were always presented in ascending (smallest to largest) order for easy learning. When a wrong selection happened, a failed attempt was recorded. Not was the experiment proceeded to next trial until a trial was done correctly. Participants were allowed to try for no more than 5 times for each trial before success, otherwise, the trial was aborted. Totally, there were: C. Liu et al. 3.5 Performance Measures The experimental measures were selection time (ST), error rate (ER, there was ONE error when participants failed at their ﬁrst attempt), number of aborted trials (AT, i.e.,number of trials that had failed for 5 times), and number of crossings (NC, the times of the cursor enters a target without selecting it, i.e., when dwell time in the target was less than the given time threshold). ST was deﬁned as the time consumed from tilting the phone to selecting a target. NC was used to reveal the diﬃculty degrees to dwell the pointer in a target district, it was 1 in a perfect selection task. In the next section, these measures were calculated to averages per trial from the primitive experimental records. Table 1: Statistical eﬀects for performance measures. Eﬀect ST ER AT NC Name df F p ηp 2 F p ηp 2 F p ηp 2 F p ηp 2 block(b) 2, 22 33 .000 .750 24.3 .000 .689 0.9 .44 .072 16.8 .000 .604 VFL(v) 2,22 119 .000 .915 197.4 .000 .947 13 .000 .541 186.3 .000 .944 TOL(o) 3,33 95.6 .000 .897 45.3 .000 .804 12.7 .000 .535 7.7 .001 .411 TML(m) 2,22 291.7 .000 .964 62.6 .000 .851 10.7 .001 .494 150.9 .000 .932 b × v 4,44 2.3 .08 .170 6.9 .000 .385 0.7 .57 .064 7.8 .000 .416 v × o 6,66 8.2 .000 .428 24.4 .000 .689 12.6 .000 .533 2.7 .022 .195 v × m 4,44 26 .000 .702 38.9 .000 .780 107 .000 .492 78.7 .000 .877 o × m 6,66 1.6 .17 .126 2.5 .03 .183 1.6 .15 .130 0.5 .79 .046 v × o × m 12,132 1.1 .41 .087 0.5 .93 .042 1.7 .07 .137 0.9 .53 .077 “×” means interaction eﬀects. Tilt Space 9 4 Results A 3 blocks × 3 V FLs × 4 TOLs × 2 TMLs RM-ANOVA was conducted on mean data of ST,ER,AT, and NC. Statistical reports for the four performance measures are shown in Table 1. 4.1 Selection Time Learning eﬀects were observed from the signiﬁcant eﬀect of block on ST. There was no signiﬁcant interaction eﬀect between feedback and block, Fig. 5 shows the similar learning eﬀects under diﬀerent visual feedback conditions. Block 3 2 1 Average ST (ms) 3000 2500 2000 1500 1000 500 0 1919 2014 2408 2421 2678 2170 2186 2381 1809 NV PV FV VFL Fig. 5: Average ST by blocks and VFLs. TOL 12 8 6 4 Average ST (ms) 3000 2500 2000 1500 1000 500 0 1876 2785 2507 2381 2336 2389 2228 2163 2202 2069 1888 1823 NV PV FV VFL Fig. 6: Average ST by TOLs and VFLs. Block 3 2 1 Average ST (ms) 3000 2500 2000 1500 1000 500 0 1919 2014 2408 2421 2678 2170 2186 2381 1809 NV PV FV VFL TOL 12 8 6 4 Average ST (ms) 3000 2500 2000 1500 1000 500 0 1876 2785 2507 2381 2336 2389 2228 2163 2202 2069 1888 1823 NV PV FV VFL Fig. 5: Average ST by blocks and VFLs. Fig. 6: Average ST by TOLs and VFLs. There was a signiﬁcant eﬀect of V F (F(2, 22) = 119.04, p < 0.001, ηp2 = 0.915) on ST. Pairwise comparisons showed signiﬁcant diﬀerences between all visual feedback levels (at p < 0.001). The participants performed the fastest under the NV condition, but the most slowly under the PV (Fig. 5). Based on our observations, we speculate that this may because, under the FV and PV conditions, the participants tended to tilt the device from a small angle little by little to a large one and kept watching the visual feedback during a trial. But under the NV condition, the participants tended to tilt the device directly to the location they thought to be appropriate based on proprioception, kinesthetic memory, and spatial perception and cognition, since it was impossible to rely on any visual feedback. Page 1 Although the participants had more practice before the trials of the sub- sequent TOLs (Fig. 6) and TMLs (Fig. 7), tests of within-subjects contrasts revealed general ST increases with those of level numbers of the two factors. These trends indicated the signiﬁcant increase of manipulation diﬃculty in tilt control when the number of divided districts increased, and the upper bounds of human ability to manipulate tilt input. 10 C. Liu et al. 4.1 Selection Time TML 4 3 2 Average ST (ms) 3000 2500 2000 1500 1000 500 0 1746 2847 2521 2139 2432 2257 2047 2092 1904 NV PV FV VFL Fig. 7: Average ST by TMLs and VFLs. Block 3 2 1 Averate ERs 40% 30% 20% 10% 0% 31% 38% 10% 16% 3% 3% 6% 25% 9% NV PV FV VFL Fig. 8: Average ERs by blocks and VFLs. 10 C. Liu et al. TML 4 3 2 Average ST (ms) 3000 2500 2000 1500 1000 500 0 1746 2847 2521 2139 2432 2257 2047 2092 1904 NV PV FV VFL C. Liu et al. 10 Block 3 2 1 Averate ERs 40% 30% 20% 10% 0% 31% 38% 10% 16% 3% 3% 6% 25% 9% NV PV FV VFL Fig. 7: Average ST by TMLs and VFLs. Fig. 8: Average ERs by blocks and VFLs. 4.2 Accuracy 10: Average ERs by TMLs and VFLs. Block 3 2 1 Average NC 3.0 2.5 2.0 1.5 1.0 .5 .0 1.4 1.4 1.5 2.4 1.3 1.2 1.3 2.5 2.9 NV PV FV VFL Fig. 11: Average NC by blocks and VFLs. Block 3 2 1 Average AT 5% 4% 3% 2% 1% 0% 3.5% 3.9% 4.8% NV PV FV VFL Fig. 12: Average AT by blocks and VFLs. Tilt Space 11 TML 4 3 2 Average ERs 40% 30% 20% 10% 0% 40% 32% 22% 14% 12% 9% 5% 4% 3% NV PV FV VFL 11 TOL 12 8 6 4 Average ERs 50% 40% 30% 20% 10% 0% 27% 31% 21% 14% 10% 10% 12% 4% 4% 5% 45% 3% NV PV FV VFL Fig. 9: Average ERs by TOLs and VFLs. TML 4 3 2 Average ERs 40% 30% 20% 10% 0% 40% 32% 22% 14% 12% 9% 5% 4% 3% NV PV FV VFL Fig. 10: Average ERs by TMLs and VFLs. TOL 12 8 6 4 Average ERs 50% 40% 30% 20% 10% 0% 27% 31% 21% 14% 10% 10% 12% 4% 4% 5% 45% 3% NV PV FV VFL Fig. 9: Average ERs by TOLs and VFLs. Fig. 10: Average ERs by TMLs and VFLs. Block 3 2 1 Average NC 3.0 2.5 2.0 1.5 1.0 .5 .0 1.4 1.4 1.5 2.4 1.3 1.2 1.3 2.5 2.9 NV PV FV VFL Fig. 11: Average NC by blocks and VFLs. Block 3 2 1 Average AT 5% 4% 3% 2% 1% 0% 3.5% 3.9% 4.8% NV PV FV VFL Fig. 12: Average AT by blocks and VFLs. Block 3 2 1 Average AT 5% 4% 3% 2% 1% 0% 3.5% 3.9% 4.8% NV PV FV VFL Fig. 12: Average AT by blocks and VFLs. Block 3 2 1 Average AT 5% 4% 3% 2% 1% 0% 3.5% 3.9% 4.8% NV PV FV VFL Fig. 12: Average AT by blocks and VFLs. Block 3 2 1 Average NC 3.0 2.5 2.0 1.5 1.0 .5 .0 1.4 1.4 1.5 2.4 1.3 1.2 1.3 2.5 2.9 NV PV FV VFL Fig. 11: Average NC by blocks and VFLs. Fig. 12: Average AT by blocks and VFLs. Number of Aborted Trials. AT indicates the diﬃcult degree to complete the experimental tasks. As shown in Fig. 4.2 Accuracy 12, the diﬀerences of AT across the blocks were not signiﬁcant (F(2, 22) = 0.85, NS). Figure 12 shows all ATs were approximately 0 under the FV and PV conditions. A minor decrease of AT, from 4.8% to 3.5%, under NV condition was observed (Fig. 12). 4.2 Accuracy We explore the eﬀects of experimental factors on accuracy from three aspects: ER, NC, and AT. Statistical results of these metrics are shown in Table 1. Page 1 Error Rate. A signiﬁcant eﬀect of block on ER (F(2, 22) = 24.34, p < 0.001, ηp2 = 0.689) revealed that the participants improved tilt input ability with limited exer- cise, as shown in Fig. 8. There was a signiﬁcant eﬀect of feedback on ER(F(2, 22) = 197.45, p < 0.001, ηp2 = 0.947). Pairwise comparisons showed there were signiﬁ- cant diﬀerences between VFLs (at p < 0.001). It was the most and least error- prone under the NV and the FV conditions, respectively. Figure 8 also reveals that there was a signiﬁcant interaction eﬀect between block and VF, and ER decreased the fastest under the NV condition. This indicated that although it was the most error-prone to perform tasks without any visual feedback, the par- ticipants improve their ability to control tilt input more quickly with an eyes-free method. The similar variation trends and human ability upper bounds with orienta- tion resolution (Fig. 9) and magnitude (Fig. 10) on MT were also observed on ER. We will further analyze these results in the subsequent subsections. Number of Crossings. NC indicates how diﬃcult to dwell the cursor (pointer) in a district. As shown in Table 1, the main and interaction eﬀects (except that of TO × TM and that of VF × TO × TM) of all the experimental factors had signiﬁcant impacts on NC. Figure 11 shows there was a signiﬁcant learning eﬀect across the three blocks (F(2, 22) = 16.75, p < 0.001, ηp2 = 0.604) and a signiﬁcant VF eﬀect (F(2, 22) = 186.32, p < 0.001, ηp2 = 0.944) on NC. A signiﬁcant interaction eﬀect between block and feedback is also observed in Fig. 11. PV possessed the largest NC among the three VFLs indicated that partially visual feedback imposed diﬃculty on keeping a mobile device still with a 3D angle. Tilt Space 11 TOL 12 8 6 4 Average ERs 50% 40% 30% 20% 10% 0% 27% 31% 21% 14% 10% 10% 12% 4% 4% 5% 45% 3% NV PV FV VFL Fig. 9: Average ERs by TOLs and VFLs. TML 4 3 2 Average ERs 40% 30% 20% 10% 0% 40% 32% 22% 14% 12% 9% 5% 4% 3% NV PV FV VFL Fig. 4.3 Orientation Eﬀects under the NV Condition TOL 12 8 6 4 Average ST (ms) 2500 2000 1500 1000 500 0 2276 2085 1945 2073 1861 1868 1815 1683 1734 1710 2061 1858 4 3 2 TML Fig. 13: Average ST by TOLs and TMLs under the NV condition. Fig. 13: Average ST by TOLs and TMLs under the NV condition. Fig. 14: Average ERs by TOLs and TMLs under the NV condition. TOL 12 8 6 4 Average NC 2.0 1.5 1.0 .5 .0 4 3 2 TML TOL 12 8 6 4 2.0 1.5 1.0 .5 .0 4 3 2 TML . 15: Average NC by TOLs and MLs under the NV condition. TOL 12 8 6 4 Average AT 12% 10% 8% 6% 4% 2% 0% 5% 3% 0% 2% 3% 12% 6% 2% 8% 2% 6% 0% 4 3 2 TML Fig. 16: Average AT by TOLs and TMLs under the NV condition. TOL 12 8 6 4 Average AT 12% 10% 8% 6% 4% 2% 0% 5% 3% 0% 2% 3% 12% 6% 2% 8% 2% 6% 0% 4 3 2 TML TOL Fig. 15: Average NC by TOLs and TMLs under the NV condition. Fig. 16: Average AT by TOLs and TMLs under the NV condition. Page 1 0.036), see Fig. 15. Figure 16 shows that AT also decreased minutely from TOL6(3.9%) to TOL8(2.8%) (F(1, 11) = 1.068, p = 0.324, ηp2 = 0.088). Figure 17 illustrates the distribution of ERs at TOL12 in detail: ERs in the device’s axial directions were lower than in the other directions, similarly Guo and Paek [9] reported the similar results on smart watches. Page 1 0.036), see Fig. 15. Figure 16 shows that AT also decreased minutely from TOL6(3.9%) to TOL8(2.8%) (F(1, 11) = 1.068, p = 0.324, ηp2 = 0.088). Figure 17 illustrates the distribution of ERs at TOL12 in detail: ERs in the device’s axial directions were lower than in the other directions, similarly Guo and Paek [9] reported the similar results on smart watches. 4.3 Orientation Eﬀects under the NV Condition Page 1 Generally, experimental task performance metrics of speed (according to ST, see Fig. 13), accuracy (according to ERs, see Fig. 14), diﬃculty to dwell in the target (according to NC, see Fig. 15), and diﬃculty to complete a selection (according to AT, see Fig. 16) decreased with the increase of TOLs and TMLs. But we observed an exception (from TOL6 to TOL8) for TO from the ﬁgures. We con- ducted another 3 blocks × 4 TOLs × 2 TMLs RM-ANOVA on data under NV condition to get further insights. Figure 13 shows MT decreased minutely from TOL6(1888ms) to TOL8(1876ms) (F(1, 11) = 0.514, p = 0.488, ηp2 = 0.045). ERs also decreased slightly from TOL6(31.3%) to TOL8(27.3%) (F(1, 11) = 3.28, p = 0.097, ηp2 = 0.23), see Fig. 14. Similarly, there was also a slight de- crease of NC from TOL6(1.42) to TOL8(1.4) (F(1, 11) = 0.406, p = 0.537, ηp2 = C. Liu et al. 12 TOL 12 8 6 4 Average ERs 60% 50% 40% 30% 20% 10% 0% 52% 36% 42% 28% 46% 27% 33% 22% 36% 19% 19% 12% 4 3 2 TML TOL 12 8 6 4 Average ERs 60% 50% 40% 30% 20% 10% 0% 52% 36% 42% 28% 46% 27% 33% 22% 36% 19% 19% 12% 4 3 2 TML Fig. 14: Average ERs by TOLs and TMLs under the NV condition. TOL 12 8 6 4 Average ST (ms) 2500 2000 1500 1000 500 0 2276 2085 1945 2073 1861 1868 1815 1683 1734 1710 2061 1858 4 3 2 TML Fig. 13: Average ST by TOLs and TMLs under the NV condition. TOL 12 8 6 4 Average ERs 60% 50% 40% 30% 20% 10% 0% 52% 36% 42% 28% 46% 27% 33% 22% 36% 19% 19% 12% 4 3 2 TML Fig. 14: Average ERs by TOLs and TMLs under the NV condition. TOL 12 8 6 4 Average NC 2.0 1.5 1.0 .5 .0 4 3 2 TML Fig. 15: Average NC by TOLs and TMLs under the NV condition. TOL 12 8 6 4 Average AT 12% 10% 8% 6% 4% 2% 0% 5% 3% 0% 2% 3% 12% 6% 2% 8% 2% 6% 0% 4 3 2 TML Fig. 16: Average AT by TOLs and TMLs under the NV condition. 4.4 Discernible Numbers of Orientation Resolution Levels and Magnitude Levels The participants had to keep watching the screen during a trial under FV and PV, but PV was defeated by the other two VFLs on both speed and accuracy. This indicates that PV made no contribution for the experimental tasks. In the following of this subsection, we determine the participants’ performance based on the data gathered when they had limited practice with tilt input, so only the data of block 3 were analyzed. A 3 V FLs × 4 TOLs × 2 TMLs RM-ANOVA was conducted on mean data of ER, AT, and NC in block 3. The largest NC in block 3 was 1.4 and 1.6 under the FV (see Fig. 18) NV conditions (see Fig. 19), respectively. So NC imposed no limitations on Tilt Space 13 Fig. 17: Average ERs at TOL12. Fig. 17: Average ERs at TOL12. discernible levels of the two factors. Similarly, all the percentages of ATs in block 3 under the FV condition were approximately 0% (see Fig. 20), and the largest percentage of AT under the NV condition was 11% (see Fig. 21). Thus AT had no inﬂuences on discernible levels of the two factors, either. TOL (Error bars: +/- 1 SE) 12 8 6 4 Average NC 2.0 1.5 1.0 .5 .0 1.4 1.3 1.4 1.4 1.3 1.2 1.2 1.3 1.2 1.2 1.1 1.1 4 3 2 TML Fig. 18: Average NC by TOLs and TMLs in block 3 under the FV con- dition. TOL (Error bars: +/- 1 SE) 12 8 6 4 Average NC 2.0 1.5 1.0 .5 .0 1.5 1.5 1.6 1.5 1.4 1.4 1.4 1.4 1.4 1.2 1.2 1.1 4 3 2 TML Fig. 19: Average NC by TOLs and TMLs in block 3 under the NV con- dition. TOL (Error bars: +/- 1 SE) 12 8 6 4 Average NC 2.0 1.5 1.0 .5 .0 1.5 1.5 1.6 1.5 1.4 1.4 1.4 1.4 1.4 1.2 1.2 1.1 4 3 2 TML TOL (Error bars: +/- 1 SE) 12 8 6 4 Average NC 2.0 1.5 1.0 .5 .0 1.4 1.3 1.4 1.4 1.3 1.2 1.2 1.3 1.2 1.2 1.1 1.1 4 3 2 TML Fig. 18: Average NC by TOLs and TMLs in block 3 under the FV con- dition. Fig. 19: Average NC by TOLs and TMLs in block 3 under the NV con- dition. 4.4 Discernible Numbers of Orientation Resolution Levels and Magnitude Levels Figure 22 shows the largest ER in block 3 was 4.2% under the FV condition. So all the explored levels for both TO and TM are discernible, i.e., TOLs were 12 and TMLs were 4. There was only one ER (8.3%) less than 10% under the NV condition, as shown in Fig. 23. In that case, TOLs and TMLs were 4 and 2, respectively. These two are discernible numbers for the two factors under NV. If we extend 14 C. Liu et al. TOL (Error bars: +/- 1 SE) 12 8 6 4 Average AT 0% 4 3 2 TML Fig. 20: Average AT by TOLs and TMLs in block 3 under the FV con- dition. TOL (Error bars: +/- 1 SE) 12 8 6 4 Average AT 14% 12% 10% 8% 6% 4% 2% 0% 11% 4% 2% 1% 7% 1% 4% 0% 7% 2% 3% 0% 4 3 2 TML Fig. 21: Average AT by TOLs and TMLs in block 3 under the NV con- dition. TOL (Error bars: +/- 1 SE) 12 8 6 4 Average ERs 6% 4% 2% 0% 4.2% 3.9% 4.2% 2.6% 3.7% 1.7% 1.9% 3.5% 1.7% .5% 2.1% 4.2% 4 3 2 TML Fig. 22: Average ERs by TOLs and TMLs in block 3 under the FV con- dition. TOL (Error bars: +/- 1 SE) 12 8 6 4 Average ERs 50% 40% 30% 20% 10% 0% 46.5% 30.2% 31.2% 20.8% 38.4% 20.5% 23.6% 16.0% 33.7% 13.5% 13.9% 8.3% 4 3 2 TML Fig. 23: Average ERs by TOLs and TMLs in block 3 under the NV con- dition. 14 C. Liu et al. TOL (Error bars: +/- 1 SE) 12 8 6 4 Average AT 0% 4 3 2 TML Fig. 20: Average AT by TOLs and TMLs in block 3 under the FV con- dition. TOL (Error bars: +/- 1 SE) 12 8 6 4 Average AT 14% 12% 10% 8% 6% 4% 2% 0% 11% 4% 2% 1% 7% 1% 4% 0% 7% 2% 3% 0% 4 3 2 TML Fig. 21: Average AT by TOLs and TMLs in block 3 under the NV con- dition. 14 C. Liu et al. 4.4 Discernible Numbers of Orientation Resolution Levels and Magnitude Levels TOL (Error bars: +/- 1 SE) 12 8 6 4 Average AT 0% 4 3 2 TML 14 TOL (Error bars: +/- 1 SE) 12 8 6 4 Average AT 14% 12% 10% 8% 6% 4% 2% 0% 11% 4% 2% 1% 7% 1% 4% 0% 7% 2% 3% 0% 4 3 2 TML Fig. 20: Average AT by TOLs and TMLs in block 3 under the FV con- dition. Fig. 21: Average AT by TOLs and TMLs in block 3 under the NV con- dition. TOL (Error bars: +/- 1 SE) 12 8 6 4 Average ERs 6% 4% 2% 0% 4.2% 3.9% 4.2% 2.6% 3.7% 1.7% 1.9% 3.5% 1.7% .5% 2.1% 4.2% 4 3 2 TML TOL (Error bars: +/- 1 SE) 12 8 6 4 Average ERs 6% 4% 2% 0% 4.2% 3.9% 4.2% 2.6% 3.7% 1.7% 1.9% 3.5% 1.7% .5% 2.1% 4.2% 4 3 2 TML Fig. 22: Average ERs by TOLs and TMLs in block 3 under the FV con- dition. TOL (Error bars: +/- 1 SE) 12 8 6 4 Average ERs 50% 40% 30% 20% 10% 0% 46.5% 30.2% 31.2% 20.8% 38.4% 20.5% 23.6% 16.0% 33.7% 13.5% 13.9% 8.3% 4 3 2 TML Fig. 23: Average ERs by TOLs and TMLs in block 3 under the NV con- dition. TOL (Error bars: +/- 1 SE) 12 8 6 4 Average ERs 50% 40% 30% 20% 10% 0% 46.5% 30.2% 31.2% 20.8% 38.4% 20.5% 23.6% 16.0% 33.7% 13.5% 13.9% 8.3% 4 3 2 TML Fig. 22: Average ERs by TOLs and TMLs in block 3 under the FV con- dition. Fig. 23: Average ERs by TOLs and TMLs in block 3 under the NV con- dition. ER tolerance to 15%, the discernible numbers of TOLs and TMLs were 8 and 2, respectively. ER tolerance to 15%, the discernible numbers of TOLs and TMLs were 8 and 2, respectively. 4.5 Subjective Feedback Page 1 The survey was to determine how the participants felt using tilt control for target acquisition and selection tasks under diﬀerent VFLs. Participant’s subjective ratings of the three levels are shown in Fig. 24. The ratings include usability, ease of learning, and hand and eye fatigue. Page 1 The participants rated FV the highest regarding measures of usability and ease of learning; while NV was rated the best for eye and hand fatigue. According to our observations, under the FV and the PV conditions, the participants tried hard to keep watching visual feedback while tilting the device, and to keep their wrists in a special posture to keep the screen in a visible range: this limited the movement of wrists and arms. While under the NV condition, the participants manipulated the device to perform trials with more comfortable gestures. Although the participants rated NV the lowest regarding usability, they believed that using a mobile with an eyes-free method is irreplaceable for 15 Tilt Space 1 2 3 4 5 6 7 Usability Ease of Learning Hand Fatigue Eye Fatigue FV PV NV Fig. 24: Likert scale ratings by VFLs. 1 2 3 4 5 6 7 Usability Ease of Learning Hand Fatigue Eye Fatigue FV PV NV Fig. 24: Likert scale ratings by VFLs. Fig. 24: Likert scale ratings by VFLs. some special application scenarios, e.g., while walking on the road. NV was rated the lowest for ease of learning, but it is conﬂicting with the quantitative statistical results. We speculate that, under the NV condition, the diﬃculty of tilt manipulation made the participants think it was also diﬃcult to obtain the ability of tilt manipulation. Other open comments include: “Tilt to the upper right corner is very un- comfortable.” Given that all the participants were right-handed, this is easy to understand the comment and consistent with the aforementioned orientation eﬀects. 5 Concept Designs of Tilt-based Interaction Using an Eyes-Free Method Building on the experimental results and our observations, we now explore the tilt-based design space using an eyes-free method. We often receive a phone call when walking across a crossroad, sometimes holding a bag in one hand. At that case, it is diﬃcult to answer the call. Fur- thermore, we might put us in danger if we watch the mobile phone on the road. According to our experimental results, we can easily deal with the phone call with tilt input using an eyes-free method and by one hand (see Fig. 25a). Typi- cally, we have four choices to deal with a phone call. We may ﬁrst get to know the calling-number by an audio prompt message, and then determine to accept or deny the call, or send an automatic message and deny the call. The division of the tilt space with four TOLs and one TML is suitable for this case (See Fig. 25a). Figure 25b shows the concept design of a music player supporting eyes-free interaction. Since it is easier to be controlled for “X-Y axial” orientation districts, more commands are assigned in these directions (see Fig. 25b). There are two TOLs in these axial directions, but only one in the diagonal. In the design, opposite functions are mirrored in one line, most functions are located alone X- Y axes. By default, the surface of the player is hidden. Users tilt a mobile phone into a certain 3D tilt district and keep its tilting posture for one second, then a function is chosen, and then the player works according to the chosen command. C. Liu et al. 16 16 (b) 1 2 3 4 5 6 8 7 — —— (a) (b) (c) (d) Fig. 25: Concept designs for tilt space using an eyes-free method. (a) Dealing with a phone call with tilt input. (b) Mapping between the tilt space districts and the music player commands. (c) Manipulating a toy car with tilt input. (d) A smart toy car that can be manipulated by a mobile APP through Blue-tooth. 1 2 3 4 5 6 8 7 — —— (a) (a) (b) (c) (d) (c) (d) Fig. 25: Concept designs for tilt space using an eyes-free method. (a) Dealing with a phone call with tilt input. (b) Mapping between the tilt space districts and the music player commands. (c) Manipulating a toy car with tilt input. 5 Concept Designs of Tilt-based Interaction Using an Eyes-Free Method (d) A smart toy car that can be manipulated by a mobile APP through Blue-tooth. 17 Tilt Space In the market, there are many smart toy cars that can be manipulated by a mobile APP through Blue-tooth, see Fig. 25d. We typically manipulate a smart toy car using the GUI of an APP. But that is not the best method, since we had better keep visually tracking with the toy car when it is running on the road, otherwise it may run into some obstacles or even be crashed. Based on our study results, we build a pie menu that is suitable for eyes-free interaction, see Fig. 25c. We employ tilt input to make a toy car run forward by tilting a mobile phone outward (at approximately twelve o’clock) or make the toy speed up by tilting the phone further in the same direction. When we want to stop the toy, we just tilt the phone backward (at approximately six o’clock); and if we tilt back the phone further, we make the toy run reversely. We also manipulate the toy turn leftward or rightward by tilting the phone leftward or rightward, respectively. During the whole process, we keep on watching the toy car and don’t need to glimpse the screen of the mobile phone. 6 Discussion The results indicate that the participants could distinguish at least 12 TOLs and 4 TMLs under the FV condition. But there exist some spectra of TM in which the usability is not good enough, e.g., the spectrum where the TM is near 90°, it is diﬃcult to keep a device in that posture and get visual feedback from the screen for our wrists and eyes, respectively. These TM spectra should not be used in applications or be used as mappings to some special least use commands, e.g., the command of APP exit. For the NV condition, although the discernible numbers are small, NV had the highest speed and were the best in terms of hand and eye fatigue. NV is not bothered by “fat ﬁnger” [31] and hand occlusion [36], which generally exist for all direct-touch screens. And the eyes-free method can reduce some safety risks when we use a mobile phone on the road. Although NV has the potential to address the aforementioned issues of a mobile phone, it limits the available number of tilt input commands. NV is more suitable for some special interaction scenarios, where one’s vision is unavailable and/or one of her/his hands is occupied and fewer interfacial commands are necessary. The previous research [26] had probed discretizing raw angular space using linear, quadratic, and sigmoid functions to improve angular tilt control. We adopted a uniform linear discrete function for better spatial sense and cognition, especially for the NV condition where there is no other cues to identify a target district in a 3D tilt space. We have found out that there are orientation eﬀects of tilt input, especially under the NV condition. We speculate that these results may be caused by the bisection method. Bisection is the simplest dividing method. We can utilize n (a positive integer) times of bisection to divide something into 2n equal parts. The numbers of 4 and 8 are both integer exponents of 2, under these dividing con- ditions the participants could perform trials more easily, so better performance was found at TOL4 and TOL8. Especially, the impact of dividing method was C. Liu et al. 18 magniﬁed under the NV condition, since the participants had no other cues be- sides dividing the space upon their own sense and cognition. As for the condition of TOL12, the axial directions also conformed to the least times of bisection. 6 Discussion The best performance in the axial directions may also be caused by our experience in other disciplines, e.g., the knowledge about system of rectangular coordinates from mathematics. 7 Conclusion and Future Work In our study, a quantitative experiment was conducted to determine the eﬀects of visual feedback on tilt control of a mobile phone and the upper bounds of human ability to control the tilt input under diﬀerent VFLs. The experimental results have some indications for tilt-based interaction design. First, partially visual feedback (PV ) performed not so well as fully visual feedback (FV ) and no visual feedback (NV ) in terms of both speed and accuracy. This reveals that PV has no help for tilt input. Second, the participants could distinguish at least 12 tilt orientation levels (TOLs) and 4 tilt magnitude levels (TMLs) under the FV condition. Third, the participants could distinguish 4 TOLs and 2 TMLs or 8 TOLs and 2 TMLs under the NV condition with an error rate tolerance of 10% or 15%, respectively. Four, the ability of the participants on tilt control was related with orientation direction. There exist some limitations of our work. First, the experiment was conducted when the participants were seated. But some external factors, e.g., motion sta- tus of the subjects, could inﬂuence the results. We had considered some other factors during the experiment design, but they had ﬁnally been excluded from the experiment to prevent it from becoming too complicated. Some other factors should be considered in our future work. Second, we only employed dwell time (1s) to conﬁrm a target selection, but diﬀerent selection modes may also have impacts on the results. In the future work, we will investigate impacts of diﬀerent selection modes. Third, tilt gestures have the potential to serve the blind, since the techniques support eyes-free interaction. We will explore the use of tilt for people with visual impairments. Fourth, we only utilized audio feedback under none visual feedback condition. But it is not clear whether there are some other kinds of feedback that are more suitable for eyes-free interaction. In our future work, we will explore diﬀerent feedback, e.g., tactile and auditory feedback, and ﬁnd out the most suitable feedback mechanism for the NV condition. References 1. 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W3031127341.txt	https://www.nature.com/articles/s41467-020-16168-x.pdf	en		hnRNP H/F drive RNA G-quadruplex-mediated translation linked to genomic instability and therapy resistance in glioblastoma	Nature communications	2,020	cc-by	16,225	ARTICLE https://doi.org/10.1038/s41467-020-16168-x OPEN hnRNP H/F drive RNA G-quadruplex-mediated translation linked to genomic instability and therapy resistance in glioblastoma 8 ✉, 1234567890():,; Pauline Herviou1,2,3,9, Morgane Le Bras1,2,3,9, Leïla Dumas1,2,3, Corinne Hieblot1,2,3, Julia Gilhodes4, Gianluca Cioci5, Jean-Philippe Hugnot6, Alfred Ameadan 7, François Guillonneau 7, Erik Dassi Anne Cammas 1,2,3 ✉ & Stefania Millevoi 1,2,3 ✉ RNA G-quadruplexes (RG4s) are four-stranded structures known to control mRNA translation of cancer relevant genes. RG4 formation is pervasive in vitro but not in cellulo, indicating the existence of poorly characterized molecular machinery that remodels RG4s and maintains them unfolded. Here, we performed a quantitative proteomic screen to identify cytosolic proteins that interact with a canonical RG4 in its folded and unfolded conformation. Our results identiﬁed hnRNP H/F as important components of the cytoplasmic machinery modulating the structural integrity of RG4s, revealed their function in RG4-mediated translation and uncovered the underlying molecular mechanism impacting the cellular stress response linked to the outcome of glioblastoma. 1 Cancer Research Center of Toulouse (CRCT), INSERM UMR 1037, 31037 Toulouse, France. 2 Université Toulouse III Paul Sabatier, 31330 Toulouse, France. d’Excellence “TOUCAN”, Toulouse, France. 4 Institut Universitaire du Cancer de Toulouse-Oncopole, 31100 Toulouse, France. 5 TBI, Université de Toulouse, CNRS, INRA, INSA, Toulouse, France. 6 INSERM U1051, Institute for Neurosciences, Hôpital Saint Eloi, Université de Montpellier 2, 34090 Montpellier, France. 7 Plateforme Protéomique 3P5, Université de Paris, Inserm U1016-institut Cochin, Labex GReX, 22 rue Méchain, 75014 Paris, France. 8 Department of Cellular, Computational and Integrative Biology (CIBIO), University of Trento Via Sommarive 9, 38123 Trento, Italy. 9These authors contributed equally: Pauline Herviou, Morgane Le Bras. ✉email: erik.dassi@unitn.it; anne.cammas@inserm.fr; stefania.millevoi@inserm.fr 3 Laboratoire NATURE COMMUNICATIONS \| (2020)11:2661 \| https://doi.org/10.1038/s41467-020-16168-x \| www.nature.com/naturecommunications 1 ARTICLE M NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-020-16168-x ost steps in the gene expression pathway are regulated by the ability of speciﬁc RNA regions to form duplexes and other types of RNA conformations involving both Watson-Crick and non-canonical interactions. RNA-binding proteins (RBPs) establish highly dynamic interactions with such RNA elements, creating functional ribonucleoprotein complexes that are essential for every step of post-transcriptional control, including mRNA processing, stability, transport and translation. Accumulating evidences showed that RBPs are dysregulated in cancers, with a major proportion altered in glioblastomas (GBM)1, one of the deadliest forms of brain cancer, and impact on the expression and function of oncogenic and tumorsuppressor proteins2. A detailed knowledge of the interactions between RBPs and their cancer-related RNA targets is vital to better understand tumor biology and potentially unveil new targets for anti-cancer therapy. Among the many unusual RNA conformations, RNA Gquadruplex (RG4) structures are intriguing not only because they possess unique properties and have been implicated in key cellular functions and gene expression mechanisms but also, and more importantly, their dysregulation has been proposed to have a tremendous impact on human diseases, including cancer3. RG4s are extremely stable structures formed by stacking of two or more G-quartets, each composed of four guanines interacting via Hoogsteen bonding. RG4 motif hotspots include both 5′ and 3′ untranslated regions (5′UTR and 3′UTR, respectively)4, suggesting an important role in mRNA translation. Their formation is regulated by intrinsic properties (e.g., the nature of the coordinating ion, the loop sequence and length, the number of Gquartets) and extrinsic interacting factors, with RBPs being critical regulators of RG4 conformation and function in cancer cells. This notion is supported by studies demonstrating the role of RG4-protein interactions on the expression of cancer-relevant genes3 as well as by afﬁnity proteomic approaches identifying RG4-binding proteins (or RG4-BPs)5–9 known to modulate multiple cancer traits. Important insights on the impact of RBPs on RG4 formation have been recently provided by highthroughput RG4 mapping studies which showed that RG4 formation is pervasive in vitro10,11 but not in cellulo10. This led to propose that RBPs might be critical to maintain RG4s unfolded in eukaryotic cells10. However, the notion of global in cellulo unfolding is in contrast with cellular imaging studies showing RG4 formation in cellulo as well as with functional in cellulo analysis of RG4-driven endogenous or reporter gene expression3. This view has been recently revisited by in vitro transcription experiments12 and in cellulo RG4s capturing approaches13 which provided evidence of transient RG4 formation. These observations, together with other ﬁndings suggesting that the rate of protein-RNA complex assembly is faster than RG4 structuration14, reinforced the view that RBPs play a major role in shifting RG4s toward an unfolded state, yet the RG4s dynamics and function remain poorly investigated. Recent unbiased afﬁnity proteomic approaches identiﬁed several RG4 interactors, including RBPs (e.g., hnRNP H, hnRNP F, FMRP) and RNA helicases (e.g., DDX21, DDX3X, DHX36)5–8. However, given that the strategy used in these studies consisted in comparing RBP binding either to folded G4s or to their mutated version (harboring substitutions of the Gs), the question of which RBPs bind the unfolded RG4s and of whether, how and by what extent they impact on post-transcriptional gene expression in cancer cells have not been fully addressed. Answering these questions is essential to gain a better understanding of the role of RBP-RG4 interactions in translational control where RG4s function as strong repressors by different poorly elucidated mechanisms15. Here, we identify hnRNP H and hnRNP F as important components of the cytoplasmic molecular machinery that 2 speciﬁcally bind RG4s in their unfolded state. Our ﬁndings establish a role for hnRNP H/F as translational regulators acting in synergy with the RNA helicase DHX36 and impacting the biology of GBM. This activity appears to be involved in the resistance mechanisms of GBM, possibly accounting for the failure of current treatments. Results Identiﬁcation of the protein machinery binding to folded or unfolded RG4s. Previous work demonstrated that the canonical RG4 sequence G3A2G3A2G3A2G3 (hereafter referred to as the G3A2) is highly prone to form a RG4 structure in vitro10,11 but remained largely unfolded when ectopically expressed in cells10. This led to propose that RNA helicases and RBPs unfold RG4s and maintain them in an unfolded state. To identify the protein machinery that recognizes RG4 forming G-rich sequences and modulates their function in mRNA translation, we used an unbiased proteomic approach based on RNA afﬁnity puriﬁcation of cytoplasmic proteins (refer to Supplementary Fig. 1a for fractionation control) with immobilized biotinylated RNAs followed by mass spectrometry (RP-MS). Unlike other studies using RP-MS to identify proteins bound to wild-type RG4-forming or mutated G-less sequences5–8, we compared afﬁnity enrichment between the G3A2 RNA (G3A2 WT) folded into a RG4 (as described in the Methods section) and its modiﬁed version (hereafter referred to as G3A2 7dG) in which replacement of guanines by 7-deaza-guanines prevented Hoogsteen base-pairing and RG4 formation (Supplementary Fig. 1b), as revealed by circular dichroism spectra (Supplementary Fig. 1c). Gel electrophoresis followed by silver staining displayed different complex protein patterns between the native and 7-deaza modiﬁed G3A2 RNAs, whereas mock pull-downs with control beads were remarkably clean (Supplementary Fig. 1d). Proteins bound to the G3A2 WT and 7dG RNAs were subjected to tryptic digestion followed by HCD-MS/MS allowing quantitative label free proteomic analysis of RNA-protein interaction data16. RG4-BPs (i.e., proteins binding to the G3A2 WT) and G-rich-BPs (i.e., proteins binding to the G3A2 7dG) were deﬁned by the ratio WT/7dG and high conﬁdence proteins (false discovery rate (FDR < 0.05)) were ranked according to an arbitrary 1.5-fold enrichment cutoff after subtraction of the background proteins resulting from nonspeciﬁc protein binding to the bait RNA sequences (Supplementary Data 1). This quantitative analysis performed with four biological replicates revealed 370 signiﬁcant G3A2 protein interactors (with 237 proteins found in all replicates), among which we experimentally characterized 328 RG4-BPs and 42 Grich-BPs. The RP-MS screen (Fig. 1a, Supplementary Data 1) selectively enriched known RG4-BPs, revealed RBPs that have not previously been reported to interact with RG4s and, more importantly, underscored the RBPs that preferentially bind folded or unfolded RG4s. As expected, RNA helicases were found preferentially associated to structured RG4s (Fig. 1a). We compared these results with a recent qualitative RP-MS data set6 identifying cytoplasmic proteins associated to the RG4 inhibiting NRAS mRNA translation17. Of the 370 high-conﬁdence proteins identiﬁed in our screen, 27 overlapped with the 80 high-conﬁdence proteins bound to the NRAS RG46, resulting in 343 additional cytoplasmic RG4 binders, of which 320 were assigned to speciﬁc functional pathways, including translation and RNA metabolism (Fig. 1b, c). In addition, the intersection of our RP-MS and the RNA-binding total proteome (using a compilation of recent RNA interactome capture methods18–22), revealed that 260 out of the 370 identiﬁed proteins were annotated as RBPs (Fig. 1b and Supplementary Data 2). It is noteworthy that several recently identiﬁed m6A-responsive RBPs (based on ref. 20) were found NATURE COMMUNICATIONS \| (2020)11:2661 \| https://doi.org/10.1038/s41467-020-16168-x \| www.nature.com/naturecommunications ARTICLE NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-020-16168-x a 10 8 RG4-BP G-rich-BP RBP 2 0 CHD4 SAMD4B LRRC59 PPIL4 IARS MATR3 RPL24 PPFIBP1 SEC61B HEXIM1 hnRNP K HMGB2 CAPRIN1 PARP12 RPS6 RPL23A DNAJC9 ZC3H4 UPF3B LARS EXOC4 AP2A1 TCOF1 AP2B1 HADHA 4 RBM12 MBNL1 KSRP FUBP1 hnRNP H3 hnRNP H2 MBNL2 HARS EIF4H HDLBP hnRNP H1 EEF1G VARS SLBP EEF1D CCT8 FUBP3 EEF1B2 CCT6A TARDBP CCT4 hnRNP F QKI SNRPD2 SRAS2 LFQ intensity (log2 WT/7dG) 6 –2 –4 RG4-BP Herdy DDX58 DDX3X SNRNP200 GBP1 DDX17 RG4-BP Herviou b DDX5 0 DDX6 Helicases DHX9 2 DHX36 LFQ intensity (log2 WT/7dG) –6 15 c 4 98 20 Perez-Perri/Gerstberger/ Queiroz/Trendel/Urdaneta 140 37.3% Reactome/KEGG pathways 120 38 Number of proteins RBP list 1 2 206 32 7 100 23.3% 80 60 12.2% 40 5% 20 G-rich-BP Herviou 4.4% 4.4% 3.8% 2.9% 0 Protein Infectious Cytosolic tRNA processing in disease aminoacylation ER (13) (42) (15) L1CAM Cell cycle PolII Metabolism interactions (17) transcription of RNA (10) termination (80) (15) Translation (128) d Input Mock G3A2 WT 7dG Mut DHX36 100 kDa DHX9 150 kDa DDX3X 75 kDa LARP1 150 kDa f 50 kDa hnRNP H/F KSRP G3A2: hnRNP H/F 75 kDa e WT Relative protein levels hnRNP I ** * * 7dG * NS NS * NS ** NS DHX9 DDX3X LARP1 * * * NS 0"! 1 ! "# " $"! $"# " Relative hnRNP H/F protein levels 7dG WT NS * ** * ( NT cPDS Mut Mut 42 21 7dG WT 6% cPDS * Mut Bait RNA NT WT Bait RNA 7dG Mut 00 DHX36 hnRNP KSRP H/F among the RG4-binders, highlighting a possible interplay between RG4s and epitranscriptomic modiﬁcations of RNAs23. To conﬁrm the RP-MS results and further validate preferential binding to the RG4 sequence or structure, RNA pull-down experiments were repeated by incubating cytoplasmic extracts with RNAs containing the G3A2 WT, the G3A2 7dG or the mutated (G-tract-less or G-less) G3A2 sequence (Mut) (Supplementary Fig. 1b). The recovered proteins, for which the silver stained SDS-PAGE gel displayed distinct protein proﬁles (Supplementary Fig. 1d), were then probed for some RBPs and NATURE COMMUNICATIONS \| (2020)11:2661 \| https://doi.org/10.1038/s41467-020-16168-x \| www.nature.com/naturecommunications 3 ARTICLE NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-020-16168-x Fig. 1 Analysis of RG4 sequence or structure binding preferences by RP-MS reveals hnRNP H/F binding to unfolded RG4s. a RNA afﬁnity chromatography using the G3A2 sequence either native (WT, which forms RG4s) or 7-deaza-modiﬁed (7dG, unable to form RG4s) and U251 cytoplasmic cell extracts, followed by mass spectrometry (RP-MS). Proteins identiﬁed from RP-MS were sorted according to the ratio G3A2 WT/7dG (top 20 were shown) and to whether they are RBPs (based on refs. 18–22) or RNA helicases. Data are presented as mean values ± SEM of n = 4 independent experiments, FDR < 0.05 (two-sided paired t-test). Highlighted in red are the different members of the hnRNP H/F subfamily. b Venn diagram showing the overlap of this study (Herviou, red and orange), which identiﬁed proteins bound to RG4s either folded (RG4-BPs) or unfolded (G-rich-BP), with the RG4-BPs identiﬁed in Herdy6 (blue) and the RBPs identiﬁed in at least 2 RNA capture methods18–22 (white). c Functional enrichment analysis of the identiﬁed high conﬁdence 343 factors not known as RG4 binders. d, e Validation of RP-MS by performing RNA afﬁnity chromatography using G3A2 WT, 7dG or Mut RNAs, followed by western blot analysis (d) quantiﬁed and normalized to the WT (e). Data are presented as mean values ± SEM of n = 5 independent experiments for hnRNP H/F and n = 3 independent experiments for the other proteins, P < 0.05, P < 0.005, NS: Non-Signiﬁcant (two-sided paired t-test). Bait RNA: RNAs retained on beads. Source data and exact P-values are provided as a Source Data ﬁle. f RNA afﬁnity chromatography using the G3A2 RNAs as in (d), treated with carboxypyridostatin (cPDS) or untreated (NT), followed by western blot analysis, quantiﬁcation and normalization of the hnRNP H/F protein levels to the control (hnRNP I). Data are presented as mean values ± SEM of n = 3 independent experiments, P-value = 0.02276 and P-value = 0.3228 for the WT and 7dG RNAs, respectively, NS: Non-Signiﬁcant (two-sided paired t-test). Shown is a representative result from n = 3 independent experiments. Source data are provided as a Source Data ﬁle. RNA helicases found to bind preferentially either the native or the 7dG G3A2 RNAs (Fig. 1d, e). We focused on proteins known to bind RG4s, as for instance DHX3624,25, DHX924, DDX3X6, and additional proteins, as KSRP or LARP1, whose function was not associated to RG4 binding. Similar bead-bound RNA elution proﬁles suggested that the differential interaction was not related to the amount of loaded RNA (Fig. 1d, f). As expected, RG4 helicases recently identiﬁed as translational regulators, speciﬁcally DHX3624, DHX924, DDX3X6, were enriched by pull-down with the G3A2 WT and showed less interaction with the G3A2 7dG or the Mut sequence, corroborating the requirement of a RG4 for RNA binding. Similar results were obtained for the translational regulator LARP1 (Fig. 1d, e), who was also found at the NRAS RG46, suggesting that this is an uncharacterized RG4-BP possibly binding high G-content 3′UTR motifs26. In contrast, the RBPs hnRNP H/F showed a remarkable selectivity towards the 7dG RNA but a weaker interaction with the G-less RNA (Fig. 1d, e), indicating that these proteins bind G-rich sequences incapable of RG4 folding. As observed for hnRNP H/F, KSRP (top 3 hit in Supplementary Data 1), a RBP previously reported to regulate miRNA biogenesis through binding G-rich motifs27, exhibited stronger binding to the 7dG RNA (Fig. 1d, e) but whether this factor is involved in the RG4 network will require further validation. Overall, these results extended the number of proteins binding the RG4-forming G-rich sequences and provided the ﬁrst comprehensive evidence of which proteins bind structured RG4s and which ones prefer to bind the G-rich sequence per se. To bring further insights into the role and mechanism of action of the machinery preferentially binding unfolded RG4s, we focused on two closely (structurally and functionally) related RBPs28–31, hnRNP H and hnRNP F (or hnRNP H/F), since these factors have been reported to regulate mRNA expression through binding RG4-forming sequences29,32,33 but their role in translation via these motifs or structures has not been investigated yet. As observed for the G3A2, hnRNP H/F binding to the NRAS RG417 depended on RG4 unfolding and the presence of Gstretches (Supplementary Fig. 2). It is interesting to note that the overall binding protein proﬁle was similar between NRAS and G3A2 but differed between cytoplasmic and total extracts (Supplementary Fig. 2b, c). The RG4 structuration-dependency of hnRNP H/F binding was further analyzed by RNA-pull down with RNA baits pre-incubated with either the small-molecule ligand carboxypyridostatin (cPDS) or pyridostatin (PDS) known to speciﬁcally stabilize cytoplasmic RG4s34 or RNA/DNA G4s35, respectively. We found that the binding of hnRNP H/F, but not that of the control polypyrimidine tract-binding protein hnRNP I (described in the Methods section), to both the G3A2 RG4 (Fig. 1f and Supplementary Fig. 3a) and NRAS RG4 4 (Supplementary Fig. 2d) was decreased upon cPDS or PDS treatment. Similar results were obtained by reversed pull-down (i.e., RBP/helicase immunoprecipitation of G3A2 RNAs (WT or 7dG), followed by RNA detection; Supplementary Fig. 3b, c) and surface plasmon resonance (Supplementary Fig. 3d, e), further validating that the binding of hnRNP H/F and helicases depend on RG4 structuration. hnRNP H/F localization and association with translationally active fractions. The ability of cytoplasmic hnRNP H/F to bind to unfolded RG4s prompted us to study the function and mechanism of action of these interactions in regulating mRNA translation in cancer cells. We focused on high-grade glioma or GBM, highly aggressive, angiogenic and treatment-resistant brain tumors, for the following reasons. First, previous studies showed that RBPs are highly dysregulated in GBM1, with hnRNP H/F being over-expressed both at the protein and mRNA level28,36. Then, the nuclear activity of these factors appeared to be involved in the pathogenesis and progression of malignant gliomas28. Finally, it is well known that mRNA translation dysregulation contributes to GBM progression and response to current therapeutic treatments37,38, yet the molecular mechanisms and therapeutic targets remain to be fully elucidated. To address whether hnRNP H/F drive translational control of genes contributing to GBM progression and treatment, we ﬁrst evaluated the expression level of hnRNP H/F in tumor and normal tissues from the TCGA database. We found that hnRNP H/F family members displayed higher expression levels in GBM compared to normal brain (Supplementary Fig. 4a), suggesting a potential role for both RBPs in GBM gene expression reprogramming. In addition, data from REMBRANDT (Repository for Molecular Brain Neoplasia Data), a publicly available dataset with information on tumor gene expression, treatment history, and survival, demonstrated that high hnRNP H or hnRNP F expression is correlated with poor survival (Supplementary Fig. 4b), indicating that hnRNP H/F are likely clinically relevant molecular target in GBM. To gain insight into the role of hnRNP H/F in translation regulation in GBMs, we ﬁrst addressed their speciﬁc localization by subcellular fractionation of three GBM cell lines (U251, LN18 and U87) that differ in their response to chemo- and radiotherapy treatments and in the mutational proﬁles (Supplementary Fig. 4c). In addition to being present in nuclear fractions, hnRNP H/F co-distributed with proteins associated with active translation (eIF4A) and was enriched in microsomal fractions, containing endoplasmic reticulum-associated proteins (Fig. 2a). This result is consistent with previous ﬁndings showing moderate to high cytoplasmic expression for both hnRNP H and hnRNP F, NATURE COMMUNICATIONS \| (2020)11:2661 \| https://doi.org/10.1038/s41467-020-16168-x \| www.nature.com/naturecommunications ARTICLE NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-020-16168-x a U251 C LN18 M N C M U87 N C M N 50 kDa hnRNP H/F 50 kDa eIF4A 150 kDa PERK 20 kDa Histone H3 Tubulin 50 kDa b c 60S 80S 80S NT NT OD 254 nm OD 254 nm 60S Puromycin 40S NP Input 7 8 9 P NP P 7 10 11 12 13 14 15 16 Puro: hnRNP H/F cPDS 40S 8 9 10 11 12 13 14 15 16 cPDS: – 50 kDa 50 kDa + 50 kDa 100 kDa – – 50 kDa + – hnRNP H/F 25 kDa RPL22 DHX36 – 150 kDa d DHX9 + – DDX3X + – 25 kDa + 100 kDa 1 150 kDa 75 kDa 75 kDa Relative protein quantity (log2) + hnRNP H/F cPDS/NT 0 –1 50 kDa –2 eIF4A + 11 50 kDa 12 13 14 15 EEA1 + 250 kDa 250 kDa 37 kDa – RPS6 37 kDa + Relative protein quantity (log2) RPL22 – 1 cPDS/NT 0 –1 11 12 13 14 15 Polysomal fractions depending on the tissues and on the normal/tumoral status39. Then, we performed polysomes proﬁling combined with immunoblotting to monitor the distribution of hnRNP H/F between translational inactive (non-polysomes, NP) and active (polysomes, P) fractions, in the absence or presence of puromycin, a drug that causes ribosome dissociation. We found that hnRNP H/ F and other RG4 helicases (e.g., DHX36, DHX9 and DDX3X) cosedimented with translating polyribosomes and that their association depended on polysome integrity (Fig. 2b). The cosedimentation proﬁles observed and their modiﬁcations after treatment with puromycin were similar to those of the initiation factor eIF4A. Speciﬁcally, the fraction of hnRNP H/F loaded on NATURE COMMUNICATIONS \| (2020)11:2661 \| https://doi.org/10.1038/s41467-020-16168-x \| www.nature.com/naturecommunications 5 ARTICLE NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-020-16168-x Fig. 2 hnRNP H/F subcellular localization and association with polysomal fractions. a Subcellular fractionation of GBM cell lines, followed by western blot analysis of hnRNP H/F, eIF4A (cytosolic and microsomal marker), PERK (microsomal marker), histone H3 (nuclear marker) and tubulin (cytosolic marker associated to microsomes). Nuclear (N), microsomal (M), and cytosolic fractions (C). Shown is a representative result from n = 2 independent experiments. Source data are provided as a Source Data ﬁle. b Polysome proﬁle of U251 cells untreated (NT) or puromycin treated (Puro), followed by western blot analysis from individual non-polysomal (NP) and polysomal (P) fractions by probing for hnRNP H/F, DHX36, DHX9, DDX3X, eIF4A. EEA1: negative control. RPS6: positive control. Shown is a representative result from n = 3 independent experiments. Source data are provided as a Source Data ﬁle. c As in b, except that cells were NT or treated with 20 μM carboxypyridostatin (cPDS) for 1 h, and probing for hnRNP H/F or RPL22 (negative control). Shown is a representative result from n = 2 independent experiments. Source data are provided as a Source Data ﬁle. d Repartition of hnRNP H/F proteins in polysomal fractions was quantiﬁed with n = 2 independent experiments. the polysomes was 5% and, similarly to Sauer et al.25, those of DHX36 and eIF4A were 9.5 and 7%, respectively. These results suggest the involvement of hnRNP H/F in the regulation of the initial steps of mRNA translation. Consistent with the observation that RG4 structuration reduced hnRNP H/F RNA-binding (Fig. 1), we observed that the cPDS- (or PDS- (Supplementary Fig. 5a)) induced stabilization of RG4s resulted in the dissociation of hnRNP H/F from translating ribosomes in U251 (Fig. 2c, d, Supplementary Fig. 5a) and U87 (Supplementary Fig. 5b) GBM cells. Taken together, these results suggest that hnRNP H/F localize to sites of active translation and associate to translating ribosomes in a way that depends on the ability of RG4s to adopt an unfolded conformation. Role for hnRNP H/F in translational regulation of DNA damage response genes. To demonstrate a functional role for hnRNP H/F in translational regulation, we transfected U87 or LN18 GBM cells with hnRNP H and/or hnRNP F speciﬁc or control siRNAs for 48 h, followed by quantiﬁcation of global protein synthesis rates by pulse-labeling with puromycin and immunoblotting using an anti-puromycin antibody (i.e., SUnSET assay). We found that hnRNP H/F silencing induced only minor reduction of global translation rates (Supplementary Fig. 6a–d). Consistent with this, the polysomal proﬁle was slightly altered by hnRNP H/F depletion (Fig. 3a, Supplementary Fig. 6e), indicating that cells deﬁcient in hnRNP H/F are not globally defective in protein synthesis. Neither apoptosis nor proliferation were affected under these treatment conditions (Supplementary Fig. 6f, g), suggesting that changes in translational efﬁciency after hnRNP H/F silencing were not directly related to these processes. Based on these results and our previous ﬁndings (Figs. 1 and 2), we reasoned that hnRNP H/F might selectively control translation of sub-groups of mRNAs containing RG4-forming sequences. To test this hypothesis, we ﬁrst mapped RG4-forming sequences within hnRNP H/F-binding regions in 5′UTRs, 3′UTRs and CDSs by combining the bioinformatic prediction of RG4 formation (using QGRS Mapper40) and the reanalysis of previously published in cellulo RNA-protein interactions using CLIP-seq (crosslinking immunoprecipitation (CLIP) combined with deep RNA sequencing) data41,42 (Supplementary Fig. 7a). Strikingly, hnRNP H/F bound an important fraction of RG4s over all the RG4s predicted in the transcriptome (11% of 5′UTR, 2.7% of CDS, and 11.4% of 3′UTR) (Fig. 3b). Similar results were obtained by intersecting experimentally validated RG4s (based on11) with hnRNP H/F CLIP-seq data (Supplementary Fig. 7b, c), although the magnitudes of the enrichment were different and reﬂected the shifted abundance of RG4s identiﬁed in the different regions of the mRNA by the rG4-seq method11 (Supplementary Fig. 7b). Overall, these results support the notion of widespread regulation of RG4-containing mRNAs by hnRNP H/F. In addition, RG4s were signiﬁcantly enriched in the binding regions of hnRNP H/F relative to random control sequences (Fig. 3c, Supplementary 6 Fig. 7d and Supplementary Data 3). Most hnRNP H/F sites in those regions contain a high-scoring RG4-forming sequence (Supplementary Fig. 7e), with hnRNP F sites being less dense but still highly enriched, especially in 5′UTRs (Fig. 3c). These results extend the notion of a physical link between hnRNP F and RG4s (recently investigated in ref. 32) to translational regulatory regions, but most notably underscore the extent of hnRNP HRG4 interactions, which has not been reported so far. Gene Ontology enrichment analysis showed that hnRNP H and hnRNP F bind RG4-containing RNAs associated with genes regulating cell stress response, including those involved in the response to DNA damage (DDR) (Supplementary Fig. 7f). This result is particularly relevant to GBM since chemo- and radio-resistance of these tumors is largely inﬂuenced by the expression of DDR genes43. We then asked whether RG4-containing mRNAs bound by hnRNP H/F were candidates for direct translational control by these factors. To this end, we performed polysomal fractionation of hnRNP H/F-depleted cells followed by RNA isolation from non-polysome (NP), light (LP) and heavy (HP) polysome fractions and RT-qPCR analysis. Based on our bioinformatic analysis (Fig. 3b, c and Supplementary Fig. 7), we selected 5 mRNAs involved in the DDR and/or playing a function in GBM that contained an hnRNP H/F binding site overlapping RG4-forming sequences. Among them, the mRNA encoding VEGF (vascular endothelial growth factor) was chosen as positive control due to its pivotal role in regulating tumor angiogenesis in human gliomas44. Also, the VEGF mRNA is regulated at the translational level by a variety of mechanisms relying on different sequence/ structure elements, including RG4s45. Furthermore, we previously demonstrated that RG4 stabilization strongly represses VEGF mRNA translation and protein expression in living cells45. The ability of these mRNAs to form RG4s was validated by performing RNA-immunoprecipitation (RIP) assays with cytoplasmic extracts and the BG4 antibody, known to recognize folded RG4s34. In agreement with the bioinformatic analysis of RG4 formation, we found that these mRNAs were prone to form RG4s in cellulo (Fig. 3d and Supplementary Fig. 8a). The translational efﬁciency of these mRNAs and the control HPRT mRNA, following hnRNP H/F silencing, was quantiﬁed either by analyzing the ratio HP/total RNA (Fig. 3e) or by measuring the distribution of each mRNA across the gradient (Supplementary Fig. 8b). We observed that hnRNP H/F depletion induced a signiﬁcant modiﬁcation in mRNAs association with translating polysomes, indicating a role of hnRNP H/F in both translational activation (MECP2 and PRR5) and repression (VEGF, USP1, CCNA2, BABAM1) (Fig. 3e and Supplementary Fig. 8b). Importantly, cPDS cellular treatments over short periods of time (1 h) also modiﬁed the translation efﬁciency of these targets (Fig. 3f), without affecting the mRNA amounts for all except the USP1 mRNA, for which the effect on transcripts levels was reversed compared to the translational effect (Supplementary Fig. 8c). For this target, and in agreement with previous ﬁndings obtained with the VEGF mRNA45, we further validated the direct effect of cPDS on RG4-dependent translation using USP1 RNA NATURE COMMUNICATIONS \| (2020)11:2661 \| https://doi.org/10.1038/s41467-020-16168-x \| www.nature.com/naturecommunications ARTICLE NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-020-16168-x a b siCtr c 6 4 2 0 NP LP 600 400 200 200 100 0 0 d 0 0 siH/F / siCtr 1.0 BG4 Relative mRNA quantity (log2 (HP)/(NP+LP+HP)) NS 4# * * NS NS NS * 0.5 NS 0.0 –0.5 0 –1.0 NT cPDS NT MECP2 10 3′UTR CDS * 6$ Fold enrichment over IgG and relative to HPRT 1 20 e * NS 30 2 HP 5′UTR IgG 3 –log10 p value 8 RG4 per Mb in CLIP F sites 10 –log10 p value 40S RG4 per Mb in CLIP H sites % of H/F-bound RG4/ total RG4 80S 2" hnRNP F 12 60S OD 254 nm hnRNP H siH/F cPDS VEGF NT cPDS NT USP1 cPDS BABAM1 NT * cPDS MECP2 PRR5 CCNA2 f g VEGF USP1 BABAM1 HPRT siCtr siH/F * * ** cPDS/NT 0.0 –0.5 * –1.0 * –1.5 * MECP2 PRR5 VEGF * NS 7dG Mut 1.0 0.5 0 WT ** USP1 BABAM1 CCNA2 Relative Rluc/Fluc activity of USP1 mRNA reporter in cellulo Relative mRNA quantity (log2 (HP)/(NP+LP+HP)) 0.5 HPRT reporters and in vitro translation assays (Supplementary Fig. 8d). It is important to note that hnRNP H/F depletion and cPDS treatment resulted in similar positive/negative effects in translation efﬁciency (Fig. 3e, f) that were consistent with the ability of cPDS to both diminish hnRNP H/F binding to RG4 forming sequences (Fig. 1 and Supplementary Figs. 1–3) and dissociate hnRNP H/F from translating ribosomes (Fig. 2c, d). To further explore the translational regulation mediated by hnRNP H/F bound to RG4 forming sequences, we transfected GBM U87 ((Fig. 3g) or U251, Supplementary Fig. 8e) cells with in vitrotranscribed USP1 RNA reporters containing the RG4 sequence RG4 in USP1 RNA reporter WT (USP1 RG4 WT), 7dG-modiﬁed (USP1 RG4 7dG) or mutated (USP1 RG4 Mut). We observed that hnRNP H/F silencing signiﬁcantly decreased the expression of the USP1 RG4 WT, but also, by a greater extent, that of the USP1 RG4 7dG, while leaving the USP1 RG4 Mut reporter unaffected (Fig. 3g, Supplementary Fig. 8e). Moreover, ligand-induced RG4-stabilization resulted in signiﬁcant inhibition of the USP1 WT, not the USP1 RG4 7dG, expression (Supplementary Fig. 8f). These functional effects fully mirrored both the efﬁciency of hnRNP H/ F binding to the RG4 RNAs (WT, 7dG, Mut) (Fig. 1d, e, Supplementary Figs. 2 and 3) and the effect of a RG4-stabilizing NATURE COMMUNICATIONS \| (2020)11:2661 \| https://doi.org/10.1038/s41467-020-16168-x \| www.nature.com/naturecommunications 7 ARTICLE NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-020-16168-x Fig. 3 hnRNP H/F drive mRNA translation of stress-response genes. a Polysome proﬁle of U87 cells treated with control (siCtr) and hnRNP H/F (siH/F) siRNAs. The positions of the 40S, 60S and 80S ribosomal subunits and non-polysomal (NP) and light (LP) and heavy (HP) polysomal fractions are indicated. b Fraction of RG4s (in 5′UTR, CDS, and 3′UTR regions) bound by hnRNP H/F over all RG4s predicted in the transcriptome. c Density of RG4s per Mb of hnRNP H and F binding sites, along with the -log10(P-value) of the enrichment with respect to random sites. d Immunoprecipitation (IP) of in cellulo RNA-protein complexes in U87 cells (cytoplasmic fraction) untreated (NT) or treated with 20 μM carboxypyridostatin (cPDS) for 2 h using the BG4 antibody or control IgG, followed by RT–qPCR analysis. The relative mRNA levels for each IP sample were normalized to the corresponding IP IgG and to the corresponding input sample and were plotted relatively to the HPRT mRNA (negative control). Data are presented as mean values ± SEM of n = 3 independent experiments. e As in a, but followed by RT–qPCR analysis from pooled NP, LP, HP fractions, for the indicated mRNAs and quantiﬁcation by analyzing the ratio HP/total mRNAs. Data are presented as mean values ± SEM of n = 3 independent experiments. f NP, LP, HP fractions were extracted from U87 cells NT or treated with 20 μM cPDS for 1 h and RT-qPCR was performed using primers for the indicated mRNAs. Quantiﬁcation and plot as in d. Data are presented as mean values ± SEM of n = 4 independent experiments. g Ratio of Renilla/Fireﬂy luciferase activities (Rluc/Fluc) determined using U87 cells treated with siCtr and siH/F siRNAs, followed by cotransfection with USP1 RNA reporters containing the RG4 unmodiﬁed (WT), 7dG-modiﬁed (7dG) or mutated (Mut) and an internal control mRNA encoding the Fluc. Data are presented as mean values ± SEM of n = 4 independent experiments. For all the panels, P < 0.05, P < 0.005, *P < 0.0005, NS: Non-Signiﬁcant (two-sided paired t-test). For a, d, e, f, g data and exact P-values are provided as a Source Data ﬁle. ligand on these interactions (Fig. 1f, Supplementary Figs. 2 and 3). The observations that the translation of the USP1 mRNA driven by the unfolded RG4 (7dG-modiﬁed) was insensitive to stabilizing ligands (Supplementary Fig. 8f) but much more responsive to hnRNP H/F loss compared to the USP1 RG4 WT (Fig. 3g, Supplementary Fig. 8e) suggest that the dynamic equilibrium between RG4s and linear G-rich sequences in cellulo results in low binding of hnRNP H/F to RG4s but, when preventing RG4 from folding, hnRNP H/F strongly bind the G-rich RNA to potentiate translation. In agreement with the dual role of RG4s in translation15, these results also demonstrate that, RG4 stabilization, resulting from either the absence of hnRNP H/ F or from the addition of RG4 stabilizing ligands, can either activate or suppress mRNA translation. Cooperation between hnRNP H/F and DHX36 in translational regulation. We then sought to deﬁne the molecular mechanism underlying the function of hnRNP H/F in translation regulation involving RG4 motifs. Previous work speculated on the possibility that hnRNP H/F-RG4 interaction could be facilitated by helicases, speciﬁcally DHX3646. This unwinding factor has been shown to bind RG4s both in vitro47 and in cellulo48, and to be required for optimal translation of two mixed lineage leukemia proto-oncogenes in synergy with Aven49. Furthermore, DHX36 is associated with translating polysomes (Fig. 2b) and regulates mRNA translation by speciﬁcally targeting RG4s24. To investigate the possibility that hnRNP H/F and DHX36 cooperate to regulate RG4-dependent translation, we ﬁrst performed coimmunoprecipitation assays using total (TE) or cytoplasmic (CE) extracts from U87 (Fig. 4a) or U251 (Supplementary Fig. 9a) GBM cells, in the presence of RNase and DNase to exclude nucleic acid-mediated interactions. In agreement with previous large-scale protein-protein interaction studies50, we found that hnRNP H/F co-immunoprecipitated with DHX36 in both total and cytoplasmic extracts, irrespective of which protein was immunoprecipitated (Fig. 4a and Supplementary Fig. 9a). Unlike DHX36, DHX9 and DDX3X were co-immunoprecipitated with hnRNP H/F in total extract, but weakly in the cytoplasmic extract, suggesting the formation of different RBP-helicase-RG4 complexes depending on their subcellular localization. However, neither hnRNP H/F nor DHX36 antibodies precipitated the translation initiation factor eIF4A (Fig. 4a and Supplementary Fig. 9a), recently proposed as an RG4 regulator51. To analyze the formation of ribonucleoprotein complexes involving hnRNP H/F, DHX36 and RG4-containing mRNAs, we performed a series of RIP assays using cytoplasmic extracts from U87 cells. In agreement with CLIP-data41,42, we found that the hnRNP H/F antibody immunoprecipitated endogenous mRNAs (Fig. 4b) 8 previously identiﬁed as hnRNP H/F translational targets (Fig. 3). Since these mRNAs were also found in DHX36 RIP samples (Fig. 4b), we concluded that hnRNP H/F-DHX36 interactions might be involved in the translation regulation of RG4-containing mRNAs. However, as shown above, even if the two proteins shared similar distribution proﬁles in polysomes (Fig. 2b) and in microsomes (Fig. 2a and Supplementary Fig. 9b), they display opposite RNA-binding preferences, with hnRNP H/F preferentially associated to unfolded RG4s while DHX36 showing an improved association to structured RG4s (Fig. 1 and Supplementary Data 1). By combining RIP with the depletion of either of these factors, we tested the possibility of a sequential mechanism that would ﬁrst unfold the RG4s and then keep them unfolded. As shown in Fig. 4c, d and Supplementary Fig. 9c, d, while DHX36 silencing reduced the binding of hnRNP H/F to RG4 targets, the recruitment of DHX36 was not affected by hnRNP H/F depletion, indicating that DHX36 is necessary for hnRNP H/F to bind to RG4s targets but not the opposite. Together, these results suggest that hnRNP H/F is recruited onto G-rich elements through direct interaction with DHX36 once the latter has bound and unfolded RG4s. To further test this model, we veriﬁed the in cellulo RG4 structuration after depletion of hnRNP H/F or DHX36 in LN18 (or U251), using the BG4 antibody and the treatment with cPDS as a positive control34. For both cell lines, we observed that depletion of either of the two factors induced a similar increase in the BG4 signal, which was RNAse-dependent and comparable in magnitude to that previously observed for DHX3625 (Fig. 4e, f and Supplementary Fig. 9e). Therefore, hnRNP H/F and DHX36 might cooperate to maintain RG4s in an unfolded conformation, thus facilitating or repressing mRNA translation depending on whether the speciﬁc RG4 plays a negative or a positive role in this process, respectively. Impact of hnRNP H/F-RG4 mediated translational regulation on the DDR. Based on the observation that a sub-group of mRNAs containing RG4 and interacting with hnRNP H/F are associated with stress response (Supplementary Fig. 7f), we hypothesized that the RG4 formation induced by hnRNP H/F silencing or RG4 stabilization (Fig. 4e, f and Supplementary Fig. 9e) could interfere with the cells’ ability to synthesize proteins playing a cytoprotective role, resulting in cellular DNA damage stress. Combined analysis of two markers of genetic instability, γH2AX (i.e., phosphorylated H2AX) and 53BP152, by immunoﬂuorescence microscopy revealed that hnRNP H/F removal from LN18 cells induced the appearance of nuclear foci of both factors (Fig. 5a). Consistent with this result, increased phosphorylation of H2AX was observed after treatment of LN18 cells with cPDS NATURE COMMUNICATIONS \| (2020)11:2661 \| https://doi.org/10.1038/s41467-020-16168-x \| www.nature.com/naturecommunications ARTICLE NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-020-16168-x a TE Input CE Mock IP IP H/F DHX36 Input Mock IP H/F IP DHX36 50 kDa hnRNP H/F DHX36 100 kDa 150 kDa DHX9 DDX3X 75 kDa eIF4A 50 kDa b IP IgG Fold enrichment over IgG and relative to HPRT IP H/F 6 IP DHX36 4 * ** * ** 2 0 MECP2 c IP hnRNP H/F: siCtr USP1 VEGF BABAM1 d siDHX36 CCNA2 IP DHX36: siCtr * *** * NS Fold enrichment over IgG and relative to HPRT Fold enrichment over IgG and relative to HPRT 2.0 1 0.5 0 siDHX36 NS NS NS 1.5 NS NS 1 0.5 0 MECP2 VEGF USP1 BABAM1 CCNA2 MECP2 e f siCtr siH/F siDHX36 VEGF USP1 – RNAse BABAM1 CCNA2 + RNAse siCtr + cPDS BG4 10 μm Normalized cytoplasmic BG4 foci per cell Cytoplasmic BG4 * * * 3 * * ** 2 NS 1 0 siCtr (Fig. 5b, c). Ionizing radiation radiotherapy (IR) and temozolomide chemotherapy (TMZ) are part of the standard treatment for GBM patients. The ability of hnRNP H/F to induce DDR markers prompted us to determine whether hnRNP H/F silencing could enhance the cytotoxic effects of IR or TMZ. To this end, we depleted hnRNP H/F in the chemo- and radio-resistant GBM cell line LN18 and either analyzed DNA damage repair by monitoring H2AX phosphorylation or measured the cell survival fraction using clonogenic assays, after treatment with IR (Fig. 5d, e and Supplementary 10a–c) or TMZ (Supplementary 10d, e). Our results showed that hnRNP H and/or hnRNP F silencing induced a marked increase in γ-H2AX after exposure to IR (Fig. 5d and siH/F siDHX36 siCtr + cPDS Supplementary Fig. 10a, b). Similar results were observed in the presence of the chemotherapeutic agent TMZ (Supplementary Fig. 10d). This reduced ability to cope with genotoxic stress correlated with reduced survival fraction upon IR (Fig. 5e and Supplementary Fig. 10c) or TMZ (Supplementary Fig. 10e). To deﬁne whether the LN18 cells’ ability to recover after genotoxic stress is dependent on translational regulation by hnRNP H/F, we analyzed the expression of γ-H2AX after treatment with TMZ, in the presence or absence of ectopically expressed hnRNP H/F and/ or the translational inhibitor cycloheximide (CHX). As indicated in Fig. 5f, we observed that γ-H2AX was increased after CHX treatment, indicating that the recovery from TMZ-induced DNA NATURE COMMUNICATIONS \| (2020)11:2661 \| https://doi.org/10.1038/s41467-020-16168-x \| www.nature.com/naturecommunications 9 ARTICLE NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-020-16168-x Fig. 4 hnRNP H/F collaborate with DHX36 to regulate RG4-dependent translation. a Immunoprecipitation (IP) of U87 total (TE) or cytoplasmic (CE) extracts, followed by western blot analysis and probing with the indicated antibodies. Shown is a representative result from n = 3 independent experiments. Source data are provided as a Source Data ﬁle. b IP of in cellulo RNA-protein complexes (RIP) in cytoplasmic extracts from U87 cells with the hnRNP H/F or DHX36 antibody, followed by RT-qPCR analysis of MECP2, VEGF, USP1, BABAM1, CCNA2, HPRT mRNAs. Data are presented as mean values ± SEM of n = 5 independent experiments for MECP2 and n = 3 independent experiments for the other mRNAs, P < 0.05, P < 0.005, *P < 0.0005, (two-sided paired t-test). c, d RIP as in b but after treatment with control (siCtr) siRNAs and either DHX36 (siDHX36) (c) or hnRNP H/F (siH/F) (d) siRNAs, followed by RT-qPCR analysis. The relative mRNA levels for each RIP sample in (b–d) were normalized to the corresponding IP IgG and input sample, and were plotted relatively to the HPRT mRNA. Data are presented as mean values ± SEM of n = 4 independent experiments, P < 0.05, P < 0.005, P < 0.0005, NS: Non-Signiﬁcant (two-sided paired t-test). e Immunoﬂuorescence experiments in LN18 cells using the BG4 antibody after treatment with siCtr, siH/F, siDHX36 siRNAs and carboxypyridostatin (cPDS). Phase contrast served to mark the cytoplasm and the nucleus. Panels with masked nuclear signal allow visualization of the BG4 signal in the cytoplasm. Shown is a single representative ﬁeld from one experiment over n = 2 independent experiments. f Quantiﬁcation of BG4 cytoplasmic foci number per cell observed in e. Number of cells counted in the -RNase conditions: 7132 cells for siCtr, 4945 cells for siH/F, 7877 cells for siDHX36, 6843 cells for siCtr+cPDS; Number of cells counted in the +RNase conditions: 6844 cells for siCtr, 5901 cells for siH/F, 6770 cells for siDHX36, 6893 cells for siCtr+cPDS. Data are presented as mean values ± SEM, statistical signiﬁcance was performed on the full cell populations P < 0.05, P < 0.005, P < 0.0005, NS: Non-Signiﬁcant (two-sided Kolmogorov–Smirnov test). For b–d, f source data and exact P-values are provided as a Source Data ﬁle. damage was dependent on protein synthesis. Overexpression of hnRNP H/F markedly reduced H2AX phosphorylation, suggesting that these factors play a role in the recovery after TMZinduced DNA damage. The observation that this effect is counteracted by CHX, led us to propose that hnRNP H/F control the cell response to a genotoxic insult by regulating the synthesis of proteins involved in the DDR. F and DHX36 (Fig. 6f). These results, together with the observation that hnRNP H/F and DHX36 interacted in the cytoplasm (Fig. 4a, Supplementary Fig. 9a), that both factors bound the USP1 mRNA and controlled its protein expression (Figs. 4b–d and 6b–d and Supplementary Fig. 11), strongly support a role for hnRNP H/F and DHX36 in coordinating USP1 expression in GBM. USP1 translational regulation by hnRNP H/F and DHX36 in GBM cells and tumors. To strengthen the notion that hnRNP H/ F and DHX36 cooperate to regulate the translation of RG4containing DDR genes, we decided to focus on the mRNA encoding USP1, an ubiquitin peptidase with important functions in DNA repair53,54. USP1 was chosen for further investigation also because its increased expression in GBM has been associated to resistance to treatments55, providing a rationale for USP1 inhibition as a potential therapeutic approach against GBM. Furthermore, the USP1 mRNA translational regulation has been recently identiﬁed as a major mechanism of cisplatin resistance in non-small-cell lung cancer, yet the molecular mechanisms remain to be investigated56. We ﬁrst validated that, as shown for hnRNP H/F (Fig. 3), USP1 is a DHX36 translational target by performing polysomal analysis combined RT–qPCR analysis of the USP1 mRNA. We found that the polysome proﬁle of U87 was only slightly altered by DHX36 depletion (Fig. 6a), in agreement with previous ﬁndings reporting a mRNA speciﬁc role of this helicase in translational regulation24. In agreement with polysomal analysis of USP1 mRNA translation regulation by hnRNP H/F (Fig. 3e and Supplementary Fig. 8b) or DHX36 (Fig. 6b and Supplementary Fig. 11a), silencing of either of these factors or treatment with cPDS reduced USP1 protein expression (Fig. 6c–e and Supplementary Fig. 11b), providing further support for a RG4-dependent translational mechanism in which both hnRNP H/F and DHX36 cooperate to activate USP1 protein synthesis. In addition, loss of DHX36 or hnRNP H/F induced an increase in protein ubiquitination, in agreement with USP1 deubiquitinating function (Fig. 6c). Finally, to investigate the potential clinical importance of our ﬁndings, we analyzed the expression of hnRNP H/F, DHX36 and USP1 in human glioma patient tissues. Gliomas are classiﬁed into low-grade (LGG) types with slow growth, and high-grade types (HGG) (or GBM), with fast growth and spread into normal brain tissue57. Analysis of the protein expression of the three factors in four LGG and three GBM human tumor samples revealed that hnRNP H/F, DHX36 and USP1 were markedly more expressed in GBM compared to LGG. In HGG, the ﬂuctuation in the protein expression of USP1 appeared to correspond to that of hnRNP H/ Discussion Recent data proposed that RG4s tend to massively form in vitro10,11, in accordance with their great stability, but their in cellulo formation was proposed to be highly dynamic due to the presence of a protein machinery that drive them to an unfolded state10,13. In contrast to previous RP-MS data sets5–9, we were able to capture and identify proteins binding to folded and unfolded RG4s by comparing native and 7dG-substituted G3A2 RNAs. Of note, incorporation of 7dG was instrumental in the identiﬁcation of functionally relevant G4s in long RNAs58. Our RP-MS screen (Supplementary Data 1) selectively enriched several RNA helicases (e.g., DHX36, DHX9, DDX3X, DDX5, DDX17) (Fig. 1), reinforcing the concept of a dynamic equilibrium between the formation and resolution of RG4 structures. Surprisingly, while eIF4A, who was previously found to be required for translation of two-quartet RG4-forming (CGG)4 motifs51, did not associate with RG4s (as in ref. 6), its cofactor, eIF4H, selectively bound the 7dG G3A2 RNA. In agreement with59, eIF4H could help to destabilize the RG4 by binding to the newly formed single-stranded region after partial strand structure unfolding by eIF4A. However, the observation that hnRNP H/F did not interact with eIF4A (Fig. 4a and Supplementary Fig. 9a) and bind G triplets60 susceptible to structure in three-quartet RG4s, suggests the intriguing possibility that the requirement of a speciﬁc helicase-RBP pairs (eIF4A-eIF4H or DHX36-hnRNP H/F) depends on the number of quartets stacked to form RG4s. The intersection of our RP-MS data with the NRAS RG4-binding cytoplasmic proteome6 and the RNAbinding total proteome18–22 (Supplementary Data 2) revealed cytoplasmic RBPs whose function was not associated to RG4binding, including known translation factors, such as LARP126. We also identiﬁed additional RG4-binding proteins, including known RNA-interactors but also proteins that have not been annotated as RBPs, thus extending the number of proteins binding the RG4-forming G-rich sequences (Fig. 1, Supplementary Data 1 and 2). Future studies will be needed to fully characterize the RG4/G-rich binding proteome in terms of speciﬁcity, selectivity, RG4/G-rich topology and mode of binding (direct or indirect). 10 NATURE COMMUNICATIONS \| (2020)11:2661 \| https://doi.org/10.1038/s41467-020-16168-x \| www.nature.com/naturecommunications ARTICLE NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-020-16168-x b a cPDS (μM): siH/F siCtr 1000 750 37 kDa c 500 250 0 10 μm γH2AX * 6 4 2 53BP1 0 cPDS (μM): e Time post-IR (h): 0 2 6 Survival fraction d Ctr H/F 24 0 2 6 10 GAPDH * DAPI siRNA : 2 15 kDa Relative γH2AX protein levels Mean Intensity 53BP1 γH2AX * siCtr siH/F 0 γH2AX 24 hnRNP H/F 50 kDa γH2AX 15 kDa GAPDH 37 kDa 2 10 siCtr 150 siH/F 100 50 * 0 0 f 0 2 IR Dose (Gy) 4 Time post-TMZ (h) CHX: 0 3 – pICE 1 0.88 +/– 0.07 1 1.05 +/– 0.38 1 0.50 +/– 0.11 1 1.20 +/– 0.08 + γH2AX Flag H/F – + pICE Flag Flag H/F 15 kDa 15 kDa 15 kDa – 50 kDa + 50 kDa – 50 kDa + 50 kDa – 37 kDa + 37 kDa – 37 kDa + 37 kDa pICE GAPDH 15 kDa Flag H/F More importantly, our work underscored the RBPs that preferentially bind unfolded RG4s, which included all hnRNP H/F family members (Supplementary Data 1). This result is consistent with RNA-protein interaction studies using either puriﬁed recombinant proteins14,61 or extracts from GBM (U87) cells33 showing that hnRNP H33 and hnRNP F14,61 preferentially binds linear G-tracts. The Drosophila hnRNP H/F homolog, Glorund, also recognizes G-tracts RNA in a single-stranded conformation62. In contrast, other sets of studies demonstrated that hnRNP H and/or hnRNP F29,32 bind RG4s, but not the mutated version, and that the small molecule TMPYP4 modulates this interaction29,32. To reconcile this whole set of results, and based on the observation that hnRNP H/F binding is modulated by DHX36 silencing but not the opposite (Fig. 4), we propose a twostep mechanism of binding in which RNA helicases ﬁrst resolve RG4s and then recruit hnRNP H/F driving their binding to the linear G-rich regions. Thus, our ﬁndings reﬁne the model of RBP recruitment by RNA helicases recently proposed49 by deﬁning the RG4 folding status in the regulatory mechanism. A key question regarding the mechanistic of translational regulation was whether hnRNP H/F simply bind unfolded RG4s or had a function once bound to the linear G-rich regions. The last hypothesis is NATURE COMMUNICATIONS \| (2020)11:2661 \| https://doi.org/10.1038/s41467-020-16168-x \| www.nature.com/naturecommunications 11 ARTICLE NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-020-16168-x Fig. 5 hnRNP H/F drive genomic instability and therapy resistance. a Immunoﬂuorescence experiments in LN18 cells using the γ-H2AX, 53BP1 antibodies and DAPI staining. Mean intensities of γ-H2AX and 53BP1 in 2322 cells were plotted; the bottom and top of the box present the ﬁrst and third quartile, respectively; the band inside the box shows the mean and the whiskers show the upper and lower extremes. Statistical signiﬁcance was performed on the full cell populations. n = 2322 cells examined. Shown is a single representative ﬁeld from one experiment over n = 2 independent experiments. For γ-H2AX: *P-value = 4.26e-10, for 53BP1: P < 2.2e-16 (two-sided Mann & Whitney test). b Western blot analysis of γ-H2AX in LN18 cells treated with dose scale of carboxypyridostatin (cPDS) for 24 h. Shown is a representative result from n = 3 independent experiments. c Quantiﬁcation of the γ-H2AX levels in LN18 treated with cPDS normalized to GAPDH levels and plotted relatively to the untreated condition. Data are presented as mean values ± SEM of n = 3 independent experiments, P-value = 0.0157 and P-value = 0.0457 for the 2 µM and 10 µM cPDS treatment respectively (two-sided paired t-test). d Quantiﬁcation of DNA repair kinetics by western blot analysis of γ-H2AX after 4 Gy γ-irradiation in LN18 cells treated with control (siCtr) or hnRNP H/F (siH/F) siRNAs. Shown is a representative result from n = 2 independent experiments. e Plating efﬁciency assays measuring the cell survival fraction in LN18 treated with siCtr or siF siRNAs and submitted to a radiation dose scale. Data are presented as mean values ± SEM of 6 wells, P-value = 0.0003 and P-value = 0.0006 for the 2 Gy and 4 Gy dose, respectively (two-sided paired t-test). f Quantiﬁcation of DNA repair kinetics by western blot analysis of γH2AX after temozolomide (TMZ) treatment in LN18 cells transfected with an empty plasmid (pICE) or a plasmid expressing Flag-hnRNP H/F. Shown is a representative result from n = 2 independent experiments. For all panels, source data are provided as a Source Data ﬁle. supported by our results showing that unfolded RG4s (7dG) still require the presence of hnRNP H/F for their function in translational regulation (Fig. 3g and Supplementary Fig. 8e). While our results suggest that hnRNP H and hnRNP F behave similarly in their interactions (RNA-protein (Fig. 1) or protein-protein (Fig. 4)) and function (Fig. 3) (as previously reported29,30), recent data showing that the two factors do not fully share the same set of protein interactors50, raise important questions about the possibility of differential translational effects discernable at the level of individual mRNAs or in speciﬁc translational compartments (cytosol versus microsomes). Finally, DHX36 and DHX9 were shown to stimulate mRNA translation by unfolding RG4s at upstream open reading frames (uORFs)24. These results together with our ﬁndings support interesting future investigations to determine whether hnRNP H/F are involved in this regulatory mechanism. In addition to highlighting the possibility that this mechanism may be important for splicing32 or polyadenylation29,46, our study extends the functions of hnRNP H/F to translational regulation and assigns to this mechanism a key role in the regulation of genes involved in resistance to treatments in GBM (Fig. 6). Although further work is needed to understand and characterize the full hnRNP H/F translatome, we found that RG4s are overrepresented in hnRNP H/F-binding sites at translational regulatory regions of mRNAs involved in pathways associated to genome instability and DNA damage and that hnRNP H/F bind an important fraction of predicted (Fig. 3b) or experimentally validated RG4s (based on ref. 11) (Supplementary Fig. 7c). Therefore, we predict that hnRNP H/F drive a substantial part of the RG4-dependent translational regulation and impact on the maintenance of genome integrity. In line with this view, RG4 stabilization by hnRNP H/F silencing or treatment with cPDS, induced the expression of markers of genome instability (Fig. 5). Although it could not be excluded that these effects are associated with the nuclear functions of hnRNP H/F29,46, we provided evidence that the link between hnRNP H/F and genome stability depends in part on mRNA translational regulation (Fig. 5). Moreover, hnRNP H/F inhibition not only induced but also enhanced chemo- and radio-therapy-induced DNA damage correlated with reduced cell survival (Fig. 5, Supplementary Fig. 10), indicating that targeting the RG4-dependent and hnRNP H/F-sensitive regulatory mechanism sensitizes cancer cells to treatments currently used to treat GBM patients (Fig. 7). Mining GBM TCGA and REMBRANDT data sets (Supplementary Fig. 4) as well as analyzing the protein expression in human glioma protein samples (Fig. 6), we found that hnRNP H/F is increased in GBM and correlates with poor survival, extending the notion of a key role of hnRNP H/F family members in cancer development and progression63. Our results support a model (Fig. 7) in 12 which hnRNP H/F overexpression in GBM coordinately regulate the translation of RG4-containing mRNAs encoding proteins involved in maintaining genome stability and in the response to genotoxic damage. The observation that 74 mRNAs coding for stress response factors are targeted by both hnRNP H/F and DHX36 (Supplementary Fig. 12) opens up new avenues for future research to investigate whether and how these regulations induce adaptive changes crucial for tumor cell survival during treatment and the development of resistance. Our results not only extend the notion of a link between G4 and genomic instability64 to mRNA translational regulation but also associates it with a role in resistance to treatments in GBM. Given that 1) our results were similar regardless of the GBM cell line (e.g., Fig. 2a, c, d and Supplementary Fig. 5b; Fig. 3g and Supplementary Fig. 8e, Fig. 5d and Supplementary Fig. 10b) or the cancer cell-type (Supplementary Fig. 13), 2) hnRNP H/F were found to be deregulated in many tumors (Supplementary Fig. 14), and 3) hnRNP H/F RG4containing mRNA targets signiﬁcantly enriched genes associated to GBM (adjusted P-value = 0.03284 and 0.001729 for H and F targets, respectively) but also to other cancers, including breast (adjusted P-value 0.033 and 1.2E-06) and ovarian cancers (adjusted P-value 0.013 and 1.8E-05), we propose that the link between hnRNP H/F and cancer mediated by RG4-dependent translational regulation could apply to other cancer cells and tumors, thus making hnRNP H/F a potential target for therapeutic intervention. Overall, our results support the notion that hnRNP H/F are an essential regulatory hub in GBM networks that drives translational control of RG4-containing genes contributing to GBM progression and response to treatments. Moreover, our RP-MS screen raises interesting future investigations to determine how modulation of RG4 structural integrity impacts cellular functions related to cancer hallmarks. Methods Cell culture and treatment. Glioblastoma cells (LN18, ATCC CRL-2610; U251MG ECACC #;09063001 U87, SIGMA, #89081402-1VL) were grown in DMEM media (4.5 g/l glucose) supplemented with 10% FBS, 2 mM L-glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin. Cells were tested for mycoplasma contamination by PCR. Cells were incubated/exposed at 37 °C with: 20 µM PDS (Selleckchem S7444) or 20 µM cPDS (Sigma-Aldrich SML1176) or 10 µM PhenDC3 (Polysciences, #26000-1) for the indicated time, 100 µg/ml Puromycin (Sigma P8833) for 1 h, 500 µM or dose scale of TMZ for 24 h, 4 Gy or dose scale of γ-irradiation (Gammacell 40 Exactor). Cell transfection. siRNAs were transfected using the Lipofectamine RNAiMAX (Life Technologies) according to the manufacturer’s instructions. In brief, cells were reverse-transfected with 2.5 nM siRNA for 48 h. siRNA oligonucleotides Control (5′-GGUCCGGCUCCCCCAAAUG dTdT-3′), against hnRNP H (5′-GG UAUUCGUUUCAUCUACA dTdT-3′), hnRNP F (5′-GGUGUCCAUUUCAU CUACA dTdT-3′) and DHX36 (5′-GGUGUUCGGAAAAUAGUAA dTdT-3′) NATURE COMMUNICATIONS \| (2020)11:2661 \| https://doi.org/10.1038/s41467-020-16168-x \| www.nature.com/naturecommunications ARTICLE NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-020-16168-x a siCtr b siDHX36 40S NP c Relative mRNA quantity (log2 (HP)/(NP+LP+HP)) 80S siRNA: LP –0.4 –0.6 HPRT d 50 kDa DHX36 100 kDa 250 kDa 100 kDa 70 kDa 55 kDa Ubiquitin 40 kDa 25 kDa GAPDH 1.0 0.8 * 0.6 0.4 0.2 0.0 siRNA: Ctr H/F DHX36 37 kDa f e GAPDH –0.2 150 kDa hnRNP H/F USP1 0 USP1 Ctr H/F DHX36 USP1 cPDS (μM): 0 0.2 HP Relative USP1 protein level normalized to USP1 mRNA level OD 254 nm 60S 2 Low Grade Gliomas High Grade GBM USP1 150 kDa DHX36 100 kDa 10 150 kDa 37 kDa 50 kDa hnRNP H/F 50 kDa Actin Fig. 6 hnRNP H/F and DHX36 regulate USP1 translation in glioblastoma cells and tumors. a Polysome proﬁle of U87 cells treated with control (siCtr) and DHX36 (siDHX36) siRNAs. b As in a, followed by RT–qPCR analysis from pooled non-polysomal (NP), light (LP) and heavy (HP) polysomal fractions, using speciﬁc primers for USP1 and HPRT mRNAs, and quantiﬁcation by analyzing the ratio HP/total mRNAs from n = 2 independent experiments. Source data are provided as a Source Data ﬁle. c Western blot analysis of USP1 and ubiquitination in U87 cells treated with siCtr, siRNAs against hnRNP H/F (siH/ F) or DHX36 (siDHX36). Source data are provided as a Source Data ﬁle. d USP1 protein levels in c were normalized ﬁrst to GAPDH protein levels and then to USP1 mRNA levels and plotted relatively to the siCtr condition. Data are presented as mean values ± SEM of n = 3 independent experiments, P-value = 0.0291 and P-value = 0.05 for siH/F and siDHX36 respectively (two-sided paired t-test). e Western blot analysis of USP1 in LN18 cells treated with carboxypyridostatin (cPDS) dose scale for 24 h. Shown is a representative result from n = 3 independent experiments. Source data are provided as a Source Data ﬁle. f Western blot analysis of USP1, DHX36 and hnRNP H/F levels in protein extracts from Diffuse Low Grade Gliomas (Grade II) and High Grade GBM (grade IV). Shown is a representative result from n = 3 independent western blot. Source data are provided as a Source Data ﬁle. were synthesized by SIGMA. For DNA plasmid transfections, 3.7 µg of plasmids was transfected in 60 mm diameter dishes using jet-PEI reagent (Polyplus) according to the manufacturer’s instructions. For Luciferase mRNA transfections, 250 ng of reporter mRNA was transfected in 48-well plates using lipofectamine 2000 reagent according to the manufacturer’s instructions. Cells were subsequently incubated at 37 °C for 48 h or 16 h following DNA plasmid or mRNA reporter transfections respectively, before harvesting and analysis. resuspended in 500 µl of lysis buffer A) was transferred to a 5-ml round-bottom tube and 50 µl of detergent mix (3.3% (w/v) sodium deoxycholate, 6.6% (v/v) Tween 40) were added. After incubation on ice for 5 min, the supernatantpostnuclear fraction was recovered (perinuclear fraction). The pellet-nuclear fraction (washed with buffer A) was resuspended in 500 µl of lysis buffer A supplemented with 0.1% SDS and sonicated. After centrifugation at 1000 g (4 °C) for 5 min, supernatant (nuclear fraction) was transferred into a fresh tube. Cell fractionation. For cell fractionation, cells were gently resuspended in 500 µl of hypotonic lysis buffer (10 mM Tris pH 8.0, 1.5 mM MgCl2, 10 mM NaCl, 1 mM DTT) and vortexed for 4 s. After centrifugation at 1000 g (4 °C) for 5 min, supernatant (cytosolic fraction) was recovered. Pellet fraction (washed twice with hypotonic lysis buffer) was resuspended in 500 µl lysis buffer A (10 mM Tris pH 8.0, 140 mM NaCl, 1.5 mM MgCl2, 0.5% NP40, 1 mM DTT). The supernatant (microsomal fraction) was recovered. Pellet-nuclear fraction (washed twice and Mass spectrometry. Proteins were lysed and denatured in Tris 50 mM pH 8.5 and SDS 2% while disulﬁde bridges were reduced using TCEP 10 mM and subsequent free thiols groups were protected using chloroacetamide 50 mM for 5 min at 95 °C. Proteins were trypsin-digested overnight using the suspension trapping (S-TRAP) method to collect peptides as described in65. Eluted peptides were vaccum-dried while centrifuged in a Speed Vac (Eppendorf). C18 liquid nanochromatography and Mass Spectrometry (MS) nanoﬂowHPLC and MS analyses were performed at NATURE COMMUNICATIONS \| (2020)11:2661 \| https://doi.org/10.1038/s41467-020-16168-x \| www.nature.com/naturecommunications 13 ARTICLE NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-020-16168-x Low hnRNP H/F hnRNP H/F High hnRNP H/F DHX36 DHX36 hnRNP H/F G G G Translation DDR mRNAs Translation DDR mRNAs Sensitive Resistant Glioblastoma Fig. 7 Model for the role of hnRNP H/F-RG4 interactions in regulating mRNA translation of mRNAs linked to GBM response to treatments. hnRNP H/F expression levels in GBM modulate the RG4-dependent mRNA translation impacting the DDR response involved in the response to standard GBM treatments (radiotherapy and chemotherapy). The underlying mechanism involves the binding of the helicase DHX36 that unwinds the RG4s, enabling hnRNP H/F to associate with unfolded RG4s and maintain them linear. the 3P5 proteomics facility (University de Paris) using an U3000 RSLC system hyphenated to an Orbitrap fusion MS (all from Thermo Fisher Scientiﬁc). All mobile phases are made with milliQ-grade H2O produced with a milliQ integral-3 (from Merck-Millipore). Peptides were solubilized in 10 µl of 0.1% triﬂuoroacetic acid (TFA) and 10% acetonitrile (ACN). 1 µl was loaded, concentrated, and washed for 3 min on a C18 reverse-phase precolumn (3-µm particle size, 100 Å pore size, 75-µm inner diameter, 2-cm length; Thermo Fischer Scientiﬁc) with loading solvent containing 0.1% TFA and 2% ACN. Peptides were separated on a C18 reverse phase resin (2-µm particle size, 100 Å pore size, 75-µm inner diameter, 25-cm length; Thermo Fisher Scientiﬁc) with a 35-min binary gradient starting from 99% of solvent A containing 0.1% formic acid and ending in 40% of solvent B containing 80% ACN, 0.085% formic acid. The mass spectrometer acquired data throughout the elution process and operated in a data-dependent scheme with full MS scans acquired with the Orbitrap, followed by as many MS/MS ion trap HCD spectra 5 s can ﬁt (data-dependent acquisition with top speed mode: 5-s cycle) using the following settings for full MS: automatic gain control (AGC) target value: 2.10e5, maximum ion injection time (MIIT): 60 ms, resolution: 6.10e4, m/z range 350–1500. For HCD MS/MS: Quadrupole ﬁltering, Normalised Collision Energy: 30. Ion trap rapid detection: isolation width: 1.6 Th, minimum signal threshold: 5000, AGC: 2.10e4, MIIT: 100 ms, resolution: 3.10e4. Peptides with undeﬁned charge state or charge state of 1 or over 7 were excluded from fragmentation, a dynamic exclusion time was set at 30 s. Identiﬁcations (protein hits) and quantiﬁcations were performed by comparison of experimental peak lists with a database of theoretical sequences using MaxQuant version 1.6.2.1066. The databases used were the human sequences from the curated Uniprot database (release June 2018) and a list of in-house frequent contaminant sequences. The cleavage speciﬁcity was trypsin’s with maximum 2 missed cleavages. Carbamidomethylation of cysteines was set as constant modiﬁcation, whereas acetylation of the protein N terminus and oxidation of methionines were set as variable modiﬁcations. The false discovery rate was kept below 5% on both peptides and proteins. The “match between runs” (MBR) option was allowed with a match time window of 1 min and an alignment time window of 30 min. For statistical analysis, data were imported into the Perseus software version 1.6.1.167. Reverse and contaminant proteins were excluded from analysis. LFQ intensity data were transformed into log2. Samples with at least 3 valid LFQ values of intensity per condition are selected. Imputation was performed on the missing values. Where initial data were insufﬁcient in one condition but enough data in the other condition, the imputation step allowed ratio calculation for all eligible hits (i.e. at least 3 valid values in at least one group). We imputed missing data using a random value comprised in the lowest range of LFQ intensities obtained in MaxQuant with the following settings: 0.3 as gaussian width relative to the standard deviation of measured values, and 1.8 as downshift factor (default perseus values).The proteins were selected as differential if their qvalues remained under 0.05 after a permuted FDR test (column x of the Supplementary Data 1). The reproducibility between each replicate was evaluated by hierarchical clustering analysis of protein expression (Euclidean distance) or 14 Principal Component Analysis (Supplementary Data 1). Log2 of the expression values were used for this analysis. RNA chromatography. 200 μg (WB analysis) or 400 μg (RP-MS analysis) of the U251 cytoplasmic (cytosolic + microsomal fractions (as described in “Cell fractionation” of the Methods section) were precleared with 20 μl of streptavidin acrylamide beads (Thermo Fisher Scientiﬁc) in the binding buffer containing 20 mM Tris pH 8, 1 mM DTT, 100 mM KCl, 0.2 mM EDTA for 1 h at 4 °C. For RG4 formation, 1 µg (WB analysis) or 3 µg (MS analysis) of in vitro-transcribed biotinylated RNAs were heated to 95 °C for 5 min in one volume of 1× phosphatebuffered saline supplemented with 2 M KCl and cooled down at room temperature. Biotinylated RNAs were then ﬁxed on 10 μl of streptavidin acrylamide beads by incubation in the binding buffer for 1 h at 4 °C. For PDS/cPDS experiments, 10 µM of PDS/cPDS were then added to the RNA-beads mix and incubated for 30 min. The RNA ﬁxed on beads was then combined to the precleared extracts for 1 or 3 h at 4 °C, for PDS/cPDS and untreated experiments respectively. The beads were collected by centrifugation, washed ﬁve times with 1 ml of the binding buffer, resuspended in 30 μl of elution buffer (50 mM Tris pH 8.0, 1% SDS), and boiled for 10 min. After centrifugation, the supernatant was collected. 1 µL was kept for RNA detection and the rest was loaded onto an SDS–PAGE gel and analyzed by western blot or used for MS analysis. In vitro transcription. RNAs used in RNA chromatography experiments were transcribed using the MEGAscript Kit (Invitrogen AM1333) as per manufacturer’s instructions. 7.5 mM ATP/CTP, 6.75 mM UTP, 0.75 mM biotinylated UTP (Biotin16-UTP, Lucigen BU6105H) and either 7.5 mM GTP or 6.75 mM 7-deazaguanine (TriLink N-1044) plus 0.75 mM GTP was used. For luciferase reporter mRNAs, m7G-cap was added using the Vaccinia capping system (M208S NEB) kit according the manufacturer’s instructions. To generate the DNA templates to synthetize the luciferase reporter mRNAs, oligonucleotides G3A2 WT, G3A2 Mut, NRAS WT, NRAS Mut were annealed and cloned in the pSC-B-amp/kan plasmid from the Strataclone Blunt PCR cloning kit, then digested by NheI and puriﬁed. All oligonucleotide sequences are available in the Supplementary Table 1. RNA concentration was determined using the Clariostar BMG and software v.5.21 R4, Labtech and MARS Clariostar Analysis Software v.3.20 R2. In vitro and in cellulo analysis of translational activity. For the in vitro translational activity analysis, 100 ng of in vitro transcribed luciferase Renilla reporter mRNAs (WT and 7dG) were preincubated 30 min at room temperature with increasing amount of cPDS. RRL (Flexi Rabbit Reticulocyte Lysate kit) were added to a ﬁnal volume of 10 µl and the lysates were incubated 90 min at 30 °C. 5 µl of the reaction were used for the luciferase assay. For the in cellulo IRES activity analysis, the U87 or U251 cells transfected with luciferase Renilla and Fireﬂy reporter mRNAs were harvested in 100 µl of Passive Lysis Buffer (Promega). 10 µl of this extract were analyzed with the luciferase assay. CD spectroscopy. For the spectroscopy measurements, RNAs were prepared in buffers containing 10 mM Tris-HCl (pH 7.4), 0.1 mM EDTA in the presence of 100 mM KCl and annealed by heating to 95 °C and then cooling slowly to room temperature. CD of RNAs was determined at 20 °C by a Jasco J-815 spectropolarimeter equipped with a temperature controller. CD spectra ranging from 190 to 350 nm was recorded in a 1-mm path length cuvette, in triplicates, averaged and buffer subtracted. Silver staining. Proteins co-puriﬁed by RNA chromatography or present in wholecell lysates (inputs) were separated by SDS-PAGE and subjected to silver staining using Pierce Silver Stain Kit (Thermo Scientiﬁc, 24612) according to manufacturer’s instructions. Reverse afﬁnity chromatography. For RG4 formation, in vitro-transcribed biotinylated RNAs were heated to 95 °C for 5 min in one volume of 1 x phosphatebuffered saline supplemented with 2 M KCl and cooled down at room temperature in presence or absence of cPDS 10 µM. hnRNP H/F or DHX36 were immunoprecipitated overnight as described in “immunoprecipitation” of the Methods section. Beads were then washed three times in wash buffer (20 mM Tris HCl pH 8, 100 mM KCl, 0.5% NP-40, 0.4 mM EDTA, 1 mM DTT) and incubated with the in vitro-transcribed biotinylated RNAs in 500 µL of binding buffer (20 mM Tris pH 8, 0.05% NP-40, 1 mM DTT, 100 mM KCl, 0.2 mM EDTA) for 1 h or 2 h when RNAs were incubated with cPDS. After ﬁve washes in binding buffer, beads were resuspended in 60 μl of elution buffer (50 mM Tris pH8, 1% SDS), and boiled for 10 min. Immunoprecipitated proteins were analyzed by western blot and biotinylated RNAs were analyzed by urea PAGE followed by biotin detection (as described in “Biotinylated RNA detection” section). Surface plasmon resonance. All binding studies based on surface plasmon resonance technology were performed on BIAcore T200 optical biosensor instrument (GE Healthcare) at 4 °C. Capture of the different biotinylated RNA (WT or 7dG) was performed on a Streptavidine (SA) sensorchip in HBS-EP + buffer NATURE COMMUNICATIONS \| (2020)11:2661 \| https://doi.org/10.1038/s41467-020-16168-x \| www.nature.com/naturecommunications NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-020-16168-x (10 mM HEPES, pH 7.4, 150 mM NaCl, 3 mM EDTA and 0.005% surfactant P20 (GE Healthcare). All immobilisation steps were performed at a ﬂow rate of 5 ml/ min with ﬁnal mRNA concentration of 10 µg/ml. Total amount of immobilized ligand was about 1100-1500 RU. The channel (Fc1) was used as a reference surface for all non-speciﬁc binding measurements. For binding analysis, cytoplasmic lysates were injected ﬁrst at 100 µg/ml over the immobilized surface for 2 min at a ﬂow rate of 30 ml/min. Thereafter, the hnRNP H/F antibody was injected at a concentration of 200 µg/ml for 1 min and with the same ﬂow rate settings. The binding of antibodies to molecules captured from lysates by the sensor chips were normalized using BIAevaluation 3.0 software (Biacore AB). Biotinylated RNA detection. Eluates from chromatography experiments were loaded on a 6% UREA polyacrylamide gel and electrophoresed at 4 °C for 1 h at 100 V in 0.5× TAE buffer, and then transferred to either a Biodyne B nylon membrane (Thermo Scientiﬁc, 77010) or Hybond-N + nylon membrane (Amersham Biosciences, RPN203B). After cross-link under UV light (UV Stratalinker 1800), signals were probed using the Chemiluminescent Nucleic Acid Detection Module (Thermo Scientiﬁc, 89880) according to the manufacturer’s instructions. Western Blot antibodies. For immunoblotting analysis, proteins were resolved on 12 or 7% denaturing polyacrylamide gels and were transferred to nitrocellulose membranes. The blots were blocked for 30 min with TBST-5% milk and then probed overnight with primary antibodies against DHX36 (1:1000, Abcam Ab70269), DHX9 (1:1000, Abcam Ab54593), DDX3X (1:1000, Santa Cruz sc365768), LARP1 (1:1000, Bethyl A302-087A), hnRNP H/F (1:1000, Abcam Ab10689), KSRP (1:500, Bethyl A302-022A), E2F1 (1:500, Santa Cruz sc-251), eIF4A (1:500, Santa Cruz sc-50354), PERK (1:1000, Cell Signaling Technology 3192), Histone H3 (1:1000, Cell Signaling Technology 4499), EEA1 (1:500, Santa Cruz sc-53939), RPS6 (1:1000, Santa Cruz sc-74459), RPL22 (1:1000, Novus Bio NBP1-06069), GAPDH (1:1000, Santa Cruz sc-32233), γH2AX (1:1000, Millipore 05-636), Flag (1:1000, Sigma F3165-2MG), USP1 (1:600, ProteinTech 14346-1-AP), Ubiquitin (1:1000, Cell signaling Technology 3936), Puromycin (1:1000, Millipore, MABE343), PARP (1 :1000, Cell signaling 9542), Caspase-3 (1 :1000, Cell signaling 8G10), Anti-Rabbit IgG (1:5000, Ozyme 7074S), Anti-Mouse IgG (1:5000, Ozyme 7076S). The blots were developed using the ECL system (Amersham Pharmacia Biotech) according to the manufacturer’s directions. Polysomes. Around 3.107 cells were treated with 0.1 mg/ml cycloheximide (CHX) for 15 min at 37 °C, washed twice with ice-cold phosphate-buffered saline supplemented with 0.1 mg/ml CHX (PBS/CHX), and scraped on ice in PBS/CHX. After centrifugation for 5 min at 200 g, the cell pellet was gently resuspended in 450 μl of hypotonic lysis buffer (5 mM Tris pH 7.5, 1.5 mM KCl, 1.5 mM MgCl2, 20 U/ml RNaseOUT (Invitrogen, 10777019), 0.1 mg/ml CHX and 10 µl/ml of Protease Cocktail Inhibitor (Sigma, P8340)). The lysate was vortexed for 5 s, incubated on ice for 5 min and 26 µl of 10 % Triton X-100 and 26 µl of 10% sodium deoxycholate were added. After incubation on ice for 5 min, the lysate was centrifuged at 16,000 g for 7 min at 4 °C and a volume of supernatant corresponding to 20 OD260 nm was layered on a 11.3 ml continuous sucrose gradient (5-50% sucrose in 200 mM HEPES pH7.6, 1 M KCl, 50 mM MgCl2). After 2 h of ultracentrifugation at 222,228 g in a SW41-Ti rotor at 4 °C, fractions were collected with an ISCO density gradient fractionation system (Foxy Jr fraction collector coupled to UA6UV detector, Lincoln, NE). The settings were as follows: fraction time, 62 s/ fraction; chart speed, 60 cm/h; sensitivity of the OD254 recorder, 0.5. The absorbance at 254 nm was measured continuously as a function of gradient depth; 16 fractions of approximately 0.8 ml were collected. The fractions recovered from the gradient were either analyzed individually or divided into three groups, fractions containing the most actively translated mRNAs, containing more than four ribosomes and called heavy polysomes (HP), fractions containing actively translated mRNAs containing two to three ribosomes, called light polysomes (LP) and fractions containing untranslated mRNAs (non-polysomes (NP)). Equal amounts of RNA from the NP, LP and HP fractions were extracted by using Trizol LS (Invitrogen), analysed by agarose gel and subjected to RT-qPCR analysis to determine the polysomal mRNA distribution. Protein from individual fractions were extracted by using isopropanol precipitation and analysed by western blot. SUnSET. Cells were treated with 10 µg/ml puromycin (Sigma P8833) for 10 min at 37 °C. Cells were washed twice in ice-cold PBS, scrapped on ice in PBS and collected by centrifugation at 200 g for 5 min. Cell were lysed in 50 mM HEPES pH7.0, 150 mM NaCl, 10% Glycerol, 1% Triton, 10 mM Na4P2O7, 100 mM NaF, 1 mM EDTA et 1.5 mM MgCl2 and 10 µl/ml Protease Cocktail Inhibitor (Sigma, P8340) buffer and puromycin incorporation was analyzed by western Blot. Immunoprecipitation. Cytoplasmic (cytosolic + microsomal fractions (as described in “cell fractionation” of the Methods section)) cell extracts were digested for 1 h at room temperature with Benzonase (Millipore E1014) and DNase I (Thermo Scientiﬁc EN0521) and precleared with protein-sepharose beads for 1 h at 4 °C. hnRNP H/F (10 µg, Abcam Ab10689), DHX36 (5 µg, Abcam Ab70269) or BG4 antibodies (0.5 µg, expressed from the pSANG10-3F-BG4 plasmid (Addgene #55756), kindly provided by S. Balasubramanian and puriﬁed based on45) were ARTICLE incubated with 20 µl of slurry beads (washed and equilibrated in cell lysis buffer) for 1 h at 4 °C. Beads were incubated with 1 mg of cell extracts overnight at 4 °C. Beads were washed three times with cell lysis buffer and co-immunoprecipitated proteins were analyzed by western blot. Puriﬁed RNA from mRNP complexes was resuspended in 10 µl of water and 4 µl was reverse transcribed using the RevertAidH Minus First (Thermo ﬁsher) according to the manufacturer’s protocol. Subsequently, a 1/5 dilution of cDNA was analyzed by qPCR with the SybrGreen (KAPA KK4605). The mRNA levels associated with these mRNP complexes were then standardized against HPRT mRNA levels (used as a reference) and compared with RNA levels in the IgG control and input sample. RT-qPCR. Reverse transcription (RT) was performed on 1 µg total RNA (quantiﬁed with the Clariostar BMG and software v.5.21 R4, Labtech and MARS Clariostar Analysis Software v.3.20 R2) using the RevertAidH Minus First (Thermo ﬁsher) according to the manufacturer’s protocol. 12.5 ng of cDNA was analyzed by qPCR with the SybrGreen (KAPA KK4605) using the StepOne software v2.2.2 (Applied Biosystems). Expression of MECP2, PRR5, VEGF, USP1, BABAM1, CCNA2 was standardized using HPRT as a reference, and relative levels of expression were quantiﬁed by calculating 2^ΔΔCT, where ΔΔCT is the difference in CT (cycle number at which the amount of ampliﬁed target reaches a ﬁxed threshold) between target and reference. All primer sequences are available in Supplementary Table 2. Immunoﬂuorescence. For the detection of markers of genetic instability, cells grown on coverslips were ﬁxed with 3% paraformaldehyde in PBS for 20 min at room temperature, washed with PBS twice for 5 min, permeabilized with 0.5% Triton X-100/1% normal goat serum in PBS for 15 min, and washed with 1% normal goat serum/PBS three times for 10 min each. The coverslips were then incubated with primary antibodies in 1% normal goat serum/PBS at room temperature for 1 hr using antibodies against γ-H2AX (JBW301 Millipore 05-636; 1:500) and 53BP1 (Cell Signaling 2675; 1:200). The coverslips were washed twice for 10 min and incubated with goat anti-mouse IgG secondary antibody coupled to ﬂuorescein isothiocyanate in 1% normal goat serum/PBS at room temperature for 1 h. The samples were then washed three times for 10 min each and mounted. For the detection of G4s, cells were seeded in 96-Multiwell plate coated with poly-Dlysine solution. 48 h post seeding cells were pre-ﬁxed with a solution 50% DMEM and 50% methanol/acetic acid (3:1) at RT for 5 min. After a brief wash with methanol/acetic acid (3:1), cells were ﬁxed with methanol/acetic acid (3:1) at RT for 10 min. Cells were then permeabilized with 0.1% Triton X-100 in PBS at RT for 3 min. For RNase treatment, coverslips were incubated with 100 μg/ml RNase A in PBS for 1 h at 37 °C. Cells were incubated with blocking solution (2% milk in PBS, pH 7.4) for 1 h at RT and then with 1 μg per slide of BG4 in blocking solution (2 h at RT). Cells were then incubated with 1:800 of a rabbit antibody against the Flag epitope (Cell Signaling ref# 2368) in blocking solution for 1 h. Next, cells were incubated at RT with 1:500 Alexa Fluor 488 goat anti-rabbit IgG (Life technologies ref# A11008) in blocking solution for 1 h and with DAPI for 10 min. After each step, cells were washed three times for 10 min with 0.1% Tween-20 in PBS under gentle rocking. Cells were visualized at room temperature by using a confocal microscope (Zeiss, LSM780) or using the high-content Operetta High-Content Imaging System (Harmony Imaging 4.8; PerkinElmer). For the high-content analysis, cytoplasmic foci detection and subsequent analyses were performed with Columbus 2.8.2 software (PerkinElmer). Plating efﬁciency, clonogenicity assay. LN18 glioblastoma cells were transfected with siRNA (siCtr or siF), after twenty-four hours, cells were harvested and plated in 6-well plates at different concentration (500, 750, 1000 cells/well for siCtr and 1500, 2500, 5000 for siF) in duplicate. Twenty-four hours later cells were irradiated with an ionizing radiation scale (from 0 to 4 Gy) using the Gammacell 40 Exactor irradiator (Nordion, Ottawa, Canada) or with TMZ dose scale (from 100 to 500 µM). Cells were then incubated for approximately 10 days until colonies were visible with the naked eye without any joining between colonies. Then, plates were washed and cells were ﬁxed with 10% formalin for 10 min, the formalin was removed and cells were covered with 10% crystal violet oxalate (RAL Diagnostics, Martillac, France) for 10 min, plates were rinsed with water until no additional color comes off the plate. Colonies were then counted to calculate the plating efﬁciency. Plating efﬁciency (%) = (number of colonies formed/number of cells plated) × 100. GBM tumour sample. The used protein extracts derived from both low and high grade gliomas were originally processed and used in the article from from Pr JP Hugnot’s lab68. Total protein lysates in RIPA Buffer (Sigma) (50 mM Tris–HCl pH 8.0, 150 mM NaCl, 1% NP40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate, 5 mM sodium ﬂuoride, 0.5 mM sodium vanadate, and 1× protease inhibitor cocktail (Roche)) were extracted from 3 GBM (grade IV), and 4 Diffuse Low Grade Gliomas (Grade II: 2 astrocytomas and oligodendrogliomas 2). Tumors samples were obtained from the Montpellier hospital (“biological resource centre”, (Collection NEUROLOGIE, DC-2013-2027/DC-2010-1185 /Authorization AC-20173055/Research Protocol P487) with patient consent. All the methods used were NATURE COMMUNICATIONS \| (2020)11:2661 \| https://doi.org/10.1038/s41467-020-16168-x \| www.nature.com/naturecommunications 15 ARTICLE NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-020-16168-x carried out in accordance with relevant guidelines and regulations of French Institut National de la Santé et de la Recherche Médicale (INSERM). All experimental protocols were approved by INSERM. The tissues were obtained from patients, who underwent surgical resection. The tissues were processed, classiﬁed and graded as described in69. The clinicopathological parameters of the patients and tumors are described in the Supplementary Table 3. CLIP data analysis. Reads were trimmed (minimum quality 25, minimum length 18nt) and adapters removed with Trim Galore (https://github.com/FelixKrueger/ TrimGalore) with UMIs extracted with UMI-tools (10.1101/gr.209601.116) when needed. Remaining reads were aligned to the hg19 assembly of the human genome with STAR (10.1093/bioinformatics/bts635). Duplicates were collapsed, using UMIs when available. CLIP sites were eventually called with clipper (10.1038/ nsmb.2699), using an FDR threshold of 0.05. Background sites were obtained by generating 10000 random sequence sites with length corresponding to the average site length. Sites were annotated for their gene and genomic region of origin with ctk (10.1093/bioinformatics/btw653). RG4 elements were predicted within CLIP sites (extended by 25nt upstream and downstream) and in whole 5′UTR, CDS, and 3′UTR by means of QGRS Mapper (https://doi.org/10.1093/nar/gkl253) with default parameters. RG4 were selected as those predicted RG4 having a score of at least 19. All enrichments were computed with the Fisher exact test, using random sites as background frequencies. Densities per Mb were obtained by dividing the number of sites/RG4 by the total length of the corresponding genomic region obtained from the genome assembly annotations. Gene Ontology analysis was performed in R with the topGO package (Alexa A, Rahnenfuhrer J (2018). topGO: Enrichment Analysis for Gene Ontology. R package version 2.34.0.) and results plotted with REVIGO (http://revigo.irb.hr/, medium similarity threshold). CLIP data for DHX36 was obtained from ref. 25. Target 5′UTRs, CDS or 3′UTRs were selected as those with at least one signiﬁcant cluster. Intersections were then performed with the list of hnRNP H/F common targets in the different mRNA regions. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. Data analysis. Data analyses were performed with Microsoft Excel, Graphpad Prism8, ImageJ v 1.52, R v 3.6.1, RStudio v 1.0.153 and ﬁgures were prepared with Microsoft power point, Inkscape v 0.92.4, Gimp v 2.10.18. Reporting summary. Further information on research design is available in the Nature Research Reporting Summary linked to this article. 23. 24. 25. Data availability The mass spectrometry proteomic data have been deposited to the ProteomeXchange Consortium via the PRIDE [1] partner repository with the dataset identiﬁer PXD015609. CLIP data for DHX36, hnRNP H and hnRNP F were obtained from GEO ID GSE105171, E-MTAB-6221, GSE34993, respectively. The REMBRANDT dataset was available at the Betastasis website (http://www.betastasis.com/glioma/rembrandt/kaplanmeier_survival_curve/). The source data underlying Figs. 1d–f, 2a–c, 3a, d–f, g, 4a–e, 5a, b, d–f, 6b, c, e, f are provided as a Source Data ﬁle. The source data underlying Supplementary Figs. 1d, 2b–d, 3a–c, 5a, b, 6a–g, 8b–f, 9a, e, 10a, d, e, 11a, b, 13a, d are provided as a Source Data ﬁle. All data is available from the authors upon reasonable request. S.M. is the lead contact for correspondence. 26. 27. 28. 29. 30. 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The 2007 WHO classiﬁcation of tumours of the central nervous system. Acta Neuropathol. 114, 97–109 (2007). Acknowledgements We are grateful to S. Queille for assistance with immunoﬂuorescence experiments; to M-J. Pillaire for discussion and materials; to A. Willis, for providing the hnRNP I antibody; S. Pautet for assistance with preliminary experiments. We acknowledge members of the S. Millevoi, S. Pyronnet and E. Moyal laboratories as well as N. Puget, D. Gomez and I. Gallouzi for discussions and advices. We thank C. Broussard (LC-MS supervision), M. Leduc (LIMS management), P. Mayeux (proteomic expertise and experimental design) from the 3P5 proteomic facility of the Université de Paris. The Orbitrap Fusion mass spectrometer was acquired with funds from the FEDER through the “Operational Programme for Competitiveness Factors and employment 2007-2013” and from the “Canceropole Ile de France”. We are grateful to M. Augustus for providing extracts of tumor samples; L. Ligat (Technology cluster of CRCT) for helping with surface plasmon resonance experiments and confocal imaging; Y. Martineau, S. Shin and A. Olichon for helping with RIP assays using the BG4 antibody; A. De Magis for helping with immunoﬂuorescence assays. This work was supported by institutional grants (from INSERM, Université Toulouse III - Paul Sabatier, CNRS) and by funding from LNCC (Ligue Nationale Contre le Cancer), ARC (Association pour la Recherche contre le Cancer), Emergence Cancéropole GSO, Laboratoire d’Excellence TOUCAN (ANR11-LABX) and ANR (ANR-17-CE120017-01). MLB was supported by the Midi-Pyrénées Region/INSERM, PH by ANR (ANR-17-CE12-0017-01) and LD by MENRT. Author contributions S.M. conceived the project. S.M. and A.C. designed and supervised the experiments. P.H. and M.L.B. performed most of the experiments, together with A.C., with assistance by L.D. and C.H. A.C. made all the ﬁgures. J.G. performed TCGA analysis. J.P.H. provided GBM samples. F.G. designed and supervised the proteomic analysis. A.A. performed proteomic analysis and statistical data treatment. E.D. performed bioinformatic analysis. G.C. performed CD experiments. S.M. wrote the manuscript with input from A.C., M.L.B., P.H., L.D., E.D. and F.G. Competing interests The authors declare no competing interests. Additional information Supplementary information is available for this paper at https://doi.org/10.1038/s41467020-16168-x. Correspondence and requests for materials should be addressed to E.D., A.C. or S.M. Peer review information Nature Communications thanks Cyril Dominguez, Katrin Paeschke, Jean-Pierre Perreault and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available. 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. 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https://openalex.org/W3088161002	https://hal.archives-ouvertes.fr/hal-03032184/document	English	null	Time Machine Biology: Cross-Timescale Integration of Ecology, Evolution, and Oceanography	Oceanography	2,020	cc-by	14,287	>10 m of sediment (Revelle, 1987). Ever since the voyage of HMS Beagle (1831–1836), ocean expeditions have pro- vided novel insights into geological and biological processes. The HMS Challenger expedition (1872–1876) was one of the first to systematically collect numer- ous marine sediment and organis- mal samples from around the world (Figure 1), setting the scene for contem- porary oceanography, paleoceanography, and marine biology (Macdougall, 2019). The Swedish Albatross expedition (1947– 1948) expanded on Challenger insights by extracting the first deep-ocean sediment cores, which encompassed hundreds of thousands of years of sedimentation and facilitated pioneering paleoceano- graphic and micropaleontological stud- ies (Arrhenius, 1952; Emiliani, 1955; Parker, 1958; Olausson, 1965; Benson and Sylvester-Bradley, 1971; Benson, 1972; Berger, 2011). Sediment cores are vertical columns of sediment recovered by tech- niques designed to penetrate the seafloor (Figure 1). The Albatross expedition made use of a prototype piston core designed by Kullenberg that was capable of recovering Since the Albatross expedition, numer- ous seafloor sediment samples have been collected from coring expeditions and accumulated at oceanographic institu- tions (Berger, 2011). These collections have facilitated global-scale analyses of past climate change, such as reconstruc- tion of global ocean conditions during the last ice age by the CLIMAP proj- ect (Climate: Long range Investigation, Mapping, and Prediction; CLIMAP Project Members, 1976, 1984). The same material also made it possible to inves- tigate species and community dynam- ics across temporal scales. For instance, Ruddiman (1969), a geologist, used planktonic foraminiferal records in sur- face sediments from the North Atlantic to reveal large-scale spatial patterns in present-day species, a pioneering con- tribution to the field now known as macroecology (Brown and Maurer, 1989; Brown, 1995; Yasuhara et al., 2017b). Ruddiman’s analysis of planktonic fora- miniferal diversity was feasible because many paleoclimatic reconstructions, such as CLIMAP, use the present-day Scientific ocean drilling began with the launch of the international Deep Sea Drilling Project (DSDP) in 1968, fol- lowed by the Ocean Drilling Program (ODP) in 1983, the Integrated Ocean Drilling Program (IODP) in 2003, and the International Ocean Discovery Program (IODP) in 2013 (Becker et al., 2019). These projects allowed scien- tists to recover sediment sequences up to several kilometers in length, span- ning ~170 million years (Figure 1; Becker et al., 2019; Clement and Malone, 2019). Marine geology and marine biology have common origins. The iconic founding hero of this connection was Charles Darwin. – Berger, 2011 distribution and relative abundance of microfossil species as environmen- tal proxies (Box 1). As a result, CLIMAP and related efforts (e.g., the mid-Pliocene PRISM project or Pliocene Research, Interpretation and Synoptic Mapping) have built comprehensive, global data sets of microfossil community censuses for several time periods in Earth his- tory, including the present day, the Last Glacial Maximum, and the Pliocene (CLIMAP Project Members, 1976, 1984; Dowsett et al., 1994, 2013). These data were seldom studied from a biologi- cal perspective initially but later proved critical for gaining insight into present (Rutherford et al., 1999; Fenton et al., 2016; Tittensor et al., 2010) and past (Yasuhara et al., 2012c, 2020) biodiversity patterns on global and regional scales.i ABSTRACT. Direct observations of marine ecosystems are inherently limited in their temporal scope. Yet, ongoing global anthropogenic change urgently requires improved understanding of long-term baselines, greater insight into the relationship between cli- mate and biodiversity, and knowledge of the evolutionary consequences of our actions. Sediment cores can provide this understanding by linking data on the responses of marine biota to reconstructions of past environmental and climatic change. Given continuous sedimentation and robust age control, studies of sediment cores have the potential to constrain the state and dynamics of past climates and ecosystems on time scales of centuries to millions of years. Here, we review the development and recent advances in “ocean drilling paleobiology”—a synthetic science with potential to illumi- nate the interplay and relative importance of ecological and evolutionary factors during times of global change. Climate, specifically temperature, appears to control Cenozoic marine ecosystems on million-year, millennial, centennial, and anthropogenic time scales. Although certainly not the only factor controlling biodiversity dynamics, the effect size of temperature is large for both pelagic and deep-sea ecosystems. TIME MACHINE BIOLOGY CROSS-TIMESCALE INTEGRATION OF ECOLOGY, EVOLUTION, AND OCEANOGRAPHY SPECIAL ISSUE ON PALEOCEANOGRAPHY: LESSONS FOR A CHANGING WORLD TIME MACHINE BIOLOGY CROSS-TIMESCALE INTEGRATION OF ECOLOGY, EVOLUTION, AND OCEANOGRAPHY SPECIAL ISSUE ON PALEOCEANOGRAPHY: LESSONS FOR A CHANGING WORLD i i i i i i i i i TIME MACHINE BIOLOGY CROSS-TIMESCALE INTEGRATION OF ECOLOGY ceanography \| June 2020 Early Online Release Oceanography \| Vol.33, No.2 6 CROSS-TIMESCALE INTEGRATION OF ECOLOGY, EVOLUTION, AND OCEANOGRAPHY Credit: Aaron O’Dea, Yuanyuan Hong, Moriaki Yasuhara, Katsuno Kimoto, Jeremy R. Young, Kotaro Hirose, Tamotsu Nagumo, Yoshiak Aita, André Rochon, Erin M. Dillon, Briony Mam By Moriaki Yasuhara, Huai-Hsuan May Huang, Pincelli Hull, Marina C. Rillo, Fabien L. Condamine, Derek P. Tittensor, Michal Kučera, Mark J. Costello, Seth Finnegan, Aaron O’Dea, Yuanyuan Hong, Timothy C. Bonebrake, N. Ryan McKenzie, Hideyuki Doi, Chih-Lin Wei, Yasuhiro Kubota, and Erin E. Saupe ceanography \| June 2020 Early Online Release EVOLUTION, AND OCEANOGRAPHY Credit: Aaron O’Dea, Yuanyuan Hong, Moriaki Yasuhara, Katsunori Kimoto, Jeremy R. Young, Kotaro Hirose, Tamotsu Nagumo, Yoshiaki Aita, André Rochon, Erin M. Dillon, Briony Mamo Marine geology and marine biology have common origins. The iconic founding hero of this connection was Charles Darwin. – Berger, 2011 >10 m of sediment (Revelle, 1987). Scientific ocean drilling has been deemed one of the most successful international scientific collaborations ever undertaken (Berger, 2011) and has provided unpar- alleled marine data on a global scale that has resulted in numerous publications (>11,000 peer-reviewed papers; Clement and Malone, 2019; Koppers et al., 2019). Integration of paleoceanographic and paleobiological data from deep-sea sed- iments has provided improved under- Oceanography \| https://doi.org/10.5670/oceanog.2020.225 Present Past a b c FIGURE 1. (a) An overview of sediment core collection and processing for micropaleontological research. Illustrations by Simon J. Crowhurst and the Godwin Laboratory. (b) Ocean-floor sediment samples collected with a dredge during the HMS Challenger expe- dition on March 21, 1876, in the South Atlantic. Sample number M.408 from the Ocean-Bottom Deposit (OBD) Collection held by the Natural History Museum in London (for more information, see Rillo et al., 2019). (c) Sand-sized residue of a Pleistocene deep-sea sed- iment from ODP Site 925 in the equatorial Atlantic Ocean consisting of numerous microfossil shells. Scale bar: 1 mm. Present Past a b c Present Past a b c Present Past a Present Past a Present Past a aa Pas Past Past bb b b c b c c FIGURE 1. (a) An overview of sediment core collection and processing for micropaleontological research. Illustrations by Simon J. Crowhurst and the Godwin Laboratory. (b) Ocean-floor sediment samples collected with a dredge during the HMS Challenger expe- dition on March 21, 1876, in the South Atlantic. Sample number M.408 from the Ocean-Bottom Deposit (OBD) Collection held by the Natural History Museum in London (for more information, see Rillo et al., 2019). (c) Sand-sized residue of a Pleistocene deep-sea sed- iment from ODP Site 925 in the equatorial Atlantic Ocean consisting of numerous microfossil shells. Scale bar: 1 mm. Oceanography \| June 2020 Early Online Release BIOTIC DYNAMICS OVER MILLIONS OF YEARS climate change and biodiversity levels across multiple marine clades suggests that climate, particularly temperature, controls diversification dynamics on long timescales (Box 2 and Figure 3). Higher temperatures generally corre- spond with higher levels of biodiversity (Box 2). However, the precise mecha- nisms responsible for this coupling are debated. One potential explanation is that higher temperatures can enhance meta- bolic efficiency and resulting reproduc- tion, with such enhancements potentially resulting in increased speciation and standing of the co-evolution of marine systems and their biota over the last ~10 years. Here, we review these efforts to understand the interaction between climate change and the marine biosphere on both long and short timescales. Our review is time structured and focuses on the influence of climate on biodiversity on million-year, millennial, centennial, and more recent timescales. We end with a discussion of how deep-sea biodiversity dynamics on the most recent timescales can inform our understanding of the changes expected in the Anthropocene. standing of the co-evolution of marine systems and their biota over the last ~10 years. Here, we review these efforts to understand the interaction between climate change and the marine biosphere on both long and short timescales. Our review is time structured and focuses on the influence of climate on biodiversity on million-year, millennial, centennial, and more recent timescales. We end with a discussion of how deep-sea biodiversity dynamics on the most recent timescales can inform our understanding of the changes expected in the Anthropocene. Quantifying how and why biodiver- sity levels have changed over Earth his- tory is fundamental to macroecology and macroevolution. Scientific ocean drill- ing samples have allowed for unprece- dented insight into these dynamics on million-year timescales, particularly in response to large-scale global climate and tectonic changes (Figure 2; Kucera and Schönfeld, 2007; Norris, 2000; Fraass et al., 2015; Lowery et al., 2020). The gen- eral correspondence between Cenozoic Oceanography \| June 2020 Early Online Release et al., 2012c; Fenton et al., 2016; Lam and Leckie, 2020; Meseguer and Condamine, 2020), potentially reflecting degree of climatic heterogeneity (Saupe et al., 2019). The standard tropical-high and extratropical-low LBG can be traced back at least to the Eocene for both deep-sea benthos (~37 million years ago [Ma]; Thomas and Gooday, 1996; Figure 3) and pelagic plankton (~48–34 Ma; Fenton et al., 2016). BIOTIC DYNAMICS OVER MILLIONS OF YEARS Notably, initiation of the LBG observed today in the deep sea predates the Eocene-Oligocene transi- tion (Thomas and Gooday, 1996), sug- gesting that it began with the opening of the Drake Passage (Scher and Martin, 2006; Figure 2) rather than with climatic changes at the Eocene-Oligocene tran- sition (Figure 3). Indeed, the opening and closing of major seaways (Figure 2) has been shown to alter the distribution of both shallow-marine and deep-sea organisms throughout the Cenozoic, therefore species richness (Allen et al., 2002; Yasuhara and Danovaro, 2016). both longitudinally (e.g., Tethyan and Central American Seaways) and latitu- dinally (e.g., Arctic gateways) (O’Dea et al., 2007; Renema et al., 2008; Yasuhara et al., 2019b). In addition to regulating temporal pat- terns of biodiversity, climate change over the Cenozoic has affected spatial pat- terns of marine biodiversity. For example, large-scale climatic shifts have modified one of the foremost patterns in ecol- ogy, the latitudinal biodiversity gradient (LBG), in which the number of species decreases from the equator to the poles (Hillebrand, 2004a,b; Saupe et al., 2019). In the ocean, LBGs are often character- ized by an equatorial dip, resulting in a bimodal biodiversity pattern (Rutherford et al., 1999; Worm et al., 2005; Chaudhary et al., 2016, 2017; Worm and Tittensor, 2018; Rogers et al., 2020; Yasuhara et al., 2020). Growing evidence suggests the LBG was flatter during warm periods (e.g., Eocene, Pliocene) and steeper during cold periods (e.g., Last Glacial Maximum of 20,000 years ago; Yasuhara Although evidence supports climate as a primary control on Cenozoic bio- diversity change, it is certainly not the only driver of biodiversity dynamics (e.g., Ezard et al., 2011; Condamine et al., 2019; Lam and Leckie, 2020). Ecological interactions in addition to climate, for example, have been found to influence the macroevolution of planktonic foramin- ifera (Ezard et al., 2011). Continued study of biotic traits will allow for examination of the relative roles of abiotic (e.g., cli- mate) versus biotic factors in shaping ecosystems and their changes through time (Schmidt et al., 2004), as well as of the relationship between biodiversity and ecosystem function (Henehan et al., 2016; Yasuhara et al., 2016; Alvarez et al., 2019). BIOTIC DYNAMICS OVER MILLIONS OF YEARS Although microfossils are ideal subjects for macroecological and macroevolutionary analyses, their study is limited by poor understanding of the life histories and ecology of most species and by evolving species concepts, limited phylogenetic hypothe- ses for most clades, and the poor preservation potential of other organisms in the community aside from microfossils (i.e., the majority of the ecosystem is not fossilized). In spite of these limita- tions, almost every trophic or functional group in marine commu- nities is represented by (at least) one well-fossilized microfossil group, providing a basis for macroecological and macroevolu- tionary synthesis. Notable exceptions are the prokaryotes and viruses, which leave no body fossils. Obtaining information on the history of these groups requires alternative approaches, such as the use of organic biomarkers in the case of some prokaryote clades or ancient DNA (Armbrecht, 2020, in this issue). spatial and temporal scales, as reviewed here. Although microfossils are ideal subjects for macroecological and macroevolutionary analyses, their study is limited by poor understanding of the life histories and ecology of most species and by evolving species concepts, limited phylogenetic hypothe- ses for most clades, and the poor preservation potential of other organisms in the community aside from microfossils (i.e., the majority of the ecosystem is not fossilized). In spite of these limita- tions, almost every trophic or functional group in marine commu- nities is represented by (at least) one well-fossilized microfossil group, providing a basis for macroecological and macroevolu- tionary synthesis. Notable exceptions are the prokaryotes and viruses, which leave no body fossils. Obtaining information on the history of these groups requires alternative approaches, such as the use of organic biomarkers in the case of some prokaryote clades or ancient DNA (Armbrecht, 2020, in this issue). Microfossils are abundant in deep-sea sediment cores and, in many cases, constitute a large portion of the sediment itself (Figure 1; e.g., see Marsaglia et al., 2015). Thus, hundreds of thou- sands to millions of plankton specimens can be found in a gram of deep-sea sediment. Microfossils are key tools for interpreting age and environment in sediment cores due to their excellent preservation and high abundance. The first and last appearance of key species and groups are used to determine the ages of sed- Microfossils are abundant in deep-sea sediment cores and, in many cases, constitute a large portion of the sediment itself (Figure 1; e.g., see Marsaglia et al., 2015). Oceanography \| https://doi.org/10.5670/oceanog.2020.225 Oceanography \| June 2020 19 Oceanography \| https://doi.org/10.5670/oceanog.2020.225 BIOTIC DYNAMICS OVER MILLIONS OF YEARS iments in cores (Berggren et al., 1995; Motoyama, 1996), whereas the trace element and isotopic compositions of microfossils are used to reconstruct the paleoceanographic history of sediment cores with regard to, for example, temperature, salinity, and polar ice volume (Figure 2; e.g., Zachos et al., 2001; Lisiecki and Raymo, 2005; Norris et al., 2013). At the same time, the high abundance and diversity of microfossil groups preserved in small amounts of sediment (Figure 1) allow for quantitative assessment of the composition and dynamics of past communities across a range of spatial and temporal scales, as reviewed here. Microfossils are microscopic remains of organisms or their parts preserved in the fossil record. The most widely studied micro- fossils are biomineralized structures with high fossilization poten- tial produced by a range of organisms, including photosynthetic plankton such as coccolithophores and diatoms, various mixo- to heterotrophic protists such as planktonic and benthic foramin- ifera and planktonic radiolarians, and small metazoans such as benthic ostracods (small bivalved crustaceans; Yasuhara et al., 2017b). Dinoflagellates (auto-, mixo-, and heterotrophic plankton) also leave abundant microfossils in the form of resistant organic (and occasionally biomineralized) resting cysts (de Vernal, 2013). Many microfossils represent parts of larger organisms, such as the scales and teeth of fish (Field et al., 2009; Sibert et al., 2017) and shark denticles (Dillon et al., 2017), referred to as ichthyoliths. Although pollen and spores have terrestrial origins, they can also be preserved in both marine and freshwater sediments (Sánchez Goñi et al., 2018). iments in cores (Berggren et al., 1995; Motoyama, 1996), whereas the trace element and isotopic compositions of microfossils are used to reconstruct the paleoceanographic history of sediment cores with regard to, for example, temperature, salinity, and polar ice volume (Figure 2; e.g., Zachos et al., 2001; Lisiecki and Raymo, 2005; Norris et al., 2013). At the same time, the high abundance and diversity of microfossil groups preserved in small amounts of sediment (Figure 1) allow for quantitative assessment of the composition and dynamics of past communities across a range of spatial and temporal scales, as reviewed here. BIOTIC DYNAMICS OVER MILLIONS OF YEARS ice sheets Antarctic ice sheets c δ18O (‰) δ18O (‰) 70 Ma 60 Ma 50 Ma 40 Ma 30 Ma 20 Ma 10 Ma 1 Ma The major tectonic events include: • Tasmanian Passage opened (33.5–33.7 Ma; Cronin, 2009) • Drake Passage opened (shallow water connection started at ~41 Ma and deepwater connection established by ~30 Ma; Scher and Martin, 2006; Yasuhara et al., 2019b) l fi ll l • MPTO: Mid-Pliocene Thermal Optimum (4.5–3 Ma; Cronin, 2009) • MPT: Mid-Pleistocene transition (1.2–0.7 Ma; Elderfield et al., 2012) • MBE: Mid-Brunhes Event (0.43 Ma; Holden et al., 2011) The major biotic events include: l d f f 70 65 60 55 50 45 40 35 30 25 20 15 10 5 0 5 4 3 2 1 0 Bering Strait opened Fram Strait opened Himalayan uplift MMCO Mi−1 E Asian Monsoon intensifed IAA hotspot O/M Caribbean coral extinction EECO PETM P/E foram extinction Mid-Miocene foram turnover AMOC onset EOT E/O foram turnover 5 4 3 2 1 0 5.0 4.0 3.0 Pliocene Quaternary MBE MPT MPTO MBE Arctic & JS ostracod turnover MPT foram extinction Caribbean extinctions a Aus-Pac-Eur collision Plio Q Panama-SA collision Formation of the Panama Isthmus complete Arabian hotspot b b Age (Ma) West-Tethys hotspot Paleoc Eocene Oligo Miocene Ara-Ana collision Tethys SW closed Drake Passage opened Tasmanian Passage opened NH. ice sheets Antarctic ice sheets c δ18O (‰) δ18O (‰) 25 20 15 10 5 0 Bering Strait opened Fram Strait opened Himalayan uplift MMCO Mi−1 E Asian Monsoon intensifed IAA hotspot O/M Caribbean l ti ti Mid-Miocene foram turnover 2 1 0 Quaternary MBE MPT MBE Arctic & JS ostracod turnover MPT foram extinction Caribbean a Aus-Pac-Eur collision Plio Q Panama-SA collision Arabian hotspot b b o Miocene Ara-Ana collision Tethys SW closed NH. BIOTIC DYNAMICS OVER MILLIONS OF YEARS ice sheets Antarctic ice sheets 70 65 60 55 50 45 40 35 30 25 20 15 10 5 0 5 4 3 2 1 0 Bering Strait opened Fram Strait opened Himalayan uplift MMCO Mi−1 E Asian Monsoon intensifed IAA hotspot O/M Caribbean coral extinction EECO PETM P/E foram extinction Mid-Miocene foram turnover AMOC onset EOT E/O foram turnover 5 4 3 2 1 0 5.0 4.0 3.0 Pliocene Quaternary MBE MPT MPTO MBE Arctic & JS ostracod turnover MPT foram extinction Caribbean extinctions a Aus-Pac-Eur collision Plio Q Panama-SA collision Formation of the Panama Isthmus complete Arabian hotspot b b Age (Ma) West-Tethys hotspot Paleoc Eocene Oligo Miocene Ara-Ana collision Tethys SW closed Drake Passage opened Tasmanian Passage opened NH. ice sheets Antarctic ice sheets δ18O (‰) δ18O (‰) 5 0 Bering Strait opened IAA hotspot 0 MBE MBE Arctic & JS ostracod turnover a Plio Q Panama-SA collision b b heets heets b b a • MPTO: Mid-Pliocene Thermal Optimum (4.5–3 Ma; Cronin, 2009) • MPT: Mid-Pleistocene transition (1.2–0.7 Ma; Elderfield et al., 2012) • MBE: Mid-Brunhes Event (0.43 Ma; Holden et al., 2011) ocene turnover 1 ternary MPT MPT foram hotspot NH. BIOTIC DYNAMICS OVER MILLIONS OF YEARS ice tarctic ice The major tectonic events include: 10 Himala ene j • Tasmanian Passage opened (33.5–33.7 Ma; Cronin, 2009) 15 intensif oce • Tasmanian Passage opened (33.5–33.7 Ma; Cronin, 200 15 inte oce Tasmanian Passage opened (33.5 33.7 Ma; Cronin, 2009) • Drake Passage opened (shallow water connection started at ~41 Ma and deepwater connection established by ~30 Ma; Scher and Martin, 2006; Yasuhara et al., 2019b) • Australia-Pacific-Eurasia (Aus-Pac-Eur) collision (~23 Ma; Renema et al., 2008) • Arabia-Anatolia (Ara-Ana) collision (~20 Ma; Renema et al., 2008) • Fram Strait opened (20–10 Ma; Yasuhara et al., 2019b) • Himalayan uplift latest phase (15–10 Ma; Tada et al., 2016) • Tethys Seaway closed (~19 Ma; Harzhauser et al., 2007; Yasuhara et al., 2019b) • Bering Strait opened (7.4–4.8 Ma; Yasuhara et al., 2019b) • Panama-South America (SA) collision (~24 Ma; Farris et al., 2011) • Formation of the Panama Isthmus complete (~3 Ma; O’Dea et al., 2016; Jaramillo, 2018) c 70 Ma 60 Ma 30 M 20 M 50 45 40 35 30 25 20 15 Fram Strait opened MMCO Mi−1 O/ co EECO for AMOC onset EOT E/O tu Aus-Pac-Eur collision Arab g ( ) Eocene Oligo Mioc Ara-Ana collision Tethys SW closed Drake Passage opened Tasmanian Passage opened • Drake Passage opened (shallow water connection started at ~41 Ma and deepwater connection established by ~30 Ma; Scher and Martin, 2006; Yasuhara et al., 2019b)i c 25 20 15 Fram Strait opened MMCO Mi−1 Aus-Pac-Eur collision A Mio Ara-Ana collision Tethys SW closed The major biotic events include: • Paleocene/Eocene (P/E) deep-sea foraminifera extinction (55.5 Ma; Thomas, 2007) • Eocene/Oligocene (E/O) deep-sea foraminifera turnover (36–31 Ma; Thomas, 2007) • Oligocene/Miocene (O/M) Caribbean coral extinction (~23 Ma; Johnson et al., 2009) • Mid-Miocene deep-sea foraminifera turnover (~15 Ma; Thomas, 2007) • West Tethys hotspot (Mesozoic to ~30 Ma; Renema et al., 2008) • Indo-Australian Archipelago (IAA) hotspot (23–0 Ma; Renema et al., 2008; Yasuhara et al., 2017a) • Arabian hotspot (23–16 Ma; Renema et al., 2008) • Caribbean extinctions (3–1 Ma; O’Dea et al., 2007; O’Dea and Jackson 2009) • MPT deep-sea foraminifera extinction (1.2–0.7 Ma; Hayward et al., 2007) • MBE Arctic (0.43 Ma; Cronin et al., 2017) and Japan Sea (JS) deep-sea ostracod turnover (0.43 Ma; Huang et al., 2018) 50 Ma 40 Ma 10 Ma 1 Ma aribbean extinction ram extinction ram ver 5 4 3 2 5 0 4 0 3 0 Pliocene Qu MPTO Caribbean extinctions Formation of Panama Isthm complete ethys hotspot A The major climatic events include: 60 55 oc • PETM: Paleocene-Eocene Thermal Maximum (55.5 Ma; Cronin, 2009) • EECO: Early Eocene Climatic Optimum (52–50 Ma; Cronin, 2009) • EOT: Eocene-Oligocene transition (~34 Ma) 65 60 Pale • EECO: Early Eocene Climatic Optimum (52–50 Ma; Cronin, 2009) EOT E Oli t iti ( 34 M ) 65 Pa • AMOC: Atlantic Meridional Overturning Circulation onset (~34 Ma; Hutchinson et al., 2019) 70 5 4 3 2 1 0 • Mi-1 event (23 Ma; Cronin, 2009 δ18O (‰) Ice sheets: • The durations of the Northern Hemisphere (NH) and Antarctic ice sheets are from Zachos et al. BIOTIC DYNAMICS OVER MILLIONS OF YEARS Thus, hundreds of thou- sands to millions of plankton specimens can be found in a gram of deep-sea sediment. Microfossils are key tools for interpreting age and environment in sediment cores due to their excellent preservation and high abundance. BIOTIC DYNAMICS OVER MILLIONS OF YEARS The first and last appearance of key species and groups are used to determine the ages of sed- The major tectonic events include: • Tasmanian Passage opened (33.5–33.7 Ma; Cronin, 2009) • Drake Passage opened (shallow water connection started at ~41 Ma and deepwater connection established by ~30 Ma; Scher and Martin, 2006; Yasuhara et al., 2019b) • Australia-Pacific-Eurasia (Aus-Pac-Eur) collision (~23 Ma; Renema et al., 2008) • Arabia-Anatolia (Ara-Ana) collision (~20 Ma; Renema et al., 2008) • Fram Strait opened (20–10 Ma; Yasuhara et al., 2019b) • Himalayan uplift latest phase (15–10 Ma; Tada et al., 2016) • Tethys Seaway closed (~19 Ma; Harzhauser et al., 2007; Yasuhara et al., 2019b) • Bering Strait opened (7.4–4.8 Ma; Yasuhara et al., 2019b) • Panama-South America (SA) collision (~24 Ma; Farris et al., 2011) • Formation of the Panama Isthmus complete (~3 Ma; O’Dea et al., 2016; Jaramillo, 2018) The major climatic events include: • PETM: Paleocene-Eocene Thermal Maximum (55.5 Ma; Cronin, 2009) • EECO: Early Eocene Climatic Optimum (52–50 Ma; Cronin, 2009) • EOT: Eocene-Oligocene transition (~34 Ma) • AMOC: Atlantic Meridional Overturning Circulation onset (~34 Ma; Hutchinson et al., 2019) • Mi-1 event (23 Ma; Cronin, 2009) • MMCO: Mid-Miocene Climatic Optimum (18–14 Ma; Cronin, 2009) • East Asian Monsoon intensified (15–10 Ma; Tada et al., 2016) • MPTO: Mid-Pliocene Thermal Optimum (4.5–3 Ma; Cronin, 2009) • MPT: Mid-Pleistocene transition (1.2–0.7 Ma; Elderfield et al., 2012) • MBE: Mid-Brunhes Event (0.43 Ma; Holden et al., 2011) The major biotic events include: • Paleocene/Eocene (P/E) deep-sea foraminifera extinction (55.5 Ma; Thomas, 2007) • Eocene/Oligocene (E/O) deep-sea foraminifera turnover (36–31 Ma; Thomas, 2007) • Oligocene/Miocene (O/M) Caribbean coral extinction (~23 Ma; Johnson et al., 2009) • Mid-Miocene deep-sea foraminifera turnover (~15 Ma; Thomas, 2007) • West Tethys hotspot (Mesozoic to ~30 Ma; Renema et al., 2008) • Indo-Australian Archipelago (IAA) hotspot (23–0 Ma; Renema et al., 2008; Yasuhara et al., 2017a) • Arabian hotspot (23–16 Ma; Renema et al., 2008) • Caribbean extinctions (3–1 Ma; O’Dea et al., 2007; O’Dea and Jackson 2009) • MPT deep-sea foraminifera extinction (1.2–0.7 Ma; Hayward et al., 2007) • MBE Arctic (0.43 Ma; Cronin et al., 2017) and Japan Sea (JS) deep-sea ostracod turnover (0.43 Ma; Huang et al., 2018) Ice sheets: • The durations of the Northern Hemisphere (NH) and Antarctic ice sheets are from Zachos et al. BIOTIC DYNAMICS OVER MILLIONS OF YEARS (2001) 70 65 60 55 50 45 40 35 30 25 20 15 10 5 0 5 4 3 2 1 0 Bering Strait opened Fram Strait opened Himalayan uplift MMCO Mi−1 E Asian Monsoon intensifed IAA hotspot O/M Caribbean coral extinction EECO PETM P/E foram extinction Mid-Miocene foram turnover AMOC onset EOT E/O foram turnover 5 4 3 2 1 0 5.0 4.0 3.0 Pliocene Quaternary MBE MPT MPTO MBE Arctic & JS ostracod turnover MPT foram extinction Caribbean extinctions a Aus-Pac-Eur collision Plio Q Panama-SA collision Formation of the Panama Isthmus complete Arabian hotspot b b Age (Ma) West-Tethys hotspot Paleoc Eocene Oligo Miocene Ara-Ana collision Tethys SW closed Drake Passage opened Tasmanian Passage opened NH. ice sheets Antarctic ice sheets c δ18O (‰) δ18O (‰) 70 Ma 60 Ma 50 Ma 40 Ma 30 Ma 20 Ma 10 Ma 1 Ma 70 65 60 55 50 45 40 35 30 25 20 15 10 5 0 5 4 3 2 1 0 Bering Strait opened Fram Strait opened Himalayan uplift MMCO Mi−1 E Asian Monsoon intensifed IAA hotspot O/M Caribbean coral extinction EECO PETM P/E foram extinction Mid-Miocene foram turnover AMOC onset EOT E/O foram turnover 5 4 3 2 1 0 5.0 4.0 3.0 Pliocene Quaternary MBE MPT MPTO MBE Arctic & JS ostracod turnover MPT foram extinction Caribbean extinctions a Aus-Pac-Eur collision Plio Q Panama-SA collision Formation of the Panama Isthmus complete Arabian hotspot b b Age (Ma) West-Tethys hotspot Paleoc Eocene Oligo Miocene Ara-Ana collision Tethys SW closed Drake Passage opened Tasmanian Passage opened NH. BIOTIC DYNAMICS ON MILLENNIAL TIMESCALES example, deep-sea benthic foramin- ifera show a prominent global extinction event during the MPT (Hayward et al., 2007). Similarly, ostracod taxa with affin- ity for warm temperatures were abundant both in the Arctic and the North Atlantic Oceans before the MBE (DeNinno et al., 2015; Cronin et al., 2017). However, after the MBE warm-adapted taxa went extinct regionally in the Arctic, with shrink- ing distributions to the south (DeNinno et al., 2015; Cronin et al., 2017). In the Sea of Japan, endemic cool water species replaced circumpolar species after the MBE, and many circumpolar species went extinct regionally (Ozawa and Kamiya, 2005; Cronin and Ikeya, 1987; Huang et al., 2018, 2019). In sum, when orbital cyclicity is consistent, microfossil species seem to show evidence of repeated com- munity assembly that matches prevailing conditions, indicating that community assembly may be deterministic. Changes in the expression of orbital forcing, how- ever, can lead to extinction. How and why such changes instigate widespread biotic disturbance is not well understood, but In addition to million-year timescales, sediment core data provide insight into biodiversity-climate dynamics on millen- nial timescales. Orbital variations have resulted in changes in climate on 10,000- to 100,000-year timescales throughout Earth history, as is well documented in benthic δ18O records (Figure 2B; Raymo et al., 2004; Lisiecki and Raymo, 2005). Surface-ocean and deep-ocean condi- tions change in pace with orbital forc- ing of global climate, as do the locations of oceanic currents and bio-provinces (Cronin, 2009). These oceanic changes are matched by changes in the composi- tion of marine microfossil communities that mirror orbital-scale climatic changes (Cronin et al., 1996, 1999; Cronin and Raymo, 1997). Orbital forcing provides repeated experiments on community assembly, with microfossil assemblages demonstrating that similar communities come together repeatedly under similar environmental conditions (Cronin et al., 1996; Beaufort et al., 1997; Yasuhara and Changes in the dominant mode of orbital cyclicity can, by contrast, per- manently disturb marine ecosystems (Hayward et al., 2007; DeNinno et al., 2015; Cronin et al., 2017; Huang et al., 2018, 2019), such as during the transi- tion from 41,000- to 100,000-year cycles in the Mid-Pleistocene Transition (MPT) and the Mid-Brunhes Event (MBE). For BOX 2. MICROFOSSIL BIODIVERSITY TRENDS OVER THE CENOZOIC 2014; Lowery et al., 2020). Cetaceans show trends similar to those of diatoms, and the two may have coevolved (Berger, 2007; Marx and Uhen, 2010). BIOTIC DYNAMICS OVER MILLIONS OF YEARS (2001) δ O (‰) • MMCO: Mid-Miocene Climatic Optimum (18–14 Ma; Cronin, 2009) • East Asian Monsoon intensified (15–10 Ma; Tada et al., 2016) ( ) c 70 Ma 60 Ma 50 Ma 40 Ma 30 Ma 20 Ma 10 Ma 1 Ma FIGURE 2. Cenozoic global changes and major events. (a) Cenozoic summary and (b) Plio-Pleistocene closeup. Global deep-sea oxygen isotope records (smaller value indicates warmer climate) are from Zachos et al. (2001) for (a), and from the LR04 stack of Lisiecki and Raymo (2005) for (b). Major tectonic (green), climatic (blue), biotic (red), and ice-sheet (purple) events are shown. (c) Paleogeographic maps from Leprieur et al. (2016). Light blue: deep tropical ocean. Yellow: tropical shallow reefs. White and light gray: deep ocean and shallow waters outside the tropical boundary, respectively. c FIGURE 2. Cenozoic global changes and major events. (a) Cenozoic summary and (b) Plio-Pleistocene closeup. Global deep-sea oxygen isotope records (smaller value indicates warmer climate) are from Zachos et al. (2001) for (a), and from the LR04 stack of Lisiecki and Raymo (2005) for (b). Major tectonic (green), climatic (blue), biotic (red), and ice-sheet (purple) events are shown. (c) Paleogeographic maps from Leprieur et al. (2016). Light blue: deep tropical ocean. Yellow: tropical shallow reefs. White and light gray: deep ocean and shallow waters outside the tropical boundary, respectively. Oceanography \| June 2020 Early Online Release Oceanography \| June 2020 Early Online Release Cronin, 2008; Huang et al., 2018). This close association supports climate, and particularly temperature, as a key driver of marine biodiversity, with the forma- tion of similar assemblages reflecting the process of species tracking their pre- ferred temperature conditions. Orbital- scale time-series studies, for example, consistently show that temperature rather than productivity is the best predictor of deep-sea biodiversity patterns (Hunt et al., 2005; Yasuhara et al., 2009, 2012b). Temperature likely controls biodiversity because fewer species can physiologi- cally tolerate conditions in colder places than in warmer places on these ecological timescales (Currie et al., 2004; Yasuhara and Danovaro, 2016). BIOTIC DYNAMICS ON CENTENNIAL TIMESCALES Recent advances in high-resolution paleoceanographic studies (Bond et al., 1997; Bianchi and McCave, 1999; deMenocal et al., 2000; Oppo et al., 2003; McManus et al., 2004; Yasuhara et al., 2019a) have improved understanding of centennial-scale biotic responses to cli- mate change, bridging the gap between geological timescales and the timescales of ecological studies. The centennial timescale has long been a “blind spot” in ecological analysis, lodged between the range of biological monitoring without historical reconstruction and the res- olution attainable in most paleontological research (Yasuhara, 2019). Paleobiological records on this timescale can be garnered from micro- fossils preserved in sediments deposited under high sedimentation rates and/or conditions that minimize post-depositional sediment mixing. Such records can be found in sediment drifts (e.g., sediments collected from a sediment drift at ODP Site 1055 on the Carolina Slope show sedimentation rates of ~23 cm per thousand years; Yasuhara et al., 2008) or in enclosed settings in marginal oceanic basins that act as natural sediment traps and where local anoxia prevents sediment mixing (such as the basins of the Californian borderland, Cariaco Basin, or silled fjords; see Yasuhara et al., 2019c). Although seasonal and annual signals are likely smoothed by bioturbation in many of these sediment cores, it is minimized in cores from anoxic basins. By using these minimally disturbed cores, decadal community changes can be reconstructed to bridge the gaps across timescales (Kuwae et al., 2017; Salvatteci et al., 2018; see the next section). Number of species Number of species Number of species 0 0 0 00 Number of species 40 60 80 100 Number of species Pioneering studies using records from the Gulf of Mexico and the Santa Barbara Basin off California have documented responses of biodiversity to centennial-scale abrupt climatic changes (Flower and Kennett, 1995; Cannariato et al., 1999). Initial findings documenting the response of benthic foraminifera to centennial-scale deoxygen- ation events in the Santa Barbara Basin (Cannariato et al., 1999) have been complemented by data from various benthic groups, includ- ing molluscs, foraminifera, ostracods, and ophiuroids (Moffitt et al., 2015; Myhre et al., 2017). BIOTIC DYNAMICS ON MILLENNIAL TIMESCALES The contrast between calcareous and siliceous microfossil biodiversity patterns may occur because calcareous microfossils tend to be dominant and diverse in tropical and sub- tropical latitudes, whereas siliceous groups are dominant in polar, high latitude seas (Rutherford et al., 1999; Powell and Glazier, 2017; Dutkiewicz et al., 2020; Lowery et al., 2020). Thus, colder periods may allow for higher biodiversity in siliceous microfossil groups, given their preference for cooler waters. Alternatively, the differences could also reflect biases resulting from preserva- tion and sampling of siliceous microfossils (Lowery et al., 2020). The Cenozoic biodiversity curve for planktonic foraminifera (Ezard et al., 2011; Fraass et al., 2015) is surprisingly similar to those for other global marine groups, such as sharks (Condamine et al., 2019) and calcareous nannofossils (Lowery et al., 2020; Rabosky and Sorhannus, 2009), and to a regional curve of Neotropical ter- restrial plants (Jaramillo et al., 2006) (Figure 3). Commonalities among these curves include: (1) peak biodiversity in the Eocene (56–34 Ma), (2) a major extinction event at the Eocene-Oligocene boundary, (3) a Miocene diversification phase (for planktonic foraminifera and sharks), and (4) a Pliocene biodiversity high (for planktonic foraminifera and sharks) (Figure 3). Dinoflagellates also show a similar Eocene diversity peak (Katz et al., 2005; Stover et al., 1996). Oceanography \| https://doi.org/10.5670/oceanog.2020.225 Oceanography \| June 2020 21 p g ( y , ) Oceanography \| https://doi.org/10.5670/oceanog.2020.225 In contrast to calcareous microfossil groups such as planktonic foraminifera and calcareous nannofossils, siliceous microfossil groups such as diatoms and radiolarians show contrasting bio- diversity trends (Lowery et al., 2020). BIOTIC DYNAMICS ON MILLENNIAL TIMESCALES For example, diatom diver- sity increased during cooling periods, peaked at the Eocene- Oligocene transition, and reached highest levels during the Plio-Pleistocene (Figure 3; Katz et al., 2005, 2007; Lazarus et al., PETM EECO EOT 5 4 3 2 1 0 −1 δ18O (‰) Planktonic foraminifera (morphospecies) 0 20 40 60 80 Number of species Planktonic foraminifera (lineages) 10 15 20 25 30 35 Number of species Neotropical palynofora 150 200 250 300 350 Number of species 40 60 80 100 120 140 160 Number of species Diatoms 40 60 80 100 Number of species Cetaceans 0 20 40 60 80 100 120 Number of genera bf$Age_ka/1000 20 30 40 50 Equatorial Pacifc Number of species Deep−sea foraminifera Maastrichtian Danian Selandian Thanetian Ypresian Lutetian Bartonian Priabonian Rupelian Chattian Aquitanian Burdigalian Langhian Serravallian Tortonian Messinian Zanclean Quaternary K Paleoc Eocene Oligo Miocene Q Plio LBG onset 70 60 50 40 30 20 10 0 5 Age (Ma) 70 60 50 40 30 20 10 0 5 Age (Ma) MMCO Southern Ocean Lamniform sharks Calcareous nannofossils ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● may be explained by the scale of environmental perturbation; larger- scale changes could exceed species’ tolerances and/or eliminate poten- tial refugia (Hayward et al., 2012). MICROFOSSILS AND THE BRAVE NEW ANTHROPOCENEh position of marine plankton commu- nities. They found that Anthropocene assemblages differ from their pre- industrial equivalents, and the observed differences in species composition are consistent with the expected effect of cur- rent temperature change trends (Jonkers et al. 2019). Similarly, Moy et al. (2009) used shells of planktonic foraminifera from surface sediments as a benchmark for calcification intensity in living plank- tonic foraminifera. These authors discov- ered that shells of modern Globigerina bulloides are about a third lighter than those from the sediments, consistent with reduced calcification induced by ocean acidification in the Anthropocene. Their results were recently confirmed by Fox et al. (2020), who observed shell thin- ning when comparing planktonic fora- minifera specimens collected from his- torical (HMS Challenger, 1872–1876) to recent (Tara Oceans, 2009–2016) plank- ton samples. Large collections of surface sediment samples, both modern and his- torical (Rillo et al., 2019), are available to study, providing an opportunity to quan- tify anthropogenic impacts on the com- position of a range of marine ecosystems and on traits of their constituent species. and on traits of their constituent species. Areas with particularly high rates of sedimentation (e.g., ~50–100 cm per hundred years in Osaka Bay and >120 cm per thousand years in the Santa Barbara Basin; Barron et al., 2010; Field et al., 2006; Yasuhara et al., 2007) can provide insight into the effects that humans are having on marine ecosystems on even finer tempo- ral scales. Study of these sediments has revealed significant marginal marine eco- system degradation caused by human- induced eutrophication and resulting bot- tom water hypoxia (Barmawidjaja et al., 1995; Cooper, 1995; Cronin and Vann, 2003; Weckström et al., 2007; Willard and Cronin, 2007; Yasuhara et al., 2007, 2019c; Tsujimoto et al., 2008). The sediment layer on the surface of the ocean floor represents a time-averaged assemblage of microfossils. Because of slow sedimentation in the deep sea (typ- ically less than 10 cm per 1,000 years) and sediment mixing down to a depth of 10 cm (bioturbation), a typical 1 cm thick surface-sediment sample represents aver- age deposition over centuries to millennia (Jonkers et al., 2019). Thus, the propor- tion of microfossils recording conditions of the Anthropocene (typically >~1950) in surface sediments is negligible, which means core-top sediments typically pro- vide a global pre-industrial baseline for the state of marine communities in fos- silized organisms (Jonkers et al., 2019; Yasuhara et al., 2020). MICROFOSSILS AND THE BRAVE NEW ANTHROPOCENEh Although anthropogenic forcing is the primary driver of current biodiversity change (Díaz et al., 2019), natural vari- ability in community composition is also at play, and its overprinting can prevent quantification of anthropogenic effects. Measuring this baseline temporal vari- ability is crucial to partition the human signal, but is difficult to accomplish without long ecological time series that precede the Anthropocene. Microfossils Recently, Jonkers et al. (2019) com- pared planktonic foraminifera assem- blages collected from surface sediments that provide a pre-industrial baseline with assemblages collected from sedi- ment traps that monitored particle flux to the seafloor over the last 40 years. The authors examined whether anthropo- genic climate change modified the com- BOX 3. AUTOMATION BOX 3. AUTOMATION lyzed at the assemblage scale (Beaufort et al., 2014; Elder et al., 2018; Hsiang et al., 2018, 2019; Kahanamoku et al., 2018). lyzed at the assemblage scale (Beaufort et al., 2014; Elder et al., 2018; Hsiang et al., 2018, 2019; Kahanamoku et al., 2018). Another advantage of the microfossil record is that it is increas- ingly possible to automate key steps in gathering and processing data, due in part to the small size of samples and specimens. Once a core is obtained, the major data-gathering steps are washing and sieving sediment; picking, identifying, and mount- ing specimens; and, for studies of phenotypic evolution, measur- ing morphological traits of specimens. Recent technological and methodological advances can substantially reduce the time and effort required for some of these steps. Automated picking systems that take sieved size fractions, sep- arate them into individual particles, and image each particle may greatly reduce picking times (de Garidel-Thoron et al., 2017; Itaki et al., 2020). With samples that have already been picked and mounted, hundreds or thousands of individual microfossils can be imaged simultaneously in three dimensions and algorithmi- cally parsed into individual images from which basic morphomet- ric traits and features can be automatically extracted and ana- These efforts build on decades of previous automation work that either extracted coarser (size related) data or was relatively more labor intensive (Bollmann et al., 2005; Knappertsbusch et al., 2009). Given sufficient training data sets, convolutional neural nets can now identify planktonic foraminifera, cocco- lithophores, and radiolarians with accuracy similar to that of taxo- nomic specialists (Beaufort and Dollfus, 2004; de Garidel-Thoron et al., 2017; Hsiang et al., 2019; Itaki et al., 2020). BIOTIC DYNAMICS ON CENTENNIAL TIMESCALES Cenozoic climate and biodiversity estimates of mul- Oceanography \| June 2020 Early Online Release Oceanography \| June 2020 Early Online Release and on traits of their constituent species. Areas with particularly high rates of sedimentation (e.g., ~50–100 cm per hundred years in Osaka Bay and >120 cm per thousand years in the Santa Barbara Basin; Barron et al., 2010; Field et al., 2006; Yasuhara et al., 2007) can provide insight into the effects that humans are having on marine ecosystems on even finer tempo- ral scales. Study of these sediments has revealed significant marginal marine eco- system degradation caused by human- induced eutrophication and resulting bot- tom water hypoxia (Barmawidjaja et al., 1995; Cooper, 1995; Cronin and Vann, 2003; Weckström et al., 2007; Willard and Cronin, 2007; Yasuhara et al., 2007, 2019c; Tsujimoto et al., 2008). BIOTIC DYNAMICS ON CENTENNIAL TIMESCALES Centennial-scale records spanning the last 20,000 years in the North Atlantic Ocean have revealed that deep- sea benthic ostracod diversity responded to changes in deepwater circulation and temperature during the abrupt climatic changes of the Heinrich I (17,000–14,600 yr BP), the Younger Dryas (12,900– 11,700 yr BP), and the 8.2 ka event (8200 yr BP) without recognizable time lags (Yasuhara et al., 2008, 2014; Yasuhara, 2019). These stud- ies also documented rapid rearrangement of local communities fol- lowing the abrupt climatic changes. Thus, even on centennial times- cales, climate, and more specifically temperature, has dramatic effects on marine biodiversity. At least locally, the dominant response to cli- mate change seems to involve range shifts and recolonization from the same species pool (Yasuhara and Cronin, 2008; Yasuhara et al., 2009; Yasuhara and Danovaro, 2016). Excessive extinctions in the future may therefore affect the resilience of these ecosystems. FIGURE 3. Cenozoic climate and biodiversity estimates of mul- tiple taxonomic groups. From top to bottom: oxygen isotopic records (Zachos et al., 2001; smaller value indicates warmer climate) and biodiversity curves of global planktonic foramin- ifera morphospecies (Lowery et al., 2020) and lineages (Ezard et al., 2011), global lamniform sharks (Condamine et al., 2019), neotropic palynoflora (Jaramillo et al., 2006), global calcareous nannofossils (Lowery et al., 2020), global diatoms (Lazarus et al., 2014), global cetacean genera (Marx and Uhen, 2010; Uhen, 2020), and Southern Ocean and equatorial Pacific deep-sea benthic foraminifera (Thomas and Gooday, 1996). The onset of the deep-sea benthic foraminifera latitudinal biodiversity gradi- ent (LBG) is indicated at ~37 million years ago. FIGURE 3. Cenozoic climate and biodiversity estimates of mul- tiple taxonomic groups. From top to bottom: oxygen isotopic records (Zachos et al., 2001; smaller value indicates warmer climate) and biodiversity curves of global planktonic foramin- ifera morphospecies (Lowery et al., 2020) and lineages (Ezard et al., 2011), global lamniform sharks (Condamine et al., 2019), neotropic palynoflora (Jaramillo et al., 2006), global calcareous nannofossils (Lowery et al., 2020), global diatoms (Lazarus et al., 2014), global cetacean genera (Marx and Uhen, 2010; Uhen, 2020), and Southern Ocean and equatorial Pacific deep-sea benthic foraminifera (Thomas and Gooday, 1996). The onset of the deep-sea benthic foraminifera latitudinal biodiversity gradi- ent (LBG) is indicated at ~37 million years ago. FIGURE 3. Oceanography \| https://doi.org/10.5670/oceanog.2020.225 Oceanography \| June 2020 23 Oceanography \| https://doi.org/10.5670/oceanog.2020.225 FUTURE OUTLOOKh The continuity and duration of marine sediment core data make it possible to assess the relative importance of abrupt versus gradual, secular changes in cli- mate to species and communities, tied to a refined (and ever improving) under- standing of past climate change. The importance of spatial and temporal scales in (macro)ecology and (macro)evolution is well known (Brown and Maurer, 1989; Benton, 2009; Blois et al., 2013), with the patterns and drivers differing across space (Chiu et al., 2019; Jöst et al., 2019; Kusumoto et al., 2020) and time (Huang et al., 2018; Yasuhara et al., 2016, 2019b). Marine sediment cores permit interroga- tion of these dynamics at multiple tem- poral scales (Lewandowska et al., 2020). Biotic interactions generally tend to con- trol dynamics on smaller spatial and tem- Fish-scale paleobiological studies have similarly provided insight on baseline variability in fish populations. For exam- ple, marginal marine and continental margin sediments in the Pacific extended population dynamics for anchovy and sardines back to the nineteenth century and past millennia (Baumgartner et al., 1992; Field et al., 2009; Checkley et al., 2017; Kuwae et al., 2017; Salvatteci et al., Yet, linking paleoecological insights to modern-day ecological change is rel- atively unexplored, and such insights do not necessarily make their way into informing global policy. Identifying and circumscribing the limits of such trans- position remain challenging, particu- larly given the taxonomic biases of pres- ervation and the rapidity of modern-day change. The opportunity remains to address such challenges and ensure that paleoecological data complement mod- ern ecological data and, where appro- priate, contribute to assessments and policy (e.g., IPBES [Intergovernmental Science-Policy Platform on Biodiversity and Ecosystem Services], the post- 2020 Global Biodiversity Framework of the CBD [Convention on Biological Diversity]). Studies of sediment cores provide a long-term perspective on climate/biodiversity links that can con- textualize modern marine ecological change and provide insights that would otherwise remain absent. “ While we lack a true time machine, the opportunity provided by this “biological time machine” remains unique.” . “ While we lack a true time machine, the opportunity provided by this “biological time machine” remains unique.” . ” . poral scales, while physical climatic fac- tors, particularly temperature as reviewed here, appear to dominate biotic dynamics on larger scales (Benton, 2009; Yasuhara et al., 2016). MICROFOSSILS AND THE BRAVE NEW ANTHROPOCENEh Given these ongoing developments, it is becoming possible to envision a near future in which the entire sample processing and data extraction workflow is streamlined and largely automated, with taxonomic experts guiding and overseeing the process but spending the majority of their time analyzing data sets that may be far larger, denser, and more data-rich than is currently feasible. provide these time-series data on com- munity composition across multiple tem- poral scales, albeit for a limited set of taxa. For example, using planktonic fora- minifera data, Lewandowska et al. (2020) compared the magnitude of biodiver- sity change across temporal scales from decades to millions of years. They found that, as expected, biodiversity change was greatest across the longest multi-million- year timescale and decreased at shorter timescales. However, they observed rela- tively large changes in community com- position, comparable to the magnitude of changes over the longest timescale, most recently. The magnitude of recent turn- over is suggestive of a large anthropogenic effect but may also reflect “noisy” annu- ally averaged sediment trap time series. especially in the tropics (Dornelas et al., 2018; Blowes et al., 2019), but those that are available are extremely valuable for comparing the magnitude of biodiversity change observed in the fossil record to that observed in response to anthropo- genic and recent climatic forcing. particularly with concerted comparisons among Anthropocene, centennial, mil- lennial, and million-year timescales. Regardless of timescale, paleobiolog- ical studies, especially those that exam- ine the relationship between climate and biodiversity and ecosystem func- tioning, provide insight into the poten- tial response of biodiversity to ongoing climate change. The pace and scale of anthropogenic impacts on ecosystems and ecosystem services remain of great concern (Díaz et al., 2019). Climate change is expected to have an accelerat- ing effect on the ocean, yet the challenges of using relatively short-term ecologi- cal data to understand long-term conse- quences to biodiversity and ecosystems remain significant. Sediment cores and associated microfossils can help elucidate links between climate and spatial bio- diversity (e.g., Yasuhara et al., 2012c, 2020), extinction risks (e.g., Harnik et al., 2012; Finnegan et al., 2015), natural base- lines (e.g., Yasuhara et al., 2012a, 2017b), and biotic consequences on evolutionary timescales (e.g., Ezard et al., 2011). REFERENCES The sedimentary record of climatic and anthropogenic influence on the Patuxent estuary and Chesapeake Bay ecosys- tems. Estuaries 26:196–209, https://doi.org/10.1007/ BF02695962. Bond, G.C., W. Showers, M. Cheseby, R. Lotti, P. Almasi, P. deMenocal, P. Priore, H. Cullen, I. 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AUTHORS Moriaki Yasuhara (moriakiyasuhara@gmail.com; yasuhara@hku.hk) is Associate Professor, School of Biological Sciences and Swire Institute of Marine Science, The University of Hong Kong, and a member of State Key Laboratory of Marine Pollution, City University of Hong Kong, Hong Kong SAR. Huai-Hsuan May Huang is Postdoctoral Researcher, School of Biological Sciences and Swire Institute of Marine Science, The University of Hong Kong, Hong Kong SAR. Pincelli Hull is Assistant Professor, Department of Geology and Geophysics, Yale University, New Haven, CT, USA. Marina C. ACKNOWLEDGMENTS We thank Katsunori Kimoto, Jeremy R. Young, Kotaro Hirose, Tamotsu Nagumo, Yoshiaki Aita, Noritoshi Suzuki, David Lazarus, André Rochon, Erin M. Dillon, and Briony Mamo for help with microfossil images; Simon J. Crowhurst and David A. Hodell for help with coring overview illustrations; Richard D. Norris, R. 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https://openalex.org/W3105313605	https://www.biorxiv.org/content/biorxiv/early/2020/11/18/2020.11.16.386086.full.pdf	English	null	Clearance of <i>Clostridioides difficile</i> colonization is associated with antibiotic-specific bacterial changes	bioRxiv (Cold Spring Harbor Laboratory)	2,020	cc-by	13,931	. CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted November 18, 2020. ; https://doi.org/10.1101/2020.11.16.386086 doi: bioRxiv preprint . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted November 18, 2020. ; https://doi.org/10.1101/2020.11.16.386086 doi: bioRxiv preprint Clearance of Clostridioides difﬁcile colonization is associated with antibiotic-speciﬁc bacterial changes Running title: Clearance of Clostridioides difﬁcile colonization Nicholas A. Lesniak1, Alyxandria M. Schubert1, Hamide Sinani1, Patrick D. Schloss1,† † To whom correspondence should be addressed: pschloss@umich.edu 1. Department of Microbiology and Immunology, University of Michigan, Ann Arbor, MI 1. Department of Microbiology and Immunology, University of Michigan, Ann Arbor, MI 1 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted November 18, 2020. ; https://doi.org/10.1101/2020.11.16.386086 doi: bioRxiv preprint . CC-BY 4.0 International license available under a which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted November 18, 2020. ; https://doi.org/10.1101/2020.11.16.386086 doi: ioRxiv preprint Abstract 1 The gut bacterial community prevents many pathogens from colonizing the intestine. Previous 2 studies have associated speciﬁc bacteria with clearing Clostridioides difﬁcile colonization across 3 different community perturbations. However, those bacteria alone have been unable to clear C. 4 difﬁcile colonization. To elucidate the changes necessary to clear colonization, we compared 5 differences in bacterial abundance between communities able and unable to clear C. difﬁcile 6 colonization. We treated mice with titrated doses of antibiotics prior to C. difﬁcile challenge which 7 resulted in no colonization, colonization and clearance, or persistent colonization. Previously, we 8 observed that clindamycin-treated mice were susceptible to colonization but spontaneously cleared 9 C. difﬁcile. Therefore, we investigated whether other antibiotics would show the same result. We 10 found reduced doses of cefoperazone and streptomycin permitted colonization and clearance 11 of C. difﬁcile. Mice that cleared colonization had antibiotic-speciﬁc community changes and 12 predicted interactions with C. difﬁcile. Clindamycin treatment led to a bloom in populations related 13 to Enterobacteriaceae. Clearance of C. difﬁcile was concurrent with the reduction of those blooming 14 populations and the restoration of community members related to the Porphyromonadaceae 15 and Bacteroides. Cefoperazone created a susceptible community characterized by a drastic 16 reduction in the community diversity, interactions, and a sustained increase in abundance of 17 many facultative anaerobes. Lastly, clearance in streptomycin-treated mice was associated with 18 the recovery of multiple members of the Porphyromonadaceae, with little overlap in the speciﬁc 19 Porphyromonadaceae observed in the clindamycin treatment. Further elucidation of how C. difﬁcile 20 colonization is cleared from different gut bacterial communities will improve C. difﬁcile infection 21 treatments. 22 2 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted November 18, 2020. ; https://doi.org/10.1101/2020.11.16.386086 doi: bioRxiv preprint Importance 23 The community of microorganisms, known as the microbiota, in our intestines prevents pathogens, 24 such as C. difﬁcile, from establishing themselves and causing infection. This is known as 25 colonization resistance. However, when a person takes antibiotics, their gut microbiota is disturbed. 26 This disruption allows C. difﬁcile to colonize. C. difﬁcile infections (CDI) are primarily treated with 27 antibiotics, which frequently leads to recurrent infections because the microbiota have not yet 28 returned to a resistant state. The infection cycle often ends when the fecal microbiota from a 29 presumed resistant person are transplanted into the susceptible person. Although this treatment 30 is highly effective, we do not understand the mechanism of resistance. We hope to improve the 31 treatment of CDI through elucidating how the bacterial community eliminates C. difﬁcile colonization. 32 We found C. difﬁcile was able to colonize susceptible mice but was spontaneously eliminated 33 in an antibiotic-treatment speciﬁc manner. These data indicate each community had different 34 requirements for clearing colonization. Understanding how different communities clear colonization 35 will reveal targets to improve CDI treatments. 36 3 3 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted November 18, 2020. ; https://doi.org/10.1101/2020.11.16.386086 doi: bioRxiv preprint Introduction 37 CC-BY 4.0 International license available under a which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted November 18, 2020. ; https://doi.org/10.1101/2020.11.16.386086 doi: ioRxiv preprint . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted November 18, 2020. ; https://doi.org/10.1101/2020.11.16.386086 doi: bioRxiv preprint elimination of C. difﬁcile colonization. We hypothesized that each colonized community has 64 perturbation-speciﬁc susceptibilities and requires speciﬁc changes to clear the pathogen. To 65 induce a less severe perturbation, we reduced the doses of cefoperazone and streptomycin. This 66 resulted in communities that were initially colonized to a high level (>106 CFU/g feces) and then 67 spontaneously cleared C. difﬁcile. We found each antibiotic resulted in unique changes in the 68 microbiota that were associated with the persistence or clearance of C. difﬁcile. These data further 69 support the hypothesis that C. difﬁcile can exploit numerous niches in perturbed communities. 70 Introduction 37 A complex consortium of bacteria and microbes that inhabits our gut, known as the microbiota, 38 prevent pathogens from colonizing and causing disease. This protection, known as colonization 39 resistance, is mediated through many mechanisms such as activating host immune responses, 40 competing for nutrients, producing antimicrobials, and contributing to the maintenance of the 41 mucosal barrier (1). However, perturbations to the intestinal community or these functions opens 42 the possibility that a pathogen can colonize (2). For example, the use of antibiotics perturb the gut 43 microbiota and can lead to Clostridioides difﬁcile infection (CDI). 44 CDI is especially problematic due to its burden on the healthcare system (3, 4). C. difﬁcile can 45 cause severe disease, such as toxic megacolon, diarrhea, and death (5). CDI is primarily treated 46 with antibiotics (6). CDIs recalcitrant to antibiotics are eliminated by restoring the community with 47 a fecal microbiota transplant (FMT), returning the perturbed community to a healthier protective 48 state (7, 8). However, FMTs are not always effective against CDI and have the risk of transferring a 49 secondary infection (9, 10). Therefore, we need to better understand how the microbiota clears the 50 infection to develop more effective treatments. 51 Previous research has shown that the microbiota affects C. difﬁcile colonization. Mouse models 52 have identiﬁed potential mechanisms of colonization resistance such as bile salt metabolism and 53 nutrient competition (11–14). However, studies that have restored those functions were unable to 54 restore complete resistance (15, 16). This could be attributed to the complexity of the community 55 and the mechanisms of colonization resistance (17, 18). We previously showed that when C. 56 difﬁcile colonizes different antibiotic-treated murine communities it modiﬁes its metabolism to ﬁt 57 each speciﬁc environment (14, 19, 20). Therefore, we have investigated the bacterial community 58 dynamics concurrent with C. difﬁcile elimination across uniquely perturbed communities. 59 Jenior et al. (20) observed that clindamycin-treated mice spontaneously cleared C. difﬁcile 60 colonization whereas mice treated with cefoperazone and streptomycin did not. Here, we continued 61 to explore the different effects these three antibiotics have on C. difﬁcile colonization. The 62 purpose of this study was to elucidate the gut bacterial community changes concurrent with 63 4 . Reduced doses of cefoperazone and streptomycin allowed communities to spontaneously 72 It is made The copyright holder for this preprint this version posted November 18, 2020. ; https://doi.org/10.1101/2020.11.16.386086 doi: ioRxiv preprint of these antibiotics uniquely changed the microbiota, we hypothesized that the microbiota varied 91 across these antibiotic treatments that resulted in colonization clearance. 92 of these antibiotics uniquely changed the microbiota, we hypothesized that the microbiota varied 91 across these antibiotic treatments that resulted in colonization clearance. 92 Reduced doses of cefoperazone and streptomycin allowed communities to spontaneously 72 Reduced doses of cefoperazone and streptomycin allowed communities to spontaneously 72 clear C. difﬁcile colonization. To understand the dynamics of colonization and clearance of C. 73 difﬁcile, we ﬁrst identiﬁed conditions which would allow colonization and clearance. Beginning with 74 clindamycin, mice were treated with an intraperitoneal injection of clindamycin (10 mg/kg) one day 75 prior to challenge with C. difﬁcile. All mice (N = 11) were colonized to a high level (median CFU = 76 3.07×107) the next day and cleared the colonization within 10 days; 6 mice cleared C. difﬁcile within 77 6 days (Figure 1A). Previous C. difﬁcile infection models using cefoperazone and streptomycin 78 have not demonstrated clearance. So we next explored whether cefoperazone and streptomycin 79 could permit colonization and subsequent clearance with lower doses. We began with replicating 80 the previously established C. difﬁcile infection models using these antibiotics (20). We treated 81 mice with cefoperazone or streptomycin in their drinking water for 5 days (0.5 mg/mL and 5 mg/mL, 82 respectively) and then challenged them with C. difﬁcile. For both antibiotics, C. difﬁcile colonization 83 was maintained for the duration of the experiment as previously demonstrated (Figure 1B-C) (20). 84 Then we repeated the C. difﬁcile challenge with reduced doses of the antibiotics (cefoperazone - 85 0.3 and 0.1 mg/mL; streptomycin - 0.5 and 0.1 mg/mL). For both antibiotic treatments, the lowest 86 dose resulted in either no colonization (N = 8) or a transient, low level colonization (N = 8, median 87 length = 1 day, median CFU/g = 2.8 × 103) (Figure 1B-C). The intermediate dose of both antibiotics 88 resulted in a high level colonization (median CFU/g = 3.5 × 106) and half (N = 8 of 16) of the mice 89 clearing the colonization within 10 days. Based on our previous research, which showed each 90 5 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted November 18, 2020. ; https://doi.org/10.1101/2020.11.16.386086 doi: bioRxiv preprint . CC-BY 4.0 International license available under a which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. C. difﬁcile was associated with antibiotic-speciﬁc changes to the microbiota 138 difﬁcile colonization in cefoperazone-treated mice was associated with a shift in the microbiota to a 139 new community structure which seemed unable to recover from the antibiotic perturbation, whereas 140 clearance occurred when the community was capable of returning to its original structure. 141 mice that cleared C. difﬁcile to those that did not. For the communities that cleared C.difﬁcile, 120 diversity was maintained throughout the experiment (Figure 2B). The mice treated with cefoperazone 121 that remained colonized experienced an increase in alpha diversity, likely driven by the decrease 122 in highly abundant populations and increase in low abundant populations (Figure 1E). These 123 persistently colonized communities also had a large shift away from the initial community structure 124 caused by the antibiotic treatment (P < 0.05), which remained through the end of the experiment (P 125 < 0.05) (Figure 2B). These data suggested that it was necessary for cefoperazone-treated mice to 126 become more similar to the initial pre-antibiotic community structure to clear C. difﬁcile. We next 127 investigated the changes in OTU abundances between the communities that cleared C. difﬁcile 128 and those that did not to elucidate the community members involved in clearance. Communities 129 that remained colonized were signiﬁcantly enriched in facultative anaerobic populations including 130 Enterococcus, Pseudomonas, Staphylococcus, and Enterobacteriaceae at the time of challenge. 131 Communities that cleared C. difﬁcile had signiﬁcant enrichment in 10 different OTUs related to the 132 Porphyromonadaceae at the end of the experiment (Figure 3A). We were also interested in the 133 temporal changes within each community so we investigated which OTUs changed due to antibiotic 134 treatment or during the C. difﬁcile colonization. The majority of signiﬁcant temporal differences in 135 OTUs for cefoperazone-treated mice occurred in persistently colonized communities. Persistently 136 colonized communities had a persistent loss of numerous relatives of the Porphyromonadaceae and 137 increases in the relative abundance of facultative anaerobes (Figure 4C, S2). Overall, persistent C. 138 difﬁcile colonization in cefoperazone-treated mice was associated with a shift in the microbiota to a 139 new community structure which seemed unable to recover from the antibiotic perturbation, whereas 140 clearance occurred when the community was capable of returning to its original structure. 141 Finally, we identiﬁed the differences in C. difﬁcile colonization for streptomycin-treated 142 mice. C. difﬁcile was associated with antibiotic-speciﬁc changes to the microbiota CC-BY 4.0 International license available under a which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted November 18, 2020. ; https://doi.org/10.1101/2020.11.16.386086 doi: ioRxiv preprint . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted November 18, 2020. ; https://doi.org/10.1101/2020.11.16.386086 doi: bioRxiv preprint mice that cleared C. difﬁcile to those that did not. For the communities that cleared C.difﬁcile, 120 diversity was maintained throughout the experiment (Figure 2B). The mice treated with cefoperazone 121 that remained colonized experienced an increase in alpha diversity, likely driven by the decrease 122 in highly abundant populations and increase in low abundant populations (Figure 1E). These 123 persistently colonized communities also had a large shift away from the initial community structure 124 caused by the antibiotic treatment (P < 0.05), which remained through the end of the experiment (P 125 < 0.05) (Figure 2B). These data suggested that it was necessary for cefoperazone-treated mice to 126 become more similar to the initial pre-antibiotic community structure to clear C. difﬁcile. We next 127 investigated the changes in OTU abundances between the communities that cleared C. difﬁcile 128 and those that did not to elucidate the community members involved in clearance. Communities 129 that remained colonized were signiﬁcantly enriched in facultative anaerobic populations including 130 Enterococcus, Pseudomonas, Staphylococcus, and Enterobacteriaceae at the time of challenge. 131 Communities that cleared C. difﬁcile had signiﬁcant enrichment in 10 different OTUs related to the 132 Porphyromonadaceae at the end of the experiment (Figure 3A). We were also interested in the 133 temporal changes within each community so we investigated which OTUs changed due to antibiotic 134 treatment or during the C. difﬁcile colonization. The majority of signiﬁcant temporal differences in 135 OTUs for cefoperazone-treated mice occurred in persistently colonized communities. Persistently 136 colonized communities had a persistent loss of numerous relatives of the Porphyromonadaceae and 137 increases in the relative abundance of facultative anaerobes (Figure 4C, S2). Overall, persistent C. C. difﬁcile was associated with antibiotic-speciﬁc changes to the microbiota Beginning with the clindamycin-treated mice, we analyzed their fecal 16S rRNA gene sequences to 94 identify the community features related to C. difﬁcile colonization and clearance. First, we compared 95 the most abundant bacterial genera of the communities at the time of C. difﬁcile challenge. The 96 clindamycin-treated mice became dominated by relatives of Enterobacteriaceae with a concurrent 97 reduction in the other abundant genera, except for populations of Lactobacillus (Figure 1D, S1). 98 These community changes permitted C. difﬁcile to colonize all of these mice, but all of the mice 99 were are also able to clear the colonization. We next investigated how the microbiota diversity 100 related to C. difﬁcile clearance. Clindamycin treatment decreased the alpha diversity (P < 0.05) and 101 similarity to the pre-clindamycin community at the time of C. difﬁcile challenge (P < 0.05) (Figure 102 2A). But it was not necessary to restore the community similarity to its initial state to clear C. difﬁcile. 103 Therefore we investigated the temporal differences in the abundance of the operational taxonomic 104 units (OTUs) between the initial untreated community and post-clindamycin treatment at the time 105 of challenge and between the time of challenge and the end of the experiment. Clindamycin 106 treatment resulted in large decreases in 21 OTUs and a bloom of relatives of Enterobacteriaceae 107 (Figure 4A). With the elimination of C. difﬁcile, we observed a drastic reduction of the relatives of 108 Enterobacteriaceae and recovery of 10 populations related to Porphyromonadaceae, Bacteroides, 109 Akkermansia, Lactobacillus, Biﬁdobacterium, Lachnospiraceae, and Clostridiales (Figure 4A). Thus, 110 clindamycin reduced most of the natural community allowing C. difﬁcile to colonize. The recovery of 111 only a small portion of the community was associated with eliminating the C.difﬁcile population. 112 We applied the same analysis to the cefoperazone-treated mice to understand what community 113 features were relevant to clearing C. difﬁcile. Increasing the dose of cefoperazone shifted the 114 dominant community members from relatives of the Porphyromonadaceae, Bacteroides and 115 Akkermansia to relatives of the Lactobacillus and Enterobacteriaceae at the time of challenge 116 (Figure 1E, S1). We saw a similar increase in relatives of Enterobacteriaceae with clindamycin. 117 However, the cefoperazone-treated mice that had larger increases in Enterobacteriaceae were 118 unable to clear C. difﬁcile. We next investigated the differences between the cefoperazone-treated 119 6 . C. difﬁcile was associated with antibiotic-speciﬁc changes to the microbiota Increasing the dose of streptomycin maintained the abundance of relatives of the 143 Porphyromonadaceae and Bacteroides, but reduced most of the other genera including populations 144 of the Lactobacillus, Lachnospiraceae, Ruminococcaceae, Alistipes, and Clostridiales (Figure 1F). 145 Both communities that cleared and those that remained colonized had similar changes in diversity. 146 Streptomycin-treated mice became mildly dissimilar (P < 0.05) and less diverse (P < 0.05) with 147 streptomycin treatment but by the end of the experiment returned to resemble the pre-antibiotic 148 7 . CC-BY 4.0 International license available under a which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted November 18, 2020. ; https://doi.org/10.1101/2020.11.16.386086 doi: ioRxiv preprint . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted November 18, 2020. ; https://doi.org/10.1101/2020.11.16.386086 doi: bioRxiv preprint community (P < 0.05) (Figure 2C). Those communities that remained colonized had slightly lower 149 alpha-diversity than those that cleared C. difﬁcile. (P < 0.05). Persistently colonized mice had 150 reduced relative abundance of relatives of Alistipes, Anaeroplasma, and Porphyromonadaceae 151 at time of challenge compared to the mice that cleared C. difﬁcle (Figure 3B). At the end of the 152 experiment the mice that were still colonized had lower abundances of Turicibacter, Alistipes, and 153 Lactobacillus. Since most of the differences were reduced relative abundances in the colonized 154 mice, we were interested to explore what temporal changes occurred between pre-antibiotic 155 treatment, the time of challenge, and the end of the experiment for the communities that cleared 156 C. difﬁcile. The temporal changes in streptomycin-treated mice were more subtle than those 157 observed with the other antibiotic treatments. At the time of challenge, the communities that 158 remained colonization had reductions in 4 OTUs related to the Porphyromonadaceae. Those that 159 cleared C. difﬁcile also had changes in OTUs related to the Porphyromonadaceae, however, 2 160 populations decreased and 2 increased in abundance (Figure 4B, D). C. difﬁcile was associated with antibiotic-speciﬁc changes to the microbiota difﬁcile were not as distinct as those observed with the cefoperazone treatment. 166 The differences between colonized and cleared streptomycin-treated mice were minimal, which 167 suggested the few differences may be responsible for the clearance. Overall, these data revealed 168 that while there were commonly affected families across the antibiotic treatments, such as the 169 Porphyromonadaceae, C. difﬁcile clearance was associated with community and OTU differences 170 speciﬁc to each antibiotic. 171 Distinct features of the bacterial community at the time of infection predicted end point 172 C. difﬁcile was associated with antibiotic-speciﬁc changes to the microbiota At the end of the experiment, 161 all communities experienced recovery of the abundance of many of the populations changed by 162 the streptomycin treatment, but the communities that remained colonized did not recover 5 of the 163 OTUs of Alistipes, Lactobacillus, and Porphyromonadaceae that were reduced by streptomycin. 164 The differences between the streptomycin-treated mice that remained colonized and those had 165 been cleared of C. difﬁcile were not as distinct as those observed with the cefoperazone treatment. 166 The differences between colonized and cleared streptomycin-treated mice were minimal, which 167 suggested the few differences may be responsible for the clearance. Overall, these data revealed 168 that while there were commonly affected families across the antibiotic treatments, such as the 169 Porphyromonadaceae, C. difﬁcile clearance was associated with community and OTU differences 170 speciﬁc to each antibiotic. 171 community (P < 0.05) (Figure 2C). Those communities that remained colonized had slightly lower 149 alpha-diversity than those that cleared C. difﬁcile. (P < 0.05). Persistently colonized mice had 150 reduced relative abundance of relatives of Alistipes, Anaeroplasma, and Porphyromonadaceae 151 at time of challenge compared to the mice that cleared C. difﬁcle (Figure 3B). At the end of the 152 experiment the mice that were still colonized had lower abundances of Turicibacter, Alistipes, and 153 Lactobacillus. Since most of the differences were reduced relative abundances in the colonized 154 mice, we were interested to explore what temporal changes occurred between pre-antibiotic 155 treatment, the time of challenge, and the end of the experiment for the communities that cleared 156 C. difﬁcile. The temporal changes in streptomycin-treated mice were more subtle than those 157 observed with the other antibiotic treatments. At the time of challenge, the communities that 158 remained colonization had reductions in 4 OTUs related to the Porphyromonadaceae. Those that 159 cleared C. difﬁcile also had changes in OTUs related to the Porphyromonadaceae, however, 2 160 populations decreased and 2 increased in abundance (Figure 4B, D). At the end of the experiment, 161 all communities experienced recovery of the abundance of many of the populations changed by 162 the streptomycin treatment, but the communities that remained colonized did not recover 5 of the 163 OTUs of Alistipes, Lactobacillus, and Porphyromonadaceae that were reduced by streptomycin. 164 The differences between the streptomycin-treated mice that remained colonized and those had 165 been cleared of C. Distinct features of the bacterial community at the time of infection pr 172 colonization. To determine whether the community composition at the time of C. difﬁcile challenge 173 could predict C. difﬁcile clearance, we built a machine learning model using L2 logistic regression. 174 We evaluated the predictive performance of the model using the area under the receiver operating 175 characteristic curve (AUROC), where a value of 0.5 indicated the model is random and 1.0 indicated 176 the model always correctly predicts the outcome. Our model resulted in a AUROC of 0.986 [IQR 177 8 . CC-BY 4.0 International license available under a which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted November 18, 2020. ; https://doi.org/10.1101/2020.11.16.386086 doi: ioRxiv preprint . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted November 18, 2020. ; https://doi.org/10.1101/2020.11.16.386086 doi: bioRxiv preprint 0.970-1.000], which suggested that the model was able to use the relative abundance of OTUs 178 at the time of challenge to accurately predict colonization clearance (Figure S3). To assess the 179 important features, we randomly permuted each OTU feature by removing it from the training set 180 to determine its effect on the prediction (Figure 5A). The most important feature was an OTU 181 related to the Enterobacteriaceae, whose abundance predicted clearance. This result appears to 182 have been strongly driven by the clindamycin data (Figure 5B, C). The remaining OTU features 183 did not have a large effect on the model performance, which suggested that the model decision 184 was spread across many features. These results revealed the model used the relative abundance 185 data of the community members and the relationship between those abundances to correctly 186 classify clearance. There were many OTUs with treatment and outcome speciﬁc abundance 187 patterns that did not agree with the odds ratio of the OTU used by the model. For example, 188 Enterobacteriaceae abundance inﬂuenced the model to predict clearance (Figure 5B), however 189 in experiments that used cefoperazone, the communities that remained colonized had higher 190 abundances of Enterobacteriaceae than the communities that cleared colonization (Figure 5C). Distinct features of the bacterial community at the time of infection pr 172 The 191 model arrived at the correct prediction through the inﬂuence of other OTUs. Therefore, the model 192 used different combinations of multiple OTUs and their relative abundances across treatments to 193 predict C. difﬁcile clearance. These data can offer a basis for hypotheses regarding the distinct 194 combinations of bacteria that promote C. difﬁcile clearance. 195 0.970-1.000], which suggested that the model was able to use the relative abundance of OTUs 178 at the time of challenge to accurately predict colonization clearance (Figure S3). To assess the 179 important features, we randomly permuted each OTU feature by removing it from the training set 180 to determine its effect on the prediction (Figure 5A). The most important feature was an OTU 181 related to the Enterobacteriaceae, whose abundance predicted clearance. This result appears to 182 have been strongly driven by the clindamycin data (Figure 5B, C). The remaining OTU features 183 did not have a large effect on the model performance, which suggested that the model decision 184 was spread across many features. These results revealed the model used the relative abundance 185 data of the community members and the relationship between those abundances to correctly 186 classify clearance. There were many OTUs with treatment and outcome speciﬁc abundance 187 patterns that did not agree with the odds ratio of the OTU used by the model. For example, 188 Enterobacteriaceae abundance inﬂuenced the model to predict clearance (Figure 5B), however 189 in experiments that used cefoperazone, the communities that remained colonized had higher 190 abundances of Enterobacteriaceae than the communities that cleared colonization (Figure 5C). The 191 model arrived at the correct prediction through the inﬂuence of other OTUs. Therefore, the model 192 used different combinations of multiple OTUs and their relative abundances across treatments to 193 predict C. difﬁcile clearance. These data can offer a basis for hypotheses regarding the distinct 194 combinations of bacteria that promote C. difﬁcile clearance. 195 Conditional independence networks revealed treatment-speciﬁc relationships between the 196 the relationship between temporal changes in the community and C. difﬁcile by building a 198 conditional independence network for each treatment using SPIEC-EASI (sparse inverse 199 covariance estimation for ecological association inference) (21). First, we focused on the ﬁrst-order 200 associations of C. difﬁcile (Figure 6A). In clindamycin-treated mice, C. difﬁcile had positive 201 associations with relatives of Enterobacteriaceae, Pseudomonas, and Olsenella and negative 202 associations with relatives of the Lachnospiraceae and Clostridium XIVa. C. difﬁcile had limited 203 associations in cefoperazone-treated mice; the primary association was positive with relatives 204 of Enterobacteriaceae. In streptomycin-treated mice, C. difﬁcile had negative associations 205 with relatives of the Porphyromonadaceae and positive associations with populations of the 206 9 . CC-BY 4.0 International license available under a which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted November 18, 2020. ; https://doi.org/10.1101/2020.11.16.386086 doi: ioRxiv preprint . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted November 18, 2020. ; https://doi.org/10.1101/2020.11.16.386086 doi: bioRxiv preprint Ruminococcaceae, Bacteroidetes, Clostridium IV and Olsenella. Next, we quantiﬁed the degree 207 centrality, the number of associations between each OTU for the whole network of each antibiotic 208 and outcome, and betweenness centrality, the number of associations connecting two OTUs that 209 pass through an OTU (Figure 6B). This analysis revealed cefoperazone treatment resulted in 210 networks primarily composed of singular associations with much lower degree centrality (P < 0.05) 211 and betweenness centrality (P < 0.05) than the other antibiotic treatments. Communities that were 212 treated with cefoperazone that resulted in cleared or persistent colonization had 10 to 100-fold 213 lower betweenness centrality values than communities treated with clindamycin or streptomycin. 214 Collectively, these networks suggest C. difﬁcile colonization was affected by unique sets of OTUs in 215 mice treated with clindamycin and streptomycin, but cefoperazone treatment eliminated bacteria 216 critical to maintaining community interactions and had few populations that associated with C. 217 difﬁcile. 218 speciﬁc changes within each community to clear C. difﬁcile. 234 Previous studies have identiﬁed microbiota associated with C. difﬁcile colonization resistance in 235 either a set of closely related murine communities or collectively across many different susceptible 236 communities (11, 15, 22). These bacteria were then tested in C. difﬁcile infection models. These 237 experiments were able to show decreased colonization but were unable to fully clear C. difﬁcile 238 (11, 23). Rather than looking for similarities across all susceptible communities, we explored 239 the changes that were associated with C. difﬁcile clearance for each antibiotic. Even though 240 these mice all came from the same breeding colony with similar initial microbiomes, C. difﬁcile 241 clearance was associated with antibiotic-speciﬁc changes in community diversity, OTU abundances, 242 and associations between OTUs. Our data suggest that the set of bacteria necessary to restore 243 colonization resistance following one antibiotic perturbation may not be effective for all antibiotic 244 perturbations. We have developed this modeling framework starting from a single mouse community. 245 It should also be relevant when considering interpersonal variation among humans (24). 246 Recent studies have begun to uncover how communities affect C. difﬁcile colonization (17–20, 24). 247 We attempted to understand the general trends in each antibiotic treatment that lead to clearance 248 of C. difﬁcile. We categorized the general changes and microbial relationships of these experiments 249 into three models. First, a model of temporary opportunity characterized by the transient dominance 250 of a facultative anaerobe which permits C. difﬁcile colonization but C. difﬁcile is not able to persist, 251 as with clindamycin treatment. We hypothesize this susceptibility is due to a transient repression 252 of community members and interventions which further perturb the community may worsen the 253 infection. Time alone may be sufﬁcient for the community to clear colonization (15, 22, 25) but 254 treating the community with an antibiotic or the bowel preparation for an FMT (26, 27) may prolong 255 susceptibility by eliminating protective functions or opening new niches. Second, a model of an 256 extensive opportunity characterized by a signiﬁcant perturbation leading to a persistent increase 257 in facultative anaerobes and exposing multiple niches, as with cefoperazone treatment. These 258 communities appear to have been severely depleted of multiple critical community members and 259 are likely lacking numerous protective functions (20). We hypothesize multiple niches are available 260 for C. difﬁcile to colonize. Discussion 219 We have shown that different antibiotic treatments resulted in speciﬁc changes to the microbiota 220 that were associated with C. difﬁcile clearance. Clindamycin-treated mice became susceptible 221 with a dominant bloom in populations related to Enterobacteriaceae. Clearance was associated 222 with the resolution of the bloom and recovery of bacteria that were reduced by the antibiotic 223 treatment. Cefoperazone-treated mice became susceptible with the expansion of numerous 224 facultative anaerobes. Communities with a sustained presence of these facultative anaerobes 225 were unable to recover from the initial antibiotic perturbation or clear the colonization, whereas 226 the communities that returned to their initial community were able to clear C. difﬁcile colonization. 227 Streptomycin-treated mice became susceptible with fewer and smaller changes than the other 228 treatments. The communities that cleared colonization had slightly higher α-diversity than those 229 that remained colonized. Additionally, all communities in mice treated with streptomycin had 230 similar numbers of OTUs changing through the experiment but the speciﬁc OTUs were different for 231 each outcome. These observations support our hypothesis that each colonized community has 232 antibiotic-speciﬁc changes that create unique conditions for C. difﬁcile colonization and requires 233 10 . CC-BY 4.0 International license available under a which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted November 18, 2020. ; https://doi.org/10.1101/2020.11.16.386086 doi: ioRxiv preprint . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted November 18, 2020. ; https://doi.org/10.1101/2020.11.16.386086 doi: bioRxiv preprint speciﬁc changes within each community to clear C. difﬁcile. 234 In this scenario, a full FMT may be insufﬁcient to provide adequate 261 diversity and abundance to outcompete and occupy all the exposed niches. Multiple FMTs (28, 262 11 . CC-BY 4.0 International license available under a which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted November 18, 2020. ; https://doi.org/10.1101/2020.11.16.386086 doi: ioRxiv preprint . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted November 18, 2020. ; https://doi.org/10.1101/2020.11.16.386086 doi: bioRxiv preprint 29) or transplant of an enriched fecal community (30) may be necessary to recover the microbiota 263 enough to outcompete C. difﬁcile for the nutrient niches and replace the missing protective functions. 264 Third, a model of a speciﬁc opportunity characterized by a perturbation that only affects a select 265 portion of the microbiota, leading to small changes in relative abundance and a slight decrease 266 in diversity, opening a limited niche for C. difﬁcile to colonize, as with streptomycin treatment. 267 We hypothesize that a few speciﬁc bacteria would be necessary to recolonize the exposed niche 268 space and eliminate C. difﬁcile colonization (13, 17). A fecal microbiota transplant may contain the 269 bacterial diversity needed to ﬁll the open niche space and help supplant C. difﬁcile from the exposed 270 niche of the colonized community. Analyzing each of these colonization models individually allowed 271 us to understand how each may clear C. difﬁcile colonization. 272 Future investigations can further identify the exposed niches of susceptible communities and 273 the requirements to clear C. difﬁcile colonization. One common theme for susceptibility across 274 treatments was the increased abundance of facultative anaerobes. These blooms of facultative 275 anaerobes could be attributed to the loss of the indigenous obligate anaerobes with antibiotic 276 treatment (31, 32). However, it is unclear what prevents the succession from the facultative 277 anaerobes back to the obligate anaerobes in cefoperazone-treated mice. Future studies should 278 investigate the relationship between facultative anaerobe blooms and susceptibility to colonization 279 as well as interventions to recover the obligate anaerobes. Another aspect to consider in future 280 experiments is C. difﬁcile colonization and developing targeted therapeutics. 292 We have shown that mice became susceptible to C. difﬁcile colonization after three different 293 antibiotic treatments and then differed in their ability to clear the colonization. These experiments 294 have shown that each antibiotic treatment resulted in different community changes leading to 295 C. difﬁcile clearance. These differences suggest that a single mechanism of infection and one 296 treatment for all C. difﬁcile infections may not be appropriate. While our current use of FMT to 297 eliminate CDI is highly effective, it does not work in all patients and has even resulted in adverse 298 consequences (7–10). The ﬁndings in this study may help explain why FMTs may be ineffective. 299 Although an FMT transplants a whole community, it may not be sufﬁcient to replace the missing 300 community members or functions to clear C. difﬁcile. Alternatively, the FMT procedure itself may 301 disrupt the natural recovery of the community. The knowledge of how a community clears C. difﬁcile 302 colonization will advance our ability to develop targeted therapies to manage CDI. 303 speciﬁc changes within each community to clear C. difﬁcile. 234 difﬁcile strain speciﬁcity. Other strains may ﬁll different niche space and ﬁll 281 other community interactions (33–35). For example, more virulent strains, like C. difﬁcile VPI 282 10463, may have a greater effect on the gut environment since it produces more toxin (15, 36). 283 Those differences could have different impacts on the susceptible community and change the 284 requirements to clear C. difﬁcile. Finally, we have shown that the functions found in communities 285 at peak colonization are antibiotic-speciﬁc (20). Here, we have shown the community changes 286 associated with C. difﬁcile clearance are antibiotic-speciﬁc. It is unknown how the community 287 functions contributing to C. difﬁcile clearance compare across antibiotics. Examining the changes 288 in transcription and metabolites during clearance will help deﬁne the activities necessary to clear C. 289 difﬁcile and if they are speciﬁc to the perturbation. This information will build upon the community 290 differences presented in this study and move us closer to elucidating how the microbiota clears C. 291 12 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted November 18, 2020. ; https://doi.org/10.1101/2020.11.16.386086 doi: bioRxiv preprint . CC-BY 4.0 International license available under a which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted November 18, 2020. ; https://doi.org/10.1101/2020.11.16.386086 doi: ioRxiv preprint Materials and Methods 304 Animal care. All mice were obtained from a single breeding colony and maintained in 305 speciﬁc-pathogen-free (SPF) conditions at the University of Michigan animal facility. All mouse 306 protocols and experiments were approved by the University Committee on Use and Care of 307 Animals at the University of Michigan and completed in agreement with approved guidelines. 308 Antibiotic administration. Mice were given one of three antibiotics, cefoperazone, clindamycin, 309 or streptomycin. Cefoperazone (0.5, 0.3, or 0.1 mg/ml) and streptomycin (5, 0.5, or 0.1 mg/ml) 310 were delivered via drinking water for 5 days. Clindamycin (10 mg/kg) was administered through 311 intraperitoneal injection. 312 C. difﬁcile challenge. Mice were returned to untreated drinking water for 24 hours before 313 challenging with C. difﬁcile strain 630∆erm spores. C. difﬁcile spores were aliquoted from a 314 single spore stock stored at 4◦C. Spore concentration was determined one week prior to the day of 315 challenge (37). 103 C. difﬁcile spores were orally gavaged into each mouse. Once the gavages 316 were completed, the remaining spore solution was serially diluted and plated to conﬁrm the spore 317 13 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted November 18, 2020. ; https://doi.org/10.1101/2020.11.16.386086 doi: bioRxiv preprint . CC-BY 4.0 International license available under a which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted November 18, 2020. ; https://doi.org/10.1101/2020.11.16.386086 doi: ioRxiv preprint concentration that was delivered. 318 Sample collection. Fecal samples were collected on the day antibiotic treatment was started, on 319 the day of C. difﬁcile challenge and the following 10 days. For the day of challenge and beyond, 320 a fecal sample was also collected and weighed. Under anaerobic conditions a fecal sample was 321 serially diluted in anaerobic phosphate-buffered saline and plated on TCCFA plates. After 24 hours 322 of anaerobic incubation at 37◦C, the number of colony forming units (CFU) were determined (38). 323 DNA sequencing. Total bacterial DNA was extracted from each fecal sample using MOBIO 324 PowerSoil-htp 96-well soil DNA isolation kit. Sequence data accession number. All 16S rRNA gene sequence data and associated metadata 355 are available through the Sequence Read Archive via accession PRJNA674858. 356 Sequence data accession number. All 16S rRNA gene sequence data and associated metadata 355 are available through the Sequence Read Archive via accession PRJNA674858. 356 Materials and Methods 304 We created amplicons of the 16S rRNA gene V4 region 325 and sequenced them using an Illumina MiSeq as described previously (39). 326 DNA sequencing. Total bacterial DNA was extracted from each fecal sample using MOBIO 324 PowerSoil-htp 96-well soil DNA isolation kit. We created amplicons of the 16S rRNA gene V4 region 325 and sequenced them using an Illumina MiSeq as described previously (39). 326 Sequence curation. Sequences were processed using mothur(v.1.43.0) as previously described 327 (39). Brieﬂy, we used a 3% dissimilarity cutoff to group sequences into operational taxonomic units 328 (OTUs). We used a naive Bayesian classiﬁer with the Ribosomal Database Project training set 329 (version 16) to assign taxonomic classiﬁcations to each OTU (41). With the fecal samples, we 330 also sequenced a mock community with a known community composition and their true 16s rRNA 331 gene sequences. We processed this mock community along with our samples to determine our 332 sequence curation resulted in an error rate of 0.019%. 333 Statistical analysis and modeling. Diversity comparisons were calculated in mothur. To compare 334 α-diversity metrics, we calculated the number of OTUs (Sobs) and the Inverse Simpson diversity 335 index. To compare across communities, we calculated dissimilarity matrices based on metric of 336 Yue and Clayton (42). All calculations were made by rarifying samples to 1,200 sequences per 337 sample to limit biases due to uneven sampling. OTUs were subsampled to 1,200 counts per sample 338 and remaining statistical analysis and data visualization was performed in R (v3.5.1) with the 339 tidyverse package (v1.3.0). Signiﬁcance of pairwise comparisons of α-diversity (Sobs and Inverse 340 Simpson), β-diversity (θYC), OTU abundance, and network centrality (betweenness and degree) 341 were calculated by pairwise Wilcoxon rank sum test and then P values were corrected for multiple 342 comparisons with a Benjamini and Hochberg adjustment for a type I error rate of 0.05 (43). Logistic 343 regression models were constructed with OTUs from all day 0 samples using half of the samples 344 to train and the other half to test the model. The model was developed from the caret R package 345 14 . CC-BY 4.0 International license available under a which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted November 18, 2020. Materials and Methods 304 ; https://doi.org/10.1101/2020.11.16.386086 doi: ioRxiv preprint . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted November 18, 2020. ; https://doi.org/10.1101/2020.11.16.386086 doi: bioRxiv preprint (v6.0-85) and previously developed machine learning pipeline (44). For each antibiotic treatment, 346 conditional independence networks were calculated from the day 1 through 10 samples of all 347 mice initially colonized using SPIEC-EASI (sparse inverse covariance estimation for ecological 348 association inference) methods from the SpiecEasi R package after optimizing lambda to 0.001 349 with a network stability between 0.045 and 0.05 (v1.0.7) (21). Network centrality measures degree 350 and betweenness were calculated on whole networks using functions from the igraph R package 351 (v1.2.4.1). 352 (v6.0-85) and previously developed machine learning pipeline (44). For each antibiotic treatment, 346 conditional independence networks were calculated from the day 1 through 10 samples of all 347 mice initially colonized using SPIEC-EASI (sparse inverse covariance estimation for ecological 348 association inference) methods from the SpiecEasi R package after optimizing lambda to 0.001 349 with a network stability between 0.045 and 0.05 (v1.0.7) (21). Network centrality measures degree 350 and betweenness were calculated on whole networks using functions from the igraph R package 351 (v1.2.4.1). 352 Code availability. Scripts necessary to reproduce our analysis and this paper are available in an 353 online repository (https://github.com/SchlossLab/Lesniak_Clearance_XXXX_2020). 354 Code availability. Scripts necessary to reproduce our analysis and this paper are available in an 353 online repository (https://github.com/SchlossLab/Lesniak_Clearance_XXXX_2020). 354 Sequence data accession number. All 16S rRNA gene sequence data and associated metadata 355 are available through the Sequence Read Archive via accession PRJNA674858. 356 Acknowledgements 357 Thank you to Begüm Topçuoglu and Sarah Tomkovich for critical discussion in the development and 358 execution of this project. This work was supported by several grants from the National Institutes 359 for Health R01GM099514, U19AI090871, U01AI12455, and P30DK034933. Additionally, NAL 360 was supported by the Molecular Mechanisms of Microbial Pathogenesis training grant (NIH T32 361 AI007528). The funding agencies had no role in study design, data collection and analysis, decision 362 to publish, or preparation of the manuscript. 363 15 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted November 18, 2020. ; https://doi.org/10.1101/2020.11.16.386086 doi: bioRxiv preprint . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted November 18, 2020. ; https://doi.org/10.1101/2020.11.16.386086 doi: bioRxiv preprint References 364 1. 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Free 472 Radical Biology and Medicine 105:93–101. doi:10.1016/j.freeradbiomed.2016.09.022. 473 33. Carlson PE, Walk ST, Bourgis AET, Liu MW, Kopliku F, Lo E, Young VB, Aronoff DM, 474 Hanna PC. 2013. The relationship between phenotype, ribotype, and clinical disease in human 475 clostridium difﬁcile isolates. Anaerobe 24:109–116. doi:10.1016/j.anaerobe.2013.04.003. 476 34. Thanissery R, Winston JA, Theriot CM. 2017. Inhibition of spore germination, growth, and 477 toxin activity of clinically relevant c. difﬁcile strains by gut microbiota derived secondary bile 478 acids. Anaerobe 45:86–100. doi:10.1016/j.anaerobe.2017.03.004. 479 35. Theriot CM, Koumpouras CC, Carlson PE, Bergin II, Aronoff DM, Young VB. 2011. 480 Cefoperazone-treated mice as an experimental platform to assess differential virulence 481 ofClostridium difﬁcilestrains. Gut Microbes 2:326–334. doi:10.4161/gmic.19142. 482 36. Rao K, Micic D, Natarajan M, Winters S, Kiel MJ, Walk ST, Santhosh K, Mogle JA, Galecki 483 36. Rao K, Micic D, Natarajan M, Winters S, Kiel MJ, Walk ST, Santhosh K, Mogle JA, Galecki 483 AT, LeBar W, Higgins PDR, Young VB, Aronoff DM. 2015. Clostridium difﬁcile Ribotype 027: 484 Relationship to age, detectability of toxins a or b in stool with rapid testing, severe infection, and 485 mortality. Clinical Infectious Diseases 61:233–241. doi:10.1093/cid/civ254. 486 37. Sorg JA, Dineen SS. 2009. Laboratory maintenance ofClostridium difﬁcile. Current Protocols 487 in Microbiology 12. doi:10.1002/9780471729259.mc09a01s12. 488 38. Winston JA, Thanissery R, Montgomery SA, Theriot CM. 2016. Cefoperazone-treated 489 mouse model of clinically-relevant ∆clostridium difﬁcile strain r20291. Journal of Visualized 490 38. Winston JA, Thanissery R, Montgomery SA, Theriot CM. 2016. Cefoperazone-treated 489 mouse model of clinically-relevant ∆clostridium difﬁcile strain r20291. Journal of Visualized 490 20 . 16. McDonald JAK, Mullish BH, Pechlivanis A, Liu Z, Brignardello J, Ka 417 It is made The copyright holder for this preprint this version posted November 18, 2020. ; https://doi.org/10.1101/2020.11.16.386086 doi: bioRxiv preprint 512 Figure 1. Reduced antibiotic doses permitted murine communities to be colonized and 513 spontaneously clear that C. difﬁcile colonization. (A-C) Daily CFU of C. difﬁcile in fecal samples 514 of mice treated with clindamycin, cefoperazone, or streptomycin from time of challenge (Day 0) 515 through 10 days post infection (dpi). The bold line is the median CFU of the group and the 516 transparent lines are the individual mice. (D-F) Relative abundance of twelve most abundant genera 517 at the time of C difﬁcile challenge all other genera grouped into Other Each column is an individual 518 512 512 Figure 1. Reduced antibiotic doses permitted murine communities to be colonized and 513 spontaneously clear that C. difﬁcile colonization. (A-C) Daily CFU of C. difﬁcile in fecal samples 514 of mice treated with clindamycin, cefoperazone, or streptomycin from time of challenge (Day 0) 515 through 10 days post infection (dpi). The bold line is the median CFU of the group and the 516 transparent lines are the individual mice. (D-F) Relative abundance of twelve most abundant genera 517 at the time of C. difﬁcile challenge, all other genera grouped into Other. Each column is an individual 518 mouse. LOD = Limit of detection. (clindamycin - 10 mg/kg N =11; cefoperazone - 0.5 mg/mL N = 5, 519 0.3 mg/mL N = 9, 0.1 mg/mL N = 2; streptomycin - 5.0 mg/mL N = 8, 0.5 mg/mL N = 7, 0.1 mg/mL 520 N = 7). 521 Figure 1. Reduced antibiotic doses permitted murine communities to be colonized and 513 spontaneously clear that C. difﬁcile colonization. (A-C) Daily CFU of C. difﬁcile in fecal samples 514 of mice treated with clindamycin, cefoperazone, or streptomycin from time of challenge (Day 0) 515 through 10 days post infection (dpi). The bold line is the median CFU of the group and the 516 transparent lines are the individual mice. (D-F) Relative abundance of twelve most abundant genera 517 at the time of C. difﬁcile challenge, all other genera grouped into Other. Each column is an individual 518 mouse. LOD = Limit of detection. 16. McDonald JAK, Mullish BH, Pechlivanis A, Liu Z, Brignardello J, Ka 417 CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted November 18, 2020. ; https://doi.org/10.1101/2020.11.16.386086 doi: bioRxiv preprint . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted November 18, 2020. ; https://doi.org/10.1101/2020.11.16.386086 doi: bioRxiv preprint Experiments. doi:10.3791/54850. 491 39. Kozich JJ, Westcott SL, Baxter NT, Highlander SK, Schloss PD. 2013. Development 492 of a dual-index sequencing strategy and curation pipeline for analyzing amplicon sequence 493 data on the MiSeq illumina sequencing platform. Applied and Environmental Microbiology 494 79:5112–5120. doi:10.1128/aem.01043-13. 495 40. Schloss PD, Westcott SL, Ryabin T, Hall JR, Hartmann M, Hollister EB, Lesniewski RA, 496 Oakley BB, Parks DH, Robinson CJ, Sahl JW, Stres B, Thallinger GG, Horn DJV, Weber 497 CF. 2009. Introducing mothur: Open-source, platform-independent, community-supported 498 software for describing and comparing microbial communities. Applied and Environmental 499 Microbiology 75:7537–7541. doi:10.1128/aem.01541-09. 500 41. Wang Q, Garrity GM, Tiedje JM, Cole JR. 2007. Naïve bayesian classiﬁer for rapid assignment 501 of rRNA sequences into the new bacterial taxonomy. Applied and Environmental Microbiology 502 73:5261–5267. doi:10.1128/aem.00062-07. 503 42. Yue JC, Clayton MK. 2005. A similarity measure based on species proportions. 504 Communications in Statistics - Theory and Methods 34:2123–2131. doi:10.1080/sta-200066418. 505 43. Benjamini Y, Hochberg Y. 1995. Controlling the false discovery rate: A practical and powerful 506 approach to multiple testing. Journal of the Royal Statistical Society: Series B (Methodological) 507 57:289–300. doi:10.1111/j.2517-6161.1995.tb02031.x. 508 44. Topçuo˘glu BD, Lesniak NA, Rufﬁn MT, Wiens J, Schloss PD. 2020. A framework for 509 effective application of machine learning to microbiome-based classiﬁcation problems. mBio 11. 510 doi:10.1128/mbio.00434-20. 511 21 . CC-BY 4.0 International license available under a which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted November 18, 2020. ; https://doi.org/10.1101/2020.11.16.386086 doi: ioRxiv preprint . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. 16. McDonald JAK, Mullish BH, Pechlivanis A, Liu Z, Brignardello J, Ka 417 (clindamycin - 10 mg/kg N =11; cefoperazone - 0.5 mg/mL N = 5, 519 0.3 mg/mL N = 9, 0.1 mg/mL N = 2; streptomycin - 5.0 mg/mL N = 8, 0.5 mg/mL N = 7, 0.1 mg/mL 520 N = 7). 521 522 523 22 . CC-BY 4.0 International license available under a which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted November 18, 2020. ; https://doi.org/10.1101/2020.11.16.386086 doi: ioRxiv preprint . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted November 18, 2020. ; https://doi.org/10.1101/2020.11.16.386086 doi: bioRxiv preprint 524 Figure 2. Microbiota community diversity showed antibiotic-speciﬁc trends associated with 525 C. difﬁcle colonization clearance. For communities colonized with C. difﬁcile from mice treated 526 with clindamycin (A), cefoperazone (B), and streptomycin (C), microbiota α-diversity (Sobs and 527 Inverse Simpson) and β-diversity (θYC) were compared at the initial pre-antibiotic treatment state, 528 time of C. difﬁcile challenge (TOC), and end of the experiment. β-diversity (θYC) was compared 529 524 524 Figure 2. Microbiota community diversity showed antibiotic-speciﬁc trends associated with 525 Figure 2. Microbiota community diversity showed antibiotic-speciﬁc trends associated with 525 C. difﬁcle colonization clearance. For communities colonized with C. difﬁcile from mice treated 526 with clindamycin (A), cefoperazone (B), and streptomycin (C), microbiota α-diversity (Sobs and 527 Inverse Simpson) and β-diversity (θYC) were compared at the initial pre-antibiotic treatment state, 528 time of C. difﬁcile challenge (TOC), and end of the experiment. β-diversity (θYC) was compared 529 C. difﬁcle colonization clearance. For communities colonized with C. difﬁcile from mice treated 526 with clindamycin (A), cefoperazone (B), and streptomycin (C), microbiota α-diversity (Sobs and 527 Inverse Simpson) and β-diversity (θYC) were compared at the initial pre-antibiotic treatment state, 528 time of C. difﬁcile challenge (TOC), and end of the experiment. β-diversity (θYC) was compared 529 23 . CC-BY 4.0 International license available under a which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted November 18, 2020. 16. McDonald JAK, Mullish BH, Pechlivanis A, Liu Z, Brignardello J, Ka 417 ; https://doi.org/10.1101/2020.11.16.386086 doi: ioRxiv preprint . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted November 18, 2020. ; https://doi.org/10.1101/2020.11.16.386086 doi: bioRxiv preprint between the initial pre-antibiotic treatment to all other initial pre-antibiotic treatment communities 530 treated with the same antibiotic, the initial community to the same community at the time of C. 531 difﬁcile challenge, and the initial community to the same community at end of the experiment. 532 (clindamycin - cleared N = 11; cefoperazone - cleared N = 7, colonized N = 9; streptomycin - cleared 533 N = 9, colonized N = 11). * indicates statistical signiﬁcance of P < 0.05, calculated by Wilcoxon 534 rank sum test with Benjamini-Hochberg correction. 535 537 24 24 . CC-BY 4.0 International license available under a which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted November 18, 2020. ; https://doi.org/10.1101/2020.11.16.386086 doi: ioRxiv preprint . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted November 18, 2020. ; https://doi.org/10.1101/2020.11.16.386086 doi: bioRxiv preprint 538 Figure 3. OTU abundance differences between communities that cleared C. difﬁcile 539 colonization and remained colonized are unique to each treatment. For cefoperazone (A) and 540 streptomycin (B), the difference in the relative abundance of OTUs that were signiﬁcantly different 541 between communities that eliminated C. difﬁcile colonization and those that remained colonized 542 within each antibiotic treatment for each time point Bold points are median relative abundance and 538 538 Figure 3. OTU abundance differences between communities that cleared C. difﬁcile 539 colonization and remained colonized are unique to each treatment. For cefoperazone (A) and 540 streptomycin (B), the difference in the relative abundance of OTUs that were signiﬁcantly different 541 between communities that eliminated C. difﬁcile colonization and those that remained colonized 542 within each antibiotic treatment for each time point. 16. McDonald JAK, Mullish BH, Pechlivanis A, Liu Z, Brignardello J, Ka 417 Bold points are median relative abundance and 543 transparent points are relative abundance of individual mice. Lines connect points within each 544 comparison to show difference in medians. Only OTUs at time points with statistically signiﬁcant 545 Figure 3. OTU abundance differences between communities that cleared C. difﬁcile 539 colonization and remained colonized are unique to each treatment. For cefoperazone (A) and 540 streptomycin (B), the difference in the relative abundance of OTUs that were signiﬁcantly different 541 between communities that eliminated C. difﬁcile colonization and those that remained colonized 542 within each antibiotic treatment for each time point. Bold points are median relative abundance and 543 transparent points are relative abundance of individual mice. Lines connect points within each 544 comparison to show difference in medians. Only OTUs at time points with statistically signiﬁcant 545 25 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted November 18, 2020. ; https://doi.org/10.1101/2020.11.16.386086 doi: bioRxiv preprint differences, P < 0.05, were plotted (calculated by Wilcoxon rank sum test with Benjamini-Hochberg 546 correction). Limit of detection (LOD). 547 548 549 549 550 Figure 4. Each antibiotic had speciﬁc sets of temporal changes in OTU abundance 551 associated with C. difﬁcile colonization and clearance. For clindamycin (A), cefoperazone (C), 552 and streptomycin (B, D), the difference in the relative abundance of OTUs that were signiﬁcantly 553 different between time points within each C. difﬁcile colonization outcome for each antibiotic 554 treatment. Bold points are median relative abundance and transparent points are relative 555 abundance of individual mice. Lines connect points within each comparison to show difference in 556 medians. Arrows point in the direction of the temporal change of the relative abundance. Only 557 OTUs at time points with statistically signiﬁcant differences, P < 0.05, were plotted (calculated 558 by Wilcoxon rank sum test with Benjamini-Hochberg correction). Bold OTUs were shared across 559 outcomes. Limit of detection (LOD). 560 550 Figure 4. Each antibiotic had speciﬁc sets of temporal changes in OTU abundance 551 associated with C. difﬁcile colonization and clearance. For clindamycin (A), cefoperazone (C), 552 and streptomycin (B, D), the difference in the relative abundance of OTUs that were signiﬁcantly 553 different between time points within each C. 16. McDonald JAK, Mullish BH, Pechlivanis A, Liu Z, Brignardello J, Ka 417 difﬁcile colonization outcome for each antibiotic 554 treatment. Bold points are median relative abundance and transparent points are relative 555 abundance of individual mice. Lines connect points within each comparison to show difference in 556 medians. Arrows point in the direction of the temporal change of the relative abundance. Only 557 OTUs at time points with statistically signiﬁcant differences, P < 0.05, were plotted (calculated 558 by Wilcoxon rank sum test with Benjamini-Hochberg correction). Bold OTUs were shared across 559 outcomes. Limit of detection (LOD). 560 562 26 . CC-BY 4.0 International license available under a which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted November 18, 2020. ; https://doi.org/10.1101/2020.11.16.386086 doi: ioRxiv preprint . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted November 18, 2020. ; https://doi.org/10.1101/2020.11.16.386086 doi: bioRxiv preprint 563 Figure 5. Distinct features of the bacterial community at the time of infection can classify 564 end point colonization. (A) L2 logistic regression model features’ importance determined by 565 the decrease in model performance when randomizing an individual feature. All OTUs affecting 566 performance shown. Dashed lines show performance range of ﬁnal model with all features 567 included. (B) Distribution of odds ratio used in L2 logistic regression model. Values above 1 568 indicate abundance predicted the community cleared colonization (red) and values below 1 indicate 569 abundance predicted C. difﬁcile remained colonized (blue). Feature label and boxplot are colored 570 to match the median odds ratio. (C) Relative abundance difference in features used by L2 logistic 571 regression model displayed by antibiotic treatment. 572 563 563 Figure 5. Distinct features of the bacterial community at the time of infection can classify 564 end point colonization. (A) L2 logistic regression model features’ importance determined by 565 the decrease in model performance when randomizing an individual feature. All OTUs affecting 566 performance shown. Dashed lines show performance range of ﬁnal model with all features 567 included. (B) Distribution of odds ratio used in L2 logistic regression model. 16. McDonald JAK, Mullish BH, Pechlivanis A, Liu Z, Brignardello J, Ka 417 Values above 1 568 indicate abundance predicted the community cleared colonization (red) and values below 1 indicate 569 abundance predicted C. difﬁcile remained colonized (blue). Feature label and boxplot are colored 570 to match the median odds ratio. (C) Relative abundance difference in features used by L2 logistic 571 regression model displayed by antibiotic treatment. 572 Figure 5. Distinct features of the bacterial community at the time of infection can classify 564 574 27 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted November 18, 2020. ; https://doi.org/10.1101/2020.11.16.386086 doi: bioRxiv preprint . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted November 18, 2020. ; https://doi.org/10.1101/2020.11.16.386086 doi: bioRxiv preprint 575 Figure 6. Conditional independence networks reveal treatment-speciﬁc relationships 576 between the community and C. difﬁcile during colonization clearance. (A) SPIEC-EASI 577 (sparse inverse covariance estimation for ecological association inference) networks showing 578 conditionally independent ﬁrst-order relationships between C. difﬁcile and the community as C. 579 difﬁcile was cleared from the gut environment. Nodes are sized by median relative abundance 580 of the OTU. A red colored edge indicates a negative interaction and blue indicates a positive 581 interaction, while edge thickness indicates the interaction strength. (B) Network centrality measured 582 with betweenness, i.e. how many paths between two OTUs pass through an individual, and degree, 583 i.e. how many connections an OTU had. * indicates statistical signiﬁcance of P < 0.05, calculated 584 by Wilcoxon rank sum test with Benjamini-Hochberg correction. 585 75 575 Figure 6. Conditional independence networks reveal treatment-speciﬁc relationships 576 between the community and C. difﬁcile during colonization clearance. (A) SPIEC-EASI 577 (sparse inverse covariance estimation for ecological association inference) networks showing 578 conditionally independent ﬁrst-order relationships between C. difﬁcile and the community as C. 579 difﬁcile was cleared from the gut environment. Nodes are sized by median relative abundance 580 of the OTU. A red colored edge indicates a negative interaction and blue indicates a positive 581 interaction, while edge thickness indicates the interaction strength. 16. McDonald JAK, Mullish BH, Pechlivanis A, Liu Z, Brignardello J, Ka 417 (B) Network centrality measured 582 with betweenness, i.e. how many paths between two OTUs pass through an individual, and degree, 583 i.e. how many connections an OTU had. * indicates statistical signiﬁcance of P < 0.05, calculated 584 by Wilcoxon rank sum test with Benjamini-Hochberg correction. 585 Figure 6. Conditional independence networks reveal treatment-speciﬁc relationships 576 between the community and C. difﬁcile during colonization clearance. (A) SPIEC-EASI 577 (sparse inverse covariance estimation for ecological association inference) networks showing 578 conditionally independent ﬁrst-order relationships between C. difﬁcile and the community as C. 579 difﬁcile was cleared from the gut environment. Nodes are sized by median relative abundance 580 of the OTU. A red colored edge indicates a negative interaction and blue indicates a positive 581 interaction, while edge thickness indicates the interaction strength. (B) Network centrality measured 582 with betweenness, i.e. how many paths between two OTUs pass through an individual, and degree, 583 i.e. how many connections an OTU had. * indicates statistical signiﬁcance of P < 0.05, calculated 584 by Wilcoxon rank sum test with Benjamini-Hochberg correction. 585 Figure 6. Conditional independence networks reveal treatment-speciﬁc relationships 576 Figure 6. Conditional independence networks reveal treatment-speciﬁc relationships 576 between the community and C. difﬁcile during colonization clearance. (A) SPIEC-EASI 577 (sparse inverse covariance estimation for ecological association inference) networks showing 578 conditionally independent ﬁrst-order relationships between C. difﬁcile and the community as C. 579 difﬁcile was cleared from the gut environment. Nodes are sized by median relative abundance 580 ure 6. Conditional independence networks reveal treatment-speciﬁc rel 586 28 28 . CC-BY 4.0 International license available under a which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted November 18, 2020. ; https://doi.org/10.1101/2020.11.16.386086 doi: ioRxiv preprint . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted November 18, 2020. ; https://doi.org/10.1101/2020.11.16.386086 doi: bioRxiv preprint 8 588 29 29 29 . CC-BY 4.0 International license available under a which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. 16. McDonald JAK, Mullish BH, Pechlivanis A, Liu Z, Brignardello J, Ka 417 It is made The copyright holder for this preprint this version posted November 18, 2020. ; https://doi.org/10.1101/2020.11.16.386086 doi: ioRxiv preprint . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted November 18, 2020. ; https://doi.org/10.1101/2020.11.16.386086 doi: bioRxiv preprint nitial microbiota relative abundance of mice prior to antibiotic treatment Figure S1. Initial microbiota relative abundance of mice prior to antibiotic treatment. 589 Relative abundance at the beginning of the experiment prior to antibiotic treatment of twelve most 590 abundant genera post antibiotic treatment, all other genera grouped into Other. Each column is an 591 individual mouse. Color intensity is log10-transformed mean percent relative abundance of each 592 day. (N = 57). 593 596 Figure S2. Temporally differing OTU for cefoperazone-treated mice that cleared C. difﬁcile 597 colonization. Bold points are median relative abundance and transparent points are relative 598 abundance of individual mice. Lines connect points within each comparison to show difference 599 in medians. Arrows point in the direction of the temporal change of the relative abundance. Only 600 OTUs at time points with statistically signiﬁcant differences, P < 0.05, were plotted (calculated by 601 Wilcoxon rank sum test with Benjamini-Hochberg correction). Limit of detection (LOD). 602 596 Figure S2. Temporally differing OTU for cefoperazone-treated mice that cleared C. difﬁcile 597 colonization. Bold points are median relative abundance and transparent points are relative 598 abundance of individual mice. Lines connect points within each comparison to show difference 599 in medians. Arrows point in the direction of the temporal change of the relative abundance. Only 600 Figure S2. Temporally differing OTU for cefoperazone-treated mice that clear Figure S2. Temporally differing OTU for cefoperazone-treated mice that cleared C. difﬁcile 597 colonization. Bold points are median relative abundance and transparent points are relative 598 abundance of individual mice. Lines connect points within each comparison to show difference 599 in medians. Arrows point in the direction of the temporal change of the relative abundance. Only 600 OTUs at time points with statistically signiﬁcant differences, P < 0.05, were plotted (calculated by 601 Wilcoxon rank sum test with Benjamini-Hochberg correction). Limit of detection (LOD). 602 30 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted November 18, 2020. ; https://doi.org/10.1101/2020.11.16.386086 doi: bioRxiv preprint 603 604 605 Figure S3. Bacterial community at the time of infection can classify endpoint colonization. 606 Classiﬁcation performance of L2 logistic regression. Area under the receiver-operator curve for 607 classifying if the community will remain colonized based on the OTUs present at the time of C. nitial microbiota relative abundance of mice prior to antibiotic treatment 608 difﬁcile infection (Day 0). Cross-validation of model performed on half of the data to tune model 609 (CV AUC) and then tuned model was tested on the held-out data (Test AUC). 610 605 Figure S3. Bacterial community at the time of infection can classify endpoint colonization. 606 Classiﬁcation performance of L2 logistic regression. Area under the receiver-operator curve for 607 classifying if the community will remain colonized based on the OTUs present at the time of C. 608 difﬁcile infection (Day 0). Cross-validation of model performed on half of the data to tune model 609 (CV AUC) and then tuned model was tested on the held-out data (Test AUC). 610 31
https://openalex.org/W2082211942	https://bmccancer.biomedcentral.com/counter/pdf/10.1186/1471-2407-10-255	English	null	The distribution of the therapeutic monoclonal antibodies cetuximab and trastuzumab within solid tumors	BMC cancer	2,010	cc-by	7,472	RESEARCH ARTICLE Open Access © 2010 Lee and Tannock; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Com- mons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduc- tion in any medium, provided the original work is properly cited. * Correspondence: ian.tannock@uhn.on.ca 1 Divisions of Applied Molecular Oncology and Medical Oncology and Hematology Princess Margaret Hospital and University of Toronto, Toronto, ON, Canada Full list of author information is available at the end of the article Abstract Background: Poor distribution of some anticancer drugs in solid tumors may limit their anti-tumor activity. Methods: Here we used immunohistochemistry to quantify the distribution of the therapeutic monoclonal antibodies cetuximab and trastuzumab in relation to blood vessels and to regions of hypoxia in human tumor xenografts. The antibodies were injected into mice implanted with human epidermoid carcinoma A431 or human breast carcinoma MDA-MB-231 transfected with ERBB2 (231-H2N) that express high levels of ErbB1 and ErbB2 respectively, or wild-type MDA-MB-231, which expresses intermediate levels of ErbB1 and low levels of ErbB2. Results: The distribution of cetuximab in A431 xenografts and trastuzumab in 231-H2N xenografts was time and dose dependent. At early intervals after injection of 1 mg cetuximab into A431 xenografts, the concentration of cetuximab decreased with increasing distance from blood vessels, but became more uniformly distributed at later times; there remained however limited distribution and binding in hypoxic regions of tumors. Injection of lower doses of cetuximab led to heterogeneous distributions. Similar results were observed with trastuzumab in 231-H2N xenografts. In MDA-MB-231 xenografts, which express lower levels of ErbB1, homogeneity of distribution of cetuximab was achieved more rapidly. Conclusions: Cetuximab and trastuzumab distribute slowly, but at higher doses achieve a relatively uniform distribution after about 24 hours, most likely due to their long half-lives in the circulation. There remains poor distribution within hypoxic regions of tumors. including those of the breast [3], colon [4], head and neck [5], kidney [6], lung [7,8], pancreas [9], prostate [10] and esophagus [11,12] and has been associated with an aggressive phenotype. Lee and Tannock BMC Cancer 2010, 10:255 http://www.biomedcentral.com/1471-2407/10/255 Lee and Tannock BMC Cancer 2010, 10:255 http://www.biomedcentral.com/1471-2407/10/255 Lee and Tannock BMC Cancer 2010, 10:255 Lee and Tannock BMC Cancer 2010, 10:255 http://www.biomedcentral.com/1471-2407/10/255 Research article The distribution of the therapeutic monoclonal antibodies cetuximab and trastuzumab within solid tumors Carol M Lee and Ian F Tannock* * Correspondence: ian.tannock@uhn.on.ca Drugs and reagents The monoclonal antibody cetuximab (IMC-C225, Erbitux) was provided by Imclone Systems, Inc. (New York, NY, USA) as a solution at a concentration of 2 mg/ ml. Trastuzumab (Herceptin) was obtained from the hos- pital pharmacy at a concentration of 21 mg/ml. The hypoxia-selective agent EF5 and Cy5-conjugated anti-EF5 antibody [29,30] were kindly provided by Dr. C. Koch, Philadelphia, PA. Blood vessels in tumor sections were visualized with a rat anti-mouse CD31 (PECAM-1) monoclonal antibody that was purchased from BD Pharmingen (Mississauga, ON, Canada) and the Cy3- conjugated goat anti-rat IgG secondary antibody was purchased from Jackson Immuno Research Laboratories, Inc. (West Grove, PA). Cetuximab and trastuzumab were recognized in tissue sections with goat anti-human IgG conjugated with horseradish peroxidase (Biosource, Montreal, Canada). Cryosections were prepared at 10 μm thickness and tri- ple stained to identify cetuximab or trastuzumab, CD31 and EF5. Horseradish peroxidase (HRP) conjugated to anti-human IgG was used to recognize the therapeutic monoclonal antibodies. DAB (3,3'-diaminobenzidine) is a chromogenic substrate for HRP and it deposits a brown specific stain in the presence of HRP. Blood vessels in tis- sue sections were recognized by the expression of CD31 on endothelial cells. Purified rat anti-mouse CD31 mono- clonal antibody was applied at a concentration of 1:500 and left overnight at 4°C. Primary antibody binding was Background Medin [31] (University of Toronto, ON, Canada). All the cell lines were maintained as monolayers in Dulbecco's Modified Eagle's Medium (DMEM), supplemented with 10% fetal calf serum (FCS), at 37°C in a humidified atmosphere of 95% air plus 5% CO2. Tests were performed routinely to ensure that cells were free of mycoplasma. Tumors were generated by injection of ~2 × 106 exponentially-growing cells into the right and left flanks of 6-8 week old female athymic nude mice, purchased from Harlan Sprague- Dawley Laboratory Animal Centre (Madison, WI, USA). Mice were housed five per cage, and sterile tap water and food were given ad libitum. All procedures were carried out following approval of the Institutional Animal Care Committee. and prevents the production of an active truncated frag- ment [20-22]. These agents have shown therapeutic activ- ity against colorectal cancer and breast cancer respectively and are in wide clinical use [21,22]. Limited penetration of drugs through tumor tissue is an important and rather neglected cause of clinical resis- tance to chemotherapy [23-25]. Drug distribution from blood vessels within tumors depends on diffusion and and/or convection, and is inhibited by consumption in proximal cells [23,25-27]; for monoclonal antibodies con- sumption is due to binding to the receptor target, which is dependent on antibody dose, number of antigenic tar- gets per cell, and the affinity of the antibody for its target [28]. Convection depends on gradients of pressure (both hydrostatic and osmotic) between the vascular space and the interstitial space, while diffusion depends on molecu- lar size, shape and concentration gradients [26,27]. Because monoclonal antibodies are large molecules they might be expected to have poor distribution from tumor blood vessels [28]. However drugs with a long half-life in the circulation may establish a more uniform distribution in tissues even if penetration of tissue is relatively slow, whereas drugs with a short half-life may have a non-uni- form distribution. Here we report a study of the distribu- tion of the monoclonal antibodies, cetuximab and trastuzumab, in tumors that express different levels of their target receptors. Expression of ErbB1 and ErbB2 receptors in the xeno- grafts was confirmed by applying cetuximab or trastu- zumab to sections of tumors ex vivo, followed by their recognition using anti-human IgG as described below. Endogenous expression of ErbB1 and ErbB2 were also confirmed and assessed by diagnostic antibodies from Zymed (Clone 31G7) and Neomarkers (Clone SP3) respectively. Experimental design Tumor-bearing mice were divided randomly into groups of 5-6, and treatment with cetuximab or trastuzumab was initiated when the diameter of tumors was approximately 7-8 mm. One group was selected randomly as the control, and the other mice received cetuximab or trastuzumab (0.01 mg to 1.0 mg) as a single intraperitoneal (i.p.) or intravenous (i.v.) injection. Control mice were given equal volumes of PBS. Animals were killed at various intervals after injection of cetuximab or trastuzumab; they received an i.p. injection of EF5 (0.2 ml of 10 mM EF5) 2 hours before they were killed in order to identify hypoxic regions of tumors [29,30]. Tumors were removed and embedded with Tissue-Tek OCT (Optimal Cutting Tem- perature, Sakura Finetek USA Inc., Torrance, CA). The tissue boxes were gently immersed in liquid nitrogen, and then stored at -70°C. Background The ErbB family of receptor kinases is a group of four trans-membrane proteins (ErbB1 - ErbB4) that share sim- ilarities in structure and are involved in signaling path- ways that stimulate cellular proliferation [1]. Ligand binding induces receptor homo- and hetero-dimeriza- tion, although no ligand has been identified for ErbB2. Dimerization of the receptors stimulates their intrinsic tyrosine kinase activity resulting in receptor autophos- phorylation [2]. These phosphorylated residues serve as binding sites for molecules involved in the regulation of intracellular signaling cascades. Overexpression of ErbB receptors may occur in a wide range of epithelial cancers, Molecular targeted agents that interact with receptor tyrosine kinases on tumor cells are used increasingly in clinical oncology. There are two classes of agents, mono- clonal antibodies and low-molecular-weight tyrosine kinase inhibitors. Cetuximab (chimeric mouse/human) and trastuzumab (humanized) are monoclonal antibodies that target the extracellular domain of the receptors ErbB1 [13-16] and ErbB2 [15,17] respectively. Binding of cetuximab and trastuzumab to ErbB1 and ErbB2 respec- tively prevents receptor phosphorylation and activation of the kinase domain, thereby inhibiting cell proliferation [18-20]. Binding of trastuzumab to its receptor also reduces shedding of the extracellular domain of ErbB2 * Correspondence: ian.tannock@uhn.on.ca 1 Divisions of Applied Molecular Oncology and Medical Oncology and Hematology Princess Margaret Hospital and University of Toronto, Toronto, ON, Canada Full list of author information is available at the end of the article Lee and Tannock BMC Cancer 2010, 10:255 http://www.biomedcentral.com/1471-2407/10/255 Page 2 of 11 the American Type Culture Collection (Manassas, VA, USA), while MDA-MB-231 cells transfected with ERBB2 (231-H2N) were kindly provided by Dr. J. Medin [31] (University of Toronto, ON, Canada). All the cell lines were maintained as monolayers in Dulbecco's Modified Eagle's Medium (DMEM), supplemented with 10% fetal calf serum (FCS), at 37°C in a humidified atmosphere of 95% air plus 5% CO2. Tests were performed routinely to ensure that cells were free of mycoplasma. Tumors were generated by injection of ~2 × 106 exponentially-growing cells into the right and left flanks of 6-8 week old female athymic nude mice, purchased from Harlan Sprague- Dawley Laboratory Animal Centre (Madison, WI, USA). Mice were housed five per cage, and sterile tap water and food were given ad libitum. All procedures were carried out following approval of the Institutional Animal Care Committee. the American Type Culture Collection (Manassas, VA, USA), while MDA-MB-231 cells transfected with ERBB2 (231-H2N) were kindly provided by Dr. J. Fluorescence microscopy Images were tiled using an Olympus BX50 upright fluo- rescent microscope linked to a Photometrics CoolSnap HQ2 CCD camera, a motorized X-Y stage connected to a computer preloaded with Media Cybernetics In Vivo and Image Pro-PLUS software (Media Cybernetics, Silver Spring, MD) and a stage controller board. Tumor sections were scanned and tiled under white light and two differ- ent filters: (i) images of Cy3 fluorescence of CD31 were visualized using 530 nm to 560 nm excitation and 573 nm to 647 nm emission filter sets, while (ii) images of the Cy5 fluorescence of EF5 were visualized with 630 nm to 650 nm excitation and 665 nm to 695 nm emission filter sets. Composite images of cetuximab, CD31, and EF5 or tras- tuzumab, CD31 and EF5 were generated using Image Pro PLUS (version 5) and subsequently pseudo-colored. To investigate the distribution of drug in relation to distance from the nearest blood vessel or hypoxic region, images displaying anti-CD31 staining or EF5 staining were con- verted to black and white binary images: each image was overlayed with the corresponding field of view displaying drug intensity, resulting in an 8-bit black and white image with blood vessels or hypoxic regions identified by an intensity of 255 (white) and drug intensity ranging from 0-254 (gray scale). Areas of interest were selected from each tissue section and were on average 1600 × 1600 μm (0.4 μm2/pixel). Areas of necrosis and staining artifact were excluded. Figure 1 Immmunohistochemical staining of sections of xeno- grafts. Immmunohistochemical staining after ex vivo application of cetuximab to identify ErbB1 expression (upper panels) or of trastuzum- ab to identify ErbB2 expression (lower panels). Scale bar = 100 μm. sion of ErbB2 in wild-type MDA-MB-231 xenografts and high expression in the ERBB2-transfected 231-H2N xenografts (Fig. 1, lower panels). In tumors that express the receptors the staining indicates their distribution on the cell membrane. Distributions of each monoclonal antibody in relation to distance from the nearest blood vessel and the nearest region of hypoxia in the tumor section were quantified utilizing Image Pro software. A minimum signal level just below threshold was set for each tissue section; this was based on an average background reading from regions without staining. Cell lines and tumor models Experiments were performed utilizing the ErbB1-overex- pressing human epidermoid carcinoma (A431) and a human breast adenocarcinoma (MDA-MB-231), using both wild-type and ERBB2 transfected (231-H2N) cell lines. A431 and MDA-MB-231 cells were obtained from Lee and Tannock BMC Cancer 2010, 10:255 http://www.biomedcentral.com/1471-2407/10/255 Page 3 of 11 disclosed using a Cy3-conjugated goat anti-rat IgG sec- ondary antibody. Hypoxic regions were recognized by cyanine-5-conjugated mouse anti-EF5 (1/50) antibody. Figure 1 Immmunohistochemical staining of sections of xeno- grafts. Immmunohistochemical staining after ex vivo application of cetuximab to identify ErbB1 expression (upper panels) or of trastuzum- ab to identify ErbB2 expression (lower panels). Scale bar = 100 μm. Fluorescence microscopy The pixel intensity and distance to the nearest vessel or region of hypoxia for all pixels within the selected region of interest above threshold were mea- sured with a customized algorithm. The intensity of cetuximab or trastuzumab signal was represented as mean ± SEM for all pixels at a given distance to the near- est vessel or region of hypoxia and plotted as a function of that distance. Expression of receptors was fairly uniform in tumors, except for regions of hypoxia (defined by EF5 staining) where there was lower expression of ErbB1 and ErbB2. We also studied receptor expression in tumors of animals that were treated with the therapeutic antibodies, and found no effect of treatment on receptor expression. Time- and dose-dependent distribution of cetuximab Dose-dependent distribution of cetuximab in A431 xeno- grafts 24 h after i.p. injection of different doses is shown in Fig. 2. After injection of 0.01 mg or 0.05 mg cetuximab, there was selective distribution closer to blood vessels, and no penetration to hypoxic regions (shown in green), but at 24 h after injection of 1.0 mg cetuximab, the distri- bution was more uniform within the tumor, although there remained minimal drug penetration to hypoxic regions identified by uptake of EF5. Staining was honey- comb in appearance, consistent with antibody binding to receptors on the outer membranes of tumor cells. There Expression of ErbB receptors Ex vivo staining using cetuximab was used to recognize expression of ErbB1 in A431 and MDA-MB-231 tumor sections; these tumors express high and intermediate lev- els of ErbB1 respectively (Fig. 1, upper panels). Similarly, ex vivo application of trastuzumab indicates low expres- Lee and Tannock BMC Cancer 2010, 10:255 Page 4 of 11 http://www.biomedcentral.com/1471-2407/10/255 Figure 2 Dose response distribution of cetuximab in relation to blood vessels and regions of hypoxia in A431 xenografts. Left panels sh the distribution of cetuximab (blue) in relation to blood vessels (red) and regions of hypoxia (green) in A431 xenografts at 24 h after an i.p. inject of (A) 0.01 mg, (B) 0.05 mg, and (C) 1.0 mg. In right panels staining intensity (mean +/- SEM) due to cetuximab is plotted against distance from th nearest blood vessel in the tumor section. Note minimal drug binding in hypoxic regions. Scale bar = 100 μm. 0 10 20 30 40 50 60 20 40 60 80 100 Distance from blood vessel (μμμμm) Fluorescent Intensity 0 10 20 30 40 50 60 20 40 60 80 100 Distance from blood vessel (μμμμm) Fluorescent Intensity 0 10 20 30 40 50 60 20 40 60 80 100 Distance from blood vessel (μμμμm) Fluorescent Intensity 0 10 20 30 40 50 60 20 40 60 80 100 Distance from blood vessel (μμμμm) Fluorescent Intensity Distance from blood vessel (μμμμm) 0 10 20 30 40 50 60 20 40 60 80 100 Distance from blood vessel (μμμμm) Fluorescent Intensity Distance from blood vessel (μμμμm) 0 10 20 30 40 50 60 20 40 60 80 100 Distance from blood vessel (μμμμm) Fluorescent Intensity Figure 2 Dose response distribution of cetuximab in relation to blood vessels and regions of hypoxia in A431 xenografts. Left panels show the distribution of cetuximab (blue) in relation to blood vessels (red) and regions of hypoxia (green) in A431 xenografts at 24 h after an i.p. injection of (A) 0.01 mg, (B) 0.05 mg, and (C) 1.0 mg. In right panels staining intensity (mean +/- SEM) due to cetuximab is plotted against distance from the nearest blood vessel in the tumor section. Note minimal drug binding in hypoxic regions. Scale bar = 100 μm. Discussion Cetuximab and trastuzumab have shown limited efficacy in causing remission in a proportion of patients with met- astatic colorectal cancer and breast cancer respectively [21,22], while trastuzumab has improved survival of women with ErbB2 positive breast cancer when given as adjuvant therapy after chemotherapy [32-34]. Monoclo- nal antibodies are large molecules, which are "consumed" by binding to receptors on the cell surface, conditions that might lead to poor penetration of tissue within solid tumors [28]. Indeed, an early study of the distribution of a radiolabeled monoclonal antibody into multicellular spheroids suggested very slow penetration of tissue, with establishment of a steep concentration gradient [35], and more recent studies of the penetration of drugs such as doxorubicin (which binds avidly to DNA) have shown quite poor distribution [23-25]. Thus limited distribution of therapeutic agents within solid tumors is a potentially important and relatively neglected cause of drug resis- tance, especially in the metastatic setting. Here we have used quantitative immunohistochemistry to study the distribution within human tumor xenografts of two ther- apeutic monoclonal antibodies in clinical use, cetuximab and trastuzumab, to determine if their efficacy might be limited by failure to reach all of the target tumor cells in an effective concentration. The distribution of cetuximab 24 h after an intravenous injection of different doses was also investigated in A431 xenografts (data not shown). There were no significant differences in the distributions of cetuximab after i.p. and i.v. injection. Time- and dose-dependent distribution of cetuximab in MDA-MB-231 xenografts (which express intermediate levels of ErbB1) is summarized in Table 1: with exclusion of the immediate perivascular region, staining intensity was relatively constant with increasing distance from the blood vessel at most times and doses, suggesting more rapid distribution than in the A431 tumors, which have higher levels of expression of ErbB1. Absolute levels of bound cetuximab increased with both dose injected and time after injection. The results of our study show that distribution of both of these therapeutic antibodies is time and dose-depen- dent. At short intervals after injection of all doses there is a concentration gradient of staining intensity of the anti- bodies with increasing distance from blood vessels within tumors that strongly express the target receptor. However there is a greater change in the gradient of cetuximab intensity in A431 xenografts than of trastuzumab inten- sity in 231-H2N xenografts. Discussion At moderate and high doses the distribution then becomes more uniform with time, while at lower doses the heterogeneous distribution is retained. Distribution of cetuximab and trastuzumab in relation to hypoxic regions provides a better understand- ing of the distribution of the antibodies distal to blood vessels. There remains minimal drug distribution to Expression of ErbB receptors Lee and Tannock BMC Cancer 2010, 10:255 http://www.biomedcentral.com/1471-2407/10/255 Page 5 of 11 selective perivascular localization of trastuzumab at 30 min and 4 h after injection, but more uniform distribu- tion after 24 h. Staining intensities at ~20 μm from blood vessels after an injection of 0.3 mg of trastuzumab varied only by a factor of ~1.5 at 30 min to 24 h after injection, again suggesting early saturation of cells proximal to blood vessels. Trastuzumab distribution in relation to hypoxic regions is plotted in green in Fig 5, staining intensity due to trastuzumab increases in regions close to hypoxia as the time interval increases. was an apparent increase in intensity at very short dis- tances from the centers of blood vessels, likely because of lack of expression of ErbB1 on endothelial cells and peri- cytes. The time-dependent distribution of cetuximab after an i.p. injection of 1.0 mg into mice bearing A431 xenografts is shown in Fig 3. With exclusion of the immediate perivascular region, there was a gradient of decreasing concentration at increasing distances from blood vessels at 30 min and 4 h after injection (Fig 3), but at 24 h and 48 h the intensity of cetuximab staining was relatively uni- form within the tumor tissue (Figs. 2 and 3). There was no staining due to cetuximab in hypoxic regions shown in green. Cetuximab distribution in relation to hypoxic regions is also plotted in Fig 3, which shows that staining intensity due to cetuximab increases as the distance from the hypoxic regions increases. The slopes of the relation- ships between staining intensity of cetuximab and dis- tance from blood vessels after various doses and times are summarized in Table 1. These data confirm relatively uni- form distribution at 24 h - 48 h after injection of the higher dose of 1 mg cetuximab, with the caveat that there is still minimal binding within hypoxic regions of the tumors. Trastuzumab was not found bound to cells of MDA- MB-231 xenografts which express low levels of ErbB2. Time- and dose-dependent distribution of trastuzumab The distribution of trastuzumab at 2 h after i.v. injection of doses of 0.1 mg, 0.3 mg or 1.0 mg into mice bearing 231-H2N xenografts (which over-express ErbB2) is shown in Fig 4. There was selective localization close to blood vessels at lower doses and uniform distribution after the 1.0 mg dose. Staining due to trastuzumab was not found in regions of hypoxia (shown in green). Stain- ing intensities at ~20 μm from blood vessels varied by a factor of ~1.5 after i.v. injection of doses of 0.1 mg - 1.0 mg (Table 1), suggesting that binding to proximal cells is close to saturated. The distribution of trastuzumab as a function of time after injection of 0.3 mg is shown in Fig 5: There was Lee and Tannock BMC Cancer 2010, 10:255 http://www.biomedcentral.com/1471-2407/10/255 Lee and Tannock BMC Cancer 2010, 10:255 Page 6 of 11 http://www.biomedcentral.com/1471-2407/10/255 Table 1: Cetuximab and trastuzumab staining intensity in different xenografts. Time- and dose-dependent distribution of trastuzumab Cell line Monoclonal antibody Dose (mg) Time after injection Staining Intensity at 20 μm from blood vessels (mean IU) ± SEM Staining Intensity at 100 μm from blood vessels (mean IU) ± SEM Gradient of Staining Intensity (IU/μm) A431 Cetuximab 0.01 24 h 26.8 ± 2.0 12.7 ± 1.4 -0.18 0.05 24 h 34.3 ± 4.8 24.5 ± 0.4 -0.12 1.0 30 min 36.1 ± 0.2 21.2 ± 1.3 -0.19 1.0 4 h 34.9 ± 2.7 25.0 ± 2.3 -0.12 1.0 24 h 35.9 ± 3.5 34.7 ± 3.9 -0.02 1.0 48 h 36.1 ± 1.2 37.8 ± 1.8 -0.02 MDA-MB-231 Cetuximab 0.01 24 h 7.0 ± 0.6 7.4 ± 1.1 0.01 0.05 24 h 7.6 ± 0.8 6.9 ± 0.9 -0.01 0.1 24 h 15.0 ± 2.8 18.5 ± 2.8 0.04 0.5 15 min 6.9 ± 1.5 6.3 ± 0.7 -0.01 0.5 30 min 8.3 ± 5.7 5.7 ± 3.7 -0.03 0.5 1 h 18.9 ± 1.1 17.0 ± 1.1 -0.02 0.5 2 h 19.1 ± 3.9 14.0 ± 2.9 -0.06 0.5 4 h 20.8 ± 1.5 24.3 ± 1.5 0.04 0.5 6 h 17.6 ± 4.0 20.3 ± 4.6 0.03 0.5 24 h 16.7 ± 2.2 20.7 ± 1.6 0.05 1.0 24 h 17.3 ± 2.7 21.2 ± 2.5 0.05 231-H2N Trastuzumab 0.1 2 h 16.8 ± 2.1 12.7 ± 1.8 -0.05 0.3 30 min 19.4 ± 0.9 16.6 ± 1.3 -0.04 0.3 2 h 23.0 ± 1.6 19.9 ± 2.1 -0.04 0.3 4 h 24.0 ± 2.1 18.5 ± 3.0 -0.07 0.3 24 h 29.0 ± 1.0 27.3 ± 0.8 -0.02 1.0 2 h 27.0 ± 1.3 26.2 ± 1.4 -0.01 Staining intensity of cetuximab and trastuzumab at 20 μm and 100 μm from blood vessels in A431, MDA-MB-231 and 231-H2N xenografts. Gradient of staining intensity is shown. Table 1: Cetuximab and trastuzumab staining intensity in different xenografts. hypoxic tumor cells under all conditions, which is proba- bly due both to limited availability of drug in these regions, and to decreased expression of the ErbB recep- tors under hypoxic conditions. higher levels of ErbB1, and homogeneity of distribution of cetuximab in MDA-MB-231 xenografts was achieved more rapidly. This is probably due to the low receptor binding of cetuximab (i.e. less consumption of drug) by proximal cells in MDA-MB-231 xenografts. Trastuzumab was not identified after injection in MDA-MB-231 xeno- grafts, which express low levels of ErbB2. Time- and dose-dependent distribution of trastuzumab The difference in time dependence of the distributions of the monoclonal antibodies as compared to that for doxorubicin, which is relatively independent of time after injection [24] is most likely due to the half-lives of the drugs in the circulation: doxorubicin has a short initial half-life [36], such that most penetration from vessels takes place quickly, whereas monoclonal antibodies have a half-life of days [37-39], allowing for a more constant process of tissue penetration. Multiple phase I and II clinical trials have established that standard weekly dosing of cetuximab or trastuzumab in humans achieves trough serum concentrations that are usually above 50 μg/ml [37,38,40-42]. We did not mea- sure serum concentration of cetuximab or trastuzumab in our mice. Others have reported maximum serum levels of cetuximab of ~65 μg/ml and ~400 μg/ml cetuximab after injection of doses of 0.25 mg and 1.0 mg into mice respec- tively [28,39], similar to those reported in patients. Injec- The gradients of cetuximab intensity in MDA-MB-231 xenografts, which express intermediate levels of ErbB1, are less steep than in A431 xenografts, which express Lee and Tannock BMC Cancer 2010, 10:255 Page 7 of 11 Lee and Tannock BMC Cancer 2010, 10:255 http://www.biomedcentral.com/1471-2407/10/255 http://www.biomedcentral.com/1471-2407/10/255 Figure 3 Time response distribution of cetuximab in relation to blood vessels and regions of hypoxia in A431 xenografts. Left panels show the distribution of cetuximab (blue) in relation to blood vessels (red) and regions of hypoxia (green) in A431 xenografts at (A) 30 min, (B) 4 h and (C) 48 h after i.p. injection of 1.0 mg. In right panels staining intensity due to cetuximab is plotted against distance to the blood vessel in red and distance to region of hypoxia in green. Scale bar = 100 μm. Figure 3 Time response distribution of cetuximab in relation to blood vessels and regions of hypoxia in A431 xenografts. Left panels show the distribution of cetuximab (blue) in relation to blood vessels (red) and regions of hypoxia (green) in A431 xenografts at (A) 30 min, (B) 4 h and (C) 48 h after i.p. injection of 1.0 mg. In right panels staining intensity due to cetuximab is plotted against distance to the blood vessel in red and distance to region of hypoxia in green. Scale bar = 100 μm. Time- and dose-dependent distribution of trastuzumab Lee and Tannock BMC Cancer 2010, 10:255 Page 8 of 11 Lee and Tannock BMC Cancer 2010, 10:255 http://www.biomedcentral.com/1471-2407/10/255 http://www.biomedcentral.com/1471-2407/10/255 ure 4 Dose response distribution of trastuzumab in relation to blood vessels and regions of hypoxia in 231-H2N xenografts. Left pan w the distribution of trastuzumab (blue) in relation to blood vessels (red) and regions of hypoxia (green) in MDA-MB-231 breast cancer xenogra sfected with ErbB2 (231-H2N) at 2 h after i.v. injection of (A) 0.1 mg, (B) 0.3 mg and (C) 1.0 mg. In right panels staining intensity (mean +/- SEM to trastuzumab is plotted against distance from the nearest blood vessel in the tumor section. Note minimal drug binding in hypoxic regions e bar = 100 μm. 0 10 20 30 40 50 60 20 40 60 80 100 Distance from blood vessel (μμμμm) F lu o re s c e n t In te n s ity 0 10 20 30 40 50 60 20 40 60 80 100 Distance from blood vessel (μμμμm) Fluorescent Intensity 0 10 20 30 40 50 60 20 40 60 80 100 Distance from blood vessel (μμμμm) Fluorescent Intensity 0 10 20 30 40 50 60 20 40 60 80 100 Distance from blood vessel (μμμμm) F lu o re s c e n t In te n s ity 0 10 20 30 40 50 60 20 40 60 80 100 Distance from blood vessel (μμμμm) Fluorescent Intensity 0 10 20 30 40 50 60 20 40 60 80 100 Distance from blood vessel (μμμμm) Fluorescent Intensity Figure 4 Dose response distribution of trastuzumab in relation to blood vessels and regions of hypoxia in 231-H2N xenografts. Left panels show the distribution of trastuzumab (blue) in relation to blood vessels (red) and regions of hypoxia (green) in MDA-MB-231 breast cancer xenografts transfected with ErbB2 (231-H2N) at 2 h after i.v. injection of (A) 0.1 mg, (B) 0.3 mg and (C) 1.0 mg. In right panels staining intensity (mean +/- SEM) due to trastuzumab is plotted against distance from the nearest blood vessel in the tumor section. Note minimal drug binding in hypoxic regions. Scale bar = 100 μm. Lee and Tannock BMC Cancer 2010, 10:255 Page 9 of 11 http://www.biomedcentral.com/1471-2407/10/255 re 5 Time response distribution of trastuzumab in relation to blood vessels and regions of hypoxia in 231-H2N xenografts. References 1. Roskoski R Jr: The ErbB/HER receptor protein-tyrosine kinases and cancer. Biochemical and biophysical research communications 2004, 319(1):1-11. 2. Weiss A, Schlessinger J: Switching signals on or off by receptor dimerization. Cell 1998, 94(3):277-280. 3. DiGiovanna MP, Stern DF, Edgerton SM, Whalen SG, Moore D, Thor AD: Relationship of epidermal growth factor receptor expression to ErbB-2 signaling activity and prognosis in breast cancer patients. J Clin Oncol 2005, 23(6):1152-1160. 4. O'Dwyer PJ, Benson AB: Epidermal growth factor receptor-targeted therapy in colorectal cancer. Seminars in oncology 2002, 29(5 Suppl 14):10-17. 4. O'Dwyer PJ, Benson AB: Epidermal growth factor receptor-targeted therapy in colorectal cancer. Seminars in oncology 2002, 29(5 Suppl 14):10-17. 5. Herbst RS, Hong WK: IMC-C225, an anti-epidermal growth factor receptor monoclonal antibody for treatment of head and neck cancer. Seminars in oncology 2002, 29(5 Suppl 14):18-30. 5. Herbst RS, Hong WK: IMC-C225, an anti-epidermal growth factor receptor monoclonal antibody for treatment of head and neck cancer. Seminars in oncology 2002, 29(5 Suppl 14):18-30. 6. Lager DJ, Slagel DD, Palechek PL: The expression of epidermal growth factor receptor and transforming growth factor alpha in renal cell carcinoma. Mod Pathol 1994, 7(5):544-548. 6. Lager DJ, Slagel DD, Palechek PL: The expression of epidermal growth factor receptor and transforming growth factor alpha in renal cell carcinoma. Mod Pathol 1994, 7(5):544-548. 7. Berger MS, Gullick WJ, Greenfield C, Evans S, Addis BJ, Waterfield MD: Epidermal growth factor receptors in lung tumours. The Journal of pathology 1987, 152(4):297-307. 7. Berger MS, Gullick WJ, Greenfield C, Evans S, Addis BJ, Waterfield MD: Epidermal growth factor receptors in lung tumours. The Journal of pathology 1987, 152(4):297-307. 8. Hirsch FR, Scagliotti GV, Langer CJ, Varella-Garcia M, Franklin WA: Epidermal growth factor family of receptors in preneoplasia and lung cancer: perspectives for targeted therapies. Lung cancer (Amsterdam, Netherlands) 2003, 41(Suppl 1):S29-42. 9. Xiong HQ, Abbruzzese JL: Epidermal growth factor receptor-targeted therapy for pancreatic cancer. Seminars in oncology 2002, 29(5 Suppl 14):31-37. 9. Xiong HQ, Abbruzzese JL: Epidermal growth factor receptor-targeted therapy for pancreatic cancer. Seminars in oncology 2002, 29(5 Suppl 14):31-37. 10. Di Lorenzo G, Tortora G, D'Armiento FP, De Rosa G, Staibano S, Autorino R, D'Armiento M, De Laurentiis M, De Placido S, Catalano G, et al.: Expression of epidermal growth factor receptor correlates with disease relapse and progression to androgen-independence in human prostate cancer. Clin Cancer Res 2002, 8(11):3438-3444. 10. Competing interests h h d l h tion of trastuzumab was reported to lead to serum levels of about 5 ng/ml at 6-24 hours after i.p injection of a sin- gle low dose of 0.3 mg/kg into mice [43]; if pharmacoki- netics were linear this would imply doses of ~15 mg/ mouse to achieve levels of 10 ug/ml in serum, but it seems unlikely that pharmacokinetics of the two antibod- ies would differ by such a large amount. Competing interests The authors declare that they have no competing interests. Authors' contributions CL designed and performed all the experiments and drafted the manuscript. IT conceived of the study, obtained funding for it and participated in its design and coordination and drafted the manuscript. Both authors read and approved the final manuscript. Acknowledgements S d b y g Several other investigators have studied the distribu- tion of various antibodies, or antibody fragments, in tumors. Their results depend on changes in blood flow [44] the affinity of the antibodies for their targets, but in general these authors have reported problems of hetero- geneity of distribution at various times after their admin- istration [45-48]. We were able to identify two other studies of the distribution of trastuzumab (but none of cetuximab) in solid tumors. Dennis et al used intravital microscopy to detect trastuzumab, conjugated to fluores- cein isothiocyanate (FITC), in relation to blood vessels of MMTV/HER2 transgenic mice (expressing high levels of ErbB2) that were constrained to grow in a transparent window chamber; they reported perivascular localization of trastuzumab at 24 hours after injection of 10 mg/kg (about 0.25 mg/mouse) [49]. Their study suggests poorer (or slower) distribution of trastuzumab than the one reported here; a possible reason is higher expression of ErbB2 in the MMTV/HER tumors as compared to the 231-H2N xenografts investigated in our study. Baker et al used similar methods to our own, and investigated time- dependent distributions of trastuzumab in xenografts (that did or did not express ErbB2) after i.p. injection doses in the range of 4-20 mg/kg (about 0.1- 0.5 mg/ mouse) [50]. They found perivascular distribution of drug at 3 h, and that tumor margins reached saturation with trastuzumab more rapidly than the (poorly-vascu- larized) interior. Drug distribution became more uniform at 24 h as compared to 8 h after injection of 4 mg/kg, but some heterogeneity of trastuzumab distribution was observed in the tumor under all conditions; this is consis- tent with our finding of poor drug uptake in hypoxic tumor regions. Supported by a research grant from the Canadian Institutes for Health Research (MOP 89762). We thank Dr Licun Wu for technical support. Supported by a research grant from the Canadian Institutes for Health Research (MOP 89762). We thank Dr Licun Wu for technical support. References Di Lorenzo G, Tortora G, D'Armiento FP, De Rosa G, Staibano S, Autorino R, D'Armiento M, De Laurentiis M, De Placido S, Catalano G, et al.: Expression of epidermal growth factor receptor correlates with disease relapse and progression to androgen-independence in human prostate cancer. Clin Cancer Res 2002, 8(11):3438-3444. Author Details f Divisions of Applied Molecular Oncology and Medical Oncology and Hematology Princess Margaret Hospital and University of Toronto, Toronto, ON, Canada Divisions of Applied Molecular Oncology and Medical Oncology and Hematology Princess Margaret Hospital and University of Toronto, Toronto, ON, Canada Received: 17 July 2009 Accepted: 3 June 2010 Published: 3 June 2010 This article is available from: http://www.biomedcentral.com/1471-2407/10/255 © 2010 Lee and Tannock; 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 unrestricte BMC Cancer 2010, 10:255 Time- and dose-dependent distribution of trastuzumab Left panels w the distribution of trastuzumab (blue) in relation to blood vessels (red) in MDA-MB-231 breast cancer xenografts transfected with ErbB2 (231- at (A) 30 min, (B) 4 h and (C) 24 h after i.v. injection of 0.3 mg trastuzumab. In right panels staining intensity due to trastuzumab is plotted against nce to the blood vessel in red and distance to region of hypoxia in green. Scale bar = 100 μm. Figure 5 Time response distribution of trastuzumab in relation to blood vessels and regions of hypoxia in 231-H2N xenografts. Left panels show the distribution of trastuzumab (blue) in relation to blood vessels (red) in MDA-MB-231 breast cancer xenografts transfected with ErbB2 (231- H2N) at (A) 30 min, (B) 4 h and (C) 24 h after i.v. injection of 0.3 mg trastuzumab. In right panels staining intensity due to trastuzumab is plotted against distance to the blood vessel in red and distance to region of hypoxia in green. Scale bar = 100 μm. Page 10 of 11 Lee and Tannock BMC Cancer 2010, 10:255 http://www.biomedcentral.com/1471-2407/10/255 15. Harris M: Monoclonal antibodies as therapeutic agents for cancer. The lancet oncology 2004, 5(5):292-302. 13. Goldberg RM: Cetuximab. Nature reviews 2005:S10-11. Conclusions 11. Itakura Y, Sasano H, Shiga C, Furukawa Y, Shiga K, Mori S, Nagura H: Epidermal growth factor receptor overexpression in esophageal carcinoma. An immunohistochemical study correlated with clinicopathologic findings and DNA amplification. Cancer 1994, 74(3):795-804. 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https://openalex.org/W2255981601	https://europepmc.org/articles/pmc4748604?pdf=render	English	null	Idiopathic pulmonary fibrosis – clinical management guided by the evidence-based GRADE approach: what arguments can be made against transparency in guideline development?	BMC medicine	2,016	cc-by	3,840	© 2016 Rochwerg et al. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Rochwerg et al. BMC Medicine (2016) 14:22 DOI 10.1186/s12916-016-0563-0 Rochwerg et al. BMC Medicine (2016) 14:22 DOI 10.1186/s12916-016-0563-0 Rochwerg et al. BMC Medicine (2016) 14:22 DOI 10.1186/s12916-016-0563-0 Rochwerg et al. BMC Medicine (2016) 14:22 DOI 10.1186/s12916-016-0563-0 Open Access Idiopathic pulmonary fibrosis – clinical management guided by the evidence- based GRADE approach: what arguments can be made against transparency in guideline development? Bram Rochwerg1,2, Holger J. Schünemann1,2 and Ganesh Raghu3,4* Bram Rochwerg1,2, Holger J. Schünemann1,2 and Ganesh Raghu3,4* Bram Rochwerg1,2, Holger J. Schünemann1,2 and Ganesh Raghu3,4* * Correspondence: graghu@u.washington.edu 3Department of Medicine (Division of Pulmonary and Critical Care Medicine), University of Washington, Seattle, WA, USA 4Center for Interstitial lung Diseases, UW Medical Center, Seattle, WA, USA Full list of author information is available at the end of the article Abstract Evidence-based guidelines have undergone an incredible transformation over the last number of years. Significant advances include explicit linkages of systematic evidence summaries to the strength and direction of recommendations, consideration of all patient-important factors, transparent reporting of the recommendation generation process including conflict of interest management strategies and the production of clinical practice guidelines which use simple and clear language. The Grading of Recommendations Assessment, Development and Evaluation (GRADE) methodology provides a framework for guideline development and was employed to produce the recently published ATS/ERS/JRS/ALAT update on treatment for idiopathic pulmonary fibrosis (IPF). Herein we discuss the advantages of using an evidence-based approach for guideline development using the IPF process and resultant document as an example. Keywords: Evidence-based, Guideline, Idiopathic pulmonary fibrosis In 2000, a selected panel of international experts in the field of interstitial lung diseases provided a guideline for the diagnosis and management of IPF [5]. Due to the paucity of evidence available and the relatively immature field of guideline methodology, this document was devel- oped using the conventional consensus-approach based on the opinions of the few expert panel members without a systematic review of the literature or formal quality of evidence evaluation. Regardless, this document, then considered state of the art for the disease, provided useful direction to clinicians in diagnosing and managing pa- tients with IPF. Over the next decade, an increasing number of studies in the field of IPF were published based on this guidance document. With accumulating evidence, it became onerous for practicing clinicians to carefully review and interpret the most current studies. In order to address this challenge and to improve on the previous document, the 2011 guideline document employed an evidence-based approach, namely the GRADE (Grading of Recommendations Assessment, Development and Evaluation) approach to guideline development [6, 7]. Background d b Evidence-based guidelines have undergone intense evolution over the last 15 years [1–4]. The main driver behind this transformation has been the change in focus from what used to be called expert- or consensus-based to evidence-driven recommendations. This distinction represents a typical misunderstanding as even in the era of evidence-based guidelines, recommendations are developed by clinical experts in the field and require consensus of panel members on the best possible treatment options. It is the transparent link between the evidence and the recommendations and the requirement of making structured expert judgments that represents a shift in the guideline development paradigm. This is true even for evidence-based guidelines that focus on complex and rare diseases such as idiopathic pulmonary fibrosis (IPF). * Correspondence: graghu@u.washington.edu 3Department of Medicine (Division of Pulmonary and Critical Care Medicine), University of Washington, Seattle, WA, USA 4Center for Interstitial lung Diseases, UW Medical Center, Seattle, WA, USA Full list of author information is available at the end of the article Page 2 of 4 Rochwerg et al. BMC Medicine (2016) 14:22 Page 2 of 4 since demonstrated the harm of this treatment inter- vention [25]. The 2011 guideline redefined IPF with precise diagnostic criteria based on clinical, radiological and histopathological features. Also, for the first time in the field of IPF, it pro- vided evidence-based treatment recommendations [8]. For the 2015 IPF treatment guideline, the McMaster University GRADE (MacGRADE; cebgrade.mcmaster.ca) team performed comprehensive systematic reviews for each of the 12 clinical questions. These were done in collaboration with clinical experts in the field to ensure proper question development and an experienced infor- mation scientist [26]. These systematic reviews provided the IPF guideline panel, including experienced IPF experts (who based on their involvement in IPF clinical trials and direct financial COI were considered conflicted panel members) and the non-conflicted members with the best available evidence summaries on which to base recom- mendations. The conflicted and non-conflicted members of the panel discussed all the evidence summaries in an open format, thereby enabling the non-conflicted panel members to deepen their understanding of the clinical relevance of the data and seek clarification as required. The evidence summaries were included as part of the guideline document to ensure the transparency of the entire process. Evidence-based Guideline Development Evidence based Guideline Development Some believe that the opinion of experts in the field is driven by their understanding of the clinical problem and their accurate interpretation of the underlying lit- erature and therefore such recommendations may be considered, in effect, evidence-based. This approach has gone wrong on many occasions [11, 12]. Using a transparent and structured process in guideline devel- opment not only mandates a linkage of evidence to recommendation development but also ensures this linkage is explicit and systematically demonstrated [13–20]. In effect, this helps to limit the potential for bias. Guideline panel members may have strong opinions or academic bias surrounding a specific area in which they have clinical ex- pertise or they may have other bias related to interactions with academic colleagues or industry partners [21, 22]. In an attempt to address this, GRADE requires systematic or pragmatic searches of the literature and the production of evidence summaries such as evidence profiles [23], ideally based on pooled treatment effects and produced by panel members without conflict of interest (COI) or independent methodologists. The latter helps to ensure a fair and reproducible assessment of the current literature address- ing a specific clinical question [21]. Certainties in evidence assessments were performed by the MacGRADE team and were then reviewed by the entire panel to ensure accuracy and agreement. In each case that the certainty in effect estimates was downgraded, explicit rationale was provided in the evidence profile and in the guideline manuscript. Including the certainty as part of the final recommendation, as we have done for the 2015 IPF evidence-based guideline, helps stakeholders in interpreting the recommendations made by the panel. Recommendations based on higher quality of evidence allow clinicians and patients to be more reassured that this intervention is beneficial. Recommendations based on lower quality of evidence provide caution to stakeholders and recognize the uncertainty that exists regarding the benefits of this intervention. Although the estimate of treatment effect and certainty in evidence are important, guideline panelists should also consider other factors when deciding on the strength of recommendations. Elements such as the balance between the desirable and undesirable effects, the resources re- quired, the impact on health equality, the acceptability and the feasibility of treatment must also be considered [20]. Background d b In this commentary, we will discuss the motivation behind this evolution in evidence-based guidelines and the benefits of developing guidelines that are linked to underlying evidence summaries along with explicit as- sessments of the quality (certainty) of the evidence. We will focus on the utility of GRADE methodology [7, 9], used for the 2011 IPF evidence-based guideline and again for the more recent 2015 IPF treatment update [10]. ALAT: Latin American Thoracic Society; ATS: American Thoracic Society; COI: conflict of interest; ERS: European Respiratory Society; EtD: evidence to decision; GRADE: Grading of Recommendations Assessment, Development and Evaluation; IPF: Idiopathic pulmonary fibrosis; JRS: Japanese Respiratory Competing interests Ganesh Raghu has declared no financial competing interests. He is a consultant for IPF studies at Boehringer Ingelheim, Biogen,FibroGen Inc, Gilead, Janssen,, MedImmune, Promedior,, Sanofi Aventis and Veracyte. Bram Rochwerg & Holger Schunemann have no conflicts of interest to declare. The final result, using the GRADE process, is a compre- hensive, systematic, and explicit evidence-based guideline. Recommendations for the 2015 IPF update were formu- lated using the common terminology of “we suggest” for conditional (also known as weak) recommendations and “we recommend” for strong recommendations [19]. Strong recommendations are those that are applicable to the vast majority of patients, understanding a small minor- ity will choose the opposite course of action. These recom- mendations are sometimes used to drive policy decisions. Conditional recommendations should apply to the majority of patients but there will be a large minority who will choose the opposite [13]. For conditional recommenda- tions, especially those based on low or very low certainty of evidence, a model of shared decision-making between clini- cians and patients is imperative considering all the factors above in addition to the individual patient’s values and pref- erences [28]. In essence, providing the recommendation in clear language along with a descriptive rationale empowers patients, clinicians, and stakeholders to better understand how the recommendations were formulated and to better apply them to their specific clinical practices and situations. This process using GRADE methodology is inherently different than that used by regulatory agencies when they consider market approval for pharmacological agents for the treatment of IPF. References Idiopathic pulmonary fibrosis: diagnosis and treatment. International consensus statement. American Thoracic Society (ATS), and the European Respiratory Society (ERS). Am J Respir Crit Care Med. 2000;161(2 Pt 1):646–64. 6. Guyatt GH, Oxman AD, Schunemann HJ, Tugwell P, Knottnerus A. GRADE guidelines: a new series of articles in the Journal of Clinical Epidemiology. J Clin Epidemiol. 2011;64(4):380–2. 6. Guyatt GH, Oxman AD, Schunemann HJ, Tugwell P, Knottnerus A. GRADE guidelines: a new series of articles in the Journal of Clinical Epidemiology. J Clin Epidemiol. 2011;64(4):380–2. Authors’ contributions BR d d h fi d BR produced the first draft of the manuscript. GR & HS provided input through subsequent edits. All authors approved the final version for submission. Evidence-based Guideline Development As opposed to consensus documents that use an ad hoc approach, GRADE mandates an explicit assess- ment of these criteria, using the Evidence-to-Decision framework (EtD), with documentation of panel judgements and rationale [19, 20, 27]. Another significant benefit of evidence-based guidelines using GRADE methodology is the formal assessment of certainty in treatment effects (also known as quality of evi- dence or confidence in evidence). GRADE mandates a sys- tematic and explicit assessment of specific methodological domains in order to evaluate how certain we are in the evidence for each outcome [24]. The certainty in treat- ment effect is then considered by the guideline panel and factored into the judgement regarding the strength and direction of recommendations. In this regard, several guidelines that followed unstructured approaches have un- fortunately made inappropriately strong recommendations without considering the certainty in the evidence. As an example from the IPF literature, the 2000 consensus statement recommended treatment with azathioprine and corticosteroids for patients with IPF without explicit quality of evidence assessment [5]. Subsequent RCTs have Input from clinical experts in the field is integral and guideline panelists must interpret the evidence sum- mary, the certainty in estimate effects and consider the factors listed above in order to arrive at a direction and strength of recommendation using the EtD. Although Rochwerg et al. BMC Medicine (2016) 14:22 Page 3 of 4 some judgements will require an element of subjectivity, GRADE ensures explicit recording of rationale in order to improve transparency and reproducibility. Discussion of the evidence summaries and other EtD criteria for the 2015 IPF guidelines included all panel members, however, in order to help ensure impartiality, only non-conflicted members of the panel were permitted to formulate the recommendations themselves [22]. Society; MacGRADE: McMaster University GRADE group; RCT: randomized controlled trials. 9. Guyatt GH, Oxman AD, Vist GE, Kunz R, Falck-Ytter Y, Alonso-Coello P, et al. GRADE: an emerging consensus on rating quality of evidence and strength of recommendations. BMJ. 2008;336(7650):924–6. Conclusion In summary, guidelines lacking the methodological com- ponents described above, especially those on topics with sufficient evidence, while conveying the (unstructured) opinions of clinical experts in the field, have a significant risk of providing biased recommendations that may be then used to guide patient care. With the increasing amount of evidence that has accumulated since the 2000 guidelines, the evolution towards evidence-based guide- lines using the methodology described for a complex disease such as IPF is a clear benefit to all and represents a true advance in clinical science and patient-centered healthcare. At the end, what arguments can be made against transparency in guideline development? 7. Schunemann HJ, Jaeschke R, Cook DJ, Bria WF, El-Solh AA, Ernst A, et al. An official ATS statement: grading the quality of evidence and strength of recommendations in ATS guidelines and recommendations. Am J Respir Crit Care Med. 2006;174(5):605–14. 7. Schunemann HJ, Jaeschke R, Cook DJ, Bria WF, El-Solh AA, Ernst A, et al. An official ATS statement: grading the quality of evidence and strength of recommendations in ATS guidelines and recommendations. Am J Respir Crit Care Med. 2006;174(5):605–14. 8. Raghu G, Collard HR, Egan JJ, Martinez FJ, Behr J, Brown KK, et al. An official ATS/ERS/JRS/ALAT statement: idiopathic pulmonary fibrosis: evidence-based guidelines for diagnosis and management. Am J Respir Crit Care Med. 2011;183(6):788–824. 8. Raghu G, Collard HR, Egan JJ, Martinez FJ, Behr J, Brown KK, et al. An official ATS/ERS/JRS/ALAT statement: idiopathic pulmonary fibrosis: evidence-based guidelines for diagnosis and management. Am J Respir Crit Care Med. 2011;183(6):788–824. 9. Guyatt GH, Oxman AD, Vist GE, Kunz R, Falck-Ytter Y, Alonso-Coello P, et al. GRADE: an emerging consensus on rating quality of evidence and strength of recommendations. BMJ. 2008;336(7650):924–6. 10. Raghu G, Rochwerg B, Zhang Y, Garcia CA, Azuma A, Behr J, et al. An Official ATS/ERS/JRS/ALAT Clinical Practice Guideline: Treatment of Idiopathic Pulmonary Fibrosis. An Update of the 2011 Clinical Practice Guideline. Am J Respir Crit Care Med. 2015;192(2):e3–19. 10. Raghu G, Rochwerg B, Zhang Y, Garcia CA, Azuma A, Behr J, et al. An Official ATS/ERS/JRS/ALAT Clinical Practice Guideline: Treatment of Idiopathic Pulmonary Fibrosis. An Update of the 2011 Clinical Practice Guideline. Am J Respir Crit Care Med. 2015;192(2):e3–19. 11. Norris SL, Burda BU, Holmer HK, Ogden LA, Fu R, Bero L, et al. Author’s specialty and conflicts of interest contribute to conflicting guidelines for screening mammography. J Clin Epidemiol. 2012;65(7):725–33. References 1. Oxman AD, Fretheim A, Schunemann HJ. Improving the use of research evidence in guideline development: introduction. Health Res Policy Syst. 2006;4:12. 1. Oxman AD, Fretheim A, Schunemann HJ. Improving the use of research evidence in guideline development: introduction. Health Res Policy Syst. 2006;4:12. 2. Schunemann HJ, Hill SR, Kakad M, Vist GE, Bellamy R, Stockman L, et al. Transparent development of the WHO rapid advice guidelines. PLoS Med. 2007;4(5):e119. 2. Schunemann HJ, Hill SR, Kakad M, Vist GE, Bellamy R, Stockman L, et al. Transparent development of the WHO rapid advice guidelines. PLoS Med. 2007;4(5):e119. 3. Schunemann HJ, Woodhead M, Anzueto A, Buist AS, Macnee W, Rabe KF, et al. A guide to guidelines for professional societies and other developers of recommendations: introduction to integrating and coordinating efforts in COPD guideline development. An official ATS/ERS workshop report. Proc Am Thorac Soc. 2012;9(5):215–8. 3. Schunemann HJ, Woodhead M, Anzueto A, Buist AS, Macnee W, Rabe KF, et al. A guide to guidelines for professional societies and other developers of recommendations: introduction to integrating and coordinating efforts in COPD guideline development. An official ATS/ERS workshop report. Proc Am Thorac Soc. 2012;9(5):215–8. 3. Schunemann HJ, Woodhead M, Anzueto A, Buist AS, Macnee W, Rabe KF, et al. A guide to guidelines for professional societies and other developers of recommendations: introduction to integrating and coordinating efforts in COPD guideline development. An official ATS/ERS workshop report. Proc Am Thorac Soc. 2012;9(5):215–8. 4. Schunemann HJ, Wiercioch W, Etxeandia I, Falavigna M, Santesso N, Mustafa R, et al. Guidelines 2.0: systematic development of a comprehensive checklist for a successful guideline enterprise. CMAJ. 2014;186(3):E123–42. 4. Schunemann HJ, Wiercioch W, Etxeandia I, Falavigna M, Santesso N, Mustafa R, et al. Guidelines 2.0: systematic development of a comprehensive checklist for a successful guideline enterprise. CMAJ. 2014;186(3):E123–42. 5. American Thoracic Society. Idiopathic pulmonary fibrosis: diagnosis and treatment. International consensus statement. American Thoracic Society (ATS), and the European Respiratory Society (ERS). Am J Respir Crit Care Med. 2000;161(2 Pt 1):646–64. 4. Schunemann HJ, Wiercioch W, Etxeandia I, Falavigna M, Santesso N, Mustafa R, et al. Guidelines 2.0: systematic development of a comprehensive checklist for a successful guideline enterprise. CMAJ. 2014;186(3):E123–42. 5. American Thoracic Society. Idiopathic pulmonary fibrosis: diagnosis and treatment. International consensus statement. American Thoracic Society (ATS), and the European Respiratory Society (ERS). Am J Respir Crit Care Med. 2000;161(2 Pt 1):646–64. g p 5. American Thoracic Society. Author details 1 1Department of Medicine, McMaster University, Hamilton, ON, Canada. 2Department of Clinical Epidemiology & Biostatistics, McMaster University, Hamilton, ON, Canada. 3Department of Medicine (Division of Pulmonary and Critical Care Medicine), University of Washington, Seattle, WA, USA. 4Center for Interstitial lung Diseases, UW Medical Center, Seattle, WA, USA. Received: 16 September 2015 Accepted: 17 September 2015 Received: 16 September 2015 Accepted: 17 September 2015 11. Norris SL, Burda BU, Holmer HK, Ogden LA, Fu R, Bero L, et al. Author’s specialty and conflicts of interest contribute to conflicting guidelines for screening mammography. J Clin Epidemiol. 2012;65(7):725–33. 10. Raghu G, Rochwerg B, Zhang Y, Garcia CA, Azuma A, Behr J, et al. An Official ATS/ERS/JRS/ALAT Clinical Practice Guideline: Treatment of Idiopathic Pulmonary Fibrosis. An Update of the 2011 Clinical Practice Guideline. Am J Respir Crit Care Med. 2015;192(2):e3–19. 8. Raghu G, Collard HR, Egan JJ, Martinez FJ, Behr J, Brown KK, et al. An official ATS/ERS/JRS/ALAT statement: idiopathic pulmonary fibrosis: evidence-based guidelines for diagnosis and management. Am J Respir Crit Care Med. 2011;183(6):788–824. Abbreviations 12. Wormser GP, Dattwyler RJ, Shapiro ED, Halperin JJ, Steere AC, Klempner MS et al. The clinical assessment, treatment, and prevention of lyme disease, human granulocytic anaplasmosis, and babesiosis: clinical practice guidelines by the Infectious Diseases Society of America. Clin Infect Dis. 2006;43(9):1089–134. 12. Wormser GP, Dattwyler RJ, Shapiro ED, Halperin JJ, Steere AC, Klempner MS, et al. The clinical assessment, treatment, and prevention of lyme disease, human granulocytic anaplasmosis, and babesiosis: clinical practice guidelines by the Infectious Diseases Society of America. Clin Infect Dis. 2006;43(9):1089–134. Page 4 of 4 13. Guyatt GH, Oxman AD, Kunz R, Falck-Ytter Y, Vist GE, Liberati A, et al. Going from evidence to recommendations. BMJ. 2008;336(7652):1049–51. 14. Schunemann HJ. Guidelines 2.0: do no net harm-the future of practice guideline development in asthma and other diseases. Curr Allergy Asthma Rep. 2011;11(3):261–8. 15. Scott IA, Guyatt GH. Clinical practice guidelines: the need for greater transparency in formulating recommendations. Med J Aust. 2011;195(1):29–33. 16. Guyatt G, Akl EA, Oxman A, Wilson K, Puhan MA, Wilt T, et al. Synthesis, grading, and presentation of evidence in guidelines: article 7 in Integrating and coordinating efforts in COPD guideline development. An official ATS/ERS workshop report. Proc Am Thorac Soc. 2012;9(5):256–61. 17. Schunemann HJ, Oxman AD, Akl EA, Brozek JL, Montori VM, Heffner J, et al. Moving from evidence to developing recommendations in guidelines: article 11 in Integrating and coordinating efforts in COPD guideline development. An official ATS/ERS workshop report. Proc Am Thorac Soc. 2012;9(5):282–92. 18. Shekelle P, Woolf S, Grimshaw JM, Schunemann HJ, Eccles MP. Developing clinical practice guidelines: reviewing, reporting, and publishing guidelines; updating guidelines; and the emerging issues of enhancing guideline implementability and accounting for comorbid conditions in guideline development. Implement Sci. 2012;7:62. 19. Andrews J, Guyatt G, Oxman AD, Alderson P, Dahm P, Falck-Ytter Y, et al. GRADE guidelines: 14. Going from evidence to recommendations: the significance and presentation of recommendations. J Clin Epidemiol. 2013;66(7):719–25. 20. Andrews JC, Schunemann HJ, Oxman AD, Pottie K, Meerpohl JJ, Coello PA, et al. GRADE guidelines: 15. Going from evidence to recommendation- determinants of a recommendation’s direction and strength. J Clin Epidemiol. 2013;66(7):726–35. 21. Guyatt G, Akl EA, Hirsh J, Kearon C, Crowther M, Gutterman D, et al. The vexing problem of guidelines and conflict of interest: a potential solution. Ann Intern Med. 2010;152(11):738–41. 22. Schunemann HJ, Osborne M, Moss J, Manthous C, Wagner G, Sicilian L, et al. Rochwerg et al. BMC Medicine (2016) 14:22 28. van der Weijden T, Boivin A, Burgers J, Schunemann HJ, Elwyn G. Clinical practice guidelines and patient decision aids. An inevitable relationship. J Clin Epidemiol. 2012;65(6):584–9. 27. Schunemann HJ, Mustafa R, Brozek J. Diagnostic accuracy and linked evidence–testing the chain. Z Evid Fortbild Qual Gesundhwes. 2012;106(3): 153–60. Abbreviations An official American Thoracic Society Policy statement: managing conflict of interest in professional societies. Am J Respir Crit Care Med. 2009;180(6):564–80. 23. Guyatt GH, Oxman AD, Santesso N, Helfand M, Vist G, Kunz R, et al. GRADE guidelines: 12. Preparing summary of findings tables-binary outcomes. J Clin Epidemiol. 2013;66(2):158–72. 24. Balshem H, Helfand M, Schunemann HJ, Oxman AD, Kunz R, Brozek J, et al. GRADE guidelines: 3. Rating the quality of evidence. J Clin Epidemiol. 2011;64(4):401–6. 25. Raghu G, Anstrom KJ, King Jr TE, Lasky JA, Martinez FJ. Prednisone, azathioprine, and N-acetylcysteine for pulmonary fibrosis. N Engl J Med. 2012;366(21):1968–77. 26. Higgins JPT, Green S (editors). Cochrane Handbook for Systematic Reviews of Interventions Version 5.1.0 [updated March 2011]. The Cochrane Collaboration, 2011. Available from www.cochrane-handbook.org. 27. Schunemann HJ, Mustafa R, Brozek J. Diagnostic accuracy and linked evidence–testing the chain. Z Evid Fortbild Qual Gesundhwes. 2012;106(3): 153–60. 28. van der Weijden T, Boivin A, Burgers J, Schunemann HJ, Elwyn G. Clinical practice guidelines and patient decision aids. An inevitable relationship. J Clin Epidemiol. 2012;65(6):584–9. 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/W4303446565	https://surgicalcasereports.springeropen.com/counter/pdf/10.1186/s40792-022-01543-1	English	null	Diagnosis and clinical implication of collision gastric adenocarcinomas: a case report	Surgical case reports	2,022	cc-by	6,667	Case presentation We reviewed a prospectively recorded database of patients with gastric cancers (GCs) who underwent gastrectomy at our department from January 2008 to December 2018. Of 1041 patients who underwent gastrectomy, 81 (7.8%) had multiple synchronous Correspondence: tsumirohi2827@gmail.com 1 Department of Gastrointestinal Surgery, Japanese Red Cross Aichi Medical Center Nagoya Daiichi Hospital, 3‑35 Michishita‑Cho, Nakamura‑Ku, Nagoya 453‑8511, Japan Full list of author information is available at the end of the article Background and only small case series that describe collision gastric adenocarcinomas (CGAs) [6–18]. A CGA, wherein two synchronous adenocarcinomas develop nearby, can have specific characteristics; however, the features and clinical implications remain to be clarified because it is rare and often difficult to diagnose. Collision tumors are a subtype of simultaneous multi- ple tumors, wherein two independent tumors collide with or partially infiltrate each other, with clear borders and without the histological transition of one tumor to another [1]. Collision tumors are rare and usually found during pathological examination of surgically excised specimens. They can be encountered in many organs, including the brain, lung, esophagogastric junction, liver, and uterus [1–5]; however, there are limited reports fi This study aimed to reveal the methods for CGA diag- nosis and provide insight into the clinical implications of CGAs. © 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/. Abstract Background: Collision tumors are a subtype of simultaneous tumors wherein two unrelated tumors collide or infil‑ trate each other. Collision gastric adenocarcinomas (CGA) are rare and difficult to diagnose, and their clinical implica‑ tions remain unclear. Herein, we aimed to reveal diagnostic methods for CGA and provide insight into its implications. Case presentation: Among 1041 cases of gastric cancers (GCs) resected between 2008 and 2018, we included cases of confirmed CGA. Patients’ backgrounds, preoperative endoscopy findings, macroscopic imaging findings, and histopathology findings [including immunostaining for CK 7, MUC2, and mismatch repair (MMR) proteins] were investigated. The incidence of CGA was 0.5%: 5 of 81 cases having simultaneous multiple GCs. Tumors were mainly in the distal stomach. The CGA in two cases was between early cancers, in two cases was between early and advanced cancers, and in one case was between advanced cancers. There were three cases of collision between differentiated and undifferentiated types and two cases between differentiated types. Immunostaining with CK7 and MUC2 was useful for diagnosing collision tumor when the histology was similar to each other. Among ten GCs comprising CGA, nine tumors (90%) exhibited deficient MMR proteins, suggesting high microsatellite instability (MSI). Conclusions: CGA is rare and usually found in the distal stomach. Close observation of shape, optimal dissection, and detailed pathological examination, including immunostaining, facilitated diagnosis. CGAs may have high MSI potential. Keywords: Collision tumor, Collision adenocarcinoma, Multiple gastric cancer, Gastric cancer Diagnosis and clinical implication of collision gastric adenocarcinomas: a case report Hiromitsu Imataki1 , Hideo Miyake1, Hidemasa Nagai1, Yuichiro Yoshioka1, Norihiro Yuasa1, Junichi Takamizawa2, Ayami Kiriyama3 and Masahiko Fujino3 Imataki et al. Surgical Case Reports (2022) 8:193 https://doi.org/10.1186/s40792-022-01543-1 Imataki et al. Surgical Case Reports (2022) 8:193 https://doi.org/10.1186/s40792-022-01543-1 CASE REPORT Open Access *Correspondence: tsumirohi2827@gmail.com 1 Department of Gastrointestinal Surgery, Japanese Red Cross Aichi Medical Center Nagoya Daiichi Hospital, 3‑35 Michishita‑Cho, Nakamura‑Ku, Nagoya 453‑8511, Japan Full list of author information is available at the end of the article Imataki et al. Surgical Case Reports (2022) 8:193 Imataki et al. Surgical Case Reports (2022) 8:193 Imataki et al. Surgical Case Reports (2022) 8:193 Page 2 of 10 lesion was adjacent to a distal, depressed lesion with marginal protrusion (Fig. 1b). Three distal and two total gastrectomies were performed. adenocarcinomas. Among them, we found five patients with CGAs (6.2%) by postoperative detailed macroscopic observation and histopathological examination. Herein, we defined CGAs as gastric adenocarcinomas that have collided with each other, with partial topographic sepa- ration and histologically clear borders and without a histological transition of one to another type of adeno- carcinoma [1]. Suspected collision tumors involving adenocarcinomas with squamous differentiation (n = 3), neuroendocrine tumors (n = 3), and lymphomas (n = 1) were excluded. The patients’ medical histories, find- ings of preoperative endoscopy, macroscopic imaging of the resected specimens, and histopathology, including immunostaining with CK 7, MUC2, and mismatch repair (MMR) proteins, were investigated.h Immunohistochemistry Immunohistochemical staining for CK 7 and MUC2 was performed in two patients. Immunohistochemical staining for mismatch repair (MMR) proteins, including MLH1, MLH2, PMS2, and MLH6, was performed for the 14 multiple gastric adenocarcinomas in five patients with CGA. Deparaffinized 4-μm-thick sections from each paraffin block were exposed to 0.3% hydrogen perox- ide for 15 min to block endogenous peroxidase activity. Antigen retrieval was performed by autoclaving sections in 10 mM citrate buffer (pH 6.0) for 10 min. Sections were stained with primary antibodies, including anti- MLH1 (ES05, 1:200 dilution; Dako, Glostrup, Denmark), anti-MSH2 (FE11, 1:200 dilution; Calbiochem, La Jolla, CA, USA), anti-PMS2 (A16-4, 1:200 dilution; Biocare Medical, Concord, CA, USA), and anti-MSH6 antibod- ies (SP93, 1:200 dilution; Spring Bioscience, Pleasanton, CA, USA). We used an automated stainer (Dako) and En Vision Detection System (Dako) according to the ven- dor’s protocol. Non-neoplastic epithelial and stromal cells served as internal positive controls. Tumors show- ing significantly reduced or the loss of expression of any MMR protein were deemed to be MMR-deficient. The immunohistochemical staining results were evaluated by two pathologists (AK and MF). We explored microsatellite instability (MSI) in the 14 GCs of the five study patients with retained MMR protein expression. Immunohistochemical findings of MMR proteins, including MLH1, MLH2, PMS2, and MLH6, in a representative case (Case 3) are shown in Fig. 4. Ten GCs showed deficient MMR proteins: MLH1(−), MLH2( +), PMS(−), and MLH6( +), while four GCs showed abundant MMR proteins: MLH1( +), MLH2( +), PMS( +) and MLH6( +). Of note, among ten GCs comprising CGA, nine (90%) exhibited defi- cient MMR proteins, suggesting high MSI (MSI-high) (Table 1). The schematic distribution of the deficient/ abundant MMR of the 14 GCs is shown in Fig. 5.i Macroscopic findings Macroscopic images of fixed, resected specimens of the five patients are presented in Fig. 2. The number of GCs in each patient ranged from two to five. The loca- tion of the CGAs was mainly in the distal stomach. The macroscopic shapes were complex or bizarre due to the clear yet ambiguous borders of the multiple compo- nents. Formalin-fixed resected specimens were divided according to the macroscopic findings of two adjacent lesions; the cutting lines were set perpendicular to the border of the two adjacent lesions (Fig. 2). The study protocol was approved by the ethics commit- tee of our hospital (Registration Number: 2020–318). All participants provided informed consent. Histopathological findings Th l i di l The two lesions displayed different histopathologies, and the border was clear without transitional tissue in all five patients (Fig. 3). The two histopathologies were diagnosed as differentiated tubular and poorly differen- tiated adenocarcinomas by hematoxylin and eosin (HE) staining in three patients (Cases 1–3, Fig. 3a-1–4, b-1– 4, c-1–4). Collision tumors were diagnosed by immu- nohistochemistry using CK 7 and MUC2 (Cases 4 and 5, Fig. 3d-1–4, e-1–4). In case 4, the two tumors were similar, well-differentiated tubular adenocarcinomas; however, immunostaining for CK 7 showed a differ- ence in positivity (Fig. 3d-2–4). In case 5, both tumors were similar, moderately differentiated tubular adeno- carcinomas; however, one tumor was CK 7-positive and MUC2-negative, and the other was CK 7-negative and focally MUC2-positive (Fig. 3e-2–4). Patient demographics and endoscopic findingsh The demographics and characteristics of the five patients with CGA are shown in Table 1. The median age was 75 years (range, 66–81), and three patients were male. Endoscopic images of the five patients are shown in Fig. 1. A CGA was preoperatively suspected in one patient (Case 2), in whom an irregular, depressed None of the five patients experienced a relapse after gastrectomy; the median relapse-free survival was 32 months. One patient died of pancreatic cancer 32 months after gastrectomy. Page 3 of 10 Imataki et al. Surgical Case Reports (2022) 8:193 Imataki et al. Surgical Case Reports Table 1 Summary of 5 cases of collision gastric adenocarcinoma CK7 cytokeratin 7, Gx gastrectomy, L lower stomach, M middle stomach, MMR mismatch repair, D deficient, P proficient No. Age Sex Surgery Number of cancer Collision cancer Other cancer Location First cancer Second cancer Macroscopic type Size (mm) Depth of invasion Histology MMR Macroscopic type Size (mm) Depth of invasion Histology MMR Location Macroscopic type, depth of invasion, histology 1 66 F Distal Gx 2 LM 2 45 mp por1 D 0–IIc 50 sm2 tub2 D 2 66 F Distal Gx 2 L 0–IIc 25 sm2 tub2 D 0–IIc 40 sm2 por1 D 3 78 M Total Gx 5 L 0–IIc 30 sm2 por2 P 1 40 mp tub1 D U M L Type 3, ss, tub2 > por, pMMR 0-IIc, m, tub1, pMMR Type 3, mp, por, pMMR 4 81 M Distal Gx 2 LM 0–IIc 20 m tub1, CK7( +) D 0–IIc 30 sm1 tub1, CK7(−) D 5 75 M Total Gx 3 L 0–IIa + IIc 25 sm2 tub2, CK7( +), MUC2(−) D 2 60 mp tub2, CK7 focal ( +), MUC2 focal( +) D L 0-IIa, m, tub1, dMMR Table 1 Summary of 5 cases of collision gastric adenocarcinoma Imataki et al. Surgical Case Reports (2022) 8:193 Imataki et al. Surgical Case Reports Page 4 of 10 Fig. 1 Endoscopic findings of five patients with collision gastric adenocarcinoma. a Case 1: a 66-year-old woman; two irregular ulcers with a marginal protrusion in the gastric angle (arrow, arrowhead). b Case 2: a 66-year-old woman; an irregular depressed lesion (arrowhead) and a distal adjacent, depressed lesion with marginal protrusion (arrow) of the gastric antrum. Patient demographics and endoscopic findingsh c Case 3: a 78-year-old man; a nodular elevated lesion (arrow) associated with a reddish depressed lesion (arrowhead) in the posterior wall of the gastric angle. d Case 4: an 81-year-old man; an irregular slight depressed lesion in the anterior wall of the gastric angle. e Case 5: a 75-year-old man; an elevated lesion with an irregular ulcer in the posterior wall of the gastric antrum Fig. 1 Endoscopic findings of five patients with collision gastric adenocarcinoma. a Case 1: a 66-year-old woman; two irregular ulcers with a marginal protrusion in the gastric angle (arrow, arrowhead). b Case 2: a 66-year-old woman; an irregular depressed lesion (arrowhead) and a distal adjacent, depressed lesion with marginal protrusion (arrow) of the gastric antrum. c Case 3: a 78-year-old man; a nodular elevated lesion (arrow) associated with a reddish depressed lesion (arrowhead) in the posterior wall of the gastric angle. d Case 4: an 81-year-old man; an irregular slight depressed lesion in the anterior wall of the gastric angle. e Case 5: a 75-year-old man; an elevated lesion with an irregular ulcer in the posterior wall of the gastric antrum Discussionh patterns and intermediate structures between the two types of tumors, to exclude tumors with suspected intra- tumor heterogeneity. Further, GCs with squamous differ- entiation, neuroendocrine tumors, and lymphomas were excluded. This study showed that the incidence of CGA was 0.5% of the 1041 patients with surgically resected GC and 6.2% of the 81 patients with multiple synchronous adenocar- cinomas. The collision tumors were identified by close macroscopic observation of their complex shapes, opti- mal division of the resected specimens, conventional HE staining, and immunostaining using CK 7 and MUC2. Among the ten collision tumors, nine exhibited deficient MMR proteins, suggesting high MSI. Our extensive search of the English and Japanese lit- erature (1996–2022) revealed 16 patients with CGAs according to the definition used in the present study (Additional file 1: Table S1) [6–18]. After the inclusion of our five patients, 21 cases were summarized in total. The median age of the patients was 70 years (interquar- tile range [IQR], 65–77 years), and 71% were men. The number of GCs in each patient ranged from two to five, and six patients (29%) had more than two adenocarci- nomas. The location of the CGAs was mainly the distal stomach (n = 12), followed by the middle stomach (n = 7). Frequent macroscopic types of tumors comprising a col- lision tumor were type 2, 0–IIc, 0–I, and 0–IIa in 11, nine, six, and five cases, respectively. The median size of tumors comprising CGAs was 35 mm (IQR, 25–50 mm). More than half of the tumors were early GCs (mucosal and submucosal invasion in 12 and 13 tumors, respec- tively). Frequent histological types were differentiated gg g g Collision tumors are generally malignant tumors that originate primarily independently of each other at two separate sites and which later, in the course of their expansion, invade each other [19]. However, the diagnos- tic criteria for collision tumors have not been defined. In 1961, Dodge described a collision tumor as having sepa- rate tumor areas of two distinct histological patterns, which lack areas of transitional patterns or intermediate structures between the two types of tumors [1]. Later, Wanke and Spagnolo accepted some transitional patterns in the areas of collision [20, 21]. Because tumor collision may represent intratumor heterogeneity, we adopted Dodge’s definition, including the absence of transitional Imataki et al. Surgical Case Reports (2022) 8:193 Imataki et al. Surgical Case Reports Page 5 of 10 Fig. Discussionh 2 Images of formalin-fixed resected specimens divided according to the macroscopic findings of two adjacent lesions. The cutting lines were set perpendicular to the border of two adjacent lesions (arrows and arrowheads). a Case 1: an irregular, depressed lesion in the lesser curvature of the middle and lower stomach (arrow and arrowhead). b Case 2: a large irregular, depressed lesion with marginal protrusion (arrow) and a proximal adjacent depressed lesion (arrowhead) in the posterior wall of the lower stomach. c Case 3: a nodular elevated lesion (arrow) and a distal adjacent depressed lesion (arrowhead) in the posterior wall in the middle stomach. d Case 4: two adjacent irregular, depressed lesions (arrow and arrowhead) in the anterior wall of the middle stomach. e Case 5: a large, well-demarcated ulcer with marginal protrusion (arrowhead) and an adjacent small depressed lesion (arrow) in the lower stomach Fig. 2 Images of formalin-fixed resected specimens divided according to the macroscopic findings of two adjacent lesions. The cutting lines were set perpendicular to the border of two adjacent lesions (arrows and arrowheads). a Case 1: an irregular, depressed lesion in the lesser curvature of the middle and lower stomach (arrow and arrowhead). b Case 2: a large irregular, depressed lesion with marginal protrusion (arrow) and a proximal adjacent depressed lesion (arrowhead) in the posterior wall of the lower stomach. c Case 3: a nodular elevated lesion (arrow) and a distal adjacent depressed lesion (arrowhead) in the posterior wall in the middle stomach. d Case 4: two adjacent irregular, depressed lesions (arrow and arrowhead) in the anterior wall of the middle stomach. e Case 5: a large, well-demarcated ulcer with marginal protrusion (arrowhead) and an adjacent small depressed lesion (arrow) in the lower stomach patients with multiple GCs [40–43]. We first investigated the MSI status in the CGAs and found a high rate (90%) of deficient MMR proteins, suggesting high MSI.hi tubular and poorly differentiated adenocarcinomas (22 and 14, respectively). Recently, cases that showed histo- pathological differences between the two components comprising a CGA by immunohistochemistry using EBER-ISH, TP53, MUC2, MUC5AC, and CK 7 have been reported [15–18]. i The Cancer Genome Atlas project classified GCs into four subtypes based on a comprehensive molecular evaluation: tumors positive for the Epstein–Barr virus, tumors with MSI, tumors with chromosomal instabil- ity, and genomically stable tumors. Discussionh MSI-type tumors exhibit hypermethylation and elevated mutation rates and account for 5–22% of all GCs [44, 45]. Cho et al. hypothesized that the acquisition of an MMR deficiency occurs in the early stage of the gastric tumorigenesis associated with Lynch syndrome [46], which is caused by germline pathogenic variants in four MMR genes: MLH1, MSH2, PMS2, and MSH6 [47]. Meanwhile, spo- radic MSI-high GCs may be related to hypermethyla- tion of the MLH1 promoter [48]. Previous studies have reported a high prevalence (17–33%) of MSI-high in synchronous multiple GCs [43, 49]. MSI-high tumors have different clinicopathologic characteristics than MSI-low or MSI-stable tumors; MSI-high GCs are CGAs are a subtype of multiple synchronous GC; therefore, several clinical characteristics overlap those of multiple GCs. Multiple synchronous GCs have been reported to account for 5–15% of all GC cases [22] and are associated with older age [23–26], being male [23, 27–29], the macroscopic type (elevated or depressed) [23, 30], the histologic type (differentiation) [24, 25, 31–33], the presence of intestinal mucin [25, 28], severe mucosal atrophy or intestinal metaplasia [25, 28, 34, 35], and sub- mucosal ectopic gastric glands [35]. Multiple GCs are frequently associated with primary malignancies in other organs [33, 36–38], and the development of a metachro- nous GC after distal gastrectomy is clinically impor- tant [24, 39]. In addition, recent genetic studies indicate that MSI-high tumors are often (17–33%) observed in Imataki et al. Surgical Case Reports (2022) 8:193 Imataki et al. Surgical Case Reports Page 6 of 10 Fig. 3 Histopathological findings of the resected specimen. a-1–a-4 Case 1: a lesion with a poorly differentiated adenocarcinoma (por) collided with a lesion with a moderately differentiated tubular adenocarcinoma (tub2). a-1 Loupe image (hematoxylin–eosin [HE]), a-2 por (HE, × 40), a-3 border of the two lesions (HE, × 100), a-4 tub2 (HE, × 40) b-1–b-4 Case 2: a lesion with tub2 collided with a lesion with poorly differentiated adenocarcinoma (solid type, por1). b-1 Loupe image (HE), b-2 tub2 (HE, × 100), b-3 border of the two lesions (HE, × 40), b-4 por1 (HE, × 100). c-1–c-4 Case 3: a lesion with poorly differentiated adenocarcinoma (non-solid type, por2) collided with a lesion with well-differentiated tubular adenocarcinoma (tub1). c-1 Loupe image (HE, c-2 por2 (HE, × 100), c-3 border of the two lesions (HE, × 40), (c-4) tub1 (HE, × 100). Discussionh d-1–d-4 Case 4: two tumors were similar tub2; however, they had a different positivity for CK 7. d-1 Loupe image (HE), d-2 CK positive (× 40), d-3 border of the two lesions (× 40), d-4 CK 7-negative (× 40). e-1–e-4 Case 5: Two tumors were similar tub2; however, one tumor was CK 7-positive and MUC2-negative; and the other was CK 7-negative and MUC2 focally positive. e-1 Loupe image (HE), e-2 CK 7 staining (× 20), e-3 border of the two lesions (HE, × 40), e-4 MUC2 staining (× 20) Fi 3 Hi h l i l fi di f h d i 1 4 C 1 l i i h l diff i d d i ( ) llid d Fig. 3 Histopathological findings of the resected specimen. a-1–a-4 Case 1: a lesion with a poorly differentiated adenocarcinoma (por) collided with a lesion with a moderately differentiated tubular adenocarcinoma (tub2). a-1 Loupe image (hematoxylin–eosin [HE]), a-2 por (HE, × 40), a-3 border of the two lesions (HE, × 100), a-4 tub2 (HE, × 40) b-1–b-4 Case 2: a lesion with tub2 collided with a lesion with poorly differentiated adenocarcinoma (solid type, por1). b-1 Loupe image (HE), b-2 tub2 (HE, × 100), b-3 border of the two lesions (HE, × 40), b-4 por1 (HE, × 100). c-1–c-4 Case 3: a lesion with poorly differentiated adenocarcinoma (non-solid type, por2) collided with a lesion with well-differentiated tubular adenocarcinoma (tub1). c-1 Loupe image (HE, c-2 por2 (HE, × 100), c-3 border of the two lesions (HE, × 40), (c-4) tub1 (HE, × 100). d-1–d-4 Case 4: two tumors were similar tub2; however, they had a different positivity for CK 7. d-1 Loupe image (HE), d-2 CK positive (× 40), d-3 border of the two lesions (× 40), d-4 CK 7-negative (× 40). e-1–e-4 Case 5: Two tumors were similar tub2; however, one tumor was CK 7-positive and MUC2-negative; and the other was CK 7-negative and MUC2 focally positive. e-1 Loupe image (HE), e-2 CK 7 staining (× 20), e-3 border of the two lesions (HE, × 40), e-4 MUC2 staining (× 20) Imataki et al. Surgical Case Reports (2022) 8:193 Page 7 of 10 Imataki et al. Surgical Case Reports Fig. 4 Immunohistochemical findings of mismatch repair (MMR) proteins in a representative colliding gastric adenocarcinoma (Case 3). a MLH1(−), MLH2( +), PMS2(−), and MLH6( +) indicating deficient MMR. Discussionh b MLH1( +), MLH2( +), PMS2( +), and MLH6( +) indicating proficient MMR using monoclonal antibodies that target programmed death receptor-1 (PD-1) has shown promising results in patients with irresectable or metastatic MSI-high GC [55]. There are several hypotheses on the pathogeneses of collision tumors: (1) a carcinogenic stimulus on two neighboring mucosal regions resulting in the coexist- ence of two distinct neoplasms that later expand into each other and collide; (2) factors generated by an orig- inal tumor, such as gastrin’s trophic effect, granulocyte colony-stimulating factor, and immunosuppression, may induce the development of a neighboring second primary tumor (tumor-to-tumor carcinogenesis) [56– 58]; (3) a common progenitor cell that grows contralat- erally during cell division and afterward differentiates into two cell types that maintain their characteristics [59, 60]; and (4) malignant transformations and changes at the edge of an original tumor promote the develop- ment of a second distinct adjacent tumor [61]. A high rate of deficient MMRs was found in CGAs, suggesting that hypermethylation of the MLH1 promotor occurs in the adjacent gastric mucosa. Fig. 4 Immunohistochemical findings of mismatch repair (MMR) proteins in a representative colliding gastric adenocarcinoma (Case 3). a MLH1(−), MLH2( +), PMS2(−), and MLH6( +) indicating deficient MMR. b MLH1( +), MLH2( +), PMS2( +), and MLH6( +) indicating proficient MMR Fig. 4 Immunohistochemical findings of mismatch repair (MMR) proteins in a representative colliding gastric adenocarcinoma (Case 3). a MLH1(−), MLH2( +), PMS2(−), and MLH6( +) indicating deficient MMR. b MLH1( +), MLH2( +), PMS2( +), and MLH6( +) indicating proficient MMR associated with older women, an intestinal-type (Lau- ren classification), middle and distal stomach locations, and fewer lymph node metastases [50–53]. In addi- tion, Janjigian et al. reported that patients with MSI- high tumors suffered rapid disease progression after first-line standard cytotoxic therapy [54]. Treatment Our study has some limitations. CGA is a rare disease (0.5% of surgically resected GCs), so our study investi- gated just five patients. In addition, to reduce the possi- bility of intratumor heterogeneity, we adopted Dodge’s definition from 1961, which required only histopatho- logical staining; therefore, it was easy to operate. Fig. 5 Schematic distribution and deficient/proficient mismatch repair protein (MMR) of 14 gastric cancers. D deficient MMR, P proficient MMR distribution and deficient/proficient mismatch repair protein (MMR) of 14 gastric cancers. D deficient MMR, P proficient MMR Fig. Discussionh 5 Schematic distribution and deficient/proficient mismatch repair protein (MMR) of 14 gastric cancers. D deficient MM Page 8 of 10 Imataki et al. Surgical Case Reports (2022) 8:193 Page 8 of 10 Imataki et al. Surgical Case Reports (2022) 8:193 Imataki et al. Surgical Case Reports Additional sequencing data may make it easier to con- firm that the two tumors are distinct and originated independently. Funding This work was supported by the Japanese Red Cross Aichi Medical Center Nagoya Daiichi Hospital Research Grant to HI (grant number NFRCH21-0003). The funder had no role in the study design, data collection, data analysis, deci‑ sion to publish, or manuscript preparation. 12. Kunisaki C, Kobayashi S, Kido Y, et al. A case of collision carcinoma of the stomach (in Japanese). Jpn J Cancer Clin. 1996;42:1141–6. 12. Kunisaki C, Kobayashi S, Kido Y, et al. A case of collision carcinoma of the stomach (in Japanese). Jpn J Cancer Clin. 1996;42:1141–6. 13. Takagi K, Iwakiri K, Shimozyu K, et al. Collision early gastric cancers, report of a case (in Japanese with English abstract). Stomach Intestine. 1997;32:1141–6. References 1. Dodge OG. Gastro-oesophageal carcinoma of mixed histological type. J Pathol Bacteriol. 1961;81:459–71. https://doi.org/10.1002/path.17008 10219. Availability of data and materials Data sharing does not apply to this article. The datasets supporting the con‑ clusions of this article are included within the article and its additional file. 14. Igari K, Tokairin Y, Kumagai Y, et al. A case of collision adenocarcinoma of the stomach (in Japanese). Geka. 2008;70:1007–10. 14. Igari K, Tokairin Y, Kumagai Y, et al. A case of collision adenocarcinoma of the stomach (in Japanese). Geka. 2008;70:1007–10. 15. Okada A, Arai T, Saeki S, et al. Gastric collision tumor of adenocarcinoma and Epstein–Barr virus-related carcinoma—a case report. Nihon Rinsho Geka Gakkai Zasshi (J Jpn Surg Assoc). 2010;71:1513–7 (Japanese with English abstract). Abbreviations CGA: Collision gastric adenocarcinoma; GCs: Gastric cancers; HE: Hematoxylin and eosin; MMR: Mismatch repair; MSI: Microsatellite instability. y 5. Jang KS, Lee WM, Kim YJ, Cho SH. Collision of three histologically distinct endometrial cancers of the uterus. J Korean Med Sci. 2012;27:89–92. https://doi.org/10.3346/jkms.2012.27.1.89. y 5. Jang KS, Lee WM, Kim YJ, Cho SH. Collision of three histologically distinct endometrial cancers of the uterus. J Korean Med Sci. 2012;27:89–92. https://doi.org/10.3346/jkms.2012.27.1.89. Acknowledgements None. 9. Hamada T, Kondo K, Itagaki Y, et al. Multiple early gastric cancers, type IIa and type IIc of the stoma in the gastric remnant, report of a case (in Japanese with English abstract). Stomach Intestine. 1994;29:1708–82. 9. Hamada T, Kondo K, Itagaki Y, et al. Multiple early gastric cancers, type IIa and type IIc of the stoma in the gastric remnant, report of a case (in Japanese with English abstract). Stomach Intestine. 1994;29:1708–82. Received: 21 August 2022 Accepted: 23 September 2022 Received: 21 August 2022 Accepted: 23 September 2022 Received: 21 August 2022 Accepted: 23 September 2022 Conclusions CGA is rare; however, its diagnosis is not difficult if close observation of the resected specimen and detailed pathological examinations are performed. 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J Clin Gastroenterol. 1988;3:1497–501. 7. Yamamoto S, Matsuo R, Matsuoka K, et al. A case of colliding carcinoma of the stomach (in Japanese). J Clin Gastroenterol. 1988;3:1497–501. Additional file 1: Table S1. Reported cases of collision gastric adenocarcinoma. Additional file 1: Table S1. Reported cases of collision gastric adenocarcinoma. 8. Aoyagi K, Hashimoto K, Kohfuji K, Tanaka T, Kodama I, Yano S, et al. Two cases of colliding carcinoma of the stomach (in Japanese with English abstract). Jpn J Gastroenterol Surg. 1992;25:2152–6. https://doi.org/10. 5833/jjgs.25.2152. 8. Aoyagi K, Hashimoto K, Kohfuji K, Tanaka T, Kodama I, Yano S, et al. Two cases of colliding carcinoma of the stomach (in Japanese with English abstract). Jpn J Gastroenterol Surg. 1992;25:2152–6. https://doi.org/10. 5833/jjgs.25.2152. Competing interests p g The authors declare that they have no competing interests. Author details Although rare, it is important to diagnose CGA accu- rately. If CGA is diagnosed with a single GC, several clinicopathological characteristics, including those of multiple GC and MSI, can be lost, affecting the choice of chemotherapy regimens, postoperative follow-up, and prognosis. Close macroscopic observation with the optimal cutting of the resected specimen and a detailed pathological examination, including immunostaining, can promote the accurate diagnosis of CGA. 1 Department of Gastrointestinal Surgery, Japanese Red Cross Aichi Medi‑ cal Center Nagoya Daiichi Hospital, 3‑35 Michishita‑Cho, Nakamura‑Ku, Nagoya 453‑8511, Japan. 2 Department of Laboratory Medicine, Japanese Red Cross Aichi Medical Center Nagoya Daiichi Hospital, 3‑35 Michishita‑Cho, Nakamura‑Ku, Nagoya 453‑8511, Japan. 3 Department of Pathology, Japanese Red Cross Aichi Medical Center Nagoya Daiichi Hospital, 3‑35 Michishita‑Cho, Nakamura‑Ku, Nagoya 453‑8511, Japan. 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https://openalex.org/W2404666139	http://koreascience.or.kr:80/article/JAKO201621650893639.pdf	English	null	Dosimetric Characteristics of 6 MV Modified Beams by Physical Wedges of a Siemens Linear Accelerator	Asian Pacific journal of cancer prevention	2,016	cc-by	5,033	Asian Pac J Cancer Prev, 17 (4), 1685-1689 target volume without creating a hotspot (Muren et al., 2009; Vinagre et al., 2009). Determination of the changes value in beam quality and dose distribution curves due to presence of PW across the radiation field is critical to calculate of dose distribution in the treatment planning systems (TPS) (Miften et al., 2000; Petrovic et al., 2010; Muhammad et al., 2011). The dosimetric properties of the hardened photon beam by PW filters may be varied between different linear accelerators (Linac) and even for the same model. Therefore the dosimetric parameters of wedge filters must be exactly known for each Linac, individually. Directly measuring of these parameters is difficult, time consuming and tedious. In the recent years, Monte Carlo (MC) calculation methods are well known to accurately survey of radiation beams used to diagnostic and therapeutic applications (Verhaegen et al., 1999; Shih et al., 2001). DOI:http://dx.doi.org/10.7314/APJCP.2016.17.4.1685 Dosimetric Characteristics of Physical Wedges with a Siemens Linear Accelerator DOI:http://dx.doi.org/10.7314/APJCP.2016.17.4.1685 Dosimetric Characteristics of Physical Wedges with a Siemens Linear Accelerator RESEARCH ARTICLE Introduction Assuming uniform clonogenic cell distribution in the tumor volume, the aim of conformal radiotherapy techniques is to obtain of high local tumor control probability (TCP) and keeping the normal tissue complication probability (NTCP) as low as possible (Muren et al., 2001; Khan, 2014). Therefore, the International Commission on Radiation Units and Measurements(ICRU) recommends a limit of minimum and maximum dose in the planning process of target volume within -5% and +7% of the prescribed dose to reach a high and uniform dose distribution in the target volume and minimal possible dose in any organ at risk (ICRU: Report 50; 1993). i The physical wedge filters (PWs) are made from steel, lead or tungsten alloys and modulate the photon intensity across the beam. They are located in beam pathway to cause substantial attenuation in the intensity of photon beams and increase the average energy of transferred beam. Due to the irregular body contour and tumor volume, the beam modifiers such as the PWs are still widely used in external beam radiation therapy to improve the dose uniformity in the planning target volume (Ahmad et al., 2010; Njeh, 2015) beside the progressive techniques such as enhanced dynamic wedge (Kowalik et al., 2013). It can be used as a missing tissue compensator as well as to shift the isodose pattern toward its thinner edge at a In this study the x-ray beam quality indexes such as photon spectrum, mean energy, half-value layers (HVL) beside the percent depth dose (PDD) and dose profile curves for 6 MV-Siemens Linac were studied in the presence of 150 and 450 PWs using BEAMnrc and DOSXYZnrc packages. 1Department of Medical Physics, Faculty of Medicine, 2Departments of Clinical Oncology, Golestan Hospital, 3Student Research Committee, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran For correspondence: mahbubefadaei@ymail.com Dosimetric Characteristics of 6 MV Modified Beams by Physical Wedges of a Siemens Linear Accelerator Mansour Zabihzadeh1,2,3, Mohammad Javad Tahmasebi Birgani1,2, Mojtaba Hoseini-Ghahfarokhi1,3, Sholeh Arvandi2, Seyed Mohammad Hoseini2, Mahbube Fadaei1 Asian Pacific Journal of Cancer Prevention, Vol 17, 2016 Abstract Physical wedges still can be used as missing tissue compensators or filters to alter the shape of isodose curves in a target volume to reach an optimal radiotherapy plan without creating a hotspot. The aim of this study was to investigate the dosimetric properties of physical wedges filters such as off-axis photon fluence, photon spectrum, output factor and half value layer. The photon beam quality of a 6 MV Primus Siemens modified by 150 and 450 physical wedges was studied with BEAMnrc Monte Carlo (MC) code. The calculated present depth dose and dose profile curves for open and wedged photon beam were in good agreement with the measurements. Increase of wedge angle increased the beam hardening and this effect was more pronounced at the heal region. Using such an accurate MC model to determine of wedge factors and implementation of it as a calculation algorithm in the future treatment planning systems is recommended. Keywords: Beam hardening - Monte Carlo calculation - physical wedge filter - radiotherapy Asian Pac J Cancer Prev, 17 (4), 1685-1689 Asian Pac J Cancer Prev, 17 (4), 1685-1689 Benchmark of simulated Linac head Open field Benchmark of simulated Linac head Open field An tuned incident electron beam with the mean energy of 6.2 MeV, the Gaussian energy spread with FWHM=1 MeV and the spatial FWHM of 1 mm resulted a good agreement between the measured and calculated data. The agreement between measurements and calculations (Figure 1) were within 1% for depth dose profile beyond the depth of maximum dose and for the lateral profile inside the field. The PDD and dose profiles were calculated in DOSXYZnrc using a water phantom of 50x50x50 cm3 positioned at SSD of 100 cm for field size of 10 × 10 cm2. The voxel size with dimensions of 1 × 1 cm2 in the lateral directions and 0.25 cm resolution in depth were considered for PDD calculations. The lateral dose profiles were calculated for depth of 10 cm with 0.2 cm resolution in X or Y direction and 1 cm in the remaining two directions. Number of histories in DOSXYZnrc was selected 4 billion particles to reach the statistical uncertainties <1% in all MC calculations. Characteristic of photons like spatial fluence, energy fluence and mean energy was extracted from the calculated phase space MC simulation MC simulations were performed by EGSnrc MC code based user codes of BEAMnrc and DOSXYZnrc (Rogers et al., 2003). The dimensions and materials of different components of Linac head were built using data from the vendor. A number of component modules (CMs) including of SLAB, FLATFILT, CHAMBER, MIRROR, JAWS and PIRAMIDS were used to modeling of exit window, target, primary collimator & flattening filter, monitoring chambers, mirror, jaws and PWs filter, respectively. In all calculations, ECUT (lower energy threshold for electron transport), similar to AE (threshold energy for electron creation) and PCUT (lower energy threshold for photon transport), similar to AP (threshold energy for photon creation) were 0.700 MeV and 0.01 MeV, respectively. Directional bremsstrahlung splitting (DBS) technique with splitting number of 1000 was used as one of the variance reduction method. In addition, electron range reduction method with ESAVE_GLOBAL= 2 MeV was applied in simulation of the whole head with the exception of the target CM where ESAVE_GLOBAL was 0.7 MeV. All other EGSnrc parameters were set to their default values. i Kc(t) Kc(0) = ∑N = Df DE Ei μen,air p (Ei)e [-μ (Ei) absorber ) DEi ∑N = Df DE Ei μen,air p (Ei)DEi n=i n=i t = 1 2 (1) (1) p All needed data for solving of equation 1 were extracted from XAAMDI database and from MC calculations and processed by homemade program in MATLAB software. All simulation processes was done in parallel mode on Ubuntu operating system installed on a computer, equipped with 21 cores of Intel Xeon® CPU X7560@2.27 GHz and 16 GB of RAM. The modes of open field (without a PW) and 15° or 45° PW modulated fields were evaluated. One of the phase space files was scored in a plan perpendicular to the beam axis which was located just below the down surface of X-jaws. Two other score plans were defined to generate phase space files at surface of the phantom for both open and wedge-filtered fields. Two steel wedges of 15° or 45° with non-linear profiles were located at 40.2 cm from the target under the X-jaws. Electron histories of 1.5 x 108 were run to collect about 300 million particles in the first phase space file. The particles in each phase space were recycled several times in order to achieve an interested statistical uncertainty. Measurements The measurements were performed for Siemens Primus 1Department of Medical Physics, Faculty of Medicine, 2Departments of Clinical Oncology, Golestan Hospital, 3Student Research Committee, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran *For correspondence: mahbubefadaei@ymail.com Mansour Zabihzadeh et al at phantom surface using the BEAMDP interface. The photon fluences vs. lateral off-axis distances were scored in 12.5 × 0.5 cm2 rectangular bins across the field. The photon energy spectra were evaluated by scoring the photon energy distribution in rectangular bins with resolution of 1 cm along the angled side of PW in toe, center and heel regions. Linac 6 MV photon beam according to recommendations of IAEA protocol, TRS-398 (Andreo et al., 2000). Measurements of the PW factors were carried out using 0.125 cm3 Farmer type ionization chamber with DOSE1 electrometer (FC65G, Scanditronix-Wellhofer, Germany) at the depth of 10 cm with source to surface distance (SSD) of 100 cm. The PDD and dose profile curves for open and PW field of 10 × 10 cm2 were measured in 50 cm3 PTW-Blue water phantom and processed by dosimetry software RFAplus (Version 5.2, Scanditronix-Wellhofer, Germany). Each measurements were repeated three times with precision of 0.2%. The off-axis variation of output factor (the ratio of absorbed doses at a reference depth with and without wedge) was investigated for both x and y directions at a depth of 10 cm and SSD of 100 cm. Spectral distribution (energy fluence in each energy interval) contains valuable data about the radiation beam, especially beam quality which can be represented by several indexes such as HVL, TPR20,10, etc. Although the measurement of spectral distribution can be so demanding, MC codes can easily produce it. Therefore, beam hardening effect was investigated at toe, center and heel regions using three 1x12.5 cm2 bins across the field in the form of half value layer (HVL) that defined as the thickness, t, of water needed to attenuate the in-air collision Kerma, Kc, to half of its measure from when no water was present (equation 1). The summation was performed over 200 equidistant energy bins with fluence, Df DE i, mid-energy, Ei, and energy width, ΔEi. The linear attenuation coefficient, μ, and the mass energy absorption coefficient, μen/p, for each energy bin were extracted from the XAAMDI database (Hubbell et al., 1995). Photon energy fluence Lateral dose profile in depth of 10 cm was normalized to its central voxel value gure 1. Comparisons of Calculated and Measured. Figure 4. The Photon Spectral Distributions of 6 MV Photon in the Toe, Central and Heal Regions of Open and 15O and 45O Physical Wedged Fields for 10 x 10 cm2 field at SSD=100 cm. The Photon energy fluences are normalized per incident electron on target Figure 1. Comparisons of Calculated and Measured. a) PDD and b) Lateral dose profile for SSD of 100 cm and open field of 10 x 10 cm2. The PDD was normalized to 100 at maximum dose depth. Lateral dose profile in depth of 10 cm was normalized to its central voxel value Figure 2. Comparison of the MC calculated and experimentally measured in-line dose profiles for a). 15oand b). 45o physical wedged fields. Lateral dose profile in depth of 10 cm was normalized to its central axis value as 100% 0 10 20 30 40 50 60 70 80 90 100 110 -‐12 -‐10 -‐8 -‐6 -‐4 -‐2 0 2 4 6 8 10 12 Rela1ve dose (%) Oﬀ axis distance (cm) a. PW 15° measurment calcula1on 0 10 20 30 40 50 60 70 80 90 100 110 120 -‐12 -‐10 -‐8 -‐6 -‐4 -‐2 0 2 4 6 8 10 Rela1ve dose (%) Oﬀ axis distance (cm) b. PW 45° measurm calcula1o 0 10 20 30 40 50 60 70 80 90 100 110 ‐ 2 -‐10 -‐8 -‐6 -‐4 -‐2 0 2 4 6 8 10 12 Rela1ve dose (%) Oﬀ axis distance (cm) a. PW 15° measurment calcula1on 0 10 20 30 40 50 60 70 80 90 100 110 120 -‐12 -‐10 -‐8 -‐6 -‐4 -‐2 0 2 4 6 8 10 12 Rela1ve dose (%) Oﬀ axis distance (cm) b. PW 45° measurment calcula1on ‐ 0 10 20 30 40 50 60 70 80 90 100 110 -‐12 -‐10 -‐8 -‐6 -‐4 -‐2 0 2 4 6 8 10 12 Rela1ve dose (%) Oﬀ axis distance (cm) a. PW 15° measurment calcula1on 0 10 20 30 40 50 60 70 80 90 100 110 120 -‐12 -‐10 -‐8 -‐6 -‐4 -‐2 0 2 4 6 8 10 Rela1ve dose (%) Oﬀ axis distance (cm) b. Photon energy fluence PW 45° measurm calcula1o 0 10 20 30 40 50 60 70 80 90 100 110 -‐10 -‐8 -‐6 -‐4 -‐2 0 2 4 6 8 10 12 Rela1ve dose (%) Oﬀ axis distance (cm) a. PW 15° measurment calcula1on 0 10 20 30 40 50 60 70 80 90 100 110 120 -‐12 -‐10 -‐8 -‐6 -‐4 -‐2 0 2 4 6 8 10 12 Rela1ve dose (%) Oﬀ axis distance (cm) b. PW 45° measurment calcula1on Figure 5. The variation of in-line and cross-line output factors across the beam line a) 15O wedge and b) 45O wedge; for 6 MV photon beam in 10 x 10 cm2 field size at SSD=100 cm 0.630 0.640 0.650 0.660 0.670 0.680 0.690 0.700 0.710 0.720 ‐ 5 -‐4 -‐3 -‐2 -‐1 0 1 2 3 4 5 6 Out put factors Oﬀ axis distance (cm) a. 15 PW line ross line 0.240 0.260 0.280 0.300 0.320 0.340 0.360 0.380 0.400 -‐6 -‐5 -‐4 -‐3 -‐2 -‐1 0 1 2 3 4 5 6 Out put factors Oﬀ axis distance (cm) b. 45 PW In line Cross line 0.630 0.640 0.650 0.660 0.670 0.680 0.690 0.700 0.710 0.720 -‐6 -‐5 -‐4 -‐3 -‐2 -‐1 0 1 2 3 4 5 6 Out put factors Oﬀ axis distance (cm) a. 15 PW In line Cross line 0.240 0.260 0.280 0.300 0.320 0.340 0.360 0.380 0.400 -‐6 -‐5 -‐4 -‐3 -‐2 -‐1 0 1 2 3 4 5 Out put factors Oﬀ axis distance (cm) b. 45 PW In line Cross line 0.630 0.640 0.650 0.660 0.670 0.680 0.690 0.700 0.710 0.720 ‐ -‐4 -‐3 -‐2 -‐1 0 1 2 3 4 5 6 Out put factors Oﬀ axis distance (cm) a. 15 PW ine oss line 0.240 0.260 0.280 0.300 0.320 0.340 0.360 0.380 0.400 -‐6 -‐5 -‐4 -‐3 -‐2 -‐1 0 1 2 3 4 5 6 Out put factors Oﬀ axis distance (cm) b. 45 PW In line Cross line 0.630 0.640 0.650 0.660 0.670 0.680 0.690 0.700 0.710 0.720 -‐6 -‐5 -‐4 -‐3 -‐2 -‐1 0 1 2 3 4 5 6 Out put factors Oﬀ axis distance (cm) a. 15 PW In line Cross line 0.240 0.260 0.280 0.300 0.320 0.340 0.360 0.380 0.400 -‐6 -‐5 -‐4 -‐3 -‐2 -‐1 0 1 2 3 4 5 Out put factors Oﬀ axis distance (cm) b. Off-axis photon fluences l fields are presented in Figure 4. From Figure 4, it can be dedicated that energy fluence for PWs decrease across the wedge angled direction from the toe to the heal region compared to open field. This effect is more significant for the 45°wedge. The off-axis photon fluences of open and 15°and 45° physical wedged fields are calculated in resolution of 5 mm as shown in Figure 3. It is clear from Figure 3 that photon fluence is nearly uniform across the field at open field while for wedged field decreases from the toe to the heel region of the wedge. This effect is more significant at larger wedge angles. Output (wedge) factor From Figure 5.a it can be found that the in-line output Output (wedge) factor From Figure 5.a it can be found that the in-line output Output (wedge) factor From Figure 5.a it can be found that the in-line output Figure 4. The Photon Spectral Distributions of 6 MV Photon in the Toe, Central and Heal Regions of Open and 15O and 45O Physical Wedged Fields for 10 x 10 cm2 field at SSD=100 cm. The Photon energy fluences are normalized per incident electron on target 0 1 2 3 4 5 6 7 0.0 1.0x10 -6 2.0x10 -6 3.0x10 -6 4.0x10 -6 5.0x10 -6 6.0x10 -6 7.0x10 -6 Fluence (1/cm2) Energy/MeV open field, toe open field, center open field, heal 15 PW, toe 15 PW, center 15 PW, heal 45 PW, toe 45 PW, center 45 PW, heal 0 1 2 3 4 5 6 7 0.0 1.0x10 -6 2.0x10 -6 3.0x10 -6 4.0x10 -6 5.0x10 -6 6.0x10 -6 7.0x10 -6 Fluence (1/cm2) Energy/MeV open field, toe open field, center open field, heal 15 PW, toe 15 PW, center 15 PW, heal 45 PW, toe 45 PW, center 45 PW, heal Physical wedged fields In order to validate the MC simulated PWs, the calculated PDD and lateral dose profiles of 15°and 45° physical wedged fields were compared to measurements. Only in-line dose profiles (in Y direction) are depicted in Figure 2. The MC calculations agreed well with the measurements within 2% inside the field for both of the wedges. Asian Pacific Journal of Cancer Prevention, Vol 17, 2016 1686 DOI:http://dx.doi.org/10.7314/APJCP.2016.17.4.1685 Dosimetric Characteristics of Physical Wedges with a Siemens Linear Accelerator d 15°and 45° solution of 5 Figure 3 that field at open fields are presented in Figure 4. From Figure 4, it can be dedicated that energy fluence for PWs decrease across the wedge angled direction from the toe to the heal region compared to open field. This effect is more significant for the 45°wedge. DOI:http://dx.doi.org/10.7314/APJCP.2016.17.4.1685 Dosimetric Characteristics of Physical Wedges with a Siemens Linear Acceleratori Off-axis photon fluences l Photon energy fluence The calculated spectral distributions of 6 MV photons from 10 x 10 cm2 at SSD=100 cm for toe, central and heal regions of open and 15°and 45° physical wedged Figure 1. Comparisons of Calculated and Measured. a) PDD and b) Lateral dose profile for SSD of 100 cm and open field of 10 x 10 cm2. The PDD was normalized to 100 at maximum dose depth. Lateral dose profile in depth of 10 cm was normalized to its central voxel value 0 10 20 30 40 50 60 70 80 90 100 110 -‐12 -‐10 -‐8 -‐6 -‐4 -‐2 0 2 4 6 8 10 12 Rela1ve dose (%) Oﬀ axis distance (cm) b. Inline dose proﬁle measurment calcula1on 0 10 20 30 40 50 60 70 80 90 100 110 0 2 4 6 8 10 12 14 16 18 20 PDD (%) Depth (cm) a. PDD curve measurment calcula1on 0 10 20 30 40 50 60 70 80 90 100 110 -‐12 -‐10 -‐8 -‐6 -‐4 -‐2 0 2 4 6 8 10 1 Rela1ve dose (%) Oﬀ axis distance (cm) b. Inline dose proﬁle measurme calcula1on 0 10 20 30 40 50 60 70 80 90 100 110 0 2 4 6 8 10 12 14 16 18 20 PDD (%) Depth (cm) a. PDD curve measurment calcula1on 0 10 20 30 40 50 60 70 80 90 100 110 -‐12 -‐10 -‐8 -‐6 -‐4 -‐2 0 2 4 6 8 10 12 Rela1ve dose (%) Oﬀ axis distance (cm) b. Inline dose proﬁle measurment calcula1on 0 10 20 30 40 50 60 70 80 90 100 110 0 2 4 6 8 10 12 14 16 18 20 Depth (cm) a. PDD curve measurment calcula1on 0 10 20 30 40 50 60 70 80 90 100 110 -‐12 -‐10 -‐8 -‐6 -‐4 -‐2 0 2 4 6 8 10 1 Rela1ve dose (%) Oﬀ axis distance (cm) b. Inline dose proﬁle measurmen calcula1on 0 10 20 30 40 50 60 70 80 90 100 110 0 2 4 6 8 10 12 14 16 18 20 PDD (%) Depth (cm) a. PDD curve measurment calcula1on Energy/MeV Figure 1. Comparisons of Calculated and Measured. a) PDD and b) Lateral dose profile for SSD of 100 cm and open field of 10 x 10 cm2. The PDD was normalized to 100 at maximum dose depth. Photon energy fluence 45 PW In line Cross line Figure 2. Comparison of the MC calculated and experimentally measured in-line dose profiles for a). 15oand b). 45o physical wedged fields. Lateral dose profile in depth of 10 cm was normalized to its central axis value as 100%‐ Figure 5. The variation of in-line and cross-line output factors across the beam line a) 15O wedge and b) 45O wedge; for 6 MV photon beam in 10 x 10 cm2 field size at SSD=100 cm Asian Pacific Journal of Cancer Prevention, Vol 17, 2016 1687 Figure 3. In-line Photon Fluences for Open and 15O and 45o Physical Wedged Fields of 10 x 10 cm2 at SSD=100 cm. The Photon fluences are normalized per incident electron on target. 0.0E+00 2.0E-‐06 4.0E-‐06 6.0E-‐06 8.0E-‐06 1.0E-‐05 1.2E-‐05 1.4E-‐05 -‐7 -‐6 -‐5 -‐4 -‐3 -‐2 -‐1 0 1 2 3 4 5 6 7 Fluence (1/cm2) Oﬀ axis distance (cm) open ﬁeld 15 wedge ﬁeld 45 wedge ﬁeld Figure 6. The HVL changes (in term of water, cm) with wedge angles at the toe, central and heal regions of 15o and 45O PWs 9.5 10 10.5 11 11.5 12 12.5 13 0 15 30 45 HVL (cm) Wedge angle (degree) Heal Center Toe 0.0E+00 2.0E-‐06 4.0E-‐06 6.0E-‐06 8.0E-‐06 1.0E-‐05 1.2E-‐05 1.4E-‐05 -‐7 -‐6 -‐5 -‐4 -‐3 -‐2 -‐1 0 1 2 3 4 5 6 7 Fluence (1/cm2) Oﬀ axis distance (cm) open ﬁeld 15 wedge ﬁeld 45 wedge ﬁeld an Pacific Journal of Cancer Prevention, Vol 17, 2016 1687 Figure 6. The HVL changes (in term of water, cm) with wedge angles at the toe, central and heal regions of 15o and 45O PWs 9.5 10 10.5 11 11.5 12 12.5 13 0 15 30 45 HVL (cm) Wedge angle (degree) Heal Center Toe 9.5 10 10.5 11 11.5 12 12.5 13 0 15 30 45 HVL (cm) Wedge angle (degree) Heal Center Toe Figure 3. In-line Photon Fluences for Open and 15O and 45o Physical Wedged Fields of 10 x 10 cm2 at SSD=100 cm. The Photon fluences are normalized per incident electron on target. Figure 6. The HVL changes (in term of water, cm) with wedge angles at the toe, central and heal regions of 15o and 45O PWs Asian Pacific Journal of Cancer Prevention, Vol 17, 2016 Mansour Zabihzadeh et al no meaningful variations vs. Half Value Layer (HVL) The beam hardening effects of the 15° and 45° PWs in term of HVL are presented in Figure 6. HVLs (absorber=water, cm) increase from the toe to the heal region across the beam for each of 15°and 45° physical wedged fields. The maximum alternation of HVL 29.77%, was obtained at the heal region due to more beam hardening effect. However, there was no significant difference between the central and heal parts of 15° PW. Discussion In this study the maximum relative error of calculations was better than 1% at in-side and out-side of the field. The comparison of PDD and dose profile curves for open field without PWs show a good agreement between MC calculated results and measurements. The source of the negligible difference in out of field of dose profile may be caused by inaccurate provided data by the vendor or non- ideal tuning of incident electron beam parameters due to its time consuming. For the wedged fields this agreement was better than 2%. The slight discrepancies may be related to little uncertainties in the real steel composition. These agreements are comparable with reported results by other groups (Zhu et al., 2000; Shih et al., 2001; Attalla et al., 2010).l In conclusion our MC modeling of physical wedge dosimetric properties are in good agreement with measurements. The results indicate that the presence of a wedge in beam line causes beam hardening. Increase of wedge angle increases the beam hardening and this effect is more pronounced at the heal region of PWs. Using of such accurate MC model to determine of wedge factors and implementation of it as a calculation algorithm in treatment planning system (TPS) is recommended. Photon energy fluence off-axis distance due to the same attenuating thickness of wedge across the cross-line direction of photon beam. factors decrease across the field from the toe to the heal region and this change is more pronounced for 45° wedge. Additionally, it is clear that output factor is not changed along the cross-line direction (Figure 5.b). As can be seen from Figure 6, the HVL increase from the toe to the heal regions for each wedges; 2.12% and 6.28% for 15° and 45° PWs, respectively. In the central region, in spite of the larger thickness of 45° PW than 15°PW, the HVLs of 15° and 45° PWs are same. It can be explained that the more production of scattered photons (due to the larger scattering mass in case of 45° PW) may compensate the hardening effect. Dependence of mean photon energy to the wedge angle for other Linac models such as Varian has been well documented by Geraily, et al. (2014). For the open field the HVL of the center region is 2.42% larger than the toe and the heal regions; It can be caused by attenuation properties of the Gaussian shape flattening filter located in Linac head that has more fluence and energy attenuating powers in its central part. This effect is clear from off-axis fluence curve of open field in Figure 3. Acknowledgements According to Figure 3, PWs reduce photon fluence entirely due to attenuation effect of PW and this reduction is higher by increasing of wedge angle. Across the beam line, the reduction of photon fluence increase from the toe to the heal region of PW due to the higher thickness of heal part than toe along the photon beam path. The average energy of photon were 1.62, 1.57 and 1.62 MV for open field, 1.85, 1.92 and 1.91 MV for 15°PW and 2.16, 2.31 and 2.35 MV for 45° PW, respectively (Figure 4). For toe, central and heal regions of the beam, the mean calculated energy shifts to higher energy with increase in wedge angle that means the presence of wedge in beam line decreases the lower energy components of beam more strongly than the higher energy photons. This is more significant for the heel region due to more thickness of steel wedge filter. These results are in consistence with other reported data (Verhaegen and Das, 1999; Zhu et al., 2000). This study was Funded by the research and technology deputy of Ahvaz Jundishapur University of Medical Sciences & Arvand international University of Medical Sciences, Ahvaz, Iran. References Ahmad M, Hussain A, Muhammad W, et al (2010). Studying wedge factors and beam profiles for physical and enhanced dynamic wedges. J Med Physics, 35, 33-41. Andreo P, Burns DT, Hohlfeld K, et al (2000). IAEA ,TRS-398: Absorbed dose determination in external beam radiotherapy: An International code of practice for dosimetry based on standards of absorbed dose to water. IAEA: international atomic energy agency. Vienna, 10, 46-80. Attalla EM, Abo-Elenein HS, Ammar H, et al (2010). Comparison of dosimetric characteristics of Siemens virtual and physical wedges for ONCOR linear accelerator. J Med Physics, 35, 164-9. As shown in Figure 5, the output factors changed across the in-line direction from 0.638 to 0.718 and from 0.246 to 0.383 for 15° and 45° PWs, respectively. The averaged value of in-line output factor was 0.313 and 0.683 for 15° and 45° PWs, respectively. These output variations are not considered in treatment planning systems (TPS) or during manual calculation to determination of dose distribution. Our calculated in-line output factors on the central axis in depth of 10 cm and 10 x 10 cm2 field size at SSD=100 were 0.693 and 0.307, that are in a good agreement with measured data of 0.675 and 0.312 for 15° and 45° PWs, respectively. As expected, the cross-line output factors (square dots in Figure 5) of wedged fields show Geraily GH, Mirzapour M, Mahdavi S.R, et al (2014). Monte Carlo study on beam hardening effect of physical wedges. Int J Radiat Res, 12, 249-56. Hubbell JH, Seltzer SM (1995). Tables of x-ray mass attenuation coefficients and mass energy absorbtion coefficients from 1 keV to 20 keV for elements Z=1 to 92 and 48 additional substances of dosimetric interest. NISTIR, 5632. IAEA, report: TRS-398 (2000). Absorbed dose determination in external beam radiotherapy. Veina, International atomic energy agency. ICRU: Report -50 (1993). Prescribing, recording and reporting photon beam therapy. Bethesda, MD: International Asian Pacific Journal of Cancer Prevention, Vol 17, 2016 1688 DOI:http://dx.doi.org/10.7314/APJCP.2016.17.4.1685 Dosimetric Characteristics of Physical Wedges with a Siemens Linear Accelerator asurements Commission on Radiation Units and Measurements.i Kowalik A, Litoborski M (2013). Multienergetic verification of dynamic wedge angles in medical accelerators using multichannel linear array. Reports Practical Oncol Radiotherapy, 18, 220-34. Miften M, Zhu XR, Takahashi K, et al (2000). Implementation and verification of virtual wedge in a three-dimensional radiotherapy planning system. Med Physics, 27, 1635-43. Muhammad W, Maqbool M, Shahid M, et al (2011). DOI:http://dx.doi.org/10.7314/APJCP.2016.17.4.1685 Dosimetric Characteristics of Physical Wedges with a Siemens Linear Accelerator Asian Pacific Journal of Cancer Prevention, Vol 17, 2016 1689 References Assessment of computerized treatment planning system accuracy in calculating wedge factors of physical wedged fields for 6 MV photon beams. Physica Med, 27, 135-43. Muren LP, Hafslund R, Gustafsson A, et al (2001). Partially wedged beams improve radiotherapy treatment of urinary bladder cancer. Radiotherapy Oncol, 59, 21-30. Njeh CF (2015). Enhanced dynamic wedge output factors for Varian 2300CD and the case for a reference database. J Applied Clinical Med Physics, 16, 5498. Petrovic B, Grzadziel A, Rutonjski L, et al (2010). Linear array measurements of enhanced dynamic wedge and treatment planning system (TPS) calculation for 15 MV photon beam and comparison with electronic portal imaging device (EPID) measurements. Radiol Oncol, 44, 199-206. Rogers DWO, Ma C-M, Walters B, et al (2003). BEAMnrc User Manual. NRCC Report PIRS-509 (National Research Council of Canada), Ottawa, Ontario, Canada. Shih R, Li XA, Chu JC (2001). Dynamic wedge versus physical wedge: a Monte Carlo study. Medical Physics, 28, 612-9. Verhaegen F, Das IJ (1999). Monte Carlo modelling of a virtual wedge. Physics Med Biol, 44, 251-9. Vinagre FL, Simoes PC, Rachinhas PJ (2009). Omni-wedge technique for increased dose homogeneity in head and neck radiotherapy. Physica Med, 25, 154-9. Zhu XR, Gillin MT, Jursinic PA, et al (2000). Comparison of dosimetric characteristics of Siemens virtual and physical wedges. Medical Physics, 27, 2267-77.
https://openalex.org/W4297965022	https://escholarship.org/content/qt75j050zw/qt75j050zw.pdf?t=rxcg8r	English	null	Demes: a standard format for demographic models	Genetics	2,022	cc-by	8,984	UC Irvine UC Irvine Previously Published Works Title Demes: a standard format for demographic models Permalink https://escholarship.org/uc/item/75j050zw Journal Genetics, 222(3) ISSN 0016-6731 Authors Gower, Graham Ragsdale, Aaron P Bisschop, Gertjan et al. Publication Date 2022-11-01 DOI 10.1093/genetics/iyac131 Peer reviewed UC Irvine UC Irvine Previously Published Works Title Demes: a standard format for demographic models Permalink https://escholarship.org/uc/item/75j050zw Journal Genetics, 222(3) ISSN 0016-6731 Authors Gower, Graham Ragsdale, Aaron P Bisschop, Gertjan et al. Publication Date 2022-11-01 DOI 10.1093/genetics/iyac131 Peer reviewed UC Irvine Journal Genetics, 222(3) ISSN 0016-6731 Authors Gower, Graham Ragsdale, Aaron P Bisschop, Gertjan et al. Publication Date 2022-11-01 DOI 10.1093/genetics/iyac131 Peer reviewed Received: June 13, 2022. Accepted: August 23, 2022 V C The Author(s) 2022. Published by Oxford University Press on behalf of Genetics Society of America. 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 reuse, distribution, and reproduction in any medium, provided the original work is properly cited. Abstract Understanding the demographic history of populations is a key goal in population genetics, and with improving methods and data, ever more complex models are being proposed and tested. Demographic models of current interest typically consist of a set of discrete popula- tions, their sizes and growth rates, and continuous and pulse migrations between those populations over a number of epochs, which can re- quire dozens of parameters to fully describe. There is currently no standard format to deﬁne such models, signiﬁcantly hampering progress in the ﬁeld. In particular, the important task of translating the model descriptions in published work into input suitable for population genetic simulators is labor intensive and error prone. We propose the Demes data model and ﬁle format, built on widely used technologies, to allevi- ate these issues. Demes provide a well-deﬁned and unambiguous model of populations and their properties that is straightforward to imple- ment in software, and a text ﬁle format that is designed for simplicity and clarity. We provide thoroughly tested implementations of Demes parsers in multiple languages including Python and C, and showcase initial support in several simulators and inference methods. An introduc- tion to the ﬁle format and a detailed speciﬁcation are available at https://popsim-consortium.github.io/demes-spec-docs/. Keywords: demographic models; inference; simulation Demes: a standard format for demographic models Graham Gower ,1,† Aaron P. Ragsdale ,2,† Gertjan Bisschop ,3 Ryan N. Gutenkunst ,4 Matthew Hartfield , Ekaterina Noskova ,5 Stephan Schiffels ,6 Travis J. Struck ,4 Jerome Kelleher ,7,,‡ Kevin R. Thornton 8,‡ 1Section for Molecular Ecology and Evolution, Globe Institute, University of Copenhagen, 1350 Copenhagen K, Denmark, 2Department of Integrative Biology, University of Wisconsin–Madison, Madison, WI 53706, USA, 1Section for Molecular Ecology and Evolution, Globe Institute, University of Copenhagen, 1350 Copenhagen K, Denmark, 2 p g gy y 3Institute of Ecology and Evolution, The University of Edinburgh, Edinburgh EH9 3FL, UK, p g gy y 3Institute of Ecology and Evolution, The University of Edinburgh, Edinburgh EH9 3FL, UK, 4 gy y g g 4Department of Molecular and Cellular Biology, University of Arizona, Tucson, AZ 85721, USA, 4Department of Molecular and Cellular Biology, University of Arizona, Tucson, AZ 85721, USA, p g y y g 6Max Planck Institute for Evolutionary Anthropology, 04103 Leipzig, Germany, 7 6Max Planck Institute for Evolutionary Anthropology, 04103 Leipzig, Germany, 7Big Data Institute, Li Ka Shing Centre for Health Information and Discovery, University of Oxford, Oxford OX3 7LF, UK, 8D f E l d E l i Bi l U i i f C lif i I i CA 92697 USA 7Big Data Institute, Li Ka Shing Centre for Health Information and Discovery, University of Oxford, Oxford OX3 7LF, UK 8Department of Ecology and Evolutionary Biology University of California Irvine CA 92697 USA 7Big Data Institute, Li Ka Shing Centre for Health Information and Discovery, University of Oxford, O 8Department of Ecology and Evolutionary Biology, University of California, Irvine, CA 92697, USA 8Department of Ecology and Evolutionary Biology, University of California, Irvine, CA 92697, USA thor: Big Data Institute, Li Ka Shing Centre for Health Information and Discovery, University of Oxford, Oxford OX3 7LF, UK. Emai di ox ac uk Corresponding author: Big Data Institute, Li Ka Shing Centre for Health Information and Discovery, University of Oxford, Oxford OX3 7LF, UK. Email jerome.kelleher@bdi.ox.ac.uk j †These authors contributed equally to this work, as joint ﬁrst authors. ‡ †These authors contributed equally to this work, as joint ﬁrst authors. ‡These authors contributed equally to this work, as joint last authors. ‡These authors contributed equally to this work, as joint last authors. GENETICS, 2022, 222(3), iyac131 https://doi.org/10.1093/genetics/iyac131 Advance Access Publication Date: 29 September 2022 Investigation Highlighted Article Received: June 13, 2022. Accepted: August 23, 2022 Powered by the California Digital Library University of California Powered by the California Digital Library University of California eScholarship.org Demes The design of Demes is a balance between two partially compet- ing requirements: that (a) models should be easy for humans to understand and manipulate; and (b) software processing Demes models should be provided with an unambiguous representation that is straightforward to process. For efficiency of understanding and avoidance of model specification error, we require a data rep- resentation without redundancy (i.e. repetition of values). However, for the simplicity of software working with the Demes model (and the avoidance of programming error, or divergence in interpretations of the specification), it is preferable to have an ex- plicit representation, in which all relevant values are readily available. Thus, Demes are composed of three entities: the Human Data Model (HDM) designed for human readability; the Machine Data Model (MDM) designed for programmatic input and processing; and the parser, which is responsible for trans- forming the former into the latter. g y Here, we present “Demes,” a data model and file format speci- fication for complex demographic models developed by the PopSim Consortium (Adrion et al. 2020). The Demes data model precisely defines the sizes and relationships of populations, and it provides a way to explicitly encode the information relevant to demography while avoiding repetition. This data model is imple- mented in the widely used YAML format (Ben-Kiki et al. 2009), which is a data serialization language that provides a good bal- ance between human and machine readability. The specification precisely defines the required behavior of implementations, en- suring that there is no ambiguity of interpretation, and includes both a reference implementation and an extensive suite of test examples and their expected output. The initial software ecosys- tem includes high-quality Python and C parser implementations, as well as utilities for verification and visualization of Demes models, and has been implemented in several popular inference and simulation methods (Table 1). We hope that this data model and file format will be widely adopted by the community, such Here, we provide a brief overview of the population genetics models that Demes supports and the components of the Demes infrastructure. Complete technical details of the MDM and HDM, and the responsibilities of the parser are provided in the online Demes specification (https://popsim-consortium.github. io/demes-spec-docs/; accessed 2022 September 12). This specifi- cation rigorously defines the data model, fully describing the en- tities and their relationships, and the required behavior of implementations. Introduction depiction, a list of key parameters in tabular form and a discus- sion within the text. Unfortunately, these descriptions are often ambiguous, and implementing the precise model inferred for later simulation is at best tedious and error prone (Adrion et al. 2020; Ragsdale et al. 2020), and occasionally impossible because of missing information. The ever-increasing amount of genetic sequencing data from ge- netically and geographically diverse species and populations has allowed us to infer complex demography and study life history at fine scales. An integral component to such population genetics studies is simulation. Software to either simulate whole-genome sequences (Thornton 2014, 2019; Staab et al. 2015; Kelleher et al. 2016; Haller and Messer 2019; Baumdicker et al. 2022) or informa- tive summary statistics of diversity (Gutenkunst et al. 2009; Jouganous et al. 2017; Kamm et al. 2017) have enabled the increas- ing complexity of genomic studies, with several software pack- ages capable of handling large sample sizes, many interacting populations, and deviations from panmictic random-mating assumptions. This ability to infer and simulate such complex de- mographic scenarios, however, has highlighted a major short- coming in community standards: the fragmented landscape of different ways to describe demographic models makes it difficult to compare inferences made by different methods and to reliably simulate from previously inferred models. Inference results are typically reported in publications via a combination of visual Simulation is a core tool in population genetics, and many methods have been developed over the past 3 decades (Carvajal- Rodrıguez 2008; Liu et al. 2008; Arenas 2012; Hoban et al. 2012; Yuan et al. 2012). Simulations are based on highly idealized popu- lation models, and one of the key uses of inferred demographic histories is to make simulations more realistic. Simulation meth- ods take three broad approaches to specifying the demographic model to simulate, using either a command line interface (e.g. Hudson 2002; Hernandez 2008; Kern and Schrider 2016), a custom input file format (e.g. Guillaume and Rougemont 2006; Excoffier and Foll 2011; Shlyakhter et al. 2014), or an Application Programming Interface (API) to allow models to be defined pro- grammatically (e.g. Thornton 2014; Hernandez and Uricchio 2015; Kelleher et al. 2016; Becheler et al. 2019; Haller and Messer \| GENETICS, 2022, Vol. 222, No. 3 2 2019; Thornton 2019; Baumdicker et al. 2022). Demes Since the online specification is definitive, we will not recapitulate the details here, but instead focus on the high-level properties of the model and the rationale behind key design decisions. Introduction Command line interfaces are a concise way of expressing demographic models, and the syntax defined by ms (Hudson 2002) is used by several simulators (e.g. Chen et al. 2009; Ewing and Hermisson 2010; Staab et al. 2015). However, this conciseness means that models of even intermediate complexity are difficult for humans to un- derstand, making errors likely. APIs are more verbose, but require a substantial time investment to learn, and as they are tied to a specific tool this knowledge is not portable to other simulators. Like APIs, input parameter file formats for simulators allow the model specification to be less terse and allow for documentation in the form of comments. Several graphical user interfaces and visualization methods have been developed, which greatly facili- tate interpretation (Mailund et al. 2005; Antao et al. 2007; Parreira et al. 2009; Ewing and Hermisson 2010; Parobek et al. 2017; Zhou et al. 2018). However, these methods currently have little traction as they are all either directly coupled to an internal simulation method or to the syntax of a specific simulator. There is currently no way in which demographic models inferred by different pack- ages can be simulated or visualized by downstream software. that users can expect to simulate directly from inferred models with little to no programming effort. We have included software infrastructure developed for working with Demes models (such as parsing, validation, and visualization) as well as downstream software that implement the speciﬁcation, at the time of writing. Table 1. Software support for Demes. Software infrastructure demes-python A Python library for loading, saving, and working with Demes models. Includes support for converting to and from ms (Hudson 2002; https://github.com/popsim-consortium/demes-python). demes-c A C library for parsing Demes YAML descriptions (https://github.com/grahamgower/demes-c). demes-rust A Demes parser in Rust (https://github.com/molpopgen/demes-rs). demes-julia A parser in Julia (https://github.com/apragsdale/Demes.jl). demesdraw A Python library for visualizing Demes models (https://github.com/grahamgower/demesdraw). Methods using Demes as input/output format dadi Optimizes parameters in models of demographic history and distributions of ﬁtness effects using SFS (Gutenkunst et al. 2009). Can simulate SFS from Demes models. demes-slim Loads Demes models into the SLiM forward simulator (Haller and Messer 2019). fwdpy11 Simulates the Wright–Fisher model forward in time (Thornton 2014, 2019). Demes are the preferred format for specifying a demographic model. GADMA Infers models of demographic history (Noskova et al. 2020). Outputs Demes models and visualizations. gIMble Fits IM-type demographic models and infers genomic barriers to geneﬂow (Laetsch et al. 2022). Outputs inferred models in Demes format. moments Optimizes parameters in models of demographic history using SFS and linkage disequilibrium statistics (Jouganous et al. 2017; Ragsdale and Gravel 2019). Models to be optimized can be speciﬁed in Demes. MSMC A script provided in the MSMC-tools repository (https://github.com/stschiff/msmc-tools) converts MSMC (Schiffels and Durbin 2014; Schiffels and Wang 2020) output to the demes format. msprime Simulates population genetic models using tree sequences (Kelleher et al. 2016; Kelleher and Lohse 2020; Baumdicker et al. 2022). Demographic history models can be speciﬁed using Demes. W h i l d d ft i f t t d l d f ki ith D d l ( h i lid ti d i li ti ) ll d t Population genetics model For inference and simulation software to meaningfully interoper- ate, there must be a shared understanding of what a demo- graphic model is. Population genetics is a large field, and rather than attempting to capture all possible within- and between- Table 1. Software support for Demes. Software infrastructure demes-python A Python library for loading, saving, and working with Demes models. Includes support for converting to and from ms (Hudson 2002; https://github.com/popsim-consortium/demes-python). demes-c A C library for parsing Demes YAML descriptions (https://github.com/grahamgower/demes-c). demes-rust A Demes parser in Rust (https://github.com/molpopgen/demes-rs). demes-julia A parser in Julia (https://github.com/apragsdale/Demes.jl). demesdraw A Python library for visualizing Demes models (https://github.com/grahamgower/demesdraw). Methods using Demes as input/output format dadi Optimizes parameters in models of demographic history and distributions of ﬁtness effects using SFS (Gutenkunst et al. 2009). Can simulate SFS from Demes models. demes-slim Loads Demes models into the SLiM forward simulator (Haller and Messer 2019). fwdpy11 Simulates the Wright–Fisher model forward in time (Thornton 2014, 2019). Demes are the preferred format for specifying a demographic model. GADMA Infers models of demographic history (Noskova et al. 2020). Outputs Demes models and visualizations. gIMble Fits IM-type demographic models and infers genomic barriers to geneﬂow (Laetsch et al. 2022). Outputs inferred models in Demes format. moments Optimizes parameters in models of demographic history using SFS and linkage disequilibrium statistics (Jouganous et al. 2017; Ragsdale and Gravel 2019). Models to be optimized can be speciﬁed in Demes. MSMC A script provided in the MSMC-tools repository (https://github.com/stschiff/msmc-tools) converts MSMC (Schiffels and Durbin 2014; Schiffels and Wang 2020) output to the demes format. msprime Simulates population genetic models using tree sequences (Kelleher et al. 2016; Kelleher and Lohse 2020; Baumdicker et al. 2022). Demographic history models can be speciﬁed using Demes. Table 1. Software support for Demes. ( ; p g p p py ) A C library for parsing Demes YAML descriptions (https://github.com/grahamgower/demes-c). A Demes parser in Rust (https://github.com/molpopgen/demes-rs). p ( p g p g j ) A Python library for visualizing Demes models (https://github.com/grahamgower/demesdraw). nput/output format p izes parameters in models of demographic history and distributions of ﬁtness effects using SFS (Gutenkunst 2009). Can simulate SFS from Demes models. ) Loads Demes models into the SLiM forward simulator (Haller and Messer 2019). izes parameters in models of demographic history using SFS and linkage disequilibrium statistics (Jouganous 2017; Ragsdale and Gravel 2019). Models to be optimized can be speciﬁed in Demes. Population genetics model ; g ) p p A script provided in the MSMC-tools repository (https://github.com/stschiff/m and Durbin 2014; Schiffels and Wang 2020) output to the demes format. g ) p Simulates population genetic models using tree sequences (Kelleher et al. 2016; Kelleher and Lohse 2020; Baumdicker et al. 2022). Demographic history models can be speciﬁed using Demes. G. Gower et al. \| 3 population processes, we have instead adopted a pragmatic ap- proach of identifying a common set of assumptions shared by many methods. We outline the processes and assumptions briefly here and in the Appendix. (a) Demographic models consist of one or more populations (or “demes”) defined by their size histories and the time intervals of their existence. Individuals can move between populations based on their ancestor-descendant relationships or by continuous or discrete migration events. Within a population, we assume Wright–Fisher dynamics (see the Appendix for more precise details). As described in the Scope of the Specification section be- low, the demographic model does not, as a deliberate simplifica- tion and separation of duties, include any information about genome biology or selection. These basic assumptions of discrete Wright–Fisher popula- tions connected by instantaneous or continuous migrations are shared by many inference methods (e.g. Gutenkunst et al. 2009; Li and Durbin 2011; Gravel 2012; Schiffels and Durbin 2014; Jouganous et al. 2017; Kamm et al. 2017; Ragsdale and Gravel 2019; Excoffier et al. 2021), and forwards- and backwards-time simulators (e.g. Hudson 2002; Gutenkunst et al. 2009; Excoffier and Foll 2011; Kelleher et al. 2016; Jouganous et al. 2017; Haller and Messer 2019; Thornton 2019). Demes therefore serves as “middleware” between inference methods and simulation soft- ware, capturing these common assumptions. (b) ( ) Fig. 1. Example isolation-with-migration Demes model. a) The HDM representation expressed as YAML. b) A visual representation of the model using demesdraw. The same model in the MDM form is provided in Fig. A1. It is important to note that the goal of describing the basic population processes precisely is not to be proscriptive about what methods may or may not use the specification, but so that we can be clear on what situations we can expect methods to agree exactly. Arbitrary population processes—for example, within-deme continuous spatial structure (Wright 1943; Barton et al. 2002, 2010; Ringbauer et al. 2017; Battey et al. Population genetics model 2020)—may be layered on top of this basic description, but as dynamics diverge from the core assumptions, then of course we can expect results to differ accordingly. Human data model The Demes HDM is focused on efficient human understanding and avoiding errors. We have adopted the widely used YAML for- mat (Ben-Kiki et al. 2009) as the primary interface for writing and interchanging demographic models (see the Appendix for ratio- nale). Demographic models provide information about global fea- tures of the model (such as time units and generation times), populations (as “demes”) and their existence intervals (as “epochs”), and gene flow between populations (as continuous “migrations” or instantaneous “pulse” events). Figure 1 shows an example isolation-with-migration model in HDM format. Fig. 1. Example isolation-with-migration Demes model. a) The HDM representation expressed as YAML. b) A visual representation of the model using demesdraw. The same model in the MDM form is provided in Fig. A1. recommended—that all models are expressed in a maximally concise form, and we wholeheartedly endorse the explicit state- ment of parameters where it increases model legibility. Structurally, the HDM encourages human understanding by avoiding redundancy in the description where possible and by providing a mechanism for specifying default values that are inherited hierarchically. For values that repeat across fields, the “defaults” mechanism may be used to implicitly assign default values to fields of the given type. A default is superseded by an explicitly provided value if given. Size values are inherited natu- rally following the progression of time. For example, if an epoch start_size is not provided (either directly, or via a defaults sec- tion), it is assumed to be equal to the end_size of the previous epoch. This also means that the first epoch of each population must specify the initial size (or it must be provided in a defaults section). Application: simulation using Demes Here, we highlight the interaction between Demes and other soft- ware, including simulation and model illustration tools. Demes allow us to specify a demographic model which can be used as the input for a growing number of simulation packages (Table 1). We implemented the human two-population demographic model from Tennessen et al. (2012) inferred from European and African- American sequencing data. This model (shown in Demes format in Fig. A2) is parameterized by an ancestral population with an ancient growth, divergence into “AFR” and “EUR” that each has multiple-epoch size histories, and multiple epochs of continuous migration between the two branches (illustrated using demes- draw in Fig. 2a). The large final sizes (500; 000 individuals each) are one to three orders of magnitude larger than ancestral popu- lation sizes, reflecting the recent explosive population size in- crease in humans. The model is therefore limited to features that we can expect many different demographic inference and simulation methods to share. The specification only describes demographic features at the population level. Features of genome biology are out of scope, including mutation and recombination rates, genome annotations, ploidy, and so on. Selection and dominance models are absent, as discussed in the Appendix. It is important to note, however, that Demes may be used in applications that include additional population genetic processes outside of what is explic- itly modeled in the specification, such as interpreting population sizes as carrying capacities, implementations of hard selection, or layering more complicated mating or spatial structure. The Demes specification is intended to provide a basic model that can be elaborated on where necessary. Demes are not a standard population genetic simulation specifi- cation, although it could be part of one. Since the standard is based on JSON, and JSON documents can be arbitrarily nested, we can imagine a simple specification of genome features such as muta- tion and recombination rates in which the demography is defined by an embedded Demes specification. Features of the simulation specification (such as defining the time and location of samples) can then refer to the Demes model. This design, in which we embed the demographic model within a larger specification rather than adding arbitrary and unrelated complexities to the demography is an essential simplification and separation of duties. We used this model to simulate 20 haploid genome copies from EUR and AFR at time zero (i.e. Scope of the specification A primary design goal of Demes is to provide a means of unam- biguously communicating the results of demographic model inferences to population genetic simulators. Since demography is defined in terms of groups of individuals and these groupings are influenced by genetics, it is difficult to find a simple definition that separates the two. Thus, we have attempted to be pragmatic, limiting the features that we include in Demes to those that are in practise regarded as part of a demographic model. Application: simulation using Demes present day) to obtain the joint site-frequency spectrum (SFS), a summary of observed allele frequencies widely used in evolutionary inference (Bustamante et al. 2001; Gutenkunst et al. 2009; Tennessen et al. 2012; Jouganous et al. 2017; Kamm et al. 2017; Kim et al. 2017). The Demes model (Figs. 2a and A2) was provided as the input demog- raphy to msprime (Baumdicker et al. 2022) to simulate a large recombining region under the mutation rate assumed in Tennessen et al. (2012), and we computed the observed SFS using tskit (Ralph et al. 2020). Using the same Demes model as input to moments (Jouganous et al. 2017), we computed the expectation of the joint SFS and compared to the msprime simulated data (Fig. 2, b and c). Figure 2d shows the code required to run the sim- ulations in msprime and moments, and demonstrates that pre- cisely the same input model, without modification, was provided to both packages. Such interoperability is a major gain for researchers, which we hope will become the expected norm as more packages adopt the Demes format. The Demes specification is static by design—we wish to unam- biguously describe a demographic model with a concrete set of parameters. This simplicity means that we cannot directly spec- ify parameter distributions or estimated confidence intervals for those parameters. While it is not difficult to imagine extending the specification in ways that would allow this, it is not clear that the benefits are worth the greatly increased parser complexity (see the Appendix). Parsers While the HDM is designed for human readability and concise- ness, the underlying data model suitable for software implemen- tation (the MDM) is redundant and exhaustive. Translation from the HDM to the MDM requires resolving hierarchically defined default values and verifying relationships between populations and the validity of specified parameter values. Because this translation and validation require significant programming ef- fort, we define a standard software entity as part of the specifica- tion to perform this task (the parser), which is intended to be shared by programs that support Demes as input. The Demes specification precisely defines the required behavior of parsers, Avoiding redundancy in this way reduces the cognitive load on readers, by highlighting necessary parameters which may be otherwise be obscured. It is not necessary—or indeed \| GENETICS, 2022, Vol. 222, No. 3 that derive their ancestry from A when it goes extinct. Demes A and X have only one epoch, in which the population sizes are con- stant, whereas deme Y has 2 epochs. Deme Y’s second epoch has a different end_size than its start_size, which indicates the size grows exponentially from 1,000 individuals at 50 generations ago to 3,000 individuals at time 0 (the present). The migration section lists one migration stanza, between demes X and Y. This migration stanza does not indicate a source or destination deme, so the mi- gration is symmetric. No migration times are specified, so migra- tions occur continuously at the given rate during the time interval over which both demes exist (from 100 generations ago until the present). We do not attempt a detailed explanation of all Demes features here, and readers are instead directed to the tutorial and detailed specification in the online documentation (https://pop sim-consortium.github.io/demes-spec-docs/; accessed 2022 September 12). and we provide a reference implementation written in Python to resolve any potential ambiguities, as well as an extensive test suite of examples and the expected outputs. In addition, we have high-quality parser implementations in the Python, C, Rust, and Julia languages (Table 1) providing a solid foundation for the soft- ware ecosystem. By maintaining high-quality Demes parsers available as libraries, we ensure consistency across simulation and inference software. Having common parsers also benefits users by providing consistent and informative error messages for missing values or issues in formatting. Data availability The Demes specification and documentation are available at https://popsim-consortium.github.io/demes-spec-docs/ Antao T, Beja-Pereira A, Luikart G. MODELER4SIMCOAL2: a user- friendly, extensible modeler of demography and linked loci for coalescent simulations. Bioinformatics. 2007;23(14):1848–1850. Arenas M. Simulation of molecular data under diverse evolutionary scenarios. PLoS Comput Biol. 2012;8(5):e1002495. Conflicts of interest None declared. None declared. Literature cited Adrion JR, Cole CB, Dukler N, Galloway JG, Gladstein AL, Gower G, Kyriazis CC, Ragsdale AP, Tsambos G, Baumdicker F, et al. A community-maintained standard library of population genetic models. eLife. 2020;9:e54967. Adrion JR, Cole CB, Dukler N, Galloway JG, Gladstein AL, Gower G, Kyriazis CC, Ragsdale AP, Tsambos G, Baumdicker F, et al. A community-maintained standard library of population genetic models. eLife. 2020;9:e54967. Discussion b, c) We compared the single-population SFS in each population, showing agreement between the simulation methods. d) Python code snippets of the interactions between demes and the simulation software. An extended script to compute the SFS shown in (b) and (c) is given in Fig. A3. Fig. 2. Illustration and simulation using Demes. a) Using an inferred demographic model from Tennessen et al. (2012) speciﬁed as a YAML ﬁle in Demes format (Fig. A2), we used demesdraw to visualize the demographic model (note the recent exponential growth resulting in present-day population sizes that greatly exceed those in the past). We then used msprime to simulate genomic data for 20 genome copies sampled from the two contemporary populations, and we used moments to compute the expected joint site-frequency spectrum for the same sample sizes (Fig. A3). b, c) We compared the single-population SFS in each population, showing agreement between the simulation methods. d) Python code snippets of the interactions between demes and the simulation software. An extended script to compute the SFS shown in (b) and (c) is given in Fig. A3. Discussion In Fig. 1, we provide an example isolation-with-migration model. Models typically start with a concise description, followed by the mandatory time_units field. This model uses the defaults sec- tion to provide a default start_size of 1,000 individuals for each epoch of each deme. There are 3 demes in the model, an ancestral deme named “A” which exists arbitrarily far back into the past then ceases to exist at 100 generations ago, and demes “X” and “Y” Stable and healthy software ecosystems require standard inter- change formats, allowing for the development of high-quality and long-lasting tools that produce and consume the standard. Demographic models are a key part of population genetics re- search, and to date, the transfer of inferred models to down- stream simulations has been ad hoc, and conversions between the many different ways of expressing such models are both G. Gower et al. \| 5 (a) (b) (d) (c) Fig. 2. Illustration and simulation using Demes. a) Using an inferred demographic model from Tennessen et al. (2012) speciﬁed as a YAML ﬁle in Demes format (Fig. A2), we used demesdraw to visualize the demographic model (note the recent exponential growth resulting in present-day population sizes that greatly exceed those in the past). We then used msprime to simulate genomic data for 20 genome copies sampled from the two contemporary populations, and we used moments to compute the expected joint site-frequency spectrum for the same sample sizes (Fig. A3). b, c) We compared the single-population SFS in each population, showing agreement between the simulation methods. d) Python code snippets of the interactions between demes and the simulation software. An extended script to compute the SFS shown in (b) and (c) is given in Fig. A3. b) (d) (c) (b) (c) (a) (b (c) (a) (c) Fig. 2. Illustration and simulation using Demes. a) Using an inferred demographic model from Tennessen et al. (2012) speciﬁed as a YAML ﬁle in Demes format (Fig. A2), we used demesdraw to visualize the demographic model (note the recent exponential growth resulting in present-day population sizes that greatly exceed those in the past). We then used msprime to simulate genomic data for 20 genome copies sampled from the two contemporary populations, and we used moments to compute the expected joint site-frequency spectrum for the same sample sizes (Fig. A3). Funding labor intensive and error-prone. The proposed Demes standard is an attempt to bridge this gap between inference and simulation, and also to provide the foundations for a sustainable ecosystem of tools built around this data model. Table 1 shows some initial infrastructure that we have built as part of developing Demes, but many other useful tools can be envisaged that produce, con- sume, or transform this format. GG was supported by a Villum Fonden Young Investigator award to Fernando Racimo (project no. 00025300). RG and TS were sup- ported by the National Institute of General Medical Sciences of the National Institutes of Health (R01GM127348 to RNG). MH was supported by a Natural Environment Research Council Independent Research Fellowship (NE/R015686/1). JK was sup- ported by the Robertson Foundation. SS was supported by fund- ing from the European Research Council under the European Union’s Horizon 2020 research and innovation program (grant agreement No 851511). GB was supported by funding from the European Research Council (ModelGenomLand, 757648). Reproducibility is a significant problem throughout the scien- ces (Baker 2016), and various measures have been proposed to in- crease the likelihood of researchers being able to replicate results in the literature (Munafo` et al. 2017). The most basic requirement for reproducibility is that we must be able to state precisely what the result in question is. 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Appendix an arbitrary point in history as “time zero.” A natural speciﬁca- tion for time units is in generations, although other time units are permitted, such as years, accompanied by the generation time so that downstream software may convert times into gener- ations as required. The Demes speciﬁcation is a formal data model for describing the properties of populations over time, along with some metadata and provenance information. The data model is based on the ubiquitous JSON (Bray 2017) standard, and formally deﬁned using JSON Schema (Wright et al. 2020). Along with the schema, full technical details of the model are provided in the online speciﬁ- cation document (https://popsim-consortium.github.io/demes- spec-docs/). There must be at least one population with an inﬁnite start_time. An inﬁnite start time may be interpreted differently depending on the simulator. In a coalescent setting, there is no up- per bound for the coalescent time of lineages in this population. In a forwards-time setting, the interval of time between inﬁnity and the oldest noninﬁnite model time (i.e. the “ﬁrst event”) is approxi- mated by the simulator’s burn-in phase—detailed guidance is pro- vided in the online speciﬁcation. Communicating editor: G. Coop Communicating editor: G. Coop an arbitrary point in history as “time zero.” A natural speciﬁca- tion for time units is in generations, although other time units are permitted, such as years, accompanied by the generation time so that downstream software may convert times into gener- ations as required. Population genetics model details In Demes, demographic models consist of one or more interact- ing populations, or “demes,” understood to be a collection of indi- viduals that can be conveniently modeled using a deﬁned set of rules and parameters (Gilmour and Gregor 1939; Gilmour and Heslop-Harrison 1954). To avoid confusion with the name of the speciﬁcation itself, we will use the term “population” in this dis- cussion, with the understanding that the terms are interchange- able. A population is deﬁned as some collection of individuals that exists for some period of time, and has a well-deﬁned size (i.e. number of individuals) during that time period. Individuals can move between populations either according to their ances- tor–descendant relationships or through processes involving migrations. Few other properties of the populations are speciﬁed in the model: we are concerned primarily with deﬁning the popu- lations, their sizes, and the movement of individuals between those populations. Population dynamics Within a population, we assume that allele frequency dynamics can be described by the Wright–Fisher model. Brieﬂy, generations are nonoverlapping (all parents reproduce and die simulta- neously), and for allele i currently at frequency pi, its frequency in the next generation (at birth) is expected to be piwi=w, where wi and w are the marginal and mean ﬁtnesses, respectively, prop- erly weighted according to ancestry proportions. In this frame- work, a forward-time simulation of ﬁnite populations is Sizes and epochs Population sizes are given as numbers of individuals, and details such as ploidy levels are considered external to the model. We therefore focus on the number of individuals as opposed to the number of genome copies. Sizes and mating system details are speciﬁed for each population within population-speciﬁc epochs. Epochs are contiguous time intervals that deﬁne the existence in- terval of the population. Each epoch speciﬁes the population size over that interval, which can be a constant value or a function deﬁned by start and end sizes that must remain positive. Only ex- ponential population size changes are currently supported, but other functions may be added to the speciﬁcation over time. Acknowledgments Wright A, Andrews H, Hutton B, Dennis G. JSON schema: a media type for describing JSON documents. 2020. https://json-schema. org/ Shlyakhter I, Sabeti PC, Schaffner SF. Cosi2: an efﬁcient simulator of exact and approximate coalescent with selection. Bioinformatics. 2014;30(23):3427–3429. Wright S. Isolation by distance. Genetics. 1943;28(2):114–138. Yuan X, Miller DJ, Zhang J, Herrington D, Wang Y. An overview of population genetic data simulation. J Comput Biol. 2012;19(1): 42–54. Staab PR, Zhu S, Metzler D, Lunter G. scrm: efﬁciently simulating long sequences using the approximated coalescent with recom- bination. Bioinformatics. 2015;31(10):1680–1682. Zhou Y, Tian X, Browning BL, Browning SR. POPdemog: visualizing population demographic history from simulation scripts. Bioinformatics. 2018;34(16):2854–2855. Tajima F. Evolutionary relationship of DNA sequences in ﬁnite popu- lations. Genetics. 1983;105(2):437–460. Communicating editor: G. Coop Time units Population and event times are written as units in the past, so that time zero corresponds to the ﬁnal generation or “now,” and event times in the past are values greater than zero with larger values corresponding to times in the more distant past. By having time values increase into the past, we avoid the need to choose 8 \| GENETICS, 2022, Vol. 222, No. 3 8 Fig. A1. Isolation-with-migration example model from Fig. 1 in MDM form. The MDM form of the model is complete and explicit, but contains much redundant information that is omitted in the HDM form. Fig. A1. Isolation-with-migration example model from Fig. 1 in MDM form. The MDM form of the model is complete and explicit, but contains much redundant information that is omitted in the HDM form. Fig. A2. The Tennessen et al. (2012) two-population demographic model in Demes format. This model includes a single ancestral population that expands in size in the past, followed by divergence between AFR- and EUR-labeled populations. The two-population phase of the model includes multiple epochs of varying size, and rapid exponential growth over the past 5,000 years in each population. cloning of an individual. More speciﬁcally, for a given epoch within a population denote the clonal rate by r and the selﬁng rate by S. S and r can take any value between zero and one and can sum to more than one. Each generation a proportion of off- spring r are expected to be generated through clonal reproduc- tion, while 1 r are expected to arise through sexual reproduction. Within the sexually reproduced offspring, a propor- tion S is born via self-fertilization while the rest have parents drawn at random from the previous generation. Depending on the simulator, this random drawing of parent may occur either with or without replacement. When drawing occurs with replace- ment, a small amount of “residual” selﬁng is expected, so that the realized selﬁng probability is ð1 rÞðS þ ð1 SÞ=NÞ instead of ð1 rÞS (so that even with r ¼ 0 and S ¼ 0, selﬁng may still occur with probability 1=N), although this effect is negligible in large populations (Nordborg and Donnelly 1997). Fig. A1. Isolation-with-migration example model from Fig. 1 in MDM form. The MDM form of the model is complete and explicit, but contains much redundant information that is omitted in the HDM form. Time units equivalent to multinomial sampling of allele frequencies each generation (Bu¨ rger 2000, pp. 29–31; Crow and Kimura 1970, pp. 179–181), and a backwards-time (coalescent) simulation follows the approximations described in Tajima (1983), Hudson (1983), and Wakeley (2008, Chapter 3). Furthermore, this model assumes “soft” selection (Christiansen 1975), meaning that the dynamics of population sizes changes are independent of the details of indi- vidual ﬁtnesses. As such, this model excludes scenarios such as “hard selection,” in which population sizes are dependent on a population’s mean ﬁtness, or stochastic ﬂuctuations in popula- tion size, such as interpreting population sizes as carrying capaci- ties. Many forwards and backwards time simulators currently implement this model (e.g. Hudson 2002; Gutenkunst et al. 2009; Excofﬁer and Foll 2011; Kelleher et al. 2016; Jouganous et al. 2017; Haller and Messer 2019; Thornton 2019). By allowing the deﬁnition of selﬁng and cloning probabilities, we allow many standard models to be deﬁned. However, by pa- rameterizing selﬁng and cloning as we have, we assume that these properties of populations can be speciﬁed independently from the genetics. In other words, mutations that cause selﬁng probabilities to ﬂuctuate within an epoch are not considered. More details of the mathematical properties of selﬁng and clon- ing rates in a coalescent context can be found in Nordborg and Donnelly (1997) and Hartﬁeld et al. (2016). Rationale for static models Other infer- ence procedures based on optimizing a loss function (Gutenkunst et al. 2009; Jouganous et al. 2017; Kamm et al. 2017; Ragsdale and Gravel 2019; Excofﬁer et al. 2021) need users to specify parameter bounds, and possibly nonlinear or conditional constraints be- tween parameters. Indeed, the choice of how to parameterize a model could be important for some inference methods (e.g. abso- lute times vs relative times between events). sampling parents from the ancestral population to contribute to offspring in the newly generated population. If more than one an- cestor is speciﬁed, the proportions of ancestry from each contrib- uting population must be provided, and those proportions must sum to one. In this case, parents are chosen randomly from each ancestral population with probability given by those proportions. Individuals in a population may have parents from a different population through migrations. These can be deﬁned as continu- ous migration rates over time intervals for which populations co- exist or through instantaneous (or pulse) migration events at a given time. Continuous migration rates are deﬁned as the proba- bility that parents in the “destination” population are chosen from the “source” population. On the other hand, pulse migration events specify the instantaneous replacement of a given fraction of individuals in a destination population by individuals with parents from a source population. sampling parents from the ancestral population to contribute to offspring in the newly generated population. If more than one an- cestor is speciﬁed, the proportions of ancestry from each contrib- uting population must be provided, and those proportions must sum to one. In this case, parents are chosen randomly from each ancestral population with probability given by those proportions. p p p y g y p p Individuals in a population may have parents from a different population through migrations. These can be deﬁned as continu- ous migration rates over time intervals for which populations co- exist or through instantaneous (or pulse) migration events at a given time. Continuous migration rates are deﬁned as the proba- bility that parents in the “destination” population are chosen from the “source” population. On the other hand, pulse migration events specify the instantaneous replacement of a given fraction of individuals in a destination population by individuals with parents from a source population. Rationale for static models The Demes speciﬁcation is designed to describe demographic models deﬁned by a ﬁxed set of model parameters. As described in the main text, it does not include information about estimated conﬁdence intervals or the joint distribution of parameter values. In this section, we describe the rationale for this design decision. The parameters of demographic models are typically tightly coupled, and cases in which distributions for different parame- ters can be simply described are rare. In this situation, the sim- plest way to describe an estimated distribution is to list a large number of samples from the posterior. While writing out a large number of Demes models in YAML format may seem inefﬁcient, it can in fact be a compact way to describe these distributions. For example, consider a one-population model with piecewise- constant sizes over 20 epochs which has 40 free parameters: the start_size and end_time values for each epoch. If we sam- ple 50,000 models from the posterior distribution, the resulting multidocument YAML ﬁle is 45 MiB. This format compresses down to 8.4 MiB when gzipped or 6.2 MiB when compressed with LZMA2, which is on par with an equivalent binary representation of the free parameters (40 50000 4 bytes 7:6 MiB). Fig. A3. Simulation of SFS for the Tennessen model. We ﬁrst load the demographic model using demes (as graph), which can then be used by msprime to create the demographic model used in msprime. sim_ancestry(). The same loaded graph can also be passed to moments to compute the expected joint SFS. To compare the SFS in Fig. 2, we marginalize the joint SFS to obtain the single-population SFS for both AFR and EUR populations. Lines interfacing demes and other software are highlighted. Fig. A3. Simulation of SFS for the Tennessen model. We ﬁrst load the demographic model using demes (as graph), which can then be used by msprime to create the demographic model used in msprime. sim_ancestry(). The same loaded graph can also be passed to moments to compute the expected joint SFS. To compare the SFS in Fig. 2, we marginalize the joint SFS to obtain the single-population SFS for both AFR and EUR populations. Lines interfacing demes and other software are highlighted. Similarly, one might be interested in running simulations in which the demographic model parameters are drawn from a dis- tribution, e.g. in ABC inference (Beaumont et al. 2002). Relationships between populations A population may have one or more ancestors, which are other populations that exist at the population’s start time. If one ances- tor is speciﬁed, the ﬁrst generation is constructed by randomly Each population has an assigned selﬁng rate and cloning rate, where each deﬁnes the probability that offspring are generated from one generation to the next by either self-fertilization or G. Gower et al. 9 with an emphasis on simplicity and which interoperates well with JSON (indeed, YAML 1.2 is a superset of JSON). We chose YAML over JSON because although JSON is an excellent format for data interchange, it is ill-suited for human understanding and manipulation. We also considered other declarative data ex- change formats such as TOML, but chose YAML because of its equivalence with JSON, popularity, and good software support. Since the Demes data model is deﬁned in JSON Schema, however, there is no formal dependency on YAML and implementations may choose to use JSON directly if they wish (e.g. for greater efﬁ- ciency). Rationale for static models Implementing the many distributions of interest and support- ing a general way to describe a model’s free parameters would greatly increase the complexity of parsers, with relatively limited beneﬁt to most users. It is unlikely that Demes could be made sufﬁciently ﬂexible without implementing many features of general-purpose programming languages, such as variables, ar- ithmetic, and ﬂow control. Such use cases are therefore better served by writing model-generating functions in an existing pro- gramming language, for example using the Demes Python API (e.g. as implemented in moments; Jouganous et al. 2017; Ragsdale and Gravel 2019). As an intriguing possibility for developments in this direction, there exist many templating solutions for YAML and JSON that are speciﬁcally designed for extending static data in arbitrarily complex ways (e.g. YTT, Jsonnet, CUE, and Dhall). Rationale for YAML We have adopted the widely used YAML format (Ben-Kiki et al. 2009) as the recommended means of interchanging Demes mod- els (e.g. Figs. 1 and A2). YAML is a data serialization language
https://openalex.org/W4224027052	https://link.springer.com/content/pdf/10.1007/s10671-022-09314-3.pdf	English	null	The KUSF and the NCAA: a comparative study of national collegiate sport organizations' academic policies	Educational research for policy and practice	2,022	cc-by	8,370	Abstract The purpose of this comparative study was to investigate and compare the academic policies of the Korean University Sport Federation (KUSF) and the National Collegiate Athletic Association (NCAA). A qualitative, multiple case study design was utilized for this study. Speciﬁcally, in-depth interviews were completed with current South Korean (N = 3) and US (N = 3) college women’s tennis players to understand their experiences and perceptions linked to the KUSF’s and NCAA’s academic policies. A total of three distinct themes emerged with US student-athletes: (a) academic advisor involvement, (b) academic regulations, and (c) study hall. One theme emerged with South Korean student-athletes: absence of academic support. The results of this study provide practical insights to administrators of South Korean intercollegiate athletics regarding their academic policies. Keywords KUSF · NCAA · College student-athletes · Academic policies B Youngjik Lee youngjik.lee@louisville.edu The KUSF and the NCAA: a comparative study of national collegiate sport organizations’ academic policies Youngjik Lee1 · Addison Pond1 · Mary Hums1 · Casey George2 Received: 5 April 2021 / Accepted: 3 March 2022 / Published online: 14 April 2022 © The Author(s) 2022 1 Department of Health and Sport Sciences, College of Education and Human Development, University of Louisville, 2100 S Floyd St, Louisville, KY 40292, USA 2 Department of Educational Leadership, Evaluation and Organizational Development, College of Education and Human Development, University of Louisville, Louisville, USA 1 Department of Health and Sport Sciences, College of Education and Human Development, University of Louisville, 2100 S Floyd St, Louisville, KY 40292, USA Educational Research for Policy and Practice (2022) 21:323–337 https://doi.org/10.1007/s10671-022-09314-3 Educational Research for Policy and Practice (2022) 21:323–337 https://doi.org/10.1007/s10671-022-09314-3 ORIGINAL ARTICLE B Youngjik Lee youngjik.lee@louisville.edu 1 Department of Health and Sport Sciences, College of Education and Human Development, University of Louisville, 2100 S Floyd St, Louisville, KY 40292, USA 2 Department of Educational Leadership, Evaluation and Organizational Development, College of Education and Human Development, University of Louisville, Louisville, USA 1 Introduction South Korean sport culture places great emphasis on elite-level athletics. Speciﬁcally, actions such as intensive training practices, excessive time commitments, and questionable coaching practices are justiﬁed if they result in a gold medal (Heo, 2011). Due to the emphasis on athletic success, many South Korean secondary school athletes are required to devote all of their time to enhance their athletic performance at the expense of pursuing any endeavors besides their sport, including academics (Heo, 2010). South Korean college athletes do not fare much better than their secondary school counter- parts. The South Korean college sport system only considers athletic performance for college admission, meaning that high school athletes can become college athlete without proper aca- demic preparation or credentials (e.g., GPA and college entrance exam) (Kim, 2011). Further, similar to their time in high school, collegiate athletes are asked to focus solely on their sport 123 324 Y. Lee et al. (Lee, 2015). Due to this imbalanced culture in South Korean sport, South Korean college athletes are often unable to recognize the importance of academics (Ham, 2003). Despite the emphasis on athletics and athletic performance, very few athletes achieve high-proﬁle elite athlete status (Ham, 2003; Huml et al., 2014; Kim et al., 2014). In addition, even if athletes do attain high-proﬁle status, their competitive life span is very short. For example, the average age of a retired South Korean professional athlete in 2016 was only 23.8 years old (Yoo, 2016). Subsequently, most college athletes will need to integrate back into society shortly after their graduation or retirement from athletic competition (Otto et al., 2019). However, many South Korean college athlete do not possess the social or educational skills necessary to successfully make this transition. In an attempt to remedy the situation South Korean college athletes’ face, the Korean University Sport Federation (KUSF) was established in 2010. The KUSF is the governing body for 116 South Korean universities that offer sports teams. Two of the KUSF’s main goals are managing and supporting South Korean college athlete’ academic development and performance (KUSF, 2020a). The KUSF’s mission is to create a South Korean version of the NCAA (KUSF, 2018). Many South Korean sport scholars and practitioners have referenced and mentioned NCAA academic policies as potential guidelines for use in the South Korean collegiate ath- letic system (Kim, 2011; Kim & Park, 2009; Lee, 2013). 1 Introduction These scholars’ works created some initial understanding in South Korea about the NCAA’s Division I academic requirements. However, most of those studies were limited to simply translating the NCAA’s Division I aca- demic requirements from the NCAA manual from English into Korean. Making NCAA-like rules applicable in the South Korean sport system is a more difﬁcult task. The purpose of this study was to investigate and compare the KUSF’s and NCAA’s academic policies. Speciﬁcally, this study examined current South Korean and US college athletes’ experiences and perceptions of KUSF and NCAA academic policies, respectively. Athletes are vital stakeholders in intercollegiate athletics, as they are directly affected by these academic policies (Freeman, 1984). The following research questions guided the study: RQ1. How do KUSF athletes describe their on-campus academic experiences? RQ1. How do KUSF athletes describe their on-campus academic experiences? RQ2. How do NCAA athletes describe their on-campus academic experiences? RQ2. How do NCAA athletes describe their on-campus academic experiences? This study is signiﬁcant because conducting ﬁeld research with KUSF and NCAA athletes will produce a better understanding of the academic policies of each country’s intercolle- giate athletics governing body. Furthermore, by comparing and understanding the differences between South Korean and US intercollegiate athletics, this study can provide valuable insight to administrators/practitioners in South Korean intercollegiate athletics who intend to adapt and implement NCAA-style academic policies in South Korea. 2.1 History of South Korean elite sport As previously mentioned, South Korean sport culture places great emphasis on high athletic achievement. This cultural reality sprang from South Korea’s recent sporting history. In the 1970s, the South Korean government wanted to enhance the image of the nation through sport. To do so, government ofﬁcials successfully bid to host the 1988 Summer Olympic Games in hopes of educating the world about South Korea (Lee, 2005; Lim & Huh, 2009). Thus, the South Korean government emphasized elite-level athletic performance through national policies focused on winning gold medals (Lee, 2006). South Korea’s policy with regards to elite-level athletes created an imbalanced sport culture, which can succinctly be explained by stating that athletic success is everything (Heo, 2011). As a result, South Korean athletes believed that achieving high-proﬁle elite status was the only way to be considered successful (Ham, 2003). Even though the South Korean government asked their athletes to become patriots by winning gold medals, they did not emphasize other aspects of athletes’ lives (Hong & Ryu, 2007). For example, South Korean secondary school athletes could not pursue a proper education during their time in the secondary school since South Korean junior athletes are required to devote all their time to training for their sport (Heo, 2010). The sporting culture which focused only on athletes’ athletic achievement still exists and affects South Korean college athlete as well. Speciﬁcally, South Korean college athlete cannot focus on their academics for two reasons. First, South Korean college athlete are expected to focus on their athletic performance and achievement to act as representatives for South Korea. Second, their athletic achievements also enhance their institution’s image (Kim et al., 2014). The current application system for South Korean colleges also encourages college athlete to place less emphasis on their academics. South Korean colleges do not evaluate high school athletes’ GPA as a part of their admission criteria (Lee, 2015). South Korean colleges also do not promote athletes’ academic progress. Athletes oftentimes receive high grades despite missing classes and assignments to participate in competitions and practices (Lee, 2015). As a result, the imbalance between academics and athletics in the South Korea college sport system negatively affects athletes’ lives after graduation. For example, South Korean college athlete ﬁnd it difﬁcult to successfully integrate into society after graduation because they only develop sport skills and lack any social or business-oriented life skills (Kim et al., 2014). 2 Literature review In order to set the stage for understanding how athletes perceive and experience KUSF and NCAA regulations, a history of collegiate sports in both countries is presented, as well as an overview of both governing bodies. We begin with South Korea and the KUSF. 123 The KUSF and the NCAA: a comparative study of national collegiate … 325 2.1 History of South Korean elite sport Given this background, the KUSF was established in an attempt to mitigate the gap between academics and athletics. 2.3 History of academic reform of the NCAA The NCAA’s academic bylaws governing athletes began in the 1940s (Smith, 2011). In 1947, the NCAA adopted and implemented the “Sanity Code,” designed to adhere to the deﬁnition of amateurism, prohibiting professional athletes from competing in college athletics, and banning all athletic scholarships to prevent recruiting and ﬁnancial abuse (Ridpath, 2002; Waller, 2003). With this Sanity Code, the Constitutional Compliance Committee was created, which served as the ﬁrst regulatory body to investigate abuse by the NCAA’s member institu- tions (Crowley, 2006; Kennedy, 2007). Initially, the Sanity Code appeared to be successful. However, colleges and universities found ways to provide athletes with secret scholarships or pay them discreetly (Waller, 2003). Consequently, the Sanity Code was repealed when the NCAA refused to expel seven institutions for violations (Oriard, 2012; Waller, 2003). As an alternative to the Sanity Code, the NCAA approved the 12-point code in a 1952 convention. Among those 12 codes, half either directly or indirectly addressed the academic aspect of athletes such as (1) Conﬁne practice sessions to the recognized season of the sport (that is, no spring football), and limit or closely supervise out-of-season practices; (2) Limit the number of games in each sport, particularly football and basketball; (3) Reconsider postseason games in light of pressure they create (i.e., a potential ban on bowl games); (4) Require normal aca- demic progress toward a degree for purposes of eligibility; (5) Admit athletes only under the institution’s published requirements; (6) Give close attention to the curriculum of the athlete, to assure that he is not diverted from his educational objective (Oriard, 2012, p. 10). In 1973, the NCAA instituted the 2.0 rule. The 2.0 rule abandoned standardized test scores and only required a 2.0 high school GPA for incoming students to compete (Waller, 2003). Despite criticisms of the 2.0 rule, such as lack of uniformity in the nation’s different high school grading systems, the 2.0 rule existed until 1986. In 1986, the NCAA established a new rule, known as Proposition 48. Proposition 48 was similar to the 2.0 rule, but also included a minimum test score requirement. Proposition 48 required incoming athletes to have a minimum 700 SAT score or a composite score of 15 on the ACT, as well as a minimum high school GPA of 2.0 (Staurowsky & Ridpath, 2005). To strengthen Proposition 48, in 1992 the NCAA created Proposition 16 (Benson, 1993). 2.2 The KUSF In 2010, to remedy the current situation South Korean athletes are facing, a group of South Korean university presidents established the KUSF as the governing body for college sports (KUSF, 2010). The main reason for establishing the KUSF was to manage South Korean college athlete’ academics (Lee, 2015). A total of 116 institutions across the nation are currently KUSF members. The KUSF operates and manages the U-league, which offers a total of six different sports: basketball, volleyball, baseball, ice hockey, soft-tennis, and soccer (KUSF, 2020b). In 2017, in an attempt to improve South Korean college athlete’ academic situation, the KUSF established and implemented a minimum required GPA rule for its member institu- tions. Based on the rule, athletes earning a GPA lower than 2.0 from the previous semester 123 12 326 Y. Lee et al. are not eligible to compete in the following semester’s U-league competitions. Compared to KUSF and college sports in Korea, college sports in the USA have a longer history. 2.3 History of academic reform of the NCAA Proposition 16 included the following changes: (a) increased the number of core high school classes from 11 to 13 and (b) required incoming students to have a minimum GPA and speciﬁc minimum SAT score balancing their GPA (Crowley, 2006). After 13 years of Proposition 16, in 2006 the NCAA implemented the Academic Progress Rate (APR), a system still in use today. The APR is a term-by-term point measurement of eligibility and retention. Athletes can earn two points per semester. For example, athletes will earn one point if they are eligible and another point if they return after the fall semester (LaForge & Hodge, 2011). Points are assigned in the spring semester with the same criteria. Athletes who are eligible and return to their universities can earn total of four points per year. APR is assessed for each team and is calculated by taking the total points earned by the team at a given time divided by the total points possible for the team and then multiplied by one thousand (Kennedy, 2007; Meyer, 2005). A perfect score of APR is 1,000 and teams that score below 930 can face sanctions such as restrictions on their scholarships and practice time (NCAA, 2021). 123 12 The KUSF and the NCAA: a comparative study of national collegiate … 327 In sum, today’s NCAA academic standards are governed by rules in two speciﬁc cate- gories—initial eligibility for freshmen and continuing eligibility, including progress toward degree. It is important to note that the NCAA not only implemented academic regulations but also suggested/required that institutions help their athletes achieve academic success on their campuses. This led to the growth in offering academic advising services for athletes. 2.4 Academic advising at NCAA institutions Academic advising is deﬁned as “providing advice to students regarding academic, social, or personal issues, and this advice could be to inform, suggest, counsel, discipline, coach, mentor, or even teach” (Kuhn, 2008, p. 3). Athletic advising in NCAA institutions began in the 1970s (Rubin, 2017). After it started, academic advising for athletes has been serving various purposes. In the 1970s, academic advising focused on scheduling classes and has since expanded to include increasing retention and graduation rates (Broughton & Neyer, 2001). In intercollegiate athletics, athletic department advisors typically play an important role in academic advising for athletes. Athletic advisors possess expertise with the various NCAA academic rules and eligibility regulations that athletes must follow (Broughton & Neyer, 2001). In terms of educational background of athletic advisors, Rubin (2017) found that most of athletic advisor in NCAA have earned a master’s degree in various programs such as higher education, athletic administration, sport management, school counseling, academic advising, and business administration. In 1991, the NCAA approved bylaw 16.3.1.1, which required academic counseling and support services for all NCAA Division I athletes. This bylaw also provided ﬁnancial sup- port for the continuation of academic support services for athletes by creating the Academic Enhancement Fund. This Fund provides ﬁnancial support for institutions to manage tutoring, hire academic support staff, and acquire new equipment that can help athletes academically (NCAA, 2019). Today, many athletic departments construct and operate state-of-the-art aca- demic facilities in order to encourage athletes to excel in the classroom (Huml et al., 2014). All of these tactics help shape the academic experiences of NCAA athletes. This section dis- cussed the overall information of KUSF and its academic policies as well as NCAA’s history of academic reform and academic advising to gain better understanding of both organizations’ academic policies for their athletes. The purpose of this study was to explore and compare the KUSF’s and NCAA’s aca- demic policies by examining current South Korean and US college athletes’ experiences and perceptions of KUSF and NCAA academic policies. Name South Korean Participants Jaehee 4 Soojung 3 Hyunjung 3 US Participants Leigha 4 Brea 4 Alisa 3 All names are pseudonyms. 3.1 Case study design A case study can be deﬁned as exploring “a real-life, contemporary bounded system (a case) or multiple bounded systems (cases) over time, through detailed, in-depth data col- lection involving multiple sources of information… and reports a case description and case themes” (Creswell, 2007, p. 97). This study utilized a case study design to examine KUSF’s and NCAA’s academic policies through multiple sources of information, such as in-depth interviews with current athletes from both the KUSF and the NCAA, as well as various doc- uments, including academic journals, news articles, and academic policy manuals from both governing organizations. Since this study includes more than one case, a multiple case study design was utilized (Gustafsson, 2017). A multiple case study design allows the researchers to understand the differences and similarities between the cases (Stake, 1995). In addition, by using a multiple case study design, the researchers were able to analyze the data both within each case and across cases (Yin, 1993). 3.2 Selection of participants The target population for this study was college athlete in South Korea and the USA. This research used purposive sampling methods to recruit participants who could describe their academic experiences as athletes either in South Korea or the USA, while maintaining a manageable sample size for in-depth qualitative analysis (Beamon, 2012). To recruit the participants, the investigators contacted current athletes from South Korean and US univer- sities (see Table 1). A total of six interviews were conducted, (N = 6), three with South Korean athletes and three with athletes competing in the USA. Since college athletes may have different experiences based on the unique characteristics of the sports they play (e.g., daily practice and tournament schedules), this study selected participants from the same sport (women’s tennis) for direct comparison. Speciﬁcally, three women’s tennis players from a large university in a major city in South Korea and three women’s tennis players from a large university in the Southern US participated in this study. 3 Method This study utilized a qualitative research design to examine and understand the academic experiences of college athlete from South Korea and the USA. According to Glesne (2016), a qualitative study design is useful in for “documenting how structures shape individual experiences, and also how individuals create, change, or penetrate the structure that exists” (p. 39). This approach does not remove participants from their everyday lives and leads to understanding their real lived experiences (Creswell, 2007; Itoh et al., 2017; Marshall & Rossman, 2014). Using a qualitative approach, this study sought to uncover the thoughts, perspectives, and feelings of the participants, which would allow readers to enter into and experience the world of the participants (Yin, 1993). 123 123 328 Y. Lee et al. Table 1 Information of study sample 3.3 Data collection Theresearchersconductedin-depth,semi-structuredinterviewswiththeparticipants.Interms of the US participants, each athlete was interviewed individually in a face-to-face setting. 123 123 The KUSF and the NCAA: a comparative study of national collegiate … 329 With the athletes in South Korea, on the other hand, telephone interviews were conducted as access to the population of participants was limited due to their locations. Each interview lasted approximately 30–45 min. Data collection occurred from December 2019 through February 2020. The investigators obtained human subjects’ approval from their university. Respondents were informed that their participation was entirely voluntary. In keeping with protocol, signed informed consent was collected from the participants. Speciﬁcally, the US participants phys- ically signed the consent form, whereas South Korean participants signed electronically and sent the forms to the researchers via email. All participants were given an information letter, along with a consent form. The interviews included several questions asking athletes about their experiences and perspectives of their institutions’ academic support services. Speciﬁcally, the researchers modiﬁed questions used in previous research which related to athlete academic services (Huml et al., 2014). Some examples of speciﬁc interview questions include (see appendix for all questions that used in the study): • Are you satisﬁed with the current academic services provided by your institution? • Are you satisﬁed with the current academic services provided by your institution? • Do you know the academic requirements for you as athletes? • Do you think you were given the necessary academic support from your institution during this academic year? • Do you think you were given the necessary academic support from your institution during this academic year? Other written materials were used for triangulating the data, as well as for gaining a better understanding of both the KUSF and NCAA’s academic policies. Speciﬁcally, news articles, the ofﬁcial website, and academic policy manuals were used to examine the KUSF’s academic policies. In terms of NCAA, news articles, the ofﬁcial website, academic policy manual, and academic journal were analyzed. 3.4 Translation Strategies Interviews with the South Korean athletes were conducted and transcribed in Korean and then translated into English. A translation strategy was utilized to minimize errors that might have arisen during the translation process (Weeks et al., 2007). First, two bilingual translators translated the transcripts from Korean to English. Second, two bilingual translators who had not previously read the manuscripts were requested to translate the English version of transcripts back to Korean (back translation). Lastly, the ﬁnal process included substantial discussions with all translators regarding any potential discrepancies identiﬁed during back translation. 3.6.1 Case #1—NCAA athletes’ academic experiences Three distinct themes emerged regarding the NCAA’s athletes’ academic experiences: (a) academic advisor involvement, (b) academic regulations, and (c) study hall. The next sections will include participants’ quotes to illustrate these themes. Theme 1: Academic advisor involvement Athletes indicated that their academic advisor helped improve their academic work in various ways. In addition, athletes indicated that their academic advisor was always available and actively communicated with them in various modes (e.g., email, phone call, text). For example, Alisa stated, “Yes, [the academic advisor] is always available, and whenever I need him, he’s there to help…I normally just text or email him.” Leigha commented “Yes, our academic advisor is always available. Sometimes I would text him at like 10 pm to ask him questions and he would always respond.” Brea stated “I think that he provided us with all the necessary support during this academic year.” Alisa added: So, to make a schedule with him, I usually give him a call, because that’s easier to do versus text. And then, I just tell him what classes I want to be taking and he’ll kind of guide you through like what’s smart to take during season, what’s not smart to take during the season in terms of like the complexity of the classes. It is important to note that the academic advisors actively motivated and encouraged the athletes to go to class and emphasized the importance of academics. Leigha commented that “Yes, our academic advisor deﬁnitely expects and motivates all of us to go to classes.” Brea concurred: We had a match recently in Indiana. It was like a two and half to three-hour drive, we weren’t leaving till later that day, and he made sure to text everyone that we needed to go to classes because we weren’t leaving until 3pm. So, if your class ended at 2pm you still had to go to class to bolster your GPA. Participants’ comments about the role of their academic advisor, which included scheduling classes, motivating athletes go to classes, and helping their assignments, supported similar ﬁndings from previous studies (Otto et al., 2019). Theme 2: academic regulations Athlete eligibility has become a greater priority for uni- versities (Huml et al., 2014) as both the NCAA and many institutions set speciﬁc academic regulations. Learning and understanding regulations such as minimum GPA or satisfactory progress toward a degree is critical. 3.5 Data Analysis The ﬁrst and second authors utilized Braun and Clarke’s (2012) thematic analysis to analyze the participants’ interview data. There was no external auditor for the analysis. Thematic analysis is a systemic process to organize, identify, and offer patterns of themes/meanings across a dataset (Braun & Clarke, 2012). Based on thematic analysis, there are a total of six procedures to analyze data: (a) familiarize one’s self with the data through reading and re- reading, (b) generate initial codes, (c) search for themes from the codes, (d) review potential themes, (e) deﬁne themes, and (f) provide the report. Analysis of the dataset led to the identiﬁcation of three distinct themes for US college athletes’ academic services experiences and one theme for South Korean college athletes’ academic services experiences. 123 123 330 Y. Lee et al. 3.6 Results The results of this study revealed a total of four themes. These themes are organized in two individual cases by NCAA and KUSF athletes’ academic experiences. 3.6.1 Case #1—NCAA athletes’ academic experiences Athletes become ineligible to compete if they cannot meet academic requirements. Study participants learned about those rules during their freshman year through various ways provided by their institution. For example, Leigha mentioned that “I read all NCAA academic regulations in the handbook, which they gave us the ﬁrst week of classes.” Brea stated: NCAA regulation rules I learned that my freshman year. I have to go to a bunch of orientations that kind of goes over all the academic rules what you can and cannot 12 3 The KUSF and the NCAA: a comparative study of national collegiate … 331 do…There is also a handbook that all athletes get in the beginning of the year and someone from the athletic department goes over the handbook with us. do…There is also a handbook that all athletes get in the beginning of the year and someone from the athletic department goes over the handbook with us. All NCAA coaches have to complete an annual exam on the rules and regulations for NCAA athletes since coaches are deemed the guardians of athletes (NCAA, 2019). Coaches also have to meet speciﬁc scores on their annual exam to remain employed in their coaching positions. With their knowledge, coaches also helped the study participants regarding aca- demic regulations. Alisa commented that “I can always ask about the academic regulations, including my eligibility, to my coaches.” It is important to note that institutions try to educate athletes regarding NCAA academic regulations through various means such as orientations and handbooks. These efforts aim to ensure that athletes acknowledge the requirements and regulations they have to follow to remain eligible. Theme 3: study hall Academic centers are typically open to every athlete 7 days a week, and include a variety of resources, such as private study carrels, group study rooms, computer labs, and quiet places to rest. The US participants’ institution recently constructed a new academic center for their athletes (Rubin & Moses, 2017). Study hall is also held in the academic center. Speciﬁcally, every freshman and selected students who do not meet the academic standards set by the institution after their freshman year are required to participate in six to eight hours of study hall per week. Study hall is not required after freshman year as long as athletes maintain good academic standing of a 2.0 GPA or higher. 3.6.1 Case #1—NCAA athletes’ academic experiences Brea stated: “As a freshman, I did participate in study hall and had to do eight hours per week, but as a senior, I do not have to participate in that” and Alisa concurred that “my ﬁrst semester, I had six hours of study hall every week that I had to meet, but since I am a junior and I got a good GPA, I don’t have to do it now.” In addition, study hall is supervised by academic advisors and study hall monitors. The study hall requirement aids the athletes, especially freshmen, with time management skills and helps them adapt to the new, challenging academic environment and expectations at their institution. In addition, seeing groups of athletes studying motivates other athletes to do the same. For example, Leigha commented that “I think study hall deﬁnitely helps you to prepare. If you go there, you see people who are studying there and it makes you want to study more.” Brea mentioned, “Usually freshmen have to go to study hall just to kind of force them to ﬁgure out how to balance school, as well as their sport.” Alisa also commented that “Yes, I think study hall is deﬁnitely monitored appropriately…like it’s quiet and it’s really nice to be able to sit down and like see other athletes working hard and doing their homework.” This section discussed the NCAA’s academic policies based on the interviews with current NCAA athletes. The following section describes the South Korean college athletes’ experiences with the KUSF’s academic policies. 3.6.2 Case #2—KUSF athletes’ academic experiences Theme: absence of academic support Even though the KUSF is the governing body for South Korean intercollegiate athletics, the organization has no speciﬁc bylaw requiring indi- vidual institutions to provide academic support for athletes. There is, however, a minimum required 2.0 GPA for South Korean athletes participating in U-league sports (i.e., soccer, basketball, baseball, ice hockey, volleyball, and soft-tennis). Hyunjung stated, “There’s no academic support for athletes, but the athletes who are participating in U-league still have to meet the minimum required GPA set by KUSF.” 123 123 332 Y. Lee et al. South Korean youth sport culture likely exacerbates the academic difﬁculties that col- lege athlete experience. As previously mentioned, the lack of balance between athletics and academics in South Korean sport culture requires athletes to devote almost all of their time and attention during secondary school to training for their sport. Athletes are not able to devote a proportionate amount of time to their education, as this would take time away from training (Heo, 2011). In addition, athletic performance is the only criteria for South Korean athlete college admission, as schools do not consider high school athletes’ GPA as a part of this process (Lee, 2015). Subsequently, South Korean college athletes commonly experience difﬁculties with classwork and would beneﬁt from academic support that can provide basic learning skills and strategies. For example, Soojung stated, “Since we skipped most classes during our time in high school to practice our sports, we are not familiar with academic learning…honestly we do not have basic learning skills, such as how to take study notes.” Jaehee also added that. even though I’ve graduated from high school and became a college student, our aca- demic abilities need to be improved signiﬁcantly…honestly, for example, most of the athletes around me only have basic math skills such as algebra, and some do not know even know the alphabet. Therefore, there is a big discrepancy between the level of the classwork and our actual academic ability. This statement showed that how lack of education from high school negatively affects South Korean college athletes’ classwork. This statement showed that how lack of education from high school negatively affects South Korean college athletes’ classwork. Since there are no academic support services speciﬁcally for athletes, they are forced to use the academic support services provided for all students at the institution. 3.6.2 Case #2—KUSF athletes’ academic experiences However, as previously discussed, athletes’ academic ability is different from non-athlete students, as athletes are unable to take advantage of the academic support services available to all students at their institution. For example, Jaehee stated that “It’s kind of hard to catch up with academic support services for all students due to our learning ability…also, most academic support services take place on weekdays, so it overlaps with our training and practice schedule.” Understandably, participants experienced difﬁculties managing their academics. 4 Discussion The purpose of this study was to investigate and compare athletes’ academic experiences at KUSF and NCAA institutions. The results illustrated that US athletes beneﬁtted from various academic services at their institution. The US athletes indicated three different types of support (i.e., academic advisor involvement, NCAA regulations, study hall) as beneﬁcial for them. On the other hand, despite the KUSF’s minimum GPA requirement to participate in the U-league, no academic services were available for South Korean college athletes. It is important to note that the NCAA not only requires a minimum GPA for athletes’ eligibility, but also that individual institutions provide academic services to support them. For example, NCAA Bylaw 16.3.1.1 requires academic support services be available for all athletes (Huml et al., 2014; Navarro et al., 2020; NCAA, 2006). In addition, the NCAA created the Academic Enhancement Fund to provide ﬁnancial support to help NCAA member institutions provide variousacademicsupportservicesfortheirathletessuchastutoring,hiringacademicadvisors, and purchasing new equipment related to academics (NCAA, 2019). The participants in this study expressed that those support services directly affected athletes’ academic experiences in positive ways. 123 123 123 333 The KUSF and the NCAA: a comparative study of national collegiate … The KUSF, meanwhile, created and implemented a minimum required GPA rule in hopes of improving athletes’ academic performance, but did not mandate that institutions set up formal programs to help them achieve academic success (Kim, 2011; Kim & Park, 2009; Lee, 2013). The results of the current study revealed, however, that South Korean college athlete need a formal support structure to achieve this standard—namely additional academic support from their institution beyond simply a required minimum GPA rule. Previous research suggested that South Korean secondary school athletes were required to spend most of their time practicing their sport (Heo, 2010). This means they do not have the opportunity to learn proper academic skills and yet are expected to meet minimum academic standards. In other words, the policy lacks a mechanism to achieve its goal. Considering this fact, requiring a minimum GPA but not providing any academic support for college athlete may not help athletes academically at all. That situation could potentially make it more challenging for athletes to meet the required standards to remain eligible and could even make them become frustrated, give up on their academics, and derail their athletic career. In addition, the results suggested that the KUSF needs to create a stronger academic climate which encourages and supports academic services for their athletes. The ﬁndings of this study revealed that the climate in South Korean college sports where athletic achievement is the only way to be considered successful makes athletes believe they need to focus solely on athletics rather than academics. Thus, as a governing body of South Korean college sports, the KUSF needs to try and change the current climate of South Korean college sport to reinforce the need for their athletes to balance academics and athletics while providing formal support to achieve these goals. Next,whenreferringtoandtryingtoapplypoliciesfromtheUStothecurrentSouthKorean system, administrators need to ﬁrst gain a better understanding of the development of those policies. For example, as noted above, the NCAA’s long history is extremely different from the KUSF’s. Additionally, there are discrepancies in the overall environments and systems between the NCAA and the KUSF. In other words, the policies for NCAA Division I could more likely be successful within the context of institutions with more than a hundred years of history. 5 Limitations and future directions Like any other research endeavor, this study was not without limitations. First, since the present study had limited number of participants, it might be hard to generalize the results of this study to a wider population. The future study will need to later examine larger sample size to obtain a holistic understanding of each country’s national collegiate sport organization’s academic policies. Speciﬁcally, future research could be broadened by using of quantitative analysis with great numbers of participants. Second, the researchers only explored athletes from one speciﬁc sport, women’s tennis. Since other sports have their own characteristics, it would be worthwhile to examine the academic experiences of athletes from various sports. Third, this study only focused on athletes’ experiences to compare the academic policies between South Korean and US colleges. Even though athletes themselves can be viewed as essential stakeholders since they are directly affected by the academic policies, other stake- holders such as academic advisors, faculty members, and athletic administrators are involved indevelopingcollegeathletes’academicpolicies.Therefore,futureresearcherswouldbewise to examine other stakeholders’ perspectives on academic policies in South Korean and US colleges to attain a holistic understanding of both countries’ academic policies. Lastly, it would be worthwhile to investigate academic policies of other countries’ collegiate govern- ing bodies. Since the system and environments of college sports vary widely by countries, future research could compare the academic policies of collegiate governing bodies among those various countries. 123 Therefore, while gleaning ideas from foreign-based policies is a good place to start, simply applying those policies to the South Korean system without modiﬁcation will be insufﬁcient to promote true change. South Korean college sport administrators will need to think harder and deeper about how to make a “South Korean” version of academic policies for college athletes. Finally, but most importantly, if new policies are developed, the welfare of athletes should be the top priority. It is imperative that South Korean athletes begin to realize the importance of balancing academics and athletics. In a culture where the imbalance between these two still reigns supreme, changing the culture will require measured and thoughtful policy generation and implementation. The results of this study provide practical insights to administrators and practitioners working in South Korean intercollegiate athletics. Since South Korean college athletes are not familiar with academic learning experiences due to the absence of a proper education during their time in secondary school, it becomes important that they receive sufﬁcient aca- demic support and resources in higher education. Speciﬁcally, the KUSF needs to focus on constructing and implementing new policies that can realistically help athletes while also supporting the goals established through their regulatory policies. For example, South Korean colleges might hire some academic advisors that can increase its athletes’ academic achieve- ment by providing regular academic support. While a model such as the NCAA’s Academic Enhancement Fund may work better than simply requiring a minimum GPA, an assessment 123 123 334 Y. Lee et al. of institutional capacity and resources required to implement such a policy should ﬁrst be conducted to assess the potential impact on institutions. of institutional capacity and resources required to implement such a policy should ﬁrst be conducted to assess the potential impact on institutions. Interview questions • How many times you met with your academic advisor in this semester? • How many times you met with your academic advisor in this semester? • Do you know the academic requirements for you as a student-athlete? • How many times you met with your academic advisor in this semester? • Do you know the academic requirements for you as a student-athlete? y y y • Do you know the academic requirements for you as a student-athlete? • Do you know the NCAA regulations regarding your academic eligibility? • Yes, from where? Who? • Yes, from where? Who? • Does your academic advisor is always available to schedule meetings with you? • What’s the process of you make a schedule with him/her? • How many you used the tutorial services in this semester? Or per week? • How many you used study hall in this semester? Or per week? • Do you think your study hall is monitored appropriately? • Do you think study hall helps you prepare for class? • Are you satisﬁed with the current academic support services that provided by your insti- tution? • Tutoring you received • Academic services provided • Personnel of the academic services (tutors, counselors) 6 Conclusion The present study aimed to explore and compare the KUSF’s and NCAA’s academic policies by examining current South Korean and US college athletes’ experiences and perceptions of KUSF and NCAA academic policies, respectively. 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https://openalex.org/W4309900354	https://zenodo.org/record/7353741/files/60%20Ergasheva%20Mahliyohon%20Maxamatsoli%20qizi%20315-320.pdf	Chinese	null	汉、乌比喻修辞格文化对比分析	Zenodo (CERN European Organization for Nuclear Research)	2,022	cc-by	999	International scientific-practical conference on the topic of “Problems and perspectives of modern technology in teaching foreign languages” VOLUME 2 \| SPECIAL ISSUE 27 ISSN 2181-1784 SJIF 2022: 5.947 \| ASI Factor = 1.7 International scientific-practical conference on the topic of “Problems and perspectives of modern technology in teaching foreign languages” VOLUME 2 \| SPECIAL ISSUE 27 ISSN 2181-1784 SJIF 2022: 5.947 \| ASI Factor = 1.7 International scientific-practical conference on the topic of “Problems and perspectives of modern technology in teaching foreign languages” International scientific-practical conference on the topic of “Problems and perspectives of modern technology in teaching foreign languages” https://doi.org/10.5281/zenodo.7353741 Ergasheva Mahliyohon Maxamatsoli qizi Xitoy tili nazariyasi va amaliyoti kafedrasi o‘qituvchisi O‘zbekiston davlat jahon tillari universiteti 摘要：比喻是人类最古老、最广泛、最活跃、最普遍因而也是最基本、最重要的一种修辞格式。我们生活中说话的时候常常用比喻, 也常常用一些动物来比喻某一些人或者事情。但据每个国家传统文化与随俗这些比喻会表达各种意思。本文章主要谈的是汉语与乌兹别克语比喻并对比喻当中常常使用的动物做了一些对比. 我们的文章帮助学习汉语的学生理解中国文化和一些象征动物关键词：比喻，汉语，乌兹别克语，象征动物，对比分析语言包含着丰富的民族文化，比喻作为语言运用现象，它深刻地呈现出民族文化的特点。由于语言涉及到各自的地域、历史、民族、宗教、民族心理等文化元素，喻体产生的喻义在不同的语言系统中被赋予或同或异的文化内涵。一、汉、乌比喻句中常用动物象征义的对比分析中国与乌兹别克斯坦都是农业国家，动物资源非常丰富，汉乌比喻句里常常出现各种动物喻体。这些喻体在汉乌不同的文化背景下，产生的联想也有异有同。分以下两种情况: 首先，喻体相同，喻义不同。 “牛”在历史上是作为中国人农耕的得力助手，中国人和牛有一种自然而然的亲近感，牛是力量的象征，也是忠诚的象征，所以很多积极的比喻句都使用“牛”。如“你是个牛人!”是说话人对对方能力的极大赞美。 “老黄牛” 比喻老老一、汉、乌比喻句中常用动物象征义的对比分析中国与乌兹别克斯坦都是农业国家，动物资源非常丰富，汉乌比喻句里常常出现各种动物喻体。这些喻体在汉乌不同的文化背景下，产生的联想也有异有同。分以下两种情况: 首先，喻体相同，喻义不同。 “牛”在历史上是作为中国人农耕的得力助手，中国人和牛有一种自然而然的亲近感，牛是力量的象征，也是忠诚的象征，所以很多积极的比喻句都使用“牛”。如“你是个牛人!”是说话人对对方能力的极大赞美。 “老黄牛” 比喻老老异有情首先，喻体相同，喻义不同。 “牛”在历史上是作为中国人农耕的得力助手，中国人和牛有一种自然而然的亲近感，牛是力量的象征，也是忠诚的象征，所以很多积极的比喻句都使用“牛”。如“你是个牛人!”是说话人对对方能力的极大赞美。 “老黄牛” 比喻老老 315 w November 2022 w November 2022 316 w www.oriens.uz November 2022 个比喻跟中文的颗老鼠屎害了锅汤是样的意思。 (2) Buzoqni yugurgani somonhonagacha. (牛只能跑到玉米秸场。) “牛只能跑到玉米秸场”比喻每个人能量有限，只能做到力所能及的事。中国人历来信奉 “龙”是一种神物。龙是中华民族封建皇权的象征，是高贵、权势、尊荣的代名词。汉语里有许多以 “龙”为喻体的比喻。如: “没有擒龙术，不敢下深海” “龙交生龙，凤生凤”等。但在乌兹别克文化中“龙”不是吉祥的象征，代表苦难，它的样子也很可怕，是长着三个头的大蛇。乌国的人们也常常用 “她是有三个头的龙”来比喻作风很轻佻的女人。 “狗”虽然是人类最好的朋友，但汉文化当中涉及“狗”的词语多是不好的意思。比如，“狗嘴里吐不出象牙”，比喻坏人说不出好话。现在在网络语言中常常称呼还没结婚、没谈恋爱的人为 “单身狗”。“狗仗人势”比喻坏人依靠某种势力欺侮别人。乌兹别克语里“狗”大部分表示好的意义，千年以来，人民认为狗是人的最好朋友。在文学作品里常常用狗比喻忠心。如“Itdek vafodor” 意思是像狗一样忠实。其次，喻体相同，喻义相同。 “马”最早被中国人驯养的动物之一，是人类的助手。马是力量和高远的象征。 “马”常常与“龙”搭配使用，例如， “龙马精神”，比喻像龙、马一样的精 316 w www.oriens.uz November 2022 (2) Buzoqni yugurgani somonhonagacha. (牛只能跑到玉米秸场。) “牛只能跑到玉米秸场”比喻每个人能量有限，只能做到力所能及的事。中国人历来信奉 “龙”是一种神物。龙是中华民族封建皇权的象征，是高贵、权势、尊荣的代名词。汉语里有许多以 “龙”为喻体的比喻。如: “没有擒龙术，不敢下深海” “龙交生龙，凤生凤”等。但在乌兹别克文化中“龙”不是吉祥的象征，代表苦难，它的样子也很可怕，是长着三个头的大蛇。乌国的人们也常常用 “她是有三个头的龙”来比喻作风很轻佻的女人。 “狗”虽然是人类最好的朋友，但汉文化当中涉及“狗”的词语多是不好的意思。比如，“狗嘴里吐不出象牙”，比喻坏人说不出好话。现在在网络语言中常常称呼还没结婚、没谈恋爱的人为 “单身狗”。“狗仗人势”比喻坏人依靠某种势力欺侮别人。乌兹别克语里“狗”大部分表示好的意义，千年以来，人民认为狗是人的最好朋友。在文学作品里常常用狗比喻忠心。如“Itdek vafodor” 意思是像狗一样忠实。其次，喻体相同，喻义相同。 “马”最早被中国人驯养的动物之一，是人类的助手。马是力量和高远的象征。 “马”常常与“龙”搭配使用，例如， “龙马精神”，比喻像龙、马一样的精 316 w November 2022 www.oriens.uz International scientific-practical conference on the topic of “Problems and perspectives of modern technology in teaching foreign languages” VOLUME 2 \| SPECIAL ISSUE 27 ISSN 2181-1784 SJIF 2022: 5.947 \| ASI Factor = 1.7 International scientific-practical conference on the topic of “Problems and perspectives of modern technology in teaching foreign languages” 实实、勤勤恳恳工作的人。鲁迅先生有一句名言“俯首甘为孺子牛”，意思是他愿意像牛一样为老百姓服务。而乌兹别克语比喻中“牛”很少用。乌国人的印象中， “牛”是个普通动物，表示比较笨。所以即使用在比喻中也是很消极的意义。例如: 义。例如: （1）Bir tirraqi buzoq podani buzar. (一只拉肚子的牛弄脏一群牛。） “一只拉肚子的牛弄脏一群牛”比喻一个坏人会危害社会。乌兹别克语中的个比喻跟中文的 “一颗老鼠屎害了一锅汤”是一样的意思。（1）Bir tirraqi buzoq podani buzar. (一只拉肚子的牛弄脏一群牛。）（1）Bir tirraqi buzoq podani buzar. (一只拉肚子的牛弄脏一群牛。） “一只拉肚子的牛弄脏一群牛”比喻一个坏人会危害社会。乌兹别克语中的个比喻跟中文的 “一颗老鼠屎害了一锅汤”是一样的意思。 www.oriens.uz International scientific-practical conference on the topic of “Problems and perspectives of modern technology in teaching foreign languages” VOLUME 2 \| SPECIAL ISSUE 27 ISSN 2181-1784 SJIF 2022: 5.947 \| ASI Factor = 1.7 International scientific-practical conference on the topic of “Problems and perspectives of modern technology in teaching foreign languages” 神。“老马识途”比喻富有经验的人在办事。还有“马到成功”，原意是征战时战马一到便获得胜利，比喻成功迅速而顺利。 “马不停蹄”比喻一刻也不停留，一直前进。乌兹别克语当中 “马”也具备好的象征意义。几千年历史以来马算是男孩子的朋友。因为古代每个乌国的男孩儿都有自己的马，有马可以跑到远的地方。乌兹别克语里有很多关于“马”的短语。比如， “Yaxshi ot keyin chopadi”(好的马后跑)，比喻努力而有能力的人慢慢成功。“Otning o ‘rnini toy bosar”(小马崽会代替马的位置)，类似汉语的“长江后浪推前浪，一代更比一代强。” 猪也是中国人非常熟悉的动物，在农村，几乎每家都养猪，而猪贪吃懒做、身体肥胖、行动笨拙的本性也是两国人对猪的印象。汉语用“像猪一样笨” 猪也是中国人非常熟悉的动物，在农村，几乎每家都养猪，而猪贪吃懒做、身体肥胖、行动笨拙的本性也是两国人对猪的印象。汉语用“像猪一样笨” 或者“大肥猪”来形容愚蠢、肥胖的人。乌兹别克语也有一些类似的比喻。如: 做、身体肥胖、行动笨拙的本性也是两国人对猪的印象。汉语用“像猪一样笨” 或者“大肥猪”来形容愚蠢、肥胖的人。乌兹别克语也有一些类似的比喻。如: (3) Cho‘chqa shu oddiy ishni ham eplolmaysan. ( 猪，这么简单的事也办不了。) (3) Cho‘chqa shu oddiy ishni ham eplolmaysan. ( 猪，这么简单的事也办不了 ) 了。) 二、汉、乌比喻句常用的事物对比分析自然环境也影响人的日常生活，两个民族在认识与体会大自然的过程中，创造了很多比喻。自然环境中的天、太阳、月亮、星星也被赋予了丰富的喻义。第一，关于天地的喻义。中国最早提出“天人合一”，认为自然与人都遵从同一规律、同一道理。“天长地久”，像天和地存在的时间那样长，比喻情感永远不变;乌语中也有类似说法:“Osmon uzoq yer qattiq”(天长地硬)。天与地距离无穷无尽，高天和平地的差异也无穷无尽，汉语中用“天壤之别” 来比喻事物之间的差别之大;乌语中比喻这种差异用“osmon bilan yercha”(天地之差)“天”对于人类来说是无穷高，够不到的，汉语中“尾巴翘到天上去了”，用一种夸张的比喻第一，关于天地的喻义。中国最早提出“天人合一”，认为自然与人都遵从同一规律、同一道理。“天长地久”，像天和地存在的时间那样长，比喻情感永远不变;乌语中也有类似说法:“Osmon uzoq yer qattiq”(天长地硬)。天与地距离无穷无尽，高天和平地的差异也无穷无尽，汉语中用“天壤之别” 来比喻事物之间的差别之大;乌语中比喻这种差异用“osmon bilan yercha”(天地之差)“天”对于人类来说是无穷高，够不到的，汉语中“尾巴翘到天上去了”，用一种夸张的比喻 317 w November 2022 w November 2022 www.oriens.uz www.oriens.uz International scientific-practical conference on the topic of “Problems and perspectives of modern technology in teaching foreign languages” VOLUME 2 \| SPECIAL ISSUE 27 ISSN 2181-1784 SJIF 2022: 5.947 \| ASI Factor = 1.7 International scientific-practical conference on the topic of “Problems and perspectives of modern technology in teaching foreign languages” 手法，说明一个人趾高气昂的骄傲神情;乌语中则用“burni osmonda”(鼻子在天)，比喻一个人很骄傲，看不起其他人。第二，关于月亮的喻义。月亮在中国文化中含丰富的比喻意义。它是一种美的象征。中国诗词当中有很多作品用月亮来比喻美丽的女子。宋玉的《神女赋》中“其少进也，皎若明月舒其光”，就是用明月来形容神女之美。宋代词人苏轼很喜欢用月亮来比喻美人。《明月几时有》中的“人有悲欢离合，月有阴晴圆缺，此事古难全。但愿人长久，千里共婵娟”，把美女婵娟比喻成月亮，在美丽的月色下借月抒怀，思念亲人。他还在《宿望湖楼再和》中则用美人来比喻月亮: “新月如佳人，出海初弄色。”此外，汉语中的成语 “花容月貌” “闭月羞花”都是来比喻美丽的女子。乌语文化中月亮也具有美的意象，很多文学作品里把女孩儿比喻成月反。通过上述对比分析，可以看出汉乌两个民族在文化、观念等方面有共性也有个性，这些异同也反映到喻体上，由此产生了丰富的比喻义。但总体来说，汉乌比喻义中的共性较多。产生这一现象的主要原因是虽然两国地理条件不同，生活习惯也不同，但文化和思维还是有相通之处的，这就奠定了两个民族对喻体的选择、认知和联想基础。汉乌两种语言都习惯选择生活中熟悉的动物作喻体;也习惯用自然现象和条件作喻体;另外，汉乌两个民族中有很多含有比喻修辞格的熟语、成语、歇后语，而且它们的意义是相同的。如汉语的“对牛弹钢琴”与乌语的“Devorga gapirmoq”(对墙说话)，汉语的 “一颗老鼠屎害了一锅汤”与乌语的“bir tirraqi buzoq podani buzar”(一只拉肚子的牛弄脏一群牛) 等。汉语比喻修辞格的思维方式和乌语比喻的思维方式有所不同，喻体词汇包含着中国文化、思维和语言习惯。在课堂上教师培养学生汉语比喻的思维方式，同时乌兹别克汉语学习者自己也应该多重视培养自己思维方式。乌兹别克学生应该摒弃原有的思维模式，意识到汉、乌两国语言比喻修辞上的差异，并将区别对待，不应该把乌语比喻修辞格的用法想当然地套用在汉语比喻手法通过上述对比分析，可以看出汉乌两个民族在文化、观念等方面有共性也有个性，这些异同也反映到喻体上，由此产生了丰富的比喻义。但总体来说，汉乌比喻义中的共性较多。产生这一现象的主要原因是虽然两国地理条件不同，生活习惯也不同，但文化和思维还是有相通之处的，这就奠定了两个民族对喻体的选择、认知和联想基础。汉乌两种语言都习惯选择生活中熟悉的动物作喻体;也习惯用自然现象和条件作喻体;另外，汉乌两个民族中有很多含有比喻修辞格的熟语、成语、歇后语，而且它们的意义是相同的。如汉语的“对牛弹钢琴”与乌语的“Devorga gapirmoq”(对墙说话)，汉语的 “一颗老鼠屎害了一锅汤”与乌语的“bir tirraqi buzoq podani buzar”(一只拉肚子的牛弄脏一群牛) 等。汉语比喻修辞格的思维方式和乌语比喻的思维方式有所不同，喻体词汇包含着中国文化、思维和语言习惯。在课堂上教师培养学生汉语比喻的思维方式，同时乌兹别克汉语学习者自己也应该多重视培养自己思维方式。乌兹别克学生应该摒弃原有的思维模式，意识到汉、乌两国语言比喻修辞上的差异，并将区别对待，不应该把乌语比喻修辞格的用法想当然地套用在汉语比喻手法 319 w November 2022 www.oriens.uz 318 w www.oriens.uz November 2022 乌语文化中月亮也具有美的意象，很多文学作品里把女孩儿比喻成月亮，来凸显女孩儿的美丽与纯洁。 (4) Ko‘ngil olmaganim, ko‘kda hilolim. 爱你，在天空中的我的月牙。例(4)中把女孩儿比喻成“新月”(hilol)，乌语中月亮叫 oy ，而新月叫 hilol。女孩的美丽和青春，只有新月的美才配得上，所以经常把漂亮的女孩儿比喻成新月。 (5) Oyning o‘n beshi yorug ‘o ‘n beshi qorong‘u. 月亮十五天是亮的，十五天是暗的。例(5)是乌兹别克人常用来安慰别人的的比喻句。月亮每隔十五天会满月，月亮时满时缺，生活中的变化也像月亮的变化一样，有的时候会遇到幸福，有的会遇到困难，但不要灰心，好的日子会来的。第三，关于乌云的喻义。汉乌比喻句中常常出现乌云，都是比喻不好的事情或者沉重的心情。汉语中的“乌云密布”常常用来比喻人的心情很沉重、很压抑。乌语中也有类似的表达。如: 乌语文化中月亮也具有美的意象，很多文学作品里把女孩儿比喻成月亮，来凸显女孩儿的美丽与纯洁。亮，来凸显女孩儿的美丽与纯洁。 (4) Ko‘ngil olmaganim, ko‘kda hilolim. 爱你，在天空中的我的月牙。例(4)中把女孩儿比喻成“新月”(hilol)，乌语中月亮叫 oy ，而新月叫 hilol。女孩的美丽和青春，只有新月的美才配得上，所以经常把漂亮的女孩儿比喻成新月。亮，来凸显女孩儿的美丽与纯洁。 (4) Ko‘ngil olmaganim, ko‘kda hilolim. 爱你，在天空中的我的月牙。例(4)中把女孩儿比喻成“新月”(hilol)，乌语中月亮叫 oy ，而新月叫 hilol。女孩的美丽和青春，只有新月的美才配得上，所以经常把漂亮的女孩儿比喻成新月。 (4) Ko ngil olmaganim, ko kda hilolim. 爱你，在天空中的我的月牙。例(4)中把女孩儿比喻成“新月”(hilol)，乌语中月亮叫 oy ，而新月叫 hilol。女孩的美丽和青春，只有新月的美才配得上，所以经常把漂亮的女孩儿比喻成新月。 (5) Oyning o‘n beshi yorug ‘o ‘n beshi qorong‘u. 月亮十五天是亮的，十五天是暗的。 (5) Oyning o‘n beshi yorug ‘o ‘n beshi qorong‘u. 月亮十五天是亮的，十五天是暗的。是暗的。例(5)是乌兹别克人常用来安慰别人的的比喻句。月亮每隔十五天会满月，月亮时满时缺，生活中的变化也像月亮的变化一样，有的时候会遇到幸福，有的会遇到困难，但不要灰心，好的日子会来的。例(5)是乌兹别克人常用来安慰别人的的比喻句。月亮每隔十五天会满月，月亮时满时缺，生活中的变化也像月亮的变化一样，有的时候会遇到幸福，有的会遇到困难，但不要灰心，好的日子会来的。月，月亮时满时缺，生活中的变化也像月亮的变化样，有的时候会遇到幸福，有的会遇到困难，但不要灰心，好的日子会来的。福，有的会遇到困难，但不要灰心，好的日子会来的。第三，关于乌云的喻义。汉乌比喻句中常常出现乌云，都是比喻不好的事情或者沉重的心情。汉语中的“乌云密布”常常用来比喻人的心情很沉重、很压抑。乌语中也有类似的表达。如: 第三，关于乌云的喻义。汉乌比喻句中常常出现乌云，都是比喻不好的事情或者沉重的心情。汉语中的“乌云密布”常常用来比喻人的心情很沉重、很压抑。乌语中也有类似的表达。如: 318 w November 2022 w November 2022 International scientific-practical conference on the topic of “Problems and perspectives of modern technology in teaching foreign languages” VOLUME 2 \| SPECIAL ISSUE 27 ISSN 2181-1784 SJIF 2022: 5.947 \| ASI Factor = 1.7 International scientific-practical conference on the topic of “Problems and perspectives of modern technology in teaching foreign languages” VOLUME 2 \| SPECIAL ISSUE 27 ISSN 2181-1784 SJIF 2022: 5.947 \| ASI Factor = 1.7 (6) Shahar osmonida aylangan qora bulutlar tarqab, quyosh chiqa boshladi. 城市天空的黑云慢慢失去，太阳开始出来。例(6) 中“qora bulutlar”(黑云或乌云)比喻不好的事情，说明城市刚刚经历了灾难，作者又用 quyosh(太阳)来比喻光明和希望，城市的未来是光明的。第四，关于蜂蜜的喻义。蜂蜜在两种语言的比喻中都包含甜蜜的意思。汉语里常说:“生活比蜜还甜”，比喻生活像蜂蜜一样甜蜜。乌语给女孩儿起名字的时候经常使用蜂蜜，很多女孩儿名字叫 Asal(蜂蜜)，父母希望女儿的生活变蜂蜜一样甜蜜。蜂蜜特别甜，但吃得多对身体不好，所以乌兹别克人说 “Asalning ozi shirin”(蜂蜜少才会甜)，比喻喜欢的东西也不要贪多，否则物极必反 (6) Shahar osmonida aylangan qora bulutlar tarqab, quyosh chiqa boshladi. 城反。 64~67。 www.oriens.uz International scientific-practical conference on the topic of “Problems and perspectives of modern technology in teaching foreign languages” VOLUME 2 \| SPECIAL ISSUE 2 ISSN 2181-1784 SJIF 2022: 5.947 \| ASI Factor = 1 上。多读一些汉语经典散文与小说，从中学习比喻修辞格，要去深入思考，与乌语比喻进行对比分析。中乌两国有历史悠久的文化交流，两国之间在历史文化、社会政治、语言思维等方面都具有共同点，但毕竟汉语与乌语是两个独立的语言，由于地理、历史、民族文化不同，选择的喻体也不尽相同。因此教学比喻时，教师应从跨文化交际的角度下给学生讲解相关的文化背景知识。如 “你真牛”这句， “牛” 是具有中国独有的文化，乌兹别克语没有这种说法和理解，对乌兹别克人来说牛是笨的动物。如果教师不解释清楚，学生就无法理解这比喻的意思。 International scientific-practical conference on the topic of “Problems and perspectives of modern technology in teaching foreign languages” VOLUME 2 \| SPECIAL ISSUE 27 ISSN 2181-1784 SJIF 2022: 5.947 \| ASI Factor = 1.7 上。多读一些汉语经典散文与小说，从中学习比喻修辞格，要去深入思考，与 International scientific-practical conference on the topic of “Problems and perspectives of modern technology in teaching foreign languages” 中乌两国有历史悠久的文化交流，两国之间在历史文化、社会政治、语言思维等方面都具有共同点，但毕竟汉语与乌语是两个独立的语言，由于地理、历史、民族文化不同，选择的喻体也不尽相同。因此教学比喻时，教师应从跨文化交际的角度下给学生讲解相关的文化背景知识。如 “你真牛”这句， “牛” 是具有中国独有的文化，乌兹别克语没有这种说法和理解，对乌兹别克人来说牛是笨的动物。如果教师不解释清楚，学生就无法理解这比喻的意思。参考文献 1. 曹铁根.汉语比喻与文化[J].湖北师范学报,2003,23(4): 117~121。 2. 陈汝东.对外汉语修辞学[M].广西教育出版社, 2000. 3. 冯广艺.汉语比喻理论的基本特征 [J].黄冈师范学院学报, 2000,20(1); 67。 4. Миртожиев Миразиз. Ўзбек тилида полисемия [M]-Т: Фан. 1975。 5. Мукаррамов М. Ўзбек тилида ўхшатиш [M] Фан. Тошкент: 1976。 320 w November 2022 w November 2022 www.oriens.uz
https://openalex.org/W2264416382	https://www.epj-conferences.org/articles/epjconf/pdf/2016/01/epjconf-ISRD2015_03001.pdf	English	null	Comparison of Analysis Results Between 2D/1D Synthesis and RAPTOR-M3G in the Korea Standard Nuclear Plant (KSNP)	EPJ web of conferences	2,016	cc-by	2,414	Comparison of Analysis Results Between 2D/1D Synthesis and RAPTOR-M3G in the Korea Standard Nuclear Plant (KSNP) Mi Joung Lim1,a, Young Jae Maeng1, Arnold H. Fero2, and Stanwood L. Anderson2 1 Korea Reactor Integrity Surveillance Technology, 168 Gajeong- ro, Yuseong-gu Daejeon, Korea 2 Westinghouse Electric Company LLC, 1000 Westinghouse Drive, Cranberry Township, Pennsylvania 16066-5228, USA Abstract. The 2D/1D synthesis methodology has been used to calculate the fast neutron (E > 1.0 MeV) exposure to the beltline region of the reactor pressure vessel. This method uses the DORT 3.1 discrete ordinates code and the BUGLE-96 cross-section library based on ENDF/B-VI. RAPTOR-M3G (RApid Parallel Transport Of Radiation-Multiple 3D Geometries) which performs full 3D calculations was developed and is based on domain decomposition algorithms, where the spatial and angular domains are allocated and processed on multi-processor computer architecture. As compared to traditional single- processor applications, this approach reduces the computational load as well as the memory requirement per processor. Both methods are applied to surveillance test results for the Korea Standard Nuclear Plant (KSNP)-OPR (Optimized Power Reactor) 1000 MW. The objective of this paper is to compare the results of the KSNP surveillance program between 2D/1D synthesis and RAPTOR-M3G. Each operating KSNP has a reactor vessel surveillance program consisting of six surveillance capsules located between the core and the reactor vessel in the downcomer region near the reactor vessel wall. In addition to the In- Vessel surveillance program, an Ex-Vessel Neutron Dosimetry (EVND) program has been implemented. In order to estimate surveillance test results, cycle-speciﬁc forward transport calculations were performed by 2D/1D synthesis and by RAPTOR-M3G. The ratio between measured and calculated (M/C) reaction rates will be discussed. The current plan is to install an EVND system in all of the Korea PWRs including the new reactor type, APR (Advanced Power Reactor) 1400 MW. This work will play an important role in establishing a KSNP-speciﬁc database of surveillance test results and will employ RAPTOR-M3G for surveillance dosimetry location as well as positions in the KSNP reactor vessel. a Corresponding author: mjlim@krist.co.kr EPJ Web of Conferences 106, 03001 (2016) DOI: 10.1051/epjconf/201610603001 C ⃝Owned by the authors, published by EDP Sciences, 2016 EPJ Web of Conferences 106, 03001 (2016) DOI: 10.1051/epjconf/201610603001 C ⃝Owned by the authors, published by EDP Sciences, 2016 This is an Open Access article distributed under the terms of the Creative Commons Attribution License 2.0, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 3. Transport Calculation Method Discussion Traditional 2D and 1D Synthesis method uses 2D and 1D transport solutions in order to obtain 3D neutron ﬂux distribution (Eq. (1)) as described in US Regulatory Guide 1.190 [2] : (r, , z) = (r, ) • (r, z) (r) · (1) (1) Where, (r, , z) is the synthesized 3D neutron ﬂux distribution, (r, ) is the 2D transport solution in r, geometry, (r, z) is the 2D solution for a cylindrical reactor model using the actual axial core power distribution, and (r) is the 1D solution for a cylindrical reactor model using the same source per unit height as that used in the r, 2D calculation. g RAPTOR-M3G (RApid Parallel Transport Of Radiation – Multiple 3D Geometries) is a parallel, deterministic radiation transport code developed by Westinghouse. The plant-speciﬁc 2D/1D and 3D geometric models are shown in Figs. 1, 2, and 3. 2. Background The Code of Federal Regulations (CFRs), Title 10, Part 50, Appendix H [1], requires that neutron dosimetry be present to monitor the reactor vessel throughout plant life and that material specimens be used to measure damage associated with the end-of-life fast neutron exposure of the reactor vessel. Currently, Ex-Vessel Neutron Dosimetry (EVND) sets are installed in 16 operating pressurized water reactors (PWRs) in Korea. Among the 16 PWRs, Hanbit (HB) Units 3, 4, 5, & 6 and Hanul (HU) Units 3, 4, 5, & 6 are Korea Standard Nuclear Plants (KSNPs). The EVND Program has been designed primarily to meet the code and to provide a long term monitoring of fast neutron exposure distributions within the reactor vessel wall that could experience signiﬁcant radiation induced increases in reference nil ductility transition temperature (RTNDT) over the service lifetime of the plant. 1. Introduction This paper presents comparisons of analysis results between 2D/1D synthesis and RAPTOR-M3G in the Korea Standard Nuclear Plant (KSNP). The KSNP is similar to the Combustion Engineering (CE) design plants where the reactor was supplied by CE and DOOSAN. Each of these operating reactors Article available at http://www.epj-conferences.org or http://dx.doi.org/10.1051/epjconf/201610603001 EPJ Web of Conferences has a reactor vessel surveillance program consisting of six surveillance capsules located between the barrel and the reactor vessel in the downcomer region near the reactor vessel inner-wall (In-Vessel). A comprehensive Ex-Vessel Neutron Dosimetry program is also implemented in all KSNP (OPR- 1000). The six surveillance capsules are located at the following azimuthal angels: 83◦, 97◦, 104◦, 263◦, 277◦, and 284◦. Those six positions correspond to ﬁrst-octant-equivalent angles of 7◦and 14◦. Ex-Vessel Neutron Dosimetry capsules located at angles of 0◦, 7◦, 14◦, and 45◦. The reasons for that positioning are to locate dosimetry at angles that correspond to the angles of the surveillance capsules (7 and 14 degrees). g To analyze In-Vessel and Ex-Vessel surveillance programs, traditional two dimensional (2D) and 1D synthesis methodologies have been widely applied to evaluate the fast neutron (E > 1.0 MeV) ﬂuence exposure to the reactor vessel. Full 3D calculations using RAPTOR-M3G are critical for KSNP EVND evaluation due to the complexity of the cavity which is ex-core detector resin canister and RPV support column. 4. Comparisons of Calculation and Measurement Results The neutron dosimetry capsule sensor reactions combined provide a means to measure the fast neutron energy spectrum inside the reactor structures and in the reactor cavity. The measurements were done by gamma-ray spectrometry using high resolution HPGe detectors. The measured speciﬁc activities of these reactions are converted to the reaction rates and then compared to the calculated. In addition to the six Ex-Vessel Neutron Dosimetry capsules that have been withdrawn for this analysis, the previously 03001-p.2 03001-p.2 15th ISRD ore Midplane Geometry R- and Axial Geometry R-Z of 2D/1D Synthesis model. 15th ISRD 15th ISRD Figure 1. Core Midplane Geometry R- and Axial Geometry R-Z of 2D/1D Synthesis model. Figure 1 Core Midplane Geometry R and Axial Geometry R Z of 2D/1D Synthesis model Figure 1. Core Midplane Geometry R- and Axial Geometry R-Z of 2D/1D Synthesis model. Figure 1. Core Midplane Geometry R- and Axial Geometry R-Z of 2D/1D Synthesis model. 03001-p.3 EPJ Web of Conferences Midplane Geometry R- and Axial Geometry R-Z of RAPTOR-M3G model. EPJ Web of Conferences EPJ Web of Conferences EPJ Web of Conferences Figure 2. Core Midplane Geometry R- and Axial Geometry R-Z of RAPTOR-M3G model. 03001-p.4 15th ISRD Figure 3. Geometry and Mesh of RAPTOR-M3G model. Figure 3. Geometry and Mesh of RAPTOR-M3G model. withdrawn in-vessel surveillance capsule dosimetry sensors have been re-analyzed using in order to provide more measurement data points to validate the transport calculation model. The dosimetry calculations were performed with the RadTrackTM [3] Code System. A plant- and cycle-speciﬁc library of ﬂux data is constructed within RadTrack using radial and axial power distributions, fuel design speciﬁcations, system pressure and temperatures. g p y p p Comparisons of the measurement results from each of previously withdrawn in-vessel and six ex-vessel sensor set irradiations with corresponding analytical predictions were used to demonstrate compliance with the requirements of Regulatory Guide 1.190. The total database summary of the measurement-to-calculation (M/C) comparisons based on the individual sensor reactions with the least- squares adjustment procedure [4] are summarized in Tables 1 to 4 for reactions sensitive in the fast energy range. Uncertainty was calculated by the standard deviation in the average. These 2D-Calculation data comparisons show similar and consistent results with the linear average M/C ratio of 0.94 to 1.09 for in-vessel and 0.96 to 1.10 for ex-vessel in good agreement. 4. Comparisons of Calculation and Measurement Results The comparisons demonstrate that the ±20% (1) for in-vessel and ±30% for ex-vessel agreement between calculation and measurement required by Regulatory Guide 1.190. These 3D-Calculation data comparisons show similar and consistent results with the linear average M/C ratio of 0.97 to 1.08 for in-vessel and 0.96 to 1.07 for ex-vessel in good agreement. The comparisons demonstrate that the ±20% (1) for in-vessel and ±30% for ex-vessel agreement between calculation and measurement required by Regulatory Guide 1.190. 03001-p.5 EPJ Web of Conferences Table 1. Summary of Measurement to 2D-Calculation for In-Vessel of KSNP. Reaction HB3* HB4* HB5 HB6 HU3 HU4 HU5 HU6 63Cu(n,)60Co 1.07 1.10 1.03 1.06 1.16 1.05 1.13 – 46Ti(n,p)46Sc 1.09 1.03 0.97 1.02 1.10 1.00 1.08 1.17 54Fe(n,p)54Mn 0.97 0.93 0.89 0.91 0.95 0.92 0.95 1.07 58Ni(n,p)58Co 0.98 0.91 0.88 0.91 0.95 0.93 0.91 1.02 Average 1.03 0.99 0.94 0.98 1.04 0.98 1.02 1.09 % Standard Deviation 6.3 9.2 7.5 7.9 10.3 6.3 10.3 7.0 * Included 2nd Surveillance Test Results. Table 1. Summary of Measurement to 2D-Calculation for In-Vessel of KSNP. Table 2. Summary of Measurement to 2D-Calculation for Ex-Vessel of KSNP. Reaction HB3 HB4 HB5 HB6 HU3 HU4 HU5 HU6 63Cu(n,)60Co 1.02 0.99 1.01 1.01 0.99 0.97 0.95 1.08 46Ti(n,p)46Sc 1.01 0.98 1.00 0.98 0.97 0.93 0.93 1.04 54Fe(n,p)54Mn 1.09 1.04 1.07 1.05 1.04 1.02 0.98 1.13 58Ni(n,p)58Co 1.03 0.96 1.01 0.99 0.98 0.94 0.91 1.05 238U(n,f)137Cs 1.14 0.98 1.16 1.11 1.08 1.07 0.97 1.15 237Np(n,f)137Cs 1.15 1.02 1.23 1.20 1.19 1.18 1.03 1.15 Average 1.07 1.00 1.08 1.06 1.04 1.02 0.96 1.10 % Standard Deviation 5.7 3.0 9.0 8.1 8.1 9.5 4.5 4.5 Note: Ex-Vessel data included mid-plane capsule. Table 3. Summary of Measurement to 3D-Calculation for In-Vessel of KSNP. Table 3. Summary of Measurement to 3D-Calculation for In-Vessel of KSNP. y Reaction HB3* HB4* HB5 HB6 HU3 HU4 HU5 HU6 63Cu(n,)60Co 1.10 1.12 1.04 1.08 1.17 1.07 1.14 – 46Ti(n,p)46Sc 1.14 1.08 0.99 1.05 1.12 1.05 1.10 1.13 54Fe(n,p)54Mn 1.03 0.98 0.93 0.96 0.99 0.98 0.99 1.07 58Ni(n,p)58Co 1.04 0.97 0.93 0.96 0.99 1.00 0.95 1.01 Average 1.08 1.04 0.97 1.01 1.07 1.03 1.05 1.07 % Standard Deviation 4.8 7.1 5.5 6.1 8.6 4.1 8.6 5.6 *Included 2nd Surveillance Test Results. 4. Summary of Measurement to 3D-Calculation for Ex-Vessel of KSNP. Table 4. Summary of Measurement to 3D-Calculation for Ex-Vessel of KSNP. 4. Comparisons of Calculation and Measurement Results Reaction HB3 HB4 HB5 HB6 HU3 HU4 HU5 HU6 63Cu(n,)60Co 1.02 0.99 1.00 1.00 0.99 0.97 0.95 1.07 46Ti(n,p)46Sc 1.01 0.98 0.99 0.98 0.97 0.92 0.93 1.03 54Fe(n,p)54Mn 1.08 1.03 1.06 1.04 1.03 1.00 0.97 1.10 58Ni(n,p)58Co 1.02 0.95 1.00 0.98 0.97 0.92 0.91 1.03 238U(n,f)137Cs 1.12 0.96 1.15 1.12 1.07 1.05 0.96 1.13 237Np(n,f)137Cs 1.14 1.01 1.22 1.19 1.19 1.17 1.02 1.14 Average 1.07 0.99 1.07 1.05 1.04 1.00 0.96 1.08 % Standard Deviation 5.3 3.1 8.9 8.3 8.2 9.3 4.2 4.5 Note: Ex-Vessel data included mid-plane capsule. ART = Initial RTNDT + RTNDT + Margin. (2) 03001 p 6 5. 3D Transport versus 2D/1D Synthesis Results Discussion Table 5 presents a summary of the comparison of the Adjusted Reference Temperature (ART) result between 2D/1D synthesis and RAPTOR-M3G. According to Regulatory Guide 1.99 Revision 2[5], the adjusted reference temperature(ART) for each material in the beltline is given by the following expression: ART = Initial RTNDT + RTNDT + Margin. (2) (2) 03001-p.6 15th ISRD Table 5. Comparison of ART Results between 2D/1D Synthesis and RAPTOR-M3G. Table 5. Comparison of ART Results between 2D/1D Synthesis and RAPTOR-M3G. p y ART ◦F (EOL) HB3 HB4 HB5 HB6 HU3 HU4 HU5 HU6 2D/1D Synthesis 74.2 54.5 11.2 41.0 83.2 33.2 24.2 32.6 3D Transport 73.2 53.9 10.6 40.6 82.6 32.4 23.7 32.0 Initial RTNDT is the reference temperature for the unirradiated materials as deﬁned in Paragraph NB- 2331 of Section III of the ASME Boiler and Pressure Vessel Code. RTNDT is the mean value of the adjustment in reference temperature caused by irradiation and should be calculated as follows: RTNDT = (CF) f(0.28−0.10 log f) (3) (3) CF(◦F) is the chemistry factor, a function of copper and nickel content. CF is given in Regulatory Guide 1.99 Revision 2 or the mean of the measured values. The neutron ﬂuence at any depth in the vessel wall, f(1019 n/cm2, E > 1 MeV), is determined as follows: f = fsurf(e−0.24x) (4) (4) where fsurf(1019 n/cm2, E > 1 MeV ) is the calculated value of the neutron ﬂuence at the inner wetted surface of the vessel at the location of the postulated defect, and x(in inches) is the depth in the vessel wall measured from the vessel inner surface. The depth is 1/4 T and 3/4 T locations in the pressure vessel wall. Margin is the quantity, ◦F, that is to be added to obtain conservative, upper-bound values of adjusted reference temperature for the calculations required by Appendix G to 10 CFR Part 50. ART of 2D/1D synthesis method show higher than RAPTOR-M3G at End of Life(32EFPY). The reason is that 2D/1D synthesis method has excessive conservatism because of having just one model of R- and R-Z separately. RAPTOR-M3G performing 3D calculation can be applied to various reactor structures, because the code can simulate the model realistically and reasonably in geometric view points. The difference of ART Results between 2D/1D Synthesis and RAPTOR-M3G is less than 5%, both method has a good assessment. References [1] The Code of Federal Regulations (CFRs), Title 10, Part 50, Appendix H, “Reactor Vessel Material Surveillance Program Requirements,” US NRC [1] The Code of Federal Regulations (CFRs), Title 10, Part 50, Appendix H, “Reactor Vessel Material Surveillance Program Requirements,” US NRC [2] US NRC Regulatory Guide 1.190, “Calculational and Dosimetry Methods for Determining Pressure Vessel Neutron Fluence” [3] G. A. Fischer, “RadTrack: An Automated Tool for Tracking the Radiation Environment in LWRS, Version 1.1.” WCAP-16558-NP, Revision 1, Westinghouse Electric Company LLC (2008) [4] S. L. Anderson, “Benchmark Testing of the FERRET Code for Least Squares Evaluation of Light Water Reactor Dosimetry,” WCAP-16558-NP-A, Revision 0, Westinghouse Electric Company LLC (May 2006) [4] S. L. Anderson, “Benchmark Testing of the FERRET Code for Least Squares Evaluation of Light Water Reactor Dosimetry,” WCAP-16558-NP-A, Revision 0, Westinghouse Electric Company LLC (May 2006) [5] Regulatory Guide 1.99, Revision 2, “Radiation Embrittlement of Reactor Vessel Materials,” (May 1988) [5] Regulatory Guide 1.99, Revision 2, “Radiation Embrittlement of Reactor Vessel Materials,” (May 1988) [6] LTR-REA-14-28, Revision 0, “KRIST/Westinghouse Training Supplemental Information” Attachment 2, “Using Tecplot 360 to Visualize the Results of Transport Analysis” (February 2014) 03001-p.7
W2327444307.txt	https://symbiosisonlinepublishing.com/nanoscience-technology/nanoscience-technology12.pdf	en		Ultra-stable dye-filled polytetrafluoroethylene thin films	Nanoscience & technology	2,014	cc-by	2,566	www.symbiosisonline.org www.symbiosisonlinepublishing.com Symbiosis Research Article Nanoscience & Technology: Open Access Open Access Ultra-stable dye-filled polytetrafluoroethylene thin films Kostyantyn Grytsenko1, Sigurd Schrader2 and Heiner Detert3 1 Institute of Semiconductor Physics, pr. Nauki 41, Kyiv, 03650, Ukraine, University of Applied Sciences Wildau, Bahnhofstrasse, 15745, Germany 3 Institute of Organic Chemistry, J.Gutenberg University, Duesbergweg10-14, D-55099, Mainz, Germany 2 Received: March 18, 2014; Accepted: May 26, 2014; Published: May 28, 2014 Corresponding author: Kostyantyn Grytsenko, Institute of Semiconductor Physics, pr. Nauki 41, Kyiv, 03650, Ukraine; email: d.grytsenko@gmail.com Abstract Polytetrafluoroethylene (PTFE) and polyparaphenylene sulphide (PPS) thin films filled with molecules of different dyes were obtained by co-deposition in vacuum. The PTFE matrix prevented aggregation of dye molecules more strongly, than PPS matrix. During heating PTFE matrix kept the dye molecules in not aggregated state up to dye decomposition temperature. Dyes of all types, buried in the PTFE matrix, exhibited high stability toward actions of external factors: light, temperature, acids. Keywords: Dye; PTF; PPS; Nanocomposite; Thin film; Optical spectra; Stability Introduction Recent years revealed breakthrough in the industrial applications of the thin organic functional films. The devices include multi layers with both active and passive layers consisted of the small organic molecules (dyes) and/or polymers. As a rule, the dye solid films are not as stable as necessary for industrial application. To enhance stability of the film optical properties, dye molecules are introduced into polymer matrix by dissolution in solvent and then film is produced by spin coating and other wet techniques. Various sensors made from the organic thin films are described in literature, for example, in the reviews [1,2]. Novel research in this field are concerned not only dye-in-polymer film [3], but three component nanocomposite film as well [4]. In many cases this is a good method to prevent dye molecule aggregation and keep film with necessary optical properties for a long time. Nevertheless, for advanced industrial applications the more stability of the film optical properties is necessary. Diaminonitro-stillbene filled CYTOP films were made by means of laser evaporation in vacuum for the use in electro-optic devices [5]. But with organic compound concentration increase the crystal domains were appeared in the film together with amorphous phase. The matrix material or deposition method gave the film with not good quality. Here we report the one way for the significant improvement of the stability of the dye molecules to action of the environmental factors. Method is based on the simultaneous evaporation and co-deposition on the substrate in vacuum of the polytetrafluoroethylene (PTFE) and dye. This is possible due to utilization of the unique method of the PTFE film Symbiosis Group deposition in vacuum. PTFE pellets are heated to decomposition temperature in the crucible. Pellets emit volatile gasses, which undergo activation by electron cloud created just above crucible [6-10]. In such deposition method the energetic species able to destroy dye molecules in the gas phase and on the substrate during film growth are absent. PPS and PTFE thin films, filled with dye molecules of various kinds at different concentrations were obtained, their optical properties were studied during light, solvent, temperature actions. The paper describes behaviour of the dyes in thin film polymer matrices during external actions. Materials and Methods PTFE pellets were used. PPS was presented by Chevron Phillips Chemical Company Ryton® Business Unit. Phthalocyanine (Pc), polymethine and squaraine (SQ) dyes were synthesized in the Institute of Organic Chemistry of National Academy of Sciences of Ukraine, photocromic compounds were given by Photochemistry Center, Moscow, Russian Federation, pyrazine type (St) dyes were presented by Institute of Organic Chemistry of Mainz University, Germany. Films were deposited using UVN-74 installation equipped with computerised control system, including Pfeiffer pressure meter, optical spectrometer StellarNet and Sigma quartz thickness monitor. Starting pressure in the chamber was 10- 3 Pa. Thin PTFE films were deposited in vacuum by the decomposition of the bulk PTFE pellets in the heated crucible and activation of the emitted gasses with cloud of accelerated electrons, localised just at the crucible outlet. The PTFE evaporation scheme is presented at Figure 1. It was shown that the treatment with electron cloud led to significant changes of the emitted gasses composition. After condensation the secondary PTFE was characterized with mainly linear macromolecular chains and amorphous structure. More details can be found elsewhere [6-10]. Dye was evaporated from thermally heated crucible. The PPS films were deposited by thermal decomposition of PPS powder in the heated crucible. Glass substrates were used. The film thickness was in the 50 - 100 nm range. *Corresponding author email: d.grytsenko@gmail.com Ultra-stable dye-filled polytetrafluoroethylene thin films Copyright: © 2014 Grytsenko et al. filled PTFE films was shown using well known stable Pc and SQ dyes [12-14]. It was suggested, that dye formed separate phase in the PTFE matrix. The suppression of the dye aggregation and transition to high temperature phase was proportional to dye concentration decrease. Annealing led to a higher order inside the dye nanoparticles (NPs), but not to NPs size increase. The PTFE matrix restricts molecular migration and NPs growth. Variation of the SQ dye side groups led to the different type of aggregation in the SQ-filled PTFE film, which is also dependent on dye concentration [12,13]. At small dye concentration and high growth rate, the separate dye molecules were frozen in the PTFE matrix. At higher dye concentration molecules formed the NPs in the PTFE matrix with the structure, equilibrium at the nano-size. Figure 1: Scheme of the PTFE film deposition: 1 – glass substrate, 2 – hot tungsten wire, 3 – metal crucible, 4 – its holder, 5 – PTFE pellets, 6 – shield, 7 – cloud of electrons. The measurements of photostationary spectra, kinetics of photocoloration, photobleaching, spontaneous relaxation and photo degradation were carried out using the Ocean Optics spectrometer [11]. The absorption and luminescence spectra of the PTFE and PPS films, filled with acidochromic dye 1 or with squaraine 3 (Figure 2), were measured after immersion of the (A-initial) films in air saturated with dichloromethane (B), followed by methanol (C), followed by acetic acid (D) and finally trifluoroacetic acid (E). All immersions in vapours were about 15 minutes. Polymer films, filled with squaraine 2 were used for studies of the films behaviour during heating. Optical spectrometer Polytec was used for spectra recording in situ during film heating in air in home-made oven. Results and Discussion At first stage of the research the probability to obtain dye- H3C N The next research was made testing various dyes with different physical chemical effects used to induce optical changes in the films for possible industrial utilization [15,16]. Photochromic thin films were prepared by co-deposition of PTFE and spirocompounds [11]. The irradiation of the film with ultraviolet light led to transition of initial transparent spiro form to merocyanine form with absorption in visible region. It was found certain differences of photochromic transformation of spiropyrans in solution and in composite films deposited in vacuum. The observed independence of the position and a shape of the absorption band of the photoinduced merocyanine form from concentration of photochromic compounds in PTFE film testified the absence of the well known aggregation for spirocompound molecules. Composite films revealed high stability toward photodegradation and irreversible photochemical transformations. The stability of the photoinduced form is important for preparation of photochromic media for three-dimensional optical memory [11]. The dye filled PTFE film was tested for thermal lithography by focused laser beam with 405 nm wavelength. The round shaped holes were obtained with their diameter far smaller than diameter of laser beam, due to high melting temperature and N H3C CH3 N N CH3 1 2 3 Figure 2: Formula of compounds used for co-deposition with polymers: 1 - 2,5-(-4-dimethylaminostyryl)-pyrazine (St); 2 – R= CH3, 2,4-Bis-(4-dimethylamino-2-hydroxyphenyl)cyylobutane-1,3-dione (SQ 2); 3 - R= C4H7, (E,E)-bis[4-(dibutylamino)-2,6-dihydroxyphenyl]squaraine (SQ 3). Citation: Grytsenko K, Schrader S, Detert H (2014) Ultra-stable dye-filled polytetrafluoroethylene thin films. Nanosci Technol 1(2): 1-5. Page 2 of 5 Ultra-stable dye-filled polytetrafluoroethylene thin films Copyright: © 2014 Grytsenko et al. Figure 3: Optical spectra changes of three-component films after vapor treatment: 1 – absorption and 2 – luminescence of the St+PPS film; 3 – absorption and 4 – luminescence of the St+Au+PTFE film; 5 - absorption and 6 – luminescence of the St+As2S3+PTFE film. Citation: Grytsenko K, Schrader S, Detert H (2014) Ultra-stable dye-filled polytetrafluoroethylene thin films. Nanosci Technol 1(2): 1-5. Page 3 of 5 Ultra-stable dye-filled polytetrafluoroethylene thin films Copyright: © 2014 Grytsenko et al. Figure 4: Evolution of the optical spectra of SQ-filled polymer films during heating: 1 – pure SQ film; 2 – SQ in PPS; 3 – SQ in PTFE. high viscosity of the PTFE melt. The film is useful for super high density optical memory [15]. Two component systems of all type of dyes in the PTFE showed no any absorption or luminescence changes in all vapors (B-E) even at high dye load. But the same dyes showed optical changes while in another polymer, for example, SQ 3 [16]. At low dye load the absence of the optical change can be explained by steric factor, but at high (30%) concentration of the dye in the film the result seems very strange. The same all dyes but in PPS matrix revealed changes in absorption and luminescence spectra under the same conditions. The next experiments were made using PTFE matrix filled with both dye and inorganic compounds. In comparison with dye-in-PTFE systems, three component films with inorganic and dye nanoparticles (NPs) in PTFE matrix are sensitive toward vapors. Figure3 presents the absorption and luminescence spectra of the films after vapors action. St+Au+PTFE film revealed changes in absorption spectrum, luminescence was suppressed. St+As2S3+PTFE film revealed no absorption change, but detectable luminescence change. These results show, that three component organic-inorganic thin film with PTFE matrix can be used for aggressive vapor optical detection and recognition. The experiments showed, that the PTFE even in thin film form has strong ability to stabilize the dyes incorporated in it toward all environmental factors. It is hardly possible to explain the results as the sum of the dye and PTFE properties in the thin nanocomposite film. The diffusion of vapors and gases should take a few second through film with thickness of several tens of nanometers. The inorganic NPs can absorb gaseous molecules by such a way supply them into PTFE film toward dye NPs. The PTFE macromolecules deposited by developed method contained electric charge [17]. Perhaps this charge does not allow molecules of the gasses penetrate into film bulk. But what the mechanism of suppression of phototransformations of the spirocompound molecules in charged PTFE environment is not clear. The discovered experimental results deserve farther studies for both scientific understanding and for industrial applications. The thermal stability of the different polymethine dyes in PTFE matrix was studied in comparison with the properties of Citation: Grytsenko K, Schrader S, Detert H (2014) Ultra-stable dye-filled polytetrafluoroethylene thin films. Nanosci Technol 1(2): 1-5. Page 4 of 5 Ultra-stable dye-filled polytetrafluoroethylene thin films the same dyes in the PPS matrix. Figure4 presents evolution of the spectra of the SQ 2 -filled polymer films with dye concentration in the range 10-15 vol. % during heating. The effect of the polymer matrix on the film optical properties changes during heating is clearly seen. It should be mentioned, that SQ optical band splitting or widening in as-deposited PPS matrix are stronger than in as-deposited PTFE matrix. The SQ molecules aggregation in PPS matrix decomposed at the temperature close to dye decomposition itself [18]. At the temperature at which SQ-filled PPS films became transparent, dye-filled PTFE films still revealed some optical absorption. The PTFE matrix kept the SQ molecules in the not aggregated state up to SQ decomposition temperature. The PTFE film filled with SQ exhibited change of 650 nm absorption band into 450 nm band. Film was not completely transparent up to 2800C. The effect can be explained by SQ molecule decomposition, while the decomposition products were kept in the PTFE matrix. Conclusions New nanocomposite thin film material was produced by codeposition of PTFE and dye in vacuum. The films revealed unique stability of the optical properties of the buried dye nanoparticles under almost all environmental actions. The nanocomposite films are perspective for various photonic applications. Acknowledgments Thanks for the financial support with STCU 5709 Grant and Project 5709-2 funded by National Academy of Sciences of Ukraine. Thanks to DAAD for supply of the Polytec spectrometer References 1. Basabe-Desmonts L, Reinhoudt DN, Crego-Calama M (2007) Design of fluorescent materials for chemical sensing. Chem Soc Rev 36(6): 993–1017. 2. Korostynska O, Arshak K, Gill E, Arshak A (2007) Review on State-of-theart in Polymer Based pH Sensors. Sensors 7: 3027-3042 . 3. Mathew RJ, Raj S, Dhesingh RS (2013) PMMA-based Stimuli-responsive Flexible Thin-films. Chemistry Letters 42(11): 1360-1362. 4. Sarkisov SS, Czarick M, Fairchild BD, Liang Y, Kukhtareva T et al. (2013) Colorimetric polymer-metal nanocomposite sensor of ammonia for the agricultural industry of confined animal feeding operations. Opt Eng 53(2): 021107. 5. Kannari F, Fujii T, Shima H, Matsumoto N (1996) Electrical and optical characteristics of organic thin films fabricated by laser ablation. Appl Surf Sci 96-98: 625-629. Copyright: © 2014 Grytsenko et al. 6. Gritsenk KP, Khimchenko Yu I, Lantoukh GV (1987) Deposition of barrier sublayer of optical carrier by evaporation of polytetrafluoroethylene in vacuum with ionisation of degradation products. Optical recording information, Naukova Dumka, Kyiv, Ukraine. 85-88. 7. Gritsenko K (1991) Mechanism of PTFE film growth in vacuum. Ukrainian Chem J 57(7): 782-784. 8. Gritsenko KP Krasovsky AM (2003) Thin film deposition of polymers by vacuum degradation. Chem Rev 103( 9): 3607-3649. 9. Gritsenko KP, Capobianchi A, Convertino A, Friedrich J, Schulze RD et al. (2005)Polymer-metal composite thin film prepared by co-evaporation in vacuum. In (Eds.) Iwamori S Polymer surface modification and polymer coatings by dry process technologies, Research Signpost, Kerala. Pp: 85109. 10. Grytsenko KP (2008) Growth mechanism, properties and applications of vacuum-deposited PTFE films. Russian J. Chem. Soc L II (3): 112123. 11. Grytsenko KP, Machulin VF, Ait AO, Gorelik AM, Kobeleva OI et al. (2010) Photochromic films prepared by vacuum сo-deposition of polymer and spiropyrans. Optical Memory and Neural Networks 19(3): 254–259. 12. Gritsenko KP (1995) Laser radiation action on phthalocyanine-filled PTFE film. Thes. Conference “Polymer composites-95”, Soligorsk 6566. 13. Gritsenko KP, Dimitriev OP, Kislyk VV, Getsko OM, Schrader S et al. (2002) Dye-fluoropolymer nanocomposite film deposition in vacuum”. Colloids & Surf A Physchem Eng Aspects 198-200: 625-632. 14. Gritsenko KP, Dimitriev OP, Grinko DO, Schrader S, Brehmer L (2003) Dye-filled polymer film formation in a vacuum. Proc SPIE 4833: 487-492. 15. Grytsenko, K., Lytvyn, P., Navozenko, O., Kolomzarov, Yu., Briks, J., Kurdukov, V., Slominskii, Yu., Tolmachev, O., Ksianzou, V., Schrader, S. “Evaporable dyes with absorption in blue region”, Conference Nanotechnology and nanomaterials, August 29 –– September 1, 2013, Bukovel, Abstr. Book: p.183. 16. Balbo Block, M. A. and Hecht S. (2004). Alternating (Squaraine-Receptor) Sensory Polymers: Modular One-Pot Synthesis and Signal Transduction via Conformationally Controlled Exciton Interaction, Macromolecules, 37: 4761-4769. 17. Grytsenko K, Kolomzarov Yu, Lytvyn P, Beyer H, Ksenzou V, et al. (2011) Effect of magnetic field on film formation by means of polytetrafluoroethylene decomposition in vacuum. Materials Technolog Tools 16(4): 56-59. 18. Grytsenko K, Navozenko O, Kolomzarov Yu, Kryuchin A, Tolmachev O ey al. (2012) Optical properties of dye-filled polymer films, deposited in vacuum. Data Record Storage & Proc 14(1): 3-9. Citation: Grytsenko K, Schrader S, Detert H (2014) Ultra-stable dye-filled polytetrafluoroethylene thin films. Nanosci Technol 1(2): 1-5. Page 5 of 5
https://openalex.org/W4285447897	https://www.researchsquare.com/article/rs-639164/latest.pdf	English	null	Avoiding the “Great Filter”: A Projected Timeframe for Human Expansion Off-World	Research Square (Research Square)	2,021	cc-by	8,101	Avoiding the “Great Filter”: A Projected Timeframe for Human Expansion Off-World Jonathan Jiang (  Jonathan.H.Jiang@jpl.nasa.gov ) California Institute of Technology https://orcid.org/0000-0002-5929-8951 Philip Rosen Retired Kristen Fahy Jet Propulsion Laboratory Physical Sciences - Article Keywords: Great Filter, Computing Power, Deep Space Missions, Human Landings, Colonization, Humanity, Survival Posted Date: July 26th, 2021 DOI: https://doi.org/10.21203/rs.3.rs-639164/v2 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Avoiding the “Great Filter”: A Projected Timeframe for Human Expansion Off-World Jonathan Jiang (  Jonathan.H.Jiang@jpl.nasa.gov ) California Institute of Technology https://orcid.org/0000-0002-5929-8951 Philip Rosen Retired Kristen Fahy Jet Propulsion Laboratory Physical Sciences - Article Keywords: Great Filter, Computing Power, Deep Space Missions, Human Landings, Colonization, Humanity, Survival Posted Date: July 26th, 2021 DOI: https://doi.org/10.21203/rs.3.rs-639164/v2 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Abstract A foundational model has been developed based on trends built from empirical data of space exploration and computing power through the first six plus decades of the Space Age which projects earliest possible launch dates for human-crewed missions from cis-lunar space to selected Solar System and interstellar destinations. The model uses computational power, expressed as transistors per microprocessor, as a key broadly limiting factor for deep space missions’ reach and complexity. The goal of this analysis is to provide a projected timeframe for humanity to become a multi-world species through off-world colonization, and in so doing all but guarantees the long-term survival of the human race from natural and human-caused calamities that could befall life on Earth. Be-ginning with the development and deployment of the first nuclear weapons near the end of World War II, humanity entered a ‘Window of Peril’ which will not be safely closed until robust off-world colonies become a reality. Our findings suggest the first human-crewed missions to land on Mars, selected Asteroid Belt objects, and selected moons of Jupiter and Saturn can occur before the end of the 21st century. Launches of human-crewed interstellar missions to exoplanet destinations within roughly 40 lightyears of the Solar System are seen as possible during the 23rd century and launch of intragalactic missions by the end of the 24th century. An aggressive and sustained space exploration program, which includes colonization, is thus seen as critical to the long-term survival of the human race. Avoiding the “Great Filter”: A Projected Timeframe for Human Expansion Off-World Jonathan H. Jiang1, Philip E. Rosen2, Kristen A. Fahy1 1. Jet Propulsion Laboratory, California Institute of Technology, Pasadena, CA, USA 2. Energy Industry Engineer (Retired), Vancouver, WA, USA Copyright © 2021, All Rights Reserved. Keywords: Great Filter, Computing Power, Deep Space Missions, Human Landings, Colonization, Humanity, Survival Expansion Off World Jonathan H. Jiang1, Philip E. Rosen2, Kristen A. Fahy1 1. Jet Propulsion Laboratory, California Institute of Technology, Pasadena, CA, USA 2. Energy Industry Engineer (Retired), Vancouver, WA, USA Copyright © 2021, All Rights Reserved. Keywords: Great Filter, Computing Power, Deep Space Missions, Human Landings, Colonization, Humanity, Survival Physical Sciences - Article Posted Date: July 26th, 2021 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 Avoiding the “Great Filter”: A Projected Timeframe for Human Expansion Off-World Jonathan H. Jiang1, Philip E. Rosen2, Kristen A. Fahy1 1. Jet Propulsion Laboratory, California Institute of Technology, Pasadena, CA, USA 2. Energy Industry Engineer (Retired), Vancouver, WA, USA Copyright © 2021, All Rights Reserved. Keywords: Great Filter, Computing Power, Deep Space Missions, Human Landings, Colonization, Humanity, Survival 1. Introduction Under existential threats and pressures such as sharp changes in population and demographics straining precious resources, climate change, nuclear war, pandemic, and the dramatic increase in the prevalence of facilities capable of producing deadly genetically engineered pathogens, it is easily understandable how one could become convinced humanity is living through an ever-expanding debris field of our own troubled history since the end of World War II. Is it just in our nature that we are bound to hurdle towards self-annihilation once the means are at hand, falling victim to the cosmic “Great Filter” [1]? The term “Great Filter” is a conceptualized probability threshold that could pose a barrier to the evolution of intelligent life. 1 Consideration of that question, which has challenged researchers since before the dawn of the Space Age, manifests analytically as the “L” factor (lifespan of technical/communicative civilizations) in the famed Drake Equation [2] and more recently found by statistical modeling to be the Equation’s most influential term [3]. Indeed, “Our transient existence has lasted for less than 10 one-billionths of cosmic history so far on a tiny rock we call Earth, surrounded by a vast lifeless space” [4]. Against this backdrop, however, we are reminded that, “We are more than just our genes” [5]. Human minds sufficiently complex to render our home planet unlivable, have also created the technology able to travel through space and investigate other worlds. Given the fact the Earth also faces natural threats such as asteroid impacts, supernovae radiation, and super- volcanic activity, “our chance for survival could improve if some people choose to move away from Earth. Currently, all our eggs are in one basket. Venturing into space offers the advantage of preserving our civilization from a single-planet disaster” [6]. In recent decades, as robotic missions throughout the Solar System progressed, much speculation has been given to the next steps including colonization [7]. Focusing on the assurance for humanity’s survival that would be afforded by permanent, self- sustaining, and genetically diverse colonization off-Earth, and deter-mining when such a Herculean task can be expected to occur, is paramount. Estimates for how long a technological civilization might last if confined to just its home world varies widely, from the pessimistic limit of destruction occurring in the immediate future, to “a few centuries but not much longer” [6] to approaching 8,000 years [8]. 1. Introduction Such a broad range is to be expected given the complexities inherent in individual and group behaviors of humans across Earth’s population of nearly 8 billion. Estimates for off-world colonization’s timeframe can be expected to vary as well, functionally dependent as they are on the rate of technological development, government and private industry priorities, and public support, among other factors. However, the more than 60 years since the start of the Space Age have brought rapid development of humanity’s off-world capability, from Sputnik in 1957 to men on the moon in 1969 to robotic probes having visited the far reaches of the Solar System and Kuiper Belt. Hence, a key parameter within humanity’s control for impacting the odds of long-term survival can be found in how many years it will take to establish at least one permanent, self-sustaining and genetically diverse off-world colony. This ‘window of peril’, which opened in 1945 for the technological civilization of Earth, will remain a threat until such colonies become a reality. But how do we meet the challenge of estimating how long until robust off-world colonies are established? Answering, even in the very approximate, such a far-reaching query requires extensive modeling involving both quantitative and qualitative data from the start of the Space Age to the present, plus projections into the coming decades of the 21st century that encompass an array of unknowns such as findings from the Moon, Mars, Jovian and Saturnian satellites already underway in this decade. Rather than trying from the outset to build a complex multi- variable model, the first steps of an incremental approach is presented here whose intent is to lay the foundation for building the complex model by first keying on a single critical parameter. The choice for this critical parameter is computing power, and is driven by the fact of its own exponential technological development has run parallel to that of space exploration technology. Cutting edge space exploration technology historically relies on computational capability for handling multitudes of complex calculations which enables rapid progress. For the purposes of this analysis, computing power will be measured as the number of transistors that can be 2 contained within a single microprocessor. 1. Introduction The logic of this choice follows directly from the clear dependence of scientists, inventors, engineers, medical researchers and others on their ability to gather, process and interpret the vast quantities of information required to conceive, design, build, test, operate and maintain the new technologies required for increasingly complex and distant human missions into deep space. “[Shawn] Domagal-Goldman and [Giada] Arney, [NASA Goddard astrobiologists] envisage future exoplanet missions where AI technologies embedded on spacecraft are smart enough to make real-time decisions, saving the many hours necessary to communicate with scientist on Earth” [9]. At this point it should be noted that computing power, expressed as transistors per microprocessor, does have an analogy to the human brain. More specifically, the human brain contains ~1011 neurons and ~1014 synaptic connections among those neurons [10]. Further, “A transistor can be thought of as an analog to the synapse (connection) between two neurons” [11]. As well, with cutting edge technology such as using ‘persistent photoconductivity’ found in some perovskite semiconductors as a kind of ‘optical memory’, this “…phenomenon can also mimic synopses in the brain that are used to store memories” [12]. Hence, computing power as described by transistor count per micro-processor can be thought of as a cousin to the human brain at the basic structural level, with both organic and synthetic computational devices being used in concert to develop the new technologies of the Space Age. Finally, it should be noted that any estimate of deep space mission timing merely projects a date of earliest launch from Earth (or, more generally, cis-lunar space, which means "not beyond the moon" in Latin), but does not include actual travel time to a given mission’s final destination or the follow-on time interval to establish human colonies once the first crewed landing is made. On this note, while chemically-powered rockets can likely suffice for missions to Mars, Jovian space and, perhaps, as far as the Saturn system, their inherent exhaust velocity limitation of about 5 km/sec precludes their consideration for practical interstellar travel [13]. Travel to interstellar destinations would require highly advanced technology such as generational star ships and/or breakthrough propulsion systems – e.g., Bussard Ramjet [14] utilizing a CNO-catalyzed nuclear fusion cycle [13, 15]. In any case, greater computing power than available today will be needed to engineer, build and operate such designs. 2. Methodology As computing power grows at an ever-accelerating rate, approximately doubling every two years during much of the second half of the 20th century [16] and continuing into the 21st century, so too has humanity’s reach into outer space. The robotic component of the Space Age, which began with Sputnik in 1957, quickly advanced to the first lunar flybys in 1959 (Luna 1 and 2), flyby missions to Venus (Mariner 2, 1962), Mars (Mariner 4, 1964), a successful soft landing on the Moon (Luna 9, 1966), and launches of later successful flybys of the outer planets by Pioneer 10 in 1972, Pioneer 11 in 1973 and Voyager 1 and 2 in 1977. The model discussed below is based on the confluence of these two exponential trends: computing power (expressed as transistors per microprocessor) as a function of time and distance (measured in astronomical units, AUs) from launch point Earth to the first robotic deep space missions’ farthest successfully achieved objective as a function of time. 3 2.1 Computational Power and Deep Space Missions Over Time As computing power grows at an ever-accelerating rate, approximately doubling every two years during much of the second half of the 20th century [16] and continuing into the 21st century, so too has humanity’s reach into outer space. The robotic component of the Space Age, which began with Sputnik in 1957, quickly advanced to the first lunar flybys in 1959 (Luna 1 and 2), flyby missions to Venus (Mariner 2, 1962), Mars (Mariner 4, 1964), a successful soft landing on the Moon (Luna 9, 1966), and launches of later successful flybys of the outer planets by Pioneer 10 in 1972, Pioneer 11 in 1973 and Voyager 1 and 2 in 1977. The model discussed below is based on the confluence of these two exponential trends: computing power (expressed as transistors per microprocessor) as a function of time and distance (measured in astronomical units, AUs) from launch point Earth to the first robotic deep space missions’ farthest successfully achieved objective as a function of time. C = 15.70226 exp(0.34821 T) (1) where C = computing power, T = time in number of years since 1957, and the correlation coefficient R2 = 0.994. See Figure 1 left-panel for the graphical representation of data corresponding to equation (1). where C = computing power, T = time in number of years since 1957, and the correlation coefficient R2 = 0.994. See Figure 1 left-panel for the graphical representation of data corresponding to equation (1). D = 0.04846 exp(0.31272 T) (2) 2.1. Computational Power and Deep Space Missions Over Time As will be seen, both trends can be easily plotted in a semi-log manner with the linear horizontal axis being the number of years since the start of the Space Age (Figure 1). Exponential trend curves are then best-fit to the plotted data, yielding: C = 15.70226 exp(0.34821 T) (1) D = 0.04846 exp(0.31272 T) (2) where D = distance from launch point Earth to farthest successfully achieved mission objective in AUs (minimal distance from Earth’s orbit to objective’s orbit), T = time in number of years since 1957, and the correlation coefficient R2 = 0.994. See right-panel of Figure 1 for the graphical representation of data corresponding to equation (2). Please refer to Figure 3 at the end of this section for a simplified flow chart of the development of this and other relationships which comprise the core of our model. With the commonly time dependent equations of these two data sets in hand, it is then a straight forward exercise to combine the two equations as to eliminate T, their time dimension, creating a new power relationship between computing power and distance from launch point Earth to farthest successfully achieved robotic mission objective: C = 457.2437 D!.!!##$ (3) (3) where the correlation coefficient R2 = 0.996. It should be noted that the computing power vs. time data set contains 24 data points over the 61 years between 1957 and 2017 [17] while the deep space missions vs. time data set includes only 5 data points. This limitation, implicit in its reliance on the relatively small number of deep space missions’ empirical data, cannot be avoided. where the correlation coefficient R2 = 0.996. It should be noted that the computing power vs. time data set contains 24 data points over the 61 years between 1957 and 2017 [17] while the deep space missions vs. time data set includes only 5 data points. This limitation, implicit in its reliance on the relatively small number of deep space missions’ empirical data, cannot be avoided. Equation 3 provides a relationship between how far distance-wise, robotic deep space missions have progressed relative to concurrent computing power. This relation between a fundamental element which underlies the technology and procedures necessary for venturing 4 into deep space for extended periods of time, and ultimately establishing off-world colonies, is coupled to the physical reach of such efforts. D = 0.04846 exp(0.31272 T) Figure 1: Left-panel; Computational power as expressed in number of transistors per microprocessor from 1971 to 2017, time expressed as number of years since start of the Space Age (1957), used in empirically derived equation (1) [17]; Right-panel: First of their kind robotic deep space missions’ (flybys) planetary objective minimum distance from Earth orbit (in astronomical units) versus number of years from start of Space Age (1957) to missions’ launch year, used in empirically derived equation (2). Figure 1: Left-panel; Computational power as expressed in number of transistors per microprocessor from 1971 to 2017, time expressed as number of years since start of the Space Age (1957), used in empirically derived equation (1) [17]; Right-panel: First of their kind robotic deep space missions’ (flybys) planetary objective minimum distance from Earth orbit (in astronomical units) versus number of years from start of Space Age (1957) to missions’ launch year, used in empirically derived equation (2). While it may appear more straightforward to estimate the timing of additional and more ex- tensive deep space missions by basing such projections solely on selected successful missions already flown – i.e., simply following the established trend further out in space and time, doing so would blunt the influence in any such estimate from the critical component of concurrent technological development. Indeed, when just the simple logarithmic extrapolation of first successful flyby missions actually flown is used as the basis – i.e., equation (2), solved for time as a function of distance, to estimate timing of far-reaching human-crewed missions, an ever- widening discrepancy arises with respect to increasing mission objective distance. As an example, for the nearest interstellar mission to Proxima Centauri, the aforementioned simplistic extrapolation predicts a first successful launch date of 2080 for human-crewed mission, 174 years sooner than predicted by our model which takes into account as well the projected time required to develop mission-necessary computing power. When one considers that Moore’s Law is expected to run up against material and engineering limitations [18], while radical new forms of computing to carry out ever more complex calculations (e.g., quantum computing) are yet to be perfected, the means to accomplish the developmental dimension of humanity’s most daunting endeavors cannot be ignored. 2.2 From Robotic Missions to Human-Crewed Landings Robotic missions have and will continue to pave the way for human crews to follow. However, the leap from relatively inexpensive (and ultimately expendable) machine voyagers to craft capable of keeping groups of highly complex living organisms alive in the most hostile of environments is an enormous leap in nearly every design and construction detail. As one aspect, humans evolved under the protection of Earth’s strong magnetic field and dense atmosphere which work in combination to screen-out the vast majority of high energy particles from interstellar space – commonly referred to as cosmic rays. Direct exposure to cosmic rays, even for relatively short periods of time, would pose grave health risks to an unprotected crew. Although shielding can be employed to meet this need, this would add substantially to the engineering requirements of crewed spacecraft along with additional mass necessitating more powerful engines, which in turn adds further to the mass of the vehicle presenting engineers with what amounts to a Newtonian conundrum. Nonetheless, the first 10 years of the Space Age brought spectacular example of bridging robotic to human landings, this beginning with Luna 1 and 2’s flyby of the Moon in 1959, Luna 9’s soft landing in 1966, and culminating in 1969 with the first crewed landing by Apollo 11. While this progression provides a critical data point for this analysis, at least one other data point would be needed to begin proposing a quantifiable time- distance relationship between robotic and human missions. It is at this point a degree of speculation is needed concerning humanity’s next likely target for ‘boots on the regolith’ – Mars. Mariner 4 first flew by Mars in 1964 and was followed by the first successful soft landers, Viking 1 and 2, in 1976. While humans have yet to step foot on the Martian surface, a first crewed landing in the 2030s is envisioned [19]. For the purposes of this analysis, we will venture a somewhat conservative assumption that such a landing successfully launches in 2038. Note that while the optimal Hohmann transfer orbit launch windows for a Mars mission in the late 2030s will actually occur in 2037 and 2039, for the purposes of our calculations and average of 2038 will be used). D = 0.04846 exp(0.31272 T) Since the first successful interplanetary robotic flyby missions in the first half of the 1960s, Mariner 2’s visit to Venus and Mariner 4’s close encounter with Mars, rapid advancement in computing have accompanied and supported ever more far reaching and complex missions. These include flybys of all the outer planets, Pluto and a Kuiper Belt Object, orbiters in the Jovian and Saturnian systems, asteroid regolith sampling and a series of semiautonomous rovers 5 scouring the surface of Mars for signs of an ancient life-supporting environment. While human- crewed missions have yet to venture beyond the Moon, advances in control and automation systems, materials and engineering techniques, as well as in space medicine (largely thanks to the database amassed from two decades of long-term crew postings to the International Space Station) made in the half century since Apollo have paved the way for humans to feasibly set foot on Mars before the close of the 2030s. 2.2 From Robotic Missions to Human-Crewed Landings When going from launch of a first actual or first projected flyby mission to a first robotic lander mission for a given destination, a fixed number of years is simply added to the actual or projected flyby launch date which corresponds to the actual delta realized for Mars, that of 11 years (Mariner 4’s flyby in 1964 to Viking 1 and 2’s launch in 1975). The implicit assumption is that the technological leaps required to go from robotic flybys to robotic landings are minor compared to going from robotic flyby to human landings. Further, for robotic missions the intermediate step between flybys and landings, that of an orbiter without a lander, is ignored per the leap from flyby to orbiter also being minor when compared to the challenges of placing a complex device safely on the surface of another world. Finally, while several of the Soviet era Venera probes to Venus did successfully place robotic landers on the surface, the severe conditions char-acteristic of Venus greatly limited their operating window and would almost certainly rule out Venus as a viable destination for human landings, much less colonization, in the foreseeable future. For these reasons only Mariner 2’s Venus flyby in 1962 is included in the construction of our model. 6 With two sets of data points for time interval and distance when going from robotic flyby to human landing missions, noting again that the second set of data points, this involving Mars, are partially speculative, it is possible to propose a mathematical relationship. While any curve can be perfectly fitted to this small a data set, this analysis will confine itself to a logarithmic relationship between time (expressed as number of years between first successful robotic flyby and human landing) vs. distance from launch point Earth to mission objective (measured in AUs). The resulting equation is as follows: T 12 824 l (D) + 86 532 (4) (4) Tfh = 12.824 ln(D) + 86.532 (4) where Tfh = number of years from launch of first successful robotic flyby mission to launch of first successful human landing. In the case of the Moon, D = 0.00256 AU while Tfh is 10 years. For Mars, D = 0.376 AU (closest orbital approach, not necessarily the distance a crewed or non-crewed vehicle would take in the instance of a Hohmann transfer orbit) while Tfh is projected to be 74 years. 2.2 From Robotic Missions to Human-Crewed Landings Figure 2: Number of years separating launch of first successful robotic flyby to robotic landing and first successful robotic landing to human landing, for Moon and Mars missions. Note: Launch of first successful human landing on Mars is assumed to occur in calendar year 2038. Figure 2: Number of years separating launch of first successful robotic flyby to robotic landing and first successful robotic landing to human landing, for Moon and Mars missions. Note: Launch of first successful human landing on Mars is assumed to occur in calendar year 2038. Figure 2: Number of years separating launch of first successful robotic flyby to robotic landing and first successful robotic landing to human landing, for Moon and Mars missions. Note: Launch of first successful human landing on Mars is assumed to occur in calendar year 2038. The rationale for using a logarithmic relationship is founded in the nature of technological development itself, specifically the familiar S-curve of development vs. time. First, there is a relatively slow run-up over decades which can be regarded here as our going from the Wright Brothers’ first powered flight of a heavier than air vehicle in 1903 to Sputnik in 1957 – i.e., the lower portion of the S-curve. Technological progress then experiences a sharp upward acceleration over a short period of time, this covering the first ~20 years of the Space Age, that took us from Sputnik to Voyager 1 and 2 – i.e., the steep middle portion of the S-curve. Finally, with much of the ‘low hanging fruit’ of technical barriers having been surmounted for relatively near-Earth mission objectives, the long and gently curving upper section of the S-curve is 7 encountered. It is the upper half of the S-curve that is relevant to our analysis and best expressed mathematically in the logarithmic form of equation (4) and as illustrated below in Figure 2. Finally, there is consideration for the time interval in going from the first crewed landing to the first self-sustaining, genetically viable (i.e., robust) colony. As no such colony has yet been established off-world, actual data for folding into this analysis is pending though the coming years with the prospect for the beginnings of said colonies (NASA, 2019). 2.2 From Robotic Missions to Human-Crewed Landings For the purposes of this analysis, it will be assumed that once the planned Artemis Base Camp is successfully established near the Moon’s south polar region, launches for true colonization can begin within a relatively short time frame from the successful first crewed landings’ launch to Mars and beyond. 3.1 Actual and projected timing for deep space missions and associated computational power In Table 1, the Solar System mission destinations were chosen per their history of robotic visits, and in the case of the Moon, the human landings that have taken place. Note again the assumption for first human landing on Mars to launch from Earth in year 2038 (averaged from 2037 and 2039). The rationale for the interstellar mission destinations are as follows: Proxima Centauri – The closest known star to the Solar System at 4.2 lightyears, this active red dwarf is strongly suspected of having a least two planets and at least one of those, Proxima Centauri b, in the star’s habitable zone [22]. Tau Ceti – The closest known G-class (Sol-like) star not part of a tandem star system, it is 11.9 lightyears away and hosts at least four planets and at least one in the star’s habitable zone (http://phl.upr.edu/press-releases/twonearbyhabitableworlds). Trappist System – A cool red dwarf star 40.5 lightyears away that is thought to host at least seven terrestrial-sized planets and several among those potentially in the star’s habitable zone [23]. Trappist System – A cool red dwarf star 40.5 lightyears away that is thought to host at least seven terrestrial-sized planets and several among those potentially in the star’s habitable zone [23]. 4 kpc from Center of the Milky Way – An annular region located approximately 4,000 parsecs from the center of the Milky Way Galaxy (closest point: ~14,000 lightyears from the Solar System, in the direction of Sagittarius and the galactic center) has been suggested by statistical modeling as the region of the Milky Way most likely to have contained complex life, including technological civilizations, in the past [3]. Table 1: Actual and projected timing of first successful robotic and human missions for selected destinations within the Solar System and interstellar space. Destination Moon Mars Asteroid Belt Jovian System Saturn System Proxima Centauri Tau Ceti Trappist System 4 kpc from CoMW Dist. 2.3 Assembling the Model Figure 3 outlines how the above empirically derived equations are combined to create the model for extended deep space exploration. It must again be emphasized that computing power, while a broadly affecting factor, is only one such factor in a highly complex interplay of dynamic influences on how far and how soon humanity will establish off-world colonies. As such, this model is best considered a starting point version of more rigorous versions to come (see 5. Future work discussion). Figure 3: Empirical equations 1 and 2, in combination with distance to mission objective and flyby mission launch year data, yield projections for extended missions’ launch years and computational power required. Note: “f”, “f’”, “g”, “h” and “i” denote functional relationships. Computational Power Needed for First Successful Human Landing Equation 1 Year Computational Power First Achieved for First Successful Human Landing Equation 1 as T = f'(C) Distance to Mission Objective Actual Launch Year of First Successful Flyby {If Already Occurred} Equation 3 C = h(D) Equation 1 as T = f'(C) {If 1st Successful Flyby Yet to Occur} Projected Earliest Launch Year of First Successful Robotic Landing Year Computational Power First Achieved for First Successful Robotic Landing Equation 1 as T = f'(C) Projected Earliest Launch Year of First Successful Human Landing Equation 4 Tfh = i(D) Equation 2 D = g(T) Equation 1 C = f(T) Equation 2 D = g(T) Equation 1 as T = f'(C) {If 1st Successful Flyby Yet to Occur} Projected Earliest Launch Year of First Successful Robotic Landing Equation 3 C = h(D) Equation 1 C = f(T) Distance to Mission Objective Year Computational Power First Achieved for First Successful Robotic Landing Equation 1 as T = f'(C) Projected Earliest Launch Year of First Successful Human Landing Equation 4 Tfh = i(D) Actual Launch Year of First Successful Flyby {If Already Occurred} Year Computational Power First Achieved for First Successful Human Landing Equation 1 as T = f'(C) Figure 3: Empirical equations 1 and 2, in combination with distance to mission objective and flyby mission launch year data, yield projections for extended missions’ launch years and computational power required. Note: “f”, “f’”, “g”, “h” and “i” denote functional relationships. 8 3. Results and Discussion The data and derived equations contained herein estimate the projected calendar year dates and associated computational power that will be required for the successful launch of initial robotic landing and human landing missions from Earth to a given set of deep space mission objectives. 3.1 Actual and projected timing for deep space missions and associated computational power from Earth (AU) 0.0026 0.3763 1.5587 3.9501 8.0412 265,486 752,526 2,562,570 882,424,035 1st Flyby 1959 1964 1972 1972 1973 Not Yet Launched Not Yet Launched Not Yet Launched Not Yet Launched 1st Robotic Lander 1966 1975 Not Yet Launched Not Yet Launched 1997 Not Yet Launched Not Yet Launched Not Yet Launched Not Yet Launched 1st Human Landing 1969 2038 Not Yet Launched Not Yet Launched Not Yet Launched Not Yet Launched Not Yet Launched Not Yet Launched Not Yet Launched Tables for LookUp Data on Pre-Chosen Destinations and Projections for Earliest Possible Mission Launch Dates Actual and Calculated Projections for Calendar Year of Launch from Earth of First Successful Mission Type and Destination and Corresponding Computational Power Requirement Launch Year of 1st Successful Flyby Mission 1959 1964 1972 1972 1973 2007 2010 2014 2032 Year Computational Power First Achieved 1948 1964 1968 1971 1973 2018 2021 2025 2043 Year Computational Power First Achieved 1959 1975 1979 1982 1984 Launch Year of 1st Successful Robotic Landing 1959 1975 1979 1982 1984 2018 2021 2025 2043 Launch Year of 1st Successful Human Landing 1969 2038 2064 2076 2086 2254 2270 2290 2383 Year Computational Power First Achieved 1969 2038 2064 2076 2086 2253 2269 2289 2381 tual and projected timing of first successful robotic and human missions for selected destinations within the Solar System and interstellar space. Table 1: Actual and projected timing of first successful robotic and human missions for selected destinations within the Solar System and interstellar space. 9 9 Utilizing the data in Table 1, Figure 4 provides a graphical representation of when, in terms of the Common Era calendar year, the first successful launches from Earth have occurred and are estimated to occur for both robotic and human landings to interplanetary and interstellar mission destinations. Figure 4: Graphical representation of data in Table 1 for launch date from Earth/cis-lunar space of Figure 4: Graphical representation of data in Table 1 for launch date from Earth/cis-lunar space of robotic and human landing missions having destinations ranging from the Moon to approximately 14,000 lightyears from the Solar System. Figure 4: Graphical representation of data in Table 1 for launch date from Earth/cis-lunar space of robotic and human landing missions having destinations ranging from the Moon to approximately 14,000 lightyears from the Solar System. 3.2 Interpretation of selected missions’ results Our model predicts first human launch to an Asteroid Belt object could happen by the mid-2060s, with speculation that any human colonies to follow may eventually take hold on some of the larger bodies such as Ceres, 2 Pallas or 4 Vesta. Jovian System: Our model predicts a first human-crewed launch to Jovian space may be possible by the mid-2070s. A more optimistic assessment was assembled by NASA in a 2003 study which detailed a human-crewed launch to the outermost Galilean moon, Callisto, by the mid-2040s [25]. While the four Galilean moons Io, Europa, Ganymede, and Callisto are the most obvious targets for robotic exploration, only the outermost moon, Callisto, is sufficiently removed from Jupiter’s intense radiation fields to consider human exploration and possible colonization within the 21st century. Further robotic explorations of the Jovian system are planned such as the European Space Agency’s Jupiter Icy Moons Explorer, set to launch in 2022, and NASA’s Europa Clipper, scheduled to launch in 2026 - the findings from which may well further the case for humans to follow or suggest other, more viable, destinations. Saturn System: Saturn and its many moons, while roughly twice as distant from Earth as the Jovian system, is projected to be reachable via a first human-crewed launch that would take place in the mid-2080s. Saturn’s largest moon, Titan (possessing of a thick atmosphere of light hydrocarbons and the only interplanetary moon to have been visited thus far by a robotic lander), is an obvious target for long range plans owing largely to its vast hydrocarbon resources. However, Enceladus is at least as intriguing for human colonization due to the suspected subsurface ocean, as indicated by observations of recurrent H2O plumes erupting from its south polar region [26]. While the Jovian moon Europa is also strongly suspected of having a subsurface ocean, the aforementioned radiation concerns may make Enceladus a superior target for human colonization. Interstellar Destinations: As discussed in Section 3.1, the first crewed launch to even the nearest interstellar destination, the Proxima system, is not envisioned until the mid-23rd century. Once, however, this great leap has been sent on its way, launches to star systems ‘moderately’ more distant such as Tau Ceti at nearly 12 lightyears away are projected to be possible within another two to three decades. 3.2 Interpretation of selected missions’ results The above results are calculated for nine pre-chosen mission destinations, the first five of which are within the Solar System and representative of actual mission destinations flown starting in the late 1950s while the remaining four destinations are interstellar and run the range from our nearest stellar neighbor to approximately halfway to the center of the Milky Way Galaxy. Continued is a brief discussion for each destination: Moon: The world closest to Earth and, so far, the only world humans traveled to and have landed upon. Having already been visited by humans, this places the Moon somewhat removed from the intent of this analysis given much of the most challenging aspects of its colonization have either been solved or are now coming into reach in the very near term with NASA’s proposed ‘Artemis Base Camp’ by the late 2020s [21], as well as plans by other nations (e.g., China) to establish crewed facilities on the lunar surface in the coming decades. Given that the Moon is only a four-day trip from LEO by existing spacecraft technology, resupply from Earth would be far easier than even the next closest world for colonization, Mars, which currently takes a minimum of roughly six months to reach. Mars: As stated in Section 2.2, an assumption has been made for a first successful human landing on Mars to occur in the late 2030s, this likely coming as an extension of the Artemis Program [21]. 10 Hence, Mars is effectively treated as a fixed point in our analysis with speculation that at least the beginnings of self-sustained colonization can be expected well before the end of the 21st century. Supporting this assumption is analysis suggesting Mars to be the most eco-nomically viable location in the Solar System for colonization [24] and the focus of at least one well-funded private venture. Asteroid Belt: Popular media references to human missions to the Asteroid Belt, which lay between the orbits of Mars and Jupiter, often involve mining operations. This is not without some scientific backing such as the potential for substantial metals recovery from 16 Psyche which is the target of a NASA robotic orbiter mission set to launch in August 2022. 4. Conclusions A simple model based on trends built from empirical data of space exploration and computing power through the first six plus decades of the Space Age has been developed which projects earliest possible launch dates of human-crewed missions to Solar System and interstellar destinations. The model algebraically folds in the expected broadly limiting factor of computational power, as quantitatively expressed in transistors per microprocessor, to the demonstrated extent of robotic and human missions to the Earth’s Moon and the other planets and moons of the Solar System. While space exploration for purely scientific ends is a noble goal and certainly worthy in and of itself of the costs and sacrifices of such great endeavors, the goal of this analysis is to provide a timeframe for humanity to become a multi-world species through off-world colonization that would logically follow on the heels of earlier human landings. By creating at least one, and preferably multiple, self-sustaining and genetically viable off-world colonies, humanity can best assure its long-term survival from both natural and human-caused calamities that threaten life on Earth. The immediacy of such threats began with the development of the first nuclear weapons and has proliferated in multiple directions in the three quarters of a century since the end of World War II. In the early 21st century we find ourselves in the midst of a ‘Window of Peril’ which, having been opened in 1945, cannot be safely closed until said colonies are realized. Per the findings of our model, launches from Earth of the first human-crewed missions to land on Mars, selected minor planet/planetoid bodies in the Asteroid Belt, at least one of the four Galilean moons of Jupiter, and at least one of the moons of Saturn should be possible before the end of the 21st century – if computational power is the only limiting factor and advances in microcircuitry continue apace. Human-crewed missions to interstellar destinations, while theoretically possible, will be dependent not only on development of onboard AI capability but also technologically advanced propulsive systems, the development of which is logically dependent as well on an ever-greater ability for computation. If undertaken and pursued to success, humanity will have not only all but assured its own survival but will have become a true spacefaring civilization [27]. 4. Conclusions Off-world colonization, like the Great Filter’s self-annihilation, is within humanity’s reach – which we choose as our future will constitute Earth’s reply to Fermi’s Paradox. 3.2 Interpretation of selected missions’ results A first human launch to still more distant solar systems such as Trappist at approximately 40 lightyears from the Solar System would entail another roughly two decades. Finally, the launch of a human-crewed voyage on the galactic scale, such as traversing the Milky Way’s disk to reach halfway to its center, remains a goal much more distant in time and would not be expected possible before the late 24th century. 11 It must again be emphasized that all projections for human-crewed missions described above are first possible launch dates from Earth/cis-lunar space. Without revolutionary breakthroughs in propulsive engineering, human voyages – and any colonization that would follow – to interstellar destinations is highly unlikely to be undertaken. Fortunately, the Solar System presents a target rich interplanetary environment for astronauts to further explore and adventuring colonists to conquer starting here in the 21st century. 1. Folding-in additional ‘Associated Empirical Functions’ (i.e., other affecting factors for equation (1) such as the trend of annual inflation-adjusted public and private sector 5. Future Work The methodology for deriving the projected results of this analysis suggests at least two avenues for further expansion. Referring to Figure 5 (a generalization of Figure 3), below, they are: 1. Folding-in additional ‘Associated Empirical Functions’ (i.e., other affecting factors for equation (1) such as the trend of annual inflation-adjusted public and private sector 12 funding for space exploration programs) to the ‘Primary Empirical Function’ of actual missions flown, expressed above as equation (2). This would yield a new version of the ‘Modified Primary Empirical Function’ for equation (3) through which the new ‘Associated Empirical Function’ is expressed in a revised ‘Objective Function’. In addition to the funding parameter, other empirically derived associated functions may include but are not limited to international cooperation (building on the historic successes of early robotic missions to Venus and Mars, Apollo-Soyuz, and the International Space Station), public support, prior robotic survey findings, political and social priorities. Note that while some of these parameters may prove difficult to quantify in the direct sense, usable trends for the purpose of this type of analysis may still be extracted from data covering the six plus decades of the Space Age. From here, multiple ‘Objective Functions’ would be generated that would then need to be either algebraically combined to a ‘Global Objective Function’ or, more simply, their individual ‘Projected Results’ consolidated via weighted averaging scenario. g g g 2. Adding an iterative feature to the calculations in which initial and intermediate model results are sequentially compared against the data set for the ‘Primary Empirical Function’. Any discrepancies above a chosen tolerance would then generate successive correction factors that would be folded into the ‘Objective Function’ for each iteration until ‘Projected Results’ are brought within the chosen tolerance. 2. Adding an iterative feature to the calculations in which initial and intermediate model results are sequentially compared against the data set for the ‘Primary Empirical Function’. Any discrepancies above a chosen tolerance would then generate successive correction factors that would be folded into the ‘Objective Function’ for each iteration until ‘Projected Results’ are brought within the chosen tolerance. Figure 5: Methodology for combining an empirically derived primary function with an empirically derived associated function (future work item 1), plus optional recursive loop for successive corrections (future work item 2). Objective Function Projected Results Modified Primary Empirical Function Correction Factors Empirically- Derived Adjustments Comparison: Projected Results vs. 5. Future Work Corresponding Primary Empirical Function Data Primary Empirical Function Associated Empirical Primary Empirical Function Modified Primary Empirical Function Objective Function Projected Results Associated Empirical Correction Factors Figure 5: Methodology for combining an empirically derived primary function with an empirically derived associated function (future work item 1), plus optional recursive loop for successive corrections (future work item 2). 13 Acknowledgments This research was supported by the Jet Propulsion Laboratory, California Institute of Technology, under the contract with NASA. We acknowledge the partial funding support from the NASA Exoplanet Research Program NNH18ZDA001N. Data Statement The data underlying this article are available in the article and in its online supplementary material. For additional questions regarding the data sharing, please contact the corresponding author at Jonathan.H.Jiang@jpl.nasa.gov. References [1] Hanson R. 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https://openalex.org/W4390847719	https://magnascientiapub.com/journals/msarr/sites/default/files/MSARR-2023-0174.pdf	English	null	Dietary exposure and risk characterization of selected toxic metals in crude palm oil (Elaeis guineensis Jacq) from six states in Niger Delta, Nigeria	Magna Scientia Advanced Research and Reviews	2,024	cc-by	9,053	Article DOI: https://doi.org/10.30574/msarr.2024.10.1.0174 Article DOI: https://doi.org/10.30574/msarr.2024.10.1.0174 Abstract The increase in toxic metal (TM) contamination in crude palm oil (CPO) due to anthropogenic activities, method of processing and handling makes it necessary to assess the contents of metals of toxicological interest to ensure safety. The contents of TMs [lead (Pb), cadmium (Cd), chromium (Cr), nickel (Ni) and arsenic (As)] were quantified in CPO samples locally produced from six selected states in Niger Delta, Nigeria using atomic absorption spectrophotometer. The TM contents and dietary intakes were compared with permitted limits of international agencies such as the European Food Safety Authority (EFSA) and Joint FAO/WHO Expert Committee on Food Additives (JECFA). The TM contents exceeded the permissible limits of JECFA with Cr in Akwa Ibom State samples (3.08 mg kg-1) being the highest. The dietary monthly intake of Cd for consumption of 25 g day-1 of CPO for adults and children were 40.36 µg kg-bw-1 month-1 (161%) and 91.6 µg kg-bw-1 month-1 (366%), respectively. The margin of exposure (MOE) values of Pb due to consumption of CPO ranged from 1.39 to 3.83 (nephrotoxic effects) and 3.32 to 9.12 (cardiotoxic effects) for adults, and 0.49 to 1.34 (neurotoxic effect), 0.61 to 1.69 (nephrotoxic effects) and 0.61 to 4.01 (cardiotoxic effects) for children. Furthermore, MOE values of inorganic arsenic (iAs) for carcinogenic effects due to consumption of CPO for adults and children ranged from 0.32 to 26.53 and 0.14 to 11.69, respectively. The results highlight the necessity to set up monitoring program for TMs in CPO to reduce exposure level. Keywords: Crude Palm Oil; Toxic Metals; Dietary Intake; Lifetime Cancer Risk; Margin of Exposure; Niger Delta Keywords: Crude Palm Oil; Toxic Metals; Dietary Intake; Lifetime Cancer Risk; Margin of Exposure; e Palm Oil; Toxic Metals; Dietary Intake; Lifetime Cancer Risk; Margin of Exposure; Niger Delta * Corresponding author: Onyegeme-Okerenta Blessing Minaopunye Copyright © 2024 Author(s) retain the copyright of this article. This article is published under the terms of the Creative Commons Attribution Liscense 4.0. Dietary exposure and risk characterization of selected toxic metals in crude palm oil (Elaeis guineensis Jacq) from six states in Niger Delta, Nigeria Vera Chituru Isiodu, Blessing Minaopunye Onyegeme-Okerenta * and Eka Bassey Essien Vera Chituru Isiodu, Blessing Minaopunye Onyegeme-Okerenta * and Eka Bassey Essien Department of Biochemistry, Faculty of Science, University of Port Harcourt, Rivers State, Nigeria. Magna Scientia Advanced Research and Reviews, 2024, 10(01), 025–037 Publication history: Received on 20 November 2023; revised on 30 December 2023; accepted on 0 Copyright © 2024 Author(s) retain the copyright of this article. This article is published under the terms of the Creative C Magna Scientia Advanced Research and Reviews, 2024, 10(01), 025–037 is therefore, of great significance to assess the levels of toxic metals in CPOs and to report possible contamination that would represent a health hazard. Food consumption had been identified as the major pathway of human exposure to toxic metals, compared with other ways of exposure such as inhalation and dermal contact. Intake of TMs from food consumption is dependent on the TM concentrations in food and the quantity of food consumed by individuals. International agencies like US Environmental Protection Agency (USEPA) and Joint FAO/WHO Expert Committee on Food Additives (JECFA) have provided guidelines on the intake of TMs by humans. The JECFA recommended permissible tolerable weekly intakes (PTWIs) and acceptable daily intakes as guidelines for food additives and certain contaminants in foods. Also, the US EPA provided reference dose (RfDo) values in ug kg-bw-1day- 1 for some TMs. The presence in vegetable oil of TMs such as Pb, Cd, Cr, Ni or As is mostly due to environmental contamination. For some of these metals, limit values in palm oil have been established by Codex Alimentarius (Codex Alimentarius Standard 193-1995) and the European Commission (EC No. 1881/2006 – contaminants in foodstuffs) [6]. Toxic heavy metals (TMs) are widely present and dispersed in the environment. Their accumulation in plants is particularly important because nutritional substances might translocate from plants in the food chain and finally, they can accumulate in humans [5]. Due to the high nutritional importance of palm oil and their key role in the diet, the increased content of TMs might pose a significant health risk for their consumers. Most of the TMs entering the human body through the consumption pathway originate from the plant products grown in soil, which as the results of the geogenic or anthropogenic factors, may pose a threat of their migration to edible plants due to increased concentration or mobility [5]. 2.1. Sampling locations and sampling Samples of CPO were collected in clean sterilized and over-dried amber glass bottles directly from local palm oil mills in the six states (Akwa, Ibom, Bayelsa, Cross, River, Delta, Edo, and Rivers) that make up the states in Niger Delta, Nigeria. Six (6) samples were collected from each state making a total of 36 in all. The sampling sites in the various states are shown in Figure 1. These CPOs were produced from the states using traditional method. Magna Scientia Advanced Research and Reviews, 2024, 10(01), 025–037 Considering the importance of palm oil and their derivatives in the human diet, the potential contamination of this food group with toxic metals and the potential health risks derived from the dietary exposure, the objectives of this study were: (i) quantification of selected five toxic heavy metals (Pb, Cd, Cr, Ni, and As) in CPOs consumed by the population of the selected states in Niger Delta, Nigeria, (ii) assessment of the exposure to these of TMs from the consumption of CPOs and via estimation of the dietary intakes in comparison with health-based guidance values (tolerable intakes) provided by European Food Safety Authority (EFSA), Joint FAO/WHO Expert Committee on Food Additives (JECFA) and United State Environmental Protection Agency (USEPA), iii) the correlation, similarity and source of these metals in samples using Pearson correlation analysis, (iv) characterization of potential health risks using lifetime cancer risk (LCR) and margin of exposure (MOE). On this regard, the work was conducted with the purposes of contributing to a better understanding and to the monitoring of the adequacy and safety of the consumption of crude red palm oil of these consumers in particular and Nigerians in general. 1. Introduction Vegetable oils are being widely used all over the world in cooking, cosmetic, pharmaceutical and chemical industries. The presence of essential fatty acids, phytosterols and a tocopherol, as well as high levels of antioxidative nutrients like monosaturated fatty acids, enhances the nutritional value of vegetable oils. Oils and fats have many vital functions in the human body as such as energy source, structural component and vitamins in the human body [1] as well as their cholesterol-lowering effect. The quality of edible oils with regard to their freshness, storability/shelf life and influence on nutritional value and human health is directly linked to the concentration of some metals [2, 3]. The toxic metals (TMs) can be incorporated naturally into the oil from the soil, use of fertilizers and by unwholesome farming practices where the raw ingredients were grown for the seed-based and/ or during the production process such as in the extraction or refining processes of edible oils [4, 5]. The toxic metals can be very deleterious even at low concentration when ingested over a long period. The presence of some trace elements like Fe, Cu, Ca, Mg, Co, Ni and Mn in oil can increase the rate of oxidation by increasing the generation of free radicals from fatty acids or hydroperoxides [4], while others like Pb, Cd, Cr, Ni, and As are very important on account of their potential toxicity and metabolic roles when they have been consumed by humans [4]. It Magna Scientia Advanced Research and Reviews, 2024, 10(01), 025–037 2.3.1. Instrument Measurements The certified standard solutions of Pb, Cd, Cr, Ni, and As (1 mg mL-1) were purchased from the National Institute of Metrology, China. The working standard solutions were prepared from the standard solutions with Milli-Q water (18.2 MΩ cm resistivity) from a Millipore Milli-Q system (Thermo Scientific EASYpure II, Waltham, MA, USA). Calibration was performed by analyzing the prepared working standard solutions and two agent blank samples. Concentrations of Pb, Cd, Cr, Ni, and As were determined using an Inductively Coupled Plasma-Atomic Emission Spectrometry instrument (SPECTRO Analytical Instruments GmbH, Kleve, Germany) with axial viewing configuration. Total concentrations of As were determined using Atomic Fluorescence Spectrometry (AFS-930) (Beijing Jitian instrument Ltd., Beijing, China). 2.3.2. Sample Treatment One gram of each homogenized sample was weighed in Teflon tubes using an analytical precision balance. Two mL of 30% hydrogen peroxide (H2O2) and 4 mL of 65% concentrated nitric acid (HNO3) were added. Samples were subjected to wet digestion in acid medium with microwave digestion system. Three replicates were prepared for each sample. The microwave digestion process is based on a one-hour program divided into stages:  Initial stage of fifteen minutes until reaching 100 ˚C maintained for five m  Second stage of ten minutes until reaching 180 ˚C, maintained for 15;  Second stage of ten minutes until reaching 180 ˚C, m g g  Third stage of fifteen minutes of lowering the temperature to ambient temperature. Finally, the digested samples are placed in 10 ml volumetric flasks and made up to volume with distilled water obtained from the distillation system (Millipore, Burlington, MA, USA) [7].  Third stage of fifteen minutes of lowering the temperature to ambient temperature. Finally, the digested samples are placed in 10 ml volumetric flasks and made up to volume with distilled water obtained from the distillation system (Millipore, Burlington, MA, USA) [7]. 2.2. Reagents (Chemicals) All chemicals and reagents used in this study were analytical grade. Nitric acid (HNO3) and hydrogen peroxide (H2O2) were purchased from Sigma Aldrich, Darmstadt, Germany. Distilled water was used throughout the work for preparation and dilutions of standard solutions. 26 Magna Scientia Advanced Research and Reviews, 2024, 10(01), 025–037 2.3. Sample Preparation and Analytical Methods 2.3. Sample Preparation and Analytical Methods Magna Scientia Advanced Research and Reviews, 2024, 10(01), 025–037 Figure 1 Map of study area Figure 1 Map of study area Figure 1 Map of study area Estimated daily intake The estimated daily intake (EDI) is defined as the amount of a chemical element/substance/contaminant that is ingested with a portion of a food or foods per body weight. The EDIs were calculated considering a 25 g day-1 consumption scenario of the CPO and the detected concentration of the toxic metal (Eqn. 1). The calculation of this value makes it possible to evaluate the risk when comparing it with the reference value established by food safety institutions. The EDI was calculated as follows: EDI (mg kg −bw −1 day −1) = Ci ∗ IRi BW … … … … (1) where Ci is the concentration of THM i, (mg kg-1), IRi is the daily consumption rate (kg day-1) and BW is the body weight (kg) of the consumer. Likewise, average body weights for an adult and a child of 63 kg and of 21.1 kg, respectively were used [9]. where Ci is the concentration of THM i, (mg kg-1), IRi is the daily consumption rate (kg day-1) and BW is the body weight (kg) of the consumer. Likewise, average body weights for an adult and a child of 63 kg and of 21.1 kg, respectively were used [9]. stimated monthly intake (EMI) was calculated as per Eqn. (2): Estimated monthly intake (EMI) was calculated as per Eqn. (2): EMI (mg kg-bw-1 month-1) = EDI * 30…………….(2) EMI (mg kg-bw-1 month-1) = EDI * 30…………….(2) The contribution percentage allows obtaining a percentage value over the reference value (or health-based guidance value). It was calculated as per Eqn. (3): The contribution percentage allows obtaining a percentage value over the reference value (or health-based guidance value). It was calculated as per Eqn. (3): Contribution (%) = DI HBGV ∗ 100 … … … … (3) Contribution (%) = DI HBGV ∗ 100 … … … … (3) where DI is dietary intake, which could be either daily (EDI) or monthly (EMI), and HBGV is health-based guidance value, which could be the corresponding tolerable daily intake (TDI) or monthly intake (TMI). Tolerable daily intake (TDI) is the highest quantity of a toxic metal that a person can consume each day throughout a lifetime without an appreciable health risk while TMI is the highest quantity of a toxic metal that a person can consume monthly throughout a lifetime without an appreciable health risk. 2.4.1. Dietary Exposure Assessment 2.4.1. Dietary Exposure Assessment Estimated daily intake Estimated daily intake For the Cd, Cr, and Ni risk characterization, the TDI and TMI were used: Cd: 25 μg kg-bw-1 month-1 [10]; Cr: 300 μg kg- bw-1 day-1 [11] and Ni: 13 μg kg-bw-1 day-1 [12]. Equation 3 was used to estimate the contribution percentage of the estimated intakes to the TDI and TMI. 2.3.4. Quality Assurance and Control All apparatus and glassware used through the entire laboratory analysis were sterilized. They were first washed with tap water and liquid detergent in order to keep the analyte from contamination and ensure a grease-free surface. Then, they were soaked with deionized water and followed by rinsing with 0.5 N HNO3, and rinsed again with distilled water and a corresponding analyte solution to be analysed based on its necessity. 2.3.3. Determination of Toxic Metals The determination of Pb, Cd, Cr, Ni and As was performed with an atomic absorption spectrophotometer (AS-800, Perkin Elmer, USA) with a graphite chamber (HGA-800, Perkin Elmer, USA) (GF-AAS). Atomic absorption spectrophotometry (AAS) is the analytical method approved by Regulation 333/2007 (CE) modified by Regulation 836/2011 [8] for the official control of levels of lead, cadmium and mercury in food products. The determination of Hg was carried out with a cold vapour atomic absorption spectrophotometer (AS-800, Perkin Elmer, USA) (CV-AAS) with a flow injection system (FIMS-400, Perkin Elmer, USA). The instrumental wavelengths (nm) were: Cd (228.8), Pb (283.3), and Hg (253.7); the instrumental limits of quantification (LOQ) of the method were: Cd (0.013 mg/kg), Pb (0.040 mg/kg), and Hg (0.10 mg/kg). Instrumental conditions are shown in Table 2. Reference material (NIST SRM 1577 27 Magna Scientia Advanced Research and Reviews, 2024, 10(01), 025–037 Magna Scientia Advanced Research and Reviews, 2024, 10(01), 025–037 BL, Sigma Aldrich, Germany) was used for the determination of Hg. For Cd and Pb, the reference material NIST1567B Wheat four (Sigma Aldrich, Germany) was used. The recovery study, subjecting the reference material to the same conditions as the samples, yielded recovery values above 97% in all cases. 2.4. Methodology for Health Risk Assessment The method for health risk assessment is based on the following: 2.4.4. Margin of Exposure The risks to human health resulting from Pb and iAs exposure in consumed palm oil were characterized by applying the Margin of Exposure (MOE) method in line with the recommendations of the European Food Safety Authority [14]. Margin of exposure is the margin between the dose that caused a ten per cent increase in tumour frequency in animals and the dose that humans are normally exposed to. The MOE is used for assessing the risk of substances with genotoxic and carcinogenic properties. It is the ratio between the lower confidence level of benchmark dose (BMDL) (mg kg-bw-1 day-1 determined in experimental animals and the CDI (mg kg-bw-1 day-1). The benchmark dose was the lower limit of 99% confidence interval of the BMDL at the dose resulting in biological effect, or a predetermined measure for toxicity resulting in an adverse effect of 10% or 1% compared with the control group. The MOE value was calculated based on the Eq. (6): MOE = BMDL CDI … … … . (6) MOE = BMDL CDI … … … . (6) As report by the EFSA Panel, MOE values <104 are interpreted as possible concern, 104-105 as low concern, >105 as negligible concern as long as actions are taken to minimize further exposure, while >106 values are translated as negligible concern. According to EFSA Panel on Contaminants in the Food Chain (CONTAM Panel) the Pb current tolerable weekly intake (TWI) of 25 μg kg-bw-1 is no longer appropriate as there is no evidence for a threshold for critical lead-induced effects and confirmed by Joint FAO/WHO Expert Committee on Food Additives (JECFA) as well. Therefore, the following benchmark dose lower confidence limits (BMDLs) for lead (Pb) [16]:  BMDL01 for children (neurotoxicity) - 0.50 μg kg-bw-1 day-1; for adults (nephrotoxicity) - 0.63 μg kg-bw-1 day-1;  BMDL10 for adults (nephrotoxicity) - 0.63 μg kg-bw-1 day-1;  BMDL01 for adults (cardiovascular disorders) - 1.50 μg kg-bw-1 day-1. The EFSA Panel on Contaminants in the Food Chain (CONTAM Panel) established the lower limit of the benchmark dose (BDML01) for adults (cancer incidence) with inorganic As (iAs) induced lung cancer as the end point of toxicity effect between 0.3 to 8 µg kg-bw-1 day-1 for a 1% increased risk of cancer of the lung, skin and bladder, as well as skin lesions [17]. 2.4.4. Margin of Exposure In this study, according to the safety protection principle of risk assessment the benchmark dose lower confidence limits (BMDLs) for inorganic arsenic (iAs): BMDL01 for children and adults (carcinogenicity) - 0.3 µg kg-bw-1 day-1 2.4.2. Risk characterization The risk was characterized by two quantitative parameters namely lifetime cancer risk (LCR) [13] and margin of exposure (MOE) [14]. The risk was characterized by two quantitative parameters namely lifetime cancer risk (LCR) [13] and margin of exposure (MOE) [14]. 2.4.3. Lifetime Cancer Risk Estimates The lifetime cancer risk (LCR) of population groups in six selected states in Niger Delta, Nigeria caused by THM dietary intake was calculated based on Eq. (5). The lifetime cancer risk (LCR) of population groups in six selected states in Niger Delta, Nigeria caused by THM dietary intake was calculated based on Eq. (5). LCR = CDI * SF ………….(5) where ILCR= the incremental lifetime cancer risk of the dietary exposure (dimensionless); SF = oral cancer slope factor (Pb: 0.0085, Cd: 0.38, Cr: 0.5, Ni: 1.7, As: 1.5 kg day mg-1) [13]. According to the magnitude of LCR values, the LCR level is divided into three grades, including acceptable or inconsequential (LCR < 1 x 10-6), Cautionary (1 x 10-6 ≤ LCR ≤ 1 x 10-4) and Unacceptable risk (LCR > 1 x 10-4) [15]. According to the magnitude of LCR values, the LCR level is divided into three grades, including acceptable or inconsequential (LCR < 1 x 10-6), Cautionary (1 x 10-6 ≤ LCR ≤ 1 x 10-4) and Unacceptable risk (LCR > 1 x 10-4) [15]. Chronic daily intake The chronic daily intake (CDI) was calculated as per Eqn. (4): The chronic daily intake (CDI) was calculated as per Eqn. (4): CDI (mg kg −bw −1 day −1) = Ci ∗ EDI ∗ EFi ∗ ED ATc … … … (4) 28 Magna Scientia Advanced Research and Reviews, 2024, 10(01), 025–037 Where CDI is chronic daily dietary intake (mg kg-bw-1 day-1), EF is exposure frequency (day), ED is exposure duration (year) and ATc is the average time for carcinogen (ED * LT) (day-year), LT is lifetime expectancy (year). 3.1. Content of Toxic Metals in Crude Palm oil The quantification of TM content in edible palm oil is vital, since several TMs play major roles in various metabolic processes in the human body. Moreover, these TMs are toxic if consumed in excessive quantities. Table 1 shows the mean average concentration of toxic metals (Pb, Cd, Cr, Ni, As) in the CPOs samples collected from local producers in six selected states of states in Niger Delta, Nigeria. The mean concentrations of Pb, Cd, Cr, Ni and As were 0.823, 0.346, 1.746, 0.344 and 0.0640 mg kg-1, respectively. All the values, except As, exceeded permissible limits of international bodies such European Food Safety Authority (EFSA) and Joint FAO/WHO Expert Committee on Food Additives (JECFA). In general, the toxic metals analyzed follow the descending order of Cr > Pb > Cd = Ni > As. The contents of Pb in the samples were found to be in the range of 0.340 to1.50 mg kg-1, with an average value of 0.823 mg kg-1. The Pb contents obtained in CPO from the study area were significantly higher than the maximum tolerable limit set by European community and Joint FAO/WHO Expert Committee on Food Additives (JECFA) in vegetable oils (0.1 mg kg-1) [18, 19]. Table 1 Mean concentration (mg kg-1) of toxic metals in the crude palm oil samples from six stat in Niger Delta, Nigeria ntration (mg kg-1) of toxic metals in the crude palm oil samples from six states, six selected states a Table 1 Mean concentration (mg kg-1) of toxic metals in the crude palm oil samples from six states, six selected states in Niger Delta, Nigeria TM mean SD min max Maximum Levels EC [19] FAO/WHO [20] Pb 0.823 0.422 0.340 1.500 0.1 0.1 Cd 0.346 0.969 0.001 3.390 0.05 0.5 Cr 1.746 0.788 0.820 3.080 Ni 0.344 0.250 0.020 0.690 - 0.2 As 0.064 0.077 0.010 0.283 0.1 0.1 The contents of Pb in the samples were found to be in the range of 0.340 to1.50 mg kg-1, with an average value of 0.823 mg kg-1. The Pb contents obtained in CPO from the study area were significantly higher than the maximum tolerable limit set by European community and Joint FAO/WHO Expert Committee on Food Additives (JECFA) in vegetable oils (0.1 mg kg-1) [18, 19]. Lead is a cumulative element that has no biological role and have carcinogenic effects. 2.5. Statistical Analysis For every sample, each parameter was measured three times, and the average values for every state were given as mean ± SD. Multiple comparisons were performed using Kruskall-Wallis ANOVAs. The degree of linear dependence between the various parameters assayed was evaluated using the Beauvais-Pearson test. 29 Magna Scientia Advanced Research and Reviews, 2024, 10(01), 025–037 Magna Scientia Advanced Research and Reviews, 2024, 10(01), 025–037 The levels of Ni in the oil samples from the studied areas ranged from 0.020-0.690 mg kg-1 with a mean value of 0.344 mg kg-1. Except in Cross River State, the levels were higher than the maximum permissible limit (0.2 mg kg-1) set by World Health Organisation [20]. Trace amounts of Ni may be beneficial as an activator of some enzyme systems, influences iron absorption and metabolism and may be an essential component of the hemopoietin process [33]. However, Ni exposure is associated with adverse health effects, such as dermatitis, vomiting and nausea, cyanosis, gastrointestinal discomfort, pulmonary fibrosis, cardiovascular and kidney diseases, increased lung and nose cancer risk, and even death [34, 35]. The As content in the samples were in the range of 0.000 to 0.10 mg kg-1 with an average value of 0.058 mg kg-1. The obtained values fell far below the action level recommended by FAO/WHO of 0.5 mg kg-1 [20]. Arsenic (As) is a non- essential element. It occurs in several forms, of which inorganic arsenic is the most toxic form. Inorganic As (iAs, i.e., sum of As+3 and As+4) form is carcinogenic. Chronic As exposure due to its metabolism process might damage each human body organ; including respiratory, cardiovascular, neurological, gastrointestinal, hematological, renal, hepatic, developmental, and reproductive disorders and carcinogenic, mutagenic, and immunological effects such as hepatocarcinoma as well as cancers of the skin, skin lesions (in the form of hyperkeratosis, hyperpigmentation or hypopigmentation) [36, 37]. Some researchers in Nigeria have conducted several studies that showed metals (Pb, Cd, Cr, Ni, and As) in palm oil produced and sold in different parts of Nigeria. Table 2 shows the mean concentrations of heavy metals of this study compared with other studies in Nigeria. Some of the detected metal contents in these studies were reported below the permissible limits [38- 43]. 3.1. Content of Toxic Metals in Crude Palm oil Acute Pb exposure can induce appetite loss, fatigue, headaches, stomach discomfort, hypertension, and insomnia [21, 22] while chronic exposure affects multiple body systems, including the gastrointestinal, neurological, cardiovascular, haematological and renal systems [1, 23]. It causes sterility, neonatal mortality, morbidity, sub-cellular changes, increased systolic blood pressure and cardiovascular diseases in adults as well as neuro-developmental disorders (reduces intellectual performance and cognitive development) in children [24, 25]. Furthermore, Pb displaces Ca in bone tissues leading to bone formation mechanisms, alteration of compositional properties, and disruption of mineralization [1]. The concentrations of Cd in the samples were found to be in the range of ND (not detected) -3.390 mg kg-1, with an average value of 0.728 mg kg-1. All the investigated samples showed lower Cd levels, except at Edo State, than the recommended maximum permissible limits of 0.5 mg kg-1 [18]. Cadmium is a nonessential toxic element that has no biological role and classified as a human carcinogen. Cadmium absorbed through intake is known to bio-accumulate in the liver and kidney and the human body takes from 10 to 30 years to excrete this element [26]. The resultant health effects are reproductive deficiencies, kidney dysfunction, bone demineralization, skeletal damage, prostate proliferative changes as well as cancer [27- 30]. Chromium, an essential element of biological interest, can exist in two oxidation states (Cr+3 and Cr+6). Cr+3 acts as a critical cofactor in insulin action and plays an important role in the metabolism of lipid, glucose, and protein. Cr+6 is carcinogenic according to the International Agency for Research on Cancer [31] and acute Cr poisoning can be mutagenic and carcinogenic [24, 32] and the toxic effects of Cr+3 intake include skin rash, nose irritation, bleeding, upset stomach, kidney and liver damage. The highest and lowest contents of Cr were observed in samples from Rivers and Delta, respectively. Cr concentrations varied from 0.820 to 2.780 mg kg-1 with an average value of 1.515 mg kg-1. 30 Magna Scientia Advanced Research and Reviews, 2024, 10(01), 025–037 Correlations support the interpretation of meaningful measurements. This approach also helps to identify common factors, inducing the observation of metal relationships. Magna Scientia Advanced Research and Reviews, 2024, 10(01), 025–037 Table 2 Mean concentration of toxic metals in crude red palm oil and other studies in Nigeria Table 2 Mean concentration of toxic metals in crude red palm oil and other studies in Nigeria Pb Cd Cr Ni As References 0.823 0.346 1.746 0.344 0.064 This study 0.032 0.050 0.025 0.052 0.002 [38] 0.178 0.022 - - - [39] 0.039 0.645 0.178 0.26 - [40] - - - 0.38 - [41] ˂0.001 0.154 0.04 - 0.163 [42] 0.001 0.006 - 0.001 - [43] Table 2 Mean concentration of toxic metals in crude red palm oil and other studies in Nigeria Pb Cd Cr Ni As References 0.823 0.346 1.746 0.344 0.064 This study 0.032 0.050 0.025 0.052 0.002 [38] 0.178 0.022 - - - [39] 0.039 0.645 0.178 0.26 - [40] - - - 0.38 - [41] ˂0.001 0.154 0.04 - 0.163 [42] 0.001 0.006 - 0.001 - [43] 3.2. Pearson Correlation coefficient Table 3 The Pearson correlation coefficients of toxic metals in palm oil Pb Cd Cr Ni As Pb 1 Cd 0.501a 1 Cr 0.734a 0.205 1 Ni 0.756a 0.465b 0.672a 1 As 0.352 -0.109 0.393 0.394 1 a Correlation is significant at the 0.01 level (2-tailed); b Correlation is significant at the 0.05 level (2-tailed) Correlations support the interpretation of meaningful measurements. This approach also helps to identify commo factors, inducing the observation of metal relationships. A high value of the coefficient reveals a high linear correlation between the contents of two metals. Table 3 shows th Pearson correlation coefficient (r) between the metals in oil samples. Pearson correlation coefficients showed moderat positive correlations between Pb and Cr (0.734), Pb and Ni (0.756), and Cr and Ni (0.672). Overall, the correlatio 3.2. Pearson Correlation coefficient 3.2. Pearson Correlation coefficient 3.2. Pearson Correlation coefficient Table 3 The Pearson correlation coefficients of toxic metals in palm oil Pb Cd Cr Ni As Pb 1 Cd 0.501a 1 Cr 0.734a 0.205 1 Ni 0.756a 0.465b 0.672a 1 As 0.352 -0.109 0.393 0.394 1 a Correlation is significant at the 0.01 level (2-tailed); b Correlation is significant at the 0.05 level (2-tailed) Table 3 The Pearson correlation coefficients of toxic metals in palm oil Correlations support the interpretation of meaningful measurements. This approach also helps to identify common factors, inducing the observation of metal relationships. Correlations support the interpretation of meaningful measurements. This approach also helps to identify common factors, inducing the observation of metal relationships. Correlations support the interpretation of meaningful measurements. This approach also helps to identify common factors, inducing the observation of metal relationships. A high value of the coefficient reveals a high linear correlation between the contents of two metals. Table 3 shows the Pearson correlation coefficient (r) between the metals in oil samples. Pearson correlation coefficients showed moderate positive correlations between Pb and Cr (0.734), Pb and Ni (0.756), and Cr and Ni (0.672). Overall, the correlation A high value of the coefficient reveals a high linear correlation between the contents of two metals. Table 3 shows the Pearson correlation coefficient (r) between the metals in oil samples. Pearson correlation coefficients showed moderate positive correlations between Pb and Cr (0.734), Pb and Ni (0.756), and Cr and Ni (0.672). Overall, the correlation 31 Magna Scientia Advanced Research and Reviews, 2024, 10(01), 025–037 behaviour of metals in the studied samples remained noticeably diverse, which may be attributed to the disproportions of the origin of samples. 3.3. Exposure assessment of toxic metals via consumption of CPOs In order to evaluate dietary-related daily or monthly contaminant exposure in humans, the computed EDIs and EMIs were compared with their corresponding PTDIs and PTMIs, respectively. Table 4 summarizes the dietary intake (EDI: μg kg-bw-1 day-1; and EMI: μg kg-bw-1 month-1) values via consumption of 25 g day-1 of CPO in children and adults. It also shows the percentage of contribution to the provisional tolerable daily and monthly intakes (PTDIs/PTMIs) for both children and adults. The EMI values of Cd for adults and children for EDS were 36.32 and 27.57 μg kg-bw-1 month-1 respectively, and 145.3 and 110.3%, respectively above the PTMI values. These will cause major health hazard to both adult and children’s consumers from that area. The EDI values for other metals (Cr and Ni) for adults and children were considerably lower than their corresponding PTDIs, and represent contributions to PTDICr and PTDINi of 0.10 to 0.37% and 0.05 to 1.87% for adults and 0.07 to 0.28% and 0.04 to 1.2% for children, respectively and pose no health risk for both age-groups of consumers. Table 4 Estimated dietary intakes of toxic metals (Cd, Cr, and Ni) in crude palm oil compared with the tolerable intakes TM Parameter AKS BAS CRS DES EDS RVS For adults of body weight of 63 kg Cd EMI (µg kg-bw-1 month-1) 0.60 0.24 0.18 0.48 40.36 1.01 %TMI 2.38 0.95 0.74 1.90 161.43 4.05 Cr (III) EDI (mg kg-bw-1 day-1) 0.00 0.00 0.00 0.00 0.00 0.00 %TDI 0.34 0.26 0.15 0.11 0.20 0.33 Ni EDI (µg kg-bw-1 day-1) 0.21 0.15 0.04 0.12 0.16 0.14 %TDI 1.63 1.18 0.27 0.93 1.22 1.07 For children of body weight of 21.1 kg Cd EMI (µg kg-bw-1 month-1) 1.35 0.54 0.42 1.08 91.58 2.30 %TMI 5.40 2.16 1.67 4.32 366.31 9.18 Cr (III) EDI (mg kg-bw-1 day-1) 2.29 1.76 1.01 0.74 1.36 2.26 %TDI 0.76 0.59 0.34 0.25 0.45 0.75 Ni EDI (µg kg-bw-1 day-1) 0.48 0.35 0.08 0.27 0.36 0.32 %TDI 3.71 2.67 0.62 2.11 2.77 2.42 EDI: estimated daily intake; EMI: estimated monthly intake; TDI: tolerable daily intake; TMI: tolerable monthly intake. y intakes of toxic metals (Cd, Cr, and Ni) in crude palm oil compared with the tolerable intakes Table 4 Estimated dietary intakes of toxic metals (Cd, Cr, and Ni) in crude palm oil compared with 3.4.1. Cancer risk Table 5 is a summary of the cancer risk (CR) and the (∑CR) values for exposure to TMs due to consumption of 25 g day- 1 of CPO. The CR for Pb, Cd, Cr, Ni and As for children and adults ranged from were 1.94 x 10-7; 1.56 x 10-6; 7.86 x 10-6; 2.77 x 10-5 and 2.26 x 10-5, and 2.26 x 10-7; 1.82 x 10-6; 9.16 x 10-6; 3.23 x 10-5 and 2.63 x 10-5, respectively. The lifetime cancer risk (LCR) values of Pb and Cd, except at EDS, for the population across the states were insignificant, 1.79 x 10-5 - 7.26 x 10-5 below 1 x 10-6. In comparison, the cancer risk (CR and LCR) values of Cr, and Ni for all the states were within the “cautionary risk” range of 1.0 x 10-6 - 1.0 x 10-4, except As (1.88 x 10-4) for consumers from Akwa Ibom State. The LCRiAs for CPOs from Akwa Ibom and Delta states are above 1 x 10-4 while the total lifetime cancer risk (except Cross River State) are above the threshold of 1 x 10-4. The LCR values were in the following descending orders: AKS > DES > EDS = RVS > BAS > CRS. Palm oil samples from Akwa Ibom State pose the highest potential LCR while those from Cross River State pose the less LCR to consumers (figure 2). Table 5 is a summary of the cancer risk (CR) and the (∑CR) values for exposure to TMs due to consumption of 25 g day- 1 of CPO. The CR for Pb, Cd, Cr, Ni and As for children and adults ranged from were 1.94 x 10-7; 1.56 x 10-6; 7.86 x 10-6; 2.77 x 10-5 and 2.26 x 10-5, and 2.26 x 10-7; 1.82 x 10-6; 9.16 x 10-6; 3.23 x 10-5 and 2.63 x 10-5, respectively. The lifetime cancer risk (LCR) values of Pb and Cd, except at EDS, for the population across the states were insignificant, 1.79 x 10-5 - 7.26 x 10-5 below 1 x 10-6. In comparison, the cancer risk (CR and LCR) values of Cr, and Ni for all the states were within the “cautionary risk” range of 1.0 x 10-6 - 1.0 x 10-4, except As (1.88 x 10-4) for consumers from Akwa Ibom State. 3.4.1. Cancer risk 32 Magna Scientia Advanced Research and Reviews, 2024, 10(01), 025–037 5 Cancer (CR) and total cancer (∑CR) risk of toxic metals due to consumption of 25 g day-1 of CPO sample ected states in Niger Delta, Nigeria TM AKS BAS CRS DES EDS RVS Adults Pb 3.13E-07 2.49E-07 2.11E-07 1.14E-07 2.49E-07 2.21E-07 Cd 2.62E-07 1.05E-07 8.13E-08 2.10E-07 1.78E-05 4.46E-07 Cr 1.33E-05 1.03E-05 5.90E-06 4.33E-06 7.95E-06 1.32E-05 Ni 5.02E-05 3.62E-05 8.45E-06 2.86E-05 3.76E-05 3.29E-05 As 6.69E-05 1.18E-05 6.21E-06 3.94E-05 1.24E-05 1.45E-05 ∑CR 1.31E-04 5.86E-05 2.09E-05 7.26E-05 6.09E-05 6.12E-05 Children Pb 2.68E-07 2.13E-07 1.81E-07 9.77E-08 2.13E-07 1.90E-07 Cd 2.25E-07 9.00E-08 6.97E-08 1.80E-07 1.53E-05 3.82E-07 Cr 1.14E-05 8.80E-06 5.06E-06 3.71E-06 6.82E-06 1.13E-05 Ni 4.31E-05 3.10E-05 7.25E-06 2.46E-05 3.22E-05 2.82E-05 As 5.74E-05 1.01E-05 5.33E-06 3.37E-05 1.07E-05 1.24E-05 ∑CR 1.12E-04 5.02E-05 1.79E-05 6.23E-05 5.22E-05 5.25E-05 AKS - Akwa Ibom State, BAS- Bayelsa State, CRS - Cross River State, DES - Delta State, EDS - Edo State, RVS - River State re 2 Lifetime cancer risk (LCR) and total lifetime cancer risk (∑LCR) due to consumption of 25 g day-1 of pa samples from six selected states in Niger Delta, Nigeria Margin of exposure of Pb and iAs OE values of Pb for adults and for children from consumption of CPO (25 g day-1) were 1.46 to 6.43 (nephr s) and 3.47 to 15.32 (cardiotoxic effects), and 1.41 to 6.21 (neurotoxic effect), 1.77 to 7.83 (nephrotoxic e 22 t 18 63 ( di t i ff t ) ti l Lik i th MOE l f iA f ti f l Magna Scientia Advanced Research and Reviews, 2024, 10(01), 025–037 5 Cancer (CR) and total cancer (∑CR) risk of toxic metals due to consumption of 25 g day-1 of CPO samples from ected states in Niger Delta, Nigeria TM AKS BAS CRS DES EDS RVS Adults Pb 3.13E-07 2.49E-07 2.11E-07 1.14E-07 2.49E-07 2.21E-07 Cd 2.62E-07 1.05E-07 8.13E-08 2.10E-07 1.78E-05 4.46E-07 Cr 1.33E-05 1.03E-05 5.90E-06 4.33E-06 7.95E-06 1.32E-05 Ni 5.02E-05 3.62E-05 8.45E-06 2.86E-05 3.76E-05 3.29E-05 As 6.69E-05 1.18E-05 6.21E-06 3.94E-05 1.24E-05 1.45E-05 ∑CR 1.31E-04 5.86E-05 2.09E-05 7.26E-05 6.09E-05 6.12E-05 Children Pb 2.68E-07 2.13E-07 1.81E-07 9.77E-08 2.13E-07 1.90E-07 Cd 2.25E-07 9.00E-08 6.97E-08 1.80E-07 1.53E-05 3.82E-07 Cr 1.14E-05 8.80E-06 5.06E-06 3.71E-06 6.82E-06 1.13E-05 Ni 4.31E-05 3.10E-05 7.25E-06 2.46E-05 3.22E-05 2.82E-05 As 5.74E-05 1.01E-05 5.33E-06 3.37E-05 1.07E-05 1.24E-05 ∑CR 1.12E-04 5.02E-05 1.79E-05 6.23E-05 5.22E-05 5.25E-05 AKS - Akwa Ibom State, BAS- Bayelsa State, CRS - Cross River State, DES - Delta State, EDS - Edo State, RVS - River State e 2 Lifetime cancer risk (LCR) and total lifetime cancer risk (∑LCR) due to consumption of 25 g day-1 of palm oil samples from six selected states in Niger Delta, Nigeria Magna Scientia Advanced Research and Reviews, 2024, 10(01), 025–037 Cancer (CR) and total cancer (∑CR) risk of toxic metals due to consumption of 25 g day-1 of CPO samples from ted states in Niger Delta, Nigeria TM AKS BAS CRS DES EDS RVS Adults Pb 3.13E-07 2.49E-07 2.11E-07 1.14E-07 2.49E-07 2.21E-07 Cd 2.62E-07 1.05E-07 8.13E-08 2.10E-07 1.78E-05 4.46E-07 Cr 1.33E-05 1.03E-05 5.90E-06 4.33E-06 7.95E-06 1.32E-05 Ni 5.02E-05 3.62E-05 8.45E-06 2.86E-05 3.76E-05 3.29E-05 As 6.69E-05 1.18E-05 6.21E-06 3.94E-05 1.24E-05 1.45E-05 ∑CR 1.31E-04 5.86E-05 2.09E-05 7.26E-05 6.09E-05 6.12E-05 Children Pb 2.68E-07 2.13E-07 1.81E-07 9.77E-08 2.13E-07 1.90E-07 Cd 2.25E-07 9.00E-08 6.97E-08 1.80E-07 1.53E-05 3.82E-07 Cr 1.14E-05 8.80E-06 5.06E-06 3.71E-06 6.82E-06 1.13E-05 Ni 4.31E-05 3.10E-05 7.25E-06 2.46E-05 3.22E-05 2.82E-05 As 5.74E-05 1.01E-05 5.33E-06 3.37E-05 1.07E-05 1.24E-05 ∑CR 1.12E-04 5.02E-05 1.79E-05 6.23E-05 5.22E-05 5.25E-05 AKS - Akwa Ibom State, BAS- Bayelsa State, CRS - Cross River State, DES - Delta State, EDS - Edo State, RVS - River State Table 5 Cancer (CR) and total cancer (∑CR) risk of toxic metals due to consumption of 25 g day-1 of CPO samples from six selected states in Niger Delta, Nigeria AKS - Akwa Ibom State, BAS- Bayelsa State, CRS - Cross River State, DES - Delta State, EDS - Edo State, RVS - River State Figure 2 Lifetime cancer risk (LCR) and total lifetime cancer risk (∑LCR) due to consumption of 25 g day-1 of palm oil samples from six selected states in Niger Delta, Nigeria Figure 2 Lifetime cancer risk (LCR) and total lifetime cancer risk (∑LCR) due to consumption of 25 g day-1 of palm oil samples from six selected states in Niger Delta, Nigeria 3.4.1. Cancer risk The LCRiAs for CPOs from Akwa Ibom and Delta states are above 1 x 10-4 while the total lifetime cancer risk (except Cross River State) are above the threshold of 1 x 10-4. The LCR values were in the following descending orders: AKS > DES > EDS = RVS > BAS > CRS. Palm oil samples from Akwa Ibom State pose the highest potential LCR while those from Cross River State pose the less LCR to consumers (figure 2). Table 5 is a summary of the cancer risk (CR) and the (∑CR) values for exposure to TMs due to consumption of 25 g day- 1 of CPO. The CR for Pb, Cd, Cr, Ni and As for children and adults ranged from were 1.94 x 10-7; 1.56 x 10-6; 7.86 x 10-6; 2.77 x 10-5 and 2.26 x 10-5, and 2.26 x 10-7; 1.82 x 10-6; 9.16 x 10-6; 3.23 x 10-5 and 2.63 x 10-5, respectively. The lifetime cancer risk (LCR) values of Pb and Cd, except at EDS, for the population across the states were insignificant, 1.79 x 10-5 - 7.26 x 10-5 below 1 x 10-6. In comparison, the cancer risk (CR and LCR) values of Cr, and Ni for all the states were within the “cautionary risk” range of 1.0 x 10-6 - 1.0 x 10-4, except As (1.88 x 10-4) for consumers from Akwa Ibom State. The LCRiAs for CPOs from Akwa Ibom and Delta states are above 1 x 10-4 while the total lifetime cancer risk (except Cross River State) are above the threshold of 1 x 10-4. The LCR values were in the following descending orders: AKS > DES > EDS = RVS > BAS > CRS. Palm oil samples from Akwa Ibom State pose the highest potential LCR while those from Cross River State pose the less LCR to consumers (figure 2). Magna Scientia Advanced Research and Reviews, 2024, 10(01), 025–037 g day-1) for adults and children ranged between 0.00 to 104.16 (carcinogenic effects) and 0.00 to 126.70 (carcinogenic effects), respectively. An MOE value of 10,000 or higher is considered of low concern from a public health view point with respect to carcinogenic effect. Hence, a small MOE represents a higher risk than a larger MOE. Considering the BMDL of 0.63 µg kg-bw-1 day-1 (nephrotoxic effects) and 1.5 µg kg-bw-1 day-1 (cardiotoxic effects), the MOE values obtained indicate potential nephrotoxicity and cardiotoxicity due to Pb and carcinogenicity due to exposure to iAs for adults, and potential neurotoxicity, nephrotoxicity and cardiotoxicity (Pb) and carcinogenicity (iAs) for children. able 6 Margin of Exposure (MOE) of Pb and iAs due to daily consumption of 25 g of palm oil from six selected stat Niger Delta, Nigeria Sample states Pb iAs CDI MOE MOE MOE CDI MOE Neurotoxicity Nephrotoxicity Cardiotoxicity Carcinogenicity For adult of body weight of 63 kg AKS 0.452 1.393 3.316 0.064 4.681 BAS 0.359 1.754 4.177 0.011 26.526 CRS 0.306 2.062 4.909 0.275 1.092 DES 0.165 3.826 9.108 0.931 0.322 EDS 0.359 1.754 4.177 0.012 25.200 RVS 0.319 1.972 4.696 0.014 21.600 For children of body weight of 21.1 kg AKS 1.027 0.487 0.614 1.461 0.145 2.063 BAS 0.815 0.614 0.773 1.841 0.026 11.690 CRS 0.693 0.721 0.909 2.163 0.014 22.211 DES 0.374 1.338 1.686 4.014 0.086 3.507 EDS 0.815 0.614 0.773 1.841 0.027 11.105 RVS 0.725 0.690 0.869 2.069 0.03 9.519 AKS - Akwa Ibom State, BAS- Bayelsa State, CRS - Cross River State, DES - Delta State, EDS - Edo State, RVS - River State posure (MOE) of Pb and iAs due to daily consumption of 25 g of palm oil from six selected states e 6 Margin of Exposure (MOE) of Pb and iAs due to daily consumption of 25 g of palm oil from six s ger Delta, Nigeria posure (MOE) of Pb and iAs due to daily consumption of 25 g of palm oil from six selected states a 3.5. Margin of exposure of Pb and iAs The MOE values of Pb for adults and for children from consumption of CPO (25 g day-1) were 1.46 to 6.43 (nephrotoxic effects) and 3.47 to 15.32 (cardiotoxic effects), and 1.41 to 6.21 (neurotoxic effect), 1.77 to 7.83 (nephrotoxic effects) and 4.22 to 18.63 (cardiotoxic effects), respectively. Likewise, the MOE values of iAs from consumption of palm oil (25 33 References [1] Amadi CN, Bocca B, Ruggieri F, Ezejiofor AN, Uzah G, Domingo JL, Rovira J, Frazzoli C, Orisakwe OE. Human dietary exposure to metals in the Niger delta region, Nigeria: Health risk assessment, Environmental Research, 2022; 207(1):1-9, Doi:10.1016/j.envres.2021.112234. [1] Amadi CN, Bocca B, Ruggieri F, Ezejiofor AN, Uzah G, Domingo JL, Rovira J, Frazzoli C, Orisakwe OE. Human dietary exposure to metals in the Niger delta region, Nigeria: Health risk assessment, Environmental Research, 2022; 207(1):1-9, Doi:10.1016/j.envres.2021.112234. [2] Chu YL, Jiang SJ. Speciation analysis of arsenic compounds in edible oil by ion 353 chromatography–inductively coupled plasma mass spectrometry. Journal of Chromatography 2011; 354 A:1218, 5175–5179. [3] Manjusha R, Shekhar R, Kumar SJ. Ultrasound assisted extraction of Pb, Cd, Cr, Mn, Fe, Cu, Zn from edible oils with tetramethylammonium hydroxide and EDTA followed by determination using graphite furnace atomic absorption spectrometer. Food chemistry, 2019; 294:384-389. doi:10.1016/j.foodchem.2019.04.104 [3] Manjusha R, Shekhar R, Kumar SJ. Ultrasound assisted extraction of Pb, Cd, Cr, Mn, Fe, Cu, Zn from edible oils with tetramethylammonium hydroxide and EDTA followed by determination using graphite furnace atomic absorption spectrometer. Food chemistry, 2019; 294:384-389. doi:10.1016/j.foodchem.2019.04.104 [4] Bakircioglu D, Bakircioglu Kurtulus Y, Yurtsever S. Comparison of extraction 340 induced by emulsion breaking, ultrasonic extraction and wet digestion procedures for determination of metals in edible oil samples in Turkey using ICP-OES. Food Chemistry, 2013; 342(138):770–775. [4] Bakircioglu D, Bakircioglu Kurtulus Y, Yurtsever S. Comparison of extraction 340 induced by emulsion breaking, ultrasonic extraction and wet digestion procedures for determination of metals in edible oil samples in Turkey using ICP-OES. Food Chemistry, 2013; 342(138):770–775. [5] Khan A, Khan S, Khan MA, Qamar Z, Waqas M. The uptake and bioaccumulation of heavy metals by food plants, their effects on plants nutrients, and associated health risk: A review. Environmental Science and Pollution Research, 2015; 22:13772–13799. https://doi.org/10. 1007/s11356-015-4881-0 [6] Lacoste F. Undesirable substances in vegetable oils: anything to declare? Oilseeds & fats Crops and Lipids, 2014; 21(1):1-9, DOI: 10.1051/ocl/2013060 [7] Rubio-Armendariz C, Paz S, Gutiérrez ÁJ, González-Weller D, Revert C, Hardisson A. Human Exposure to Toxic Metals (Al, Cd, Cr, Ni, Pb, Sr) from the Consumption of Cereals in Canary Islands. Foods, 2021; 10(6):1158. https://doi.org/10.3390/foods10061158 [8] European Commission (EC). 4. Conclusion The results obtained from the samples of edible crude oil palm oil from six selected states of the Niger Delta, Nigeria exceed the maximum permissible limits for toxic metal contents established by the World Health Organization and European Union. The Pb exposure from consumption of crude palm oil could have toxic effects such as nephrotoxicity and cardiotoxicity in both adults and children as well as neurotoxicity in children. Also, consumption of crude palm oil could have carcinogenicity due to exposure to iAs for both adults and children. The calculated total cancer risk (∑CR) (for both children and adult consumers for both age groups from Akwa Ibom State) and total lifetime cancer (∑LCR) values from exposure to Pb, Cd, Cr, Ni, and iAs exceeded the benchmark (10-5), which illustrated potential carcinogenesis among consumers. It can be concluded that the crude palm oil samples were not free of any chemical element contamination. The results highlight the need to set up educational for the artisanal or local producers as well as monitoring and surveillance programs for the safety of consumers of palm oil in these selected states in Niger Delta, in particular and Nigeria in general. 34 34 Magna Scientia Advanced Research and Reviews, 2024, 10(01), 025–037 Disclosure of conflict of interest The Authors declare that there is no conflict of interest The Authors declare that there is no conflict of interest Compliance with ethical standards Acknowledgement Acknowledgement The authors express their profound appreciation to the locals who provided security and transportation during sample collections from the six states in South-South zone of the Niger Delta. 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https://openalex.org/W3014183306	https://www.mdpi.com/1424-8220/20/7/2055/pdf?version=1586769659	English	null	Game Theory in Mobile CrowdSensing: A Comprehensive Survey	Sensors	2,020	cc-by	13,781	Received: 20 February 2020; Accepted: 2 April 2020; Published: 6 April 2020 Abstract: Mobile CrowdSensing (MCS) is an emerging paradigm in the distributed acquisition of smart city and Internet of Things (IoT) data. MCS requires large number of users to enable access to the built-in sensors in their mobile devices and share sensed data to ensure high value and high veracity of big sensed data. Improving user participation in MCS campaigns requires to boost users effectively, which is a key concern for the success of MCS platforms. As MCS builds on non-dedicated sensors, data trustworthiness cannot be guaranteed as every user attains an individual strategy to benefit from participation. At the same time, MCS platforms endeavor to acquire highly dependable crowd-sensed data at lower cost. This phenomenon introduces a game between users that form the participant pool, as well as between the participant pool and the MCS platform. Research on various game theoretic approaches aims to provide a stable solution to this problem. This article presents a comprehensive review of different game theoretic solutions that address the following issues in MCS such as sensing cost, quality of data, optimal price determination between data requesters and providers, and incentives. We propose a taxonomy of game theory-based solutions for MCS platforms in which problems are mainly formulated based on Stackelberg, Bayesian and Evolutionary games. We present the methods used by each game to reach an equilibrium where the solution for the problem ensures that every participant of the game is satisfied with their utility with no requirement of change in their strategies. The initial criterion to categorize the game theoretic solutions for MCS is based on co-operation and information available among participants whereas a participant could be either a requester or provider. Following a thorough qualitative comparison of the surveyed approaches, we provide insights concerning open areas and possible directions in this active field of research. Keywords: mobile crowdsensing; Internet of things; trustworthiness; user incentives; game theo sensors sensors sensors Review Game Theory in Mobile CrowdSensing: A Comprehensive Survey Venkat Surya Dasari 1,†, Burak Kantarci 1,,† , Maryam Pouryazdan 2,†, Luca Foschini 3,† and Michele Girolami 4,† Venkat Surya Dasari 1,†, Burak Kantarci 1,,† , Maryam Pouryazdan 2,†, Luca Foschini 3,† and Michele Girolami 4,† 1 School of Electrical Engineering and Computer Science, University of Ottawa, Ottawa, ON K1N 6N5, Canada; vdasa047@uottawa.ca 2 Watts Water Technologies, North Andover, MA 01845, USA; maryam.pouryazdan@wattswater.com 3 Department of Computer Science at the University of Bologna, 40136 Bologna, Italy; luca.foschini@unibo.it 4 National Council of Research ISTI-CNR Italy, 56124 Pisa, Italy; michele.girolami@isti.cnr.it * Correspondence: burak.kantarci@uottawa.ca; Tel.: +1-613-562-5800 † All authors contributed equally to this work. School of Electrical Engineering and Computer Science, University of Ottawa, Ottawa, ON K1N 6N5, Canada; vdasa047@uottawa.ca Watts Water Technologies, North Andover, MA 01845, USA; maryam.pouryazdan@wattswater.com Department of Computer Science at the University of Bologna, 40136 Bologna, Italy; luca.foschini@unibo.i National Council of Research ISTI-CNR Italy, 56124 Pisa, Italy; michele.girolami@isti.cnr.it Correspondence: burak.kantarci@uottawa.ca; Tel.: +1-613-562-5800 All th t ib t d ll t thi k † All authors contributed equally to this work. 1. Introduction With the massive deployment and wide adoption of the Internet of Things (IoT), the number of connected devices is tremendously increasing. This phenomenon enables monitoring and control of almost all physical infrastructures without dedicating signiﬁcant amount of (ﬁxed) sensing and computing resources. Sensed data plays a predominant role in IoT: According to Cisco, 2.5 quintillion bytes of daily data generation and 30 billion IoT-connected devices are forecast by the year 2020 [1]. Thus, it is vital to supplement the existing dedicated sensing infrastructures via sustainable and Sensors 2020, 20, 2055; doi:10.3390/s20072055 www.mdpi.com/journal/sensors 2 of 23 Sensors 2020, 20, 2055 cost-efﬁcient non-dedicated sensing solutions such as participatory or opportunistic sensing via smartphone sensors [2]. By taking advantage of embedded sensors in smart phones, sensed data can be collected in high volumes, and can be processed in real time in support of the IoT-based services. Widely known as Mobile CrowdSensing (MCS), this process is envisioned to be an integral component of IoT systems [3,4]. MCS empowers citizens with smart devices participate in the collection of sensed data by rewarding them for their contribution/effort. In MCS campaigns, sensed data is collected from various locations through built-in sensors of the mobile devices by either implicitly (opportunistic) or explicitly (participatory) recruiting users (see Figure 1). Acquired data is aggregated and processed, analyzed and visualized to support various services that lead to smart and sustainable spaces. As an example, a project by Microsoft, namely Nericell, aims to monitor trafﬁc and driving conditions of roads through smart phone sensors [5]. In another project, called Mobile Century, GPS enabled mobile phones are recruited for trafﬁc monitoring purposes [6]. Wide usage of social networking applications and skyrocketing number of mobile phones pave the way for MCS applications to be employed for various types of services such as quality of living, emergency preparedness, health care, smart transportation, environmental monitoring and public safety [7–10]. In Reference [11], an indoor application of MCS is considered where positioning and orientation information of landmark objects are estimated through mobile crowd-sensed data so to obtain indoor ﬂoor maps. Figure 1. An illustration of data acquisition in a Mobile CrowdSensing (MCS) environment, border encompassing task Ti denotes sensing task coverage. User with smart device in hand represents their preference to participate in sensing campaign. Figure 1. An illustration of data acquisition in a Mobile CrowdSensing (MCS) environment, border encompassing task Ti denotes sensing task coverage. 1. Introduction User with smart device in hand represents their preference to participate in sensing campaign. Despite the beneﬁts listed above, the realization of robust MCS systems is still challenging. From the standpoint of an MCS platform, dependability/trustworthiness and veracity assurance of crowd-sensed data is a grand challenge as data acquisition involves anonymous and mainly unreliable sources. It is worth mentioning that lack of dependability or veracity of crowd-sensed data may occur due to various reasons such as intentional manipulation (i.e., malicious actions by adversaries) or 3 of 23 Sensors 2020, 20, 2055 malfunctioning of sensors, which might just be a temporary situation. Lack of ground truth at the MCS platform makes this problem further challenging. Nevertheless, to prevent MCS campaigns from vulnerabilities, effective user recruitment is the only tool that an MCS platform can use. More speciﬁcally, we consider that some possible solutions to prevent the lack of dependability [12–14] are: to design systems able to manage and update the reputation of the involved users by adopting optimal user’s recruiting policies; to dynamically assess and keep updated the trustworthiness of the involved users; and to adopt recruitment policies able to detect or event to predict the existence of non-cooperative users, that is, users not providing useful data for the MCS system. Reputation based selection schemes are widely used by researchers in such a way that reputation level became an added attribute/constraint for recruiting a participant [15–19]. From the standpoint of the participants, privacy is a primary concern that may de-incentivize users against participating in MCS campaigns. To overcome that potential issue and improve participation in sensing campaigns, related work proposes effective methods of incentivization, which can be in the form of either monetary or non-monetary (e.g., entertainment, badges, etc.) [20,21]. While the incentive’s being monetary or non-monetary is not within the scope of this study, it should be recognized that sensing, computing and transmission costs incurred during MCS campaigns call for compensation. Moreover, MCS requires diversity of participants in a campaign so as to have the fused sensory data as accurate as possible with respect to the actual value to increase the quality of crowdsensed data. Reddy et al. [22] examine participant behaviour in a range of monetary payoffs, and results indicate that provoking competition between participants may not help obtain high quality information through collaboratively sensed data. It is reported that proper design of micro-payments can lead the useful participation. 1. Introduction A recent work introduced the concept of trading of tasks between users by considering the time required to tackle a task by a traded user [23]. Results supported the hypothesis that proposed mechanism could reduce total sensing time. Finally, another important issue from the participant perspective is energy consumption. It is anticipated that in the near future it will be possible to power up smart devices through wireless signals. Vamsi et al. [24] were successful in delivering power to battery less temperature and camera sensors using Wi-Fi signals. This might extremely reduce the cost of sensing and facilitates data collection process by encouraging more number of users to participate in sensing campaigns without worry of battery. Moreover mobility of users in MCS improves sensing coverage area. To tackle all above contrasting goals, several works in the recent literature are proposing to adopt a Game Theory approaches, where each participant aims to maximize its proﬁt, namely the objective function of each player. Game theory analyzes the situation where multiple players are involved in, and a player’s gain does not only depend on their own strategy. In MCS, game can be formulated either among participants or between the platform and the participants. In the former case, participants of CrowdSensing compete among themselves to achieve higher proﬁt and the decision of each participant will be inﬂuenced by the actions of others. In the latter case, decisions of the platform over user incentives are affected by participants’ strategies. In either case, the proﬁt can apply in the form of maximizing the score, the reputation or the beneﬁts obtained from users. In this survey, we review various game theoretical methods applied in MCS that help to reach an equilibrium in a competition for gaining higher utility between service requester and service provider. We start with presenting a brief introduction to the typical game theory models. Next, we classify the game models applied in MCS on the basis of co-operation. This classiﬁcation is further extended by the level of information, that is, complete or incomplete information, available among players involved in the formulated game. Finally, the paper presents open research areas that can be considered for future research in this ﬁeld. The rest of the paper is organized as follows. 2.1. Privacy and Security Privacy is one of the most crucial issue that every participant is concerned with. For instance, information gathered from search history of an individual plays a crucial role in displaying advertisements in social networking applications. According to Hewlett Packard study there exist an average of 25 vulnerabilities that a device can be attacked through, and 70% of IoT devices are vulnerable to these attacks [25]. The shared data may contain sensitive information of mobile device as well as private information of users [26] which leads to privacy leaks. Access control should be permitted based on pre-established trust between user and service provider [27]. Moreover most of the MCS campaigns involve location-based tasks such as trafﬁc congestion control, which reveal daily mobility patterns of users. Many location based services need to access the location of a user, Sastry et al. [28] proposed a simple technique to avoid malicious activities that utilize location based comforts without the physical presence. Most popular location spotting service is done by GPS (Global Positioning System) with the accuracy of less than 5 m, and can introduce vulnerabilities for attackers sending strong radio signals [29]. To illustrate potential privacy breaches, a route prediction model was proposed by Patterson et al. [30] based on historical behaviour of a user. It is worth noting that, contextual pattern analysis do not only reveal residential pattern by can lead to inferring the residential address of a user [31]. Yang et al. presented various privacy and security threats that might occur from crowd-sourced information. As a countermeasure, a CrowdSourcing architecture was proposed where task is fragmented before CrowdSourcing it [26]. In order to deal with disclosure of location information, the authors of Reference [32] proposed a model which distorts the precision of user location, and results show that privacy of user can be achieved with the cost of quality of data. In another work, Talasila et al. [33] proposed a framework to improve reliability of data based on location by eradicating malicious user with the help of Bluetooth discovery (i.e., every user should reside in range of each other to sense the same task). In an empirical study on human participants, the authors of Reference [34] found out that an obvious user discomfort in terms of privacy while majority of users seemed to prefer legal regulations due to lack of trust in human-initiated or automated mechanisms that claim to ensure privacy preservation. 2.1. Privacy and Security At this point, it is worth noting that there exists a trade-off between privacy preservation and data trustworthiness. Thus, obtaining trustworthy data from participant requires the crowd-sensed information to be analyzed by exploiting many features, which may cause de-incentivizing privacy concerns [35]. In order to cope with the requirements, Wang et al. [36] proposed a framework named ARTSense (Anonymity, Reputation, Trust) to preserve private data–particularly location and identity of users– while ensuring trustworthiness based on reputation of recruited users. On the other hand, the authors of References [37,38] argue that most of the users do not oppose to share their location. 2. Background and Challenges in Mobile Crowdsensing Despite of potential beneﬁts of MCS, few primary challenges must be considered for building a sustainable system: privacy and security, data quality, trustworthiness, energy consumption and incentives for participants. This section presents needed background material together with main emerging guidelines and solutions against those main ﬁve challenges under diverse scenarios. 1. Introduction In Section 2, we give some needed background material about the MCS paradigm, while in Section 3 we introduce a general model for Game theory that can be applied to MCS focusing, especially, on some existing models for cooperative and non-cooperative games. We then present in Section 4 a possible adaptation of the Game theory Sensors 2020, 20, 2055 4 of 23 model to the MCS paradigm by presenting a survey of existing works addressing such approach. In Section 5, we envision new directions and main open research areas in Game theory in MCS. Finally, Section 6 concludes the paper reporting ﬁnal discussions. 2.2. Quality of Data Quality of data is difﬁcult to maintain in MCS due to various reasons for example, malfunctioning of sensors, environmental conditions. Low quality data costs hundreds of million USD loss only for US business as reported by the Data Warehousing Institute [39]. Moreover to obtain sensor data over a large area, high number of participants should be recruited; which comes at the expense of 5 of 23 Sensors 2020, 20, 2055 increased sensing budget for the CrowdSensing platform as the users expect incentives for providing sensing as a service. On the other hand data collected more than certain threshold may not help in the improvement of the data quality [40]. To overcome this problem, the authors of Reference [41] proposed an estimation strategy for the locations that cannot be covered by recruited participants. The proposed approach uses the neighbouring cell values however data obtained may not be accurate which may lead to the degradation of quality. The degradation of quality may result in poor decisions. The authors of Reference [42] showed that the higher the quality of data, the lesser the storage space required in the cloud. Liu et al. [43] proposed a mechanism in which a task is assigned to a group that has high credit index, and that can deliver the required quality denoted by the Quality of Information (QoI) satisfaction index. Gong et al. [44] presented task assignment mechanisms under various scenarios to improve data quality and reported that the quality of data is associated to the task duration, data collection times and spatiotemporal coverage. On the other side missing data inference should be done in an error-free manner at the server to meet the quality requirements. Re-sampling techniques such as bootstrapping could be used to approximate the quality from sparse data to a reasonable value [45]. Kong et al. [46] used spatiotemporal correlations to deduce missing environmental data. Furthermore, the size of participant population can be reduced by selecting minimum number of cells to sense and pursuing inference of missing data [45]. Moreover, in the case of participatory sensing in MCS campaigns, data quality may vary due to lack of experience in participants (e.g., capturing blur photos, recording audio with microphone covered etc.). In addition to all, computational power and sensory capabilities of each smart phone varies, thus not all participants might be able to deliver the same level of quality of sensed data [47]. 2.2. Quality of Data As most of the quality estimation mechanisms do not treat sensor error and user reliability differently, this naive assumption of not decoupling user and the smartphone in terms of reliability may lead to inaccurate evaluation of participants during the recruitment process. To address this challenge, the authors of Reference [48] proposed an algorithm that exploits the confusion matrix and separates user and device errors to quantify the quality of a participant. 2.3. Trustworthiness Trustworthiness plays a vital role in participatory sensing, since data obtained from participants influence decision making. Trust can be established in the form of either trust by reliability or trust by decision [49]. The former is computed on an entity, and denotes the probability by which that entity reports truthfully whereas the latter stands for the situation where an entity is trusted irrespective of its possible errors. For example trust on anonymous participants could be considered as reliability trust whereas trust on anchored sources/dedicated sensors would be analogous to decision trust. For this reason MCS needs more reliable trusted participants to pursue an action since it is based on non-dedicated sensors, and the acquired data can be used for the development of critical CrowdSensing applications such as health care [50], and pollution monitoring [51]. Sybil attacks are also possible in MCS. Chang et al. [52] proposed a framework which detects changes towards heavy traffic intensity through active and passive tests so as to conduct trust credit assessment based on previous records to recommend whether or not the traffic intensity is injected by Sybil nodes. Another issue in CrowdSensing is the level of reliability on security mechanisms, that is, to which extent can the security mechanism be trusted? Huang et al. used Gompertz function to design a reputation system that ensures trustworthiness in the data received [53]. Pouryazdan et al. [54] presented a detailed comparison among various trustworthy mechanisms where users are assumed to be static (i.e., not mobile). One of the other proposed approaches to tackle trustworthiness is the use of collaborative reputation scores that is, the combination of statistical reputation [55] as well as vote-based reputation score [56] along with anchor assistance. Chen et al. [35] proposed supervised and unsupervised trust weight calculation methods for crowdsourcing, which could also be adopted by MCS systems. Another work related to trustworthiness [48] shows the proposed model ’mPASS’ could perform effectively even under malicious environments. Reliability/trustworthiness was evaluated 6 of 23 Sensors 2020, 20, 2055 by analyzing the credibility of a user with sensor accuracy. Wengdong et al focused on considering attentiveness/interest of user along with quality of the data submitted, in order to evaluate the most trusted user. Attentiveness/willingness is deﬁned as the shortest time window in which a user joins an MCS campaign to undertake sensing tasks [57]. 2.4. Energy Battery is the source of energy, driving force of a mobile device. Regulation of energy consumption has almost equal concern in MCS with the protection of security and privacy of users. Sensing, raw data processing and transferring of collected data require battery power. Battery level reduction of a device depends on many factors such as Operating System (OS) used by the device, type and number of sensors used for sensing and moreover GPS consumes different amount of energy whether device is indoor or outdoor [59]. Energy consumption and rate of data transfer varies depending on the type of network mode we choose (3G, WiFi, LTE, etc.) [60] and sampling frequency [61]. Liu et al. [43] proposed a distributed energy management scheme in which users are penalized for using high energy even though they might be maintaining the quality of data. Anjomshoa et al. [62] considered residual battery level of devices in user recruitment since low residual energy is one of the reason for opting out from a sensing campaign. In Reference [63], Liu et al. claimed that region of interest change from time to time and there may be a case where the platform cannot ﬁnd users having sufﬁcient battery life, which would eventually result in low Quality of Information (QoI). In order to acquire better QoI in the aforementioned case, an energy aware selection model was proposed where users’ sampling rate varied based on battery level so as to enable the platform to recruit low-battery users to meet the required quality of information. The authors of Reference [64] dealt with network protocols to prevail energy efﬁcient transmission with the deployment of static nodes in sensing environment. Tomasoni et al. [65] analyzed energy conserving capabilities of different data collection frameworks and claimed that energy consumption during reporting of data is higher compared to sensing of data. As reported by Tomasoni et al., battery draining occurs not only while sensing and transferring data but also while waking up the phone so Lane et al. [66] proposed the Piggyback CrowdSensing (PCS) to overcome this challenge. In PCS, sensing begins when other applications such as location detection during operation of Google maps or phone calls to avoid energy overhead due to waking up the device, which consumes considerable amount of energy at high sensing cost. 2.3. Trustworthiness On the other hand, a participant may not exhibit the same level of effectiveness in all types of tasks. That being said, it is possible to experience situations when recruitment of a highly reputed participant might be inadequate. Amintoos and Kanhere considered this challenge and calculated various reputation based parameters such as quality of data, region of interest, ﬁeld of expertize and also response time. Their simulation results showed 15% increase of trust in proposed scheme when compared to baseline mechanisms [58]. 2.5. Incentives Participants tend to drop from sensing campaign because of not being selected. Lee et al. [70] proposed a participant virtual credit concept in support of non-winners so that they have high chances of winning (i.e., being selected) in future rounds. Ofﬂine incentive mechanisms may not work in real time as they assume user could wait until the completion of biding by other users. To overcome this challenge, the authors of References [71,72] proposed an online incentive mechanism. As most of the users are busy in rush hours, participants for sensing campaign may not be sufﬁcient which increases dependence on available participants. An attempt to attract users in aforementioned case, Wang et al. [73] proposed a two level heterogeneous incentive mechanism by allocating rewards based on spatio-temporal popularity of certain task. 2.6. Agent-Based Strategies Besides leveraging user-provided data, it is also imperative to incorporate agents with MCS campaigns [74]. Normally, mobile agents would be deployed in a crowdsensing setting to collect and/or query data [75] or perform other tasks as overviewed below. With the advent of augmented and virtual reality communications, the authors of Reference [76] propose to consolidate an agent-based approach with crowdsensing campaigns so to enable virtual and augmented reality in a MCS setting. Leveraging agents is also considered to cope with the untrusted nature of MCS servers. With this in mind, the authors of Reference [77] propose a distributed agent-based strategy to ensure a certain level of privacy where crowd-sourced data goes through every agent following perturbation of aggregated statistics at the agents. 2.5. Incentives CrowdSensing participants utilize their own resources to sense and send the required information. It is obvious that no participant wish to sacriﬁce their time, energy and cost without receiving any beneﬁt. The system should be designed to motivate crowd to participate by offering incentives. Moreover design of an incentive mechanism in any CrowdSensing environment is crucial because of ﬁxed budgets in most of the MCS platforms. Every user in system should not be incentivized fairly as quality of service or contribution provided to the task varies from user to user. The following question remains—How could one quantify the quality of data received from a user without any ground truth? The authors of Reference [67] estimated quality of data and level of contribution using information theory on top of Expectation Maximization (EM) algorithm. Some tasks do not compromise in data quality, other may need more coverage area. Effective incentive mechanisms can tackle this situation up to a great extent. In case of data quality, incentivizing users based on their reputation might be a possible approach whereas in the case of large coverage, availability of user is of great concern. 7 of 23 Sensors 2020, 20, 2055 To attract users to a sensing location, incentives offered should not be less than the actual sensing costs, this price imbalance is one of the challenges presented in a detailed survey of incentive mechanisms in To attract users to a sensing location, incentives offered should not be less than the actual sensing costs, this price imbalance is one of the challenges presented in a detailed survey of incentive mechanisms in MCS [68]. To address this situation, recruiting participants based on mobility patterns is proposed by Reddy et al. [69]. One of the popular approaches to recruit participants for lower budget is through reverse auction. Participants tend to drop from sensing campaign because of not being selected. Lee et al. [70] proposed a participant virtual credit concept in support of non-winners so that they have high chances of winning (i e being selected) in future rounds this price imbalance is one of the challenges presented in a detailed survey of incentive mechanisms in MCS [68]. To address this situation, recruiting participants based on mobility patterns is proposed by Reddy et al. [69]. One of the popular approaches to recruit participants for lower budget is through reverse auction. 3. Presentation of Common Game Theory Models This section provides an overview of main Game Theory approaches, by also elaborating on the possible advantages of their application in the MCS context and introducing the main taxonomy directions used to analyze all proposals surveyed in the next section. Game theory has been a useful tool to model the behavior of participants and platforms to maximize their utilities [78]. Primary reason behind formulating these interactions in the format of games stems from the selﬁshness and rationality of participants. Thus, in the existence of selﬁsh and rational users, cooperativeness of the participants can be ensured through effective games [79]. A typical use case that leverage game theory is the design of incentives to recruit a sufﬁcient size of participant population for the sensing campaigns [80]. In addition, pricing strategies for the value of the data so to eliminate untrutful participants in MCS campaigns [81] is to address the security and trustworthiness concerns in MCS campaigns. As mentioned earlier, energy consumption is one of the roadblocks against wide adoption of MCS. With this in mind, game theory serves as a useful tool to make a reasonable compromise between the utility of the MCS platform (i.e., delivery ratio of crowd-sensed data) and energy consumption of participating devices [82]. Another challenge for MCS systems is the privacy concerns of participants. To this end, games need to be formulated between participants and the MCS platform in order to maximize the privacy requirements of the participants while maximizing the utility of the platform [83]. The three major components that make up the game models are players, strategies and payoffs. A player denotes a decision making entity whereas a strategy is analogous to a set of rules for decision under various situations. Payoff can be either a reward or loss that players experience when they implement their respective strategies. Figure 2 presents the popular models in game theoretic solutions. Game theoretic solutions are examined under ﬁve categories: co-operative/non-co-operative games, information games, evolutionary games, static/dynamic games, and zero/non-zero games. 8 of 23 Sensors 2020, 20, 2055 Among these ﬁve categories, information games can further be broken down into two sub-categories: complete/incomplete information games and perfect/imperfect information games. In the following, we decided to organize the section structure according to the above categorization; hence, each subsection will detail a category, and we further split the information games subsection into two parts. Figure 2. Classiﬁcation of game theoretic models. 3. Presentation of Common Game Theory Models Figure 2. Classiﬁcation of game theoretic models. 3.1. Co-Operative and Non-Co-Operative Games The primary criterion for the classiﬁcation of games is based on the co-operation between players. Winning strategy of a player varies from one game to another. When users co-operate with each other in order to receive a better payoff, a co-operative game is formulated. In a co-operative game, players receive assistance from third parties in order to enable co-operation ensuring through sanctions and incentives between players instead of defecting others. This is to avoid any dishonesty in participation. Co-operative game assumes that participants can achieve certain outcomes among themselves through co-operative agreements. Participants/players aim at common goals and interests that enable them to establish trust and co-operate. Co-operation will be successful when payoff or gain obtained by co-operation is considerably higher than the individual efforts of the players. In mobile CrowdSensing campaigns, participants are encouraged/incentivized to co-operate by the data collector/platform so as to maximize their utility [84]. One way to impose co-operation in the prisoner’s dilemma is to repeat the game [85] which results in better equilibrium than competing with each other. On the other hand, a game is said to be non-co-operative if players cannot form clusters required to enable co-operation. Non-co-operative game treats each participant as a unit of analysis as it assumes each participant acts with respect to their self interest whereas participants of a co-operative game form groups to obtain better payoffs, and the unit of analysis is more often a group/subgroup of participants. 3.2.1. Perfect and Imperfect Information Games In games with perfect information, participants have knowledge about previous decisions made by other participants of a game [86]. Tic-tac-toe and ultimatum games are possible examples of perfect information games. In an ultimatum game [87], one of the players (say player 1) receives payment 9 of 23 Sensors 2020, 20, 2055 and proposes a split ratio to another player involved (say player 2). Player 2 can choose to accept or reject the proposed offer, and if rejected by player 2, none of the players receives the payment. In perfect information games only one player moves at each timestamp by observing the decision of the other player. Chess can be one of the popular example for perfect information game. In the mobile networks context, with the help of the perfect information games, the problem of having selﬁsh users in multi-packet slotted aloha was analyzed in Reference [88], whereas an imperfect information game would constitute common knowledge of possible strategies and type of the player but not the actions opted by others. 3.2.2. Complete and Incomplete Information Games As stated earlier, information is the major source of reference while making a decision. Participants of games with complete information can be equipped with the knowledge of possible strategies and the payoffs of others. Unlike the complete information games, in incomplete information games, payoffs and strategies of other participants are not completely known. Incomplete information games can be further classiﬁed as symmetric and asymmetric. Asymmetric incomplete information is a case where every player has some private information which is unaware of others. In a game of asymmetric information, a player’s gain mainly depends on amount of information possessed by the player [89]. 3.4. Static and Dynamic Games Games are typically represented in either normal or extensive form [92]. Games in which players make decisions simultaneously without knowledge of others decisions are known as static games. Prisoners dilemma, sealed auction bid are some of the examples of a static game [93]. This form is comprised of list of participants, list of strategies for each player and payoffs for each participant and represented in matrix as follows. In a dynamic game, choices are made sequentially over time, or game is repeated [93]. Best example to illustrate dynamic game is the checkers game where each player have multiple possibilities to make a move based on the previous move of an opponent [94]. Players engaged in a sequential game make a move by anticipating the final outcome, that is, prefer to obtain final long term gain rather than short term. Participants in a game make decisions based on the information available to them. 3.3. Evolutionary Games Evolutionary games are highly competitive where players can update their strategies to increase their gain/payoff. A repeated game does not guarantee that a winning strategy remain winning. For example in classical game theory either defectors (in a non-co-operative game ) or co-operators (in co-operative game) will get higher payoff. But in the case of evolutionary games, this phenomenon may vary from time to time. The change in strategies may sound evolutionary games as unstable. There can be an evolutionary stable strategy when adopted by almost every member of the group, and no other external person can disrupt the system with a new strategy. It might be possible to predict user behavior when a player repeats his/her set of strategies [90]. Repeated game falls under category of evolutionary games. Maintaining good reputation may be an asset and helps to get better payoff under repeated games [91]. 3.5. Zero Sum and Non Zero-Sum Games Zero-sum games are one of the cases in constant sum games, where gain of one participant has a negative effect on other participants’ payoff [95]. At any point of the game, the overall gain of participants is always summed up to zero whereas in constant sum game, it is a ﬁxed value. Since each participant in zero-sum games completely oppose others’ interests, there is no scope of collaborative strategies, moreover it lists under strictly competitive games. Most of the sporting games come under strictly competitive in zero-sum games. Some of the classic examples of zero sum game are chess followed by boxing and so forth. In contrast, if proﬁt and loss of all players do not add up to 10 of 23 Sensors 2020, 20, 2055 zero/constant sum, then it ends up being a non-zero sum game. Here, participants strategies are neither collaborative nor completely opposite, also called mixed strategies that is, participants partially compete with each other and partially co-operate for better payoffs. In mixed strategy games, one participants gain does not necessarily result in others’ loss [96]. 4. Game Theory in MCS This section proceeds by stating the motivation of using Game Theory in MCS followed by subsections that present surveyed works dividing the in those adopting co-operative and non-co-operative game approaches under complete and incomplete information. Let us also anticipate that Table 1 will provide and outline pros and cons of each surveyed solution with the type of game used. Figure 3, instead, synthesize all main issues addressed by surveyed solutions according to the game theoretic approaches. Figure 3. Main issues addressed by surveyed solutions according to the game theoretic approaches. Figure 3. Main issues addressed by surveyed solutions according to the game theoretic approaches. 11 of 23 Sensors 2020, 20, 2055 Table 1. An overview of research publications with the description of game involved. 4. Game Theory in MCS Reference Type of Game Co-Operation Information Pros Cons [97] Bayesian game Non-co-operative Incomplete Considered Arbitrary selection asymmetric with allpay auction users [98] Repeated game Non-co-operative Incomplete Recruitment Requestors of speciﬁc task forced to expertees pay for poor service [99] Coalition game Yes Complete Energy, cost Truthfulness in effective sensing reputation is left as future work [100] Co-operative game between- Yes Incomplete Budget depend Assumes every user and platform on quality of device have equal data obtained sensing capabilities [101] Thesus (based on Bayesian- Non-co-operative Complete and Incentives are Believed that efforts Nash equilibrium) Incomplete based on effort, exerted by user is truthfulness same for every task [102] CSRS (Non-co-operative game) Non co-operative Complete Adaptable for Believed transmission crowded tasks cost is same for all [103] Stackelberg Game Non-co-operative Complete and Maximum User Assumes all users Incomplete Utility on par target for longterm with Platform gains [104] Stackelberg Game Non-co-operative Incomplete Uses WiFi to Not considered reduce cost user trustworthiness [105] Evolutionary game Non-co-operative Incomplete Examined Data reporting delay dynamic nature may occur due late of users convergence [106] Stackelberg game Yes Complete and Effective online Considered sensing Incomplete incentive cost for any task is mechanisms equal [107] Stackelberg game Yes Complete, Various Incentive Not considered Symmetrically, mechanisms for negligence of users Asymmetrically CrowdSensing, while sensing the Incomplete computing information [108] One-shot repeated game Yes Complete Low sensing cost All users in sensing with adequate campaign receive users part of payments [109] Multi-leader Stackelberg game Non-co-operative Incomplete Analyzes pricing Not considered competition resource variation between crowd among crowd sourcers sourcers [110] Stackelberg Bayesian game Non-co-operative Complete and Promotes early Agressive payments Incomplete contributions [111] Three stage stackelberg game Yes Complete Promotes Relies largely on co-operation social relationships with low cost of users The motivation in applying game theory in MCS is to cope with the competitive behavior exhibited Table 1. An overview of research publications with the description of game involved. The motivation in applying game theory in MCS is to cope with the competitive behavior exhibited by participants to get higher utilities/beneﬁts. Data requester aims to get high quality, trustworthy data within low budget whereas users aim to be compensated for their sensing service. Moreover, gain and loss in opportunistic sensing does not entirely depend on oneself rather it also relies on other participants’ strategies and moves. 4. Game Theory in MCS Hence game theory could be beneﬁcial in order to analyze this competitiveness among sensing participants, sensing requesters. Sensors 2020, 20, 2055 12 of 23 CrowdSourcing takes advantage of peoples interest in playing games and formulates games through which required information is acquired. Dion et al. investigated the impact of a virtual reward on data quality and satisfaction of the user [112]. Three types of virtual rewards (badges, points and no reward) were considered. Results show that users are well motivated when rewarded through badges and moreover quality of data has improved due to virtual rewards in crowdsourcing. Cyber physical social systems (CPSS) play a crucial role in improving the quality of life, some of the prominent examples of cyber physical systems are smart grids, autonomous vehicles and medical monitoring. With this in mind, the authors of Reference [113] took a step forward and introduced an incentive mechanism for improvement in CrowdSourcing for CPSS, where users were classiﬁed under three categories (malicious, speculative and honest users), and an auction game was modeled where compensation was provided based on user reputation while users aim to reduce their sensing costs in order for them to be selected. Authors assume that faults from sensors are negligible. Zhi-Gang et al. focused on the impact of game history on users’ preference to co-operate in a game [84]. In evolutionary games, participants study strategies of predecessor and modify their decisions for the next round of game. Indeed, formulating an evolutionary game is at the expense of extra memory usage overhead. In order to investigate the memory effect on co-operation, authors consider three different dilemma games (Prisoner’s Dilemma, Snowdrift game and Stag Hunt game ) and make the following conclusion. The users/participants with memory unconditionally follow the strategy of a participant whose payoff is higher than his/her own, and posses less co-operation ratio compared to the agents whose history is randomly initialized. As massive connectivity will leverage the concept of Industrial Internet of Things, Industry 4.0 can also leverage the concept of mobile CrowdSensing as stated in Reference [114]. Since users are eager to increase their payoff data quality and overall performance would be demolished. To maintain social welfare in games between requester and sensing user, the authors of Reference [115] proposed a zero-determinant strategy to introduce a linear relationship between co-players’ pay-offs without them considering each other’s strategy. 4. Game Theory in MCS The results show that the proposal outperforms co-operative, defective and random strategies adopted by the requester. 4.1. Co-Operative Games in MCS Co-operation among players of game can happen only if both players believe in beneﬁt of collaborating with each other, Figure 4 depicts the same. Here information among players might be sensing cost, quality of data, user biding, payoffs, strategies and so forth, depending the context. Co-operation can occur between users or between data requesters and users since all the participants who are involved in MCS are parts of the game. Co-operation between requester and user leads to ﬂexibility in the budget resulting in veracity of the data obtained [100]. In addition, conﬁguring co-operative games in MCS helps in maintaining the balance in the number of participants [99,108]. A detailed and synthetic view of all solutions surveyed in the following is available in Table 1 and Figure 3. 13 of 23 Sensors 2020, 20, 2055 Figure 4. Figure describing types of co-operation among participants. Figure 4. Figure describing types of co-operation among participants. 4.1.1. Co-Operative Games in MCS with Complete Information Jaimes et al. [108] study the problem of participants dropping out from sensing system when the platform persistently excludes them from sensing campaigns. This is not a desired situation for the system since it is preferred to have more number of participants for high value in the crowd-sensed data. To overcome this issue, a co-operative incentive mechanism was introduced in which former winner tries to share some of their proﬁt with neighbors in order to keep their bids higher and as a consequence making the winner win again. If one neighbour rejects the offer, the winner reduces their bid however rejection can happen if and only if the minimum proﬁt of user i is greater than the proposed offer or at least one neighbour’s bid is less than the winner’s bid. Proﬁt is shared among the winner’s neighbours based on their bids. Here, participants co-operate rather than competing with each other so that each of them can beneﬁt from participating in the game. However this approach can be limited to only repeated games. In co-operative spectrum sensing secondary users try to be rational and do not join co-operative sensing campaign unless motivated by some incentives. To address this issue, the authors of Reference [99] proposed a co-operative game based time optimization model where each player alter their own time strategy to maximize co-operative sensing and users are recruited based on their current reputation value. Yang et al. 4.1. Co-Operative Games in MCS [111] proposed a social incentive mechanism by promoting co-operation between users for better quality of information. A three stage Stackelberg game is designed where in additional stage, players exert pressure on their friends based on difference between current strategy and strategy at Nash equilibrium to get high payoff. Also proved that proposed social co-operative mechanism is cost effective where decrease in quality of information effects everyone since gathered information is shared by everyone, e.g., Live trafﬁc updates. 4.1.2. Co-Operative Games in MCS with Incomplete Information 4.1.2. Co-Operative Games in MCS with Incomplete Information In order to improve data quality in mobile CrowdSensing Yang et al. [100] proposed a quality estimation model through unsupervised learning followed by surplus sharing module designed as a co-operative game between users and platform where payments for users are calculated based on Shapley value and. The main motivation here is platform does not have ﬁxed budget but surplus Sensors 2020, 20, 2055 14 of 23 14 of 23 earned by platform depend on users quality of contributions. In oder to evaluate data trustworthiness authors proposed a truth estimation model that performed better compared with other heuristic models. Moreover they assume quality data providers dominate at the start of truth estimation model and all users have homogeneous devices. Formulation of co-operative game between platform and users helps for realistic dynamic ﬂow budget. 4.1.3. Co-Operative Games in MCS Considering Both Complete and Incomplete Information Luo et al. [106] state that traditional incentive mechanisms have not taken into account users performing in diverse multiple tasks according to priority of task. Four incentive mechanisms were introduced with the help of stackelberg game based on information( complete and incomplete) of user and type of tasks (homogeneous or heterogeneous). They took a step ahead and calculated time complexity for their proposed algorithms. Results show that reward function based on number of users favors the platform, whereas rewards based on the value of a task is proﬁtable for users. Duan et al. [107] focused on with incentive mechanisms for both acquisition of data and for distributed computing. They assumed that users are homogeneous in reporting data but differ in their computational power then considered two cases (i) complete information/symmetrically incomplete information( knowledge about cost of sensing is same for client and user), (ii) asymmetrical incomplete information (only client is unaware of sensing cost). They formulated reward based incentive mechanism for case-(i) with the help of the two stage Stackelberg game, where in stage-1 client advertise total reward and minimum number of collaborators and users under a game probably reach Nash equilibrium. Besides that contract based incentive mechanism was used for case-(ii) to incentivize users based on their efﬁciency in which contract is proposed by client and users accept contract only if it is proﬁtable. 4.2.1. Non-Co-Operative Games in MCS with Complete Information The authors of Reference [102] took advantage of concept of peer to peer communication to overshadow the draw back of high cost and poor adaptability of traditional centralized server with increase in number of tasks. User store the sensed data and share among themselves for a reasonable incentive, assuming that different tasks have same size of data interpreting that upload and download costs are almost same for every user which may not be true in practical scenario. Since every user would like to get maximum beneﬁt in every transaction they formulated a non-co-operative game using Wardrop equilibrium (notation of Nash equilibrium for games over networks) and user payoffs are determined based on quality, revenue sharing scheme. 4.2. Non-Co-Operative Games in MCS Non-co-operation among players appear when co-operation does not yield to maximum payoffs in a competitive games. Co-operation among players may not solve optimal price determination [102] for data collected, that satisfy both user and requester. Non- co-operation among users indirectly compel users to provide high quality data [103,109], improving the effort of users [101]. Researchers determine solutions from Equilibrium obtained from Non-co-operative game. 4.2.1. Non-Co-Operative Games in MCS with Complete Information 4.2.2. Non-Co-Operative Games in MCS with Incomplete Information Peng et al. [105] anticipate the possibility of competition between crowd sourcers and proposed an evolutionary based algorithm where users choose a crowd sourcer dynamically to sense and share data. Accordingly if the payment received is less than the average payoff by that particular crowd sourcer, user selects another probable crowd sourcer until he settle. Where in case of crowd sourcers rivalry in process of attracting users, they tend to increase the budget which leads to decrease in proﬁt. Authors treated this rivalry as non-co-operative game and solution is presented as Unique Nash equilibrium, where every crowd sourcer have similar budget and equal distribution of users among themselves. Sensors 2020, 20, 2055 15 of 23 Former incentive mechanisms haven’t took a detailed look into the cost of wiﬁand its availability in order to provide adequate incentives to the users. Data providers( users) using mobile data have different sensing cost and coverage area compared to Wi-Fi users, which are to be considered to make an effective payoff. Cheung et al. [104] introduced delay sensitive MCS by formulating a two stage stackelberg game to model interactions between platform and user. However user recruitment strategies are not clearly mentioned and it is hard to platform to have complete information of wiﬁ availability and costs for each and every user in real scenarios. Usually, sensing campaigns contain diverse users with enclosed personal information (for example, sensing cost). The intuitive game formed is, platform would try to increase its utility by recruiting users for cheaper price if complete information of user is known, but users make sure that it is conﬁned. The authors of Reference [97] analysed this challenge and came with an optimal solution where users payoff is determined by his amount of contribution that helps in increase of platform /cloud sourcer utility and rewards are based on prize tuple. They also mention that heterogeneous users with incomplete information behave like if they have homogeneous setting that is, individually rational. y Another incentive mechanism was proposed by Xie et al. [98] based on requesters rating on received data. They formulated reputation protocol which eliminates users by considering history of low quality data. In order to quantify the minimum payoff offered to the high quality data, authors used repeated game to characterize users behaviour by enclosing users skill as a private information. 5.1. Evolutionary Games Reaching to an equilibrium state in evolutionary games may take too long as strategies of user change dynamically with the outcome of every iteration until they get a satisﬁed payoff. This nature of game may sometimes leads to unstable system. It is important to consider the time taken to acquire stability while designing game. Outcome of game could be unpredictable due to changing strategies of rational users. Opted strategy should be robust such that it can compete with itself along with succeeding against others strategy [116]. 5.4. Co-Operative Games Enabling complete information within users in a co-operative game may lead to loss of platform utility. An appropriate example of this scenario could be [108] where in a reverse auction, neighbours bidding information is accessible to users. This may lead to combined attack strategy by users in desire of receiving higher rewards. However complete information with co-operation would be fruitful if game is between people who are familiar with each other. Most of the sensing participants may not be comfortable to co-operate with strangers. On the other hand user may not be completely sure of beneﬁt from collaboration because of dubious rationality of others. 4.2.2. Non-Co-Operative Games in MCS with Incomplete Information Effect of requesters bias in rating the received data was mentioned as future work. Dong et al. [109] subdivided crowd sourcer into sensing platform, task originator considering that both are taken care by different companies in real world. Authors represented the competition between multiple platforms as dynamic non-co-operative game. Strategies of other players in this game are unknown, each platform resolve to a pricing strategy based on response of other platforms and task originators. The interaction between platform and task originator is formulated as multi leader stackelberg game (since there exist multiple platforms). At ﬁrst platforms propose the price and then task originator have a strategy to maximize its utility by opting the optimal platform, based on quality of service. The solution is given as Nash equilibrium at which no player can improve his utility by changing his strategy alone and is determined by the proposed iterative algorithm. 4.2.3. Non-Coperative Games in MCS Considering Both Complete and Incomplete Information Jin in Reference [101] introduce an incentive mechanism named Theseus which generates a non-co-operative game where users opt different level of effort in sensing. User payoffs are based on their quality of data which is paramaterized by error probability (calculated from truth discovery algorithm) . By this they assure that at Bayesian Nash Equilibrium all users try to utilize their maximum efforts to provide high quality data. Many incentive mechanisms were proposed in CrowdSensing, out of which contract based model is one of them. Li et al. [103] designed two different quality aware incentive mechanisms for full information(QUAC-F) and incomplete information(QUAC-I). In case of full information ( cost function, ability distribution and risk attitude, etc.) stackelberg game was induced in which platform is leader and a contract based approach for incomplete information. Xu et al. [110] proposed an incentive mechanism based on the time stamp of joining in sensing task. Motive behind this model is to receive more efforts from the early user by providing better incentives to the early user. They formulated two games one with complete information and other is incomplete (i.e., contributors joining time is a private information). In order to demonstrate the competition between users, stackelberg bayesian game is designed and incentive mechanisms were introduced to show the disparity between early and late users with unique bayesian equilibrium for each mechanism. Sensors 2020, 20, 2055 16 of 23 16 of 23 5.2. Nash Equilibrium Game theory assumes users always behave rationally, each and every individual is sufﬁciently intelligent to take an optimal self-interested decision. This assumption may be impractical in CrowdSensing, as it deals with several human beings each of them having different level of thinking ability. For instance most of the users would like to adopt best strategy whereas some greedy, naive users may be inclined to risk so there would be a chance of getting higher payoffs, which leads to unpredictable equilibrium. 5.3. Stackelberg Game Most of the non-co-operative games in CrowdSensing have incorporated stackelberg game. Similar to other games stackelberg game also have limitation of assuming that players behave in bounded rationality. Moreover it assumes that players can perfectly observe leaders strategy, which may not be true in dealing with humans [117] due to difference in abilities to observe. Followers deviation from optimal strategy could degrade utility of platform. In addition to that [118], assume that players opt a strategy which favours the platform when tie between strategies appear. At equilibrium follower could try to understand, decrypt the strategy of leader [119], which leads to loss for leader in following game. 5. Open Research Areas This section, without any pretence of being exhaustive, highlights open research areas that we deem crucial to address still open issues in applying game theory to MCS. In particular, the following section covers ﬁve possible research areas in the ﬁeld of game theory, namely Evolutionary games, Nesh Equilibrium, Stackelberg Game, Co-operative games and Non-cooperative games. 5.1. Evolutionary Games 5.5. Non-Cooperative Games One of the major barrier for building non-co-operative games in MCS is excessive competition with non-co-operation may lead to destruction in motivation of user to participate in sensing campaigns. This effects availability of sensing users for CrowdSensing. Furthermore the trade-off between individual beneﬁt( by non-co-operation) of user and collaboration beneﬁt should be addressed because of various inﬂuential factors that effect rational thinking. Sensors 2020, 20, 2055 17 of 23 17 of 23 6. Conclusion and Discussion Mobile CrowdSensing (MCS) has recently become an emerging paradigm for large-scale distributed sensing campaign. MCS takes advantages of several advancements in the ﬁeld of sensing devices, network capabilities, communication infrastructures and data analytic knowledge. This survey focuses on the adoption of the Game theory in order to model how users can be involved into the MCS loop. Game theory provides interesting comparison between a player and a data provider. Users are players whose goal is to maximize a possible utility function by performing actions that beneﬁt the MCS campaign and, at the same time, increase this function. Of course, the Game theory approach can fail in predicting the human behaviour since he/she may suddenly act in different way irrespective of the better outcome. Moreover, players might fail to apply the best of all possible strategies to get higher payoff, this happens since a sensing user (ordinary people) may not understand game, as a result a platform may lose a high trustworthy user. Apart from exceptions, Game theory provides a better solution in a self-intended competitive environment. Furthermore, co-operation could not be a better option for tasks which require data instantaneously without any delay since establishment of co-operation between user might cause delay. The combination of a Game theory with the MCS paradigm represents a promising approach for increasing the effectiveness of the data collection campaign. However, we consider that some existing barriers still represent the major barrier for a massive recruitment of uses. As a meaningful example, we consider issues related to the management of personal information and how such data are preserved by the MCS organizers. Moreover, the user of a MCS mobile app for collecting data might induce scepticism from the end-users by limiting their adoption. 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W2919544927.txt	https://hal.science/hal-02101661/document	en		The host galaxy of GRB 980425/SN1998bw: a collisional ring galaxy	Monthly Notices of the Royal Astronomical Society	2,019	cc-by	11,622	The host galaxy of GRB 980425/SN1998bw: a collisional ring galaxy M. Arabsalmani, S. Roychowdhury, T.K. Starkenburg, L. Christensen, E. Le Floc’H, N. Kanekar, F. Bournaud, M.A. Zwaan, J.P.U. Fynbo, P. Møller, et al. To cite this version: M. Arabsalmani, S. Roychowdhury, T.K. Starkenburg, L. Christensen, E. Le Floc’H, et al.. The host galaxy of GRB 980425/SN1998bw: a collisional ring galaxy. Monthly Notices of the Royal Astronomical Society, 2019, 485 (4), pp.5411-5422. �10.1093/mnras/stz735�. �hal-02101661� HAL Id: hal-02101661 https://hal.science/hal-02101661 Submitted on 1 Mar 2022 HAL is a multi-disciplinary open access archive for the deposit and dissemination of scientific research documents, whether they are published or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. 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. Distributed under a Creative Commons Attribution 4.0 International License MNRAS 485, 5411–5422 (2019) doi:10.1093/mnras/stz735 Advance Access publication 2019 March 14 The host galaxy of GRB 980425/SN1998bw: a collisional ring galaxy M. Arabsalmani,1,2,3‹ S. Roychowdhury ,4,5 T. K. Starkenburg ,6 L. Christensen ,7 E. Le Floc’h,1,2 N. Kanekar ,8 F. Bournaud,1,2 M. A. Zwaan,9 J. P. U. Fynbo ,10 P. Møller 9 and E. Pian11 1 IRFU, CEA, Université Paris-Saclay, F-91191 Gif-sur-Yvette, France Paris Diderot, AIM, Sorbonne Paris Cité, CEA, CNRS, F-91191 Gif-sur-Yvette, France 3 School of Physics, University of Melbourne, Victoria 3001, Australia 4 Jodrell Bank Centre for Astrophysics, School of Physics & Astronomy, The University of Manchester, Oxford Road, Manchester M13 9PL, UK 5 Institut d’Astrophysique Spatiale, CNRS, Université Paris-Sud, Université Paris-Saclay, Bât. 121, F-91405 Orsay Cedex, France 6 Flatiron Institute, 162 Fifth Avenue, New York NY 10010, USA 7 Dark Cosmology Centre, Niels Bohr Institute, University of Copenhagen, Juliane Maries Vej 30, DK-2100 Copenhagen Ø, Denmark 8 National Centre for Radio Astrophysics, Tata Institute of Fundamental Research, Pune University, Pune 411007, India 9 European Southern Observatory, Karl-Schwarzschild-Strasse 2, D-85748 Garching bei München, Germany 10 Cosmic Dawn Center, Niels Bohr Institute, University of Copenhagen, Juliane Maries Vej 30, DK-2100 Copenhagen Ø, Denmark; DTU-Space, Technical University of Denmark, Elektrovej 327, DK-2800 Kgs. Lyngby, Denmark 11 INAF, Astrophysics and Space Science Observatory, via P. Gobetti 101, 40129 Bologna, Italy 2 Université ABSTRACT We report Giant Metrewave Radio Telescope (GMRT), Very Large Telescope (VLT), and Spitzer Space Telescope observations of ESO 184−G82, the host galaxy of GRB 980425/SN 1998bw, that yield evidence of a companion dwarf galaxy at a projected distance of 13 kpc. The companion, hereafter GALJ193510-524947, is a gas-rich, star-forming galaxy with a star formation rate of 0.004 M yr−1 , a gas mass of 107.1±0.1 M , and a stellar mass of 107.0±0.3 M . The interaction between ESO 184−G82 and GALJ193510-524947 is evident from the extended gaseous structure between the two galaxies in the GMRT H I 21 cm map. We find a ring of high column density H I gas, passing through the actively star-forming regions of ESO 184−G82 and the GRB location. This ring lends support to the picture in which ESO 184−G82 is interacting with GALJ193510-524947. The massive stars in GALJ193510524947 have similar ages to those in star-forming regions in ESO 184−G82, also suggesting that the interaction may have triggered star formation in both galaxies. The gas and star formation properties of ESO 184−G82 favour a head-on collision with GALJ193510-524947 rather than a classical tidal interaction. We perform state-of-the-art simulations of dwarf– dwarf mergers and confirm that the observed properties of ESO 184−G82 can be reproduced by collision with a small companion galaxy. This is a very clear case of interaction in a gammaray burst host galaxy and of interaction-driven star formation giving rise to a gamma-ray burst in a dense environment. Key words: gamma-ray burst: general – galaxies: interactions – galaxies: ISM – galaxies: kinematics and dynamics – galaxies: star formation – radio lines: galaxies. 1 I N T RO D U C T I O N Long-duration gamma-ray bursts (GRBs) are luminous explosions in the Universe, with powerful energy releases that make them detectable back to when the first stars and galaxies were formed E-mail: maryam.arabsalmani@cea.fr, maryam.arabsalmani@unimelb. edu.au (e.g. Tanvir et al. 2009). For a few seconds, these extremely bright explosions release the energy that our Sun emits in its whole lifetime (see Piran et al. 2013). Their short durations (a few seconds to minutes) and enormous energy releases can be explained by radiation from highly relativistic outflowing particles with Lorentz factors >100 (see Piran 2004, and references therein). Such outflowing jets can be powered by rotational energy tapped from the compact remnants, magnetars or black holes, of the corecollapse of massive stars (Usov 1992; Woosley 1993; MacFadyen & C 2019 The Author(s) Published by Oxford University Press on behalf of the Royal Astronomical Society Downloaded from https://academic.oup.com/mnras/article/485/4/5411/5380812 by CNRS - ISTO user on 01 March 2022 Accepted 2019 February 25. Received 2019 February 16; in original form 2018 December 12 5412 M. Arabsalmani et al. MNRAS 485, 5411–5422 (2019) 2017), this WR region appears to have been formed in a recent episode of star formation (Hammer et al. 2006; Le Floc’h et al. 2012). GRB 980425 occurred in an H II region 800 pc from this WR region. The GRB H II region contains young and massive stars with estimated ages between 5 and 8 Myr (Krühler et al. 2017). Fynbo et al. (2000) proposed that interactions could have triggered the recent star formation episode in ESO 184−G82. However, extensive multiwavelength studies ESO 184−G82 and its surroundings did not yield any sign of interactions or a companion galaxy. Christensen et al. (2008) mapped the H α emission from the host and found its velocity field to show ordered rotation without any signature of a disturbance. Foley et al. (2006) studied the field of ESO 184−G82 to search for possible companions interacting with the GRB host, but found all of the observed galaxies in the field to lie at significantly greater distances than ESO 184−G82. They concluded that ESO 184−G82 is an isolated dwarf galaxy and interactions with other galaxies are not responsible for its star formation. H I 21 cm mapping studies with radio interferometers allow the possibility of tracing the spatial distribution and velocity fields of the neutral hydrogen in nearby galaxies. Such H I 21 cm studies of GRB host galaxies provide a powerful tool to directly test the hypothesis that an interaction might have triggered the star formation that gave rise to the GRB. In Arabsalmani et al. (2015), we used the Giant Metrewave Radio Telescope (GMRT) to map the H I 21 cm emission from ESO 184−G82, finding its gas disc to be disturbed, while the global gas properties of the galaxy appeared similar to those of local dwarfs. This was the first tentative evidence that interactions or a merger event might indeed have played a role in the recent star formation activity of ESO 184−G82. In this paper, we present deep GMRT H I 21 cm emission observations of ESO 184-G82, which allow us to study the structure of the atomic gas in the vicinity of GRB 980425 in detail, with high spatial resolution. We combine our H I 21 cm mapping data with optical and infrared imaging studies to glean further information on the galaxy’s star formation history. We also use state-of-the-art simulations to compare the observed properties with a model of a merger event. This paper is organized as follows. The observations and data reduction are presented in Section 2. The H I 21 cm mapping results are discussed in Section 3, while Section 4 provides details on the companion galaxy identified in this paper. Next, Section 5 compares the observed H I 21 cm morphology of ESO 182-G82 and its companion to that expected in a simulation of the merger of two disc galaxies. Sections 6 and 7 contain, respectively, a general discussion and a summary of our results. 2 O B S E RVAT I O N S A N D DATA R E D U C T I O N 2.1 H I 21 cm emission observations We used the L-band receivers of the GMRT to map the H I 21 cm emission of ESO 184−G82 on six consecutive days between 2016 March 17 and 2016 March 22 (proposal no: 29076; PI: Arabsalmani). The observations used the GMRT Software Backend, with a bandwidth of 4.167 MHz, centred at 1408.246 MHz, and sub-divided into 512 channels, yielding a velocity resolution of 1.7 km s−1 and a total velocity coverage of ≈885 km s−1 . The southern declination of ESO 184−G82 (δ ∼ −53◦ ) implies that it is visible from the GMRT for only ≈2.5 h per day. Our total on-source time from the six runs was hence only 9.6 h; however, we note that this was significantly larger than the on-source time (≈2.5 hours) of our earlier run (Arabsalmani et al. 2015). The bright flux calibrator Downloaded from https://academic.oup.com/mnras/article/485/4/5411/5380812 by CNRS - ISTO user on 01 March 2022 Woosley 1999; Aloy et al. 2000; Zhang, Woosley & MacFadyen 2003; Zhang, Woosley & Heger 2004; Yoon & Langer 2005; Woosley & Heger 2006). This is observationally supported by the occurrence of GRBs in actively star-forming regions, which links GRB formation to massive stars (Paczyński 1998; Fynbo et al. 2000; Bloom, Kulkarni & Djorgovski 2002; Le Floc’h et al. 2003; Christensen, Hjorth & Gorosabel 2004; Fruchter et al. 2006; Fynbo et al. 2006a; Lyman et al. 2017). The association of GRBs with type Ib,c supernovae (SNe; e.g. Galama et al. 1998; Hjorth et al. 2003; Stanek et al. 2003; Malesani et al. 2004; Pian et al. 2006), and also the young ages of stellar populations in GRB environments (Chary, Becklin & Armus 2002; Christensen et al. 2004) strengthen the connection between GRBs and massive stars. GRBs typically occur in low-mass and metal poor (dwarf) galaxies (Fynbo et al. 2006b; Prochaska et al. 2008; Savaglio, Glazebrook & Le Borgne 2009; Castro Cerón et al. 2010; Graham & Fruchter 2013; Krühler et al. 2015; Cucchiara et al. 2015; Perley et al. 2016). This is often interpreted as an indication that a low metallicity is needed for the formation of GRB progenitors. Such a hypothesis is consistent with the single-star progenitor model for GRB formation, where the low metallicity of the progenitor star is critical. However, the detection of several GRBs in metalrich environments (Savaglio et al. 2012; Elliott et al. 2013; Schady et al. 2015), and particularly, the identification of a large number of massive and metal-rich GRB host galaxies (dark/dust-obscured GRB hosts, Svensson et al. 2012; Hunt et al. 2014; Perley et al. 2013) has raised questions about whether a low metallicity is indeed necessary for the formation of GRB progenitor stars. While the low-metallicity requirement is debated, high star formation densities do appear to play a critical role for the formation of GRB progenitors. Studies in both the local and the high-z Universe show that massive stars (and hence GRB progenitors) are more likely to be found in regions with high star formation rate (SFR) densities (Dabringhausen, Kroupa & Baumgardt 2009; Dabringhausen et al. 2012; Banerjee & Kroupa 2012; Marks et al. 2012; Peacock et al. 2017; Schneider et al. 2018; Zhang et al. 2018). GRB hosts are indeed found to have high surface densities of SFR (Kelly et al. 2014). Interactions are known to enhance the star formation activities of galaxies (Renaud et al. 2014) and also trigger the formation of massive and compact clumps (see Renaud 2018, and references therein). It would be therefore interesting to investigate whether interactions and mergers are common in GRB host galaxies. Evidence for interactions is likely to be easier to obtain in the closest GRB host galaxies, as their proximity allows us to identify both very faint companion galaxies and weak disturbances in their velocity fields. Perhaps the best system in this regard is ESO 184−G82, the host of GRB 980425 and its associated supernova, SN 1998bw (Galama et al. 1998). At z = 0.0087, this has the lowest redshift of any known GRB till date. ESO 184−G82 is a barred spiral (Sbc-type) galaxy, and has several H II regions that are actively forming stars (Fynbo et al. 2000). It has an SFR of 0.2–0.4 M yr−1 (Christensen et al. 2008; Krühler et al. 2017) and a stellar mass of 108.7 M (Michałowski et al. 2014) that place it on the galaxy main-sequence relation in the M∗ –SFR plane (Brinchmann et al. 2004). Most interestingly, there is a very bright star-forming region in the host galaxy with an sSFR more than an order of magnitude larger than the overall sSFR of the host (Hammer et al. 2006; Christensen et al. 2008). This is one of the most luminous and infrared-bright H II regions identified to date in the nearby Universe (Le Floc’h et al. 2012). With a high density of young and massive Wolf–Rayet (WR) stars with ages less than 3 Myr (Krühler et al. ESO 184−G82: a collisional ring galaxy Table 1. Parameters of the GMRT H I data cubes used in this paper. Synthesized beam (arcsec × arcsec) 3.0 × 9.3 11.9 × 16.8 17.2 × 23.9 25.3 × 44.1 Channel width (km s−1 ) Noise in line-free channel (mJy Bm−1 ) 7.0 7.0 7.0 7.0 0.7 1.0 1.2 1.7 2.2 Ancillary data We obtained ancillary data for the field of ESO 184−G82 at several wavelengths. These include (i) optical broad-band images of the field obtained with the FOcal Reducer and low dispersion Spectrograph (FORS) on the Very Large Telescope (VLT) on 1999 October 10, 14, and 15, using the BBESS , VBESS , IBESS , and RBESS filters, with a total exposure time of 300 s in each filter [Programme IDs: 064.H-0375(A) and 066.D-0576(A), PI: F. Patat], (ii) a narrowband image of the field with the H α filter obtained with VLT/FORS on 2000 August 03, with a total exposure time of 300 s [Programme ID: 165.H-0464(A), PI: Van Den Heuvel], (iii) a 4.5 μm continuum data set obtained with the InfraRed Array Camera (IRAC) on the Spitzer Space Telescope on 2004 April 03, with a total exposure time of 100 s [as a part of the IRS Guaranteed Time, Houck et al. 2004, Programme ID: 76], and (iv) a Hubble Space Telescope (HST) image with the MIRVIS/Clear filter centred at 5737.453 Å 2000 on June 11 with a total exposure time of 295 s (Programme ID: GO-8640, PI: Holland) The VLT/FORS data were analysed following the procedure described by Sollerman et al. (2005). The details of the IRAC data reduction are presented in Le Floc’h et al. (2006), while the HST data analysis is described in Fynbo et al. (2000). 3 T H E AT O M I C G A S I N E S O 1 8 4 −G 8 2 In Fig. 1, we show the H I 21 cm total intensity and velocity maps of ESO 184−G82 at three different angular resolutions. The synthesized beams are shown in the bottom-left corner of each panel, and have the FWHMs of 25 arcsec × 44 arcsec, 12 arcsec × 17 arcsec, and 3 arcsec × 9 arcsec for the left, middle, and right panels, respectively (see Table 1 for the properties of the H I cubes). The H I 21 cm intensity map at our lowest resolution (25 arcsec × 44 arcsec) is shown in grey-scale in the top left-hand panel. The corresponding contours are overlaid on the optical VLT/FORS (Bband) image of the galaxy in the middle left-hand panel. These maps show the extent of the diffuse gas, since we are sensitive to low H I column densities, ≈3.6 × 1019 cm−2 , at this resolution. We clearly see that the diffuse H I is much more extended than the optical disc of the galaxy (at least twice as large in diameter). We derive a total H I mass of 108.90±0.04 M for the main gas disc from the total H I flux measured at this resolution, consistent with the value we reported in Arabsalmani et al. (2015). This is comparable to the stellar mass of the galaxy, M∗ = 108.7 M . The velocity field at the same coarse resolution (bottom left-hand panel) shows that the atomic gas disc has ordered rotation, but that the gas to the north-east corner seems not to be following the rotation of the gas disc. The H I intensity map at a resolution of 12 arcsec × 17 arcsec in grey-scale, and with contours overlaid on the VLT/FORS Bband image, are shown in the middle panels of the top two rows. From these maps, we find that, in addition to the main mass of gas coincident with the optical disc of ESO 184−G82, the gas disc extends at least 5 kpc to the north of the optical disc. We clearly detect the presence of an H I knot to the north-east of the galaxy, about an arcminute from the optical centre of ESO 184−G82 (middle panels). The extension of gas towards this knot is strong evidence of tidal interaction between ESO 184−G82 and the object associated with the H I knot. The velocity field at the same resolution (bottom-middle panel) shows that whereas the main gas disc appears to have regular rotation along an axis running through the centre of the optical disc from south-east to north-west, the extension to the north contains kinematically disturbed gas. The presence of disturbed gas strengthens the case for an ongoing interaction between the GRB host and a companion galaxy. The highest resolution H I map, with a resolution of 3 arcsec × 9 arcsec, is shown in grey-scale in the top right-hand panel. The corresponding contours, overlaid on the HST image of the MNRAS 485, 5411–5422 (2019) Downloaded from https://academic.oup.com/mnras/article/485/4/5411/5380812 by CNRS - ISTO user on 01 March 2022 3C 48 was observed at the start and end of each observing run to calibrate the system bandpass. ‘Classic’ AIPS was used for the analysis of the data (Greisen 2003). After initial data editing and bandpass calibration, a ‘channel-0’ visibility data set was created by averaging together line-free channels. The flux scale of the data was set by an initial calibration to a sky model based on our earlier GMRT continuum image of the field (Arabsalmani et al. 2015). This was followed by a standard self-calibration, imaging and data-editing procedure on the same channel-0 data set, until no further improvement was seen in the continuum image on further self-calibration. The antenna-based gains derived from the above procedure were then applied to all visibilities of the original multichannel data set. At the end of the loop, the final calibration was applied to all the visibilities. The radio continuum image made using the line-free channels at the end of the self-calibration cycle was used to subtract the continuum from the calibrated visibilities, using the task UVSUB. The residual visibilities were mapped with different U– V tapers to produce spectral cubes at different angular resolutions using the task IMAGR. The velocity resolution was optimized to be 7 km s−1 by averaging groups of four channels together. This was done to improve the statistical significance of the detected H I 21 cm emission in independent velocity channels while still having sufficient resolution to accurately trace the velocity field. The task MOMNT was then applied to the spectral cubes in order to obtain maps of the H I total intensity and the intensityweighted velocity field at different angular resolutions. MOMNT works by masking out pixels in the spectral data cube that lie below a threshold flux in a secondary data cube created by smoothing the original cube both spatially and along the velocity axis – the smoothing ensures that any localized noise peaks are ignored and only emission correlated spatially and in velocity is chosen. We created the secondary data cube by applying Hanning smoothing across blocks of three consecutive velocity channels, whereas spatially a Gaussian kernel of full width at half-maximum (FWHM) equal to six pixels was applied. The threshold flux used to select pixels was approximately 1.5 times the noise in a line-free channel of the original cube. We produced four spectral cubes with different angular resolutions. The synthesized beam FWHMs for these cubes are 3.0 arcsec × 9.3 arcsec, 11.9 arcsec × 16.8 arcsec, 17.2 arcsec × 23.9 arcsec, and 25.3 arcsec × 44.1 arcsec. The properties of the four cubes are listed in Table 1. 5413 5414 M. Arabsalmani et al. MNRAS 485, 5411–5422 (2019) Downloaded from https://academic.oup.com/mnras/article/485/4/5411/5380812 by CNRS - ISTO user on 01 March 2022 Figure 1. Top row: H I 21 cm total intensity maps in grey-scale at three different angular resolutions. The beam FWHMs (same in all panels of a column) are shown in the bottom-left corner of each panel, and are 25 arcsec × 44 arcsec, 12 arcsec × 17 arcsec, and 3 arcsec × 9 arcsec from left to right, respectively. The cyan squares in the left and middle panels show the area covered in the panel immediately to the right. The black bars represent a physical scale of 5 kpc in each panel. Middle row: Contours of the same H I 21 cm total intensity shown in the top row, overlaid on optical images of ESO 184−G82. The first contour of each H I intensity map is at the 3σ level of a single channel of the respective data cube. The first contour is at √ 3.6 × 1019 cm−2 (left-hand panel), 1.2 × 1020 cm−2 (middle panel), and 6.0 × 1020 cm−2 (right-hand panel), with each subsequent contour in multiples of 2. The optical images are the VLT/FORS B-band image in the left and middle panels, and the HST image (MIRVIS filter, centred at 5737.453 Å) in the right-hand panel. In the left-hand panel, the galaxy to the south-east of the GRB host in the FORS image is at z = 0.044 and hence the extension of gas in that direction is not related to it. The GRB location is marked with the red circles. The location of the WR region is marked with a yellow circle in the right-hand panel. Bottom row: H I 21 cm velocity field covering the same spatial area as covered in the respective top and middle row panels. The colourbar below the middle panel shows the velocity of the gas with respect to the centre of the H I 21 cm emission line in units of km s−1 . ESO 184−G82: a collisional ring galaxy 5415 4 T H E C O M PA N I O N G A L A X Y O F E S O 1 8 4 −G 8 2 The structure of the atomic gas in ESO 184−G82 shows clear evidence for an ongoing interaction between the galaxy and a companion object, probably associated with the H I knot of Fig. 1. In order to identify the optical counterpart of the H I knot, we searched the multiple VLT/FORS optical images. In all these images, we clearly detect optical emission spatially coincident with the H I knot. We also identify this object in the Spitzer/IRAC2 4.5 μm image and the VLT/FORS H α image. The optical and infrared images of the field are shown in Fig. 3 (see also the top-middle panel of Fig. 1). These confirm the association of the H I knot with a galaxy centred on RA = 19h 35m 09.s 6 and Dec. = −52h 49m 46.s 9 and at a projected Table 2. Properties of GALJ193510-524947, the companion of ESO 184−G82. B 18.84 ± 0.10 V R I F4.5 μm (μJy) FH α (erg s−1 cm−2 ) SFR (M yr−1 ) M∗ (M ) MH I (M ) 18.33 ± 0.10 18.05 ± 0.15 17.93 ± 0.20 17 ± 7 3.4 × 10−15 0.004 107.0 ± 0.3 107.1 ± 0.1 MNRAS 485, 5411–5422 (2019) Downloaded from https://academic.oup.com/mnras/article/485/4/5411/5380812 by CNRS - ISTO user on 01 March 2022 distance of 13 kpc from the centre of ESO 184−G82. The H I 21 cm emission from this galaxy is centred at a redshift of z = 0.00867 ± 0.00002, consistent with the redshift of ESO 184−G82 (z = 0.00860 ± 0.00002 obtained from the H I 21 cm emission line); i.e. the centres of the H I 21 cm emission from the two galaxies are separated by about 20 km s−1 . We name this newly identified galaxy GALJ193510-524947. (see Table 2 for a summary of its properties). GALJ193510-524947 appears to be a star-forming dwarf galaxy. We obtain an H α emission flux of 3.4 × 10−15 erg s−1 cm−2 , implying that the galaxy has an SFR of 0.004 M yr−1 (based on the calibration of Kennicutt 1998). The VLT/FORS images yield AB magnitudes of B = 18.84 ± 0.10, V = 18.33 ± 0.10, R = 18.05 ± 0.15, and I = 17.93 ± 0.20 for the galaxy (corresponding to absolute magnitudes of −13.66, −14.17, −14.45, and −14.75, respectively). The Spitzer/IRAC2 photometry is complicated by contamination from two foreground stars in the 4.5 μm image (see the right-hand panel of Fig. 3). We hence first measured the contributions of the two stars using aperture photometry, and subtracted their emission to obtain the flux density of GALJ193510524.947. This yielded a 4.5 μm flux density of 17 ± 7μJy, equivalent to an AB magnitude of 20.8+0.6 −0.4 . Note that while GALJ193510524947 is clearly detected in the Spitzer/IRAC2 image, the presence of the two bright stars in the image lead to the large uncertainty in the IRAC photometry. We estimated the stellar mass of GALJ193510-524947 by modelling its spectral energy distribution (SED) with LePhare (Arnouts et al. 1999) based on our optical and near-infrared (NIR) photometry. We use the stellar population synthesis templates developed by Bruzual & Charlot (2003), assume a Chabrier initial mass function (IMF; Chabrier 2003), and consider an exponentially declining star formation history (SFR ∝ e−t/τ ). From this, we estimate a stellar mass of 107.0±0.3 M for GALJ193510-524947. We note that our SED modelling is dominated by the optical photometry that has lower errors than the 4.5 μm photometry. In order to confirm the stellar mass derived from the SED modelling, we obtain an independent estimate of the stellar mass of GALJ193510-524947 from its NIR photometry alone. For this, we assume a ratio of 0.6 for the Spitzer/IRAC 3.6–4.5 μm fluxes (Zhu et al. 2010) and use the calibration of Leroy et al. (2008). We then obtain a stellar mass of ∼106.7 M from the NIR photometry which is consistent with our estimate from the SED modelling. We measure an H I mass of 107.1±0.1 M for GALJ193510524947. This is comparable to its stellar mass, implying that GALJ193510-524947 is a gas-rich galaxy. We detect the H I 21 cm emission line from GALJ193510-524947 in five channels, corresponding to a velocity width of ≈35 km s−1 (see Fig. 2). This places the galaxy on the baryonic Tully–Fisher relation for low-mass dwarfs in the local Universe (McGaugh 2012). Based on our SED modelling, we derive the continuum emission of GALJ193510-524947 at the rest-frame H α wavelength (6562.8 Å) to be 1.2 × 10−16 erg s−1 cm−2 Å−1 . With this and the H α flux measured from the VLT/FORS narrow-band image we estimate the equivalent width of the H α line (EWH α , the ratio of H α flux to the continuum level at the wavelength of the H α emission) to galaxy, are shown in the middle right-hand panel. At this resolution, we are sensitive to only high H I column density gas, with N(H I) 1020.8 cm−2 . We find the high column density H I to have formed a ring around the optical centre of the galaxy. This ring passes across the actively star-forming regions in the galaxy and encircles the stellar bar in ESO 184−G82. The locations of both the SN/GRB and the WR region are situated in the Western part of this high column density ring, portions of which were also picked up in our previous GMRT H I 21 cm image (Arabsalmani et al. 2015). Gas rings can form due to resonances with bars or resonances caused by a mild tidal interaction with a companion galaxy (resonance rings, Buta & Combes 1996; Buta 1999). But high-density gas rings, which are also the sites of enhanced star formation in galaxies, are usually formed due to collisions with small companions (see Appleton & Struck-Marcell 1996 for a review on collisional ring galaxies). In Section 5, we discuss the likely cause for the formation of the high column density gas ring in ESO 184−G82. The high resolution velocity field (bottom right-hand panel) shows that this high column density ring of gas follows the rotation of the main H I disc of the galaxy, though within the ring there are regions with velocity gradients as high as 40 km s−1 over sub-kpc scales. In order to take a detailed look at the velocity distribution of the H I gas, we use the spectral cube with an angular resolution of 17 arcsec × 24 arcsec. This intermediate resolution allows us to both be sensitive to relatively low gas column densities and spatially distinguish the H I knot to the north-east from the main gas disc of ESO 184−G82. Fig. 2 shows the H I fluxes per 7 km s−1 velocity channel at this resolution. There is H I around the optical centre of the galaxy that shows ordered rotation – the peak emission shifts from south-east to north-west with increasing velocity. But there appears to be a substantial amount of H I that does not follow the ordered rotation, located to the north–north-east of the optical centre. Emission from the previously mentioned spatially distinct north-eastern knot is detected in the velocity channels between 2591.7 and 2612.6 km s−1 . Note that much of the kinematically disturbed gas in various velocity channels is extended towards the location of this north-eastern knot, reminiscent of gas being dragged out of a galactic halo by the passage of another galaxy during a merger event. 5416 M. Arabsalmani et al. be ∼ 28 Å for GALJ193510-524947. Using Starburst99 models with instantaneous star formation laws (Leitherer et al. 1999), this corresponds to an age of ∼ 8 Myr, somewhat larger than, but comparable to, those of the H II regions in ESO 184−G82 (see Christensen et al. 2008; Krühler et al. 2017). 5 I N T E R AC T I O N B E T W E E N E S O 1 8 4 −G 8 2 AND GALJ193510-524947 The structure of atomic gas and also the star formation activity in ESO 184−G82 and GALJ193510-524947 are reminiscent of an interacting system. In addition to the main mass of gas which is spatially coincident with the optical disc of ESO 184−G82, the atomic gas in ESO 184−G82 has an extension over at least 5 kpc (projected size) to the north of the optical disc. The eastern part MNRAS 485, 5411–5422 (2019) of the extended gas looks like a bridge between ESO 184−G82 and GALJ193510-524947, suggestive of a tidal interaction or a collision between the two galaxies. While the main mass of gas in ESO 184−G82 shows regular rotation, a substantial amount of H I gas in the extension is disturbed and does not follow the ordered rotation. This is reminiscent of gas being dragged out of ESO 184−G82 by the passage of GALJ193510-524947. The H I gas with the highest column density appears to have formed a ring around the optical centre of ESO 184−G82, passing across its actively star-forming regions. This structure, resembling a cartwheel-like ring, is suggestive of a collisional ring formed due to a head-on collision with a small galaxy like GALJ193510-524947 that passed through the disc of ESO 184−G82 close to its centre (a drop-through collision, see Wong et al. 2006). The large velocity gradients of the atomic gas in the gas ring of ESO 184−G82 and also the presence Downloaded from https://academic.oup.com/mnras/article/485/4/5411/5380812 by CNRS - ISTO user on 01 March 2022 Figure 2. The H I 21 cm flux in successive 7 km s−1 velocity channels of the 17 arcsec × 24 arcsec resolution data cube. Contours √ are overlaid for clarity. The first contour for each channel (positive: solid, negative: dashed) is at the 2σ level, with each subsequent contour in multiples of 2. The beam is shown in the bottom-left corner of each panel. The optical centre of ESO 184−G82 and the centre of the H I knot are marked with black and red squares, respectively. ESO 184−G82: a collisional ring galaxy 5417 of the large H α knots coincident with the H I ring lend support to this hypothesis. In a drop-through collision, the inner annular shells of gas will have larger velocities compared to the outer shells. The catching up of the inner shells with the outer shells creates shock and compression of gas and leads to the formation of a ring of highdensity gas with a large velocity dispersion (Appleton & StruckMarcell 1996; Bournaud & Combes 2003). The high-velocity dispersion of gas in the ring increases the Jeans mass that leads to the formation of massive molecular gas clumps and hence large knots of star formation (Horellou & Combes 2001; Renaud et al. 2018). Unlike in classical tidal interactions, the lack of large gas inflows towards the galaxy centre during a collision results in the absence (or negligible amount) of star formation enhancement in the central regions (Renaud et al. 2018). This picture is consistent with the star formation activity in ESO 184−G82. As is clear from its H α emission-line observations, the recent star formation in ESO 184−G82 is not concentrated in the centre of the galaxy, but arises in a number of H II regions coincident with the high column density H I ring (see the middle panel of Fig. 3; see also Christensen et al. 2004; Krühler et al. 2017). Moreover, the velocity map presented in the bottom right-hand panel of Fig. 1 clearly shows large velocity gradients (as large as 40 km s−1 on sub-kpc scales) along the radius of the high column density gas ring, typical of collisional rings. The gas ring in ESO 184−G82 is also asymmetric, with its centre offset from the optical centre of the galaxy. It also has higher column densities in its north-west region compared to those in the rest of the ring. These asymmetric features too are typical of collisional rings. The identification of GALJ193510-524947, a companion galaxy associated with the H I knot to the north-east of ESO 184−G82, and at a projected distance of 13 kpc from the GRB host, strengthens the case for an interaction between the two galaxies. We use state-of-the-art simulations of dwarf–dwarf mergers (Starkenburg, Helmi & Sales 2016) to test whether the observed gas and star formation properties of ESO 184−G82 and its companion can be reproduced by a dwarf–dwarf interaction model. These are controlled (isolated) simulations, performed with the OWLS version (Schaye et al. 2010) of the N-body/smoothed particle hydrodynamic code GADGET-3 (Springel, Yoshida & White 2001; Springel 2005). In these simulations, both dwarf galaxies have a Hernquist dark matter halo and exponential stellar and gas discs. The primary dwarf contains 5 × 106 particles in its dark matter halo and 106 particles in its baryonic matter. The secondary dwarf contains 106 particles in its dark matter halo and 2 × 105 particles in its baryons. The gravitational softening length is 10 pc for dark matter, and 3 pc for baryonic particles, and the smoothing is done over 48 neighbours. Gas above a density of 0.1 cm−3 is governed by an effective equation of state and forms stars following a Kennicutt– Schmidt relation while at lower densities it follows an isothermal equation of state (see Schaye & Dalla Vecchia 2008). We consider both the non-star-forming gas with temperature <2 × 104 K, and the least dense three-fourths of the star-forming gas to be in the atomic phase (following Genel et al. 2014) and assume a mass ratio of molecular gas to atomic gas of ≈1/3 (Saintonge et al. 2011). Feedback is implemented based on the kinetic stellar wind prescription of Dalla Vecchia & Schaye (2008) and is calibrated to ensure self-regulated star formation. For a detailed description of the simulations, we refer the readers to Starkenburg et al. (2016). Our main criteria are to simultaneously reproduce the following observed features of ESO 184−G82: (i) the extension of H I gas from ESO 184−G82 towards GALJ193510-524947, (ii) the asymmetric ring of high column density H I gas in ESO 184−G82, (iii) the large velocity gradients of the H I in the ring, (iv) the enhancement of star formation in the location of the gas ring, and finally (v) the lack of enhanced star formation activity in the central regions of ESO 184−G82. We run two sets of simulations, one for classical tidal interactions and the other for collisional interactions. In both, we start with two discs with smoothly distributed stellar and gas components. In the tidal interaction simulations, the secondary galaxy passes through the outskirts of the primary galaxy disc at the first pericentre on an orbit that has a small inclination with respect to the plane of the disc of the primary dwarf (covering a range of −10 to 30 deg). In the collisional interaction simulations, the secondary galaxy has a direct collision with the primary galaxy with angles between 70 and 90 deg with respect to the plane of the primary galaxy. We find that the tidal interaction simulations, within the ranges of the initial configurations mentioned below, fail to reproduce the observed criteria (ii) to (v) mentioned above. The explored initial MNRAS 485, 5411–5422 (2019) Downloaded from https://academic.oup.com/mnras/article/485/4/5411/5380812 by CNRS - ISTO user on 01 March 2022 Figure 3. Ancillary observations of the field around ESO 184−G82 show a companion galaxy co-spatial with the north-eastern H I knot (at a projected distance of 13 kpc from the centre of ESO 184−G82), marked with a white circle in each of the panels. We name this newly identified galaxy GALJ193510-524947. Panels from left to right: FORS B band, VLT/FORS narrow-band image centred on H α, Spitzer/IRAC 4.5 μm. Note that there is a bright star in the foreground of GALJ193510-524947 in the three images, with a second NIR-bright foreground star in the Spitzer image. The GRB location is marked with the green circles. 5418 M. Arabsalmani et al. MNRAS 485, 5411–5422 (2019) has been dragged out from the primary galaxy is visible as the extension of gas between the two galaxies. The overall stellar and gas distributions of the dwarf–dwarf system at this snapshot matches those of ESO 184−G82 and GALJ193510-524947. The velocity field of the atomic gas too resembles the velocity map of the atomic gas in ESO 184−G82, presented in the bottom-middle panel of Fig. 1. The dense gas in the region of the ring demonstrates velocity gradients of a few tens of km s−1 over sub-kpc scales. Such large velocity gradients result in the formation of massive gas clouds and hence large knots of star formation. These massive gas clouds as well as large star formation knots are resolved in our simulations and are visible in the third row of Fig. 4. The large velocity gradients can also be clearly seen in the velocity distribution of the gas ring shown separately in Fig. 5. The radius of the gas ring continues to increase with time and its density contrast decreases as can be seen in the fourth snapshot (the fourth row in Fig. 4) that corresponds to 150 Myr after the first collision. At this time, the secondary galaxy is moving further away from the primary dwarf. The fifth snapshot (the fifth row in Fig. 4), corresponding to 100 Myr before the second collision, when the secondary galaxy, after reaching its apocentre, is moving back towards the primary galaxy for the second collision. By this time, the distribution of atomic gas in the primary galaxy has become more uniform, with no depression in the central regions. Also, the star formation activity is only present in the centre of the galaxy. 6 DISCUSSION The gas and star formation properties of ESO 184−G82, along with the identification of a companion galaxy, GALJ193510-524947, in its vicinity provide clear evidence for interaction between the two galaxies. Our simulations support the paradigm in which ESO 184−G82 had a head-on collision with its companion that led to the formation of the high column density gas ring observed in our H I map. This ring, with large velocity gradients (as high as 40 km s−1 over sub-kpc scales), is an ideal site for formation of massive giant molecular clouds and hence superstar clusters (SSCs). This, consequently, increases the probability of formation of massive stars in the star-forming regions within the gas ring and in turn increases the chance of a GRB progenitor to form (see also Roychowdhury, Arabsalmani & Kanekar 2019, who find the recently discovered transient AT2018cow to be located within an asymmetric high column density ring of atomic gas in its host galaxy). Note that this paradigm is for the formation of the GRB progenitor, and may not have any bearing on the stellar explosion mechanism that ultimately produces the GRB. A comparison between the stellar mass distributions of GRB hosts and the cosmic SFR density shows that most GRBs occur in galaxies with stellar masses lower than those of galaxies responsible for the bulk of star formation (e.g. galaxies with M∗ = 1010.0−10.7 M at z < 1, Conroy & Wechsler 2009). This is especially the case for GRBs at z 2 (see Perley et al. 2016). Hence, the processes that create these energetic explosions are not linked in a trivial way to the SFRs of their host galaxies. There must be other factors that drive the formation of GRB progenitors in galaxies. The typical low mass and metallicity of GRB hosts has been widely interpreted as low metallicity being the primary requirement for GRB formation. However, this has been called into question by the detection of a large number of GRBs in massive, metal-rich galaxies (Savaglio et al. 2012; Svensson et al. 2012; Elliott et al. 2013; Perley et al. 2013; Hunt et al. 2014; Schady et al. 2015). A less addressed, but more likely, factor is high SFR densities. Downloaded from https://academic.oup.com/mnras/article/485/4/5411/5380812 by CNRS - ISTO user on 01 March 2022 configurations for this set of simulations are a virial mass range of 5.0 × 1010 –1.0 × 1011 M (in three steps) for the primary dwarf and 8.0 × 108 –5.0 × 1010 M (in seven steps) for the secondary dwarf galaxy; the range of 0.001–0.02 (in five steps) for the ratio of baryonic to virial mass, 0.3–0.7 (in five steps) for the ratio of gas to baryonic mass, and 1–4 (in five steps) for the ratio of gas to stellar disc size for both the dwarf galaxies; the range of −10 to 30 deg (in six steps) for the inclination of the orbit of the secondary galaxy with respect to the disc of the primary dwarf; and the range of 28– 66 kpc (in three steps) for the initial separation between the two dwarf galaxies. In particular, in tidal interactions the star formation enhancement in the central regions of the galaxy (central kpc) should contribute significantly (if not dominantly) to the total star formation enhancement of the galaxy (Barnes & Hernquist 1991; Di Matteo et al. 2007, 2008; Teyssier, Chapon & Bournaud 2010; Powell et al. 2013; Renaud et al. 2014; Hibbard & van Gorkom 1996). The fact that this is not the case in ESO 184−G82 suggests that it is unlikely that a tidal interaction has taken place in the system. Conversely, the collisional interaction simulations simultaneously reproduce all the above observational constraints. Fig. 4 presents the outcome of one of these simulations at five different times; for each time sample, the different results are plotted in a single row. The four columns (from left to right) present, respectively, the distributions of the stellar surface brightness, the SFR, the H I column density, and the H I 21 cm velocity field. The viewing angle for the simulation presented in this figure is 45 deg from the plane of the primary dwarf galaxy. The primary and secondary dwarfs have halo masses of 8 × 108 M and 3 × 1010 M , respectively. The dark matter particle masses for the primary and secondary dwarf are 2.2 × 104 M and 4.0 × 104 M , while the baryonic particle masses are 1.6 × 103 M and 1.0 × 103 M , respectively. The top row shows the configuration of the system in a snapshot corresponding to 90 Myr before the first collision, when the secondary dwarf is at a distance of 20 kpc (projected distance of 16 kpc) from the primary galaxy. The primary dwarf at this time has a stellar mass of ≈ 7 × 108 M and a similar atomic gas mass. The secondary dwarf is moving with a velocity of 100 km s−1 , on a direct collision course with the disc of the primary galaxy and at an angle of 70 deg with respect to the plane of the primary galaxy. The first collision occurs at a point 2 kpc away from the centre of the primary dwarf disc, and results in the formation of annular shells moving outwards from the collision point. The inner shells have larger velocities compared to the outer shells. The shock caused by this difference in velocities results in the compression of gas and, as a consequence, in the enhancement of star formation. The highdensity gas and the enhancement of star formation in the primary dwarf can be seen in the second snapshot (the second row of Fig. 4) that shows the system 30 Myr after the first collision. By this time, star formation in the primary dwarf has increased by more than a factor of 3 compared to that before the collision. With time, the shock wave moves outwards from the point of collision and forms an asymmetric ring of dense atomic gas in the primary dwarf. This ring is clearly visible in the third snapshot (the third row of Fig. 4) that corresponds to 80 Myr after the first collision. By this time, the formation of molecular gas in the dense atomic gas ring has enhanced the star formation in the ring. At the same time, the lack of sufficient inflow of gas to the centre of the primary dwarf has resulted in very little or no star formation enhancement in the centre of the galaxy. The secondary dwarf is at a distance of 17 kpc (projected distance of 13 kpc) from the primary galaxy and is moving away from it. The atomic gas that ESO 184−G82: a collisional ring galaxy 5419 Downloaded from https://academic.oup.com/mnras/article/485/4/5411/5380812 by CNRS - ISTO user on 01 March 2022 Figure 4. An example of a simulated head-on collision between a dwarf galaxy and a small companion, simultaneously reproducing the main observed features in ESO 184−G82 (see Section 5 for details). The viewing angle for the simulation presented in this figure is 45 deg from the plane of the primary dwarf galaxy. Each row corresponds to a snapshot in time of the dwarf–dwarf simulation, with time increasing from top to bottom. The four columns, from left to right, contains the distributions of, respectively, the stellar surface brightness, the SFR, the H I column density, and the H I 21 cm velocity field. The third row, corresponding to 80 Myr after the first collision, resembles the observed properties of ESO 184−G82 and its companion, GALJ193510-524947. Note that in the first and last rows the secondary dwarf is moving towards the primary dwarf for a collision, and in the other three rows, it is moving away from the primary dwarf after a collision. MNRAS 485, 5411–5422 (2019) 5420 M. Arabsalmani et al. Kelly et al. (2014) found the SFR surface densities of GRB hosts to be higher than those of field galaxies, implying that they have high SFR densities. Regions with high star formation densities are expected to power strong gas outflows with large velocities (Lagos, Lacey & Baugh 2013; Sharma et al. 2017). The large galaxy outflow velocities deduced from the velocity widths of the interstellar medium absorption lines in GRB hosts (detected in GRB afterglows, see Arabsalmani et al. 2018b and references therein) are therefore consistent with the hypothesis of GRBs originating in regions with high-SFR densities. This picture is in agreement with GRB formation models. There are two main models proposed for the generation of relativistic jets in GRBs through core-collapse of massive stars. In one, a single progenitor star with anomalously rapid rotation forms the GRB (MacFadyen & Woosley 1999; Hirschi, Meynet & Maeder 2005; Yoon & Langer 2005; Woosley & Heger 2006), while in the other, the GRB is associated with the core collapse of a massive star stripped by a companion in a close binary system (Usov 1992; Izzard, Ramirez-Ruiz & Tout 2004; Podsiadlowski et al. 2004; Fryer & Heger 2005; Detmers et al. 2008; Podsiadlowski et al. 2010; Tout et al. 2011; Kinugawa & Asano 2017). The single-star model requires the progenitor to retain angular momentum (necessary for GRB formation) and, at the same time, lose substantial mass (to develop into a hydrogen- and helium-poor star). While the presence of metals helps the mass-loss (and the removal of the hydrogen/helium envelope), this also carries away angular momentum. This contradiction is avoided in chemically homogeneous evolved progenitors, which however require much lower metallicities than is observed in GRB hosts. On the other hand, the metallicity constraints on the progenitor stars when GRBs are produced in close binaries are more relaxed; although these models predict a higher probability of GRB formation in metal-poor progenitors, they do not rule out high (e.g. solar or supersolar) metallicities in the progenitors (e.g. see Podsiadlowski et al. 2010; Tout et al. 2011). It is notable that Sana et al. (2014) found massive stars to form nearly exclusively in multiple systems (see also Mason et al. 2009; Sana et al. 2012). Formation of GRB progenitors in massive and dense SSCs is particularly in agreement with the models in MNRAS 485, 5411–5422 (2019) Downloaded from https://academic.oup.com/mnras/article/485/4/5411/5380812 by CNRS - ISTO user on 01 March 2022 Figure 5. The simulated velocity field of the gas ring in the primary dwarf 80 Myr after the first collision. This ring contains the atomic gas with column densities above 2 × 1020 cm−2 . Velocity gradients of a few tens of km s−1 can be seen to exist within sub-kpc scales, similar to what is observed in ESO 184−G8 (see the bottom right-hand panel of Fig. 1). which GRBs form through dynamical interactions and collisions of massive stars in dense environments (see e.g. van den Heuvel & Portegies Zwart 2013). The link between high SFR densities and GRB progenitors is also supported by the observed top heavy IMF in regions with high SFR densities. There are several lines of evidence in the local Universe indicating that more massive stars are found in regions with high SFR densities than would be expected from a standard IMF (e.g. Salpeter, Chabrier, etc; Dabringhausen et al. 2009, 2012; Banerjee & Kroupa 2012; Marks et al. 2012; Peacock et al. 2017). Schneider et al. (2018) studied a compact and bright H II region in the Large Magellanic Cloud whose properties may closely replicate starbursts at high redshifts. They found it to contain 32 per cent more stars with masses larger than 30 M than expected from a standard IMF. This is in line with the findings of Zhang et al. (2018) who recently investigated four submillimetre galaxies at z = 2–3 and found evidence for a top heavy IMF in all of them. Therefore, regions with compact and intense star formation, such as massive SSCs, are the likely birth-place of GRB progenitors (also see Chen, Prochaska & Bloom 2007). Massive SSCs are known to be common in interacting systems (Elmegreen, Kaufman & Thomasson 1993; de Grijs et al. 2003; Bastian 2008; Renaud 2018). The absence of gravitational shear and the increased turbulence in interacting systems are thought to aid the gravitational collapse of massive amounts of gas into massive and compact GMCs, which may subsequently form SSCs (see Elmegreen et al. 2000; Teyssier et al. 2010; Weidner, Bonnell & Zinnecker 2010; Elmegreen et al. 2017). The large velocity dispersion of the interstellar gas in interacting systems not only increases the Jeans mass (which in turn results in the formation of massive clumps), but also heightens the temperature of the clouds, thus shifting the IMF towards more massive stars (see Elmegreen et al. 1993). Collisional encounters between galaxies, though rare compared to tidal interactions, are more efficient in triggering the formation of massive and compact SSCs (Struck et al. 1996; Burkert, Brodie & Larsen 2005; Elmegreen & Elmegreen 2006; Pellerin et al. 2010). In a recent study, Renaud et al. (2018) showed that head-on collisions produce fewer, but larger SSCs, compared to tidal interactions. The above evidence suggests a natural link between the host galaxies of GRBs and interacting systems. Such a link seems to be especially likely at z 1, since interactions appear to play a dominant role in the formation of massive and compact SSCs at low redshifts. At higher redshifts, the high-gas fractions of galaxies can cause gravitational instability, leading to the collapse of large amounts of gas into massive and dense clumps. Recent studies however indicate that, at high redshifts too, violent mechanisms such as major or minor mergers are required to generate strong concentrations of gas (see Elbaz et al. 2018, and references therein). The typical high sSFR values of GRB host galaxies (e.g. Sokolov et al. 2001; Chary et al. 2002; Christensen et al. 2004; Savaglio et al. 2009; Svensson et al. 2010; Perley et al. 2015), suggesting a recent boost in their star formation, are consistent with the existence of a link between GRB hosts and interacting systems. Interactions are known to enhance the star formation activities of galaxies and increase their SFR up to an order of magnitude higher (e.g. Renaud et al. 2014; Pan et al. 2018). Earlier studies have found indications of interactions and mergers in GRB host galaxies, but the evidence has not been unambiguous. Chary et al. (2002) found 6 of the 11 GRB hosts in their sample to be disturbed or to have candidate companion galaxies. Spectroscopic studies are required in order to, as the first step, confirm whether the candidate companions are ESO 184−G82: a collisional ring galaxy 7 S U M M A RY We have used the GMRT to map the H I 21 cm emission from ESO 184−G82, the z = 0.0087 host galaxy of GRB 980425/SN1998bw. The H I 21 cm intensity images and velocity distribution yield clear evidence that ESO 184−G82 is undergoing an interaction with a companion galaxy: these include the detection of an H I knot to the north-east of ESO 184–G82, an extended H I structure extending from ESO 184–G82 towards the H I knot, the disturbed H I velocity field, and finally the presence of a high column density H I ring, likely a collisional ring, around the optical centre of ESO 184−G82, passing through the actively star-forming regions of the galaxy. We use VLT/FORS, HST, and Spitzer optical and NIR imaging to identify a small galaxy coincident with the H I knot detected in the GMRT H I 21 cm image, at a projected distance of 13 kpc from the centre of the GRB host galaxy. We find the companion galaxy to be a gas-rich star-forming dwarf galaxy, with an SFR of 0.004 M yr−1 , a gas mass of 107.1±0.1 M , and a stellar mass of 107.0±0.3 M . Head-on collisions produce star-forming gaseous rings with high surface densities and velocity dispersions, leading to the formation of massive SSCs in the ring. At the same time, they cause little (or no) star formation enhancement in the centre of the galaxies, unlike classical tidal interactions. Our simulations of dwarf–dwarf mergers illustrate this process and show that a head-on collision can reproduce the main observed gas and star formation features of ESO 184−G82 simultaneously. This is while it is difficult to explain the observed properties such as the presence of the gas ring and the absence of the star formation enhancement in the centre of ESO 184−G82 via a tidal encounter. Our findings therefore suggest that the collision between ESO 184−G82 and its companion galaxy has led to the formation of dense and massive SSCs in which the GRB progenitor must have formed. AC K N OW L E D G E M E N T S MA and SR would like to thank Bruce Elmegreen, Francoise Combes, Diane Cormier, and David Elbaz for valuable discussions. We thank the staff of the GMRT for making these observations possible. The GMRT is run by the National Centre for Radio Astrophysics of the Tata Institute of Fundamental Research. We acknowledge using data based on observations collected at the European Southern Observatory under ESO programmes 064.H0375(A), 066.D-0576(A), and 165.H-0464(A). This work is based in part on observations made with the Spitzer Space Telescope, which is operated by the Jet Propulsion Laboratory, California Institute of Technology under a contract with NASA, and also data made with the NASA/ESA Hubble Space Telescope, obtained from the data archive at the Space Telescope Science Institute. MA acknowledges support from UnivEarthS Labex programme at Sorbonne Paris Cité (ANR-10-LABX-0023 and ANR-11-IDEX0005-02). 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https://openalex.org/W2069663025	https://escholarship.org/content/qt5vn9h8qd/qt5vn9h8qd.pdf?t=ooem2v	English	null	The environmental psychology of child sexual abuse	Journal of environmental psychology	1,994	cc-by	13,151	UC Irvine UC Irvine Previously Published Works Title The environmental psychology of child sexual abuse Permalink https://escholarship.org/uc/item/5vn9h8qd Journal Journal of Environmental Psychology, 14(3) ISSN 0272-4944 Authors Holman, E Alison Stokols, Daniel Publication Date 1994-09-01 DOI 10.1016/s0272-4944(94)80059-6 Copyright Information This work is made available under the terms of a Creative Commons Attribution License, availalbe at https://creativecommons.org/licenses/by/4.0/ Peer reviewed UC Irvine UC Irvine Previously Published Works Title The environmental psychology of child sexual abuse Permalink https://escholarship.org/uc/item/5vn9h8qd Journal Journal of Environmental Psychology, 14(3) ISSN 0272-4944 Authors Holman, E Alison Stokols, Daniel Publication Date 1994-09-01 DOI 10.1016/s0272-4944(94)80059-6 Copyright Information This work is made available under the terms of a Creative Co at https://creativecommons.org/licenses/by/4.0/ Peer reviewed Introduction In order to understand and prevent such negative consequences as revictimization, we must identify the contextual circumstances, as well as the cog- nitive, emotional, and behavioral processes, that contribute to their development. In order to understand and prevent such negative consequences as revictimization, we must identify the contextual circumstances, as well as the cog- nitive, emotional, and behavioral processes, that contribute to their development. It is estimated that 15-35% of all American women, and 6-10% of all American males are sexually abused as children (Finkelhor, 1979; Russell, 1986; Hartman & Burgess, 1989). Nearly one half (47%) of these children experience this abuse at the hands of a family member, and another 40% are abused by an acquaintance (Hartman & Burgess, 1989; Dare & McKurdy, 1992). Comparative data documenting prevalence rates for child sexual abuse in 20 different countries suggest that international rates of abuse are similar to those reported in the United States--7-36% for women, 3-29% for men (see Finkelhor, 1994). p Several conceptual models have been proposed to explain the development of various long-term effects of CSA (Finkelhor & Browne, 1985; Hartman & Burgess, 1989; Starr et al., 1991; Briere, 1992; Cole & Putnam, 1992; Trickett & Putnam, 1993). What is noticeably missing from these models, however, is an awareness of how the sociophysical environment influences the etiology, experience, and develop- mental consequences of sexual abuse. For example, how do the temporal patterns of household activi- ties and the environmental design of family resi- dences increase or decrease opportunities for the perpetration of CSA? How might sexual abuse influence the development of a child's interactions with her physical surroundings? In what ways might sociocultural and demographic forces moder- ate the incidence and consequences of CSA for the individual, her family, and the community? These questions can only be addressed by expanding the conceptual and methodological scope of current research on CSA. In recent years, sexual abuse has been associated with a variety of psychological and interpersonal adjustment problems (Russell, 1986; Cole & Putnam, 1992; see Kendall-Tackett et al., 1993 for a review) and, consequently, this topic has received a great deal of public and professional attention. Yet, despite the increasing volume of research on the adverse impacts of child sexual abuse (CSA), little is known about the mechanisms facilitating individual adaptation and the reasons underlying successful adjustment in some individuals. E. ALISON HOLMAN AND DANIEL STOKOLS School of Social Ecology, University of California, Irvine, CA 92717, U.S.A. E. ALISON HOLMAN AND DANIEL STOKOLS School of Social Ecology, University of California, Irvine, CA 92717, U.S.A. Abstract This paper offers an environmental psychological analysis of child sexual abuse, a pervasive and disruptive societal problem. Earlier analyses of child sexual abuse have emphasized clinical, social, and developmental concepts and methodologies, while neglecting the environmental context of the problem. The proposed con- ceptualization of child sexual abuse suggests that a broader understanding of the etiology and psychosocial consequences of this problem can be achieved by integrating theoretical constructs drawn from clinical, social, developmental, and environmental psychology. First, some of the key findings and analytical perspectives from earlier studies of child sexual abuse are reviewed and then those findings are linked to a broader analysis of people-environment transactions. This transactional approach emphasizes contextual influences on the etiology and psychosocial outcomes of child sexual abuse, and suggests both clinical and environmental design strategies to reduce the prevalence and disruptive impacts of this problem. Powered by the California Digital Library University of California eScholarship.org 0272-4944/94/030237+16508.00/0 Journal of Environmental Psychology (1994) 14, 237-252 © 1994 Academic Press Limited Clinical, Social and Developmental Research on Child Sexual Abuse environmental factors in moderating the occurrence and long-term consequences of this problem. While the ecological context of child maltreatment has been examined in earlier analyses (e.g. Garbarino, 1977; Belsky, 1980), the role of the physical environ- ment as it influences the occurrence and conse- quences of CSA has not been addressed in prior theoretical and empirical research. Hence, we aim to integrate clinical, social, developmental, and environmental psychological perspectives in order to achieve a broader understanding of the dele- terious long-term effects of CSA, with special atten- tion given to the unusually high rates of sexual revictimization among sexually abused individuals (see Gorcey et al., 1986; Russell, 1986). A variety of psychological, somatic, and interpersonal symptoms have been associated with sexual abuse. These symptoms include cognitive and emotional difficulties such as low self-esteem (Finkelhor, 1979; Morrow & Sorell, 1989), rumination and flashbacks (Silver et al., 1983; Courtois, 1988), dissociative processes (Chu & Dill, 1990; Meiselman, 1990), de- pression (Bagley & Ramsey, 1986; Briere & Runtz, 1988) and anxiety (Murphy et al., 1988; Greenwald et al., 1990); interpersonal problems such as in- ability to trust (Herman, 1981; Meiselman, 1990), and difficulties in establishing and maintaining interpersonal relationships (Russell, 1986; Alexander & Lupfer, 1987); behavioral problems such as highly sexualized, aggressive, and/or self-destruc- tive behaviors (Sedney & Brooks, 1984; Briere, 1988; Friedrich, 1988); and high risk revictimiza- tion (Gorcey et al., 1986; Russell, 1986). Because subsequent victimization may exacerbate other negative effects identified among abused individuals (Murphy et al., 1988), preventing revictimization is a crucial first step in promoting positive adaptation. To do this, however, we must understand the pro- cesses underlying the development of long-term adjustment problems, especially, how sexual abuse survivors become vulnerable to revictimization. ( y , ; , ) Our analysis focuses on theoretical concepts and research findings from environmental psychology that are relevant to an understanding of sexually abusive experiences and the long-term difficulties encountered by some abuse survivors. Constructs from environmental psychology pertaining to per- sonal space (e.g. Hall, 1966; Sommer, 1969; Alello, 1987), territoriality (e.g. Altman, 1975; Brown, 1987; Taylor, 1988), and place identity (Proshansky et al., 1983; Korpela, 1989), for example, suggest pro- cesses by which sexually abusive acts can engender patterns of relating to the physical environment that render abused individuals vulnerable to future danger. Introduction For example, why is it that as many as 68% of incest survivors, compared with 38% of non-abused women, report being sexually assaulted as adults (Russell, 1986)? The major goal of this paper is to develop an environmental psychological analysis of CSA that highlights the role of sociocultural and physical 237 E.A. Holman and D. Stokols 238 Clinical, Social and Developmental Research on Child Sexual Abuse Moreover, these personal and interpersonal processes are embedded within a broader social, spatial, and cultural context that can either encour- age or constrain opportunities for the perpetration of, and recovery from, CSA. Various theoretical perspectives have been used to explain the development of long-term effects among sexually abused individuals. In a recent review of studies on the effects of sexual abuse on children, these perspectives were described as either core-symptom theories or multifaceted models of trauma (Kendall-Tackett et al., 1993). The focus of core-symptom theories is on identifying specific symptomatology that distinguishes sexual abuse survivors from non-abused and/or clinical populations. For example, post-traumatic stress disorder is an organized framework that accounts for intrapsychic cognitive (e.g. rumination, flash- backs), physiological (autonomic hyperarousal), and behavioral (avoidant, phobic) responses to the traumatic stress of sexual abuse (Deblinger et al., 1989). Another core-symptom approach suggests that the abuse damages the child's developing sense of self which, in turn, gives rise to subsequent psychosocial difficulties (Cole & Putnam, 1992; cf. McCann & Pearlman, 1990). Finally, some researchers have suggested that premature stimulation of the child's sexuality may initiate patterns of inappro- priate sexual behavior that adversely influence subsequent development (Friedrich, 1988). We recognize the similarities in response to various forms of childhood victimization, such as psy- chological, physical, and sexual abuse (Garbarino, 1977; Emery, 1989; cf. Briere, 1992). The experience of sexual abuse, however, has the unique character- istic of violating a most intimate aspect of a child's body boundary--sexuality. Hence, although the con- cepts outlined in this paper may be broadly applica- ble to situations in which a child's body boundaries are generally violated, we focus here on developing the analysis as it applies to the CSA experience. 1 Because our analysis builds on earlier research concerning the long-term impacts of CSA, we begin with an overview of the patterns of psychological and social adjustment identified in sexually abused individuals. The contributions and limitations of earlier models developed to explain these findings are examined, and an environmental psychological analysis of CSA is proposed as a framework for future research. Finally, we discuss the clinical and environmental design implications of our analysis. Environmental Psychology of Child Sexual Abuse 239 conditions, a trauma encapsulation phase that involves processing the experience, disclosure of the abuse, and post-trauma responses to the abuse. The authors propose a set of information processing steps that highlights important aspects of adjust- ment. Clinical, Social and Developmental Research on Child Sexual Abuse First, encapsulation of the event involves maintaining defensive silence while currently experiencing conscious memories of the abuse. Second, dissociation from the event is described as a self-preservation strategy that shifts attention away from the abuse experience, even though the abuse may be ongoing. Finally, dissociation may precipitate 'splitting' in which the child's ego frag- ments resulting in self-blame and justification of the abuse using self-deprecatory explanations. At a behavioral level, splitting is thought to result in unusual patterns of stimulation and inhibition of sexual behavior. Multifaceted explanations for the long-term effects of sexual abuse have drawn from clinical research findings to develop an overarching theo- retical model of adjustment to sexual abuse. For example, Finkelhor and Browne (1985) suggest that the dysfunctions manifested in abused individuals' lives stem from four traumagenic dynamics surround- ing the abuse: traumatic sexualization, betrayal, stigmatization, and powerlessness. They further suggest that the co-occurrence of these experiences characterizes response to sexual abuse, but that any one alone may indicate many different forms of childhood trauma. In Finkelhor and Browne's model, traumatic sexualization is a process in which a child's sexuality is shaped in a developmentally inappropriate and interpersonally dysfunctional manner. As a child is taught to exchange sexual favors for the love, affec- tion, and attention s/he needs, s/he learns sexually inappropriate behavior and develops a distorted view of his/her own sexual self. Betrayal results when the child becomes aware that a person s/he is dependent on has harmed him/her and violated his/her trust- hence, feelings of betrayal would be expected in cases of parental incest or sexual abuse by a known trusted adult. Powerlessness refers to a process in which the child's sense of personal will, desire, and self-efficacy are constantly undermined. Finally, stigmatization refers to the negative implications about the child (i.e. being bad, shameful, or guilty for the abuse) that may become part of the child's self-image. These four dynamic processes are used as an organizing scheme for explaining the various effects of sexual abuse, and specific symptoms com- monly described by survivors are grouped according to this framework (Finkelhor & Browne, 1985). There are at least two limitations inherent in each of these analyses of the consequences of CSA. First, they focus almost entirely on the damage done to the abused individual as the source of sub- sequent adjustment problems. Thus, the 'problem' ultimately resides within the abused individual. Clinical, Social and Developmental Research on Child Sexual Abuse Stokols and its disclosure rather than specific characteristics of the abuse, or the child's personal response to the abuse• These authors suggest that the stress associated with CSA alters the child's hormonal environment which induces behavioral, emotional, and biological changes in the child's social, familial, and physio- logical experience• This model is focused within the individual child, with family and peer support modi- fying the relationship between psychological distress and the child's developing competencies. In sum, this model specifically posits that sexual abuse has negative effects on female development because it intensifies the pubertal experience by: (a) creating painful and/or uncomfortable feelings about sexual- ity that complicate the onset of sexual maturation; (b) threatening the interpersonal relationships that are normally used to deal with the changes brought about by puberty; and (c) hormonally initiating an early onset of puberty• Most relevant to our analysis, however, is the impact of CSA on a child's body- image while coping with the rapid, uncontrollable biological changes brought on by puberty• As we discuss later, the child's developing relationship with his/her physical body is central to learning to negotiate safely in the sociophysical environment• More recently, however, a call has been made for the application of life span developmental theory in studies of adaptation to abuse (Friedrich, 1988; Starr et al., 1991; Cole & Putnam, 1992; Kendall- Tacket et al., 1993; Finkelhor & Dziuba-Leather- man, 1994). For example, Cole and Putnam (1992) adopted the perspective of developmental psycho- pathology to articulate specific processes underlying the adjustment problems identified among incest survivors. This model traces adjustment problems to the potentially disruptive effects incest may have on one's sense of self and interpersonal relations across developmental transitions• Starr et al. (1991) extend this model by suggesting that development occurs within a larger social ecological context: • . . in order to explore these connections (between child maltreatment and adult functioning) it will be necessary to employ a conceptual model that recog- nizes the organizational properties of the self- system, . . . the social organization of the ecological setting . . . the critical nature of developmental timing as key features• (Starr et al., 1991, p. 21) To summarize, several models have been offered to account for the range of psychosecial symptoms found among sexual abuse survivors. Clinical, Social and Developmental Research on Child Sexual Abuse With a few exceptions, these models have focused on psycho- logical symptoms of the abused individual, rather than on either the underlying developmental pro- cesses giving rise to these symptoms, or the socio- cultural and physical-environmental factors that may encourage sexually abusive behavior and in- fluence the duration and severity of an individual's response to such events. Thus, we turn now to an analysis of CSA that addresses these previously neglected aspects of the problem• One of the crucial features that distinguishes this model from the others is its attention to the ongoing social environment which surrounds the abuse experience• Thus, these authors provide one of the few contextually based approaches to understand- ing long-term adaptation to CSA. Another developmentally-oriented analysis attri- butes psychosocial adjustment problems to parent- child transactions that give rise to insecure and disorganized patterns of attachment behavior in the child (Alexander, 1992). Attachment theorists have proposed that internal working models of inter- personal relationships develop from early parent- child interactions and may influence subsequent social development (Bowlby, 1988; see also Main et al., 1985). Accordingly, an abused individual's functioning in the social environment may be power- fully influenced by his/her experiences of parental abuse• This application of attachment theory helps to explain, at least in part, the internal and inter- personal processes that mediate a child's subse- quent adjustment to sexually abusive experiences• Integrating Clinical, Social, Developmental, and Environmental Psychological Perspectives on Child Sexual Abuse Clinical, Social and Developmental Research on Child Sexual Abuse Even though this may not be intended, reducing the long-term effects of CSA to the individual's dysfunc- tion may inadvertently pathologize abused individ- uals. Second, the exclusive focus on negative out- comes such as persistent distress or dysfunction reflects an assumption that incest necessarily en- genders long-term emotional distress and behavioral dysfunction. This approach neglects the possibility that negative consequences of abusive events may be short-lived and that individuals are often able to cope effectively with those events. Given that 40- 65% of non-clinical samples of abused individuals do not manifest chronic symptomatology (Russell, 1986; Stein et al., 1988), the explanatory power of these models is limited. Ideally, theoretical models of child sexual abuse should explore the full range of potential responses, look beyond individual dys- function, and encompass those contextual factors that may influence the occurrence of abuse and its impact on psychosocial adaptation. Another model of post-abuse adjustment organizes the characteristic psychological symptoms of sexual abuse into six broad categories: (a) negative self- evaluation, (b) chronic perception of danger or injustice, (c) powerlessness or preoccupation with control, (d) dissociative control over awareness, (e) impaired self-reference, and (f) reduction of painful internal states (Briere, 1992). These themes represent the underlying cognitive, emotional, and behavioral mechanisms that explain abused indi- viduals' symptomatology. Several authors have discussed the sociocultural and/or developmental contexts that may affect post-abuse adjustment. For example, Summit (1988) argues that the social rejection, denial, and blame that often accompany disclosure of abuse may perpetuate sexual victimization of children by encouraging social complacency and indifference to their plight. This explanation for the negative consequences of sexual abuse focuses on the role of the social environment that surrounds the abuse A third model specifies phases of abuse and recovery, as well as conceptual processes to explain adjustment (Hartman & Burgess, 1989). Unlike the other models, this approach considers the abuse in relation to pre-abuse and post-abuse experiences that may influence the long-term consequences of CSA. This stage model of CSA includes pre-trauma 240 E.A. Holman and D. • . . in order to explore these connections (between child maltreatment and adult functioning) it will be necessary to employ a conceptual model that recog- nizes the organizational properties of the self- system, . . . the social organization of the ecological setting . . . the critical nature of developmental timing as key features• (Starr et al., 1991, p. 21) Environmental Psychological Perspectives Environmental Psychological Perspectives physical environment (Saegert, 1987; Stokols, 1988). Theories of transformational change suggest that opportunities for both growth and deviance are cre- ated by these pivotal events and experiences, through a process of deviation amplification (Maruyama, 1963). According to deviation amplification theory, any event (either planned or fortuitous) can initiate a chain of events that takes adaptation in an in- creasingly positive or negative direction (Aldwin & Stokols, 1988). For example, Rutter's (1987) analy- sis of psychological resilience suggests that stressful childhood events may guide development toward deviance or adaptation, depending on the socio- cultural context in which the event occurs. A major assumption of our analysis is that the physical environment influences the etiology, expres- sion, and consequences of CSA. Our examination of these environmental influences on CSA begins with a discussion of certain core themes or meta- theoretical principles of environmental psychology (Stokols &Altman, 1987; Saegert & Winkel, 1990). We then discuss the relevance of more specific constructs drawn from several research paradigms of environmental psychology, including personal space, territoriality, place identity, and environ- mental stress. Considering these themes of human-environment transaction, optimization, and transformation, CSA can be construed as a life event or series of life experiences that has enormous potential to trans- form the quality of a child's transactions with his/her environment. Specifically, the potentially coercive and emotionally distressing nature of CSA may diminish a child's active efforts to optimize his/ her environment, and to promote a predominantly passive stance toward the environment. Thus, one form of transformational change triggered by CSA would be reflected in the child's shift from active modes of dealing with the sociophysical environ- ment to a more passive and reactive stance toward her surroundings. One theme of environmental psychology is the dynamic and reciprocal quality of people-environ- ment transactions. The transactional perspective (Altman & Rogoff, 1987; Wohlwill & Heft, 1987) assumes that people's interactions with their environ- ments sometimes involve reactive adjustments to existing environmental conditions and, at other times, more active and goal-directed efforts to modify the environment in accord with specified preferences and plans. Typically, people shift back and forth between active and reactive orientations toward the environment. In uncontrollable or inflexible environ- ments, however, opportunities for changing one's surroundings are restricted and the individual's stance toward the environment thus becomes pre- dominantly passive or reactive. Environmental Psychological Perspectives A fourth theme of environmental psychology is that the etiology, intensity, and consequences of one's experiences with the environment can be best understood by considering the spatial, temporal, and sociocultural contexts in which those experi- ences and events occur. In the following section we apply these themes in an analysis of the etiology, experience, and aftermath of CSA. A second theme of environmental psychology is human--environment optimization (Stokols, 1978). The optimization concept posits that people ideally strive to achieve 'optimal environments', i.e. those that maximize the fulfillment of their goals and plans. In many instances, though, people are con- strained by situational factors to accept undesirable environmental conditions or, at best, to 'satisfice' (Simon, 1957; Dubios, 1965). Whereas the concept of optimization refers to ideal conditions, it is useful at a heuristic level in emphasizing the goal-directed and reciprocal nature of people-environment transactions. Transformational Processes Within the Temporal, Spatial and Sociocultural Contexts of CSA The temporal context of CSA can be viewed in relation to at least three major phases of abusive experiences: the pre-abuse phase, the abuse phase from the onset of CSA to its termination, and the post-abuse phase. The child's sociophysical environment plays an important role in each of these phases of CSA. A third theme of environmental psychology relates to the tension between equilibrium vs transforma- tional processes in people-environment transactions. Non-transformational research emphasizes the con- ditions under which people's interactions with their environments are essentially stable and unchanging. Transformational studies, on the other hand, empha- size the power of certain life events (e.g. relocation to a novel environment or sudden change in an existing one) to dramatically restructure the pre- existing pattern of a person's social relationships and the quality of his or her experiences with the Environmental Psychology of Child Sexual Abuse 241 Integrating Clinical, Social, Developmental, and Environmental Psychological Perspectives on Child Sexual Abuse A great deal of our knowledge about the processes affecting long-term adjustment to child sexual abuse has come from clinically based studies of abused individuals (Herman, 1981; Meiselman, 1990; Putnam, 1990; Briere, 1992) and socially- oriented studies of students or community members (Finkelhor, 1979; Silver et al., 1983; Russell, 1986; Alexander & Lupfer, 1987; Stein et al., 1988). Our interest lies in integrating clinical, social, and developmental approaches to CSA with the concep- tual and methodological perspectives of environ- mental psychology to gain a broader understanding of the processes underlying long-term adaptation• Finally, Trickett and Putnam (1993) have pro- posed a psychobiological/developmental model for explaining long-term adjustment to CSA. Their model offers a unique integration of the psychological and physiological responses to sexual abuse by conceptualizing CSA as a stressor that may directly affect the quality and timing of pubertal experiences• Environmental Psychology of Child Sexual Abuse The abuse phase In order to understand an individual's interpreta- tion of, and response to CSA we must consider the sociocultural context surrounding the abuse. Both the immediate social environment and the prevailing sociocultural norms are likely to influence what the CSA experience means to the child. Over the last few decades, sociocultural norms for sexual behavior have varied greatly, with the recent AIDS epidemic serving as a catalyst for intense public debate about sexual mores. Moreover, the last decade has seen a great increase in the public awareness about CSA. Hence, we would expect the sociocultural and moral landscape of different periods in history to affect both the nature of the CSA experience and the indi- vidual's response to it. That is, CSA that occurs in the 1990s may have very different meaning and impact for the child than abuse that took place 20, 30, 40, or 50 years ago. With this in mind, we now turn to a discussion of the transformational pro- cesses that may occur during the CSA experience. p , ) While these characteristics of the broader, macro- level sociophysical environment have been linked theoretically to sexually abusive behavior, little attention has been given to micro-level sociospatial factors that may increase the opportunity or motivation to commit CSA. In accord with earlier ecological analyses of crime occurrence and distri- bution within urban communities (e.g. Cohen & Felson, 1979; Archea, 1985; Fisher & Nasar, 1992), it seems reasonable to assume that some physical environments may 'afford' sexually abusive behavior through their spatial layout and design features. For example, just as family isolation may increase a child's vulnerability to CSA (Finkelhor, 1979), so might the physical isolation of a child within a home (see e.g. Tierney & Corwin, 1983). Conversely, the degree to which the layout of a home incorporates 'defensible space' may also affect the etiology of CSA by influencing patterns of territorial and self-protec- tive behavior (e.g. Holahan & Wandersman, 1987; Newman, 1972; Taylor, 1987). A home that allows individual control over personal space with- out promoting extreme physical, visual, or auditory isolation may reduce opportunities for a motivated perpetrator to commit CSA. In sum, to understand fully the etiology of CSA, it is important to consider opportunities for CSA that are afforded by aspects of the physical environment (Gibson, 1977). The pre-abuse phase We cannot expect to understand all the fortuitous events that initiate CSA, but we can identify periods of time and characteristics of sociophysical environ- E.A. Holman and D. Stokols 242 ments that may encourage it. Toward this end, researchers have outlined four preconditions associ- ated with the onset of CSA: motivation to commit CSA, lack of internal inhibiting factors, lack of external inhibitors, and lack of resistance from the child (Finkelhor, 1986). Sociocultural factors thought to be associated with higher rates of CSA include high rates of social change, immigration, geographic mobility (Tierney & Corwin, 1983), overcrowded or disorganized conditions (Bagley, 1969), a diminished sense of belonging to a group, household density levels, and social or physical isolation (Finkelhor, 1979; see also Tierney & Corwin, 1983; Haugaard & Repucci, 1988). behavior. For example, it is conceivable that the presence of adult book stores or displays of por- nography may foster the development of cognitive schema for sexually exploitative interpersonal rela- tionships and undermine internal sanctions against abusive behavior. It would be useful to explore the nature and prevalence of sexually abusive behavior in sociophysical environments that condone sexual exploitation. The abuse phase y ( , ) Applying this theory to CSA, it is hypothesized that CSA decreases the child's primary control over interpersonal relationships and forces the child to become disproportionately more passive than active in his or her encounters with the environment. This transformation is likely to be exacerbated when the child is physically or emotionally dependent on the offending person. Under these circumstances, the child may experience feelings of helplessness, power- lessness, and confusion (Finkelhor & Browne, 1985). Such dependency may pressure the child to modify his/her natural responses (e.g. fight or flight) in order to survive (Summit, 1983). For example, many abuse survivors become hypervigilant, learn- ing to assess the needs and desires of others around them. This hypervigilance includes a tendency to be so 'other-directed' (Briere, 1992) that the individual becomes preoccupied with accommodating his/herself to others while neglecting her own personal safety and dignity. These behaviors, however, may in fact, be attempts to gain secondary control by accommo- dating the abusive, dangerous situation. p Empirical evidence regarding boundary regulating processes indicates that humans normally flee situa- tions in which privacy boundaries are transgressed (Felipe & Sommers, 1966; Dosey & Meisels, 1969; McDowell, 1972; Aiello, 1987). However, children are often physically or emotionally coerced into complying with CSA, making it difficult to flee the situation (Russell, 1986; Silver et al., 1983). Thus, instead of fleeing from the situation physically, 2 the child may cope with the violation of personal space and body by mentally leaving--dissociating from the physical self (Briere & Runtz, 1988; Chu & Dill, 1990; Putnam, 1993). In essence, the person retreats from conscious, active participation in the sociophysical world. g , g An adult's abuse of power over a child can also lead to traumatic bonding (deYoung & Lowry, 1992; Dutton & Painter, 1981), which involves a powerful emotional dependency between two people that develops when one person intermittently mistreats the other (Dutton & Painter, 1981). Individuals involved in these relationships tend to be socially isolated, with the aggressor of the pair exerting tremendous control over the social activities of the 'victim' (Dutton & Painter, 1981). The frequent use of force or intimidation to maintain secrecy about the abuse may encourage social withdrawal, isola- tion, and alienation by creating a barrier between the abused individual and others (see Silver et al., 1983). The abuse phase y g p CSA commonly involves an opportunistic viola- tion of personal space that occurs when the child's vulnerability converges with an offender's motiva- tion to abuse. The experience can be thought of as a deviation amplifying process that transforms a child's relationship with the sociophysical environ- ment through its undermining effects on perceived control. The coercive force that commonly accom- panies CSA (Finkelhor, 1979; Russell, 1986) violates a most fundamental form of control learned in early life--control over one's own body and bodily sensa- tions. As the offender asserts power over the child, he creates a passive or withdrawn role for the child. The child's transactions within his/her sociophysical space become limited when his/her attempts to control the environment are overpowered by an offending adult. Control theorists suggest that when individ- uals are unable to exert active, primary control in a situation, they may resort to secondary methods of control such as lowering expectations to prevent disappointment and affiliating with powerful others (Rothbaum et al., 1982). On the surface, these tech- Characteristics of the sociocultural environment may also influence the onset of CSA by shaping people's attitudes and beliefs about sexual behavior. Researchers studying the effects of pornographic violence on sexual attitudes have provided convinc- ing evidence that sexual violence in movies and television enhances both aggressive behavior toward women (Donnerstein, 1980) and acceptance of sexu- ally.aggressive behavior in males (Malamuth, 1983; 1986). Little is known, however, about the relation- ship between characteristics of the sociophysical environment and the expression of sexually abusive Environmental Psychology of Child Sexual Abuse 243 niques look like passive acquiescence, however, the individual is thought to experience greater perceived control despite his/her inability to influence external forces directly (Rothbaum et al., 1982). to one's group' and serves to help people protect themselves from the undue external influence of others (Altman, 1975). Regulation of personal space and territorial behavior are two means by which individuals maintain privacy boundaries (Hall, 1966; Altman, 1975). The 'primary' territories we inhabit help shape and reinforce our identities, and provide a sense of security and freedom to express our 'true selves'. Therefore, violations of primary territorial boundaries are likely to be experienced as personal affronts that would normally provoke a defensive response (Altman, 1975). The abuse phase Thus, by interfering with a child's ability to control his/her body, CSA sets a standard for control beliefs and behavior that may be generalized to the child's t'~'ansactions within the broader sociophysical environment. Initially, this transformation may manifest itself as a difficulty in establishing and protecting appropriate interpersonal spatial bound- aries. Ultimately, this transformation may lead a child to unwittingly accommodate his/herself to dangerous social and physical environments. These dissociative processes are thought to pro- tect the individual from overwhelming feelings and other sensory input that accompany abuse (Briere & Runtz, 1988; Speigel, 1988, 1989). ~Iowever, the need to mentally dissociate from his/her physical body to flee from invasive, abusive contact may have negative consequences if it encourages the child to view the body as something separate from the 'self (Young, 1992). Objectification of the physical form may render the child out of touch with the physical sensations associated with unwanted, uncomfortable touching, the very experiences s/he should learn to avoid. To the extent that dissociation removes con- scious awareness of the internal physical sensations triggered by external environmental stimuli, it may weaken an individual's ability to identify danger in the physical environment. This is the Achilles heel for revictimization, leaving the individual at greater risk for subsequent sexual assault, a The post-abuse phase Having identified some of the general themes of environmental psychology that apply to the etiology and experience of CSA, we now focus on specific areas of research in environmental psychology that may help to explain the long-term negative consequences of sexual abuse. As a violation of At the most intimate level, CSA is a transgression of privacy and personal space that violates physical and psychosexual body boundaries. Privacy has been defined as 'selective control of access to self or 244 E. A~ Holman and D. Stokols interpersonal spatial boundaries, CSA disrupts boundary-regulating abilities by denying children the right to control their interactions with others, thereby undermining their developing self-respect and identity formation (see e.g. Goffman, 1961). As a violation of primary territory, CSA disrupts the development of attachments to physical places, starting with the physical body, the smallest space a child can occupy. The tendency to dissociate in response to these violations may, over time, generalize beyond the physical body to other uncomfortable settings, making it difficult for the child to stay mentally present and fully aware in non-abuse re- lated sociophysical settings as well. Moreover, as a form of interpersonal violence that takes place within traditionally 'safe' environments, CSA may undermine a child's developing assumptions about 'safety' and 'safe' places. Instead of learning that home and com- munity are 'safe', s/he may internalize a view of these places as dangerous, unsafe places in general (see Garbarino et al., 1992). Thus, in response to CSA, the child may develop a generalized alienation from many places in his/her immediate sociophysical world. CSA can also disrupt a child's relationship with the physical environment by creating memories that later evoke feelings of place aversion (Proshan- sky et al., 1983; Rubinstein, 1993). Aspects of the physical environment may become permanent, painful reminders of the abuse through classical conditioning of psychological responses to the trauma (van der Kolk, 1993). As mentioned earlier, the Trickett and Putnam (1993) model emphasizes the role of CSA in altering the child's hormonal pro- cesses especially as they influence the onset of puberty. Another area of psychobiological research has identified the important role played by stress- related hormones in intensifying learning and memory processes, in that memories of highly stress- ful events become more enduring and vivid than those associated with lower levels of psychophysio- logic arousal (McGaugh, 1989). The post-abuse phase Hence, the physio- logic response associated with CSA may precipitate the formation of memories that hinder the individ- ual's subsequent ability to form positive attachments to people and specific places. The more arousing the event, the more enduring this effect is likely to be. g y The powerful influence of sexually traumatic events in the formation of painful and persistent memories is evidenced by the frequently observed tendency of abused individuals to 'flashback' to some aspect of the CSA experience (Silver et al., 1983; van der Kolk, 1993). Flashbacks are com- monly described by clinicians as sensory stimuli classically conditioned at the time of the abuse that subsequently remind the survivor of the feelings associated with abusive events (Meiselman, 1990 van der Kolk, 1993). The stimuli may include an~ smells, sounds, sights, touches, or tastes that wer~ experienced during the abuse. Re-experiencinl these sensations triggers feelings of distress tha are seemingly unwarranted to the individual. Tha is, the distress has no clear basis in present circu~ stances. If the child originally responded to th abuse by dissociating, then re-experiencing certai environmental sensory cues associated with tl~ abusive setting may unknowingly trigger dissoci~ tion at a later time. Thus, dissociation, a on~ successful coping strategy for adjusting to the u~ controllable abuse (Putnam, 1993), may transfor the child's way-of-being in the physical world al render the child more vulnerable than others being targeted by perpetrators. The net result unusually high rates of revictimization amo: sexually abused individuals. p p y Dissociation can also be thought of as a deviation- amplifying process that initiates a negative (or posi- tive) chain of events, and affects a child's ability to develop an optimal relationship with the socio- physical environment. Specifically, CSA may disrupt a survivor's attachments to primary territories that provide a sense of belonging in the physical world (Proshansky et al., 1983). Through violations of the child's primary territory, such as the perpetrator's unwanted entry into his/her room, bed, or body, the child learns s/he doesn't have the right to own or control private space. Instead, the child learns that private personal spaces are where intimate people hurt one another, hence generating feelings of mis- trust about these places. Aversion to the physical space associated with the abuse creates alienation from primary territories, such as a bedroom or home environment, that undermines the ongoing develop- ment of place attachments (Proshansky et al., 1983). The post-abuse phase Moreover, since identification with a primary terri- tory is thought to impart feelings of security, the need to dissociate from the physical environment may undermine the child's ability to establish phys- ical anchor points that strengthen one's sense of personal security (Wapner, 1981). If a sense of 'rootedness' or attachment to personal places strengthens a child's ability to interpret and control physical surroundings, then experiences that weaken these attachments undermine the child's ability to navigate safely through the physical environment (Proshansky et al., 1983). Finally, the dissociative process and the impli disrespect of the body that is encouraged by the abl serves as the basis for self-destructive behavi, Environmental Psychology of Child Sexual Abuse 245 whereas others are suggested by prior studies of child physical abuse and neglect (Garbarino, 1977; Belsky, 1980, 1993). The physical and social environ- mental antecedents of CSA are grouped in Table 1 within three contextual levels: the immediate resi- dential environment of the child; the child's neigh- borhood; and conditions within the broader commu- nity. These environmental contexts correspond to the microsystem, mesosystem, and macrosystem levels within Bronfenbrenner's (1979) ecology of human development model. commonly seen among survivors in clinical settings (Meiselman, 1990; Young, 1992). Such behavior may represent an attempt to either destroy the source of uncomfortable feelings (one's body or sexuality) or numb the sensations arising from within the body (Briere, 1992; Young, 1992). For example, self- mutilation has been described as a way to allow the pain to come out of the body as it bleeds (anony- mous incest group member, personal communica- tion, November, 1987). This description is consistent with previous assertions that the physical body becomes a separate object, an encapsulated source of pain for the individual, that cannot be comfort- ably 'reoccupied' until the pain is let out. For some individuals, this pain leads to suicidal behavior, making sexual abuse one of the risk factors for adolescent suicide (Deykin et al., 1985; Garland & Zigler, 1993). Within the child's residential environment, a number of physical and social factors are hypothe- sized to increase the likelihood of CSA. First, accord- ing to the routine activities perspective on crime (Cohen & Felson, 1979; Brantingham & Brantingham, 1993), the spatial layout and interior design features of the home can create opportunities for sexual abuse, provided that a motivated offender is present in the household. The post-abuse phase Specifically, physical isolation of the child's bedroom from other shared areas of the home, and auditory and visual separation of the child's room from these areas, can constrain family surveillance processes and render the child more vulnerable to victimization. Toward an Environmental Psychological Theory of Child Sexual Abuse The preceding discussion outlined three temporal phases of child sexual abuse and identified environ- mental factors that are associated with CSA within each of these phases. In this section, we discuss some of the next steps that need to be taken toward developing an environmental psychological theory of CSA. We begin by examining the proposed envi- ronmental antecedents of CSA and the intervening processes that link these factors to the occurrence of CSA. Working hypotheses regarding the direct and interactive influence of these environmental factors on CSA are proposed. Next, we present additional hypotheses about the person-environment trans- actions that are most closely associated with the psychological experience and consequences of CSA, and strategies for altering these processes through both clinical and environmental design interventions. The preliminary hypotheses offered in this section remain to be tested empirically and organized into a more coherent theoretical framework in future research. The temporal patterning of family activities and the number of individuals present in the household can similarly influence the child's physical isolation and vulnerability, while the geographic separation of the residence from other homes and neighbors can further weaken informal social controls over potential perpetrators of CSA. Also, physical stres- sors in the home such as high noise and density levels, as well as financial strains, may provoke stress and negative affect among family members, making potential offenders more likely to engage in deviant and abusive behavior (Burgess & Conger, 1978; Pelton, 1978). At the neighborhood level, several environmental factors have been found to be closely associated with child physical abuse and neglect, although the direct links among these factors and sexual abuse remain to be empirically established (Belsky, 1993). For ex- ample, high rates of residential mobility and neigh- borhood transience can undermine social cohesion and supportive social contacts among neighbors, rendering potential perpetrators more isolated and likely to abuse their children (Garbarino & Sherman, 1980; Creighton, 1985; Garbarino & Kostelny, 1992). Similarly, the presence of social and physical 'in- civilities' in the neighborhood (e.g. public drunken- ness, gangs, prostitution, graffiti, vandalism, dilapi- dated buildings) reflect community disorganization and non-supportive social climates that are con- E.A. Holman and D. Stokols TABLE 1 Physical and social environmental factors and intervening process hypothesized to increase the likelihood of child sexual abuse Environmental contexts of child sexual abuse Physical and social environmental antecedents of child sexual abuse Intervening processes linking environmental factor and child sexual abuse Residential environment Neighborhood level Community context l spatial layout and interior design of the home temporal patterning of household activities family size and residential density geographic isolation of the home l average length of residence among families in the neighborhood social and physical incivilities such as public drunkenness, gangs, prostitution, graffiti, vandalism, dilapidated buildings presence of pornographic stores high volume of vehicular traffic l societal tolerance of violence and aggressive sexual behaviour societal norms regarding corporal punishment, authoritarianism, and family privacy high rates of economic and social change within the community environmental design features afford opportunities for CSA by constraining children's privacy and defensible space household activity patterns may influence surveillance processes family size and household density may increase stress and negative affect isolation may reduce informal social controls over potential perpetrators high levels of transience, and the presence of physical and social incivilities, can undermine supportive social networks among neighborhood residents the presence of pornographic stores may symbolize sexual mores that are conducive to CSA high traffic volume can further undermine neighborhood cohesion societal tolerance of violence, aggressive sexuality, and corporal punishment of children may weaken constraints against CSA; privacy norms may further delimit societal interventions among families at risk high levels of economic and social change in in the broader community may provoke psychological strains that foster deviant sexual behavior TABLE 1 Intervening processes linking environmental factor and child sexual abuse environmental design features afford opportunities for CSA by constraining children's privacy and defensible space household activity patterns may influence surveillance processes family size and household density may increase stress and negative affect isolation may reduce informal social controls over potential perpetrators societal tolerance of violence, aggressive sexuality, and corporal punishment of children may weaken constraints against CSA; privacy norms may further delimit societal interventions among families at risk high levels of economic and social change in in the broader community may provoke psychological strains that foster deviant sexual behavior ducive to deviant behavior (Nasar & Fisher, 1993; Perkins et al., 1993). The presence of pornographic businesses in the neighborhood also are symbolic of norms and values associated with various forms of sexual deviance. Physical and social environmental antecedents of CSA Several physical and social environmental factors, and a series of intervening processes that link these factors with sexually abusive events, are summa- rized in Table 1. Some of the environmental factors and intervening processes listed in Table I have been mentioned, in earlier sections of the paper 246 E.A. Holman and D. Stokols E.A. Holman and D. Stokols Moreover, certain environmental conditions such as a high volume of vehicular traffic have been found to reduce informal contacts among neighbors (Appleyard, 1981) and may, thereby, weaken social controls over potential child abusers. private property, these norms may create a poten- tially dangerous precedent of social reticence to intervene in these 'private' affairs of a family--even when a child is being hurt. Furthermore, high rates of economic and social change within the commu- nity have been found to provoke psychological dis- tress and social strains at both family and community levels (Dooley &Catalano, 1984). These strains can further increase the likelihood of child abuse among those individuals who are already predisposed to- ward sexual deviance (e.g. by negative emotional states, neuroticism, or personal experiences of abuse during their own childhoods; Belsky, 1993). ducive to deviant behavior (Nasar & Fisher, 1993; Perkins et al., 1993). The presence of pornographic businesses in the neighborhood also are symbolic of norms and values associated with various forms of sexual deviance. Moreover, certain environmental conditions such as a high volume of vehicular traffic have been found to reduce informal contacts among neighbors (Appleyard, 1981) and may, thereby, weaken social controls over potential child abusers. Within the broader community, societal tolerance of violence and aggressive sexual behavior, social norms regarding individual and family rights to privacy, corporal punishment of children, and authoritarian values are hypothesized to weaken social constraints against CSA and other forms of sexual deviance (Ziegert, 1983; Belsky, 1993). For example, the individual's right to privacy has been extended to the family so that events taking place within the family are viewed as private family busi- ness, not subject to any forms of social scrutiny. When coupled with a tendency to view children as The working hypotheses outlined above pertain to the direct and separate links between specific physical and social environmental factors and the perpetration of sexual abuse. Considering that 'child physical abuse and neglect are multiply deter- mined by factors operating at multiple levels of analysis' (Belsky, 1993, p. 427), efforts to develop an environmental psychological theory of CSA will Environmental Psychology of Child Sexual Abuse 247 need to address the interactive relationships between multiple environmental predictors and moderators of CSA, along with a host of intrapersonal pre- disposing factors including personality dispositions and parental histories of childhood abuse. E.A. Holman and D. Stokols For example, we would expect that the likelihood of CSA incidents increases additively to the extent that environmental conditions within residential, neighborhood, and community settings (1) maxi- mize opportunities for predatory behavior among motivated offenders; (2) promote psychological stress and negative affect among family members; (3) isolate the family from other neighborhood residents; (4) undermine community cohesion; and (5) convey norms and values that are conducive to sexual deviance and abusive behavior. On the other hand, the strength of association between any one of these factors and CSA is expected to decrease to the extent that other environmental and intrapersonal predictors of CSA are absent. boundary and personal space; (2) mental dissocia- tion from one's body; (3) disruption of territorial control and privacy regulation; (4) the development of traumatic memories of particular places; (5) a stigmatized sense of place identity and an aversion rather than attachment to places; and (6) a general- ized alienation from one's sociophysical world. p y In order to fully understand how CSA transforms a child's relationship within the sociophysical envi- ronment, it is important to consider these and other mechanisms mediating children's transactions with the environment. In an extension of previous work (see Garbarino et al., 1992), we would suggest that CSA carries the potential for seriously damaging a child's developing core assumptions about self and the world (see Cole & Putnam, 1992; Janoff-Bulman, 1992). We suggest that CSA places affected children at risk for developing views of the world that are organized around assumptions of persistent danger, interpersonal deviance, and self-deprecating thoughts (see Garbarino et al., 1992; Janoff-Bulman, 1992). In so doing, CSA may potentially alter a child's 'way-of-being' in the sociophysical world by tainting his/her perceptions and assumptions about the world around his/her. Person × environment transactional processes associated with the experience and consequences of CSA It is hypothesized that the adverse psychological and health impacts of these traumatic transactions between the abused child and his/her immediate surroundings will be more severe and persistent to the extent that CSA experiences (1) occur over a prolonged period; (2) are perpetrated by a family member on whom the child is highly dependent (Russell, 1986; Kendall-Tackett et al., 1993); and (3) occur across a number of developmental stages in the child's life. An important task for future research is to develop methods to assess the rela- tionship between CSA and an individual's use of personal space, privacy-regulating abilities, and sense of alienation within varying sociophysical spaces. Moreover, research that addresses the role of trau- matic events in altering world views, with specific attention paid to how these views influence the individual's transactions within the sociophysical environment, would be useful in furthering our understanding of the long-term consequences of CSA. For example, one methodology that has proven useful in identifying adults' traumatic memories associated with childhood places is the use of environmental autobiographies (Rubinstein, 1993), although these retrospective accounts of earlier experiences are subject to potential recall biases and questions about the authenticity of traumatic memories (Loftus, 1993). Multi-method strategies • for documenting CSA experiences during childhood, The preceding hypotheses pertain to the pre-abuse phase of CSA. The development of a more inte- grated, environmental psychological theory of CSA also requires an understanding of the person- environment transactions that are activated during the abuse and post-abuse phases of CSA. More- over, an environmental psychological theory of CSA must not only be able to identify these core trans- actional processes, but also specify how they can be moderated (e.g. through clinical and environmental design interventions) so as to hasten recovery and minimize the adverse emotional and physical health impacts of childhood sexual abuse. Some of the major categories of person-environ- ment transaction associated with the abuse and post-abuse phases of CSA are listed in Table 2. Clinical implications In the preceding discussion, we described a devia- tion-amplifying process of increasingly negative consequences, triggered by the abuse experience and eventuating in revictimization and self-destruc- tive behaviors• Deviation amplification theory, how- ever, posits that these processes can be redirected toward more positive outcomes given the right set of circumstances (Maruyama, 1963). A very similar point has been made by Rutter (1989) who states that: Having established a modicum of safety and self- awareness of one's body, individuals can then be encouraged to identify cognitive and affective cues in the sociophysical environment that seem to trigger dissociation from the body. Once these triggers are recognized, the individual can consider alternative choices for responding to threatening cues besides dissociation• Encouraging the individual to con- sciously attend to such cues has a dual function: (1) to become aware of the threatening stimuli that initiate dissociation; and (2) to heighten the person's tendency to consciously scan both the inner and outer environment. These functions may enable the individual to identify danger by attending to and linking internal signals of threat with external environmental cues. In sum, as individuals begin to establish a sense of belonging in the physical world, their heightened awareness of bodily and environ- mental cues in the context of a safe interpersonal relationship can encourage the development of self- protective behavior• Self-protective behavior then becomes the abused individual's source of power to prevent future revictimization and promote adapta- tion. We now consider certain environmental design approaches that may enhance adaptation among sexually abused individuals. • . . the impact of some factor in childhood may lie less in the immediate behavioural change it brings about than in the fact that it sets in motion a chain reaction in which one 'bad' thing leads to another or, conversely, that a good experience makes it more likely that another one will be encountered (page 27)• To promote the health and well-being of abused individuals, it is important to identify and strengthen the positive deviation-countering experi- ences that can optimize the person's relationships with the sociophysical environment• Restoring personal control and reciprocity in these relation- ships may be a crucial ingredient for countering the negative effects initiated by CSA. Person × environment transactional processes associated with the experience and consequences of CSA These include (1) traumatic violations of the body TABLE 2 Person-environment transaction associated with the abuse and post-abuse phases of child sexual abuse TABLE 2 Person-environment transaction associated with the abuse and post-abuse phases of child sexual abuse Violation of body boundary and personal space Mental dissociation from one's physical body Disruption of territorial control and privacy regulation Development of traumatic environmental memories Experiences of place aversion rather than attachment Stigmatized sense of place identity Generalized alienation from one's sociophysical world Violation of body boundary and personal space Mental dissociation from one's physical body Disruption of territorial control and privacy regulation Development of traumatic environmental memories Experiences of place aversion rather than attachment Stigmatized sense of place identity Generalized alienation from one's sociophysical world E.A. Holman and D. Stokols 248 and for cross-validating adults' recollections of these traumatic experiences, remain to be developed in future research. tive spiral of traumatic bonding (Bowlby, 1988; Garmezy, 1991). As these positive interpersonal experiences occur, the perceived threat of inter- personal violation diminishes, thereby minimizing the need to mentally flee from one's own body. In the context of a trusting relationship with another person, an abused individual can begin to feel a sense of belonging and learn how to regulate privacy needs through individuation and separation from an attachment figure (see Korpela, 1989; Proshan- sky et al., 1983). Our analysis suggests that the disruptive emo- tional and health impacts of CSA can be reduced through clinical and environmental design inter- ventions that alter or, in effect, 'repair' the trau- matic transactional processes instigated by sexual abuse. In the remaining portions of the paper, we propose a number of clinical and environmental design strategies that may counteract the long-term negative consequences of CSA. An important direc- tion for future research is to empirically test the effectiveness of these strategies in facilitating recovery from sexually abusive experiences during childhood• y ) The re-establishment of personal control over privacy needs is, perhaps, the most important clini- cal application of our analysis• Privacy regulation can be strengthened through the development of skills to manage primary territory and personal space• For the sexual abuse survivor, this process will often need to begin with developing a sense of belonging and identity in the physical body. Toward this end, clinicians can encourage self-awareness of bodily sensations, while simultaneously respecting and advocating the abused individual's right to control this awareness• Clinical implications Specifically, experiences that (1) help the individual unlearn the helpless, passive/reactive mode of relating with the environment; (2) weaken the traumatic interpersonal bond with the perpetrator (Dutton & Painter, 1981); and (3) teach self-protective privacy-regulating skills are most likely to transform the person-environ- ment relationship in a positive direction• Conclusion CSA has been conceptualized as a transactional pro- cess in which a child's natural tendency to mentally and/or physically flee the violation of abusive events subsequently inhibits his or her development of positive self identity and attachment to the physical environment. Over time, this once useful coping strategy predisposes the individual to revictimiza- tion and perhaps self-destructive behavior. By apply- ing principles of privacy regulation and place attachment to the study of the long-term effects of abuse, researchers may be better able to under- stand revictimization. Moveover, the application of these principles in clinical therapy with abuse survivors may provide techniques that can prevent further victimization of survivors and, hopefully, increase the rate of recovery. Finally, by considering the role of the physical environment in sexual abuse recovery, guidelines for environmental designs that promote the optimal outcomes for survivors can be developed. p g Environmental design strategies also can be used to enhance the perceived safety and comfort of a setting For example, shelters, clinical settings, and homes can be built and decorated so that they: (1) enable individuals to have high visual access without excessive exposure to others (Archea, 1977); (2) permit flexibility in establishing and removing privacy-regulating boundaries (e.g. in bathrooms, bedrooms, etc.); (3) incorporate aspects of the natural environment into building designs; and (4) encour- age individuals to personalize their space so that it reflects positive aspects of their self and social identities (Proshansky et al., 1983; Deaux, 1993). These positive symbols of one's self can directly counter the depersonalizing effects of dissociative processes (see Putnam, 1985) and strengthen personal identity. In this paper, we have attempted to explain patterns of long-term adjustment to sexual abuse using the conceptual and methodological perspec- tives of environmental psychology. This integration offers a broader theoretical view of adaptation to abuse that can enhance efforts to prevent revictim- ization and self-destructive behaviors. Most impor- tantly, by examining the sociocultural and physical- environmental contexts of CSA, we can reframe the abuse experience so that future scientific research can contribute toward improved outcomes for survivors of sexual abuse. Also, to the extent that therapeutic settings can be spatially configured and decorated to match clients' needs, the capacity of those settings to support the development of interpersonal security will be greater. The psychobiological model of adap- tation to CSA (Trickett & Putnam, 1993) suggests yet another strategy for personalizing the thera- peutic environment. Environmental design implications Environmental design implications For example, socially supportive relationships, including professional therapy, may create opportu- nities for establishing healthy emotional attachments to people and places, thereby interrupting a nega- Environmental psychologists have identified the potentially restorative effects of the physical Environmental Psychology of Child Sexual Abuse 249 environment on mental and emotional well-being (Kaplan, 1983; Ulrich, 1983; Hartig et al., 1991; Parsons, 1991). They propose that the natural environment promotes cognitive and affective restoration by capturing one's involuntary attention and blocking the experience of unpleasant thoughts. This process is thought to restore mental concen- tration (Kaplan, 1983) and combat the deleterious effects of stress (Ulrich, 1983). These models focus almost exclusively on the restorative effects of the natural environment, ignoring the restorative potential of human-made or built environments (Parsons, 1991). Preliminary evidence suggests, however, that certain aspects of one's primary terri- tory may produce a restorative effect on the indi- vidual when it is associated with positive relaxing experiences (Korpela, 1989). For an abused person, it would be important to identify aspects of primary territory that have positive meanings to counter the negative, abuse-related emotions associated with home environments. To the extent that the physical environment can be structured to reinforce positive associations and minimize distressing ones, it becomes a coping resource for the individual. for an individual who experiences low levels of physiological arousal, environmental stimulation that enables the individual to learn competence in negotiating the physical environment may be use- ful. Thus, to the extent that the environment is designed to afford low levels of vulnerability, match the stimulatory needs of the individual, and promote high levels of individual control over privacy regula- tion, it can facilitate effective coping in sexually abused individuals. Conclusion It may be worthwhile to design therapeutic environments so that they accommodate the client's stimulatory needs. For an individual whose emotional distress involves physiologic hyper- arousal, an environment that presents low levels of stimulation may be most appropriate. Conversely, References Chu, J. A. & Dill, D. L. (1990). 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https://openalex.org/W3198539875	https://hts.org.za/index.php/hts/article/download/6548/19562	English	null	Change agency and urban vulnerability: Theological-ecclesial paralysis or deep solidarity	HTS teologiese studies	2,021	cc-by	9,482	Copyright: Copyright: © 2021. The Authors. Licensee: AOSIS. This work is licensed under the Creative Commons Attribution License. Some hopeful narratives are retrieved – often not ecclesial nor theological – as clues for theological-ecclesial-diaconal transformation, if it is to participate as change agents in vulnerable urban contexts. Finally, a deliberate theological praxis and deep diaconate are proposed as essential ingredients for (faith-based) change agency, which is able to transform urban vulnerability. This is done with reference to Harvey Cox’s (1965:116) assertion that ‘the church’s task in the secular city is to be the diakonos of the city, the servant who bends himself to struggle for its wholeness and health’. Read online: Scan this QR code with your smart phone or mobile device to read online. Read online: Scan this QR code with your smart phone or mobile device to read online. HTS Teologiese Studies/Theological Studies ISSN: (Online) 2072-8050, (Print) 0259-9422 Page 1 of 10 Original Research Original Research Page 1 of 10 Page 1 of 10 Change agency and urban vulnerability: Theological- ecclesial paralysis or deep solidarity Globally, cities respond differently to their most vulnerable urban populations, notably so during the coronavirus disease 2019 (COVID-19) pandemic. In the City of Tshwane, there seems to be a general paralysis of the church and theological education in relation to urban vulnerability. If the church and theological education are to participate as change agents to help transform urban vulnerability, a deliberate theological praxis and deep urban diaconate – in solidarity with the city’s most vulnerable realities – are proposed. This is done with reference to Harvey Cox’s assertion that the task of the church in the city is that of Diakonos and the provocations of Philippino liberation theologian, Daniel Pilario. 2Department of Practical Theology and Mission Studies, Faculty of Theology and Religion, University of Pretoria, Pretoria, South Africa Contribution: This article proposes that urban vulnerability and various responses to it need to be reflected upon as a priority, much more deliberately, considering how the entire urban household (oikos) is at risk. Keywords: urban vulnerability; urban diaconate; theological praxis; theological education; ecclesial and theological liberation; change agency. Dates: Globally, cities respond differently to their most vulnerable populations, also in the face of the coronavirus disease 2019 (COVID-19). Here, urban vulnerability – referring to vulnerable urban places, people and eco-systems – is framed as a risk to the sustainability of the entire urban household. How to cite this article: De Beer, S., 2021, ‘Change agency and urban vulnerability: Theological- ecclesial paralysis or deep solidarity’, HTS Teologiese Studies/Theological Studies 77(3), a6548. https://doi. org/10.4102/hts.v77i3.6548 In the context of urban vulnerability, this article then considers change agency as the deliberate processes, practices and interventions, aimed at addressing, overcoming and transforming vulnerability into resilience. The contention of this article is that there is a general paralysis of theological education and the church in relation to urban vulnerability, also in the City of Tshwane.1 It argues that such a paralysis flows from the ongoing absence of (1) a deliberate theological praxis focusing on, or immersed in, contexts of urban vulnerability, as well as (2) a deep diaconate connected with urban vulnerability in systematic, strategic and transformative ways. Such a paralysis serves to deepen urban vulnerability, as it expresses a lack of vision, will, resources and agency to help address and overcome diverse forms of urban vulnerability innovatively and decisively. The primacy of the city’s most vulnerable populations and places: A theological and moral imperative One of liberation theology’s core premises is a preferential option for the poor (Gutierrez 1988:xxv). It considers the well-being of society through the lenses of those who are mostly Read online: Scan this QR code with your smart phone or mobile device to read online. Read online: Scan this QR code with your smart phone or mobile device to read online. 1.The City of Tshwane is the name of the metropolitan municipality of which Pretoria forms a part. This is also the administrative capital city of South Africa. Note: Special Collection: Being a Change Agent in a (Post-) Covid South Africa, sub-edited by Erna Oliver (University of South Africa). Open Access http://www.hts.org.za Original Research Page 2 of 10 Page 2 of 10 Page 2 of 10 excluded from society. It considers the faithfulness of the church through the lenses of how faithful the church is to Christ in the poor and the stranger. Urban vulnerability is often the result of systemic socio- economic-spatial exclusions, over shorter or longer periods of time, mediated by policies, planning and priorities that fail to foster radical inclusivity or opt, in a central manner, for the most vulnerable urban places and populations, beyond political or other forms of rhetoric only (cf. also Bhanjee 2019). An urban theology of liberation will: (1) locate itself with the city’s most vulnerable, marginalised and oppressed populations and places; (2) work actively for the liberation of the city’s most vulnerable through action, reflection, dialogue, research and activism; and (3) evaluate the well-being or success of the city in terms of how well it does to include vulnerable populations and places meaningfully and sustainably into the life and fibre of the city, in ways that mediate integral liberation and multiple freedoms. It is an expression of people’s lack of access to essential sources of livelihood, well-being or freedom, and the lack of resistance of those forces that render people or neighbourhoods vulnerable. Bohle (2001:3/6) distinguished between internal and external aspects of vulnerability. There are forces or processes external to a person or neighbourhood that determine their conditions and futures, often in spite of them; but there are also internal aspects such as abilities or resourcefulness of persons or neighbourhoods to anticipate, cope with or recover from external pressures; or, the internal inabilities or deficits of persons or neighbourhoods, rendering their vulnerability even more absolute. The primacy of the city’s most vulnerable populations and places: A theological and moral imperative The point of departure of this article therefore is the primacy of the city’s most vulnerable populations and places, both as a theological imperative and as a moral and human imperative, ensuring that the weakest among us are bestowed with greater honour in order for the abundance of Christ to be shared equitably. To ensure the ongoing and full liberation – and therefore integration and participation – of the city’s most vulnerable populations in the life of the city, deep change is required. If the church and theological education are largely paralysed, in the face of deep urban vulnerabilities, where will the agency come from to effect such a deep change? Can change agency be fostered in local faith communities and congregations, in more deliberate ways, and what could be the sources of such formational works? Can theological education, generally, and practical theology, more specifically, be a source of liberating change, flowing from deep solidarity with very vulnerable populations? Can such liberating theology be possible without the primacy of deep, embodied solidarity? Rather concretely, in the Gauteng City-Region or the City of Tshwane, urban vulnerability or vulnerable urban populations could refer to whole regions or neighbourhoods, specific buildings or properties, or specific groups of people or populations. Informal settlement neighbourhoods are often experiencing different forms of vulnerability, partly because of the spontaneous formation of settlements, without pre-planning and formal inclusion in urban structures. Parts of Alexandra Township, for example, are perpetually at risk of flooding and loss of life, when the Jukskei River comes down because of heavy rainfalls. Many hijacked buildings in Johannesburg or Tshwane have all the characteristics of vulnerability – both physically and in terms of the well-being of the people living there – because of poor or no maintenance, fire hazards or criminal activity. Framing urban vulnerability Urban vulnerability is defined differently by different people, depending on their vantage point. Globally, cities are home to very vulnerable populations, sometimes even constituting the majority of a city’s population. Cities where 80% – 90% of the population live in urban slums, characterised by layers of vulnerability, are an example. Cities and associated urban vulnerabilities affect the entire planet (cf. Srinivas 2021). Specific people groups or populations characterised often by absolute forms of vulnerability – exclusion and marginality – are homeless communities, people living with chronic mental illness, people living with disability, certain groups among the LGBTIQ+ communities, impoverished and isolated older persons, young unemployed people, girl children, commercial sex workers, in particular, street workers and those forced into prostitution. One definition of urban vulnerability, which features most prominently, is to describe it as the exposure of urban residents (particularly poor populations) to climate hazards and their ability to adapt or mitigate risks in order to reduce negative impacts (cf. Lankao & Quinn 2011; Rubin 2011; Srinivas 2021). A very tangible example for demonstrating how human, economic, social and environmental vulnerabilities inter- relate includes landfill sites in the entire Gauteng City- Region, which have also become home to poor people who are making a living off the sites. The relationship between urban vulnerability and environmental toxicity in these sites – both directly for those living on landfill sites being exposed to their hazardous nature permanently and for surrounding neighbourhoods because of poor air quality – has devastating consequences. I consider urban vulnerability in a more holistic manner as described by Birkmann et al. (2013:199–201), considering human-social, physical, cultural, economic, health, environmental and institutional factors. To address, reduce or even overcome urban vulnerability, all these factors – or the totality of the urban household (oikos) – need to be considered simultaneously. Open Access http://www.hts.org.za Original Research Original Research Urban vulnerability therefore should be understood as multidimensional (i.e. different types of vulnerability as seen above), dynamic (i.e. changing over time), site-specific (i.e. each location differs) and at different scales (from individual or household, to neighbourhood city or even country) (cf. Van Westen 2014). Vulnerability and resilience In a number of definitions, vulnerability and resilience are indicated as two extremes on a spectrum (cf. Birkmann 2006; Birkmann et al. 2013; Bohle 2001; Pelling 2003). Absolute vulnerability will denote multiple forms of exclusion, a lack of access to resources that mediate well-being or freedom and a lack of capacity to resist multiple forces dealing with vulnerability. Resilience, on the other hand, would be ‘the capacity of individuals or communities to withstand the impact’ (Pelling 2003) of external or internal forces of exclusion, crisis or risk and adaptive mechanisms to address and creatively overcome such forces. Vulnerable urban contexts call for radical change. The harshness of urban vulnerability – and oft invisibility and exclusion of vulnerable people and places – requires not aloof forms of theological reflection and action, but the kind of theological praxis that stands in close solidarity with vulnerable people and places, fusing reflection and action, analysis and discernment, allowing for the one to shape the other, with the city’s most vulnerable groups as its primary interlocutors. Birkmann et al. (2013) have developed the so-called MOVE framework to understand and address vulnerability. This framework regards vulnerability as exposure to various risks or hazards, susceptibility of persons and communities to vulnerability amidst such risks, fragility of persons or communities, and the lack of resilience to cope or adapt when facing risks (cf. Birkmann et al. 2013:195). Building resilience will therefore have to address conditions that will help reduce exposure to hazards or risks; and in doing so reduce susceptibility and fragility of populations or neighbourhoods to vulnerability, whilst building their capacity to cope, adapt or deal creatively with risks or threatening changes facing them (cf. Birkmann et al. 2013:194). This is, or should be, a very holistic endeavour, addressing social-human, economic, cultural, physical, environmental and institutional elements that contribute to vulnerability. Without such solidarity – allowing urban vulnerability to be visible, central and disruptive – our theological work will be untransformed. Without such solidarity, deep vulnerabilities will continue to be regarded as inevitable, and our contributions failing to make deep change. Instead, making urban vulnerability visible in very concrete ways, whilst simultaneously challenging its perceived inevitability, will help prioritise the urban poor, theologically, and help ensure that urban vulnerability also receives a central place on the city’s agenda. Vulnerability and resilience The change that vulnerable urban contexts demand, even though urgent, often occurs best when facilitated and effected incrementally, over longer periods, with the ownership and active participation of those most affected (cf. Swilling, Tavener-Smith & Keller 2015:261–277). Incrementality should not be an excuse to minimise urgency however. Imaginaries of radical urban change – in the direction of greater resilience, freedom and well-being – should be constructed, made to be non-negotiable and then facilitated collaboratively through multiple preventative, protective and reconstructive actions. In the Centre for Faith and Community at the University of Pretoria, we designed a 3-day course entitled ‘Approaches to community transformation’. One of the central thrusts in this course suggests that transforming communities is about accompanying communities from vulnerability to resilience, in holistic and integrated ways. Framing urban vulnerability theological vision on urban vulnerability, but submits that important work needs to be performed in this regard, both to support systemic transformation that will reduce the conditions that enable vulnerability, and to develop measurable indicators for concrete local transformations of urban vulnerability in the direction of an increasing sense of resilience, flourishing and well-being. The works of people such as Van Schalkwyk (2014), Castillo (2019) and Holden, Nadeau and Porio (2017) need to be considered in this regard, fusing ecological, feminist and liberationist perspectives and exploring implications for local faith-based responses. The challenge is to contextualise these insights in relation to very localised expressions of urban vulnerability. Urban vulnerability – an eco-theological challenge And by the end of March 202 spaces for homeless persons were offered in California (Hutt 2020). Not one hotel in Ts willing to avail itself, and I am unaware o anywhere in South Africa. In Paris, the Red Cross expressed its commitmen government’s interventions: ‘As in every cris fragile, the most isolated and destitute will exposed and the most forgotten’ (Blanquine 202 pledged to stand in solidarity with exactly thes Lima, Peru, a traditional bull ring was conv homeless shelter during COVID-19 (Ruiz et al. 2 These global examples, as in the Tshwane exam signs of hope and lessons to glean from. The possibility for impactful, coordinated responses urban vulnerability through diverse strategi government, civil society and universit collaborate. Street homelessness in South Africa: Lo responses to urban vulnerability The way in which street homeless communities Page 4 of 10 Original Research devises. Fifteen-year old Karim Mohamed is isolated from his friends and support structures: devises. Fifteen-year old Karim Mohamed is isolated from his friends and support structures: care, as – through a lens of urban vulnerability – the paralysis of church and theological education is severely exposed. care, as – through a lens of urban vulnerability – the paralysis of church and theological education is severely exposed. Before the outbreak, Karim used to go to the al-Fateh Mosque nearby the square for breakfast, lunch and dinner. (n.p.) Different cities in response to urban vulnerability during COVID-19 – and the paralysis of church and theological education in the City of Tshwane Places of worship are shut down, gatherings are not permitted and places where the most vulnerable could wash, eat and congregate are not off limits. Different cities, globally, respond to urban vulnerability in their midst, rather differently. During COVID-19, the face of vulnerability has often been accentuated, particularly as the well-to-do had ways and means to isolate themselves, leaving the excluded rather visible, for once. In places like Kampala, Human Rights Watch (2020) reported multiple incidents where vulnerable populations – informal traders, homeless persons and the LGBTIQ+ community – were seriously violated: On March 29 (2020), community residents and police raided a shelter for homeless lesbian, gay, bisexual and transgender youth in Wakiso, outside of Kampala, and beat and arrested 23 people, including shelter residents. (n.p.) These different approaches varied from harsh marginalisation to innovative protection. Roy (2020) described how the poor in India were marched from Delhi during the hard lockdown imposed by Narendra Modi: None of the above should be made off as isolated incidents. These are the daily experiences of the city’s most vulnerable populations, pre- and during COVID-19. In most instances, vulnerable urban people also lack solidarity from churches and theological institutions, allowing the image of God to be violated on the streets and marketplaces of the world’s cities every day. As an appalled world watched, India revealed herself in all her shame – her brutal, structural, social and economic inequality, her callous indifference to suffering… As shops, restaurants, factories and the construction industry shut down, as the wealthy and the middle classes enclosed themselves in gated colonies, our towns and megacities began to extrude their working-class citizens – their migrant workers – like so much unwanted accrual. Many driven out by their employers and landlords, millions of impoverished, hungry, thirsty people, young and old, men, women, children, sick people, blind people, disabled people, with nowhere else to go, with no public transport in sight, began a long march home to their villages… Some died on the way. (n.p.) There are also some examples with signs of hope. The Mayor of London availed 300 hotel rooms for homeless people to self-isolate (Hutt 2020). The Accor Group in Paris provided 600 hotel beds for homeless people, and government in France invested € 50 000 (almost R1 billion) (Cook 2020). Different cities in response to urban vulnerability during COVID-19 – and the paralysis of church and theological education in the City of Tshwane And by the end of March 2020, 2000 bed spaces for homeless persons were offered in the state of California (Hutt 2020). Not one hotel in Tshwane was willing to avail itself, and I am unaware of any such anywhere in South Africa. As they walked, some were beaten brutally and humiliated by the police, who were charged with strictly enforcing the curfew… A few days later, worried that the fleeing population would spread the virus to villages, the government sealed state borders even for walkers. People who had been walking for days were stopped and forced to return to camps in the cities they had just been forced to leave. (n.p.) In Paris, the Red Cross expressed its commitment to support government’s interventions: ‘As in every crisis, the most fragile, the most isolated and destitute will be the most exposed and the most forgotten’ (Blanquine 2021:n.p.). They pledged to stand in solidarity with exactly these groups. In Lima, Peru, a traditional bull ring was converted into a homeless shelter during COVID-19 (Ruiz et al. 2020). Similar reports came from the Philippines. Pilario (2020) wrote about the socially excluded in the cities and towns of the Philippines: Now that people have some mobility and can go to work in limited capacities… there will be always people who remain excluded. They are suffering the same hunger and pain as they did during the total lockdown… street people, the sick, the elderly, those living under bridges, those who have no homes. (n.p.) These global examples, as in the Tshwane example, serve as signs of hope and lessons to glean from. They prove the possibility for impactful, coordinated responses to overcome urban vulnerability through diverse strategies in which government, civil society and university partners collaborate. Coronel (2020) reports how government officials in certain parts of Metro Manila forced shelters, also those run by churches or priests, to close down. In response, a restaurant, two Catholic universities, and a number of churches opened their doors to shelter homeless persons. The De La Salle University opened its sport centre to accommodate 79 homeless people, working with the Divine Word Missionaries. Urban vulnerability – an eco-theological challenge If urban vulnerability reduces the ability of the urban oikos – the household of humanity, the economy and the environment – to mediate well-being and freedom; placing human dignity and the integrity of creation at risk; and results from socio- economic spatial-environmental injustices, it is indeed a theological concern, requiring not only deep theological analysis and reflection, but also theological action. I speak about it as an eco-theological challenge, not in a narrow environmental sense only, but as a theological challenge that involves the totality of the urban household (oikos). This article is not meant to provide a comprehensive eco- I pose the question as to the possible role of theological praxis and deep diaconate as ingredients of urban change agency. As a critical part of the urban citizenry, the church and theological education could potentially contribute as a servant of collaborative processes, which stand in solidarity with urban vulnerabilities, broker tables of collaboration between various sectors in a city and facilitate own resources as contributions towards urban reconstruction. It requires a complete, rigorous and penetrating assessment and deconstruction of current notions of diaconate or pastoral http://www.hts.org.za Original Research devises. Fifteen-year old Karim Mohamed is is his friends and support structures: Before the outbreak, Karim used to go to the al- nearby the square for breakfast, lunch and dinner. Places of worship are shut down, gatherings are n and places where the most vulnerable could w congregate are not off limits. In places like Kampala, Human Rights Watch (20 multiple incidents where vulnerable population traders, homeless persons and the LGBTIQ+ c were seriously violated: On March 29 (2020), community residents and p shelter for homeless lesbian, gay, bisexual and youth in Wakiso, outside of Kampala, and beat an people, including shelter residents. (n.p.) None of the above should be made off as isolat These are the daily experiences of the city’s mos populations, pre- and during COVID-19. In mo vulnerable urban people also lack solidarity fro and theological institutions, allowing the image violated on the streets and marketplaces of the w every day. There are also some examples with signs o Mayor of London availed 300 hotel rooms f people to self-isolate (Hutt 2020). The Accor Gr provided 600 hotel beds for homeless p government in France invested € 50 000 (almos (Cook 2020). Street homelessness in South Africa: Local responses to urban vulnerability The way in which street homeless communities are engaged during COVID-19 is a barometer for how municipalities, the church and institutions generally embrace, or marginalise, vulnerable populations. In South Africa, various In Cairo (Al-Youm 2020), as lockdown was enforced, many homeless people and street children were left to their own http://www.hts.org.za http://www.hts.org.za Original Research Page 5 of 10 from varied backgrounds into collective action to find solutions to COVID-related issues’. municipalities responded in various ways to street homelessness, both during and after hard lockdown. In eThekwini, 1000 people were housed in 10 temporary shelters within two days. In the City of Tshwane, 25 temporary COVID-19 shelters were created, housing 1500 people within 10 days, and doing 12 000 health screenings. The City of Johannesburg was not able to create more than five temporary shelters. And in Cape Town, 2000 people were forced into a makeshift shelter in Strandfontein, which immediately drew the ire of homeless communities, human rights activists and the community of Strandfontein, fast evolving into a potential humanitarian disaster. The city was forced to shut down Strandfontein, and only a small percentage of those who were accommodated here were provided with alternative accommodation. With reference to their own CAN, in the neighbourhood of Woodstock, they wrote (Scheepers et al. 2020): [A]s a CAN we are trying to experiment – as are a host of other organisers and activists – with new and old ways of community building so that we might create transformation in our neighbourhood that lasts beyond our response to the pandemic. (n.p.) What started in one neighbourhood was soon spreading across the City of Cape Town, from Woodstock to places such as Constantia, Muizenberg and Khayelitsha. The concept also spread to other parts of the country because its efficacy was soon becoming clear. By July 2020, there were already more than 100 CANs across the Gauteng City-Region (Heywood 2020). In spite of very constructive collaboration in the City of Tshwane between the municipality, non-governmental organisations (NGOs) and researchers from two universities, in the aftermath of hard lockdown, many of the temporary shelters closed down, with the city failing to sustain its commitment to a post-lockdown strategy drafted in collaboration with the research community and the Tshwane Homelessness Forum (cf. Ndlazi 2021). It leads to the question of why lessons learnt during COVID-19 are not optimally taken advantage of in shaping alternative urban futures. Street homelessness in South Africa: Local responses to urban vulnerability Could it indeed be that the city’s most vulnerable populations – who require deliberate and additional support for their viable inclusion in the city’s fabric – are regarded as lesser citizens of the city, not ‘deserving’ of being prioritised? Drawing from the examples and templates of CANs, the South African Council of Churches motivated its constituencies to form what it called Local Ecumenical Action Networks (LEANs). According to Craig Stewart, who was closely involved in the framing of the LEAN vision and template, more than 100 LEANs were formed across the country, including ‘brand new’ formations, ‘existing groups that took on a new shape’, very active LEANs, ‘whilst others didn’t get much further than starting’.2 In some instances, collaborations deepened significantly, for example, between different academic disciplines engaging homelessness in Tshwane through an evidence-based approach. In eThekwini, different faith formations found remarkable ways to collaborate in supporting homeless shelters. It became clear that instances where government, NGOs and the research community collaborated, evidence-based interventions were made possible and vulnerable communities were welcomed into spaces where significant alternatives were available. These hopeful signs should become the imaginaries that determine our policy and investment priorities. Across the country, a number of temporary shelters became permanent housing facilities, and two new housing projects for older homeless persons were opened during hard lockdown in the City of Tshwane, seeing concrete collaboration between a social housing company and a social development agency. Such permanent housing options for vulnerable groups during COVID-19 serve as success stories arising from collaborative conversations and partnerships, committed to long-term, sustainable and local solutions. COVID-19 not only revealed serious fault lines in how vulnerable urban populations are excluded from policy and strategic investment, and how both state and church are not really geared towards ongoing or crisis interventions, but also offered new opportunities for collaboration and innovation, and progressive NGOs, citizen groups and academics modelled possible interventions that could become the ‘new normal’, if the political and moral will existed (cf. De Beer 2020b). In the majority of cases where such innovative and transformative responses to urban vulnerability were seen, government played an enabling and not a stifling role. The role of government ‘is to help create the conditions where good outcomes are more likely to emerge’ (Brown 2019). 2.From e-mail correspondence with the author of this article, enquiring about the status of LEANs. Pilario (2016) says: Pilario (2016) says: Bereft of social, political, and economic networks, the Eucharist is the poor’s only connection that works … the Eucharistic table is a symbol of hope for the battered, the lost, the hungry, the broken, the excluded, the poor. (n.p.) Over a period of 10 days in the City of Tshwane, 25 temporary COVID-19 shelters were opened for homeless persons. Elsewhere, I wrote a piece that was entitled ‘The miracle of Tshwane’ (De Beer 2020a), because of the remarkable collaborative effort undertaken to house homeless persons in a short space of time. Taking away this communal and sacramental experience, which proclaims and embodies God being with us, as we gather in communion, is like taking away the only connection to life for those facing absolute vulnerability. Eleven of these shelters were run from the facilities of churches or faith-based organisations. Although the embrace of homeless persons by these churches and faith-based organisations (FBOs) was heartening and often mutually life- changing, the fact that only 11 shelters were opened from church bases, in a city the size of Tshwane with literally thousands of churches lining its streets, raises urgent questions. It makes one think about Taylor’s (1996) conclusion that only a small remnant actually responds faithfully to the gospel. In the face of deep urban vulnerability, there are places and people in need of deep, embodied solidarity, to resist life- denying forces of this and other pandemics. The sacramental solidarity of practising presence, in a challenging time of physical distancing and self-isolation, revealed a theological poverty that now demands urgent attention. I acknowledge that theological education lost a significant opportunity to shape a new generation of leaders, able to respond to catastrophe in appropriate, tangible and life- affirming ways. Instead of discerning innovative ways of practising our vocation together in response to COVID-19, particularly, and urban vulnerability, more generally, theological schools by and large also shut our doors, went online and prevented students, generally speaking, from amazing learning experiences at the coal face of humanity. Encouraged to research and write more during this time, it revealed a theological posture that finds it possible to write from discarnate places, instead of urging deep solidarity that would transform the ways in which we teach and write. Pilario (2016) says: Sadly, mostly they came from other disciplines – as diverse as accounting and architecture – whilst theological students and educators kept themselves at a safe distance. How does one foster solidarity – through our theological education – in the face of debilitating vulnerability without being in solidarity ourselves? I raise the question from two perspectives: on the one hand, it seems as if the current theological education system fails to understand as part of its mandate to produce ‘frontline workers’ able to confront death in vulnerable urban places; on the other hand, students of theology and ministry themselves seem to lack a desire to be seen as ‘frontline workers’, unlike health workers, for example, raising serious questions, about the nature of vocation. The isolation of the city’s vulnerable was often more accentuated during this time, as places of refuge where they would ordinarily find comfort now shut its doors. It has been relatively easy for well-resourced churches to move their worship events online, but for the majority of worshippers in poor neighbourhoods of Tshwane, this was not similarly possible. Moreover, whilst some lauded the possibilities of virtual worship, the ways in which those in need of care were Pilario (2016) says: A number of churches considered opening their facilities but sought guarantees that their properties would be safe and that homeless persons will not want to stay there permanently. Instead of considering long-term journeys, whereby they could become partners in the effort to overcome street homelessness more decisively, they took a short-term view and mostly opted out. Many churches used government’s closure of worship spaces during hard lockdown as a reason for why they could not provide care to the most vulnerable among us. They were waiting for government to give them a mandate to care for the poor. There were, of course, churches that did not open their facilities, but invested generously – financially or in the form of other resources – to assist those who fell through the cracks when lockdown disabled people from accessing their normal sources of income or livelihoods. One church opened its facility as a central resource hub from where donations could be distributed to shelters across the city. Township churches across Tshwane had to deal with the reality of hunger and food insecurity, but they themselves being ill-resourced could not necessarily respond as boldly and generously as they would want to. Solidarity between resource-poor and resource-rich churches also lacks considerably, and – whilst we know it – the pandemic highlights our disconnectedness from each other under a magnifying glass. In shelters across the city, there were students, from time to time, acting as shelter managers, social workers or health volunteers. Sadly, mostly they came from other disciplines – as diverse as accounting and architecture – whilst theological students and educators kept themselves at a safe distance. How does one foster solidarity – through our theological education – in the face of debilitating vulnerability without being in solidarity ourselves? I raise the question from two perspectives: on the one hand, it seems as if the current theological education system fails to understand as part of its mandate to produce ‘frontline workers’ able to confront death in vulnerable urban places; on the other hand, students of theology and ministry themselves seem to lack a desire to be seen as ‘frontline workers’, unlike health workers, for example, raising serious questions, about the nature of vocation. In shelters across the city, there were students, from time to time, acting as shelter managers, social workers or health volunteers. The paralysis of church and theological education in the City of Tshwane A very innovative and exciting response – to mobilise and connect good will and resources locally, in order to address local vulnerabilities – was the creation of Community Action Networks (CANs). Scheepers, Lakhani and Armstrong (2020:n.p.) described CANs in the following way: ‘Community Action Networks in South Africa bring residents Although my assertion might be met with contention in many quarters, I acknowledge that COVID-19 in the City of Tshwane, especially from the perspective of particularly 2.From e-mail correspondence with the author of this article, enquiring about the status of LEANs. http://www.hts.org.za Open Access Original Research Original Research often locked out revealed a theological disposition void of deep solidarity. often locked out revealed a theological disposition void of deep solidarity. vulnerable populations, revealed a general paralysis of church and theological education, in response. I say ‘general’ paralysis because in isolated instances, there were remarkable outpourings of support, demonstrating ‘unusual kindness’ (cf. De Beer 2020a). A transformative urban diaconate Although there is much debate in diaconal discourse about how contemporary understandings of diaconate are not necessarily consistent to the original use of the Greek term diakonia, here I will not dwell on that debate (cf. Latvus 2017). Instead, I consider diakonia as ‘a social and contextual construct’ (Latvus 2017:98), in the way described by Latvus, and consistent to, what he calls, ‘the old and truly original task of the church’ (Latvus 2017:98) which is ‘to love the other, open closed doors, make communities inclusive and work against oppressive structures’ (Latvus 2017:4). It is a caritative and justice-seeking diaconate that has evolved over time as a ‘reaction against abused and mistreated groups’ (Latvus 2017:98), seeking to be faithful to the Christ who, in Mark, identifies himself ultimately as diakonos. When I speak about theological praxis, I speak about the interplay between reflection and action, socio-ecclesial analysis and deconstruction, and creative and provocative imagination of something radically new, grounded in a spirituality of deep change. I consider theological praxis as ‘a praxis of solidarity’ that is ‘inspired by the gospel’ and working ‘in the interests of liberation’ (Gutierrez 1988:xxx). It is not neutral nor a-political, as it is in solidarity with places and people discarded, marginalised and rendered invisible, by urban political processes. It is a deeply discerning praxis, together with those facing vulnerable conditions in existential, detrimental and sometimes deadly ways. As an evolving concept and ‘a social and contextual construct’, I therefore propose that we fundamentally revisit our notion or praxis of an urban diaconate, if we are to engage constructively transformatively in vulnerable urban contexts in contemporary African societies. Tetty and Nel (2020) have reflected on such a transformational diaconal praxis as belonging to the core of truly missional congregations. Nurturing, very intentionally, such a theological praxis in contexts of urban vulnerability – putting on hold our preconceived ideas of what good news should look like, and through which wineskins it should be mediated – might give us a chance to discern and birth socio-diaconal pastoral responses, which are much more appropriately and deeply connected to urban vulnerability. The kind of change required in contexts of urban vulnerability includes uprooting unjust and exclusionary urban systems and arrangements, whilst building and modelling new – more just, inclusive and liberating – systems and arrangements, until it takes root. Original Research Peddling platitudes will not transform vulnerable communities, but indeed serves only as religious opium to pacify the people. Original Research Original Research Original Research lacking tools for deep theological reflection, replacing strategic action with ad hoc relief, and generally, lacking long- term, humble and costly solidarity. Instead of those experiencing vulnerability becoming the central agents of change, we often centre our own relief work rather. If our urban diaconate is only about making the pain a little bearable, and postponing the inevitability of death with a few more months, it fails to imagine with the prophets of old rebuilt walls, repaired streets, overcoming of vulnerability and liberation of the oppressed. I lament what I call the absence of deliberate theological praxis and deep urban diaconate in relation to urban vulnerability. Obviously, I generalise, as there are profound examples to the contrary. But, generally speaking, I argue that such an absence contributes to the reinforcement of life- denying urban vulnerabilities. On the other hand, I hold that a deliberate theological praxis deeply connected to specific urban populations and places facing exclusionary and life-denying vulnerabilities, and urban diaconal responses flowing from that might provide the substance for (faith-based) change agency required in such contexts. I acknowledge that shallow or unreflective diaconal form, void of deep theological praxis, is reflective of our theological education. Should solidarity of the kind proposed here be embodied in the curriculum, postures and pedagogies of theological education and theological educators, urban vulnerability will be engaged through radically different diaconal and pastoral forms. I acknowledge, namely that – in the face of urban vulnerability – a theological praxis needs to be nurtured, which is deliberately immersed in conditions of such vulnerability, and shaped in its orientation, commitments, reflections and solidarities, by those who experience the harshness of urban vulnerability first hand. Fostering change agency in contexts of urban vulnerability, at least in faith communities, needs to be deliberate about a theological praxis and urban diaconate that is contextually appropriate, in deep solidarity with urban vulnerabilities, and grounding itself in a spirituality of urban liberation or transformation, in which vulnerable people or places become our central guides. Hundreds of churches are present in contexts where urban vulnerability is rife. Their presence, sadly, does not necessarily guarantee the development of an intentional theological praxis, connected to and transforming urban vulnerability. Instead, their presence is often simply recycling ecclesial rituals and practices a-contextually, hardly expressing the depth and cry of vulnerability they are surrounded with. Urban vulnerability, theological praxis and urban diaconate The complexities and life-denying realities of urban vulnerability, in its multi-layered expressions, call for deliberate, sophisticated, well-reflected and strategic responses. http://www.hts.org.za Open Access Page 7 of 10 The ‘bending servant’ struggling for urban wholeness: Transforming urban vulnerability It is a radical spirituality of struggle and commitment for transformation of sinful social structures and for the liberation of their victims. (p. 6) In exploring a transformational urban diaconate, immersed in the ‘rough grounds’, I concur with Dulles (2002:84), asserting the importance of the church recognising herself as part of ‘the total human family’, with a mandate, like Christ himself, not to be served but to serve. Dulles (2002:87) speaks about a ‘secular-dialogic’ ecclesiology in which the world is the locus of our theological reflection or action, and our theological praxis is in ongoing dialogue with many others. The moment we understand the church as part of ‘the total human family’ changes the shape of how we do theology. The deafening silences (of word and deed) of the majority of local churches and theological education in the City of Tshwane, and elsewhere in South Africa, in response to life-denying urban vulnerabilities, belie something of a lost vocation. When people with disabilities are denied their grants because the relevant department ran out of money; people queueing for their social relief grants are pepper-sprayed by law enforcement agencies; and very vulnerable people are evicted from their homes in the middle of hard lockdown, in breach of COVID-19 regulations, in the middle of winter; one hears hardly a whisper from the ecclesial and theological corridors of power. Perhaps, our lacks of solidarity have removed us to far from where the cries of urban vulnerability grow louder by the day. The church as a servant ‘must share in the secular problems or ordinary human life, not dominating, but helping and serving’ (Bonhoeffer 1971:382; cf. also Kgatle 2019:71–83). Winter (1963:55) speaks about a servant church – in The New Creation as Metropolis – that is ‘no longer an institutional structure of salvation alongside the worldly structures’, but rather a ‘community within the worldly structures’, engaging with humanity ‘in the struggle against the powers of evil’. It is a church not aloof from humanity and the struggles of the city, but in deep solidarity, as part of ‘the total human family’, working to discern, erect and celebrate signs of the new creation in the contemporary and unfolding metropolis. Transformational diaconia extends beyond the church seeking to end suffering and injustice, however. It rather seeks to build up communities in order for people to practise own agency in dealing with life-denying forces. A transformative urban diaconate These are not ad hoc charitable processes, but evidence-based and measurable I lament the general absence of a deliberate theological praxis in places of urban vulnerability, because such a praxis can start to articulate and design new socio-diaconal and pastoral responses, not yet seen. Whilst the church engages socially or diaconally, it often does so without sound social analysis, Open Access Open Access http://www.hts.org.za Original Research Page 8 of 10 The ‘bending servant’ struggling for urban wholeness: Transforming urban vulnerability interventions, over long periods of time, with multiple partners, to effect deep change. Here is the question: how many forms of urban diaconate in the South African context are we aware of that are actually engaging this dual challenge, of uprooting systems and building new ones, in ways that are concrete, strategic and measurable? Unless our diaconal intentions are such, they will hardly reflect the inaugural mandate of Jesus in Luke 4 – to be good news to the poor and to liberate the oppressed – as they will continue to treat symptoms through short-term relief, thereby having accepted the inevitability of the status quo. A transformative urban diaconate will go beyond a charitable approach to urban vulnerability, seeking rather to transform urban vulnerability into resilient, viable and sustainable urban forms of living – indeed, indicating measures of well- being and wholeness not known before the diaconal intervention(s). Danny Pilario (in an interview with Justaert 2012) made a case for returning to the ‘rough grounds’ of a ‘praxis approach’. I take it as a call to do theology in the ‘rough grounds’ of messy urban contestations and life-denying vulnerabilities, there to discern and animate irruptions of good news, concretely: The World Council of Churches (2013) describes diakonia as: Service that makes the celebration of life possible for all. It is faith effecting change, transforming people and situations so that God’s reign may be real in the lives of all people, in every here and now. (p. 108) The rough grounds… are the locus from which all theology should start. It is also the place where all theology ends. For God chose to locate Him/Herself on the rough grounds, among the margins of society. (n.p.) Diakonia, in their definition, is transforming people, places and situations in ways that overcome death-dealing conditions. It is diaconal ministry that includes ‘comforting the victim’ and ‘confronting “the powers and principalities” (Ephesians 6:12)’ (Ham 2013:7). I submit that our diaconal response to urban vulnerability should depart from a posture of immersed solidarity, should help animate small acts of kindness and support as well as larger interventions demonstrating measurable transformation. Death requires not whimsical acts of piety only but bold confrontations, both arresting deadly forces and calling forth life. Ham (2013) speaks about such a transformative diaconate in this way: It must heal the victim as well as the one who victimizes. The ‘bending servant’ struggling for urban wholeness: Transforming urban vulnerability Or, it comes alongside those who already stand tall in the face of death, refusing to succumb, learning from them and being transformed into a different kind of servanthood in which we are not central but supportive companions. Building on the work of Winter and others, in his 1965 classic, The Secular City, Harvey Cox goes even further, articulating it as such: ‘[T]he church’s task in the secular city is to be the diakonos of the city, the servant who bends himself http://www.hts.org.za http://www.hts.org.za Open Access Page 9 of 10 Original Research Original Research Page 9 of 10 Connected to places of urban vulnerability, I propose to create ‘multiple centres in what was deemed marginal; make visible what dominant narratives sought to hide; and connect these “new centres” to build synergy and consensus from below’ (De Beer 2018:37). It is the political process of making vulnerable visible, centring vulnerable places and people as priority and organising and connecting such centres of action, into movements of urban change, from below. This, however, requires a different ecclesial form, and a different theological pedagogy. It asks for a shifting ‘geography of reason’, which will allow the city’s most vulnerable systems, people and places to transform the way we do and think theology. to struggle for its wholeness and health’ (Cox 1965:116) (emphasis mine). This line of Cox’s could be the subject of prolonged meditation, with the potential to animate innovative and radical forms of servanthood, not ordinarily evident in our mediocre ecclesial landscapes. A ‘servant who bends himself to struggle’ for the ‘wholeness and health’ of the city is a rather poignant and visual image – it speaks of a Christ-like solidarity, bending under the many crosses of urban vulnerability placed upon her shoulders, holding on with many others, for the sake of wholeness, health and well-being, particularly for those parts of the city excluded from such. It makes me think about my dear friend, Jember Teferra,3 an Orthodox woman of faith, who recently died, but laid down her life many years ago as she tirelessly served in the slums of Addis Ababa. Conclusion This article sought to frame urban vulnerability as an eco- theological challenge. It argued that immersed theological praxis and deep urban diaconate are two prerequisites for faith-based change agency, able to transform urban vulnerability. It lamented, however, a theological-ecclesial paralysis in relation to urban vulnerability, to such an extent that even diaconal acts are unable to facilitate change, as they often merely serve to sustain (and protect even) the status quo. Instead, it calls for deep theological and ecclesial solidarity with vulnerable urban communities, allowing their cries to shape our theological thinking and action. It suggests the church as ‘bending servant’, struggling for urban wholeness through healing urban fractures. It is my contention that the majority of ecclesial forms today, and even the ways in which we educate (practising) theologians of the future, fail to heed the calls of Bonhoeffer, Dulles, Winter or Cox, or the examples of Jember Teferra and others like her. Their calls to become servants in deep solidarity with human frailty and vulnerability are seldom heeded. For such an urban diaconate to unfold – able to contribute in ways that will transform urban vulnerability – our very notion and forms of diaconate need to be transformed, as well as the ways in which we educate future leaders for Africa’s urban contexts. In the ‘rough grounds’ of urban vulnerability, our diaconal forms will have to look, feel and smell like the contexts we seek to serve in. Transforming urban vulnerability requires more radical forms of solidarity, subversive action to outwit bad power, dismantling of unjust structures, erection of bold alternatives to the status quo, and, at times, hopeful-humble revolt. 3.Jember Teferra (1943–2021) was an Ethiopian community organiser, community developer and faith-based leader, who believed in bottom-up urban development. She founded an organisation called Integrated Holistic Approach – Urban Development Project (IHA-UDP), a project committed to the regeneration of urban slums through the active involvement and prioritisation of beneficiary communities themselves. Later, she founded the Birhan Social Development College, providing training and education to community development workers. Healing urban fractures through the lens of transformational diaconal service What I propose is a transformational urban diaconate (or socio-diaconic pastorate), which is fully committed to ‘healing urban fractures’ (Cox 1965:114–125). That is the purpose of the urban diaconate according to Cox – to heal urban fractures in order to mediate an increasing sense of urban wholeness, particularly in the most vulnerable urban places and amongst the most vulnerable urban populations. Elsewhere, I suggested (De Beer 2018:): If urban vulnerability truly deals death, we cannot but become wiser and shrewder in how we engage, arrest and overcome it – through more strategic and collaborative approaches, which are evidence-based, measurable and having the future in mind; and, through more subversive political approaches, which expose, dismantle and replace edifices of death-dealing power. We need to vigilantly develop tools to discern the fractures, responsive spiritualities, and appropriate and bold strategies, networks and resources to overcome fractures. This would imply a deep and caring presence in fractured places, abandoned by state, market or church. (p. 37) The ‘bending servant’ struggling for urban wholeness: Transforming urban vulnerability Jember and the organisations she created accompanied the poorest communities of her beloved city, brokering all their resources and networks in the interest of communities experiencing absolute forms of vulnerability, very strategically, until measurable evidence of change became visible – in areas ranging from healthcare, water and sanitation, housing, early childhood development, youth care, care of the elderly and people living with disabilities. Her faith did not set itself apart from the city, but found expression in depth of servanthood, working for the ‘wholeness and health’ of her city. Theirs is a transformative urban diaconate that literally rebuilt communities one block and system at a time. 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https://openalex.org/W2152117821	https://academicjournals.org/journal/AJB/article-full-text-pdf/0DC6D7E9535.pdf	English	null	Enzymatic hydrolysis of esters from 2-carboxy-6-methoxy-2,3-dihydrobenzofuran acid	African journal of biotechnology	2,009	cc-by	4,321	Corresponding author. E -mail: rtovar@uv.mx. Tel. +1 228 841- 89-00, Ext. 13168. Fax: +1 228 841-89-32. 1Instituto de Ciencias Básicas, Universidad Veracruzana, Av. Dr. Rafael Sánchez Altamirano s/n Col. Industrial Anima C.P. 91190 Xalapa, Ver. México. 1Instituto de Ciencias Básicas, Universidad Veracruzana, Av. Dr. Rafael Sánchez Altamirano s/n Col. Industrial Animas C.P. 91190 Xalapa, Ver. México. p , 2Departamento de Sistemas Biológicos. Universidad Autónoma Metropolitana-Xochimilco. Calz. del Hueso No. 1100, Col. Villa Quietud, Coyoacán, México, D.F. 04960 México. Accepted 21 September, 2009 In this work we describe the results of the enzymatic hydrolysis of esters from 2-carboxy-6-methoxy- 2,3-dihydrobenzofuran acid using Candida antarctica lipase supported on acrylic resin, with enantiomeric excesses ranging between 80 to 99%. Key words: 2-Carboxy-6-methoxy-2,3-dihydrobenzofuran acid, Candida antarctica lipase, enantioselective hydrolysis. INTRODUCTION phenylethylamine and amphetamines (Harada et al., 1968; Tovar-Miranda et al., 1999). This last procedure is very tedious because the methodology requires firstly the salt to be a solid; then this salt must be recrystallized several times to reach a constant optical rotation. After this, the salt must be treated with HCl to recover the enantiomerically pure acid. The present study describes the results when supported on acrylic resin Lipase B from Candida antarctica is used in the enzymatic resolution of a substantially flat structure as 2,3-dihydrobenzofuran acid that is the core of a large number of compounds showing important pharmacological activities, which by other methods are very difficult to carry out. The 2, 3-dihydrobenzofuran system is the core of a large number of important compounds which are present in nature or have been obtained through synthesis (Pieters et al., 1999). These compounds have a variety of signifi- cant pharmacological activities, some of which are cyto- toxic and can be found in both roots and leaves of plants (Tsai et al., 1998). Other 2,3-dihydrobenzofuran deriva- tives are used in the treatment of diabetic retinopathy and arthritis (Apers et al., 2002); others are use in the therapy for arteriosclerosis, liver diseases and in the treatment of vascular diseases of the brain (Kuethe et al., 2005). Some synthetic dihydrobenzofurans have even proved to be better antioxidants than vitamin E (Nicholas et al., 2006; Park et al., 2001). On other hand, some com- pounds with the 2, 3-dihydrobenzofuran system have been tested in the production of liquid crystal displays in television sets of 25 inches or larger (Bremer and Lietzau, 2005). African Journal of Biotechnology Vol. 8 (21), pp. 5958-5963, 2 November, 2009 Available online at http://www.academicjournals.org/AJB DOI: 10.5897/AJB09.1092 ISSN 1684–5315 © 2009 Academic Journals African Journal of Biotechnology Vol. 8 (21), pp. 5958-5963, 2 November, 2009 Available online at http://www.academicjournals.org/AJB DOI: 10.5897/AJB09.1092 ISSN 1684–5315 © 2009 Academic Journals Full Length Research Paper Enzymatic hydrolysis of esters from 2-carboxy-6- methoxy-2,3-dihydrobenzofuran acid Ricardo Tovar-Miranda1, Raúl Cortés-García1, Samuel Cruz-Sánchez1, Rodolfo Méndez Bellido1, Arturo Coaviche1 and Héctor Luna2 Full Length Research Paper General procedure for preparative experiments In an Erlenmeyer flask were dissolved 700 mg of 5d or 5e in 14 ml of D-tB (9:1) and 350 mg of acrylic supported enzyme were added. The reaction mixture was stirred at room temperature and the progress of the reaction was monitored by TLC. Then the reaction mixture was filtered through a cotton plug and rinsed with dichloro- methane. The organic layer was washed with 5% NaOH (2 × 20 ml), followed by water (2 × 20 ml). The solvent was dried and evaporated to yield 314 mg (45%) of unreacted ester. The aqueous layers were combined and acidified with HCl and extracted with ethyl ether; the organic layer was washed with water (2 × 20 ml) and brine (2 × 20 ml), dried with anhydrous Na2SO4 and evaporated to give 297 mg (54%) of the corresponding acid as a brown solid. To determine the influence of the amount of water on the enantioselectivity of the enzymatic hydrolysis, known amounts water were added to the anhydrous solvents and the enantioselectivity was determined by using the ethyl ester as substrate because it showed the greatest enantioselectivity in an anhydrous mixture of D:tB (9:1). The quantities of added water were 2.5, 5.0 and 7.5% (v/v) and the results were 50, 74 and 59% ee respec- tively. It was clear that some amount of water (around 5%; v/v) was necessary for the adequate activity of the enzyme. Typical procedure for the enzymatic hydrolysis To a solution of 100 mg of ester in 2 ml of dioxane-ter-butanol (9:1), were added 50 mg of C. antarctica lipase B supported on acrylic resin. The reaction mixture was stirred at 180 or 300 rpm at room temperature. The reaction progress was followed by Thin Layer Chromatography (TLC). After 60 min the reaction mixture was filtered through a cotton plug and the enzyme washed with dichloro- methane. The organic layer was washed twice with 5% NaOH (15 ml), followed by water (2 × 15 ml) and dried with anhydrous sodium sulfate and evaporated to yield the corresponding ester as a colorless liquid. The acid was recovered from the aqueous phase by acidification with HCl followed by extraction with ethyl ether. The organic layer was washed with water until the pH was near 7, dried with anhydrous sodium sulfate and evaporated to give a brown solid. The obtained acid was esterified by refluxing with methanol and p-toluenesulfonic acid for 2 h, to yield the corresponding methyl ester which was purified by column chromatography using hexane- ethyl acetate (90:10) for its chromatographic analysis. ) Interestingly, the hydrolysis took place in an apparent absence of water, because no water was added to the reaction medium and the solvents were supposed to be pure. It is worth mentioning that the solvents used in these reactions were taken directly from the bottle without any additional purification; consequently they may have contained some water which could be the responsible for the hydrolytic reaction. The enzyme used was the fraction B of C. antarctica lipase (CAL B) and it was also used without any additional treatment. These facts prompted us to carry out an experiment using anhydrous solvents; dioxane was dried from sodium-benzophenone, while ter- butanol was distilled from sodium. Under equivalent reaction conditions, but under a nitrogen atmosphere, the ethyl ester gave an enantiomeric excess(ee) of 59%. Analytical of 5 with the corresponding alcohols: methyl, ethyl, isopropyl, n-butyl, isobutyl, benzyl and heptyl, in the presence of p-toluenesulfonic acid. The reaction products were identified by Nuclear Magnetic Resonance, 1H and 13C NMR measurements were performed on a Varian Mercury Plus 300BB using deuterated chloroform (CDCl3) solutions containing TMS as internal standard and compared with literature reports (Tovar-Miranda et al., 1999). Enantiomeric excesses were determined as the corresponding methyl esters by High Performance Liquid Chromatography (HPLC) using a Chiracel-OD column (250 mm length x 4.6 mm i.d.) with a DAD detector and a solvent system (mobile phase) of hexane/isopropyl alcohol (90:10); the flow rate was kept at 0.8 ml/min during the analysis. The optical rotation was determined in a Perkin-Elmer polarimeter, Mod. 341 and compared with those previously descry- bed (Tovar-Miranda et al., 1999). TLC were performed on aluminium sheets (5 × 5 cm) silica gel 60 F254 from Merck, eluted with hexane-ethyl acetate (80:20) and detected with UV-lamp at 254 nm. p p From a previous work (Villanueva-Lendechy et al., 2000), dioxane (D), ter-butanol (tB) or mixtures thereof were chosen as solvent for carrying out the enzymatic reactions. It was also decided to use the ester 6b to determine the solvent or solvents mixture that could give the best enantioselectivity by the enzymatic hydrolysis. Dioxane was initially used and then, in an attempt to improve the enantioselectivity, the following mixtures of dioxane (D) and ter-Butanol (tB) were tested; 9:1, 7:3, 1:1, 3:7 and 1:9. These reactions were carried out at room temperature; for these experiments a 2:1 ratio subtrate/C. antarctica lipase (Novozym)(w/w) were used. Based on these results, it was possible to establish that the mixture of dioxane-ter-butanol 9:1 (D-tB 9:1) was the most adequate for evaluating the behavior of the other esters during the enzymatic reaction, in order to deter- mine the effect of the chain length on the resolution process. The absolute configuration of the obtained acid was determined by the comparison of the optical rotation of the methyl esters with reported data (Tovar-Miranda et al., 1999). Chemicals ) Enantiomerically pure 2, 3-dihydrobenzofuran acids have been obtained by resolution through the corres- ponding diastereoisomeric salts using chiral amines like The solvents, dioxane (D), ter-butyl alcohol (tB) and methanol were purchased from Sigma-Aldrich and used without any treatment, at least it is indicated. The esters were purified on column chromato- graphy using silica gel 60 (0.040 - 0.063 mm) purchased from Merck. p-Toluenesulfonic acid and anhydrous sodium sulfate were obtained from JT Baker. Lipase B from C. antarctica, Novozym was obtained from Novo Nordisk Co. and employed without any further treatment. Tovar-Miranda et al. 5959 p p y y y eE values of the isolated acid 7; calculated as methyl ester using the program “Selectivity” by K. Faber and H. Hoening, http//borgc185.kfunigraz.ac.at. f etermined by HPLC. Optical purity of the acid was determined as the methyl ester by HPLC. RESULTS AND DISCUSSION In other experiments, the influence of the ester chain on enantioselectivity was studied. Consequently the hydrolysis of these all esters (6a-g) was carried at room temperature, but the enantioselectivity of the reaction was also assayed at low temperature, specifically at 0°C. Table 1 summarizes the results of these experiments. The acid 5 was prepared following the procedure shown in Figure 1, which has been described in the literature (Fuson et al., 1944; Witiak et al., 1971), starting with the umbeliferone 2. For this study, the esters (6a-g) for the enzymatic resolution were obtained through the reaction Afr. J. Biotechnol. 5960 Figure 1. Synthesis and enzymatic hydrolysis of substrates 6a - g. Figure 1. Synthesis and enzymatic hydrolysis of substrates 6a - g. Figure 1. Synthesis and enzymatic hydrolysis of substrates 6a - g. Table 1. Enzymatic hydrolysis of the estersa at room temperature and 0°C. Table 1. Enzymatic hydrolysis of the estersa at room temperature and 0°C. Compound Solvent ee (ester)c ee (acid)d Conversion (%) Ee 6ab D-tB 9:1 65.9 27.0 27.2 3 6bb D-tB 9:1 66.6 75.5 45.1 14 6cb D-tB 9:1 10.7 43.8 14.6 3 6db D-tB 9:1 48.8 83.7 52.6 18 6eb D-tB 9:1 7.0 88.8 34.9 18 6fb D-tB 9:1 9.7 33.2 20.9 2 6gb D-tB 9:1 41.3 78.9 38.5 12 6af D-tB 9:1 63.6 28.8 57.2 3 6bf D-tB 9:1 64.9 72.1 48.3 11 6cf D-tB 9:1 11.6 40.4 17.5 3 6df D-tB 9:1 44.7 79.4 41.1 13 6ef D-tB 9:1 16.1 83.0 35.2 12 6ff D-tB 9:1 12.8 46.6 25.9 3 6gf D-tB 9:1 42.4 77.8 36.0 12 aReaction cond.:100 mg of ester and 50 mg of enzyme; magnetic stirring, 1 h. bReaction carried out at room temperature. cDetermined by HPLC. dOptical purity of the acid was determined as the methyl ester by HPLC. eE values of the isolated acid 7; calculated as methyl ester using the program “Selectivity” by K. Faber and H. Hoening, http//borgc185.kfunigraz.ac.at. fReaction carried out at 0°C. 5961 Tovar-Miranda et al. Table 2. Enzymatic hydrolysisa at room temperature and 0°C. Table 2. Enzymatic hydrolysisa at room temperature and 0°C. RESULTS AND DISCUSSION Compound Solvent ee (ester)c ee (acid)d Conversion (%) Ee 6ab D-tB 1:9 71.5 24.8 59.1 3 6bb D-tB 1:9 75.3 21.5 64.1 3 6cb D-tB 1:9 10.8 12.2 35.7 1 6db D-tB 1:9 71.5 41.3 54.5 5 6eb D-tB 1:9 1.7 53.2 49.9 3 6fb D-tB 1:9 59.2 21.3 57.1 3 6gb D-tB 1:9 69.2 41.0 53.6 5 6af D-tB 1:9 71.8 18.5 61.3 3 6bf D-tB 1:9 74.8 31.4 56.4 4 6cf D-tB 1:9 15.9 17.4 40.5 2 6df D-tB 1:9 68.8 48.2 48.0 6 6ef D-tB 1:9 2.3 52.6 48.5 3 6ff D-tB 1:9 57.4 15.1 57.2 2 6gf D-tB 1:9 62.0 33.1 52.6 4 aReaction cond.:100 mg of ester and 50 mg of enzyme; magnetic stirring, 1 h. bReaction carried out at room temperature. cDetermined by HPLC. dOptical purity of the acid was determined as the methyl ester by HPLC. eE values of the isolated acid 7; calculated as methyl ester. fReaction carried out at 0°C. and solvents); this may be due to steric effect. From the Table 1 it can be established that the best results were obtained with the n-butyl (6d) and isobutyl (6e) esters, which showed 83.7 and 88.8% ee respec- tively, for the acid, at room temperature in D-tB (9:1). Similar enantioselectivity was observed when the reaction took place at 0°C, the acid being produced in 79.4 and 83.0% of ee, respectively. Without a doubt, this optical purity can be rise to values higher than 90% ee through just one recrystallization of the obtained acid. A direct relationship between the enantioselectivity and the kind and length of the ester chain also seems to exist, since the heptyl ester also showed acceptable values (79 and 78% of ee), both at room temperature and at 0°C, respectively. So, these results show that the reaction and the enantioselectivity are better with linear chain esters. The effect of mixture of solvents was re-evaluated, but this time involving all esters. The experiments were carried out inverting the ratio of solvents to D-tB 1:9. The reactions were carried out once again at room tempe- rature and at 0°C and the results are detailed in Table 2. The enzyme in this solvent mixture (D-tB 1:9) behaved in a similar way at both room temperature and 0°C. Under these conditions, the enzyme showed greater selectivity toward short-chain esters (6a and 6b, 72 and 75% ee, respectively). RESULTS AND DISCUSSION It was also observed that when the mixture D-tB (1:9) was used, the conversions were greater than with D-tB (9:1), comparing Tables 1 and 2. From the results it can also established that for esters 6c and 6f the hydrolysis did not show good enantioselectivity under any of the reaction conditions studied (temperature From the Table 1 it can be established that the best results were obtained with the n-butyl (6d) and isobutyl (6e) esters, which showed 83.7 and 88.8% ee respec- tively, for the acid, at room temperature in D-tB (9:1). Similar enantioselectivity was observed when the reaction took place at 0°C, the acid being produced in 79.4 and 83.0% of ee, respectively. Without a doubt, this optical purity can be rise to values higher than 90% ee through just one recrystallization of the obtained acid. A direct relationship between the enantioselectivity and the kind and length of the ester chain also seems to exist, since the heptyl ester also showed acceptable values (79 and 78% of ee), both at room temperature and at 0°C, respectively. So, these results show that the reaction and the enantioselectivity are better with linear chain esters. The ethyl ester (6b) was the derivative that showed the best enantioselectivity of all the esters in this study under any of the reaction conditions. In addition, there appears to be a solvent effect on 6b, because when D-tB 1:9 was used, the ester was recovered in an acceptable ee, 75.3 and 74.8% of ee, at room temperature and 0°C, respectively (Table 2). In contrast, when D-tB 9:1 was used, the remaining ester 6b, at room temperature and 0°C (Table 1), gave 66.6 and 64.9 % ee and the pro- duced acid showed the greatest optical purity, 75.5 and 72.1% of ee (Table 1). This could be interpreted as an effect of the bulkiness of the alcohol on the active site of the enzyme during the transition state. On the other hand, conversion and yields differ in each solvent mixture, being highest in D-tB 9:1; this suggests that the reaction time becomes very important and that some- times an hour of reaction is too long for this experiment. The effect of mixture of solvents was re-evaluated, but this time involving all esters. The experiments were carried out inverting the ratio of solvents to D-tB 1:9. RESULTS AND DISCUSSION Entry Ester Time (min) Conversión (%) ee (ester) ee (acid)b Ed 1c 6d 30 27.3 41.8 88.0 23 2c 6d 60 19.6 64.4 89.9 36 3c 6d 90 21.3 7.8 85.6 14 4c 6d 120 31.9 58.6 74.8 12 5c 6d 150 30.3 59.7 68.8 10 6c 6e 30 11.6 16.5 92.5 32 7c 6e 60 26.9 5.6 90.9 22 8c 6e 90 19.5 46.1 83.5 18 9c 6e 120 22.7 25.0 98.7 254 10c 6e 150 22.6 21.4 84.2 14 11d 6d 30 38.5 43.4 83.0 16 12d 6d 60 40.5 51.4 74.8 11 13d 6d 90 43.0 66.0 73.3 12 14d 6d 120 44.5 59.2 62.5 8 15d 6d 150 49.0 64.7 54.3 6 16d 6e 30 29.6 27.1 85.4 16 17d 6e 60 36.9 4.6 84.3 12 18d 6e 90 40.0 17.3 97.1 77 19d 6e 120 43.1 16.1 78.2 10 20d 6e 150 46.5 2.4 85.0 12 aReaction cond.:100 mg of ester and 50 mg of enzyme. bOptical purity of acid was determined as methyl ester. cStirred at 180 rpm. dStirred at 300 rpm. eE values of the isolated acid 7; calculated as methyl ester. Table 3, the good enantioselectivity was observed from the first minutes of the reaction onward. combined effect between the structure of the ester alkyl chain and the reaction solvent, for example, the ester 6a, being the smallest, did not suffer any influence from the solvent or the temperature of reaction; on the contrary, ester 6f showed a substantial improvement in enantio- selectivity due to an increase in conversion, when the 9:1 solvent mixture changed to 1:9 (Tables 1 and 2). The results of the reactions taking place at the higher speed of stirring, 300 rpm are also shown in Table 3. The optical purities of the isolated acids were good but slightly lower than those obtained at 180 rpm; an example of this was the hydrolysis of 6d, which had a good enantio- selectivity after 30 min (entry 11, Table 3). It is also noticeable that, in general, the conversions were almost duplicated in the reactions at higher stirring speed. Similarly, in the case of ester 6e (entry 18), 90 min was the reaction time required to obtain the product of hydrolysis in its best optical purity, 97% of ee, with an E = 77. RESULTS AND DISCUSSION The reactions were carried out once again at room tempe- rature and at 0°C and the results are detailed in Table 2. Interesting results were observed when the length of the hydrocarbonated chain increased, as in the case of 6d. The experiments indicated that the solvent intervened again in the selectivity, because in D-tB 1:9 the remaining ester was isolated in good selectivity, 71.5% of ee (Table 2), at room temperature. When the solvent was changed (D-tB 9:1), the selectivity changed too and the acid acquired the greatest enantiomeric excess, with value of 83.7% of ee (Table 1), for the reaction at room tempe- rature, whereas for the reaction at 0°C, the acid was isolated in 79.4% of ee (Table 1). The enzyme in this solvent mixture (D-tB 1:9) behaved in a similar way at both room temperature and 0°C. Under these conditions, the enzyme showed greater selectivity toward short-chain esters (6a and 6b, 72 and 75% ee, respectively). It was also observed that when the mixture D-tB (1:9) was used, the conversions were greater than with D-tB (9:1), comparing Tables 1 and 2. From the results it can also established that for esters 6c and 6f the hydrolysis did not show good enantioselectivity under any of the reaction conditions studied (temperature From these results, it becomes evident that there is a Afr. J. Biotechnol. 5962 Table 3. Enzymatic hydrolysis of estersa 6d and 6e at room temperature with stirring at 180 and 300 rpm. Table 3. Enzymatic hydrolysis of estersa 6d and 6e at room temperature with stirring at 180 and 300 rpm. REFERENCES Apers S, Paper D, Bürgermeister J, Baronikova S, Van Dyck S, Lemiére G, Vlietinck A, Pieters L (2002). Antiangiogenic Activity of Synthetic Dihydrobenzofuran Lignans. J. Nat. Prod. 65: 718-720. Bremer M, Lietzau L (2005). 1, 1, 6, 7-Tetrafluoroindanes: improved liquid crystals for LCD-TV application. New J. Chem. 29: 72-74. Fuson RC, Kneisley JW, Kaiser EW (1944). Coumarilic acid. Org. Synth. 24: p. 33. or Org. Synth. Coll. (1955), 3: 209-211. Harada I, Hirose Y, Nakaozaki M (1968). The absolute configurations of (+)-marmesin and (−)-hydroxytrementone. Tetrahedron Lett. 9: 5463- 5466. Kuethe JT, Wong A, Journet M, Davies IW (2005). A Rapid Synthesis of 2-Aryl-5-substituted-2, 3-dihydrobenzofurans. J. Org. Chem. 70: 3727-3729. RESULTS AND DISCUSSION From these results seems possible that the speed of agitation was not decisive in regard to the enantio- selectivity, but it did influence the rate of the hydrolysis reaction. Undoubtedly, the size of the alcohol chain was more important and the results indicated the ester 6e as the derivative with the best stereoselectivity. According to the obtained results, 6d and 6e were the derivatives for which the enzyme showed greater enan- tioselectivity regarding the produced acid, 7. Therefore it was decided to carry out experiments to determine the reaction time to get the maximum optical purity of the hydrolysis product. These experiments were carried out at two speeds of stirring, 180 and 300 rpm, in order to also evaluate the effect of agitation on the reaction. From the results of the reaction conducted at 180 rpm (Table 3), it was observed that 6d at 60 min. (entry 2) gave the isolated acid in good optical purity (89.9% of ee, E = 36), while the remaining isolated ester in this experi- ment showed an acceptable ee (64.4%). In the case of 6e, truly amazing results were obtained because the acid was isolated with an optical purity of 98.7% of ee, with an E = 254, after 120 min of reaction (entry 9); as seen in Preparative scale experiments were carried out to determine the influence of the enzyme and substrate concentration on the enantioselectivity of the reaction with esters 6d and 6e; the results of these experiments 5963 Tovar-Miranda et al. Table 4. Effect of reaction concentration on the enzymatic hydrolysis of esters 6d and 6ea. Entry Ester Subst. conc. (mg/ml) Conversion (%) ee (%) ester ee (%)e acid Ef 1 6db 50 53.6 58.9 49.9 5 2 6dc 25 45.0 74.0 64.3 10 3 6dd 100 43.8 38.9 41.6 4 4 6eb 50 52.3 95.3 64.3 16 5 6ec 25 40.0 89.3 48.2 8 6 6ed 100 45.1 91.7 76.5 24 aReaction: Subst/enzyme 2:1(w/w) at 25°C, at 180 rpm, in D-tB (9:1), for 4 h. b Reaction with 700 mg of ester. c Diluted reaction with100 mg of ester. d Concentrated reaction with100 mg of ester. e Optical purity of the acid was determined as the methyl ester by HPLC. fE values of the isolated acid 7; calculated as methyl ester. RESULTS AND DISCUSSION in these preparative reactions was the isolated ester; whereas in the experiments carried out with 100 mg, it was the isolated acid that showed the highest optical purity, under the same conditions. are summarized in Table 4. Diluted reactions (entries 2 and 5) as well as concentrated ones (entries 3 and 6) were carried out. The best conditions resulted in the concentrated reaction with the ester 6e (entry 6), both products of the reaction showed the high enantio- selectivity (91.7 and 76.5% ee). It is worth to mention that in entry 4, the isolated remaining ester showed with an excellent optical purity, 95% of ee. The isolated acids from the preparative reactions were recrystallized and then esterified to determine their optical purity; thus, the acid from 6d increased its ee from 49.9 to 71.2%, whereas the acid from 6e increased from 64.3 to 82.3%; these increases in optical purity were obtained with just one recrystallization. Conclusion The best selectivity was observed for the n-butyl and isobutyl esters at both temperatures room and 0°C; in both cases the acid showed the highest optical purity, although the methyl and ethyl esters showed an acceptable selectivity at both temperatures. It was found that the composition of the solvent mixture had a slight influence on the selectivity, especially for the short-chain esters. As previously noted, 6b was the ester that most constantly showed high selectivity under all reaction conditions; it was observed that 6d and to a lesser extent 6g, showed even higher selectivity, but only with certain solvent mixtures. Although, for this case the recovered esters had the highest optical purity. The experiments also suggest that the speed of agitation does not signifi- cantly influence the enantioselectivity of the enzyme. When the substrate concentration in the reaction was changed, only for the 6d and 6e derivatives, 6e presented the best behavior in all cases; this apparently indicates that the concentration of the reaction is not important, even during preparative experiments. Interestingly, the compound with the highest optical purity Nicholas WR, Yang CG, Shi Z, He C (2006). Gold(I)-Catalyzed Synthesis of Dihydrobenzofurans from Aryl Allyl Ethers. Synlett. pp. 1278-1280. Park NS, Jung YS, Park CH, Seong CM, Lim HJ (2001). Synthesis of Novel 2-Aryl-2-methyl-2, 3-dihydrobenzofurans. Bull. Korean Chem. Soc. 22: 139-140. Pieters L, Van Dyck S, Gao M, Bai R, Hamel E, Vlietinck A, Lemiére G (1999). Synthesis and Biological Evaluation of Dihydrobenzofuran Lignans and Related Compounds as Potential Antitumor Agents that Inhibit Tubulin Polymerization. J. Med. Chem. 42: 5475-5481. Tovar-Miranda R, Cortés-García R, Trinidad-Nino LR, Joseph-Nathan P (1999). Synthesis and absolute configuration of (S)-(−)- and (R)-(+)- 2,3-dihydro-2-(1-methylethenyl)-6-methoxybenzofuran. J. Nat. Prod. 62: 1085-1087. Tsai IL, Hsieh CF, Duh CY (1998). Additional cytotoxic neolignans from Persea obovatifolia. Phytochemistry, 48: 1371-1375. Villanueva-Lendechy MA, Cortés-García R, Tovar-Miranda R, Luna H (2000). Evaluación de los Polvos de Acetona de Hígado Animal en la Resolución Enzimática del Acido 2,3-Dihidro-6-Methoxibenzofuran-2- Carboxílico. Rev. Soc. Quím. Méx. 44: 200-203. Witiak DT, Feller DR, Stratford ES, Hackney RE, Nazareth R, Wagner G (1971). Inhibitory action of α-(4-chlorophenoxy)-α-methylpropionic acid analogs on cholesterol biosynthesis and lipolysis in vitro. J. Med. Chem. 14: 754-757.
https://openalex.org/W4390912867	https://bmcoralhealth.biomedcentral.com/counter/pdf/10.1186/s12903-024-03852-3	English	null	Oral cancer cell to endothelial cell communication via exosomal miR-21/RMND5A pathway	BMC oral health	2,024	cc-by	6,093	© The Author(s) 2024. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Abstract Required for meiotic nuclear division 5 homolog A (RMND5A), a novel ubiquitin E3 Ligase, has been reported to correlate with poor prognosis of several cancers. However, its role in endothelial cells has not been reported. In this study, overexpression of RMND5A in human umbilical vein endothelial cells (HUVECs) was performed via lentiviral infection, followed by MTT, would healing and tube formation assay as well as signaling analysis. Moreover, crosstalk between HUVECs and oral squamous cell carcinoma (OSCC) cells was investigated by indirect co-culture with condition medium or tumor cell derived exosomes. Our results showed that overexpression of RMND5A reduced the proliferation, migration and tube formation ability of HUVECs by inhibiting the activation of ERK and NF-κB pathway. Interestingly, OSCC cells can inhibit RMND5A expression of endothelial cells via exosomal miR-21. In summary, our present study unveils that OSCC cells can activate endothelial cells via exosomal miR-21/RMND5A pathway to promote angiogenesis, which may provide novel therapeutic targets for the treatment of OSCC. Keywords Required for meiotic nuclear division 5 homolog A, Oral cancer, Endothelial cells, Exosome, miR-21 Finally, the E2 enzyme collaborates with an E3 ubiquitin ligase to transfer the ubiquitin molecule onto the target protein. The multi-subunit carboxy-terminal to LisH (CTLH) complex is a newly discovered E3 ligase, which is composed of at least Ran-binding protein M (RanBPM), muskelin, WD repeat-containing protein 26 (WDR26), armadillo repeat-containing protein 8 (ARMC8) α/β, GID4, required for meiotic nuclear division 5 A (RMND5A) and macrophage erythroblast attacher (MAEA) [2, 3]. The E3 ligase activity of the CTLH com plex is dependent on RMND5A and MAEA. RMND5A harbors the LisH/CTLH motifs and contributes to micro tubule dynamics, cell migration, nuclear motility and chromosome segregation. RMND5A has been known to be important for HeLa cell migration by interacting with RanBPM and stabilizing Exportin-5 protein [4]. Mean while, deletion of RMND5A promotes HEK293 cell pro liferation via c-RAF/ERK signaling [5]. Also, RMND5A Oral cancer cell to endothelial cell communication via exosomal miR-21/ RMND5A pathway Yu-qi Sun1†, Bing Wang1†, Lin-wei Zheng1, Ji-hong Zhao1,2* and Jian-gang Ren1,2* Yu-qi Sun1†, Bing Wang1†, Lin-wei Zheng1, Ji-hong Zhao1,2* and Jian-gang Ren1,2* Introduction Ubiquitination is a complex process that involves the sequential actions of several enzymes [1]. First, ubiqui tin is activated in an ATP-dependent process by an E1 ubiquitin-activating enzyme. Next, the activated ubiqui tin is transferred to an E2 ubiquitin-conjugating enzyme. †Yu-qi Sun and Bing Wang authors contributing equally to this article. Correspondence: Ji-hong Zhao jhzhao988@whu.edu.cn Jian-gang Ren rjg19870708@whu.edu.cn 1The State Key Laboratory Breeding Base of Basic Science of Stomatology (Hubei-MOST) & Key Laboratory of Oral Biomedicine Ministry of Education, School & Hospital of Stomatology, Wuhan University, No. 237 Luoyu Road, Wuhan 430079, China 2Department of Oral and Maxillofacial Surgery, School & Hospital of Stomatology, Wuhan University, Wuhan 430079, China © The Author(s) 2024. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. BMC Oral Health BMC Oral Health Sun et al. BMC Oral Health (2024) 24:82 https://doi.org/10.1186/s12903-024-03852-3 Open Access Real-time quantitative PCR (qPCR) q (q ) Total RNA of HUVECs was extracted using FastPure Cell/Tissue Total RNA Isolation Kit (Vazyme, Jiangsu, China). As we previously described [14], for mRNA detection, 1000 ng of RNA was reversely transcribed to cDNA by using the HiScript III RT SuperMix for qPCR (+ gDNA wiper) (Vazyme, Jiangsu, China). Obtained cDNA was amplified with Taq Pro Universal SYBR qPCR Master Mix (Vazyme, Jiangsu, China). GAPDH was selected as an internal control. MiRNA was synthesized into cDNA with specific RT primers and combined with specific forward primers for amplification by using Bulge- Loop™ miRNA qRT-PCR Starter Kit (RiboBio, Guang zhou, China). U6 was selected as an internal control. The primers of GAPDH, RMND5A, and RanBPM were syn thesized by Tsingke Biotechnology Co., Ltd. U6 and miR- 21 primers were synthesized by Guangzhou RiboBio Co., Ltd. Quant Studio 6 Flex Real-Time PCR System (Applied Biosystems, CA, USA) was used for real-time qPCR. All the primers’ sequences were shown in Table 1. Transient miRNA transfection Angiogenesis is the formative process of new blood vessels from the pre-existing vasculature [8]. Angiogen esis is a critical event in tumor progression by supplying oxygen and nutrients to tumor cells [9]. It is well known that endothelial cell (EC) proliferation and migration are required for angiogenesis [10]. Of interest, many studies have found that tumor cells can regulate angiogenesis via direct or indirect communication with endothelial cell [11, 12]. Based on the reported function of RMND5A, in this study, we investigated the role of RMND5A in endo thelial cells and the underlying mechanisms. In addition, we found that oral squamous cell carcinoma (OSCC) cells could regulate RMND5A expression in endothelial cells via exosomal microRNA-21 (miR-21). Hsa-miR‑21‑5p mimics, hsa-miR‑21‑5p inhibitor and negative control (NC) were purchased from Guang zhou RiboBio Co., Ltd. (Guangzhou, China). HUVECs and OSCC cell lines CAL27 and SCC25 were transiently transfected with riboFECTTMCP Transfection Kit (Ribo Bio) at the concentration of 50 nM (miR-21 mimic and NC) or 100 nM (miR-21 inhibitor and NC). Materials and methods Cell culture As we previously described, human umbilical vein endo thelial cells (HUVECs) were isolated from human umbili cal cord veins [13]. This study has been approved by the review board of the ethics committee of the Hospital of Stomatology, Wuhan University, and the formed consent was signed by the parents of newborns. HUVECs were cultured in endothelial cell medium (ECM, Sciencell) containing 10% fetal bovine serum (FBS) and 1% endo thelial cell growth supplement (ECGs). OSCC cell lines CAL27 and SCC25 were purchased from the American Tissue Culture Collection (ATCC). CAL27 cells were cultured in dulbecco’s modified eagle medium (DMEM) supplemented with 10% FBS. SCC25 cells were cultured in the mixture of DMEM and Ham’s F12 in equal pro portion supplemented with 10% FBS. All the cells were grown in a humidified incubator at 37 °C and 5% CO2. Constructs and viral infection of HUVECs Human RMND5A cDNA (NM_022780.4) was amplified by PCR and subcloned into a lentiviral pHS-AVC vec tor (pLV-hef1a-mNeongreen-P2A-Puro-WPRE-CMV- 3xFlag) containing 3xFlag (Beijing Syngentech Co., Ltd.) (Beijing, China). 293FT cells and polyethylenimine (PEI) reagent was used for virus packaging. For RMND5A overexpression, HUVECs were infected with viral par ticles packaged with the recombinant plasmid, and 10 µg/ ml polybrene (Sigma-Aldrich, Darmstadt, Germany) was Table 1 Primer sequences used for real-time PCR Gene Forward (5’-3’) Reverse (5’-3’) RMND5A TTTACACGGGATGCTTGTGC ACTCCAGTACACTGCCTCTG RanBPM GCCCAGTTGGAAATCAGCTT CTGATCGAGCCATCAGTCCT GAPDH GCTCTCTGCTCCTCCTGTTC ACGACCAAATCCGTTGACTC Western blot analysis (WB) According to our previous study [15], 20 µg of total protein was loaded for SDS-PAGE electrophoresis and transferred to the PVDF membrane at a voltage of 110 V (90 min). After that, the membrane was blocked in 5% skimmed milk for 1 h, and incubated with primary anti bodies (anti Flag: #2064, Dia-An Biotechnology, 1:3000; anti-NF-κB: #8242, Cell Signaling Technology, 1:1000; anti-phos NF-κB: #3033, Cell Signaling Technology, 1:1000; anti-ERK: #4695, Cell Signaling Technology, 1:2000; anti-phos ERK: #4370, Cell Signaling Technology, 1:3000; anti-GAPDH: sc-365,062, Santa Cruz Biotechnol ogy, 1:2000) at 4 oC overnight, followed by incubation with HRP-conjugated secondary antibodies at room tem perature for 1 h. The protein signals were detected with ECL kit. GAPDH was used as a loading control. Sun et al. BMC Oral Health (2024) 24:82 Page 2 of 8 Sun et al. BMC Oral Health has been proven as a potential prognostic marker in breast cancer and pancreatic adenocarcinoma [6, 7]. However, the role of RMND5A in endothelial cells has not been reported. used to enhance the efficiency of viral infection. After 48-h infection, the positively infected cells were selected with puromycin (InvivoGen, CA, USA). Isolation and size-measurement of exosomes CAL27 and SCC25 cells were cultured in exosome- depleted serum. The collected media was centrifuged at 2,000 g for 10 min, and the supernatant was centrifuged at 16,000 g for 1 h at 4 °C to remove the cell debris and big vesicles. After that, the supernatant was ultracentri fuged at 100,000 g for 2 h at 4 °C. The obtained pallet was suspended in PBS and stocked at -80 °C. Meanwhile, a little bit of pallet solution was diluted and loaded into a NanoSight NS300 to measure the particle size. Wound-healing assay According to our previous study [16], HUVECs were seeded in 6-well plates and vertically scraped with a 200- ul sterile micropipette tip at 80-90% confluence. After that, the cells were gently rinsed with PBS and cultured with serum-free ECM media at 37 °C and 5% CO2. At 0 and 24 h, the cells were photographed under a phase microscope and the migrated cells were counted. MTT assay HUVECs were seeded in a 96-well plate at 5 × 103 cells per well and cultured in ECM plus 10% FBS and different Sun et al. BMC Oral Health (2024) 24:82 Page 3 of 8 Page 3 of 8 (2024) 24:82 Sun et al. BMC Oral Health 1% (Fig. 1c). By comparison with vector control group, overexpression of RMND5A significantly inhibited the proliferation of HUVECs under the condition of ECGs (Fig. 1c). Would healing assay showed that overexpres sion of RMND5A significantly inhibited the migration ability of HUVECs (Fig. 1d). In addition, overexpres sion of RMND5A reduced the tube formation ability of HUVECs evidenced by decreased capillary-like struc tures (Fig. 1e). 1% (Fig. 1c). By comparison with vector control group, overexpression of RMND5A significantly inhibited the proliferation of HUVECs under the condition of ECGs (Fig. 1c). Would healing assay showed that overexpres sion of RMND5A significantly inhibited the migration ability of HUVECs (Fig. 1d). In addition, overexpres sion of RMND5A reduced the tube formation ability of HUVECs evidenced by decreased capillary-like struc tures (Fig. 1e). 1% (Fig. 1c). By comparison with vector control group, overexpression of RMND5A significantly inhibited the proliferation of HUVECs under the condition of ECGs (Fig. 1c). Would healing assay showed that overexpres sion of RMND5A significantly inhibited the migration ability of HUVECs (Fig. 1d). In addition, overexpres sion of RMND5A reduced the tube formation ability of HUVECs evidenced by decreased capillary-like struc tures (Fig. 1e). doses of ECGs for 3 days. Subsequently, 20 µl of 3-(4,5- dimethylthiazole-2-yl)-2,5-diphenyl tetrazolium bromide solution (MTT) (5 mg/ml) was added to each well at 37 °C for 4 h away from light. After removing the super natant, 150 µl of dimethyl sulfoxide (DMSO) was added to each well. The absorbance was measured on Bio-Tek microplate reader at 490 nm. OSCC cell-derived exosomes inhibit RMND5A gene expression of HUVECs Crosstalk between tumor cells and endothelial cells is important to tumor development [19, 20]. Based on above results, we further investigated the effects of OSCC cells on RMND5A expression in HUVECs. Inter estingly, the cell culture supernatant (condition media, CM) from either CAL27 or SCC25 cells dose-depend ently decreased RMND5A gene expression in HUVECs after 24-h treatment. However, the gene expression of RanBPM, another subunit of CTLH complex, was not changed (Fig. 3a and b). Previous studies have reported that tumor-derived exosomes can target endothelial cells to promote angiogenesis [21, 22]. Therefore, we isolated the exosomes from the condition media by differen tial centrifugation and confirmed the size by Nanosight (Fig. 3c). Our data showed that the exosomes from either CAL27 or SCC25 cells significantly decreased RMND5A gene expression in HUVECs after 24-h treatment, while the exosomes depleted CM from OSCC cells cannot inhibit RMND5A expression (Fig. 3d and e). These results demonstrated that the inhibitory effect of OSCC cells on RMND5A expression of endothelial cells was exosome dependent. In vitro tube formation assay According to our previous study [16], the cool 96-well plate was coated with 50 ul of Matrigel (BD Biosci ences) and placed in a 37 °C incubator for 45 min. Next, HUVECs were seeded in triplicate for 10 h and capillary- like structures were quantified under a phase microscope (Olympus). Statistical analysis All data are presented as mean ± SD and analyzed by using GraphPad Prism 9 (San Diego, CA, USA). Student’s t-test was used when comparing two groups, while one- way ANOVA followed by post hoc tests was used if more than two groups. P < 0.05 was considered statistically significant. RMND5A inhibits the activation of ERK and NF-κB in HUVECs Numerous studies have demonstrated the critical role of ERK and NF-κB pathways in the regulation of cell prolif eration and migration [17, 18]. Recently, it was reported that RMND5A can regulate ERK activation via c-RAF ubiquitination in HeLa cells [5]. To explore the mecha nism on the effects of RMND5A in HUVECs, we com pared the activation of ERK and NF-κB. Our WB data showed that the phosphorylation levels of both ERK and NF-κB were significantly decreased in the HUVECs over expressing RMND5A compared with vector control cells in the ECM media with 1% ECGs (P < 0.01) (Fig. 2a and b) or without ECGs (P < 0.001) (Fig. 2a and c) for 4 h at 37 oC. These results revealed that RMND5A may inhibit endothelial cell proliferation, migration and tube forma tion by regulating ERK and NF-κB pathways. RMND5A inhibits the proliferation, migration and tube formation ability of HUVECs To investigate the role of RMND5A in endothelial cells, we overexpressed Flag-tag RMND5A in HUVECs via len tiviral infection. Real-time PCR and WB data proved the elevated expression of RMND5A in HUVECs (Fig. 1a and b). Our MTT data showed ECGs dependent growth of HUVECs (Fig. 1c). ECGs induced concentration depen dent proliferation of HUVECs under a concentration of Page 4 of 8 Sun et al. BMC Oral Health (2024) 24:82 (2024) 24:82 Sun et al. BMC Oral Health Fig. 1 RMND5A inhibits the proliferation, migration and tube formation of HUVECs. (a, b) Overexpression of Flag-tag RMND5A in HUVECs via lentiviral infection. Real-time PCR (a) and WB (b) data proves the elevated expression of RMND5A in HUVECs. (c) MTT assay shows ECGs dependent growth of HU VECs. By comparison with vector control group, overexpression of RMND5A significantly inhibits the proliferation of HUVECs under the condition of ECGs. (d) Would healing assay shows that overexpression of RMND5A significantly inhibited the migration ability of HUVECs. (e) Overexpression of RMND5A re duces the tube formation ability of HUVECs evidenced by decreased capillary-like structures. Data is expressed as mean ± SD. ns, not significant; P < 0.05; P < 0.01; P < 0.001 Fig. 1 RMND5A inhibits the proliferation, migration and tube formation of HUVECs. (a, b) Overexpression of Flag-tag RMND5A in HUVECs via lentiviral infection. Real-time PCR (a) and WB (b) data proves the elevated expression of RMND5A in HUVECs. (c) MTT assay shows ECGs dependent growth of HU VECs. By comparison with vector control group, overexpression of RMND5A significantly inhibits the proliferation of HUVECs under the condition of ECGs. (d) Would healing assay shows that overexpression of RMND5A significantly inhibited the migration ability of HUVECs. (e) Overexpression of RMND5A re duces the tube formation ability of HUVECs evidenced by decreased capillary-like structures. Data is expressed as mean ± SD. ns, not significant; P < 0.05; P < 0.01; P < 0.001 Fig. 2 RMND5A inhibits the activation of ERK and NF-κB in HUVECs. WB data shows that RMND5A overexpression in HUVECs significantly decreases both ERK and NF-κB phosphorylation levels under the condition of ECM media with 1% ECGs (a, b) or without ECGs (a, c). Data is expressed as mean ± SD. ns, not significant; P < 0.01; P < 0.001 Fig. 2 RMND5A inhibits the activation of ERK and NF-κB in HUVECs. RMND5A inhibits the proliferation, migration and tube formation ability of HUVECs WB data shows that RMND5A overexpression in HUVECs significantly decreases both ERK and NF-κB phosphorylation levels under the condition of ECM media with 1% ECGs (a, b) or without ECGs (a, c). Data is expressed as mean ± SD. ns, not significant; P < 0.01; **P < 0.001 OSCC cell-derived exosomal miR-21 inhibits RMND5A gene expression of HUVECs OSCC cell-derived exosomal miR-21 inhibits RMND5A gene expression of HUVECs OSCC cell-derived exosomal miR-21 inhibits RMND5A gene expression of HUVECs exosomes [26]. Our data showed that RMND5A gene expression was significantly decreased after overexpres sion of miR-21 using miR-21 mimic in HUVECs (Fig. 4a and b). Importantly, the condition media of miR-21 inhibitor transfected CAL27 and SCC25 cannot inhibit RMND5A expression of HUVECs (Fig. 4c and d). Con sistently, the exosomes derived from miR-21 inhibitor transfected CAL27 and SCC25 cells had no significant effects on RMND5A gene expression of HUVECs (Fig. 4e Exosomal miRNAs have been proven to play a criti cal role in tumor microenvironment by inhibiting gene expression [23]. Previous studies demonstrated that miR- 21 is consistently overexpressed in a variety of cancers including OSCC [24], and RMND5A is a strong candi date target of miR-21 [25]. Meanwhile, miR-21 is found to be highly expressed in the CAL27- and SCC25-derived Page 5 of 8 Page 5 of 8 (2024) 24:82 Sun et al. BMC Oral Health h Fig. 3 OSCC cell-derived exosomes inhibit RMND5A gene expression of HUVECs. (a, b) CAL27 (a) and SCC25 (b) cell culture supernatant (condition media, CM) dose-dependently decreases RMND5A gene expression in HUVECs after 24-h treatment, but does not change RanBPM gene expression. (c) The exosomes are purified from the condition media by differential centrifugation and confirmed by Nanosight. (d, e) The exosomes from CAL27 (d) and SCC25 (e) cells, but not exosome-depleted CM, significantly decrease RMND5A gene expression in HUVECs after 24-h treatment. Data is expressed as mean ± SD. ns, not significant; P < 0.05; *P < 0.01 Fig. 3 OSCC cell-derived exosomes inhibit RMND5A gene expression of HUVECs. (a, b) CAL27 (a) and SCC25 (b) cell culture supernatant (condition media, CM) dose-dependently decreases RMND5A gene expression in HUVECs after 24-h treatment, but does not change RanBPM gene expression. (c) The exosomes are purified from the condition media by differential centrifugation and confirmed by Nanosight. (d, e) The exosomes from CAL27 (d) and SCC25 (e) cells, but not exosome-depleted CM, significantly decrease RMND5A gene expression in HUVECs after 24-h treatment. Data is expressed as Fig. 3 OSCC cell-derived exosomes inhibit RMND5A gene expression of HUVECs. (a, b) CAL27 (a) and SCC25 (b) cell culture supernatant (condition media, CM) dose-dependently decreases RMND5A gene expression in HUVECs after 24-h treatment, but does not change RanBPM gene expression. (c) The exosomes are purified from the condition media by differential centrifugation and confirmed by Nanosight. OSCC cell-derived exosomal miR-21 inhibits RMND5A gene expression of HUVECs (d, e) The exosomes from CAL27 (d) and SCC25 (e) cells, but not exosome-depleted CM, significantly decrease RMND5A gene expression in HUVECs after 24-h treatment. Data is expressed as mean ± SD. ns, not significant; P < 0.05; *P < 0.01 and f). These data revealed that OSCC cells can inhibit RMND5A expression of endothelial cells via exosomal miR-21. and thymoma compared to normal tissues, respectively [7]. In addition, RMND5A is reported to positively reg ulate the migration of HeLa and pancreatic adenocarci noma cells [4, 7]. However, the function of RMND5A in endothelial cells has not been reported. In this study, we found that reversely, overexpression of RMND5A inhib ited the proliferation, migration and tube formation of endothelial cells. Mechanistically, our results showed that overexpression of RMND5A in endothelial cells attenuated ERK and NF-κB activities, which are classi cal positive regulators in the cell cycle, motility and tube formation of endothelial cells [27–29]. These data on the signaling pathway is very consistent with a recent study that RMND5A is an E3 ligase of c-RAF to regulate c-RAF degradation thereby ERK activation [5]. Furthermore, we found that CAL27-derived exosomes significantly enhanced the proliferation, migration and tube formation as well as ERK activation in HUVECs (Supplemental Fig. 1). More importantly, we noted a sub stantial decrease in these effects after silencing miR-21 in CAL27 cells, suggesting that miR-21 plays a critical role in modulating the pro-angiogenic properties of OSCC- derived exosomes. Discussion Previous studies have demonstrated that RMND5A expression is significantly higher in the tumor tissues of pancreatic adenocarcinoma, stomach adenocarcinoma Page 6 of 8 Page 6 of 8 (2024) 24:82 Sun et al. BMC Oral Health (2024) 24:82 Sun et al. BMC Oral Health Fig. 4 OSCC cell-derived exosomal miR-21 inhibits RMND5A gene expression of HUVECs. (a) Real-time PCR proves that miR-21 expression in HUVECs is upregulated after transfection with miR-21 mimic. (b) RMND5A gene expression is significantly decreased after overexpression of miR-21 using miR-21 mimic in HUVECs. (c, d) The condition media of miR-21 inhibitor transfected CAL27 (c) and SCC25 (d) cells cannot inhibit RMND5A expression of HUVECs. (e, f) The exosomes derived from miR-21 inhibitor transfected CAL27 (e) and SCC25 (f) had no significant effects on RMND5A gene expression of HUVECs. Data is expressed as mean ± SD. ns, not significant; P < 0.05; P < 0.01; P < 0.001 Fig. 4 OSCC cell-derived exosomal miR-21 inhibits RMND5A gene expression of HUVECs. (a) Real-time PCR proves that miR-21 expression in HUVECs is upregulated after transfection with miR-21 mimic. (b) RMND5A gene expression is significantly decreased after overexpression of miR-21 using miR-21 mimic in HUVECs. (c, d) The condition media of miR-21 inhibitor transfected CAL27 (c) and SCC25 (d) cells cannot inhibit RMND5A expression of HUVECs. (e, f) The exosomes derived from miR-21 inhibitor transfected CAL27 (e) and SCC25 (f) had no significant effects on RMND5A gene expression of HUVECs. Data is expressed as mean ± SD. ns, not significant; P < 0.05; P < 0.01; P < 0.001 Endothelial cell is a key player in angiogenesis [30]. Under normal conditions, most of endothelial cells and vascular system remain quiescent [31]. However, in tumor microenvironment, tumor cells can activate normal quiescent endothelial cells for angiogenesis in a paracrine manner such as cytokine, growth factor and extracellular vesicle (EV) [32]. Exosomes, a small size subset of EVs around 30 ~ 150 nm in diameter, can trans port proteins and nucleic acids including miRNAs as mediators in cell-to-cell communication [33]. So far, sev eral exosomal miRNAs have been identified to influence angiogenesis and extracellular matrix remodeling in the tumor microenvironment via multiple signaling path ways. OSCC derived exosomal miR-221 can target phos phoinositide-3-kinase regulatory subunit 1 to enhance tube formation ability of HUVECs [34]. Discussion MiR-138 downregulates miRNA processing in HeLa cells by targeting RMND5A and decreasing Exportin-5 stability. Nucleic Acids Res. 2014;42:458–74. 5. McTavish CJ, Bérubé-Janzen W, Wang X, Maitland MER, Salemi LM, Hess DA, Schild-Poulter C. Regulation of c-Raf Stability through the CTLH Complex. Int J Mol Sci. 2019;20:934. 5. McTavish CJ, Bérubé-Janzen W, Wang X, Maitland MER, Salemi LM, Hess DA, Schild-Poulter C. Regulation of c-Raf Stability through the CTLH Complex. Int J Mol Sci. 2019;20:934. Discussion Meanwhile, another study reveals that OSCC derived exosomes can promote tumor angiogenesis by transfer miR-210-3p and targeting PI3K/AKT pathway [35]. Besides, OSCC derived exosomes are rich in miR-21, which displays oncogenic activity and acts as an angiogenesis inducer via activating AKT and ERK signaling pathway [36]. In this study, we proved that miR-21 can downregulate RMND5A expression in endothelial cells as predicated in previous study [37]. Moreover, we found that OSCC cells can regulate RMND5A expression in endothelial cells by secreting exosomal miR-21. For the function assay, silencing miR-21 in OSCC cells led to a marked reduc tion in the pro-angiogenic activities of OSCC derived exosomes. The residual pro-angiogenic effects of OSCC derived exosomes may be attributed to other enriched miRNAs. There are still some limitations in this study. For exam ple, RMND5A KO mice could be generated to investi gate the effects of RMND5A on vascular system in vivo. Furthermore, we are generating a specific and sensitive antibody against RMND5A to explore the role of miR- 21/RMND5A in OSCC progression and elucidate the clinical significances of RMND5A expression, such as the correlation among endothelial RMND5A expression with microvessel density in OSCC tissues and prognosis of OSCC patients. Page 7 of 8 Page 7 of 8 Sun et al. BMC Oral Health (2024) 24:82 Sun et al. BMC Oral Health (2024) 24:82 Sun et al. BMC Oral Health 2. Maitland MER, Kuljanin M, Wang X, Lajoie GA, Schild-Poulter C. Proteomic analysis of ubiquitination substrates reveals a CTLH E3 ligase complex- dependent regulation of glycolysis. FASEB J. 2021;35:e21825. In summary, our present study is the first to report inhibitory effects of RMND5A on the proliferation, migration and tube formation of endothelial cells via inhibiting ERK and NF-κB activation. Moreover, OSCC cells can activate endothelial cells via exosomal miR-21/ RMND5A pathway to promote angiogenesis. Our pres ent study unveils new mechanisms of tumor angiogenesis and may provide novel therapeutic targets for the treat ment of OSCC. dependent regulation of glycolysis. FASEB J. 2021;35:e21825. 3. Maitland MER, Onea G, Chiasson CA, Wang X, Ma J, Moor SE, Barber KR, Lajoie GA, Shaw GS, Schild-Poulter C. The mammalian CTLH complex is an E3 ubiquitin ligase that targets its subunit muskelin for degradation. Sci Rep. 2019;9:9864. 4. Li J, Chen Y, Qin X, Wen J, Ding H, Xia W, Li S, Su X, Wang W, Li H, Zhao Q, Fang T, Qu L, Shao N. Declarations 19. Choi H, Moon A. Crosstalk between cancer cells and endothelial cells: implications for tumor progression and intervention. 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Author contributions YS, JZ and JR contributed to conception and experiment design; YS and BW performed the experiments; YS, BW, LZ and JR contributed to data acquisition, analysis and interpretation; YS and JR drafted the manuscript; JZ and JR revised the manuscript. All authors gave final approval and agree to be accountable for all aspects of the work. 14. Xia HF, Ren JG, Zhu JY, Yu ZL, Zhang W, Sun YF, Zhao YF, Chen G. Downregula tion of miR-145 in venous malformations: its association with disorganized vessels and sclerotherapy. Eur J Pharm Sci. 2017;100:126–31. 15. Li ZZ, Wang HT, Lee GY, Yang Y, Zou YP, Wang B, Gong CJ, Cai Y, Ren JG, Zhao JH. Bleomycin: a novel osteogenesis inhibitor of dental follicle cells via a TGF- β1/SMAD7/RUNX2 pathway. Br J Pharmacol. 2021;178:312–27. Funding Thi k This work was funded by the grants from National Natural Science Foundation of China to Dr. J.G. Ren (81600385) and Prof. J.H. Zhao (81671008). 16. 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https://openalex.org/W3028232326	https://europepmc.org/articles/pmc7238661?pdf=render	English	null	Yield of household contact investigation of patients with pulmonary tuberculosis in southern Ethiopia	BMC public health	2,020	cc-by	6,082	Yassin et al. BMC Public Health (2020) 20:737 https://doi.org/10.1186/s12889-020-08879-z Yassin et al. BMC Public Health (2020) 20:737 https://doi.org/10.1186/s12889-020-08879-z Open Access © The Author(s). 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Yield of household contact investigation of patients with pulmonary tuberculosis in southern Ethiopia Mubarek A. Yassin1, Kesetebirhan D. Yirdaw2, Daniel G. Datiko1, Luis E. Cuevas3 and Mohamm Mubarek A. Yassin1, Kesetebirhan D. Yirdaw2, Daniel G. Datiko1, Luis E. Cuevas3 and Mohammed A. Yassin4 Abstract Background: Household Contacts (HHCs) of patients with pulmonary tuberculosis (PTB) have a higher risk of developing TB. Contact investigation is recommended to reach this group and identify undiagnosed cases. In this study, we have determined the yield of contact investigation among HHCs of patients with smear-positive PTB, and estimated TB burden. Methods: We conducted retrospective record review for the occurrence of TB among HHCs of Index PTB+ cases treated between November 2010 and April 2013 in 12 public health facilities in Boricha district. HHCs were followed up monthly and revisited between March and June 2015. Information on additional TB cases diagnosed and treated among HHCs were documented. HHCs who were diagnosed as having TB after the index cases were diagnosed and treated were considered as ‘incident cases’. Presumptive TB case was defined as those having cough for ≥2 weeks or enlarged lymph node. Diagnosis of TB among HHCs were made using smear-microscopy and/or X-rays, and clinically for Extra-pulmonary TB (EPTB). Results: One thousand five hundred and seventeenth HHCs of 344 index cases were visited and screened for TB and followed up for a median of 37 months. 77 (5.1% - 72 with PTB and 5 with EPTB) HHCs developed TB during 4713 person-years of follow-up with an estimated incidence of 1634 (95% CI: 1370-2043) per 100,000 person-years follow-up which is much higher than the estimated TB incidence for the general population in Ethiopia of 210/100, 000. Half (41/77) of incident TB cases were diagnosed within the first year of diagnosis of the index cases and 88% (68/77) were adults (Hazard Ratio: 4.03; 95% CI: 2.00–8.12). Conclusion: HHCs of index PTB+ cases have high risk of developing active TB. Long term follow-up of HHCs could help improve TB case finding depending on country contexts. Further studies on effectiveness and feasibility of the approach and integration in routine settings are needed. Keywords: Tuberculosis, Index case, Household contacts, Contact investigation, Case finding Keywords: Tuberculosis, Index case, Household contacts, Contact investigation, Case finding or diagnosed and treated, but not reported to national TB programs (NTP) [1]. Missed opportunities to identify these cases include case finding among contacts of index cases in the community. Background To view a co The Creative Commons Public Domain Dedication waiver (h data made available in this article, unless otherwise stated in * Correspondence: mubahmd@gmail.com 1TB REACH Project, Awassa, Ethiopia Full list of author information is available at the end of the article * Correspondence: mubahmd@gmail.com 1TB REACH Project, Awassa, Ethiopia Full list of author information is available at the end of the article * Correspondence: mubahmd@gmail.com 1TB REACH Project, Awassa, Ethiopia Full list of author information is available at the end of the article * Correspondence: mubahmd@gmail.com 1TB REACH Project, Awassa, Ethiopia Full list of author information is available at the end of the article Background Tuberculosis (TB) case finding strategies mostly rely on passive case finding. According to World Health Organization (WHO) over 30% of the estimated new TB cases are missed every year due to being not diagnosed WHO recommends contact investigation (CI) for household contacts (HHCs) of patients with bacterio- logically confirmed TB in low and middle income coun- tries [2]. CI identifies individuals at high risk for logically confirmed TB in low and middle income c tries [2]. CI identifies individuals at high risk © The Author(s). 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/ The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. * Correspondence: mubahmd@gmail.com 1TB REACH Project, Awassa, Ethiopia Full list of author information is available at the end of the article logically tries [2] © The Author(s). 2020 Open Access This article is licensed u which permits use, sharing, adaptation, distribution and rep appropriate credit to the original author(s) and the source, p changes were made. The images or other third party materi licence, unless indicated otherwise in a credit line to the ma licence and your intended use is not permitted by statutory permission directly from the copyright holder. Yassin et al. BMC Public Health (2020) 20:737 Page 2 of 9 Page 2 of 9 household visits immediately after index cases initiated treatment, registered and screened consenting HHCs for active TB and conducted contact screening and moni- tored adherence to Isoniazid preventive therapy (IPT) at community level. HEWs also conducted regular follow up visits to households of index cases as part of routine case finding activity to identify symptomatic cases and facilitate diagnosis. Household contacts who didn’t have TB during initial contact investigation visit were followed-up by HEWs monthly. Any person having cough for two or more weeks and/or having enlarged lymph nodes was considered as presumptive TB case [14]. Diagnosis of TB among HHCs was made using smear-microscopy and/or X-rays, and clinically for Extra-pulmonary TB (EPTB). Two sputum samples, spot and morning, were collected and light-emitting-diodes- fluorescent microscopes (LED-FM) were used for diag- nosis [15]. TB cases identified among HHCs were regis- tered and received the same treatment and followed up as the index cases. developing tuberculosis [3–7] and offers an opportunity for early diagnosis and treatment of contacts with active TB, to interrupt transmission and reduce morbidity and mortality in affected individuals [8, 9]. However, CI is not often conducted routinely or not conducted well [10]. In Ethiopia, yield from CI has been assessed in two dif- ferent ways. The first is CI within 3 months of diagnosis of index TB cases [11]. A yield of 2500/100,000 HHCs was reported in this approach in Amhara and Oromia regions of Ethiopia where 15,527 HHCs were screened for TB. While this approach identified undiagnosed cases, some of them may be prevalent cases which were not diagnosed in a timely manner and others could be new cases. In the second approach, CI was done for household (HH) members of previously diagnosed index cases [12]. The study by Zewdu et al. in Amhara and Oromia regions was conducted from June–October 2014 and indicated that in this approach, of 272,441 close contacts of 47,021 index cases diagnosed 2 years before the study started, TB notification was 768/100,000 after contact investigation. This study demonstrated that CI could identify additional TB cases (most likely new) long after index cases were diagnosed and treated. In this study, we estimated additional TB cases diagnosed among HHCs of patients with smear positive pulmonary TB (PTB+) during long term follow-up. Index PTB+ cases diagnosed between November 2010 and April 2013 were selected and their HHCs were retrospectively studied. Identification and address of the index cases were collected from TB registers of the health centers. Between March and June, 2015, house- holds of all index cases were visited by the study team to check for the occurrence of additional TB cases among HHCs between November 2010 and April 2015 and vali- dated the data. Trained health professionals working in the health centers and health posts collected the data at household level using pre-tested questionnaire. All household members of index TB case diagnosed be- tween Nov 2010 and April 2013 were recorded. ‘Index TB cases’ are those who were diagnosed first and all TB cases diagnosed in a household after the diagnosis of index cases were considered as ‘TB cases among HHCs’. Household contact is defined as a person who shared the same enclosed living space for one or more nights or for frequent or extended periods during the day with the index case during the 3 months before the diagnosis of TB [16]. Data collection at the households was done from March to June 2015; household head or index case was interviewed about TB status of HHCs. Data collec- tion was supervised on daily basis to assure data quality. Data in completed questionnaire from the households was cross-checked with TB DOTS registers in the health facilities where category of TB and exact date the HHCs started treatment were collected. Methods We conducted retrospective record review for the occur- rence of TB among household contacts of index PTB+ cases treated between November 2010 and April 2013 in 12 public health facilities in Boricha district, Sidama Zone, Southern Ethiopia. The district has a population of 314,296 and is located 33 kms from Hawassa, which is the regional capital. The district has 12 health centers providing TB diagnosis and treatment services [13]. Each health center provides support to health posts in their catchment areas, which are run by two health extension workers (HEWs). Between 2010 and 2015, a community-based TB project implemented in the dis- trict and trained HEWs who visited all households in their villages (Kebeles) and collected sputum samples from presumptive TB cases, prepared smears, fixed slides, and sent slides to nearest health facility laboratory for staining and smear microscopy. Their supervisors fa- cilitated transportation of smears to health facilities and delivery of results back to HEWs. Smear negative cases were referred to health facilities for further investigation including chest X-rays and clinical follow-up. Supervi- sors also brought anti-tuberculosis drugs to HEWs whenever a person was diagnosed with TB. HEWs sup- ported treatment and followed up patients during treat- ment. HEWs with District TB supervisor conducted Independent variables collected included age, gender, educational status, marital status, occupation, religion, family size and treatment start date for index cases while for HHCs age, gender and treatment start date. The dependent variable was TB disease among HHCs of PTB+ index cases after treatment start by the index Yassin et al. BMC Public Health (2020) 20:737 Page 3 of 9 Page 3 of 9 Page 3 of 9 cases. Data were entered into Epi Info 3.5.4 and exported to and analyzed using Stata statistical software version 12. The data can be accessed in the supplemen- tary material (Additional file 1). cases among HHCs were adults (68/77 = 88.3%, p value < 0.001). As shown in Table 2, TB incidence was highest in the first year with a magnitude of 2740 (95% CI: 2018-3772) (Figs. 2, 3). y HHCs diagnosed with active TB during contact in- vestigation visits were considered as ‘incident TB cases.’ Person-years at risk of TB disease was calcu- lated as time between date index case started treat- ment until date of TB diagnosis made among HHCs or for those who did not develop the disease, till last follow-up date. Methods Household contacts that changed resi- dence or died were excluded from the analysis. TB incidence density was calculated as the number of in- cident TB cases among HHCs divided by the total number of HHCs’ follow-up person-years observed (PYO). A subgroup analysis of TB incidence among HHCs of index cases diagnosed between years 2010– 2011 was calculated to determine TB incidence trend among people with longer follow-up. Hazard ratios, generated using Cox regression, were used to identify determinants of incident TB. P-value < 0.05 was con- sidered statistically significant. Clustering effect of data because contacts to index cases are household members was adjusted by using household ID when calculating confidence interval, hazard ratio, and p value. The median time between the index cases and HHCs cases diagnosed with active TB was 11 months (inter- quartile range: 7–29). Figure 4 indicates the distribution of index and incident TB cases among HHCs by calen- dar time. Discussion The estimated TB burden among household contacts of index cases with PTB was high in our setting with 1634 (95% CI: 1370-2043) HHCs diagnosed with TB per 100, 000 PYO. This was almost eight times higher than the estimated incidence of TB in Ethiopia for the general population which was 210 (95% confidence interval 168–250) per 100,000) in 2015 [17]. HHCs of people with PTB could develop symptoms of TB at any time and long term and regular follow up of this high risk group could be an opportunity to improve TB case find- ing and reach the ‘missing’ people with TB.. Overall, 77 TB cases were identified in the house- holds of index cases at the end of follow-up period. This is 22% (77/344) or 22 TB cases identified among HHCs per 100 index PTB+ cases followed for a me- dian of 3 years. Compared to short term prospective studies that reported 8.5% in South Africa [18] and 6.4% in Ethiopia [11], the yield from a long term fol- low up (average 2.2 years) was 14% in Turkey [19], 4.4% in Ethiopia [12] and 1.6% in Ghana [20] al- though the studies have used different approaches and duration of follow up. This indicates that long term follow-up of contacts of index cases is a poten- tial intervention that needs consideration for imple- mentation in routine program settings, especially in settings where community-based house-to-house visits are done as part of a routine program as it is the case in Ethiopia. Result Of the 420 index cases identified from TB DOTS regis- ters from November 2010 to April 2013 for whom initial contact investigation was conducted, 44 (10%) couldn’t be located while 25 (6%) had incomplete address and were excluded. Thus, households of 351 index cases were revisited in 2015. Seven of these were excluded be- cause there were no eligible HHCs (n = 4) or there was a second index case in the household (n = 3). From the remaining 344 index cases, 1543 HHCs were enumer- ated for follow-up but only 1517 were analyzed as 19 did not fulfill the definition of a HHC, five changed resi- dence after follow-up started while two died (See Fig. 1). Of the 344 index cases, majority were adults (96.5%), 273 (79.4%) were married and 217 (63.1%) illiterate. In 215 (62.5%) of households, there were five or more people living together. Median age (inter-quartile range) of index and HHCs were 35 (26–45) and 18 (12–30) years, respectively. Men constituted slightly higher pro- portion of both index cases and HHCs (~ 57% for both). HHCs were followed-up for a median of 37 months (in- ter-quartile range: 30–47 months) (Table 1). In our study, majority of TB cases among HHCs were diagnosed during the first few months after diagnosis of the index cases and then gradually declining with time. A similar trend was reported elsewhere [12, 19, 21]. In Ethiopia, there is huge potential to conduct CI and other targeted prevention interventions using the health extension worker program and health develop- ment armies [22]. Contact investigations should be in- tensified through active case finding and integrated with provision of preventive therapy to eligible con- tacts coupled with provision of education and social protection to maximize access to TB services. Sustain- ing household level interventions depends on cost- effectiveness. One model that was reported to be cost-effective in Vietnam for case finding was a q g 77 (5.1%) HHCs developed TB during the 4713 person-years (PYs) of follow-up with an estimated inci- dence of 1634 (95% CI: 1370-2043) per 100,000 PYs. 54/ 77 (70.1%) had PTB+, 18/77 (23.4%) smear-negative TB and 5 had extra-pulmonary TB (Fig. 1). Majority of TB g 77 (5.1%) HHCs developed TB during the 4713 person-years (PYs) of follow-up with an estimated inci- dence of 1634 (95% CI: 1370-2043) per 100,000 PYs. Result 54/ 77 (70.1%) had PTB+, 18/77 (23.4%) smear-negative TB and 5 had extra-pulmonary TB (Fig. 1). Majority of TB Yassin et al. BMC Public Health (2020) 20:737 Page 4 of 9 Fig. 1 Flow chart indicating selection of study participants effectiveness with optimal yield of case finding while provision of TB preventive treatment (TPT) should be prioritized for eligible groups. system where the contacts were invited to come to health facility for screening and/or diagnosis than through house visits [23]. While this is cost-effective, it is likely to result in less number of household con- tacts visiting health facilities and getting screened as reported by another study in Ethiopia [11]. In the Vietnam study, only 2.6 contacts were screened per index case compared to 4.4 and 5.8 contacts screened in our study and the study by Gashu et al. [12] which were done through household visits. Therefore, it is important to strike the right balance between cost- effectiveness and yield of contact investigation ap- proaches. The intensive household level follow-up could be cost effective if it is tailored to those at greatest risk of developing active TB and done through a community-based follow-up approaches [24]. Further studies are warranted to find out if facility-based and community-based contact investiga- tion approaches could be combined to maximize cost- The period shortly after diagnosis of index cases and adult age were the only independent risk factors for diagnosis of TB among HHCs. The latter could be due to the difficulty of diagnosing TB among children rather than low disease incidence in this age group, as espe- cially for under five children where obtaining appropri- ate specimen including through gastric lavage was not feasible in our setting where screening was conducted by HEWs [25–27]. As in our study, family size was not a determinant in another study [18]; rather it was over- crowding or close relationship that put contacts at greater risk of developing the disease [28]. Diagnostic challenges are not only for children, but also for adults. Since contact investigation is an active case finding approach, this is an opportunity for early case finding [29, 30]. While smear microscopy could be Page 5 of 9 Yassin et al. Result BMC Public Health (2020) 20:737 Table 1 Socio-demographic characteristics of index TB cases treated for TB between Nov 2010-Apr 2013) and relation to Incident TB among household contacts (treated for TB between Nov 2010-Apr 2015) Index case or household characteristic Response # Index TB cases # of TB cases among HHCsa Person years for HHCs (PY) TB incidence for HHCs per 100,000 PY (95% CI) Adjusted hazard ratiob (95% CI) for HHCs P valueb Age Children < 15 12 (3.49%) 4 147 2718 (1020–7242) 1 0.288 Adults ≥15 332 (96.51%) 73 4566 1599 (1271–2011) 0.55 (0.18–1.65) Gender Female 149 (43.31%) 34 1896 1793 (1281–2510) 1 0.619 Male 195 (56.69%) 43 2817 1527 (1132–2058) 0.89 (0.55–1.43) Marital status Single 71 (20.64%) 21 965 2177 (1420–3339) 1 0.207 Married 273 (79.36%) 56 3749 1494 (1150–1941) 0.71 (0.41–1.21) Education Illiterate 217 (63.08%) 42 3078 1365 (1009–1847) 1 0.180 Elementary 94 (27.33%) 27 1189 2270 (1557–3311) 1.61 (0.97–2.69) High school or above 33 (9.59%) 8 446 1793 (897–3585) 1.32 (0.6–2.94) Occupation Housewife 99 (28.78%) 24 1211 1982 (1328–2957) 1 0.691 Farmer 163 (47.38%) 34 2478 1372 (980–1920) 0.72 (0.42–1.25) Daily laborer 55 (15.99%) 1 87 1152 (162–8178) 1.06 (0.53–2.88) Student 6 (1.74%) 15 715 2099 (1266–3482) 0.59 (0.08–4.66) Others 21 (6.1%) 3 222 1348 (435–4181) 0.72 (0.21–2.49) Religion Christian 307 (89.24%) 72 4185 1721 (1366–2168) 1 0.591 Muslim 33 (9.59%) 5 467 1072 (446–2575) 0.61 (0.24–1.57) Others 4 (1.16%) 0 62 0 – Family size < 5 129 (37.5%) 21 952 2207 (1439–3384) 1 0.262 5–8 190 (55.23%) 48 3064 1567 (1181–2079) 0.71 (0.41–1.21) > 8 25 (7.27%) 8 698 1146 (573–2292) 0.52 (0.22–1.24) Total 344 (100%) 77 4713 1634 (1307–2043) aHHC H h ld t t Yassin et al. Result BMC Public Health (2020) 20:737 Page 6 of 9 Table 2 Determinants of incident tuberculosis among household contacts of index TB cases (Nov 2010-Apr 2015) in Boricha District, Sidama zone Characteristic Response # TB cases among HHCsa Person years TB incidence per 100,000 (95% CI) Hazard ratiob (95% CI), adjusted P valueb HHC Age Children < 15 9 1638 549 (286–1056) 1 < 0.001 Adults ≥15 68 3075 2211 (1743–2804) 4.03 (2.00–8.12) HHC Gender Female 34 2124 1601 (1144–2241) 1 Male 43 2589 1661 (1232–2239) 0.99 (0.63–1.57) 0.971 Time to Diagnosis 0–12 41 1496 2740 (2018–3722) 1 0.002 13–24 12 1469 817 (464–1439) 0.30 (0.16–0.57) 25–36 12 1151 1043 (592–1837) 0.38 (0.20–0.72) 36+ 12 597 2007 (1140–3534) 0.68 (0.36–1.32) aHHCs Household contacts bAdjusted to clustering within household Table 2 Determinants of incident tuberculosis among household contacts of index TB cases (Nov 2010-Apr 2015) in Boricha District, Sidama zone Characteristic Response # TB cases among a Person years TB incidence per 100,000 Hazard ratiob (95% CI), P valueb vulnerability of HHCs [31–33]. Some indexes could not be located because of poor documentation of address and this should be taken into consideration not only when contact investigation is considered but when index cases are registered in TB DOTS registers. Reporting of episodes by HHCs and recall bias, quality of screening by HEWs, and more importantly, difficulties in categor- izing cases diagnosed among HHCs as prevalent or new cases, and whether the source of infection is the index case or not. There was also no comparison group to as- sess if long term follow-up of HHCs resulted in identifi- cation of new cases that may not have been detected through passive case finding. As the screening and diag- nostic tests used in this study were less sensitive and available in peripheral centers and could detect the most infectious form of TB, additional and more sensitive screening and diagnostic tests such as chest x-ray and GeneXpert are required to enable early diagnosis among high risk people such as contacts with expanded criteria for ‘presumptive TB’ than symptom-based screening. y g The strength of this study comes from the fact that this was an active case finding activity that was con- ducted among high risk groups (HHCs). Authors’ contributions MuAY originated the research idea, collected and analyzed the data and wrote the manuscript; KDY, LEC, DGD, & MoAY contributed to data analysis and writing the manuscript; All authors read and approved the final manuscript. The study was conducted in a high TB burden setting where at the time of the study TB case notification of as high as 210 per 100,000 people was reported due to im- plementation of innovative community-based TB care through engagement of HEWs [14]. Since contact inves- tigation is dependent on number of index cases followed, case finding may be lower in settings where there is low index case identification. Hence, the contribution of contact investigation to case finding in routine setting where TB case notification is as low as 140 per 100,000 population could be smaller [34]. Received: 7 October 2019 Accepted: 10 May 2020 Received: 7 October 2019 Accepted: 10 May 2020 Funding h h g The authors received no funding to conduct this study. References 1. World Health Organisation. Global tuberculosis report. 2017. 1. World Health Organisation. Global tuberculosis report. 2017. 2. Organization WH. Recommendations for investigating contacts of persons with infectious tuberculosis in low- and middle-income countries. ISBN; 2012. Result As our study is based on retrospective data, it has a number of limita- tions including completeness and quality of data on monthly follow-up and certain important determinants like IPT use, HIV status, malnutrition, overcrowding, and ventilation which are likely to contribute to Fig. 2 Incident Tuberculosis among Household Contacts of Index Cases Diagnosed between November 2010–April 2013 Fig. 2 Incident Tuberculosis among Household Contacts of Index Cases Diagnosed between November 2010–April 2013 ident Tuberculosis among Household Contacts of Index Cases Diagnosed between November 2010–April 2013 Yassin et al. BMC Public Health (2020) 20:737 Page 7 of 9 Fig. 3 Incident Tuberculosis among Household Contacts to Index Cases Diagnosed between November 2010 and December 2011. Tuberculosis incidence was high in the first year and decreased during year two and beyond. In contrast to Fig. 2, tuberculosis incidence after year two continued to be low and the reason is that this analysis included higher proportion of household contacts that were followed for three or more years. But, note that even if TB incidence was lower for years two to four, it remained higher than that for the general population Fig. 3 Incident Tuberculosis among Household Contacts to Index Cases Diagnosed between November 2010 and December 2011. Tuberculosis incidence was high in the first year and decreased during year two and beyond. In contrast to Fig. 2, tuberculosis incidence after year two continued to be low and the reason is that this analysis included higher proportion of household contacts that were followed for three or more years. But, note that even if TB incidence was lower for years two to four, it remained higher than that for the general population Fig. 4 Occurrences of Index and Incident TB cases among Household Contacts across Calendar Time in Boricha District, Sidama Zone, November 2010 – April 2015 Yassin et al. BMC Public Health (2020) 20:737 Page 8 of 9 Page 8 of 9 Page 8 of 9 Page 8 of 9 specific (symptom-based, clinical and smear micros- copy), TB incidence is likely to be a little underestimated since GeneXpert or culture tests were not used for diagnosis. Conclusion 3. Fok A, Numata Y, Schulzer M, FitzGerald MJ. Risk factors for clustering of tuberculosis cases: a systematic review of population-based molecular epidemiology studies. Int J Tuberc Lung Dis. 2008;12(5):480–92. HHCs of index TB cases remain at a higher risk of de- veloping TB disease even after years of diagnosis of index cases. Regular follow up of HHCs is an opportun- ity to identify undiagnosed TB cases and should be con- sidered as part of TB case finding strategy wherever this is feasible. To accelerate the effort to end TB, any con- tact investigation approach should also ensure provision of TPT for eligible HHCs. y 4. Kenyon TA, Valway SE, Ihle WW, Onorato IM, Castro KG. Transmission of multidrug-resistant mycobacterium tuberculosis during a long airplane flight. N Engl J Med. 1996;334(15):933–8. 5. Yim JJ, Selvaraj P. Genetic susceptibility in tuberculosis. Respirology. 2010; 15(2):241–56. https://doi.org/10.1111/j.1440–1843.2009.01690.x. 6. Kenyon TA, Creek T, Laserson K, Makhoa M, Chimidza N, Mwasekaga M, Tappero J, Lockman S, Moeti T, Binkin N. Risk factors for transmission of mycobacterium tuberculosis from HIV-infected tuberculosis patients, Botswana. Int J Tuberc Lung Dis. 2002;6(10):843–50. 7. Triasih R, Rutherford M, Lestari T, Utarini A, Robertson CF, Graham SM. Contact investigation of children exposed to tuberculosis in South East Asia: a systematic review. J Trop Med. 2012;2012:301808. https://doi.org/10.1155/ 2012/301808 Epub 302011 Nov 301815. Availability of data and materials The dataset supporting the conclusions of this article is included within the article and its additional file (Additional file 1). Ethics approval and consent to participate The study was approved by the Institutional Review Board of Medicine and Health Science College of Hawassa University. Written informed consent was obtained from index cases or parents or head of the household for children and adolescents < 18 years for information gathering during household visit. Despite all the limitations, we think that the findings could be relevant for NTPs to consider as part of their strategy/approach to find the missing people with TB depending on country contexts and availability of re- sources while further studies on cost effectiveness of the strategy in different programmatic and routine settings would be needed. It is also important to note that most new TB cases identified were among the general popula- tion and not among household contacts. This means in- terventions to reduce transmission at community level including early diagnosis, treatment and prevention are far more important to end TB by 2030 in line with the targets in the Sustainable Development Goals [35]. Consent for publication Not applicable. Consent for publication Not applicable. Not applicable. Author details 1 1TB REACH Project, Awassa, Ethiopia. 2FHI360, Addis Ababa, Ethiopia. 3Liverpool School of Tropical Medicine (LSTM), Liverpool, UK. 4The Global Fund, Geneva, Switzerland. 1TB REACH Project, Awassa, Ethiopia. 2FHI360, Addis Ababa, Ethiopia. 3Liverpool School of Tropical Medicine (LSTM), Liverpool, UK. 4The Global Fund, Geneva, Switzerland. Additional file 1. 10. Fox GJ, Barry SE, Britton WJ, Marks GB. Contact investigation for tuberculosis: a systematic review and meta-analysis. Eur Respir J. 2013;41(1):140–56. https://doi.org/10.1183/09031936.00070812 Epub 09032012 Aug 09031930. Competing interests Competing interests The authors declare that they have no competing interests. Competing interests The authors declare that they have no competing interests. p g The authors declare that they have no competing interests. Supplementary information Supplementary information accompanies this paper at https://doi.org/10. 1186/s12889-020-08879-z. 8. Fox GJ, et al. Contact investigation in households of patients with tuberculosis in Hanoi, Vietnam: a prospective cohort study. PLoS One. 2012;7(11):e49880. Abbreviations CI: Contact investigation; DOTS: Directly observed short course treatment; EPTB: Extra-pulmonary tuberculosis; HEWs: Health extension workers; HH: Household; HHCs: Household contacts; IPT: Isoniazid preventive therapy; PTB: Pulmonary TB; PTB+: Smear-positive pulmonary tuberculosis; PYO: Person-years observed; TB: Tuberculosis; WHO: World Health Organization 11. Jerene D, Melese M, Kassie Y, Alem G, Daba S, Hiruye N, Girma B, Suarez P. The yield of a tuberculosis household contact investigation in two regions of Ethiopia. Int J Tuberc Lung Dis. 2015;19(8):898–903. 11. Jerene D, Melese M, Kassie Y, Alem G, Daba S, Hiruye N, Girma B, Suarez P. The yield of a tuberculosis household contact investigation in two regions of Ethiopia. Int J Tuberc Lung Dis. 2015;19(8):898–903. 12. Gashu Z, Jerene D, Ensermu M, Habte D, Melese M, Hiruy N, Shibeshi E, Hamusse SD, Nigussie G, Girma B. The yield of community-based “retrospective” tuberculosis contact investigation in a high burden setting in Ethiopia. PLoS One. 2016;11(8):e0160514. Acknowledgements Not applicable. Acknowledgements Not applicable. 13. Southern Nations Nationalities, & Peoples Regional State Health Bureau. Health sector annual performance review report, Hawassa. 2015. Page 9 of 9 Yassin et al. BMC Public Health (2020) 20:737 Yassin et al. BMC Public Health (2020) 20:737 14. Yassin MA, Datiko DG, Tulloch O, Markos P, Aschalew M, Shargie EB, Dangisso MH, Komatsu R, Sahu S, Blok L. Innovative community-based h d bl d t b l i tifi ti d i t t . Yassin MA, Datiko DG, Tulloch O, Markos P, Aschalew M, Shargie EB 14. Yassin MA, Datiko DG, Tulloch O, Markos P, Aschalew M, Shargie EB, Dangisso MH, Komatsu R, Sahu S, Blok L. Innovative community-based approaches doubled tuberculosis case notification and improve treatment outcome in southern Ethiopia. PLoS One. 2013;8(5):e63174. outcome in southern Ethiopia. PLoS One. 2013;8(5):e63174. 15. Federal Ministry of Health. Guidelines for clinical and programmatic management of TB, Leprosy and TB/HIV in Ethiopia. Addis Ababa, Ethiopia. 2012. 15. Federal Ministry of Health. Guidelines for clinical and program 16. Federal Ministry of Health. Guidelines on programtic managment of drug resistant tuberculosis in Ethiopia. 2013. 17. World Health Organisation. Global tuberculosis report, 2015. 2015. 18. Little KM, Msandiwa R, Martinson N, Golub J, Chaisson R, Dowdy D. Yield of household contact tracing for tuberculosis in rural South Africa. BMC Infect Dis. 2018;18(1):299. 19. Fox GJ, Nhung NV, Sy DN, Hoa NL, Anh LT, Anh NT, Hoa NB, Dung NH, Buu TN, Loi NT. Household-contact investigation for detection of tuberculosis in Vietnam. N Engl J Med. 2018;378(3):221–9. 20. 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https://openalex.org/W2135805898	https://boris.unibe.ch/74785/1/Beysard_Krebs.pdf	English	null	Tracing reinforcement through asymmetrical partner preference in the European common vole Microtus arvalis	BMC evolutionary biology	2,015	cc-by	7,465	© 2015 Beysard et al. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. RESEARCH ARTICLE Open Access Beysard et al. BMC Evolutionary Biology (2015) 15:170 DOI 10.1186/s12862-015-0455-5 Beysard et al. BMC Evolutionary Biology (2015) 15:170 DOI 10.1186/s12862-015-0455-5 Abstract Background: The mechanistic basis of speciation and in particular the contribution of behaviour to the completion of the speciation process is often contentious. Contact zones between related taxa provide a situation where selection against hybridization might reinforce separation by behavioural mechanisms, which could ultimately fully isolate the taxa. One of the most abundant European mammals, the common vole Microtus arvalis, forms multiple natural hybrid zones where rapidly diverging evolutionary lineages meet in secondary contact. Very narrow zones of hybridization spanning only a few kilometres and sex-specific gene flow patterns indicate reduced fitness of natural hybrids and incipient speciation between some of the evolutionary lineages. In this study, we examined the contribution of behavioural mechanisms to the speciation process in these rodents by fine-mapping allopatric and parapatric populations in the hybrid zone between the Western and Central lineages and experimental testing of the partner preferences of wild, pure-bred and hybrid female common voles. Results: Genetic analysis based on microsatellite markers revealed the presence of multiple parapatric and largely non-admixed populations at distances of about 10 km at the edge of the area of natural hybridization between the Western and Central lineages. Wild females from Western parapatric populations and lab-born F1 hybrids preferred males from the Western lineage whereas wild females of Central parapatric origin showed no measurable preference. Furthermore, wild and lab-born females from allopatric populations of the Western or Central lineages showed no detectable preference for males from either lineage. Conclusions: The detected partner preferences are consistent with asymmetrical reinforcement of pre-mating reproductive isolation mechanisms in the European common vole and with earlier results suggesting that hybridization is more detrimental to the Western lineage. As a consequence, these differences in behaviour might contribute to a further geographical stabilization of this moving hybrid zone. Such behavioural processes could also provide a mechanistic perspective for frequently-detected asymmetrical introgression patterns in the largely allopatrically diversifying Microtus genus and other rapidly speciating rodents. source: https://doi.org/10.7892/boris.74785 * Correspondence: gerald.heckel@iee.unibe.ch 1Computational and Molecular Population Genetics (CMPG), Institute of Ecology and Evolution, University of Bern, Baltzerstrasse 6, CH 3012 Bern, Switzerland 2Swiss Institute of Bioinformatics, Genopode, CH 1015, Lausanne, Switzerland Full list of author information is available at the end of the article Tracing reinforcement through asymmetrical partner preference in the European common vole Microtus arvalis Mathias Beysard1,2, Rebecca Krebs-Wheaton1,3 and Gerald Heckel1,2* Background thus promote speciation. It has been argued, though controversial, that pre-zygotic isolation could be rein- forced when two taxa have returned to parapatry and experienced hybridization [1–6]. If the outcome of cross- fertilization between incipient species results in unfit hybrids, natural selection could favour the reinforcement of pre-zygotic isolation to avoid costly maladaptive hybridization [2, 3, 7, 8]. The progressive reinforcement of pre-zygotic isolation mechanisms could ultimately achieve speciation, thus fully isolating the two taxa [2, 8]. This holds true if the cost of hybridization is symmetrical, with sufficient selection pressure acting on both taxa involved. The mechanisms by which speciation progresses and is completed remain elusive for most organisms. Separation in allopatric ranges provides a plausible condition where selection and/or genetic drift may lead to differences in morphological, physiological or behavioural phenotypes which increase reproductive isolation between taxa and source: https://doi. Page 2 of 8 Beysard et al. BMC Evolutionary Biology (2015) 15:170 In the presence of asymmetrical maladaptive hybridization ([9 and references therein, 10, 11]), the reinforcement process is expected to evolve in an analogous asymmet- rical manner. Yukilevich [12] has recently shown in a meta-analysis of species pairs of Drosophila that the direc- tion and the strength of hybrid dysfunction are indicative of the direction of reinforcement, suggesting that asym- metrical post-zygotic isolation could lead to a concordant increase of pre-zygotic isolation. In the presence of asymmetrical maladaptive hybridization ([9 and references therein, 10, 11]), the reinforcement process is expected to evolve in an analogous asymmet- rical manner. Yukilevich [12] has recently shown in a meta-analysis of species pairs of Drosophila that the direc- tion and the strength of hybrid dysfunction are indicative of the direction of reinforcement, suggesting that asym- metrical post-zygotic isolation could lead to a concordant increase of pre-zygotic isolation. is possible to assess if pre-mating isolation evolved potentially in reaction to maladaptive hybridization or if it existed before the secondary contact. We focus here on female rather than male partner preference because females are usually considered to be the sex with higher physiological costs of reproduction in mammals and should thus be choosier in mate choice [29, 30], and sex-specific gene flow patterns in the Western-Central hybrid zone of the common vole suggest selection in particular against males [19, 25]. The rodent genus Microtus has probably experienced the fastest radiation in extant mammals in the last 1.5 million years [13]. Background Strong genetic differentiation within many nominal species suggests ongoing speciation pro- cesses with different levels of reproductive isolation or the presence of cryptic species [14–16]. In the common vole (Microtus arvalis), four main parapatric evolution- ary lineages (Western, Central, Italian and Eastern) defined by mtDNA, Y-chromosomal and autosomal DNA are spread across Central Europe with additional lineages (Balkans, M. obscurus) in the east [17–23]. The origin of the divergence between these evolutionary line- ages probably dates back to the Last Glacial Maximum [24]. The recolonization of Europe from their allopatric glacial refugia has led to multiple secondary contacts where hybrid zones have formed which differ in the age of divergence between the evolutionary lineages involved and the level of gene flow between them [18–20, 25]. Investigations of the sex-specific genetic structure of these hybrid zones have shed light on ongoing speciation processes between the M. arvalis lineages, with indirect evidence for partial reproductive isolation between the Central, Western and Italian lineages [20, 25]. The absence of Y-chromosome introgression between the Western and Central lineages relative to autosomal markers despite male-biased dispersal in the species [26–28] and the very narrow area of hybridization (a few kilometres) support a lack of fitness at least for some male hybrids in their natural environment [25]. We have suggested that hybridization should be more detrimental to the Western lineage, as this lineage lost ground to the Central lineage since the initial secondary contact but it remains unknown if potential asymmetrically-acting pre-mating mechanisms are involved in the dynamics of the hybrid zone [25]. In this study, we assess directly the preferences of female common voles from populations across the zone of asymmetrical hybridization for males from the Western or Central evolutionary lineages. Nuclear genetic markers were used to determine the extent of the area of hybridization and to localise parapatric, non-admixed vole populations. To detect potential reinforcement of partner preferences in the area of hybridization, we tested then experimentally the partner preferences of females from these parapatric populations for males from the two evolutionary lineages, and compared this to the prefer- ences of females from allopatric populations outside of the area of recent and past hybridization. The partner preferences of the wild individuals were further com- pared to those of lab-born females derived from allopatric populations in the non-admixed ranges of the Western and Central lineages and those of first gener- ation hybrids. Parapatric and allopatric populations in the hybrid zone area The lakes and stream in the valley are represented in blue, while the crests of the valley are the dotted lines Fig. 1 Genetic background and localisation of the parapatric Microtus arvalis populations (crosses) analysed in the hybrid zone between the evolutionary lineages Western and Central. The probability of membership for genetic clusters obtained with Geneland is given in light yellow (>0.9) for the Western and in red (<0.1) for the Central lineage. The distance between the two experimental populations is 8.4 km. Black dots represent other populations included in the admixture analyses but not in tests of partner preferences. The allopatric populations for partner preference tests were localised farther to the west and east of the area shown. The lakes and stream in the valley are represented in blue, while the crests of the valley are the dotted lines Our analyses showed that partner preference of female common voles varied according to their origin across the zone of admixture in nature (Fig. 3). Time spent with the males was quantified with the partner prefer- ence index (PPind) where 1 would indicate a complete preference for a Western male, −1 a complete preference for a Central male and 0 would indicate an absence of preference (see Material and Methods). The Western females of parapatric origin showed a strong preference for Western males (mean PPind = 0.33; p =0.02, N = 17). Western females from allopatric populations chose a male regardless of their lineage of origin (wild: mean PPind = 0.12; p = 0.35, N = 17, lab-born: mean PPind = −0.11; p = 0.39, N = 15). Partner preferences of the wild Western females from parapatric populations were significantly stronger than those from allopatric populations (Mann– Whitney test, p = 0.04). No significant preference for West- ern or Central males was detected in the Central females from wild parapatric (mean PPind = 0.06; p = 0.89, N =14) or allopatric populations (mean PPind = 0.07; p = 0.63, N = 15) or in the lab-born Central females of allopatric origin (mean PPind = −0.04; p = 0.59, N = 24). However, a strong preference for Western males was also detected in the F1 hybrid females (mean PPind = 0.3; p =0.002, N = 20). Parapatric and allopatric populations in the hybrid zone area Dedicated sampling provided a refined characterization of the zone of admixture between the Western and Cen- tral evolutionary lineages in the Swiss Jura mountain range. Targeted trapping around the centre of the hybrid zone yielded 60 individuals additional to the 371 de- scribed in Beysard & Heckel [25] for which genotypes were obtained at 14 highly variable microsatellite loci. Bayesian clustering analyses of all 431 individuals showed the presence of the two pure evolutionary lineages at lo- calities of less than ten kilometres distance on either side of the crest of a valley (Figure 1). Based on the genetic landscape map and admixture proportions smaller than 0.1 at the localities, we then selected populations (distance: 8.4 km) on either side of the nuclear admixture area for the partner preference tests of females of parapatric origin. We obtained 17 females from the Western parapatric population and 14 females from the Central parapatric population for preference tests. These were complemented by individuals from allopatric populations (17 and 15 females, respectively) farther outside of the area of hybridization (distance: 64.2 km). The specific structure and dynamics of the hybrid zone between the Western and Central lineages of the com- mon vole offer the opportunity to study the role and evolution of pre-mating mechanisms across a secondary contact. If hybridization has resulted in the establish- ment of specific asymmetrical partner preferences, these could have arisen relatively soon after admixture [5] and should be detectable in populations close to the centre of hybridization. By comparing the partner preference of females from parapatric populations in the hybrid zone to the preferences of females from allopatric regions, it Beysard et al. BMC Evolutionary Biology (2015) 15:170 Page 3 of 8 Fig. 1 Genetic background and localisation of the parapatric Microtus arvalis populations (crosses) analysed in the hybrid zone between the evolutionary lineages Western and Central. The probability of membership for genetic clusters obtained with Geneland is given in light yellow (>0.9) for the Western and in red (<0.1) for the Central lineage. The distance between the two experimental populations is 8.4 km. Black dots represent other populations included in the admixture analyses but not in tests of partner preferences. The allopatric populations for partner preference tests were localised farther to the west and east of the area shown. Partner preference tests Females spent their time in the preference test mostly with unfamiliar and unrelated males from the Western or Central lineages rather than alone in the central com- partment of the apparatus (Fig. 2). All females visited both males in the respective test already during the initial discovery time. Also thereafter, females had always the opportunity to avoid social contact and remain in the central compartment without being seen by the males, but the proportion of time spent in the central compartment was low (13.8 % on average, sd = 0.22). The experimental situation appeared not to prevent nor- mal social behaviour of males, since they showed typical signs of interest in the visiting females by sniffing, lick- ing, following them around, huddling and sometimes mounting them. Some huddling periods and other social interactions were followed by repeated mating in the test apparatus. Fig. 2 Experimental setup for the evaluation of female partner preference in common voles (view from above). A plastic box was divided into three chambers by opaque plastic rectangles with a gap in the middle which allows the female to visit the stimulus males. The central chamber is inaccessible to the leashed males, providing therefore a safe zone for the female. The setup was recorded by a video camera 1 m above the central chamber Fig. 2 Experimental setup for the evaluation of female partner preference in common voles (view from above). A plastic box was divided into three chambers by opaque plastic rectangles with a gap in the middle which allows the female to visit the stimulus males. The central chamber is inaccessible to the leashed males, providing therefore a safe zone for the female. The setup was recorded by a video camera 1 m above the central chamber Parapatric and allopatric populations in the hybrid zone area Ten of these females were mater- nally of Central and ten of Western origin but only a single individual showed a preference (PPind = −0.56) for a Central male. Discussion Our analyses show a stronger partner preference of female common voles from the Western edge of the sec- ondary contact zone compared to females of allopatric origin. This is consistent with a signature of asymmetrical reinforcement of partner preference, suggesting that Beysard et al. BMC Evolutionary Biology (2015) 15:170 Page 4 of 8 Fig. 3 Partner preference index (PPind) of female M. arvalis of allopatric, parapatric or hybrid origin for males from the Western or Central lineages. A PPind of 1 would indicate a complete preference for Western males, −1 a complete preference for Central males and 0 the absence of a preference. Each female category is represented by a boxplot, the bold line being the median and the grey circle being the mean. A star above a boxplot marks a significant deviation from zero Fig. 3 Partner preference index (PPind) of female M. arvalis of allopatric, parapatric or hybrid origin for males from the Western or Central lineages. A PPind of 1 would indicate a complete preference for Western males, −1 a complete preference for Central males and 0 the absence of a preference. Each female category is represented by a boxplot, the bold line being the median and the grey circle being the mean. A star above a boxplot marks a significant deviation from zero It has been argued that reinforcement requires some gene flow, but that an excess of it could erode its effects ([5, 31] and references therein). In the present situation, it is possible that the asymmetrical dynamics of gene flow detected with autosomal, Y-chromosomal and mtDNA markers at the contact zone [25] have provided the conditions for reinforcement in the Western popula- tion only. After initial secondary contact and local replacement of the Western lineage, the advance of the Central lineage was apparently stopped in the Vallée de Joux, resulting in a narrow area of admixture on its inner slope [25]. Thus the tested Central voles from the edge of the admixture area on the outer slopes of the valley might not have enough contact at present with Western individuals to induce or maintain reinforcement. Hybridization during the initial contact of the M. arvalis lineages in the area might have potentially led also to a reinforcement of the partner preference of Central females. Discussion However, incoming migrants from the non- reinforced Central populations would lead to a dilution of the effects of reinforcement until disappearance [5]. premating isolation mechanisms did not evolve in allop- atry but after hybridization in secondary contact. The presence of a similar pattern among first generation hy- brid females supports a potential role of the hybridization process in the evolution of partner preference in the com- mon vole. Conclusions h d d The detected partner preferences in female common voles are consistent with asymmetrical reinforcement of pre-mating reproductive isolation mechanisms. As a consequence, these differences in behaviour might con- tribute to a reduction in gene flow between the evolu- tionary lineages and a further geographical stabilization of this particular moving hybrid zone (see also [25]). However, if specific partner preferences may evolve rapidly after secondary contact and hybridization be- tween allopatric rodent lineages, similar processes could also provide a mechanistic perspective for the asym- metrical introgression patterns that have been detected in multiple other Microtus taxa (e.g. [14–16] and refer- ences therein). The contribution of such processes to the extremely rapid rates of speciation in Microtus and many other groups of rodents [52] thus deserves fur- ther exploration. Signature of reinforcement of pre-mating mechanisms Signature of reinforcement of pre-mating mechanisms Previous investigations of the secondary contact zone between the evolutionary lineages Western and Central in M. arvalis revealed a very narrow area of current hybridization, which coupled with an absence of Y- chromosome introgression compared to other parts of the genome, suggests a lack of fitness for some hybrid males [25]. The extended data set analysed here con- firmed the localisation of the narrow area of contact. The current position of the hybrid zone is the result of the replacement of the Western lineage by the Central lineage, likely due to an asymmetric deficit of fitness afflicting the Western lineage [25]. If post-zygotic isola- tion affects one of the taxa more, this taxon is expected to exhibit stronger reinforcement [12]. Thus, the detec- tion of a stronger preference for Western males in the Western parapatric population is consistent with an evolutionary response to asymmetrical maladaptive hybridization and may represent an asymmetrical sig- nature of reinforcement of pre-zygotic isolation. Comparable studies on other rodents are very rare des- pite their enormous number of species and rate of evolu- tionary diversification. A case of apparently similar asymmetrical reinforcement was detected in the European house mouse hybrid zone, where maladaptive hybridization between subspecies is present but varies strongly according to geography [32–34]. Evidence for reinforcement in the Beysard et al. BMC Evolutionary Biology (2015) 15:170 Beysard et al. BMC Evolutionary Biology (2015) 15:170 Page 5 of 8 pulchellum [44]. On the contrary, the movement of a water strider hybrid zone is likely driven by a strong preference of female Limnoporus notabilis for male L. dissortis [45]. In the common vole, the preference of fe- male F1 hybrids for Western males is at odds with the direction of movement of the contact zone detected in Beysard & Heckel [25]. Overall, the absence of a detect- able preference for any lineage in the allopatric voles does not support a key role for partner preferences in the replacement of the Western by the Central lineage since this would probably require a particular partner preference also in pure populations. Nevertheless, the existence of a preference for Western males in the Western population at the edge of the contact zone might contribute to the stabilization of the hybrid zone, preventing (or slowing down) further advancement of the Central lineage. Signature of reinforcement of pre-mating mechanisms house mouse system stems also from the border of the hybrid zone, where it was shown that urinary signals permit Mus musculus musculus individuals to avoid mice from a M. m. domesticus or hybrid ori- gin [6, 35–37]. Ganem and colleagues [38] suggested that the dominant behaviour of domesticus males over musculus males may have resulted in asymmet- rical effective migration favouring asymmetrical reinforcement in the hybrid zone, and differences in explorative behaviour have been described [39]. The data presented here do not allow us to address a potential role of dominant behaviour of Central or Western males in the detected pattern of asymmetrical reinforcement, but this factor could be tested in the future. Conditions for establishing reinforcement The outcome of hybridization often shows intermediate traits compared to the parents, but hybridization has also the potential to be a creative force leading via trans- gressive segregation to evolutionary novelties like differ- ent behaviours or other new phenotypes in hybrids ([39], recently reviewed in 40). In the context of mate prefer- ences, hybrids may then favour or ignore different traits compared to the parents, which might explain the strong partner preference of the first-generation hybrids for Western M. arvalis males (Fig. 3). In the house mouse system, Christophe and Baudoin [41] found overall a sig- nificant preference of lab-born F1 hybrid females for musculus males, and wild caught hybrids may also show a preference for musculus traits [37]. On the mechanistic side, our results could be explained parsimoniously by epistasis involving at least two loci. An allele of Central origin would activate a preference for Western males by interacting with an allele of Western origin at a different locus. This would also permit the fixation of this Central allele at the Western edge of the contact zone by repeti- tive backcrosses, consistent with the observed pattern. Recent studies in the house mouse system have shown that epistatic genes could indeed be involved in repro- ductive isolation mechanisms at the post-and pre-zygotic level [42]. Given the polygynous mating system of the common vole with frequent multiple paternity [46, 47], the influ- ence of mate choice of female M. arvalis on realized reproduction should be considered. Multiple paternity might result from coercive mating or from a female’s choice of one (or several) other partners with favourable traits [48, 49]. However, successive mating with different males may lead to sperm competition, another pre- zygotic mechanism potentially involved in reproductive isolation and the dynamics of secondary contact ([50 and references therein, 51]). Asymmetries in fertilization success of sperm from males from the parental lineages or an advantage of sperm from males of pure parental lineages over hybrids (see [51]) may at least contribute to the structure and dynamics of gene flow in the hybrid zones of M. arvalis and form testable hypotheses for future analyses. Partner preference testing setup We used an experimental setup for the partner preference tests that is widely-used for assaying social and sexual behaviour in rodents (see [57] and references therein). The setup consisted of a plastic box (65 cm x 37 cm x 34 cm) divided into three chambers by opaque plastic rectangles, with a gap in the middle which allows the female to travel between the chambers (Fig. 2). Stimulus males were prevented from entering the central compart- ment by a collar around the neck which was attached to their respective compartments at the short sides of the box with steel fishing wire (Flexonit 0.45 mm diameter, Cebra Plochingen, Germany). The central compartment was accessible only to the females and provided them a non-choice/safe zone. The voles were provided with water in sipper tubes and food pellets in all three compartments. The floor was covered by a layer of wood chips. Boxes were cleaned after each experiment and washed with ethanol (70 %). The impact of partner preference on the dynamics of the hybrid zone The movement of the Western-Central hybrid zone in M. arvalis was hypothesized to have been driven by par- ticular partner preference in addition to asymmetrical post-zygotic isolation [25]. Two different kinds of part- ner preferences could lead to a replacement of one taxon by another: an asymmetric conspecific mate preference or a particular preference for non-conspecifics (reviewed in [43]). For example, Orchelimum nigripes katydid females prefer to mate with conspecific males which results in the replacement of the non-discriminating O. Beysard et al. BMC Evolutionary Biology (2015) 15:170 Page 6 of 8 Admixture in the common vole hybrid zone In order to precisely test voles from populations at the edge of the area of hybridization where pre-mating isola- tion would be most relevant, we extended the sampling around the centre of recent hybridization described in Beysard & Heckel [25] by genotyping 60 newly trapped individuals (added to an initial data set of 371 voles) with 14 microsatellite markers [53]. We then ran Geneland 2.0.12 [54] to obtain a detailed description of the distribution of the lineages in the geographic centre of admixture. Analogous to Beysard & Heckel [25], we assumed two genetic clusters and performed 10 runs of 1 000 000 iterations with 50 000 burn-in. After checking for consistency between the 10 runs, we displayed the run with the best likelihood on a map of probability of membership for each lineage (Fig. 1). Partner preferences of wild females were compared to lab-born females which were derived from allopatric populations in the non-admixed ranges of the Western and Central lineages. The Western voles stemmed from several populations of the Département du Jura and Département de Saone-et-Loire (France) and the Central voles from various populations in the cantons Bern and Schaffhausen (Switzerland). We tested unrelated (i.e. stemming from crosses with different parents and no known kin-relationship) virgin lab-born females after the age of 40 days. Since female common voles are preco- cious breeders and are capable of reproducing before they are weaned (14–18 days old), we were certain that the females were of reproductive age [55]. The lab born females were either pure Central (first or second gener- ation, 24 individuals), pure Western (first or second generation, 15 individuals), or F1 hybrids between pure Central and Western lineages (20 individuals). Among these F1 hybrids, ten females were offspring from crosses between Central males and Western females and the others from the reverse combination. Localization of parapatric and allopatric populations h l d h d f h l d Localization of parapatric and allopatric populations We then selected sites at the edge of the nuclear admix- ture area to trap females for the partner preference tests. These sites were the first suitable habitats outside the area of detectable nuclear admixture which showed signs for the presence of many voles (numerous burrows). We refer to these two sites as Western and Central parapa- tric populations. The Western parapatric population (17 females) was located on the inner slope of the Vallée de Joux (46°38'12"North, 6°17'53"East) whereas the Central parapatric population (14 females) was on the outer slope (46°39'13"North, 6°24'47"East). For a com- parison with allopatric females, we selected populations outside of the area of recent and past hybridization (allo- patric Western population (17 females) 46°41'11"North, 6°8'46"East; allopatric Central population (15 females) 46°35'34"North, 6°34'36"East; see [25]). Voles were trapped with Longworth small mammal traps (Penlon). The males used as stimuli for the partner preference tests were first or second generation lab-born from the same pure allopatric Central and Western stock but unfamiliar and unrelated to the experimental females and to each other (i.e. no close kin relation). Males were kept with their siblings until 30 days of age and were then housed individually to avoid the establishment of dominance behaviour, which might influence partner preference tests [56]. Materials and methods partner preference tests. During the acclimatization time, potential ecto- and endo-parasites were eliminated with Ivomec (Merial, Derendingen Switzerland) in order to avoid infection during the experiments. Experimental animals Animal experimentation for this study followed the guidelines of the Association for the Study of Animal Behaviour and trapping occurred under permits BE-08/ 10 and BE-90/10 issued by the cantonal veterinary offices after approval by the Bernese cantonal commission on animal experimentation. We were committed to reduce stress for the animals as much as possible. Voles were kept before partner preference tests in 17 x 28 x 13 cm polycar- bonate cages (Indulab, Gams Switzerland) with a thick layer of wood chips and paper tubes as structural enrich- ment. They were supplied with rodent pellets (Provimi Kliba, Kaiseraugst Switzerland), carrots and water ad libi- dum. The room was maintained on a 14:10 light:dark schedule at an ambient temperature of 21 °C. Wild-caught voles were acclimatized for at least two weeks before Beysard et al. BMC Evolutionary Biology (2015) 15:170 Page 7 of 8 Page 7 of 8 Beysard et al. BMC Evolutionary Biology (2015) 15:170 Stimulus males were placed in the apparatus three hours prior to the introduction of the experimental female in order to give them time to adjust to the situation and explore their compartment [57, 58]. Females were intro- duced to the central compartment, and behaviour was re- corded for 4.5 h using a Logitech C510 camera positioned 1 m above the centre of the experimental setup. The first 30 min were considered as discovery time to allow the females to explore the test apparatus and not scored. Vid- eos were recorded in .mov format and behaviour was manually scored using JWATCHER v1.0 [59]. vole research. This study was supported by a grant from the Swiss National Science Foundation (21003A-278 127377) to GH. vole research. 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Acknowledgements Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color ﬁgure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color ﬁgure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color ﬁgure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and take full advantage of: Submit your next manuscript to BioMed Central and take full advantage of: 45. Klingenberg CP, Spence JR, Mirth CK. Introgressive hybridization between two species of waterstriders (Hemiptera : Gerridae : Limnoporus): geographical structure and temporal change of a hybrid zone. J Evol Biol. 2000;13:756–65. 45. Klingenberg CP, Spence JR, Mirth CK. Introgressive hybridization between two species of waterstriders (Hemiptera : Gerridae : Limnoporus): geographical structure and temporal change of a hybrid zone. J Evol Biol. 2000;13:756–65. • Convenient online submission 46. Borkowska A, Ratkiewicz M. Promiscuity, male reproductive success and mate relatedness in a natural population of the common vole. J Zool. 2010;280:195–201. 46. Borkowska A, Ratkiewicz M. Promiscuity, male reproductive success and mate relatedness in a natural population of the common vole. J Zool. 2010;280:195–201. • Thorough peer review 47. Fink S, Excoffier L, Heckel G. Mammalian monogamy is not controlled by a single gene. Proc Nat Acad Sci USA. 2006;103:10956–60. 47. Fink S, Excoffier L, Heckel G. Mammalian monogamy is not controlled by a single gene. Proc Nat Acad Sci USA. 2006;103:10956–60. 48. Berteaux D, Bety J, Rengifo E, Bergeron J-M. Multiple paternity in meadow voles (Microtus pennsylvanicus): investigating the role of the female. Behav Ecol Sociobiol. 1999;45:283–91. 48. Berteaux D, Bety J, Rengifo E, Bergeron J-M. Multiple paternity in meadow voles (Microtus pennsylvanicus): investigating the role of the female. Acknowledgements Explorative strategies in two house mouse subspecies and their hybrids. Biol J Linn Soc. 2013;108:225–35. 39. Hiadlovská Z, Vošlajerová Bímová B, Mikula O, Piálek J, Macholán M. Transgressive segregation in a behavioural trait? Explorative strategies in two house mouse subspecies and their hybrids. Biol J Linn Soc. 2013;108:225–35. 40. Abbott R, Albach D, Ansell S, Arntzen JW, Baird SJE, Bierne N, et al. Hybridization and speciation. J Evol Biol. 2013;26:229–46. 40. Abbott R, Albach D, Ansell S, Arntzen JW, Baird SJE, Bierne N, et al. Hybridization and speciation. J Evol Biol. 2013;26:229–46. 41. Christophe N, Baudoin C. Olfactory preferences in two strains of wild mice, Mus musculus musculus and Mus musculus domesticus, and their hybrids. Anim Behav. 1998;56:365–9. 41. Christophe N, Baudoin C. Olfactory preferences in two strains of wild mice, Mus musculus musculus and Mus musculus domesticus, and their hybrids. Anim Behav. 1998;56:365–9. 42. Janoušek V, Wang LY, Luzynski K, Dufková P, Vyskočilová MM, Nachman MW, et al. Genome-wide architecture of reproductive isolation in a naturally occurring hybrid zone between Mus musculus musculus and M. m. domesticus. Mol Ecol. 2013;21:3032–47. 42. Janoušek V, Wang LY, Luzynski K, Dufková P, Vyskočilová MM, Nachman MW, et al. Genome-wide architecture of reproductive isolation in a naturally occurring hybrid zone between Mus musculus musculus and M. m. domesticus. Mol Ecol. 2013;21:3032–47. 43. Buggs RJA. Empirical study of hybrid zone movement. Heredity. 2007;99:301–12. 43. Buggs RJA. Empirical study of hybrid zone movement. Heredity. 2007;99:301–12. 44. Shapiro LH. Asymmetric assortative mating between two hybridizing Orchelimum katydids (Orthoptera : Tettigoniidae). Amer Midl Nat. 2001;145:423–7. 44. Shapiro LH. Asymmetric assortative mating between two hybridizing Orchelimum katydids (Orthoptera : Tettigoniidae). Amer Midl Nat. 2001;145:423–7. Acknowledgements Behav Ecol Sociobiol. 1999;45:283–91. 49. Jennions MD, Petrie M. Why do females mate multiply? A review of the genetic benefits. Biol Reviews. 2000;75:21–64. 49. Jennions MD, Petrie M. Why do females mate multiply? A review of the genetic benefits. Biol Reviews. 2000;75:21–64.
W2002196943.txt	https://zenodo.org/records/1814464/files/article.pdf	de		Ueber Arthigonbehandlung der gonorrhoischen Vulvovaginitis kleiner Mädchen	Deutsche medizinische Wochenschrift/Deutsche Medizinische Wochenschrift	1,911	public-domain	1,259	DEuTSCHE MEDIZINISCHE WOCHENSOfflIFT. No. 50 Aus der Dermatologischen Universitätsklinik in Breslau. (Direktor : Geh. Med.-Rat Prof. Dr. A. Neisser.) Deber Arthigonbehandlung der gonorrhoischen Vulvovaginitis kleiner Mädchen. Von Dr. Stephanie Rygier. Jeder Dermatologe und jeder Kinderarzt weiß, mit welchen Schwierigkeiten die lokale Behandlung der gonorrhoischen Kindervulvovaginitiden verknüpft ist. Abgesehen davon, daß die Kinder bei der Behandlung unruhig sind und man immer dabei gute Assistenz haben muß, sind auch ihre unentwickelten Geschlechtsteile so klein, eng und zart , daß man nur schwer etwas einführen oder einspritzen kann. Die zarte Schleimhaut blutet sehr leicht und ist so reizbar, daß man schon nach einigen Tagen der Behandlung gezwungen ist, die ganze Therapie auf einige Zeit auszusetzen. Die Gonokokken aber vermehren sich in den zahlreichen Falten der kindlichen, lockeren Schleimhaut um so schneller, je jünger der Organismus ist. Sie sitzen in solchen Fällen auf oder tief zwischen dem lEpithel monate-, ja sogar jahrelang und fühlen sich daselbst sicher vor jeglichem Angriff. Sie finden so ausgezeichneten Nährboden, daß sie alle anderen Bakterien überwuchern, und es ist nichts leichthr, als von einem gonorrhoischen Kinde Gonokokkenreinkulturen zu bekommen Fast in jeder medizinischen Zeitung liest man bald von den glänzenden Erfolgen eines Argentumpraparates, bald von den Erfolgen einer Salbe gegen die Vulvovaginitis der Kinder. Daraus aber ergibt sich, daß sie alle mehr oder weniger unsicher in thren Wirkungen sind. Wir schreiben und sprechen von Vulvovaginitiden viel, weil wir mit allen Mitteln, die wir gegen diese Krankheit zu unserer Verfügung haben, unzufrieden sind. Ich muß offen gestehen, daß mir fast keine lokale Behandlung ein gutes Resultat gegeben hat. Daher möchte ich glauben, daß die Autoren, die so glänzende Erfolge publizieren, ihre Patientinnen vielleicht nicht lange genug nach dem Verschwinden der Gonokokken kontrolliert haben und so ein passa- gerer Erfolg einen definitiven vorgetäuscht hat. Auch uns ist es natürlich in vielen Fällen geglückt, durch lokale Behandlung eine Vulvovaginitis gonokokkenfrei zu machen. Konnte aber aus äußeren Gründen nun, nachdem die Gonokokken verschwunden waren, die mikroskopische Kontrolle nicht längere Zeit fortgesetzt werden, so kamen die kleinen Patientinnen fast regelmäßig nach wenigen Wochen mit den vollen Symptomen und dem gleichen positiven Gonokokkenbefund wieder, wie er bei Beginn der Behandlung bestanden hatte. Eine seit wenigen Jahren erst in Aufnahme kommende Therapie ist die Serum- und Vakzinbehandlung der TJrethro- vaginitis der Kinder. DieVakzinbehandlung der Vulvovaginitis wurde zuerst im Anschluß an die Wrightschen Arbeiten in Amerika mit gutem Erfolge betrieben und wurde in Deutschland von Bruck eingeführt, der hierfür das von ihm angegebene Gonokokkenvakzin , ,Arthigon" (Chemische Fabrik vorm. E. Schering, Charlottenburg) empfahl. Allerdings finden wir bei der Therapie der kindlichen Vulvovaginitiden mit Arthigon nicht Bedingungen, um so günstige Erfolge zu erhalten, wie sie bei Epididymitis und Arthritis mit Arthigoninjektionen erzielt worden sind. Auf die Gründe will ich hier nicht näher eingehen (s. die vorstehende Arbeit von Schultz) und nur daran erinnern, daß die Gonokokkenvakzinbehandlung, deren Wirkungsmecha- nismus demjenigen des Tuberkulins ähnlich zu sein scheint, die besten Resultate bei geschlossenen gonorrhoischen Her- den gibt, während der offene Schleimhautkatarrh gar- nicht (Urethritis) oder weniger. sicher darauf reagiert, (Vulvovaginitis). Nach den Untersuchungen von Sc h mid t scheinen die Verhältnisse beim Zervixkatarrh der Erwachsenen allerdings ähnlich zu liegen wie in den ,,geschlossenen" Herden. Entsprechend dem in dieser Beziehung geringen Material unserer Klinik habe ich bisher nur vier Vulvovaginitiden kleiner Mädchen!mit Arthigon behandeln können. Wenn auch von diesen nur zwei völlig geheilt, einer wesentlich gebessert ist, Heruntergeladen von: NYU. Urheberrechtlich geschützt. 2334 14. Dezember 1911. DETJTSCKE MEDIZINISCHE WOOHENSOHRIFT. 2335 also kein gliinzender Erfolg vorhanden ist, so ist dieses Re- bei der nötigen Ausdauer zur Heilung hätten gebracht sultat im Vergleiche zu dem der sonst üblichen lokalen Ther- werden können. apie, über die ich eine recht reichliche Erfahrung besitze, so Nebenerscheinungen außer den gewünschten Terngünstig, daß ich glaube im Anschluß an die günstigen Berichte peraturanstiegen oder irgend welche Schädigungen wurden der früheren Autoren (s. bei Bruck) auch über diese wenigen nie beobachtet. Fülle berichten zu sollen: Schluß. Wenn wir also auch nicht den optimistischen StandFall 1. M. P., 7 Jahre alt. Vulvovaginitis chronica sieben Monate (!) punkt mancher amerikanischen Autoren teilen, so g]auben in zwei Krankenhäusern erfolglos behandelt. Befund bei der Aufnahme (22. April) Gonokokken ± +, Eiterkörperchen + +. Vom 22. April bis 4. August klinisch und poliklinisch sorgfältige Lokalbehandlung (Protargol, Hegonon etc.); ohne Erfolg. Lokalbehandlung ausgesetzt. 5. bis 10. August Gonokokken + +, Eiterkörperchen + +. lo. August: Erste Arthigoninjektion 0,5 cern in die Glutiien (ohne Allgemeinreaktion). 10. bis 15. August: Gonokokken + +, Eiterkörperchen + +. August: ZweiteArthigoninjektion 1,Occm. Allgemeinreaktion 37,2. bis 19. August: Gonokokken -, Eiterkörperchen spärlich, doch auch wir nach unseren Erfahrungen, daß die Gonokokkenvakzintherapie die Vulvovaginitis kleiner Mädchen spezifisch zu beeinflussen und zu heilen imstande ist, und zwar mit bedeutend größerer Sicherheit und Schnelligkeit, als dies jede andere Heilmethode zu leisten vermag. Es sollte daher in jedem Falle die völlig unschädliche Behandlung mit Gonokokkenvakzin eventuell neben den Lokaiprozeduren Anwendung finden. Epithel + +. Weitere poliklinische Beobachtung: Bei einer Untersuchung am 30. August sollen spärliche Gonokokken gefunden worden sein. Patientin hat sich dann der weiteren Beobachtung entzogen. Obgleich ich diesen Befund nicht selbst kontrollieren konnte, so nehme ich in diesem Falle ein Rezidiv an und betrachte daher den Fall durch die Arthigon. behandlung als gebessert, nicht als geheilt. Vielleicht wäre in Anbetracht der ganz deutlichen Beeinflussung des Prozesses durch die spezifische Behandlung bei genügender Fortsetzung eine definitive Heilung erzielt worden. Fall 2. A. G., 4 Jahre ait. Infektion durch den Vater Anfang Februar. Vom 13. Februar bis 31. August erfoiglose poliklinische und klinische Behandlung mit lokaler Therapie (Protargol, Hegonon, Pyozyanase, Kai. pergaman. etc.) 31. August Wiederaufnahme in die Klinik Gonokokken + +, Eiterkörperchen + +; keine Lokaitherapie. 1. September: 1. Arthigoninjektion 0,5, Reaktion 37,5. ,, 1,0, 4. September: 2. ,, 37,7. 7. bis 15. September: Ausfluß hat nachgelassen, keine Gonokokken, spärliche Eiterkörperchen. 9. September: 3. Arthigoninjektion 1,5, Reaktion 37,7. ,, 2,0, 13. September: 4. ,, 37,7. ,, 2,5, 16. September: 5. ,, 37,4. ,, 3,0, ,, 37,7. 20. September: 6. 21. September: Wieder spärlich Gonokokken, wieder Eiterkörperchen zahlreicher. 25. September: 7. Arthigoninjektion 3,5, keine Reaktion. ,, 4,0, Reaktion 37,8. 28. September: 8. 4,5, keine Reaktion. ,, 2. Oktober: 9. Vom 2. Oktober ab dauernd gonokokkenfrei, auch nach beendigter Kur. 7. Oktober: 10. Arthigoninjektion 5,0, Reaktion 37,9. Fall 3. Ch. Seh., 11 jahre alt. Akute Vulvo-vaginitis seit acht Tagen. Vom 10. August bis 1. September acht Arthigoninjektionen (0,25, 0,5, 1,0, 1,5, 2,0, 3,0, 4,0, 5,0,); keine Lokaltherapie. Bis zum 31. August, wenn auch immer spärlicher werdend, Gono- kokken nachweisbar; von da ab dauernd gonokokkenfrei (kontrolliert bis Ende Oktober). Fall II und Ill sind also sichere, durch Arthigon bedingte Heilungen. Einen Mißerfolg zeigt Fall 4. S. E., 7 Jahre alt, Stupruin vor zehn Tagen. Akute Vulvo- vaginitis. 4. bis 30. September: Sechs Arthigoninjektionen (0,5, 1,0, 1,5, 2,0, 2,5, 3,0) und Lokaltherapie. Patientin verläßt aus äußeren Gründen die Klinik; Gonokokken, wenn auch spärlich, noch vorhanden. Wir haben also unter diesen vier Fällen zwei volle }leilerfolge, einen zweifelhaften und einen Mißerfolg zu verzeichnen. Es muß jedoch bemerkt werden, daß die beiden mit Erfolg behandelten Fälle bis zur Höchstdose von 5,0 pro dosi und zu einer Gesamtdose von 27,5 bezw. 17,25 Arthi- gon gelangten, während die beiden anderen Patientinnen nur bis 3,0 pro dosi und bis zur Gesamtmenge von 8 bezw. 10,5 Arthi- gon behandeltwerden konnten. Es ist also wohl möglich, daß auch diese bisher unbefriedigt verlaufenen Fälle Heruntergeladen von: NYU. Urheberrechtlich geschützt. 19. August: Dritte Arthigoninjektion 1,5 corn. Reaktion 37,1. 20. bis 24. August: Gonokokken-, Eiterkörperchen spärlich, sehr geringer Fluor. 21. August: Vierte Arthigoninjektion 2 cern. Reaktion 37,8. August: Fünfte Arthigoninjektion 3 cern. Reaktion 37,7. August: Keine Gonokokken, Entlassung.
https://openalex.org/W4387188008	https://www.frontiersin.org/articles/10.3389/fcvm.2023.1221541/pdf?isPublishedV2=False	English	null	A physics-based machine learning technique rapidly reconstructs the wall-shear stress and pressure fields in coronary arteries	Frontiers in cardiovascular medicine	2,023	cc-by	9,463	A physics-based machine learning technique rapidly reconstructs the wall-shear stress and pressure ﬁelds in coronary arteries Benjamin Morgan 1, Amal Roy Murali 2, George Preston 1, Yidnekachew Ayele Sima 1, Luis Alberto Marcelo Chamorro 1, Christos Bourantas 3, Ryo Torii 4, Anthony Mathur 3, Andreas Baumbach 3, Marc C. Jacob 2, Sergey Karabasov 1 and Rob Krams 1* EDITED BY Monica Sigovan, CREATIS, France REVIEWED BY Xiao Yun Xu, Imperial College London, United Kingdom Simone Saitta, Polytechnic University of Milan, Italy Bruno Sixou, INSA Lyon, France CORRESPONDENCE Rob Krams r.krams@qmul.ac.uk RECEIVED 12 May 2023 ACCEPTED 11 September 2023 PUBLISHED 29 September 2023 CITATION Morgan B, Murali AR, Preston G, Sima YA, Marcelo Chamorro LA, Bourantas C, Torii R, Mathur A, Baumbach A, Jacob MC, Karabasov S and Krams R (2023) A physics-based machine learning technique rapidly reconstructs the wall-shear stress and pressure ﬁelds in coronary arteries. Front. Cardiovasc. Med. 10:1221541. d i 10 3389/f 2023 1221541 EDITED BY Monica Sigovan, CREATIS, France REVIEWED BY Xiao Yun Xu, Imperial College London, United Kingdom Simone Saitta, Polytechnic University of Milan, Italy Bruno Sixou, INSA Lyon, France CORRESPONDENCE Rob Krams r.krams@qmul.ac.uk RECEIVED 12 May 2023 ACCEPTED 11 September 2023 PUBLISHED 29 September 2023 CITATION Morgan B, Murali AR, Preston G, Sima YA, Marcelo Chamorro LA, Bourantas C, Torii R, Mathur A, Baumbach A, Jacob MC, Karabasov S and Krams R (2023) A physics-based machine learning technique rapidly reconstructs the wall-shear stress and pressure ﬁelds in coronary arteries. Front Cardiovasc Med 10:1221541 1Department of Science and Engineering, Queen Mary University of London, London, United Kingdom, 2Laboratoire de Mécanique des Fluides et d’Acoustique UMR5509, INSA Lyon, Ecole Centrale de Lyon, University of Lyon, Ecully, France, 3Bart’s Heart Centre, London, United Kingdom, 4Department of Mechanical Engineering, University College London, London, United Kingdom With the global rise of cardiovascular disease including atherosclerosis, there is a high demand for accurate diagnostic tools that can be used during a short consultation. In view of pathology, abnormal blood ﬂow patterns have beendemonstrated to be strong predictors of atherosclerotic lesion incidence, location, progression, and rupture. Prediction of patient-speciﬁc blood ﬂow patterns can hence enable fast clinical diagnosis. However, the current state of art for the technique is by employing 3D- imaging-based Computational Fluid Dynamics (CFD). The high computational cost renders these methods impractical. In this work, we present a novel method to expedite the reconstruction of 3D pressure and shear stress ﬁelds using a combination of a reduced-order CFD modelling technique together with non-linear regression tools from the Machine Learning (ML) paradigm. Speciﬁcally, we develop a proof-of-concept automated pipeline that uses randomised perturbations of an atherosclerotic pig coronary artery to produce a large dataset of unique mesh geometries with variable blood ﬂow. TYPE Original Research PUBLISHED 29 September 2023 DOI 10.3389/fcvm.2023.1221541 KEYWORDS arterial blood ﬂow, shear stress, pressure drop, reduced order modelling, machine learning Frontiers Frontiers in Cardiovascular Medicine arterial blood ﬂow, shear stress, pressure drop, reduced order modelling, machine learning Frontiers A physics-based machine learning technique rapidly reconstructs the wall-shear stress and pressure ﬁelds in coronary arteries Benjamin Morgan 1, Amal Roy Murali 2, George Preston 1, Yidnekachew Ayele Sima 1, Luis Alberto Marcelo Chamorro 1, Christos Bourantas 3, Ryo Torii 4, Anthony Mathur 3, Andreas Baumbach 3, Marc C. Jacob 2, Sergey Karabasov 1 and Rob Krams 1* A total of 1,407 geometries were generated from seven reference arteries and were used to simulate blood ﬂow using the CFD solver Abaqus. This CFD dataset was then post-processed using the mesh-domain common-base Proper Orthogonal Decomposition (cPOD) method to obtain Eigen functions and principal coefﬁcients, the latter of which is a product of the individual mesh ﬂow solutions with the POD Eigenvectors. Being a data-reduction method, the POD enables the data to be represented using only the ten most signiﬁcant modes, which captures cumulatively greater than 95% of variance of ﬂow features due to mesh variations. Next, the node coordinate data of the meshes were embedded in a two-dimensional coordinate system using the t-distributed Stochastic Neighbor Embedding (t-SNE) algorithm. The reduced dataset for t-SNE coordinates and corresponding vector of POD coefﬁcients were then used to train a Random Forest Regressor (RFR) model. The same methodology was applied to both the volumetric pressure solution and the wall shear stress. The predicted pattern of blood pressure, and shear stress in unseen arterial geometries were compared with the ground truth CFD solutions on “unseen” meshes. The new method was able to reliably reproduce the 3D coronary artery haemodynamics in less than 10 s. Front. Cardiovasc. Med. 10:1221541. doi: 10.3389/fcvm.2023.1221541 Front. Cardiovasc. Med. 10:1221541. doi: 10.3389/fcvm.2023.1221541 1. Introduction coherent structures using essential information of the ﬂow solution ﬁeld (e.g. 3D velocity and pressure) while reducing dimensionality of the data. Initial studies used both temporal and spatial information of the velocity ﬁeld to reduce its dimensions in non-health related areas (7). The ﬁrst health applications used these methods to study coherent structures in the velocity ﬁeld of idealised phantoms of bifurcations, saccular and aortic aneurysm (10). Patient-speciﬁc applications, which are noisier, have been successfully studied by accounting for such noise in the signal (11). In order to apply these reduced order ﬂow solution ﬁelds to novel objects, an interpolation needs to be carried out. Atherosclerosis is the leading cause of death in the developed world, accounting for more than 40% of total mortalities per year. While it has been accepted that risk factors like hypertension, high cholesterol and diabetes play a pivotal role in the progression of the disease, they do not explain the prediliction of atherosclerotic plaque formation near sites of arterial bifurcation, side branching and curvature (1). These predilection sites have been associated with disturbed blood ﬂow and endothelial shear stress patterns (2). Numerous experimental and clinical studies in the last few decades have posited an essential role for disturbed shear stress in initiating atherosclerosis, in progression from simple to advanced plaques, and in rupture of advanced, vulnerable plaques (2). Furthermore, disturbed shear stress patterns are also associated with in-stent restenosis and atherosclerosis (3). Despite the overwhelming number of studies demonstrating the decisive role of blood ﬂow in clinical atherosclerosis, disturbed shear stress patterns have not yet been considered whilst making clinical decisions during catheterization or surgery. This is mainly due to the high computational cost and long convergence times required for sufﬁciently accurate numerical solutions. Several propositions have been made to reduce time requirements, of which one of the earliest attempts was by applying supercomputers to the numerical solvers (4). While this reduced convergence time from a full day to a few hours, a condition now met by standard modern computers, this is still not sufﬁcient to aid in diagnostics. Clinical decisions depend on data which can be reliably obtained within minutes, preferably seconds. Hence, newer statistical modelling methods were used to further reduce convergence time of Computational Fluid Dynamics (CFD) simulations based on machine learning (5). These can roughly be divided into two categories, the classical machine learning methods and physics-based machine learning methods. 2. Outline of methodology We have developed an automatic pipeline which generates synthetic data from existing 3D reconstructed blood vessels (12), performs proper orthogonal decomposition (POD) on the shear stress and pressure ﬁeld solutions, and t-distributed Stochastic Neighbour Embedding (t-SNE) on the mesh coordinate data to enable feature reduction. The reduced mesh and ﬂow parameter ﬁelds are then used to train, validate and test a RFR model to perform interpolation; thereby enabling a fast reconstruction of CFD solution in a given geometry. In the case of an unseen geometry as test input, the position of the corresponding geometry in the t-SNE space is calculated analytically, and the mode coefﬁcients are predicted using RFR. Recombination of the previously extracted mesh-wise modes along with the newly predicted POD mode coefﬁcients is then used to produce the ﬂow ﬁeld solutions for the new geometry. The pipeline is summarised in the form of a ﬂowchart as shown in Figure 1, and the methods are described in Sections 3, 4, 5 and 6. 1. Introduction Classical machine learning methods use the power of deep learning to estimate wall shear stress proﬁles (5). The advantage of these methods is the ﬂexibility of the feature space to predict these wall shear proﬁles primarily due to the high expressivity of Deep Neural Networks (DNN) and their ability to identify high dimensional features. However, such methods are not based on capturing the inherent physical conservation laws of the governing ﬂuid ﬂow. Consequently, any change in feature space will necessitate a DNN recalibration cycle. In light of these advances in closely related ﬁelds of research, this paper establishes the foundation of our novel method amalgamating these techniques and applies it to a well- characterised experimental dataset of atherosclerotic pig coronary arteries (12). We will show how to modify classical POD, introduce a shape optimizer for blood vessels, and present a suitable Random Forest Regressor (RFR) model to predict ﬂow ﬁelds in novel arteries. COPYRIGHT © 2023 M COPYRIGHT © 2023 Morgan, Murali, Preston, Sima, Marcelo Chamorro, Bourantas, Torii, Mathur, Baumbach, Jacob, Karabasov and Krams. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. Frontiers in Cardiovascular Medicine 01 frontiersin.org Morgan et al. 10.3389/fcvm.2023.1221541 10.3389/fcvm.2023.1221541 Frontiers in Cardiovascular Medicine 3. Creating a well-annotated synthetic data repository To overcome the above, physics-based machine learning technologies have raised interest recently. These methods are predicated on capturing the underlying physics either via incorporation of the actual conservation laws (6) or by data- driven extraction of physically interpretable ﬂow characteristics (7) as features for regression. For instance, Reduced-order modelling of CFD simulations are motivated by the presence of coherent structures, identiﬁed from their statistical moments in the datasets available from short duration simulations (8, 9). By applying orthogonal decomposition theory, it is possible to identify high energy Eigenvectors, also known as modes, of these Synthetic data has been proposed to meet the huge data requirement of artiﬁcial intelligence (AI) (13). Here, we developed a hybrid technique which uses a combination of realistic and synthetic data. The realistic data was obtained from a validated 3D reconstruction method of coronary arteries based upon a pullback of OCT images and angiography (Figure 2). This 3D vessel anatomy was then used as a seed to generate synthetic data by applying random spatial perturbations to the original mesh. To prevent unnatural, discontinuous geometric differences within each mesh phantom, the perturbations are Frontiers in Cardiovascular Medicine 02 frontiersin.org Morgan et al. 10.3389/fcvm.2023.1221541 FIGURE 1 The data processing pipeline is summarized in this ﬂowchart. OCT images are obtained in the cath. lab. and used to extrapolate a 3D contour. Mesh generation and Computational Fluid Dynamics are done through an automatic pipeline. The velocity proﬁles obtained from CFD will act as the ground truth. Synthetic data generation (n = 1407) is done by random purturbation of the length-wise diameter of each independent blood vessel (n = 7). Data reduction is performed on the shear stress, and pressure ﬁelds obtained from CFD, via POD (see text for details), and on the input meshes through t-SNE (see text for details). These reduced data sets are used to train (using 90% of the data) and validate (using 10% of data) the machine learning learning module FIGURE 2 10 randomly selected phantom geometries from the dataset are visualised. All phantoms shown were generated from the same OCT image. Variation in shape is due to random synthetic perturbations applied to the artery diameter, the function of which is a composite of two sinusoids with randomised amplitude, frequency, phase and vertical displacement. This ensures smooth, continuous variation along the length of the artery regardless of input parameters. 3. Creating a well-annotated synthetic data repository Frontiers in Cardiovascular Medicine 03 frontiersin.org FIGURE 1 The data processing pipeline is summarized in this ﬂowchart. OCT images are obtained in the cath. lab. and used to extrapolate a 3D contour. Mesh generation and Computational Fluid Dynamics are done through an automatic pipeline. The velocity proﬁles obtained from CFD will act as the ground truth. Synthetic data generation (n = 1407) is done by random purturbation of the length-wise diameter of each independent blood vessel (n = 7). Data reduction is performed on the shear stress, and pressure ﬁelds obtained from CFD, via POD (see text for details), and on the input meshes through t-SNE (see text for details). These reduced data sets are used to train (using 90% of the data) and validate (using 10% of data) the machine learning learning module FIGURE 1 The data processing pipeline is summarized in this ﬂowchart. OCT images are obtained in the cath. lab. and used to extrapolate a 3D contour. Mesh generation and Computational Fluid Dynamics are done through an automatic pipeline. The velocity proﬁles obtained from CFD will act as the ground truth. Synthetic data generation (n = 1407) is done by random purturbation of the length-wise diameter of each independent blood vessel (n = 7). Data reduction is performed on the shear stress, and pressure ﬁelds obtained from CFD, via POD (see text for details), and on the input meshes through t-SNE (see text for details). These reduced data sets are used to train (using 90% of the data) and validate (using 10% of data) the machine learning learning module FIGURE 2 10 randomly selected phantom geometries from the dataset are visualised. All phantoms shown were generated from the same OCT image. Variation in shape is due to random synthetic perturbations applied to the artery diameter, the function of which is a composite of two sinusoids with randomised amplitude, frequency, phase and vertical displacement. This ensures smooth, continuous variation along the length of the artery regardless of input parameters. FIGURE 2 10 randomly selected phantom geometries from the dataset are visualised. All phantoms shown were generated from the same OCT image. Variation in shape is due to random synthetic perturbations applied to the artery diameter, the function of which is a composite of two sinusoids with randomised amplitude, frequency, phase and vertical displacement. This ensures smooth, continuous variation along the length of the artery regardless of input parameters. 3. Creating a well-annotated synthetic data repository Blood rheology was modelled as a non-Newtonian ﬂuid following the Carreau- Yasuda model, which at high strain rates incorporates the effect of shear thinning in the deﬁnition of kinematic viscosity as: n ¼ n1 þ (n0 n1)(1 þ (t_g)a)(n1)=a, where _g ¼ @u=@y is the ﬂow shear gradient near the wall, and the model coefﬁcients are summarised in Table 1. For turbulence modelling, the standard k– 1 RANS (Reynolds Averaged Navier Stokes) model was used. All calculations were performed using APOCRITA, the HPC cluster of Queen Mary University of London (14). 3. Creating a well-annotated synthetic data repository FIGURE 2 10 randomly selected phantom geometries from the dataset are visualised. All phantoms shown were generated from the same OCT image. Variation in shape is due to random synthetic perturbations applied to the artery diameter, the function of which is a composite of two sinusoids with randomised amplitude, frequency, phase and vertical displacement. This ensures smooth, continuous variation along the length of the artery regardless of input parameters. Frontiers in Cardiovascular Medicine 03 frontiersin.org 10.3389/fcvm.2023.1221541 10.3389/fcvm.2023.1221541 Morgan et al. performed on the same CFD mesh), this is known as common base POD (cPOD) (15). In our methodology for obtaining common mode functions underlying multiple meshes, the time domain is replaced with the domain of the mesh geometries. It is assumed that a few smoothly varying variables can be used to represent the mesh cases. The goal is to obtain the hidden common modes in the stationary solutions, on multiple meshes, while the mesh is smoothly varied. To obtain the modes underlying the variations in pressure and shear stress ﬁelds, we use the method of SVD. We begin with a dataset of CFD simulated steady-state ﬂow solutions. For one simulation, the chosen output variable (e.g. pressure and wall shear) is organised into a N-length vector, where N is the number of nodes in the mesh. These vectors are oriented horizontally and then stacked vertically. With M meshes, the resulting 2D solution matrix A has the dimensions M N. Our application of SVD follows the theory of snapshots (16), similar to other use cases. However, each snapshot (stacked vector) in our solution matrix is not a different time frame of the same simulation, but rather a steady state solution ran with identical conditions on a different, uniquely shaped mesh. SVD factors the matrix into a product of three matrices A ¼ UDVT, where the columns of U and V are orthonormal (V is transpose), and the singular matrix D is diagonal with positive real numbers, organised by magnitude in descending order. The sum of the singular values represents the total amount of information in the system. They are analogous to the Eigenvalues of the Eigen decomposition, and represent the magnitude, or signiﬁcance, of each Eigenvector, or POD mode. The singular values can then be used to estimate the number of modes needed to reconstruct the ﬂow solutions without signiﬁcant loss of information (16). 4. Data reduction of the CFD solution ﬁelds using proper orthogonal decomposition POD is a tool in CFD post processing and is derived from the Singular Value Decomposition (SVD) method for matrix factorisation commonly used in statistical analysis. The method ﬁnds correlations in the vector ﬂow solution ﬁeld, which contains small linear perturbations, to obtain an Eigenbasis onto which the mesh ﬂow data can be projected. In classical POD, the correlations are obtained in the time domain to identify ﬂow structures that are most dynamically important in time during the evolution of turbulence. The same methodology is also extended to varying ﬂow cases based on different experimental setups (e.g. considering a number of unsteady ﬂow experiments 3. Creating a well-annotated synthetic data repository Both vector matrices U and V are organised in terms of the singular values, from most to least signiﬁcant. The summed energy of each leading mode, being their corresponding singular values, are then used to deﬁne a tolerance threshold for information loss. Due to spatial coherence of particular modes of variation of the ﬂow with respect to the mesh shape, the number of modes that capture the majority of useful information are the ﬁrst few, as compared to the full dataset. Modes that fall outside of a chosen threshold in terms of correlative signiﬁcance can be truncated from the dataset, drastically reducing the dimensionality of the data whilst incurring a tolerable underestimation of the concerned node-wise ﬂow parameter. Additionally, although not implemented in the current case, explicit smoothing can also be applied in the correlation matrix space to enhance numerical properties of the meh-wise POD coefﬁcients (8). In this case, the leading 10 modes were found to capture >95% of total information about both the pressure and wall shear stress, and thus were deemed sufﬁcient for accurate reconstruction TABLE 1 Parameters of the Carreau-Yasuda model. based on the amplitude of a sinusoid, which distributed the perturbation lengthwise. The sinusoid components have independently randomised amplitude, frequency, phase and vertical offset. With this method, 200 phantom meshes per each of the 7 unique blood vessels available were generated. Including the 7 natural artery shapes, this results in a total of 1407 3D meshes in this preliminary dataset. These geometries were then input to the CFD solver Abaqus (v16.2) to obtain the pressure and shear stress ﬁeld by solving the governing steady-state incompressible Navier-Stokes equations. In the solver, the governing equations were discretised on ≏100,000 mixed hexahedral and triangular prismatic elements in accordance with the second order of approximation. The advection term in the momentum equation was discretised using second-order least squares. To accelerate convergence of the steady solution with imposing the divergence free velocity ﬁeld, the pressure- correction method (SIMPLE) was used with an efﬁcient solution of the Poisson pressure equation. Boundary conditions were imposed as constant inﬂow (100 cm/s), and zero pressure outﬂow. On all vessel walls, a zero velocity and logarithmic wall function boundary condition was speciﬁed. Frontiers in Cardiovascular Medicine 5. Data reduction of the synthetic meshes of coronary blood vessels Several shape optimizers have been proposed in the literature, of which t-SNE has acquired a lot of attention (17). The t-SNE is a Frontiers in Cardiovascular Medicine 04 frontiersin.org 10.3389/fcvm.2023.1221541 Morgan et al. compared. It was found that the RFR algorithm combined with the Regressor Chain algorithm were best suited for this task. compared. It was found that the RFR algorithm combined with the Regressor Chain algorithm were best suited for this task. statistical method for visualising high-dimensional data by embedding each N-dimensional data point in a reduced space, typically of two or three dimensions. A higher number of embedding dimensions will retain a greater accuracy of clustering, but also increase the sparsity of data within the space. More speciﬁcally, t-SNE generates the joint Gaussian distribution of the conditional chance that a nearby mesh coordinate is sufﬁciently close in terms of Euclidean distance to an initial mesh coordinate. The unknown variance of the Gaussian distribution is obtained from the Shannon entropy. This step creates a matrix of each mesh coordinate with all other mesh coordinates where a chance is provided on the basis of distance. g g The RFR algorithm is a supervised machine learning technique that integrates multiple independent decision trees on a training data set: the obtained results are ensembled to obtain a more robust single model compared to the results of each tree separately (18). RFR is a supervised learning method in the sense that during training it identiﬁes mappings between inputs and outputs. In our setup, the t-SNE coordinates of the meshes are the input and the cPOD principal coefﬁcients are the output. In our approach, an independent RFR regressor is employed for each of the 10 coefﬁcients. The Random Forest Regression algorithm utilised in our work is obtained from the popular Machine Learning library Scikit-learn. Scikit-learn is built to facilitate the use of Artiﬁcial Intelligence and Machine Learning algorithms, and is used in regression, classiﬁcation, and clustering tasks. The model is imported as “sklearn.ensemble.RandomForestRegressor.” Additionally, a Regressor Chain architecture is used to obtain a multiple output model that organises the regression of individual modes in a chained fashion. Thus, RFR creates a regression model for each pressure coefﬁcient, where each model makes a prediction for its coefﬁcient speciﬁed by the chain by using all the t-SNE features provided to the model and the predictions of previous outputs in the chain. 7. Results An automatic pipeline was implemented to perform highly accurate 3D reconstruction from biplane angiograms and an OCT pullback (19), to automatically generate a mesh and on basis thereof, and to generate small perturbations in the topology of meshes. The latter was then used to generate a full stationary solution of the shear stress and pressure ﬁelds using the Navier- Stokes solver in Abaqus. The perturbation parameters were bounded to induce small but signiﬁcant changes in the accompanying geometry of the meshes (Figure 2). This also resulted in appreciable changes to the pressure and wall shear ﬁelds (Figure 3). The cumulative wall shear stress and pressure ﬁelds were then further analysed with the cPOD procedure. The ﬁrst 10 modes of the pressure and shear stress ﬁelds were sufﬁcient to reproduce >95% of the variance of both ﬁelds, leading to modest errors in the reproduction of the original ﬁelds of <1% (Figures 4 and 5). 5. Data reduction of the synthetic meshes of coronary blood vessels This ensures that the correlation between the features are taken into account to enhance the regression. As a next step, a reduced order mapping is obtained by minimizing the Kullback-Leibler divergence between the Gaussian distribution of the original points and a Student’s t-distribution of points in a reduced dimensional space. The resulting vectors are then used to ﬁll the feature space. In a sense, the space is “seeded” with the meshes produced from the natural OCT images. The space around each image is then populated with the synthetic mesh vectors, which have a small but signiﬁcant geometrical difference from the parent mesh. The goal being to ﬁll the feature space and bridge the empty regions between the clusters. Given that the principal coefﬁcients are physics-based, they should maintain a causal link to the values of the embedding coordinates, which represent variability in mesh shape. A ﬁlled feature space with an intact causal link will aid an interpolative machine learning model to make accurate coefﬁcient predictions for an unseen geometry (Figures 5 and 6). It is worth noting that what constitutes a “ﬁlled” feature space is highly dependent on the chosen t-SNE parameters and the natural limits of the data that is being reduced. The “natural limit” is in reference to the fact that a hypothetical dataset containing all possible natural variations of the artery shape will produce a “ﬁlled” feature space, and the regions that are not populated will represent shapes that do not occur naturally, and thus may not be useful for a diagnostic tool. Hence, we aim to produce synthetic data, which is not so different from the natural data as to have its shape fall outside of this hypothetical set. It is for this same reason that it is better to bolster the dataset with natural shapes wherever possible, with synthetic data playing a supplemental role. Integration of human OCT patient data is forthcoming in future research. 6. Random forest regressor and regressor chain Next was a reduction in the dimensions of the mesh topology using t-SNE (Figures 6 and 7) for utilisation in a low-dimensional regression task. The t-SNE algorithm enables control over the clustering behaviour based on similarity through its perplexity parameter. This was ﬁne tuned to obtain an approximately homogeneous distribution of the mesh cases, whilst preserving noticeable clustering features. This allows for a smooth geometrical representation suitable for regression. As can be observed, the t-SNE features resolve to seven clusters corresponding to seven natural artery shapes. To which, random perturbations are introduced to generate quantitatively distinct SVD re-organizes the modes based on their energy level content and the number of modes are truncated when >95% of the variance of the ﬁeld is preserved. This resulted in the ﬁrst 10 modes for the pressure ﬁeld and the shear stress ﬁeld for the dataset we use for this study, which when used for reconstructing the solution leads to a root mean squared error less than 5%. In order to interpolate the POD principal coefﬁcient ﬁeld that enables predictions of future objects, simple feed-forward neural networks and classical machine learning methods were Frontiers in Cardiovascular Medicine 05 frontiersin.org Morgan et al. 10.3389/fcvm.2023.1221541 FIGURE 3 A collection of meshes generated using various OCT images and perturbation parameters, coloured by the pressure (left) and wall shear (right) solutions from CFD simulations. The mesh dimensions are normalised for the sake of visualisation. FIGURE 3 A collection of meshes generated using various OCT images and perturbation parameters, coloured by the pressure (left) and wall shear (right) solutions from CFD simulations. The mesh dimensions are normalised for the sake of visualisation. FIGURE 4 (left) Root-mean-squared error for the reconstruction of the original mesh-wise pressure solution from a truncated set of 10 principal coefﬁcients per mesh. The error is normalised against the range of pressure values across all meshes. (right) Singular values for the decomposition of the pressure solution, normalised against the largest value. These singular values are ordered by magnitude and represent the relative contribution of each POD mode to the energy of the overall pressure solution. Subsequent values quickly decay to <1% of the highest value, as the ﬁrst several modes represent the overwhelming majority of the information in the pressure ﬁeld. This indicates that many of these trailing modes can be safely discarded from the dataset without losing a signiﬁcant amount of information. Frontiers in Cardiovascular Medicine 6. Random forest regressor and regressor chain FIGURE 4 (left) Root-mean-squared error for the reconstruction of the original mesh-wise pressure solution from a truncated set of 10 principal coefﬁcients per mesh. The error is normalised against the range of pressure values across all meshes. (right) Singular values for the decomposition of the pressure solution, normalised against the largest value. These singular values are ordered by magnitude and represent the relative contribution of each POD mode to the energy of the overall pressure solution. Subsequent values quickly decay to <1% of the highest value, as the ﬁrst several modes represent the overwhelming majority of the information in the pressure ﬁeld. This indicates that many of these trailing modes can be safely discarded from the dataset without losing a signiﬁcant amount of information. synthetic datapoints. Additionally, within each of the t-SNE clusters, the variation of the principal coefﬁcients are also smooth and continuous since their values are correlated with variation in mesh shape. to be 20, and the best maximum number of trees for the model was found to be 70. The machine learning model was applied for the test data set as well. Figures 8 and 9 show the results for shear stress and pressure for the two most signiﬁcant POD modes respectively. The mean Root Mean Square Error (RMSE) of the prediction of the dominant mode coefﬁcient was 15.2% for pressure and 19.7% for shear stress. The 1407 t-SNE data points with their respective pressure and shear stress modes were shufﬂed and divided into a training data set (80% of the overall data) and a validation data set (remaining 20%). The training dataset was used for ten iterations to train the RFR model, where the best maximum tree depth was found With the regression for cPOD principal coefﬁcients completed, the mesh-wise modes previously generated by the cPOD method 06 frontiersin.org Morgan et al. 10.3389/fcvm.2023.1221541 FIGURE 5 The mesh-wise reconstruction error for wall shear (left) is much lower than pressure reconstruction using the same number of coefﬁcients. Additionally, the singular values (right) decay to 0 in a fewer number of modes compared to the pressure decomposition. These factors are indicative of the wall shear solution being easier for the POD method to decompose than static pressure, possibly due to the fewer number of CFD nodes for which it is computed. 6. Random forest regressor and regressor chain 10.3389/fcvm.2023.1221541 FIGURE 8 Predictions of POD principal coefﬁcients of shear stress for ﬁrst two modes using the proposed framework, compared to the ground truth for the test data set. The ﬁrst part of the same data set was used for training via the RFR. The regression was performed on the 2D t-SNE representation of the meshes against the principal coefﬁcients. FIGURE 8 Predictions of POD principal coefﬁcients of shear stress for ﬁrst two modes using the proposed framework, compared to the ground truth for the test data set. The ﬁrst part of the same data set was used for training via the RFR. The regression was performed on the 2D t-SNE representation of the meshes against the principal coefﬁcients. standard deviations of computed errors are summarised in Table 2. It should be noted that the range of NMAE and NRMSE for pressure is within the accuracy reported for the machine learning models of pressure in aortic ﬂows based on autoencoders and Deep Neural Networks (DNNs) (20). It can also be noticed that the standard deviation and the mean error values are of the same order of magnitude in all cases, which suggests that the populated parameter space for the considered coronary artery problem is relatively sparse. The latter is in agreement with sparsity of the t-SNE maps (Figures 6 and 7). The error variation is particularly large for the shear stresses, which can be explained by a much smaller statistical ensemble of the wall shear surface points in comparison with the volume points where pressure was computed. This is supported by an estimate based on the central limit theorem (21), which suggests that the ratio of statistical errors of the pressure and wall shear stresses should scale as a square root of the ratio of the number of surface points to that of the volume points, and which is about 1:4.5 for all considered meshes. together with the newly predicted coefﬁcients are used to reconstruct the ﬂow ﬁeld. Results of the 3D reconstruction of the shear stress and pressure ﬁelds for the CFD method (“ground truth”) the cPOD reconstruction alone, and the RFR prediction are shown in Figure 10. 6. Random forest regressor and regressor chain FIGURE 6 The distribution of all meshes in the database embedded in 2D t-SNE space with colours representing the principal coefﬁcients of the static pressure solutions for the ﬁrst (left) and second (right) mesh wise POD modes. FIGURE 5 The mesh-wise reconstruction error for wall shear (left) is much lower than pressure reconstruction using the same number of coefﬁcients. Additionally, the singular values (right) decay to 0 in a fewer number of modes compared to the pressure decomposition. These factors are indicative of the wall shear solution being easier for the POD method to decompose than static pressure, possibly due to the fewer number of CFD nodes for which it is computed. FIGURE 5 The mesh-wise reconstruction error for wall shear (left) is much lower than pressure reconstruction using the same number of coefﬁcients. Additionally, the singular values (right) decay to 0 in a fewer number of modes compared to the pressure decomposition. These factors are indicative of the wall shear solution being easier for the POD method to decompose than static pressure, possibly due to the fewer number of CFD nodes for which it is computed. FIGURE 6 The distribution of all meshes in the database embedded in 2D t-SNE space with colours representing the principal coefﬁcients of the static pressure solutions for the ﬁrst (left) and second (right) mesh wise POD modes. FIGURE 6 The distribution of all meshes in the database embedded in 2D t-SNE space with colours representing the principal coefﬁcients of the static pressure solutions for the ﬁrst (left) and second (right) mesh wise POD modes. FIGURE 6 FIGURE 6 The distribution of all meshes in the database embedded in 2D t-SNE space with colours representing the principal coefﬁcients of the static pressure solutions for the ﬁrst (left) and second (right) mesh wise POD modes. FIGURE 7 The distribution of all meshes in the database embedded in 2D t-SNE space with colours representing the principal coefﬁcients of the wall shear solutions for the ﬁrst (left) and second (right) mesh wise POD modes. FIGURE 7 The distribution of all meshes in the database embedded in 2D t-SNE space with colours representing the principal coefﬁcients of the wall shear solutions for the ﬁrst (left) and second (right) mesh wise POD modes. Frontiers in Cardiovascular Medicine Frontiers in Cardiovascular Medicine 07 frontiersin.org Morgan et al. 6. Random forest regressor and regressor chain These were used for further error quantiﬁcation of the ﬂow solution in the physical space, relative L1 and L2 norm errors, which are analogues to the normalised mean absolute errors (NMAE) and normalised root mean square errors (NRMSE), respectively, considered in other studies (20). The errors were calculated using the dominant 10 POD modes for the test dataset of 20% of the meshes in accordance with the following deﬁnitions: NMAE(i ¼ 1, . . . , imeshmax) ¼ P j¼ jnodemax j¼1 jf ML ij f GT ij j jnodemax ( max (f) min (f)) 100% NRMSE(i ¼ 1, . . . , imeshmax) ¼ ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ P j¼ jnodemax j¼1 (f ML ij f GT ij )2 q jnodemax ( max (f) min (f)) 100% where jnodemax is the total number of CFD data points in the considered volumetric/surface distributions, imeshmax is the number of meshes in the test dataset, ML and GT denote the machine learning and the ground truth (CFD) solutions respectively, and f stands for the pressure or wall shear stress solution component. The mean values and the corresponding Frontiers in Cardiovascular Medicine 8. Discussion It was noted that full feature space homogenisation would require signiﬁcantly more drastic and exotic synthetic manipulation of the OCT data, which would likely negatively impact the ability of the data to represent reality. A better balance between number of real data versus synthetic data is required to bring this technique closer to real- world application. In future, a systematic procedure can be adapted to generate the synthetic meshes in an optimal way by exploiting sensitivity of the coronary ﬂow response to perturbations of the baseline vessel geometry, similar to the deformation matrix method recently developed for aortic ﬂow simulations (23). 2:96 + 2:84 3:51 + 3:19 11:21 + 11:81 11:23 + 11:81 Current developments in physics-based artiﬁcial intelligence allow us to accelerate these methods so that clinical interventions in the cath lab can be evaluated on novel parameters such as shear stress, pressure drop, and/or velocity ﬁeld. The main ﬁndings of the current paper are that a) synthetic perturbation is an effective way to generate additional surrogate data, which can help satisfy the large volumes required by AI algorithms, b) cPOD, a time- independent variation of POD, can be used to substantially reduce the dimensions of pressure and shear stress ﬁeld data in simulated blood vessels, c) metrics for quantifying the shape of a blood vessel mesh, such as t-SNE, are effective schemes to drastically reduce the degrees of freedom corresponding to variations in vessel geometry, and d) an interpolative method based on a RFR model was able to predict new pressure ﬁelds within seconds, with mean relative L1 and L2 errors (NMAE and NRMSE) of 2.96% and 3.51% respectively. The errors of the wall shear stress reconstruction show an approximately 4 times larger scatter in comparison with the pressure calculation, in statistical agreement with the smaller number of mesh surface points in comparison with the volume points. In unsteady ﬂuid mechanics problems on a ﬁxed mesh, a 1D time coordinate is typically used as an evolutionary variable to characterise the snapshots of the POD method. Here, this approach is generalised to a set of 2D t-SNE coordinates, which are cognate with time for the purpose of POD snapshots and were found sufﬁcient to reconstruct the pressure and wall shear stress ﬁelds in any speciﬁed blood vessel shape. The t-SNE technique was applied to reduce the complexity of each mesh whilst preserving their characteristic features. 8. Discussion In doing so, their relative similarity necessarily remains intact (25) due to the fact that, prior to the embedding step, t-SNE computes the difference between the input meshes based on Euclidean distance between the node coordinates. Therefore, the clustering of the variable phantom meshes around their respective reference shapes arises naturally. Notably, the entire process of meshing the OCT contour domain, embedding this geometry in 2D t-SNE space, predicting the coefﬁcients and constructing the pressure and wall shear stress ﬁelds cumulatively takes no more than 2 min, which underpins the success of this method. Furthermore, the applicability of 2D t-SNE coordinates to describe ≏100,000 degrees of freedom corresponding to the number of CFD mesh elements implies a factor of 105 dimensionality reduction. In the future, to model multiple solution components in space and time, use of a higher dimensional t-SNE space instead of 2D t-SNE may be reconsidered, and the relationship between clustering accuracy and data sparsity will be investigated. p p p Synthetic manipulations have recently been introduced to Machine Learning to overcome the excessive requirement of well annotated data for AI algorithms (13). We have developed a hybrid approach which took into account the natural variation between blood vessels and applied random synthetic perturbations to produce variants of this original data, with the aim of populating the t-SNE feature space (Figure 2). It was noted that full feature space homogenisation would require signiﬁcantly more drastic and exotic synthetic manipulation of the OCT data, which would likely negatively impact the ability of the data to represent reality. A better balance between number of real data versus synthetic data is required to bring this technique closer to real- world application. In future, a systematic procedure can be adapted to generate the synthetic meshes in an optimal way by exploiting sensitivity of the coronary ﬂow response to perturbations of the baseline vessel geometry, similar to the deformation matrix method recently developed for aortic ﬂow simulations (23). The standard RFR algorithm was found to be a suitable option for non-linear regression to reconstruct the POD signals from the t-SNE space. Despite the simplicity of the RFR model, the accuracy of predictions was encouraging. Essentially, the model uses the calculated t-SNE co-ordinates and their associated principal coefﬁcients to interpolate the coefﬁcient values over the whole embedding space. 8. Discussion Rheological theories of Atherosclerosis have been shown to successfully predict plaque location, plaque progression, and plaque rupture (22). They have not been used to infer clinical decisions. Frontiers in Cardiovascular Medicine frontiersin.org 08 Morgan et al. 10.3389/fcvm.2023.1221541 FIGURE 9 Predictions of POD principal coefﬁcients of pressure for ﬁrst two modes using the proposed framework, compared to the ground truth for the test data set. Training and testing of the RFR model for pressure utiised the same algorithm, conﬁguration, and optimization as shear stress. FIGURE 9 FIGURE 9 Predictions of POD principal coefﬁcients of pressure for ﬁrst two modes using the proposed framework, compared to the ground truth for the test data set. Training and testing of the RFR model for pressure utiised the same algorithm, conﬁguration, and optimization as shear stress. FIGURE 10 A visualisation of the ﬂow ﬁeld solution for pressure (left) and wall shear (right) of two test meshes. Shown is the ground truth CFD simulation data (top), the reconstructed POD solution using the 10 most dominant coefﬁcients calculated from the CFD solution (middle) and the reconstruction using the RFR predicted coefﬁcients (bottom). FIGURE 10 A visualisation of the ﬂow ﬁeld solution for pressure (left) and wall shear (right) of two test meshes. Shown is the ground truth CFD simulation data (top), the reconstructed POD solution using the 10 most dominant coefﬁcients calculated from the CFD solution (middle) and the reconstruction using the RFR predicted coefﬁcients (bottom). 09 Frontiers in Cardiovascular Medicine frontiersin.org 10.3389/fcvm.2023.1221541 10.3389/fcvm.2023.1221541 Morgan et al. of DNN parameters, while the optimal choice of the latter is application dependent. Differently to the mainstream approach, our method is based on the generalisation of proper orthogonal decomposition (cPOD). This allows for treatment of multiple vectors of the solution matrix of interest simultaneously, which is largely analogous with multiple unsteady ﬂow experiments in ﬂuid mechanics. An important advantage of the POD framework is that it sorts the individual modes in terms of correlative signiﬁcance. In the current coronary ﬂow simulations, we have considered mesh shape variability as an evolutionary factor for each steady solution component of interest. This is similar to the recent application of Principle Component Analysis (PCA) to data-driven modelling of aortic ﬂows (23), where separate DNN models were used for pressure and absolute velocity. frontiersin.org 8. Discussion However, in comparison to the standard PCA and DNN techniques, the suggested cPOD approach allows for extension of the solution matrix from single scalars to 3D velocity vectors and pressure components simultaneously on different meshes in space and time. TABLE 2 Mean errors and standard deviations of reconstructed pressure solution (Left), and of the reconstructed shear stress solution (Right). All values are normalized against corresponding range of values in the full dataset. TABLE 2 Mean errors and standard deviations of reconstructed pressure solution (Left), and of the reconstructed shear stress solution (Right). All values are normalized against corresponding range of values in the full dataset. NMAE, % NRMSE, % NMAE, % NRMSE, % 2:96 + 2:84 3:51 + 3:19 11:21 + 11:81 11:23 + 11:81 Current developments in physics-based artiﬁcial intelligence allow us to accelerate these methods so that clinical interventions in the cath lab can be evaluated on novel parameters such as shear stress, pressure drop, and/or velocity ﬁeld. The main ﬁndings of the current paper are that a) synthetic perturbation is an effective way to generate additional surrogate data, which can help satisfy the large volumes required by AI algorithms, b) cPOD, a time- independent variation of POD, can be used to substantially reduce the dimensions of pressure and shear stress ﬁeld data in simulated blood vessels, c) metrics for quantifying the shape of a blood vessel mesh, such as t-SNE, are effective schemes to drastically reduce the degrees of freedom corresponding to variations in vessel geometry, and d) an interpolative method based on a RFR model was able to predict new pressure ﬁelds within seconds, with mean relative L1 and L2 errors (NMAE and NRMSE) of 2.96% and 3.51% respectively. The errors of the wall shear stress reconstruction show an approximately 4 times larger scatter in comparison with the pressure calculation, in statistical agreement with the smaller number of mesh surface points in comparison with the volume points. Synthetic manipulations have recently been introduced to Machine Learning to overcome the excessive requirement of well annotated data for AI algorithms (13). We have developed a hybrid approach which took into account the natural variation between blood vessels and applied random synthetic perturbations to produce variants of this original data, with the aim of populating the t-SNE feature space (Figure 2). Frontiers in Cardiovascular Medicine 8. Discussion The RFR segregates feature data into groups before interpolating within each group, which is particularly suitable for the clustered t-SNE features. Notably, the distribution of mode coefﬁcients in the t-SNE space (Figures 6 and 7) demonstrates smooth variations due to the inherent correlation between the shape of a mesh and the major ﬂow patterns captured by the dominant POD modes. Dimensionality reduction helps retain deﬁning features whilst drastically reducing the volume of data required to represent them. This makes machine learning algorithms more likely to identify such features, along with being more computationally efﬁcient. Additionally, it aids in removing noise and extraneous features which can confound important signals (24). In many bio- mechanical applications, autoencoders in combination with DNNs have been a very popular technique to reduce the geometrical complexity to a small set of scalars, which be learnt from the training data. Depending on the DNN calibration, such approaches can be tuned to reproduce the ground truth CFD solution within a few percent relative error (20). However, it can be argued that performance of such methods is strongly dependent on the choice Frontiers in Cardiovascular Medicine 10 frontiersin.org Morgan et al. 10.3389/fcvm.2023.1221541 10.3389/fcvm.2023.1221541 Morgan et al. 9. Limitations of the method and conclusion modelling of coronary artery ﬂows. The speed and accuracy obtained were highly motivating and were able to calculate the pressure and shear stress ﬁelds of an unknown vessel within seconds. Rheological theories of Atherosclerosis have been shown to successfully predict plaque location, plaque progression, and plaque rupture (22), but they have not been used to infer clinical decisions. Current developments in physics-based AI allow us to accelerate these methods such that clinical interventions in the cath lab can be evaluated on novel parameters such as shear stress, pressure drop and 3D velocity ﬁeld. To translate the current method to clinical applications, several limitations must be addressed. First, the current implementation assumes that shape variations are the most important factor affecting velocity ﬁelds and their derived parameters. This is corroborated by theoretical arguments, as well as observations that velocity, shear stress and pressure drop strongly scale with diameter. However, the artery ﬂow ﬁeld also scales with the inﬂow velocity, which changes throughout the cardiac cycle. To systematically account for the unsteady velocity variation, future developments include extending the scope of the AI model by re-adding the time evolution input. In the meantime, the current simpliﬁed steady model may already be sufﬁcient if the ﬂow features of interest are slow compared to the viscous effects, i.e. the ﬂow in the coronary vessel is quasi-steady. In this case, the time history of inﬂow velocity variation can be decoupled into a series of time frames, where each frame may be represented by a steady process at a different inlet velocity scale. In turn, the shear stress and pressure ﬁelds at each frame can be rapidly reconstructed from the inﬂow velocity and the shear stress and pressure ﬁelds of a baseline dataset using the scaling law introduced by Taylor et al. (26). To conclude, we developed a method to produce a very fast solution to the Navier-Stokes equations, as we aimed to focus on applying this method in a clinical environment with high demand for rapid solutions. We are currently working towards newer methods enabling time dependent ﬂows that incorporate solid state interactions, as well as higher accuracy AI modelling functions with corresponding error estimates. Conﬂict of interest Finally, in line with many recent works devoted to the proof- of-concept data-driven modelling of cardio-vascular ﬂows (20), we simpliﬁed the model by considering the vessel without side branches. However, it is known that bifurcations occur in the main stem of the left coronary artery, which might affect the inﬂow conditions. Hence, to reduce the effect of the bifurcation in the current study, the starting site of the 7 catheterised segments was deliberately located 5 vessel diameters downstream of the main stem. Nevertheless, to account for general topology of coronary vessels, which may be of practical interest, the suggested reduced order modelling approach will be extended to side branches in future work. 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. Data availability statement The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation. A more serious limitation of the current study is the neglect of the natural ﬂexibility and heterogeneity of vessel walls in the ﬂow modelling process. Whilst the rigid wall assumption signiﬁcantly accelerates the solution of the governing Navier-Stokes equations, modelling of the Fluid Structure Interaction (FSI) is essential to correctly capture the coronary artery ﬂow behaviour (27). Hence, future developments will incorporate the FSI model into the simulation driven dataset of the suggested cPOD-tSNE framework. Acknowledgments Despite the overall salutary results of the RFR method, to further reﬁne accuracy of the machine learning model predictions in future, the RFR algorithm may be replaced by more advanced methods such as those based on Gaussian processes; one advantage of which being uncertainty quantiﬁcation to provide an overall error estimate for the user. Such estimations would be an invaluable addition to a model that is intended for use as a diagnostic tool for clinicians. The BHF (FS/PhD/22/29316) is appreciated for their funding of this project. This study was conducted with the assistance of the Research Software Engineering team in ITS Research at Queen Mary University of London. Author contributions All authors listed have made a substantial, direct, and intellectual contribution to the work and approved it for publication. Frontiers in Cardiovascular Medicine 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. Despite the above-mentioned limitations of the current work, it can be concluded, using t-SNE and cPOD to perform interpolation by Machine Learning was very successful for the proof-of-concept frontiersin.org 11 10.3389/fcvm.2023.1221541 Morgan et al. Morgan et al. Morgan et al. References 14. King T, Butcher S, Zalewski L. Apocrita - High Performance Computing Cluster for Queen Mary University of London (2017). Available from: https://doi.org/10.5281/ zenodo.438045 1. Wentzel JJ, Corti R, Fayad ZA, Wisdom P, Macaluso F, Winkelman MO, et al. 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https://openalex.org/W4241562267	https://www.solid-earth.net/6/1157/2015/se-6-1157-2015.pdf	English	null	Analysis of soil moisture condition under different land uses in arid region of Horqin Sandy Land, northern China	null	2,015	cc-by	9,149	1 Introduction Abstract. Land use plays an important role in controlling spatial and temporal variations of soil moisture by inﬂuenc- ing inﬁltration rates, runoff and evapotranspiration, which is important to crop growth and vegetation restoration in semi- arid environments, such as Horqin sandy land in north China. However, few studies have been conducted comparing differ- ences of dynamics of soil water conditions and the responses of soil to inﬁltration under different land use types in semi- arid area. Five different land use types were selected to ana- lyze soil moisture variations in relation to land use patterns during the growing season of 2 years. Results showed that soil moisture condition was affected by different land uses in semi-arid sandy soils. The higher soil moisture content among different land uses was exhibited by the grassland, followed by cropland, poplar land, inter-dunes and shrub land. The temporal variations of soil moisture in different land uses were not always consistent with the rainfall due to the dry sequence. Moreover, soil water at the surface, in the root zone and at the deep soil layer indicated statistical dif- ferences for different types of land cover. Meanwhile, tempo- ral variations of soil moisture proﬁle changed with precipita- tion. However, in the deep soil layer, there was a clear lag in response to precipitation. In addition, seasonal variations of proﬁle soil moisture were classiﬁed into two types: increas- ing and waving types. And the stable soil water layer was at 80–120 cm. Furthermore, the inﬁltration depth exhibited a positive correlation with precipitation under all land uses. This study provided an insight into the implications for land and agricultural water management in this area. Soil is an important source of goods service and resources that essential to humankind. Soils have the ability to clean water sources, thus improving human health (Helmke and Losco, 2013; Keesstra et al., 2012). Most importantly, the soil system is the key component of the Earth system that controls the water, mineral and organic matter cycles and there is a need to research different aspects of the soil and land degradation that affect the fate of the Earth system (Gan- jegunte et al., 2014; Mukhopadhyay and Maite, 2014; Brevik et al., 2015). Soil water content is essential in controlling the soil pro- cesses involving the physical, chemical, and biological pro- cesses of the soil system (Brevik et al., 2015). Analysis of soil moisture condition under different land uses in the arid region of Horqin sandy land, northern China C. Y. Niu1,2, A. Musa1, and Y. Liu1,2 1Institute of Applied Ecology, Chinese Academy of Sciences, Shenyang 110016, China 2College of Resources and Environment, University of Chinese Academy of Sciences, Beijing 100049, China C. Y. Niu1,2, A. Musa1, and Y. Liu1,2 1Institute of Applied Ecology, Chinese Academy of Sciences, Shenyang 110016, China 2College of Resources and Environment, University of Chinese Academy of Sciences, Beijing 100049, China Correspondence to: A. Musa (alamusa@iae.ac.cn) Received: 5 June 2015 – Published in Solid Earth Discuss.: 21 July 2015 Revised: 15 September 2015 – Accepted: 9 October 2015 – Published: 23 October 2015 Received: 5 June 2015 – Published in Solid Earth Discuss.: 21 July 2015 Revised: 15 September 2015 – Accepted: 9 October 2015 – Published: 23 October 2015 Received: 5 June 2015 – Published in Solid Earth Discuss.: 21 July 2015 Revised: 15 September 2015 – Accepted: 9 October 2015 – Published: 23 October 2015 1 Introduction In general, these processes that take place in soil strongly depend on the quantity and composition of water. In the case of the water in- ﬁltration process, soil water content dictates that water ﬂows across the soil surface, reaches the soil proﬁle, or, ﬁnally, per- colates to recharge aquifers, which is essential to understand the hydrological cycle (Cerdà, 1999). It is known that the irrational land uses may impact on soil water, resulting in decreased and uneven inﬁltration (Markus et al., 1994), poor germination and reduced yields (Abadi Ghadim, 2000), accelerated leaching of agrochem- icals (Taumer et al., 2006), increased runoff and enhanced erosion (Doerr et al., 2000). Li et al. (2009) showed that land use change from woodland to grassland decreased soil water by 18 % during 1981–2000 in an agricultural catchment of the Loess Plateau. Cerdà (2000) reported that soil erodibility was greater under agricultural land use compared with scrub- land under arid environments, indicating that cultivating the 2.1 Study area This study was conducted at the Ulan’aodu Station in the west of the Horqin sandy land (43◦02′ N, 119◦39′ E), north- east of China. Ulan’aodu Station, built in 1975 and af- ﬁliated with the Institute of Applied Ecology of the Chi- nese Academy of Sciences, is one of the monitoring net- work stations of the Department of Desertiﬁcation Control, State Forestry Administration of China. Horqin sandy land is located in the semi-arid environmental region of northern China. Due to the long-term inﬂuences of human activities (e.g., extensive ﬁrewood consumption, heavy grazing and land reclamation for agriculture) and climate changes (e.g., changes of precipitation regimes and temperature), this re- gion has suffered serious desertiﬁcation over recent decades, resulting in the scattered tree grassland to be the Horqin sandy grassland (Zuo et al., 2009). To date, most sandy grass- lands have degenerated into ﬁxed, semi-ﬁxed, semi-mobile and mobile sandy lands (Liu et al., 2009; Zuo et al., 2009). The elevation of this region is about 480 m, and the climate is temperate, semiarid continental and monsoonal, and it is characterized by low precipitation and frequent occurrence of strong winds. The mean annual precipitation is 284.4 mm, of which 70 % occurs from June to August. The mean annual open-pan evaporation ranges from 2000 to 2500 mm. The av- erage aridity index is 1.99, the relative humidity varies be- tween 50 ∼55 %, and drought is serious in the spring (Ala- musa et al., 2014). The mean annual temperature is 6.2 ◦C, annual frost-free period is 140 d and the mean annual wind velocity is 4.2 m s−1. The landscape is characterized by gen- tly undulating, mobile, semi-mobile, and ﬁxed sand dunes with inter-dune bottomlands (Alamusa et al., 2014). Soil properties are characterized by their coarse texture and loose structure with high proportion of sand (85–95 %) and low or- ganic matter content (0.15–0.5 % organic C) (Su et al., 2004). The soils are vulnerable to wind erosion, particularly after cultivation. The most widely distributed sandy vegetation type was Mongolian ﬂora including grasses (e.g., Pennise- tum centrasiaticum L., Cleistogenes squarrosa L., Phrag- mites australis L.), shrubs (e.g., Caragana microphylla kom, Salix gordejevii, Hedysarum fruticosum pall.), and subshrubs (e.g., Artemisia halodendron L., Artermisia frigida L.). Moreover, land use can affect precipitation inﬁltration, which is of great importance to vegetation restoration and crop production in arid areas (Wang et al., 2008; Ziadat and Taimeh, 2013). 2 Materials and methods Soil water exhibits a tremendous heterogeneity in space and time (Gomez-Plaza et al., 2000). Therefore, spatial and temporal variations of soil moisture have always been the critical issues in vegetation restoration and water resource management, especially in the semi-arid and arid ecosystems (Brevik et al., 2015; Yu et al., 2015). Additionally, land use change contributes to the variability of soil moisture, result- ing in soil deterioration, agricultural productivity decline and land degradation (Biro et al., 2013; Fu et al., 2003; Leh et al., 2013; Parras-Alcántara and Lozano-García, 2014). 2.1 Study area Inﬁltration is the movement of water into the soil from the surface by downward or gravitational ﬂow (Thompson et al., 2010; Zhang et al., 2010). It is the feedback between the inﬁltration of water from precipita- tion and the water use characteristics of the particular veg- etation community that ecologically predominates in an area that determines the moisture state of the soil in the root zone (Sandvig and Phillips, 2006). Some studies have shown that plant biomass and productivity increased signiﬁcantly with increasing soil inﬁltration rates, which have close relation- ships with covered vegetation types (Benegas et al., 2014; Finley and Glenn, 2010; Fu et al., 2015; Zhang et al., 2014; Zheng et al., 2015). In addition to types and coverage de- grees of vegetation (Cerdà, 1998; Molina et al., 2007), pre- cipitation patterns (Wang et al., 2008), antecedent soil water content (Guo et al., 2014) and soil properties (Neris et al., 2012) also play the inﬂuential role in the water inﬁltration process. Researches have focused on the dynamics of soil water conditions and the responses of soil water to precipitation in- ﬁltration under the land-surface processes in semi-arid region (Huang et al., 2012). However, previous studies have investi- gated that spatial and temporal variation of soil water under a certain land use type, and drawing signiﬁcant research atten- tion is lacking on the differences of dynamics of soil water conditions under different land use types. Thus, it is neces- sary to understand the comparisons of the dynamics of soil water conditions under different land use types. This not only lays the foundation for the effective use of water resources, but also plays an important role in the land productivity eval- uation and desertiﬁcation recovery in arid land. C. Y. Niu et al.: Analysis of soil moisture condition in arid region of northern China 1158 ture distribution as well as the inﬂuence of precipitation on proﬁle soil water for different land uses. ture distribution as well as the inﬂuence of precipitation on proﬁle soil water for different land uses. land under certain conditions might contribute to increasing soil erosion and runoff. Therefore, understanding soil water variations under different land use types is critical for agri- cultural water management and land erosion control (Gao et al., 2014; Starr, 2005; Ziadat and Taimeh, 2013). Published by Copernicus Publications on behalf of the European Geosciences Union. Published by Copernicus Publications on behalf of the European Geosciences Union. 2.2 Experimental design The overall aim of this paper was to describe how sandy soil moisture responds to different vegetation types among different land uses types in Horqin sandy land. The speciﬁc objectives of this study were the following: (1) to examine patterns and dynamics of soil moisture under different vege- tation types; (2) to analyze differences in vertical soil mois- Five typical land use types were selected to be research plots in Horqin sandy land, including poplar land (poplar), grass- land (fenced grassland), cropland (maize), inter-dunes and shrub land (Caragana korshinskii kom), respectively (Fig. 1). Solid Earth, 6, 1157–1167, 2015 www.solid-earth.net/6/1157/2015/ C. Y. Niu et al.: Analysis of soil moisture condition in arid region of northern China C. Y. Niu et al.: Analysis of soil moisture condition in arid region of northern China 1159 y g Figure 1. Land use types and soil water sampling points in the experiment area. Figure 1. Land use types and soil water sampling points in the experiment area. Figure 1. Land use types and soil water sampling points in the experiment area. 16 years ago, there was 400 m × 600 m of poplar land in the ﬂat sandy land. The plant density was 1.5 m × 2.0 m, and the average height and diameter was 22.6 and 16.3 cm, re- spectively. Herbaceous plants and litter were observed on the ground. Measured vegetation cover and soil properties were shown in Table 1 for different land uses. Precipitation and potential evaporation were measured at the meteorological station in Ulan’aodu. Grassland was present in the ﬂat sandy land with an over- all area of about 2.4 × 107 m2 without shrubs and arbor. The community was mainly composed of perennial grass, includ- ing Calamagrostis epigejos and Spodiopogon sibiricus Trin with an average height of 52.6 cm. 2.3 Field methods The 20 m × 20 m research plots were set in the center of ﬁve types of land uses, and ﬁve sample plots (4 m × 4 m) were set randomly in each type of ﬁve land uses. Soil moisture mea- surements were taken at a depth of 0–120 cm at each plot, and proﬁles were divided into 0–20, 20–40, 40–60, 60–80, 80– 100 and 100–120 cm at 20 cm intervals. Soil samples were taken out by a drill, and soil moisture was measured gravi- metrically (the soil samples were oven-dried at 105 ◦C for 12–14 h based on oven drying method). At each plot, three points were randomly chosen to calculate the average value of soil moisture. The measuring time was from 15 April to 15 October in 2002 and 2003 with a 15 day interval. Cropland was present in the ﬂat sandy land with an area of about 5.0 × 104 m2. Cropland was reclaimed from grassland over the past decades. The annual maize was the main crop in this rainfed cropland. Inter-dune research plots were selected between ﬁxed sandy dunes and mobile sandy dunes with 400 m × 500 m area. The vegetation species consisted of Agriophyllum squarrosum, Calamagrostis epigejos, Salix ﬂavida, Potentilla chinensis, Caragana korshinskii kom and so on. Shrub land was mainly located in the dunes based on mo- bile dunes, which were one or three consecutive dunes area between 1.6–2.4 × 106 m2. Caragana korshinskii kom com- munity land was selected as the shrub land with a slop of 30◦, which was planted by 1.0 m × 1.5 m 23 years ago, and the average height and crown was 1.8 and 2.2 m, respectively. The groundwater table was observed using groundwater observation well in each area of land uses. In addition, plant roots were generally distributed at soil layers of 0–80 cm. Therefore, soil layers of 0–20, 0–80 and 80–120 cm were de- ﬁned as surface horizon, root zone and deep soil layer, re- spectively. In this study, surface soil water was the value of 3.1 Rainfall and evapotranspiration Precipitation of June, July and August was higher than the other months in 2002 and 2003, and evapotranspiration was always higher than precipitation for each month. More- over, according to the meteorological data recorded for each month, the annual precipitation from June to August was over 70% of the whole year. The annual potential evapotran- spiration is about 1500 mm in growing season (from April to October ), about 5 times larger than the precipitation (300 mm) (Fig. 2). First, the low rainfall was taken from 2.6 mm precipitation event on 24 April 2002. To show proﬁle soil water variations for ﬁve land uses, soil water was observed at 0–120 cm on 23, 25 April 2002. Second, the medium rainfall was taken from 16.2 mm pre- cipitation events on 9 and 10 June 2002. To indicate proﬁle soil water variations for ﬁve land uses, soil water was mea- sured at 0–120 cm on 8, 11 June 2002. Third, the high rainfall was taken from 40.5 mm precipita- tion events on 11–13 July 2002. To explore proﬁle soil water variations for ﬁve land uses, soil water was surveyed at 0– 120 cm on 11, 15 July 2002. 2.4 Data analysis 0–20 cm soil water; root zone soil water was the mean value of 0–20, 20–40, 40–60 and 60–80 cm soil water; and deep layer soil water was the mean value of 80–100 and 100– 120 cm soil water (Gao et al., 2014). Statistical analyses were performed to test the inﬂuence of land use on soil moisture using a one-way ANOVA (Analy- sis of Variance), and comparisons of the difference between mean water content in different land uses were implemented using the least signiﬁcant difference (LSD) method (at the p < 0.05 level). The analyses were conducted through SPSS Statistics v17.0© 2008 SPSS Inc. To indicate the inﬁltration depth of rainfall and its effects on vertical soil moisture changes, we measured soil water at the day (1–2 days) before rainfall events, after rainfall events (1–2 days), and before the next rainfall events, respectively. Considering the hysteresis of rainfall inﬁltration process, soil water was measured with an interval of days after rainfall events. The lengths of time intervals were dependent on the precipitation; in general, the larger the amount of precipita- tion, the longer the time interval. Four types of rainfall were selected to evaluate vertical soil water changes derived from precipitation events in 2002 (Gao et al., 2014). www.solid-earth.net/6/1157/2015/ www.solid-earth.net/6/1157/2015/ Solid Earth, 6, 1157–1167, 2015 C. Y. Niu et al.: Analysis of soil moisture condition in arid region of northern China 1160 1160 C. Y. Niu et al.: Analysis of soil moisture condition in arid region of northern Chi Table 1. Vegetation and soil characteristics at 0–20 cm for the ﬁve land uses in study area. Land use Land cover type Canopy cover Buck density Soil texture (plant species) percent (%) (g cm−3) Sand percent Clay percent (> 0.01 mm) (< 0.002 mm) Poplar land Populus davidiana 78 1.32 87.3 12.7 Grassland Calamagrostis epigejos and Spodi- opogon sibiricus Trin 90 1.23 80.85 19.15 Cropland Zea mays 86 1.19 93.05 6.95 Inter-dunes Agriophyllum squarrosum, Calam- agrostis epigejos, Salix ﬂavida, and Potentilla chinensis 80 1.55 84.7 15.3 Shrub land Caragana korshinskii kom 65 1.46 94.5 5.5 Table 1. Vegetation and soil characteristics at 0–20 cm for the ﬁve land uses in study area. www.solid-earth.net/6/1157/2015/ 3.2 Comparisons of vertical soil water in different land uses Signiﬁcant differences of soil water were observed among three layers in ﬁve land uses (Table 2). First, the soil wa- ter content of grassland was signiﬁcantly (p < 0.05) higher than that of the other land uses in comparison of surface soil water, while, in cropland and poplar land, surface soil water was markedly higher than that of inter-dunes and shrub land. However, there were no signiﬁcant differences on surface soil water between inter-dunes and shrub land. Second, soil mois- ture of grassland was also remarkably higher than the other land uses in root zone, while the shrub land was evidently lower than that in the other land uses. Furthermore, the soil water of grassland and inter-dunes was conspicuously higher than the other land uses in the deep soil layer, while shrub Fourth, the extreme-high precipitation was taken from 102.4 mm precipitation events on 4, 5 and 6 August 2002. To explore proﬁle soil water variations for ﬁve land uses, soil water was observed at 0–120 cm on 3 and 15 August 2002. In order to determine the effect of precipitation on soil wa- ter changes, the absolute differences were calculated between soil water content at 0–120 cm intervals after precipitation and that before precipitation for each land use. We hypothe- sized that the absolute difference was deﬁned as a signiﬁcant change resulting from precipitation inﬁltration if it was posi- tive and more than 0.5 %, or else, was deﬁned as a stochastic change (Gao et al., 2014). Solid Earth, 6, 1157–1167, 2015 www.solid-earth.net/6/1157/2015/ www.solid-earth.net/6/1157/2015/ C. Y. Niu et al.: Analysis of soil moisture condition in arid region of northern China C. Y. Niu et al.: Analysis of soil moisture condition in arid region of northern China 1161 0 50 100 150 200 250 300 2003/10 2003/8 precipitation and ET0(mm) precipitation（mm） ET0（mm） 2002/4 2002/6 2002/8 2002/10 2003/6 2003/4 Figure 2. Monthly precipitation and potential evapotranspiration (ET0) during the study period. Table 2 Mean water content at different layers of ﬁve land uses Figure 3. The temporal variations of mean soil moisture within 0– 120 cm in ﬁve land uses. 1161 C. Y. Niu et al.: Analysis of soil moisture condition in arid 0 50 100 150 200 250 300 2003/10 2003/8 precipitation and ET0(mm) precipitation（mm） ET0（mm） 2002/4 2002/6 2002/8 2002/10 2003/6 2003/4 Figure 2. Monthly precipitation and potential evapotranspiration (ET0) during the study period. Figure 3. 3.2 Comparisons of vertical soil water in different land uses The temporal variations of mean soil moisture within 0– 120 cm in ﬁve land uses. Figure 2. Monthly precipitation and potential evapotranspiration (ET0) during the study period. Figure 3. The temporal variations of mean soil moisture within 0– 120 cm in ﬁve land uses. Table 2. Mean water content at different layers of ﬁve land uses. P value refers to the probability of same soil water values of analy- sis of variance in the 95 % percent signiﬁcance level. Values in each column with the same letter are not signiﬁcantly (p < 0.05, LSD) different among land uses. Table 2. Mean water content at different layers of ﬁve land uses. P value refers to the probability of same soil water values of analy- sis of variance in the 95 % percent signiﬁcance level. Values in each column with the same letter are not signiﬁcantly (p < 0.05, LSD) different among land uses. of 2.42 and 2.38 %, respectively, which was consistent with rainfall. Variations of soil water proﬁles changed with time and were dependent on rainfall (Fig. 4a–c). Under different land uses, surface soil water varied with the changes of precip- itation patterns (Fig. 4a). Surface soil water of each land use pattern presented higher soil moisture content during the peak of precipitation between July and August. For instance, soil surface water of grassland reached a peak value (14.78 %) in August 2002 when a heavy rainfall event (112.6 mm) occurred. However, soil water presented a com- plicated variation in root zone (Fig. 4b). For example, soil water of ﬁve land use patterns presented an increasing trend in August 2002 due to the heavier rainfall. On the contrary, soil water presented a decreasing trend from June to Septem- ber when heavier rainfall still continued during this period. There were not obviously changes in deep soil layers under different land uses with the changes of precipitation (Fig. 4c), and deep soil water presented a smooth temporal change dur- ing the whole growing season. Soil water content ( %) Land use Surface Root zone Deep layer (0–20 cm) (0–80 cm) (80–120 cm) Poplar land 3.77b 5.97b 12.1b Grassland 11.54a 12.55a 21.65a Cropland 3.72b 6.68b 9.06bc Inter-dunes 1.66c 4.32c 14.37ab Shrub land 1.93c 1.95d 1.86c P value < 0.001 < 0.001 0.00781 land was signiﬁcantly lower than the other land use patterns in deep soil layer, too. 3.3 Temporal variations of soil water in different land uses Temporal variations of soil water content for surface horizon (a), root zone (b) and deep soil layer (c) in different land uses. Figure 4. Temporal variations of soil water content for surface horizon (a), root zone (b) and deep soil layer (c) in different land uses. Figure 4. Temporal variations of soil water content for surface horizon (a), root zone (b) and deep soil la 3.3 Temporal variations of soil water in different land uses Furthermore, in spring (Fig. 5a), soil water in different land uses, except cropland, exhibited a low–high–low trend from 0 to 120 cm, and the peak values appeared at 60 cm (grassland), 110 cm (inter-dunes and poplar land) and 40 cm (shrub land) with the values of 13.55, 17.09, 11.17 and 2.80 %, respectively. However, soil moisture in cropland showed an increasing trend from 0 to 120 cm. In summer (Fig. 5b), soil moisture in poplar land, grassland and inter- dunes showed a single peak with depth increasing, and the peak value appeared at 90, 90 and 110 cm, respectively. Meanwhile, in cropland and shrub land, soil water exhib- ited two peaks, and the peak values appeared at 60, 110 cm and 20, 110 cm, respectively. In autumn (Fig. 5c), soil mois- ture of all the land uses were observed similar stable trend with depth increasing, except that soil moisture of grassland showed a relatively smooth trend at 0–100 cm and gradually declined from 16.15 % at 100 cm to 3.09 % at 120 cm. First, the poplar land soil moisture content was increasing after rainfall and was consistent with the temporal variations of rainfall (Fig. 3). Second, grassland soil water variations showed two and three peaks in 2002 and 2003, respectively, and the peaks appeared in May, August and May, and in July and September, respectively; this was not synchronous with the rainfall temporal variations. Third, cropland showed a peak in May and September, respectively, which was not con- current with rainfall in 2002, when the only peak appeared in August due to the lack of relevant data in 2003, which was accordant with rainfall. Fourth, the inter-dune soil water vari- ations showed two peaks at the beginning of June and Au- gust, which was consistent with rainfall changes; inter-dune soil moisture showed a decreasing trend in 2003. Moreover, shrub land soil water variations showed single peak both in 2002 and 2003, and appeared in July and June with a value Solid Earth, 6, 1157–1167, 2015 www.solid-earth.net/6/1157/2015/ www.solid-earth.net/6/1157/2015/ C. Y. Niu et al.: Analysis of soil moisture condition in arid region of northern China 1162 Figure 4. Temporal variations of soil water content for surface horizon (a), root zone (b) and deep soil layer (c) in different land uses. Figure 4. 4 Discussion 4.1 Soil moisture variability for different land uses 4.1.1 Temporal variations of mean soil moisture within 0–120 cm precipitation events (Fig. 4a); in root zone (0–80 cm), soil water showed a complicated trend due to evapotranspiration (Fig. 4b). There are two potential explanations. First, the ef- fects of the root distribution on soil moisture may contribute to this difference (Sala et al., 1992). Second, vegetation pos- sibly transforms the soil’s physical properties, such as soil bulk density, physical composition and porosity (Garcia- Ruiz, 2010). These changes, in turn, inﬂuence the inﬁltration rate, storage, and redistribution of soil water (Lipiec et al., 2006). However, the deep soil layer (80–120 cm) had a rel- atively smooth temporal change, a clear lag in response to precipitation. The variations of mean soil moisture in different land uses were not always consistent with the rainfall (Fig. 3). This is because the “dry” sequence appeared (Fu et al., 2003). In arid land, the soil moisture content often reached its highest value after a heavier rain, and when the rain had stopped for a cou- ple of days, the soil moisture content decreased sharply due to evapotranspiration, absorbed by roots or runoff etc. While several small rain events occurred, the soil moisture con- tent did not increase and kept decreasing in some land uses. Then we called it “dry” sequence. Although several small rain events occurred in July 2002 and 2003, they did not in- terrupt the dry trend. Further, soil moisture content reaching peak value corresponded to the amount of precipitation, and that the higher mean moisture contents appear after heavier rain. However, there were differences in response to the rain due to land uses. For example, the peak in mean soil mois- ture content for cropland in 2002 and poplar land in 2003 showed a lag effect following a rain event (Fig. 3), because of the interception by canopy and the buffering inﬂuence of groundcover. Under different land uses, however, the temporal varia- tions of soil water in surface, root zone, and deep soil layer were observed following rainfall patterns, similar temporal evolutions of soil water at different proﬁles existed (Fig. 4). This implied that land use inﬂuenced on spatial patterns how- ever insubstantial on temporal variations of soil water (Gao et al., 2014). Accordingly, precipitation was the main factor that affected soil water and caused similar trends of tempo- ral patterns for different land uses. 3.4 Inﬂuences of precipitation inﬁltration on vertical soil water changes for different land uses 120 100 80 60 40 20 0 0 3 6 9 121518 0 3 6 9 121518 0 3 6 9 121518 (a) Soil water content (%) Soil depth (cm) Poplar land Grassland Cropland Inter-dunes Shrubland (b) (c) Figure 5. Seasonal patterns of vertical soil water variations for dif- ferent land uses: spring (a), summer (b) and autumn (c). Soil water content (%) Soil water inﬁltration after four rainfall events was differ- ent under ﬁve land uses (Table 3). Soil water at 0–20 cm of ﬁve land uses increased importantly after light (2.6 mm) and medium (16.2 mm) rainfall. After a heavy rainfall event (40.5 mm), an increasing trend in soil water was observed at 0–60 cm. Remarkably, the whole proﬁle (0–120 cm) of soil water was supplied by a heavier rainfall event (102.4 mm). Different land uses showed different responses to the four types of rainfall. Under the low rainfall, the higher incre- mental soil water was exhibited by grassland, followed by cropland, inter-dunes, shrub land and poplar land at 0–20 cm depth. Similarly, under the medium rainfall, the higher in- cremental soil water was exhibited by grassland (with the value of 5.08 %), followed by cropland, inter-dunes, poplar land and shrub land at 0–20 cm. Moreover, under the heavy rainfall (40.5 mm), the highest incremental soil water at 0– 60 cm was exhibited by grassland, followed by cropland, poplar land, inter-dunes and shrub land. Furthermore, under the extreme heavy rainfall (102.4 mm), the highest incremen- tal soil water at 0–120 cm was grassland (with the value of 18.25 %), followed by cropland, poplar land, inter-dunes and shrub land. Figure 5. Seasonal patterns of vertical soil water variations for dif- ferent land uses: spring (a), summer (b) and autumn (c). Solid Earth, 6, 1157–1167, 2015 www.solid-earth.net/6/1157/2015/ www.solid-earth.net/6/1157/2015/ C. Y. Niu et al.: Analysis of soil moisture condition in arid region of northern China 1163 Table 3. Soil water variations (%) for different depth at 0–120 cm after four precipitation events. The values in the table represent the absolute difference between soil water content after rainfall and that before rainfall. Signiﬁcant changes are bold, and negative values represent soil water content decreases. Precipitation event Low (2.6 mm) Medium (16.2 mm) Soil depth (cm) Poplar Grass Crop Inter-dunes Shrub Poplar Grass Crop Inter-dunes Shrub 0–20 0.55 4.75 1.34 0.71 0.64 0.85 5.08 1.83 0.76 0.69 20-40 −0.07 −0.14 −0.06 −0.08 −0.02 −0.05 −0.31 −0.9 −0.59 −0.15 40–60 0.03 −0.15 −0.06 −0.09 −0.07 −0.05 −0.03 −0.99 −0.12 −0.13 60–80 −0.1 −0.07 −0.04 −0.13 0.09 −0.01 0.15 −0.03 −0.2 −0.03 80–100 −0.05 −0.04 −0.01 −0.04 −0.05 −0.01 −0.02 −0.02 −0.12 −0.01 100–120 −0.03 0.01 −0.01 −0.01 −0.02 −0.01 −0.01 0.01 0 0 Incremental (%) 0.58 4.75 1.34 0.71 0.64 0.85 5.08 1.83 0.76 0.69 Precipitation event High (40.5 mm) Extreme high (102.4 mm) Soil depth (cm) Poplar Grass Crop Inter-dunes Shrub Poplar Grass Crop Inter-dunes Shrub 0–20 1.85 5.13 2.58 1.92 1.25 2.98 4.83 3.95 2.33 2.18 20–40 0.68 3.77 1.9 1.63 1.02 2.82 3.72 2.41 2.21 2 40–60 0.56 1.45 0.96 0.41 0.35 2.52 3.2 2.37 2.15 1.35 60–80 −0.29 −0.97 −0.65 −0.68 0.04 2.33 2.46 2.17 2.36 1.29 80–100 −0.06 −0.04 −0.08 −0.06 −0.02 1.57 2.11 1.88 2.03 1.06 100–120 −0.01 0.01 −0.01 0 −0.01 1.05 1.93 1.44 1.65 1.78 Incremental (%) 3.09 10.35 5.44 3.96 2.58 13.27 18.25 14.22 12.73 9.66 www.solid-earth.net/6/1157/2015/ 4.1.2 Proﬁle variations of soil moisture in different land uses There is a signiﬁcant difference on the surface (0–20 cm) soil water among different land uses (Table 2). This is not consistent with the results that showed variations of 0–20 cm soil water in different land uses were low over the growing seasons in the hilly area of the Loess Plateau (Chen et al., 2007). Because most of the time soil moisture is controlled by factors such as soil texture, vegetation, aspect of the hill- slope or local topography, and sometimes in semiarid land, it seems that there are other factors, such as topographical and vegetation presence, with a major inﬂuence on soil wa- ter content (Gómez-Plaza et al., 2000). Therefore, the dif- ferences of soil moisture variations between these two areas might due to the different soil texture, topography and veg- etation cover. Moreover, soil surface moisture of grassland was signiﬁcantly higher than the other land uses (p < 0.05), because of the higher clay content (Table 1) and the high water holding capacity of the surface soil. And soil surface moisture of inter-dunes was signiﬁcantly lower than the other land uses, because of the poor permeability result from heavy clay soil, and the effective water content was low (Li et al., 2010). Also, there were statistical differences (p < 0.05) of soil water in both root zone and deep soil layer for different land uses (Table 2). This may be the result of different evap- otranspiration patterns due to various distributions of plant root under different land uses (Gao et al., 2014). In general, the inﬁltration process is controlled by precipi- tation patterns, ground cover, soil characteristics, slop and initial soil moisture (Gabarrón-Galeote et al., 2013; Gómez- Plaza et al., 2000; Wang et al., 2013), which in turn affects soil water. In our study, the amount of medium precipitation was 6.2 times that of low rainfall; however the increase of soil wa- ter after medium precipitation was only 1.2 times that after low precipitation. The reason is that vegetation just begin to come into leaf in the early growing season (low precipita- tion), and that plants can intercept part of precipitation with thick branches and luxuriant foliage in mid-June (medium precipitation) (Gao et al., 2014). This is consistent with the result reported by Wang et al. (2005) that shrub community (C. korshinskii kom) canopy interception was 11.7 % of the precipitation. 4.1.2 Proﬁle variations of soil moisture in different land uses Furthermore, the study suggested that inﬁltra- tion depth increased with the increase of precipitation, indi- cating that inﬁltration depth will be greater than 120 cm after heavier precipitation (Table 3). Similarly, Yao et al. (2013) reported that inﬁltration depth increased with increasing pre- cipitation amount. Although a positive correlation was indi- cated between precipitation and inﬁltration depth, it is difﬁ- cult to quantify the relationship due to the varying initial soil water content, rainfall intensity, vegetation cover, and me- teorological conditions among the four precipitation events (Gao et al., 2014). Clearly, the proﬁle of soil moisture indicated distinct verti- cal patterns for various seasons (Fig. 5). In spring, two types were classiﬁed as increasing and waving types; these are based on soil moisture changes with depth. Cropland showed the increasing type, likely due to the low evapotranspiration in spring (Qiu et al., 2011; Stéfanon et al., 2014; Yang et al., 2014) and the loose soil characteristic for rainfall inﬁltrating. The waving type consisted of poplar land and shrub land, grassland and inter-dunes, where soil moisture presented a low–high–low change in proﬁle. A possible explanation was the different soil bulk density and root distribution. Moreover, vegetation affecting on precipitation interrup- tion for soil was to inﬂuence soil water by changing precipi- tation and soil properties (Li et al., 2013). In our study, there were no obvious differences on inﬁltration depth after four types of precipitation intensity, but the incremental soil wa- ter was different in different land uses. Under the low rainfall condition, incremental soil water of poplar land was less than that of the other land uses. The possible reason was that the vegetation canopy and litter stopped precipitation from enter- ing the soil. Furthermore, under other three types of precip- itation, incremental soil water in shrub land (C. korshinskii kom) was smaller than that of the other land uses. The ﬁrst reason was that soil water runoff was strong on shrub land due to the effect of slope (Adekalu et al., 2007) and the sec- ond reason was that shrub community was set up on mobile dunes where the soil evaporation was intense and vegetation cover was low. On the contrary, higher vegetation cover of other land uses prevented from direct solar radiation to sur- face soil and high clay (Table 1) led to higher water-holding capability. C. Y. Niu et al.: Analysis of soil moisture condition in arid region of northern China 1164 4 Discussion 4.1 Soil moisture variability for different land uses 4.1.1 Temporal variations of mean soil moisture within 0–120 cm Therefore, land use was the main reason that affected the spatial variations, and pre- cipitation was the main factor that inﬂuenced the temporal patterns, which was consistent with the recent ﬁndings from Yao et al. (2012). Moreover, the temporal variations of soil moisture proﬁle changed with precipitation (Fig. 4). Surface (0–20 cm) soil water for all different land uses correlated positively with Solid Earth, 6, 1157–1167, 2015 www.solid-earth.net/6/1157/2015/ 5 Conclusions Climate change affects the hydrological cycle (Parry et al., 2007) and is likely to reduce summer precipitation over the central parts of arid and semi-arid Asia. This will aggravate water conditions, leading to serious desertiﬁcation. There- fore, it is vitally important to make efﬁcient use of the lim- ited water resources to improve the ecological environment and agricultural water management in these regions (Gao et al., 2014). Different land uses contributed to the soil moisture variations along with rainfall, topography and soil properties. The tem- poral variations of mean soil moisture in different land uses were not always consistent with the rainfall due to the dry sequence. The cropland and poplar land showed a lag effect following a rain event due to the canopy interception and the buffering inﬂuence of groundcover. Moreover, soil water in surface, root zone and deep soil layer indicated statistical dif- ferences under different types of land cover. Meanwhile, Soil water proﬁles under different land uses that changed with precipitation, soil water in surface, and root zone horizons were driest during April to September, and soil water lag was observed in deep soil layer. Vertical soil water proﬁles exhib- ited seasonal patterns for different land uses, and the stable layer of soil moisture was found at 80–120 cm. Moreover, a positive correlation existed between precipitation and inﬁl- tration depth for all land uses. Those results give us an insight into land and water management, an irrigation scheme must be worked out to meet water requirements based on soil wa- ter content and evapotranspiration, thereby improving water use efﬁciency. All of this could also help to control soil ero- sion and land degradation better. Grassland represented more adaptive vegetation in this area. Therefore, the local govern- ment should establish fenced meadow and decrease grazing properly to protect natural grassland and plant trees with the reasonable density in the ﬂowing sandy land and inter-dune land. Because cropland could induce severer soil erosion and land degradation, other land uses in terms of artiﬁcial forest and shrub (e.g., poplar and C. korshinskii kom in our site) and natural rehabilitation (e.g., fenced grassland in our site) have been suggested to plant and protect by fence (Zhang et al., 2012). In addition, soil moisture of deep layer in shrub land was signiﬁcantly lower than that in the other land uses (Table 2), soil moisture would decline in C. 5 Conclusions korshinskii kom shrub land for the reason soil water could not get the supple- ment from the deep soil water resulting from C. korshinskii kom had deep and enormous roots for water uptake (She et al., 2013). Moreover, surface soil water of poplar land was signiﬁcantly (P < 0.001) lower than that in grassland (Ta- ble 2), and water consumption of poplar was higher than grassland vegetation (Kang et al., 2008), therefore, the sur- face soil layer could be dry and deteriorate due to precipita- tion interruption and deep-root water uptake (Wang et al., 2013). Consequently, this would result in the competitive disadvantage of deep-rooted trees and shrub. If this situa- tion continued, “low, thin, old trees” and further degrada- tion might occur (Zhao et al., 2009). In the light of high soil moisture maintained in grassland and inter-dunes, herba- ceous plants are likely to be an adaptive and stable vegetation type under this environmental condition. Acknowledgements. We thank Yongming Luo and Hongmei Wang for assistance with ﬁeld and laboratory work. The project was supported by the State Key Laboratory of Forest and Soil Ecology (LFSE2014-06) and the National Basic Research Program of China (2013CB429902). The authors gratefully acknowledge the ﬁnancial support and Ulan’aodu Station for providing climate data of this region. Furthermore, catchment water balance and the factors that affected water balance were inﬂuenced by different land uses. Dai et al. (2006) reported that land use was the main factor that affected water balance and evapotranspiration was the largest expenditure in land water balance. Moreover, comparison (from high to low) of the evapotranspiration in different land uses was cropland, arbor, shrub land and grass- land (Kang et al., 2008). In addition, our study indicated that surface soil moisture exhibited relatively low values (< 5 %) during the growing season (May–July) (Fig. 4a). Thus, an irrigation scheme must be worked out to meet the water re- quirement and then increase soil water utilization according to soil water content and evapotranspiration. Edited by: P. Pereira C. Y. Niu et al.: Analysis of soil moisture condition in arid region of northern China above, both grassland and inter-dunes could maintain higher soil moisture, which would be beneﬁcial for the stable devel- opment of plant community in this landscape. We should also reasonably manage efﬁcient water use to maintain the water balance and the development of a stable vegetation community. 4.1.2 Proﬁle variations of soil moisture in different land uses Moreover, in summer, soil moisture in the poplar land, grassland, and inter-dunes presented a low–high–low change with depth, while the cropland and shrub land presented a low–high–low–high–low change, likely due to the different evapotranspiration of different vegetation (Li et al., 2009; Wagendorp et al., 2006). Furthermore, soil moisture showed a stable (high–low– high–low) trend with wave-changing type at 0–120 cm ex- cept grassland in autumn (Fig. 5c). Grassland soil moisture gradually decreased with depth from 100 to 120 cm, which was not consistent with the recent ﬁndings (Gao et al., 2014). The possible reason was the differences in topography, which was the main factor controlling time stability (Gómez-Plaza et al., 2000). Furthermore, land type of inter-dunes had a higher soil bulk density and clay content (Table 1), leading to a slow in- ﬁltration rate, and the incremental soil water was smaller than cropland, poplar land and grassland (Fig. 3). Therefore, it could maintain a high soil water level for a long time, which contributed to the growing herbaceous plants. As discussed Solid Earth, 6, 1157–1167, 2015 www.solid-earth.net/6/1157/2015/ www.solid-earth.net/6/1157/2015/ 1165 References Abadi Ghadim, A. K.: Water repellency: a whole-farm bio- economic perspective, J. Hydrol., 231–232, 396–405, 2000. Adekalu, K. O., Olorunfemi, I. A., and Osunbitan, J. A.: Grass mulching effect on inﬁltration, surface runoff and soil loss of three agricultural soils in Nigeria, Bioresource Technol., 98, 912–917, 2007. 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https://openalex.org/W2165139285	https://europepmc.org/articles/pmc4090653?pdf=render	English	null	Omental whirl associated with bilateral inguinal hernia: a case report	Journal of medical case reports	2,014	cc-by	2,808	Open Access Open Access © 2014 Silva 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. Silva et al. Journal of Medical Case Reports 2014, 8:239 http://www.jmedicalcasereports.com/content/8/1/239 Silva et al. Journal of Medical Case Reports 2014, 8:239 http://www.jmedicalcasereports.com/content/8/1/239 JOURNAL OF MEDICAL CASE REPORTS JOURNAL OF MEDICAL CASE REPORTS Abstract Introduction: Torsion of the omentum is a rare cause of abdominal pain. It is clinically similar to common causes of acute surgical abdomen and is often diagnosed during surgery. Inguinal hernia is a common condition but not frequently related with torsion of the omentum. Case presentation: A 40-year-old Caucasian man came to our emergency department with abdominal pain of the left quadrant and abdominal distension for 2 days. His medical history included an untreated left inguinal hernia in the last year. Computed tomography revealed densification of mesocolon with left omentum “whirl” component and other signs of omental torsion. During an exploratory laparoscopy, a wide twist of his omentum with necrotic alterations that extended to the bilateral inguinal hernial content was observed. Omentectomy and surgical repair of bilateral inguinal hernia were performed. Conclusions: Torsion of the omentum is a rare entity and usually presents a diagnostic challenge. The use of abdominal computed tomography can help diagnosing torsion of the omentum preoperatively and, thus, prevents a surgical approach. Nonetheless, some cases of torsion of the omentum require surgical repair. Accordingly, a laparoscopic approach is minimally invasive and efficient in performing omentectomy. Keywords: Abdominal pain, Omentum, Torsion, Whirl sign Omental whirl associated with bilateral inguinal hernia: a case report Elsa Silva1, Ana Franky Carvalho1,2,3, Diogo Rocha4, António Mesquita Rodrigues1,2, Ricardo Pereira1, Ana João Rodrigues2,3 and Pedro Leão1,2,3* * Correspondence: pedroleao@ecsaude.uminho.pt 1General Surgery, Hospital of Braga, 4701-965, Braga, Apartado 2242, Portugal 2Life and Health Sciences Research Institute (ICVS), School of Health Sciences, University of Minho, Braga, Portugal Full list of author information is available at the end of the article Introduction other organs such as acute cholecystitis, pancreatitis, and adnexitis, tumors, and postsurgical adhesions [1]. There are also predisposing factors such as sex, obesity, sudden strong increase in intra-abdominal pressure (brought on by coughing or violent exercise), traumas, autonomics (large pedicle, larger or more twisted than normal epiploic blood vessels), hyperperistalsis, surgical adherences, or some acute process in an intracavitary organ causing displace- ment of the omentum [5,6]. Torsion of the omentum is a clinical condition in which the organ rotates on its long axis compromising its vascularity. Segmental torsion of the greater omentum is more common; it was first described by Bush in 1896 [1]. By 2001, slightly fewer than 300 cases had been reported, almost all misdiagnosed as acute appendicitis and discovered during an exploratory laparotomy [2,3]. The condition is more common in males, with a ratio of 2:1 in the third decade of life and 5:1 in the fourth dec- ade of life [1,4], but it can occur at any age and it can be primary or secondary [5]. Primary torsion (idiopathic) is less common [1], whereas secondary torsion is associated with pre-existing conditions including longer than normal omentum, internal hernias, inflammatory pathologies of Torsion of the omentum presents nonspecific clinical symptoms, namely moderate abdominal pain that may be similar to acute appendicitis. Thus, it is difficult to obtain a preoperative clinical diagnosis. According to the literature, ultrasound and computed tomography (CT) are useful tools for establishing a preoperative diagnosis [7,8]. The purpose of this publication is to present a case report of a patient with a rare condition of unusual clinical presentation. We report a case of torsion of the greater omentum diagnosed preoperatively associated with bilateral inguinal hernias. Moreover, we also show * Correspondence: pedroleao@ecsaude.uminho.pt 1General Surgery, Hospital of Braga, 4701-965, Braga, Apartado 2242, Portugal 2Life and Health Sciences Research Institute (ICVS), School of Health Sciences, University of Minho, Braga, Portugal Full list of author information is available at the end of the article Page 2 of 4 Silva et al. Journal of Medical Case Reports 2014, 8:239 http://www.jmedicalcasereports.com/content/8/1/239 Silva et al. Journal of Medical Case Reports 2014, 8:239 http://www.jmedicalcasereports.com/content/8/1/239 reducing the hernia content, omentectomy was performed. His abdominal cavity was reached using a 10mm tro- car under his umbilicus. Two accessory trocars were placed in his right flank (5mm). Introduction A median minilapar- otomy (6cm) through the umbilicus port was created for exteriorization and resection of his large necrotic omentum (Figure 2B). Surgical repair of bilateral in- guinal hernia was performed by Lichtenstein technique, since the success of laparoscopic inguinal hernia repair techniques (such as transabdominal preperitoneal) in in- carcerated hernias is still controversial [10,11]. A histo- pathological examination revealed large areas of interstitial hemorrhage, vascular congestion and thrombosis compat- ible with omental torsion. that minimally invasive surgery can help to confirm the diagnosis, while providing treatment and a better post- operative recovery. Case presentation A 40-year-old Caucasian man was admitted to our emer- gency department with moderate pain of his left ab- domen for 2 days. He also complained of abdominal distension but presented no other signs and symptoms like fever, nausea, vomiting, diarrhea or intestinal ob- struction. His medical history included an untreated left inguinal hernia in the last year. A physical examination revealed pain in his left abdomen associated with a palp- able mass on his left flank, without rebound tenderness or guarding. His postoperative period occurred without complica- tions and he was discharged on the third day after surgery. One month later he yielded no complaint. Two bilateral reducible inguinal hernias were found. Laboratory results revealed a C-reactive protein of 124mg/L. Given this unspecific clinical presentation, an abdominal and pelvic CT scan were performed, re- vealing an intra-abdominal abnormality, suggestive of pathologic infiltration of his omentum and the pres- ence of the whirl sign suggesting torsion of his greater omentum (Figure 1A and 1B). The distal end of his omentum seemed to be alongside the spermatic cord into a bilateral inguinal hernia sac. Since there was no evidence of acute abdomen or incarcerated hernia and he was stable, we decided on conservative treatment because it has been reported that spontaneous derota- tion of the omentum can occur [9]. After he had spent 2 days as an in-patient of our hospital, his pain per- sisted and his left inguinal hernia became incarcerated. A preoperative diagnosis of secondary torsion of his greater omentum associated with an inguinal bilateral hernia (particularly left inguinal hernia) was established and he submitted to surgery. Authors’ contributions S d P l d h ES and PL evaluated the patient. AFC, AJR, AMR and PL were involved in drafting the manuscript and revising it critically for important intellectual content. DR was involved in CT scanning and imaging evaluation. PL, RP and ES performed the laparoscopic omentectomy and hernia repair. PL and AMR were involved in revising it critically and have given final approval of the version to be published. All authors read and approved the final manuscript Discussion l Omental torsion is a benign rare cause of acute abdo- men, easily misdiagnosed as acute appendicitis, acute cholecystitis or diverticulitis. It is defined as axial twist- ing along the long axis of the omentum to such an extent that its vascularity is compromised. It most frequently affects the right side because the right side of the omentum is longer than the left side, more mobile and less richly vascularized with poor collateralization [12,13]. However, in this case, we found a complete tor- sion of the greater omentum with necrosis secondary to incarceration in a bilateral inguinal hernia, more evident on the left side. The predisposing factors identified in this case were sex (more frequent in males), a large ped- icle and history of untreated inguinal hernia. Given the few clinical signs, the preoperative diagnosis was largely based on radiological findings. CT findings of greater omental torsion include a well circumscribed, oval, or cake-like fatty mass with heterogeneous attenuation, containing strands of soft tissue attenuation [12] and particularly the presence of concentric linear strands Laparoscopy was performed to evaluate the extent of the damage to his omentum. A wide twist of omentum with large necrotic vascular changes that extended to the inguinal hernia content was found (Figure 2A). After Figure 1 Computed tomography scans showing the whirl (arrow) of the omentum. A) caudal view. B) cranial view. Figure 1 Computed tomography scans showing the whirl (arrow) of the omentum. A) caudal view. B) cranial view. Page 3 of 4 Silva et al. Journal of Medical Case Reports 2014, 8:239 http://www.jmedicalcasereports.com/content/8/1/239 Figure 2 Intraoperatory view of the whirl of the omentum. A) Exteriorization of the omentum after minilaparotomy showing the precise point of torsion. B) Complete exeresis of the twisted omentum. Figure 2 Intraoperatory view of the whirl of the omentum. A) Exteriorization of the omentum after minilaparotomy showing the precise point of torsion. B) Complete exeresis of the twisted omentum. Conclusions (the “whirl sign”). Of notice, the whirl sign may not be as apparent if the axis of rotation is not perpen- dicular to the transverse scanning plane [14]. Although a CT scan is helpful in diagnosing torsion of the omentum and may prevent an unnecessary surgery, the extension of omental torsion may not be clearly visualized through this technique, rendering close vigilance of clinical resolution/ deterioration. Segmental omental torsion is usually a benign and self-limiting disease [15], capable of evolv- ing to resolution within 2 weeks with conservative mea- sures. This fact was taken into consideration since the patient experienced no clinical signs of deterioration in the emergency service. The treatment of complete tor- sion of the greater omentum with secondary necrosis is usually surgical [8]. In this case, a laparoscopic approach should be considered because it is less invasive and associated with lower morbidity. Moreover, when pre- operative diagnosis is not clear by imaging techniques, laparoscopy is useful for both diagnosis and treatment [13,16]. The criticism to this approach is the surgical treatment that was used for the treatment of the hernias, since laparoscopic repair of incarcerated, non-reducible groin hernias has to be done urgently and can be performed with an endoscopic technique, as advised by the European Association for Endoscopic Surgery [17]. However, a surgeon should not endanger the patient's life. In this perspective, there is controversy regarding the laparoscopic approach to large incarcerated inguinal hernias. Conservative treatment of primary torsion has been described in cases of partial omental torsion. However, the treatment of complete torsion of the greater omen- tum with secondary necrosis is surgical. The presence of precipitating factors for torsion of the omentum (secondary torsion) should render close vigilance of the patient as the situation can worsen. It is still not clear whether treatment of the precipitating factor should be the initial approach. Nonetheless, the use of a laparo- scopic approach seems to be successful in such a benign situation. Consent Written informed consent was obtained from the patient for publication of this case report and any accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal. Competing interests The authors declare that they have no competing interests. Author details 1 The combination of imaging techniques and minimally invasive surgery were helpful in making a correct diag- nosis and avoiding a more invasive surgical approach such as laparotomy. 1General Surgery, Hospital of Braga, 4701-965, Braga, Apartado 2242, Portugal. 2Life and Health Sciences Research Institute (ICVS), School of Health Sciences, University of Minho, Braga, Portugal. 3ICVS/3B’s - PT Government Associate Laboratory, Braga/Guimarães, Portugal. 4Radiology Department, Hospital of Braga, 4701-965 Braga, Portugal. Page 4 of 4 Page 4 of 4 Silva et al. Journal of Medical Case Reports 2014, 8:239 http://www.jmedicalcasereports.com/content/8/1/239 References 1. Montiel-Jarquin A: Clinical characteristics of torsion of the omentum. Gastroenterol Res 2009, 2:220–223. 1. Montiel-Jarquin A: Clinical characteristics of torsion of the omentum. Gastroenterol Res 2009, 2:220–223. 2. 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References Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color ﬁgure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color ﬁgure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and take full advantage of: Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission
https://openalex.org/W4385954968	https://www.researchsquare.com/article/rs-2972030/latest.pdf	English	null	Bibliometric Analysis on Mercury Emissions from Coal-fired Power Plants: A Systematic Review and Future Prospect	Research Square (Research Square)	2,023	cc-by	15,254	Research Article Version of Record: A version of this preprint was published at Environmental Science and Pollution Research on February 21st, 2024. See the published version at https://doi.org/10.1007/s11356-024-32369-z. Page 1/33 Abstract Coal-fired power plants (CFPPs) are one of the most significant sources of mercury emissions certified by the Minamata Convention, which has attracted much attention in recent years. In this study, we used the Web of Science (WOS) and CiteSpace to analyze the knowledge structure of this field from 2000 to 2022 and then reviewed it systematically. The field of mercury emissions from coal-fired power plants has developed steadily. The research hotspots can be divided into three categories: 1) emission characterization research focused on speciation changes and emission calculations; 2) emission control research focused on control technologies; 3) environmental impact research focused on environmental pollution and health risk. In conclusion, using an oxygen-rich atmosphere for combustion and installing high-efficiency air pollution control devices (APCDs) helped to reduce the formation of Hg0. The average mercury removal rates of APCDs and modified adsorbents after ultra-low emission retrofit were distributed in the range of 82-93% and 41-100%, respectively. The risk level of mercury in combustion by-products was highest in desulfurization sludge (RAC>10%) and followed by fly ash(10<RAC<30%) and desulfurization gypsum(1<RAC<10%). Additionally, we found that the implementation of pollution and carbon reduction policies in China had reduced mercury emissions from CFPPs by 45% from 2007 to 2015, increased the efficiency of mercury removal from APCDs to a maximum of 96%, and reduced global transport and health risk of atmospheric mercury. The results conjunctively achieved by CiteSpace and the literature review will enhance understanding of CFPPs mercury emissions research and provide new perspectives for future research. 1 Introduction Nowadays, mercury emissions from the coal combustion process are an international research hotspot; due to its toxicity, long-range transport, and bioaccumulation properties (Giang et al., 2015; Pavlish et al., 2003). In response to global mercury emission controls, the United Nations adopted the Minamata Convention on January 19, 2013, which has been in force since August 16, 2017. Currently, 91 countries, including China, have signed the Minamata Convention. The Minamata Convention identified that coal-fired power plants (CFPPs) are one of the five significant anthropogenic sources of mercury and its compounds. Additionally, atmospheric mercury emissions were about 603 tons in China, approximately 30% of the world's total mercury emissions in 2015 (Yu et al., 2021), and of which CFPPs accounted for 33% of China's emissions in the same year (Guo et al., 2021). The prevention and control of mercury pollution in China's CFPPs is of great significance to global mercury emission reduction. Research on mercury emissions from CFPPs has been strengthened in recent years, and the research system covers a wide range of research topics from sources to emissions and deposition. The research status of mercury emissions from CFPPs can be revealed by exploring the evolution, hotspots, and knowledge clusters, which can assist scholars identify and develop new research frontiers. The bibliometric method is a quantitative analysis method of literature based on statistics. Collecting external features such as publication year, keywords, authors, and institutions, and analyzing their distribution and change patterns, helps to grasp the research hotspots and development trend of a knowledge field as a whole(Liu et al., 2023). The commonly used bibliometric tools include CiteSpace, Bibexcel, WOS Viewer, Gephi, SATI, etc. (Moral-Muñoz et al., 2020). CiteSpace, also known as "citation space", is a visual analysis tool for Page 2/33 Page 2/33 visualizing and describing trends in research data by constructing collaborative information and co- occurrence networks in the literature (Chen, 2006). It has been widely used in many research fields such as information science, medicine, and environmental science (Chen et al., 2012; Hou et al., 2018; Zhu and Hua, 2017). Chao et al. reviewed heavy metal pollution in CFPPs using bibliometric studies and the results showed that the recent hotspots were harmful trace elements and speciation analysis (Chao et al., 2021). Although atmospheric mercury is the most frequent keyword in this knowledge field, there is still a gap in the metrological analysis of the subcategory of mercury emissions from CFPPs. 1 Introduction In this study, we utilized the bibliometric software CiteSpace and the date from the Web of Science (WOS) core collection to analyze the knowledge structure of mercury emissions from CFPPs from 2000 to 2022. The aim was to identify the research power network in the knowledge area and to identify the research hotspots and evolutionary pathways of mercury emissions from CFPPs. Based on this analysis, we conducted a literature review to explore the hotspots and suggest future research trends and directions in the context of current policies for pollution and carbon reduction. We believed that our findings would provide references and a fresh perspective for future research. In this study, we utilized the bibliometric software CiteSpace and the date from the Web of Science (WOS) core collection to analyze the knowledge structure of mercury emissions from CFPPs from 2000 to 2022. The aim was to identify the research power network in the knowledge area and to identify the research hotspots and evolutionary pathways of mercury emissions from CFPPs. Based on this analysis, we conducted a literature review to explore the hotspots and suggest future research trends and directions in the context of current policies for pollution and carbon reduction. We believed that our findings would provide references and a fresh perspective for future research. 2 Data and methods The data for this article come from the Web of Science (WOS) system, one of the most widely used databases for academic and bibliometric research. The "Web of Science Core Collection" database was chosen, which includes six sub-databases (SCIE, SSCI, A&HCI, ESCI, CCR-EXPANDED, and IC) covering academic papers from core journals. The search term topic consists of two parts: A and B. A encompasses different types of coal- fired power generation facilities, such as "coal-fired power plants," "coal-fired power stations," and "coal-fired power industry." Meanwhile, B includes various ways to refer to mercury emissions, such as "mercury emissions" or "Hg emissions". The retrieval period was set as "2000–2022" and the system language as "English". After removing non-compliant data, filling in missing information, and eliminating duplicate literature, a total of 564 documents were obtained. CiteSpace uses scientometric methods and data visualization to identify key information in the literature. To highlight the core structure of the network, it sets a threshold for the number of network nodes and only visualizes citations that meet the threshold conditions. Threshold setting methods include Top N, Top N%, Threshold Interpolation, and Select Citers. "Top N" was selected as the setting method, with N set to 50, representing the 50 citations with the highest number of citations each year that were selected to appear in the visualization mapping. In addition, the relationship between research objects can be reflected by the number of nodes, the number of links, and link density. Keywords clustering is a tool that identifies closely related groups of keywords by analyzing their co- occurrence patterns. Its primary objective is to reveal thematic clusters containing multiple sets of keywords. The effectiveness of keywords clustering can be evaluated using two measures: the clustering module value Modularity (Q) and the clustering mean silhouette (S). When Q is higher than 0.3 and S is higher than 0.7, it generally indicates convincing clustering results and significant clustering structure. Page 3/33 Page 3/33 However, CiteSpace can only visually analyze the external characteristics of literature. Therefore, this paper combines the content of the literature and carried out a further literature review based on bibliometric analysis. However, CiteSpace can only visually analyze the external characteristics of literature. Therefore, this paper combines the content of the literature and carried out a further literature review based on bibliometric analysis. 3.1 Publications and journals Figure 1 illustrates the posting volume trends of studies on mercury emissions from CFPPs during 2000– 2022, showing an overall increasing trend. Some researchers started to focus on mercury emissions from CFPPs in the early 21st century. The first peak was reached in 2007 and the number of publications exceeded 30 for the first time. Since then, mercury emissions from CFPPs had received significant attention from scholars, with the number of publications remaining above 25 in most years. The highest number of publications was recorded in 2021, reaching 44. Based on the posting volume trends, we divided the research phases on mercury emissions from CFPPs during 2000–2022 into three periods. The first period (2000–2005) was the embryonic period, during which scholars recognized the importance of CFPPs as an anthropogenic source of mercury emissions and studied the distribution and conversion of mercury during coal burning. The second period (2006–2014) was the development period, which followed the enactment of the Clean Air Mercury Act (2005) by the U.S. Based on the posting volume trends, we divided the research phases on mercury emissions from CFPPs during 2000–2022 into three periods. The first period (2000–2005) was the embryonic period, during which scholars recognized the importance of CFPPs as an anthropogenic source of mercury emissions and studied the distribution and conversion of mercury during coal burning. The second period (2006–2014) was the development period, which followed the enactment of the Clean Air Mercury Act (2005) by the U.S. Environmental Protection Agency. It was mainly focused on mercury control technology research about activated carbon materials and elemental mercury removal, as well as the strengthening of establishing mercury emission inventories. The last period (2015–2022) was the continuous development period, which followed the implementation of ultra-low emission retrofits for CFPPs in China (2014) and the entry into force of the United Nations Minamata Convention on Mercury (2017). During this period, the emission reduction options for these policies, emission reduction potential, and environmental impact of the policies were proposed and analyzed. Environmental Protection Agency. It was mainly focused on mercury control technology research about activated carbon materials and elemental mercury removal, as well as the strengthening of establishing mercury emission inventories. The last period (2015–2022) was the continuous development period, which followed the implementation of ultra-low emission retrofits for CFPPs in China (2014) and the entry into force of the United Nations Minamata Convention on Mercury (2017). 3.1 Publications and journals During this period, the emission reduction options for these policies, emission reduction potential, and environmental impact of the policies were proposed and analyzed. Table 1 displays the number of publications from the top 10 journals that contribute to the field of knowledge on mercury emissions from CFPPs from 2000 to 2022. Fuel topped the list with 54 publications (9.6%). The second is Environmental Science & Technology, which published 48 publications (8.5%). Chemical Engineering Journal (IF = 16.744) was the journal with the highest impact factors. The top 10 journals reflected the interdisciplinary nature of the field and encompassed a variety of disciplines, such as environmental science, chemistry, and energy science. Page 4/33 Table 1 Top 10 productive journals from 2000 to 2022. Top 10 productive journals from 2000 to 2022. Rank Journal aTP b% cIF 1 Fuel 54 9.6 8.035 2 Environmental Science & Technology 48 8.5 11.357 3 Energy & Fuels 39 6.9 4.654 4 Fuel Processing Technology 28 5.0 8.129 5 Atmospheric Environment 21 3.7 5.755 6 Science of The Total Environment 19 3.4 10.754 7 Journal of Hazardous Materials 19 3.4 14.224 8 Journal of the Air & Waste Management Association 14 2.5 1.567 9 Chemical Engineering Journal 13 2.3 16.744 10 Environmental Pollution 12 2.1 9.988 11 Atmospheric Chemistry and Physics 12 2.1 7.197 ber of total articles; b%, The proportion of journal articles in the total articles; cTF, Impact facto aTP, Number of total articles; b%, The proportion of journal articles in the total articles; cTF, Impact factor in 2021. aTP, Number of total articles; b%, The proportion of journal articles in the total articles; cTF, Impact factor in 2021 Table 2 provides an overview of the top 10 highly cited publications in the field, covering the period between 2000 and 2015, with the most frequently cited article garnering 895 citations. These publications contained mainly topics from energy, atmospheric science, and environmental protection research, which reflected that the authoritative publications on mercury emissions from CFPPs were mainly focused on research related to energy, atmospheric science, and environmental protection, and the economic and social dimensions have not been fully developed. In addition, the top 10 highly cited publications were published in top-tier journals, including Fuel Processing Technology and Environmental Science & Technology, which were among the top 10 journals in the field of mercury emission from CFPPs (see Table 1). 3.1 Publications and journals Rank Title aTC Year Journal 1 Status review of mercury control options for coal-fired power plants 895 2003 Fuel Processing Technology 2 Anthropogenic mercury emissions in China 644 2005 Atmospheric Environment 3 Mercury emission and speciation of coal-fired power plants in China 360 2010 Atmospheric Chemistry and Physics 4 Updated Emission Inventories for Speciated Atmospheric Mercury from Anthropogenic Sources in China 344 2015 Environmental Science & Technology 5 Trace elements in coal: Associations with coal and minerals and their behavior during coal utilization - A review 312 2010 Fuel 6 Adsorbents for capturing mercury in coal-fired boiler flue gas 311 2007 Journal of Hazardous Materials 7 Gas-phase transformations of mercury in coal-fired power plants 305 2000 Fuel Processing Technology 8 Trends and characteristics of atmospheric emissions of Hg, As, and Se from coal combustion in China, 1980–2007 278 2010 Atmospheric Chemistry and Physics 9 Towards the development of a chemical kinetic model for the homogeneous oxidation of mercury by chlorine species 247 2000 Fuel Processing Technology 10 Control of mercury emissions from coal-fired electric utility boilers 241 2006 Environmental Science & Technology aTC, Total number of citations by other literature 3.2 Contributions of countries and institutions Figure 2(a) depicts the network of relations between countries and regions involved in the publication of articles on mercury emissions from CFPPs during 2000–2022, with 52 countries and territories represented. Except for China, the majority of publications originated from developed countries and regions, which consisted of the current global economic and cultural development patterns. China and the United States were central to this network, with Canada, Spain, South Korea, Poland, Australia, Japan, and the United Kingdom among the countries and regions that work more closely with them. Figure 2(b) presented the number and Figure 2(a) depicts the network of relations between countries and regions involved in the publication of articles on mercury emissions from CFPPs during 2000–2022, with 52 countries and territories represented. Except for China, the majority of publications originated from developed countries and regions, which consisted of the current global economic and cultural development patterns. China and the United States were central to this network, with Canada, Spain, South Korea, Poland, Australia, Japan, and the United Kingdom among the countries and regions that work more closely with them. 3.1 Publications and journals Overall, Fuel Processing Technology, Atmospheric Chemistry and Physics, Environmental Science & Technology, Atmospheric Environment, Fuels, and Journal of Hazardous Materials were highly cited and published journals in the field of mercury emissions from CFPPs and may be of particular significance. Page 5/33 Table 2 Table 2 Top 10 cited literature from 2000 to 2022. Rank Title aTC Year Journal 1 Status review of mercury control options for coal-fired power plants 895 2003 Fuel Processing Technology 2 Anthropogenic mercury emissions in China 644 2005 Atmospheric Environment 3 Mercury emission and speciation of coal-fired power plants in China 360 2010 Atmospheric Chemistry and Physics 4 Updated Emission Inventories for Speciated Atmospheric Mercury from Anthropogenic Sources in China 344 2015 Environmental Science & Technology 5 Trace elements in coal: Associations with coal and minerals and their behavior during coal utilization - A review 312 2010 Fuel 6 Adsorbents for capturing mercury in coal-fired boiler flue gas 311 2007 Journal of Hazardous Materials 7 Gas-phase transformations of mercury in coal-fired power plants 305 2000 Fuel Processing Technology 8 Trends and characteristics of atmospheric emissions of Hg, As, and Se from coal combustion in China, 1980–2007 278 2010 Atmospheric Chemistry and Physics 9 Towards the development of a chemical kinetic model for the homogeneous oxidation of mercury by chlorine species 247 2000 Fuel Processing Technology 10 Control of mercury emissions from coal-fired electric utility boilers 241 2006 Environmental Science & Technology aTC, Total number of citations by other literature 3.2 Contributions of countries and institutions Figure 2(a) depicts the network of relations between countries and regions involved in the publication of articles on mercury emissions from CFPPs during 2000–2022, with 52 countries and territories represented. Except for China, the majority of publications originated from developed countries and regions, which consisted of the current global economic and cultural development patterns. China and the United States were central to this network, with Canada, Spain, South Korea, Poland, Australia, Japan, and the United Kingdom among the countries and regions that work more closely with them. Figure 2(b) presented the number and proportion of publications from the top 10 productive countries and regions in the field of mercury emissions f CFPP b t 2000 d 2022 Chi th l di t i t f bli ti ith 264 Top 10 cited literature from 2000 to 2022. Top 10 cited literature from 2000 to 2022. 3.1 Publications and journals Overall, recent articles from countries such as China, the USA, and Canada and from institutions such as Huazhong University of Science and Technology, Tsinghua University, Chinese Academy of Sciences, North China Electric Power University, and Southeast University may be of particular significance in this field. 3.3 Research hot topics Keywords play a vital role in articles as they condense the topic and essence of the article into a few words. In the study of mercury emissions from CFPPs, we excluded keywords such as "coal-fired power plants", "mercury emissions", and "emissions" to determine the hot topics in this field. Figure 3(a) showed the keyword network from 2000 to 2022, with 483 nodes and 2488 connections. Statistical high-frequency keywords were often used to identify hot issues in a research field. Figure 3(b) shows the top 10 keywords for occurrences, with 6 keywords having a frequency of ≥ 60 times. These high-frequency keywords included "Coal combustion", "flue gas", "elemental mercury" and "fly ash", which were the main focus of scholars in this field. To further understand the knowledge structure and research hotspots of mercury emissions from CFPPs, we used the clustering results of CiteSpace to generate a cluster graph shown in Fig. 3(c). The research in this field mainly revolved around CFPPs and mercury emissions, resulting in the formation of nine keyword clusters (#0 elemental mercury, #1 Air, #2 emission factor, #3 trace elements, #4 air pollution control devices, #5 fly ash, #6 mercury control, #7 Air Pollution, and #8 mercury measurement) that form three main hot topics: characteristics of mercury emissions from CFPPs, control of mercury emissions from CFPPs, and environmental impacts of mercury emissions from CFPPs. 3.1 Publications and journals Figure 2(b) presented the number and proportion of publications from the top 10 productive countries and regions in the field of mercury emissions from CFPPs between 2000 and 2022. China was the leading country in terms of publications, with 264 articles (38.2%), followed by the United States with 183 articles (26.5%), accounting for over 60% of the total Page 6/33 Page 6/33 number of publications. All published in Canada, Spain, South Korea, Poland, Australia, and Japan were more than 10 articles. Figure 2(c) illustrates the cooperation among the major issuing institutions involved in publishing articles on mercury emissions from CFPPs during 2000–2022. The network consisted of 365 nodes and 514 connections, with a connection density of 0.08. Huazhong University of Science and Technology had the highest number of publications and more links, indicating extensive collaboration with other institutions, although not closely. 71% of institutions had only one publication, indicating that there were more mercury- emitting research institutions in CFPPs, while they had previously collaborated independently. Figure 2(d) listed the top 10 productive research institutions in terms of global publications from 2000 to 2022. Huazhong University of Science and Technology ranked first with 36 publications, followed by Tsinghua University, Chinese Academy of Sciences, and North China Electric Power University, all with more than 20 papers. Overall, recent articles from countries such as China, the USA, and Canada and from institutions such as Huazhong University of Science and Technology, Tsinghua University, Chinese Academy of Sciences, North China Electric Power University, and Southeast University may be of particular significance in this field. Figure 2(c) illustrates the cooperation among the major issuing institutions involved in publishing articles on mercury emissions from CFPPs during 2000–2022. The network consisted of 365 nodes and 514 connections, with a connection density of 0.08. Huazhong University of Science and Technology had the highest number of publications and more links, indicating extensive collaboration with other institutions, although not closely. 71% of institutions had only one publication, indicating that there were more mercury- emitting research institutions in CFPPs, while they had previously collaborated independently. Figure 2(d) listed the top 10 productive research institutions in terms of global publications from 2000 to 2022. Huazhong University of Science and Technology ranked first with 36 publications, followed by Tsinghua University, Chinese Academy of Sciences, and North China Electric Power University, all with more than 20 papers. 4.1 Emission characteristics Studies on mercury emissions from anthropogenic sources consistently pointed out that CFPPs were the primary contributor. Consequently, numerous investigations had been conducted on the emissions and characteristics of mercury from CFPPs. Related clusters included the #2 emission factor and the #8 mercury measurement. The main thematic keywords included were mercury emission, speciation, behavior, emission inventory, etc. Page 7/33 Page 7/33 The research work on this topic could be divided into two levels. At the macro level, the amount of mercury emitted to the atmosphere from CFPPs in a region was accounted for and an inventory of atmospheric mercury emissions was established, and the spatial and temporal distribution characteristics of atmospheric mercury emissions from CFPPs are identified to provide data to support the formulation of policies and standards to constrain atmospheric mercury emissions. At the microscopic level, fuel coal, bottom ash, fly ash, desulfurization gypsum, desulfurization wastewater, and flue gas were sampled and analyzed to investigate the migration patterns and influencing factors of mercury throughout the entire process, from coal combustion to flue gas emission. The research work on this topic could be divided into two levels. At the macro level, the amount of mercury emitted to the atmosphere from CFPPs in a region was accounted for and an inventory of atmospheric mercury emissions was established, and the spatial and temporal distribution characteristics of atmospheric mercury emissions from CFPPs are identified to provide data to support the formulation of policies and standards to constrain atmospheric mercury emissions. At the microscopic level, fuel coal, bottom ash, fly ash, desulfurization gypsum, desulfurization wastewater, and flue gas were sampled and analyzed to investigate the migration patterns and influencing factors of mercury throughout the entire process, from coal combustion to flue gas emission. 4.1.1 Emissions of mercury There are three models that are used for estimating flue gas mercury emissions from CFPPs: emission factor models, mass balance models, and flue gas calculation models. For the emission factor model, the mercury content in coal ( ), coal consumption ( ) of CFPPs, and the comprehensive emission factors of the boiler and air pollution control device combination ( ) is used to estimate mercury emissions. The calculation formula by using the emission factor model was as follows: Hgcoal Mcoal ∏CEFi Hgair = Mcoal × Hgcoal × ∏CEFi#（1） For the mass balance model, the mass balance equation of mercury based on its transport characteristics was established to estimate mercury emissions from flue gas or to study the mercury distribution behavior of individual CFPPs. The sources of mercury input to CFPPs were coal and limestone, and the destinations are mainly flue gas and CFPP by-products (bottom ash, fly ash, gypsum, and desulfurization wastewater). The calculation formula by using the mass balance model was as follows: Hgair = Hgin −Hgsolidandliquid = （Hgcoal + Hglimestone）−（Hgbottomash + Hgflyash + Hggypsum + Hgwastewater）#（2） For the flue calculation model, the low-level heat of coal ( ) and excess air ratio (α) to calculate the actual flue gas volume (VY). Then, combined with the coal combustion volume (Mcoal) and the mercury emission concentration (B), the emissions of mercury are estimated. The calculation formula by using the flue calculation mode was as follows: Qnet Hgair = VY × B × Hgcoal = ( + 0.77 + 1.0161(α −1) × ( + 0.61) × B × Mcoal#（3） 1.04Qnet 4187 Qnet 41415 The flue calculation model relies on empirical values and does not consider the impact of plant equipment operating conditions, resulting in rough numerical results (Diao et al., 2018). Therefore, there were limited applications of the flue calculation model in related research. In contrast, the mass balance model and emission factor model are widely used in studies, with the former mainly used to study the mercury partitioning behavior and to calculate mercury emissions(Gao et al., 2017), while the latter was widely used to The flue calculation model relies on empirical values and does not consider the impact of plant equipment operating conditions, resulting in rough numerical results (Diao et al., 2018). Therefore, there were limited applications of the flue calculation model in related research. 4.1.1 Emissions of mercury In contrast, the mass balance model and emission factor model are widely used in studies, with the former mainly used to study the mercury partitioning behavior and to calculate mercury emissions(Gao et al., 2017), while the latter was widely used to Page 8/33 Page 8/33 estimate mercury emissions from a CFPP or a regional plants and to establish mercury emission inventories(Dabrowski et al., 2008). Mercury emission inventories can be developed using both top-down and bottom-up approaches. Top-down emission inventory methods estimate emissions using emission factor methods based on data such as the amount of coal burned and the mercury content of coal in a region. However, as it relies on official data, the top-down method introduces uncertainty in the emission inventory(Wu et al., 2010). In contrast, the bottom-up method involves collecting fine-grained activity data from point sources, including boiler type, boiler capacity, coal consumption, APCDs, and latitude and longitude, to calculate mercury emissions from each source and estimate emissions for the region(Wang et al., 2014). In theory, the bottom-up method should yield more accurate results than the top-down method. However, both methods suffer from uncertainties due to inaccuracies in parameters such as mercury content in coal and mercury removal efficiency of APCDs, which can introduce uncertainties into the emission inventory. Refining coal types, APCD types, and mercury removal efficiency can effectively reduce inventory uncertainty(Liu et al., 2018), which required more supporting measurement data. Since 1997, the United States, Australia, China, and South Africa have established their own mercury emission inventories for CFPPs(Dabrowski et al., 2008; Hassett-Sipple et al., 1997; Nelson, 2007; Tian et al., 2014). The establishment of long-term emission databases is important for capturing the changes in mercury emissions, supporting the formulation and adjustment of mercury control policies, and testing their effectiveness. For example, Australia's mercury emission inventory for the past two decades shows that nearly one-third of CFPPs in Australia had been shut down in recent years, resulting in a significant reduction in mercury emissions from CFPPs(MacFarlane et al., 2022). In 2005, China established a preliminary inventory of mercury emissions from CFPPs, estimating 56.56 tons of emissions in 2000(Jiang et al., 2005). However, by 2007, the number had surged to 132 tons(Tian et al., 2011). In response, China has introduced a series of policies since 2011 aimed at clean energy substitution, decommissioning of small-capacity units, and replacing small-capacity units with larger ones. 4.1.1 Emissions of mercury Since 2014, China has implemented ultra-low emission upgrades for CFPPs, adopting stricter energy efficiency and environmental protection standards for atmospheric emissions. Within seven years, the proportion of coal consumption in China's primary energy consumption decreased by 9.6%. As a result, the mercury emissions from CFPPs in China showed a downward trend, with the mercury emissions decreasing to 73 tons in 2015(Liu et al., 2018), a 45% decrease compared to 2007. 4.1.2 Migration and transformation of mercury The combustion of coal can result in the release of three different mercury speciation, namely gaseous elemental mercury (Hg0), gaseous oxidized mercury (Hg2+), and particulate mercury (HgP). Figure 4(a) illustrates the mass distribution and speciation transformation pattern of mercury from coal combustion to flue gas emissions. The technical route of APCDs in the figure was the most typical ultra-low emission technology retrofit route in China: SCR + LLT-ESP + WFGD. Under this technical route, 0.11% of the mercury was in the bottom ash produced after coal combustion, about 58% of mercury fell into the ash hopper with the fly ash after passing through LLT-ESP, and 27% of mercury can be collected in the products such as gypsum and WFGD wastewater produced by WFGD, leaving only about 15% of the mercury was emitted from Page 9/33 Page 9/33 the flue gas. The average Hg emission concentration from CFPPs stacks in China was about 1.54 ± 1.58µg/m3(Cao et al., 2020; Chen et al., 2020; Guo et al., 2020; Hua et al., 2016; Liming et al., 2017; Qian et al., 2016; C. Song et al., 2017; Song C. et al., 2017; Wang et al., 2018; Wang, 2020; Wen et al., 2020; Y. Zhang et al., 2017; Zhao and Han, 2019), a value much lower than the current industry standard (30µg/m3). Figure 4(b) and Fig. 4(c) illustrates mercury speciation distribution in different coal types and combustion modes. Two types of coal-fired boilers commonly used in CFPPs are pulverized coal boilers (PC) and circulating fluidized bed boilers (CFB). Combustion in a CFB boiler generally produces a higher proportion of HgP than in a PC which is attributed to the lower combustion temperature range of the CFB (800–950°C) absorbing more Hg0 and Hg2+ into the fly ash and converting it into HgP (X. Li et al., 2019). Figure 4(d) shows the speciation changes of mercury as flue gases pass through APCDs. The combustion flue gas was first through the selective catalytic reduction (SCR) device, which oxidized a portion of Hg0 to Hg2+ through the catalytic action of the SCR catalyst. It not only reduced the Hg0 concentration by 44% but also increased the Hg2+ concentration at the inlet of the wet flue gas desulfurization (WFGD) unit, facilitating the subsequent removal of Hg2+. 4.1.2 Migration and transformation of mercury It was anticipated that the percentage of mercury that migrates to fly ash and desulfurization by- Page 10/33 Page 10/33 Page 10/33 products will continue to rise. Therefore, it is crucial to give more consideration to the appropriate handling and disposal of by-products containing mercury in the future. In conclusion, the temporal distribution of mercury emissions from Chinese CFPPs tended to increase before decreasing. This trend was closely related to the implementation of more targeted and stringent policies in recent years, such as clean energy substitution, small unit phase-out and substituted, and ultra-low emission retrofit. Burning low sulfur and high chlorine coal, using an oxygen-rich atmosphere for combustion, and installing high-efficiency APCDs helped reduce the formation of Hg0. Besides, as flue gas treatment technology continues to develop, the proportion of mercury migrating into fly ash and desulfurization by- products will gradually increase. It is important to pay more attention to the proper treatment and disposal of mercury-containing waste in the future. Finally, after undergoing ultra-low emission retrofit, the average Hg emission concentration CFPPs in China was about 1.54 ± 1.58µg/m3, which was well below the 30 µg/m3 limit value. In addition, compared to the United States' mercury emission limits (1.70 µg/m3 for non-low-rank coal from active CFPPs and 5.10 µg/m3 for low-rank from all CFPPs), China should consider tightening the relevant emission standards of mercury in the future. 4.2 Environmental impact The mercury generated by CFPPs can migrate, transform, and accumulate in the environment, leading to bioaccumulation in organisms. Studying the environmental impacts of mercury emissions from CFPPs and understanding their hazards and behavior patterns are crucial for developing more scientifically effective strategies for mercury emissions control. This is also a hot research topic among scholars in the field of mercury emissions from CFPPs. The relevant clusters for this theme include #1 Air and #7 Air Pollution, and keywords such as deposition, environmental impact, health, and contamination. The research on this topic mainly analyzes the adverse effects of mercury emissions from CFPPs on the atmospheric environment, soil environment, water environment, and biological health, as well as the economic costs. The mercury generated by CFPPs can migrate, transform, and accumula bioaccumulation in organisms. Studying the environmental impacts of understanding their hazards and behavior patterns are crucial for develo strategies for mercury emissions control. This is also a hot research top mercury emissions from CFPPs. The relevant clusters for this theme inc keywords such as deposition, environmental impact, health, and contam mainly analyzes the adverse effects of mercury emissions from CFPPs environment, water environment, and biological health, as well as the ec 4.1.2 Migration and transformation of mercury After SCR, the flue gas passed through a high-efficiency dust collector, where Hg2+ was converted to HgP, and a small amount of Hg0 was continually oxidized to Hg2+. Most of the HgP in the flue gas was trapped and collected in the ash hopper. Subsequently, the flue gas carrying a large amount of Hg2+ entered the WFGD, which had an 18% higher Hg2+ removal rate compared to a CFPP with only an electrostatic precipitator (ESP)(Lee et al., 2006; X. Yang et al., 2007), due to the capture efficiency of water- soluble Hg2+ by the WFGD unit. Finally, after WFGD, the flue gas contained about 88% of Hg0 and 12% of Hg2+ 。 Figure 5 shows the factors influencing the mercury in flue gas. The oxidation and adsorption of mercury in the gas phase were significantly influenced by the fly ash, resulting in an increased concentration of HgP in the flue gas. Generally, the adsorption of mercury by fly ash was negatively correlated with temperature, particle size, and concentration of sulfur oxides (SO2 and SO3), while being positively correlated with the unburned carbon content(Kim et al., 2010; Su et al., 2017; Sung et al., 2017). The adsorption of mercury by fly ash was also affected by factors such as the concentration of halogens and the concentration and form of mercury in the flue gas(Burmistrz et al., 2016; X. Li et al., 2019). Additionally, O2 and NOx in the flue gas had a certain catalytic oxidation effect on mercury, promoting the formation of Hg2+ in the flue gas(X. Yang et al., 2007). However, the presence of H2O in the flue gas had been shown to hinder the oxidation of Hg0 to Hg2+. Some studies suggested that this may be due to the effect of H2O on catalyst activity(Li et al., 2021), while others suggested that H2O can react directly with HgCl2 in an SO2 atmosphere, promoting the generation of Hg0(Zhao et al., 2006). In China, the ultra-low emission retrofit of CFPPs since 2014 has led to the implementation of stricter emission limits for particulate matter, SO2, and NOx, with values of 10, 35, and 50 mg/m³ respectively. As a result, APCDs have been upgraded and their efficiency in removing mercury from flue gas has been improving (see Fig. 6). 4.2.1 Environmental pollution of mercury Figure 6 shows the migration and conversion of mercury from a CFPP stack. Mercury is emitted into the flue gas in the form of total gaseous mercury (TGM, containing Hg0, CH3Hg, and (CH3)2Hg)), reactive gaseous mercury (RGM, containing Hg2+), and total particulate mercury (TPM). The fugitive form of mercury is one of the key factors affecting the extent of atmospheric deposition(Song et al., 2021). The atmospheric lifetime of RGM and TPM is only a few weeks or even days, thus they can easily settle within about 10–18 km from the CFPPs, resulting in high concentrations of mercury in the topsoil layer, up to 267.1 ng/g(Kang et al., 2019; F. Li et al., 2020; Li et al., 2017; Martin et al., 2021; Martin and Nanos, 2016). In contrast, the atmospheric lifetime of TGM is about 1–2 years, thus it has a global impact on the environment due to its long-distance transportation. For example, about 75% of the flue gas emitted from a CFPP in Inner Mongolia, China, is Hg0, resulting in mercury pollution over 150 km to 1000 km of the area (Cheng et al., 2020). In the presence of reducing agents such as sulfur dioxide, Hg2+ in the stack emissions from CFPPs are converted to Hg0 in the plume, exacerbating the global impact of mercury from CFPPs(Deeds et al., 2013). Page 11/33 Mercury emissions from CFPPs accumulate in soil and water bodies through atmospheric deposition. For instance, Soil samples taken near CFPPs in Spain and China revealed mercury concentrations of over 1000 µg/kg (Martin and Nanos, 2016; Yang and Wang, 2008); In Serbia, soil mercury concentrations near the Nikola Tesla CFPP were even as high as 2100 µg/kg(Goodarzi, 2004). Mercury concentration in the soil had been found to show a negative correlation with distance from the CFPPs and was also influenced by factors such as organic matter content, pH, and soil lithology type(Cheng et al., 2020; Martin and Nanos, 2016; Sherman et al., 2012). Additionally, mercury emissions from CFPPs can enter water bodies through atmospheric deposition and soil erosion, leading to contamination of both fibers and sediments in water bodies(Hylander and Goodsite, 2006). Moreover, mercury in solid by-products from CFPPs has an impact on the environment via migration. 4.2.1 Environmental pollution of mercury Based on existing studies using the risk assessment code (RAC), the risk level of mercury in CFPPs desulfurization sludge to the environment was moderate to high (RAC > 10%)(Chang et al., 2021; Wang et al., 2022); fly ash presented the second-highest potential risk, indicating a medium risk (10 < RAC < 30%)(Han et al., 2021); FGD gypsum had a low-risk level (1 < RAC < 10%)(Zhao et al., 2018). Furthermore, Zhao et al. found that mercury concentration in wastewater from WFGD and WESP exceeded the permissible value of 0.001 mg/L in the aqueous environment in a sampling study of a CFPP in China that underwent ultra-low emission retrofit(Zhao et al., 2017b). Consequently, the environmental mercury pollution from CFPPs desulfurization and dust removal by-products should be taken into account in the future. 4.2.2 Health risk of mercury Mercury contamination can have adverse health effects on local organisms, particularly when it is in the form of methylmercury. In general, methylmercury toxicity > inorganic mercury > monomeric mercury(Zhao et al., 2019). Sulfur dioxide from CFPPs increases the rate of inorganic mercury methylation during deposition, and methylmercury is enriched along the food chain, causing health hazards to local flora and fauna(Wiener et al., 2012). Research has shown that mercury concentrations in areas close to CFPPs exceed the toxicity threshold for raptors(Badry et al., 2019), and mosquitoes around CFPPs in Ohio, USA, have almost all methylmercury in their bodies(Konkler and Hammerschmidt, 2012). To demonstrate the correlation between CFPPs and mercury levels in organisms, Subhavana et al. conducted a comparative study of total mercury concentrations in the hair of citizens in South India(Subhavana et al., 2019). Their study included one city with four active CFPPs and two cities without major mercury sources. The results showed that hair mercury concentrations were lower in rice and fish consumers in the city with CFPPs than in consumers in the other two cities. Methylmercury enters the human body primarily through ingestion and can cause damage to various organs, including the central nervous system, digestive and immune systems, lungs, kidneys, skin, and eyes(George et al., 2015). This can lead to the development of cardiovascular disease, autism, mental decline, and other diseases (Kimakova et al., 2019; Lewandowski, 2010; Sunderland et al., 2016). In particular, prenatal exposure to mercury poses a particular threat to the development of the newborn(Kimakova et al., 2019). For instance, prenatal exposure to methylmercury was associated with 1,566 cases of mental retardation per year in the United States, contributing 3.2% of the disease; and CFPPs caused 41% of cases of mental retardation from anthropogenic sources, with a lifetime economic cost of up to $1.3 billion per year to the birth cohort(Trasande et al., 2006). Page 12/33 In 1972, the United States adopted the World Health Organization's (WHO) TDI, a safe dose for humans, which was set at 0.47µg/kg/d, revised to 0.30µg/kg/d in 1985, and changed to 0.10µg/kg/d in 1995. China also formulated the "12th Five-Year Plan" for the comprehensive prevention and control of heavy metal pollution, which included mercury as one of the five major heavy metals and included it in the scope of total control. 4.2.2 Health risk of mercury During the 12th Five-Year Plan period in China (2011–2015), retrofitting measures for CFPPs dropped 30,484.77 IQ points and prevented 114 deaths, reducing the health impacts of mercury emissions(J. Li et al., 2020). Mercury emissions from CFPPs still posed a threat to biological health, and more stringent control measures for CFPPs are necessary. Lipfert al. showed that removing 70% of the mercury from CFPPs would reduce mercury deposition in the United States by 5–10% and reduce methylmercury in freshwater fish(Lipfert et al., 2005). Zhang et al. found that more stringent mercury control measures would reduce mercury deposition by more than 15% in East Asia(W. Zhang et al., 2017). In addition, because of the lag in the biological impact of mercury reduction, even if the world is actively reducing mercury emissions from CFPPs, mercury emissions from CFPPs will continue to be deposited in soil and water for some time to come, posing a threat to biological health. In general, the spatial extent of deposition resulting due to mercury emissions from CFPPs depended on the proportion of Hg0 emissions. Large emissions of Hg0 exacerbated the global impact of mercury, and SO2 emissions from CFPPs promoted the formation of Hg0. Since the environmental risk of mercury in fly ash was moderate (10% < RAC < 30%), together with the large percentage of mercury in fly ash (16–97%), secondary emissions of mercury during fly ash curing should be taken into account. However, while global controls on mercury from CFPPs had reduced mercury deposition, there was also a lag in the biological impact of mercury abatement. Therefore, mercury emissions from CFPPs would continue to be deposited in soil and water bodies, posing a threat to biological health for some time to come. 4.3 Emission control Mercury has adverse effects on ecological environment and human health, and it is important to control mercury emissions from CFPPs. Therefore, the development and adaptation of efficient and low-cost mercury control technologies had been a hot topic in the field of mercury emissions in CFPPs. The related clusters are #0 elemental mercury, #4 air pollution control devices, #5 fly ash, and #6 mercury control. Key topics mentioned include elemental mercury, fly ash, activated carbon, mercury control, air pollution control devices, adsorption, and oxidation. Mercury control can be classified into three stages based on the life cycle of coal: pre-combustion, in- combustion, and post-combustion. Currently, post-combustion flue gas treatment technologies are receiving more research attention, as they have demonstrated effective mercury control. However, pre-combustion and in-combustion control methods are also being explored due to their potential to reduce the cost per unit calorific value of coal and improve boiler efficiency while also removing mercury. 4.3.1 Pre-combustion and in-combustion control techniques Besides, low-NOx combustion technology, originally designed for NOx control, can also facilitate Hg0 conversion due to changes in boiler temperature and fly ash properties(Luo et al., 2009). In addition, furnace injection technology involves adding oxidants such as alkali metal oxides and halogenated salts to the coal during combustion, which can reduce Hg0 in flue gas by 25–80%(Min-qiang, 2009; Yang et al., 2016; Zhuang et al., 2007). In-combustion control of mercury involves modifying the form of mercury in flue gas by adding an oxidant or regulating coal powder temperature and particle size during combustion to facilitate the conversion of Hg0 into Hg2+ and HgP, which are more easily removable. Common technologies for mercury control during combustion include fluidized bed combustion technology, low-NOX combustion technology, and furnace injection technology. Among them, fluidized bed combustion technology provides a long flue gas residence time and low-temperature atmosphere, which increases the chance of conversing Hg0 to Hg2+ and HgP(Xun et al., 2014). Besides, low-NOx combustion technology, originally designed for NOx control, can also facilitate Hg0 conversion due to changes in boiler temperature and fly ash properties(Luo et al., 2009). In addition, furnace injection technology involves adding oxidants such as alkali metal oxides and halogenated salts to the coal during combustion, which can reduce Hg0 in flue gas by 25–80%(Min-qiang, 2009; Yang et al., 2016; Zhuang et al., 2007). 4.3.1 Pre-combustion and in-combustion control techniques Pre-combustion control of mercury refers to the treatment of coal before combustion, and commonly used control techniques include coal washing and mild pyrolysis. Coal washing aimed to remove sulfur-based Page 13/33 Page 13/33 inorganic contaminants, particularly pyrite, whic mercury can be partial removed during coal was Coal-washing technologies include physical, che washing technologies are based on the differen selective agglomeration, column froth flotation, 40–82% for raw coal(Brown et al., 1999). Besides, chemical coal-washing technology ma sulfur in raw coal in different forms and can ach efficiency of mercury removal by one-step wash possibility of two-step washing based on one-st improved the rate of mercury removal from lign microbial desulfurization process involves the u thiobacillus for coal washing, which reduced the coal(Thomas Klasson et al., 2006). Gentle heating technology takes advantage of t allowing the mercury in the coal to be volatilized adsorbent. The temperature at which different c varies from 275 to 500°C, and the maximum me 2004; Wang et al., 2000). In practical technical a and coal quality changes. Therefore, the choice factors into account while achieving the greates Chmielniak et al. demonstrated that a mild pyro more cost and removal efficiency advantages(C In-combustion control of mercury involves mod regulating coal powder temperature and particle into Hg2+ and HgP, which are more easily remov combustion include fluidized bed combustion te injection technology. Among them, fluidized bed time and low-temperature atmosphere, which in al., 2014). Besides, low-NOx combustion techno Hg0 conversion due to changes in boiler temper furnace injection technology involves adding ox the coal during combustion, which can reduce H inorganic contaminants, particularly pyrite, which is the most common carrier of mercury in coal. Thus, mercury can be partial removed during coal washing, with an average mercury removal rate of about 50%. Coal-washing technologies include physical, chemical, and biological methods. Among them, physical coal- washing technologies are based on the differences in physical properties of the components in coal, including selective agglomeration, column froth flotation, and cyclone, and they have mercury removal efficiencies of 40–82% for raw coal(Brown et al., 1999). inorganic contaminants, particularly pyrite, which is the most common carrier of mercury in coal. Thus, mercury can be partial removed during coal washing, with an average mercury removal rate of about 50%. Coal-washing technologies include physical, chemical, and biological methods. 4.3.1 Pre-combustion and in-combustion control techniques Among them, physical coal- washing technologies are based on the differences in physical properties of the components in coal, including selective agglomeration, column froth flotation, and cyclone, and they have mercury removal efficiencies of 40–82% for raw coal(Brown et al., 1999). Besides, chemical coal-washing technology mainly uses a series of chemical reactions to quickly separate sulfur in raw coal in different forms and can achieve 30–90% of the efficiency of mercury removal. The efficiency of mercury removal by one-step washing was about 30%, but Dronen et al. investigated the possibility of two-step washing based on one-step washing with concentrated HCl, which significantly improved the rate of mercury removal from lignite and bituminous coal(Dronen et al., 2004). In addition, the microbial desulfurization process involves the use of microorganisms such as pseudomonas, sulfurous, and thiobacillus for coal washing, which reduced the emission of SO2 and removes 20% of mercury from coal(Thomas Klasson et al., 2006). Gentle heating technology takes advantage of the fact that mercury was volatile when the coal was heated, allowing the mercury in the coal to be volatilized and then removed by adsorption using a mercury removal adsorbent. The temperature at which different coal speciation achieved the maximum mercury removal rate varies from 275 to 500°C, and the maximum mercury removal rate was about 70–80%(Guffey and Bland, 2004; Wang et al., 2000). In practical technical applications, excessive operating temperature will bring cost and coal quality changes. Therefore, the choice of combustion temperature was based on taking these factors into account while achieving the greatest possible removal of mercury from coal. For instance, Chmielniak et al. demonstrated that a mild pyrolysis technology for lignite at a temperature of 300°C has more cost and removal efficiency advantages(Chmielniak et al., 2017). In-combustion control of mercury involves modifying the form of mercury in flue gas by adding an oxidant or regulating coal powder temperature and particle size during combustion to facilitate the conversion of Hg0 into Hg2+ and HgP, which are more easily removable. Common technologies for mercury control during combustion include fluidized bed combustion technology, low-NOX combustion technology, and furnace injection technology. Among them, fluidized bed combustion technology provides a long flue gas residence time and low-temperature atmosphere, which increases the chance of conversing Hg0 to Hg2+ and HgP(Xun et al., 2014). 4.3.2 Post-combustion control technology Post-combustion mercury control technology refers to the addition of one or more chemical reagents and facilities to the flue gas of CFPPs, which helps to transform mercury compounds (such as HgCl2 and HgO) into forms that are easier to remove. This is through a series of devices, including synergistic mercury removal Page 14/33 Page 14/33 technology represented by APCDs and specialized mercury removals technology such as adsorption and catalytic oxidation. APCDs equipped with CFPPs had a synergistic effect on Hg removal while controlling particulate matter, SO2, and NOx (Li et al., 2022). There were two main ways for the APCDs to synergistically remove mercury: 1) through the use of high-efficiency dust removal facilities (ESP, FF, ESP-FF, LTESP, WESP) to remove Hgp in the flue gas, 2) through the use of WFGD to remove Hg2+ in the flue gas. The type and performance of SCR catalysts, dust removal equipment, and desulfurization processes are important factors affecting the mercury removal efficiency of APCDs. In 2014, China implemented flue gas ultra-low emissions of CFPPs and carried out two routes of transformation for the existing APCDs(Li, 2015). One is based on low-temperature electrostatic precipitation technology: SCR + WHR (waste heat recovery) + LLT-ESP (low-temperature electrostatic precipitator) + WFGD + WESP (optional) + FGR (optional); the other is based on wet electrostatic precipitation technology: SCR + AH (air heat exchanger) + ESP/FF/ESP-FF + WFGD + WESP (wet electrostatic precipitator) + FGR (optional). As of 2020, 88% of China's CFPPs had achieved ultra-low emission transformation. SCR + LLT-ESP + WFGD was the dominant route, which increased the average mercury removal efficiency of APCDs from 63–82% (see Fig. 7). Furthermore, the mercury removal efficiency of SCR + FF + WFGD after ultra-low emission retrofit is 90%, and the mercury removal efficiency of ESP-FF + WFGD after ultra-low emission retrofit is increased from 66–96%. SCR + ESP + WFGD + WESP, the less applied ultra-low emission technology route, can reach 96% mercury removal efficiency. The adsorption of mercury by adsorbents is often the result of the combined action of physical adsorption and chemical reaction. Mercury adsorption technology refers to the use of adsorbents, such as carbon-based adsorbents, calcium-based adsorbents, mineral-based adsorbents, and fly ash, to adsorb Hg0 in flue gas through injection into the flue or fixed bed adsorption(Liu et al., 2019; Zhao et al., 2019). 4.3.2 Post-combustion control technology Among them, carbon- based adsorbents, represented by activated carbon, biomass coke, and petroleum coke, have excellent surface properties and well-developed micropores, which have good adsorption effects on mercury(Li et al., 2018). Especially for activated carbon, its highly efficient adsorption capacity can help CFPPs improve their mercury removal efficiency by 10%(Sung et al., 2018), and has been widely used. However, due to its high price, more research turned to more economical non-carbon-based adsorbents. In contrast, calcium-based adsorbents have a low cost and can remove a small amount of mercury while adsorbing sulfur oxides (H. Yang et al., 2007). Furthermore, Mineral-based adsorbents are cost-effective and environmentally friendly and have been favored by scholars. The most promising material for mercury adsorption is fly ash from CFPPs, which offers significant cost advantages. Fly ash can effectively remove mercury by oxidizing flue gas mercury and enriching the mercury into HgP. Currently, research efforts are primarily focused on modifying existing adsorbents to enhance the efficiency of mercury removal. Table 3 presents the various modification methods and their effects on mercury adsorbents. These methods mainly involve adding halide, acid, metal and metal oxides, microwave, plasma and so on, which usually improve mercury removal efficiency by enhancing adsorption sites, increasing the surface area, enhancing running cycle and so on. The average mercury removal efficiency of the modified adsorbents were distributed in the range of 41–100%. The adsorption of mercury by adsorbents is often the result of the combined action of physical adsorption and chemical reaction. Mercury adsorption technology refers to the use of adsorbents, such as carbon-based adsorbents, calcium-based adsorbents, mineral-based adsorbents, and fly ash, to adsorb Hg0 in flue gas through injection into the flue or fixed bed adsorption(Liu et al., 2019; Zhao et al., 2019). Among them, carbon- based adsorbents, represented by activated carbon, biomass coke, and petroleum coke, have excellent surface properties and well-developed micropores, which have good adsorption effects on mercury(Li et al., 2018). properties and well-developed micropores, which have good adsorption effects on mercury(Li et al., 2018). Especially for activated carbon, its highly efficient adsorption capacity can help CFPPs improve their mercury removal efficiency by 10%(Sung et al., 2018), and has been widely used. However, due to its high price, more research turned to more economical non-carbon-based adsorbents. In contrast, calcium-based adsorbents have a low cost and can remove a small amount of mercury while adsorbing sulfur oxides (H. Yang et al., 2007). 4.3.2 Post-combustion control technology Furthermore, Mineral-based adsorbents are cost-effective and environmentally friendly and have been favored by scholars. The most promising material for mercury adsorption is fly ash from CFPPs, which offers significant cost advantages. Fly ash can effectively remove mercury by oxidizing flue gas mercury and enriching the mercury into HgP. Currently, research efforts are primarily focused on modifying existing adsorbents to enhance the efficiency of mercury removal. Table 3 presents the various modification methods and their effects on mercury adsorbents. These methods mainly involve adding halide, acid, metal and metal oxides, microwave, plasma and so on, which usually improve mercury removal efficiency by enhancing adsorption sites, increasing the surface area, enhancing running cycle and so on. The average mercury removal efficiency of the modified adsorbents were distributed in the range of 41–100%. Page 15/33 Table 3 Modification methods and effect of mercury adsorbent. The data were taken from (G. Li et al., 2019; Qi et al., 2015; Xu et al., 2018). aMM bMR Modification effect Raw adsorbents cMRE Halide modification Iodide, bromide, chloride, and so on ①Add active adsorption sites; ② Synergy effect of catalytic oxidation and mercury adsorption Active carbon 78% (10– 100%) Coke 67% (18– 100%) Fly ash 56% (8- 100%) Mineral- based 72% (38– 100%) calcium- based 90% Metal oxides 89% (53– 100%) Acid modification HNO3, H2SO4, HClO4, H2O2, aqua regia and so on ①Add active adsorption sites Active carbon 60% (9– 98%) Coke 72% (35– 93%) Metal and metal oxides modification Noble metals, transition metal oxides, and so on ①Maintain good Hg0 removal performance in the higher temperature range; ②Enhance running cycle;③Enhance active adsorption sites Active carbon 77% (10– 100%) Coke 66% (9– 95%) Metal oxides 75% (3- 100%) Mineral- based 77% (20– 98%) Fly ash 43% (4– 98%) Microwave modification Microwave ①Increase the surface area, micropore volume, and total pore volume Coke 41% (13%-88%) Active carbon 68% Plasma modification Plasma ①Increase the active sites on the surface of various adsorbents Active carbon 77% (60– 90%) Metal 62% (30 Page 16/33 aMM bMR Modification effect Raw adsorbents cMRE Miscellaneous modification CO2, high temperature ① Improve the surface area; ②Promote initial mercury adsorption Active carbon 100% aMM is a modification method. bMM is modification reagents. cMRE is mercury removal efficiency. aMM is a modification method. bMM is modification reagents. cMRE is mercury removal efficiency. 4.3.2 Post-combustion control technology In China, 88% of CFPPs achieved ultra-low emission transformation from 2014 to 2020, which achieved an average mercury removal efficiency of 82–93% for the different technology routes. In addition, the mercury removal efficiency of the modified adsorbent and SCR catalyst is 41–100% and 83–100%, respectively. 4.3.2 Post-combustion control technology Mercury catalytic technology involves the catalytic oxidation of Hg0 to the more easily removable form of Hg2+. The mercury catalysts can be divided into SCR catalysts and specialized mercury removal catalysts (after dust collectors) depending on the input location. Existing research on specialized mercury removal catalyst materials had focused on precious metals and transition metals, and their efficiency in oxidizing mercury ranges from 4-100% (Dranga et al., 2012). Simultaneous denitrification and mercury removal catalysts encompass photocatalysts, SCR catalysts, and others. Among these, photocatalytic technology is advantageous due to its low cost, environmental friendliness, and mild conditions. However, it suffers from low efficiency and weak stability. Therefore, many scholars were focusing on the development of SCR catalysts that can achieve denitrification efficiencies of 65–96% and mercury removal efficiencies of 83– 100%(Yanbing et al., 2021). Nonetheless, vanadium-based catalysts, which are commercial SCR catalysts for CFPPs, have certain problems, including high dependence on hydrochloric acid, poor NH3 resistance, and low Hg0 oxidation ability. As a result, transition metal-based and rare earth metal-based catalysts, which have acceptable Hg0 oxidation capacity, are more environmentally friendly, and have lower prices relative to vanadium-based catalysts, are the current research hotspots for SCR catalysts. In general, mercury control technologies applied for CFPPs include synergistic methods (such as coal washing, fluidized bed combustion, low-NOx combustion, and APCDs) and specialized removal techniques (such as low-temperature pyrolysis, the addition of oxidants, adsorbents, and catalysts). Among these, the removal pathway of APCDs usually involves converting Hg0 to more easily removable Hg2+ and HgP, which can achieve good mercury removal, especially SCR + ESP-FF + WFGD (about 96%). In China, 88% of CFPPs achieved ultra-low emission transformation from 2014 to 2020, which achieved an average mercury removal efficiency of 82–93% for the different technology routes. In addition, the mercury removal efficiency of the modified adsorbent and SCR catalyst is 41–100% and 83–100%, respectively. In general, mercury control technologies applied for CFPPs include synergistic methods (such as coal washing, fluidized bed combustion, low-NOx combustion, and APCDs) and specialized removal techniques (such as low-temperature pyrolysis, the addition of oxidants, adsorbents, and catalysts). Among these, the removal pathway of APCDs usually involves converting Hg0 to more easily removable Hg2+ and HgP, which can achieve good mercury removal, especially SCR + ESP-FF + WFGD (about 96%). 5 Conclusion and outlook Under the current global environmental management requirements of reducing pollution and carbon and actively addressing climate change, new requirements are placed on the development of the coal power industry. Through literature review, this study suggests that future research on mercury emissions from CFPPs can focus on the following aspects. 1. The current mercury emission limits for China's CFPPs do not along with the ultra-low emission requirements, indicating the need for further improvement. 2. Studies have indicated that fossil energy will still play an important role by 2050, accounting for 10–15% of China's energy consumption(Liu et al., 2021). Thus, it is crucial to study the combined impact of carbon reduction policies and the implementation of CCUS technology on reducing air pollutants, including mercury emissions from CFPPs. 3. Considering the harmful environmental impact of mercury emissions from CFPPs, particularly on the health of fetuses and children, there is an urgent need for enhanced real-time monitoring of mercury emissions from these plants and a comprehensive risk assessment of the surrounding environment. By considering the China's carbon peaking and neutrality goals as well as the current coal-based energy situation, CFPPs will continue to play an important role for some time to come. We believe that our research is instructive for future mercury reduction, environmental protection, and human health. 5 Conclusion and outlook After 22 years of development, the knowledge area of mercury emissions from CFPPs has been further improved and a core group of authors has been formed. Based on the results of the bibliometric analysis, the research stages on mercury emissions from CFPPs during 2000–2022 can be roughly divided into the embryonic period (2000–2005), development period (2006–2015), and continuous development period (2016–2022). Emission calculation, synergistic removal of mercury, and environmental risks are the focus of scholars. China (38.2%), Huazhong University of Science and Technology (8.0%), and Fuel (9.6%) were the countries, institutions, and journals with the most publications. According to the results of keyword clustering Page 17/33 Page 17/33 Page 17/33 analysis, the research hotspots of mercury emission from CFPPs mainly include three major research themes: emission characteristics, emission control, and environmental impact. analysis, the research hotspots of mercury emission from CFPPs mainly include three major research themes: emission characteristics, emission control, and environmental impact. Through a systematic literature review, it can be concluded that the speciation transformation patterns of mercury in coal combustion and APCDs control processes have been revealed. Fly ash and desulfurization byproducts were the main (about 58%) destinations of mercury emissions. The efficiency of mercury removal technologies varies greatly depending on factors such as temperature and materials, and more economical and environmentally efficient technology development is the direction of future research. APCDs could synergistically remove mercury and could remove 82–93% of mercury after ultra-low modification. The environmental impact of atmospheric mercury emissions from CFPPs could be influenced by the proportion of Hg0 and SO2 emissions. The risk level of mercury in combustion by-products was highest in desulfurization sludge (RAC > 10%) and followed by fly ash(10 < RAC < 30%) and desulfurization gypsum(1 < RAC < 10%). Furthermore, SO2 emissions from CFPPs exacerbated the rate of methylation of inorganic mercury during deposition, leading to methylmercury enriched in the food chain and causing human health and economic and cultural harm. Currently, countries such as China, the United States, Canada, and Japan have set carbon reduction targets for 2030. The United States and Canada have committed to reducing national greenhouse gas emission levels by 50–52% and 40–45%, respectively, in 2030 compared to 2005 levels, while Japan has committed to reducing greenhouse gas emissions by 46–50% in 2030 compared to 2013 levels. In 2019, China pledged to achieve carbon peaking in 2030 and achieve carbon neutrality by 2060. Declarations Ethical Approval Page 18/33 Not applicable. Consent to Participate Not applicable. Consent to Publish All authors have consented to publish this work. Authors Contributions Qi Liu: Conceptualization, Data Curation, Writing - Original Draft. Jiajia Gao: Methodology, Writing - Review & Editing. Guoliang Li: Funding acquisition. Yang Zheng: Supervision. Rui Li: Methodology. Tao Yue: Supervision, Conceptualization. Funding The authors gratefully acknowledge financial support from National Nature Science Foundation of China (NO 52200121) Competing Interests The authors declare no competing interests. Availability of data and materials The data supporting the findings of this study are publicly available in the paper cited in the text Not applicable. Consent to Participate All authors have consented to publish this work. onceptualization, Data Curation, Writing - Original Draft. Jiajia Gao: Methodology, Writing - Review & Guoliang Li: Funding acquisition. Yang Zheng: Supervision. Rui Li: Methodology. Tao Yue: C li i The authors gratefully acknowledge financial support from National Nature Science Foundation of China (NO. 52200121) The authors declare no competing interests. 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Page 28/33 Figure 2 (a-d) Contribution of countries, regions, and institutions from 2000 to 2022. Figure 2 (a-d) Contribution of countries, regions, and institutions from 2000 to 2022. (a-d) Contribution of countries, regions, and institutions from 2000 to 2022. (a-d) Contribution of countries, regions, and institutions from 2000 to 2022. Page 29/33 Page 29/33 Figure 7 Mercury removal efficiency of different APCDs. The bars represent the range of values, and the points represent the average. ULE means that have been retrofitted with ultra-low emissions. The data was taken from (X. Li et al., 2019; Liu et al., 2018; Su et al., 2017; Wen et al., 2020; Zhao et al., 2017a). Graphicalabstract.jpg Figure 3 (a-c) Keywords from 2000-2022. Page 30/33 Figure 4 (a). Mercury emission characteristics of typical ultra-low emission technology routes and (b-d) speciation distribution of mercury in different coal species, boilers, and APCDs. The data were taken from (Gao et al., 2022; Guo et al., 2020; Song C. et al., 2017; Su et al., 2017; Wang et al., 2010; Wen et al., 2020; Zhang et al., 2015; Zhao and Han, 2019). gure 4 Figure 4 (a). Mercury emission characteristics of typical ultra-low emission technology routes and (b-d) speciation distribution of mercury in different coal species, boilers, and APCDs. The data were taken from (Gao et al., 2022; Guo et al., 2020; Song C. et al., 2017; Su et al., 2017; Wang et al., 2010; Wen et al., 2020; Zhang et al., 2015; Zhao and Han, 2019). (a). Mercury emission characteristics of typical ultra-low emission technology routes and (b-d) speciation distribution of mercury in different coal species, boilers, and APCDs. The data were taken from (Gao et al., 2022; Guo et al., 2020; Song C. et al., 2017; Su et al., 2017; Wang et al., 2010; Wen et al., 2020; Zhang et al., 2015; Zhao and Han, 2019). Page 31/33 (a-f) Influencing factors of mercury in the flue gas. The data were taken from (Fu et al., 2022; Huang and Luo, 2010; Lee and Wilcox, 2017; Wang et al., 2008). Page 32/33 Figure 6 Figure 6 Page 32/33 Atmospheric transport and deposition of mercury. Supplementary Files This is a list of supplementary files associated with this preprint. Click to download. Graphicalabstract.jpg Page 33/33 Page 33/33
https://openalex.org/W2152778657	https://his.diva-portal.org/smash/get/diva2:641038/FULLTEXT01	English	null	The Movement to a New Understanding: A Life-World-Based Study about How People Learn to Live with Long-Term Illness	Journal of nursing & care	2,012	cc-by	6,231	Journal of Nursing & Care The International Open Access Journal of Nursing & Care Executive Editors Stephanie M. Chalupka Worcester State College, USA Martha Craft-Rosenberg University of Iowa, USA Linda Honan Pellico Yale University School of Nursing, USA Judith Lloyd Storfjell University of Illinois, USA Dott Federico Bilotta Albert Einstein College of Medicine, USA The International Open Access Journal of Nursing & Care Dott Federico Bilotta Albert Einstein College of Medicine, USA ISSN: 2167-1168 Journal of Nursing & Care The International Open Access Journal of Nursing & Care Executive Editors Stephanie M. Chalupka Worcester State College, USA Martha Craft-Rosenberg University of Iowa, USA Linda Honan Pellico Yale University School of Nursing, USA Judith Lloyd Storfjell University of Illinois, USA Dott Federico Bilotta Albert Einstein College of Medicine, USA T his article was originally published in a journal by OMICS vailable online at: OMICS Publishing Group (www.omicsonline.org) ISSN: 2167-1168 ISSN: 2167-1168 Introduction a person suffers from long-term illness, the body is changed, and thus her access to the world is changed as well. The experiences of people living with long-term illness are seen in this research as constituting a possibility for learning, while learning is seen as a complex phenomenon that involves the whole person. Patient experiences of living with long-term-illness have been described in several studies [1,2]. Suffering from such an illness is a life-changing process [3] and generates a need to learn. This article highlights the learning process of individuals suffering from long-term illness. A major starting point for this study is that patients’ learning has not previously been focussed on and, consequently, has not formed the basis for the creation of study programs and other forms of health services-teaching material. Previous research shows that the starting point for patient education often was a bio-medical or the so-called external perspective on illness [4]. This has been shown in research where the effects of patient education have been studied in the form of measureable values [5]. One consequence of this approach is that patients who do not follow the advice they receive are considered as noncompliant [6]. In previous research, an imminent risk also emerged that patients were excluded from participation in the care and treatment in their own health process [4,7]. Thus, a number of factors from the life-world approach are problematical and create difficulties for patients learning to live with a long-term illness [8]. Far too little attention has been given to the individual living with a long-term illness and whose experiences should be considered valuable and important- experiences that, if utilized, can constitute a foundation for formulating a teaching material. A significant part of research in the educational/ didactic field has taken place in disciplines other than the caring sciences, for example, sociology [9] and psychology [10]. The theories and programs that underpin patient education are most often based on theories from outside of the caring sciences; in particular, a life- world didactic perspective is missing [6,8]. Thus, the motives exist for carrying out research of the learning process inpatients from a caring science perspective based on the life-world approach. Dahlberg et al. [14] developed a research approach, termed Reflective Life-world Research (RLR), which is based on Giorgi’s [15] phenomenological approach. Nursing & Care Nursing & Care Open Access J Nurs Care ISSN: 2167-1168 JNC, an open access journal Mia Berglund* and Susanne Källerwald Mia Berglund* and Susanne Källerwald S h l f Lif S i U i it f Skö d Skö d S d School of Life Sciences, University of Skövde, Skövde, Sweden Interview Qualitative interviews are described by Dahlberg et al. [16] as being a dialogue whose aim is to get the informant to reflect on her experiences. The reflective process is initialized by the use of questions [16], which lead to new thoughts. The questions are based on what is said in the interview, and their purpose is to direct attention towards the phenomenon and gain a greater understanding of its meanings. The interview in the present study is seen as a dialogue, in which the aim is to achieve openness and flexibility. The informant’s attention has been directed towards the phenomenon, and the reflective process was encouraged with the use of more profound questions. Corresponding author: Mia Berglund, School of Life Sciences, University of Skövde, Skövde, Sweden, E-mail: mia.berglund@his.se Corresponding author: Mia Berglund, School of Life Sciences, University of Skövde, Skövde, Sweden, E-mail: mia.berglund@his.se Received December 07, 2012; Accepted December 26, 2012; Published December 31, 2012 Abstract Objective: The objective of the present study was to analyze and describe the phenomenon of learning to live with long-term illness. Method: The design and implementation of the research was based on a reflective lifeworld approach. The study consisted of interviews with people who live with different types of long-term illness. Results: Learning to live with a long-term illness happens in such a way as to respond to the will to live everyday life. The essential meaning of learning to live with long-term illness is constituted by the following elements: learning to know and live with a stranger, the driving forces of learning, learning methods are a balancing act, making the illness visible, as well as seeking knowledge and understanding. The result of the learning process can be understood as movement to a new understanding that is shown in the way the person with the illness acts and gives herself with the illness more space in life. Conclusion: The results show that genuine learning is something that differs from learning information and that the learning must be supported by the sufferer’s situation for a long period of time at an existential level. Conclusion: The results show that genuine learning is something that differs from learning information and that the learning must be supported by the sufferer’s situation for a long period of time at an existential level. Methods Received December 07, 2012; Accepted December 26, 2012; Published December 31, 2012 Introduction The overall aim of the reflective life-world approach is to describe and clarify lived experiences in such a way that a person’s knowledge and understanding of her existence and experiences is increased. The focus in this study was the experiences of people/ individuals with long-term illness. What does learning entail when living with long-term illness? What does learning contain? How does the learning process occur? What is the result of the learning process? Available online at: OMICS Publishing Group (www.omicsonline.org) T his article was originally published in a journal by OMICS Publishing Group, and the attached copy is provided by OMICS Publishing Group for the author’s benefit and for the benefit of the author’s institution, for commercial/research/educational use including without limitation use in instruction at your institution, sending it to specific colleagues that you know, and providing a copy to your institution’s administrator. T All other uses, reproduction and distribution, including without limitation commercial reprints, selling or licensing copies or access, or posting on open internet sites, your personal or institution’s website or repository, are requested to cite properly. Digital Object Identifier: http://dx.doi.org/10.4172/2167-1168.1000125 Digital Object Identifier: http://dx.doi.org/10.4172/2167-1168.1000125 Digital Object Identifier: http://dx.doi.org/10.4172/2167-1168.1000125 Berglund and Källerwald, J Nurs Care 2012, 1:6 http://dx.doi.org/10.4172/2167-1168.1000125 J Nurs Care ISSN: 2167-1168 JNC, an open access journal Findings The essential meaning of learning to live with long-term illness concerns movement towards a changed understanding of access to the world. In this movement, in which everyday life as well as relationships Analysis in accordance with the reflective life-world approach with oneself and others is affected, a continual renegotiation is needed. Learning includes developing an understanding of the relationship between the demands of life and those of the illness. Learning happens as a response to the will to live an ordinary life. In order to be able to push the illness away in favour of the patient`s pre-illness life, a learning process is needed where the sufferer paradoxically allows the illness more space. To learn to balance on life’s tightrope is hard work that necessitates profound self-reflection. The self-reflection constitutes the instrument of learning and the learning process can be both hampered and supported by other people. Learning entails that the individual develops a new understanding of herself. The essential meaning of learning to live with long-term illness is constituted by the following elements of meaning; The approach can be described as a dialectic process, starting with the whole, analysing its parts followed by the synthetic reconstitution of the whole with the aim to reach an understanding of the essence of the phenomenon [14,16]. The analysis starts with the initial whole, where the researcher acquaints herself with the text. Subsequently the focus of the reading is directed to the parts, which means seeking units of meaning: a word, a sentence, or a longer piece of text. The meanings are unpacked in this part of the process, and each meaning is reflected against the background of the whole. The next phase involves the building of clusters, which are the same as groups of meanings. When all the units of meaning in the data have been utilized and clustered into groups, it is time to move on to the next phase, the forming of the essence. Dahlberg et al. [14] describes the essence text as an abstraction and synthesis of the structure of meanings that makes the phenomenon into the actual phenomenon and nothing else. The essence can thus be understood as a new whole. • Learning to know and live with a stranger • The driving forces of learning • Learning methods are a balancing act • Making the illness visible Ethical considerations The ethical considerations for this study have been based on ethical principles of research [17,18] and laws [19] in Sweden at the time for data collection (September 2007–January 2008). Based on the ethical principles, the requirements of information consent, confidentiality, and use have been applied. All participants received a letter where he or she was invited to participate in the study. The participants were guaranteed confidentiality and were informed that participation was voluntary. After the participant had given their informed (oral and written) consent the interview was conducted. In the interview situation the researchers strove to remain open and flexible to the informants needs. It is a delicate task to get the informant to divulge more about their experience of this phenomenon without submitting him or her to pressure. The informants were all very grateful for the dialogue and they afterwards said that they had a good conversation and that they learned some things about themselves. Professional support was available if needed. The stranger causes problems for those trying to live their familiar life. The stranger and the problems it imposes mean that the person does not recognize herself in her own body and must learn to access the world in new ways; the process is also about dealing with the fear of an uncertain future. This problem is expressed as follows by a person who is blind: When you cannot see, then you lose companionship with people. You cannot go out as you want. You are limited pretty much to the home. One cannot decide that tomorrow I’m doing this, because it depends on how I feel. The problems that the stranger causes in life mean that people do not feel at home in their living situation, which is expressed in self- doubt and doubts about their capability. Learning to know and live with a stranger The stranger is to be understood as the illness and the changed body. Learning involves getting to know the stranger, understanding how it affects the body, and learning to handle the changed life and constantly occurring problems. When the stranger enters the person’s life, the person does not want to let it in, which is done by refusing to acknowledge the stranger’s existence. One way is by not naming the illness. One woman says that it took her 24 years before she pronounced the name of her illness. The fact that it is hard to let the stranger into one’s life is shown through people finding other causes for their symptoms, causes which feel more familiar to their individual life-worlds. The ages of the participants varied between 21 and 84. Five men and four women participated in the study. The participants were from four different municipalities in western Sweden. The interviews lasted between 35 and 117 minutes (with a mean of 60 minutes). The interviews were conducted in conjunction with dissertation work [8]. Yes, it was that I refused to admit that I had rheumatoid arthritis. [...] I do not know; I was not sick. Sometimes when it was too hard, then I wrapped it with an elastic bandage and then people asked: ‘What have you done? It’s dislocated’, I replied. And when my brother called, he always asked, ‘Hello, how is it with the things that you do not have?’ No, I did not want to talk about it; I did not want to admit it. Citation: Berglund M, Källerwald S (2012) The Movement to a New Understanding: A Life-World-Based Study about How People Learn to Live with Long-Term Illness. J Nurs Care 1:125. doi:10.4172/2167-1168.1000125 Participants • Seeking knowledge and understanding The study’s participants had different long-term illnesses. The common denominator of these illnesses was that they affected the participants’ daily lives. The informants themselves named their illnesses. The following illnesses and functional impairments were described (numerals in parentheses are used to indicate that two informants had the same illness or functional impairment): Rheumatoid Arthritis (RA), Bechterew’s Disease, Chronic Obstructive Pulmonary Disease (COPD), varicose ulcer [2], heart attack [2], cardiac insufficiency [2], thigh-bone amputation without prosthesis, double lower leg amputation with two prostheses, hip arthritis [2] Multiple Sclerosis (MS) [2], diabetes, type 1 [2], renal failure [2], blindness, impaired vision [2], neuropathy [2], gastric paresis [2], sub-cortical white-matter dementia, Binswanger’s disease (blood vessel abnormality), back pain, and alcoholism. • Care that impedes learning • The result of learning Scientific approach and method Citation: Berglund M, Källerwald S (2012) The Movement to a New Understanding: A Life-World-Based Study about How People Learn to Live with Long-Term Illness. J Nurs Care 1:125. doi:10.4172/2167-1168.1000125 Citation: Berglund M, Källerwald S (2012) The Movement to a New Understanding: A Life-World-Based Study about How People Learn to Live with Long-Term Illness. J Nurs Care 1:125. doi:10.4172/2167-1168.1000125 The ontological and epistemological suppositions in this article are based on life-world theory [11-13]. In life-world theory, a person is seen as a lived body [13]. The lived body is simultaneously physical, mental, and existential. A person is a provider and a seeker of knowledge when she wants to take care of, comprehend, and understand life [11]. When Copyright: © 2012 Berglund M. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Volume 1 • Issue 6 • 1000125 Page 2 of 5 Page 2 of 5 Making the illness visible pursuit of a familiar life. This endeavour emerges as a key driving force towards learning as demonstrated by the desire, tenacity, and ability to influence one`s own health processes. In order for learning to take place, it is important to direct attention to the illness and allow it to become visible. This visibility is enabled by ability to express the thoughts and experiences of the illness in words. This includes being honest and open both to oneself and others. The visibility of experiences creates opportunities for reflection on one’s own learning. The will is a force that triggers learning and thus constitutes the engine of learning. This engine, in addition to the pursuit of the familiar life, is fuelled by the idea of maintaining independence. Refusing to be more sick, along with the desire to slow the deterioration, are the driving forces of the learning process, as a man with dementia says: Being honest and open about the illness is described as an active choice that becomes part of the learning process. A man describes how he has previously found it difficult to talk about his illness, but that he ‘now is more open [...].when I meet a new person, I usually say that I have diabetes, so they know it’. If others have knowledge of the problem, it is easier to allow the illness and treatment to take place. ‘I believe that if I am not honest and open about it, then I’m almost there again, so then I might as well be honest and open’. No, I did not want to accept that I would sit and not remember anything. It was difficult for me. Therefore, I had to learn to do little thingsto maintain my memory. Obstinacy allows for the continuation of an active life during long- term illness. The person learns new strategies in order to be active. ‘Yes, for the obstinacy I have, it’s not of this world. But perhaps that is what makes you go forward’. Obstinacy is an engine for learning, but at the same time it can be a problem for realizing the limitations the illness actually has. ‘Yes, it’s clear that this has been a force helping me to succeed in life; yes, obstinacy in the right amount is good but not as much as I have’. J Nurs Care ISSN: 2167-1168 JNC, an open access journal Learning methods are a balancing act Learning to live with long-term illness can also be described as an act of balancing, where the person learns to balance between the demands of both life and illness. To find a decent existence, one must weigh the pros and cons. The balancing act is about developing an understanding of life with the illness while highlighting the positive parts of life. Yes, the illness itself makes life look like a downhill battle. I have diabetes and have to live with it. Interviewer: What do you do when that feeling comes? Yes, I might think about it a little bit, and then I think of somethingthat is positive in my life. My family. The driving forces of learning Learning to live with long-term illness can be characterized by the Volume 1 • Issue 6 • 1000125 Citation: Berglund M, Källerwald S (2012) The Movement to a New Understanding: A Life-World-Based Study about How People Learn to Live with Long-Term Illness. J Nurs Care 1:125. doi:10.4172/2167-1168.1000125 Page 3 of 5 Page 3 of 5 pursuit of a familiar life. This endeavour emerges as a key driving force towards learning as demonstrated by the desire, tenacity, and ability to influence one`s own health processes. Seeking knowledge and understanding Learning to live with long-term illness is a process of seeking knowledge and understanding to manage the situation. Knowledge is applied when coming to terms with several illnesses simultaneously and increasing one understands of a threatening future. Sometimes, caregivers seem to hinder the patient’s learning process. The need for information is described in different ways, ranging from not wanting to know more to having a great need for more knowledge. A search for knowledge involves seeking a manifold image of the future at a time when the illness pictured by the general public, the media, and loved ones often is dark and frightening. This picture does not always correspond with one’s own experiences. In this way, thoughts and discrepancies initiate the search for information. The ability to influence the health process is a force for learning. This is demonstrated as the individual has a possibility to influence restrictions in life and damage to the body. This drive is described by someone who will soon be in need of dialysis: ‘It is only now when they wanted to put me in dialysis that I began to change my diet’. That these changes made an impact on this person’s health is evidenced by his strengthened desire to learn more in order to further improve her health. The feeling of gain makes it easier to sustain changes and not revert to old habits. The information given by professionals can be difficult to understand, especially in relation to one’s own situation. ’... No, it was not me ....’ If the professional does not offer the help that is needed; or, when the advice does not match patients’ experiences, patients seek information on their own. When asked what information a man with MS had received from health care providers, he said ‘Nothing at all, about what it was and what I should do so that it will not get any worse, and so I know nothing more than that I probably will be sitting in a wheelchair in the future’. This man received information about the illness from friends, relatives, and television. ‘Yes, on House [TV series]’. This lack of information and ability to see the patient’s needs cost patients uneasiness, anxiety and an inability to take charge of, and gain from, the improvements that the patient can achieve for his or her own benefit. Making the illness visible One dimension of obstinacy is that it promotes the perseverance and courage required in situations where learners understand how to use their bodies in a whole new way, for example, learning to walk with prostheses. Citation: Berglund M, Källerwald S (2012) The Movement to a New Understanding: A Life-World-Based Study about How People Learn to Live with Long-Term Illness. J Nurs Care 1:125. doi:10.4172/2167-1168.1000125 Care that impedes learning Living as normally as possible while observing what causes the problems requires knowledge. A decreased energy level constitutes a lesson in itself, compelling the individual to consider which may lead to new insights: ‘To learn where the limits are, how much you manage and how much you dare’. By observing physical reactions in different situations, those who are learning understand more about both themselves and the illness and thereby may gain a greater understanding of what causes certain reactions. Adding observations like pieces of a puzzle allows one to reach an understanding of the importance of balancing life with illness. Learning is impeded when caregivers do not see the patient as a person and do not believe what the patient says or see the patients fear. The ability to understand and cooperate with doctors and nurses are hampered when professionals do not speak a language the patient understands: ‘Yes, they say, those strange damn words that I don’t have a clue what they mean’. Learning becomes more difficult when the patient is not allowed to ask questions: I was terribly disappointed and I wanted to ask some questions and then he looked at me and said, here I am the one asking the questions. Hope is balanced against realistic expectations. The balancing act is about not giving up hope and at the same time not putting too much faith in it. ‘One would of course like to feel healthy but I know that I cannot’. The professionals impede patients’ learning when they do not see their fear. Fear contributes to the patient’s inclination to evade from both disease and health care. Fear is an obstacle for asking for and Volume 1 • Issue 6 • 1000125 Page 4 of 5 receiving help. The patient’s fear is covered in the healthcare context. One informant said: ‘No, I tried to look happy while I was at the hospital, but I was afraid and I didn’t ask any questions because I was anxious to get away’. No one had asked him if he was afraid. very little that has a distinct life-world theoretical foundation. Ekebergh [20] maintains that most studies do not reach the core of the complex problems inherent in learning not venturing beyond the how-questions to the what-questions. J Nurs Care ISSN: 2167-1168 JNC, an open access journal The results of learning Learning to live with long-term illness gives insight into the importance of allowing the illness more space in one’s life. It is an insight that the affected obtain by learning to listen to himself, to his own body. When a person learns to live with a long-term illness this often results in development of maturity and self-awareness enabling an inner calm. Learning is about making the best of the life that the person has. Genuine learning takes place at a deeper level. Our results have shown that genuine learning has taken place when the sufferers have gone from having ignored their situation of being ill to letting the illness be a part of their lives. This is shown in the results as hard work that takes time. The patient’s genuine learning certainly grows gradually into knowledge. The problems are, however, that this extended learning process, in a life with long-term illness, contributes to the advancement of the illness and that complications can occur. In the results, this has been seen to cost the sufferers a great deal. Speeding up the learning process thus has many virtues. Long term illness prevent a full life, but a correct diagnosis, that is, a diagnosis that the person trusts, gives incentive to make life changes. Previous high expectations can be adapted to a reasonable level. This means that the illness is allowed more space in their lives, and thus also creates the all-important prerequisite for well-being. Learning is about understanding the significance in taking the time to get to know the stranger. It is easy to regret a detrimental former way of life when it’s results becomes noticeable. A paradox can be seen in learning how to live with long-term illness. This paradox entails that if the illness is not given space in a person’s life, it will intrude and demand much greater space later on. Patients need help to see and understand this paradox. There are ethical problems in patient education that need to be addressed. The questions that can be asked are the following: Do professionals have the right to force new knowledge onto the patient? Has the professions’ duty to inform and supervise been correctly formulated? The ethical questions also raise musings about personal responsibility in connection with long-term illness. The results of learning When I think of how my body has been damaged, I regret [...]not my life but that I have not taken care of myself properly. To not give the illness space in one’s life gives freedom, but a freedom that is described as ‘costly’. When the person has learned to listen to the signals, she often has reached the insight that the learning process has taken far too long, as demonstrated in expressions such as ‘understood too late’. The consideration of risks for the future are weighed against what is most valued for the moment. These include wishes for a long life and the person choosing to give the illness room in her life. What is the actual aim in learning to live with long-term illness, and who sets this aim? Do patients, professionals, and society have the same aim? Is the aim a transmission of information, a change in behaviour and treatment compliance, as previous research has shown? Does the aim concern the sufferers being able to gain insight into their actual situation, accept it, and reconcile to their situation? According to this study, the aim concerns a changed understanding, insight, personal development, and an ability to take charge of one’s life with a long-term illness. Learning means to grow and change. It results in feelings of peace and quiet, making it easier to enjoy life despite the illness. The knowledge that one has reached provides the opportunity to take advantage of good times, which is described as a ‘mature life’, to extend one’s perspective, and to preserve healthy relations. In life with long- term illness, achieved knowledge is good for oneself and one’s relatives. Learning is about seeing what life has been like, what has changed, and trying to make the best of today and the future. Giving one’s illness more space results in personal growth and the opportunity to enjoy more of the life that the person actually has. Conclusion Learning to live with long-term illness is a very complex phenomenon; it is incorporated in life as a whole and is thus difficult to distinguish from living. The learning affects the whole person, the body, cognitive functions, emotions, practical matters, and social life. This complexity has to be understood in order for genuine learning to be possible. Care that impedes learning The patient’s learning ability is lost when the patient is excluded from communication and decision making regarding their treatment. Care that impedes learning The aim in the present study, however, has been to describe the meaning of learning to live with a long-term illness in its deepest sense. This means that the aim has been to get answers to the what-questions, that is, what learning entails for persons with long-term illness. The chosen approach [15] and way of studying the phenomenon has contributed to getting answers to both the what-questions and the how-questions and provides important new knowledge. receiving help. The patient’s fear is covered in the healthcare context. One informant said: ‘No, I tried to look happy while I was at the hospital, but I was afraid and I didn’t ask any questions because I was anxious to get away’. No one had asked him if he was afraid. Doubts about the caregivers’ knowledge and skills lead to a search for an alternative trustworthy explanation or the choice to reject the diagnosis and consequently to ignore the treatment. Advice and prescriptions given by caregivers, who neglect the patients’ own knowledge and past experiences, will often be considered as not worth following. This increases the gap between caregiver and the patient and increases the distrust of health care. A woman describes how she was prescribed medication although she did not believe in the doctors’ diagnosis: The study has focused on the learning process dealing mainly with genuine learning, something which I maintain is rare in the present day caregiver patient relation and much differs from the learning of information [8,21]. The information society of today makes it easy for patients and relatives themselves to seek and obtain information about illness and treatment. There is nothing that emerges in the results that would indicate a lack of information. Previous research [7,8,22,23] has shown that patients do not lack information about illness and treatment, but that they still have problems in translating this information into their own situation. From the results of the present study, it can be seen that information and teaching on an ‘admonishing level’, a level where patients are expected to do what they are told, is not the same as a genuine learning. I do not have it I said. But then I thought that maybe I should not go to Spain if I have TB, so it’s best to keep quiet so I took all the drugs, and then I buried them somewhere around here [pointing out to the yard]. Citation: Berglund M, Källerwald S (2012) The Movement to a New Understanding: A Life-World-Based Study about How People Learn to Live with Long-Term Illness. J Nurs Care 1:125. doi:10.4172/2167-1168.1000125 J Nurs Care ISSN: 2167-1168 JNC, an open access journal Discussion There is a significant amount of research about patient education, but considerably less that describes patients’ learning and, above all, Volume 1 • Issue 6 • 1000125 Page 5 of 5 The need for learning does not concern learning merely about an illness, it concerns the person’s whole health situation. deepen understanding of the concept of compliance? A theoretical discussion. Scand J Caring Sci 19: 274-279. 7. Johansson A, Ekebergh M (2006) The meaning of well-being and participation in the process of health and care women’s experiences following a myocardial infarction. Int J Qual Stud Health Well-being 1:100-108. Learning to live with long-term illness has to be seen as a lifelong quest in the person’s life. 8. Berglund M (2011) Att ta rodret i sitt liv-lärande utmaningar vid långvarig sjukdom. Linnaeus University Dissertations. The learning process thus contributes to the possibilities for changes and new priorities in life. The result of learning can be understood as a movement to a new understanding demonstrated by the way the person acts and allows the illness more space in her life. 9. Ruderfelt D, Axelsson L (2004) Diabetes patient education—factors and interventions of significance for changeing [sic] diet.Vård i Norden [Nordic Journal of Nursing Research & Clinical]. 24: 9-14. Learning strategies are to be developed to support patients’ genuine learning. 10. Boardman T, Catley D, Grobe JE, Little TD, Ahluwalia JS (2006) Using motivational interviewing with smokers: do therapist behaviors relate to engagement and therapeutic alliance? J Subst Abuse Treat 31: 329-339. 1. Thorne SE, Paterson BL (2000) Two decades of insider research: what we know and don’t know about chronic illness experience. Annu Rev Nurs Res 18: 3-25. Practical Implications 11. Heidegger M (2008) Being and time. HarperCollins, London. The new perspective on learning to live with long-term illness, which is presented here, places many demands on care organizations to provide care that is based on the patient’s needs and not simply the diagnosis of the illness. In order for patients to be seen and supported as whole and learning individuals, a change of paradigm is needed, one in which professionals see their roles being changed from giving information to providing support for the patients’ genuine learning. This study presents a possibility to understand patients’ learning processes as well as increasing their health and wellbeing. Learning strategies have to be developed that focuses on the patient’s experiences as a starting point and results in a life-world and caring perspective. 12. Husserl E (1975) Experience and Judgment. Northwestern University Press, IL. 13. Merleau-Ponty M (2002) Phenomenology of Perception. Routledge, London. 14. Dahlberg K, Dahlberg H, Nyström M (2008) Reflective Lifeworld Research. (2ndedn). Professional Publishing Svc, Berlin. 15. Giorgi A (2009) The descriptive phenomenological method in psychology. A modified Husserlian approach. Duquesne University Press, Pittsburgh. 16. Dahlberg K (2006) The essence of essences - the search for meaning structures in phenomenological analysis of lifeworld phenomena. Int J Qual Stud Health Well-being 1:11-19. 17. The Swedish Research Council (2002) Forskningsetiskaprinciper inom humanistiska-samhällsvetenskaplig forskning. Research Council. Citation: Berglund M, Källerwald S (2012) The Movement to a New Understanding: A Life-World-Based Study about How People Learn to Live with Long-Term Illness. J Nurs Care 1:125. doi:10.4172/2167-1168.1000125 2. Thorne S, Paterson B, Acorn S, Canam C, Joachim G, et al. (2002) Chronic illness experience: insights from a metastudy. Qual Health Res 12: 437-452. 5. Newman S, Steed L, Mulligan K (2004) Self-management interventions for chronic illness. Lancet 364: 1523-1537. 3. Toombs SK (1993) The Meaning of illness-a phenomenological account of the different perspectives of physician and patient. Kluwer Academic Publisher, Boston. 6. Friberg F, Scherman MH (2005) Can a teaching and learning perspective References 18. World Medical Association (2013) Declaration of Helsinki world medical association declaration of Helsinki. 19. SFS (2003:460).Lagenometikprövningav forskning som avser människor (The law on ethical review of research involving humans). 2. Thorne S, Paterson B, Acorn S, Canam C, Joachim G, et al. (2002) Chronic illness experience: insights from a metastudy. Qual Health Res 12: 437-452. 20. Ekebergh M (2001) Tillägnandet av vårdvetenskaplig kunskap. Reflexionens betydelse för lärandet. Doktorsavhandling. Institutionen för vårdvetenskap, Åbo Akademi, Vasa. 3. Toombs SK (1993) The Meaning of illness-a phenomenological account of the different perspectives of physician and patient. Kluwer Academic Publisher, Boston. 21. Colaizzi PF (1978) Learning an existence. Oxford University Press, New York. 4. Nordberg L (2008) När kroppen sätter gränser-en studie omatt leva med hjärtsvikt I medelåldern. Växjö University Press. 22. Skalla KA, Bakitas M, Furstenberg CT, Ahles T, Henderson JV (2004) Patients’ need for information about cancer therapy. Oncol Nurs Forum 31: 313-319. 5. Newman S, Steed L, Mulligan K (2004) Self-management interventions for chronic illness. Lancet 364: 1523-1537. 23. Suhonen R, Nenonen H, Laukka A, Välimäki M (2005) Patients’ informational needs and information received do not correspond in hospital. J Clin Nurs 14: 1167-1176. 6. 4. Nordberg L (2008) När kroppen sätter gränser-en studie omatt leva med hjärtsvikt I medelåldern. Växjö University Press. References Friberg F, Scherman MH (2005) Can a teaching and learning perspective Submit your next manuscript and get advantages of OMICS Group submissions Unique features: • User friendly/feasible website-translation of your paper to 50 world’s leading languages • Audio Version of published paper • Digital articles to share and explore Special features: • 200 Open Access Journals • 15,000 editorial team • 21 days rapid review process • Quality and quick editorial, review and publication processing • Indexing at PubMed (partial), Scopus, DOAJ, EBSCO, Index Copernicus and Google Scholar etc • Sharing Option: Social Networking Enabled • Authors, Reviewers and Editors rewarded with online Scientific Credits • Better discount for your subsequent articles Submit your manuscript at: http://www.omicsonline.org/submission/ Submit your next manuscript and get advantages of OMICS Group submissions Unique features: • User friendly/feasible website-translation of your paper to 50 world’s leading languages • Audio Version of published paper • Digital articles to share and explore Special features: • 200 Open Access Journals • 15,000 editorial team • 21 days rapid review process • Quality and quick editorial, review and publication processing • Indexing at PubMed (partial), Scopus, DOAJ, EBSCO, Index Copernicus and Google Scholar etc • Sharing Option: Social Networking Enabled • Authors, Reviewers and Editors rewarded with online Scientific Credits • Better discount for your subsequent articles Submit your manuscript at: http://www.omicsonline.org/submission/ Volume 1 • Issue 6 • 1000125
https://openalex.org/W2012604059	https://periodicos.uem.br/ojs/index.php/ActaSciBiolSci/article/download/11451/pdf	English	null	Molecular variants in populations of Bryconamericus aff. iheringii (Characiformes, Characidae) in the upper Paraná river basin	Acta Scientiarum. Biological Sciences	2,013	cc-by	5,464	Variantes moleculares em populações de Bryconamericus aff. iheringii (Characiformes, Characidae) da bacia do alto rio Paraná RESUMO. Há indícios de que Bryconamericus aff. iheringii represente um complexo de espécies. Os marcadores moleculares de DNA têm sido eficazes em estudos de filogenia, taxonomia e identificação de espécies crípticas. Neste estudo, as seqüências parciais de genes da ATPase 6 e 8 foram utilizados para avaliar a diversidade genética dentro e entre populações de B. aff. iheringii das sub-bacias dos rios Tibagi, Pirapó e Ivaí, pertencentes a bacia do Alto Rio Paraná. As análises das seqüências dos genes apresentaram alta diversidade genética em B. aff. iheringii das sub-bacias estudadas, com valores de distâncias genéticas semelhantes às encontradas entre espécies diferentes. Houve uma divisão dos indivíduos em cinco grupos. A comparação com outras espécies de Bryconamericus que têm seqüências disponíveis no GenBank confirmou que os indivíduos estudados possuem valores relevantes de distância genética encontrados entre espécies diferentes. No entanto, com os dados disponíveis não é possível descartar a hipótese de que as populações correspondem a um grupo resultante de hibridação, nem que houve introgressão de DNA mitocondrial entre espécies diferentes. Palavras-chave: characiformes, Bryconamericus, mtDNA, genes da ATPase 6 e 8. Camila Montoro Mazeti, Thiago Cintra Maniglia, Sônia Maria Alves Pinto Prioli and Alberto José Prioli sidade Estadual de Maringá, Av. Colombo, 5790, 87020-900, Maringá, Paraná, Brazil. Author for correspondence. E-mail: mazeti@hotmail.com ABSTRACT. There are evidences that Bryconamericus aff. iheringii represents a species complex. DNA molecular markers have been effective in studies on phylogeny, taxonomy, and identification of cryptic species. In this study, partial sequences of genes of ATPase 6 and 8 were used to assess genetic diversity within and among populations of B. aff. iheringii of sub-basins of Tibagi, Pirapó and Ivaí rivers, belonging to the Upper Paraná river basin. The analysis of the sequences of genes pointed out high genetic diversity in B. aff. iheringii from the sub-basins studied with genetic distance values comparable to those found among different species. There was a division of the individuals into five groups. The comparison with other species of Bryconamericus that have sequences available in GenBank confirmed that the individuals studied have relevant values of genetic distance, found among different species. Nevertheless, with the available data it is not possible to refute the hypothesis that the populations correspond to a group resulting from hybridization or that there might have been introgression of mitochondrial DNA among different species. Keywords: characiformes, Bryconamericus, mtDNA, genes of ATPase 6 and 8. http://www.uem.br/acta ISSN printed: 1679-9283 ISSN on-line: 1807-863X Acta Scientiarum Doi: 10.4025/actascibiolsci.v35i2.11451 http://www.uem.br/acta ISSN printed: 1679-9283 ISSN on-line: 1807-863X Acta Scientiarum Doi: 10.4025/actascibiolsci.v35i2.11451 Material and methods Cytogenetic studies have been undertaken with individuals of B. aff. iheringii. In specimens of Água da Floresta river, at the sub-basin of Tibagi river, individuals showed a diploid number (2n) of 52 chromosomes distributed as 8M+22SM+10ST+12A, with a fundamental number (FN) of 92 (PAINTNER-MARQUES et al., 2003). The same 2n was found in a population of Keller stream, at the sub-basin of Ivaí river (PORTELA-CASTRO; JULIO-JUNIOR, 2002). However, three different cytotypes were detected in this population: I, with 12M+18SM+8ST+14A; II, with 10M+22SM+8ST+12A; and III, with 8M+28SM+6ST+10A. Capistano et al. (2008) examined specimens of B. aff. iheringii of three streams belonging to the Upper Paraná river basin (Keller stream, Maringá stream and Tatupeba stream) have registered 2n = 52, but three different karyotypes have been observed. As cytotype I (population of Maringá stream), the karyotype is composed of 12M+18SM+8ST+14A with FN of 90; the II (population of Keller stream) had 8M+28SM+10ST+6A, FN equal to 94 ; the cytotype III (species of Tatupeba stream) with 8M+20SM+8ST+16A with FN of 88. Although the 2n= 52 chromosomes is a characteristic of Bryconamericus sp., as described previously, the karyotypes for different species in this genus vary, suggesting that chromosomal rearrangements may be involved in the karyotypic evolution of this group of fish (PAINTNER-MARQUES et al., 2003). Portela-Castro and Julio-Junior (2002) suggests that these changes may have been the result of pericentric inversions and that perhaps these cytotypes correspond to different species of Bryconamericus. Specimens of B. aff. iheringii were collected in three localities of Paraná river Basin (Table 1 and Figure 1) and were deposited in the fish collection of Molecular Genetics laboratory at the Center for Research in Limnology, Ichthyology and Aquaculture (NUPÉLIA) of the Maringá State University. Total genomic DNA was obtained from tissue samples according to Monesi et al. (1998), with modifications. Table 1. Geographical coordinates of the collection points of specimens of the Bryconamericus aff. iheringii in the upper Paraná river basin. river basin. Sampling Localities Coordinates mtDNA samples 1. Tibagi river, Pitangui Stream, Ponta Grossa city, Paraná State 25° 01’ S – 50° 04’ W 12 2. Pirapó river, Maringá Stream, Maringá city, Paraná State 23° 20’ S – 51° 51’ W 5 3. Ivaí river, Keller Stream, Marialva city, Paraná State 23° 38’ S – 51° 51’ W 6 Figure 1. Collection areas of Bryconamericus aff. iheringii in the upper Paraná river basin. Introduction Characiformes, has been divided into subfamilies by different authors, due to the large number of species and the similarities among groups of genera. Thus, the species with poorly known evolutionary relationships were considered Incertae sedis, including Bryconamericus genus (LIMA et al., 2005). The taxon B. iheringii is described as having its type- locality in São Lourenço City (Rio Grande do Sul State - Brazil), thus the species that occur in the Paraná river is probably new to science, so that it would be more appropriate to call them as B. aff. iheringii (GRAÇA; PAVANELLI, 2007) One of the major groups of freshwater fish worldwide is formed by representatives of the Characiformes order (NELSON, 2006). Genetic and cytogenetic studies have confirmed the hypothesis shared by systematists that it is a non- monophyletic group (SAITOH et al., 2003). Many studies have indicated problems of classification from specific (CAPISTANO et al., 2008; PAINTNER-MARQUES et al., 2003; PRIOLI et al., 2004) to superorder levels (SAITOH et al., 2003). The family Characidae, included into Maringá, v. 35, n. 2, p. 241-248, Apr.-June, 2013 Acta Scientiarum. Biological Sciences 242 Mazeti et al. Mazeti et al. Results With the PCR amplification, it was possible to have a sequence of approximately 1,500 base-pairs (bp). However, a shorter sequence with 800 bp, covering the partial regions of the genes of ATPase 6 and 8, was selected for analysis due to its better quality sequencing after manual editing. The proportion of bases found in this sequence was A= 0.2910; C= 0.2857; G= 0.1074; T= 0.3159, with the transition/transversion rate ratios (Ti/Tv) of 4.099, the estimate of global genetic differentiation (Nst) equal to 6, with rate of invariant nucleotides (I) of 0.6693. Based on the characteristics found in the sequences studied, the evolutionary model that best applies to explain the genetic model was the Tamura Nei plus I (TrN+I) model. The alignment sequences analyzed showed nucleotide substitutions of 143 points, being 28 transversion and 125 transitions. There were a high number of substitutions associated with groups of individuals, indicating genetic similarity among them. In this way, individuals were classified into five groups. The neighbor-joining and maximum likelihood trees have confirmed the formation of five groups (Figure 2). The sequences amplified were aligned with the program Clustal W (THOMPSON et al., 1994) and edited in Bioedit (HALL, 1999). Procedures using the corrected Akaike Information Criterion (AIC) and Bayesian Information Criterion (BIC) of the program Modeltest 3.7 (POSADA et al., 1998) were determined by maximum likelihood. The differentiation among populations of different sub- basins was inferred from neighbor-joining and maximum likelihood trees, and Bayesian Inference using the model defined by the program Modeltest 3.7. The neighbor-joining tree, with 10,000 bootstrap samplings, was performed with the program MEGA 4.0 and the maximum likelihood tree, also using 10,000 bootstrap samplings, was obtained with the program PAUP4.0.b10b4 (SWOFFORD, 2002). The Phylogeny of Bayesian inference, which calculated the posterior probability of genealogical relationships in a better model of development, was obtained by the Markov Chain Monte Carlo Simulation (MCMC) using the program MrBayes 3.0 (HUELSENBECK; RONQUIST, 2001). The tree was done with 300,000 generations, with the retention of 10 generations. The first 20,000 generations were discarded because the number was determined for the parameters converging to stability. Consequently, only 280,000 generations were used to calculate the consensus tree. Table 2 lists the values of the average nucleotide diversity calculated with the TrN+I model among the five groups of B. aff. iheringii and the outgroup B. scleroparius. Material and methods The numbers represent the collection points: 1 – Tibagi river, 2 – Pirapó river and 3 – Ivaí river. A segment that corresponds to the complete Sampling Localities Coordinates mtDNA samples 1. Tibagi river, Pitangui Stream, Ponta Grossa city, Paraná State 25° 01’ S – 50° 04’ W 12 2. Pirapó river, Maringá Stream, Maringá city, Paraná State 23° 20’ S – 51° 51’ W 5 3. Ivaí river, Keller Stream, Marialva city, Paraná State 23° 38’ S – 51° 51’ W 6 The genetic variations can be analyzed by molecular markers, such as the sequences of mitochondrial DNA (mtDNA) (PRIOLI et al., 2002). The mitochondrial DNA molecule is highly conserved; however, the mitochondrial genes ATPase 6 and 8 have the characteristic of accumulating nucleotide substitutions that can detect genetic variations among species or even among populations of the same species (BERMINGHAM; MARTIN, 1998; MACHORDON; DOADRIO, 2001; PERDICES; DOADRIO, 2001; WONG et al., 2004). Therefore, the molecular analysis, comparing different populations of B. aff. iheringii, can provide important information in elucidating the condition of these taxonomic species. Figure 1. Collection areas of Bryconamericus aff. iheringii in the upper Paraná river basin. The numbers represent the collection points: 1 – Tibagi river, 2 – Pirapó river and 3 – Ivaí river. A segment that corresponds to the complete sequence of the genes of ATPase 6 and 8 and partial sequence of the genes tRNALys and COIII were amplified by PCR using primers H9236 (5´- GTTAGTGGTCAKGGGCTTGGRTC-3´) and L8331 (5´-AAAGCRTYRGCCTTTTAAGC-3´) Maringá, v. 35, n. 2, p. 241-248, Apr.-June, 2013 Acta Scientiarum. Biological Sciences Molecular variants of Bryconamericus 243 samplings was conducted with the program MEGA 4.0. The dendrogram of maximum likelihood was obtained with 500 bootstrap samplings of the program PAUP4.0.b10b4 (SWOFFORD, 2002). described by Lovette et al. (1998). Amplifications were carried out according to Prioli et al. (2002). The samples were purified after the amplification, according to Rosenthal et al. (1993). The final product of PCR reaction was used in sequencing reactions of nucleotides in a MegaBace Automatic Sequencer (Amersham) following manufacturer's instructions. Acta Scientiarum. Biological Sciences Results iheringii of the sub-basin of Tibagi river (BRCN TB), sub-basin of Pirapó river (BRCN PI) and sub-basin of Ivaí river (BRCN IV), located in the upper Paraná river basin. One individual of B. scleroparius was used in the analysis as an outgroup. The bootstrap analysis was based on 10,000 samplings, only values higher than 65 were represented. Figure 3. Scatter plot of haplotypes along the Principal Coordinates with three values of eigenvectors of the individuals of five groups of Bryconamericus aff. iheringii of Tibagi, Pirapó and Ivaí rivers. Figure 3. Scatter plot of haplotypes along the Principal Coordinates with three values of eigenvectors of the individuals of five groups of Bryconamericus aff. iheringii of Tibagi, Pirapó and Ivaí rivers. Figure 3. Scatter plot of haplotypes along the Principal Coordinates with three values of eigenvectors of the individuals of five groups of Bryconamericus aff. iheringii of Tibagi, Pirapó and Ivaí rivers. Figure 3. Scatter plot of haplotypes along the Principal Coordinates with three values of eigenvectors of the individuals of five groups of Bryconamericus aff. iheringii of Tibagi, Pirapó and Ivaí rivers. Figure 2. Neighbor-joining (A) and maximum likelihood trees (B) constructed with the TrN+I model, from partial sequences of the genes of ATPase 6 and 8 of individuals of B. aff. iheringii of the sub-basin of Tibagi river (BRCN TB), sub-basin of Pirapó river (BRCN PI) and sub-basin of Ivaí river (BRCN IV), located in the upper Paraná river basin. One individual of B. scleroparius was used in the analysis as an outgroup. The bootstrap analysis was based on 10,000 samplings, only values higher than 65 were represented. Figure 4. Tree of phylogeny of Bayesian inference calculated using the program Mr. Bayes 3.0 by the Markov Chain Monte Carlo Simulation (MCMC). The values in the branches are the percentage of posterior probability of the last 280,000 generations simulated using the model of nucleotide substitution TrN + I Table 2. Average nucleotide diversity calculated with the TrN+I model among the five groups of Bryconamericus. aff. iheringii and the outgroup Bryconamericus scleroparius. Table 2. Average nucleotide diversity calculated with the TrN+I model among the five groups of Bryconamericus. aff. iheringii and the outgroup Bryconamericus scleroparius. Group 1 Group 2 Group 3 Group 4 Group 5 Group 1 0.001 Group 2 0.045 NS* Group 3 0.040 0.043 0.008 Group 4 0.124 0.120 0.110 0.000 Group 5 0.132 0.128 0.119 0.064 0.001 B. Results The values obtained were at least 0.04, between groups 1 and 3, and a maximum of 0.132, between groups 1 and 5. The scatter plot of haplotypes confirmed the formation of five distinct groups within the species (Figure 3). For the phylogenetic analysis, an individual of the species B. scleroparius was used as outgroup. Scatter plots of the haplotypes were made in main coordination, with the program Statistica 6.0 (STATSOFT Inc., 2001). The Bayesian Inference of Phylogeny tree has confirmed the groups trained in neighbor-joining and maximum likelihood trees (Figure 4). The high values of a posteriori probability indicate the trend of branches generated when it is assumed a large number of generations. GenBank sequences of other species of Bryconamericus (AF412573 - AF412627) were selected and analyzed to be compared to the sequences of B. aff. iheringii obtained in this study. In order to ensure greater reliability in the analysis, the sequences were aligned, and only those pairs of bases in the region of the genes of ATPase 6 and 8, were considered. The differentiation among populations of different species of Bryconamericus was inferred from neighbor-joining and maximum likelihood dendrograms, using the same model previously defined by the program Modeltest 3.7. The neighbor-joining clustering based on 10,000 bootstrap The neighbor-joining groups, with 10,000 bootstrap samplings, and maximum likelihood, with 500 bootstrap samplings, constructed with the TrN+I model among the five groups of B. aff. iheringii and the species B. scopiferus, B. emperador, B. terrabensis, B. scleroparius, B. ricae and B. bayano also supported the division of individuals of B. aff. iheringii into five groups (Figure 5). It was observed Maringá, v. 35, n. 2, p. 241-248, Apr.-June, 2013 Acta Scientiarum. Biological Sciences 244 Mazeti et al. genetic distance were achieved when comparing the five groups of B. aff. iheringii with the species available in GenBank (0.18 to 0.217), evidencing the high differentiation of B. aff. iheringii in relation to the other species of Bryconamericus. that individuals of B. aff. iheringii analyzed in this study are genetically distant from the other species of Bryconamericus, whose sequences are available in GenBank. Figure 2. Neighbor-joining (A) and maximum likelihood trees (B) constructed with the TrN+I model, from partial sequences of the genes of ATPase 6 and 8 of individuals of B. aff. Acta Scientiarum. Biological Sciences Discussion Several studies with different approaches have been performed using the genes of ATPase 6 and 8 in fish. At lower taxonomic levels, with close phylogenetic relationships, such as populations of the same species and subspecies, the surveys have shown that the genetic distances, in absolute values, vary between 0.002 and 0.054 (FAULKS et al., 2008; KINZIGER et al., 2007; MACHORDOM; DOADRIO, 2001; PERDICES; DOADRIO, 2001; SIVASUNDAR et al., 2001). Among species of the same genus, the observed values are between 0.015 and 0.13 (FROUFE et al., 2005; MACHORDON; DOADRIO, 2001; PERDICES; DOADRIO, 2001; REID; WILSON, 2006; SIVASUNDAR et al., 2001). For higher taxonomic levels, the values of genetic diversity are also high, ranging from 0.062 to 0.16 (SIVASUNDAR et al., 2001; FROUFE et al., 2005). In the analysis with the model of nucleotide substitution TrN+I, the distances obtained have showed five groups of B. aff. iheringii. The distances TrN+I between groups 1 and 3 (0.040) and groups 1 and 5 (0.132) are comparable with the distances TrN+I among Bryconamericus species with sequences available in GenBank, whose values are distributed from 0.030 to 0.093. In this distribution, the lowest values are compatible with intra-specific levels found in the literature. In contrast, the highest values are at levels found in congeneric species. Considering that, in this study it was used only representatives of B. aff. iheringii from sub-basins located in the upper Paraná river, it was not expected high levels of molecular polymorphism. However, diversity could be expected at low levels in populations of B. aff. iheringii of the upper Paraná river, as found in previous studies, in some cytotypes, with the same diploid number, but with different karyotypic forms (PORTELA-CASTRO; JULIO-JUNIOR, 2002; CAPISTANO et al., 2008). In cytogenetic studies, the individuals of Ivaí river had four different cytotypes, while in the molecular analysis, only two haplotypes. One of these haplotypes was also found in individuals of the Pirapó river, belonging to group 3, the only group that was distributed in all sub-basins studied. This fact can be justified due to the greater proximity between the collection points in the Ivaí and Pirapó rivers, with a greater possibility of connection between fish populations with a lower genetic diversity due to the increased gene flow. Figure 5. Molecular variants of Bryconamericus Molecular variants of Bryconamericus Molecular variants of Bryconamericus Acta Scientiarum. Biological Sciences Figure 5. Neighbor-joining (A) and maximum likelihood (B) dendrograms constructed with the TRN + I model, based on partial nucleotide sequences of the genes for ATPase 8 and 6 of B. aff. iheringii, B. scopiferus, B. emperador, B. terrabensis, B. scleroparius, B. ricae and B. bayano. Analyses were based on 500 bootstrap re-samplings, only values higher than 65 were represented. Discussion Acta Scientiarum. Biological Sciences Maringá, v. 35, n. 2, p. 241-248, Apr.-June, 2013 Results scleroparius 0.199 0.212 0.185 0.205 0.201 NS* - non-significant. simulated using the model of nucleotide substitution TrN + I Table 3. Average genetic distance between groups of individuals of Bryconamericus aff. iheringii of Tibagi, Pirapó and Ivaí rivers, and individuals of other species of the genus Bryconamericus, calculated with TrN + I from the 800 bp partial fragment of the genes ATPase 8 and 6. Gp 1 Gp 2 Gp 3 Gp 4 Gp 5 I II III IV V VI Group 1 0.001 Group 2 0.046 NS* Group 3 0.041 0.044 0.008 Group 4 0.123 0.119 0.109 0.000 Group 5 0.130 0.126 0.116 0.062 0.001 B. scopiferus (I) 0.207 0.217 0.200 0.216 0.209 0.005 B. emperador (II) 0.202 0.213 0.194 0.214 0.208 0.054 0.016 B. terrabensis (III) 0.193 0.202 0.183 0.201 0.201 0.082 0.086 0.003 B. scleroparius (IV) 0.194 0.207 0.180 0.200 0.196 0.091 0.093 0.030 0.000 B. ricae (V) 0.194 0.207 0.182 0.204 0.191 0.090 0.091 0.034 0.040 0.007 B. bayano (VI) 0.205 0.217 0.191 0.215 0.211 0.091 0.091 0.032 0.042 0.042 0.001 NS* - non-significant. Table 3 contains the values of average nucleotide diversity calculated with the TrN+I model among the five groups of B. aff. iheringii and the species of the genus Bryconamericus available in GenBank. The lowest values of genetic distances were observed among the five groups of B. aff. iheringii (0.04 to 0.13) and among the six species available in GenBank (0.03 to 0.093). The higher values of Maringá, v. 35, n. 2, p. 241-248, Apr.-June, 2013 Acta Scientiarum. Biological Sciences 245 Molecular variants of Bryconamericus Discussion Neighbor-joining (A) and maximum likelihood (B) dendrograms constructed with the TRN + I model, based on partial nucleotide sequences of the genes for ATPase 8 and 6 of B. aff. iheringii, B. scopiferus, B. emperador, B. terrabensis, B. scleroparius, B. ricae and B. bayano. Analyses were based on 500 bootstrap re-samplings, only values higher than 65 were represented. Possibly this haplotype has arisen from populations in Tibagi river, which presents several Maringá, v. 35, n. 2, p. 241-248, Apr.-June, 2013 Acta Scientiarum. Biological Sciences 246 Mazeti et al. waterfalls that vary from 1.5 to 115 meters, along an altitude variation of 762 meters (FRANÇA, 2002). This condition favors the isolation of populations and the emergence of different haplotypes. study for the groups are pertinent to those found for different species. So, the analyses suggest that the levels of genetic differentiation of B. aff. iheringii of the upper Paraná river are consistent in indicating divergences compatible with a species complex, with up to five different species. g p yp Until recently, there were no anthropogenic barriers that could have prevented the displacements source-mouth, thus it is plausible that regular migrants from group 3 have reached the basin of Paranapanema, Paraná and Ivaí rivers. Apparently, the haplotypes of groups 2 and 5 were restricted to Tibagi river basin (Figure 2). A probable explanation for the differences in the geographic distribution would be the greater aggressiveness and/or dispersal ability of the group 3. As a result of the wider geographic distribution, the average intra-group distance (d = 0.008) shows that the group 3 is more heterogeneous. Morphologically, it can be suggested that the populations of B. aff. iheringii belong to a single species. However, the neighbor- joining and maximum likelihood trees indicate the formation of two clades, because of the consistent values of genetic differences, which may indicate the presence of at least two ancestral species. The clade A with the groups 1, 2 and 3, and the clade B with the groups 4 and 5 (Figure 2). This configuration is confirmed by high bootstrap values in the trees. The scatter plots of haplotypes (Figure 3) corroborate the information provided by the group, and Bayesian inference (Figure 4) evidences a high value of a posteriori probability (1.00) for the branch between these two clades. p p The nucleotide differences found among the haplotypes strongly indicate interspecific levels. Conclusion In this way, taking into account that B. aff. iheringii shows molecular evidences of formation of species complexes, it is likely that analysis of samples from other regions and other basins could reveal many other groups genetically differentiated at interspecific level. This analysis pointed out remarkable evidences of the diversity under the name B. aff. iheringii, but for now, only available in molecular analyses. Because of its magnitude, it is imperative that taxonomic studies, supported by molecular methods, promote the mapping of this diversity. The distances found between clades A and B of B. aff. iheringii (0.11 to 0.132) (Table 2) are at levels equivalent to those found for different species of Bryconamericus, showing high genetic differentiation. Moreover, within each clade, the genetic distances among groups correspond to interspecific distances. Discussion However, with the data available it is not possible to discard the hypothesis that the populations may correspond to a group resulting from the hybridization of two or more species of Bryconamericus. Another possibility would be the introgression of mitochondrial DNA among different species. Regardless the explanation, it seems inevitable that there should be events of speciation. Nevertheless, for a greater understanding of the genetic overview of this group, further studies are required, using molecular markers more conserved than ATPase. Acknowledgements We would like to thank to COMCAP (Complexo de Centrais de Apoio à Pesquisa – UEM), for the sequencing of samples, to Dra. Carla Simone Pavanelli, for the taxonomic identification of fish, to Dra. Ana Maria Geahl for the collection of fish from the Tibagi river and, to the Post-Graduate Program in Comparative Biology of the State University of Maringá. The genetic distances among groups and other species of Bryconamericus were higher, ranging from 0.18 to 0.217 (Table 3). Values are within a range of distances often found among species of the same family but of different genera. This result could be expected, since the sequences of Bryconamericus available in GenBank are of Central American species (REEVES; BERMINGHAM, 2006). Neighbor-joining and maximum likelihood groups (Figure 5) corroborate these results. 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https://openalex.org/W4281252788	https://www.nature.com/articles/s41598-022-12451-7.pdf	English	null	Refractive astigmatism in phaco-canaloplasty vs phaco-non-penetrating deep sclerectomy	Scientific reports	2,022	cc-by	7,290	Refractive astigmatism in phaco‑canaloplasty vs phaco‑non‑penetrating deep sclerectomy OPEN Anna Byszewska, Jacek Rudowicz, Katarzyna Lewczuk, Joanna Jabłońska & Marek Ręka This study aimed to assess refractive astigmatism, in phaco-canaloplasty (PC) vs phaco-non- penetrating deep sclerectomy (PDS) in a randomized, prospective study within 24 months. Patients were randomized pre-operatively, 37 underwent PC and 38 PDS. The following data was collected: BCVA, IOP, number of antiglaucoma medications, refraction with autokeratorefractometry. The assessment of astigmatism was simple arithmetic and vector analysis (calculations included cylinder with axis in form of centroids) and included double angle plots and cumulative refractive astigmatism graphs. Pre-operative mean BCVA in PC was 0.40 ± 0.43 and was comparable to BCVA in PDS 0.30 ± 0.32logMAR (P = 0.314). In the sixth month follow-up, mean BCVA showed no difference (P = 0.708) and was 0.07 ± 0.13 and 0.05 ± 0.11, respectively. However, 2 years after the intervention mean BCVA was better in PC 0.05 ± 0.12 than in PDS 0.12 ± 0.23 and it was statistically significant (P = 0.039). Mean astigmatism in PC at baseline was 1.13 ± 0.73Dcyl, at 6 months it was 1.09 ± 0.61 and at 2 years 1.17 ± 0.51. In PDS at baseline 1.35 ± 0.91 at 6 months 1.24 ± 0.86 and at 2 years 1.24 ± 0.82. There were no differences between the groups in mean astigmatism throughout the study. Centroids (mean of a cylinder with axis) in PC were pre-operatively 0.79D@172˚ ± 1.10Dcyl, at 6 months 0.75D@166˚ ± 1.01 and at 24-months 0.64D@164˚ ± 1.11 and in PDS pre-operatively 0.28D@10˚ ± 1.63D at 6 months 0.26D@11˚ ± 1.5 and at 24-months 0.47D@20˚ ± 1.43. The direction of mean astigmatism was against the rule in all analyzed time points. The mean baseline IOP in PC was 19.4 ± 5.8 mmHg and 19.7 ± 5.4 mmHg in PDS(P = 0.639). From the 6-month IOP was lower in PC, at 24-months it was 13.8 ± 3.3 mmHg in PC and 15.1 ± 2.9 mmHg in PDS(P = 0.048). In both groups preoperatively patients used median(Me) of three antiglaucoma medications(P = 0.197), at 24-months in PC mean 0.5 ± 0.9 Me = 0.0 and 1.1 ± 1.2 Me = 1.0 in PDS(P = 0.058). Both surgeries in mid-term observation are safe and effective. They do not generate vision-threatening astigmatism and do not even change the preoperative direction of mean astigmatism. Refractive astigmatism is stable throughout the observation. www.nature.com/scientificreports www.nature.com/scientificreports Scientific Reports \| (2022) 12:8604 Ophthalmology Department, Military Institute of Medicine, Warsaw, Poland. email: ania.byszewska@gmail.com Subjects and methodsh j Subjects. This paper represents further aspects of a previously published study, where patients and methods were described in detail5,11. The methods and results are presented in line with Guidelines on Design & Report- ing Glaucoma Trials12. This study is in line with European Union entitled Good Clinical Practice for Trials on Medical Products in the European Community and the tenets of the World Medical Association Declaration of Helsinki. The project received approval from the Institutional Review Board of the Military Institute of Medicine in Warsaw (9/WIM/2011) and was registered at https://www.clinicaltrials.gov NCT01726543 on 15/11/2012.ii g p g Patients with coexisting glaucoma and cataract (NC1 and NC2) classified according to the Lens Opacificities Classification System LOCS III were qualified for the study. In all cases glaucoma procedure was performed with cataract extraction and monofocal IOL implantation. Glaucoma types. Types of glaucoma eligible for the study were primary open-angle glaucoma (POAG), pseudoexfoliation glaucoma (PEX), and pigmentary glaucoma. Apart from the type of glaucoma, patients must have had at least one of the following features: well-documented progression of the visual field; non-compliance in anti-glaucoma therapy or allergy to topical medications, daily fluctuations in pressure. Both PC and PDS as well as surgical alternatives were explained in detail to candidates. After declaring willingness to participate in the study, each patient signed an informed consent. Design. The design of the study was randomized and prospective. Randomization into groups was carried out by a random sorting algorithm with an allocation ratio set to 1.0 on the day of surgery. y g g y g y A single physician (AB) was responsible for the preoperative examination, randomization, and postoperative care. Also, a single surgeon (MR) performed all the surgical procedures and then was excluded from any further medical involvement in this study to avoid bias. y In the course of the study the intraocular pressure (IOP), the number of antiglaucoma medications, Best Corrected Visual Acuity (BCVA), autokeratorefractometry (AKR), Humphrey 24-2 visual fields (VF), Optical Coherent Tomography (OCT) morphology of the surgical site, Quality of life (QoL) assessment- were collected prospectively5,11. Preoperative examination. At the baseline visit, ophthalmic and general medical history was taken. Dur- ing the visit the AKR measurement was carried out as the first examination, to avoid any influence from other, especially contact examinations. www.nature.com/scientificreports/ Although both surgeries are non-penetrating, there are many differentiating factors intra and postoperative which may influence the postoperative refraction. During PC cautery is used as little as possible, to spare the outflow vessels, while it is not that important in PDS- this may vastly change the architecture of the wound. In PDS the outflow depends on well-functioning filtering bleb, to achieve that only two loose fixating sutures are used on the superficial scleral flap, whereas in PC there are five tight sutures and the bleb is aimed to be watertight and flat. Additionally, in PDS to maintain outflow though the bleb , we used 5-fluouracil (5-FU) in subconjunctival injections. This substance may cause transient irregular astigmatism, due to toxicity to the corneal epithelium. And last but not least in PC a prolene suture is inserted and tightened inward in Schlemm’s canal. Such tension changes the anatomical relations, which was already shown with ultrabiomicroscopy (UBM) or anterior segment OCT. g In this paper, we want to concentrate on the refractive aspects of both non-penetrating surgeries. Refractive astigmatism may be considered as one of the safety indicators, as it directly influences visual acuity, and quite frequently a correction with glasses is poorly tolerated.hh This analysis aimed to describe the 24-month postoperative course of astigmatism for both procedures. The period in which postoperative astigmatism stabilizes is still under research. Cunliffe et al.6 showed data 2 months after penetrating procedures, Dietze et al.7 after 3 months, Willekens et al.8 observed stabilization after 3 months, Claridge et al.9 and Hong et al.10 reported changes up to 12 months. Many authors suggested the need for long- term observation. In this paper we analyze preoperative data, then after 6 and 24 months.t p p y p pt To our knowledge, there is little literature describing astigmatic changes after glaucoma procedures, and none in 24 months period concerning canaloplasty and PDS, compared to other aspects of glaucoma surgery. Refractive astigmatism in phaco‑canaloplasty vs phaco‑non‑penetrating deep sclerectomy OPEN Nowadays glaucoma surgery is under constant innovation. Gold standard—trabeculectomy is the most often performed glaucoma procedure worldwide1, however, is associated with certain risks, due to its’ perforating character. Well-known complications such as postoperative hypotony, macular edema, hyphema, shallow anterior chamber, or filtering bleb associated problems such as avascularity, late bleb leakage leading to infections or bleb insufficiency, cause qualifications for surgeries in more advanced stages of glaucoma. The ratio risk of the surgery to benefit is relatively high to perform procedures in early and moderate glaucoma patients.h i y g p p y g p Then the non-penetrating procedures were developed. Non-penetrating deep sclerectomy although not such IOP lowering as compared to trabeculectomy, enables a much better safety profile with avoidance of most of the serious, sight-threatening complications. Its safety and efficacy are thoroughly described in the literature2,3.i Canaloplasty is a relatively new procedure in glaucoma surgery, introduced in scientific publications for the first time in 2007 by Lewis4. It represents one step further in safety and quality of life for a glaucoma patient. It is also a non-penetrating procedure and the surgical approach compromises of similar steps as DPS, up to the dissection of the trabeculo-Descemet’s membrane5. Both procedures, although technically challenging for surgeons, enabled a change of attitude in referring patients for glaucoma surgeries in early and moderate stages. \| https://doi.org/10.1038/s41598-022-12451-7 Scientific Reports \| (2022) 12:8604 www.nature.com/scientificreports/ www.nature.com/scientificreports/ anesthesia (2% xylocaine and 0.5% bupivacaine) by one experienced surgeon (MR). Classic canaloplasty was carried out with a standard canaloplasty set (iTrack from Ellex Medical Lasers Pty Ltd., Adelaide, Australia). Nonpenetrating deep sclerectomy was carried out with a Healaflow implant, a slowly resorbable crosslinked viscoelastic gel (Anteis Ophthalmology, Geneva, Switzerland). In both procedures, a fornix-based superficial scleral flap was dissected, followed by a deep scleral flap and a TDM dissection. During the next step, a 2.2 mm clear corneal temporal incision was made, the cataract was phacoemulsified (Infiniti Vision System, Alcon Surgi- cal, Fort Worth, TX), and a monofocal IOL was implanted. The deep scleral flap was excised. In PC, 360° of the Schlemm’s canal circumference was catheterized and viscodilated with 10.0 Prolene suture left under tension to distend the trabecular meshwork inward. In PDS, after dissection of TDM, the roof of Schlemm’s canal was removed. The superficial scleral flap was then loosely sutured to the sclera, and HealaFlow was injected under the flap to create a filtering bleb. In PC, the superficial flap was sutured tightly to prevent leakage and subsequent bleb formation with interrupted 10-0 monofilament nylon suture. The conjunctiva was sutured down over the limbus with one interrupted 8.0 Vicryl suture. Postoperative protocol. The postoperative visits were scheduled for days 1 and 7 and 1, 3, 6, 12, 18, and 24 months after surgery. However, patients were informed that they can show up any time when needed. A topi- cal steroid and antibiotic combination was prescribed for 4 weeks after surgery. During postoperative examina- tions, AKR data was collected as the first one, followed by BCVA assessment and then IOP was determined and the number of hypotensive medications was noted. Afterwards the anterior segment examination including gonioscopy (in the case of PC, to assess any possible complications) and at the end of visit a dilated pupil fun- doscopy was performed. Glaucoma drugs were discontinued on the day of surgery. In the course of the study, medications were administered again, when required, under the guidelines of the European Glaucoma Society. Complications that occurred within 30 days were analyzed as early, whereas after 30 days were considered as late. To keep IOP at a sufficiently low level, additional procedures were carried out. www.nature.com/scientificreports/ In the case of PDS, they were associated with filtering bleb maintenance such as 5-FU subconjunctival injections (when signs of bleb failure were noticed—new, tortuous vessels, hyperemia, or encapsulation), suture lysis, and needling. 5-FluoroUracil was injected in a dose of 0.2 ml (5 mg) in the lower fornix of the operated eye. If needling of filtering bleb was required (encapsulated and flat blebs, which caused elevated IOP), the patient was anesthetized with proxymeta- caine eye drop, and needling was performed at the slit lamp, followed by 5-FU injection. Goniopuncture, which is laser puncture of TDM, was performed in both PC and PDS, when filtration through TDM was suspected to be insufficient (with an Nd: YAG laser, about 3–20 shots were applied using energy ranging from 2 to 4 mJ). Refractive parameters. The refraction was recorded at the central 3-mm diameter by Auto-kerato-refrac- tometry (Topcon TRK 2P), which is serviced according to manufacturer recommendations. The AKR data was analysed at the baseline, then 6 months and 24 months post-surgery. All calculations were performed after the transposition of the cylinder values to the plus form. p y p Two types of analysis were performed- refractive and vector. Arithmetic. The first one is the simple arithmetic calculation of the mean of the cylinder, without consider- ing its axis. This analysis was performed to discuss the numerical results available in the literature. Such a dif- ference in the mean value of the cylinder allows reporting the mean change in the magnitude of astigmatism. Aggregate data of astigmatism values are depicted in the cumulative data plots. Vector. The vector analysis—the second method presented is a calculation of the cylinder change with con- sideration of its axis, which results in a calculation of centroid. The preoperative and postoperative refractive measurements (cylinder with its axis) were evaluated by vector analysis, according to the method proposed by Holladay et al13 Centroid is a form of astigmatism, which is calculated in a certain way (see below), so that it can be presented as a set of x and y values on a cartesian graph (standard polar data are converted to cartesian values suitable for calculations, but still including the axis). What is most important centroid incorporates the magnitude and axis of astigmatism. It can be then included in the standard descriptive statistics (means, stand- ard deviations). Subjects and methodsh Then uncorrected distance visual acuity and BCVA were checked, followed by parameters required for IOL calculation such as axial length and keratometry. Central corneal thickness was also evaluated. We collected data of IOP with the number of antiglaucoma medications taken. Routinely a gonios- copy was performed. All subjects had a dilated slit-lamp examination. IOP measurements. The IOP was measured with a Goldmann applanation tonometer and all measure- ments included in the analysis were taken between 8 and 10 am. During the qualification visit, a diurnal curve of IOP was assessed and a single measurement on the day of surgery was taken. Routinely, two measurements were taken, if they varied more than 1 mmHg, the third one was taken, and the outcome was the average of the three measurements. Based on IOP values, the course of mean IOP was defined. BCVA. A standard ETDRS chart was used to measure the BCVA. The calculations were performed using log- MAR -a logarithm of the minimum angle of resolution. SRK T formula was used to calculate the IOL. Surgical procedure. Surgical procedures were previously described in detail5,11. For a better understand- ing of the outcomes, the description is cited below. All surgical procedures were performed under retrobulbar Scientific Reports \| (2022) 12:8604 \| https://doi.org/10.1038/s41598-022-12451-7 www.nature.com/scientificreports/ www.nature.com/scientificreports/ cyl = √ x2 + y2 Angle = 1/2 ∗( tan −1 y / x if x > 0 and x > 0 angle = axis if x < 0 axis = angle + 90◦ if x > 0 and y < 0 axis = angle + 180◦ if x = 0 a y < 0 axis = 135◦ if x = 0 a y > 0 axis = 45◦ if x = 0 i y = 0 axis = 0◦ if y = 0 a x < 0 axis = 90◦ if y = 0 a x > 0 axis = 0◦ cyl = √ x2 + y2 Angle = 1/2 ∗( tan −1 y / x if x > 0 and x > 0 angle = axis if x < 0 axis = angle + 90◦ if x > 0 and y < 0 axis = angle + 180◦ if x = 0 a y < 0 axis = 135◦ if x = 0 a y > 0 axis = 45◦ if x = 0 i y = 0 axis = 0◦ if y = 0 a x < 0 axis = 90◦ if y = 0 a x > 0 axis = 0◦ The calculation was performed for each individual, to compute individual surgically induced refractive change. The mean of all x and y allowed to calculate aggregate refractive change for the analyzed groups.h gh y gg g g y g p The data was displayed in the double angle plots (the angles had to be doubled as the astigmatism vector eturns to the same value when it traverses an angle of 180 degrees).h g g ) The major and minor axes of the ellipse surrounding the centroid were determined by standard deviations of x and y coordinates. The trend of astigmatism was evaluated depending on centroid values and axis.h yh g g The double-angle plots were depicted with the help of a double-angle plot tool for astigmatism available on the ASCRS website14. Statistical analyses. The Shapiro–Wilk was used for the assessment of the normality of the data. Non- parametric data were calculated with a χ2 test with corrections. Comparisons between the groups (IOP, BCVA, refractive astigmatism) were performed with U Mann–Whitney test, and the Student’s T-test. Results Subjects. Throughout the study, 37 patients were randomized for PC and 38 for PDS. All patients were Caucasian. Mean age was comparable for both groups, in PC was 75.1 ± 8 years and 73.6 ± 6.2 years (P = 0.079). The sex structure and side of the surgery did not differ between both arms of the study. The glaucoma types were primary open-angle and pseudoexfoliation glaucoma (27/10 in PC and 34/4 in PDS), P = 0.124 (Table 1). BCVA. The BCVA data is presented in the logMAR scale. Baseline BCVA in the PC and PDS group was comparable and was 0.40 ± 0.43 and 0.30 ± 0.32 , respectively (P = 0.314). After 6 months visual acuity improved significantly to 0.07 ± 0.13 and 0.05 ± 0.11 (P = 0.708). A 24-month follow-up revealed a better mean BVCA in PC, which was 0.05 ± 0.12 than in PDS 0.12 ± 0.23 (P = 0.039). For both groups improvement was significant in all-time points during the study (P < 0 001). Two years after surgery, stable vision or improvement of one or more Snellen lines was present in all patients after PC and in the majority of PDS (85.3%). A decline of one line was noted in three PDS (8.8%) patients. The decline of two lines was observed in two subjects (5.9%). One developed diabetic macular edema and the second had transient visual acuity instability, as the good vision was regained at the following examination (0.0 logMAR). No changes in BCVA were observed in 8.8% PDS and 13.3% PC eyes. Astigmatism. Refractive data was available from 35 PC and 37 PDS patients. Mean astigmatism at the base- line in PC was 1.13 ± 0.73 Dcyl, whereas in PDS 1.35 ± 0.91 Dcyl, at 6 months post-surgery it was stable 1.09 ± 0.61 Dcyl in PC and respectively 1.24 ± 0.86 for PDS. At the end of the 24-month observation, it was still around one Diopter and precisely for PC 1.17 ± 0.49 Dcyl and 1.24 ± 0.82 Dcyl for PDS. Throughout the whole observation, there were no differences found between the studied groups in mean astigmatism. Also, Friedman ANOVA analyses showed no statistical differences within the groups during this 24 months timespan (Table 2). yf g p g p Cumulative astigmatism data is depicted in the diagrams (Figs. 1, 2). www.nature.com/scientificreports/ Friedman’s analy- sis of variance (ANOVA) for matched groups, mean ranks, and rank sums were also used for posthoc compari- sons. A P-value of 0.05 or less was considered significant. Calculations were performed using the Statistica 10.0 PL software. www.nature.com/scientificreports/ When a larger set of data is presented, centroid means the mean of all values, and a trend for astigmatism can be described (with the rule, against the rule, or oblique).h g ( g q ) The data were converted from standard polar values (cylinder and axis) to Cartesian values (point with x,y coordinates) to evaluate trends in astigmatism (against the rule, with the rule or oblique) and to define mean astigmatism in centroid form. g For conversion from polar to cartesian values the following mathematical formula was applied: x = cyl ∗cos(2 ∗axis) y = cyl ∗sin(2axis) x = cyl ∗cos(2 ∗axis) y = cyl ∗sin(2axis) Then the Cartesian coordinates were converted to standard polar values, with formulas: https://doi.org/10.1038/s41598-022-12451-7 Scientific Reports \| (2022) 12:8604 \| www.nature.com/scientificreports/ Table 1. Patients’ demographic data. * Chi2. # Mann–Whitney U. Phacocanaloplasty Phaco-deep sclerectomy P-value Data Mean ± SD ratio N 37 38 0.908 * Age (years) 75.1 ± 8.1 73.6 ± 6.2 0.079# Sex (female/male) 15/22 21/17 0.202 * Eye (right/left) 15/22 17/21 0.713 * Glaucoma type: POAG/PEX 27/10 34/4 0.124 * Table 1 Patients’ demographic data Chi2 #Mann Whitney U Phacocanaloplasty Phaco-deep sclerectomy P-value Data Mean ± SD ratio N 37 38 0.908 Age (years) 75.1 ± 8.1 73.6 ± 6.2 0.079# Sex (female/male) 15/22 21/17 0.202 * Eye (right/left) 15/22 17/21 0.713 * Glaucoma type: POAG/PEX 27/10 34/4 0.124 * Table 1. Patients’ demographic data. * Chi2. # Mann–Whitney U. www.nature.com/scientificreports/ Results of statistical analysis between the groups (U-Mann Whitney Test) as well as for separate groups in analyzed time points (Friedman ANOVA). Statistical significance for P < 0.05. all of the subjects had astigmatism lower than 2D, whereas in DS there was 6% of patients who had astigmatism higher than 3D. Vector analysis results. Mean centroid before the surgery was 0.79D @172˚ ± 1.10 for PC and 0.28D @ 10˚ ± 1.63 for PDS (P = 0.364). Six months later it was 0.75D@166˚ ± 1.01 and 0.26D@11˚ ± 1.50 respectively (P = 0.828). At the end of observation mean astigmatism in PC was 0.64D@164˚ ± 1.11 and 0.47D@20˚ ± 1.43D in PDS (P = 0.874). Also, Friedman_ANOVA analyses for separate groups showed no statistical differences within study time. The centroids are depicted graphically in double-angle plots below. In both groups, preopera- tive astigmatism was against the rule (ATR) and the trend was stable throughout the study. (Figs. 3, 4, 5, 6, 7 and 8). IOP The mean baseline IOP in PC was 19 4±5 8mmHgand 19 7±5 4 mmHg in PDS and did not differ Figure 1. Cumulative astigmatism in PC. Figure 1. Cumulative astigmatism in PC. Figure 1. Cumulative astigmatism in PC. Figure 1. Cumulative astigmatism in PC. all of the subjects had astigmatism lower than 2D, whereas in DS there was 6% of patients who had astigmatism higher than 3D. all of the subjects had astigmatism lower than 2D, whereas in DS there was 6% of patients who had astigmat higher than 3D. Vector analysis results. Mean centroid before the surgery was 0.79D @172˚ ± 1.10 for PC and 0.28D @ 10˚ ± 1.63 for PDS (P = 0.364). Six months later it was 0.75D@166˚ ± 1.01 and 0.26D@11˚ ± 1.50 respectively (P = 0.828). At the end of observation mean astigmatism in PC was 0.64D@164˚ ± 1.11 and 0.47D@20˚ ± 1.43D in PDS (P = 0.874). Also, Friedman_ANOVA analyses for separate groups showed no statistical differences within study time. The centroids are depicted graphically in double-angle plots below. In both groups, preopera- tive astigmatism was against the rule (ATR) and the trend was stable throughout the study. (Figs. 3, 4, 5, 6, 7 and 8). IOP. The mean baseline IOP in PC was 19.4 ± 5.8mmHgand 19.7 ± 5.4 mmHg in PDS and did not differ between the groups (P = 0 639). www.nature.com/scientificreports/ www.nature.com/scientificreports/ all of the subjects had astigmatism lower than 2D, whereas in DS there was 6% of patients who had astigmatism higher than 3D. Vector analysis results. Mean centroid before the surgery was 0.79D @172˚ ± 1.10 for PC and 0.28D @ 10˚ ± 1.63 for PDS (P = 0.364). Six months later it was 0.75D@166˚ ± 1.01 and 0.26D@11˚ ± 1.50 respectively ( ) h d f b d Table 2. Arithmetic mean of astigmatism. Results of statistical analysis between the groups (U-Mann Whitney Test) as well as for separate groups in analyzed time points (Friedman ANOVA). Statistical significance for P < 0.05. Dcylinder_month N Mean [Dcyl][minimum; maximum] SD Median 1st, 3rd quartile N Mean [Dcyl][minimum; maximum] SD Median 1st, 3rd quartile U-Mann–Whitney test p-value PC PDS Dcyl_0 35 1.13 [0; 3] 0.73 1.0 [0.5; 1.5] 37 1.35 [0.25; 4] 0.91 1.25 [0.75; 1.5] P = 0.544 Dcyl_6 34 1.09 [0; 3.5] 0.61 1.0 [0.5; 1.25] 37 1.24 [0; 3.95] 0.86 1.0 [0.75;1.5] P = 0.595 Dcyl_24 31 1.17[0;2.75] 0.49 1.0 [0.75;1.5] 37 1.24 [0; 3.5] 0.82 1.25 [0.75;1.75] P = 0.917 Friedman_ANOVA p-value Chi2 ANOVA (N = 32, df = 2) = 1.407407 P = 0.49475 Chi2 ANOVA (N = 36, df = 2) = 0.5909091 P = 0.74419 Figure 1. Cumulative astigmatism in PC. Table 2. Arithmetic mean of astigmatism. Results of statistical analysis between the groups (U-Mann Whitney Test) as well as for separate groups in analyzed time points (Friedman ANOVA). Statistical significance for P < 0.05. Dcylinder_month N Mean [Dcyl][minimum; maximum] SD Median 1st, 3rd quartile N Mean [Dcyl][minimum; maximum] SD Median 1st, 3rd quartile U-Mann–Whitney test p-value PC PDS Dcyl_0 35 1.13 [0; 3] 0.73 1.0 [0.5; 1.5] 37 1.35 [0.25; 4] 0.91 1.25 [0.75; 1.5] P = 0.544 Dcyl_6 34 1.09 [0; 3.5] 0.61 1.0 [0.5; 1.25] 37 1.24 [0; 3.95] 0.86 1.0 [0.75;1.5] P = 0.595 Dcyl_24 31 1.17[0;2.75] 0.49 1.0 [0.75;1.5] 37 1.24 [0; 3.5] 0.82 1.25 [0.75;1.75] P = 0.917 Friedman_ANOVA p-value Chi2 ANOVA (N = 32, df = 2) = 1.407407 P = 0.49475 Chi2 ANOVA (N = 36, df = 2) = 0.5909091 P = 0.74419 Table 2. Arithmetic mean of astigmatism. Results of statistical analysis between the groups (U-Mann Whitney Test) as well as for separate groups in analyzed time points (Friedman ANOVA). Statistical significance for P < 0.05. Table 2. Arithmetic mean of astigmatism. Results At baseline, 47% of the PC patients and 36% of PDS had astigmatism ≤ 0,75D. Six months postoperatively in both groups about half of the patients had astigmatism ≤ 1D, whereas ≤ 1,5 D, 91% of PC, and 78% of DS patients. At the end of observation, almost https://doi.org/10.1038/s41598-022-12451-7 Scientific Reports \| (2022) 12:8604 \| Conclusion/discussion We found only one paper describing astigmatic changes in canaloplasty. Moelle et al.15 observed 26 canaloplasty patients and the mean preoperative astigmatism was 0.77 ± 0.5 D, then it was higher in the initial healing phase to reach stabilization at 6 months (0.86 ± 0.52D). There was no significant difference in preoperative astigmatism and astigmatism at 6 months (P > 0.05). Findings are similar to those presented in this paper. 6t Egrilmez16 assessed astigmatism in 10 patients following deep sclerectomy and 12 after viscocanalostomy and confronted this data with trabeculectomy results. The lowest arithmetic averages of induced vectors were in viscocanalostomy, then deep sclerectomy, being both statistically lower at 3 and 6 months compared to trab- eculectomy. In our study arithmetic averages are stable within the groups throughout the study. Preoperatively the mean astigmatism value was 1.13 ± 0.73 in PC and 1.35 ± 0.91 in PDS and it stayed on a similar level throughout the study, which is confirmed in Friedman ANOVA analysis. i On the contrary, El-Saied17 compared a relatively large group of trabeculectomy and deep sclerectomy subjects and concluded that both after 6 months induce significant postoperative astigmatism, due to flattening along the vertical meridian, more with trabeculectomy than deep sclerectomy. y y Vector analysis allowed us to access the trend of astigmatism. In both groups throughout the study, it was against the rule. As it was against the rule also preoperatively we are not trying to look for the causes of the postoperative trend, as the surgery did not significantly influence the trend. And such a trend describes the studied population.fh p p In our paper, both analyzed procedures, although non-penetrating, characterize certain differences. The principle of canaloplasty is enhanced outflow through distal outflow pathways-dilated Schlemm’s canal, collec- tor channels18,19, while dissection of the scleral flap is performed to get access to Schlemm’s canal. On the other hand, NPDS functioning is based to a vast degree on the subconjunctival outflow, apart from transscleral and suprachoroidal pathways20.lf Nonetheless, the presence of the scleral flap itself and the quantity and strength of suturing may affect the direction of postoperative astigmatism. In the analyzed PDS group there are only two scleral sutures on the superficial scleral flap, one on each side, which allows a controlled flow of aqueous humor to subconjunctival space. The flap is sutured loosely. www.nature.com/scientificreports/ Within the first 3 post-op months, no difference in mean IOP was found. Start- ing from the 6th month, the mean IOP was lower in PC, and the difference lasted until 24 months (P = 0.048). After 6 months mean IOP for PC was 13.02 ± 3.24 and in PDS 14.2 ± 2.9. At the end of observation, mean IOP significantly decreased to 13.8 ± 3.3 mmHg (P = 0.001) and 15.1 ± 2.9 mmHg (P = 0.001), respectively. Mean IOP was reduced by 25.7% in PC and 18.9% in PDS. https://doi.org/10.1038/s41598-022-12451-7 Scientific Reports \| (2022) 12:8604 \| www.nature.com/scientificreports/ Figure 2. Cumulative astigmatism in PDS. Figure 2. Cumulative astigmatism in PDS. Figure 2. Cumulative astigmatism in PDS. Medications. Fewer medications were used after surgery than before in both groups (P < 0 05). Baseline mean number of meds in PC was 2.6 ± 0.9, Me = 3.0, and in PDS, 2.9 ± 0.9, Me = 3.0 (P = 0.197). After 6 months in PC it was 0.2 ± 0.5 ME = 0 and for PDS 0.2 ± 0.6 ME = 0 (P = 0.639). At the 24 months follow up it was still without statistically significant difference in PC 0.5 ± 0.9 Me = 0.0 and 1.1 ± 1.2 Me = 1.0 (P = 0.058). Medications. Fewer medications were used after surgery than before in both groups (P < 0 05). Baseline mean number of meds in PC was 2.6 ± 0.9, Me = 3.0, and in PDS, 2.9 ± 0.9, Me = 3.0 (P = 0.197). After 6 months in PC it was 0.2 ± 0.5 ME = 0 and for PDS 0.2 ± 0.6 ME = 0 (P = 0.639). At the 24 months follow up it was still without statistically significant difference in PC 0.5 ± 0.9 Me = 0.0 and 1.1 ± 1.2 Me = 1.0 (P = 0.058). Conclusion/discussion In PC, on the contrary, five tight sutures (one to the apex and two to each side) were put to secure the watertight closure of the flap.l p gl p In neither of the procedure, a full-thickness scleral gap was dissected, the partial thickness of scleral flaps was comparable in both groups. The layer of the trabeculoDescemet’s window enabled the uninterrupted continuity of the tissues, which may also contribute to lesser change in refraction16. Scientific Reports \| (2022) 12:8604 \| https://doi.org/10.1038/s41598-022-12451-7 www.nature.com/scientificreports/ Figure 3. Preoperative astigmatism on the double angle plot in PC. The trend is ATR. The yellow dots represent a single subject’s data. Figure 3. Preoperative astigmatism on the double angle plot in PC. The trend is ATR. The yellow dots represent a single subject’s data. Figure 3. Preoperative astigmatism on the double angle plot in PC. The trend is ATR. The yellow dots repre a single subject’s data. Figure 3. Preoperative astigmatism on the double angle plot in PC. The trend is ATR. The yellow dots represent a single subject’s data. The shape of the scleral flap in PDS is a rectangle, whereas in PC it is ellipsoid. Suturing a flap to the sclera can create a redistribution of mechanical tensions. The possible effect on astigmatism concerning the shape of the scleral flap was previously discussed in the literature. Tanito et al.21 suggested that that the triangular flap may characterize better readaptation than a rectangular one, thus creating little probability for an unsupported corneal edge. The same situation can be addressed to PC, as there are relatively many tight sutures along the whole edge of the flap, which enables good adaptation. gl g Another aspect is the cauterization of the scleral wound, which may contract the tissue, then cause the reshaping of the sclera. In PDS cauterization is used routinely, whereas in PC it should be avoided when possible. Cauterization closes superficial distal outflow pathways, which are indirectly a target in this surgery22.i il Well-functioning filtering blebs in PDS rarely cause dysesthesia23. When the surgery achieves a stable state, the filtering blebs are relatively flat and usually do not cause vertical steepening of the cornea. Conclusion/discussion In contrary to trabeculectomy, where with the rule astigmatism is often induced24–26 followed by against the rule shift.l tt Induction of postoperative astigmatism may be influenced by various intraocular pressure levels, especially when the hypotony is present, the eye is more vulnerable to shape changes. It plays a vital role in penetrating surgeries, with a higher risk of a long-lasting profound hypotony. In our study, the mean IOPs were lower in PC, which was statistically significant, but we had no cases of long-standing hypotony, which would persist until a https://doi.org/10.1038/s41598-022-12451-7 Scientific Reports \| (2022) 12:8604 \| www.nature.com/scientificreports/ Figure 4. Astigmatism on the double angle plot 6 months post-op in PC. The trend is ATR. The yellow dots represent a single subject’s data. Figure 4. Astigmatism on the double angle plot 6 months post-op in PC. The trend is ATR. The yellow dots represent a single subject’s data. 6-month follow-up. A regression analysis was calculated however, it showed no relationship between astigmatism values and IOP levels.tf 6-month follow-up. A regression analysis was calculated however, it showed no relationship between astigmatism values and IOP levels.tf Additionally, we analysed data for separate right and left eyes. However, no differences were found between eyes for single surgery (PC and PDS) in all time points (results are not included in the paper).i Additionally, we analysed data for separate right and left eyes. However, no differences were found between eyes for single surgery (PC and PDS) in all time points (results are not included in the paper).i During the first postoperative month, 95% of PDS patients received 5-FU subconjunctival injections, ranging from 1 to 10, with the mean number 3.75 injections per person. 5-FU is a well-known antimetabolite to improve success in bleb-dependent procedures. However, it is also known to induce intermittent irregular astigmatism. H l ft d h d d h f d ff During the first postoperative month, 95% of PDS patients received 5-FU subconjunctival injections, ranging from 1 to 10, with the mean number 3.75 injections per person. 5-FU is a well-known antimetabolite to improve success in bleb-dependent procedures. However, it is also known to induce intermittent irregular astigmatism. However, in a longer perspective, after 6 and 24 months, we did not see the refractive difference.h g p ptf The most important conclusion of the conducted analysis is that both non penetrating surgeries, do not induce significant refractive change. Received: 5 September 2021; Accepted: 30 March 2022 Received: 5 September 2021; Accepted: 30 March 2022 References References 1. Conlon, R., Saheb, H. & Ahmed, I. I. K. Glaucoma treatment trends: A review. Can. J. Ophthalmol. 52, 114–124 (2017).fi 1. Conlon, R., Saheb, H. & Ahmed, I. I. K. Glaucoma treatment trends: A review. Can. J. Ophthalmol. 52, 114–124 (2017). 2. Gabai, A., Cimarosti, R., Battistella, C., Isola, M. & Lanzetta, P. Efficacy and safety of trabeculectomy versus nonpenetrating surger- ies in open-angle glaucoma: A meta-analysis. J. Glaucoma 28, 823–833 (2019).i p ( ) 2. Gabai, A., Cimarosti, R., Battistella, C., Isola, M. & Lanzetta, P. Efficacy and safety of trabeculectomy versus nonpenetrating surger- ies in open-angle glaucoma: A meta-analysis. J. Glaucoma 28, 823–833 (2019).i p g g y 3. Eldaly, M. A., Bunce, C., Elsheikha, O. Z. & Wormald, R. Non-penetrating filtration surgery versus trabeculectomy for open-angle glaucoma. Cochrane Database Syst. Rev. 2014 (2014).l g y 4. Lewis, R. A. et al. Canaloplasty: Circumferential viscodilation and tensioning of Schlemm’s canal using a flexible microcatheter for the treatment of open-angle glaucoma in adults: Interim clinical study analysis. J. Cataract Refract. Surg. 33, 1217–1226 (2007). 4. Lewis, R. A. et al. Canaloplasty: Circumferential viscodilation and tensioning of Schlemm’s canal using a flexible microcatheter for the treatment of open-angle glaucoma in adults: Interim clinical study analysis. J. Cataract Refract. Surg. 33, 1217–1226 (2007). 5. Rękas, M., Byszewska, A., Petz, K., Wierzbowska, J. & Jünemann, A. Canaloplasty versus non-penetrating deep sclerectomy—A prospective, randomised study of the safety and efficacy of combined cataract and glaucoma surgery; 12-month follow-up. Graefe’s Arch. Clin. Exp. Ophthalmol. 253, 591 (2015).ffhf 6. Cunliffe, I. A., Dapling, R. B., West, J. & Longstaff, S. The effect of trabeculectomy on corneal topography. Ophthalmic Surg. 24 135 (1993).f 7. Dietze, P. J. et al. Visual function following trabeculectomy: Effect on corneal topography and contrast sensitivity. J. Glaucoma 6 99–103 (1997).i 8. Willekens, K., Pinto, L. A., Delbeke, H., Vandewalle, E. & Stalmans, I. Trabeculectomy with Moorfields conjunctival closure tech- nique offers safety without astigmatism induction. J. Glaucoma 25, e531–e535 (2016).hf f 9. Claridge, K. G., Galbraith, J. K., Karmel, V. & Bates, A. K. The effect of trabeculectomy on refraction, keratometry and corneal topography. Eye (Lond). 9(Pt 3), 292–298 (1995).hf g y y 0. Hong, Y. J., Choe, C. M., Lee, Y. G., Chung, H. S. & Kim, H. K. The effect of mitomycin-C on postoperative corneal astigmatism in trabeculectomy and a triple procedure. Ophthalmic Surg. Conclusion/discussion This is true for the within-group analysis as well between the groups.hh g p ptf The most important conclusion of the conducted analysis is that both non penetrating surgeries, do not induce significant refractive change. This is true for the within-group analysis as well between the groups.hh ih This study although prospective, randomized has its limitations. The surgery itself combined cataract extrac- tion with glaucoma and cataract surgery may influence the outcomes. At baseline, there is a lenticular component of astigmatism, whereas postoperatively IOL component. This analysis was based on AKR data, which evaluates whole astigmatism, not only corneal changes. On the other hand, it presents a real-life data.h g y g p The study is a single-center study and the sample number is limited. https://doi.org/10.1038/s41598-022-12451-7 Scientific Reports \| (2022) 12:8604 \| www.nature.com/scientificreports/ Figure 5. Astigmatism on the double angle plot 24 months post-op in PC. The trend is ATR. The yellow dots represent a single subject’s data. Figure 5. Astigmatism on the double angle plot 24 months post-op in PC. The trend is ATR. The yellow dots represent a single subject’s data. https://doi.org/10.1038/s41598-022-12451-7 Scientific Reports \| (2022) 12:8604 \| www.nature.com/scientificreports/ Figure 6. Preop astigmatism on the double angle plot PDS. The trend is ATR. The yellow dots represent a single subject’s data. Figure 6. Preop astigmatism on the double angle plot PDS. The trend is ATR. The yellow dots represent a single subject’s data. https://doi.org/10.1038/s41598-022-12451-7 Scientific Reports \| (2022) 12:8604 \| www.nature.com/scientificreports/ Figure 7. Astigmatism on the double angle plot 6 months post-op in PDS. The trend is ATR. The yellow dots represent a single subject’s data. Figure 7. Astigmatism on the double angle plot 6 months post-op in PDS. The trend is ATR. The yellow dots represent a single subject’s data. https://doi.org/10.1038/s41598-022-12451-7 Scientific Reports \| (2022) 12:8604 \| www.nature.com/scientificreports/ Figure 8. Astigmatism on the double angle plot 24 months post-op in PDS. The trend is ATR. The yellow dots represent a single subject’s data. Figure 8. Astigmatism on the double angle plot 24 months post-op in PDS. The trend is ATR. The yellow dots represent a single subject’s data. www.nature.com/scientificreports/ G., Galbraith, J. K., Karmel, V. & Bates, A. K. The effect of trabeculectomy on refraction, keratometry and cornea topography. Eye 9, 292–298 (1995). 26. Claridge, K. G., Galbraith, J. K., Karmel, V. & Bates, A. K. The effect of trabeculectomy on refraction, keratometry and corneal topography. Eye 9, 292–298 (1995). Competing interests h p g The authors declare no competing interests. References Lasers 29, 484–489 (1998). in trabeculectomy and a triple procedure. Ophthalmic Surg. Lasers 29, 484–489 (1998). 11. Byszewska, A., Jünemann, A. & Rękas, M. Canaloplasty versus nonpenetrating deep sclerectomy: 2-year results and quality assessment. J. Ophthalmol. 2018, 2347593 (2018). y p p p g 11. Byszewska, A., Jünemann, A. & Rękas, M. Canaloplasty versus nonpenetrating deep sclerectomy: 2-year results and quality of life assessment. J. Ophthalmol. 2018, 2347593 (2018). J p , ( ) 12. Guidelines on Design & Reporting Glaucoma Trials, World Glaucoma Association. (Kugler Publications, 2009). https://doi.org/10.1038/s41598-022-12451-7 Scientific Reports \| (2022) 12:8604 \| Author contributions A.B., J.R. and M.R. wrote the main manuscript text. K.L. and J.J. prepared the tables. A.B. and J.R. collected the data and prepared the figures. All authors reviewed the manuscript. A.B., J.R. and M.R. wrote the main manuscript text. K.L. and J.J. prepared the tables. A.B. and J.R. collected the data and prepared the figures. All authors reviewed the manuscript. Additional information Correspondence and requests for materials should be addressed to A.B. Correspondence and requests for materials should be addressed to A.B. © The Author(s) 2022 www.nature.com/scientificreports/ www.nature.com/scientificreports/ 13. Holladay, J. T., Moran, J. R. & Kezirian, G. M. Analysis of aggregate surgically induced refractive change, prediction error, and intraocular astigmatism. J. Cataract Refract. Surg. 27(1), 61–79 (2001).i 4. Abulafia, A., Koch, D. D., Holladay, J. T., Wang, L. & Hill, W. Pursuing perfection in intraocular lens calculations: IV. Rethinking astigmatism analysis for intraocular lens-based surgery: Suggested terminology, analysis, and standards for outcome reports. J Cataract Refract. Surg. 44, 1169–1174 (2018).t f g 5. Moelle, M. C., Cursiefen, C., Rejdak, R., Horn, F. K. & Junemann, A. G. M. Time course of induced astigmatism after canaloplasty J. Glaucoma 23, e53–e59 (2014). 6. Egrilmez, S., Ates, H., Nalcaci, S., Andac, K. & Yagci, A. Surgically induced corneal refractive change following glaucoma surgery Nonpenetrating trabecular surgeries versus trabeculectomy. J. Cataract Refract. Surg. 30, 1232–1239 (2004). 7. El Sayyad, F., Helal, M., El-Kholify, H., Khalil, M. & El-Maghraby, A. Nonpenetrating deep sclerectomy versus trabeculectomy in bilateral primary open-angle glaucoma. Ophthalmology 107, 1671–1674 (2000). p y p g g p gy ( ) 18. Körber, N., Hermann, C., Peckar, C. & Pavlidis, M. Fluorescein channelography in canaloplasty: quantitative approach. Spektrum Augenheilkd. 30(1), 23–30 (2016).ll g 9. Grieshaber, M. C., Pienaar, A., Olivier, J. & Stegmann, R. Channelography: Imaging of the aqueous outflow pathway with flexible microcatheter and fluorescein in canaloplasty. Klin. Monbl. Augenheilkd. 226, 245–248 (2009).l l p y g 0. Johnson, D. H. & Johnson, M. How does nonpenetrating glaucoma surgery work? Aqueous outflow resistance and glaucoma surgery. J. Glaucoma 10, 55–67 (2001).f g y 21. Tanito, M., Matsuzaki, Y., Iikeda, Y. & Fujihara, E. Comparison of surgically induced astigmatism following different glaucoma operations. Clin. Ophthalmol. 11, 2113–2120 (2017).l p p 2. Grieshaber, M. C. M., Pienaar, A., Olivier, J. & Stegmann, R. Clinical evaluation of the aqueous outflow system in primary open- angle glaucoma for canaloplasty. Vis. Sci. 51, 1498–1504 (2010). g g p y 3. Cheng, J., Hu, K. & Anand, N. Nonpenetrating glaucoma surgery (deep sclerectomy, viscocanaloplasty, and canaloplasty). in Managing Complications in Glaucoma Surgery. 51–72. https://doi.org/10.1007/978-3-319-49416-6_3 (Springer, 2017).hf 24. Delbeke, H., Stalmans, I., Vandewalle, E. & Zeyen, T. The effect of trabeculectomy on astigmatism. J. Glaucoma 25, e308–e312 (2016). ( ) 5. Hugkulstone, C. E. Changes in keratometry following trabeculectomy. Br. J. Ophthalmol. 75, 217–218 (1991).hf 25. Hugkulstone, C. E. Changes in keratometry following trabeculectomy. Br. J. Ophthalmol. 75, 217–218 (1991).hf 6. Claridge, K. Reprints and permissions information is available at www.nature.com/reprints. 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. 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/. https://doi.org/10.1038/s41598-022-12451-7 Scientific Reports \| (2022) 12:8604 \|
https://openalex.org/W3045144133	https://hal.umontpellier.fr/hal-02953024/document	English	null	Coffinite formation from UO2+x	Scientific reports	2,020	cc-by	8,639	To cite this version: Stephanie Szenknect, Delhia Alby, Marta López García, Chenxu Wang, Renaud Podor, et al.. Coﬀinite formation from UO 2+x. Scientific Reports, 2020, 10 (1), pp.12168. 10.1038/s41598-020-69161-1. hal-02953024 Coﬀinite formation from UO 2+x Stephanie Szenknect, Delhia Alby, Marta López García, Chenxu Wang, Renaud Podor, Frédéric Miserque, Adel Mesbah, Lara Duro, Lena Zetterström Evins, Nicolas Dacheux, et al. To cite this version: Stephanie Szenknect, Delhia Alby, Marta López García, Chenxu Wang, Renaud Podor, et al.. Coﬀinite formation from UO 2+x. Scientific Reports, 2020, 10 (1), pp.12168. 10.1038/s41598-020-69161-1. hal-02953024 Coﬀinite formation from UO 2+x Stephanie Szenknect, Delhia Alby, Marta López García, Chenxu Wang, Renaud Podor, Frédéric Miserque, Adel Mesbah, Lara Duro, Lena Zetterström Evins, Nicolas Dacheux, et al. Coffinite formation from UO2+x PEN Most of the highly radioactive spent nuclear fuel (SNF) around the world is destined for final disposal in deep-mined geological repositories. At the end of the fuel’s useful life in a reactor, about 96% of the SNF is still UO2. Thus, the behaviour of UO2 in SNF must be understood and evaluated under the weathering conditions of geologic disposal, which extend to periods of hundreds of thousands of years. There is ample evidence from nature that many uranium deposits have experienced conditions for which the formation of coffinite, USiO4, has been favoured over uraninite, UO2+x, during subsequent alteration events. Thus, coffinite is an important alteration product of the UO2 in SNF. Here, we present the first evidence of the formation of coffinite on the surface of UO2 at the time scale of laboratory experiments in a solution saturated with respect to amorphous silica at pH = 9, room temperature and under anoxic conditions. Uraninite, UO2+x is the most common U4+ mineral in nature followed by coffinite, USiO4, which is found as a primary phase or an alteration product in many uranium deposits. Coffinite, tetragonal, is isostructural with zircon (ZrSiO4) and thorite (ThSiO4); however, coffinite can contain some water either as H2O or OH groups1. Altered uraninite and coffinite have been documented from Oklo, Gabon2–5, deposits in the Athabasca Basin4,6,7 and Elliot Lake, Canada8. Other examples include Jachymov, Czech Republic9 or La Crouzille district, France10. For many years, coffinite had gone unrecognized in most uranium deposits, particularly uranium roll-front deposits, as a distinct phase because of its fine grain size and intimate association with uraninite5,6,11,12. The alteration of uraninite to coffinite is a key event for UO2 in nature and UO2 in spent fuel in a geologic repository. Coffinite, being a U4+-silicate, is associated with reducing environments, with sulphides and organic matter1, where it likely precipitated from neutral to weakly alkaline fluids. Coffinite formation in sedimentary uranium deposits is associated with relatively low temperatures, 80–130 °C. A detailed investigation of meteoric roll-front deposits in the Athabasca basin, suggest an estimated temperature of coffinite precipitation in the uranium front of no greater than 50 °C13. HAL Id: hal-02953024 https://hal.umontpellier.fr/hal-02953024v1 Submitted on 29 Sep 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. www.nature.com/scientificreports www.nature.com/scientificreports Coffinite formation from UO2+x Stéphanie Szenknect1, Delhia Alby1, Marta López García2, Chenxu Wang4, Renaud Podor1, Frédéric Miserque5, Adel Mesbah1, Lara Duro2, Lena Zetterström Evins3, Nicolas Dacheux1, Jordi Bruno2 & Rodney C. Ewing4 OPEN Scientific Reports \| (2020) 10:12168 1ICSM, Univ Montpellier, CEA, CNRS, ENSCM, 30207 Bagnols sur Cèze, France. 2Amphos 21, Consulting, Carrer Veneçuela, 103, Planta 2, 08019 Barcelona, Spain. 3Swedish Nuclear Fuel and Waste Management Co, Blekholmstorget 30, 101 24 Stockholm, Sweden. 4Department of Geological Sciences, Stanford University, Stanford, CA 94305‑2115, USA. 5DES‑Service de la Corrosion et du Comportement des matériaux dans leur Environnement (SCCME), CEA, Université Paris-Saclay, 91191 Gif‑Sur‑Yvette, France. email: stephanie.szenknect@cea.fr Coffinite formation from UO2+x PEN Even though laboratory experiments report coffinite formation at 150–250 °C14,15, it appears that these elevated temperatures are not required to form coffinite in nature.fi pp p qfi Although coffinite is abundant in uranium ore deposits, its synthesis has been a major challenge since its initial description as a mineral in 195516. A number of investigators have sought to obtain pure synthetic coffinite, but only a few have succeeded14,15,17–21. Synthetic coffinite was always obtained under hydrothermal conditions. Systematically, the samples obtained were a mixture of phases, mainly composed of fine grains of USiO4, nano- particles of UO2 and amorphous SiO2. All of these attempts to synthesize coffinite indicate that there is only a narrow range in terms of temperature, pH, uranium and silicate ions concentrations and oxygen fugacity for which the formation of coffinite over UO2 is favored. More recently, the determination of thermodynamic data has been made possible thanks to the preparation of a single-phase USiO4 sample22,23. These data confirm the relative stability of coffinite and UO2 as a function of groundwater composition. Thermodynamic calculations indicate unambiguously that coffinite is less stable than the quartz and UO2 (cr) mixture at 25 °C. However, coffinite precipitates in solutions undersaturated with respect to amorphous UO2⋅2H2O (am) in silicate solu- tions with concentrations typical of groundwater (i.e.[Si]tot between 7 × 10–5 and 5 × 10–3 mol L−1)24. This result supports the idea that the uraninite-coffinite transformation requires a prior destabilization of uraninite and that this could be caused by self-irradiation, leading to metamictization of the solid and radiolysis of water and/ or surface oxidation at moderate oxygen fugacities. Non-stoichiometry is also common in natural uraninite and 1ICSM, Univ Montpellier, CEA, CNRS, ENSCM, 30207 Bagnols sur Cèze, France. 2Amphos 21, Consulting, Carrer Veneçuela, 103, Planta 2, 08019 Barcelona, Spain. 3Swedish Nuclear Fuel and Waste Management Co, Blekholmstorget 30, 101 24 Stockholm, Sweden. 4Department of Geological Sciences, Stanford University, Stanford, CA 94305‑2115, USA. 5DES‑Service de la Corrosion et du Comportement des matériaux dans leur Environnement (SCCME), CEA, Université Paris-Saclay, 91191 Gif‑Sur‑Yvette, France. email: stephanie.szenknect@cea.fr \| https://doi.org/10.1038/s41598-020-69161-1 Scientific Reports \| (2020) 10:12168 www.nature.com/scientificreports/ Figure 1. (a) U-4f core levels XPS spectrum of UO2 pellet before leaching. (b) SEM micrograph (BSE mode) of the surface of the UO2 pellet before leaching experiment. Scale bar 50 µm. Figure 1. (a) U-4f core levels XPS spectrum of UO2 pellet before leaching. Coffinite formation from UO2+x PEN 2 2 ( ) • We show that coffinite precipitation could lower the uranium release from the UO2 matrix of SNF through oxidative weathering in the presence of oxygen in the geological repository. g p yg g g p y • Dissolution assisted by silicate ions and precipitation under slightly oxidative conditions (i.e., Eh between -100 and + 100 mV) explains the coexistence of uraninite and coffinite in uranium ore deposits. g p yg g g p y • Dissolution assisted by silicate ions and precipitation under slightly oxidative conditions (i.e., Eh between -100 and + 100 mV) explains the coexistence of uraninite and coffinite in uranium ore deposits. Coffinite formation from UO2+x PEN (b) SEM micrograph (BSE mode) of the surface of the UO2 pellet before leaching experiment. Scale bar 50 µm. could have a significant effect on uraninite reactivity and solubility9. Coffinite could thus be preferentially formed at the interface between UO2+x resulting from the oxidation of UO2 surface layer and the silicate-bearing fluids.ifi Similar to natural uraninite, recent findings regarding the thermodynamic stability of coffinite have renewed the interest in considering coffinite as a potential alteration product of SNF in a geologic repository, particularly under reducing conditions. During in-reactor irradiation UO2 fuel pellets experience many chemical modifica- tions and considerable radiation-induced defect formation. Such microstructural changes in UO2 matrix occur from the nanometer up to the macroscopic scale25 and, by similar to uraninite, could enhance the possibility of the formation of coffinite26. Most of the geologic sites under investigation for underground repositories are located in undisturbed clay-rich rock or granite, with silica-rich groundwaters ([Si]tot ~ 10–4 mol/L), deep enough to have reducing conditions (typical Eh range from − 50 to − 300 mV)27,28. Understanding the interaction of used fuel with the silicate-rich groundwaters is critical to evaluate the safety of different disposal strategies, as the coffinitization process has not been considered until now. In this paper: • We show for the first time, at laboratory time scale, the formation of coffinite from UO2 in the presence of solution saturated with respect to SiO2(am) under conditions typical of near-surface uranium deposits and deep-mined geologic repositories for SNF. • We show for the first time, at laboratory time scale, the formation of coffinite from UO2 in the presence of solution saturated with respect to SiO2(am) under conditions typical of near-surface uranium deposits and deep-mined geologic repositories for SNF.fi p g g p • We have constrained the conditions of formation in an Eh–pH diagram where the precipitation of coffinite is favoured over UO2⋅2H2O (am).fi p g g p • We have constrained the conditions of formation in an Eh–pH diagram where the precipitation of coffinite is favoured over UO2⋅2H2O (am). 2 2 ( ) • We show that coffinite precipitation could lower the uranium release from the UO2 matrix of SNF through oxidative weathering in the presence of oxygen in the geological repository. Experimental results L−1), but oversaturated with respect to USiO4 coffinite (i.e.[U]tot = 10–5 mol L−1). pH, Eh, Si and U elemental concentrations were monitored during the leaching experiment (Fig. 2). Th l d h h d b l d ft d f h h f ll The results in Fig. 2 indicate that the conditions stabilized after 100 days of contact with the following average and standard deviation values: pH = 8.76 ± 0.03; Eh = − 55 ± 35 mV; [Si]tot = (1.41 ± 0.05) × 10–3 mol L−1. However, the uranium elemental concentration decreased regularly in solution until 350 days of contact with solution. Experimental data obtained at steady state are plotted together within the predominance diagram of the uranium system (Fig. 3). Compared to the literature, the introduction of silicate ions in solution strongly modifies the usual predominance domains of the major uranium aqueous species and minerals31. As can be seen in this diagram, experimental data fall within the stability range of U6+ species, UO2(OH)3 − and (UO2)3(OH)7 −. Regarding the solid phases, experimental data fall in the stability domain of coffinite, which indicates that anoxic conditions led to Eh values of an appropriate range to form coffinite. Examination of the Pourbaix diagram for U (Fig. 3) shows a narrow domain in Eh values where coffinite can be formed coexisting with meta-schoepite, UO3⋅0.9H2O under slight reducing conditions, UO2⋅2H2O (am) being stable under more reducing potentials.h g g g g p The solid/solution interface was characterized by Environmental-SEM (ESEM) and Grazing Incidence-XRD (GI-XRD). Finally, the experiment was stopped after 371 days and the UO2 pellet was withdrawn from the solu- tion for characterization by High Resolution Transmission Electron Microscopy (HR-TEM). During exposure to the silicate solution, GI-XRD patterns were collected for an incident angle, θi of 1° at various times and checked for the appearance of new peaks that would indicate the formation of coffinite at the surface of the UO2 pellet. The X-ray penetration depth in UO2 at this grazing incident angle was estimated to be 120 nm by Tracy et al.32 GI-XRD diffractograms (Fig. 4a) showed the appearance of peaks characteristic of the tetragonal (I41/amd) coffinite phase after 155 days of solution contact. The presence of the coffinite peaks became obvious after 210 days, whereas patterns obtained showed no evidence of meta-schoepite formation. Particular analysis of the (111) diffraction maximum of UO2 (Fig. Experimental results p UO2 powder was synthesized, then sintered under reducing conditions to maintain uranium in the tetravalent oxidation state. UO2 individual pellet was characterized by X-ray diffraction (XRD), scanning electron micros- copy (SEM) and X-ray photoelectron spectroscopy (XPS). Details of the synthesis and characterization are included in the “Experimental methods” section. p For the pellet treated at high temperature under vacuum, the value of the unit cell parameter obtained by Rietveld refinement was: a = 546.95(1) pm (Fig. S1 of the supporting information). This value was compared with the unit cell parameter determined by Leinders et al.29 for stoichiometric UO2 (a = 547.127 (8) pm). This indicates that the sintered pellet has not oxidized to UO2+x. However, three main contributions were needed to fit the experimental U-4f7/2 core level XPS spectrum of UO2 pellet (Fig. 1a). These contributions were attributed to U4+, U5+ and U6+ oxidation states with U-4f7/2 peak binding energies of 379.7 ± 0.3 eV; 380.8 ± 0.3 eV and 382.3 ± 0.3 eV, respectively14,30. The presence of shake-up satellite peaks at 6.8 eV and 8.1 eV from the main U-4f7/2 peaks showed that uranium oxidation states were mainly U4+ and U5+. In situ Ar+ ion etching led to U-4f core levels spectrum with only one U4+ contribution. This confirmed that uranium in the bulk material was U4+, while U5+ and U6+ were only present as a thin oxidation layer at the pellet surface. SEM images of the pellet before leaching (Fig. 1b) showed large grains of 10–25 µm in size. Grain “pull-out” was also observed and attributed to the polishing step. This grain pull-out contributed to the significant increase of the open porosity in the pellet, and thus an increased reactive surface area.h This pellet was leached at room temperature, under anoxic conditions (pO2 ≤ 1 ppm), with a solution slightly undersaturated with respect to amorphous silica at 25 °C and at pH = 8.76 (i.e.[Si]tot = (1.77 ± 0.03) × 10–3 mol Scientific Reports \| (2020) 10:12168 \| https://doi.org/10.1038/s41598-020-69161-1 www.nature.com/scientificreports/ Figure 2. Eh (a); pH (b); uranium (c) and silicate (d) elemental concentrations during the leaching of the UO2 pellet (open symbols represent data obtained after ultrafiltration of the solution). Figure 2. Eh (a); pH (b); uranium (c) and silicate (d) elemental concentrations during the leaching of the UO2 pellet (open symbols represent data obtained after ultrafiltration of the solution). Experimental results Predominance domains of the major aqueous species and solid phases are shown as a function of the reduction potential, Eh(V) and pH for total U, [U]tot = 5 × 10–6 mol L−1 in water containing silicate ions, [Si]tot = 2 × 10–3 mol L−1 and in equilibrium with the atmosphere. Calculations were made considering coffinite stability domain proposed by Szenknect et al.22 and the formation constant of the hydroxosilicate complex, U(OH)3(H3SiO4)3 2− proposed by Mesbah et al.15 UO2(cr) is not allowed to be present in the calculations. Symbol correspond to experimental data at equilibrium (≥ 100 days). Calculations performed by using the Thermochimie database (https://www.thermochimie-tdb.com). Figure 4. (a) GI-XRD patterns obtained at θi = 1° for different leaching times (in days). The Bragg peak positions characteristic of UO2 (PDF: 00-005-0550) and USiO4 (PDF: 00-011-0420) are shown with black and orange bars, respectively. (b) Extract of the (111) diffraction peak of UO2. Figure 4. (a) GI-XRD patterns obtained at θi = 1° for different leaching times (in days). The Bragg peak positions characteristic of UO2 (PDF: 00-005-0550) and USiO4 (PDF: 00-011-0420) are shown with black and orange bars, respectively. (b) Extract of the (111) diffraction peak of UO2. The surface of the UO2 pellet was observed regularly by ESEM. Selected micrographs recorded at high mag- nification highlight the evolution of the UO2 grains at the solid/solution interface (Fig. 5). Selected micrographs recorded at low magnification are presented in Fig. S3 of the supporting data to illustrate massive grain detach- ment at the pellet surface. These results showed that silicate ions had a deleterious effect on the UO2 pellet microstructure. As a consequence, the surface area of the pellet in contact with the solution increased through the development of grains roughness and numerous cavities.t After a short contact time with the silicate solution, grain detachment was observed (Fig. 5 at 15 days), the surface of the some grains was altered and small particles were observed in pores. After longer contact times and the appearance of XRD lines associated to coffinite on the GI-XRD patterns (Fig. 5 at 217; 254 and 339 days), the surface of altered grains was covered by small particles embedded in a gel. Some of these particles exhibited a bipyramidal morphology characteristic of coffinite15. fi In the TEM image (Fig. 6a), the morphology of the grains indicated by the blue dotted circle and the red dotted quadrangle differ. Experimental results 4b) suggests the evolution of the fluorite-structure. Patterns obtained showed a shift of the positions of the XRD lines to smaller 2θ angles, corresponding to larger unit-cell parameters. With time, asymmetric peak broadening decreased. Qualitatively, these results indicated an increase of unit cell volume, caused by the decreasing contribution of the oxidized surface layer. This UO2+x layer being more soluble than stoichiometric UO2, it most likely has preferentially dissolved at the beginning of the dissolution experiments33. Nevertheless, U concentration in solution decreased. This result shows that under the experimental conditions investigated, available U is removed from solution. The solution was initially spiked with U to reach conditions oversaturated with respect to coffinite. The precipitation of coffinite would explain the decrease of Si and U concentration in the bulk solution. This mechanism could also trigger the dissolution of the UO2+x layer by creating undersaturated conditions for UO2+x at the pellet/solution interface. Scientific Reports \| (2020) 10:12168 \| https://doi.org/10.1038/s41598-020-69161-1 www.nature.com/scientificreports/ www.nature.com/scientificreports/ Figure 3. Pourbaix diagram for U. Predominance domains of the major aqueous species and solid phases are shown as a function of the reduction potential, Eh(V) and pH for total U, [U]tot = 5 × 10–6 mol L−1 in water containing silicate ions, [Si]tot = 2 × 10–3 mol L−1 and in equilibrium with the atmosphere. Calculations were made considering coffinite stability domain proposed by Szenknect et al.22 and the formation constant of the hydroxosilicate complex, U(OH)3(H3SiO4)3 2− proposed by Mesbah et al.15 UO2(cr) is not allowed to be present in the calculations. Symbol correspond to experimental data at equilibrium (≥ 100 days). Calculations performed by using the Thermochimie database (https://www.thermochimie-tdb.com). Figure 3. Pourbaix diagram for U. Predominance domains of the major aqueous species and solid phases are shown as a function of the reduction potential, Eh(V) and pH for total U, [U]tot = 5 × 10–6 mol L−1 in water containing silicate ions, [Si]tot = 2 × 10–3 mol L−1 and in equilibrium with the atmosphere. Calculations were made considering coffinite stability domain proposed by Szenknect et al.22 and the formation constant of the hydroxosilicate complex, U(OH)3(H3SiO4)3 2− proposed by Mesbah et al.15 UO2(cr) is not allowed to be present in the calculations. Symbol correspond to experimental data at equilibrium (≥ 100 days). Calculations performed by using the Thermochimie database (https://www.thermochimie-tdb.com). Figure 3. Pourbaix diagram for U. Conclusionhi The first evidence of coffinite formation from UO2 has been obtained at low temperature under conditions relevant for geological disposal of SNF and in uranium ore deposits. In a solution slightly undersaturated with respect to SiO2(am) at 25 °C (i.e.[Si]tot = (1.77 ± 0.03) × 10–3 mol L−1 and pH = 9), anoxic conditions provided Eh values in a range that caused the dissolution of UO2 through oxidative weathering, followed by precipitation of coffinite. In the studied conditions, the precipitation of coffinite was thermodynamically favored as compared with the mixture of UO2⋅2H2O (am) and SiO2 (am). Using current thermodynamic databases, geochemical calculations showed that coffinite coexisted with UO2(OH)3 − predominant species in solution. From a kinetic point of view, the formation of coffinite occurred rapidly and the precipitation of coffinite was unambiguously evidenced after 155 days of leaching at the surfaces of the UO2 pellet.hfi t These results explained the common occurrence of coffinite in sedimentary uranium ore deposits and showed that coffinite should be considered in modeling the long-term behavior of SNF in a geologic repository. The formation of coffinite is a mechanism that reduces the amount of uranium released from the SNF, especially in the event of an increase of the redox potential of the groundwater. This mechanism may also reduce the release of tetravalent actinides, such as plutonium, which form solid solutions with tetragonal structure of coffinite34,35 but it could also trigger the release of other radionuclides which do not fit into the coffinite structure, as dem- onstrated by the release of radiogenic Pb occuring during coffinitization3. Experimental results Additionally, in the Energy-Dispersive X-ray (EDX) spectrum obtained from the for- mer, there appeared to be a strong signal from Si that did not appear in the spectrum from the latter (Fig. 6b). This suggests that the grain indicated by the blue circle may be a neoformed coffinite particle. To determine the structure of this particle, the high-resolution TEM image was analyzed (Fig. 6c). The d-spacings between the two sets of white lines were 0.469 nm and 0.281 nm, respectively, which were identified as the (011) and (121) in coffinite14,18. Neither of these two d-spacings belonged to UO2. The fast Fourier transform (FFT) of the image agreed well with the electron diffraction pattern of this grain, as shown in Fig. 6d. These results clearly indicated the formation of the neoformed coffinite. Scientific Reports \| (2020) 10:12168 \| https://doi.org/10.1038/s41598-020-69161-1 www.nature.com/scientificreports/ Figure 5. SEM micrographs (BSE mode) of the UO2 pellet recorded at different leaching times and high magnification. The blue dotted circles indicate neoformed grains with bipyramidal morphology characteristic of zircon-type crystals. Scale bars 2 µm. Figure 5. SEM micrographs (BSE mode) of the UO2 pellet recorded at different leaching times and high magnification. The blue dotted circles indicate neoformed grains with bipyramidal morphology characteristic of zircon-type crystals. Scale bars 2 µm. Experimental methods Preparation of UO2 pellet. The oxalic acid used to perform the synthesis was purchased from Sigma- Aldrich in analytical grade. The uranium (IV) chloride solution was prepared by dissolving metal chips provided by CETAMA (CEA France) in concentrated HCl solution (6 mol L−1). The final concentration of the stock solution was determined by Inductively Coupled Plasma-Atomic Emission Spectroscopy (ICP-AES) and was found to [U]tot = 0.515 ± 0.001 mol L−1. The uranium oxalate precursor was obtained by direct precipitation in an opened vessel in application of the protocol reported by Hingant et al.36 The weighed oxalic acid was dissolved in about 50 mL of deionized water at 60 °C, then the uranium solution was added slowly leading to the direct precipitation of the oxalate precursor. The molar ratio of oxalic acid/uranium was equal to 3. The mixture was left under continuous stirring for 30 min, then centrifuged twice with deionized water and finally once with ethanol in order to eliminate the excess of oxalic acid. The resulting powder was dried overnight in an oven at 90 °C. The prepared oxalate was calcined at 600 °C for 6 h under Ar/H2 (5%) atmosphere in order to maintain uranium under its tetravalent oxidation state. The calcination at 600 °C was considered as a compromise to ensure the complete conversion of the powder with less residual carbon and to obtain a powder with a specific surface area https://doi.org/10.1038/s41598-020-69161-1 Scientific Reports \| (2020) 10:12168 \| www.nature.com/scientificreports/ Figure 6. (a) TEM images of the sample after 339 days leaching. The blue dotted circle indicates a neoformed coffinite grain, while the red dotted quadrangle indicates a UO2 grain. Scale bar 200 nm. (b) EDX spectrum obtained from the coffinite and the UO2 grain, respectively. Carbon and Copper signals originate from the TEM grid with lacey carbon film. (c) High-resolution TEM image of the coffinite grain, as indicated by the blue circle in (a). Scale bar 2 nm. (d) Fast Fourier Transform of the HRTEM image in (c) and the diffraction pattern of this coffinite grain. Scale bar 21/nm. Figure 6. (a) TEM images of the sample after 339 days leaching. The blue dotted circle indicates a neoformed coffinite grain, while the red dotted quadrangle indicates a UO2 grain. Scale bar 200 nm. (b) EDX spectrum obtained from the coffinite and the UO2 grain, respectively. www.nature.com/scientificreports/ Then, the pH was adjusted to pH = 9 with the help of 8 mol L−1 of NaOH solution (Carlo Erba, ACS reagent). g During the experiment, the container was closed most of the time, except to monitor the pH and Eh values and to sample the solution. The pH was measured using a Mettler Toledo InLab Expert Pro-ISM electrode against pH buffers (Inlab Solutions, Mettler Toledo, pH = 2.00; 4.01, 7.00 and 9.21 at 25 °C). The redox potential of the solution was monitored using a Pt combined electrode (Mettler Toledo InLab Redox) stored in the glove box. According to the value of the potential vs. NHE of the Ag/AgCl, KCl (3 mol L−1) electrode, Eh was calculated as: Eh = Emeas(Pt) + 207 mV. At regular time intervals, 1–4 mL of the solution were taken off, then centrifuged at 14,000 rpm for 5 min, acidified up to 2% using HNO3 (69% from VWR Chemicals) and stored at 4 °C before analysis. After 328 days of leaching, an additional centrifugation step was performed at 9,000 rpm for 20 min using ultra-filtration PES membrane of 3 KDa in Vivaspin tubes. The samples were analyzed either by ICP-AES (Spectro Arcos EOP) or by ICP-MS (Thermo Scientific iCAP RQ). The calibration was performed using Plasma- CAL (SCP Science) single element calibration standards ([U]tot or [Si]tot = 1,000 ppm) diluted in 1% HNO3 solu- tion. Concentrations and associated uncertainties were respectively the average and twice the standard deviation of three replicates. For ICP-MS analysis of U elemental concentration, Bi and Ir were used as internal standards.h p y The pellet was removed several times from the leaching solution to perform Grazing Incidence XRD (GI- XRD) and Environmental Scanning Electron Microscope (ESEM) analyses. For that purpose, the pellet was softly rinsed with deionized water and gently dried on an absorbing towel, then it was introduced in airtight sample-holder sealed in the Ar glove box before analyses. The sample-holder was not opened during GI-XRD analysis to avoid long-term exposure to ambient atmosphere. Characterizations of UO2 pellet. The UO2 samples were characterized by powder X-ray diffraction (PXRD). PXRD patterns were recorded using a Bruker D8 advance diffractometer with copper radiation (λCu Kα1,2 = 1.54184 Å) in a parallel mode and using the reflection geometry. The patterns were recorded between 5° and 100° (2θ) with a step of 0.02° and a counting time of 3 h. www.nature.com/scientificreports/ and effective densities. Then, the surface area associated to the pores was obtained assuming that the pore size distribution was representative of the whole sample and that the pores were cylindrical. The resulting surface was divided by the mass of the sample to evaluate the specific surface area. An average value of the specific surface area was deduced from the analysis of 5 images recorded at low magnification37. From SEM images analysis, the specific surface area of the pellet was estimated to be (1.6 ± 0.9) × 10–3 m2 g−1. and effective densities. Then, the surface area associated to the pores was obtained assuming that the pore size distribution was representative of the whole sample and that the pores were cylindrical. The resulting surface was divided by the mass of the sample to evaluate the specific surface area. An average value of the specific surface area was deduced from the analysis of 5 images recorded at low magnification37. From SEM images analysis, the specific surface area of the pellet was estimated to be (1.6 ± 0.9) × 10–3 m2 g−1. Leaching experiment. Leaching test was performed under anoxic conditions at 25 °C by contacting the UO2 pellet with a solution slightly undersaturated with respect to amorphous silica but oversaturated with respect to USiO4 coffinite at pH = 9. With this aim, 500 mL of the leaching solution were first prepared by adding sodium metasilicate (Na2SiO3 Sigma-Aldrich, analytical grade) to deionized water. The deionized water used for the preparation of the solution was previously out-gassed by boiling it for 2 h then cooled under bubbling with N2. The leaching solution was then stored for several days in the Ar-flushed glove box before being used in order to reach equilibrium with the partial pressure of O2(g) in the glove box (pO2 ≤ 1 ppm). The silicate concentration in the solution analyzed by ICP-AES reached [Si]tot = (1.77 ± 0.03) × 10–3 mol L−1 whereas the inorganic carbon (IC) content was found to [IC] = (1.6 ± 0.1) × 10–4 mol L−1 from measurement with TOC-meter apparatus (Shi- madzu). ) 100 mL of the solution was introduced in a teflon container (Savillex) with the UO2 pellet. The solution was immediately spiked with 1.8 µL of the uranium stock solution in order to increase the uranium elemental concentration to 10–5 mol L−1. www.nature.com/scientificreports/ The resulting data were refined using the Fullprof_ suite38 by applying the Rietveld method and using the Thomson Cox profile function39. Pure silicon was used as a standard to determine instrumental parameters. Zero shift, unit cell parameters, overall displacement, preferred orientation and anisotropic size model for the microstructural characteristics were considered for all the refine- ments. The PXRD pattern of the prepared powder showed UO2+x (fluorite structure-type, space group Fm 3 m) with lattice parameter: a = 546.77(1) ppm.f p ( ) pp Grazing Incidence XRD (GI-XRD) patterns were recorded using a Bruker D8 Advance diffractometer equipped with a motorized reflectivity stage which allowed vertical translation of the sample. The complete pri- mary optics setup was already described by Szenknect et al.40,41 and was composed of a Cu Kα1,2 (λ = 1.54184 Å) source, a Göbel mirror, a motorised divergence slit, a fixed 0.2 mm slit, an automatic absorber, a fixed 0.1 mm slit after the absorber, and 2.5° Sollers slits. The secondary optics included a motorised anti-scattering slit, a graphite monochromator, 2.5° Sollers slits, a 0.05 mm receiving slit and a point detector. GI-XRD diffractograms were obtained at θi = 1° to evidence the presence of coffinite phase at the pellet surface.hi fi The microstructure of the prepared pellet was first observed using a Quanta 200 Environmental Scanning Electron Microscope (ESEM-FEG, FEI Company) equipped with a backscattered electron detector (BSED) in high vacuum conditions with a 8 kV accelerating voltage and a 7 mm working distance. During the leaching experiment, the surface of the pellet was also regularly observed under environmental conditions. According to Podor et al.42, the pellet was directly introduced in the ESEM chamber equipped with a Peltier stage without any further preparation. The Peltier stage was cooled down to 2 °C prior the introduction of the sample. A great cau- tion was paid to the pumping sequence in order to avoid any dehydration of the sample. This pumping sequence consisted in 5 differential pumping steps between 50 and 200 Pa of water. Finally, the water vapor pressure in the chamber was adjusted to 40 Pa which corresponded to a relative humidity of 5.7%. With this experimental procedure, the sample was never dried. Experimental methods Carbon and Copper signals originate from the TEM grid with lacey carbon film. (c) High-resolution TEM image of the coffinite grain, as indicated by the blue circle in (a). Scale bar 2 nm. (d) Fast Fourier Transform of the HRTEM image in (c) and the diffraction pattern of this coffinite grain. Scale bar 21/nm. that promoted the densification by sintering. The specific surface area (SSA) of the UO2 powder was analyzed using 10 points krypton adsorption isotherm and the B.E.T. method (ASAP 2020, Micromeritics). The SSA of the starting powder reached 8 m2 g−1. UO2 pellet was prepared by uniaxial pressing at 500 MPa of 1.2 g of the obtained UO2 powder in a dye of 13 mm in diameter. The pellet was placed in a carbon furnace then sintered at 1,700 °C for 8 h under vacuum. The pellet obtained was then polished with successive grain size of 10 µm, 5 µm and 1 µm. Finally, a polishing step using colloidal silica was achieved to eliminate micro-scratches and to obtain optical grade polished surface. The pellet was then placed in an ethanol bath and sonicated in order to remove all traces of silica particles that could remain at the surface of the sample. The mass of the pellet after polishing was equal to 1.105 ± 0.001 g. The densification rate of the pellet was determined by combining geometric measurements thanks to a caliper splint and helium pycnometry. The apparent density of each pellet was evaluated by geometrical measurements and compared to the calculated density of UO2 (dcalc = 11 g.cm-3). Measurement of the effective density by helium pycnometry allowed the differentiation between the open and closed porosity. The densification rate of the pellet was 93 ± 1% (indicating 7% porosity of which 3% was determined to be closed).hih g y The specific surface area of the pellet was too low to be measured using Kr adsorption. Thus, it was estimated from SEM images recorded at low magnification and He pycnometry. SEM images of 92 × 62 µm2 were binarized using the FiJi software to determine the surface area of the pores in each investigated domain. The pore diam- eter distribution was evaluated from these images using the “analyse particles” plugin implemented in the FiJi software. Experimental methods The height of the pores was calculated to meet the volume of open porosity deduced from apparent Scientific Reports \| (2020) 10:12168 \| https://doi.org/10.1038/s41598-020-69161-1 www.nature.com/scientificreports/ www.nature.com/scientificreports/ www.nature.com/scientificreports/ Table 1. Equilibrium constants of selected uranium phases and hydroxocomplexes. TC stands for ThermoChimie Database. Solubility Log10K°s,0 Reference Coffinite + 4H+ ⇆ U4+ + H4SiO4 − 7.80 Coffinite (TC)48 − 5.25 Coffinite22 Coffinite (am) + 4H+ ⇆ U4+ + H4SiO4 − 1.5 Coffinite (am) Estimated from NEA guidelines UO2⋅2H2O (am) + 4H+ ⇆ U4+ + 4H2O 1.5 49 Hydroxocomplexes Log10K°(1,n) U4+ + 3H2O + 3H4SiO4 ⇆ U(OH)3(H3SiO4)3 2− + 6H+ − 18.39 15 Table 1. Equilibrium constants of selected uranium phases and hydroxocomplexes. TC stands for ThermoChimie Database. Table 1. Equilibrium constants of selected uranium phases and hydroxocomplexes. *TC stands for ThermoChimie Database. charge compensation flood gun. The instrument was calibrated to the silver Fermi level (0 eV) and to the 3d5/2 core level of metallic silver (368.3 eV). The C-1s signal for adventitious carbon was used to correct the charge effect. The C–C/C–H component of C-1s spectra was fixed at 285.0 eV. The analysis zone was 900 µm diameter spot. The pass energy for overview and high resolution spectra was 150 eV and 20 eV, respectively. The data processing was performed using the commercial Avantage software. For the fitting procedure, a Shirley back- ground has been used. charge compensation flood gun. The instrument was calibrated to the silver Fermi level (0 eV) and to the 3d5/2 core level of metallic silver (368.3 eV). The C-1s signal for adventitious carbon was used to correct the charge effect. The C–C/C–H component of C-1s spectra was fixed at 285.0 eV. The analysis zone was 900 µm diameter spot. The pass energy for overview and high resolution spectra was 150 eV and 20 eV, respectively. The data processing was performed using the commercial Avantage software. For the fitting procedure, a Shirley back- ground has been used. g Transmission Electron Microscopy (TEM) characterization were carried out using a FEI Tecnai G2 F20 X-TWIN Transmission Electron Microscope operated at 200 kV, equipped with an energy dispersive spectros- copy (EDS) system. Fast Fourier transformation (FFT) and image filtering were performed using DigitalMicro- graph software. The samples after leaching were ultrasonicated in acetone for 30 min at room temperature to separate coffinite particles from the surface of the pellets. The solutions after ultrasonication were dripped onto TEM grids with lacey carbon thin film using pipettes. For each TEM sample, only thin area on the edge of each particle was observed for ideal high-resolution imaging conditions. References 1. Deditius, A. P., Utsunomiya, S. & Ewing, R. C. The chemical stability of coffinite, USiO4⋅nH2O; 0 < n < 2, associated with organic matter: A case study from Grants uranium region, New Mexico, USA. Chem. Geol. 251, 33–49 (2008).i 1. Deditius, A. P., Utsunomiya, S. & Ewing, R. C. The chemical stability of coffinite, USiO4⋅nH2O; 0 < n < 2, associated with organic matter: A case study from Grants uranium region, New Mexico, USA. Chem. Geol. 251, 33–49 (2008).i y g ( ) 2. Janeczek, J. Mineralogy and geochemistry of natural fission reactors in Gabon. Rev. Mineral. Geochem. 38, 321–392 (1999).i i 3. Evins, L. Z., Jensen, K. A. & Ewing, R. C. Uraninite recrystallization and Pb loss in the Oklo and Bangombe natural fission reactor Gabon. Geochim. Cosmochim. Acta 69, 1589–1606 (2005). 4. Janeczek, J. & Ewing, R. C. Dissolution and alteration of uraninite under reducing conditions. J. Nucl. Mater. 190, 157–173 (1992 4. Janeczek, J. & Ewing, R. C. Dissolution and alteration of uraninite under reducing conditions. J. Nucl. Mater. 190, 157 173 (1992). 5. Deditius, A. P., Utsunomiya, S., Wall, M. A., Pointeau, V. & Ewing, R. C. Crystal chemistry and radiation-induced amorphization of P-coffinite from the natural fission reactor at Bangombe, Gabon. Am. Mineral. 94, 827–836 (2009). fii g 6. Fayek, M., Harrison, T. M., Ewing, R. C., Grove, M. & Coath, C. D. O and Pb isotopic analyses of uranium minerals by ion micro- probe and U-Pb ages from the Cigar Lake deposit. Chem. Geol. 185, 205–225 (2002).f fii 6. Fayek, M., Harrison, T. M., Ewing, R. C., Grove, M. & Coath, C. D. O and Pb isotopic analyses of uranium minerals by ion micro- probe and U-Pb ages from the Cigar Lake deposit. Chem. Geol. 185, 205–225 (2002).f probe and U-Pb ages from the Cigar Lake deposit. Chem. Geol. p g g p 7. Alexandre, P. & Kyser, T. K. Effects of cationic substitutions and alteration in uraninite, and implications for the dating of uranium deposits. Can. Mineral. 43, 1005–1017 (2005). 8. Ono, S. & Fayek, M. Decoupling of O and Pb isotope systems of uraninite in the early Proterozoic Conglomerates in the Elliot Lake district. Chem. Geol. 288, 1–13 (2011).fi 8. Ono, S. & Fayek, M. Decoupling of O and Pb isotope systems of uraninite in the early Proterozoic Conglomerates in the Elliot Lake district. Chem. Geol. 288, 1–13 (2011).fi 9. www.nature.com/scientificreports/ Modeling of the U speciation in solution. Phreeqc44 and GibbsStudio45 codes were used to model the aqueous chemistry of the leaching experiment carried out with UO2 pellets. ThermoChimie Database vs 1046,47 was used with some modifications. Selected log K° values used for calculations are summarized in Table 1. i g Experimental data used for numerical modelling correspond to that obtained at equilibrium (> 100 days) fter ultrafiltration of the samples. Received: 11 May 2020; Accepted: 6 July 2020 Received: 11 May 2020; Accepted: 6 July 2020 References Janeczek, J. & Ewing, R. C. Coffinitization—a mechanism for the alteration of UO2 under reducing conditions. MRS Proc. 257, 487–504 (1991). 9. Janeczek, J. & Ewing, R. C. Coffinitization—a mechanism for the alteration of UO2 under reducing conditions. MRS Proc. 257, 487–504 (1991). 10. Leroy, J. & Holliger, P. Mineralogical, chemical and isotopic (U-Pb method) studies of Hercynian uraniferous mineraliza (Margnac and Fanay mines, Limousin, France). Chem. 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Update on the chemical thermodynamics of uranium, neptunium, plutonium, americium and techn In Chemical Thermodynamics Vol. 5 (Ed. OECD-NEA) (Elsevier, 2003) Acknowledgementsh g This project was initiated and partially funded by the Swedish Nuclear Fuel and Waste Management Co. (SKB). R.C.E and C.W. were supported by funding from the U.S. Department of Energy, National Nuclear Security Administration, Office of Defense Nuclear Nonproliferation Research and Development under Contract No. DE-AC02-76SF00515.”Authors acknowledge Béatrice Baus-Lagarde and Bruno Corso at ICSM for the technical support during ICP-MS and XRD analyzes, respectively. References 44, 1057–1063 (1959).fifi h p p p p ( ) ( ) 0. Costin, D. T. et al. How to explain the difficulties in the coffinite synthesis from the study of uranothorite?. Inorg. Chem. 50 11117–11126 (2011). Scientific Reports \| (2020) 10:12168 \| https://doi.org/10.1038/s41598-020-69161-1 www.nature.com/scientificreports/ 21. Labs, S. et al. 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L., Chen, C. H., Park, S., Davisson, M. L. & Ewing, R. C. Measurement of UO2 su X-ray diffraction: Implications for nuclear forensics. J. Nucl. Mater. 502, 68–75 (2018). Author contributions S.S., L.D., J.B. L.Z.E. and N.D. conceived the study. D.A. and S.S. carried out the leaching experiments. M.L.R. carried out geochemical modelling. C.W. provided the HRTEM results. R.P. recorded the SEM images. A.M. contributed to XRD data interpretation and performed Rietveld refinement. F.M. performed XPS analyses and undertook XPS data interpretation. R.C.E. participated in the interpretation of the results. All authors contributed to writing and editing of the manuscript. References y g 2 X-ray diffraction: Implications for nuclear forensics. J. Nucl. Mater. 502, 68–75 (2018). f 33. Shoesmith, D. W. Fuel corrosion processes under waste disposal conditions. J. Nucl. Mater. 282, 1–31 (2000). ll h b d d l k d d l Th f 33. Shoesmith, D. W. Fuel corrosion processes under waste dispo 34. Keller, C. 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https://openalex.org/W2096424822	https://europepmc.org/articles/pmc2836277?pdf=render	English	null	Daptomycin for methicillin-resistant Staphylococcus epidermidis native-valve endocarditis: a case report	Annals of clinical microbiology and antimicrobials	2,010	cc-by	3,294	Duah Annals of Clinical Microbiology and Antimicrobials 2010, 9:9 http://www.ann-clinmicrob.com/content/9/1/9 Duah Annals of Clinical Microbiology and Antimicrobials 2010, 9:9 http://www.ann-clinmicrob.com/content/9/1/9 Duah Annals of Clinical Microbiology and Antimicrobials 2010, 9:9 http://www.ann-clinmicrob.com/content/9/1/9 Duah Annals of Clinical Microbiology and Antimicrobials 2010, 9:9 http://www.ann-clinmicrob.com/content/9/1/9 Open Access Daptomycin for methicillin-resistant Staphylococcus epidermidis native-valve endocarditis: a case report Marylene Duah* * Correspondence: mduah@shsny.com Samaritan Medical Center, Watertown, NY, USA © 2010 Duah; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Abstract Coagulase-negative staphylococci (CoNS) have been increasing in importance as a cause of native valve endocardi- tis (NVE). Most cases of NVE caused by CoNS are attributable to Staphylococcus epidermidis. NVE caused by CoNS acquired in a nosocomial setting may differ from cases acquired in the community in several ways. It may be asso- ciated with hemodialysis, the presence of a long-term indwelling central catheter or pacemaker, or a recent inva- sive procedure; nosocomial cases may have a higher rate of methicillin resistance among CoNS isolates, and so be more likely to be treated with vancomycin. Unfortunately, NVE caused by methicillin-resistant CoNS has been asso- ciated with significantly higher rates of persistent bacteremia and in-hospital mortality than methicillin-susceptible isolates. The poor outcomes in these cases point to the need for alternative therapies with potent activity against methicillin-resistant CoNS. In our medical center, a 76-year-old man presented with native-valve endocarditis and positive blood cultures for methicillin-resistant Staphylococcus epidermidis (MRSE). During each of three 6-week courses of treatment with vancomycin, blood cultures were negative, but they once again became positive for MRSE when vancomycin was discontinued. The minimum inhibitory concentration of the MRSE isolates for vanco- mycin remained stable at 2 μg/mL. Eventually, treatment with daptomycin was initiated (500 mg [7 mg/kg]) 3 times/week for 6 weeks. Over the following year, no positive cultures for MRSE were detected. Background CoNS acquired in a nosocomial setting differed from cases acquired in a community-based setting in several ways. Nosocomial cases were associated with several predisposing factors, including hemodialysis, the pre- sence of a long-term indwelling central catheter or pace- maker, or a recent invasive procedure [4]. Notably, nosocomial cases had a much higher rate of methicillin resistance among CoNS isolates (58 versus 22%; P = 0.05), and consequently were more likely to be treated with vancomycin (65 versus 25%; P < 0.01). In turn, NVE caused by methicillin-resistant CoNS was asso- ciated with significantly higher rates of persistent bacter- emia (25 versus 9%; P = 0.01) and in-hospital mortality (40 versus 16%; P = 0.03) than methicillin-susceptible isolates. The poor outcomes in these cases point to the need for alternative therapies with potent activity against methicillin-resistant CoNS. Coagulase-negative staphylococci (CoNS) have been increasing in importance as a cause of native valve endocarditis (NVE) [1-3]. According to recent data from the International Collaboration of Endocarditis-Prospec- tive Cohort Study (ICE-PCS), CoNS account for 7.8% of all cases of NVE and cause death in one quarter of these cases, even though a majority of patients undergo surgical treatment [4]. In the ICE-PCS cohort, which included 1635 patients from 61 centers in 28 countries with definite NVE and no history of injection drug use, the mortality rate for NVE caused by CoNS was com- parable to that for NVE caused by Staphylococcus aur- eus and significantly higher than that for NVE caused by viridans group streptococci [4]. Most cases of NVE caused by CoNS are attributable to Staphylococcus epidermidis, including 80% of those in the ICE-PCS cohort [4]. In this cohort, NVE caused by Daptomycin is a novel cyclic lipopeptide with activity against most aerobic Gram-positive pathogens, including strains resistant to methicillin, vancomycin, and other antibiotics [5,6]. Daptomycin is highly active against Duah Annals of Clinical Microbiology and Antimicrobials 2010, 9:9 http://www.ann-clinmicrob.com/content/9/1/9 Page 2 of 4 CoNS, including methicillin-resistant S. epidermidis (MRSE) [7-9]. Most clinical isolates of MRSE and other methicillin-resistant CoNS are susceptible to daptomy- cin at a minimum inhibitory concentration (MIC) of 0.5 μg/mL or less [10,11]. Daptomycin produces rapid, concentration-dependent bactericidal activity, but its mechanism differs from other antibiotics; it damages the bacterial cell membrane but causes only minimal cell lysis [12-14]. Low-grade fever was present. Initial treatment course The patient was hospitalized for NVE on December 27, 2006. Two sets of blood cultures were positive for MRSE (Figure 1). Vancomycin 500-1000 mg IV was started after hemodialysis. Repeat blood cultures obtained on January 16, 2007, showed S. epidermidis (on one of two sets) that was susceptible (determined by VITEK 2) to trimethoprim/sulfamethoxazole, linezolid, vancomycin, and rifampin. The vancomycin MIC was 2 μg/mL. Rifampin 300 mg bid was added to vancomy- cin on January 16. Four blood cultures obtained between January 18 and February 8, while the patient was receiv- ing vancomycin and rifampin, were negative. The first course of antibiotics was discontinued on February 8. Twelve days later, on February 20, two sets of blood cul- tures were positive for S. epidermidis. The bacterium was sensitive to linezolid, rifampin, and vancomycin, with the vancomycin MIC remaining at 2 μg/mL. The preclinical and clinical profiles of daptomycin suggest that it should be useful in treating NVE caused by CoNS, even after the failure of treatment with vanco- mycin. This report describes a case of NVE caused by MRSE that was treated successfully with daptomycin after the patient failed multiple courses of vancomycin. Background The patient reported hav- ing some headaches and malaise but denied any chest pain, shortness of breath, or other symptoms. Labora- tory test results were: C-reactive protein, 1.9 mg/dL (normal, < 0.5 mg/dL); erythrocyte sedimentation rate, 13 mm/hr; hemoglobin, 13.1 g/dL; hematocrit, 38.9%; platelet count, 281,000/μL; and white blood cell count, 9,000/μL, with a differential of 43% neutrophils, 29% lymphocytes, 16% monocytes, and 10% eosinophils. Daptomycin is approved by the US Food and Drug Administration for the treatment of bloodstream infec- tions, including right-sided infective endocarditis caused by methicillin-resistant and -susceptible strains of S. aureus as well as for complicated skin and skin structure infections caused by susceptible Gram-positive patho- gens [15]. In a randomized clinical trial, daptomycin was as effective as a control regimen (low-dose gentamicin plus either an antistaphylococcal penicillin or vancomy- cin) in patients with bacteremia caused by S. aureus, including the subset with right-sided endocarditis [16] and those with methicillin-resistant strains [17]. A sub- sequent systematic review of published case reports and case series identified 19 patients with endocarditis who had been treated with daptomycin, mostly after failure of previous vancomycin therapy [18]. In one of these cases, endocarditis with bacteremia was due to CoNS and was treated successfully with daptomycin after van- comycin failure [19]. In an experimental model of endo- carditis caused by MRSE, daptomycin produced > 99.9% kill rates by 8 hours after dosing and reduced bacterial load to a greater degree than vancomycin at 1 to 3 days [20]. Medical history The patient’s medical history was significant for end- stage renal disease caused by diabetic nephropathy, and congestive heart failure with severely decreased left-ven- tricular systolic function. The patient had undergone hemodialysis 3 times per week for 4 years. The medical history also showed coronary artery disease, gout, and hyperlipidemia. In 1993, the patient had undergone cor- onary artery bypass grafting followed by multiple angio- plasties, but he was no longer a candidate for cardiac surgery. Case Presentation y g μg The patient was started on a second course of IV van- comycin on February 22, while continuing with his dia- lysis treatment. Trough drug levels were maintained in the range of 15 to 20 μg/mL. The patient had minimal symptoms, except for intermittent low-grade fever and shortness of breath. Blood cultures obtained during the second course of vancomycin were negative. Antibiotics were discontinued on April 30, but 8 days later, blood cultures were again positive for S. epidermidis. The patient was started on a third course of IV vancomycin, with a 500-mg dose given after each dialysis session. Trough drug levels were still in the range of 15 to 20 μg/mL. Repeat blood cultures on May 10, June 25, and June 27 were negative. The third course of vancomycin A 76-year old man receiving dialysis for end-stage renal disease was referred to our medical center because of blood cultures repeatedly positive for CoNS (MRSE). Over the previous 4 months, two infected hemodialysis catheters had been removed, and for each episode, the patient had been treated with a 2-week course of intra- venous (IV) vancomycin, with trough drug levels main- tained in the range of 15 to 20 μg/mL. After the second episode, blood cultures became persistently positive for CoNS within 1 week of discontinuing vancomycin. A transesophageal echocardiogram revealed moderate aor- tic valve sclerosis and mild aortic regurgitation and showed stringy masses attached to the aortic cusps sug- gestive of a bacterial infection (left-sided endocarditis). Page 3 of 4 Duah Annals of Clinical Microbiology and Antimicrobials 2010, 9:9 http://www.ann-clinmicrob.com/content/9/1/9 Duah Annals of Clinical Microbiology and Antimicrobials 2010, 9:9 http://www.ann-clinmicrob.com/content/9/1/9 Figure 1 Timeline showing blood culture results for methicillin-resistant Staphylococcus epidermidis in relation to treatment. was completed on June 27, but blood cultures on July 12 and July 14 were again positive for CoNS (MRSE). The vancomycin MIC was 2 μg/mL, and the daptomycin MIC was 0.75 μg/mL. On July 14, vancomycin was restarted with rifampin 300 mg three times daily. week after vancomycin was withdrawn. In contrast, blood cultures were negative during daptomycin treat- ment and have remained negative for more than 1 year after the 6-week course of daptomycin was completed. Discussion This patient is typical of those who are likely to develop NVE caused by methicillin-resistant CoNS. Consistent with the ICE-PCS cohort [4], the patient had several fac- tors predictive of a poor outcome from NVE caused by CoNS (persistent bacteremia, congestive heart failure, and chronic illness). He also had predisposing factors for methicillin-resistant CoNS (hemodialysis, long-term indwelling catheter) that confer higher mortality. Dapto- mycin successfully eradicated MRSE after the patient had failed several courses of vancomycin. Although blood cultures were negative during vancomycin treat- ment, cultures positive for CoNS were seen within 1 Conclusions Treatment with daptomycin 500 mg (7 mg/kg) 3 times/ week for 6 weeks enabled the patient, a 76-year old man on hemodialysis who had presented with native valve endocarditis, to clear blood cultures positive for MRSE, after 3 courses of vancomycin did not. Treatment with daptomycin offers patients like these an option even after vancomycin failure. Case Presentation The patient was treated with daptomycin at a dose of 500 mg (7 mg/kg) after each dialysis (3 times/week) for 6 weeks, which is generally consistent with the dosing recommendation for daptomycin in patients with bacter- emia and endocarditis caused by S. aureus [15]. For the approved indication, daptomycin is recommended at a dose of 6 mg/kg for 2 to 6 weeks based on the physi- cian’s working diagnosis. Because daptomycin is elimi- nated primarily by the kidneys, the once-daily dosing schedule should be adjusted to once every 48 hours for patients with significant renal impairment, including those undergoing hemodialysis [15]. In this case, as dap- tomycin was given with hemodialysis, the drug was given 3 times/week. Further treatment course The patient was admitted on July 27 with fever, chills, and rigor. He was diagnosed with a central-line infection caused by Enterobacter cloacae and received lock therapy with IV ciprofloxacin 400 mg daily through his dialysis catheter for 2 weeks. Vancomycin was discontinued. Because the cultures on July 12 and 14 were positive for MRSE, IV daptomycin 500 mg (7 mg/kg) after each dialy- sis (3 times/week) was started and continued for 6 weeks, until September 6. Blood cultures obtained on September 13 and 20 and October 2 were negative. Over the follow- ing year, the patient did not have any recurrence of blood cultures positive for S. epidermidis. The patient has remained stable and continues to do well at 18 months after discontinuing daptomycin. Authors’ contributions The author conceived of this case report, gathered the source material, and drafted the manuscript. The author conceived of this case report, gathered the source material, and drafted the manuscript. Abbreviations 13. Hobbs JK, Miller K, O’Neill AJ, Chopra I: Consequences of daptomycin- mediated membrane damage in Staphylococcus aureus. J Antimicrob Chemother 2008, 62:1003-1008. CoNS: Coagulase-negative staphylococci; ICE-PCS NVE: International Collaboration of Endocarditis-Prospective Cohort Study; IV: intravenous; MIC: minimum inhibitory concentration; MRSE: methicillin-resistant Staphylococcus epidermidis; NVE: native valve endocarditis. 14. Cotroneo N, Harris R, Perlmutter N, Beveridge T, Silverman JA: Daptomycin exerts bactericidal activity without lysis of Staphylococcus aureus. . Antimicrob Agents Chemother 2008, 52:2223-2225. Acknowledgements 15. Cubicin® (daptomycin for injection): Package insert. Lexington, MA: Cubist Pharmaceuticals Inc 2008. 15. Cubicin® (daptomycin for injection): Package insert. Lexington, MA: Cubist Pharmaceuticals Inc 2008. Phase Five Communications, New York provided editorial services funded by Cubist Pharmaceuticals. Phase Five Communications, New York provided editorial services funded by Cubist Pharmaceuticals. 16. Fowler VG Jr, Boucher HW, Corey GR, Abrutyn E, Karchmer AW, Rupp ME, Levine DP, Chambers HF, Tally FP, Vigliani GA, Cabell CH, Link AS, DeMeyer I, Filler SG, Zervos M, Cook P, Parsonnet J, Bernstein JM, Price CS, Forrest GN, Fätkenheuer G, Gareca M, Rehm SJ, Brodt HR, Tice A, Cosgrove SE, S. aureus Endocarditis and Bacteremia Study Group: Daptomycin versus standard therapy for bacteremia and endocarditis caused by Staphylococcus aureus. N Engl J Med 2006, 355:653-665. Competing interests 17. Rehm SJ, Boucher H, Levine D, Campion M, Eisenstein BI, Vigliani GA, Corey GR, Abrutyn E: Daptomycin versus vancomycin plus gentamicin for treatment of bacteremia and endocarditis due to Staphylococcus aureus : subset analysis of patients infected with methicillin-resistant isolates. J Antimicrob Chemother 2008, 62:1413-1421. The author declares that she has no competing interests. Received: 7 October 2009 Accepted: 18 February 2010 Published: 18 February 2010 Received: 7 October 2009 Accepted: 18 February 2010 Published: 18 February 2010 Received: 7 October 2009 18. Falagas ME, Giannopoulou KP, Ntziora F, Vardakas KZ: Daptomycin for endocarditis and/or bacteremia: a systematic review of the experimental and clinical evidence. J Antimicrob Chemother 2007, 60:7-19. Consent Oral informed consent for publication of this case was obtained from the patient. Written informed consent was obtained from the patient’s next of kin (his wife) for publication of this case report after the patient expired. A copy of the written consent is available for review from the Editor-in-Chief of this journal. Page 4 of 4 Duah Annals of Clinical Microbiology and Antimicrobials 2010, 9:9 http://www.ann-clinmicrob.com/content/9/1/9 References 1. Etienne J, Eykyn SJ: Increase in native valve endocarditis caused by coagulase negative staphylococci: an Anglo-French clinical and microbiological study. Br Heart J 1990, 64:381-384. 19. Segreti JA, Crank CW, Finney MS: Daptomycin for the treatment of gram- positive bacteremia and infective endocarditis: a retrospective case series of 31 patients. Pharmacotherapy 2006, 26:347-352. 2. Miele PS, Kogulan PK, Levy CS, Goldstein S, Marcus KA, Smith MA, Rosenthal J, Croxton M, Gill VJ, Lucey DR: Seven cases of surgical native valve endocarditis caused by coagulase-negative staphylococci: an underappreciated disease. Am Heart J 2001, 142:571-576. 20. Cha R, Rybak MJ: Daptomycin against multiple drug-resistant staphylococcus and Enterococcus isolates in an in vitro pharmacodynamic model with simulated endocardial vegetations. Diagn Microbiol Infect Dis 2003, 47:539-546. 3. Chu VH, Cabell CH, Abrutyn E, Corey GR, Hoen B, Miro JM, Olaison L, Stryjewski ME, Pappas P, Anstrom KJ, Eykyn S, Habib G, Benito N, Fowler VG Jr, International Collaboration on Endocarditis Merged Database Study Group: Native valve endocarditis due to coagulase-negative staphylococci: report of 99 episodes from the International Collaboration on Endocarditis merged database. Clin Infect Dis 2004, 39:1527-1530. 3. Chu VH, Cabell CH, Abrutyn E, Corey GR, Hoen B, Miro JM, Olaison L, Stryjewski ME, Pappas P, Anstrom KJ, Eykyn S, Habib G, Benito N, Fowler VG Jr, International Collaboration on Endocarditis Merged Database Study Group: Native valve endocarditis due to coagulase-negative staphylococci: report of 99 episodes from the International Collaboration on Endocarditis merged database. Clin Infect Dis 2004, 39:1527-1530. doi:10.1186/1476-0711-9-9 Cite this article as: Duah: Daptomycin for methicillin-resistant Staphylococcus epidermidis native-valve endocarditis: a case report. Annals of Clinical Microbiology and Antimicrobials 2010 9:9. doi:10.1186/1476-0711-9-9 Cite this article as: Duah: Daptomycin for methicillin-resistant Staphylococcus epidermidis native-valve endocarditis: a case report. Annals of Clinical Microbiology and Antimicrobials 2010 9:9. g 4. Chu VH, Woods CW, Miro JM, Hoen B, Cabell CH, Pappas PA, Federspiel J, Athan E, Stryjewski ME, Nacinovich F, Marco F, Levine DP, Elliott TS, Fortes CQ, Tornos P, Gordon DL, Utili R, Delahaye F, Corey GR, Fowler VG Jr, International Collaboration on Endocarditis-Prospective Cohort Study Group: Emergence of coagulase-negative staphylococci as a cause of native valve endocarditis. Clin Infect Dis 2008, 46:232-242. 4. References Chu VH, Woods CW, Miro JM, Hoen B, Cabell CH, Pappas PA, Federspiel J, Athan E, Stryjewski ME, Nacinovich F, Marco F, Levine DP, Elliott TS, Fortes CQ, Tornos P, Gordon DL, Utili R, Delahaye F, Corey GR, Fowler VG Jr, International Collaboration on Endocarditis-Prospective Cohort Study Group: Emergence of coagulase-negative staphylococci as a cause of native valve endocarditis. Clin Infect Dis 2008, 46:232-242. 5. Sader HS, Streit JM, Fritsche TR, Jones RN: Antimicrobial activity of daptomycin against multidrug-resistant Gram-positive strains collected worldwide. Diagn Microbiol Infect Dis 2004, 50:201-204. g 6. Steenbergen JN, Alder J, Thorne GM, Tally FP: Daptomycin: a lipopeptide antibiotic for the treatment of serious Gram-positive infections. J Antimicrob Chemother 2005, 55:283-288. 7. Critchley IA, Draghi DC, Sahm DF, Thornsberry C, Jones ME, Karlowsky JA: Activity of daptomycin against susceptible and multidrug-resistant Gram-positive pathogens collected in the SECURE study (Europe) during 2000-2001. J Antimicrob Chemother 2003, 51:639-649. 8. Fluit AC, Schmitz FJ, Verhoef J, Milatovic D: Daptomycin in vitro susceptibility in European Gram-positive clinical isolates. Int J Antimicrob Agents 2004, 24:59-66. 9. Streit JM, Jones RN, Sader HS: Daptomycin activity and spectrum: a worldwide sample of 6737 clinical Gram-positive organisms. J Antimicrob Chemother 2004, 53:669-674. 10. Zhanel GG, DeCorby M, Nichol KA, Wierzbowski A, Baudry PJ, Karlowsky JA, Lagacé-Wiens P, Walkty A, Mulvey MR, Hoban DJ, Canadian Antimicrobial Resistance Alliance: Antimicrobial susceptibility of 3931 organisms isolated from intensive care units in Canada: Canadian National Intensive Care Unit Study, 2005/2006. Diagn Microbiol Infect Dis 2008, 62:67-80. doi:10.1186/1476-0711-9-9 Cite this article as: Duah: Daptomycin for methicillin-resistant Staphylococcus epidermidis native-valve endocarditis: a case report. Annals of Clinical Microbiology and Antimicrobials 2010 9:9. References Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color ﬁgure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color ﬁgure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color ﬁgure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and take full advantage of: 11. Hope R, Livermore DM, Brick G, Lillie M, Reynolds R, on behalf of the BSAC Working Parties on Resistance Surveillance: Non-susceptibility trends among staphylococci from bacteraemias in the UK and Ireland, 2001-06. J Antimicrob Chemother 2008, 62(suppl 2):ii65-ii74. 12. Silverman JA, Perlmutter NG, Shapiro HM: Correlation of daptomycin bactericidal activity and membrane depolarization in Staphylococcus aureus. Antimicrob Agents Chemother 2003, 47:2538-2544.
https://openalex.org/W3180549268	https://discovery.ucl.ac.uk/id/eprint/10146925/1/2022%20-%20Morris%20-%20planning%20a%20method%20for%20covariate%20adjustment%20-%20trials.pdf	English	null	Planning a method for covariate adjustment in individually randomised trials: a practical guide	Trials	2,022	cc-by	14,537	© 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://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. METHODOLOGY Open Access Abstract Background: It has long been advised to account for baseline covariates in the analysis of confirmatory randomised trials, with the main statistical justifications being that this increases power and, when a randomisation scheme balanced covariates, permits a valid estimate of experimental error. There are various methods available to account for covariates but it is not clear how to choose among them. Methods: Taking the perspective of writing a statistical analysis plan, we consider how to choose between the three most promising broad approaches: direct adjustment, standardisation and inverse-probability-of-treatment weighting. Results: The three approaches are similar in being asymptotically efficient, in losing efficiency with mis-specified covariate functions and in handling designed balance. If a marginal estimand is targeted (for example, a risk difference or survival difference), then direct adjustment should be avoided because it involves fitting non-standard models that are subject to convergence issues. Convergence is most likely with IPTW. Robust standard errors used by IPTW are anti-conservative at small sample sizes. All approaches can use similar methods to handle missing covariate data. With missing outcome data, each method has its own way to estimate a treatment effect in the all-randomised population. We illustrate some issues in a reanalysis of GetTested, a randomised trial designed to assess the effectiveness of an electonic sexually transmitted infection testing and results service. Conclusions: No single approach is always best: the choice will depend on the trial context. We encourage trialists to consider all three methods more routinely. Keywords: Covariate adjustment, Estimands, Standardisation, Inverse probability of treatment weighting, Randomised controlled trials, Clinical trials, Missing data Confounding does not affect trials that are properly randomised, since confounding is a systematic bias and any imbalances in covariates in randomised trials are due to chance, which introduces non-systematic error that is reflected in inference [1]. However, it remains desirable from a statistical perspective to account for covariates in the analysis of a trial. Planning a method for covariate adjustment in individually randomised trials: a practical guide Tim P. Morris1,2* , A. Sarah Walker1, Elizabeth J. Williamson2 and Ian R. White1 Morris et al. Trials (2022) 23:328 https://doi.org/10.1186/s13063-022-06097-z Morris et al. Trials (2022) 23:328 https://doi.org/10.1186/s13063-022-06097-z Motivating example: the GetTested trial The GetTested trial was designed to assess the effective- ness of an Internet-accessed sexually transmitted infec- tion (STI) testing and results service (chlamydia, gonor- rhoea, HIV, and syphilis) on STI testing uptake and STI cases diagnosed. It has further been argued that it is illegitimate to ignore covariates that have been measured; see ‘myth 6’ of refer- ence [1]. Briefly, 2072 participants were recruited in the London boroughs of Southwark and Lambeth. Participants were randomised to an invitation to use an Internet-based STI testing and results service (intervention) or a standard test from a walk-in sexual health clinic (control). A premise of this article is therefore that accounting for covariates in the analysis of trials is a good idea. We are concerned with how trial statisticians and clinical inves- tigators should agree on a method before seeing the data. This perspective is taken to help statisticians working on trials make sensible, informed decisions when writing a statistical analysis plan. Treatment allocation involved a minimisation proce- dure balancing for gender (male/female/trans), number of sexual partners in the 12 months before randomisation (categorised as 1, 2+; note that one-or-more was part of the eligibility criteria) and sexual orientation (men who have sex with men vs. other groups). Each of the covariates was weighted equally when determining marginal imbal- ance and intervention or control were assigned at random with 80% probability of assignment to the favoured arm. We will focus most heavily on binary outcome data since this is where some of the issues are most acute. When the outcome measure is continuous and the analy- sis aims to estimate a difference in means, some — though not all — of the considerations of this paper become redundant. In particular, the discussion below about non- collapsibility is not relevant to the mean difference, which is collapsible (which will be discussed further for binary outcomes). Desirable properties of analysis of covariance using ordinary least squares for continuous outcomes are well appreciated, particularly when treatment–covariate interactions are assumed to be negligible (discussed fur- ther below). The two outcomes of principal interest were both binary. The first was whether participants took an STI test within 6 weeks of randomisation, with the control arm proportion anticipated to be around 10%. Background Randomised controlled trials are designed to estimate average treatment effects. This article considers the han- dling of covariates in the analysis of individually ran- domised trials. By covariates, we mean measurements on participants recorded at baseline that are thought to be prognostic. Typical examples are age at randomisation and disease severity at randomisation. Covariate adjustment is desirable because, if a covari- ate predicts outcome, accounting for its effect on outcome will improve power to detect a treatment effect [2–4] unless none of the covariates in a model are prognostic Correspondence: tim.morris@ucl.ac.uk 1MRC Clinical Trials Unit at UCL, London, UK 2Department of Medical Statistics, LSHTM, London, UK Correspondence: tim.morris@ucl.ac.uk 1MRC Clinical Trials Unit at UCL, London, UK 2Department of Medical Statistics, LSHTM, London, UK Morris et al. Trials (2022) 23:328 Morris et al. Trials (2022) 23:328 Morris et al. Trials (2022) 23:328 Page 2 of 17 Part of the motivation for this article is that statistical research papers have frequently recommended covariate adjustment due to improvements in power, with little thought given to the implications of different adjustment methods (TPM being one culpable author). The approach generally favoured in clinical trials is direct adjustment using an outcome regression model. This is so prevalent that reviews of practice have not needed to discuss which methods were actually used [7, 11]. This article works through the implications and aims to contrast its proper- ties with two other methods of adjustment better known in the epidemiology literature: standardisation and inverse probability of treatment weighting. [2]. This is sometimes explained as ‘accounting for chance imbalance’, though we view this differently: a large imbal- ance (as might be expected in an observational study) can make inference less precise; adjustment gains power by acknowledging chance covariate balance. Covariate adjustment is also desirable because, if a covariate is balanced by the randomisation scheme, for example by using stratified block randomisation or min- imisation, adjustment is necessary to obtain a valid esti- mate of experimental error. An unadjusted analysis mis- takenly assumes that chance imbalance in covariates could have occurred; an extremely useful property for covariates that were unmeasured. However, when randomisation has been balanced according to measured covariates, the fact that imbalance could not have occurred must be acknowl- edged in our statistical inference [5]. This was pointed out by Fisher almost a century ago but is frequently ignored in practice [6, 7]. Motivating example: the GetTested trial The sec- ond outcome, and the outcome for which sample size was calculated, was STI diagnosis (following a positive STI test) within 6 weeks of randomisation, with control arm proportion anticipated to be around 0.6%. The pri- mary analysis was planned to account for the following covariates: gender, age, number of sexual partners in the 12 months before randomisation (10 categories where the final category is > 10), sexual orientation (four categories) and ethnicity (five categories) [12]. The chosen covariates were all assumed to be prognostic. For adjustment to be worthwhile, the covariates to be included in the analysis must be prognostic: adjustment for a covariate that is not prognostic is essentially equiv- alent to an unadjusted analysis, though it can lose power in small samples. It is not the purpose of this article to discuss which covariates to choose. The Committee for Proprietary Medicinal Products Points to consider on adjustment for baseline covariates document gives some guidance [8]. It does caution against approaches that select covariates most strongly associated with the out- come in the trial; however, subsequent work (for example [9, 10]) has shown that such a procedure can be pre- specified in a principled manner. Direct adjustment ‘Direct adjustment’ refers to fitting an outcome regres- sion model including terms for randomised treatment Z (an indicator equal to 1 if assigned to treatment and 0 if assigned to control) and covariates X, but no interaction between Z and X. The treatment effect and its standard error are estimated directly as the treatment coefficient from the model. This might be done, for example, using a generalised linear model, which may be standard (with canonical link function) or non-standard, or a Cox model [13, 14]. Comments on methods in the next section. Note that the estimand targeted is discussed there. in the next section. Note that the estimand targeted is discussed there. There are many possible specific ways to implement each of the three broad approaches. For example, Tsiatis and colleagues used a form of standardisation but the within- arm outcome models are defined to yield ‘as good predic- tions as possible without concerns over bias’. However, for the purposes of this article, we will generally refer to or use simple implementations of the three broad approaches. For standardisation with binary outcomes, we will (by default) use a logistic regression working model fitting main effects of covariates to produce predictions. For IPTW, we will use a logistic regression including only the main effects of covariates to model the probability of ran- domised group given covariates. For direct adjustment, we will use generalised linear models with a link func- tion that permits parameter estimation on the scale of the summary measure of interest, for example a binomial gen- eralised linear model with identity link function for a risk difference. Standardisation Standardisation fits an outcome regression model includ- ing Z and X, possibly including interactions with Z, and then standardises the results by summing or integrating over the distribution of covariates observed in the trial. One intuitive way to achieve this is by making predic- tions if all participants were assigned to the intervention arm and then if all participants were assigned to the con- trol arm and forming a suitable contrast of the predictions [15–18]. Standardisation builds on outcome regression by fixing the summary of the treatment effect but allowing flexible modelling to estimate it; for example, a risk ratio can be estimated using logistic regression. Standardisation is sometimes termed marginalisation or G-computation and is implemented in Stata’s margins command and R’s stdReg package [19]. Besides the three broad approaches, there exist hybrid methods; in particular, estimators based on the influ- ence function target marginal summaries and include both a model for treatment (as does IPTW) and a model for outcome (as does standardisation). Furthermore, the superficial relationship between this form of IPTW and propensity scores suggests other estimators, for exam- ple matching rather than weighting. However, we regard the possibility of discarding data from some randomised individuals as unpalatable and we do not consider it further. Three broad approaches to accounting for covariates Three broad approaches to covariate adjustment are described below. We outline the generic procedures for estimation in this section and discuss their properties Page 3 of 17 Page 3 of 17 Page 3 of 17 Morris et al. Trials (2022) 23:328 Morris et al. Trials (2022) 23:328 Inverse probability of treatment weighting We now work through some properties that are important to consider when writing a statistical analysis plan. These properties are mainly — but not solely — statistical. We also report results of applying the different methods to the GetTested trial. Inverse probability of treatment weighting (IPTW) involves fitting two models. The first uses participants’ covariate values to predict the probability of being ran- domised to the arm they were in fact randomised to. The second then fits a simple model contrasting the treat- ment arms, weighted according to the inverse probability of treatment estimated by the first model. This effec- tively creates a weighted trial sample (pseudo-population) in which both trial arms have the same distribution of observed covariate values. This may sound odd since, if we have used simple randomisation, we know that the ‘true’ weight for all participants is identical, so trying to predict it with covariates appears futile [20]. However, the goal is not to obtain an estimate of this probability but instead to either reweight to a better balanced trial or acknowledge the balance observed. Summary measure of the estimand: marginal or conditional The topic of estimands has become increasingly promi- nent in clinical trials since the publication of the ICH E9(R1) addendum [21]. The addendum lists five attributes of an estimand: the treatment condition of interest, target population, outcome variable, handling of intercurrent events and population-level summary. Here, we focus on the population-level summary (the later section on handling missing data will focus on the tar- get population). Table 1 lists some population-level sum- maries commonly used in clinical trials and, in partic- ular, notes whether conditional and marginal estimands coincide. The method is computationally the same as using propensity scores in observational data but the motiva- tion and considerations for variable inclusion are different (just as the motivation for covariate adjustment would be different in randomised trials vs. observational studies). It is not our purpose to argue for any particular sum- mary measure but it would be remiss to pass over how the choice should be made. Some statisticians assume that Page 4 of 17 Morris et al. Trials (2022) 23:328 Morris et al. Trials (2022) 23:328 Morris et al. Trials Morris et al. Inverse probability of treatment weighting Trials Table 1 Some population-level summaries commonly used in clinical trials with binary outcome measures Outcome type Summary measures Collapsible?* Continuous Mean difference Yes Binary Risk difference Yes Risk ratio Yes Odds ratio No Time-to-event Hazard ratio No Restricted mean survival time difference Yes Do conditional and marginal summary measures always coincide? ome population-level summaries commonly used in clinical trials with binary outcome measures Do conditional and marginal summary measures always coincide? marginal), the odds of dying in the control arm becomes (1 + 5)/(5 + 9) = 3/7 and the marginal odds ratio is 5.4. the correct approach must be to choose a measure as a parameter of a model that might have generated the data. Permutt argues that the choice of scale should be ‘linear in utility’: a hypothetical value of treatment effect of 2 should be twice as attractive as 1 whatever the potential outcome on control [22]. Others argue for a measure that can be easily interpreted. We regard the first view as misguided even if the true model were known and the second as too strict, since it rules out any relative measure. The choice of measure should be a trial-specific tradeoff between ease of interpretation, close relation to average patient benefit and potential transportability to other settings. the correct approach must be to choose a measure as a parameter of a model that might have generated the data. At first sight, this is astonishing! Both strata have odds ratios equal to 9 but, when put together, the odds ratio changes. It is not a weighted average of the within-stratum odds ratios. Treatment is exactly balanced within strata, so this is not due to imbalance. Neither is it effect mod- ification, since the log odds ratio is identical in both strata. The general phenomenon is known as ‘non- collapsibility’, which describes the relationship between the marginal and conditional summary measure: the true marginal odds ratio is attenuated towards 1 com- pared with the conditional odds ratio. It occurs because the average of the logit is not the logit of the average. While the odds ratio is non-collapsible, the risk ratio and risk difference are collapsible. For insights into why non-collapsibility occurs, see Daniel, Zhang and Farewell [23]; see also [24] and [25] for more technical discussions, particularly into the relation between collapsibility and confounding. The following discussion relates to non-collapsible sum- maries [23]. Inverse probability of treatment weighting treatment comparing people in the same stratum?’ The odds ratio of 5.4 is a between-strata or ‘marginal’ estimand formed by comparing the effect of treatment for groups made up of half stratum A and half B, relating to the ques- tion ‘what would be the odds ratio in a population made up of half stratum A and half stratum B’. In non-inferiority studies, where the null is a non-zero difference between arms on some scale, it is frequently argued that the margin of non-inferiority is easier to understand, define and interpret on a marginal than a con- ditional scale. This is in line with our own experiences in collaborations. Had the notional trial recruited a different proportion of patients from each stratum, the conditional odds ratio would have remained 9 but the marginal odds ratio would not have remained 5.4. For example, suppose that in stra- tum A we had recruited three times as many people (so that the numbers in that row are all multiplied by three). The marginal odds ratio would then be 6. Its true value depended on the proportion of participants in each stra- tum. In general, a marginal summary depends on the covariate distribution when we have non-collapsibility. This is uncomfortable; after all, regression models con- dition on covariate values rather than modelling their distribution, but we see that the distribution nonetheless matters. The choice of marginal or conditional estimand is clearly not simple: the true value of the estimand may depend on the distribution of observed covariates (always marginal and sometimes conditional), on which covariates are conditioned-on in the model (conditional), and fur- ther on the distribution of omitted prognostic covariates (both). Note that these aspects have implications for the quantities being combined in meta-analysis and for appar- ent heterogeneity in meta-analysis. We will not comment further on these points here. For non-inferiority studies with a non-collapsible sum- mary measure, it is worth noting the scale on which the non-inferiority margin is defined. Suppose the margin is specified as a marginal hazard ratio, then the correspond- ing non-inferiority margin on the scale of the conditional hazard ratio is further from 1. If this fact is forgotten and a conditional hazard ratio is estimated without changing the margin, we could expect to lose power compared with an unadjusted analysis (which targets a marginal estimand). Inverse probability of treatment weighting For readers not familiar with this term, we provide a numerical example in Table 2. The reader should suppose that the frequencies given are ‘true’ in the sense that, had we recruited a very large sample size, the cells of Table 2 would contain exact multiples of the frequencies shown. Consider a trial in a condition that includes partici- pants from two measurable strata, A and B, which have a substantial effect on prognosis. The trial team recruits 40 participants — 20 from each stratum — and, within strata, randomises 10 to intervention and 10 to control. In stratum A, the odds of dying on the control arm is 5/5 = 1. In stratum B, the outlook is far more favourable, with the odds of dying on the control arm just 1/9. Despite these differences, the treatment effect (a condi- tional odds ratio) is 9 in each stratum. We might have conducted a trial recruiting patients from just one of the strata or from both. If we put all 40 people together, as shown at the right-most block of Table 2 (hence the term Rather than collapsing our strata into ‘both’, as in the right-hand panel of Table 2, it is possible to adjust for strata and recover the conditional odds ratio of 9 (using logistic regression adjusted for strata, or stratified Mantel–Haenszel). Note that non-collapsibility is not ‘bias’ as sometimes supposed, but a case of different estimands. The odds ratio of 9 is a within-stratum or conditional estimand formed by comparing the effect of treatment within a stratum, relating to the question ‘what would be the odds ratio for Table 2 An illustration of non-collapsibility of the odds ratio Stratum A B Both Allocation Dead Alive Dead Alive Dead Alive Intervention 9 1 5 5 14 6 Control 5 5 1 9 6 14 Odds ratio 9 9 5.4 Table 2 An illustration of non-collapsibility of the odds ratio 5.4 Page 5 of 17 Morris et al. Trials (2022) 23:328 Morris et al. Trials (2022) 23:328 of conditional distributions, though the word marginal is a preference rather than necessary to the definition. Inverse probability of treatment weighting This discomfort may lead us to conclude that the condi- tional odds ratio is obviously preferable. This is misled for two reasons. First, suppose there were a second covariate to stratify on Table 2 that also strongly predicted out- come but it was unknown (or at least unmeasured). The true value of the conditional odds ratio still depends on the distribution of a covariate, but since the covariate is unmeasured its distribution is unknown: we cannot know what we are marginalising over. Second, this example is contrived such that the conditional odds ratios within strata were identical. When there is effect modification on the scale of the population summary measure, the true value of the conditional measure will also depend on the distribution of observed covariates. In terms of the three broad methods considered, direct adjustment always targets a conditional summary mea- sure; standardisation typically — but not necessarily — targets a marginal summary measure; and IPTW always targets a marginal summary measure. Standardisation and IPTW are rarely used in trials but receive more atten- tion in the epidemiological literature. One justification given for this seems be that the notional ‘target trial’ would always target a marginal summary [15]. Ironically, trials which do adjust for covariates tend to use direct adjustment and so target a conditional summary. Either a marginal or conditional estimand may be desir- able and this depends on context. For example, a patient may wish to know ‘what would happen if someone similar to me were to choose this intervention vs. not?’ Mean- while, for policy makers, the average difference an inter- vention would make if offered to a group of people might be of more interest, though they might equally wish to know about the effect for specific groups. Note that a dif- ferent covariate distribution in the target group changes the value of the marginal estimand. Some authors have explored on how to extend inference to a different tar- get population [26, 27]. Interestingly, marginal estimands appear to be favoured for causal inference from observa- tional data: Hernán and Robins define a population causal effect as ‘a contrast of any functional of the marginal distributions of counterfactual outcomes under different actions or treatment values’ (emphasis added) [15]. By this definition, the within-stratum odds ratio of 9 would not target a population causal estimand, since it is a contrast Efficiency/precision/power A key reason to account for covariates in the analysis is to increase power. Note that for non-collapsible summary measures it is wrong to attempt to compare precision of marginal and conditional estimators but in general it is possible to compare power when the null is zero difference, since collapsibility then holds [23]. Marginal adjusted estimators have been shown to be more efficient than marginal unadjusted estimators [33, 34]. We are concerned with choosing a procedure for analy- sis prior to seeing data, so it would be unwise to jeopardise the analysis by choosing a procedure that may not con- verge [29, 30]. While it may be possible to specify a backup procedure, it would need to target the same estimand. This may prompt the question why not specify the backup procedure as the first choice (one good reason may be due to lower power). Because adjustment separates the effect of a treatment from the effects of covariates, we can typically infer the effect of a treatment with a little more precision, though it is possible to lose precision in small samples with non- or weakly prognostic covariates. It is therefore usually desir- able to use an efficient method of accounting for covariate effects, or the potential gains in power may not be fully realised. Convergence requires particular attention when infer- ence relies on (for example) bootstrapping or re- randomisation tests. Both involve augmenting the data using simulation and analysing the resulting dataset. We now need to be confident that not only will conver- gence be achieved in one dataset but in every dataset constructed by the procedure. While it is sometimes argued that weighting estima- tors are inefficient, Williamson, White and Forbes showed that, in the trial context with a continuous outcome mea- sure, IPTW is asymptotically as efficient as direct adjust- ment [20], backed up by simulation results using finite samples. Any ‘inefficiency’ of IPTW tends to arise due to extreme weights, just as the variance reduction it achieves is a result of similar estimated weights for all individuals. As with convergence, thinking about the weighting model makes clear that this will not be a problem when using this method in the analysis of trials. Note that a closely related method, overlap weighting, has recently been shown to be more efficient in finite samples and is worth consideration for covariate adjustment [35, 36]. Convergence d f Having defined an estimand, we require an estimator to compute an estimate. For many estimators, parameter estimation proceeds through some iterative technique. In maximum likelihood estimation, for example, an algo- rithm is used to find parameters that maximise the likeli- hood of the data. This involves finding parameter values that maximise a function. Once an algorithm has found a maximum, it is said to have ‘converged’. It sometimes hap- pens that the algorithm fails to converge to a maximum or that the maximum to which it converges is local (that is, a small bump rather than the true maximum) or not unique. This is clearly an issue. In the analysis of randomised trials, non-convergence tends to occur for one of two reasons: first, problems that Morris et al. Trials (2022) 23:328 Morris et al. Trials (2022) 23:328 Page 6 of 17 isation may mitigate possible issues associated with direct adjustment (given the same estimand such as a risk ratio). occur with certain models (e.g. generalised linear mod- els with binomial outcome distribution and identity- or log-link function); second, including too many parame- ters compared with the effective sample size (e.g. fitting fixed centre intercepts with few participants per centre) [28]. Essentially, the observed data are not consistent with a model that fits within the given constraints. Efficiency/precision/power At the time of writing, the lack of a general implementation in statistical software means, here, we do not further consider this otherwise attractive approach. Direct adjustment and standardisation may involve fit- ting different outcome models. For example, to estimate a risk difference, direct adjustment may use a generalised linear model with binomial outcome with identity link function. In terms of convergence, this would be a risky plan. It is possible to estimate an adjusted risk difference using other methods. This could use standardisation fol- lowing estimation through a logistic regression model, which comes with the guarantee of converging to a unique maximum. A popular technique for estimating the risk ratio without incurring convergence problems is to use a Poisson model with robust standard errors [31], where convergence is likely due to the canonical link function. g y IPTW involves specifying a model for treatment P(Z \| X) to estimate weights. This may be any model for binary data (regardless of the trial outcome type). Due to randomisation, it is always true that P(Z \| X) = P(Z). This means that the model for a binary treatment would not be misspecified regardless of how covariates X are modelled, provided parameters were not constrained to be wrong. Allocation ratios in trials are most frequently 1:1 but rarely more extreme than 1:2. For 1:1 allocation, the ‘outcome’ proportion in the treatment model will be approximately 50%, and never near 0 or 1, and its distri- bution given covariates is random, making ‘separation’ (or ‘perfect prediction’) unlikely [32]. All this means that the treatment model has a good chance of converging. The subsequent outcome model has no covariates and so is certain to converge. Handling covariates balanced by design h h d f b l There are many methods of balancing covariates at the design stage. The most popular seem to be stratified blocks and minimisation [7]. When a covariate-balancing method is used, it is necessary to account for the randomi- sation scheme in the analysis, or the estimated standard error for the treatment effect will be biased upwards, pro- ducing confidence intervals that are too wide (meaning they have greater than 1 −α coverage) and miscalibrated p-values [5]. For some intuition, suppose a trial uses stratified blocks, with stratification by a single, binary, prognostic covari- ate. That covariate will then always be distributed equally across the randomised groups (provided each block is completed). It is then impossible for any difference seen to be due to this covariate. Effectively, the variability in IPTW therefore seems to be the safest broad approach if convergence is anticipated to be an issue, while standard- Page 7 of 17 Morris et al. Trials (2022) 23:328 Morris et al. Trials (2022) 23:328 slight undercoverage in small samples [38]. In this type of setting, non-parametric bootstrap may be required, until a closed variance formula with a small-sample correction has been developed. the treatment effect due to possible chance imbalances is eliminated, since imbalances can never occur under this design. An analysis that ignores this systematic bal- ance will assume that imbalance in a covariate could have occurred by chance and calculate a standard error accord- ingly. This would be too large, since an imbalance in this particular covariate could not in fact have occurred [1]. Adjustment for the covariate separates the effect of covari- ate/s on outcome from the effect of treatment on outcome, and this is acknowledged in calculation of the standard error. While non-parametric bootstrap is a useful tool, we regard it as not being ideal due to inherent Monte Carlo error — though it may sometimes be the only option. Monte Carlo error can be made small with a suitably large number of bootstrap repetitions. An important but often neglected point about the bootstrap is that the resam- pling procedure must mimic the sampling used in the study itself. A simple bootstrap procedure invokes simple randomisation and will return upwardly biased standard errors or confidence intervals that are too wide if the trial did not in fact use simple randomisation. Misspecification of the covariate model We consider misspecification of the mean function relat- ing covariates to outcome rather than misspecification more generally. We illustrate the ideas using a continuous outcome, which lends itself to this visual explanation, but expect similar results for other outcome types. Consider a study with a single covariate Xi, randomised treatment Zi and the model that generates outcomes Yi is Yi = α + θZi + γ X2 i . (1) (1) Note that there is no residual error here; the mean func- tion determines the outcome exactly. The analyst plans to fit to the observed data a model (which is misspecified) with mean function Handling covariates balanced by design h h d f b l So, if the trial used blocked randomisation within strata, the bootstrap procedure should be restricted to do the same, else it tar- gets the wrong sampling distribution. Ensuring that the bootstrap procedure mimics the design actually used may be awkward if for example the trial used minimisation. There is literature on this going back at least to Fisher, who seemed to regard the point as obvious for analysis of variance in agricultural experiments [6]. It is gener- ally accepted that direct adjustment and standardisation can be specified to provide valid standard errors. This also holds for IPTW, though has not previously been commented on. Suppose we again have a single binary covariate which is perfectly balanced across treatment groups and estimate P(Z = z \| X = x). Then this prob- ability will be identical for every individual in the trial. Fitting the weighted regression to contrast the treatment effect will then return an identical estimate to a model that ignores the covariate. What is perhaps surprising is that the IPTW estimator still has a smaller standard error than the unweighted model. The variance formula, which does not say anything about the design, effectively ‘sees’ and acknowledges the balance after estimating the weights and rewards itself accordingly [20]. It is also clear that this hap- pens by analogy to direct adjustment, since asymptotically the two methods have the same standard errors. Variance estimation f Some variance formulas rely on approximations and some are asymptotic. For direct adjustment based on maxi- mum likelihood estimation, formulas are available for all commonly used models. For standardisation, the standard error from a fitted model is transformed using the delta method (asymptotic) [37]. Due to non-linearity, this could lead to p-values and confidence intervals that do not quite agree, if the p-value is taken from the model on the estima- tion scale; one possibility is to use test-based confidence intervals for measures with the same null. For example, suppose the outcome model is a logistic regression and the summary measure of the treatment effect is a risk dif- ference. The logistic regression returns a test-statistic of z for treatment. A 95% confidence interval for the risk dif- ference can then be constructed by taking ±1.96/z times the distance between the estimated risk difference and 0, and adding the result to the estimated risk difference. yi = ˆα + ˆθzi + ˆλxi. (2) (2) Suppose that this notional study is run and the observed x among those recruited is perfectly uniform on (−0.5, 0.5), as depicted by the first horizontal grey bar at the top of Fig. 1. When the misspecified model is fitted, ˆλ = 0. Next consider a trial where observed xi values are uniform on (0, 1) or (0.5, 1.5), also depicted by grey horizontal bars in Fig. 1. It is now clear that when the analyst fits their model (2), ˆλ > 0. In the first case, the sample correla- tion of x with x2 is zero, but in second and third cases, it is greater than zero. The analyst’s adjustment for x thus partially adjusts for x2 despite the model being misspeci- fied. This will generally be true when a covariate actually adjusted for is correlated with covariates not adjusted for. Suppose that this notional study is run and the observed x among those recruited is perfectly uniform on (−0.5, 0.5), as depicted by the first horizontal grey bar at the top of Fig. 1. When the misspecified model is fitted, ˆλ = 0. Next consider a trial where observed xi values are uniform on (0, 1) or (0.5, 1.5), also depicted by grey horizontal bars in Fig. 1. It is now clear that when the analyst fits their model (2), ˆλ > 0. Variance estimation f In the first case, the sample correla- tion of x with x2 is zero, but in second and third cases, it is greater than zero. The analyst’s adjustment for x thus partially adjusts for x2 despite the model being misspeci- fied. This will generally be true when a covariate actually adjusted for is correlated with covariates not adjusted for. IPTW estimators use robust standard errors that acknowledge the estimation of weights in the first step. These robust standard errors are asymptotically valid but recent work has demonstrated that they can produce The lower panel of Fig. 1 gives the estimated stan- dard errors after linear adjustment, showing that linear adjustment is always as efficient as no adjustment. Morris et al. Trials (2022) 23:328 Page 8 of 17 Morris et al. Trials (2022) 23:328 Fig. 1 Upper panel: Data from four notional trials where individuals recruited have different distributions of X. The two quadratic curves show the data in the two arms. Lower panel: SE after no adjustment and after linear adjustment for each of the three trials Fig. 1 Upper panel: Data from four notional trials where individuals recruited have different distributions of X. The two quadratic curves show the data in the two arms. Lower panel: SE after no adjustment and after linear adjustment for each of the three trials When using direct adjustment using the data in Fig. 1, the model is misspecified. Meanwhile, when using IPTW, the model used to form weights is by definition correctly specified. Despite this, the two return nearly identical results. The criteria for good specification of IPTW are slightly different than usual: what matters is not the cor- rectness of the specification of the model for Z \| X but how well the model for Z \| X models the predictors of Y; doing this better will result in a more suitable ‘rebalancing’. By attempting to balance X instead of X2, the covariate will be well balanced at certain points but less so at (for example) particularly high or low values of X. the outcome definition) but sometimes occurs in one or more covariates. Meanwhile, data on the randomised arm should never be missing. We will first discuss the issues when outcomes are incomplete and then when covari- ates are incomplete, along with some solutions for each of the three broad approaches. Variance estimation f As with any inference from incomplete data, it is important to understand the mech- anisms under which bias will and will not be introduced and so we discuss missingness dependent on randomised arm, covariates and outcome separately. With missing outcome data, a good starting point is to consider implications of missingness for the simplest analysis: including only those individuals with observed outcomes (complete-case analysis). When the probability of data being missing depends only on randomised arm, a complete-case estimator is unbiased and efficient. When missingness depends on the outcome, complete-case anal- ysis and multiple imputation under missing-at-random Handling data missing at random with the three adjustment approaches Some data will inevitably be missing for some partici- pants in the majority of randomised trials. This is most often in the outcome (unless ‘missing’ is somehow part of Morris et al. Trials (2022) 23:328 Page 9 of 17 Page 9 of 17 are biased in general; we will return to this point under sensitivity analysis. weighting, with missingness predicted from covariates separately by randomised arm. These are not our primary recommendations because we want a principal analy- sis that can be readily extended to principled sensitivity analyses. When outcome missingness depends on the observed value/s of covariates, a complete-case estimator may or may not be biased. If the covariate/s were not adjusted for, data would be missing not at random, which is a more dif- ficult statistical problem; if the covariate/s causing miss- ingness are adjusted for in the analysis, this becomes a missing-at-random problem. It is sometimes said that multiple imputation is not needed when outcomes are missing-at-random. However, estimation based on the complete cases is unbiased for a population represented by the complete cases. This estimand does not in general equal the estimand that targets the population actually randomised, unless the treatment effect is the same for these two populations, a potentially strong assumption. Supposing that the all-randomised population is of inter- est, a complete-cases estimator is then potentially biased, though the magnitude of bias will be small in practice. For a worked numerical example explaining this point, see the supplementary material. y Missing covariate values are not inevitable in ran- domised trials but do sometimes occur. As with miss- ing outcome data, analysis based only on the complete cases may inadvertently target a complete-cases popula- tion rather than all randomised and may be biased if the all-randomised population is intended. Unlike with miss- ing outcome data, discarding individuals with observed data on treatment and outcome does not follow the intention-to-treat principle and does throw away infor- mation. However, it is sometimes simple to target the all-randomised population: for any method that targets a marginal or collapsible summary measure, simple mean imputation (across arms, not within) and the missing indicator method are generally appropriate methods [41]. When using direct adjustment with a non-collapsible summary measure, it is more difficult to deal with incom- plete covariate data, and this typically requires a correct model [41]; if covariates are missing not at random, this can be very difficult. Handling data missing at random with the three adjustment approaches The message is then that more care is required to collect all covariate data if direct adjustment is to be used with a non-collapsible summary measure. If the aim of accounting for covariates in the analysis is simply to increase precision or to estimate a conditional summary measure, and not to target the all-randomised population, the remainder of this section and the appen- dices can be skipped. With outcomes missing according to this mechanism (depending on covariates), performing multiple imputa- tion by-randomised-arm and then analysing the imputed data sets by any of the three approaches to adjustment tar- gets the all-randomised population [39]. For direct adjust- ment, this is straightforward but there are subtleties in terms of statistical inference for the other two approaches: Sensitivity analysis with outcomes missing-not-at-random There are rarely cases where we know the true missing- ness mechanism. The assumption of missing-at-random depending on randomised arm and covariates is a con- venient starting point but it is important to examine the extent to which inferences are sensitive to alternative missingness mechanisms. This prompts sensitivity anal- ysis [29]. To obtain valid inference, missingness mecha- nisms that represent departures from missing-at-random then need to be explicitly invoked. • For standardisation, the question is whether to apply Rubin’s rules before or after the standardisation step. Since approximate normality is more likely on the estimation scale (for example log-odds) than the summary scale (for example risk difference), this is likely to be the appropriate scale for combining. In this situation, we view multiple imputation as a gen- eral and convenient framework for statistical inference under various departures for each of the three broad approaches, though not the only one [42]. Suppose for example that the planned approach to covariate adjust- ment was standardisation, and under missing-at-random, we planned to standardise to the all-randomised sam- ple (which is valid). There is no extension of this con- cept under missing-not-at-random mechanisms. Multiple imputation by-arm is a convenient way to do sensitivity analysis. It makes little sense to have a mismatch between the primary analysis and sensitivity analyses other than the missingness mechanism invoked, since we want to ensure that sensitivity of results are attributable to the change of missingness mechanism rather than the change of method. Analyses of GetTested This article focuses on planning a statistical analysis but it is nonetheless instructive to consider some of the issues discussed when different approaches are used. This helps to illustrate what may happen and prompts us to reflect on how we might plan. As described previously, our analysis of the GetTested trial is for two outcome measures: any test, which occurred in 35%, and any diagnosis, which occurred in 1.6% [43]. For both outcomes, two summary measures were of interest to the investigators: the risk ratio and the risk difference. Recall that there are five categorical covari- ates to adjust for, which in the direct adjustment model use 18 parameters in addition to the intercept and ran- domised arm. Table 3 gives the estimated treatment effect and standard error from various analyses estimating each measure on the two outcomes. For the Any test and Any diagnosis outcomes, there were 612 and 27 events respec- tively from 1739 observed outcomes (324 had missing outcomes). Table 3 presents results for the complete-cases population. Results targeting the all-randomised estimand are presented and discussed in Appendix 2. Covariate data were fully observed. Three of the 14 analyses in Table 3 failed to produce any sensible estimate. Two of these instances were due to the use of an identity-link-function binomial model to esti- mate a risk difference, leading to non-convergence, which happened for both outcomes. This emphasises the point that it would have been unwise to plan this as the adjust- ment model. The last was when directly estimating the log risk ratio for any test using a Poisson model. It did produce an estimate, which was 541, which indicated separation of outcome and a clearly untrustworthy estimate. y y Of the methods that did converge, the estimated treat- ment effects and standard errors tended to be similar across methods. The most notable difference in estimates is the complete-cases log risk ratio for any diagnosis, where the direct and standardisation analyses estimated a larger value than the IPTW analysis. This turns out to be due to the covariate men who have sex with men, The results presented are intended to compare adjust- ment methods for the same outcome and summary mea- Table 3 Results of analyses of the GetTested trial. All models included main effects only. Link function is canonical unless otherwise specified. Handling data missing at random with the three adjustment approaches The results presented are intended to compare adjust- ment methods for the same outcome and summary mea- su ar th eff co an co lo ob in se ra se us m ha th m ris an ou m ac is wh a to Table 3 Results of analyses of the GetTested trial. All models included m specified. The dash symbol - means model did not converge, except for separation for one or more covariates Outcome measure Summary measure Adjustment method M Any test (occurred in 35%) Risk difference Direct I Standardisation L IPTW L Log risk ratio Direct P Direct L Standardisation L IPTW L Any diagnosis (occurred in 1.6%) Risk difference Direct I Standardisation L IPTW L Log risk ratio Direct P Direct L Standardisation L IPTW L Handling data missing at random with the three adjustment approaches The dash symbol - means model did not separation for one or more covariates Outcome measure Summary measure Adjus Any test (occurred in 35%) Risk difference Direct Stand IPTW Log risk ratio Direct Direct Stand IPTW Any diagnosis (occurred in 1.6%) Risk difference Direct Stand IPTW Log risk ratio Direct Direct Stand IPTW sure. For the risk ratio, two direct adjustment methods are used: a log-binomial model and a Poisson model. In the analyses presented, the methods used included ‘main’ effects of covariates only. Of course, interactions between covariates could have been included for any method, and interactions between covariates and randomised arm could have been included for standardisation. Given the low number of diagnosis events — both anticipated and observed — including these interactions would have been inadvisable for that outcome. For these illustrative analy- ses, missing outcome data were assumed to be missing-at- random given covariates. ent result to all-randomised standardisation. Sensitivity analyses are important enough that we regard coherence between the primary and sensitivity analysis as worthy of consideration. ent result to all-randomised standardisation. Sensitivity analyses are important enough that we regard coherence between the primary and sensitivity analysis as worthy of consideration. Analyses of GetTested This article focuses on planning a statistical analysis but it is nonetheless instructive to consider some of the issues discussed when different approaches are used. This helps to illustrate what may happen and prompts us to reflect on how we might plan. As described previously, our analysis of the GetTested trial is for two outcome measures: any test, which occurred in 35%, and any diagnosis, which occurred in 1.6% [43]. For both outcomes, two summary measures were of interest to the investigators: the risk ratio and the risk difference. Recall that there are five categorical covari- ates to adjust for, which in the direct adjustment model use 18 parameters in addition to the intercept and ran- domised arm. Table 3 gives the estimated treatment effect and standard error from various analyses estimating each measure on the two outcomes. For the Any test and Any diagnosis outcomes, there were 612 and 27 events respec- tively from 1739 observed outcomes (324 had missing outcomes). Table 3 presents results for the complete-cases population. Results targeting the all-randomised estimand are presented and discussed in Appendix 2. Covariate data were fully observed. Handling data missing at random with the three adjustment approaches It is possible that multiple imputation under missing-at-random may have delivered a slightly differ- • For IPTW, it is however possible that Rubin’s variance formula will be inconsistent due to uncongeniality [40]. Furthermore, attempting to use multiple imputation may involve fitting the direct adjustment model — the first step of standardisation — so using multiple imputation may imply that IPTW is not needed. We would lose, for example, the advantages in terms of convergence. • For IPTW, it is however possible that Rubin’s variance formula will be inconsistent due to uncongeniality [40]. Furthermore, attempting to use multiple imputation may involve fitting the direct adjustment model — the first step of standardisation — so using multiple imputation may imply that IPTW is not needed. We would lose, for example, the advantages in terms of convergence. There are alternatives to multiple imputation. Under covariate-dependent missingness, standardisation can be applied to the all-randomised sample rather than only those with complete outcome data. Meanwhile, IPTW can be combined with inverse probability of missingness Morris et al. Trials (2022) 23:328 Morris et al. Trials (2022) 23:328 Page 10 of 17 Page 10 of 17 ent result to all-randomised standardisation analyses are important enough that we regar between the primary and sensitivity analysis consideration. Analyses of GetTested This article focuses on planning a statistical it is nonetheless instructive to consider some discussed when different approaches are use to illustrate what may happen and prompts on how we might plan. As described previously, our analysis of th trial is for two outcome measures: any occurred in 35%, and any diagnosis, which 1.6% [43]. For both outcomes, two summa were of interest to the investigators: the risk risk difference. Recall that there are five catego ates to adjust for, which in the direct adjust use 18 parameters in addition to the interce domised arm. Table 3 gives the estimated trea and standard error from various analyses esti measure on the two outcomes. For the Any diagnosis outcomes, there were 612 and 27 ev tively from 1739 observed outcomes (324 outcomes). Table 3 presents results for the com population. Results targeting the all-randomis are presented and discussed in Appendix 2. C were fully observed. The results presented are intended to com ment methods for the same outcome and sum Table 3 Results of analyses of the GetTested trial. A specified. Discussion We have compared the properties of three broad methods for estimating and adjusted treatment effect: direct adjust- ment, standardisation and inverse probability of treatment weighting. Our impression is that direct adjustment is the most commonly used approach in clinical trials and that standardisation and IPTW are better appreciated in observational epidemiology and warrant more considera- tion by trialists. In particular, it is clear that many investi- gators are interested in summarising the treatment effect as a risk ratio or risk difference. Direct adjustment is noto- riously unstable for both measures and so an unadjusted estimate is frequently reported, which will be inefficient. Tension between methods is typically greatest with binary outcome data, so we chose to focus on this setting. In particular, for continuous data analysed using linear models, none of the issues around collapsibility are rele- vant. Covariate adjustment using analysis of covariance is then typically suitable (direct regression of the outcome on treatment and covariates) unless treatment–covariate interactions are to be modelled, when standardisation would be the appropriate approach. Having discussed some properties of the three approa- ches, we provide Table 4 for reference, listing some of the points to consider for each approach. Table 4 Points to consider on properties of each approach Issue Direct adjustment Standardisation Inverse probability weighting Estimand for non-collapsible summary measures Conditional Marginal Marginal For non-collapsible summary measures, true β depends on... Covariates conditioned on in out- come model In-trial distribution of covariates In-trial distribution of covariates Misspecification of covariate effects Loses efficiency vs. correctly spec- ified model but expected to gain vs. no adjustment. True β changes under non-collapsibility Loses efficiency vs. correctly speci- fied model but expected to gain vs. no adjustment Loses efficiency vs. correctly speci- fied model but expected to gain vs. no adjustment Convergence Vulnerable Reasonable (but see GetTested experience) Solid Stratification/minimisation handled by variance estimator Yes Yes Yes Efficiency Asymptotically optimal Asymptotically optimal Asymptotically optimal Standard error calculation Direct Delta method Robust, accounting for estimation of weights via joint estimating equations. Analyses of GetTested The dash symbol - means model did not converge, except for * where the log risk ratio estimated as 541, indicating separation for one or more covariates Outcome measure Summary measure Adjustment method Model (variable modelled) Treatment effect estimate (SE) Any test (occurred in 35%) Risk difference Direct Identity-link binomial (outcome) - Standardisation Logistic (outcome) 0.260 (0.021) IPTW Logistic (treatment) 0.262 (0.021) Log risk ratio Direct Poisson, robust SE (outcome) -* Direct Log-link binomial (outcome) 0.797 (0.075) Standardisation Logistic (outcome) 0.796 (0.074) IPTW Logistic (treatment) 0.806 (0.075) Any diagnosis (occurred in 1.6%) Risk difference Direct Identity-link binomial (outcome) - Standardisation Logistic (outcome) 0.015 (0.006) IPTW Logistic (treatment) 0.013 (0.006) Log risk ratio Direct Poisson, robust SE (outcome) 0.972 (0.433) Direct Log-link binomial (outcome) 0.915 (0.412) Standardisation Logistic (outcome) 0.959 (0.412) IPTW Logistic (treatment) 0.855 (0.412) Table 3 Results of analyses of the GetTested trial. All models included main effects only. Link function is canonical unless otherwise specified. The dash symbol - means model did not converge, except for * where the log risk ratio estimated as 541, indicating separation for one or more covariates Morris et al. Trials (2022) 23:328 Morris et al. Trials (2022) 23:328 Page 11 of 17 We have described the methods in general terms with- out giving recommendations about specific implemen- tations. In our analysis of GetTested, the standardisa- tion estimates were computed from the direct adjust- ment model (logistic regression including main effect terms for each covariate) but could have been com- puted from a model including interactions among covari- ates and interactions of covariates with randomised group. Similarly, we used IPTW on the probability of treatment given covariates, which was estimated using logistic regression with a main effect term for each covariate. which in the direct model based on Poisson regression estimated extremely large risk ratios (around 15). Remov- ing this covariate from the two models resulted in very similar estimates, close to the IPTW result shown. which in the direct model based on Poisson regression estimated extremely large risk ratios (around 15). Remov- ing this covariate from the two models resulted in very similar estimates, close to the IPTW result shown. Treatment–covariate interac- tions Discussion Standard error can be biased downwards in small sam- ples [38] Treatment–covariate interac- tions Can be fitted but does not pro- duce an estimate of an average treatment effect Naturally handled this and pro- duces an estimate of the average treatment effect Does not handle Handling of missing covari- ate data in order to target all-randomised population Missing indicator or single mean imputation (though neither is suit- able with non-collapsible popula- tion summary measures) Missing indicator or single mean imputation Missing indicator or single mean imputation Handling of missing outcome data in order to target all- randomised population Multiple imputation by-arm (or inverse probability of missingness weighting) Standardisation to all-randomised rather than complete-case sam- ple; alternatively multiple imputa- tion by-arm or inverse probability of missingness weighting Inverse probability of missingness weighting (or multiple imputation by-arm ) Table 4 Points to consider on properties of each approach Reasonable (but see GetTested experience) Soli Stratification/minimisation handled by variance estimator Efficiency Standard error calculation Yes Yes Yes Asymptotically optimal Delta method Asymptotically optimal Delta method Asymptotically optimal Direct Asymptotically optimal Robust, accounting for estimation of weights via joint estimating equations. Standard error can be biased downwards in small sam- ples [38] Does not handle Treatment–covariate interac- tions Treatment–covariate interac- tions Naturally handled this and pro- duces an estimate of the average treatment effect Missing indicator or single mean imputation Can be fitted but does not pro- duce an estimate of an average treatment effect Missing indicator or single mean imputation Missing indicator or single mean imputation Handling of missing covari- ate data in order to target all-randomised population Missing indicator or single mean imputation Missing indicator or single mean imputation (though neither is suit- able with non-collapsible popula- tion summary measures) Inverse probability of missingness weighting (or multiple imputation by-arm ) Standardisation to all-randomised rather than complete-case sam- ple; alternatively multiple imputa- tion by-arm or inverse probability of missingness weighting Inverse probability of missingness weighting (or multiple imputation by-arm ) Handling of missing outcome data in order to target all- randomised population Multiple imputation by-arm (or inverse probability of missingness weighting) Page 12 of 17 Morris et al. Trials (2022) 23:328 Binary outcomes are common in non-inferiority trials and the scale of the population summary should match the scale on which the non-inferiority margin is defined. Conclusions We hope that this work stimulates statisticians working in trial teams to think carefully about adjustment methods, particularly by placing the estimand — which requires clinical investigators’ input — first, followed by consid- eration of the more statistical aspects. None of the three approaches is always best and the choice will depend on the trial context. Standardisation and IPTW are largely unused in trials, but have many advantages which mean they warrant routine consideration. It is possible to estimate the covariate-adjusted differ- ence in survival proportion at a time t using standardisa- tion or IPTW, which some trials are beginning to do in sta- tistical analysis plans, or the difference in restricted-mean survival times [44]. A variance estimator has been derived for standardisation [19], though not (to our knowledge) for IPTW. Improved power is frequently the main motivation for covariate adjustment, so a natural question arises regard- ing how to account for this improved power in sample size calculations. The increased power through covariate adjustment depends on the unknown prognostic value of covariates. If this is assumed to be appreciable but turns out to be modest, the trial will be underpowered. We therefore advise a cautious approach and would generally calculate sample size without accounting for covariates. If sample size calculation were to take account of covari- ate adjustment, we do not believe this would affect the choice between approaches, given that they have similar efficiency. Discussion In our experience, the margin is never defined as a con- ditional odds ratio, suggesting that the use of standardisa- tion or IPTW will be necessary. around convergence are to be expected in smaller subgroups. g 3. For IPTW, should we fit weighting models separately within subgroups or overall? We believe that this is an open question but note that direct adjustment and standardisation would adjust for covariates separately within subgroups. For time-to-event outcome data, the issues will depend on the chosen summary measure, as with binary out- come data. In clinical trials, this is frequently the hazard ratio, which is non-collapsible. A further important issue specific to hazard ratios is that even if hazards are pro- portional on the conditional scale they may not be pro- portional on the marginal scale, so an adjusted marginal hazard ratio (estimated by standardisation or IPTW) may be an inappropriate summary of the treatment effect, though this is also true of an unadjusted hazard ratio. 4. Subgroup analyses should be supported by interaction tests. These are straightforward for direct adjustment and IPTW. For standardisation, one could test for interaction on the scale of the working model, but it is more appropriate to test on the scale of the estimand. 4. Subgroup analyses should be supported by interaction tests. These are straightforward for direct adjustment and IPTW. For standardisation, one could test for interaction on the scale of the working model, but it is more appropriate to test on the scale of the estimand. Appendix 1: targeting the complete-case and all-randomised populations in the presence of missing data c) True value of summary measure within levels of X Table 5 a) Full data from a notional randomised trial. b) Complete cases, where all individuals with X = 0 and half of individuals with X=1 are complete cases. c) True value of summary measure within levels of X a) Full data b) Complete cases X = 0 X = 1 X = 0 X = 1 Z = 0 Z = 1 Z = 0 Z = 1 Z = 0 Z = 1 Z = 0 Z = 1 Y = 1 42 26 180 140 Y = 1 21 13 180 140 Y = 0 458 474 320 360 Y = 0 229 237 320 360 500 500 500 500 250 250 500 500 c) Value of summary measures within X (both in all randomised and among complete cases) Summary measure X = 0 X = 1 Odds ratio 0.598 0.691 Risk ratio 0.619 0.778 Risk difference −0.032 −0.080 b) Complete cases X = 0 X = 1 Z = 0 Z = 1 Z = 0 Z = 1 Y = 1 21 13 180 140 Y = 0 229 237 320 360 250 250 500 500 a) Full data X = 0 X = 1 Z = 0 Z = 1 Z = 0 Z = 1 Y = 1 42 26 180 140 Y = 0 458 474 320 360 500 500 500 500 c) Value of summary measures within X (both in all randomised and among complete cases) c) Value of summary measures within X (both in all randomised and among complete cases) c) Value of summary measures within X (both in all randomised and among complete cases) Summary measure X = 0 X = 1 Odds ratio 0.598 0.691 Risk ratio 0.619 0.778 Risk difference −0.032 −0.080 Summary measure X = 0 X = 1 Odds ratio 0.598 0.691 Risk ratio 0.619 0.778 Risk difference −0.032 −0.080 the full data from a notional randomised trial in which 2000 participants are recruited. Of these, 1000 have the covariate X = 1 and 1000 have X = 0. Treatment Z is stratified by covariate X so that 500 participants have each combination of values of X and Z. Finally, Table 5c gives the within-stratum odds ratios, risk ratios and risk dif- ferences. Appendix 1: targeting the complete-case and all-randomised populations in the presence of missing data Note that we have ensured there is some effect modification by X on each of these summary scales, since we will not in general be able to choose a scale on which there is no effect modification (particularly before seeing the data). sider four: a risk ratio, a risk difference, a conditional odds ratio and a marginal odds ratio. In these examples, con- ditional odds ratios are estimated by logistic regression, while marginal odds ratios, risk ratios and risk differences are estimated in two ways: first by standardisation after logistic regression and second by IPTW. The standardis- ation analysis does not include an interaction of X with Z in the logistic regression model, though it could do. Note that when two or more approaches can be used for one summary measure (the marginal odds ratio, risk ratio and risk difference are all estimated using standardisation and IPTW), the estimates produced will be identical (see [15], chapters 12 and 13). Suppose now that among those with X = 0, exactly half are a complete case, while among those with X = 1, all are a complete case. The complete cases are depicted in Table 5b. The probability of being a complete case depends only on the covariate X, meaning that the fre- quencies in the cells of the 2 × 2 table for X = 1 are exactly half what they were in Table 5a. Table 5c thus correctly represents the true values of the within-stratum summaries from Table 5b and a. Table 6 gives results of analyses for all randomised and for the complete cases. The key point is that the true value of the complete-cases estimand differs from the true value of the all-randomised estimand for all summary measures. This is because there is effect modification by X on each scale and the relative proportion with X = 1 changes in the complete cases under covariate-dependent missing- ness, so the relative contribution of each stratum-specific effect to the overall effect changes. As previously, we do not dictate which summary of the treatment effect should be used for the trial. Appendix 1: targeting the complete-case and all-randomised populations in the presence of missing data The received wisdom is that, when missingness depends only on covariates, a complete-case estimator is unbiased and efficient. There is however a subtlety to this, which impacts on our three adjustment methods: The estimand no longer targets a population with the empirical distri- bution of covariates among those randomised but among the complete cases. This is true unless the treatment effect is identical within strata on the chosen summary scale, as we will show. Many randomised trials report subgroup analyses, which we have not discussed. This raises some interesting issues and questions: As with non-collapsibility, the fact that the value of the estimand for all-randomised and complete-cases may dif- fer is not bias but a case of targeting different estimands: the target population attribute of the estimands differs. It is perhaps difficult to contrive an argument for the complete-case estimand. 1. When adjustment covariates are correlated with the variable defining the subgroup, the expected precision gains from covariate adjustment will diminish. At the most extreme, if we consider adjusting for the variable that defines the subgroup (where the ‘adjustment’ and ‘subgroup’ variables have perfect correlation), adjustment gains nothing: since X does not vary within subgroup, adjustment cannot improve predictions about Y. To demonstrate this point and outline some solutions, we will work through a simple numerical example with covariate-dependent missingness, show that a standard complete-case estimator changes the estimand, and note one possible advantage of standardisation over the other methods in this respect. The numerical example can be regarded as deterministic in the sense that estimators that fail to recover the exact value of the estimand seen in the true data do target a different estimand. 2. Should we choose the same approach to adjustment as for the main analyses? Ideally yes, with the caveat that any subgroup analysis is inherently conditional on subgroup membership, so the estimand is conditional-on-subgroup. Furthermore, issues Consider a trial with binary X, Y and Z, where the data can be represented in a 2 × 2 × 2 table. Table 5a gives Page 13 of 17 Morris et al. Trials (2022) 23:328 Morris et al. Trials (20 Table 5 a) Full data from a notional randomised trial. b) Complete cases, where all individuals with X = 0 and half of individuals with X=1 are complete cases. Appendix 1: targeting the complete-case and all-randomised populations in the presence of missing data With covariate-dependent missingness and outcome Y incomplete, a simple method can be used to target stan- dardisation to the all-randomised estimand: y For standardisation, the all-randomised estimand was targeted by standardising over the all-randomised popu- lation, as described in Appendix 1. 1. Retain in the data all randomised individuals 2. Fit the estimation model for standardisation in the complete cases (possibly including additional or different interactions between Z and covariates) For IPTW, inverse-probability-of-missingness weight- ing was used. The probability of missingness was esti- mated using a logistic regression model for missingness including all covariates that are present in the primary analysis, and interactions with randomised arm. Since the two outcomes were always missing simultaneously, esti- mated missingness probabilities were identical for both outcomes. This approach is straightforward to imple- ment due to the monotone missingness pattern (baseline covariates were fully observed and outcomes were missing on the same individuals). 3. Standardise over the all-randomised distribution of X (implemented in Stata’s margins command with the noesample option) The result of applying this method to the numerical exam- ple gives results identical to the left hand column of Table 6, indicating that the procedure can target the all- randomised estimand. However, as seen in the analysis of the GetTested study (see Appendix 2), this neat ‘trick’ is not fool-proof. Additional file 2 contains the Stata code used, which can be inspected to resolve any ambiguity in the description of the three methods. Note that multiple imputation or inverse-probability-of-missingness weighting could have been used for any of the three adjustment methods. For direct adjustment and IPTW, estimators of the all- randomised estimand are slightly less straightforward, but can be achieved by using multiple imputation with a sepa- rate imputation model for each randomised arm or using inverse probability of missingness weighting with the weighting model based on randomised arm and covari- ates. It would seem most natural to pair direct adjustment with by-arm multiple imputation, because both posit a model for the outcome data. IPTW therefore pairs more naturally with inverse probability of missingness weight- ing, where neither of the weighting models involve the value of the outcome. Table 7 shows the results from these analyses. The main point to note is that some analyses which returned an esti- mate for the complete-case population (see Table 3) do not for the all-randomised. Appendix 1: targeting the complete-case and all-randomised populations in the presence of missing data We con- Table 6 Summary of the true value of the estimand in all randomised and among the complete cases under covariate-dependent missingness Summary measure All-randomised Complete cases Conditional odds ratio 0.670 0.679 Marginal odds ratio 0.698 0.700 Risk ratio 0.748 0.761 Risk difference −0.056 −0.064 This method is possible with complete X and incomplete Y e value of the estimand in all randomised and among the complete cases under covariate-dependent Page 14 of 17 Page 14 of 17 Morris et al. Trials (2022) 23:328 It would seem that there is little to choose between the methods in terms of handling of missing data. How- ever, there are different modifications to the adjustment methods that we can used to target the all-randomised estimand in several cases. complete-case population, but here we consider the all- randomised population. To target this population, we retain the assumption that outcomes are missing at ran- dom and then use a different method for each adjustment approach. We have until now talked about ‘complete cases’ without specifying whether X or Y is incomplete. With X incom- plete, the simple-mean-imputation or missing indicator methods described by White and Thompson can be used to target the all-randomised estimand for marginal sum- mary measures [41]. Moving beyond complete cases is imperative here since the incomplete cases have impor- tant information in observed Y. However, neither method is appropriate if the summary measure of interest is con- ditional and non-collapsible and data are missing-not-at- random, in which case there is no method to target the all-randomised estimand besides correct modelling of the not-at-random missingness mechanism. For direct adjustment, we target the all-randomised population using multiple imputation ‘by-arm’. Each out- come was multiply imputed separately (not jointly), since they are always observed or missing simultaneously and so there is not auxiliary information. The imputation pro- cedure used a logistic regression model, separately for each arm, to impute outcome, including main effects of covariates. The imputation model for both outcomes was ‘augmented’ to handle separation of outcome given covariates [45]. Ten imputations were used. Note that it is the separate imputation model by-arm that targets the all-randomised population. Simply including randomised arm as a covariate in the imputation model would not achieve this unless there were no treatment–covariate interactions on the scale of the summary measure. Appendix 1: targeting the complete-case and all-randomised populations in the presence of missing data This turns out to be due to collinearity or perfect prediction, described below. How- ever, this affects the methods in different ways, as we see below. For direct adjustment, the multiple imputation step suf- fered from perfect prediction due to collinearity and the imputation model for any diagnosis had to be augmented [45]. The subsequent estimation of risk differences with an identity-link binomial model failed to converge for both outcomes. For any diagnosis risk ratios, the log-link bino- mial model also failed to converge; the Poisson model did Appendix 2: Issues with estimation for the all-randomised population in GetTested We now consider further results from re-analysis of GetTested. Table 3 considers an estimand for the Page 15 of 17 Morris et al. Trials (2022) 23:328 Morris et al. Trials (2022) 23:328 Table 7 Appendix 2: Results of analyses of the GetTested trial targeting the all-randomised population. All models included main effects only. Link function is canonical unless otherwise specified. The dash symbol - means model did not converge, with reasons described in the text of Appendix 2 Table 7 Appendix 2: Results of analyses of the GetTested trial targeting the all-randomised population. All models included main effects only. Link function is canonical unless otherwise specified. The dash symbol - means model did not converge, with reasons d ib d i th t t f A di 2 Table 7 Appendix 2: Results of analyses of the GetTested trial targeting the all-randomised population. All models included main effects only. Link function is canonical unless otherwise specified. The dash symbol - means model did not converge, with reasons described in the text of Appendix 2 Table 7 Appendix 2: Results of analyses of the GetTested trial targeting the all-randomised population. All models included main effects only. Link function is canonical unless otherwise specified. The dash symbol - means model did not converge, with reasons described in the text of Appendix 2 Outcome measure Summary measure Adjustment method Model (variable modelled) Treatment effect estimate (SE) Any test (occurred in 35%) Risk difference Direct Identity-link binomial (outcome) - Standardisation Logistic (outcome) 0.258 (0.021) IPTW Logistic (treatment) 0.259 (0.021) Log risk ratio Direct Poisson, robust SE (outcome) 0.804 (0.069) Direct Log-link binomial (outcome) - Standardisation Logistic (outcome) 0.799 (0.075)* IPTW Logistic (treatment) 0.802 (0.075) Any diagnosis (occurred in 1.6%) Risk difference Direct Identity-link binomial (outcome) - Standardisation Logistic (outcome) - IPTW Logistic (treatment) 0.013 (0.006) Log risk ratio Direct Poisson, robust SE (outcome) - Direct Identity-link binomial (outcome) - Standardisation Logistic (outcome) - IPTW Logistic (treatment) 0.866 (0.414) Almost-all-randomised. Estimate was returned only after omitting four participants affected by collinearity Almost-all-randomised. Estimate was returned only after omitting four participants affected by collinearity of methods targeting the all-randomised population is ongoing. The ideal solution is of course to avoid missing outcome data as far as possible during the conduct of a trial. Acknowledgements We are grateful to Jonathan Bartlett, Richard Riley, Leanne McCabe, Brennan Kahan, Andrew Althouse, Maarten van Smeden and Babak Choodari-Oskooei for discussions relating to this work. Our acknowledgement of these individuals does not imply their endorsement of this article. Supplementary Information Th li i t i l t For standardisation, no estimate was immediately returned for either outcome or summary measure. This was due to collinearity in the logistic regression mod- els predicting outcome, which meant that predictions were not produced for four transgender individuals. By omitting these individuals in the standardisation step, an almost-all-randomised estimate could be obtained for any test but not for any diagnosis (marked with asterisks in Table 7). Supplementary Information The online version contains supplementary material available at https://doi.org/10.1186/s13063-022-06097-z. The online version contains supplementary material available at https://doi.org/10.1186/s13063-022-06097-z. Additional file 1: Stata code to generate Fig. 1. Additional file 2: Stata code for the analysis of the GetTested trial (Assumes the data file journal.pmed.1002479.s001.xls has been downloaded from https://journals.plos.org/plosmedicine/article?id= 10.1371/journal.pmed.1002479#sec020). Additional file 1: Stata code to generate Fig. 1. Additional file 2: Stata code for the analysis of the GetTested trial (Assumes the data file journal.pmed.1002479.s001.xls has been downloaded from https://journals.plos.org/plosmedicine/article?id= 10.1371/journal.pmed.1002479#sec020). IPTW returned estimates for both summary mea- sures and both outcomes. The main threat to it obtain- ing an estimate for the all-randomised population is perfect prediction of missingness in the missingness model. Authors’ contributions Given the above issues, there is little to say in terms of comparing the estimated log risk ratios obtained across approaches. When approaches did return an estimate and standard error, they were very similar. It is hard to say what differences can be attributed to the populations tar- geted, the specific assumptions about missing data, or the approach to modelling assumptions specific to the approach to covariate adjustment. Further investigation TPM, ASW, EJW and IRW conceived of the article, planned the work and interpreted the results. TPM drafted the article. All authors have approved the submitted version. Abbreviations IPTW: Inverse probability of treatment weighting; STI: Sexually transmitted infection IPTW: Inverse probability of treatment weighting; STI: Sexually transmitted infection Appendix 2: Issues with estimation for the all-randomised population in GetTested return an estimate but we do not regard this as a valid all-randomised estimate because many parameters had to be dropped from the imputation models in order for them to fit. For any test, there was severe collinearity in the imputation model. Even after augmentation, only 916 individuals could be used in the imputation model. This again represents a failure to return an estimate relevant to the all-randomised population. References Kahan BC, Morris TP. 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ASW is an National Institute for Health Research Senior Investigator; as such, the views expressed are those of the authors and not necessarily those of the NHS, the NIHR, or the Department Page 16 of 17 Morris et al. Trials (2022) 23:328 Morris et al. Trials (2022) 23:328 of Health. EJW was supported by the MRC Network of Hubs for Trials Methodology HTMR Award MR/L004933/2/N96 and MRC project grant MR/S01442X/1. 20. Williamson EJ, Forbes A, White IR. Variance reduction in randomised trials by inverse probability weighting using the propensity score. Stat Med. 2014;33(5):721–37. 21. International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use. Addendum on estimands and sensitivity analysis in clinical trials to the guideline on statistical principles for clinical trials. 2019. https://www.ema.europa.eu/en/documents/scientific- guideline/ich-e9-r1-addendum-estimands-sensitivity-analysis-clinical- trials-guideline-statistical-principles_en.pdf. Accessed 17 Feb 2021. Received: 13 July 2021 Accepted: 10 February 2022 26. Dahabreh IJ, Robertson SE, Steingrimsson JA, Stuart EA, Hernán MA. Extending inferences from a randomized trial to a new target population. Stat Med. 2020;39(14):1999–2014. 27. Cole SR, Stuart EA. Generalizing evidence from randomized clinical trials to target populations: the ACTG 320 trial. Am J Epidemiol. 2010;172(1): 107–15. Consent for publication N/A Consent for publication N/A 23. Daniel R, Zhang J, Farewell D. Making apples from oranges: comparing noncollapsible effect estimators and their standard errors after adjustment for different covariate sets. Biom J. 2021;63(3):528–57. Ethics approval and consent to participate 22. Permutt T. Do covariates change the estimand? Stat Biopharm Res. 2020;12(1):45–53. Competing interests Tim Morris consults for Kite Pharma, Inc. Ian White has provided consultancy services or courses to Exelixis, AstraZeneca, GSK and Novartis, for which his employer has received funding. Elizabeth Williamson declares personal income from providing training to AstraZeneca. 24. Didelez V, Stensrud MJ. On the logic of collapsibility for causal effect measures. Biom J. 2021;64(2):235–42. 25. Huitfeldt A, Stensrud MJ, Suzuki E. On the collapsibility of measures of effect in the counterfactual causal framework. Emerg Themes Epidemiol. 2019;16(1):1–5. References J R Stat Soc Ser B. 1972;34: 187–220. 41. White IR, Thompson SG. Adjusting for partially missing baseline measurements in randomised trials. Stat Med. 2005;24(7):993–1007. 15. Hernán MA, Robins JM. Causal Inference: What If. Boca Raton: Chapman & Hall/CRC; 2020. 42. Cro S, Morris TP, Kenward MG, Carpenter JR. Sensitivity analysis for clinical trials with missing continuous outcome data using controlled multiple imputation: a practical guide. Stat Med. 2020;39(21):2815–42. 16. Cummings P. The relative merits of risk ratios and odds ratios. Arch Pediatr Adolesc Med. 2009;163(5):438. 16. Cummings P. The relative merits of risk Pediatr Adolesc Med. 2009;163(5):438. 16. Cummings P. The relative merits of risk ratios and odds ratios. 17. Snowden JM, Rose S, Mortimer KM. Implementation of g-computation on a simulated data set: Demonstration of a causal inference technique. Am J Epidemiol. 2011;173(7):731–8. 43. Wilson E, Free C, Morris TP, Syred J, Ahamed I, Menon-Johansson AS, Palmer MJ, Barnard S, Rezel E, Baraitser P. Internet-accessed sexually transmitted infection (e-STI) testing and results service: a randomised, single-blind, controlled trial. PLOS Med. 2017;14(12):e1002479. 18. Lee Y, Nelder JA. Conditional and marginal models: another view. Stat Sci. 2004;19(2):219–38. 44. Royston P, Altman D. External validation of a Cox prognostic model: principles and methods. BMC Med Res Methodol. 2013;13(1):33+. 19. Sjölander A. Regression standardization with the R package stdReg. Eur J Epidemiol. 2016;31(6):563–74. Page 17 of 17 Morris et al. Trials (2022) 23:328 Morris et al. Trials 45. White IR, Daniel R, Royston P. Avoiding bias due to perfect prediction in multiple imputation of incomplete categorical variables. Comput Stat Data Anal. 2010;54(10):2267–75. 45. White IR, Daniel R, Royston P. Avoiding bias due to perfect prediction in multiple imputation of incomplete categorical variables. Comput Stat Data Anal. 2010;54(10):2267–75. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
https://openalex.org/W2281350673	https://europepmc.org/articles/pmc4158480?pdf=render	Latin	null	4-(2-Fluorophenyl)-2-methoxy-5,6,7,8,9,10-hexahydrocycloocta[<i>b</i>]pyridine-3-carbonitrile	Acta crystallographica. Section E	2,014	cc-by	3,294	4-(2-Fluorophenyl)-2-methoxy- 5,6,7,8,9,10-hexahydrocycloocta[b]- pyridine-3-carbonitrile Data collection Bruker Kappa APEXII diffractometer Absorption correction: multi-scan (SADABS; Sheldrick, 1996) Tmin = 0.977, Tmax = 0.981 37690 measured reﬂections 3475 independent reﬂections 2812 reﬂections with I > 2(I) Rint = 0.027 R. Vishnupriya,a J. Suresh,a S. Maharani,b R. Ranjith Kumarb and P. L. Nilantha Lakshmanc* aDepartment of Physics, The Madura College, Madurai 625 011, India, bDepartment of Organic Chemistry, School of Chemistry, Madurai Kamaraj University, Madurai 625 021, India, and cDepartment of Food Science and Technology, University of Ruhuna, Mapalana, Kamburupitiya 81100, Sri Lanka Correspondence e-mail: plakshmannilantha@ymail.com Reﬁnement R[F 2 > 2(F 2)] = 0.053 wR(F 2) = 0.155 S = 1.08 3475 reﬂections 220 parameters 10 restraints H-atom parameters constrained max = 0.65 e A˚ 3 min = 0.61 e A˚ 3 Received 1 May 2014; accepted 15 July 2014 Edited by O. Blacque, University of Zu¨rich, Switzerland Edited by O. Blacque, University of Zu¨rich, Switzerland Data collection: APEX2 (Bruker, 2004); cell reﬁnement: SAINT (Bruker, 2004); data reduction: SAINT; program(s) used to solve structure: SHELXS97 (Sheldrick, 2008); program(s) used to reﬁne structure: SHELXL97 (Sheldrick, 2008); molecular graphics: PLATON (Spek, 2009); software used to prepare material for publication: SHELXL97. Key indicators: single-crystal X-ray study; T = 293 K; mean (C–C) = 0.003 A˚; disorder in main residue; R factor = 0.053; wR factor = 0.155; data-to-parameter ratio = 15.8. In the title compound, C19H19FN2O, the cyclooctene ring adopts a twisted boat–chair conformation. The dihedral angle between the plane of the ﬂuorophenyl substituent and that of the pyridine ring is 76.39 (8). The F and ortho-H atoms of the ﬂuorobenzene ring are disordered, with occupancy factors of 0.226 (5) and 0.774 (5). In the crystal, no signiﬁcant inter- actions are observed between the molecules beyond van der Waals contacts. JS and RV thank the management of Madura College for their encouragement and support. RRK thanks the University Grants Commission, New Delhi, for funds through Major Research Project F. No. 42–242/2013 (SR) Supporting information for this paper is available from the IUCr electronic archives (Reference: ZQ2223). organic compounds Experimental Crystal data C19H19FN2O Mr = 310.36 Monoclinic, P21=n a = 9.5219 (3) A˚ b = 13.8808 (4) A˚ c = 12.1140 (3) A˚ = 97.829 (1) V = 1586.20 (8) A˚ 3 Z = 4 Mo K radiation = 0.09 mm1 T = 293 K 0.28 0.25 0.23 mm Data collection Bruker Kappa APEXII diffractometer Absorption correction: multi-scan (SADABS; Sheldrick, 1996) Tmin = 0.977, Tmax = 0.981 37690 measured reﬂections 3475 independent reﬂections 2812 reﬂections with I > 2(I) Rint = 0.027 Reﬁnement R[F 2 > 2(F 2)] = 0.053 wR(F 2) = 0.155 S = 1.08 3475 reﬂections 220 parameters 10 restraints H-atom parameters constrained max = 0.65 e A˚ 3 min = 0.61 e A˚ 3 Acta Crystallographica Section E Structure Reports Online ISSN 1600-5368 Acta Crystallographica Section E Structure Reports Online ISSN 1600-5368 V = 1586.20 (8) A˚ 3 Z = 4 Mo K radiation = 0.09 mm1 T = 293 K 0.28 0.25 0.23 mm S1. Comment The synthesis of hydrogenated compounds has been extensively studied due to their interesting biological properties. For example, derivatives of 1,4-dihydropyridine exhibit high biological activities as calcium channel blockers (Bossert et al., 1981) and as calcium agonists or antagonists (Bossert & Vater, 1989; Wang et al.,1989; Alajarin et al., 1995). Our interest in preparing pharmacologically active pyridine-related compounds led us to the title compound, derived from a 1,4-di- hydropyridine and we have undertaken X-ray crystal structure determination of substituted pyridine scaffolds in order to establish its molecular conformation. The molecular structure of the title compound is shown in Fig 1. The cyclooctane ring (C1–C8) adopts twisted boat chair conformation. The central pyridine component is planar, with a maximum deviation from the mean plane that of 0.0207 (1) Å for atom C1. The phenyl substituent at C9 of the pyridine ring has a (+) synclinal conformation, which is evidenced by the C15–C14–C9–C10 torsion angle 77.10 (18)°. The shortening of the C–N distances [1.347 (2) and 1.312 (2) Å] and the opening of the N1–C11–C10 angle [122.98 (16)°] may be attributed to the size of the substituent at C1. There is a long Csp2—Csp1 bond (C10–C12 ═1.433 (3) Å), due to conjugation as found in similar related structures (Ramesh et al., 2009a, 2009b). The dihedral angle between the pseudo-axial phenyl substituent and the plane of the pyridine ring is 76.39 (8)°. Due to conjugation, the bond length C11—O1 (1.342 (2) Å) is shorter than the bond length C13—O1 (1.434 (2) Å). No significant intermolecular hydrogen bonds, π—π stacking interactions between neighboring aromatic rings or C— H···π interactions towards them are observed. No significant intermolecular hydrogen bonds, π—π stacking interactions between neighboring aromatic rings or C— H···π interactions towards them are observed. S2. Experimental A mixture of cyclooctanone (1 mmol), 2-fluorobenzaldehyde (1 mmol) and malononitrile (1 mmol) were taken in methanol (10 ml) to which lithium ethoxide (1 equiv) was added. The reaction mixture was heated under reflux for 2–3 h. After completion of the reaction (TLC), the reaction mixture was poured into crushed ice and extracted with ethyl acetate. The excess solvent was removed under vacuum and the residue was subjected to column chromatography using petroleum ether/ethyl acetate mixture (95:5 v/v) as eluent to obtain pure product. Melting point: 161–162 °C, yield: 67%. References For the biological activities of substituted pyridine derivatives, see: Bossert & Vater (1989); Bossert et al. (1981); Wang et al. (1989); Alajarin et al. (1995). For similar structures, see: Ramesh et al. (2009a,b). Alajarin, R., Vaquero, J. J., Alvarez-Builla, J., Pastor, M., Sunkel, C., de Casa- Juana, M. F., Priego, J., Statkow, P. R., Sanz-Aparicio, J. & Fonseca, I. (1995). J. Med. Chem. 38, 2830–2841. Bossert, F., Meyer, H. & Wehinger, E. (1981). Angew. Chem. Int. Ed. Engl. 20, 762–764. Bossert, F. & Vater, W. (1989). Med. Res. Rev, 9, 291–324. Bruker (2004). APEX2 and SAINT. Bruker AXS Inc., Madison, Wisconsin, USA. Ramesh, P., Subbiahpandi, A., Thirumurugan, P., Perumal, P. T. & Ponnuswamy, M. N. (2009a). Acta Cryst. E65, o450. y ( ) Ramesh, P., Sundaresan, S. S., Thirumurugan, P., Perumal, P. T. & Ramesh, P., Sundaresan, S. S., Thirumurugan, P., Perumal, P. T. & Ponnuswamy, M. N. (2009b). Acta Cryst. E65, o996–o997. a es , ., Su da esa , S. S., u u uga , ., e u a , . . & Ponnuswamy, M. N. (2009b). Acta Cryst. E65, o996–o997. Ponnuswamy, M. N. (2009b). Acta Cryst. E65, o996–o997. Sheldrick, G. M. (1996). SADABS. University of Go¨ttingen, Germany. Sheldrick, G. M. (2008). Acta Cryst. A64, 112–122. Spek, A. L. (2009). Acta Cryst. D65, 148–155. Spek, A. L. (2009). Acta Cryst. D65, 148–155. Wang, S. D., Herbette, L. G. & Rhodes, D. G. (1989). Acta Cryst. C45, 1748– 1751. o872 Vishnupriya et al. Acta Cryst. (2014). E70, o872 doi:10.1107/S1600536814016365 supporting information Acta Cryst. (2014). E70, o872 [doi:10.1107/S1600536814016365] Acta Cryst. (2014). E70, o872 [doi:10.1107/S1600536814016365] Acta Cryst. (2014). E70, o872 [doi:10.1107/S1600536814016365] Acta Cryst. (2014). E70, o872 [doi:10.1107/S1600536814016365] Figure 1 Figure 1 The molecular structure of the title compound showing 30% probability displacement ellipsoids and the atom-numbering scheme. g The molecular structure of the title compound showing 30% probability displacement ellipsoids and the atom-numbering scheme. 4-(2-Fluorophenyl)-2-methoxy-5,6,7,8,9,10-hexahydrocycloocta[b]pyridine-3-carbonitrile 4-(2-Fluorophenyl)-2-methoxy-5,6,7,8,9,10-hexahydrocycloocta[b]pyridine-3-carbonitril S3. Refinement Crystal data, data collection and structure refinement details are summarized in Table 1. H atoms were placed in calculated positions and allowed to ride on their carrier atoms with C—H = 0.93 (aromatic CH), 0.96 (methyl CH3) and 0.97 Å (methylene CH2). Isotropic displacement parameters for H atoms were calculated as Uiso = 1.5Ueq(C) for CH3 groups and Uiso = 1.2Ueq(carrier atom) for all other H atoms. The F and H atoms of the fluorobenzene rings are disordered over two sets of sites in the ratio 0.226(): 0.774 (5). sup-1 Acta Cryst. (2014). E70, o872 supporting information Figure 1 The molecular structure of the title compound showing 30% probability displacement ellipsoids and the atom-numbering scheme supporting information Refinement Refinement on F2 Least-squares matrix: full R[F2 > 2σ(F2)] = 0.053 wR(F2) = 0.155 S = 1.08 3475 reflections 220 parameters 10 restraints Primary atom site location: structure-invariant direct methods Secondary atom site location: difference Fourier map Hydrogen site location: inferred from neighbouring sites H-atom parameters constrained w = 1/[σ2(Fo2) + (0.0648P)2 + 0.7644P] where P = (Fo2 + 2Fc2)/3 (Δ/σ)max < 0.001 Δρmax = 0.65 e Å−3 Δρmin = −0.61 e Å−3 Extinction correction: SHELXL97 (Sheldrick, 2008), Fc=kFc[1+0.001xFc2λ3/sin(2θ)]-1/4 Extinction coefficient: 0.010 (2) Refinement Refinement on F2 Least-squares matrix: full R[F2 > 2σ(F2)] = 0.053 wR(F2) = 0.155 S = 1.08 3475 reflections 220 parameters 10 restraints Primary atom site location: structure-invariant direct methods Secondary atom site location: difference Fourier map Hydrogen site location: inferred from neighbouring sites H-atom parameters constrained w = 1/[σ2(Fo2) + (0.0648P)2 + 0.7644P] where P = (Fo2 + 2Fc2)/3 (Δ/σ)max < 0.001 Δρmax = 0.65 e Å−3 Δρmin = −0.61 e Å−3 Extinction correction: SHELXL97 (Sheldrick, 2008), Fc=kFc[1+0.001xFc2λ3/sin(2θ)]-1/4 Extinction coefficient: 0.010 (2) Refinement Refinement on F2 Least-squares matrix: full R[F2 > 2σ(F2)] = 0.053 wR(F2) = 0.155 S = 1.08 3475 reflections 220 parameters 10 restraints Primary atom site location: structure-invariant direct methods Secondary atom site location: difference Fourier map Hydrogen site location: inferred from neighbouring sites H-atom parameters constrained w = 1/[σ2(Fo2) + (0.0648P)2 + 0.7644P] where P = (Fo2 + 2Fc2)/3 (Δ/σ)max < 0.001 Δρmax = 0.65 e Å−3 Δρmin = −0.61 e Å−3 Extinction correction: SHELXL97 (Sheldrick, 2008), Fc=kFc[1+0.001xFc2λ3/sin(2θ)]-1/4 Extinction coefficient: 0.010 (2) Extinction correction: SHELXL97 (Sheldrick, 2008), Fc=kFc[1+0.001xFc2λ3/sin(2θ)]-1/4 Extinction coefficient: 0.010 (2) 4-(2-Fluorophenyl)-2-methoxy-5,6,7,8,9,10-hexahydrocycloocta[b]pyridine-3-carbonitrile Crystal data C19H19FN2O Mr = 310.36 Monoclinic, P21/n Hall symbol: -P 2yn a = 9.5219 (3) Å b = 13.8808 (4) Å c = 12.1140 (3) Å β = 97.829 (1)° V = 1586.20 (8) Å3 Z = 4 F(000) = 656 Dx = 1.300 Mg m−3 Mo Kα radiation, λ = 0.71073 Å Cell parameters from 2000 reflections θ = 2–27° µ = 0.09 mm−1 T = 293 K Block, colourless 0.28 × 0.25 × 0.23 mm Data collection Bruker Kappa APEXII diffractometer Radiation source: fine-focus sealed tube Graphite monochromator Detector resolution: 0 pixels mm-1 ω and φ scans Absorption correction: multi-scan (SADABS; Sheldrick, 1996) Tmin = 0.977, Tmax = 0.981 37690 measured reflections 3475 independent reflections 2812 reflections with I > 2σ(I) Rint = 0.027 θmax = 27.0°, θmin = 2.2° h = −12→12 k = −17→17 l = −15→15 sup-2 Acta Cryst. (2014). E70, o872 supporting information Special details Geometry. All e.s.d.'s (except the e.s.d. in the dihedral angle between two l.s. planes) are estimated using the full covariance matrix. The cell e.s.d.'s are taken into account individually in the estimation of e.s.d.'s in distances, angles and torsion angles; correlations between e.s.d.'s in cell parameters are only used when they are defined by crystal symmetry. An approximate (isotropic) treatment of cell e.s.d.'s is used for estimating e.s.d.'s involving l.s. planes. Refinement. Refinement of F2 against ALL reflections. The weighted R-factor wR and goodness of fit S are based on F2, conventional R-factors R are based on F, with F set to zero for negative F2. The threshold expression of F2 > σ(F2) is used only for calculating R-factors(gt) etc. and is not relevant to the choice of reflections for refinement. R-factors based on F2 are statistically about twice as large as those based on F, and R- factors based on ALL data will be even larger. Fractional atomic coordinates and isotropic or equivalent isotropic displacement parameters (Å2) x y z Uiso/Ueq Occ. (<1) C1 0.48826 (19) 0.14942 (13) 0.47836 (14) 0.0386 (4) C2 0.5667 (2) 0.18410 (14) 0.38693 (15) 0.0454 (4) H2A 0.5635 0.1344 0.3303 0.054 H2B 0.6653 0.1942 0.4170 0.054* C3 0.5073 (2) 0.27694 (16) 0.33279 (17) 0.0537 (5) H3A 0.5695 0.2983 0.2806 0.064* H3B 0.4156 0.2631 0.2903 0.064* C4 0.4896 (2) 0.35887 (16) 0.41229 (19) 0.0575 (5) H4A 0.4163 0.3413 0.4569 0.069* H4B 0.4566 0.4152 0.3689 0.069* C5 0.6229 (3) 0.38643 (15) 0.49078 (18) 0.0566 (5) H5A 0.7047 0.3636 0.4588 0.068* H5B 0.6286 0.4562 0.4943 0.068* C6 0.6324 (3) 0.34781 (16) 0.60938 (18) 0.0593 (6) H6A 0.5458 0.3651 0.6384 0.071* H6B 0.7098 0.3807 0.6546 0.071* C7 0.6549 (2) 0.23933 (16) 0.62511 (16) 0.0496 (5) H7A 0.7308 0.2200 0.5839 0.059* H7B 0.6871 0.2274 0.7034 0.059* C8 0.52873 (18) 0.17526 (13) 0.58972 (14) 0.0384 (4) C9 0.45081 (18) 0.13641 (12) 0.66895 (14) 0.0370 (4) C10 0.33770 (18) 0.07500 (12) 0.63392 (14) 0.0380 (4) C11 0.30274 (19) 0.05759 (13) 0.51926 (14) 0.0396 (4) Fractional atomic coordinates and isotropic or equivalent isotropic displacement parameters (Å2) x y z Uiso/Ueq Fractional atomic coordinates and isotropic or equivalent isotropic displacement parameters (Å2) sup-3 Acta Cryst. (2014). Special details E70, o872 supporting information supporting information C12 0.2597 (2) 0.02745 (14) 0.71119 (15) 0.0438 (4) C13 0.1466 (3) −0.01094 (17) 0.37142 (17) 0.0565 (5) H13A 0.0670 −0.0539 0.3596 0.085 H13B 0.2237 −0.0373 0.3376 0.085* H13C 0.1203 0.0505 0.3385 0.085* C14 0.48547 (16) 0.15856 (14) 0.79050 (14) 0.0421 (4) C15 0.55536 (16) 0.09561 (12) 0.86460 (16) 0.0557 (5) H15 0.5783 0.0356 0.8380 0.067* 0.226 (5) F1B 0.3841 (3) 0.3087 (4) 0.7588 (5) 0.115 (4) 0.226 (5) C16 0.5944 (2) 0.1143 (2) 0.97544 (19) 0.0741 (8) H16 0.6419 0.0686 1.0227 0.089* C17 0.5606 (3) 0.2033 (3) 1.0142 (2) 0.0807 (9) H17 0.5861 0.2185 1.0891 0.097* C18 0.4901 (3) 0.2696 (2) 0.9439 (2) 0.0737 (8) H18 0.4672 0.3296 0.9708 0.088* C19 0.4532 (2) 0.24716 (17) 0.83331 (18) 0.0571 (5) H19 0.4054 0.2926 0.7859 0.069* 0.774 (5) F1A 0.5857 (2) 0.00848 (12) 0.82521 (14) 0.0769 (8) 0.774 (5) N1 0.37525 (17) 0.09332 (11) 0.44417 (12) 0.0417 (4) N2 0.1995 (2) −0.01308 (16) 0.77174 (16) 0.0628 (5) O1 0.18988 (15) 0.00092 (11) 0.48868 (11) 0.0518 (4) Atomic displacement parameters (Å2) U11 U22 U33 U12 U13 U23 C1 0.0420 (9) 0.0375 (9) 0.0364 (9) 0.0037 (7) 0.0055 (7) 0.0002 (7) C2 0.0526 (11) 0.0467 (10) 0.0383 (9) −0.0006 (8) 0.0116 (8) −0.0020 (8) C3 0.0626 (12) 0.0571 (12) 0.0401 (10) −0.0043 (10) 0.0021 (9) 0.0066 (9) C4 0.0647 (13) 0.0491 (11) 0.0588 (13) 0.0085 (10) 0.0090 (10) 0.0078 (10) C5 0.0719 (14) 0.0432 (10) 0.0570 (12) −0.0088 (10) 0.0170 (10) −0.0035 (9) C6 0.0699 (14) 0.0607 (13) 0.0486 (11) −0.0243 (11) 0.0127 (10) −0.0139 (10) C7 0.0431 (10) 0.0664 (13) 0.0378 (9) −0.0106 (9) 0.0002 (7) 0.0045 (9) C8 0.0380 (9) 0.0400 (9) 0.0366 (8) 0.0014 (7) 0.0025 (7) 0.0022 (7) C9 0.0380 (9) 0.0381 (8) 0.0340 (8) 0.0039 (7) 0.0019 (6) 0.0003 (7) C10 0.0405 (9) 0.0383 (9) 0.0349 (8) 0.0019 (7) 0.0043 (7) 0.0005 (7) C11 0.0427 (9) 0.0374 (9) 0.0374 (9) −0.0001 (7) 0.0010 (7) −0.0009 (7) C12 0.0447 (10) 0.0489 (10) 0.0377 (9) −0.0029 (8) 0.0058 (7) −0.0039 (8) C13 0.0626 (13) 0.0613 (13) 0.0414 (10) −0.0116 (10) −0.0081 (9) −0.0042 (9) C14 0.0396 (9) 0.0516 (10) 0.0350 (9) −0.0050 (8) 0.0046 (7) −0.0034 (7) C15 0.0576 (12) 0.0679 (14) 0.0404 (10) 0.0027 (10) 0.0024 (9) 0.0012 (9) F1B 0.133 (8) 0.091 (6) 0.122 (7) 0.054 (5) 0.026 (6) −0.025 (5) C16 0.0668 (15) 0.113 (2) 0.0402 (11) −0.0049 (15) −0.0012 (10) 0.0109 (13) C17 0.0796 (17) 0.123 (3) 0.0404 (12) −0.0294 (17) 0.0112 (11) −0.0250 (15) C18 0.0838 (17) 0.0808 (17) 0.0614 (15) −0.0219 (14) 0.0281 (13) −0.0317 (14) C19 0.0610 (13) 0.0617 (13) 0.0516 (11) −0.0084 (10) 0.0183 (10) −0.0141 (10) F1A 0.1046 (16) 0.0693 (12) 0.0548 (11) 0.0386 (10) 0.0034 (9) 0.0068 (8) N1 0.0484 (9) 0.0409 (8) 0.0350 (7) −0.0002 (6) 0.0034 (6) −0.0010 (6) N2 0.0648 (12) 0.0756 (13) 0.0504 (10) −0.0147 (10) 0.0164 (9) 0.0009 (9) C12 0.2597 (2) 0.02745 (14) 0.71119 (15) 0.0438 (4) C13 0.1466 (3) −0.01094 (17) 0.37142 (17) 0.0565 (5) H13A 0.0670 −0.0539 0.3596 0.085* H13B 0.2237 −0.0373 0.3376 0.085* H13C 0.1203 0.0505 0.3385 0.085* C14 0.48547 (16) 0.15856 (14) 0.79050 (14) 0.0421 (4) C15 0.55536 (16) 0.09561 (12) 0.86460 (16) 0.0557 (5) H15 0.5783 0.0356 0.8380 0.067* 0.226 (5) F1B 0.3841 (3) 0.3087 (4) 0.7588 (5) 0.115 (4) 0.226 (5) C16 0.5944 (2) 0.1143 (2) 0.97544 (19) 0.0741 (8) H16 0.6419 0.0686 1.0227 0.089* C17 0.5606 (3) 0.2033 (3) 1.0142 (2) 0.0807 (9) H17 0.5861 0.2185 1.0891 0.097* C18 0.4901 (3) 0.2696 (2) 0.9439 (2) 0.0737 (8) H18 0.4672 0.3296 0.9708 0.088* C19 0.4532 (2) 0.24716 (17) 0.83331 (18) 0.0571 (5) H19 0.4054 0.2926 0.7859 0.069* 0.774 (5) F1A 0.5857 (2) 0.00848 (12) 0.82521 (14) 0.0769 (8) 0.774 (5) N1 0.37525 (17) 0.09332 (11) 0.44417 (12) 0.0417 (4) N2 0.1995 (2) −0.01308 (16) 0.77174 (16) 0.0628 (5) O1 0.18988 (15) 0.00092 (11) 0.48868 (11) 0.0518 (4) sup-4 Acta Cryst. Special details (2014). E70, o872 supporting information supporting information supporting informatio O1 0.0554 (8) 0.0587 (8) 0.0388 (7) −0.0169 (6) −0.0025 (6) −0.0006 (6) Geometric parameters (Å, º) C1—N1 1.347 (2) C9—C14 1.496 (2) C1—C8 1.398 (2) C10—C11 1.404 (2) C1—C2 1.497 (2) C10—C12 1.433 (3) C2—C3 1.520 (3) C11—N1 1.312 (2) C2—H2A 0.9700 C11—O1 1.342 (2) C2—H2B 0.9700 C12—N2 1.139 (3) C3—C4 1.514 (3) C13—O1 1.434 (2) C3—H3A 0.9700 C13—H13A 0.9600 C3—H3B 0.9700 C13—H13B 0.9600 C4—C5 1.527 (3) C13—H13C 0.9600 C4—H4A 0.9700 C14—C15 1.360 (3) C4—H4B 0.9700 C14—C19 1.385 (3) C5—C6 1.524 (3) C15—F1A 1.3459 (10) C5—H5A 0.9700 C15—C16 1.368 (3) C5—H5B 0.9700 C15—H15 0.9300 C6—C7 1.529 (3) F1B—C19 1.3477 (10) C6—H6A 0.9700 C16—C17 1.375 (4) C6—H6B 0.9700 C16—H16 0.9300 C7—C8 1.509 (3) C17—C18 1.367 (4) C7—H7A 0.9700 C17—H17 0.9300 C7—H7B 0.9700 C18—C19 1.374 (3) C8—C9 1.399 (2) C18—H18 0.9300 C9—C10 1.394 (2) C19—H19 0.9300 N1—C1—C8 123.26 (16) C9—C8—C7 120.56 (15) N1—C1—C2 114.61 (15) C10—C9—C8 119.14 (15) C8—C1—C2 122.11 (16) C10—C9—C14 118.89 (15) C1—C2—C3 113.46 (16) C8—C9—C14 121.97 (15) C1—C2—H2A 108.9 C9—C10—C11 118.40 (16) C3—C2—H2A 108.9 C9—C10—C12 122.05 (16) C1—C2—H2B 108.9 C11—C10—C12 119.52 (16) C3—C2—H2B 108.9 N1—C11—O1 120.49 (16) H2A—C2—H2B 107.7 N1—C11—C10 122.98 (16) C4—C3—C2 115.43 (16) O1—C11—C10 116.53 (16) C4—C3—H3A 108.4 N2—C12—C10 177.8 (2) C2—C3—H3A 108.4 O1—C13—H13A 109.5 C4—C3—H3B 108.4 O1—C13—H13B 109.5 C2—C3—H3B 108.4 H13A—C13—H13B 109.5 H3A—C3—H3B 107.5 O1—C13—H13C 109.5 C3—C4—C5 115.42 (19) H13A—C13—H13C 109.5 C3—C4—H4A 108.4 H13B—C13—H13C 109.5 C5—C4—H4A 108.4 C15—C14—C19 115.90 (18) C3—C4—H4B 108.4 C15—C14—C9 122.68 (17) C5—C4—H4B 108.4 C19—C14—C9 121.36 (18) O1 0.0554 (8) 0.0587 (8) 0.0388 (7) −0.0169 (6) −0.0025 (6) −0.0006 (6) 4 (8) 0.0587 (8) 0.0388 (7) −0.0169 (6) −0.0025 (6) −0.0006 (6) O1 Geometric parameters (Å, º) sup-5 Acta Cryst. (2014). supporting information E70, o872 supporting information pp g H4A—C4—H4B 107.5 F1A—C15—C14 116.92 (17) C6—C5—C4 115.89 (18) F1A—C15—C16 118.4 (2) C6—C5—H5A 108.3 C14—C15—C16 124.7 (2) C4—C5—H5A 108.3 C14—C15—H15 117.7 C6—C5—H5B 108.3 C16—C15—H15 117.7 C4—C5—H5B 108.3 C15—C16—C17 117.5 (3) H5A—C5—H5B 107.4 C15—C16—H16 121.3 C5—C6—C7 116.94 (17) C17—C16—H16 121.3 C5—C6—H6A 108.1 C18—C17—C16 120.7 (2) C7—C6—H6A 108.1 C18—C17—H17 119.7 C5—C6—H6B 108.1 C16—C17—H17 119.7 C7—C6—H6B 108.1 C17—C18—C19 119.5 (3) H6A—C6—H6B 107.3 C17—C18—H18 120.2 C8—C7—C6 116.88 (17) C19—C18—H18 120.2 C8—C7—H7A 108.1 F1B—C19—C18 123.1 (4) C6—C7—H7A 108.1 F1B—C19—C14 115.2 (4) C8—C7—H7B 108.1 C18—C19—C14 121.8 (2) C6—C7—H7B 108.1 C18—C19—H19 119.1 H7A—C7—H7B 107.3 C14—C19—H19 119.1 C1—C8—C9 117.45 (16) C11—N1—C1 118.64 (15) C1—C8—C7 121.95 (16) C11—O1—C13 116.85 (15) N1—C1—C2—C3 87.0 (2) C10—C9—C14—C15 77.10 (18) C8—C1—C2—C3 −91.8 (2) C8—C9—C14—C15 −102.71 (18) C1—C2—C3—C4 52.0 (2) C10—C9—C14—C19 −105.84 (18) C2—C3—C4—C5 54.9 (3) C8—C9—C14—C19 74.3 (2) C3—C4—C5—C6 −100.7 (2) C19—C14—C15—F1A 178.92 (13) C4—C5—C6—C7 69.8 (3) C9—C14—C15—F1A −3.88 (17) C5—C6—C7—C8 −74.8 (3) C19—C14—C15—C16 −0.02 (14) N1—C1—C8—C9 3.2 (3) C9—C14—C15—C16 177.18 (17) C2—C1—C8—C9 −178.04 (16) F1A—C15—C16—C17 −179.13 (17) N1—C1—C8—C7 −179.21 (17) C14—C15—C16—C17 −0.2 (2) C2—C1—C8—C7 −0.4 (3) C15—C16—C17—C18 0.4 (3) C6—C7—C8—C1 80.7 (2) C16—C17—C18—C19 −0.3 (3) C6—C7—C8—C9 −101.8 (2) C17—C18—C19—F1B −179.75 (17) C1—C8—C9—C10 −0.3 (2) C17—C18—C19—C14 0.1 (3) C7—C8—C9—C10 −177.92 (17) C15—C14—C19—F1B 179.93 (8) C1—C8—C9—C14 179.53 (16) C9—C14—C19—F1B 2.7 (2) C7—C8—C9—C14 1.9 (3) C15—C14—C19—C18 0.08 (18) C8—C9—C10—C11 −2.5 (3) C9—C14—C19—C18 −177.16 (17) C14—C9—C10—C11 177.63 (16) O1—C11—N1—C1 −179.64 (16) C8—C9—C10—C12 175.57 (17) C10—C11—N1—C1 −0.1 (3) C14—C9—C10—C12 −4.2 (3) C8—C1—N1—C11 −3.0 (3) C9—C10—C11—N1 2.9 (3) C2—C1—N1—C11 178.14 (16) C12—C10—C11—N1 −175.29 (17) N1—C11—O1—C13 −5.3 (3) C9—C10—C11—O1 −177.58 (16) C10—C11—O1—C13 175.14 (17) C12—C10—C11—O1 4.2 (3) sup-6 Acta Cryst. (2014). E70, o872
https://openalex.org/W3097143782	https://www.mdpi.com/2543-6031/88/5/443/pdf?version=1656407686	English	null	Short-Acting Inhaled β2-Agonists: Why, Whom, What, How?	Advances in Respiratory Medicine	2,020	cc-by	5,311	Andrzej Emeryk 1, Justyna Emeryk-Maksymiuk 2 1Department of Pediatric Pulmonology and Rheumatology Medical University in Lublin, Poland 2Chair of Internal Medicine and Department of Internal Medicine in Nursing Medical University in Lublin, Poland 1Department of Pediatric Pulmonology and Rheumatology Medical University in Lublin, Poland 2Chair of Internal Medicine and Department of Internal Medicine in Nursing Medical University in Lublin, Poland Introduction after administration of inhaled salbutamol, but the duration of action does not exceed 4–6 hours (Figure 1) [8]. Short-acting b2-agonists (SABA) stimulate b2-adrenergic receptor (b2-AR). They are called b2-mimetics or b2-agonists. First selective SABA widely used in clinical practice appeared more than 50 years ago. They were introduced to the global market in the following order: terbutaline (1966 yr.), salbutamol (1968 yr.) and fenoterol (1970 yr.) [1]. Next drugs, such as levalbuterol, reproterol, rimiterol, klenbuterol and pirbuterol, were introduced afterwards [2–4]. Short-act ing b2-agonists are selective agonists of b2-AR, however they differ in their degree of selectivity. Salbutamol (albuterol) is the most used SABA in the world — in US it took 9 th place on the list of prescribed medicines in 2016 (70 million of prescriptions) [5]. According to World Health Organization (WHO) salbutamol ranks among the most effective and safest medicines essential to health care systems [6]. Racemic salbutamol is an equal (1:1) mixture of R-salbutamol (levalbuterol) and S-salbutamol isomers. R-isomer of salbutamol is a pharmacologically active compound which exhibits many clinical effects, including potent bronchodilation [7]. Suppression of bronchoc onstriction and bronchodilation occur 5 minutes Drugs from this group provide effective protection against exercise-induced bronchoc onstriction within 0.5–2.0 hours also against bronchoconstriction triggered by exposure to sensitizing allergen [9, 10]. Clinical studies show more potent bronchodilation and less side effects of R-salbutamol in comparison with racemic sal butamol [11–13]. High cost of levalbuterol justi fies, however, its administration only in selected clinical conditions [13]. Abstract We showed the present data about the efficacy and safety of inhaled short-acting b2-agonists (SABA), such as salbutamol and fenoterol, in the management of obstructive diseases in children and adults. Our work discusses major mechanisms of action, clinical effects, possible side effects and indications of inhaled SABA. We presented current recommendations for the position of SABA in the therapy of obstructive diseases in children and adults, particularly in asthma and chronic obstructive pulmonary disease. Key words: short-acting b2-agonist, salbutamol, fenoterol, inhalation, nebulization, asthma, COPD Key words: short-acting b2-agonist, salbutamol, fenoterol, inhalation, nebulization, asthma, COPD Adv Respir Med. 2020; 88: 443–449 REVIEW REVIEW www.journals.viamedica.pl Address for correspondence: Andrzej Emeryk, Department of Pediatric Pulmonology and Rheumatology Medical University in Lublin, Lublin, Poland; e-mail: andrzejemeryk@plusnet.pl DOI: 10.5603/ARM.a2020.0132 Received: 08.02.2020 Copyright © 2020 PTChP ISSN 2451–4934 Salbutamol versus fenoterol Two inhaled drugs from SABA group are available in Poland: salbutamol and fenoterol. Table 1 shows their most important properties. Data in the table demonstrate that b2/b1 (se lectivity index) stimulation index is 10 times greater for salbutamol than fenoterol. Having in mind similar stimulation of b2 receptors by both drugs (0.55 salbutamol vs 0.60 fenoterol), it means that salbutamol exerts more selective b2-AR stimulation vs fenoterol and both cause similar bronchodilation. Studies from the 1990s 443 www.journals.viamedica.pl Advances in Respiratory Medicine 2020, vol. 88, no. 5, pages 443–449 Figure 1. Effect of single dose of salbutamol, formoterol, and salmeterol on FEV1 value in asthma patients [8]. FEV1 — forced expiratory volume in the first second Figure 1. Effect of single dose of salbutamol, formoterol, and salmeterol on FEV1 value in asthma patients [8]. FEV1 — forced expiratory volume in the first second Table 1. Comparison of short-acting b2-agonists’ (salbutamol and fenoterol vs isoproterenol) ability to stimulate b-adrener gic receptors ( b1, b2, b3) [14, 15] b1 inotropic activity (atrium) b2 dilatory activity (bron chi)* b3 lipolytic activity (adi pocytes) b2/b1 index Isoproterenol 1,0 1,0 1,0 1,0 Salbutamol 0,0004 0,55 0,002 1375 Fenoterol 0,005 0,6 0,02 120 Increase in FEV1 (forced expiratory volume in the first second) ≥ 15% from baseline arison of short-acting b2-agonists’ (salbutamol and fenoterol vs isoproterenol) ability to stimulate b-adrener ceptors ( b1, b2, b3) [14, 15] — reduction of capillary permeability (reduc tion of plasma exudate); — suppression of sensory nerves activation; — improvement of mucociliary clearance; — dilatation of pulmonary vascular bed (de crease in pO2); proved similar or better clinical efficacy and better safety of salbutamol in comparison with inhaled fenoterol in different age groups of asth ma patients [16, 17]. Better safety is also a result of the fact that salbutamol shows more features of b2-AR partial agonist than fenoterol. It deter mines that asthma patients using salbutamol have lower risk of death vs patients using feno terol [17, 18]. It should be stressed that adverse effects of fenoterol increase in hypoxemia, which occurs during severe and prolonged episode of bronchoconstriction [19]. — increased release of surfactant. They can depend on the polymorphism of gene encoding b2-AR located on chromosome 5q31-q32 [20, 21]. Some studies showed rela tionship between response to SABA, course of asthma and the polymorphism of gene encoding b2-AR [22]. Salbutamol versus fenoterol It is mainly about 2 polymorphisms: genotype Arg/Arg at codon 16 of gene encoding b2-AR and Gln/Gln at codon 27 of this gene. It was revealed that homozygotes for Arg/Arg at codon 16 of gene encoding b2-AR with chronic obstructive pulmonary disease (COPD) are pre disposed to more severe clinical course of the disease [23]. Similar relationship was shown in patients with cystic fibrosis with altered response to SABA [24]. Clinical effects of SABA Short-acting b2-agonists in conditions with bronchoconstriction exert many following clini cal effects [1, 15]: Short-acting b2-agonists in conditions with bronchoconstriction exert many following clini cal effects [1, 15]: — bronchodilation (removal of bronchial smooth muscles constriction); — prevention of bronchoconstriction induced by different bronchoconstrictive factors; 444 www.journals.viamedica.pl Andrzej Emeryk, Justyna Emeryk-Maksymiuk, Short-acting inhaled b2-agonists: why, whom, what, how? 38], including inhalers transmitting information to the health care system in real time [39]. Short-acting b2-agonists usage can be associ ated with many side effects described may years ago [25]: tachycardia, skeletal muscle tremor, hypokalaemia, increased level of lactic acid in plasma (lactic acidosis), headaches, hypergly caemia. Systemic side effects are observed rarely after inhalation administration and increased risk of cardiovascular side effects appear in patients with comorbid cardiovascular disease, especially in the elderly [26, 27]. It is worth mentioning that paradoxical bronchoconstriction after in halation of SABA occurs in 4.4% of the general population [28]. Salbutamol in pressurised metered-dose inhaler in comparison to other pharmaceutical forms of SABA Short-acting b2-agonists have different routes of administration (inhalation, oral and intrave nous), because they are available in different pharmaceutical forms. Many forms of salbutamol are available [40]: — inhalation aerosol (suspension) from pres surised metered-dose inhaler (pMDI) (chil dren and adults); There are additional possible side effects and adverse clinical effects when SABA are used in asthma. These effects occur in patients receiving SABA as monotherapy or/and if SABA are used very often or regularly without inhaled cortico steroids (ICS). It can lead to increased risk of the following adverse outcomes [29–35]: — powder from dry powder inhaler (DPI), types: Diskus (children over 4 years and adults), Turbuhaler (children over 3 years and adults) and Easyhaler and (children over 4 years, adults); — inhalation solution for nebulizers (children and adults); — decrease in the number and sensitivity of b2-AR; — sirup (children over 2 years, adults); — tablets (children over 2 years, adults); — diminished bronchial response to SABA or/ /and LABA; — solution for injection (adults). Inhalation is the most effective way of SABA therapy in airway diseases. Oral therapy can be alternative only exceptionally in small children, who do not accept inhalation or cannot inhale properly [38]. Additional parenteral therapy (salbutamol) is necessary rarely in patients with severe exacerbation of asthma, who do not re spond do proper inhalation therapy [41]. Clinical effects of SABA — increased bronchial hyperresponsiveness; — increased allergic reactions and eosinophilic airway inflammation; — increased risk for asthma exacerbation (with regular or frequent use: ≥ 3 SABA canisters/ /year, average 1.7 puffs/day); — increased risk of death in patients with asth ma (≥ 11 SABA canisters/year); Inhalation formulations of SABA most often used are listed in Table 2 [43]. Inhalation formulations of SABA most often used are listed in Table 2 [43]. — deterioration in spirometric parameters. Th f hi h k f According to table 2, four inhalation formu lations of salbutamol are available: pMDI, pres surised metered-dose inhaler — breath actuated pMDI (pMDI-BA), DPI and inhalation solution for nebulizers; fenoterol is available only as pMDI. The expected therapeutic clinical effects and probability of side effects can depend on the choice of SABA inhalation method. Below we present the most important rules of SABA inhalation therapy: These facts which are known for many years and other new clinical evidences for efficacy and safety of SMART therapy (Single Maintenance and Reliver Therapy) changed the perception of the role and place of SABA in the management of asthma in last Global Initiative for Asthma report (GINA) 2019 [36], which will be discussed further below. Another way of limiting the SABA overuse relies on monitoring use of SABA by patients preferably with electronic inhalers [37, Table 2. Inhalation formulations of SABA and SABA/ipratropium bromide combinations. Abbreviations according to [44] Type of inhaler pMDI pMDI-BA MDLI (respimat) DPI Nebulization Salbutamol + + – + + Fenoterol + – – – – Salbutamol + ipratropium bromide + – + – + Fenoterol + ipratropium bromide + – + – + pMDI — pressurised metered-dose inhaler; pMDI-BA — pressurised metered-dose inhaler — breath actuated); MDLI — metered dose liquid inhaler; DPI — dry powder inhaler Table 2. Inhalation formulations of SABA and SABA/ipratropium bromide combinations. Abbreviations according to [44] Type of inhaler pMDI pMDI-BA MDLI (respimat) DPI Nebulization Salbutamol + + – + + Fenoterol + – – – – Salbutamol + ipratropium bromide +* – + – + Fenoterol + ipratropium bromide + – + – + pMDI — pressurised metered-dose inhaler; pMDI-BA — pressurised metered-dose inhaler — breath actuated); MDLI — metered dose liquid inhaler; DPI — dry powder inhaler le 2. Inhalation formulations of SABA and SABA/ipratropium bromide combinations. Abbreviations accordin ulations of SABA and SABA/ipratropium bromide combinations. Clinical effects of SABA Abbreviations according to [44] 445 Advances in Respiratory Medicine 2020, vol. 88, no. 5, pages 443–449 Studies on administration of salbutamol from other DPI are ongoing [47]. 1. pMDI with properly fitted inhalation cham ber is preferred method of SABA inhalation in children below 6 years, irrespective of severity of asthma attack, place of adminis tration (home, admission ward, clinical ward, intensive care unit) [36, 45, 46]. 4. Nebulization with pneumatic nebulizer — both intermittent and continuous — should be used in case of insufficient response or lack of re sponse to SABA and life-threatening bronchoc onstriction [36, 48, 49]. In adults with severe exacerbation of asthma continuous nebulized salbutamol more effectively improves lung function than intermittent nebulization [50]. 2. Dose of salbutamol from pMDI depends main ly on the severity of asthma attack, not on patient age. According to GINA 2019 report during the first hour children below 6 years can receive up to 6 puffs, older children and adults can receive up to 12–30 puffs [36]. 5. 5. Clinical effects of salbutamol nebulization to a considerable degree depend on the type of nebulizer: breath actuated pneumatic nebu lizer provides better effect than continuous nebulization and mesh nebulizer in compar ison to pneumatic nebulizer [51, 52]. 3. Salbutamol can be inhaled from DPI (e.g: Diskus and Easyhaler) in children above 6 years and adult with asthma, which pro vides similar clinical efficacy to pMDI [36]. Table 3. Clinical effects of SABA Current indications for SABA in children and adults [36, 55–64]* Disease/state Indications Comment Asthma Attacks of dyspnoea, cough, and wheeze Disease exacerbations Prevention of exercise-induced bronchoconstriction Prevention of bronchoconstriction triggered by allergen exposure Alternative to SMART therapy in patients < 12 years Always with ICS — evidence A Obstructive bronchitis — so-called early childhood asthma Exacerbation of bronchoconstriction First-line treatment Number of doses should be adjusted to the patient’s clinical condition Stable COPD Initial treatment — dyspnoea attack or/and respiratory difficulties As-needed SABA — Only patients from group A — Reduction in symptoms and an increase in FEV1 — evidence A — Combination of SABA + SAMA is superior to SABA or SAMA alone (symptoms and FEV1) — evidence A COPD — exacerbation Acute exacerbation of disease SABA added to other medication Increase the dose or frequency of SABA or combine SABA and SAMA in the initial treatment of acute exacerbation — evidence C Bronchiolitis Selected cases with confirmed positive clinical response to treatment In most cases there are no indications to routine therapy Cystic fibrosis Pulmonary exacerbation with features of bronchoconstriction and confirmed positive clinical response or in patients with positive BDR test, before inhalation of hypertonic saline Rather commonly used, however ecommendations are not explicit Transient tachypnoea of the newborns Very poor evidences for the efficacy Chronic lung disease in premature babies Prevention and treatment Poor evidences for the efficacy Familial dysautonomia SABA + SAMA 1 study confirming SABA efficacy Other diseases with reversible bronchoconstriction Acute chest syndrome in sickle cell disease Further studies are needed Bronchodilator reversibility (BDR) test Spirometric features of bronchoconstriction (FEV1%FVC < 80 % predicted value, FEV1%VC < 80% predicted value, PEF < 80% predicted value) 2–4 puffs of salbutamol pMDI + inhalation chamber as the standard of BDR test One shouldn’t be afraid of administration of SABA (salbutamol) in the elderly (> 90 years) [65] FEV1 — forced expiratory volume in the first second; ICS — inhaled corticosteroids; COPD — chronic obstructive pulmonary disease; SABA — short-acting b2-agonists; SAMA — short-acting muscarinic antagonists; SMART — Single Table 3. Clinical effects of SABA Current indications for SABA in children and adults [36, 55–64] — Only patients from group A — Reduction in symptoms and an increase in FEV1 — evidence A — Combination of SABA + SAMA is superior to SABA or SAMA alone (symptoms and FEV1) — evidence A One shouldn’t be afraid of administration of SABA (salbutamol) in the elderly (> 90 years) [65] FEV1 — forced expiratory volume in the first second; ICS — inhaled corticosteroids; COPD — chronic obstructive pulmonary disease; SABA — short-acting b2-agonists; SAMA — short-acting muscarinic antagonists; SMART — Single Maintenance and Reliver Therapy 446 www.journals.viamedica.pl www.journals.viamedica.pl Andrzej Emeryk, Justyna Emeryk-Maksymiuk, Short-acting inhaled b2-agonists: why, whom, what, how? Table 4. Initial emergency (as-needed) pharmacotherapy of asthma according to GINA 2019 report [36] Age group Preferred management Alternative management Comments Patients ≥ 12 years Low dose ICS-formoterol — Step 1–5 treatment SABA pMDI, pMDI-BA SABA pMDI + IC SABA DPI— Step 1–5 treatment ICS: budesonide or beclometasone Patients 6–11 years SABA DPI SABA pMDI + IC SABA DPI — Step 1–5 treatment Low dose ICS-formoterol — Step 3–5 treatment *Children receiving ICS-formoterol combination as maintenance Patients 5 years and younger SABA “as-needed” pMDI + IC — Step 1–4 treatment SABA “as-needed” by nebulizer — Step 1–4 treatment Proper use of the equipment and estimation of appropriate dose of the drug are required DPI — dry powder inhaler; ICS — inhaled corticosteroids; IC — inhalation chamber; pMDI — pressurised metered-dose inhaler; SABA — short-acting b2-agonists Table 4. Initial emergency (as-needed) pharmacotherapy of asthma according to GINA 2019 report [36] 6. Parenteral, oral, or nebulized SABA are as sociated with increased risk of side effects (tachycardia, muscle tremor, headaches, hy pokalaemia). In this respect inhalation from pMDI is the safest method [53, 54]. 6. Parenteral, oral, or nebulized SABA are as sociated with increased risk of side effects (tachycardia, muscle tremor, headaches, hy pokalaemia). In this respect inhalation from pMDI is the safest method [53, 54]. DPI remains alternative option (worse regarding the efficacy and safety). In children 6–11 years preferred emergency management is administra tion of SABA from pMDI + inhalation chamber in combination with ICS (any medication) or oral corticosteroid [36]. Administration of SABA from pMDI + inhalation chamber: 4–10 puffs for every 20 minutes for the first hour of symp toms. References: 1. Tattersfield A.E. Current issues with beta2-adrenoceptor ago nists: historical background. Clin Rev Allergy Immunol. 2006; 31(2–3): 107–118, doi: 10.1385/CRIAI:31:2:107, indexed in Pubmed: 17085787. 1. Tattersfield A.E. Current issues with beta2-adrenoceptor ago nists: historical background. Clin Rev Allergy Immunol. 2006; 31(2–3): 107–118, doi: 10.1385/CRIAI:31:2:107, indexed in Pubmed: 17085787. Clinical effects of SABA Budesonide in combination with formoterol in SMART therapy model is alternative option for some children [36]. In group of children up to 5 years the only option of emergency treatment is SABA „as-needed” — from pMDI + inhalation chamber (preferred management) or in nebuli zation (alternative management) in all asthma steps [36]. 7. Short-acting b2-agonist (alternatively in com bination with ipratropium bromide) in pMDI + IC or in nebulization is the first-line initial treatment of acute exacerbation of COPD [55]. 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Cochrane Database Syst Rev. 2014(6): CD001266, doi: 10.1002/14651858.CD001266.pub4, indexed in Pubmed: 24937099. 50. Rodrigo G. Asthma in adults (acute). BMJ Clin Evid. 2011; 4: 1513. 51. Lin Y.Z., Huang F.Y. Comparison of breath-actuated and con ventional constant-flow jet nebulizers in treating acute asth matic children. Acta Paediatr Taiwan. 2004; 45(2): 73–76, in dexed in Pubmed: 15335114. 61. Dunn M, Muthu N, Burlingame CC, et al. Reducing albuterol use in children with bronchiolitis. Pediatrics. 2020; 145(1), doi: 10.1542/peds.2019-0306, indexed in Pubmed: 31810996. 62. Bar-Aluma BE, Efrati O, Kaufmann H, et al. A controlled trial of inhaled bronchodilators in familial dysautonomia. Lung. 2018; 196(1): 93–101, doi: 10.1007/s00408-017-0073-7, indexed in Pubmed: 29234869.i 52. Lipworth B.J., Sims E.J., Taylor K. i wsp. Dose-response to salbutamol via a novel palm sized nebuliser (Aerodose Inhal er), conventional nebuliser (Pari LC Plus) and metered dose inhaler (Ventolin Evohaler) in moderate to severe asthmatics. Br J Clin Pharmacol. 2005; 59(1): 5–13, doi: 10.1111/j.1365- 2125.2005.02168.x, indexed in Pubmed: 15606434. 63. Barry PJ, Flume PA. Bronchodilators in cystic fibrosis: a crit ical analysis. Expert Rev Respir Med. 2017; 11(1): 13–20, doi: 10.1080/17476348.2017.1246358, indexed in Pubmed: 27718752. 53. Ducharme F.M., Dell S.D., Radhakrishnan D. i wsp. Diagnosis and management of asthma in preschoolers: A Canadian Tho racic Society and Canadian Paediatric Society position paper. Can Respir J. 2015; 22(3): 135–143, doi: 10.1155/2015/101572, indexed in Pubmed: 25893310. 64. Knight-Madden J, Hambleton I. Inhaled bronchodilators for acute chest syndrome in people with sickle cell dis ease. Cochrane Database of Systematic Reviews. 2016, doi: 10.1002/14651858.cd003733.pub4. 54. Hartman S., Merkus P., Maseland M. i wsp. Hypokalaemia in children with asthma treated with nebulised salbutamol. Arch Dis Child. 2015; 100(10): 970–972, doi: 10.1136/archdis child-2015-308427, indexed in Pubmed: 26044135. 65. Melo SM, Oliveira LA, Rocha RD, et al. Bronchodilator test in extreme old age: Adverse effects of short-acting beta-2 adren ergic agonists with clinical repercussion and bronchodilator response. Rev Assoc Med Bras (1992). 2019; 65(11): 1343– 1348, doi: 10.1590/1806-9282.65.11.1343, indexed in Pubmed: 31800894. child-2015-308427, indexed in Pubmed: 26044135 55. Global Strategy for the Diagnosis, Management, andPreven tion of Chronic Obstructive Pulmonary Disease 2020 Report. 449 www.journals.viamedica.pl
https://openalex.org/W4361241664	https://figshare.com/articles/journal_contribution/Supplementary_Figure_1_from_Effects_of_Tetramethoxystilbene_on_Hormone-Resistant_Breast_Cancer_Cells_Biological_and_Biochemical_Mechanisms_of_Action/22368083/1/files/39813155.pdf	Malayalam	null	Supplementary Figure 3 from Effects of Tetramethoxystilbene on Hormone-Resistant Breast Cancer Cells: Biological and Biochemical Mechanisms of Action	null	2,023	cc-by	13	ure 1 DMSO 3 µM TMS ure 1 DMSO 3 µM TMS C
https://openalex.org/W2613767867	https://acquire.cqu.edu.au/ndownloader/files/25838843	English	null	From Phoenix Wright to Atticus Finch	null	2,017	cc-by	3,820	1. INTRODUCTION Play is a compelling form of learning, particularly where it allows exploration of an otherwise high risk environment, whether it be lion cubs play fighting or in simulations of a high pressure courtroom environment1. Soaring legal costs and cutbacks to legal aid mean that large percentages of clients are forced to represent themselves in court. The rates of self-representation vary by jurisdiction, type of case and year2 but are consistently increasing, especially for middle-income earners3 who are unable to obtain legal aid. This paper investigates the feasibility of a using a courtroom learning sim to teach these self-represented litigants basic advocacy skills and by doing so facilitate access to justice. The problems associated with self-represented persons are well known and include:  Lack of legal skill - ignorance of legal issues, court procedures, forms and legal language;  Lack of objectivity and emotional distance from their case.9  Impact on court staff – following irrelevant precedents, incorrect or incomplete documents, filing irrelevant materials, ignorance of procedural issues and penalties, consuming library time and resources, misunderstanding procedural advice, frustration and abuse. While many learning games focus on memorising and testing recall of facts, interactive media has the potential to develop more complex capabilities through the exploration of meaningful choices and the consequences that flow from them. Whilst self- represented litigants (and law students) might benefit from a better understanding of detailed court process and etiquette, the real focus of concern is more a broader range of skills such as structuring an argument, choosing between relevant and irrelevant issues, and connecting evidence to assertions; skills that interactive media can cultivate.4 Further, simulations can build confidence in the use of courtroom language and cultivate cultural capital around presenting an authentic voice that anchors learning in emotionally relevant experience.5 p ,  Impact on the judiciary - disruption to the usual flow of court proceedings. Information collected in Australian Federal courts suggests representation is relevant to outcome, negotiated settlements are more likely with representation and the failure rate for self- presented parties is high.10 This impacts on the justice system in several ways. Immediately there is an issue of access to justice for the self-represented individuals. Beyond this there are other costs that spill over, these clients require more time to present their case than a trained professional which drive up the costs of the other parties and of administering the system generally. From Phoenix Wright to Atticus Finch: Legal Simulation Games as an Aid to Self-represented Litigants Scott Beattie CQ University 120 Spencer St, Melbourne Victoria, Australia +61 3 8662 0584 s.beattie@cqu.edu.au Stephen Colbran CQ University 160 Ann St, Brisbane Queensland, Australia +61 409 305 235 stephen.colbran@gmail.com Stephen Colbran CQ University 160 Ann St, Brisbane Queensland, Australia +61 409 305 235 stephen.colbran@gmail.c Stephen Colbran CQ University 160 Ann St, Brisbane Queensland, Australia +61 409 305 235 stephen.colbran@gmail.com Keywords y Law; simulation; self-represented litigants; advocacy 2. THE PROBLEMS FACED BY SELF- REPRESENTED LITIGANTS This paper examines the feasibility of a courtroom simulation being used to assist self-represented litigants in learning basic advocacy skills. A review of legal and forensic mechanics in games, particularly the Phoenix Wright games, provides design roadmap for a courtroom learning simulation. Around the world, litigants often represent themselves for various reasons including the high cost of legal services, cutbacks to legal aid or denial of service, individual choice, that no legal representative is willing to act and jurisdictions which discourage or prevent legal representation.6 The extent of the problem is considerable. In 2015-2016, the Australian High Court reports rates of around 46% of self-represented clients and 76% self- representation in immigration cases.7 In British Columbia, the Court of Appeal in 2015 faced 27% self-representative litigants with the percentage rising to 57% in Family law appeals.8 © 2017 International World Wide Web Conference Committee (IW3C2), published under Creative Commons CC BY 4.0 License. WWW 2017 Companion, April 3-7, 2017, Perth, Australia. ACM 978-1-4503-4914-7/17/04. DOI: http://dx.doi.org/10.1145/3041021.3054171 3.1 3.1 The Phoenix Wright Games (Nintendo, 2001-2016)15 3.1 3.1 The Phoenix Wright Games (Nintendo, 2001-2016)15 The most extensive implementation of legal-themed gameplay is the Phoenix Wright series of games on the Nintendo handheld game systems. Similar in format to the Japanese Visual Novel adventure game genre, the storytelling and characters of these games have a generated a strong international fanbase, despite being technically quite simple with limited animation and rigidly structured player choices. Learning games have been used to provide coaching in some legal arenas, such as Party for Your Rights, an online game created to teach consumer rights and advocacy (http://partyforyourrights.vic.gov.au) or the Law Dojo (http://www.lawschooldojo.com) a set of online games which tests knowledge of the law aimed primarily at law students. While these projects have positive outcomes regarding transfer of knowledge, they are, at their foundation, well presented quizzes and provide little scope for the interaction and experimentation by which play becomes deep learning. They also have limited audience scope because each is bound to jurisdiction-specific detail of laws and regulatory provisions. Learning games have been used to provide coaching in some legal arenas, such as Party for Your Rights, an online game created to teach consumer rights and advocacy (http://partyforyourrights.vic.gov.au) or the Law Dojo (http://www.lawschooldojo.com) a set of online games which tests knowledge of the law aimed primarily at law students. While these projects have positive outcomes regarding transfer of knowledge, they are, at their foundation, well presented quizzes and provide little scope for the interaction and experimentation by which play becomes deep learning. They also have limited audience scope because each is bound to jurisdiction-specific detail of laws and regulatory provisions. The Phoenix Wright games take place in an abstracted version of the Western court room, one where drama and wild storytelling are more important than accurate simulation. Lawyers are involved in criminal investigation, care little for the rules of evidence and sometimes employ psychic powers. Nevertheless, the core of the gameplay is useful in that it involves teasing out fact and relevant data from the mass of contradictory and misleading evidence. Some client interviewing is involved, but the game focuses on physical evidence in a way that is consistent with fictional crime dramas, but less useful in the simulation of everyday court process where testimony and credibility are far more important. One of the strengths of play is the ability to facilitate experimentation and exploration within a safe environment. 1. INTRODUCTION Self-represented litigants need to focus primarily on presentation of facts, not on understanding the law. An understanding of legal documents and regulatory instruments is important, but knowledge of precedent and statutory interpretation is seldom important at first instance and in any case legally complex cases are generally going to be the domain of legal specialists in the appellate courts. 425 feedback on choices made. Gamist exploration could incentivise play by scoring and providing feedback on recommended strategies. Narrative exploration could engage learners by solving mysteries and understanding the twists and turns of a particular case. Exploration through simulation builds confidence and allows the player to take on the identity of a professional and cultivate cultural capital via a quite literal ‘feel for the game’.14 Self-represented litigants therefore do not to be trained as lawyers, but in the manner of lay advocates with a focus on arranging an argument, understanding the way in which regulatory documents work, filtering relevant from irrelevant information, placing emphasis and providing evidence for arguments. While these concepts can be described in explanatory documents, the practical aspects can only be understood through actual application, preferably in a low risk hypothetical simulation, something which has a long history even in pre-digital legal education.11 While self-represented litigants may, or may not elect to undertake more formal training in advocacy skills, a learning game that simulates these processes might provide a more engaging and practical way to explore the issues and build confidence. A simulation of this kind could service a number of audiences in addition to the self- represented litigant including law students and might also be of use as microgaming activities in broader training contexts.12 3. COMPARATIVE ANALYSIS OF GAMES THEMED AROUND LEGAL AND FORENSIC PROCESSES Inspiration for this project can be derived from games which have dealt with legal process, particularly the aspect of detection and mystery solving. Entertainment games are not constrained by fidelity to jurisdictional and procedural detail, but nevertheless provide useful examples of implementation of legal/forensic problem solving through game mechanics. Educational simulations can sometimes place too much focus on simulationist aspects, in providing an authentic verisimilitude, at the cost of engaging players through game elements and storytelling. In traditional law moots, for example, too much emphasis might be placed on details of court procedure at the expense of deeper understanding of how legal arguments are constructed as narratives. It is important that each of these aspects find the best balance and it is relevant to consider design ideas in entertainment games too understand what drives player engagement. A self represented litigant should feel some motivation to play irrespective of the educational benefits if they are to be properly engaged. The key aptitudes that such a learning game might foster could include:  Understanding how to apply generalised rules and regulations to specific fact circumstances.  Judging the relevance of different situational facts or arguments.  Framing an argument, selecting key points, sequencing these in a logical manner and providing appropriate emphasis to each aspect.  Connecting facts and claims to credible evidence.  Basic understanding of procedures and etiquette, not tied to a particular jurisdiction but providing appropriate transferable knowledge.  Confidence in presenting an argument and the resilience to listen to criticism or counter-argument and then respond appropriately. 3.5 Until Dawn (Supermassive Games, 2015) and the Telltale Games Series 3.5 Until Dawn (Supermassive Games, 2015) and the Telltale Games Series 3.2 LA Noire (Rockstar Games, 2011) While a high budget, ‘triple A’ game such as LA Noire occupies the opposite end of the technical spectrum from the Phoenix Wright games, it has at is core a similar game mechanics in separating truth from lies and finding the correct evidence to apply for each assertion. LA Noire is a forensic police drama rather than a legal drama and takes place in a 1940’s Los Angeles which is heavily inspired by the fiction of James Ellroy. 3.2 LA Noire (Rockstar Games, 2011) ( , ) While a high budget, ‘triple A’ game such as LA Noire occupies the opposite end of the technical spectrum from the Phoenix Wright games, it has at is core a similar game mechanics in separating truth from lies and finding the correct evidence to apply for each assertion. LA Noire is a forensic police drama rather than a legal drama and takes place in a 1940’s Los Angeles which is heavily inspired by the fiction of James Ellroy. These games are worth briefly mentioning as contemporary examples of branched storytelling games. Until Dawn takes inspiration from slasher horror films. The Telltale Games cross a number of genres including well received adaptations of A Game of Thrones and The Walking Dead. Each of these games explore, to different degrees, multiple pathways through a narrative where different choices lead to different consequences. While many of the choices available may have limited effect on the overall narrative flow, these games establish the importance of exploration and the incorporation of choice in branching plotlines. The Telltale Games have a useful online feature where players can compare their choices to the statistically aggregated choices of other players, something which might be used as a reflective point in a law simulation, especially if this can be leveraged for further feedback on advocacy skills. As a high profile console game it contains gameplay elements which are beyond the scope of a straightforward learning sim including elaborate 3D modeling of the city, driving mechanics and combat systems. The most pertinent elements of design involve the interrogation of suspects and the process by which truth can be ascertained through threaded dialogue. 3.5 Until Dawn (Supermassive Games, 2015) and the Telltale Games Series The game boasts impressive facial motion capture technology and use of recognisable film and television actors to augment the detection process but in actuality it is the comparison of testimony to other evidence in the detective’s notebook the provides the most fruitful field of play. This approach might usefully applied to the less- adversarial lawyer-client interview using mechanics that explore different versions of truth, perception filters and faulty witness memory. LA Noire also provides examples of how bad player decisions might be modeled and explored, indeed at one stage the game requires the player to make a mistake in a way that advances the plot (and references the Ellroy source material). 3.1 3.1 The Phoenix Wright Games (Nintendo, 2001-2016)15 Ron Edwards ‘GNS Theory’ of play holds that games allow exploration of three connected, sometimes contradictory domains: Gamism (inter-player competition within a rules framework), Narrativism (storytelling) and Simulationism (providing a credible simulation of a particular environment.13 Many learning games do not foster exploration as they focus on quizzes and recall of facts on the foundation that there is a single correct answer. Interactive media has a greater potential to explore more ambiguous situations where there are multiple solutions to a problem. Any law based game must manage the Phoenix Wright legacy, by building on the game series’ successes, but also in marking itself out as different and distinctive in ways that are crucial to success as a learning tool. The game structure and economical use of animation indicate a useful way in which court process can be transformed into an interactive model, but closer attention must be paid to core skills in structuring argument, applying rules and filtering information for relevance to the case. Courtroom skills and advocacy are seldom a matter of a single correct approach and learning via play might allow novice advocates to experiment with different strategies and obtain 426 4. APPLICATION OF GAME DESIGN CONCEPTS TO A LEGAL SIMULATION A review of these very different entertainment game products provides a range of game design tools that can be used to create a compelling, but also educational legal simulation that emphasises the importance of foundational advocacy skills. Such a game would be best delivered through a web interface and via mobile devices on order to reach the broadest audience. Use of an online delivery mode would allow the incorporation of weblinks for additional resources and help. As a service to the community the game would be provided for free (with perhaps some appropriate sponsorship) and should be designed in a way that is independent of any specific jurisdiction and appropriate to a wide audience. Any laws, regulatory provisions, documents and rules would be fictional and designed specifically for the game’s generic setting. This would also mean that the game would not be mistaken for concrete legal advice specific to any particular jurisdiction. 3.3 Her Story (Sam Barlow, 2015) y ( , ) In stark contrast to the high production values of LA Noire, Her Story is an indie game which uses a simple game interface to explore a complex narrative connected by videos of a police interview with an accused suspect. By piecing together these interview fragments, which take place at different times in the game’s chronology, players construct the overall narrative in a way which is compelling and often surprising. The authoring design interface for such a game would also be crucial as this would allow a broad range of contributors, including authors and legal specialists, without the need for programming knowledge.16 In projects such as this there is a need to draw on writers who can craft compelling and interesting stories that drive players toward resolution, rather than simply modeling what occurs in the courtroom. A diversity of authors would also allow for stories with distinct cultural voices, something which may be important for maximising impact on communities who lack access to the law and individuals of lack confidence in dealing with the justice system. Unlike the Phoenix Wright games, the game should also maintain a fidelity toward legal advocacy techniques and authenticity in documentary artefact design,17 but should not lose sight of the importance of player engagement. Her Story uses actual film video of an actor providing testimony which creates strong verisimilitude, but which might not be practical in a simulation that seeks to focus on the construction and presentation of courtroom argument. The game is noteworthy in its use of individual perspective, unreliable storytelling and the simple database interface through which the player explores the narrative. The mechanics of this game might not be directly transferrable to a legal simulation, but the overall approach to narrative and the complex understanding of testimony and memory indicate allows an exploration of the nature of evidence beyond simple binaries of true and false. 3.4 Papers, Please (Lucas Pope, 2013) 3.4 Papers, Please (Lucas Pope, 2013) Described as “a Dystopian Document Thriller”, this indie game simulates bureaucratic border crossing with the aim of illustrating the plight of displaced persons. The game uses documents and bureaucratic paperwork as the means by which narrative is constructed and truth is determined. Papers, Please illustrates the role of bureaucracy in supporting totalitarian government, but its design approach might also be useful for understanding the role of documentary evidence in more liberal legal environments. The focus on documentation is useful in conveying its central role in legal process and the importance of creating a clear narrative and argument through written documents. The Phoenix Wright games establish a strong precedent for the use of strong character design, but minimal animation, so that the costs of a legal simulation could be managed, especially via the reuse of core game assets. Rather than focus on development of complex graphic systems, the focus of the game would be on the exploration of choice and the consequences of choice. Feedback on these choices could be provided in a mimetic way (via case success or failure, comments from the judge) or through direct feedback to the players themselves on their advocacy choices. The primary site of exploration would be in the construction of an argument. Prioritisation of different argument elements would be 427 [5] DeCaporale-Ryan, L. N., Dadiz, R. and Peyre, S. E. 2016. Simulation-Based Learning: From Theory to Practice, Families. Systems & Health, 34, 2 (April 2016), 159-162. a fundamental aspect of this exploration. Novice advocates often use a ‘shotgun approach’ to courtroom argument, something which is time consuming for the court and likely to distract from the strongest elements of their argument. By reconfiguring legal argument within a game structure a simulation might emphasise the importance of weighing different alternatives and making strong choices. Individual cases should be re-playable so that player might test out different approaches and enhance their overall success ratings. [6] Australian Institute of Judicial Administration. 2001. Litigants in Person Management Plans: Issues for Courts and Tribunals. 2-3. [7] High Court of Australia. 2016. Annual Report 2015-2016. [8] Court of Appeal for British Columbia. 2015. Annual Report 2015. Additional game mechanics might also be used to enhance player engagement. 3.4 Papers, Please (Lucas Pope, 2013) [15] The English editions of the Phoenix Wright games include Phoenix Wright: Ace Attorney (2001), Phoenix Wright: Ace Attorney: Justice for All (2002), Phoenix Wright: Ace Attorney: Trials and Tribulations (2004), Apollo Justice: Ace y ( ) p Attorney (2007), Phoenix Wright: Ace Attorney – Dual Destinies (2013) and Phoenix Wright: Ace Attorney – Spirit of Justice (2016). 3.4 Papers, Please (Lucas Pope, 2013) The ability to customise a persona, earn achievements/badges and to carry forward success from one case to another would provide hooks for ongoing participation.18 These mechanisms could also be implemented in a way that is simple and cost effective and ties player identity to a Google or Facebook login. Creating a framework through which different cases can be delivered would also facilitate ongoing development of complexity in advocacy and problem solving skills.19 [9] McInnes v R (1979) 143 CLR 575, 590 per Murphy J. [10] Australian Law Reform Commission. 2000. Managing Justice: A Review of the Federal Civil Justice System. 12.218, 12, 221. [11] Hollander, P. A. 1977. The Uses of Simulation in Teaching Law and Lawyering Skills. Simulations and Games 8, 3 (September 1977), 319-340. [12] Lukosch, H., Kurapati, S., Groen, D., and Verbraeck, A. 2016. Microgames for Situation Learning: A Case Study in Independent Planning. Simulation & Gaming, 47, 3 (June 2016), 346-367. Games allow players to experience high-risk situations and may therefore familiarise the unfamiliar. While this proposition is open to debate in the field of combat simulations, a court simulation may be a way of exposing lay people to the language, processes and setting of the courtroom in a way that enhances advocacy skills rather than simply as a vehicle for drama. While rhythm games such as Guitar Hero (Activision 2005) or Rock Band (Harmonix Music System 2007) may not train actual musical skills, these can provide Bourdieu’s ‘feel for the game’, in a way that cultivates cultural capital and lay the foundation for teaching simulations such as Rocksmith (Ubisoft 2011) or actual music lessons. Likewise, by making the player feel like a lawyer a courtroom simulation might establish basic advocacy skills, but also become the vehicle for further formal or informal learning. [13] Edwards, R. 2003. GNS and other matters of Role-Playing Theory, The Forge (2003), http://www.indie- rpgs.com/articles/1. [14] Bourdieu, P. 1992. The Logic of Practice, Stanford University Press, 66. [15] The English editions of the Phoenix Wright games include Phoenix Wright: Ace Attorney (2001), Phoenix Wright: Ace Attorney: Justice for All (2002), Phoenix Wright: Ace Attorney: Trials and Tribulations (2004), Apollo Justice: Ace Attorney (2007), Phoenix Wright: Ace Attorney – Dual Destinies (2013) and Phoenix Wright: Ace Attorney – Spirit of Justice (2016). 5. REFERENCES [1] Huizinga, J. 1950. Homo Ludens: A Study of the Play Element in Culture, The Beacon Press, Boston, MA. 76-88. [16] Klemke, R., van Rosmalen, P., Ternier, S. and Westera, W. 2015. Keep It Simple: Lowering the Barrier for Authoring Serious Games. Simulation & Gaming, 46, 1 (February 2015), 40-67. [2] Richardson et al, 2012. Self-Represented Litigants, Gathering Useful Information , Final Report June 2012 Appendix E, Macfarlane, J. 2013. The National Self-Represented Litigants Project: Identifying and Meeting the Needs of Self- Represented Litigants, Final report, 2013. [17] Dixon, R. J. 2002. Toward greater authenticity: A case for divergent simulations. Simulation & Gaming, 33, 3 (September 2002), 360-66. [3] Australian Government Productivity Commission. 2014. Access to Justice Arrangements. Productivity Commission Inquiry Report. No 72 (September 2014). [18] McDaniel, R. and Fanferelli, J. 2016. Building Better Digital Badges: Pairing Completion Logic with Psychological Factors. Simulation & Gaming, 47, 1 (February 2016), 732- 102. [4] Prensky, M. 2001. Digital Game-Based Learning. Paragon House, St Paul, MN, 157. [19] Wardaszko, M. 2016. Building Simulation Game-Based teaching Program for Secondary School Students. Simulation & Gaming, 47, 3 (June, 2016), 287-303. 428
W4298073649.txt	https://zenodo.org/records/7127848/files/2022_Masterarbeit_Burdukat_74115_19SAM_HTWK_Leipzig.pdf	de		Herrschaft vs. Emanzipation: Raubt die Theorielosigkeit von Jugendarbeit, im Kontext Sozialer Arbeit, ihr die Kraft?	Zenodo (CERN European Organization for Nuclear Research)	2,022	cc-by	38,334	Hochschule für Technik, Wirtschaft und Kultur Leipzig Fachbereich für Sozialwissenschaften Fakultät Architektur und Sozialwissenschaften Masterarbeit Herrschaft vs. Emanzipation Raubt die Theorielosigkeit von Jugendarbeit, im Kontext Sozialer Arbeit, ihr die Kraft? Tobias Burdukat Matrikelnr. 74115 Erstgutachter Prof. Dr. phil. Friedemann Affolderbach Zweitgutachterin Prof. Dr. phil. Heike Förster 2. März 2022 „Ganz grob gesehen besteht mein Leben aus zwei Teilen. Aus meiner Kindheit und dem Rest.“ Rocko Schamoni (* 1966) Musiker, Schriftsteller, Schauspieler Prolog Zum Ende des Jahres 2018 eskalierte eine Auseinandersetzung zwischen dem Jugendamt des Landkreises Leipzig und dem Förderverein für Jugendkultur und Zwischenmenschlichkeit e.V. (kurz: FJZ e.V.). Dabei drehte es sich um die Anerkennung als Freier Träger der Jugendhilfe nach § 75 SGB VIII. Der von mir und guten Freundinnen im Jahr 2006 gegründete Verein veranstaltete über viele Jahre hinweg Projekte der Jugendarbeit, Konzerte und Festivals in Grimma und trug dadurch zur Belebung und der Gestaltung von jugend(sub)kulturellen Angeboten im ländlichen Raum bei. Zum damaligen Zeitpunkt war der FJZ e.V. Träger des Projektes Dorf der Jugend (vgl. Burdukat 2021) und versuchte durch die Anerkennung in eine reguläre institutionelle Förderung, durch das Jugendamt, für die Offene Jugendarbeit nach § 11 SGB VIII zu rutschen. Schlussendlich wurde die Anerkennung damals nach einer enormen Öffentlichkeitsarbeit1 und einer rechtlichen Auseinandersetzung ausgesprochen und der Förderung somit vorläufig stattgegeben. Zum 31.12.2021 schaffte es dann das Jugendamt, unter dem Vorwand der fehlenden Haushaltsmittel, die Förderung nicht mehr weiter zu bewilligen und somit existiert aktuell keine professionelle Jugendarbeit mehr, welche im Rahmen der Konzeption Dorf der Jugend durchgeführt wird. Im Rahmen dieser Auseinandersetzungen mit dem Jugendamt, den politischen Vertreterinnen und den großen Wohlfahrtsverbänden wurde deutlich, dass es nicht ausreichend ist, sich Gedanken über eine funktionierende Konzeption der Jugendarbeit zu machen und diese dann praktisch und erfolgreich durchzuführen. Vielmehr zeigte die Auseinandersetzung, dass die Praxis engagierte, selbstständige und vor allem selbstbewusste junge Menschen zusammen gebracht hat, denen das Projekt und die damit verbundenen Möglichkeiten zur Gestaltung von Gesellschaft und die Kommunikation von gesellschaftlichen Konflikten ein eigenes inneres Bedürfnis waren. Für mich persönlich wurde damals eine Grenze sichtbar, die ich in ihrer derartigen Form bisher unterschätzt hatte. Denn vielmehr ging es nicht darum, die Jugendarbeit nicht zu finanzieren, sondern es ging darum zu verhindern, dass kritische junge Menschen sich in die festgefahrenen und etablierten ländlichen Strukturen der Erwachsenen einbringen und offen und intensiv kommunizieren, was sie an diesen konservativen Strukturen stört. Besonders in ländlichen Regionen sind Jugendliche nicht erwünscht und werden als störend wahrgenommen, ob es nun die zu laute Musik einer Party oder die hörbaren Räder von Skateboards sind, Erwachsene finden immer einen Grund, um ihre Ablehnung gegenüber jungen Menschen auszudrücken und ihnen zu signalisieren, dass sie nicht einverstanden sind mit der Art und Weise, wie junge Menschen ihre Freizeit gestalten. Durch die aufgebrochenen Konflikte war es unumgänglich, nach Möglichkeiten zu suchen, wie die Arbeit mit Jugendlichen gestaltet werden kann, ohne sich dabei permanent der Norm von Erwachsenen und der Gesellschaft zu unterwerfen. 1 Nähere Informationen und Pressemeldungen unter: https://dorfderjugend.de/saveyourhinterland/ verfügbar am 02.03.2022 Dadurch musste natürlich die Frage gestellt werden, welche Rolle Jugendarbeit dabei spielt und wie Jugendarbeit dazu beitragen kann, dass Gesellschaft sich wandelt und wie es möglich wird, das junge Menschen diesen Wandel gestalten können. Ich bin sehr dankbar für den Rat von Prof. Dr. phil. Stephan Beetz von der Hochschule Mittweida, mich diesen Fragen im Rahmen eines späten Masterstudiums anzunehmen und im Rahmen des Studiums an der HTWK Leipzig diese Fragestellungen vertieft zu haben. Ohne eine finanzielle Unterstützung durch die Rosa Luxemburg Stiftung wäre es durch die während des Studiums einsetzende Corona Pandemie nicht möglich gewesen, diese Arbeit zu schreiben, weshalb ich hier meine besondere Dankbarkeit für das Stipendium ausdrücken möchte. Ich selbst komme nicht aus einer akademischen Familie und das wissenschaftliche Arbeiten fällt mir schwer, jedoch hat meine Familie mich in all den Jahren, ob nun während des Bachelorstudiums oder jetzt mit dem Masterstudium immer unterstützt und dabei die nie enden wollenden prekären Lebensverhältnisse, in denen ich mich bewege, toleriert. Sie haben mir ermöglicht jung zu sein um heraus zu finden wer ich bin. Ich frag mich nicht mehr wer ich bin Ich frag mich nur wer will ich sein Eins werd ich ganz sicher nie Ich werd nie so sein wie die Tex Brasket - Straßenmusiker und Sänger von SLIME Ich selbst möchte nicht so sein wie der überwiegende Teil unserer Erwachsenen Gesellschaft und ich möchte und kann kein Teil mehr von dieser sein. Ich bin deshalb unheimlich dankbar dafür, dass ich heute noch Teil einer (Sub)Kultur sein kann und dass sich viele junge Menschen, ausgehend vom Projekt Dorf der Jugend in Grimma, zusammen gefunden haben, die Gesellschaft zu verändern und die Hegemonie der Erwachsenen durchbrechen zu wollen und sich nicht dem Zwang der gesellschaftlichen Norm unterwerfen. Dank euch kann ich immer wieder sagen: ...another world is possible!!! Herrschaft vs. Emanzipation Tabellenverzeichnis 1 In der Jugendarbeit tätige Personen . . . . . . . . . . . . . . . . . . . . . . . . . 19 2 Auswertung der Jugendlichen 14 - 18 Jahre pro Jugendarbeiterin . . . . . . . . 20 3 Jugend - Typen der Disziplinen . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38 4 Auswertung Häufigkeit der Kategorien . . . . . . . . . . . . . . . . . . . . . . . . 45 5 Auswertung Matching der Kategorien . . . . . . . . . . . . . . . . . . . . . . . . 47 6 Auswertung der Kategorien mit dem jeweiligen Stabilisierungsattribut . . . . . . 69 7 Sinnzusammenhang Emanzipationshierarchie . . . . . . . . . . . . . . . . . . . . 72 8 Sinnzusammenhang Herrschaftshierarchie . . . . . . . . . . . . . . . . . . . . . . 72 Abbildungsverzeichnis 1 Graph »Jugend« gesamt . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44 2 Graph Kategorie Alter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45 3 Bedeutung Disziplinen für Kategorien . . . . . . . . . . . . . . . . . . . . . . . . 46 4 Verteilung Disziplin Alter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48 5 Wissenschaftlerinnen mit Emanzipationsperspektive . . . . . . . . . . . . . . . . 83 6 Graph Verhalten - Handlungsfähigkeit - Kultur - Sozialisation 84 . . . . . . . . . . 1 Herrschaft vs. Emanzipation Inhaltsverzeichnis 1 Einleitung 4 2 Der grundlegende Widerspruch 9 2.1 Herrschaft vs. Emanzipation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 2.1.1 Emanzipation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11 2.1.2 Herrschaft . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14 3 2.2 Viele Führerinnen - keine Führung . . . . . . . . . . . . . . . . . . . . . . . . . 18 2.3 Klingt gut - Zur Idee der Selbstverwaltung . . . . . . . . . . . . . . . . . . . . . . 21 Viel Wissen, wenig Struktur - eine Annäherung an die Methodik 23 3.1 Grundimpuls der Verallgemeinerung . . . . . . . . . . . . . . . . . . . . . . . . . 23 3.2 Emanzipation durch Selbstreflexion . . . . . . . . . . . . . . . . . . . . . . . . . . 24 3.3 Von der Anomie zur Ontologie . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26 3.3.1 Struktur als Wesensmerkmal . . . . . . . . . . . . . . . . . . . . . . . . . 27 3.3.2 Die Methode zur Struktur . . . . . . . . . . . . . . . . . . . . . . . . . . . 29 3.4 4 32 34 4.1 Dateneigenschaften . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36 4.1.1 Identität . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36 4.1.2 Kontrolle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36 4.1.3 Disziplin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37 Sinnzusammenhang durch Kategorien . . . . . . . . . . . . . . . . . . . . . . . . 38 Auswertung der Daten 44 5.1 Darstellung der Auswertungsgrundlage . . . . . . . . . . . . . . . . . . . . . . . . 44 5.2 Sinn oder Unsinn - die Kategorien . . . . . . . . . . . . . . . . . . . . . . . . . . 47 5.2.1 Alter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47 5.2.2 Entwicklungsstufe . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51 5.2.3 Rechtsfähigkeit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54 5.2.4 Verhalten . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56 5.2.5 Handlungsfähigkeit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60 5.2.6 Kultur - Sozialisation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65 Der Zusammenhang von Sinn und Unsinn . . . . . . . . . . . . . . . . . . . . . . 68 5.3.1 Kontrolle der Identitäten . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68 5.3.2 Sichtbarkeit durch Hierarchiemodelle . . . . . . . . . . . . . . . . . . . . . 69 5.3 6 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Die Daten des Graphen 4.2 5 Zusammenfassung Es ist an der Zeit 74 6.1 74 Von »Jugend« zu Jugend . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 Herrschaft vs. Emanzipation 6.2 6.3 7 Herrschaft vs. Emanzipation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77 6.2.1 Die Ebene der Jugendlichen . . . . . . . . . . . . . . . . . . . . . . . . . . 77 6.2.2 Die professionelle Ebene . . . . . . . . . . . . . . . . . . . . . . . . . . . . 78 Theorielosigkeit im Kontext Sozialer Arbeit . . . . . . . . . . . . . . . . . . . . . 80 Wie weiter? - Ausblick 82 Literatur 86 Anlagen 92 Anlage A - Erstauswertung Jugendarbeit OST/WEST in Zahlen . . . . . . . . . . . . 92 3 Herrschaft vs. Emanzipation 1 Einleitung Diese Arbeit ist möglich durch die langjährige praktische Tätigkeit und die dabei, auf verschiedensten Ebenen, gesammelten Erfahrungen, im Arbeitsfeld der Jugendarbeit. Durch die Erkenntnisse der Praxis und Erfahrungen mit der Aus- und Weiterbildung von Sozialarbeiterinnen im Themenbereich »Jugend«2 , wird sie gleichzeitig auch zwingend nötig. Erst durch die positiven und negativen praktischen Erkenntnisse, die festgestellten Grenzen der Arbeit und vor allem die dadurch sichtbar gewordenen Macht- und Herrschaftstrukturen war es schlussendlich möglich diese Arbeit zu schreiben. Was ist Jugendarbeit? ist der Buchtitel des Klassikers einer Jugendarbeitstheorie von C.W. Müller u. a. 1972, der 1964 erstmals veröffentlicht wurde. Gleichzeitig stellt man in der Praxis und Theorie fest, dass diese Frage nicht einfach zu beantworten ist und es keine eindeutige und allgemeingültige Antwort auf diese Frage gibt. Deshalb ist es grundsätzlich positiv zu bewerten wenn, fast 60 Jahre später, am 21. und 22. Februar 2022 eine Tagung unter dem gleichnamigen Titel stattgefunden hat, um sich der Beantwortung wieder annähern zu können. Da die vorliegende Masterarbeit ebenfalls einen Beitrag zur Beantwortung der Frage leistet, war es selbstverständlich auf den Call zu eben dieser Tagung eine Ergebnispräsentation einzureichen, um die gewonnenen Erkenntnisse vor einem Fachpublikum vorzustellen. Ein Interesse an einer selbstkritischen Fachdiskussion zu Jugendarbeit bestand jedoch von Seiten der Tagungsleitung nicht, da Praktikerinnen und Masterstudentinnen nicht den Ansprüchen der theoretischen Fachdiskussion genügen: „[...] manchmal müssen wir auch intern unsere Fachdiskussionen führen können und uns an den wissenschaftlichen Qualitätsansprüchen messen, ohne immer Praxisbezug herstellen zu müssen.“ (Prof. Dr. Benedikt Sturzenhecker)3 Das gewählte Zitat steht damit exemplarisch für den Zustand von »Jugend« - Theorie und Jugendarbeitstheorie. Die Arbeit stellt kritisch in Frage ob die Fachdiskussion überhaupt Erkenntnisse für die Jugendarbeit hervor bringt oder ob sie heute vielmehr eine ideologisch geprägte Diskussion ist. Es ist auffällig geworden, das die verschiedensten wissenschaftlichen Qualitätsansprüche nicht gegeben sind und sich über die letzten Jahrzehnte eine interne „Deutungscommunity“ heraus gebildet hat, die sagt was Jugendarbeit ist oder tun soll und ständig neues Wissen für die Praxis produziert, welches von außen betrachtet ein riesiges Wirrwarr an Konzepten, Ideen und Lesarten darstellt. Diese Community ist nicht allein dafür verantwortlich das Jugendarbeit ist wie sie aktuell ist, jedoch trägt sie einen erheblichen Anteil daran. Denn für die Jugendlichen stellen sich keine spürbaren Veränderungen mehr ein. Es fühlt sich ein bisschen an, als drehen 2 Um herauszustellen das es keinen einheitlichen Begriff von Jugend gibt, wird im Rahmen dieser Arbeit bis zum Pkt. 6.1 eine Abgrenzung zum bisherigen Verständnis von Jugend durch die »« ausgedrückt. 3 Zitat aus einem Mailverlauf im Dezember 2021 mit Prof. Dr. Sturzenhecker auf die Anfrage einer Ergebnispräsentation der Erkenntnisse aus der hier vorliegenden Masterarbeit. 4 Herrschaft vs. Emanzipation sich die Fachdiskussionen im Kreis, da sich die Wissenschaft, die Wohlfahrtsverbände, die Politik und mittlerweile auch die Studiengänge im Bereich der Sozialen Arbeit immer weiter davon entfernen, was im Rahmen dieser Arbeit als Ausgangspunkt für eine Theorie von Jugendarbeit identifiziert wird. In den letzten Jahren und während des Masterstudiums habe ich mich oft gefragt was denn Jugendarbeit überhaupt ist, obwohl ich ausgehend von dem erfolgreich durchgeführten Praxisprojekt Dorf der Jugend (vgl. Burdukat 2021) glaubte zu wissen wie ich Jugendarbeit, im Kontext Sozialer Arbeit, beschreiben und definieren kann. Bei Gesprächen und bei Vorträgen stellte ich oft fest, dass meine Vorstellung und mein Bild von Jugendarbeit nicht deckungsgleich ist mit dem was andere darunter verstehen. Im Rahmen des Masterstudiums stellte sich zudem heraus, dass auch Soziale Arbeit als Profession eine andere Auffassung von Jugendarbeit hat. Verdeutlicht wurde, dass Soziale Arbeit in ein sozialstaatliches System eingebunden ist, welches klare Vorstellung davon hat wie mit Jugendlichen zu arbeiten ist und was der Auftrag von Sozialer Arbeit und im Besonderen, der Jugendarbeit, ist. Die Frage dabei ist, was denn im wissenschaftlichen Kontext »Jugend« und damit verbunden Jugendarbeit bedeutet. Ausgehend von dem Begriff »Jugend«, dessen Deutung und Erforschung bilden sich Konzepte der Jugendarbeit heraus, welche dann als Jugendarbeitstheorie bezeichnet werden. Diese Jugendarbeitstheorie bettet sich ein in den Kontext der Profession Sozialer Arbeit und damit entstehen für die Jugendarbeit professionelle Handlungsstrategien, welche sich aus den Methoden und Konzepten der Sozialen Arbeit und ihrer Bezugswissenschaften ergeben. Diese beeinflussen wiederum die Jugendarbeitstheorie, wie z.B. die Debatten über das Doppelte oder Tripple Mandat4 oder die unterschiedlichsten Konzepte der Entwicklungspsycholgie. Es drängt sich somit die Frage auf, welchen Status Jugendarbeit innerhalb der Sozialen Arbeit einnimmt und welchen Status Jugendarbeit und »Jugend« außerhalb von Sozialer Arbeit hat. Diese Arbeit soll die Notwendigkeit verdeutlichen diesen Status grundsätzlich zu hinterfragen und ihn zu klären. In meiner Bachelorarbeit (vgl. Burdukat 2014, S. 35ff) komme ich zu dem Ergebnis das Jugendarbeit nur Selbstständigkeit und Eigenständigkeit vermitteln kann, wenn sie selbst, innerhalb Sozialer Arbeit, eigen- und selbstständig ist. Ob dafür eine reale Möglichkeit und Chance besteht, möchte diese Arbeit beantworten. Das Anliegen dieser Arbeit ist, der Jugendarbeit einen Platz als eigenständige Disziplin einzuräumen und ihr damit den Status einer, von Sozialer Arbeit, unabhängigen Handlungstheorie mit eigener Forschung, Theorie und Praxis einzurichten. Die vorliegende Arbeit begibt sich deshalb auf die Suche, was im wissenschaftlichen Kontext als »Jugend« zu verstehen ist, welchen Einfluss dies wiederum auf die damit verbundene Theorie4 In den Arbeitsfeldern der Sozialen Arbeit nimmt eine besondere Debatte zum Doppel und Tripple Mandat der Sozialarbeiterinnen viel Raum ein. Das Doppelte Mandat beschreibt eine anwaltschaftliche Funktion der Sozialarbeiterinnen zwischen Staat und Klientinnen, beim Tripple Mandat wird diese anwaltschaftliche Funktion um ein Mandat für die Menschenrechte erweitert. 5 Herrschaft vs. Emanzipation bildung einer Jugendarbeitstheorie hat und wie sich darüber die Eigenständigkeit erklären lässt. Das Erkenntisinteresse liegt darauf eine Begrifflichkeit zu finden und diese mit universellen Kategorien und Attributen zu versehen mit der eine Theoriebildung möglich wird. Diese von der individuellen Perspektive oder dem Normverständnis der jeweiligen Jugendarbeitstheorie oder Praxis, der Sozialen Arbeit, der jeweiligen Bezugswissenschaft, der Gesellschaft oder der die Arbeit finanzierenden Institutionen zu trennen, ist Untersuchungsgegenstand und Ziel zugleich. Dies wird nur möglich, indem der Begriff »Jugend«, auf dem sich dann die existierende Jugendarbeitstheorie aufbaut, untersucht und von der Vielfalt der Perspektiven zu »Jugend« abstrahiert wird. Die aktuelle Tendenz von JugendEN zu sprechen, wie es u.a. auch im aktuellen „Handbuch Offene Kinder- und Jugendarbeit“ (Deinert u. a. 2021) vorgeschlagen wird, wirkt dabei wenig zufriedenstellend für einen wissenschaftlichen Diskurs. In den letzten Jahren setzt sich in der wissenschaftlichen Debatte, rund um den Begriff »Jugend«, die Verwendung und die Beschreibung von JugendEN durch. Dies wird mit den immer vielfältiger werdenden Lebenswelten die „[...] durch Entstrukturierung, Entgrenzung, Verdichtung und Pluralisierung gekennzeichnet.“ sind (vgl. Witte, C. Schmitt und Niekrenz 2021, S. 376) erklärt. Darüber hinaus lässt sich feststellen, dass eine Verwendung von JugendEN vorgeschlagen wird, da eine passende Definition von »Jugend« fehlt, welche der Heterogenität jugendlicher Lebenswelten gerecht wird. Diese Heterogenität wird hingegen in zahlreichen Publikationen immer weiter ausdifferenziert und spezialisiert, so dass durch das Fehlen eines universellen Begriffes von »Jugend« mit JugendEN die Auseinandersetzung mit dem Begriff umgangen wird. Damit wird das Problem und der zu untersuchende Widerspruch zwischen Herrschaft und Emanzipation verschleiert. Dadurch entstehen Kommunikationsdefizite zwischen der Theorie und der Praxis und die Konzept- und Methodenentwicklung für die Praxis stagniert. Eine gute Theorie hilft der Praxis und muss im Handlungstheoretischen Zusammenhang flexibel bleiben. Durch eine Beschreibung von JugendEN wird es hingegen möglich eine Vielzahl von unterschiedlichsten Konzepten zu entwickeln, die sich ad hoc der sich stetig wandelnden »Jugend« anpassen können. Für Karl Popper (vgl. Popper 1997, S. 188) war eine Theorie, die nicht falsifizierbar ist nicht wissenschaftlich und dies passiert im Kontext der Jugendarbeit. Die Definitionen und Beschreibungen sind so vage und auf den jeweiligen Fall angepasst, dass man immer etwas richtiges schreiben kann und wenn die Jugendarbeitstheorie nicht mehr passt, dann ändert sich die Definition von »Jugend« ohne die bisherige kritisch zu hinterfragen. „Wissenschaft, die nicht kritisch das Vorhandene hinterfragt, die nicht nach den Gründen, den verborgenen Ursachen sucht, ist nach meinem Verständnis keine Wissenschaft, sondern Ideologie: sie klärt nicht auf, sie verschleiert.“ (Hirschfeld 2015a, S. 17f) Der Anspruch dieser Arbeit ist es deshalb einen Beitrag für eine Systematik und Ordnung in der Theoriebildung rund um den Begriff »Jugend« und die daran anknüpfende Jugendarbeitstheorie 6 Herrschaft vs. Emanzipation zu leisten. Es geht darum Gemeinsamkeiten der unterschiedlichsten Beschreibungen, Erzählungen und Bilder von »Jugend« zu identifizieren, um die Sprache, Forschung und somit die Theoriebildung und das Erkenntisinteresse über »Jugend« herauszustellen, sowie bestimmte Macht- und Herrschaftsstrukturen aufzudecken die schlussendlich Emanzipation5 verhindern. Diese Herangehensweise ergibt sich aus meiner langjährigen praktischen Tätigkeit auf diesem Gebiet und folgt somit nach Habermas 2013 einem emanzipatorischen Erkenntnisinteresse, welches die Besonderheiten von »Jugend« der Theoriebildung zugänglich macht. Die vorliegende Arbeit erhebt keinen Anspruch auf Vollständigkeit, die bisher erhobenen Daten stellen einen Anfang dar. Es wird damit ein Beitrag zur Konkretisierung und Systematisierung von dem Begriff »Jugend« und der damit verbundenen Jugendarbeit und einer Jugendarbeitstheorie geleistet. Um dies zu leisten wird im Kapitel Zwei Der grundlegende Widerspruch, ausgehend vom Titel der Arbeit dargestellt. Dabei wird im Wesentlichen erläutert wie Herrschaft und Emanzipation für die vorliegende Untersuchung zu verstehen sind und wie sich aus diesem Verständnis die Hypothese einer Theorielosigkeit von Jugendarbeit ergibt. Die Diversität und Komplexität des Begriffes »Jugend« und des Widerspruches der Arbeit machen es in Kapitel Drei notwendig eine Methode vorzustellen, welche dem entsprechen kann. Viel Wissen, wenig Struktur - eine Annäherung an die Methodik ist dabei die Schaffung einer Grundlage für die vorliegende wissenschaftliche Arbeit, welche darüber hinaus ein weiterführendes wissenschaftliches Arbeiten ermöglicht. Die Methode muss einem interdisziplinären Anspruch gerecht werden und betrachtet deshalb die existierende Jugendarbeit, innerhalb der Profession Sozialer Arbeit, kritisch. Die an der Netzwerk- und Graphentheorie von Norbert Elias und Harrison White orientierte und durch Talcott Parsons inspirierte Methode einer phänomenologischen Netzwerkanalyse machen es notwendig Die Daten des Graphen in Kapitel Vier, näher zu erläutern. Die Eigenschaften und Zusammenhänge der Daten werden gemäß der Methode mit einem eigenen Vokabular ausgestattet, welches in der Auswertung als Grundlage und für ein besseres Verständnis gebraucht wird. Die Auswertung der Daten in Kapitel Fünf stellt dabei den zentralen und wesentlichen Bestandteil der vorliegenden Arbeit dar. Nach einer exemplarischen Visualisierung und Darstellung der Auswertungsgrundlage werden die Daten gemäß der gebildeten Kategorien und entsprechend dem Widerspruch zwischen Herrschaft und Emanzipation interpretiert und ausgewertet. 5 Die Verwendung und Bedeutung des Begriffes Emanzipation, im Rahmen dieser Arbeit, wird unter Pkt. 2.1.1 näher erläutert. Ursprünglich wird unter Emanzipation die Freilassung von unterdrückten Individuen oder Gruppen aus der Familie oder aus Herrschaftsverhältnissen, wie z.b. bei Sklaven im römischen Recht, verstanden (vgl. Oelschlägel 2017, S. 239). Es geht dabei um die Neugestaltung und Erweiterung von Gleichheit und Gleichberechtigung. 7 Herrschaft vs. Emanzipation Es ist an der Zeit dem wissenschaftlichen Diskurs über »Jugend«, ausgehend von der vorliegenden Untersuchung, eine Definition von Jugend vorzuschlagen. Im Kapitel Sechs wird deshalb die Kennzeichnung des Begriffes »Jugend« aufgehoben und durch die vorliegende Definition der Widerspruch und die Hypothese der Theorielosigkeit beantwortet. Dieses Kapitel ist dadurch Fazit und Ergebnisdarstellung der vorliegenden Arbeit, zugleich aber auch Auftakt und Impulsgeber für weitere Forschungen. Weshalb es erforderlich wurde die Frage: Wie weiter? - Ausblick in Kapitel Sieben umfassend zu stellen und damit weiterführende Arbeiten zu ermöglichen. Die Arbeitsaufträge und Fragestellungen für Theorie und Praxis die sich mit der vorgeschlagenen Definition ergeben, werden hier kurz vorgestellt und sind als Vorschlag für den wissenschaftlichen Diskurs zu verstehen. 8 Herrschaft vs. Emanzipation 2 Der grundlegende Widerspruch Ausgehend vom Titel der Arbeit wird in diesem Kapitel erläutert wie Herrschaft und Emanzipation im Rahmen dieser zu verstehen sind. Die begriffliche Rahmung und der grundlegende Widerspruch zwischen Herrschaft und Emanzipation wird ergänzt durch eine exemplarische Kontextualisierung der Begriffe und wie dies zu der These einer Theorielosigkeit von Jugendarbeit führt. In einer romantischen Verklärung wird in »Jugend« der Wandel unserer Gesellschaft gesehen. Es wird auf die sogenannten Jugend(sub)kulturen geblickt und was diese mit uns selbst und auch mit der Gesellschaft gemacht haben. Gleichzeitig wird versucht dies zu steuern und zu lenken. Dabei wird darauf gehofft das der Sozialstaat durch die finanzielle Unterstützung der Jugendarbeit, und der »Jugend« an sich, diesen Wandel finanziert. Dafür bewegt die Jugendarbeit sich in Abhängigkeiten, welche sich verselbstständigt haben, sodass es schwer ist den gesellschaftlichen Wandel mit professioneller Arbeit zu fördern. Sofern der Wandel das Ziel professioneller Jugendarbeit ist. Auf der anderen Seite gibt es die Probleme und Defizite, die viele junge Menschen haben, es wird für viele immer schwieriger sich innerhalb der Gesellschaft zu entwickeln und eigene Vorstellungen und Träume vom Leben zu realisieren. Diese Defizite werden erforscht, da die Ergebnisse der Forschung die Grundlage für die Bereitstellung von Finanzen für die praktische sowie wissenschaftliche Arbeit rund um das Thema »Jugend« bilden. Als Praktikerinnen der Jugendarbeit arbeitet man innerhalb eines komplexen Konstruktes, welches von staatlichen Vorgaben und den sich ständig wandelnden Rahmenbedingungen beeinflusst wird, dass es unheimlich schwierig ist, eine möglichst am Jugendlichen orientierte Arbeitsweise zu entwickeln und praktisch umzusetzen. Die Jugendarbeitstheorie hat sich innerhalb der Sozialen Arbeit entwickelt, oder besser gesagt die Soziale Arbeit hat sich der Jugendarbeit angenommen und mit Hilfe der Bezugswissenschaften wie z.B. Erziehungswissenschaft, Pädagogik, Soziologie, Kriminologie u.v.a. eine Art Theorie entworfen. Diese Kombination von Jugendarbeit und Sozialer Arbeit hat seinen Ursprung in der Feststellung, dass der »Jugend« eine bestimmte Devianz oder ein Risiko innewohnt, welches sich als problematisch für den gesamtgesellschaftlichen Kontext heraus stellen kann. In Deutschland z. B. lässt sich die Einführung einer finanziell durch den Staat unterstützten Jugendpflege auf die Angst vor dem inneren Feind Sozialismus zurückführen, der die staatliche Ordnung gefährdet. Dieser Feind wurde in der im 19. Jahrhundert starken Zunahme von Gesprächskreis und Vereinsgründungen unter Jugendlichen vermutet, sodass der Staat eine Million Reichsmark für die Gründung von Jugendverbänden und Vereinen im Jahr 1911 bereitstellte, die sich mit der Inanspruchnahme einer finanziellen Zuwendung jedoch unter die Obhut der staatlich organisierten Jugendpflege begeben mussten und somit kontrollierbar wurden (vgl. Mitterauer 1986, S. 219). Das Denken von Devianz, Risiko und Gefahr stellt sich in einem hierarchisch 9 Herrschaft vs. Emanzipation gedachten Aufbau unserer Gesellschaft dar. Dieser lässt sich durch die verschiedensten Entwicklungsstufen, vom Embryo im Mutterleib bis hin zum erfahrenen und nahezu allwissenden Seniorinnen mit der Erkenntnis über den Sinn des Lebens kurz vor ihrem Ableben, darstellen. Diesem fiktiven Aufbau wohnen zahlreiche Momente und Ebenen der Herrschaft und somit einer Macht über Perspektive6 inne, die für den Inhalt der Arbeit näher benannt werden müssen. Gleichzeitig wird in den Unterschiedlichsten Zusammenhängen der Sozialen Arbeit und noch viel öfter in der Jugendarbeit der Begriff der Emanzipation verwendet. Emanzipation als positiv konnotiertes Wort vermittelt ein besseres Gefühl, weil sich dies wie die Verbesserung der Norm und dem innere Streben nach Freiheit anfühlt. Allein schon die Verwendung der Begrifflichkeit impliziert eine ihr innewohnende Kritik an der Norm und den bestehenden Verhältnissen. Dabei bleibt es gleich, ob wir Freiheit und Macht im Sinne von Hannah Arendt7 oder ob wir frei nach Kants kategorischen Imperativ8 die Befreiung aus den sozialen, ökonomischen, politische und geistigen Abhängigkeits- und Strukturverhältnissen meinen, welchen eine Unmündigkeit inne wohnt die aufgrund des fehlenden Verständnisses für die Verhältnisse entsteht (vgl. Emanzipation in Hillmann 2007). Die Arbeit ist überschrieben mit einem grundsätzlichen Widerspruch zwischen der Herrschaft und der Emanzipation. Dass es diesen Widerspruch gibt, scheint sich grundhaft aus den Wörtern von Herrschaft und Emanzipation zu ergeben, jedoch ist dadurch noch nicht offensichtlich, was überhaupt dieser Widerspruch ist. Vielmehr lässt sich der Widerspruch als Konflikt, Dissens, Streit und dadurch als Prozess beschreiben. In diesem Kapitel wird deshalb betrachtet, wer Träger der Herrschaft und Träger der Emanzipation ist, um den tatsächlichen Widerspruch bearbeiten zu können. 2.1 Herrschaft vs. Emanzipation Zunächst werde ich mit dem zu verwendenden Emanzipationsbegriff beginnen, da dieser für die Betrachtung von Herrschaft wichtige Schlussfolgerungen enthält. Darüber hinaus soll verdeutlicht werden, dass der Blick in dieser Arbeit von der Emanzipation in Richtung der Herrschaft und nicht im Sinne eines Empowermentgedankens für bestimmte Gruppen gedacht ist. Da ich Teil der zu beschreibenden Prozesse im Bereich der Jugendarbeit bin, nehme ich selbst keine neutrale Rolle ein, womit die Arbeit keine vollständig neutrale Außenperspektive darstellt. Jedoch ist es 6 Hier ist gemeint das von Älteren über Jüngere und im Besonderen durch Wissen, Strukturen und Institutionen Macht über die einzelnen Individuen und auch Gruppen ausgeübt wird. 7 Die Fähigkeit Initiative zu ergreifen und gemeinsam mit anderen handeln und dadurch Neues zu beginnen, welches dann sichtbar im öffentlichen Raum mit anderen ermöglicht wird, bedeutet Freiheit und damit eine Form von Macht welche der Gewalt gegenüber steht. (Sagebiel und Pankofer 2015, S. 60) 8 Kant definierte den Menschen als Vernunftbegabtes Wesen. Der kategorische Imperativ diente dazu Handlungen zu beschreiben und drückt vereinfacht aus das der Mensch nur so handeln soll das sein Handeln auch gleichzeitig für alle anderen Menschen gelten kann. Dadurch vereinte er Erziehungsfragen mit der Philosophie, da dadurch die Fragen nach Moral und Gerechtigkeit für die Handlung relevant werden. 10 Herrschaft vs. Emanzipation dadurch möglich eine kritische und reflexive, im Sinne einer Selbstreflexion nach Habermas (vgl. Pkt. 3.2), Position einzunehmen welche das Erkenntnisinteresse der Arbeit bestimmt. 2.1.1 Emanzipation Emanzipation hat in der Sozialen Arbeit den Aspekt von einem „[...] pädagogisch unterstützten, schrittweisen Lernprozess des (jungen) Menschen in Richtung auf seine Mündigkeit“ (Oelschlägel 2017, S. 239). Also die Entwicklung und Herausbildung des jugendlichen Individuums in die existierende Gesellschaft. Jeder Jugendliche durchläuft grundsätzlich einen solchen individuellen Emanzipationsprozess. In einer einfachen Beschreibung ist dies die natürliche Emanzipation von den Eltern. In einer komplexeren Betrachtung durchlaufen alle Jugendlichen diesen Prozess und somit ist dies ein Gruppenprozess, der nicht mehr einzig über das Individuum betrachtet werden kann. Das heißt wenn nicht nur das Elternhaus sondern soziale, ökonomische, politische oder geistige Abhängigkeitsverhältnisse betrachtet werden, entsteht dadurch ein relevantes Veränderungspotenzial für unsere Gesellschaft. Diese Faktoren bedeuten dann eine wesentlich komplexere Betrachtungsweise von Emanzipation. Das Potenzial einen kollektiven Veränderungsprozess in Gang zu setzen wohnt hier der Emanzipation inne. Der Emanzipationsprozess kann also als eine Veränderung des bestehenden Machtgefüges beschrieben werden, welcher in Form einer Auseinandersetzung zwischen den Machtstarken und den Machtschwachen hervortritt. Emanzipation bezieht sich „auf jene Vielzahl historisch spezifischer, zumeist generationenübergreifender sozialer Prozesse, in denen sich Individuen bzw. Gruppen aus wirtschaftlichen, sozialen oder kulturellen Zwangs- und Abhängigkeitsverhältnissen selbst befreien.“ (vgl. Berger 2014). Besonders brisant stellt sich der Emanzipationsprozess dar, wenn man auf zwei große globale und gesellschaftlich relevante Emanzipationsbewegungen schaut, welche wir aus der jüngeren Geschichte kennen. Eine ist die Frauenbewegung, welche heute leider in Institutionen und Projekten versandet ist und ihre Ziele noch nicht vollständig erreichen konnte und die andere ist die der sozialistischen Arbeiterinnenbewegung, von der heute überhaupt keine Rede mehr ist und die nahezu keine Rolle mehr spielt (vgl. Behrend 1995, S. 16ff). Bewegungen können also verschwinden oder Wirkungslos werden. Dies kann nach Behrend auf die fehlende Beschäftigung und Fortschreibung der eigenen Bewegungs- oder Emanzipationsgeschichte und einer fehlenden Vernetzung zurück geführt werden. „So bedeutet Geschichte aus der Sicht der Machtlosen zu betreiben, die Gründe des Aufstieges und die Ursachen des Scheiterns, der Vereinnahmung oder der Niederlage ihrer Bewegungen und die Defizite und bleibenden Leistungen ihrer Ideen zu untersuchen.“ (ebd., S. 12). 11 Herrschaft vs. Emanzipation Diese Arbeit betrachtet Emanzipation aus diesen Gründen in einem gänzlich anderen Kontext als es, wie zuvor beschrieben, sonst im Zusammenhang mit »Jugend« und Jugendarbeit der Fall ist. Wir müssen die Emanzipation und folglich auch den Emanzipationsprozess auf zwei Ebenen untersuchen. Aus der Perspektive der Sozialarbeiterinnen gedacht stellen sich diese Ebenen, im Sinne von Kunstreich und Peters, als „Kampfplatz der verschiedenen Klassen und sozialen Gruppen“ (Hirschfeld 2015a, S. 70) dar: Die Erste Ebene ist die der Jugendlichen, welche mit dem Ablösungsprozess aus dem Elternhaus oder der Elterlichen Sorge bereits einen natürlichen Prozess der Emanzipation durchlaufen.9 Um diese Ebene und die soziale Gruppe der Jugendlichen beschreiben und greifen zu können wird es notwendig den existierenden Begriff der »Jugend« so zu beschreiben, dass er dem innewohnenden Emanzipationsprozess gerecht wird. Es geht dabei darum »Jugend« nicht aus Sicht der Herrschenden oder orientiert an Zuschreibungen und Defiziten zu fassen und zu erklären, sondern sie als eigenständige soziale Gruppe und ein System auf Grundlage von Differenzen (vgl. Luhmann 2004, S. 61) zu anderen sozialen Gruppen, wie z. B. den Erwachsenen, darzustellen. Es wird dabei unterstellt und angenommen, dass aufgrund des natürlichen Emanzipationsprozesses, der »Jugend« innewohnt und der im Grunde die menschliche, individuelle wie kollektive Selbstveränderung beschreibt, mehr ist als nur die Beschreibung einer zum Gegenstand von Sozialer Arbeit gewordenen Lebensphase. Aus dieser Grundannahme heraus ergeben sich die noch folgenden Erläuterungen zu Herrschaft, dort wird beschrieben was oder wer herrscht und vor allem wie dies passiert und sich folglich in Opposition zur sich emanzipierenden Gruppe befindet. Es wird dabei von einer grundsätzlichen Gleichheit der Menschen ausgegangen, die jedoch real nicht existiert und immer wieder neu verifiziert werden muss, wodurch sich die bisherige Gleichheit neu definiert. Diese Verifizierung der Gleichheit stellt gleichzeitig den Emanzipationsprozess dar (Jacques Rancière in Genel und Deranty 2021, S. 122). Die zweite Ebene ergibt sich aus der bereits angesprochenen Profession der Sozialen Arbeit, welche ihre Legitimation als Wissenschaft aus den zahlreichen Bezugswissenschaften und dem damit verbundenen Alleinstellungsmerkmal der Allzuständigkeit (vgl. Seithe 2012, S. 48ff) bezieht. Meine persönlichen, praktischen und wissenschaftlichen Erfahrungen basieren darauf, dass ich Teil einer Jugend(sub)kultur bin, dann in der Jugendarbeit aktiv war und schlussendlich darüber in der Profession Sozialer Arbeit landete. Ich selbst habe keine Biografie der Akzeptanz durch die Gesellschaft und der Passgenauigkeit in die Welt und Norm der Erwachsenen durchlaufen. Im Fachbereich der Sozialen Arbeit, der damit verbundenen Wissenschaft und Forschung stoße ich immer wieder auf Probleme, die verursacht sind durch eine Disfunktionalität oder Kommunikationsstörung zwischen Jugendarbeit und Sozialer Arbeit und schlussendlich mit den Bezugswissenschaften. Das der persönlich von mir praktizierte Ansatz von Jugendarbeit Wirkung 9 Das dieser natürliche Prozess sich auch auf andere Art und Weise erklären lässt wird uns später noch beschäftigen. 12 Herrschaft vs. Emanzipation hat, zeigt sich durch die langjährige und heute noch wirkende Praxis. Dadurch entsteht gleichzeitig das Interesse nach den Ursachen für die Disfunktionalität und Kommunikationsstörung zu fragen. Deshalb soll Jugendarbeit und eine eigenständige Jugendarbeitstheorie sich selbst von Sozialer Arbeit emanzipieren und was sie vielleicht auch zwingend muss, wenn sie als solche ernst genommen werden möchte. Das Soziale Arbeit als Profession zum Teil des Widerspruches geworden ist, kann auf unterschiedlichste Ursachen zurück geführt werden. Eine viel zu wenig diskutierte Ursache ist die Unterstützung des aktivierenden Sozialstaates, durch Konzepte und Ideen wie „Fordern und Fördern“ (im Kontext von Hartz IV) oder im Bereich der Jugendarbeit die Debatte über Jugendbeteiligung, Aktivierung und Eigeninitiative. Dadurch werden gesellschaftliche und sozialpolitische Probleme unkenntlich gemacht und verdeckt (vgl. Seithe 2012, S. 273ff), die es im Sinne einer Emanzipation zu verändern gilt. Es verschwinden also durch die radikalisierte Individualisierung10 und eine auf die Spitze getriebene Subjektorientierung, im Rahmen des Konzeptes der Lebensweltorientierung, gänzlich die Interessen und Bedürfnisse der jeweiligen Gruppen, bzw. werden diese verdeckt. Die mit den Jugendlichen verbundene Jugendarbeit wird maßgeblich in ihren Konzepten, den dafür zugrunde liegenden Studien und Betrachtungen eben genau durch diesen, der Sozialen Arbeit eigenen, individualisierenden Ansatz der Lebensweltorientierung erfasst, beschrieben und in ihrem Handeln bestimmt. Thiersch, auf den das Konzept der Lebensweltorientierung zurück zu führen ist, warnte „schon 1995 ... davor ..., dass das Konzept der Lebensweltorientierung ... als Einfallstor für ... die ... Verdrängung der sozialpolitischen Fragen durch phänomenologisch-subjektive Diskussionen über Lebensführung und Lebensbewältigungsmuster“ (Thiersch in ebd., S. 274) missbraucht werden könnte. Die auf der ersten Ebene näher zu beschreibende Gruppe der Jugendlichen hat somit einen fundamentalen Einfluss auf den Emanzipationsprozess der zweiten, der professionellen, Ebene. Dies als Abfolge und als Einheit zu verstehen, ist eine Konsequenz, die sich aus der Beschäftigung mit der auf die Gruppe der Jugendlichen wirkenden Macht ergibt. „Macht analytisch zu fokussieren, heißt damit, historisch-spezifische Machtverhältnisse zu verstehen - und das wiederum heißt dann sehr wohl auch, die Tätigkeiten der darin eingebundenen Akteur:innen [...] systematisch in den Blick zu nehmen.“ (Kessl 2021, S. 29) Es ist deshalb unumgänglich, sich zuerst genau die Ebene der Jugendlichen anzuschauen, da ein überwiegender Teil der Jugendarbeit nicht durch die Jugendlichen, sondern durch die Jugendarbeit und damit durch die Soziale Arbeit entwickelt wurde. Dadurch entsteht hier ein Macht über - Verhältnis welches dem Emanzipationsprozess grundhaft entgegen steht. Ich behaupte das Soziale Arbeit demnach ein Konstrukt der Herrschaft ist, welches über die Jugendarbeit und 10 Damit ist eine sich ständig weiter zuspitzende Verantwortungsübergabe auf den einzelnen Menschen gemeint, die dazu führt das die Probleme, die einzelne Individuen haben auch eine sozialpolitische und gesamtgesellschaftliche Ursache haben. 13 Herrschaft vs. Emanzipation damit über eine, noch zu definierende, Jugendarbeitstheorie herrscht. Sie ist dadurch eher an einem Integrations- anstatt an einem Emanzipationsprozess interessiert. Soziale Arbeit wird als Teil des Staates und des Macht- und Herrschaftsgefüges verstanden, dadurch entsteht eine Definitionsmacht vom Staat durch die Soziale Arbeit gegenüber der machtschwachen »Jugend«. Das zeigt wie wichtig es ist sehr genau zu beschreiben was unter »Jugend« zu verstehen ist, damit sich daran ein Diskurs über Jugendarbeitstheorie anschließen kann. Wenn im Rahmen dieser Arbeit von Emanzipation die Rede ist, wird es um diese beiden Ebenen gehen. Das Hauptaugenmerk liegt jedoch auf der Beschreibung von »Jugend«, die auf natürliche Art und Weise mit der Emanzipation verbunden ist. Darauf aufbauend kann eine Jugendarbeitstheorie, beschrieben werden, welche u.a. kritische machttheoretische Fragen entwickelt. Es wird darum gehen, wo überhaupt und vor allem wie ein Emanzipationsprozess verläuft, an dem sich eine Theorie der Jugendarbeit orientieren kann. Da der Analyse- und Beschreibungsprozess der ersten Ebene komplex ist, wird im Rahmen dieser Masterarbeit auf eine tiefere und analytische Betrachtung der zweiten Ebene verzichtet, aber es wird im Ausblick (vgl. Pkt. 7) noch einmal darauf eingegangen. 2.1.2 Herrschaft Der Begriff der Herrschaft geht stets einher mit dem Begriff der Macht und seit Hannah Arendt11 geht der Begriff der Macht einher mit dem Begriff der Gewalt, wobei Herrschaft nicht gleich Macht und Macht nicht gleich Gewalt ist. Es sind heute Machtphänomene, die Herrschaft und eine Bestimmung von machtvollen und machtschwachen Gruppen die untersucht werden. Diese Arbeit untersucht einen Widerspruch zwischen Herrschaft und Emanzipation, bzw. will diesen genauer beschreiben um damit aussagekräftige Ableitungen zur Jugendarbeitstheorie zu bilden. Es ist also unumgänglich zu erklären was dabei unter dem Begriff Herrschaft zu verstehen ist. Die Diversität des Machtbegriffes zu erörtern passt nicht in den Rahmen dieser Arbeit. Einen sehr guten Überblick über die unterschiedlichsten Beschreibungen von Macht liefern uns Sagebiel und Pankofer 2015. Zudem werden in der Arbeit die unterschiedlichsten Beschreibungen von Macht und Herrschaft aufgegriffen. Ich denke, dass alle Versuche ob nun von Weber, Parson, Alinsky, Arendt, Foucault, Bourdieu, Marx, Popitz, Luhmann oder neuere von Staub-Bernasconi, Kraus, Butler und Rancière uns dabei helfen können, die Macht und damit die Herrschaft zu identifizieren. Einer Tradition von Machtbeschreibung zu folgen, führt dazu, dass der Blick auf eine mögliche Stärkung der Machtschwachen oder Anteilslosen12 und die Etablierung von Gegen11 Hannah Arendt trennt die Begriffe Macht und Gewalt und bezeichnet sie als Gegensätze. Wenn eine von beiden absolut herrscht ist es nach ihr nicht möglich das die andere vorhanden ist. Darüber hinaus kann Gewalt keine Macht hervorbringen und sie greift nur dann wenn eigentliche Macht in Gefahr ist. (vgl. Sagebiel und Pankofer 2015, S. 56ff) 12 Im Rahmen der Arbeit wird auch der Begriff der Anteilslosen für Machtschwache gebraucht. Dieser entspringt den Überlegungen von Jacques Rancière (vgl. Rancière 2018) 14 Herrschaft vs. Emanzipation macht13 verzerrt wird. Alle Forscherinnen und Denkerinnen die sich damit befassten hatten eine bestimmte Perspektive als sie versuchten, Herrschaft oder Macht zu beschreiben. Auffallend ist, dass bei einigen »Jugend« und Emanzipation eine außerordentliche Rolle gespielt haben. Popitz erläutert z.B. die Prozesse der Macht an der Entwicklung von Jugendlichen oder schreibt 1992: „Emanzipationsbestrebungen und Befreiungskämpfe finden sich mannigfach in der neueren Geschichte: Klassenkämpfe, Frauenemanzipationsbewegungen, Befreiungskämpfe aus kolonialer Unterdrückung, antirassistische Bewegungen und alle zu beobachtenden Demokratiebewegungen in diktatorisch regierten Staaten. Immer ist es die Konfrontation von Macht und Freiheit, die jede Form der Machtausübung als Eingriff in die Selbstbestimmung und Freiheitsbeschränkung entlarvt. Da alle Macht fragwürdig ist, bedarf es ihrer Rechtfertigung und Begründung. „Macht in allen Zusammenhängen, in allen Formen ist unlösbar verknüpft mit der Frage nach dem Warum“ “ (Popitz in Sagebiel und Pankofer 2015, S. 44) Auch Parsons und Luhmann bauen »Jugend« in ihre Beschreibung von Gesellschaft ein und besonders Luhmann stellt damit die Beschreibung von »Jugend«, als homogene Lebensphase, in Frage (vgl. Scherr 2009, S. 67ff). Der Begriff der Herrschaft in dieser Arbeit, entsteht aus der persönlichen Wahrnehmung aus der Praxis der Jugendarbeit und der Sozialen Arbeit. Einige der gemachten Erfahrungen sind deckungsgleich mit den Beschreibung von Macht, welche die oben genannten Wissenschaftlerinnen etabliert haben. Die Fördermittel und Projektstruktur in der Jugendarbeit zeigt, dass dort so viele Kontrollinstanzen und Regularien eingebaut wurden das eine entweder - oder - Entscheidung eine Entscheidung über den eigenen Arbeitsplatz ist. Wenn eine Jugendarbeiterin sich der Macht und der Kontrolle entziehen möchte oder es nicht verantworten kann das Jugendliche dieser ausgesetzt werden, weil er/sie aus professioneller Perspektive zu dem Schluss gekommen ist, dass ein Förderprojekt, die Förderrichtlinie oder das Handeln des Jugendamtes, des Trägers oder der Kommune an den Bedürfnissen der Zielgruppe vorbei geht, dann ist dies gekoppelt an die Streichung der Finanzierung. „Destruktive Kontrolle zielt auf das Einhalten von Vorgaben mittels destruktiver Macht solche Kontrollbestrebungen können nur auf Verhinderung unerwünschter Verhaltensweisen zielen und deren Erfolg ist ebenso wenig vom Eigensinn der Adressatinnen abhängig, wie die eingesetzte destruktive Macht.“ (Kraus 2021, S. 108) Jugendarbeiterinnen haben somit einen sehr begrenzten Handlungsspielraum und Einflußbereich und die soziale Gruppe der Jugendlichen hat diese Möglichkeiten noch viel weniger. Es 13 Eine Gegenmacht beschreibt den Aufbau eines alternativen Machtsystems, welches dem real existierenden und herrschenden System gegenüber steht. Es ist darauf ausgerichtet die eigene individuelle Handlungsfähigkeit und die der Gruppe wieder herzustellen und Einschränkungen dieser zu überwinden. Dies führt zu der Möglichkeit die Kontrolle über die eigenen Lebensbedingungen zu gewinnen. (vgl. Winter 2012, S. 451ff) 15 Herrschaft vs. Emanzipation ist ein ständiges Bewegen in prekären Verhältnissen von Entscheidungen, es entstehen Abhängigkeiten mit denen versucht wird, die eigene Arbeit und auch die Wünsche der Jugendlichen zu legitimieren. Dadurch beginnt ein Kreislauf, in welchem man für die Bedürfnisse von Jugendlichen kämpft, diese kommuniziert und damit verbunden Kompromisse eingeht um die Bedürfnisse befriedigen zu können. Damit blendet man jedoch aus, dass durch das Bekanntwerden der Bedürfnisse, die Macht der bereits Herrschenden gestärkt wird, weil diese als Bedürfnissbefriedigerinnen dann wiederum Forderung stellen können. Die Macht legitimiert und verfestigt sich dadurch. Staub-Bernasconi beschreibt dies z.B. im Bedürfnis und Abhängigkeitstheorem und dem damit eng verbundenen Theorem der organisationellen Überlegenheit (vgl. Staub-Bernasconi 2021, S. 375ff). „Macht hängt von der Bedürfnislage derjenigen ab, über die Macht ausgeübt werden soll. Wer keine Bedürfnisse und Wünsche hat, über den kann keine Macht ausgeübt werde.“ (Wagener in ebd., S. 375) Wenn wir nun die gegenwärtige Forschung und die sich daraus ergebende Jugendarbeitstheorie anschauen, dann wird deutlich, wie sehr wir als Erwachsene und Sozialarbeiterinnen daran interessiert sind, zu wissen welche Bedürfnisse junge Menschen haben. Es werden Konstrukte geschaffen mit denen Jugendliche als Kompetenter und wichtiger Teil unserer Gesellschaft beschrieben werden, aber auch als besonders gefährdete Gruppe welche die Gesellschaftsordnung der Erwachsenen Welt gefährden könnte. Diese Konstrukte werden strukturiert und darauf aufbauend werden Handlungsstrategien entwickelt um mit den Konstrukten der Jugendlichen umgehen zu können und um den Erwachsenen das Gefühl zugeben - alles im Griff - zu haben. „Herrschaft ist eine strukturelle Eigenschaft der Machtbeziehung: Sie spiegelt die grundlegende Asymetrie der Ressourcen wider, auf die sich die Akteure bei ihren Transaktionen miteinander stützen können. Diese Herrschaft macht aber ihre Auswirkungen nicht in einer einzigen Richtung geltend (sie ist von ihrem Wesen her umkehrbar). Vor allem aber braucht sie für ihren Erhalt das Spiel der Machtbeziehungen. Herrschaft steht somit im Widerspruch zu Macht, sie ist im Gegenteil eine Bedingung für die Ausübung von Macht und läßt sich nur durch sie wiederherstellen.“ (Friedberg in Krieger 2021, S. 46) Friedbergs Beschreibung von Herrschaft entspricht dem Begriff der im Rahmen dieser Arbeit gemeint ist. Es wird zum einen kritisch die Herrschaft angesprochen, die Erwachsene mit ihren Institutionen und ihren Vorstellungen von Norm, Werten und Gerechtigkeit auf Emanzipation ausüben. Zum anderen wird gleichzeitig die Herrschaft von Sozialer Arbeit, der Forschung und damit der Jugendarbeit und Jugendarbeitstheorie angesprochen, welche strukturelle Machtbeziehungen herstellt und damit Herrschaft implementiert. Für diese Arbeit und den Widerspruch zwischen Herrschaft und Emanzipation ist wichtig, dass die Macht im Widerspruch zur Herrschaft steht und Macht vielfältiger zu betrachten ist. Nicht einem Dualismus von Macht und 16 Herrschaft vs. Emanzipation Ohnmacht zu verfallen, schafft die Möglichkeit nach Modellen zu fragen wie Emanzipation von Machtschwachen ermöglicht werden kann. Durch die strukturelle Betrachtungsweise wird indirekt auch die nicht mehr umkehrbare Entwicklung der Sozialen Arbeit als Dienstleistungssektor angesprochen. Die historisch gewachsenen Institutionen bündeln die Interessen der unterschiedlichsten Gruppen in großen Wohlfahrtsverbänden, welche für nicht organisierte soziale Gruppen, wie z.B. Jugendliche sprechen. Ob die Einbettung von Jugendarbeit in einen korporativen - konservativen Wohlfahrtsstaat wie Deutschland zu Emanzipation führt, ist infrage zu stellen. Es ist somit offen, ob für Jugendliche Gerechtigkeit im Verhältnis zu Erwachsenen im Rahmen des existierenden Systems hergestellt werden kann, da im Zentrum tradierte hierarchische soziale Gemeinschaften wie Familie, Volk und vor allem der Mittelstand stehen (vgl. Benz 2021, S. 245ff). Es muss deshalb bei der Frage nach Herrschaft auch darum gehen, wie politische und ökonomische Machtressourcen verteilt sind und durch »Jugend« und Jugendarbeit in Anspruch genommen werden können, um Emanzipation von »Jugend« zu ermöglichen und Gegenmacht herzustellen. „Um diese potenzielle Gegenmacht zu domestizieren und zu legitimieren, entwickeln Organisationen Partizipations- und Mitbestimmungssysteme, die den Mitarbeitern suggerieren, sie seien an den Entscheidungen gleichberechtigt beteiligt. So wird ,Emanzipation´ zum letzten Trick des Managements: den Unterschied von Vorgesetzten und Untergebenen zu leugnen und damit dem Untergebenen seine Machtbasis zu entziehen. Unter Vorgabe des Machtausgleichs wird aber nur die Macht reorganisiert, die die Untergebenen im großen und ganzen schon haben.“ (Luhmann in Sagebiel und Pankofer 2015, S. 71) Luhmann, der Macht entsprechend seiner Systemtheorie dem politischen System zuordnet, welchem auch Wohlfahrtsverbände und damit im weitesten Sinne auch die Soziale Arbeit angehört, spricht in diesem, eigentlich auf Organisationen bezogenen, Zitat einen für uns sehr wichtigen „letzten Trick“, die Emanzipation, an. In Projekten wird versucht Jugendlichen Beteiligung im politischen System zu ermöglichen und sie einzubinden. Die Praxis zeigt, dass durch Formen wie Jugendparlamente o.ä. nur ein ganz bestimmter und oftmals der Mittelschicht angehöriger Teil von Jugendlichen angesprochen wird, aber eben nicht alle. Gleichzeitig kann dies als ein konstruiertes Mitbestimmungs- und Partizipationsmodell betrachtet werden, mit dem verhindert wird das jugendliche Gegenmacht entsteht da ein bestimmtes Milieu von Jugendlichen dem Gesamtprozess entzogen wird. Dies könnte durchaus daran liegen, dass wir überhaupt keine wirkliche Vorstellungen davon haben, was die »Jugend« ist und die Jugendarbeit dadurch Teil dieser Spaltung und Domestizierung ist. Aktuell spricht die Jugendarbeitstheorie eben von JugendEN (vgl. Pkt. 1) und nach wie vor von unterschiedlichen Generationen14 . Häufig werden damit immer nur Einzelne einer gesamten sozialen Gruppe angesprochen und mit diesen Einzelnen wird von außen die Großgruppe von Jugendlichen konstruiert. 14 In der Literatur aber auch verstärkt in den Medien wird oft von Generation X, Y, Z oder Digital Natives, etc. gesprochen. 17 Herrschaft vs. Emanzipation „Soziale Konstruktionen von ‘Jugend’ sind daher immer auch eingebettet in machtförmig gestaltete soziale Strukturen, wie zum Beispiel zwischen den Generationen.“ (King 2013, S. 35) Womit auch schon der Betrachtungsweise ein Herrschaftsmoment unterstellt werden kann. Darüber hinaus werden durch derartige Betrachtungen eben genau die Möglichkeiten geschaffen, gegenüber allen Jugendlichen dann wiederum sagen zu können, dass Erwachsene und das politische System daran interessiert sind, dass sie mitbestimmen können und gleichberechtigt sind. Blenden jedoch unsere in anderen Kontexten konstruierte Macht über Perspektive gegenüber den Jugendlichen aus und wollen damit unsere Macht wiederum stabilisieren. Den Jugendlichen wird damit die Möglichkeit entzogen, sich zu organisieren und eine wirkmächtige Gegenmacht zu entwickeln, mit derer Hilfe Emanzipation möglich wird, wir herrschen. Ob wir sie der »Jugend« oder nur den einzelnen Jugendlichen entziehen, bleibt hier offen, da wir noch nicht sicher sagen können, was »Jugend« bedeutet. Herrschaft im Kontext dieser Arbeit spielt auf die durch Erwachsene konstituierte und historisch gewachsene Gesellschaft und das damit verbundene politische System an, dem die Vorstellungen einer Utopie von Gesellschaft durch Jugendliche entgegensteht. Die Gesellschaft der Erwachsenen meint zum einen die Institutionen, die Jugendarbeit organisieren, wie z. B. eine Kommune, ein Jugendamt oder ein Wohlfahrtsverband, aber auch die einzelnen Freien Träger welche den Zwängen der vorgenannten unterliegen. Gleichzeitig versucht die Vertretung des politischen Systems in Gesetzen, Programmen, Studien und Berichten mithilfe der Wissenschaft und Forschung »Jugend« zu beschreiben und zu erreichen, um sie für die Durchsetzung ihrer Ziele zu gewinnen und um die Herrschaft der Gesellschaft der Erwachsenen zu stabilisieren. Zum anderen ist die Jugendarbeit Teil der Sozialen Arbeit und Soziale Arbeit Teil des Wohlfahrtsstaates, welche dadurch ebenfalls einen Anteil daran haben, dass junge Menschen keine Emanzipationsprozesse durchlaufen können. Es wird somit die grundsätzliche Frage nach der Rolle und vor allem der Funktion von Sozialer Arbeit im Widerspruch von Herrschaft und Emanzipation gestellt. Jedoch will diese Arbeit einen Beitrag für die Theorie und Forschung zu »Jugend« und insbesondere für die Jugendarbeit leisten, an welchem sich andere Arbeitsfelder, die ebenfalls mit bestimmten Milieus und sozialen Gruppen arbeiten, orientieren können. 2.2 Viele Führerinnen - keine Führung Die Frage im Titel der Arbeit: „Raubt die Theorielosigkeit von Jugendarbeit, im Kontext Sozialer Arbeit, ihr die Kraft?“ möchte auf ein immer wiederkehrendes Problem aufmerksam machen, an welchem man nicht vorbei kommt, wenn man sich mit »Jugend« und Jugendarbeit beschäftigt. Im Rahmen der Jugendarbeit ist es nicht ungewöhnlich mit den unterschiedlichsten Beschreibungen von »Jugend« konfrontiert zu werden. Man kann sich heraus suchen, welche einem entweder 18 Herrschaft vs. Emanzipation am Besten passt für das, was der/die Jugendarbeiterinnen machen will oder welche am ehesten zutreffend ist auf das Bild, welches der Träger, bei dem die Praxis angesiedelt ist oder das politische System, welches die Arbeit finanziert, von »Jugend« haben. Überspitzt gesagt wird für jede Lesart und für jedes Bedürfnis der sozialarbeiterischen oder politischen Perspektive etwas dabei sein. Dadurch entsteht eine Deutungshoheit von Einzelnen oder einer Community, welche eine Theorie über Jugendarbeit schwer zugänglich machen für eine Überprüfung und den klassisch wissenschaftlichen Diskurs und die damit verbundenen Methoden. Erstmalig wird in der Literatur schon 2001, also vor 20 Jahren, von einer Krise in der Jugendarbeit gesprochen. Was genau diese Krise jedoch ist und ausmacht bleibt bis heute immer noch ungeklärt. Ein möglicher Erklärungsansatz, von Werner Thole und Jens Pothmann im Kontext der Offenen Kinder- und Jugendarbeit, geht von einer enormen Flexibilisierung der Arbeitsverhältnisse in diesem Bereich aus, was in einem direktem Zusammenhang mit dem Dienstleistungscharakter von Sozialer Arbeit steht. Es wird dabei festgestellt, dass die Qualifizierungen durch Fachkräftegebote und den Ausbau von Studiengängen zwar zugenommen hat, es aber keine Nachweise oder Kriterien gibt, ob die formale Qualifizierung durch ein Studium der Sozialen Arbeit auch zu einer fachlichen Eignung für das Arbeitsfeld der Jugendarbeit geeignet ist (vgl. Thole und Pothmann 2013, S. 567). Wenn man sich daran anschließend die Daten der in der Jugendarbeit Tätigen (vgl. Tabelle 1), im Bundesdeutschen Vergleich, veranschaulicht. Kann dadurch gezeigt werden, dass sich die Beschäftigtenzahlen nicht signifikant verschlechtert haben, auch wenn man dies in ein Verhältnis zur Altersgruppe der 14 - 18 Jährigen setzt, fällt auf, dass es keine quantitative Verschlechterung im Feld der Jugendarbeit zu beobachten gibt. Bundesland 1994 2002 2018 Baden-Württemberg Bayern Berlin Brandenburg Bremen Hamburg Hessen Mecklenburg-Vorpommern Niedersachsen Nordrhein-Westfalen Rheinland-Pfalz Saarland Sachsen Sachsen-Anhalt Schleswig-Holstein Thüringen 4.283 3.319 2.980 1.875 346 1.229 3.202 985 3.517 10.282 1.705 253 1.615 833 2.077 1.112 4.860 4.850 3.064 1.421 293 1.267 4.260 1.657 3.598 8.667 1.579 281 2.873 1.505 2.307 1.927 4.153 4.596 2.051 1.008 332 923 3.607 739 4.330 9.568 2.207 173 2.022 817 1.281 1.136 Tabelle 1: In der Jugendarbeit tätige Personen Der Personalschlüssel der in der Jugendarbeit Tätigen bezogen auf die Jugendlichen, welche auf 19 Herrschaft vs. Emanzipation eine tätige Person entfallen, (vgl. Tabelle 2)15 hat sich im bundesdeutschen Vergleich der Schlüssel von 1994 auf 2018 von 123 auf 105 Jugendliche pro Jugendarbeiterin, sogar verbessert. In OST ist diese Verbesserung von 155 auf 91 Jugendliche pro Jugendarbeiterin, noch viel deutlicher zu sehen, wohingegen in WEST die Zahlen über die Letzten 30 Jahre relativ konstant geblieben sind. Bundesland 1994 2002 2018 Baden-Württemberg Bayern Berlin Brandenburg Bremen Hamburg Hessen Mecklenburg-Vorpommern Niedersachsen Nordrhein-Westfalen Rheinland-Pfalz Saarland Sachsen Sachsen-Anhalt Schleswig-Holstein Thüringen 124 182 58 93 89 61 92 140 113 86 117 206 181 211 63 150 123 139 58 133 109 61 74 81 123 115 146 204 98 116 64 85 131 134 70 104 92 84 82 89 93 91 88 251 81 106 112 76 Durchschnitt BUND Durchschnitt OST (ohne Berlin) Durchschnitt WEST (ohne Saarland) 123 155 103 108 103 106 105 91 101 Tabelle 2: Auswertung der Jugendlichen 14 - 18 Jahre pro Jugendarbeiterin Wichtig ist, anzumerken, dass diese Werte in einigen ländlichen Region deutlich unterschritten werden und besonders in ländlichen Regionen in OST gravierende Abweichungen von den Durchschnittswerten festzustellen waren, was die Beobachtung aus der eigenen Praxis von nahezu Jugendarbeiterinnen - Freien - Regionen 16 belegen konnte (vgl. Anlage A). Wie auch schon Thole und Pothmann angemerkt haben geben uns diese Zahlen keinerlei Auskunft über die Qualität von Jugendarbeit. Wenn also von einer Krise der Jugendarbeit gesprochen wird, dann muss die Qualität von Jugendarbeit hinterfragt werden und dafür benötigt es eine fundamentale theoretische Diskussion über »Jugend« und Jugendarbeit. Die Qualität, welche sich dann durch einen Theorie Praxis Transfer in der direkten Arbeit widerspiegelt, können wir nur überprüfen, wenn eine grundlegende Theorie von »Jugend« und von Jugendarbeit existiert. Durch die Allzuständigkeit der Sozialen Arbeit muss diese Theorie auch wissenschaftsübergreifend17 ak15 Bei der Durchschnittsberechnung wurde in OST auf Berlin und in WEST auf das Saarland verzichtet, da diese deutlich vom Durchschnitt abwichen und somit das Ergebnis verzerrt hätten. 16 u.a. in Sachsen: Landkreis Leipzig oder Nordsachsen 17 Damit sind die Bezugswissenschaften gemeint aus denen sich die Grundlagen der Sozialen Arbeit ableiten und die der Profession als Allzuständige Wissenschaft ihr Existenzberechtigung als Wissenschaft geben. 20 Herrschaft vs. Emanzipation zeptiert werden. Wenn die Jugendarbeit also als selbst- und eigenständig wahrgenommen werd will und Jugendliche begleiten möchte, ob nun auf einem integrativen oder emanzipatorischen Weg, dann muss sie „[...]sich zunächst selbst im Denken orientieren,...“ (Helmut Kentler in C.W. Müller u. a. 1972). Diese Orientierung beginnt damit, sich auf einen Begriff »Jugend« zu einigen. Darauf aufbauend kann dann über Jugendarbeit gesprochen werden und daraus lässt sich eine Theorie von Jugendarbeit als Jugendarbeitstheorie entwickeln. Die Beantwortung der Frage: Was ist Jugend? und Was ist Jugendarbeit? bildet die Führung oder den Kompass zur Orientierung durch das Meer an Konzepten, Theorien und Ideen zu »Jugend« und Jugendarbeit in Wissenschaft, Forschung und Praxis. Um herausfinden zu können, welchen Führerinnen aus Wissenschaft oder Praxis wir Aufmerksamkeit entgegenbringen und die bei einer Theoriebildung zu beachten sind, ist es notwendig und überfällig, den Begriff »Jugend« zu bestimmen. Durch die fehlende Begriffsbestimmung verblassen viele praktische Konzepte der Jugendarbeit in dem weiten Feld der Sozialen Arbeit und sie nimmt eine nachrangige Rolle in der Lehre ein, was die fehlende Qualität und die fehlende Qualifizierung der Sozialarbeiterinnen für Jugendarbeit erklären könnte. 2.3 Klingt gut - Zur Idee der Selbstverwaltung Ein sehr eindrückliches Beispiel der durch Theorielosigkeit entstandenen Worthülsen möchte ich im Folgenden erläutern. Ein zentrale Rolle innerhalb der fortwährenden Debatte über Jugendarbeit hat stets die Selbstverwaltung eingenommen. Sie ist in sehr vielen Ausführungen zum Thema Jugendarbeit zu finden, dabei ist die Literatur zum ursprünglichen Konzept und der Idee von Selbstverwaltung recht dünn, dennoch taucht sie immer wieder als erstrebenswertes Ideal und Kernelement der Selbstveränderung auf. Anhand der Idee der Selbstverwaltung lässt sich zeigen, wie ein Wort ohne dahinterliegende Theorie dazu führen kann, dass von einer Theorielosigkeit im Bezug auf die Jugendarbeit gesprochen werden kann. Inga Enderle 2019 zeigt in einer umfassenden Auseinandersetzung mit der Methode, dass sich kein einheitliches und praktikables Konzept der kollegialen Selbstverwaltung 18 finden lässt, mit welchem ad hoc im Rahmen der Jugendarbeit gearbeitet werden könnte. Da sich der/die Jugendarbeiterin stets in Abhängigkeitsstrukturen mit Trägern oder der Kommune befindet, sind somit dem Zulassen von tatsächlicher Selbstverwaltung und dem Erreichen der damit verbundenen emanzipatorischen Bildungsziele, wie z. B. der freien Entfaltung des Individuums, natürliche Grenzen gesetzt. Eine mögliche konzeptionelle 18 Als kollegiale Selbstverwaltung wird die Form der Selbstverwaltung bezeichnet die auch im Rahmen von Jugendarbeit prakiziert werden soll. Eine davon abweichende Form der Selbstverwaltung ist z. B. die kommunale Selbstverwaltung auf die sich das in Deutschland verankerte Subsidaritätsprinzip beruft, durch das den Bundesländern, Landkreisen und Kommunen bestimmte eigenständige Verantwortungsbereiche und auch eigene Länderspezifische Gesetzgebungsverfahren ermöglicht werden. 21 Herrschaft vs. Emanzipation Beschreibung der Selbstverwaltung, im Rahmen der Jugendarbeit, hätte vielleicht die Jugendzentrumsbewegung der 1960er/1970er Jahre in der BRD liefern können. Leider wurden die Versuche von tatsächlicher Selbstverwaltung im Rahmen der emanzipatorischen und vor allem selbstorganisierten Jugendarbeit durch die Pädagogisierung der Bewegung und dem damit einsetzenden Marsch durch die Institutionen 19 zerstört, bevor sie ihre gesamte Kraft entfalten konnte. Die Soziale Arbeit wurde als verlängerter Arm der Kommunen und Institutionen angesehen und die „formell weiterbestehende Selbstverwaltung“ (Templin 2015, S. 456) wurde immer weiter ausgehöhlt und verkommt dadurch zu einer wirkunmächtigen Worthülse. Damit Selbstverwaltung, als ein Bestandteil von Jugendarbeit, also nicht nur zum Selbstzweck existiert, braucht es für eine vollkommene Form der Selbstverwaltung auch das ideale und befreite Individuum (vgl. Enderle 2019, S. 328), welches sich bereits in sozialen Strukturverhältnissen bewegt, die frei sind von Abhängigkeiten und der bereits angesprochenen Unmündigkeit (vgl. Pkt. 2). Wer weiß, vielleicht könnte es heute ein Konzept der Selbstverwaltung geben, wonach Jugendarbeiterinnen arbeiten, wenn die Jugendzentrumsbewegung sich weiter entwickelt hätte. Da dem nicht so ist und die Soziale Arbeit einen erheblichen Anteil am Niedergang dieser Bewegung hat (vgl. Templin 2015, S. 461ff), ist es notwendig die Soziale Arbeit und die aktuelle Jugendarbeit zu hinterfragen und somit die Anwendung einer Konzeption von Selbstverwaltung. Es wird in der alltäglichen Arbeit auf ein idealisiertes Konzept ohne dahinter liegende Theorie zurückgegriffen und es werden damit die unterschiedlichsten praktischen Herangehensweisen an die Arbeit begründet. Im Umkehrschluss bedeutet dies, dass kritisch jedwede Konzeption der Jugendarbeit infrage gestellt werden kann, die sich auf das Konzept der Selbstverwaltung beruft, da es dazu schlichtweg keine wissenschaftlich begründete Systematik und Methode gibt. Vielmehr liegt in diesem konkreten Fall eine Sammlung aus Erfahrung und Beobachtungen vor, mit denen wissenschaftlich gearbeitet werden kann, jedoch im vorliegenden Fall, wie später noch festgestellt wird, unklar ist, wo und wie dieses Erfahrungswissen tatsächlich angewandt werden kann oder ob es wissenschaftlich ist (vgl. Popper in Pfister 2016, S. 200). 19 Diente als Methode der Studentinnenbewegung und wurde von Rudi Dutschke ausgerufen. Im Kontext der Jugendzentrumsbewegung bedeutete es, dass immer mehr junge Menschen Sozialpädagogik studierten um ihr eigenes Zentrum leiten zu können. Im Rahmen meines Forschungsprojektes, im Rahmen des Masterstudiums an der HTWK Leipzig, wurde eine fast deckungsgleiche Aussage, kritisch reflexiv, durch eine Interviewpartnerin, die Teil der damaligen Jugendzentrumsbewegung war, ebenso getroffen. Zum Zeitpunkt des Interviews war sie Leiterin der kommunalen Jugendarbeit. 22 Herrschaft vs. Emanzipation 3 Viel Wissen, wenig Struktur - eine Annäherung an die Methodik Durch die Diversität des Themas »Jugend« und die Komplexität des Widerspruches soll im Rahmen dieses Kapitels eine Methode vorgestellt werden, welche dem gerecht wird und eine Grundlage für ein weiteres wissenschaftliches Arbeiten zu diesem Thema ermöglicht. Der Fokus lag dabei darauf, einen kritischen Blick auf die Soziale Arbeit und die existierende Jugendarbeit zu ermöglichen, der auch interdisziplinär betrachtet, überprüft und angewandt werden kann. Die Untersuchung des grundlegenden Widerspruchs stellte sich von Beginn an als schwieriges Unterfangen dar, weil man an den Punkt gelangt, an dem man sich für eine Lesart von »Jugend« entscheiden muss. Somit ergab sich die Notwendigkeit, eine eigene Methode der Analyse zu finden, um dem Thema »Jugend« und gleichzeitig dem wissenschaftlichen sowie interdisziplinären Anspruch gerecht zu werden. In den zahlreichen Bezugswissenschaften, wie Soziologie, Philosophie, den Erziehungswissenschaften und der Pädagogik wurde sich weit vor der Entstehung der Sozialarbeitswissenschaft mit dem Thema »Jugend« auseinandergesetzt, wodurch sich die Notwendigkeit einer phänomenologischen Betrachtung ergab. Ziel ist es, eine Methodik der Analyse und Darstellung zu finden, welche es ermöglicht, sich am Ende mit unterschiedlichen wissenschaftstheoretischen Herangehensweisen der Fragestellung anzunehmen. 3.1 Grundimpuls der Verallgemeinerung Wenn die Rede von Jugendarbeitstheorie ist, musste die Frage: „Was ist eine Theorie?“ grundsätzlich beantwortet werden. Dem methodischen Vorgehen und der Annäherung liegt eine neopositivistisch, oder kritisch rational geprägte Überlegung zugrunde. Die über »Jugend« existierenden Bilder, Definitionen, Wahrnehmungen oder Beschreibungen sind Hypothesen (vgl. Popper 1997, S.212f), aus denen sich ein sehr eigenwilliges Theoriegebäude entwickelt hat. Das Theoriegebäude der Jugendarbeit stützt sich auf empirische Forschung und leitet immer wieder neue Nuancen zur Theoriebildung ab. Dies führt dazu, dass immer neues Wissen entsteht, was die Praxis und Forschung beeinflusst. Gleichzeitig passiert dies ebenso in der Praxis durch neue Erfahrungen und Beobachtungen. Dieses neu entstehende Wissen wird der Jugendarbeitstheorie zugeführt, die in der Forschung und Praxis gemachten Beobachtungen werden dann in die Theorie eingearbeitet. Es entsteht dadurch ein immer größeres, mittlerweile unüberschaubares, Theoriegebäude. Wenn die Beobachtungen zu »Jugend«, wissenschaftsübergreifend lediglich Hypothesen sind, dann basiert das Theoriegebäude nicht auf einer Theorie sondern lediglich auf Vermutungen. Wenn Popper schreibt: „So wie die Hauptaufgabe des naturbearbeitenden Ingenieurs darin besteht, dass er Maschinen konstruiert, umbaut und in Gang hält, so ist es die Aufgabe des Sozialingeneurs, [...], soziale Institutionen zu entwerfen, umzugestalten und die schon bestehenden in Funktion zu 23 Herrschaft vs. Emanzipation erhalten. Der Terminus ’soziale Institutionen’ wird hier in einem sehr weiten Sinn verwendet [...]“ (Popper 1997, S. 297). Dann kann man die Sozialarbeiterinnen als die Sozialingeneurinnen betrachten, dabei taucht dann zugleich die Frage auf, wer die Architektinnen an der sich die Sozialarbeiterinnen orientieren sollen, sind. Wer liefert also den theoretischen Unterbau für die alltägliche Arbeit? Eine möglicher theoretischer Unterbau kann somit ein bestimmtes Bild, eine bestimmte Definition von »Jugend« oder gar von der Gesellschaft sein, welche wiederum ein bestimmtes Normverständnis oder bestimmte Prozesse der Vergesellschaftung beinhaltet. Diese Entscheidung, für ein Bild im Rahmen einer Beschreibung von Gesellschaft, bedeutet jedoch, sich für eine bestimmte Denktradition zu entscheiden, was die Diversität und Heterogenität einschränkt. Demnach müssten wir uns mit jeder einzelnen Beschreibung von »Jugend« in jeder einzelnen Bezugswissenschaft auseinandersetzen, um sie im Sinne Poppers einer Falsifizierung zu unterziehen. Da es sich aber um eine Vielzahl von Wissenschaften und somit um eine Vielzahl von Hypothesen handelt, mit denen eine Auseinandersetzung notwendig ist, wird dies zu einem schwierigen Unterfangen. Den Widerspruch zwischen Herrschaft und Emanzipation zu bearbeiten, wird noch unmöglicher. Die Methodik von Popper und das damit verbundene wissenschaftliche Infragestellen von existenten Theoriegebäuden ist sehr reizvoll und stellt einen wesentlichen Anhaltspunkt für die Arbeit dar. In der vorliegenden Untersuchung greift diese Methode jedoch nicht, da keine interdisziplinäre Jugendarbeitstheorie innerhalb der Sozialarbeitswissenschaften erkennbar ist und die Frage: „Was ist die »Jugend«?“ zuerst beantwortet werden muss, da eine Jugendarbeitstheorie nur darauf aufbauen kann. 3.2 Emanzipation durch Selbstreflexion »Jugend« zu beschreiben und theoretisch zu greifen, ohne dabei die Gesellschaft, die historische Entwicklung, die Ökonomie und vor allem die Politik mit aufzunehmen, ist grob fahrlässig und blendet die Besonderheiten von »Jugend« vollständig aus. Die Jugendarbeitstheorie zielt auf ein permanentes praktisches Erkenntnisinteresse ab, was in vielen Teilen auf dem tradierten Selbstverständnis der bereits benannten Deutungscommunity und den in ihrer Tradition Handelnden aufbaut. Dadurch wird der/die Jugendliche und die »Jugend« immer mehr zum Objekt der Betrachtung. Theodor Litt schreibt dazu bereits 1927 in seinem während des 2. Weltkrieges verbotenem Buch: „Je mehr die Objektbetrachtung die Oberhand gewinnt, um so sicherer geht der erzieherische Wille in jene pseudopädagogische Herrschsucht über, der es nur darauf ankommt, am Zögling bestimmte, aus welchen Gründen auch immer, erwünschte Wirkungen hervorzubringen.“ (Litt 1967) 24 Herrschaft vs. Emanzipation Mit historischem Blick auf den Nationalsozialismus und die Rolle von Sozialer Arbeit in ihm ist es mehr als verständlich, dass diese kritische Perspektive von Litt damals nicht erwünscht war und heute eine unzureichend gewürdigte Rolle einnimmt. Litt beschreibt schon damals gut die Schattenseite der Pädagogik, der Erziehung und damit auch der Sozialen Arbeit. Die angesprochene Herrschsucht und damit die Herrschaft über bestimmte Menschen und Gruppen stellt einen zentralen Punkt in der Auseinandersetzung mit »Jugend« dar. Deshalb ist es unumgänglich, die einzelnen Theorien und Konzepte einer kritischen Selbstreflexion zu unterziehen, welche „von einem emanzipatorischen Erkenntnisinteresse“ (Habermas 2013, S. 66) geprägt sein müssen. In diesem Zusammenhang weist Habermas auf ein weiteres Problem hin, welches den heutigen Zustand der Jugendarbeit sehr gut zusammenfasst. Die weite Landschaft der »Jugend« Beschreibungen und die der Jugendarbeitstheorie sowie die damit verbundene Forschung lässt die „erkenntnisleitenden Interessen verschwinden, festigt die Fiktion, als sei der sokratische Dialog allgemein und jederzeit möglich. [...] Gerade die reine Theorie, die alles aus sich selber haben will, fällt dem verdrängten Äußeren anheim und wird ideologisch.“ (ebd., S. 71). Die einzelnen Theorien der Bezugswissenschaften, welche einer Jugendarbeitstheorie zugrunde liegen, sind im Sinne von Habermas einer Kritik zugänglich und können ein emanzipatorisches Erkenntnisinteresse fördern, um zu erforschen, wo »Jugend« mittels Sozialisationsmechanismen entmündigt, fremdbestimmt, verdinglicht und instrumentalisiert wird. Offen ist hingegen, wo im Rahmen der Sozialen Arbeit eine Kritik an Jugendarbeitstheorie und »Jugend« platziert werden kann. Diese Objektivierung durch Beschreibungen von »Jugend« zeigt sich besonders deutlich z. B. im 12. Kinder- und Jugendbericht (Bundesministerium für Familie 2006). Er schafft es auf über 400 Seiten zu beschreiben, welche unterschiedlichen Anforderungen es an die Bildung durch die unterschiedlichsten Lebens- und Lernwelten von Jugendlichen gibt und wie „gute“ Bildung im Sinne einer zukunftsfähigen Gesellschaft aussehen könnte. Er schafft es allerdings nicht, im Rahmen der unterschiedlichsten Beschreibungen zu benennen, was unter »Jugend« zu verstehen ist, sondern nur wie Bildung eben auf Unterschiede reagieren kann. Für die aktuellen Fragen und Beschreibungen trifft eine Analyse von Helmuth Schelsky sehr gut zu: „Diese neue Selbstentfremdung des Menschen, die ihm die Identität seiner selbst und des anderen rauben kann [...], ist die Gefahr, das der Schöpfer sich sein Werk, der Konstrukteur in seine Konstruktion verliert. Der Mensch schaudert zwar davor zurück, sich restlos in die selbstproduzierte Objektivität, in ein konstruiertes Sein zu transzendieren, und arbeitet doch unaufhörlich am Fortgang dieses Prozesses der wissenschaftlichen Selbstobjektivierung.“ (Helmuth Schelsky in Habermas 2013, S. 73) Die Jugendarbeitstheorie wirkt wie hier durch Schelsky und Habermas beschrieben, als hat sie sich in ihrer Konstruktion von »Jugend« verloren, womit das grundsätzliche Problem einer Theo- 25 Herrschaft vs. Emanzipation rie von »Jugend« verdeckt wird. Ähnlich wie bei Popper führt jedoch eine striktes Folgen der Kritischen Theorie von Habermas dazu, dass wir uns quer durch die Bezugswissenschaften und alle möglichen Lesarten von »Jugend« arbeiten müssen, um diese einzeln mit einem gesellschaftskritischen Blick im Sinne der Emanzipation auf ihre Herrschafts- und die Machtphänomene hin zu beleuchten. Jedoch ist in diese Arbeit die Perspektive der Selbstreflexion aus eigenen praktischen Erfahrungen und Beobachtungen in die Bearbeitung mit eingeflossen, um das eigene Denken von den herrschenden Institutionen und Wissenschaften emanzipieren zu können. Diesem emanzipatorischen Anspruch folgend, ist es deshalb wichtig, zu versuchen, aus der Perspektive der Beherrschten, Machtschwachen und Anteilslosen auf den Begriff »Jugend« und damit auf die Jugendarbeitstheorie zu schauen. Die Grundgedanken von Habermas helfen bei der Orientierung und Prüfung, von welchen Seiten oder Institutionen Herrschaft auf »Jugend« und Jugendarbeit ausgeübt wird und was wir demnach als Herrschaftsstruktur zur Bearbeitung des Widerspruches definieren müssen. 3.3 Von der Anomie zur Ontologie Die Beschreibung einer Anomie im Kontext von Gesellschaft geht auf die Überlegungen von Emile Durkheim zurück, welcher sich im Rahmen einer der ersten und sehr wegweisenden empirischen Studien im 19. Jahrhundert mit den Ursachen von Selbstmorden auseinandersetzt. Er findet dabei einen Typus, welchen er den anomischen Selbstmord nennt und der „[...] ein Indikator für den krisenhaften Zustand eines gesellschaftlichen Gemeinwesens.“ (Brock, Junge und Krähnke 2015, S. 122) ist. Dieser Zustand einer Gesellschaft, der auch als Chaos und als Normlosigkeit bezeichnet werden kann, wird heute im Allgemeinen Sprachgebrauch mit der utopischen Idee einer herrschaftsfreien Gesellschaft (Anarchie) verwechselt, was ein fataler Fehler ist und der Idee von Herrschaftsfreiheit, i.S. einer Hierarchiefreiheit, nicht gerecht wird. Wie die Anomie gehen auf Durkheims Überlegungen auch die Implementierung bestimmter Regeln für die Erschließung eines Soziologischen und damit sozialwissenschaftlichen Zusammenhangs zurück. „Der erste Schritt des Soziologen muß also darin bestehen, die Dinge, die er behandelt, zu definieren, damit man weiß, und genau weiß, um welches Problem es sich handelt. ... Immer ist zum Gegenstande der Untersuchung nur eine Gruppe von Erscheinungen zu wählen, die zuvor durch gewisse äußere gemeinsame Merkmale definiert worden ist.“ (Durkheim 1895 in ebd., S. 119) Dieser Schritt aus Definition und genauer Bestimmung des zu analysierenden Widerspruches ist unumgänglich, da sonst die Gefahr besteht, dass man sich in der Vielzahl an Jugendarbeitstheorien, Jugendbildern und Deutungen oder Beschreibungen von »Jugend« quer über alle Bezugswissenschaften, die sich damit auseinandersetzen, verlieren würde und eine Analyse nicht mehr möglich wird. 26 Herrschaft vs. Emanzipation Die Frage nach der Theorielosigkeit von Jugendarbeit und ob diese ihr die Kraft im Kontext von Sozialer Arbeit raubt, lässt sich nur über die Begriffsbestimmung beantworten. Zentraler Gegenstand der Untersuchung muss es demzufolge sein, den Begriff »Jugend« zu definieren. Auch wenn Durkheim mit der Anomie einen gesellschaftlichen Zustand beschrieben hat, passt diese Beschreibung sehr gut, wenn man sich vor Augen führt, welche Unmengen an Forschung und Wissen rund um den Begriff »Jugend« existieren. Von außen betrachtet stellt sich die Jugendarbeitstheorie als eigenes System dar, welches in sich geschlossen wirkt und nur bestimmte Expertinnen dieses System definieren, deuten und, wie bereits angesprochen aus- und mitgestalten können. Wenn nun also von einer Krise der Jugendarbeit die Rede ist (vgl. Pkt. 2.2), dann sollte eine Jugendarbeitstheorie auch im Stande sein, einen Lösungsvorschlag für diese Krise zu entwickeln, was nach Durkheim das Ergebnis seines Dreischrittes aus Funktionsanalyse, Kausalerklärung und Pathologieanalyse (vgl. Brock, Junge und Krähnke 2015, S. 120) ist. Um »Jugend« zu definieren, um darauf aufbauend den anomischen Zustand der Jugendarbeitstheorie zu überwinden, wird die Bildung einer entsprechenden Ontologie, mit der ein weiteres Arbeiten möglich wird, unumgänglich. Dabei bilden die Bezugswissenschaften und gesetzliche bzw. programmatische Beschreibungen im Bezug auf »Jugend« die Entitäten der zu bildenden Ontologie. Es wird nur Bezug auf die ursprünglichen Definitionen innerhalb der Entitäten genommen. Etwaige Definitionen in jugendarbeitstheoretischen Kontexten werden vorerst ausgeblendet, da diese sich im Regelfall auf einzelne Bezugswissenschaften oder auf einen bestimmten historischen Kontext beziehen, wie es z. B. bei den Jugendbewegungen oder Jugend(sub)kulturen der Fall ist. Die Vorgehensweise dabei ist, dass die jeweilige Beschreibung einen allgemeingültigen Begriff von »Jugend« entwickelt und bestimmt, der dann entsprechend des Kategoriensystems in der Ontologie platziert werden kann. 3.3.1 Struktur als Wesensmerkmal Die Methode, mit Strukturen bestimmte Phänomene und wissenschaftlich verwertbare Zusammenhänge zu beschreiben, geht zurück auf Ferdinand de Saussure, der als Sprachwissenschaftler versuchte, das Verhältnis von Zeichen innerhalb eines Systems zu analysieren. Diese Methoden mündeten dann in dem, was wir heute als Strukturalismus kennen, deren bekanntester Vertreter Claude Levi-Strauss war. Er stellte u. a. bereits in den 1960er Jahren fest, das: „Je umfangreicher unsere Kenntnisse werden, desto mehr verdunkelt sich das Grundschema, weil die Dimensionen sich vervielfachen und weil das Anwachsen der Bezugsachen über eine gewisse Schwelle hinaus die intuitive Methode lähmt: es gelingt einem nicht mehr, sich ein System vorzustellen, wenn dessen Darstellung ein Kontinuum erfordert, das drei oder vier Dimensionen überschreitet.“ (Claude Levi-Strauss 1962 in Walitschke 2017, S. 64) 27 Herrschaft vs. Emanzipation Die Dimension an Wissen, Kenntnissen und Beobachtungen zu »Jugend« ist mittlerweile so umfangreich, sodass der eigentliche Kern von »Jugend« sehr tief verborgen in all dem ist. Die Vielzahl an Studien und Beschreibungen münden oft in der Ausformulierung ganz eigener Beschreibungen, passend zum Forschungsgegenstand der Studie, den gewünschten Typen oder Klassifizierungen von Jugendlichen, die allerdings nur wenig über die »Jugend« an sich und vor allem deren soziale und normative Funktion verraten. Hartmut Griese konstatiert, dass er keinen weiteren Versuch auf der Suche nach einer sozialwissenschaftlichen »Jugend« - Theorie unternehmen wird. „a) Die Flut ‘jugendthematischer Publikationen’ hat [...] noch zugenommen, ist m.E. nicht mehr überschaubar und aufzuarbeiten; [...] b) In Sachen ‘Sozialwissenschaftliche Jugendtheorien’ [...] ist in dieser Zeit wenig Innovatives entwickelt worden; [...] c) Trotz der Fülle der Studien - in der Regel Auftragsstudien [...] und konjunkturell orientierte Publikationen [...] - ist der theoretische Ertrag insgesamt mager und nicht jugendspezifisch; [...]“ (Griese 1987, S. 223). Was Griese für den sozialwissenschaftlichen Bereich bereits 1987 feststellte, hat für den Bereich der Jugendforschung und die Pädagogik Reinders 2006 ebenfalls feststellen müssen. Über eine Beschreibung von vier unterschiedlichen Jugendtypen sollte erreicht werden, dass gleichsam den Erziehungswissenschaften und der Pädagogik handlungstheoretische Ableitungen ermöglicht werden. „Das Programm enthält eine ganze Reihe von Anknüpfungspunkten an erziehungswissenschaftliche Forschung, nicht aber an pädagogische Theorien des Jugendalters. Dies liegt daran, dass es letztere in der Pädagogik der Nachkriegszeit kaum gibt.“ (Reinders 2006, S. 266). Die enorme historisch gewachsene Lücke in der Forschung zu »Jugend« - Theorie nicht nur im Allgemeinen der Sozialen Arbeit, sondern auch bereits in den einzelnen Bezugswissenschaften, erfordert einen methodischen Ansatz, mit dem diese Lücke geschlossen und ein Prozess angestoßen werden kann, der eine mögliche Definition von »Jugend« zum einen nachzeichnen und zum anderen fortlaufend durch neue Erkenntnisse auch zukünftig aktualisieren kann. Ausgehend von Levi-Strauss bewegt sich die Untersuchung deshalb im Rahmen einer strukturellen Analyse, um die unterschiedlichen Vorschläge und Definitionsansätze von »Jugend« abstrahiert zu betrachten, um hauptsächlich die Beziehungen zwischen den unterschiedlichen Bildern und besonders die Beziehungen zwischen einzelnen Kategorien der Bilder sichtbar zu machen (vgl. Walitschke 2017, S. 149). Aus der Abstraktion soll dann durch die Beziehungen und Verbindungen eine Determination von »Jugend« in die jeweiligen Bezugswissenschaften möglich gemacht werden, sodass der jeweilige Standpunkt nicht mehr „die Frage- und Problemstellung und die Ergebnisse der jeweiligen jugendtheoretischen Diskussion vorwegnimmt.“ (Griese 1987, S. 207) Dadurch ergibt sich die Notwendigkeit der interdisziplinäre Herangehensweise. Die Struktur der Beziehungen soll somit eine gemeinsame Kommunikation und Diskussion wieder möglich machen. Der 28 Herrschaft vs. Emanzipation interdisziplinäre handlungstheoretische Ansatz geht zurück auf Talcot Parsons, der eine Analysemethode entwickelte, mit der er soziale Systeme und Gesellschaft beschreibt. Die Chance zur Sichtbarmachung der Beziehungen und damit der Struktur zwischen den Bezugswissenschaften der Sozialen Arbeit ist gleichzeitig auch die Kritik, da dass notwendige Abstraktionsniveau und die dadurch darstellbaren Beziehungen immer auch abhängig sind von der Auswahl der Literatur (vgl. Brock, Junge und Krähnke 2015, S. 213). Jedoch ist dieser Kritikpunkt zu umgehen, wenn die Datensammlung und die damit verbundene Beziehungsdarstellung dem wissenschaftlichen Diskurs frei zugänglich ist und man sie dadurch erweitern kann. Eine solche Erweiterung führt zu anderen Beziehungen der einzelnen Daten, jedoch würden wir eben nicht wie bisher „[...]gewisse Erkenntnisse einfach aufgegeben haben...“ (Luhmann 2004, S. 18), sondern sie werden dem weiteren wissenschaftlichen Diskurs zur Verfügung gestellt. Der weiterführenden Feststellung von Luhmann „[...] das wir noch nicht wieder in der Lage sind, diese Gewinne in einen Theoriesatz einzubauen.“ (ebd., S. 18) stimme ich nicht zu. Ein Arbeiten mit der in dieser Arbeit beschriebenen Methode kann die gewonnenen Erkenntnisse in den Datensatz einarbeiten, sodass sie eine neue Beziehung darstellen. Es wird einige Zeit dauern, um einen möglichst hohen Grad der Vollständigkeit zu erreichen, jedoch ist es unter Zuhilfenahme von methodischen Verfahren der Informatik möglich, die Erkenntnisse der unterschiedlichsten Bezugswissenschaften zu erfassen und somit durch die Strukturierung des Wissens auch die Beziehungen zu verdeutlichen. Dies führt zu einer wissenschaftsvereinenden Theorie von »Jugend«, die gleichzeitig erweiterbar und im Sinne der Allzuständigkeit einer Jugendarbeitstheorie als Teil Sozialer Arbeit anwendbar ist. Dabei bleibt es zum gegenwärtigen Zeitpunkt gleich, ob wir im Sinne Luhmanns das Wort Kommunikation oder im Sinne Parsons das Wort Handlung verwenden20 , um damit das System bzw. den Prozess zu beschreiben. Auch haben die Einwände von Habermas, das System und Handlung nicht miteinander kompatibel sind (Brock, Junge und Krähnke 2015, S. 214), für den vorliegenden Versuch »Jugend« interdisziplinär zu fassen keine Relevanz, da dieser Ansatz möglichst umfassend und unvoreingenommen eine strukturelle Beschreibung zu finden erstmalig, verfolgt wird. 3.3.2 Die Methode zur Struktur Aus der Perspektive von Handlungstheoretikerinnen wie Talcot Parsons, der seine Wissenschaftstheorie als „analytischen Realismus“ 21 bezeichnete, liegt in der Beschreibung der Bedin20 Luhmanns Theorie von Gesellschaft basiert darauf, dass sie ein Konstrukt von unterschiedlichen Systemen ist. Die Kommunikation ist bei ihm der Entstehungsprozess dieser einzelnen Systeme. Bei Parsons hingegen ist das was Luhmann als Kommunikation bezeichnet die Handlung, weshalb Parsons Gesellschaft als Handlungssystem darstellt. Da eine Untersuchung dieser Theorien nicht Bestandteil der vorliegenden Arbeit ist, wird hier darauf nur Bezug genommen, um auszudrücken, dass die Methode beide Theorien berücksichtigen kann und eine Klärung für die Untersuchung irrelevant ist. 21 Parsons schließt sich den Überlegungen von Kant an und betrachtet Begriff und Anschauung als untrennbar. Dies bedeutet, dass eine Gesellschaft immer dann eine Gesellschaft ist, wenn sie als solche bezeichnet wird. In einem anderen Kontext könnte diese Gesellschaft aber auch z. B. die Aktienbörse oder ebenso eine Bewegung 29 Herrschaft vs. Emanzipation gungen des Handelns die Möglichkeit, die Handlung und die ihr zugrunde liegenden Elemente, darzustellen (Brock, Junge und Krähnke 2015, S. 193). Für ihn stellte sich in seinen Überlegungen die Balance zwischen der individuellen Freiheit und der sozialen Ordnung als großes Problem dar, um Handlungszusammenhänge und damit Gesellschaft beschreiben zu können. Sein Erklärungsansatz zur Beschreibung von Gesellschaft und besonders von gesellschaftlicher Entwicklung ist jedoch unzureichend, da sich z. B. ein emanzipatorischer Prozess darin nur schwer greifen und beschreiben lässt. Er löste für sich dieses Balanceproblem, indem er der Handlung selbst eine bestimmte normative Orientierung zwischen der individuellen Freiheit und der sozialen Ordnung zuschrieb und damit eine Art Brücke, durch die Orientierung als Beziehung zwischen individueller Freiheit und sozialer Ordnung, baute. Diese Orientierung fasste er in bestimmten Handlungseinheiten zusammen, welche für sich wieder in ein funktionales Subsystem aufteilbar wären. Die schematische Darstellungsweise wird als AGIL Schema bezeichnet und versucht unterschiedliche gesellschaftliche Variablen zusammenzuführen. Wie bereits beschrieben entstehen die Bilder, Beschreibung und Theorieansätze zu »Jugend« innerhalb der Bezugswissenschaften, welche im AGIL Schema wiederum als ein eigenes Subsystem darstellbar wären. Der Begriff selbst entwickelt sich dabei aus einer bestimmten Orientierung heraus. Um mit Talcott Parsons zu sprechen, richtet sich das Interesse also darauf, welche Brücken es interdisziplinär zwischen den Bezugswissenschaften gibt. Die Überschneidungen von unterschiedlichsten Orientierungen bilden dann Knotenpunkte, die eine interdisziplinäre Erkenntnis darstellen. Es geht also um die Relationen und Überschneidungen der Erkenntnisse, die Besonderheit bei unserem Gegenstand ist die große Menge an Publikationen und Studien, welche den Kern unserer Untersuchung in sich tragen. Mit Parsons „action is system“ 22 weiterzuarbeiten bietet sich deshalb nicht an. Die Handlung muss offen, also dynamisch und flexibel gestaltet sein, sodass neue Erkenntnisse eingearbeitet werden können, ohne das bisherige aufzugeben. Nach Parsons muss darüber hinaus angenommen werden, dass der Ordnung der Subsysteme Bezugswissenschaften eine übergeordnete Ordnung zugrunde liegt. Es braucht hier jedoch diese Dynamik und Flexibilität abseits einer übergeordneten Ordnung, da wir die heutigen aktuellen Beschreibungen von »Jugend« nicht mehr in eine bestimmbare Relation zu »Jugend« - Theorien setzen können. Es muss somit erst einmal begonnen werden, ein Netzwerk darzustellen, um über sinnhafte und unsinnige Relationen und Zusammenhänge diskutieren zu können. Dieser Problematik der Offenheit der Forschung begegnet u. a. die relationale Soziologie, welche sich jedoch grundsätzlich auf Individuen bezieht, mit der phänomenologischen Netzwerktheorie (kurz: PNT) nach Harrison White. Nach White ist also davon auszugehen, dass die unterschiedlichen Beziehungen, welche zwischen den Ausführungen zu »Jugend« existieren, „grundlegend mit Sinn verwoben“ (M. Schmitt 2015, 22 sein. (vgl. Brock, Junge und Krähnke 2015, S. 193) Eine Kritik an Parsons war und ist, dass er seine Handlungstheorie so weit vorantrieb das schlussendlich die Handlung (action) zur Gesellschaft (system) wurde. Dabei wurden gewisse Standards für die Handlung definiert, die z. B. unterschiedliche Klassen oder Milieus nicht berücksichtigten. 30 Herrschaft vs. Emanzipation S. 2) sind, die Aufgabe wird nun sein, diesen Sinn darzustellen. Ein Arbeiten mit den strukturalistischen Elementen ermöglicht über die Netzwerktheorie ein Einbinden der unterschiedlichen Ansätze der Wissenschaftstheorie23 , mit denen dann die Sinnzusammenhänge interpretiert und beschrieben werden können. Der aktuelle Schwerpunkt der Arbeit stellt somit vielmehr einen Anfang für weiterführende Forschung dar. Um das an Popper orientierte Ingenieurinnen und Architektinnen Beispiel (vgl. Pkt. 3.1) aufzugreifen: es herrscht gegenwärtig eine Situation, in der die Architektur (Sinn) scheinbar mit der Art und Weise (Handlung) eins ist und die Handlung auch den Sinn bestimmt. Diese Situation soll über die Darstellung als Netzwerk entzerrt werden. Es soll der Unordnung kein übergeordnetes Strukturprinzip unterstellt werden, jedoch kann davon ausgegangen werden, dass es kleinere, lokalere Ordnungen gibt, wie es u. a. an dem Beispiel von Reinders24 deutlich gemacht werden konnte (vgl. M. Schmitt 2015, S. 71). Die Beziehungen und Knotenpunkte ergeben sich dann aus dem Zusammenspiel dieser, womit der verdunkelte Kern von »Jugend« sichtbar wird. Whites Überlegungen zu Netzwerken und die damit verbundene Theorie wird als Teilbereich der Graphentheorie beschrieben, welche im Wesentlichen auf Leonard Euler und seinen Überlegungen zum Königsberger Brückenproblem zurückgehen. Euler suchte nach einer Möglichkeit, einen Stadtrundgang durch Königsberg zu finden, bei dem jede Brücke über den Fluss Pregel nur einmal genutzt wird. Auch das Problem der Arbeit sind die Brücken (Beziehungen), deren Anfang und Ende als Knotenpunkte und die Verbindung als Kante definiert werden (vgl. Mönius, Steuding und Stumpf 2021, S. 1ff), was einen Unterschied z. B. zu Parsons darstellt. Allerdings ist im Kontext von »Jugend«, im Gegensatz zu Euler wiederum die Schwierigkeit vorhanden, dass noch nicht bekannt ist, wo überhaupt die Brücken sind. Die Mathematik und die Informatik haben es möglich gemacht, dass sich die Lösung von bestimmten Problemen und Fragestellungen, die sich aus Graphen und somit dem Netzwerk ergeben, dargestellt und berechnet werden können. Durch eine stete Weiterentwicklung können hier mittlerweile auch große Netzwerke dargestellt und innerhalb derer Probleme gelöst werden. Für den vorliegenden Untersuchungsgegenstand interessieren besonders die drei folgenden Aspekte der Graphentheorie: – Zusammenhang: Für die Fragestellung nach einer Basistheorie von »Jugend« ist es wichtig, bestimmte Zusammenhänge der unterschiedlichsten Beschreibungen sichtbar zu machen. In der Graphentheorie kann nur von einem Zusammenhang gesprochen werden, wenn die Kantenfolge (Beziehung) nicht unterbrochen wird. (vgl. Euler-Kreis ebd., S. 5) – Cliquenproblem: Besonders spannend für die Daten und eine weitere Arbeit damit wird die Frage nach bestimmten Cliquen innerhalb des Graphen sein, also die Frage nach gemeinsam und vollständig miteinander verbundenen Beziehungen, Abhängigkeiten und Identitäten von »Jugend«. (vgl. Party-Problem ebd., S. 16) 23 24 vgl. z. B. den Ansatz von Popper Pkt. 3.1 oder Habermas Pkt. 3.2 vgl. Pkt. 3.3.1 31 Herrschaft vs. Emanzipation – Matching: Damit sollen Gemeinsamkeiten zwischen den Zusammenhängen und den einzelnen Beschreibungen von »Jugend« innerhalb der bisherigen Anomie gefunden werden. (vgl. Schubfachprinzip Mönius, Steuding und Stumpf 2021, S. 9 und vgl. Ramsey-Theorie ebd., S. 17) Besonders wird das Matching noch einmal berücksichtigt, wenn Kategorien, welche den Sinnzusammenhang herstellen, beschrieben sind. Da davon auszugehen ist, dass bestimmte Kategorien gemeinsam in Erscheinung treten, wird interessieren, welche dies sind und ob sich daraus eine Basistheorie zu »Jugend« ableiten lässt. Unter Bezugnahme auf Talcott Parsons AGIL-Schema und die Netzwerktheorie von Harrison White werden unter Pkt. 4.2 die Kategorien gebildet werden, welche den Sinnzusammenhang und damit den gesamten Prozess sichtbar machen können. 3.4 Zusammenfassung Die Aufgabe einer basistheoretischen und zukunftsorientierten Beschreibung von »Jugend« ähnelt der Prozessbeschreibung von Zivilisation durch Norbert Elias aus dem Jahr 1936, auf die dann später auch Habermas aufbaute, um »Jugend« als „Produkt einer historischen Entwicklung“ (Habermas in Mitterauer 1986, S. 32) zu beschreiben. Elias verzichtete dabei auf die verzweifelte Suche nach dem Anfang einer theoretischen Definition und auf das Konzept der sich gegenüberstehenden Pole. Damit können wir dem „Hier Individuum, dort Gesellschaft“ (Elias in Figuration 1986/2006 S. 103) entgehen (vgl. Treibel 2008, S. 70). Dies ist vergleichbar mit der Zwickmühle der einzelnen Bezugswissenschaften und wissenschaftstheoretischen Traditionen, welcher entgangen werden soll, indem der Begriff »Jugend« als Geflecht oder Netzwerk in Form eines Graphen dargestellt wird. Die von Elias begründete Prozess- oder Figurationssoziologie sucht ebenfalls nach den Zusammenhängen und der Balance, um ausgehend davon wissenschaftlich beschreiben zu können. Was bei ihm die Balance und die Zusammenhänge zwischen Individuum und Gesellschaft sind, sind für die vorliegende Arbeit die Zusammenhänge zwischen den unterschiedlichen Beschreibungen von »Jugend« in den unterschiedlichsten Bezugswissenschaften. „ ... das, was hier als ,Verflechtung‘ bezeichnet wird, und damit das ganze Verhältnis von Individuum und Gesellschaft, kann niemals verständlich werden, solange man sich, wie es heute oft der Fall ist, die ,Gesellschaft‘ im wesentlichen als eine Gesellschaft von Erwachsenen vorstellt, von ,fertigen‘ Individuen, die niemals Kinder waren und niemals sterben. Eine wirkliche Klarheit über das Verhältnis von Individuum und Gesellschaft vermag man erst dann zu gewinnen, wenn man das beständige Werden von Individuen inmitten einer Gesellschaft, wenn man den Individualisierungsprozeß in die Theorie der Gesellschaft mit einbezieht“ (Gesellschaft der Individuen 1987/2001, 46)" (ebd., S. 91) 32 Herrschaft vs. Emanzipation Ein Rückgriff auf Norbert Elias kann eine mögliche weitere Einflechtung von Studien, Publikationen etc. in den Graphen besser ermöglichen und damit der Komplexität des Themas gerecht werden. Denn, wie durch das eben angeführten Zitat deutlich wird, es geht genau um diese Einbeziehung und Verflechtung, denn der Individualisierungsprozess und nicht die Individualisierung oder die fertigen Individuen in der Gesellschaft sind der elementare Gegenstand unserer Untersuchung. Diese Möglichkeit der Einbeziehung wird jede Neuerscheinung und Interpretation wieder als Beziehung erscheinen lassen, womit einer isolierten Betrachtung begegnet werden kann und eine Balance im Bezug auf die Objektivierung durch die radikalisierte Individualisierung hergestellt werden kann. Das Hauptaugenmerk liegt also darauf, zu beginnen, diesen Prozess in seinen Grundelementen und Verflechtungen innerhalb der Vielzahl von Wissen und Erkenntnissen sichtbar zu machen. Die Perspektive von Norbert Elias, welche mit Talcott Parsons und Harrison White erweitert wird und dabei an den Überlegungen von Habermas und Luhmann orientiert ist, „... öffnet den Blick über die Fachgrenzen [...] hinweg [..] kann helfen, die vielfach geforderte interdisziplinäre Flexibilität aufzubauen; und sie hilft, sich in den ganz alltäglichen Verstrickungen [...] nicht zu verlieren. Die Arbeitsinstrumente [...] tragen zu einem besseren Verständnis der gesellschaftlichen Zusammenhänge und insbesondere der Integration historischer, individueller und sozialer Aspekte bei. Wir wissen dann nicht, ob etwas gut oder schlecht ist – aber wir wissen, wie es dazu gekommen ist [...].“ (Treibel 2008, S.101). Aufbauend darauf kann Herrschaft und Emanzipationen eingeordnet werden und es entsteht eine Aussagekraft zum Widerspruch. Um so dann die Beziehungen von einer Jugendarbeitstheorie innerhalb des Prozesses zu prüfen und zu wissen, welche Kraft und welchen Stellenwert diese im Rahmen der Sozialen Arbeit einnehmen. 33 Herrschaft vs. Emanzipation 4 Die Daten des Graphen Durch die unter Pkt. 3 beschriebene Methode sind die Daten nicht einfach nur Zitate, Zahlen, Autorinnen und Quellen, sondern erfüllen für die Auswertung eine Funktion. Es werden deshalb in diesem Kapitel die Eigenschaften und Zusammenhänge der Daten erläutert, um somit auch das Vokabular der Auswertung darzustellen. Die Daten des Graphen und die damit verbundene Sichtbarmachung der Sinnzusammenhänge sind alle bisher eingepflegten Literaturquellen, welche als dataset mit den jeweiligen verwendeten Zitaten und beschriebenen Eigenschaften veröffentlicht wurden (vgl. Burdukat und Tramp 2022). Diese Datensammlung hat für die vorliegende Arbeit in der Version 1.0 den Stand vom 01.03.2022. Die Daten zu sammeln, stellte sich von Anfang an als schwierig dar, da durch die bereits beschriebene Publikationsmenge (vgl. Pkt. 3.3.1) in unterschiedlichsten Zusammenhängen es nur schwer möglich ist eigenständige Literatur für den Untersuchungsgegenstand zu finden, welche eine verwertbare Allgemeingültigkeit enthält und nicht schon handlungsorientierte Jugendarbeitstheorie oder Sozialarbeitstheorie ist. „Die Jugendfrage ist - als eine „never ending story“ - in diesem Jahrhundert in den geisteswissenschaftlichen Disziplinen [..] und interdisziplinär wiederholt diskutiert .“ (Hafeneger 1995, S. 20) Die Publikationsmenge führt darüber hinaus nicht mehr zu Erkenntnissen, sondern spezialisiert sich. In jedem Sachbuch, in dem »Jugend« eine Rolle spielt, findet sich erst einmal eine mehrseitige Abhandlung, wie der/die Autorin den Begriff versteht, oder es wird darauf verzichtet da ein Verständnis vorausgesetzt wird. Dies führt zum einen zu einer Unschärfe des Begriffes und wie bei der Datenerhebung festgestellt wurde, auch zu einer gewissen Beliebigkeit der Begriffsverwendung. Darüber hinaus ist die Veröffentlichungspraxis in den sozialwissenschaftlichen Bereichen eher problemzentriert, es gibt z. B. einem Sammelband zu Arbeitslosigkeit, einen Sammelband zur Sucht und dann wieder eine Veröffentlichung zu Jugend(sub)kulturen die Beiträge von Interesse enthalten. All diese Beiträge rekurrieren den Begriff »Jugend« in einer bestimmten Orientierung. Die Datenerhebung mit der Bedingung der Allgemeingültigkeit soll die Publikationsmenge, sowie deren Vielfalt bewältigen, dabei wurde Literatur wieder aus der Datensammlung heraus genommen, da die Allgemeingültigkeit nicht gegeben oder eine zu differenzierte Typologisierung vorgenommen wurde. Es konnte bei insgesamt 14 Wissenschaften, welche als origin bezeichnet werden, ein allgemeingültiger Bezug zu »Jugend« hergestellt werden. Die meisten Bezüge fanden sich innerhalb der Soziologie und in der Sozialwissenschaft. Zudem wurde eine Gruppe Law-Acts-Programm-Policy eingefügt, mit der versucht wurde für eine Kategorie einen Überblick der Unterschiede, für Europa, zu geben. Ebenfalls in diese Gruppe eingeordnet wurden programmatische Auftragsstudien und Erhebungen wie z. B. die letzten fünf Kinder und 34 Herrschaft vs. Emanzipation Jugendberichte der Bundesregierung, die zwar ein sozialwissenschaftliches Fundament haben, jedoch einen programmatischen Blickwinkel aufweisen. Da einige Beschreibungen nicht genau zuordenbar waren, wurden teilweise zwei Bezüge als origin erfasst25 . Die aktuellen Daten geben den Ursprungsautor der Beschreibung von »Jugend« an und sind somit eher Sekundärliteratur. Ein Ersetzen der bisherigen Literaturquellen durch die primären Literaturquellen kann im Rahmen dieser Arbeit nicht geleistet werden. Für die Sichtbarmachung von bestimmten Traditionslinien in der Denkweise wäre dies ein erheblicher Zugewinn für den Graphen. Auch die Jahreszahl der Veröffentlichung entspricht nicht immer dem Entstehungszeitraum der Beschreibung. Oft waren einige Beschreibungen auch für länger Zeit gültig und sind es noch, sodass hier das Jahr der Beschreibung teilweise durch die Attribute Century (Jahrhundert), Decade (Jahrzehnt) und Year from Study erweitert wurde. Um die Beschreibungen nicht nur zeithistorisch, sondern auch räumlich zu platzieren, wurde das Wirkungsland des Ursprungsautors bzw. das Land, für welches die Beschreibung gültig ist, eingepflegt. Die Daten wurden so erhoben, dass zu einem späteren Zeitpunkt weitere Daten eingepflegt werden können, sodass sich irgendwann eine internationale Perspektive ergibt. Diese offene Datenbankstruktur ist wichtig, um einen umfassenden Vorschlag auch für weitere Forschung gewährleisten zu können. Die Bedingungen und die Lebenswelten, in denen »Jugend« stattfindet, unterscheiden sich in einer globalen Betrachtung. Deshalb ist die Grundstruktur des Graphen und der Daten so aufgebaut, dass die Beschreibung zu einer transnationalen Beschreibung ausgedehnt werden kann, die in der Lage ist: „globale Ungleichheiten in ihrer komplexen Verschränkung zum Gegenstand zu machen und die diskursiven Konstruktionen von Jugend in ihrer Bedeutung für gesellschaftliche Ordnung (und den Erhalt dieser Ungleichheiten) analytisch zu erfassen.“ (Pfaff 2020, S. 89) Als möglicher weiterer Forschungsgegenstand ist auch spannend zu erfahren, ob die lange Tradition der Einbettung von Jugendarbeit und Jugendhilfe im Kontext Sozialer Arbeit in den Wohlfahrtsstaat einen Einfluss auf die Beschreibung von »Jugend« hat bzw. hatte. Da die Besonderheiten des Wohlfahrtsstaates26 und die gesellschaftlichen Umstände einen Einfluss auf die Entwicklung der Gesellschaft und damit auf »Jugend« hatte. Der Widerspruch zwischen Emanzipation und Herrschaft verläuft somit transnational gesehen, nicht gleich. »Jugend« existiert global, stellt sich dennoch aber unterschiedlich dar. 25 Die Kinder- und Jugendberichte wurden z. B. als Ursprungsbezug mit Law-Acts-Programm-Policy und Sozialwissenschaft bestimmt, hingegen eine Altersangabe aus z. B. einem Gesetz nur Law-Acts-Programm-Policy wär. 26 Die Systeme der Wohlfahrtsstaaten unterscheiden sich global betrachtet im Aufbau und der Zusammensetzung, weshalb davon auszugehen ist, dass diese Unterschiede einen Einfluss auf »Jugend« haben. 35 Herrschaft vs. Emanzipation 4.1 Dateneigenschaften Wie unter Pkt. 3.3.2 ausgeführt, sollen die verwendeten Daten mithilfe eines Graphen dargestellt und ausgewertet werden. Da der Graph ein Netzwerk erfassen soll, ist es erforderlich, die Daten im Rahmen der phänomenologischen Netzwerktheorie und relationalen Soziologie nach White zu beschreiben. Die wesentliche Grundstruktur der unterschiedlichsten Netzwerke hat White in seinem Hauptwerk Identity and Control im Jahr 1992 beschrieben, folgend sollen die Daten mit den drei wesentlichen Begriffen Identität, Kontrolle und Disziplin beschreiben werden. 4.1.1 Identität „Identität steht für Zurechnungspunkte, die Beobachter als nicht-bio-physische Ursache von Prozessen sehen (und sich aus der Beobachtung entsprechende Erwartungen hinsichtlich der Identität entwickeln).“ (M. Schmitt 2015, S. 66) Identität ist in den erhobenen Daten die erfasste Definition oder Beschreibung von »Jugend«, welche entsprechend der Allgemeingültigkeit extrahiert werden konnte27 . Kommen die Beschreibungen in ähnlicher Art und Weise vor, kann man daraus zu einem späteren Zeitpunkt neue Bedeutungseinheiten bilden, welche dann ebenfalls einen Punkt bzw. eine weitere Identität darstellen. Auf die Bildung derartiger Einheiten wurde zum gegenwärtigen Zeitpunkt verzichtet, da die bisherigen Daten dies noch nicht zulassen. Aus den bereits benannten Gründen (vgl. Pkt. 4) wurde auf Selbstbeschreibungen von »Jugend« durch Jugendliche28 und auch auf reproduzierte Identitäten in Studien und Publikationen zur Jugendarbeit verzichtet. Wenn diese noch ergänzt werden und die Forschungslage und Datenerhebung sich ändert, bietet sich eine solche Bildung von Bedeutungseinheiten als eigenständige Identität an. Durch die Erfassung der Jahreszahlen und der länderspezifischen Verankerung kann ein erster Einblick in den Kontext der räumlichen und zeitlichen Dimension gegeben werden, anhand dessen sich ein Wechsel von Identitäten auch in einem historischen Kontext darstellen lässt. Hier können z. B. noch gesellschaftliche Ereignisse oder Revolten und Jugendbewegungen eingepflegt werden welche die Dynamik und eventuelle Abhängigkeiten der Identität verdeutlichen können. 4.1.2 Kontrolle „Kontrolle steht für die Verflechtung zwischen Identitäten, die diese als Identität stabilisiert.“ (ebd., S. 69) 27 28 Im Weiteren der Arbeit wird Identität nach White für »Jugend« verwendet. Wie dies bei einer Jugend(sub)kultur oder einer Jugendbewegung der Fall ist. 36 Herrschaft vs. Emanzipation Dieser Ansatz unterscheidet sich von den „Makro-Theorien von Parsons, Luhmann und Bourdieu, die alle auf ihre Weise eine recht geordnete und ständig reproduzierende Gesellschaft entwerfen.“ (M. Schmitt 2015, S. 70) und stellt die Verflechtung der Identitäten nach Elias dar. Durch die Dynamik der Verflechtung kann die ständige Veränderung der sozialen Ordnung sichtbar gemacht werden, wohingegen »Jugend« darin gleich bleiben kann. Wir können so beantworten, ob sich »Jugend« mit Gesellschaft ändert, oder Gesellschaft mit »Jugend«. Karl Mannheim schrieb 1952: „Die Jugend ist ihrer Natur nach weder fortschrittlich noch konservativ, doch zufolge der in ihr schlummernden Kräfte zu allem Neuen bereit.“ (Mannheim in Böhnisch 2020, S. 153). Kontrolle kann hier auch doppeldeutig ausgelegt werden: zum einen kann Kontrolle, mit Blick auf den Widerspruch, eine Dimension der Herrschaft sein, zum anderen ist es aber möglich das Kontrolle eine Dimension der Emanzipation ist. Kontrolle versteht White als eine Art Platzhalter der Identität, in erster Linie auch ein Platzhalter von außen, also kurzum von der Gesellschaft. Um Kontrolle besser zu beschreiben und sie im Netzwerk und im Graphen sichtbar zu machen, werden hier die Kategorien platziert, welche darüber hinaus noch mit dem Label Herrschaft oder Emanzipation versehen werden. 4.1.3 Disziplin „Eine Disziplin steht für eine relativ dauerhafte Molekülbildung im Sozialen. Diese reproduzieren sich in internen Kontrollprozessen, bei denen Identitäten nach spezifischen Wertmaßstäben beobachtet und zueinander in Beziehung gesetzt werden.“ (M. Schmitt 2015, S. 72) Die Disziplinen stellen die Anomie dar, welchen kein fiktives übergeordnetes Strukturprinzip unterstellt werden kann und was deshalb die fundamentale Schwierigkeit in der bisherigen Auseinandersetzung mit »Jugend« darstellt. Es kann davon ausgegangen werden, dass die Disziplinen innerhalb eine „lokale Ordnung“ (ebd., S. 71) besitzen. Die Disziplinen sind demzufolge in unserem Netzwerk die Bezugswissenschaften und die Programme oder Gesetze. Die Disziplinen lassen sich nach White in drei Typen einteilen, diese sind interface, arena und council. Für die vorliegende Arbeit wurde auf den Typ interface verzichtet, da es dafür notwendig gewesen wär, noch eine Vielzahl von Konzepten und praktischen Handlungsmethoden der Jugendarbeit zu erfassen. Dies ist jedoch nicht Inhalt der Arbeit. Der Fokus lag erst einmal auf dem Begriff »Jugend« und zu einem späteren Zeitpunkt kann der Graph ergänzt werden. Spannend ist dieser Typ, weil mit dem Einfügen einer Unterkategorie hier sichtbar gemacht werden könnte, welche Formen der Jugendarbeit sich in einer Ordnungs- und Kontrollfunktion verstehen und woher dieser Anspruch kommt oder welche Formen sich eher in einer emanzipatorische Linie sehen. Es könnte darüber ein Qualitätsdialog von Jugendarbeit in Theorie und Praxis ermöglicht werden, der wie unter Pkt. 2.2 dargestellt, aktuell nicht möglich ist. Die Erkenntnisse der Forschung und die der Praxis würden dadurch zu einer gemeinsamen Wissenserweiterung beitragen, was sich in beide Bereiche 37 Herrschaft vs. Emanzipation positiv auswirkt. Der Typ arena beinhaltet die bisher eingepflegten Bezugswissenschaften und unter council werden die Politik, die Gesetze, Förderprogramme aber auch Institutionen der Jugendarbeit zusammengefasst (vgl. Tabelle 3). Disziplin-Typ Kontrollprozess Beispiele interface Bindung - commit Jugendarbeitstheorie, technologische Handlungsanweisungen arena Auswahl - select Professionen council vermitteln - mediate Politik, Gesetzgebung (Parlament), Förderprogramme, Institutionen wie Wohlfahrtsverbände, Träger der Jugendhilfe, Jugendamt, Kommune, etc. Tabelle 3: Jugend - Typen der Disziplinen Die Tabelle ist wie bei White noch um Bewertungskriterien erweiterbar, welche dann im Netzwerk sichtbar werden. Das Spannende für den zukünftigen Umgang mit den Daten ist, dass diese schon „passende Identitäten aufgrund von je eigener Auswahlkriterien“ (M. Schmitt 2015, S. 75) inkludieren. Dies bestätigt die bisherige Beschreibung der je eigenen Lesart und Interpretation der einzelnen Bezugswissenschaften (vgl. Pkt. 3.3.2). White beschreibt ebenfalls eine Art Hackordnung die als Dominanzordnung der einzelnen Typen der Disziplinen zugrunde liegt und die mit Werten und einer Hierarchie einhergehen (vgl. ebd., S. 72). Diese Dominanz kann z.B. zwischen den Bezugswissenschaften und der Politik oder der Gesetzgebung festgestellt werden. 4.2 Sinnzusammenhang durch Kategorien Der Sinnzusammenhang und der Prozess, der als »Jugend« beschrieben wird, wird im Rahmen unserer Methode durch Kategorien sichtbar, sie bilden die Architektur für unseren Untersuchungsgegenstand. Die Kategorien wurden in etwa nach der Hälfte (ca. 100) der erfassten Datensätze phänomenologisch gebildet, da zu Beginn nicht eindeutig klar war, welche Kategorien sinnvoll aus dem Datenmaterial zu bilden sind. Auf die Bildung von Bedeutungseinheiten wurde im Sinne der Auswertung verzichtet, jedoch könnte dies zu einem späteren Zeitpunkt noch nachgeholt werden. Um die jeweiligen Kategorien im Sinne der Methode zu betrachten und Bedeutungseinheiten zu bilden, wäre eine eigene Ontologie und ein eigener Datensatz für jede der Kategorien notwendig gewesen. Eine derartige Betrachtung konnte im Rahmen der Arbeit nicht geleistet werden. Für die aktuelle Fragestellung ist es daher ausreichend, die Kategorien in ihrer Eigenheit zu beschreiben. Wesentlich ist hingegen, dass im Sinne der Netzwerktheorie von White jeder Sinnzusammenhang die Identität mittels der Dimension (Herrschaft oder Emanzipation) stabilisiert. White bezeichnete dies als Kontrolle der Identität (vgl. Pkt. 4.1.2). 38 Herrschaft vs. Emanzipation Folgende Kategorien wurden für den Datensatz gebildet: Alter: Die Alterskategorie wurde als Erste gebildet, da abzusehen war, dass im Rahmen der Datenerhebung immer wieder neue Lebensalter als Beginn und Ende von »Jugend« herangezogen werden. Die Kategorie führt im Wesentlichen zu einer Objektivierung und Verdinglichung der Lebensphase, sie steht damit einer prozesshaften und dynamischen Betrachtung entgegen und dient häufig einer quantitativen Annäherung an das Thema. Sie kann als Herrschaftsdimension identifiziert werden. Entwicklungsstufe: Hier wird versucht, verschiedene Phasen von »Jugend« zu beschreiben, welche unterschiedlich ausgelegt und interpretiert werden. Als Beispiele können hier Pubertät oder Adoleszenz oder die Beschreibungen wie die Trennung in Jugendlicher und junger Erwachsener genannt werden. Hier ist ebenfalls eine starke Individualisierung des Jugendlichen und die „phänomenologische-subjektive Diskussion“ (Thiersch in Seithe 2012, S. 274) über die Bewältigung der Lebensphase eingeschlossen, die nicht nur die sozialpolitischen, sondern auch gesellschaftlichen Fragen und Rahmenbedingungen ausblendet. Diese Kategorie kann durch ihre integrative Absicht als Platzhalter der Identitäten unter einem herrschenden Kontext verstanden werden. Rechtsfähigkeit: Die Rechtsfähigkeit meint die Übertragung und Gleichstellung eines Jugendlichen mit einem Erwachsenen. Durch die - gesetzlich/rechtlich definierte - Gleichstellung wird ebenso das Ende von individuellen Entwicklungsmöglichkeiten eingeschränkt. Entwicklung kann im weiten Sinne auf individueller (siehe Kategorie: Entwicklungsstufe dieses Kapitel), aber auch auf gesellschaftlicher und sozialer Ebene verstanden werden. Die Rechtsfähigkeit definiert einen Menschen als fertigen Erwachsenen innerhalb der Gesellschaft, dadurch ist sie ebenfalls ein Platzhalter der Identität im Sinne der Dimension Herrschaft. Verhalten: Eine der spannendsten Kategorien, die sich mit Blick auf »Jugend« völlig unterschiedlich ausprägen kann und eine stark entgegengesetzte Selbst- und Fremdperspektive besitzt, ist das Verhalten. Betrachten wir »Jugend« als potenziell gefährdete Lebensphase, die durch abweichendes und deviantes Verhalten charakterisiert wird, soll sie meist ad hoc und in vielen Konzepten reguliert oder erzogen werden. Die Erziehung und Beeinflussung des Verhaltens bedient sich dann der Methoden einer Schwarzen Pädagogik wie „Drohen, Locken, Bedrängen, Überreden, Manipulieren“ (vgl. ebd., S. 62f), die auch in der Jugendarbeit - auch heute noch angewandt werden. Fraglich ist, ob nicht schon z. B. die zur Verfügungstellung von Fördermitteln für bestimmte Projekte auch ein Locken im Sinne der Schwarzen Pädagogik ist. Zudem kann Verhalten einfach nur eine Reaktion auf und der Ausdruck von Konflikten, Problem und Krisen sein, mit denen Jugendliche konfrontiert sind. Diese Situationen ergeben sich aus den realen sozialen und gesellschaftlichen Rahmenbedingungen und den fiktiven Deutungen von »Jugend«, weshalb ein bestimmtes Verhalten somit vielleicht eine Abgrenzung darstellt (vgl. Griese 1987, S. 41ff). 39 Herrschaft vs. Emanzipation „Soziologisch gesehen ist die Jugend die Periode im Leben eines Menschen, in welcher die Gesellschaft, in der er lebt, ihn ... nicht mehr als Kind ansieht, ihm aber den vollen Status, die Rollen und Funktionen des Erwachsenen noch nicht zuerkennt.“ (August Hollinghead in Mitterauer 1986, S. 25) Was Hollinghead in der 1. Hälfte des 20. Jahrhunderts feststellte, formuliert z. B. Karl Hurrelmann in gleicher Art und Weise. Hurrelmann bezeichnet »Jugend« damit als Übergang zwischen der „unselbstständigen Kindheit in das selbstständige Erwachsenenalter“ (vgl. Hurrelmann und Quenzel 2016, S. 39). Daraus ergibt sich eine Statuspanik und: „Personen in Statuspanik sind in ihren Verhaltensweisen häufig nicht »wählerisch« und nehmen keine Rücksicht darauf, welche Konsequenzen ihr Handeln für andere bzw. für die Gesellschaft hat.“ (Heitmeyer 2018, S. 109) Kurt Lewin29 , der über die Beschreibung von jugendlicher Panik eine ganze Theorie von »Jugend« aufstellte, schrieb: „Der unsichere Charakter seiner Ideale und Werte hält den Jugendlichen in einem Zustand von Konflikt und Spannung, der umso größer ist, je zentraler die Probleme sind.“ (Lewin in Griese 1987, S. 43) Da die Probleme der »Jugend« auch die Probleme der Gesellschaft sind und sie diese durch ihr Verhalten anzeigen, liegt in eben diesem immer neuen Verhalten der »Jugend« auch eine emanzipatorische Dimension verborgen. “Wie dieses Neue allerdings ausfällt, [...] hängt von den gesellschaftlichen Rahmenbedingungen ab; vor allem von der Bereitschaft der maßgeblichen gesellschaftlichen Kräfte, das Neue und damit Konflikthafte in der Jugend anzuerkennen. An der Generationengestalt der Jugend symbolisiert sich also das jeweils historisch Neue, ob es nun die Jugend selbst durch Konflikte, Protest, abweichendes Verhalten ausdrückt oder ob sich lediglich die gesellschaftliche Diskussion um den Wandel an Bestand, Integration oder Zerfall der Gesellschaft an dem Zustand der jungen Generation entzündet.“ (Böhnisch 2020, S. 37) Daraus ergibt sich eine seismographische Funktion der Kommunikation von »Jugend« durch das Verhalten, welches von Relevanz für einen emanzipatorischen Blickwinkel ist. Handlungsfähigkeit: Hier wird im Wesentlichen die Identitätsbildung des Individuums angesprochen, welche im Sinne einer Handlungsfähigkeit an Selbstständigkeit und Eigenständigkeit erkennbar ist. 29 War ein deutscher Sozialpsychologe Anfang des 20. Jahrhunderts. Er erforschte mit Feldanalysen das Verhalten von Jugendlichen. Dabei erweiterte er die bisherige Analyse von Verhalten durch die äußeren Einflüsse der Lebenswelt und erweiterte dadurch die Psychoanalyse. 40 Herrschaft vs. Emanzipation „Ausgangspunkt wären nunmehr die sozialen Prozesse, in denen junge Menschen eine Stärkung oder Beschränkung ihrer Handlungsmächtigkeit [...] erfahren. Mit dem Fokus [...] kann also theoretisch und empirisch der soziale und politische Ermöglichungsraum thematisiert werden, in dem junge Menschen ihren Alltag bewältigen und ihre Handlungsfähigkeit ausbilden.“ (Schröer 2009) Die Kategorie kann der Emanzipation zugeordnet werden, wobei sie sich auch auf eine eingeschränkte individuelle Form der Emanzipation in bestimmten Grenzen beziehen kann. Diese eingeschränkte Form kann als eine Art „Scheinemanzipation“ bezeichnet werden, da sie sich einzig auf individueller Ebene abspielt. “Entscheidend ist vielmehr eine Handlungsfähigkeit im System, um über dieses hinauszugehen. Und das geht nur sehr begrenzt individuell, verlangt vielmehr eine kollektive Bewegung.“ (Paulo Freier in Hirschfeld 2015b, S. 16) Diese begrenzte individuelle Handlungsfähigkeit führt noch nicht zu gesellschaftlichen Emanzipationsprozessen, sondern ist eher mit einer integrativen Note versehen. Die Handlungsfähigkeit ist im Sinne einer Jugendarbeitstheorie deshalb besonders von Interesse, da genau dieser Entwicklungsprozess zum einen etwas über die Qualität von Jugendarbeit aussagen kann und zum anderen gesetzlich verankert ist im § 11 SGB VIII30 . Hier wird deutlich, dass es sinnvoll ist eine eigene Ontologie und einen eigenen Graphen zur besseren Beschreibung der Handlungsfähigkeit zu entwickeln, um der Diversität der interdisziplinären Begriffsverwendung gerecht zu werden. Kultur und Sozilisation: Die Kategorie der Kultur und Sozialisation umfassend darzustellen, übersteigt den Rahmen dieser Arbeit. Entscheidend ist, dass sie für den Widerspruch eine Kontextualisierung der Emanzipation in der Jugendarbeitstheorie sowie für die Begriffsbestimmung eine sehr zentrale Rolle einnimmt. Im Rahmen dieser Kategorie wird die gesellschaftliche Relevanz von »Jugend« beschrieben. „Jugend ist ein gesellschaftliches bzw. sozio-kulturelles Produkt.“ (Griese 1987, S. 30) Im Umkehrschluss ist »Jugend« damit auch in der Lage Gesellschaft zu verändern, um nicht nur Produkt sondern auch Produzentin zu sein. Es geht hier um die grundsätzliche Auffassung und Vorstellung von Gesellschaft, die in der Welt der Erwachsenen existiert und die von »Jugend« nicht gleich reproduzierend übernommen wird. Damit gehen die der Gesellschaft innewohnenden Phänomene von Herrschaft und Macht einher, weshalb sich in der Kategorie auch der grundsätzliche Widerspruch heraus kristallisiert und darstellen lässt. Gleichzeitig entsteht rund um die Kategorie das, was in der Fachliteratur oft als Mythos beschrieben wird, der sich rund um 30 In diesem § wird Eigen- und Selbstständigkeit als wesentliches Ziel von Jugendarbeit benannt. Dabei ist offen, wie dies gemeint ist, ob im Sinne der individuellen oder einer emanzipatorischen Handlungsfähigkeit. 41 Herrschaft vs. Emanzipation „die unterschiedlichen (zeitgeistbezogenen) Konstruktionen [...] und rhetorisch-kulturellen Muster (Klischees) über Jugend und Jugendlichkeit“ (Hafeneger 1995, S. 61) bildet. Für diese Arbeit ist es nicht erforderlich tiefer in die einzelnen Beschreibungen von Gesellschaft einzusteigen. Die Relevanz der Kategorie für den Untersuchungsgegenstand lässt sich auf den Begriff Entfremdung reduzieren, welcher mal direkt, fragmentiert oder umschrieben in den unterschiedlichsten Beschreibungen von Gesellschaft enthalten ist. An ihm entlädt sich der grundsätzliche Widerspruch zwischen Herrschaft und Emanzipation und wird somit sichtbar. „Entfremdung bedeutet Indifferenz und Entzweiung, Machtlosigkeit und Beziehungslosigkeit sich selbst und einer als gleichgültig und fremd erfahrenen Welt gegenüber. Entfremdung ist das Unvermögen, sich zu anderen Menschen, zu Dingen, zu gesellschaftlichen Institutionen und damit auch - so eine Grundintuition des Entfremdungsmotives - zu sich selbst in Beziehung zu setzen. Eine entfremdete Welt präsentiert sich dem Individuum als sinn- und bedeutungslos, erstarrt oder verarmt, als eine Welt, die nicht »die seine« ist, in der es nicht »zu Hause« ist oder auf die es keinen Einfluss nehmen kann.“ (Jaeggi 2019, S. 21) Dies ist für die Kategorie Kultur - Sozialisation bedeutend, da sich ablesen lässt, welchen Umständen »Jugend« im Spannungsfeld der Entfremdung begegnet, ob in der unbewussten Formierung zu Jugend(sub)kulturen oder Jugendbewegungen oder in der individuellen Bewältigung der eigenen Lebensrealität. Es drückt sich „in den Freizeitbedürfnissen der Wunsch nach Veränderung und Überwindung der Entfremdung“ (Kollan 1980, S. 118) aus. Dieser Ausdruck, der entweder zu einer Reproduktion der gesellschaftlichen Verhältnisse führt oder auf Veränderung drängt, ist als emanzipatorische Dimension für unsere Betrachtung zu verstehen. Betrachtet man darüber hinaus die Kategorie global, zeigt sich das »Jugend« „globalisierte Ästhetiken und ökonomische Trends auf ihre konkreten sozialen Lagen und soziokulturellen Ressourcen vor Ort“ (Pfaff 2020, S. 84) bezieht. Womit durch diese Kategorie und die entstehenden Sinnzusammenhänge eine Makro- sowie eine Mikroperspektive von Gesellschaft und damit verbunden von Jugendarbeitstheorie ermöglicht wird. Anmerkungen: Es lässt sich für die Daten und die Betrachtung dieser eine semantische Abfolge aus den Kategorien ableiten und beschreiben. Diese Abfolge kann Auskunft darüber geben, welches Bild sich von »Jugend« in einer Definition und darauf aufbauend in einer Jugendarbeitstheorie oder innerhalb eines Konzeptes von Jugendarbeit verbirgt. Die einzelnen Unterschiede werden im Rahmen dieser Arbeit nicht untersucht, jedoch soll der Graph und damit die Methode die Möglichkeit dazu bereits eröffnen. Es wird immer einzeln, je nach Definition bewertet, ob die Kategorie eine Herrschafts- oder Emanzipationsdimension besitzt. Dies ist erforderlich, da sich besonders in den frühen Deutungen von »Jugend« die Herrschaftdimension in Form von Altersangaben und Beschreibungen von Entwicklungsstufen verbirgt, obwohl der Beschreibung ein emanzipatorischer Anspruch zugesprochen werden kann. Ein Beispiel dafür ist das Gene- 42 Herrschaft vs. Emanzipation rationenmodell von Karl Mannheim31 . Umgekehrt gibt es Beschreibungen, in denen zwar ein emanzipatorischer Anspruch verborgen sein könnte, aber das darüber gebaute Gesamtbild von »Jugend« eher der Dimension von Herrschaft entspricht. Deutlich wird dies u. a. an Beschreibungen, die sich eher um ein Erziehen und Integrieren der »Jugend« oder der Machtschwachen in die machtstarke Gesellschaft der Erwachsenen bemühen. Deshalb muss in den Daten auf eine je einzelne Bewertung der Kategorien geachtet werden, da z. B. das Verhalten ein Indikator für eine integrierende Lesart sein kann, aber auch als Sichtbarmachung eines emanzipatorischen Anspruches hilfreich ist. Diesen Sinnzusammenhängen soll deshalb eine besondere Aufmerksamkeit entgegengebracht werden, weshalb die Dimension der Kategorien einzeln codiert werden muss. 31 Das Generationenkonzept und das sprechen über bestimmte Alterskohorten geht im Wesentlichen zurück auf Karl Mannheim. Es geht davon aus, dass bestimmte Jahrgänge nur an bestimmten Ausschnitten der Geschichte partizipieren können. Kulturelle und politische Rahmenbedingungen der Gesellschaft bestimmen die jeweilige Generation und charakterisieren den Generationenübergang. (vgl. dazu Karl Mannheim in Hafeneger 1995, S. 45f und Griese 1987, S. 78ff) Heute werden in Studien immer noch Begriffe wie Generation X, Y, Z oder Digital Natives etc. genutzt um bestimmte Alterskohorten dementsprechend einzuteilen und zu beschreiben. 43 Herrschaft vs. Emanzipation 5 Auswertung der Daten Durch die erarbeitete Methode und die gesammelten Daten wird in diesem Kapitel der Auswertung und der Interpretation der Daten Raum gegeben. Zum besseren Verständnis wird dabei die Untersuchung exemplarisch visualisiert. Die Daten, welche in den Graphen eingepflegt wurden, bilden die Grundlage für die Auswertung (vgl. Burdukat und Tramp 2022). Es wird hier noch einmal betont, dass kein Anspruch auf Vollständigkeit der Erfassung erhoben wird, es gilt folglich der Erhebungsstand (vgl. Pkt. 4). Die Daten wurden mit Google Spreadsheat erfasst und bearbeitet, mit Eccenca Corporate Memory erfolgte die Transformation in einen Wissengraphen und die Dashboardvisualisierung wurde mithilfe von Redash durchgeführt. Durch die Erfassung der Definitionen, Beschreibungen und Bilder mit allen Eigenschaften (vgl. Pkt. 4.1) und den Sinnzusammenhängen (vgl. Pkt. 4.2) ergibt sich ein Graph zu »Jugend« wie in Abbildung 1 zu sehen. Abbildung 1: Graph »Jugend« gesamt 5.1 Darstellung der Auswertungsgrundlage Es wurden insgesamt 174 Beschreibungen, Bilder oder Definitionen von »Jugend« erfasst. Jeder einzelnen Auffassung konnte bis zu drei Kategorien zugeordnet werden. Die Menge der Kategorien hing von der Genauigkeit der Beschreibung ab, denn eine genaue Altersangabe (z. B. in einem Gesetz) spricht nur die Kategorie Alter an, wohingegen eine Altersangabe mit Verweis auf die Entwicklungsstufe (durch Erwähnung von Pubertät oder Adoleszenz) zwei Kategorien anspricht. Dadurch entstehen insgesamt 302 Sinnzusammenhänge (vgl. Tabelle 4) aus den 174 Beschreibungen, welche die Grundlage der Interpretation und für weitere Auswertungen bilden. 44 Herrschaft vs. Emanzipation Kategorie Häufigkeit Alter Entwicklungsstufe Rechtsfähigkeit Verhalten Handlungsfähigkeit Kultur - Sozialisation 71 44 13 48 59 67 Summe 302 Tabelle 4: Auswertung Häufigkeit der Kategorien Die 174 Beschreibungen verteilen sich mit den 302 Sinnzusammenhängen auf insgesamt 14 Bezugswissenschaften und die zusätzlich gebildete Disziplin Law-Acts-Program-Policy. Aus der Verteilung der Kategorien auf die Disziplinen ergibt sich eine Gewichtung dieser, die »Jugend« beschreiben und definieren. Aus dem unter Abbildung 1 dargestellten Gesamtgraphen lassen sich dann die einzelnen Graphen für die jeweiligen Kategorien bilden (z. B. Alter vgl. Abbildung 2), die uns die Informationen liefern, welche für die Untersuchung benötigt werden. Abbildung 2: Graph Kategorie Alter 45 Herrschaft vs. Emanzipation Einige Beschreibungen konnten nicht nur einer Disziplin zugeordnet werden, weshalb sich die Häufigkeit der Disziplinen von der Häufigkeit der Sinnzusammenhänge unterscheidet. Es wird auf eine tabellarische Darstellung verzichtet, da diese Betrachtung im Rahmen der Arbeit nachrangig erfolgte. Die interdisziplinäre Bedeutung wird durch die gewählte Darstellungsgrafik (vgl. Abbildung 3) deutlich. Abbildung 3: Bedeutung Disziplinen für Kategorien Für die Auswertung der Sinnzusammenhänge war es von besonderem Interesse herauszufinden, in welcher Häufigkeit die Kategorien gemeinsam auftauchen und wie sich dieses Matching verteilt. Zur besseren Darstellung der Pivot-Tabelle (vgl. Tabelle 5) werden folgende Abkürzungen für die Kategorien verwendet: Alter - A; Entwicklungsstufe - EWS; Rechtsfähigkeit - RF; Verhalten V; Handlungsfähigkeit - HF; Kultur - Sozialisation - KS. Die Summe erfasst nur die Überschneidungen und nicht wie unter Tabelle 4 die Gesamthäufigkeit der Kategorie. Durch die Möglichkeit von bis zu drei Kategorien ist eine Darstellung in einer Tabelle nicht möglich. Deutlich wird dies am Beispiel der Handlungsfähigkeit, welche insgesamt 83 Mal mit anderen Kategorien, aber insgesamt nur 59 Mal vorkommt. 46 Herrschaft vs. Emanzipation Kategorie A EWS RF V HF KS Alter Entwicklungsstufe Rechtsfähigkeit Verhalten Handlungsfähigkeit Kultur - Sozialisation 13 4 2 4 3 13 4 4 2 9 2 4 16 4 23 3 16 4 9 16 16 Summe 26 58 2 4 14 23 25 61 36 83 25 36 80 Tabelle 5: Auswertung Matching der Kategorien 5.2 Sinn oder Unsinn - die Kategorien Es wird zuerst eine Betrachtung der Kategorien vorgenommen und dort die jeweiligen Besonderheiten dargestellt, dabei wird auf bestimmte Übereinstimmungen mit anderen Kategorien hingewiesen. Nach der Einzelbetrachtung wird noch einmal gesondert unter Einbeziehungen der Dimensionen Herrschaft und Emanzipation ein Gesamtzusammenhang im Sinne der Untersuchung hergestellt. 5.2.1 Alter In der allgemeinen Wahrnehmung und im alltäglichen Gebrauch wird gedacht, dass »Jugend« über das Alter gut bestimmbar und beschreibbar ist. Sie wird beschrieben als „Lebensphase/Abschnitt zwischen Kindheit und Erwachsenenalter (12 - 27 Jahre)“ (Jugend in Schröer 2009) oder als eine zeitlich greifbare Phase im Lebenslauf, in der eine „Anpassung an die Welt der Erwachsenen“ (Jean Piaget 1901 in Griese 1987, S. 28) stattfindet. Eine Beschreibung durch bestimmte Altersstufen und damit die Festlegung von Altersgrenzen gehen zurück auf Rousseau und andere Pädagogen, die die heutige Erziehungswissenschaft und Pädagogik bestimmten. Im 18. Jahrhundert entwickelte Jean-Jaques Rousseau (1762) ein Stufenmodell, um bestimmte Erziehungsetappen darzustellen, und setzte dabei Altersstufen für die Pubertät das 12. - 15. Lebensjahr und für die daran anschließende Adoleszenz vom 15. - 20. Lebensjahr fest (vgl. ebd., S. 20f). Die Einteilung in Altersstufen in der Entwicklung findet sich noch heute in der Literatur und bei insgesamt 13 Datensätzen taucht eine Altersdefinition gemeinsam mit der Beschreibung von Entwicklungsstufen auf, z. B. in den Kinder- und Jugendberichten oder einigen Veröffentlichungen zur Adoleszenzforschung32 und den frühen Überlegungen zum Generationenmodell33 . Das Konzept der Adoleszenz und einer Erweiterung des Stufenmodells von Rousseau geht zurück auf Stanley Hall (1904) und andere Wissenschaftlerinnen zu Beginn des 20. Jahrhunderts, 32 33 z.B. bei Stanley Hall (vgl. King 2013) z.B. bei Karl Mannheim (vgl. Griese 1987) 47 Herrschaft vs. Emanzipation Abbildung 4: Verteilung Disziplin Alter welche einzelne Phasen des Aufwachsens mit Alterskategorien versahen und Begriffe wie Postadoleszenz einführten (vgl. King 2013, S. 29ff). Karl Mannheim (1928), auf den das Generationenkonzept zurückgeht, bezieht sich für seine Altersangabe auf Eduard Spranger, um das Einsetzen des „selbstexperimentierenden Lebens“ (Mannheim in Griese 1987, S. 86) zu beschreiben. Dabei ist auffällig, dass die Vertreterinnen der Generationenkonzepte wie Margaret Mead oder Karl Mannheim nachrangig mit Altersangaben umgehen und sie als eine ca. Angabe verwenden. Die Altersgrenzen tauchen in den Daten, abseits der Disziplin Law-Acts-Programm-Policy, grundsätzlich eher weniger auf (ca. 33%). Es ergeben sich aus der Beziehung zu den Entwicklungsund Generationenkonzepten Überschneidungen zu den anderen Kategorien, sodass Handlungsfähigkeit fünf Mal und Rechtsfähigkeit und Kultur - Sozialisation je vier Mal gemeinsam mit dem Alter auftauchen (vgl. Tabelle 5). Der überwiegende Anteil der Altersangaben (ca. 67%) im Rahmen der aktuellen Daten geht auf gesetzliche oder programmatische Regelungen zurück (vgl. Abbildung 4). Schließlich lässt sich feststellen, dass das Alter eine untergeordnete und tatsächlich nachrangige Rolle in der Beschreibung von »Jugend« im wissenschaftlichen Kontext einnimmt. Mit einer Erhebung der Europäischen Union zu Altersdefinitionen von »Jugend« in Europa konnten von insgesamt 47 europäischen Mitgliedsländern sowie Weißrussland Altersangaben in den Graphen 48 Herrschaft vs. Emanzipation eingepflegt werden (vgl. Perovic 2016). Dabei reicht die Spanne der erhobenen Daten von null Jahren als Beginn, und bis 35 Jahre als Ende. Es zeigt sich kein einheitliches Bild einer verwertbaren Altersangabe, die »Jugend« allgemeingültig beschreiben kann. Auch in der umfassenden historischen Betrachtung von Mitterauer 1986 lassen sich keine konkreten und verwertbaren Altersangaben finden, die einzige Orientierung als Ende der »Jugend« bildet dort der sogenannte European marriage pattern 34 , der sogar bis in die Zeit des Römischen Reiches rekonstruiert werden kann. Das Alter ist, im Kontext von »Jugend«, historisch betrachtet, als ambivalent zu verstehen. Denn in der damaligen (Vorindustriellen) Zeit ist ein lediger 40-jähriger Knecht noch Jugendlicher im Sinne von »Jugend«, hingegen ein 20-jähriger verheirateter Bauer mit eigenem Hof ist ein Erwachsener. Die Festschreibungen von Altersgrenzen in gesetzlichen Regelungen sind ein relativ neues Phänomen, sie tauchen ab dem 18. und 19. Jahrhundert gemeinsam mit der Aufklärung und der Entstehung der Erziehungswissenschaften sowie der Pädagogik auf35 . Im gesamten Datenbestand fällt die Alterskategorie selten mit der Kategorie der Handlungsfähigkeit, der Kultur - Sozialisation und dem Verhalten zusammen. Zum Beispiel ist dies der Fall bei einem der ersten Vordenker der heutigen Erziehungswissenschaften, Cajetan von Weiller (1800). Der schreibt: „Die Jugend strebt gegen alle Beschränkungen ihrer sich entfaltenden Menscheit. Sie sucht jedes Hinderniß wegzurücken, das irgendeine Knospe der manigfaltigen Menschenkräfte, die sich jetzt in ihr zur Blüthe anschicken ersticken will.“ (Ruberg 2002, S. 138) Dass er dieses Streben ab dem 14. Lebensjahr beginnen lässt, aber kein Ende definiert, passt zu den anderen Herangehensweisen, die Alter in ihre Überlegungen einbeziehen. Es wird zwar ein Anfang der »Jugend« festgelegt, das Ende wird hingegen offengelassen. Auch der Anfang fällt in allen Daten sehr unterschiedlich aus und es scheint, dass er abhängig ist von der untersuchten Gruppe, ein präzises Alter lässt sich nicht bestimmen. Der Durchschnitt aus allen 71 vorkommenden Altersbestimmungen von »Jugend« liegt bei einer Altersangabe von 11,9 Jahren als Beginn. Wobei zu beachten ist, dass nur bei 63 ein genauer Anfang gesetzt wurde. Davon entfallen 52 auf Law-Acts-Programm-Policy, wovon wiederum 48 die länderspezifischen Angaben der Europäischen Union sind. Das Ende wird ebenfalls überwiegend von den gesetzlichen oder programmatischen Schriften bestimmt und fällt mit der Rechtsfähigkeit zusammen, welche auf Mündigkeit oder das Wegbrechen von Anspruchsvoraussetzungen abzielt. Ob diese Anspruchsvoraussetzungen sich auf Unterstützung oder die vollwertige Gleichbehandlung im Sinne des Strafgesetzbuches etc. beziehen, spielt keine Rolle. 34 Gibt das Heiratsalter an, welches historisch in Europa sehr hoch war. Dies lässt sich darauf zurückführen, „dass erst dann geheiratet werden darf, wenn der Mann die Stellung als Hausherr bzw. als Vorstand des Haushalts übernommen hat.“ (Mitterauer 1986, S. 28) 35 Im Zuge der Aufklärung definieren und etablieren sich die Erziehungswissenschaften und die Pädagogik als eigenständige Wissenschaften. Sie differenzieren sich zunehmend, durch zahlreiche Veröffentlichungen, aus (vgl. dazu Grunert 2020, S. 16). 49 Herrschaft vs. Emanzipation Als durchschnittliches Ende der »Jugend« kann hier 27,46 Jahre angegeben werden. Wenn dies mit dem bereits angesprochenen European marriage pattern in Verbindung gebracht wird, zeigt sich ein Wandel dieser Kategorie. Das durchschnittliche Heiratsalter betrug für 2020 in Deutschland bei Männern 34,9 Jahre und bei Frauen 32,4 Jahre36 . Diese Durchschnittswerte passen zu der überwiegend verbreiteten Auffassung von »Jugend« als Lebensphase innerhalb eines bestimmten Zeitraumes.37 Von besonderem Interesse ist, dass sich eine Festlegung von Altersgrenzen für »Jugend« nicht aus der bestehenden wissenschaftlichen Literatur der Bezugswissenschaften herleiten lässt, sondern dass der überwiegende Teil der Altersangaben (52 von 71) durch staatliche Definitionen oder damit verbundenen Auftragsstudien definiert wird. Zusammenfassend kann gesagt werden, dass erst ab Mitte des 18. Jahrhunderts und durch die aufkommende Erziehungswissenschaft von einem besonderen Ereignis oder wie Rousseau sagen würde von der Zweiten Geburt (vgl. Grunert 2020, S. 16), im Zusammenhang mit »Jugend«, Altersangaben gemacht werden. Davor wurde das Ende von »Jugend« maßgeblich über Initiationsriten38 oder über bestimmte Definitionen von Selbstständigkeit durch wirtschaftliche Unabhängigkeit oder Heirat bestimmt. Das, was wir über »Jugend« aus der Zeit wissen, entspringt hauptsächlich den Erzählungen von jungen Männern und war dort wiederum den jungen gebildeten Studenten39 (vgl. Mitterauer 1986, S. 248) vorbehalten. Der überwiegenden Lehrmeinung, dass »Jugend« mit der Aufklärung und der Industriellen Revolution entsteht, kann somit für die Angabe von Altersgrenzen zugestimmt werden. Im Wesentlichen gibt die Kategorie des Alters aufgrund der Dominanz des Disziplintyps council (zu dem Law-Acts-Programm-Policy unter Pkt. 4.1 zugeordnet wurde) keine Auskunft über das was »Jugend« ist. Es kann festgehalten werden, dass eine Trennung der Lebensphase vor dem Erwachsensein vorgenommen wurde, um die Erziehung und Menschwerdung als pädagogisch und erzieherisch zu begleitenden Abschnitt abzugrenzen, um nicht alles mit Kind zu bezeichnen. Denn als die Jugendlichen, im Sinne von noch nicht erwachsen, wurden in der Vergangenheit (vor Rousseau) alle bezeichnet, die noch nicht Erwachsene im Sinne der Gesellschaft waren. Es findet sich keine wirkliche Differenzierung der Begriffe vor der Aufklärung. In den frühen Jahren der Pädagogik und Erziehungswissenschaften tauchen die Begriffe Kind und »Jugend« oft auch gemeinsam auf. Deutlich wird dies an Formulierungen wie z. B. von Friedrich Adolph Wilhelm Diesterweg (ca. 1840) der die Gültigkeit der Aufgabe einer „menschlichen Bildung“ durch die 36 https://www.destatis.de/DE/Themen/Gesellschaft-Umwelt/Bevoelkerung/EheschliessungenEhescheidungen-Lebenspartnerschaften/Tabellen/eheschliessungen-heiratsalter.html verfügbar am 07.02.2022 37 Wie es das erwähnte Zitat von Schröer aus dem Handwörterbuch für Erziehungswissenschaften oder die Ausführungen von Zinnecker im aktuellen Handbuch für Offene Kinder- und Jugendarbeit (vgl. Witte, C. Schmitt und Niekrenz 2021) verdeutlichen. 38 Darunter werden alle Ereignisse gefasst die einen Übergang von dem Kindes- ins Erwachsenenalter beschreiben. In Religionen sind dies z. B. Ereignisse wie die Firmung, Konfirmation, Barmitzwa o. ä. Ereignisse. In vielen anderen Kulturkreisen werden dazu auch Ereignisse wie z. B. Wanderungen (Walk througs) oder diverse Mutproben gezählt. 39 Wie z. B. in der Zeit des Sturm und Drang oder der Burschenschaftsbewegung gut sichtbar. 50 Herrschaft vs. Emanzipation Schule „für alle Kinder des Volkes, für die Gesamtjugend des Staates, ohne Ansehen der Eltern“ (vgl. Geißler 2006, S. 139) beschreibt. Er verwendet dabei „Kinder“ und „Gesamtjugend“ nahezu gleichbedeutend, um die Zielgruppe von Bildung zu definieren. Es lassen sich auch keine wirklich greifbaren Altersangaben, abseits von staatlichen Festsetzungen finden, die einem wissenschaftlichen Konsens entsprechen. Vielmehr kann eine Beschreibung des Beginns einer Jugendphase eine Anfangszäsur darstellen, in der bestimmte neue und andere Herrschaftsstrukturen greifen müssen, da die Struktur der Familie zunehmend an Bedeutung verliert. Dies wird notwendig, um den Jugendlichen auf dem Weg zum vollwertigen und rechtlich gleichgestellten Erwachsenen, in eben diese Erwachsenengsellschaft gemäß der Vorstellung der Erwachsen, integrieren zu können (vgl. Mitterauer 1986, S. 79). Dadurch, dass ein Ende nicht wirklich definierbar ist und es sich vielmehr aus der Mündigkeit und der rechtlichen Gleichstellung ergibt, werfen die Kategorie und die Altersangaben vielmehr Fragen auf, als sie Antworten geben. Im Sinne unseres Widerspruches platziert sie die Identitäten von »Jugend« eindeutig im Sinne der Erwachsenengesellschaft. Als Dimension der Herrschaft lässt sich aus den Daten somit keine wirkliche Bedeutung für eine Theorie der Jugendarbeit feststellen. 5.2.2 Entwicklungsstufe Wie unter Alter (Pkt. 5.2.1) schon erwähnt, taucht die Kategorie der Entwicklungsstufe häufig mit der Bestimmung des Alters auf, wobei eine Einteilung von Entwicklungsstufen sich wesentlich differenzierter mit dem Phänomen der »Jugend« auseinandersetzt und sich in fast allen Disziplinen der Bezugswissenschaften Verweise auf Entwicklungsstufen finden lassen. Ihren Ursprung hat diese Einteilung ebenfalls in der Mitte des 18. Jahrhunderts und in den Werken der schon angesprochenen Pädagogen Rousseau oder Weiller. Auch Immanuel Kant (1790) spricht von einer sich in Stufen vollziehenden Entwicklung zwischen Kind und Erwachsenem, welche eine darauf angepasste Bildung und Erziehung notwendig macht (vgl. Riefling 2014). Ein auf Kant aufbauender Pädagoge und früher Erziehungswissenschaftler ist Friedrich Schleimacher (1826), er versucht Pädagogik und Erziehung so zu beschreiben, dass sie sich an den: „[...] jungen Menschen anschmiegt, mithin unter Berücksichtigung des jeweils schon vollzogenen individuellen Bildungsprozesses (der übrigens ebenfalls als Einigung von Natur und Vernunft zu verstehen ist) die äußere Welt so zugänglich macht, dass in ihr das strukturelle Nebeneinander sozialer und kultureller Lebenssphären, mithin die Gütergemeinschaften gleichsam nacheinander zugänglich werden - Erziehung wird also so organisiert, dass nach der Familie und der Schulzeit die entscheidenden Gütergemeinschaften, nämlich Kirche, Staat, geselliges Leben und Wissenschaft erlebt und erfahren werden.“ (Schleimacher in Winkler 2006, S. 93). 51 Herrschaft vs. Emanzipation Dies fällt zusammen mit der Kategorie - Kultur - Sozialisation - und setzt den Bildungsprozess in einen gesamtgesellschaftlichen Kontext, sodass auch Theodor Litt oder Klaus Mollenhauer 150 Jahre später sich auf Schleimacher beziehen und seinen Ausführungen eine gewisse emanzipatorische Perspektive zuschreiben. Dies führt dazu, dass die Entwicklungsstufe 16 Mal gemeinsam mit Kultur - Sozialisation auftaucht, was sogar drei Matches mehr (13 von 44) als in der Kategorie Alter sind. Es verwundert auch nicht, dass sich am meisten die Disziplin der Soziologie (39,3 %) und die der allgemeinen Sozialwissenschaften (14,8 %) mit Entwicklungsstufen auseinandergesetzt haben. Auffällig ist, dass ein Match von den Kategorien Entwicklungsstufe und Kultur - Sozialisation häufig mit integrierenden Beschreibungen und der klaren Beschreibung einer Übergangsphase „[...] des verlängerten Probierens, des vorläufigen Fußfassens und der aktiven, mobilen Integration in die Gesellschaft“ (nach Rosenmayr in King 2013, S. 30), zusammenfällt. Hier kann ein kausaler Zusammenhang zwischen dem Herausbilden der sogenannten ICH-Identität40 , wie sie unter Bezugnahme auf die Psychoanalyse von Freud in den Sozialwissenschaften u. a. bei Charlotte Bühler (1929) (vgl. Hafeneger 1995, S. 39ff) oder später bei Erik H. Erikson (1971) (vgl. Griese 1987, S. 64ff) eine große Rolle bei der Beschreibung von »Jugend« und den sich vollziehenden Prozessen, über mehrere Entwicklungsstufen hinweg, gespielt haben, hergestellt werden. Ausgeblendet wird bei einer derartigen Betrachtung häufig der gesamtgesellschaftliche Kontext und es kommt zu einer eher biologischen Beschreibung von »Jugend«, aus derer auch Verhalten und Krisen erklärt werden sollen. Was dazu führt, dass eine überschneidende Beschreibung von Verhalten (9 von 44) und im Besonderen von Handlungsfähigkeit (18 von 44) mit der Kategorie der Entwicklungsstufe zusammen fällt. Diese beiden Kategorien gehen im Rahmen der Beschreibung eines Entwicklungskonzeptes für Jugendliche davon aus, dass es aufgrund körperlicher Veränderungen zu [...] Unsicherheit, Orientierungslosigkeit, Konflikten und Spannungen in seinem Verhalten und in seiner Gesamtbefindlichkeit führen muss.“ (Kurt Lewin 1950 in ebd., S. 64ff). Das psychoanalytische Entwicklungskonzept beschreibt die Phase der »Jugend« als Übergangsoder Durchgangsphase, was heute noch so betrachtet wird, wobei es zu immer feineren Spezifikationen, besonders unter Bezugnahme auf die Lebenswelten der Jugendlichen, in der Darstellung kommt. Eine sehr wichtige Erkenntnis aus der Beschäftigung mit dem Konzept der Entwicklungsstufen ist, dass es einen bestimmten Moment in der Entwicklung eines Menschen gibt, an dem bestimmte psychische Aktivitäten stattfinden, die diese Phase zu etwas Besonderem macht. Dabei spielt die Handlungsfähigkeit eine wesentliche Rolle, da in eben dieser Phase Handlungsfähigkeit ausgebildet werden soll. Deshalb wird dieser Kategorie in manchen Lesarten eine emanzipatorische Komponente zugesprochen, z. B. Vera King, die sich intensiv und umfassend mit der Adoleszenz beschäftigt ausführt: 40 Beschreibt den psychischen Prozess bei dem das Kind sich über den Jugendlichen zu einem selbstständigen Individuum wandelt, welches in Abhängigkeit zu seiner Umwelt steht. Mit diesem psychoanalytischen Prozess bildet sich ein neues Selbstbewusstsein heraus. 52 Herrschaft vs. Emanzipation „Ohne Adoleszenz gebe es nicht nur keine Möglichkeit der Ablösung aus familialen Determinationen. Vor allem gebe es - so Erdheims Pointierung in einer kulturanalytischen Perspektive - ohne Adoleszenz keine Geschichte, keinen Kulturwandel. Mit der Adoleszenz erhalte der Mensch in seiner Ontogenese gleichsam eine „zweite Chance“, insofern die Prägung der Kindheit transformiert und somit, zumindest partiell und potenziell, überwunden werden könnten. Erst dadurch werden, so Erdheim, auf individueller Ebene die Voraussetzungen geschaffen für kulturellen Wandel, der - potenziell - mehr sein kann als nur eine Reproduktion des Alten in neuem Gewande. (King 2013, S. 11). Zusammenfassend lässt sich aus den Daten herauslesen, dass eine Beschreibung von »Jugend« als bestimmte Lebensphase, ob nun als Übergang oder ob sie „...durch die spezifische Verflechtung von Individuations- und Integrationsprozessen „abgrenzbar“ bleibt und einen „eigenständigen Stellenwert im menschlichen Lebenslauf (behält)“ (Hurrelmann in ebd., S. 42) überwiegend mit einer integrierenden Bedeutung versehen ist. Es wird versucht, bestimmte Verhaltensweisen auf fehlende Handlungsfähigkeit zurückzuführen, begründet mit bestimmten psychoanalytischen Erkenntnissen. Es ist aber nicht entscheidend „wie man diese Phase nennt, sondern ob es sie überhaupt gibt“ (Mitterauer 1986, S. 23). Aus historischer Perspektive lässt sich keine wirkliche Übergangsphase herauslesen, sondern vielmehr geht es darum, bestimmte Rollen einzunehmen. Dies ist nach Erik H. Erikson mit bestimmten Unsicherheiten und Krisen verbunden und führt zu Identitätsproblemen (vgl. Griese 1987, S. 71). Schlussendlich bedeutet dies wiederum eine „Anpassung an die Welt der Erwachsenen“ (ebd., S. 28), die beabsichtigt wird. Diese Anpassung wird mit einer gefährlichen Individualisierungtendenz aufgeladen, wodurch sich durch die „Verdinglichungs- bzw. Naturalisierungsphänomene“ (vgl. Jaeggi 2019, S. 88) die Beschreibung zunehmend von der tatsächlichen Realität der »Jugend« entfernt und damit auch entfremdet. Es werden durch eine solche Eigendynamik der immer spezielleren Beschreibung praktische Fragen des Umgangs, mit den für den jungen Menschen neu entstehenden Situationen, verdeckt. Durch die Entfremdung wird in gewisser Weise das Wachsen an den Widersprüchen und den Situationen und somit auch die eigenständige Entwicklung einer selbstbestimmten Handlungsfähigkeit von den Jugendlichen selbst entfernt. Auch wenn in der Vergangenheit einige Wissenschaftlerinnen die Entwicklungsstufen mit einer kritischen Perspektive betrachtet haben und darin eine Chance für gesellschaftlichen Wandel sahen, bleibt festzustellen, dass die Beschreibung von außen einer Identität »Jugend« dabei hilft, eben diese zu kontrollieren und damit verbunden auch in das existente gesellschaftliche System zu integrieren. Es kann entsprechend der gesammelten Daten festgestellt werden, dass es einen Veränderungsprozess im menschlichen Körper im Anschluss an die Kindheit gibt, wie man diesen jedoch nennt und ob eine Einteilung in Phasen hilfreich ist, ist für eine Begriffsbestimmung und die Frage nach der Jugendarbeitstheorie fraglich. Die vorliegenden Daten zeigen deutlich, dass es aus der Perspektive von Erwachsenen eine Interesse gibt, diesen Prozess zu beschreiben, um die Menschen, die sich in dieser Phase bewegen, zu kontrollieren, um sie darauf aufbauend in 53 Herrschaft vs. Emanzipation die Gesellschaft mit all den vorgegeben Normen und Werten zu integrieren. Dadurch dient diese Kategorie und die vorhanden Daten weniger dazu, »Jugend« zu beschreiben, sondern sie nützen vielmehr dazu, die scheinbar gewünschte Reproduktion der gesellschaftlichen Verhältnisse sicher zu stellen. 5.2.3 Rechtsfähigkeit Im Rahmen der erfassten Daten taucht die Rechtsfähigkeit insgesamt nur 14 Mal unter allen 174 erfassten Beschreibungen auf, gleichzeitig bildet sie eine sehr relevante Kategorie, die einen unmittelbaren Einfluss auf die Lebensrealität von »Jugend« darstellt. Wenn man die Benennung von »Jugend« und damit die Berücksichtigung von bestimmten Rechten für eben diese global betrachtet, lässt sich feststellen, dass von 198 UN Staaten 122 (im Jahr 2014) ein nationales Jugendprogramm haben oder sich mit »Jugend« beschäftigen (Perovic 2016, S. 7). Ein genauer Blick zeigt, dass die Rechtsfähigkeit jedoch nicht darauf abzielt, dass Jugendliche Rechte haben, sondern die Rechtsfähigkeit drückt im Kontext von »Jugend« vielmehr aus, wann sie bestimmte Rechte bekommen, um als gleichwertig und gleich innerhalb der Gesellschaft anerkannt zu werden. Dies drückt sich dann in der Festlegung und Beschreibung von Altersstufen aus, sodass es nicht verwundert, dass diese Kategorie je vier Mal mit einer Altersfestlegung und vier Mal mit der Beschreibung von Entwicklungsstufen zusammen fällt. Der 14. Kinder- und Jugendbericht definiert entsprechend dieser Kategorie dann »Jugend« auch als: „... diejenige Phase im Lebenslauf [...], in der der Prozess der Integration in die zentralen erwachsenenspezifischen Funktionen und Rollen bewältigt und zum Abschluss gebracht werden [...] [muss]. Dabei werden den Jugendlichen bestimmte, den Erwachsenen vorbehaltene Rechte und Pflichten vorenthalten - bzw. aus anderer Perspektive formuliert: Jugendliche werden vor den Anforderungen und Zumutungen bestimmter erwachsenenspezifischer Rollen- und Verhaltenszumutungen geschützt.“ (Bundesministerium für Familie 2013, S. 137) Es werden durch die Rechtsfähigkeit und die stufenweise Übergabe von vollwertigen Rechten, welche auch mit der Übernahme von Pflichten einhergehen, bestimmte hierarchische Ordnungen in der Gleichheit aufgebaut. Durch diesen Übergang soll dann eine Integration in die Gesellschaft der Erwachsenen stattfinden und dafür, dass diesen Anforderungen entsprochen werden kann, bedarf es der Herausbildung von Handlungsfähigkeit, welche auch vier Mal in Verbindung mit Rechtsfähigkeit auftaucht. Es kann hier also unterstellt werden, dass »Jugend« aufgrund von einer Abhängigkeit sich den erwachsenen Autoritäten unterordnen muss und deshalb eingeschränkte Rechte besitzt. Damit verbunden scheint es einen Integrations- und Schutzauftrag zu geben, da »Jugend« als potenziell gefährdet für sich und für die soziale Ordnung gilt (vgl. Lutz Roth in Scherr 2009, S. 20). Aus dieser Gefährdung heraus lassen sich auch die beiden Übereinstimmungen dieser Kategorie 54 Herrschaft vs. Emanzipation mit dem Verhalten erklären, denn wenn es eine potenzielle Gefährdung von Jugend gibt, gibt es auch einen Auftrag dieser Gefährdung entgegen zu wirken und damit kann die stufenweise Übertragung von Rechten und damit die Konstruktion eines Herrschaftsverhältnisses legitimiert werden. Historisch betrachtet stellt die Rechtsfähigkeit eher die Definition des elterlichen Herrschaftsverhältnisses dar (vgl. Mitterauer 1986, S. 43) oder markiert die Grenze und das Ende des Übergangs zur selbstverantwortlichen und realitätsangepassten Person (Theodor Scharrmann (1965) in ebd., S. 27). Die Kategorie der Rechtsfähigkeit stellt somit im Kontext von »Jugend« auf mehreren Ebenen eine sehr widersprüchliche Kategorie dar. Es kann davon ausgegangen werden, dass sich die Rechtsfähigkeit aus dem Gedankengang von »Jugend« als Übergangsphase herleitet und bedingt durch bestimmte Entwicklungsstufen das einzelne Individuum ab einem bestimmten Alter Rechte für bestimmte Sachen in Anspruch nehmen kann. Ab dem 18. Lebensjahr trägt es die vollständigen Rechte und auch Pflichten eines Erwachsenen, der gesellschaftlichen Norm, kann aber aufgrund von fehlender Anpassung, Reife oder Defiziten noch als Jugendlicher mit dem Status des Heranwachsenden (bis Vollendung 21. Lebensjahr in Deutschland)41 betrachtet werden. Es wird damit eine Art weiterführender Erziehungsauftrag eingeführt, der sich an der Reife und einer Erweiterung der Unmündigkeit und Gleichstellung gegenüber Erwachsenen bemisst. Diese hierarchische Struktur einer Unter- und Überordnung der Mündigkeiten und daraus abgeleiteten Erziehungsaufträgen steht u. a. einer Menschenrechtserklärung entgegen, in der jeder Mensch frei und gleich an Würde und Rechten geboren wird (vgl. Artikel 1 AEMR). In der neueren Konfliktauseinandersetzung wird dies häufig als das ‘Gleichheits’ - Paradox (vgl. Kallenbach und Christina Müller 2018, S. 107f) benannt und bietet viel Raum für eine philosophische Betrachtung. Wenn davon auszugehen ist, dass »Jugend« eine gesonderte Betrachtung von Rechten benötigt, eröffnet sich dadurch ein Raum, in welchem die Rechte eingefordert werden können, da der eigentlichen Gleichheit eine „Verrechnung“ (vgl. Rancière 2018, S. 19) zugrunde liegt. Es wird somit, zumindest für die festgelegte Altersgrenze, ein Verhandlungsraum eröffnet, in welchem dezidierte Rechte für Jugendliche, gegenüber der Gesellschaft der Erwachsenen verhandelt werden können42 . Wenn man der Logik von Rancière folgt, kann Emanzipation, im Sinne einer Definition von »Jugend« gemäß der Rechtsfähigkeit, gegenüber den Erwachsenen nur bis zur Vollendung des 18. Lebensjahres passieren, da ab dem 18. Lebensjahr der Mensch die vollen Rechte eines Erwachsenen erhält und somit alle gleich sind. Dies bringt nun die verschiedenen widersprüchlichen Ebenen der Kategorie zum Vorschein. Auf der einen Seite scheint es einen gewissen Zeitraum zu geben in welchem Jugendliche Rechte einfordern können, da sie, obwohl sie gleich geboren sind, 41 Das Jugendgerichtsgesetz (JGG) regelt noch einmal den Umgang mit Straftaten für Menschen zwischen dem 14. und 21. Lebensjahr. Die darin als Jugendliche (bis 18) oder Heranwachsende (bis 21) bezeichnet werden. Dort heißt es: „Die Anwendung des Jugendstrafrechtes soll vor allem erneuten Straftaten eines Jugendlichen oder Heranwachsenden entgegenwirken. Um dieses Ziel zu erreichen, sind die Rechtsfolgen [...] am Erziehungsgedanken auszurichten.“ (§2 Abs. 1 JGG ) 42 Ein Beispiel wäre hier die Herabsetzung des Wahlalters in Deutschland auf 16, da hier eine Ungleichbehandlung zwischen der rechtlich definierten »Jugend« und den Erwachsenen existiert. 55 Herrschaft vs. Emanzipation nicht gleich sind und sie sich darüber in den Diskurs bringen und die Grenzen der Gleichheit verhandeln können. Im Umkehrschluss bedeutet dies jedoch auch, dass mit dem Erreichen der Rechtsfähigkeit keine Verhandlung von Ungleichheiten mehr möglich wär, da die Gleichheit von Jugendlichen und Erwachsenen formal hergestellt wurde. Auf der anderen Seite nutzt die Kategorie Altersangaben, die den Erkenntnissen der Entwicklungsstufen und einer damit verbunden eingeschränkten Rechtsfähigkeit, bis zur Vollendung des 14. Lebensjahres, entspringen und führt diese fort. Zusammenfassend lässt sich auch aus der Kategorie der Rechtsfähigkeit keine wirkliche Beschreibung von »Jugend« entnehmen, die dem Begriff gerecht wird. Sie baut auf den Kategorien Alter und Entwicklungsstufe auf und verwendet diese in einem organisatorischen Zusammenhang, bezogen auf eine staatliche Ordnung von Rechten und Pflichten. Es kann auch kein wirklicher wissenschaftlicher Kontext zwischen der Rechtsfähigkeit und den erfassten Beschreibungen von »Jugend« festgestellt werden, dafür kommt sie in den gesammelten Daten zu selten vor. Wenn sie vorkommt, dann taucht sie eher als eine Feststellung in der Soziologie und der historischen Betrachtung und nicht als eine relevante empirische Größe auf. Jedoch gibt sie viel Auskunft über die Herrschaftsmechanismen, welche der Begriffsverwendung zugrunde liegen, denn wenn man den aufgegriffenen Gedanken von Jacques Rancière weiter spinnt, endet jedwedes emanzipatorische Potenzial von »Jugend« mit dem Erlangen der Rechtsfähigkeit, da »Jugend« de facto dann nicht mehr als ungleiche und eigenständige Gruppe, Kohorte oder Generation existiert. Da selbst aber die Kategorie Alter einen Begriff von »Jugend« verwendet, der weit über das 18. Lebensjahr hinausreicht und auch die Entwicklungsstufenkonzepte davon sprechen das „[...] eine „postadoleszente Phase“ [...] bis weit in das dritte, zuweilen sogar bis in das vierte Lebensjahrzehnt hineinreichen“ (Thole und Hornstein 2017, S. 463), hilft uns die Rechtsfähigkeit nicht bei einer Beschreibung. Sie zeigt vielmehr, dass dem Menschen zwar ein Übergang zwischen Kind und Erwachsenem zugestanden wird, und bezeichnet diesen dann als »Jugend«, ansonsten aber eine absolute und unveränderbare Gesellschaft der Erwachsenen existiert, welche aus »Jugend« heraus nicht mehr veränderbar ist. Wenn man Rechtsfähigkeit emanzipatorisch denken möchte, muss man es auf die Möglichkeit und das Recht, ein eigenständiges Leben abseits von der Kontrolle durch Erwachsene und pädagogischen Institutionen (vgl. Scherr 2009, S. 20), zu führen umdenken. Dabei steht hier jedoch die Mündigkeit und die Gleichheit, welche altersbedingt einsetzt, im Weg und platziert die Identitäten von »Jugend« eindeutig im Sinne der Herrschaft als eine sehr kurze Übergangsphase im Leben. 5.2.4 Verhalten In den Beschreibungen taucht die Kategorie des Verhaltens von »Jugend« insgesamt 48 Mal auf. Im Vergleich zu den anderen Kategorien ist im Besonderen zu berücksichtigen, dass sich an ihr die Kritik an der Begriffsverwendung und den damit verbundenen weiterführenden Kategorien 56 Herrschaft vs. Emanzipation sowie den Umgang mit »Jugend« durch Gesellschaft und im Besonderen durch Soziale Arbeit und Jugendarbeit ablesen lässt. Ihre Verwendung geht dabei insgesamt neun Mal mit einer Kritik der allgemeinen Beschreibung von »Jugend« einher, was ansonsten bei keiner der anderen Kategorien zu beobachten ist, außer sie stehen in direkter Verbindung mit dem Verhalten. Diese kritischen Beiträge fallen dann zusammen mit dem Auftauchen der Kategorie im Allgemeinen, ob es nun „jener Kampf ums Ganze“ (Gustav Wyneken 1914 in Hafeneger 1995, S. 33) oder die Beschreibung von »Jugend« als „... Kämpfer um den Sinn des Lebens [...] für eine zukünftige Ordnung der Welt“ (Siegfried Bernfeld 1914 in ebd., S. 37) ist. Es wird über diese Verwürfnisse zwischen einer sich formierenden »Jugend« und der existenten Gesellschaft der Erwachsenen versucht, im Sinne der Psychoanalyse das Verhalten von jungen Menschen zu erklären. Später wird das Verhalten in den Generationenkonzepten als die „unterschiedliche Antwort [..] auf generationentypische Erfahrungen [...] nach weltanschaulichen, sozialen, kulturellen und politischen Orientierungen“ (nach Karl Mannheim 1928 in ebd., S. 45) beschrieben. Margaret Mead (1971) sieht in dem raschen sozio-kulturellen Wandel und einer damit verbundenen Bindungslosigkeit, Desorientierung und grundhaften Unsicherheit die Erklärung für „typische“ jugendliche Verhaltensweisen (vgl. Griese 1987, S. 60f). Dieser negativ Trend der Beschreibung von Verhalten setzt sich mit einer zunehmenden wissenschaftlichen und forschenden Beschäftigung mit dem Thema fort. Die Vielzahl von Studien und ad hoc Theorien ab den 1980er Jahren haben daran einen erheblichen Anteil. Der in der deutschen Soziologie mit einer nicht zuordenbaren Sonderrolle vom einstigen Liberalen zum späteren konservativen versehene Helmut Schelsky (1963) beschreibt entstehendes deviantes und risikobehaftetes Verhalten bei Jugendlichen ebenfalls aus einer Verhaltensunsicherheit heraus. Diese entsteht durch die „voneinander verhaltensmäßig unabhängigen Sozialrollen des modernen Menschen“ (vgl. ebd., S. 107) und der Suche nach Sicherheit. Mit dieser Perspektive nimmt in den folgenden Jahren die Betrachtung des Verhaltens enorm zu, wollte die Erziehung und Pädagogik noch durch die Beschreibung des Verhaltens den Versuch unternehmen »Jugend« anzupassen und in die Gesellschaft der Erwachsenen zu integrieren (vgl. Kategorie Entwicklungsstufe, die sich neun Mal mit Verhalten überschneidet) nimmt nun die „Defizit- bzw. Störungsperspektive“ (Anhorn 2010, S. 40) den überwiegenden Raum bei der Betrachtung von »Jugend« ein. Somit kann z. B. eine Studie versuchen herauszufinden, welche Schwierigkeiten eine Jugendlicher bewältigen muss und eine Jugendarbeitstheorie entwickelt dann eine dazu passende Bewältigungsstrategie. Aus dieser Perspektive der Bewältigungsstrategien heraus ergibt sich mehr oder weniger ein sich auch in den Daten ablesbarer und eindeutiger Zusammenhang zwischen dem Verhalten und der Kategorie Handlungsfähigkeit, welche 23 Mal gemeinsam auftauchen. Wenn es in den 1970er Jahren noch die „mangelnde Handlungskompetenz [...] der Jugend“ (nach Ralf Bohnsack in Griese 1987, S. S. 165ff) war, dann sind es heute die radikal individualisierten Lebensläufe gemäß der kritischen Modernisierungstheorie (Ulrich Beck) die Risiken entstehen lassen, die Jugendliche ausbalancieren müssen (vgl. Thole und Hornstein 2017, S. 463f). 57 Herrschaft vs. Emanzipation Damit »Jugend« all dem begegnen kann, was die sozialwissenschaftliche und soziologische Forschung als problematisches Verhalten beschreibt, ist es notwendig, sie handlungsfähig zu machen, damit das Verhalten sich entsprechend der gesellschaftlichen Vorstellungen entwickelt. Womit auch eine starke Verbindung zwischen Verhalten und der Kategorie der Kultur - Sozialisation als logische Abfolge entsteht, denn Verhalten und Kultur - Sozialisation tauchen in den Daten sogar 25 Mal gemeinsam auf. Denn wenn wir davon ausgehen, dass sich durch »Jugend« das „jeweils historisch Neue [...] durch [...] abweichendes Verhalten ausdrückt“ (Böhnisch 2020, S. 37) dann entstehen durch den Blick der Jugendarbeit zwei grundhaft widersprüchliche Identitätsperspektiven zu »Jugend«, die durch die jeweilige Dimension (Herrschaft oder Emanzipation) bestimmt werden. Die emanzipatorische Identität möchte die Gleichheit mit der Welt der Erwachsenen nicht anerkennen da sie sich emanzipieren möchte und etwas Neues entstehen soll. Hier ist Verhalten emanzipatorisch mit der Kategorie Kultur - Sozialisation verbunden. Rancière beschreibt dies mit einer „Absage an das Soziale“ (Genel und Deranty 2021, S. 28) dem jedoch eine ästhetische Dimension innewohnt und sich in eben dieser ausdrückt. Diese Ästhetik könnte man bei einer genaueren Betrachtung in den sogenannten Jugend(sub)kulturen feststellen oder in bereits schon erwähnten Emanzipationsbewegungen identifizieren. Wenn sich diese Perspektive jedoch durchsetzt, wird eine eh schon krisengeschüttelte Gesellschaft mit einer ungewissen Zukunft in ihren grundsätzlichen Macht- und Herrschaftsverhältnissen infrage gestellt, sodass »Jugend« zwingend notwendig als Gefährdung und negativ betrachtet werden muss (vgl. Anhorn 2010, S. 40ff). Diese Betrachtung kann als die zweite, die herrschaftliche Identitätsperspektive, bezeichnet werden, wo die Kategorie Kultur - Sozialisation durch Herrschaft mit dem Verhalten verknüpft ist. Diese lässt sich über eine möglichst problematische Beschreibung des Verhaltens am besten ausdrücken und somit „... versucht die jeweils ältere Generation zwangsläufig immer auch, in die adoleszenten Neugestaltungsprozesse einzugreifen und auch intergenerational hegemoniale Vormachtstellungen zu behalten.“ (King 2013, S. 67). Womit Jugendarbeit eine Form der Integrationshilfe in die bestehenden gesellschaftlichen Verhältnisse leistet. Zusammenfassend lässt sich sagen, dass an dem Sinnzusammenhang der Platzierung von Verhalten eine bestimmte Richtung und Tendenz der Sinnzusammenhänge für die Kategorien Handlungsfähigkeit und Kultur - Sozialisation ablesbar ist. Die seismografische Eigenschaft der Kategorie zeigt sich somit in den Daten. Wenn Verhalten mit einem Defizitblick beschrieben wird, mündet dies meist in den Übergangsbeschreibungen, wo dem abweichenden Verhalten mit Herausbildung von Handlungsfähigkeit begegnet werden kann, um den Menschen dann in die bestehende Gesellschaft zu integrieren. Es kann dadurch eine bessere Reproduktion der gesellschaftlichen Verhältnisse stattfinden, die wiederum die bestehenden Macht- und Herrschaftsstrukturen stabilisiert, diese Logik ist mittlerweile sehr tief verankert in der Sozialpolitik und damit verbunden auch in der Sozialen Arbeit und der Jugendarbeit. Die Deutung von Verhalten als jugendspezifische Normabweichung hat sich somit von einem altersspezifischen, episodenhaft-passageren, entwicklungsbedingten und bisweilen gar als ent- 58 Herrschaft vs. Emanzipation wicklungsnotwendig erachteten psychosozialen Faktum gewandelt. Heute werden selbst die geringfügigsten Verhaltensabweichungen bzw. die unscheinbarsten Zeichen ihrer möglichen ‘Ausgeburt’ oder Realisierung [...] als erste, grundsätzliche ernstzunehmende Indizien gedeutet, denen als nur vordergründig harmlose ‘Symptome’ bei der Einleitung einer abweichenden ‘Karriere’ eine große prognostische Bedeutung zugeschrieben wird. Es entspringt über das Verhalten eine Straf-Politik und Maßregel-Pädagogik, die unter der eingängigen Prämisse des „Wehret-den-Anfängen“und der „Null-Toleranz“ danach trachtet, mit strikten, frühzeitigen, unmittelbaren, lückenlosen und harten strafenden Eingriffen der unterstellten ‘grassierenden Regellosigkeit’ unter Jugendlichen und jungen Erwachsenen wirksam einhalt zu gebieten. (vgl. dazu Anhorn und Stehr 2018, S. 12ff). Diese Feststellung ist nicht neu, Siegfried Bernfeld äußerte 1926 ähnliches, wenngleich ihn das seine Anerkennung innerhalb der pädagogischen und soziologischen Community kostete. Er schrieb: „Die bürgerliche Pädagogik erfüllt ihre Aufgabe eben wegen ihrer Unwissenschaftlichkeit. Sie ist ein Instrument des Klassenkampfes zugunsten des herrschenden Bürgertums.“ (Niemeyer und Naumann 2006, S. 272). Diese damals erwähnte Unwissenschaftlichkeit kann heute auf die einseitigen und sehr naturaliserenden und individualisierenden Betrachtungen und Beschreibungen von jugendlichem Verhaltensweisen bezogen werden, welche ihren Ursprung eher in der Sozialpolitik und nicht in der tatsächlichen Beschreibung von »Jugend« haben. Wenn demgegenüber Verhalten jedoch einfach nur als Anzeiger des krisenhaften gesellschaftlichen Zustandes gedeutet wird, dann entsteht eine völlig andere Perspektive, und es lässt sich ausgehend von dem Verhalten eine emanzipatorische Dimension eröffnen. Denn die noch zu betrachtende Handlungsfähigkeit bedeutet dann plötzlich eine kritische Reflexion der gesellschaftlichen Prozesse (vgl. Hirschfeld 2015a, S. 181). Das Verhalten enthüllt somit Widersprüche die ansonsten verdeckt werden (vgl. Heydorn 2004, S. 37), wenn diese Widersprüche offen daliegen entsteht ein komplett neuer Sinnzusammenhang, der ein emanzipatorisches Bildungspotenzial in sich trägt. Verhalten eignet sich deshalb als erste Kategorie, um tatsächlich »Jugend« beschreiben zu können. Aus der genauen Betrachtung des Verhaltens in einem gesellschaftlichen und soziokulturellen Kontext ergeben sich folglich die verdeckten Widersprüche und entfremdeten Lebenszusammenhänge innerhalb unserer Gesellschaft. Sofern die Objektbetrachtung nicht die Oberhand gewinnt und damit verbunden in eine pseudopädagogische Herrschsucht übergeht (vgl. Litt 1967), die schlussendlich die existierenden Macht und Herrschaftsverhältnisse reproduzieren will, wohnt dem Verhalten die Stabilisierungsdimension der Emanzipation inne. Dieses Innewohnen lässt sich anhand der vorliegenden Daten auch aus der bisherigen Theorie zu »Jugend« rekonstruieren und wenn der Betrachtungswinkel von Herrschaft und Emanzipation ausgeblendet wird, 59 Herrschaft vs. Emanzipation zeigt sich wissenschaftsübergreifend ein kausaler Zusammenhang zwischen dem Verhalten und den gesellschaftliche Zustände, die dadurch angezeigt werden. 5.2.5 Handlungsfähigkeit Dadurch, dass die eben beschriebene Kategorie des Verhaltens sowohl als Herrschafts- und als Emanzipationsdimension eine Platzierungsfunktion für die Identität »Jugend« erfüllen kann, verwundert es nicht, dass die Handlungsfähigkeit in den 174 erfassten Beschreibungen 59 Mal vorkommt. Darüber hinaus ist sie die am häufigsten mit anderen Kategorien interagierende, was sich ähnlich wie beim Verhalten aus einer Doppelrolle der Kategorie ergibt. Dabei fällt auf, dass sie so stark wie keine andere Kategorie zusammen mit der Kultur - Sozialisation (37 Mal) in Erscheinung tritt. Die Häufigkeit und die starke Interaktion lassen sich wesentlich besser und umfassender historisch rekonstruieren als bei den bisherigen Kategorien. Diese gingen meist nur mit der Auseinandersetzung der Menschwerdung und den damit verbundenen Überlegungen zu Erziehung und Pädagogik seit der Aufklärung einher. Die Bildung von Gemeinschaften, um im Sinne einer autonomen Jugendgruppe43 aktiv und besser handlungsfähig zu sein, als allein, kann bis ins frühe Mittelalter rekonstruiert werden. Jugendliche Gemeinschaften lassen sich bei den Zünften der Gesellen oder Bruderschaften bis ins 13. Jahrhundert (vgl. Mitterauer 1986, S. 196f) rekonstruieren. Dazu zählen auch Burschenschaften der Studenten und die informellen Zusammenschlüsse von jungen Menschen zur gemeinsamen Organisation von Tanzveranstaltungen, welche sich durch das Brauchtum und bestimmte Traditionen bis weit ins Mittelalter rekonstruieren und mit »Jugend« in Verbindung bringen lassen (vgl. ebd., S. 236ff). Diese Zusammenschlüsse dienten letztendlich dazu als Gruppe handlungsfähiger zu sein und um sich auf individueller Ebene von Eltern oder dem Handwerks- oder Lehrmeister zu emanzipieren. Da diese Überlieferungen stark mit einer männlichen Erzählung von »Jugend« verbunden ist, ist es wichtig und von besonderer Bedeutung, dass sich eine weibliche Erzählung von »Jugend« wahrscheinlich noch viel weiter rekonstruieren lässt. Dadurch kann dem Mythos, einer Entstehung von »Jugend« als eher männliches Phänomen, im Sinne eines geschlechtsneutralen Begriffes von »Jugend« widersprochen werden. Michael Mitterauer kann keine wirkliche Datierung machen, als er von den wahrscheinlich ältesten informellen Jugendgruppen spricht, aber er verweist darauf, dass es archäologische Expertise bräuchte um dies besser zu bestimmen. Es handelt sich dabei um die sogenannten Spinnstuben, welche als informelle Mädchengruppen bezeichnet werden können (vgl. ebd., S: 190ff) und die durch den Ausschluss der Mädchen und 43 Meint eine auf Freiwilligkeit basierende und unabhängig von den Organisationseinheiten der Erwachsenenwelt entstehende Gruppe. Dabei können im vorindustriellen Europa (dank des hohen Heiratsalters) drei unterschiedliche Typen ausgemacht werden. Zum einen die Gruppen, welche sich regional auf Gemeinde/Kommune/Stadt begrenzen, welche die sich als eigenständige Gliederung bei Vereinen ansiedeln und lose informelle Gruppen. (vgl. Mitterauer 1986, S. 96ff) 60 Herrschaft vs. Emanzipation Frauen aus dem sozialen und öffentlichen Freizeitleben, zu einer Art Ersatz für dieses gemeinschaftliche Zusammensein geworden sind. Mit diesem Blickwinkel erscheint die Entwicklung einer autonomen Persönlichkeit, wie sie später Rousseau beschreibt, plötzlich noch einmal anders, und die überwiegend männliche geprägte Beschreibung von »Jugend« wird durch eine historisch über die Handlungsfähigkeit rekonstruierbare weibliche Beschreibung ergänzt. Die dann später häufig mit »Jugend« einhergehende Beschreibung von Jugend(sub)kulturen lässt sich zudem eher über informelle Jugendgruppen als über die formellen Jugendgruppen in Vereinen, Verbänden oder Burschenschaften charakterisieren. Wesentlich für die Bildung und das Zustandekommen von informellen Gruppen kann die „Deinstitutionalisierung und Entritualisierung des jugendlichen Gemeinschaftslebens“ (Mitterauer 1986, S. 236f) ausgemacht werden. Diese Platzierung von eigenständigem sowie selbstständigem und damit verbunden dem kollektiven Erwerb von gemeinschaftlicher Handlungsfähigkeit bei den informellen Jugendgruppen wird als wesentlich für die Beschreibung der Kategorie erachtet. Zum einen bekommt die Beschreibung der Kategorie und darauf aufbauend eine allgemeine Beschreibung von »Jugend« dadurch eine historisch rekonstruierbare weibliche Entstehungsgeschichte und zum anderen ermöglicht es in zukünftigen Bearbeitung die Integration von jugend(sub)kulturellen Erscheinungen. Betrachtet man die Handlungsfähigkeit im Kontext von »Jugend«, dann zeigt sich ähnlich wie beim Verhalten, dass wir hier über zwei Denktraditionen sprechen. Eine zeigt sich heute durch eine robuste Sozialpolitik, die Normabweichungen als eine Art soziale Sicherheitslücke (vgl. Anhorn und Stehr 2018) betrachten und somit darauf bedacht ist, Handlungsfähigkeit so auszubilden, dass die eigene Existenz gesichert werden kann. Diese Existenzsicherung der Erwachsenen reicht so weit, dass es im Sozialgesetzbuch §11 Abs. 1 SGB VIII heißt: [...] sie zur Selbstbestimmung befähigen und zu gesellschaftlicher Mitverantwortung und zu sozialem Engagement anregen und hinführen. Dass es sich dann hierbei jedoch um die existente Gesellschaft mit ihren Normen und Werten und ihrer Zusammensetzung handelt ergibt sich aus zahlreichen Veröffentlichungen und Beschreibungen die »Jugend« eine mangelnde Kompetenz unterstellen. Dabei spielt die Gestaltung eines angeblichen Übergangs und die Bewältigung und Integration „[...] des jungen Gesellschaftsmitgliedes in ein zunehmend komplexer werdendes Netz von sozialen Erwartungen und Verpflichtungen, die mit der Herausbildung entsprechender Kompetenzen zur Teilnahme an den sozialen Interaktionsprozessen einhergeht.“ (Hurrelmann und Quenzel 2016, S. 33) eine tragende und leitlinienartige Rolle. Diese Lesart der Bewältigung „[...] von ökonomischer und sozialer Abhängigkeit, eingeschrenkten Rechten, pädagogischer Einwirkung und Qualifizierungszwängen [...]“ mündet in der praktischen Hilfestellung zur „sozialen Platzierung“ (Scherr 2009, S.24). Wichtig ist, dass eine derartige Betrachtung zusammen fällt mit der Problematisierung von 61 Herrschaft vs. Emanzipation Verhalten zu Beginn des 20. Jahrhundert. »Jugend« wird im Kontext der Generationenverhältnisse als zukünftiger „Träger von Kontinuität und Kultur (sowie des Geistes)“ beschrieben und es ist dort bereits Ziel durch pädagogisches Einwirken von „(fremden) Erwachsenen“ den „erwachsenen Willen“ freiwillig aufzunehmen und sich dann freiwillig unterzuordnen (vgl. Herman Nohl 1914 in Hafeneger 1995, S. 34f). Eine derartige Perspektive steht einer Handlungsfähigkeit im Sinne der informellen Gruppen diametral entgegen, denn sie soll sich „frei von der Einflussnahme durch Erwachsene“ (vgl. Hornstein nach Gills 1901 in Scherr 2009, S. 22) entwickeln können. Vielmehr sollte »Jugend« glauben: „[...] daß sie Flügel habe und daß alles Rechte auf ihre herbrausende Ankunft warte, ja erst durch sie gebildet, mindestens durch sie befreit werde. [...] Die Stimmen des Andersseins, Besserseins, Schönerseins ist in diesen Jahren so laut wie unabgenutzt, das Leben heißt ‘Morgen’, die Welt ‘Platz für uns’“ (Bloch 2019, S. 132) Dadurch ergibt sich mit der Entwicklung und Herausbildung von Handlungsfähigkeit eine unheimliche emanzipatorische Chance. Wenn »Jugend« nicht nur mit einer Übergangsphase erklärt wird, dann muss sie als Handlungs- bzw. Interaktionszusammenhang begriffen werden, der sich im Wesentlichen innerhalb von gleichaltrigen Gruppen vollzieht (vgl. Friedrich H. Tenbruck 1962 in Griese 1987, S. 126ff). Daraus ergibt sich, dass „heranwachsende [...] Potential gesellschaftlicher Veränderung hervorzubringen“ (Klaus Mollenhauer 1964 in Niemeyer und Rautenberg 2006, S. 336), welches zu einer emanzipatorischen Jugendarbeit und der Entwicklung einer emanzipatorisch betrachteten Handlungsfähigkeit geführt hat. Die Handlungsfähigkeit bewältigt demnach die aus den gesellschaftlichen Wirrungen, der Entstrukturierung und Entfremdung der Lebenswelten entstehenden Bedürfnisse der Jugendlichen. Damit schließt sich auch der Zusammenhang zum Verhalten, denn: „haben die Jugendlichen erst einmal zu ihren eigenen Bedürfnissen gefunden, sie quasi eingestanden, so können sie schlecht ihr prinzipielles Interesse an einer Verbesserung ihrer Lebenslage leugnen, wie sie es zuvor als eine Form von Abwehr getan haben“ (Kollan 1980, S. 103) Denn wenn darüber hinaus: „[...] klar ist, daß die Probleme Jugendlicher die Probleme der Gesellschaft, also Ausdruck der selben sind, umgekehrt eine Emanzipation der Jugendlichen von diesen Problemen und dieser Gesellschaft letztendlich nur möglich ist als eine allgemeine gesellschaftliche Emanzipation.“ (ebd., S. 16) Somit ist die Handlungsfähigkeit nicht zufällig eine der am stärksten in den Daten vorkommende und mit anderen Kategorien interagierende Platzierungskategorie, denn in ihr verbirgt sich die Art und Weise, wie der Widerspruch zwischen Herrschaft und Emanzipation bewältigt werden kann. 62 Herrschaft vs. Emanzipation Zusammenfassend kann festgestellt werden, dass die Handlungsfähigkeit stark mit der Kultur Sozialisation in Verbindung steht und dass diese starke Ausprägung auf die zentrale Rolle dieser Kategorie zurückzuführen ist. Eine Platzierung über die Handlungsfähigkeit von »Jugend« ist notwendig für eine Stabilisierung der Identität im Sinne der Herrschaft und eine damit verbundene Reproduktion der gesellschaftlichen Verhältnisse. Daraus ergeben sich eine Vielzahl an Theorien und Konzepten, die darauf abzielen, dass sich Jugendliche mit den durch die Institutionen44 zur Verfügung gestellten Ressourcen integrieren oder es zu einem Ausschluss und einer damit häufig einhergehenden Problembiografie kommt. Wer also, im Sinne der integrativen Funktion, diese Ressourcen nutzt „... um sich zu reproduzieren, unterwirft sich gleichzeitig der Selektivität der Institution.“ (Cremer-Schäfer 2001, S. 63). Diese Betrachtung von Handlungsfähigkeit könnte zukünftig auch eine Antwort liefern, warum es aktuell eine so geringe Teilhabe von jungen Menschen an den Angeboten der Jugendarbeit gibt. Durch die Überschneidung mit der Kategorien der Entwicklungsstufe (18 Mal) und dem Verhalten (23 Mal) kann daraus geschlossen werden, dass Jugendliche diese integrierenden und damit verbunden auch ausschließenden Mechanismen erkannt bzw. erfühlt haben und sich somit dem Zugriff durch die Institutionen entziehen wollen. Ob dies bewusst oder unbewusst passiert spielt dabei keine Rolle. Die Handlungsfähigkeit stellt sich somit als eine Art Homonym dar, wie es Rancière auch für die Politik anhand Platons Unterscheidung in Politeia und Politeiai (vgl. Rancière 2018, S. 75f) und einer damit verbundenen Differenzierung von la politique (dt. Emanzipation) und le politique (dt. das Politische) darlegt. Darauf baut er seine Theorie von Gleichheit und Ungleichheit innerhalb der Gesellschaft auf und markiert den Dissens als wesentlichen Faktor damit Politik existiert. Wenn nun Handlungsfähigkeit herausgebildet werden soll, um die Verhältnisse zu reproduzieren, wird der »Jugend« ein gewisser Rahmen zur Verfügung gestellt in welchem sie diesen Dissens kommunizieren können. Damit die Reproduktion der Macht- und Herrschaftsverhältnisse jedoch sicher gestellt werden kann, darf dies nur innerhalb des vorgegebenen Rahmens passieren. Ein besonders gutes Beispiel zur Konstruktion eines solchen Rahmens ist die umfassende Beschreibung von politischer Bildung und Beteiligung der »Jugend« im Rahmen des 16. Kinder- und Jugendberichtes der Bundesregierung. Dort heißt es: „Der Hintergrund für das Berichtsthema war und ist, dass demokratische Gesellschaften vor der dauerhaften Herausforderung stehen, Kinder und Jugendliche nicht nur in staatliche und soziale Strukturen zu integrieren (bzw. zu „sozialisieren“), sondern auch ihre Entwicklung als mündige Bürgerinnen und Bürger zu fördern, damit sie ihre Interessen in Politik und 44 Meint hier die Institutionen der Gesellschaft der Erwachsenen, zu denen auch Soziale Arbeit und Jugendarbeit als Teile des Staates verstanden werden. „Institutionen erfüllen [...] eine Reproduktionsfunktion für eine Ordnung; sie werden gleichzeitig von sozialen Akteuren benutzt, um an gesellschaftlich erzeugten Ressourcen zu partizipieren und um sich (als gesellschaftliche Subjekte) in einer Struktur zu reproduzieren.“ (Cremer-Schäfer 2001, S. 63) 63 Herrschaft vs. Emanzipation Gesellschaft vertreten und diese aktiv mitgestalten können.“ (Bundesministerium für Familie 2020, S. 41). Dies steht in einem harten Widerspruch zum Verständnis von Emanzipation und Dissens im Sinne von Rancière. Für ihn existiert Politik „... wenn die natürliche Ordnung der Herrschaft unterbrochen ist durch die Einrichtung eines Anteils der Anteilslosen.“ (Rancière 2018, S. 24). Die Anteilslosigkeit wird bei »Jugend« weit über die eigentliche Rechtsfähigkeit hinaus durch die Sozialwissenschaften dargelegt, indem sie aus der „[...] Ungleichheit eine Realität und aus der Gleichheit ein von diesem Ausgangspunkt aus zu erreichendes Ziel“ (Rancière in Genel und Deranty 2021, S. 120) definiert. Diese Zieldefinition legitimiert sämtliche Jugendforschung im Sinne von Problembeschreibungen und damit verbunden jedwede Konstruktion und Erfindung von immer neuen ad hoc Konzepten, die sich der Bewältigung annehmen. Damit also die natürliche Ordnung nicht gefährdet wird, wird für »Jugend« eine Anteilslosigkeit konstruiert, um einen politischen und emanzipatorischen Anspruch vorwegzunehmen. In dieser Denkperspektive bedeutet eine Handlungsfähigkeit Ausbilden, mit der konstruierten Anteilslosigkeit bzw. Ungleichheit zu brechen und die Gleichheit (welche sich z. B. aus §1 AEMR ergibt) einzufordern, bzw. durch das Erlangen von Handlungsfähigkeit in der Lage zu sein, dies gegenüber der Gesellschaft überhaupt zu können. Die Handlungsfähigkeit und das damit verbundene autonome jugendliche Individuum, welches sich, um handlungsfähig zu sein, in Gruppen zusammen findet, eignet sich somit sehr gut, um »Jugend« zu beschreiben und unseren Widerspruch zwischen Herrschaft und Emanzipation zu bearbeiten. Denn die Antwort auf diesen Widerspruch verbirgt sich in ihr. Wenn beschrieben werden kann, welche Stabilisierungsfunktion die Handlungsfähigkeit im Bezug auf die Identität »Jugend« einnimmt, dann entsteht in dessen Folge die Fähigkeit, »Jugend« zu beschreiben. Hier zeigt sich, dass »Jugend« nicht diese oft beschriebene Lebensphase und der Übergang ist, denn sonst wäre es in der Vergangenheit nicht zur Bildung von Jugend(sub)kulturen und weltweiten Jugendbewegungen gekommen. Diese haben sich, ohne jetzt alle einzeln zu betrachten, stets aus einer subjektiv empfundenen Ungleichheit heraus entwickelt und innerhalb der (Sub)kultur oder Bewegung wurden eigene individuelle Bewältigungsstrategien entworfen. Es wurde somit Handlungsfähigkeit abseits der Institutionen hergestellt und die Alltagsbewältigung und Bedürfnisbefriedigung hat zur Konstruktion eigener, bis ins hohe Alter anhaltenden Strategien geführt. Je mehr Handlungsfähigkeit also in »Jugend« ausgebildet wird, womit die eigenen Bedürfnisse befriedigt werden können, desto mehr „[...] wird sich bei den Menschen, die nicht von der Fäulnis der Gestrigen angesteckt und ihm verschworen sind, immer erhalten [...]“ (Bloch 2019, S. 132). Es schließt sich an die Handlungsfähigkeit auch eine Erklärung an, warum heute teilweise von extrem angepassten Jugendlichen gesprochen wird und es auf der anderen Seite Menschen weit über die »Jugend« hinaus gibt, die Teil einer Jugendbewegung oder (Sub)kultur waren bzw. es noch sind, die viel weniger an die Gesellschaft angepasst sind als andere Jugendliche. Somit lässt sich an der Handlungsfähigkeit völlig altersunabhängig »Jugend« erkennen. Wenn gleich durch 64 Herrschaft vs. Emanzipation die Entwicklungsstufen gut beschrieben ist, dass zu einem bestimmten Zeitpunkt im Leben eines jeden Menschen die Suche nach Handlungsmöglichkeiten unter Gleichaltrigen besonders groß ist. 5.2.6 Kultur - Sozialisation Wie anhand der bereits beschriebenen Kategorien - hier besonders Verhalten und Handlungsfähigkeit - sichtbar wurde, erhöht sich durch diese die Bedeutung der Kategorie Kultur - Sozialisation, sodass diese in den Daten 67 Mal vorkommt und fast so stark wie die Handlungsfähigkeit mit den anderen Kategorien interagiert. Besonders auffällig ist dabei, dass sie mit der Rechtsfähigkeit gar nicht und mit dem Alter lediglich vier Mal gemeinsam auftaucht. Wie aus den vorhergehenden Kategorien sichtbar wurde, steht sie besonders mit dem Verhalten (25 Mal) und der Handlungsfähigkeit (36 Mal) in Interaktion. Dass sie insgesamt 18 Mal mit der Entwicklungsstufe auftaucht, kann damit begründet werden, dass: „[...] die Pubertät auf der Ebene der Gehirnentwicklung eine der vitalsten und reichsten Phasen darstellt: Niemals vorher und hinterher hat man ein schärferes Sensorium für Gerechtigkeit und Ungerechtigkeit, für Wahrhaftigkeit und Lüge, für Klugheit und Dummheit“ (Welzer 2014, S. 248) Dies kommt dem zukünftig möglichem besseren Zustand von Immanuel Kant (Riefling 2014), dem Hindernisse Wegrücken von Cajetan von Weiller (Ruberg 2002) und dem Ursprung in der zweiten Geburt von Jean-Jacques Rousseau (Mitterauer 1986, S. 34) nahe. Zwischen der Aufklärung und der Gegenwart entstehen dann unzählige Theorien und Beschreibungen, die heute zu der Erkenntnis kommen, das »Jugend« eine bestimmte soziale Gruppe innerhalb der Gesellschaft ist, die enttraditionalisiert ist (Thole und Hornstein 2017) und die in völlig entstrukturierten, entgrenzten und pluralisierten Lebenswelten (Witte, C. Schmitt und Niekrenz 2021) unterwegs ist, in denen sie sich zurechtfinden muss. Darüber hinaus sind Jugendliche scheinbar mit der Vielfalt der Angebote überfordert und brauchen Hilfe durch die Erwachsenen. So zumindest lesen sich aktuelle Kinder- und Jugendberichte oder andere Studien, die sich mit »Jugend« beschäftigen. Die umfassende Ausgabe, des aktuellen Handbuchs für Offene Kinder- und Jugendarbeit (Deinert u. a. 2021) mit 2.030 Seiten in drei Bänden kommt da schon einem Manifest gleich. Dieses soll den Jugendarbeiterinnen eine Art Leitfaden sein, um die »Jugend« durch eben diese Angebotsvielfalt und die zerrüttete Welt zu führen, bis sie sich selbst darin zurechtfinden und Teil dieser geworden sind. Ranciére hingegen schreibt: „Wir müssen uns der Logik der Ungleichheit entledigen, die im wissenschaftlichen Diskurs am Werk ist, das heißt im Diskurs der Disziplinen, die solche Schilderungen angeblich erklären. Denn wenn Soziologen oder Sozialhistoriker sich im Namen ihrer Wissenschaft der 65 Herrschaft vs. Emanzipation Schilderung des Schreiners bemächtigen, machen sie seine egalitären Auftritt (performance) wieder zunichte. Sie heben die Abspaltung des Blicks des Parkettlegers von seinen Armen wieder auf und setzen eine andere Abspaltung an deren Stelle, die Ungleichheit verursacht. Denn sie ziehen eine Trennungslinie zwischen dem, was seine Sätze sagen, und dem, was sie bedeuten, zwischen ihrer nackten Materialität und der sozialen Situation, die sie zum Ausdruck bringen. Sie versagen dem Diskurs des Schreiners mithin einen eigenen Sinn.“ (Genel und Deranty 2021, S. 130) Wenn die Forschung und die insgesamt 14 Bezugswissenschaften, welche in den vorliegenden Daten vertreten sind, so weiter machen wie bisher, dann besteht das nächste Handbuch Offene Kinder- und Jugendarbeit womöglich aus neun Bänden und umfasst 6000 Seiten, in denen die Sätze des Schreiners oder eben das Verhalten der »Jugend« gedeutet und interpretiert werden. Der eigentliche Diskurs, den »Jugend« führen möchte und der sich über die Ästhetik und den Sinn von Verhalten ausdrückt, wird nicht geführt und wurde es vielleicht noch nie. Auch wenn es mit den bereits erwähnten Wissenschaftlerinnen wie z. B. Bernfeld, Litt, Mollenhauer, Heydorn oder Kollan durchaus Ansätze gab, die auf die in der »Jugend« wohnende Kraft zur Veränderung der gesellschaftlichen Verhältnisse geblickt haben, geht dieser Prozess durch die Wissenschaft und die ständige Problematisierung vollständig verloren. »Jugend« ist somit nicht nur eine gesellschaftliche Kategorie (vgl. Zinnecker in Witte, C. Schmitt und Niekrenz 2021), sondern »Jugend« ist im Sinne der Kategorie von Kultur - Sozialisation dazu in der Lage eine eigene Form von Gesellschaft zu sein, die mit den Macht- und Herrschaftsverhältnissen und damit mit der Gesellschaft der Erwachsenen im Diskurs um Gleichheit und Ungleichheit steht. »Jugend« ist somit Träger des kulturellen Wandels (vgl. Eduard Spranger 1924 in Hafeneger 1995, S. 41) und in der Lage soziokulturelle Konflikte zum Vorschein zu bringen. Sie wird damit im Rahmen der Kategorie zum Träger von Emanzipation, da so „die natürliche Ordnung [...] durch eine Freiheit unterbrochen ist, die die Gleichheit aktualisiert.“ (Rancière 2018, S. 29). »Jugend« ist auch nicht auf einen demokratischen Konsens aus, denn sie wollen aufs Ganze gehen (Gustav Wyneken in Hafeneger 1995, S. 33) und sind zu allem Neuen bereit (Karl Mannheim in Böhnisch 2020, S. 153). Konsens und Integration wird versucht, durch Soziale Arbeit und Jugendarbeit herzustellen, welche dadurch nicht emanzipatorisch ausgerichtet sein kann. Das »Jugend« in der Lage ist, den Prozess der Vergesellschaftung zu gestalten, konnte anhand der Handlungsfähigkeit historisch rekonstruiert werden und lässt sich an den Jugend(sub)kulturen und den Jugendbewegungen der letzten 100 Jahre ablesen. Interessant ist, dass Margaret Mead diese Platzierung und die Zuschreibung einer (Sub)Kultur der »Jugend« als Kritik beschreibt, da durch diese Platzierung einer (Sub)Kultur noch einmal eine Grenze zwischen der institutionalisierten Kultur der Erwachsenen und der der »Jugend« gezogen wird (vgl. Hafeneger 1995, S. 56f). Mead beschreibt auch, dass die »Jugend« durch die Performance, welche sie wie der Schreiner bei Rancière an den Tag legen zum Kultobjekt wird und es stylish und attraktiv ist, jugendlich zu wirken. Dadurch kommen sie als „authentischer Träger des Kultes in eine durchaus relevante soziokulturelle 66 Herrschaft vs. Emanzipation Machtposition“ (Heinzlmaier 2013, S. 126), mit der sie Veränderungen und Wandel leisten können. Dies ist ein weiteres Indiz für die Begründung einer sich ständig wandelnde Forschung mit den immer neuen Beschreibungen und der grenzenlosen Vermarktung von »Jugend«. Denn dadurch wird versucht, diese mögliche Machtposition der »Jugend« durch jugendlich wirkende Erwachsene nicht entstehen zu lassen. Darüber hinaus könnte unter »Jugend« mit einem solchen Blickwinkel auch die Arbeiterinnenbewegung und die Frauenrechtsbewegung gefasst werden. Denn in beiden Bewegungen empfanden junge Menschen etwas was eigentlich gleich sein müsste als ungleich. Durch Wissen und Bildung erlangten sie Kenntnis von der ursprünglichen Gleichheit und wollten diese verifizieren. Dies wurde möglich, indem sie handlungsfähig wurden und somit aktiv den sozi-kulturellen Wandel mitgestalteten. Zusammenfassend kann gesagt werden, dass die Kategorie der Kultur - Sozialisation für den meisten Diskussionsstoff sorgt und dass sie sicherlich unterschiedlich ausgelegt werden kann. Nach Heinz Joachim Heydorn (1970) lässt sich sagen, dass Bildung und somit Wissen über die eigenen Lebensrealitäten grundsätzlich im Widerspruch zur Reproduktion der gesellschaftlichen Verhältnisse und der Herrschaft steht. Denn mit und durch dieses Wissen entsteht ein Gefühl für einen gemeinschaftlichen Lebenszusammenhang unter allen die »Jugend« sind, diesen Prozess der Wissensaneignung und Menschwerdung bezeichnet er als emanzipatorischen Prozess (Heydorn 2004, S. 295). Durch diesen Prozess entstanden und entstehen Jugendbewegungen und sie werden weiter entstehen und einen Einfluss auf die Gesellschaft haben. Es kommt also nicht darauf an, der »Jugend« etwas Neues zu geben oder sie mit besonderen pädagogischen Ideen zu umgarnen. Es kommt darauf an, ihr Verhalten und ihr Leben kritisch werden zu lassen (Hirschfeld 2015a, S. 179) und somit Vergesellschaftung und Diskurs zwischen der Gesellschaft der Erwachsenen und der Gesellschaft der »Jugend« zu ermöglichen. Die vorliegenden Daten zeigen, dass »Jugend« eindeutig einen relevanten Einfluss auf die zukünftige Gesellschaft hat, damit jedoch dieser Prozess der gesellschaftlichen Entwicklung nicht unkontrolliert abläuft, braucht es genaue Beschreibungen und Erzählungen von »Jugend«. Diese gingen sogar so weit, dass der geschaffene Mythos von »Jugend« zum Motor des gesellschaftlichen Wandels auserkoren wurde (vgl. Theodor Litt in Hafeneger 1995, S. 73) und über die »Jugend« versucht wurde, die Fehler der Erwachsenen Gesellschaft auszubügeln. Es kann aus den Daten eine Steuerung und Kontrolle des sozialen Wandels und der Gesellschaft heraus gelesen werden, was an den unzähligen Beschreibungen des Übergangs in eine schwierige Welt deutlich wird. Die Stabilisierungsfunktion von »Jugend« bei dieser Kategorie ergibt sich meist aus der Funktion der Kategorie der Handlungsfähigkeit oder der des Verhaltens. In insgesamt 42 von 67 Fällen wird im Rahmen dieser Kategorie die »Jugend« sogar im Sinne der Emanzipation betrachtet, was nichts anderes bedeutet, als dass der »Jugend« grundhaft die Fähigkeit eingeschrieben ist, gesellschaftliche Veränderungen und sozialen Wandel zu gestalten, soziale Konflikte zu bearbeiten und sich somit von der übrigen Gesellschaft zu emanzipieren. Warum dieses Potenzial nicht genutzt wird, kann diese Arbeit nicht aufklären, aber um die „besondere Erzählung von der ‘Jugend’“ als Le- 67 Herrschaft vs. Emanzipation bensphase, als problematischer und schwieriger Übergang oder gar als Risiko „zu durchbrechen, heißt letztlich nichts anderes, als die vorgegebenen Macht- und Herrschaftsverhältnisse in Frage zu stellen.“ (Anhorn 2010, S. 41). 5.3 Der Zusammenhang von Sinn und Unsinn Wie im vorangegangen Abschnitt schon angedeutet, wurden die einzeln gebildeten Kategorien mit Sinnzusammenhängen versehen, welche die jeweilige Identität von »Jugend« im Rahmen der Arbeit entweder bei Herrschaft oder bei Emanzipation platzieren und somit das Stabilisierungsattribut darstellen. 5.3.1 Kontrolle der Identitäten Insgesamt konnten die einzelnen Definitionen 302 Mal mit den sechs Kategorien beschrieben werden, wovon 172 die »Jugend« im Sinne der Herrschaft und 130 im Sinne der Emanzipation stabilisieren (vgl. Tabelle 6). Durch die Erfassung der Altersgrenzen in den unterschiedlichen Ländern wird sie überproportional oft nach dem Alter (insgesamt 66 Mal) bei der Herrschaft platziert. Wenn allerdings die Kategorie Alter weggelassen wird, entsteht für die Herrschaft eine Summe von 106 und für die Emanzipation von 125. Weiterhin ist festzustellen, dass die Rechtsfähigkeit, bis auf eine Ausnahme, ausschließlich in der Herrschaft platziert ist und im Verhältnis zu allen anderen Kategorien relativ selten in den Definitionen auftaucht. Darüber hinaus baut die Kategorie der Rechtsfähigkeit nur auf der des Alters und der Entwicklungsstufe auf und versucht »Jugend« in einen gesetzlichen und damit institutionellen Rahmen zu pressen. Die Rechtsfähigkeit und das Alter sind trotz alledem sehr relevant für die zur Verfügungstellung von Mitteln im Rahmen des Wohlfahrtsstaates und damit für die Ausstattung und die Beschäftigung mit dem Thema »Jugend« im Rahmen der Sozialen Arbeit sowie in den Arbeitsfeldern der Jugendhilfe und der Jugendarbeit. Bei einem Verzicht auf Alter und Rechtsfähigkeit als die Extreme ergibt sich in der Betrachtung, dass »Jugend« dann von 218 Kategorien insgesamt nur noch 94 Mal durch die Herrschaft stabilisiert wird, hingegen aber 124 Mal durch die Emanzipation. Bei der Auswertung der Kategorien konnte festgestellt werden, dass die überwiegenden Erzählungen sich ausgehend vom Verhalten unterschiedlich auf die Betrachtung von Handlungsfähigkeit und Kultur - Sozialisation auswirken. Festzustellen ist allerdings, dass trotzdessen das Verhalten je 24 Mal gleich vorkommt, es zu unterschiedlichen weiterführenden Betrachtungen bei der Handlungsfähigkeit und der Kultur - Sozialisation kommt. Betrachtet man also diese drei Kategorien in Interaktion ergibt sich eine Summe von 174 wobei hier nur 64 Mal die Stabilisierungsfunktion der Herrschaft, aber fast doppelt so viel (110 Mal) die Emanzipation als Sinnzusammenhang angesprochen wird. 68 Herrschaft vs. Emanzipation Kategorie Emanzipation Herrschaft Summe Alter Entwicklungsstufe Rechtsfähigkeit Verhalten Handlungsfähigkeit Kultur - Sozialisation 5 14 1 24 44 42 66 30 12 24 15 25 71 44 13 48 59 67 Summe Summe ohne Alter 130 125 172 106 302 231 Tabelle 6: Auswertung der Kategorien mit dem jeweiligen Stabilisierungsattribut Es kann somit festgestellt werden, dass die grundsätzliche Identität von »Jugend« keine Identität ist, die sich eindeutig durch Herrschaft platzieren lässt. Es scheint vielmehr eher eine Grundtendenz hin zu einem emanzipatorischen Blick auf »Jugend« zu geben. Dadurch das sich die Realität von Jugendarbeit aber nicht emanzipatorisch darstellt und die Regelungen in den Gesetzen sowie die klare Strukturierung und Beschreibung als risikobehaftete Übergangsphase in der Gesellschaft dominiert, muss den Kategorien eine Hierarchie zugrunde liegen. 5.3.2 Sichtbarkeit durch Hierarchiemodelle Um diese Hierarchie darstellen zu können, wurden für die jeweiligen Kategorien modellhaft Faktoren eingeführt, um die jeweiligen Stabilisierungsattribute zu gewichten. Wenn allerdings die Logik der Herrschaft eine Machtstruktur darstellt, dann ist es notwendig, eine Hierarchie für die Dimension der Herrschaft zu bilden, welche sich an den Darlegungen zu eben dieser unter Punkt 2.1.2 orientiert. Demgegenüber sollte dann eine Hierarchie im Sinne einer emanzipatorischen Gegenmachtstruktur nach Punkt 2.1.1 entgegengesetzt werden. Beide Hierarchien orientieren sich dann an den jeweiligen Ausführungen zu den einzelnen Kategorien. Daraus ergeben sich die beiden folgenden Hierarchiemodelle: Herrschaft: Die Beschreibungen von Alter und Rechtsfähigkeit sind eindeutig dem Staat und seinen Institutionen zuzuordnen, somit können diese als oberste Ebene betrachtet werden und erhalten dadurch den Faktor vier. Die damit verbundenen wohlfahrtsstaatlichen Institutionen wie Wohlfahrtsverbände, Jugendämter und schlussendlich die Soziale Arbeit richten ihren Fokus und ihre Arbeit nach den Vorgaben des Staates und stehen mit diesem in Interaktion. Dies führt dazu, dass ein besonderer Fokus auf das einzelne Individuum gelegt wird und der Übergang anhand von verschiedenen Entwicklungsstufen und entsprechend dem jugendlichen Verhalten dargestellt wird. Die Entwicklungsstufen helfen dabei, »Jugend« als Verdinglichungs- und Naturalisierungsphänomen zu betrachten. Wohingegen durch das Verhalten ein problemzentrierter und sehr individualistischer Blick erzeugt wird. Dadurch, dass an diesen Kategorien Erziehungs- 69 Herrschaft vs. Emanzipation und Bildungsinhalte sowie bestimmte Maßnahmen festgemacht werden, welche die anschließenden Kategorien und deren Ausrichtung betreffen, erhalten sie den Faktor drei. Die Handlungsfähigkeit richtet sich stark nach den zuvor festgestellten Beschreibungen und bildet somit die praktische Realisierung und die Möglichkeit, Macht- und Herrschaftsstrukturen zu stabilisieren. Sie ist dadurch Dreh- und Angelpunkt von Jugendarbeit und Sozialer Arbeit und dem weiten Feld der Sozialwissenschaften, wo die grundsätzlichen Handlungsparameter erarbeitet werden und Konzepte und Methoden entstehen. Allerdings ist sie, eingebettet in diese Macht- und Herrschaftsstrukturen, sehr stark abhängig von den Vorgaben der vorgenannten Kategorien, sodass sie keine wirkliche eigenständige Rolle spielt und sich stets und immer wieder neu an dem sich ändernden Verhalten orientiert. Sie zeigt sich in einer Herrschaftshierarchie wie eine Fahne im Wind und erhält deshalb hier den Faktor zwei, auch wenn sie der zentrale methodische Arbeitsplatz von Jugendarbeit ist. Die für Diskussionsstoff sorgende Kategorie Kultur - Sozialisation wird mit dem Faktor eins ausgestattet, da hier deutlich zu erkennen ist, dass die Daten, welche bisher gesammelt wurden, sich eher darauf beziehen, festzustellen, dass diese Kategorie zwar einen Einfluss hat, es »Jugend« aber grundhaft nicht zugetraut wird, hier relevanten Einfluss zu nehmen. Wahrscheinlicher ist allerdings, dass diese Einflussnahme auf die Entwicklung unserer Gesellschaft abzielt. Damit diese koordiniert und in strukturierten Bahnen der bisherigen Gesellschaft abläuft, weshalb diese Kategorie in der Hierarchie der Herrschaft an letzter Stelle steht. Emanzipation: In der Betrachtung der Kategorien konnte festgestellt werden, dass sich Alter und Rechtsfähigkeit nicht eignen, um »Jugend« zu beschreiben und sie durch die Erwachsenen und somit auch durch die Wissenschaft teilweise beliebig konstruiert werden. Besonders deutlich lässt sich dies an den unterschiedlichen Altersangaben quer über Europa darlegen. In einer emanzipatorischen Hierarchie bilden sie somit die irrelevanteste und unterste Ebene und werden mit dem Faktor eins bestimmt. Die Entwicklungsstufe konnte als real existent identifiziert werden und es ist de facto so, dass in einem jeden Menschenleben ein biologischer Prozess der Veränderung stattfindet, der einen Wandel (die zweite Geburt) darstellt. Dadurch, dass diese real existente Gegebenheit vorhanden ist, erhält die Entwicklungsstufe den Faktor zwei, wenngleich durch die Adoleszenzforschung gut dargelegt werden kann, dass es sich hier um verschiedene Stufen handelt, beschreiben sie dennoch eine Tatsache, die auf alle Menschen zutrifft. Dadurch kommt es dazu, dass »Jugend« als Übergangsphase und als Abschnitt beschrieben wird, jedoch völlig individuell zu betrachten ist, womit die beschriebene radikalisierte Individualisierungslogik einsetzt. Deshalb ist es unter emanzipatorischen Gesichtspunkten wichtig, die Existenz der Phase und damit der Kategorie anzuerkennen, ihr aber eine nachrangige Rolle zuzuschreiben. Da sich das Verhalten als eine mit seismografischen Eigenschaften ausgestattete Kategorie und mit einer zufälligen Gleichverteilung der Dimensionen gezeigt hat, erhält diese wie bei Herrschaftshierarchie den Faktor drei, um die mit ihr verbundene Schlüsselrolle besser herausstellen zu können. Das Verhalten zeigt im Grunde die Missstände der Gesellschaft an und wirkt somit als eine Art Spiegel in beide Richtungen, ob nun integrierend oder emanzipierend, als sehr bestimmen- 70 Herrschaft vs. Emanzipation der Handlungsleitfaden. Anders als bei der Herrschaftshierarchie wird in einer emanzipatorischen Hierarchie die Kategorie der Handlungsfähigkeit als zentraler Moment für einen möglichen gesellschaftlichen und soziokulturellen Wandel betrachtet. Handlungsfähigkeit bedeutet hier kritisch die Verhältnisse zu hinterfragen und durch eine emanzipatorische Bildung innerhalb der Widersprüche handlungsfähig zu werden. Deshalb wird sie ebenso wie die Kultur - Sozialisation mit dem Faktor vier betrachtet. Wenn Emanzipation nach Rancière als eine Verifizierung der Gleichheit zu verstehen ist, dann ergibt sich ein Diskurs und ein Konflikt zwischen der Gesellschaft der Erwachsenen und der Gesellschaft der »Jugend«. Dieser Konflikt, der immer wieder neu durch die Verifizierung von Gleichheit ausgetragen wird, endet erst, wenn wir in einer utopischen Gesellschaft der reinen Gleichheit und Hierarchiefreiheit leben, in der es keinerlei anteilslose Individuen mehr gibt. Die »Jugend« lässt sich als Träger dieses Konfliktes beschreiben, weshalb diese Kategorie auch anders als bei der Herrschaft mit dem höchsten Faktor bewertet werden muss. Zusammenfassung: Durch die Einarbeitung der jeweiligen Faktoren im Sinne der Hierarchisierung der Kategorien erscheinen die vorliegenden Daten anders und drücken wesentlich deutlicher aus, was »Jugend« ist und vor allem, wie sie durch die Gesellschaft platziert wird. Damit wird der in den Daten im Besonderen durch Anhorn, Mitterauer, Mead, Hafeneger, Griese, Bühler (Johannes-Christoph), Bernfeld, King, Litt und Olk geäußerten kritischen Perspektive auf die Beschreibung von »Jugend« Rechnung getragen und es können dadurch die bereits identifizierten Kategorien für eine Beschreibung noch einmal verifiziert werden. Obwohl die Datenmengen, welche in dem aktuellen Datensatz zur allgemeingültigen Beschreibung von »Jugend« vorhanden sind, relativ unterschiedlich verteilt sind (insbesondere die vielen Daten zum Alter). Die Auswertung der Daten entsprechend der unterschiedlichen Hierarchiemodelle zeigt deutlich, dass eine emanzipatorische Hierarchie das Alter (trotz der vielen Daten) gemeinsam mit der Rechtsfähigkeit und den eigentlich in den Grunddaten stark vertretenen Entwicklungsstufen in den Summen weit hinter sich lässt (vgl. Tabelle 7). Eine Implementierung der Hierarchie zeigt das Verhalten, Handlungsfähigkeit und die Kultur - Sozialisation eine enorme Bedeutung einnehmen, hingegen in der Hierarchie der Herrschaft das Verhalten und die Entwicklungsstufen alles beschreiben, was dann im Rahmen der Handlungsfähigkeit umgesetzt wird (vgl. Tabelle 8). Diese Umsetzung sollte dann am besten im Sinne einer Reproduktion erfolgen, da die Kultur - Sozialisation in der Hierarchie der Herrschaft nur eine nachrangige Rolle einnimmt. Besonders fällt auf, dass in der Herrschaftshierarchie die Sinnzusammenhänge, selbst ohne eine Berücksichtigung des Alters, die emanzipatorischen Zusammenhänge (248 zu 265) knapp dominieren und sie somit mehr oder weniger unterdrücken. Dies ist eine sehr spannende Feststellung, denn bei einer Betrachtung unter der Emanzipationshierarchie zeigt sich, dass mit Alter (450 zu 370) und folglich auch ohne (445 zu 304) die emanzipatorischen Sinnzusammenhänge deutlich überwiegen. 71 Herrschaft vs. Emanzipation Kategorie Alter Entwicklungsstufe Rechtsfähigkeit Verhalten Handlungsfähigkeit Kultur - Sozialisation Faktor Emanzipation Herrschaft Summe 1 2 1 3 4 4 5 28 1 72 176 168 66 60 12 72 60 100 71 88 13 144 236 268 Summe Summe ohne Alter 820 749 Tabelle 7: Sinnzusammenhang Emanzipationshierarchie Kategorie Alter Entwicklungsstufe Rechtsfähigkeit Verhalten Handlungsfähigkeit Kultur - Sozialisation Faktor Emanzipation Herrschaft Summe 4 3 4 3 2 1 20 42 4 72 88 42 264 90 48 72 30 25 284 132 52 144 118 67 Summe Summe ohne Alter 797 513 Tabelle 8: Sinnzusammenhang Herrschaftshierarchie Es kann also davon ausgegangen werden, dass die Hierarchie der Sinnzusammenhänge einen starken Einfluss darauf hat, wie »Jugend« zu betrachten ist. Wenn davon auszugehen ist, dass sich das Verhalten, egal unter welchem Betrachtungswinkel, immer gleich zeigt, dann ist es ausschlaggebend, wie auf das Verhalten geschaut wird und welcher Sinnzusammenhang dafür hergestellt wird. Die aktuelle Zusammensetzung unserer Perspektive auf »Jugend« kann als eine eindeutig herrschaftliche betrachtet werden, was dazu führt, dass »Jugend« immer wieder anders beschrieben wird und sich aus den Beschreibungen immer wieder neue Erklärungen für die Jugendarbeit ergeben. Dies führt dazu, dass die Kategorie Kultur - Sozialisation eine absolut nachrangige Rolle (Summe 67) innerhalb einer Herrschaftshierarchie einnimmt, hingegen in einer Emanzipationshierarchie eine absolut Dominanz (Summe 268) entfaltet. Durch die Modelle soll gezeigt werden, dass das Verhalten von Jugendlichen ist wie es ist und dass es in einem direkten Zusammenhang mit der Kategorie Kultur - Sozialisation steht. Denn völlig unabhängig vom Verhalten (immer 142) wird die Ausprägung der Kategorie Kultur - Sozialisation eindeutig durch den Blickwinkel auf eben dieses bestimmt. Ist das Verhalten ein Problemanzeiger von individuellen Problemen, die bearbeitet werden müssen, oder ist es ein Anzeiger von gesellschaftlichen und sozialen Konflikten, die einen sozialen Wandeln implementieren. Spannend ist dabei auch, dass eine Herrschaftshierarchie anscheinend mit viel weniger Handlungsfähigkeit (118) umgehen kann, dies lässt sich ggf. erklären durch einen Ausschluss, der 72 Herrschaft vs. Emanzipation stattfindet, wenn das Individuum sich nicht integrieren möchte. Dieser Ausschluss wird dann abseits von Sozialer Arbeit, Jugendarbeit, Bildung und Erziehung reguliert und bestätigt die Erläuterungen von Ahnhorn, von einem ständig robuster werdenden Sozialstaat, der sich mit Straf-Politik in einen Sicherheitsstaat verwandelt (vgl. Pkt. 5.2.4). Auch knüpft diese Schlussfolgerung an die Logik von Rancière45 und seinen Ausführungen zu Politik und Polizei an. Denn tatsächliche Emanzipationsprozesse können nur stattfinden, wenn der polizeilichen Ordnung eine neue Gemeinschaft oder Gruppe hinzugefügt wird. Genau in diesem Prozess entsteht für ihn Politik. Wenn also gemäß der Herrschaftshierarchie die Entstehung von Handlungsfähigkeit der »Jugend« in einem geringen und durchaus kontrollierbarem Maß beschrieben werden kann, kann die polizeiliche Ordnung die damit entstehenden Prozesse abfedern und der gesellschaftliche Wandel (Kultur - Sozialisation 67 in Herrschaftshierarchie) bleibt kontrollierbar und kann durch die Institutionen begleitet werden. Wenn sich die Handlungsfähigkeit hingegen gemäß der Emanzipationshierarchie (236) entfalten kann und sich wie am Beispiel der Jugend(sub)kulturen und Jugendbewegungen eigene Vergesellschaftungsprozesse mit eigenen Gemeinschaften etablieren, dann wird der soziale und gesellschaftliche Wandel (268) nicht mehr kontrollierbar durch die polizeiliche Ordnung. »Jugend« wird somit hier zum Initiator von Politik. Damit dies aber kontrollierbar bleibt, wird »Jugend« von den Institutionen der Wissenschaft und Forschung eben hauptsächlich über das Verhalten und die Entwicklungsstufen in einer Herrschaftshierarchie beschrieben, somit bleibt »Jugend« und der gesellschaftliche Wandel organisierbar. Eine authentische und auch entsprechend der gesammelten Daten nachvollziehbare Beschreibung von »Jugend« muss eine Emanzipationshierarchie der Sinnzusammenhänge anerkennen und »Jugend« als eigenständiges soziokulturelles Wesen mit einer eigenen Form der Vergesellschaftung akzeptieren. Damit geht jedoch einher, dass soziale und gesellschaftlichen Konflikte aufbrechen und die etablierten Macht- und Herrschaftsstrukturen durch sich neu bildende Strukturen angegriffen werden, weil nach Jacques Rancière der polizeilichen Ordnung eine nicht vorgesehene Gemeinschaft hinzugefügt wird oder nach Silvia Staub-Bernasconi oder Björn Kraus eine Gegenmacht entsteht. 45 Neben den grundsätzlichen Überlegungen zu Emanzipation und der Politik, trennt Rancière grundsätzlich in Politik und Polizei. Polizei ist bei ihm die grundsätzliche Ordnung aller Körper in einer Gesellschaft mit Institutionen und dem vorhanden sein einer polizeilichen Ordnung (Polizei versteht er nicht im Sinne der Exekutive). Politik hingegen tritt hervor wenn die Gleichheit verifiziert werden soll. Also wenn grundsätzlich alle Menschen frei sind, Frauen aber kein Wahlrecht besitzen dann führt die Verifikation von dem angeblich gleichen, was real nicht so ist, zu einem Emanzipationsprozess. Dort entsteht für ihn Politik. Ein Streik der nur Reformen und keine Verbesserungen fordert oder der Autoritäten anprangert ohne bessere Gehälter zu fordern ist für ihn noch nicht politisch, da kein Streit entsteht. (vgl. Rancière 2018, S. 40ff) 73 Herrschaft vs. Emanzipation 6 Es ist an der Zeit Im folgenden Kapitel soll zu aller erst möglichst kurz und präzise eine eigenständige Definition von »Jugend« entsprechend der Ergebnisse dieser Arbeit entworfen werden. Darüber hinaus soll dann mit dieser Definition der Widerspruch und die Eingangsfrage der Theorielosigkeit beantwortet werden. 6.1 Von »Jugend« zu Jugend In der vorliegenden Arbeit sollte die Uneinigkeit bezüglich der Begrifflichkeit von »Jugend« dargestellt werden, indem diese stets markiert wurde, um herauszustellen, dass es scheinbar keinen eindeutigen Begriff von »Jugend« gibt. Durch die Erfassung von möglichst allgemeingültigen Beschreibungen, Erzählungen und Bildern wurde versucht, herauszufinden, ob es möglich ist, einen eben solchen einheitlichen Begriff mittels einer phänomenologischen Netzwerkanalyse unter Beachtung der Grundgedanken von Norbert Elias und Harrison White zu entwickeln. Der Analyse lag dabei stets ein kritischer und reflexiver Blick zugrunde, mit dem versucht wurde, sich nicht auf eine Wissenschaft oder eine praktische Beschreibung festzulegen, sondern eben auch diese Vielseitigkeit und die damit verbundenen Probleme zu berücksichtigen. Es wurde möglich, durch insgesamt sechs Kategorien die einzelnen Beschreibungen zu bewerten und darauf aufbauend Sinnzusammenhänge zu bilden und diese modellhaft in zwei unterschiedliche Hierarchien einzubetten. Schlussendlich ergab bereits die Kategorieauswertung, dass sich bestimmte Kategorien zur Beschreibung von »Jugend« eignen und bestimmte nachrangig zu betrachten sind. Eine Betrachtung des Alters, um »Jugend« zu beschreiben, ergab, dass dies mit einer gewissen Willkür versehen ist, welche sich dann ebenfalls in der Rechtsfähigkeit niederschlägt. Es ließen sich keinerlei wissenschaftliche Fakten zur Sinnhaftigkeit von Altersgrenzen finden. Die Rechtsfähigkeit konnte als eine Art Macht- und Herrschaftsübergabe von dem elterlichen zum individuellen und damit auch zum gesellschaftlichen Herrschaftsverhältnis identifiziert werden und eignet sich am wenigsten, um »Jugend« zu beschreiben. In den Beschreibungen zu den Entwicklungsstufen und damit verbunden auch zu den Generationenmodellen konnte ein real existierender und biologisch begründbarer Anfang von »Jugend« identifiziert werden. Denn auch historisch lässt sich feststellen, dass im Leben eines jeden Menschen ein bestimmter Wandel stattfindet. Wenn man sich die Daten genauer anschaut, lässt sich dieser psychoanalytisch und auch biologisch erklären. Es ist also eine Tatsache, dass »Jugend« im Anschluss an die Kindheit definitiv einen Anfang hat, der sich jedoch vollkommen individuell bei jedem Menschen zu einem anderen Zeitpunkt abspielt. Ein Endpunkt ließ sich auch über die Entwicklungsstufen nicht feststellen und wirkte wie beim Alter sehr beliebig und willkürlich. Es ist also ein bisschen wie raten, wenn man für den Anfang oder das Ende ein Alter festlegt, besser ist zu sagen, es passiert im Anschluss an die Kindheit, 74 Herrschaft vs. Emanzipation ohne dies genau einzugrenzen. Die dann daran anknüpfenden individuellen Entwicklungsstufen, welche in Summe als Adoleszenz bezeichnet werden, helfen dabei, die menschliche Entwicklung zu verstehen, für eine Beschreibung von »Jugend« erscheinen sie jedoch irrelevant. Durch die vorhandene Tatsache der zweiten Geburt 46 entwickelt der Mensch ein bestimmtes und sehr viel feineres Gespür für die Welt, die ihn umgibt. Das Individuum testet ab diesem Zeitpunkt anders und intensiver seine Grenzen aus, wie weit es gegenüber vorhanden Strukturen der erwachsenen Gesellschaft gehen kann und versucht sich vehement von diesen durch Verhalten abzugrenzen. In dem Verhalten steckt jedoch viel mehr als nur die bloße Abgrenzung von der Gesellschaft der Erwachsenen. In ihm verbergen sich tief liegende Bedürfnisse, die durch die ihn umgebende Welt nicht befriedigt werden können, diese Bedürfnisse können rein individueller Natur sein können, aber auch auf einen soziokulturellen und gesellschaftlichen Konflikt hinweisen, den der junge Mensch noch nicht versteht, aber in dieser Zeit spüren und fühlen kann. Das Verhalten dient demnach als eine Art von Kommunikation, von der der junge Mensch Gebrauch macht, um mit der Welt um ihn herum in Interaktion zu treten. Diese Form der Kommunikation und Interaktion wandelt sich genau wie die Welt, die den jungen Menschen während dieser Zeit umgibt, weshalb eine sich ständig wandelnde Welt auch eine sich ständig wandelnde Lebenswelt47 bedeutet. Das Verhalten kann somit hier als eine Stimme oder auch viele Stimmen von jungen Menschen verstanden werden, die zwar laut sind, aber noch keine Form der Sprache darstellen. Die Sprache ist jedoch wichtig, um entweder den Konflikt aufbrechen zu lassen, um somit Widerstand gegen die Herrschaft und die mit Konflikten belastete Gesellschaft der Erwachsenen zu leisten und um den Konflikt ggf. zu beseitigen bzw. die Gleichheit aus der eigenen Lebenswelt heraus zu verifizieren. Oder aber es geht darum, sich selbst mit der Gesellschaft und den Konflikten durch Sprache zu verständigen, um selbst Teil dieser Gesellschaft werden zu können. Beide Möglichkeiten des Sprechens benötigen Handlungsfähigkeit, die sich ausgehend vom Verhalten dann entwickeln muss. Da wir eine Definition von »Jugend« und nicht eine von der Verständigung mit der Gesellschaft der Erwachsenen suchen, wird folgend nur die Sprache des Widerstandes für den jungen Menschen relevant sein. Handlungsfähig zu sein und somit im Sinne des Widerstandes zu sprechen, bedeutet die eigene Lebenswelt durch Bildung kritisch werden zu lassen. Ausgehend von der kritischen Lebenswelt bedarf es zur Handlungsfähigkeit hier die Kooperation mit anderen jungen Menschen, die ähnlich sprechen und nicht nur laut sein wollen. Es müssen somit Handlungsgemeinschaften gebildet werden, in denen man die nun durch die kritische emanzipatorische Bildung bekannten Konflikte für sich bearbeiten kann, um diese 46 Der von Rousseau eingeführte Begriff beschreibt nach wie vor sehr gut diesen Moment und damit den nicht definierbaren Zeitpunkt. 47 Es wird hier der Begriff der Lebenswelt nach Habermas bewusst aufgegriffen, da dieser Kommunikation und auch Interaktion anhand dieses Begriffes beschrieben hat. Darüber hinaus ermöglicht der Begriff als Ressource Widerstand gegen Herrschaft und auch gleichzeitig eine Verständigung mit dieser (Genel und Deranty 2021, S. 13) 75 Herrschaft vs. Emanzipation dann der Gesellschaft der Erwachsenen einschreiben zu können. Diese Handlungsgemeinschaften gewinnen durch Größe als Bewegungen oder (Sub)Kulturen an Macht, welche hier als emanzipatorische Gegenmacht bezeichnet werden soll. Wird diese Einschreibung ohne eine Gemeinschaft oder nur eine kleine Gemeinschaft von ebenfalls jungen Menschen gemacht, hat diese wenig Aussicht auf Erfolg, um den Konflikt zu bearbeiten und damit die Gleichheit zu verifizieren. Die Einschreibung der Konfliktbewältigung in die Gesellschaft der Erwachsenen entfaltet dann die soziokulturelle und gesellschaftliche Veränderungskraft, da die Bewältigungsstrategie der Handlungsgemeinschaft die geltende Gleichheit der Erwachsenengesellschaft neu festlegt. Dieser Prozess kann nicht mit einer Altersangabe festgeschrieben werden, denn er beginnt immer wieder neu und mit jedem neu geborenen Menschen besteht die Möglichkeit, dass sich der Prozess wiederholt. Je mehr Bewältigungsstrategien für Konflikte durch die Handlungsgemeinschaften gefunden werden, desto länger kann der junge Mensch an der Emanzipation und damit der Verifizierung von Gleichheit teilhaben, auch wenn er vom Lebensalter her schon als alt betrachtet wird. Durch die kritische Selbstreflexion besteht immer wieder aufs Neue die Möglichkeit, Konflikte mit der Gesellschaft der Erwachsenen aufzudecken und sich Handlungsgemeinschaften zur Konfliktbewältigung anzuschließen. Voraussetzung ist dabei jedoch, dass der Weg der Verständigung gemieden wird, denn dieser führt zu einer schrittweisen Integration in die Gesellschaft der Erwachsenen und dem Ende von »Jugend«. Das Ende von »Jugend« bemisst sich demnach an der Menge von Integrationsprozessen in die Gesellschaft der Erwachsen und einer damit erfolgten Unterordnung in die bestehenden Macht- und Herrschaftsverhältnisse. Ich schlage deshalb die folgende Definition von Jugend vor: Die Jugend ist eine Form der Vergesellschaftung und der Bewältigung von individuellen und sozialen Konflikten. Sie beginnt aufgrund biologischer und psychischer Veränderungensprozessse im menschlichen Körper im Anschluss an die Kindheit und endet mit der Integration in die Gesellschaft der Erwachsenen. Die Integration zeichnet sich aus durch eine Akzeptanz der bestehenden Macht- und Herrschaftsverhältnisse und den damit verbundenen Konflikten. Jugend wird sichtbar durch verschiedenste Verhaltensweisen, die innen liegende und noch unbekannte Bedürfnisse nach außen kommunizieren. Diese Verhaltensreaktionen sind in den ersten Jahren nach der Kindheit besonders stark ausgeprägt und erfüllen den Zweck der Abgrenzung gegenüber der Gesellschaft der Erwachsenen. Gleichzeitig repräsentieren sie individuelle und soziale Konflikte, welche in diesen ersten Jahren unbewusst gespürt und durch das Verhalten ausgedrückt werden. Sie ist gekennzeichnet durch das Erlangen von Handlungsfähigkeit und der damit einhergehenden selbstständigen Bewältigung der individuellen und sozialen Konflikte. Die Handlungsfähigkeit entsteht in Gemeinschaften von Menschen, welche sich in einer ähnlichen 76 Herrschaft vs. Emanzipation Lebensrealität und Lebenswelt befinden, sie wird dadurch zu einer kollektiven Bewältigungsstrategie. Diese Gemeinschaften können sich als kleine informelle Gruppen oder aber als globale Bewegungen und (Sub)Kulturen mit einer besonderen Ästhetik und Sprache zeigen, über die sie mit der bereits vorhandenen und etablierten Gesellschaft kommunizieren. Ihr wesentliches Merkmal ist die Fähigkeit, Emanzipationsprozesse zu gestalten, aus denen sich Forderungen ergeben, die dazu dienen, Gleichheit zu verifizieren, um dadurch die bisherige Gleichheit der Erwachsenen zu erweitern. Durch die Fähigkeit, sich von den Erwachsenen zu emanzipieren, werden soziale Konflikte bewältigt und Gesellschaft neu gestaltet. 6.2 Herrschaft vs. Emanzipation Unter Pkt. 2.1.1 wurde beschrieben, dass sich der Widerspruch zwischen Herrschaft und Emanzipation auf zwei unterschiedlichen Ebenen darstellt. Mit der nun vorliegenden Beschreibung von Jugend soll der Widerspruch entsprechend der Ebenen dargestellt werden. 6.2.1 Die Ebene der Jugendlichen Wenn Jugend eine Form der Vergesellschaftung und damit eine Form der Konfliktbewältigung durch das Erlangen von Handlungsfähigkeit ist, dann stellt sich auf dieser Ebene der Widerspruch als ein fortwährender Konflikt zwischen der Gesellschaft der Erwachsenen (Herrschaft) und der Gesellschaft der Jugend (Emanzipation) dar. Was nichts anderes bedeutet, als dass dieser Widerspruch existiert, seitdem es Menschen gibt und das er fortwährend existieren wird. Der Widerspruch zwischen Herrschaft und Emanzipation ist auf dieser Ebene also ein Gesellschaftlicher zwischen Jugend und Erwachsenen. Auch wenn der Begriff Jugend erst seit ca. 150 Jahren aktiv verwendet wird, ist durch den fortwährenden gesellschaftlichen Wandel und damit einhergehende Emanzipationsprozesse davon auszugehen, dass es Jugend schon immer gab und sie damit kein Kind der Moderne ist. Die Frage, welche gestellt wurde, wollte wissen, wie dieser Widerspruch zum Vorschein kommt und wie er sich zeigen lässt. Offensichtlich wurde, dass die bisherigen und dargestellten Beschreibungen von Jugend sich nur sehr selten eigneten, um eben genau diesen Widerspruch zum Vorschein zu bringen. Es konnte vielmehr festgestellt werden, dass es zwar derartige Beschreibungen des gesellschaftlichen Wandels gab, diese aber eine nachrangige Rolle einnahmen oder Jugend darin eher als Motor für den gesellschaftlichen Wandel und die Bewältigung der sozialen Konflikte gesehen wurde, der durch die nun herrschenden Erwachsenen selbst nicht geleistet werden konnte. Da festgestellt wurde, dass ein überwiegender Teil der Beschreibungen von Jugend darauf abzielt, sie über die Erlangung von Handlungsfähigkeit in die bestehende Gesellschaft zu integrieren, kann davon ausgegangen werden, dass der Widerspruch gemieden werden soll. 77 Herrschaft vs. Emanzipation Wenn er sich zeigt, könnten dadurch nicht mehr kontrollierbare Prozesse der gesellschaftlichen Veränderung einsetzen und dies würde der grundhaften Ausrichtung der aktuellen sozialstaatlichen Politik entgegenstehen. Für unsere Betrachtung ist es somit noch nicht einmal relevant, in welcher philosophischen und machttheoretischen Erscheinungsform sich der Widerspruch zeigt, sondern es kommt momentan darauf an zu akzeptieren, dass es diesen Widerspruch überhaupt gibt. „Conflict is the essential core of a free and open society.“ (Alinsky 1989, S. 62) Mit den Worten von Saul D. Alinsky, ist der Konflikt damit der wesentliche Kern unserer freiheitlichen Gesellschaft und nur wenn dieser Kern immer wieder neu verhandelt oder durch neue Gleichheitseinschreibungen verifiziert wird, wie es Rancière ausdrücken würde, können die sozialen und verdeckten Konflikte unserer Gesellschaft bearbeitet werden. Wenn Erwachsene Jugend als Gefahr, Risiko oder Problem verstehen, dann werden sie den Widerspruch nicht anerkennen. Sie gehen vielmehr davon aus, dass das Wesen und die Werte von Jugend negativ sind und meinen das Jugend durch ihr Verhalten und „[...] wilde Kleidungs- und Haarstile mit der Vorliebe für ohrenbetäubenden Lärm, sei es Rap oder Rock oder nur Disco-Sound, und mit der ständigen Suche nach Mitteln und Wegen um den Abstand zur etablierten Welt deutlich zu machen“ (Dahrendorf 1994, S. 275) nicht in der Lage ist die Konflikte zu bearbeiten. Die vorliegende Arbeit hält dem entgegen, dass Jugend Träger von Bewältigungsprozessen der sozialen Konflikte ist und in der Lage ist, diese sich entfaltende Handlungsmacht zur Bewältigung der Konflikte der Gesellschaft von Erwachsenen einzuschreiben. Den Widerspruch und die Konflikte drückt sie aus durch ihr Verhalten und mit einer eigenen Ästhetik (Kleidung, Haarstil, Kunst, Medien, etc.) und was für Erwachsene wie Lärm scheint ist einfach nur eine eigene Form der Sprache (Rap, Rock, Disco, Musikstile allgemein, etc.). Die Voraussetzung für eine Kommunikation ist schlussendlich die Anerkennung des Widerspruches und Konfliktes aufseiten der Herrschaft, was jedoch impliziert Jugend als relevante Größe und Form der Vergesellschaftung anzuerkennen, ihre Ästhetik zu akzeptieren und ihre Sprache zu verstehen. 6.2.2 Die professionelle Ebene Durch die vorliegende Definition von Jugend lässt sich der Widerspruch zwischen Sozialer Arbeit und Jugendarbeitstheorie logisch darlegen und beschreiben. Die Soziale Arbeit als allzuständige und in Deutschland den Abhängigkeiten des Sozialstaates unterworfene Profession kann, wie zuvor die Erwachsenen als Herrschaft und mächtig beschrieben werden. Wie anhand der Daten gezeigt werden konnte, zielt sie mit der Beschreibung von Jugend als Lebensphase und Übergang darauf ab, Jugend in die bestehende Gesellschaft der Erwachsenen 78 Herrschaft vs. Emanzipation zu integrieren, sodass diese reproduziert und stabilisiert wird. Dieser integrierende Ansatz kann auf den Ursprung der Sozialen Arbeit in der Fürsorge zurückgeführt werden und hat sich durch die bessere Kenntnis über die Problemlagen und Lebenswelten zunehmend individualisiert. Es konnte festgestellt werden, dass die Beschreibungen von Jugend überwiegend darauf abzielen, die Verhaltensweisen von Jugendlichen zu erforschen, um darauf aufbauend individuelle Handlungsfähigkeit herzustellen, damit sich die Jugendlichen in der Gesellschaft der Erwachsenen zurechtfinden und Kompromisse mit den Konflikten eingehen. Die Jugendarbeit als Arbeitsfeld der Sozialen Arbeit spielt dabei eine wesentliche Rolle und passt ihre Handlungsstrategien an die Gesellschaft an, um die Integration der Jugendlichen zu ermöglichen. Es konnte jedoch ebenfalls festgestellt werden, dass genau diese Integration in bestehende Macht- und Herrschaftsverhältnisse von Anfang an einer Kritik unterworfen war und dass sich diese Kritik wie ein roter Faden durch die Beschreibungen von Jugend gezogen hat. Dabei fällt auf, dass die kritischen Perspektiven, wie z. B. die in dem Klassiker Was ist Jugendarbeit? (vgl. C.W. Müller u. a. 1972) vorgestellten Ideen für eine emanzipatorische Jugendarbeit von Müller, Giesecke, Mollenhauer und Kentler, der Ansatz der emanzipatorischen Bildung von Heydorn (vgl. Heydorn 2004) oder aber die Bedürfnisorientierte Jugendarbeit von Kollan (vgl. Kollan 1980), ihre Handlungstheorie zwar um Emanzipation, als zentralen Begriff entwickelten, aber keinen eigenen Begriff von Jugend entwarfen oder festschrieben, an welchem ihre Definition ansetzte. Sie grenzten sich jedoch deutlich von der damaligen Praxis der Jugendarbeit und Pädagogik ab, da sie darin kein emanzipatorisches Potenzial sahen. Dies führte dazu, dass ihre Ideen zwar dem jeweiligen Zeitgeist entsprachen und damit die zu der Zeit wirkenden Sozialarbeiterinnen und noch viel mehr die Jugendlichen beeinflussten, ebenso wie es zurzeit der Wandervögel und großen Jugendverbände Bernfelds Ideen taten (vgl. Niemeyer und Naumann 2006), sie aber in der sich ständig wandelnden Gesellschaft und somit der sich ständig wandelnden Sozialen Arbeit an Relevanz verloren. Die praktische Jugendarbeit ist somit in einen Strudel geraten, der fortwährend aktualisiert wurde und heute kaum noch zu durchschauen ist, dadurch werden die essenziellen Konflikte innerhalb der Wissenschaft nahezu vollständig verdeckt. Dabei sind die Überlegungen zu einem theoretischen Unterbau vernachlässigt worden und wurden von der Sozialarbeitsforschung und den Problembeschreibungen, die zu normativen Handlungsansätzen führten, verdrängt. Um einen kritischen Diskurs über Jugendarbeit führen zu können, bedarf es einer empirisch-analytischen Basis, von der aus der Diskurs dann grundsätzlich geführt werden kann. Deshalb sollte sich eine Jugendarbeitstheorie als eine eigen- und selbstständige Theorie verstehen, die von einem Jugendbegriff aus gedacht wird, welcher nicht Teil der existierenden Macht- und Herrschaftsverhältnisse ist. „Jugendarbeit ist also [...] eine Sphäre, welche sich nicht emanzipieren kann, ohne sich von allen übrigen Sphären der Gesellschaft zu emanzipieren.“ (Kollan 1980, S. 16) Demnach kann Jugendarbeit und eine sich entwickelnde Jugendarbeitstheorie nicht Teil von 79 Herrschaft vs. Emanzipation Sozialer Arbeit sein, denn sie verfolgt im Sinne der in dieser Arbeit herausgestellten Beschreibung von Jugend grundhaft andere Ziele. Sie zeigt sich als ein durchaus lebenslanger Prozess, der sich an der Entstehung von Emanzipationsprozessen orientiert und diese dann begleitet. Dabei sind Handlungsmethoden wie z. B. die unter Punkt 2.3 beschriebene kollegiale Selbstverwaltung, deren Entwicklung, Konkretisierung, praktische Erprobung, Beobachtung und wissenschaftliche Analyse ein wesentlicher Bestandteil. Eine Jugendarbeitstheorie ist dann nicht mehr nur eine Randerscheinung in dem breiten Feld der Sozialen Arbeit, in welchem sie stets um Anerkennung und Legitimation kämpft, sie wird damit zu einer eigenständigen Wissenschaft und hat sich im Denken orientiert (vgl. Pkt. 2.2). Aus dieser Perspektive wird auch ein interdisziplinäres Arbeiten in Theorie und Praxis verbessert und sicherlich ist ein Austausch zwischen der Sozialen Arbeit und der Jugendarbeit notwendig, jedoch im Sinne der Emanzipation und nicht mehr im Sinne der Integration einer Jugendhilfe48 . Der Widerspruch stellt sich somit „[...] als Moment der Diskontinuität, als eine - wenn auch zunächst nur gedankliche - „Unterbrechung“ in der Kontinuität einer eingespielten Praxis Sozialer Arbeit“ dar. Die Unterbrechung und der Widerspruch sind „[...] auf eine grundsätzliche Problematisierung von Macht- und Herrschaftsverhältnissen gerichtet [...], d.h. auf gesellschaftlich erzeugte Unterdrückungs- Ausbeutungs- und Ausschließungsverhältnisse, auf ungerechtfertigte Beschränkungen kollektiver und individueller Selbstbestimmungsmöglichkeiten, auf Mechanismen der Disziplinierung und Normalisierung etc.“ (Anhorn, Bettinger u. a. 2012, S. 7). Eine Jugendarbeitstheorie ist folglich ein Teil einer kritischen Sozialarbeitswissenschaft, da sie darauf ausgerichtet ist, Methoden der Handlungsfähigkeit zu entwickeln, welche die eben genannten Verhältnisse und Beschränkungen problematisiert und in den gesellschaftlichen Diskurs um Gleichheit und Gerechtigkeit immer wieder neu einschreibt. 6.3 Theorielosigkeit im Kontext Sozialer Arbeit Es ergibt sich aus der Definition von Jugend und den dargelegten Widersprüchen, dass es zum gegenwärtigen Zeitpunkt keine eigenständige Jugendarbeitstheorie gibt. Es gibt vielmehr einen Bereich in Wissenschaft und Praxis, der sich entsprechend einem sich ständig wandelnden und bisher völlig undurchsichtigen Bild von Jugend mit eben dieser beschäftigt. Da die bisherige Auseinandersetzung sehr diffus stattgefunden hat und im Rahmen dieser Arbeit aus insgesamt 14 Wissenschaften Informationen zusammen getragen wurden, kann festgestellt werden, dass es sehr viel Wissen über Jugend und damit verbunden auch über Jugendarbeit gibt. Dieses Wissen gilt es zukünftig mithilfe der vorliegenden oder einer ähnlichen Methode zu analysieren, um 48 Die Jugendhilfe mit ihren Bereichen wie z. B. Hilfen zur Erziehung, Inobhutnahme, etc. wirkt übergreifend auch für Kinder und entspricht einem Schutzauftrag des Kindeswohls, welcher hier nicht infrage gestellt werden soll. Jedoch soll hier klar zwischen diesen Aufträgen unterschieden werden. 80 Herrschaft vs. Emanzipation dadurch das gesammelte Wissen entsprechend des Jugendbegriffes zu strukturieren. Dabei sollte eine Strukturierung unter Bezugnahme auf die wesentlichen Kategorien Verhalten, Handlungsfähigkeit und Kultur - Sozialisation stattfinden. Es ist grundlegend davon auszugehen, dass in den entstandenen Handlungstheorien viel Wissen enthalten ist, was einer Jugendarbeitstheorie zugeführt werden kann und damit zur Theoriebildung beiträgt. Das Hauptaugenmerk muss jedoch zwingend auf einer kritischen Reflexion und einem emanzipatorischen Erkenntnisinteresse liegen, ob die bisherigen Daten aus Studien und sonstigen Erhebungen aus der Sozialarbeitswissenschaft zu verwenden sind, muss überprüft werden. Sicherlich müssen die Rohdaten entsprechend der Kategorien anders interpretiert werden. Die Chance für eine Jugendarbeitstheorie liegt darin, dass der gesamte Bereich der (Sub)Kultur und die Bewegungen global zahlreiche Handlungsmethoden entwickelt haben, die Auskunft darüber geben, wie Emanzipationsprozesse historisch verlaufen sind und vor allem was funktioniert und was nicht funktioniert hat. Eine Theorie gemäß des entwickelten Begriffes von Jugend unterscheidet sich insofern grundlegend von Sozialer Arbeit, dass sie das Individuum mitdenkt und durchaus auch entsprechend seiner Bedürfnisse in den Vordergrund rücken kann, grundsätzlich die Handlungsfähigkeit aber darauf ausgerichtet ist, kollektive Gemeinschaften zu entwickeln, welche in der Lage sind, Gleichheit neu zu verhandeln. 81 Herrschaft vs. Emanzipation 7 Wie weiter? - Ausblick Die vorliegende Arbeit beinhaltet mehrere Arbeitsaufträge und Fragestellungen, die sich aus der Erarbeitung der Methode und der nunmehr vorliegenden Definition von Jugend ergeben. Für den wissenschaftlichen Kontext bildet diese Masterarbeit einen Auftakt für weiterführende Forschungen. Eine genauere Darstellung der Methode und wissenschaftstheoretische Vertiefung wird als notwendig erachtet, da hier natur- und geisteswissenschaftliche Methoden ineinanderfließen. Darüber hinaus ergeben sich aus den unterschiedlichen wissenschaftstheoretischen Ansätzen vertiefende Aufträge für die Interpretation. Da es in der Arbeit auch um einen Theorie - Praxis Transfer ging, sollten die Erkenntnisse der Arbeit in Verbindung mit der bereits erarbeiteten Konzeption Dorf der Jugend (vgl. Burdukat 2021) noch einmal für die Praxis aufgearbeitet werden. Durch die Bezugnahme auf Beschreibungen, die im Rahmen der Arbeit, mit einem emanzipatorischen Sinnzusammenhang versehen wurden, konnten Wissenschaftlerinnen identifiziert werden, welche bei der Erarbeitung von emanzipatorischen Konzepten hilfreiche Impulse geben können (vgl. Abbildung 5). Ein genauerer Blick lohnt sich bei den Wissenschaftlerinnen, die sich in einer emanzipatorischen Perspektive mit dem Verhalten und der Handlungsfähigkeit auseinandergesetzt haben. Die grafische Darstellung dessen war im Rahmen der Arbeit schwer möglich, jedoch ist dies bei der Untersuchung des Graphen am Computer interaktiv angelegt, sodass eine Auswahl zu jedem/r beliebigen Wissenschaftlerin oder Wissenschaft möglich ist und durch Auswahlkriterien und Suchfunktionen die einzeln dargestellten Ergebnisse gut auffindbar sind. Ein Graph, welcher die priorisierten Kategorien Verhalten, Handlungsfähigkeit und Kultur - Sozialisation enthält, zeigt sich entsprechend dem aktuellen Arbeitsstand noch unübersichtlich und es fehlen ihm noch Verlinkungen und die Einbindung in einen allgemeinen Wissensgraphen (vgl. Abbildung 6). Im Rahmen eines Forschungsprojektes an der Technischen Informationsbibliothek in Hannover49 sollen die in der Arbeit gewonnenen, Erkenntnisse auch für eine weitere Arbeit zugänglich gemacht werden. Durch die Einbindung in das ORKG Projekt wird es möglich, die jeweiligen Wissenschaftlerinnen auch in einem größeren und allgemeineren Kontext darzustellen. Darüber wird es möglich, die unterschiedlichsten Kontexte zu Gesellschaftstheorien und die Betrachtung von Macht- und Herrschaftsverhältnissen sichtbar zu machen. Darüber hinaus könnte eine Veröffentlichung erarbeitet werden, welche die Definition und die Notwendigkeit von Emanzipationsprozessen in einfacher Sprache formuliert, um sie Jugend zur 49 Die TIB Hannover und die Leibniz Universität Hannover erarbeiten mit weiteren Kooperationspartnerinnen einen Open Research Knowledge Graphen. Dieser soll es ermöglichen, durch die Auswahlfuntionen und die Darstellung auch die passende Literaturquelle und damit verbundenes Wissen anzuzeigen. Das Wissen wird darüber als Netzwerk dargestellt. Darüber hinaus soll sichtbar gemacht werden, mit welchen anderen Fragestellungen und Research Problemen sich die unterschiedlichen Wissenschaftlerinnen auseinandergesetzt haben. Ziel ist es, die enorm aufwendige Literaturrecherche zu bestimmten wissenschaftlichen Fragestellungen zu optimieren und wesentlich zu verbessern. Nähere Informationen unter: http://orkg.org oder vgl. Jaradeh 2020 82 Herrschaft vs. Emanzipation Abbildung 5: Wissenschaftlerinnen mit Emanzipationsperspektive Verfügung zu stellen. Mit einer solchen dreiteiligen Veröffentlichung für Jugendtheorie, Jugendarbeit und Jugend können alle Zielgruppen erreicht werden, über die im Rahmen dieser Arbeit geschrieben wurde. Dadurch kann ein Diskurs entstehen und die Definition kann nicht nur von Theoretikerinnen und Praktikerinnen bewertet und eingeschätzt werden, sondern auch von Jugend, über die schlussendlich geschrieben wurde. Um die Kategorien noch besser beschreiben zu können und damit verbunden auch noch mehr Daten gerade aus Studien mit zu integrieren, bietet sich die Konstruktion einer eigenen Ontologie für die Kategorien Verhalten, Handlungsfähigkeit und Kultur - Sozialisation an. Über diese eigene Ontologie können dann gemäß der phänomenologischen Analyse noch weitere Bedeutungseinheiten (vgl. Pkt. 4.2) hinzugefügt werden, welche sich aus den Kategorien ergeben. Durch die Erweiterung mit den Bedeutungseinheiten wird es möglich, z. B. bei der Kategorie Kultur - Sozialisation historische Ereignisse mit in den Graphen aufzunehmen, oder aber über die Kategorie der Handlungsfähigkeit Konzepte aus der Praxis von Jugendarbeit in den Graphen einzupflegen. 83 Herrschaft vs. Emanzipation Abbildung 6: Graph Verhalten - Handlungsfähigkeit - Kultur - Sozialisation Des Weiteren entsteht so die Möglichkeit, die umfangreiche Literatur und Forschungsarbeit zu Jugendbewegungen und Jugend(sub)kulturen innerhalb des Graphen abzubilden, auf welche bisher verzichtet wurde. Diese Erweiterung ermöglicht einen notwendigen Qualitätsdialog, welcher mit einer Wirkungsmessung für Handlungskonzepte verbunden werden kann. Durch diesen Dialog wird es möglich die Ergebnisse aus Praxis50 und Theorie gleichsam in dem Graphen sichtbar zu machen und dadurch keine Hierarchie zwischen Theorie und Praxis entstehen zu lassen. Da Jugend global stattfindet und es, wie unter Pkt. 4 angedeutet, relevante Unterschiede zwischen den Wohlfahrtsstaaten und zusätzlich eine Vielzahl an Ländern ohne wohlfahrtsstaatliche Grundlage gibt, ist es notwendig, diese Besonderheiten ebenfalls in dem Graphen abzubilden, dadurch kann sich eine transnationale Perspektive auf Jugend eröffnen, die Jugend selbst bereits hat (vgl. Pfaff 2020, S. 79ff). Diese Betrachtung im wohlfahrtsstaatlichen Kontext kann darüber hinaus Erkenntnisse liefern, welche Auswirkungen bestimmte staatliche Ordnungssysteme auf die Entwicklung der Gesellschaft haben und wie stark ausgeprägt die Macht- und Herrschaftsstruk50 Praxis meint gemäß der gefundenen Definition nicht nur eine Praxis von Jugendarbeit, sondern auch eine Praxis in Jugendbewegungen und Jugend(sub)kulturen sowie in den bisher nicht berücksichtigten Emanzipationsbewegungen. 84 Herrschaft vs. Emanzipation turen sind. Eine solche Analyse bedarf dann wiederum, den in der Arbeit vernachlässigten, tiefen Blick auf die unterschiedlichen Theorien von Gesellschaft und die unterschiedlichen Theorien zu Macht. Dies führt zu einer interdisziplinären Betrachtung von Macht- und Herrschaftsverhältnissen im Kontext des Untersuchungsgegenstandes. Ein im Rahmen des Graphen noch nicht bewältigtes Problem ist der Umgang mit kritischen Beiträgen. In der Abbildung 5 konnte der Graph sichtbar machen welche Wissenschaftlerinnen einzelne Sinnzusammenhänge unter dem Blickwinkel der Emanzipation betrachtet haben. Die kritischen Beiträge wie z. B. von Roland Anhorn, Hartmut Griese, Benno Hafeneger, u. a. (vgl. Pkt. 5.3.2) sind bisher noch unter Herrschaft platziert, da sie einen IST - Zustand der Betrachtungsweise darstellen. Diese Beiträge weisen jedoch besonders auf die Probleme hin und sollten deshalb in die weitere Betrachtung zwingend aufgenommen werden. Weiterhin stellt eine bessere Differenzierung zwischen Jugendhilfe, im Sinne einer Fürsorge, und einer sich bildenden Jugendarbeitstheorie, im Sinne der Emanzipation, eine zukünftige Aufgabe dar. Beide Felder können durchaus interagieren und sich dadurch gegenseitig ergänzen, jedoch sollten die Aufträge und Grenzen deutlicher heraus gearbeitet werden. Darüber könnten auch, mit der Sozialen Arbeit einhergehende, Fragestellungen wie z. B. das Doppelte oder Tripple Mandat bearbeitet werden. Mit der vorliegenden Definition von Jugend kann hier konstatiert werden, das eine Jugendarbeiterin im Sinne der Definition kein doppeltes Mandat hat sondern ausschließlich ein Mandat der Jugend. Daran knüpfen sich jedoch komplexe Fragestellungen an, angefangen bei der Finanzierung von Jugendarbeit und die Frage ob Jugendarbeit im Feld der Sozialen Arbeit richtig platziert ist. Kurzum stellt die vorliegende Arbeit einen Auftakt einer intensiv zu führenden Debatte über Jugend und Jugendarbeit dar. 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Sozialgeschichte der Jugend. ger. 1. Auflage, Erstausgabe. Edition Suhrkamp 1278 = N.F., 278 Neue historische Bibliothek. Frankfurt am Main: Suhrkamp. Mönius, Katja, Jörn Steuding und Pascal Stumpf (2021). Einführung in die Graphentheorie: Ein farbenfroher Einstieg in die Diskrete Mathematik. ger. essentials. Wiesbaden: Springer Fachmedien. Müller, C.W. u. a. (1972). Was ist Jugendarbeit? Vier Versuche zu einer Theorie. 6. Auflage. Juventa-Paperback. München: Juventa-Verlag. Niemeyer, Christian und Marek Naumann (2006). „Siegfried Bernfeld (1892-1953): Vom Außenseiter zum Idol des Mainstream“. ger. In: Klassiker der Pädagogik: Die Bildung der modernen Gesellschaft. Hrsg. von Bernd Dollinger. 1. Auflage. Wiesbaden: VS Verlag für Sozialwissenschaften, S. 265 –286. Niemeyer, Christian und Michael Rautenberg (2006). „Klaus Mollenhauer (1928-1998): Pädagogik als vergessener Zusammenhang“. ger. In: Klassiker der Pädagogik: Die Bildung der modernen Gesellschaft. Hrsg. von Bernd Dollinger. 1. Auflage. Wiesbaden: VS Verlag für Sozialwissenschaften, S. 331 –352. Oelschlägel, Dieter (2017). „Emanzipation“. ger. In: Wörterbuch Soziale Arbeit : Aufgaben, Praxisfelder, Begriffe und Methoden der Sozialarbeit und Sozialpädagogik. Hrsg. von Dieter Kreft und Ingrid Mielenz. 8., vollständig überarbeitete und aktualisierte Auflage. Edition Sozial. Weinheim: Beltz Juventa, S. 239 –241. Perovic, Bojana (März 2016). Defining Youth in Contemporary National Legal and Policy Frameworks Across Europe. Techn. Ber. European Commission und the Council of Europe. Pfaff, Nicole (2020). „Jugendforschung in den Fallstrickendes methodologischen Nationalismus?!“ ger. In: Erziehungswissenschaftliche Jugendforschung: Ein Aufbruch. Hrsg. von Cathleen Grunert u. a. 1st ed. 2020. Springer eBook Collection. Wiesbaden: Springer Fachmedien. Pfister, Jonas, Hrsg. (2016). Texte zur Wissenschaftstheorie. ger. 2. Auflage. Reclams UniversalBibliothek Nr. 19421. Stuttgart: Reclam. Popper, Karl R (1997). Karl Popper Lesebuch: Ausgewählte Texte zur Erkenntnistheorie, Philosophie der Naturwissenschaften, Metaphysik, Sozialphilosophie. Hrsg. von Miller David. 2. durchges. Aufl. Tübingen: UTB / Mohr Siebeck. Rancière, Jacques (2018). Das Unvernehmen. 7. Auflage. Frankfurt am Main: Suhrkamp Verlag. Reinders, Heinz (2006). Jugendtypen zwischen Bildung und Freizeit: theoretische Präzisierung und empirische Prüfung einer differenziellen Theorie der Adoleszenz. ger. Münster: Waxmann. Riefling, Markus (2014). „Erziehung als Entwicklungshelfer der Moralität. Zur Bedeutung von Kants Moralphilosophie, Anthropologie und Geschichtsphilosophie für seine Theorie der Erziehung“. In: Kant-Studien 105.3, S. 406–417. doi: doi:10.1515/kant-2014-0018. 89 Herrschaft vs. Emanzipation Ruberg, Christiane (2002). Wie ist Erziehung möglich?: Moralerziehung bei den frühen pädagogischen Kantianern. Bad Heilbrunn: Julius Klinkhardt. Sagebiel, Juliane und Sabine Pankofer (2015). Soziale Arbeit und Machttheorien: Reflexionen und Handlungsansätze. ger. Freiburg im Breisgau: Lambertus. Scherr, Albert (2009). Jugendsoziologie: Einführung in Grundlagen und Theorien. ger. 9., erw. u. umfassend überarb. Aufl. Lehrbuch. Wiesbaden: VS Verl. für Sozialwissenschaften. Schmitt, Marco (2015). Zur Aktualität von Harrison White: Einführung in sein Werk. ger. Aktuelle und klassische Sozial- und Kulturwissenschaftler innen. Wiesbaden: Springer VS. Schröer, Wolfgang (2009). „Jugend“. ger. In: Handwörterbuch Erziehungswissenschaft. Hrsg. von Sabine Andresen u. a. Weinheim: Beltz, S. 452 –463. Seithe, Mechthild (2012). Schwarzbuch Soziale Arbeit. ger. 2., durchges. und erw. Aufl. Wiesbaden: VS Verlag für Sozialwissenschaften. Staub-Bernasconi, Silvia (2021). „Macht und (kritische) Soziale Arbeit“. ger. In: Macht in der Sozialen Arbeit: Interaktionsverhältnisse zwischen Kontrolle, Partizipation und Freisetzung. Hrsg. von Björn Kraus und Wolfgang Krieger. 5., überarb. und erw. Aufl. Detmold: JacobsVerlag. Templin, David (2015). Freizeit ohne Kontrollen: die Jugendzentrumsbewegung in der Bundesrepublik der 1970er Jahre. Hamburger Beiträge zur Sozial- und Zeitgeschichte. Wallstein Verlag. Thole, Werner und Walter Hornstein (2017). „Jugend“. In: Wörterbuch Soziale Arbeit : Aufgaben, Praxisfelder, Begriffe und Methoden der Sozialarbeit und Sozialpädagogik. Hrsg. von Dieter Kreft und Ingrid Mielenz. 8. vollständig überarbeitete und aktualisierte Auflage. Edition Sozial. Weinheim: Beltz Juventa, S. 462 –467. Thole, Werner und Jens Pothmann (2013). „Die MitarbeiterInnen in der Offenen Kinder- und Jugendarbeit“. In: Handbuch Offene Kinder- und Jugendarbeit. Hrsg. von Ulrich Deinert und Benedikt Sturzenhecker. 4., überarbeitete und aktualisierte Auflage. Wiesbaden: VS Verlag für Sozialwissenschaften \| Springer Fachmedien. Kap. 67, S. 559 –579. Treibel, Annette (2008). Die Soziologie von Norbert Elias: Eine Einführung in ihre Geschichte, Systematik und Perspektiven. ger. SpringerLink Bücher. Wiesbaden: VS Verlag für Sozialwissenschaften. Walitschke, Michael (2017). Im Wald der Zeichen: Linguistik und Anthropologie. Das Werk von Claude Lévi-Strauss. ger. Reprint 2017. Linguistische Arbeiten; 331. Berlin: De Gruyter. Welzer, Harald (2014). Selbst Denken: Eine Anleitung zum Widerstand. 2. Aufl. Frankfurt am Main: Fischer Verlag GmbH. Winkler, Michael (2006). „Friedrich Schleiermacher (1768-1834)“. ger. In: Klassiker der Pädagogik: Die Bildung der modernen Gesellschaft. Hrsg. von Bernd Dollinger. 1. Auflage. Wiesbaden: VS Verlag für Sozialwissenschaften, S. 75–99. Winter, Rainer (2012). „Macht, Kultur und soziale Intervention. CulturalStudies als kritische Theorieperspektivein der Sozialen Arbeit“. In: Kritik der Sozialen Arbeit - kritische Soziale 90 Herrschaft vs. Emanzipation Arbeit. Hrsg. von Roland Anhorn u. a. Bd. 12. Perspektiven kritischer Sozialer Arbeit. Wiesbaden: VS Verlag für Sozialwissenschaften, S. 449 –462. Witte, Matthias D., Caroline Schmitt und Yvonne Niekrenz (2021). „Jugendliche“. In: Handbuch Offene Kinder- und Jugendarbeit. Hrsg. von Ulrich Deinert u. a. 5., vollständig neugestaltete Auflage. Wiesbaden: VS Verlag für Sozialwissenschaften \| Springer Fachmedien, S. 375 –386. 91 HTWK Leipzig Fachbereich Soziale Arbeit Studiengang: 19SAM Modul: 3.4 Praxisprojekt II Dozentin: Frau Prof. Dr. phil. Heike Förster Anlage Erstbetrachtung Ausstattung Jugendarbeit in Zahlen 9. April 2021 Inhaltsverzeichnis 1 2 Auswertung und Erstbetrachtung 2 1.1 1.2 1.3 2 2 6 Falsche Fährte durch Wahrnehmungsinterpretation . . . . . . . . . . . . . . . . . Ausstattung der Jugendarbeit in OST und WEST Deutschland . . . . . . . . . . Der Blick auf die tiefere Ebene . . . . . . . . . . . . . . . . . . . . . . . . . . . . Fazit 7 Literatur 7 Name: Tobias Burdukat Matrikelnr.: 74115 Mail: tobias.burdukat@stud.htwk-leipzig.de 1 1 Auswertung und Erstbetrachtung 1.1 Falsche Fährte durch Wahrnehmungsinterpretation Die Argumentationskette der schlechteren Ausstattung von Jugendarbeit in Ostdeutschland und besonders in Sachsen, ist eine weit verbreitete welcher ich ebenfalls folgte um dadurch die mangelhafte Qualität, welche sich oft in einem nicht Vorhandensein von Angeboten der Jugendarbeit widerspiegelt, zu rechtfertigen. In der 1. Arbeit zu dem Forschungsprojekt führte ich dies als relevanten Unterschied innerhalb der Jugendarbeit an, begrenzte mich selbst jedoch auf die Aussage das es sichtbar weniger selbstverwaltete Projekte innerhalb der Jugendarbeit gibt. Nachweislich falsch lag ich mit der Aussage, dass die Jugendarbeit stetig beschnitten wurde1 , was durchaus der Wahrnehmung vieler in der Jugendarbeit tätiger entspricht, so auch meiner. Ich begründete dieses Legitimationsproblem und die damit verbundenen Kürzungen mit einem mangelndem Verständnis von Jugend und Jugendarbeit, welches seinen Ursprung in der Sozialisation der in der Jugendarbeit Tätigen und besonders bei den für die Jugendarbeit Verantwortlichen und bei den großen Trägern der Jugendhilfe (Freie und Institutionelle), hat. Darüber hinaus unterstellte ich der Politik ein mangelndes Verständnis von Jugend und Jugendarbeit. Diese Unterstellung bezüglich der Politik möchte ich bewusst, aufgrund der aktuellen Kürzungen von Fördergeldern in Plauen2 , der Stigmatisierung von Jugendarbeit durch die Verwaltung in Hamburg3 oder den immer währenden Angriffen gegenüber der Jugendarbeit und der Jugend von Erwachsenen und Politikerinnen die ich vor der eigenen „Haustür“ wahrnehme gern aufrecht erhalten, solange bis Politik hier das Gegenteil beweisen kann. Auch kann die Gestaltung und finanzielle Ausstattung von Jugendarbeit nicht abschließend geklärt werden, da die Praxis sich mittlerweile auf die, durch die Politik verursachten Bedingungen, der sich ständig wandelnden Fördermitteltöpfe, eingestellt hat. Die Tatsache, und im Forschungsprojekt für OST eindeutig an Aussagen belegbare Feststellung, dass sich Jugendarbeit hier klare Vorgaben machen lässt passt in die Annahme, das die Herrschaft die Emanzipation junger Menschen behindert. Die klaren Vorstellungen von Fördermitteln sind nur selten an den tatsächlichen Bedürfnissen, meint von und durch Jugendliche kommuniziert, orientiert. Vielmehr orientieren sie sich an dem was Erwachsene, Politik und Jugendarbeit(Hier verstanden als Teil der Sozialen Arbeit und nicht im emanzipatorischen Sinn) denkt was Jugendliche brauchen. 1.2 Ausstattung der Jugendarbeit in OST und WEST Deutschland Es ist nur ehrlich wenn ich schreibe das ich die folgende Auswertung gemacht habe um nachweisen zu können das ich mit meiner Annahme des Beschneidens der Jugendarbeit in Ostdeutschland richtig lag und somit für die Auswertung des Forschungsprojektes nicht nur mit einer Annahme bezüglich der Ausstattung heran gehe, sondern diese auch in Zahlen untersetzen kann. Ich wollte eine Wahrnehmung nachzeichnen, welche verteilt über die letzten 30 Jahre, eine negative Entwicklung, in der personellen Ausstattung der in der Jugendarbeit tätigen im Verhältnis zu den durch die Jugendarbeiterinnen begleiteten Jugendlichen, in Zahlen darstellbar macht. 1 vgl. S. 10 Seminararbeit Burdukat 1. Semester 19SAM Methodiken der empirischen Sozialforschung https://www.spiegel.de/politik/deutschland/plauen-cdu-streicht-geld-fuer-demokratieprojektmit-stimmen-von-afd-und-iii-weg-a-1c72fced-a212-4cb6-92f2-6c029c093ad4 verfügbar am 03.04.2021 3 https://www.entschlossen-offen.de/2021/01/28/entschlossen-offene-arbeit-extrem-wichtighamburger-sozialbehoerde-richtet-ihren-blick-in-zeiten-rechten-terrors-nach-links/ verfügbar am 03.04.2021 2 2 Dort vermutete ich ablesen zu können, dass ich zusätzlich zur negativ Entwicklung auch noch OST - WEST Unterschiede ablesen kann, um meine Annahme und die mit ihr verbundenen Arbeitshypothesen zu untersetzen. Ich entschied mich anhand der verfügbaren Zahlen über das Bundesamt für Statistik für eine genauere Betrachtung der Jahre 1994, 2002 und 2018. Dabei habe ich für die Erhebung der Daten der in der Jugendarbeit tätigen Personen auf die Regionaldatenbank Deutschland der Statistischen Ämter des Bundes und der Länder4 und für die Erhebung der in dem jeweiligen Bundesland oder Landkreis lebenden Jugendlichen auf die GENESIS Datenbank des Statistischen Bundesamtes (Destatis)5 zurück gegriffen. Bundesland 1994 2002 2018 Baden-Württemberg Bayern Berlin Brandenburg Bremen Hamburg Hessen Mecklenburg-Vorpommern Niedersachsen Nordrhein-Westfalen Rheinland-Pfalz Saarland Sachsen Sachsen-Anhalt Schleswig-Holstein Thüringen 4.283 3.319 2.980 1.875 346 1.229 3.202 985 3.517 10.282 1.705 253 1.615 833 2.077 1.112 4.860 4.850 3.064 1.421 293 1.267 4.260 1.657 3.598 8.667 1.579 281 2.873 1.505 2.307 1.927 4.153 4.596 2.051 1.008 332 923 3.607 739 4.330 9.568 2.207 173 2.022 817 1.281 1.136 Tabelle 1: In der Jugendarbeit tätige Personen Die verfügbaren Zahlen für die in der Jugendarbeit tätigen (vgl. Tabelle 1) könnten für eine tiefere Betrachtung noch genauer sein, um sich ein tatsächliches Bild über die Ausstattung im Sinne einer Offenen Kinder- und Jugendarbeit zu machen. Jedoch kann davon ausgegangen werden das bei politischen Entscheidungen zu Debatten der Finanzierung auf derartige Erhebungen zurück gegriffen wird, weshalb ein Wissen über diese Zahlen für jedwede Finanzierungsdebatte welche man* führen möchte von einer enormen Relevanz ist. Zusätzlich sind die Zahlen ausreichend um meine These der Beschneidung von Jugendarbeit zu betrachten, wenngleich die durch das Statistische Bundesamt erfassten Zahlen neben den Vordergründigen Mitarbeiterinnen in der Jugendarbeit wie z.B. in Jugendzentren, in Häusern der offenen Tür, der mobilen Jugendarbeit, kulturpädagogische oder kulturelle Einrichtungen (worunter z.B. Soziokulturelle Zentren fallen) auch die Mitarbeiterinnen in Jugendherbergen oder auf Jugendzeltplätzen mit einrechnen. Eine Schärfung der Zahlen wäre hier sicherlich sinnvoll, um eine bessere Betrachtung zu ermöglichen. Diesen Zahlen wurde die Anzahl der Jugendlichen im Alter von 14 - 18 Jahren in den jeweils exemplarisch ausgewählten Jahren entgegen gesetzt (vgl. Tabelle 2) um eine Zahl der Jugendlichen, pro in der Jugendarbeit hauptamtlich tätigen (vgl. Tabelle 3), zu ermitteln. 4 5 https://www.regionalstatistik.de https://www-genesis.destatis.de 3 Bundesland Baden-Württemberg Bayern Berlin Brandenburg Bremen Hamburg Hessen Mecklenburg-Vorpommern Niedersachsen Nordrhein-Westfalen Rheinland-Pfalz Saarland Sachsen Sachsen-Anhalt Schleswig-Holstein Thüringen 1994 2002 2018 531.319 605.225 174.180 174.624 31.098 75.252 295.117 138.455 399.333 892.480 200.651 52.348 293.690 175.950 131.421 166.926 601.442 677.625 179.533 189.360 32.084 77.535 316.405 134.457 445.022 1.002.173 231.082 57.446 284.303 175.986 148.279 165.266 546.962 617.449 144.466 105.478 30.673 77.997 299.175 66.206 403.945 880.003 194.366 43.484 165.374 87.357 143.630 87.432 Tabelle 2: Jugendlichen im Alter von 14 - 18 Jahren Es wurde speziell die Altersgruppe der 14 - 18 jährigen betrachtet da diese Altersspanne für eine gelingende emanzipatorische Jugendarbeit und die Entwicklung von Eigenständigkeit und Selbstständigkeit innerhalb der Jugendphase als relevanteste Altersspanne bezeichnet werden kann, wenngleich sich die Adoleszenzphase nicht genau bestimmen lässt (vgl. King 2013), können wir sie mehrheitlich in dieser Alterskohorte beobachten. Zusätzlich wird die Arbeit mit den Zahlen nach Vollendung des 18. Lebensjahres schwieriger da durch die Beendigung der Schule und eine, besonders in ländlichen Räumen, einhergehende Fluktuation der jungen Menschen, keine wirklich Aussagekräftigen Rückschlüsse mehr möglich macht. Abbildung 1: Graphische Darstellung der Entwicklung Setzen wir diese Zahlen nun in ein Verhältnis stellt sich im Bundesdeutschen Durchschnitt eine 4 Bundesland 1994 2002 2018 Baden-Württemberg Bayern Berlin Brandenburg Bremen Hamburg Hessen Mecklenburg-Vorpommern Niedersachsen Nordrhein-Westfalen Rheinland-Pfalz Saarland Sachsen Sachsen-Anhalt Schleswig-Holstein Thüringen 124 182 58 93 89 61 92 140 113 86 117 206 181 211 63 150 123 139 58 133 109 61 74 81 123 115 146 204 98 116 64 85 131 134 70 104 92 84 82 89 93 91 88 251 81 106 112 76 Durchschnitt BUND: Durchschnitt OST (ohne Berlin): Durchschnitt WEST (ohne Saarland): 123 155 103 108 103 106 105 91 101 Tabelle 3: Auswertung der Jugendlichen 14 - 18 Jahre pro Jugendarbeiterin Verbesserung der in der Jugendarbeit tätigen, pro Jugendliche von 1994 zu 2018 dar. War 1994 eine in der Jugendarbeit Tätiger noch durchschnittlich für 123 Jugendliche Ansprechpartnerin hat sich die Situation zu 2018 auf 105 Jugendliche verbessert. Bei der Berechnung der Durchschnittswerte für OST und WEST Deutschland wurde in WEST auf die Verwendung der Zahlen vom Saarland verzichtet, da diese überdurchschnittlich schlecht im Verhältnis zu allen anderen Bundesländern waren. In OST wurde bei der Durchschnittsberechnung auf die Zahlen von Berlin verzichtet, da diese zumindest für 1994 und 2002 überdurchschnittlich gut waren. Der Verzicht auf die beiden Extreme sollte dabei helfen ein realistischeres Bild der Durchschnittswerte, welche für die vorliegende Arbeit von Interesse sind, zu erhalten. Dabei hat sich gezeigt, dass sich der Durchschnitt in den OST Bundesländern von 155 (1994) Jugendlichen, pro in der Jugendarbeit tätigen, auf 103 (2002) und schlussendlich auf 91 (2018) Jugendliche deutlich verbessert hat. Wenn man* die regionalen und Bundesland spezifischen Schwankungen ausblendet, kann in WEST in etwa von einer gleichbleibenden Ausstattung gesprochen werden (1994 - 103; 2002 - 106; 2018 - 101). Die Bundesländer OST liegen somit in Summe deutlich unter dem Bundesdurchschnitt (vgl. Abbildung 1), was meine ursprünglich getroffene Aussage zur Beschneidung der Jugendarbeit, zumindest anhand der Zahlen, widerlegt. Besonders hart habe ich es immer, auch aus persönlicher Betroffenheit, in Sachsen empfunden, jedoch liegt Sachsen mit 81 Jugendlichen pro in der Jugendarbeit tätigen noch weit vor dem Ostdeutschen Durchschnitt und hat laut den Zahlen gemeinsam mit Thüringen (76 in 2018) die Bundesdeutsche Spitzenposition in der personellen Ausstattung inne (Berlin mit 70 in 2018 wurde außen vor gelassen). 5 1.3 Der Blick auf die tiefere Ebene Da mich meine Wahrnehmung trotz der Zahlen nicht ganz loslassen wollte blickte ich zumindest für Sachsen noch einmal genauer hinein, um zu erfahren ob diese Zahlen sich durch die Ausstattung in den Großstädten (Leipzig, Chemnitz, Dresden) so gut darstellen. Als Vergleichsbundesland wählte ich Hessen, welches mit 82 Jugendlichen, pro in der Jugendarbeit tätigen, in etwa gleich aufgestellt ist wie Sachsen. Schwierig war es Vergleichswerte für die Landkreise und kreisfreien Städte, bezogen auf die Bevölkerungsstruktur, zu erhalten, so dass ich eine neue Alterskohorte bilden musste und nur die Ausstattung für die 15 - 18 jährigen prüfen konnte. 2018 Jugendliche 15 - 18 Jahre in Jugendarbeit tätige Jugendarbeiterin pro Jugendlicher Sachsen 131.651 2.022 65 Leipzig Stadt Landkreis Leipzig Nordsachsen 11.946 6.550 4.988 347 79 66 34 82 75 Chemnitz Stadt Erzgebirgskreis Mittelsachsen 5.540 8.426 7.552 157 176 129 35 47 58 241.058 3.607 66 Kassel Stadt Landkreis Kassel Werra Meißner Kreis 5.100 6.722 2.736 136 225 43 37 29 63 Wiesbaden Stadt Groß-Gerau Rheingau Taunus Kreis 7.838 8.011 5.608 160 144 85 48 55 65 Land/Stadt/Landkreis Hessen Tabelle 4: Landkreisvergleich - ausgewählte Kreise Sachsen und Hessen Der Durchschnitt in Sachsen (65) und Hessen (66) lag für diese Alterskohorte im Jahr 2018 in etwa gleich. Spannender wird es wenn man nun auf die genauen Zahlen blickt, dort zeichnet sich ein erwartetes Gefälle zwischen den Städten und den ländlich geprägten Landkreisen ab. Spannend dabei war das ich mit meiner Wahrnehmung für den Landkreis Leipzig gar nicht so falsch lag, denn blickt man* auf Chemnitz und die umliegenden Landkreise wie den Erzgebirgskreis oder Mittelsachsen so ist dort das Gefälle zwischen Stadt und Land zwar vorhanden, aber nicht annähernd in einer solchen Dimension wie im Leipziger Umland in den Landkreisen Nordsachsen und dem Landkreis Leipzig, der eine absolute Spitzenposition im Negativranking einnimmt. Kommen z.B. in Leipzig 34 Jugendliche der Alterskohorte auf einen Tätigen, so sind es im Landkreis Leipzig 82, was auch weit unter dem Durchschnitt für das Land Sachsen für diese Alterskohorte liegt. In den zufällig ausgewählten Städten und Landkreisen in Hessen lassen sich diese Unterschiede nicht in einer derartigen Größenordnung fest stellen, wenn gleich sie vorhanden sind. 6 2 Fazit Die Frage ob die von mir häufig geäußerte Aussage, der schlechteren Ausstattung, auch stimmt, lässt sich oberflächlich betrachtet mit dieser Aufstellung beantworten. Es stimmt auf den ersten Blick nicht und die Jugendarbeit stellt sich anhand der Zahlen sogar durchschnittlich als besser ausgestattet in den OST Bundesländern dar. Bei näheren Überlegungen wird jedoch deutlich das sich zum einen starke regionale Unterschiede ausmachen lassen und es wird in Zukunft zu prüfen sein ob diese Auswertung in ein Verhältnis mit anderen Zahlen, wie z.B. dem pro Kopf Einkommen in den Regionen oder auch anderen attraktiven Freizeitangeboten für junge Menschen, zu setzen ist. Diese tiefere Betrachtung könnte in ihrer Gesamtdarstellung dazu beitragen einen Gesamtdeutschen Blick für Jugendarbeit zu entwicklen und ggf. auch Aufschluss darüber geben was es zum Beispiel für Rahmenbedingungen braucht die eine qualitativ hochwertige Arbeit, trotz geringer Ausstattung ermöglichen. Literatur King, Vera (2013). Die Entstehung des Neuen in der Adoleszenz: Individuation, Generativität und Geschlecht in modernisierten Gesellschaften. Adoleszenzforschung : Zur Theorie und Empirie der Jugend aus transdisziplinärer Perspektive. Wiesbaden: VS Verlag für Sozialwissenschaften. 7 Diese Arbeit wurde mit LATEX über die Webplattform https://overleaf.com geschrieben. Das Tabellen-Management wurde mit Google Spreadsheat (https://drive.google.com) durchgeführt. Die Wissengraph-Transformation fand mit freundlicher Unterstützung der eccenca GmbH durch die Software eccenca Corporate Memory (https://eccenca.com) statt. Die Dashboard-Visualisierung, für die finale Darstellung der Daten, wurde mit Redash (https://redash.io) realisiert. Ich bedanke mich bei all den Menschen die mich während des Masterstudiums und bei der Erstellung der Masterarbeit unterstützt, mich motiviert, mir Kraft und Halt gegeben und mich ausgehalten haben. Ein besonderer Dank gilt der für die finanzielle Unterstützung, im Rahmen eines Stipendiums von April 2020 - März 2022. Erklärung über die selbstständige Verfassung Hiermit versichere ich, Tobias Burdukat, geboren am 05.05.1983, gegenüber der Fakultät Architektur und Sozialwissenschaften der HTWK Leipzig, Fachbereich Soziale Arbeit, dass diese Arbeit mit dem Titel „Herrschaft vs. Emanzipation – Raubt die Theorielosigkeit von Jugendarbeit, im Kontext Sozialer Arbeit, ihr die Kraft?“ selbstständig verfasst wurde. Es wurden keine anderen als die angegebenen Quellen und Hilfsmittel benutzt sowie alle Stellen, die wörtlich oder sinngemäß aus den benutzten Quellen entnommen worden sind, als solche kenntlich gemacht wurden. Diese Arbeit wurde in gleicher oder ähnlicher Form noch keiner anderen Prüfungsbehörde vorgelegt. Grimma, am Datum Tobias Burdukat
W2897459464.txt	https://journal.uii.ac.id/intervensipsikologi/article/download/10871/9376	en		PENERAPAN PRINSIP - PRINSIP PARENT CHILD INTERACTION THERAPY UNTUK MENURUNKAN SIBLING RIVALRY PADA REMAJA	JIP/JIP (Jurnal Intervensi Psikologi)	2,017	cc-by-sa	4,822	Penerapan Prinsip-prinsip Parent-child Interaction Therapy untuk Menurunkan .... PENERAPAN PRINSIP-PRINSIP PARENT-CHILD INTERACTION THERAPY UNTUK MENURUNKAN SIBLING RIVALRY PADA REMAJA APPLICATION OF THE PARENT-CHILD INTERACTION THERAPY PRINCIPLES TO DECREASE SIBLING RIVALRY IN ADOLESCENT Septhi Karlina Utami Hendriati Agustiani Langgersari Elsari Novianti Fakultas Psikologi Universitas Padjadjaran,Bandung Email: septhiutami.psi@gmail.com ABSTRACT This research was carried out to obtain an intervention program design by using Parent-Child Interaction Therapy (PCIT) principles which could decrease sibling rivalry in adolescent who has younger sibling with intellectual disability.This research involved two pairs of mother and child (adolescent) who were sampled through use of purposive sampling and tried out by using quasi experimental method with A-B-A design. The data were obtained through interviews, observation, and questionnaire. The measurement tools were Sibling Rivalry questionnaire and Dyadic Parent-Child Interaction Coding System III (DPICS-III). There was decline in sibling rivalry and increase in mother’s ability to apply PCIT skills. Meanwhile the qualitative data showed that PCIT skills mastered and applied by the mothers in daily basis parenting made the children to feel respected and could decrease jealousy, anger, and rivalry feeling with their siblings. Keywords: Parent-Child Interaction Therapy (PCIT), Sibling Rivalry, Sibling Relationship, Adolescent, Intellectual Disability. ABSTRAK Penelitian ini dilakukan untuk memperoleh suatu rancangan program intervensi menggunakan prinsip-prinsip Parent-Child Interaction Therapy (PCIT) yang dapat menurunkan sibling rivalry remaja yang memiliki adik dengan intellectual disability. Orang tua dilatih untuk dapat membentuk kelekatan yang positif dan memberikan perintah secara efektif kepada anak remajanya. Pada penelitian ini dilibatkan dua pasangan ibu dan anak (remaja) yang diperoleh dengan menggunakan teknik purposive sampling dan diuji coba dengan menggunakan metode kuasi eksperimen dengan rancangan A-B-A design. Data diperoleh melalui wawancara, observasi, dan alat ukur berupa kuesioner. Alat ukur yang digunakan adalah Kuesioner Sibling Rivalry dan Dyadic Parent-Child Interaction Coding System III (DPICS-III). Dari data kuantitatif hasil uji coba program intervensi, terlihat adanya penurunan sibling rivalry dan peningkatan kemampuan ibu dalam menerapkan keterampilan-keterampilan PCIT. Dari data kualitatif didapatkan bahwa keterampilan PCIT yang dikuasai ibu dan diterapkan dalam pengasuhan sehari-hari membuat anak merasa dihargai dan mengurangi rasa cemburu, marah, dan bersaing dengan adiknya. Kata kunci : Parent-Child Interaction Therapy (PCIT), Sibling Rivalry, Sibling Relationship, Remaja, Intellectual Disability. Jurnal Intervensi Psikologi Vol. 9 No. 2 Desember 2017 \| 219 Septhi Karlina Utami, Hendriati Agustiani & Langgersari Elsari Novianti Hubungan dengan saudara kandung merupakan hubungan yang paling bertahan lama sepanjang hidup seseorang, sebab hubungan ini didapatkan sejak kelahiran (Cicirelli, 1995). Hubungan antara seorang anak dengan saudara kandungnya melibatkan ikatan fisik dan emosional pada tahap-tahap kritis sepanjang kehidupan mereka. Saudara memiliki peran penting sejak masa kanak-kanak, mulai menjadi teman bermain hingga menjadi sumber dukungan sosial (Furman & Buhrmester, 1985; McHale, Updegraff, & Whitemann, 2012). Hubungan saudara sekandung (sibling relationship) merupakan interaksi total antara fisik, verbal, dan non verbal dari dua atau lebih individu yang memiliki keterkaitan dalam pengetahuan, persepsi, sikap, kepercayaan dan perasaan sepanjang masa, sejak individu menyadari kehadiran saudara kandungnya (Cicirelli, 1995; McHale, Kim, & Whiteman, 2006). Dalam interaksinya, mereka akan saling memengaruhi kemampuan kognitif dan sosialisasi yang dipergunakan dalam kehidupan mereka seharihari (Furman & Buhrmester, 1985; McHale, Updegraff, & Whitemann, 2012). Hubungan saudara sekandung memiliki dampak positif dan negatif bagi individu. Seorang anak dapat belajar untuk mengekspresikan sejumlah emosi yang dimilikinya, seperti sayang, loyalty, marah, dan bersaing dengan saudara kandungnya. Mereka akan membangun sebuah hubungan, saling mendukung dan saling berbagi satu sama lain serta belajar saling mengembangkan kemampuan sosial mereka. Namun, keberadaan dua orang anak membuat orang tua harus membagi perhatian untuk anak-anaknya. Adanya pembagian perhatian ini yang dapat menimbulkan sibling rivalry pada anak terhadap saudaranya, terutama rasa marah dan cemburu (Strohm, 2006). Shaffer mendefinisikan sibling rivalry sebagai kecemburuan, kompetisi atau kemarahan antara dua atau lebih individu bersaudara (Shaffer & Kipp, 2010). Sibling rivalry terjadi jika anak merasa mulai kehilangan perhatian serta kasih sayang dari orang tua dan merasa saudara kandungnya adalah saingan dalam mendapatkan perhatian dan kasih sayang tersebut. Cemburu adalah reaksi normal yang dialami oleh anak (Leung, 1991; Volling, Kennedy, & Jackey, 2010). Freud (Newman, 2016) mengungkapkan bahwa persaingan serta kecemburuan yang muncul dalam sibling rivalry didorong oleh keinginan untuk merebut kasih sayang dari orang tuanya. Rasa marah dan cemburu kepada saudara kandung ini juga dapat dimanifestasikan dalam perilaku agresif anak (Newman, 2016). Sawicki (1997) menyebutkan 4 bentuk manifestasi perilaku dari sibling rivalry yang dialami oleh seorang anak adalah 220 \| Jurnal Intervensi Psikologi Vol. 9 No. 2 Desember 2017 Penerapan Prinsip-prinsip Parent-child Interaction Therapy untuk Menurunkan .... agresi, regresi, tingkah laku mencari perhatian orang tua, atau dapat juga berkurangnya kemandirian. Sibling rivalry adalah kompetisi antara dua orang yang bersaudara untuk memperoleh cinta, kasih sayang, dan perhatian dari orang tua sebagai suatu pengakuan (Dorland, 1988 dalam Leung, 1991; Chaulagain, PU, Moras, Aranha, & Shetty, 2016). Perbedaan perlakuan dari orang tua dapat menjadi pemicu munculnya sibling rivalry pada anak. Pada kondisi-kondisi tertentu, misalnya pada anak dengan diabilitas intelektual, seorang anak memang akan membutuhkan perhatian yang lebih dibandingkan saudaranya. Diabilitas intelektual adalah gangguan selama periode perkembangan termasuk gangguan intelektual dan kurangnya fungsi adaptasi konseptual, sosial, dan praktikal (American Psychiatric Association, 2013). diabilitas intelektual ditandai dengan kekurangan dalam kemampuan mental secara umum, seperti penalaran, pemecahan masalah, planning, berpikir abstrak, pembelajaran akademis, dan belajar dari pengalaman. Anak yang mengalami diabilitas intelektual akan memperoleh perhatian yang lebih dari orang tua. Orang tua yang berbagi perhatian kepada adik atau saudara kandung yang sedang sakit, dipersepsikan sebagai perhatian yang berlebihan oleh anak yang lainnya. Persepsi ini menimbulkan rasa cemburu pada anak karena merasa terancam. Anak menganggap bahwa kehadiran adik yang memiliki keterbatasan tersebut sebagai penyebab hilangnya perhatian orang tua yang selama ini didapatkan olehnya. Anak memiliki persepsi negatif, bahwa orang tua lebih menyayangi adik dibandingkan dirinya, yang membuat timbulnya perasaan marah, cemburu, dan keinginannya untuk bersaing dengan adik. Oleh sebab itu, dapat dikatakan bahwa sibling rivalry muncul akibat dari persepsi anak terhadap sikap orang tua yang mungkin tidak sama dengan yang dimaksud oleh orang tua. Rutter (Febriyani, 2010) mengemukakan terdapat 6 peranan orang tua, salah satunya adalah berperan memberikan disiplin dan membentuk tingkah laku anak. Orang tua membantu membentuk tingkah laku anak dengan memberikan dorongan atau larangan, belajar bertanggung jawab, mengetahui mana yang baik dan tidak, serta belajar cara bertingkah laku terhadap ayah, ibu, saudara, tetangga ataupun orang lain. Oleh karena itu, sebagai orang tua, baik untuk anak normal maupun ABK, sikapnya harus tetap sama. McNeil dan Hembree-Kigin (2010) menyatakan bahwa Parent-Child Interaction Therapy (PCIT) dapat mengatasi permasalahan sibling rivalry dengan cara membangun relasi yang positif antara orang tua dan anak-anaknya. PCIT adalah intervensi yang diberikan melalui dua tahap (McNeil & Hembree-Kigin, 2010). Jurnal Intervensi Psikologi Vol. 9 No. 2 Desember 2017 \| 221 Septhi Karlina Utami, Hendriati Agustiani & Langgersari Elsari Novianti Tahap pertama adalah Child Directed Interaction (CDI), yaitu orang tua belajar untuk dapat berinteraksi dengan anak melalui sebuah permainan. Interaksi itu diwujudkan dalam tingkah laku mengapresiasi anak ketika anak menampilkan tingkah laku positif dan mengacuhkan anak ketika menampilkan perilaku negatif. Tahap kedua adalah Parent Directed Interaction (PDI), yaitu orang tua diajarkan bagaimana cara mengajarkan anak untuk dapat patuh terhadap aturannya. Jika biasanya PCIT digunakan hanya kepada satu orang anak, untuk masalah sibling rivalry, maka orang tua harus dapat melakukannya kepada dua anaknya sekaligus. Orang tua harus dapat bersikap adil dalam penerapan aturan kepada kedua anaknya. Penelitian ini menyusun dan menguji coba rancangan program intervensi yang menerapkan prinsip-prinsip PCIT untuk menurunkan sibling rivalry pada remaja yang memiliki adik kandung diabilitas intelektual. METODE PENELITIAN Subjek Penelitian Partisipan dalam penelitian ini berjumlah 2 pasang ibu dan anak remaja berusia 10-15 tahun serta memiliki adik dengan diabilitas intelektual. Menurut Brinkmeyer dan Eyberg (McNeil-Kigin, 2010), PCIT dapat melibatkan salah satu atau kedua orang tua atau orang lainnya yang berperan penting dalam pengasuhan anak. Dalam hal ini, orang tua yang paling berperan dalam pengasuhan anak adalah ibu. Oleh karena ibu, penelitian ini melibatkan 2 anak remaja beserta masingmasing ibu. Desain Penelitian Desain penelitian yang digunakan adalah A-B-A design, yang bertujuan untuk melihat pengaruh dari perlakuan (intervensi) yang diberikan dengan cara membandingkan kondisi sebelum dan sesudah diberikan intervensi. Metode Pengumpulan Data Alat ukur yang digunakan dalam penelitian ini adalah kuesioner sibling rivalry dan panduan observasi Dyadic Parent-Child Interaction Coding SystemIII (DPICS-III). Kuesioner sibling rivalry disusun oleh peneliti berdasarkan teori/konseptual Schaefer & Millman (1981), terdiri dari 40 item dalam bentuk skala 1-4 (sangat tidak setuju, tidak setuju, setuju, dan sangat setuju). Hasilnya masuk menjadi 3 kategori, rendah, sedang dan tinggi. Reliabilitas kuesioner ini adalah 0,868, yang artinya alat ukur dapat diandalkan. DPICS-III ini merupakan alat ukur formal observasional yang mengukur perilaku orang tua dan anak untuk menilai kualitas interaksi orang tua dan anak. Alat ukur ini digunakan sebagai penilaian 222 \| Jurnal Intervensi Psikologi Vol. 9 No. 2 Desember 2017 Penerapan Prinsip-prinsip Parent-child Interaction Therapy untuk Menurunkan .... untuk melihat ada tidaknya perubahan kualitas interaksi orang tua-anak sebagai hasil intervensi. Penilaian melalui alat ukur ini dilakukan selama 5 menit pertama di setiap pertemuan baik saat menilai keterampilan CDI (coding CDI) maupun PDI (coding PDI). Melalui alat ukur ini dapat dinilai penguasaan keterampilan orang tua ketika berinteraksi dengan anak. Alat ukur ini diambil dari buku McNeil-Kigin (2010) yang berjudul Parent-Child Interaction Therapy. Rancangan Intervensi Program intervensi yang diberikan adalah pemahaman mengenai prinsipprinsip PCIT, yaitu mengenai cara memperkuat hubungan antara orang tua dan anak, membangun harga diri (self esteem) anak, meningkatkan tingkah laku positif anak, teknik pengelolaan tingkah laku anak, dan pemberian instruksi yang efektif kepada anak. Capaian terciptanya interaksi yang baik antara ibu dan anak, diharapkan dapat mengubah persepsi negatif anak terhadap ibu yang berdampak pada menurunnya tingkat sibling rivalry anak. Program intervensi ini terdiri atas 2 tahap, yaitu tahap CDI (Child Directed Interaction) dan PDI (Parent Directed Interaction). Tahap CDI dimulai dengan sesi pengajaran dasar-dasar keterampilan CDI yang dilaksanakan melalui presentasi, diskusi, pemberian contoh, dan bermain peran. Setelah sesi pengajaran, dilakukan sesi pelatihan keterampilan CDI. Jumlah sesi CDI secara spesifik bergantung pada kecepatan orang tua dalam memperoleh keterampilan. Penguasaan orang tua dalam keterampilan CDI menjadi persyaratan bagi orang tua untuk dapat melanjutkan ke tahap PDI. Hal yang penting pula adalah memperhatikan keberlangsungan orang tua untuk hadir dalam sesi terapi secara lengkap. Namun, untuk kepentingan anak, akan lebih baik apabila orang tua beralih ke tahap PDI, meskipun orang tua belum menguasai keterampilan tahap CDI (Nurlita, 2012). Tahap PDI diawali dengan sesi pengajaran dan dilanjutkan dengan sesi pelatihan keterampilannya. Rancangan program yang telah disusun kemudian direview oleh psikolog sekaligus praktisi yang telah memiliki legalitas untuk melaksanakan PCIT. HASIL PENELITIAN Rancangan program yang disusun peneliti kemudian ditelaah oleh ahli, seorang Psikolog Anak yang memiliki legalitas untuk melakukan PCIT, untuk kemudian diperbaiki. Hasil revisi rancangan program diujicobakan kepada subjek. Ahli memberikan saran mengenai subjek penelitian yang mengalami sibling rivalry sebaiknya adalah usia remaja dibandingkan kanak-kanak. Seperti yang Jurnal Intervensi Psikologi Vol. 9 No. 2 Desember 2017 \| 223 Septhi Karlina Utami, Hendriati Agustiani & Langgersari Elsari Novianti dijelaskan sebelumnya, rancangan program intervensi ini terdiri dari dua tahap, yaitu CDI dan PDI. Ibu harus memenuhi kriteria penguasaan keterampilan CDI terlebih dahulu sebelum memasuki tahap PDI. Prinsip yang mendasari tahapan CDI adalah attachment theory, dengan tujuan meningkatkan interaksi antara ibu dan anak, agar tercipta hubungan yang hangat diantara ibu dan anak. Berdasarkan attachment theory, pondasi yang kuat untuk menciptakan sebuah hubungan yang hangat harus terjadi sejak masa kanak-kanak. Namun, ikatan antara orang tua dan anak ini pun perlu terus dipelihara. Attachment security mengarah pada kapasitas individu untuk mendapatkan kenyamanan pada sosok yang dianggapnya dapat memberikan rasa aman ketika sedang merasa tertekan (stress). Salah satu kondisi stressfull adalah masa transisi dari anak-anak ke remaja. Pada masa remaja, peran orang tua sebagai pelindung, pemberi rasa nyaman, dan pengetahuan masih tetap ada, namun sudah disesuaikan dengan kebutuhan mereka sebagai remaja (Dubois-Comtois et al., 2013). Secara kognitif, remaja sudah memasuki tahapan concrete operational, saat ia sudah dapat menerima alasan logis. Misalnya, larangan untuk melakukan suatu kegiatan mesti disertai alasan logisnya. Karenanya, orang tua perlu menyertakan alasan logis dalam setiap arahan kepada anak remajanya. Masa remaja juga disebut sebagai masa sensitif bagi perkembangan emosi individu. Sering kali terjadi perbedaan persepsi atas apa yang diterimanya dari lingkungan, yang membuatnya menunjukkan tingkah laku negatif. Misalnya, ketika orang tua sering mendahulukan kebutuhan adiknya, oleh remaja dipersepsikan sebagai rasa sayang orang tua yang lebih besar untuk adik atau bahkan ia merasa sudah tidak disayang oleh orang tuanya, sehingga ia menunjukkan sikap memberontak kepada orang tua atau menyakiti adiknya. Penerapan prinsip attachment pada tahap CDI ini dinilai tepat untuk dapat menciptakan hubungan yang hangat antara orang tua dengan anak remajanya. Kriteria penguasaan kemampuan CDI untuk ibu dengan anak remaja lebih rendah dibandingkan untuk ibu dengan anak usia kurang dari 7 tahun. Hal ini terkait dengan karakteristik remaja yang sedang mengembangkan identitas diri dan cenderung untuk tidak ingin didominasi oleh orang tua. Oleh karenanya, dalam penelitian ini ibu, sebagai orang tua, lebih banyak diam memperhatikan kegiatan anaknya. Meskipun demikian, tetap perlu diajarkan kepada ibu untuk mengapresiasi anak melalui non verbal behavior, seperti acungan jempol, kedipan mata, tos, dan gestur tubuh lainnya yang menunjukkan apresiasi ibu kepada anak. Meskipun remaja ingin memiliki kebebasan, namun 224 \| Jurnal Intervensi Psikologi Vol. 9 No. 2 Desember 2017 Penerapan Prinsip-prinsip Parent-child Interaction Therapy untuk Menurunkan .... Oleh karena itu, jumlah kriteria pencapaian ibu dalam keterampilan CDI dengan anak remaja lebih rendah dibandingkan keterampilan CDI ibu dengan anak-anak. Perbaikan berikutnya yang diusulkan oleh ahli PCIT adalah jumlah sesi dan materi yang disajikan di setiap sesinya. Menurut ahli PCIT, untuk remaja, materi mengenai memberikan perintah yang efektif dan menerapkan aturan kepada anak dipisahkan. Hal ini terkait dengan karakteristik remaja yang biasanya sensitif secara emosional, sehingga perlu diperhatikan mengenai strategi penyampaian perintah dan penerapan aturan kepadanya. Caranya adalah menerapkan aturan secara bertahap, agar anak dapat bekerja sama dengan orang tua (McNeil-Kigin, adalah mengenai pemberian perintah yang efektif. Pada umumnya, dimulai pada usia anak akhir (late childhood) anak sudah dapat menerima 2-3 perintah dalam satu kalimat. Misalkan seperti, “simpan tasnya dengan rapi, ganti baju, lalu segera makan!” Hanya saja yang perlu diperhatikan adalah bentuk kalimat memiliki makna yang jelas sehingga mudah dimengerti. Jenis permainan perlu disesuaikan dengan minat remaja. Jenis permainan seperti boneka, alat mewarnai (krayon dan pensil warna), dan kitchen sets sudah tidak diminati oleh anak usia 8 tahun ke atas. Dibandingkan menggunakan krayon, lebih baik siapkan peralatan melukis seperti cat, lem, glitter, atau lainnya untuk anak yang memiliki minat di bidang seni. Selain itu mainan yang bersifat konstruktif level advanced, misalnya lego yang diperuntukkan bagi remaja, atau mainan lain yang membutuhkan strategi, seperti monopoli, ludo, catur, otello, dan ular tangga. Hal ini sesuai dengan karakteristik remaja yang sedang mengeksplorasi kemampuannya, mencoba hal baru, dan menyukai tantangan. Setelah seluruh masukan para ahli diterapkan dalam rancangan intervensi, rancangan ini kemudian diujicobakan terhadap subjek penelitian, yaitu dua 2010). Materi pertama dari intervensi pasang ibu dan anak remajanya. pada dasarnya mereka pun memiliki kebutuhan untuk diperhatikan oleh orang tua, dalam hal ini adalah ibu. Pembentukan attachment antara orang tua dan anak di masa remaja ini bukan lagi mengenai kuantitas, melainkan tentang kualitas (Dubois-Comtois et al., 2013). Jadi, bukan lagi seberapa banyak ibu mampu hadir di hadapan anak atau seberapa sering ibu memuji anak, namun tentang seberapa dalam perbincangan di antara keduanya atau seberapa paham orang tua terhadap kebutuhan anaknya. Partisipan 1 Jurnal Intervensi Psikologi Vol. 9 No. 2 Desember 2017 \| 225 Septhi Karlina Utami, Hendriati Agustiani & Langgersari Elsari Novianti Tabel 1. Rangkuman Hasil Coding CDI berdasarkan DPICS-III* Sesi Tanggal Pre 13/4 Coaching CDI 21/4 25/4 28/4 5/5 Coaching PDI 12/5 16/5 19/5 23/5 Mastery** Pujian Spesifik 0 2 4 4 4 3 5 5 5 4 (Labeled Praise-LP) Refleksi (R) 3 2 6 11 10 12 9 11 8 7 Behavioral 1 2 3 5 4 5 5 4 4 4 Description (BD) Komentar Netral 3 2 3 1 0 0 0 1 1 Pujian Umum 4 1 1 0 0 0 0 1 0 (Unlabeled Praise-UP) Perintah 3 2 1 0 1 1 1 0 0 (Langsung/Tidak 0 Langsung) ≤3 Pertanyaan 2 2 1 1 1 1 1 1 1 0 (Question) Komentar Negatif 2 1 0 0 0 0 1 0 0 0 Angka menunjukkan kemampuan partisipan untuk menampilkan perilaku-perilaku yang diharapkan. * Mastery : Batas minimal pencapaian kemampuan partisipan. Tabel 2. Rangkuman Hasil Coding PDI berdasarkan DPICS-III* Sesi Modul 1 Session-2 Session-4 Tgl 5/5 12/5 Perint Lang Tidak Σ Langs Tidak ah sung Langs ung Langs ung ung Patuh 2 0 2 2 1 Tidak 1 1 2 1 1 Patuh Σ 3 1 4 3 2 Perin tah Σ 50 2/4 3/5 % Patuh Modul 2 Σ 3 Session-5 16/5 Langs Tidak ung Langsu ng 3 0 Σ 3 Modul 3 Session-6 Session-7 19/5 23/5 Lang Tidak Σ Lang Tidak Σ sung Lang sung Lang sung sung 3 1 4 4 0 4 2 0 1 1 0 1 1 0 1 1 5 3 1 4 3 2 5 4 1 5 60 % 3/4 226 \| Jurnal Intervensi Psikologi Vol. 9 No. 2 Desember 2017 75 % 4/5 80 % 4/5 80 % Penerapan Prinsip-prinsip Parent-child Interaction Therapy untuk Menurunkan .... Nilai Axis Title 150 100 50 0 13-Apr 13Apr Nilai 120 20-Apr 21Apr 118 27-Apr 25Apr 116 28Apr 114 04-Mei 05Mei 107 11-Mei 12Mei 103 16Mei 99 18-Mei 19Mei 93 23Mei 90 25-Mei 25Mei 85 Gambar 1. Sibling Rivalry Partisipan 1 Partisipan 2 Tabel 3. Rangkuman Hasil Coding CDI berdasarkan DPICS-III* Sesi Pre Coaching CDI Tanggal 13/4 23/4 Coaching PDI 1/5 8/5 11/5 17/5 21/5 Mastery** 4 4 4 4 5 5 Praise-LP) Refleksi (R) 5 Behavioral Description (BD) 2 10 5 10 4 9 5 10 5 9 4 10 7 7 4 Komentar Netral 5 Pujian Umum (Unlabeled 3 1 1 1 1 1 0 2 0 1 0 0 0 - Praise-UP) Perintah (Langsung/Tidak 3 Langsung) 0 1 1 1 1 1 Pertanyaan (Question) Komentar Negatif 1 1 1 0 0 0 0 0 1 0 0 0 Pujian Spesifik (Labeled 2 2 0 4 0 0 0 ≤3 Jurnal Intervensi Psikologi Vol. 9 No. 2 Desember 2017 \| 227 Septhi Karlina Utami, Hendriati Agustiani & Langgersari Elsari Novianti Tabel 4. Rangkuman Hasil Coding PDI berdasarkan DPICS-III Sesi Tgl Perintah Patuh Tidak Patuh Σ Perintah Σ Patuh Modul 1 Session-2 1/5 Lang Tidak Σ sung Lang sung 3 0 3 0 1 0 3 1 3/4 4 Modul 2 Session-4 Session-5 8/5 11/5 Lang Tidak Σ Lang Tidak sung Lang sung Lang sung sung 3 1 4 4 0 0 1 1 0 0 3 75 % 2 4/5 5 4 80 % Σ 4 0 0 4/4 Modul 3 Session-6 Session-7 17/5 21/5 Lang Tidak Lang Tidak Σ Σ sung Lang sung Lang sung sung 4 1 5 4 0 4 0 0 0 0 0 0 4 4 100 % 1 5/5 5 4 100% 0 4/4 4 100 % Skor Axis Title 150 100 50 0 13-Apr 20-Apr 27-Apr 04-Mei 11-Mei 18-Mei 13-Apr 23-Apr 01-Mei 08-Mei 11-Mei 17-Mei 21-Mei 24-Mei Skor 137 124 103 101 90 86 76 69 Gambar 2. Sibling Rivalry Partisipan 2 Dilihat dari hasil uji coba yang dilakukan kepada dua pasangan ibu dan anak tersebut, dapat dilihat bahwa penurunan skor sibling rivalry terjadi ketika ibu sudah menguasai kemampuan PRIDE, yaitu memberikan pujian secara spesifik, mampu merefleksikan, meniru- ketika mengerjakan tugas dari peneliti, dan mulai diterapkan pada setiap kegiatan di rumah. PEMBAHASAN kan, dan mendeskripsikan kegiatan anak dengan jelas, serta adanya rasa antusias selama menemani anak bermain. Hal ini Sibling rivalry memiki tiga dimensi, yaitu kecemburuan, kompetisi dan rasa marah. Pada kasus kakak dengan adik yang mengalami diabilitas intelektual, ibu lakukan baik ketika berkegiatan bersama peneliti di ruang terapi, di rumah kehadiran adik beserta keterbatasannya membuat orang tua terlihat lebih 228 \| Jurnal Intervensi Psikologi Vol. 9 No. 2 Desember 2017 Penerapan Prinsip-prinsip Parent-child Interaction Therapy untuk Menurunkan .... memperhatikan adik dan merasa dirinya diabaikan, menganggap dirinya tidak berharga dibandingkan adik karena adik yang terlihat selalu menjadi prioritas, dan sebagainya. Persepsi ini yang kemudian menimbulkan rasa cemburu atau iri pada kakak. Menyikapi hal demikian, individu berusaha untuk merebut kembali perhatian orang tuanya melalui berbagai cara. Seperti yang diyakini oleh Sigmund Freud dan Melanie Klein (Hiles, 2012) bahwa perhatian dan kasih sayang orang tua merupakan suatu kepuasan (desire of needs) bagi anak yang secara alamiah tumbuh sejak ia bayi. Karenanya, ketika perhatian orang tua seolah hanya tertuju pada adik, secara alamiah ada rasa ketidaknyamanan pada diri kakak yang dinamakan cemburu, lalu menimbulkan rasa marah yang kemudian memunculkan usahanya merebut perhatian orang tua, dengan berbagai cara. Hal ini juga yang terjadi pada kedua partisipan penelitian ini. Partisipan 1 (I) mengakui bahwa ibu terlihat lebih memperhatikan kebutuhan adik dibandingkan dirinya. Sebelum si adik diketahui memiliki keterbatasan, perhatian itu dirasakan sama besarnya. Sementara itu, partisipan 2 (N) mengakui ibunya cukup dapat berlaku adil dalam memenuhi kebutuhan dirinya dan adik. Adil yang dimaksud adalah sama baik dari durasi ataupun jumlahnya. Hanya saja, N merasa ibu tidak cukup adil ketika menerapkan aturan kepada dirinya dan adik. Adik dirasakan N mendapatkan banyak keistimewaan, seperti waktu bermain yang tidak terbatas, mengerjakan tugas sekolah yang selalu ditemani, dan pembelaan yang dirasakan berlebihan ketika ia dan adiknya bertengkar. Baik I maupun N mengakui bahwa mereka iri terhadap perlakuan ibu terhadap adiknya. Baik I maupun N mengaku bahwa masingmasing dirinya sayang kepada adik, mengetahui kondisi adik, namun rasa iri tersebut tidak bisa meredam rasa marah yang sering muncul ketika adik mulai mendapat keistimewaan dari ibu. Dari pemaparan tersebut terlihat bahwa anak merasa diabaikan oleh ibunya karena perhatian dan kasih sayang ibu dinilai lebih banyak diberikan kepada adik yang mengalami diabilitas intelektual. Melalui program intervensi yang diberikan di tahap pertama, ibu diberikan materi mengenai Child Directed Interaction (CDI), yang memiliki sasaran membangun kehangatan, meningkatkan hubungan kasih sayang antara anak dan ibu, serta meningkatkan pengalaman positif antara anak dan ibu. Hasil penelitian menyatakan bahwa skor sibling rivalry menurun dengan cukup besar ketika ibu sudah menguasai keterampilanketerampilan yang diberikan pada tahap ini. Ibu yang menguasai keterampilan untuk mendengarkan anak, memberikan apresiasi terhadap apa yang dilakukan Jurnal Intervensi Psikologi Vol. 9 No. 2 Desember 2017 \| 229 Septhi Karlina Utami, Hendriati Agustiani & Langgersari Elsari Novianti anak, mengikuti kegiatan anak dengan antusias, menyebabkan perasaan-perasaan tidak dihargai dan diabaikan oleh orang tua yang dialami anak mulai menurun. Tahap kedua dari program intervensi yang diberikan adalah Parent Directed Interaction (PDI). Pada tahap ini ibu diajarkan untuk dapat memberikan perintah yang efektif kepada anak. Terdapat perbedaan cara memberikan perintah yang efektif untuk kakak dan adik. Namun, pada intinya adalah perintah harus berupa kalimat langsung yang jelas dan pemberian konsekuensi yang konsisten atas kepatuhannya. Hal ini harus dilakukan baik kepada kakak ataupun adik, agar rasa cemburu yang dialami kakak dapat menurun. Perubahan terbesar terlihat pada partisipan 2 (N). Hasil skor sibling rivalry N menurun dengan drastis ketika ibu sudah diberikan materi mengenai PDI ini. PCIT merupakan intervensi yang membantu memperbaiki kualitas hubungan antara ibu dan anak. Dalam penelitian ini, melalui intervensi yang menerapkan prinsip-prinsip PCIT kakak (yang berusia remaja) berhasil membentuk secure attachment dengan ibunya, memiliki hubungan yang hangat dengan ibu, dapat bekerja sama dalam suatu kegiatan dengan ibu, sehingga menimbulkan perasaan senang, diperhatikan, dan disayangi. Terlebih ketika ibu berhasil menerapkan keterampilan yang diajarkan dalam berinteraksi dengan kedua anaknya secara berimbang. Sesuai dengan yang yang dinyatakan McNeil-Kigin (2010), PCIT yang diberikan kepada orang tua dengan anak lebih dari satu orang, membuat orang tua tersebut harus dapat menerapkan keterampilannya tersebut kepada seluruh anaknya. Pada penelitian ini, kakak mulai merasakan bahwa kasih sayang dan perhatian ibu sama besar kepada dirinya dan adiknya, setelah intervensi diberikan. Persepsi negatif mengenai perbedaan perlakuan yang sebelumnya dimiliki oleh kakak berubah menjadi positif. Hal ini terjadi karena emosi dapat mengarahkan persepsi yang dimiliki oleh suatu individu dengan suatu stimulus yang sangat berkaitan secara emosional. Persepsi individu terhadap lingkungannya akan sangat bergantung pada kebutuhan, tujuan, dan nilai-nilai yang dianutnya (Brosch, 2013). Keberhasilan program intervensi ini karena beberapa faktor, misalnya kerja sama yang baik antara ibu, anak, dan peneliti sebagai pelaksana program intervensi. Kedua ibu yang menjadi partisipan tidak segan untuk mengkomunikasikan segala kendala yang mereka temui di rumah ketika melakukan tugas yang diberikan untuk sama-sama mencari solusi, sehingga pembelajaran terus berlanjut. Selain itu adanya upaya dan semangat ibu untuk menangani konflik 230 \| Jurnal Intervensi Psikologi Vol. 9 No. 2 Desember 2017 Penerapan Prinsip-prinsip Parent-child Interaction Therapy untuk Menurunkan .... yang terjadi di keluarganya, dalam hal ini adalah sibling rivalry yang dialami anak, membuat para ibu ini terlihat berupaya untuk mengubah dirinya sendiri. Kedua ibu memiliki proses belajar yang berbeda dalam hal kecepatan. Namun, keduanya sama-sama memiliki semangat yang besar untuk berubah, terlihat dari kehadirannya yang tepat waktu di setiap pertemuan yang dijanjikan. Hal ini mendukung jalannya program intervensi sehingga dapat efektif untuk melatihkan keterampilan PCIT pada ibu dalam berinteraksi dengan anaknya masing-masing. Dengan PCIT, ibu belajar gaya pengasuhan yang otoritatif, memenuhi kebutuhan rasa aman untuk membentuk attachment positif, dan prinsip-prinsip belajar sosial untuk mengubah perilaku anak (McNeilKigin, 2010). Pada partisipan 2 (ibu N), proses belajar terjadi dengan cepat. Hal ini tidak terlepas dari dukungan suami yang diperolehnya, sedangkan partisipan 1 (ibu A) merupakan orang tua tunggal. Menurut Brinkmeyer dan Eyberg (McNeil-Kigin, 2010), PCIT dapat melibatkan salah satu atau kedua orang tua atau orang lainnya yang berperan penting dalam pengasuhan anak. Ibu N, memberikan ilmu yang dipelajarinya selama intervensi kepada suaminya di rumah, sehingga keduanya bisa saling mengingatkan dan memiliki pola asuh yang sama kepada kedua anaknya. Hasilnya, menurut remaja N, perubahan terjadi bukan hanya pada cara ibu berbicara kepadanya, namun juga pada ayahnya. SIMPULAN DAN SARAN Simpulan Program intervensi dengan menggunakan prinsip-prinsip Parent-Child Interaction Therapy yang dirancang dapat digunakan untuk menurunkan sibling rivalry pada remaja yang memiliki adik dengan diabilitas intelektual. Uji coba terhadap dua pasangan ibu dan anak, diperoleh data kuantitatif bahwa program intervensi yang dirancang dengan menggunakan prinsip-prinsip Parent-Child Interaction Therapy dapat menurunkan sibling rivalry pada remaja yang memiliki adik dengan diabilitas intelektual. Pada partisipan 1, program intervensi dengan menggunakan prinsipprinsip PCIT ini membuat anak merasa dihargai dan diperhatikan, sehingga dapat mengurangi rasa cemburu, marah, dan persaingan anak kepada adiknya. Pada partisipan 2, program intervensi dengan menggunakan prinsip-prinsip PCIT ini membuat ibu dan ayah bekerjasama untuk mengubah sikap dalam menerap-kan peraturan di rumah. Hal ini membuat anak merasa dihargai. Jurnal Intervensi Psikologi Vol. 9 No. 2 Desember 2017 \| 231 Septhi Karlina Utami, Hendriati Agustiani & Langgersari Elsari Novianti Saran Orang tua perlu menerapkan keterampilan yang sudah dikuasainya selama program intervensi kepada seluruh anak yang dimilikinya agar tidak ada seseorang yang merasa diistimewakan atau diabaikan. Orang tua perlu menerapkan keterampilan yang sudah dikuasainya secara konsisten agar perubahan perilaku anak dapat bersifat permanen. DAFTAR PUSTAKA American Psychiatric Association. (2013). diagnostic and statistical manual of mental disorders, fifth edition. Arlington, VA: American Psychiatric Association. Brosch, T. et al. (2013). The impact of emotion, on perception, attention, memory and decision making. Swiss Med Wkly. 2013; 143: w13786 Chaulagain S., PU S., Moras SM., Aranha PR, & Shetty, AP. (2016). A study on knowledge regarding sibling rivalry in children among mothers in selected hospital at mangaluru. JSIR, 5(4), 122-124. Cicirelli, V. G. (1995). 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Pendekatan pcit (parent-child interaction therapy) pada anak usia sekolah dengan masalah perilaku disruptive. Tesis. Depok: Fakultas Psikologi Universitas Indonesia. Sawicki, J. A. (1997). Sibling rivalry and the new baby: anticipatory guidance and management strategies. Pediatric Nursing. Strohm, K. (2006). Sibling of children with special needs. Retrieved 2015, from www.learninglinks.org.au. Volling, B. L., Kennedy, D. E., & Jackey, L. M. (2010). The development of sibling jealousy. in s. l. hart, & m. legerstee, handbook of jealousy, theory research and multidisciplinary approaches (pp. 387412). UK: John Wiley & Sons. Jurnal Intervensi Psikologi Vol. 9 No. 2 Desember 2017 \| 233
https://openalex.org/W3167826362	https://pure.knaw.nl/ws/files/230017704/Derksen2021.pdf	English	null	Animal studies in clinical MRI scanners: A custom setup for combined fMRI and deep-brain stimulation in awake rats	Journal of neuroscience methods	2,021	cc-by	14,839	Take down policy If b li h h 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. Royal Netherlands Academy of Arts and Sciences (KNAW) Animal studies in clinical MRI scanners Derksen, Maik; Rhemrev, Valerie; van der V Mulder, Tosca; Reneman, Liesbeth; Nederv Denys, Damiaan published in Journal of Neuroscience Methods 2021 DOI (link to publisher) 10.1016/j.jneumeth.2021.109240 document version Publisher's PDF, also known as Version of record document license CC BY Link to publication in KNAW Research Portal Link to publication in KNAW Research Portal citation for published version (APA) Derksen, M., Rhemrev, V., van der Veer, M., Jolink, L., Zuidinga, B., Mulder, T., Reneman, L., Nederveen, A., Feenstra, M., Willuhn, I., & Denys, D. (2021). Animal studies in clinical MRI scanners: A custom setup for combined fMRI and deep-brain stimulation in awake rats. Journal of Neuroscience Methods, 360, 109240. https://doi.org/10.1016/j.jneumeth.2021.109240 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. 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Take down policy Maik Derksen a,b, Valerie Rhemrev a, Marijke van der Veer a, Linda Jolink a, Birte Tosca Mulder a, Liesbeth Reneman c, Aart Nederveen c, Matthijs Feenstra a,b,1, Ingo Willuhn a,b,1,, Damiaan Denys a,b,1 a The Netherlands Institute for Neuroscience, Royal Netherlands Academy of Arts and Sciences, Amsterdam, the Netherlands b Department of Psychiatry, Amsterdam University Medical Centers (location AMC), University of Amsterdam, Amsterdam, the Netherlands c Department of Radiology and Nuclear Medicine, Amsterdam University Medical Centers (location AMC), University of Amsterdam, Amsterdam, the Netherlands Contents lists available at ScienceDirect Contents lists available at ScienceDirect A B S T R A C T Keywords: Rodent fMRI Human MRI scanner Deep-brain stimulation Novel electrodes Translational research Custom-made MRI coils Keywords: Rodent fMRI Human MRI scanner Deep-brain stimulation Novel electrodes Translational research Custom-made MRI coils Background: In humans, functional magnetic resonance imaging (fMRI) cannot be used to its full potential to study the effects of deep-brain stimulation (DBS) on the brain due to safety reasons. Application of DBS in small animals is an alternative, but was hampered by technical limitations thus far. New Method: We present a novel setup that extends the range of available applications by studying animals in a clinical scanner. We used a 3 T-MRI scanner with a custom-designed receiver coil and a restrainer to measure brain activity in awake rats. DBS electrodes made of silver were used to minimize electromagnetic artifacts. Before scanning, rats were habituated to the restrainer. Results: Using our novel setup, we observed minor DBS-electrode artifacts, which did not interfere with brain- activity measurements significantly. Movement artifacts were also minimal and were not further reduced by restrainer habituation. Bilateral DBS in the dorsal part of the ventral striatum (dVS) resulted in detectable in creases in brain activity around the electrodes tips. Comparison with Existing Methods: This novel setup offers a low-cost alternative to dedicated small-animal scanners. Moreover, it can be implemented in widely available clinical 3 T scanners. Although spatial and temporal resolution was lower than what is achieved in anesthetized rats in high-field small-animal scanners, we obtained scans in awake animals, thus, testing the effects of bilateral DBS of the dVS in a more physiological state. Conclusions: With this new technical setup, the neurobiological mechanism of action of DBS can be explored in awake, restrained rats in a clinical 3 T-MRI scanner. in specific neural circuits (Bourne et al., 2012; Figee et al., 2012). Corresponding author at: The Netherlands Institute for Neuroscience, Royal Netherlands Academy of Arts and Sciences, Amsterdam, the Netherlands. E-mail address: i.willuhn@nin.knaw.nl (I. Willuhn). 1 authors contributed equally. E-mail address: pure@knaw.nl E-mail address: pure@knaw.nl E-mail address: pure@knaw.nl Download date: 24. Oct. 2024 Journal of Neuroscience Methods 360 (2021) 109240 Available online 5 June 2021 0165-0270/© 2021 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). * Corresponding author at: The Netherlands Institute for Neuroscience, Royal Netherlands Academy of Arts and Sciences, Amsterdam, the Netherlands. E-mail address: i.willuhn@nin.knaw.nl (I. Willuhn). 1 authors contributed equally. https://doi.org/10.1016/j.jneumeth.2021.109240 Received 6 September 2020; Received in revised form 30 March 2021; Accepted 1 June 2021 Available online 5 June 2021 0165-0270/© 2021 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). https://doi.org/10.1016/j.jneumeth.2021.109240 Received 6 September 2020; Received in revised form 30 March 2021; Accepted 1 June 2021 Animal studies in clinical MRI scanners: A custom setup for combined fMRI and deep-brain stimulation in awake rats Maik Derksen a,b, Valerie Rhemrev a, Marijke van der Veer a, Linda Jolink a, Birte Zuidinga a, Tosca Mulder a, Liesbeth Reneman c, Aart Nederveen c, Matthijs Feenstra a,b,1, Ingo Willuhn a,b,1,, Damiaan Denys a,b,1 2.1. Electrodes and fabrication Our overall aim is to use fMRI in awake animals to study the effects of high-frequency DBS in different target locations, such as the ventral striatum/nucleus accumbens and the internal capsule (Denys et al., 2020; van Dijk et al., 2013; Pinhal et al., 2018). Here, we present our findings using the novel setup while using the dorsal part of the ventral striatum (dVS) as target for DBS. Scanning awake animals is an advantage because anesthetics have been reported to suppress and alter neural activity and functional con nectivity (compared to awake animals), while increasing cerebral blood flow because of the vasodilatory effect of the anesthetics (Pan et al., 2015; Paasonen et al., 2018). Different anesthetics tend to uniquely modulate functional connectivity in MRI settings, all distinct from that in awake animals (Paasonen et al., 2018) possibly reducing the trans lational nature of these experiments (Chuang and Nasrallah, 2017). For these reasons, and with the reported possibility of immobilizing rodents without overt signs of stress (King et al., 2005; Febo, 2011; Yee et al., 2016), we decided to perform fMRI acquisition in awake rats that were restrained in a custom-designed combination of a body tube, head-stage and nose cone. We have adopted a training protocol aiming to habituate the animals to, and minimize stress caused by the restraining and MRI environment. The effectiveness of our restraining training protocol was examined by looking at the changes in blood-plasma corticosterone levels over time and by comparing motion parameters during fMRI acquisition between trained and untrained animals. The impedance of each electrode was tested by placing the electrode tips in saline solution (0.9 % sodium chloride in water) while connected Fig. 1. Silver electrode induces smaller MRI artifact than standard platinum/ iridium electrode. A) Custom-made platinum/iridium (Pl/Ir) electrode commonly used in animal research. Both two-channel electrodes are made of insulated, twisted wires. Insulation was removed from the lower 0.2 mm. B) T2- weighted MR image of platinum/iridium electrode suspended in saline solution. C) Custom-made MRI-compatible silver (Ag) electrode. D) T2-weigthed MR image of silver electrode suspended in saline solution. The silver electrode in duces a smaller MRI artifact compared to the standard platinum/ iridium electrode. We explored the possibilities of using a standard 3 T clinical MRI scanner. 2.1. Electrodes and fabrication Stimulation electrodes were custom-made from either platinum/ iridium wires (90 % platinum, 10 % iridium, 75 μm bare diameter, 18 μm PFTE insulation coating, Advent Research Materials LTD, Oxford, United Kingdom), a paramagnetic alloy commonly used for electrodes in animal research with a magnetic susceptibility of 27 × 10−5 at 20 de grees Celsius, or silver wires (99.99 % pure silver, 125 μm bare diameter, 26 um PFTE insulation coating, Advent Research Materials LTD, Oxford, United Kingdom), a diamagnetic alloy with a relatively lower magnetic susceptibly of -2.6 × 10-5. Where MRI studies in DBS patients suffer from electrode imaging artifacts because of the materials used, and in addition are limited to low-field MRI scanners with the DBS turned off during acquisition due to safety considerations (Tagliati et al., 2009; Medtronic, 2015), studying the effects of DBS on the brain in a rodent model has several advantages. For instance, combining DBS and fMRI in an animal model allows tar geting of different and multiple brain areas, varying the properties of the DBS current applied to the brain, and comparing effects in disease models and normal controls (Feenstra and Denys, 2012). In addition, higher filed strengths can be applied. The wires were twisted into a two-channel electrode lead and cut to the appropriate length, with a distance of approximately 250 μm be tween the two contact points in the longitudinal direction in order to enlarge the stimulation area and prevent short-circuiting (Fig. 1A&C). From each contact point, approximately 200 μm of insulation material was removed to enlarge the contact surface. At the opposite end, the electrode connector was created by stripping approximately 3.5 mm of the leads’ insulation and inserting each end into a 7 mm long sterling silver tube (92.5 % silver, 7.5 % copper, 1.5/1.0 mm outer/inner diameter, Bijou Moderne, Bleiswijk, the Netherlands). The end of the tube where the lead was inserted into was closed off with pliers; the flattened tube end now clasping onto the lead inside and ensuring electrical conduction, and the open tube end serving as a connector. Both silver tubes were then glued into a small plastic block holding them in place (Fig. 2). i In the present study, we describe a novel DBS-MRI setup for inves tigating the effects of DBS on brain activity in awake rats, using fMRI. 2. Methods and analyses improvements on this matter have been made, for instance, by reducing the amount of ferromagnetic material used and the programming of specific absorption rate (SAR) reducing MR-sequences, MRI in DBS pa tients at 1.5 T and pulse-sequences with limited RF power are recom mended (Medtronic, 2015). 1. Introduction i With functional magnetic resonance imaging (fMRI) it is possible to investigate DBS-induced regional as well as global changes in brain function. However, fMRI in patients with DBS implants is a safety haz ard. The interaction between the MRI scanner’s radio frequency (RF) fields and the implanted electrodes, the conductive leads, and other DBS devices, can induce electric currents on the lead wires, which increase the energy absorption in the tissue surrounding the DBS electrodes (Golestanirad et al., 2019). Furthermore, the static magnetic field and gradient system of the MR machine might cause movement of the implanted electrodes or malfunction of the DBS device. Even though Deep-brain stimulation (DBS) is an intervention that relies on implanted electrodes delivering high-frequency currents into targeted brain areas. It is increasingly being examined as a treatment for neuropsychiatric disorders (Harmsen et al., 2020), such as obsessive compulsive disorder (Denys et al., 2020), major depressive disorder (van der Wal et al., 2020), and drug and alcohol addiction (Bari et al., 2018). While there is still a limited understanding of the underlying mecha nisms of action of DBS and its therapeutic effect, it is often hypothesized that DBS results in restoring normal activity and functional connections Journal of Neuroscience Methods 360 (2021) 109240 Journal of Neuroscience Methods 360 (2021) 109240 M. Derksen et al. 2.1. Electrodes and fabrication The choice for a clinical MRI scanner was prompted by our wish to perform fMRI experiments in rats when access to a high-field small animal scanner is not available. This meant that we had to use a newly designed MRI receiver coil intended for scanning rat brains. This two- channel surface coil consists of three coil loops covering the top and sides of the rat brain and enables parallel imaging. The coil fits closely around the head of the animal within the restrainer tube. Both the restrainer and coil designs incorporate special openings for connecting gas tubes and wires. As our standard platinum/iridium DBS electrodes are suboptimal for use in an MRI scanner, we used custom-made, MRI compatible elec trodes made of 99.99 % pure silver wires. The MRI artifacts generated by magnetic susceptibility of our silver electrodes were compared to the artifacts seen when using platinum/iridium wires, a material commonly used in preclinical animal research and some patient DBS systems (Medtronic, 2009). We assessed the effectiveness of our custom-made electrodes by comparing cFOS expression at the electrode tip of an active DBS electrode in the dVS, with that of a non-active electrode. Fig. 1. Silver electrode induces smaller MRI artifact than standard platinum/ iridium electrode. A) Custom-made platinum/iridium (Pl/Ir) electrode commonly used in animal research. Both two-channel electrodes are made of insulated, twisted wires. Insulation was removed from the lower 0.2 mm. B) T2- weighted MR image of platinum/iridium electrode suspended in saline solution. C) Custom-made MRI-compatible silver (Ag) electrode. D) T2-weigthed MR image of silver electrode suspended in saline solution. The silver electrode in duces a smaller MRI artifact compared to the standard platinum/ iridium electrode. With this setup, we have successfully acquired BOLD fMRI mea surements of bilateral DBS, in animals that were awake during data acquisition, and with minimal electrode artifacts. 2 Journal of Neuroscience Methods 360 (2021) 109240 M. Derksen et al. anesthetics (3% induction, 1.8-2.5% maintenance, 1:1 air in oxygen at 0.3 L/min, Isoflo, Zoetis, Rotterdam, the Netherlands) and their tem perature was kept at 37 degrees Celsius by means of a heating pad. After a sagittal incision, the skull was exposed and two burr holes were drilled to allow for bilateral electrode implants. 2.1. Electrodes and fabrication Two custom-made silver elec trodes were lowered into the brain, targeting the dVS (AP + 1.72 mm, ML +/- 1.60 mm from bregma and DV – 7.0 mm) (Paxinos and Watson, 2007). Four stainless steel screws were placed rostrally and caudally to the electrodes, to secure the electrodes to the skull with acrylic dental cement (Simplex Rapid, Kemdent, Swindon, United Kingdom). Then, the wound was closed with sutures and the animals were given a subcu taneous meloxicam injection (1 mg/kg, Metacam, Boehringer Ingelheim Vetmedica Ltd., Duluth, Georgia, United States) for post-op pain man agement. The animals were allowed a minimum recovery time of one week. Fig. 2. Materials and manufacturing of custom-made MRI-compatible silver electrode. A) Insulated silver wire twisted into two-channel electrode lead (99.99 % pure silver, 125 μm bare diameter, 26 μm PTFE insulation coating; Advent Research Materials LTD, Oxford, United Kingdom). B) Silver tubes, used as connector (92.5 % sterling silver, Bijou Moderne, Bleiswijk, The Netherlands). C) Plastic holder for connector. D) Fully assembled electrode. 2.2. MRI In order to determine the MRI artifacts due to the magnetic suscep tibility of the different materials, the electrodes were lowered into a small container completely filled with saline solution (0.9% sodium chloride in water) which was closed off. T2-weighted images were ob tained using a multi-shot turbo spin echo (TSE) sequence (TSE factor = 8, TR =4000 ms, TE =56 ms, acquired voxel size = 0.2 × 0.2x1.5 mm, acquired matrix size = 176 × 168, 12 slices). The images were acquired on a 3.0 T Philips Ingenia MR system (Philips Healthcare, Best, the Netherlands) using the custom-made two-channel receiver coil (MRCoils, Zaltbommel, the Netherlands), part of the restrainer setup described below. 2.3.1. Subjects Fifteen male Wistar rats (225 – 250 grams, Charles River Labora tories, Saint-Germainsur-l’Arbreste, France) were included in this study. Four animals were excluded on basis of incorrect electrode placement. The animals were housed socially during the acclimatization period of one-week minimum before surgery and individually after surgery, on a normal day-night cycle (lights on 7am to 7pm) with ad libitum food and water. The experiments were approved by the Animal Experimentation Committee of the Royal Netherlands Academy of Arts and Sciences (KNAW) and carried out in agreement with Dutch law (Wet op de Dierproeven, 1996) and European regulations (Guideline 2010/63/EU). 2.3.3. Unilateral DBS To protect the leads connected to the DBS electrodes from being damaged by the animals or getting intertwined, animals were tethered to a counterbalanced arm, part of a Stand-Alone Raturn System (BASi, West Lafayette, Indiana, United States), allowing the animal to move freely inside a plastic bowl with cage bedding and access to water, in a non-illuminated box. Both implanted electrodes were connected to a digital stimulator (DS8000, World Precision Instruments, Berlin, Ger many) through constant current digital linear stimulation isolators (DSL100, World Precision Instruments, Berlin, Germany) with carbon fiber wires (CPVC4050, 18 American wire gauge, 1.0 mm diameter, 3000 strands, World Precision Instruments, Berlin, Germany). If the impedance of the left hemisphere electrode did not exceed 50kΩ, it received biphasic, constant current pulses (80 μs pulse width at 130 Hz, 300 μA stimulation, as described in van Dijk et al., 2011) for 30 minutes continuously, with the right electrode functioning as sham. If the left hemisphere electrode did exceed 50kΩ, sham and stimulation electrode sides were reversed. Ninety minutes after DBS, the animals were deeply anesthetized with sodium pentobarbital (100 mg/kg) and transcardially perfused with phosphate-buffered saline (PBS) and fixated with 4% paraformaldehyde (PFA). The brain was put in PFA for 24 hours, transferred to a 30% sucrose in PBS solution until saturated, and flash frozen in isopentane (HoneyWell, Seelze, Germany) before stored at -80 degrees Celsius. Fig. 2. Materials and manufacturing of custom-made MRI-compatible silver electrode. A) Insulated silver wire twisted into two-channel electrode lead (99.99 % pure silver, 125 μm bare diameter, 26 μm PTFE insulation coating; Advent Research Materials LTD, Oxford, United Kingdom). B) Silver tubes, used as connector (92.5 % sterling silver, Bijou Moderne, Bleiswijk, The Netherlands). C) Plastic holder for connector. D) Fully assembled electrode. to an impedance meter (IMP-1, BAK Electronics, Inc., Umatilla, Florida, United States). Measured impedances varied between 10 and 20 kΩ at 1 kHz. 2.3.4. cFOS immunohistochemistry y Coronal sections of the animals’ brains were taken using a cryostat (Leica Biosystems, Wetzlar, Germany), collected free-floating in cryo protectant (30% glycerol, 30% ethylene glycol, 40% 0.1 M PBS), and stored at -20 degrees Celsius. Sections containing the electrode tips were selected for cFOS immunohistochemistry, with two consecutive slices per electrode tip. The sections were stained using the avidin-biotin complex (ABC) method. First, the sections were pretreated for ten mi nutes in a solution of 10% methanol (HoneyWell, Seelze, Germany) and 3% H2O2 (30%, Merck, Darmstadt, Germany) in TBS to suppress endogenous peroxidase. Then, after immersing them in blocking solu tion (10% horse serum (Invitrogen, Carlsbad, California, United States) in SuperMix (SUMI, 0.5% Triton-X 100 (Sigma-Aldrich, Zwijndrecht, the Netherlands) and 0.25% gelatin in TBS)), the sections were incubated in a solution of the primary antibody, rabbit anti-cFOS (1:5000, ab190289, Abcam Biotechnologies, Cambridge, United Kingdom), and 4% horse serum in SUMI for 24 hours at 4 degrees Celsius; then incubated in a solution of the secondary antibody, biotinylated horse-anti-rabbit IgG (1:600, Vector Laboratories Inc., Burlingame, California, United States), and 4% horse serum in SUMI for 1 hour; then placed in a avidin-biotin horseradish peroxidase complex solution (1:800, ABC Elite Kit, Vector Laboratories Inc., Burlingame, California, United States) for one hour for signal amplification; and finally incubated in a solution of dia minobenzidine (DAB, 0.05%, Sigma-Aldrich, Zwijndrecht, the Netherlands) with 0.03% H2O2 and 0.23% nickel/ammonium sulphate 2.3. cFOS 2.4. Restrainer setup with copper wiring, effectively forming a single loop. The connecting copper wiring of the second loop creates a 10-percent overlap with the first, decoupling the two channels. Together, the coil loops’ coverage encompasses the entire brain of the rodent. The coil loops were placed on the inside of an acrylic arch, with an opening in the top, that will fit tightly around an adult rat’s head. Coil tuning is achieved by fixed ca pacitors, while matching can be done with variable capacitors on-site if needed. The leads running from the surface coil are covered by ceramic floating cable traps, suppressing shield currents. The coil can be con nected to the scanner-bed interface through a custom-made interface box (MRCoils, Zaltbommel, the Netherlands). This sixteen-channel interface box, containing low noise factor amplifiers and transmit/ receive switches, serves to connect custom made MRI coils through BNC- connectors. The interface box itself connects to the scanner bed The custom-build, MRI-compatible restrainer setup, was designed for the purpose of MR image acquisition of the brains of awake rats, while retaining access to the animal’s head and tail (Fig. 3). This offers a wide variety of experimental possibilities during MRI acquisition, such as electrical or optical stimulation, access to cranial windows and/or in fusions into or sampling from the brain, as well as from the tail. The restrainer setup consists of three main parts – a body tube, a bite bar with anesthetics nose cone, and a fully adaptable head stage holder – which are placed onto a clear plastic base plate. Inside the setup, a custom-made two-channel surface receiver coil array (MRCoils, Zalt bommel, the Netherlands) can be placed and secured (Fig. 4). This surface coil consists of two loops, one covering the top of the rodent’s head (channel one). The second loop (channel two) is created by con necting two loops located to the left and right side of the rodent’s head Fig. 3. Custom-made MRI restrainer setup for simultaneous scanning and DBS in awake rodents. A) Top-down view: 1- oxygen and isoflurane supply-hose, 2- adjustable horizontal bar (black) for head fixation, 3- fully adjustable nose piece including bite bar and nose cone for isoflurane supply (blue), 4- custom made MRI coil (see Fig. 4 for more details), 5- shoulder bar to restrict forward movement (black), 6- clear body tube, 7- disc to restrict backward movement (white). 2.3.5. cFOS detection The sections were digitized at 10x magnification with a CCD camera (D-sight bright-field slide scanning microscope, A. Menarini Diagnostics, Italy) at equal lighting settings between images. Analyses were per formed using Image Pro (Image Pro Plus, Media Cybernetics Inc., Rockville, Maryland, United States). A circular ROI of a fixed size (350 μm diameter) was manually placed around the electrode tip, and a background measurement area was set over white matter. A low-pass filtered image was created from the green channel of the 8-bit RGB image, in which pixels that exceeded the optical density (OD) threshold (set to produce the optimal SNR in relation to the background area) were marked as cFOS positive. Correction for difference in background staining between sections was done by calculating the relative density (RD): if the OD measured in the background would exceed the OD threshold, the background OD was subtracted from the OD in the ROI. The RD was averaged over the two selected section per animal, for each ROI. Fig. 4. Custom-made two-channel head surface coil for rats. A) The coil inside its casing. Electrode and head-fixation connectors protrude through an access hole (arrow), allowing the coil to be lowered fully onto the animal’s head. B) Top-down view of the coil, with casing removed: 1- access hole, 2- the coil loop covering the top of the rodent’s head. C) Inside, bottom-up view of the two- channel surface coil. Arrows indicate the placement of the three coil loops constituting the coil array: 1- loop covering the top of the rodent’s head (channel 1), 2 and 3- loops covering the sides of the rodent’s head (channel 2). D) Side view: 1- the coil loop covering the top of the head, 2- the coil loop covering the left side of the head (same as right side). 2.3.6. Analysis The difference between the cFOS expression in DBS versus sham stimulated hemispheres was analyzed by performing a paired samples t- test on the average RDs, obtained as described above (n = 9). The analysis was done using the open-source statistics software JASP (JASP Team, 2019, version 0.9.1). 2.3.2. Surgery 2.3.2. Surgery For electrode implantation, animals were put under isoflurane g y For electrode implantation, animals were put under isoflurane 3 Journal of Neuroscience Methods 360 (2021) 109240 M. Derksen et al. Fig. 4. Custom-made two-channel head surface coil for rats. A) The coil inside its casing. Electrode and head-fixation connectors protrude through an access hole (arrow), allowing the coil to be lowered fully onto the animal’s head. B) Top-down view of the coil, with casing removed: 1- access hole, 2- the coil loop covering the top of the rodent’s head. C) Inside, bottom-up view of the two- channel surface coil. Arrows indicate the placement of the three coil loops constituting the coil array: 1- loop covering the top of the rodent’s head (channel 1), 2 and 3- loops covering the sides of the rodent’s head (channel 2). D) Side view: 1- the coil loop covering the top of the head, 2- the coil loop covering the left side of the head (same as right side). (Sigma-Aldrich, Zwijndrecht, the Netherlands) for 4 minutes – with washing steps preceding all incubations. Finally, the sections were mounted on gelatin coated slides, dehydrated with increasing EtOH concentrations, cleared in xylene, and coverslipped using Entellan mountant (Merck, Darmstadt, Germany). 2.5.3. Restraining-habituation protocol h l f h At this point the first blood sample was collected (200 μL, indicated as ‘before restraining sample’ as the animal is still anesthetized) in a blood collection capillary tube (Microvette CB 300 Z, Sarstedt AG & Co. KG, Nümbrecht, Germany), and the tubing and dead space of the catheter were flushed with a heparin solution (2.5 I.U./mL in 0.9 % saline solution). Then the animal was placed inside a sound insulated box, in which two audio speakers were placed. Isoflurane anesthetics were turned off, and a second blood sample was taken at this time. By means of an USB camera placed inside the box, the animals could be observed. Ten minutes after the anesthetics were turned off, the animals were awake - as indicated by whisker movements. At this point the third blood sample was taken (indicated as ‘beginning of restraining sample’). Finally, the animal’s head is secured into place. This is done by attaching a head stage (a 3D-printed plastic block which is attached onto the animal’s skull) to the head stage holder of the restrainer setup. The head stage holder is composed of four vertical carbon fiber rods, two on the left and two on the right of the setup, connected by two horizontal, plastic, 3D-printed beams. From the center of those beams, a single carbon fiber rod runs from left to right with the center of the rod located over the animal’s head. This rod can be lowered onto the animal’s head, and a plastic hood, glued into the rod, can be placed over the head stage that is fixed onto the animal’s skull and locked down with a plastic screw. The head stage holder is fully adjustable and can be moved in all directions to fixate the animal’s head and align it correctly for MR image acquisition. i With the animal awake, MRI sounds consisting of the noises of a variety of sequences were played through the speakers inside the box for 60 min. The power level measured at the location of the animal’s head reached a maximum of 100 decibels. During this period, blood samples were collected every ten minutes until the end of the training session (indicated as ‘end of restraining sample’). 2.5.3. Restraining-habituation protocol h l f h The training protocol for the purpose of habituating the animals to being restrained, consisted of six consecutive days of the animals being restrained and exposed to MRI noises, aiming at acclimatization to the restraining and noises and thereby reducing the stress the animals experience. During the training, blood samples were taken at regular intervals, in order to later assess the stress levels of the animals by analyzing the amount of corticosterone in the blood plasma. l The animal’s incisor teeth are placed over a bite bar. This piece of 3D-printed plastic is designed after a typical stereotaxic bite bar, and can be moved forwards, backwards and up and down. A 3D-printed nose cone can be placed over the animal’s nose, to provide inhalation anes thetics or other gasses. After fixating the animal’s incisors properly in side the bite bar it can be locked into place with screws. The part of the bite bar that actually gets covered by the incisors, can easily be taken out and replaced if damaged. Rats were mildly sedated using isoflurane anesthesia to be placed in the fMRI-compatible restrainer device (3% induction, 1.5-2.0 % during placement, in medical air (70 % NO2, 30 % O2) at 0.6 L/min). The paws of the animal were fixated against its body before it being placed inside a clear plastic tube. Then the rat was fixated as described before. The custom-made, two-channel arch-shaped receiver coil is then lowered onto the animal’s head and secured into the setup with screws on the left and right side. The coil can be moved down as to touch the head of the animal; the body tube and the bite bar can be moved for wards or backwards to locate the animal’s head exactly in the center of the coil. In the top of the coil is a 1.5x1.0 cm large opening, allowing access to the animal’s skull. There is room for items protruding from, or being lowered into the skull, such as cannulas or connectors. pi After the animal was placed inside the restrainer, a catheter, with a length of clear tubing which could be closed off connected to it, was placed inside the tail vein in order to take blood samples during the training sessions (Vasofix Safety, 22 G, B. Braun Melsungen AG, Mel sungen, Germany). 2.4. Restrainer setup B) Head implants: 1- Silver connectors of two bilaterally implanted custom-made silver electrodes, 2- Plastic connector for head fixation. C) Close-up front view: 1- shoulder bar to restrict forward movement, 2- clear body tube, 3- screw running through the adjustable horizontal bar, used to attach and fixate animal’s head, 4- electrode connectors, 5- custom-made MRI coil, 6- animal’s nose protruding into the nose cone (blue). Fig. 3. Custom-made MRI restrainer setup for simultaneous scanning and DBS in awake rodents. A) Top-down view: 1- oxygen and isoflurane supply-hose, 2- adjustable horizontal bar (black) for head fixation, 3- fully adjustable nose piece including bite bar and nose cone for isoflurane supply (blue), 4- custom made MRI coil (see Fig. 4 for more details), 5- shoulder bar to restrict forward movement (black), 6- clear body tube, 7- disc to restrict backward movement (white). B) Head implants: 1- Silver connectors of two bilaterally implanted custom-made silver electrodes, 2- Plastic connector for head fixation. C) Close-up front view: 1- shoulder bar to restrict forward movement, 2- clear body tube, 3- screw running through the adjustable horizontal bar, used to attach and fixate animal’s head, 4- electrode connectors, 5- custom-made MRI coil, 6- animal’s nose protruding into the nose cone (blue). 4 Journal of Neuroscience Methods 360 (2021) 109240 M. Derksen et al. interface. The MR scanner’s body coil was used as the transmit coil. interface. The MR scanner’s body coil was used as the transmit coil. induction, 1.8-2.5% maintenance, 1:1 air in oxygen at 0.3 L/min, Isoflo, Zoetis, Rotterdam, the Netherlands) and their temperature was kept at 37 degrees Celsius by means of a heating pad. After a sagittal incision, the skull was exposed, the plastic pedestal was placed onto the skull and two burr holes were drilled to allow for plastic, MRI-compatible screws to lock the pedestal in place using the screw holes of the pedestal. A third screw was placed caudally to the pedestal, and acrylic dental cement (Simplex Rapid, Kemdent, Swindon, United Kingdom) was used to secure the pedestal in place. Then, the wound was closed with sutures and the animals were given a subcutaneous meloxicam injection (1 mg/ kg, Metacam, Boehringer Ingelheim Vetmedica Ltd., Duluth, Georgia, United States) for post-op pain management. The animals were allowed a minimum recovery time of one week. The animal’s body is secured into place inside a clear plastic tube. 2.5.3. Restraining-habituation protocol h l f h As the clear tubing connected to the tail vein catheter was running from inside the box to the outside, the box could remain closed throughout the training sessions, and the tubing and dead space of the catheter were flushed after every sampling with a pre-calculated volume of heparin solution to ensure no blood remained in the tubing and catheter. Blood plasma was prepared by centrifuging the blood samples for 15 min at 2000 x g at 4 degrees Celcius. The plasma was stored at -20 degrees Celsius until corticoste rone immunoassay. After the animal is fixated in the restrainer setup, the whole setup can be placed on the scanner bed and put inside the bore. By placing sand bags against the sloping sides of the base plate, movement of the setup inside the scanner can be prevented. 2.5.4. Corticosterone immunoassay Plasma corticosterone concentrations were assessed in triplicate using enzyme-linked immunosorbent assay (ELISA) kits (DetectX, K014- H, Arbor Assays, Ann Arbor, Michigan, United States) with a standard range from 10,000 to 78.125 pg/mL assessed in duplicate. A Varioskan microplate reader (ThermoFisher Scientific, Waltham, Massachusetts, United States) was used to acquire the raw optical density (OD) values. 2.4. Restrainer setup After the animal’s front and hind paws are fixated against its body with adhesive tape or a Rodent Snuggle (Lomir Biomedical Inc, Notre-Dame- de-l’ˆIle-Perrot, Canada), it can be put into the body tube where a plastic disc closes off the tube at the rear of the animal, preventing the animal to move backwards. The animal’s tail is put through the hole in the disc, and the disc is placed against the animal’s rear and locked in place with a screw. The hole in the disc leaves room for tubing or wires next to the tail. At the other end of the body tube, the animal’s head is sticking out from the neck up. A u-shaped plastic rod is put through the tube, from top to bottom, in between the head and shoulders of the animal on the left and right side, preventing the animal from moving forwards or backwards. The tube is then placed onto the base plate: holes in the bottom of the tube align with screws on the base plate and the tube can be screwed down. 2.5.1. Subjects Fourteen male Wistar rats (250 – 350 grams, Charles River Labora tories, Saint-Germainsur-l’Arbreste, France) were included in this study. The animals were housed socially during the acclimatization period of one-week minimum before surgery and individually after surgery, on a normal day-night cycle (lights on 7am to 7pm) with ad libitum food and water. The experiments were approved by the Animal Experimentation Committee of the Royal Netherlands Academy of Arts and Sciences (KNAW) and carried out in agreement with Dutch law (Wet op de Dierproeven, 1996) and European regulations (Guideline 2010/63/EU). 2.6.4. MRI preparation The values for each point in the standard range were averaged, as well as the values for each plasma sample resulting in mean raw ODs. The mean of the blank replicates was subtracted from these mean raw OD values as background correction, resulting in net ODs. Then, with use of a four- parameter logistic curve fit, the plasma corticosterone concentration of each plasma sample was quantified from the net ODs in ng/mL. p p The animals were shortly put under isoflurane anesthesia (3%, 1:1 air in oxygen at 0.3 L/min) and placed in the custom-made MRI rat restrainer (see 2.4 Restrainer setup) with the addition of a pneumatic pillow placed under the animal’s body for respiration monitoring (Small Animal Instruments, Inc., Stony Brook, New York, United States). The custom-made, two-channel receiver coil (MRCoils, Zaltbommel, the Netherlands) was placed over the animal’s head, after which the setup was placed inside a 3.0 T clinical scanner (Philips Healthcare, Best, the Netherlands) (Fig. 5). The DBS electrodes were connected to a digital stimulator (DS8000, World Precision Instruments, Berlin, Germany) through constant current digital linear stimulus isolators (DSL100, WPI, Berlin, Germany), both placed outside of the scanner room, connected to the patch bay. Ferrite chokes were used on the cables coming from the stimulus isolators to the patch bay outside the scanner room, to suppress possible high frequency interference. Carbon fiber wires leading from the electrodes to the patch bay were used inside the scanner room. 2.6.5. Practical considerations When performing MRI scans in awake animals, it is of importance to keep the preparatory period preceding the scans, during which the an imals are anesthetized, as short as possible. The animals were secured in the setup on a table with a dedicated isoflurane setup, close to the MRI- room. Preparing the animals for MRI scans was done by two well-trained researchers: one securing the animal inside the setup with the other one assisting. To ensure that fixing the head pedestal to the setup, as well as connecting the leads to the DBS-electrodes was a smooth and easy procedure, the head pedestal was placed as perpendicular to the skull as possible, and a mold the size of the hole in the MRI-coil was used to make sure the placement of the pedestal and electrode connectors stayed within bounds. This way, securing the animal in the setup was done within minutes. Transporting the animal to the MRI scanner, connecting it to the isoflurane setup for the MR room, connecting the coil, and the DBS leads to the electrodes was all done within a few minutes as well. When planning experiments, one must take in account the extra time setting up and cleaning will take; often clinical MRI sites will not allow equipment for animal experiments to be left on site, and all surfaces and rooms that have been in contact with animals will need thorough cleaning after the experiments. 2.6.2. Surgery The surgery consisted of bilateral electrode implantation into the dVS, and the placement of a MRI-compatible head pedestal on the ani mal’s skull. The procedures for electrode implantation and placing the pedestal are described in the ‘cFOS’- and ‘Training and corticosterone’- sections respectively – with the exception of the use of stainless steel screws. Here, only plastic screws were used. The animals were allowed a minimum recovery time of one week before taking part in the DBS/MRI experiment. 2.5.5. Analysis The effect of the restrainer training was analyzed by looking at the difference in plasma corticosterone concentrations on the first day of training (day 1) and the final day of training (day 6) by comparing the concentrations before restraining (with the animal anesthetized), at the beginning of the restraining and at the end of the restraining session between day 1 and 6, using paired samples t-tests in JASP (JASP Team, 2019, version 0.9.1). Also, the difference in corticosterone concentra tion from beginning to end of the restraining session was compared between day 1 and 6. This was done by subtracting the concentration measured at the beginning of the restraining session from the end con centration of that session, after correcting for the concentration measured before restraining (end−before before ∗100 −beginning−before before ∗100). Then, a paired samples t-test was used to compare the resulting values of day 1 with day 6. 2.6.1. Subjects Twenty-three male Wistar rats (250–350 grams, Charles River Lab oratories, Saint-Germainsur-l’Arbreste, France) were included in this study. Seven animals were excluded based on incorrect electrode placements, leaving 16 animals. Eight of these animals received training with the purpose of habituating them to MR noises and being restrained (“trained animals”), while the 8 others did not (“untrained animals”). The MRI-data of the untrained animals was further analyzed; of those 8 animals, 2 showed non-optimal EPI data and were discarded, leaving 6 animals for the analysis of DBS-induced BOLD activation. The animals were housed socially during the acclimatization period of one-week minimum before surgery and individually after surgery, on a normal day-night cycle (lights on 7am to 7pm) with ad libitum food and water. The experiments were approved by the Animal Experimentation Com mittee of the Royal Netherlands Academy of Arts and Sciences (KNAW) and carried out in agreement with Dutch law (Wet op de Dierproeven, 1996) and European regulations (Guideline 2010/63/EU). 2.6.6. MRI acquisition and simultaneous DBS MR images were acquired on a 3.0 T Philips Ingenia MR system 2.6.6. MRI acquisition and simultaneous DBS MR images were acquired on a 3.0 T Philips Ingenia MR system Fig. 5. Placement of the custom-made restrainer and DBS-MRI setup in a clinical scanner. 1- custom-made connector box connecting the setup’s MRI coil to the scanner’s interface on the scanner bed, 2- restrainer (containing an albino rat) and DBS MRI setup placed on scanner bed, secured between sandbags to minimize movement artifacts, 3- carbon fiber leads running from the DBS electrodes to the patch panel in the scanner room’s wall, 4- tubing for oxygen and isoflurane supply. 2.5.2. Surgery For placing and securing a plastic, MRI-compatible pedestal to the animals’ skull, animals were put under isoflurane anesthetics (3% 5 Journal of Neuroscience Methods 360 (2021) 109240 M. Derksen et al. 2.6.4. MRI preparation 2.6.3. Restrainer habituation Following the fMRI acquisition during which DBS was delivered, the animals again received isoflurane anesthetics (3%, 1:1 air in oxygen at 0.3 L/min) and a T2-weighted anatomical image was obtained (multishot turbo spin echo (TSE) sequence, TSE factor = 8, TR =4000 ms, TE =56 ms, voxel size = 0.2 × 0.2 × 1.5 mm, matrix size = 176 × 168, 12 slices). The restrainer setup was then removed from the scanner. While still under anesthetics, the DBS elec trodes were connected to a stimulus generator (WPI A365, World Pre cision Instruments, Berlin, Germany) and electrical lesions were made for determining the electrode tip location during subsequent histology (500 μA direct current for 20 s). The animals were euthanized by ter minal cardiac puncture after which the brains were removed, flash frozen, and stored at -80 degrees Celsius for further processing. (Philips Healthcare, Best, the Netherlands). During the preparatory scout scans, the animals were kept under an isoflurane anesthetics maintenance dose (1.8 %–2.0 %, in medical air at 0.6 L/min) to prevent possible motion of the animal. Then, anesthetics were discontinued and functional MR image acquisition (multishot spin-echo (SE) echo-planar imaging (EPI) with interleaved slice acquisition, EPI factor = 9, TR =1000 ms, TE =45 ms, dynamic acquisition time =5000 ms, voxel size = 0.55 × 0.55 × 1.5 mm, matrix size = 64 × 57, 12 slices) was started when the respiration rate of the animal passed 100 breaths per minute. (cutoff 90 s), and FILM pre-whitening was applied. The implemented GLM was a block design modeling stimulation ON (4 volumes during DBS) and stimulation OFF (8 volumes during DBS), repeated 10 times, and preceded by a base line period of 8 volumes. A double-gamma HRF and temporal derivative were added onto the model. Group analyses was subsequently performed by using FMRIB’s Local Analysis of Mixed Ef fects (FLAME 1 + 2). The statistical significance was set to P < 0.05 and cluster correction was applied. DBS-induced responses are expressed in z-score units and overlaid as a heat-map on the custom template. Furthermore, to assess the DBS effect in the ventral striatum, we expressed induced activation as percentage signal change in the nucleus accumbens, as region of interest (ROI). An MRI rat brain atlas was registered to the custom-made template of this study, and an ROI-mask of the nucleus accumbens was made. 3. Results Motion correction parameters were graphically displayed for visual inspection per direction. For each animal, the absolute, cumulative amount of motion correction needed for the entirety of the fMRI sequence acquisition (10 min) was calculated, and with an independent samples t-test the difference between the amount of correction needed in the untrained versus the trained group was analyzed for each direction. 2.6.9. Temporal signal-to-noise ratio Th t l i l t i t 2.6.9. Temporal signal-to-noise ratio The temporal signal-to-noise ratio (tSNR) within our BOLD fMRI- data was calculated from the same 16 animals used to analyze the mo tion correction parameters, using the same 10-minute period of data during which the animals had received DBS. For each animal, the tem poral mean was divided by the temporal standard deviation for the non- pre-processed (but, motion corrected and brain extracted) whole brain 4D BOLD-image, using the FSLmaths toolkit. From the resulting image, the mean tSNR was calculated by averaging all non-zero voxels. Then, overall tSNR and SD were calculated from the individual 16 tSNRs. In order to compare the tSNR between animals that had received restrainer training (n = 8) and those that had not (n = 8), an independent t-test was performed. Anatomical and EPI data were converted from DICOM to NifTI format, using the MRIcron/dcm2nii tool (Chris Rorden, 4AUGUST2017 (Debian) 64-bit BSD License). ImageJ (version 1.50d, National Institutes of Health, Bethesda, Maryland, USA) was used to modify the voxel sizes to ten times their original size to better accommodate the processing algorithms of the FMRIB Software Library (FSL, Analysis Group, FMRIB, Oxford, United Kingdom). FMRIB’s Linear Image Registration Tool (MCFLIRT), with a 6 degrees of freedom (DOF) transformation was used for motion correction of the fMRI time series, using a hand-picked reference volume in the middle of the time series. The motion correc tion parameters for the left – right, inferior – superior, and anterior – posterior directions were saved for later inspection (trained animals n = 8, untrained animals n = 8). 3.2. cFOS Nine rats with bilateral electrodes in the dVS were stimulated unilaterally (Fig. 6A&B). A paired samples t-test of the relative cFOS density in the areas around the electrode tips, showed a significant difference between the two hemispheres, with a higher relative cFOS density in the hemisphere with DBS ON (M = 0.2564, SD = 0.05923) compared to the hemisphere with DBS OFF (M = 0.1969, SD = 0.05422) (t(8) = 4.387, p < 0.01) (Fig. 6C). 2.6.3. Restrainer habituation With use of the featquery – FEAT results interrogation tool (FMRIB Software Library 6.0) the time-series of the ROI was extracted from each of the 6 animals’ functional data. For each animal, the signal intensity during the DBS ON periods were averaged, and of the DBS OFF periods all the second halves were aver aged (omitting the first 4 volumes directly after each DBS ON period), as mean signal intensity during DBS ON and DBS OFF, respectively. The 8 volumes preceding the first DBS ON period were averaged as the signal intensity during baseline. The percentage signal change for DBS ON and DBS OFF were subsequently calculated for each animal, and a paired samples t-test was performed to analyze the difference in percentage signal change between the DBS OFF and DBS ON periods in the ROI, using the open-source statistics software JASP (JASP Team, 2020, version 0.14.0.0) During fMRI acquisition, bilateral DBS (a biphasic, constant current, 80 μs pulse width at 130 Hz, 300 μA stimulation) was delivered to the animals. The stimulation protocol consisted of 20 s of DBS (which equals 4 volumes), followed by 40 s of no stimulation (8 volumes), repeated 10 times. Preceding the 10 repetitions, 8 dummy scans and 40 s of baseline recordings were obtained. Following the fMRI acquisition during which DBS was delivered, the animals again received isoflurane anesthetics (3%, 1:1 air in oxygen at 0.3 L/min) and a T2-weighted anatomical image was obtained (multishot turbo spin echo (TSE) sequence, TSE factor = 8, TR =4000 ms, TE =56 ms, voxel size = 0.2 × 0.2 × 1.5 mm, matrix size = 176 × 168, 12 slices). The restrainer setup was then removed from the scanner. While still under anesthetics, the DBS elec trodes were connected to a stimulus generator (WPI A365, World Pre cision Instruments, Berlin, Germany) and electrical lesions were made for determining the electrode tip location during subsequent histology (500 μA direct current for 20 s). The animals were euthanized by ter minal cardiac puncture after which the brains were removed, flash frozen, and stored at -80 degrees Celsius for further processing. 2.6.8. DBS-induced brain activation analysis 2.6.8. DBS-induced brain activation analysis To investigate DBS-induced brain activation, all anatomical and BOLD images of 6 animals were prepared for processing by extracting the brain segment using the FMRIB’s Brain Extraction Tool (FSL/BET). An anatomical template was custom-made, by registering the individual brain-extracted anatomical images to one representative anatomical image and taking the median of those images. The BOLD EPI images of each of the animals were registered to their corresponding anatomical images, with a combination of linear and non-linear registration (using FMRIB’s Linear Image Registration Tool (FLIRT) and FMRIB’s Nonlinear Image Registration Tool (FNIRT), respectively) and carefully checked. The individual anatomical images were then registered linearly to the custom template, after which the resulting transformation matrix was applied to the corresponding functional data. 3.1. Electrodes Visual inspection of the T2-weighted images of the custom-made, two-channel electrodes of different materials, indicates that electrodes made of silver induce smaller MRI artifacts than electrodes made of the commonly used platinum/iridium (Fig. 1). 2.6.3. Restrainer habituation Leading up to the MRI acquisition, 8 of the animals received restrainer training for 6 consecutive days, as described above. On the day following the last restrainer training, the animals were taken from the stables in the morning and brought to the MRI facility where they remained in their home cages until they went into MRI preparation. The other 8 animals which did not receive restrainer training, were taken to the MRI facility after a minimum one week of recovery from surgery. The batch of 8 trained animals were put in the experiment first, after which the batch of 8 untrained animals followed. All other procedures regarding the DBS/MRI experiment did not differ between the trained and untrained animals. Fig. 5. Placement of the custom-made restrainer and DBS-MRI setup in a clinical scanner. 1- custom-made connector box connecting the setup’s MRI coil to the scanner’s interface on the scanner bed, 2- restrainer (containing an albino rat) and DBS MRI setup placed on scanner bed, secured between sandbags to minimize movement artifacts, 3- carbon fiber leads running from the DBS electrodes to the patch panel in the scanner room’s wall, 4- tubing for oxygen and isoflurane supply. 6 Journal of Neuroscience Methods 360 (2021) 109240 M. Derksen et al. (Philips Healthcare, Best, the Netherlands). During the preparatory scout scans, the animals were kept under an isoflurane anesthetics maintenance dose (1.8 %–2.0 %, in medical air at 0.6 L/min) to prevent possible motion of the animal. Then, anesthetics were discontinued and functional MR image acquisition (multishot spin-echo (SE) echo-planar imaging (EPI) with interleaved slice acquisition, EPI factor = 9, TR =1000 ms, TE =45 ms, dynamic acquisition time =5000 ms, voxel size = 0.55 × 0.55 × 1.5 mm, matrix size = 64 × 57, 12 slices) was started when the respiration rate of the animal passed 100 breaths per minute. During fMRI acquisition, bilateral DBS (a biphasic, constant current, 80 μs pulse width at 130 Hz, 300 μA stimulation) was delivered to the animals. The stimulation protocol consisted of 20 s of DBS (which equals 4 volumes), followed by 40 s of no stimulation (8 volumes), repeated 10 times. Preceding the 10 repetitions, 8 dummy scans and 40 s of baseline recordings were obtained. 3.3. Corticosterone B, C, D, and E: mean + SD, = p < 0.05, n = 14. Fig. 6. cFOS expression at the tip of the MRI-compatible silver electrode is increased after DBS. A) cFOS expression (black dots) at the electrode tip (dorsal part of the ventral striatum; dVS) in the DBS OFF condition (control). B) cFOS expression at the electrode tip after DBS ON (for 30 min). C) Difference of cFOS expression between DBS OFF and DBS ON periods at the electrode tip, measured by relative density (mean + SD). = p < 0.01, n = 9. Fig. 6. cFOS expression at the tip of the MRI-compatible silver electrode is increased after DBS. A) cFOS expression (black dots) at the electrode tip (dorsal part of the ventral striatum; dVS) in the DBS OFF condition (control). B) cFOS expression at the electrode tip after DBS ON (for 30 min). C) Difference of cFOS expression between DBS OFF and DBS ON periods at the electrode tip, measured by relative density (mean + SD). = p < 0.01, n = 9. g. 7. Restrainer habituation does not decrease stress measured via blood-plasma corticosterone. A) Timeline indicating when blood-plasma samples were collecte uring restrainer habituation: Before restraining (animal anesthetized), at the beginning and the end of the restraining period (animal awake at both time points). B omparison of blood corticosterone concentration before restraining on days 1 and 6. C) Corticosterone comparison at the beginning of the restraining periods o ays 1 and 6. D) Comparison of corticosterone levels at the end of the restraining periods on days 1 and 6. No significant differences were found for comparisons anels B, C, and D. E) Significant difference between relative, ‘within-day’ changes in corticosterone levels (between beginning and end of restraining) of days 1 an This indicates that the stress response during the restraining period on day 1 did not decrease after repeated restraining, but instead increased. Values are displaye percentage of corticosterone levels before restraining on the respective day: end−before before ∗100 −beginning−before before ∗100. Results demonstrate a greater increase in stre vels during the restraining period on day 6 compared to day 1, and indicate that restrainer training failed to diminish the stress experienced by the rats. B, C, D, an mean + SD, * = p < 0.05, n = 14. Fig. 7. 3.3. Corticosterone The FMRI Expert Analysis Tool (FEAT) was used to apply a general linear model (GLM) to the functional data of the six animals. First, the functional data was smoothened (FWHM 10 mm), high-pass filtered The effectiveness of the restrainer training was assessed by analyzing blood plasma corticosterone levels, from samples taken before and during the restrainer session on day 1 and day 6 of training (n = 14) 7 Journal of Neuroscience Methods 360 (2021) 109240 M. Derksen et al. (Fig. 7A). N i ifi t diff f d h i th l When looking at the difference in plasma corticosterone levels at the b i i th d f t i i d 1 d t d 6 f Fig. 6. cFOS expression at the tip of the MRI-compatible silver electrode is increased after DBS. A) cFOS expression (black dots) at the electrode tip (dorsal part of the ventral striatum; dVS) in the DBS OFF condition (control). B) cFOS expression at the electrode tip after DBS ON (for 30 min). C) Difference of cFOS expression between DBS OFF and DBS ON periods at the electrode tip, measured by relative density (mean + SD). ** = p < 0.01, n = 9. Fig. 7. Restrainer habituation does not decrease stress measured via blood-plasma corticosterone. A) Timeline indicating when blood-plasma samples were collected during restrainer habituation: Before restraining (animal anesthetized), at the beginning and the end of the restraining period (animal awake at both time points). B) Comparison of blood corticosterone concentration before restraining on days 1 and 6. C) Corticosterone comparison at the beginning of the restraining periods on days 1 and 6. D) Comparison of corticosterone levels at the end of the restraining periods on days 1 and 6. No significant differences were found for comparisons in panels B, C, and D. E) Significant difference between relative, ‘within-day’ changes in corticosterone levels (between beginning and end of restraining) of days 1 and 6. This indicates that the stress response during the restraining period on day 1 did not decrease after repeated restraining, but instead increased. Values are displayed as percentage of corticosterone levels before restraining on the respective day: end−before before ∗100 −beginning−before before ∗100. Results demonstrate a greater increase in stress levels during the restraining period on day 6 compared to day 1, and indicate that restrainer training failed to diminish the stress experienced by the rats. 3.3. Corticosterone Restrainer habituation does not decrease stress measured via blood-plasma corticosterone. A) Timeline indicating when blood-plasma samples were collected during restrainer habituation: Before restraining (animal anesthetized), at the beginning and the end of the restraining period (animal awake at both time points). B) Comparison of blood corticosterone concentration before restraining on days 1 and 6. C) Corticosterone comparison at the beginning of the restraining periods on days 1 and 6. D) Comparison of corticosterone levels at the end of the restraining periods on days 1 and 6. No significant differences were found for comparisons in panels B, C, and D. E) Significant difference between relative, ‘within-day’ changes in corticosterone levels (between beginning and end of restraining) of days 1 and 6. This indicates that the stress response during the restraining period on day 1 did not decrease after repeated restraining, but instead increased. Values are displayed as percentage of corticosterone levels before restraining on the respective day: end−before before ∗100 −beginning−before before ∗100. Results demonstrate a greater increase in stress levels during the restraining period on day 6 compared to day 1, and indicate that restrainer training failed to diminish the stress experienced by the rats. B, C, D, and E: mean + SD, * = p < 0.05, n = 14. 3.4.2. Temporal signal-to-noise ratio The average tSNR within our BOLD fMRI datasets is 15.47 ± 1.90 (n = 16). There was no significant difference in tSNR found between the animals that had received restrainer training prior to going into the scanner (n = 8) and those that had not received training (n = 8) (15.80 ± 2.22 and 15.15 ± 1.60, respectively). By visual inspection, it can be concluded that for both the trained and the untrained animals, the motion correction parameters do not exceed the acquired voxel dimensions (indicated by grey dotted horizontal lines), and DBS (indicated by grey vertical bars) does not affect the motion correction parameters. Furthermore, no significant difference 9 tion-correction parameters for fMRI scan sessions in awake rats during DBS in the dorsal part of the ventral striatum (dVS) stay within voxel dime differ between animals which received restrainer habituation training (trained) and those that did not (untrained). Panels A, C, and E display m parameters in left–right (L–R), superior–inferior (S–I), and anterior–posterior (A–P) planes, respectively. fMRI data were analyzed with m software (MCFLIRT, FMRIB Software Library 6.0). Data were collected during a ten-minute period and are shown as mm displacement from the sults from individual animals are shown (green = untrained rats (n = 8), purple = trained rats (n = 8)). Gray vertical bars indicate periods of dVS D BS on the movement correction parameters are observed. Voxel dimensions in the L-R, S–I, and, A-P directions are 0.55, 0.55, and 1.50 mm, respec nsions are indicated in each panel by dotted lines. The motion-correction parameters did not exceed voxel dimensions, indicating that the data co animals can be corrected for movement. Panels B, D, and F represent the sum of movement corrections applied by the software over the entire 10-m ndividual values (icons) and group averages (bars). These data show that the total, cumulative amount of motion correction applied to the fMRI d e, does not differ between the group of trained and untrained animals, in any direction (L–R, S–I, and A–P) (mean + SD). Fig. 8. Motion-correction parameters for fMRI scan sessions in awake rats during DBS in the dorsal part of the ventral striatum (dVS) stay within voxel dimensions and do not differ between animals which received restrainer habituation training (trained) and those that did not (untrained). (Fig. 7A). N ii When looking at the difference in plasma corticosterone levels at the beginning versus the end of restraining on day 1 compared to day 6 of training (corrected for the level of corticosterone before restraining on that respective day) a significant difference was found, indicating a larger increase of plasma corticosterone during restraining on day 6 (M = 203.15, SD = 199.7) compared to day 1 (M = 91.66, SD = 160.1) (t (13) = -2.204, p < 0.05, Fig. 7E). (Fig. 7A). No significant differences were found when comparing the plasma corticosterone levels (ng/mL) on day 1 with day 6, looking at the sam ples taken before restraining (day 1: M = 106.42, SD = 56.55; day 6: M = 96.39, SD = 66.39; Fig. 7B), looking at the samples taken at the beginning of restraining (day 1: M = 112.06, SD = 113.53; day 6: M = 75.00, SD = 57.66; Fig. 7C), and looking at the samples taken at the end of restraining (day 1: M = 181.30, SD = 87.44; day 6: M = 209.30, SD = 176.61; Fig. 7D). 8 Journal of Neuroscience Methods 360 (2021) 109240 M. Derksen et al. M. Derksen et al. 3.4. DBS and MRI was found between the total amount of motion correction (defined as the sum of movement corrections applied by the software over the entire 10- minute period) needed to correct for motion during fMRI acquisition between the trained and untrained animals, when comparing the two groups on the corrections for left – right, superior – inferior, and anterior – posterior directions (Fig. 8B, D and F). 3.4.1. Motion correction Motion correction parameters for DBS scan session of awake animals that had been subjected to restrainer training (n = 8) and that had not received restrainer training prior to the MRI scan (n = 8) were collected for the left – right, superior – inferior, and anterior – posterior directions for the duration of 10 min, and are shown in Fig. 8A, C and E. The po sition at time point 0 was set to 0 mm movement in the graphs with all other time points relative to that. 4. Discussion We have described the successful combination of bilateral DBS with fMRI measurements in rats that were awake during data acquisition in a clinical 3 T MRI scanner. By using silver as a material for our DBS electrodes, we minimized magnetic susceptibility artifacts in BOLD signal. Stimulation of the ventral striatum affected brain activity near the tip of the DBS electrodes as evidenced by cFOS induction and changes in fMRI BOLD signal, thereby validating successful delivery of electrical current to surrounding brain tissue. functional connectivity in animal models of psychiatric and neurological disorders, without the confounding effects of anesthetics, such as its influence on neurovascular coupling, suppression, and alteration of neural activity and functional connectivity, and changes in cerebral blood flow, compared to awake animals. The custom-made surface coil enabled data collection in a 3 T clinical scanner functioning as receiver coil with coil loops encompassing the entire brain, and a two-channel setup enabling parallel imaging. The coil setup combines easy access to the animals’ electrodes and head stage with securing the animals in the restrainer that restricts head move ments to an acceptable minimum during MRI scans. Our readouts of stress experienced by the animal and head movements indicated that with our restrainer setup, habituation training prior to MRI acquisition is neither necessary nor beneficial. l Previous fMRI studies in rodent DBS have applied a variety of MRI sequences and electrode materials, but thus far only in anesthetized animals (e.g. Dunn et al., 2009; Young et al., 2011; Lai et al., 2015). Using this approach, Albaugh et al. (2016) reported brain activation induced by stimulation of the ventral striatum. fMRI studies in awake animals (see Ferris et al., 2011, for a review), on the other hand, have i Together, the combination of DBS and awake fMR imaging, provides a promising technique to study the effects of DBS on brain activity and Fig. 9. DBS-induced activation in awake animals. Bilateral DBS applied through silver electrodes targeted at the dorsal part of the ventral striatum (dVS), induces BOLD-activation in awake rats (n = 6). Notable activation was found in the area around the electrode tips, the ventral striatum including the accumbal area, the caudate putamen, parts of the primary and cingulate cortices, and prefrontal areas (prelimbic, infralimbic, orbitofrontal and insular regions). Electrode locations are indicated by black lines, with arrows indicating the electrode tips. 3.4.3. DBS-induced activation To investigate the effect of DBS targeted at the dVS in awake rats, a GLM approach was used to analyze the DBS-induced brain activation in a group of 6 animals. The group analysis shows patterns of activation in the general vicinity of the electrode tips, as well as effects of stimulation outside of that region (Fig. 9). Most specifically, DBS-induced activation can be seen around the stimulation area, the ventral striatum including the nucleus accumbens, in brain areas associated with executive func tions such as the caudate putamen, and in prefrontal areas (prelimbic, infralimbic, insular and orbitofrontal cortex), and parts of the primary and cingulate cortices. To assess the percentage signal change during DBS in the ventral striatum, the nucleus accumbens was chosen as a representative ROI to investigate. When looking at the average percentage signal change during DBS OFF compared to the average percentage signal change during DBS ON (within the group), a significant difference was found using a paired samples t-test (Fig. 10) (t(5) = -6.509, p = 0.001). The results indicate the presence of a higher BOLD signal in the nucleus accumbens during DBS ON periods (M = 1.05 % signal change, SD = 0.345) and a lower BOLD signal during DBS OFF periods (M = -0.59 % signal change, SD = 0.412) compared to baseline. Fig. 10. DBS targeted at the dorsal part of the ventral striatum (dVS) induces a significantly higher percentage signal change compared to intermittent periods of no stimulation in the nucleus accumbens of awake rats. A significant dif ference in percentage signal change in the chosen ROI, the nucleus accumbens (NAc), was found when comparing the DBS ON periods with DBS OFF periods in a group of 6 awake rats. Signal intensity was extracted from the data using featquery (FMRIB Software Library 6.0) with the nucleus accumbens as ROI, from each animal, and the percentage signal change was calculated for DBS ON and the latter half of the DBS OFF periods relative to baseline. A paired sample t-test was performed on the percentages signal change of the DBS ON versus the DBS OFF periods. Mean + SD, * - p = 0.001. 3.4.2. Temporal signal-to-noise ratio Panels A, C, and E display motion- correction parameters in left–right (L–R), superior–inferior (S–I), and anterior–posterior (A–P) planes, respectively. fMRI data were analyzed with motion- correction software (MCFLIRT, FMRIB Software Library 6.0). Data were collected during a ten-minute period and are shown as mm displacement from the center of mass. Results from individual animals are shown (green = untrained rats (n = 8), purple = trained rats (n = 8)). Gray vertical bars indicate periods of dVS DBS; no effect of DBS on the movement correction parameters are observed. Voxel dimensions in the L-R, S–I, and, A-P directions are 0.55, 0.55, and 1.50 mm, respectively. Voxel dimensions are indicated in each panel by dotted lines. The motion-correction parameters did not exceed voxel dimensions, indicating that the data collected from these animals can be corrected for movement. Panels B, D, and F represent the sum of movement corrections applied by the software over the entire 10-minute period, as individual values (icons) and group averages (bars). These data show that the total, cumulative amount of motion correction applied to the fMRI data by the software, does not differ between the group of trained and untrained animals, in any direction (L–R, S–I, and A–P) (mean + SD). 9 Journal of Neuroscience Methods 360 (2021) 109240 M. Derksen et al. Fig. 10. DBS targeted at the dorsal part of the ventral striatum (dVS) induces a significantly higher percentage signal change compared to intermittent periods of no stimulation in the nucleus accumbens of awake rats. A significant dif ference in percentage signal change in the chosen ROI, the nucleus accumbens (NAc), was found when comparing the DBS ON periods with DBS OFF periods in a group of 6 awake rats. Signal intensity was extracted from the data using featquery (FMRIB Software Library 6.0) with the nucleus accumbens as ROI, from each animal, and the percentage signal change was calculated for DBS ON and the latter half of the DBS OFF periods relative to baseline. A paired sample t-test was performed on the percentages signal change of the DBS ON versus the DBS OFF periods. Mean + SD, * - p = 0.001. 4. Discussion (2009) used (0.23 × 0.23 × 1.5) or Lehto et al. (2017) (0.55 × 0.55 × 1), but sizes of approximately 0.25 × 0.25 × 1 have been reported by a number of other groups. The performance of the system we describe (in awake rats) is at the lower end of performance compared to dedicated small animal scanners using anesthetized animals. However, we believe that the advantages of our novel method in using awake animals, making preclinical MR imaging experiments more accessible to both animal and clinical researchers, and lowering costs outweigh the disadvantage of lower scan resolution. Our results demonstrate that basal concentration of blood-plasma corticosterone in animals that receive restrainer training, remained the same across repeated training sessions, indicating that the basal stress-level of the animals is not affected by such habituation sessions (in contrast to previous findings, Keim and Sigg, 1976; Watanabe et al., 1992). We observed no differences between corticosterone levels on days 1 and 6, neither at the start, nor at the end of the restraining period. Instead, we found a mild increase of corticosterone levels in response to the 60-minute restraining. Remarkably, this increase of corticosterone levels during a single training session did not habituate over repeated sessions. On the contrary: comparing the observed increase of cortico sterone during day 6 of habituation training with that observed on day 1, shows this increase has become larger over repeated sessions. g p Furthermore, looking at the amount of head movements during the actual MRI scan sessions, we found no differences in amount of motion correction needed between trained and untrained animals, indicating that the training of the animals was not beneficial in decreasing head movements and that this variable seems to have no relation to our corticosterone data. Importantly, motion correction parameters did never exceed the acquired voxel size during a given 10-minute scan session, and the periods of DBS during those 10 min did not increase head movement. This is also reflected in the tSNR, which was not different between control and trained animals. In other studies, it has been reported that the most dramatic movements occur in the vertical (S–I) direction, possibly as a result of the setup design, and that restrainer training is most effective there (King et al., 2005; Febo 2001). 4. Discussion First, we pioneered the use of silver electrodes for DBS in rodents, which has not been reported before to our knowledge. Magnetic susceptibility artifacts (distortions or local signal changes due to local magnetic field in homogeneities) are lower for silver than for platinum/iridium. Our re sults (Fig. 1) illustrate the higher suitability of silver in this respect. Stimulation using these electrodes readily induces cFOS expression and BOLD activation around the electrodes (Figs. 6&9). Why silver has not been used before is unclear – possibly the relative flexibility of the material was considered a potential cause for imprecise electrode placement. However, in our experience this is not the case. The second innovation is the use of a custom-designed head coil that enabled scanning in a standard clinical 3 T scanner. This coil had to be compatible with the machine’s field strength and connector interface, and allows manual matching and tuning prior to each series of experi ments. The combination of our restrainer setup, coil and the 3 T clinical scanner let us effectively conduct our experiments, and was in daily practice straightforward to work with, as setting up the coil was of a plug-and-play nature. The scan settings and sequences were chosen after numerous pilot scans and were optimized and adapted to rat-brain anatomy and hemodynamic responses. After properly placing the coil over the animal’s head and securing it into the setup, it can easily be connected to the interface box. The performance (in terms of temporal signal-to-noise ratio (tSNR), and temporal and spatial resolution) is to a large extent determined by the field strength and -homogeneity of the scanner, and the gradient system. The average tSNR of our datasets was 15.5 ± 1.9, which is above the average reported by Welvaert and Rosseel (2013). For a comparison to other small animal DBS-MRI data, temporal and spatial resolution (expressed as TR and voxel size, respectively) may be more useful. The repetition time we used (TR =4000 ms) was not substantially different from other studies, which combined a 9.4 T scanner and DBS (3000 ms in Young et al., 2011; Lehto et al., 2018; 2260 ms in Dunn et al., 2009), but others reported TRs of 1000 ms or shorter (e.g. Lai et al., 2015; Zhao et al., 2020; Albaugh et al., 2016). Our voxel size of 0.55 × 0.55 × 1.5 is somewhat larger than that what Dunn et al. 4. Discussion Numbers below the slices indicate relative location to bregma (mm). Color bars denote z score values at a significance threshold of p < 0.05 (corrected). Fig. 9. DBS-induced activation in awake animals. Bilateral DBS applied through silver electrodes targeted at the dorsal part of the ventral striatum (dVS), induces BOLD-activation in awake rats (n = 6). Notable activation was found in the area around the electrode tips, the ventral striatum including the accumbal area, the caudate putamen, parts of the primary and cingulate cortices, and prefrontal areas (prelimbic, infralimbic, orbitofrontal and insular regions). Electrode locations are indicated by black lines, with arrows indicating the electrode tips. Numbers below the slices indicate relative location to bregma (mm). Color bars denote z score values at a significance threshold of p < 0.05 (corrected). 10 M. Derksen et al. Journal of Neuroscience Methods 360 (2021) 109240 2011; Yee et al., 2016). The motivation to conduct such acclimation procedures is based on findings that suggest that repeated exposure to a stressor results in habituation. More specifically, after multiple expo sures, a decrease of overt signs of stress (such as HPA reactivity (i.e. high levels of corticosterone), high heart rate, behavioral struggle during restraint) compared to first-time exposure was reported (Keim and Sigg, 1976; Natelson et al., 1988; review: Grissom and Bhatnagar, 2008). The impact of restraint is not only dictated by the severity of the restraint, but also by its duration, repetition, past stress experience, handling, and intrinsic biological factors (Keim and Sigg, 1976). Previous protocols applied varied strongly (Low et al., 2016) and yielded widely varying results, most notably a lack of habituation to repeated restraint in some cases (e.g. Pitman et al., 1988; Marin et al., 2007). Furthermore, habituation of the corticosterone response does not necessarily imply a reduction or absence of other measures of stress (Low et al., 2016). We examined the effectiveness of restrainer training in our animals, and compared movement of trained and untrained animals during their MRI scan sessions. been developed and applied to study connectivity in neuronal networks (e.g. Chuang and Nasrallah, 2017) or effects of pharmacological and sensory or motivational stimuli (e.g. Febo et al., 2005; Yee et al., 2016). Since DBS in awake rats had not been described yet, we designed a setup that made it possible to perform (f)MRI acquisition in awake rats. Our method incorporates a number of technical innovations. 4. Discussion Our data shows no such large movements, in neither the trained nor untrained groups; occasional erratic movements were observed in in dividual animals, but never exceeding voxel dimensions. i As the restrainer training did not have a significant effect on the head movements during MRI acquisition; did not lead to a habituation of corticosterone increases; and might even result in long-lasting changes in other physiological measures (Low et al., 2016), we conclude that it is not preferred or needed to expose the animals to repeated restraint before MRI scan sessions. However, different approaches to restrainer training exist, e.g. increasing the duration of restraining gradually over sessions (Febo, 2011; Low et al., 2016), and may lead to more favorable results. The third innovation is creating the possibility to study DBS with MRI in awake animals. In order to do so, a restrainer and head stage- combination was designed to fixate the awake animal in our setup, while also accommodating the custom-designed head coil and leads connecting the DBS electrodes with the stimulator (Figs. 3–5). After an initial phase of getting familiar with this setup, working with it allowed for a smooth and rapid execution of the experiments – an important feat as it is of importance to keep the time the animals are under anesthesia prior to the awake scans as short as possible. With our setup, a wide range of brain MRI experiments becomes available in rodents that are either anesthetized or awake during acquisition. The pilot study in a group of six animals shows that the set-up is applicable to a typical experimental question and provides data of us able quality. The average activation pattern after bilateral dVS stimu lation showed bilateral activation of basal-ganglia and cortical areas in the forebrain (Fig. 9). Comparison with published results is challenging, as the only other study of ventral striatal DBS used unilateral stimulation with a more anteromedial placement (Albaugh et al., 2016). The per centage change in BOLD response cannot be compared to that reported by those authors as they used CBV with contrast agent injections. However, the changes we report are within the range of those reported One of the challenges of performing MRI-scans in awake, conscious animals is preventing movement – in neuroscientific experiments, that is especially motion of the rodents’ heads. References Paasonen, J., Stenroos, P., Salo, R.A., Kiviniemi, V., Gr¨ohn, O., 2018. Functional connectivity under six anesthesia protocols and the awake condition in rat brain. NeuroImage 172, 9–20. https://doi.org/10.1016/j.neuroimage.2018.01.014. Albaugh, D.L., Salzwedel, A., Van Den Berge, N., Gao, W., Stuber, G., Shih, Y.I., 2016. 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Discussion Other groups that performed awake animal MRI in combination with a restrainer setup have resorted to acclimating/habituating the animals to being restrained in a series of training sessions prior to the MRI-scan session (King et al., 2005; Febo, 11 Journal of Neuroscience Methods 360 (2021) 109240 M. Derksen et al. chronically implanted in rats. Magn. Reson. Med. 61 (1), 222–228. https://doi.org/ 10.1002/mrm.21803. by Chao et al. (2014) after unilateral stimulation of the ventro-posterior thalamus. In conclusion, the combination of the custom-made coil and the design of our restrainer enables animal fMRI inside a clinical MRI-scanner environment. Clinical scanners, and scanners for humans in general, are much more abundant than pre-clinical small-animal MR sites. Therefore, the possibility to perform animal scanning at clinical scanners is more cost-efficient and requires less effort to set up, compared to outsourcing the experiment or acquiring and maintaining a dedicated small-animal scanner. As the scanner hardware and software will be familiar to trained staff, using our coil and setup combination at an existing clinical MR site makes animal MRI research easily available not only to animal researchers but also to clinical researchers. By following our methods, pre-clinical research can be conducted at any site with MRI facilities for humans, enabling researchers within the same institute to conduct complementary translational animal research in parallel to clinical research, narrowing the gap between the two. chronically implanted in rats. Magn. Reson. Med. 61 (1), 222–228. https://doi.org/ 10.1002/mrm.21803. Febo, M., 2011. Technical and conceptual considerations for performing and interpreting functional MRI studies in awake rats. Front. 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Deep brain stimulation restores frontostriatal network activity in obsessive-compulsive disorder. Nat. Neurosci. 16 (4), 386–387. https:// doi.org/10.1038/nn.3344. CRediT authorship contribution statement Keim, K.L., Sigg, E.B., 1976. Physiological and biochemical concomitants of restraint stress in rats. Pharmacol. Biochem. Behav. 4 (3), 289–297. https://doi.org/10.1016/ 0091-3057(76)90244-6. Maik Derksen: Investigation, Methodology, Formal analysis, Visu alization, Writing - original draft. Valerie Rhemrev: Investigation. Marijke van der Veer: Investigation, Visualization. Linda Jolink: Investigation. Birte Zuidinga: Investigation, Visualization. Tosca Mulder: Investigation. Liesbeth Reneman: Conceptualization, Writing - review & editing. Aart Nederveen: Conceptualization, Methodology, Writing - review & editing. Matthijs Feenstra: Conceptualization, Methodology, Supervision, Writing - original draft. Ingo Willuhn: Conceptualization, Methodology, Supervision, Writing - original draft. Damiaan Denys: Conceptualization, Writing - review & editing. King, J.A., Garelick, T.S., Brevard, M.E., Chen, W., Messenger, T.L., Duong, T.Q., Ferris, C.F., 2005. Procedure for minimizing stress for fMRI studies in conscious rats. J. Neurosci. Methods 148 (2), 154–160. https://doi.org/10.1016/j. jneumeth.2005.04.011. j Lai, H., Albaugh, D.L., Kao, Y.J., Younce, J.R., Shih, Y.I., 2015. Robust deep brain stimulation functional MRI procedures in rats and mice using an MR-compatible tungsten microwire electrode. Magn. Reson. Med. 73 (3), 1246–1251. https://doi. org/10.1002/mrm.25239. g Lehto, L.J., Idiyatullin, D., Zhang, J., Utecht, L., Adriany, G., Garwood, M., et al., 2017. MB-SWIFT functional MRI during deep brain stimulation in rats. NeuroImage 159, 443–448. https://doi.org/10.1016/j.neuroimage.2017.08.012. Lehto, L.J., Filip, P., Laakso, H., Sierra, A., Slopsema, J.P., Johnson, M.D., et al., 2018. Tuning neuromodulation effects by orientation selective deep brain stimulation in the rat medial frontal cortex. Front. Neurosci. 21, 899. https://doi.org/10.3389/ fnins.2018.00899. Low, L.A., Bauer, L.C., Pitcher, M.H., Bushnell, M.C., 2016. Restraint training for awake functional brain scanning of rodents can cause long-lasting changes in pain and stress responses. Pain 157 (8), 1761–1772. https://doi.org/10.1097/j. pain.0000000000000579. Funding j g Grissom, N., Bhatnagar, S., 2008. Habituation to repeated stress: get used to it. Neurobiol. Learn. Mem. 92 (2), 215–224. https://10.1016/j.nlm.2008.07.001. This research did not receive any funding from agencies in the for- profit sector. Harmsen, I.E., Elias, G.J.B., Beyn, M.E., Boutet, A., Pancholi, A., Germann, J., et al., 2020. Clinical trials for deep brain stimulation: current state of affairs. Brain Stimul. 13 (2), 378–385. https://doi.org/10.1016/j.brs.2019.11.008. The authors report no declarations of interest. p Marin, M.T., Cruz, F.C., Planeta, C.S., 2007. Chronic restraint or variable stresses differently affect the behavior, corticosterone secretion and body weight in rats. Physiol. Behav. 90 (1), 29–35. https://doi.org/10.1016/j.physbeh.2006.08.021. Acknowledgements y g j y Medtronic, 2009. Reclaim DBS Therapy for OCD. Lead Kit for Deep Brain Stimulation. Retrieved from. http://www.accessdata.fda.gov/cdrh_docs/pdf5/H050003D.pdf. We thank Birte Forstmann (University of Amsterdam) for her insightful comments on the manuscript, and Ralph Hamelink (Netherlands Institute for Neuroscience) for his technical assistance and practical contributions to this study. Medtronic, 2015. MRI Guidelines for Medtronic Deep Brain Stimulation Systems. Retrieved from. http://manuals.medtronic.com/content/dam/emanuals/neuro /CONTRIB_228155.pdf. Natelson, B.H., Ottenweller, J.E., Cook, J.A., Pitman, D., McCarthy, R., Tapp, W.N., 1988. Effect of stressor intensity on habituation of the adrenocortical stress response. Physiol. Behav. 43 (1), 41–46. https://doi.org/10.1016/0031-9384(88)90096-0. Declaration of Competing Interest The authors report no declarations of interest. 4. Discussion Golestanirad, L., Kirsch, J., Bonmassar, G., Downs, S., Elahi, B., Martin, A., et al., 2019. RF-induced heating in tissue near bilateral DBS implants during MRI at 1.5 T and 3T: the role of surgical lead management. Neuroimage 184, 566–576. https://doi.org/ 10.1016/j.neuroimage.2018.09.034. affective neuroscience. NeuroImage 138, 221–232. https://doi.org/10.1016/j. neuroimage.2016.05.046. Young, C.K., Brown, A.R., Robinson, J.H.B., Tuor, U.I., Dunn, J.F., Bland, B.H., Teskey, G. C., 2011. Functional MRI response and correlated electrophysiological changes during posterior hypothalamic nucleus deep brain stimulation. NeuroImage 56, 35–44. https://doi.org/10.1016/j.neuroimage.2011.02.023. Zhao, S., Li, G., Tong, C., Chen, W., Wang, P., Dai, J., et al., 2020. Full activation pattern mapping by simultaneous deep brain stimulation and fMRI with graphene fiber electrodes. Nat. Commun. 11, 1788. https://doi.org/10.1038/s41467-020-15570-9. p Yee, J.R., Kenkel, W.M., Kulkarni, P., Moore, K., Perkeybile, A.M., Toddes, S., et al., 2016. BOLD fMRI in awake prairie voles: a platform for translational social and Zhao, S., Li, G., Tong, C., Chen, W., Wang, P., Dai, J., et al., 2020. Full activation pattern mapping by simultaneous deep brain stimulation and fMRI with graphene fiber electrodes. Nat. Commun. 11, 1788. https://doi.org/10.1038/s41467-020-15570-9. Young, C.K., Brown, A.R., Robinson, J.H.B., Tuor, U.I., Dunn, J.F., Bland, B.H., Teskey, G. C., 2011. Functional MRI response and correlated electrophysiological changes during posterior hypothalamic nucleus deep brain stimulation. NeuroImage 56, 35–44. https://doi.org/10.1016/j.neuroimage.2011.02.023. 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BOLD fMRI in awake prairie voles: a platform for translational social and 13 13
https://openalex.org/W4225282913	https://statecon.rea.ru/jour/article/download/1590/1308	Russian	null	Mortality in the Volgograd Region Against the COVID-19 Pandemic	Statistika i èkonomika	2,022	cc-by	9,330	Mortality in the Volgograd Region Against the COVID-19 Pandemic The COVID-19 pandemic, which began in Russia in March 2020, had a huge impact on socio-economic processes. In numerous studies analyzing mortality caused by coronavirus infection, it is concluded that the number of deaths is underestimated. The high morbidity and mortality caused by coronavirus infection has far-reaching consequences for the economy of the regions and the country as a whole: deterioration in health, a decrease in the working-age population, a change in the structure of consumption of goods and services, etc. In this regard, it is relevant to analyze the processes associated with mortality from coronavirus infection. mortality rates in absolute and relative (per 1000 people) terms. The processing of statistical data was carried out using the Microsoft Excel application package and matplotlib, pandas, numpy (Python programming language), pyramid (R programming language) libraries. Results. In 2020, the number of deaths in the Volgograd region turned out to be more than in 2019 by 6647 people. If the trends in the dynamics of the intensity of mortality would persist in the year of the pandemic, then the total number of deaths in the Volgograd region would be equal to 32044 people. In this case, the excess mortality would have amounted to 7368 people. mortality rates in absolute and relative (per 1000 people) terms. The processing of statistical data was carried out using the Microsoft Excel application package and matplotlib, pandas, numpy (Python programming language), pyramid (R programming language) libraries. Results. In 2020, the number of deaths in the Volgograd region turned out to be more than in 2019 by 6647 people. If the trends in the dynamics of the intensity of mortality would persist in the year of the pandemic, then the total number of deaths in the Volgograd region would be equal to 32044 people. In this case, the excess mortality would have amounted to 7368 people. Conclusion. As a result of the study, it was revealed that a significant increase in the number of deaths in the Volgograd region during the pandemic is explained by Rosstat as the cause of coronavirus infection by only 33.2%. This discrepancy may be the result of incorrect accounting of deaths from coronavirus infection. Another factor in the increase in mortality during a pandemic may be a decrease in the quality of medical care. Demographic statistics Demographic statistics А.В. Алпатов Statistics and Economics  V. 19. № 2. 2022 А.В. Алпатов УДК 314.48 DOI: http://dx.doi.org/10.21686/2500-3925-2022-2-23-35 Волжский политехнический институт (филиал), Волжский, Россия Смертность в Волгоградской области на фоне пандемии Covid-19 Пандемия COVID-19, которая началась в России в марте 2020 года, оказала огромное воздействие на социально-экономические процессы. В многочисленных исследованиях, посвящённых анализу смертности, вызванной коронавирусной инфекцией, делается вывод о занижении числа умерших. Высокая заболеваемость и смертность, обусловленная коронавирусной инфекцией, имеет далеко идущие последствия для экономики регионов и страны в целом: ухудшение здоровья, снижение численности трудоспособ- ного населения, изменение структуры потребления товаров и услуг и т.д. В связи с этим актуальным является анализ про- цессов, связанных со смертностью от коронавирусной инфекции. Целью исследования является выявление основных тенденций в нозологической и половозрастной структуре смертности насе- ления Волгоградской области в годы, предшествующие пандемии COVID-19, оценка вклада смертности от коронавирусной инфекции в общую смертность в 2020 году. Оценка избыточ- ной смертности проводилась с учетом динамики возрастных коэффициентов смертности. статистических данных проводилась с помощью пакета прикладных программ Microsoft Excel и библиотек matplotlib, pandas, numpy (язык программирования Python), pyramid (язык программирования R). Результаты. В 2020 году число умерших в Волгоградской области оказалось больше, чем в 2019 году на 6647 человек. Если бы тенденции в динамике интенсивности смертности сохранились бы и в год пандемии, то общее количество умер- ших по Волгоградской области оказалось бы равным 32044 человек. При этом избыточная смертность составила бы 7368 человек. Заключение. В результате исследования было выявлено, что существенный рост числа умерших в Волгоградской области в период пандемии объясняется Росстатом в качестве причины коронавирусной инфекцией только на 33,2%. Данное несоот- ветствия может являться следствием неправильного учета смертности от коронавирусной инфекции. Другим фактором увеличения смертности в период пандемии может быть снижение качества медицинского обслуживания. Произошла переориентация работы медицинских учреждений на лечение пациентов с коронавирусной инфекцией, увеличилась нагрузка на скорую медицинскую помощь. Материалы и методы. Основными источниками информации для исследования смертности была Российская база данных по рождаемости и смертности и данные Росстата. В работе при анализе смертности от COVID-19 использовались также данные оперативного штаба. Анализ динамики смертности про- водился с помощью таких показателей как средняя ожидаемая продолжительность жизни при рождении, общий коэффициент смертности, возрастные показатели смертности в абсолютном и относительном (на 1000 человек) выражении. Обработка Ключевые слова: COVID-19, коронавирусная инфекция, смерт- ность, общий коэффициент смертности, средняя ожидаемая продолжительность жизни при рождении, Волгоградская об- ласть, избыточная смертность. Введение тате сопоставления данных о смертности между регионами России и другими странами в период пандемии автор в рабо- те [2] делает предположение о «существенном расхождении в методологии учета смертности в различных субъектах Россий- ской Федерации». Это как ми- нимум, а как максимум «речь может идти о намеренном ис- кажении данных». Пандемия COVID-19, ко- торая началась в России в марте 2020 года, существенно повлияла на характер социаль- но-экономических процессов. Произошла перестройка прак- тически всех сфер человече- ской деятельности. Наиболь- шее негативное воздействие пандемия оказала на протека- ние демографических процес- сов: увеличение смертности и снижение рождаемости [1]. р [ ] Научные публикации, в ко- торых анализируется смерт- ность в регионах России в период пандемии COVID-19, нацелены прежде всего на вы- явление общих закономерно- стей. При этом не уделяется существенное внимание осо- бенностям отдельных регио- нов. Понимание демографи- ческих процессов в настоящее время крайне важно при пла- нировании социально-эконо- мического развития. Высокая заболеваемость и смертность, обусловленная коронавирус- ной инфекцией, имеет далеко идущие последствия: ухудше- ние здоровья, снижение чис- ленности трудоспособного на- селения, изменение структуры потребления товаров и услуг и т.д. Анализ публикаций на мо- мент написания статьи пока- зал отсутствие подробных де- мографических исследований по Волгоградской области. В связи с этим актуальным яв- ляется анализ процессов, свя- занных со смертностью от ко- ронавирусной инфекции на региональном уровне. При международном срав- нении смертности от корона- вирусной инфекции Россия показывает не самые лучшие результаты. В работе [5] была проведена оценка снижения средней ожидаемой продолжи- тельности жизни при рождении (далее – ОПЖ) в 2020 году на фоне пандемии COVID-19 для 37 стран мира с уровнем жизни выше среднего и для которых есть надежные и полные дан- ные о смертности в период с 2005 по 2020 год. Уровень сни- жения ОПЖ определялся как разница между фактическим значением ОПЖ и прогноз- ным. При прогнозировании ОПЖ использовалась модель Ли-Картера [6]. Наихудший результат был зафиксирован в России. Снижение ОПЖ у мужчин составило 2,33 лет, у женщин – 2,14 лет. Второй по степени снижения оказались США (мужчины – 2,27, жен- щины – 1,37). В таких странах как Дания, Исландия и Юж- ная Корея уменьшение ОПЖ не выявлено. В научных исследованиях, в которых рассматривается влияние коронавирусной ин- фекции на смертность, наи- большее внимание уделяется следующим вопросам: подсчет числа умерших от причин, свя- занных с COVID-19, их струк- тура по возрасту и полу, вли- яние пандемии на изменение общих показателей смертно- сти, выявление факторов, ко- торые увеличивают риск смер- ти от COVID-19. Введение В публикациях [2,3,4] дела- ется вывод о занижении числа умерших вследствие коронави- русной инфекцией. Например, в работе [2] отмечается, что в России в 2020 году итоговая смертность оказалась выше по сравнению с 2019 годом на 323,7 тыс. человек или на 18%. При этом число умер- ших от COVID-19 по данным Росстата в избыточной смерт- ности составляет 50,2%. В ре- гионах России доля смертей, обусловленной коронавирус- ной инфекцией в избыточной смертности, существенно раз- личается. В Москве доля ко- видных смертей почти совпада- ет с избыточной смертностью и составляет 98,4%. Довольно высокий уровень в Еврейской АО (92,4%) и Санкт-Петербур- ге (91,0%). К регионам, в ко- торых избыточная смертность меньше всего была объяснена коронавирусной инфекци- ей относятся Ленинградская область (5,8%), Рязанской область (4,5%), Чеченская Республика (3,9%). В резуль- Целью настоящей работы является выявление основных тенденций в нозологической и половозрастной структуре смертности населения Вол- гоградской области в годы, предшествующие пандемии COVID-19, оценка вклада смертности от коронавирус- ной инфекции в общую смерт- ность в 2020 году. Измерение избыточной смертности про- водилось с учетом динамики возрастных коэффициентов смертности. В работах [7,8,9] исследо- вались факторы, влияющие на повышение смертности в ус- ловиях пандемии в России. К экономическим показателям, которые влияют на смертность, относятся валовой региональ- ный продукт на душу населе- ния [8], уровень безработицы [7]. Среди демографических показателей наибольшее влия- ние оказывает доля городского населения [9], а также возраст- ная структура населения: уро- вень смертности повышается с увеличением возраста. Это оценивалось с помощью доли В работах [7,8,9] исследо- вались факторы, влияющие на повышение смертности в ус- ловиях пандемии в России. К экономическим показателям, которые влияют на смертность, относятся валовой региональ- ный продукт на душу населе- ния [8], уровень безработицы [7]. Среди демографических показателей наибольшее влия- ние оказывает доля городского населения [9], а также возраст- ная структура населения: уро- вень смертности повышается с увеличением возраста. Это оценивалось с помощью доли Mortality in the Volgograd Region Against the COVID-19 Pandemic There has been a reorientation of the work of medical institutions to the treatment of patients with coronavirus infection; the burden on emergency medical care has increased. The purpose of the study is to identify the main trends in the nosological and age-sex structure of mortality in the Volgograd region in the years preceding the COVID-19 pandemic, to assess the contribution of mortality from coronavirus infection to total mortality in 2020. Estimation of excess mortality was carried out taking into account the dynamics of age-specific mortality rates. Materials and methods. The main sources of information for the study of mortality were the Russian database on fertility and mortality and Rosstat data. In the work, when analyzing mortality from COVID-19, data from the operational headquarters were also used. The analysis of the mortality dynamics was carried out using such indicators as the average life expectancy at birth, the crude death-rate, age-specific Keywords: COVID-19, coronavirus infection, mortality, crude death- rate, life expectancy at birth, Volgograd region, excess mortality. Statistics and Economics  V. 19. № 2. 2022 23 Демографическая статистика пенсионеров [8], доли лиц старше трудоспособного воз- раста [7]. 1. Статистические данные и методы исследования Основным источником ин- формации для исследования смертности была Российская база данных по рождаемости и смертности [10]. Оперативные данные по учету числа умер- Статистика и экономика  Т. 19. № 2. 2022 24 Demographic statistics 2017 год оказала благоприят- ная половозрастная структура населения — наличие большой когорты женщин фертильно- го возраста, которые родились во второй половине 80-х годов прошлого века. Кроме того, принятие Федерального закона № 256-ФЗ от 29.12.2006 «О до- полнительных мерах государ- ственной поддержки семей, имеющих детей» повлияло на календарный сдвиг рождаемо- сти. Это проявилось в росте суммарного коэффициента рождаемости в 2007–2015 го- дах [13]. В ближайшее время число родившихся будет сокра- щаться, поскольку в возраст с наибольшей интенсивностью деторождений будут вступать малочисленные поколения женщин, родившихся в сере- дине 90-х годов. Это можно увидеть из рисунка 1, на кото- ром показана возрастно-поло- вая структура населения Вол- ших за 2019, 2020 и 2021 год, а также динамика смертности по основным причинам были взяты c сайта Федеральной службы государственной ста- тистики [11]. В работе при ана- лизе смертности от COVID-19 использовались также данные оперативного штаба [12]. ляется мерой оценки влияния пандемии на итоговую смерт- ность в регионе. Рис. 1. Возрастно-половая структура населения Волгоградской области за 2021 год. Темная заливка слева показывает перевес мужчин над женщинами в данной возрастной группе, справа показывает, соответственно перевес женщин над мужчинами. Источник: рассчитано автором на основе данных [11]. Fig. 1. Sex and age structure of the population of the Volgograd region for 2021. The dark shading on the left shows the predominance of men over women in this age group, on the right shows the predominance of women over men, respectively. Source: calculated by the author based on the data [11]. 2. Динамика смертности в Волгоградской области 2. Динамика смертности в Волгоградской области В настоящее время одной из острейших проблем Вол- гоградской области является продолжающаяся депопуляция населения. Сокращение чис- ленности населения происхо- дит вследствие естественной убыли, а также за счет мигра- ционного оттока населения в другие регионы России. Ру- ководство страны и области предпринимает попытки для улучшения демографической ситуации. Принимаются зако- ны и проекты, которые долж- ны стимулировать рождае- мость и снижать смертность. Анализ динамики показа- телей смертности населения Волгоградской области про- водился при сопоставлении с общероссийскими показателя- ми. Смертность исследовалась в целом по региону, в разрезе мужчины/женщины, по основ- ным классам причин смерти. Использовались такие показа- тели как ОПЖ, общий коэф- фициент смертности (далее – ОКС), возрастные показатели смертности в абсолютном и относительном (на 1000 чело- век) выражении. Существенное влияние на увеличение показателей рож- даемости в период с 2007 по Расчет ОПЖ по Волгоград- ской области и в целом по России проводился автором на основе однолетних таблиц смертности. Если сравнивать эти результаты с данными, опубликованными Росстатом, то есть небольшие отличия. Для мужчин среднее арифме- тическое различий в показа- теле ОПЖ равно ОПЖ ∆ = 0,00 со средним квадратическим отклонение σ = 0,04. Для жен- щин, соответственно, ОПЖ ∆ = = –0,02, σ = 0,06. Оценка влияния панде- мии, вызванной COVID-19, на смертность в 2020 году осу- ществлялась следующим обра- зом. Сначала были рассчитаны среднегодовые темпы роста возрастных коэффициентов смертности для мужчин и жен- щин во временном диапазоне с 2015 по 2019 год. Затем на основе среднегодовых темпов роста был сделан прогноз по каждой возрастной группе: ка- ким было бы число умерших, если бы тенденции в сокраще- нии смертности сохранились. Разница между прогнозным значением числа умерших в 2020 году и фактическим ко- личеством умерших в 2019 яв- Рис. 1. Возрастно-половая структура населения Волгоградской области за 2021 год. Темная заливка слева показывает перевес мужчин над женщинами в данной возрастной группе, справа показывает, соответственно перевес женщин над мужчинами. Источник: рассчитано автором на основе данных [11]. Fig. 1. Sex and age structure of the population of the Volgograd region for 2021. The dark shading on the left shows the predominance of men over women in this age group, on the right shows the predominance of women over men, respectively. Source: calculated by the author based on the data [11]. Statistics and Economics  V. 19. № 2. 2022 25 Демографическая статистика Рис. 2. 2. Динамика смертности в Волгоградской области Динамика общего числа умерших всего населения Волгоградской области (кривая 1) и доли мужчин в общем числе умерших (кривая 2). Источник: рассчитано автором на основе данных [10]. Fig. 2. Dynamics of the total number of deaths of the entire population of the Volgograd region (curve 1) and the proportion of men in the total number of deaths (curve 2). Source: calculated by the author based on the data [10]. гоградской области на начало 2021 года. Возрастно-половая структура имеет волнообраз- ную форму, что является ха- рактерным для России и для большей части ее регионов. На рисунке приведена допол- нительная шкала с указанием года рождения поколения. Наиболее существенные результаты, связанные с де- мографическими процессами в регионе, были достигнуты в области снижения смертности. ОПЖ в период с 2003 по 2015 год выросла у всего населения Волгоградской области на 5,8 лет и составила 71,9 лет [14]. Рис. 2. Динамика общего числа умерших всего населения Волгоградской области (кривая 1) и доли мужчин в общем числе умерших (кривая 2). Источник: рассчитано автором на основе данных [10]. На рис. 2. показана динами- ка числа умерших по региону в период с 1989 по 2020 год (оба пола). Можно отметить, что в течение рассматриваемого пе- риода данный показатель су- щественно варьируется. Значе- ния изменяются от 29,72 тыс. умерших в 1989 году до 42,97 тыс. в 2002. Такая противоре- чивая динамика числа умерших обусловлена волнообразной половозрастной структурой населения (см. рис. 1), а также высокой смертностью в 90-х го- дах XX столетия, прежде всего среди мужчин трудоспособного возраста, что стало предметом многочисленных исследований Fig. 2. Dynamics of the total number of deaths of the entire population of the Volgograd region (curve 1) and the proportion of men in the total number of deaths (curve 2). Fig. 2. Dynamics of the total number of deaths of the entire population of the Volgograd region (curve 1) and the proportion of men in the total number of deaths (curve 2). Source: calculated by the author based on the data [10]. щую смертность вносит сверх- смертность мужчин, особенно в сложные периоды развития России. C 1993 по 2016 год доля умерших в течение года мужчин составляла более 50%. С 2017 года наметился гендер- ный разворот в сторону преоб- ладания женщин в структуре общей смертности. Данный факт, очевидно, обусловлен как на уровне России в целом, так и на региональном уровне [14]. Кривая, показывающая на рис. 2. Динамика смертности в Волгоградской области 2 динамику доли мужчин в общем числе умерших, доволь- но хорошо повторяет характер временного ряда абсолютного показателя смертности. Это во многом отражает тот факт, что существенный вклад в об- Рис. 3. Динамика средней ожидаемой продолжительности жизни при рождении ОПЖ, лет (а) и разница в ОПЖ между женщинами и мужчинами ΔОПЖ (b). Источник: рассчитано автором на основе данных [10]. Fig. 3. Dynamics of average life expectancy at birth, years (a) and the difference in life expectancy between women and men (b). Source: calculated by the author based on the data [10] Рис. 3. Динамика средней ожидаемой продолжительности жизни при рождении ОПЖ, лет (а) и разница в ОПЖ между женщинами и мужчинами ΔОПЖ (b). Источник: рассчитано автором на основе данных [10]. Источник: рассчитано автором на основе данных [10]. Fig. 3. Dynamics of average life expectancy at birth, years (a) and the difference in life expectancy between women and men (b). Source: calculated by the author based on the data [10] Статистика и экономика  Т. 19. № 2. 2022 26 Demographic statistics ΔОПЖ. Наибольшая гендерная разница в ОПЖ по региону наблюдалась в 2000 году и со- ставляла 13,3 лет. К 2020 году вследствие того, что снижение уровня смертности у мужчин происходило более высокими темпами, ΔОПЖ уменьшилась и составила 9,6 лет. в целом по стране и в Волго- градской области в частности, является бытовое пьянство. Подробные исследования о связи смертности и потребле- нии алкоголя проводятся Нем- цовым А. В., например, [16, 17]. В работе [17] отмечается, что с 2004 года происходит за- метное уменьшение потребле- ния алкоголя в России. Это согласуется с кривыми, пред- ставленными на рис. 3. в целом по стране и в Волго- градской области в частности, является бытовое пьянство. Подробные исследования о связи смертности и потребле- нии алкоголя проводятся Нем- цовым А. В., например, [16, 17]. В работе [17] отмечается, что с 2004 года происходит за- метное уменьшение потребле- ния алкоголя в России. Это согласуется с кривыми, пред- ставленными на рис. 3. тем, что длительный период вымирания мужчин привел к существенной гендерной дис- пропорции в старших возраст- ных группах. ру На рис. 3a показана дина- мика интегрального показате- ля смертности – средней ожи- даемой продолжительности жизни, дифференцированной по полу. Для сравнения при- ведены временные ряды ОПЖ всего населения Волгоградской области и России в целом. Общее улучшение социаль- но-экономической ситуации в стране позитивно отразилось и на динамике ОПЖ. 2. Динамика смертности в Волгоградской области С 2004 по 2019 год включительно наблю- дался практически стабильный рост данного показателя у обо- их полов. При этом ОПЖ муж- чин выросла с 60,1 лет (2003 год) до 69,3 (2019 год), т.е. на 15,3%, а у женщин в течение рассматриваемого временного интервала рост составил 8,2% (с 72,9 лет в 2003 до 78,9 лет в 2019 году). При сравнении ОПЖ насе- ления Волгоградской области с общероссийскими значениями видно, что кривая для насе- ления региона на протяжении всего исследуемого периода остается выше значений по- казателя ОПЖ для России в целом. Из рис. 3b следует, что величина ΔОПЖ по Волгоград- ской области меньше чем сред- ний показатель по России. Это различие особенно существен- но в 1994 году (ΔОПЖ для Рос- сии было равным 13,7 лет) и в 2005 году (ΔОПЖ = 13,3 лет), когда по Волгоградской обла- сти уже несколько лет проис- ходило сокращение ΔОПЖ. Снижение количества умер- ших, которое с 2004 года про- должалось до 2019 года, обу- словлено, как было показано выше, уменьшением смертно- сти. В течение данного пери- ода население Волгоградской области сократилось с 2673,1 до 2507,5 тыс. человек. Это яв- ляется дополнительным факто- ром снижения числа умерших. Однако общая положительная динамика была остановлена в 2020 году. Это год начала пандемии, вызванной корона- вирусной инфекцией. Число умерших в 2020 году в регио- не составило 39410 человек, а это на 6647 человек больше, чем в предыдущем году. Та- кого однолетнего прироста числа умерших не было даже в самые кризисные 90-е годы, тем более на фоне ежегодного Необходимо отметить, что большой гендерный разрыв в показателе ОПЖ является ха- рактерной особенностью Рос- сии и ряда государств пост- советского пространства [15]. Одним из самых существен- ных факторов, который влия- ет на сверхсмертность мужчин Таким образом, мужское население существенно сокра- тило свое отставание в средней ожидаемой продолжительно- сти жизни. Это особенно вид- но из рисунка 3б, на котором показана разница в ОПЖ меж- ду женщинами и мужчинами – Рис. 4. Динамика ОКС: a) мужчин и женщин по Волгоградской области; b) всего населения Волгоградской области и России. Источник: рассчитано автором на основе данных [10]. Fig. 4. Dynamics of the crude death-rate: a) men and women in the Volgograd region; b) the entire population of the Volgograd region and Russia. Source: calculated by the author based on the data [10] Рис. 4. Динамика ОКС: a) мужчин и женщин по Волгоградской области; b) всего населения Волгоградской области и России. Источник: рассчитано автором на основе данных [10]. Fig. . Dynamics of the crude death-rate: a) men and women in the Volgograd region; b) the entire population of t Volgograd region and Russia. 2. Динамика смертности в Волгоградской области основных участка: 2003–2012 и 2012–2019. На первом из них коэффициент смертности снижается, а на втором демон- стрирует довольно продолжи- тельный рост. мики смертности в течение 10–15 лет, которые предше- ствовали началу пандемии. На рис. 4 показана дина- мика общего коэффициента смертности для мужского и женского населения Волго- градской области, а также для обоих полов по региону и по России в целом. Общий коэф- фициент смертности применя- ется для сопоставления дан- ных о смертности в различных статистических совокупностях. Показатель ОКС не зависит от общей численности населе- ния, а зависит от возрастной структуры населения, а также от распределения интенсив- ности смертей по возрастным группам. Характер кривых с 2003 по 2019 год для мужчин и женщин несколько отличает- ся друг от друга. В частности, динамика ОКС у населения мужского пола в целом поло- жительная. Выпадают из об- щей тенденции только 2013 и 2017 годы, в которых снижени- ем ОКС было несколько завы- шенным. На кривой ОКС для женского населения есть два Обращает на себя внимание аномально высокое значение ОКС в 2010 году для населения женского пола. Это согласуется и с другими данными: на рис. 2 в 2010 доля смертей мужчин су- щественно сократилась, на рис 3 можно увидеть небольшое снижение ОПЖ женщин. Лето 2010 года был очень жарким во многих регионах России, в том числе и в Волгоградской области. Существует ряд ис- следований, посвященных вли- янию климатических условий на динамику смертности. Уве- личение смертности летом 2010 года в России анализировалось, например, в работе [18]. В част- ности, было отмечено, что в ав- густе 2010 года по сравнению с августом 2009 года, смертность выросла в целом по стране на 27,4%. В таблице 1 приведены оперативные данные по числу умерших в течение четырех ме- Анализ динамики возраст- ных коэффициентов смерт- ности женщин (см. рис. 5) показывает, что в 2010 году наблюдается всплеск интенсив- ности смертности в возрастных группах старше 70 лет. Данные смерти и внесли наиболее су- щественный вклад в снижение ОПЖ. У мужчин снижения ожидаемой продолжительно- сти жизни не произошло. При этом каких-либо существен- ных трансформаций в возраст- ном профиле коэффициентов смертности не выявлено. На рис. 6. показана дина- мика общих коэффициентов смертности по основным клас- сам причин смерти (оба пола). Как видно из рисунка характер Рис. 6. 2. Динамика смертности в Волгоградской области 4. Dynamics of the crude death-rate: a) men and women in the Volgograd region; b) the entire population of the Volgograd region and Russia. S l l d b h h b d h d [10] Statistics and Economics  V. 19. № 2. 2022 27 Демографическая статистика Таблица 1 (Table 1) сокращения населения в реги- оне. ОПЖ в Волгоградской об- ласти за 2020 год уменьшилась и у мужчин, и у женщин, со- ответственно, на 1,9 и 1,8 лет (см. рис. 3a). Такая же тенден- ция наблюдается и в целом по стране. На резкий рост смерт- ности, безусловно, повлияла эпидемия. Однако, данный во- прос будет более детально рас- смотрен в следующем разделе работы. Сейчас остановимся на некоторых аспектах дина- Таблица 1 (Table 1) Оперативные данные по числу умерших в течение четырех месяцев для населения Волгоградской области (оба пола), человек Operational data on the number of deaths within four months for the population of the Volgograd region (both sexes), people Месяц 2009 2010 2011 июнь 3246 3416 2901 июль 3584 3427 3156 август 2667 4673 3229 сентябрь 2809 2841 2680 Источник: составлено автором на основе данных [11] Source: compiled by the author based on the data [11] Source: compiled by the author based on the data [11] Рис. 5. Динамика пятилетних возрастных коэффициентов смертности населения Волгоградской области старше 30 лет в разрезе мужчин/женщин. Источник: рассчитано автором на основе данных [10]. Fig. 5. Dynamics of five-year age-specific mortality rates of the population of the Volgograd region over 30 years old for men and women Рис. 5. Динамика пятилетних возрастных коэффициентов смертности населения Волгоградской област старше 30 лет в разрезе мужчин/женщин. Источник: рассчитано автором на основе данных [10]. Fig. 5. Dynamics of five-year age-specific mortality rates of the population of the Volgograd region over 30 years old for men and women. Source: calculated by the author based on the data [10] Источник: рассчитано автором на основе данных [10]. Fig. 5. Dynamics of five-year age-specific mortality rates of the population of the Volgograd region over 30 years old for men and women. Статистика и экономика  Т. 19. № 2. 2022 28 Demographic statistics сяцев за 2009, 2010 и 2011 год по Волгоградской области для обоих полов. В июле-августе 2010 года регион попал в зону с аномально высокой темпера- турой. Результатом воздействия неблагоприятных факторов окружающей среды стала вы- сокая смертность в августе 2010 по сравнению с аналогичным периодом в 2009 и 2011 годах. 2. Динамика смертности в Волгоградской области Динамика коэффициентов смертности по основным классам причин смерти (оба пола), на 1000 человек: 1 – новообразования, 2 – внешние причины, 3 – болезни органов дыхания, 4 – болезни органов пищеварения, 5 – болезни системы кровообращения, 6 – инфекционные и паразитарные болезни. Источник: рассчитано автором на основе данных [11]. Fig. 6. Dynamics of mortality rates by main classes of causes of death (both sexes), per 1000 people: 1 – neoplasms, 2 – external causes, 3 – diseases of the respiratory system, 4 – diseases of the digestive system, 5 – diseases of the circulatory system, 6 – infectious and parasitic diseases. Source: calculated by the author based on the data [11] Рис. 6. Динамика коэффициентов смертности по основным классам причин смерти (оба пола), на 1000 человек: 1 – новообразования, 2 – внешние причины, 3 – болезни органов дыхания, 4 – болезни органов пищеварения, 5 – болезни системы кровообращения, 6 – инфекционные и паразитарные болезни. Источник: рассчитано автором на основе данных [11]. Fig. 6. Dynamics of mortality rates by main classes of causes of death (both sexes), per 1000 people: 1 – neoplasms, 2 – external causes, 3 – diseases of the respiratory system, 4 – diseases of the digestive system, 5 – diseases of the circulatory system, 6 – infectious and parasitic diseases. Fig. 6. Dynamics of mortality rates by main classes of causes of death (both sexes), per 1000 people: 1 – neoplasms, 2 – external causes, 3 – diseases of the respiratory system, 4 – diseases of the digestive system, 5 – diseases of the circulatory system, 6 – infectious and parasitic diseases. Fig. 6. Dynamics of mortality rates by main classes of causes of death (both sexes), per 1000 people: 1 neoplasms, 2 – external causes, 3 – diseases of the respiratory system, 4 – diseases of the digestive system, 5 – diseases of the circulatory system, 6 – infectious and parasitic diseases. Source: calculated by the author based on the data [11]. Statistics and Economics  V. 19. № 2. 2022 29 Демографическая статистика ших только изменениями в возрастной структуре населе- ния не получится. Поэтому из- быточную смертность можно связать с пандемией. Однако, если разделить смертность от COVID-19 по данным Росста- та на избыточную смертность, то окажется, что по Волгоград- ской области доля составля- ет 36,8%, а по России 42,5%. Таким образом, более 50% избыточных смертей остают- ся необъясненными. Данное расхождение характерно прак- тически для всех регионов. 3. Вклад COVID-19 в смертность за 2020 год Перейдем теперь к рас- смотрению наиболее важно- го в настоящее время вопро- са – влиянию пандемии на смертность в Волгоградской области. В таблице 2 приве- дены общие данные по числу умерших от коронавирусной инфекции оперативного штаба и Росстата за 2020 год. Наблю- дается существенное расхож- дение между этими данными как в целом по России, так и по Волгоградской области. Коэффициент смертности от новообразований (кривая 1) в течение исследуемого пери- ода времени оставался прак- тически на одном уровне и лишь в последние шесть лет наметилось снижение данного показателя с 2,37‰ в 2015 до 2,08‰ в 2020 году. Практи- чески в этот же период прои- зошло снижение смертности от болезней органов дыхания (кривая 3): с 0,67‰ в 2014 до 0,36‰ в 2019 году. Пандемия, вызванная коронавирусной инфекцией, привела к тому, что 2020 году анализируемый показатель смертности вырос до 0,46‰ (на 25,7%). В работе [19] исследовалась заболеваемость, вызванная COVID-19 в Волгоградской области в период с 24.03.2020 по 18.10.2020. Отмечается, что все пациенты со смертельны- ми случаями имели несколько сопутствующих заболеваний. При этом «в подавляющем большинстве случаев леталь- ные исходы от COVID-19 были ассоциированы с заболевания- ми сердечно-сосудистой си- стемы (65,3%) и патологией эндокринной и сердечно-со- судистой системы (21,1%)». Это коррелирует с ростом смертности вследствие болез- Избыточная смертность в 2020 году по сравнению с 2019 годом для Волгоградской об- ласти составила 6647 человек, а по России 340286 человек. Ранее было отмечено, что в целом уровень смертности в регионе снижался. При этом уменьшалась и общая числен- ность населения. Объяснить такое увеличение числа умер- Наиболее существенно ва- рьировался в течение исследу- емого периода коэффициент смертности от внешних при- чин (кривая 2). В 2001 году данный показатель достиг сво- его пикового значения и со- ставил 2,04‰. Начиная с 2002 года смертность от внешних причин стала сокращаться и в 2020 коэффициент смертности был уже равен 0,88‰. С 2000 года позитивную тенденцию демонстрирует смертность вследствие инфекционных и паразитарных болезней (кри- вая 6). Коэффициент смерт- ности снизился с 0,39% в 1999 году до 0,18% в 2020. Таблица 2 (Table 2) Смертность от COVID 19 за 2020 год Таблица 2 (Table 2) Статистика и экономика  Т. 19. № 2. 2. Динамика смертности в Волгоградской области В работе [2] показано, что доля смертности от COVID-19 в избыточной смертности в декабре 2020 года варьируется по регионам России от 98,4% (г. Москва) до 3,9% (Чеченская республика). В данном «рей- тинге» Волгоградская область занимает среднее положение. динамики для различных клас- сов причин смерти отличается. Наибольший вклад в смерт- ность на протяжении всего ис- следуемого периода вносят бо- лезни системы кровообращения (кривая 5). Можно отметить, что во временном диапазоне с 1993 по 2010 год именно дан- ный класс причин обеспечивал высокий уровень смертности, который наблюдался в Волго- градской области. После 2010 года наблюдается снижения смертности от болезней систе- мы кровообращения. В 2020 году произошел резкий рост со- ответствующего коэффициента смертности с 7,01‰ до 8,57‰. зывали существенного влияния ни кризисные годы, ни перио- ды социально-экономического подъема. Коэффициент смерт- ности от болезней органов пи- щеварения практически весь период рос. Начиная с 2015 года можно отметить стабили- зацию данного коэффициента и даже небольшое снижение. В 2020 году смертность от болез- ней органов пищеварения уве- личилась по сравнению с 2019 годом на 22,2%. 3. Вклад COVID-19 в смертность за 2020 год Статистика и экономика  Т. 19. № 2. 2022 3. Вклад COVID-19 в смертность за 2020 год 2022 Таблица 2 (Table 2) Смертность от COVID-19 за 2020 год Deaths from COVID-19 in 2020 Регион Число умерших от COVID-19, человек Общее число умерших в течение года, человек Избыточная смертность по сравнению с 2019 годом, человек Доля смертей, связанной с COVID-19, % Данные оперштаба Данные Росстата 2019 2020 Россия 56271 144699 1798307 2138586 340279 42,5 Волгоградская область 458 2448 32763 39410 6647 36,8 Источник: составлено автором на основе данных [10, 11, 12]. Source: compiled by the author based on the data [10, 11 and 12]. Смертность от COVID-19 за 2020 год Deaths from COVID-19 in 2020 На динамику смертности по причине болезней органов пи- щеварения (кривая 4) не ока- 30 Demographic statistics Таблица 3 (Table 3) Таблица 3 (Table 3) Таблица 3 (Table 3) ней системы кровообраще- ния (см. рис. 6) в 2020 году по сравнению с 2019 годом. В от- носительном выражении рост коэффициента смертности со- ставил 22,2%. Сильнее всего вырос коэффициент смертно- сти по классу «Болезни орга- нов дыхания» – на 25,7%. Рост коэффициентов смертности по данным причинам был отме- чен и в целом по России. Таблица 3 (Table 3) Оперативные данные по числу умерших в Волгоградской области (оба пола) Operational data on the number of deaths in the Volgograd region (both sexes) Месяц Число умерших, человек Индекс, % 2019 2020 2021 2020/2019 2021/2020 Январь 3041 2 934 3 838 96,5 130,8 Февраль 2680 2 655 3 165 99,1 119,2 Март 2847 2 643 3 263 92,8 123,5 Апрель 2834 2 793 3 323 98,6 119,0 Май 2924 3 119 3 237 106,7 103,8 Июнь 2608 2 753 3 145 105,6 114,2 Июль 2693 3 566 3 883 132,4 108,9 Август 2637 2 813 4 543 106,7 161,5 Сентябрь 2338 3 220 4384 137,7 136,1 Октябрь 2912 4 139 – 142,1 – Ноябрь 2634 4 137 – 157,1 – Декабрь 2704 4 342 – 160,6 – Источник: составлено автором на основе данных [11] Source: compiled by the author based on the data [11] Таблица 3 (Table 3) Оперативные данные по числу умерших в Волгоградской области (оба пола) Operational data on the number of deaths in the Volgograd region (both sexes) В 2020 году в России были две волны роста заболеваемости от COVID-19. Периоды разви- тия этих волн в разных регио- нах отличались. Раньше всех эпидемия затронула Москву: пик заболеваемости пришелся на начало мая. 3. Вклад COVID-19 в смертность за 2020 год В Волгоградской области первая волна началась в середине апреля и продолжи- лась до конца июля. Следующий всплеск заболеваемости произо- шел в октябре и продлился до конца 2020 года. Вторая волна сопровождалась увеличением числа заболевших и умерших по сравнению с первой волной [19]. Пиковые значения ежесу- точно выявляемых заболевших во время второй волны превы- шали примерно в три раза чис- ло заболевших во время первой волны. В таблице 3 приведены оперативные данные по коли- чество умерших в течение ме- сяца по Волгоградской обла- сти за 2019, 2020 и 2021 годы. В пятом столбце приведены индексы, которые показывают отношение числа умерших в те- р [ Source: compiled by the author based on the data [11] Проанализируем половоз- растной профиль умерших в Волгоградской области от ко- ронавирусной инфекции по данным Росстата. Отметим, что до возраста 35 лет число умерших по данной причине небольшое. При этом избы- точная смертность была отри- цательной. Поэтому в первую возрастную группу включены умершие в возрасте до 35 лет; интервал следующих возраст- ных групп составил 10 лет. Наибольшее число смертей от COVID-19 у мужчин приходит- ся на возрастную группу 65–74 лет, а у женщин – 75–84 лет. В этих же возрастных катего- чение месяца в 2020 году к чис- лу умерших за аналогичный пе- риод в 2019 году. Видно, что до апреля включительно в регионе наблюдалось ежемесячное сни- жение умерших по сравнению с 2019 годом. Однако уже с на- чалом первой волны пандемии наблюдается рост смертности: в мае – на 6,7%, июне – 5,6%, июле – 32,7%. Во время второй волны прирост числа умерших был существенно выше, на- пример, в декабре он составил 60,6%. Таким образом, опера- тивные данные по учету числа умерших согласуются с особен- ностями протекания пандемии COVID-19. Statistics and Economics  V. 19. № 2. 2022 3. Вклад COVID-19 в смертность за 2020 год В ряде иссле- дований также был выявлен бо- лее высокий уровень смертности от коронавирусной инфекции у мужчин по сравнению с жен- щинами. Например, в работе [1] отмечается, что «Мужчины по сравнению с женщинами поте- ряли на 45% больше лет жизни. Гендерная разница в потерянных годах потенциальной жизни свя- зана с двумя закономерностями: во-первых, от COVID-19 умира- ет больше мужчин и, во-вторых, мужчины умирают в более моло- дом возрасте». р Общая избыточная смерт- ность в Волгоградской обла- сти за 2020 год по сравнению с 2019 составляет, как было отмечено выше, 6647 человек. Данное значение не учитывает динамику возрастных коэффи- циентов смертности. В годы предшествующие пандемии смертность во всех возрастных группах в целом снижалась (см. рис. 5). Косвенно на снижение смертности указывают также оперативные данные за пер- вые четыре месяца 2020 года: количество зарегистрирован- ных умерших оказалась мень- ше, чем за аналогичный период предыдущего года (см. таблица 3). В 2020 году у мужчин и жен- щин старше 45 лет возрастные коэффициенты смертности за- метно увеличились (см. рис. 5). Можно отметить, что жен- щин умерло от COVID-19 больше, чем мужчин. При этом до 75-летнего возраста перевес был на стороне мужского насе- ления. Итоговое превышение числа умерших женщин над мужчинами произошло благо- даря возрастным группам от 75 лет и старше. Такое половоз- растное распределение числа умерших от коронавирусной инфекции обусловлено осо- бенностями возрастной струк- туры населения региона. Можно отметить, что жен- щин умерло от COVID-19 больше, чем мужчин. При этом до 75-летнего возраста перевес был на стороне мужского насе- ления. Итоговое превышение числа умерших женщин над мужчинами произошло благо- даря возрастным группам от 75 лет и старше. Такое половоз- растное распределение числа умерших от коронавирусной инфекции обусловлено осо- бенностями возрастной струк- туры населения региона. Чтобы оценить интенсив- ность смертности, вызванной COVID-19 в различных по- ловозрастных группах были рассчитаны коэффициенты смертности. На рис. 7 показа- ны возрастные коэффициенты смертности вследствие коро- навирусной инфекции (AMC) в разрезе мужчины/женщины, а также доля AMC в возраст- ном коэффициенте смертно- сти от всех причин KAM. Однако, несмотря на то, что возрастные коэффициен- ты смертности от COVID-19 у мужчин больше, чем у жен- щин следует отметить, что пандемия оказала существен- В данной работе была про- ведена оценка избыточной смертности с учетом динамики пятилетних возрастных коэф- фициентов смертности во вре- менном интервале с 2015 по Рис. 7. 3. Вклад COVID-19 в смертность за 2020 год Таблица 4 (Table 4) ц ( ) Половозрастной профиль умерших от COVID-19 Sex and age profile of those, who died from COVID-19 Возраст, лет Число умерших от COVID-19, человек Избыточная смертность в 2020 году по сравнению с 2019 годом, человек Доля умерших в данной возрастной группе от COVID-19 в избыточной смертности, % мужчины женщины оба пола мужчины женщины оба пола мужчины женщины оба пола 0–34 11 6 17 –3 –7 –10 – – – 35–44 30 21 51 119 67 186 25,2 31,4 27,4 45–54 96 85 181 145 134 279 66,2 63,5 64,9 55–64 256 233 489 651 477 1128 39,3 48,9 43,3 65–74 383 360 743 1281 1036 2316 29,9 34,8 32,1 75–84 305 420 725 607 1084 1691 50,2 38,8 42,9 85+ 94 148 242 410 647 1057 22,9 22,9 22,9 Всего 1175 1273 2448 3210 3438 6647 36.6 37.0 36.8 Источник: составлено автором на основе данных [10] Source: compiled by the author based on the data [10] Половозрастной профиль умерших от COVID-19 Sex and age profile of those, who died from COVID-19 Statistics and Economics  V. 19. № 2. 2022 31 Демографическая статистика ное влияние и на смертность женщин. Коронавирусная ин- фекция вносит существенный вклад в интенсивность смерт- ности в возрастных группах от 45 до 75 лет и составляет порядка 10–12% (см. рис. 7). У мужчин этот вклад в про- центном соотношение ниже: 6–7% в возрасте 45 лет и выше. Условные обозначения: AMC – возрастные коэффи- циенты смертности от корона- вирусной инфекции, где A – возраст (age); M – смертность (mortality); C – COVID-19. КАМ – доля AMC в возраст- ных коэффициентах смертно- сти по всем причинам. риях наблюдается наибольшая избыточная смертность. Доля умерших от COVID-19 в избы- точной смертности выше всего в возрастном интервале 45–54 лет и составляет 66,2% для мужчин и 63,5% для женщин. В меньшей степени избыточная смертность объясняется коро- навирусной инфекцией в воз- растных группах 35–44 и 85+. риях наблюдается наибольшая избыточная смертность. Доля умерших от COVID-19 в избы- точной смертности выше всего в возрастном интервале 45–54 лет и составляет 66,2% для мужчин и 63,5% для женщин. В меньшей степени избыточная смертность объясняется коро- навирусной инфекцией в воз- растных группах 35–44 и 85+. Интенсивность смертности от COVID-19 у мужчин выше, чем у женщин. При увеличении возраста это различие еще боль- ше усиливается. В возрастной группе 80–84 лет с наивысшей интенсивностью смертности у мужчин AMC равен 9,8‰, а у женщин – 5,2‰. 3. Вклад COVID-19 в смертность за 2020 год В 2020 году имеет место значительной рост смертности по таким классам как болезни системы кровообращения и бо- лезни органов дыхания. В ряде исследований отмечается, что умершие от коронавирусной ин- фекции часто имеют сопутству- ющие заболевания, связанные с сердечно-сосудистой системой, хроническими болезнями ниж- них дыхательных путей [19, 20]. теканием болезни и высокой смертностью. Очевидно, что за 2021 год уровень смертности вследствие пандемии окажет- ся выше, чем в 2020 году. На основе оперативных данных учета смертей, приведенных в таблице 3, можно оценить сколько умрет по Волгоград- ской области в 2021 году. По- следние данные – за сентябрь. Если предположить, что в оставшиеся до конца года три месяца численность умерших будет ежемесячно фиксиро- ваться на уровне сентября, то общее число умерших за 2021 год составит 45933. Это зна- чение существенно выше, чем в 2020 году, в котором было учтено 39410 умерших. В ус- ловиях снижения рождаемости и продолжающейся миграци- онной убыли в Волгоградской области получится демографи- ческий кризис сопоставимый с кризисом, который был в 90-е годы прошлого столетия. 2019 год. В дальнейшим избы- точную смертность, оцененную на основе динамики возрастных коэффициентов смертности, будем называть потенциальной избыточной смертностью. В та- блице 5 приведены результаты оценки данного показателя. 2019 год. В дальнейшим избы- точную смертность, оцененную на основе динамики возрастных коэффициентов смертности, будем называть потенциальной избыточной смертностью. В та- блице 5 приведены результаты оценки данного показателя. Итоговые результаты сле- дующие. Если бы тенденции в динамике интенсивности смертности сохранились бы и в 2020 году, то общее количе- ство умерших по Волгоград- ской области составило бы 32044 человек. Таким образом, избыточную смертность мож- но оценить в 7368 человек. Доля умерших от COVID-19 в потенциальной избыточной смертности равна 33,2%. Таким образом, возможной причиной анализируемого не- соответствия является непра- вильный учет смертности от коронавирусной инфекции. Другим фактором увеличения смертности в период пандемии может быть снижение качества медицинского обслуживания. Произошла переориентация ра- боты медицинских учреждений на лечение пациентов с коро- навирусной инфекцией, увели- чилась нагрузка на скорую ме- дицинскую помощь. Наиболее чувствительными к качеству ме- дицинских услуг являются люди преклонного возраста. В табли- це 4 приведено половозрастное распределение доли умерших от COVID-19 в избыточной смерт- ности. В возрастной группе 85+ данный показатель имеет наи- меньшее значение и составляет 22,9%, что в некоторой степени подтверждает сделанное выше предположение. Сравнение возрастного про- филя потенциальной избыточ- ной смертности с фактической (таблица 4) показывает, что до 65-летнего возраста потери на- селения оказались гораздо су- щественней, чем это следует из фактического распределения избыточной смертности. 3. Вклад COVID-19 в смертность за 2020 год Дан- ный факт обусловлен тем, что смертность снижалась прежде всего у населения трудоспо- собного возраста. 3. Вклад COVID-19 в смертность за 2020 год Возрастные коэффициенты смертности от коронавирусной инфекции за 2020 год по Волгоградской области (AMC), ‰ и доля AMC в возрастных коэффициентах смертности по всем причинам (KAM), %. Источник: рассчитано автором на основе данных [10]. Fig. 7. Age-specific mortality rates from coronavirus infection in 2020 for the Volgograd region (AMC: A – age; M – mortality; C – COVID-19), ‰ and the share of AMC in age-specific mortality rates for all causes (СAM), %. Source: calculated by the author based on the data [10]. Рис. 7. Возрастные коэффициенты смертности от коронавирусной инфекции за 2020 год по Волгоградской области (AMC), ‰ и доля AMC в возрастных коэффициентах смертности по всем причинам (KAM), %. Источник: рассчитано автором на основе данных [10]. Fig. 7. Age-specific mortality rates from coronavirus infection in 2020 for the Volgograd region (AMC: A – age; M – mortality; C – COVID-19), ‰ and the share of AMC in age-specific mortality rates for all causes (СAM), %. Source: calculated by the author based on the data [10]. Статистика и экономика  Т. 19. № 2. 2022 32 Demographic statistics Таблица 5 (Table 5) Таблица 5 (Table 5) Результаты оценки избыточной смертности с учетом динамики возрастных коэффициентов смертности по Вол- гоградской области Results of excess mortality assessment taking into account the dynamics of age-specific mortality rates in the Volgograd region Возраст, лет Прогноз числа умерших в 2020 году на основе среднего темпа роста mx, человек Разница между прогнозным числом умерших в 2020 году и фактическим числом умерших в 2019, человек мужчины женщины оба пола мужчины женщины оба пола 0–34 586 247 833 71 21 92 35–44 1073 364 1437 151 85 236 45–54 1577 628 2205 206 146 352 55–64 3294 1420 4714 806 555 1361 65–74 4154 2814 6968 1095 934 2029 75–84 3494 5561 9055 889 1411 2300 85+ 1596 5236 6832 416 582 998 Всего 15774 16270 32044 3634 3734 7368 Источник: составлено автором на основе данных [10] Source: compiled by the author based on the data [10] Таблица 5 (Table 5) Результаты оценки избыточной смертности с учетом динамики возрастных коэффициентов смертности по Вол- гоградской области Results of excess mortality assessment taking into account the dynamics of age-specific mortality rates in the Volgograd region р [ Source: compiled by the author based on the data [10] Source: compiled by the author based on the data [10] тей? 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Epidemic manifestations of COVID-19 on the territory of the Volgograd region: intermediate results. Vestnik Vol- GMU = Bulletin of the Volgograd State Medical University. 2020; 4(76): 30-36. DOI 10.19163/1994- 9480-2020-4(76)-30-36. (In Russ.) 11. Dannyye Federal’noy sluzhby gosudarstvennoy statistiki = Data of the Federal State Statistics Service [Internet]. Available from: https://rosstat. gov.ru/. (cited 01.11.2021). (In Russ.) 20. Sabgayda T.P., Zubko A.V., Semenova V.G., Change in the structure of causes of death in the second year of the COVID-19 pandemic in Moscow [Internet]. Sotsial’nyye aspekty zdorov’ya naseleniya = Social aspects of public health. 2021; 67(4): 1. Statistics and Economics  V. 19. № 2. 2022 Information about the author Alexey V. Alpatov Cand. Sci. (Physics and Mathematics), Associate Professor of the Department of «Computer science and programming technology» Volgograd State Technical University, branch of the Volga Polytechnical Institute, Volzhskiy, Russia E-mail: alpatov80@mail.ru Alexey V. Alpatov Cand. Sci. (Physics and Mathematics), Associate Professor of the Department of «Computer science and programming technology» Volgograd State Technical University, branch of the Volga Polytechnical Institute, Volzhskiy, Russia E-mail: alpatov80@mail.ru Алексей Викторович Алпатов К.ф.-м.н., доцент кафедры «Информатика и технология программирования» Волжский политехнический институт (филиал), Волжский, Россия Эл. почта: alpatov80@mail.ru Алексей Викторович Алпатов К.ф.-м.н., доцент кафедры «Информатика и технология программирования» Волжский политехнический институт (филиал), Волжский, Россия Эл. почта: alpatov80@mail.ru Алексей Викторович Алпатов К.ф.-м.н., доцент кафедры «Информатика и технология программирования» Волжский политехнический институт (филиал), Волжский, Россия Эл. почта: alpatov80@mail.ru 35
https://openalex.org/W4385437077	https://revistas.upt.edu.pe/ojs/index.php/arquitek/article/download/634/630	es		Índice de caminabilidad en las centralidades urbanas de los distritos de Huánuco, Amarilis y Pillco Marca	Arquitek	2,022	cc-by	4,354	ARQUITEK \| N°21 \| Enero - Junio 2022 \| Edición online ISSN-2617-0892 \| DOI: https://doi.org/10.47796/ra.2022i21 ÍNDICE DE CAMINABILIDAD EN LAS CENTRALIDADES URBANAS DE LOS DISTRITOS DE HUÁNUCO, AMARILIS Y PILLCO MARCA1 WALKABILITY INDEX IN THE URBAN CENTRALITIES OF THE DISTRICTS OF HUÁNUCO, AMARILIS AND PILLCO MARCA PRESENTADO : 24.04.22 ACEPTADO : 20.06.22 DOI: https://doi.org/10.47796/ra.2022i21.634 RENATO EDU BARZOLA GOMEZ 2 Universidad de Huánuco, Huánuco - Perú https://orcid.org/0000-0002-0745-3534 renato.barzola@udh.edu.pe RESUMEN ABSTRACT Este trabajo explora el entorno urbano construido de Huánuco donde la infraestructura de espacios viales ene casi exclusividad para los autos, motos y trimotos. Así, el trabajo se propone medir el entorno urbano construido de tres centralidades urbanas (Huánuco, Amarilis y Pillco Marca). Mediante el análisis del índice de caminabilidad, basado en cuatro variables: calidad ambiental, densidad, confort peatonal y entropía, y con instrumentos como la observación, mapeo y análisis de bases de datos espaciales, se construye un índice de caminabilidad que permite entender las condiciones del entorno construido urbano existente. Los resultados nos permiten iden ﬁcar que la centralidad urbana del distrito de Amarilis presenta mejores condiciones en la variable de calidad ambiental, densidad y confort peatonal que garan zan un mejor entendimiento a una escala local. Esto permite plantear polí cas locales para la mejora de los espacios urbanos y recuperar la caminabilidad en las centralidades. This work explores the urban built environment of Huánuco where the infrastructure of road spaces is almost exclusively for cars, motorcycles and tricycles. Thus, the work proposes to measure the urban built environment of three urban centers (Huánuco, Amarilis and Pillco Marca). Through the analysis of the walkability index based on four variables: environmental quality, density, pedestrian comfort and entropy, with instruments such as observa on, mapping and analysis of spa al databases. A walkability index is constructed which allows us to understand the condi ons of the exis ng urban built environment. The results allow us to iden fy that the urban centrality of the district of Amarilis presents be er condi ons in the variable of Environmental Quality, Density and Pedestrian Comfort that guarantee a be er understanding at a local scale, this allows us to propose local policies for the improvement of urban spaces and to recover walkability in the centrali es. Palabras clave: caminabilidad, indicadores, transitabilidad, centralidad, movilidad urbana. Key words: Walkability, indicators, walkability, centrality, urban mobility. 1 2 El artículo recoge los resultados de la investigación “Índice de caminabilidad de los distritos de Huánuco, Amarilis y Pillco Marca”. Es un análisis en torno a la caminabilidad, accesibilidad y transitabilidad en Huánuco (Perú), nanciado por el Vicerrectorado de Investigación de la Universidad de Huánuco, mediante el concurso interno de proyectos de investigación 2021. Arquitecto por la Universidad Nacional del Centro del Perú; magíster en Gerencia Pública por la Universidad Continental; maestrando en Arquitectura, Urbanismo y Desarrollo Territorial Sostenible por la Ponticia Universidad Católica del Perú; docente del programa académico de Arquitectura de la Universidad de Huánuco. 88 RENATO EDU BARZOLA GOMEZ INTRODUCCIÓN En los centros urbanos de América La na, se ha mantenido un modelo urbanís co que está basado en el transporte motorizado. El proceso de urbanización construyó ciudades dispersas con una preferencia en las vías amplias y solo vehiculares. Este modelo de crecimiento de la ciudad en América La na ha derivado en una movilidad urbana ausente, por ello, es importante la creación de entornos transitables y de la mejora de la caminabilidad en la ciudad (Forsyth, 2015). Muchas ins tuciones vienen proponiendo rever r aquella tendencia como al Nueva Agenda Urbana de la ONU- Hábitat, que propone una visión de ciudad con un sistema de movilidad urbana de calidad (Siclari, 2017). Resalta la importancia de la movilidad no motorizada, especíﬁcamente la caminata (Forsyth, 2015), entre cuyos beneﬁcios están la menor conges ón vehicular y las mejores condiciones de salud. La movilidad urbana es considerada como un derecho ciudadano (Tapia, 2018), por lo que es necesario promover ciudades accesibles y conectadas con acceso para todos. La movilidad urbana ha encontrado, en el desplazamiento del peatón, el medio óp mo de sostenibilidad en el transporte. Esta beneﬁcia a la ciudad, además de permi r el aprovechamiento del espacio por sus habitantes sin ningún po de dis nciones. La reconstrucción del espacio recupera los derechos de los ciudadanos y permite el disfrute de un espacio que cubra las necesidades de ac vidad y sociabilidad en las calles (Machín Gil, H. J., y Ghidini, R. ,2013). Por ese mo vo, la ciudad requiere de una planiﬁcación urbana con una mejor movilidad, asimismo, de una organización integrada de la distribución espacial, la densidad y la estructura de las calles. La transitabilidad se desarrolló para explicar los modos de transporte ac vos, ahora se usará para determinar el entorno construido del caminar (Gerlinde Grasseret al., 2016). Se trata de que la caminabilidad responda al diseño que favorece al traslado a pie proporcionando accesiblilidad a los espacios urbanos (MedinaRuiz, 2020). En ese marco, la caminabilidad se puede entender como las caracterís cas del entorno construido y uso del suelo que favorecen realizar recorridos peatonales para ir al trabajo, pasear, realizar ejercicios u otros; estos pueden ser favorables o desfavorables para la población local (Leslie et al. 2006). Por tal mo vo, el modo de entender la caminabilidad será a través de la medición de la calidad del entorno (Bradshaw, 2013). Esta caminabilidad debe garan zar el traslado del peatón de un si o a otro; las caracterís cas sicas y las rutas deben ser transitables con espacios más agradables y sociales que op micen las opciones de transitabilidad y que fomenten la ac vidad sica en su traslado. El contexto a estudiar es el entorno construido de tres centralidades urbanas de la provincia de Huánuco. El estudio se basa en la metodología de Chris Bradshaw y Evandro Cardoso dos Santos, para quienes la medición del índice de caminabilidad está basado en cuatro variables: calidad ambiental, densidad, confort y entropía, elegidas para este estudio. Teniendo en cuenta el marco teórico precedente de la caminabilidad y transitabilidad, el obje vo de este estudio es medir el entorno urbano construido de tres centralidades urbanas de los distritos de Huánuco, Amarilis y Pillco Marca (Figura 1). A s i m i s m o, s e p re te n d e p ro p o n e r u n a alterna va de medición de la caminabilidad. Se eligió como unidad espacial la ciudad de Huánuco que está ubicada en la zona centro oriental del Perú. Las centralidades urbanas seleccionadas pertenecen a los distritos de Huánuco, Amarilis y Pillco Marca, zonas de áreas urbanas nacidas a par r del crecimiento de la ciudad (Figura 2). Se resalta que estas 89 ARQUITEK \| N°21 \| Enero - Junio 2022 \| Edición online ISSN-2617-0892 \| DOI: https://doi.org/10.47796/ra.2022i21 Figura 1. Distritos Huánuco, Amarilis y Pilco Marca Urbano de Huánuco 2021-2031); sin embargo, este instrumento técnico administra vo aun considera parámetros en beneﬁcio del automóvil en todos los distritos. En términos metodológicos, el ar culo propone una metodología cuan ta va. Primero, se basa en la observación directa de las caracterís cas espaciales de las centralidades, el otro es la medición de los componentes sicos a través de las medidas del entorno construido con los indicadores en todos los trayectos de cada centralidad urbana. Los resultados del estudio nos ayudan a iden ﬁcar las condiciones en que se encuentra la infraestructura de las centralidades para caminar. Esta información servirá para iniciar propuestas de polí cas locales de movilidad urbana y espacio público en los distritos de Huánuco, Amarilis y Pillco Marca, y así restablecer la caminabilidad en las centralidades. MATERIALES Y MÉTODOS Nota: 1=veredas de Jr. Dos de Mayo, distrito de Huánuco; 2= veredas del Jr. Túpac Yupanqui, distrito de Amarilis; 3= veredas del Jr. Prol. Los Álamos, distrito de Pillco Marca. centralidades se consolidaron en base a un plan d i re c to r d e 1 9 8 3 , h a sta u n a re c i e nte actualización del PDU (Plan de Desarrollo 90 Este trabajo realiza un estudio de la caminabilidad a través de la medición de la transitabilidad. El diseño de la inves gación propone una estrategia metodológica de base cuan ta va (usos de suelo, áreas de parque, dimensiones, can dad de árboles, veredas, ediﬁcaciones, densidad, vías y altura de ediﬁcación) para generar datos ú les dentro del urbanismo para Huánuco. Se u lizaron diferentes instrumentos para medir el entorno construido. Para la recolección de datos se consideró diversas fuentes: observación directa, mapeos e imágenes satelitales. La selección de las tres centralidades urbanas se basó en la capital del departamento y las ciudades con mayor dinámica urbana (se encuentran en el rango de 40 000 a 85 000 habitantes), por lo cual fue seleccionado como objeto de estudio.la centralidad Huánuco, Amarilis y Pillco Marca. RENATO EDU BARZOLA GOMEZ Para la construcción del índice de caminabilidad, este estudio u lizó la base metodológica de Chris Bradshaw y Evandro Cardoso dos Santos. Se propuso cuatro variables que con enen índices a desarrollar (ver Tabla 1). Este estudio se realizó en dos etapas. Primero, se recolectaron los datos urbanos de los distritos de Huánuco, Amarilis y Pillco Marca del PDU (Plan de Desarrollo Urbano de Huánuco 2021-2031), y se midieron a través del apoyo del ArcGis 10.4 e imágenes satelitales de alta resolución. Se realizaron mapeos de cada centralidad de forma semimanual. Luego, se llevó a cabo la observación no par cipante, donde el inves gador se limita solo a tomar nota de los hechos. Esto se desarrolló durante los meses de noviembre y diciembre del 2021, y de enero a febrero del 2022. Se visitó cada centralidad para registrar las caracterís cas sico-espaciales mediante anotaciones y fotogra as. Las visitas se realizaron en dos rangos de horario. De acuerdo a lo anterior, se propone cuatro variables necesarias para estudiar las centralidades de Huánuco por las caracterís cas sico–espaciales, las cuales son las siguientes: calidad ambiental densidad confort peatonal entropía Esta categoría de calidad ambiental agrupa dos índices. Para este caso, se considera densidad de arbolado urbano y proporción de áreas de parque. La calidad ambiental para este caso se calcula como la relación entre las dos variables, asignando el mismo peso para cada una, de modo que el índice de calidad ambiental se deﬁne como: Calidad Ambiental = Arbolado urbano + Áreas de parque 2 Densidad de arbolado urbano. La densidad de arbolado urbano considera el número de árboles por hectárea. Arbolado urbano = Número de árboles Área total de la centralidad (ha) Proporción de área de parque. La proporción de área de parque se considera tomando como fuente el PDU de Huánuco (2021). Se calcula la proporción de área de parque con respecto al área total del terreno. Se deﬁne como: 2 Área total de parque = Área total de parque (m ) 2 Área total del centro urbano (m ) Variable de densidad Cada variable posee dos índices, excepto la entropía. Se detalla en la Tabla 1. Tabla 1. Variables e Índices Variable Variable de calidad ambiental. Índice Calidad ambiental Arbolado urbano Proporción de área de parque Densidad Densidad de población Índice construcción Confort peatonal Sección peatonal Ratio entre la anchura y altura Entropía Usos de suelo Esta categoría agrupa dos indicadores: la densidad y el índice de construcción. La densidad para este caso se calcula como la relación entre los dos índices, asignando el mismo peso para cada una, de modo que el índice de densidad se deﬁne como: Densidad = Densidad poblacional + Índice de construcción 2 Densidad poblacional. La densidad poblacional se considera tomando como fuente el INEI (2017). Se calcula por habitantes por hectárea. Se deﬁne como: 91 ARQUITEK \| N°21 \| Enero - Junio 2022 \| Edición online ISSN-2617-0892 \| DOI: https://doi.org/10.47796/ra.2022i21 Densidad poblacional = Intensidad Área total del centro urbano (ha) precisa como: Ra o entre la anchura y la altura= W/H Índice de construcción. Donde: U lizando el análisis espacial, se calcula el área construida como: W= Sección de calle H= Altura de los ediﬁcios Área construida = C * (NP + S +SS) Variable de entropía. Donde: C: Área del terreno ocupado por la construcción NP: Número de pisos S: Sótano o SS: Semisótanos En esta categoría, el indicador es la mezcla de uso de suelo. Se calcula en relación con la siguiente fórmula: Mezcla de usos = Conseguidos del área construida, se calcula el índice de construcción como el cociente de área construida sobre su área del barrio. Se precisa como: Índice de construcción = Área de construida 2 Área total del centro urbano (m ) Variable de confort peatonal. Esta variable agrupa dos indicadores: la sección peatonal y el ra o entre la anchura y la altura. El confort para este caso se calcula como la relación entre los dos indicadores, asignando el mismo peso para cada una. De modo que el índice de confort se deﬁne como: Confort = Sección peatonal + ratio entre anchura y altura peatonal 2 Sección peatonal. La sección peatonal es una medida simple de la anchura que posee la plataforma peatonal como: Sección peatonal = Anchura de la plataforma peatonal Ra o entre la anchura y la altura. La ra o entre la anchura y la altura considera los datos como la anchura de la sección de la calle y la altura de los ediﬁcios. Se 92 -∑k+pk*In(pk) Ink Donde: k: corresponde a la categoría del uso del suelo. p: corresponde al porcentaje de la superﬁcie de cada uso especíﬁco del suelo. n: corresponde al número de categorías del uso de suelo. RESULTADOS El área de estudio está ubicada en la provincia de Huánuco, departamento de Huánuco del Perú. De los componentes considerados para diferenciar los distritos seleccionados, se consideró las redes de conexión, densidad de población, composición del uso de suelo, densidad de construcción y dinámicas sociales y económicas. Esta es la base para la elección de las centralidades urbanas. Los lugares de estudio están en los distritos de Huánuco, Amarilis y Pillco Marca. Cada centralidad ene caracterís cas y limitaciones urbanas para el futuro desarrollo de la ciudad. Los resultados se obtuvieron mediante las medidas del entorno construido que permi eron conocerlo. A con nuación, se detalla el cálculo de cada variable. RENATO EDU BARZOLA GOMEZ Figura 2. Centralidad de Huánuco, Amarilis y Pillco Marca Tabla 2. Superﬁcies de los usos en las centralidades Uso del distrito Comercio Educación Recreación Pública Salud Usos especiales Vivienda Vivienda Comercio Huánuco 241934.10 57947.10 23943.52 21089.38 66579.42 25118.69 74564.75 Amarilis 17359.51 4129.99 21410.01 319.99 5821.08 74941.53 36947.79 Pilco Marca 11828.37 21410.01 13707.20 988.83 1432.79 119679.41 102899.31 93 ARQUITEK \| N°21 \| Enero - Junio 2022 \| Edición online ISSN-2617-0892 \| DOI: https://doi.org/10.47796/ra.2022i21 Cálculo de la calidad ambiental. En la Tabla 3, se muestra el índice de arbolado y área de parque en las centralidades estudiadas. Tabla 3. Índice de arbolado y área de parque Centralidad Índice Arbolado Área de Parque Índice Huánuco 1.04 0.038 0.54 Amarilis 1.87 0.095 0.98 Pilco Marca 1.31 0.037 0.67 Con respecto a la variable índice de arbolado, nos permite observar que en Huánuco con 1.04 ene el valor más bajo que muestra superﬁcies bajas de arbolado, y el valor más alto 1.87 lo ene Amarilis (Tabla 3). Del índice, podemos deducir que las calles son poco atrac vas porque hay poca sombra, a excepción del malecón en Huánuco donde la densidad de arbolado es alta. En relación a las áreas de parque, se puede iden ﬁcar que la centralidad urbana de Amarilis 0.095 es la más alta. Se obtuvo como resultado que el índice de calidad ambiental es favorable para el distrito de Amarilis 0.98 donde existe un po de vivienda residencial y áreas urbanas con caracterís cas de su entorno urbano adecuado. Cálculo de la densidad. Tabla 4. Densidad de población y índice de construcción Centralidad Densidad de población Índice de construcción Índice Huánuco 0.28 1.59 0.93 Amarilis 0.55 1.20 0.87 En la Tabla 4, se muestra la densidad poblacional hallada mediante una escalada de variable Se obtuvo un valor más bajo con menor densidad en Pillco Marca de 0.17, en contraste, el valor más alto de Amarilis 0.55 representa mayor interacción entre densidad poblacional. El índice de construcción de mayor valor es 94 Huánuco 1.59, donde existen múl ples ediﬁcaciones republicanas, la de menor valor es Pillco Marca 1.20. En los tres distritos se iden ﬁca una altura de dos pisos mayoritariamente. Se obtuvo como resultado que el índice de densidad reﬂeja que la centralidad de Huánuco de 0.93, por ser el centro más comercial de la provincia, posee un mayor valor. Cálculo de confort peatonal. Tabla 5. Sección peatonal y ra o entre anchura y altura Centralidad Sección peatonal Ratio Índice Huánuco 1.04 1.77 1.41 Amarilis 1.14 2.20 1.67 Pilco Marca 0.99 1.74 1.37 La variable sección peatonal es de mucha importancia. Los valores encontrados son muy bajos en la mayoría de las calles. En este caso, el índice de sección peatonal presenta valores bajos en Pillco Marca 0.99 donde se observa calles sin pavimento y deterioradas. Amarilis 1.14 es la de mayor valor, pero en términos técnicos aún es baja. El indicador de ra o entre anchura y altura presenta calles en la que la altura de ediﬁcación es baja en relación con la sección de la calle. Así, se observa Pillco Marca 1.74 con el valor más bajo de este indicador, debido a la existencia de casas en adobe; en contraste con Amarilis 2.20 donde existen ediﬁcaciones de tres pisos. Se obtuvo como resultado que el índice de confort peatonal es favorable para el distrito de Amarilis con 1.67 (Tabla 5). Cálculo entropía. Tabla 6. Extensión de los usos mixtos del suelo en las tres centralidades Distrito Huánuco Índice 0.0972 Amarilis Pilco Marca 0.0725 0.0541 RENATO EDU BARZOLA GOMEZ En el cuadro, se puede observar que el índice de entropía de la medida establecida va de 0 a 1. El valor 0 representa un uso del suelo perfectamente homogéneo y el valor 1 perfecto heterogéneo. El índice de entropía alto representa una alta variedad de uso de suelo. Las caracterís cas de los dis ntos pos de uso de suelo y sus cambios son los componentes que aportan este estudio. Para el cálculo del índice de caminabilidad, se analizó la interacción de las cuatro variables. Este índice se deﬁne como el promedio ponderado de las categorías de calidad ambiental, densidad, confort peatonal y entropía, como se puede visualizar en la siguiente ﬁgura, donde se muestran los siguientes resultados. En la Figura 3, se iden ﬁcan los valores calculados de los índices de caminabilidad de las tres centralidades. Figura 3. Índice de caminabilidad en las tres centralidades 1 0.8 0.89 0.73 0.71 0.6 0.4 0.2 0 Huánuco Amarilis Pilco Marca Con respecto al índice de caminabilidad, se puede observar la centralidad de Amarilis de 0.89, con el valor más alto; una caracterís ca importante de esta centralidad es que se desarrolló en base a la planiﬁcación urbana y ene un uso de suelo de vivienda en alta densidad. Por el contrario, la centralidad del distrito de Pillco Marca obtuvo el índice más bajo de 0.71, debido a la construcción, autoconstrucción y ocupación del suelo agrícola sin planiﬁcación. Se concluye que los espacios p eato n a les s o n má s a p ro p ia d o s en la centralidad urbana del distrito de Amarilis y son más adecuados para la caminabilidad. DISCUSIÓN De acuerdo con los resultados de las centralidades analizadas, lo hallado nos permite entender que el entorno construido de estos presenta dis ntas deﬁciencias y calidades del entorno construido urbano, por lo que requiere de una intervención basada en la aplicación de los índices para la caminabilidad por parte del gobierno local. De acuerdo con los resultados obtenidos, la centralidad del distrito de Huánuco presenta índices bajos en la variable de calidad ambiental de 0.54; las caracterís cas del arbolado y áreas verdes son pocas, debido que existen solo dos áreas de recreación en esta centralidad. La centralidad de Amarilis posee un valor alto en los índices de calidad ambiental de 0.98, densidad de 0.87 y confort peatonal de 1.67; las caracterís cas urbanas de esta centralidad pueden incluir la caminabilidad en s u s ento rn o s y es p a c io s u rb a n o s . L a s caracterís cas urbanas de la centralidad de Pillco Marca presenta más índices bajos, producto de ser un distrito nuevo; la inversión en infraestructura para caminar dentro de este distrito es muy baja: varias manzanas no poseen veredas y algunas fueron autoconstruidas; la ausencia del gobierno local es más evidente en Pillco Marca. La propuesta metodológica es fácil de aplicar esencialmente porque no es necesario u lizar ningún so ware. Tampoco se u lizaron técnicas estadís cas complejas para hallar los resultados, por lo que facilita su uso en los instrumentos técnicos norma vos de planiﬁcación urbana. Además, los discursos sobre la caminabilidad, donde el peatón debe tener un espacio saludable y generar la 95 ARQUITEK \| N°21 \| Enero - Junio 2022 \| Edición online ISSN-2617-0892 \| DOI: https://doi.org/10.47796/ra.2022i21 integración social, toma un alto valor en el lugar de estudio, debido a que su crecimiento urbano de dos centralidades presenta bajos valores y solo uno puede inmediatamente dar la importancia del peatón en su entorno construido. Por lo tanto, entendemos que todos caminamos, pero actualmente no existen p o l í ca s u r b a n a s c l a ra s a favo r d e l a caminabilidad en áreas de alta demanda. A diferencia de lo observado en otras inves gaciones sobre la caminabilidad donde se iden ﬁca los índices, en el caso analizado, la escala barrial y las caracterís cas contextuales sico-espaciales, con la comparación de centralidades dentro de un área urbana consolidada, permite comprender cómo se maneja la movilidad urbana en los distritos. El estudio de la caminabilidad en las centralidades en escala micro profundizó en aspectos del entorno urbano y espacial. Cada área de centralidad urbana no considera caracterís cas del entorno construido fuera de esta. A par r de ello, es importante ampliar el radio de acción. Cabe señalar también que no se consideró variables como contaminación acús ca y ambiental, densidad de empleo, áreas iluminadas y otros en esta inves gación, las cuales permi rían realizar futuras inves gaciones considerando esos índices. CONCLUSIONES El análisis realizado nos permi ó entender el entorno construido de las centralidades urbanas, las cuales resultan importantes para comprender la ciudad y las formas de desplazamiento a pie. En los tres distritos de estudio, existen limitaciones para la caminabilidad donde el entorno construido está ausente, deteriorado y en malas condiciones, sobre todo en los distritos de Huánuco y Pillco Marca. El análisis de los índices permi ó vislumbrar caracterís cas relevantes de las 96 centralidades estudiadas. Adicionalmente, puede aﬁrmarse que la centralidad urbana de Amarilis resultó ser la que presenta mejores condiciones para la caminabilidad. La propuesta de instrumentos de planeamiento urbano requiere incluir y fortalecer los criterios de caminabilidad para mejorar las condiciones de la movilidad urbana. Este trabajo inicia una importante ver ente de inves gaciones urbanas en la ciudad de Huánuco. Este estudio ha pretendido ser un aporte para los estudios urbanos, por ende, se pone a disposición información relevante para los gestores urbanos para poder trasladarlas a polí cas locales urbanas. Finalmente, se puede aﬁrmar, que la ciudad puede modiﬁcarse, transformarse y diseñarse para ser caminable, y la inves gación aporta con los índices que deben ser considerados en toda planiﬁcación e intervención urbana. REFERENCIAS BIBLIOGRÁFICAS Bradshaw, C. (2013). A ra ng system for neighbourhood walkability. In Boulder CO. Anais, 14th Interna onal Pedestrian Conference. Forsyth A. (2015). ¿Qué es un lugar transitable? El debate sobre la transitabilidad en el diseño urbano. Int Urbano Des., 20(4), 274–292. doi: 10.1057/udi.2015.22. Gerlinde Grasser, Delﬁen van Dyck, Sylvia Titze, Willibald J. Strongegger (2017). Una perspec va europea sobre la caminabilidad basada en SIG y los modos ac vos de transporte. European Journal of Public Health, 27(1), 145–151. h ps://doi.org/10.1093/eurpub/ckw118 Leslie, E., Bu erworth, I. y Edwards, M. (2006, octubre). Medición de la transitabilidad de las comunidades locales u lizando datos de RENATO EDU BARZOLA GOMEZ Sistemas de Información Geográﬁca. En Walk21VII, “Los Próximos Pasos”, 7ª Conferencia Internacional sobre Caminar y Comunidades H a b i t a b l e s . M e l b o u r n e . h p : / / w w w. caminar21. com/papers/m (Vol. 6). Raﬁemanzelat, R., Emadi, M. y Kamali, A. (2016). City Sustainability: the inﬂuence of walkability on built environments. In 3rd Conference on Sustainable Urban Mobility, Greese, pp. 107-104. Machín, H. y Ghidini, R. (2013). Buenas condiciones para el peatón. Recogida de información técnica. Revista dos Transportes Públicos-ANTP, (134), 81-102. Dos Santos, E. (2003). Calçadas seguras: responsabilidade de todos. In 1º Seminário Paranaense De Calçadas. Anais. Marquet, O. y Miralles-Guasch, C. (2015). La ciudad caminable y la importancia de los entornos de proximidad para la movilidad co diana de Barcelona. Ci es, 42, 258 – 266.10.1016/j.ci es.2014.10.012 Medina-Ruiz, M. (2020). La caminabilidad como estrategia proyectual para las redes peatonales del borde urbano. Barrio Sierra Morena. Usme. Revista de Arquitectura, 22(2), 78-93. Moura, F., Cambra, P. y Gonçalves, AB (2017). Medición de la caminabilidad para dis ntos grupos de peatones con un método de evaluación par cipa vo: un estudio de caso en Lisboa. 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https://openalex.org/W3037647629	https://www.studiapsychologica.com/uploads/Colombo_SP_2_vol.62_2020_pp.123-137.pdf	English	null	Personal Impulsivity Mediates the Effects of Neuromodulation in Economic Intertemporal Choices: A Pilot Study	Studia psychologica	2,020	cc-by	9,202	Personal Impulsivity Mediates the Effects of Neuromodulation in Economic Intertemporal Choices: A Pilot Study Barbara Colombo1, Paola Iannello2, Gugliemo Puglisi3, Alessandro Antonietti2 1 Department of Psychology, Neuroscience Lab, Champlain College, 163 South Willard Street, Burlington, 05401 VT, USA 2 Department of Psychology, Università Cattolica del Sacro Cuore, Largo Gemelli 1, 20122 Milano, Italy 3 Laboratory of Motor Control, Department of Medical Biotechnology and Translational Medicine, Università degli Studi di Milano and Humanitas Research Hospital, IRCCS, Via Viotti, 3/5 - 20133, Milano, Italy Barbara Colombo1, Paola Iannello2, Gugliemo Puglisi3, Alessandro Antonietti2 1 Department of Psychology, Neuroscience Lab, Champlain College, 163 South Willard Street, Burlington, 05401 VT, USA 2 Department of Psychology, Università Cattolica del Sacro Cuore, Largo Gemelli 1, 20122 Milano, Italy 3 Laboratory of Motor Control, Department of Medical Biotechnology and Translational Medicine, Università degli Studi di Milano and Humanitas Research Hospital, IRCCS, Via Viotti, 3/5 - 20133, Milano, Italy The involvement of the prefrontal cortex in intertemporal choices has been long recognized. Using neurostimulation techniques, recent studies have indicated that the left dorsolateral prefrontal cortex (DLPFC) influences performance on intertemporal choice tasks. The present pilot study is aimed to explore further the DLPFC’s role in intertemporal choices by assessing the influence of individual levels of impulsivity on modulating the stimulation’s effects. Thirteen subjects participated in a within-subjects experiment. During the three sessions, participants received 20 minutes of transcranial direct current stimulation (tDCS; either sham, anodal, or cathodal) and were administered the Intertemporal Choice Task. Then, they completed the Barratt Impulsivity Scale and the Dickman Impulsivity Inventory. Using a re- peated-measure generalized linear model, we explored the effects of stimulation on intertemporal choice (either immediate or delayed reward) on impulsive responses, defined as quick answers. The individual level of impulsivity was included in the model as a covariate. According to the results, participants made a greater number of impulsive choices favoring immediate rewards after cathodal stimulation of the left DLPFC. Additionally, a moderating role of individual impulsivity emerged. This study provides support for the involvement of the left DLPFC in intertemporal choices. We contend that the role of individual differ- ences should be further explored to obtain a better understanding of intertemporal choice behavior. Key words: intertemporal choice, neuromodulation, transcranial direct current stimulation, individual differ- ences, impulsivity, delay discounting 2017; Berns, Laibson, & Loewenstein, 2007; Frederick, Loewenstein, & O’Donoghue, 2002), have been studied from various perspectives within the fields of economics, psychology, and neuroscience. Studia Psychologica, Vol. 62, No. 2, 2020, 123-137 https://doi.org/10.31577/sp.2020.02.795 Studia Psychologica, Vol. 62, No. 2, 2020, 123-137 https://doi.org/10.31577/sp.2020.02.795 Correspondence concerning this article should be addressed to Barbara Colombo, Neuroscience Lab, Champlain College, 163 South Willard Street, Burlington, 05401 VT, USA. E-mail: bcolombo@champlain.edu ORCID https://orcid.org/0000-0002-4095-9633 Personal Impulsivity Mediates the Effects of Neuromodulation in Economic Intertemporal Choices: A Pilot Study Evidence from the research lit- erature suggests that when asked to choose between an immediate and a delayed reward (and not only when economic behavior is tar- geted; see Eikemo & Leknes, 2019), people Intertemporal Choices in Neuroscience Re- search One system is linked to limbic and paralimbic responses and values immediate rewards, whereas the other one relies on frontoparietal areas and evaluates deferred gratifications. This picture is consistent with the data derived from studying both primates and human patients with lesioned DLPFC and LOFC, as they exhibited lower judgment and temporal planning skills and more frequent impulsive behaviors (Floden, Alexander, Kubu, Katz, & Stuss, 2008; Kalenscher, 2006; Noonan et al., 2010; Wallis, 2012). It also supports the idea that the choice of immediate rewards is associated with and can be interpreted as impulsive behavior. Neuroeconomic studies based on noncli- nical samples often report an online record- ing of cortical activity while people are making decisions. The main limitation of this approach is that the correlational nature of the findings does not allow researchers to prove causal hypotheses regarding the actual relationships between areas and functions (Stewart & Walsh, 2006). In recent years, neurostimu- lation techniques have provided a method to overcome this limitation. While brain stimula- tion is applied, a target area is either inhibited or activated to explore its specific influence on intertemporal choices. More specifically, transcranial direct current stimulation (tDCS) produces a continuous low-intensity electric current on the scalp to increase or decrease cortical excitability by depolarizing or hyperpo- larizing, respectively, cortical neurons at a sub- threshold level (Paulus, Peterchev, & Ridding, 2013). Overall, anodal stimulation, which in- creases the spontaneous firing frequency of cortical neurons, has been reported to enhance the performance of cognitive tasks (Fregni et al., 2005; Javadi, Cheng, & Walsh, 2012; Metuki, Sela, & Lavidor, 2012; Straube, Wolk, & Chatterjee, 2011; Wirth et al., 2011), whereas cathodal stimulation causes an inhibition of cognitive processes (Boehringeretal, 2013; Pope & Miall, 2012; Straubeetal, 2011). There- Within the field of neuroscience, functional magnetic resonance imaging (fMRI) studies have shown that several interacting neural sys- tems are involved in the process of making intertemporal choices (McClure, Laibson, Loewenstein, & Cohen, 2004; McClure, Marzilli Ericson, Laibson, Loewenstein, & Cohen, 2007; Tanaka et al., 2004; Xu, Liang, Wang, Li, & Jiang, 2009; Wittmann, Leland, & Paulus, 2007). McClure and colleagues (2004) ob- served that the possibility of receiving imme- diate rewards instead of deferred alternatives elicits greater activation of some paralimbic structures, such as the ventral striatum, orbital frontal cortex, and medial prefrontal cortex. Intertemporal Choices in Neuroscience Re- search Intertemporal choices, that is decisions involv- ing consequences at different points in time (for a review, see Becker, Walker, & McCord, Received April 17, 2019 Received April 17, 2019 Studia Psychologica, Vol. 62, No. 2, 2020, 123-137 124 tend to undervalue the postponed reward (Lempert & Phelps, 2016; Loewenstein & Prelec, 1992; Myerson & Green, 1995; Raineri & Rachlin, 1993). The delayed reward is gen- erally preferred when it is substantially higher than the proximal one. Choosing the second alternative depends on several factors: the length of the delay, the amount of money, and the devaluation rate of the postponed reward. This last factor, known as the temporal dis- counting effect, has become the principal framework used in psychology to explain how people make intertemporal choices (e.g., Benzion, Rapoport, & Yagil, 1989; Bickel, Koffarnus, Moody, & Wilson, 2014; Charlton et al., 2013; Green, Fristoe, & Myerson, 1994; Story, Vlaev, Seymour, Darzi, & Dolan, 2014). This paradigm assumes that delayed rewards are discounted more as the length of delay in- creases, and the rate at which delayed conse- quences lose value is referred to as the dis- count rate. Discount rates are sometimes used as a measure of impatience (Marzilli Ericson, White, Laibson, & Cohen, 2015). tend to undervalue the postponed reward (Lempert & Phelps, 2016; Loewenstein & Prelec, 1992; Myerson & Green, 1995; Raineri & Rachlin, 1993). The delayed reward is gen- erally preferred when it is substantially higher than the proximal one. Choosing the second alternative depends on several factors: the length of the delay, the amount of money, and the devaluation rate of the postponed reward. This last factor, known as the temporal dis- counting effect, has become the principal framework used in psychology to explain how people make intertemporal choices (e.g., Benzion, Rapoport, & Yagil, 1989; Bickel, Koffarnus, Moody, & Wilson, 2014; Charlton et al., 2013; Green, Fristoe, & Myerson, 1994; Story, Vlaev, Seymour, Darzi, & Dolan, 2014). This paradigm assumes that delayed rewards are discounted more as the length of delay in- creases, and the rate at which delayed conse- quences lose value is referred to as the dis- count rate. Discount rates are sometimes used as a measure of impatience (Marzilli Ericson, White, Laibson, & Cohen, 2015). neural systems (De Martino, Kumran, Seymour, & Dolan, 2006; McClure et al., 2004; Sanfey, Rilling, Aronson, Nystrom, & Cohen, 2003; Sanfey, Loewenstein, McClure, & Cohen, 2006). Intertemporal Choices in Neuroscience Re- search Hecht, Walsh, and Lavidor (2013) found that individuals were more likely to choose smaller immediate gains instead of a larger delayed benefit when the left DLPFC was stimulated and the right DLPFC was in- hibited through tDCS, when compared to the sham stimulation. In a recent study, He and colleagues (2016) used anodal high-definition tDCS (HD-tDCS) to investigate the causal role of the left DLPFC in performing the intertem- poral choice (ITC) task and showed that HD- tDCS over the left DLPFC lowered the delay- discounting rate (k). Intertemporal Choices in Neuroscience Re- search Frontoparietal areas, such as the dorsolateral prefrontal cortex (DLPFC), lateral orbitofrontal cortex (LOFC), and posterior parietal cortex (PPC), are activated by greater amounts of delayed rewards. These areas also play a key role in tasks requiring executive control (Miller & Cohen, 2001; Platt & Glimcher, 1999). These findings support the hypothesis that the outcome of an intertemporal choice results from an interaction between two competing Studia Psychologica, Vol. 62, No. 2, 2020, 123-137 125 fore, when a variation in choices is observed, one can presume that the stimulated area is playing a major role within the decision-mak- ing process (Stewart & Walsh, 2006). This hy- pothesis has been confirmed by several stud- ies that used brain stimulation to moderate participants’ behavior during decision-making tasks entailing immediate or impulsive re- sponses (Colombo, Balzarotti, & Mazzuchelli, 2016; Iannello, Colombo, & Antonietti, 2014, Oldrati, Patricelli, Colombo, & Antonietti, 2016). has been argued that several aspects of ex- ecutive functioning (for example, working memory, inhibition, and task switching) may provide the basis for successful self-regu- lation (Blair & Ursache, 2011; Hofmann, Schmeichel, & Baddeley, 2012). Following the implications derived from these studies, we can also argue that self-regulation, which in- volves the inhibition of impulsive tendencies (Baumeister, Bratslavsky, Muraven, & Tice, 1998), may be responsible for the preference for greater distal (i.e., temporally delayed) re- wards instead of smaller proximal (i.e., imme- diate) ones (Fujita & Carnevale, 2012). This pattern provides additional support for the claim that choosing immediate rewards can be interpreted as impulsive behavior because it is associated with a lack of inhibition of im- pulsive behavior. A few studies have used this methodology to examine intertemporal choices. For example, Figner and colleagues (2010) showed that repetitive transcranial magnetic stimulation (rTMS) for 15 minutes at 1 Hz on the left PFC led to a greater frequency of preferences for immediate rewards. These data have been interpreted as being driven by a lower level of self-regulation, i.e., being unable to resist the temptation of an immediate reward. Cho and colleagues (2010) reported that the inhibition of the right DLPFC using continuous theta burst stimulation (cTBS, an rTMS protocol wherein pulses are applied in bursts of three, deliv- ered at a frequency of 50 Hz and with an interburst interval of 200 ms) enhanced im- pulsive responses. Individual Differences in the Level of Impul- sivity Impulsivity has been considered a multifacto- rial construct and literature provides evidence of multiple varieties of impulsivity (Avila et al., 2004; Dickman, 1990; Evenden, 1999), these varieties being united by a suboptimal way to handle time (Kim & Lee, 2011). Impulsivity has been considered a multifacto- rial construct and literature provides evidence of multiple varieties of impulsivity (Avila et al., 2004; Dickman, 1990; Evenden, 1999), these varieties being united by a suboptimal way to handle time (Kim & Lee, 2011). Impulsive decision-making can be viewed as a failure to appropriately consider certain types of temporal factors. Impulsivity may refer to the tendency to weigh immediate outcomes strongly and to discount the value of delayed rewards precipitously (Frederick, Loewenstein, & O’Donoghue, 2002; Kalenscher & Pennartz, 2008). In addition to temporal discounting, impulsivity has been also defined as a lack of inhibitory control, which implies the inability to suppress an action and thus results in a rapid and sometimes premature response (Kim & Lee, 2011). Lack of inhibition of impulsive be- havior is linked to what has historically been called “reflection impulsivity,” that is, the ten- dency toward rapid action before sufficient in- formation is gathered (Kagan, Rosman, Day, Albert, & Phillips, 1964). Framing impulsivity according to this perspective leads to assess- ing impulsivity levels by comparing reaction These results can be explained by referring to research that supports the key role of the DLPFC in guiding executive processes (for a review, see Tanji & Hoshi, 2008). Many neuro- physiological studies on both animals and humans have confirmed that the bilateral DLPFC can be viewed as the neural correlate of the central executive system (Osaka et al., 2007). This information is relevant because it Studia Psychologica, Vol. 62, No. 2, 2020, 123-137 126 times; individuals who are more impulsive will tend to choose an answer quickly and without thinking (Moeller, Barratt, Dougherty, Schmitz, & Swann, 2001). If considering impulsivity as resulting from failures in successfully handling temporal factors in decision making, it implies that individuals may vary substantially in their strategies to deal with time during decision making. specific role of individual impulsivity in modu- lating the tDCS effect. The data from this initial study enable a preliminary assessment of our hypotheses and procedure; however, the re- sults should be confirmed by a larger study. Individual Differences in the Level of Impul- sivity To date, within economic temporal discount- ing literature, impulsivity has been measured as the tendency to discount the value of delayed rewards prematurely (Frederick, Loewenstein, & O’Donoghue, 2002; Kalenscher & Pennartz, 2008). The present study shifts the focus to a slightly different facet of impulsivity, thus intro- ducing in this field of research a measure of speed responses, which correspond to the (in)ability to suppress a premature response. Literature suggests that there are individual differences in self-control (e.g., Paschke et al., 2016) and this is consistent, indicating the appropriateness of assessing self-con- trol as a trait. For example, Tangney, Boone, and Baumeister (2018) developed a scale to measure trait self-control based on the sur- vey of self-control problems and failures. Thus, we can hence assume that individuals have different levels of impulsivity depending on their self-regulation skills. This inference has been also confirmed by findings of a re- lationship between higher impulsivity and lower self-control, which leads to addiction related behavior (Dahlen, Martin, Ragan, & Kuhlman, 2005; Hair & Hampson, 2006). Moreover, impulsive individuals tend to un- dervalue delayed rewards more heavily (Crean, de Wit, & Richards, 2000; Hinson, Jameson, & Whitney, 2003; Iannello, Biassoni, Nelli, Zugno, & Colombo, 2015; Wittman & Paulus, 2008). ( ) y pp p p After reviewing the results reported in the lit- erature, we hypothesized that the subjects would be more impulsive – less able to con- trol their impulsive behavior – and would have faster reaction times (as discussed above) after cathodal stimulation and less impulsive after anodal stimulation, if compared to the sham condition. Given the fact that, to the best of our knowledge, no previous study has ex- plored the moderating role of personal impul- sivity in decision-making tasks, this part of the study is exploratory. However, as impulsivity is often associated with intolerance to delay and sensitivity to immediate rewards (e.g., Robbins, 2007; Whiteside & Lynam, 2003), we can hypothesize that a higher level of individual impulsivity should enhance the effect of cathodal stimulation and decrease the effect of anodal stimulation. Present Study Given the promising results of brain stimula- tion of the left DLPFC and the interesting in- sights gained regarding the relationship be- tween the DLPFC and impulsivity, the present study aimed to further explore the DLPFC’s role in influencing intertemporal choices and the function of individual levels of impulsivity in modulating the effects of stimulation. We aimed to a) investigate whether people with low impulsivity levels respond to tDCS differ- ently from those with high impulsivity, b) ex- plore the DLPFC’s role in influencing intertem- poral choices by using tDCS and comparing the effects of anodal and cathodal stimulations to a control (sham) condition, and c) study the Participants Thirteen participants (6 men and 7 women, mean age = 24.3; SD = 5.9) joined the study. A Power analysis aimed at assessing the achieved power (using GPower software), considering the sample size, the within sub- ject design and 24 measurements for the 3 conditions returned a power (1 – β) of .97. The power analysis calculation dealt with the main effects and was based on expected effect size of Cohen’s f = .25. Studia Psychologica, Vol. 62, No. 2, 2020, 123-137 127 The participants included undergraduate and graduate students recruited through ad- vertising on university message boards. All participants were right-handed and had no metallic implants. Individuals affected by at- tention deficit hyperactivity disorder (ADHD) or neuropsychiatric disorders as well as smokers, alcoholics, and people with sub- stance abuse – all of whom usually display atypical preferences for immediate gratifica- tion (Wittman & Paulus, 2007) – were ex- cluded. Tools Transcranial Direct Current Stimulation. tDCS was delivered through two saline-soaked sponge electrodes (25 cm2) using a constant- current stimulator (HDC Series by Newronika S.r.l). The active electrode was placed over the F3 position of the EEG 10-20 System (corre- sponding to the left DLPFC), and the control electrode was placed over the right triceps muscle. As part of the experiment, participants un- derwent three different stimulation conditions – anodal, cathodal, and sham – on three dif- ferent days (once a week) in a counterbalanced manner. To prevent subjects from differentiat- ing between real and sham stimulations, the current was slowly increased to 1.5 mA over a period of 30 seconds. For real stimulation (i.e., either cathodal or anodal), after 30 seconds, the current was maintained at 1.5 mA for 20 minutes, whereas during the sham stimula- tion, the current was slowly decreased back to zero over 30 seconds. Procedure The IRB of the Catholic University approved the research project. Each participant read and signed an informed consent form prior to the experiment. Information concerning the reward system for participants was included in the informed consent form. The design procedure, a within-subjects design, consisted of sessions lasting 40 min- utes, which occurred once a week for three consecutive weeks. Intertemporal Choice Task (ITC). We used the task proposed by McClure et al. (2004). Participants were presented with a series of 24 binary choices. Each pair consisted of two alternative monetary outcomes, which differed in both specific amounts (5–80 €) and times of availability (ranging from the day of the ex- perimental session to six weeks later). Spe- cifically, in each trial, participants had to choose between a smaller amount of money that would be immediately available and a larger amount that would be received after some time delay. The earlier rewards could be 5 € or 40 €, and the latter could exceed the former by 15%, 25%, 30%, or 50%. The second reward could be delayed by two, four, or six weeks. Each session had the same structure, which is as follows: 1. Twenty minutes of tDCS stimulation in three conditions and in counter-balanced se- quence for each of the three sessions (i.e., sham, anodal, cathodal); 2. ITC task; ) 2. ITC task; 2. ITC task; 3. The third session had two additional phases: a. Application of the impulsivity inventories; b. Reward (one reward among the ones selected by the participants during the three section was randomly sorted and awarded to the participant) Self-report impulsivity inventories were ad- ministered during the last session – rather than at the beginning of the entire experiment – to avoid the possibility that the participants’ ex- plicit and conscious answers about their own level of impulsivity might affect their perfor- mance on the subsequent behavioral task (functioning as a prime) by causing them to reflect on their impulsivity. The task was administrated using a laptop computer, and the two alternatives were pre- sented on the two sides of the screen. Partici- pants indicated their responses by clicking the mouse to select their chosen reward, and there was no time limit. In accordance with our defi- nition of impulsivity as discussed in the Intro- Studia Psychologica, Vol. 62, No. 2, 2020, 123-137 128 duction, the response time (RT) was recorded. Statistical Analyses All statistical analyses were performed using IBM SPSS software (IBM, Armonk, NY, USA, version 20). The level of significance was set at α = 0.05. We used the Kolmogorov–Smirnov test to check for the normal distribution of data. A repeated-measures generalized linear model (GLM) was used to explore the within- subjects effects of the type of stimulation on the different dependent variables (see below). Follow-up regressions were used to explore the specific role of individual differences. Barratt Impulsivity Scale. At the end of the last session, the Italian version of the 30-item Barratt Impulsivity Scale Version 11 (BIS-11; Fossati, Di Ceglie, Acquarini, & Barratt, 2001) was administered. Related to impulsivity, it measures six primary factors (attention, mo- tor, self-control, cognitive complexity, persever- ance, and cognitive instability) as well as three secondary factors (attentional, motor, and non- planning). All items are rated on a 4-point scale (1 = rarely or never; 2 = occasionally; 3 = often; 4 = almost always or always). The BIS-11 has demonstrated an acceptable level of internal consistency (Cronbach’s alpha = .79). We computed scores for each of the three sub- scales assessing secondary factors (at- tentional impulsivity: Cronbach’s alpha = .73; motor impulsivity: Cronbach’s alpha = .78; non- planning impulsivity: Cronbach’s alpha = .70). Dickman Impulsivity Inventory. Individual impulsivity was also assessed by using the short version of the Dickman Impulsivity Inven- tory (DII-S; Claes, Vertommen, & Braspenning, 2000; Dickman, 1990). We translated the origi- nal scale into Italian using a standardized pro- cedure including two independent transla- tions, followed by determination of a consen- sus translation by an expert panel. The trans- lated scale demonstrated good internal con- Barratt Impulsivity Scale. At the end of the last session, the Italian version of the 30-item Barratt Impulsivity Scale Version 11 (BIS-11; Fossati, Di Ceglie, Acquarini, & Barratt, 2001) was administered. Related to impulsivity, it measures six primary factors (attention, mo- tor, self-control, cognitive complexity, persever- ance, and cognitive instability) as well as three secondary factors (attentional, motor, and non- planning). All items are rated on a 4-point scale (1 = rarely or never; 2 = occasionally; 3 = often; 4 = almost always or always). The BIS-11 has demonstrated an acceptable level of internal consistency (Cronbach’s alpha = .79). Statistical Analyses We computed scores for each of the three sub- scales assessing secondary factors (at- tentional impulsivity: Cronbach’s alpha = .73; motor impulsivity: Cronbach’s alpha = .78; non- planning impulsivity: Cronbach’s alpha = .70). Di k I l i it I t I di id l Procedure The lower the RT, the more impulsive the sub- jects. sistency in the present study (Cronbach’s al- pha = .75). This inventory was designed to assess the personality trait of impulsiveness by using 23 true-false items organized into two subscales: functional impulsivity (the tendency to act with relatively little forethought when do- ing so is optimal; Cronbach’s alpha = .70) and dysfunctional impulsivity (the tendency to act with relatively little forethought when doing so causes problems; Cronbach’s alpha = .71). At the end of the experiment (i.e., the conclu- sion of the third weekly session), participants were given a reward. One of the trials from the three sessions was randomly selected, and the participant received the reward according to the choice he or she had made in that trial. If the subject had chosen the immediate reward, the corresponding amount of money was given before he or she left the session; if the partici- pant had chosen the delayed reward, he or she was asked to come back to the lab two, four, or six weeks later (according to the time interval specified in that delayed option) to re- ceive the corresponding amount of money. Participants were informed about how the re- wards would be assigned when they signed the informed consent form. Main Effect of Stimulation We first tested for possible gender differences. As no significant difference emerged within our sample, we did not add gender as a between- subjects variable in the subsequent analyses. We then ran a repeated measures ANOVA aimed at exploring possible differences among tDCS conditions depending on the type of reward (immediate vs. postponed): results (non significant but coherent with previous lit- erature findings) are reported in the supple- mentary materials. Dickman Impulsivity Inventory. Individual impulsivity was also assessed by using the short version of the Dickman Impulsivity Inven- tory (DII-S; Claes, Vertommen, & Braspenning, 2000; Dickman, 1990). We translated the origi- nal scale into Italian using a standardized pro- cedure including two independent transla- tions, followed by determination of a consen- sus translation by an expert panel. The trans- lated scale demonstrated good internal con- Because we were interested in exploring different impulsive behaviors, as reflected by RTs, we used a repeated-measures GLM to explore the effects of the type of stimulation (independent within-subjects variable) and the kind of reward chosen (immediate versus post- poned reward) on the RT (faster responses were considered as more impulsive) for the different choices (small or big reward – we Studia Psychologica, Vol. 62, No. 2, 2020, 123-137 129 (F1;7 = 1.66; p = .24; η2 = .19) on RT emerged. However, the results highlighted an interac- tion effect of the stimulation, the kind of reward, and functional impulsivity: F2;6 = 8.27; p < .05; η2 = .73. An interaction effect among stimula- tion, kind of reward, and attentional impulsivity emerged: F2;6 = 10.95; p < .01; η2 = .78. A third interaction effect among stimulation, kind of retained this distinction when looking at RTs because we were interested in exploring the differences in behavior depending on the choice). All the BIS-11 and DII-S main subscales were included in the model as covariates. No main effects of the stimulation (F2;14 = 1.243; p = .27; η2 = .17) or the type of reward Figure 1 Response Time depending on tDCS condition, the type of reward and level of impuls ity. Errors bars show Standard Errors. Figure 1 Response Time depending on tDCS condition, the type of reward and level of impuls ity. Errors bars show Standard Errors. Figure 1 Response Time depending on tDCS condition, the type of reward and level of impulsiv- ity. Errors bars show Standard Errors. Studia Psychologica, Vol. 62, No. Note. b represents unstandardized regression weights, SEb corresponds to the standard error for the unstandardized regression weights,  indicates the standardized regression weights. indicates p <.05 Note. b represents unstandardized regression weights, SEb corresponds to the standard error for the unstandardized regression weights indicates the standardized regression weights Main Effect of Stimulation 2, 2020, 123-137 130 Table 1 Follow-up regressions using impulsive responses as the criterion Predictors b SEb  BIS - Motor Impulsivity Anodal Immediate Reward Constant 1.14 8.08 BIS MI .72 .42 .46 R 2 = .46; p = .11 Anodal Postponed Reward Constant -.13.34 9.13 BIS MI 1.28 .47 .64* R 2 = .40; p = .02 Sham Immediate Reward Constant -3.68 8.27 BIS MI .89 .46 .50 R 2 = .25; p = .08 Sham Postponed Reward Constant -8.15 10.72 BIS MI .99 .55 .48 R 2 = .23; p = .09 Cathodal Immediate Reward Constant -.82 7.45 BIS MI .86 .49 .54* R 2 = .54; p = .05 Cathodal Postponed Reward Constant -12.94 9.09 BIS MI 1.31 .47 .65* R 2 = .42; p = .02 DII - Functional Impulsivity b SEb  Anodal Immediate Reward Constant 6.79 4.44 DII – FI 1.96 1.01 .51 R 2 = .26; p = .08 Anodal Postponed Reward Constant -1.53 5.29 DII – FI 3.05 1.20 .61* R 2 = .37; p = .03 Sham Immediate Reward Constant 5.52 5.29 DII - FI 1.89 1.20 .43 R 2 = .18; p = .14 Sham Postponed Reward Constant -2.35 5.44 DII - FI 3.18 1.23 .61* R 2 = .38; p = .02 Cathodal Immediate Reward Constant 6.51 4.37 DII - FI 2.21 .99 .56* R 2 = .31; p = .04 Cathodal Postponed Reward Constant .07 5.51 DII - FI 2.89 1.25 .57* R 2 = .33; p = .04 Note. b represents unstandardized regression weights, SEb corresponds to the standard error for the unstandardized regression weights,  indicates the standardized regression weights. * indicates p <.05 Studia Psychologica, Vol. 62, No. 2, 2020, 123-137 131 left DLPFC in moderating impulsive intertem- poral choice by considering a possible mod- erating effect of individual levels of impulsivity. reward, and motor impulsivity was observed: F2;6 = 5.66; p < .05; η2 = .65. Finally, an interac- tion effect among stimulation, kind of reward, and non-planning impulsivity emerged: F2;6 = 7.07; p < .05; η2 = .70. Findings previously reported in the literature (Hecht, Walsh, & Lavidor, 2013) indicated that a greater number of choices favoring immedi- ate rewards occurred after cathodal stimula- tion of the left DLPFC. Main Effect of Stimulation Our results are in line with these findings, even if we focused on a different facet of impulsivity, as measured through response speed (reaction time). Spe- cifically, even if a main effect of the stimulation did not emerge – possibly because of our small sample size – we noted the participants’ tendency to report more impulsive (faster) re- sponses after cathodal stimulation when pre- sented with immediate rewards. Postponed rewards tended to elicit more impulsive (faster) answers. Whereas anodal stimulation appeared to have levelled the dif- ference between immediate vs. postponed re- wards, after cathodal stimulation individuals reported faster responses when presented with postponed rewards, and the extent of these differences varied depending on the type of impulsivity (see Figure 1). A follow-up regression (Table 1) to explore the specific role of functional impulsivity in modu- lating impulsive responses highlighted a sig- nificant effect after the anodal stimulation when the reward was delayed (b = 3.05; SEb = 1.20; b = .61; p < .05). The same effect emerged in the sham condition for delayed rewards (b = 3.18; SEb = 1.23; b = .61; p < .05). After the cathodal stimulation, functional impulsivity appeared to modulate impulsive answers both when the reward was immediate (b = 2.21; SEb = .99; b = .56; p < .05) and when it was postponed (b = 2.90; SEb = 1.25; b = .57; p < .05). Our study added a further level of complexity by including the role of individual impulsivity in the model. We found significant interaction ef- fects, thereby supporting the notion of a mod- erating role played by individual differences in impulsivity. Especially, we found that motor impulsivity (the tendency to act without think- ing) and functional impulsivity (the tendency to act with little forethought in situations where such behavior is beneficial) moderated the effect of cathodal stimulation (by way of in- creasing its effect), both when the reward was immediate and when it was postponed. The same two types of impulsivity also moderated the effect of anodal stimulation (again increas- ing the effect of stimulation), but only for de- layed rewards. These data suggest that when the DLPFC is inhibited, people become more impulsive, favor immediate rewards, and make faster and less mediated decisions; moreover, this effect is enhanced for individuals who al- ready tend to act without thinking. Main Effect of Stimulation To better understand the role of attentional impulsivity in modulating impulsive re- sponses, we ran a follow-up regression. No significant effect emerged. A follow-up regression to explore the specific role of motor impulsivity in moderating impul- sive responses showed a significant effect af- ter anodal stimulation when the reward was delayed (b = 1.28; SEb = .47; b = .64; p < .01). After cathodal stimulation, motor impulsivity appeared to moderate impulsive answers both when the reward was immediate (b = .86; SEb = .40; b = .54; p = .05) and when it was post- poned (b = 1.31; SEb = .47; b = .65; p < .01). No effect emerged under the sham condition. By contrast, when the activity of the DLPFC was enhanced, people tended to ponder their decision longer and chose delayed rewards more often. The fact that being more impul- sive increased the effect of anodal stimulation for delayed rewards implies that people, who are more impulsive, tend to change their be- havior significantly and become more reflec- tive after anodal stimulation but only in case of delayed rewards. In other words, the tendency A follow-up regression focusing on non-plan- ning impulsivity found no significant effect. Discussion The within-subjects pilot study presented in this paper explored the effect of tDCS on the Studia Psychologica, Vol. 62, No. 2, 2020, 123-137 132 to delay discounting per se is not affected, but if an impulsive person considers the delayed rewards, then he or she is more likely to give more thought to the decision and eventually choose the delayed reward option after anodal stimulation. These results confirm previous findings suggesting that impulsive people tend to have an altered sense of time (Wittman & Paulus, 2007), implying that they opt for smaller and immediate rewards because they over- estimate the duration of time intervals. This finding explains these individuals’ tendency to discount the value of delayed rewards and pre- fer immediate benefits. In short, time seems to take longer for them and is thus perceived as a higher cost, leading them to choose the immediate reward. The fact that after anodal stimulation people are more likely to choose the delayed rewards and spend a longer time making their decision could be explained as a possible effect of the anodal stimulation, which leads individuals to consider more carefully the exact weight of time when evaluating the value of the rewards. to delay discounting per se is not affected, but if an impulsive person considers the delayed rewards, then he or she is more likely to give more thought to the decision and eventually choose the delayed reward option after anodal stimulation. These results confirm previous findings suggesting that impulsive people tend to have an altered sense of time (Wittman & Paulus, 2007), implying that they opt for smaller and immediate rewards because they over- estimate the duration of time intervals. This finding explains these individuals’ tendency to discount the value of delayed rewards and pre- fer immediate benefits. In short, time seems to take longer for them and is thus perceived as a higher cost, leading them to choose the immediate reward. The fact that after anodal stimulation people are more likely to choose the delayed rewards and spend a longer time making their decision could be explained as a possible effect of the anodal stimulation, which leads individuals to consider more carefully the exact weight of time when evaluating the value of the rewards. about their own behavior. Specifically, individu- als’ beliefs about their tendency to behave in certain ways have been formed through nu- merous experiences over time. Discussion When partici- pants are asked to complete a questionnaire about their level of impulsivity, they refer to these consolidated and established beliefs about their own impulsivity. Apparently, the manner in which individuals tended to rate their own behavior was affected by the stimulation, lead- ing them to be more focused on actual behav- ioral performance beyond the immediate task- related experience, whereas the same pattern did not arise in the sham condition. This is an interesting discovery that should be explored in depth in future studies. Another possible explanation of this con- flict between our results and previous find- ings could be due to the different scales used to assess impulsivity. We used fine scales that were probably able to discriminate quite extensively among different kinds of impul- sivity, thus providing more sophisticated re- sults. This fact highlights an interesting inter- action effect that should be investigated in depth in future studies. Overall, our data sug- gest that the effect of brain stimulation alone cannot explain individuals’ behavior; rather, we must consider individual differences as well to acquire a better understanding of the complicated mechanisms underlying intertemporal choices. An additional consideration related to these results, which should be addressed further in future studies, concerns the age of our sub- jects. Steinberg and colleagues (2009) re- ported that teenagers tend to be less oriented toward the future and prefer to focus on the immediate present. This perspective would lead to a stronger preference for immediate rewards, regardless of any other consider- ation. Although our participants were older than teenagers, the mean age of 24 could justify the possibility that a stronger-than-average cognitive focus on the present might still play a role in their reasoning strategies. Conclusion doi.org/10.1037/0022-3514.74.5.1252 Baumeister, R. F., Heatherton, T. F., & Tice, D. M. (1994). Losing control: How and why people fail at self-regulation. San Diego, CA, US: Academic Press. The findings of the present study, although they will have to be confirmed in follow-up studies, offer several interesting implications. Low be- havioral inhibition and high reward sensitivity are generally considered major predictors of risk-taking behavior (Gullo & Dawe, 2008). Conversely, there is a positive correlation be- tween the ability to resist impulsive reactions and the achievement of positive life outcomes (Keough et al., 1999). Taken together, these findings suggest that delay-discounting behav- ior is influenced by brain stimulation, but not in a direct and deterministic manner. What seems crucial is the interaction between stim- ulation and individuals’ impulsivity levels, which indicates that behavior is affected by different factors that pertain to distinct domains. Our data, by highlighting how the tDCS affected behavior differently depending on the individual level of impulsivity, suggest an important role of individual impulsivity that should be further explored to better understand the effect of tDCS itself. Our results suggest that a tDCS-based protocol could potentially be used both in re- search activity and as a form of support for populations prone to risk-taking behavior. How- ever, the protocol and the expected outcomes should be calibrated according to individual levels of impulsivity. This recommendation is in line with a growing body of research within the tDCS literature that emphasizes consider- ing individual differences, such as gender, physiological differences, and cognitive abili- Becker, C., Walker, D., & McCord, C. (2017). A sys- tematic literature review on intertemporal choice in software engineering-protocol and results. arXiv preprint arXiv:1701.08310. Berns, G. S., Laibson, D., & Loewenstein, G. (2007). Intertemporal choice – toward an integrative framework. Trends in Cognitive Science, 11(11), 482–488. https://doi.org/10.1016/j.tics.2007.08.011 Bickel, W. K., Koffarnus, M. N., Moody, L., & Wilson, A. G. (2014). The behavioral-and neuro-economic process of temporal discounting: A candidate be- havioral marker of addiction. Neuropharmacol- ogy, 76, 518–527. https://doi.org/10.1016/ j.neuropharm.2013.06.013 Benzion, U., Rapoport, A., & Yagil, J. (1989). Dis- count rates inferred from decisions: An experi- mental study. Management Science, 35(3), 270– 284 Blair, C., & Ursache, A. (2011). A bidirectional model of executive functions and self-regulation. Hand- book of self-regulation: Research, theory, and applications, 2, 300–320. Boehringer, A., Macher, K., Dukart, J., Villringer, A., & Pleger, B. (2013). Cerebellar transcranial direct current stimulation modulates verbal working memory. Limitations The present study, which constitutes a first promising step toward understanding the role of individual differences in modulating the ef- fect of brain stimulation on specific cognitive tasks, has some limitations that should be addressed in future studies. First, the small sample size of the pilot study represents an obvious limitation and may limit the general- izability of the results. This study, however, should be considered an initial exploratory in- quiry; we recognize that replication with a larger sample would be needed to further corrobo- rate these preliminary, though encouraging, findings. Replication should also occur with Interestingly enough, no effect of individual levels of impulsivity was found when we con- sidered impulsive responses under the sham condition. This result partially contradicts pre- vious findings indicating that personal impul- sivity was associated with a preference for proximal reward in temporal discounting tasks (Kirby & Hernstein, 1995; Pine, Shiner, Seymour & Dolan, 2010). One possible expla- nation could lie in the nature of self-report scales, which assess what individuals believe Studia Psychologica, Vol. 62, No. 2, 2020, 123-137 133 different age cohorts to examine the possible effect of age. ties (Jones & Berryhill, 2012; Krause & Kadosh, 2014), to thoroughly understand the differen- tial effects of stimulation on participants. ties (Jones & Berryhill, 2012; Krause & Kadosh, 2014), to thoroughly understand the differen- tial effects of stimulation on participants. ties (Jones & Berryhill, 2012; Krause & Kadosh, 2014), to thoroughly understand the differen- tial effects of stimulation on participants. A second limitation concerns the exclusive reliance on self-reported rating scales to mea- sure individual impulsivity. Although literature on individual differences has relied mainly on self-reported measures, integrating other types of measures (such as direct observa- tions or behavioral measures) could provide a more comprehensive and multifaceted picture of the construct. Future research could address this issue by combining different methods of measuring individual impulsivity. References Avila, C., Cuenca, I., Felix, V., Parcet, M., & Miranda, A. (2004). Measuring impulsivity in school-aged boys and examining its relationship with ADHD and ODD ratings. Journal of Abnormal Child Psy- chology, 32(3), 295–304. Baumeister, R. F., Bratslavsky, E., Muraven, M., & Tice, D. M. (1998). Ego depletion: Is the active self a limited resource?. 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https://openalex.org/W3002266072	https://www.mdpi.com/2075-163X/10/2/87/pdf?version=1581908233	English	null	Immobilization of Potentially Toxic Elements (PTE) by Mineral-Based Amendments: Remediation of Contaminated Soils in Post-Industrial Sites	Minerals	2,020	cc-by	10,014	Received: 14 December 2019; Accepted: 20 January 2020; Published: 21 January 2020 Abstract: In many post-industrial sites, the high contents and high mobility of diﬀerent potentially toxic elements (PTEs) make the soils unsuitable for eﬀective management and use. Therefore, immobilization of PTE seems to be the best remediation option for such areas. In the present study, soil samples were collected in post-industrial areas in Northeastern Poland. The analyzed soil was characterized by especially high contents of Cd (22 mg·kg−1), Pb (13 540 mg·kg−1), and Zn (8433 mg·kg−1). Yellow lupine (Lupinus luteus L.) and two types of mineral-based amendments were used to determine their combined remediation eﬀect on PTE immobilization. A greenhouse pot experiment was conducted to evaluate the inﬂuence of chalcedonite and halloysite on plant growth, chlorophyll a ﬂuorescence, the leaf greenness index (SPAD), PTE uptake, and the physicochemical properties and toxicity of soil. The application of chalcedonite resulted in the greatest increase in soil pH, whereas halloysite contributed to the greatest reduction in the contents of Ni, Pb, Zn, and Cr in soil, compared with the control treatment. The addition of halloysite signiﬁcantly increased plant biomass. The application of mineral-based amendments increased the ratio of variable ﬂuorescence to maximum chlorophyll ﬂuorescence (Fv/Fm) in yellow lupine leaves. The leaf greenness index was highest in plants growing in soil amended with chalcedonite. The results of this study suggest that mineral-based amendments combined with yellow lupine could potentially be used for aided phytostabilization of multi-PTE contaminated soil in a post-industrial area. Keywords: soil amendments; immobilization; halloysite; chalcedonite Maja Radziemska 1,* , Agnieszka B˛e´s 2 , Zygmunt M. Gusiatin 3 , Grzegorz Majewski 1, Zbigniew Mazur 2, Ayla Bilgin 4 , Iwona Jaskulska 5 and Martin Brtnický 6,7 Maja Radziemska 1,* , Agnieszka B˛e´s 2 , Zygmunt M. Gusiatin 3 , Grzegorz Majewski 1, Zbigniew Mazur 2, Ayla Bilgin 4 , Iwona Jaskulska 5 and Martin Brtnický 6,7 1 Institute of Environmental Engineering, Warsaw University of Life Sciences, Nowoursynowska 159, 02-776 Warsaw, Poland; grzegorz_majewski@sggw.pl 1 Institute of Environmental Engineering, Warsaw University of Life Sciences, Nowoursynowska 159, 02-776 Warsaw, Poland; grzegorz_majewski@sggw.pl 2 Faculty of Environmental Management and Agriculture, University of Warmia and Mazury in Olsztyn Łódzki 4, 10-727 Olsztyn, Poland; agnieszka.bes@uwm.edu.pl (A.B.); zbigniew.mazur@uwm.edu.pl (Z 2 Faculty of Environmental Management and Agriculture, University of Warmia and Mazury in Olsztyn, Pl. Łódzki 4, 10-727 Olsztyn, Poland; agnieszka.bes@uwm.edu.pl (A.B.); zbigniew.mazur@uwm.edu.pl (Z.M.) 3 Faculty of Geoengineering, University of Warmia and Mazury in Olsztyn, Słoneczna St. 45G, 10 719 Olsztyn, Poland; mariusz.gusiatin@uwm.edu.pl 2 Faculty of Environmental Management and Agriculture, University of Warmia and Mazury in Olsztyn, Pl. Łódzki 4, 10-727 Olsztyn, Poland; agnieszka.bes@uwm.edu.pl (A.B.); zbigniew.mazur@uwm.edu.pl (Z.M.) 3 Faculty of Geoengineering, University of Warmia and Mazury in Olsztyn, Słoneczna St. 45G, 10 719 Olsztyn, Poland; mariusz.gusiatin@uwm.edu.pl Łódzki 4, 10 727 Olsztyn, Poland; agnieszka.bes@uwm.edu.pl (A.B.); zbigniew.mazur@uwm.edu.pl (Z.M. 3 Faculty of Geoengineering, University of Warmia and Mazury in Olsztyn, Słoneczna St. 45G, 10 719 Olsztyn, Poland; mariusz.gusiatin@uwm.edu.pl y g p 4 Faculty of Engineering, Artvin Coruh University, Seyitler Campus, 08000 Artvin, Turkey; ayla.bilgin@artvin.edu.tr y g 5 Faculty of Agriculture and Biotechnology, UTP University of Science and Technology, Bernardy´nska 6/8, 85-029 Bydgoszcz, Poland; jaskulska@utp.edu.pl y g j p p 6 Faculty of AgriSciences, Mendel University in Brno, Department of Agrochemistry, Soil Science, Microbiology and Plant Nutrition, 61300 Brno, Czech Republic; martin.brtnicky@seznam.cz 7 Institute of Chemistry and Technology of Environmental Protection, Faculty of Chemistry, Brno Unive of Technology, 61200 Brno, Czech Republic * Correspondence: maja_radziemska@sggw.pl; Tel.: +48-2259-353-70 minerals minerals www.mdpi.com/journal/minerals 1. Introduction Rapid industrialization and population growth in the last century have led to adverse changes in the natural environment [1]. Soil, water, and air pollution can pose considerable threats for human www.mdpi.com/journal/minerals Minerals 2020, 10, 87; doi:10.3390/min10020087 2 of 13 Minerals 2020, 10, 87 health and distort the natural balance of ecosystem components [2]. Anthropogenic pressure disrupts biogeochemical cycles and increases the contents of PTE in the environment, which creates serious problems on the local and global scale [3]. Eﬀorts are being made to restore degraded soils and decrease the content of contaminating substances below the established threshold values [4]. In many cases, soil quality standards cannot be met for technical or ﬁnancial reasons; therefore, the main aim of restoration is to reduce contamination availability and its adverse environmental impact. Phytoremediation poses a viable alternative to other restoration methods [5]. It can be used independently or in combination with conventional methods of soil and water remediation. Phytoremediation is eﬀective in purifying not only small but also large degraded areas [6]. Phytostabilization has attracted considerable interest in recent years because it is a technique which relies on plants to immobilize pollutants in soil and reduce their bioavailability in the environment [7]. Contaminants like PTE are immobilized when they are absorbed and accumulated in plant roots, adsorbed on root surfaces, and precipitated in the rhizosphere [8]. The chemical compounds secreted by plant roots into the rhizosphere decrease the bioavailability of PTE. This remediation method protects soil from further degradation, including erosion, and the immobilized compounds are less likely to migrate to other links in the food chain [9]. Phytostabilization eﬀectively diminishes ecological risk in soil containing both organic and inorganic pollutants. Another advantage is the fact that in areas where the phytostabilization is applied, the above-ground plant parts have a low content of PTE; therefore, they can be eﬀectively used to generate energy in all biomass conversion processes (combustion, co-combustion, fermentation). Compounds that deactivate and immobilize PTE ions in soil can be additionally used in phytostabilization. Mineral-based amendments such as phosphogypsum [10], calcium carbonate, bentonite [11], sulfo-calcic ﬂy ash [12], mixtures of diammonium phosphate and hydroxyapatite [13], calcium carbonate [14], hydrated lime [15], diatomite, and dolomite [16] have been described in the literature. The eﬀectiveness of phytostabilization is determined by the type and quantity of soil amendments. 2.1. Mineral-Based Amendments and Experimental Design 2. Materials and Methods 2.1. Mineral-Based Amendments and Experimental Design 2. Materials and Methods Two mineral-based amendments, i.e., chalcedonite (limited liability company Chalcedon Poland, Inowłódz, Poland) and halloysite (“Dunino” strip mine, Intermark Company, Legnica, Poland), were mixed with soil in the amount of 3.0% (w/w). Soil without the amendments (0.0%, w/w) was the control treatment. Soil samples were thoroughly mixed, and pots ﬁlled with soil with a moisture content at 60% of water holding capacity were left in a dark room for over 3 weeks to equilibrate the soil mixture. Each treatment was replicated three times. Scanning electron microscope (SEM) images of mineral-based amendments are presented in Figure 1. The oxides content in soil amendments was measured using X-ray diﬀraction (XRD) analysis (Philips X-ray Diﬀractometer with X’Celerator Detector), while the speciﬁc surface area was measured with a Micromeritics ASAP 2020 BET N2 (Norcross, GA, USA). Table 1 presents selected properties of the mineral-based amendments used in the experiment. 2.1. Mineral‐Based Amendments and Experimental Design Two mineral‐based amendments, i.e., chalcedonite (limited liability company Chalcedon Poland, Inowłódz, Poland) and halloysite (“Dunino” strip mine, Intermark Company, Legnica, Poland), were mixed with soil in the amount of 3.0% (w/w). Soil without the amendments (0.0%, w/w) was the control treatment. Soil samples were thoroughly mixed, and pots filled with soil with a moisture content at 60% of water holding capacity were left in a dark room for over 3 weeks to equilibrate the soil mixture. Each treatment was replicated three times. Scanning electron microscope (SEM) images of mineral‐based amendments are presented in Figure 1. The oxides content in soil amendments was measured using X‐ray diffraction (XRD) analysis (Philips X‐ray Diffractometer with X’Celerator Detector), while the specific surface area was measured with a Micromeritics ASAP 2020 BET N2 (Norcross, GA, USA). Table 1 presents selected properties of the mineral‐based amendments used in the experiment. Figure 1. Scanning electron microscope (SEM) images of chalcedonite (a) and halloysite (b). Figure 1. Scanning electron microscope (SEM) images of chalcedonite (a) and halloysite (b). Figure 1. Scanning electron microscope (SEM) images of chalcedonite (a) and halloysite (b). Figure 1. Scanning electron microscope (SEM) images of chalcedonite (a) and halloysite (b). Table 1. Properties of mineral‐based amendments. Table 1. Properties of mineral-based amendments. Table 1. Properties of mineral‐based amendments. Table 1. Properties of mineral-based amendments. Table 1. Properties of mineral‐based amendments. ameter Chemical Composition in Oxide (wt. 1. Introduction Therefore, eﬀective and inexpensive compounds of natural origin are being researched, including mineral-based amendments such as chalcedonite and halloysite. Chalcedonite is a siliceous rock, and its chemical and phase composition make it highly suitable for a wide range of applications [17]. Chalcedonite has a homogenous chemical composition with an estimated 94% content of silicon dioxide (SiO2). Chalcedonite rock is composed mainly of chalcedony with small amounts of quartz, opal, iron hydroxides, pyrite, manganese compounds, and clay minerals. Chalcedonite is a mesoporous material with a relatively uniform pore structure and a total pore volume of 0.03–0.04 cm3·g−1 [18]. Halloysite is composed of nanotubes, and it is characterized by high porosity and speciﬁc surface area, high cation exchange capacity, and ease of chemical and mechanical processing. Although it contains trace amounts of contaminants, it is highly stable across a wide pH range [19]. The chemical composition of halloysite is highly similar to kaolin. The main structural component in this group of minerals is the 1:1 kaolinite group of clay minerals with the crystal-chemical formula Al4Si4O10(OH)8 [20]. Halloysite has many applications in engineering and environmental protection, and it is widely used as a mineral adsorbent for removing toxic substances and PTE from water and wastewater [21,22]. Halloysite has not commonly been tested because, although a large amount of it is available, there are only three halloysite mines in the world. Halloysite is characterized by a large speciﬁc surface area (approximately 65,000–400,000 m2·kg−1) and considerable porosity (60–70%) [23]. Despite the favorable properties of chalcedonite and halloysite, there has been little research into their application in assisted phytostabilization of chemically degraded soils. In view of the above, the aim of this study was to evaluate the eﬀect of mineral-based amendments on assisted phytostabilization of soil heavily contaminated with mixed PTE. The phytotoxic and phytochemical properties of the soil and the chemical composition of the plants were analyzed, and chlorophyll a ﬂuorescence and the leaf greenness index (SPAD) were determined. 3 of 13 Minerals 2020, 10, 87 2.1. Mineral-Based Amendments and Experimental Design 2. Materials and Methods %) Specific Surface Area (m2∙g−1) cedonite SiO2—89.06 ± 0.46; Al2O5—9.33 ± 0.14; K2O—1.21 ± 0.08; MgO—0.40 ± 0.011 7.44 loysite SiO2—39.6, Al2O3—37.0, Fe2O3—16.1, TiO2—2.30, CaO—0.66, MgO—0.13, Na2O—0.04, K2—0.05; P2O5—0.52 75.0 The experiment was conducted in a naturally ventilated greenhouse. The tested plant spe ll l i (L l t ) P k hi h l t d i 5 0 k l th l t Th l Table 1. Properties of mineral-based amendments. Parameter Chemical Composition in Oxide (wt. %) Speciﬁc Surface Area (m2·g−1) pH Chalcedonite SiO2—89.06 ± 0.46; Al2O5—9.33 ± 0.14; K2O—1.21 ± 0.08; MgO—0.40 ± 0.011 7.44 7.12 Halloysite SiO2—39.6, Al2O3—37.0, Fe2O3—16.1, TiO2—2.30, CaO—0.66, MgO—0.13, Na2O—0.04, K2—0.05; P2O5—0.52 75.0 7.66 y p ( ) p g p y y p p were watered every other day with deionized water to maintain 60% of the maximum water holding capacity of the soil. At the end of the experiment (approximately 54 days after sowing), lupine plants were harvested, weighed, and separated into the roots and above‐ground parts. 2.2. Soil from Post‐Industrial Areas Used in the Experiment I thi t d l f il h il t i t d ith Cd Pb d Z ll t d f The experiment was conducted in a naturally ventilated greenhouse. The tested plant species was yellow lupine (L. luteus) cv. Perkoz which was planted in 5.0 kg polyethylene pots. The plants were watered every other day with deionized water to maintain 60% of the maximum water holding capacity of the soil. At the end of the experiment (approximately 54 days after sowing), lupine plants were harvested, weighed, and separated into the roots and above-ground parts. In this study, samples of soil heavily co contaminate a depth of 0–20 cm in post‐industrial areas in Northeas 2.2. Soil from Post-Industrial Areas Used in the Experiment using the method described by Pan et al. [24]. The study area mainly stores ferrous and non‐ferrous metal waste since 1946. About 150 kg of soil were sampled from several dozen points located within an area of 1000 m2. At each sampling point, 5 soil samples were collected using a soil auger and mixed to achieve a composite soil sample representing a given sampling point. Before the pot In this study, samples of soil heavily co-contaminated with Cd, Pb, and Zn were collected from a depth of 0–20 cm in post-industrial areas in Northeastern Poland (53◦47′3.48” N; 20◦30′52.56” E) using the method described by Pan et al. [24]. The study area mainly stores ferrous and non-ferrous metal waste since 1946. About 150 kg of soil were sampled from several dozen points located within 4 of 13 Minerals 2020, 10, 87 an area of 1000 m2. At each sampling point, 5 soil samples were collected using a soil auger and mixed to achieve a composite soil sample representing a given sampling point. Before the pot experiment, the samples were air-dried and passed through a 2 mm nylon sieve. The soil, classiﬁed as loamy ﬁne sand, was composed of 76% sand, 23% silt, and 1% clay fractions. The selected physicochemical properties of the soil are presented in Table 1. 2.3. Soil Analysis Before the experiment, soil samples were assayed for pH and electrical capacity (EC) in distilled water extracts (1:2.5 w/v) using a pH-meter (HI 221) and cation exchange capacity (CEC), calculated as the sum of hydrolytic acidity (in 1M Ca(CH3COO)2) and exchangeable bases (in 0.1 M HCl). Soil organic matter was determined by combustion at 550 ◦C. The total content of PTE (Cd, Cr, Cu, Ni, Pb, Zn) was determined with a ﬂame atomic absorption spectrometer (FAAS) (Varian AA28OFS, Mulgrave, Australia) using the U.S. Environmental Protection Agency Method 3050B after soil digestion in a mixture of concentrated HCl, HNO3, and H2O2 in a microwave oven (MARSXpress, CEM Corporation, Matthews, NC, USA) using a single-stage program (ramp to temperature for 30 min, hold samples at 170 ◦C for 45 min). The extracts after mineralization were ﬁltered through Whatman ﬁlters into 50 mL glass ﬂasks. The accuracy of PTE analysis by FAAS was validated by analyzing the reference material, CRM 142 R. 2.4. Plant Material Analysis At the end of the experiment, the harvested L. luteus plants were divided into shoot and root parts. The plant material was carefully washed with ultrapure water to remove soil particles, and then air-dried at room temperature. Before chemical analyses, the plants were powdered using an analytical mill (Retsch type ZM300, Hann, Germany) and kept at ambient temperature. The contents of PTE in the above-ground parts and roots of plants were determined by mineralizing them in a microwave oven with concentrated HNO3. Chlorophyll a ﬂuorescence in the plants was measured during the growing season with the Handy-PEA portable chlorophyll ﬂuorimeter (Hansatech Instruments Ltd., Pentney, UK). Measurements were conducted on two randomly selected, fully developed leaves, and the results were averaged (ﬁve plants per pot, two measurements per plant). The maximum quantum eﬃciency of PSII (Fv/Fm ratio; Fv is the variable ﬂuorescence; Fm is the maximum ﬂuorescence), as an indicator of plant adaptation to dark, was determined on lupine leaves three times during the experiment. Before the measurements, leaves were covered with clips for 30 min for dark adaptation. Fluorescence emission was induced on 4 mm2 of leaf surface area with a light intensity of 3000 µmol m−2 s−1. Maximum ﬂuorescence (MF) was measured for 100 ms. Fluorescence kinetics were analyzed in the Pocket PEA Plus V1.10 program (Hansatech Instruments Ltd., Pentney, UK). The leaf greenness index (SPAD) was determined on ten leaves in each plant with the SPAD 502 chlorophyll meter (Konica Minolta, Tokyo, Japan). The leaf greenness index was expressed in dimensionless SPAD units. All measurements were conducted in triplicate. 2.5. Ecotoxicological Analysis The phytotoxicity of contaminated soils was determined with the Phytotoxkit seed germination biotest and an early growth microbiotest (Microbiotests Inc., Ghent, Belgium). Phytotoxicity analyses were also carried out in soils with mineral-based amendments (halloysite and chalcedonite). The analyses were conducted in triplicate, and Phytotoxkit soil was the control treatment. Phytotoxicity tests were performed with the use of sorghum (Sorghum saccharatum L.) seeds. Observations were conducted for seven days, and digital images were acquired every day. Root length was measured by digital image analysis in the Image Tool 3.0 program (UTHSCSA, San Antonio, TX, USA). The results Minerals 2020, 10, 87 5 of 13 were used to calculate the percentage inhibition of seed germination and root growth with the use of the following formula: (1) I = ((A −B))/A × 100, (1) where: I—inhibition (%), A—seed germination or the length of roots in control soil, B—seed germination or the length of roots in the tested soils (with and without mineral-based amendments). where: I—inhibition (%), A—seed germination or the length of roots in control soil, B—seed germination or the length of roots in the tested soils (with and without mineral-based amendments). 2.6. Statistical Analysis The results of the experiment were processed statistically in the Statistica 13.3 program (San Diego, CA, USA). The normality of data distribution in every independent group was determined in the Shapiro–Wilk test, and the residuals were additionally checked for normality. The homogeneity of variance was veriﬁed by Levene’s test. The signiﬁcance of diﬀerences between means was determined by one-way ANOVA (F-test). Signiﬁcant diﬀerences (p < 0.05) between the mean values of diﬀerent treatments were evaluated and compared in Tukey’s range test. The results were also subjected to Principal Component Analysis (PCA) in the XLStat program (Addinsoft, Paris, France). 3.1. Soil Parameters The analyzed soils had a pH of 7.54 ± 0.4, pointing to alkaline conditions. The mean contents of PTE are presented in Table 2. Soil contamination was evaluated based on the classiﬁcation set forth in the Regulation of the Polish Ministry of the Environment [25]. The contents of Cd, Pb, and Zn in degraded soils from post-industrial areas exceeded the thresholds values given in the above classiﬁcation. The contents of the remaining PTE were within the safe range. Table 2. Selected properties of the analyzed soil. Parameter Unit Value Acceptable Values a pH - 7.54 ± 0.4 - Electrical conductivity mS·cm−1 0.84 ± 0.3 - Organic matter %·d.m. 11 ± 2.1 - Cation Exchange Capacity C mol (+)·kg−1 51.9 ± 2.7 - Cadmium (Cd) mg·kg−1 22 ± 2.5 15 Copper (Cu) mg·kg−1 496 ± 144.4 600 Chromium (Cr) mg·kg−1 352 ± 61.1 500 Nickel (Ni) mg·kg−1 113 ± 24.5 300 Lead (Pb) mg·kg−1 13,540.0 ± 669.6 600 Zinc (Zn) mg·kg−1 8430 ± 1376.5 1000 a threshold values set forth by the Regulations of the Polish Ministry of the Environment [25]. The values in bold signiﬁcantly exceed threshold contents. Table 2. Selected properties of the analyzed soil. Table 2. Selected properties of the analyzed soil. a threshold values set forth by the Regulations of the Polish Ministry of the Environment [25]. The values in bold signiﬁcantly exceed threshold contents. between treatments. 3.3. Plant Growth after the Addition of Mineral-Based Amendments 3.3. Plant Growth After the Addition of Mineral‐Based Amendments 3.3. Plant Growth After the Addition of Mineral‐Based Amendments The yield of the above‐ground biomass of L. luteus is presented in Figure 4. The analyses of plant growth and development indicate that chalcedonite and halloysite increased the yield of the above‐ground biomass of L. luteus. Halloysite contributed to the greatest increase in the yield of above ground biomass relative to the control treatment The yield of the above-ground biomass of L. luteus is presented in Figure 4. The analyses of plant growth and development indicate that chalcedonite and halloysite increased the yield of the above-ground biomass of L. luteus. Halloysite contributed to the greatest increase in the yield of above-ground biomass relative to the control treatment. f f The yield of the above‐ground biomass of L. luteus is presented in Figure 4. The analyses of plant growth and development indicate that chalcedonite and halloysite increased the yield of the above‐ground biomass of L. luteus. Halloysite contributed to the greatest increase in the yield of above‐ground biomass relative to the control treatment. Figure 4. The effect of mineral‐based amendments on the yield of above‐ground biomass of L. luteus. Error bars ± standard error (n = 3). Different letters denote significant differences between treatments. Figure 4. The effect of mineral‐based amendments on the yield of above‐ground biomass of L. luteus. Error bars ± standard error (n = 3). Different letters denote significant differences between treatments. Figure 4. The eﬀect of mineral-based amendments on the yield of above-ground biomass of L. luteus. Error bars ± standard error (n = 3). Diﬀerent letters denote signiﬁcant diﬀerences between treatments. Figure 4. The effect of mineral‐based amendments on the yield of above‐ground biomass of L. luteus. Error bars ± standard error (n = 3). Different letters denote significant differences between treatments. Figure 4. The effect of mineral‐based amendments on the yield of above‐ground biomass of L. luteus. Error bars ± standard error (n = 3). Different letters denote significant differences between treatments. Figure 4. The eﬀect of mineral-based amendments on the yield of above-ground biomass of L. luteus. Error bars ± standard error (n = 3). Diﬀerent letters denote signiﬁcant diﬀerences between treatments. 3.2. Soil Parameters after the Addition of Mineral-Based Amendments The pH of soil from post-industrial areas was aﬀected by the type of mineral-based amendments (Figure 2). Chalcedonite induced the greatest increase in soil pH, from 8.14 in the control treatment to 8.25. The PTE content of phytostabilized soil is presented in Figure 3. The total contents of Ni, Pb, Zn, and Cr were most eﬀectively reduced by halloysite, relative to non-amended control soil. Chalcedonite decreased the total amounts of Cu and Cd in the analyzed soil. 6 of 13 on‐amen Minerals 2020, 10, 87 ents of Ni, Pb, Zn rol soil Chalcedo ure 2. Soil pH after phytostabilization. Error bars ± standard error (n = 3). Different letters den nificant differences between treatments. Figure 2. Soil pH after phytostabilization. Error bars ± standard error (n = 3). Diﬀerent letters denote signiﬁcant diﬀerences between treatments. Minerals 2020, 10, 87 6 of 13 Minerals 2020, 10, 87 6 of 13 re 2. Soil pH after phytostabilization. Error bars ± standard error (n = 3). Different letters de ficant differences between treatments. Figure 2. Soil pH after phytostabilization. Error bars ± standard error (n = 3). Diﬀerent letters denote signiﬁcant diﬀerences between treatments. Minerals 2020, 10, 87 6 of 1 Minerals 2020, 10, 87 6 of 1 Figure 3. Total potentially toxic elements (PTE) contents in soil after the application of mineral‐based amendments. Error bars ± standard error (n = 3). Different letters denote significant differences Figure 3. Total potentially toxic elements (PTE) contents in soil after the application of mineral-based amendments. Error bars ± standard error (n = 3). Diﬀerent letters denote signiﬁcant diﬀerences between treatments. Figure 3. Total potentially toxic elements (PTE) contents in soil after the application of mineral‐based amendments. Error bars ± standard error (n = 3). Different letters denote significant differences between treatments. Figure 3. Total potentially toxic elements (PTE) contents in soil after the application of mineral‐based amendments. Error bars ± standard error (n = 3). Different letters denote significant differences b Figure 3. Total potentially toxic elements (PTE) contents in soil after the application of mineral-based amendments. Error bars ± standard error (n = 3). Diﬀerent letters denote signiﬁcant diﬀerences between treatments. Figure 3. Total potentially toxic elements (PTE) contents in soil after the application of mineral‐based amendments. Error bars ± standard error (n = 3). Different letters denote significant differences between treatments. denote significant differ 3.5. Chlorophyll Fluorescence 3.5. Chlorophyll Fluorescence 3.5. Chlorophyll Fluorescence Chalcedonite and halloysite increased the value of Fv/Fm in lupine leaves relative to the control treatment (Figure 6). The Fv/Fm ratio was determined at 0.73–0.74 in control soil, 0.78–0.8 in soil ith th dditi f h ll it d 0 77 0 81 i il d d ith h l d it Chalcedonite and halloysite increased the value of Fv/Fm in lupine leaves relative to the control treatment (Figure 6). The Fv/Fm ratio was determined at 0.73–0.74 in control soil, 0.78–0.8 in soil with the addition of halloysite, and 0.77–0.81 in soil amended with chalcedonite. Chalcedonite and halloysite increased the value of Fv/Fm in lupine leaves relative to the control treatment (Figure 6). The Fv/Fm ratio was determined at 0.73–0.74 in control soil, 0.78–0.8 in soil with the addition of halloysite, and 0.77–0.81 in soil amended with chalcedonite. y Figure 6. Leaf greenness index (SPAD). Different letters above the columns indicated significant Figure 6. Leaf greenness index (SPAD). Different letters above the columns indicated significant difference at p < 0.05. Figure 6. Leaf greenness index (SPAD). Diﬀerent letters above the columns indicated signiﬁcant diﬀerence at p < 0.05. Figure 6 Leaf greenness index (SPAD) Different letters above the columns indicated significant Figure 6. Leaf greenness index (SPAD). Different letters above the columns indicated significant difference at p < 0.05. Figure 6. Leaf greenness index (SPAD). Diﬀerent letters above the columns indicated signiﬁcant diﬀerence at p < 0.05. 3 4 PTE Accumulation in L luteus 3.4. PTE Accumulation in L. luteus 3.4. PTE Accumulation in L. luteus 3.4. PTE Accumulation in L. luteus The effect of mineral‐based amendments on PTE accumulation in the roots and above‐ground biomass of L. luteus is presented in Figure 5. The analyzed PTE were accumulated mostly in lupine The effect of mineral‐based amendments on PTE accumulation in the roots and above‐ground biomass of L. luteus is presented in Figure 5. The analyzed PTE were accumulated mostly in lupine t I i ith th d d t l t t t h ll it l d t th t t The eﬀect of mineral-based amendments on PTE accumulation in the roots and above-ground biomass of L. luteus is presented in Figure 5. The analyzed PTE were accumulated mostly in lupine roots. 7 of 13 Minerals 2020, 10, 87 In comparison with the non-amended control treatment, halloysite led to the greatest accumulation of Ni and Cr, whereas chalcedonite resulted in the greatest accumulation of Cd in lupine roots. Minerals 2020, 10, 87 7 of 13 Minerals 2020, 10, 87 7 of 13 Figure 5. The effect of mineral‐based amendments on potentially toxic elements accumulation in the roots and above‐ground biomass of L. luteus. Error bars ± standard error (n = 3). Different letters d t i ifi t diff b t t t t Figure 5. The eﬀect of mineral-based amendments on potentially toxic elements accumulation in the roots and above-ground biomass of L. luteus. Error bars ± standard error (n = 3). Diﬀerent letters denote signiﬁcant diﬀerences between treatments. Figure 5. The effect of mineral‐based amendments on potentially toxic elements accumulation in the roots and above‐ground biomass of L. luteus. Error bars ± standard error (n = 3). Different letters denote significant differences between treatments. Figure 5. The effect of mineral‐based amendments on potentially toxic elements accumulation in the roots and above‐ground biomass of L. luteus. Error bars ± standard error (n = 3). Different letters Figure 5. The eﬀect of mineral-based amendments on potentially toxic elements accumulation in the roots and above-ground biomass of L. luteus. Error bars ± standard error (n = 3). Diﬀerent letters denote signiﬁcant diﬀerences between treatments. Figure 5. The effect of mineral‐based amendments on potentially toxic elements accumulation in the roots and above‐ground biomass of L. luteus. Error bars ± standard error (n = 3). Different letters denote significant differences between treatments. p denote significant d denote significant 3.8. Statistical Analysis 3.8. Statistical Analysis The suitability of the data for factor analysis was assessed using Kaiser‐Meyer‐Olkin (KMO) and Barlett Tests. The value of KMO = 0.518 confirmed the suitability of the data for factor analysis. Two factors with eigenvalues greater than 1 were identified, where the ratio of the variance explained by each factor to the total variance decreased gradually (Table 3 and Figure 9). These two factors explained 100% of total variance. The first factor explained 58.73% of total variance. Biomass and the content of Cd, Ni, and Cr in the roots had strong negative loads; the content of Cu, Ni, and Zn in the above‐ground parts of the tested plants, the content of Cu and Ni in the roots, and the content of Ni and Zn in the soil had strong positive loads, whereas the Cd content of the 3.8. Statistical Analysis The suitability of the data for factor analysis was assessed using Kaiser‐Meyer‐Olkin (KMO) and Barlett Tests. The value of KMO = 0.518 confirmed the suitability of the data for factor analysis. Two factors with eigenvalues greater than 1 were identified, where the ratio of the variance explained by each factor to the total variance decreased gradually (Table 3 and Figure 9). These two factors explained 100% of total variance. The first factor explained 58.73% of total variance. Biomass and the content of Cd, Ni, and Cr in the roots had strong negative loads; the content of Cu, Ni, and Zn in the above‐ground parts of the tested plants, the content of Cu and Ni in the roots, and the content of Ni and Zn in the soil had strong positive loads, whereas the Cd content of the The suitability of the data for factor analysis was assessed using Kaiser-Meyer-Olkin (KMO) and Barlett Tests. The value of KMO = 0.518 conﬁrmed the suitability of the data for factor analysis. Two factors with eigenvalues greater than 1 were identiﬁed, where the ratio of the variance explained by each factor to the total variance decreased gradually (Table 3 and Figure 9). These two factors explained 100% of total variance. The ﬁrst factor explained 58.73% of total variance. 3.7. Phytotoxicity 3.7. Phytotoxicity 3.7. Phytotoxicity saccharatum before the phytostabilization experiment Error bars represent standard deviation error (SD n = 6 determinations) Different letters Figure 8. Growth inhibition (GI) and root growth (RI) of S. saccharatum before the phytostabilization experiment. Error bars represent standard deviation error (SD, n = 6 determinations). Different letters d i ifi diff b Figure 8. Growth inhibition (GI) and root growth (RI) of S. saccharatum before the phytostabilization experiment. Error bars represent standard deviation error (SD, n = 6 determinations). Diﬀerent letters denote signiﬁcant diﬀerences between treatments. 3.7. Phytotoxicity 3.7. Phytotoxicity 3.7. Phytotoxicity In phytotoxicity analyses of contaminated soil, the percentual inhibition of seed germination (GI) was generally lower (16.6% on average for all samples) than the percentage inhibition of root growth (RI) (41.9% on average for all samples) (Figure 8). The tested mineral‐based amendments had no significant effect on the germination of S. saccharatum seeds. Chalcedonite added to contaminated soil significantly differentiated the length of S. saccharatum roots relative to non‐amended contaminated soil. The differences in root length observed in the tested plants growing in soil amended with halloysite were not statistically significant. In phytotoxicity analyses of contaminated soil, the percentual inhibition of seed germination (GI) was generally lower (16.6% on average for all samples) than the percentage inhibition of root growth (RI) (41.9% on average for all samples) (Figure 8). The tested mineral-based amendments had no signiﬁcant eﬀect on the germination of S. saccharatum seeds. Chalcedonite added to contaminated soil signiﬁcantly diﬀerentiated the length of S. saccharatum roots relative to non-amended contaminated soil. The diﬀerences in root length observed in the tested plants growing in soil amended with halloysite were not statistically signiﬁcant. In phytotoxicity analyses of contaminated soil, the percentual inhibition of seed germination (GI) was generally lower (16.6% on average for all samples) than the percentage inhibition of root growth (RI) (41.9% on average for all samples) (Figure 8). The tested mineral‐based amendments had no significant effect on the germination of S. saccharatum seeds. Chalcedonite added to contaminated soil significantly differentiated the length of S. saccharatum roots relative to non‐amended contaminated soil. The differences in root length observed in the tested plants growing in soil amended with halloysite were not statistically significant. Figure 8. Growth inhibition (GI) and root growth (RI) of S. saccharatum before the phytostabilization experiment. Error bars represent standard deviation error (SD, n = 6 determinations). Different letters Figure 8. Growth inhibition (GI) and root growth (RI) of S. saccharatum before the phytostabilization experiment. Error bars represent standard deviation error (SD, n = 6 determinations). Different letters denote significant differences between treatments Figure 8. Growth inhibition (GI) and root growth (RI) of S. saccharatum before the phytostabilization experiment. Error bars represent standard deviation error (SD, n = 6 determinations). Diﬀerent letters denote signiﬁcant diﬀerences between treatments. Figure 8. Growth inhibition (GI) and root growth (RI) of S. g g difference at p < 0.05. p 3 6 Leaf Greenness Index (SPAD 3.6. Leaf Greenness Index (SPAD) 3.6. Leaf Greenness Index (SPAD) The leaf greenness index of lupine plants differed significantly across treatments containing the tested mineral‐based amendments (Figure 7). In the first month of the experiment, the leaf greenness index was highest in soil containing chalcedonite (37.555 SPAD). The analyzed parameter was 22.0% lower on average (30.82 SPAD) in non‐amended control soil. Similar trends were observed during 3.6. Leaf Greenness Index (SPAD) The leaf greenness index of lupine plants differed significantly across treatments containing the tested mineral‐based amendments (Figure 7). In the first month of the experiment, the leaf greenness index was highest in soil containing chalcedonite (37.555 SPAD). The analyzed parameter was 22.0% lower on average (30.82 SPAD) in non‐amended control soil. Similar trends were observed during the second and the third measurement. The leaf greenness index of lupine plants diﬀered signiﬁcantly across treatments containing the tested mineral-based amendments (Figure 7). In the ﬁrst month of the experiment, the leaf greenness index was highest in soil containing chalcedonite (37.555 SPAD). The analyzed parameter was 22.0% lower on average (30.82 SPAD) in non-amended control soil. Similar trends were observed during the second and the third measurement. 8 of 13 of 13 Minerals 2020, 10, 87 Minerals 2020 10 87 Figure 7. Chlorophyll a fluorescence—the Fv/Fm ratio. Different letters above the columns indicated significant difference at the p < 0.05. Figure 7. Chlorophyll a ﬂuorescence—the Fv/Fm ratio. Diﬀerent letters above the columns indicated signiﬁcant diﬀerence at the p < 0.05. Figure 7. Chlorophyll a fluorescence—the Fv/Fm ratio. Different letters above the columns indicated significant difference at the p < 0.05. Figure 7. Chlorophyll a fluorescence—the Fv/Fm ratio. Different letters above the columns indicated significant difference at the p < 0.05. Figure 7. Chlorophyll a ﬂuorescence—the Fv/Fm ratio. Diﬀerent letters above the columns indicated signiﬁcant diﬀerence at the p < 0.05. Figure 7. Chlorophyll a fluorescence—the Fv/Fm ratio. Different letters above the columns indicated significant difference at the p < 0.05. p denote significant d denote significant 3.8. Statistical Analysis Variables VF1 VF2 Biomass −0.941 *** 0.339 pH −0.010 −1.000 * Cu a 0.604 0.797 * Cu b 0.935 * 0.355 Cu c 0.808 * 0.589 Ni a 0.970 * 0.241 Ni b 0.995 * 0.097 Ni c −0.983 * 0.182 Cd a 0.382 0.924 * Cd b 0.711 0.703 Cd c −0.980 * 0.198 Pb a 0.934* 0.356 Pb b 0.410 −0.912 * Pb c −0.243 0.970 * Zn a 0.918 * 0.396 Zn b 0.995 * −0.103 Zn c 0.411 0.912 * Cr a 0.723 0.690 Cr b −0.263 0.965 * Cr c −0.874 * 0.486 Eigenvalue 12.634 7.366 Variability (%) 58.734 41.266 Cumulative % 58.734 100.000 * Significant factor loading is bold faced (* strong > 0.75; medium 0.50–0.75; * weak 0.50–0.30) [26] a total content in soil; b metal contents in the above‐ground plant material; c metal contents in the root. Table 3. Varimax rotated factor matrix for the analyzed dataset. Variables VF1 VF2 Biomass −0.941 *** 0.339 pH −0.010 −1.000 * Cu a 0.604 0.797 * Cu b 0.935 * 0.355 Cu c 0.808 * 0.589 Ni a 0.970 * 0.241 Ni b 0.995 * 0.097 Ni c −0.983 * 0.182 Cd a 0.382 0.924 * Cd b 0.711 0.703 Cd c −0.980 * 0.198 Pb a 0.934* 0.356 Pb b 0.410 −0.912 * Pb c −0.243 0.970 * Zn a 0.918 * 0.396 Zn b 0.995 * −0.103 Zn c 0.411 0.912 * Cr a 0.723 0.690 Cr b −0.263 0.965 * Cr c −0.874 * 0.486 Eigenvalue 12.634 7.366 Variability (%) 58.734 41.266 Cumulative % 58.734 100.000 * Signiﬁcant factor loading is bold faced (* strong > 0.75; medium 0.50–0.75; * weak 0.50–0.30) [26] a total content in soil; b metal contents in the above-ground plant material; c metal contents in the root. p denote significant d denote significant 3.8. Statistical Analysis Biomass and the content of Cd, Ni, and Cr in the roots had strong negative loads; the content of Cu, Ni, and Zn in the above-ground parts of the tested plants, the content of Cu and Ni in the roots, and the content of Ni and Zn in the soil had strong positive loads, whereas the Cd content of the above-ground biomass and the Cr content of soil had moderately positive loads. The second factor explained 41.26% of total variance. pH and the Pb content of the above-ground parts had strong negative loads; the content of 9 of 13 rong e Cr l Minerals 2020, 10, 87 explained 41.2 negative loads; Cu and Cd in the soil, the content of Pb and Zn in the roots, and the Cr content of the above-ground parts had strong positive loads, whereas biomass had a moderately negative load. negative load. Table 3. Varimax rotated factor matrix for the analyzed dataset. Table 3. Varimax rotated factor matrix for the analyzed dataset. Variables VF1 VF2 Biomass −0.941 *** 0.339 pH −0.010 −1.000 * Cu a 0.604 0.797 * Cu b 0.935 * 0.355 Cu c 0.808 * 0.589 Ni a 0.970 * 0.241 Ni b 0.995 * 0.097 Ni c −0.983 * 0.182 Cd a 0.382 0.924 * Cd b 0.711 0.703 Cd c −0.980 * 0.198 Pb a 0.934* 0.356 Pb b 0.410 −0.912 * Pb c −0.243 0.970 * Zn a 0.918 * 0.396 Zn b 0.995 * −0.103 Zn c 0.411 0.912 * Cr a 0.723 0.690 Cr b −0.263 0.965 * Cr c −0.874 * 0.486 Eigenvalue 12.634 7.366 Variability (%) 58.734 41.266 Cumulative % 58.734 100.000 * Signiﬁcant factor loading is bold faced (* strong > 0.75; medium 0.50–0.75; * weak 0.50–0.30) [26] a total content in soil; b metal contents in the above-ground plant material; c metal contents in the root. 4. Discussion In the present study, the total contents of Cd, Pb, and Zn considerably exceeded the threshold levels set forth by the Regulation of the Polish Ministry of the Environment [25]. The eﬀectiveness of phytoremediation in degraded areas can be improved through the addition of amendments that decrease erosion and/or the transfer of PTE to deep soil horizons and reduce the quantity of phytoavailable metals in soil [27]. The applied amendments immobilize PTE in soil, thus contributing to the reinstatement of the physiochemical and functional properties of degraded soils. In the current study, halloysite enhanced the immobilization of metals (Ni, Pb, Zn, and Cr) in soil, which could be attributed to its large speciﬁc surface area and cation exchange capacity [22]. Halloysite’s ability to immobilize PTE in contaminated soil was also reported in our previous studies which demonstrated that this mineral-based amendment eﬀectively immobilized Ni [28], Pb, Zn [29], and Cu [30]. PTE-binding aﬃnity is inﬂuenced by the granulometric composition, organic matter content, and pH of soil [31]. The mobility of PTEs decreases with an increase in soil pH. Potential toxic elements are less bioavailable in alkaline soils. According to Blake and Goulding [32], the bioavailability of Cd increases at pH > 5.5, the bioavailability of Zn, Ni, and Cu increases at pH 5.0, and the bioavailability of Pb at pH < 4.5. In the present study, the greatest increase in soil pH was observed in the treatment containing chalcedonite. A signiﬁcant increase in soil pH was also reported by Jin et al. [33] and Abad-Valle et al. [34] who analyzed the eﬀectiveness of mineral-based amendments (nano-hydroxyapatite, sepioloite) in the remediation of PTE-polluted soils, which is consistent with the present ﬁndings. Mineral-based amendments can promote assisted phytostabilization by inducing the immobilization of pollutants in soil [29]. In the present study, halloysite and chalcedonite considerably decreased the total PTE content of soil by increasing soil pH and, consequently, reducing the mobility and bioavailability of PTE. High contents of PTE have toxic eﬀects on plants, and they compromise or inhibit plant growth and development [35]. Heavily contaminated soils can be sown with plant species that are highly tolerant of pollution [36]. In the current study, soils contaminated with high levels of PTE were eﬀectively remediated through the combined application of L. luteus and mineral-based amendments. p denote significant d denote significant 3.8. Statistical Analysis Variables VF1 VF2 Biomass −0.941 *** 0.339 pH −0.010 −1.000 * Cu a 0.604 0.797 * Cu b 0.935 * 0.355 Cu c 0.808 * 0.589 Ni a 0.970 * 0.241 Ni b 0.995 * 0.097 Ni c −0.983 * 0.182 Cd a 0.382 0.924 * Cd b 0.711 0.703 Cd c −0.980 * 0.198 Pb a 0.934* 0.356 Pb b 0.410 −0.912 * Pb c −0.243 0.970 * Zn a 0.918 * 0.396 Zn b 0.995 * −0.103 Zn c 0.411 0.912 * Cr a 0.723 0.690 Cr b −0.263 0.965 * Cr c −0.874 * 0.486 Eigenvalue 12.634 7.366 Variability (%) 58.734 41.266 Cumulative % 58.734 100.000 * Significant factor loading is bold faced (* strong > 0.75; medium 0.50–0.75; * weak 0.50–0.30) [26] a total content in soil; b metal contents in the above‐ground plant material; c metal contents in the root. Table 3. Varimax rotated factor matrix for the analyzed dataset. ariables VF1 Bi 0 941 *** * Signiﬁcant factor loading is bold faced (* strong > 0.75; medium 0.50–0.75; * weak 0.50–0.30) [26] a total content in soil; b metal contents in the above-ground plant material; c metal contents in the root. * Significant factor loading is bold faced (* strong > 0.75; medium 0.50–0.75; * weak 0.50–0.30) [26] a total content in soil; b metal contents in the above‐ground plant material; c metal contents in the root. Figure 9. Plot of principal component scores (variables after varimax rotation). Figure 9. Plot of principal component scores (variables after varimax rotation). Figure 9. Plot of principal component scores (variables after varimax rotation). Figure 9. Plot of principal component scores (variables after varimax rotation). The correlation matrix of PCA data is shown in Table 2. Signiﬁcant negative correlations were found between biomass vs. the content of Cu in the above-ground parts of plants, Cu in the roots, Ni and Pb in the soil, Pb in the above-ground parts, Zn in the soil, and Zn in the above-ground parts (r = −0.560 to −0.971), whereas signiﬁcant positive correlations were noted between biomass vs. the content of Ni, Cd, Pb, and Cr in the roots and Cr in the above-ground parts of L. luteus (r = −0.575 10 of 13 Minerals 2020, 10, 87 to −0.989). Signiﬁcant negative correlations were also observed between pH vs. p denote significant d denote significant 3.8. Statistical Analysis the content of Cu and Cd in the soil, Cu in the roots, Cd in the above-ground parts, Pb and Zn in the roots, Cr in the soil, and Cr in the above-ground parts (r = −0.597 to −0.968). The correlation analysis revealed that PTE parameters were strongly correlated with one another. 4. Discussion In other studies, mineral-based amendments such as zeolite, CaO, bentonite, and dolomite eﬀectively immobilized PTE in soil and increased the yields of the tested plants [37]. The mechanism by which plant roots absorb PTE is highly complex and involves several processes, including cation exchange across cell walls, intracellular transport and rhizosphere processes [38,39]. Plant cover can limit the migration of PTE to deeper soil horizons and above-ground plant parts by stabilizing pollutants in the rhizosphere [40]. The results of this study validate the above observation. According to the literature, PTE absorbed from the soil solution are more likely to be accumulated in roots than in shoots, proportionally to their content in the soil [41,42]. The analyzed PTE were accumulated mainly in lupine roots, in particular in soil amended with halloysite. In the extensive root system, the mineralization of old roots also increases the organic matter content of soil and promotes the immobilization of contaminating PTE [43]. Other studies have demonstrated that plants used for phytostabilization eﬀectively absorb PTE, and the absorption of Zn, Pb, Cd, and Ni by plant roots generally increases with a rise in pollution levels [29,30]. The applied plants should be characterized by low fertilizer, water, and pesticide requirements, high eﬃciency of the photosynthetic apparatus, and they should be well adapted to the local climate [44]. Previous research [45,46] and the present ﬁndings indicate that PTE impair chlorophyll synthesis and decrease the chlorophyll content of plants. Plant resistance to toxic levels of PTE varies across species and varieties. Despite the fact that PTE are bound by various organic substances (proteins and amino acids), these pollutants decrease the chlorophyll content of plants, thus 11 of 13 Minerals 2020, 10, 87 reducing photosynthetic eﬃciency [47]. The roots are the ﬁrst plant organs to come into contact with and respond to PTE in soil, which was also observed in S. saccharatum in this study. The percentual inhibition of seed germination and root growth indicates that the analyzed soil was characterized by low toxicity for the tested plant species. 5. Conclusions Degraded post-industrial areas are characterized by adverse physicochemical properties, including high contents of PTE, which inhibit the development of vegetation. In the present study, the addition of halloysite to contaminated soil signiﬁcantly increased L. luteus biomass. The above mineral-based amendment was also most eﬀective in reducing the total contents of Ni, Pb, Zn, and Cr in soil. Chalcedonite led to the highest increase in soil pH relative to the control treatment. The uptake of PTE by L. luteus plants depended on the type of metal and the applied mineral-based amendments. The total content of PTE was higher in the roots than in the above-ground parts of L. luteus plants. The Ni and Cr content in roots was signiﬁcantly higher in soil amended with halloysite than in the control treatment. The application of mineral-based amendments increased the Fv/Fm ratio in yellow lupine leaves. The leaf greenness index was highest in plants growing in soil amended with chalcedonite. Chalcedonite signiﬁcantly diﬀerentiated the length of S. saccharatum roots. Author Contributions: Author Contributions: Conceptualization, M.R. and Z.M.G.; methodology, M.R.; software, A.B. (Agnieszka B˛e´s), G.M., A.B. 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Phytostabilization—Management strategy for stabilizing t elements in contaminated soils. Int. J. Environ. Res. Public Health. 2017, 14, 958. [CrossRef] 31. Hou, S.; Zheng, N.; Tang, L.; Ji, X.; Li, Y. Eﬀect of soil pH and organic matter content on heavy metals availability in maize (Zea mays L.) rhizospheric soil of non-ferrous metals smelting area. Environ. Monit. Asses. 2019, 191, 634. [CrossRef] 32. Blake, L.; Goulding, K.W.T. Eﬀects of atmospheric deposition, soil pH and acidiﬁcation on heavy metal contents in soils and vegetation of semi-natural ecosystems at Rothamsted Experimental Station, UK. Plant Soil 2002, 240, 235–251. [CrossRef] 33. Jin, Y.; Liu, W.; Li, H.-L.; Shen, S.-G.; Liang, S.-X.; Liu, C.; Shan, L. References Exploration of chlorophyll a ﬂuorescence and plant gas exchange parameters as indicators of drought Tolerance in Perennial Ryegrass. Sensors 2019, 19, 2736. [CrossRef] [PubMed] 45. Dabrowski, P.; Pawluskiewicz, B.; Kalaji, H.M.; Baczewska, A.H. The eﬀect of light availability on leaf area index, biomass production and plant species composition of park grasslands in Warsaw. Plant Soil Environ. 2013, 59, 543–548. [CrossRef] 46. Bes, A.; Warminski, K.; Adomas, B. Long-term responses of Scots pine (Pinus sylvestris L.) and European beech (Fagus sylvatica L.) to the contamination of light soils with diesel oil. Environ. Sci. Pollut. Res. 2019, 26, 10587–10608. [CrossRef] 47. Hashemi, S.A. Studying the eﬀects of heavy metal on chlorophyll and sugar in one year-old seedlings organs of Acer velutinum specie. Acta Ecol. Sin. 2018, 38, 224–227. [CrossRef] © 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
https://openalex.org/W2804144642	https://europepmc.org/articles/pmc6100001?pdf=render	English	null	Molecular Diversity by Olefin Cross-Metathesis on Solid Support. Generation of Libraries of Biologically Promising β-Lactam Derivatives	Molecules/Molecules online/Molecules annual	2,018	cc-by	12,262	Received: 7 May 2018; Accepted: 14 May 2018; Published: 16 May 2018 Abstract: The application of the reagent-based diversiﬁcation strategy for generation of libraries of biologically promising β-lactam derivatives is described. Key features are the versatility of the linker used and the cross-metathesis functionalization at the cleavage step. From an immobilized primary library, diversity was expanded by applying different cleavage conditions, leading to a series of cholesterol absorption inhibitor analogues together with interesting hybrid compounds through incorporation of a chalcone moiety. Keywords: diversity oriented synthesis; solid-phase organic synthesis; β-lactam derivatives; oleﬁn metathesis; cholesterol absorption inhibitors Received: 7 May 2018; Accepted: 14 May 2018; Published: 16 May 2018 Article Molecular Diversity by Oleﬁn Cross-Metathesis on Solid Support. Generation of Libraries of Biologically Promising β-Lactam Derivatives Luciana Méndez, Andrés A. Poeylaut-Palena and Ernesto G. Mata * ID Instituto de Química Rosario, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario-CONICET, Suipacha 531, Rosario S2002LRK, Argentina; mendez@iquir-conicet.gov.ar (L.M.); andrespoeylaut@gmail.com (A.A.P.-P.) * Correspondence: mata@iquir-conicet.gov.ar; Tel.: +54-9-341-437-0477 Luciana Méndez, Andrés A. Poeylaut-Palena and Ernesto G. Mata * ID Instituto de Química Rosario, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario-CONICET, Suipacha 531, Rosario S2002LRK, Argentina; mendez@iquir-conicet.gov.ar (L.M.); andrespoeylaut@gmail.com (A.A.P.-P.) * Correspondence: mata@iquir-conicet.gov.ar; Tel.: +54-9-341-437-0477 * Correspondence: mata@iquir-conicet.gov.ar; Tel.: +54-9-341-437-0477 Molecules 2018, 23, 1193; doi:10.3390/molecules23051193 molecules molecules molecules 1. Introduction The β-lactam skeleton is an active acylating agent with numerous applications in clinical therapy [1]. Besides the well-known use as antibiotics [2], β-lactam ring has inhibitory effects on prostate speciﬁc antigen [3,4], thrombin [5], human cytomegalovirus protein [6], human leukocyte elastase [7], cysteine protease [8,9], and human fatty acid amide hydrolase [10], as well as anticancer properties [11–15] and neuroprotective action [16,17]. A new area in β-lactam-based drugs has been established by development of β-lactam derivatives with strong cholesterol absorption inhibitor reducing LDL concentration [18–21]. An example is the commercial drug ezetimibe, which is one of the most prescribed drugs in the US. Recently, simvastatin has been shown to provide an incremental beneﬁt on reducing cardiovascular events in acute coronary syndrome patients, when co-administered with ezetimibe [22]. Furthermore, β-lactams have also been considered as peptidomimetic species for mimicking certain properties of proteins and, from a synthetic point of view, they are key synthons for the preparation of various heterocyclic compounds of biological importance [23–25]. In drug discovery related organic synthesis, one of the tools for a rapid and efﬁcient construction of diversity-based small molecules is parallel solid-phase synthesis. Particularly, solid-phase chemistry has recently aroused interest in metal-catalyzed cross-coupling reactions since undesirable soluble homodimers can be washed away during puriﬁcation providing chemoselectivity, while immobilization of one of the substrates makes its homodimerization a less favorable process due to site isolation (Scheme 1) [26]. In this regard, we and others have recognized the usefulness of solid-supported oleﬁn cross-metathesis for generation of biologically relevant molecules [27–31], including a comprehensive study to understand the process [32,33]. From the viewpoint of green chemistry, using solid-phase synthetic sequences allows a signiﬁcant reduction in solvent waste, since puriﬁcation is performed by phase separation, avoiding chromatographic isolation of products which requires a large consumption of organic solvents [34]. Molecules 2018, 23, 1193; doi:10.3390/molecules23051193 www.mdpi.com/journal/molecules www.mdpi.com/journal/molecules 2 of 15 Molecules 2018, 23, 1193 Scheme 1. Metal-catalyzed solid-phase synthesis. Scheme 1. Metal-catalyzed solid-phase synthesis. In the search of diversity, a bunch of very ingenious strategies have been developed, among which diversity-oriented synthesis (DOS) is the most widespread [35–37]. According to DOS, skeletal diversity can be basically achieved by two main approaches: one known as “substrate-based diversiﬁcation” where the same reagent generates different structures by reaction with selected substrates; and “reagent-based diversiﬁcation” in which different products are obtained when the same substrate is subjected to different reaction conditions [38]. 1. Introduction Generally, the point of attachment to the resin in solid-phase organic synthesis (SPOS) limits the possibilities of diversity generation. In previous work, we have developed a series of β-lactam compounds linked to the resin at position 1 or 3 of the ring. Only the substituents on the two remaining positions could be combined [27,39]. In order to maximize structural variation from a library of compounds, we have studied a strategy based on a linker, which ensures release by different reaction conditions to obtain diverse products using a DOS reagent-based diversiﬁcation approach (Scheme 2). Scheme 2. Reagent-based diversiﬁcation approach in solid-phase synthesis. Scheme 2. Reagent-based diversiﬁcation approach in solid-phase synthesis. 3 of 15 Molecules 2018, 23, 1193 We have shown that high molecular diversity in β-lactam scaffolds can be obtained using a linker cleavable by two orthogonal conditions: one by treatment with 10% TFA, and the other using a set of oleﬁn cross-metathesis reactions. While Seeberger developed oleﬁn linkers for solid-phase synthesis of glycosides [40], we decided to introduce a new twist using them to generate diversity during cleavage step. Thus, a series of cholesterol absorption inhibitor analogues, together with interesting β-lactam-chalcone hybrid compounds, have been generated. Our results are summarized in this extended paper [41]. 2. Results and Discussion Synthesis started with immobilization of 4-pentenoic acid to Wang resin by standard coupling conditions, to afford the corresponding resin 1 which, in turn, was the substrate for a solid-supported oleﬁn cross-metathesis by reaction with 1-(chloromethyl)-4-vinylbenzene in the presence of second generation Grubbs precatalyst, according to the methodology reported by us (Scheme 3) [32]. Then, oxidation of the supported benzyl chloride 2 with DMSO/NaHCO3 at 155 ◦C [42] yielded the aldehyde 3, which was characterized by 13C gel-phase NMR (Figure 1). Aldehyde 3 was used as substrate for the synthesis of libraries of β-lactam derivatives. Scheme 3. Synthesis of versatile linker 3. Figure 1. 13C gel-phase NMR of compounds 2 and 3. Scheme 3. Synthesis of versatile linker 3. Scheme 3. Synthesis of versatile linker 3. Figure 1. 13C gel-phase NMR of compounds 2 and 3. Figure 1. 13C gel-phase NMR of compounds 2 and 3. Figure 1. 13C gel-phase NMR of compounds 2 and 3. Molecules 2018, 23, 1193 4 of 15 2.1. Primary β-Lactam Library Geometric optimization employing a semi-empirical method such as AM1 [49] indicated 5 of 15 Molecules 2018, 23, 1193 a radical change in the arrangement of the aromatic ring at position 1 (Figure 3). In the N-aryl β-lactam, H-3 and H-4 were in the same plane of the N-1 aromatic ring, while in N-benzyl β-lactam these protons were near the axis of the ring. Due to anisotropic effect, protons H-3 and H-4 in the N-aryl-β-lactam were in a deshielding area, whereas in N-benzyl-β-lactam those protons were in a shielding environment. The absence of an oxygen atom directly linked to position 3 of the β-lactam ring in 8bb, leading to a drastic shift of the signal to higher ﬁelds. Molecules 2018, 23, x FOR PEER REVIEW 5 of 15 these protons were near the axis of the ring. Due to anisotropic effect, protons H‐3 and H‐4 in the N‐aryl‐β‐lactam were in a deshielding area, whereas in N‐benzyl‐β‐lactam those protons were in a shielding environment. The absence of an oxygen atom directly linked to position 3 of the β‐lactam Figure 2. Comparative 1H-NMR spectra of 3,4-cis- and trans-substituted β-lactams. ring in 8bb, leading to a drastic shift of the signal to higher fields. Figure 2. Comparative 1H‐NMR spectra of 3,4‐cis‐ and trans‐substituted β‐lactams. Molecules 2018, 23, x FOR PEER REVIEW 5 of 15 β-lactam were in a deshielding area, whereas in N-benzyl-β-lactam those protons were in a shielding environment. The absence of an oxygen atom directly linked to position 3 of the β-lactam ring in 8bb, leading to a drastic shift of the signal to higher fields. Deleted: a Deleted: a Deleted: s Molecules 2018, 23, x FOR PEER REVIEW 5 of 15 β-lactam were in a deshielding area, whereas in N-benzyl-β-lactam those protons were in a shielding environment. The absence of an oxygen atom directly linked to position 3 of the β-lactam ring in 8bb, leading to a drastic shift of the signal to higher fields. Deleted: a Deleted: a Deleted: s Molecules 2018, 23, x FOR PEER REVIEW 5 of 15 β-lactam were in a deshielding area, whereas in N-benzyl-β-lactam those protons were in a shielding environment. The absence of an oxygen atom directly linked to position 3 of the β-lactam ring in 8bb, leading to a drastic shift of the signal to higher fields. 2.1. Primary β-Lactam Library The secondary “TFA” library of β–lactams. Comment Figure 2. Comparative 1H-NMR spectra of 3,4-cis- and trans-substituted β-lactams. Table 1. The secondary “TFA” library of β–lactams. Comment Figure 2. Comparative 1H-NMR spectra of 3,4-cis- and trans-substituted β-lactams. Table 1. The secondary “TFA” library of β–lactams. Comment Figure 2. Comparative 1H-NMR spectra of 3,4-cis- and trans-substituted β-lactams. Table 1. The secondary “TFA” library of β–lactams. Comment Figure 2. Comparative 1H-NMR spectra of 3,4-cis- and trans-substituted β-lactams. Table 1. The secondary “TFA” library of β–lactams. Comment Figure 2. Comparative 1H-NMR spectra of 3,4-cis- and trans-substituted β-lactams. Table 1. The secondary “TFA” library of β–lactams. Comment Figure 2. Comparative 1H-NMR spectra of 3,4-cis- and trans-substituted β-lactams. Table 1. The secondary “TFA” library of β–lactams. 2.1. Primary β-Lactam Library For the solid-phase version of the classical Staudinger reaction between imines and ketenes [43–45], we ﬁrst developed the synthesis of a set of immobilized imines 5a–d by treatment of the aldehyde with different amines 4a–d in reﬂuxing benzene using a Dean–Stark trap (Scheme 4). For the in situ generation of ketenes, we found that stable acid chlorides such as phenoxyacetyl chloride were ideal, however, when aliphatic carboxylic acids derivatives were required, the best choice was the activation of the free acid by Mukaiyama’s reagent. Thus, immobilized imines 5a–d were treated with either acid chloride or carboxylic acids (6a–d) and Mukaiyama’s reagent to obtain the primary β-lactam library (7aa–dd) (Scheme 4). Scheme 4. Primary immobilized β-lactam library. Scheme 4. Primary immobilized β-lactam library. The relative conﬁguration of C-3 and C-4 substituents was determined taking the coupling constant between H-3 and H-4 nucleus in 1H-NMR experiments after triﬂuoroacetic acid (TFA) cleavage into account. Coupling constants greater than 4 Hz were indicative of a cis conﬁguration [46], so β-lactams 8aa and 8ab had a cis conﬁguration while 8bb had a trans conﬁguration (Figure 2 and Table 1). During Staudinger reaction, the diastereoselectivity for cis or trans conﬁguration of the resulting β-lactams was governed by substituents on the starting building blocks (imine and ketene) [47,48]. Ketenes that were activated by heteroatoms had strong preference for formation of cis products. This was the case for β-lactams 8aa (J = 5.0 Hz) and 8ab (J = 4.4 Hz) that have been constructed from ketenes derived from dehydrohalogenation of phenoxyacetyl chloride. On the other hand, β-lactam 8bb, which was synthesized from an alkyl-ketene derivative obtained from 5-phenylvaleric acid, gave a trans conﬁguration (J = 2.2 Hz). The presence of a heteroatom tethered to the α-position of the ketene and tended to form the cis products by conrotatory ring closure of the zwitterionic intermediate, formed through a nucleophilic attack of imine nitrogen on the electrophilic carbonyl carbon of the ketene [47]. Conversely, thermodynamically more stable trans-β-lactam 8bb was generated by isomerization of the zwitterion, when a hydrocarbon chain was tethered to the ketene. 1H-NMR signals corresponding to protons H-3 and H-4 showed a deshielding effect when substituent of position 1 changed from N-benzyl to N-aryl, respectively (compare 8aa and 8ab in Figure 2). 2.1. Primary β-Lactam Library Deleted: a Deleted: a Deleted: s Molecules 2018, 23, x FOR PEER REVIEW 5 of 15 β-lactam were in a deshielding area, whereas in N-benzyl-β-lactam those protons were in a shielding environment. The absence of an oxygen atom directly linked to position 3 of the β-lactam ring in 8bb, leading to a drastic shift of the signal to higher fields. Deleted: a Deleted: a Deleted: s Molecules 2018, 23, x FOR PEER REVIEW 5 of 15 β-lactam were in a deshielding area, whereas in N-benzyl-β-lactam those protons were in a shielding environment. The absence of an oxygen atom directly linked to position 3 of the β-lactam ring in 8bb, leading to a drastic shift of the signal to higher fields. Deleted: a Deleted: a Deleted: s Molecules 2018, 23, x FOR PEER REVIEW 5 of 15 β-lactam were in a deshielding area, whereas in N-benzyl-β-lactam those protons were in a shielding environment. The absence of an oxygen atom directly linked to position 3 of the β-lactam ring in 8bb, leading to a drastic shift of the signal to higher fields. Deleted: a Deleted: a Deleted: s Molecules 2018, 23, x FOR PEER REVIEW 5 of 15 β-lactam were in a deshielding area, whereas in N-benzyl-β-lactam those protons were in a shielding environment. The absence of an oxygen atom directly linked to position 3 of the β-lactam ring in 8bb, leading to a drastic shift of the signal to higher fields. Deleted: a Deleted: a Deleted: s Molecules 2018, 23, x FOR PEER REVIEW 5 of 15 β-lactam were in a deshielding area, whereas in N-benzyl-β-lactam those protons were in a shielding environment. The absence of an oxygen atom directly linked to position 3 of the β-lactam ring in 8bb, leading to a drastic shift of the signal to higher fields. Deleted: a Deleted: a Deleted: s Molecules 2018, 23, x FOR PEER REVIEW 5 of 15 β-lactam were in a deshielding area, whereas in N-benzyl-β-lactam those protons were in a shielding environment. The absence of an oxygen atom directly linked to position 3 of the β-lactam ring in 8bb, leading to a drastic shift of the signal to higher fields. Deleted: a Deleted: a Deleted: s g g g g Molecules 2018, 23, x FOR PEER REVIEW 5 of 15 β-lactam were in a deshielding area, whereas in N-benzyl-β-lactam those protons were in a shielding environment. 2.1. Primary β-Lactam Library The absence of an oxygen atom directly linked to position 3 of the β-lactam ring in 8bb, leading to a drastic shift of the signal to higher fields. Deleted: a Deleted: a Deleted: s Molecules 2018, 23, x FOR PEER REVIEW 5 of 15 β-lactam were in a deshielding area, whereas in N-benzyl-β-lactam those protons were in a shielding environment. The absence of an oxygen atom directly linked to position 3 of the β-lactam ring in 8bb, leading to a drastic shift of the signal to higher fields. Deleted: a Deleted: a Deleted: s Molecules 2018, 23, x FOR PEER REVIEW 5 of 15 β-lactam were in a deshielding area, whereas in N-benzyl-β-lactam those protons were in a shielding environment. The absence of an oxygen atom directly linked to position 3 of the β-lactam ring in 8bb, leading to a drastic shift of the signal to higher fields. Deleted: a Deleted: a Deleted: s Molecules 2018, 23, x FOR PEER REVIEW 5 of 15 β-lactam were in a deshielding area, whereas in N-benzyl-β-lactam those protons were in a shielding environment. The absence of an oxygen atom directly linked to position 3 of the β-lactam ring in 8bb, leading to a drastic shift of the signal to higher fields. Deleted: a Deleted: a Deleted: s Molecules 2018, 23, x FOR PEER REVIEW 5 of 15 β-lactam were in a deshielding area, whereas in N-benzyl-β-lactam those protons were in a shielding environment. The absence of an oxygen atom directly linked to position 3 of the β-lactam ring in 8bb, leading to a drastic shift of the signal to higher fields. Deleted: a Deleted: a Deleted: s Molecules 2018, 23, x FOR PEER REVIEW 5 of 15 β-lactam were in a deshielding area, whereas in N-benzyl-β-lactam those protons were in a shielding environment. The absence of an oxygen atom directly linked to position 3 of the β-lactam ring in 8bb, leading to a drastic shift of the signal to higher fields. Deleted: a Deleted: a Deleted: s Molecules 2018, 23, x FOR PEER REVIEW 5 of 15 β-lactam were in a deshielding area, whereas in N-benzyl-β-lactam those protons were in a shielding environment. The absence of an oxygen atom directly linked to position 3 of the β-lactam ring in 8bb, leading to a drastic shift of the signal to higher fields. 9 7db trans Ph 8db 55 10 7d t a 4 FPh 8d 52 11 7dd trans 4-MeOPh 8dd 33 a Overall isolated yield after flash column chromatography (five steps, based on loading of resin 2 (0.40 mmol/g, determined by elemental analysis of chlorine)). Delete 10 7dc trans 4-FPh 8dc 52 11 7dd trans 4-MeOPh 8dd 33 9 7db trans Ph 8db 55 10 7dc trans 4-FPh 8dc 52 11 7dd trans 4 MeOPh 8dd 33 a Overall isolated yield after flash column chromatography (five steps, based on loading of resin 2 (0.40 mmol/g, determined by elemental analysis of chlorine)). Delete D l t a 11 7dd trans 4-MeOPh 8dd 33 a Overall isolated yield after flash column chromatography (five steps, based on loading of resin 2 Delete 10 7dc trans 4-FPh 8dc 52 11 7dd trans 4-MeOPh 8dd 33 a Overall isolated yield after flash column chromatography (five steps, based on loading of resin 2 (0.40 mmol/g, determined by elemental analysis of chlorine)). Delete Delete 11 7dd trans 4-MeOPh 8dd 33 a Overall isolated yield after flash column chromatography (five steps, based on loading of resin 2 (0 40 l/ d i d b l l l i f hl i )) Delete 10 7dc trans 4-FPh 8dc 52 11 7dd trans 4-MeOPh 8dd 33 a Overall isolated yield after ﬂash column chromatography [ﬁve steps, based on loading of resin 2 (0.40 mmol/g, determined by elemental analysis of chlorine)]. 10 7dc trans 4‐FPh 8dc 52 y g p y ( p , g (0.40 mmol/g, determined by elemental analysis of chlorine)). Delete Delete 11 7dd trans 4-MeOPh 8dd 33 a Overall isolated yield after flash column chromatography (five steps based on loading of resin 2 10 7dc trans 4-FPh 8dc 52 (0.40 mmol/g, determined by elemental analysis of chlorine)). Delete a Overall isolated yield after flash column chromatography (five steps, based on loading of resin 2 Delete 11 7dd trans 4-MeOPh 8dd 33 (0.40 mmol/g, determined by elemental analysis of chlorine)). Delete a Overall isolated yield after flash column chromatography (five steps, based on loading of resin 2 (0 40 mmol/g, determined by elemental analysis of chlorine)) Delete 11 7dd trans 4-MeOPh 8dd 33 a Overall isolated yield after flash column chromatography (five steps based on loading of resin 2 D l t determined by elemental analysis of chlorine)]. 9 7db trans Ph 8db 55 10 7dc trans 4‐FPh 8dc 52 a Overall isolated yield after flash column chromatography (five steps, based on loading of resin 2 (0.40 mmol/g, determined by elemental analysis of chlorine)). Deleted: Deleted: 11 7dd trans 4-MeOPh 8dd 33 a Overall isolated yield after flash column chromatography (five steps based on loading of resin 2 D l d 10 7dc trans 4-FPh 8dc 52 a Overall isolated yield after flash column chromatography (five steps, based on loading of resin 2 (0.40 mmol/g, determined by elemental analysis of chlorine)). Deleted: Deleted: 11 7dd trans 4-MeOPh 8dd 33 a Overall isolated yield after flash column chromatography (five steps, based on loading of resin 2 Deleted: 10 7dc trans 4 FPh 8dc 52 11 7dd trans 4-MeOPh 8dd 33 (0.40 mmol/g, determined by elemental analysis of chlorine)). Deleted: a Overall isolated yield after flash column chromatography (five steps, based on loading of resin 2 (0.40 mmol/g, determined by elemental analysis of chlorine)). Deleted: 11 7dd trans 4-MeOPh 8dd 33 a Overall isolated yield after flash column chromatography (five steps based on loading of resin 2 D l t d y g p y [ p , g ( /g, determined by elemental analysis of chlorine)]. 2.1. Primary β-Lactam Library Commen y y Figure 2. Comparative 1H-NMR spectra of 3,4-cis- and trans-substituted β-lactams. Table 1. The secondary “TFA” library of β–lactams. Commen Figure 2. Comparative 1H-NMR spectra of 3,4-cis- and trans-substituted β-lactams. Table 1. The secondary “TFA” library of β–lactams. Commen Figure 2. Comparative 1H-NMR spectra of 3,4-cis- and trans-substituted β-lactams. Table 1. The secondary “TFA” library of β–lactams. Commen Figure 2. Comparative 1H-NMR spectra of 3,4-cis- and trans-substituted β-lactams. Table 1. The secondary “TFA” library of β–lactams. Commen Figure 2. Comparative 1H-NMR spectra of 3,4-cis- and trans-substituted β-lactams. Table 1. The secondary “TFA” library of β–lactams. Commen Figure 2. Comparative 1H-NMR spectra of 3,4-cis- and trans-substituted β-lactams. Table 1. The secondary “TFA” library of β–lactams. Commen Figure 2. Comparative 1H-NMR spectra of 3,4-cis- and trans-substituted β-lactams. Table 1. The secondary “TFA” library of β–lactams. Commen Figure 2. Comparative 1H-NMR spectra of 3,4-cis- and trans-substituted β-lactams. Table 1. The secondary “TFA” library of β–lactams. Commen Figure 2. Comparative 1H-NMR spectra of 3,4-cis- and trans-substituted β-lactams. Table 1. The secondary “TFA” library of β–lactams. Commen Figure 2. Comparative 1H-NMR spectra of 3,4-cis- and trans-substituted β-lactams. Table 1. The secondary “TFA” library of β–lactams. Commen Figure 2. Comparative 1H-NMR spectra of 3,4-cis- and trans-substituted β-lactams. Table 1. The secondary “TFA” library of β–lactams. Commen Figure 2. Comparative 1H-NMR spectra of 3,4-cis- and trans-substituted β-lactams. Table 1. The secondary “TFA” library of β–lactams. Commen Figure 2. Comparative 1H-NMR spectra of 3,4-cis- and trans-substituted β-lactams. Table 1. The secondary “TFA” library of β–lactams. Commen Figure 2. Comparative 1H-NMR spectra of 3,4-cis- and trans-substituted β-lactams. Table 1. The secondary “TFA” library of β–lactams. Commen Figure 2. Comparative 1H-NMR spectra of 3,4-cis- and trans-substituted β-lactams. Table 1. The secondary “TFA” library of β–lactams. Commen Figure 2. Comparative 1H-NMR spectra of 3,4-cis- and trans-substituted β-lactams. Table 1. The secondary “TFA” library of β–lactams. Commen Figure 2. Comparative 1H-NMR spectra of 3,4-cis- and trans-substituted β-lactams. Table 1. The secondary “TFA” library of β–lactams. Commen Figure 2. Comparative 1H-NMR spectra of 3,4-cis- and trans-substituted β-lactams. Table 1. The secondary “TFA” library of β–lactams. Commen Figure 2. Comparative 1H-NMR spectra of 3,4-cis- and trans-substituted β-lactams. Table 1. The secondary “TFA” library of β–lactams. Comment Figure 2. Comparative 1H-NMR spectra of 3,4-cis- and trans-substituted β-lactams. Table 1. The secondary “TFA” library of β–lactams. Comment Figure 2. Comparative 1H-NMR spectra of 3,4-cis- and trans-substituted β-lactams. Table 1. 2.1. Primary β-Lactam Library Deleted: a Deleted: a Deleted: s Molecules 2018, 23, x FOR PEER REVIEW 5 of 15 β-lactam were in a deshielding area, whereas in N-benzyl-β-lactam those protons were in a shielding environment. The absence of an oxygen atom directly linked to position 3 of the β-lactam ring in 8bb, leading to a drastic shift of the signal to higher fields. Deleted: a Deleted: a Deleted: s Molecules 2018, 23, x FOR PEER REVIEW 5 of 15 β-lactam were in a deshielding area, whereas in N-benzyl-β-lactam those protons were in a shielding environment. The absence of an oxygen atom directly linked to position 3 of the β-lactam ring in 8bb, leading to a drastic shift of the signal to higher fields. Deleted: a Deleted: a Deleted: s Figure 2. Comparative 1H-NMR spectra of 3,4-cis- and trans-substituted β-lactams. Figure 2. Comparative 1H‐NMR spectra of 3,4‐cis‐ and trans‐substituted β‐lactams. Table 1. The secondary “triﬂuoroacetic acid (TFA)” library of β-lactams. Table 1. The secondary “trifluoroacetic acid (TFA)” library of β–lactams. Table 1. The secondary “triﬂuoroacetic acid (TFA)” library of β-lactams. Table 1. The secondary “trifluoroacetic acid (TFA)” library of β–lactams. Table 1. The secondary “triﬂuoroacetic acid (TFA)” library of β-lactams. Table 1. The secondary “trifluoroacetic acid (TFA)” library of β–lactams. Table 1. The secondary triﬂuoroacetic acid (TFA) library of β-lactams. Table 1. The secondary trifluoroacetic acid (TFA) library of β–lactams. Figure 2. Comparative 1H-NMR spectra of 3,4-cis- and trans-substituted β-lactams. Table 1. The secondary “TFA” library of β–lactams. Commen Figure 2. Comparative 1H-NMR spectra of 3,4-cis- and trans-substituted β-lactams. Table 1. The secondary “TFA” library of β–lactams. Commen Figure 2. Comparative 1H-NMR spectra of 3,4-cis- and trans-substituted β-lactams. Table 1. The secondary “TFA” library of β–lactams. Commen Figure 2. Comparative 1H-NMR spectra of 3,4-cis- and trans-substituted β-lactams. Table 1. The secondary “TFA” library of β–lactams. Commen Figure 2. Comparative 1H-NMR spectra of 3,4-cis- and trans-substituted β-lactams. Table 1. The secondary “TFA” library of β–lactams. Commen Figure 2. Comparative 1H-NMR spectra of 3,4-cis- and trans-substituted β-lactams. Table 1. The secondary “TFA” library of β–lactams. Commen Figure 2. Comparative 1H-NMR spectra of 3,4-cis- and trans-substituted β-lactams. Table 1. The secondary “TFA” library of β–lactams. Commen Figure 2. Comparative 1H-NMR spectra of 3,4-cis- and trans-substituted β-lactams. Table 1. The secondary “TFA” library of β–lactams. Commen Figure 2. Comparative 1H-NMR spectra of 3,4-cis- and trans-substituted β-lactams. Table 1. The secondary “TFA” library of β–lactams. 2.1. Primary β-Lactam Library Comment Entry Starting Material 3,4 Configuration R1 R2 Product Yield (%) a 1 7aa cis PhO Bn 8aa 58 2 7ab cis PhO Ph 8ab 43 3 7bb trans Ph 8bb 28 4 7bc trans 4‐FPh 8bc 38 5 7bd trans 4‐MeOPh 8bd 53 6 7cb trans Ph 8cb 26 7 7cc trans 4‐FPh 8cc 42 8 7cd trans 4‐MeOPh 8cd 60 9 7db t Ph 8db 55 Entry Starting Material 3,4 Conﬁguration R1 R2 Product Yield (%) a 1 7aa cis PhO Bn 8aa 58 2 7ab cis PhO Ph 8ab 43 3 7bb trans Entry Starting Material 3,4 Configuration R1 R2 Product Yield (%) a 1 7aa cis PhO Bn 8aa 58 2 7ab cis PhO Ph 8ab 43 3 7bb trans Ph 8bb 28 4 7bc trans 4-FPh 8bc 38 5 7bd trans 4-MeOPh 8bd 53 6 7cb trans Ph 8cb 26 7 7cc trans 4-FPh 8cc 42 8 7cd trans 4-MeOPh 8cd 60 9 7db trans Ph 8db 55 10 7dc trans 4-FPh 8dc 52 11 7dd trans 4-MeOPh 8dd 33 Ph 8bb 28 4 7bc trans Entry Starting Material 3,4 Configuration R1 R2 Product Yield (%) a 1 7aa cis PhO Bn 8aa 58 2 7ab cis PhO Ph 8ab 43 3 7bb trans Ph 8bb 28 4 7bc trans 4-FPh 8bc 38 5 7bd trans 4-MeOPh 8bd 53 6 7cb trans Ph 8cb 26 7 7cc trans 4-FPh 8cc 42 8 7cd trans 4-MeOPh 8cd 60 9 7db trans Ph 8db 55 10 7dc trans 4-FPh 8dc 52 4-FPh 8bc 38 5 7bd trans Entry Starting Material 3,4 Configuration R1 R2 Product Yield (%) a 1 7aa cis PhO Bn 8aa 58 2 7ab cis PhO Ph 8ab 43 3 7bb trans Ph 8bb 28 4 7bc trans 4-FPh 8bc 38 5 7bd trans 4-MeOPh 8bd 53 6 7cb trans Ph 8cb 26 7 7cc trans 4-FPh 8cc 42 8 7cd trans 4-MeOPh 8cd 60 9 7db trans Ph 8db 55 4-MeOPh 8bd 53 6 7cb trans Entry Starting Material 3,4 Configuration R1 R2 Product Yield (%) a 1 7aa cis PhO Bn 8aa 58 2 7ab cis PhO Ph 8ab 43 3 7bb trans Ph 8bb 28 4 7bc trans 4-FPh 8bc 38 5 7bd trans 4-MeOPh 8bd 53 6 7cb trans Ph 8cb 26 7 7cc trans 4-FPh 8cc 42 8 7cd trans 4-MeOPh 8cd 60 9 7db trans Ph 8db 55 10 7dc trans 4-FPh 8dc 52 11 7dd trans 4-MeOPh 8dd 33 Ph 8cb 26 7 7cc trans Entry Starting Material 3,4 Configuration R1 R2 Product Yield (%) a 1 7aa cis PhO Bn 8aa 58 2 7ab cis PhO Ph 8ab 43 3 7bb trans Ph 8bb 28 4 7bc trans 4-FPh 8bc 38 5 7bd trans 4-MeOPh 8bd 53 6 7cb trans Ph 8cb 26 7 7cc trans 4-FPh 8cc 42 8 7cd trans 4-MeOPh 8cd 60 9 7db trans Ph 8db 55 10 7dc trans 4-FPh 8dc 52 4-FPh 8cc 42 8 7cd trans Entry Starting Material 3,4 Configuration R1 R2 Product Yield (%) a 1 7aa cis PhO Bn 8aa 58 2 7ab cis PhO Ph 8ab 43 3 7bb trans Ph 8bb 28 4 7bc trans 4-FPh 8bc 38 5 7bd trans 4-MeOPh 8bd 53 6 7cb trans Ph 8cb 26 7 7cc trans 4-FPh 8cc 42 8 7cd trans 4-MeOPh 8cd 60 9 7db trans Ph 8db 55 d h d 4-MeOPh 8cd 60 9 7db trans Entry Starting Material 3,4 Configuration R1 R2 Product Yield (%) a 1 7aa cis PhO Bn 8aa 58 2 7ab cis PhO Ph 8ab 43 3 7bb trans Ph 8bb 28 4 7bc trans 4-FPh 8bc 38 5 7bd trans 4-MeOPh 8bd 53 6 7cb trans Ph 8cb 26 7 7cc trans 4-FPh 8cc 42 8 7cd trans 4-MeOPh 8cd 60 9 7db trans Ph 8db 55 10 7dc trans 4-FPh 8dc 52 11 7dd trans 4-MeOPh 8dd 33 a Overall isolated yield after flash column chromatography (five steps based on loading of resin 2 D l t d Ph 8db 55 10 7dc trans Entry Starting Material 3,4 Configuration R1 R2 Product Yield (%) a 1 7aa cis PhO Bn 8aa 58 2 7ab cis PhO Ph 8ab 43 3 7bb trans Ph 8bb 28 4 7bc trans 4-FPh 8bc 38 5 7bd trans 4-MeOPh 8bd 53 6 7cb trans Ph 8cb 26 7 7cc trans 4-FPh 8cc 42 8 7cd trans 4-MeOPh 8cd 60 9 7db trans Ph 8db 55 10 7dc trans 4-FPh 8dc 52 11 7dd trans 4-MeOPh 8dd 33 4-FPh 8dc 52 11 7dd trans Entry Starting Material 3,4 Configuration R1 R2 Product Yield (%) a 1 7aa cis PhO Bn 8aa 58 2 7ab cis PhO Ph 8ab 43 3 7bb trans Ph 8bb 28 4 7bc trans 4-FPh 8bc 38 5 7bd trans 4-MeOPh 8bd 53 6 7cb trans Ph 8cb 26 7 7cc trans 4-FPh 8cc 42 8 7cd trans 4-MeOPh 8cd 60 9 7db trans Ph 8db 55 10 7dc trans 4-FPh 8dc 52 4-MeOPh 8dd 33 a Overall isolated yield after ﬂash column chromatography [ﬁve steps based on loading of resin 2 (0 40 mmol/g Molecules 2018, 23, 1193 6 of 15 6 of 15 Figure 3. 2.1. Primary β-Lactam Library AM1 geometric optimization of N-aryl and N-benzyl β-lactams. Figure 3. AM1 geometric optimization of N-aryl and N-benzyl β-lactams. Generation of this primary library was monitored by IR and 13C gel-phase NMR. A representative example of 13C gel-phase NMR of resin 7bd is shown in Figure 4. Signals assigned to the immobilized β-lactam are marked (*). The most representative signals were 55.1 ppm (MeO-), and 60.7/60.2 ppm corresponding to C-3 and C-4 of the β-lactam ring. Figure 4. 13C gel-phase NMR of immobilized β-lactam 7bd. Figure 4. 13C gel-phase NMR of immobilized β-lactam 7bd. 2.2. Secondary β-Lactam Libraries b Yield of the cross-metathesis step calculated from the ratio between product yield and the yield of the corresponding β-lactams 8bb–bd (see Table 1). c Product was esteriﬁed with diazomethane prior to puriﬁcation. 6 7bd OMe 2 BrPh 10bdc 16 41 a Overall isolated yield after flash column chromatography [four steps, based on loading of resin 2 (0.40 mmol/g, determined by elemental analysis of chlorine)]. b Yield of the cross‐metathesis step calculated from the ratio between product yield and the yield of the corresponding β‐lactams a Overall isolated yield after ﬂash column chromatography [four steps, based on loading of resin 2 (0.40 mmol/g, determined by elemental analysis of chlorine)]. b Yield of the cross-metathesis step calculated from the ratio between product yield and the yield of the corresponding β-lactams 8bb–bd (see Table 1). c Product was esteriﬁed with diazomethane prior to puriﬁcation. Hybrid structures, obtained from combining at least two biologically significant moieties, have emerged as a novel approach in finding new chemical entities [51–53]. The molecular hybridization strategy has proven to be helpful in many aspects related to drug discovery, such as overcoming drug‐resistance problems or improving active transport mechanisms. In particular, β‐lactam‐based hybrids have recently acquired importance owing to the fact that many of them exhibit very promising biological activity [54–59]. In order to further increase diversity and also obtain biologically interesting β‐lactam‐based hybrids, metathesis conditions, previously developed for the synthesis of chalcones, were applied for the cleavage step (Table 3) [60]. In this secondary library, called “Ru2”, cleavage conditions were performed by reaction of β‐lactams 7bc and 7bd with non‐immobilized α,β‐unsaturated ketones, being the most efficient precatalyst of Hoveyda‐Grubbs Hybrid structures, obtained from combining at least two biologically signiﬁcant moieties, have emerged as a novel approach in ﬁnding new chemical entities [51–53]. The molecular hybridization strategy has proven to be helpful in many aspects related to drug discovery, such as overcoming drug-resistance problems or improving active transport mechanisms. In particular, β-lactam-based hybrids have recently acquired importance owing to the fact that many of them exhibit very promising biological activity [54–59]. In order to further increase diversity and also obtain biologically interesting β-lactam-based hybrids, metathesis conditions, previously developed for the synthesis of chalcones, were applied for the cleavage step (Table 3) [60]. 2.2. Secondary β-Lactam Libraries Taking advantage of the multiple possibilities of cleavage, a series of secondary libraries was generated. Thus, TFA treatment of the primary library, followed by diazomethane methylation gave an eleven compounds’ secondary “TFA” library (Table 1). The efﬁciency of the solid-phase synthetic strategy has proven to be excellent. Yields ranged from 26% to 60% for ﬁve synthetic steps. Analyzing the reaction outcome of the obtained trans-β-lactams (entries 3–11), no clear tendency could be observed. While yields increase from R2 = Ph to R2 = 4–FPh and 4–MeOPh, in the cases of R1 = phenylpropyl and vinyl, this tendency was reverted in case of R1 = allyl (Figure 5). In order to increase diversity during cleavage step, we studied the oleﬁn cross-metathesis on the immobilized β-lactams. Thus, a new secondary library was created when β-lactams 7bb–bd, bearing a 3-phenylpropyl group at C-3 position, reacted with different oleﬁns to afford structural 7 of 15 Molecules 2018, 23, 1193 diversiﬁcation at C4 position (Table 2). This secondary library, called “Ru1”, was present in six different alternative β-lactams (10bba–bdc) obtained by reaction of the oleﬁns in presence of second generation Grubbs precatalyst. In accordance with our previous results in the area of oleﬁn cross-metathesis [32], trans-crotonic acid (entries 1 and 2), a type I oleﬁn [50] and allylbenzene (entries 3 and 4), a type II oleﬁn, performed the cleavage process with high efﬁciency, while 2-bromostyrene (entries 5 and 6), a type IV oleﬁn, provided a more poor yield. Molecules 2018, 23, x FOR PEER REVIEW 7 of 15 generation Grubbs precatalyst. In accordance with our previous results in the area of olefin cross‐metathesis [32], trans‐crotonic acid (entries 1 and 2), a type I olefin [50] and allylbenzene (entries 3 and 4), a type II olefin, performed the cleavage process with high efficiency, while 2 b t ( t i 5 d 6) t IV l fi id d i ld Yield Yield Yield Yield Yield Yield Figure 5. Yields of R1 and R2 β-lactam substituents. Figure 5. Yields of R1 and R2 β‐lactam substituents. Figure 5. Yields of R1 and R2 β-lactam substituents. Figure 5. Yields of R1 and R2 β‐lactam substituents. Table 2. The secondary “Ru1” library of β-lactams. Table 2. The secondary “Ru1” library of β‐lactams. Table 2. The secondary “Ru1” library of β-lactams. Table 2. The secondary “Ru1” library of β‐lactams. 2.2. Secondary β-Lactam Libraries Entry Starting Material R2 R3 Product Yield (%) a CM Yield (%) b 1 7bb –H MeO2C– c 10bba 25 87 2 7bd –OMe MeO2C– c 10bda 47 88 3 7bd –OMe Bn 10bdb 49 92 4 7bc –F Bn 10bcb 31 81 5 7bc –F 2‐BrPh 10bcc 27 50 6 7bd –OMe 2‐BrPh 10bdc 16 41 a Overall isolated yield after flash column chromatography [four steps, based on loading of resin 2 (0.40 mmol/g, determined by elemental analysis of chlorine)]. b Yield of the cross‐metathesis step calculated from the ratio between product yield and the yield of the corresponding β‐lactams Entry Starting Material R2 R3 Product Yield (%) a CM Yield (%) b 1 7bb –H MeO2C– c 10bba 25 87 2 7bd –OMe MeO2C– c 10bda 47 88 3 7bd –OMe Bn 10bdb 49 92 4 7bc –F Bn 10bcb 31 81 5 7bc –F 2-BrPh 10bcc 27 50 6 7bd –OMe 2-BrPh 10bdc 16 41 a Overall isolated yield after ﬂash column chromatography [four steps, based on loading of resin 2 (0.40 mmol/g, determined by elemental analysis of chlorine)]. b Yield of the cross-metathesis step calculated from the ratio between product yield and the yield of the corresponding β-lactams 8bb–bd (see Table 1). c Product was esteriﬁed with diazomethane prior to puriﬁcation. Entry Starting Material R2 R3 Product Yield (%) a CM Yield (%) b 1 7bb –H MeO2C– c 10bba 25 87 2 7bd –OMe MeO2C– c 10bda 47 88 3 7bd –OMe Bn 10bdb 49 92 4 7bc –F Bn 10bcb 31 81 5 7bc –F 2‐BrPh 10bcc 27 50 6 7bd –OMe 2‐BrPh 10bdc 16 41 a Overall isolated yield after flash column chromatography [four steps, based on loading of resin 2 (0.40 mmol/g, determined by elemental analysis of chlorine)]. b Yield of the cross‐metathesis step calculated from the ratio between product yield and the yield of the corresponding β‐lactams Entry Starting Material R2 R3 Product Yield (%) a CM Yield (%) b 1 7bb –H MeO2C– c 10bba 25 87 2 7bd –OMe MeO2C– c 10bda 47 88 3 7bd –OMe Bn 10bdb 49 92 4 7bc –F Bn 10bcb 31 81 5 7bc –F 2-BrPh 10bcc 27 50 6 7bd –OMe 2-BrPh 10bdc 16 41 a Overall isolated yield after ﬂash column chromatography [four steps, based on loading of resin 2 (0.40 mmol/g, determined by elemental analysis of chlorine)]. 2.2. Secondary β-Lactam Libraries In this secondary library, called “Ru2”, cleavage conditions were performed by reaction of β-lactams 7bc and 7bd with non-immobilized α,β-unsaturated ketones, being the most efﬁcient precatalyst of Hoveyda-Grubbs Molecules 2018, 23, 1193 8 of 15 8 of 15 8 of 15 Molecules 2018, 23, 1193 carbene ruthenium complex [61–63]. Thus, immobilized β-lactams 7bc–bd were treated with substituted vinyl phenyl ketones 9d–f in the presence of Hoveyda-Grubbs precatalyst to yield the soluble compounds 10bcd–bdg which combined two recognized pharmacophoric moieties such as azetidinone and chalcone in one molecule. Molecules 2018, 23, x FOR PEER REVIEW 8 of 15 soluble compounds 10bcd–bdg which combined two recognized pharmacophoric moieties such as azetidinone and chalcone in one molecule. Table 3. The secondary “Ru2” hybrid β–lactam library. Table 3. The secondary “Ru2” hybrid β–lactam library. Table 3. The secondary “Ru2” hybrid β–lactam library. Table 3. The secondary “Ru2” hybrid β–lactam library. Table 3. The secondary “Ru2” hybrid β–lactam library. Table 3. The secondary “Ru2” hybrid β–lactam library. Entry Starting Material R2 R3 Product Yield (%) a CM Yield (%) b 1 7bd –OMe –CH3 10bdd 20 38 2 7bd –OMe –OCH3 10bde 15 28 3 7bc –F –I 10bcf 22 58 4 7bc –F –CH2Ph 10bcg 20 c 53 a Overall isolated yield after flash column chromatography [four steps, based on loading of resin 2 (0.40 mmol/g, determined by elemental analysis of chlorine)]. b Yield of the cross‐metathesis step calculated from the ratio between product yield and the yield of the corresponding β‐lactams 8bc–bd (see Table 1) c Product could not be completely purified because of impurities Entry Starting Material R2 R3 Product Yield (%) a CM Yield (%) b 1 7bd –OMe –CH3 10bdd 20 38 2 7bd –OMe –OCH3 10bde 15 28 3 7bc –F –I 10bcf 22 58 4 7bc –F –CH2Ph 10bcg 20 c 53 a Overall isolated yield after ﬂash column chromatography [four steps, based on loading of resin 2 (0.40 mmol/g, determined by elemental analysis of chlorine)]. b Yield of the cross-metathesis step calculated from the ratio between product yield and the yield of the corresponding β-lactams 8bc–bd (see Table 1). c Product could not be completely puriﬁed because of impurities. Table 3. The secondary “Ru2” hybrid β–lactam library. Table 3. The secondary “Ru2” hybrid β–lactam library. Table 3. The secondary “Ru2” hybrid β–lactam library. Table 3. The secondary “Ru2” hybrid β–lactam library. 2.2. Secondary β-Lactam Libraries Entry Starting Material R2 R3 Product Yield (%) a CM Yield (%) b 1 7bd –OMe –CH3 10bdd 20 38 2 7bd –OMe –OCH3 10bde 15 28 3 7bc –F –I 10bcf 22 58 4 7bc –F –CH2Ph 10bcg 20 c 53 a Overall isolated yield after flash column chromatography [four steps, based on loading of resin 2 (0.40 mmol/g, determined by elemental analysis of chlorine)]. b Yield of the cross‐metathesis step calculated from the ratio between product yield and the yield of the corresponding β‐lactams 8bc–bd (see Table 1) cProduct could not be completely purified because of impurities Entry Starting Material R2 R3 Product Yield (%) a CM Yield (%) b 1 7bd –OMe –CH3 10bdd 20 38 2 7bd –OMe –OCH3 10bde 15 28 3 7bc –F –I 10bcf 22 58 4 7bc –F –CH2Ph 10bcg 20 c 53 a Overall isolated yield after ﬂash column chromatography [four steps, based on loading of resin 2 (0.40 mmol/g, determined by elemental analysis of chlorine)]. b Yield of the cross-metathesis step calculated from the ratio between product yield and the yield of the corresponding β-lactams 8bc–bd (see Table 1). c Product could not be completely puriﬁed because of impurities. Table 3. The secondary Ru2 hybr y y β y β y a Overall isolated yield after flash column chromatography [four steps, based on loading of resin 2 (0.40 mmol/g, determined by elemental analysis of chlorine)]. b Yield of the cross‐metathesis step calculated from the ratio between product yield and the yield of the corresponding β‐lactams 8bc–bd (see Table 1) cProduct could not be completely purified because of impurities a Overall isolated yield after ﬂash column chromatography [four steps, based on loading of resin 2 (0.40 mmol/g, determined by elemental analysis of chlorine)]. b Yield of the cross-metathesis step calculated from the ratio between product yield and the yield of the corresponding β-lactams 8bc–bd (see Table 1). c Product could not be completely puriﬁed because of impurities. a Overall isolated yield after flash column chromatography [four steps, based on loading of resin 2 (0.40 mmol/g, determined by elemental analysis of chlorine)]. 3.1. General Information Chemical reagents were purchased from commercial suppliers and used without further puriﬁcation, unless otherwise noted. Solvents were analytical grade or were puriﬁed by standard procedures prior to use. Reactions requiring inert atmosphere were carried out under high-purity dry nitrogen atmosphere. Solvents from these reactions were transferred under high-purity dry nitrogen using syringes. All reactions were monitored by thin layer chromatography (Merck, Darmstadt, Germany) performed on silica gel 60 F254 pre-coated aluminum sheets, visualized by a 254 nm UV lamp, and stained with an ethanolic solution of 4-anisaldehyde. Column ﬂash chromatography (Merck, Darmstadt, Germany) was performed using silica gel 60 (230–400 mesh). The purity criteria were (i) the appearance of a single spot by thin layer chromatography (ii) the presence of the corresponding signals in 1H and 13C-NMR and (iii) the range of melting points in case of solid samples. Molecular modeling was performed with HyperChem v8.03 (Hypercube, Gainesville, FL, USA) using the AM1 method Solid-phase reactions were carried out in polypropylene cartridges equipped with a frit (Supelco, Bellefonte, PA, USA), unless reﬂux conditions were required. In that case, standard glassware was used. All solid-phase reaction mixtures were stirred at slowest rate. Compounds 8bb–8dd and 10bba–10bdc have been previously reported [41]. 2.2. Secondary β-Lactam Libraries 9 of 15 Molecules 2018, 23, 1193 3.2. Instrumental and Physical Data 1H-NMR spectra were recorded in a Bruker Avance spectrometer (Bruker Analytik GmbH, Karlsruhe, Germany) at 300 MHz in CDCl3 with tetramethylsilane (TMS) as internal standard (0 ppm). 13C-NMR spectra were recorded on the same apparatus at 75 MHz with CDCl3 as solvent and reference (76.9 ppm). Chemical shifts (δ) were reported in ppm upﬁeld from TMS and coupling constants (J) were expressed in Hertz. The following abbreviations were used to indicate multiplicities: s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet, and bs = broad singlet. For preparation of the samples for 13C gel-phase NMR, 50–80 mg of resin was placed in a standard NMR tube and 0.5 mL of CDCl3 was slowly added in order to obtain a gel, which was homogenized by sonication. Spectra were run according to the literature [64,65]. Infrared spectra were recorded on a Shimadzu FT-IR spectrometer model 8101 (Shimadzu, Tokyo, Japan). The resin samples were measured as dispersions in KBr discs, made by compression of a mixture ﬁnely powdered in an agate mortar. Approximately 3 mg sample was used in 100 mg KBr. 2.2. Secondary β-Lactam Libraries b Yield of the cross‐metathesis step calculated from the ratio between product yield and the yield of the corresponding β‐lactams 8bc–bd ( bl 1) d ld b l l f d b f a Overall isolated yield after ﬂash column chromatography [four steps, based on loading of resin 2 (0.40 mmol/g, determined by elemental analysis of chlorine)]. b Yield of the cross-metathesis step calculated from the ratio between product yield and the yield of the corresponding β-lactams 8bc–bd (see Table 1). c Product could not be completely puriﬁed because of impurities. Through the analysis of the 13C‐NMR spectra of the components of obtained libraries, a clear pattern could be determined (Figure 6). Signals corresponding to substituents of all positions are slightly affected by remaining substituents. For instance, 4‐methoxy‐phenyl substituent at position 1 in azetidinones 8bd, 8cd, 8dd, 10bda, 10bdb, 10bdc, 10bdd and 10bde showed similar chemical shifts regardless the remaining substituents of β‐lactam. Signals of the three carbons of the β‐lactam ring (C‐2, C‐3 and C‐4) could clearly be identified, having only a remarkable and expected low‐field chemical shift of C‐3 in case of 3‐phenoxy‐cis‐β‐lactams (8aa and 8ab). Although there was the possibility that other signals could interfere, the patterns shown in Figure 6 were useful for a rapid detection of β‐lactam ring containing compounds obtained by high throughput parallel synthesis. Through the analysis of the 13C-NMR spectra of the components of obtained libraries, a clear pattern could be determined (Figure 6). Signals corresponding to substituents of all positions are slightly affected by remaining substituents. For instance, 4-methoxy-phenyl substituent at position 1 in azetidinones 8bd, 8cd, 8dd, 10bda, 10bdb, 10bdc, 10bdd and 10bde showed similar chemical shifts regardless the remaining substituents of β-lactam. Signals of the three carbons of the β-lactam ring (C-2, C-3 and C-4) could clearly be identiﬁed, having only a remarkable and expected low-ﬁeld chemical shift of C-3 in case of 3-phenoxy-cis-β-lactams (8aa and 8ab). Although there was the possibility that other signals could interfere, the patterns shown in Figure 6 were useful for a rapid detection of β-lactam ring containing compounds obtained by high throughput parallel synthesis. Figure 6. Comparative chart of the 13C‐NMR signals of the library components. Figure 6. Comparative chart of the 13C-NMR signals of the library components. Figure 6. Comparative chart of the 13C‐NMR signals of the library components. Figure 6. Comparative chart of the 13C-NMR signals of the library components. 3.3. Synthetic Procedures General procedure for the synthesis of 3-phenoxy-β-lactam derivatives (8aa–ab) and β-lactam-chalcone hybrids (10bdd–bcg): a mixture of 4-pentenoic acid (420 µL, 4.15 mmol, 5.0 eq.) in anhydrous DMF (10 mL) and DCC (830 µL, 4.15 mmol, 5.0 eq.) was stirred for 30 min at room temperature and transferred via cannula to Wang resin (750.0 mg, 1.1 mmol/g, 0.83 mmol), which was previously rinsed with anhydrous DMF (5.0 mL). After adding DMAP (100.0 mg, 0.83 mmol, 1.0 eq.), the reaction mixture was stirred for 16 h at room temperature. Then, resin was washed with DMF (3 × 10 mL), 1% AcOH in AcOEt (3 × 10 mL), AcOEt (3 × 10 mL), MeOH (3 × 10 mL) and CH2Cl2 (3 × 10 mL), and dried in vacuo. The immobilized pentenoate (1) (820 mg, 0.83 mmol) was suspended in anhydrous CH2Cl2 (20 mL) and 4-vinylbenzyl chloride (590 µL, 4.15 mmol, 5.0 eq.) was added via syringe under a nitrogen atmosphere. After that, Grubbs’ second generation pre-catalyst (35.0 mg, 41.5 µmol, 5 mol %) was added and the reaction was reﬂuxed for 20 h. The mixture was then ﬁltered, washed with CH2Cl2 (3 × 10 mL), MeOH (3 × 10 mL), CH2Cl2 (1 × 10 mL), and dried under high vacuum. The procedure was repeated once to ensure complete reaction. In the next step, NaHCO3 (68.5 mg, 2.2 eq.) in DMSO (25.0 mL) was added to the immobilized benzyl chloride (2) (915.0 mg, 0.37 mmol) and the mixture was heated to 155 ◦C for 6 h. After that, the suspension was ﬁltered, washed with DMSO (3 × 10 mL), CH2Cl2 (3 × 10 mL), MeOH (3 × 10 mL), CH2Cl2 (1 × 10 mL) and dried in vacuo to obtain the Wang 10 of 15 Molecules 2018, 23, 1193 resin-linked aldehyde 3. In the next step, the immobilized aldehyde 3 (121.2 mg; 0.13 mmol) was placed in a round-bottom ﬂask and suspended in anhydrous benzene (20 mL) and the corresponding amine (10 equiv.) was added. A Dean-Stark trap was then ﬁtted, ﬁlled with molecular sieves 4 Å and the suspension was heated in reﬂux for 14 h. After that, the resin was ﬁltered, washed with benzene (3 × 4 mL), DCM (3 × 4 mL), MeOH (3 × 4 mL) and CH2Cl2 (1 × 4 mL), and dried in vacuo to obtain the resin-bound imine 5. 3.3. Synthetic Procedures For 3-phenoxy-β-lactam derivatives (8aa–ab): to an aliquot of immobilized imine 5a–b (0.11 mmoles), triethylamine (0.30 mL, 2.2 mmoles, 20 eq.) and phenoxyacetyl chloride (0.23 mL, 1.65 mmoles, 15 equiv.) was added at 0 ◦C. The suspension was stirred for 16 h at room temperature. After ﬁltration, the resin was washed with CH2Cl2 (3 × 4 mL), AcOEt (3 × 4 mL), MeOH (3 × 4 mL), CH2Cl2 (1 × 4 mL), and dried under high vacuum. Then, a 10% solution of TFA in CH2Cl2 (3 mL) was added to the polymer-bound β-lactam 7aa–ab. The reaction mixture was stirred for 50 min at room temperature, ﬁltered, and washed with CH2Cl2 (3 mL). The ﬁltrate was evaporated under reduced pressure. Esteriﬁcation with diazomethane afforded the crude product that was then puriﬁed by column chromatography (hexane-AcOEt). g p y For β-lactam-chalcone hybrids (10bdd–bcg): 5-phenylvaleric acid (49 mg, 0.28 mmoles, 2.5 equiv.) and triethylamine (90 µL, 0.66 mmoles, 6 equiv.) were dissolved in anhydrous chloroform (3 mL) and added to a suspension of immobilized imine 5b–d (0.11 mmoles) in anhydrous chloroform (1.5 mL) under nitrogen atmosphere. After one minute, 2-chloro-1-methylpyridinium iodide (Mukaiyama’s reagent, 84.3 mg, 0.33 mmoles, 3 equiv.) was added and the suspension was stirred at room temperature for 24 h. Then, the reaction mixture was ﬁltered and the resin was washed successively with CH2Cl2 (3 × 4 mL), AcOEt (3 × 4 mL), MeOH (3 × 4 mL), and CH2Cl2 (1 × 4 mL). Resin 7bc–bd (0.11 mmol) was placed in a 25 mL round-bottom ﬂask, purged with dry nitrogen, suspended in anhydrous toluene (3 mL) and oleﬁn 9d–f (0.55 mmol, 5 eq.) dissolved in anhydrous toluene (3 mL) was added via syringe. After addition of Hoveyda-Grubbs precatalyst (3.4 mg, 5.5 µmol, 5 mol %), the ﬂask was ﬁtted with a reﬂux condenser with a cannula adapted to allow the elimination of generated ethylene during the reaction. The system was heated to 75 ◦C for one hour under nitrogen atmosphere. The mixture was ﬁltered and the ﬁltrate was evaporated under reduced pressure to afford the crude product. The solvent was evaporated under reduced pressure and the crude material was puriﬁed by ﬂash column chromatography (hexane-AcOEt). 3.4. 3.4. Analytical Data 3.3. Synthetic Procedures Analytical Data Methyl (E)-5-(4-(1-benzyl-4-oxo-3-phenoxyazetidin-2-yl)phenyl)pent-4-enoate: RMN de 1H (CDCl3, 300 MHz): δ 7.37–7.05 (m, 11H), 6.90–6.82 (m, 1H), 6.77–6.68 (m, 2H), 6.39 (d, J = 15.9 Hz, 1H), 6.20 (dt, J1 = 15.9 Hz, J2 = 6.6 Hz, 1H), 5.39 (d, J = 4.4 Hz, 1H), 4.89 (d, J = 14.6 Hz, 1H), 4.72 (d, J = 4.4 Hz, 1H), 3.85 (d, J = 14.6 Hz, 1H), 3.69 (s, 3H), 2.60–2.40 (m, 4H). RMN de 13C (CDCl3, 75 MHz): δ 173.2, 165.4, 156.9, 137.6, 134.6, 131.4, 130.3, 129.1, 129.0, 128.8, 128.7, 128.5, 127.8, 125.9, 121.9, 115.4, 82.1, 61.1, 51.5, 44.0, 33.6, 28.1. Methyl (E)-5-(4-(1-benzyl-4-oxo-3-phenoxyazetidin-2-yl)phenyl)pent-4-enoate: RMN de 1H (CDCl3, 300 MHz): δ 7.37–7.05 (m, 11H), 6.90–6.82 (m, 1H), 6.77–6.68 (m, 2H), 6.39 (d, J = 15.9 Hz, 1H), 6.20 (dt, J1 = 15.9 Hz, J2 = 6.6 Hz, 1H), 5.39 (d, J = 4.4 Hz, 1H), 4.89 (d, J = 14.6 Hz, 1H), 4.72 (d, J = 4.4 Hz, 1H), 3.85 (d, J = 14.6 Hz, 1H), 3.69 (s, 3H), 2.60–2.40 (m, 4H). RMN de 13C (CDCl3, 75 MHz): δ 173.2, 165.4, 156.9, 137.6, 134.6, 131.4, 130.3, 129.1, 129.0, 128.8, 128.7, 128.5, 127.8, 125.9, 121.9, 115.4, 82.1, 61.1, 51.5, 44.0, 33.6, 28.1. Molecules 2018, 23, 1193 11 of 15 Methyl (E)-5-(4-(4-oxo-3-phenoxy-1-phenylazetidin-2-yl)phenyl)pent-4-enoate: RMN de 1H (CDCl3, 300 MHz): δ 7.40–7.02 (m, 11H), 6.98–6.90 (m, 1H), 6.86–6.75 (m, 2H), 6,37 (d, J = 15.9 Hz, 1H), 6.18 (dt, J1 = 15.9 Hz, J2 = 6.2 Hz, 1H), 5.56 (d, J = 5.0 Hz, 1H), 5.37 (d, J = 5.0 Hz, 1H), 3.68 (s, 3H), 2.55–2.40 (m, 4H). RMN de 13C (CDCl3, 75 MHz): δ 173.2, 163.0, 156.9, 137.7, 136.8, 131.3, 130.3, 129.2, 129.0, 128.2, 126.0, 124.5, 122.1, 117.5, 115.7, 81.2, 61.8, 51.5, 33.6, 28.1. 11 of 15 Molecules 2018, 23, 1193 Methyl (E)-5-(4-(4-oxo-3-phenoxy-1-phenylazetidin-2-yl)phenyl)pent-4-enoate: RMN de 1H (CDCl3, 300 MHz): δ 7.40–7.02 (m, 11H), 6.98–6.90 (m, 1H), 6.86–6.75 (m, 2H), 6,37 (d, J = 15.9 Hz, 1H), 6.18 (dt, J1 = 15.9 Hz, J2 = 6.2 Hz, 1H), 5.56 (d, J = 5.0 Hz, 1H), 5.37 (d, J = 5.0 Hz, 1H), 3.68 (s, 3H), 2.55–2.40 (m, 4H). RMN de 13C (CDCl3, 75 MHz): δ 173.2, 163.0, 156.9, 137.7, 136.8, 131.3, 130.3, 129.2, 129.0, 128.2, 126.0, 124.5, 122.1, 117.5, 115.7, 81.2, 61.8, 51.5, 33.6, 28.1. 3.3. Synthetic Procedures 1-(4-Methoxy-phenyl)-4-[4-(3-oxo-3-p-tolyl-propenyl)-phenyl]-3-(3-phenyl-propyl)-azetidin-2-one: 1H-NMR: 7.92 (d, J = 8.1 Hz, 2H), 7.76 (d, J = 15.9 Hz, 1H), 7.62 (d, J = 8.1 Hz, 2H), 7.51 (d, J = 15.9 Hz, 1H), 7.37 (d, J = 8.1 Hz, 2H), 7.31–7.14 (m, 7H), 6.78 (d, J = 9 Hz, 2H), 4.62 (d, J = 2.1 Hz, 1 H), 3.73 (s, 3H), 3.13–3.07 (m, 1H), 2.66 (t, J = 7 Hz, 2H), 2.43 (s, 3H), 1.99–185 (m, 4H). 13C-NMR: 189.8, 166.7, 156.0, 143.8, 143.3, 141.5, 140.6, 135.5, 135.2, 131.1, 129.3, 129.1, 128.6, 128.4, 126.4, 125.9, 122.5, 118.1, 114.3, 60.8, 60.6, 55.4, 35.7, 28.9, 28.4, 21.6. HRMS calcd. for: C35H33NNaO3+; (M + Na+, m/z): 538.23527; found: 538.23673. 1-(4-Methoxy-phenyl)-4-{4-[3-(4-methoxy-phenyl)-3-oxo-propenyl]-phenyl}-3-(3-phenyl-propyl)-azetidin-2-one: 1H-NMR: 8.02 (d, J = 8.8 Hz, 2H), 7.76 (d, J = 15.7 Hz, 1H), 7.63 (d, J = 8.2 Hz, 2H), 7.52 (d, J = 15.7 Hz, 1H), 7.36 (d, J = 8.2 Hz, 2H) 7.30–7.14 (m, 7H), 6.97 (d, J = 8.8 Hz, 2H), 6.77 (d, J = 9.0 Hz, 2H), 4.62 (d, J = 2.1 Hz, 1H), 3.89 (s, 3H), 3.73 (s, 3H), 3.12–2.99 (m, 1H), 2.68–2.63 (m, 2H), 1.98–1.82 (m, 4H). 13C-NMR: 188.5, 166.8, 163.5, 156.0, 142.9, 141.5, 140.5, 135.3, 131.1, 130.9, 130.8, 129.1, 128.4, 126.4, 125.9, 122.3, 118.1, 114.3, 113.9, 60.8, 60.5, 55.5, 55.4, 35.7, 28.9, 28.4. HRMS calcd. for C35H33NNaO4+ (M + Na+, m/z): 554.23018; found: 554.22853. 1-(4-Methoxy-phenyl)-4-{4-[3-(4-methoxy-phenyl)-3-oxo-propenyl]-phenyl}-3-(3-phenyl-propyl)-azetidin-2-one: 1H-NMR: 8.02 (d, J = 8.8 Hz, 2H), 7.76 (d, J = 15.7 Hz, 1H), 7.63 (d, J = 8.2 Hz, 2H), 7.52 (d, J = 15.7 Hz, 1H), 7.36 (d, J = 8.2 Hz, 2H) 7.30–7.14 (m, 7H), 6.97 (d, J = 8.8 Hz, 2H), 6.77 (d, J = 9.0 Hz, 2H), 4.62 (d, J = 2.1 Hz, 1H), 3.89 (s, 3H), 3.73 (s, 3H), 3.12–2.99 (m, 1H), 2.68–2.63 (m, 2H), 1.98–1.82 (m, 4H). 13C-NMR: 188.5, 166.8, 163.5, 156.0, 142.9, 141.5, 140.5, 135.3, 131.1, 130.9, 130.8, 129.1, 128.4, 126.4, 125.9, 122.3, 118.1, 114.3, 113.9, 60.8, 60.5, 55.5, 55.4, 35.7, 28.9, 28.4. HRMS calcd. for C35H33NNaO4+ (M + Na+, m/z): 554.23018; found: 554.22853. 4. Conclusions An interesting application of DOS of biologically promising compounds has been developed. A key feature was the application of the reagent-based diversiﬁcation approach using a linker with different possibilities of cleavage. Starting from an immobilized primary library, diversity was expanded through many alternatives according to the reaction conditions used. A series of cholesterol absorption inhibitor analogues was obtained, as well as interesting hybrid compounds through incorporation of a chalcone group via cross-metathesis. Here, we have demonstrated the reliability of the methodology which can be suitable for generating large libraries of analogue structures. Author Contributions: A.A.P.-P. and E.G.M. conceived and designed the experiments; L.M. and A.A.P.-P. performed the experiments and analyzed the data; L.M., A.A.P.-P., and E.G.M. wrote the paper. Acknowledgments: Support from CONICET, ANPCyT, Agencia Santafesina de Ciencia, Técnica e Innovación (ASACTEI) and Universidad Nacional de Rosario from Argentina is gratefully acknowledged. Conﬂicts of Interest: The authors declare no conﬂict of interest. The funding sponsors had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the decision to publish the results. 3.3. Synthetic Procedures Molecules 2018, 23, 1193 12 of 15 1-(4-Fluoro-phenyl)-4-{4-[3-(4-iodo-phenyl)-3-oxo-propenyl]-phenyl}-3-(3-phenyl-propyl) azetidin-2-one: 1H-NMR: 7.87 (d, J = 8.5 Hz, 2H), 7.78 (d, J = 15.6 Hz, 1H), 7.70 (d, J = 8.5 Hz, 2H), 7.63 (d, J = 8.1 Hz, 2H), 7.44 (d, J = 15.6 Hz, 1H), 7.37 (d, J = 8.1 Hz, 2H), 7.28–7.14 (m, 7H), 6.93 (m, 2H), 4.63 (d, J = 2.1 Hz, 1H), 3.10 (m, 1H), 2.67 (m, 2H), 2.0–1.82 (m 4H). 13C-NMR: 189.4, 167.0, 144.2, 141.4, 140.5, 137.9, 137.2, 135.1, 133.8, 129.8, 129.3, 128.4, 128.3, 127.9, 126.8, 126.5, 126.0, 125.6, 122.0, 118.3, 118.2, 116.0, 115.7, 100.8, 60.9, 60.8, 35.6, 28.9, 28.4. HRMS calcd. For C33H27FINNaO2+ (M + Na+, m/z): 638.09627; found: 638.09727. 12 of 15 12 of 15 Molecules 2018, 23, 1193 1-(4-Fluoro-phenyl)-4-{4-[3-(4-iodo-phenyl)-3-oxo-propenyl]-phenyl}-3-(3-phenyl-propyl) azetidin-2-one: 1H-NMR: 7.87 (d, J = 8.5 Hz, 2H), 7.78 (d, J = 15.6 Hz, 1H), 7.70 (d, J = 8.5 Hz, 2H), 7.63 (d, J = 8.1 Hz, 2H), 7.44 (d, J = 15.6 Hz, 1H), 7.37 (d, J = 8.1 Hz, 2H), 7.28–7.14 (m, 7H), 6.93 (m, 2H), 4.63 (d, J = 2.1 Hz, 1H), 3.10 (m, 1H), 2.67 (m, 2H), 2.0–1.82 (m 4H). 13C-NMR: 189.4, 167.0, 144.2, 141.4, 140.5, 137.9, 137.2, 135.1, 133.8, 129.8, 129.3, 128.4, 128.3, 127.9, 126.8, 126.5, 126.0, 125.6, 122.0, 118.3, 118.2, 116.0, 115.7, 100.8, 60.9, 60.8, 35.6, 28.9, 28.4. HRMS calcd. For C33H27FINNaO2+ (M + Na+, m/z): 638.09627; found: 638.09727. 1-(4-Fluoro-phenyl)-4-{4-[3-(4-iodo-phenyl)-3-oxo-propenyl]-phenyl}-3-(3-phenyl-propyl) azetidin-2-one: 1H-NMR: 7.87 (d, J = 8.5 Hz, 2H), 7.78 (d, J = 15.6 Hz, 1H), 7.70 (d, J = 8.5 Hz, 2H), 7.63 (d, J = 8.1 Hz, 2H), 7.44 (d, J = 15.6 Hz, 1H), 7.37 (d, J = 8.1 Hz, 2H), 7.28–7.14 (m, 7H), 6.93 (m, 2H), 4.63 (d, J = 2.1 Hz, 1H), 3.10 (m, 1H), 2.67 (m, 2H), 2.0–1.82 (m 4H). 13C-NMR: 189.4, 167.0, 144.2, 141.4, 140.5, 137.9, 137.2, 135.1, 133.8, 129.8, 129.3, 128.4, 128.3, 127.9, 126.8, 126.5, 126.0, 125.6, 122.0, 118.3, 118.2, 116.0, 115.7, 100.8, 60.9, 60.8, 35.6, 28.9, 28.4. HRMS calcd. For C33H27FINNaO2+ (M + Na+, m/z): 638.09627; found: 638.09727. References and Note 1. Arya, N.; Jagdale, A.Y.; Patil, T.A.; Yeramwar, S.S.; Holikatti, S.S.; Dwivedi, J.; Shishoo, C.J.; Jain, K.S. The chemistry and biological potential of azetidin-2-ones. Eur. J. Med. Chem. 2014, 74, 619–656. [CrossRef] [PubMed] 1. 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References and Note This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/). © 2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
https://openalex.org/W2018305051	https://hal-pasteur.archives-ouvertes.fr/pasteur-00455158/file/journal.ppat.1000521.pdf	English	null	NRP/Optineurin Cooperates with TAX1BP1 to Potentiate the Activation of NF-κB by Human T-Lymphotropic Virus Type 1 Tax Protein	PLOS pathogens	2,009	cc-by	10,889	To cite this version: Chloé Journo, Josina Filipe, Frédégonde About, Sébastien A. Chevalier, Philippe V. Afonso, et al.. NRP/Optineurin Cooperates with TAX1BP1 to potentiate the activation of NF-kappaB by human T-lymphotropic virus type 1 tax protein.. PLoS Pathogens, 2009, 5 (7), pp.e1000521. 10.1371/jour- nal.ppat.1000521. pasteur-00455158 NRP/Optineurin Cooperates with TAX1BP1 to potentiate the activation of NF-kappaB by human T-lymphotropic virus type 1 tax protein. Chloé Journo, Josina Filipe, Frédégonde About, Sébastien A. Chevalier, Philippe V. Afonso, John N. Brady, David Flynn, Frédéric Tangy, Alain Israël, Pierre-Olivier Vidalain, et al. Abstract Nuclear factor (NF)-kB is a major survival pathway engaged by the Human T-Lymphotropic Virus type 1 (HTLV-1) Tax protein. Tax1 activation of NF-kB occurs predominantly in the cytoplasm, where Tax1 binds NF-kB Essential Modulator (NEMO/IKKc) and triggers the activation of IkB kinases. Several independent studies have shown that Tax1-mediated NF-kB activation is dependent on Tax1 ubiquitination. Here, we identify by co-immunoprecipitation assays NEMO-Related Protein (NRP/Optineurin) as a binding partner for Tax1 in HTLV-1 infected and Tax1/NRP co-expressing cells. Immunofluorescence studies reveal that Tax1, NRP and NEMO colocalize in Golgi-associated structures. The interaction between Tax1 and NRP requires the ubiquitin-binding activity of NRP and the ubiquitination sites of Tax1. In addition, we observe that NRP increases the ubiquitination of Tax1 along with Tax1-dependent NF-kB signaling. Surprisingly, we find that in addition to Tax1, NRP interacts cooperatively with the Tax1 binding protein TAX1BP1, and that NRP and TAX1BP1 cooperate to modulate Tax1 ubiquitination and NF-kB activation. Our data strongly suggest for the first time that NRP is a critical adaptor that regulates the assembly of TAX1BP1 and post-translationally modified forms of Tax1, leading to sustained NF-kB activation. Citation: Journo C, Filipe J, About F, Chevalier SA, Afonso PV, et al. (2009) NRP/Optineurin Cooperates with TAX1BP1 to Potentiate the Activation of NF-kB by Human T-Lymphotropic Virus Type 1 Tax Protein. PLoS Pathog 5(7): e1000521. doi:10.1371/journal.ppat.1000521 Editor: Susan Ross, University of Pennsylvania School of Medicine, United States of America Editor: Susan Ross, University of Pennsylvania School of Medicine, United States of America Editor: Susan Ross, University of Pennsylvania School of Medicine, United States of America Received March 31, 2009; Accepted June 22, 2009; Published July 17, 2009 Received March 31, 2009; Accepted June 22, 2009; Published July 17, 2009 This is an open-access article distributed under the terms of the Creative Commons Public Domain declaration which stipulates that, once placed in the public domain, this work may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. This is an open-access article distributed under the terms of the Creative Commons Public Domain declaration which stipu domain, this work may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone Funding: RM was supported by INSERM and is now supported by E´cole Normale Supe´rieure de Lyon. RW is supported by CNRS. CJ, SAL and PVA were supported by the Ministe`re de la Recherche. Abstract JF is a recipient of the Fundac¸a˜o para a Cieˆncia e a Tecnologia PhD fellowship. DF was supported by a Fulbright Fellowship through the U.S. Department of State Bureau of Educational and Cultural Affairs. This work was supported by PTR214 from the Pasteur Institute and by the Rubicon FP6 European Network of Excellence to AI. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Competing Interests: The authors have declared that no competing interests exist. * E-mail: renaud.mahieux@ens-lyon.fr * E-mail: renaud.mahieux@ens-lyon.fr . These authors contributed equally to this work. " These authors are joint senior authors on this work. { Deceased. HAL Id: pasteur-00455158 https://pasteur.hal.science/pasteur-00455158v1 Submitted on 11 Feb 2010 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. NRP/Optineurin Cooperates with TAX1BP1 to Potentiate the Activation of NF-kB by Human T-Lymphotropic Virus Type 1 Tax Protein Chloe´ Journo1,2,3,4., Josina Filipe5., Fre´de´gonde About1, Se´bastien A. Chevalier6, Philippe V. Afonso1, John N. Brady6{, David Flynn7, Fre´de´ric Tangy7, Alain Israe¨l5, Pierre-Olivier Vidalain7, Renaud Mahieux1,2,3,4"*, Robert Weil5" 1 Unite´ d’Epide´miologie et Physiopathologie des Virus Oncoge`nes, CNRS URA 3015, Institut Pasteur, Paris, France, 2 Oncogene`se Re´trovirale, INSERM U758, Lyon, France, 3 Ecole Normale Supe´rieure, Lyon, France, 4 IFR 128 BioSciences Lyon-Gerland, Lyon, France, 5 Unite´ de Signalisation Mole´culaire et Activation Cellulaire, CNRS URA 2582, Institut Pasteur, Paris, France, 6 Laboratory of Cellular Oncology, NIH/NCI, Bethesda, Maryland, United States of America, 7 Laboratoire de Ge´nomique Virale et Vaccination, Institut Pasteur, Paris, France PLoS Pathogens \| www.plospathogens.org Both Tax1 and Tax2 proteins co-precipitate with NRP In order to confirm Tax/NRP interaction, Tax1 and Tax2 were co-expressed in 293T cells with VSV-tagged NRP and co- immunoprecipitations were performed. Both Tax2 (Figure 1A, lane 2) and Tax1 (Figure 1B, left panel, lane 3) were detected in VSV-NRP immunoprecipitates. IRAK1 respectively, which are required for binding to NEMO and subsequent activation of NF-kB [7,8]. One of the main mechanisms restricting this process is the NF-kB-mediated induction of deubiquitinases such as A20 and CYLD [9,10]. The reverse experiment (i.e. immunoprecipitation of Tax1 followed by immunoblotting for VSV-NRP) confirmed the interaction between the two proteins (Figure 1B, right panel, lane 3). We then aimed to demonstrate the interaction between Tax1 and endogenous NRP in two HTLV1-infected cell lines, C8166 and C91PL. The uninfected cell line CEM was used here as a negative control. As expected, Tax1 could be specifically recovered from NRP immunoprecipitates in both HTLV-1- infected cell lines (Figure 1C, compare lanes 3 and 5 with lane 1). The specificity of these interactions was controlled using an irrelevant antibody for the immunoprecipitation (Figure 1C, lanes 2, 4 and 6). Hence, NRP interacts with Tax1 both in transfected and in infected cells. NF-kB activation plays a critical role in HTLV-1-mediated oncogenesis. This process occurs predominantly in the cytoplasm where HTLV-1 Tax1 binds NEMO and triggers the activation of IKKa and IKKb [11–13]. Tax1 can also stimulate the alternative pathway of NF-kB activation through the IKKa-dependent processing of NF-kB p100 precursor protein [14]. Independent studies have shown that Tax1 ubiquitination is dependent on the E2 ubiquitin-conjugating enzyme Ubc13 and is critical for Tax1 binding to NEMO and the subsequent NF-kB activation [15–18]. In addition, Tax1 binding protein TAX1BP1 [19,20] is involved in the recruitment of A20 deubiquitinase and the negative control of TNF-a-, IL-1- and LPS-mediated NF-kB activation [17], suggesting that Tax1-dependent activation of NF-kB could also be more complex than originally thought. Because cellular factors other than NEMO and Ubc13 could contribute to the activation of NF-kB by Tax1, we searched for novel interactors of Tax1 and Tax2, the equivalent of Tax1 for HTLV-2. Here we report the identification of NEMO-Related Protein (NRP) as a novel Tax interactor. NRP is ubiquitously expressed and exhibits strong homologies to NEMO (53% sequence similarity), but its function is still unknown [21]. Introduction and antigens can activate NF-kB. NF-kB activity is tightly regulated by inhibitory IkB proteins. Upon stimulation, signals are transduced that lead to the degradation of IkB, allowing NF- kB to translocate into the nucleus and to activate its target genes. IkB degradation by the 26S proteasome is triggered by its phosphorylation by a multisubunit IkB kinase (IKK) complex that contains two homologous catalytic subunits (IKKa and IKKb) and a regulatory subunit, NF-kB Essential Modulator (NEMO/IKKc). An important mechanism in the NF-kB pathway is the interaction between NEMO and K63-linked polyubiquitin chains. In the case of TNF-a stimulation, the attachment of the polyubiquitin chains to RIP1 serves to bring the NEMO/IKK complex to the TNF-a receptor and is required for NF-kB activation [6]. Other studies have shown that TCR and IL-1 stimulations induce the attachment of K63-linked polyubiquitin chains to Bcl10 and Human T-Lymphotropic Virus type 1 (HTLV-1) is the etiological agent of Adult T cell Leukemia/Lymphoma (ATL) and of HTLV-Associated Myelopathy/Tropical Spastic Parapa- resis (HAM/TSP) [1–3]. HTLV-1 contains a unique pX region in the 39 portion of its genome, which encodes regulatory and accessory proteins that are involved in viral replication and cell proliferation. Among them, Tax1 plays a critical role by triggering cell immortalization through various mechanisms [4], including activation of signaling pathways such as NF-kB [5]. The NF-kB family of transcription factors plays an important role in the regulation of cellular activation, proliferation, and survival. A large number of stimuli including bacterial lipopoly- saccharide (LPS), tumor necrosis factor (TNF)-a, interleukin (IL)-1 1 July 2009 \| Volume 5 \| Issue 7 \| e1000521 NRP Potentiates Tax Activity or Tax2 proteins were fused to Gal4 DNA binding domain (Gal4- BD) and used as baits to screen a library of human spleen cDNA fused to Gal4 transactivation domain (Gal4-AD). Screens were performed by mating to reach a five-time coverage of the cDNA library complexity, and yielded 59 positive yeast colonies with Tax1 and 36 with Tax2. Using Tax1 as bait, we retrieved three previously known interactors of Tax1 [TAX1BP1 (2 colonies), TAX1BP3 (37 colonies) and SRF (3 colonies)], which demon- strates the specificity of the screen. NEMO was only found using Tax2 as bait (2 colonies). This corresponds to a known limitation of the two-hybrid system since numerous pairs of interacting proteins fail to rebuild a functional transcription factor in yeast when fused to Gal4-DB and Gal4-AD. Introduction From the screen performed with Tax2, we essentially selected yeast colonies expressing a new interactor of Tax: NEMO-Related Protein NRP (23 colonies). Although various lengths of the NRP protein were encoded, the smallest NRP fragment shown to interact with Tax2 was spanning amino acid 411 to the C-terminus end of the protein. This led us to test the ability of Tax1 to interact with NRP using a different binding assay. PLoS Pathogens \| www.plospathogens.org Results Tax1 has been described as having distinct localizations depending on its post-translational modification status [15,16]. Ubiquitinated Tax1 was reported to localize in the cytoplasm, more specifically in Golgi/centrosome-associated structures [18], Both Tax1 and Tax2 proteins co-precipitate with NRP Mutations in its sequence have been associated with primary open-angle glaucoma (POAG), and for this reason, NRP was also named Optineurin for ‘‘optic neuropathy inducing’’ protein [22]. In order to confirm the interaction with an alternative biochemical method, we determined whether Tax1 and NRP could be found in the same fractions after gel filtration (Figure 1D). Experiments using glycerol gradients were also performed (Figure S1). Tax1 and HA-tagged NRP were co-expressed in HeLa cells. After gel filtration, extracts were analyzed by western blot. As shown in Figure 1D, Tax1 was recovered from fractions 33 to 35 (Figure 1D, lower panel), corresponding to low molecular mass fractions. This subset of Tax1 molecules essentially represents free molecules. Tax1 was also recovered from fraction 25, suggesting that a subset of Tax1 molecules was present in high molecular mass complexes. HA-NRP was recovered from fractions 22 to 26 (Figure 1D, upper panel), showing that a majority of HA-NRP molecules were found in high molecular mass complexes. Co- fractionation of Tax1 and HA-NRP in fraction 25 indicated that both proteins could be found in the same complexes. We show that both NRP and TAX1BP1 form a functional complex with Tax1, and we demonstrate that a synergistic interaction between TAX1BP1 and NRP contributes to Tax1- mediated NF-kB activation. These results obtained with distinct biochemical methods support the interaction between Tax1 and NRP. Author Summary Oncogenic viruses (i.e., viruses that can induce cancer) have usually been found to deregulate several cellular signaling pathways controlling cell survival and prolifera- tion. Among those, the NF-kB pathway is particularly important. In this study, we focus on the Human T- Lymphotropic Virus type 1 (HTLV-1), which infects immune T cells, and is associated with the development of a severe hematological disease, termed adult T cell leukemia. The viral Tax oncoprotein is known to activate the NF-kB pathway, but the precise mechanism is still under investigation. In cells, proteins can undergo modifications that can modulate their function. In the case of Tax, a modified form of the protein (ubiquitinated Tax) is able to activate the NF-kB pathway. Our aim was to identify cellular proteins that participate in the modification of Tax, and in turn in the regulation of its function. We show for the first time that the cellular protein NRP/Optineurin interacts with Tax and increases its ubiquitination, thus leading to an enhanced NF-kB activation. We further demonstrate that TAX1BP1, another cellular protein that had been previously identified as a partner of Tax, also participates in this regulation. Thus, this study uncovers new actors of the virally induced cell signaling. Tax2 interacts with NRP in a yeast two-hybrid screen As previously described, Tax1-GFP showed a discrete granular appearance in the cytoplasm, which was more intense at the Golgi apparatus as shown by colocalization with GM130 staining (Figure 2A, green and blue). Staining for NEMO indicated that expression of Tax1-GFP induced the recruitment of this protein at the Golgi apparatus where both proteins colocalize (Figure 2B, green and blue). As expected, NRP was predominantly localized at the Golgi apparatus, and its localiza- tion was not affected by Tax1-GFP expression (Figure 2A and B, red). Interestingly, we also observed colocalization between Tax1- GFP and NRP in these Golgi-associated structures (Figure 2A and B). These results are consistent with the observed interaction between Tax1 and NRP in vivo, and suggest that these interactions occur at the Golgi apparatus where NEMO is recruited. Because it has been reported that Tax1 interacts with NEMO at the Golgi apparatus in an ubiquitin-dependent manner, we hypothesized that a similar mechanism could be involved in the Tax1/NRP interaction. NRP ubiquitin-binding domain (UBD) mediates binding to Tax1 Tax2 interacts with NRP in a yeast two-hybrid screen Since Tax1 was shown to promote the relocaliza- tion of the NEMO/IKK complex to the Golgi apparatus [23], we suspected that NRP might also interact with Tax1 at the Golgi apparatus. We performed a series of immunofluorescence stainings in HeLa cells expressing a C-terminal GFP-tagged Tax1 plasmid, which was previously demonstrated to have a subcellular localization and an ability to activate NF-kB similar to those of untagged Tax1 ([24] and data not shown). To evaluate whether NRP colocalizes with Tax1-GFP and NEMO at the Golgi apparatus, we performed a double staining for NRP and either NEMO or GM130 in order to visualize the Golgi apparatus (Figure 2). As previously described, Tax1-GFP showed a discrete granular appearance in the cytoplasm, which was more intense at the Golgi apparatus as shown by colocalization with GM130 staining (Figure 2A, green and blue). Staining for NEMO indicated that expression of Tax1-GFP induced the recruitment of this protein at the Golgi apparatus where both proteins colocalize (Figure 2B, green and blue). As expected, NRP was predominantly localized at the Golgi apparatus, and its localiza- tion was not affected by Tax1-GFP expression (Figure 2A and B, red). Interestingly, we also observed colocalization between Tax1- GFP and NRP in these Golgi-associated structures (Figure 2A and B). Tax1-GFP, which colocalized with NRP (Figure S2A and B, red) and NEMO (Figure S2B, blue) in perinuclear structures that were associated with the Golgi apparatus (Figure S2A, blue). As in HeLa cells, we observed that Tax1-GFP expression had no effect on NRP localization (Figure S2, compare A and C). where it interacts with NEMO and induces NF-kB activation. SUMOylated Tax1, however, was found in the nucleus [15,16]. NRP, on the other hand, is predominantly localized at the Golgi apparatus [21]. Since Tax1 was shown to promote the relocaliza- tion of the NEMO/IKK complex to the Golgi apparatus [23], we suspected that NRP might also interact with Tax1 at the Golgi apparatus. We performed a series of immunofluorescence stainings in HeLa cells expressing a C-terminal GFP-tagged Tax1 plasmid, which was previously demonstrated to have a subcellular localization and an ability to activate NF-kB similar to those of untagged Tax1 ([24] and data not shown). To evaluate whether NRP colocalizes with Tax1-GFP and NEMO at the Golgi apparatus, we performed a double staining for NRP and either NEMO or GM130 in order to visualize the Golgi apparatus (Figure 2). PLoS Pathogens \| www.plospathogens.org Tax2 interacts with NRP in a yeast two-hybrid screen Tax2 interacts with NRP in a yeast two-hybrid screen To identify novel binding partners of Tax proteins, we used a standard yeast two-hybrid screening procedure. Full-length Tax1 July 2009 \| Volume 5 \| Issue 7 \| e1000521 2 NRP Potentiates Tax Activity Figure 1. Co-immunoprecipitation and co-fractionation of Tax and NRP. (A) 293T cells were transfected with Tax2 and VSV-NRP as indicated. Total lysates were immunoprecipitated with an anti-VSV antibody and western blot analysis was performed using anti-Tax2 and anti-VSV antibodies. (B) 293T cells were transfected with Tax1 and VSV-NRP as indicated. Total lysates were immunoprecipitated with an anti-VSV or anti-Tax1 antibody and western blot analysis was performed using anti-Tax1 and anti-VSV antibodies. (C) Total lysates from CEM, C8166 and C91PL cell lines were immunoprecipitated with an anti-NRP antibody (+) or an irrelevant anti-His antibody (2) and western blot analysis was performed using anti- NRP and anti-Tax1 antibodies. (D) HeLa cells were transfected with Tax1 and HA-NRP, and cell extracts were analyzed by gel filtration chromatography. Fractions 16 to 40 as well as total extracts (input) were then analyzed by western blotting using antibodies directed against HA (upper panel) or Tax1 (lower panel). Precalibration of the column is indicated beneath the fractions count (kDa). doi:10.1371/journal.ppat.1000521.g001 Figure 1. Co-immunoprecipitation and co-fractionation of Tax and NRP. (A) 293T cells were transfected with Tax2 and VSV-NRP as indicated. Total lysates were immunoprecipitated with an anti-VSV antibody and western blot analysis was performed using anti-Tax2 and anti-VSV antibodies. (B) 293T cells were transfected with Tax1 and VSV-NRP as indicated. Total lysates were immunoprecipitated with an anti-VSV or anti-Tax1 antibody and western blot analysis was performed using anti-Tax1 and anti-VSV antibodies. (C) Total lysates from CEM, C8166 and C91PL cell lines were immunoprecipitated with an anti-NRP antibody (+) or an irrelevant anti-His antibody (2) and western blot analysis was performed using anti- NRP and anti-Tax1 antibodies. (D) HeLa cells were transfected with Tax1 and HA-NRP, and cell extracts were analyzed by gel filtration chromatography. Fractions 16 to 40 as well as total extracts (input) were then analyzed by western blotting using antibodies directed against HA (upper panel) or Tax1 (lower panel). Precalibration of the column is indicated beneath the fractions count (kDa). doi:10.1371/journal.ppat.1000521.g001 where it interacts with NEMO and induces NF-kB activation. SUMOylated Tax1, however, was found in the nucleus [15,16]. NRP, on the other hand, is predominantly localized at the Golgi apparatus [21]. July 2009 \| Volume 5 \| Issue 7 \| e1000521 Binding of NRP stabilizes Tax1 polyubiquitination Tax1 ubiquitination is critical for its binding to the NEMO/ IKK complex and its subsequent activation [15,16,18]. Given that NRP binds to polyubiquitinated Tax1, we wondered whether it could modulate Tax1 polyubiquitination status, and hence its ability to activate the NEMO/IKK complex. We thus analyzed the effect of silencing NRP on the level of polyubiquitinated Tax1. His-tagged Tax1 and HA-tagged ubiquitin were co-expressed in 293T cells, with a control siRNA (Figure 4A, lane 1) or with a siRNA targeting NRP (Figure 4A, lane 2). Ni-NTA pulldown was then performed in highly reducing and denaturating conditions, in order to avoid any deubiquitination and to ensure that only products covalently linked to Tax1 would be purified. By blotting for HA-ubiquitin, we assessed the level of polyubiquitinated Tax1 in each sample, which appears as high-molecular-weight products. When compared to control cells, the level of polyubiquitinated Tax1 was strikingly reduced in NRP-silenced cells, (Figure 4A, Figure 2. Colocalization of Tax1, NRP and NEMO in Golgi-associated structures in HeLa cells. HeLa cells were transfected with Tax1-GFP. Cells were stained with an anti-NRP antibody (red) and either (A) an anti-GM130 or (B) an anti-NEMO antibody (blue). Cells were observed as described in the Materials and Methods section. Differential interference contrast is shown on the left merge image. Scale bar = 10 mm. doi:10.1371/journal.ppat.1000521.g002 Figure 2. Colocalization of Tax1, NRP and NEMO in Golgi-associated structures in HeLa cells. HeLa cells were transfected with Tax1-GFP. Cells were stained with an anti-NRP antibody (red) and either (A) an anti-GM130 or (B) an anti-NEMO antibody (blue). Cells were observed as described in the Materials and Methods section. Differential interference contrast is shown on the left merge image. Scale bar = 10 mm. doi:10.1371/journal.ppat.1000521.g002 acceptor sites for ubiquitin is critical for the interaction with NRP. Taken together, these results further support the hypothesis that NRP binds to Tax1 through polyubiquitin chains on Tax1. (Figure 3C). Interestingly, a single point mutation in this domain known to disrupt the binding to K63-linked polyubiquitin (D474N) [25] was sufficient to severely reduce the interaction of NRP with Tax1 (Figure 3C, compare lane 1 and 2). This result suggests that NRP binding to Tax1 is mediated by an interaction between NRP UBD and K63-linked polyubiquitin chains conjugated to Tax1. PLoS Pathogens \| www.plospathogens.org July 2009 \| Volume 5 \| Issue 7 \| e1000521 NRP ubiquitin-binding domain (UBD) mediates binding to Tax1 In order to map the domain(s) of NRP involved in the binding to Tax1, we performed co-immunoprecipitation experiments using a series of NRP mutants. We first co-expressed Tax1 with either a N-terminal or a C-terminal deletion mutant of NRP (Figure 3A). Immunoprecipitation of NRP DN alone led to the recovery of Tax1 from cell lysates (Figure 3B, upper panel, lane 3) while NRP DC was unable to interact with Tax1 (Figure 3B, upper panel, lane 2), showing that Tax1 binds to the C-terminal part of NRP. Immunoblotting for VSV-NRP confirmed that these mutants were expressed at similar levels when compared to the wild-type NRP (Figure 3B, lower panel), and both were efficiently immunopre- cipitated with the anti-VSV antibody (Figure 3B, middle panel). Because HTLV-1 infects mainly T cells in vivo, we performed similar immunofluorescence microscopy studies using Jurkat T cells (Figure S2). Consistent with the results obtained in HeLa cells, a subset of Tax1-positive cells harbored a cytoplasmic staining for Because the C-terminal domain of NRP encompasses an ubiquitin-binding domain (UBD) (Figure 3A), we tested the contribution of this domain to the interaction with Tax1 July 2009 \| Volume 5 \| Issue 7 \| e1000521 3 NRP Potentiates Tax Activity Ubiquitination-defective Tax1 mutants exhibit impaired binding to NRP Tax1 displays 10 lysine residues to which ubiquitination chains may be linked. It has been shown that lysines 4 to 8 significantly contribute to Tax1 polyubiquitination [15,16,26]. 293T cells were therefore transfected with Tax1 mutants in which all (K1–10R) or only a subset (K7–8R) of lysines were mutated into arginines. Interestingly, lysine-less Tax1 was impaired for NRP binding (Figure 3D, upper panel, lane 2), although this mutant was efficiently precipitated by the anti-Tax1 antibody (Figure 3D, middle panel, lane 2). Furthermore, the defect in NRP binding was not observed with mutant K7–8R (Figure 3D, upper panel, lane 3). These results strongly suggest that the integrity of Tax1 PLoS Pathogens \| www.plospathogens.org July 2009 \| Volume 5 \| Issue 7 \| e1000521 4 NRP Potentiates Tax Activity Figure 3. Involvement of NRP UBD and Tax1 ubiquitination in NRP/Tax1 interaction. (A) Schematic representation of NRP constructs used in the experiment. NRP DC construct is deleted from amino acid 279 to the C-terminus of the protein, whereas NRP DN is deleted from amino acid 1 to 299. Substitution at position 474 is known to abrogate the function of the ubiquitin-binding domain (UBD). 293T cells were transfected with (B) wild-type Tax1 and either wild-type VSV-NRP (wt), VSV-NRP DC, or VSV-NRP DN; (C) wild-type Tax1 and either wild-type VSV-NRP or VSV-NRP D474N; (D) wild-type VSV-NRP and either wild-type Tax1 (wt) or mutant Tax1 in which the indicated lysine residues were mutated into arginine (K7–8R and K1–10R). Lysates were immunoprecipitated with an anti-VSV or anti Tax-1 antibody as indicated and western blot analyses were performed using anti-Tax1 and anti-VSV antibodies. doi:10.1371/journal.ppat.1000521.g003 Figure 3. Involvement of NRP UBD and Tax1 ubiquitination in NRP/Tax1 interaction. (A) Schematic representation of NRP constructs used in the experiment. NRP DC construct is deleted from amino acid 279 to the C-terminus of the protein, whereas NRP DN is deleted from amino acid 1 to 299. Substitution at position 474 is known to abrogate the function of the ubiquitin-binding domain (UBD). 293T cells were transfected with (B) wild-type Tax1 and either wild-type VSV-NRP (wt), VSV-NRP DC, or VSV-NRP DN; (C) wild-type Tax1 and either wild-type VSV-NRP or VSV-NRP D474N; (D) wild-type VSV-NRP and either wild-type Tax1 (wt) or mutant Tax1 in which the indicated lysine residues were mutated into arginine (K7–8R and K1–10R). Ubiquitination-defective Tax1 mutants exhibit impaired binding to NRP Lysates were immunoprecipitated with an anti-VSV or anti Tax-1 antibody as indicated and western blot analyses were performed using anti-Tax1 and anti-VSV antibodies. doi:10.1371/journal.ppat.1000521.g003 upper panel, compare lane 1 and 2). As control, we analyzed the level of NRP in cell lysates, and showed that it was indeed reduced in cells transfected with NRP-directed siRNA (Figure 4A, lower panel, compare lane 1 and 2). Thus, in the absence of NRP, polyubiquitinated Tax1 is less abundant. ubiquitinated Tax1 in this lane (data not shown). As expected, we observed a correlation between the ability of NRP variants to bind to Tax1, and the level of polyubiquitinated Tax1 (Figure 4B, upper panel, compare lanes 5 and 9 with 7 and 11). However, we also observed that the levels of polyubiquitinated Tax1 were enhanced in cells over-expressing NRP DC or NRP D474N when compared to cells expressing endogenous NRP only (Figure 4B, compare lanes 7 and 11 to lane 3). This might be the consequence of a potential residual interaction between these mutants and Tax1. One model that could account for this observation is that polyubiquitinated Tax1 is stabilized through its interaction with NRP. To test whether the effect of NRP on Tax1 polyubiquitina- tion was dependent upon the interaction between both proteins, we performed the same type of experiments in cells over- expressing wild-type or mutant forms of NRP (NRP DN, DC and D474N) (Figure 4B). We predicted that wild-type NRP or NRP DN, which are able to bind to Tax1, would stabilize polyubiquitinated Tax1, whereas NRP DC and NRP D474N, which have lost the potential to bind to Tax1, would not. In order to highlight the differences among the lanes, a very short exposure time is shown, which accounts for the weak HA signal obtained in the absence of over-expressed NRP (Figure 4B, upper panel, lane 3). However, longer exposures revealed the presence of poly- To determine the linkage specificity of Tax1 polyubiquitination, we used an ubiquitin mutant (HA-Ub K0), in which all lysine residues are mutated to arginine (HA-Ub K0) and that can therefore no longer build conventional polyubiquitin chains. As expected, the HA-Ub K0 did not support Tax1 ubiquitination, indicating that the high-molecular-weight products of Tax1 indeed represented polyubiquitin chains (Figure 4B, lanes 4, 6, 8, 10 and 12). Altogether, these data suggest that NRP binds and stabilizes the polyubiquitin chains linked to Tax1. PLoS Pathogens \| www.plospathogens.org NRP potentiates Tax1-induced NF-kB activation was impaired for the binding to Tax1 (Figure 3C) as well as for the stabilization of Tax1 polyubiquitination (Figure 4B), we compared the ability of wild-type NRP and NRP-D474N to potentiate Tax1 activity on the NF-kB reporter plasmid. As expected, NRP- D474N had no effect on Tax1 activity when compared to wild- type NRP, although expression levels of both constructs were similar (Figure 5C). Taken together, these results indicate that NRP specifically modulates Tax1-induced NF-kB activation, possibly by stabilizing Tax1 polyubiquitination in an interaction- dependent manner. Because previous studies have suggested that Tax1 ubiquitina- tion is critical for its ability to activate the NF-kB pathway [15,16], we hypothesized that stabilization of Tax1 polyubiquitination by NRP would enhance activation of this pathway. We therefore tested the effect of increasing or decreasing NRP expression on Tax1-induced NF-kB activation using an NF-kB reporter gene assay (Figure 5). Jurkat cells were transfected with an NF-kB reporter gene together with Tax1 and increasing amounts of VSV- tagged NRP. As previously reported [5], NF-kB activity was induced upon Tax1 over-expression, and VSV-NRP further enhanced NF-kB activity in a dose-dependent manner (Figure 5A, left panel). We then evaluated the effect of NRP knockdown on Tax1-induced NF-kB activity. Compared with control siRNA, a two-fold decrease in Tax1-mediated NF-kB activity was observed when expression of NRP was silenced (Figure 5B, left panel), whereas no significant impact on the basal activity of the promoter could be measured in the absence of Tax1. Ubiquitination-defective Tax1 mutants exhibit impaired binding to NRP PLoS Pathogens \| www.plospathogens.org July 2009 \| Volume 5 \| Issue 7 \| e1000521 5 NRP Potentiates Tax Activity Figure 4. Modulation of Tax1 ubiquitination by NRP. (A) 293T cells were transfected with Tax1-His and HA-ubiquitin (HA-Ub), and either with an irrelevant siRNA directed against b-globin (control) or NRP-directed siRNA. (B) 293T cells were transfected with Tax1-His, and either wild-type HA- Ub or lysine-less HA-Ub (HA-Ub K0), together with wild-type VSV-NRP, VSV-NRP DC, VSV-NRP DN or VSV-NRP D474N, as indicated. Ubiquitinated forms of Tax1 were retained on Ni-NTA beads and processed for western blot analysis using anti-HA antibodies. Levels of expression of Tax1 and NRP in total lysates were determined by western blot using anti-Tax1 and (A) anti-NRP or (B) anti-VSV antibodies. doi:10.1371/journal.ppat.1000521.g004 Figure 4. Modulation of Tax1 ubiquitination by NRP. (A) 293T cells were transfected with Tax1-His and HA-ubiquitin (HA-Ub), and either with an irrelevant siRNA directed against b-globin (control) or NRP-directed siRNA. (B) 293T cells were transfected with Tax1-His, and either wild-type HA- Ub or lysine-less HA-Ub (HA-Ub K0), together with wild-type VSV-NRP, VSV-NRP DC, VSV-NRP DN or VSV-NRP D474N, as indicated. Ubiquitinated forms of Tax1 were retained on Ni-NTA beads and processed for western blot analysis using anti-HA antibodies. Levels of expression of Tax1 and NRP in total lysates were determined by western blot using anti-Tax1 and (A) anti-NRP or (B) anti-VSV antibodies. doi:10.1371/journal.ppat.1000521.g004 PLoS Pathogens \| www.plospathogens.org TAX1BP1 cooperates with NRP for binding to Tax1 and modulation of Tax1 ubiquitination Since we observed that NRP stabilizes Tax1 poly-ubiquitina- tion, we wondered whether Tax1-binding protein 1 (TAX1BP1), which is also involved in ubiquitin-dependent regulation of NF-kB, could participate in this process. TAX1BP1 was originally identified as a binding partner of Tax1 [19,20]. More recently, TAX1BP1 was reported to interact with A20, Itch and RNF11 to form a functional ubiquitin-editing complex that regulates the ubiquitination of RIP1 and TRAF6 [27]. Thus, we hypothesized that TAX1BP1 acts together with NRP to modulate Tax1 ubiquitination. To ensure the specificity of NRP’s effect on Tax1-mediated NF- kB activation, we performed the same experiments using a HTLV-1-LTR-luc reporter plasmid, which is known to be under the control of CREB rather than NF-kB. Using this construct, no or only a limited effect of NRP over-expression or silencing was observed (Figure 5A, right panel, and Figure 5B, right panel). As a control, NRP expression was determined in the presence or absence of siRNA directed against NRP (Figure 5B, left and right panels). Thus, NRP specifically enhances the activity of Tax1 on the NF-kB pathway. 293T cells were cotransfected with VSV-tagged NRP, Flag- tagged TAX1BP1 and Tax1, and immunoprecipitation of either Tax1, Flag-TAX1BP1 or VSV-NRP was performed (Figure 6A, B and C, respectively). Immunoprecipitates were then blotted with anti-Flag, anti-VSV or anti-Tax1 antibodies. These experiments confirmed that TAX1BP1 interacts with Tax1 (Figure 6A, lane 2). In addition, we observed that TAX1BP1 also interacts with NRP (Figure 6C, lane 4). More interestingly, the amount of TAX1BP1 associated with Tax1 was increased when NRP was co-expressed We also tested whether the potentiating effect of NRP on Tax1- dependent NF-kB activity was dependent upon the interaction between Tax1 and NRP. Because we showed that NRP-D474N July 2009 \| Volume 5 \| Issue 7 \| e1000521 6 NRP Potentiates Tax Activity Figure 5. Potentiation of Tax1-dependent NF-kB activation by over-expressed and endogeneous NRP. (A) Jurkat T cells were transfected with either an Igk-(kB)3-luc construct (left), or an HTLV-1-LTR-luc construct (right), together with Tax1 (1 mg) and VSV-NRP (range 0.25 to 1 mg) as indicated. (B) 293T cells were transfected with either an Igk-(kB)3-luc construct (left), or an HTLV-1-LTR-luc construct (right), together with Tax1 (100 ng) and an irrelevant siRNA directed against b-globin (2) or NRP-targeted siRNA (+), as indicated. TAX1BP1 cooperates with NRP for binding to Tax1 and modulation of Tax1 ubiquitination (C) 293T cells were transfected with an Igk-(kB)3-luc construct together with Tax1 (400 ng) and either wild-type VSV-NRP or VSV-NRP D474N (400 ng) as indicated. (A, B, C) Luciferase activity was measured and normalized, and is shown as fold induction compared to basal promoter activity. (B, C) Cell lysates were run and probed for NRP and Tax1. doi:10.1371/journal.ppat.1000521.g005 Figure 5. Potentiation of Tax1-dependent NF-kB activation by over-expressed and endogeneous NRP. (A) Jurkat T cells were transfected with either an Igk-(kB)3-luc construct (left), or an HTLV-1-LTR-luc construct (right), together with Tax1 (1 mg) and VSV-NRP (range 0.25 to 1 mg) as indicated. (B) 293T cells were transfected with either an Igk-(kB)3-luc construct (left), or an HTLV-1-LTR-luc construct (right), together with Tax1 (100 ng) and an irrelevant siRNA directed against b-globin (2) or NRP-targeted siRNA (+), as indicated. (C) 293T cells were transfected with an Igk-(kB)3-luc construct together with Tax1 (400 ng) and either wild-type VSV-NRP or VSV-NRP D474N (400 ng) as indicated. (A, B, C) Luciferase activity was measured and normalized, and is shown as fold induction compared to basal promoter activity. (B, C) Cell lysates were run and probed for NRP and Tax1. doi:10 1371/journal ppat 1000521 g005 doi:10.1371/journal.ppat.1000521.g005 doi:10.1371/journal.ppat.1000521.g005 (Figure S3A and S3C). Silencing NRP did not impair the localization of Tax1-GFP to the Golgi apparatus (compare Figure S3B and S3C, upper panel), where it was still able to colocalize with NEMO (Figure S3C, lower panel). We then tested the effect of depleting either TAX1BP1 alone (Figure S3A and S3D) or together with NRP (Figure S3A and S3E) on the localization of Tax1 and NEMO to the Golgi apparatus. Preventing the expression of TAX1BP1 or of both TAX1BP1 and NRP had no effect on the subcellular distribution of Tax1 and NEMO (compare Figure S3B, S3D and S3E). Collectively, these results suggest that NRP and TAX1BP1 are not critical for the localization of Tax1 and for the recruitment of NEMO/IKK complex to the Golgi apparatus. (Figure 6A, compare lanes 2 and 4). Similarly, the interaction between NRP and Tax1 was strongly induced in the presence of TAX1BP1 (Figure 6A, compare lanes 3 and 4) and the interaction between NRP and TAX1BP1 was also increased by the expression of Tax1 (Figure 6B, compare lanes 2 and 3, and Figure 6C, compare lanes 4 and 6). PLoS Pathogens \| www.plospathogens.org July 2009 \| Volume 5 \| Issue 7 \| e1000521 TAX1BP1 cooperates with NRP for binding to Tax1 and modulation of Tax1 ubiquitination Thus, these results demonstrate that these three proteins interact with each other and suggest that NRP can be part of a ternary complex with Tax1 and TAX1BP1. To study the functionality of this complex, we used several approaches. First, we performed immunofluorescence imaging to determine whether NRP or TAX1BP1 alone or in association could affect the localization of Tax1 to the Golgi apparatus and the recruitment of the NEMO/IKK complex by Tax1 to this organelle. Because NRP is localized at the Golgi apparatus, we first asked whether Tax1 localization to the Golgi-associated structures was dependent upon NRP expression. NRP-specific siRNA was used to specifically knockdown NRP expression in HeLa cells and Tax1-GFP localization was then investigated In another approach, we determined whether depleting TAX1BP1 could affect the regulatory effect of NRP on Tax1 ubiquitination and NF-kB activation (Figure 7). Interestingly, silencing TAX1BP1 expression precluded the stabilization of Tax1 ubiquitination that was observed when over-expressing NRP PLoS Pathogens \| www.plospathogens.org July 2009 \| Volume 5 \| Issue 7 \| e1000521 7 NRP Potentiates Tax Activity Figure 6. Synergistic interaction between Tax1, TAX1BP1 and NRP. 293T cells were transfected with Tax1, VSV-NRP and Flag-TAX1BP1 as indicated. Total lysates were immunoprecipitated with (A) anti-Tax1, (B) anti-Flag or (C) anti-VSV antibodies. Immunoblot analyses of immunoprecipitates and lysates were performed using antibodies directed against Flag, VSV and Tax1, as indicated. doi:10.1371/journal.ppat.1000521.g006 Figure 6. Synergistic interaction between Tax1, TAX1BP1 and NRP. 293T cells were transfected with Tax1, VSV-NRP and Flag-TAX1BP1 as indicated. Total lysates were immunoprecipitated with (A) anti-Tax1, (B) anti-Flag or (C) anti-VSV antibodies. Immunoblot analyses of immunoprecipitates and lysates were performed using antibodies directed against Flag, VSV and Tax1, as indicated. doi:10.1371/journal.ppat.1000521.g006 (Figure 7A, compare lanes 2 and 4). In addition, over-expressed TAX1BP1 up-regulated Tax1-dependent NF-kB activation (Figure 7B, left panel, lane 2) and this effect was decreased by silencing NRP (Figure 7B, left panel, lane 4). Furthermore, to determine whether NRP and TAX1BP1 act synergistically or not, we examined the effect of double-siRNA knock-down of NRP and TAX1BP1 on the activation of NF-kB mediated by Tax1. As expected from the ubiquitination assay (Figure 7A), we observed that depletion of TAX1BP1 decreased Tax1-mediated NF-kB activation (Figure 7B, right panel, lane 2). Interestingly, following the double depletion, the level of NF-kB activation was not further decreased as compared to the single depletion of TAX1BP1 (Figure 7B, right panel, lane 4). TAX1BP1 cooperates with NRP for binding to Tax1 and modulation of Tax1 ubiquitination How this interaction is regulated is not completely understood. To gain insight into this, we searched for additional Tax-interacting proteins. This led to the identification of NRP, also named Optineurin, as an interacting protein for Tax. This interaction is mediated through the ubiquitin-binding domain of NRP. Interestingly, mutations of the ubiquitination sites of Tax1 prevented its association with NRP, strongly suggesting that Tax1 ubiquitination is required for its interaction with NRP. Ubiquitination of Tax1 provides an important regulatory mechanism that promotes Tax1-mediated activation of NF-kB [15–17,23]. Tax1 polyubiquitin chains are composed predominantly of K63-linked chains and the ubiquiti- nation of Tax1 is dependent on the E2 ubiquitin-conjugating enzyme Ubc13 [17,18]. The NF-kB activation process occurs through the direct binding of the ubiquitinated form of Tax1 to NEMO [16] in a specific subcellular compartment [18,23,28]. Different studies have suggested that Tax ubiquitination regulates TAX1BP1 cooperates with NRP for binding to Tax1 and modulation of Tax1 ubiquitination As controls, NRP, TAX1BP1 and Tax1 expression levels were determined by western blot (Figure 7A and 7B). Altogether, these results suggest that NRP and TAX1BP1 cooperate in Tax1-mediated NF-kB activation. several models have been suggested to explain how this occurs. An important advance came first from experiments showing that Tax1 directly interacts with NEMO, which then functionally recruits Tax1 into the large NEMO/IKKa/IKKb complex that phosphorylates IkB molecules [11–13]. How this interaction is regulated is not completely understood. To gain insight into this, we searched for additional Tax-interacting proteins. This led to the identification of NRP, also named Optineurin, as an interacting protein for Tax. This interaction is mediated through the ubiquitin-binding domain of NRP. Interestingly, mutations of the ubiquitination sites of Tax1 prevented its association with NRP, strongly suggesting that Tax1 ubiquitination is required for its interaction with NRP. Ubiquitination of Tax1 provides an important regulatory mechanism that promotes Tax1-mediated activation of NF-kB [15–17,23]. Tax1 polyubiquitin chains are composed predominantly of K63-linked chains and the ubiquiti- nation of Tax1 is dependent on the E2 ubiquitin-conjugating enzyme Ubc13 [17,18]. The NF-kB activation process occurs through the direct binding of the ubiquitinated form of Tax1 to NEMO [16] in a specific subcellular compartment [18,23,28]. Different studies have suggested that Tax ubiquitination regulates (Figure 7A, compare lanes 2 and 4). In addition, over-expressed TAX1BP1 up-regulated Tax1-dependent NF-kB activation (Figure 7B, left panel, lane 2) and this effect was decreased by silencing NRP (Figure 7B, left panel, lane 4). Furthermore, to determine whether NRP and TAX1BP1 act synergistically or not, we examined the effect of double-siRNA knock-down of NRP and TAX1BP1 on the activation of NF-kB mediated by Tax1. As expected from the ubiquitination assay (Figure 7A), we observed that depletion of TAX1BP1 decreased Tax1-mediated NF-kB activation (Figure 7B, right panel, lane 2). Interestingly, following the double depletion, the level of NF-kB activation was not further decreased as compared to the single depletion of TAX1BP1 (Figure 7B, right panel, lane 4). As controls, NRP, TAX1BP1 and Tax1 expression levels were determined by western blot (Figure 7A and 7B). Altogether, these results suggest that NRP and TAX1BP1 cooperate in Tax1-mediated NF-kB activation. several models have been suggested to explain how this occurs. An important advance came first from experiments showing that Tax1 directly interacts with NEMO, which then functionally recruits Tax1 into the large NEMO/IKKa/IKKb complex that phosphorylates IkB molecules [11–13]. PLoS Pathogens \| www.plospathogens.org PLoS Pathogens \| www.plospathogens.org Constructs and siRNA Constructs and siRNA pSG5M-Tax1, pSG5M-Tax2, Tax1-6His, Tax1-GFP plasmids were previously described [18,36]. Tax mutants harboring substitutions of all (K1–10R) or some (K7–8R) lysines into arginines were described elsewhere and were kindly provided by C. Pique [26]. HA- and VSV-tagged NRP plasmids were obtained by cloning NRP ORF (aa 1–577) into pT7link-HA or pcDNA3/ pT7-link-GVSV vectors at EcoRI sites. To identify the domains of NRP required for in vivo interaction with Tax1, modified forms of NRP (VSV-NRP-DC (aa 1–278) and VSV-NRP-DN (aa 300–577)) (Figure 3A) were generated by site-directed mutagenesis with a PCR-based strategy and inserted at EcoRI sites into pcDNA3/ pT7-link-GVSV vector. VSV-NRP D474N plasmid was generated by site-directed mutagenesis. HA-tagged wild-type and lysine-less (K0, in which all lysine residues are mutated into arginine) ubiquitin constructs were obtained from P. Jalinot [37]. Flag- tagged TAX1BP1 plasmid was a kind gift from E. Harhaj [33]. NRP double-stranded siRNA (GGAGACUGUUGGAAGC- GAAGU) and b-globin double-stranded siRNA (control, GGU- GAAUGUGGAAGAAGUU) were purchased from Proligo (Sig- ma). SMART pool siRNA directed against TAX1BP1 was purchased from Dharmacon. Concerning the regulation of Tax1 ubiquitination by NRP, the simplest interpretation of our experiments is that NRP either interacts directly or indirectly with an ubiquitin ligase, or that it prevents the interaction of Tax1 with a deubiquitinase. The E3 ubiquitin ligase(s) responsible for K63-linked polyubiquitination and the deubiquitinase responsible for the cleavage of these ubiquitin chains on Tax1 remain to be identified. Discussion It is well established that one of the primary actions of Tax1 is to permanently activate the NF-kB signaling in the cytoplasm, and PLoS Pathogens \| www.plospathogens.org July 2009 \| Volume 5 \| Issue 7 \| e1000521 8 NRP Potentiates Tax Activity Figure 7. Cooperative modulation of Tax1 ubiquitination and NF-kB activation by NRP and TAX1BP1. (A) 293T cells were transfected with Tax1-His, HA-Ub, and VSV-NRP and either b-globin- (control) or TAX1BP1-directed siRNA, as indicated. Ubiquitinated forms of Tax1 were retained on Ni-NTA beads and processed for western blot analysis using anti-HA antibodies. Levels of expression of Tax1, VSV-NRP and TAX1BP1 in total lysates were verified by western blot using anti-Tax1, anti-VSV and anti-TAX1BP1 antibodies, respectively. (B) 293T cells were transfected with an Igk- (kB)3-luc construct and Tax1, together with (left panel): Flag-TAX1BP1 and irrelevant b-globin (2) or NRP-targeted (+) siRNA; or (right panel): b-globin, NRP or TAX1BP1-specific siRNA, or both siRNA, as indicated. Luciferase activity was measured and normalized, and is shown as fold induction compared to basal activity. Levels of NRP, TAX1BP1 and Tax1 were assessed in cell lysates. doi:10.1371/journal.ppat.1000521.g007 NRP Potentiates Tax Activity Figure 7. Cooperative modulation of Tax1 ubiquitination and NF-kB activation by NRP and TAX1BP1. (A) 293T cells were transfected with Tax1-His, HA-Ub, and VSV-NRP and either b-globin- (control) or TAX1BP1-directed siRNA, as indicated. Ubiquitinated forms of Tax1 were retained on Ni-NTA beads and processed for western blot analysis using anti-HA antibodies. Levels of expression of Tax1, VSV-NRP and TAX1BP1 in total lysates were verified by western blot using anti-Tax1, anti-VSV and anti-TAX1BP1 antibodies, respectively. (B) 293T cells were transfected with an Igk- (kB)3-luc construct and Tax1, together with (left panel): Flag-TAX1BP1 and irrelevant b-globin (2) or NRP-targeted (+) siRNA; or (right panel): b-globin, NRP or TAX1BP1-specific siRNA, or both siRNA, as indicated. Luciferase activity was measured and normalized, and is shown as fold induction compared to basal activity. Levels of NRP, TAX1BP1 and Tax1 were assessed in cell lysates. doi:10.1371/journal.ppat.1000521.g007 July 2009 \| Volume 5 \| Issue 7 \| e1000521 PLoS Pathogens \| www.plospathogens.org 9 NRP Potentiates Tax Activity IKK relocalization to the Golgi apparatus [23] or to the centrosome [18]. This relocalization involves the accumulation of Tax1 in Golgi-associated lipid rafts allowing the recruitment of the NEMO/IKK complex to these microdomains [28]. Antibodies The following antibodies were used: anti-Tax1 (Tab172), anti- Tax2 (GP3738) [36], anti-NRP (100 000, Cayman Chemical), anti-HA (MMS-101R, Covance), anti-VSV (V 5507, Sigma- Aldrich, or ascite fluid of clone P5D4), anti-GM130 (610823, BD Transduction Laboratories), anti-NEMO (611306, BD Transduc- tion Laboratories), anti-Flag M2 (F-3165, Sigma-Aldrich), anti- TAX1BP1 (sc-81390, Santa Cruz Biotechnology), anti-His (sc-804, Santa Cruz Biotechnology). TAX1BP1 is a cellular protein that binds to Tax1 and acts as an ubiquitin-dependent negative regulator of NF-kB signaling in response to TNF-a stimulation [27]. It has recently been shown that this negative regulation is mediated by a quaternary complex containing TAX1BP1, A20, Itch and RNF11 [31–34]. Our results indicate that TAX1BP1 interacts with NRP and Tax1 individually and also with both proteins together to form a ternary complex, raising the possibility that TAX1BP1 participates in NRP-mediated enhancement of Tax1 ubiquitina- tion. We have observed that the stabilization of Tax1 ubiquitination by NRP was completely impaired in the absence of TAX1BP1 (Figure 7A) and that NRP and TAX1BP1 cooperated to positively regulate Tax1-induced NF-kB activa- tion (Figure 7B). Thus, we can propose that the negative regulation of TNF-a-induced NF-kB activation is mediated by a quaternary A20/TAX1BP1/Itch/RNF11 complex, as opposed to the positive regulation of Tax1-induced NF-kB activation, which is mediated by a ternary complex containing Tax1, TAX1BP1 and NRP. PLoS Pathogens \| www.plospathogens.org Cell culture HeLa and 293T cell lines were grown in DMEM medium. HTLV-1-infected C8166 and C91PL and uninfected CEM and Jurkat cell lines were grown in RPMI 1640 medium. In all cases, the medium was supplemented with fetal bovine serum (10%) and antibiotics (100 units/ml penicillin and 100 mg/ml streptomycin), and cells were maintained at 37uC in 5% CO2. p [ ] The interaction of NEMO with polyubiquitinated substrates is involved in transporting the NEMO/IKK complex towards its site of activation. This has been well described for the stimulation of NF-kB by the TNF-R and TCR [6,29], allowing the recruitment of NEMO to these receptors and the subsequent phosphorylation of IKKb by the upstream kinase TAK1. Similarly, DNA damage causes the translocation of NEMO from the cytoplasm to the nucleus and its phosphorylation by ATM [30]. Although previous studies have suggested that Tax1 ubiquitination is correlated with the localization of the NEMO/IKK complex at the Golgi apparatus, modulation of this process is still poorly understood. Our model is that NRP is a positive player in Tax1-induced NF- kB activation by increasing the polyubiquitination of Tax1. This model is supported by our results showing that increasing the level of NRP increases Tax1 polyubiquitination (Figure 4) and that the level of NRP expression correlates with Tax1-induced NF-kB activation (Figure 5). Whether NEMO also exerts a similar effect on Tax1 ubiquitination still remains unknown. Of note, a different effect of NRP was obtained with TNF-a-induced NF-kB activation, where NRP was shown to compete with NEMO for the binding to polyubiquitinated RIP and consequently to inhibit NF-kB activation [25]. The situation with Tax1 is unusual since the polyubiquitinated substrate that binds to NEMO and NRP is Tax1 itself, an NF-kB activator. Supporting Information Cells were observed as described in the Materials and Methods section. Differential interference contrast (DIC) is shown. Scale bar = 10 mm. Figure S2 Colocalization of Tax1, NRP and NEMO in Golgi- associated structures. Jurkat cells were transfected with (A and B) Tax1-GFP or (C) GFP alone and stained with an anti-NRP antibody (red) and either (A and C) an anti-GM130 or (B) an anti- NEMO antibody (blue). Cells were observed as described in the Materials and Methods section. Differential interference contrast (DIC) is shown. Scale bar = 10 mm. Found at: doi:10.1371/journal.ppat.1000521.s002 (5.89 MB TIF) Figure S3 Effect of NRP or TAX1BP1 silencing on Tax1 localization and colocalization with NEMO. HeLa cells were transfected with Tax1-GFP and with siRNA directed against either (A and B) b-globin (control, -), (A and C) NRP, (A and D) TAX1BP1, or (A and E) both NRP and TAX1BP1. (A) Lysates were analyzed by western blot to control NRP and/or TAX1BP1 depletion. (B to E) Cells were stained with either an anti-GM130 Yeast two-hybrid screen Yeast culture mediums were prepared and screens were performed as previously described [38]. Tax1 and Tax2 coding sequences were cloned by in vitro recombination using the GatewayH technology (Invitrogen) into Gal4-BD yeast two-hybrid vector pDEST32 (Invitrogen), and transformed into AH109 yeast strain (Clontech) using a standard Lithium/Acetate procedure. GAL4-BD-Tax1 and -Tax2 fusion proteins did not induce autonomous transactivation of HIS3 reporter gene, and screens were performed on synthetic medium lacking histidine (2His medium) and supplemented with 10 mM of 3-amino-1,2,4-triazole (3-AT, Sigma-Aldrich). A mating strategy was used for screening the human spleen cDNA library cloned in the GAL4-AD pPC86 vector (Invitrogen) and previously transformed into Y187 yeast strain (Clontech). After 6 days of culture on selective medium, [His+] colonies were selected and purified over 3 weeks by culture on selective medium to eliminate false-positives. AD-cDNAs were amplified by PCR from zymolase-treated yeast colonies using primers that hybridize within the pPC86 regions flanking cDNA inserts. PCR products were sequenced and cellular interactors were identified by BLAST analysis. Since it has been shown that Tax1 inactivates A20 by disrupting the TAX1BP1/Itch/A20 complex, thus counteracting its negative function [33], we speculate that NRP also is involved in this process. Furthermore, since RNF11 has been shown to interact with NRP [35], it will be important to determine whether this protein is also present in the TAX1BP1/Tax1/NRP complex and whether it regulates Tax1 ubiquitination. Future studies are needed to specifically address the mechanism whereby the Tax1/ NRP/TAX1BP1 complex positively regulates Tax1 ubiquitination and subsequent NF-kB activation. July 2009 \| Volume 5 \| Issue 7 \| e1000521 10 NRP Potentiates Tax Activity in Guanidium Buffer (10 mM Tris HCl pH 8.0, 100 mM Na2HPO4/NaH2PO4, 6 M Guanidium) [26]. Cell lysates were then incubated with Ni-NTA beads (His-select HF Agarose Beads, Sigma-Aldrich) at room temperature. Beads were then extensively washed with Guanidium Buffer, Urea Buffer (10 mM Tris HCl pH 6.4, 100 mM Na2HPO4/NaH2PO4, 8 M Urea) and cold PBS. Bound proteins were finally eluted and processed for immunoblot analysis. in Guanidium Buffer (10 mM Tris HCl pH 8.0, 100 mM Na2HPO4/NaH2PO4, 6 M Guanidium) [26]. Cell lysates were then incubated with Ni-NTA beads (His-select HF Agarose Beads, Sigma-Aldrich) at room temperature. Beads were then extensively washed with Guanidium Buffer, Urea Buffer (10 mM Tris HCl pH 6.4, 100 mM Na2HPO4/NaH2PO4, 8 M Urea) and cold PBS. Bound proteins were finally eluted and processed for immunoblot analysis. Luciferase assays Jurkat and 293T cells were transiently transfected with either an HTLV-1-LTR-luc or an Igk-(kB)3-luc plasmid together with the indicated plasmids or siRNA. The amount of total DNA was equalized using a pSG5M backbone vector, as previously reported [36]. All transfections were carried out in the presence of a renilla luciferase vector (phRG-TK) in order to normalize the results for transfection efficiency. Luciferase activity was assayed 18 h post- transfection using the Dual-Luciferase Reporter Assay System (Promega) on a Berthold LB9500C luminometer as reported previously [39]. Glycerol gradient fractionation and gel filtration analysis Glycerol gradient fractionation and gel filtration analysis HeLa cells were lysed in a lysis buffer (50 mM Tris-HCl pH 7.4, 120 mM NaCl, 5 mM EDTA, 0.5% Nonidet-P40, 0.2 mM Na3VO4, 1 mM DTT, 1 mM PMSF) in the presence of protease inhibitors (Complete, Boehringer) by 15 passages through a 24- gauge needle. Transient transfections 293T cells were transfected using either the Polyfect reagent (Qiagen) or Lipofectamine 2000 (Invitrogen). For luciferase assays, 293T were first transfected with siRNA using Icafectin 442 (Eurogentec), followed 48 h later by DNA and siRNA transfection using Lipofectamine 2000. Jurkat cells were transfected using the Superfect reagent (Qiagen), except in Figure S2 where they were nuleofected using the Amaxa Nucleofector Technology (Amaxa Biosystems). HeLa cells were transfected using the Effectene reagent (Qiagen). Immunoprecipitations and immunoblots 293T, CEM, C8166 and C91PL cell lines cells were lysed in Chris buffer (50 mM Tris, pH 8.0, 0.5% Nonidet P-40, 200 mM NaCl, and 0.1 mM EDTA) supplemented with a cocktail of protease inhibitors (Complete, Roche), and the phosphatase inhibitors sodium fluoride (100 mM) and sodium orthovanadate (2 mM). Proteins were then recovered by immunoprecipitation from an equivalent amount of proteins, using one of the following antibodies: anti-Tax, anti-VSV, anti-NRP, anti-Flag. Immune complexes were recovered with magnetic Staphylococcus aureus Protein A or Protein G beads (Bio-Adembeads, Ademtech). Immunoprecipitates were then washed with lysis buffer, eluted and resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Subsequent immunoblots were performed according to a previously described protocol [36] and proteins transferred to nitrocellulose (I-Blot, Invitrogen) or Immobilon membranes (Millipore) were revealed with ECL Westen Blotting Substrate (Pierce) or ECL Plus Western Blotting Detection Reagent (Amersham). Supporting Information Whole cell extracts were fractionated by centrifugation at 39000 rpm for 24 hrs on a 10–40% glycerol gradient (12 ml), using an Sw 41 rotor (Beckmann). The glycerol-containing buffers were prepared with the same composition as the lysis buffer. Twenty-four fractions (0.5 ml each) were then collected and 20 ml of each fraction were processed for immunoblot analysis. Figure S1 Glycerol gradient analysis of Tax1 and NRP. HeLa cells were transfected with Tax1 and HA-NRP, and cell extracts were separated through a glycerol gradient. All fractions as well as cell extracts (input) were analyzed by western blotting using antibodies directed against HA (upper panel) or Tax1 (lower panel). Precalibration of the glycerol gradient is indicated beneath the fractions count (kDa). Found at: doi:10.1371/journal.ppat.1000521.s001 (5.09 MB TIF) Figure S1 Glycerol gradient analysis of Tax1 and NRP. HeLa cells were transfected with Tax1 and HA-NRP, and cell extracts were separated through a glycerol gradient. All fractions as well as cell extracts (input) were analyzed by western blotting using antibodies directed against HA (upper panel) or Tax1 (lower panel). Precalibration of the glycerol gradient is indicated beneath the fractions count (kDa). For the gel filtration analysis, whole cell extracts were subjected to another centrifugation at 15000 rpm for 15 min, and then purified using Quick Spin Sephadex G25 columns (Roche). Gel filtration chromatography was carried out on a Superpose 6 column (Pharmacia) in a FPLC running buffer (50 mM Tris-HCl pH 7.4, 120 mM NaCl, 5 mM EDTA, 0.1% Nonidet-P40, 0.2 mM Na3VO4, 1 mM DTT, 1 mM PMSF, 10% glycerol) in the presence of protease inhibitors (Complete, Boehringer). The columns were precalibrated with albumin (67 kDa), aldolase (158 kDa), catalase (232 kDa), ferritin (440 kDa), thyroglobulin (669 kDa) and blue dextran (2000 kDa). Several glycerol gradients were loaded and analyzed with the same proteins, with the addition of the ovalbumin (43 kDa). Forty fractions of 0.5 ml each were then collected and 20 ml of fractions 16 to 40 were processed for immunoblot analysis. ( ) Found at: doi:10.1371/journal.ppat.1000521.s001 (5.09 MB TIF) Found at: doi:10.1371/journal.ppat.1000521.s001 (5.09 MB TIF) Figure S2 Colocalization of Tax1, NRP and NEMO in Golgi- associated structures. Jurkat cells were transfected with (A and B) Tax1-GFP or (C) GFP alone and stained with an anti-NRP antibody (red) and either (A and C) an anti-GM130 or (B) an anti- NEMO antibody (blue). Indirect immunofluorescence Twenty-four hours post-transfection, cells were fixed with 4% paraformaldehyde, rinsed and permeabilized in PBS containing 0.5% Triton X-100. Following pre-incubation with PBS contain- ing 5% BSA, cells were incubated with primary antibodies in PBS containing 1% BSA for 1 h at room temperature. Samples were then stained with Alexa Fluor 568-conjugated goat anti-rabbit IgG (A-11010, Invitrogen), Cy5-conjugated donkey anti-mouse IgG (715-175-150, Jackson ImmunoResearch Laboratories), or AMCA-conjugated horse anti-mouse IgG (CI-2000, Vector Laboratories) for 1 h at room temperature. Where indicated, an additional staining of nuclei was performed with DAPI (Sigma) for 5 min. The coverslips were washed, mounted with Vectashield Mounting Medium (H-1000, Vector Laboratories), and examined under a Zeiss Axioplan 2 microscope, using the Zeiss ApoTome system and the Zeiss Axiovision 4.4 software. PLoS Pathogens \| www.plospathogens.org References Chiari E, Lamsoul I, Lodewick J, Chopin C, Bex F, et al. (2004) Stable ubiquitination of human T-cell leukemia virus type 1 tax is required for proteasome binding. J Virol 78: 11823–11832. 8. Ordureau A, Smith H, Windheim M, Peggie M, Carrick E, et al. 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Kovalenko A, Wallach D (2006) If the prophet does not come to the mountain: dynamics of signaling complexes in NF-kappaB activation. Mol Cell 22: 433–436. 25. Zhu G, Wu CJ, Zhao Y, Ashwell JD (2007) Optineurin negatively regulates TNFalpha- induced NF-kappaB activation by competing with NEMO for ubiquitinated RIP. Curr Biol 17: 1438–1443. 7. Wu CJ, Ashwell JD (2008) NEMO recognition of ubiquitinated Bcl10 is required for T cell receptor-mediated NF-kappaB activation. Proc Natl Acad Sci U S A 105: 3023–3028. q 26. Nickel pull-down Forty-eight hours after transfection, cells were harvested in cold PBS and lysed under highly denaturating and reductive conditions PLoS Pathogens \| www.plospathogens.org July 2009 \| Volume 5 \| Issue 7 \| e1000521 11 NRP Potentiates Tax Activity with the image acquisition and analyses. This article is dedicated to the memory of JN Brady. or an anti-NEMO antibody as indicated (red). Nuclei were stained using DAPI (blue). Cells were observed as described in the Materials and Methods section. Scale bar = 10 mm. Acknowledgments We thank C. Pique, P. Jalinot and E. Harhaj for the generous gift of reagents. We also thank the Pasteur PFID platform (Imagopole) for help We thank C. Pique, P. Jalinot and E. Harhaj for the generous gift of reagents. We also thank the Pasteur PFID platform (Imagopole) for help Author Contributions Found at: doi:10.1371/journal.ppat.1000521.s003 (9.35 MB TIF) Found at: doi:10.1371/journal.ppat.1000521.s003 (9.35 MB TIF) Conceived and designed the experiments: CJ JF FA SAC PVA JNB DF POV RM RW. Performed the experiments: CJ JF FA SAC DF POV RW. Analyzed the data: CJ JF SAC JNB DF FT AI POV RM RW. Wrote the paper: CJ JF AI POV RM RW. 20. Jin DY, Jeang KT (1995) GenBank accession no. U33821. References Virology 368: 351–362. g 19. Gachon F, Peleraux A, Thebault S, Dick J, Lemasson I, et al. 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https://openalex.org/W3046625382	https://nottingham-repository.worktribe.com/preview/5247745/54832.pdf	English	null	TransVis: Integrated Distant and Close Reading of Othello Translations	IEEE transactions on visualization and computer graphics	2,022	cc-by	19,262	1 1 JOURNAL OF LATEX CLASS FILES, VOL. 14, NO. 8, AUGUST 2015 TransVis: Integrated Distant and Close Reading of Othello Translations Mohammad Alharbi, Robert S Laramee, and Tom Cheesman Abstract—Studying variation among time-evolved translations is a valuable research area for cultural heritage. Understanding how and why translations vary reveals cultural, ideological, and even political inﬂuences on literature as well as author relations. In this paper, we introduce a novel integrated visual application to support distant and close reading of a collection of Othello translations. We present a new interactive application that provides an alignment overview of all the translations and their correspondences in parallel with smooth zooming and panning capability to integrate distant and close reading within the same view. We provide a range of ﬁltering and selection options to customize the alignment overview as well as focus on speciﬁc subsets. Selection and ﬁltering are responsive to expert user preferences and update the analytical text metrics interactively. Also, we introduce a customized view for close reading which preserves the history of selections and the alignment overview state and enables backtracing and re-examining them. Finally, we present a new Term-Level Comparisons view (TLC) to compare and convey relative term weighting in the context of an alignment. Our visual design is guided by, used and evaluated by a domain expert specialist in German translations of Shakespeare. ! • Mohammad Alharbi is with the Department of Computer Science at Swansea University, United Kingdom. E-mail: m.alharbi.508205@swansea.ac.uk. • Robert Laramee is with the School of Computer Science at the University of Nottingham, United Kingdom. E-mail: robert.laramee@nottingham.ac.uk. • Tom Cheesman is with College of Arts and Humanities, Swansea Univer- sity, United Kingdom. E-mail: t.cheesman@swansea.ac.uk 1 INTRODUCTION AND MOTIVATION • A novel visual design that supports comparison of an arbitrary number of parallel translations. • A novel visual design that supports comparison of an arbitrary number of parallel translations. T EXT visualization is a popular subﬁeld of information visualization due to the rapid increase in digital text data over the last two decades [3], [54]. In this project, researchers with an expert background in the Arts and Hu- manities prepared 38 translations of Shakespeare’s play The Tragedy of Othello, the Moor of Venice (1604). The translations were originally written over a span of 244 years from the Christoph Martin Wieland translation [88] in 1766 to the Christian Leonard translation [55] in 2010. Our data set contains the 38 translations as well as the English base text of a sample of the full play –Act 1 Scene 3. T • Customized mechanisms for rapid and interactive ﬁlter- ing and selection of a large number of German transla- tions of Shakespeare. • Interactive and dynamic analysis of similarity metrics to support comparisons and analysis of customized parallel translations. • Examples, detailed observations, a case study, and do- main expert feedback from a specialist in German transla- tions of Shakespeare. Based on the notion of close and distant reading of texts [11], [59], we attempt to create a novel interactive visual design that combines distant and close views of the parallel translations of Shakespeare’s Othello. Guided by the visual information seeking mantra [78], inspiration from previous work on this topic, and close collaboration with the domain expert we derive six requirements and ﬁve tasks that our application must support (discussed in Section 4). The proposed visual design is guided closely and reviewed by a domain expert from Arts and Humanities. The rest of this paper is organized as follows: Section 2 introduces and deﬁnes the most important terms used in this paper and introduces the parallel data as well as the similarity metrics. Section 3 discusses previous work related to our approach and the challenge domain. Section 4 outlines the design requirements and tasks that our ap- proach supports. Section 5 introduces our proposed appli- cation components. Section 6 provides the domain expert feedback and case studies. In Section 7, we discuss the study outcome and report on the design process utilizing guidance provided by Sedlmair et al [77]. We ﬁnish with conclusions and future work directions. 1 INTRODUCTION AND MOTIVATION y p Contributions: In this paper, we contribute the follow- ing: • We support integrated distant and close reading in the same view and implement them with smooth zooming and panning. Index Terms—Text visualization, Othello, Parallel Translations. g • Tom Cheesman is with College of Arts and Humanities, Swansea Univer- sity, United Kingdom. E-mail: t.cheesman@swansea.ac.uk y g E-mail: m.alharbi.508205@swansea.ac.uk. E-mail: robert.laramee@nottingham.ac.uk. 2 DEFINITIONS AND BACKGROUND The text of Othello is a play for theatrical performance. Like most play texts, it contains three kinds of segment: ‘speeches’ (words to be spoken by actors), ‘speaker identiﬁers’ (words indicating the character in the play who speaks), and ‘stage directions’ (words which instruct the director and actors). A speech is always preceded by a speaker identiﬁer. A speech can consist of one to many sentences, and one to many words. In our dataset, all three kinds of text are predeﬁned as segments. The word forms are important at this stage. In the previ- ous work, the formulations of Eddy and Viv do not consider advanced linguistics algorithms to reduce inﬂectional forms of words such as lemmatization or stemming [53]. This is due to the nature of the German language which is con- sidered inﬂected. Special challenges appear with German Shakespeare texts due to the use of antiquated language and poetic orthography. However, we build up a lemmatization dictionary for our corpus using Cascaded Analysis Broker “CAB” [46] which is developed for the German Text Archive (Deutsches Text Archiv, DTA) [10]. CAB is an HTTP-based web service morphological normalization tool developed for historical German text especially for the 18th and 19th centuries. An alignment is a meta-data object which links a given translation segment with its corresponding text in the En- glish base text. In our dataset, alignments have been created (in a machine-assisted manual process) between each seg- ment in the English base text, and each segment in all the translations which has a meaning which corresponds to the base text segment. Some translations omit, transpose, and add new words, sentences, and even speeches, so aligning is a complex task. However, the majority of translations in our dataset are ‘faithful’, ‘close’ and complete translations. With those, making speech-by-speech alignments is relatively straightforward. As a dimensionality reduction algorithm, each segment is represented by a ﬁxed length of vector of word weights TF-IDF (term frequency–inverse document frequency) [67], [71]. Then, the similarity coefﬁcient between segments vec- tors can be obtained by the Euclidean Distance between each pair of segments. Euclidean distance is usually the default metric used to measure the distance between two points or vectors. It is the default distance metric used with the K- means algorithm [40]. 2 DEFINITIONS AND BACKGROUND This section provides background information on terminol- ogy, the text data, and translation meta-data. • Deﬁnitions: In this section, we explain the notions of close and distant reading. Close reading generally deﬁnes the process of carefully reading word-for-word and interpreting a passage to develop a deep understanding of the ideas contained in the text [11]. Close reading signiﬁes the critical analysis of small and speciﬁc components of the text over the general theme. Close reading may involve annotation and highlighting techniques to increase the comprehension JOURNAL OF LATEX CLASS FILES, VOL. 14, NO. 8, AUGUST 2015 2 process. In a literary context, close reading is deﬁned by Nancy Boyles [11] as “reading to uncover layers of meaning that lead to deep comprehension.” The practice of close reading is inherently subject to the reader, and interpretations of the text vary according to readers and context [57]. document using a number of <blockquote> nodes. Each <blockquote> has a number of <q> nodes which contain the actual text segments. 2. <segmentdefinitions>: stores the meta-data as- sociated with each segment such as segment ID, length, speaker, etc. On the other hand, distant reading aims to provide an overview of the text by moving from an in-depth explo- ration of the individual components of the text to presenting the global features of the text(s) [47]. In contrast to close reading, distant reading is technically a more objective pro- cess because most of the context is hidden and the reader is left with the result of computationally and analytically abstracted visualization. 3. <alignments>: is included in all documents except the base text. It consists of a number of <alignment> nodes which match a given segment of a translation with the corresponding segment of the base text using the segment ID elements stored in the <segmentdefinitions> node. • Similarity Metrics “Eddy and Viv”: The Eddy and Viv metrics were introduced by Cheesman et al. [21] to quantify how a given base segment (English in our case) is interpreted and translated between parallel texts (German in our case). Eddy characterizes the translated segments in terms of distinctiveness. A higher Eddy value indicates higher dissimilarity from other translations. Throughout the paper, some special terms are used. A segment is a meta-data object. A segment can be any con- tinuous sequence of words within a text, but generally, it is a meaningful unit. 2 DEFINITIONS AND BACKGROUND • Description of Parallel Translation Data: A group from the Arts and Humanities working on a project called “Trans- lation Arrays: Version Variation Visualization” [20] collected 55 translations of Shakespeare’s play Othello (1604) Act 1, Scene 3 into German. The translations span 1766–2010. So far, 38 translations of the collection were optically scanned from paper prints, corrected from OCR errors, segmented, and aligned with the English base text to create a parallel corpus. The set of translations we study was collected over a time-span of 2-3 years from various sources, such as libraries, second-hand book-sellers, archives, theater pub- lishers and theater companies. The translation data is stored in XML format on the project’s website [19]. After obtaining the similarity values between each pair of aligned segments, a weight value “Eddy value” is com- puted by averaging the sum of similarity values between each pair, such that: Eddy(Sj i ) = Pn k=1 Sj i −Sk i n (1) (1) where Sj i denotes segment i in translation j and n denotes the number of translations. Each <document> node in the dataset XML ﬁle rep- resents a German translation of the base English text and is associated with a variety of meta-data such as, au- thors <authortranslator> and description of the trans- lation <description>. The <document> node consists of three nodes: <doccontent>, <segmentdefinitions> and <alignments>. The base English text is also repre- sented as a node of <document>, and does not have the <alignments> sub-node. Viv, on the other hand, is the average pairwise distance between every two segments projected on the base segment, also known as the diameter of a cluster [70]. Viv represents the stability of the base segment. A high Viv value indicates low stability (high variability) which means the segment’s translations vary considerably between authors. We com- pute the Viv value for a given base segment i as: V iv(i) = Pn k=1 Eddy(Si k) n (2) (2) 1. <doccontent>: contains the actual content of the n 3 JOURNAL OF LATEX CLASS FILES, VOL. 14, NO. 8, AUGUST 2015 There are many approaches developed to perform com- parative tasks. However, users and systems can often per- form comparative tasks even if the systems are not devel- oped in the context of comparison [29]. Gleicher et al. 3.3 Visual Designs for Comparison of Parallel Texts There are a number of different visual representations that can facilitate visual comparison between multiple texts. The most common layout to visualize parallel texts is juxta- position. For example ItLv [58] combines a timeline chart with multiple bar charts stacked vertically to represent documents. Another example is the Versioning Machine tool 2 DEFINITIONS AND BACKGROUND [30] group comparative visualizations into three representations: juxtaposition which shows objects side-by-side, superposi- tion which overlays objects in the same visual space, and explicit representation of relationships. We can distinguish between the three categories in different ways. The sepa- rated object’s design relies on the memory of the user to conduct a complete comparison and there is no correspon- dence between them. Overlay designs use the same visual coordinate system to layout objects and proximity is needed to represent the connection. Explicit encoding facilitates computational tasks to investigate relationships. Gleicher et al. believe that interaction techniques such as brushing and linking can be helpful when applied to facilitate visual comparison. Also, using animation to show, for example, transitions can be helpful in understanding the connection between objects. Animation approaches can be useful to aid comparison between related objects, however, they can be problematic when not implemented carefully [37]. High Eddy and Viv values are interesting to arts and humanities researchers because if a translation has high Eddy values, it indicates that the translator is working in a more unusual, possibly a more creative way relative to others, maybe interpreting the text in a new way. This might be due to circumstances such as historical changes in the language and/or the culture, as a result of political, economic and social change for example. It may be due to an individual translator developing their own new approach to the translation task. Or it may have to do with a new market developing for a new kind of text –in this case, new kind of theatrical drama. High Viv values indicate which base text segments are associated with variation among translations, which enables research into the textual factors (such as complexity, ambiguity, polysemy, semantic salience or affective intensity) which may provoke translators to deviate from one another. Eddy and Viv metrics can be computed interactively and dynamically, that means every user customization derives new similarity metrics. Text There are approaches designed to support the analysis of a single document which, however, can be extended to facili- tate comparison between multiple documents. Fingerprint- ing approaches [51], [62] are used to highlight semantic text properties on different hierarchy levels such as the develop- ment of relationships between characters in literature. The Text Variation Explorer [79] provides linguistically-assisted visualization to examine a user-selected text window. Vari- focalReader [52] presents an interactive multi-layer visual design based on the hierarchy level of a text, such as chapters, pages, and sentences. It incorporates multiple visual presentations to support the analyst exploring the document, such as bar charts, pictograms, and word clouds. We discuss general comparison as a task and the design that supports it based on Gleicher et al. [30] in Section 3.1. Then, we group the related literature into four groups. The ﬁrst category in Section 3.2 presents the general design that supports comparison of text in general. The second group in Section 3.3 discusses the designs that support visual comparison between parallel text. The third group in Section 3.4 presents visualization solutions that enable digital humanities tasks. The fourth group in Section 3.5 introduces previous visualizations of Shakespeare’s Othello. Many visualization systems are designed to analyze collections of documents without explicit support of inner relationships between documents. For example, Brehmer et al. [12] present an open-source platform that analyzes user-uploaded documents. It also allows the user to create custom visual layouts. Also, there are many techniques that visualize results of queries, such as Sparkler [36] which plot the results on a radar-like view and the search-engine similarity (SES) tool [81] which visualizes the results of multiple web search engines using multiple views. In Table 1, we summarize the visual comparative ap- proaches that facilitate parallel text comparison tasks. We indicate what type of documents they study, the maximum number of documents viewed in parallel in both close and distant views, the visual designs used in the distant views, as well as the language studied. If the meta-data is not provided explicitly, we extract it from the examples provided. Our work is different from the aforementioned work. We present a specially customized visualization of explicit parallel texts that are strongly related to each other, i.e. versions or editions of the same text. In the next section, we examine the related work in the area of parallel text. 3.2 Visual Designs for Visual Comparison of General Text 3.2 Visual Designs for Visual Comparison of General Text McNabb and Laramee [56] survey the surveys in informa- tion visualization literature and feature a text-focus cate- gory in their classiﬁcation. Alharbi and Laramee [3] also review the existing surveys that speciﬁcally review text visualization literature. They include and classify 13 surveys that review text visual analytic approaches. Wanner et al. [85] review the literature of text event detection techniques that are used along with visual analytics approaches. Also, Kucher and Kerren [54] survey the literature of the text visualization and classify them on a novel text visualization taxonomy. J¨anicke et al. [47], [48] are the only surveys that consider Digital Humanities as well as the data visualization community. 3.1 Visual Comparison as a General Task 8, AUGUST 2015 4 Summary of visual design characteristics for related work discussed in the sections: 3.3, 3.4 and 3.5. The “-” sign in the table indicates that an approach does not provide a distant or a close reading view. “Arbitrary” means the number is open based on the author’s claims and “Not-speciﬁed” means the number of parallel documents is not mentioned in the paper and is not exempliﬁed. The references are ordered based on publication date. [74] which enables the user to investigate multiple docu- ments side-by-side and integrates linking functionality to highlight corresponding text fragments. Similarly, multiple approaches use a side-by-side layout to represent compared objects, such as Jong et al. [34], Welsh and Hooper [84], Behrisch et al. [8], Wheeles and Jensen [86], the text view by Geng et al [27], the text reader by J¨anicke et al. [49], Howell et al. [39], Cheesman et al. [21], and J¨anicke et al [50]. Asokarajan et al [5] visualize the variation in a pixel- base matrix where the x-axis represents the offset in the text and the y-axis represents the variation (witnesses). They also visualize the summary of variation at the pages, lines, and words level. We extend the discussion of some of these approaches in Section 3.4. [74] which enables the user to investigate multiple docu- ments side-by-side and integrates linking functionality to highlight corresponding text fragments. Similarly, multiple approaches use a side-by-side layout to represent compared objects, such as Jong et al. [34], Welsh and Hooper [84], Behrisch et al. [8], Wheeles and Jensen [86], the text view by Geng et al [27], the text reader by J¨anicke et al. [49], Howell et al. [39], Cheesman et al. [21], and J¨anicke et al [50]. Asokarajan et al [5] visualize the variation in a pixel- base matrix where the x-axis represents the offset in the text and the y-axis represents the variation (witnesses). They also visualize the summary of variation at the pages, lines, and words level. We extend the discussion of some of these approaches in Section 3.4. links. Explicit encoding of relationships can be implemented using a dot plots representation to detect similarity and dissimilarity patterns, such as in Ribler and Abrams [64], in J¨anicke et al [49], and in Abdul-Rahman et al. [2]. Collins et al. (Parallel Tag Clouds) [23] also uses links and word clouds to encode the relationships between documents. 3.1 Visual Comparison as a General Task The system we present uses juxtaposition but with up to 38 close as well as distant reading of parallel translations. It also features explicit alignment curves. The exploration and interaction techniques are customized and implemented to satisfy the user requirements and tasks stated in Section 4. 3.1 Visual Comparison as a General Task Comparison is included in most task taxonomies [13], [68], [90]. It facilitates the exploration of the data in order to un- derstand the similarities or differences between comparison elements [4], [29]. JOURNAL OF LATEX CLASS FILES, VOL. 14, NO. 8, AUGUST 2015 4 References Source of text studied Max number of documents viewable simultaneously Visual design of distant reading view Languages Close Reading Distant Reading Ribler and Abrams [64] Programming codes - Arbitrary Patterngram English Monroy et al. [58] Literature (Don Quixote) 1 5 Multiple bar charts Spanish Schreibman et al. [74] Poetry Arbitrary - - English Jong et al. [34] Unspeciﬁed documents 9 14 Multiple views+pixel-based visualization English Collins et al. [23] U.S. Circuit Court Decisions 1 13 Parallel coordinates+tag cloud Englsih B¨uchler et al. [16] Ancient Greek text 3 - - Greek Welsh and Hooper [84] Newton Alchemical corpus 2 - - English Geng et al. [28] Literature (Othello) 8 8 Parallel coordinates German Behrisch et al. [8] News 2 33 Heatmap matrix English Howell et al. [39] Literature (The Secret Scripture) 2 - - English J¨anicke et al. [49] The Bible translations 2, 7 2 Text Re-use grid, Dot Plot view English J¨anicke et al. [42] The Bible translations - 24 Variant graphs English Geng et al. [27] Literature (Othello) 10 10 Parallel coordinates, heat maps, scatter plots German Riehmann et al. [65] PhD theses and Wikis documents 2 Not-speciﬁed Slope graph, glyph-based visualization English Asokarajanet al [5] Classical Latin texts - 22 Multiple views+pixel-based visualization, dot plot English Cheesman et al. [21] Literature (Othello) 1 2 , 40 Juxtaposed text versions, stylometrics diagram German Silvia et al. [80] Classical Latin texts - 12 Storyline visualization Latin J¨anicke et al. [50] Medieval texts 2 2 Juxtaposed text versions French Abdul-Rahman et al. [2] 18th-century literature 2 2 Parallel Coordinates, dot plot French TABLE 1 Summary of visual design characteristics for related work discussed in the sections: 3.3, 3.4 and 3.5. The “-” sign in the table indicates that an approach does not provide a distant or a close reading view. “Arbitrary” means the number is open based on the author’s claims and “Not-speciﬁed” means the number of parallel documents is not mentioned in the paper and is not exempliﬁed. The references are ordered based on publication date. JOURNAL OF LATEX CLASS FILES, VOL. 14, NO. 3.4 Examples of Visualizations from Digital Humanities There are many visualization solutions that enable digital humanities’ primary tasks. In this section, we focus on visual approaches that feature alignments between parallel texts. Jong et al. [34] present an interactive tool that conveys the structure of parallel texts using color-coded boxes repre- senting words. The reader can switch between the structural view and the textual view to facilitate close reading. Plagiarism detection is an application of visual text comparison as in White and Joy [87]. Also, Riehmann et al. [65] combine an overview slope graph and glyph-based de- tailed representations to explore given text against multiple sources. Different approaches overlay parallel texts in the same coordinate system in order to communicate comparative objectives. For example, the variant graph in J¨anicke et al. [43], Storylines in Silvia et al. [80], and Geng et al. [27], [28]. The variant graphs and parallel coordinates visualization represent each object as a line in the visual space. In the variant graphs, the y-axis illustrates the offset in the text or time. B¨uchler et al. [16] introduce a graph visualization to illustrate citation variation among documents in a corpus. They provide a distant reading view of the citation in the corpus using an interactive bar chart. Howell et al. [39] propose a close reading visual design driven by digital humanities methodologies of the novel The Secret Scripture (2008). They visually compare two different encodings of the same novel using different color-coded highlights. There are approaches which extract relationships be- tween parallel documents and explicitly visualize them to support visual comparison. The Stylometric representation of versions in Cheesman et al. [21] encodes the similarity be- tween connected texts using the thickness and length of the J¨anicke et al. [49] introduce distinctive contributions to this ﬁeld. They introduce multiple visual designs to depict text re-use between collections of text. For distant reading, JOURNAL OF LATEX CLASS FILES, VOL. 14, NO. 8, AUGUST 2015 5 exploration and investigation, such as brushing, selection, and linking. The second component of the visual design is the close view which shows the actual text and highlights the selected keywords. they design a visual matrix to discover the type and amount of text re-use between pairs of texts. Additionally, they intro- duce the text re-use reader which consists of two panels: a dot plot view and a text reader. The former view depicts the type of text re-use between two texts, e.g. 3.4 Examples of Visualizations from Digital Humanities a diagonal pattern indicates sections repetition while vertical and horizontal patterns indicate phrase re-use. The text reader shows two panels aligning two documents, both panels are linked and respond to one another. They also introduce the text variant graph [44] to detect variations between versions at the sentence level. The graph uses color-coded links for each version and font size to encode the number of occurrences among all versions. They demonstrate a graph applied to ﬁve versions of the Bible. Geng et al. [27] integrate multiple visual designs to illustrate the similarity between subsets of translations of Shakespeare’s play Othello. They provide visual designs to support distant reading, such as heat maps to illustrate seg- ment structure, and parallel coordinates to depict similarity among versions. In the text view, close reading is obtained by showing the text segments in multiple versions of the play. Cheesman et al. [21] present a web-based tool that en- ables the user to create parallel, segment-aligned multi- version corpora. The main goal of their project is to digitally explore patterns of variation among multiple translations. They present two overview designs which provide a distant reading view of the corpus. A small multiples pairwise alignment map of 35 German translations is used. Each translation is aligned with the base English text. Each speech is represented by a vertical rectangle and the height of the rectangle encodes the length of the speech. The edges between each translation and the base text represent align- ments between segments. The viewer can identify different attributes of each text and make comparisons, such as the variation in length between translations and the base text. In the same overview context, they provide more analytical and statistical aggregations of the corpus data represented by a stylometric network. The network diagram shows translation clusters which depict the similarities among versions. The connection edges represent the similarities in particular sets of frequency counts, and the thickness of the edges reﬂects the degree of similarity. J¨anicke et al. [42] propose an extended distant view of the variant graph to support analysis on higher text abstractions such as sections or chapters. They exemplify their method with a distant reading of 24 Bible editions. J¨anicke and Wrisley [50] propose a visual analytics en- vironment that supports aligning two versions, or more, computationally. 3.4 Examples of Visualizations from Digital Humanities Also, the tool integrates different interac- tive methods that analyze textual alignments, along with an intermediate view between close and distant reading which they call “Meso reading”. The Meso reading view combines the text and the statistical features together within the same visual ﬁeld. The difference between our work and the related work discussed in this section is that our proposed design con- nects a distant reading view of all 38 translations with the close reading view in a novel way by smooth zooming and panning. Interactive zooming and aligning facilitate comparison of the related speeches across a number of parallel texts (Section 5). Previous work separates the close and distant reading views in multiple windows. The user is required to cognitively integrate the two. Also, we encode means to help the user validate alignment or translation of the segments using similarity metrics. The Eddy and Viv metrics are calculated interactively and dynamically to reﬂect the similarity among the current selection of parallel translations. Among all of the related work, our proposed design deals with a cross-language dataset and presents a macro (distant) view of the entire collection. Previous work does not generally, support comparison of over 30 aligned translations and is generally restricted to the English language or a single language. Additionally, Cheesman et al. [21] provide a detailed interface which aligns segments of the base text with trans- lated versions along with similarities metrics (Eddy and Viv) (Explained in Section 2). The difference between the work presented in this sec- tion and our work is that Geng et al. [27], [28] present only a subset of the collection and is difﬁcult to scale accordingly. On the other hand, Cheesman et al.’s [21] alignment map aligns only one translation with the base text and does not encode any similarity features. The Eddy and Viv in- terface provides only a close reading and does not allow ﬁltering and selection of translations. Our work supports the comparison of the whole collection and incorporates the encoding of similarity metrics. It provides a variety of interaction and exploration techniques that facilitate the analysis and visualization of the collection. There is some overlap of co-authorship between this current work and previous work on Shakespeare’s Othello. We exploit the previous studies to guide the current work. 3.4 Examples of Visualizations from Digital Humanities g g g g g In Section 3.5, we introduce the related literature that analyses and provides visual designs of Shakespeare’s Oth- ello collection. 5 TRANSVIS’S DESIGN The original question that was posed for the application to address is: how can the variation between any number of parallel translations of a given source text be represented visually so as to enable users (a) to identify overlaps, absences, additions, and variation between parallel trans- lations and (b) to study the ﬁndings of various kinds of algorithmic, comparative text analyses. So, the rationale behind our visual design is to enable users to interactively explore the translation collection to answer this question. To achieve this, we established and incrementally reﬁned a list of requirements. The requirements that our proposed implementation fulﬁlls are as follows: In this section, we introduce our proposed interactive visual design of parallel translations and relate our choices to the tasks from Section 4. Our system is composed of four main constituents starting with an overview. g The ﬁrst window offers the alignment overview of parallel translations of Shakespeare’s Othello (T1, T5). It provides a general context for understanding the collection and conveys the whole dataset in one visual layout. Fur- thermore, it leverages interactive capabilities to enable the user to explore and ﬁnd interesting patterns and features within the collection. The alignment overview allows users to examine signiﬁcant, larger patterns in the translations which are not readily viewable from narrow or detailed views. Window (A) in Figure 1 shows the distant reading view of 38 parallel translations aligned with the base text. The curved edges between translations depict alignments between speeches. The zoomed-in portion in Figure 1 shows a close view of the curved edges and segments. R1 An application that enables comparison of translated parallel text. R1 An application that enables comparison of translated parallel text. p R2 A visual design that supports both close and distant reading. g R3 A layout that supports close reading for further detailed analysis. y R4 A visual design that considers stable versus unstable translations. Design justiﬁcation of the alignment overview: Our data is high-dimensional. We present 38 texts and each text contains multiple levels of abstraction (term, segment, speech, and books). Thus, we present a parallel view of translations and in each translation we present the encap- sulated structure. Juxtaposition supports visual compari- son of alignments intuitively, when comparing different manuscripts the user places them spatially next to each other and performs comparison. 5 TRANSVIS’S DESIGN Also, we incorporate a number of exploration techniques to help the user customize the alignment overview to their preference such as by zoom- ing, ﬁltering, or selecting. R5 Interaction that enables customization of the translated texts. R5 Interaction that enables customization of the translated texts. R6 Interaction that supports general exploration and anal- ysis. R6 Interaction that supports general exploration and anal- ysis. To action this list of requirements, we established a list of associated tasks for implementation. We derived ﬁve main tasks based on the typology of visualization tasks by Brehmer and Munzner [14] in order achieve the aforemen- tioned requirements and motivate our visual design: T1 In this discovery process, the user should be enabled to explore the alignment overview in order to identify a region of interest (R1). [discover→explore→identify] The second main component is the options panel, shown in Figure 1 (B). It provides the user with a range of layout functions in order to facilitate exploration of the collection and comparison of translations (T2, T5). The user can per- form a query-based search and the results are visualized using focus+context in the main window (A). It features a number of tabs that support different tasks. The ﬁrst tab is for color properties which enables the user to modify the color mapping schemes and color-map different properties of the speeches, such as individual speech length and lan- guage similarity values. The second tab is the options tab where the user may alter the properties of the visual design, such as the order of the translations and length of time tooltips are shown over speeches. The third tab is the ﬁlter tab. Different ﬁlters are provided for the user to reduce the complexity of the visual design, such as stage direction and speaker ﬁlters. These ﬁlters are discussed in Section 5.2.3. The last tab is the time-oriented thumbnail view. In this section, all of the translations are depicted using color-coded thumbnails. If the translation is shown in the main window (A), the thumbnail is green otherwise it is red. These ﬁltering and selection options are the result of our feedback sessions with the domain expert. This is described in more detail in Section 6.1. T2 After identifying a region of interest (T1), the user may navigate the space leveraging smooth zooming and panning. As the user navigates, multiple levels of details are aggregated and rendered (R2, R3, R6). 3.5 Previous Work on Shakespeare’s Othello In this section, we discuss previous visualizations of Shake- speare’s Othello. Geng et al. [28] introduce a focus+context parallel coordinates layout for comparing eight translations of Shakespeare’s Othello. Their design consists of two main components. The ﬁrst is a distant view represented by par- allel coordinates to show the variation between translations and the use of the most frequent words. A collective concor- dance of the most frequent words is shown in the column on the far left. Each coordinate represents a word-translation pair, and the thickness of the bar encodes the similarity rank. They support various interaction techniques to aid In summary, the current work is unique in that it in- cludes 38 translations along with the source text. The current work supports integrated distant and close reading in the same view and implements them with smooth zooming and panning. Also, it allows the user to explore different regions of interest and stores them for further analysis. The current design allows the user to interactively customize the alignment overview, and subsequently update the similarity metrics based on the user’s preference. Although, the TLC 6 JOURNAL OF LATEX CLASS FILES, VOL. 14, NO. 8, AUGUST 2015 to align texts side-by-side to support comparison tasks. Ad- ditionally, the design is also guided by careful collaboration with the domain expert. (Term Level Comparison) design is not novel, including it in the process of exploring the dataset is novel and proves useful. See Sections: 5.1.3 and 6.2 for practical use of the TLC view. 5 TRANSVIS’S DESIGN The zoomed-in rectangle is part of the ﬁgure, not of the visualization itself. A B C D Fig. 1. The alignment overview (A) shows the parallel alignment of translations with the original base text. The highlighted path in (A) shows the distant alignments of a segment of the “Othello” speech starting with “I ran it through...”. In the left-bottom, a zoomed-in view magniﬁes the curved edges. Window (B) shows the options panel that facilitate exploration of the collection and comparison of translations. View (C) is a close reading view (the detailed view) that corresponds to a user-selected speech and each aligned speech. Window (D) shows the Term-Level Comparison (TLC) view. The zoomed-in rectangle is part of the ﬁgure, not of the visualization itself. Design justiﬁcation of time-oriented thumbnail view: Most of the user options are implemented in close collabo- ration with the domain expert. The thumbnail view clusters the translations chronologically which makes it intuitive and quick to explore and navigate. Adding and removing translations is simply performed either by toggling a trans- lation on or off or dragging and dropping. It is important to let the user customize the starting point when there are so many translations. The view aids the user by visually informing him of the selected translations (green buttons) and deselected ones (red buttons). to revisit any previously selected paths for further analysis (T5). Also, the text in this view is accessible to user and can be copied to be used beyond our system. The detailed view is discussed in Section 5.1.2. Design justiﬁcation of the detailed view: It is rec- ommended to allow the user to have access to the actual text particularly when developing visualization for literary scholars [34], [48]. This close reading gives complete access to the text for further analysis with other software. This view aligns the segments in a compact and simple context for further analysis. It incorporates a list of previously selected paths to facilitate comparison between paths and also to enhance the user experience by saving the actions history (T5). We provide this addition of close reading to support close and distant reading simultaneously without losing the user’s ﬁrst (or previous) choice of speech for close reading. The third main component of the system is the detailed view which is the focus subset that the user is interested in after performing ﬁltering and selection (T4). 5 TRANSVIS’S DESIGN [T1→navigate→aggregate] T3 As the user performs T1 and/or T2, the user may apply different ﬁltering and selection tasks to assess the exploration task (R5). [T1/T2→ﬁlter/select] p T4 As the user performs T2, the user may select a segment to obtain details-on-demand (i.e. a close reading view) (R3). [T2→select] T5 As the user performs T1, T2 and/or T4, the user may perform interactive comparisons of the parallel translations exploiting meta-data based on the align- ment of speeches and text similarity metrics (R4, R6). [T1/T2/T4→compare] T5 As the user performs T1, T2 and/or T4, the user may perform interactive comparisons of the parallel translations exploiting meta-data based on the align- ment of speeches and text similarity metrics (R4, R6). [T1/T2/T4→compare] We relate to these tasks in the discussion of our pro- posed design (Section 5). Our design is inﬂuenced by the visual information seeking mantra by Shneiderman [78] that suggests providing an overview ﬁrst, then zooming and ﬁltering options, and ﬁnally details-on-demand. The design is also inﬂuenced by previous work on this topic which aims NAL OF LATEX CLASS FILES, VOL. 14, NO. 8, AUGUST 2015 A B D C . The alignment overview (A) shows the parallel alignment of translations with the original base text. The highlighted path in (A) shows t nt alignments of a segment of the “Othello” speech starting with “I ran it through...”. In the left-bottom, a zoomed-in view magniﬁes the curv s. Window (B) shows the options panel that facilitate exploration of the collection and comparison of translations. View (C) is a close read (the detailed view) that corresponds to a user-selected speech and each aligned speech. Window (D) shows the Term-Level Comparis view. The zoomed-in rectangle is part of the ﬁgure, not of the visualization itself. JOURNAL OF LATEX CLASS FILES, VOL. 14, NO. 8, AUGUST 2015 7 A B D C The alignment overview (A) shows the parallel alignment of translations with the original base text. The highlighted path in (A) sho alignments of a segment of the “Othello” speech starting with “I ran it through...”. In the left-bottom, a zoomed-in view magniﬁes the Window (B) shows the options panel that facilitate exploration of the collection and comparison of translations. View (C) is a close he detailed view) that corresponds to a user-selected speech and each aligned speech. Window (D) shows the Term-Level Com view. 5.1.2 Detailed View For Close Reading In addition to smooth zooming in the alignment overview that enables the user compare speciﬁc speeches, the user is able to select any speech to analyze and compare it along with the aligned speeches in a dedicated detailed view as shown in Figure 2 (T4, T5). The detailed view provides another close reading option for the speeches such as trans- lators names, speech identiﬁer and Eddy value (discussed in Section 2). The close view shows the user-selected segment using a highlighted red border and each aligned speech in a scrollable window that is easy to read and investigate (T5). Each speech is paired with a colored bar showing the similarity distance to indicate the distinctiveness between aligned speeches. The longer the bar, the more distinctive the speech is with respect to the other translations. Design justiﬁcation of the TLC view: As TLC view could be considered as parallel coordinates, it presents strength when exploring and processing multi-variate data [41]. Such techniques are useful to explore anomalies in the data even without an extended outlier-detecting mechanism [61]. As a result of the TF-IDF, the commonly used terms are assigned lower values and vice versa. Thus, the distinctive terms will stand out very clearly in the TLC view which is the main motivation behind this design. To overcome the limitation of the view when there are cluttered lines, the view provides a list of words in alphabetical order. When the user selects any word, the TLC highlights the line corresponding to this word. The user also can enable brushing to highlight multiple lines. The color opacity of the unselected terms is reduced in order to remain as context. The terms list updates to reﬂect the brushed terms. The terms list also assigns the same color of the term’s line to the list item to visually identify the line’s correspondence as shown in Figure 3. The colors of the lines are automatically generated to uniquely assign a color to each term. In order to improve the user experience and to ease the exploration and analysis task for the researchers, we maintain a history of all previously investigated speeches in a list as shown in Figure 2 (bottom-left corner). When the user revisits any of the previously examined speeches in the list, the corresponding speech and the edges of alignments are highlighted in the alignment overview. 5 TRANSVIS’S DESIGN As shown in Figure 1 (C), the detailed view shows a close reading of the user-selected speech along with the aligned speech translations. It stores both the interesting aligned segments (path) and the alignment overview state, so the user is able The fourth component is the Term-Level Comparison (TLC) view (Figure 1 (D)). The TLC is an interactive ana- JOURNAL OF LATEX CLASS FILES, VOL. 14, NO. 8, AUGUST 2015 8 explained it results in ﬁltered alignments as the path moves left-to-right. However, the user-chosen order of translations affects the results in the alignment overview and the user needs to choose the sorting function carefully. lytical tool that assigns weights to each word in each user- chosen segment aligned with a base English segment. We use TF-IDF as a term-weighting to measure the signiﬁcance of each original word or lemma in a segment with respect to the word occurrence in the whole corpus (the segments aligned with the base English text in our case). The view clearly justiﬁes the Eddy metrics and signiﬁes the terms (original or lemmas) that deﬁne segments. Also, it aids the user in ﬁnding terms and translation variance (T5). To show this plot, the user can drag an interesting path, usually because of an Eddy value distinctiveness pattern, then drops it onto the TLC view area to see the word’s contribution in this path. This view is also motivated by the on-going discussion with the domain expert requesting close analysis and details. This mechanism can be useful, particularity when com- paring translations and as a ﬁltering option (T3, T5). How- ever, in some cases, it might not yield results when the translations are not related, such as when translations stem from a different era or author. 5.1 Visual Design Factors The TLC View illustrates the weighting of each term using line charts. The y axis shows the normalized weighting of the terms. Along the x axis, we render vertical lines to indicate the translations. In this view, we can explore each term weight across all translations. The TLC view (Figure 3) facilitates comparisons and analysis tasks (T5). For the term- weighting, as discussed in Section 2, we use the TF-IDF weight for each word. We separate the primarily visual and interactive design features to facilitate reading. 5.1.2 Detailed View For Close Reading The interactive history list identiﬁes each user-selected alignment using the segment identiﬁcation and the translation name such that the user may remember their own user provenance with respect to the alignment selection. 5.1.1 Filtering based on Derived Alignments Each speech is paired with with a colored bar (annotated using a green border) to indicate the similarity distance. In the bottom-left corner, a list of all previously selected speeches. If the user selects any speech from the list, the corresponding text is highlighted in the alignment overview and the edges of alignments are presented above. Fig. 3. An example of aligned segments of the base English segment starting with “Thou art sure of me...” depicted by term-weighting matrices using the TLC view. In this example, the user brushes the three peaks that reﬂect the distinctive translations of the word “Moor” in the context: “I hate the Moor”. The highlighted translations are “Neger” in Buhss (1996) [17], “Schwarze” in G¨unther (1992) [33], and “Maure” in Schwarz (1941) [76]. The terms list reﬂects the brushing result and assigns the same colors to the terms. The user-chosen terms are rendered in the focus while the rest are rendered as context. Fig. 3. An example of aligned segments of the base English segment starting with “Thou art sure of me...” depicted by term-weighting matrices using the TLC view. In this example, the user brushes the three peaks that reﬂect the distinctive translations of the word “Moor” in the context: “I hate the Moor”. The highlighted translations are “Neger” in Buhss (1996) [17], “Schwarze” in G¨unther (1992) [33], and “Maure” in Schwarz (1941) [76]. The terms list reﬂects the brushing result and assigns the same colors to the terms. The user-chosen terms are rendered in the focus while the rest are rendered as context. (a) (a) (b) Fig. 4. (a) An example of two corresponding sub-sets of six editions of Baudissin (1832) [7]. The variation of colors in the dashed rectangle in the original version (a) is clearer than the corrsponding rectangles in the lemmatized version (b). (b) (a) is clearly a result of inﬂected words that share the same lemma. Although, technically, TLC view is not a parallel coordi- nates view, it inherits some of its limitations. The TLC view can be difﬁcult to interpret and explore in the case of long segments due to over-plotting. Much research focuses on interaction techniques to overcome the visual clutter chal- lenge in parallel coordinates plots by reducing the dataset or by reducing or modifying the order of the dimensions [45]. 5.1.1 Filtering based on Derived Alignments The dataset contains alignment information between each speech in each translation and the English base text. How- ever, it is useful to have an alignment between any two arbitrary translations. We derive meta-data that aligns two arbitrary translations with respect to the base text. We align two speeches from different translations if they correspond to the same base English speech. Alignment overview with TLC view: The rationale of this view is to observe and explore the aligned segments in the term level and highlight outliers and uncommon terms. The TLC shows the terms that contribute to the pattern discovered in the alignment view when using the similarity metrics. g p With the alignment meta-data, we have the alignment between the source text segments and the translations’ segments. The derived alignment is the alignment between a translation and another translation. If the translation is not adjacent to the source text, we derive the alignment between two segments (a and b) of two translations (A and B) if b has an actual alignment and a has an actual alignment, we connect them. The path of alignment disconnects if one of these conditions break. For instance, if b has an actual alignment with the source segment, and a which belongs to translation A that occurs before translation B does not have an actual alignment, the path of alignment disconnects. This mechanism is implemented as a user option, and as To explore aligned segments, the user can drag-and- drop any segment of the path from the alignment overview into the TLC view. A list of the terms is also presented on the right. If the user selects any word in the list, the corresponding line is highlighted, and vice-versa. In Figure 3, three distinctive terms are highlighted in the TLC view: “Neger”, “Schwarze”, and “Maure”. These translations correspond to the word “Moor” in the context: “I hate the Moor.” These terms are illustrated in the three brushed peaks shown in the ﬁgure. The terms list shows the JOURNAL OF LATEX CLASS FILES, VOL. 14, NO. 8, AUGUST 2015 9 Fig. 2. The detailed view shows the user-selected speech highlighted using a red border and the aligned speeches ordered consistent with the translations appearing in the alignment overview. Each speech is paired with with a colored bar (annotated using a green border) to indicate the similarity distance. 5.1.1 Filtering based on Derived Alignments In our case, the combination of TF-IDF as a term- weighting metric causes the common terms to clutter in the areas of low x values. However, as this can be problematic when searching for common terms, it helps to identify rare vocabulary in each translation. (a) (b) Fig. 4. (a) An example of two corresponding sub-sets of six editions of Baudissin (1832) [7]. The variation of colors in the dashed rectangle in the original version (a) is clearer than the corrsponding rectangles in the lemmatized version (b). The alphabetically-ordered word list that accompanies the view can also help the user ﬁnd term line by clicking on a word and highlighting the corresponding line. The stop words removal, or lemmatization can make the plot less cluttered. only selected terms and the color facilitates identifying the correspondence between the lines and the terms. This se- lected terms are rendered in focus and the rest are rendered in context. See the domain expert feedback in Section 6.1 for further discussion on this. 5.1.1 Filtering based on Derived Alignments In the bottom-left corner, a list of all previously selected speeches. If the user selects any speech from the list, the corresponding text is highlighted in the alignment overview and the edges of alignments are presented above. Fig. 3. An example of aligned segments of the base English segment starting with “Thou art sure of me...” depicted by term-weighting matrices JOURNAL OF LATEX CLASS FILES, VOL. 14, NO. 8, AUGUST 2015 9 Fig. 2. The detailed view shows the user-selected speech highlighted using a red border and the aligned speeches ordered consistent with the translations appearing in the alignment overview. Each speech is paired with with a colored bar (annotated using a green border) to indicate the similarity distance. In the bottom-left corner, a list of all previously selected speeches. If the user selects any speech from the list, the corresponding text is highlighted in the alignment overview and the edges of alignments are presented above. JOURNAL OF LATEX CLASS FILES, VOL. 14, NO. 8, AUGUST 2015 JOURNAL OF LATEX CLASS FILES, VOL. 14, NO. 8, AUGUST 2015 9 Fig. 2. The detailed view shows the user-selected speech highlighted using a red border and the aligned speeches ordered consistent with the translations appearing in the alignment overview. Each speech is paired with with a colored bar (annotated using a green border) to indicate the similarity distance. In the bottom-left corner, a list of all previously selected speeches. If the user selects any speech from the list, the corresponding text is highlighted in the alignment overview and the edges of alignments are presented above. Fig. 3. An example of aligned segments of the base English segment starting with “Thou art sure of me...” depicted by term-weighting matrices using the TLC view. In this example, the user brushes the three peaks that reﬂect the distinctive translations of the word “Moor” in the context: “I hate the Moor”. The highlighted translations are “Neger” in Buhss (1996) [17], “Schwarze” in G¨unther (1992) [33], and “Maure” in Schwarz (1941) [76]. The terms list reﬂects the brushing result and assigns the same colors to the terms. The user-chosen terms are rendered in the focus while the rest are rendered as context. Fig. 2. The detailed view shows the user-selected speech highlighted using a red border and the aligned speeches ordered consistent with the translations appearing in the alignment overview. 5.2.1 Smooth Zooming of Translations In our design, we start with the alignment overview of the whole collection of translations. The user is able to zoom in smoothly and ﬂuidly to explore the dataset and investigate (T1, T2). When zooming in smoothly, more details fade in gradually, such as the actual text content of each speech to support close reading. See Figure 5. We compute the average time (milliseconds) of ren- dering the scene while performing the zooming. For each number of translations, we record the average time for the process. See Figure 7 in the supplementary ﬁle. Our system takes about 5.6 ms to render the scene. To demonstrate the scalability and the linearity between the number of translations and performance time, we modiﬁed the dataset to increase the number of texts to 76 translations and 1 base English text. We can see from the ﬁgure that the time in general increases as the number of translation increases. We perform this experiment using a machine with the following speciﬁcation: Processor: 3.3 GHz Intel Core i5, operating system: Mac OS version 10.14, memory: 8 GB DDR3, and an AMD Radeon graphics card with 2 GB of memory. With zooming as an option, the user is able to investigate a region of interest in the same view and has the ability to transition between close and distant reading without switching between multiple windows. When zoomed in, the user is able to pan smoothly for comparison of translations (T4, T5). During the zooming, we maintain certain levels of detail as shown in Figure 6 in the supplementary ﬁle. The ﬁrst level illustrates the speech and depicts the length of the segments. Also, the relative thickness of the border high- lighting the user-selected segment is increased relatively. We decrease the thickness of the highlighting border as the user zooms in. In the second level, more segment details are revealed, such as local colors and the length of the text. Fi- nally, at the third level, the text is readable and the thickness of the highlighting border is decreased considerably. This functionality is implemented in the alignment overview and it zooms along both x and y axes with respect to the mouse position. Zooming in on only y axis could cause only a single word to appear on each line which is difﬁcult to read. 5.2 Interaction Design Factors In this section, the primarily interaction design factors that our system encompasses are elaborated. The design is heav- ily based on user preference and interaction to customize the output. In general, the interaction starts with the alignment overview, the system by default shows the entire collection. The user can interact with the system to modify the align- ment overview using the options panel, shown in Figure 1 (B). For example, the user can change the order of transla- tions based on a range of sorting presets, ﬁlter the alignment overview based on a speaker, or change the presented translation using the time-oriented thumbnail view (Section 5.2.3). Then, the user can use the smooth zooming and panning (Section 5.2.1) to explore and identify passages of interests. Once the user ﬁnds an interesting pattern guided by the predeﬁned customization and the similarity-based color-coded segments, the user can use the detailed view Lemmatization (as discussed in Section 2) is a normaliza- tion algorithm used to reduce inﬂected words and identify the base word [53]. We use CAB [46] to create a dictionary of the lemmas for our corpus. Processing such an antiquated language is challenging due to many missing terms. Example–lemmatized versus original text: We compare the results between a lemmatized and non-lemmatized ver- sion of the translations. Our corpus includes several editions of the classic, canonical Baudissin translation, ﬁrst published in 1832 and often re-published (100s of times so far): printed editions from 1851, 1923, 1926, and 1947, and a digital edition from 2000. In Figure 4, we can see in the original version (a) that the variation of colors in the dashed rectan- gles is more distinctive than the corresponding areas in the rectangles in the lemmatized version (b). The variation in JOURNAL OF LATEX CLASS FILES, VOL. 14, NO. 8, AUGUST 2015 Fig. 5. An example of three levels of integrated zooming and the smooth changes to the level of detail. (left) Distant reading without zooming. (middle and right) close reading after the user zooms in. The textual content of the speeches fades in smoothly at an increasing level of detail. The grey curved path indicates a user-selected alignment. JOURNAL OF LATEX CLASS FILES, VOL. 14, NO. 8, AUGUST 2015 JOURNAL OF LATEX CLASS FILES, VOL. 14, NO. 8, AUGUST 2015 10 (a) (b) Fig. 6. Two snapshots of the same region of the collection. 5.2 Interaction Design Factors In (a) the segments aligned with the user-selected segment are out of view and not visible. In (b) the segments are horizontally aligned with the user- selected segment. (a) (b) Fig. 5. An example of three levels of integrated zooming and the smooth changes to the level of detail. (left) Distant reading without zooming. (middle and right) close reading after the user zooms in. The textual content of the speeches fades in smoothly at an increasing level of detail. The grey curved path indicates a user-selected alignment. (b) (a) Fig. 6. Two snapshots of the same region of the collection. In (a) the segments aligned with the user-selected segment are out of view and not visible. In (b) the segments are horizontally aligned with the user- selected segment. (Section 5.1.2) to explore or save the path of alignment by clicking on it, or dragging the path and dropping it on the TLC view (Section 5.1.3) to examine the terms contained in the path. When the user clicks on individual segments, the system saves them and allows the user to revisit them and retrieve the alignment overview. Both of the case studies (Section 6.2) demonstrate the interactive overall process that facilitates the ﬁnding. (Section 5.1.2) to explore or save the path of alignment by clicking on it, or dragging the path and dropping it on the TLC view (Section 5.1.3) to examine the terms contained in the path. When the user clicks on individual segments, the system saves them and allows the user to revisit them and retrieve the alignment overview. Both of the case studies (Section 6.2) demonstrate the interactive overall process that facilitates the ﬁnding. There are other approaches that utilize interactive lenses [18], [66]. We believe that magic lens and interactive mag- niﬁcation techniques that allow the user to obtain a close picture of the interested area could be useful. However, they also introduce new drawbacks such as either distortion or discontinuity between the focus and context areas. We discuss these techniques with the domain expert and believe such alternatives could be explored in future through task- and design-driven studies. We did not incorporate them because the user requirements are fulﬁlled with our current design choices. This discussion also leads to the identiﬁca- tion of an important pitfall in design studies. See Section 7 for details. 5.2.3 Filtering and Selection of Speeches and Translations in Options Panel Some of the segments in the collection are not spoken in the play but provide directions to the characters. These segments are called stage directions and can be toggled on or off (T3, T5). Most of the time, researchers are not interested in studying them and removing them can reduce clutter from the scene. In the options panel (Figure 1 (B)), the user is able to ﬁlter and compare the translations in a variety of different ways (T3, T5). We found that this option was always the ﬁrst user-option chosen by the domain expert (see case studies in Section 6.2). One option is ﬁltering the data based on a custom query. The user is able to search for a given speaker, a speciﬁc segment ID or word. Also, the collection can be ﬁltered based on a speaker name if the user is interested in a speciﬁc character. The speaker search is non-trivial since the speakers are translated differently from one translation to another, e.g. the speaker “Duke of Venice” is translated for example into: “Der Doge”, “Herzog”, and “Doge” and the speaker “First Senator” is translated into: “1 Senator”, “Senator” and more commonly “Erster Senator”. However, to address this issue and to reveal more relevant results across both the base English text and the translations, we populate the speaker list with all of the characters in the original text along with those not aligned with the original text. When the user looks up a speaker, we can eliminate all characters not aligned with it. Thus, we are able to depict all translated speakers in the translations. See Section 6.1 for the domain expert feedback. The researcher is able to use the thumbnail view tab to add or remove translations. Adding and removing transla- tions is achieved using a smooth drag and drop interaction or simply by selecting and deselecting translations (T5). The translations in the options panel as seen in Figure 1 (B) are color-coded green or red according to the translation pres- ence or absence in the alignment overview (A) respectively. See case studies Section 6.2 for an example of this used in practice. Additionally, we implement various translation order- ing and sorting options to aid researchers in ﬁnding the best layout that supports comparison of translations (T5). The options provide an initial layout that suits the researcher’s needs. 5.2.1 Smooth Zooming of Translations In this ﬁgure, a segment of speech by “Othello” was mistakenly aligned with a segment of speech by “Duke of Venice”. Fig. 7. The placement of the unstable translation by B¨arfuss (2001) [6] results in multiple disconnected edges between non-adjacent transla- tion. The differently colored edges illustrate the alignment between the two non-adjacent translations. Fig. 8. A subset of a ﬁltered focus+context rendering of alignment overview. The view is ﬁltered by the speaker “Duke of Venice”. The high number of matches is due to mistaken correspondence during the original segment alignment process which is easily discovered by the visualization result. In this ﬁgure, a segment of speech by “Othello” was mistakenly aligned with a segment of speech by “Duke of Venice”. may also smoothly re-arrange the order of the translations manually through a drag-and-drop mouse movement. In our feedback and evaluation sessions with domain expert from the Arts and Humanities, this feature was used exten- sively. Example–Juxtapositioning Aligned Speeches: We im- plement a mechanism to smoothly translate corresponding speeches vertically as seen in Figure 6. In Figure 6 (a), the user-chosen segment and the aligned segments are not in the alignment overview as a result of their original placement within the translation sequence. In Figure 6 (b) the trans- lations slide vertically to line up and with the user-chosen segment. Thus, the user is able to compare and explore the segments in the same close view (T5). is transformed differently into “Erster Senator: Tot?” as in Flatter (1952) [26], “Die Senatoren: Tot?” as in Zeynek (1948) [91] and “Senator: Tot?” as in Wachsmann (2005) [83]. In some cases, speaker transformation is accepted since the source text might indicate different speakers for the same speech and translators interpret this with a variation. However, in other cases, the process of the alignment is not accurate and the speaker ﬁlter can facilitate the discov- ery of errors during the original, labor-intensive alignment process. For example, in Figure 8, the “Duke of Venice” speaker is selected to show only the corresponding seg- ments. However, a great volume of matching translated segments are connected to our surprise (≈1500 segments). After investigating, we ﬁnd that the aligner has mistakenly linked a speech by “Othello” to the speaker “Duke of Venice” which causes this volume of unexpected results. A conﬁguration can occur, if two adjacent translations are not aligned. This might lead to confusion. 5.2.1 Smooth Zooming of Translations Zooming in on x axis could result in long, difﬁcult to read lines and difﬁculty comparing alignments. 5.2.2 Filtering, Selection, and Positioning in the Alignment Overview Filtering and selection aim to reduce the complexity of the scene by abstracting some elements of the translations to help the user focus and ﬁnd regions of interest. The user can click on an individual speech and see the corresponding translated and aligned speeches interactively. Within each translation, horizontal bars represent a speech or a segment depending on the user preference. The vertical order of bars implicates the position of the corresponding speech or segment in the text. The thickness illustrates the length of the speech or segment calculated in words. However, when adjacent translations are distant from one another vertically, it may become difﬁcult to compare the aligned speeches, speciﬁcally in zoomed-in views, as illustrated in Figure 6 (a). Thus, the user can choose to interactively align them vertically side-by-side (T5), as shown in Figure 6 (b). Multiple works link different abstractions of single text using zoomable layouts. VarifocalReader [52] uses a com- bination of x and y axes zooming to show the multiple layers of abstraction. However, it becomes challenging when comparing more documents, and the number of abstractions layers needs to be decreased. Gold et al. [31] integrates the close and distant reading of a single text using zooming along the y-axis. On the other hand, Asokarajan et al. [5] use the zooming along the x axis to examine the view closely and do not provide a level of abstraction based on the zooming. We connect the corresponding segments using curved edges as illustrated in Figure 6. The user-selected alignment is highlighted to be distinct from other alignments. The user JOURNAL OF LATEX CLASS FILES, VOL. 14, NO. 8, AUGUST 2015 Fig. 7. The placement of the unstable translation by B¨arfuss (2001) [6] results in multiple disconnected edges between non-adjacent transla- tion. The differently colored edges illustrate the alignment between the two non-adjacent translations. 11 Fig. 8. A subset of a ﬁltered focus+context rendering of alignment overview. The view is ﬁltered by the speaker “Duke of Venice”. The high number of matches is due to mistaken correspondence during the original segment alignment process which is easily discovered by the visualization result. 5.2.1 Smooth Zooming of Translations To address this limitation, we implement a user interaction technique to render such edges that connect non-adjacent translations using a different color and rendering order as shown in Figure 7 which illustrates the problem when examining an unstable translation such as B¨arfuss (2001) [6]. However, this is still can be challenging particularly when dealing with unstable translations. In all of the above ﬁlters, the results are shown using focus+context. The results are visually highlighted while the context is preserved in greyscale. 5.2.3 Filtering and Selection of Speeches and Translations in Options Panel 5.2.3 Filtering and Selection of Speeches and Translations in Options Panel Some of the options have been suggested by the domain expert, such as sorting the translations chronolog- ically. Other options sort based on the aggregation of the similarity metrics, e.g. average and total of Eddy values. See case studies Section 6.2 for an example of this used in practice. Example–Filtering Based on Speaker: In some seg- ments, we are able to see transformation in speaker names, e.g. the speaker in the speech: “Duke of Venice: Dead?” The proposed visual design implements different color mapping schemes to assist researchers in investigating the JOURNAL OF LATEX CLASS FILES, VOL. 14, NO. 8, AUGUST 2015 12 Fig. 9. The alignment overview of the translation collection reveals an increase of variation between translations particularly after the second world war, with the exception of Engel (1939) [25]. 12 parallel translations. There are different primary color map- ping schemes as shown in the accompanying video. In the center, there are three translations attributes that we color code the text based on (segment length, speech length, and Eddy and Viv value). In the bottom section, we provide the user with a range of color mapping scheme presets for the Viv values. The user is able to customize speciﬁc translation attributes which are multiple-aligned segments and non-aligned segments. See Section 6.1 for a case study demonstrating the utility of these color-mapping schemes. The sequential color schemes are generated using Color Brewer [35]. The rainbow-style color scheme is generated using Telea’s algorithm [82]. 6 EVALUATION We work closely with the domain expert and the project is driven by a real-world historical investigation. See Figure 10 in the supplementary ﬁle. With the close observation by the domain expert, we designed the application to satisfy the user requirements in Section 4 and facilitate exploration and interaction to enhance the user experience. Fig. 9. The alignment overview of the translation collection reveals an increase of variation between translations particularly after the second world war, with the exception of Engel (1939) [25]. The domain expert feedback demonstrated in Section 6.1 is based on the 9 in-person, regular sessions to demonstrate the integrated visual design features over 5 months. All of the sessions are video-recorded for post-analysis and archiving gathering. Our semi-structured interview ques- tions were previously planned and guided by Hogan et al. [38]. The total number of hours of feedback is around 9 hours. The ﬁrst few sessions consisted mostly of software demonstrations to guide the development of features. See Figure 10 in the supplementary ﬁle. However, these gradu- ally turned into an active hands-on use of the software by the domain expert. The software also evolved due to feature requests. We implemented features that give researchers more control over the exploration, comparison, and analysis of parallel translations. During the face-to-face feedback sessions, there are different patterns that were observed, such as, the discovery of software bugs, and the discovery of data-level errors. Relevant online outputs, presentations, and published ar- ticles by him and his collaborators are listed on the project’s website [19]. The articles include publications in Digital Scholarship in the Humanities [21] and Journal of Data Mining and Digital Humanities [22]. 6.2 Case Studies Using the Design and the Integrated Similarity metrics Detailed View Feedback: The domain expert ﬁnds this view useful because it fulﬁlls tasks T4, T5. He experi- mented with this feature and shortly afterwards he started searching for an alignment path he examined previously. He states: “How do I ﬁnd it now?” The provenance list that stores previously selected paths enables the user to trace back all of the user’s actions whilst exploring the alignment overview. The domain expert ﬁnds this helpful to retrace his previous selections. The technical limit to the number of archived user-interactions is the same as that of a web browser with bookmarks. The domain expert might ﬁnd it cognitively difﬁcult to remember the location of an alignment in the list when there too many archived user-interactions. This cognitive limit can be addressed in future work by enabling the user to personally rename the archived user-interaction labels. Also, the domain expert suggested a potential feature that enables them to keep notes in each alignment. This discussion leads to a new pitfall (PF-33) called feature creep. See Section 7 for details. We can select a particular set of translations (a sub-corpus) within our corpus on the basis of known features such as type of translation, or date. The Eddy color mapping iden- tiﬁes segments which are of interest because they diverge from others which are more similar. Eddy is calculated on the basis of words, not semantics, so different values do not necessarily predict differences of understanding or interpretation. But Eddy color mapping in this interface encourages an exploratory kind of reading which shifts between scales and between following the course of a single text (vertically) and comparing between texts (horizontally). It also encourages exploring what comparative juxtaposi- tions produce interesting results for a humanistic reader. Do apparently identical texts differ? Discovering the work of editors/rewriters: As mentioned earlier, our corpus includes different editions of the Baudissin translation, ﬁrst published in 1832 and often re-published (100s of times so far): printed editions from 1851, 1923, 1926, and 1947, and a digital edition from 2000. Using the interactive drag-and- drop thumbnail view, we can select those ﬁve translations. 6.1 Domain Expert Feedback “Again, intensifying and bringing back the hint of homo-eroticism in the original, which the classic text censored.” Filtering Feedback: The domain expert ﬁlters out the outlier translations, and stage direction segments then chooses ﬁve different editions of Baudissin (1832). The do- main expert discovers that Wolf (1926) [89] and Brunner (1947) [15] show high-Eddy values. The domain expert stated, “These editors ‘intervened’ quite often, altering the text they had received from earlier editions –usually to improve it, i.e. remove bits of poor writing. Both make changes which are not ‘signiﬁcant’ but just make the text more readable (and actable).” we discover that there are three translations that stand out from the rest. The three translations have three recognizable words with high values which can be easily observed in the TLC view. These words are translations of the English word “Moor” in the context: “I hate the Moor ...”. The translations are “Maure” in Schwarz (1941) [76], “Schwarze” in G¨unther (1992) [33], and “Neger” in Buhss (1996) [17]. Most of the translations translate the word into “Mohren”. “That is a good result, that is interesting,” states the domain expert as he looks at the resulting images. He validates the discovery and reasons that as, “That word is translated in different ways in different times with different political implications.” 6.1 Domain Expert Feedback The Alignment Overview Feedback: The alignment overview enables the researcher to capture global patterns and direct attention to a region of interest for a more detailed investigation. When demonstrating the camera-positioning features in the alignment overview, the domain expert states, “In a birds eye view, the visual design shows how versions differ in length, and the number of alignments, and the Eddy color mapping neatly highlights (a) versions which are generally a high- Eddy and (b) segments and passages in the run of text which are high-Eddy. This is great.” The color mapping used to illustrate different translation attributes in combination with sorting options may reveal new insight to the domain expert. As seen in Figure 9, the domain expert notices that the variation between trans- lations increases distinctively after the second world war. Also, he stated that, “modern translators increasingly diverge from the norms of theatrical Shakespeare language established in the 19th century and early 20th versions.” In Section 6.2, we present two case studies conducted to validate our visual design and the integrated similarity metrics. Domain Expertise: The domain expert is Professor Tom Cheesman, in the Department of Modern Languages, Translation and Interpreting in the College of Arts and Humanities, Swansea University. He is the principal inves- tigator on the “Version Variation Visualisation” project. The project is responsible for collecting, aligning, and warehous- ing the dataset that we are examining, and other ‘multi- retranslation’ datasets and the team has developed proto- type online tools [19] for managing such datasets and visual- ising comparative analyses of them. Professor Cheesman is a specialist in modern and contemporary German literature and culture. He has been researching German culture and translating German literature since the early 1980s. Profes- sor Cheesman has been investigating the history of German translations of Shakespeare’s play Othello since 2009, using both traditional qualitative methods (contextualised close reading) and experimental, quantitative, digital methods. Search Feedback: The focus+context rendering of alignment overview of search results can help the user Fig. 10. Focus+context rendering of alignment overview of the results of the search for the word “Lust”. Fig. 10. Focus+context rendering of alignment overview of the results of the search for the word “Lust”. JOURNAL OF LATEX CLASS FILES, VOL. 14, NO. 8, AUGUST 2015 Bau. 1851 Bau. 1923 Bau. 1926 Bau. 1947 Bau. 2000 Fig. 11. An example of a translation variation between ﬁve editions of Baudissin (1832). 6.1 Domain Expert Feedback Wolff (1926) stands out to be the most distinctive translation among the editions. JOURNAL OF LATEX CLASS FILES, VOL. 14, NO. 8, AUGUST 2015 13 (a) (b) Fig. 12. (a) Five classic canonical Baudissin translations. We can see the Wolff translation stands out due to the high values of Eddy. (b) After de-selecting the 1926 translation, we can see more variation among the four remaining translations. Fig. 11. An example of a translation variation between ﬁve editions of Baudissin (1832). Wolff (1926) stands out to be the most distinctive translation among the editions. to discover patterns or uniqueness in the collection. For example, the domain expert was interested in the word “Lust”. The results are appealing and uncover two main stream of paths that use the word. Most of the translations use the word once (13 translations), four translations did not use this term. As shown in Figure 10, above the top and below bottom main patterns we can see outlier usage of the word. “That is very good because I can see straight away some patterns and most of the translators are using the word in the same segments and some translators are using the word unexpectedly,” the domain expert stated. (b) (a) Fig. 12. (a) Five classic canonical Baudissin translations. We can see the Wolff translation stands out due to the high values of Eddy. (b) After de-selecting the 1926 translation, we can see more variation among the four remaining translations. Filtering Feedback: The domain expert ﬁlters out the outlier translations, and stage direction segments then chooses ﬁve different editions of Baudissin (1832). The do- main expert discovers that Wolf (1926) [89] and Brunner (1947) [15] show high-Eddy values. The domain expert stated, “These editors ‘intervened’ quite often, altering the text they had received from earlier editions –usually to improve it, i.e. remove bits of poor writing. Both make changes which are not ‘signiﬁcant’ but just make the text more readable (and actable).” From this focus and similar context, we can depict trans- lation variation easily. For example, in a speech Iago says to Roderigo: “If thou dost, I shall never love thee after. Why, thou silly gentleman!” All other Baudissin use the word “Fre- undschaft” which means ‘friendship’ and Wolff (1926) uses the word “Liebe” which means ‘love’ (Figure 11). 6.2 Case Studies Using the Design and the Integrated Similarity metrics Looking at a distance at this set of ﬁve texts, the Eddy value coloring immediately conveys that the 1926 edition is very different from the other four: high Eddy in almost every segment, while the other four show low Eddy values, in nearly all segments as shown in Figure 12(a). This reveals See Section 7 for details. Term-Level Comparisons View Feedback: The aligned translations of the base English speech starting with “Thou art sure of me...” show different segments with noticeable higher Eddy values. We easily plot the path of alignments interactively using a drag-and-drop of any seg- ment from the path onto the TLC view. As seen in Figure 3, JOURNAL OF LATEX CLASS FILES, VOL. 14, NO. 8, AUGUST 2015 Muthmaßung schätzungsweise Vermutung Gerüchte Baud. 1851 Baud. 1923 Baud. 1947 Baud. 2000 Fig. 13. The TLC view of the path containing the phrase “the aim reports”. The colored lines represent terms and each vertical line rep- resents a translation. The annotated words and arrows illustrate the corresponding terms and lines. JOURNAL OF LATEX CLASS FILES, VOL. 14, NO. 8, AUGUST 2015 14 Muthmaßung schätzungsweise Vermutung Gerüchte Baud. 1851 Baud. 1923 Baud. 1947 Baud. 2000 Fig. 14. A sub-set of the translation selected based on the domain expert knowledge shows three periods of generally high Eddy values. The three periods are highlighted by borders. Baud. 1923 Fig. 13. The TLC view of the path containing the phrase “the aim reports”. The colored lines represent terms and each vertical line rep- resents a translation. The annotated words and arrows illustrate the corresponding terms and lines. Fig. 14. A sub-set of the translation selected based on the domain expert knowledge shows three periods of generally high Eddy values. The three periods are highlighted by borders. Ihr, Vater schätzte gut Fig. 15. Three uses of the TLC view to help the domain expert ﬁnd variation between different translation. The top shows a common use of the words ‘Ihr’ and ‘Vater’. The middle and bottom views show that the words: ‘sch¨atzte’ and ‘gut’ which are used only in speciﬁc translations. that the 1926 so-called ‘edition’ (‘revised’ by Max Wolff) [89] is not really an ‘edition’ of the Baudissin translation at all: it’s almost a completely new translation, created by editing Baudissin. Interactively zooming in for close in- spection shows that Wolff consistently modernizes (rewrites Baudissin in 1920s language, including some slang) and frequently intensiﬁes meanings for dramatic effect, includ- ing making the play’s homo-erotic subtext more prominent. This reﬂects a relaxing of sexual inhibitions in the Weimar Republic. Next, we can interactively de-select Wolff’s 1926 trans- lation from the sub-corpus under investigation, leaving four actual editions of the Baudissin translation. See Section 7 for details. Now, the updated Eddy coloring shows variation among them –as shown in Figure 12(b), particularly in Brunner (1947) [15] but also others, in the majority of segments. Some of this variation is due to historical spelling changes (lemmatiza- tion aims to ﬁlter these out, but can still have trouble with some antiquated and poetic, unusual forms of spelling). Some changes are more signiﬁcant. They are caused by editors aiming to ‘improve’ the Baudissin text they have received, making it easier for readers, actors, and audiences. We can explore variation between these translations at the term level using the TLC view, for example Shakespeare’s phrase “the aim reports” was translated by Baudissin as: “Muthmaßung berichtet” (conjecture reports) (1851, in accord with the 1832 text). This is a very compressed expression (meaning: ‘people making conjectures report’), not easy to follow and also difﬁcult to speak. Some later editors changed this: “sch¨atzungsweise man berichtet” (estimating, people report) (1923), “Vermutung meldet” (supposition re- ports) (1947), and “Ger¨uchte melden” (rumours report) (2000). The TLC view (Figure 13) illustrates the translation varia- tion between these four editions. The changes in 1923 and 2000 make the text more like conversational German, less ‘literary’. The change in 1947 substitutes a much commoner word. Such small changes are referred to as ‘silent emenda- tions’ in the history of edition-making. Identifying them is a hideously tedious task for traditional scholarship. Fig. 14. A sub-set of the translation selected based on the domain expert knowledge shows three periods of generally high Eddy values. The three periods are highlighted by borders. Ihr, Vater schätzte gut Fig. 15. Three uses of the TLC view to help the domain expert ﬁnd variation between different translation. The top shows a common use of the words ‘Ihr’ and ‘Vater’. The middle and bottom views show that the words: ‘sch¨atzte’ and ‘gut’ which are used only in speciﬁc translations. Fig. 15. Three uses of the TLC view to help the domain expert ﬁnd variation between different translation. The top shows a common use of the words ‘Ihr’ and ‘Vater’. The middle and bottom views show that the words: ‘sch¨atzte’ and ‘gut’ which are used only in speciﬁc translations. See Section 7 for details. Schwarz was a pioneer in using the variety of available German terms in order to dramatize race attitudes within in the play. Her use of ‘Maure’ is clearly intended to dignify Othello as a black man, which is perhaps surprising given when and where she was writing. Interactively reading up, down and diagonally in the corpus enables us to see this. Maure Schw. 1941 Fig. 16. Two TLC views that show words used distinctively in the translations. In top the word “Maure” was ﬁrstly used by Schwarz (1941). In bottom, we can see the distinctive words in the translations of Shake- speare’s odd phrase: “Of love, of worldly matters and direction”. A further reduced sub-set, using the thumbnail view, can be investigated to compare Schwarz with Baudissin (a translation she knew), Gundolf (1909) [32], one which she may have known, and Zeynek (1947), who also trans- lated during the Nazi dictatorship. Her version is shown to be generally lowest on distinctiveness. She is generally conservative. But Eddy is high for her segment including Shakespeare’s odd phrase ‘Of love, of worldly matters and direction’. We can justify the distinctiveness pattern using the TLC view as shown in Figure 16(bottom). Baudissin had: ‘Der Lieb und unsrem h¨auslichen Gesch¨afte’ (love and our household business); Gundolf: ‘Zuliebe weltlichem Gesch¨aft’ (for the sake of worldly business) (a typical poetically condensed translation by him); Zeynek: ‘zu Liebe, irdschem Tun und Treiben’ (for love, earthly activities); Schwarz: ‘f¨ur die Liebe, f¨ur die praktischen Gesch¨afte’ (for love, for practical business). The example illustrates her rather practical, easily comprehensible, modern language style. Zeynek, on the other hand, uses antiquated and ornate language, and so scores generally very high Eddy values. In the interface, de-selecting the earlier 19th-century translations heightens the visibility of the distinctiveness in the 1950s–1960s and 1990s in terms of Eddy coloring. Inspecting segments with high Eddy in the 1960s and 90s reveals interesting patterns in textual detail. 7 STUDY OUTCOME AND DESIGN GUIDELINES J¨anicke et al. [48] provide a valuable discussion on collabora- tion experience with humanities scholars when developing visualization solutions. They report some successful designs and approaches that outline the collaboration process [1]. J¨anicke et al. present insights and practices that are carried out during the initial project start, the iterative development and the involvement of the digital humanities in it, and the evaluation of the design with the humanities scholars. Most of the translators are unknown –their work has never been studied. One is of particular interest because she is the only woman who has translated a number of Shakespeare’s plays into German: Hedwig Schwarz. Her version of Othello is from 1941 [76]. By selecting a subset including other full-length performance versions from her lifetime (1898-1985), we can highlight segments where her version is distinctive. There are not many. In several short segments, she is unusually concise and informal. A feature which stands out is her unusual use of the term ‘Maure’ for ‘Moor’. Othello is a ‘Moor’ –a controversial term, meaning African, variously understood as Black African or North African/Arab/Berber (as in the ‘Moors’ of Moslem Spain). German has two traditional words - ‘Mohr’ meaning Black African and ‘Maure’ meaning North African, with contrary connotations of ‘barbarian’ versus ‘civilized’. All transla- tions before Schwarz used ‘Mohr’. She was the ﬁrst transla- tor to use ‘Maure’ for ‘Moor’, in the play’s subtitle and in the text. She uses ‘Maure’ when the speaker is respectful, e.g. Othello’s wife, Desdemona. Racist characters (Brabantio, Iago, Rodrigo) use ‘Mohr’ but sometimes ‘Maure’. Rothe g This tool has not been fully integrated with the do- main expert’s previously used framework due to limited resources. This challenge is reported by Sedlmair et al. [77] as pitfall (PF-5: Insufﬁcient time available from potential col- laborators). As the domain expert is familiar with visualiza- tion projects related to the domain, the project encountered another pitfall (PF-17: Experts focusing on visualization design vs. domain problem) where the domain expert some- times focuses on the visualization design problems rather than the domain problem. The project also encountered a pitfall (PF-30: Too much domain background in paper) in the earlier stage where we presented unbalanced background towards the domain side. See Section 7 for details. periods of generally high Eddy value: the early 19th cen- tury (Schiller (1805) [73], Benda (1826) [9], Ortslepp (1839) [63]), the 1950s-60s (Schr¨oder (1962) [75], Rothe (1963) [69]) and the 1990s (G¨unther (1992) [33], Motschach (1992) [60], Buhss (1996) [17]). The translations created before Baudissin (1832) were experimenting with varied ways of translating Shakespeare’s plays. Baudissin’s version of Othello (for the ‘Schlegel-Tieck’ edition of Shakespeare’s plays) soon became canonical - the standard, the one which ‘everybody knows’, even today. Until the 1950s, there were many other German translations of Shakespeare but all were heavily inﬂuenced by the ‘Schlegel-Tieck’ style, and Eddy values are low. After the Second World War, new ways of translating Shakespeare began to be tried, as part of a general breaking away from tradition. But this was a complicated process. Innovation and tradition co-existed, tradition becoming stronger again in the 1970s-80s. Then in the 1990s, experimentalism took over. The individual distinctiveness of translators: In the same way, the interface makes it possible to explore com- paratively sets of independent translations. Our corpus includes a majority of full-length, poetic translations for theatrical performance –26 of these (including the variant Baudissin texts); also four ‘prose’ translations (for read- ing, not for performance) and eight theatrical adaptations (shortened, and much freer in the ways they translate). From the interactive thumbnail view, we select the 26, and view them at a distance in chronological order. As seen in Figure 14, the Eddy value colorings clearly show three JOURNAL OF LATEX CLASS FILES, VOL. 14, NO. 8, AUGUST 2015 15 Maure zuliebe, weltlichem praktischen irdschem, treiben häuslichen Gund. 1909 Schw. 1941 Baud. 1947 Zeyn. 1948 Fig. 16. Two TLC views that show words used distinctively in the translations. In top the word “Maure” was ﬁrstly used by Schwarz (1941). In bottom, we can see the distinctive words in the translations of Shake- speare’s odd phrase: “Of love, of worldly matters and direction”. (1963) used the same double-word tactic. No other transla- tors have used ‘Maure’ since. Figure 16(top) shows the TLC view with the word ‘Maure’ highlighted. Some more recent translators use provocatively offensive terms, or neutral terms intended to minimize the racism theme: different responses to ongoing developments in the politicisation of race language and awareness of its emotional force: one of the themes of Othello. See Section 7 for details. After zoom- ing in for a closer view and with the assistance of TLC view as shown in Figure 15, we can see: Othello’s line beginning ‘Her father loved me’ is translated by all as ‘Ihr Vater liebte mich’ (‘Her father loved me’) except: ‘sch¨atzte mich’ (thought highly of me) ( Zeynek (1948) [91], Schaller (1959) [72], Motschach (1992)), ‘war mir gut’ (was fond of me) (Rothe (1963)), ‘mochte mich’ (liked me) (G¨unther 1992, Buhss (1996)), ‘schien mich zu m¨ogen’ (seemed to like me) (Leonard (2010) [55]). The periods of more dif- ferent, distinctive translation are evident here. Love and hate, liking and disliking, and their ambiguities, between men and women and between men, are major themes in Othello. Desdemona’s father, Brabantio, hates Othello, his son-in-law. When translators change the kind and intensity of emotions in this way, it matters a great deal. The interface makes it relatively easy to discover patterns in translators’ treatment of emotions and other themes. Exactly why partic- ular translators make particular choices at particular times requires a lot more discussion. 7 STUDY OUTCOME AND DESIGN GUIDELINES Such a project opens many future possibilities in terms of the scale of the corpus, the use of more sophisticated linguistics or evaluation methods and more languages. We encourage more in-depth research to address the scalability challenge when dealing with a large parallel text dataset. Future possibilities include the application of translation training. • Support close and distant reading for a large number of texts. We encourage further research that supports distant and close reading for an even larger number of texts. • Establish good communication with the domain expert(s) in order to guide the project and to provide useful feed- back. We also offer some caution that working on text analysis for multiple languages is very challenging because different languages have different analysis tools associated with them requiring a variety of specialized language-speciﬁc knowl- edge. The alignment process is preprocessed and manually generated and is a time-consuming job. Most of the align- ment applications use basic and inaccurate algorithms to align two or more segments. Thus, we would like to inves- tigate ways to accelerate the alignment process. 7 STUDY OUTCOME AND DESIGN GUIDELINES The (+) and (-) indicate if we encountered the pitfall during the development of our tool or not. A table of the encountered pitfalls identiﬁed by Sedlmair et al. [77] and their relevance to this project. The (+) and (-) indicate if we encountered the pitfall during the development of our tool or not. is the options panel which enables the user to customize the alignment overview. The third is the detailed view which interacts with the main window and displays a close reading of the alignments of the selected segment. Also, it saves the history of user-selected segments and reveals previous alignments. Finally, we present the TLC view, which is a novel and interactive technique to examine the word level variation within the translations. Our proposed application is driven and evaluated by experts from the Arts and Hu- manities. We provide the domain feedback on the applica- tion features. Please see the supplementary video for a soft- ware demonstration: https://youtu.be/FnA1YbWdiNQ. In order to communicate some of the lessons learned throughout the project lifespan, we derive some general visualization guidelines to facilitate transferring the expe- rience to the general visualization audience: • Implement support for customization of the texts. Giving the user control of adding or removing translation, and to sort based on a variety of options is always appreciated. y p y pp • Provide a mechanism to save the user actions. We think this supports the user better while exploring the applica- tion. Future work includes generalizability. Our design is applied to a speciﬁc corpus and generalizing the platform to adapt different corpus and different languages is prob- lematic because each corpus has different encoding schemes and annotations. Another direction of future work is to incorporate semantic clustering and structural similarity between translations. The rendering could be updated to reﬂect this when zooming in or out of the scene. • Users appreciate smooth zooming due to the context-and- detail provided in the same window. • Close reading is always preferable to be available [34], [48]. Also providing users with complete access to the text is beneﬁcial to enable them to analyse and explore text beyond the visual tool. y • Implement a keyword or sentence search functionality. We found this feature useful during the feedback session. 9 ACKNOWLEDGEMENTS In this paper, we present a unique, integrated visual de- sign to support distant and close reading of the collec- tion of parallel translations of Othello. The visual design aims to present a smooth interactive experience for digital humanities scholars. We identify ﬁve main tasks that our proposed application addresses. The application consists of four components. The ﬁrst one is the context view (alignment overview) of the collection which leverages a range of exploration and interaction techniques. It facilitates smooth zooming which can integrate distant and close reading within the same window. The second component The authors would like to thank Liam McNabb, Dylan Rees and Matthew Roach for their help in proofreading the manuscript. The authors also thank the Technical and Voca- tional Training Corporation (TVTC) and the Saudi Cultural Bureau for funding and supporting this research endeavour. [1] A. Abdul-Rahman, J. Lein, K. Coles, E. Maguire, M. Meyer, M. Wynne, C. R. Johnson, A. Trefethen, and M. Chen. Rule-based visual mappings–with a case study on poetry visualization. In 7 STUDY OUTCOME AND DESIGN GUIDELINES Although we think the domain ex- pert feedback and the case studies are balanced, we observe more emphasis on positive feedback which could indicate to another pitfall (PF-26: Liking necessary but not sufﬁcient for JOURNAL OF LATEX CLASS FILES, VOL. 14, NO. 8, AUGUST 2015 16 Pitfall # Pitfall How? PF-5 Insufﬁcient time available from potential collaborators No time/support for full deployment PF-17 Experts focusing on visualization design vs. domain problem Domain expert focuses in design issues PF-25 Usage scenario not case study: non-real task/data/user Some reported descoveries made by de- velopers PF-26 Liking necessary but not sufﬁcient for validation Domain experts were linked closely dur- ing design, however, some criticisms were still reported PF-30 Too much domain background in paper At earlier stage, the background was dominant PF-32 Premature end: win race vs. practice mu- sic for debut Attempts to publish early submissions too soon before serious and thoughtful reﬁnement PF-33 Feature creep The domain expert requests features be- yond the deﬁned requirements PF-34 Domain expert high/low expectation The domain expert might lack the knowl- edge of the development cost TABLE 2 A table of the encountered pitfalls identiﬁed by Sedlmair et al. [77] and their relevance to this project. The (+) and (-) indicate if we encountered the pitfall during the development of our tool or not. validation). Table 2 compares and reports our experiences and reﬂections on the pitfalls reported by Sedlmair et al. Another possible pitfall which is related to the presentation of the work is that attempting to publish the work too soon before serious and thoughtful reﬁnement (PF-32: Premature end: win race vs. practice music for debut). p We also encountered other challenges which are not always a pitfall and might relate to some of the pitfalls reported in Table 2. For example, feature creep [24] (PF-33) is a challenge we encountered, where the domain expert re- quests features beyond the previously deﬁned requirements and are difﬁcult to incorporate in the current design because it might need another design approach. 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https://openalex.org/W4307371149	https://authors.library.caltech.edu/114793/1/2205.01112.pdf	English	null	Kepler and the Behemoth: Three Mini-Neptunes in a 40 Million Year Old Association	The Astronomical journal	2,022	cc-by	17,617	ABSTRACT arXiv:2205.01112v1 [astro-ph.EP] 2 Stellar positions and velocities from Gaia are yielding a new view of open cluster dispersal. Here we present an analysis of a group of stars spanning Cepheus (l = 100◦) to Hercules (l = 40◦), hereafter the Cep-Her complex. The group includes four Kepler Objects of Interest: Kepler-1643 b (Rp = 2.32 ± 0.14R⊕, P = 5.3 days), KOI- 7368 b (Rp = 2.22 ± 0.12R⊕, P = 6.8 days), KOI-7913 Ab (Rp = 2.34 ± 0.18R⊕, P = 24.2 days), and Kepler- 1627 Ab (Rp = 3.85±0.11R⊕, P = 7.2 days). The latter Neptune-sized planet is in part of the Cep-Her complex called the δ Lyr cluster (Bouma et al. 2022). Here we focus on the former three systems, which are in other regions of the association. Based on kinematic evidence from Gaia, stellar rotation periods from TESS, and spectroscopy, these three objects are also ≈40 million years (Myr) old. More speciﬁcally, we ﬁnd that Kepler- 1643 is 46+9 −7 Myr old, based on its membership in a dense sub-cluster of the complex called RSG-5. KOI-7368 and KOI-7913 are 36+10 −8 Myr old, and are in a diffuse region that we call CH-2. Based on the transit shapes and high resolution imaging, all three objects are most likely planets, with false positive probabilities of 6 × 10−9, 4×10−3, and 1×10−4 for Kepler-1643, KOI-7368, and KOI-7913 respectively. These planets demonstrate that mini-Neptunes with sizes of ≈2 Earth radii exist at ages of 40 million years. arXiv:2205.01112v1 [astro-p Keywords: exoplanet evolution (491), open star clusters (1160), stellar ages (1581) Kepler and the Behemoth: Three Mini-Neptunes in a 40 Million Year Old Association Kepler and the Behemoth: Three Mini-Neptunes in a 40 Million Year Old Association L. G. BOUMA,1, ∗R. KERR,2 J. L. CURTIS,3 H. ISAACSON,4 L. A. HILLENBRAND,1 A. W. HOWARD,1 A. L. KRAUS,2 A. BIERYLA,5 D. W. LATHAM,5 E. A. PETIGURA,6 AND D. HUBER7 L. G. BOUMA,1, ∗R. KERR,2 J. L. CURTIS,3 H. ISAACSON,4 L. A. HILLENBRAND,1 A. W. HOWARD,1 A. L. KRAUS,2 A. BIERYLA,5 D. W. LATHAM,5 E. A. PETIGURA,6 AND D. HUBER7 L. G. BOUMA,1, ∗R. KERR,2 J. L. CURTIS,3 H. ISAACSON,4 L. A. HILLENBRAND,1 A. W. HOWARD,1 A. L. KRAUS,2 A. BIERYLA,5 D. W. LATHAM,5 E. A. PETIGURA,6 AND D. HUBER7 1Cahill Center for Astrophysics, California Institute of Technology, Pasadena, CA 91125, USA 2Department of Astronomy, The University of Texas at Austin, Austin, TX 78712, USA 3Department of Astronomy, Columbia University, 550 West 120th Street, New York, NY 10027, USA 4Astronomy Department, University of California, Berkeley, CA 94720, USA 5Center for Astrophysics \| Harvard & Smithsonian, 60 Garden St, Cambridge, MA 02138, USA 6Department of Physics & Astronomy, University of California Los Angeles, Los Angeles, CA 90095, USA 7Institute for Astronomy, University of Hawai‘i, 2680 Woodlawn Drive, Honolulu, HI 96822, USA 05.01112v1 [astro-ph.EP] 2 May 2022 arXiv:2205.01112v1 [astro-ph.EP] 2 May 2022 3Department of Astronomy, Columbia University, 550 West 120th Street, New York, NY 10027, USA 4Astronomy Department, University of California, Berkeley, CA 94720, USA Center for Astrophysics \| Harvard & Smithsonian, 60 Garden St, Cambridge, MA 02138, USA 6Department of Physics & Astronomy, University of California Los Angeles, Los Angeles, CA 90095, USA 7Institute for Astronomy, University of Hawai‘i, 2680 Woodlawn Drive, Honolulu, HI 96822, USA Corresponding author: L. G. Bouma luke@astro.caltech.edu ∗51 Pegasi b Fellow ∗51 Pegasi b Fellow DRAFT VERSION MAY 4, 2022 Typeset using LATEX twocolumn style in AASTeX63 DRAFT VERSION MAY 4, 2022 Typeset using LATEX twocolumn style in AASTeX63 DRAFT VERSION MAY 4, 2022 Typeset using LATEX twocolumn style in AASTeX63 2.1. Previous Related Work Our focus is on a region of the Galaxy 200 to 500 pc from the Sun, above the galactic plane, and spanning galactic lon- gitudes of 40◦to 100◦. Two rich clusters in this region are the δ Lyr cluster (Stephenson 1959) and RSG-5 (Röser et al. 2016). Each of these clusters was known before Gaia. Their reported ages are between 30 and 60 Myr. Early empirical evidence that these two clusters could be part of a large and more diffuse population was apparent in the Gaia-based pho- tometric analysis of pre-main-sequence stars by Zari et al. (2018, see their Figures 11 and 13). Further kinematic con- nections and complexity were highlighted by Kounkel & Covey (2019), who included these previously known groups in the larger structures dubbed “Theia 73” and “Theia 96”1. The connection made by Kounkel & Covey (2019) between the previously known open clusters and the other groups in the region was made as part of an unsupervised clustering analysis of the Gaia DR2 positions and on-sky velocities with a subsequent manual “stitching” step. Their results support the idea that there is an overdensity of 30 to 60 Myr old stars in this region of the Galaxy. Kerr et al. (2021), in a volume- limited analysis of the Gaia DR2 point-source catalog out to one third of a kiloparsec, identiﬁed three of the nearest sub- populations, dubbed “Cepheus-Cygnus”, “Lyra”, and “Cer- berus”. Kerr et al. (2021) reported ages for each of these subgroups between 30 and 35 Myr. The prime Kepler mission (Borucki et al. 2010) found most of the currently known transiting exoplanets, and it was con- ducted before Gaia. It is therefore sensible to revisit the Ke- pler ﬁeld, given our new constraints on the stellar ages. Here, we expand on our earlier study of a 38+7 −6 Myr old Neptune-sized planet in the Kepler ﬁeld (Kepler-1627 Ab; Bouma et al. 2022). This planet’s age was derived based on its host star’s membership in the δ Lyr cluster. While our analysis of the cluster focused on the immediate vicin- ity of Kepler-1627 in order to have a reasonable scope, it became clear that the δ Lyr cluster seems to also be part of a much larger group of similarly aged stars. 1. INTRODUCTION et al. 2012; Owen & Wu 2013; Fulton et al. 2017; Ginzburg et al. 2018; Lee & Connors 2021). The discovery and characterization of planets younger than a billion years is a major frontier in current exoplanet re- search. The reason is that the properties of young planets pro- vide benchmarks for studies of planetary evolution. For in- stance, young planets can inform our understanding of when hot Jupiters arrive on their close-in orbits (Dawson & John- son 2018), how the sizes of planets with massive gaseous en- velopes evolve (Rizzuto et al. 2020), the timescales for close- in multiplanet systems to fall out of resonance (Izidoro et al. 2017; Arevalo et al. 2022; Goldberg & Batygin 2022), and whether and how mass-loss explains the radius valley (Lopez The discovery of a young planet requires two claims to be true: the planet must exist, and its age must be secured. Spaced-based photometry from K2 and TESS has yielded a number of exemplars for which the planetary evidence comes from transits, and the age is based on either cluster member- ship (Mann et al. 2017; David et al. 2019; Newton et al. 2019; Bouma et al. 2020; Nardiello et al. 2020) or else on correlates of youth such as stellar rotation, photospheric lithium con- tent, x-ray activity, and emission line strength (Zhou et al. 2021; Hedges et al. 2021). In this work, we leverage recent analyses of the Gaia data, which have greatly expanded our knowledge of stellar groups (e.g., Cantat-Gaudin et al. 2018; Kounkel & Covey 2019; Kerr et al. 2021). To date these analyses have mostly clus- tered on stellar positions and 2D velocities. One important 2 result has been the discovery of diffuse streams and tidal tails comparable in stellar mass to the previously known cores of nearby open clusters (Meingast et al. 2019; Meingast et al. 2021; Gagné et al. 2021). Even though these streams are spread over tens to hundreds of parsecs, their velocity dis- persions can remain coherent at the ∼1 km s−1 level. Internal dynamics and projection effects can also drive them to be much larger: in the Hyades, stars in the tidal tails are ex- pected to span up to ±40kms−1 in velocity relative to the cluster center (Jerabkova et al. 2021). 2.1. Previous Related Work This associa- tion, which is at an average distance of 330 pc from the Sun, spans Cepheus to Hercules (galactic longitudes, l, between 40◦and 100◦), at galactic latitudes between 0◦and 20◦. An assessment of its membership, substructure, and age distri- bution will be provided as part of the 1 kpc expansion of the SPYGLASS project (R. Kerr et al. in prep), where it is given the name Cep-Her, after the endpoint constellations. 1 See their visualization online at http://mkounkel.com/mw3d/mw2d.html (accessed 15 March 2022) 1. INTRODUCTION The stars in such dif- fuse regions can be veriﬁed to be the same age as the core cluster members through analyses of color–absolute magni- tude diagrams (Kounkel & Covey 2019), stellar rotation pe- riods (Curtis et al. 2019; Bouma et al. 2021), and chemical abundances (Hawkins et al. 2020). While there are implica- tions for our understanding of star formation and cluster evo- lution (Dinnbier & Kroupa 2020), a separate consequence is that we now know the ages of many more stars, including previously known planet hosts. 2. THE CEP-HER COMPLEX 2.1. Previous Related Work 2.2. Member Selection This step yielded 1,097 TRIO OF MINI-NEPTUNES IN CEP-HER 3 150 200 250 300 350 400 Y [pc] 0 100 Z [pc] 40 50 60 70 80 90 100 l [deg] 0 10 20 b [deg] -100 -50 0 50 100 150 200 250 X [pc] 150 200 250 300 350 400 Y [pc] Kepler-1627 A KOI-7368 KOI-7913 A KOI-7913 B Kepler-1643 40 50 60 70 80 90 100 l [deg] 15 10 5 0 5 10 15 vl * [km s 1] -100 -50 0 50 100 150 200 250 X [pc] 0 100 Z [pc] 15 10 5 0 5 10 15 vl * [km s 1] 10 8 6 4 2 0 vb [km s 1] Figure 1. Positions and velocities of candidate members of the Cep-Her complex. Top row: On-sky positions in galactic coordinates. Black points are stars for which group membership is more secure than for gray points. Kepler-1627 is in the outskirts of the δ Lyr cluster (Bouma et al. 2022), which is centered at (l,b) ≈(66◦,12◦). The Kepler footprint is shown in gray. Middle row: Galactic positions. The Sun is at (X,Y,Z) = (0,0,20.8) pc; lines of constant heliocentric distance are shown between 250 and 400 pc, spaced by 50 pc. Bottom row: Galactic tangential velocities (left) and galactic longitudinal velocity versus galactic longitude (right). The gray band in the lower-right shows the ±1-σ projection of the Solar velocity with respect to the local standard of rest (Schönrich et al. 2010). There is a strong spatial and kinematic overlap between Kepler-1643 and RSG-5 (magenta). The local population of candidate young stars around KOI-7368 and KOI-7913 is more diffuse – we call this region “CH-2” (lime-green). The selection method for all of the stars is described in Section 2.2. 2.2. Member Selection The possibility that the δ Lyr cluster, RSG-5, and the sub- populations identiﬁed by Kerr et al. (2021) share a common origin has yet to be fully substantiated, but preliminary clus- tering results from the 1 kpc SPYGLASS analysis (R. Kerr et al. in prep) suggest the presence of contiguous stellar pop- ulations connecting each of these groups in space-velocity coordinates. In other words, the stars appear to be comoving, though with a continuous gradient in velocity as a function of position (Figure 1, lower panels). In this work, our primary interest in the region stems from the fact that a portion of it was observed by Kepler (Figure 1, top panel). To further ex- plore this sub-population, we select candidate Cep-Her mem- bers through four steps, the ﬁrst three being identical to those described in Section 3 of Kerr et al. (2021). We brieﬂy sum- marize them here. Our focus is on the intersection of the Cep-Her complex with the Kepler ﬁeld. Cross-matching the stars thought to be in Cep-Her against known Kepler Objects of Interest (KOIs; Thompson et al. 2018) yielded four candidate cluster mem- bers: Kepler-1627, Kepler-1643, KOI-7368, and KOI-7913. Given our earlier analysis of Kepler-1627, we focus here on the latter three objects. After analyzing the relevant prop- erties of Cep-Her (Section 2), we derive the stellar proper- ties (Section 3) and validate the planetary nature of each sys- tem using a combination of the Kepler photometry and high- resolution imaging (Section 4). We conclude with a discus- sion of mini-Neptune size evolution, and point out possible directions for future work (Section 5). The ﬁrst step is to select stars that are photometrically distinct from the ﬁeld star population based on Gaia EDR3 magnitudes {G,GRP,GBP}, parallaxes and auxiliary redden- ing estimates (Lallement et al. 2019). 2.2. Member Selection These stars are either pre-main-sequence K and M dwarfs due to their long contraction timescales, or massive stars near the zero-age main sequence due to their rapid evolutionary timescales. The second step is to perform an unsupervised HDB- SCAN clustering on the photometrically selected population (Campello et al. 2015; McInnes et al. 2017). The parame- ters we use in the clustering are {X,Y,Z,cvb,cvl∗}, where c is the size-velocity corrective factor, which is taken as c = 6pc/kms−1 to ensure that the spatial and velocity scales have identical standard deviations. Positions are computed assum- ing the astropy v4.0 coordinate standard (Astropy Col- laboration et al. 2018). As input parameters to HDBSCAN, we set the minimum ϵ threshold past which clusters cannot be fragmented as 25 pc in physical space, and 25/c km s−1 in velocity. The minimum cluster size N is set to 10, as is k, the parameter used to deﬁne the “core distance” density metric. Figure 1 shows the positions of these KOIs along various projections. Kepler-1643 is near the core RSG-5 population both spatially and kinematically. KOI-7368 and KOI-7913 are in a diffuse region ≈40 pc above RSG-5 in Z and ≈100 pc closer to the Sun in Y. In tangential galactic velocity space, there is some kinematic overlap between the region contain- ing the latter two KOIs and the main RSG-5 group. We deﬁne two sets of stars in the local vicinity of our ob- jects of interest. For candidate RSG-5 members near Kepler- 1643, we require: The unsupervised clustering in this case yielded 8 distinct subgroups. These groups are then used as the “seed” popula- tions for the third step, which is to search for objects at least as close to the 10th nearest HDBSCAN-identiﬁed member in space-velocity coordinates. This third step yields stars that are spatially and kinematically close to the photometrically young stars, but which cannot be identiﬁed as young based on their positions in the color–absolute magnitude diagram. X/pc ∈[45,75] Y/pc ∈[320,350] Z/pc ∈[40,80] vb/kms−1 ∈[−4,−2.5] vl∗/kms−1 ∈[3.5,6], X/pc ∈[45,75] Y/pc ∈[320,350] Z/pc ∈[40,80] vb/kms−1 ∈[−4,−2.5] vl∗/kms−1 ∈[3.5,6], p g g The outcome of the analysis up to the point of the third step is shown in Figure 1. 2.2. Member Selection TRIO OF MINI-NEPTUNES IN CEP-HER 3 3 40 50 60 70 80 90 100 l [deg] 0 10 20 b [deg] -100 -50 0 50 100 150 200 250 X [pc] 150 200 250 300 350 400 Y [pc] Kepler-1627 A KOI-7368 KOI-7913 A KOI-7913 B Kepler-1643 -100 -50 0 50 100 150 200 250 X [pc] 0 100 Z [pc] 150 200 250 300 350 400 Y [pc] 0 100 Z [pc] 15 10 5 0 5 10 15 vl * [km s 1] 10 8 6 4 2 0 vb [km s 1] 40 50 60 70 80 90 100 l [deg] 15 10 5 0 5 10 15 vl * [km s 1] Figure 1. Positions and velocities of candidate members of the Cep-Her complex. Top row: On-sky positions in galactic coordinates. Black points are stars for which group membership is more secure than for gray points. Kepler-1627 is in the outskirts of the δ Lyr cluster (Bouma et al. 2022), which is centered at (l,b) ≈(66◦,12◦). The Kepler footprint is shown in gray. Middle row: Galactic positions. The Sun is at (X,Y,Z) = (0,0,20.8) pc; lines of constant heliocentric distance are shown between 250 and 400 pc, spaced by 50 pc. Bottom row: Galactic tangential velocities (left) and galactic longitudinal velocity versus galactic longitude (right). The gray band in the lower-right shows the ±1-σ projection of the Solar velocity with respect to the local standard of rest (Schönrich et al. 2010). There is a strong spatial and kinematic overlap between Kepler-1643 and RSG-5 (magenta). The local population of candidate young stars around KOI-7368 and KOI-7913 is more diffuse – we call this region “CH-2” (lime-green). The selection method for all of the stars is described in Section 2.2. 4 this yielded 11 known false positives, 6 conﬁrmed planets, and 8 candidate planets (see Appendix A). To determine whether these objects were potentially consistent with being i) planets, and ii) ≲108 years old, we inspected the Kepler data validation reports and Robovetter classiﬁcations. Youth was assessed based on the presence of rotational modula- tion at the expected period and amplitude (e.g., Rebull et al. 2020). Planetary status was assessed through the Robovetter ﬂags, and by requiring non-grazing transits with S/N > 10. Four objects passed both cuts: Kepler-1627, Kepler-1643, KOI-7368, and KOI-7913. stars with high-quality photometry and astrometry. 2.2. Member Selection To enable a selection cut that ﬁlters out ﬁeld-star contaminants, we also compute a weight metric, D, deﬁned such that the group member with the smallest core distance has D = 1, the group member with the greatest core distance has D = 0, and weights for the other group members are log-normally distributed between these two extremes. After applying a set of quality cuts on the astrometry and photometry2 this procedure yields D ∼log10 N(−1.55,0.61). To visualize the results, in Figure 1 we show 12,436 ob- jects with D > 0.02 as gray points, and 4,763 objects with D > 0.10 as black points. The δ Lyr cluster is visible at (l,b) = (68◦,15◦) and (vl′,vb) = (−4.5,−4)kms−1. RSG-5 is visible at (l,b) = (83◦,6◦), (vl′,vb) = (5.5,−3.5)kms−1. Most of the other subclusters, including in Cep-Cyg (l,b = 90◦,7◦) and Cerberus (l,b = 48◦,18◦) are too small or dispersed to have previously been analyzed in great detail. though RSG-5 does have a greater spatial extent toward smaller X (Figure 1, middle panels). For the diffuse stars near KOI-7368 and KOI-7913, we require X/pc ∈[20,70] Y/pc ∈[230,270] Z/pc ∈[75,105] vb/kms−1 ∈[−3.5,−1.5] vl∗/kms−1 ∈[2,6] and we call this latter set of stars “CH-2”, using the prelim- inary Cep-Her (CH) subgroup identiﬁer from R. Kerr et al. (in prep). These cuts yielded 173 candidate RSG-5 mem- bers, and 37 candidate CH-2 members. These stars are listed in Appendix A, as are the Cep-Her candidates that were ob- served by Kepler. Our fourth and ﬁnal step was to cross-match the candidate Cep-Her member list against all known Kepler Objects of In- terest. We used the Cumulative KOI table from the NASA Exoplanet Archive from 27 March 2022, and also compared against the q1_q17_dr25 table (Thompson et al. 2018). From the candidate members with weights exceeding 0.02, 2 ϖ/σϖ > 5; G/σG > 50; GRP/σGRP > 20; GBP/σGBP > 20 2.3.1. Color–Absolute Magnitude Diagram Color–absolute magnitude diagrams (CAMDs) of the can- didate RSG-5 and CH-2 members are shown in the upper row of Figure 2. The stars from the δ Lyr cluster are from Bouma et al. (2022), and the ﬁeld stars are from the Gaia 2 ϖ/σϖ > 5; G/σG > 50; GRP/σGRP > 20; GBP/σGBP > 20 TRIO OF MINI-NEPTUNES IN CEP-HER 5 5 1.0 1.5 2.0 2.5 3.0 (GBP GRP)0 [mag] 5 6 7 8 9 10 11 Absolute MG, 0 [mag] Kepler-1643 Lyr candidates RSG-5 candidates Field K2 K5 M0 M2 M3 M4 Spectral Type 1.0 1.5 2.0 2.5 3.0 (GBP GRP)0 [mag] 5 6 7 8 9 10 11 Absolute MG, 0 [mag] KOI-7368 KOI-7913 A KOI-7913 B Lyr candidates CH-2 candidates Field K2 K5 M0 M2 M3 M4 Spectral Type 0.5 1.0 1.5 2.0 2.5 (GBP GRP)0 [mag] 0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 Rotation Period [days] Kepler-1643 Pleiades Praesepe RSG-5 candidates F0 F5 G0 K0 K3 K5 K7 M0 M1 M2 M3 Spectral Type 0.5 1.0 1.5 2.0 2.5 (GBP GRP)0 [mag] 0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 Rotation Period [days] KOI-7368 KOI-7913 A KOI-7913 B Pleiades Praesepe CH-2 candidates F0 F5 G0 K0 K3 K5 K7 M0 M1 M2 M3 Spectral Type Figure 2. Age-diagnostic diagrams from the stellar groups near Kepler-1643, KOI-7368, and KOI-7913. Top row: Color–absolute magnitude diagram of candidate Cep-Her members, plotted over candidate members of the δ Lyr cluster (≈38 Myr; Bouma et al. 2022) and the Gaia EDR3 Catalog of Nearby Stars (gray background). The left and right columns shows stars in RSG-5 and CH-2, respectively. The range of colors is truncated to emphasize the pre-main-sequence. Stars that fall far below the cluster sequences are ﬁeld interlopers. Bottom row: TESS and ZTF-derived stellar rotation periods, with the Pleiades (≈112 Myr) and Praesepe (≈650 Myr) shown for reference (Rebull et al. 2016; Douglas et al. 2017). The detection efﬁciency for reliable rotation periods falls off beyond (GBP −GRP)0 ≳2.6. 2.3.2. Stellar Rotation Periods For ZTF, we used the same color cut to focus on FGKM stars, but restricted the sample to 13 < G < 18 to avoid the saturation limit on the bright end and ensure sufﬁcient photometric precision at the faint end. We followed the procedure outlined in Curtis et al. (2020): we downloaded 8′ × 8′ image cutouts, ran aperture photom- etry for the target and neighboring stars identiﬁed with Gaia, and used them to deﬁne a systematics correction to reﬁne the target light curves. The CAMDs show that for RSG-5, all but one of the can- didate members are on a tight pre-main-sequence locus. This implies a false positive rate of a few percent, at most. By comparison, our control sample (the δ Lyr candidates) has a false positive rate of ≈12%, based on the number of stars that photometrically overlap with the ﬁeld population, while being outliers from the bulk cluster population. For CH-2, our membership selection gives 27 objects in the color range displayed, and 23 of them appear to be consistent with being on the pre-main-sequence. This implies a false positive rate in CH-2 of ≈15%. Figure 2 also shows that most RSG-5 and CH-2 mem- bers overlap with the δ Lyr cluster, and that the groups are therefore roughly the same age. To quantify this, we use the method introduced by Gagné et al. (2020, their Sec- tion 6.3). The idea is to ﬁt the pre-main-sequence loci of a set of reference clusters, and to then model the locus of the target cluster as a linear combination of these reference clus- ter loci. For our reference clusters, we used UCL, IC 2602, and the Pleiades, with the memberships reported by Dami- ani et al. (2019) and Cantat-Gaudin et al. (2018) respec- tively. We adopted ages of 16 Myr for UCL (Pecaut & Mama- jek 2016), 38 Myr for IC 2602 (David & Hillenbrand 2015; Randich et al. 2018) and 112 Myr for the Pleiades (Dahm 2015). These assumptions and the subsequent processing steps taken to exclude ﬁeld stars and binaries were identi- cal to those described in Bouma et al. (2022). The mean and uncertainty of the resulting age posterior are 46+9 −7 Myr for RSG-5, and 36+10 −8 Myr for CH-2. For comparison, this pro- cedure yields an age for the δ Lyr cluster of 38+6 −5 Myr. 2.3.2. Stellar Rotation Periods EDR3 Catalog of Nearby Stars (Gaia Collaboration et al. 2021b). To make these diagrams, we imposed the data ﬁlter- ing criteria from Gaia Collaboration et al. (2018a), which in- clude binaries while omitting artifacts from for instance low photometric signal to noise, or a small number of visibility periods. We then corrected for extinction using the Lalle- ment et al. (2018) dust maps and the extinction coefﬁcients kX ≡AX/A0 from Gaia Collaboration et al. (2018a), assum- ing that A0 = 3.1E(B−V). This yielded a mean and standard deviation for the reddening of E(B −V) = 0.036 ± 0.002 for RSG-5, and E(B −V) = 0.017 ± 0.001 for CH-2. By way of comparison, in Bouma et al. (2022) the same query for the δ Lyr cluster yielded E(B −V) = 0.032 ± 0.006. Finally, for the plots we set the color range to best visualize the region of maximal age information content: the pre-main-sequence. An independent way to assess the age of the candidate cluster members is to measure their stellar rotation periods. This approach can be achieved using surveys such as TESS (Ricker et al. 2015) and the Zwicky Transient Facility (ZTF, Bellm et al. 2019); it leverages a storied tradition of measur- ing rotation periods of stars in benchmark open clusters (see e.g., Skumanich 1972; Curtis et al. 2020). The TESS data in our case are especially useful, since they provide 3 to 5 lu- nar months of photometry for all of our candidate CH-2 and RSG-5 members. We selected stars suitable for gyrochronology by requiring (GBP −GRP)0 ≥0.5 to focus on FGKM stars that experience magnetic braking. For TESS, we also restricted our sample to G < 16, to ensure the stars are bright enough to extract usable light curves from the full-frame images. The magni- tude cut corresponds to (GBP −GRP)0 < 2.6 (∼M3V) at the relevant distances. These cuts gave 19 stars in CH-2 and 47 stars in RSG-5. We extracted light curves from the TESS images using the unpopular package (Hattori et al. 2021), and regressed them against systematics with its causal pixel model. We measured rotation periods using Lomb-Scargle periodograms and visually vetted the results using an interac- tive program that allows us to switch between TESS Cycles, select particular sectors, ﬂag stars with multiple periods, and correct half-period harmonics. 2.3.1. Color–Absolute Magnitude Diagram By way of comparison, in Bouma et al. (2022) the same query for the δ Lyr cluster yielded E(B −V) = 0.032 ± 0.006. Finally, for the plots we set the color range to best visualize the region of maximal age information content: the pre-main-sequence. 2.3.1. Color–Absolute Magnitude Diagram 1.0 1.5 2.0 2.5 3.0 (GBP GRP)0 [mag] 5 6 7 8 9 10 11 Absolute MG, 0 [mag] KOI-7368 KOI-7913 A KOI-7913 B Lyr candidates CH-2 candidates Field K2 K5 M0 M2 M3 M4 Spectral Type 1.0 1.5 2.0 2.5 3.0 (GBP GRP)0 [mag] 5 6 7 8 9 10 11 Absolute MG, 0 [mag] Kepler-1643 Lyr candidates RSG-5 candidates Field K2 K5 M0 M2 M3 M4 Spectral Type Absolute MG, 0 [mag] (GBP GRP)0 [mag] (GBP GRP)0 [mag] 0.5 1.0 1.5 2.0 2.5 (GBP GRP)0 [mag] 0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 Rotation Period [days] Kepler-1643 Pleiades Praesepe RSG-5 candidates F0 F5 G0 K0 K3 K5 K7 M0 M1 M2 M3 Spectral Type 0.5 1.0 1.5 2.0 2.5 (GBP GRP)0 [mag] 0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 Rotation Period [days] KOI-7368 KOI-7913 A KOI-7913 B Pleiades Praesepe CH-2 candidates F0 F5 G0 K0 K3 K5 K7 M0 M1 M2 M3 Spectral Type Spectral Type Figure 2. Age-diagnostic diagrams from the stellar groups near Kepler-1643, KOI-7368, and KOI-7913. Top row: Color–absolute magnitude diagram of candidate Cep-Her members, plotted over candidate members of the δ Lyr cluster (≈38 Myr; Bouma et al. 2022) and the Gaia EDR3 Catalog of Nearby Stars (gray background). The left and right columns shows stars in RSG-5 and CH-2, respectively. The range of colors is truncated to emphasize the pre-main-sequence. Stars that fall far below the cluster sequences are ﬁeld interlopers. Bottom row: TESS and ZTF-derived stellar rotation periods, with the Pleiades (≈112 Myr) and Praesepe (≈650 Myr) shown for reference (Rebull et al. 2016; Douglas et al. 2017). The detection efﬁciency for reliable rotation periods falls off beyond (GBP −GRP)0 ≳2.6. 6 EDR3 Catalog of Nearby Stars (Gaia Collaboration et al. 2021b). To make these diagrams, we imposed the data ﬁlter- ing criteria from Gaia Collaboration et al. (2018a), which in- clude binaries while omitting artifacts from for instance low photometric signal to noise, or a small number of visibility periods. We then corrected for extinction using the Lalle- ment et al. (2018) dust maps and the extinction coefﬁcients kX ≡AX/A0 from Gaia Collaboration et al. (2018a), assum- ing that A0 = 3.1E(B−V). This yielded a mean and standard deviation for the reddening of E(B −V) = 0.036 ± 0.002 for RSG-5, and E(B −V) = 0.017 ± 0.001 for CH-2. 2.3.2. Stellar Rotation Periods Parameter Value Uncertainty Comment Kepler-1643 Stellar parameters: Gaia G [mag] 13.836 ±0.003 A Teff [K] 4916 ±110 B logg⋆[cgs] 4.502 ±0.035 C R⋆[R⊙] 0.855 ±0.044 C M⋆[M⊙] 0.845 ±0.025 C ρ⋆[g cm−3] 1.910 ±0.271 C Prot [days] 5.106 ±0.044 D Li EW [mÅ] 130 +6, −5 E Transit parameters: P [days] 5.3426258 ±0.0000101 D Rp/R⋆ 0.025 ±0.001 D b 0.58 ±0.05 D Rp [R⊕] 2.32 ±0.14 D t14 [hours] 2.41 ±0.06 D KOI-7368 Stellar parameters: Gaia G [mag] 12.831 ±0.004 A Teff [K] 5241 ±100 F logg⋆[cgs] 4.499 ±0.030 C R⋆[R⊙] 0.876 ±0.035 C M⋆[M⊙] 0.879 ±0.018 C ρ⋆[g cm−3] 1.840 ±0.225 C Prot [days] 2.606 ±0.038 D Li EW [mÅ] 236 +16, −14 E Transit parameters: P [days] 6.8430341 ±0.0000125 D Rp/R⋆ 0.023 ±0.01 D b 0.50 ±0.06 D Rp [R⊕] 2.22 ±0.12 D t14 [hours] 2.79 ±0.07 D KOI-7913 Stellar parameters: Gaia G [mag] 14.200 ±0.003 A Teff,A [K] 4324 ±70 B Teff,B [K] 4038 ±70 B logg⋆,A [cgs] 4.523 ±0.043 C R⋆,A [R⊙] 0.790 ±0.049 C M⋆,A [M⊙] 0.760 ±0.025 C ρ⋆,A [g cm−3] 2.172 ±0.379 C Prot,A [days] 3.387 ±0.016 D Prot,B [days] 2.642 ±0.067 D (Li EW)A [mÅ] 65 +8, −6 E (Li EW)B [mÅ] 42 +12, −19 E ∆GAB [mag] 0.51 ±0.01 G Apparent sep. [au] 959.4 ±1.9 G Transit parameters: P [days] 24.278571 ±0.000263 D Rp/R⋆ 0.027 ±0.001 D b 0.30 ±0.15 D Rp [R⊕] 2.34 ±0.18 D t14 [hours] 4.40 0.21 D NOTE— (A) Gaia Collaboration et al. (2021a). (B) HIRES SpecMatch-Emp (Yee et al. 2017). (C) Cluster isochrone (Choi et al. 2016; Bressan et al. 2012). (D) Kepler light curve. (E) HIRES/TRES (Bouma et al. 2021). (F) TRES SPC (Buchhave et al. 2012; Bieryla et al. 2021). (G) Magnitude difference and ap- parent physical separation between primary and secondary; from Gaia EDR3. (H) HIRES SpecMatch-Synth (Petigura et al. 2017). Table 1. Selected system parameters of Kepler-1643, KOI-7368, and KOI-7913. with rotation periods between 10 and 12.5 days. As previ- ously noted, the age interpretation for these M-dwarfs is not obvious. Regardless, the ≈15% false positive rate for CH-2 determined from the CAMD seems consistent with our frac- tion of detected rotation periods; RSG-5 was missing rotation periods for ≈15% of its candidate members, even though all of its stars appear photometrically consistent with being on a single pre-main-sequence locus. 2.3.2. Stellar Rotation Periods The older isochronal age of RSG-5 is consistent with its location relative to the δ Lyr cluster in the upper left panel of Fig- ure 2. Generally speaking, this method is expected to be ac- curate provided that the metallicities of IC 2602 and the Cep- Her groups (RSG-5, CH-2, and the δ Lyr cluster) are roughly identical. The spectroscopic metallicities that we ﬁnd in Sec- tion 3 suggest that this is indeed the case. The lower panels of Figure 2 show the results. In RSG- 5, 41/47 stars have rotation periods faster than the Pleiades (87%). This numerator omits the two stars with periods >12 days visible in the lower-left panel of Figure 2. The age interpretation for these latter stars, particularly the ≈M2.5 dwarf, is not obvious. Rebull et al. (2018) for instance have found numerous M-dwarfs with 10-12 day rotation periods at ages of USco (∼8 Myr), and some may still exist at ages of LCC (∼16 Myr; L. Rebull submitted). Regardless, since nearly no ﬁeld star outliers seem to be present on the RSG- 5 CAMD, the fact that we do not detect rotation periods for ≈13% of stars should perhaps be taken as an indication for the fraction of stars for which rotation periods might not be detectable, due to e.g., pole-on stars having lower amplitude starspot modulation. For CH-2, 13/19 stars have rotation periods that are obvi- ously faster than their counterparts in the Pleiades (68%). 4 stars, not included in the preceding numerator, are M-dwarfs TRIO OF MINI-NEPTUNES IN CEP-HER 7 7 Table 1. Selected system parameters of Kepler-1643, KOI-7368, and KOI-7913. 3. THE STARS Many of the salient properties of the Kepler objects of in- terest in Cep-Her can be gleaned from Figure 2. The stars span spectral types of G8V (Kepler-1627) to K6V (KOI-7913 A). The secondary in the KOI-7913 system has spectral type ≈K8V. And since a star with Solar mass and metallicity ar- rives at the zero-age main sequence at t ≈40 Myr (Choi et al. 2016), these stars are all in the late stages of their pre-main- sequence contraction. The adopted stellar parameters are listed in Table 1. The stellar surface gravity, radius, mass, and density are found by interpolating against the MIST isochrones (Choi et al. 2016). The statistical uncertainties from this technique mostly orig- inate from the parallax uncertainties; the systematic uncer- tainties are taken to be the absolute difference between the PARSEC (Bressan et al. 2012) and MIST isochrones. Re- ported uncertainties are a quadrature sum of the statistical and systematic components. To verify these parameters and to analyze youth proxies such as the Li 6708 Å doublet and Hα, we acquired spectra. We also acquired high resolution imaging for each system, to constrain the existence of visual companions, including pos- sible bound binaries. We give the system-by-system details in Sections 3.1 through 3.3, and summarize their implications for the youth of the stars in Section 3.4. 2.3.2. Stellar Rotation Periods It is challenging to convert these stellar rotation periods to a precise age estimate, since on the pre-main-sequence the stars are spinning up due to thermal contraction rather than down due to magnetized braking. Regardless, the rota- tion period distributions of both CH-2 and RSG-5 seem con- sistent with other 30 Myr to 50 Myr clusters (e.g., IC 2602 and IC 2391; Douglas et al. 2021). They also seem consis- tent with the false positive rates estimated from the color– absolute magnitude diagrams. 3.1. Kepler 1643 Spectra —For Kepler-1643, we acquired two iodine-free spectra from Keck/HIRES on the nights of 2020 Aug 16 and 2021 Oct 25. The acquisition and analysis fol- lowed the usual techniques of the California Planet Survey (Howard et al. 2010). We derived the stellar parameters (Teff,logg,R⋆) using SpecMatch-Emp (Yee et al. 2017), which yielded values in <1-σ agreement with those from 8 the cluster-isochrone method. This approach also yielded [Fe/H] = 0.13 ± 0.09. Using the broadened synthetic tem- plates from SpecMatch-Synth (Petigura et al. 2017), we found vsini = 9.3 ± 1.0 kms−1. The systemic radial velocity at the two epochs was −9.1±1.9 kms−1 and −7.8±1.2 kms−1 respectively (Chubak et al. 2012). To infer the equivalent width of the Li I 6708 Å doublet, we followed the procedure described by Bouma et al. (2021). This yielded a strong de- tection: EWLi = 130+6 −5 mÅ, with values consistent at <1-σ between the two epochs. The quoted value does not correct for the Fe I blend at 6707.44 Å. Given the purported age and effective temperature of the star, the lithium equivalent width is somewhat low. We discuss this in greater depth in Sec- tion 3.4. in optical passbands. The two stars are separated in Gaia EDR3 by 3.′′5 on-sky, and have parallaxes consistent within 1-σ (with an average ϖ = 3.66 ± 0.01 mas). The apparent on-sky separation is 959±2 au. The Gaia EDR3 proper mo- tions are also very similar. Since two stars were resolved in the Kepler Input Catalog and are roughly one Kepler pixel apart, an accurate crowding metric has already been applied in the NASA Ames data products to correct the mean ﬂux level (Morris et al. 2017). This is important for deriving ac- curate transit depths. Spectra —We acquired Keck/HIRES spectra for KOI-7913 A on the night of 2021 Nov 13, and KOI-7913 B on the night of 2021 Oct 26. The SpecMatch-Emp machin- ery yielded Teff,A = 4324 ± 70K, and Teff,B = 4038 ± 70K. The remaining parameters were in agreement with those from the cluster isochrone. For the primary, we also found [Fe/H] = −0.06±0.09, vsini = 13.3±1.0 kms−1, and RVsys = −17.8±1.1 kms−1. For the secondary, these same parameters were [Fe/H] = −0.01 ± 0.09, vsini = 10.7 ± 1.0 kms−1, and RVsys = −18.8 ± 1.1 kms−1. 3.4. Spectroscopic Youth Indicators High-Resolution Imaging —We acquired adaptive optics imag- ing of KOI-7368 on the night of 2019 June 12, again using NIRC2. The observational conﬁguration and reduction were identical as for Kepler-1643. No companions were detected, and the analysis of the image residuals yielded contrast limits of ∆K′ = 5.2 mag at ρ = 150 mas, ∆K′ = 6.7 mag at ρ = 300 mas, and ∆K′ = 8.7 mag at ρ > 1000 mas. Figure 3 shows key portions of the HIRES and TRES spec- tra for the Kepler objects in Cep-Her. Lithium absorption is obvious at 6708Å in all stars except KOI-7913 B. Hα is in emission for both components of KOI-7913, and in absorp- tion for the hotter stars. Here, we compare these observations against benchmark open clusters in order to assess their im- plications for the stellar ages. 3.1. Kepler 1643 Neither of the KOI-7913 com- ponents shows strong lithium absorption, but this is expected given their ≈K6V and ≈K8V spectral types. They do how- ever both show Hα in emission. Broadly speaking, both ob- servations are consistent with a ≈40 Myr age for KOI-7913 (see Section 3.4). High-Resolution Imaging —We acquired adaptive optics imag- ing of Kepler-1643 on the night of 2019 June 28 using the NIRC2 imager on Keck-II. Using the narrow camera (FOV = 10.2′′), we obtained 4 images in the K′ ﬁlter (λ = 2.12µm) with a total exposure time of 320 s. The images did not show any additional visual companions. We analyzed the data following Kraus et al. (2016), and determined the detec- tion limits by analyzing the residuals after subtracting an em- pirical PSF template. This procedure yielded contrast limits of ∆K′ = 4.1 mag at ρ = 150 mas, ∆K′ = 5.8 mag at ρ = 300 mas, and ∆K′ = 8.3 mag at ρ > 1000 mas. 3.2. KOI-7368 High-Resolution Imaging —We acquired adaptive optics imag- ing of KOI-7913 on the night of 2020 Aug 27 using the NIRC2 imager. The observational conﬁguration and reduc- tion were identical as before. The images showed KOI-7913 A, KOI-7913 B, and an additional faint neighbor ≈0.′′99 due East of KOI-7913 B. Applying the PSF-ﬁtting routines from Kraus et al. (2016), the tertiary object has a separa- tion ρ = 4397 ± 3 mas from the primary, at a position angle 231.17◦±0.02◦, with ∆K′ = 6.97±0.04. While it is too faint to affect the interpretation of the transit signal, it would be amusing if this faint neighbor were comoving and therefore part of the system, because it would have a mass between 10 and 15 MJup. Additional imaging epochs will tell. Spectra —For KOI-7368, we acquired a spectrum on 2015 June 1 using the echelle spectrograph (TRES; F˝urész et al. 2008) mounted at the Tillinghast 1.5m at the Fred Lawrence Whipple Observatory. The Stellar Parameter Classiﬁcation pipeline for TRES has been described by Bieryla et al. (2021). It is based on the synthetic template library con- structed by Buchhave et al. (2012). The resulting stellar pa- rameters (Teff,logg,R⋆) agreed with those from the cluster- isochrone method within 1-σ. Auxiliary spectroscopic pa- rameters included the metallicity [Fe/H] = −0.02±0.08, the equatorial velocity vsini = 20.21 ± 0.50 kms−1, and the sys- temic velocity RVsys = −10.9±0.2 kms−1. The Li 6708Å EW measurement procedure yielded EWLi = 236+16 −14 mÅ. 3.3. KOI-7913 Binarity —KOI-7913 is a binary. The north-west primary is ≈0.5 magnitudes brighter than the south-east secondary Figure 4 compares the measured lithium equivalent widths of the Kepler objects against a few reference populations. TRIO OF MINI-NEPTUNES IN CEP-HER TRIO OF MINI-NEPTUNES IN CEP-HER 9 9 6705 6710 0.8 1.0 Li-I Kepler-1627 A 6705 6710 0.8 1.0 Li-I KOI-7368 6705 6710 0.8 0.9 1.0 Li-I Kepler-1643 6705 6710 0.9 1.0 Li-I KOI-7913 A 6705 6710 0.9 1.0 Li-I KOI-7913 B 6560 6565 0.8 1.0 H 6560 6565 0.8 1.0 H 6560 6565 0.8 1.0 H 6560 6565 1.0 1.2 H 6560 6565 0.9 1.2 1.5 H Relative flux Wavelength [Å] Figure 3. Spectroscopic youth diagnostics for Kepler-1627, KOI-7368, Kepler-1643, and KOI-7913 AB. The spectra are shown in the observed frame, and the stars are sorted left-to-right in order of decreasing effective temperature. Figure 3. Spectroscopic youth diagnostics for Kepler-1627, KOI-7368, Kepler-1643, and KOI-7913 AB. The spectra are shown in the observed frame, and the stars are sorted left-to-right in order of decreasing effective temperature. We selected reference studies from the literature only when upper limits were explicitly reported. KOI-7368 and KOI- 7913 A have secure lithium detections, while for KOI-7913 B the detection is marginal (EWLi = 42+12 −19 mÅ). For all three stars, as well as for Kepler-1627 A, the observed lithium equivalent width is consistent with the stellar effective tem- peratures and a ≈40 Myr age. sidering the top panels of Figure 4, it is remarkable that in 50 million years, stars between 4500 K and 5200 K go from hav- ing a tight lithium sequence to one with a dispersion ≈10× greater. The existence of the Li dispersion in Pleiades-age K- dwarfs has been known for decades; it has also been known that the stars with the largest lithium abundances are also the most rapidly rotating (Butler et al. 1987; Soderblom et al. 1993). More recent analyses of this correlation have been re- viewed by Bouvier (2020). The conclusion of that work was that the origin of the rotation-lithium correlation likely lies within pre-main-sequence stellar physics. If so, one would expect the IC 2602 and Tuc-Hor K-dwarfs to show a larger in- trinsic lithium dispersion. A recent analysis of the ≈40 Myr NGC 2547 by Binks et al. 3.3. KOI-7913 (2022) suggests that this may be the case, though that study only had ≈10 stars in the relevant effective temperature range. This suggests that RSG-5 and Cep-Her could be worthy objects for a closer analysis of the lithium-rotation correlation near the zero-age main sequence. Kepler-1643, in RSG-5, is conspicuously below the 40- 50Myr sequence in the left-panel of Figure 4, though above the ﬁeld stars (EWLi = 130+6 −5 mÅ). The right panel shows the comparison against the Pleiades, where Kepler-1643 is more consistent with the observed dispersion in lithium. One explanation for the lack of Li in Kepler-1643 could be that it is a ﬁeld interloper; another could be that RSG-5 is much older than 50 Myr. We do not favor either expla- nation. RSG-5 cannot be much older than 50 Myr based on its proximity to the δ Lyr cluster and IC 2602 in the CAMD, and because it is below the Pleiades in the rotation versus color diagram (Figure 2). Kepler-1643 also seems highly unlikely to be a ﬁeld interloper, because we demonstrated a ≈1% false positive probability in our spatio-kinematic selec- tion of RSG-5 members, and there is a similar independent chance (≈1%) of a ﬁeld K2V star having a rotation period below the Pleiades (McQuillan et al. 2014). This yields a puzzle: how could a star have spatial, kinematic, and rota- tional evidence consistent with being in a ≈50 Myr cluster, but a low lithium content? 3.4.2. Hα As shown in Figure 3, Hα is in emission for both com- ponents of KOI-7913, and in absorption for the hotter stars. Additionally, the emission appears double-peaked for both of the KOI-7913 components. An important note is that KOI- 7913 A and KOI-7913 B were spatially resolved from each other during data acquisition. Performing a cross-correlation between each of the stars and the nearest matches in the Keck/HIRES template library, we also found that the CCFs for both components of KOI-7913 showed no indications of double-lined binarity (Kolbl et al. 2015). Our preferred explanation for Kepler-1643’s meager lithium content is that the reference sample of IC 2602 and Tuc-Hor stars may not fully explore all possible stellar rota- tion periods and lithium equivalent widths at this age. Con- 10 3500 4000 4500 5000 5500 6000 6500 Effective Temperature [K] 0 100 200 300 400 Li6708 EW [mÅ] Kepler Field 40-50 Myr Kepler-1627 A KOI-7368 KOI-7913 A KOI-7913 B Kepler-1643 3500 4000 4500 5000 5500 6000 6500 Effective Temperature [K] 0 100 200 300 400 Li6708 EW [mÅ] Kepler Field 112 Myr Kepler-1627 A KOI-7368 KOI-7913 A KOI-7913 B Kepler-1643 K2 K4 K6 K8 M0 M2 M4 M6 Spectral Type 10 8 6 4 2 0 2 H EW [Å] TucHor (Kraus+14) Field KOI-7913 A KOI-7913 B Kepler-1643 Figure 4. Lithium 6708Å and Hα equivalent widths for the objects of interest compared to young open clusters and ﬁeld stars. Positive equivalent width means absorption; negative equiv- alent width means emission. Top & middle: The ﬁeld stars are KOIs from Berger et al. (2018). The “40-50 Myr” reference stars (left) are from IC 2602 (Randich et al. 2018) and Tuc-Hor (Kraus et al. 2014). The “112 Myr” stars are from the Pleiades (Soderblom et al. 1993; Jones et al. 1996; Bouvier et al. 2018). The statistical un- certainties on the equivalent widths are shown, or else are smaller than the markers. Bottom: The Hα comparison is against Tuc-Hor (≈40 Myr; Kraus et al 2014) 10 Balmer line emission, particularly in Hα, is expected for low-mass stars of this age. Kraus et al. (2014) for instance, in their survey of Tuc-Hor (≈40 Myr), observed that all cluster members with spectral types >K4.5V had Hα in emission. 4.1. Kepler Data The Kepler space telescope observed Kepler-1643, KOI- 7913, and KOI-7368 at a 30-minute cadence between May 2009 and April 2013. For all three systems quarters 1 through 17 were observed with minimal data gaps. The top panel of Figure 5 shows a 50-day slice of the PDCSAP light curves for the three new Cep-Her candidates, along with Kepler- 1627. In PDCSAP, non-astrophysical variability is removed through a cotrending approach that uses a set of basis vectors derived by applying singular value decomposition to a set of systematics-dominated light curves (Smith et al. 2017). In our analysis, we used the PDCSAP light curves with the de- fault optimal aperture (Smith et al. 2016). Cadences with non-zero quality ﬂags were omitted. In all cases, the stars are dominated by spot-induced modulation with peak-to-peak variability between 2% and 10%. These signals are much larger than the transits, which have depth ≈0.1%. To quantify the stellar rotation periods, we calculated the Lomb-Scargle periodogram for each Kepler quarter independently. The re- sulting means and standard deviations are in Table 1. Effective Temperature [K] Effective Temperature [K] K2 K4 K6 K8 M0 M2 M4 M6 Spectral Type 10 8 6 4 2 0 2 H EW [Å] TucHor (Kraus+14) Field KOI-7913 A KOI-7913 B Kepler-1643 3.4.2. Hα Ths is consistent with our observations: KOI-7913 shows Hα in emission for both components, and in absorption for all of our other Kepler objects (Figure 3, lower panel). The double- peaked nature of the emission, though not always present, is also common for active stars. Proxima Centauri, for instance, has double-peaked Hα emission (Collins et al. 2017). Given that we have ruled out spectroscopic binarity, the most likely explanation is self-absorption: photons near the center of the line see a greater optical depth from higher layers of the chro- mosphere, while photons on the wings are too far from the rest-wavelength to excite electrons and be re-absorbed in the upper layers. The exact details of when a star’s atmosphere reaches the conditions for such self-absorption require non- local thermal equilibrium models of the chromosphere (Short & Doyle 1998; Fuhrmeister et al. 2005). 3500 4000 4500 5000 5500 6000 6500 Effective Temperature [K] 0 100 200 300 400 Li6708 EW [mÅ] Kepler Field 40-50 Myr Kepler-1627 A KOI-7368 KOI-7913 A KOI-7913 B Kepler-1643 Effective Temperature [K] Effective Temperature [K] 3500 4000 4500 5000 5500 6000 6500 Effective Temperature [K] 0 100 200 300 400 Li6708 EW [mÅ] Kepler Field 112 Myr Kepler-1627 A KOI-7368 KOI-7913 A KOI-7913 B Kepler-1643 4.2. Transit and Stellar Variability Model Figure 4. Lithium 6708Å and Hα equivalent widths for the objects of interest compared to young open clusters and ﬁeld stars. Positive equivalent width means absorption; negative equiv- alent width means emission. Top & middle: The ﬁeld stars are KOIs from Berger et al. (2018). The “40-50 Myr” reference stars (left) are from IC 2602 (Randich et al. 2018) and Tuc-Hor (Kraus et al. 2014). The “112 Myr” stars are from the Pleiades (Soderblom et al. 1993; Jones et al. 1996; Bouvier et al. 2018). The statistical un- certainties on the equivalent widths are shown, or else are smaller than the markers. Bottom: The Hα comparison is against Tuc-Hor (≈40 Myr; Kraus et al. 2014) Our goals in ﬁtting the Kepler light curves are twofold. First, we want to derive accurate planetary sizes and orbital properties. Second, we want to remove the spot-induced vari- ability signal to enable a statistical assessment of the proba- bility that the transit signals are planetary. We ﬁtted the data as follows. Given the transit ephemeris from Thompson et al. (2018), we ﬁrst trimmed the light curve to a local window around each transit that spanned three tran- sit durations before and after each transit midpoint. The out- TRIO OF MINI-NEPTUNES IN CEP-HER 11 50 0 50 Kepler-1643 50 0 50 KOI-7368 50 0 50 KOI-7913 550 560 570 580 590 600 50 0 50 Kepler-1627 Relative flux [ppt] Days from start 4 2 0 2 4 Hours from mid-transit 4 3 2 1 0 1 Relative flux [ppt] Kepler-1643 4 2 0 2 4 Hours from mid-transit 5 4 3 2 1 0 1 Relative flux [ppt] KOI-7913 4 2 0 2 4 Hours from mid-transit 4 3 2 1 0 1 Relative flux [ppt] KOI-7368 4 2 0 2 4 Hours from mid-transit 6 5 4 3 2 1 0 1 Relative flux [ppt] Kepler-1627 5. Raw and processed light curves for the Kepler Objects of Interest in Cep-Her. Top: 50 day light curve segment from th Kepler data. The ordinate shows the PDCSAP median-subtracted ﬂux in units of parts-per-thousand (×10−3). The dominant starspots; planetary transit times are indicated but the individual transits are not visible at this scale. Bottom: Phase-folded tran 1643, KOI-7913, KOI-7368, and Kepler-1627 with stellar variability removed. The maximum a posteriori model is shown with th d the residual after subtracting the transit model is vertically displaced. 4.2. Transit and Stellar Variability Model Windows over 10 hours are shown. Gray points are indiv asurements; black points are binned to 20 minute intervals and have a representative 1 error bar in the center right of each pan 50 0 50 Kepler-1643 50 0 50 KOI-7368 50 0 50 KOI-7913 550 560 570 580 590 600 50 0 50 Kepler-1627 Relative flux [ppt] Days from start 550 560 570 580 590 600 50 0 50 Kepler-1627 Re Days from start 4 2 0 2 4 Hours from mid-transit 4 3 2 1 0 1 Relative flux [ppt] Kepler-1643 4 2 0 2 4 Hours from mid-transit 5 4 3 2 1 0 1 Relative flux [ppt] KOI-7913 4 2 0 2 4 Hours from mid-transit 4 3 2 1 0 1 Relative flux [ppt] KOI-7368 4 2 0 2 4 Hours from mid-transit 6 5 4 3 2 1 0 1 Relative flux [ppt] Kepler-1627 Figure 5. Raw and processed light curves for the Kepler Objects of Interest in Cep-Her. Top: 50 day light curve segment from the 3.9 years of Kepler data. The ordinate shows the PDCSAP median-subtracted ﬂux in units of parts-per-thousand (×10−3). The dominant signal is from starspots; planetary transit times are indicated but the individual transits are not visible at this scale. Bottom: Phase-folded transits of Kepler-1643, KOI-7913, KOI-7368, and Kepler-1627 with stellar variability removed. The maximum a posteriori model is shown with the gray line, and the residual after subtracting the transit model is vertically displaced. Windows over 10 hours are shown. Gray points are individual ﬂux measurements; black points are binned to 20 minute intervals, and have a representative 1-σ error bar in the center-right of each panel. 4.2. Transit and Stellar Variability Model Figure 5 shows the justiﬁcation: the transit is ﬂat and has a high S/N (≈47). The shape is therefore nearly impossible to reproduce with eclips- ing binary models. We ﬁtted the data using exoplanet (Foreman-Mackey et al. 2020). We assumed a Gaussian likelihood, and sam- pled using PyMC3’s No-U-Turn Sampler (Hoffman & Gel- man 2014), after having initialized to the the maximum a pos- teriori (MAP) model. We used the Gelman & Rubin (1992) statistic, ˆR, as our convergence diagnostic. The resulting ﬁts are shown in the lower panels of Figure 5, and the important derived parameters are in Table 1. The set of full parameters and their priors are given in Appendix B. Intriguingly, Kepler-1643 failed one of the data validation centroid shift tests (see the q1_q17_dr25_koi data re- lease): the angular distance between the target star’s cata- log position and the position of the transiting source was measured as 1.′′0 at 4.4-σ. The reports show however that two outlying quarters (2 and 6) drive the offset — the cen- troid locations from the other Kepler quarters are consistent at ≲0.′′4. This is an instructive exercise in how stellar vari- ability complicates centroid-based vetting tests. The shifts measured by these tests are determined from the in- and out- of-transit ﬂux-weighted centroids. For stars with signiﬁcant spot-induced variability there is no static baseline in either the in- or out-of-transit phases. A potential drawback of our approach is that to remove the starspot-induced variability, we ﬁxed 5 parameters per tran- sit to their MAP values. An alternative could be to ﬁt the planetary transits simultaneously with the starspot-induced variability using a quasiperiodic Gaussian process (GP). We explored this approach, but found that it often required ﬁne- tuning of the hyperparameter priors, otherwise the GP would overﬁt ﬂares, instrumental noise, and any other variability that was not part of the transit. This failure mode is perni- cious, in that it yields an ill-founded sense of conﬁdence in a visually clean ﬁt. Our model is simpler, and it has the beneﬁt that the white noise jitter never trades off with any parameter equivalent to a damping timescale for the coherence of the GP. It is also computationally efﬁcient, and it captures the planetary parameters about which we care the most. KOI-7368 —KOI-7368.01 is listed on the NASA Exoplanet Archive as a “candidate” planet. Morton et al. 4.2. Transit and Stellar Variability Model 4 2 0 2 4 Hours from mid-transit 4 3 2 1 0 1 Relative flux [ppt] Kepler-1643 4 2 0 2 4 Hours from mid-transit 4 3 2 1 0 1 Relative flux [ppt] KOI-7368 4 2 0 2 4 Hours from mid-transit 5 4 3 2 1 0 1 Relative flux [ppt] KOI-7913 4 2 0 2 4 Hours from mid-transit 6 5 4 3 2 1 0 1 Relative flux [ppt] Kepler-1627 Relative flux [ppt] Relative flux [ppt] 4 2 0 2 4 Hours from mid-transit 5 KOI 7913 4 2 0 2 4 Hours from mid-transit 6 5 4 3 2 1 0 1 Relative flux [ppt] Kepler-1627 Relative flux [ppt] Relative flux [ppt] Hours from mid-transit Hours from mid-transit Hours from mid-transit Hours from mid-transit Figure 5. Raw and processed light curves for the Kepler Objects of Interest in Cep-Her. Top: 50 day light curve segment from the 3.9 years of Kepler data. The ordinate shows the PDCSAP median-subtracted ﬂux in units of parts-per-thousand (×10−3). The dominant signal is from starspots; planetary transit times are indicated but the individual transits are not visible at this scale. Bottom: Phase-folded transits of Kepler-1643, KOI-7913, KOI-7368, and Kepler-1627 with stellar variability removed. The maximum a posteriori model is shown with the gray line, and the residual after subtracting the transit model is vertically displaced. Windows over 10 hours are shown. Gray points are individual ﬂux measurements; black points are binned to 20 minute intervals, and have a representative 1-σ error bar in the center-right of each panel. 12 foreground eclipsing binaries, hierarchical eclipsing binaries, and background eclipsing binaries. of-transit points in each local window were then ﬁtted with a fourth-order polynomial, which was divided out from the light curve. The resulting ﬂattened transits were then ﬁt- ted with a transit model that assumed quadratic limb darken- ing. The model therefore included 8 free parameters for the transit ({P,t0,logRp/R⋆,b,u1,u2,R⋆,logg}), 2 free parame- ters for the light curve normalization and a white noise jitter ({⟨f⟩,σ f }), and 5 ﬁxed parameters for each transit. Kepler-1643 —Kepler-1643 b (KOI-6186.01) was already val- idated as a transiting planet by Morton et al. (2016), who found a probability for any of the aforementioned false posi- tive scenarios of 9×10−6. Repeating the calculation with our own stellar-variability correction and the new NIRC2 imag- ing constraints, we ﬁnd FPP = 6 × 10−9. 4.2. Transit and Stellar Variability Model (2016) did not compute a false positive probability for the system be- cause their default trapezoidal ﬁtting routine failed, presum- ably due to the spot-induced variability. Our ﬁtting approach rectiﬁes this point, and our new NIRC2 images revealed no new stellar companions. Performing the relevant calculation, we ﬁnd FPP = 4×10−3. Though not as convincing as Kepler- 1643, this clears the usual threshold for calling the planet statistically validated (Morton 2012). The S/N of the tran- sit is ≈32, which indicates that it is unlikely to be caused by systematic noise in the light curve (see Figure 5). The positional probability score also meets the requirements for transiting sources thought to share positions with their target stars (Bryson & Morton 2017). 4.3. Planet Validation Though the transit has the lowest S/N of any of the objects discussed (≈14), its lower FPP rela- tive to KOI-7368 can be understood through its ﬂat-bottomed shape, combined with its long transit duration relative to most eclipsing binary models (Figure 5). The host star probabil- ity usability score (Bryson & Morton 2017) also meets the usual threshold, and so the planet is statistically validated. Its disposition has however previously ﬂuctuated from “false positive” to “candidate” (see Appendix C). The most likely explanation is the presence of KOI-7913 B, which is located ≈0.9 Kepler pixels away from Kepler-7913 A. While the ≈1.5 pixel FWHM of the Kepler pixel response function im- plies that there is blending between the two stars, the target- pixel level data for KOI-7913 B reveals an entirely different stellar rotation period (Table 1), and no hint of the transit sig- nal. This implies that KOI-7913 B cannot host the planet. a signiﬁcant role (Owen 2020). As an example, Rogers & Owen (2021) predicted that given a core mass distribution peaked at ≈4 M⊕, an ice-poor rock/iron core composition, and a typical H/He mass fraction of ≈4%, there should be a local maximum in planet occurrence rates at 2 to 3 R⊕, at times between 10 and 100 Myr. The models advanced by Gupta & Schlichting (2020) and Lee & Connors (2021) agree; their differences lie in the mechanism for producing the radius valley, and in whether a population of rocky plan- ets is predicted to exist at the time of disk dispersal. Systems such as K2-25, V1298 Tau, HIP-67522, TOI-837, and TOI-1227 have sizes that are anomalously large rela- tive to the predicted peak in planet occurrence at 2 to 3 R⊕. However, their large sizes can be accommodated by invoking any of i) larger core masses, ii) more volatile-rich compo- sitions, iii) larger initial atmospheric mass fractions, or iv) longer thermal cooling times. Secure mass measurements would help constrain this parameter space, but the ∼1 km s−1 spot-induced radial velocity semi-amplitudes make measur- ing the Doppler orbits very difﬁcult (Cale et al. 2021; Zicher et al. 2022; Klein et al. 2022). Regardless, the new Kepler- 1643, KOI-7368, and KOI-7913 systems do demonstrate that at least some planets at 40 Myr have sizes that are consis- tent with theoretical expectations for mini-Neptunes. 4.3. Planet Validation In the future, it may be possible to obtain independent ev- idence for the planetary nature of the Cep-Her planets, for instance by observing spectroscopic transits. For now, it is of interest whether the transit signals might be astrophys- ical false positives, or whether they are statistically more likely to be planetary. We adopt the Bayesian framework implemented in VESPA to assess the relevant probabilities (Morton 2012, 2015). Brieﬂy summarized, the priors in VESPA assume the binary star occurrence rate from Ragha- van et al. (2010), direction-speciﬁc star counts from Girardi et al. (2005), and planet occurrence rates as described by Morton (2012, Section 3.4). The likelihoods are then eval- uated by forward-modeling a synthetic population of eclips- ing bodies for each astrophysical model class, in which each population member has a known trapezoidal eclipse depth, total duration, and ingress duration. These summary statis- tics are then compared against the actual photometric data to evaluate the probabilities of false positive scenarios such as It bears mentioning that KOI-7368 shows a centroid shift in the q1_q17_dr25_koi validation reports, similar to Kepler-1643. For KOI-7368, the reported offset is smaller, and less signiﬁcant (0.′′2; 3.0-σ). Again, the data validation reports show that the shift is caused by a few outlying quar- ters (4, 5, 8, and 12). Since the remaining data show consis- tent scatter in their centroid locations, these outlying quarters are likely also caused by the stellar variability. Our NIRC2 imaging independently shows that there are no neighboring sources that could cause an offset of the observed amplitude. KOI-7913 —KOI-7913.01 is also currently listed on the NASA Exoplanet Archive as a “candidate” planet. The Mor- TRIO OF MINI-NEPTUNES IN CEP-HER 13 ton et al. (2016) analysis was of Q1-Q17 KOIs from DR24, and therefore spanned KOI-1.01 to KOI-7620.01 (omitting KOI-7913.01). However the results of the subsequent DR25 analysis by Morton et al. are listed at the NASA Exoplanet Archive. The relevant table gives a probability for the sys- tem being an astrophysical false positive of 1.4 × 10−4, with the most likely false positive scenario being a blended eclips- ing binary. Repeating the calculation with our new detrend- ing and NIRC2 contrast curves, we ﬁnd a similar result: FPP = 1.3 × 10−4. 5.1. Normal-Sized Mini-Neptunes Exist at 40Myr The most signiﬁcant novelty about the planets in Kepler- 1643, KOI-7368, and KOI-7913 is that their sizes (2.2 to 2.3 R⊕) are normal relative to the known population of mini- Neptunes from Kepler. At ﬁeld star ages, mini-Neptune sizes span 1.8 R⊕to 3.6 R⊕, with the most common size being ≈2.4R⊕(Fulton et al. 2017). The known planets younger than 108 years are almost all larger, with sizes between 4 and 10R⊕(Mann et al. 2016; David et al. 2016; Benatti et al. 2019; David et al. 2019; Newton et al. 2019; Rizzuto et al. 2020; Bouma et al. 2020; Mann et al. 2022). Figure 6 ex- plores this by showing the sizes, orbital periods, and ages of the known transiting planets, emphasizing planets with precise ages. The smallest previously known planets com- parable to the new Cep-Her mini-Neptunes are AU Mic c (3.0 ± 0.2R⊕, see Martioli et al. 2021 and Gilbert et al. 2022), Kepler-1627 Ab (3.8 ± 0.2R⊕; Bouma et al. 2022), and AU Mic d (4.2±0.2R⊕; Plavchan et al. 2020). 4.3. Planet Validation While selection effects imposed by spot-induced photometric vari- ability are a likely explanation for why planets this small have not previously been identiﬁed (e.g., Zhou et al. 2021), future work should quantify this bias more carefully, in order to en- able empirical studies of how the planetary size distribution changes at early times. 5. DISCUSSION & CONCLUSION 5.1. Normal-Sized Mini-Neptunes Exist at 40Myr 5.2. Is CH-2 a Coeval Population? 10 1 100 101 102 103 Orbital period [days] 100 101 Planet radius [Earths] 107 108 109 Age [years] Orbital period [days] Orbital period [days] Figure 6. Radii, orbital periods, and ages of transiting exoplanets. Planets younger than a gigayear with ages more precise than a factor of three are emphasized. The Cep-Her planets are Kepler-1643 b (□), KOI-7368 b (▽), KOI-7913 Ab (X), and Kepler-1627 Ab (+). Interesting trends in the population of planets younger than 108 years old include i) their large sizes and ii) the lack of hot Jupiters. The new objects of interest in Cep-Her have normal mini-Neptune sizes between 2 and 3 R⊕, which is a novelty given their ages. Parameters are from the NASA Exoplanet Archive (2022 Apr 5). Figure 6. Radii, orbital periods, and ages of transiting exoplanets. Planets younger than a gigayear with ages more precise than a factor of three are emphasized. The Cep-Her planets are Kepler-1643 b (□), KOI-7368 b (▽), KOI-7913 Ab (X), and Kepler-1627 Ab (+). Interesting trends in the population of planets younger than 108 years old include i) their large sizes and ii) the lack of hot Jupiters. The new objects of interest in Cep-Her have normal mini-Neptune sizes between 2 and 3 R⊕, which is a novelty given their ages. Parameters are from the NASA Exoplanet Archive (2022 Apr 5). Cen, do not (Wright & Mamajek 2018). This complicates the feasibility of deriving kinematic ages through traceback, as well as through the expansion itself (see Crundall et al. 2019). A more minimal approach is that suggested by Tofﬂemire et al. (2021): search for coeval, phase-space neighbors, mea- sure their ages, and determine if they share a common age. This approach can demonstrate whether a star is currently associated with a set of coeval stars, though it falls short of determining what the association looked like in the past. Our analysis of CH-2 meets the latter standard for demonstrating the existence of a ≈40 Myr stellar association. Given its low density, in CH-2 truly a star cluster? For this discussion, we adopt the deﬁnition that a star cluster is a group of at least 12 stars that was physically associated at its time of formation. The “12” is set to distinguish star clusters from high-order multiples (see Krumholz et al. 2019). 5.3. Future work 5.2. Is CH-2 a Coeval Population? RSG-5, and Kepler-1643’s membership inside it, meet typ- ical expectations for a star claimed to be in an open clus- ter. RSG-5 is an obvious overdensity relative to the local ﬁeld, and our membership selection easily yielded a clean pre-main-sequence locus (Figure 2). CH-2, and KOI-7913 and KOI-7368’s membership inside it, do not meet these ex- pectations in as obvious a manner. This is because the CH-2 association is diffuse. To quantify the density difference between CH-2 and RSG- 5, we can compare the spatial and velocity volumes searched for each group. For RSG-5, we drew 173 candidate mem- bers from a 30pc × 30pc × 40pc rectangular prism, given a 1.5kms−1×2.5kms−1 rectangle in apparent galactic velocity. For CH-2, our 37 candidate members came from a rectangu- lar prism of dimension 50pc × 40pc × 30pc, and a rectan- gular box of 2kms−1 × 4kms−1. If we deﬁne the searched volume in units of pc3 km2 s−2, then the volume ratio of CH-2 to RSG-5 is 3.5 to 1. The ratio of number densities (candi- date members per unit searched volume) in RSG-5 relative to CH-2 is 16 to 1. The theoretical expectation is that mini-Neptunes with sizes of 2 to 3 R⊕should be common at ages of 107 to 108 years. This expectation is tied to inferences about the initial distributions of planetary core mass, core composition, and atmospheric mass fraction (Owen & Wu 2017). The Kelvin- Helmholtz cooling timescale, which is tied to the entropy of the planetary interior shortly after disk dispersal, also plays 14 10 1 100 101 102 103 Orbital period [days] 100 101 Planet radius [Earths] 107 108 109 Age [years] Figure 6. Radii, orbital periods, and ages of transiting exoplanets. Planets younger than a gigayear with ages more precise than a factor of three are emphasized. The Cep-Her planets are Kepler-1643 b (□), KOI-7368 b (▽), KOI-7913 Ab (X), and Kepler-1627 Ab (+). Interesting trends in the population of planets younger than 108 years old include i) their large sizes and ii) the lack of hot Jupiters. The new objects of interest in Cep-Her have normal mini-Neptune sizes between 2 and 3 R⊕, which is a novelty given their ages. Parameters are from the NASA Exoplanet Archive (2022 Apr 5). 5.4. Summary We have shown that Kepler-1643 b, KOI-7368 b, and KOI- 7913 Ab are 40 to 50 million years old, and that each system is most likely planetary. The evidence for the planetary in- terpretation comes from an application of VESPA to the Ke- pler data, alongside new imaging from NIRC2. The valid- ity of the VESPA framework rests on the premise that non- astrophysical false positives can be rejected. This seems to be the case for all three objects, even though Kepler-1643 and KOI-7368 both show weak centroid offsets between the in and out-of-transit phases. For both cases, the observed shifts are consistent with being caused by starspot-induced variability in speciﬁc quarters spuriously moving the stellar center-of-light. Independently, our imaging rules out com- panion stars with the brightnesses and positions that would be needed to explain the reported shifts. All three objects are therefore most likely planets. A number of worthy photometric projects also seem pos- sible given the new understanding of Cep-Her. One is aster- oseismology of the δ Sct stars, using either TESS or Kepler data (Bedding et al. 2020). For cases in which the modes are resolved, this might yield age or metallicity estimates for the subgroups independent of other methods. Other projects could include a more comprehensive analysis of the stellar rotation periods, searches of the Kepler light curves for exo- comets (Zieba et al. 2019), and searches for missed planets around the most rapid rotators. Each system has multiple indicators of youth that support the reported ages. For Kepler-1643, the strongest youth in- dicator is its physical and kinematic association with RSG-5. Based on the color–absolute magnitude diagram, we are able to select members of this cluster with a false positive rate of a few percent (Figure 2). Kepler-1643 is one such mem- ber. While the stellar rotation period period agrees with this assessment, the star’s lithium equivalent width is marginally low, which might motivate future exploration of lithium de- pletion across FGKM stars in RSG-5 (see Section 3.4). Exoplanet demographics at early times —Our main motivation for ﬁnding new young planets is to help benchmark models for planetary evolution. However demographic analyses of the known planets between 107 and 109 years have so far been rather limited. Approximately 40 such planets are now known (Figure 2). About half come from K2, a quarter from TESS, and now a quarter from Kepler. 5.2. Is CH-2 a Coeval Population? We explicitly do not require a “star cluster” to be gravitationally bound: dissolved clusters as well as their tidal tails are in- cluded in our adopted deﬁnition of “clusters”. We similarly do not require a threshold number of stars per unit spatial vol- ume. The latter point acknowledges that an important factor in cluster identiﬁcation is also density in velocity space. Per- haps once stellar rotation periods and chemical abundances reach the same level of ubiquity as stellar proper motions, they might enable further reﬁnement of our ability to dis- cover stars that formed as part of the same event. It would be a worthy exercise to perform a similar search for coeval phase-space neighbors on the entire dataset of known exoplanet hosts. For the time being, we can offer the anecdotal point that in our experience, most stars do not have dozens of 40 Myr neighbors within a local volume of a few km s−1 and tens of parsecs. From a data-driven perspective, demonstrating that a group of stars was physically associated at its time of formation is challenging. While some young groups show kinematic evi- dence for expansion (Kuhn et al. 2019), many, including Sco- 5.3. Future work 5.3. Future work TRIO OF MINI-NEPTUNES IN CEP-HER 15 Cep-Her —Our analysis to date has focused only on portions of Cep-Her that were observed by Kepler: RSG-5, CH-2, and the δ Lyr cluster. In Bouma et al. (2022) as well as this work, we have shown that these groups share similar ages, and have kinematic correlations that suggest a common ori- gin. With that said, the membership and kinematics of the other Cep-Her groups shown in Figure 1 deserve independent attention. An important aspect of the remaining work will be to acquire radial velocities of a large subset of the stars, and to determine whether the traceback approach could be applicable. Wide-ﬁeld spectroscopic surveys such as LAM- OST (Zhao et al. 2012) or SDSS-V (Kollmeier et al. 2017) could enable such analyses, while also providing sensitivity to the Li 6708 Å line. If Gaia DR3 provides RVS spectra for the brighter F and G dwarfs, these would also contain the calcium infrared triplet as another age indicator. These indicators, combined with better kinematics, would help in deﬁnitively unraveling the formation history of the complex. Jupiter occurrence rate is strongly dependent on stellar mass and metallicity (Petigura et al. 5.2. Is CH-2 a Coeval Population? 2018, 2022), particular care would be needed to select a sample of well-studied FGK dwarfs for the measurement, likely using stars in Sco OB2, Cep-Her, and Orion. For demographic studies focused on how mini-Neptune sizes evolve, the combined K2 and Ke- pler dataset would be the better primary source. ACKNOWLEDGMENTS L.G.B. is supported by the Heising-Simons Foundation 51 Pegasi b Fellowship and the NASA TESS GI Program (80NSSC21K0335 and 80NSSC22K0298). R.K. is sup- ported by the Heising-Simons Foundation. J.L.C. is sup- ported by NSF AST-2009840 and the NASA TESS GI Pro- gram (80NSSC22K0299). D.H. is supported by the Alfred P. Sloan Foundation and NASA (80NSSC19K0597). Facilities: Astrometry: Gaia. Imaging: Second Genera- tion Digitized Sky Survey. Keck:II (NIRC2). Spectroscopy: Tillinghast:1.5m (TRES). Keck:I (HIRES). Photometry: Ke- pler, TESS, ZTF. Software: astropy (Astropy Collaboration et al. 2018), astroquery (Ginsburg et al. 2018), exoplanet REFERENCES Campello, R. J. G. B., Moulavi, D., Zimek, A., & Sander, J. 2015, ACM Transactions on Knowledge Discovery from Data, 10, 5:1 Agol, E., Luger, R., & Foreman-Mackey, D. 2020, AJ, 159, 123 Arevalo, R. 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Summary Given the current state of the ﬁeld, a few reﬂections regard- ing experimental design of a demographic survey focused on planetary evolution over the ﬁrst gigayear might be use- ful. The ﬁrst is that such a project requires a set of target stars with known ages. A promising way to compile rele- vant stars could be to combine automated spatio-kinematic clustering from Gaia with rotation periods measured using TESS (see the appendices of Bouma et al. 2022). The sec- ond consideration is that all the known young planets smaller than 3 R⊕come from either K2 or Kepler. Demographic in- ferences based on TESS are therefore limited to planetary sizes ≳4 R⊕, for planets close-in to their host stars. It would be worthwhile to compare the occurrence rates of both types of planets with those from the main Kepler sample. One speciﬁc question that seems within reach would be to clar- ify whether enough young stars have been searched for the dearth of young hot Jupiters to be signiﬁcant. Since the hot The spatio-kinematic argument for the youth of KOI-7368 and KOI-7913 is weaker because they are in an association of stars, CH-2, that is more diffuse. For KOI-7913, stronger indicators of its age come from its binary. Both stellar com- ponents in KOI-7913 have isochronal ages consistent with 40 Myr. Both components also show Hα in emission, which for the ≈K6V primary is a strong indicator that the star is ≲100Myr old. KOI-7368 is slightly more massive, and the Li 6708 Å measurement and stellar rotation period provide independent veriﬁcation of the star’s youth. The astrophysical implication of these considerations is that planets ≈2 Earth radii in size exist at ages of 40 mil- lion years. It will be interesting to continue the push down to smaller planetary sizes at comparable ages – the planetary detections we have presented are well above the average de- tection signiﬁcance for Kepler planets. 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The complete catalog of can- didate Cep-Her members will be provided by R. Kerr et al. in prep. using Gaia DR3; Table 2 is from an early version of that analysis based on Gaia EDR3. Note that more restrictive weight cuts should be imposed if one wishes to remove the majority of ﬁeld star interlopers. Table 2 was created by cross-matching candidate Cep-Her members (selected using Gaia EDR3; Section 2.2) against a Kepler to Gaia DR2 cross-match (the gaia-kepler.fun crossmatch database created by Megan Bedell). The kic_dr2_ang_dist column is from the latter table. The EDR3 to DR2 match was performed using the gaiaedr3.dr2_neighbourhood table, and the closest proper motion and epoch-corrected angular distance neighbor was taken as the single best match. The edr3_dr2_mag_diff column gives some indication of the reliability of this EDR3 to DR2 conversion, as there are a few cases between Gaia DR2 and EDR3 where partially resolved binaries became fully resolved. Candidate matches between Cep-Her and the Kepler Objects of Interest: —The full list of candidate matches between Cep-Her and the Kepler Objects of Interest is as follows – the objects are listed in order of descending weights, D. Objects designated as conﬁrmed planets included Kepler-1627, Kepler-1643, Kepler-1331, Kepler-1062, and Kepler-1933. Objects designated as candidate planets included KOI-5264, KOI-8007, KOI-7572, KOI-7375, KOI-7368, KOI-7638, KOI-5632, and KOI-7913. Objects designated known false positive planet candidates included KOI-6437, KOI-5988, KOI-7871, KOI-7655, KOI-5024, KOI-61, KOI-4336, KOI-6812, KOI-3399, and KOI-6277. Finally, Kepler-1902 (KOI-3090) has one conﬁrmed planet (KOI-3090.02), and one false positive (KOI-3090.01). Of these objects, only Kepler-1627, Kepler-1643, KOI-7368, and KOI-7913 met our requirements for potentially both i) having real planets, and ii) being ≲108 years old, based on the presence of rotational modulation at the expected period and amplitude. Of the 14 conﬁrmed and candidate planets, 6 failed ﬁrst ﬁlter, and 7 independently failed the second. One object was ambiguous: Kepler-1933. This system has a conﬁrmed ≈1.4R⊕planet, a stellar rotation period of 6.5 days, and an effective temperature of ≈5750K. This places it near the upper envelope of the rotation period vs. color distribution for the Pleiades, making it unlikely to be ≈40 Myr old. Nonetheless, we acquired a reconnaissance HIRES spectrum, and it yielded EWLi = 93±5 mÅ. Combined with the rotation period, this suggests an age for Kepler-1933 between 100 and 300 Myr. TRIO OF MINI-NEPTUNES IN CEP-HER TRIO OF MINI-NEPTUNES IN CEP-HER 19 B. TABLE OF TRANSIT FIT PARAMETERS Table 4 gives the full set of ﬁtted and derived parameters from the model described in Section 4.2. Priors and convergence statistics are also listed. A. CANDIDATE CEP-HER MEMBERS Based on these indicators, the system is unlikely to be part of Cep-Her, but could merit further study. Candidate matches between Cep-Her and the Kepler Objects of Interest: —The full list of candidate matches between Cep-Her and the Table 3 —contains spatial, kinematic, astrometric, and rotation period information for the 173 candidate RSG-5 members and 37 candidate CH-2 members described in Section 2.2. These are the data used to make the lower panels of Figure 2; as with Table 2, these are from a preliminary version of the SPYGLASS 1 kpc expansion (R. Kerr et al. in prep). We adopted the ZTF period over the TESS period in three cases: (1) Gaia EDR3 2081755809272821248: the Lomb-Scargle periodogram favored 6.67 days, consistent with the ZTF period of 6.61 days; however, we ﬂagged it as a candidate double-dipper, which appears to have inaccurately doubled the TESS period to 13.34 days; (2) Gaia EDR3 2081737529891330560: we found 3.06 days with TESS and 6.64 days with ZTF; we suspect that TESS captured the 1/2-period harmonic and adopt the approximately double value from ZTF; (3) 2134851775526125696: for this star, we measured 1.91 days with TESS from Cycle 2, but noted that the signal appeared to be missing in Cycle 4; ZTF found a strong signal at 12.23 days and we adopt this as the star’s period. In the remaining overlap cases, we adopted the average between TESS and ZTF as the ﬁnal period. For these overlap stars, the median absolute deviation is 0.01 days, showing remarkable consistency between the surveys. For three stars, we failed to detect a period in TESS but recovered one from ZTF; in all cases the periods appear to be 13–16 days. These stars were: (1) Gaia EDR3 2129930258400157440, for which TESS showed a ﬂat light curve while ZTF yielded a 15.3-day period; (2) Gaia EDR3 2082376861542398336, LS found a 7.6-day period which we rejected during visual validation; we found 15.4 days with ZTF, and we suspect that the weak/rejected signal form TESS might have been a 1/2 period harmonic; (3) Gaia EDR3 2082397099429013120, similar to the previous case, we rejected a 6.7-day signal from TESS and recovered a 12.8-day period with ZTF. e-prints, arXiv:1711.03234 2018, A&A, 620, A172 Skumanich, A. 1972, ApJ, 171, 565 Skumanich, A. 1972, ApJ, 171, 565 Zhao, G., Zhao, Y.-H., Chu, Y.-Q., Jing, Y.-P., & Deng, L.-C. 2012, Research in Astronomy and Astrophysics, 12, 723 Zhao, G., Zhao, Y.-H., Chu, Y.-Q., Jing, Y.-P., & Deng, L.-C. 2012, R h i A d A h i 12 723 Smith, J. C., Morris, R. L., Jenkins, J. M., et al. 2016, PASP, 128, 124501 Zhou, G., Quinn, S. N., Irwin, J., et al. 2021, AJ, 161, 2 Smith, J. C., Stumpe, M. C., Jenkins, J. M., et al. 2017, Kepler Science Document, 8 Zicher, N., Barragán, O., Klein, B., et al. 2022, arXiv:2203.01750 [astro-ph] Soderblom, D. R., Jones, B. F., Balachandran, S., et al. 1993, AJ, Soderblom, D. R., Jones, B. F., Balachandran, S., et al. 1993, AJ, 106, 1059 Stephenson, C. B. 1959, PASP, 71, 145 Zieba, S., Zwintz, K., Kenworthy, M. A., & Kennedy, G. M. 2019, A&A, 625, L13 Stephenson, C. B. 1959, PASP, 71, 145 C. DISPOSITION HISTORY OF KOI-7913 The disposition of KOI-7913.01 has been debated: in q1_q17_dr25_koi the source was ﬂagged as a false positive, with the comment “cent_kic_pos—halo_ghost”. This comment and disposition were removed in the q1_q17_dr25_sup_koi data 20 Table 2. Candidate Cep-Her members observed by Kepler Table 2. Candidate Cep-Her members observed by Kepler Parameter Example Value Description dr2_source_id 2073765172933035008 Gaia DR2 source identiﬁer. dr3_source_id 2073765172933035008 Gaia (E)DR3 source identiﬁer. kepid 5641711 KIC identiﬁer. ra 297.40986 Gaia EDR3 right ascension [deg]. dec 40.89719 Gaia EDR3 declination [deg]. weight 0.041 Strength of connectivity to other candidate cluster members. v_l -0.51 Longitudinal galactic velocity [km s−1]. v_b -8.23 Latitudinal galactic velocity [km s−1]. x_pc -8035.4 Galactocentric X position coordinate [pc]. y_pc 331.4 Galactocentric Y position coordinate [pc]. z_pc 65.3 Galactocentric Z position coordinate [pc]. kic_dr2_ang_dist 0.298 Separation between KIC and Gaia DR2 positions [arcsec]. edr3_dr2_mag_diff 0.002 G-band difference between EDR3 and DR2 source match [mag]. NOTE—Table 2 is published in its entirety in a machine-readable format. One entry is shown for guidance regarding form and content. NOTE—Table 2 is published in its entirety in a machine-readable format. One entry is shown for guidance regarding form and content. Table 3. Rotation periods and kinematics for candidate RSG-5 and CH-2 members. Table 3. Rotation periods and kinematics for candidate RSG-5 and CH-2 members. Parameter Example Value Description dr3_source_id 2127562009133684480 Gaia (E)DR3 source identiﬁer. ra 291.02306 Gaia EDR3 right ascension [deg]. dec 46.43843 Gaia EDR3 declination [deg]. parallax 3.7099 Gaia EDR3 parallax [milliarcsec]. ruwe 0.981 Gaia EDR3 renormalized unit weight error. weight 0.087 Strength of connectivity to other candidate cluster members. v_l 2.78 Longitudinal galactic velocity [km s−1]. v_b -2.87 Latitudinal galactic velocity [km s−1]. x_pc -8068.5 Galactocentric X position coordinate [pc]. y_pc 256.0 Galactocentric Y position coordinate [pc]. z_pc 86.3 Galactocentric Z position coordinate [pc]. (BP-RP)0 -0.115 Gaia GBP-GRP color, minus E(GBP-GRP). (M_G)0 0.442 Absolute G-band magnitude, corrected for extinction. cluster CH-2 RSG-5 or CH-2. Prot_Adopted NaN Adopted rotation period if available, else NaN [days]. Prot_TESS NaN TESS rotation period if available, else NaN [days]. Prot_ZTF NaN ZTF rotation period if available, else NaN [days]. Prot_Confused NaN Boolean ﬂag; true when stars are photometrically blended. NOTE—Table 3 is published in its entirety in a machine-readable format. One entry is shown for guidance regard- ing form and content. release, which renamed the planet a “candidate”. NOTE—Table 3 is published in its entirety in a machine-readable format. One entry is shown for guidance regard- ing form and content. C. DISPOSITION HISTORY OF KOI-7913 In this note, we discuss the interpretation of these ﬂags (which do not apply to the system, according to the latest analysis). We also discuss how the relative on-sky positions of KOI-7913 A and KOI-7913 B affect the interpretation of the Kepler data. release, which renamed the planet a “candidate”. In this note, we discuss the interpretation of these ﬂags (which do not apply to the system, according to the latest analysis). We also discuss how the relative on-sky positions of KOI-7913 A and KOI-7913 B affect the interpretation of the Kepler data. release, which renamed the planet a “candidate”. In this note, we discuss the interpretation of these ﬂags (which do not apply to the system, according to the latest analysis). We also discuss how the relative on-sky positions of KOI-7913 A and KOI-7913 B affect the interpretation of the Kepler data. As described by Thompson et al. (2018), the “cent_kic_pos” ﬂag is an indication that the measured source centroid is offset from its expected location in the Kepler Input Catalog. The ﬁnal Kepler data validation reports, generated 2016 Jan 30, do not show this to be the case for KOI-7913. Moreover, the statistical signiﬁcance of any centroid offset is lower than for KOI-7368 and Kepler-1643 (which both show centroid offsets that are likely explained by the stellar variability). As described by Thompson et al. (2018), the “cent_kic_pos” ﬂag is an indication that the measured source centroid is offset from its expected location in the Kepler Input Catalog. The ﬁnal Kepler data validation reports, generated 2016 Jan 30, do not show this to be the case for KOI-7913. Moreover, the statistical signiﬁcance of any centroid offset is lower than for KOI-7368 and Kepler-1643 (which both show centroid offsets that are likely explained by the stellar variability). What of the “halo_ghost” ﬂag? This test measures the transit strength for the pixels inside the aperture, and compares it to that measured in the ring of pixels around said aperture (the “halo”). One usually expects the transit signal to be strongest in the What of the “halo_ghost” ﬂag? This test measures the transit strength for the pixels inside the aperture, and compares it to that measured in the ring of pixels around said aperture (the “halo”). One usually expects the transit signal to be strongest in the TRIO OF MINI-NEPTUNES IN CEP-HER 21 central aperture, rather than the halo. C. DISPOSITION HISTORY OF KOI-7913 Two types of false positive scenarios can change this and trigger the ﬂag: the ﬁrst is when optical ghosts from bright eclipsing binaries reﬂect off the CCD, and contaminate the target star. The second is when the PRF of nearby stars directly overlaps with the PRF of the target star (see Thompson et al. 2018, Section A.5.2). The most obvious explanation for KOI-7913 is the latter case, given that KOI-7913 B is ≈0.9 Kepler pixels away from Kepler-7913 A and so it usually part of the “halo”. Due to the on-sky orientation of KOI-7913 A and KOI-7913 B, the default “optimal aperture” selected in quarters 3, 7, 11, and 15 in fact included both stars, while for the remaining quarters KOI-7913 B was excluded from the optimal aperture but was included as part of the halo (see pages 35 through 71 of the data validation reports.) Given the orientation of the stars and the ≈1.5 pixel FWHM of the Kepler pixel response function, some blending between the two stars is present. The pointing geometries from quarters 3, 7, 11, and 15 however did not affect the observed transit depths, which is an indication that the crowding metric applied in the data products accurately correct the mean ﬂux level (Morris et al. 2017). Analysis of the target-pixel data that was separately acquired for KOI-7913 B also reveals a different stellar rotation period, and no hint of the transit signal. Given the orientation of the stars and the ≈1.5 pixel FWHM of the Kepler pixel response function, some blending between the two stars is present. The pointing geometries from quarters 3, 7, 11, and 15 however did not affect the observed transit depths, which is an indication that the crowding metric applied in the data products accurately correct the mean ﬂux level (Morris et al. 2017). Analysis of the target-pixel data that was separately acquired for KOI-7913 B also reveals a different stellar rotation period, and no hint of the transit signal. 22 Table 4. Priors and posteriors for the transit models with local polynomials removed. Param. Unit Prior Median Mean Std. Dev. C. DISPOSITION HISTORY OF KOI-7913 3% HDI 97% HDI ESS ˆR −1 Kepler-1643 P d N (5.34264;0.01000) 5.3426257 5.3426258 0.0000101 5.3426071 5.3426454 7884 1.1e-03 t(1) 0 d N (134.38;0.02) 134.3820 134.3820 0.0011 134.3799 134.3841 7390 3.7e-04 logRp/R⋆ – U(−6.215;0.000) -3.688 -3.689 0.021 -3.728 -3.653 4449 -7.8e-05 b – U(0;1 + Rp/R⋆) 0.583 0.578 0.051 0.485 0.673 4705 1.9e-04 u1 – Kipping (2013) 0.26 0.29 0.21 0.00 0.68 5324 7.9e-04 u2 – Kipping (2013) 0.32 0.31 0.32 -0.26 0.88 4908 8.4e-04 R⋆ R⊙ N (0.855;0.044) 0.851 0.851 0.045 0.766 0.933 7473 7.2e-04 logg cgs N (4.502;0.035) 4.507 4.507 0.035 4.442 4.576 6530 -1.4e-04 logσf – N (log⟨σ f ⟩;2.000) -8.520 -8.520 0.019 -8.556 -8.486 7966 2.1e-04 ⟨f⟩ – N (1.000;0.100) 1.000 1.000 0.000 1.000 1.000 7488 3.2e-04 Rp/R⋆ – – 0.025 0.025 0.001 0.024 0.026 4449 -7.8e-05 ρ⋆ g cm−3 – 1.94 1.95 0.19 1.60 2.31 6081 9.4e-05 Rp RJup – 0.207 0.207 0.012 0.184 0.231 6326 2.5e-04 Rp REarth – 2.32 2.32 0.13 2.06 2.59 6326 2.5e-04 a/R⋆ – – 14.31 14.32 0.47 13.49 15.23 6081 8.2e-05 cosi – – 0.041 0.040 0.005 0.032 0.049 4929 2.4e-04 T14 hr – 2.41 2.41 0.06 2.30 2.53 4774 5.3e-04 T13 hr – 2.23 2.23 0.07 2.11 2.36 4561 6.2e-04 KOI-7368 P d N (6.84294;0.01000) 6.8430344 6.8430341 0.0000125 6.8430107 6.8430574 10045 6.5e-05 t(1) 0 d N (137.06;0.02) 137.0463 137.0463 0.0014 137.0437 137.0489 10303 9.2e-05 logRp/R⋆ – U(−4.605;0.000) -3.760 -3.763 0.031 -3.819 -3.708 4043 6.3e-04 b – U(0;1 + Rp/R⋆) 0.508 0.500 0.064 0.380 0.612 4434 3.5e-04 u1 – Kipping (2013) 0.98 0.95 0.27 0.43 1.42 5809 -5.6e-05 u2 – Kipping (2013) -0.19 -0.16 0.31 -0.66 0.42 4387 2.6e-04 R⋆ R⊙ N (0.876;0.035) 0.874 0.874 0.036 0.804 0.938 9902 7.3e-04 logg cgs N (4.499;0.030) 4.503 4.502 0.030 4.445 4.557 7527 2.7e-05 logσf – N (log⟨σ f ⟩;2.000) -8.314 -8.314 0.012 -8.337 -8.292 10636 1.3e-03 ⟨f⟩ – N (1.000;0.100) 1.000 1.000 0.000 1.000 1.000 9742 -2.9e-04 Rp/R⋆ – – 0.023 0.023 0.001 0.022 0.025 4043 6.3e-04 ρ⋆ g cm−3 – 1.87 1.88 0.15 1.59 2.16 6829 3.4e-04 Rp RJup – 0.198 0.198 0.011 0.177 0.218 5676 2.8e-04 Rp REarth – 2.22 2.22 0.12 1.98 2.44 5676 2.8e-04 a/R⋆ – – 16.67 16.68 0.45 15.86 17.54 6829 3.3e-04 cosi – – 0.030 0.030 0.004 0.022 0.038 4518 5.4e-04 T14 hr – 2.79 2.79 0.07 2.65 2.93 4845 5.0e-04 T13 hr – 2.62 2.62 0.08 2.47 2.78 4575 3.1e-04 KOI-7913 P d N (24.27838;0.01000) 24.278553 24.278571 0.000263 24.278112 24.279085 4413 1.5e-03 t(1) 0 d N (154.51;0.05) 154.5121 154.5124 0.0063 154.4998 154.5237 5612 6.0e-04 logRp/R⋆ – U(−5.298;0.000) -3.599 -3.602 0.046 -3.689 -3.519 4290 5.6e-04 b – U(0;1 + Rp/R⋆) 0.312 0.298 0.153 0.005 0.523 2373 1.8e-03 u1 – Kipping (2013) 0.27 0.34 0.28 0.00 0.86 4491 -6.1e-05 u2 – Kipping (2013) 0.21 0.23 0.32 -0.31 0.86 5935 7.0e-04 R⋆ R⊙ N (0.790;0.049) 0.788 0.788 0.049 0.699 0.881 6847 2.8e-04 logg cgs N (4.523;0.043) 4.526 4.527 0.042 4.450 4.606 5714 6.6e-04 logσf – N (log⟨σ f ⟩;2.000) -7.197 -7.197 0.019 -7.230 -7.161 6976 1.4e-04 ⟨f⟩ – N (1.000;0.100) 1.000 1.000 0.000 1.000 1.000 6998 2.8e-04 Rp/R⋆ – – 0.027 0.027 0.001 0.025 0.030 4290 5.6e-04 ρ⋆ g cm−3 – 2.20 2.21 0.25 1.78 2.70 5357 5.6e-04 Rp RJup – 0.209 0.209 0.016 0.179 0.238 4882 1.3e-03 Rp REarth – 2.34 2.34 0.18 2.01 2.67 4882 1.3e-03 a/R⋆ – – 40.92 40.95 1.54 38.14 43.84 5357 6.6e-04 cosi – – 0.008 0.007 0.004 0.000 0.013 2344 1.9e-03 T14 hr – 4.39 4.40 0.21 3.98 4.76 3952 5.6e-04 T13 hr – 4.13 4.13 0.22 3.72 4.55 3632 7.6e-04 NOTE— ESS refers to the number of effective samples. NOTE— ESS refers to the number of effective samples. ˆR is the Gelman-Rubin convergence diagnostic. Logarithms in this table ar denotes a uniform distribution, and N a normal distribution. (1) The ephemeris is in units of BJKD (BJDTDB-2454833). C. DISPOSITION HISTORY OF KOI-7913 ˆR is the Gelman-Rubin convergence diagnostic. Logarithms in this table are base-e. U denotes a uniform distribution, and N a normal distribution. (1) The ephemeris is in units of BJKD (BJDTDB-2454833). Table 4. Priors and posteriors for the transit models with local polynomials removed.
https://openalex.org/W2798197276	http://www.sportedu.org.ua/html/journal/2015-N4/pdf-en/15mipicl.pdf	English	null	Quickness and endurance fitness of pedagogic college girl students under influence of cheer-leading	Physical education of students	2,015	cc-by	3,905	© Masliak I.P., 2015 http://dx.doi.org/10.15561/20755279.2015.0304 PHYSICAL EDUCATION OF STUDENTS PHYSICAL EDUCATION OF STUDENTS PHYSICAL EDUCATION OF STUDENTS QUICKNESS AND ENDURANCE FITNESS OF PEDAGOGIC COLLEGE GIRL STUDENTS UNDER INFLUENCE OF CHEER-LEADING Masliak I.P. Kharkiv State Academy of Physical Culture Kharkiv State Academy of Physical Culture Abstract. Purpose: to determine dynamic of quickness and endurance indicators of pedagogic college girl students under influence of cheer-leading. Material: in the research 385 girl students participated, who composed 3 control and 3 experimental groups. Quickness fitness was registered by indicators of 60 meters’ run (sec.); latent time of motor response (msec.); run on the sport during 5 sec (quantity of steps); tapping test (quantity of points). Level of endurance fitness was registered by results of 2000 meters’ run (min). Results: the most effective cheer-leading exercises, which positively influence on girl students’ endurance, have been determined. The most favorable age periods for training of quickness and endurance under influence of cheer-leading exercises have been found. The higher increment in quickness indicators was registered in 15 years old girls. The most substantial increment of endurance was registered in 16 years old girls. Conclusions: it is recommended to include cheer-leading exercises: basic movements, jump elements, constants, in variable components of girl students’ academic training program. Key words: physical education, students, quickness, endurance, cheer-leading. Introduction1 Physical education is an integral component of teaching, education and vocational training at higher educational establishments [10]. In process of future specialists’ training significant attention shall be paid to mastering of professional knowledge and skills, development of physical qualities and formation of healthy life style [15]. Modern students endure much less physical and much higher emotional-psychological loads, stress states, hypodynamia. It results in reduction of adaptation potentials of young people’s organisms and increase of morbidity. That is why the problem of increasing of students’ physical education effectiveness at the account of introduction of new and non traditional motor functioning’s kinds is rather topical. A number of authors note improvement of students’ health and motor fitness at the account of priority usage of volleyball [19, 23], light athletic [1], football [8], body-flex and pilates [20], swimming [17], aqua-fitness [6], futsal [22] in academic training program. Cheer-leading is one of innovative kinds of motor functioning. Cheer-leading, as a kind of sports, has appeared comparatively not long age and has acquired wide acclaim among USA youth, in European countries and in Ukraine. It has show character, renders different impacts; it is dynamic and can be practiced by wide age range of people [4, 16, 18]. With it cheer-leading does not require special equipment and apparatuses. It is especially important in conditions of social-economical crisis. A number of authors note in their works positive influence of cheer-leading of pre school age children’s physical condition [12]; on physical health, motor fitness and interest to physical culture of secondary schools’ pupils [2, 3, 13, 14]; physical condition and physical workability of first years girl students of technical university [11]; on formation of wide arsenal of motor skills of higher educational establishments’ students [5]; on development of specific physical qualities of college girl students [7, 21]. With it influence of cheer-leading exercises on complex manifestation of college girl students’ motor abilities has still been studied insufficiently. Purpose, tasks of the work, material and methods Purpose, tasks of the work, material and methods The purpose of the research is to determine dynamic of quickness and endurance indicators of pedagogic college girl students under influence of cheer-leading. The methods of the research: theoretical analysis and generalization of scientific-methodic literature; pedagogic experiment; pedagogic testing;; methods of mathematical statistic. Quickness fitness was registered by indicators of 60 meters’ run (sec.); latent time of motor response (msec.); run on the sport during 5 sec (quantity of steps); tapping test (quantity of points). Level of endurance fitness was registered by results of 2000 meters’ run (min). 24 The researches were conducted on the base of pedagogic college of Kharkov humanitarian-pedagogic institute. 385 first - third years girls students of physical culture profile participated in the researches. They composed 3 control and 3 experimental groups: 1st group – 1st year girl students; 2nd group – 2nd year girl students and 3rd group – 3rd year girl students. All girl students were the members of main and preparatory health group. The research was approved by ethic committee of Kharkov humanitarian pedagogic institute. In the course of experiment control groups’ girl students were trained by traditional physical education program. In variable component of experimental groups’ training process cheer-leading exercises were introduced (basic movements, jump elements, constants and so on). PHYSICAL EDUCATION OF STUDENTS Analysis of the received data and normative [9] showed that indicators of 60 meters’ run, latent period of motor response and run on the spot correspond to marl 2 points in all tested groups; tapping test corresponded to 4 points. Thus, level of quickness of 1st – 3rd year girl students in average corresponds to mark “unsatisfactory”. Analysis of data after experiment (see fig.1) showed that all quickness indicators of experimental groups’ girl students significantly and confidently improved (р <0.05 – 0.001). Fig 1. Indicators of quickness level in experimental groups before and after experiment: a – 30 meters’ run (sec.), b – run on the spot (quantity of steps), c – tapping test (quantity of points). Fig. 2. Indicators of motor response in experimental groups before and after experiment 0 5 0 5 0 5 0 5 0 5 0 5 0 5 0 5 0 a b c a b c a b c 1 группа 2 группа 3 группа до эксперимента посля эксперимента 0.23 0.24 0.25 0.26 0.27 0.28 0.29 1 группа 2 группа 3 группа мс до эксперимента после эксперимента 1st group 2nd group 3rd group before experiment after experiment before experiment after experiment ms 1st group 2st group 3st group 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 a b c a b c a b c 1 группа 2 группа 3 группа до эксперимента посля эксперимента с 1st group 2nd group 3rd group before experiment after experiment до эксперимента before experiment Fig 1. Indicators of quickness level in experimental groups before and after experiment: a – 30 meters’ run (sec.), b – run on the spot (quantity of steps), c – tapping test (quantity of points). Fig. 2. Indicators of motor response in experimental groups before and after experiment 0.23 0.24 0.25 0.26 0.27 0.28 0.29 1 группа 2 группа 3 группа мс до эксперимента после эксперимента before experiment after experiment ms 1st group 2st group 3st group 0.23 0.24 0.25 0.26 0.27 0.28 0.29 1 группа 2 группа 3 группа мс до эксперимента после эксперимента before experiment after experiment ms 1st group 2st group 3st group гру 3st group Fig. 2. Results of the researches Analysis of the received results of quickness testing (see table 1) showed absence of confident distinctions between indicators of girl students of experimental and control groups (р>0.05). Analysis of initial data permitted to detect the absence of definite system of distinctions in results (р>0.05). The exclusion were indicators of motor response and run on the spot, where distinctions in results of 1st and 2nd as well as between 2nd and 3rd groups were confident (р <0.05 – 0.001). Quickness indicators of experimental and control groups girl students before experiment Groups I II III Indicators X ± m 60 meters’ run (sec.) n 75 108 95 Experimental 10.75+0.08 10.70+0.07 10.84+0.05 n 23 34 50 Control 10.81+0.13 10.73+0.10 10.91+0.08 t 0.38 0.24 0.71 р >0.05 >0.05 >0.05 Latent time of motor response (msec.) Experimental 0.29+0.00 0.27+0.00 0.28+0.00 Control 0.29+0.01 0.27+0.00 0.28+0.00 t 0.62 0.30 0.73 р >0.05 >0.05 >0.05 Run on the spot (quantity of steps) Experimental 18.25+0.35 17.95+0.18 17.59+0.19 Control 18.74+0.38 17.74+0.28 17.22+0.27 t 0.94 0.65 1.10 р >0.05 >0.05 >0.05 Tapping test (quantity of points) Experimental 68.67+0.62 66.34+1.05 67.36+0.87 Control 67.39+1.63 66.74+1.29 66.54+1.34 t 0.73 0.24 0.51 р >0.05 >0.05 >0.05 25 PHYSICAL EDUCATION OF STUDENTS PHYSICAL EDUCATION OF STUDENTS Indicators of motor response in experimental groups before and after experiment Analysis of experimental data showed that in 60 meters’ run results of 1st year girl students improved by 2.32%, 2nd year girl students – by 2.33%, 3rd – by 2.39%; latent period of motor response improved by 10.34%; 7.40%; 7.14% accordingly; run on the spot – by 17.26%; 17.32%; 1.,99% accordingly; tapping test – by 6.61%; 8.90%; 3.88% accordingly. Thus, substantial changes took place in frequency of legs’ movements. The highest increment in quickness was observed in girls of 15 years old age. 26 Analysis of control groups’ data after experiment showed that their results also improved to some extent. But these changes were less significant than in experimental groups and were not confident (р>0.05). Results of tests were as follows: 60 meters’ run of 1st group girl students – improved by 1.48%, 2nd group – by 2.05%; 3rd group – by 1.83%); latent period of motor response – by 3.44%; 3.70%; 3.57% accordingly; run on the spot – by 6.24%; 4.79%; 4.52% accordingly; tapping test – by 2.83%; 2.68%; 4.68% accordingly. Analysis of repeated data in age aspect revealed confident difference in indicators of tapping test between 1st and 2nd as well as between 2nd and 3rd experimental groups (р <0.05-0.001). The rest indicators do not significantly differ in comparison with initial data. Comparison of repeated data of experimental groups with normative [9] showed increasing of results by 1 point of assessment scale in the following indicators: 60 meters’ run and tapping test (1st group’s girl students) and latent period of motor response (1st and 2nd groups’ girl students). As for the other indicators we observed their significant and confident improvement. But there were no changes by assessment scale. In our opinion it is explained by imperfectness of available assessment criteria. Comparison of control groups’ results showed analogous to experimental groups’ character of changes by assessment scale in the following indicators: 60 meters’ run and latent period of motor response. Thus, application of specially selected cheer-leading exercises in process of girl students’ physical education rendered positive influence on quickness level. The most substantial increment of results was noted in girls of 15 years old age. Analysis of endurance data revealed absence of confident differences between indicators of control and experimental groups (р> 0.05) (see table 2). PHYSICAL EDUCATION OF STUDENTS In age aspect we observed worsening of results with age (but statistically not confident р> 0.05). Exclusion were the data of 1st and 2nd experimental groups’ girl students, whose data were statistically different (р <0.001). Table 2 Endurance indicators of experimental and control groups’ girl students before experiment Groups I II III Indicators X ± m 2000 meters’ run (min.) n 75 108 95 Experimental 16.69+0.12 17.69+0.93 17.41+0.14 n 23 34 50 Control 16.89+0.21 16.70+0.17 17.03+0.19 t 0.81 1.04 1.59 р >0.05 >0.05 >0.05 T Endurance indicators of experimental and control groups’ girl students before experiment Comparison of 2000 meters’ run results with normative (governmental physical education program) showed that in all tested groups they are significantly lower than standards and are far from reaching the lowest limit. That is why their mark was 0 points. Analysis of results after application of cheer-leading (see fig.3) showed that endurance indicators in experimental groups significantly and confidently improved (р <0.001). For example, results of 1st year girl students improved by 6.59%, 2nd years – by 16.05%, 3rd year – by 11.37%. The highest increment in results of endurance training was observed in 16 years’ old girls. Analysis of analogous data of control groups’ girl students showed confident improvement of results in 2nd and 3rd groups (р <0.05 – 0.01). For example, 1st group girl students’ results improved by 2.13%; 2nd group – by 2.93%; 3rd group – by 4.46%. Percent increment of indicators in control groups was significantly lower than in experimental groups. It should be noted that in repeated results of control and experimental groups’ girl students we observed significant and confident domination of experimental groups’ results (р <0.001). 27 EDUCATION OF STUDENTS Fig.3. Indicators of endurance fitness of experimental groups’ girl students before and after experiment 13 14 15 16 17 181 группа2 группа3 группа мин. до эксперимента после эксперимента before experiment after experiment 1st group 2st group 3st group Min. мин. Min. Fig.3. Indicators of endurance fitness of experimental groups’ girl students before and after experimen Analysis of repeated results in age aspect showed that character of differences changed significantly in experimental groups. For example, indicators of 1st and 3rd groups became much better and it was statistically confident (р <0.001). Analysis of analogous indicators of control groups did not reveal significant changes in comparison with initial data. Conclusions: As a result of initial testing we detected “low” level of quickness and endurance of pedagogic college gi 1. As a result of initial testing we detected “low” level of quickness and endurance of pedagogic college girl students. 2 A li ti f h l di i i h i l d ti i fl d iti l i k d students. 2. Application of cheer-leading exercises in physical education influenced positively on quickness and students. 2. Application of cheer-leading exercises in physical education influenced positively on quickness and endurance of the tested contingent. 2. Application of cheer-leading exercises in physical education influenced positively on endurance of the tested contingent. The further researches in this direction can be realized by means of determination of cheer-leading influence on physical abilities of higher educational establishments’ girl students. The further researches in this direction can be realized by means of determination of cheer-leading influence on physical abilities of higher educational establishments’ girl students. A k l d PHYSICAL EDUCATION OF STUDENTS Comparison of repeated results of endurances testing with normative showed absence of changes by assessment scale: they equal to zero in all tested groups. Thus, application of specially selected cheer-leading exercises in physical training of girl students positively influenced on endurance fitness. The highest increment of results was observed in 16 years’ old girl students. Discussion Analysis of scientific-methodic literature showed that there is quite few works, devoted to cheer-leading implementation in physical education process in different educational establishments. Also quite a few works on problems of cheer-leading’s influence on different aspect of training and formation of students’ motor competence was found. In available works influence of cheer-leading exercises on pre school age children’s physical condition was analyzed [12];on physical health and motor qualities’ condition of secondary schools pupils [2, 3, 13, 14]; on level of physical condition and physical workability of technical university girl students [11]; on power and coordination training of college students [7, 21]. base of analysis and generalization of the conducted research’s results we supplement the data of T.M. On the base of analysis and generalization of the conducted research’s results we supplement the data of T.M. Bala [13] about positive influence of cheer-leading exercises on motor skills’ training; expanded the data of N.V. Kryvoruchko, I.P. Masliak [7, 21] about influence of cheer-leading on students’ motor abilities. For the first time we determined: the most suitable for training by cheer-leading exercises forms of girl students’ quickness; the most favorable age periods for training of quickness and endurance under influence of cheer- leading exercises. For the first time we determined: the most suitable for training by cheer-leading exercises forms of girl students’ quickness; the most favorable age periods for training of quickness and endurance under influence of cheer- leading exercises. References: 1. Andres A, Serbo E, Festriga S. Sportivno-orientovane fizichne vikhovannia studentiv [Sport oriented physical education of students]. Moloda sportivna nauka Ukraini, 2015;19:6-12. (in Ukrainian) 1. Andres A, Serbo E, Festriga S. Sportivno-orientovane fizichne vikhovannia studentiv [Sport oriented physical education of students]. Moloda sportivna nauka Ukraini, 2015;19:6-12. (in Ukrainian) 2. Bala TM, Maslyak IP. Zmina rivnia fizichnogo zdorov’ia shkoliariv 5-6-kh klasiv pid vplivom vprav cherlidinga [Change of physical health of 5-6 forms’ pupils under influence of cheer-leading exercises]. Moloda sportivna nauka Ukraini, 2011;15:10-15. (in Ukrainian) 2. Bala TM, Maslyak IP. Zmina rivnia fizichnogo zdorov’ia shkoliariv 5-6-kh klasiv pid vplivom vprav cherlidinga [Change of physical health of 5-6 forms’ pupils under influence of cheer-leading exercises]. Moloda sportivna nauka Ukraini, 2011;15:10-15. (in Ukrainian) 3. Bala TM, Maslyak IP. Zmina rivnia fizichnogo zdorov’ia shkoliariv 7-9-kh klasiv pid vplivom vprav cherlidinga [Change of physical health of 7-9 forms’ pupils under influence of cheer-leading exercises]. Sportivnij visnik Pridniprov’ia, 2011;2:21-23. (in Ukrainian) 3. Bala TM, Maslyak IP. Zmina rivnia fizichnogo zdorov’ia shkoliariv 7-9-kh klasiv pid vplivom vprav cherlidinga [Change of physical health of 7-9 forms’ pupils under influence of cheer-leading exercises]. Sportivnij visnik Pridniprov’ia, 2011;2:21-23. (in Ukrainian) Bala TM, Maslyak IP. Chirliding u fizichnomu vikhovanni shkoliariv [Cheer-leading in physical e schoolchildren], Kharkov; 2014 (in Ukrainian) 5. Zinchenko IA. Pobudova trenuval'nogo procesu sportsmeniv u cherlidingu na etapi specializovanoi bazovoi pidgotovki. Cand. Diss. [Building of sportsmen’s training process in cheer-leading at stage of specialized basic training. Cand. Diss.], Kharkov; 2013 (in Ukrainian) 5. Zinchenko IA. Pobudova trenuval'nogo procesu sportsmeniv u cherlidingu na etapi specializovanoi bazovoi pidgotovki. Cand. Diss. [Building of sportsmen’s training process in cheer-leading at stage of specialized basic training. Cand. Diss.], Kharkov; 2013 (in Ukrainian) 6. Kozina ZhL, Iermakov SS, Bazylyuk TA, Voloshina EV. Innovacionnye tekhnologii akvafitnesa igrovoj napravlennosti s primeneniem tekhnicheskikh ustrojstv v fizicheskom vospitanii studentok [Aqua-fitness innovative technologies of game orientation with application of technical devices in girl students’ physical education]. Physical education of students, 2012;1:42-46. (in Ukrainian) 7. Krivoruchko N, Maslyak I. Dinamika pokaznikiv rozvitku koordinacijnikh zdibnostej studentiv VNZ pid vplivom vprav chirlidingu [Dynamic of coordination indicators of HEE students under influence of cheer-leading exercises]. Moloda sportivna nauka Ukraini, 2013; 7: 87-91. (in Ukrainian) 7. Krivoruchko N, Maslyak I. Dinamika pokaznikiv rozvitku koordinacijnikh zdibnostej studentiv VNZ pid vplivom vprav chirlidingu [Dynamic of coordination indicators of HEE students under influence of cheer-leading exercises]. Moloda sportivna nauka Ukraini, 2013; 7: 87-91. (in Ukrainian) 8. Malyar EI. Acknowledgement The researches have been fulfilled in compliance with topical plan of scientific-research works of Kharkov State Academy of Physical Culture for 2013 - 2015 by topic 3.5.29. “Theoretical and applied principles of organization of physical training, physical fitness and physical condition of different population strata monitoring”. 28 Conflict of interests The author declares that there is no conflict of interests. References: Rozvitok profesijno vazhlivikh iakostej studentiv special'nosti «Opodatkuvannia» zasobami futbolu. Cand. Diss. [Training of professionally significant qualities of students (specialization “Taxation”) by means of football. Cand. Diss.], Lviv; 2008 (in Ukrainian) 9. Romanenko VA. Diagnostika dvigatel'nykh sposobnostej [Diagnostic of motor abilities], Donetsk: Don NU; 2005 (in Ukrainian) 10. Terenteva NM, Mameshina MA, Maslyak IP. Teoriia ta metodika fizichnogo vikhovannia [Theory a physical education], Kharkov, KSAPC; 2010. (in Ukrainian) 11. Timofeeva OV. Chirliding v sisteme fizicheskogo vospitaniia studentok [Cheer-leading in system physical education]. Teoriia i praktika fizicheskoj kul'tury, 2008;11:36-38. (in Russian) 12. Timofeeva TN,. Tatarinova OO, Vladimirova SM. Cherliding dlia doshkol'nikov [Cheer-leading for pre school age children]. Detskij sad: teoriia i praktika, 2011;10:68-75. (in Russian) 13. Bala T. Change in the level of strength and endurance development of 5-6 grades pupils under cheerleading exercises influence. Slobozhanskyi herald of science and sport , 2015; 3: 14-18. 13. Bala T. Change in the level of strength and endurance development of 5-6 grades pupils under cheerleading exercises influence. Slobozhanskyi herald of science and sport , 2015; 3: 14-18. 14. Bala TM. The influence of cheerleading exercises on the schoolchildren’s physical health of 5-9th forms. 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Pedagogics, psychology, medical biological problems of physical training and sports, 2012; 11: 90-93. http://dx.doi:10.6084/ m9.figshare.97369. Information about the author: Masliak I.P.; http://orcid.org/0000-0003-1306-0849; ira.maslyak@mail.ru; Kharkiv State Academy of Physical Culture; Klochkivska str. 99, Kharkiv, 61058, Ukraine. Cite this article as: Masliak I.P. Quickness and endurance fitness of pedagogic college girl students under influence of cheer-leading. Physical education of students, 2015;4:24-30. http://dx.doi.org/10.15561/20755279.2015.0404 The electronic version of this article is the complete one and can be found online at: http://www.sportpedu.org.ua/html/arhive-e.html This is an Open Access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited (http://creativecommons.org/licenses/by/3.0/deed.en). Received: 22.08.2015 Accepted: 29.08.2015; Published: 30.08.2015 Information about the author: Masliak I.P.; http://orcid.org/0000-0003-1306-0849; ira.maslyak@mail.ru; Kharkiv State Academy of Physical Culture; Klochkivska str. 99, Kharkiv, 61058, Ukraine. Cite this article as: Masliak I.P. Quickness and endurance fitness of pedagogic college girl students under influence of cheer-leading. Physical education of students, 2015;4:24-30. http://dx.doi.org/10.15561/20755279.2015.0404 The electronic version of this article is the complete one and can be found online at: http://www.sportpedu.org.ua/html/arhive-e.html This is an Open Access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited (http://creativecommons.org/licenses/by/3.0/deed.en). Received: 22.08.2015 Accepted: 29.08.2015; Published: 30.08.2015 30
https://openalex.org/W3012597117	https://air.unimi.it/bitstream/2434/713940/2/s41747-019-0130-5.pdf	English	null	Patient organ and effective dose estimation in CT: comparison of four software applications	European radiology experimental	2,020	cc-by	9,343	* Correspondence: angelo.vanzulli@ospedaleniguarda.it 3Department of Radiology, ASST Grande Ospedale Metropolitano Niguarda, Piazza Ospedale Maggiore, 3, 20162 Milan, Italy Full list of author information is available at the end of the article © The Author(s). 2020 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. Abstract Background: Radiation dose in computed tomography (CT) has become a topic of high interest due to the increasing numbers of CT examinations performed worldwide. Hence, dose tracking and organ dose calculation software are increasingly used. We evaluated the organ dose variability associated with the use of different software applications or calculation methods. Methods: We tested four commercial software applications on CT protocols actually in use in our hospital: CT-Expo, NCICT, NCICTX, and Virtual Dose. We compared dose coefficients, estimated organ doses and effective doses obtained by the four software applications by varying exposure parameters. Our results were also compared with estimates reported by the software authors. Results: All four software applications showed dependence on tube voltage and volume CT dose index, while only CT-Expo was also dependent on other exposure parameters, in particular scanner model and pitch caused a variability till 50%. We found a disagreement between our results and those reported by the software authors (up to 600%), mainly due to a different extent of examined body regions. The relative range of the comparison of the four software applications was within 35% for most organs inside the scan region, but increased over the 100% for organs partially irradiated and outside the scan region. For effective doses, this variability was less evident (ranging from 9 to 36%). Conclusions: The two main sources of organ dose variability were the software application used and the scan region set. Dose estimate must be related to the process used for its calculation. Keywords: Radiation (ionizing), Radiation dosage, Radiation protection, Software, Tomography (x-ra Key points Organ dose results obtained using different software applications are not always comparable. There is an increased interest in the risk associated with medical x-ray exposure associated with com- puted tomography (CT) scans. (2020) 4:14 (2020) 4:14 De Mattia et al. European Radiology Experimental (2020) 4:14 https://doi.org/10.1186/s41747-019-0130-5 De Mattia et al. European Radiology Experimental https://doi.org/10.1186/s41747-019-0130-5 European Radiology Experimental Open Access Patient organ and effective dose estimation in CT: comparison of four software applications Cristina De Mattia1, Federica Campanaro1, Federica Rottoli1, Paola Enrica Colombo1, Andrea Pola2, Angelo Vanzulli3* and Alberto Torresin1 Background Radiation dose in x-ray computed tomography (CT) has become a topic of high interest due to the increasing numbers of CT examinations performed worldwide [1– 5]. Studies underlined the increase over the years in the number of CT examinations resulting in an increase in the dose per capita for the population. For the USA, Brenner et al. [6, 7] reported that the number of CT ex- aminations per year rose from 2.8 million in 1981 to 20 million in 1995 and to 62 million in 2007. In Germany, from 1996 to 2012, the annual effective dose per capita Several commercial software applications allow estimating organ dose for CT examinations. Organ dose calculation software differs on the phantom and the calculation algorithm. The National Cancer Institute CT (NCICT) dosimetry system (National Cancer Institute, Bethesda, USA) [22– 24] uses hybrid voxel computational phantoms (Univer- sity of Florida family). In general, voxel phantoms are defined starting from the segmentation of CT images of patients with dimension close to the reference. Non- uniform rational basis-splines surfaces are introduced in hybrid phantoms to maintain the flexibility of stylised phantoms for anatomy modifications. In this way, it is possible to adapt the stylised phantom to the reference dimension indicated by the International Commission on Radiological Protection (ICRP) for both genders [25, 26]. In addition to the adult phantoms, the software also allows to select paediatric phantoms for newborn, 1, 5, 10 or 15 years of age. The NCICTX software (National Cancer Institute, Bethesda, USA) implements the same NCICT hybrid voxel computational phantoms family but enhanced to better adapt to the size of the patient under study, following the National Health and Nutrition Examination Survey (NHANES) IV database [27]. The NCICTX phantom li- brary contains 100 adult males, 93 adult females, 85 males and 73 females of paediatric age, with different mass and height combinations [28], defined starting from the NCICT phantom. The NEXO[DOSE]® software (Bracco Imaging, Milan, Italy) integrates NCICTX directly in the application, without external Internet connection. In this context, the first software applications for organ dose calculation were born. In general, all applications are based on the same principle: they use a set of organ doses, pre-calculated on single sections, typically 1-cm scans, which are combined to obtain the entire scan region and adjusted according to the exposure parameters in use [15, 17]. g p p [ ] To date, several software have been introduced to calcu- late the organ dose in CT. Since the 1990s, there has been an evolution of calculation methods and graphic presenta- tions, thus allowing for an easier use. When choosing a software application for organ dose CT calculation, we should consider phantoms, algorithms, reference device and validation (if available). Phantoms include the most elementary mathematical types up to hybrid voxel compu- tational ones, which allow more reliable estimates [18]. The calculation algorithm, combined with the scanner modelling, allows to create a set of dose coefficients used to calculate the organ dose. Page 2 of 16 De Mattia et al. European Radiology Experimental (2020) 4:14 De Mattia et al. European Radiology Experimental (2020) 4:14 for CT examinations has more than doubled [8]. A retrospective analysis carried out in Italy (Lombardy dis- trict) between 2004 and 2014 [9] showed a 39% increase in the number of CT examinations per 1,000 residents. Among the software applications we analysed, the CT- Expo (G. Stamm, Hannover and H.D. Nagel, Buchholz, Germany) is the only one that uses a family of mathem- atical phantoms (Adam, Eva, Child, Baby), for which the body surface and organs are expressed by equations. It is also able to simulate the modulation of the beam in the CT scan and to choose between axial and spiral mode [20]. It is an application usable in the Excel® (Microsoft Corporation, Redmond, United States) environment, based on the computational method developed by Stamm and Nagel [21] for the analysis of data collected in the survey conducted in Germany in 1999 and 2002. A CT scan involves a dose larger than the most com- mon radiographic procedures. Depending on the acquisi- tion setup, the dose to the organs included in the scan region was reported to range from 15 mSv for an adult to 30 mSv for a newborn, with an average of 2–3 scans per study [7]. With the increase in the collective dose for med- ical exposures, there has been an increase in publications focused on radiological risk estimation [10–12]. g [ ] Exposure from a diagnostic CT examination is referred to have a stochastic effect. An epidemiological study of radiation-induced tumour risk for patients undergoing CT procedures first requires an assessment of the dose deliv- ered to the organs and tissues exposed. The organ dose is defined as the dose received by the specific organ per unit of mass. It mainly depends on patient’s anatomy, scan re- gion, and scanner’s output. Its estimate is the basis for risk analysis. However, the dose to the organs is not an imme- diate information easy to be obtained. Samei et al. [13] de- fined its determination as a Holy Grail [13]. In the 1990s, the European Commission (Council Recommendation 1999/519/EC) encouraged the research of new methods to estimate the patient dose in CT. The general approach was the use of a Monte Carlo algorithm associated with an anthropomorphic phantom [14–16]. Some software use a limited set of these coefficients and a number of correction factors to adapt the result to the reference conditions variation, such as tube voltage or phantom [19]. By reference device we mean, the scanner model used to simulate the photons histories in the Monte Carlo code. Generally, these soft- ware applications present a list of devices from which the user can select the one of interest. Obviously, older soft- ware does not include new generation scanners. Virtual Dose, an application funded by the National In- stitute of Biomedical Imaging and Bioengineering (NIBIB, USA; https://www.nibib.nih.gov/), presents a ‘software as a service’ (SaaS) architecture, for which the application can be accessed remotely through a web-based interface, with- out the need to install the software locally [29, 30]. NEX- O[DOSE]® integrates the Virtual Dose functionality through a RESTful application program interface. Its li- brary includes a set of voxel phantoms representing men, women and children of different ages (newborn, 5, 10, and 15 years of age) [31, 32]. It also represents pregnant women, considering the three gestation trimesters, and obese patients with different mass index [33, 34]. The aim of this study was to compare these four com- mercial software applications (CT-Expo and NCICT as De Mattia et al. European Radiology Experimental (2020) 4:14 Page 3 of 16 Page 3 of 16 De Mattia et al. European Radiology Experimental stand-alone software applications, NCICTX and Virtual Dose, integrated within the NEXO[DOSE]® radiation dose monitoring system) in terms of dosimetric data variability, both as organ dose and as effective dose, using different calculation methods, including the simu- lation of different exposure CT parameters. stand-alone software applications, NCICTX and Virtual Dose, integrated within the NEXO[DOSE]® radiation dose monitoring system) in terms of dosimetric data variability, both as organ dose and as effective dose, using different calculation methods, including the simu- lation of different exposure CT parameters. NCICT and NCICTX report the dose for shallow mar- row tissue as opposed to the active marrow and not for the bone surface. These differences have to be taken into account in the software comparison. We analysed the dose results at four levels. First, we estimated how the organ dose changes by varying CT exposure parameters. As reported by Hall and Brenner [10], for a CT study, the organ dose depends on a num- ber of factors, such as the tube current and scanning time, the scan pitch, the tube voltage and the specific de- sign of the scanner. In the organ dose calculation for single-phase protocols, we investigated the results change modifying the exposure parameters one at a time, while keeping the others fixed. Specifically, we in- vestigated the influence of voltage, pitch, collimation and slice thickness on organ dose results. For each organ, we calculated the dose discrepancies as difference of the values obtained changing the analysed parameter. Discrepancies were then normalised to the results ob- tained using the parameter mainly set in the CT proto- cols used in our hospital and reported as percentage. For the voltage effect, we used these couples of values: 100 and 120 kVp, 80 and 100 kVp, 120 and 140 kVp. The values of 120, 100, and 120 kVp were taken as reference, respectively. To assess the impact of slice thickness, we compared the values of 1 and 3 mm, keeping as refer- ence the latter value, mostly used in our clinical routine. We compared the organ dose estimations resulting from setting the pitch values of 0.8, 1.0, and 1.4, the last one taken as reference. For the collimation we set 40 and 19.2 mm, keeping as reference 19.2 mm. We also investi- gated the influence of the scanner model. All the soft- ware analysed allowed to select vendor and scanner model. We selected the following scanner: Sensation 64 (Siemens, Syngo CT 2009E), Brilliance 64 (Philips, Host Version 3.5.5.1000), Brilliance 16 (Philips, Host Version 2.3.0.1781 ), SOMATOM Definition (Siemens, Syngo Variability due to exposure parameters change Variability due to exposure parameters change Variability due to exposure parameters change At the first level of analysis, we tested the effect of changing voltage, collimation, pitch, slice thickness, and scanner model. All the four software applications showed a depend- ence on the tube voltage, while only CT-Expo showed a de- pendence on the other parameters. The relative discrepancies found for each couple of voltages are shown in Table 3. Discrepancies were calculated for each organ but we reported in the table only the minimum and the maximum discrepancies found for each class of organs. For the CT-Expo software, the discrepancies found were always of the same magnitude, less than 1%, independent of the position of the organ in relation to the scan region. CTDIvol Volume computed tomography dose index CT 2012B). For each of them, we set pitch and collima- tion actually in use on that device. Only for the CT- Expo software, we were able to study the organ dose dis- crepancies between helical and axial mode, taking the helical mode as reference. For all four software applications, the discrepancies ob- tained moving from 120 to 100 kVp were within 10% for all the organs located inside the scan region or partially ir- radiated. From 100 to 80 kVp, the discrepancies were greater, especially for the organs outside the scan region and the distributed ones, with variations up to 17%. Using 140 kVp, organ dose results for CT-Expo, NCICT and NCICTX were the same obtained at 120 kVp. Only Virtual Dose showed variations by setting this voltage (Table 3). For the other three levels of analysis, the dose results were normalised to the volume CT dose index (CTDIvol) used in the calculation. Thus, dimensionless values were obtained, analogous to the dose coefficients on which the software applications here considered are based [29, 37]. At the second level of analysis, NCICT and Virtual Dose results were compared with dose coefficients re- ported by their authors. We used as reference the organ dose coefficients reported by Lee et al. [37] and by Ding et al. [29] for head, chest and abdomen-pelvis scans, for the ICRP reference male and female adult phantoms, with 120 kVp. For each body part and gender, the paired Student t test was used. The same test was used to com- pare NCICT and NCICTX. Only the CT-Expo software showed a dependence on the other exposure parameters. Methods European Radiology Experimental (2020) 4:14 Page 4 of 16 Table 1 Input data required by the software CT-Expo, NCICT, NCICTX, and Virtual Dose to estimate the organ dose for a CT study Data CT-Expo NCICT NCICTX Virtual Dose Gender (male/female) X X X X Age X X X X Patient habitus (weight, height) X X Scan start, scan end X X X X Scanner vendor and model X X X X Voltage (kVp) X X X X Filter (head/body) X X X X Axial or spiral mode X Current × time (mAs) X X X X Collimation (mm) X X X X Pitch X X X X CTDIvol (mGy) X X X Slice thickness (mm) X Current modulation X CTDIvol Volume computed tomography dose index Fourth, we calculated the effective dose, starting from organ dose results and multiplying our coefficients by the median CTDIvol for each area [39]. We used the weighting factors for organs and tissues provided by the ICRP 103 (2007) [40]. We added for comparison the ef- fective dose obtained multiplying the median dose- length product (DLP) by the conversion factors pro- posed by Huda et al. (k coefficients) [41–43]. Methods The study was evaluated by our Institutional Review Board and the requirement for informed consent was waived. We calculated the organ doses starting from single-phase CT protocols mainly used in our hospital for head, maxillofacial, chest and abdomen-pelvis exami- nations. The simulated scan region was derived from a representative sample of images stored in our picture ar- chiving and communication system while exposure pa- rameters were extracted from the dose tracking software NEXO[Dose]®. An example of the first and last slice of a chest CT study as well as of the scan regions set on the phantom are shown in Fig. 1. To calculate the organ dose, the software had to cap- ture acquisition parameters such as the tube voltage (kVp) and the tube current (mAs). The information re- quired by each software is reported in Table 1. For NCICTX and Virtual Dose software, where the user can select patient height and weight, we set the adult reference phantom given by ICRP 110 (2009) [35]. The four software applications do not consider the same organs for dose calculation. In addition, there are differences in organ definitions (Table 2). For example, the bone surface, one of the tissues for which the ICRP specifies the weighting factor in the effective dose calcu- lation, has a different meaning in each of the software applications analysed. CT-Expo refers to bone surfaces, but the phantom implemented does not actually have a different bone structure for the marrow and the surface [36]. Virtual Dose considers bone endosteum instead. Fig. 1 Examples of scan region on the different phantoms implemented in CT-Expo, NCICT, NCICTX, and Virtual Dose. The region to be set, in this case for a chest study, was defined by selecting images archived in our Picture Archiving and Communication System (PACS). We can see that the anatomical landmarks, used to fix the scan start and end, change according to the phantom used. In particular, CT-Expo phantom lacks anatomical details Fig. 1 Examples of scan region on the different phantoms implemented in CT-Expo, NCICT, NCICTX, and Virtual Dose. The region to be set, in this case for a chest study, was defined by selecting images archived in our Picture Archiving and Communication System (PACS). We can see that the anatomical landmarks, used to fix the scan start and end, change according to the phantom used. In particular, CT-Expo phantom lacks anatomical details De Mattia et al. Variability due to exposure parameters change Table 4 shows the discrep- ancies found. Variations were larger for organs at margins and outside the scan region. The major discrepancies were associated with the change of pitch and scanner model; as previously explained, the scanner model change involved the pitch and collimation adjustment. Using the CT-Expo software, the helical mode was as- sociated with a higher organ dose than that obtained with the axial mode, more marked for partially irradiated organs or those located outside the scan region, with a relative rise from 15 to 50%. Third, we compared the organ dose results obtained from the four software applications, setting the same pa- rameters, in particular the main used in our hospital for CT exam of head, chest, abdomen-pelvis and maxillo- facial. We calculated the range (difference between max- imum and minimum value) for each organ and reported it as a percent of the mean value. Organs were distin- guished between those completely located inside the scan region, those only partially irradiated, those just outside the scan region and those distributed throughout the body (e.g., skin, muscles), as already proposed by other authors [38]. For organs outside the scan region, only dose coefficients above 0.01 were reported. Comparison with authors’ dose coefficients Comparison with authors’ dose coefficients Comparison between dose coefficients calculated by NCICT software and dose coefficients reported by its authors [37] revealed differences as reported in Table 5. The same comparison for dose coefficients calculated by Virtual Dose and dose coefficients reported by its au- thors [29] is also reported in Table 5. De Mattia et al. European Radiology Experimental (2020) 4:14 Page 5 of 16 Table 2 List of the body parts considered by CT-Expo, NCICT, NCICTX, and Virtual Dose for organ dose estimation Body part CT-Expo NCICT NCICTX Virtual Dose Adrenals X X X X Bladder X X X X Blood vessels X X Bone endosteum X Bone marrow X X X X Bone surface X Shallow marrow X X Brain X X X X Breast X (F) X (M, F) X (M, F) X (M, F) Colon X X X Effective dose ICRP 103 X X X Effective dose ICRP 60 X X X X Extra-thoracic tissue X X Eye balls X X Gallbladder X X X X Gonads X X X X Heart X X (wall) X (wall) X Kidneys X X X X Lens X X X Liver X X X X Low large intestine X Lungs X X X X Lymph nodes X X Muscle X X X X Oesophagus X X X X Oral mucosa X X (oral cavity) X (oral cavity) X Pancreas X X X X Pituitary gland X X Rectum and sigmoid X X Remainder ICRP 103 X Remainder ICRP 60 X Salivary glands X X X X Skin X X X X Small intestine X X X X Spinal cord X X Spleen X X X X Stomach X X (wall) X (wall) X Thymus X X X X Thyroid X X X X Trachea X X Upper large intestine X Uterus (F)/prostate (M) X X X X F Female, ICRP International Commission on Radiological Protection, M Male De Mattia et al. European Radiology Experimental (2020) 4:14 Page 6 of 16 Page 6 of 16 De Mattia et al. European Radiology Experimental (2020) 4:14 Table 3 Organ dose variation according to tube voltage. We compared 100 and 120 kVp, 80 and 100 kVp, and 120 and 140 kVp keeping as reference respectively 120, 100, and 120 kVp. Comparison between NCICT and NCICTX Comparison between NCICT and NCICTX The results of the paired Student t test using these two software applications are shown in Table 5. They were not significantly different only for head female (p = 0.219) and abdomen-pelvis male areas (p = 0.127). Table 6 summarises the mean value and the relative range obtained using the four software applications ac- cording to body parts, taking into consideration the lo- cation of the organs with respect to the scan region. The range was wider for the partially irradiated organs and external to the scan region; for the organs inside the scan region, excluding oesophagus, spleen, and stomach, the range was generally within 35%. Comparison with authors’ dose coefficients For each organ implemented in the four software studied, we calculated the discrepancies as difference of the values obtained for each couple of voltages. These discrepancies are then normalised to the organ dose at reference voltage. We reported here only the minimum and the maximum values of discrepancy found for each class of organs subdivided according to the position relative to the scan region o o ga s subd ded acco d g to t e pos t o e at e to t e sca eg o CT-Expo (%) NCICT (%) NCICTX (%) Virtual Dose (%) 100–120 kVp (120 kVp as reference) Organs inside the scan region < 1 1–5 0–6 1–4 Partially irradiated organs < 1 3–8 3–9 0–6 Organs outside the scan region < 1 1–8 2–9 0–13 Distributed organs < 1 1–9 1–9 3–6 80–100 kVp (100 kVp as reference) Organs inside the scan region < 1 1–6 0–11 0–4 Partially irradiated organs < 1 3–8 2–16 1–6 Organs outside the scan region < 1 1–17 2–17 1–14 Distributed organs < 1 2–17 2–13 3–6 120–140 kVp (120 kVp as reference) Organs inside the scan region 0 0 0 2–8 Partially irradiated organs 0 0 0 6–11 Organs outside the scan region 0 0 0 8 Distributed organs 0 0 0 3–9% The disagreement obtained is higher for the organs at the edge of the scan region and it ranges generally be- tween 10 and 50%, but it can exceed the 100%. completely irradiated. Only NCICTX and NCICT con- sider the pituitary gland. Figure 3 shows the histogram of the dose coefficients obtained simulating the CT max- illofacial protocol. Figure 4 shows the histogram of the dose coefficients obtained simulating the CT chest protocol. Finally, Fig. 5 shows the histogram of the dose coefficients obtained simulating the CT abdomen-pelvis protocol. Comparison among the four software applications Figure 2 shows the histogram of the organ dose per CTDIvol unit (dose coefficient), obtained simulating the CT head protocol with the four software applications, using male and female reference phantoms. Among the considered organs, brain, pituitary gland and lens were Table 4 First level of analysis using CT-Expo: organ dose variations found according to slice thickness, pitch, collimation and scanner model. For the slice thickness, we compared 1 and 3 mm, keeping the latter as reference. We set pitch at 0.8, 1.0, and 1.4 (with 1.4 as reference), collimation at 19.2 and 40.0 mm (with 19.2 mm as reference). For each organ, we calculated the discrepancies as difference of the values obtained. These discrepancies were then normalised to the organ dose at reference condition. We reported here only the minimum and the maximum values of discrepancy found for each class of organs subdivided according to the position relative to the scan region p g Slice thickness (%) Pitch (%) Collimation (%) Scanner model (%) Organs inside the scan region 0–1 < 1 < 1 3–13 Partially irradiated organs 0–3 6–40 1–4 0–50 Organs outside the scan region 1–3 7–30 1–10 5–40 Distributed organs 0 2–6 < 2 1–2 Page 7 of 16 De Mattia et al. European Radiology Experimental (2020) 4:14 Table 5 Second level of analysis for NCICT and Virtual Dose software: comparison with authors’ dose coefficients and between NCICT and NCICTX, for head, chest, and abdomen-pelvis scans (120 kVp). For each body part and gender, we reported the results of the paired Student t test (top) and the range of the relative discrepancies for each class of organs (bottom). In the CT abdomen- pelvis scan, the authors of Virtual Dose software do not report the dose coefficient for the organs considered as outside the scan region eg o . Comparison among the four software applications NCICT/authors’ dose coefficient Virtual Dose/authors’ dose coefficient NCICT/NCICTX M F M F M F CT head study 0.117 0.109 0.343 0.089 0.047 0.219 CT chest study < 0.001 < 0.001 0.027 0.239 0.012 < 0.001 CT abdomen-pelvis study 0.054 0.015 0.089 0.002 0.127 0.018 CT head study Organs inside the scan region 1–2% 2–6% 1% 3% 13–25% 4–13% Partially irradiated organs 24–40% 44–72% 25–29% 16–22% 4–33% 6–180% Distributed organs 10–12% 21–30% 2–14% 10–13% 5–19% 0–21% CT chest study Organs inside the scan region 2–30% 2–33% 3–9% 3–8% 3–20% 6–17% Partially irradiated organs 19–260% 43–190% 0–4% 1–6% 20–75% 0–32% Organs outside the scan region 21–230% 80–200% 0–16% 4–19% 0–82% 1–100% Distributed organs 22–27% 32–34% 5–7% 4–5% 4–6% 1–5% CT abdomen-pelvis study Organs inside the scan region 0–400% 0–62% 0–640% 0–22% 0–24% 0–17% Partially irradiated organs 0% 0% 57–110% 47–83% 7–28% 10–200% Organs outside the scan region 0-3% 2% - - 9–11% 19–36% Distributed organs 19–52% 12–18% 19–33% 1–18% 14% 1–10% F Female, M Male Among the software applications analysed, only NCICTX and Virtual Dose allow to adapt the phantom to the habitus of the patient, while CT-Expo is the only one able to consider the current modulation and the mode (axial/spiral), taking into account the overranging effect. Contrary to what was expected [10], only CT-Expo de- pends on exposure parameters such as pitch, collimation and scanner model. Though this software is more detailed in the scanner-based modelling respect to the others, at the same time, it is the least realistic from the point of view of phantom anatomy, by using stylised phantoms. It reported the same dose value for oral cavity and salivary glands, for thymus and oesophagus, for pancreas and gallbladder. For the bone marrow, CT-Expo estimated a dose much higher than those estimated by the other three software applications. This is because the phantom imple- mented in CT-Expo does not have a specific representa- tion for the bone marrow and the bone surface, but it applies a correction factor to the dose received from the entire bone structure [36]. For each class of organs of Table 6, the median range of the dose coefficients and the first and third quartiles (in brackets) were as follows: organs inside the scan region, 26% (16–33%); partially irradiated organs, 80% (53–114%); organs outside the scan region, 94% (60–110%); distrib- uted organs, 33% (25–67%). Effective dose comparison Starting from the results of the four software applica- tions, we calculated the effective dose. In Fig. 6 we com- pare, through a histogram of effective doses, the software used with the value estimated using k coeffi- cient multiplied by the DLP for each area. We did not reported the study of maxillofacial since there are no k coefficients for this exam. Considering all the results for each body part, the standard deviation normalised to the mean value was 36% for head, 22% for chest, and 9% for abdomen-pelvis. Discussion Based on our results, we can note that for all the four software applications, the only decisive exposure parameters are tube voltage and CTDIvol, even if we have to stress that for CT-Expo, the discrepancies This study showed that the variability related to the use of different software applications and methods to calcu- late the organ dose in CT exams is significant. De Mattia et al. European Radiology Experimental (2020) 4:14 Page 8 of 16 found changing the tube voltage were always of the same magnitude and lower than 1%. This is probably explained by the use of conversion factors applied to a set of dose coefficients obtained at a reference voltage, while the other software applications have a set for each voltage. In the comparison with authors’ dose coefficient, the disagreement is due basically to the different Fig. 2 Organ dose per volume computed tomography dose index (CTDIvol) unit, obtained simulating the CT Head protocol with CT-Expo, NCICTX, NCICT, and Virtual Dose, using a male and a female phantom. These values are dose coefficients, obtained as organ dose (mGy) and CTDIvol (mGy) ratio. Brain, pituitary gland, and lens are completely irradiated. Only NCICTX and NCICT consider the pituitary gland. Virtual Dose does not consider lens Fig. 2 Organ dose per volume computed tomography dose index (CTDIvol) unit, obtained simulating the CT Head protocol with CT-Expo, NCICTX, NCICT, and Virtual Dose, using a male and a female phantom. These values are dose coefficients, obtained as organ dose (mGy) and CTDIvol (mGy) ratio. Brain, pituitary gland, and lens are completely irradiated. Only NCICTX and NCICT consider the pituitary gland. Virtual Dose does not consider lens found changing the tube voltage were always of the same magnitude and lower than 1%. This is probably explained by the use of conversion factors applied to a set of dose coefficients obtained at a reference voltage, while the other software applications have a set for each voltage. In the comparison with authors’ dose coefficient, the disagreement is due basically to the different De Mattia et al. European Radiology Experimental (2020) 4:14 Page 9 of 16 extent of the scan regions simulated by the software authors compared with those in use in our hospital. Discussion For example, in the abdomen-pelvis exam, NCICT au- thors specify that their scan extends from the liver up to the femoral heads, thus sparing gonads and rec- tum while in our hospital the scan extends beyond than ours for bladder, rectum, prostate/uterus, and go- nads, with discrepancies that exceed 100%. For Virtual Dose, gonads dose discrepancy exceeds 600%, only for male phantom, while for female, it is within 10%. These discrepancies depend on the different positions of male and female gonads in relation to the end of Fig. 3 Organ dose per volume computed tomography dose index (CTDIvol) unit, obtained simulating the CT maxillofacial protocol with CT-Expo, NCICTX, NCICT, and Virtual Dose, using a male and a female phantom. These values are dose coefficients, obtained as organ dose (mGy) and CTDIvol (mGy) ratio. Pituitary gland and lens are completely irradiated. Only NCICTX and NCICT consider the pituitary gland. Virtual Dose does not consider lens Fig. 3 Organ dose per volume computed tomography dose index (CTDIvol) unit, obtained simulating the CT maxillofacial protocol with CT-Expo, NCICTX, NCICT, and Virtual Dose, using a male and a female phantom. These values are dose coefficients, obtained as organ dose (mGy) and CTDIvol (mGy) ratio. Pituitary gland and lens are completely irradiated. Only NCICTX and NCICT consider the pituitary gland. Virtual Dose does not consider lens extent of the scan regions simulated by the software authors compared with those in use in our hospital. For example, in the abdomen-pelvis exam, NCICT au- thors specify that their scan extends from the liver up to the femoral heads, thus sparing gonads and rec- tum, while in our hospital, the scan extends beyond the pubic symphysis. Therefore, their values are lower than ours for bladder, rectum, prostate/uterus, and go- nads, with discrepancies that exceed 100%. For Virtual Dose, gonads dose discrepancy exceeds 600%, only for male phantom, while for female, it is within 10%. These discrepancies depend on the different positions of male and female gonads in relation to the end of the scan region. De Mattia et al. European Radiology Experimental (2020) 4:14 Page 10 of 16 Fig. 4 Organ dose per volume computed tomography dose index (CTDIvol) unit, obtained simulating the CT Chest protocol with CT-Expo, NCICTX, NCICT, and Virtual Dose, using a male and female phantom. These values are dose coefficients, obtained as organ dose (mGy) and CTDIvol (mGy) ratio. Discussion Lungs, heart, breast, thymus, oesophagus, and spleen are completely irradiated. CT-Expo considers the breasts only for the female phantom Fig. 4 Organ dose per volume computed tomography dose index (CTDIvol) unit, obtained simulating the CT Chest protocol with CT-Expo, NCICTX, NCICT, and Virtual Dose, using a male and female phantom. These values are dose coefficients, obtained as organ dose (mGy) and CTDIvol (mGy) ratio. Lungs, heart, breast, thymus, oesophagus, and spleen are completely irradiated. CT-Expo considers the breasts only for the female phantom Fig. 4 Organ dose per volume computed tomography dose index (CTDIvol) unit, obtained simulating the CT Chest protocol with CT-Expo, NCICTX, NCICT, and Virtual Dose, using a male and female phantom. These values are dose coefficients, obtained as organ dose (mGy) and CTDIvol (mGy) ratio. Lungs, heart, breast, thymus, oesophagus, and spleen are completely irradiated. CT-Expo considers the breasts only for the female phantom De Mattia et al. European Radiology Experimental (2020) 4:14 Page 11 of 16 Fig. 5 Organ dose per volume computed tomography dose index (CTDIvol) unit, obtained simulating the CT abdomen-pelvis protocol with CT- Expo, NCICTX, NCICT, and Virtual Dose, using a male and female phantom. These values are dose coefficients, obtained as organ dose (mGy) and CTDIvol (mGy) ratio. Liver, stomach, colon, bladder, prostate/uterus, gonads, spleen, pancreas, adrenals, kidneys, small intestine, gallbladder, and rectum are completely irradiated. Only NCICTX and NCICT consider trachea and rectum. CT-Expo considers the breasts only for the female phantom Fig. 5 Organ dose per volume computed tomography dose index (CTDIvol) unit, obtained simulating the CT abdomen-pelvis protocol with CT- Expo, NCICTX, NCICT, and Virtual Dose, using a male and female phantom. These values are dose coefficients, obtained as organ dose (mGy) and CTDIvol (mGy) ratio. Liver, stomach, colon, bladder, prostate/uterus, gonads, spleen, pancreas, adrenals, kidneys, small intestine, gallbladder, and rectum are completely irradiated. Only NCICTX and NCICT consider trachea and rectum. CT-Expo considers the breasts only for the female phantom Fig. 5 Organ dose per volume computed tomography dose index (CTDIvol) unit, obtained simulating the CT abdomen-pelvis protocol with CT- Expo, NCICTX, NCICT, and Virtual Dose, using a male and female phantom. These values are dose coefficients, obtained as organ dose (mGy) and CTDIvol (mGy) ratio. Liver, stomach, colon, bladder, prostate/uterus, gonads, spleen, pancreas, adrenals, kidneys, small intestine, gallbladder, and rectum are completely irradiated. Only NCICTX and NCICT consider trachea and rectum. Discussion European Radiology Experimental (2020) 4:14 Page 13 of 16 Table 6 Dose coefficients obtained as an average of the values calculated by the software CT-Expo, NCICT, NCICTX, and Virtual Dose. In brackets, the range is shown as the difference between maximum and minimum, divided by the mean value (Continued) Table 6 Dose coefficients obtained as an average of the values calculated by the software CT-Expo, NCICT, NCICTX, and Virtual Dose. In brackets, the range is shown as the difference between maximum and minimum, divided by the mean value (Continued) Table 6 Dose coefficients obtained as an average of the values calculated by the software CT-Expo, NCICT, NCICTX, and Virtual Dose. In brackets, the range is shown as the difference between maximum and minimum, divided by the mean value (Continued) DC (% relative range) DC (% relative range) Male Female Organs outside the scan region Maxillofacial Thyroid 0.046 (144%) 0.068 (131%) Chest Thyroid 1.449 (117%) 1.509 (111%) Salivary glands 0.138 (45%) 0.149 (100%) Oral cavity 0.119 (78%) 0.149 (97%) Pancreas 0.633 (108%) 0.825 (49%) Colon 0.132 (255%) 0.074 (85%) Gallbladder 0.654 (103%) 0.938 (54%) Abdomen-pelvis Thymus 0.064 (91%) 0.065 (76%) Trachea 0.037 (12%) 0.043 (20%) Distributed organs Head Bone marrow 0.060 (121%) 0.065 (112%) Skin 0059 (27%) 0.061 (30%) Maxillofacial Bone marrow 0.057 (130%) 0.064 (131%) Skin 0.05 (34%) 0.053 (32%) Chest Bone marrow 0.392 (20%) 0.458 (34%) Skin 0.328 (46%) 0.355 (52%) Abdomen-pelvis Bone marrow 0.56 (0%) 0.613 (30%) Skin 0.51 (17%) 0.47 (20%) Distributed organs parts, head, and chest, but the received dose is not so negligible. From NCICT and NCICTX applications, we expected comparable results: they were created by the same research group, with similar calculation algorithm but different phantom library. The differences found underline the importance of the phantom used. In fact, it is not easy to fix the same scan region on different phantoms, as evidenced by Fig. 1. Furthermore, the same organs can be represented with different shape, size, and position in the various phantoms, resulting in a different fractions of the irradiated organ if this is not completely included into the scan region. Several studies already confirmed that the dose estima- tion is very difficult for organs at the borders of the scan region [38, 44]. Discussion CT-Expo considers the breasts only for the female phantom De Mattia et al. European Radiology Experimental (2020) 4:14 Page 12 of 16 De Mattia et al. European Radiology Experimental (2020) 4:14 Page 12 of 16 Table 6 Dose coefficients obtained as an average of the values calculated by the software CT-Expo, NCICT, NCICTX, and Virtual Dose. In brackets, the range is shown as the difference between maximum and minimum, divided by the mean value Table 6 Dose coefficients obtained as an average of the values calculated by the software CT-Expo, NCICT, NCICTX, and Virtual Dose. In brackets, the range is shown as the difference between maximum and minimum, divided by the mean value Table 6 Dose coefficients obtained as an average of the values calculated by the software CT-Expo, NCICT, NCICTX, and Virtual Dose. In brackets, the range is shown as the difference between maximum and minimum, divided by the mean value DC (% relative range) DC (% relative ran Male Female Organs inside the scan region Head Brain 0.722 (33%) 0.777 (16%) Pituitary gland 0.589 (29%) 0.603 (12%) Lens 0.863 (28%) 0.908 (16%) Maxillofacial Lens 0.869 (27%) 0.909 (18%) Pituitary gland 0.541 (34%) 0.565 (6%) Chest Lungs 1.397 (25%) 1.498 (22%) Heart 1.386 (6%) 1.427 (30%) Breast 1.25 (29%) 1.368 (44%) Thymus 1.458 (13%) 1.507 (12%) Oesophagus 1.153 (70%) 1.266 (57%) Spleen 0.938 (67%) 1.192 (59%) Abdomen-pelvis Liver 1.215 (10%) 1.33 (27%) Stomach 1.18 (33%) 1.291 (56%) Colon 1.35 (25%) 1.451 (30%) Bladder 1.227 (28%) 1.323 (19%) Prostate/uterus 1.158 (14%) 1.173 (37%) Gonads 1.504 (26%) 1.169 (8%) Spleen 1.212 (13%) 1.378 (21%) Pancreas 1.115 (21%) 1.274 (42%) Adrenals 1.162 (26%) 1.255 (26%) Kidney 1.369 (10%) 1.533 (27%) Small intestine 1.276 (26%) 1.399 (24%) Gallbladder 1.085 (33%) 1.169 (45%) Rectosigmoide 1.165 (27%) 1.123 (16%) Partially irradiated organs Head Salivary glands 0.420 (92%) 0.416 (40%) Thyroid 0.032 (134%) 0.038 (97%) Oral cavity 0.376 (142%) 0.306 (165%) Maxillofacial Brain 0.56 (53%) 0.577 (46%) Salivary glands 0.571 (68%) 0.635 (77%) Oral cavity 0.477 (112%) 0.561 (68%) Chest Liver 0884 (70%) 1.077 (79%) Stomach 0.799 (82%) 0.971 (53%) Kidney 0.327 (153%) 0.462 (79%) Adrenals 0.745 (92%) 0.983 (51%) Abdomen-pelvis Breast 0.272 (85%) 0.265 (120%) Oesophagus 0.183 (163%) 0.184 (143%) Lungs 0.326 (47%) 0.334 (52%) Heart 0.407 (50%) 0.371 (87%) Page 13 of 16 De Mattia et al. Discussion For these organs, the scattering contribu- tion becomes important and a statistical error has to be taken into account, due to the Monte Carlo calculation uncertainties which increase while reducing the number of photons [45]. A difference of a few millimetres in the scan extent can change the dose result by some factors [36]. In terms of effective dose, variability due to the use of different software applications is less evident, except for head study for which the great percentage variation (36%) was due to the low mean effective dose. In fact, the larger dose ranges are for organs partially irradiated. Moreover, we can note that Virtual Dose returns the lowest effective dose for each scan area and that k coeffi- cients seems to be too small for head and chest area. The comparison among software applications showed that the variability of organ dose is lower for the completely irradiated organs than for organs partially irradiated or outside the scan region, for which the organ dose range increases. In this regard, the maxillo- facial CT study is very interesting since the principal organs involved are only partially irradiated. This study has some limitations. First, we must consider the choice of the scan region. In fact, the anatomical landmarks, useful to fix the scan start and end, changed with the phantom used. This can involve a scan region of different lengths and positions on the software applications analysed. Second, using CT-Expo, The range is very broad for thyroid in the chest CT study, and for salivary glands and oral mucosa in head and maxillofacial CT studies. These organs are at the border of the scan region and the scan margin definition is very critical by using different phantoms. The thyroid is always peripheral for the two mainly scanned body De Mattia et al. European Radiology Experimental (2020) 4:14 Page 14 of 16 Fig. 6 Effective dose histogram. For each software application, we estimated the effective dose starting from the organ dose coefficients multiplied by the median volume computed tomography dose index (CTDIvol) for head, chest, and abdomen-pelvis exams. The effective dose was obtained as sum of the organ doses weighted according to International Commission on Radiological Protection 103. We have added the values estimated using dose-length product conversion factors (k coefficients) Fig. 6 Effective dose histogram. Abbreviations CT C d CT: Computed tomography; CTDIvol: Volume CT dose index; DLP: Dose- length product; ICRP: International Commission on Radiological Protection; NCICT: National Cancer Institute CT Funding The authors state that this work has not received any funding. In conclusion, our study showed that (1) the organ dose value must be related to the software used and to the scan region set; (2) the dose coefficients reported in the literature for different anatomical areas represent a scan condition not always represen- tative of the protocols used in clinical practice; (3) the acquisition parameters, such scanner model, collimation, pitch and layer thickness, do not signifi- cantly influence the dose estimation made by the software; (4) the variation in the results related to the acquisition tube voltage is lower than that due to the use of different software. Discussion For each software application, we estimated the effective dose starting from the organ dose coefficients multiplied by the median volume computed tomography dose index (CTDIvol) for head, chest, and abdomen-pelvis exams. The effective dose was obtained as sum of the organ doses weighted according to International Commission on Radiological Protection 103. We have added the values estimated using dose-length product conversion factors (k coefficients) Fig. 6 Effective dose histogram. For each software application, we estimated the effective dose starting from the organ dose coefficients multiplied by the median volume computed tomography dose index (CTDIvol) for head, chest, and abdomen-pelvis exams. The effective dose was obtained as sum of the organ doses weighted according to International Commission on Radiological Protection 103. We have added the values estimated using dose-length product conversion factors (k coefficients) the user cannot directly enter the CTDIvol value. This is calculated on the basis of the mAs set. This implies that the CTDIvol value used in CT-Expo may be slightly different from the other software applications. Third, a possible bias could be due to the effect of scanner model variation on dose calculation, because of our choice to change also the other parameters such as pitch or colli- mation, based on the aim to recreate the real-world application of the scanners in analysis. Finally, we compared the dose values in relation to the voltage set, without changing the phantom used for the calculation. However, in clinical practice, the tube voltage is linked to the constitution of the patient. Acknowledgements The authors wish to thank Marco Ciboldi, Elena Magalotti, Steve Massey, and Gianluca Viganò for their contributions to this project. The authors wish to thank Marco Ciboldi, Elena Magalotti, Steve Massey, and Gianluca Viganò for their contributions to this project. Authors’ contributions CDM and FR analysed and interpreted the data and drafted the work. PEC, AT, and AP designed the work and interpreted the data. FC and FR designed and revised the work. AP, AT, and AV revised the work. All authors read and approved the final manuscript. Availability of data and materials The datasets used and analysed during the current study are available from the corresponding author on reasonable request. Ethics approval and consent to participate The study was evaluated by the Institutional Review Board (ASST Grande OspedaleMetropolitano Niguarda) and the requirement for informed consent was waived. Consent for publication l bl Competing interests The authors declare that they have no competing interests. References Brenner DJ, Hall EJ (2007) Computed tomography- an increasing source of radiation exposure. N Engl J Med 357:2277–2284. https://doi.org/10.1056/ NEJMra072149 7. Brenner DJ, Hall EJ (2007) Computed tomography- an increasing source of radiation exposure. N Engl J Med 357:2277–2284. https://doi.org/10.1056/ NEJMra072149 30. 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Turner AC, Zankl M, De Marco JJ et al (2010) The feasibility of a scanner- independent technique to estimate organ dose from MDCT scans: using CTDIvol to account for differences between scanners. Med Phys 37:1816– 1825. https://doi.org/10.1118/1.3368596 39. Turner AC, Zankl M, De Marco JJ et al (2010) The feasibility of a scanner- independent technique to estimate organ dose from MDCT scans: using CTDIvol to account for differences between scanners. Med Phys 37:1816– 1825. https://doi.org/10.1118/1.3368596 40. (2007). The 2007 Recommendations of the International Commission on Radiological Protection. 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.
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